WO1996030353A1 - Reversible protease inhibitors - Google Patents

Reversible protease inhibitors Download PDF

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Publication number
WO1996030353A1
WO1996030353A1 PCT/US1996/003844 US9603844W WO9630353A1 WO 1996030353 A1 WO1996030353 A1 WO 1996030353A1 US 9603844 W US9603844 W US 9603844W WO 9630353 A1 WO9630353 A1 WO 9630353A1
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WIPO (PCT)
Prior art keywords
optionally substituted
alkyl
hydroxy
hydrogen
group
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PCT/US1996/003844
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French (fr)
Inventor
James T. Palmer
David Rasnick
Jeffrey L. Klaus
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Arris Pharmaceutical Corporation
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Publication date
Application filed by Arris Pharmaceutical Corporation filed Critical Arris Pharmaceutical Corporation
Priority to JP8529509A priority Critical patent/JPH11503417A/en
Priority to NZ305626A priority patent/NZ305626A/en
Priority to PL96322409A priority patent/PL322409A1/en
Priority to AU53674/96A priority patent/AU713492B2/en
Priority to EP96910499A priority patent/EP0817778A1/en
Publication of WO1996030353A1 publication Critical patent/WO1996030353A1/en
Priority to NO19974403A priority patent/NO311573B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/20—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carbonic acid, or sulfur or nitrogen analogues thereof
    • C07D295/215—Radicals derived from nitrogen analogues of carbonic acid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/26—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C317/28—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to acyclic carbon atoms of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07F9/6533—Six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to novel reversible protease inhibitors
  • the inhibitors are selective for cysteine proteases
  • cysteine proteases contain a cysteine residue at the active site responsible for proteolysis Since cysteine proteases have been implicated in a number of diseases, including arthritis, muscular dystrophy, inflammation, tumor invasion, glomeruloneph ⁇ tis, malaria, and other parasite-borne infections, methods for selectively and irreversibly inactivating them provide opportunities for new drug candidates See, for example, Esser, R E et al , Arthritis & Rheumatism (1994) 37, 236, Meyers, M H M et al , Agents Actions (1993), 39 (Special Conference Issue), C219, Machleidt, W et al, Fibnnolysis (1992), 6 Suppl 4, 125, Sloane, B F et al , Biomed Biochim Acta (1991), 50, 549, Duffy, M J , Clin Exp Metastasis (1992), 10, 145, Rosenthal, P J , Wollish, W S , Palmer, J T
  • Aldehydes have been transformed into ⁇ , ⁇ -unsaturated esters and sulfones by means of the Wadsworth-Emmons-Horner modification of the Wittig reaction, shown below (Wadsworth, W S and Emmons, W D (J Am Chem Soc (1961), 83, 1733)
  • R alkyl, aryl, etc
  • EWG COOEt, SO j Me. etc
  • cysteine protease inhibitors include epoxysuccinyl peptides, including E-64 and its analogs (Hanada, K e a/., Agr ⁇ c Biol Chem (1978), 42, 523, Sumiya, S ef a/., Chem Pharm.
  • An aspect of this invention is a protease inhibitor comprising a targeting group linked through a two carbon atom chain to an electron withdrawing group, wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 ⁇ M
  • An additional aspect of this invention is a protease inhibitor comprising a targeting group linked either directly or through a linker selected from the group consisting of an intermediate carbon atom or a two carbon atom chain to a sulfone group group, wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 ⁇ M
  • a further aspect of this invention is a compound, preferably a protease inhibitor, of Formula I
  • n 0 to 13
  • A-B represents a linkage selected from -C(0)NR 3 -, -CH 2 NR 3 -, -C(0)CH 2 - and -NR 3 C(0)-, wherein R J is hydrogen or as defined below,
  • X represents a bond, methylene or the linkage -CH 2 CH(R 4 )-, wherein R 4 is hydrogen, alkyl or arylalkyl
  • Y is -CH(R 5 )- or -NR 5 -, wherein R 5 is hydrogen or as defined below,
  • Z is -(CH 2 ) 2 -, -C(R 6 )(R 7 )- or -N(R 7 )-, wherein R 6 is hydrogen or methyl and R 7 is as defined below,
  • Z 1 is -(CH 2 ) 2 -, -C(R 6 )(R 8 )- or -N(R 8 )-, wherein R 6 is hydrogen or methyl and R 8 is as defined below,
  • R 1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylaminosulfonyl, arylsulfonyl or heteroarylsulfony
  • R 7 and R ⁇ are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylamino, dialkylamino, u ⁇ edo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R 3 or R 5 forms a divalent radical selected from (C 3 ⁇ )m
  • R 2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, guanidino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, preferably wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 ⁇ M
  • An additional aspect of this invention is a compound, preferably a protease inhibitor, of Formula II
  • R 9 is cyano, -C(0)OR 1 °, -P(O)(OR 10 ) 2 , -S(O)(NR 10 )R 10 , C(0)R 11 , -S(0)R 11 , -C(0)NR 12 R 13 , -S(0) 2 NR 12 R 13 , -C(0)NHR 14 or -S(0) 2 NHR 14 , wherein each R 10 is independently hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyl
  • n, A, B, Y, Z, R 1 and R 10 are as defined above, and the pharmaceutically acceptable salts; individual isomers and mixtures of isomers thereof, preferably wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 ⁇ M.
  • a further aspect of this invention is a compound, preferably a protease inhibitor, of Formula III:
  • R 5 is hydrogen, methyl, fluoro or a group selected from Formulae (a) and (b) as defined above, and R 16 is a group selected from phenyl or (C 5 ⁇ )heteroaryl (which group is optionally substituted with at least one radical selected from aikylcarbamoyl, dialkylcarbamoyi, alkyloxycarbonyl, alkylsulfmamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, carbamoyi, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkylsulfamoyl, dialkylsulfamoyl, alkyloxysulfonyl, aryl, hetero
  • An additional aspect of this invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a cysteine protease inhibitor of the invention, or of an individual isomer, a mixture of isomers, or the pharmaceutically acceptable salt or salts thereof, in combination with one or more pharmaceutically acceptable excipients.
  • a further aspect of this invention is a method for treating a condition capable of amelioration by inhibition of a cysteine protease in an animal in need thereof, which method comprises administering to such animal a therapeutically effective amount of a cysteine protease inhibitor of the invention, or of an individual isomer, mixture of isomer, or the pharmaceutically acceptable salt or salts thereof.
  • Another aspect of this invention is a method for detecting a cysteine protease in a sample, which method comprises
  • An aspect of this invention are the processes for preparing the cysteine protease inhibitors of this invention
  • Figure 1 depicts Scheme I, the synthesis of Formula I compounds when X is a bond
  • the synthetic steps are as follows a) HC0 2 H, H 2 0, b) HBr/acetic acid, c) 4-methylmorphol ⁇ ne, isobutyl chloroformate, Mu-ROH, and d) chromatographic purification
  • the groups are as defined herein
  • Figure 2 depicts Scheme 2, the synthesis of Formula I compounds when X is a methylene group
  • the synthetic steps are as follows a) 4-methylmorphol ⁇ ne, isobutyl chloroformate, followed by NaBH 4 reduction in water/THF, b) CH 3 S0 2 CI, t ⁇ ethylamine, CH 2 CI 2 , c) R,SH, NaH, CH 3 OH, THF, heat, d) 4-chloroperbenzo ⁇ c acid, CH 2 CI 2 , e) HCI/dioxane or p-CH 3 C 6 H 4 S0 3 H/ether, and f) Mu-ROH, 4- met ylmorpholine, isobutyl chloroformate
  • Figure 3 depicts Scheme 3, the synthesis of Formula I compounds when X is a methylene group
  • the synthetic steps are as follows a) (CH 3 ) 3 CH 2 CH 2 SH, NaH, MeOH, THF, heat, b) 4-chloroperbenzo ⁇ c acid, c) (n-C 4 H 9 ) 4 N , THF, followed by BrCH 2 CI, heat, d) HCI/dioxane or 4-CH 3 C 6 H 4 S0 3 H/ether, and, e) 4-methylmorphol ⁇ ne, isobutyl chloroformate, Mu-PheOH
  • Figure 4 depicts Scheme 4, the synthesis of Formula II compounds
  • the synthetic steps are as follows a) Cl H 2 N * (CH 3 )OCH 3 , dicyclohexylcarboiimide, Et 3 N/CH 2 CI 2 , b) LiAIH ⁇ F, c) NaH/THF, d) Hcl/d ⁇ oxane/CH 2 CI 2 , e) 4-methylmorphol ⁇ ne, isobutyl chloroformate/THF, and f) H 2 , 5% Pd/C
  • Figure 5 depicts Scheme 5, the synthesis of Formula I compounds when X is an ethylene
  • Figure 6 depicts the synthesis of compounds of Formula II in which R 9 is -COOH
  • Figure 7 depicts the synthesis of compounds of Formula II in which R 9 is -P(O)(R 10 ) 2 .
  • the synthetic scheme is as follows: a) NaH THF; b) anhydrous p-CH 3 C 6 H 4 S0 3 H/ether; c) 4-methylmorpholine, isobutyl chloroformate/THF, and; d) H 2 , Pd/C.
  • Figure 8 depicts the synthesis of compounds of Formula II in which R 9 is -C(0)NHR 14 .
  • the synthetic scheme is as follows: a) NaOH/EtOH, followed by Hcl/H 2 0; b) benzylamine, dicyclohexylcarbodiimide, CH 2 CI 2 ; c) NaH/THF, diethyl benzylamidomethylenephosphonate; d) HCI/dioxane; e) 4-methylmorpholine, isobutyl chloroformate, THF; f) H 2 , Pd/C, and as an alternative preparation from carboxylates as synthesized via Scheme 6, above; and g) aniline, dicyclohexylcarbodiimide, CH 2 CI 2 .
  • Figure 9 depicts the general synthesis of compounds of Formula II.
  • Figure 10 depicts the synthesis of compounds of Formula III.
  • the synthetic steps are as follows: a) CH 3 CN or other suitable solvent, reflux; b) H 2 0, NaOH, followed by extraction into organic medium; c) phosphorane, THF (Wittig reaction); d) p-CH 3 C 6 H 4 S0 3 H, ether; e) Mu-PheOH, 4-methylmorpholine, isobutyl chloroformate, THF; and f) H 2 , Pd/C.
  • Alkyl as in alkyl, alkyloxy, alkylthio, alkylsulfonyl, aikylcarbamoyl, dialkylcarbamoyi, heteroarylalkyl, arylalkyl, and the like, means a straight or branched, saturated or unsaturated hydrocarbon radical having from 1 to 10 carbon atoms or the number of carbon atoms indicated (e.g., methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, ferf-butyl, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylallyl, ethynyl, 1-propynyl, 2-propynyl, etc.).
  • Alkyloxyphosphinyl and “dialkyloxyphosphinyl” mean the radicals -P(0)(OH)OR and -P(0)(OR) 2 , respectively, wherein R is alkyl as defined above.
  • Alkanoyl as in alkanoyl, alkanoyloxy, heterocycloalkylalkanoylamino, and the like, means the radical -C(0)R, wherein R is alkyl as defined above, having overall from 1 to 11 carbon atoms or the number of carbon atoms indicated (e.g., (C, ⁇ )alkanoyl includes the radicals formyl, acetyl, propionyl, isopropionyl, butyryl, isobutyryl, crotonoyl, isocrotonyl, etc.).
  • Aryl means an aromatic monocyclic or polycyclic hydrocarbon radical containing 6 to 14 carbon O 96/30353 US96/03844
  • aryl includes phenyl, naphthyl, anthracenyl, phenanthrenyl, 1 ,2,3,4-tetrahydro-5-naphthyl, 1 -oxo-1 , 2-d ⁇ hydro-5-naphthyl, 1-th ⁇ oxo-1 ,2-d ⁇ hydro-5-naphthyl, etc )
  • Aroyl means the radical -C(0)Ar, wherein Ar is aryl as defined above, having overall from 7 to 15 carbon atoms or the number of carbon atoms indicated (e g , (C 7 n)aroyl includes benzoyi, naphthoyi, etc )
  • Cycloalkyl as in cycloalkyl and cycloalkylalkyl, means a saturated or unsaturated, monocyclic or polycyclic hydrocarbon radical containing 3 to 20 carbon atoms or the number of carbon atoms indicated, wherein the carbon atom with the free valence is a member of a non-aromatic ring and any carbocyclic ketone and thioketone derivative thereof (e g , the term cycloalkyl is meant to include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, b ⁇ cyclo[2 2 2]octyl, 1 ,2,3,4-tetrahydro- 1 -naphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 9-fluorenyl, etc )
  • Halo means fluoro, chloro, bromo or lodo
  • Heterocycloalkyl as in heterocycloalkyl, heterocycloalkylalkanoylamino, heterocycloalkylcarbonyl, heterocycloalkylcarbonyl, and the like, means cycloalkyl as defined above wherein 1 to 5 of the indicated carbon atoms is replaced by a heteroatom chosen from N, O, S, P or As, wherein the atom with the free valence is a member of a non-aromatic ring, and any heterocychc ketone, thioketone, sulfone or sulfoxide derivative thereof, (e g , the term heterocycloalkyl is meant to include pipe ⁇ dyl, pyrrolidinyl, pyrrolinyl, imidazo dinyl, indolmyl, quinuclidinyl, morpholinyl, piperazinyl, ⁇ /-methylp ⁇ peraz ⁇ nyl, piperadinyl, 4,4-d ⁇ oxo
  • Heteroaryl means an aromatic monocyclic or polycyclic hydrocarbon radical containing overall from 5 to 14 atoms or the number of atoms indicated, wherein 1 to 5 of the indicated carbon atoms are replaced by a heteroatom chosen from N, O, S, P or As, wherein the atom with the free valence is a member of an aromatic ring, and any heterocychc ketone and thioketone derivative thereof (e g , the term heteroaryl is meant to include thienyl, furyl, pyrrolyl, py ⁇ midmyl, isoxazolyl, oxaxolyl, dolyl, benzo[b]th ⁇ enyl, isobenzofuranyl, punnyl, isoqumolyl, pterdinyl, py ⁇ midinyl, imidazolyl, py ⁇ dyl, pyrazolyl, pyrazinyl, 4-oxo-1 ,2-d ⁇ hydro-1 -
  • R ⁇ -Z-A- in which Y is -N(R 5 ), Z is -CH(R 7 )-, A is carbonyl and R 7 together with R 5 forms 1 ,2-diphenylenedimethylene means a group of following formula:
  • Substituted derivatives of the 1 ,2-phenylenedimethylene divalent radical may contain a hydroxy group on any carbon within the ring system or an oxo group on either of the unsaturated ring carbon atoms.
  • Phosphono means the radical -P(0)(OH) 2 .
  • Methylene as in “(C 3J) )methylene”and “(C 3 . 7 )methylene” mean a straight, saturated divalent radical having the number of carbon atoms indicated; "(C 3 ⁇ )methylene” includes trimethylene (-(CH 2 ) 3 -) and tetramethylene (-(CH 2 ) 4 -)
  • a preferred embodiment herein utilizes a proline residue as an A-B-Z group, wherein A-B represents CH 2 -NR 3 and R 3 together with either R 7 or R ⁇ form a C3 methylene.
  • the group R 1 -Y-Z-A- in which Y is -(NR 5 )-, Z is -CH(R 7 )-, A is carbonyl and R 7 together with R 5 forms trimethylene means a group of following formula:
  • Substituted derivatives of the trimethylene and tetramethylene divalent radicals may contain a hydroxy group, or a protected derivative thereof, or an oxo group on any of the ring carbon atoms. Suitable hydroxy protective groups are defined below.
  • Oxa(C 3 . 7 )methylene and "aza(C 3 . 7 )methylene” mean methylene as defined above wherein one of the indicated carbon atoms is replaced by an oxygen or nitrogen atom, respectively.
  • oxa(C 5 )methylene includes 3-oxapentamethylene (-CH 2 CH 2 OCH 2 CH 2 -) and 2-oxapentamethylene (-CH 2 OCH 2 CH 2 CH 2 -).
  • -CfOJNR ⁇ R 22 means the radical 4-morpholinylcarbonyl when R 21 and R 22 together form 3-oxapentamethylene and the radical 1-piperazinylcarbanoyl when R 21 and R 22 together form 3-azapentamethylene.
  • Adjacent as use in the phrase “R 7 together with an adjacent R 3 ", means that the atoms to which the R 7 and R 3 groups are respectively attached are in turn attached to one another
  • Animal includes humans, non-human mammals (e g , dogs, cats rabbits, cattle, horses, sheep, goats, swine, deer, etc ) and non-mammals (e g , birds, etc )
  • Disease specifically includes any unhealthy condition of an animal or part thereof and includes an unhealthy condition which may be caused by, or incident to, medical or veterinary therapy applied to that animal, i e , the "side effects" of such therapy
  • EWG Electrode withdrawing group
  • Preferred electron withdrawing groups are those which would similarly stabli ze ylides of the general formula (RO) 2 P(0)C(R)EWG Suitable electron withdrawing
  • Leaving group has the meaning conventionally associated with it in synthetic organic chemistry, i e , an atom or group displaceable under alkylatmg conditions, and includes halo and alkane- or arenesulfonyloxy, sucha mesyloxy, ethanesulfonyloxy, benzenesulfonyloxy and tosyloxy, and alkaesulfonylamino, alkanecarbonylamino, aminosulfonylamino, aminocarbonylamino and the like.
  • Isomerism is the phenomenon wherein compounds have identical molecular formulae but differ in the nature or sequence of bonding of their atoms or in the arrangement of theri atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “steroisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and stereoisomers that are nonsuperimposable mirror images are termed “enantiomers” or sometimes "optical isomers”. A carbon atom bonded to four nonidentical substituents is termed a "chiral center".
  • a compound with one chiral center has two enantiomeric forms of opposite chirality is termed a "racemic mixture".
  • a compound that has more than one chiral center has 2 ⁇ 1 enantiomeric pairs, where n is the number of chiral centers.
  • Compounds with more than one chiral center may exist as ether an individual diasteromer or as a mixture of diastereomers, termed a "diastereomeric mixture”.
  • Compounds of Formulae I, II and III can exist as individual steroisomers or mixtures of stereoisomers.
  • compounds of Formulae I, II and III contain a chiral center at the carbon to which the substituent R ⁇ is attached.
  • compounds of Formulae I, II and III in which Z is -C(R 6 )(R 7 ) contain a chiral center at the carbon to which the R 7 substituent is attached.
  • compounds of Formulae I, II and III in which n is 0 and Z is -C(R 6 )(R 7 ) will have two chiral centers and can exist as four individual stereoisomers or any mixture thereof.
  • Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center.
  • the substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog and then the absolute descriptor R is assigned if the three highest ranked substituents are arranged in space (with the fourth lowest ranked substituent directed away from the observer) from high to low priority in a clockwise sequence and the absolute descriptor S is assigned for a counterclockwise arrangement.
  • the absolute descriptor R or S is cited in parenthesis followed by a hyphen and the chemical name of the compound.
  • the absolute descriptor R or S is cited immediately after the appropriate locant.
  • Acyl radicals derived from naturally occurring amino acids are referred to as their amino acid radicals preceded by the descriptor L (e.g., L-phenylalanine).
  • the nonnatural enantiomers of amino acid acyl radicals are preceded by the descriptor D.
  • the amino acid side chains are the (S) or L-form, due to the stereospecificity of enzymes, although the D-forms may be used in some cases.
  • R 1 is 4-morphol ⁇ nylcarbonyl
  • R 8 is 2-phenylethyl and lies on the same side of the reference plane as the R 7 substituent
  • R 7 is benzyl
  • R 19 is phenylsulfonyl
  • R 8 is 2-phenylethyl and lies on the same side of the reference plane as the R 7 substituent, R 7 is benzyl and R 19 is ethoxycarbonyl
  • compositions of the invention are pure diasteromers
  • compositions contain mixtures of diasteromers
  • Preferred embodiments have greater than about 70% of a single disasteromer, with at least about 90% being particularly preferred
  • Protective group has the meaning conventially associated with it in synthetic organic chemistry, i e , a group which blocks a reactive site in a compound See for example Greene et al , Protective Groups in Organic Synthesis, 2nd Ed , John Wiley & Sons, 1991 , hereby incorporated by reference
  • hydroxy protective groups include heterocycloalkyl-carbonyl such as 4-morphol ⁇ nylcarbonyl and the like, aroyl such as benzoyi and arylalkyl such as benzyl and the like
  • am o protective groups include aryloxycarbonyl such as benzyloxycarbonyl and the like, aroyl such as benzoyi and the like and oxycarbonyl such as ethoxycarbonyl and 9-fluorenylmethoxycarbonyl and the like
  • guanidino protective groups include sulfonyl such as 2,3,5-t ⁇ methyl-4-methoxyphenyl-sulfon
  • cysteine protease-associated disorders pathological conditions associated with cysteine proteases
  • the condition is associated wtih increased levels of cysteine proteases, for example, arthritis, muscular distrophy, inflammation, tumor invasion, and glomerulonephritis are all associated with increased levels of cysteine proteases
  • the condition is associated with the appearance of an extracellular cysteine protease activity that is not present in normal tissue
  • a cysteine protease is associated with the ability of a pathogen, such as a virus, to infect or replicate in the host organism
  • cysteine protease associated disorders include, but are not limited to, arthritis, muscular distrophy, inflammation, tumor invasion, glomeruloneph ⁇ tits, malaria, Alzheimer's disease, cancer metastasis, trauma, inflammation, gingivitis, leishmaniasis, fila ⁇ asis, and other bacterial and parasite-borne infections
  • disorders associated with interleukin 1 ⁇ converting enzyme (ICE) are included
  • “Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use
  • “Pharmaceutically acceptable salts” means salts which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity
  • Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfunc acid, nitric acid, phosphoric acid, and the like, or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succtnic acid, malic acid, maleic acid, fumaric acid, tartatic acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzo ⁇ c acid, cmnamic acid, madelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethaned ⁇ sulfon ⁇ c acid, 2-hydroxyethanesulfon ⁇ c acid, benzene
  • Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases
  • Acceptabale inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydoxide
  • Acceptable organic bases include ethanolamine, diethanolamine, t ⁇ ethanolamine, tromethamine, ⁇ /-methylglucam ⁇ ne and the like
  • “Therapeutically effective amount” means that amount which when administered to an animal for treating a disease includes
  • the present invention relates to novel cysteine protease inhibitors Without being bound by theory, it is believed that the inhibitors bind to cysteine proteases based on the following scheme
  • the enzyme is thus reversibly inhibited by means of interactions between the R, Y and Z moieties of the inhibitor and the surface of the binding sites of the enzyme, and by means of hydrogen bonding interactions between the sulfone and active site ammo acid side chains
  • the inhibitors of the present invention inhibit cysteine proteases and do not inhibit serine, aspartyl, and zinc proteases
  • the protease inhibitors of the present invention may have activity against other types of proteases, such as serine, aspartyl or other metalloproteases, but to a lesser extent
  • the electron withdrawing properties of the sulfone group of Formula I polarize the electrons between the sulfone group and the carbon to which it is attached, thus permitting hydrogen bonding between itself and active site residues of a cysteine protease, to allow tight binding between the inhibitor and the cysteine protease, as is generally described below It is to be understood that there is presumably additional electron withdrawing or electron polarization occurring between the sulfur atom and the oxygen atoms, which allows the oxygen atoms to participate in hydrogen bonding with active site residues of the protease and thus contributing even further to the inhibition of the enzyme
  • cysteine protease inhibitor an inhibitor which inhibits cysteine proteases
  • cysteine protease inhibitors are specific to cysteine proteases, that is, they do not inhibit other types of protease such as serine, aspartyl, or other metalloproteases
  • cysteine protease inhibitors of the invention may inhibit other types of proteases as well
  • reversible herein is meant that the inhibitor binds non-covalently to the enzyme, and is to be distinguished from irreversible inhibition See Walsh, Enzymatic Reaction Mechanisms, Freeman & Co , N Y , 1979 "Reversible” in this context is a term understood by those skilled in the art
  • the reversible cysteine protease inhibitors are competitive inhibitors, that is, they compete with substrate in binding reversibly to the enzyme, with the binding of inhibitor and substrate being mutually exclusive
  • the stoichiometry of inhibition is 1 1 , that is, a single inhibitor molecule is sufficient to inhibit a single enzyme molecule
  • cysteine protease inhibitors herein are designed to bind reversibly to cysteine proteases This binding is accomplished by using peptide-based or peptidomimetic structures as targeting groups that mimic naturally occurring substrates and/or inhibitors "Peptidomimetic", for the purposes of this invention, means ammo acid or peptide-hke in structure but wherein one or more of the peptide linkages (i e , -C(O)NR-) is substituted by an isoste ⁇ c form, i e -CH 2 NR-, -C(0)CH 2 - or -NRC(O)- and/or wherein non-naturally occurring ammo acid substituents are present
  • Targeting group for the purposes of this application, means a peptide or peptidomimetic residue of the cysteine protease inhibitor that allows the binding of the inhibitor to a cysteine protease
  • the targeting group of a cysteine protease inhibitor comprises at least two ammo acid side chains or side chain analogs, linked via a peptide bond or isostere
  • the targeting group may comprise up to about 15 am o acids or analogs, although inhibitors are generally from about 1 to 7 ammo acids or analogs, since smaller inhibitors are usually desired in therapeutic applications
  • n is preferably from 0 to 13, with from 0 to 5 being preferred and from 0 to 3 being particularly preferred
  • the targeting group can be represented by a naturally or non-naturally occurring peptide residue of the following formula
  • R 8 and R 7 components represent naturally or non-naturally occurring am o acid analogs or substituents as is more fully described below
  • the targeting group of the inhibitor may also contain additional functional groups, as depicted by R 1 and described herein
  • ammo acid substituents of the targeting group interact with the surface binding sites of the protease to promote binding It is also believed that the ammo acid substituent proximal to the electron withdrawing group (e g , R 8 of the above formula) will occupy the S, position of the substrate binding site and therefore is designated the P, residue of the inhibitor Similarly, the next adjacent ammo acid substituent (e g , R 7 of the above formula) will occupy the S 2 position of the substrate binding site and is designated the P 2 residue of the inhibitor If present, additional am o acid substituents will occupy the S 3 , S 4 , etc positions of the substrate binding site and be designated as the P 3 , P 4 , etc residues of the inhibitor An additional targeting group may be attached to the electron withdrawing group and, if present, its ammo acid substituents will occupy the S,', S 2 ', etc positions of the substrate binding sites and are designated the P 3 ', P 4 ',
  • targeting groups for specific enzymes are determined by rules governing substrate specificity in cysteine proteases (e g , see “Protemase Inhibitors", in Research Monographs in Cell and tissue Physiology (1986), ed Barret ef a/ , Vol 12, Chapter 4 Inhibitors of Cysteine Proteinases, Daniel Rich, Elsevier, New York, and Thornberry ef al , supra , hereby expressly incorporated by reference)
  • ICE ⁇ nterleuk ⁇ n-1 converting enzyme accepts an aspartic acid substituent (i e , 2-carboxyethyl) at the P, position and an alanme (methyl), val e (isopropyl) or histidme (4- ⁇ m ⁇ dazolylmethyl) substituent at the P 2 position
  • Papain accepts a argmine, lysine, N- benzyloxycarbonyllysine (i e 4-benzyloxycarbonylam ⁇ nobuty
  • R 7 and R 8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylamino, dialkylamino, u ⁇ edo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, am o, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R 3 or R s forms a divalent radical selected from (C 3 ⁇ )
  • preferred R 7 and R 8 groups are the naturally occunng ammo acid side chains and homologous derivatives These include, but are not limited to, alanme (methyl), argmine (3- guanidmopropyl), asparagme (carbamoylmethyl), citurlline (3-ure ⁇ dopropyl), aspartic acid (carboxymethyl), cysteine (mercaptomethyl), glutamic acid (2-carboxyethyl), glutamine (2- carbamoylethyl), glycine (hydrogen), histidme (4- ⁇ m ⁇ dazolylmethyl), homophenylalanine (2- phenylethyl), homose ⁇ ne (2-hydroxylethyl), isoleucine ((1-methylpropyl), leucme (isobutyl), lysme (4- aminobutyl), methionine (2-methylth ⁇ oethyl), ⁇ -(1-naphthyl)alan ⁇ n
  • n 0 to 5
  • A-B represents a linkage selected from -C(0)NR 3 -, wherein R 3 is hydrogen or as defined below, Y is -N(R 5 )-, wherein R 5 is hydrogen or as defined below, Z is -(CH 2 ) 2 - or -C(R 6 )(R 7 )-, Z 1 is -CH(R 8 )-, R is hydrogen, alkyloxycarbonylalkanoyl of overall 3 to 10 carbon atoms, (C, 9 )alkoxycarbonyl, (C 2 10 )alkanoyl (optionally substituted with a radical selected from carboxy, (C, 9 )alkyloxycarbonyl and hetero(C 4 ⁇ )cycloalkyl(C 2 10 )alkanoylam ⁇ no),
  • alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylam o, dialkylammo, unedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino or a protected derivative thereof) (C 3 7 )cycloalkyl, (C 3 7 )cycloalkyl(C 1 5 )alkyl, py ⁇ dyl.
  • a radical selected from hydroxy, ammo, alkylam o, dialkylammo, unedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino or a protected derivative thereof
  • More preferred compounds of Formulae I, II and III are those in which n is 0 to 2, A-B represents a linkage selected from -C(0)NR 3 -, wherein R 3 is hydrogen or as defined below, Y is -N(R 5 )-, wherein R 5 is hydrogen or as defined below, Z is -(CH 2 ) 2 - or -C(R 6 )(R 7 )- (with the proviso that when n is 0, Z is not -(CH 2 ) 2 -), Z 1 is -CH(R 8 )-, R 1 is hydrogen, (C 4 ⁇ )alkoxycarbonyl, (C 2 ⁇ )alkanoyl (optionally substituted with a radical selected from carboxy, (C, 5 )alkyloxycarbonyl and hetero(C 4 ⁇ )cycloalkyl(C 4 ⁇ ) alkanoylam o), -C(0)NR 21 R 22 wherein R 21 and R 22 together form aza(C 2 - 6 )
  • R 8 and R 7 are independently (C 5 ⁇ )cycloalkyl, (C 5-6 )cycloalkylmethyl, 3-py ⁇ dyl, 2-th ⁇ enyl, 2-furyl, 4- ⁇ m ⁇ dazolyl, 3- ⁇ ndolyl, 3-pyr ⁇ dylmethyl, 2-th ⁇ enylmethyl, 2-furylmethyl,4- ⁇ m ⁇ dazolylmethyl, 3- ⁇ ndolylmethyl, (C*.
  • alkyl (optionally substituted with a radical selected from mercapto, carboxy, ammo, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyl, guanidino and hydroxy, or a protected derivative thereof), a group selected from phenyl, 1-naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, ammo, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R 3 or R 5 forms a divalent radical selected from (C 3Jl )methylene and 1,2-phenylened ⁇ methylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo)
  • Particularly preferred compounds of Formulae I, II and III are those in which n is 0 to 1, A-B represents a linkage selected from -C(0)NR 3 -, Y is -N(R 5 )-, wherein R 5 is hydrogen or as defined below, Z is -C(R ⁇ )(R 7 )-, is -CH(R 8 )-, R 1 is hydrogen, fert-butoxycarbonyl, benzyloxycarbonyl, acetyl, 3-carboxyprop ⁇ onyl, 3-methoxycarbonylprop ⁇ onyl, biotinylaminohexanoyl, phenylacetyl, benzoyi, dimethylammosulfonyl, benzylsulfonyl, 1-p ⁇ peraz ⁇ nylcarbonyl, 4-methylp ⁇ peraz ⁇ n- 1-ylcarbonyl or 4-morphol ⁇ nylcarbonyl, R 7 is 3-pyr ⁇ dylmethyl, 2-th ⁇ enyl
  • More particularly preferred compounds of Formulae I, II and III are those in which n is 0, A-B represents a linkage selected from -C(0)NH-, Y is -NH-, Z is -CH(R 7 )-, T is -CH(R 8 )-, R 1 is hydrogen, fer -butxoycarbonyl, benzyloxycarbonyl, biotinylaminohexanoyl, benzoyi, p ⁇ per ⁇ z ⁇ n-1-ylcarbonyl, 4-methylp ⁇ peraz ⁇ n-1-ylcarbonyl or 4-morphol ⁇ nylcarbonyl, R 7 is (C, 5 )alkyl, optionally substituted benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 3-pyr ⁇ d ⁇ nylmethyl or 2-methylsulfonylethyl, and R 8 is butyl, 2-phenylethyl or 2-methylsulfonylethyl
  • A-B represents a linkage selected from -C(0)NH-, Y is -NH-, Z is -CH(R 7 )-, Z 1 is -CH(R 8 )-, R 1 is 1-p ⁇ per ⁇ z ⁇ nylcarbonyl, 4-methyl- 1-p ⁇ peraz ⁇ nylcarbonyl or 4-morphol ⁇ nylcarbonyl; R 7 is optionally substituted benzyl, 1-naphthylmethyl or 2-naphthylmethyl, and R 8 is 2-phenylethyl
  • R 2 is independently (C,. 5 )alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro, hydroxy and methoxy, or a protected derivative thereof).
  • R 4 is hydrogen, (C, 5 )alkyl or (C 6 , 0 )aryl(C, 5 )alkyl
  • R 2 is (C, 5 )alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro and hydroxy, or a protected de ⁇ vative thereof), perfluoro(C, 5 )alkyl, (C 5 ⁇
  • R 9 is -C(0)OR 10 , -P(O)(OR 10 ) 2 , -S(O)(NR 10 )R 10 , -C(0)NHC(0)R 10 or -S(0) 2 NHC(0)R 10 are those in which each R 10 is independently (C, 5 )alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro, hydroxy and methoxy or a protected derivative thereof), (C 37 )cycloalkyl, (C 3 7 )cycloalkyl(C, 5 )alkyl, or a group selected from phenyl or phenyl(C 1-6 )alkyl (which group is optionally substituted at its phenyl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl, or a protected derivative thereof) More preferred compounds of
  • R 9 is C(0)R 11 or -S(0)R 11 are those in which R 11 is (C, 5 )alkyl, (C 3 7 )cycloalkyl, (C 3 7 )cycloalkyl(C 1 5 )alkyl or a group selected from phenyl and phenyl(C, ⁇ )alkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methyl, t ⁇ fluoromethyl and methoxy) More preferred compounds of Formula II in which R" is C(0)R" or -S(0)R 11 are those in which in which R 11 is ethyl, cyclo(C 5 ⁇ )alkyl, cyclo(C 5 ⁇ )alkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from ammo hydroxy
  • R 9 is -C(0)NR 12 R 13 or -S(0) 2 NR 1 R 13 are those in which R 12 and R 13 are independently (Chalky!, (C 3 . 7 )cycloalkyl, (C 37 )cycloalkyl (C, 5 )alkyl or a group selected from phenyl and pheny ⁇ C ⁇ alkyl (which group is optionally substituted at its phenyl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl) More preferred compounds of Formula II in which R 9 is -C(0)NR 12 R 13 or O 96/30353
  • -S(0) 2 NR 12 R 13 are those in which R 12 and R 13 are independently ethyl, (C 5 ⁇ )cycloalkyl, (C- ⁇ cycloalkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from amino hydroxy, chloro, bromo or fluoro, or a protected derivative thereof).
  • Preferred compounds of Formula II in which R 9 is -C(0)NHR 14 or -S(0) 2 NHR 14 wherein R 14 is a group selected from Formulae (a) and (b) are those in which each n, A, B, Y, Z, R 1 and R 10 are as defined above with respect to preferred compounds of Formulae I, II and III.
  • R 15 is a group selected from 2-furyl, 2-thienyl, 2-pyrrolyl, 2-phospholyl, 2-arsoyl, 3-pyridyl or 3-phosphorinyl (which group is optionally substituted with at least one radical selected from (C L ⁇ alkylcarbamoyl, di(C 1 . 5 )alkylcarbamoyl, (C 1 . 5 )alkyloxycarbonyl, (C,. 5 )alkylsulfinamoyl, di(C,.
  • R 16 is a group selected from 2-furyl, 2-thienyl, 2-pyrrolyl, 2-phosholyl, 2-arsolyl, 3-pyridyl or 3-phosphorinyl (which group is optionally substituted with at least one radical selected from methylcarbamoyl, dimethylcarbamoyl, methyloxycarbonyl, methylsulfinamoyl, dimethylsulfinamoyl, methylsulfonyl, carboxy, nitro, sulfmamoyl, sulfo, carbamoyi, phosphono, methyloxyphosphinyl, dimethyloxyphosphinyl, formyl, cyano, methylsulfinyl, sulfamoyl, methylsulfamoyl, dimethylsulfamoyl, methoxysulfonyl, methylsulfonimidoyl, phenyl
  • R 16 is a group selected from Formulae (a) and (b) are those in which each n, A, B, Y, Z, R 1 and R 10 are as defined above with respect to preferred compounds of Formulae I, II and III.
  • preferred cysteine protease inhibitors of the invention are those in which the absolute configuration of each chiral center present is the (S)-conf ⁇ guration.
  • preferred compounds of Formula I in which n is 0 are those in which the absolute configuration of chiral center to which the R 7 substituent is attached is in the (/ ⁇ -configuration.
  • preferred compounds of Formula I include: /V 2 -(4-mo ⁇ holinylcarbonyl)- ⁇ / 1 -(3-phenyl-1R-phenylsulfonylpropyl)-L-phenylalaninamide (compound 1 ), ⁇ / 2 -(4-morpholinylcarbonyl)- ⁇ / 1 -(3-phenyl-1 S-phenylsulfonylpropyl)-L-phenyalaninamide (compound 2), ⁇ / 2 -(4-morphol ⁇ nylcarbonyl)- ⁇ / 1 -(3-phenyl-1-phenylsulfonylpropyl)-L-phenylalan ⁇ nam ⁇ de (compound 3), ⁇ / 2 -(4-morphol ⁇ nylcarbonyl)-/V 1 -(3-phenyl-1-benzylsulfonylpropyl)-L-leuc ⁇ nam ⁇ de (compound 1
  • Formula I includes structures represented by preferred Species IV as depicted below.
  • M is zero, one or two carbon atoms, A-B are as defined above, R ⁇ R 2 , R 7 and R 8 are as defined above, and Q is NH or CH 2 .
  • Preferred embodiments utilize A-B linkages which contain nitrogen at the B position.
  • the number of carbon atoms between the carbon to which the R 8 group is attached and the sulfur atom of the sulfone group determines whether the compound is an ⁇ -am ⁇ nosulfone, a ⁇ -aminosulfone, or a ⁇ -aminosulfone.
  • compounds may be named as aminosulfones using the names of the amino acids or using the chemical names.
  • Species V is an ⁇ -aminosulfone:
  • Species VI is a ⁇ -aminosulfone:
  • Species VII is a ⁇ -aminosulfon
  • Formula II includes structures of Species VIII, referred to as ⁇ -amino groups, particularly when R 9 is an electron withdrawing group:
  • the dissociation constant for inhibition of a protease with an inhibitor of the invention is at most 100 ⁇ M.
  • binding constant or "dissociation constant” or grammatical equivalents herein is meant the equilibrium dissociation constant for the reversible association of inhibitor with enzyme.
  • the dissociation constants are defined and determined as below.
  • E-l enzyme-inhibitor complex
  • k is the second order rate constant for the formation of the E-l reversible complex
  • k 2 is the first order rate constant for the disassociation of the reversible E-l complex
  • K, k 2 /k
  • K, values may be estimated using the Dixon plot as described by Irwin Segel in Enzyme Kinetics 1 Behavior and analysis of rapid equilibrium and steady-state enzyme systems, 1975, Wiley-lnterscience Publication, John Wiley & Sons, New York, or for competitive binding inhibitors from the following calculation
  • v 0 is the rate of substrate hydrolysis in the absence of inhibitor, and v, is the rate in the presence of competitive inhibitor.
  • dissociation constants are a particularly useful way of quantifying the efficiency of an enzyme with a particular substrate or inhibitor, and are frequently used in the art as such If an inhibitor exhibits a very low K*. it is an efficient inhibitor Accordingly, the cysteine protease inhibitors of the present invention have dissociation constants, K,, of at most about 100 ⁇ M Preferred embodiments have inhibitors that exhibit dissociation constants of at most about 10 ⁇ M, with the most preferred embodiments having dissociation constants of at most about 1 ⁇ M
  • a) is a) CI-H 2 N+(Me)OMe, dicyclohexylcarbodiimide, t ⁇ ethylamine, and b) lithium aluminum hydride
  • N-tert-butoxycarbonyl ammo acid or peptidomimetic derivative thereof is converted to the corresponding ammomethyl aldehyde (e g , see method of Fehrentz, J-A and Castro, B (Synthesis, (1983), 676, Equation 5)
  • the aldehyde is converted to the corresponding vmylogous compound via aWittig reaction or a Wadsworth-Emmons-Horner modification of the Wittig reaction (e g , see Wadsworth et al , J Amer Chem Soc 83 1733 (1991), Equation 6)
  • the vmylogous compound is reduced by catalytic hydrogenation (e g , see Equation 7) and then deprotection and coupling with a suitable N-protected ammo acid or peptide or a peptidomimetic derivative thereof gives the corresponding compound of Formula I or II
  • the vmylogous compound is deprotected and coupled with the N
  • the conversion of N-tert-butoxycarbonyl ammo acid or peptidomimetic derivative thereof to the corresponding ammomethyl aldehyde is carried out with N,0-d ⁇ methylhydroxylam ⁇ ne hydrochlo ⁇ de in the presence of t ⁇ ethylamine and dicyclohexylcarbodiimide in dicloromethane
  • the conversion is carried out by treating the ammo acid or peptidomimetic derivative with t ⁇ ethylamine and the coupling agent benzot ⁇ azol-1-yloxyt ⁇ s(d ⁇ methylam ⁇ no)phosphon ⁇ um hexafluorophosphate (BOP) and then reducing with lithium aluminum hydride to give the corresponding aldehyde (e g , see methos of Fehrentz, J-A and Castro, B , Synthesis, (1983), 676-678)
  • the conversion of the aldehyde to the corresponding vmylogous ester can be carried out with the
  • a suitable N-tert-butoxycarbonyl- ⁇ -ammoaldehyde prepared as described in Equation 5, with the sodium anion of an appropriate sulfonylmethanephosphonate (SMP) (e g, diethyl phenylsulfonylmethanephosphonate, diethyl 2-naphthylsulfonylmethane-phosphonate, diethyl methylsulfonylmethanephosphonate, etc ) gives the corresponding vmylogous sulfone
  • SMP sulfonylmethanephosphonate
  • sulfonylmethanephosphonate e g, diethyl phenylsulfonylmethanephosphonate, diethyl 2-naphthylsulfonylmethane-phosphonate, diethyl methylsulfonylmethanephosphonate, etc
  • SMP s
  • Suitable amidomethylenephosphonates can be prepared by reacting the saponification product of tnethyl phosphonoacetate with an appropriate amme
  • compounds of Formula II in which R 9 is -C(0)NHR 14 can be prepared by reacting a compound of Formula I in which R 9 is -COOH with an appropriate amme
  • the reaction can be carried out in the presence of dicyclohexylcarbodiimide in dichloromethane or by any other peptide coupling reaction sequences known to those of skill in the art.
  • ketones is synthesized by means of the Wadsworth-Emmons reaction between Boc- ⁇ - ammo aldehydes and the appropriate phosphonate, followed by catalytic reduction with hydrogen in the presence of palladium.
  • the aldehyde portion is synthesized as outlined above.
  • the phosphonate if not commercially available, is synthesized by treatment of the enolate anion of methyl or substituted methyl ketones, such as acetone or acetophenone, with diethyl chlorophosphonate.
  • the enolate anion is generated, for example, by treatment of a tetrahydrofuran solution of diisopropylamine with butyllithium, followed by addition of the ketone to the lithium diisopropylamide (LDA) solution (H.O. House, Modern Synthetic Reactions, 2nd Ed. (W. Benjamin, Inc., Menlo Park, CA, Chapter 9). Following formation of the enolate, diethyl chlorophosphonate is added. The Wadsworth-Emmons reagent forms as a consequence of coupling of the enolate with diethyl chlorophosphate.
  • LDA lithium diisopropylamide
  • Synthesis of sulfoxides is performed by means of the Wadsworth-Emmons reaction between Boc- ⁇ - ammo aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst
  • the aldehyde portion is synthesized as outlined above
  • the phosphonate is synthesized by treatment of the anion of methyl sulfoxides with diethyl chlorophosphate The anion is generated by addition of BuLi to diisopropylam e, followed by addition of the methyl sulfoxide
  • Synthesis of sulfonamides is performed by means of the Wadsworth-Emmons reaction between Boc- ⁇ -ammo aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst
  • the aldehyde portion is synthesized as outlined above
  • the phosphonate is synthesized, for instances, by a method such as the following a) diethylphosphoryl methanesulfonates, as prepared by the method of Carretero and Ghosez (Tetrahedron Lett , 28 1104- 1108 (1987)), are converted to sulfonyl chlorides by treatment with phosphorus pentachlo ⁇ de (M Quaedvlieg, in "Methoden der Organische Chemic (Houben-Weyl)", ed.
  • Synthesis of sulfoximines is performed by means of the Wadsworth-Emmons reaction between Boc- ⁇ - ammo aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst.
  • the aldehyde portion is synthesized as outlined above
  • the phosphonate may be synthesized in several ways. For example, N-alkyl or N-aryl phenyl methyl sulfoximines are made by the methods described by Johnson, in "Comprehensive Organic Chemistry (Pergamon Press), supra, Chapter 11.11.
  • the lithium anion of compounds such as N-alkyl phenyl methyl sulfoximine is prepared by the treatment of the neutral compound with buthyl lithium in THF (Cram ef al., J. Amer. Chem. Soc. 92:7369 (1970)). Reaction of this lithium anion with dialkyl chlorophosphates such as the commercially available diethyl chlorophosphate (Aldrich) results in the Wadsworth-Emmons reagent necessary for synthesis of the sulfoximme compounds
  • sulfonates is performed by means of the Wadsworth-Emmons reaction between Boc- ⁇ - ammo aldehydes and the appropriate phosphonate, for instance diethylphosphoryl methanesulfonate, followed by hydrogenation in the presence of a suitable catalyst, such as Raney nickel
  • the phosphonate may be synthesized as follows The anion of methyl dialkyl phosphonates such as the commercially available methyl diethyl phosphonate (Aldrich) is generated by treatment of said phosphonate with a strong base such as LDA The resulting anion is sulfonated with sulfur t ⁇ oxide/trimethylamine complex (Carreto ef al , Tetrahedron Lett , 28 1104-1108 (1987)) to form diethylphosphoryl methanesulfonate, which is capable of reacting in the Wadsworth-Emmons procedure with aldehydes to form ⁇ , ⁇ -unsaturated sulfon
  • the chloride compounds containing R 8 and R 9 groups are generally made using commercially available reagents and products using techniques well known in the art
  • the reaction generally produces a mixture of cis and trans configurations, favoring the trans isomer
  • the cysteine protease inhibitors of this embodiment Upon reduction to the cysteine protease inhibitors of this embodiment, the cis-trans isome ⁇ sm disappears by definition as a single compound is formed
  • cysteine protease inhibitors of the invention are further purified if necessary after synthesis, for example to remove unreacted materials
  • the cysteine protease inhibitors of the present invention may be crystallized, or passed through silica chromatography columns using solvent mixtures to elute the pure inhibitors
  • R 20 is cyano, -S(0) 2 R 2 , -CH 2 S(0) 2 R 2 , -CH 2 CH(R 4 )S(0) 2 R 2 , -(CH 2 ) 2 C(0)OR 10 , -(CH 2 ) 2 P(O)(OR 10 ) 2 , -(CH 2 ) 2 S(O)(NR 10 )R 10 , -CH 2 ) 2 C(0)R 11 , -(CH 2 ) 2 S(0)R 11 , -(CH 2 ) 2 C(0)NR 12 R 13 , -(CH 2 ) 2 S(0) 2 NR 12 R 13 , -(CH 2 ) 2 C(0)NHR 14 , -(CH 2 ) 2 S(0) 2 NHR 14 or -CH 2 CHR 15 R 16 and each A, B, X, Y, Z, R 1 , R 8 R 1 , R 8 , R 2 , R 10 , R 11 , R 12 , R 13
  • cysteine protease inhibitors of the present invention are labelled.
  • a labelled cysteine protease inhibitor herein is meant a cysteine protease inhibitor that has at least one element, isotope or chemical compound attached to enable the detection of the cysteine protease inhibitor or the cysteine protease inhibitor bound to a cysteine protease.
  • labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes
  • the labels may be incorporated into the cysteine protease inhibitor at any position
  • a label may be attached as the "R 1 " group in Formula 1 , or a radioisotope incorporated into any position
  • useful labels include 14 C, 3 H, biotm, and fluorescent labels as are well known in the art
  • cysteine protease inhibitors of the present invention may be easily screened for their inhibitory effect
  • the inhibitor is first tested against the cysteine protease for which the targeting group of the inhibitor was chosen, as outlined above
  • many cysteine proteases and their corresponding chromogenic substrates are commercially available
  • cysteine proteases are routinely assayed with synthetic chromogenic substrates in the presence and absence of the cysteine protease inhibitor, to confirm the inhibitory action of the compound, using techniques well known in the art
  • the effective inhibitors are then subjected to kinetic analysis to calculate the K, values and the dissociation constants determined
  • a compound inhibits at least one cysteine protease, it is a cysteine protease inhibitor for the purposes of the invention
  • Preferred embodiments have inhibitors that exhibit the correct kinetic parameters against at least the targeted cysteine protease
  • cysteine protease is not commercially available in a purified form
  • the cysteine protease inhibitors of the present invention may also be assayed for efficacy using biological assays
  • the inhibitors may be added to cells or tissues that contain cysteine proteases, and the biological effects measured
  • the cysteine protease inhibitors of the present invention are synthesized or modified such that the in vivo and in vitro proteolytic degradation of the inhibitors is reduced or prevented Generally, this is done through the incorporation of synthetic ammo acids, derivatives, or substituents into the cysteine protease inhibitor Preferably, only one non-naturally occurring am o acid or am o acid side chain is incorporated into the cysteine protease inhibitor, such that the targeting of the inhibitor to the enzyme is not significantly affected
  • some embodiments that use longer cysteine protease inhibitors containing a number of targeting residues may tolerate more than one synthetic derivative
  • non-naturally occurring ammo acid substituents may be designed to mimic the binding of the naturally occurring side chain to the enzyme, such that more than one synthetic substituent is tolerated
  • peptide isosteres are used to reduce or prevent inhibitor degradation
  • the resistance of the modified cysteine protease inhibitors may be tested against a variety of known commercially
  • cysteine proteases that may be inhibited by the inhibitors of the present invention are those of the family of cysteine proteases that bear a thiol group at the active site These proteases are found in bacteria, viruses, eukaryotic microorganisms, plants, and animals Cysteine proteases may be generally classified as belonging to one of four or more distinct superfamilies
  • cysteine proteases that may be inhibited by the novel cysteine protease inhibitors of the present invention include, but are not limited to, the plant cysteine proteases such as papain, ficin, aleuram, oryzam and actmidam, mammalian cysteine proteases such as cathepsms B, H, J, L, N, S, T, O, and C, (cathepsin C is also known as dipeptidyl peptidase I), interleukm converting enzyme (ICE), calcium-activated neutral proteases, calpam I and II, bleomy ⁇ n hydro
  • inhibitors of cysteine proteases are useful in a wide variety of applications
  • the inhibitors of the present invention are used to quantify the amount of cysteine protease present in a sample, and thus are used in assays and diagnostic kits for the quantification of cysteine proteases in blood, lymph, saliva, or other tissue samples, in addition to bacterial, fungal, plant, yeast, viral or mammalian cell cultures
  • the sample is assayed using a standard protease substrate
  • a known concentration of cysteine protease inhibitor is added, and allowed to bind to a particular cysteine protease present
  • the protease assay is then rerun, and the loss of activity is correlated to cysteine protease activity using techniques well known to those skilled in the art
  • the cysteine protease inhibitors are also useful to remove or inhibit contaminating cysteine proteases in a sample
  • the cysteine protease inhibitors of the present invention are added to samples where proteolytic degradation by contaminating cysteine proteases is undesirable
  • the cysteine protease inhibitors of the present invention may be bound to a chromatographic support, using techniques well known in the art, to form an affinity chromatography column A sample containing an undesirable cysteine protease is run through the column to remove the protease
  • the cysteine protease inhibitors are useful for inhibiting cysteine proteases implicated in a number of diseases
  • cathepsms B, L, and S, cruzain, calpains I and II, and mterleukin 1 ⁇ converting enzyme are inhibited
  • These enzymes are examples of lysosomal cysteine proteases implicated in a wide spectrum of diseases characterized by tissue degradation
  • diseases include, but are not limited to, arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, parasite-borne infections, Alzheimer's disease, pe ⁇ odontal disease, and cancer metastasis
  • mammalian lysosomal thiol proteases play an important role in intracellular degradation of proteins and in the processing of some peptide hormones
  • Enzymes similar to cathepsms B and L are released from tumors and may be involved in tumor metastasis
  • Cathepsin L is present in diseased human synovial fluid and transformed tissues
  • the cysteine protease inhibitors also find application in a multitude of other diseases, including, but not limited to, gingivitis, malaria, leishmaniasis, fila ⁇ asis, and other bacterial and parasite-borne infections
  • the compounds also offer application in viral diseases, based on the approach of inhibiting proteases necessary for viral replication
  • many picornoviruses including poliovirus, foot and mouth disease virus, and rh ovirus encode for cysteine proteases that are essential for cleavage of viral polyprotems
  • ICE ⁇ nterleuk ⁇ n-1 ⁇ converting enzyme
  • ICE cysteine protease responsible for processing mterleukin 1 ⁇
  • cardiovascular system including the pericardium, gastrointestinal and urogenital systems, the skin and the mucosal membranes
  • infectious diseases where active infection exists at any body site, such as meningitis and salpmgitis, complications of infections including septic shock, disseminated mtravascular coagulation, and/or adult respiratory distress syndrome, acute or chronic inflammation due to antigen, antibody and/or complement deposition, inflammatory conditions including arthritis, chalangitis, colitis, encephalitis, endocarditis, glomerulonephritis, hepatitis, myocarditis, pancreatitis, pericarditis, reperfusion injury and vascu
  • infectious diseases where active infection exists at any body site, such as meningitis and salpmgitis, complications of infections including septic shock, disseminated mtravascular coagulation, and
  • cysteine protease inhibitors of the present invention find use in drug potentiation applications
  • therapeutic agents such as antibiotics or antitumor drugs can be inactivated through proteolysis by endogeneous cysteine proteases thus rendering the administered drug less effective or inactive
  • cysteine protease inhibitors of the invention may be administered to a patient in conjunction with a therapeutic agent in order to potentiate or increase the activity of the drug This co-administration may be by simultaneous administration, such as a mixture of the cysteine protease inhibitor and the drug, or by separate simultaneous or sequential administration
  • cysteine protease inhibitors have been shown to inhibit the growth of bacteria, particularly human pathogenic bacteria (see Bjorck et al , Nature 337 385 (1989)) Accordingly, the cysteine protease inhibitors of the present invention may be used as antibacterial agents to retard or inhibit the growth of certain bacteria
  • the cysteine protease inhibitors of the invention also find use as agents to reduce the damage of bacterial cysteine proteases to host organisms
  • staphylococcus produces a very active extracellular cysteine protease which degrades insoluble elastin, possibly contributing to the connective tissue destruction seen in bacterial infections such as septicemia, septic arthritis and otitis
  • the cysteine protease inhibitors of the invention may be used to treat bacterial infections to prevent tissue damage
  • cysteine protease inhibitors of this invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with another cysteine protease inhibitor of the invention or with another therapeutic agent
  • a therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors.
  • Therapeutically effective amounts of the cysteine protease inhibitors of this invention may range from 10 micrograms per kilogram body weight ( ⁇ g/kg) per day to 10 milligram per kilogram body weight (mg/kg), typically 100 ⁇ g/kg/day to 1 mg/kg/day.
  • a therapeutically effective amount for a 80 kg human may range from 1 mg/day to 1000 mg/day, typically 10 mg/day to 100 mg/day.
  • cysteine protease inhibitors of this invention will be administered as pharmaceutical compositions by one of the following routes: oral, systemic (e.g., transdermal, intranasal, intrapulmonary, or by suppositiory) or parenteral (e.g., intramuscular, intravenous, intrapulmonary or subcutaneous).
  • routes e.g., oral, systemic (e.g., transdermal, intranasal, intrapulmonary, or by suppositiory) or parenteral (e.g., intramuscular, intravenous, intrapulmonary or subcutaneous).
  • Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixiers, aerosols or any other appropriate composiiton and are comprised of, in general, a cysteine protease inhibitor of the invention in combination with at least one pharmaceutically acceptable excipient.
  • Acceptable excipients are non-toxic, aid administration, and don not adversely affect the therapeutic benefit of the cysteine protease inhibitor of this invention.
  • excipient may be any solid, liquid, semisolid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.
  • Solid pharmaceutical excipients include starch, cellulose, talc, glucolse, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium sterate, glycerol monostearate, sodium chloride, dried skim milk and the like.
  • Liguid and semisolid excipients may be selected from water, ethanol, glycerol, propylene glycol and various oils, includingthose of petroleum, animal, vegetable or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.).
  • Preferred liquid carriers, particularly for injectable solutions include water, saline, aqueous dextrose and glycols.
  • Compressed gases may be used to disperse the cysteine protease inhibitor of this invention in aerosol form.
  • Inert gases suitable for this purpose are nitrogen, carbon dioxide, nitrous oxide, etc.
  • Other suitable pharmaceutical carriers and their formulations are described in A.R. Alfonso Reminton's Pharmaceutical Sciences 1985, 17th ed. Easton, Pa.: Mack Publishing Company, hereby expressly incorporated by reference.
  • the amount of a cysteine protease inhibitor of this invention in the composition may vary widely depending upon the type of formulation, size of a unit dosage, kind of excipients and other factors known to those of skill in the art of pharmaceutical sciences In general, the final composition will comprise from 0 1%w to 10%w of the cysteine protease inhibitor, preferably 1%w to 10%w, with the remainder being the excipient or excipients
  • the pharmaceutical composition is administered in a single unit dosage form for continuous teatment or in a single unit dosage form ad libitum when relief of symptoms is specifically required
  • Representative pharmaceutical formulations containing a cysteine protease inhibitor of the invention are described in Example 20, infra
  • Xaa 2 ammo acid at P2 position relative to active site of the enzyme
  • ⁇ -C0 2 Et ⁇ -am ⁇ no ethyl ester
  • ⁇ -S0 2 Ph ⁇ -am ⁇ nosulfone with phenyl terminus
  • ⁇ -C0 2 H ⁇ -am ⁇ nocarboxylate
  • ⁇ -PEt ⁇ -am ⁇ nophosphonate
  • ⁇ -AM ⁇ -am ⁇ noam ⁇ de
  • ⁇ -Ar(sub) ⁇ -am ⁇ noaromat ⁇ c compound (substituted as appropriate)
  • ⁇ -S0 2 Ph ⁇ -aminosulfone with phenyl substituent
  • ⁇ -S0 2 Ph ⁇ -aminosulfone with phenyl substituent
  • Hph homophenylalanine
  • PSMP diethyl phenylsulfonylmethylenephosphonate
  • Np2 2-naphthylalan ⁇ ne
  • MeOSuc methoxysuccinyl
  • Xaa 2 Phe (phenylalanine)
  • Xaa, Hph (homophenylalanine,)
  • the diastereomers were separated by flash chromatography on 230-400 mesh silica gel (20- 50% ethyl acetate/CH 2 CI 2 , gradient elution)
  • a representative solution for oral administration contains
  • Cysteine protease inhibitor 100 to 1000 mg Citric Acid Monohydrate 105 mg Sodium Hydroxide 18 mg Flavoring Water q s to 100 mL
  • a representative solution for intravenous admmstration contains
  • Cysteine protease inhibitor 10 to 100 mg Dextrose Monohydrate q s to make isotonic Citric Acid Monohydrate 1 05 mg Sodium Hydroxide 0 18 mg Saline for Injection q s to 1 0 mL
  • a representative tablet form may contain:

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Abstract

The invention relates to novel reversible protease inhibitors. The inhibitors are specific to cysteine proteases. Examples of such inhibitors include compounds with structure (1).

Description

REVERSIBLE PROTEASE INHIBITORS
FIELD OF THE INVENTION
The invention relates to novel reversible protease inhibitors The inhibitors are selective for cysteine proteases
BACKGROUND OF THE INVENTION
Cysteine or thiol proteases contain a cysteine residue at the active site responsible for proteolysis Since cysteine proteases have been implicated in a number of diseases, including arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephπtis, malaria, and other parasite-borne infections, methods for selectively and irreversibly inactivating them provide opportunities for new drug candidates See, for example, Esser, R E et al , Arthritis & Rheumatism (1994) 37, 236, Meyers, M H M et al , Agents Actions (1993), 39 (Special Conference Issue), C219, Machleidt, W et al, Fibnnolysis (1992), 6 Suppl 4, 125, Sloane, B F et al , Biomed Biochim Acta (1991), 50, 549, Duffy, M J , Clin Exp Metastasis (1992), 10, 145, Rosenthal, P J , Wollish, W S , Palmer, J T , Rasnick, D , J Clin Investigations (1991), 88, 1467, Baricos. W H et al, Arch Biochem Biophys (1991 ), 288, 468, Thornberry, N A et al . Nature (1992), 356, 768
Low molecular weight inhibitors of cysteine proteases have been described by Rich, Proteinase Inhibitors (Chapter 4, "Inhibitors of Cysteine Proteinases"), Elsevier Science Publishers (1986) Such inhibitors include peptide aldehydes, which form hemithioacetals with the cysteine of the protease active site See, for instance, Cheng, H , Keitz, P , and Jones, J B , J Org Chem (1994), 59, 7671 The disadvantage of aldehydes is their in vivo and chemical instabilities
Aldehydes have been transformed into α,β-unsaturated esters and sulfones by means of the Wadsworth-Emmons-Horner modification of the Wittig reaction, shown below (Wadsworth, W S and Emmons, W D (J Am Chem Soc (1961), 83, 1733)
Figure imgf000004_0001
where R = alkyl, aryl, etc EWG = COOEt, SOjMe. etc
α,β-unsaturated esters (Hanzlik ef al , J Med Chem , 27(6) 711-712 (1984), Thompson ef a/ , J Med Chem 29 104-111 (1986), Liu ef al , J Med Chem , 35(6) 1067 (1992)) and α,β-unsaturated sulfones (Thompson ef al , supra. Liu ef al , supra) were tested as inhibitors of two cysteine proteases, papain and dipeptidyl amino-peptidase I (also called cathepsin C) However, the inhibition of papain by these α,β-unsaturated compounds showed poor inhibition, evidenced by second order rate constants from less than 1 M 'sec ' to less than 70 M 1sec 1 for the σ,β-unsaturated esters, and from less than 20 M 1sec 1 to less than 60 M 'sec 1 for the sulfone
In addition, this chemistry has not been demonstrated with derivatives of σ-amino acids other than those corresponding to glycine, or in the case of the ester, phenylalanine Thus the chirality of these compounds is non-existent for the glycine derivatives and unclear for the phenylalanine derivatives This is significant since inhibition of an enzyme generally requires a chiral compound
Alpha-ammo sulphonic acids were suggested as potential inhibitory compounds, and several were made, although their inhibitory effects were not reported (Mcllwain et al , J Chem Soc 75 (1941 ))
In addition, the Mannich condensation of sulfinic acid, aldehyde, and ethyl carbamate, to form urethanes has been reported (Engberts et al , Recueil 84 942 (1965)
Additional methods for selectively and irreversibly inhibiting cysteine proteases have relied upon alkylation by peptide α-fluoromethyl ketones (Rasnick, D., Anal Biochem (1985), 149, 416), diazomethyl-ketones (Kirschke, H , Shaw, E Biochem Biphys Res. Commun (1981), 101, 454), acyloxymethyl ketones (Krantz, A et al , Biochemistry, (1991), 30, 4678, Krantz, A et al., U S Patent 5,055,451 , issued October 8, 1991), and ketosulfonium salts (Walker, B , Shaw, E , Fed Proc Fed Am Soc Exp Biol , (1985), 44, 1433)
Other families of cysteine protease inhibitors include epoxysuccinyl peptides, including E-64 and its analogs (Hanada, K e a/., Agrιc Biol Chem (1978), 42, 523, Sumiya, S ef a/., Chem Pharm. Bull ((1992), 40, 299 Gour-Salin, B J e a/ , J Med Chem , (1993), 36, 720), α-dicarbonyl compounds, reviewed by Mehdi, S , Bioorganic Chemistry, (1993), 21, 249, and N-peptidyl-O-acyl hydroxamates (Bromme, D , Neumann, U , Kirschke, H , Demuth, H-U , Biochim Biophys Acta, (1993), 1202, 271 An additional summary of methods for reversibly and irreversibly inhibiting cysteine proteases has recently been compiled see Shaw, E , Advances in Enzymology and Related Areas of Molecular Biology (1990), 63, 271
SUMMARY OF THE INVENTION
An aspect of this invention is a protease inhibitor comprising a targeting group linked through a two carbon atom chain to an electron withdrawing group, wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 μM
An additional aspect of this invention is a protease inhibitor comprising a targeting group linked either directly or through a linker selected from the group consisting of an intermediate carbon atom or a two carbon atom chain to a sulfone group group, wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 μM
A further aspect of this invention is a compound, preferably a protease inhibitor, of Formula I
Figure imgf000005_0001
in which n is 0 to 13,
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein RJ is hydrogen or as defined below,
X represents a bond, methylene or the linkage -CH2CH(R4)-, wherein R4 is hydrogen, alkyl or arylalkyl
Y is -CH(R5)- or -NR5-, wherein R5 is hydrogen or as defined below,
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below,
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below,
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylaminosulfonyl, arylsulfonyl or heteroarylsulfonyl,
R7 and Rβ are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylamino, dialkylamino, uπedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3^)methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), and
R2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, guanidino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, preferably wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 μM
An additional aspect of this invention is a compound, preferably a protease inhibitor, of Formula II
Figure imgf000006_0001
II in which the groups are as defined above and
R9 is cyano, -C(0)OR1°, -P(O)(OR10)2, -S(O)(NR10)R10, C(0)R11, -S(0)R11, -C(0)NR12R13, -S(0)2NR12R13, -C(0)NHR14 or -S(0)2NHR14, wherein each R10 is independently hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R11 is hydrogen, alkyl, perfluoroalkyl, cycloalkyl, cycloalkylalkyl, perfluoroaryl, perfluoroarylakyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R12 and R13 are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl or aralkyl, and R14 is -C(0)OR1°, in which R10 is as defined above, or a group selected from Formulae (a) and (b):
Figure imgf000007_0001
(a)
wherein each n, A, B, Y, Z, R1 and R10 are as defined above, and the pharmaceutically acceptable salts; individual isomers and mixtures of isomers thereof, preferably wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 μM.
A further aspect of this invention is a compound, preferably a protease inhibitor, of Formula III:
Figure imgf000007_0002
in which: the groups are as defined above and
R 5 is hydrogen, methyl, fluoro or a group selected from Formulae (a) and (b) as defined above, and R16 is a group selected from phenyl or (C5^)heteroaryl (which group is optionally substituted with at least one radical selected from aikylcarbamoyl, dialkylcarbamoyi, alkyloxycarbonyl, alkylsulfmamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, carbamoyi, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkylsulfamoyl, dialkylsulfamoyl, alkyloxysulfonyl, aryl, heteroaryl, hydroxy, alkyloxy, optionally halo-substituted alkyl, arylalkyl, halo, -*N(R17)3, wherein each R17 is independently alkyl, aryl or arylalkyl, or -N(R18)2, wherein each R1B is independently hydrogen, alkyl, aryl or arylalkyl); and the pharmaceutically acceptable salts; individual isomers and mixtures of isomers thereof, preferably wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 μM.
An additional aspect of this invention is a pharmaceutical composition comprising a therapeutically effective amount of a cysteine protease inhibitor of the invention, or of an individual isomer, a mixture of isomers, or the pharmaceutically acceptable salt or salts thereof, in combination with one or more pharmaceutically acceptable excipients.
A further aspect of this invention is a method for treating a condition capable of amelioration by inhibition of a cysteine protease in an animal in need thereof, which method comprises administering to such animal a therapeutically effective amount of a cysteine protease inhibitor of the invention, or of an individual isomer, mixture of isomer, or the pharmaceutically acceptable salt or salts thereof. Another aspect of this invention is a method for detecting a cysteine protease in a sample, which method comprises
(a) assaying said sample for protease activity using a protease substrate
(b) assaying for protease activity in the presence of a known concentration of cysteine protease inhibitor on the invention, and
(c) calculating the difference between a) and b) to determine the protease activity due to cysteine protease
An aspect of this invention are the processes for preparing the cysteine protease inhibitors of this invention
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts Scheme I, the synthesis of Formula I compounds when X is a bond The synthetic steps are as follows a) HC02H, H20, b) HBr/acetic acid, c) 4-methylmorpholιne, isobutyl chloroformate, Mu-ROH, and d) chromatographic purification The groups are as defined herein
Figure 2 depicts Scheme 2, the synthesis of Formula I compounds when X is a methylene group The synthetic steps are as follows a) 4-methylmorpholιne, isobutyl chloroformate, followed by NaBH4 reduction in water/THF, b) CH3S02CI, tπethylamine, CH2CI2, c) R,SH, NaH, CH3OH, THF, heat, d) 4-chloroperbenzoιc acid, CH2CI2, e) HCI/dioxane or p-CH3C6H4S03H/ether, and f) Mu-ROH, 4- met ylmorpholine, isobutyl chloroformate
Figure 3 depicts Scheme 3, the synthesis of Formula I compounds when X is a methylene group The synthetic steps are as follows a) (CH3)3CH2CH2SH, NaH, MeOH, THF, heat, b) 4-chloroperbenzoιc acid, c) (n-C4H9)4N , THF, followed by BrCH2CI, heat, d) HCI/dioxane or 4-CH3C6H4S03H/ether, and, e) 4-methylmorpholιne, isobutyl chloroformate, Mu-PheOH
Figure 4 depicts Scheme 4, the synthesis of Formula II compounds The synthetic steps are as follows a) Cl H2N*(CH3)OCH3, dicyclohexylcarboiimide, Et3N/CH2CI2, b) LiAIH ΪΗF, c) NaH/THF, d) Hcl/dιoxane/CH2CI2, e) 4-methylmorpholιne, isobutyl chloroformate/THF, and f) H2, 5% Pd/C
Figure 5 depicts Scheme 5, the synthesis of Formula I compounds when X is an ethylene The synthetic steps are as follows a)(CH20)n, HCI, dioxane, for instance where Ar = 2-naphthyl, b) (EtO)3P, c) CH3CO3H, CH2CI2, d) NaH, THF, e) p-CH3C6H4S03H, Et20, f) 4-methylmorpholιne, isobutyl chloroformate, and g) H2, Pd/C
Figure 6 depicts the synthesis of compounds of Formula II in which R9 is -COOH Figure 7 depicts the synthesis of compounds of Formula II in which R9 is -P(O)(R10)2. The synthetic scheme is as follows: a) NaH THF; b) anhydrous p-CH3C6H4S03H/ether; c) 4-methylmorpholine, isobutyl chloroformate/THF, and; d) H2, Pd/C.
Figure 8 depicts the synthesis of compounds of Formula II in which R9 is -C(0)NHR14. The synthetic scheme is as follows: a) NaOH/EtOH, followed by Hcl/H20; b) benzylamine, dicyclohexylcarbodiimide, CH2CI2; c) NaH/THF, diethyl benzylamidomethylenephosphonate; d) HCI/dioxane; e) 4-methylmorpholine, isobutyl chloroformate, THF; f) H2, Pd/C, and as an alternative preparation from carboxylates as synthesized via Scheme 6, above; and g) aniline, dicyclohexylcarbodiimide, CH2CI2.
Figure 9 depicts the general synthesis of compounds of Formula II.
Figure 10 depicts the synthesis of compounds of Formula III. The synthetic steps are as follows: a) CH3CN or other suitable solvent, reflux; b) H20, NaOH, followed by extraction into organic medium; c) phosphorane, THF (Wittig reaction); d) p-CH3C6H4S03H, ether; e) Mu-PheOH, 4-methylmorpholine, isobutyl chloroformate, THF; and f) H2, Pd/C.
DEFINITIONS
Unless otherwise stated, the following terms used in the specification and claims are defined for the purposes of this application and have the meanings given below:
"Alkyl", as in alkyl, alkyloxy, alkylthio, alkylsulfonyl, aikylcarbamoyl, dialkylcarbamoyi, heteroarylalkyl, arylalkyl, and the like, means a straight or branched, saturated or unsaturated hydrocarbon radical having from 1 to 10 carbon atoms or the number of carbon atoms indicated (e.g., methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, ferf-butyl, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylallyl, ethynyl, 1-propynyl, 2-propynyl, etc.).
"Alkyloxyphosphinyl" and "dialkyloxyphosphinyl" mean the radicals -P(0)(OH)OR and -P(0)(OR)2, respectively, wherein R is alkyl as defined above.
"Alkanoyl", as in alkanoyl, alkanoyloxy, heterocycloalkylalkanoylamino, and the like, means the radical -C(0)R, wherein R is alkyl as defined above, having overall from 1 to 11 carbon atoms or the number of carbon atoms indicated (e.g., (C,^)alkanoyl includes the radicals formyl, acetyl, propionyl, isopropionyl, butyryl, isobutyryl, crotonoyl, isocrotonyl, etc.).
"Aryl" means an aromatic monocyclic or polycyclic hydrocarbon radical containing 6 to 14 carbon O 96/30353 US96/03844
atoms or the number of carbon atoms indicated and any carbocylic ketone or thioketone derivative thereof, wherein the carbon atom with the free valence is a member of an aromatic ring, (e g , aryl includes phenyl, naphthyl, anthracenyl, phenanthrenyl, 1 ,2,3,4-tetrahydro-5-naphthyl, 1 -oxo-1 , 2-dιhydro-5-naphthyl, 1-thιoxo-1 ,2-dιhydro-5-naphthyl, etc )
"Aroyl" means the radical -C(0)Ar, wherein Ar is aryl as defined above, having overall from 7 to 15 carbon atoms or the number of carbon atoms indicated (e g , (C7 n)aroyl includes benzoyi, naphthoyi, etc )
"Cycloalkyl", as in cycloalkyl and cycloalkylalkyl, means a saturated or unsaturated, monocyclic or polycyclic hydrocarbon radical containing 3 to 20 carbon atoms or the number of carbon atoms indicated, wherein the carbon atom with the free valence is a member of a non-aromatic ring and any carbocyclic ketone and thioketone derivative thereof (e g , the term cycloalkyl is meant to include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, bιcyclo[2 2 2]octyl, 1 ,2,3,4-tetrahydro- 1 -naphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 9-fluorenyl, etc )
"Halo ' means fluoro, chloro, bromo or lodo
"Heterocycloalkyl", as in heterocycloalkyl, heterocycloalkylalkanoylamino, heterocycloalkylcarbonyl, heterocycloalkylcarbonyl, and the like, means cycloalkyl as defined above wherein 1 to 5 of the indicated carbon atoms is replaced by a heteroatom chosen from N, O, S, P or As, wherein the atom with the free valence is a member of a non-aromatic ring, and any heterocychc ketone, thioketone, sulfone or sulfoxide derivative thereof, (e g , the term heterocycloalkyl is meant to include pipeπdyl, pyrrolidinyl, pyrrolinyl, imidazo dinyl, indolmyl, quinuclidinyl, morpholinyl, piperazinyl, Λ/-methylpιperazιnyl, piperadinyl, 4,4-dιoxo-4-thιapιpeπdιnyl, 1 ,2,3,4-tetrahydro-3-ιsoquιnolyl, 2,4-dιaza-3-oxo-7-thιa-6-bιcyclo[3 3 OJoctyl, etc ) Thus, hetero(C6)cycloalkyl includes the radicals morpholinyl, piperazinyl, pipendinyl and the like
"Heteroaryl" means an aromatic monocyclic or polycyclic hydrocarbon radical containing overall from 5 to 14 atoms or the number of atoms indicated, wherein 1 to 5 of the indicated carbon atoms are replaced by a heteroatom chosen from N, O, S, P or As, wherein the atom with the free valence is a member of an aromatic ring, and any heterocychc ketone and thioketone derivative thereof (e g , the term heteroaryl is meant to include thienyl, furyl, pyrrolyl, pyπmidmyl, isoxazolyl, oxaxolyl, dolyl, benzo[b]thιenyl, isobenzofuranyl, punnyl, isoqumolyl, pterdinyl, pyπmidinyl, imidazolyl, pyπdyl, pyrazolyl, pyrazinyl, 4-oxo-1 ,2-dιhydro-1 -naphthyl, 4-thιoxo-1,2-dιhydro-1 -naphthyl, etc ) Thus, hetero(C6)aryl includes the radicals pyπdyl, pyπmidinyl, and the like "1,2-Phenylenedimethylene" means a divalent radical of the formula -CH2C6H4CH2-. For example, the group RΥ-Z-A- in which Y is -N(R5), Z is -CH(R7)-, A is carbonyl and R7 together with R5 forms 1 ,2-diphenylenedimethylene" means a group of following formula:
Figure imgf000011_0001
and substituted derivatives and individual stereoisomers and mixture of stereoisomers thereof. Substituted derivatives of the 1 ,2-phenylenedimethylene divalent radical may contain a hydroxy group on any carbon within the ring system or an oxo group on either of the unsaturated ring carbon atoms.
"Phosphono" means the radical -P(0)(OH)2.
"Methylene" as in "(C3J))methylene"and "(C3.7)methylene" mean a straight, saturated divalent radical having the number of carbon atoms indicated; "(C3^)methylene" includes trimethylene (-(CH2)3-) and tetramethylene (-(CH2)4-) For example, a preferred embodiment herein utilizes a proline residue as an A-B-Z group, wherein A-B represents CH2-NR3 and R3 together with either R7 or Rβ form a C3 methylene. Thus, the group R1-Y-Z-A- in which Y is -(NR5)-, Z is -CH(R7)-, A is carbonyl and R7 together with R5 forms trimethylene means a group of following formula:
Figure imgf000011_0002
and the individual stereoisomers and mixtures of stereoisomers thereof. Substituted derivatives of the trimethylene and tetramethylene divalent radicals may contain a hydroxy group, or a protected derivative thereof, or an oxo group on any of the ring carbon atoms. Suitable hydroxy protective groups are defined below.
"Oxa(C3.7)methylene" and "aza(C3.7)methylene" mean methylene as defined above wherein one of the indicated carbon atoms is replaced by an oxygen or nitrogen atom, respectively. For example, "oxa(C5)methylene" includes 3-oxapentamethylene (-CH2CH2OCH2CH2-) and 2-oxapentamethylene (-CH2OCH2CH2CH2-). Thus, -CfOJNR^R22 means the radical 4-morpholinylcarbonyl when R21 and R22 together form 3-oxapentamethylene and the radical 1-piperazinylcarbanoyl when R21 and R22 together form 3-azapentamethylene. "Adjacent", as use in the phrase "R7 together with an adjacent R3", means that the atoms to which the R7 and R3 groups are respectively attached are in turn attached to one another
"Animal" includes humans, non-human mammals (e g , dogs, cats rabbits, cattle, horses, sheep, goats, swine, deer, etc ) and non-mammals (e g , birds, etc )
"Disease" specifically includes any unhealthy condition of an animal or part thereof and includes an unhealthy condition which may be caused by, or incident to, medical or veterinary therapy applied to that animal, i e , the "side effects" of such therapy
"Electron withdrawing group" (EWG) means a functional group that in its broadest sense is a group able to exert a polarizing force on the bond between itself and the carbon to which it is attached, such that electrons are polarized in favor of the electron withdrawing group While not being limited to any particular theory, it is believed that the polarizing property enables the electron withdrawing group to participate in hydrophobic or hydrogen bonding interactions with an active site of the cysteine protease, resulting in inhibition of the enzyme In general, a moiety is suitable as an electron withdrawing group if when present in the α-position of a phosphonium ylide of the general structure Ph3P=C(R)EWG it exerts sufficient polarization to stablize the ylide against undergoing decomposition reactions with oxygen, water, hydroha c acids, alcohols, and the like Preferred electron withdrawing groups are those which would similarly stabli ze ylides of the general formula (RO)2P(0)C(R)EWG Suitable electron withdrawing groups include cyano, -S(0)2R2, -C(0)OR1°, -P(O)(OR10)2, -S(O)(NR10)R10, C(0)R11, -S(0)R11, -C(0)NR12R13, -S(0)2NR12R13, -C(0)NHR14, -S(0)2NHR14, phenyl and (C5^)heteroaryl, wherein each R2, R10, R11, R12, R13 and R14 are as defined in their broadest definitions set forth in the Summary of the Invention When the electron withdrawing group is phenyl or (C5^)heteroaryl the ring may be substituted with one or more meta directing groups (e g , alkyloxycarbonyl, alkylsulfinamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfmamoyl, sulfo, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkyisulfamoyl, dialkylsulfamoyi, alkyloxysulfonyl, disubstituted ammo, tnsubstituted ammonio, and the like), ortho and para directing groups (e g , hydroxy, alkyloxy, optionally halo-substituted alkyl, aryl, arylalkyl, halo, and the like) and electron withdrawing moieties (e g , aikylcarbamoyl, dialkylcarbamoyi, alkyloxycarbonyl, alkylsulfinamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfmamoyl, sulfo, carbamoyi, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkyisulfamoyl, dialkylsulfamoyi, alkyloxysulfonyl, aryl, heteroaryl, and the like)
"Leaving group" has the meaning conventionally associated with it in synthetic organic chemistry, i e , an atom or group displaceable under alkylatmg conditions, and includes halo and alkane- or arenesulfonyloxy, sucha mesyloxy, ethanesulfonyloxy, benzenesulfonyloxy and tosyloxy, and alkaesulfonylamino, alkanecarbonylamino, aminosulfonylamino, aminocarbonylamino and the like.
Isomerism is the phenomenon wherein compounds have identical molecular formulae but differ in the nature or sequence of bonding of their atoms or in the arrangement of theri atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "steroisomers". Stereoisomers that are not mirror images of one another are termed "diastereomers" and stereoisomers that are nonsuperimposable mirror images are termed "enantiomers" or sometimes "optical isomers". A carbon atom bonded to four nonidentical substituents is termed a "chiral center".
A compound with one chiral center has two enantiomeric forms of opposite chirality is termed a "racemic mixture". A compound that has more than one chiral center has 2π 1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as ether an individual diasteromer or as a mixture of diastereomers, termed a "diastereomeric mixture".
Compounds of Formulae I, II and III can exist as individual steroisomers or mixtures of stereoisomers. For example, compounds of Formulae I, II and III contain a chiral center at the carbon to which the substituent Rβ is attached. Furthermore, compounds of Formulae I, II and III in which Z is -C(R6)(R7) contain a chiral center at the carbon to which the R7 substituent is attached. Thus, for example, compounds of Formulae I, II and III in which n is 0 and Z is -C(R6)(R7) will have two chiral centers and can exist as four individual stereoisomers or any mixture thereof.
Individual stereoisomer may be characterized by the absolute configuration of their chiral centers. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog and then the absolute descriptor R is assigned if the three highest ranked substituents are arranged in space (with the fourth lowest ranked substituent directed away from the observer) from high to low priority in a clockwise sequence and the absolute descriptor S is assigned for a counterclockwise arrangement. When an individual stereoisomer containing one chiral center is described the absolute descriptor R or S is cited in parenthesis followed by a hyphen and the chemical name of the compound. For the purposes of this invention, when an individual stereoisomer or mixture of stereoisomers containing two or more chiral centers is described, the absolute descriptor R or S is cited immediately after the appropriate locant. Acyl radicals derived from naturally occurring amino acids are referred to as their amino acid radicals preceded by the descriptor L (e.g., L-phenylalanine). The nonnatural enantiomers of amino acid acyl radicals are preceded by the descriptor D. Preferably, the amino acid side chains are the (S) or L-form, due to the stereospecificity of enzymes, although the D-forms may be used in some cases. When no absolute descriptor is cited for a chiral center, the description is meant to include both configurations and mixtures thereof, racemic or otherwise Thus, for example, a compound of the following formula
Figure imgf000014_0001
is named
Λ/2-(4-morpholιnylcarbonyl)-Λ 1-[3-phenyl-1 S-(2-phenylsulfonylethyl)propyl]-L-phenylalanιnamιde, when
R1 is 4-morpholιnylcarbonyl, R8 is 2-phenylethyl and lies on the same side of the reference plane as the R7 substituent, R7 is benzyl and R19 is phenylsulfonyl,
Λ/2-4-morpholιnylcarbonyl-Λ 1-[3-phenyl-1-(2-phenylsulfonylethyl)propyl]-L-phenylalanιnamιde, when R1 is 4-morpholιnylcarbonyl, R8 is 2-phenylethyl and lies on either or both sides of the reference plane, R7 is benzyl and R 9 is phenylsulfonyl,
Λ/2-4-morpholιnylcarbonyl-Λ/-[3-phenyl-1 S-(2-phenylsulfonylethyl)propyl]-β-(2-naphthyl)-L-alanιnamιde, when R1 is 4-morpholιnylcarbonyl, R8 is 2-phenylethyl lies on the same side of the reference plane as the R7 substituent, R7 is 2-naphthylmethyl and R19 is phenylsulfonyl and ethyl 4S-(Λ/-4-morpholιnylcarbonyl-L-phenylalanylamιno)-6-phenylhexanoate, when R1 is
4-morpholιnylcarbonyl, R8 is 2-phenylethyl and lies on the same side of the reference plane as the R7 substituent, R7 is benzyl and R19 is ethoxycarbonyl
In a preferred embodiment, the compositions of the invention are pure diasteromers Alternatively, the compositions contain mixtures of diasteromers Preferred embodiments have greater than about 70% of a single disasteromer, with at least about 90% being particularly preferred
"Protective group" has the meaning conventially associated with it in synthetic organic chemistry, i e , a group which blocks a reactive site in a compound See for example Greene et al , Protective Groups in Organic Synthesis, 2nd Ed , John Wiley & Sons, 1991 , hereby incorporated by reference Examples of hydroxy protective groups include heterocycloalkyl-carbonyl such as 4-morpholιnylcarbonyl and the like, aroyl such as benzoyi and arylalkyl such as benzyl and the like Examples of am o protective groups include aryloxycarbonyl such as benzyloxycarbonyl and the like, aroyl such as benzoyi and the like and oxycarbonyl such as ethoxycarbonyl and 9-fluorenylmethoxycarbonyl and the like Examples of guanidino protective groups include sulfonyl such as 2,3,5-tπmethyl-4-methoxyphenyl-sulfonyl and the like Examples of suitable carboxy protective groups that form ester moieties are alkoxylcarbonyl of overall 4 to 8 carbon atoms, particularly fe t-butoxycarbonyl (BOC) or benzyloxycarbonyl (CBZ, Z), especially cycloalkylammocarbonyl or oxacycloalkylammocarbonyl of overal 4 to 8 atoms in the ring, particularly 4-morpholιnecarbonyl (Mu) and the like. "Protected" in reference to a compound of a group means a derivative of a compound or group in which a reactive site or sites are blocked with protective groups
"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances wherein the event or circumstance occurs and instances in which it does not For example, "optionally further substituted with one or more functional groups" means that the substituents may or may not be present in order for the compound described to fall within the invention, and the invention includes those compounds wherein one or more functional groups are present and those compounds in which no functional groups are present
By "cysteine protease-associated disorders" herein is meant pathological conditions associated with cysteine proteases In some disorders, the condition is associated wtih increased levels of cysteine proteases, for example, arthritis, muscular distrophy, inflammation, tumor invasion, and glomerulonephritis are all associated with increased levels of cysteine proteases In other disorders or diseases, the condition is associated with the appearance of an extracellular cysteine protease activity that is not present in normal tissue In other embodiments, a cysteine protease is associated with the ability of a pathogen, such as a virus, to infect or replicate in the host organism
Specific examples of cysteine protease associated disorders include, but are not limited to, arthritis, muscular distrophy, inflammation, tumor invasion, glomerulonephπtits, malaria, Alzheimer's disease, cancer metastasis, trauma, inflammation, gingivitis, leishmaniasis, filaπasis, and other bacterial and parasite-borne infections In particular, disorders associated with interleukin 1β converting enzyme (ICE) are included
"Pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use
"Pharmaceutically acceptable salts" means salts which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfunc acid, nitric acid, phosphoric acid, and the like, or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succtnic acid, malic acid, maleic acid, fumaric acid, tartatic acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoιc acid, cmnamic acid, madelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedιsulfonιc acid, 2-hydroxyethanesulfonιc acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonιc acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbιcyclo[2 2 2]oct-2-ene-1-carboxylιc acid, glucoheptonic aicd, 4,4'-methylenebιs(3-hydroxy-2-ene-1-carboxylιc acid), 3-phenylpropιonιc acid, tnmethylacetic acid, tertiary butylacetic acid, lauryl sulfunc acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like
Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases Acceptabale inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydoxide Acceptable organic bases include ethanolamine, diethanolamine, tπethanolamine, tromethamine, Λ/-methylglucamιne and the like
"Therapeutically effective amount" means that amount which when administered to an animal for treating a disease includes
(1) preventing the disease from occurring in an animal which may be predisposed to the disease but does not yet experience or display symptoms of the disease,
(2) inhibiting the disease, i e , arresting its development, or
(3) ameliorate the disease, i e causing regression of the disease
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel cysteine protease inhibitors Without being bound by theory, it is believed that the inhibitors bind to cysteine proteases based on the following scheme
enzyme inhibitor complex
Figure imgf000016_0001
It is believed that the enzyme is thus reversibly inhibited by means of interactions between the R, Y and Z moieties of the inhibitor and the surface of the binding sites of the enzyme, and by means of hydrogen bonding interactions between the sulfone and active site ammo acid side chains
This mechanism of reversible inhibition permits specificity of the enzyme inhibitors for cysteine proteases Generally, the inhibitors of the present invention inhibit cysteine proteases and do not inhibit serine, aspartyl, and zinc proteases However, in some embodiments, the protease inhibitors of the present invention may have activity against other types of proteases, such as serine, aspartyl or other metalloproteases, but to a lesser extent
In addition, the electron withdrawing properties of the sulfone group of Formula I polarize the electrons between the sulfone group and the carbon to which it is attached, thus permitting hydrogen bonding between itself and active site residues of a cysteine protease, to allow tight binding between the inhibitor and the cysteine protease, as is generally described below It is to be understood that there is presumably additional electron withdrawing or electron polarization occurring between the sulfur atom and the oxygen atoms, which allows the oxygen atoms to participate in hydrogen bonding with active site residues of the protease and thus contributing even further to the inhibition of the enzyme
The present invention generally provides new peptide-based and peptidomimetic cysteine protease inhibitors for use as reversible cysteine protease inhibitors By "cysteine protease inhibitor" herein is meant an inhibitor which inhibits cysteine proteases In a preferred embodiment, the cysteine protease inhibitors are specific to cysteine proteases, that is, they do not inhibit other types of protease such as serine, aspartyl, or other metalloproteases However, in alternative embodiments, the cysteine protease inhibitors of the invention may inhibit other types of proteases as well
By "reversible" herein is meant that the inhibitor binds non-covalently to the enzyme, and is to be distinguished from irreversible inhibition See Walsh, Enzymatic Reaction Mechanisms, Freeman & Co , N Y , 1979 "Reversible" in this context is a term understood by those skilled in the art In addition, the reversible cysteine protease inhibitors are competitive inhibitors, that is, they compete with substrate in binding reversibly to the enzyme, with the binding of inhibitor and substrate being mutually exclusive In addition, the stoichiometry of inhibition is 1 1 , that is, a single inhibitor molecule is sufficient to inhibit a single enzyme molecule
The cysteine protease inhibitors herein are designed to bind reversibly to cysteine proteases This binding is accomplished by using peptide-based or peptidomimetic structures as targeting groups that mimic naturally occurring substrates and/or inhibitors "Peptidomimetic", for the purposes of this invention, means ammo acid or peptide-hke in structure but wherein one or more of the peptide linkages (i e , -C(O)NR-) is substituted by an isosteπc form, i e -CH2NR-, -C(0)CH2- or -NRC(O)- and/or wherein non-naturally occurring ammo acid substituents are present
"Targeting group", for the purposes of this application, means a peptide or peptidomimetic residue of the cysteine protease inhibitor that allows the binding of the inhibitor to a cysteine protease In a preferred embodiment, the targeting group of a cysteine protease inhibitor comprises at least two ammo acid side chains or side chain analogs, linked via a peptide bond or isostere The targeting group may comprise up to about 15 am o acids or analogs, although inhibitors are generally from about 1 to 7 ammo acids or analogs, since smaller inhibitors are usually desired in therapeutic applications Thus, in Formulae I, II and III, n is preferably from 0 to 13, with from 0 to 5 being preferred and from 0 to 3 being particularly preferred
As depicted in Formulae I, II and III, the targeting group can be represented by a naturally or non-naturally occurring peptide residue of the following formula
Figure imgf000018_0001
wherein the R8 and R7 components represent naturally or non-naturally occurring am o acid analogs or substituents as is more fully described below The targeting group of the inhibitor may also contain additional functional groups, as depicted by R1 and described herein
While not being limited to any particular theory, it is believed that the am o acid substituents of the targeting group interact with the surface binding sites of the protease to promote binding It is also believed that the ammo acid substituent proximal to the electron withdrawing group (e g , R8 of the above formula) will occupy the S, position of the substrate binding site and therefore is designated the P, residue of the inhibitor Similarly, the next adjacent ammo acid substituent (e g , R7 of the above formula) will occupy the S2 position of the substrate binding site and is designated the P2 residue of the inhibitor If present, additional am o acid substituents will occupy the S3, S4, etc positions of the substrate binding site and be designated as the P3, P4, etc residues of the inhibitor An additional targeting group may be attached to the electron withdrawing group and, if present, its ammo acid substituents will occupy the S,', S2', etc positions of the substrate binding sites and are designated the P3', P4', etc residues of the inhibitor, respectively
In general, targeting groups for specific enzymes are determined by rules governing substrate specificity in cysteine proteases (e g , see "Protemase Inhibitors", in Research Monographs in Cell and tissue Physiology (1986), ed Barret ef a/ , Vol 12, Chapter 4 Inhibitors of Cysteine Proteinases, Daniel Rich, Elsevier, New York, and Thornberry ef al , supra , hereby expressly incorporated by reference) For example, ιnterleukιn-1 converting enzyme (ICE) accepts an aspartic acid substituent (i e , 2-carboxyethyl) at the P, position and an alanme (methyl), val e (isopropyl) or histidme (4-ιmιdazolylmethyl) substituent at the P2 position Papain accepts a argmine, lysine, N- benzyloxycarbonyllysine (i e 4-benzyloxycarbonylamιnobutyl), homophenylalanme (i e 2-phenylethyl), Guanidmo-phenylalnine (i e , 4-guanιdιnobenzyl) or norluecme (i e , butyl) substitutents at the P, position and phenylalnme, tyrosme, β-)2-naphthyl)alanιne (i e , 2-naphthyl), leucme, norleucme isoleucine or alanme substituents at the P2 position Cathepsin B accepts a argmine, lysme, N- benzyloxycarbonyllysme, guanidmo-phenylalanine, homophenylalanine or norleucme substituents at the P, position and phenylalanine, tyrosme, 3,5-dιιodotyrosιne (i e , 3,5-dιιodo-4-hydroxybenzyl), β-(2- naphthyl)alanme, argmine, guanidmo-phenylalanine or citrulline (i e , 3-ureιdopropyl) sybstituents at the P2 position Cathepsin L and cruzam accept argmine, lysme, homophenylalanine, guanmdino- phenylalanme, citrulline or norleucme substituents at the P, position and phenylalanine, tyrosme or β-(2-naphthyl)alanιne substituents at the P2 position Cathepsin S accepts a argmine, lysme, homophenylalanine, guanidmo-phenylalanine, citrulline or norleucme substituents at the P, position and phenylalanine, tyrosme, β-(2-naphthyl)anιne, valme, leucme, norleucme, isoleucine or alanme substituents at the P2 position DPP-1 accepts phenylalanine or tyrosme substituents at the P, position and no subsutituent or alanme at the P2 position Calpain accepts phenylalanine, tyrosme, methionine, β-methylsulfonylmethylalanine (i e , 2-methylsulfonylethyl) or valme substituent at the P, position and valme, leucme, norleucme or isoleucine substituents at the P2 position
Thus, R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylamino, dialkylamino, uπedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, am o, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or Rs forms a divalent radical selected from (C3^)methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo)
Accordingly, preferred R7 and R8 groups are the naturally occunng ammo acid side chains and homologous derivatives These include, but are not limited to, alanme (methyl), argmine (3- guanidmopropyl), asparagme (carbamoylmethyl), citurlline (3-ureιdopropyl), aspartic acid (carboxymethyl), cysteine (mercaptomethyl), glutamic acid (2-carboxyethyl), glutamine (2- carbamoylethyl), glycine (hydrogen), histidme (4-ιmιdazolylmethyl), homophenylalanine (2- phenylethyl), homoseπne (2-hydroxylethyl), isoleucine ((1-methylpropyl), leucme (isobutyl), lysme (4- aminobutyl), methionine (2-methylthιoethyl), β-(1-naphthyl)alanιne (1-napthylmethyl), β-(2- naphthyl)alanιne (2-napthylmethyl), norleucme (butyl), norval e (propyl), ornithine (3-amιnopropyl), phenylalanine (benzyl), proline (as described herein), sarcosme (methylaminomethyl), serine (hydroxymethyl), threonme (1-hydroxyethyl), tryptophan (3-ιndolymethyl), tyrosme (4-hydroxybenzyl), and valme (isopropyl)
While the broadest definition of this invention is set forth in the Summary of the Invention, certain 18 compounds of the invention are preferred For example, generally preferred compounds of Formulae I, II and 111 are those in which n is 0 to 5, A-B represents a linkage selected from -C(0)NR3-, wherein R3 is hydrogen or as defined below, Y is -N(R5)-, wherein R5 is hydrogen or as defined below, Z is -(CH2)2- or -C(R6)(R7)-, Z1 is -CH(R8)-, R is hydrogen, alkyloxycarbonylalkanoyl of overall 3 to 10 carbon atoms, (C, 9)alkoxycarbonyl, (C2 10)alkanoyl (optionally substituted with a radical selected from carboxy, (C, 9)alkyloxycarbonyl and hetero(C4 β)cycloalkyl(C2 10)alkanoylamιno), (C4 9)cycloalkyl- carbonyl, hetero(C4 B)cycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, (C, 5)alkyl, (C, 5)alkanoyl, (C, 5)alkyloxycarbonyl, (C6 10)aryl(C, s)alkyloxycarbonyl and hetero(C4^)cycloalkylcarbonyl), (C6 10)aryl(C, 5)alkyloxycarbonyl, carbamoyi, (C, 5)alkylcarbamoyl, dι(C, 5)alkylcarbamoyl, (C6 10)arylcarbamoyl, (C6 10)aryl(C, 5)alkylcarbamoyl, (Cg ^ary C, 5)alkanoyl, (C7 n)aroyl, (C, 5)alkylsulfonyl, dι(C, 5)alkylammosulfonyl, (C6 ,0)arylsulfonyl or hetero(C5 8)arylsulfonyl, and R7 and R8 are independently (C. 5)alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylam o, dialkylammo, unedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino or a protected derivative thereof) (C3 7)cycloalkyl, (C3 7)cycloalkyl(C1 5)alkyl, pyπdyl. thienyl, furyl, imidazolyl, mdolyl, pyπdyl(C1^)alkyl,
Figure imgf000020_0001
a group selected from or a group selected from phenyl, naphthyl, phenyl(C,^)alkyl, naphthyl(C,^)alkyl, (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, chloro, bromo, lodo, fluoro, methyl, tπfluoromethyl, methoxy and phenyl, or a protected derivative thereof) or together with an adjacent R3 or R4 forms a divalent radical selected from (C3j,)methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo)
More preferred compounds of Formulae I, II and III are those in which n is 0 to 2, A-B represents a linkage selected from -C(0)NR3-, wherein R3 is hydrogen or as defined below, Y is -N(R5)-, wherein R5 is hydrogen or as defined below, Z is -(CH2)2- or -C(R6)(R7)- (with the proviso that when n is 0, Z is not -(CH2)2-), Z1 is -CH(R8)-, R1 is hydrogen, (C4 β)alkoxycarbonyl, (C2^)alkanoyl (optionally substituted with a radical selected from carboxy, (C, 5)alkyloxycarbonyl and hetero(C4^)cycloalkyl(C4^) alkanoylam o), -C(0)NR21R22 wherein R21 and R22 together form aza(C2-6)methylene, oxa(C2^)methylene or (C3.7)methylene, (C4^)cycloalkylcarbonyl, benzyloxycarbonyl, acetyl, benzoyi or dimethylammosulfonyl, and R8 and R7 are independently (C5^)cycloalkyl, (C5-6)cycloalkylmethyl, 3-pyπdyl, 2-thιenyl, 2-furyl, 4-ιmιdazolyl, 3-ιndolyl, 3-pyrιdylmethyl, 2-thιenylmethyl, 2-furylmethyl,4-ιmιdazolylmethyl, 3-ιndolylmethyl, (C*.5)alkyl (optionally substituted with a radical selected from mercapto, carboxy, ammo, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyl, guanidino and hydroxy, or a protected derivative thereof), a group selected from phenyl, 1-naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, ammo, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3Jl)methylene and 1,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo)
Particularly preferred compounds of Formulae I, II and III are those in which n is 0 to 1, A-B represents a linkage selected from -C(0)NR3-, Y is -N(R5)-, wherein R5 is hydrogen or as defined below, Z is -C(Rβ)(R7)-, is -CH(R8)-, R1 is hydrogen, fert-butoxycarbonyl, benzyloxycarbonyl, acetyl, 3-carboxypropιonyl, 3-methoxycarbonylpropιonyl, biotinylaminohexanoyl, phenylacetyl, benzoyi, dimethylammosulfonyl, benzylsulfonyl, 1-pιperazιnylcarbonyl, 4-methylpιperazιn- 1-ylcarbonyl or 4-morpholιnylcarbonyl, R7 is 3-pyrιdylmethyl, 2-thιenylmethyl, 2-furylmethyl, 4-ιmιdazolylmethyl, 3-ιndolylmethyl, (C, 5)alkyl (optionally substituted with a radical selected from mercapto, carboxy, ammo, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyl, guanidino and hydroxy, or a protected derivative thereof), a group selected from benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, am o, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3-4)methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), and R8 is butyl, 2-phenylethyl, 2-methylsulfonylethyl, 2-fert-butoxycarbonylethyl, 2-fert-butoxycarbonylmethyl, 4-fert-butoxycarbonylamιnobutyl, 4-benzoylamιnobutyl or benzyloxymethyl
More particularly preferred compounds of Formulae I, II and III are those in which n is 0, A-B represents a linkage selected from -C(0)NH-, Y is -NH-, Z is -CH(R7)-, T is -CH(R8)-, R1 is hydrogen, fer -butxoycarbonyl, benzyloxycarbonyl, biotinylaminohexanoyl, benzoyi, pιperιzιn-1-ylcarbonyl, 4-methylpιperazιn-1-ylcarbonyl or 4-morpholιnylcarbonyl, R7 is (C, 5)alkyl, optionally substituted benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 3-pyrιdιnylmethyl or 2-methylsulfonylethyl, and R8 is butyl, 2-phenylethyl or 2-methylsulfonylethyl
Most preferred compounds of Formula I, II and III are those in which n is 0, A-B represents a linkage selected from -C(0)NH-, Y is -NH-, Z is -CH(R7)-, Z1 is -CH(R8)-, R1 is 1-pιperιzιnylcarbonyl, 4-methyl- 1-pιperazιnylcarbonyl or 4-morpholιnylcarbonyl; R7 is optionally substituted benzyl, 1-naphthylmethyl or 2-naphthylmethyl, and R8 is 2-phenylethyl
Generally preferred compounds of Formula I are those in which R2 is independently (C,.5)alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro, hydroxy and methoxy, or a protected derivative thereof). perhalo(C,.5)alkyl, (C3.7)cycloalkyl, (C 3-7)cycloalkyl(C,^)alkyl or a group selected from phenyl, pentafluorophenyl, naphthyl and phenyl(C,^)alkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl, or a protected derivative thereof) and R4 is hydrogen, (C, 5)alkyl or (C6 ,0)aryl(C, 5)alkyl More preferred compounds of Formula I are those in which in which R2 is (C, 5)alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro and hydroxy, or a protected deπvative thereof), perfluoro(C, 5)alkyl, (C5^)cycloalkyl, (C5^)cycloalkylmethyl or a group selected from phenyl, naphthyl and benzyl (which group is optionally substituted with one radical selected from ammo hydroxy, chloro, bromo or fluoro, or a protected derivative thereof) and R4 is hydrogen or methyl Particularly preferred compounds of Formula I are those in which R2 is methyl, tπfluoromethyl, optionally substituted phenyl, 2-naphthyl or 2-phenylethyl Most preferred compounds of Formula I in which R2 is phenyl, 2-naphthyl or 2-phenylethyl, particularly phenyl or 2-naphthyl, and R4 is hydrogen
Generally preferred compounds of Formula II in which R9 is -C(0)OR10, -P(O)(OR10)2, -S(O)(NR10)R10, -C(0)NHC(0)R10 or -S(0)2NHC(0)R10 are those in which each R10 is independently (C, 5)alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro, hydroxy and methoxy or a protected derivative thereof), (C37)cycloalkyl, (C3 7)cycloalkyl(C, 5)alkyl, or a group selected from phenyl or phenyl(C1-6)alkyl (which group is optionally substituted at its phenyl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl, or a protected derivative thereof) More preferred compounds of Formula II in which R9 is -C(0)OR10, -P(0)(OR1°)2, -S(O)(NR10)R10, -C(0)NHC(0)R10 or -S(0)2NHC(0)R10 are those in which in which R10 is ethyl, (C5^)cycloalkyl, (C5^)cycloalkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from ammo hydroxy, chloro, bromo or fluoro, or a protected derivative thereof)
Generally preferred compounds of Formula II in which R9 is C(0)R11 or -S(0)R11 are those in which R11 is (C, 5)alkyl, (C3 7)cycloalkyl, (C3 7)cycloalkyl(C1 5)alkyl or a group selected from phenyl and phenyl(C,^)alkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methyl, tπfluoromethyl and methoxy) More preferred compounds of Formula II in which R" is C(0)R" or -S(0)R11 are those in which in which R11 is ethyl, cyclo(C5^)alkyl, cyclo(C5^)alkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from ammo hydroxy, chloro, bromo or fluoro, or a protected derivative thereof)
Generally preferred compounds of Formula II in which R9 is -C(0)NR12R13 or -S(0)2NR1 R13 are those in which R12 and R13 are independently (Chalky!, (C3.7)cycloalkyl, (C37)cycloalkyl (C, 5)alkyl or a group selected from phenyl and pheny^C^alkyl (which group is optionally substituted at its phenyl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl) More preferred compounds of Formula II in which R9 is -C(0)NR12R13 or O 96/30353
21
-S(0)2NR12R13 are those in which R12 and R13 are independently ethyl, (C5<)cycloalkyl, (C-^cycloalkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from amino hydroxy, chloro, bromo or fluoro, or a protected derivative thereof).
Preferred compounds of Formula II in which R9 is -C(0)NHR14 or -S(0)2NHR14 wherein R14 is a group selected from Formulae (a) and (b) are those in which each n, A, B, Y, Z, R1 and R10 are as defined above with respect to preferred compounds of Formulae I, II and III.
Generally preferred compounds of Formula III are those in which R15 is a group selected from 2-furyl, 2-thienyl, 2-pyrrolyl, 2-phospholyl, 2-arsoyl, 3-pyridyl or 3-phosphorinyl (which group is optionally substituted with at least one radical selected from (CL^alkylcarbamoyl, di(C1.5)alkylcarbamoyl, (C1.5)alkyloxycarbonyl, (C,.5)alkylsulfinamoyl, di(C,.5)alkylsulfιnamoyl, (C^Jalkylsulfonyl, carboxy, nitro, sulfmamoyl, sulfo, carbamoyi, phosphono, (C,.5)alkyloxyphosphιnyl, d C^alkyloxyphosphinyl, (CLj alkanoyl, cyano, (C1.5)alkylsulfinyl, sulfamoyl, (CLj alkylsulfamoyl, d^CLsJalkylsulfamoyl, (C,.5)alkyloxysulfonyl, (C,.5), phenyl, naphthyl, pyridyl, thienyl, fury!, imidazolyl, indolyl, hydroxy, (C^sJalkyloxy, optionally halo-substituted (C,.5)alkyl, benzyl, halo, -*N(R17)3, wherein each R17 is independently (Chalky!, phenyl or benzyl, or -N(R18)2, wherein each R 8 is independently hydrogen, (C,.5)alkyl, phenyl or benzyl). More preferred compounds of Formula III are those in which R16 is a group selected from 2-furyl, 2-thienyl, 2-pyrrolyl, 2-phosholyl, 2-arsolyl, 3-pyridyl or 3-phosphorinyl (which group is optionally substituted with at least one radical selected from methylcarbamoyl, dimethylcarbamoyl, methyloxycarbonyl, methylsulfinamoyl, dimethylsulfinamoyl, methylsulfonyl, carboxy, nitro, sulfmamoyl, sulfo, carbamoyi, phosphono, methyloxyphosphinyl, dimethyloxyphosphinyl, formyl, cyano, methylsulfinyl, sulfamoyl, methylsulfamoyl, dimethylsulfamoyl, methoxysulfonyl, methylsulfonimidoyl, phenyl, naphthyl, pyridyl, thienyl, furyl, imidazolyl, indolyl, hydroxy, methoxy, methyl, trifluromethyl, benzyl, halo, -*N(R17)3, wherein each R17 is independently methyl, phenyl or benzyl, or -N(R18)2, wherein each R18 is independently hydrogen, methyl, phenyl or benzyl). Generally preferred compounds of Formula III in which R16 is a group selected from Formulae (a) and (b) are those in which each n, A, B, Y, Z, R1 and R10 are as defined above with respect to preferred compounds of Formulae I, II and III.
In general, preferred cysteine protease inhibitors of the invention are those in which the absolute configuration of each chiral center present is the (S)-confιguration. However, preferred compounds of Formula I in which n is 0 are those in which the absolute configuration of chiral center to which the R7 substituent is attached is in the (/^-configuration. For example, preferred compounds of Formula I include: /V2-(4-moφholinylcarbonyl)-Λ/1-(3-phenyl-1R-phenylsulfonylpropyl)-L-phenylalaninamide (compound 1 ), Λ/2-(4-morpholinylcarbonyl)-Λ/1-(3-phenyl-1 S-phenylsulfonylpropyl)-L-phenyalaninamide (compound 2), Λ/2-(4-morpholιnylcarbonyl)-Λ/1-(3-phenyl-1-phenylsulfonylpropyl)-L-phenylalanιnamιde (compound 3), Λ/2-(4-morpholιnylcarbonyl)-/V1-(3-phenyl-1-benzylsulfonylpropyl)-L-leucιnamιde (compound 4), Λ^^-morpholinylcarbony -rV S-phenyl-l-tnfluoromethylsulfonylpropyl)- L-phenylalanmamide (compound 5), Λ/2-(4-morpholιnylcarbonyl)-Λ 1-(3-phenyl-1 -benzylsulfonylpropyl)- L-phenylalanmamide (compound 6), Λ/2-(4-morpholιnylcarbonyl)-Λ 1-(3-phenyl-1-phenylsulfonylpropyl)- L-leucmamide (compound 7), Λ/2-(4-morpholιnylcarbonyl)-Λ/1-(3-phenyl-1-fluoromethylsulfonylpropyl)- L-phenylalaninamide (compound 8), Λ/2-(4-morpholιnylcarbonyl)-Λ 1-(3-phenyl- 1 S-phenylsulfonylmethylpropyl)-L-phenylalanιamιde (compound 9), Λr?-(4-morpholιnylcarbonyl)- Λ/1-{3-phenyl-1 S-[2-(2-phenylethylsulfonyl)ethyl] propyl}-L-phenylalanιnamιde (compound 10), Λ/2-(4-morpholιnylcarbonyl)-Λ/1-{3-phenyl-1S-[2-(2-naphthylsulfonyl)ethyl]propyl}-β-(2-naphthyl)- L-alanmamide (compound 11),
Figure imgf000024_0001
L-phenylalaninamide (compound 12), Λ/2-(Λ/-benzyloxycarbonyl-β-alanyl)-Λ/1-[3-phenyl- 1 S-(2-phenylsulfonylethyl)propyl]-L-phenylalanιnamιde (compound 13), 3-{2-phenyl-1 S-[3-phenyl- 1 S-(2-phenylsulfonylethyl)propylcarbamoyl]ethylcarbamoyl}propιonιc acid (compound 14), 3-{2-naphthyl-1 S-[3-phenyl-1 S-(2-phenylsulfonylethyl)propylcarbamoyl] ethylcarbamoyl}propιonιc acid (compound 15), Λ/2-(4-morpholιnylcarbonyl)-/V1-{3-pheny!-1 S-[2-(2-naphthylsulfony l)ethyl]propyl}- L-tyrosinamide (compound 16), methyl 3-{2-phenyl-1 S-[3-phenyl- 1 S-(2-phenylsulfonylethyl)propylcarbamoyl]ethylcarbamoyl}propιonate (compound 17), Λ^-(4-morpholιnylcarbonyl)-Λ 1-[3-phenyl-1 S-(2-phenylsulfonylethyl)propyl]-L-phenylalanιnamιde (compound 18), Λ 2-(β-alanyl)-Λ 1-[3-phenyl-1S-(2-phenylsulfonylethyl) propyl]-L-phenylalanιnamιde (compound 19), and 5-phenylsulfonyl-3S-{Λ/-[Λ/-(Λ/-acetyl-L-tyrosyl)-L-valyl]-L-alanylamιno}valeπc acid (compound 20) Preferred compounds of Formula II include ethyl 4S-(Λ/-benzylsulfonyl- β-(2-naphthyl)-L-alanylamιno)-6-phenylhexanoate (compound 21 ), ethyl 4S-(Λ/-benzylcarbamoyl- β-(2-naphthyl)-L-alanylamιno)-6-phenylhexanoate (compound 22), ethyl
4S-[Λ/-(4-morpholιnylcarbonyl)-β-2-(naphthyl-L-alanylamιno]-6-phenylhexanoate (compound 23), ethyl 4S-(Λ/-benzylcarbamoyl-L-phenylalanylamιno)-6-phenylhexanoate (compound 24), ethyl 4S-[Λ/-(4-morpholιnylcarbonyl)-L-phenylalanylamιno]-6-phenylhexanoate (compound 25), Λ/2-(4-morpholιnylcarbonyl)-Λ/'-[3-phenyl-1 S-(2-phenylcarbamoylethyl)propyl]-L-phenylalanιnamιde (compound 26), and Λ/^-morpholinylcarbonylHV-β-phenyl-l S-(2-benzylcarbamoylethyl)propyl]- L-phenylalanmamide (compound 27) Preferred compounds of Formula III include Λ/2-(4-morpholιnylcarbonyl)-Λ 1'{3-phenyl-1S-[2-(4-methoxyphenyl)ethyl]propyl}-L-phenylalanιnamιde (compound 28), and Λ/2-(4-morpholιnylcarbonyl)-ΛT"{3-phenyl-1S-[2-(4-amιnophenyl)ethyl]propyl}- L-phenylalanmamide (compound 29)
As will be appreciated by those in the art, Formula I includes structures represented by preferred Species IV as depicted below.
Figure imgf000025_0001
wherein M is zero, one or two carbon atoms, A-B are as defined above, R\ R2, R7 and R8 are as defined above, and Q is NH or CH2. Preferred embodiments utilize A-B linkages which contain nitrogen at the B position. In this embodiment, the number of carbon atoms between the carbon to which the R8 group is attached and the sulfur atom of the sulfone group determines whether the compound is an α-amιnosulfone, a β-aminosulfone, or a γ-aminosulfone. As is discussed below in the Examples, compounds may be named as aminosulfones using the names of the amino acids or using the chemical names.
Thus, for example, Species V is an α-aminosulfone:
Figure imgf000025_0002
Species VI is a β-aminosulfone:
Species VII is a γ-aminosulfon
Figure imgf000025_0003
Formula II includes structures of Species VIII, referred to as γ-amino groups, particularly when R9 is an electron withdrawing group:
Figure imgf000025_0004
VIII In a preferred embodiment, the dissociation constant for inhibition of a protease with an inhibitor of the invention, generally referred to by those in the art as K,, is at most 100 μM. By the term "binding constant" or "dissociation constant" or grammatical equivalents herein is meant the equilibrium dissociation constant for the reversible association of inhibitor with enzyme. The dissociation constants are defined and determined as below.
The determination of dissociation constants is known in the art. For example, for reversible inhibition reactions such as those of the present invention, the reaction scheme is as follows:
Equation 3
E + I * E k
The enzyme and the inhibitor combine to give an enzyme-inhibitor complex, E-l This step is assumed to be rapid and reversible, with no chemical changes taking place; the enzyme and the inhibitor are held together by non-covalent forces In this reaction, k, is the second order rate constant for the formation of the E-l reversible complex k2 is the first order rate constant for the disassociation of the reversible E-l complex In this reaction, K, = k2/k,
The measurement of the equilibrium constant K* proceeds according to techniques well known in the art, as described in the examples For example, assays generally use synthetic chromogenic or fluorogenic substrates
The respective K, values may be estimated using the Dixon plot as described by Irwin Segel in Enzyme Kinetics1 Behavior and analysis of rapid equilibrium and steady-state enzyme systems, 1975, Wiley-lnterscience Publication, John Wiley & Sons, New York, or for competitive binding inhibitors from the following calculation
Equation 4
1-(v/v0) = [l]/([l] + K,(1+([S]/KM)))
wherein v0 is the rate of substrate hydrolysis in the absence of inhibitor, and v, is the rate in the presence of competitive inhibitor.
It is to be understood that dissociation constants are a particularly useful way of quantifying the efficiency of an enzyme with a particular substrate or inhibitor, and are frequently used in the art as such If an inhibitor exhibits a very low K*. it is an efficient inhibitor Accordingly, the cysteine protease inhibitors of the present invention have dissociation constants, K,, of at most about 100 μM Preferred embodiments have inhibitors that exhibit dissociation constants of at most about 10 μM, with the most preferred embodiments having dissociation constants of at most about 1 μM
CHEMISTRY
The synthesis of the inhibitors of the invention proceeds as follows Compounds of Formula I in which X represents a bond can be prepared by the process depicted in Scheme 1 of Figure 1
Treatment of fert-butylcarbamate or benzyl carbamate with an appropriate aldehyde, such as isobutyraldehyde or hydrocinnamaldehyde, along with the sodium salt of a suitable sulfinic acid, such as benzenesulfmic acid (Aldrich Chemical Co ), in the presence of aqueous formic acid affords the corresponding N-protected ammomethyl sulfone Benzyloxycarbonyl protected ammomethyl sulfones are deprotected with hydrogen bromide in acetic acid Coupling with a suitable N-protected am o acid or peptide or a peptidomimetic derivative thereof affords a compound of Formula I in which X represents a bond Alternatively, an appropriate N-termmal protected ammo acid or peptide of peptidomimetic derivative thereof, such as N-(4-morpholιnylcarbonyl)phenylalanιnamιde, is reacted with an appropriate aldehyde along with the sodium salt of a suitable sulfinic acid, in the presence of aqueous formic acid to afford a compound of Formula I in which X represents a bond
Compounds of Formula 1 in which X represents a methylene bond can be prepared by the processes depicted in Schemes 2 and 3, Figures 2 and 3, respectively
Treatment of a suitable N-protected am o acid or peptidomimetic derivative thereof with sodium borohydπde affords the corresponding β-ammoethanol Treatment of the alcohol with methanesulfonyl chloride in the presence of tnethylamine affords the corresponding mesylate Nucleophi c displacement with the anion of a thiol, such as thiophenol, according to the method of Spaltenstein, A , Carpion, P , Miyake, F , and Hopkmgs, P B , J Org Chem (1987) 52, 3759, affords the corresponding β-ammosulfide The sulfide is oxidized by means of 4-chloroperbenzoιc acid to give the corresponding N-protected β-ammoethyl sulfone In a special instance, the mesylate is treated with thiolate ion such as that derived from 2-(trιmethylsιlyl)ethanethιol, the synthesis of which is described by Anderson, M B , Ranas ghe, M B , Palmer, J T , Fuchs, P.L , J Org Chem (1988) 53, 3125, to give the corresponding β-ammoethyl 2-trιmethylsιlylethyl sulfide The 2-tnmethylsιlylethyi sulfide is reduced to the corresponding β-ammoethyl 2-tπmethylsιlylethyl sulfone, which is then subjected to fluoride-mediated cleavage, extruding tπmethylsilyl fluoride and ethene as gaseous by-products, and an intermediate sulfmate, which sulfmate is alkylated in situ with an appropriate halogen-containing species such as bromochloromethane to give the corresponding N-protected β-aminoethyl halomethyl sulfone The N-protected β-aminoethyl sulfones are deprotected and then coupled with a suitable N-protected ammo acid or peptide or a peptidomimetic derivative thereof to afford a compound of Formula I in which X is methylene
Compounds of Formula II and Formula I in which X represents ethylene can be prepared by the processes depicted in Equations 5, 6 and 7
Equation 5
Figure imgf000028_0001
wherein a) is a) CI-H2N+(Me)OMe, dicyclohexylcarbodiimide, tπethylamine, and b) lithium aluminum hydride
Equation 6
Figure imgf000028_0002
Equation 7
Figure imgf000028_0003
An appropriate N-tert-butoxycarbonyl ammo acid or peptidomimetic derivative thereof is converted to the corresponding ammomethyl aldehyde (e g , see method of Fehrentz, J-A and Castro, B (Synthesis, (1983), 676, Equation 5) The aldehyde is converted to the corresponding vmylogous compound via aWittig reaction or a Wadsworth-Emmons-Horner modification of the Wittig reaction (e g , see Wadsworth et al , J Amer Chem Soc 83 1733 (1991), Equation 6) The vmylogous compound is reduced by catalytic hydrogenation (e g , see Equation 7) and then deprotection and coupling with a suitable N-protected ammo acid or peptide or a peptidomimetic derivative thereof gives the corresponding compound of Formula I or II Alternatively, the vmylogous compound is deprotected and coupled with the N-protected am o acid or peptide or a peptidomimetic derivative thereof to give the corresponding vmyloguous condensation product, which is then reduced to give the corresponding compound of Formula I or II
Compounds of Formula II can be prepared by the processes depicted in Scheme 4, Figure 4
Preferably, the conversion of N-tert-butoxycarbonyl ammo acid or peptidomimetic derivative thereof to the corresponding ammomethyl aldehyde is carried out with N,0-dιmethylhydroxylamιne hydrochloπde in the presence of tπethylamine and dicyclohexylcarbodiimide in dicloromethane Alternatively, the conversion is carried out by treating the ammo acid or peptidomimetic derivative with tπethylamine and the coupling agent benzotπazol-1-yloxytπs(dιmethylamιno)phosphonιum hexafluorophosphate (BOP) and then reducing with lithium aluminum hydride to give the corresponding aldehyde (e g , see methos of Fehrentz, J-A and Castro, B , Synthesis, (1983), 676-678) The conversion of the aldehyde to the corresponding vmylogous ester can be carried out with the sodium anion of tπethyl phosphonoacetate Deprotection of the vmylogous ester can be carried out with hydrogen chloride in dioxane The hydrogenation is typically carried out in the presence of palladium
Compounds of Formula I in which X is ethylene are conveniently prepared by the process depicted in Scheme 5, Figure 5
Treatment of a suitable N-tert-butoxycarbonyl-α-ammoaldehyde, prepared as described in Equation 5, with the sodium anion of an appropriate sulfonylmethanephosphonate (SMP) (e g, diethyl phenylsulfonylmethanephosphonate, diethyl 2-naphthylsulfonylmethane-phosphonate, diethyl methylsulfonylmethanephosphonate, etc ) gives the corresponding vmylogous sulfone The sulfone is deprotected with anhydrous p-toulenesulfonic acid in ether and then coupled with N-protected ammo acid or peptide or a peptidomimetic derivative thereof to give the corresponding vmyloguous condensation product, which is then reduced to give the corresponding compound of Formula I Suitable arylsulfonylmethanephsophonates can be prepared by treating arylthiols with paraformaldehyde in the presence of hydrogen chloride and reacting with tnethyl phosphite to give the corresponding diethyl phsophonomethyl aryl sulfide and then oxidizing the sulfide Alternatively, suitable sulfides can be obtained commercially (e g , diethylphosphonomethyl methyl sufide obtained from Aldrich Chemical Co , diethylphosphonomethyl phenyl sulfide, etc ) and oxidized to their corresponding sulfones
Compounds of Formula II in which R9 is -COOH can be prepared by the process depicted in Scheme 6, Figure 6 28
Generally, saponification of a compound of Formula II in which R9 is -COOR10 results in the cooresponding carboxylate, which upon treatment with acid gives the corresponding carboxylic acid.
Compounds of Formula II in which R9 is -P(O)(R10)2 can be prepared as depicted in Scheme 7 (Figure 7) by proceeding as in Scheme 6, but substituting for the SMP the sodium anion of an appropriate methylenediphosphonate (e.g., tetraethyl methylenediphosphonate, etc.)
Compounds of Formula II in which R9 is -C(0)NHR14 can be prepared as depicted in Scheme 8 (Figure 8) by proceeding as in Scheme 5, but substituting for the SMP an appropriate diethyl amidomethylenephsophonate (e.g , diethyl benzylamidomethylenephosphonate, etc.).
Suitable amidomethylenephosphonates can be prepared by reacting the saponification product of tnethyl phosphonoacetate with an appropriate amme Altenatively, compounds of Formula II in which R9 is -C(0)NHR14 can be prepared by reacting a compound of Formula I in which R9 is -COOH with an appropriate amme For example, the reaction can be carried out in the presence of dicyclohexylcarbodiimide in dichloromethane or by any other peptide coupling reaction sequences known to those of skill in the art.
In general, compounds of Formula II can be prepared by the process depicted in Scheme 9 (Figure 9) and substituting the starting materials represented by Structures l-VII
Structure I
Figure imgf000030_0001
Synthesis of ketones is performed by means of the Wadsworth-Emmons reaction between Boc-α- ammo aldehydes and the appropriate phosphonate, followed by catalytic reduction with hydrogen in the presence of palladium. Generally, the aldehyde portion is synthesized as outlined above. The phosphonate, if not commercially available, is synthesized by treatment of the enolate anion of methyl or substituted methyl ketones, such as acetone or acetophenone, with diethyl chlorophosphonate. The enolate anion is generated, for example, by treatment of a tetrahydrofuran solution of diisopropylamine with butyllithium, followed by addition of the ketone to the lithium diisopropylamide (LDA) solution (H.O. House, Modern Synthetic Reactions, 2nd Ed. (W. Benjamin, Inc., Menlo Park, CA, Chapter 9). Following formation of the enolate, diethyl chlorophosphonate is added. The Wadsworth-Emmons reagent forms as a consequence of coupling of the enolate with diethyl chlorophosphate. For the synthesis of cysteine protease inhibitors with nitπles as the EWG, structure II is used
Structure II
Figure imgf000031_0001
Synthesis of nitπles is performed by means of the Wadsworth-Emmons reaction between Boc-α- ammo aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst Generally, the aldehyde portion is synthesized as outlined above The phosphonate is commercially available
For the synthesis of cysteine protease inhibitors with sulfoxides as the EWG, structure III is used
Structure III
Figure imgf000031_0002
Synthesis of sulfoxides is performed by means of the Wadsworth-Emmons reaction between Boc-α- ammo aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst Generally, the aldehyde portion is synthesized as outlined above The phosphonate is synthesized by treatment of the anion of methyl sulfoxides with diethyl chlorophosphate The anion is generated by addition of BuLi to diisopropylam e, followed by addition of the methyl sulfoxide
For the synthesis of cysteine protease inhibitors with sulfonamides as the EWG, structure IV is used
Structure IV
Figure imgf000031_0003
Synthesis of sulfonamides is performed by means of the Wadsworth-Emmons reaction between Boc- α-ammo aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst Generally, the aldehyde portion is synthesized as outlined above The phosphonate is synthesized, for instances, by a method such as the following a) diethylphosphoryl methanesulfonates, as prepared by the method of Carretero and Ghosez (Tetrahedron Lett , 28 1104- 1108 (1987)), are converted to sulfonyl chlorides by treatment with phosphorus pentachloπde (M Quaedvlieg, in "Methoden der Organische Chemic (Houben-Weyl)", ed. E. Muller, Thieme Verlag, Stuttgart, 4th Ed., 1955, Vol. IX, Chapter 14), or b) treatment of the sulfonyl chloride with an amme, such as ammonia, a primary amme (including an am o acid derivative), or a secondary amine, that results in the formation of the sulfonamide (Quaedvlieg, supra, Chapter 19) The sulfonamide- phosphonate is then reacted with Boc-α-amιnoaldehydes to form the target compounds as per the Wadsworth-Emmons reaction
For the synthesis of cysteine protease inhibitors with sulfinamides as the EWG, structure V is used
Structure V
Figure imgf000032_0001
Synthesis of sulfinamides is performed by means of the Wadsworth-Emmons reaction between Boc-α- amino aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst Generally, the aldehyde portion is synthesized as outlined above The phosphonate may be synthesized using one of the following methods Treatment of methyl dialkyl phosphonates such as the commercially available methyl diethyl phosphonate (Aldrich), with thionyl chloride in the presence of aluminum chloride gives the dialkylphosphoryl methanesulfinyl chloride (Vennstra ef al , Synthesis (1975) 519 See also Anderson, "Comprehensive Organic Chemistry (Pergamon Press)", Vol 3, Chapter 11 18, (1979) Alternatively, treatment of the dialkyl phosphoryl sulfinyl chloride with amines (Stirling, Internat J. Sulfur Chem (B) 6.277 (1971)), yields the dialkyl phosphoryl sulfinamide
For the synthesis of cysteine protease inhibitors with sulfoximines as the EWG, structure VI is used
Structure VI
Figure imgf000032_0002
Synthesis of sulfoximines is performed by means of the Wadsworth-Emmons reaction between Boc-α- ammo aldehydes and the appropriate phosphonate, followed by hydrogenation in the presence of a suitable catalyst. Generally, the aldehyde portion is synthesized as outlined above The phosphonate may be synthesized in several ways. For example, N-alkyl or N-aryl phenyl methyl sulfoximines are made by the methods described by Johnson, in "Comprehensive Organic Chemistry (Pergamon Press), supra, Chapter 11.11. Alternatively, the lithium anion of compounds such as N-alkyl phenyl methyl sulfoximine is prepared by the treatment of the neutral compound with buthyl lithium in THF (Cram ef al., J. Amer. Chem. Soc. 92:7369 (1970)). Reaction of this lithium anion with dialkyl chlorophosphates such as the commercially available diethyl chlorophosphate (Aldrich) results in the Wadsworth-Emmons reagent necessary for synthesis of the sulfoximme compounds
For the synthesis of cysteine protease inhibitors with sulfonates as the EWG, structure VII is used
Structure VII
Figure imgf000033_0001
Synthesis of sulfonates is performed by means of the Wadsworth-Emmons reaction between Boc-α- ammo aldehydes and the appropriate phosphonate, for instance diethylphosphoryl methanesulfonate, followed by hydrogenation in the presence of a suitable catalyst, such as Raney nickel The phosphonate may be synthesized as follows The anion of methyl dialkyl phosphonates such as the commercially available methyl diethyl phosphonate (Aldrich) is generated by treatment of said phosphonate with a strong base such as LDA The resulting anion is sulfonated with sulfur tπoxide/trimethylamine complex (Carreto ef al , Tetrahedron Lett , 28 1104-1108 (1987)) to form diethylphosphoryl methanesulfonate, which is capable of reacting in the Wadsworth-Emmons procedure with aldehydes to form α,β-unsaturated sulfonates
Compounds of Formula II can be prepared by the process depicted in Scheme 10 (Figure 10)
The chloride compounds containing R8 and R9 groups are generally made using commercially available reagents and products using techniques well known in the art The reaction generally produces a mixture of cis and trans configurations, favoring the trans isomer Upon reduction to the cysteine protease inhibitors of this embodiment, the cis-trans isomeπsm disappears by definition as a single compound is formed
In one embodiment, the cysteine protease inhibitors of the invention are further purified if necessary after synthesis, for example to remove unreacted materials For example, the cysteine protease inhibitors of the present invention may be crystallized, or passed through silica chromatography columns using solvent mixtures to elute the pure inhibitors
In summary, the processes for preparing compounds of the invention are as follows (A) for the preparation of Formula IV
Figure imgf000033_0002
IV in which n is 0 to 12, R20 is cyano, -S(0)2R2, -CH2S(0)2R2, -CH2CH(R4)S(0)2R2, -(CH2)2C(0)OR10, -(CH2)2P(O)(OR10)2, -(CH2)2S(O)(NR10)R10, -CH2)2C(0)R11, -(CH2)2S(0)R11, -(CH2)2C(0)NR12R13, -(CH2)2S(0)2NR12R13, -(CH2)2C(0)NHR14, -(CH2)2S(0)2NHR14 or -CH2CHR15R16 and each A, B, X, Y, Z, R1, R8 R1, R8, R2, R10, R11, R12, R13, R14, R15 and R16 are as defined in the Summary of the Invention with respect to compounds of Formulae I, II and III, and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, reacting an amme of Formula V
Figure imgf000034_0001
with a compound of the Formula VI
Figure imgf000034_0002
VI
in which each n, A, B, X, Y, Z, R1, R8 and R20 are as defined above, (B) for the preparation of a compound of Formula IV in which R20 is -S(0)2R2, and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, reacting a compound of Formula VII
Figure imgf000034_0003
VII
with an aldehyde of the formula R8CHO and a sodium sulfmate of the formula R2S(0)ONa, in which each n, A, B, X, Y, Z, R1 and R8 are as defined above,
(C) for the preparation of a compound of Formula IV in which R20 is -S(0)2R2, and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof,
(1) reacting a compound of the formula NH2P, wherein P is a protective group, with an aldehyde of the formula R8CHO and a sodium sulfinate of the formula R2S(0)ONa and then deprotecting to give a compound of Formula VIII
H2N S(0) 2R in which each R2 and R8 are as defined in the Summary of the Invention with respect to Formula I; and (2) reacting the compound of Formula VIM with a compound of Formula VI in which each n, A, B, X, Y, Z and R1 are as defined above;
(D) for the preparation of a compound of Formula IV in which R20 is -CH2S(0)2R2 and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, (1) reacting a compound of Formula IX:
Figure imgf000035_0001
with a thiolate anion of the formula R2S", in which L is a leaving group R2 and R8 are as defined above, to give a compound of Formula X:
Figure imgf000035_0002
(2) oxidizing the compound of Formula X to give a compound of Formula XI:
, S(0) 2R- PHN ' ^*-' and
(3) reacting the compound of Formula XI with a compound of Formula VI in which each n, A, B, X, Y, Z and R are as defined above;
(E) for the preparation of a compound of Formula IV in which R20 is cyano, -(CH2)2S(0)2R2, -(CH2)2C(0)OR10, -(CH2)2P(O)(OR10)2, -(CH2)2S(O)(NR10)R10, -(CH2)2C(0)R11, -(CH2)2S(0)R11, -(CH2)2C(0)NR1 R13, -(CH2)2S(0)2NR1 R13, -(CH2)2C(0)NHR14or -(CH2)2S(0)2NHR14, and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof,
(1) reacting an aldehyde of Formula XII:
Figure imgf000035_0003
with a compound selected from Formulae XIII and XIV:
Figure imgf000035_0004
in which each R8 and R20 are as defined above, and then deprotecting to give a compound of Formula XV
Figure imgf000036_0001
XV
(2) reacting the compound of Formula XV with a compound of Formula VI in which each n, A, B, X, Y, Z and R1 are as defined above, and
(3) reducing,
(F) for the preparation of a compound of Formula IV in which R20 is -CH2CHR15R16 and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, (1 ) reacting an aldehyde of Formula XII with compound of Formula XVI
Figure imgf000036_0002
XVI
in which each R8, R 5 and R16 are as defined above, and then deprotectmg to give a compound of Formula XVII
Figure imgf000036_0003
XVII
(2) reacting the compound of Formula XVII with a compound of Formula VI in which each n, A, B, X, Y, Z and R1 are as defined above, and
(3) reducing,
(G) optionally further converting a non-salt form of a compound of Formula IV into a pharmaceutically acceptable salt,
(H) optionally further converting a salt form of a compund of Formula IV into non-salt form; and (H) optionally further separating a compound of Formula IV into individual stereoisomers
In one embodiment, the cysteine protease inhibitors of the present invention are labelled. By a "labelled cysteine protease inhibitor" herein is meant a cysteine protease inhibitor that has at least one element, isotope or chemical compound attached to enable the detection of the cysteine protease inhibitor or the cysteine protease inhibitor bound to a cysteine protease. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes The labels may be incorporated into the cysteine protease inhibitor at any position For example, a label may be attached as the "R1" group in Formula 1 , or a radioisotope incorporated into any position Examples of useful labels include 14C, 3H, biotm, and fluorescent labels as are well known in the art
PHARMACOLOGY AND UTILITY
Once produced, the cysteine protease inhibitors of the present invention may be easily screened for their inhibitory effect The inhibitor is first tested against the cysteine protease for which the targeting group of the inhibitor was chosen, as outlined above Alternatively, many cysteine proteases and their corresponding chromogenic substrates are commercially available Thus, a variety of cysteine proteases are routinely assayed with synthetic chromogenic substrates in the presence and absence of the cysteine protease inhibitor, to confirm the inhibitory action of the compound, using techniques well known in the art The effective inhibitors are then subjected to kinetic analysis to calculate the K, values and the dissociation constants determined
If a compound inhibits at least one cysteine protease, it is a cysteine protease inhibitor for the purposes of the invention Preferred embodiments have inhibitors that exhibit the correct kinetic parameters against at least the targeted cysteine protease
In some cases, the cysteine protease is not commercially available in a purified form The cysteine protease inhibitors of the present invention may also be assayed for efficacy using biological assays For example the inhibitors may be added to cells or tissues that contain cysteine proteases, and the biological effects measured
In one embodiment, the cysteine protease inhibitors of the present invention are synthesized or modified such that the in vivo and in vitro proteolytic degradation of the inhibitors is reduced or prevented Generally, this is done through the incorporation of synthetic ammo acids, derivatives, or substituents into the cysteine protease inhibitor Preferably, only one non-naturally occurring am o acid or am o acid side chain is incorporated into the cysteine protease inhibitor, such that the targeting of the inhibitor to the enzyme is not significantly affected However, some embodiments that use longer cysteine protease inhibitors containing a number of targeting residues may tolerate more than one synthetic derivative In addition, non-naturally occurring ammo acid substituents may be designed to mimic the binding of the naturally occurring side chain to the enzyme, such that more than one synthetic substituent is tolerated Alternatively, peptide isosteres are used to reduce or prevent inhibitor degradation In this embodiment, the resistance of the modified cysteine protease inhibitors may be tested against a variety of known commercially available proteases in vitro to determine their proteolytic stability Promising candidates may then be routinely screened in animal models, for example using labelled inhibitors, to determine the in vivo stability and efficacy
Specific cysteine proteases that may be inhibited by the inhibitors of the present invention are those of the family of cysteine proteases that bear a thiol group at the active site These proteases are found in bacteria, viruses, eukaryotic microorganisms, plants, and animals Cysteine proteases may be generally classified as belonging to one of four or more distinct superfamilies Examples of cysteine proteases that may be inhibited by the novel cysteine protease inhibitors of the present invention include, but are not limited to, the plant cysteine proteases such as papain, ficin, aleuram, oryzam and actmidam, mammalian cysteine proteases such as cathepsms B, H, J, L, N, S, T, O, and C, (cathepsin C is also known as dipeptidyl peptidase I), interleukm converting enzyme (ICE), calcium-activated neutral proteases, calpam I and II, bleomyαn hydrolase, viral cysteine proteases such as picornian 2A and 3C, aphthovirus endopeptidase, cardiovirus endopeptidase, comovirus endopeptidase, potyvirus endopeptidases I and II, adenovirus endopeptidase, the two endopeptidases from chestnut blight virus, togavirus cysteine endopeptidase, as well as cysteine proteases of the polio and rhinoviruses, and cysteine proteases known to be essential for parasite lifecycles, such as the proteases from species of Plasmodia, Entamoeba, Onchocera, Trypansoma, Leishmanta, Haemonchus, Dictyostelium, Thenleπa, and Schtstosoma, such as those associated with malaria (P falcipanum), trypanosomes (T cruzi, the enzyme is also known as cruzain or cruzipam), murine P vinckei, and the C elegans cysteine protease For an extensive listing of cysteine proteases that may be inhibited by the cysteine protease inhibitors of the present invention, see Rawlmgs ef al , Biochem J 290 205-218 (1993), hereby expressly incorporated by reference
Accordingly, inhibitors of cysteine proteases are useful in a wide variety of applications For example, the inhibitors of the present invention are used to quantify the amount of cysteine protease present in a sample, and thus are used in assays and diagnostic kits for the quantification of cysteine proteases in blood, lymph, saliva, or other tissue samples, in addition to bacterial, fungal, plant, yeast, viral or mammalian cell cultures Thus in a preferred embodiment, the sample is assayed using a standard protease substrate A known concentration of cysteine protease inhibitor is added, and allowed to bind to a particular cysteine protease present The protease assay is then rerun, and the loss of activity is correlated to cysteine protease activity using techniques well known to those skilled in the art
The cysteine protease inhibitors are also useful to remove or inhibit contaminating cysteine proteases in a sample For example, the cysteine protease inhibitors of the present invention are added to samples where proteolytic degradation by contaminating cysteine proteases is undesirable Alternatively, the cysteine protease inhibitors of the present invention may be bound to a chromatographic support, using techniques well known in the art, to form an affinity chromatography column A sample containing an undesirable cysteine protease is run through the column to remove the protease
In a preferred embodiment, the cysteine protease inhibitors are useful for inhibiting cysteine proteases implicated in a number of diseases In particular, cathepsms B, L, and S, cruzain, calpains I and II, and mterleukin 1β converting enzyme are inhibited These enzymes are examples of lysosomal cysteine proteases implicated in a wide spectrum of diseases characterized by tissue degradation Such diseases include, but are not limited to, arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, parasite-borne infections, Alzheimer's disease, peπodontal disease, and cancer metastasis For example, mammalian lysosomal thiol proteases play an important role in intracellular degradation of proteins and in the processing of some peptide hormones Enzymes similar to cathepsms B and L are released from tumors and may be involved in tumor metastasis Cathepsin L is present in diseased human synovial fluid and transformed tissues Similarly, the release of cathepsin B and other lysosomal proteases from polymorphonuclear granulocytes and macrophages is observed in trauma and inflammation
The cysteine protease inhibitors also find application in a multitude of other diseases, including, but not limited to, gingivitis, malaria, leishmaniasis, filaπasis, and other bacterial and parasite-borne infections The compounds also offer application in viral diseases, based on the approach of inhibiting proteases necessary for viral replication For example, many picornoviruses including poliovirus, foot and mouth disease virus, and rh ovirus encode for cysteine proteases that are essential for cleavage of viral polyprotems
Additionally, these compounds offer application in disorders involving ιnterleukιn-1β converting enzyme (ICE), a cysteine protease responsible for processing mterleukin 1β, for example, in the treatment of inflammation and immune based disorders of the lung, airways, central nervous system and surrounding membranes, eyes, ears, joints, bones, connective tissues, cardiovascular system including the pericardium, gastrointestinal and urogenital systems, the skin and the mucosal membranes These conditions include infectious diseases where active infection exists at any body site, such as meningitis and salpmgitis, complications of infections including septic shock, disseminated mtravascular coagulation, and/or adult respiratory distress syndrome, acute or chronic inflammation due to antigen, antibody and/or complement deposition, inflammatory conditions including arthritis, chalangitis, colitis, encephalitis, endocarditis, glomerulonephritis, hepatitis, myocarditis, pancreatitis, pericarditis, reperfusion injury and vascuhtis Immune-based diseases include but are not limited to conditions involving T-cells and/or macrophages such as acute and delayed hypersensitivity, graft rejection, and graft-versus-host disease, auto-immune diseases including Type I diabetes mellitus and multiple sclerosis Bone and cartilage reabsorption as well as diseases resulting in excessive deposition of extracellular matrix such as interstitial pulmonary fibrosis, cirrhosis, systemic sclerosis, and keloid formation may also be treated with the inhibitors of the present invention The inhibitors may also be useful in the treatment of certain tumors that produce IL 1 as an autocπne growth factor and in preventing the cachexia associated with certain tumors Apoptosis and cell death are also associated with ICE and ICE-hke activities and may be treated with the inhibitors of the present invention
Furthermore, the cysteine protease inhibitors of the present invention find use in drug potentiation applications For example, therapeutic agents such as antibiotics or antitumor drugs can be inactivated through proteolysis by endogeneous cysteine proteases thus rendering the administered drug less effective or inactive For example, it has been shown that bleomycm, an antitumor drug, can be hydrolyzed by bleomycm hydrolase, a cysteine protease (see Sebti et al , Cancer Res January 1991 , pages 227-232) Accordingly, the cysteine protease inhibitors of the invention may be administered to a patient in conjunction with a therapeutic agent in order to potentiate or increase the activity of the drug This co-administration may be by simultaneous administration, such as a mixture of the cysteine protease inhibitor and the drug, or by separate simultaneous or sequential administration
In addition, cysteine protease inhibitors have been shown to inhibit the growth of bacteria, particularly human pathogenic bacteria (see Bjorck et al , Nature 337 385 (1989)) Accordingly, the cysteine protease inhibitors of the present invention may be used as antibacterial agents to retard or inhibit the growth of certain bacteria
The cysteine protease inhibitors of the invention also find use as agents to reduce the damage of bacterial cysteine proteases to host organisms For example, staphylococcus produces a very active extracellular cysteine protease which degrades insoluble elastin, possibly contributing to the connective tissue destruction seen in bacterial infections such as septicemia, septic arthritis and otitis See Potempa et al , J Biol Chem 263(6) 2664-2667 (1988) Accordingly, the cysteine protease inhibitors of the invention may be used to treat bacterial infections to prevent tissue damage
ADMINISTRATION AND PHARMACEUTICAL COMPOSITION
In general, cysteine protease inhibitors of this invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with another cysteine protease inhibitor of the invention or with another therapeutic agent A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. Therapeutically effective amounts of the cysteine protease inhibitors of this invention may range from 10 micrograms per kilogram body weight (μg/kg) per day to 10 milligram per kilogram body weight (mg/kg), typically 100 μg/kg/day to 1 mg/kg/day. Thus, a therapeutically effective amount for a 80 kg human may range from 1 mg/day to 1000 mg/day, typically 10 mg/day to 100 mg/day.
One of ordinary skill in the art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of a cysteine protease inhibitor of this invention for a given disease.
In general, the cysteine protease inhibitors of this invention will be administered as pharmaceutical compositions by one of the following routes: oral, systemic (e.g., transdermal, intranasal, intrapulmonary, or by suppositiory) or parenteral (e.g., intramuscular, intravenous, intrapulmonary or subcutaneous). Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixiers, aerosols or any other appropriate composiiton and are comprised of, in general, a cysteine protease inhibitor of the invention in combination with at least one pharmaceutically acceptable excipient. Acceptable excipients are non-toxic, aid administration, and don not adversely affect the therapeutic benefit of the cysteine protease inhibitor of this invention. Such excipient may be any solid, liquid, semisolid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.
Solid pharmaceutical excipients include starch, cellulose, talc, glucolse, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium sterate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liguid and semisolid excipients may be selected from water, ethanol, glycerol, propylene glycol and various oils, includingthose of petroleum, animal, vegetable or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose and glycols.
Compressed gases may be used to disperse the cysteine protease inhibitor of this invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, nitrous oxide, etc. Other suitable pharmaceutical carriers and their formulations are described in A.R. Alfonso Reminton's Pharmaceutical Sciences 1985, 17th ed. Easton, Pa.: Mack Publishing Company, hereby expressly incorporated by reference.
The amount of a cysteine protease inhibitor of this invention in the composition may vary widely depending upon the type of formulation, size of a unit dosage, kind of excipients and other factors known to those of skill in the art of pharmaceutical sciences In general, the final composition will comprise from 0 1%w to 10%w of the cysteine protease inhibitor, preferably 1%w to 10%w, with the remainder being the excipient or excipients
Preferably the pharmaceutical composition is administered in a single unit dosage form for continuous teatment or in a single unit dosage form ad libitum when relief of symptoms is specifically required Representative pharmaceutical formulations containing a cysteine protease inhibitor of the invention are described in Example 20, infra
The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention It is understood that these examples in no way serve to limit the true scope of this invention but rather are presented for illustrative purposes All references cited herein are expressly incorporated by reference
EXAMPLES
The following abbreviation conventions have been used to simplify the examples
Mu = morpho ne urea
Xaa, = ammo acid at P1 position relative to active site of the enzyme
Xaa2 = ammo acid at P2 position relative to active site of the enzyme γ-C02Et= γ-amιno ethyl ester γ-S02Ph= γ-amιnosulfone with phenyl terminus γ-C02H = γ-amιnocarboxylate γ-PEt = γ-amιnophosphonate γ-AM = γ-amιnoamιde γ-Ar(sub)= γ-amιnoaromatιc compound (substituted as appropriate) β-S02Ph= β-aminosulfone with phenyl substituent α-S02Ph= α-aminosulfone with phenyl substituent
Hph = homophenylalanine
PSMP = diethyl phenylsulfonylmethylenephosphonate
Np2 = 2-naphthylalanιne
S022Np = sulfone with 2-maphthyl terminus
Phac = phenylacetyl β-Aia = β-alanine
MeOSuc = methoxysuccinyl For instance, Mu-Phe-Hph-β-S02Ph where Xaa2 = Phe (phenylalanine) and Xaa, = Hph (homophenylalanine,), transformed to the β-ammo phenyl sulfone according to the procedure described in the Examples
Example 1 Synthesis of Cysteine Protease Inhibitor Containing a γ-amιnoester as the EWG
Unless otherwise indicated, all reactions were performed under an inert atmosphere of argon or nitrogen at room temperature THF was distilled from sodium benzophenone ketyl All other solvents and commercially available reagents were used without further purification
Synthesis of Ethyl (S)-4-(4-morpholιnecarbonyl-phenylalanyl)-amιno-6-phenylhexanoate, abbreviated Mu-Phe-Hph-γ-C02Et, was as follows Unless otherwise noted, all reagents were obtained from Aldrich, Inc 0 393 g of a 60% mineral oil dispersion (9 82 mmol) of sodium hydride was added to a solution of tnethyl phosphonoacetate (2 20 g, 9 82 mmol) in THF (50 mL) at -10°C The mixture was stirred for 15 minutes, whereupon a solution of Boc-homophenylalanmal (Boc-HphH) (2 35 g, 9 82 mmol, prepared by conversion of Boc-homophenylalanine (Synthetech) to its N,0-dιmethylhydroxamιde, using the Fehrentz method followed by lithium aluminum hydride reduction) in THF (20 mL) was added The mixture was stirred for 45 minutes 1M HCI (30 mL) was added The product was extracted with ethyl acetate (50 mL), washed with saturated aqueous NaHC03 (30 mL) dried over MgS04, filtered, and evaporated to dryness The dried material was dissolved in CH2CI2 (10 mL), and a 4 0 M solution of HCI in dioxane (20 mL) was added The mixture was stirred for 30 minutes The solvents were removed under reduced pressure and the residue, ethyl (S)-4-amιno-6-phenyl-2-hexenoate hydrochloπde, was pumped dry
4-Morpholιnecarbonylphenylalanιne (Mu-PheOH, 2 74 g, 9 82 mmol, prepared according to the method described in Esser, R et al , Arthritis & Rheumatism (1994), 37, 236) was dissolved in THF (50 mL) at -10°C 4-methylmorpholιne (1 08 mL, 9 82 mmol) was added, followed by isobutyl chloroformate (1 27 mL, 9 82 mmol) The mixed anhydride was stirred for 10 minutes, whereupon a solution of ethyl (S)-4-amιno-6-phenyl-2-hexenoate hydrochloπde from the previous step in DMF (10 mL) was added, followed by 4-methylmorpholιne (1.08 mL, 9.82 mmol) The mixture was stirred for 1 hour 1 M HCI (50 mL) was added The product was extracted with ethyl acetate (100 mL), washed with saturated aqueous NaHC03 (50 mL), dπed over MgS04 and decolorizing charcoal (DARCO), filtered, and evaporated to dryness, giving 3 80 g of intermediate (80% yield from Boc-homo- phenylalanmal) To a solution of this intermediate (1 45 g, 3 09 mmol) in ethanol (25 mL) was added 5% palladium on active carbon (0 5 g) The mixture was reduced on a Parr hydrogenator for 36 hours The solution was filtered and the solvent was removed under reduced pressure, giving 1 19 g (82%) of the product
Thin-layer chromatography (TLC) was performed on each sample Visualization was accomplished by means of UV light at 254 nm, followed by ninhydnn, bromocresol green, or p-anisaldehyde stain The retention factor (RO of the Mu-Phe-Hph-γ-C02Et was 0 35 (5% MeOH/CH2CI2)
NMR spectra were recorded on a Vaπan Gemini 300 MHz instrument All 1H NMR data of this and subsequent examples are reported as delta values in parts per million relative to internal tetramethylsilane, peak assignments in boldface The following abbreviations are used s, singlet, d, doublet, t triplet, q, quartet, br, broad An asterisk (*) implies that a signal is obscured or buried under another resonance
Example 2
Synthesis of a Cysteine Protease Inhibitor containing a γ-amιnosulfone as the EWG
Synthesis of (S)-3-tert-butoxycarbonylamιno-5-phenyl-1-pnenylsulfonylpentane (Boc-Hph-γ-S02Ph) To a solution of PSMP (8 87 g, 30 34 mmol) in THF (150 mL) at 0°C was added sodium hydride (1 21 g of a 60% mineral oil dispersion) The mixture was stirred for 20 minutes, whereupon a solution of Boc-homophenylalaninal, synthesized by the method of Fehrentz and Castro, above, (7 99 g, 30 34 mmol) in THF (20 mL) was added The solution was stirred for 30 minutes at 0°C 1M HCI (100 mL) was added The product was extracted into ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate (100 mL), brine (50 mL), dried over MgS04 filtered, and the solvent was removed under reduced pressure The residue was dissolved in ethanol (100 mL) and transferred to a Parr bottle charged with 5% palladium on active charcoal (0 92 g) The mixture was reduced on a Parr apparatus for 24 hours The solution was filtered through Celite and the solvent was removed under reduced pressure TLC of the product indicated a single product in quantitative yield, R, = 0 29 (30% ethyl acetate/hexane) that stained white with paraanisaldehyde spray
Example 3
Synthesis of a Cysteine Protease Inhibitor containing a γ-amιnosulfone as the EWG
Synthesis of (S)-3-amιno-5-phenyl-1-phenylsulfonyl-pentane hydrochloπde (HCI Hph-γ-S02Ph) To a solution of Boc-Hph-γ-S02Ph (12 24 g, 30 34 mmol) in dichloromethane (20 mL) was added hydrogen chloride in dioxane (50 mL of a 4 0M solution) The mixture was stirred for 90 minutes The solvent was removed under reduced pressure, and the residue was dissolved in CH2CI2 (50 mL) The solution was carefully added to ether (500 mL) with stirring The solid was filtered, washed with ether (50 mL) and dried in vacuo
Example 4
Synthesis of a Cysteine Protease Inhibitor containing a γ-amιnosulfone as the EWG
Synthesis of (S)-3-(4-morpholιnecarbonylphenylalanyl)-amιno-5-phenyl-1 -phenylsulfonylpentane (Mu- Phe-Hph-γ-S02Ph) To a solution of Mu-PheOH (2 94 g, 10 56 mmol) in THF (75 mL) at -10°C were added 4-methylmorpholιne (1 16 mL, 10 56 mmol) and isobutyl chloroformate (1 37 mL, 10 56 mmol) The mixture was stirred for 5 minutes (S)-(E)-3-amιno-5-phenyl-1-phenylsulfonyl-1-pentene p- toluenesulfonate, synthesized by Wadsworth-Emmons condensation between Boc- homophenylalaninal and p-toluenesulfonic acid deprotection (5 00 g, 10 56 mmol) was added, followed by 4-methylmorpholιne (1 16 mL, 10 56 mmol) The mixture was stirred for 45 minutes The solution was diluted with ethyl acetate (100 mL), washed with 1 M HCI (2x50 mL), saturated aqueous sodium bicarbonate (50 mL), brine (50 mL), dried over MgS04, filtered, and the solvent was removed under reduced pressure The residue was crystallized from CH2CI2/ether to give 4 27 g (72%) of intermediate 1 17 g of this material (2 08 mmol) was dissolved in ethanol (25 mL) The solution was transferred to a Parr bottle charged with 5% palladium on active charcoal (0 30 g) The mixture was hydrogenated at room temperature overnight on a Parr shaker Ethyl acetate was added to the suspension of product, which had crystallized from the reaction mixture 1 he solution was filtered and concentrated in vacuo, and then was recrystallized from CH2CI2/hexane M p = 176-178°C TLC (50% ethyl acetate/CH2CI2) R, = 0 24
Example 5
Synthesis of a Cysteine Protease Inhibitor containing a γ-amιnosulfone as the EWG
Synthesis of (S)-3-(4-morpholιnecarbony Ity rosyl)-amιno-5-phenyl-1 -phenylsulfonylpentane (Mu-Tyr- Hph-γ-S02Ph) To a solution of 4-morpholιnecarbonyltyrosιne (Mu-TyrOH, synthesized according to the method described in Esser, R et al , Arthritis & Rheumatism (1994), 37, 236, 0 50 g, 1 70 mmol) in THF (10 mL) at -10°C were added 4-methylmorpholιne (0 187 mL, 1 70 mmol) and isobutyl chloroformate (0.220 mL, 1 70 mmol) After 5 minutes, HCI Hph-γ-S02Ph (0 577 g, 1 70 mmol, descπbed in Example 3) was added, followed by 4-methylmorpholιne (0 187 mL, 1 70 mmol) The mixture was stirred for 45 minutes Ethyl acetate (50 mL) was added The solution was washed with 1M HCI, saturated aqueous sodium bicarbonate, and brine (30 mL each), dried over MgS04, filtered, and the solvent was removed under reduced pressure The residue was precipitated from C^CIj ether to give 0.58 g (59%) of Mu-Tyr-Hph-γ-S02Ph M p 104-107°C TLC (10% Me0H/CH2CI2) R, = 0 59 Example 6
Synthesis of a Cysteine Protease Inhibitor containing a γ-amιnosulfone as the EWG
Synthesis of (S)-3-(4-morphlιnecarbonyl-2-naphthyl-alanyl)amιno-5-pheny 1-1 -(2- naphthylsulfonyl)pentane (Mu-Np2-Hph-γ-S022Np) 2-naphthalenethιol (9 64 g, 60 16 mmol) was dissolved in toluene (75 mL) Paraformaldehyde (3 97 g, 132 mmol) and HCI/dioxane (33 mL of a 4 0M solution) were added The mixture was stirred for several days at room temperature The solvent was removed under reduced pressure, and the residue was suspended in hexane (200 mL), dried over MgS04, filtered, and evaporated to dryness This material, crude chloromethyl 2-naphthyl sulfide, was combined with tnethyl phosphite (10 93 g, 65 mmol) and was heated at reflux for 4 hours The mixture was cooled to room temperature, diluted with ether (200 mL), washed with 1M HCI, saturated aqueous sodium bicarbonate, and brine (150 mL each), dried over MgS04, filtered, and concentrated in vacuo to give 17 35 g (93% crude yield) of diethyl 2-naphthylthιomethylene phosphonate This material was dissolved in CH2CI2 (300 mL) and cooled to 0°C Peracetic acid (23 5 mL of a 32% dilute acetic acid solution (Aldrich Chemical Co ) was carefully added The mixture was stirred overnight while warming to room temperature The solution was washed with freshly prepared, saturated aqueous sodium bisulfite solution (100 mL), then with several portions of saturated aqueous sodium bicarbonate, until the aqueous phase became basic The organic phase was dried over MgS04, filtered, and the solvent was removed under reduced pressure Chromatography on 60-200 mesh silica gel (0-10% ethyl acetate'CH2CI2) afforded 6 5 g (34%) of the pure Wadsworth-Emmons reagent, diethyl 2-naphthylsulfonyl-methylene phosphonate along with an approximately equal mass of impure material TLC (20% ethyl acetate/CH2CI2) R, = 0 37
To a solution of diethyl 2-naphthylsulfonylmethylene phosphonate (3 91 g, 11 42 mmol) in THF (60 mL) at 0°C was added sodium hydride (0 457 g of a 60% mineral oil dispersion The mixture was stirred for 15 minutes, whereupon a solution of Boc-homophenylalanmal (3 00 g, 11 42 mmol) in THF (5 mL) was added The mixture was stirred for 30 minutes 1 M HCI (100 mL) was added The product was extracted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate (75 mL), brine (50 mL), dried over MgS04, filtered, and the solvent was removed under reduced pressure The residue was dissolved in dichloromethane (10 mL), to which was added HCI/dioxane (25 mL of a 4 0M solution) The mixture was stirred at room temperature for 1 hour, poured into ether (300 mL), and filtered The solids were washed with ether (2x50 mL) and dried in vacuo to give 3 30 g (74% from Boc-homophenylalanmal) of (S)-(E)-3-amιno-5-phenyl-1-(2- naphthylsulfonyl)-1-pentene
To a solution of Boc-2-naphthylalanιne (2 68 g, 8 51 mmol, (Synthetech, Oregon) in THF (50 mL) at - 10°C were added 4-methylmorpholιne (0.936 mL, 8 51 mmol) and isobutyl chloroformate (1 103 mL, 8 51 mmol) The mixture was stirred for 5 minutes, whereupon (S)-(E)-3-amιno-5-phenyl-1-(2- naphthylsulfonyl)-1-pentene (3 30 g, 8 51 mmol) was added, followed by 4-methylmorpholιne (0 936 mL, 8 51 mmol) The mixture was stirred for 45 minutes, diluted with ethyl acetate (100 mL), washed with 1M HCI (50 mL), saturated aqueous sodium bicarbonate (50 mL), and brine (50 mL), dried over MgS04, filtered, and the solvent was removed under reduced pressure The intermediate, (S)-(E)-3- (tert-butoxycarbonyl-2-naphthylalanyl)amιno-5-phenyl-1-(2-naphthylsulfonyl)-1-pentene, was crystallized from a suitable mixture CH2CI2/ether/hexane in 69% yield The resulting material (3 83 g, 5 90 mmol) was dissolved in CH2CI2 (5 mL) and was treated with HCI/dioxane (15 mL of a 4 0M solution The mixture was stirred at room temperature for 1 hour The solution was poured, with stirring, into ether (500 mL) and filtered The solids were washed with ether (2x50 mL) and dried in vacuo to give the intermediate, (S)-(E)-3-(2-naphthylalanyl)-amιno-5-phenyl-1-(2-naphthylsulfonyl)-1- pentene 3 41 g, 99% yield
2 00 g of this material (3 42 mmol) was dissolved in THF (15 mL), and cooled to 0°C 4- methylmorpholine-carbonyl chloride (0 400 mL, 342 mmol) and tπethylamine (0 953 mmol) were added The mixture was stirred for 1 hour at 0°C, and then at room temperature for 2 hours Ethyl acetate (50 mL) was added The solution was washed with 1 HCI (30 mL), saturated aqueous sodium bicarbonate (30 mL), brine (30 mL), dried over MgS04, filtered, and evaporated to dryness, giving 1 58 g (69%) of intermediate, (S)-(E)-3-(4-morpholιnecarbonyl-2-naphthylalanyl)amιno-5- phenyl-1-(2-naphthylsulfonyl)-1-pentene TLC (50% ethyl acetate/hexane) R, = 0 37
0 73 g (1 10 mmol) of this material was dissolved in ethanol (20 mL) and transferred to a Parr bottle charged with 5% palladium on carbon (0 30 g) The mixture was reduced on a Parr hydrogenator for 36 hours The solution was filtered and the solvent was removed under reduced pressure The product was purified by column chromatography on 60-200 mesh silica gel (50% ethyl acetate/CH2CI2 as eluent) to give 0 14 g (19%) of pure product, Mu-Np2-Hph-γ-S022Np, along with impure material TLC (50% ethyl acetate/CH2CI2) R, = 0 34
Example 7
Synthesis of a Cysteine Protease Inhibitor containing a γ-amιnosulfone as the EWG
Synthesis of 3-acetyltyrosylvalylalany lamιno-4-hydroxy-carbony 1-1 -phenylsulfonylbutane (Ac-Tyr-Val- Ala-Asp-γ-S02Ph) Sodium hydride (0 489 g of a 60% mineral oil dispersion, 12 23 mmol) was added to a solution of diethyl phenylsulfonylmethylene phosphonate (3 58 g, 12 23 mmol) in 50 mL of THF at 0°C The mixture was stirred for 15 minutes A solution of Boc-AspH(β-Ot-Bu), (3 04 gm, 11 12 mmol, prepared by converting Boc-Asp(β-O-t-Bu) to its N,0-dιmethyldroxamιde and reducing with lithium aluminum hydπde), in THF (10 mL) was added The mixture was stirred for 1 hour, whereupon 1 M HCI (30 mL) was added The product was extracted with ethyl acetate (100 mL), washed with saturated aqueous NaHC03 (30 mL), bπne (30 mL), dried over MgS04 filtered, and evaporated to dryness, giving the intermediate, Chromatography on silica gel (20-30% ethyl acetate/hexane, gradient elution) afforded 2 07 g 45%) of the intermediate, (S)-(E)-3-tert-butoxycarbonylamιno-4-tert- butoxycarbonyl-1-phenylsulfonyl-1-butene This material was dissolved in ether (2 mL) and was treated with a solution of anhydrous p-toluenesulfonic acid (1 0 g, 5 87 mmol) in ether (2 mL) The mixture was stirred at room temperature overnight, then diluted with ether (25 ml) The white precipitate, was filtered, washed with ether, and dried in vacuo to give 0 80 g (95%) of the next intermediate, (S)-(E)-3-amιno-4-tert-butoxycarbonyl-1-phenylsulfonyl-1-butene-p-toluenesulfonate
This material was coupled, using mixed anhydride chemistry, to Ac-Tyr-Val-AlaOH, itself prepared by standard peptide chemistry, giving the next intermediate (S)-(E)-3-acetyltyrosylvalylalanylamιno-4- tert-butoxycarbonyl-1-phenylsulfonyl-1-butene
This mateπal was treated with tπfluoroacetic acid to remove the t-butyl ester of the aspartic acid side chain, giving (E)-3-acetyltyrosylvalylalanylamιno-4-hydroxycarbonyl-1-phenylsulfonyl-1-butene 0 28 g (0 444 mmol) of this material was dissolved in ethanol (10 mL) The solution was transferred to a Parr bottle, charged with 5% palladium on carbon (0 1 g) The solution was reduced on a Parr hydrogenator overnight The solution was filtered and the solvent was removed under reduced pressure The residue, when dissolved in methanol (5 mL) and diluted with 40x 1 1 CH2CI2/ether, formed a gelatinous precipitate, which was collected on a Buchner funnel to give 0 18 g (64%) yield The isomer ratio of S to R with respect to the Asp residue was estimated as approximately 3 1 based on the integration of the doublets associated with the aromatic region of the NMR as pertains to the Tyr residue
Example 8
Synthesis of a Cysteine Protease Inhibitor with a γ-amιnocarboxylate as the EWG
Synthesis of (S)-4-(4-morpholιnecarbonylphenylalanyl)amιno-6-phenylhexanoιc acid, Mu-Phe-Hph-γ- C02H To a solution of Mu-Phe-Hph-γ-C02Et. prepared according to the procedure described in Example 1 (0 5g, 1 06 mmole) was added aqueous NaOH (1 mL of a 2M solution) After 4 hr the reaction was complete 1 M HCI (4mL) was added along with water (10 mL) The product was extracted with CH2CI2 (2 x 10 mL), THF (15dπed over MgS04 the solvent was removed under reduced pressure, and the residue, Mu-Phe-Hph-γ-C02H, was pumped to a solid Yield = 0 30g (60%)
Example 9
Synthesis of a Cysteine Protease Inhibitor with a γ-amιnophosphonate as the EWG Synthesis of diethyl (S)-4-(4-morpholinecarbonyl-phenylalanyl)amino-6-phenylhexanephosphonate (Mu-Phe-Hph-γ-S02Ph) was as follows. To a solution of tetraethyl methylenediphosphonate (2.00 g, 6.94 mmol) in THF (30 mL) was added sodium hydride (0.278 g of a 60% mineral oil dispersion, 6.94 mmol). The mixture effervesced rapidly and then clarified. After 5 minutes, a solution of Boc-HphH (1.83 g, 6.94 mmol) in THF (5 mL) was added. The mixture was stirred for 1 hour. 1M HCI (20 mL) was added. The product was extracted into ethyl acetate (50 mL), washed with saturated aqueous NaHC03 (20 mL), brine (10 mL), dried over MgS04, filtered, and evaporated to dryness, giving 2.46 g (89%) of intermediate, diethyl (S)-(E)-4-tert- butoxycarbonylamino-6-phenyl-2-hexenephosphonate. To a solution of this material in CH2CI2 (3 mL) was added 10 mL of a 4.0 M solution of HCI in dioxane. The mixture was stirred at room temperature for 1.5 hours. The solvents were removed under reduced pressure and the residue was dissolved in methanol (10 mL). The solution was poured into ether (400 mL). The precipitate was collected on a Buchner funnel, washed with ether (2x20 mL), and was pumped dry to give 1.25 g (60%) of intermediate, diethyl (S)-(E)-4-amino-6- phenyl-2-hexenephosphonate hydrochloride. To a solution of Mu-PheOH (1.04 g, 3.74 mmol) in THF (15 mL) at -10°C was added 4-methylmorphoiine (0.412 mL, 3.74 mmol), followed by isobutyl chloroformate (0.486 mL, 3.74 mmol). The mixed anhydride was stirred for 5 minutes, whereupon a solution of (S)-(E)-4-amino-6-phenyl-2-hexene-phosphonate hydrochloride (1.25 g, 3.74 mmol) in DMF (5 mL) was added, followed by 4-methylmorpholine (0.412 mL, 3.74 mmol). The mixture was stirred for 1 hour. Ethyl acetate (50 mL) was added. The solution was washed with 1M HCI (25 mL), saturated aqueous NaHC03 (25 mL), and brine (10 mL), dried over MgS04, filtered, and evaporated to dryness. The product, upon treatment with CH2CI2/ether/hexane (315 mL in a 15:200:100 ratio) formed an oil that solidified on drying in vacuo to give 1.44 g (69%) of diethyl (S)- (E)-4-(4-morpholinecarbonyl-phenylalanyl)-amino-6-phenyl-2-hexenephosphonate. 0.85 g of this material was dissolved in ethanol (10 mL) and was transferred to a Parr bottle charged with 5% palladium on active charcoal. The solution was reduced on a Parr hydrogenator for 36 hours. The solution was then filtered through Celite, and the solvent was removed under reduced pressure to give 0.66 g (76%) of the final product as an oil. TLC: (5% MeOH/CH2CI2) R, = 0.27.
Example 10
Synthesis of a Cysteine Protease Inhibitor with a γ-aminoamide as the EWG.
Synthesis of benzyl (S)-3-(4-mo holinecarbonylphenyl-alanyl)-amino-6-phenylhexanamide (Mu-Phe- Hph-γ-AMBzl). The Wadsworth-Emmons reagent diethyl benzylamido-carbonylmethylenephosphonate was synthesized in two steps, first by saponification of triethyl phosphonoacetate to diethyl phosphonoacetic acid, which then was dissolved in ethyl acetate to a 0.2 M concentration, treated with an equivalent of benzylamine, 0.1 equivalents of 4-dimethylamino-pyridine, and one equivalent of dicyclohexyl-carbodiimide. To a solution of this Wadsworth-Emmons reagent (2.59 g, 9.08 mmol) in THF (40 mL) at 0°C was added sodium hydride (0.363 g of a 60% mineral oil dispersion, 9.08 mmol) The mixture was stirred at room temperature for 15 minutes, whereupon a solution of Boc- homophenylalanmal (2 39 g, 9.08 mmol) in THF (10 mL) was added The mixture was stirred for 1 hour 1 M HCI (30 mL) was added The product was extracted into ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate (50 mL), bπne (30 mL), dried over MgS04, filtered, concentrated, and crystallized from ether/hexane to give 1.81 g (51%) of benzyl (S)-(E)-3- tertbutoxycarbonylamιno-6-phenyl-2-hexenamide This material was dissolved in CH2CI2 (5 mL) To the solution was added HCI/dioxane (10 mL of a 4.0M solution). The mixture was stirred for 3 hours at room temperature The solvents were removed under reduced pressure The residue was dissolved in methanol (5 mL) and poured into ether (300 mL), whereupon the intermediate, benzyl (S)-(E)-3- amιno-6-phenyl-2-hexenamιde hydrochloride, separated out as an oil, in 82% yield (1.25 g) To a solution of Mu-PheOH (1 05 g, 3 78 mmol) in THF (15 mL) at -10°C were added 4-methylmorpholιne (0 416 mL, 3 78 mmol) and isobutyl chloroformate (0.490 mL, 3 78 mmol) The mixture was stirred for 10 minutes, whereupon a solution of benzyl (S)-(E)-3-amιno-6-phenyl-2-hexenamιde hydrochloride (1 25 g, 3 78 mmol) in THF (3 mL) was added, followed by 4-methylmorpholιne (0 416 mL, 3 78 mmol) The mixture was stirred for 45 minutes Ethyl acetate (40 mL) was added The solution was washed with 1M HCI (10 mL), saturated aqueous sodium bicarbonate (10 mL), brine (5 mL), dried over MgS04, filtered, and evaporated to dryness. The intermediate, benzyl (S)-(E)-3-(4- morpholιnecarbonyl-phenylalanyl)amιno-6-phenyl-2-hexenamιde, was precipitated from CH2CI2/ether in 56% yield 0 48 g (0 865 mmol) of this material was dissolved in ethanol (10 mL) and transferred to a Parr bottle charged with 5% palladium on active carbon The mixture was reduced on a Parr hydrogenator for 4 hours The solution was filtered through Celite, and the solvent was removed under reduced pressure The final product (Mu-Phe-Hph-γ-AMBzl) was crystallized from ethanol/hexane, giving 0 25 g (52%) TLC- (50% ethyl acetate/hexane) Rf = 0.45
Example 11
Synthesis of a Cysteine Protease Inhibitor with a γ-amιnoamιde as the EWG
Synthesis of phenyl (S)-3-(4-morpholιnecarbonylphenyl-alanyl)-amιno-6-phenylhexanamιde (Mu-Phe- Hph-γ-AMPh) To a solution of Mu-Phe-Hph-γ-C02H, (0.30g, as prepared according to Example 8), in THF (5 mL) at -10°C was added tπethylamine (90 μL, 1 eq ) followed by addition of isobutyl chloroformate (0.083 mL, 1 eq.) After 5 min, aniline (0.058 mL) was added. The cooling bath was removed and the reaction stirred at room temp for 2 hr. CH2CI2(30 mL) was added. The solution was washed with 1M HCI and saturated aqueous sodium bicarbonate (10 mL each), dried over MgS04, filtered, and the solvent was removed under reduced pressure. The residue was triturated with Et20, filtered, and dried in vacuo to give 0.29 g (85%) of the product, Mu-Phe-Hph-γ-AMPh. TLC (10% MeOH/CH2CI2) Rf = 0.70, strongly absorbs UV (254 nm), l2. Example 12
Synthesis of a Cysteine Protease Inhibitor with a γ-aromatιc as the EWG
Synthesis of (S)-4-amιnophenyl-3-(4-morpholιne-carbonylphenylalanyl)amιno-5-phenylpentane hydrochloride, (Mu-Phe-hPhe-γ-C6H4NH2 HCI)
Tπphenylphosphine (38 17 g, 0 146 mole) and 4-nιtrobenzyl chloride (25g, 0 146 mole) were dissolved in CH3CN (100 mL) and heated at reflux for 2 hours, and then allowed to cool to room temperature The reaction mixture was diluted with Et20 (300 mL), the white solid was filtered, washed with Et20 (200 mL), and dried in vacuo, giving 53 3 g (84%) of 4- nitrobenzyltπphenylphosphonium chloride as a single spot on TLC (Rf=0 71 , 4 1 1 butanol acetic acid water) 1H-NMR (d6-DMSO) 5 40-5 50 (2H, d, CH2P, J=20Hz), 7 20-7 40 (2H, dd, aromatic), 7 40-7 80 (12H, m, aromatic), 7 90-8 00 (3H, m, aromatic), 8 10-8 20 (2H, d, aromatic)
To a stirred suspension of 4-nιtrobenzyltπphenylphosphonιum chloride (10 02 g, 23 1 mmol) in CH2CI2 (100 mL) was added 4-methylmorpholιne (2 54 mL, 23 1 mmol) was added When all the solid had dissolved, Boc-HphH (4 04 g, 15 4 mmol) was added After 24 hrs , the reaction mixture was diluted with CH2CI2 (200 mL), and filtered The filtrate was washed with 1M HCI (200 mL) saturated aqueous sodium bicarbonate (200 mL), dried over MgS04 , filtered and concentrated under reduced pressure, giving 4 00 g of crude intermediate, a portion of which was purified by chromatography (gradient elution 10-30% ethyl acetate/hexane) to permit NMR analysis of the intermediate, (S)-t-butoxycarbonyl-3-amιno-1-(4-nιtrophenyl)-5-phenyl-1-pentene TLC (30% EtOAc /hexane) Rf=0 49 To a solution of this material (2 76 g, 7 2 mmol) in Et20 (25 mL), was added a solution of anhydrous β-toluenesulfonic acid (2 76 g, 16 0 mmol) in Et20 (10 mL) The reaction was left to stir 16 hrs, filtered, washed solid with Et20 (25 mL), and dried in vacuo, giving 2 g (61%) of (S)-3-amιno-1-(4-nιtrophenyl)-5-phenyl-1-pentene as a single spot on TLC (Rf=049, 10% MeOH/CH2CI2)
To a solution of Mu-PheOH (1 29 g, 4 63 mmol) in THF (20 mL) were added 4-methylmorpholιne (0 51 mL, 4 63 mmol) and isobutyl chloroformate (0 61 mL, 4 63 mmol) After 3 minutes, a solution of (S) 3-amιno-1-(4-nιtrophenyl)-5-phenyl-1-pentene hydrochloride, prepared by HCI/dioxane-mediated deprotection of (S) 3-tert-butoxycarbonylamιno-1-(4-nιtrophenyl)-5- phenyl-1-pentene precursor, 1 34 g, 4 20 mmol) in CH2CI2 (20 mL), followed by 4- methylmorpholine (0 51 mL, 4 63 mmol) The mixture was stirred overnight while warming to room temperature The solution mixture was diluted with CH2CI2 (100 mL), washed with 1M HCI (200 mL), saturated aqueous sodium bicarbonate (200 mL), dried over MgS04, filtered, and concentrated under reduced pressure to give a yellow oil This material was crystallized from CH2CI2/ether (2 100, 20 mL) to give 1 00 g (40%) of (S)-3-(4- morpholιnecarbonylphenylalanyl)amιno-1 -(4-nιtrophenyl)-5-phenyl-1 -pentene as an approximately 4 1 E/Z mixture 0 27 g (0 49 mmol) of this material was dissolved in ethanol (50 mL), transferred to a Parr bottle charged with 5% palladium on carbon (0 10 g) and reduced on a Parr hydrogenator for 8 hours/ The mixture was filtered and the solvent was removed under reduced pressure The residue was dissolved in in 4 1 ether/CH2CI2 (100 mL), to which HCI/dioxane (0 136 mL of a 4 0 M solution) was added The product, Mu-Phe-Hph-γ- C6H4NH2 HCI, was filtered and dried in vacuo Yield = 0 15 g (54%) TLC (10% methanol/CH2CI2) R, = 0 31
Example 13
Synthesis of a Cysteine Protease Inhibitor with a β-ammosulfone as the EWG
Synthesis of (S)-2-(4-morpholmecarbonyIphenyl-alanyl)amιno-4-phenyl-1-phenylsulfonylbutane (Mu-Phe-Hph-β-S02Ph) Preparation of Boc-homophenylalanmol (Boc-Hph-β-OH) and (S)-2- tert-butoxycarbonylamιno-1-methanesulfonyloxy-1-phenylbutane (Boc-Hph-β-OMs or Boc- homophenylalanmol mesylate) followed a similar scheme to that reported by Spaltenstem, Carpmo, Miyake, and Hopkins, above To a solution of Boc-homophenylalanine (10 29 g, 36 84 mmol) in THF (100 mL) at -10°C were added 4-methylmorpholιne (4 05 mL, 36 84 mmol) and isobutyl chloroformate (4 78 mL, 36 84 mmol) The solution was stirred for 10 minutes, and then was filtered The filtrate was carefully added to a stirred solution of sodium borohydπde (2 77 g, 73 67 mmol) in water (100 mL) at 0°C The mixture was stirred for 30 minutes Saturated aqueous sodium bicarbonate (200 mL) was added The product was extracted with CH2CI2 (2x100 mL), dried over MgS04, filtered, and the solvent was removed under reduced pressure to give 9 78 g (100%) Boc-homophenyl-alaninol TLC (30% ethyl acetate/hexane) Rt = 0 15 5 83 g (21 97 mmol) of this material was dissolved in CH2CI2 (150 mL), cooled to 0°C, and treated with methanesulfonyl chloride (4 15 mL, 53 71 mmol), and tπethylamine (9 24 mL, 66 3 mmol) The mixture was stirred for 30 minutes Water (100 mL) was added, the mixture was stirred vigorously The organic phase was separated, dried over MgSO, filtered, and the solvent was removed under reduced pressure, giving 7 31 g (97%) yield TLC (30% ethyl acetate/hexane) R, = 0 21 A similar procedure was employed to prepare the corresponding benzenesulfonate ester of Boc-Hph-β-OH
To a solution of thiophenol (0 653 mL, 6 36 mmol) in THF (5 mL) was added sodium hydπde (0 254 g, 6 36 mmol as a 60% mineral oil dispersion The mixture was stirred for 10 minutes A solution of Boc-homophenylalanmol benzenesulfonate (2 58 g, 6 36 mmol) in THF (5 mL) was added. The solution was stirred at room temperature for 10 minutes. Methanol (2 mL) was then added, and the mixture was heated at reflux for 1 hour. The solution was cooled, diluted with 1M NaOH (25 mL), extracted with CH2CI2 (100 mL), dried over MgS04, filtered, and the solvent was removed under reduced pressure. The residue was dissolved in CH2CI2 (35 mL) and cooled to 0°C. To the solution was added 4-chloroperbenzoic acid (3.71 g, 13.99 mmol, estimated peracid content 65% by weight). The mixture was stirred for 1 hour, whereupon 10% NaOH (35 mL) and saturated aqueous NaHS03 (35 mL) were added. The mixture was extracted with CH2CI2 (3x50 mL portions), dried over MgS04, filtered, and the solvent was removed under reduced pressure to give a waxy solid. (S)-2-tert-butoxycarbonylamino-4-phenyl-1-phenylsulfonylbutane. TLC: (30% ethyl acetate/hexane) R, = 0.32. 1.25 g of this material was dissolved in CH2CI2 (5 mL) and treated with HCI/dioxane (5 mL of a 4.0M solution). The mixture was stirred for 2 hours at room temperature. The solution was poured into ether (200 mL), forming an oily residue. The supernatant was discarded. The residue was again dissolved in CH2CI2 (10 mL), and poured into ether (200 mL). The intermediate, (S)-2-amino-4-phenyl-1-phenylsulfonylbutane hydrochloride precipitated out. The solid was filtered and dried in vacuo to give 0.40 g of material (38% yield from Boc-homophenylalaninol benzenesulfonate.
To a solution of Mu-PheOH (0.342 g, 1.23 mmol) in THF (10 mL) at -10°C were added 4- methylmorpholine (0.135 mL, 1.23 mmol) and isobutyl chloroformate (0.159 mL, 1.23 mmol). The mixture was stirred for 10 minutes, whereupon (S)-2-amino-4-phenyl-1-phenylsulfonylbutane hydrochloride (0.40 g, 1.23 mmol) was added, followed by 4-methylmorpholine (0.135 mL, 1.23 mmol). The mixture was stirred for 45 minutes. 1M HCI (15 mL) was added. The product was extracted with ethyl acetate (30 mL), washed with saturated aqueous sodium bicarbonate (15 mL), brine (15 mL), dried over MgS04, filtered, and the solvent was removed under reduced pressure. The final product, Mu-Phe-Hph-β-S02Ph, weighed 0.68 g (100% yield).
Example 14
Synthesis of a Cysteine Protease Inhibitor with a β-aminosulfone as the EWG.
Synthesis of (S)-2-tert-butoxycarbonylamino-4-phenyl-1-(1 '-trimethylsilylethyl)-sulfonylbutane (Boc-Hph-β-S02CH2CH2TMS). To a solution of 2-trimethylsilylethanethiol (0.86 g, 6.41 mmol), synthesis described by Anderson, Ranasinghe, Palmer, and Fuchs, above) in THF (10 mL) was added sodium hydride (0.256 g, 6.41 mmol as a 60% mineral oil dispersion). The mixture was stirred for 10 minutes. Boc-homophenylalaninol mesylate (2.00 g, 5.82 mmol, synthesis described in Example 13, above) was added. The solution was stirred for 2 hours. Ethyl acetate (50 mL) was added. The solution was washed with 30 mL each of 1M HCI, saturated aqueous sodium bicarbonate, and brine, dried over MgS04, filtered, and the solvent was removed under reduced pressure, giving the intermediate (S)-2-tert-butoxycarbonylamιno-4-phenylbutyl tnmethylsilylethyl sulfide TLC (5% ethyl acetate/hexane) Rf = 0 22 This material was dissolved in CH2CI2 (50 mL), cooled to -10°C, and treated with 4-chloroperbenzoιc acid (3 24 g, 12 22 mmol, estimated 65% peracid content) The mixture was stirred overnight The suspension was filtered, and saturated aqueous NaHS03 (40 mL) and saturated aqueous sodium bicarbonate (50 mL) were carefully added to the filtrate The organic phase was separated, dried over MgS04, filtered, and the solvent was removed under reduced pressure to give the product, Boc-Hph-β-S02CH2CH2TMS in quantitative mass recovery from the mesylate TLC (30% ethyl acetate/hexane) Rf = 049
Example 15
Synthesis of a Cysteine Protease Inhibitor with a β-ammosulfone as the EWG
Synthesis of (S)-2-(4-morpholιnecarbony lphenylalanyl)-amιno-1 -chloromethy Isulfony I-4- phenylbutane (Mu-Phe-Hph-β-S02CH2CI To a solution of Boc-Hph-β-S02CH2CH2TMS (0 90 g, 2 18 mmol as described in Example 14) in THF (2 mL) were added tetrabutylammonium fluoride (8 7 mL of a 1 0M THF solution) and several molecular sieves The mixture was stirred overnight at room temperature Bromochloromethane (5 mL) was added The mixture was heated at reflux for 1 hour, cooled, and the volatile components were removed under reduced pressure The residue was dissolved in ethyl acetate (75 mL), washed with 1M HCI (50 mL), dried over MgS04 filtered, and the solvent was removed under reduced pressure The residue, crude (S)- 2-tert-butoxycarbonylamιno-1-chloromethylsulfonyl-4-phenylbutane, was dissolved in ether (3 mL) A solution of anhydrous 4-toluenesulfonιc acid (0 80 g, 4 70 mmol) in ether (3 mL) was added The mixture was stirred at room temperature overnight Ether (100 mL) was added The solid intermediate, (S)-2-amιno-1-chloromethyl-sulfonyl-4-phenylbutane 4-toluenesulfonate (TsOH Hph-β-S02CH2CI), was filtered, the solids were washed with ether (2x20 mL), and dried in vacuo to give 0 193 g of material (24% from Boc-Hph-β-S02CH2CH2TMS)
To a solution of Mu-PheOH (0 109 g, 0 392 mmol) in THF (3 mL) at -10°C were added 4- methylmorpholine (43 μl, 0 392 mmol) and isobutyl chloroformate (51 μl, 0 392 mmol) The mixture was stirred for 10 minutes, whereupon TsOH Hph-β-S02CH2CI (0 17 g, 0 392 mmol) was added, followed by 4-methylmorpholιne (43 μL, 0 392 mmol) The mixture was stirred for 45 minutes Ethyl acetate (20 mL) was added The solution was washed with 1 M HCI, saturated aqueous sodium bicarbonate, and brine (2 mL each) dried over MgS04, filtered, and the solvent was removed under reduced pressure, to give the final product, Mu-Phe-Hph-β-S02CH2CI (90 mg, 48% yield Example 16
Synthesis of a Cysteine Protease Inhibitor with an α-aminosulfone as the EWG
Synthesis of 1-(tert-butoxycarbonyl)amιno-2-methyl-1-phenylsulfonylpropane (Boc-Val-α- S02Ph) To a stirred suspensionof t-butylcarbamate (2 34 g, 20 mmol) and sodium benzenesulfinate (3 28 g, 20 mmol) in water (20 mL) was added a solution of isobutyraldehyde (2 00 mL, 22 mmol) in formic acid (5 mL) The mixture was stirred at room temperature overnight The precipitate was filtered, washed with water (2x50 mL) and crystallized from isopropanol/water to give 4 72 g (75%) of the product
Example 17
Synthesis of a Cysteine Protease Inhibitor with an α-aminosulfone as the EWG
Synthesis of 1-benzyloxycarbonylamιno-3-phenyl-1-phenylsulfonylpropane (Z-Hph-α-S02Ph) To a suspension of sodium benzenesulfinate (10 g, 60 9 mmol) and benzyl carbamate (9 21 g, 60 9 mmol) in water (40 mL) was added hydrocmnamaldehyde (8 8 mL, 67 mmol) in formic acid (10 mL) The mixture was heated at 70°C for 1 hour, then permitted to cool to room temperature overnight The product crystallized out, it was filtered and recrystallized from hot isopropanol, giving 23 g (100%) yield TLC (30% ethyl acetate/hexane) R, = 0 37
Example 18
Synthesis of a Cysteine Protease Inhibitor with an α-aminosulfone as the EWG
Synthesis of (R)-1 -(4-morpholιnecarbonylphenylalanyl)amιno-3-phenyl-1 -phenylsulfonylpropane and (S)-1-(4-morpholιnecarbonylphenylalanyl)amιno-3-pheny 1-1 -phenylsulfonylpropane (Mu- Phe-Hph-α-S02Ph, epimers separated) Method A Z-Hph-α-S02Ph (1 0 g, 2 44 mmol) was treated with 30% hydrogen bromide in acetic acid (5 mL) After 30 minutes, the mixture was diluted with ether (300 mL), filtered, washed with ether (2x30 mL), and dπed in vacuo to give 0 74 g (86%) 1-amιno-3-phenyM -phenylsulfonylpropane hydrobromide (HBr Hph-α-S02Ph) To a solution of Mu-PheOH (0 64 g, 2 3 mmol) in THF (15 mL) were added 4-methylmorpholιne (0 302 mL, 2 3 mmol) and isobutyl chloroformate (0 312 mL, 2 3 mmol) The mixture was stirred for 10 minutes HBr Hph-α-S02Ph (074 g, 2 1 mmol) was added, followed by 4- methylmorpholine (0 302 mL, 2 3 mmol) After 45 minutes, the mixture was diluted with ethyl acetate (30 mL), washed with 15 L each of 1M HCI, saturated aqueous sodium bicarbonate, and brine, dried over MgS04, filtered, and the solvent was removed under reduced pressure to give 0 75 g (65%) of the product, Mu-Phe-Hph-α-S02Ph Method B: To a solution of phenylalanine amide hydrochloride (10 g, 50 mmol) in DMF (50 mL) and CH2CI2 (50 mL) were added tπethylamine (13 9 mL, 100 mmol) and 4-morpholιnecarbonyl chloride (5 9 mL, 50 mmol) The mixture was stirred overnight The solvents were removed under reduced pressure The residue was dissolved in ethyl acetate (50 mL), and filtered Ether was added to the filtrate until the solution became turbid 7 2 g (80% yield) of the intermediate, 4-morpholιnecarbonylphenylalanιne amide (Mu-Phe-NH2) crystallized from the solution after 3 days To a solution of Mu-PheNH2 (2 24 g, 8 1 mmol) in formic acid (5 mL) was added, with stirring, hydrocinnamaldehyde (1 17 mL, 8 9 mmol) The mixture was stirred for 5 hours, whereupon sodium benzenesulfinate (1 33 g, 8 1 mmol) was added The mixture was quickly heated to reflux over a five minute period, and was allowed to cool to room temperature The solution was then permitted to stir for three days An equal volume of water was added The product was extracted with CH2CI2 (3x100 mL), dried over MgS04, filtered, and the solvent was removed under reduced pressure The yield of product, diastereomeric (R)- and (S)-1-(4- morpholιne-carbonylphenylalanyl)amιno-3-phenylsulfonyl-1-phenyl-propane, was 3 9 g (90%) TLC (50% ethyl acetate/CH2CI2) Rf = 0.27,0 34
The diastereomers were separated by flash chromatography on 230-400 mesh silica gel (20- 50% ethyl acetate/CH2CI2, gradient elution)
Example 19
Inhibition of Cysteine Proteases with the
Inhibitors of the Invention
Conditions for cathepsin B 50 mM phosphate, pH 6 0, 2 5 mM EDTA, 2 5 mM DTT substrate [Z-Arg-Arg-AMC] = 50 mM (Km = 190 mM) The assay at 25° was started by the addition of cat B (final concentration approx 10 nM) and the increase in fluorescence at 450 nm with excitation at 380 nm was followed over 2 mm The depression in the rate of substrate hydrolysis following addition of varying concentrations of inhibitors was noted The assay was linear throughout the range observed Duplicate runs were measured
Conditions for cathepsin L 50 mM acetate, pH 5 5, 2 5 mM EDTA, 2 5 mM DTT substrate [Z-Phe-Arg-AMC] = 5 mM (Km = 2 mM) The assay at 25°was started by the addition of cat L (final concentration approx 1 nM) and the increase in fluorescence at 450 nm with excitation at 380 nm was followed over 2 mm The depression in the rate of substrate hydrolysis following addition of varying concentrations of inhibitors was noted The assay was linear throughout the range observed Duplicate runs were measured Conditions for cathepsin S: 50 mM phosphate, pH 6.5, 2.5 mM EDTA, 2.5 mM DTT. substrate : [Z-Val-Val-Arg-AMC] = 10 mM (Km = 18 mM). The assay at 25° was started by the addition of cat S (final concentration approx 30 pM) and the increase in fluorescence at 450 nm with excitation at 380 nm was followed over 2 min. The depression in the rate of substrate hydrolysis following addition of varying concentrations of inhibitors was noted. The assay was linear throughout the range observed. Duplicate runs were measured.
Conditions for cruzain were the same as for cathepsin L with the exception that the Km for the substrate was 1 mM.
The respective K, values were estimated by using the Dixon plot as described by Irwin Segel in Enzyme Kinetics: Behavior and analysis of rapid equilibrium and steady-state enzyme systems, 1975, Wiley-lnterscience Publication, John Wiley & Sons, New York.
The results are shown in Table 2.
Table 2
Figure imgf000057_0001
Z-β-Ala-Phe-HphγS02Ph 52 0 79 3 0 0 54 β-Ala-Phe-HphγS02Ph »50 14 11 14
Mu-Tyr-HphγS02Ph - 2 3 9 5 20
Mu-Phe-HphγC02Et 1 6 0 48 0 19 0 91
Mu-Phe-HphγCONHPh 2 6 2 0 1 3 0 13
Mu-Phe-HphγCONHBzl »50 19 30 7 7
Mu-Phe-HphγPO(02Et)2 17 3 0 1 4 15
Mu-Phe-Hph-γPh-OMe 4 8 0 94 0 37 0 89
Mu-Phe-Hph-γPh-NH2 »50 2 9 9 1 3 0
EXAMPLE 20
The following are representative phamaceutical formulation containing a cysteine protease inhibitor of this invention
ORAL FORMULATION
A representative solution for oral administration contains
Cysteine protease inhibitor 100 to 1000 mg Citric Acid Monohydrate 105 mg Sodium Hydroxide 18 mg Flavoring Water q s to 100 mL
INTRAVENOUS FORMULATION
A representative solution for intravenous admmstration contains
Cysteine protease inhibitor 10 to 100 mg Dextrose Monohydrate q s to make isotonic Citric Acid Monohydrate 1 05 mg Sodium Hydroxide 0 18 mg Saline for Injection q s to 1 0 mL
TABLET FORMULATION A representative tablet form may contain:
Cysteine protease inhibitor 1 %
Microcrystalline Cellulose 73%
Stearic Acid 25%
Colloidal Silica 1%

Claims

CLAIMS We Claim
1 A protease protease inhibitor comprising a targeting group linked through a two carbon atom chain to an electron withdrawing group, wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 μM
2 A protease inhibitor comprising a targeting group linked either directly or through a linker selected from the group consisting of an intermediate carbon atom or a two carbon atom chain to a sulfonyl group, wherein the dissociation constant for inhibition of the protease with said inhibitor (K,) is no greater than about 100 μM
3 A compound of Formula I
Figure imgf000060_0001
I in which
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below,
X represents a bond, methylene or the linkage -CH2CH(R4)-, wherein R4 is hydrogen, alkyl or arylalkyl,
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below,
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below,
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below, R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylammosulfonyl, arylsulfonyl or heteroarylsulfonyl,
R7 and Rβ are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylammo, dialkylamino, uπedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, amino, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C-jJmethyiene and 1 ,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); and R2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from amino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, guanidino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof); and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof.
4. A compound according to claim 3 wherein the dissociation constant for inhibition of a protease with said inhibitor (K,) is no greater than about 100 μM.
5. The compound of Claim 3 in which n is 0 to 5; A-B represents a linkage selected from -C(0)NR3-; Y is -N(R5)-; Z is -(CH2)2- or -C(R6)(R7)-; is -CH(R8)-; R1 is hydrogen, alkyloxycarbonylalkanoyl of overall 3 to 10 carbon atoms, (C,.9)alkoxycarbonyl, (C2.10)alkanoyl (optionally substituted with a radical selected from carboxy, (C,.9)alkyloxycarbonyl and hetero(C4.β)cycloalkyl(C2.10)alkanoylamin), (C4.9)cycloalkylcarbonyl, hetero(C4.8)cycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, (C,.5)alkyl, (C^alkanoyl, (C1.5)alkyloxycarbonyl, (C^oJarylfCLsJalkyloxycarbonyl and hetero(C4.B)cycloalkylcarbonyl), (C6.l0)aryl(C1.5)alkyloxycarbonyl, carbamoyi, (C1.5)alkylcarbamoyl, di(C1.5)alkylcarbamoyl, (Cβ.10)arylcarbamoyl, (C6.l0)aryl(C1.5)alkylcarbamoyl, (C6.10)aryl(C1.5)alkanoyl, (C7.1,)aroyl, (C,.s)alkylsulfonyl, di(C,.5)alkylaminosulfonyl, (C6.,0)arylsulfonyl or heterofC-^arylsulfonyl; R8 and R7 are independently (C3.7)cycloalkyl,
Figure imgf000061_0001
pyridyl, thienyl, furyl, imidazolyl, indolyl, pyridyl(C1-6)alkyl, thienyl(C,^)alkyl, furyl(C1.6)alkyl, imidazoly C^Jalkyl, indolyl(C,^)alkyl, (C1.5)alkyl, (optionally substituted with a radical selected from mercapto, carboxy, amino, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyl, guanidino and hydroxy, or a protected derivative thereof), a group selected from phenyl, naphthyl, pheny C^alkyl, naphthyl(C,^)alkyl, (which group is optionally substituted at its aryl ring with one to three radicals selected from amino, hydroxy, chloro, bromo, fluoro, methyl, trifluoromethyl, methoxy and phenyl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C-j methylene and 1 ,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), R2 is (C^alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro, lodo, hydroxy and methoxy, or a protected derivative thereof), perhalo(C, 5)alkyl, (C3 7)cycloalkyl, (C3 7)cycloalkyl(C* 5)alkyl or a group selected from phenyl, pentafluorophenyl, naphthyl and phenyl(C,^)alkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl, or a protected derivative thereof) and R4 is hydrogen, (C, 5)alkyl or (C6 10)aryl(C, 5)alkyl
6 The compound of Claim 5 in which n is 0 to 2, Z is -(CH2)2- or -C(R6)(R7)- (with the proviso that when n is 0, Z is not -(CH2)2-), R1 is hydrogen, (C4 8)alkoxycarbonyl, (C2^)alkanoyl (optionally substituted with a radical selected from carboxy, (C, 5)alkyloxycarbonyl and hetero(C4^)cycloalkyl(C4^)alkanoylamιno), -C(0)NR 1R22 wherein R21 and R22 together form aza(C2-6)methylene, oxa(C2^)methylene or (C3 7)methylene, (C4 β)cycloalkylcarbonyl, benzyloxycarbonyl, acetyl, benzoyi or dimethylaminosulfonyl, R8 and R7 are independently (C5^)cycloalkyl, (C5^)cycloalkylmethyl, 3-pyπdyl, 2-thιenyl, 2-furyl, 4-ιmιdazolyl, 3-ιndolyl, 3-pyrιdylmethyl, 2-thιenylmethyl, 2-furylmethyl, 4-ιmιdazolylmethyl, 3-ιndolylmethyl, methoxy, acetoxy, (C, s)alkyl (optionally substituted with a radical selected from mercapto, carboxy, ammo, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyl, guanidino and hydroxy, or a protected derivative thereof), a group selected from phenyl, 1 -naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, ammo, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3j4)methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), R2 is (C, 5)alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro and hydroxy, or a protected derivative thereof), perfluorc C, 5)alkyl, (C5^)cycloalkyl, (C5^)cycloalkylmethyl or a group selected from phenyl, naphthyl and benzyl (which group is optionally substituted with one radical selected from ammo hydroxy, chloro, bromo or fluoro, or a protected derivative thereof) and R4 is hydrogen or methyl
7 The compound of Claim 4 in which n is 0 to 1, Z is -C(R6)(R7)-, R1 is hydrogen, fert-butoxycarbonyl, benzyloxycarbonyl, acetyl, 3-carboxypropιonyl, 3-methoxycarbonylpropιonyl, biotinylaminohexanoyl, phenylacetyl, benzoyi, dimethylaminosulfonyl, benzylsulfonyl, 1-pιperizιnylcarbonyl, 4-methyl-1-pιperazιnylcarbonyl or 4-morpholιnylcarbonyl, Rβ is butyl, 2-phenylethyl, 2-methylsulfonylethyl, 2-ferf-butoxycarbonylethyl, 2-fert-butoxycarbonylmethyl, 4-fert-butoxycarbonylamιnobutyl, 4-benzoylamιnobutyl or benzyloxymethyl, R2 is methyl, tπfluoromethyl, optionally substituted phenyl, 2-naphthyl or 2-phenylethyl; R4 is hydrogen; and R7 is 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, 3-indolylmethyl, (C,.s)alkyl (optionally substituted with a radical selected from mercapto, carboxy, amino, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyi, guanidino and hydroxy, or a protected derivative thereof), a group selected from benzyl, 1-naphthylmethyl, 2-πaphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, amino, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (Cj methylene and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo).
8. The compound of Claim 7 in which n is 0; R3, R5 and R6 are each hydrogen; R1 is hydrogen, fert-butxoycarbonyl, benzyloxycarbonyl, biotinylaminohexanoyl, benzoyi, 1-piperiziny-carbonyl, 4-methyl-1-piperazinylcarbonyl or 4-morpholinylcarbonyl; R8 is butyl, 2-phenylethyl or 2-methylsulfonylethyl; R2 is phenyl, 1 -naphthyl or 2-phenylethyl; and R7 is (C,.5)alkyl, 2-methylsulfonylethyl, optionally substituted benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 3-pyridinylmethyl or 2-methylsulfonylethyl.
9. The compound of Claim 8 in which R1 is 1-piperizinylcarbonyl, 4-methyl- 1-piperazincarbonyl or 4-morpholinylcarbonyl; R8 is 2-phenylethyl; R2 is phenyl or naphth-2-yl; and R7 is optionally substituted benzyl, 1-naphthylmethyl or 2-naphthylmethyl.
10. The compound of Claim 9 in which X represents a bond, R is 4-morpholinylcarbonyl, R8 is 2-phenylethyl, R2 is phenyl and R7 is benzyl, namely Λ/2-(4-morpholinylcarbonyl)-rV-(3-phenyl-1-phenylsulfonylpropyl)-L-phenylalaninamide.
11. The compound of Claim 9 in which X represents methylene, R1 is 4-morpholinylcarbonyl, Rβ is 2-phenylethyl, R2 is phenyl and R7 is benzyl, namely /V2-(4-morpholinylcarbonyl)-Λ 1-(3-phenyl-1 S-phenylsulfonylmethylpropyl)-L-phenylalaniamide.
12. The compound of Claim 9 in which X represents -CH2CH(R4) wherein R4 is hydrogen, R is 4-morpholinylcarbonyl, R8 is 2-phenylethyl, R2 is 2-naphthyl and R7 is 2-naphthylmethyl, namely /v^-morpholinylcarbony -ΛT- -phenyl- 1S-[2-(2-naphthylsulfonyl)ethyl]propyl}-β-(2-naphthyl)-L-alaninamide. 13 The compound of Claim 9 in which X represents -CH2CH(R4) wherein R4 is hydrogen, R1 is 4-morpholιnylcarbonyl, Rβ is 2-phenylethyl, R2 is phenyl and R7 is 4-hydroxybenzyl, namely Λ/2-(4-morpholιnylcarbonyl)-Λ '-{3*-phenyl- 1 S-[2-(2-naphthylsulfonyl)ethyl]propyl}-L-tyrosιnamιde
14 The compound of Claim 9 in which X represents -CH2CH(R4) wherein R4 is hydrogen, R1 is 4-morpholιnylcarbonyl, R8 is 2-phenylethyl, R2 is phenyl and R7 is benzyl, namely Λ/2-(4-morpholιnylcarbonyl)-Λ/1-[3-phenyl-1 S-(2-phenylsulfonylethyl)propyl]-L-phenylalanιnamιde
15 A compound of Formula II
Figure imgf000064_0001
in which
II
A-B represents a linkage selected from -C(0)NR3- -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below,
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below,
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below, is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below,
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylammo), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylaminosulfonyl, arylsulfonyl or heteroarylsulfonyl,
R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, am o, alkylamino, dialkylammo, uπedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl
(which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (C3^)methylene and 1 ,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); and
R9 is cyano, -C(0)OR1°, -P(O)(OR10)2, -S(O)(NR10)R10, C(0)R11, -S(0)R11, -C(0)NR12R13, -S(0)2NR12R13, -C(0)NHR14 or -S(0)2NHR14, wherein each R10 is independently hydrogen, alkyl (optionally substituted with one or more radicals selected from amino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R11 is hydrogen, alkyl, perfluoroalkyl, cycloalkyl, cycloalkylalkyl, perfluoroaryl, perfluoroarylakyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R12 and R13 are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl or aralkyi and R14 is -C(0)OR1°, in which R10 is as defined above, or a group selected from Formula (a) and (b):
Figure imgf000065_0001
wherein each n, A, B, Y, Z, R1 and R10 are as defined above; and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof.
16. The compound of Claim 15 in which each n is 0 to 5; each A-B represents a linkage selected from -C(0)NR3-; each Y is -N(R5)-; each Z is -(CH2)2- or -C(R6)(R7)-; Z1 is -CH(R8)-; each R1 is independently hydrogen, alkyloxycarbonylalkanoyl of overall 3 to 10 carbon atoms, (C1.9)alkoxycarbonyl, (C2.10)alkanoyl (optionally substituted with a radical selected from carboxy, (C**.9)alkyloxycarbonyl and hetero(C4^)cycloalkyl(C2.10)alkanoylamino), (C4.9)cycloalkylcarbonyl, hetero(C44)cycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, (Chalky!, (C^alkanoyl, (CLsJalkyloxycarbonyl, (C6.10)aryl(C,.5)alkyloxycarbonyl and hetero(C4^)cycloalkylcarbonyl), (C6-ιo)aryl(C1.5)alkyloxycarbonyl, carbamoyi, (CLsJalkylcarbamoyl, di(C,.5)alkylcarbamoyl, (Cf- oJarylcarbamoyl, (C6.10)aryl(C,.5)alkylcarbamoyl, (C6-1o)aryl(C1.5)alkanoyl, (C^Oaroyl, (C^alkylsulfonyl, d CLsJalkylaminosulfonyl, (C^oJarylsulfonyl or hetero(C5^)arylsulfonyl; Rβ and R7 are independently (C3.7)cycloalkyl, (C3.7)cycloalkyl(C**.5)alkyl, pyridyl, thienyl, furyl, imidazolyl, indolyl, pyridyl(C1-6)alkyl, thienyl(C1-6)alkyl, furyKC-^alkyl, imidazolyl(C^)alkyl, indolyl(C,^)alkyl, a group selected from (C1.5)alkyl, (C2^)alkyloxy and (C,.5)alkanoyloxy (which group is optionally substituted with a radical selected from mercapto, carboxy, ammo, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyi, guanidino and hydroxy, or a protected derivative thereof), a group selected from phenyl, naphthyl, phenyl(C,^)alkyl, naphthyl(C,^)alkyl, (which group is optionally substituted at its aryl ring with one to three radicals selected from ammo, hydroxy, chloro, bromo, fluoro, methyl, tπfluoromethyl, methoxy and phenyl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3.,) methylene and 1,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), each R10 is independently (C, 5)alkyl (optionally substituted with one or two radicals selected from ammo, chloro, bromo, fluoro, hydroxy and methoxy or a protected derivative thereof), (C3 7)cycloalkyl, (C3 7)cycloalkyl(C, 5)alkyl, or a group selected from phenyl or phenyl(C,^)alkyl (which group is optionally substituted at its phenyl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl, or a protected derivative thereof), R11 is independently (C, 5)alkyl, (C3 7)cycloalkyl, (C3 7)cycloalkyl(C1 5)alkyl or a group selected from phenyl, and phenyl(C,^)alkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methyl, tπfluoromethyl and methoxy), and R12 and R13 are independently (C, 5)alkyl, (C3 7)cycloalkyl, (C3 7)cycloalkyl (C, 5)alkyl or a group selected from phenyl and phenyl(C,^)alkyl (which group is optionally substituted at its phenyl ring with one to two radicals selected from ammo, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl)
17 The compound of Claim 16 in which each n is 0 to 2, Z is -(CH2)2- or -C(R6)(R7)- (with the proviso that when n is 0, Z is not -(CH2)2-), each R1 is hydrogen, (C4 8)alkoxycarbonyl, (C2^)alkanoyl (optionally substituted with a radical selected from carboxy, (C, 5)alkyloxycarbonyl and hetero(C4^)cycloalkyl(C4^)alkanoylamιno), -C(0)NR21R22 wherein R2 and R22 together form aza(C2-6)methylene, oxa(C2^)methylene or (C3.7)methylene, (C4^)cycloalkylcarbonyl, benzyloxycarbonyl, acetyl, benzoyi or dimethylaminosulfonyl, R8 and R7 are independently (C-wsJcycloalkyl, (CM)cycloalkylmethyl, 3-pyπdyl, 2-thιenyl, 2-furyl, 4-ιmιdazolyl, 3-ιndolyl, 3-pyrιdylmethyl, 2-thιenylmethyl, 2-furylmethyl, 4-ιmιdazolylmethyl, 3-ιndolylmethyl, methoxy, acetoxy, a group selected from (C1-S)alkyl (which group is optionally substituted with a radical selected from mercapto, carboxy, ammo, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyi, guanidino and hydroxy, or a protected derivative thereof), a group selected from phenyl, 1 -naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, am o, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C-j methylene and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); each R10 is ethyl, (C^cycloalkyl, (C^Jcycloalkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from amino hydroxy, chloro, bromo or fluoro, or a protected derivative thereof); R11 is ethyl, cyclo(CM)alkyl, cyclo(CM)alkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from amino hydroxy, chloro, bromo or fluoro, or a protected derivative thereof); and R12 and R13 are independently ethyl, (C5^)cycloalkyl, (C5^)cycloalkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from amino hydroxy, chloro, bromo or fluoro, or a protected derivative thereof)*
18. The compound of Claim 17 in which each n is 0 to 1; Z is -C(R6)(R7)-; each R1 is hydrogen, fer -butoxycarbonyl, benzyloxycarbonyl, acetyl, 3-carboxypropionyl, 3-methoxycarbonylpropionyl, biotinylaminohexanoyl, phenylacetyl, benzoyi, dimethylaminosulfonyl, benzylsulfonyl, 1-piperizinylcarbonyl, 4-methy-1-lpiperazinylcarbonyl or 4-morpholinylcarbonyl; R8 is butyl, 2-phenylethyl, 2-methylsulfonylethyl, 2-fert-butoxycarbonylethyl, 2-fert-butoxycarbonylmethyl, 4-fert-butoxycarbonylaminobutyl, 4-benzoylaminobutyl or benzyloxymethyl; and R7 is 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, 3-indolylmethyl, a group selected from (C,.5)alkyl (which group is optionally substituted with a radical selected from mercapto, carboxy, amino, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyi, guanidino and hydroxy, or a protected derivative thereof), a group selected from benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, amino, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3-4)methylene and 1 ,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo).
19. The compound of Claim 18 in which each n is 0; each R3, R5 and R6 are hydrogen; each R is hydrogen, fert-butxoycarbonyl, benzyloxycarbonyl, biotinylaminohexanoyl, benzoyi, 1-piperizinylcarbonyl, 4-methyl-1-piperazinylcarbonyl or 4-morpholinylcarbonyl; R8 is butyl, 2-phenylethyl or 2-methylsulfonylethyl; and R7 is (Chalky!, 2-methylsulfonylethyl, optionally substituted benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 3-pyridinylmethyl or 2-methylsulfonylethyl.
20. The compound of Claim 19 in which each R1 is 1-piperizinylcarbonyl, 4-methyl- 1-piperazinylcarbonyl or 4-morpholinylcarbonyl; R8 is 2-phenylethyl; and R7 is optionally substituted benzyl, 1-naphthylmethyl or 2-naphthylmethyl. 21. The compound of Claim 20 in which R1 is 4-morpholinylcarbonyl, Rβ is 2-phenylethyl, R7 is 2-naphthylmethyl and R9 is ethoxycarbonyl namely ethyl 4S-[Λ/-(4-morpholinylcarbonyl)-β-(2-naphthyl)-L-alanylamino]-6-phenylhexanoate.
22. The compound of Claim 20 in which R1 is 4-morpholinylcarbonyl, R8 is 2-phenylethyl, R7 is benzyl and R9 is ethoxycarbonyl namely ethyl 4S-[Λ/-(4-morpholinylcarbonyl)-L-phenylalanylamino]-6-phenylhexanoate.
23. The compound of Claim 20 in which R1 is 4-morpholinylcarbonyl, R8 is 2-phenylethyl, R7 is benzyl and R9 is phenylcarbamoyl namely Λ/2-(4-morpholinylcarbonyl)- Λ M3-phenyl-1 S-(2-phenylcarbamoylethyl)propyl]-L-phenylalanιnamide.
24. The compound of Claim 20 in which R1 is 4-morpholinylcarbonyl, R8 is 2-phenylethyl, R7 is benzyl and R9 is benzylcarbamoyl namely Λ -4-(morpholinylcarbonyl)- Λ/1-[3-phenyl-1 S-(2-benzylcarbamoylethyl)propyl]-L-phenylalaninamide.
Figure imgf000068_0001
n is O to 13;
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below;
Y is -CH(R5)- or -NR5-, wherein R5 is hydrogen or as defined below;
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below;
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below;
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylaminosulfonyl, arylsulfonyl or heteroarylsulfonyl;
R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, amino, alkylamino, dialkylamino, uriedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, amino, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (Cw)methylene and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); R15 is hydrogen, methyl, fluoro or a group selected from Formulae (a) and (b):
Figure imgf000069_0001
(a)
wherein each n, A, B, Y, Z and R1 are as defined above and R10 is hydrogen, alkyl (optionally substituted with one or more radicals selected from amino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof); and R16 is a group selected from phenyl or (C^heteroaryl (which group is optionally substituted with at least one radical selected from aikylcarbamoyl, dialkylcarbamoyi, alkyloxycarbonyl, alkylsulfinamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, carbamoyi, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkyisulfamoyl, dialkylsulfamoyi, alkyloxysulfonyl, alkylsulfonimidoyl, aryl, heteroaryl, hydroxy, alkyloxy, optionally halo-substituted alkyl, arylalkyl, halo, -*N(R17)3, wherein each R17 is independently alkyl, aryl or arylalkyl, or -N(R18)2, wherein each R 8 is independently hydrogen, alkyl, aryl or arylalkyl); and the pharmaceutically acceptable salts; individual isomers and mixtures of isomers thereof.
26. The compound of Claim 25 in which each n is 0 to 5; each A-B represents a linkage selected from -C(0)NR3-; each Y is -NR5-; each Z is -(CH2)2- or -C(R6)(R7)-; Z1 is -CH(R8)-; each R1 is hydrogen, alkyloxycarbonylalkanoyl of overall 3 to 10 carbon atoms, (C^alkoxycarbonyl, (C2.10)alkanoyl (optionally substituted with a radical selected from carboxy, (C,.9)alkyloxycarbonyl and hetero(C4<)cycloalkyl(C2.10)alkanoylamino), (C4.9)cycloalkylcarbonyl, hetero(C4.β)cycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, (C^alkyl, (C^alkanoyl, (C,.5)alkyloxycarbonyl, (C6-10)aryl(C1.5)alkyloxycarbonyl and hetero(C .e)cycloalkylcarbonyl), (C^oJary CLsJalkyloxycarbonyl, carbamoyi, (C^alkylcarbamoyl, d CL^alkylcarbamoyl, (C^oJarylcarbamoyl, (Ce. ary C^alkylcarbamoyl, (C6.ιo)aryl(C,.s)alkanoyl, (C7..,,)aroyl, (C,.5)alkylsulfonyl, di(C1.5)alkylaminosulfonyl, (C6.,0)arylsulfonyl or hetero(C5^)arylsulfonyl; R8 and R7 are independently (C3.7)cycloalkyl, (C3.7)cycloalkyl(C*.5)alkyl, pyridyl, thienyl, furyl, imidazolyl, indolyl, pyridyl(C,^)alkyl, thienyl(C1-6)alkyl, furyl(C1.β)alkyl, imidazolyKC^alkyl, indolyl(C1-6)alkyl, a group selected from (C,.5)alkyl, (C2^)alkyloxy and (C^sJalkanoyloxy (which group is optionally substituted with a radical selected from mercapto, carboxy, amino, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyi, guanidino and hydroxy, or a protected derivative thereof), a group selected from phenyl, naphthyl, pheny C^alkyl, naphthyl(C,^)alkyl, (which group is optionally substituted at its aryl ring with one to three radicals selected from amino, hydroxy, chloro, bromo, fluoro, methyl, trifluororπethyl, methoxy and phenyl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3-4)methylene and 1 ,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); R10 is (C,.5)alkyl (optionally substituted with one or two radicals selected from amino, chloro, bromo, fluoro, hydroxy and methoxy or a protected derivative thereof), (C3.7)cycloalkyl, (C3.7)cycloalkyl(C1.5)alkyl, or a group selected from phenyl or phenyl(C,^)alkyl (which group is optionally substituted at its phenyl ring with one to two radicals selected from amino, chloro, bromo, fluoro, hydroxy, methoxy and optionally halo-substituted methyl, or a protected derivative thereof); and R16 is a group selected from 2-furyl, 2-thienyl, 2-pyrrolyl, 2-phosholyl, 2-arsolyl, 3-pyridyl or 3-phosphorinyl (which group is optionally substituted with at least one radical selected from (CLjJalkylcarbamoyl, di(C1.5)alkylcarbamoyl, (C,.5)alkyloxycarbonyl, (C^alkylsulfinamoyl, di(C,.5)alkylsulfinamoyl, (C,.5)alkylsulfonyl, carboxy, nitro, sulfmamoyl, sulfo, carbamoyi, phosphono, (C,.s)alkyloxyphosphinyl, di(C,.5)alkyloxyphosphinyl, (C,.5)alkanoyl, cyano, (C^alkylsulfinyl, sulfamoyl, (C1.5)alkylsulfamoyl, di(C,.5)alkylsulfamoyl, (C1.5)alkyloxysulfonyl, (C,.5)alkylsulfonimidoyl, phenyl, naphthyl, pyridyl, thienyl, furyl, imidazolyl, indolyl, hydroxy, (CLsJalkyloxy, optionally halo-substituted (C-*.5)alkyl, benzyl, halo, -+N(R17)3, wherein each R17 is independently (Chalky!, phenyl or benzyl, or -N(R18)2, wherein each R18 is independently hydrogen, (Chalky!, phenyl or benzyl).
27. The compound of Claim 26 in which each n is 0 to 2; Z is -(CH2)2- or -C(R6)(R7)- (with the proviso that when n is 0, Z is not -(CH2)2-); each R1 is hydrogen, (C4.8)alkoxycarbonyl, (C2^)alkanoyl (optionally substituted with a radical selected from carboxy, (C,.5)alkyloxycarbonyl and hetero(C4^,)cycloalkyl(C4^)alkanoylamino), -C(0)NR21R22 wherein R21 and R22 together form aza(C2.6)methylene, oxa(C2^)methylene or (C3.7)methylene, (C^cycloalkylcarbonyl, benzyloxycarbonyl, acetyl, benzoyi or dimethylaminosulfonyl; R8 and R7 are independently (Cjs^cycloalkyl, (Cw)cycloalkylmethyl, 3-pyridyl, 2-thienyl, 2-furyl, 4-imidazolyl, 3-indolyl, 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, 3-indolylmethyl, methoxy, acetoxy, (C,.5)alkyl (optionally substituted with a radical selected from mercapto, carboxy, amino, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyi, guanidino and hydroxy, or a protected derivative thereof) a group selected from phenyl, 1-naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, amino, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C3Jl)methylene and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); each R10 is ethyl, (C^Jcycloalkyl, (C5-6)cycloalkylmethyl or a group selected from phenyl and benzyl (which group is optionally substituted at its phenyl ring with one radical selected from amino hydroxy, chloro, bromo or fluoro, or a protected derivative thereof); and R16 is a group selected from 2-furyl, 2-thienyl, 2-pyrrolyl, 2-phosholyl, 2-arsolyl, 2-pyridyl or 3-phosphorinyl (which group is optionally substituted with at least one radical selected from methylcarbamoyl, dimethylcarbamoyi, methyloxycarbonyl, methylsulfinamoyl, dimethylsulfinamoyl, methylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, carbamoyi, phosphono, methyloxyphosphinyl, dimethyloxyphosphinyl, formyl, cyano, methylsulfinyl, sulfamoyl, methylsulfamoyl, dimethylsulfamoyl, methoxysulfonyl, methylsulfonimidoyl, phenyl, naphthyl, pyridyl, thienyl, furyl, imidazolyl, indolyl, hydroxy, methoxy, methyl, trifluromethyl, benzyl, halo, -*N(R17)3, wherein each R17 is independently methyl, phenyl or benzyl, or -N(R1β)2, wherein each R1β is independently hydrogen, methyl, phenyl or benzyl).
28. The compound of Claim 27 in which each n is 0 to 1; Z is -C(R6)(R7)-; each R1 is hydrogen, fert-butoxycarbonyl, benzyloxycarbonyl, acetyl, 3-carboxypropionyl, 3-methoxycarbonylpropionyl, biotinylaminohexanoyl, phenylacetyl, benzoyi, dimethylaminosulfonyl, benzylsulfonyl, 1-piperizinylcarbonyl, 4-methyl-1-piperazinylcarbonyl or 4-morpholinylcarbonyl; R8 is butyl, 2-phenylethyl, 2-methylsulfonylethyl, 2-fert-butoxycarbonylethyl, 2-ferf-butoxycarbonylmethyl, 4-fert-butoxycarbonylaminobutyl, 4-benzoylaminobutyl or benzyloxymethyl; and R7 is 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, 3-indolylmethyl, (C^alkyl (optionally substituted with a radical selected from mercapto, carboxy, amino, methylthio, methylsulfonyl, carbamoyi, dimethylcarbamoyi, guanidino and hydroxy, or a protected derivative thereof), a group selected from benzyl, 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl (which group is optionally substituted at its aryl ring with one radical selected from hydroxy, amino, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from (C^)methylene and 1 ,2-pheny lenedimethy lene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo)
29 The compound of Claim 28 in which each n is 0, each R3, R5 and R6 are hydrogen, each R1 is hydrogen, fert-butxoycarbonyl, benzyloxycarbonyl, biotinylaminohexanoyl, benzoyi, 1-pιpeπzιnylcarbonyl, 4-methyl-1-pιperazιnylcarbonyl or 4-morpholιnylcarbonyl, R8 is butyl, 2-phenylethyl or 2-methylsulfonylethyl, and R7 is (C, 5)alkyl, 2-methylsulfonylethyl, optionally substituted benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 3-pyπdιnylmethyl or 2-methylsulfonylethyl
30 The compound of Claim 29 in which each R1 is 1-pιpeπzιnylcarbonyl, 4-methyl- 1-pιperazιnylcarbonyl or 4-morpholιnylcarbonyl, R8 is 2-phenylethyl, and R7 is optionally substituted benzyl, 1-naphthylmethyl or 2-naphthylmethyl
31 The compound of Claim 30 in which R1 is 4-morpholιnylcarbonyl, R8 is 2-phenylethyl, R7 is benzyl, R15 is hydrogen and R 6 is 4-methoxyphenyl, namely
Figure imgf000072_0001
L-phenylalaninamide
32 The compound of Claim 30 in which R1 is 4-morpholιnylcarbonyl, R8 is 2-phenylethyl, R7 is benzyl, R15 is hydrogen and R16 is 4-amιnophenyl, namely /2-(4-morpholιnylcarbonyl)-V {3-phenyl-1S-[2-(4-amιnophenyl)ethyl]propyl}- L-phenylalanmamide
33 A method for inhibiting a cysteine protease comprising reversibly binding a cysteine protease inhibitor to a cysteine protease, wherein said mhibitior comprises the cysteine protease inhibitor of Claim 1
34 A method for inhibiting a cysteine protease comprising reversibly binding a cysteine protease inhibitor to a cysteine protease, wherein said mhibitior comprises the cysteine protease inhibitor of Claim 2
35 A method for inhibiting a cysteine protease comprising reversibly binding a cysteine protease inhibitor to a cysteine protease, wherein said mhibitior comprises the cysteine protease inhibitor of Claim 3
36 A method for inhibiting a cysteine protease comprising reversibly binding a cysteine protease inhibitor to a cysteine protease, wherein said mhibitior comprises the cysteine protease inhibitor of Claim 14.
37. A method for inhibiting a cysteine protease comprising reversibly binding a cysteine protease inhibitor to a cysteine protease, wherein said inhibitior comprises the cysteine protease inhibitor of Claim 24.
38. A method for treating a condition capable of amelioration by inhibition of a cysteine protease in an animal in need thereof, which method comprises administering to such animal a therapeutically effective amount of the cysteine protease inhibitor of Claim 1.
39. A method for treating a condition capable of amelioration by inhibition of a cysteine protease in an animal in need thereof, which method comprises administering to such animal a therapeutically effective amount of the cysteine protease inhibitor of Claim 2.
40. A method for treating a condition capable of amelioration by inhibition of a cysteine protease in an animal in need thereof, which method comprises administering to such animal a therapeutically effective amount of the cysteine protease inhibitor of Claim 3.
41. A method for treating a condition capable of amelioration by inhibition of a cysteine protease in an animal in need thereof, which method comprises administering to such animal a therapeutically effective amount of the cysteine protease inhibitor of Claim 14.
42. A method for treating a condition capable of amelioration by inhibition of a cysteine protease in an animal in need thereof, which method comprises administering to such animal a therapeutically effective amount of the cysteine protease inhibitor of Claim 24.
43. A pharmaceutical composition comprising a therapeutically effective amount of the cysteine protease inhibitor of Claim 1 , or of an individual isomer, a mixture of isomers, or the pharmaceutically acceptable salt or salts thereof, in combination with one or more pharmaceutically acceptable excipients.
44. A pharmaceutical composition comprising a therapeutically effective amount of the cysteine protease inhibitor of Claim 2, or of an individual isomer, a mixture of isomers, or the pharmaceutically acceptable salt or salts thereof, in combination with one or more pharmaceutically acceptable excipients.
45. A pharmaceutical composition comprising a therapeutically effective amount of the cysteine protease inhibitor of Claim 3, or of an individual isomer, a mixture of isomers, or the pharmaceutically acceptable salt or salts thereof, in combination with one or more pharmaceutically acceptable excipients
46 A pharmaceutical composition comprising a therapeutically effective amount of the cysteine protease inhibitor of Claim 14, or of an individual isomer, a mixture of isomers, or the pharmaceutically acceptable salt or salts thereof, in combination with one or more pharmaceutically acceptable excipients
47 A pharmaceutical composition comprising a therapeutically effective amount of the cysteine protease inhibitor of Claim 24, or of an individual isomer, a mixture of isomers, or the pharmaceutically acceptable salt or salts thereof, in combination with one or more pharmaceutically acceptable excipients
48 A process for the preparation of a compound of Formula IV
Figure imgf000074_0001
in which
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below,
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below,
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below,
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below,
R6 is hydrogen or methyl and R7 is as defined below,
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycioalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylammosulfonyl, arylsulfonyl or heteroarylsulfonyl; R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, amino, alkylamino, dialkylamino, uriedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, amino, guanidino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (C-^Jmethylene and 1 ,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); and
R20 is cyano, -S(0)2R2, -CH2S(0)2R2, -CH2CH(R4)S(0)2R2, -(CH2)2C(0)OR10, -(CH2)2P(O)(OR10)2, -(CH2)2S(O)(NR10)R10, -(CH2)2C(0)R11, -(CH2)2S(0)R11, -(CH2)2C(0)NR12R13, -(CH2)2S(0)2NR 2R13, -(CH2)2C(0)NHR14, -(CH2)2S(0)2NHR14 or -CH2CHR15R16, wherein R2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from amino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R4 is hydrogen, alkyl or arylalkyl, each R10 is independently hydrogen, alkyl (optionally substituted with one or more radicals selected from amino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R 1 is hydrogen, alkyl, perfluoroalkyl, cycloalkyl, cycloalkylalkyl, perfluoroaryl, perfluoroarylakyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R12 and R 3 are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl or aralkyi, R14 is -C(0)OR1°, in which R10 is as defined above, or a group selected from Formula (a) and (b):
Figure imgf000075_0001
(»)
wherein each n, A, B, Y, Z, R1 and R10 are as defined above, R15 is hydrogen, methyl, fluoro or a group selected from Formulae (a) and (b) as defined above, and R16 is a group selected from phenyl or (C^heteroaryl (which group is optionally substituted with at least one radical selected from aikylcarbamoyl, dialkylcarbamoyi, alkyloxycarbonyl, alkylsulfinamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, carbamoyi, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkyisulfamoyl, dialkylsulfamoyi, alkyloxysulfonyl, alkylsulfonimidoyl, aryl, heteroaryl, hydroxy, alkyloxy, optionally halo-substituted alkyl, arylalkyl, halo, -*N(R17)3, wherein each R17 is independently alkyl, aryl or arylalkyl, or -N(R18)2, wherein each R1β is independently hydrogen, alkyl, aryl or arylalkyl); and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof; which process comprises: (A) reacting an amine of Formula V:
H,N ' - R "
with a compound of Formula VI:
Figure imgf000076_0001
VI
in which each n, A, B, X, Y, Z, R1, R8 and R20 are as defined above; and
(B) optionally further converting a non-salt form of a compound of Formula IV into a pharmaceutically acceptable salt;
(C) optionally further converting a salt form of a compund of Formula IV into non-salt form; and
(D) optionally further separating a compound of Formula IV into individual stereoisomers.
49. A process for the preparation of a compound of Formula IV:
Figure imgf000076_0002
in which: n is O to 12;
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below;
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below;
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below;
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below;
R6 is hydrogen or methyl and R7 is as defined below;
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylaminosulfonyl, arylsulfonyl or heteroarylsulfonyl;
R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, amino, alkylamino, dialkylamino, uriedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, amino, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (C-^methylene and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo); and R20 is -S(0)2R2, wherein R2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from amino, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof; which process comprises: reacting a compound of Formula VII:
Figure imgf000077_0001
VII
with an aldehyde of the formula R8CHO and a sodium sulfinate of the formula R S(0)ONa, in which each n, A, B, X, Y, Z, R and RB are as defined above;
(B) optionally further converting a non-salt form of a compound of Formula IV into a pharmaceutically acceptable salt; (C) optionally further converting a salt form of a compund of Formula IV into non-salt form, and
(D) optionally further separating a compound of Formula IV into individual stereoisomers
50 A process for the preparation of a compound of Formula IV
Figure imgf000078_0001
in which
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3- -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below,
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below,
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below,
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below,
R6 is hydrogen or methyl and R7 is as defined below,
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylammosulfonyl, arylsulfonyl or heteroarylsulfonyl,
R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylamino, dialkylammo, uπedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl
(which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected deπvative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (Cj methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), and
R20 is -S(0)2R2, wherein R2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from amino, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof); and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof; which process comprises:
(A) (1 ) reacting a compound of the formula NH2P, wherein P is a protective group, with an aldehyde of the formula RβCHO and a sodium sulfinate of the formula R2S(0)ONa and then deprotecting to give a compound of Formula VIII:
R"
H,N S(0) ,R-
in which R2 and R8 are as defined above; and
(2) reacting the compound of Formula VIM with a compound of Formula VI:
Figure imgf000079_0001
VI
in which each n, A, B, X, Y, Z, and R1 are as defined above;
(B) optionally further converting a non-salt form of a compound of Formula IV into a pharmaceutically acceptable salt;
(C) optionally further converting a salt form of a compund of Formula IV into non-salt form; and
(D) optionally further separating a compound of Formula IV into individual stereoisomers.
51. A process for the preparation of a compound of Formula IV:
in which:
Figure imgf000079_0002
n is 0 to 12;
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below;
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below;
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below; Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below,
R6 is hydrogen or methyl and R7 is as defined below,
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylammosulfonyl, arylsulfonyl or heteroarylsulfonyl,
R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylamino, dialkylamino, uπedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl
(which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (C3-4)methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), and
R20 is -CH2S(0)2R2, wherein R2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, which process comprises
(A) (1) reacting a compound of Formula IX
Figure imgf000080_0001
with a thiolate anion of the formula R2S , in which L is a leaving group and R2 and R8 are as defined above, to give a compound of Formula X
,SR'
PHN (2) oxidizing the compound of Formula X to give a compound of Formula XI:
Figure imgf000081_0001
and
(3) reacting the compound of Formula XI with a compound of Formula VI:
Figure imgf000081_0002
VI
in which each n, A, B. X. Y, Z and R are as defined above;
(B) optionally further converting a non-salt form of a compound of Formula IV into a pharmaceutically acceptable salt;
(C) optionally further converting a salt form of a compund of Formula IV into non-salt form; and
(D) optionally further separating a compound of Formula IV into individual stereoisomers.
52. A process for the preparation of a compound of Formula IV:
Figure imgf000081_0003
in which: n is O to 12;
A-B represents a linkage selected from -C(0)NR3-, -CH2NR3-, -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below;
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below;
Z is -(CH2)2-, -C(Rβ)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below;
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below;
R6 is hydrogen or methyl and R7 is as defined below;
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylaminosulfonyl, arylsulfonyl or heteroarylsulfonyl,
R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylammo, dialkylamino, unedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (C^methylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy or a protected derivative thereof, or oxo), and R20 is cyano, -(CH2)2S(0)2R2, -(CH2)2C(0)OR1°, -(CH2)2P(O)(OR10)2, -(CH2)2S(O)(NR10)R10, -(CH2)2C(0)R11, -(CH2)2S(0)R11, -(CH2)2C(0)NR12R13, -(CH2)2S(0)2NR1 R13, -(CH2)2C(0)NHR1 or -(CH2)2S(0)2NHR14 , wherein R2 is hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, halo hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from am o, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), each R10 is independently hydrogen, alkyl (optionally substituted with one or more radicals selected from ammo, halo, hydroxy, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R11 is hydrogen, alkyl, perfluoroalkyl, cycloalkyl, cycloalkylalkyl, perfluoroaryl, perfluoroarylakyl or a group selected from aryl and arylalkyl (which group is optionally substituted at its aryl ring with one to two radicals selected from ammo, halo, hydroxy, optionally halo-substituted alkyl, alkyloxy, nitro, alkylsulfonyl and arylsulfonyl, or a protected derivative thereof), R12 and R13 are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl or aralkyi and R14 is -C(0)OR °, in which R10 is as defined above, or a group selected from Formula (a) and (b)
Figure imgf000082_0001
(a)
wherein each n, A, B, Y, Z, R and R10 are as defined above, and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, which process comprises (A) (1) reacting an aldehyde of Formula XII:
Figure imgf000083_0001
with a compound selected from Formulae XIII and XIV:
Figure imgf000083_0002
XIII XIV
in which each R8 and R20 are as defined above, and then deprotecting to give a compound of Formula XV:
Figure imgf000083_0003
xv
(2) reacting the compound of Formula XV with a compound of Formula VI:
Figure imgf000083_0004
VI
in which each n, A, B, X, Y, Z and R1 are as defined above, and (3) reducing;
(B) optionally further converting a non-salt form of a compound of Formula IV into a pharmaceutically acceptable salt;
(C) optionally further converting a salt form of a compund of Formula IV into non-salt form; and
(D) optionally further separating a compound of Formula IV into individual stereoisomers.
53. A process for the preparation of a compound of Formula IV:
Figure imgf000083_0005
in which: A-B represents a linkage selected from -C(0)NR3-, -CH2NR3- -C(0)CH2- and -NR3C(0)-, wherein R3 is hydrogen or as defined below,
Y is -CH(R5)- or -N(R5)-, wherein R5 is hydrogen or as defined below,
Z is -(CH2)2-, -C(R6)(R7)- or -N(R7)-, wherein R6 is hydrogen or methyl and R7 is as defined below,
Z1 is -(CH2)2-, -C(R6)(R8)- or -N(R8)-, wherein R6 is hydrogen or methyl and R8 is as defined below,
R6 is hydrogen or methyl and R7 is as defined below,
R1 is hydrogen, alkyloxycarbonylalkanoyl, alkyloxycarbonyl, alkanoyl (optionally substituted with a radical selected from carboxy, alkyloxycarbonyl and heterocycloalkylalkanoylamino), cycloalkylcarbonyl, heterocycloalkylcarbonyl (optionally substituted with a radical selected from hydroxy, alkyl, alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and heterocycloalkylcarbonyl) arylalkyloxycarbonyl, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, arylcarbamoyi, arylaikylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, dialkylaminosulfonyl, arylsulfonyl or heteroarylsulfonyl,
R7 and R8 are independently hydrogen, alkyl (optionally substituted with a radical selected from hydroxy, ammo, alkylamino, dialkylamino, uπedo, mercapto, alkylthio, carboxy, carbamoyi, aikylcarbamoyl, dialkylcarbamoyi, alkylsulfonyl and guanidino, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, a group selected from aryl and arylalkyl
(which group is optionally substituted at its aryl ring with one to three radicals selected from hydroxy, ammo, halo, optionally halo-substituted alkyl, alkyloxy and aryl, or a protected derivative thereof) or together with an adjacent R3 or R5 forms a divalent radical selected from a divalent radical selected from (C^Jmethylene and 1 ,2-phenylenedιmethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, or oxo), and
R20 is -CH2CHR15R16, wherein R15 is hydrogen, methyl, fluoro or a group selected from
Formulae (a)and (b)
Figure imgf000084_0001
(a)
wherein each n, A, B, Y, Z, R1 and R10 are as defined above, and R16 is a group selected from phenyl or (Cs^heteroaryl (which group is optionally substituted with at least one radical selected from aikylcarbamoyl, dialkylcarbamoyi. alkyloxycarbonyl, alkylsulfinamoyl, dialkylsulfinamoyl, alkylsulfonyl, carboxy, nitro, sulfinamoyl, sulfo, carbamoyi, phosphono, alkyloxyphosphinyl, dialkyloxyphosphinyl, alkanoyl, cyano, alkylsulfinyl, sulfamoyl, alkyisulfamoyl, dialkylsulfamoyi, alkyloxysulfonyl, alkylsulfonimidoyl, aryl, heteroaryl, hydroxy, alkyloxy, optionally halo-substituted alkyl, arylalkyl, halo, -*N(R17)3, wherein each R17 is independently alkyl, aryl or arylalkyl, or -N(R18)2, wherein each R18 is independently hydrogen, alkyl, aryl or arylalkyl), and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, which process comprises (A) (1 ) reacting an aldehyde of Formula XII
R *
PHN CHO with compound of Formula XVI
Figure imgf000085_0001
XVI
in which each R8, R15 and R16 are as defined above, and then deprotecting to give a compound of Formula XVII
Figure imgf000085_0002
XVII
(2) reacting the compound of Formula XVII with a compound of Formula VI
Figure imgf000085_0003
VI
in which each n, A, B, X, Y, Z and R1 are as defined above, and (3) reducing,
(B) optionally further converting a non-salt form of a compound of Formula IV into a pharmaceutically acceptable salt,
(C) optionally further converting a salt form of a compund of Formula IV into non-salt form, and
(D) optionally further separating a compound of Formula IV into individual stereoisomers
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CZ298197A3 (en) 1998-03-18
AU713492B2 (en) 1999-12-02
CN1071751C (en) 2001-09-26
CN1184472A (en) 1998-06-10
MY113489A (en) 2002-03-30
IL117638A0 (en) 1996-07-23
ZA962336B (en) 1996-07-31
JPH11503417A (en) 1999-03-26
EP0817778A1 (en) 1998-01-14
TW470750B (en) 2002-01-01
NO974403D0 (en) 1997-09-23
NZ305626A (en) 2000-01-28
PL322409A1 (en) 1998-01-19
KR19980703261A (en) 1998-10-15
AU5367496A (en) 1996-10-16
NO974403L (en) 1997-11-17
NO311573B1 (en) 2001-12-10
CA2216151A1 (en) 1996-10-03

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