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WO2008134429A1 - Inhibiteurs de la cystéine protéinase et leurs utilisations - Google Patents

Inhibiteurs de la cystéine protéinase et leurs utilisations Download PDF

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WO2008134429A1
WO2008134429A1 PCT/US2008/061422 US2008061422W WO2008134429A1 WO 2008134429 A1 WO2008134429 A1 WO 2008134429A1 US 2008061422 W US2008061422 W US 2008061422W WO 2008134429 A1 WO2008134429 A1 WO 2008134429A1
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alkyl
compound
compounds
halogen
disease
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William Roush
Yen Ting Chen
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Scripps Research Institute
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Scripps Research Institute
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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
    • 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

  • This invention relates to the proteinase inhibitor compounds, compositions and methods of preventing and treatment of diseases.
  • Proteases have evolved to participate in an enormous range of biological processes, mediating their effect by cleavage of peptide amide bonds within the myriad of proteins found in nature.
  • This hydrolytic action is performed by initially recognizing, then binding to, particular three-dimensional electronic surfaces displayed by a protein, which aligns the bond for cleavage within the protease catalytic site.
  • Catalytic hydrolysis then commences through nucleophilic attack of the amide bond to be cleaved either via an amino acid side-chain of the protease itself, or through the action of a water molecule that is bound to and activated by the protease.
  • cysteine proteases Proteases in which the attacking nucleophile is the thiol side-chain of a Cys residue are known as cysteine proteases.
  • the general classification of "cysteine protease” contains many members found across a wide range of organisms from viruses, bacteria, protozoa, plants and fungi to mammals. Biological investigation of Trypanosoma cruzi infection has highlighted a number of specific enzymes that are crucial for the progression of the parasite's life cycle.
  • cruzipain a cathepsin L-like cysteine protease, is a clear therapeutic target for the treatment of Chagas' disease ((a) Cazzulo, J. J. et al, Curr. Pharm. Des.
  • cysteine proteinases are encode cysteine proteinases.
  • ehcpl ehcp2, andehcp5
  • EhCP5 EhCP2, and EhCPl 12 are membrane-associated, while EhCP3 is intracellular.
  • EhCPl localizes to large cytoplasmic vesicles, a site distinct from the vesicles containing EhCP3. These cysteine proteases are presumed targets for development of anti-amebiasis drugs.
  • Compounds of formulae (I) to (III), derivatives, prodrugs, substitutes, metabolites, stereoisomers, tautomers and analogs thereof, are potent proteases inhibitors. These compounds can be used to treat diseases such as those diseases caused by foreign organisms, e.g. fungi, bacteria, protozoa, and virus. These compounds can be formulated as a pharmaceutical composition.
  • a compound of the following formula (I) comprises:
  • the compound is in a pharmaceutical composition.
  • a compound of the following formula (II) comprises: wherein P 1 comprises:
  • P 2 comprises:
  • R 5 is H, Me
  • X 1 is OH, NHR 2 ;
  • R 2 H, acyl, -CO alkyl;
  • X 2 halogen, alkyl, alkoxy
  • R6 H, OCH 3 , alkyl, halogen, -N0 2; .
  • the R group in Formulae (I) to (III) is optionally independently selected from the group hydrogen, formyl, carboxyl, cyano, hydroxy, amino, nitro, thiol, halo and the following optionally substituted moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, alkanoyl, aroyl, heterocycloyl, heteroaroyl, alkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroarylaminocarbonyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyloxy, alkoxy, aryloxy, alkanoate, aryloate, heterocyclyloate, heterocyclylo
  • the R groups comprise hydrogen, (C 1 -C 6 )alkyl or (C 1 -C 6 )alkoxy; halo or (C 1 -C 6 )haloalkyl; hydrogen, halo or (C 1 -C 6 )haloalkyl; (C 1 - C 6 )alkyl, (C 3 -C 10 )cycloalkyl or (optionally substituted)aryl; (C 2 -C 6 )alkenyl, (C 2 - C 6 )alkynyl, (C 3 -C 10 )cycloalkyl, (C 3 -C 10 )cycloalkenyl, (C 3 -C 10 )cycloalkyl(C 1 -C 6 )alkyl, (C 3 -C 10 )cycloalkenyl(C[-C 6 )alkyl, and the following optionally substituted moieties: aryl, aryl(C 1 -C 6 )alkyl or
  • the R group comprises hydrogen, methyl or methoxy.
  • the R group comprises hydrogen, fluorine, chlorine, fluorine, bromine, cyano, trifluoromethoxy or trifluoromethyl.
  • R is hydrogen, fluorine, chlorine, bromine or trifluoromethyl. In another preferred embodiment, R is hydrogen, methyl, ethyl, n-propyl, i- propyl, t-butyl, cyclohexyl or phenyl.
  • R is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, i-butyl, t-butyl, sec-butyl, phenyl, — CH(CH 2 CH 3 ) 2 , allyl, propargyl, cyclopentyl, cyclohexyl, 3-cyclohexenyl, trans-cinnamyl, -CH 2 CN, - CH(CH 3 )CN, -(CHJ) 2 OCH 3 , -CH 2 SCH 3 , --CH 2 CF 3 .
  • R groups comprise hydrogen or (C 1 -C 6 )alkyl; (C 1 -C 6 )haloallyl; (C 1 -C 6 )haloalkyl; (optionally substituted)aryl; cyano, halo or (C 1 -C 6 )haloalkyl; (optionally substituted)aryloxy, (optionally substituted)heteroaryloxy, (C 3 - C 10 )cycloalkyl, (CrC 6 )alkoxycarbonyl, cyano, (C 1 -C 6 )alkoxy, nitro, halo and (C 1 - C 6 )haloalkyl.
  • R, X, P 1 , P 2 , P 3 , P ⁇ ' are independently selected from the following optionally substituted moieties: alkyl, alkenyl, arylalkyl, hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, heterocyclyloxyalkyl, heteroaryloxyalkyl, alkylcarbonyloxyalkyl, arylcarbonyloxyalkyl, heterocyclylcarbonyloxyalkyl, heteroarylcarbonyloxyalkyl, alkoxycarbonyloxy alkyl, aryloxycarbonyloxyalkyl, heterocyclyloxycarbonyloxyalkyl, heteroaryloxycarbonyloxyalkyl, alkylaminocarbonyloxyalkyl, arylaminocarbonyloxyalkyl, heterocyclylaminocarbonyloxyalky], heteroarylaminocarbonyloxyalkyl, alkylcarbonylaminoalkyl, arylcarbonylamino
  • a pharmaceutical composition comprises any one or more compounds of formulae (I) to (III).
  • a method of preventing or treating diseases in which the disease pathology is modified by inhibiting a cysteine protease comprises administering to a patient in need thereof, a therapeutically effective amount of any one or more compounds of formulae (I) to (III) in a pharmaceutically acceptable carrier.
  • a method of preventing or treating Chagas disease comprises administering to a patient in need thereof, a therapeutically effective amount of any one or more compounds of formulae (I) to (III) in a pharmaceutically acceptable carrier.
  • a method of preventing or treating a disease caused by an organism comprises administering to a patient in need thereof, a therapeutically effective amount of any one or more compounds of formulae (I) to (III) in a pharmaceutically acceptable carrier; inhibiting one or more proteases; and, preventing or treating the disease.
  • the protease is a cysteine protease.
  • the organism is a parasite, bacterium, virus, fungus, or protozoan.
  • any one or more of compounds of formula (I) to (III) are co-administered to patients, with other protease inhibitors or drugs.
  • Figure 1 is a schematic representation showing an outline of synthesis of the compound of formula I.
  • Compounds refers to compounds of Formulae (I ) to (III), and includes any specific compounds encompassed by generic formulae disclosed herein.
  • the compounds may be identified either by their chemical structure and/or chemical name. When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound.
  • the compounds are intended to include all salts, hydrates, solvates, complexes and prodrugs, unless the context requires otherwise.
  • the compounds may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers.
  • stereoisomerically pure form e.g., geometrically pure, enantiomerically pure or diastereomerically pure
  • enantiomeric and stereoisomeric mixtures and their racemates e.g., geometrically pure, enantiomerically pure or diastereomerically pure
  • This invention comprises racemic mixtures as well as pure enantiomers.
  • Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan.
  • the compounds may also exist in several tautomeric forms including cyclic imine form, cyclic iminium form, amino-keto form, ammonium-ketone form, and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds.
  • the compounds also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds include, but are not limited to, 2 H, 3 H, 13 C, 14 C, 15 N, 17 O and 18 O.
  • Compounds may exist in unsolvated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, the hydrated, solvated and N-oxide forms are within the scope of the present disclosure. Certain compounds may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present disclosure.
  • brackets indicate the point of attachment of the partial structure to the rest of the molecule.
  • “Halo” means fluoro, chloro, bromo, or iodo.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, butyric acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, valeric acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthaIenesulfonic acid
  • Examples of appropriate pharmaceutically and veterinarily acceptable salts of the compounds include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesul
  • “Pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient or vehicle with which a compound is administered.
  • Protecting group refers to a grouping of atoms that when attached to a reactive functional group in a molecule masks, reduces or prevents reactivity of the functional group. Examples of protecting groups can be found in Green et al, “Protective Groups in Organic Chemistry", (Wiley, 2nd ed. 1991) and Harrison et ah, “Compendium of Synthetic Organic Methods", VoIs. 1-8 (John Wiley and Sons, 1971-1996).
  • Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl (“SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro- veratryloxycarbonyl (“NVOC”) and the like.
  • hydroxy protecting groups include, but are not limited to, those where the hydroxy group is either acylated or alkylated such as benzyl, and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, and branched-chain alkyl groups.
  • alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), preferably 26 or fewer, and more preferably 20 or fewer, and still more preferably 4 or fewer.
  • alkyl as used throughout the specification and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoro
  • alkyl also includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyl s described above, but that contain at least one double or triple bond respectively.
  • An “alkylaryl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • alkoxy refers to alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
  • alkenyl and “alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively.
  • the invention contemplates cyano and propargyl groups.
  • aralkyl means an aryl group that is attached to another group by a (C1-C6) alkylene group.
  • Aralkyl groups may be optionally substituted, either on the aryl portion of the aralkyl group or on the alkylene portion of the aralkyl group, with one or more substituents.
  • aryl refers to the radical of aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms (heteroaryl), for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "heteroaryls" or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkyl car bonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylaryl amino), acylamino (including alky lcarbony lam ino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,
  • cyclyl refers to a hydrocarbon 3-8 membered monocyclic or 7-14 membered bicyclic ring system having at least one non-aromatic ring, wherein the non- aromatic ring has some degree of unsaturation. Cyclyl groups may be optionally substituted with one or more substituents. In one embodiment, 0, 1, 2, 3, or 4 atoms of each ring of a cyclyl group may be substituted by a substituent.
  • cycloalkyl refers to a hydrocarbon 3-8 membered monocyclic or 7-14 membered bicyclic ring system having at least one saturated ring. Cycloalkyl groups may be optionally substituted with one or more substituents.
  • each ring of a cycloalkyl group may be substituted by a substituent
  • Cycloalkyls can be further substituted, e.g., with the substituents described above.
  • Preferred cyclyls and cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.
  • Those cyclic groups having heteroatoms in the ring structure may also be referred to as "heterocyclyl,” “heterocycloalkyl” or “heteroaralkyl.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above.
  • cyclyl or “cycloalkyl” refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls).
  • rings e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings”. Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, aryicarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), am ⁇ dino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl
  • halogen designates -F, -Cl, -Br or -I.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
  • methyl refers to a CH 3 group.
  • mercapto refers to a SH group.
  • sulfhydryl or "thiol” means -SH.
  • the compounds of the invention encompass various isomeric forms. Such isomers include, e.g., stereoisomers, e.g., chiral compounds, e.g., diastereomers and enantiomers.
  • chiral refers to molecules which have the property of non-superimpos ability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
  • diastereomers refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
  • enantiomers refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.
  • An equimolar mixture of two enantiomers is called a “racemic mixture” or a “racemate.”
  • isomers or “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • Natural amino acids represented by the compounds utilized in the present invention are in the “L” configuration, unless otherwise designated.
  • Unnatural or synthetic amino acids represented by the compounds utilized in the present invention may be in either the “D” or “L” configurations.
  • glycosidic bonds may be in either alpha- or beta- configuration
  • radiolabeled compound of any of the formulae delineated herein.
  • Such compounds have one or more radioactive atoms (e.g., 3 H, 2 H, 14 C, 13 C, 35 S, 32 P, I 25 I, 131 I) introduced into the compound.
  • radioactive atoms e.g., 3 H, 2 H, 14 C, 13 C, 35 S, 32 P, I 25 I, 131 I.
  • Such compounds are useful for drug metabolism studies and diagnostics, as well as therapeutic applications.
  • prodrug includes compounds with moieties, which can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. ScL 66:1-19; Silverman (2004) The Organic Chemistry of Drug Design and Drug Action, Second Ed., Elsevier Press, Chapter 8, pp. 497-549).
  • the prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent.
  • prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl- amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetyloxym ethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halogen, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amide
  • Substituted refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s).
  • Treating all refer to obtaining a desired pharmacologic and/or physiologic effect, e.g., inhibiting cysteine proteases.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • disease treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a disease or condition from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, e.g., arresting its development; or (c) relieving the disease.
  • “Diagnostic” or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives").
  • false negatives Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed “true negatives.” The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • patient or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. "Sample” is used herein in its broadest sense.
  • a sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
  • “Therapeutically effective amount” means the amount of a compound that, when administered to a patient for inhibiting cysteine proteases and treating the disease, is sufficient to effect such control.
  • the “therapeutically effective amount” will vary depending on the compound, the severity of the condition and the age, weight, etc., of the patient to be treated.
  • Suitable compounds of the invention include, but are not limited to, compounds of the formula (I):
  • R 3 , R 4 is alkyl, aralkyl, heteroalkyl, hydrogen, O-alkyl, O-aryl; wherein P 1 comprises:
  • P 1 is: wherein P 2 comprises:
  • R 5 is H, Me
  • X 1 is OH, NHR 2 ;
  • R 2 H, acyl, -CO alkyl;
  • X 2 halogen, alkyl, alkoxy
  • R6 H, OCH 3 , alkyl, halogen, -N0 2; .
  • a compound of formula (III) comprises:
  • the R, P 1 , P 2 , P 3 , P' 1 , and X are independently selected from the following: hydrogen, formyl, carboxyl, cyano, hydroxy, amino, nitro, thiol, halo and the following optionally substituted moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, alkanoyl, aroyl, heterocycloyl, heteroaroyl, alkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroarylaminocarbonyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyloxy, alkoxy, aryloxy, heterocyclyloxy, alkanoate
  • the above described optionally substituted moieties have the following size ranges: (C 1 -C 20 )alkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, (C 3 -C 10 )cycloalkyl, (C 3 -C 10 )cycloalkenyl, aryl, heterocyclyl, heteroaryl, (C 1 -C 6 )alkanoyl, aroyl, heterocycloyl, heteroaroyl, (C 1 -C 6 )alkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaryloxycarbonyl, (C 1 -C 6 )alkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroarylaminocarbonyl, (C 1 -C 6 )alkoxy, (C 2 -C 6 )alkenyloxy, (C 1 -
  • alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, heterocyclyl, haloalkyl, haloalkenyl and haloalkynyl have the following size ranges: (C 1 -C 20 )alkyl, (C 2 -C 6 )alkenyl, (C 2 - C 6 )alkynyl, (C 3 -C 10 )cycloalkyl, (C 3 -C 10 )cycloalkenyl, (C 3 -C 10 )cycloalkyl(C 1 -C 6 )aIkyl, (C 3 -C 10 )cycloalkenyl(C 1 -C 6 )alkyl, aryl, aryl(C 1 -C 6 )alkyl, (
  • R is selected from the group consisting of methyl, ethyl, i-propyl, i-butyl, t-butyl, sec-butyl, --CH(CH 2 CH 3 ) 2 , allyl, propargyl, cyclopentyl, cyclohexyl, 3-cyclohexenyl, trans-cinnamyl, --CH 2 CN, -CH(CH 3 )CN, ⁇ (CH 2 ) 2 OCH 3 , -CH 2 SCH 3 , -CH 2 CF 3 , a halogen, e.g., chlorine, iodine, bromine or fluorine, R.
  • a halogen e.g., chlorine, iodine, bromine or fluorine
  • sub.5 is independently hydrogen or alkyl, e.g., a substituted or unsubstituted C 1 to C 6 alkyl or cycloalkyl, including, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclohexyl, and the like.
  • R is also contemplated to be a substituted or unsubstituted aryl.
  • the aryl can be a substituted benzyl or substituted phenyl, e.g., including a halo or cyano substituted alkyl, benzyl or phenyl.
  • cysteine proteases include, but not limited to: Cruzain, Falcipain, Rhodesain, Cathepsin B, Cath.B "like", Leishmania Cath. L "like", Leishmania Papain, Cathepsin B, Cathepsin X, Cathepsin L.
  • the compound of formula (II) is substituted with N- alkyl piperdine and N-alkyl piperdine analogs. If different structural isomers are present, and/or one or more chiral centers are present, all isomeric forms are intended to be covered. Enantiomers are characterized by the absolute configuration of their chiral centers and described by the R- and S- sequencing rules of Cahn, Ingold and Prelog. Such conventions are well known in the art (e.g. see Advanced Organic Chemistry, 3 rd edition, ed. March, J,, John Wiley and Sons, New York, 1985).
  • Appropriate pharmaceutically and veterinarily acceptable salts of the compounds include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenes
  • Prodrugs are any covalently bonded compounds which release the active parent drug according to the compounds in vivo.
  • a prodrug may for example constitute an acetal or hemiacetal derivative of the exocyclic ketone functionality present in the (2- alkyl-4-oxo-tetrahydrofuran-3-yl)amide, (2-alkyl-4-oxo-tetrahydrothiophen-3-yl) amide and (2-alkyl-5-oxocyclopentyl)amide scaffold. If a chiral centre or another form of isomeric centre is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereoisomers, are intended to be covered herein.
  • Compounds of the invention containing a chiral centre may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone.
  • the compounds are useful for the in vivo treatment or prevention of diseases in which participation of a cysteine protease is implicated.
  • the protease is a cysteine protease.
  • cysteine protease see, for example, X. Que et ai, Clin. Microbiol. Reviews, 2000, Vol. 13(2): 196-206, incorporated herein in its entirety.
  • a compound for use in medicine especially for preventing or treating diseases in which the disease pathology may be modified by inhibiting a cysteine protease.
  • a compound in the preparation of a medicament for preventing or treating diseases in which the disease pathology may be modified by inhibiting a cysteine protease is provided.
  • cyste protease or “cysteine proteinase” or “cysteine peptidase” intend any enzyme of the sub-subclass EC 3.4.22, which consists of proteinases characterized by having a cysteine residue at the active site and by being irreversibly inhibited by sulfhydryl reagents such as iodoacetate.
  • cysteine proteases form a covalent intermediate, called an acyl enzyme that involves a cysteine and a histidine residue in the active site (Cys25 and Hisl59 according to papain numbering, for example).
  • cysteine protease targets for the present invention include papain, cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16), cathepsin L (EC 3.4.22.15), cathepsin K, cathepsin S (EC 3.4.22.27), cruzain or cruzipain, rhodesain, brucipain, congopain, falcipain and CPB2.8 Delta CTE.
  • Clan CA proteases are characterized by their sensitivity to the general cysteine protease inhibitor, E64 (L-trans-epoxysuccinyl-leucyl-amido (4-guanidino) butane) and by having substrate specificity defined by the S 2 pocket.
  • Cysteine proteases of the present invention can be "cathepsin L-like” or "cathepsin B-like.”
  • a cathepsin L-like cysteine protease shares structural and functional similarity with a mammalian cathepsin L, and comprises an "ERFNIN” motif (Sajid and McKerrow, MoI Biochem Parasitol (2002) 120: 1), Cathepsin L-like cysteine proteases prefer as a substrate the dipeptide sequence -Phe-Arg- 1 -Xaa-.
  • cathepsin L-like cysteine proteases include cathepsin L, cathepsin K, cathepsin S, cruzain, rhodesain and congopain, T. cruzi-L, T. rangeli-L, T. congolense-L, T. brucei-L, P. falciparum-L1, P. falciparum-L 2, P. falciparum-L3, P. vivax-L1, P. cynomolgi-L1 , P. vinckei-L and L. major-h.
  • a cathepsin B-like cysteine protease shares structural and functional similarity with a mammalian cathepsin B, and comprises an "occluding loop" (Sajid and McKerrow, supra).
  • Cathepsin B-like cysteine proteases cleave as a substrate the dipeptide sequences -Arg-Arg- 1 -Xaa- and -Phe-Arg- 1 -Xaa-.
  • Representative cathepsin B-like proteases include cathepsin B, T. cruzi-B, L. mexicana-B and L. major- B.
  • inhibitors refers to inhibitory compounds identified using in vitro and in vivo assays for cysteine protease function.
  • inhibitors refer to compounds that decrease or obliterate the catalytic function of the target cysteine protease, thereby interfering with or preventing the infectious life cycle of a parasite or in other cells where cysteine proteases play a role, such as, for example, the migratory capacity of a cancer cell or an inflammatory cell.
  • In vitro assays evaluate the capacity of a compound to inhibit the ability of a target cysteine to catalyze the cleavage of a test substrate.
  • Cellular assays evaluate the ability of a compound to interfere with the infectious life cycle of a parasite or the migration of a cancer or inflammatory cell ex vivo, while not exhibiting toxicity against the host cell.
  • Cellular assays measure the survival of a parasite-infected cell in culture. Preferred inhibitors allow for extended survival of an infected cell, either by delaying the life cycle of the parasite, or by killing the parasite.
  • In vivo assays evaluate the efficacy of test compounds to prevent or ameliorate disease symptoms, such as those associated with parasitic infection, cancer invasion or growth, or inflammatory cell migration.
  • Inhibitors are compounds that eliminate or diminish the catalytic function of a cysteine protease.
  • preferred inhibitors delay, interfere with, prevent or eliminate the completion of the infectious life cycle of a parasite or the migratory ability of a cancer cell or an inflammation cell. Additionally, preferred inhibitors prevent or diminish a parasitic infection in an individual or the migration of cancer cells or inflammatory cells in an individual, thereby preventing or ameliorating the pathogenic symptoms associated with such infections or the migration of rogue cells.
  • samples, assays, cultures or test subjects comprising a target cysteine protease are treated with a potential inhibitor compound and are compared to negative control samples without the test compound, and positive control samples, treated with a compound known to inhibit the target cysteine protease.
  • Negative control samples are assigned a relative cysteine protease activity level of 100%. Inhibition of a cysteine protease is achieved when the cysteine protease activity relative to the control is about 90%, preferably 75% or 50%, more preferably 25-0%.
  • an amount of compound that inhibits a cysteine protease is “an amount sufficient to inhibit a "cysteine protease", or a “cysteine protease inhibiting amount” of compound, thereby preventing or treating a parasitic infection, inflammation, or cancer invasion or growth in an individual.
  • cysteine proteases function in the normal physiological process of protein degradation in animals, including humans, e.g. in the degradation of connective tissue. However, elevated levels of these enzymes in the body can result in pathological conditions leading to disease. Thus, cysteine proteases have been implicated in various disease states, including but not limited to, infections by Entamoeba histolytica, Pneumocystis carinii, Trypanosoma cruzi, Trypanosoma brucei brucei and Crithidia fascicu ⁇ ata; as well as in osteoporosis, autoimmunity, schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like. See WO-A-9404172 and EP-A-0603873 and references cited in both of them.
  • staphylopain a secreted bacterial cysteine protease from S. aureus called staphylopain has been implicated as a bacterial virulence factor (Potempa, J., et a J. Biol. Chem., 262(6), 2664 2667, 1998).
  • the subject or patient is preferably an animal.
  • the animal is preferably a vertebrate, and more preferably a mammal, avian or fish.
  • the compound or composition may be administered directly to the animal subject and/or indirectly by applying it to its feed.
  • Appropriate animal subjects include those in the wild, livestock (e.g., raised for meat, milk, butter, eggs, fur, leather, feathers and/or wool), beasts of burden, research animals, companion animals, as well as those raised for/in zoos, wild habitats and/or circuses.
  • the animal subject is a mammal (including great apes such as humans).
  • mammalian subjects include primates (e.g., monkeys), bovine (e.g., cattle or dairy cows), porcine (e.g., hogs or pigs), ovine (e.g., goats or sheep), equine (e.g., horses), canine (e.g., dogs), feline (e.g., house cats), camels, deer, antelopes, rabbits, and rodents (e.g., guinea pigs, squirrels, rats, mice, gerbils, and hamsters).
  • primates e.g., monkeys
  • bovine e.g., cattle or dairy cows
  • porcine e.g., hogs or pigs
  • ovine e.g., goats or sheep
  • equine e.g., horses
  • canine e.g., dogs
  • feline
  • Avians include Anatidae (swans, ducks and geese), Columbidae (e.g., doves and pigeons), Phasianidae (e.g., partridges, grouse and turkeys), Thesienidae (e.g., domestic chickens), Psittacines (e.g., parakeets, macaws, and parrots), game birds, and ratites, (e.g., ostriches). Birds treated or protected by the inventive compounds can be associated with either commercial or noncommercial aviculture.
  • Anatidae such as swans, geese, and ducks
  • Columbidae e.g., doves and pigeons, such as domestic pigeons, Phasianidae, e.g., partridge, grouse and turkeys
  • Thesienidae e.g., domestic chickens, Psittacines, e.g., parakeets, macaws, and parrots, e.g., raised for the pet or collector market, among others.
  • fish shall be understood to include without limitation, the Teleosti grouping of fish, i.e., teleosts. Both the Salmoniformes order (which includes the Salmonidae family) and the Perciformes order (which includes the Centrarchidae family) are contained within the Teleosti grouping. Examples of potential fish recipients include the Salmonidae family, the Serranidae family, the Sparidae family, the Cichlidae family, the Centrarchidae family, the three- Line Grunt (Parapristipoma trilineatum), and the Blue-Eyed Plecostomus (Plecostomus spp), among others.
  • inventive compounds include marsupials (such as kangaroos), reptiles (such as farmed turtles) and other economically important domestic animals for which the inventive compounds are safe and effective in treating or preventing parasite infection or infestation.
  • marsupials such as kangaroos
  • reptiles such as farmed turtles
  • other economically important domestic animals for which the inventive compounds are safe and effective in treating or preventing parasite infection or infestation.
  • the invention is useful in the prevention and/or treatment of the disease states mentioned or implied herein.
  • the present invention also is useful in a method of treatment or prevention of diseases caused by pathological levels of cysteine proteases, particularly cysteine proteases of the papain superfamily, which methods comprise administering to an animal, particularly a mammal, most particularly a human, in need thereof a compound of the present invention.
  • the present invention particularly provides methods for treating diseases in which cysteine proteases are implicated, including infections by Entamoeba histolytica; Pneumocystis carinii, Trypanosoma cruzi, Trypanosoma brucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus, foot-and-mouth disease virus and Crithidia fasciculata; as well as in osteoporosis, autoimmunity, schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy and amytrophy.
  • veterinary and human pathogenic protozoas intracellular active parasites of the phylum Apicomplexa or Sarcomastigophora, Trypanosoma, Plasmodia, Leishmania, Babesia and Theileria, Cryptosporidia, Sacrocystida, Amoeba, Coccidia and Trichomonadia.
  • These compounds are also suitable for the treatment of Malaria tropica, caused by Plasmodium falciparum, Malaria tertiana, caused by Plasmodium vivax or Plasmodium ovale and for the treatment of Malaria quartana, caused by Plasmodium malariae.
  • Toxoplasmosis caused by Toxoplasma gondii
  • Coccidiosis caused for instance by Isospora belli
  • intestinal Sarcosporidiosis caused by Sarcocystis suihominis
  • dysentery caused by Entamoeba histolytica
  • Cryptosporidiosis caused by Cryptosporidium parvum
  • Chagas' disease caused by Trypanosoma cruzi
  • sleeping sickness caused by Trypanosoma brucei
  • veterinary pathogenic protozoa like Theileria parva, the pathogen causing bovine East coast fever, Trypanosoma congolense congolense or Trypanosoma vivax vivax, Trypanosoma brucei brucei, pathogens causing Nagana cattle disease in Africa, Trypanosoma brucei evansi causing Surra, Babesia bigemina, the pathogen causing Texas fever in cattle and buffalos, Babesia bovis, the pathogen causing European bovine Babesiosis as well as Babesiosis in dogs, cats and sheep, Sarcocystis ovicanis and ovifelis pathogens causing Sarcocystiosis in sheep, cattle and pigs, Cryptosporidia, pathogens causing Cryptosporidioses in cattle and birds, Eimeria and Isospora species, pathogens causing Coccidiosis in rabbits
  • veterinary pathogenic protozoa like Theil
  • Inhibitors of cruzipain are useful for the treatment of Chagas' disease.
  • the compounds of the invention can be administered to patients for prevention or treatment, in the case of an infection, with combinations of one or more the compounds.
  • the compounds of the invention can be administered to patients for prevention or treatment, in the case of an infection, with combinations of one or more compounds and other protease inhibitors.
  • protease inhibitors include peptidomimetic vinyl sulfone inhibitors and their derivatives. See, for example, CR. Caffrey et al. Molecular & Biochemical Parasitology 1 18 (2001) 62 61-73; Z. B. Mackey et al. Chem. Biol Drug Des 2006; 67: 355-363; Z. B. Mackey et al. J. Biol. Chem. Vol. 279, No. 46, pp. 48426-48433, 2004, which are incorporated by reference, herein.
  • an effective amount of one or more of the compounds may be administered to inhibit the protease implicated with a particular condition or disease.
  • this dosage amount will further be modified according to the type of administration of the compound.
  • parenteral administration of a compound of formulae (I) to (III) are preferred.
  • An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful.
  • the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit a cysteine protease.
  • the compounds may be administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day.
  • the precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
  • Prodrugs of compounds of the present invention may be prepared by any suitable method.
  • the conversion may be effected in accordance with conventional methods.
  • the compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein.
  • a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient.
  • the oral dose would be about 0.5 to about 20 mg/kg.
  • the compounds of this invention which may have good bioavailability, may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect.
  • a pharmaceutical or veterinary composition comprising one or more compounds of formulae (I) to (III) and a pharmaceutically or veterinarily acceptable carrier.
  • Other active materials may also be present, as may be considered appropriate or advisable for the disease or condition being treated or prevented.
  • each of the carriers must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • the compounds described herein are suitable for use in a variety of drug delivery systems described above. Additionally, in order to enhance the in vivo serum half-life of the administered compound, the compounds may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended serum half-life of the compounds. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., U.S. Pat. Nos. 4,235,871 , 4,501,728 and 4,837,028 each of which is incorporated herein by reference. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.
  • the formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation.
  • the formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.
  • Such methods include the step of bringing into association the above defined active agent with the carrier.
  • the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of general formula (I) in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.
  • Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.
  • the term "acceptable carrier" includes vehicles such as common excipients e.g.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring and the like can also be used. It may be desirable to add a coloring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface -active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
  • compositions suitable for oral administration include lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
  • parenteral formulations will generally be sterile.
  • inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
  • Example 1 Synthesis of compound of formula I. l-Boc-4-cyclopropylpiperazine (3). To a solution of 1-Boc-piperazine (1, 0.867 g, 4.65 mmol) in methanol (10 niL) were added [(1- ethoxycyclopropyl)oxy]triraethylsilane (2, 1.86 rnL, 9.3 mmol), acetic acid (0.83 mL, 14.5 mmol), and NaCNBH 3 (7 mL, 1 M solution in THF, 7 mmol). The mixture was stirred at 45 °C for 4 days and concentrated to dryness.
  • Phenylalanine t-butyl ester (4, 0.295 g, 1.14 mmol) was dissolved CH 2 Cl 2 (12 mL) and a saturated solution of aqueous NaHCO 3 (12 mL) at 0 °C and the mixture was stirred vigorously for 20 min. A solution of triphosgene (0.113 g, 0.38 mmol) in 2 mL of methylene chloride was added and the reaction mixture was stirred at 0 °C for another 30 min. The layers were separated and the aqueous layer was further extracted twice with methylene chloride (5 mL).
  • Proteinase activity assay The proteinase activity was determined by measuring the release of the fluorescent leaving group, 4-amino-7 -methyl coumarin (AMC) from synthetic peptide substrates (Bachem, Torrance, CA). Substrates tested included those commonly cleaved by cysteine proteinases, including Carboxybenzyloxy-Arginine- Arginine-4-amino-7-methyl coumarin (Z-Arg-Arg-AMC), Z-Ala-Arg-Arg-AMC, Z-Phe- Ala-Arg-AMC, and Z-Phe-Arg-AMC at a final concentration of 10 ⁇ M in 25 mM Tris, 2 mM EDTA, 2 mM DTT (or 5 mM cysteine), pH 7.5, in a Fluoroskan-Ascent fluorometer (Labsystems, USA).
  • AMC 4-amino-7 -methyl coumarin
  • Enzyme activity, initial velocity, and relative fluorescence units were calculated with Ascent software.
  • REU relative fluorescence units
  • Protocol enzyme concentration, instrument: 96well fluorimeter; inhibitor ( ⁇ M): 0.01 to 10; pre -incubation time temperature: 5 min @25C; substrate: Km l( ⁇ M) ZFR AMC (lOuM); buffer: 10OmM NaAc pH5.5, 5 mM DTT; comments: DMSO ⁇ 1%. Cruzain (rec) k inact /K i , k ass , k obs /I. Protocol: enzyme concentration, source: rec, (4-
  • T brucei rhodesiense Rhodesain (rec) k inact /K,, k ass , k 0bs /I.
  • Protocol enzyme concentration, source: rec ,(4-8nM); instrument: 96well fluorimeter; inhibitor ( ⁇ M): 0.01 to 10; pre-incubation time, temperature: 0 min; substrate: Km l( ⁇ M) ZFR AMC (5 ⁇ M); buffer: 10OmM NaAc pH5.5, 5 mM DTT; comments: DMSO ⁇ 1%, Prism for curve fit.
  • T. cruzi In vitro assay: Protocol: Irradiated J774 macrophages cultured in RPMI- 1640 medium with 5% heat inactivated fetal calf serum are plated onto 12 well tissue culture plates for 24h. After infection with 10 5 T. cruzi (Y strain) trypomastigotes per well for 2 hours, monolayers are washed and medium replaced with the addition of cysteine protease inhibitor (CPI). Inhibitor stocks are made to 10 mM in DMSO and diluted prior to use. Unless otherwise stated, inhibitors are tested at 10 ⁇ M by triplicate. All assays include untreated, Kl 1777-treated, and uninfected macrophage controls. Medium is replaced every 48h. Under these culture conditions, T.
  • CPI cysteine protease inhibitor
  • Treatment duration is up to 27 days as such regime results in cure of macrophages treated with 10 ⁇ M Kl 1777 (Positive control).
  • Macrophages are subsequently cultured in normal medium for up to 40 days to elucidate if effective CPIs are cidal (cure host macrophages) or trypanostatic (delay intracellular cycle of the parasite). Only effective trypanocidal CPI are tested further in animal models of acute Chagas' disease.
  • T. cruzi. Acute Chagas' Disease. Protocol Three to four week old female C3H mice weighing normally between 17-20 g are used. Animals (5 per lot) are infected with 10 6 trypomastigotes of the Y strain or the CA-I/72 clone via i.p. and treated twice daily with 30-100 mg compound/kg/weight in two daily doses via i.p. or oral gavage as indicated. Compounds are solubilized in 100 microliters (30-40% DMSO: 60-70% dH 2 O). Controls always include infected, untreated animals and a lot treated daily with 100 mg Kl 1777 (N-Pip-F-hF-VS-Phenyl)/kg weight in two daily doses.
  • Treatment is initiated 12-24 h post-infection and continued until cure, death of the animals, or for up to 27 days depending on pharmaco-kinetic and in vitro results.
  • Parasitemias are determined at the end of the experiment when animals are euthanized.
  • Tissues processed for histopahology include skeletal muscle, heart, liver, spleen, and colon.
  • Blood (5-50 microliters) is used for hemocultures. Hemocultures are considered negative if no parasites are observed for 60 days.
  • Treatment is considered effective if life expectancy is increased in treated animals vs. untreated controls, symptoms of acute Chagasic infection are absent, and histopathological observation shows normal tissues and no parasites. Compunds are considered toxic if life expectancy is lower than untreated controls (negative values).
  • PCR of blood and tissues is performed to confirm effectiveness of treatment and/or cure of treated animals. Results are expressed as survival (days) of treated animals minus untreated controls.
  • T. brucei in vivo drug screen survival mouse.

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Abstract

La présente invention concerne des composés, leurs dérivés et analogues, qui sont des inhibiteurs puissants des protéases. Ces composés peuvent être utilisés pour traiter des maladies provoquées par des organismes étrangers, par exemple, des champignons, des bactéries, des protozoaires et des virus.
PCT/US2008/061422 2007-04-24 2008-04-24 Inhibiteurs de la cystéine protéinase et leurs utilisations Ceased WO2008134429A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5317086A (en) * 1992-03-09 1994-05-31 The Regents Of The University Of California Cysteine proteinase inhibitors and inhibitor precursors
US6635621B1 (en) * 1999-07-31 2003-10-21 Naeja Pharmaceutical Inc. Cysteine protease inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5317086A (en) * 1992-03-09 1994-05-31 The Regents Of The University Of California Cysteine proteinase inhibitors and inhibitor precursors
US6635621B1 (en) * 1999-07-31 2003-10-21 Naeja Pharmaceutical Inc. Cysteine protease inhibitors

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