WO2008134429A1

WO2008134429A1 - Cysteine proteinase inhibitors and uses thereof

Info

Publication number: WO2008134429A1
Application number: PCT/US2008/061422
Authority: WO
Inventors: William Roush; Yen Ting Chen
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2007-04-24
Filing date: 2008-04-24
Publication date: 2008-11-06
Anticipated expiration: 2009-10-24

Abstract

Compounds described herein, derivatives 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.

Description

CYSTEINE PROTEINASE INHIBITORS AND USES THEREOF

FIELD OF THE INVENTION

This invention relates to the proteinase inhibitor compounds, compositions and methods of preventing and treatment of diseases.

BACKGROUND

Classically, the identification of enzymes found to be crucial for the establishment or propagation of an infectious disease has been instrumental in the development of successful drugs such as antivirals (e.g. HIV aspartyl protease inhibitors) and anti-bacterials (e.g. β-lactam antibiotics). The search for a similar Achilles heel in parasitic infections has examined numerous enzymes (e.g. parasitic dihydrofolate reductase, see Chowdhury, S. F. et al, J. Med. Chem., 42(21), 4300-4312, 1999; trypanothione reductase, see Li, Z. et al, Bioorg. Med. Chem. Lett., 11(2), 251 -254, 2001; parasitic glyceraldehydes-3 -phosphate dehydrogenase, see Aranov, A. M. et al, J. Med. Chem,, 41(24), 4790-4799, 1998). A particularly promising area of research has identified the role of cysteine proteases, encoded by the parasite, that play a pivotal role during the life cycle of the parasite (McKerrow, J. H., et al, Bioorg. Med. Chem., 1, 639- 644, 1999). Proteases form a substantial group of biological molecules which to date constitute approximately 2% of all the gene products identified following analysis of several genome sequencing programs (e.g. see Southan, C. J. Pept. Sci, 6, 453-458, 2000). 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. 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. One such enzyme, 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. 7, 1143-1 156, 2001; (b) Caffrey, C. R. et al, Curr. Drug Targets, 1, 155- 162, 2000). Although the precise biological role of cruzipain within the parasite's life cycle remains unclear, elevated cruzipain messenger RNA levels in the epimastigote developmental stage indicate a role in the nutritional degradation of host-molecules in lysosomal-like vesicles (Engel, J. C. et al, J. Cell. Sci, 1597-1606, 1998). The validation of cruzipain as a viable therapeutic target has been achieved with increasing levels of complexity. Addition of a general cysteine protease inhibitor, Z-Phe-Ala-FMK to Trypanosoma cruzi-infected mammalian cell cultures blocked replication and differentiation of the parasite, thus arresting the parasite life cycle (Harth, G., et al, MoI. Biochem. Parasitol. 58, 17-24, 1993). Administration of a vinyl sulfone-based inhibitor in a Trypanosoma cruzi-infected murine animal model not only rescued the mice from lethal infections, but also produced a complete recovery (Engel, J. C. et al, J. Exp. Med, 188(4), 725-734, 1998). Numerous other in vivo studies have confirmed that infections with alternative parasites such as Leishmania major (Selzer, P. M. et al, Proc. Natl. Acad. Sci. U.S.A., 96, 1 1015-1 1022, 1999), Schistosoma mansoni and Plasmodium falciparum (Olson, J. E. et al, Bioorg. Med. Chem., 7, 633-638, 1999) can be halted or cured by treatment with cysteine protease inhibitors. The trypanosomal family of parasites has a substantial worldwide impact on human and animal healthcare (McKerrow, J. H., et al, Ann. Rev. Microbiol. 47, 821-853, 1993). One parasite of this family, Trypanosoma cruzi, is the causative agent of Chagas' disease, which affects in excess of twenty million people annually in Latin and South America, is the leading cause of heart disease in these regions and results in more than 45,000 deaths per annum (Centers for Disease Control and prevention website). In addition, with the increase in migration of the infected population from rural to urban sites and movements from South and Central America into North America, the infection is spreading via blood transfusions, and at birth. The present treatments of choice for Trypanosoma cruzi infection, nifurtimox and benznidazole (an NADH fumarate reductase inhibitor, Turrens, J F, et al, MoI Biochem Parasitol. , 82(1), 125-129, 1996) are at best moderately successful, achieving about 60% cure during the acute phase of infection (see Docampo, R. Curr. Pharm. Design, 1, 1157-1 164, 2001 for a general discussion) whilst not being prescribed at all during the chronic phase where cardiomyopathy associated heart failure often occurs (Kirchhoff, L. V. New Engl. J. Med, 329, 639-644, 1993). Additionally, these two drugs have serious adverse toxic effects, requiring close medical supervision during treatment, and have been shown to induce chromosomal damage in chagastic infants (Gorla, N. B. et al, Mutat. Res. 206, 217-220, 1988). Other parasites, such as Entamoeba histolytica infect more than 50 million people with 1 10,000 deaths. Amebiasis is defined as the intestinal or extraintestinal infection with E. histolytica (X. Que et al., Clin. Microbiol. Reviews, 2000, Vol. 13(2): 196-206). Released cysteine proteinases play a fundamental role in invasion of target tissues by E, histolytica trophozoites. Analysis of the E. histolytica genome reveals that 40 genes encode cysteine proteinases. Studies on the expression of the cysteine proteinase genes have shown that only three, ehcpl, ehcp2, andehcp5, account for more than 90% of the cysteine proteinase specific transcripts and more than 95% of secreted amebic proteinases in vitro. The intracellular localization is known for only a few amebic cysteine proteinases. 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.

There is a strong medical need for new effective drugs for the chemotherapeutic treatment of infection and other diseases in which the pathology or the disease is treated or preventable by use of protease inhibitors.

SUMMARY

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.

In a preferred embodiment, a compound of the following formula (I) comprises:

In a preferred embodiment, the compound is in a pharmaceutical composition. In another preferred embodiment, a compound of the following formula (II) comprises:

wherein P₁ comprises:

wherein P'j comprises:

wherein R¹ is H, OCH₃, alkyl, halogen, -NO₂; and X = halogen, alkyl, alkoxy,

wherein X¹ is OH, NHR₂; R² H, acyl, -CO alkyl; X¹ = halogen, alkyl, alkoxy,

wherein R³ is alkyl, aralkyl, heteroalkyl, hydrogen, O-alkyl, O-aryl; wherein P₁ comprises:

wherein P₂ comprises:

and R⁵ is H, Me;

wherein X¹ is OH, NHR²; R² = H, acyl, -CO alkyl; X² = halogen, alkyl, alkoxy R6 = H, OCH₃, alkyl, halogen, -N0_2;.

wherein X² = O, S, NH; wherein P₃ comprises:

wherein R⁷ = CH₂R⁸ (1 to 6 C); R⁷ = CHR⁹ ₂ (branched) ( e.g. -CHMeAr); R⁷ = cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, R⁷ = t-Bu, -CF₃, -CH₂CF₃, - CH₂(CF₂)₂CF_3j -CH₂CH₂CF₃; X³ = -OR (carbamates), -OCH₂Ph, -OCH₂pyridyl, - OCH₂heteroaryl; alkyl, aryl or heteroaryl (amides), substituted aryl, heteroaryl, alkyl, aralkyl.

In another preferred embodiment, 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, heteroaryloate, alkylsulfonate, aryl sulfonate, heterocyclylsulfonate, heteroarylsulfonate, alkylamino, alkenylamino, arylamino, heterocyclylamino, heteroarylamino, alkylcarbonylamino, arylcarbonylamino, heterocyclylcarbonylamino, heteroarylcarbonylamino, alkylthio, alkenylthio, cycloalkylthio, cycloalkenylthio, arylthio, heterocyclylthio, heteroarylthio, alkylsulfinyl, alkenylsulfinyl, cycloalkylsulfinyl, cycloalkenylsulfinyl, arylsulfinyl, heterocyclylsulfinyl, heteroarylsulfmyl, alkylsulfonyl, alkenylsulfonyl, cycloalkylsulfonyl, cycloalkenylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, heteroarylsulfonyl, haloalkyl, haloalkenyl, haloalkynyl, haloalkoxy, haloalkenyloxy, haloalkylsulfonate, haloalkylcarbonylamino, haloalkylthio, haloalkylsulfinyl and haloalkylsulfonyl; hydrogen, halogen, cyano and the following optionally substituted moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, heterocyclyl, haloalkyl, haloalkenyl and haloalkynyl; and the following optionally substituted moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, arylalkenyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, alkanoyl, aroyl, heterocycloyl, heteroaroyl, cyanoalkyl, alkoxyalkyl, cycloalkoxyalkyl, aryloxyalkyl, alkylthioalkyl, cycloalkylthioalkyl, arylthioalkyl, alkylsulfinylalkyl, cycloalkylsulfinylalkyl, arylsulfinylalkyl, alkylsulfonylalkyl, cycloalkylsulfonylalkyl, arylsulfonylalkyl, haloalkyl, haloalkenyl and haloalkynyl.

In other preferred embodiments, the R groups comprise hydrogen, (C₁-C₆)alkyl or (C₁-C₆)alkoxy; halo or (C₁-C₆)haloalkyl; hydrogen, halo or (C₁-C₆)haloalkyl; (C₁- C₆)alkyl, (C₃-C₁₀)cycloalkyl or (optionally substituted)aryl; (C₂-C₆)alkenyl, (C₂- C₆)alkynyl, (C₃-C₁₀)cycloalkyl, (C₃-C₁₀)cycloalkenyl, (C₃-C₁₀)cycloalkyl(C₁-C₆)alkyl, (C₃-C₁₀)cycloalkenyl(C[-C₆)alkyl, and the following optionally substituted moieties: aryl, aryl(C₁-C₆)alkyl, aryl (C₂ -C₆)alkenyl, heterocyclyl(C₁-C₆)alkyl, heteroaryl, heteroaryl(C₁-C₆)alkyl, cyano(C₁-C₆)alkyl, (C₁-C₆)alkoxy(C,-C₆)alkyl, aryloxy(C₁- C₆)alkyl, (C₁-C₆)alkylthio(C₁-C₆)alkyl, (C,-C₆)haloalkyl and (C₂-C₆)haloalkenyl.

In another preferred embodiment, the R group comprises hydrogen, methyl or methoxy.

In another preferred embodiment, the R group comprises hydrogen, fluorine, chlorine, fluorine, bromine, cyano, trifluoromethoxy or trifluoromethyl.

In another preferred embodiment, 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.

In another preferred embodiment, R is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, i-butyl, t-butyl, sec-butyl, phenyl, — CH(CH₂CH₃)₂, allyl, propargyl, cyclopentyl, cyclohexyl, 3-cyclohexenyl, trans-cinnamyl, -CH₂CN, - CH(CH₃)CN, -(CHJ)₂OCH₃, -CH₂SCH₃, --CH₂CF₃.

In another preferred embodiment, R groups comprise hydrogen or (C₁-C₆)alkyl; (C₁-C₆)haloallyl; (C₁-C₆)haloalkyl; (optionally substituted)aryl; cyano, halo or (C₁-C₆)haloalkyl; (optionally substituted)aryloxy, (optionally substituted)heteroaryloxy, (C₃- C₁₀)cycloalkyl, (CrC₆)alkoxycarbonyl, cyano, (C₁-C₆)alkoxy, nitro, halo and (C₁- C₆)haloalkyl.

In another preferred embodiment, R, X, P₁, P₂, P₃, P ι' 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, arylcarbonylaminoalkyl, heterocyclycarbonylaminoalkyl, heteroarylcarbonylaminoalkyl, alkylsulfonylalkyl, arylsulfonyl alkyl, heterocyclylsulfonyl alkyl, heteroarylsulfonylalkyl, alkanoyl, aroyl, heterocycloyl, heteroaroyl, alkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaryloxycarbonyl, alkyl am inocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroarylaminocarbonyl, alkylaminothiocarbonyi, ary laminothi ocarbony 1, heterocycly lam inothiocarbony 1 , heteroary 1 aminothiocarbonyl , alkylsulfonyl, arylsulfonyl, heterocyclylsulfonyl and heteroarylsulfonyl.

In another preferred embodiment, a compound of formula (III) comprising:

salts, hydrates, solvates, complexes, analogs and prodrugs thereof. In another preferred embodiment, a pharmaceutical composition comprises any one or more compounds of formulae (I) to (III).

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In one aspect, the protease is a cysteine protease.

In another preferred embodiment, the organism is a parasite, bacterium, virus, fungus, or protozoan.

In another preferred embodiment, any one or more of compounds of formula (I) to (III) are co-administered to patients, with other protease inhibitors or drugs.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims. The above and further advantages of this invention may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which:

Figure 1 is a schematic representation showing an outline of synthesis of the compound of formula I.

DETAILED DESCRIPTION Compounds of formula (I) to (III), derivatives, prodrugs, metabolites, stereoisomers, tautomers, substituents and analogs thereof are potent proteases inhibitors, in particular, cysteine proteases. These compounds can be used to treat diseases such as those diseases caused by foreign organisms, e.g. fungi, bacteria, protozoa, and virus . Definitions

"Compounds" as used herein 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. Accordingly, when stereochemistry at chiral centers is not specified, the chemical structures depicted herein encompass all possible configurations at those chiral centers including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures and their racemates. 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, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O and ¹⁸O.

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.

Further, it should be understood, when partial structures of the compounds are illustrated, that 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, 4-toluene sulfonic acid, camphorsulfonic acid, A- methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like, made by conventional chemical means; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like, made by conventional chemical means. 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-naphthalenesulphonate, benzene sulphonate, p- chlorobenzenesulphonate and p-toluene sulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids. Salts which are not pharmaceutically or veterinarily acceptable may still be valuable as intermediates.

"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. Representative 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.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, and branched-chain alkyl groups. The term 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. In preferred embodiments, 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.

Moreover, the term 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. Such 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, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

The term "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)).

The terms "alkoxy," "aminoalkyl" and "thioalkoxy" refer 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. The terms "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. For example, the invention contemplates cyano and propargyl groups.

The term "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.

The term "aryl" as used herein, 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.

Those 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, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, halogenated alkyl (including trifluorom ethyl, difluoromethyl and fluroromethyl), halogenated alkoxy (including trifluoromethoxy, difluoromethoxy and fluroromethoxy), cyano, azido, heterocyclyl, alkylaryl, arylalkyl or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).

The term "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. The term "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. In one embodiment, 0, 1 , 2, 3, or 4 atoms of 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.

The terms "cyclyl" or "cycloalkyl" refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls). In some cases, 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), amϊdino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamide, nitro, halogenated alkyl (including trifluoromethyl, difluoromethyl and fluroromethyl), halogenated alkoxy (including trifluoromethoxy, difluoromethoxy and fluroromethoxy), cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety. The term "haloalkyl" is intended to include alkyl groups as defined above that are mono-, di- or polysubstituted by halogen, e.g., fluoromethyl and trifluoromethyl.

The term "halogen" designates -F, -Cl, -Br or -I.

The term "hydroxyl" means -OH. The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.

The term "methyl" refers to a CH₃ group.

The term "mercapto" refers to a SH group.

The term "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.

The term "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.

The term "diastereomers" refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.

The term "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."

The term "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.

Furthermore the indication of configuration across a carbon-carbon double bond can be "Z" referring to what is often referred to as a "cis" (same side) conformation whereas "E" refers to what is often referred to as a "trans" (opposite side) conformation. Regardless, both configurations, cis/trans and/or Z/E are contemplated for the compounds for use in the present invention.

With respect to the nomenclature of a chiral center, the terms "d" and "1" configuration are as defined by the IUPAC Recommendations. As to the use of the terms, diastereomer, racemate, epimer and enantiomer, these will be used in their normal context to describe the stereochemistry of preparations.

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. Similarly glycosidic bonds may be in either alpha- or beta- configuration

Another aspect is a radiolabeled compound of any of the formulae delineated herein. Such compounds have one or more radioactive atoms (e.g., ³H, ²H, ¹⁴C, ¹³C, ³⁵S, ³²P, I²⁵I, ¹³¹I) introduced into the compound. Such compounds are useful for drug metabolism studies and diagnostics, as well as therapeutic applications.

The term "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. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of 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 amides, di-lower alkyl amides, and hydroxy amides. Other prodrug moieties include propionoic and succinic acid esters, acyl esters and substituted carbamates. Prodrugs which are converted to active forms through other mechanisms in vivo are also included.

"Substituted" refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s).

"Treat", "treating" and "treatment" 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. For embodiments of the invention involving "disease treatment" as used herein 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"). 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. The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, 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. Compounds

Suitable compounds of the invention include, but are not limited to, compounds of the formula (I):

The above formula can comprise numerous substitutions. For example:

Wherein P₁ is

Wherein P¹I is:

wherein R¹ is H, OCH₃, alkyl, halogen, -NO_2; and X = halogen, alkyl, alkoxy;

wherein X' is OH, NHR²; R²= H, acyl, -CO alkyl; X¹ = halogen, alkyl, alkoxy,

wherein R³, R⁴ is alkyl, aralkyl, heteroalkyl, hydrogen, O-alkyl, O-aryl; wherein P₁ comprises:

Wherein P₁ is: wherein P₂ comprises:

and R⁵ is H, Me;

wherein X² = O, S, NH; wherein P₃ comprises:

wherein R⁷ = CH₂R⁸ (1 to 6 C); R⁷ = CHR⁹ ₂ (branched) ( e.g. -CHMeAr); R⁷ = cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, R⁷ = t-Bu, -CF₃, -CH₂CF₃, - CH₂(CF₂)₂CF₃, -CH₂CH₂CF₃; X³ = -OR (carbamates), -OCH₂Ph, -OCH₂pyridyl, - OCH₂heteroaryl; alkyl, aryl or heteroaryl (amides), substituted aryl, heteroaryl, alkyl, aralkyl. In another preferred embodiment, a compound of formula (III) comprises:

Other examples of compounds include:

Wherein:

In another preferred embodiment, the R, P₁, P₂, P₃, 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, aryloate, heterocyclyloate, heteroaryl oate, alkyl sulfonate, arylsulfonate, heterocyclylsulfonate, heteroarylsulfonate, alkylamino, alkenylamino, arylamino, heterocyclylamino, heteroarylamino, alkylcarbonylamino, arylcarbonylamino, heterocyclyicarbonylamino, heteroarylcarbonylamino, alkylthio, alkenylthio, cycloalkylthio, cycloalkenylthio, arylthio, heterocyclylthio, heteroarylthio, alkylsulfinyl, alkenylsulfinyl, cycloalkylsulfinyl, cycloalkenylsulfinyl, arylsulfϊnyl, heterocyclylsulfinyl, heteroarylsulfinyl, alkylsulfonyl, alkenylsulfonyl, cycloalkylsulfonyl, cycloalkenylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, heteroarylsulfonyl, haloalkyl, haloalkenyl, haloalkynyl, haloalkoxy, haloalkenyloxy, haloalkylsulfonate, haloalkylcarbonylamino, haloalkylthio, haloalkylsulfinyl and haloalkylsulfonyl. Preferably, the above described optionally substituted moieties have the following size ranges: (C₁-C₂₀)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₁₀)cycloalkyl, (C₃-C₁₀)cycloalkenyl, aryl, heterocyclyl, heteroaryl, (C₁-C₆)alkanoyl, aroyl, heterocycloyl, heteroaroyl, (C₁-C₆)alkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaryloxycarbonyl, (C₁-C₆)alkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroarylaminocarbonyl, (C₁-C₆)alkoxy, (C₂-C₆)alkenyloxy, (C₃-C₁₀)cycloalkoxy, (C₃-C₁₀)cycloalkenyloxy, (C₁- C₆)alkoxy(C₁-C₆)alkoxy, aryloxy, heterocyclyloxy, (C₁-C₆)alkanoate, aryloate, heterocyclyloate, heteroaryloate, (C₁-C₆)alkylsulfonate, arylsulfonate, heterocyclylsulfonate, heteroarylsulfonate, (C₁-C₆)alkylamino, (C₂-C₆)alkenylamino, arylamino, heterocyclylamino, heteroarylamino, (C₁-C₆)alkylcarbonylamino, arylcarbonylamino, heterocyclyicarbonylamino, heteroarylcarbonylamino, (C₁- C₆)alkylthio, (C₂-C₆)alkenylthio, (C₃-C₁₀)cycloalkylthio, (C₃-C₁₀)cycloalkenylthio, arylthio, heterocyclylthio, heteroarylthio, (C₁-C₆)alkylsulfinyl, (C₂-C₆)alkenylsulfinyl, (C₃-C₁₀)cycloalkylsulfinyl, (C₃-C₁₀)cycloalkenylsulfinyl, arylsulfinyl, heterocyclylsulfinyl, heteroarylsulfinyl, (C₁-C₆)alkylsulfonyl, (C₂-C₆)alkenylsulfonyl, (C₃-C₁₀)cycloalkylsulfonyl, (C₃-C₁₀)cycloalkenylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, heteroarylsulfonyl, (C₁-C₆)haloalkyl, (C₂-C₆)haloalkenyl, (C₂- C₆)haloalkynyl, (C₁-C₆)haloalkoxy, (C₂-C₆)haloalkenyloxy, (Q-Ce^aloalkylsulfonate, (C₁-C₆)haloalkylcarbonylamino, (C₁-C₆)haloalkylthio, (C₁-C₆)haloalkylsulfinyl and (C₁-C₆)haloalkylsulfinyl .

Optionally substituted moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, arylalkyl, heterocyclyl, haloalkyl, haloalkenyl and haloalkynyl have the following size ranges: (C₁-C₂₀)alkyl, (C₂-C₆)alkenyl, (C₂- C₆)alkynyl, (C₃-C₁₀)cycloalkyl, (C₃-C₁₀)cycloalkenyl, (C₃-C₁₀)cycloalkyl(C₁-C₆)aIkyl, (C₃-C₁₀)cycloalkenyl(C₁-C₆)alkyl, aryl, aryl(C₁-C₆)alkyl, (C,-C₆)haloalkyl, (C₂- C₆)haloalkenyl and halo(C₂-C₆)alkynyl; heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, alkanoyl, aroyl, heterocycloyl, heteroaroyl, cyanoalkyl, alkoxyalkyl, cycloalkoxyalkyl, aryloxyalkyl, alkylthioalkyl, cycloalkylthioalkyl, arylthioalkyl, alkylsulfinylalkyl, cycloalkylsulfinylalkyl, arylsulfinylalkyl, alkylsulfonylalkyl, cycloalkylsulfonylalkyl, arylsulfonylalkyl.

In another preferred embodiment, R is selected from the group consisting of methyl, ethyl, i-propyl, i-butyl, t-butyl, sec-butyl, --CH(CH₂CH₃)₂, allyl, propargyl, cyclopentyl, cyclohexyl, 3-cyclohexenyl, trans-cinnamyl, --CH₂CN, -CH(CH₃)CN, ~ (CH₂)₂OCH₃, -CH₂SCH₃, -CH₂CF₃, a halogen, e.g., chlorine, iodine, bromine or fluorine, R. sub.5 is independently hydrogen or alkyl, e.g., a substituted or unsubstituted C₁ to C₆ 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. For example, 2- methylbutyl, 2-methylpropyl, 2-chlorobenzyl, 4-chlorobenzyl, 2,3-dichIorobenzyl, 2,4- dichlorobenzyl, 4-bromobenzyl, 3,4-dichlorobenzyl, 4-cyanobenzyi, 2,4-difluorobenzyl, 3-chlorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl, 4-cyanophenyl, 3,4- dichlorophenyl, 2-methylphenyl, 4-methylphenyl, 3-chloro-4-fluorophenyl, 3,4- difluorophenyl, cyanomethyl, propyl, e.g., 1- or 2-propyl, cycloalkyls, including, e.g., cyclopentyl, cyclohexyl, cyclohexylmethyl, 3-cyclohexenyl, alkenyls, e.g., 1 - or 2- propenyl, 3-pentyl, ethyl [single isomer or isomeric mixture of syn(Z) or anti(E) isomer], as well as trihaloaryl moieties, e.g., 2-(trifluoromethyl)benzyl, 3-(trifiuoromethyl)benzyl, 4-(trifluoromethyl)benzyl, 2,4-bis(trifluoromethyl)benzyl, and the like.

The compounds are useful for inhibiting proteases such as cysteine proteases. Examples of 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.

In one preferred embodiment, 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-naphthalenesulphonate, benzene sulphonate, p- chlorobenzenesulphonate and p-toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids. Salts which are not pharmaceutically or veterinarily acceptable may still be valuable as intermediates.

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. Treatment of Disease

The compounds are useful for the in vivo treatment or prevention of diseases in which participation of a cysteine protease is implicated. In one preferred embodiment, the protease is a cysteine protease. For a review of cysteine protease, see, for example, X. Que et ai, Clin. Microbiol. Reviews, 2000, Vol. 13(2): 196-206, incorporated herein in its entirety.

In one embodiment of the invention, there is provided 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. In another embodiment, there is provided the use of 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.

The terms "cysteine 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. Mechanistically, in catalyzing the cleavage of a peptide amide bond, 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). Representative 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 S₂ 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-. Representative 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" or "inhibition" of cysteine proteases refers to inhibitory compounds identified using in vitro and in vivo assays for cysteine protease function. In particular, 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. Further, 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.

To examine the extent of inhibition, 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 (not treated with a test compound), 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, as described above, 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.

Certain 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 fascicuϊata; 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.

Additionally, 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.

In a particular embodiment, the animal subject is a mammal (including great apes such as humans). Other 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). 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. These include e.g., 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.

For purposes of the present invention, the term "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. Other animals are also contemplated to benefit from the inventive compounds, including 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.

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.

Other examples include 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. They are also suitable for the treatment of 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 rhodesiense or gambiense, the cutaneous and visceral as well as other forms of Leishmaniosis. They are also suitable for the treatment of animals infected by 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, cattle, sheep, goats, pigs and birds, especially in chickens and turkeys. In the treatment of these diseases, the compounds of the present invention may be combined with other agents.

Inhibitors of cruzipain, particularly cruzipain-specific compounds, are useful for the treatment of Chagas' disease.

In another preferred embodiment, 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.

In another preferred embodiment, 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. Examples 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. In accordance with this invention, an effective amount of one or more of the compounds may be administered to inhibit the protease implicated with a particular condition or disease. Of course, this dosage amount will further be modified according to the type of administration of the compound. For example, to achieve an "effective amount" for acute therapy, 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. Typically, 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. For those compounds in which the prodrug moiety is a ketone functionality, specifically ketals and/or hemiacetals, 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. Typically, 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. Preferably the oral dose would be about 0.5 to about 20 mg/kg.

No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention. 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.

In another preferred embodiment, there is provided 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.

The carrier, or, if more than one be present, 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. In general, 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. For compositions for oral administration (e.g. tablets and capsules), 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.

Other formulations 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.

Embodiments of 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.

EXAMPLES

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 NaCNBH₃ (7 mL, 1 M solution in THF, 7 mmol). The mixture was stirred at 45 °C for 4 days and concentrated to dryness. The residue was treated with 0.25 N HCl (30 mL) and washed three times with ethyl acetate (20 mL). The aqueous layer was basified with a concentrated K₂CO₃ solution and extracted three times with ethyl acetate (20 mL). The organic layer was washed with brine (40 mL), dried over Na₂SO₄, filtered, and concentrated to dryness to give 0.601 g (57%) of the title compound as a white solid which was used without further purification. ¹H NMR (400 MHz, CDCl₃) δ 3.34 (t, J= 5.2 Hz, 4 H), 2.55 (t, J- 4.8 Hz, 4 H), 1.60 (ddd, J= 10.0, 6.4, 3.6 Hz, 1 H), 1.46 (s, 9 H), 0.46 (m, 2 H), 0.42 (m, 2 H).

Cpp-Phe-OtBu (5). Piperazine 3 (0.288 g, 1.14 mmol) was dissolved in CH₂Cl₂ (4 mL) and TFA (4 mL) and the solution was stirred in an ice bath for 1.5 h. The solvent was removed under reduced pressure and excess trifluoroacetic acid was removed by repeated evaporation from toluene in vacuo. Without further purification, the crude TFA salt was used in the next reaction.

Phenylalanine t-butyl ester (4, 0.295 g, 1.14 mmol) was dissolved CH₂Cl₂ (12 mL) and a saturated solution of aqueous NaHCO₃ (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). The combined layers were dried over Na₂SO₄, filtered, and added to the crude 1 -cyclopropylpiperadine TFA salt prepared above at 0 °C. N - methylmorpholine (0.150 mL, 1.36 mmol) was added and the solution was stirred for 16 h. After removing the solvent in vacuo, the residue was purified by flash column chromatography (2-6% methanol in methylene chloride) to yield 0.401 g (94%) of the title compound as colorless foam: ¹H NMR (400 MHz, CDCl₃) δ 7.22-7.29 (m, 3 H), 7.14-7.17 (m, 2 H), 4.93 (d, J= 6.8 Hz, 1 H), 4.68 (dd, J= 13.0, 5.5 Hz, 1 H), 3.36 (br s, 4 H), 3.10 (d, J = 6 Hz, 2 H), 2.65 (br s, 4 Hz), 1.50 (m, 1 H), 1.41 (s, 9 H), 0.53 (br s, 4 H); ¹³C NMR (100 MHz, CDCl₃) δ 171.5, 156.0, 136.3, 129.5, 128.4, 127.0, 82.5, 54.6, 52.6, 41.4, 40.1, 38.4, 28.0, 4.2; LRMS (ES+) m/z for C₂₁H₃₁NaN₃O₃ [M+Na]⁺ calcd 396.2258, found 396.2261.

Compound Formula (I) (8). Piperazine 5 (0.378 g, 1.01 mmol) was dissolved in

40% trifluoroacetic acid in methylene chloride (6 mL) and the solution was stirred in an ice bath for 6 h. The solvent was removed and the excess acid was removed by repeated evaporation from toluene in vacuo. The crude acid (6) was used in the next reaction without further purification. Vinyl sulfone 7 (0.406 g, 1.01 mmol) prepared as described in Roush et al., J.

Am. Chem. Soc. 1998, 120, 10994, was dissolved in a solution of 40% trifluoroacetic acid in methylene chloride (6 mL) and the solution was stirred in an ice bath for 2 h. The solvent was removed, and the residue was dried over vacuum. Without further purification, the crude amine was dissolved in methylene chloride (5 mL) with enough DMF to dissolve the residue. To this solution was added acid 6 (0.320 g, 1.01 mmol) in methylene chloride (1 mL), HOBT (0.170 g, 1.1 1 mmol), N-methylmorpholine (0.222 mL, 2.02 mmol), and EDC (0.214 g, 1.12 mmol). The resulting mixture was stirred at 0 °C then warmed up to room temperature overnight. After removal of solvent with rotary evaporation, the residue was dissolved in ethyl acetate (40 mL) and a saturated solution of NaHCO₃ (15 mL). The solutions were separated and the organic layer was washed with another portion of saturated NaHCO₃ (15 mL) solution and brine (15 mL), dried over Na₂SO₄, filtered, and concentrated to dryness. Purification by flash column chromatography (1,8-3% methanol in methylene chloride) afforded the title compound (8, 0.438 g, 0.682 mmoi, 72%) as a colorless solid: ¹H NMR (400 MHz, CDCl₃) δ 7.84 (d, J = 7.6 Hz, 2 H), 7.62 (t, J = 7.6 Hz, 1 H), 7.56 (t, J = 7.6 Hz, 2 H), 7.25-7.16 (m, 6 H), 7.13 (m, 2 H), 7.03 (d, J= 6.8 Hz, 2 H), 6.78 (dd, J= 15.2, 5.0 Hz, 1 H), 6.74 (br s, 1H), 6.09 (dd, J- 15.2, 1.6 Hz, 1 H) , 5.01 (d, J= 7.2 Hz, 1 H), 4.59 (m, 1 H), 4.52 (q, J= 7.2 Hz, 1 H), 3.24 (m, 4 H), 3.02 (d, J = 7.6 Hz, 2 H), 2.51 (m, 6 H), 1.85 (m, 1 H), 1.74 (in, 1 H), 1.55 (m, 1 H), 0.45 (m, 2 H), 0.38 (m, 2 H); ¹³C NMR (100MHz, CDCl₃) δ 172.2, 157.3, 146.0, 140.8, 140.5, 137.0, 133.8, 130.8, 129.6, 129.5, 129.1, 128.9, 128.7, 128.0, 127.5, 126.6, 56.3, 53.1, 49.4, 44.2, 38.6, 36.0, 32.1, 28.3, 6.2; HRMS (ES+) m/z for C₃₄H₄₀N₄NaO₄S [M+Na]⁺ calcd 623.2668, found 623.2679.

Example 2: Inhibition of cysteine proteinases

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 μM in 25 mM Tris, 2 mM EDTA, 2 mM DTT (or 5 mM cysteine), pH 7.5, in a Fluoroskan-Ascent fluorometer (Labsystems, USA). Enzyme activity, initial velocity, and relative fluorescence units (RFU, the amount of proteinase activity needed for the release of 1 pmole of AMC per minute) were calculated with Ascent software. To determine the Michaelis constant (Km) of rEhCPl for the synthetic peptide substrates Z-Arg-Arg-AMC and Z-Ala-Arg-Arg- AMC, aliquots of purified active rEhCPl were assayed as described above with increasing concentrations of synthetic peptide substrates (0.5-20 μM) in triplicate, and the Km determined using the Enzfitter software (Biosoft, Cambridge, UK). Cruzain (rec) IC₅₀ (μM): Protocol: Instrument: 96well fluorimeter, inhibitor (μM): 0.01 to 10; pre-incubation time temperature: 5 min @25C; substrate: Km l(μM) ZFR AMC (lOμM); buffer: 10OmM NaAc pH5.5, 5 mM DTT; comments: DMSO < 1%. T. brucei rhodesiense Rhodesain (rec) IC₅₀ (μM). Protocol: enzyme concentration, instrument: 96well fluorimeter; inhibitor (μM): 0.01 to 10; pre -incubation time temperature: 5 min @25C; substrate: Km l(μM) 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-

8nM); instrument: 96well fluorimeter; inhibitor (μM): 0.01 to 10; pre-incubation time, temperature: 0 min; substrate: Km l(μM) ZFR AMC (5μM); buffer: 100 mM NaAc pH 5.5, 5 mM DTT; comments: DMSO < 1%, Prism for curve fit.

T, brucei rhodesiense Rhodesain (rec) k_inact/K,, k_ass, k_0bs/I. Protocol: enzyme concentration, source: rec ,(4-8nM); instrument: 96well fluorimeter; inhibitor (μM): 0.01 to 10; pre-incubation time, temperature: 0 min; substrate: Km l(μM) ZFR AMC (5μM); buffer: 10OmM NaAc pH5.5, 5 mM DTT; comments: DMSO < 1%, Prism for curve fit. T. brucei (in vitro) % Growth-Inhibition HTS (lμM). Protocol: ATP Measured by CellTiter-Glo luminescence; Assay conducted during cell log growth phase; DMSO final concentration: 0.5%; Max incubation: 48 hrs; measure: % cell death; controls: DMSO and known inhibitor.

T. brucei Cathepsin B (rec) % Inhibition (1 μM) HTS. Protocol: rec T brucei is preincubated with lμM inhibitor for 5 minutes at room temperature. 1 μl of inhibitor in DMSO in 100 μl of buffer. Buffer is 10OmM NaAcetate pH5.5, with 5 mM DTT. The concentration of T brucei is ~500nM. The assay is started by adding an equal volume of 40 μM ZFRAMC in buffer, and the maximum rate determined in each well.

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 10⁵ 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 μM by triplicate. All assays include untreated, Kl 1777-treated, and uninfected macrophage controls. Medium is replaced every 48h. Under these culture conditions, T. cruzi completes the intracellular cycle in 5 days in untreated controls. Treatment duration is up to 27 days as such regime results in cure of macrophages treated with 10 μM 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 10⁶ 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% dH₂O). 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). If necessary, 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. Protocol: Host: Female 2Og BALB/c mice (n = 5 for controls and test); Parasite species and strain: Trypanosoma brucei brucei 90:13; Infection dose: Mice were infected with 600 cells intra-peritoneally. Table 1. Inhibition of cysteine proteases and T. brucei

Example 3: Synthesis of Compound of Formula (III)

Cpp-Leu-OBn (10). Piperazine 3 (45 mg, 0.198 mmol) was dissolved in CH₂Cl₂

(1 mL) and TFA (1 mL) and the solution was stirred in an ice bath for 1.5 h. The solvent was removed under reduced pressure and excess trifluoroacetic acid was removed by repeated evaporation from toluene in vacuo. Without further purification, the crude TFA salt was used in the next reaction. Leucine benzyl ester (9, 78 mg, 0.198 mmol) was dissolved CH₂Cl₂ (5 mL) and a saturated solution of aqueous NaHCO₃ (5 mL) at 0 °C and the mixture was stirred vigorously for 20 min. A solution of triphosgene (20 mg, 0.067 mmol) in 1 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). The combined layers were dried over Na₂SO₄, filtered, and added to the crude 1-cyclopropylpiperadine TFA salt prepared above at 0 °C. JV- methylmorpholine (45 μL, 0.409 mmol) was added and the solution was stirred for 16 h. After removing the solvent in vacuo, the residue was purified by flash column chromatography (2-3.3% methanol in methylene chloride) to yield 73.0 mg (99%) of the title compound as colorless foam: ¹H NMR (400 MHz, CDCl₃) δ 7.31-7.39 (m, 5 H), 5.20 (d, J= 12.4 Hz, 1 H), 5.12 (d, J= 12.4 Hz, 1 H), 4.79 (d, J- 8.4 Hz, 1 H), 4.54-4.59 (m, 1 H), 3.36 (br s, 4 H), 2.63 (br s, 4 Hz), 1.60-1.72 (m, 2 H), 1.48-1.53 (m, 1 H), 0.92 (dd, J = 6.4, 2.4 Hz, 4 H), 0.43-0.50 (m, 4 H).

Compound of Formula (III) (12). Ester 10 (73 mg, 0.195 mmol) was dissolved in ethyl acetate (2.5 mL) and methanol (2.5 mL). To this solution was added 10% palladium on carbon (40 mg) and the reaction was held under a hydrogen atmosphere using a balloon overnight at room temperature. After removal of catalyst by filtration through a pad of celite, the filtrate was concentrated in vacuo. The crude acid (11) was used in the next reaction without further purification. Vinyl sulfone 7 (73 mg, 0.182 mmol) was dissolved in a solution of 33% trifluoroacetic acid in methylene chloride (3 mL) and the solution was stirred in an ice bath for 2 h. The solvent was removed, and the excess acid was removed by repeated evaporation from toluene in vacuo. Without further purification, the crude amine was dissolved in methylene chloride (3 mL) with enough DMF to dissolve the residue. To this solution was added acid 11 (52 mg, 0.183 mmol) in methylene chloride (1 mL),

HOBT (31 mg, 0.202 mmol), N-methylmorpholine (40 μL, 0.363 mmol), and EDC (38.3 mg, 0.200 mmol). The resulting mixture was stirred at 0 °C then warmed up to room temperature overnight. After removal of solvent with rotary evaporation, the residue was dissolved in ethyl acetate (15 mL) and a saturated solution OfNaHCO₃ (5 mL). The solutions were separated and the organic layer was washed with another portion of saturated NaHCO₃ (5 mL) solution and brine (5 mL), dried over Na₂SO₄, filtered, and concentrated to dryness. Purification by flash column chromatography (2-5% methanol in methylene chloride) afforded 50 mg of the title compound (49%) as a colorless solid: ¹H NMR (400 MHz, CDCl₃) δ 7.86 (d, J= 7.2 Hz, 2 H), 7.62 (t, J= 7.2 Hz, 1 H), 7.53 (t, J= 7.4 Hz, 2 H), 7.26 (t, J= 7,4 Hz, 2 H), 7.19 (t, J= 7.2 Hz, 1 H), 7.1 1 (d, J= 7.6 Hz, 2 H), 6.90 (dd, J= 15.2, 4.8 Hz, 1 H), 6.80 (d, J= 8.8 Hz, 1 H), 6.44 (dd, J= 15.0, 5.0 Hz, 1 H), 4.65 (m, 2 H), 4.23 (dt, J= 7.8, 6.8 Hz, 1 H), 3.31 (br t, J= 5.0 Hz, 4 H), 2.54-2.65 (m, 6 H), 1.91-1.99 (m, 1 H), 1.81-1.89 (m, 1 H), 1.64-1.70 (m, 1 H), 1.54-1.60 (m, 2 H), 1.42-1.49 (m, 1 H), 0.90 (dd, J= 13.2, 6.4 Hz, 6 H), 0.43-0.47 (m, 2 H), 0.37-0.40 (m, 2 H); HRMS (ES+) m/z for C₃₁H₄₂N₄NaO₄S [M+Na]⁺ calcd 589.2819, found 589.2840.

Claims

What is claimed is:

1. A compound of the following formula (I):

2. The compound of claim 1, wherein said compound is in a pharmaceutical composition.

3. A compound of the following formula (II):

wherein P₁ comprises:

wherein P'₁ comprises:

wherein R is R¹ is H, OCH₃, alkyl, halogen, -NO_2;

wherein X¹ is OH, NHR²; R² = H, acyl, -CO alkyl; X¹ = halogen, alkyl, alkoxy;

wherein P₂ comprises:

and R⁵ is H, Me;

wherein X¹ is OH, NHR²; R² = H, acyl, -CO alkyl; X² = halogen, alkyl, alkoxy;

R⁶ = H, OCH₃, alkyl, halogen, -NO₂,

wherein X² = O, S, NH; wherein P₃ comprises:

wherein R⁷ = CH₂R⁸ (1 to 6 C); R⁷ = CHR⁹ ₂ (branched) ( e.g. -CHMeAr); R⁷ - cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, R⁷ = t-Bu, -CF₃, -CH₂CF₃, - CH₂(CF₂)₂CF₃, -CH₂CH₂CF₃; X³ = -OR (carbamates), -OCH₂Ph, -OCH₂pyridyl, - OCH₂heteroaryl; alkyl, aryl or heteroaryl (amides), substituted aryl, heteroaryl, alkyl, aralkyl.

4. A compound of formula (III) comprising:

salts, hydrates, solvates, complexes, analogs and prodrugs thereof.

5. A pharmaceutical composition comprising any one or more compounds of general formulae (I) to (III), salts, solvates, metabolites, prodrugs, analogs, isomers and substituents thereof.

6. The pharmaceutical composition of claim 5, wherein a compound of general formula (I) comprises:

7. The pharmaceutical composition of claim 5, wherein a compound of general formula (II) comprises:

wherein P₁ comprises:

wherein P'₁ comprises:

wherein R is H, OCH₃, alkyl, halogen, -NO₂;

wherein X is OH, NHR; R = H, acyl, -OCO alkyl; X = halogen, alkyl, alkoxy, tryptophan and substituted tryptophan;

wherein R is alkyl, aralkyl, heteroalkyl, hydrogen, O-alkyl, O-aryl; wherein P₁ comprises:

wherein P₂ comprises:

and R is H, Me;

wherein X is OH, NHR; R - H, acyl, -OCO alkyl; X = halogen, alkyl, alkoxy.

wherein X = O, S, NH; wherein P₃ comprises:

wherein R = CH₂R (1 to 6 C); R = CHR'₂ (branched) ( e.g. -CHMeAr); R = cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, R = t-Bu, R = CF₃, CH₂CF₃, -- CH₂(CF₂)₂CF₃, - CH₂(CY₂)_mCY₃ wherein Y is a halogen and m is 0 to 10, ~ CH₂CH₂CF₃; X = -OR (carbamates), -OCH2Ph, -OCH2pyridyl, ~OCH2heteroaryl; alkyl, aryl or heteroaryl (amides), substituted aryl, heteroaryl, alkyl, aralkyl.

8. The pharmaceutical composition of claim 5, wherein a compound of general formula (III) comprises:

salts, hydrates, solvates, complexes, analogs and prodrugs thereof.

9. A method of preventing or treating diseases in which the disease pathology is modified by inhibiting a cysteine protease, comprising: 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; and, treating or preventing the disease.

10. The method of claim 9, wherein the compound is a compound of formula (I).

11. A method of preventing or treating Chagas disease, comprising: 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; and, treating or preventing Chagas disease.

12. A method of preventing or treating a disease caused by an organism comprising; 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.

13. The method of claim 12, wherein the protease is a cysteine protease.

14. The method of claim 12, wherein the organism is a parasite, bacterium, virus, fungus, or protozoan.

15. The method of claim 12, wherein any one or more of compounds of formula (I) to (III) are co-administered with other protease inhibitors.