METHODS OF INHIBITING CRUZAIN AND RHODESAIN AND

METHODS OF TREATING CHAGAS DISEASE AND AFRICAN

TRYPANOSOMIASIS

This application claims the benefit of U.S. Provisional Application No. 61/199,763, filed November 19, 2008, which is incorporated herein by reference.

TECHNICAL FIELD The invention provides for novel triazine and purine compounds that are useful for the treatment and prevention of mammalian protozoal diseases, including African trypanosomiasis, Chagas disease and other opportunistic infections.

BACKGROUND OF THE INVENTION Parasitic protozoa are responsible for a wide variety of infections in both humans and animals. Trypanosomiasis poses health risks to millions of people across multiple countries in Africa and North and South America Visitors to these regions, such as business travelers and tourists, are also at risk for contracting parasitic diseases. There are two types of African trypanosomiasis, also known as sleeping sickness. One type is caused by the parasite Trypanosoma brucei gambiense, and the other is caused by the parasite Trypanosoma brucei rhodesiensi. If left untreated, African sleeping sickness results in death. Chagas disease, caused by Trypanosoma cruzi, affects millions of people in Mexico and South and Central America. Untreated Chagas disease causes decreased life expectancy and can also result in death.

The trypanosomal family of parasites have 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), 125-9, 1996) are at best moderately successful, achieving about 60% cure during the acute phase of infection (see Docampo, R. Curr. Pharm. Design, 7, 1157-1164, 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). 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. .beta.-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., 7, 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 programmes (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 recognising, then binding to, particular three-dimensional electronic surfaces displayed by a protein, which aligns the bond for cleavage precisely 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, 1143-1156, 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, 111, 597- 606, 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 sulphone-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. Nat'l. Acad. Sci. U.S.A., 96, 11015-11022, 1999), Schistosoma mansoni and Plasmodium falciparium (Olson, J. E. et al, Bioorg. Med. Chem., 7, 633-638, 1999) can be halted or cured by treatment with cysteine protease inhibitors. The risk of parasitic diseases is also present outside developing countries.

Opportunistic infections in immunocompromised hosts caused by Pneumocystis carinii, Toxoplasma gondii, and Cryptosporidium sp. are becoming increasingly prevalent in developed countries. For example, toxoplasmosis, which is caused by the parasite Toxoplasma gondii, is found in countries throughout the world, including the United States. Pregnant women and those with weak immune systems are particularly susceptible to health risks resulting from Toxoplasma infection. Severe toxoplasmosis can result in damage to the brain, eyes, and other organs.

Cruzain is the primary cysteine protease of T. cruzi that has been shown to be essential for the survival of the parasite in host cells, and thus is a validated drug target for the treatment of Chagas' disease (Harth, G., et al. MoI. Biochem. Parasitol. 1993, 58, 1; McGrath, M. E.; et al. /. MoI. Biol. 1995, 247, 251). Several groups have demonstrated that irreversible inhibition of cruzain by small molecules (i.e. 1 and 2) eradicates infection of the parasite in cell culture and animal models (Barr, S. C; et al. Antimicrob. Agents Chemother. 2005, 49, 5160; Brak, K.; et al. /. Am. Chem. Soc. 2008, 130, 6404; Doyle, P. S.; et al. Antimicrob. Agents Chemother. 2007, 51, 3932; Jacobsen, W.; et al. Drug Metab. Dispos. 2000, 28, 1343). Irreversible inhibitors 1 and 2 contain a reactive electrophilic functional group (i.e. vinyl sulfone or 2,3,5,6-tetrafluorophenoxymethyl ketone) that can covalently bind to cruzain via nucleophilic attack of the active site cysteine. Interestingly, several non-covalent inhibitors of cruzain, though highly potent, have proven ineffective at killing the parasite in comparable in vivo models, implying that covalent modification (irreversible or reversible) of the enzyme may be essential. It should be noted however, that, to date only irreversible inhibitors of cruzain have successfully cured parasitic infection (Brak, K.; et al. /. Am. Chem. Soc. 2008, 130, 6404; Doyle, P. S.; et al. Antimicrob. Agents Chemother. 2007, 51, 3932). Despite these recent advances in the development of novel treatments for the disease the development of a small molecule reversible covalent inhibitor of cruzain was desired. This approach provides the benefit of the potential of less off-targets side- effects often associated with irreversible enzyme inhibitors (Hickey, E. R.; et al. U.S. Patent 6,982,263).

There are currently no satisfactory treatments available for these diseases. Currently available treatments are not entirely effective, and can even be toxic to the patient. For some diseases, drug-resistant strains of the protozoa can develop. A need thus exists for an antiparasitic compound for humans that is more effective and less toxic than those currently available. It is thus a goal of the invention to provide novel compounds and compositions for the treatment of parasitic disorders, including African trypanosomiasis and Chagas disease.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound of Formula I, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, each X is independently O, S, or NR _A ; each R ₁ is independently halogen, nitro, an optionally substituted Ci ₁₂ alkyl, an optionally substituted C _{3 12} aryl, an optionally substituted C ₃ ^heteroaryl, an optionally substituted Ci alkoxy, an optionally substituted Ci ₁₂ acyl, or N(R _A )(R _B );

R is an optionally substituted Ci ₁₂ alkyl, an optionally substituted C _{3 12} cycloalkyl, an optionally substituted C ₃ ^heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂₀ aralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Z is CN, C(O)CH ₂ Y, or CH=CH-Y' ;

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ _ 1 ₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl;

Y' is an optionally substituted S(O) _p C _1-12 alkyl, an optionally substituted S(O) _p C _3-12 aryl, an optionally substituted S(O) _p C _3-12 heteroaryl, an optionally substituted S(0) _p C ₇ _ ₂ oaralkyl, or an optionally substituted S(O) _p C _1-12 haloalkyl, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, an optionally substituted OC _1-12 haloalkyl, an optionally substituted NR'C _1-12 alkyl, an optionally substituted NR'C _3-12 cycloalkyl, an optionally substituted NR'C ₃ _ ₁₂ heterocycloalkyl, an optionally substituted NR'C _3-12 aryl, an optionally substituted NR'C _3-12 heteroaryl, an optionally substituted NR' C ₇ _ ₂ oaralkyl, or an optionally substituted NR'C _1-12 haloalkyl; each R' is independently an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; p is 0, 1, or 2, and n is O, 1, 2, 3, 4, or 5.

In another aspect, the invention provides a compound of Formula II, or pharmaceutically acceptable salt, solvate or hydrate thereof: (II);

wherein,

W is N, CH, or CR';

X is O, S, or NR _A ; each R' is independently an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; R ₁ is H, halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, or N(R _A )(R _B );

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _7-2 oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy; wherein R is optionally substituted by R ₁ ;

Z is CN, C(O)CH ₂ Y, or CH=CH-Y' ;

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ - ₁₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl;

Y' is an optionally substituted S(O) _p C _1-12 alkyl, an optionally substituted S(O) _p C _3-12 aryl, an optionally substituted S(O) _p C _3-12 heteroaryl, an optionally substituted S(0) _p C _7-2 oaralkyl, or an optionally substituted S(O) _p C _1-12 haloalkyl, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, an optionally substituted OC _1-12 haloalkyl, an optionally substituted NR'C _1-12 alkyl, an optionally substituted NR'C _3-12 cycloalkyl, an optionally substituted NR'C ₃ _ ₁₂ heterocycloalkyl, an optionally substituted NR'C _3-12 aryl, an optionally substituted NR'C _3-12 heteroaryl, an optionally substituted NR' C ₇ _ ₂ oaralkyl, or an optionally substituted NR'C _1-12 haloalkyl; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; and p is 0, 1, or 2.

Also disclosed herein are compounds of Formula (III), or a pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, X is O, S, or NR _A ; R ₁ is H, halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, or N(R _A )(R _B );

Y is XR; R is an optionally substituted C _3-12 cycloalkyl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ - C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _{1- 12} haloalkyl, or an optionally substituted C _1-12 alkoxy; wherein R is optionally substituted by R ₁ ; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; and n is O, 1, 2, 3, 4, or 5. In another aspect, the invention provides a pharmaceutical composition comprising one or more compounds of any of the formulae herein and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a kit comprising an effective amount of one or more compounds of the formulae herein in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to Chagas disease or African trypanosomiasis.

In one aspect, the invention provides a method of inhibiting cruzain or rhodesain in a subject, comprising administering to the subject an effective amount of one or more compounds of any of the formulae herein, wherein the administration of said compound inhibits cruzain or rhodesain.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder related to cruzain or rhodesain, wherein the subject is determined to be in need of inhibition of cruzain or rhodesain, the method comprising the step of administering to the subject an effective amount of one or more compounds of any of the formulae herein.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to parasite infection, Chagas disease, African trypanosomiasis, malaria, toxoplasmosis, or coccidiosis, comprising administering to the subject an effective amount of one or more compounds of any of the formulae herein.

In certain aspects, the present invention is directed to compounds of the formulae herein that are useful for the treatment of protozoal diseases in mammals. The present invention is also directed to compositions comprising such compounds, either alone or in combination with one or more antiprotozoal agents. The compounds of the present invention are also useful for the prevention and treatment of protozoal diseases in mammals, including toxoplasmosis, malaria, and opportunistic infections.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group. The term "alkyl," as used herein, refers to saturated, straight- or branched- chain hydrocarbon radicals containing, in certain embodiments, between one and six, or one and twelve carbon atoms, respectively. Examples of C ₁ -C ₆ alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-buty\, neopentyl, n-hexyl radicals; and examples of C ₁ -C ₁₂ alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-buty\, neopentyl, n- hexyl, heptyl, octyl radicals.

The term "alkenyl," as used herein, denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six, or two to twelve carbon atoms having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1- methyl-2-buten-1-yl, heptenyl, octenyl and the like.

The term "alkynyl," as used herein, denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six, or two to twelve carbon atoms having at least one carbon-carbon triple bond. The alkynyl group may or may not be the point of attachment to another group. Representative alkynyl groups include, but are not limited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and the like.

The term "alkoxy" refers to an -O-alkyl radical. The term "aryl," as used herein, refers to a mono- or poly-cyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. The term "aralkyl," as used herein, refers to an alkyl residue attached to an aryl ring. Examples include, but are not limited to, benzyl, phenethyl and the like. The term "cycloalkyl," as used herein, denotes a monovalent group derived from a monocyclic or polycyclic saturated or partially unsatured carbocyclic ring compound. Examples of C ₃ -Cs-cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and examples of C ₃ -C ₁₂ -cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl. Also contemplated are a monovalent group derived from a monocyclic or polycyclic carbocyclic ring compound having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Examples of such groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term "heteroaryl," as used herein, refers to a mono- or poly-cyclic (e.g., bi-, or tri-cyclic or more) fused or non-fused, radical or ring system having at least one aromatic ring, having from five to ten ring atoms of which one ring atoms is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term "heteroaralkyl," as used herein, refers to an alkyl residue residue attached to a heteroaryl ring. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl and the like.

The term "heterocycloalkyl," as used herein, refers to a non-aromatic 3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused of non-fused system, where (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above rings may be fused to a benzene ring. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. The term "alkylamino" refers to a group having the structure --NH(C ₁ -Cn alkyl) where C ₁ -C ₁₂ alkyl is as previously defined.

The term "acyl" includes residues derived from acids, including but not limited to carboxylic acids, carbamic acids, carbonic acids, sulfonic acids, and phosphorous acids. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates and aliphatic phosphates. Examples of aliphatic carbonyls include, but are not limited to, acetyl, propionyl, 2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.

In accordance with the invention, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group. Aromatic groups can be substituted or unsubstituted.

The terms "halo" and "halogen," as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.

As described herein, compounds of the invention may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. It will be appreciated that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted. " In general, the term "substituted", whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent . Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The term "optionally substituted" as used herein, refers to groups that are substituted or unsubstituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to: -F, -Cl, -Br, -I,

-OH, protected hydroxy,

-NO ₂ , -CN,

-NH ₂ , protected amino, -NH -C ₁ -C ₁₂ -alkyl, -NH -C ₂ -C ₁₂ -alkenyl, -NH -C ₂ - C ₁₂ -alkenyl, -NH -C ₃ -C ₁₂ -cycloalkyl, -NH -aryl, -NH -heteroaryl, -NH - heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino,

-O-C ₁ -C ₁₂ -alkyl, -O-C ₂ -C ₁₂ -alkenyl, -O-C ₂ -C ₁₂ -alkenyl, -0-C ₃ -C ₁₂ - cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl,

-C(O)- C ₁ -C ₁₂ -alkyl, -C(O)- C ₂ -C ₁₂ -alkenyl, -C(O)- C ₂ -C ₁₂ -alkenyl, -C(O)- C ₃ -C ₁₂ -cycloalkyl, -C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl,

-CONH ₂ , -CONH- C ₁ -C ₁₂ -alkyl, -CONH- C ₂ -C ₁₂ -alkenyl, -CONH- C ₂ -C ₁₂ - alkenyl, -CONH-C ₃ -C ₁₂ -cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH- heterocycloalkyl,

-OCO ₂ - C ₁ -C ₁₂ -alkyl, -OCO ₂ - C ₂ -C ₁₂ -alkenyl, -OCO ₂ - C ₂ -C ₁₂ -alkenyl, - OCO ₂ -C ₃ -C ₁₂ -cycloalkyl, -OCO ₂ -aryl, -OCO ₂ -heteroaryl, -OCO ₂ -heterocycloalkyl, - OCONH ₂ , -OCONH- C ₁ -C ₁₂ -alkyl, -OCONH- C ₂ -C ₁₂ -alkenyl, -OCONH- C ₂ -C ₁₂ - alkenyl, -OCONH- C ₃ -C ₁₂ -cycloalkyl, -OCONH- aryl, -OCONH- heteroaryl, - OCONH- heterocycloalkyl,

-NHC(O)- C ₁ -C ₁₂ -alkyl, -NHC(O)-C ₂ -C ₁₂ -alkenyl, -NHC(O)-C ₂ -C ₁₂ -alkenyl, -NHC(O)-C ₃ -C ₁₂ -cycloalkyl, -NHC(0)-aryl, -NHC(O)-heteroaryl, -NHC(O)- heterocycloalkyl, -NHCO ₂ - C ₁ -C ₁₂ -alkyl, -NHCO ₂ - C ₂ -C ₁₂ -alkenyl, -NHCO ₂ - C ₂ - C ₁₂ -alkenyl, -NHCO ₂ - C ₃ -C ₁₂ -cycloalkyl, -NHCO ₂ - aryl, -NHCO ₂ - heteroaryl, - NHCO ₂ - heterocycloalkyl, -NHC(O)NH ₂ , -NHC(O)NH- C ₁ -C ₁₂ -alkyl, - NHC(O)NH-C ₂ -C ₁₂ -alkenyl, -NHC(O)NH-C ₂ -C ₁₂ -alkenyl, -NHC(O)NH-C ₃ -C ₁₂ - cycloalkyl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, -NHC(O)NH- heterocycloalkyl, NHC(S)NH ₂ , -NHC(S)NH- C ₁ -C ₁₂ -alkyl, -NHC(S)NH-C ₂ -C ₁₂ - alkenyl, -NHC(S)NH-C ₂ -C ₁₂ -alkenyl, -NHC(S)NH-C ₃ -C ₁₂ -cycloalkyl, -NHC(S)NH- aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH ₂ , - NHC(NH)NH- C ₁ -C ₁₂ -alkyl, -NHC(NH)NH-C ₂ -C ₁₂ -alkenyl, -NHC(NH)NH-C ₂ -C ₁₂ - alkenyl, -NHC(NH)NH-C ₃ -C ₁₂ -cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH- heteroaryl, -NHC(NH)NH-heterocycloalkyl, -NHC(NH)-C ₁ -C ₁₂ -alkyl, -NHC(NH)- C ₂ -C ₁₂ -alkenyl, -NHC(NH)-C ₂ -C ₁₂ -alkenyl, -NHC(NH)-C ₃ -C ₁₂ -cycloalkyl, - NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl,

-C(NH)NH-C ₁ -C ₁₂ -alkyl, -C(NH)NH-C ₂ -C ₁₂ -alkenyl, -C(NH)NH-C ₂ -C ₁₂ - alkenyl, -C(NH)NH-C ₃ -C ₁₂ -cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, - C(NH)NH-heterocycloalkyl,

-S(O)-C ₁ -C ₁₂ -alkyl, - S(O)-C ₂ -C ₁₂ -alkenyl, - S(O)-C ₂ -C ₁₂ -alkenyl, - S(O)-C ₃ - C ₁₂ -cycloalkyl, - S(O)-aryl, - S(O)-heteroaryl, - S(O)-heterocycloalkyl -SO ₂ NH ₂ , - SO ₂ NH- C ₁ -C ₁₂ -alkyl, -SO ₂ NH- C ₂ -C ₁₂ -alkenyl, -SO ₂ NH- C ₂ -C ₁₂ -alkenyl, -SO ₂ NH- C ₃ -C ₁₂ -cycloalkyl, -SO ₂ NH- aryl, -SO ₂ NH- heteroaryl, -SO ₂ NH- heterocycloalkyl, -NHSO ₂ -C _r C ₁₂ -alkyl, -NHSO ₂ -C ₂ -C ₁₂ -alkenyl, - NHSO ₂ -C ₂ -C ₁₂ -alkenyl, -

NHSO ₂ -C ₃ -C ₁₂ -cycloalkyl, -NHS0 ₂ -aryl, -NHSO ₂ -heteroaryl, -NHSO ₂ - heterocycloalkyl,

-CH ₂ NH ₂ , -CH ₂ SO ₂ CH ₃ , -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, - heterocycloalkyl, -C ₃ -C ₁₂ -cycloalkyl, polyalkoxyalkyl, polyalkoxy, - methoxymethoxy, -methoxyethoxy, -SH, -S-C ₁ -C ₁₂ -alkyl, -S-C ₂ -C ₁₂ -alkenyl, -S-C ₂ - C ₁₂ -alkenyl, -S-C ₃ -C ₁₂ -cycloalkyl, -S-aryl, -S-heteroaryl, -S-heterocycloalkyl, or methylthiomethyl.

It is understood that the optionally substituted groups and the like can be further substituted. The term "subject" as used herein refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.

"Treat", "treating" and "treatment" refer to a method of alleviating or abating a disease and/or its attendant symptoms.

The term "therapeutically effective amount" means an amount effective to treat, cure or ameliorate a disease, illness or sickness.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts of the compounds formed by the process of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable include, but are not limited to, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

The term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds formed by the process of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present invention. "Prodrug", as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to afford any compound delineated by the formulae of the instant invention. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). "Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1- 38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, "Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology," John Wiley and Sons, Ltd. (2002).

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

Compounds of the Invention

In one aspect, the invention provides a compound of Formula I, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, each X is independently O, S, or NR _A ; each R ₁ is independently halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, or N(R _A )(R _B );

R is an optionally substituted C _1-12 alkyl, an optionally substituted C _{3- 12} cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Z is CN, C(O)CH ₂ Y, or CH=CH-Y' ;

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ - ₁₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl;

Y' is an optionally substituted S(O) _p C _1-12 alkyl, an optionally substituted S(O) _p C _3-12 aryl, an optionally substituted S(O) _p C _3-12 heteroaryl, an optionally substituted S(0) _p C ₇ _ ₂ oaralkyl, or an optionally substituted S(O) _p C _1-12 haloalkyl, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, an optionally substituted OC _1-12 haloalkyl, an optionally substituted NR'C _1-12 alkyl, an optionally substituted NR'C _3-12 cycloalkyl, an optionally substituted NR'C ₃ _ ₁₂ heterocycloalkyl, an optionally substituted NR'C _3-12 aryl, an optionally substituted NR'C _3-12 heteroaryl, an optionally substituted NR' C ₇ _ ₂ oaralkyl, or an optionally substituted NR'C _1-12 haloalkyl; each R' is independently an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; p is 0, 1, or 2, and n is O, 1, 2, 3, 4, or 5.

In certain embodiments, Z is CN, C(O)CH ₂ Y, or CH=CH-Y'.

In a further embodiment, Z is C(O)CH ₂ Y and Y is halogen, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, or an optionally substituted OC ₇ _ ₂ oaralkyl.

In a further embodiment, Z is CH=CH-Y' and Y' is an optionally substituted S(0) _p C _1-12 alkyl, an optionally substituted S(O) _p C _3-12 aryl, an optionally substituted S(O) _p C _3-12 heteroaryl, an optionally substituted S(0) _p C ₇ _ ₂ oaralkyl, or an optionally substituted S(O) _p C _1-12 haloalkyl.

In a first embodiment, the invention provides a compound of Formula I- a, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, X is O, S, or NR _A ;

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy; and R _A is H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-

₁₂ acyl.

In one embodiment, the invention provides a compound wherein X is NR _A and R _A is H.

In one embodiment, the invention provides a compound R is an optionally substituted C _1-12 alkyl or an optionally substituted C _3-12 cycloalkyl.

In other embodiments, R is an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₇ _ ₂ oaralkyl, or an optionally substituted C _1-12 haloalkyl. In a further embodiment, R is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 4-hydroxybutyl, or 2- hydroxyethyl.

In a second embodiment, the invention provides a compound formula I-b, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

X is O, S, or NR _A ; each R ₁ is independently halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, or N(R _A )(R _B ); each R _A and R _B is independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; and n is O, 1, 2, 3, 4, or 5. In one embodiment, X is NR _A and R _A is H.

In certain embodiments, each R ₁ is independently H, halogen, nitro, an optionally substituted C _1-12 alkyl, or an optionally substituted C _3-12 aryl and n is 1, 2, or 3.

In a further embodiment, each R ₁ is independently H, methyl, phenyl, trifluoromethyl, nitro, fluoro, chloro, or bromo, and n is 1, 2, or 3.

In other embodiments, the invention provides for compounds of the following formulae:

wherein each of X, R, R _b Y, Y' and n are defined as disclosed above.

In another aspect, the invention provides a compound of Formula II, or pharmaceutically acceptable salt, solvate or hydrate thereof:

(II);

wherein,

W is N, CH, or CR'; X is O, S, or NR _A ; each R' is independently an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; R ₁ is H, halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C _3-12 heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy, or hydroxy; wherein R is optionally substituted by R ₁ ; Z is CN, C(O)CH ₂ Y, or CH=CH-Y' ;

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ - ₁₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC _7-2 oaralkyl, or an optionally substituted OC _1-12 haloalkyl;

Y' is an optionally substituted S(O) _p C _1-12 alkyl, an optionally substituted S(O) _p C _3-12 atyl, an optionally substituted S(O) _p C _3-12 heteroaryl, an optionally substituted S(0) _p C _7-2 oaralkyl, or an optionally substituted S(O) _p C _1-12 haloalkyl, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC _7-2 oaralkyl, an optionally substituted OC _1-12 haloalkyl, an optionally substituted NR'C _1-12 alkyl, an optionally substituted NR'C _3-12 cycloalkyl, an optionally substituted NR'C ₃ _ ₁₂ heterocycloalkyl, an optionally substituted NR'C _3-12 atyl, an optionally substituted NR'C _3-12 heteroaryl, an optionally substituted NR' C _7-2 oaralkyl, or an optionally substituted NR'C _1-12 haloalkyl; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C _{3- 12} cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and p is 0, 1, or 2.

In one embodiment, W is N. In another embodiment, W is CH, or CR' .

In a first embodiment, the invention provides for a compound of Formula II- a, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

X is O, S, or NR _A ; each R ₁ is independently H, halogen, nitro, an optionally substituted C _{1- 12} alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C _3-12 heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy, or hydroxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is O, 1, 2, 3, 4, or 5.

In one embodiment, X is NR _A and R _A is H.

In another embodiment, each R ₁ is independently F, Cl, Br, I, or an optionally substituted C _1-12 alkyl. In a further embodiment, each R ₁ is independently

wherein,

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ 1 ₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C _{3- 12} cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ 1 ₂ aryl or an optionally substituted C _3-12 heteroaryl; and m is 1, 2, or 3.

In another embodiment, R is an optionally substituted C _1-12 alkyl or an optionally substituted C _3-12 cycloalkyl. In a further embodiment, R is ethyl, hydroxyalkyl, cyclopentyl, or difluoroethyl.

In a second embodiment, the invention provides for a compound of Formula II-b, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

X is O, S, or NR _A ; R' is an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; each R ₁ is independently H, halogen, nitro, an optionally substituted C _{1- 12} alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C _3-12 heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is O, 1, 2, 3, 4, or 5.

In certain embodiments, X is NR _A and R _A is H. In other embodiments, R' is an optionally substituted C _1-12 alkyl.

In still another embodiment, each R ₁ is independently F, Cl, Br, or I. In a third embodiment, the invention provides a compound of formula II-c, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ _ 1 ₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl;

X is O, S, or NR _A ; each R ₁ is independently H, halogen, nitro, an optionally substituted C _{1- 12} alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C _3-12 heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

or ; ;

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl, an optionally substituted C _3-12 heteroaiyl, an optionally substituted C _7-2 oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy, or hydroxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is O, 1, 2, 3, 4, or 5.

In one embodiment, Y is halogen, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC ₃ - ₁₂ aryl, an optionally substituted OC _3-12 heteroaryl, or an optionally substituted OCγ- ₂ oaralkyl, wherein Y is optionally substituted by R ₁ .

In a further embodiment, Y is Cl, or

In a fourth embodiment, the invention provides a compound of Formula II-d, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, Y' is an optionally substituted S(O) _p C _1-12 alkyl, or an optionally substituted S(O) _p C _3-12 aiyl;

X is O, S, or NR _A ; R ₁ is H, halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C ₃ _i2heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

;

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy, or hydroxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, n is 0, 1, 2, 3, 4, or 5; and p is 0, 1, or 2.

In certain embodiments, Y' is an optionally substituted S(O) ₂ Me or an optionally substituted S(O) ₂ Ph.

In a fifth embodiment, the invention provides a compound of Formula II-e, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ _ 1 ₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl; X is O, S, or NR _A ;

R' is an optionally substituted C _3-12 aryl or an optionally substituted C ₃ - ₁₂ heteroaryl; each R ₁ is independently H, halogen, nitro, an optionally substituted C ₃ - ₁₂ aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _{1- 12} alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C ₃ _ ₁₂ heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _{3- 12} aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is 0, 1, 2, 3, 4, or 5.

In one embodiment, Y is halogen, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC ₃ - ₁₂ aryl, an optionally substituted OC _3-12 heteroaryl, or an optionally substituted OCγ- ₂ oaralkyl, wherein Y is optionally substituted by R ₁ . In a further embodiment, Y is Cl, or

In a sixth embodiment, the invention provides a compound of Formula II- f, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

Y' is an optionally substituted S(O) _p C _1-12 alkyl, or an optionally substituted S(O) _p C _3-12 aiyl; X is O, S, or NR _A ;

R' is an optionally substituted C _3-12 aryl or an optionally substituted C ₃ - ₁₂ heteroaryl; each R ₁ is independently H, halogen, nitro, an optionally substituted C ₃ _ ₁₂ aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _{1- 12} alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C ₃ _ 1 ₂ heterocycloalkyl, or N(RA)(RB); or R ₁ is selected from

;

each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _

₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl; R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ -

₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _{3- 12} aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, p is 0, 1, or 2, and n is O, 1, 2, 3, 4, or 5.

In another aspect, the invention provides a pharmaceutical composition comprising one or more compounds of any of the formulae herein and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a kit comprising an effective amount of one or more compounds of the formulae herein in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to Chagas disease or African trypanosomiasis.

Quantitative high-throughput screening (qHTS) was conducted on -300,000 small molecules as part of the Molecular Libraries Screening Center Network (MLSCN) initiative (Austin, C. P.; et al. Science 2004, 306, 1138; Inglese, J.; et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 11473). Several diverse chemotypes emerged as promising chemically tractable hits, including various triazine nitriles. Triazine nitriles, including compounds 3 and 5-9, have been explored for many years as potential treatments for a variety of diseases, including cancer, HIV, arthritis, atherosclerosis and osteoporosis, though never for Chagas' disease. The nitrile moiety in this class of compounds is particularly electrophilic as a result of the strong electron-withdrawing nature of the triazine ring and thus is thought to form a covalent but reversible bond with the active site cysteine of the respective protease (Hickey, E. R.; et al. U.S. Patent 6,982,263; Oballa, R. M.; et al. Bioorg. Med. Chem. Lett. 2007, 17, 998; Ferreria, R.; Shoichet, B., 2007). Though reversible, it was hypothesized that the mechanism of covalent modification would prove beneficial to the inhibition of cruzain {vide supra). Moreover, the aforementioned cysteine protease inhibitory activity of triazine nitriles 6-9 coupled with the privileged nature of this scaffold should provide high hit rates of active compounds, allowing for smaller libraries to be synthesized as compared to non-privileged chemotypes. All of these factors, in addition to the molecular simplicity of this chemotype prompted the pursuance of the triazene nitriles for the treatment of Chagas' disease.

The top actives from qHTS, compounds 3, 4, and 5 all showed comparable in vitro potency against cruzain in the low μM range. Compound 4 was of particular interest because its synthesis is precedented and straightforward, allowing rapid access to analogues (Scheme 1) (WO2005/011703). Equimolar amounts of either alkyl/cycloalkyl-amines or anilines (depending on modification strategy) were added to a solution of cyanuric chloride and Hunig's base in dichloromethane at 0 °C. Depending on the starting material used in the first step, either anilines or amines were added using the procedure described above. Finally, the nitrile was then installed using KCN in DMSO at 120 °C for 10 minutes. Importantly, careful monitoring of the reaction was required as prolonged reaction times led to decomposition products.

R' = various amines (see table 1)

Scheme 1. Reagents: (a) (iPr) ₂ NEt, CH ₂ Cl ₂ , 0 °C, various anilines or primary and secondary amines; (b) (iPr) ₂ NEt, CH ₂ Cl ₂ , 0 °C, various anilines or primary and secondary amines; (c) KCN, DMSO, 120 °C.

A synthetic strategy was to modify the alkyl amine chain length and functionality. Incorporation of the cyclopentyl group maintained potency of the lead compounds (Table 1). Also, some hydrophilicity was tolerated, which, if necessary, could be exploited at a later stage to improve pharmacokinetic properties. Phenyl ring modification provided a library of compounds was synthesized to explore the effects of electron-donating/withdrawing groups in addition to incorporation of bulky hydrophobic moieties around the phenyl ring. This revealed that meta- substituted halogens on the phenyl ring proved most fruitful, increasing potency 10- fold. (Table T). A nitro group at the 3 -position (22) resulted in the most potent analogue, supporting the notion that electron-withdrawing groups at this position were most beneficial. However from a lead development standpoint this compound was not pursued due to known toxicity issues associated with the aromatic nitro groups (Adams, G. E.; et al. Biochem. Biophys. Res. Commun. 1976, 72, 824). To solidify the size requirements/tolerance around the triazine scaffold, fused and pendant phenyl rings were incorporated.

Table 1. Alkyl moeity modifications

a Activity concentration determined at N1H Chemical Genomics Center

To further explore the effect of 3-subsituted halogens, a series of compounds were synthesized including bis-meta substituted anilines (Table T). Interestingly, 3,5-difluoro triazine nitrile 21 displayed comparable potency as the mono-meta- substituted triazine 22 (both 63 nM).

Table 2. Phenyl ring moeity modifications

a Activity concentration determined at N1H Chemical Genomics Center

A further compound was synthesized similar to the compounds of Table 2, except that the cyclopentyl ring was replaced with 2,2-difluoroethyl and R was 3,5-difluoro (the AC ₅₀ was 0.025). Two different synthetic strategies were employed for the preparation of purine isomers of Type A and Type B below.

Starting from 2,6-dichloropurine, the 9-position was alkylated using the requisite alkyl halides in the presence of K ₂ CO ₃ in DMF at 60 °C (Scheme T). Two regioisomers were obtained providing alkylation primarily at N9. The ratio of the product distribution was dependent on the nature of the alkyl group, and the two isomers were easily separable by column chromatography. Displacement of the 6- position chlorine was accomplished by heating various anilines in DMF at 160 °C in the presence of Hunig's base. Finally, the nitrile was installed at the 2-position using KCN in DMSO at 120 °C. Of note, performing both of these transformations in the microwave greatly accelerated the reaction.

Scheme 2. Reagents: (a) K ₂ CO ₃ , DMF, 60 °C, various primary and secondary amines; (b) (iPr) ₂ NEt, DMF, 160 °C (μW), various anilines; (c) KCN, DMSO, 120 °C (μW).

Analogs of Type B required a modified first step. The aromatic ring was first installed at the 9-position using a Buchwald-Hartwig-like copper catalyzed cross coupling between the aromatic amine and desired boronic acids (Scheme 3). Alkyl amines could then be installed at the 6-position in the presence of Hunig's base in DMF at elevated temperature (120 °C). These analogues were also completed by displacing the final 2-Cl with KCN in DMSO at 120 °C in the microwave.

Scheme 3. Reagents: (a) phenanthroline, Cu(OAc) ₂ , 4 mol. sieves, CH ₂ Cl ₂ , rt, various aromatic boronic acids; (b) (iPr) ₂ NEt, DMF, 120 °C, various primary and secondary amines; (c) KCN, DMSO, 120 °C (μW).

Purine compounds of the invention include: 6-(3-chlorophenylamino)-9-ethyl-9H-purine-2-carbonitrile (32)

6-(3-chlorophenylamino)-9-cyclopentyl-9H-purine-2-carboni trile (33)

9-(2,2-difluoroethyl)-6-(3,5-difluorophenylamino)-9H-puri ne-2-carbonitrile (34)

9-(3,5-difluorophenyl)-6-(ethylamino)-9H-purine-2-carboni trile (35)

The purine core provided an additional 6-fold increase in potency for structural analogues of Type A. The purine analogue containing the previously most active substituents (3,5-difluoroaniline and cyclopentyl amine) improved the potency from 63 nM to 18 nM (compounds 21 and 34, respectively). Incorporation of the ethyl moiety (compound 32) gave a slight potency enhancement (IC ₅₀ of 10 nM) as did the 2,2-difluoroethyl moiety (compound 33) (IC ₅₀ of 13 nM). There was a noticeable decrease in potency for analogues of Type B. Incorporation of the matching substitution patterns for this chemotype (for instance 3,5-difluoroaniline at position 9 and ethyl amine substitution at position 6 in compound 40) gave a marked decrease in potency (251 nM). Overall, the trend in potency for each substitution pattern correlated well across the two different scaffolds, with the purine core generally showing enhanced potency. Table 3. In vitro potency of purine nitrile analogs

Once optimization was complete, various compounds were tested in additional assays. The compounds were subsequently tested against cruzain and two important cysteine proteases; rhodesain and cathepsin-B-like (TbCatB) from the Trypanosoma brucei (T. bruceϊ) parasite, the causative agent of African sleeping sickness. As shown in Table 3, the activity of compounds 3 and 21-31, 32, 35 and 36 against the primary cysteine protease of T. brucei, rhodesain, were quite comparable to those observed for cruzain. Given the structural homology of these two enzymes this result is not surprising. However, Renslo and co-workers recently showed that through small structural modifications to the phenyl alanine moiety of 1 they were able to improve rhodesain-selectivity over cruzain (Jaishankar, P.; et al. Bioorg. Med. Chem. Lett. 2008, 18, 624). Also, the compounds were consistently more potent in these assays, varying from 5-50 fold.

The same collection of potent compounds, along with qHTS top actives 3, 4, and 5, were tested against the T brucei parasite (Tbb). Several of the lead compounds (21, 24-25) showed potent activity against T. brucei with IC ₅₀ values of 5 μM. This data indicates that reversible covalent inhibitors are useful for the treatment of typanosomiasis.

The in vitro and in vivo potency of purine nitrile compounds 32, 35 and 36 against cysteine protease homologs was also tested with the following results:

Triazines 21, 24, 25, 27, 28, 30, 31 and purine nitriles 32, 35, and 36 had limited activity against the T. cruzi parasite in cell culture despite the potent in vitro activity. The T. cruzi assay is substantially more stringent than the T. brucei assay, as the former measures parasite infection of human macrophages over several weeks, demanding penetration of the host cell membrane and stability over a long period of time, whereas the T. brucei assay is conducted on free living parasites over a few days. Given the disparity in activity of these compounds in the in vitro and cell-based assays, it is likely that cell permeability and solubility is lacking. The following purine nitrile compounds may provide improved cell permeability and/or solubility:

These compounds may be made via the following scheme: The lead compounds 21, 24, 32 and 40 were also screened against a human protease panel to identify possible off-target activity. As expected, these compounds were highly active against several cathepsins, which are highly homologous human papain cysteine proteases. These results, however, are not necessarily adverse. Cathepsins are located in the lysosymes of cells, whereas the parasites are located in the more accessible cytoplasm. Potent compounds are likely to preferentially inhibit the target parasite as a result. Aside from the cathepsins, the lead compounds gave little to no inhibition against a panel of caspases, peptidases, matrix metalloproteinases (MMPs), and serine proteases. This protease profile demonstrates the specificity and broad utility of these triazine and purine nitriles.

The screen of the compounds of the invention therefore focused on identification of novel chemotypes, whether reversible or irreversible, for the inhibition of cruzain. The screen identified the covalent reversible triazine nitriles, which are known inhibitors of several human cysteine proteases. To better understand the mechanism of action of these covalent but reversible inhibitors of cruzain, an x-ray crystal structure of the cruzain/32 complex, to 1.1 A resolution was determined. The ultra-high resolution of this structure allowed analysis of the enzyme-inhibitor interactions in detail. Even before fitting and refining compound 32, the unbiased Fo-Fc electron density was unambiguous for the inhibitor. The covalent adduct with the catalytic Cys25 is clear, with the formally linear nitrile becoming a planar imino-moiety, with a Cysteine- sulfur to inhibitor carbon distance of 1.75 A and a sulfur-carbon- nitrogen angle of 125°, in agreement with standard values. Several polar interactions are observed between nitrogens in the purine ring in 32 and waters or cruzain residues. The nitrogen in the newly formed iminothioether hydrogen-bonds to water 281 (3.17 A) and to the Nε in Glnl9 (2.96 A), which is part of the oxyanion hole in cysteine proteases of the papain family, such as cruzain. The anilinic nitrogen and N7 are solvent exposed and form hydrogen bonds to water 294 (distances 2.94 A and 3.16 A, respectively). N7 is also involved in dipole-dipole interactions with waters 292 (3.14 A) and 368 (3.36 A). The inhibitor purine packs against the surface between Sl and S2 pockets of the cruzain active site, while the aromatic ring off the 6-position extends into the S2 pocket of the enzyme. Good van der Waals complementarity is observed in this mostly hydrophobic pocket, which is completely filled by the 3,5-difluorophenyl ring from 32. This supported our observation that larger, more hydrophobic groups (i.e. 3,5-dichloro) in this region result in a decrease in potency. Double conformation is observed for the terminal carbon atom in the ethyl substituent at N9. This alkyl moiety reaches toward the surface of the enzyme with no direct interactions with the binding site being observed. As such, a wide variety of substituents could be tolerated at this position and ultimately exploited for the improvement of pharmacokinetic properties.

Another embodiment is a method of making a compound of any of the formulae herein using any one, or combination of, reactions delineated herein. The method can include the use of one or more intermediates or chemical reagents delineated herein.

Another aspect is an isotopically labeled compound of any of the formulae delineated herein. Such compounds have one or more isotope atoms which may or may not be radioactive (e.g., ³ H, ² H, ¹⁴ C, ¹³ C, ³⁵ S, ³² P, ¹²⁵ I, and ¹³¹ I) introduced into the compound. Such compounds are useful for drug metabolism studies and diagnostics, as well as therapeutic applications.

A compound of the invention can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.

Alternatively, the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention can be prepared from the corresponding base addition salt or acid addition salt form, respectively. For example a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.). Prodrug derivatives of the compounds of the invention can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs can be prepared by reacting a non- derivatized compound of the invention with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, "Protecting Groups in Organic Chemistry", 3rd edition, John Wiley and Sons, Inc., 1999.

Compounds of the present invention can be conveniently prepared, or formed during the process of the invention, as solvates (e.g., hydrates). Hydrates of compounds of the present invention can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol.

Acids and bases useful in the methods herein are known in the art. Acid catalysts are any acidic chemical, which can be inorganic (e.g., hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic (e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid, ytterbium triflate) in nature. Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. Bases are any basic chemical, which can be inorganic (e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g., triethylamine, pyridine) in nature. Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. In addition, some of the compounds of this invention have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z- double isomeric forms, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- , or as (D)- or (L)- for amino acids. All such isomeric forms of these compounds are expressly included in the present invention. Optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. The configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states; thus a carbon-carbon double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion. All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.

The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. In addition, the solvents, temperatures, reaction durations, etc. delineated herein are for purposes of illustration only and one of ordinary skill in the art will recognize that variation of the reaction conditions can produce the desired bridged macrocyclic products of the present invention. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

The compounds of this invention may be modified by appending various functionalities via any synthetic means delineated herein to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

The compounds of the invention are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Methods of the Invention In one aspect, the invention provides a method of inhibiting cruzain or rhodesain in a subject, comprising administering to the subject an effective amount of one or more compounds of any of the formulae herein, wherein the administration of said compound inhibits cruzain or rhodesain.

In another aspect, the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder related to cruzain or rhodesain, wherein the subject is determined to be in need of inhibition of cruzain or rhodesain, the method comprising the step of administering to the subject an effective amount of one or more compounds of any of the formulae herein.

In one embodiment, the disease or disorder is parasite infection, Chagas disease, African trypanosomiasis, malaria, toxoplasmosis, or coccidiosis. In another aspect, the invention provides a method of treating a subject suffering from or susceptible to parasite infection, Chagas disease, African trypanosomiasis, malaria, toxoplasmosis, or coccidiosis, comprising administering to the subject an effective amount of one or more compounds of any of the formulae herein. In one embodiment, the compound is a compound of Formula I-a, I-b, I-c, I- d, I-e, I-f, II-a, II-b, II-c, II-d, II-e, or II-f.

In certain embodiments, the disorder is Chagas disease or African trypanosomiasis. In a further embodiment, the compound is a compound of

Formula I-a, I-b, I-c, I-d, I-e, I-f, II-a, II-b, II-c, II-d, II-e, or II-f. In another embodiment, the invention provides a method disclosed herein, further comprising administering an additional therapeutic agent.

The instant compounds are also useful for the prevention and treatment of mammalian protozoal diseases, including toxoplasmosis, malaria, African trypanosomiasis, Chagas disease, and opportunistic infections. In one aspect, the invention provides for compounds that are useful for the in vivo treatment or prevention of diseases in which participation of cruzain, rhodesain, or a cysteine protease is implicated.

In another aspect, the invention provides a method for preventing or treating diseases in which the disease pathology may be modified by inhibiting cruzain, rhodesain, or a cysteine protease.

In other aspects, the invention provides the use of a compound of the invention in the preparation of a medicament for preventing or treating diseases in which the disease pathology may be modified by inhibiting cruzain, rhodesain, or a cysteine protease. 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 Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei brucei and Crithidia fisiculata; as well as in osteoporosis, autoimmunity, schistosomiasis, malaria, tumour 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 al. J. Biol. Chem, 262(6), 2664-2667, 1998). The invention is useful in the prevention and/or treatment of each of the disease states mentioned or implied above. The present invention also is useful in a methods 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 Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus, foot-and-mouth disease virus and Crithidia fisiculata; as well as in osteoporosis, autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromatic leukodystrophy, muscular dystrophy and amytrophy.

Inhibitors of cruzipain, particularly cruzipain-specific compounds, are useful for the treatment of Chagas' disease. In another aspect, the invention provides a method of inhibiting dihydrofolate reductase (DHFR) activity.

DHFR inhibitors of the invention have a broad range of therapeutic applications as antibacterial, antiparasitic, and anticancer agents. Among the parasitic diseases that can be treated with these compounds are malaria, trypanosomiasis, Chagas disease, leprosy, toxoplasmosis, leishmaniasis (kala-azar, american leishmaniasis or Aleppo boil or diffuse cutaneous leishmaniasis) and Pneumocystis carinii pneumonia. The latter two are major opportunistic infections in AIDS patients and individuals with other severe immunodeficiency. DHFR inhibitors are also of potential inhibitors as agricultural chemicals (herbicides, fungicides, etc.)

In certain embodiments, the compounds of the invention include those compounds having an IC50 of 50 μM or less against a target or enzyme delineated herein. More preferably, compounds of the invention have an IC50 of 25 μM, 10 μM, 5 μM, 1 μM, or 0.5 μM or less against a target or enzyme delineated herein. Most preferred compounds have an IC50 of 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, or 0.5 μM or less against a target or enzyme delineated herein. Particularly preferred compounds include those with an IC50 of 25 μM, 10 μM, 5 μM, 1 μM, or 0.5 μM or less against a target or enzyme delineated herein capable of a parasite capable of causing a disease or infection in a mammal. Typical parasites which compounds of the invention are preferably active against include those parasites causing malaria, trypanosomiasis, Chagas disease, leprosy, toxoplasmosis, and Pneumocystis carinii pneumonia such as Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium. Preferred parasitic diseases which may be treated by administration to a patients suffering from or susceptible to a parasitic disease of one or more compounds of the invention include malaria, trypanosomiasis, Chagas disease, leprosy, toxoplasmosis, and Pneumocystis carinii pneumonia. Compounds of the invention are useful to combat parasitic infections which are known to inflict HIV-positive or patients suffering from or susceptible to AIDS.

Other protozoal diseases which can be treated by the agent according to the present invention are for instance malaria, trypanosomiasis, toxoplasmosis, babesiosis, amoebic dysentery and lambliasis. The agents according to the present invention are in particular suitable for those diseases in which the pathogen is present in organs such as the liver, spleen or kidney.

Pharmaceutical Compositions Compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Pharmaceutical compositions comprising a compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons. Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

According to the methods of treatment of the present invention, disorders are treated or prevented in a subject, such as a human or other animal, by administering to the subject a therapeutically effective amount of a compound of the invention, in such amounts and for such time as is necessary to achieve the desired result. The term "therapeutically effective amount" of a compound of the invention, as used herein, means a sufficient amount of the compound so as to decrease the symptoms of a disorder in a subject. As is well understood in the medical arts a therapeutically effective amount of a compound of this invention will be at a reasonable benefit/risk ratio applicable to any medical treatment.

The dosage for the instant compounds can vary according to many factors, including the type of disease, the age and general condition of the patient, the particular compound administered, and the presence or level of toxicity or adverse effects experienced with the drug. A representative example of a suitable dosage range is from as low as about 0.025 mg to about 1000 mg. However, the dosage administered is generally left to the discretion of the physician.

A wide variety of pharmaceutical dosage forms for mammalian patients can be employed. If a solid dosage is used for oral administration, the preparation can be in the form of a tablet, hard gelatin capsule, troche or lozenge. The amount of solid carrier will vary widely, but generally the amount of the present compound will be from about 0.025 mg to about 1 g, with the amount of solid carrier making up the difference to the desired tablet, hard gelatin capsule, troche or lozenge size. Thus, the tablet, hard gelatin capsule, troche or lozenge conveniently would have, for example, 0.025 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 25 mg, 100 mg, 250 mg, 500 mg, or 1000 mg of the present compound. The tablet, hard gelatin capsule, troche or lozenge is given conveniently once, twice or three times daily.

In general, compounds of the invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per body weight. An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g. in divided doses up to four times a day or in retard form. Suitable unit dosage forms for oral administration comprise from ca. 1 to 50 mg active ingredient.

In certain embodiments, a therapeutic amount or dose of the compounds of the present invention may range from about 0.1 mg/Kg to about 500 mg/Kg, alternatively from about 1 to about 50 mg/Kg. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. Upon improvement of a subject's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long- term basis upon any recurrence of disease symptoms. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The invention also provides for a pharmaceutical combinations, e.g. a kit, comprising a) a first agent which is a compound of the invention as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co- agent. The kit can comprise instructions for its administration. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non- fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g. a compound of the invention and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g. a compound of the invention and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene -block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The protein kinase inhibitors or pharmaceutical salts thereof may be formulated into pharmaceutical compositions for administration to animals or humans . These pharmaceutical compositions, which comprise an amount of the compound effective to treat or prevent a disorder disclosed herein and a pharmaceutically acceptable carrier, are another embodiment of the present invention.

Examples

The compounds and processes of the present invention will be better understood in connection with the following examples, which are intended as an illustration only and not to limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims.

General Methods:

Unless otherwise stated, all reactions were carried out under an atmosphere of dry argon or nitrogen in dried glassware. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (rt) is noted as 25 °C. All solvents were of anhydrous quality purchased from Aldrich Chemical Co. and used as received. Commercially available starting materials and reagents were purchased from Aldrich and were used as received.

Analytical thin layer chromatography (TLC) was performed with Sigma Aldrich TLC plates (5 x 20 cm, 60 A, 250 μm). Visualization was accomplished by irradiation under a 254 nm UV lamp. Chromatography on silica gel was performed using forced flow (liquid) of the indicated solvent system on Biotage KP-SiI prepacked cartridges and using the Biotage SP- 1 automated chromatography system. ¹ H- and ¹³ C NMR spectra were recorded on a Varian Inova 400 MHz spectrometer. Chemical shifts are reported in ppm with the solvent resonance as the internal standard (CDCl ₃ 7.26 ppm, 77.00 ppm, DMSO-J ₆ 2.49 ppm, 39.51 ppm for ¹ H, ¹³ C respectively). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, and number of protons. Low resolution mass spectra (electrospray ionization) were acquired on an Agilent Technologies 6130 quadrupole spectrometer coupled to an Agilent Technologies 1200 series HPLC. High resolution mass spectral data was collected in-house using and Agilent 6210 time-of- flight mass spectrometer, also coupled to an Agilent Technologies 1200 series HPLC system. Example 1 4-(cyclopentylamino)-6-(3,5-difluorophenylamino)-1,3,5-triaz ine-2-carbonitrile

(21)

General procedure for the formation of 4,6-dichloro-N-alkyl-1,3,5-triazine-2- amines (Step 1): 4,6-dichloro-jV-cylopentyl-1,3,5-triazin-2-amine - To a solution of cyanuric chloride (1.25 g, 6.78 mmol) in CH ₂ Cl ₂ (50 mL) at 0 °C was added Hϋnig's base (1.12 mL, 6.78 mmol). After 5 minutes, cyclopentylamine (0.67 mL, 6.78 mmol) was added and the reaction mixture was allowed to stir for 15 minutes at 0 °C, upon which time the ice bath was removed. The reaction mixture was stirred at rt for 30 min, then concentrated under reduced pressure and directly purified on silica column. Gradient elution with ethyl acetate (2— >40%) in hexanes provided 4,6-dichloro-N-cylopentyl-1,3,5-triazin-2-amine as a colorless solid: yield (1.62 g, 5.85 mmol, 86 %).

General procedure for the formation of 6-chloro- N ² -alkyl- N ⁴ -phenyl-1,3-5- triazine-2,4-diamines (Step 2): 6-chloro- N ² -cyclopentyl- N ⁴ -(3,5-difluorophenyl)-

1,3,5-triazine-2,4-diamine - To a solution of 4,6-dichloro-Ν-cyclopentyl-1,3,5- triazin-2-amine (1.5 g, 6.44 mmol) in CH ₂ Cl ₂ (50 mL) at 0 °C was added Hϋnig's base (1.12 mL, 6.44 mmol). After 5 minutes, 3,5-difluoroaniline (0.831 g, 6.44 mmol) was added and the reaction mixture was allowed to stir for 15 minutes at 0 °C, upon which time the ice bath was removed. The reaction mixture was stirred at rt for 30 min, then concentrated under reduced pressure and directly purified on silica column. Gradient elution with ethyl acetate (2— >40%) in hexanes provided 6- chloro-N ² -cyclopentyl-N ⁴ -(3,5-difluorophenyl)-1,3,5-triazine-2,4-diamine as a colorless solid: yield (1.9 g, 5.83 mmol, 91%).

General procedure for the formation of 4-alkylamino-6-phenylamino-1,3,5- triazine-2-carbonitriles (Step 3): 4-(cyclopentylamino)-6-(3,5- difluorophenylamino)-1,3,5-triazine-2-carbonitrile (21). To a solution of 6- chloro-N2-cyclopentyl-N4-(3,5-difluorophenyl)-1,3,5-triazine -2,4-diamine (1.0 g, 3.07 mmol) in DMSO (25 niL) was added KCN (0.220 g, 3.38 mmol). The reaction mixture was sealed and heated to 120 °C for 10 minutes. Upon completion, the reaction mixture was diluted with ethyl acetate and washed several times with saturated sodium chloride solution. The organic layer was dried on magnesium sulfate, filtered, and concentrated. The resulting residue was purified on silica column. Gradient elution with ethyl acetate (2→40%) in hexanes provided 4- (cyclopentylamino)-6-(3,5-difluorophenylamino)-1,3,5-triazin e-2-carbonitrile (21) as a colorless solid: yield (863 mg, 2.73 mmol, 89%); ¹ H (DMSO-J ₆ ) δ 1.45-1.63 (m, 4H), 1.63-1.79 (m, 2H), 1.84-2.02 (m, 2H), 4.06 - 4.27 (m, 1H), 6.82 - 6.92 (m, 1H) 7.49 (d, / = 8.7 Hz, 1H), 7.56 (d, / = 8.1 Hz, 1H), 8.48 (d, / = 6.3 Hz, 1H), 8.61 (brs, 1H), 10.39 (brs, 1H), and 10.52 (brs, 1H); ¹³ C NMR (DMSO-J ₆ ) δ 23.82, 23.87, 32.15, 32.41, 52.57, 98.39, 103.43, 115.67, 141.85, 151.50, 151.93, 161.63, and 164.10; HRMS (ESI) mlz 317.1319 (M+H) ⁺ (Ci ₅ H ₁₄ F ₂ N ₆ requires 317.1316).

Example 2 4-(cyclopentylamino)-6-(3-nitrophenylamino)-1,3,5-triazine-2 -carbonitrile (22)

The title compound was prepared using a procedure similar to that detailed for 21, substituting 3-nitroaniline in Step 2, providing 22 as a colorless solid: yield (800 mg, 87%); ¹ H (DMSO-J ₆ ) δ 1.47 - 1.63 (m, 4H), 1.64 - 1.78 (m, 2H), 1.93 - 2.06 (m, 2H), 4.22 - 4.33 (m, 1H), 7.53 - 7.66 (m, 1H), 7.79 - 7.94 (m, 1H), 8.63 (brs, 1H), 9.12 (brs, 1H), and 10.66 (brs, 1H); ¹³ C (DMSO-J ₆ ) δ 23.83, 32.18, 32.27, 32.36, 39.32, 39.43, 39.53, 39.74, 39.95, 40.16, 40.36, 40.57, 117.83, 130.34, and 140.50; HRMS (ESI) ml z 326.1363 (M+H) ⁺ (C ₁₅ H ₁₄ N ₇ O ₂ requires 326.1360).

Example 3 4-(3-chlorophenylamino)-6-(cyclopentylamino)-1,3,5-triazine- 2-carbonitrile

(24)

The title compound was prepared using a procedure similar to that detailed for 21, substituting 3-chloroaniline in Step 2, providing 24 as a colorless solid: yield (820 mg, 88%); ¹ H (DMSO-J ₆ ) δ 1.38 - 1.63 (m, 4H), 1.63 - 1.82 (m, 2H), 1.82 - 2.09 (m, 2H), 4.15 (brs, 1H), 6.95 - 7.19 (m, 1H), 7.33 (q, J = 8.0 Hz, 1H), 7.51 (d, / = 8.2 Hz, 1H), 8.5 (brs, 1H), and 10.37 (brs, 1H); ¹³ C (DMSO-J ₆ ) δ 23.79, 32.15, 32.26, 32.38, 52.55, 65.34, 130.56, 130.61, 130.61, 133.37, 140.65, 151.49, and 164.12; HRMS (ESI) ml z 314.1134 (M+H) ⁺ (C ₁₅ H ₁₅ ClN ₆ requires 315.1119).

Example 4 4-(3-bromophenylamino)-6-(cyclopentylamino)-1,3,5-triazine-2 -carbonitrile

(25)

The title compound was prepared using a procedure similar to that detailed for 21, substituting 3-bromoaniline in Step 2, providing 25 as a colorless solid: yield (870 mg, 92%); ¹ H (DMSO-J ₆ ) δ 1.44 - 1.63 (m, 4H), 1.63 - 1.80 (m, 2H), 1.82 - 2.11 (m, 2H), 4.03 - 4.25 (m, 1H), 7.14 - 7.35 (m, 2H), 7.52 (d, / = 7.2 Hz, 1 H), 8.54 (brs, 1H), and 10.37 (brs, 1H); ¹³ C (DMSO-J ₆ ) δ 23.82, 23.85, 32.17, 32.38, 52.55, 115.59, 121.89, 122.89, 125.93, 130.93, 140.78, and 151.46; HRMS (ESI) mlz 359.0621 (M+H) ⁺ (Ci ₅ H ₁₅ BrN ₆ requires 359.0614).

Example 5 4-(cyclopentylamino)-6-(m-tolylamino)-1,3,5-triazine-2-carbo nitrile (27)

The title compound was prepared using a procedure similar to that detailed for 21, substituting 3-methylaniline in Step 2, providing 27 as a colorless solid: yield (620 mg, 84%); ¹ H (DMSO-J ₆ ) δ 1.43 - 1.62 (m, 4 H), 1.63 - 1.79 (m, 2H), 1.80 - 2.03 (m, 2H), 2.28 (s, 3H), 4.05 - 4.32 (m, 1H), 6.87 (d, / = 7.5 Hz, 1H), 7.19 (t, / = 7.8 Hz, 1H), 7.36 - 7.55 (m, 1H), 8.30 (s, 1H), and 10.03 (s, 1H); ¹³ C (DMSO-J ₆ ) δ 21.67, 23.82, 32.21, 32.42, 52.49, 115.67, 124.33, 128.81, 138.09, 138.98, 151.44, and 164.19; HRMS (ESI) mlz 295.1662 (M+H) ⁺ (Ci ₆ H ₁₈ N ₆ requires 295.1652).

Example 6

6-(3-chlorophenylamino)-9-ethyl-9H-purine-2-carbonitrile (32)

General procedure for the formation of 2,6-dichloro-9-alkyl-9H-purines (Step 1): 2,6-dichloro-9-ethyl-9H-purine - To a solution of 2,6-dichloro-9H-purine (2 g, 10.58 mmol) in acetone (45 ml) was added sodium carbonate (2.25 g, 21.16 mmol). The reaction vessel was equipped with a reflux condenser, and the mixture was heated under reflux conditions for 20 minutes. After that time, iodoethane (0.855 ml, 10.58 mmol) was added in one portion, and the reaction mixture was allowed to stir for 5 hrs. Upon completion, the reaction mixture was concentrated under reduced pressure and directly purified on silica column. Gradient elution with ethyl acetate (2→40%) in hexanes provided regioisomers 2,6-dichloro-9-ethyl-9H-purine and 2,6-dichloro-7-ethyl-7H-purine as pale yellow solids: yield (1.5 g, 6.91 mmol, 82 %; 0.4 g, 1.843 mmol, 87 %, respectively).

General procedure for the formation of 2-chloro-N-phenyl-9-ethyl-9H-purin-6- amines (Step 2): 2-chloro-N-(3-chlorophenyl)-9-ethyl-9H-purin-6-amine - To a solution of 2,6-dichloro-9-ethyl-9H-purine (0.5 g, 2.304 mmol) in DMF (2 ml) was added 3-chloroaniline (0.294 g, 2.304 mmol) and Hunig'sBase (0.402 ml, 2.304 mmol). The reaction mixture was sealed in a microwave tube (2-5ml) and heated to 110 °C for 30 min at 90 W. Upon completion, the reaction mixture was diluted with ethyl acetate and washed several times with 3N lithium chloride solution. The organic layer was separated, dried on magnesium sulfate, filtered, concentrated under reduced pressure and directly purified on silica column. Gradient elution with ethyl acetate (5→50%) in hexanes provided 2-chloro-N-(3-chlorophenyl)-9-ethyl- 9H-purin-6-amine as a pale yellow solid: yield (.640 g, 2.077 mmol, 90 %).

General procedure for the formation of 6-phenylamino-9-ethyl-9H-purine-2- carbonitriles (Step 3): 6-(3-chlorophenylamino)-9-ethyl-9H-purine-2- carbonitrile (32) - To a solution of 2-chloro-N-(3-chlorophenyl)-9-ethyl-9H-purin- 6-amine (.05 g, 0.162 mmol) in DMSO (1 ml) was added KCN (10.57 mg, 0.162 mmol). The reaction mixture was sealed in a microwave tube (0.5 -2ml) and heated in a microwave to 140 °C for lhr at 90 W. Upon completion, the reaction mixture was dilute with ethyl acetate and washed several times with saturated sodium chloride solution. The organic layer was separated, dried on magnesium sulfate, filtered, concentrated under reduced pressure and directly purified on silica column. Gradient elution with ethyl acetate (10— >70%) in hexanes provided 6-(3- chlorophenylamino)-9-ethyl-9H-purine-2-carbonitrile as a colorless solid: yield (45mg, 0.151 mmol, 93 %); ¹ H (CDCl ₃ ) δ 1.60 (t, / = 7.34 Hz, 3 H), 4.34 (q, / = 7.37 Hz, 2 H), 7.15 (ddd, / = 8.05, 1.98, 0.93 Hz, 1 H), 7.35 (t, / = 8.12 Hz, 1 H), 7.75 (ddd, J = 8.24, 2.18, 0.88 Hz, 1 H), 7.87 (t, J = 2.05 Hz, 1 H), 7.89 (s, 1 H), 8.03 (s, 1 H).

Example 7

6-(3-chlorophenylamino)-9-cyclopentyl-9H-purine-2-carboni trile (33)

The title compound was prepared using a procedure similar to that detailed for 32, substituting cyclopentylbromide in Step 1, providing the product as a colorless solid: yield (42mg, 87%); ¹ H (CDCl ₃ ) δ 1.80 - 1.93 (m, 2 H), 1.94 - 2.10 (m, 4 H), 2.31 - 2.44 (m, 2 H), 4.99 (dq, / = 7.29, 7.14 Hz, 1 H), 7.15 (ddd, / = 8.00, 2.01, 0.95 Hz, 1 H), 7.35 (t, / = 8.12 Hz, 1 H), 7.76 (ddd, / = 8.27, 2.20, 0.93 Hz, 1 H), 7.90 (t, / = 2.05 Hz, 1 H), 8.12 (s, 1 H), 8.49 (s, 1 H); ¹³ C NMR (CDCl ₃ ) δ 23.86, 32.79, 57.10, 116.45, 118.51, 120.40, 124.37, 130.13, 138.94, 140.96, 148.98, 151.42 .

Example 8

9-(2,2-difluoroethyl)-6-(3,5-difluorophenylamino)-9H-puri ne-2-carbonitrile (34)

General procedure for the formation of 2,6-dichloro-9-fluoroalkyl-9H-purines (Step 1): 2,6-dichloro-9-(2,2-difluoroethyl)-9H-purine - To a solution of 2,6- dichloro-9H-purine (0.5 g, 2.65 mmol) 2,2-difluoroethanol (0.260 g, 3.17 mmol), and triphenylphosphine (0.763 g, 2.91 mmol) in THF (10.58 ml) was added diisopropylazadicarboxylate (0.566 ml, 2.91 mmol) dropwise at rt. The reaction mixture was allowed to stir for 16 hr, upon which time the solvent was removed under reduced pressure. The remaining residue was directly purified on silica column. Gradient elution with ethyl acetate (10→70%) in hexanes provided 2,6- dichloro-9-(2,2-difluoroethyl)-9H-purine as an off-white solid: (577mg, 2.28 mmol, 86 %).

Step 2: 2-chloro-9-(2,2-difluoroethyl)-N-(3,5-difluorophenyl)-9H-pur in-6-amine

- The title compound was prepared using a procedure similar to Example 6 above, substituting 3,5-difluoroaniline and increasing the reaction temperature to 150°C, providing the product as a pale yellow solid: yield (85 mg, 62%).

9-(2,2-difluoroethyl)-6-(3,5-difluorophenylamino)-9H-puri ne-2-carbonitrile (34) - The title compound was prepared using a procedure similar to Example 6 above, providing the product as a colorless solid: yield (30 mg, 36%). ¹ H (CDCI ₃ ) 54.66 (td, / = 14.38, 3.62 Hz, 2 H), 6.06 (t, / = 3.62 Hz, 1 H), 6.20 (t, / = 3.62 Hz, 1 H), 6.33 (t, J = 3.64 Hz, 1 H), 6.65 (tt, J = 8.84, 2.29 Hz, 1 H), 7.46 (dd, J = 8.88, 2.23 Hz, 2 H), 7.86 (s, 1 H), 8.10 (s, 1 H) ; HRMS (ESI) mlz 337.08271 (M+H) ⁺ (Ci ₄ H ₈ F ₄ N ₆ requires 337.08252).

Example 9 9-(3,5-difluorophenyl)-6-(ethylamino)-9H-purine-2-carbonitri le (35)

General procedure for the formation of 2,6-dichloro-9-fluoroalkyl-9H-purines (Step 1): 2,6-dichloro-9-(3,5-difluorophenyl)-9H-purine - To a solution of 2,6- dichloro-9H-purine (0.6 g, 3.17 mmol) in CH ₂ Cl ₂ (15 ml) was added 3,5- difluorophenylboronic acid (1.003 g, 6.35 mmol), copper (II) acetate (1.153 g, 6.35 mmol), 4 A molecular sieves (~250mg), and TEA (1.3 ml, 9.5 mmol). The reaction mixture was stirred at 100 °C for 16 hr. Upon completion, the reaction mixture was dilute with ethyl acetate, filtered through a pad of celite, washed with water and brine (3 X 3OmL), and the solvent was removed under reduced pressure. The remaining residue was directly purified on silica column. Gradient elution with ethyl acetate (5— >65%) in hexanes provided 2,6-dichloro-9-(3,5-difluorophenyl)-9H- purine as a pale yellow solid: (200mg, .664 mmol, 21 %).

2-chloro-9-(3,5-difluorophenyl)-N-ethyl-9H-purin-6-amine - The title compound was prepared using a procedure similar to Example 6 above, substituting ethylamine, providing the product as a pale yellow solid: yield (60 mg, 83%).

9-(3,5-difluorophenyl)-6-(ethylamino)-9H-purine-2-carboni trile (35) - The title compound was prepared using a procedure similar to Example 6 above, providing the product as a colorless solid: yield (23 mg, 48%).

Cruzain enzymatic assays in 1536-well format. The compounds were initially prepared as 10 mM DMSO stock solutions and were arrayed for testing as serial twofold dilutions at 5 μL per well in 1,536-well Greiner polypropylene compound plates following previously described protocols (REF: Yasgar, A., Shinn, P., Michael, S., Zheng, W., Jadhav, A., AuId, D., Austin, C, Inglese, J. & Simeonov, A. (2008). Compound Management for Quantitative High-Throughput Screening. J Assoc Lab Automat 13, 79-89.). Cruzain activity was assayed in freshly prepared 100 mM acetate buffer pH 5.5, containing 5 mM dithiothreitol (DTT) and 0.01 % Triton X-100. Three μl of reagents (buffer as negative control and cruzain at 1.5 nM final concentration in the remainder of the plate) were dispensed by Flying Reagent Dispenser™ (FRD) (Beckman Coulter.Inc, Fullerton, CA) into a black solid-bottom 1,536-well plate (Greiner Bio-One, Monroe, NC). Inhibitors were delivered as 23 nL of DMSO solutions via pintool transfer; vehicle-only control consisted of 23 nL DMSO. The plate was incubated for 15 min at room temperature, and then 1 μL of cruzain fluorogenic substrate (Z-FR-AMC, Bachem, 2 uM final concentration) was added to start the reaction. Following substrate dispense, the plate was immediately transferred into ViewLux High-throughput CCD imager (Perkin-Elmer, Waltham, MA) for kinetic fluorescence data collection utilizing standard 340 nm excitation and 450 nm emission filter set. Percent inhibition was calculated relative to the no- enzyme and uninhibited controls (48 wells averaged per condition) by using the fluorescence intensity change recorded during the first 60 seconds of reaction. Cruzain, rhodesain and tbcatB enzymatic assays in 96-well plate format. 100 μL per well of recombinant enzyme cruzain, rhodesain or tbcatB (in a buffer solution consists of 100 mM sodium acetate pH 5.5, 5 mM DTT and 0.001% Triton X-100) was added to a 96-well black plate that contained 1 μL of test compound (in DMSO). The enzyme-compound mixture was incubated for 5 min at room temperature. Then 100 μL per well of substrate Z-FR-AMC (in the same buffer solution as above) was added to the enzyme-compound mixture to initiate the reaction. The rate of increase in fluorescence (units / sec), resulting from the proteolytic cleavage of the substrate leading to the release of fluorogenic AMC was monitored as with an automated microtiter plate spectrofluorimeter (SpectraMax M5, Molecular Devices) with fluorescence readout setting of excitation at 355 nm and emission at 460 nm. The assay concentrations of enzyme and substrate are 4 nM (cruzain), 4 nM (rhodesain), 258 nM (tbcatB) and 10 μM Z-Phe-Arg-AMC, respectively. Positive control wells contained 1 μL of DMSO. IC ₅₀ curve fitting was performed with Prism 4 software (GraphPad, San Diego, CA).

Competition assays and Ki determination. Cruzain activity was conducted in 96- well plates as described above, for varying concentrations of compound 5 and Z-FR- AMC, in presence of 0.01% Triton. Each concentration of 5 (0, 25, 50, 100, 200 and 500 nM), was tested in seven concentrations of substrate (0.31 to 20 uM, in 2-fold increments). Compound was incubated with cruzain for 15 minutes prior to addition of substrate and enzyme activity was then monitored for five minutes. Given the covalent reversible mechanism of this compound, initially an increase in rates of activity was observed, until equilibrium was reached. Calculations of rates of cruzain activity were based on time points after equilibrium, when a uniform rate was observed with time. All assays were performed in duplicate. Lineaweaver-Burk plot was built in Prism 4.

Trypanosoma brucei brucei assay. Trypanosoma brucei brucei strain 221 was grown in complete HMI-9 medium containing 10% FBS, 10% Serum Plus medium (Sigma Inc. St. Louis Mo. USA) and IX penicillin/streptomycin. The trypanosomes were diluted to IxIO ⁵ per ml in complete HMI-9 medium. 95 μL per well of the diluted trypanosomes was added to sterile Greiner 96-well flat white opaque culture plates that contained 5 μL of test samples (in 10% DMSO). Control wells contained 95 μL of the diluted trypanosomes and 5 μL of 10% DMSO while control wells for 100% inhibition contained 95 μL of the diluted trypanosomes and 5 μL of ImM Thimerosal (in 10% DMSO). Trypanosomes were incubated with test samples for 48 h at 37 °C with 5% CO2 before monitoring viability. Trypanosomes were then lysed in the wells by adding 50 μl of CellTiter-GloTM (Promega Inc., Madison, WI, USA). Lysed trypanosomes were placed on an orbital shaker at room temperature for 2 min. The resulting ATP-bioluminescence of the trypanosomes in the 96-well plates was measured at room temperature using an Analyst HT plate reader

(Molecular Devices, Sunnyvale, CA, USA). All IC ₅₀ curve fittings were performed with Prism 4 software (GraphPad, San Diego, CA).

Trypanosoma cruzi assay. Assays were conducted as previously described. Seven triazine nitriles (compounds 22, 23, 25, 27-30) were tested at 1, 5 and 10 uM. All triazines were toxic against macrophages at 10 μM, inactive against T. cruzi at 1 μM and either toxic (23 and 29) or inactive (22, 25, 27, 28, 30) at 5 μM. Purines 32, 35, and 36 were tested at 10 μM and were inactive.

Cruzain expression and purification. Procruzain truncated at the C-terminal was expressed and purified using a modified protocol (Lee, Balouch and Craik, unpublished results). A 0.5 mg/mL solution of procruzain (in 100 mM sodium acetate pH 5.5, 10 mM EDTA, 5 mM DTT and 1 M NaCl, pH adjusted to 5.3) was activated at 37 C for three hours. After activation, cruzain was dialyzed in 10 mM Tris buffer pH 7.5, inhibited with the covalent reversible inhibitor methyl methanethiosulfonate (MMTS) and dialyzed again in the same buffer. Finally, the protein was purified in a MONO-Q anion exchange column, using a 0 to 500 mM NaCl gradient in 10 mM Tris buffer (pH 7.5).

Crystallography. MMTS inhibited cruzain was concentrated in to 1 mg/mL in 2 mM bis tris pH 5.8. In order to reverse MMTS inhibition, 5 mM DTT was added, followed by addition of 400 uM of compound 32. The solution was stirred for a few hours at 4 C, until cruzain was completely inhibited, and the protein was concentrated down to 7.5 mg/mL. Hanging drops for 384 crystallography conditions (Joint Center Structure Genomics screens I- IV, Qiagen) were set up using a Mosquito ^® (TTP Labtech). Each condition was screened in 1:1 and 2:1 ratio between protein solution and mother liquour. After two weeks incubation at 18 C a 200 urn crystal was obtained in 0.1 M Tris pH 8.5, 2.0 M NH ₄ H ₂ PO ₄ , in the (200 nL protein solution) : (100 nL mother liquor) drop. Crystals were then reproduced in the same conditions in 2-4 uL hanging drops, were transferred to a solution of 25% glycerol in mother liquor and crycooled in liquid nitrogen. Data collection was performed on frozen crystals in beamline 7-1 in the

Stanford Synchrotron Radiation Lightsource at the SLAC National Accelerator Laboratory. Reflections were indexed and integrated using Ipmosflm. PHASER was used for the initially phasing, based on a previously reported structure of cruzain bound to a non-covalent inhibitor (PDB entry 1ME3), with the ligand, water molecules and ions removed. The space group was P6s22. Data refinement was performed using REFMAC5 package and models were built and waters placed using Coot.

Incorporation by Reference The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended with be encompassed by the following claims. Several embodiments of the compositions and methods disclosed herein are described in the following numbered paragraphs:

1. A compound of Formula I, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, each X is independently O, S, or NR _A ; each R ₁ is independently halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, or N(R _A )(R _B );

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl, an optionally substituted C _3-12 heteroaiyl, an optionally substituted C _7-2 oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; Z is CN, C(O)CH ₂ Y, or CH=CH-Y' ;

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ - ₁₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC _7-2 oaralkyl, or an optionally substituted OC _1-12 haloalkyl;

Y' is an optionally substituted S(O) _p C _1-12 alkyl, an optionally substituted S(O) _p C _3-12 aryl, an optionally substituted S(O) _p C _3-12 heteroaryl, an optionally substituted S(0) _p C ₇ _ ₂ oaralkyl, or an optionally substituted S(O) _p C _1-12 haloalkyl, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, an optionally substituted OC _1-12 haloalkyl, an optionally substituted NR'C _1-12 alkyl, an optionally substituted NR'C _3-12 cycloalkyl, an optionally substituted NR'C ₃ _ ₁₂ heterocycloalkyl, an optionally substituted NR'C _3-12 aryl, an optionally substituted NR'C _3-12 heteroaryl, an optionally substituted NR' C ₇ _ ₂ oaralkyl, or an optionally substituted NR'C _1-12 haloalkyl; each R' is independently an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; p is 0, 1, or 2, and n is O, 1, 2, 3, 4, or 5.

2. The compound of paragraph 1 , of Formula I-a, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein, X is O, S, or NR _A ;

R is an optionally substituted C _1-12 alkyl, an optionally substituted C _{3- 12} cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _7-2 oaralkyl, an optionally substituted C _1-12 haloalkyl, or an optionally substituted C _1-12 alkoxy; and R _A is H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-

₁₂ acyl. 3. The compound of paragraph 2, wherein X is NR _A and R _A is H.

4. The compound of paragraph 2, wherein R is an optionally substituted C _1-12 alkyl or an optionally substituted C _3-12 cycloalkyl.

5. The compound of paragraph 4, wherein R is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 4- hydroxybutyl, or 2-hydroxyethyl.

6. The compound of paragraph 1, of formula I-b, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

X is O, S, or NR _A ; each R ₁ is independently halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, or N(R _A )(R _B ); each R _A and R _B is independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; and n is 0, 1, 2, 3, 4, or 5.

7. The compound of paragraph 6, wherein X is NR _A and R _A is H.

8. The compound of paragraph 6, wherein each R ₁ is independently H, halogen, nitro, an optionally substituted C _1-12 alkyl, or an optionally substituted C ₃ _ ₁₂ aryl and n is 1, 2, or 3. 9. The compound of paragraph 8, wherein each R ₁ is independently H, methyl, phenyl, trifluoromethyl, nitro, fluoro, chloro, or bromo, and n is 1, 2, or 3.

10. A compound of Formula II, or pharmaceutically acceptable salt, solvate or hydrate thereof:

( II);

wherein,

W is N, CH, or CR';

X is O, S, or NR _A ; each R' is independently an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; R ₁ is H, halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C ₃ _i2heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, an optionally substituted C _1-12 alkoxy, or hydroxy; wherein R is optionally substituted by R ₁ ;

Z is CN, C(O)CH ₂ Y, or CH=CH-Y' ; Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ _ 1 ₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl; Y' is an optionally substituted S(O) _p C _1-12 alkyl, an optionally substituted

S(O) _p C _3-12 aryl, an optionally substituted S(O) _p C _3-12 heteroaryl, an optionally substituted S(0) _p C ₇ _ ₂ oaralkyl, or an optionally substituted S(O) _p C _1-12 haloalkyl, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, an optionally substituted OC _1-12 haloalkyl, an optionally substituted NR'C _1-12 alkyl, an optionally substituted NR'C _3-12 cycloalkyl, an optionally substituted NR'C ₃ _ ₁₂ heterocycloalkyl, an optionally substituted NR'C _3-12 aryl, an optionally substituted NR'C _3-12 heteroaryl, an optionally substituted NR' C ₇ _ ₂ oaralkyl, or an optionally substituted NR'C _1-12 haloalkyl; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ - ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and p is 0, 1, or 2.

11. The compound of paragraph 10, of Formula II-a, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

X is O, S, or NR _A ; each R ₁ is independently H, halogen, nitro, an optionally substituted C _{1- 12} alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C _3-12 heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

;

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, an optionally substituted C _1-12 alkoxy, or hydroxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C _{3- 12} cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is O, 1, 2, 3, 4, or 5.

12. The compound of paragraph 11, wherein X is NR _A and R _A is H.

13. The compound of paragraph 11, wherein each R ₁ is independently F, Cl, Br, I, or an optionally substituted C _1-12 alkyl.

14. The compound of paragraph 13, wherein each R ₁ is independently

wherein,

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _{3- 12} aryl or an optionally substituted C _3-12 heteroaryl; and m is 0, 1, 2, or 3.

15. The compound of paragraph 11, wherein R is an optionally substituted C _1-12 alkyl or an optionally substituted C _3-12 cycloalkyl.

16. The compound of paragraph 14, wherein R is ethyl, hydroxyalkyl, cyclopentyl, or difluoroethyl.

17. The compound of paragraph 10, of Formula II-b, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

X is O, S, or NR _A ; R' is an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl or an optionally substituted C _3-12 heteroaryl, wherein R' is optionally substituted by R ₁ ; each R ₁ is independently H, halogen, nitro, an optionally substituted C _{1- 12} alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C _3-12 heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from ^'

each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _

₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ - ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is 0, 1, 2, 3, 4, or 5.

18. The compound of paragraph 17, wherein X is NR _A and R _A is H.

19. The compound of paragraph 17, wherein R' is an optionally substituted C _1-12 alkyl.

20. The compound of paragraph 17, wherein each R ₁ is independently F,

Cl, Br, or I.

21. The compound of paragraph 10, of Formula II-c, or pharmaceutically acceptable salt, solvate or hydrate thereof: wherein,

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ _ 1 ₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl;

X is O, S, or NR _A ; each R ₁ is independently H, halogen, nitro, an optionally substituted C _{1- 12} alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C _3-12 heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

;

R is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl, an optionally substituted C _3-12 heteroaiyl, an optionally substituted C _7-2 oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, an optionally substituted C _1-12 alkoxy or hydroxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is O, 1, 2, 3, 4, or 5.

22. The compound of paragraph 21, wherein Y is halogen, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, or an optionally substituted OC ₇ _ ₂ oaralkyl, wherein Y is optionally substituted by R ₁ .

23. The compound of paragraph 22, wherein Y is Cl, or

24. The compound of paragraph 10 of Formula II-d, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

Y' is an optionally substituted S(O) _p C _1-12 alkyl, or an optionally substituted S(O) _p C _3-12 aiyl; X is O, S, or NR _A ; R ₁ is H, halogen, nitro, an optionally substituted C _1-12 alkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _1-12 alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C ₃ - ₁₂ heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

R is an optionally substituted C _1-12 alkyl, an optionally substituted C _{3- 12} cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C ₇ _ ₂ oaralkyl, an optionally substituted C ₂ -C ₁₂ alkenyl, an optionally substituted C ₃ -C ₁₂ alkynyl, an optionally substituted C _1-12 acyl, an optionally substituted C _1-12 haloalkyl, an optionally substituted C _1-12 alkoxy or hydroxy; each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl; Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C _{3- 12} cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, n is 0, 1, 2, 3, 4, or 5; and p is 0, 1, or 2.

25. The compound of paragraph 24, wherein Y' is an optionally substituted S(O^Me or an optionally substituted S(O^Ph.

26. The compound of paragraph 10, of Formula II-e, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

Y is halogen, nitro, cyano, an optionally substituted OC _1-12 alkyl, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC ₃ _ 1 ₂ heterocycloalkyl, an optionally substituted OC _3-12 aryl, an optionally substituted OC _3-12 heteroaryl, an optionally substituted OC ₇ _ ₂ oaralkyl, or an optionally substituted OC _1-12 haloalkyl; X is O, S, or NR _A ; R' is an optionally substituted C _3-12 aryl or an optionally substituted C ₃ _ ₁₂ heteroaryl; each R ₁ is independently H, halogen, nitro, an optionally substituted C ₃ _ ₁₂ aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _{1- 12} alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C ₃ - ₁₂ heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ - ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl; R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 atyl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ - ₁₂ &ryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, and n is O, 1, 2, 3, 4, or 5.

27. The compound of paragraph 26, wherein Y is halogen, an optionally substituted OC _3-12 cycloalkyl, an optionally substituted OC _3-12 heterocycloalkyl, an optionally substituted OC _3-12 atyl, an optionally substituted OC _3-12 heteroaryl, or an optionally substituted OC _7-2 oaralkyl, wherein Y is optionally substituted by R ₁ . 28. The compound of paragraph 27, wherein Y is Cl, or

29. The compound of paragraph 10, of Formula II-f, or pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein,

Y' is an optionally substituted S(O) _p C _1-12 alkyl, or an optionally substituted S(O) _p C _3-12 aiyl; X is O, S, or NR _A ;

R' is an optionally substituted C _3-12 aryl or an optionally substituted C _{3- 12} heteroaryl; each R ₁ is independently H, halogen, nitro, an optionally substituted C _{3- 12} aryl, an optionally substituted C _3-12 heteroaryl, an optionally substituted C _{1- 12} alkoxy, an optionally substituted C _1-12 acyl, an optionally substituted C ₃ _ 1 ₂ heterocycloalkyl, or N(R _A )(R _B ); or R ₁ is selected from

each of R _A and R _B are independently H, an optionally substituted C _1-12 alkyl, or an optionally substituted C _1-12 acyl;

Rc is H, an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl;

R _D is an optionally substituted C _1-12 alkyl, an optionally substituted C ₃ _ ₁₂ cycloalkyl, an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C _3-12 aryl; or Rc and R _D together with the nitrogen to which each is connected, may form an optionally substituted C _3-12 heterocycloalkyl, an optionally substituted C ₃ _ ₁₂ aryl or an optionally substituted C _3-12 heteroaryl; m is 0, 1, 2, or 3, p is 0, 1, or 2; and n is O, 1, 2, 3, 4, or 5.

30. A method of inhibiting cruzain or rhodesain in a subject, comprising administering to the subject an effective amount of one or more compounds of Formula I of paragraph 1 or Formula II of paragraph 10, wherein the administration of said compound inhibits cruzain or rhodesain.

31. A method of treating a subject suffering from or susceptible to a disease or disorder related to cruzain or rhodesain, wherein the subject is determined to be in need of inhibition of cruzain or rhodesain, the method comprising the step of administering to the subject an effective amount of one or more compounds of Formula I of paragraph 1 or Formula II of paragraph 10.

32. The method of paragraph 31, wherein the disease or disorder is parasite infection, Chagas disease, African trypanosomiasis, malaria, toxoplasmosis, or coccidiosis.

33. A method of treating a subject suffering from or susceptible to parasite infection, Chagas disease, African trypanosomiasis, malaria, toxoplasmosis, or coccidiosis, comprising administering to the subject an effective amount of one or more compounds of Formula I of paragraph 1 or Formula II of paragraph 10.

34. The method of paragraph 33 wherein the compound is a compound of Formula I-a, I-b, II-a, II-b, II-c, II-d, II-e, or II-f.

35. The method of paragraph 33, wherein the disorder is Chagas disease or African trypanosomiasis.

36. The method of paragraph 35 wherein the compound is a compound of

Formula I-a, I-b, II-a, II-b, II-c, II-d, II-e, or II-f..

37. The method of any one of paragraphs 30, 31 or 33, further comprising administering an additional therapeutic agent.

38. A pharmaceutical composition comprising one or more compounds of any one of paragraphs 1-29 and a pharmaceutically acceptable carrier.

39. A kit comprising an effective amount of one or more compounds of any one of paragraphs 1-29 in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to Chagas disease or African trypanosomiasis.