BACKGROUND OF THE INVENTION Malaria is the fourth largest infectious disease in the world, behind diarrheal diseases, tuberculosis and measles. In humans, the disease is caused by four species of protozoal parasites in the genus Plasmodium. Plasmodium vivax and Plasmodium falciparum are the most common, while Plasmodium ovale and Plasmodium malariae occur less frequently. The majority of deaths (-3 million per year) are children under the age of five. Approximately 90% of the cases (300-500 million per year) are reported from tropical Africa, an area with very limited economic resources.

Historically, the pharmaceutical industry has not invested significantly in developing therapeutics for diseases that occur primarily in this area (Gibbons 1992). Although there are ongoing efforts directed towards development of a malaria vaccine, no suitable vaccine has been produced as yet.

Infection of humans by Plasmodium occurs with a bite from an infected Anopheles mosquito, which injects the parasite in its sporozoite stage into the host.

The parasite migrates through the blood stream to the liver, where it passes through a hepatic trophozoite stage before developing into a merozoite. Merozoites are released into the blood stream where they invade erythrocytes (red blood cells) and develop from a"ring stage"into a more metabolically active trophozoite. The trophozoite divides asexually to become a schizont, which ruptures the host erythrocyte, releasing daughter merozoites that invade other erythrocytes and reinitiate the cycle. The P. falciparum parasite, which causes the most severe form of malaria and is responsible for by far the greatest proportion of deaths, has a 48 hour life cycle within host erythrocytes that is responsible for all of the clinical manifestations of falciparum malaria. During the trophozoite stage, hemoglobin within the host erythrocyte is degraded and utilized as a source of nutrition by the parasite. Hemoglobin degradation is mediated by the action of several malarial proteases including the aspartic proteases plasmepsin I and II and the cysteine protease falcipain.

Recently several factors have heightened concern over the transmission and spread of malaria. One factor is the unprecedented movement and mobility of naive populations into endemic areas. Secondly, environmental degradation is of concern.

Global warming has the potential for both disrupting current patterns of transmission and potentially expanding the range of the Anopheles mosquito. The primary concern however is the increasing resistance to current antimalarial chemotherapy exhibited by Plasmodium. For the past 50 years chloroquine has been the mainstay for both treatment and prophylaxis of malaria. However, chloroquine resistance is now widespread in most areas where P. falciparum is endemic (Olliaro P., et al., JAMA, 275,230-233,1996). Resistance to chloroquine of P. vivax, the second most lethal malaria parasite, is also a growing threat.

Although other antimalarial drugs exist, all of them have significant drawbacks in terms of efficacy and/or safety. Quinine (or IV quinidine) is fairly toxic, and resistance to it is increasing. The use of mefloquine, a compound related to chloroquine, is limited by toxicity and is not approved in the U. S. In addition, its high cost makes mefloquine use in the developing world problematic. Amiodiaquine and fansidar can cause severe toxicity, and resistance to these agents is becoming more common. Primaquine, proguanil, maloprim, and tetracyclines have a limited range of uses, primarily in prophylaxis rather than treatment.

In addition to the geographic spread of drug resistance, the speed of resistance development is increasing as well-chloroquine resistance took 20 to 30 years to develop, whereas resistance against mefloquine developed after only four years of use. Indeed, some P. falciparum strains have been identified that are resistant to all known antimalarial drugs. There is a need for new compounds for the treatment and/or prophylaxis of malaria, particularly for malaria caused by Plasmodium falciparum. In particular, there is a need for new compounds for malaria treatment and/or prophylaxis that are effective against Plasmodium, especially P. falciparum, that are resistant to established antimalarial drugs such as chloroquine.

Efforts have been made to overcome the rapid development of resistance by using combination therapies and/or by identifying compounds with new mechanisms of action. However, even if such compounds with new modes of action could be identified, there remains a risk that the parasites would soon develop resistance to the new agents. Thus, there remains a need for the identification of novel antimalarial agents, preferably those with reduced risk of resistance development.

SUMMARY OF THE INVENTION The present invention provides a novel class of compounds and pharmaceutically acceptable derivatives thereof that are bifunctional inhibitors of aspartic and cysteine proteases. In particular, the compounds are bifunctional inhibitors of aspartic and cysteine proteases found in Plasmodium, namely plasmepsins I and II and falcipain. In other words, the compounds are characterized in that they incorporate features of plasmepsin inhibitors and features of falcipain inhibitors so that a single molecule exhibits inhibitory activity against both types of proteases. The compounds can be used alone or in combination with other agents including antimalarials, antibiotics, and vaccines, for the prophylaxis and treatment of malaria.

The invention provides a class of compounds represented by formula (I): in which X represents either CH2, NH, O, or S and Y represents either NH, O, or S; in which the conformation around the central double bond between carbon atoms numbered 3 and 4 can be either cis (E) or trans (Z) and the stereochemical conformations around the four chiral centers numbered 1,2,5, and 6 can be any combination of R and S; in which R'represents but is not limited to a residue of the following list: hydrogen, alkoxycarbonyl, arylkoxycarbonyl, alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl, cycloalkylalkanoyl, arylkanoyl, aroyl, aryloxycarbonyl, aryloxycarbonylalkyl, aryloxyalkanoyl, heterocyclylalkanoyl, heterocyclyloxycarbonyl, heterocyclylalkanoyl, heterocyclylalkoxycarbonyl, heteroarylkanoyl, heteroarylkoxycarbonyl, heteroaryloxycarbonyl, heteroaryl, alkyl, alkenyl, cycloalkyl, aryl, arylkyl, aryloxyalkyl, heteroaryloxyalkyl, hydroxyalkyl, aminocarbonyl, aminoalkanoyl, mono-and di-substituted aminocarbonyl and mono- and di-substituted aminoalkyl, alkoxyalkyl, alkylthioalkyl, mono-and di-substituted aminoalkanoyl radicals wherein the substitutions are selected from alkyl, aryl, arylkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylkyl, heterocycloalkyl, heterocycloalkylalkyl radicals, or where said aminoalkanoyl radical is di-substituted, said substituents along with the nitrogen atom to which they are attached from a heterocycloalkyl or heteroaryl radical; in which R"and R"'can each be independently selected from, but are not limited to, the following list: lower aLkyl, lower alkoxy, lower alkylthio, mono-or di-lower alkyl amino, aralkyl, arylkoxy, arylkylthio, arylkylamino, cycloalkylalkyl, cycloalkylalkoxy, cycloalkylalkylthio, cycloalkylalylamino, lower cycloalkyl, aryl, arylkyl, and heteroaryl. R"and R"'can also represent the side chain (R group) of an amino acid residue remaining after formation of the linkage bonds, the side chain being optionally substituted with any of the groups in the previous list, i. e., lower alkyl, lower alkoxy, lower alkylthio, mono-or di-lower alkyl amino, aralkyl, arylkoxy, arylkylthio, arylkylamino, cycloalkylalkyl, cycloalkylalkoxy, cycloalkylalkylthio, cycloalkylalylamino, lower cycloalkyl, aryl, arylkyl, and heteroaryl. Also encompassed are compounds where R"and R"'are connected by a bridging moiety having 1-10 atoms comprised of any stable combination of C, N, O, or S. This chain may be optionally substituted by a functionality including, but not limited to, halo, hydroxy, amino, lower alkyl, lower alkoxy, lower alkylthio, mono-or di-lower alkyl amino, oxo, thiono, alkylimino, mono-or di-alkylmethylidene. The bridging group may also be unsaturated so as to include residues of alkenes, imines, alkynes and allenes. Furthermore, any part of the bridging moiety may comprise part of an optionally substituted aromatic, heteroaromatic, or cycloalkyl, or heterocycloalkyl ring. Amino acids from which the residues containing R"and R"'can be derived include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, arginine, phenylalanine, homo-phenylalanine, tyrosine, tryptophane, and histidine; and in which R""represents but is not limited to a residue of the following list: alkyl, arylkyl, aryl, aryloxyalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, which are optionally substituted by halo, hydroxy, amino, lower alkyl, lower alkoxy, lower alkylthio, mono-or di-lower alkyl amino, oxo, thiono, alkylimino, mono-or di- alkylmethylidene. Although in certain embodiments of the invention R"and R"' represent the side chain of an amino acid naturally found in proteins, R"and R"'can also represent the side chain of an amino acid not naturally found in proteins, including an optical isomer or a modified form of an amino acid found in proteins. In either case, R"and R"'can be optionally substituted as indicated above.

In certain embodiments the present invention provides a set of novel bifunctional protease inhibitors having stereochemical diversity that are useful for the treatment and/or prophylaxis of parasitic infection, particularly malaria.

In another aspect, the invention provides pharmaceutical compositions including novel bifunctional aspartic and cysteine protease inhibitors. The pharmaceutical compositions can be used alone or in combination with other agents for the prophylaxis and treatment of malaria. The inventive compositions comprise an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, such as a diluent or excipient.

In another aspect, the invention provides methods for prophylaxis and/or treatment of conditions associated with parasitic infection, in particular malaria, by administering an effective amount of a bifunctional inhibitor of parasite aspartic and cysteine proteases to a host or patient. In certain preferred embodiments of the inventive methods an effective amount of a compound of the present invention is administered to the host or patient. In certain preferred embodiments a compound of the present invention and an effective amount of one or more other compounds useful in the treatment of malaria are administered to the host or patient.

DEFINITIONS To more clearly and concisely describe the invention, the following definitions and discussions of terms are provided.

The phrase,"pharmaceutically acceptable derivative", as used herein, denotes any pharmaceutically acceptable salt, ester, or salt of such ester, of such compound, or any other adduct or derivative which, upon administration to a patient, is capable of providing (directly or indirectly) a compound as otherwise described herein, or a metabolite or residue thereof. Pharmaceutically acceptable derivatives thus include among others pro-drugs. A pro-drug is a derivative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety which is susceptible to removal in vivo yielding the parent molecule as the pharmacologically active species. An example of a pro-drug is an ester that is cleaved in vivo to yield a compound of interest. Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs, are known and may be adapted to the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail herein below.

Certain compounds of the present invention and definitions of specific functional groups are also described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in"Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito, 1999, the entire contents of which are incorporated herein by reference.

It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term"substituted"whether preceded by the term"optionally"or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. 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. As used herein, the term"substituted"is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment of and/or prevention of parasitic infections, particularly malaria. The term "stable", as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein.

The term"aliphatic", as used herein, includes both saturated and unsaturated, straight chain (i. e., unbranched), branched, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups.

As will be appreciated by one of ordinary skill in the art,"aliphatic"is intended herein to include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term"alkyl"includes both straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl","alkynyl"and the like. Furthermore, as used herein, the terms"alkyl", "alkenyl","alkynyl"and the like encompass both substituted and unsubstituted groups.

Unless otherwise specified, alkyl and other aliphatic groups preferably contain 1-6, or 1-3, contiguous aliphatic carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,-CH2-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl,-CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl,- CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,-CH2-cyclohexyl moieties and the like, which again, may bear one or more substituents.

In certain embodiments of the present invention Cl-C3 or Cl-C6 alkyl moieties are employed. As used herein, the terms"Cl-C3-alkyl"and"Cl-C6-alkyl"refer to saturated, substituted or unsubstituted, straight-or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and three, and one and six carbon atoms, respectively, by removal of a single hydrogen atom.

Examples of Cl-C3-alkyl radicals include, but are not limited to, methyl, ethyl, propyl and isopropyl. Examples of Cl-C6-alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl and n-hexyl.

In certain embodiments of the present invention, C2-C6 alkenyl moieties are employed. The term"C2-C6-alkenyl"denotes a monovalent group derived from a hydrocarbon moiety containing from two to six carbon atoms and having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Additionally, the C2-C6 alkenyl moieties, as used herein, may be substituted or unsubstituted.

Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

In certain embodiments of the present invention, C2-C6 alkynyl moieties are employed. The term"Cl-C6-alkynyl"as used herein refers to a monovalent group derived from a hydrocarbon containing from two to six carbon atoms and having at least one carbon-carbon triple bond by the removal of a single hydrogen atom.

Additionally, the C2-C6 alkenyl moieties, as used herein, may be substituted or unsubstituted. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.

The term"Cl-C6-alkoxy"as used herein refers to a Ci-C6-alkyi group, as previously defined, attached to the parent molecular moiety through an oxygen atom.

Examples of C1-C6-alkoxy, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy.

The term"alkylamino"refers to a group having the structure-NHR'wherein R'is alkyl, as defined herein. Examples of alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino and the like. In certain embodiments, Cl- C3 alkylamino groups are utilized in the present invention. The term"Cl-C 3- alkylamino"as used herein refers to one or two Cl-C3-alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. Examples of Cl-C3-alkylamino include, but are not limited to methylamino, dimethylamino, ethylamino, diethylamino, and propylamino.

Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to: F, Cl, Br, I, OH, NO2, CN, C (O)-Cz-C6-alkyl, C (O)-aryl, C (O)-heteroaryl, C02-alkyl, C02-aryl, C02-heteroaryl, CONH2, CONH-Ci-C6-alkyl, CONH-aryl, CONH-heteroaryl, OC (O)-Cl-C6-alkyl, OC (O)-aryl, OC (O)-heteroaryl, OCO2-alkyl, OC02-aryl, OC02- heteroaryl, OCONH2, OCONH-Cl-C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC (O)-Cl-C6-alkyl, NHC (O)-aryl, NHC (O)-heteroaryl, NHCO2-alkyl, NHCO2-aryl, NHCONH-heteroaryl, S02-Cl-C6-alkyl, SO2-aryl, C3-C6-cycloalkyl, CF3, CH2CF3, CHC12, CH20H, CH2CH2OH, CH2NH2, CH2S02CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, Cl-C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, Cl-C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, Cl-C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.

The term"amino acid"as used herein refers to an organic acid containing both a basic amino group (NH2) and an acidic carboxyl group (COOH). Amino acids include both a-amino acids, in which the-NH2 group is attached to the carbon atom adjacent to the-COOH group and also acids in which the-NH2 and-COOH groups are separated by more than one intervening carbon atom. Amino acids frequently exist as dipolar ions in aqueous solution. As used herein,"amino acid"may refer to the unionized or ionized form.

The term"aprotic solvent"as used herein refers to a solvent that is relatively inert to proton activity, i. e., not acting as a proton-donor. Examples include, but are not limited to, hydrocarbons, such as hexane and toluene, for example, halogenated hydrocarbons, such as, for example, methylene chloride, ethylene chloride, chloroform, and the like, heteroaryl compounds, such as, for example, tetrahydrofuran and N-methylpyrrolidinone, and ethers such as diethyl ether, bis-methoxymethyl ether. Such compounds are well known to those skilled in the art, and it will be obvious to those skilled in the art that individual solvents or mixtures thereof may be preferred for specific compounds and reaction conditions, depending upon such factors as the solubility of reagents, reactivity of reagents and preferred temperature ranges, for example. Further discussions of aprotic solvents may be found in organic chemistry textbooks or in specialized monographs, for example: Organic Solvents Physical Properties and Methods of Purification, 4th ed., edited by John A. Riddick et al., Vol. II, in the Techniques of Chemistry Series, John Wiley & Sons, NY, 1986.

In general, the terms"aryl"and"heteroaryl", as used herein, refer to stable mono-or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously mentioned substitutents, i. e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound.

In certain embodiments of the present invention,"aryl"refers to a mono-or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like. In certain embodiments of the present invention, the term"heteroaryl", as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom 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, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups (including bicyclic aryl groups) can be unsubstituted or substituted, wherein substitution includes replacement of one, two or three of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: F, Cl, Br, I, OH, N02, CN, C (O)-Cl-C6-alkyl, C (O)-aryl, C (O)-heteroaryl, CO2-alkyl, CO2-aryl, C02- heteroaryl, CONH2, CONH-Cl-C6-alkyl, CONH-aryl, CONH-heteroaryl, OC (O)-Cl- C6-alkyl, OC (O)-aryl, OC (O)-heteroaryl, OC02-alkyl, OC02-aryl, OC02-heteroaryl, OCONH2, OCONH-Cl-C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC (O)-Cl-C6- alkyl, NHC (O)-aryl, NHC (O)-heteroaryl, NHCO2-alkyl, NHC02-aryl, NHCONH- heteroaryl, SO2-Ci-C6-alkyl, S02-aryl, C3-C6-cycloalkyl, CF3, CH2CF3, CHC12, CH20H, CH2CH20H, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, Cl-C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, Cl-C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, Cl-C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples which are described herein.

The term"cycloalkyl", as used herein, refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic or hetercyclic moieties, may optionally be substituted. F, Cl, Br, I, OH, N02, CN, C (O)-Cl-C6-alkyl, C (O)-aryl, C (O)-heteroaryl, CO2-alkyl, C02-aryl, C02-heteroaryl, CONH2, CONH- Cl-C6-alkyl, CONH-aryl, CONH-heteroaryl, OC (O)-Cl-C6-alkyl, OC (O)-aryl, OC (O)-heteroaryl, OC02-alkyl, OC02-aryl, OCO2-heteroaryl, OCONH2, OCONH- Cl-C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC (O)-Ci-C6-alkyl, NHC (O)-aryl, NHC (O)-heteroaryl, NHC02-alkyl, NHC02-aryl, NHCONH-heteroaryl, SO2-Cl-C6- alkyl, SO2-aryl, C3-C6-cycloalkyl, CF3, CH2CF3, CHC12, CH20H, CH2CH20H, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, Cl-C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, Cl-C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, Cl- C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

The term"heteroaliphatic", as used herein, refers to aliphatic moieties which contain one or more oxygen, sulfur, nitrogen, phosphorous or silicon atoms, e. g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, or cyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to: F, Cl, Br, I, OH, N02, CN, C (O)-Cl-C6- alkyl, C (O)-aryl, C (O)-heteroaryl, CO2-alkyl, C02-aryl, C02-heteroaryl, CONH2, CONH-Cl-C6-alkyl, CONH-aryl, CONH-heteroaryl, OC (O)-Cl-C6-alkyl, OC (O)-aryl, OC (0)-heteroaryl, OC02-alkyl, OC02-aryl, OC02-heteroaryl, OCONH2, OCONH- Ci-C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC (O)-Cl-C6-alkyl, NHC (O)-aryl, NHC (0)-heteroaryl, NHC02-alkyl, NHCO2-aryl, NHCONH-heteroaryl, SO2-Cl-C6- alkyl, S02-aryl, C3-C6-cycloalkyl, CF3, CH2CF3, CHC12, CH20H, CH2CH20H, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, Cl-C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, Cl-C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, Cl- C6-alkyl-thio, or methylthiomethyl. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.

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

The term"haloalkyl"denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term"heterocycloalkyl", as used herein, refers to a non-aromatic 5-, 6-or 7-membered ring or a bi-or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a"substituted heterocycloalkyl"group is utilized and as used herein, refers to a heterocycloalkyl group, as defined above, substituted by independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to: F, Cl, Br, I, OH, N02, CN, C (O)-Cl-C6-alkyl, C (O)-aryl, C (O)-heteroaryl, CO2-alkyl, COx-aryl, CO2-heteroaryl, CONH2, CONH- Cl-C6-alkyl, CONH-aryl, CONH-heteroaryl, OC (O)-Cl-C6-alkyl, OC (O)-aryl, OC (O)-heteroaryl, OCO2-alkyl, OCO2-aryl, OCO2-heteroaryl, OCONH2, OCONH- Cl-C6-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC (O)-Cl-C6-alkyl, NHC (O)-aryl, NHC (O)-heteroaryl, NHCO2-alkyl, NHC02-aryl, NHCONH-heteroaryl, SO2-Cl-C6- alkyl, S02-aryl, C3-C6-cycloalkyl, CF3, CH2CF3, CHC12, CHzOH, CH2CH20H, CH2NH2, CH2SO2CH3, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy, Cl-C6-alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino, Cl-C3-alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, Cl- C6-alkyl-thio, or methylthiomethyl.

"Hydroxy-protecting group", as used herein, refers to an easily removable group which is known in the art to protect a hydroxyl group against undesirable reaction during synthetic procedures and to be selectively removable. The use of hydroxy-protecting groups is well known in the art for protecting groups against undesirable reactions during a synthetic procedure and many such protecting groups are known, cf., for example, T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nid edition, John Wiley & Sons, New York (1991). Examples of hydroxy-protecting groups include, but are not limited to, methylthiomethyl, tert-dimethylsilyl, tert- butyldiphenylsilyl, ethers such as methoxymethyl, and esters including acetyl benzoyl, and the like.

The term"oxo"denotes a group wherein two hydrogen atoms on a single carbon atom in an alkyl group as defined above are replaced with a single oxygen atom (i. e. a carbonyl group).

The term"protected-hydroxy"refers to a hydroxy group protected with a hydroxy protecting group, as defined above, including benzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl groups, for example.

The term"protogenic organic solvent"as used herein refers to a solvent that tends to provide protons, such as an alcohol, for example, methanol, ethanol, propanol, isopropanol, butanol, t-butanol, and the like. Such solvents are well known to those skilled in the art, and it will be obvious to those skilled in the art that individual solvents or mixtures thereof may be preferred for specific compounds and reaction conditions, depending upon such factors as the solubility of reagents, reactivity of reagents and preferred temperature ranges, for example. Further discussions of protogenic solvents may be found in organic chemistry textbooks or in specialized monographs, for example: Organic Solvents Physical Properties and Methods of Purification, 4th ed., edited by John A. Riddick et al., Vol. II, in the Techniques of Chemistry Series, John Wiley & Sons, NY, 1986.

The term"treating", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term"treatment", as used herein, refers to the act of treating, as "treating"is defined immediately above.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS As mentioned above, degradation of hemoglobin provides nutrition for the malaria parasite during a portion of the life cycle. Within the red blood cell, parasites engulf erythrocyte cytoplasm, which is transported to the food vacuole. Considerable evidence suggests that hemoglobin degradation within the food vacuole is necessary for growth of the parasite. Three proteases have been isolated from food vacuoles of P. falciparum, one cysteine protease, falcipain (Rosenthal, McKerrow et al., 1988 ; Salas, Fichmann et al. 1995), and two aspartic proteases, plasmepsin I and plasmepsin II (Goldberg, Slater et al. 1991; Goldberg 1992). Plasmepsins I and II are 73% identical to each other and exhibit a similar structure (Silva, A. M., et al., PNAS, USA, 93,10034-10039,1996). The individual roles of the three proteases in the degradation pathway remains unclear in that the order in which they cleave hemoglobin has not been conclusively established (Berry, C., Proteases of Infectious Agents, Academic Press, 1999).

Cysteine and aspartic proteases appear to act cooperatively to degrade hemoglobin, and inhibitors of either class of protease have been shown to have antimalarial activity. The ability of cysteine protease inhibitors to kill parasites in culture was well correlated with their effectiveness at inhibiting cysteine proteinase activity in trophozoite extracts (Rosenthal, P. J., W. S. Wollish, et al. J Clin Invest 88 (5): 1467-72,1991; Li, Chen et al., 1996; Rosenthal, Olson et al., 1996). In a mouse model of infection with P. vinckei, the analogous murine malarial parasite, treatment with cysteine protease inhibitors resulted in a long-term curative effect (>75 days) in 80% of animals (Rosenthal, Lee et al. 1993) although high doses were required. The ability of aspartic protease inhibitors that inhibit plasmepsins to kill malaria parasites has also been demonstrated (Francis, S. E., et al., EMBO J., 13,306- 317,1994; Moon, R. P., et al., Eur. J. Biochem., 244,552-560,1997).

Semenov, et al. studied cysteine and aspartic acid protease inhibitors in cultured P. falciparum parasites and in a murine malaria model. The combination of these inhibitors exhibited a synergistic effect in blocking parasite metabolism and development, and was much more effective for the treatment of murine malaria than higher concentrations of either compound alone. A combination of two inhibitors- pepstatin and N-methyl piperazine urea-leucine-homophenylalanine-phenyl vinyl sulfone (N-Me-pipa-Leu-homoPhe-VSPh)-at doses of 20 and 10 mg/kg/dose, respectively, cured 80% of mice infected with a lethal dose of Plasmodium vinckei, whereas single drug application in higher doses offered minimal benefit in the case of N-Me-pipa-Leu-homoPhe-VSPh (20 mg/kg/dose) and cured 25% of the animals in the case of pepstatin (50 mg/kg/dose) (Semenov, Olson et al. 1998). While the exact mechanism of the antimalarial activity is not clear at this point, it is indisputable that effective drugs may be developed by targeting the proteolytic enzymes of the food vacuole of the parasite.

Potent inhibitors have been synthesized against the plasmepsin aspartic proteases as well as against the cysteine protease falcipain. Plasmpepsins are very similar to other known aspartyl proteases that are targets of drug discovery and development, i. e. HIV protease, renin and cathepsin D. In particular, plasmepsin I shows high homology with cathepsin D, and inhibitors of cathepsin D have been found to be active against plasmepsin I as well (Ring, Sun et al. 1993; Carroll, Johnson et al. 1998; Carroll and Orlowski 1998 ; Haque, Skillman et al. 1999). The most potent compounds reported were peptidic in nature and are very hydrophobic, i. e., their pharmacological profile is not very favorable.

Similar features characterize the mechanism-based, time-dependent and irreversible inhibitors of the cysteinyl protease falcipain. Inhibitors towards this protease covalently bind to the protease through a mechanism-based process (Rosenthal, Wollish et al. 1991; Rosenthal, Olson et al. 1996; Scheidt, Roush et al.

1998). The general recognition motif of the falcipain protease closely resembles the recognition motifs of the plasmepsin aspartyl proteases, i. e. hydrophobic side chains, and the chemical structure of the peptidic inhibitors for this cysteine protease bears striking similarities to inhibitors of plasmepsin.

The present invention encompasses the recognition that inhibition of both cysteine and aspartic proteases, specifically falcipain and plasmepsin (s) is a more effective strategy against malaria parasites than inhibition of either class alone. The invention recognizes both that inhibitors of falcipain and plasmepsin (s) exert synergistic effects and also that by targeting two different essential parasitic enzymes, the likelihood of resistance development is reduced. In addition, the invention encompasses the recognition that it is desirable to accomplish dual inhibition by administering a single compound rather than a combination of agents. The compounds of the present invention, therefore, incorporate features of cysteine and aspartic protease inhibitors within a single molecule to provide bifunctional inhibitors capable of inhibiting both of these classes of protease. In particular, the compounds of the present invention incorporate features of inhibitors of falcipain and plasmepsin (s) to provide bifunctional inhibitors capable of inhibiting both falcipain and plasmepsin (s). A bifunctional inhibitor provides significant advantages over a combination of two separate inhibitors in terms of drug development, allowing for a lower overall dose, decreased expense, and increased convenience for the patient.

Combination therapy with agents active against multiple targets has proven to be an effective strategy in treating HIV and tuberculosis, infectious diseases in which resistance to a single drug is extremely common. However, in both these diseases it has been noticed that lack of compliance with complicated drug regimens has led to diminished efficacy and further contributed to the development of resistant organisms.

The approach adopted in the present invention, namely providing a novel class of compounds directed against two targets, addresses this issue.

The approach adopted in the present invention towards combining characteristics of plasmepsin inhibitors with characteristics of falcipain inhibitors to construct bifunctional anti-malaria compounds is shown below. W°S fui tu 0 0 Plasmepsin 11 specific Falcipain-specific, mechanism-based, irreversible R/t H dz S-R" 0 Replacement of peptide bond CONHR ou OH OU BifunctionalInhibitor The inventive bifunctional inhibitor shown above includes a vinyl sulfone moiety, characteristic of a class of potent inhibitors of falcipain (Rosenthal, et al., 1996) and also retains features characteristic of plasmepsin inhibitors. The compound is also characterized in that the amide bond present in both of the class-specific inhibitors is replaced. In general, peptidic compounds are not desirable pharmaceuticals, particularly for oral administration, because the peptide bond is highly susceptible to cleavage. While not wishing to be bound by any theory, it is likely that removal of the peptide bond results in increased stability and oral bioavailability of the inventive protease inhibitors.

As discussed above, the invention provides compounds and pharmaceutical compositions for the prophylaxis and treatment of conditions associated with parasitic infection, particularly malaria. The pharmaceutical compositions can be used in combination with other agents. Compounds of this invention comprise those of Formula (I) set forth above in the Summary, and are illustrated in part by the various classes, subgenera and species disclosed elsewhere herein.

In certain embodiments of the invention R'and R""are 3-Cl-Ph-O-CH2-and- S02Ph, respectively, where Ph indicates a phenyl group. In certain embodiments of the invention R"and R"'are side chains of phenylalanine and homophenylalanine, respectively.

In certain embodiments of the invention R'is where R represents a halogen, and wherein the phenyl ring may be substituted at any available position but bears at most a single R group.

In certain embodiments of the invention R"is the side chain of a hydrophobic amino acid, e. g., phenylalanine, leucine, isoleucine, or valine. In certain particular embodiments, R"is one of the following: where R represents an OH group, a CH3 group, or a halogen, and wherein the phenyl ring may be substituted at any available position but bears at most a single R group.

In certain embodiments of the invention R"'is the side chain of a hydrophobic amino acid, e. g., phenylalanine, leucine, isoleucine, or valine. In certain particular embodiments of the invention R"'is one of the following: ring may be substituted at any available position but bears at most a single R group.

In certain embodiments of the invention R""is one of the following: The inventive compositions encompass any and all combinations of the specific sets of selections for R', R", R"', and R""mentioned above. It will be appreciated by one of ordinary skill in the art that numerous asymmetric centers may exist in the compounds of the present invention. Thus, inventive compounds and pharmaceutical compositions thereof may be in the form of an individual enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers. In a preferred embodiment, the inventive compounds are available in pure form.

Additionally, the present invention provides pharmaceutically acceptable derivatives of the foregoing compounds, and methods of treating animals using these compounds, pharmaceutical compositions thereof, or either of these in combination with one or more additional therapeutic agents. While the compounds and methods of the present invention are chiefly directed towards inhibition of the malarial cysteine and aspartic proteases, plasmepsin and falcipain, it is noted that a variety of other proteases are important in the life cycle of Plasmodium. In addition a number of other parasites utilize cysteine and/or aspartic proteases. Among these are Trypanosoma cruzi,Trypanosoma brucei, Leishmania mexicana, Leishmania major, Schistosoma mansoni, Onchocerca volvulus, Brugia species, and a variety of other protozoa and helminths. These proteases and their roles in the respective organisms are described in Berry, C., Proteases of Infectious Agents, Academic Press, 1999, which is herein incorporated by reference. Thus compounds of the present invention may also be useful in treatment of diseases caused by these parasites, which include sleeping sickness, Chagas disease, leishmaniasis, schistosomiasis, onchocerciasis, etc., many of which are major illnesses in the developing world.

The compounds of the present invention can be tested in a variety of assays to determine their potency as inhibitors of cysteine and aspartic proteases, specifically as inhibitors of plasmepsin (s) and falcipain. For example, the compounds can be tested using in vitro assays in trophozoite extracts or using recombinantly derived proteases.

Assays to assess the effects of inventive protease inhibitors on the degradation of hemoglobin by parasites can be performed as described in Rosenthal et al., 1996, the contents of which are herein incorporated by reference. Briefly, one such assay consists of incubating trophozoites in the presence of an inventive compound, followed by microscopic examination to detect the marked food vacuole abnormality that has been correlated with a block in hemoglobin degradation. Analysis of hemoglobin accumulation within trophozoites can also be assessed by SDS-PAGE electrophoresis. The effects of the inventive inhibitors on parasite growth and development can also be assessed by using a modified standard [3H] hypoxanthine assay as described in Silva, et al., 1996. In addition, the inventive inhibitors can be tested using in vivo models of malaria, for example in mice infected with the murine malaria parasite, Plasmodium vinckei as described in Semenov, et al., 1998, the contents of which are herein incorporated by reference. Appropriate control compounds for use in these assays include known inhibitors of cysteine and aspartic proteases, particularly inhibitors of plasmepsin (s) and falcipain, described in the references cited herein and well known in the art.

A preferred assay for testing the ability of the inventive compounds to inhibit plasmepsin is performed as described in Silva, et al., 1996, the contents of which are herein incorporated by reference. Briefly, plasmepsin I and II are cloned into the pET vector under the control of the T7 promoter. The proteins are expressed in E. coli, purified from inclusion bodies, refolded, and activated. Kinetic assays are performed using a fluorogenic substrate, DABCYL-QRMFLSFP-EDANS, which mimics the primary hemoglobin cleavage site common to both plasmepsins I and II.

To obtain protein for use in a preferred assay to test the ability of the inventive compounds to inhibit falcipain, a baculovirus expression system is used. Briefly the gene for preprofalcipain is cloned into a pBlueBac2 baculovirus transfer vector (Invitrogen) and cotransfected with wild type Autographa californica nuclear polyhedrosis virus into insect cells, e. g., High-5 cells. Recombinant viruses are plaque purified and used for high level expression in insect cells. Methods for identifying recombinant baculoviruses are well known in the art and will be described further in the Examples. Likewise, methods for obtaining a homogeneous population of recombinant baculoviruses and generating viral seed stocks and high titer viral stocks are well known in the art. Discussions of these methods may be found, for example, in O'Reilly et al., Baculovirus Expression Vectors ; A Laboratory Manual, New York: 1992 and Richardson, Christopher D. (ed.), Baculovirus Expression Protocols-Methods in Molecular Biology, Vol. 39, Totowa, N. J., 1998. The contents of these two publications are incorporated herein by reference. Purification of falcipain follows published protocols (Rosenthal, et al., 1988 ; Salas, et al., 1995), which are herein incorporated by reference. The enzymatic activity of purified falcipain in the presence and absence of inventive compounds and/or appropriate controls is assessed using the fluorogenic substrate Z-Phe-Arg-AMC as described in Rosenthal, et al., 1996.

While in preferred embodiments of the invention the bifunctional inhibitors display significant inhibitory activity towards both cysteine and aspartic proteases, in particular towards the malarial proteases, plasmepsin (s) and falcipain, it is to be understood that the inventive compounds need not inhibit these proteases to the same extent. In certain embodiments an inventive compound may display greater inhibitory activity against plasmepsin (s), while in other embodiments an inventive compound may display greater inhibitory activity against falcipain. The invention includes those inventive compounds that may display inhibitory activity towards falcipain but substantially no inhibitory activity towards plasmepsin (s) or vice versa. In certain preferred embodiments the inventive compounds display sub-micromolar activity in the enzymatic assays described above.

Recombinant DNA and other molecular biology techniques necessary for these assays are well known in the art and are, in general, performed as described in Sambrook, J., et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, the contents of which are herein incorporated by reference.

Pharmaceutical Compositions As described above, the present invention provides compounds useful for the treatment of parasitic diseases, in particular for the treatment of malaria. Thus, in another aspect of the present invention, compounds of the present invention, or pharmaceutical compositions, are utilized to treat or prevent an infection or disorder as described above. It will be appreciated that the compounds of the present invention can exist in free form for treatment, or, where appropriate, in salt form, as discussed in more detail below. Additionally, it will be appreciated that one or more of the inventive compounds can be formulated with a pharmaceutically acceptable carrier to provide a pharmaceutical composition. Compounds to be utilized in the pharmaceutical compositions include compounds existing in free form or pharmaceutically acceptable derivatives thereof, as defined herein, such as pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative, which upon administration to a patient in need, is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof, e. g., a prodrug. The present invention also contemplates the use of inventive compounds, in conjunction with known therapies to provide combination therapies for the treatment of infections and disorders.

Thus, as used herein, the term"pharmaceutically acceptable salt"refers to those salts 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. describe pharmaceutically acceptable salts in detail in J.

Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference. 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, 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, oxalic 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 adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hernisulfate, 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, lower alkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term"pharmaceutically acceptable ester" refers to esters that hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular suitable esters includes formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.

Furthermore, the term"pharmaceutically acceptable prodrugs"as used herein refers to those prodrugs of the compounds of the present invention that 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, 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 invention. The term"prodrug"refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series, and in Edward B.

Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

As described above, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are 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; 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 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), bucally, 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.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

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. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

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 dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or calcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar--agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

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 solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient (s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymer substances and waxes.

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 polethylene 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. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient (s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

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 and ear drops 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 propellants known in the art 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.

In yet another aspect, the present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, and in certain embodiments, includes an additional approved therapeutic agent for use as a combination therapy. Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the compound (s) may also be included.

Uses of compounds and pharmaceutical compositions According to the methods of treatment of the present invention, parasitic infections, particularly malaria, are treated or prevented in a patient such as a human or other mammal by administering to the patient 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. By a"therapeutically effective amount"of a compound of the invention is meant a sufficient amount of the compound to treat a parasitic infection, in particular malaria, at a reasonable benefit/risk ratio applicable to any medical treatment. 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 judgement.

The specific therapeutically effective dose level 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 total daily dose of the compounds of this invention administered to a human or other mammal in single or in divided doses can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. 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 2000 mg of the compound (s) of this invention per day in single or multiple doses.

While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more antiparasitic agents or vaccines. By"in combination", it is not intended to imply that the agents must be administered at the same time or formulated for delivery together, although these methods of delivery are within the scope of the invention. In general, each agent will be administered at a dose and on a time schedule determined for that agent. Particular combinations can be selected to maximize synergy between the agents. Preferred agents for administration in combination with the inventive compounds include antimalarial drugs such as chloroquine, mefloquine, quinine, quinidine, fansidar, and atovaquone. Preferred agents also include the less widely used drugs primaquine, proguanil, maloprim, and tetracyclines. In addition, drugs such as artemisinin (de Vries, et al., Drugs, 52,818-836,1996) and related compounds such as artelinic acid, tafenoquine, pyronaridine, fosmidomycin, desferroxamine, and azithromycin currently under study for malaria treatment and/or prophylaxis are preferred agents for use together with the compounds of the present invention. Finally, one or more of the inventive compounds described herein may be administered with each other or with other inhibitors of parasitic proteases.

EXAMPLES The method of synthesis and experimental conditions of the bifunctional inhibitors are described in schemes 1 through 3 below. The starting materials are commercially available, e. g., from suppliers such as Sigma (St. Louis, MO), Aldrich (Milwaukee, WI), etc. Predicted yields are based on homologous syntheses. In the descriptions of synthetic procedures, eq. stands for equivalent. In the schemes below, R'and R""stand for R"stands for-CH2Ph, and R"'stands for-CH2CH2Ph, where Ph represents a phenyl group.

Scheme 1 J H H a) (Me) NH-oM H H tBXHN> NMM, CH2C12 Ht v/\MaDr tb c) DIRAL-H, eU12r,/z Rz -20-C-1 O-C THF. O*C 78'C o _2p _> OC THF 0'C-78'C 1. HCI, oJ p. R'R'p Z R. O p I S 2 2 4 NMM, CH2C12 Compound 2 A solution of compound 1 (1 eq.) in methylene chloride is cooled to-20° C with a dry ice/acetone bath. N-methyl morpholine (1 eq.) is added dropwise, followed by isobutyl chloroformate (1 eq.) and the reaction is warmed to 0° C over 1 h. A solution of methyl (methoxy) amine (1 eq.) and N-methyl morpholine (1 eq.) in DMF is added to the reaction mixture. The mixture is warmed to ambient temperature and stirred for 3 h., after which it is quenched with 1N HC1 and extracted with ethyl acetate. After drying over MgS04 and concentration in vacuo, compound 2 is obtained in approximately 97% yield.

Compound 3 A solution of compound 2 (1 eq.) in anhydrous THF is cooled to 0 C with an ice/water bath and freshly prepared vinyl magnesium bromide (3 eq.) is added dropwise maintaining the temp at 0° C. The reaction is stirred at 0° C for 5 h, quenched with 1 N HC1 and extracted with ethyl acetate. Drying over MgS04 and concentration in vacuo affords compound 3 in approximately 72% yield.

Compound 4 A solution of compound 3 (1 eq.) in anhydrous ether is cooled to-78° C with a dry ice/acetone bath and DIBAL (3 eq.) is added dropwise. The reaction is stirred at-78° C for 3 h and then quenched with MeOH. After warming up to ambient temperature, 1N sodium potassium tartarate is added and the reaction is stirred for 1 h. The residue is extracted with methylene chloride and the combined organic layers are washed with brine. After drying over MgS04 and concentration in vacuo, flash chromatography affords compound 4 in approximately 71% yield.

Compound 5 4 N HC1 in dioxane is added to compound 4 (1 eq.) and the reaction is stirred at room temperature for 30 min. After concentration in vacuo, the residue is triturated with ether and re-dissolved in methylene chloride. N-methyl morpholine (8 eq.) and the acyl chloride (1 eq.) are added, and the reaction is stirred at room temperature overnight. Afterwards, the reaction is diluted with ethyl acetate and water and the organic layer is washed with saturated NaHC03 and brine. Drying over MgS04, concentration in vacuo, and purification by flash chromatography affords compound 5 in approximately 74% yield.

Scheme 2 Compound 7 A solution of compound 6 (1 eq.) in anhydrous THF is cooled to-78° C with a dry ice/acetone bath. Butyllithium (1.1 eq.) is added dropwise over a period of 5 min, followed by the addition of the acyl chloride (1 eq.). The reaction is stirred at-78° C for 2 h, warmed up to ambient temperature and quenched with saturated ammonium chloride. Afterwards, the mixture is diluted with methylene chloride and water and the organic layer is washed with brine. After drying over MgS04 and concentration in vacuo, compound 7 is obtained in approximately 86% yield.

Compound 8 A solution of compound 7 (1 eq.) in methylene chloride is cooled to 0° C with an ice water bath and dibutylboron triflate (1.1 eq.) is added dropwise, followed by diisopropylethyl amine (1.5 eq.). The reaction is stirred at 0° C for 30 min and then cooled to-78° C with a dry ice/acetone bath. Acrolein (1.5 eq.) is added dropwise, and the reaction is stirred at-78° C for 2h and then quenched with 5/1 MEOH/H202 for 10 min at-78° C and 30 min at 0° C. Afterwards, the reaction is concentrated in vacuo and the residue diluted with methylene chloride and saturated NaHC03. The aqueous layer is extracted with methylene chloride and the combined organic layers are dried over MgS04. Concentration in vacuo affords compound 8 in approximately 76% yield.

Compound 9 To a solution of compound 8 (1 eq.) in anhydrous THF at 0° C is added hydrogen peroxide (4 eq.) dropwise, followed by LiOH (2 eq., dissolved in water). The reaction is stirred at 0° C for 2 h and then quenched with Na2S03. The solution is diluted with saturated NaHC03 and extracted with methylene chloride. The aqueous layer is acidified to pH 1 with 1N HC1 and extracted with ethyl acetate. The combined organic extracts are dried over MgS04 and after concentration in vacuo afford compound 9 in good yield.

Compound 10 A solution of compound 9 (1 eq.) in methylene chloride is cooled to-20 °C with a dry ice/acetone bath. N-methyl morpholine (1 eq.) is added dropwise, followed by isobutyl chloroformate (1 eq.), and the reaction is warmed up to 0° C over 1 h. A solution of methyl (methoxy) amine (1 eq.) and N-methyl morpholine (1 eq.) in DMF is added to the reaction mixture. The mixture is warmed to ambient temperature and stirred for 3 h. Afterwards, it is quenched with 1N HC1 and extracted with ethyl acetate. After drying over MgS04 and concentration in vacuo, compound 10 is obtained in approximately 97% yield.

Compound 11 A solution of compound 10 (1 eq.) in anhydrous THF is cooled to 0° C with an ice/water bath, and LAH (3 eq.) is added and the reaction stirred for 3h. It is then quenched with 1N HC1 and extracted with ethyl acetate. The organic extracts are washed with brine and concentrated in vacuo to give compound 11 in good yield.

Compound 12 A solution of compound 16 (1.5 eq.) in anhydrous THF is cooled to 0° C, and NaH (1.5 eq.) is added in one batch. Compound 11 (1 eq.) is added, and the reaction is stirred at ambient temperature for 30 min and then quenched with NH4C1. The solution is extracted with ethyl acetate, and the organic extracts are washed with brine. After concentration in vacuo and purification, compound 12 is isolated in good yield.

Scheme 3 H IOH k) 1. CIaMe2Si, (13a) 2.20 mol % Nolan cat. (1,) in toluene, 75 °C, (136) 3. HF, pyridine, 0°C, (13c) o R- H H R 02 1. 13c 13c H H OI H OI H 12 1) 20 mol% Nolan cat. (16) 02 in toluene, 55'C 14 O R 14 02 O R°", I CYa 13a CI /RCHPh ClPCY c MeS-N, \N-SMe . l 15 Ruz l H 2 R 13b Compound 13a To a solution of dichlorodimethylsilane (20 eq.) in pyridine is added a solution of compound 5 (1 eq.) in pyridine, and the resulting mixture is stirred at ambient temperature for 8 h. Afterwards, the volatiles are distilled via a short path distillation, and the residue is re-dissolved in pyridine. Compound 12 (1 eq.), as a solution in pyridine, is added, and the reaction is stirred at ambient temperature for 8 h.

Afterwards it is concentrated in vacuo and diluted with saturated NaHCO3 and ethyl acetate. The aqueous layer is extracted with ethyl acetate and the combined organic extracts are washed with brine and dried over MgS04. Concentration in vacuo and purification by flash chromatography affords compound 13a in approximately 56% yield.

Compound 13b To a solution of compound 13a (1 eq.) in toluene) is added Nolan catalyst (compound 15,20 mol%), and the reaction is heated at 75° C for 2 h. After concentration in vacuo and purification by flash chromatography, compound 13b is isolated in approximately 61 % yield.

Compound 13c A solution of compound 13b (1 eq.) in anhydrous THF is cooled to 0 °C with an ice/water bath. HF/pyridine (10 eq.) is added, and the reaction is stirred at 0° C for 1 h. Afterwards the reaction is quenched with 1M Na2C03 and extracted with ethyl acetate. The organic extracts are dried over MgS04 and concentrated in vacuo.

Purification by flash chromatography affords compound 13c in approximately 62% yield.

Compound 14 To a solution of compound 5 (1 eq.) and compound 12 (1 eq.) in toluene (0.06 M) is added Nolan catalyst (compound 15,20 mol%), and the reaction is heated at 55° C for 2 h. After concentration in vacuo and purification by flash chromatography, compound 14 is isolated in approximately 27% yield.

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