WO2010054382A1

WO2010054382A1 - Compounds, compositions, and methods for treating malaria or leishmaniasis

Info

Publication number: WO2010054382A1
Application number: PCT/US2009/063894
Authority: WO
Inventors: Rory Curtis; Peter Distefano
Original assignee: Elixir Pharmaceuticals, Inc.
Priority date: 2008-11-10
Filing date: 2009-11-10
Publication date: 2010-05-14

Abstract

Compounds, compositions and methods for the treatment of malaria and/or leishmaniasis are described herein.

Description

Compounds, Compositions, and Methods for Treating Malaria or Leishmaniasis

CLAIM OF PRIORITY

This application claims priority from U.S. S.N. 61/113,203 and U.S. S.N. 61/113,205, filed November 10^th, 2008 which are incorporated herein by reference in their entirety.

BACKGROUND

Malaria is a mosquito-borne disease caused by the parasite Plasmodium falciparum. People with malaria often experience fever, chills, and flu-like illness. Left untreated, they may develop severe complications and die. Each year 350-500 million cases of malaria occur worldwide, and over one million people die, most of them young children in sub-Saharan Africa.

Leishmaniasis is a disease caused by protozoan parasites that belong to the genus Leishmania and is transmitted by the bite of certain species of sand fly, including flies in the genus Lutzomyia and Phlebotomus. This disease is also known as Leichmaniosis, Leishmaniose, leishmaniose, and formerly, Orient Boils, Baghdad Boil, kala azar, black fever, sandfly disease, Dum-Dum fever or espundia.

Most forms of the disease are transmissible only from animals (zoonosis), but some can be spread between humans. Human infection is caused by about 21 of 30 species that infect mammals. These include the L. donovani complex with three species (L. donovani, L. infantum, and L. chagasi); the L. mexicana complex with 3 main species (L. mexicana, L. amazonensis, and L. venezuelensis); L. tropica; L. major; L. aethiopica; and the subgenus Viannia with four main species (L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) panamensis, and L. (V.) peruviana). The different species may be morphologically indistinguishable, but can be differentiated by isoenzyme analysis, DNA sequence analysis, or monoclonal antibodies.

Visceral leishmaniasis is a severe form in which the parasites have migrated to the vital organs. There remains a need to develop medicines for the treatment and/or prevention of malaria and/or leishmaniasis.

SUMMARY

The invention relates to substituted heterocyclic compounds (e.g., a compound described herein), compositions comprising the compounds, and methods of using the compounds and compound compositions for treating malaria and/or leishmaniasis. In some embodiments, the compounds and compositions comprising them are useful for treating disease or disease symptoms of malaria and/or leishmaniasis by inhibition of sirtuin (e.g., SIRTl), e.g, by inhibition of sirtuin mediated deacetylation. Without being bound by theory, one model for the effectiveness of a SIRTl inhibitor for the treatment or prevention of malaria and/or leishmaniasis is that the SIRTl inhibitor (e.g., a compound described herein) decreases the ability of the parasite to develop resistance to conventional treatments, and/or the SIRTl inhibitor decreases the viability and/or infectivity of the parasite. In one aspect, this invention relates to a method for treating or preventing malaria and/or leishmaniasis in a subject, e.g., a disorder described herein. The method includes administering to the subject an effective amount of a compound having a formula (I):

(D wherein,

R¹ and R², together with the carbons to which they are attached, form C₅-C₁₀ cycloalkyl, C₅-C₁₀ heterocyclyl, C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆- Cio aryl, or C5-C10 heteroaryl, each of which may be optionally substituted with 1-5 R⁵; or R¹ is H, S-alkyl, or S-aryl, and R² is amidoalkyl wherein the nitrogen is substituted with alkyl, aryl, or arylalkyl, each of which is optionally further substituted with alkyl, halo, hydroxy, or alkoxy; R³ and R⁴, together with the carbons to which they are attached, form C₅-C₁₀ cycloalkyl, C5-C₁0 heterocyclyl, C5-C₁0 cycloalkenyl, C5-C₁0 heterocycloalkenyl, C₆- Cio aryl, or C₅-C₁₀ heteroaryl, each of which may be optionally substituted with 1-5

R⁶; each of R⁵ and R⁶ is, independently, halo, hydroxy, C₁-C₁O alkyl, Ci-C₆ haloalkyl, C₁-C₁O alkoxy, Ci-C₆ haloalkoxy, C₆-CiO aryl, C5-C10 heteroaryl, C7-C12 aralkyl, C₇-C₁₂ heteroaralkyl, C₃-Cg heterocyclyl, C₂-Ci₂ alkenyl, C₂-Ci₂ alkynyl, C₅- Cio cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, carboxy, carboxylate, cyano, nitro, amino, Ci-C₆ alkyl amino, Ci-C₆ dialkyl amino, mercapto, SO₃H, sulfate, S(O)NH₂, S(O)₂NH₂, phosphate, C₁-C₄ alkylenedioxy, oxo, acyl, aminocarbonyl, Ci-C₆ alkyl aminocarbonyl, C₁-C₆ dialkyl aminocarbonyl, C₁-C_1O alkoxycarbonyl, C₁-C_1O thioalkoxycarbonyl, hydrazinocarbonyl, C₁-C₆ alkyl hydrazinocarbonyl, C₁-C₆ dialkyl hydrazinocarbonyl, hydroxyaminocarbonyl; alkoxyaminocarbonyl; or one of R⁵ or R⁶ and R⁷ form a cyclic moiety containing 4-6 carbons, 1-3 nitrogens, 0-2 oxygens and 0-2 sulfurs, which may be optionally substituted with oxo or C₁-C₆ alkyl; X is NR⁷, O, or S; Y is NR^7', O or S; represent optional double bonds; each of R⁷ and R⁷ is, independently, hydrogen, Ci-C₆ alkyl, C₇-C₁₂ arylalkyl, C₇-C₁₂ heteroarylalkyl; or R⁷ and one of R⁵ or R⁶ form a cyclic moiety containing 4-6 carbons, 1-3 nitrogens, 0-2 oxygens and 0-2 sulfurs, which may be optionally substituted with oxo or Ci-C₆ alkyl; and n is 0 or 1. In certain embodiments, n can be 1.

X can be NR⁷ and Y can be NR⁷ . R⁷ and R⁷ can each be, e.g., hydrogen or CH₃. One of R⁷ and R⁷ can be hydrogen and the other can be CH₃. R¹ and R² can form C5-C₁0 cycloalkenyl.

R¹ and R² can form C₆-CiO aryl.

R¹ and R² can form C₅-C₁₀ cycloalkenyl, which may be substituted with R⁵, and R³ and R⁴ can form C₆-Ci_O aryl, which may be substituted with R⁶.

In certain embodiments, the cycloalkenyl double bond can be between the carbon attached to R¹ and the carbon attached to R². C₅-C₁₀ cycloalkenyl, e.g., C₆ or C₇ cycloalkenyl, can be substituted with R⁵ and C₆-Ci_O aryl can be substituted with

R^b

R⁶ can be halo (e.g., chloro or bromo), Ci-C₆ alkyl (e.g., CH₃), Ci-C₆ haloalkyl (e.g., CF₃) or Ci-C₆ haloalkoxy (e.g., OCF₃). R⁵ can be for example, Ci-C₆ alkyl substituted with a substituent such as an amino substituent, or aminocarbonyl (for example a substituted aminocarbonyl, substituted with substituents such an aryl, heteroaryl, cycloalkyl, heterocylcloalkyl, aminocarbonyl, alkylaminocarbonyl, alkoxycarbonyl or other substituents. In each instances, the substituents can be further sub stituted with other sub stituents . ) . n can be 0.

R¹ and R² can form C₅-C₁₀ cycloalkenyl.

R¹ and R² can form C₆-Ci_O aryl.

X can be NR⁷, and R⁷ can be, e.g., hydrogen or CH₃. R¹ and R² can form C₅-C₁₀ cycloalkenyl, which may be substituted with R⁵, and R³ and R⁴ can form C₆-Ci_O aryl, which may be substituted with R⁶.

R^b

R⁶ can be halo (e.g., chloro), C₁-C₆ alkyl (e.g., CH₃), C₁-C₆ haloalkyl (e.g.,

CF₃) or Ci-C₆ haloalkoxy (e.g., OCF₃). R⁵ can be aminocarbonyl. n can be 0.

R¹ and R² can form C₅-C₁₀ cycloalkenyl. R¹ and R² can form C₆-Ci₀ aryl.

X can be NR⁷, and R⁷ can be, e.g., hydrogen or CH₃.

In certain embodiments, the cycloalkenyl double bond can be between the carbon attached to R¹ and the carbon attached to R². C₅-C₁₀ cycloalkenyl, e.g., C₆ or C₇ cycloalkenyl, can be substituted with R⁵ and C₆-Ci_O aryl can be substituted with R⁶. These compounds may have formula (II) or formula (III):

(ID R can be halo (e.g., chloro or bromo), Ci-C₆ alkyl (e.g., CH₃), Ci-C₆ haloalkyl (e.g., CF₃) or Ci-C₆ haloalkoxy (e.g., OCF₃). R⁵ can be aminocarbonyl. The compound may be a compound selected from Figure 1 or compounds (IV), (V), (VI), or (VII).

(VI) (VII)

In one instance, the compound can be a compound of formula (VI) having a high enantiomeric excess of a single isomer, wherein the optical rotation of the predominant isomer is negative, for example, -14.1 (c=0.33, DCM) or, for example, M_D ²⁵ -41.2° (c 0.96, CH₃OH). In a second instance, the compound can be a compound of formula (IV) having a high enantiomeric excess of a single isomer, wherein the optical rotation of the predominant isomer is negative. In some instances, a compound of formula (IV), (V), or (VII) is administered having a high enantiomeric excess of a single isomer, where the predominant isomer has the same absolute configuration as the negative isomer of the compound of formula (VI) as corresponds to the asterisk carbon shown above.

The compound can preferentially inhibit SIRTl relative to a non-SIRTl sirtuin, e.g., at least a 1.5, 2, 5, or 10 fold preference. The compound can have a Ki for SIRTl that is less than 500, 100, 50, or 40 nM. The amount can be effective to ameliorate at least one symptom of malaria, e.g., a symptom described herein. Exemplary symptoms include, fever, chills, sweats, headaches, muscle pains, nausea, vomiting, elevated temperature, perspiration, tiredness, confusion, coma, neurologic focal signs, anemia (e.g., severe anemia), and respiratory difficulties.

The method can include administering the compound more than once, e.g., repeatedly administering the compound. The compound can be administered in one or more boluses or continuous. The compound can be administered from without (e.g., by injection, ingestion, inhalation, etc), or from within, e.g., by an implanted device.

The amount can be effective to increase acetylation of a sirtuin substrate (e.g., a nuclear protein, e.g., a histone or a transcription factor, e.g., p53, FoxOl, or FoxCβ) in at least some cells of the subject.

The subject can be a mammal, e.g., a human.

The subject can be identified as being in need of such treatment or prevention.

The method can further include monitoring the subject, e.g., evaluating the subject for side effects, e.g., renal function and/or efficacy of treatment. In some embodiments, the method includes administering a compound described herein to a subject with a symptom of malaria (e.g., fever, chills, sweats, headaches, muscle pains, nausea, vomiting, elevated temperature, perspiration, tiredness, confusion, coma, neurologic focal signs, anemia (e.g., severe anemia), and respiratory difficulties). In some embodiments, the method includes administering a compound described herein to a subject with malaria caused by a Plasmodium falciparum infection.

In some embodiments, the method includes administering a compound described herein to a subject with malaria caused by a Plasmodium vivax infection. In some embodiments, the method includes administering a compound described herein to a subject with malaria caused by a Plasmodium ovale infection.

In some embodiments, the method includes administering a compound described herein to a subject with malaria caused by a Plasmodium malariae infection. In some embodiments, the method includes administering a compound described herein to a subject at risk for malaria (e.g., a subject living in or traveling in a country in which a malaria infection can be contracted). In some embodiments, the method includes administering a compound described herein to a subject at risk for malaria caused by a Plasmodium falciparum infection.

In some embodiments, the method includes administering a compound described herein to a subject at risk for malaria caused by a Plasmodium vivax infection.

In some embodiments, the method includes administering a compound described herein to a subject at risk for malaria caused by a Plasmodium ovale infection. In some embodiments, the method includes administering a compound described herein to a subject at risk for malaria caused by a Plasmodium malariae infection.

In some embodiments, the method includes administering a compound described herein to a subject with severe malaria (e.g., with one or more of the following symptoms: impaired consciousness/coma, severe normocytic anemia, renal failure, pulmonary edema, acute respiratory distress syndrome, circulatory shock, disseminated intravascular coagulation, spontaneous bleeding, acidosis, hemoglobinuria, jaundice, repeated generalized convulsions, and/or parasitemia of > 5%). In some embodiments, the method includes administering a compound described herein to a subject with complicated malaria.

In some embodiments, the method includes administering a compound described herein to a pregnant subject with malaria.

In some embodiments, the method includes administering a compound described herein in combination with a second treatment.

In some embodiments, the method includes administering a compound described herein in combination with a second compound described herein.

In some embodiments, the method includes administering a compound described herein in combination with an anti-malarial agent. In some preferred embodiments, the second treatment is selected from the group consisting of: chloroquine (e.g., chloroquine phosphate), hydroxychloroquine, quinine sulfate, doxycycline, tetracycline, clindamycin, quinine sulfate plus doxycycline, quinine sulfate plus tetracycline, quinine sulfate plus clindamycin; atovaquone-proguanil (Malarone), mefloquine, primaquine (e.g., primaquine phosphate), quinidine gluconate, quinidine/quinine combination, quinidine/quinine plus doxycycline, quinidine/quinine plus tetracycline, and quinidine/quinine plus clindamycin. The compound described herein and the second treatment can be administered simultaneously or sequentially. In some embodiments, the second treatment is a bednet or an insecticide.

In some embodiments, the method includes administering a compound described herein to a subject with a symptom of leishmaniasis (e.g., irregular fever, hepatosplenomegaly, pancytopenia, polyclonal hypergammaglobulinemia with reversed albumin: globulin ratio, twice-daily temperature spikes, or emaciation). The amount can be effective to ameliorate at least one symptom of leishmaniasis.

In some embodiments, the method includes administering a compound described herein to a subject at risk for leishmaniasis (e.g., a subject living in or traveling in a country in which leishmaniasis can be contracted).

In some embodiments, the method includes administering a compound described herein to a subject with leishmaniasis caused by an infection by a parasite of the genus Lutzomyia.

In some embodiments, the method includes administering a compound described herein to a subject with leishmaniasis caused by an infection by a parasite of the genus Phlebotomus. In some embodiments, the method includes administering a compound described herein to a subject with leishmaniasis caused by an infection of a parasite of the L. donovani complex with three species (e.g., L. donovani, L. infantum, and L. chagasϊ).

In some embodiments, the method includes administering a compound described herein to a subject with leishmaniasis caused by an infection of a parasite of the L. mexicana complex (e.g., L. mexicana, L. amazonensis, and L. venezuelensis).

In some embodiments, the method includes administering a compound described herein to a subject with leishmaniasis caused by an infection of a parasite of the subgenus Viannia (e.g., L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) panamensis, and L. (V.) peruviana).

In some embodiments, the method includes administering a compound described herein to a subject with leishmaniasis caused by an L. tropica; L. major; or L. aethiopica infection. In some embodiments, the method includes administering a compound described herein to a subject with visceral leishmaniasis or a symptom thereof.

In some embodiments, the method includes administering a compound described herein to a subject with cutaneous leishmaniasis or a symptom thereof. In some embodiments, the method includes administering a compound described herein to a subject with diffuse cutaneous leishmaniasis or a symptom thereof.

In some embodiments, the method includes administering a compound described herein to a subject with mucocutaneous leishmaniasis or a symptom thereof. In some embodiments, the method includes administering a compound described herein in combination with a second treatment.

In some embodiments, the method includes administering a compound described herein in combination with another agent used to treat leishmaniasis.

In some preferred embodiments, the second treatment is selected from the group consisting of: pentavalent antimony, sodium stibogluconate (e.g., Na antimony gluconate), meglumine antimonate(e.g., 20 mg/kg slowly injected IV or IM once/day for 20 to 28 days), amphotericin B (e.g., not to exceed 1.5 mg/kg) (e.g., liposomal amphotericin B) (e.g., ABELCET, AMBISOME, AMPHOCIN, AMPHOTEC,

Amphotericin B (conventional), Amphotericin B (lipid complex), Amphotericin B (liposomal), Amphotericin B Cholesteryl sulfate complex), e.g., 3 mg/kg once/day for 5 days, then 3 mg/kg once/day on days 14 and 21, deoxycholate (e.g., 0.5 to 1 mg/kg by slow infusion every day or every other day for up to 8 wk), pentamidine (e.g., NEBUPENT, PENTAM 300), isethionate (e.g., 2 to 4 mg/kg IV once/day or every other day for up to 15 doses), miltefosine (e.g., IMPAVIDO), e.g., given 100 mg once/day (or, e.g., 2.5 mg/kg for children 2 to 11 yr) for 28 days, fluconazole (e.g., DIFLUCAN), itraconazole (e.g., SPORANOX), and paromomycin (e.g., topical paromomycin (e.g., HUMATIN)). In some embodiments, the second treatment is a supportive measure, e.g., adequate nutrition, transfusions, or antibiotics (e.g., for secondary bacterial infection).

In one aspect, this invention relates to a conjugate that includes: a targeting agent and a compound, wherein the targeting agent and the compound are covalently linked, and the compound has a formula (I). Embodiments can include one or more of the following.

The targeting agent can be an antibody, e.g., specific for a cell surface protein.

The targeting agent can be a synthetic peptide.

The targeting agent can be a domain of a naturally occurring protein. 5 In another aspect, this invention relates to a kit which includes: a compound described herein, and instructions for use for treating or preventing malaria and/or leishmaniasis. The kit may further include a printed material comprising a rendering of the structure of the name of the compound.

In other aspects, the invention relates to a composition comprising a o compound of any of the formulae herein, and a pharmaceutically acceptable carrier. The composition may contain an additional therapeutic agent, e.g., an anti-malarial agent and/or an anti-leishmaniasis agent. Also within the scope of this invention is the use of such a composition for the manufacture of a medicament for the just- mentioned use. 5 Also within the scope of this invention is a packaged product. The packaged product includes a container, one of the aforementioned compounds in the container, and a legend (e.g., a label or insert) associated with the container and indicating administration of the compound for treating or preventing malaria and/or leishmaniasis. 0 The subject can be a mammal, preferably a human. The subject can also be a non-human subject, e.g., an animal model. In certain embodiments the method can further include identifying a subject. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). 5 The term "mammal" includes organisms, which include mice, rats, cows, sheep, pigs, rabbits, goats, and horses, monkeys, dogs, cats, and preferably humans.

The term "treating" or "treated" refers to administering a compound described herein to a subject with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a disease, e.g., an infection, the symptoms of the0 disease or the predisposition toward the disease.

An effective amount of the compound described above may range from about 0.1 mg/Kg to about 500 mg/Kg, alternatively from about 1 to about 50 mg/Kg. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine or iodine.

The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C₁- C₁₂ alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). The terms "arylalkyl" or "aralkyl" refer to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3- phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

The term "alkylene" refers to a divalent alkyl, e.g., -CH₂-, -CH₂CH₂-, and - CH₂CH₂CH₂-. The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and having one or more double bonds. Examples of alkenyl groups include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent.

The terms "alkylamino" and "dialkylamino" refer to -NH(alkyl) and - NH(alkyl)₂ radicals respectively. The term "aralkylamino" refers to a -NH(aralkyl) radical. The term alkylaminoalkyl refers to a (alkyl)NH-alkyl- radical; the term dialkylaminoalkyl refers to a (alkyl)₂N-alkyl- radical The term "alkoxy" refers to an - O-alkyl radical. The term "mercapto" refers to an SH radical. The term "thioalkoxy" refers to an -S-alkyl radical. The term thioaryloxy refers to an -S-aryl radical. The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted (e.g., by one or more substituents). Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl. The term "cycloalkyl" as employed herein includes saturated cyclic, bicyclic, tricyclic,or polycyclic hydrocarbon groups having 3 to 12 carbons. Any ring atom can be substituted (e.g., by one or more substituents). The cycloalkyl groups can contain fused rings. Fused rings are rings that share a common carbon atom. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclohexyl, methylcyclohexyl, adamantyl, and norbornyl.

The term "heterocyclyl" refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). The heteroatom may optionally be the point of attachment of the heterocyclyl substituent. Any ring atom can be substituted (e.g., by one or more substituents). The heterocyclyl groups can contain fused rings. Fused rings are rings that share a common carbon atom. Examples of heterocyclyl include, but are not limited to, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl, pyrimidinyl, quinolinyl, and pyrrolidinyl.

The term "cycloalkenyl" refers to partially unsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons. The unsaturated carbon may optionally be the point of attachment of the cycloalkenyl substituent. Any ring atom can be substituted (e.g., by one or more substituents). The cycloalkenyl groups can contain fused rings. Fused rings are rings that share a common carbon atom. Examples of cycloalkenyl moieties include, but are not limited to, cyclohexenyl, cyclohexadienyl, or norbornenyl. The term "heterocycloalkenyl" refers to a partially saturated, nonaromatic 5-

10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). The unsaturated carbon or the heteroatom may optionally be the point of attachment of the heterocycloalkenyl substituent. Any ring atom can be substituted (e.g., by one or more substituents). The heterocycloalkenyl groups can contain fused rings. Fused rings are rings that share a common carbon atom. Examples of heterocycloalkenyl include but are not limited to tetrahydropyridyl and dihydropyranyl.

The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom can be substituted (e.g., by one or more substituents).

The term "oxo" refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted (e.g., by one or more substituents). The terms "aminocarbonyl," alkoxycarbonyl," hydrazinocarbonyl, and hydroxyaminocarbonyl refer to the radicals -C(O)NH₂, -C(O)O(alkyl), - C(O)NH₂NH₂, and -C(O)NH₂NH₂, respectively.

The term "amindo "refers to a -NHC(O)- radical, wherein N is the point of attachment. The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Any atom can be substituted. Suitable substituents include, without limitation, alkyl (e.g., Cl, Cl, C3, C4, C5, C6, C7, C8, C9, ClO, CIl, C12 straight or branched chain alkyl), cycloalkyl, haloalkyl (e.g., perfluoroalkyl such as CF₃), aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, alkoxy, haloalkoxy (e.g., perfluoroalkoxy such as OCF₃), halo, hydroxy, carboxy, carboxylate, cyano, nitro, amino, alkyl amino, SO₃H, sulfate, phosphate, methylenedioxy (-0-CH₂-O- wherein oxygens are attached to vicinal atoms), ethylenedioxy, oxo, thioxo (e.g., C=S), imino (alkyl, aryl, aralkyl), S(O)_nalkyl (where n is 0-2), S(O)_n aryl (where n is 0-2), S(O)_n heteroaryl (where n is 0-2), S(O)_n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof). In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents. In another aspect, a substituent may itself be substituted with any one of the above substituents. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All references cited herein, whether in print, electronic, computer readable storage media or other form, are expressly incorporated by reference in their entirety, including but not limited to, abstracts, articles, journals, publications, texts, treatises, internet web sites, databases, patents, patent applications and patent publications.

DESCRIPTION OF DRAWINGS

FIG. 1 is a table of representative compounds and data. FIG. 2 is a computer-generated model showing one possible orientation of compound 8 bound in the active site of SIRT.

FIG. 3 a is a graph depicting the inhibition of mammalia SirTl by compound 8. FIG. 3b is a Western blot of NCI-H460 cells treated with etoposide only or etoposide and compound 8. FIG. 4 is a graph depicting that enantiomer 8(-) of compound 8 preferentially inhibits yeast sir2 relative to enantiomer 8(+).

DETAILED DESCRIPTION

Malaria

Malaria is a mosquito-borne disease. Methods of treating and/or preventing malaria are described herein, including compounds and compositions for the treatment and/or prevention of malaria. In some embodiments, treatment eliminates or reduces the severity of one or more symptoms of malaria. Clinical Diagnosis

Clinical diagnosis of malaria is generally based on the patient's symptoms and on physical findings at examination.

The first symptoms of malaria (most often fever, chills, sweats, headaches, muscle pains, nausea and vomiting) are often not specific and are also found in other diseases (such as the "flu" and common viral infections). Likewise, the physical findings are often not specific (elevated temperature, perspiration, tiredness).

In severe malaria (caused by Plasmodium falciparum), clinical findings (confusion, coma, neurologic focal signs, severe anemia, respiratory difficulties) are more striking and may increase the suspicion index for malaria.

Thus, in most cases the early clinical findings in malaria are not typical and need to be confirmed by a laboratory test.

Microscopic Diagnosis Malaria parasites can be identified by examining under the microscope a drop of the patient's blood, spread out as a "blood smear" on a microscope slide. Prior to examination, the specimen is stained (most often with the Giemsa stain) to give to the parasites a distinctive appearance. This technique is a high standard for laboratory confirmation of malaria. However, it depends on the quality of the reagents, of the microscope, and on the experience of the lab technician.

Alternate methods for laboratory diagnosis include the following: Antigen Detection

Various test kits are available to detect antigens derived from malaria parasites. Such immunologic ("immunochromatographic") tests most often use a dipstick or cassette format, and provide results in 2-15 minutes. These "Rapid

Diagnostic Tests" (RDTs) offer a useful alternative to microscopy in situations where reliable microscopic diagnosis is not available. Malaria RDTs are currently used in some clinical settings and programs. However, before malaria RDTs can be widely adopted, several issues remain to be addressed, including improving their accuracy; lowering their cost; and ensuring their adequate performance under adverse field conditions. The World Health Organization's Regional Office for the Western Pacific (WHOAVPRO) provides technical information, including a list of commercially available malaria RDTs, at http : //www . wpro . who . int/rdt/ . On June 13, 2007, the U.S. Food and Drug Administration (FDA) approved the first RDT for use in the United States. This RDT is approved for use by hospital and commercial laboratories, not by individual clinicians or by patients themselves. It is recommended that all RDTs are followed-up with microscopy to confirm the results and if positive, to quantify the proportion of red blood cells that are infected. The use of this RDT may decrease the amount of time that it takes to determine that a patient is infected with malaria.

Molecular Diagnosis Parasite nucleic acids are detected using polymerase chain reaction (PCR).

This technique is more accurate than microscopy. However, it is expensive, and requires a specialized laboratory (even though technical advances will likely result in field-operated PCR machines).

Other techniques related to malaria diagnosis are:

Serology

Serology detects antibodies against malaria parasites, using either indirect immunofluorescence (IFA) or enzyme-linked immunosorbent assay (ELISA). Serology does not detect current infection but rather measures past experience.

In some instances, the malaria is resistant to available therapies for treatment. Accordingly, in some instances drug resistance tests are used.

Drug resistance tests are performed in specialized laboratories to assess the susceptibility to antimalarial compounds of parasites collected from a specific patient. Two main laboratory methods are available:

In vitro tests: The parasites are grown in culture in the presence of increasing concentrations of drugs; the drug concentration that inhibits parasite growth is used as endpoint;

Molecular characterization: molecular markers assessed by PCR or gene sequencing allow also the prediction, to some degree, of resistance to some drugs; however, the predictive values of these molecular tests are still being evaluated. Evaluation and Diagnosis

Symptoms of malaria are generally non-specific and most commonly consist of fever, malaise, weakness, gastrointestinal complaints (nausea, vomiting, diarrhea), neurologic complaints (dizziness, confusion, disorientation, coma), headache, back pain, myalgia, chills, and/or cough. The diagnosis of malaria should also be considered in any person with fever of unknown origin regardless of travel history. Patients suspected of having malaria infection are generally be urgently evaluated. Treatment for malaria is not generally initiated until the diagnosis has been confirmed by laboratory investigations. "Presumptive treatment" without the benefit of laboratory confirmation is generally reserved for extreme circumstances (strong clinical suspicion, severe disease, impossibility of obtaining prompt laboratory confirmation).

As noted above, laboratory diagnosis of malaria can be made through microscopic examination of thick and thin blood smears. Thick blood smears are more sensitive in detecting malaria parasites because the blood is more concentrated allowing for a greater volume of blood to be examined; however, thick smears are more difficult to read. Laboratories that have limited experience may prefer to use thin smears, which can aid in parasitic species identification. Blood films need to be read immediately; off-hours, qualified personnel who can perform this function should be on-call. A negative blood smear makes the diagnosis of malaria unlikely. However, because non-immune individuals may be symptomatic at very low parasite densities that initially may be undetectable by blood smear, blood smears should be repeated every 12-24 hours for a total of 48-72 hours.

After the presence of malaria parasites on a blood smear is detected, the parasite density should then be estimated. The parasite density can be estimated by looking at a monolayer of red blood cells (RBCs) on the thin smear using the oil immersion objective at 10Ox. The slide should be examined where the RBCs are more or less touching (approximately 400 RBCs per field). The parasite density can then be estimated from the percentage of infected RBCs. In addition to microscopy, other laboratory diagnostic tests are available.

Several antigen detection tests using a "dipstick" format exist but are not yet approved for general diagnostic use in the United States. Parasite nucleic acid detection using polymerase chain reaction (PCR) are more sensitive and specific than microscopy but can be performed only in reference laboratories and should be reserved for specific instances (e.g., back up or confirmation of microscopy). Serologic tests, also performed in reference laboratories, can be used to assess past malaria experience but not current infection by malaria parasites.

Treatment of malaria

Treatment for malaria is not generally initiated until the diagnosis has been confirmed by laboratory investigations. "Presumptive treatment" without the benefit of laboratory confirmation should be reserved for extreme circumstances (strong clinical suspicion, severe disease, impossibility of obtaining prompt laboratory confirmation).

Once the diagnosis of malaria has been confirmed, appropriate antimalarial treatment must be initiated immediately. Treatment should be guided by three main factors: the infecting Plasmodium species, the clinical status of the patient, and the drug susceptibility of the infecting parasites as determined by the geographic area where the infection was acquired. A compound described herein can be used in place of or in combination with another antimalarial treatment, e.g., an antimalarial treatment described herein.

Determination of the infecting Plasmodium species for treatment purposes is important for three main reasons:

P. falciparum infections can cause rapidly progressive severe illness or death while the non-falciparum ( P. vivax, P. ovale, or P. malariae) species rarely cause severe manifestations;

P. vivax and P. ovale infections require treatment for the hypnozoite forms that remain dormant in the liver and can cause a relapsing infection; and P. falciparum and P. vivax species have different drug resistance patterns in differing geographic regions. For P. falciparum infections, the urgent initiation of appropriate therapy is especially critical. The second factor affecting treatment is the clinical status of the patient. Patients diagnosed with malaria are generally categorized as having either uncomplicated or severe malaria. Patients diagnosed with uncomplicated malaria can be effectively treated with oral antimalarials. However, patients who have one or more of the following clinical criteria (impaired consciousness/coma, severe normocytic anemia, renal failure, pulmonary edema, acute respiratory distress syndrome, circulatory shock, disseminated intravascular coagulation, spontaneous bleeding, acidosis, hemoglobinuria, jaundice, repeated generalized convulsions, and/or parasitemia of > 5%) are generally considered to have manifestations of more severe disease and are generally be treated aggressively with parenteral antimalarial therapy.

Finally, knowledge of the geographic area where the infection was acquired can provide information on the likelihood of drug resistance of the infecting parasite and can enable the treating clinician to choose an appropriate drug or drug combination and treatment course. If the diagnosis of malaria is suspected and cannot be confirmed, or if the diagnosis of malaria is confirmed but species determination is not possible, antimalarial treatment effective against P. falciparum is generally initiated immediately.

Treatment Guidelines table from the United States Center for Disease Control (CDC) and can be used as a guide for treatment of malaria in the United States. The drug or drug combinations recommended for treatment are listed in bold on the first line of each box in the adult and pediatric "drug and dose" columns. Each drug and its recommended dose are then listed individually on the lines below in the same box. It is important to note that the base/salt conversions for antimalarials are a continual source of confusion and can contribute to treatment errors. In this treatment table (where appropriate), the antimalarial dose is expressed in base with the salt equivalency noted in parenthesis. A compound described herein can be used in place of or in combination with one or more of the treatment regimens shown in Table 1 of the Treatment Guidelines. After initiation of treatment, the patient's clinical and parasitologic status is generally monitored. In infections with P. falciparum or suspected chloroquine- resistant P. vivax, blood smears is generally made to confirm adequate parasitologic response to treatment (decrease in parasite density followed by clearance).

Treatment: Uncomplicated Malaria

P. falciparum or Species Not Identified

For P. falciparum infections acquired in areas without chloroquine-resistant strains, which include Central America west of the Panama Canal, Haiti, the Dominican Republic, and most of the Middle East, patients should be treated with oral chloroquine. A chloroquine dose of 600 mg base (= 1,000 mg salt) should be given initially, followed by 300 mg base (= 500 mg salt) at 6, 24, and 48 hours after the initial dose for a total chloroquine dose of 1,500 mg base (=2,500 mg salt). As a 2nd line alternative for treatment, a hydroxychloroquine dose of 620 mg base (=800 mg salt) po can be given immediately, followed by 310 mg base (=400 mg salt) po at 6, 24, and 48 hours after the initial does for a total hydroxychloroquine dose of 1,550 mg base (=2,000 mg salt). A compound described herein can be used in combination with either of these treatment regimens. For P. falciparum infections acquired in areas with chloroquine-resistant strains, three treatment options are available. The first two treatment options are quinine sulfate plus doxycycline, tetracycline, or clindamycin; or atovaquone- proguanil (Malarone). Both of these options are very efficacious. For the quinine sulfate combination options, quinine sulfate plus either doxycycline or tetracycline is generally preferred to quinine sulfate plus clindamycin because there are more data on the efficacy of quinine plus doxycycline or tetracycline. Quinine treatment should continue for 7 days for infections acquired in Southeast Asia and for 3 days for infections acquired in Africa or South America. The third option, mefloquine, is associated with a higher rate of severe neuropsychiatric reactions when used at treatment doses. This third option is currently recommended only when the quinine sulfate combination or atovaquone-proguanil options cannot be used. A compound described herein can be used in place of or in combination with any of these treatment regimens.

For pediatric patients, the treatment options are the same as for adults except the drug dose is adjusted by patient weight. The pediatric dose should never exceed the recommended adult dose. For children less than eight years old, doxycycline and tetracycline are generally not indicated; therefore, quinine (given alone for a full 7 days regardless of where the infection was acquired or given in combination with clindamycin as recommended above) and atovaquone-proguanil are recommended treatment options for chloroquine-resistant P. falciparum infections; mefloquine can be considered if these options are not available. In rare instances, doxycycline or tetracycline can be used in combination with quinine in children less than eight years old if other treatment options are not available or are not tolerated, and the benefit of adding doxycycline or tetracycline is judged to outweigh the risk. A compound described herein can be used in place of or in combination with any of these treatment regimens.

If infections initially attributed to "species not identified" are subsequently diagnosed as being due to P. vivax or P. ovale, additional treatment with primaquine is generally administered (see P. vivax and P. ovale, below).

P. malariae

There has been no widespread evidence of chloroquine resistance in P. malariae species; therefore, chloroquine remains the drug of choice for all P. malariae infections. As a 2nd line alternative for treatment, hydroxychloroquine may be given instead. A compound described herein can be used in place of or in combination with either of these treatment regimens.

P. vivax and P. ovale

Chloroquine (hydroxychloroquine as 2nd line alternative for treatment) remains the treatment of choice for all P. vivax and P. ovale infections except for P. vivax infections acquired in Papua New Guinea or Indonesia. Reports have confirmed a high prevalence of chloroquine-resistant P. vivax in these two specific areas. Rare case reports of chloroquine-resistant P. vivax have also been documented in Burma (Myanmar), India, and South America. Persons acquiring P. vivax infections from regions other than Papua New Guinea or Indonesia should initially be treated with chloroquine. If the patient does not respond to chloroquine, treatment should be changed to one of the two regimens recommended for chloroquine-resistant P. vivax infections. Persons acquiring P. vivax infections in Papua New Guinea or Indonesia should initially be treated with a regimen recommended for chloroquine-resistant P. vivax infections. The two treatment regimens for chloroquine-resistant P. vivax infections are quinine sulfate plus doxycycline or tetracycline, or mefloquine. These two treatment options are equally recommended. There are no adequate, well- controlled studies to support the use of atovaquone-proguanil to treat chloroquine- resistant P. vivax infections. A compound described herein can be used in place of or in combination with any of these treatment regimens.

In addition to requiring blood stage treatment, infections with P. vivax and P. ovale can relapse due to hypnozoites that remain dormant in the liver. To eradicate the hypnozoites, patients should be treated with a 14-day course of primaquine phosphate. CDC has recently changed its recommendations for treating hypnozoites by increasing the recommended primaquine phosphate dose to 30 mg (base) by mouth daily for 14 days. Because primaquine can cause hemolytic anemia in persons with glucose-6-phosphate-dehydrogenase (G6PD) deficiency, persons must be screened for G6PD deficiency prior to starting primaquine treatment. For persons with borderline G6PD deficiency or as an alternate to the above regimen, primaquine may be given at the dose of 45 mg (base) orally one time per week for 8 weeks ; consultation with an expert in infectious disease and/or tropical medicine is advised if this alternative regimen is considered in G6PD-deficient persons. Primaquine must not be used during pregnancy. A compound described herein can be used in place of or in combination with any of these treatment regimens.

For pediatric patients, the treatment options are the same as for adults except the drug dose is adjusted by patient weight. The pediatric dose should never exceed the adult recommended adult dose. For children less than eight years old, doxycycline and tetracycline are generally not indicated; therefore, quinine (given alone for 7 days) or mefloquine are recommended treatment options for chloroquine- resistant P. vivax infections. In rare instances, doxycycline or tetracycline can be used in combination with quinine in children less than 8 years old if other treatment options are not available, are not being tolerated, and the benefit of adding doxycycline or tetracycline is judged to outweigh the risk. Primaquine should be given to pediatric patients only after they have been screened for G6PD deficiency. A compound described herein can be used in place of or in combination with any of these treatment regimens.

Alternatives For Pregnant Women

Malaria infection in pregnant women is associated with high risks of both maternal and perinatal morbidity and mortality. While the mechanism is poorly understood, pregnant women have a reduced immune response and therefore less effectively clear malaria infections. Pregnant women are three times more likely to develop severe disease than non-pregnant women acquiring infections from the same area. In addition, malaria parasites sequester and replicate in the placenta. Malaria infection during pregnancy can lead to miscarriage, premature delivery, low birth weight, congenital infection, and/or perinatal death. For pregnant women diagnosed with uncomplicated malaria caused by P. malariae, P. vivax, P. ovale, or chloroquine- sensitive P. falciparum infection, prompt treatment with chloroquine (treatment schedule as with non-pregnant adult patients) is recommended. As a 2nd line alternative for treatment, hydroxychloroquine may be given instead. For pregnant women diagnosed with uncomplicated malaria caused by chloroquine-resistant P. falciparum infection, prompt treatment with quinine sulfate and clindamycin is recommended. Quinine treatment should continue for 7 days for infections acquired in Southeast Asia and for 3 days for infections acquired in Africa or South America; clindamycin treatment should continue for 7 days regardless of where the infection was acquired. For pregnant women diagnosed with uncomplicated malaria caused by chloroquine-resistant P. vivax infection, prompt treatment with quinine for seven days is recommended regardless of where the infection was acquired. There are no adequate, well-controlled studies to support the addition of clindamycin to quinine when treating chloroquine-resistant P. vivax infections. A compound described herein can be used in place of or in combination with any of these treatment regimens.

Doxycycline and tetracycline are generally not indicated for use in pregnant women. However, in rare instances, doxycycline or tetracycline can be used in combination with quinine if other treatment options are not available or are not being tolerated, and the benefit of adding doxycycline or tetracycline is judged to outweigh the risks. A compound described herein can be used in place of or in combination with any of these treatment regimens.

According to its U.S. label, atovaquone/proguanil is classified as a pregnancy category C medication and is generally not indicated for use in pregnant women because there are no adequate, well-controlled studies of atovaquone and/or proguanil hydrochloride in pregnant women. However, for pregnant women diagnosed with uncomplicated malaria caused by chloroquine-resistant P. falciparum infection, atovaquone-proguanil may be used if other treatment options are not available or are not being tolerated, and if the potential benefit is judged to outweigh the potential risks. There are no data on the efficacy of atovaquone/proguanil in the treatment of chloroquine-resistant P. vivax infections. A compound described herein can be used in place of or in combination with any of these treatment regimens.

Mefloquine is also a pregnancy category C medication and is generally not indicated for treatment in pregnant women. Mefloquine has not been associated with an increased risk of congenital abnormalities; however, a possible association with mefloquine treatment during pregnancy and an increase in stillbirths has been reported. CDC recommends mefloquine only when no other treatment options are available and if the potential benefit is judged to outweigh the potential risks. A compound described herein can be used in place of or in combination with any of these treatment regimens.

For P. vivax or P. ovale infections, primaquine phosphate for radical treatment of hypnozoites should not be given during pregnancy. Pregnant patients with P. vivax or P. ovale infections should be maintained on chloroquine prophylaxis for the duration of their pregnancy. The chemoprophylactic dose of chloroquine phosphate is 300mg base (=500 mg salt) orally once per week. After delivery, pregnant patients with P. vivax or P. ovale infections who do not have G6PD deficiency should be treated with primaquine. Pregnant women diagnosed with severe malaria should be treated aggressively with parenteral antimalarial therapy as described below. A compound described herein can be used in place of or in combination with any of these treatment regimens.

Treatment: Severe Malaria

Patients who are considered to have manifestations of more severe disease should be treated aggressively with parenteral antimalarial therapy. Oral antimalarial drugs (such as oral quinine, chloroquine, or mefloquine) are not recommended for the initial treatment of severe malaria. If severe malaria is strongly suspected but the first blood smear does not demonstrate parasites, a trial of parenteral antimalarial drugs should be given. If there is clinical evidence of severe malaria but the blood smear is reported as P. vivax, P. ovale or P. malariae, the patient should be treated for falciparum malaria in case of a mixed infection or misdiagnosis.

Since 1991, quinidine gluconate has been the only parenterally administered antimalarial drug available in the United States. It is recommended to give a loading dose of 6.25 mg base/kg (=10 mg salt/kg) of quinidine gluconate infused intravenously over 1-2 hours followed by a continuous infusion of 0.0125 mg base/kg/min (=0.02 mg salt/kg/min). An alternative regimen is an intravenous loading dose of 15mg base/kg (=24 mg salt/kg) of quinidine gluconate infused intravenously over 4 hours, followed by 7.5mg base/kg (=12 mg/kg salt) infused over 4 hours every 8 hours, starting 8 hours after the loading dose (see package insert). Quinidine levels should be maintained in the range of 3-8 mg/L. At least 24 hours of quinidine gluconate infusion are recommended (or 3 intermittent doses); once the parasite density is < 1% and the patient can take oral medication, the patient can complete the treatment course with oral quinine at a dosage of 10 mg salt/kg every 8 hours (for a combined treatment course of quinidine/quinine for 7 days in Southeast Asia and 3 days in Africa and South America). A compound described herein can be used in place of or in combination with any of these treatment regimens.

Initial (including loading) doses of parenteral quinine or quinidine do not need to be reduced in persons with renal failure. If renal failure persists or the patient does not improve clinically, the maintenance dosage should be reduced by one third to one half on the third treatment day. A compound described herein can be used in place of or in combination with any of these treatment regimens.

As with treatment of uncomplicated P. falciparum, quinidine/quinine therapy should be combined with doxycycline, tetracycline, or clindamycin. If the patient is unable to tolerate oral therapy, doxycycline hyclate (100 mg every 12 hours) or clindamycin (5 mg base/kg every 8 hours) may be given intravenously until the patient can be switched to oral therapy. Rapid intravenous administration of doxycycline or clindamycin should be avoided. If the patient can tolerate oral therapy, doxycycline (100 mg every 12 hours), tetracycline (250 mg every 6 hours), or clindamycin (20 mg base/kg/day divided three times per day) for 7 days are options. A compound described herein can be used in place of or in combination with any of these treatment regimens.

Parenteral quinidine gluconate is cardiotoxic and should be administered in an intensive care setting with continuous cardiac and frequent blood pressure monitoring. At the dosages required for the treatment of falciparum malaria, quinidine gluconate may cause ventricular arrhythmia, hypotension, hypoglycemia, and prolongation of the QTc interval. The quinidine gluconate infusion should be slowed or stopped for an increase in the QRS complex by > 50%, a QTc interval > 0.6 seconds, a QTc interval that is prolonged by more than 25% of the baseline value, or hypotension unresponsive to fluid challenge. Because most deaths from severe malaria occur within the first 24-48 hours, the goal of a loading dose is to quickly reach therapeutic concentrations at a time when they are needed most. Recent use of other drugs that may prolong the QTc interval (e.g., quinine or mefloquine) should be considered when determining whether a patient should receive a loading dose of quinidine gluconate. Because there is less collected experience on which to base decisions with quinidine gluconate, recommendations for administration of a loading dose are based on experience with loading doses of quinine. A loading dose of quinidine gluconate should be given unless the patient has received more than 40 mg/kg quinine in the previous 2 days or has received mefloquine in the previous 12 hours. Consulting a cardiologist and a physician with experience in treating malaria is advised when treating malaria patients in the United States with quinidine gluconate. 16 Glucose must be monitored closely as quinidine- (or quinine-) induced hyperinsulinemic hypoglycemia can occur. A compound described herein can be used in place of or in combination with any of these treatment regimens.

On June 21, 2007, CDCs Investigational New Drug Application (IND) for intravenous artesunate went into effect. This IND allows for use of an investigational antimalarial medication (intravenous artesunate) to be used for the treatment of severe malaria. A compound described herein can be used in place of or in combination with any of these treatment regimens.

While exchange transfusion has not been proven beneficial in an adequately powered randomized controlled trial, it has been an option in the treatment of severe malaria since 1974. CDC recommends that exchange transfusion be strongly considered for persons with a parasite density of more than 10% or if complications such as cerebral malaria, non-volume overload pulmonary edema, or renal complications exist. Exchange transfusion is thought to have beneficial effects by removing infected red cells, improving the rheological properties of blood, and reducing toxic factors such as parasite derived toxins, harmful metabolites, and cytokines. The risks of exchange transfusion include fluid overload, febrile and allergic reactions, metabolic disturbances (e.g., hypocalcemia), red blood cell alloantibody sensitization, transmissible infection, and line sepsis. Thus, the potential benefits of exchange transfusion should be weighed against the risks. The parasite density should be monitored every 12 hours until it falls below 1%, which usually requires the exchange of 8-10 units of blood in adults. A compound described herein can be used in place of or in combination with this treatment regimen.

Leishmaniasis Geography and epidemiology

Methods of treating and/or preventing leishmaniasis are described herein, including compounds and compositions for the treatment and/or prevention of leishmaniasis. In some embodiments, treatment eliminates or reduces the severity of one or more symptoms of malaria. For example, the compounds described herein (e.g., a compound of formula (I)) can be used to treat (e.g., eliminate or reduce the severity of one or more symptoms) or prevent leishmaniasis.

Leishmaniasis is present worldwide in tropical and some temperate areas. Leishmania are transmitted by tiny sand flies (Phlebotomus sp and Lutzomyia sp.) and survive in the vertebrate host as intracellular amastigotes. Vector flies are infected by biting humans or animals. Animal reservoirs vary with the Leishmania species and location and include canines, rodents, humans, and other animals. Infection is spread rarely by blood transfusion, shared needles, congenitally, or sexually.

Life cycle

Leishmaniasis is transmitted by the bite of female phlebotomine sandflies. The sandflies inject the infective stage, metacyclic promastigotes, during blood meals. Metacyclic promastigotes that reach the puncture wound are phagocytized by macrophages and transform into amastigotes. Amastigotes multiply in infected cells and affect different tissues, depending in part on which Leishmania species is involved. These differing tissue specificities cause the differing clinical manifestations of the various forms of leishmaniasis. Sandflies become infected during blood meals on an infected host when they ingest macrophages infected with amastigotes. In the sandfly's midgut, the parasites differentiate into promastigotes, which multiply, differentiate into metacyclic promastigotes and migrate to the proboscis.

Types of leishmaniasis There are generally four main forms of leishmaniasis. The compounds, compositions, and methods described herein can be applied to all forms of leishmaniasis. • Visceral leishmaniasis - the most serious form and potentially fatal if untreated. Visceral leishmaniasis (kala-azar; Dumdum fever) is typically caused by L. donovani or L. infantumlL. chagasi and typically occurs in India, Africa (particularly the Sudan), Central Asia, the Mediterranean basin, South and Central America, and infrequently China. Parasites disseminate from the skin to the lymph nodes, spleen, liver, and bone marrow and cause symptoms. Subclinical infections are common; only a minority of infected persons develop progressive visceral disease.

• Cutaneous leishmaniasis - the most common form which causes a sore at the bite site, which heal in a few months to a year, leaving an unpleasant looking scar. This form can progress to any of the other three forms. Cutaneous leishmaniasis is also known as oriental or tropical sore, Delhi or Aleppo boil, uta or chiclero ulcer, or forest yaws. The causative agents are L. major and L. tropica in southern Europe, Asia, and Africa; L. mexicana and related species in Mexico and Central and South America; and L. braziliensis and related species in Central and

South America. Isolated cases have been reported in travelers to endemic areas in Central and South America, Israel, and elsewhere.

• Diffuse cutaneous leishmaniasis - this form produces widespread skin lesions which resemble leprosy. • Mucocutaneous leishmaniasis - commences with skin ulcers which spread causing tissue damage, e.g., to the nose and mouth. Mucocutaneous leishmaniasis (espundia) is caused mainly by L. braziliensis, but it occasionally occurs with other Leishmania species.

Symptoms and Signs

For visceral leishmaniasis, the clinical manifestations usually develop gradually over weeks to months after inoculation of the parasite. Irregular fever, hepatosplenomegaly, pancytopenia, and polyclonal hypergammaglobulinemia with reversed albumin: globulin ratio generally occur. In some patients, there are twice- daily temperature spikes. Emaciation and death can occur within 1 to 2 yr in 80 to 90% of untreated symptomatic patients. Those with asymptomatic, self -resolving infections and survivors (after successful treatment) are resistant to further attacks unless cell-mediated immunity is impaired (e.g., in patients with AIDS). After treatment for visceral leishmaniasis, patients in the Sudan and India may develop post kala-azar dermal leishmaniasis with flat or nodular cutaneous lesions full of parasites. These lesions develop at the end of or within 6 months of therapy in patients in the Sudan and 1 to 2 years later in India. The lesions persist for a few months to a year in most patients in the Sudan but can last for years in India. Cutaneous leishmaniasis produces a well demarcated skin lesion at the site of a sand fly bite after several weeks to months. Multiple lesions may occur after multiple infective bites or with metastatic spread. The initial lesion is often a papule that slowly enlarges, ulcerates centrally, and develops a raised, erythematous border where intracellular parasites are concentrated. Ulcers are painless and cause no systemic symptoms unless secondarily infected. Leishmanial lesions generally heal spontaneously after months but may persist for years. They leave a depressed, burn- like scar. The course depends on the species and the host's immune status. In the Americas, skin lesions can be followed by metastatic mucocutaneous lesions if they are caused by L. braziliensis or related species (see mucocutaneous leishmaniasis, below). Diffuse cutaneous leishmaniasis is an uncommon form characterized by widespread nodular skin lesions resembling those of lepromatous leprosy. It is presumed to result from cell-mediated anergy to the organism.

Mucocutaneous leishmaniasis starts with a primary cutaneous ulcer. This skin lesion heals spontaneously, but parasites can metastasize to nasopharyngeal tissues. Months to years later, mucosal lesions develop, sometimes resulting in gross mutilations of the nose, palate, and face.

Diagnosis

Leishmaniasis is generally diagnosed in the hematology laboratory by direct visualization of the amastigotes (Leishman-Donovan bodies). Buffy-coat preparations of peripheral blood or aspirates from marrow, spleen, lymph nodes or skin lesions are spread on a slide to make a thin smear, and stained with leishman's or Giemsa's stain (p_H 7.2) for 20 minutes. Amastigotes are seen with monocytes or, less commonly in neutrophils, in peripheral blood and in macrophages in aspirates. Amastigotes are small, round bodies 2-4 microns in diameter with indistinct cytoplasm, a nucleus, and a small rod shaped kinetoplast. Occasionally amastigotes may be seen lying free between cells. Organisms causing simple cutaneous leishmaniasis can be differentiated from those capable of causing mucocutaneous leishmaniasis with specific DNA probes or monoclonal antibodies, or by analysis of isoenzyme patterns of cultured parasites.

Serologic tests are available. A recombinant antigen (rk39) is positive in patients with visceral leishmaniasis, but not in subclinical cases or persons with cutaneous leishmaniasis. Skin tests may be available in some areas.

Biology

The genomes of three Leishmania species (L. major, L. infantum and L. braziliensis) have been sequenced, providing information about the biology of the parasite. For example, in Leishmania, protein-coding genes are organized as large polycistronic units in a head-to-head or tail-to-tail manner; RNA polymerase II transcribes long polycistronic messages in the absence of defined RNA pol II promoters; and Leishmania has unique features with respect to the regulation of gene expression in response to changes in the environment.

Vaccines

Vaccines are not currently available. However, the genomic sequence of several Leishmania species has provided a rich source of vaccine candidates.

Structure of Compounds

Compounds that can be used in practicing the invention have a general formula (I) and contain a substituted five or six membered ring core containing one or two, respectively, oxygen, nitrogen, or sulfur atoms as a constituent atom of the ring,

e.g., X and Y in formula (I). Any ring carbon atom can be substituted. For example, R¹, R², R³, and R⁴ may include without limitation substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, heteroaryl, etc. The five or six membered ring core may be saturated, i.e. containing no double bonds, or partially or fully saturated, i.e. one or two double bonds respectively. When n =0,

"X" may be oxygen, sulfur, or nitrogen, e.g., NR⁷. The substituent R⁷ can be without limitation hydrogen, alkyl, e.g., Cl, C2, C3, C4 alkyl, Sθ₂(aryl), acyl, or the ring nitrogen may form part of a carbamate, or urea group. When n =1, X can be NR⁷, O, or S; and Y can be NR⁷ , O or S. X and Y can be any combination of heteroatoms, e,g,. N,N, N,O, N, S, etc.

A preferred subset of compounds of formula (I) includes those having one, or preferably, two rings that are fused to the five or six membered ring core, e.g., R¹ and R², together with the carbons to which they are attached, and/or R³ and R⁴, together with the carbons to which they are attached, can form, e.g., C₅-C₁₀ cycloalkyl (e.g., C5, C6, or Cl), C₅-Ci₀ heterocyclyl (e.g., C5, C6, or Cl), C₅-Ci₀ cycloalkenyl (e.g., C5, C6, or Cl), C₅-Ci₀ heterocycloalkenyl (e.g., C5, C6, or Cl), C₆-Ci₀ aryl (e.g., C6, C8 or ClO), or C₆-Ci₀ heteroaryl (e.g., C5 or C6). Fused ring combinations may include without limitation one or more of the following:

B E

C D

Preferred combinations include B, e.g. having C₆ aryl and C₆ cycloalkenyl (Bl), and C, e.g. having C₆ aryl and C₇ cycloalkenyl (Cl):

Each of these fused ring systems may be optionally substituted with substituents, which may include without limitation halo, hydroxy, C₁-C_1O alkyl (Cl,C2,C3,C4,C5,C6,C7,C8,C9,C10) , Ci-C₆ haloalkyl (C1,C2,C3,C4,C5,C6,), C₁- Cio alkoxy (Cl₅C2₅C3₅C4₅C5₅C6₅CV₅C8₅C9₅C10)₅ CI-C₆ haloalkoxy (C1,C2,C3,C4,C5,C6,), C₆-Ci₀ aryl (C6,C7,C8,C9,C10), C₅-Ci₀ heteroaryl (CS₅CO₅CV₅CS₅CQ₅CIO), C₇-C_I2 aralkyl (CV₅C8₅C9₅C10₅Cll₅C12), C₇-Ci₂ heteroaralkyl (CV₅C8₅C9₅C10₅Cll₅C12), C₃-C₈ heterocyclyl (C3,C4₅C5₅C6₅CV₅C8), C₂-Ci₂ alkenyl (C2₅C3₅C4₅C5₅C6₅CV₅C8₅C9₅C10₅Cll₅C12), C₂-Ci₂ alkynyl (C2₅C3₅C4₅C5₅C6₅CV₅C8₅C9₅C10₅Cll₅C12), C₅-Ci₀ cycloalkenyl (C5₅C6₅CV₅C8₅C9₅C10), C₅-Ci₀ heterocycloalkenyl (C5,C6,CV,C8,C9,C10), carboxy, carboxylate, cyano, nitro, amino, Ci-C₆ alkyl amino (C1,C2,C3,C4,C5,C6,), Ci-C₆ dialkyl amino (C1,C2,C3,C4,C5,C6,), mercapto, SO₃H, sulfate, S(O)NH₂, S(O)₂NH₂, phosphate, C₁-C₄ alkylenedioxy (C1,C2,C3,C4), oxo, acyl, aminocarbonyl, C₁-C₆ alkyl aminocarbonyl (C1,C2,C3,C4,C5,C6,), C₁-C₆ dialkyl aminocarbonyl (C1,C2,C3,C4,C5,C6,), C₁-C₁₀ alkoxycarbonyl (Cl,C2,C3,C4,C5,C6,CV,C8,C9,C10), Ci-Cio thioalkoxycarbonyl (Cl,C2,C3,C4,C5,C6,CV,C8,C9,C10), hydrazinocarbonyl, Ci-C₆ alkyl hydrazinocarbonyl (C1,C2,C3,C4,C5,C6,), Ci-C₆ dialkyl hydrazinocarbonyl (C1,C2,C3,C4,C5,C6,), hydroxyaminocarbonyl, etc. Preferred substituents include halo (e.g., fluoro, chloro, bromo), C₁-C₁₀ alkyl (e.g., Cl, C2, C3, C4, C5, C6, CV, C8, C9, ClO), Ci-C₆ haloalkyl (e.g., Cl₅ C2, C3, C4, C5, C6, e.g.,

CF₃), Ci-C₆ haloalkoxyl (e.g., Cl₅ C2, C3, C4, C5, C6, e.g., OCF₃), or aminocarbonyl. The substitution pattern on the two fused rings may be selected as desired, e.g., one ring may be substituted and the other is not, or both rings may be substituted with 1-5 substitutents (1,2,3,4,5 substitutents). The number of substituents on each ring may be the same or different. Preferred substitution patterns are shown below:

In certain embodiments, when n is 0 and X is NR⁷, the nitrogen substituent R⁷ can form a cyclic structure with one of the fused rings containing, e.g., 4-6 carbons, 1- 3 nitrogens, 0-2 oxygens and 0-2 sulfurs. This cyclic structure may optionally be substituted with oxo or Ci-C₆ alkyl.

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).

Exemplary compounds include those depicted in Table 1 below*:

Table 1: Exemplary compounds

* Compounds having activity designated with an A have an IC₅₀ of less than 1.0 μM. Compounds having activity designated with a B have an IC₅₀ between 1.0 μM and 10.0 μM. Compounds having activity designated with a C have an IC₅₀ greater than 10.0 μM. Compounds designated with a D were not tested in this assay. Compounds that can be useful in practicing this invention can be identified through both in vitro (cell and non-cell based) and in vivo methods. A description of these methods is described in the Examples.

Exemplary compounds described herein may inhibit activity of SIRTl or a functional domain thereof by at least 10, 20, 25, 30, 50, 80, or 90%, with respect to a natural or artificial substrate described herein. For example, the compounds may have a Ki of less than 500, 200, 100, or 50 nM.

A compound described herein may also modulate a complex between a sirtuin and a transcription factor, e.g., increase or decrease complex formation, deformation, and/or stability. Exemplary sirtuin-TF complexes include Sir2-PCAF, SIR2-MyoD, Sir2-PCAF-MyoD, Sir2-p53, Sir2-Foxθl, and Sir2-FoxO3. A compound described herein may also modulate expression of a Sir2 regulated gene, e.g., a gene described in Table 1 of Fulco et al. (2003) MoI. Cell 12:51-62.

Assays, e.g., in vitro assoays, for sitruins (e.g., SIRTl), are known in the art, for example, as described in US 20060074124, the contents of which is incorporated by reference in its entirety.

Synthesis of Compounds

The compounds described herein can be obtained from commercial sources (e.g., Asinex, Moscow, Russia; Bionet, Camelford, England; ChemDiv, SanDiego, CA; Comgenex, Budapest, Hungary; Enamine, Kiev, Ukraine; IF Lab, Ukraine; Interbioscreen, Moscow, Russia; Maybridge, Tintagel, UK; Specs, The Netherlands; Timtec, Newark, DE; Vitas-M Lab, Moscow, Russia) or synthesized by conventional methods as shown below using commercially available starting materials and reagents. For example, exemplary compound 4 can be synthesized as shown in Scheme 1 below.

Scheme 1

hydrolysis NaOH

PyAOP

Brominated β-keto ester 1 can be condensed with 4-chloroaniline followed by cyclization can afford indole 2. Ester saponification can afford acid 3. Finally amination with PyAOP can yield the amide 4. Other methods are known in the art, see, e.g., U.S. Patent 3,859,304, U.S. Patent 3,769,298, /. Am.Chem. Soc. 1974, 74, 5495. The synthesis above can be extended to other anilines, e.g., 3,5-dichloroaniline, 3-chloroaniline, and 4-bromoaniline. Regioisomeric products, e.g., 5, may be obtained using N-substituted anilines, e.g., 4-chloro-N-methylaniline.

The compounds described herein 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. 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 contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of this invention may also contain linkages (e.g., carbon-carbon bonds) or substituents that can restrict bond rotation , e.g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis/trans and E/Z isomers are expressly included in the present invention. 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, even though only a single tautomeric form may be represented (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). 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.

Techniques useful for the separation of isomers, e.g., stereoisomers are within skill of the art and are described in Eliel, E.L.; Wilen, S. H.; Mander, L.N. Stereochemistry of Organic Compounds, Wiley Interscience, NY, 1994. For example compound 3 or 4 can be resolved to a high enantiomeric excess (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 99% or greater) via formation of diasteromeric salts, e.g. with a chiral base, e.g., (+) or (-) α-methylbenzylamine, or via high performance liquid chromatography using a chiral column. In some embodiments, the crude product 4, is purified directly on a chiral column to provide enantiomerically enriched compound. For purposes of illustration, enantiomers of compound 4 are shown below.

4 4

In some instances, the compounds disclosed herein are administered where one isomer (e.g., the R isomer or S isomer) is present in high enantiomeric excess. In general, the isomer of compound 4 having a negative optical rotation, e.g. ,-14.1 (c=0.33, DCM) or [α]_D ²⁵ -41.18° (c 0.960, CH₃OH) has greater activity against the SirTl enzyme than the enantiomer that has a positive optical rotation of +32.8 (c=0.38, DCM) or [α]_D ²⁵ +22.72° (c 0.910, CH₃OH). Accordingly, in some instances, it is beneficial to administer to a subject a compound 4 having a high enantiomeric excess of the isomer having a negative optical rotation to treat a disease.

While the enantiomers of compound 4 provide one example of a stereoisomer, other stereoisomers are also envisioned, for example as depicted in compounds 6 and 7 below.

7 7

As with the compound of formula 4, in some instances it is beneficial to administer to a subject an isomer of compounds 6 or 7 that has a greater affinity for SirTl than its enantiomer. For example, in some instances, it is beneficial to administer a compound 7, enriched with the (-) optical rotamer, wherein the amide (or other substituent) has the same configuration as the negative isomer of compound 4.

In some instances, it is beneficial to administer a compound having the one of the following structures where the stereochemical structure of the amide (or other substituent) corresponds to the amide in compound 4 having a negative optical rotation.

(n is an integer from 0 to 4.) The compounds of this invention include the compounds themselves, as well as their salts and their prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active compounds.

The compounds of this invention may be modified by appending appropriate functionalities to enhance selected biological properties, e.g., targeting to a particular tissue. Such modifications are known in the art and include those which increase biological penetration into a given biological compartment (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.

In an alternate embodiment, the compounds described herein may be used as platforms or scaffolds that may be utilized in combinatorial chemistry techniques for preparation of derivatives and/or chemical libraries of compounds. Such derivatives and libraries of compounds have biological activity and are useful for identifying and designing compounds possessing a particular activity. Combinatorial techniques suitable for utilizing the compounds described herein are known in the art as exemplified by Obrecht, D. and Villalgrodo, J.M., Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon- Elsevier Science Limited (1998), and include those such as the "split and pool" or "parallel" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, A.W., Curr. Opin. Chem. Bio., (1997) 1, 60. Thus, one embodiment relates to a method of using the compounds described herein for generating derivatives or chemical libraries comprising: 1) providing a body comprising a plurality of wells; 2) providing one or more compounds identified by methods described herein in each well; 3) providing an additional one or more chemicals in each well; 4) isolating the resulting one or more products from each well. An alternate embodiment relates to a method of using the compounds described herein for generating derivatives or chemical libraries comprising: 1) providing one or more compounds described herein attached to a solid support; 2) treating the one or more compounds identified by methods described herein attached to a solid support with one or more additional chemicals; 3) isolating the resulting one or more products from the solid support. In the methods described above, "tags" or identifier or labeling moieties may be attached to and/or detached from the compounds described herein or their derivatives, to facilitate tracking, identification or isolation of the desired products or their intermediates. Such moieties are known in the art. The chemicals used in the aforementioned methods may include, for example, solvents, reagents, catalysts, protecting group and deprotecting group reagents and the like. Examples of such chemicals are those that appear in the various synthetic and protecting group chemistry texts and treatises referenced herein.

Additional compounds and methods of treatment or prevention of leishmaniasis

The compounds described herein can be used alone or in combination with additional compounds, compositions, and methods for the treatment of leishmaniasis. Some compounds and methods for the treatment of leishmaniasis are provided below.

The drug of choice for treatment of leishmaniasis generally depends on the infecting species and the geographic locations. Pentavalent antimony compounds have been used for visceral and cutaneous disease. Drugs include sodium stibogluconate (Na antimony gluconate) or meglumine antimonate(e.g., 20 mg/kg slowly injected IV or IM once/day for 20 to 28 days). Adverse effects include nausea, vomiting, malaise, and elevated amylase and liver enzymes. If cardiotoxicity develops, administration should be stopped. In industrialized countries, amphotericin B (e.g., not to exceed 1.5 mg/kg) (e.g., liposomal amphotericin B) (ABELCET, AMBISOME, AMPHOCIN, AMPHOTEC, Amphotericin B (conventional), Amphotericin B (lipid complex), Amphotericin B (liposomal), Amphotericin B Cholesteryl sulfate complex), e.g., 3 mg/kg once/day for 5 days, then 3 mg/kg once/day on days 14 and 21, is the drug of choice for immunocompetent patients. Higher doses and longer regimens are used in those with AIDS. Alternatives are deoxycholate (e.g., 0.5 to 1 mg/kg by slow infusion every day or every other day for up to 8 wk) or pentamidine (NEBUPENT, PENTAM 300), isethionate (e.g., 2 to 4 mg/kg IV once/day or every other day for up to 15 doses). A compound described herein can be used in combination with another leishmaniasis treatment, e.g., a leishmaniasis treatment described herein.

Drug resistance has become a problem with antimonials, particularly in India in persons with visceral leishmaniasis. Miltefosine (IMPAVIDO), is a drug that can be used for visceral and cutaneous leishmaniasis. Miltefosine, e.g., given 100 mg once/day ( or 2.5 mg/kg for children 2 to 11 yr) for 28 days, is effective. Adverse effects include nausea and vomiting, transient transaminase elevations, and dizziness. A compound described herein can be used in place of or in combination with another leishmaniasis treatment, e.g., a leishmaniasis treatment described herein.

Treatment of cutaneous disease depends on several factors, including the causative Leishmania species, extent of lesion, and whether dissemination to the mucosa is a concern. Parenteral pentavalent antimonials are often used, particularly for Leishmania species that can disseminate to cause mucosal leishmaniasis. Fluconazole (DIFLUCAN) or itraconazole (SPORANOX) is effective in some cases. Paromomycin (topical paromomycin (HUMATIN)) has been used for L. major infections. Mucosal disease frequently relapses, as does visceral disease in patients with AIDS. Diffuse cutaneous leishmaniasis is relatively resistant to treatment. . A compound described herein can be used in place of or in combination with another leishmaniasis treatment, e.g., a leishmaniasis treatment described herein.

Drug-resistant leishmaniasis may respond to immunotherapy (inoculavtion with parasite antigens plus an adjuvant) which aims to stimulate the body's own immune system to kill the parasite.

Supportive measures may be needed for patients with visceral leishmaniasis, including adequate nutrition, transfusions, and antibiotics for secondary bacterial infection. Reconstructive surgery may be required for mucocutaneous disease with gross distortion of the nose or palate, but surgery should be delayed for 6 to 12 months after therapy to avoid loss of grafts to relapses. For prevention, treatment of cases in a geographic area, reduction of the vector population, and elimination of nonhuman reservoirs where appropriate may help. Insect repellents containing DEET provide protection. Insect screens, bed nets, and clothing are more effective if treated with permethrin (ELIMITE, NIX, RID SPRAY) or pyrethrum, because the tiny flies can penetrate mechanical barriers. A compound described herein can be used in combination with another leishmaniasis treatment, e.g., a preventative leishmaniasis treatment described herein.

Compounds, compositions, and methods of administration Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄ salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Salt forms of the compounds of any of the formulae herein can be amino acid salts of carboxy groups (e.g. L-arginine, -lysine, -histidine salts). The compounds of the formulae described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, with a dosage ranging from about 0.5 to about 100 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.

Upon improvement of a patient'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. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

The compositions delineated herein include the compounds of the formulae delineated herein, as well as additional therapeutic agents if present, in amounts effective for achieving a modulation of disease or disease symptoms, including those described herein. The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α- tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, 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, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as OC-, β-, and γ- cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically- acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic 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 mannitol, water, Ringer's solution 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 may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch.

Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of this invention is useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention. The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. A composition having the compound of the formulae herein and an additional agent (e.g., a therapeutic agent) can be administered using an implantable device. Implantable devices and related technology are known in the art and are useful as delivery systems where a continuous, or timed-release delivery of compounds or compositions delineated herein is desired. Additionally, the implantable device delivery system is useful for targeting specific points of compound or composition delivery (e.g., localized sites, organs). Negrin et al., Biomaterials, 22(6):563 (2001). Timed-release technology involving alternate delivery methods can also be used in this invention. For example, timed-release formulations based on polymer technologies, sustained-release techniques and encapsulation techniques (e.g., polymeric, liposomal) can also be used for delivery of the compounds and compositions delineated herein.

Also within the invention is a patch to deliver active chemotherapeutic combinations herein. A patch includes a material layer (e.g., polymeric, cloth, gauze, bandage) and the compound of the formulae herein as delineated herein. One side of the material layer can have a protective layer adhered to it to resist passage of the compounds or compositions. The patch can additionally include an adhesive to hold the patch in place on a subject. An adhesive is a composition, including those of either natural or synthetic origin, that when contacted with the skin of a subject, temporarily adheres to the skin. It can be water resistant. The adhesive can be placed on the patch to hold it in contact with the skin of the subject for an extended period of time. The adhesive can be made of a tackiness, or adhesive strength, such that it holds the device in place subject to incidental contact, however, upon an affirmative act (e.g., ripping, peeling, or other intentional removal) the adhesive gives way to the external pressure placed on the device or the adhesive itself, and allows for breaking of the adhesion contact. The adhesive can be pressure sensitive, that is, it can allow for positioning of the adhesive (and the device to be adhered to the skin) against the skin by the application of pressure (e.g., pushing, rubbing,) on the adhesive or device. When the compositions of this invention comprise a combination of a compound of the formulae described herein and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents may be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents may be part of a single dosage form, mixed together with the compounds of this invention in a single composition.

Kits

A compound described herein described herein can be provided in a kit. The kit includes (a) a compound described herein, e.g., a composition that includes a compound described herein, and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of a compound described herein for the methods described herein, e.g., methods for treating or preventing malaria.

The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for administering the compound.

In one embodiment, the informational material can include instructions to administer a compound described herein in a suitable manner to perform the methods described herein such as the treatment or prevention of malaria , e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer a compound described herein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disorder described herein. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a compound described herein and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats. In addition to a compound described herein, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a flavoring agent (e.g., a bitter antagonist or a sweetener), a fragrance or other cosmetic ingredient, and/or a second agent for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than a compound described herein. In such embodiments, the kit can include instructions for admixing a compound described herein and the other ingredients, or for using a compound described herein together with the other ingredients.

A compound described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that a compound described herein be substantially pure and/or sterile. When a compound described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When a compound described herein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the composition containing a compound described herein. In some embodiments, the kit contains separate 5 containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that o has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a compound described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a 5 compound described herein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device.0 In a preferred embodiment, the device is a medical implant device, e.g., packaged for surgical insertion.

EXAMPLES

In all of the Examples below, compounds are referred to as they correspond to5 their designation in Table 1 (i.e., exemplified compounds).

Example 1 : HeLa Apoptosis Assay

The following exemplary compounds were evaluated for their effect on a HeLa cell apoptosis assay using the Cell Death Detection ELISA plus kit from Roche Applied Science.

0 1 .12 0.15

0.5 1 .23 0.04

2.5 1 .85 0.24 8 10 2.1 1 0.25

8 25 2.27 0.20

5 O 0.92 0.07

5 0.5 1 .00 0.08

5 2.5 0.97 0.1 1

5 10 1 .07 0.02

5 25 0.91 0.07

Resveratrol O 0.73 0.08

Resveratrol 0.5 0.83 0.05

Resveratrol 2.5 0.84 0.02

Resveratrol 10 1 .01 0.07

Resveratrol 25 0.56 0.08

DMSO O 0.72 0.09

DMSO 0.5 0.79 0.12

DMSO 2.5 0.91 0.13

DMSO 10 0.76 0.09

DMSO 25 1 .18 0.20

Example 2

EX-0000635 inhibition of SirT enzymes with P53 Fluor de Lys Substrate

[EX000635] μM

SirT1 SirT2 SirTβ

Sigmoidal dose-response (variable slope)

Best-fit values

BOTTOM 17.24 156.1 248.2

TOP 261 .7 966.4 499.2

LOGEC50 -0.4290 0.4890 1 .1 1 1

HILLSLOPE -1 .080 -0.9944 -1 .146

EC50 0.3724 3.083 12.90

20

eGFP rπSI RT2 rπSIRT2 rπSI RT2 eGFP rπSIRT2 No TSA +TSA +TSA +TSA +TSA + TSA

DMSO EX- 527 EX-540 EX-566

List of Reagents:

Name of Reagent Supplied As Source Catalog Storage Number human SirT1 2.5 or 3.5U / ul Biomol SE-239 -20C

Fluor de Lys Substrate 5OmM in DMSO Biomol KI-104 -20C

Fluor de Lys Developer 2Ox concentrate Biomol KI-105 -20C

NAD Solid Sigma N-1636 -20C

Nicotinamide Solid Calbiochem 481907 RT

Trizma-HCI Solid Sigma T-5941 RT

Sodium Chloride Solid Sigma S-9888 RT

Magnesium Chloride Solid Sigma M-2393 RT

Potassium Chloride Solid Sigma P-391 1 RT

Polyoxyethylene sorbitan 100% Sigma P-7949 RT monolaurate (Tween-20)

Fluor de Lys 1 OmM in DMSO Biomol KI-142 -20C

Deacetylated Standard

List of Equipment:

Tool Name Tool Source Catalog Number

Fluorescence Plate Reader BIO-TEK SIAFR

Synergy HT

Matrix Impact2 16 Channel Apogent Discoveries 2069 pipet

37C Incubator VWR 1540

List of Disposables:

Disposable Source Catalog Number

1 384 white low volume plates Greiner / Bellco 4507-84075

2 Tips for matrix 16 chan pipet Apogent Discoveries 7421

3 25ml divided reagent Apogent Discoveries 8095 reservoirs

4 Plate Sealing Films Apogent Discoveries 4418 Standard Reagent Formulations:

Prepared Component M .W. Component Final Storage

Reagent Name Name Quantity Component

(in water) Concentration

1 Tris-HCI, pH 8.0 Trizma-HCI 157.6 157.6 g / L 1 M RT

HCI to pH 8.0 pH 8.0

2 Sodium Chloride NaCI 58.44 292 g / L 5M RT

3 Magnesium MgCI₂ 203.3 20.33 g / L 10OmM RT

Chloride

4 Potassium KCI 74.55 20.13 g / L 27OmM RT

Chloride

5 Polyoxyethylene Tween-20 1 ml / 10ml 10% RT sorbitan monolaurate

6 NAD NAD 717 0.0717g / ml 10OmM -20C

7 Nicotinamide Nicotinamide 122 0.0061g / ml 5OmM -20C

8 Assay Buffer Tris-HCI, pH 25ml of 1 M 25mM 4C

8.0 stock /L

NaCI 27.4ml of 5M 137mM stock /L

KCI 10ml of 27OmM 2.7mM stock /L

MgCI₂ 10ml of 10OmM 1 mM stock /L

Tween-20 5ml of 10% 0.05% stock /L

** Prepare working stocks below just The following before use are prepared in assay buffer

9 2x Substrates Flour de Lys 6ul /ml 30OuM ice substrate

NAD 2OuI of 10OmM 2mM stock /ml

10 Enzyme Mix Biomol SirT1 ^**depends upon 0.125U/ul ice specific activity (0.5U/well) of lot. Ex: 3.511/ul, 35.71 Ul /ml

1 1 Developer / stop 2Ox 5OuI / ml 1 x in assay ice reagent developer buffer concentrate

Nicotinamide 2OuI of 5OmM 1 mM stock /ml

Example 3: In order to determine if the mammalian enzyme is inhibited by compound 8, 293T cells were transfected with a construct designed to express human SIRTl fused to glutathione-S-transferase to allow for rapid purification from cell extracts. Following lysis cell extracts were incubated with glutathione-Sepharose beads followed by several washes in lysis buffer and a final wash in SIRTl enzyme assay buffer. Beads with bound GST-SIRTl were added to the Fleur-de-lys assay (Biomol) in the presence of a range of concentrations of compound 8. As can be seen in Fig. 2a, the EC₅₀ value of compound 8 for mammalian SIRTl is comparable to that obtained for the recombinant bacterially produced human enzyme.

As can be seen in Figure 2B, compound 8 enters cells and increases p53 acetylation (at lysine 382) after etoposide treatment. In the experiment depicted in Figure 2B, NCI-H460 cells were treated with 2OuM etoposide (a DNA damaging agent) in the presence or absence of SIRTl inhibitors, either compound 8 or nicotinamde and the amount of acetylated p53 (at lysine 382) was visualized by Western blot. Compound 8 is able to increase p53 acetylation significantly relative to DMSO alone and IuM and lOuM is equally effective.

Example 4: Enantiomers of compound 8 were tested, where each enantiomer had a purity of greater than 90% enantiomeric excess, to determine if a single enantiomer was more potent than a mixture of enantiomers. NCI- H460 cells were treated for 6 hours with compounds 8(+) and 8(-) in the presence of 20 micromolar etoposide followed by lysis and immunoprecipitaion of p53 using Ab-6 (Oncogene Science). Extracts were probed with an antibody that recognizes acetylated lysine 382 of p53 (Cell Signaling). Figure 3 demonstrates that there are active and inactive enantiomers of compound 8. Specifically the inactive enantiomer, compound 8(+), does not lead to increased acetylation of p53 in the presence of etoposide whereas compound 8(-) leads to a significant increase in acetylation and satbilization of p53 protein.

Example 5: Compound 8 inhibits the SIRTl enzyme in additional cells. Cell lines U2OS and MCF7 cell lines were treated with compound 8 in the presence of 20 micromolar etoposide (TOPO) for 6 hours followed by lysis and immunoprecipitation with p53 Ab-6 conjugated to agarose beads. Samples were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes acetylated lysine 382 of p53.

The results depicted in Figure 4 demonstrate that compound 8 is competent to inhibit SIRTl in a variety of cell lines with similar effects on P53 acetylation.

Example 6: Synthesis of 2-chloro-5,6,7,8,9J0-hexahydrocycloheptarblindole- 6-carboxamide

Preparation of methyl 3-bromo-2-oxocvcloheptanecarboxylate: Methyl 2-oxo- 1-cycloheptanecarboxylate (50 g, 294 mmole) was dissolved in carbon tetrachloride (200 mL) and chilled to 0 ⁰C. Bromine (46.8 g, 294 mmole) was added via addition funnel over -30 minutes. The cooling bath was then removed and the reaction was allowed to warm to rt. The reaction was stirred under nitrogen at room temperature for 4 days. After 4 days the solution was poured into a separatory funnel containing water (IL). The organic was washed with water and dried over sodium sulfate. Removal of the solvent in vacuo gave (72.4 g, 291mmole, 99% crude yield). The material was carried on without further purification. Preparation of methyl 2-chloro-5,6 J,8,9 JO-hexahydrocycloheptarblindole-6- carboxylate: Methyl 3-bromo-2-oxocycloheptanecarboxylate (16.8 g, 67.4 mmol) and 4-chloroaniline (18.4 g, 98%, 2.13 eq, 143.6 mmol) were added to flask with thermometer, nitrogen inlet and mechanical stirring machine. As the temperature of the mixture passed 140 ⁰C a relatively rapid exotherm and vigorous evolution of gas occurred. The reaction was cooled with water immediately. The reaction mixture was dissolved in DCM (200 mL). The material was transferred into a separatory funnel and washed with water (2 x 50 mL), 3 HCl (3x 50 mL), water (2 x 50 mL), brine, dried over Na₂SO₄, and the solvent was removed by vacuo. The crude residue was applied to a Biotage and eluted with 9/1 heptane/ ethyl acetate to afford product 10 g (53%) as an off white solid, which was used for next reaction without further purification.

Preparation of 2-chloro-5,6,7,8,9,10-hexahvdrocvcloheptarblindole-6- carboxamide: Methyl 2-chloro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-6- carboxylate (10 g, 36 mmol) was dissolved in 7 N ammonia in methanol (350 mL) and transferred to Parr pressure reactor. The reaction vessel was purged briefly to displace any air with Nitrogen. The reaction was then heated to 90 ⁰C for 48 h. The reaction was cooled to r.t. and the solvent removed in vacuo, the crude residue was applied to a Biotage and eluted with grading (1/1 heptane/ ethyl acetate to 0/1 heptane/ ethyl acetate) to afford product as an off white foam, which was triturated with DCM to afford pure product 2g (21%) as an off white solid.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Other embodiments are in the claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing malaria and/or leishmaniasis, the method comprising administering to a subject an effective amount of a compound having a formula (I):

wherein,

R¹ and R², together with the carbons to which they are attached, form C₅-C₁₀ cycloalkyl, C5-C₁0 heterocyclyl, C5-C₁0 cycloalkenyl, C5-C₁0 heterocycloalkenyl, C₆-CiO aryl, or C₆-Ci_O heteroaryl, each of which may be optionally substituted with 1-5 R⁵; or R¹ is H, S-alkyl, or S-aryl, and R² is amidoalkyl wherein the nitrogen is substituted with alkyl, aryl, or arylalkyl, each of which is optionally further substituted with alkyl, halo, hydroxy, or alkoxy;

R³ and R⁴, together with the carbons to which they are attached, form C₅-C₁₀ cycloalkyl, C₅-C₁₀ heterocyclyl, C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-Ci_O aryl, or C₆-Ci_O heteroaryl, each of which are optionally substituted with 1-5 R⁶; each of R⁵ and R⁶ is, independently, halo, hydroxy, C₁-C₁₀ alkyl, Ci-C₆ haloalkyl, Ci-Cio alkoxy, Ci-C₆ haloalkoxy, C₆-CiO aryl, C5-C10 heteroaryl, C7-C12 aralkyl, C7-C12 heteroaralkyl, C₃-Cs heterocyclyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C5-C₁0 cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, carboxy, carboxylate, cyano, nitro, amino, Ci-C₆ alkyl amino, Ci-C₆ dialkyl amino, mercapto, SO₃H, sulfate, S(O)NH₂, S(O)₂NH₂, phosphate, Ci-C₄ alkylenedioxy, oxo, acyl, aminocarbonyl, Ci-C₆ alkyl aminocarbonyl, Ci-C₆ dialkyl aminocarbonyl, C₁-C₁₀ alkoxycarbonyl, C₁-C₁₀ thioalkoxycarbonyl, hydrazinocarbonyl, Ci-C₆ alkyl hydrazinocarbonyl, Ci-C₆ dialkyl hydrazinocarbonyl, hydroxy aminocarbonyl; alkoxyaminocarbonyl; or one of R⁵ or R⁶ and R⁷ form a cyclic moiety containing 4-6 carbons, 1-3 nitrogens, 0-2 oxygens and 0-2 sulfurs, which are optionally substituted with oxo or Ci-C₆ alkyl; X is NR⁷, O, or S; Y is NR^7', O or S; represent optional double bonds; each of R⁷ and R⁷ is, independently, hydrogen, Ci-C₆ alkyl, C₇-C₁₂ arylalkyl, C₇- C₁₂ heteroarylalkyl; or R⁷and one of R⁵ or R⁶ form a cyclic moiety containing 4-6 carbons, 1-3 nitrogens, 0-2 oxygens and 0-2 sulfurs, which are optionally substituted with oxo or Ci-C₆ alkyl; and n is 0 or 1.

2. The method of claim 1, wherein R¹ and R², together with the carbons to which they are attached, form C₅-C₁₀ cycloalkyl, C₅-C₁₀ heterocyclyl, C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-Ci_O aryl, or C₆-Ci_O heteroaryl, each of which may be optionally substituted with 1 -5 R .

3. The method of claim 1, wherein R and R , together with the carbons to which they are attached, form C₅-C₁₀ cycloalkenyl.

4. The method of claim 3, wherein R and R are substituted with R .

5. The method of claim 4, wherein R is, Ci-C₆ alkyl substituted with a substituent or amino carbonyl, substituted with a substituent.

6. The method of claim 5, wherein the substituent is an amino substituent, or aminocarbonyl.

7. The method of claim 1, wherein R and R , together with the carbons to which they are attached, form C₆-CiO aryl.

The method of claim 5, wherein R³ and R⁴ are substituted with R⁶.

9. The method of claim 6 wherein R⁶ is halo or Ci-C₆ alkyl.

(50

10. The method of claim 1, wherein n is 0.

11. The method of claim 1 wherein X is NR⁷.

12. The method of claim 1 wherein n is 0 and X is NR⁷.

13. The method of claim 1, having the formula (X) below:

formula (X).

14. The method of claim 13, wherein R is halo or Ci-C₆ alkyl.

15. The method of claim 13, wherein R is aminocarbonyl.

16. The method of claim 13, having the formula (XI) below:

17. The method of claim 16, wherein R⁶ is halo or alkyl.

18. The method of claim 16, wherein R⁵ is aminocarbonyl.

19. The method of claim 16, wherein wherein R⁶ is halo or alkyl and wherein

R⁵ is aminocarbonyl.

20. The method of claim 13 wherein the compound is 6-Chloro-2,3,4,9- tetrahydro-lH-carbazole-l-carboxylic acid amide.

21. The method of claim 20 wherein the compound comprises greater than a 60% enantiomeric excess of the enantiomer having an optical rotation of -14.1 (c=0.33 DCM).

22. The method of claim 21, wherein the compound comrises greater than a 90% enantiomeric excess of the enantiomer having an optical rotation of -14.1 (c=0.33 DCM).

23. The method of claim 1, comprising reducing the severity of at least one symptom of malaria.

24. The method of claim 23, wherein the symptom is fever, chills, sweats, headaches, muscle pains, nausea, vomiting, elevated temperature, perspiration, tiredness, confusion, coma, neurologic focal signs, anemia, or respiratory difficulties.

25. The method of claim 1, further comprising administering an additional treatment.

26. The method of claim 25, wherein the additional treatment is chloroquine

(e.g., chloroquine phosphate), hydroxychloroquine, quinine sulfate, doxycycline, tetracycline, clindamycin, quinine sulfate plus doxycycline, quinine sulfate plus tetracycline, quinine sulfate plus clindamycin; atovaquone-proguanil (Malarone), mefloquine, primaquine (e.g., primaquine phosphate), quinidine gluconate, quinidine/quinine combination, quinidine/quinine plus doxycycline, quinidine/quinine plus tetracycline, or quinidine/quinine plus clindamycin.

27. The method of claim 25, wherein the second treatment is a bednet or an insecticide.

28. The method of claim 1, comprising reducing the severity of at least one symptom of leishmaniasis.

29. The method of claim 28, wherein the symptom is irregular fever, hepatosplenomegaly, pancytopenia, polyclonal hypergammaglobulinemia with reversed albumin: globulin ratio, twice-daily temperature spikes, or emaciation.

30. The method of claim 1, further comprising administering an additional treatment.

31. The method of claim 30, wherein the additional treatment is pentavalent antimony, sodium stibogluconate (e.g., Na antimony gluconate), meglumine antimonite (e.g., 20 mg/kg slowly injected IV or IM once/day for 20 to 28 days), amphotericin B (e.g., not to exceed 1.5 mg/kg) (e.g., liposomal amphotericin B) (e.g., ABELCET, AMBISOME, AMPHOCIN, AMPHOTEC, Amphotericin B (conventional), Amphotericin B (lipid complex), Amphotericin B (liposomal), Amphotericin B

(Cholesteryl sulfate complex), e.g., 3 mg/kg once/day for 5 days, then 3 mg/kg once/day on days 14 and 21, deoxycholate (e.g., 0.5 to 1 mg/kg by slow infusion every day or every other day for up to 8 weeks), pentamidine (e.g., NEBUPENT, PENTAM 300), isethionate (e.g., 2 to 4 mg/kg IV once/day or every other day for up to 15 doses), miltefosine (e.g., IMPAVIDO), e.g., given 100 mg once/day (or, e.g., 2.5 mg/kg for children 2 to 11 yrs) for 28 days, fluconazole (e.g., DIFLUCAN), itraconazole (e.g., SPORANOX), and paromomycin (e.g., topical paromomycin (e.g., HUMATIN)).

32. The method of claim 30, wherein the additional treatment is a supportive measure, e.g., adequate nutrition, tranfusions, or antibiotics (e.g., for seconday bacterial infection).