Host-directed therapies for leishmania infections

Abstract

The invention relates to small molecule compounds that are useful as antimicrobial agents, pharmaceutical compositions comprising the compounds, and the use of the compounds in the treatment of subjects with microbial infections.

Description

HOST-DIRECTED THERAPIES FOR LEISHMANIA INFECTIONS FIELD OF THE INVENTION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 684,190 filed on August 16, 2024, the entire contents of which are hereby incorporated by reference.

[0002] The invention relates to small molecule compounds that are useful as antimicrobial agents. The invention also relates to the use of pharmaceutical compositions comprising these compounds in the treatment of patients with parasitic infections. STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY- SPONSORED RESEARCH

[0003] The invention was made with government support under AI125147 and AI123692, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0004] Antimicrobial resistance is a major healthcare problem worldwide. The World Health Organization explains that the emergence and spread of drug-resistant pathogens threatens the world’s ability to treat common infections and to perform life-saving procedures (e.g., chemotherapy, caesarean sections, organ transplantation). Drug-resistant pathogens also threaten our food source with food-borne contamination and could be applied as the highest-level bioterrorism agents, according to the Centers for Disease Control. Examples of such problematic pathogens are the parasites of the Leishmaia genus, which can cause major illnesses in humans. Treatment of infections caused by Leishmania parasites have serious side effects. Moreover, studies have shown that some Leishmania strains have developed resistance to known treatments.

[0005] Thus, there is a critical need for the development of novel antimicrobial compounds that can treat Leishmania infections.Leishmania Infections

[0006] Leishmaniasis is caused by protozoan parasites of the Leishmania genus, which are transmitted through the bite of infected sandflies. There are approximately 12 million active cases of leishmaniasis worldwide, with an estimated 1 million new cases occurring each year.

[0007] There are three main types of Leishmaniasis. The first and most common is cutaneous leishmaniasis, which causes skin lesions on exposed parts of the body. The second is mucocutaneous leishmaniasis, which results in destruction of mucous membranes of the nose, mouth, and throat. The third and most serious is visceral leishmaniasis (VL), which is characterized by fever, anemia, and enlarged spleen and liver. VL is fatal if untreated.

[0008] Common treatments for VL have serious side effects and have decreasing efficacy as a result of resistant strains of Leishmania. The most common treatment for VL for the past 70 years has been the systemic injection of antimonial therapies, i.e., metalloid-based treatments, such as sodium stibogluconate (SSG) or meglumine antimoniate formulations. Antimonial therapies are highly toxic and cause severe adverse side effects, including pancreatitis and cardiac arrhythmia. Further, many strains of L. donovani, one species responsible for VL, have become resistant to antimonial therapies.

[0009] To account for the antimonial resistance of Leishmania strains, new treatments have been developed for VL. Two of these are miltefosine and amphotericin b (AmpB, commonly used as an antifungal drug). However, miltefosine is not a preferred treatment, because it is teratogenic. Further, AmpB, although highly effective in treating leishmaniasis, is highly toxic and requires encapsulation in a liposomal formulation (AmBisome). Moreover, the proposed mechanism of action of AmpB on Leishmania, binding to ergosterol found in the Leishmania membrane and preventing promastigote entry into the macrophage, is thought to drive selective pressure in the development of drug resistance towards AmpB treatment. In fact, researchers have isolated a strain of Leishmania that demonstrates resistance to AmpB. Certain Leishmania strains have also shown an inherent broad-spectrum resistance against drugs they have not previously encountered, illustrating that there would be some resistance to any proposed parasite- specific therapy. Thus, new treatment options that are effective against Leishmania infections are needed.Host-Directed Therapeutics

[0010] One strategy to overcome pathogenic drug resistance is through host directed therapies (HDTs; also known as host-targeted therapies), which improve the host cell’s ability to clear infection. Because HDTs target host cells instead of pathogens, there is no direct selective pressure on the pathogens, which can mitigate further drug resistance. And, by targeting the host cells instead of the pathogens, HDTs can also potentially treat drug resistant strains. Additionally, because many pathogens take advantage of similar infectious pathways, HDTs could potentially target a broad spectrum of pathogens.

[0011] A drug that is being evaluated for HDTs is AR-12 (originally named OSU-03012):Although AR-12 was initially developed as a cancer chemotherapeutic agent and reached clinical trials and FDA IND approval for that application, studies have shown that AR-12 has broad spectrum host-directed activity against a range of pathogens. For example, AR- 12 has been shown to induce autophagy, which can disrupt the life-cycle of some intracellular pathogens. AR-12 has also been shown to inhibit expression of Glucose Regulated Protein (GRP78), which is induced in Leishmania-infected macrophages. With regard to LV, AR-12 has been shown to decrease the parasite burden of L. donovani in infected macrophages while having no direct effect on the promastigote. Further, co- treatment of AR-12 with AmpB has been shown to result in significant reduction of LV parasite burden compared to treatment with AR-12 alone or treatment with AmpB alone, which indicates the potential of AR-12 in sensitizing parasites to AmpB.

[0012] The present invention provides novel chemical compounds that are analogs of AR-12 for HDTs against leishmaniasis. These novel compounds demonstrate significantly improved HDT characteristics compared to AR-12.BRIEF SUMMARY OF THE INVENTION

[0013] One aspect of the invention is directed to novel chemical compounds of Formula (I):wherein: A is N or CR2; B is N or CR3; D is N or CR4; E is N or CR5; R1is -OR6, -NR7R8, a substituted or unsubstituted aryl, or a substituted or unsubstituted heterocyclic moiety; R2 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R3 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R4 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R5is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R6is -H, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10cycloalkyl; R7 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; andR8is -H, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10cycloalkyl; or a pharmaceutically acceptable salt thereof.

[0014] Another aspect of the invention is directed to novel pharmaceutical compositions comprising compounds of Formula (I).

[0015] Another aspect of the invention is methods of treating infections caused by bacterial pathogens, comprising administration of a pharmaceutical composition comprising compounds of Formula (I). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG 1. Process flow of screening methodology and compound selection against Leishmania.

[0017] FIG 2. Dose responses of AR-12 analog compounds and promastigote viability after treatment with L. donovani and L. mexicana.

[0018] FIG.3A. Plot of dose response of AmpB on intracellular Leishmania burden in bone marrow-derived macrophages.

[0019] FIG 3B. Plot of dose response of a compound of Formula (I) on intracellular Leishmania burden in bone marrow-derived macrophages. DETAILED DESCRIPTION OF THE INVENTION

[0020] One aspect of the invention is directed to novel chemical compounds of Formula (I):, wherein: A is N or CR2; B is N or CR3;D is N or CR4; E is N or CR5; R1 is -OR6, -NR7R8, a substituted or unsubstituted aryl, or a substituted or unsubstituted heterocyclic moiety; R2 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R3is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R4is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R5 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R6 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; R7is -H, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10cycloalkyl; and R8is -H, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10cycloalkyl; or a pharmaceutically acceptable salt thereof.

[0021] The novel chemical compounds of Formula (I) are analogs of AR-12. These compounds of Formula (I) have improved activity as compared to AR-12. Without wishing to be bound by theory, it is believed that the compounds of Formula (I) target host cells instead of pathogens, which can mitigate development of drug resistance.

[0022] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR2;B is N or CR3; D is N or CR4; E is N or CR5; R1is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety; R2is -H, -F, a substituted or unsubstituted C1-C6alkyl, a substituted or unsubstituted C3- C10 cycloalkyl, or a substituted or unsubstituted aryl; R3is -H, -F, a substituted or unsubstituted C1-C6alkyl, a substituted or unsubstituted C3- C10 cycloalkyl, or a substituted or unsubstituted aryl; R4 is -H, -F, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3- C10cycloalkyl, or a substituted or unsubstituted aryl; R5 is -H, -F, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3- C10cycloalkyl, or a substituted or unsubstituted aryl; R6is -H, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10cycloalkyl; R7is -H, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10cycloalkyl; and R8 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; or a pharmaceutically acceptable salt thereof.

[0023] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR2; B is N or CR3; D is N or CR4;E is N or CR5; R1 is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, thianyl, morpholinyl, and thiomorpholinyl; R2 is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; R3 is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; R4 is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; R5is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; R6is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; andR8is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

[0024] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR2; B is N or CR3; D is N or CR4; E is N or CR5; R1 is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, thianyl, morpholinyl, and thiomorpholinyl; R2 is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R3 is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R4is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R5is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R6 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl,aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; and R8is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

[0025] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR2; B is N or CR3; D is N or CR4; E is N or CR5; R1is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, and morpholinyl; R2 is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl;R3is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl; R4 is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl; R5 is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl; R6is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7is -H, methyl, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; and R8 is -H, methyl, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

[0026] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N; B is CR3; D is CR4; E is CR5;R1is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of pyrrolidinyl, pyrrolinyl, pyrrolinyl, tetrahydrofuranyl, piperidinyl, tetrahydropyranyl, and morpholinyl; R3 is -H or -CF3; R4 is -H or -CF3; R5is methyl, ethyl, cyclohexyl, or phenyl; R6is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7 is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; and R8 is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

[0027] One embodiment of the invention is directed to compounds of Formula (I) selected from the group consisting of:; a a pharmaceutically acceptable salt thereof.

[0028] Another aspect of the invention is directed to novel pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of Formula (I) andat least one pharmaceutically acceptable excipient. Examples of suitable pharmaceutically acceptable excipients are antioxidants; preservatives (e.g., ethylenediaminetetraacetic acid, “EDTA”; ethyleneglycol-bis -aminoethyl)-N,N,N’,N’-tetraacetic acid, “EGTA”;butylated hydroxyanisole, “BHA”; and butylated hydroxytoluene, “BHT”); coloring agents; flavoring agents; emulsifying agents; suspending agents; solvents; fillers; bulking agents; buffers (e.g., phosphates, carbonates, and citrates); delivery vehicles; binders; disintegrants; diluents; glidants; lubricants; pharmaceutical adjuvants; and the like, which are known to a person skilled in the art.

[0029] The pharmaceutical compositions of the invention may be prepared by any of the well- known techniques of pharmacy, such as effective formulation and administration procedures.

[0030] The compounds of Formula (I) exhibit resensitization of drug resistance with co- delivery with conventional antimicrobial therapies, including antimicrobial therapies used in the treatment of leishmaniasis. The compounds of Formula (I) also exhibit mathematical synergy with conventional antimicrobial therapies, permitting a reduced concentration of the conventional antimicrobial therapies. Accordingly, the invention also includes pharmaceutical compositions comprising (a) a therapeutically effective amount of a compound of Formula (I); (b) a therapeutically effective amount of at least one other pharmaceutically active agent selected from the group consisting of antibacterial compounds, antiviral compounds, antifungal compounds, antiparasitic compounds, and combinations thereof; and (c) a pharmaceutically acceptable excipient. Methods for evaluating synergy between HDR therapies and conventional therapies (e.g., amphotericin B, miltefosine, and paromomycin) can be found in M.S.H. Zahid et al., “Evaluation of synergy between host and pathogen-directed therapies against intracellular Leishmania donovani,” Int. J. Parasitol. Drugs Drug Resist., 10, 2019, 125-132. The entire contents of this disclosure are incorporated by reference herein.

[0031] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the pharmaceutical compositions of the invention may be selected from the group consisting of antimonial therapies, such as sodium stibogluconate and meglumine antimoniate; aminoglycosides, such as paromomycin; triazoles, such as ketoconazole, fluconazole, and itraconazole; phospholipids, such as miltefosine; polyenes, such as amphotericin b; antiprotozoals, such as pentamidine; and mixtures thereof.

[0032] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the pharmaceutical compositions of the invention may be selected from the group consisting of sodium stibogluconate; meglumine antimoniate; paromomycin; ketoconazole; fluconazole; itraconazole; miltefosine; amphotericin b; pentamidine; and mixtures thereof.

[0033] Yet another aspect of the invention is a new method of treating infections caused by a microbial species, comprising administration of a therapeutically effective dose of a compound of Formula (I).

[0034] The compounds of Formula (I) may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The compounds of Formula (I) and pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of Formula (I) may be administered orally, rectally, parenterally, intramuscularly, intravenously, subcutaneously, transdermally, vaginally, topically, or by inhalation.

[0035] The dosage regimen for the compounds of the invention or the pharmaceutical compositions of the invention is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed.

[0036] In many instances, the administration of the compounds of the invention or the pharmaceutical compositions of the invention will be repeated a plurality of times in a day. Multiple doses per day typically may be used to increase the total daily dose, if desired.

[0037] The compounds of Formula (I) can be used, alone or in combination with other pharmaceutically active agents, in the treatment of an infection caused by a microbial species. At least one compound of Formula (I) and at least one other pharmaceutically active agent may be may be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially.

[0038] The phrases “concurrent administration,” “co-administration,” “simultaneous administration,” and “administered simultaneously” mean that the compounds are administered in combination.

[0039] In one embodiment of the invention, a subject in need of treatment of an infection caused by a microbial species with a compound of Formula (I) is an animal. Examples of animals that may be treated according to the invention include fish, amphibians, and mammals. Examples of mammals that may be treated according to the invention include humans, primates, horses, sheep, pigs, cows, mice, rats, rabbits, dogs, and cats.

[0040] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a therapeutically effective amount of at least one compound of Formula (I).

[0041] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of Formula (I).

[0042] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a therapeutically effective amount of at least one compound of Formula (I) and a therapeutically effective amount of at least one other pharmaceutically active compound selected from the group consisting of an antibacterial compound, an antiviral compound, an antifungal compound, an antiparasitic compound, and combinations thereof.

[0043] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of Formula (I) and therapeutically effective amount of at least one other pharmaceutically active compound selected from the group consisting of an antibacterial compound, an antiviral compound, an antifungal compound, an antiparasitic compound, and combinations thereof.

[0044] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises co-administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of Formula (I) and a pharmaceutical composition comprising a therapeutically effective amount of at least one other pharmaceutically active compoundselected from the group consisting of an antibacterial compounds, an antiviral compound, an antifungal compound, an antiparasitic compound, and combinations thereof.

[0045] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the methods of the invention may be selected from the group consisting of antimonial therapies, such as sodium stibogluconate and meglumine antimoniate; aminoglycosides, such as paromomycin; triazoles, such as ketoconazole, fluconazole, and itraconazole; phospholipids, such as miltefosine; polyenes, such as amphotericin b; antiprotozoals, such as pentamidine; and mixtures thereof.

[0046] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the methods of the invention may be selected from the group consisting of sodium stibogluconate; meglumine antimoniate; paromomycin; ketoconazole; fluconazole; itraconazole; miltefosine; amphotericin b; pentamidine; and mixtures thereof.

[0047] In one embodiment of the invention, the infection is caused by parasites in the Leishmania genus.

[0048] In one embodiment of the invention, the infection is caused by parasites in the Leishmania genus that cause cutaneous leishmaniasis. Species of parasites that cause cutaneous leishmaniasis include L. major, L. tropica, L. aethiopica, and L. mexicana.

[0049] In one embodiment of the invention, the infection is caused by parasites in the Leishmania genus that cause mucocutaneous leishmaniasis. Species of parasite that cause mucocutaneous leishmaniasis include L. braziliensis.

[0050] In one embodiment of the invention, the infection is caused by parasites in the Leishmania genus that cause visceral leishmaniasis. Species of parasites that cause visceral leishmaniasis include L. donovani and L. chagasi.

[0051] In one embodiment of the invention, the infection is caused by parasites selected from L. major, L. tropica, L. aethiopica, L. mexicana, L. braziliensis, L. donovani, L. chagasi, and mixtures thereof.Definitions

[0052] The term “alkyl” refers to straight or branched chain alkyl radicals having 1 to 20 carbon atoms, and “substituted alkyl” refers to alkyl radicals further bearing one or more substituents.

[0053] The term “C1-C6 alkyl” refers to straight or branched chain alkyl radicals having 1 to 6 carbon atoms, and “substituted C1-C6 alkyl” refers to C1-C6 alkyl radicals further bearing one or more substituents.

[0054] The term “cycloalkyl” refers to cyclic ring-containing hydrocarbon moieties containing 3 to 20 carbon atoms, and “substituted cycloalkyl” refers to cycloalkyl moieties further bearing one or more substituents.

[0055] The term “C3-C10 cycloalkyl” refers to cyclic ring-containing hydrocarbon moieties containing 3 to 10 carbon atoms, and “substituted C3-C10cycloalkyl” refers to C3-C10cycloalkyl moieties further bearing one or more substituents.

[0056] The term “heterocyclic moiety” refers to cyclic moieties containing one or more heteroatoms (e.g., O, N, or S) as part of the ring structure and having 3 to 10 carbon atoms, and “substituted heterocyclic moiety” refers to heterocyclic moieties further bearing one or more substituents. Examples of heterocyclic moieties are aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, thianyl, morpholinyl, and thiomorpholinyl.

[0057] As used herein, the term “aryl” refers to aromatic groups having 6 to 24 carbon atoms, and “substituted aryl” refers to aryl groups further bearing one or more substituents. Examples of aryl groups are phenyl, naphthyl, fluorenyl, anthryl, phenanthryl, and pyrenyl.

[0058] The term “pharmaceutically acceptable” refers to a non-toxic material that does not interfere with the effectiveness of the active ingredient(s).

[0059] The term “therapeutically effective amount” means an amount of a compound of the invention that (i) treats or prevents a particular disease, condition, or disorder described herein, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particulardisease, condition, or disorder described herein, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0060] Substituted alkyl groups are substituted with one or more substituents selected from the group consisting of -F, -Cl, -Br, -I, -CN, -NO2, -ORa, -SRb, -NRcRd, phenyl, pyridyl, -CHO, -COORe, -CO(NRfRg); wherein each of Ra, Rb, Rc, Rd, Re, Rf, and Rgare independently selected from H or C1-C6 alkyl.

[0061] Substituted cycloalkyl groups, substituted aryl groups, and substituted heterocyclic moieties are substituted with one or more substituents selected from the group consisting of C1-C6alkyl, -F, -Cl, -Br, -I, -CN, -NO2, -ORa, -SRb, -NRcRd, phenyl, o-toluyl, m-toluyl, p-toluyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 4-(trifluoromethyl)phenyl, pyridyl, -CF3, -CCl3, -CBr3, -CI3, -CHO, -COORe, -CO(NRfRg); wherein each of Ra, Rb, Rc, Rd, Re, Rf, and Rg are independently selected from H or C1-C6 alkyl.

[0062] The term “salt” refers to acid addition salts, including Cl-, Br-, I-, NO2 -, HSO4 -,SO4-, HPO4-, PO42-, ethanesulfonate, trifluromethane sulfate, p-toluenesulfonate, benzenesulfonate, salicylate, propionate, ascorbate, aspartate, fumarate, galactarate, maleate, citrate, glutamate, glycolate, lactate, malate, maleate, tartrate, oxalate, succinate, and the like.

[0063] Where a salt is intended to be administered to a patient (as opposed to, for example, being used in an in vitro context), the salt preferably is pharmaceutically acceptable. The term “pharmaceutically acceptable salt” refers to a salt prepared by combining a compound of Formula (I) with an acid whose anion, or a base whose cation, is generally considered suitable for human consumption. For use in medicine, the salts of the compounds of this invention are non-toxic “pharmaceutically acceptable salts.” Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.

[0064] As used herein, the term “Formula (I)” may be referred to as a “compound(s) of the invention,” “the invention,” and “compound of Formula I.” Such terms are also defined to include all forms of the compound of Formula (I), including hydrates, solvates, isomers, crystalline and non-crystalline forms, isomorphs, polymorphs, and metabolites thereof. For example, the compounds of the invention, or pharmaceutically acceptable salts thereof,may exist in unsolvated and solvated forms. When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm. Examples

[0065] In the development of the compounds of Formula (I), various AR-12 analogs were evaluated for their activity against L. donovani. The evaluation of the AR-12 analogs considered activity (MIC) and toxicity compared to AR-12. Example 1 – Evaluation of AR-12 Analogs for L. donovani

[0066] The process flow of screening methodology and compound selection of AR-12 analog compounds for L. donovani is summarized in FIG.1. FIG.1 shows that the concentration at which intracellular Leishmania burden is reduced by 50% in THP-1 macrophages (Lum IC50) is identified by a luminescence assay described below. FIG. 1 also shows that the concentration where THP-1 macrophage cell viability is 50% (LC50) after incubation with an AR-12 analog compound is determined by MTT assay. FIG. 1 also shows that the concentration at which intracellular Leishmania burden is reduced by 50% in bone marrow derived macrophages (IC50) is identified by image-based Giemsa staining. Further, FIG.1 shows that the minimum inhibitory concentration (MIC) where extracellular Leishmania promastigote viability is reduced by 50% (MIC50) after 72-hour incubation with an AR-12 analog compound is measured by resazurin assay.

[0067] Mammalian and Parasitic Cell Lines.

[0068] Human monocytes, THP-1 (ATCC, TIB002) were used as host cells for Leishmania infection and to assess cytotoxicity of the AR-12 analog compounds tested. The cells were cultured at 37 °C, 5% CO2in RPMI medium (ATCC) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin (P / S) and 0.05 -mercaptoethanol. Cells were used in experiments up to cell passage 10.

[0069] Luminescent strains, L. donovani LV82 expressing firefly luciferase (a gift from Dr. Abhay Satoskar of Ohio State University) and L. mexicana (NR-51210, ATCC) expressingrenilla luciferase were used to evaluate the effect of the AR-12 analog compounds in intracellular and extracellular conditions. Wild-type L. donovani LV82 (ATCC) were used for Giemsa staining-based experiments. All parasites were cultured at 25 °C and 5% CO2in M199 media (Corning) supplemented with 10% FBS, 1% P / S, and Hemin (0.01 mg / mL), and used in experiments up to cell passage 15.

[0070] Luminescent-Based Evaluation of Intracellular Anti-Leishmanial Activity.

[0071] THP-1 cells were seeded in a 96-well plate (25,000 cells / well) overnight, then differentiated with 150 nM phorbol 12-myristate 13-acetate (PMA) over 72 hours. The resulting macrophages were infected with Leishmania promastigotes at a multiplicity of infection of 1:10. Promastigotes of a known cell density were resuspended in RPMI media (with 10% FBS, 1% P / S and 0.05 -mercaptoethanol) and incubated with adhered THP-1 cells over 18 hours. After infection, cells were washed three times with fresh media to remove extracellular promastigotes.

[0072] Infected macrophages were then treated with AR-12 analog compounds solubilized in DMSO ranging from 0.1 - 10 µM in concentrations. After 72 hours of treatment, the viability of the amastigotes within macrophages was determined using Promega firefly luminescence assay (Cat E1500) for L. donovani strains and Pierce Renilla luciferase assay (Cat 16166) for L. mexicana strains. Media was removed, and cells were lysed with the assay lysis buffer. The luciferase substrate was then incubated with the lysed cells for 10 min at room temperature, and luminescence of live Leishmania was measured in a white opaque 96-well plate using a Biotek plate reader. The IC50value for each AR-12 analog compound was obtained from the best fit curves obtained by plotting the relative luminescence units against drug concentration of the AR-12 analog compounds used.

[0073] Effect of AR-12 Analog Compounds on Host Cell Viability. Effect of the AR-12 analog compounds on THP-1 cell viability was measured using thiazolyl blue tetrazolium bromide, which measures cell metabolic activity (MTT). THP-1 cells were seeded in a 96- well plate (25,000 cells / well) overnight, then differentiated with 150 nM PMA over 72 hours. Macrophages were then treated with AR-12 analog compounds and resuspended in RPMI media (1 - 50 µM) for 24 hours. Media was replaced with thiazolyl blue tetrazolium bromide solubilized in RPMI media (0.5 mg / mL) and incubated at 37 °C for 2 hours. The resulting reduced formazan crystals were solubilized with isopropyl alcohol, and theabsorbance of the resulting solution was measured at 560 nm with a background subtraction at 670 nm. The concentration required to reduce host cell viability by 50% (24hr LC50) value was measured from best fit curves plotting the relative absorbance values against drug concentration of the AR-12 analog compounds used. This process was repeated with a 72-hr incubation and extended concentration range (1 - 300 µM) for select AR-12 analog compounds.

[0074] Image-Based Evaluation of Intracellular Anti-Leishmanial Activity. Bone marrow-derived macrophages (BMDMs) were isolated from BALB / c mice and cultured. Bone marrow was harvested from long bones of mice, and the isolated cells were seeded at a concentration of 2 x 106cells / mL in petri dishes with RPMI media supplemented with 10% FBS, 1% P / S, and 10% L929 conditioned media (LCM). Completely differentiated BMDMs were obtained after 7 days of culture with media change every 2 days. Harvested cells were seeded onto 10 mm glass cover slips at 5 x 105cells / well of a 24-well plate in DMEM media (without LCM) and allowed to adhere overnight. BMDM cells were then infected overnight with LV82 L. donovani or L. mexicana (NR-51210) at a multiplicity of infection of 10. After infection, cells were washed three times with fresh media to remove extracellular promastigotes and treated with AR-12 analog compounds for a 72-hour incubation. BMDM cells were then washed with phosphate buffered saline, fixed with ice- cold methanol, and stained with Giemsa (5% v / v in water). The cover slips with stained cells were mounted onto glass slides and imaged on EVOS XL (100X, Thermo Fisher Scientific). Leishmania amastigotes per 100 macrophages was determined in a blinded manner. The concentration required to reduce intracellular amastigote viability by 50% (IC50) value was measured from best fit curves plotting the normalized values against drug concentration of the AR-12 analog compound used.

[0075] Effect of AR-12 Analog Compounds on Promastigote Viability. The effect of AR- 12 analog compounds on extracellular promastigotes was evaluated using a resazurin-based assay. Late log phase Leishmania promastigotes were seeded in a 96-well plate at 1 x 105parasites per well and treated with AR-12 analog compounds (0.5 - 200 µM) for 72 hours at 25 °C. Resazurin (10 0.02% w / v) was added to the treated parasites to achieve a final resazurin concentration of 0.002% w / v and incubated for 24 hours. Viability of the promastigotes was assessed by fluorescence (excitation 544 nm, emission 590 nm, SpectraMax M2, Molecular Devices). The minimum inhibitory concentration required toreduce promastigote viability by 50% (MIC50) value was determined from best fit curves plotting the relative fluorescence values against drug concentration of the AR-12 analog compound used.

[0076] Results of Primary Screen. Primary screening of 343 AR-12 analog compounds was performed by medium throughput luminescence-based screening assay using THP-1 macrophages and luminescent L. donovani. A summary of the process is shown in FIG.1. Additionally, the effect of the compounds on host cell viability was evaluated in uninfected THP-1 cells using a colorimetric assay. These two values were used to calculate “Selectivity” of the compounds (24hr LC50 / Lum IC50). Of 343 AR-12 analog compounds screened, 66 were more selective than the parent compound AR-12. Sixteen of these compounds (Table 1) were identified with either a high potency (Lum IC50< 1 µM) or a high selectivity (>15) and were selected for secondary screening. Screening results for all compounds are shown in Table A. Table 1. . .Hits determined by potent host-directed activity (IC50 < 1 µM) or high selectivity (>15). Lum IC50 is the concentration at which intracellular L. donovani burden is reduced by 50% in THP1 macrophages as identified by luminescence assay. Concentration where THP-1 macrophage cell viability is 50% (LC50) after 24- hour incubation with compound as determined by MTT assay. Selectivity between host-directed effect and cytotoxicity, defined as 24h LC50 / Lum IC50. Parental compound AR-12 provided for reference. Table A. . . .. . ... .. .. . .

[0077] Results of Secondary Screen. Additional screening of the selected 16 AR-12 analog compounds was performed to confirm drug activity. Specifically, the effect of the AR-12 analog compounds on macrophage viability was evaluated over a longer range of time (72 hrs). Furthermore, the direct effect of the compounds on extracellular L. donovani promastigotes was measured using resazurin assay. Lastly, activity of compounds to reduce intracellular L. donovani was confirmed using Giemsa staining and image-based analysis in BMDMs was assessed. The host-directed therapeutic index was calculated by the 72hr LC50 / IC50. This characterization is detailed in Table 2 for all sixteen AR-12 analog compounds. Structures of AR-12 and the sixteen AR-12 analog compounds are shown in Table B. From this characterization, four AR-12 analog compounds (Fig 2) were identified with a therapeutic index > 40: compounds 53, 134, 197, and 354. These were selected for tertiary screening in a cutaneous Leishmania strain, L. mexicana, to ensure broad host-directed activity. Table 2Table B

[0078] Results of Tertiary Screen. Four AR-12 analog compounds identified in the secondaryscreen were further screened in L. mexicana using the same assays described for L.donovani. The ability of compounds to reduce intracellular Leishmania was evaluated byboth luminescent and image-based assays. Additionally, the activity of compounds on extracellular L. mexicana promastigotes was measured using resazurin assay (FIG. 2,Table 3). The ability of these four AR-12 analog compounds to reduce intracellular burden was similar for both L. mexicana and L. donovani as measured by luminescent and image- based assays. However, the direct effect of compounds on promastigotes varied betweenthe visceral and cutaneous strains. This suggests that the host-directed effect is the driving factor in intracellular pathogen clearance and is less susceptible to differences in Leishmania strains.

[0079] Tertiary screening showed that two AR-12 analog compounds, RTI-53 and RTI-197, did not show a reduction in macrophage viability over a 72-hr incubation up to 300 µM (Table 3, FIG. 2) but decreased intracellular parasite burden by 50% with less than 2 µM in both visceral and cutaneous leishmania strains (Table 3, FIG.3B). FIG.3A shows the dose response of AmpB on intracellular Leishmania burden in bone -marrow derived macrophages derived from wildtype C57BL6 (black circle) or lysozyme knockout mice (gray triangle) as identified by image-based Giemsa staining. FIG. 3B shows the dose response of RTI-197 on intracellular Leishmania burden in bone-marrow derived macrophages derived from wildtype C57BL6 (black circle) or lysozyme knockout mice (gray triangle) as identified image-based Giemsa staining.

[0080] The dose response plots in FIG.2 show the dose response of intracellular Leishmania burden in THP-1 macrophages as identified by luminescence-based screen (gray triangle), the dose response of intracellular Leishmania burden in bone marrow derived macrophages as identified by image-based Giemsa staining (black circle), and the dose response of extracellular Leishmania promastigote viability after 72-hour incubation with AR-12 analog compounds as measured by resazurin assay (open square). Dose responses of parental compound AR-12 are provided for reference. The data is presented as mean ± standard deviation of biological triplicates.

[0081] For the dose response plots shown in FIGS. 3A and 3B, BMDMs were isolated as described in the “Image-based Evaluation of Intracellular Anti-Leishmanial Activity” section above from B6.129P2-Lyz2tm1(cre)Ifo / J mice (stock #004781, “Lys k / o”) obtained from Jackson Laboratory and from Wildtype C57BL6 / J mice (stock #000664) . The Lys k / o mice allele strain has a nuclear-localized Cre recombinase inserted into the first coding ATG of the lysozyme 2 gene (Lyz2) which eliminates endogenous Lyz2 gene function.

[0082] BMDMs were seeded onto glass cover slips, infected with LV82 L. donovani at a multiplicity of infection of 1:10. Cells were washed to remove extracellular promastigotes and treated with RTI-197 or with AmpB for a 72-hour incubation. BMDM cells were then washed with PBS, fixed with ice-cold methanol, and stained with Giemsa (5% v / v in water).The cover slips with stained cells were mounted onto glass slides and imaged on EVOS XL (100X, Thermo Fisher Scientific). Leishmania amastigotes per 100 macrophages was determined in a blinded manner. The concentration required to reduce intracellular amastigote viability by 50% (IC50) value was measured from best fit curves plotting the normalized values against drug concentration.

[0083] RTI-53 had no direct effects on extracellular promastigote viability, indicating that all anti-leishmanial effects are host-directed. RTI-197 has some direct reduction in L. donovani extracellular promastigote viability, but host-directed effects are 5 times more significant. Additionally, RTI-197 had no effect on L. mexicana extracellular promastigote viability. Table 3 5 1 1 3d 5 c ( hy y, . .

[0084] Structures of AR-12 analogs evaluated for activity against Leishmania are depicted in Table C.Table CF

[0085] The foregoing description and examples have been set forth merely to illustratethe invention and are not meant to be limiting. Since modifications of the describedembodiments incorporating the spirit and the substance of the invention may occurto persons skilled in the art, the invention should be construed broadly to include allvariations within the scope of the claims and equivalents thereof.

Claims

CLAIMS 1. A compound of Formula (I):wherein: A is N or CR2; B is N or CR3; D is N or CR4; E is N or CR5; R1is -OR6, -NR7R8, a substituted or unsubstituted aryl, or a substituted or unsubstituted heterocyclic moiety; R2is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R3is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R4is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R5is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R6 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3- C10 cycloalkyl; R7 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3- C10cycloalkyl; and R8 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3- C10cycloalkyl; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein: A is N or CR2; B is N or CR3; D is N or CR4;E is N or CR5; R1 is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety; R2is -H, -F, a substituted or unsubstituted C1-C6alkyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R3 is -H, -F, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R4 is -H, -F, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R5 is -H, -F, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R6 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3- C10cycloalkyl; R7 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3- C10cycloalkyl; and R8 is -H, a substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted C3- C10cycloalkyl; or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein: A is N or CR2; B is N or CR3; D is N or CR4; E is N or CR5; R1 is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, thianyl, morpholinyl, and thiomorpholinyl; R2is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; R3is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; R4is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl;R5is -H, -F, -CF3, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; R6is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, - CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, - CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; and R8 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, - CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, wherein: A is N or CR2; B is N or CR3; D is N or CR4; E is N or CR5; R1is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, thianyl, morpholinyl, and thiomorpholinyl;R2is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R3 is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R4is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R5 is -H, -F, -CF3, methyl, ethyl, cyclopentyl, cyclohexyl, or phenyl; R6 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, - CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, - CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; and R8 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, -CF3, - CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopropyl, (dimethylamino)cyclobutyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1, wherein: A is N or CR2; B is N or CR3; D is N or CR4; E is N or CR5;R1is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of pyrrolidinyl, pyrrolinyl, pyrrolinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, and morpholinyl; R2 is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl; R3 is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl; R4is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl; R5 is -H, -F, -CF3, methyl, ethyl, cyclohexyl, or phenyl; R6is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7 is -H, methyl, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; and R8is -H, methyl, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH(NH2)CH3, -CH(NH2)CH2CH3, -CH2CH(NH2)CH3, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, -CH(NMe2)CH3, -CH(NMe2)CH2CH3, or -CH2CH(NMe2)CH3, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein: A is N; B is CR3; D is CR4; E is CR5; R1 is -OR6, -NR7R8, or a substituted or unsubstituted heterocyclic moiety selected from the group consisting of pyrrolidinyl, pyrrolinyl, pyrrolinyl, tetrahydrofuranyl, piperidinyl, tetrahydropyranyl, and morpholinyl; R3is -H or -CF3; R4 is -H or -CF3;R5is methyl, ethyl, cyclohexyl, or phenyl; R6 is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; R7 is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; and R8is -H, methyl, -CF3, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2NMe2, -CH2CH2NMe2, -CH2CH2CH2NMe2, cyclopentyl, cyclohexyl, aminocyclopentyl, aminocyclohexyl, (dimethylamino)cyclopentyl, or (dimethylamino)cyclohexyl; or a pharmaceutically acceptable salt thereof.

7. A compound of claim 1 selected from the group consisting of:a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound of claim 1 and at least one pharmaceutically acceptable excipient.

9. The pharmaceutical composition of claim 8, further comprising at least one other pharmaceutically active agent selected from the group consisting of antibacterial compounds, antiviral compounds, antifungal compounds, antiparasitic compounds, and combinations thereof.

10. The pharmaceutical composition of claim 9, wherein the at least one other pharmaceutically active agent is selected from the group consisting of sodium stibogluconate; meglumine antimoniate; paromomycin; ketoconazole; fluconazole; itraconazole; miltefosine; amphotericin b; pentamidine; and mixtures thereof.

11. A method of treating an infection caused by a microbial species, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1.

12. The method of claim 11, wherein the compound is selected from the group consisting of:a pharmaceutically acceptable salt thereof.

13. The method of claim 11, further comprising administering to a subject in need thereof a therapeutically effective amount of the at least one other pharmaceutically active agent selected from the group consisting of antibacterial compounds, antiviral compounds, antifungal compounds, antiparasitic compounds, and combinations thereof.

14. The method of claim 13, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered simultaneously.

15. The method of claim 14, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered in the same dosage form.

16. The method of claim 14, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered in separate dosage forms.

17. The method of claim 13, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered sequentially.

18. The method of claim 13, wherein the at least one other pharmaceutically active agent is selected from the group consisting of sodium stibogluconate; meglumine antimoniate; paromomycin; ketoconazole; fluconazole; itraconazole; miltefosine; amphotericin b; pentamidine; and mixtures thereof.

19. The method of claim 13, wherein the infection is caused by parasites in the Leishmania genus.

20. The method of claim 19, wherein the infection is caused by parasites selected from the group consisting of L. major, L. tropica, L. aethiopica, L. mexicana, L. braziliensis, L. donovani, L. chagasi, and mixtures thereof.

21. The method as in claim 13, wherein the subject is a mammal selected from the group consisting of humans, primates, horses, sheep, pigs, cows, mice, rats, rabbits, dogs, and cats.

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