Antileishmanial compounds, compositions and use thereof

Provided herein are antileishmanial compounds, compositions comprising the antileishmanial compounds, and use thereof.

TECHNICAL FIELD

The disclosure relates generally to antileishmanial compounds, compositions comprising the anti-leishmanial compounds and uses thereof.

BACKGROUND

Leishmaniasis is a neglected disease caused by protozoan parasites from the genusLeishmaniasp. It is transmitted by the sandfly vector and manifests in different clinical forms including skin ulcers, mucosa destruction, damage to visceral organs such as the liver and spleen, and bone marrow damage. The clinical outcome is determined primarily by the species of the parasite and the immune system of the host. There are 98 countries affected by leishmaniasis with more than 2 million people currently infected and 350 million people at risk.Leishmaniaparasites predominately infect monocytes in the reticulo-endothelial system.

Chemotherapy options for leishmaniasis are limited. Antimonials have been the first line drug for decades in most endemic countries, despite antimony's notorious adverse effects, hospitalization requirements and increasing cases of antimony-resistant parasites. Amphotericin B, the main alternative treatment, also causes significant harmful side effects. Liposomal formulations are better tolerated, but are prohibitively expensive for most affected populations. Miltefosine, an anti-cancer drug, was recently repurposed to treat leishmaniasis and is the only oral treatment available and approved for use in the US. Miltefosine also has toxicity limitations, teratogenicity and lack of efficacy against certainLeishmaniaspecies.

There remains a need for effective antileishmanial compounds, compositions, and methods for treating leishmaniasis. The present disclosure addresses these needs.

SUMMARY

In one aspect provided herein is a composition comprising an antileishmanial compound and a polymer. In some embodiments of any one of the aspects described herein, an anti-leishmanial compound is a compound of Formula (I) or (II):

or a pharmaceutically acceptable salt thereof.

In compounds of Formula (I), (I-A), (II) or (II-A), the carbon to which the R12group is attached can have the R or S stereochemistry. For example, the carbon to which the R12group is attached has the R stereochemistry. Preferably, the carbon to which the R12group is attached has the S stereochemistry.

In some embodiments of any one of the aspects described herein, an anti-leishmanial compound is a compound of Formula (I) or (II), where Y is N—R4, e.g., the anti-leishmanial compound is of Formula (I-A) or (II-A):

The composition can be formulated for topical, intravenous (iv) or oral. Accordingly, in some embodiments, the composition is formulated for iv administration. In some other embodiments, the composition is formulated for topical administration.

In some cases, the polymer and the antileishmanial compound are comprised in a particle comprising the polymer and the antileishmanial compound. Generally, the particle is from about 5 nm to about 1500 nm in size, e.g., from about 10 nm to about 1,000 nm in size, optionally from about 50 nm to about 200 nm in size.

In some embodiments, the particle is an expansile particle. For example, the particle comprises a first volume at a neutral pH and a second volume at an acidic pH, wherein the second volume is at least 1× greater than the first volume. Without wishing to be bound by a theory, the change in volume allows the particle to accumulate in the liver of a subject after administration and release the antileishmanial compound in the liver. In some embodiments, the antileishmanial compound is released at higher rate at an acidic pH relative to a release at neutral pH.

In some embodiments of any one of the aspects described herein, the particle is biodegradable.

The particle comprising the antileishmanial compound can be included in formulation comprising the particles and a pharmaceutically acceptable carrier or excipient.

In some embodiments of any one of the aspects described herein, the composition is formulated for topical administration. When the composition is formulated for topical administration, the composition can be in the form of is in the form of a film, a sheet, a dressing, a cream, a spray, a liquid, a gel, a hydrogel, an emulsion, or a suspension. For example, the composition can be in the form of an adhesive.

Embodiments of the various aspects described herein include a polymer. In some embodiments, the polymer can be selected from the group consisting of polycarbonates, polyesters, polyacrylates, polyamindes, and copolymers and mixtures thereof. In some cases, the polymer comprises one or more monomers of Formula (A), Formula (B), and/or Formula (C):

In monomers of Formula (C), each RCis independently:

where each Q is O;each R2is independently selected from the group consisting of hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, arylalkyl, or fluorocarbon chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents;one R3is selected from the group consisting of methoxy, ethoxy, amino, nitro, cycloalkyl, aryl, and olefin;and the remaining R3are each independently selected from the group consisting of hydrogen, methoxy, ethoxy, amino, a straight and branched alkyl, cycloalkyl, aryl, and olefin;R4, R5, and R6are each independently selected from the group consisting of a straight or branched alkyl, cycloalkyl, aryl, and olefin; andR7and R8are each independently selected from the group consisting of hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, alkylaryl, and arylalkyl.

In some embodiments of any one of the aspects described herein, the polymer comprises one or more monomers of Formula (A) and/or Formula (B). For example, the polymer comprises at least one monomer of Formula (A). In another example, the polymer comprises at least one monomer of Formula (B).

In some embodiments of any one of the aspects described herein, the polymer is a poly(glycerol carbonate) or a copolymer thereof. For example, the polymer is poly(1,3-glycerol carbonate), poly(1,2-glycerol carbonate), or a copolymer thereof. In some embodiments, the polymer is copolymer comprising poly(glycerol carbonate) and one of polycaprolactone, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(trimethylene carbonate), polyester, polycarbonate, or polyamide. For example, the polymer is copolymer comprising poly(glycerol carbonate) and polycaprolactone. In some embodiments, the copolymer is poly(1,3-glycerol carbonate)-C18-co-poly(ε-caprolactone).

In some embodiments of any one of the aspects described herein, the composition is formulated for topical administration and the polymer comprises one or more monomers of Formula (A) and/or Formula (B).

In some embodiments of any one of the aspects described herein, the composition is formulated for iv or oral administration and the polymer comprises one or more monomers of Formula (C).

In some embodiments of any one of the aspects described herein, the composition is formulated for iv or oral administration and the polymer comprises one or more monomers of Formula (A) and/or Formula (B).

In some embodiments of any one of the aspects described herein, the composition is formulated for iv or oral administration and the polymer comprises one or more monomers of Formula (C).

In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or excipient.

In some embodiments of any one of the aspects described herein, the composition further comprises an active agent. Exemplary active agents include, but are not limited to, a second antileishmanial compound. Some exemplary antileishmanial compound include, but are not limited to, antimonials, miltefosine, dronedarone, stibogluconate, meglumine antimonite, pentamidine, amphotericin B, paromomycin, reversed amidines, and pharmaceutically acceptable salts thereof), and/or the active agent is selected from the group consisting of wound-healing agents, anti-scarring agents, antioxidant agents, cooling agents, soothing agents, anti-inflammatory-agents, antibiotics, topical analgesics, counter irritants, penetration enhancers, and permeation enhancers.

In some embodiments of any one of the aspects described herein, the composition can further comprise one or more of binders, viscosity modifiers, preservatives, humectants, emollients, pH stabilizing agents, chelating agents, gelling agents, thickening agents, emulsifiers, buffers, and carriers.

In some embodiments of any one of the aspects described herein, the compound of Formula (I) or (II) has a C Log P from about 3.2 to about 4.3.

Also provided herein is a method for treating leishmaniasis or a disease or disorder associated with leishmaniasis. Generally, the method comprises: administering a therapeutically effective amount of an antileishmanial compound of Formula (I) or (II), or a composition described herein to a subject in need thereof. The leishmaniasis can be cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) or mucosal leishmaniasis (ML).

In another aspect, provided herein is a method for reducing, suppressing or inhibiting aLeishmaniaparasite in a vector. The method comprises: administering to the vector an effective amount of Formula (I) or Formula (II).

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

Compounds

Embodiments of the various aspects described herein include a compound of Formula (I) or (II):

Inventors have discovered inter alia that compounds of Formula (I) or (II) have antileishmanial activity, i.e., compounds are antileshmanial compounds. As used herein, an “antileishmanial” compound is a compound that restrict growth and/or activity of any species that is a part of theLeishmaniagenus.Leishmaniais a parasitic protozoan, a single-celled organism of the genusLeishmaniathat are responsible for the disease leishmaniasis. They are spread by sandflies of the genusPhlebotomusin Africa, Europe, and Asia, and of the genusLutzomyiain North and South America.Leishmaniacurrently affects 6 million people in 98 countries. About 0.9-1.6 million new cases occur each year, and 21 species are known to cause disease in humans.Leishmaniaamastigote growth in infected cells can be evaluated using microscopy (e.g., fluorescence, confocal), fluorescence emission spectroscopy, and luminescence spectroscopy.Leishmaniapromastigote growth can be evaluated using standard metabolism-based cell growth assays (e.g., CellTiter-Glo, MTT [3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]).

In some embodiments of any one of the aspects described herein, Y is N—R14. When Y is N—R14, the compound is of Formula (I-A) or (II-A):

In some embodiments of any one of the aspects described herein, R15is a nucleophile. As used herein, a nucleophile is a is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Some exemplary nucleophiles include but, are not limited to, hydroxyl, amino, thiol, carboxyl, cyanide, azide, and nitrite groups.

Narrower Genus

All structures of Formula (I), (I-A), (II) and (II-A) are provided herein for illustrative purpose and disclose a particular isomer. However, one of ordinary skill in the art will recognize all possible isomers of the structures of any of Formula (I), (I-A), (II) and (II-A). Therefore, other isomers such as enantiomers of any of Formula (I), (I-A), (II) and (II-A) are considered to fall within the scope of the invention. As used herein, the term “isomer” refers to a compound having the same molecular formula but differing in structure. Isomers which differ only in configuration and/or conformation are referred to as “stereoisomers.” The term “isomer” is also used to refer to an enantiomer.

The term “enantiomer” is used to describe one of a pair of molecular isomers which are mirror images of each other and non-superimposable. The designations “R” and “S” are used to denote the absolute configuration of the molecule about its chiral center. The designations may appear as a prefix or as a suffix; they may or may not be separated from the isomer by a hyphen; they may or may not be hyphenated; and they may or may not be surrounded by parentheses. The designations “(+)” and “(−)” are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) meaning that the compound is levorotatory (rotates to the left). A compound prefixed with (+) is dextrorotatory (rotates to the right). Other terms used to designate or refer to enantiomers include “stereoisomers” (because of the different arrangement or stereochemistry around the chiral center; although all enantiomers are stereoisomers, not all stereoisomers are enantiomers) or “optical isomers” (because of the optical activity of pure enantiomers, which is the ability of different pure enantiomers to rotate planepolarized light in different directions). Enantiomers generally have identical physical properties, such as melting points and boiling points, and also have identical spectroscopic properties. Enantiomers can differ from each other with respect to their interaction with plane-polarized light and with respect to biological activity.

It is noted that in compounds of Formula (I), (I-A), (II) or (II-A), the carbon to which the R12group is attached can have the R or S stereochemistry. For example, the carbon to which the R12group is attached has the R stereochemistry. Preferably, the carbon to which the R12group is attached has the S stereochemistry.

Derivatives and Prodrugs

In various embodiments, compounds of Formula (I), (I-A), (II) or (II-A) include enantiomers, derivatives, prodrugs, and pharmaceutically acceptable salts thereof. The term “derivative” as used herein refers to a chemical substance related structurally to another, i.e., an “original” substance, which can be referred to as a “parent” compound. A “derivative” can be made from the structurally-related parent compound in one or more steps. The general physical and chemical properties of a derivative are also similar to the parent compound.

Exemplary Compounds

Without wishing to be bound by a theory, inventors have discovered inter alia that compounds of Formula (I) or (II) having a C Log P from about 3.0 to about 4.5, unexpectedly and surprisingly have antileishmanial activity. Accordingly, in some embodiments of any one of the aspects described herein, the compound of Formula (I) or (II) has a C Log P from about 3.0 to about 4.5, e.g., from about 3.2 to about 4.3.

As used herein, C Log P is also known as “fragment/compound log P”. A C Log P is a method that uses a dataset from full compounds, or fragments, which are experimentally determined, and then modelled using quantitative structure-activity relationship or other regression techniques in small fragments rather than per atom. Fragment contributions are then added up, with correction factors. This method tends to be better for systems with complex aromaticity, and larger molecules—on the condition that the molecule contains features that are similar to those from which the modelling was conducted. In the case of very obscure motifs in the molecules, then the model from which the prediction is made may not have a very good correlation.

Polymers

Embodiments of the various aspects described herein include a polymer. It is noted the polymer can be homopolymer, copolymer or blockpolymer. Some exemplary polymers include, but are not limited to, polycarbonates, polyesters, polyacrylates, polyamindes, and copolymers, blockpolymers and mixtures thereof.

In some embodiments, the polymer comprises one or more monomers of Formula (A), Formula (B), Formula (C):

Exemplary monomers of Formula (A) and polymers comprising same are described in US patent publication US20200056038, content of which is incorporated herein by reference in its entirety.

Exemplary monomers of Formula (B) and polymers comprising same are described in US patent publication US20120107365 content of which is incorporated herein by reference in its entirety.

In some embodiments of any one of the aspects described herein, each R2is independently straight or branched alkyl, hydrogen or cycloalkyl. For example, R2is H or C1-C6alkyl, hydrogen. In some embodiments, R2is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or pentyl. For example, R2is methyl.

In some embodiments of any one of the aspects described herein, R4, R5, and R6are each independently selected from the group consisting of a straight or branched alkyl, cycloalkyl, aryl, and olefin.

In some embodiments of any one of the aspects described herein, each R7is independently hydrogen, a straight or branched alkyl, cycloalkyl, aryl, alkylaryl, or arylalkyl. For example, each R7is independently hydrogen, a straight or branched alkyl, or cycloalkyl. For example, each R7is independently hydrogen or C1-C6alkyl. For example, each R7is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or pentyl. In some cases, each R7is independently hydrogen or methyl.

In some embodiments of any one of the aspects described herein, each R8is independently R8is absent, alkylene or alkenylene. For example, each R8is absent or C1-C6alkylene. In some embodiments, each R8is independently absent, methylene, ethylene, propylene, butylene or pentylene. For example, each R8is absent or methylene.

In some embodiments, of any one of the aspects described herein, RCis

For example, a monomer of Formula (C) is:

Exemplary monomers of Formula (B) and polymers comprising same are described in US patent publication US20120107365 content of which is incorporated herein by reference in its entirety.

In some embodiments of any of the aspects, the polymer comprises one or more monomers of Formula (A) and/or Formula (B). For example, the polymer comprises one or more monomers of Formula (A). In another example, the polymer comprises one or more monomers of Formula (B).

In some embodiments of any one of the aspects described herein, the polymer comprises one or more monomers of Formula (C).

In some embodiments of any one of the aspects described herein, the polymer is poly(glycerol carbonate) or a copolymer thereof. For example, the polymer is poly(1,3-glycerol carbonate), poly(1,2-glycerol carbonate), or a copolymer thereof. In some embodiments, the polymer is copolymer comprising poly(glycerol carbonate) and one of polycaprolactone, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(trimethylene carbonate), polyester, polycarbonate, or polyamide. For example, the polymer is copolymer comprising poly(glycerol carbonate) and polycaprolactone. In some embodiments, the copolymer is poly(1,3-glycerol carbonate)-C18-co-poly(ε-caprolactone)

The polymer can be cross-linked. Accordingly, in some embodiments of any one of the aspects described herein, the composition further comprises a cross-linker, e.g., a cross-linker linking the polymer chains. Suitable crosslinkers include compounds with at least two (meth)acryloyl groups. As used herein, the term “(meth)acryloyl” refers to an acryloyl group, a methacryloyl group, or both. Likewise, the term “(meth)acrylate” refers to an acrylate, a methacrylate, or both; the term “(meth)acrylamide” refers to an acrylamide, a methacrylamide, or both; and the term “(meth)acrylic acid” refers to acrylic acid, methacrylic acid, or both. The crosslinkers can be di(meth)acrylates, tri(meth)acrylates, tetra(meth)acrylates, penta(meth)acrylates, and the like. These crosslinkers can be formed, for example, by reacting (meth)acrylic acid with a polyhydric alcohol (i.e., an alcohol having at least two hydroxyl groups). The polyhydric alcohol often has two, three, four, or five hydroxyl groups. Mixtures of crosslinkers can be used. In some embodiments, the crosslinkers contain at least two acryloyl groups. Exemplary crosslinkers with two acryloyl groups include 1,2-ethanediol diacrylate, 1,3-propanediol diacrylate, 1,9-nonanediol diacrylate, 1,12-dodecanediol diacrylate, 1,4-butanediol diacrylate, 1,6-hexanediol diacrylate, butylene glycol diacrylate, bisphenol A diacrylate, diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, tripropylene glycol diacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, polyethylene/polypropylene copolymer diacrylate, and neopentylglycol hydroxypivalate diacrylate modified caprolactone. Exemplary crosslinkers with three or four (meth)acryloyl groups include, but are not limited to, trimethylolpropane triacrylate (e.g., commercially available under the trade designation TMPTA-N from Surface Specialties, Smyrna, Ga. and under the trade designation SR-351 from Sartomer, Exton, Pa.), pentaerythritol triacrylate (e.g., commercially available under the trade designation SR-444 from Sartomer), tris(2-hydroxyethylisocyanurate)triacrylate (commercially available under the trade designation SR-368 from Sartomer), a mixture of pentaerythritol triacrylate and pentaerythritol tetraacrylate (e.g., commercially available from Surface Specialties under the trade designation PETIA with an approximately 1:1 ratio of tetraacrylate to triacrylate and under the trade designation PETA-K with an approximately 3:1 ratio of tetraacrylate to triacrylate), pentaerythritol tetraacrylate (e.g., commercially available under the trade designation SR-295 from Sartomer), di-trimethylolpropane tetraacrylate (e.g., commercially available under the trade designation SR-355 from Sartomer), and ethoxylated pentaerythritol tetraacrylate (e.g., commercially available under the trade designation SR-494 from Sartomer). An exemplary crosslinker with five (meth)acryloyl groups includes, but is not limited to, dipentaerythritol pentaacrylate (e.g., commercially available under the trade designation SR-399 from Sartomer).

In some embodiments, the crosslinkers are polymeric material that contains at least two (meth)acryloyl groups. For example, the crosslinkers can be poly(alkylene oxides) with at least two acryloyl groups (e.g., polyethylene glycol diacrylates commercially available from Sartomer such as SR210, SR252, and SR603) or poly(urethanes) with at least two (meth)acryloyl groups (e.g., polyurethane diacrylates such as CN9018 from Sartomer). As the higher molecular weight of the crosslinkers increases, the resulting acrylic copolymer tends to have a higher elongation before breaking Polymeric crosslinkers tend to be used in greater weight percent amounts compared to their non-polymeric counterparts.

In some embodiments, the cross-linker is 1,4-O-methacryloylhydroquinone or 1,4-phenylene-bis(2-methylacrylate).

Particles

In some embodiments of any one of the aspects described herein, the polymer and the antileishmanial compound can be comprised in a particle. The particle can be between about 5 nm and about 1500 nm in size. For example, the particle can be between about 10 nm, and about 1000 nm in size. In some embodiments, the particle can be between about 50 nm and about 200 nm in size. Polymer nanoparticles of similar size and polymer compositions are known to demonstrate significant uptake in the liver following injection.

In some embodiments, the particle is a an expansile particle. As used herein, an “expansile particle” is a particle and expansile in the presence of a particular stimulus. The expansile particle can expand in the presence of a particular pH. For example, the particle comprises a first volume at a first pH and a second volume at a second pH, optionally, the second volume is at least 1× greater than the first volume. In some embodiments, the particle comprises a first volume at a higher pH and a second volume at a lower pH. For example, the particle comprises a first volume at a neutral pH and a second volume at an acidic pH. In some embodiments, the particle comprises a first volume at a pH from about 6.5 to about 7.5 (e.g., a pH of about 7) and a second volume at a pH from about 4 to about 6 (e.g., pH of about 5), and where the second volume is at least 1× greater than the first volume. Expansile particles are known to show significant uptake in the liver (˜40%) and spleen (˜2%) following i.v. injection.

Without wishing to be bound by a theory, the change in volume allows the particle to accumulate in the liver of a subject after administration and release the antileishmanial compound in the liver. In some embodiments, the antileishmanial compound is released from the particle at higher rate at an acidic pH relative to a release at neutral pH.

The particles can be formed through water-in-oil ultrasonication methods and purified via dialysis. Particle size, monodispersity can be controlled by varying cross-linker, surfactant (e.g., polymer to surfactant ratio) and sonication parameters (e.g., sonication time and sonication pulse parameters) during preparation of the particles.

Pharmaceutical Compositions

The compositions described herein can be in form of a pharmaceutical composition. For example, the composition comprising the particle and the antileishmanial can further comprise with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents. It is noted that the compounds of Formula (I) or (II) described herein can be formulated into pharmaceutical compositions for therapeutic use without the polymer. Accordingly, in another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising an effective amount of a compound of Formula (I) or (II) (e.g., without a polymer) formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.

The compositions can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; or (3) topical application, for example, as a film, sheet, dressing, cream, ointment, liquid, gel, hydrogel, emulsion, suspension or a controlled-release patch or spray applied to the skin. Delivery using topical, oral or intravenous methods can be particularly advantageous. Accordingly, in some embodiments, the composition is formulated for oral or intravenous administration.

In some embodiments, the composition is formulated for topical administration. For topical administration, the composition can be in the form of a film, sheet, dressing, cream, ointment, liquid, gel, hydrogel, emulsion, suspension or a controlled-release patch or spray applied. In some embodiments, the composition formulated for topical administration is in the form of an adhesive.

In some embodiments, the composition is formulated for oral or intravenous administration.

Generally, the composition, e.g., pharmaceutical composition comprises an effective or therapeutically effective amount of the antileishmanial compound. The phrase “therapeutically effective amount” as used herein means that amount of a compound, material, or composition comprising a compound described herein which is effective for producing some desired therapeutic effect in a subject at a reasonable benefit/risk ratio applicable to any medical treatment, e.g., treatment ofLeishmaniaor a disease or disorder due toLeishmania.

In some embodiments, the composition is formulated as a unit dosage formulation.

Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, antileishmanial compounds described herein can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the antileishmanial compound described herein can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical composition can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., pharmaceutically acceptable oils, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use as described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The antileishmanial compound can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. For topical administration, the antileishmanial compound can be formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.

The compositions can, if desired, be presented in a pack or dispenser device which can contain one or more-unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

Second Active Agent

In some embodiments, the active agent is selected from the group consisting of wound-healing agents, anti-scarring agents, antioxidant agents, cooling agents, soothing agents, anti-inflammatory-agents, antibiotics, topical analgesics, counter irritants, penetration enhancers, and permeation enhancers.

Method of Treatment

In some embodiments, the leishmaniasis or a disease or disorder associated with leishmaniasis is caused by aLeishmaniaparasite selected from the group consisting ofLeishmania major, Leishmania tropica, Leishmania Mexicana, Leishmania braziliensis, Leishmania amazonensis, Leishmania aethiopica, Leishmania panamensis, Leishmania guyanensis, Leishmania infantum, Leishmania donovani. For example, leishmaniasis or a disease or disorder associated with leishmaniasis is caused by aLeishmaniaparasite selected from the group consisting ofLeishmania major, Leishmania braziliensis, andLeishmania donovani.

It is noted that the leishmaniasis can be cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) or mucosal leishmaniasis (ML). In some embodiments, the leishmaniasis is cutaneous leishmaniasis (CL). In other embodiments, the leishmaniasis is visceral leishmaniasis (VL).

In some embodiments, the leishmaniasis is cutaneous leishmaniasis. Cutaneous leishmaniasis is a more common form of leishmaniasis that affects 1.5 million to 2 million worldwide per year. CL is a skin infection that is characterized by symptoms which include skin sores. Skin sores typically develop within a few weeks or months of the sand fly bite. The sores can change in size and appearance over time. The sores may start out as papules (bumps) or nodules (lumps) and may end up as ulcers (like a volcano, with a raised edge and central crater); skin ulcers might be covered by scab or crust. The sores usually are painless but can be painful. Additional symptoms can include swollen lymph nodes near the sores (e.g., under the arm, if the sores are on the arm or hand).

In some embodiments, the leishmaniasis is visceral leishmaniasis. Viserceral leishmaniasis is characterized by symptoms which affects several internal organs (usually spleen, liver, and bone marrow) and can be life threatening. The illness typically develops within months (sometimes as long as years) of the sand fly bite. Affected people usually have fever, weight loss, enlargement (swelling) of the spleen and liver, and low blood counts—a low red blood cell count (anemia), a low white blood cell count (leukopenia), and a low platelet count (thrombocytopenia).

In some embodiments, the leishmaniasis is mucosal leishmaniasis. Mucosal leishmaniasis is a less common form of leishmaniasis. This form can be a consequence of infection with some of the species of the parasite that cause cutaneous leishmaniasis in parts of Latin America: certain types of the parasite might spread from the skin and cause sores in the mucous membranes of the nose, which is the most common location, mouth, or throat.

Diagnosis of leishmaniasis includes, but is not limited to, examination of tissue specimens through a skin biopsy (e.g., from skin sores (in suspected cases of cutaneous leishmaniasis) or from bone marrow (in suspected cases of visceral leishmaniasis)) for presence of the parasite, serological tests, and needle biopsy. As disclosed herein, antileishmanial compounds can reduce, suppress, or inhibit the expression or presence of aLeishmaniaparasite in a vector by at least 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, 95%, 99% or even 100%, as compared to the expression in the absence of antileishmanial compounds.

Current treatments of leishmaniasis include, but are not limited to, amphotericin B, miltefosin, pentavalent antimonials (e.g., sodium stibogluconate and meglumine antimoniate), ambisome, pentamidine isethionate, and paromomycin. Pentavalent antimonials can only be given by injection and there are no oral preparations currently available. Additional treatments include thermotherapy, cryotherapy, and laser therapy. Complications from leishmaniasis include, but are not limited to bacterial infections, scarring, relapse, septal perforation or collapse, pneumonia or gastrointestinal tract infections, Post kala-azar dermal leishmaniasis (PKDL), severe bleeding, hemophagocytic lymphohistiocytosis, and sepsis.

In some embodiments, compounds and compositions described herein can inhibit or decrease the development and/or presence of symptoms of CL, VL or ML by at least 10%, relative to the development and/or presence of symptoms in the absence of antileishmanial compounds, e.g., at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, 95%, 99% or even 100%.

Vectors

The compounds and compositions described herein can be useful in reducing, suppressing or inhibiting aLeishmaniaparasite in aLeishmaniaparasite vector. Thus, in another aspect provided herein is a method of reducing, suppressing or inhibiting aLeishmaniaparasite in a vector. The method comprises administering to the vector an effective amount of Formula (I) or Formula (II).

Administration of an effective amount of Formula (I) or Formula (II) to the vector can comprise including an effective amount of Formula (I) or Formula (II) as part of an insect repellent or insecticide. Insect repellents that can include Formula (I) or Formula (II) include, but are not limited to benzaldehyde, butopyronoxyl, N,N-diethyl-m-toluamide (DEET), dimethyl carbate, dimethyl phthalate, ethyl butylacetylaminopropionate, ethylhexanediol, icaridin, methyl anthranilate, N,N-dimethylanthranilic acid (DMA), ethyl anthranilate (EA), butyl anthranilate (BA), metofluthrin, permethrin, SS220, tricyclodecenyl allyl ether, beautyberry (Callicarpa) leaves, nepetalactone, citronella oil, essential oil ofCorymbia citriodora(lemon eucalyptus) and its active compound p-menthane-3,8-diol (PMD), lemongrass, neem oil, tea tree oil from the leaves ofMelaleuca alternifolia, and tobacco. Formula (I) or Formula (II) can also be a part of bed netting and/or clothing that has been treated with a pyrethroid-containing insecticide.

As used herein, a “vector” is a living organism that can transmit infectious pathogens between humans, or from animals to humans. Many of these vectors are bloodsucking insects, which ingest disease-producing microorganisms during a blood meal from an infected host (human or animal) and later transmit it into a new host, after the pathogen has replicated. Often, once a vector becomes infectious, they are capable of transmitting the pathogen for the rest of their life during each subsequent bite/blood meal. In some embodiments, the vector that transmits aLeishmaniaparasite is Phlembotominae. The Phlebotominae are a subfamily of the family Psychodidae. Phlebotominae are commonly known as sandflies, but the name is applied to other flies. The genera that is found in Phlebotominae subfamily include Australophlebotomus, Bichromomyia, Brumptomyia, Chinius, Dampfomyia, Datzia, Deanemyia, Evandromyia, Edentomyia, Expapillata, Hertigia, Idiophlebotomus, Libanophlebotomus,Lutzomyia, Mandalayia, Martinsmyia, Mesophlebotomites, Micropygomyia, Migonemyia, Nyssomyia, Oligodontomyia, Palaeomyia, Phlebotomites, Phlebotoiella,Phlebotomus, Pintomyia, Pressatia, Protopsychodinae, Protopsychoda, Psathyromyia, Psychodopygus, Sciopemyia, Sergentomyia, Trichophoromyia, Viannamyia, and Warileya. In North, Central, and South America, leishmaniasis is spread by the genusLutzomyia. In Africa, Europe, and Asia, leishmaniasis is spread by the genusPhlebotomus.

Some Selected Definitions

For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials can be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.

As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. In other words, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.

The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

As used herein, the term “subject” or “patient” refers to any organism to which a compound or composition disclosed herein can be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female.

Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders. In addition, compounds, compositions and methods described herein can be used to with domesticated animals and/or pets.

A subject can be one who has been previously diagnosed with or identified as suffering from or having leishmaniasis or a disease or disorder associated with leishmaniasis. Alternatively, a subject can also be one who has not been previously diagnosed. A “subject in need” of treatment for leishmaniasis or a disease or disorder associated with leishmaniasis can be a subject having leishmaniasis or a disease or disorder associated with leishmaniasis, diagnosed as having that condition, or at risk of developing that condition.

In some embodiments, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is further intended to include transgenic species. In some embodiments, the subject can be of European ancestry. In some embodiments, the subject can be of African American ancestry. In some embodiments, the subject can be of Asian ancestry.

In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.

As used herein, the term “parenteral administration,” refers to administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.

As used herein, the term “subcutaneous administration” refers to administration just below the skin. “Intravenous administration” means administration into a vein.

As used herein, the term “dose” refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.

As used herein, the term “dosage unit” refers to a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial comprising lyophilized composition or compound described herein. In certain embodiments, a dosage unit is a vial comprising reconstituted composition or compound descried herein.

By the terms “treat,” “treating” or “treatment of” (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of leishmaniasis or a disease or disorder associated with leishmaniasis.

The terms “prevent,” “preventing” and “prevention” (and grammatical variations thereof) refer to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present invention.

As used herein, the term “aliphatic” means a saturated or unsaturated and straight, branched, and/or cyclic hydrocarbon having the defined number of carbon atom. Examples include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, and cycloalkylalkynyl, having the defined number of carbon atoms.

As used herein, the term “alkyl” refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms. The alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes halo, amino, aryl, hydroxyl, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl. Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.

A “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH2group to an NH group or an O group). The term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof. In certain embodiments, the heteroatom(s) is placed at any interior position of the heteroalkyl group. Examples include, but are not limited to, —CH2—O—CH3, —CH2—CH2—O—CH3, —CH2—NH—CH3, —CH2—CH2—NH—CH3, —CH2—N(CH3)—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2, —S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, and —CH═CH—N(CH3)—CH3. In some embodiments, up to two heteroatoms are consecutive, such as, by way of example, —CH2—NH—OCH3and —CH2—O—Si(CH3)3

As used herein, the term “alkenyl” refers to an alkyl group containing at least one carbon-carbon double bond. The alkenyl group can be optionally substituted with one or more “alkyl group substituents.” Exemplary alkenyl groups include vinyl, ally, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-1-yl and heptadec-8,11-dien-1-yl.

As used herein, the term “alkynyl” refers to an alkyl group containing a carbon-carbon triple bond. The alkynyl group can be optionally substituted with one or more “alkyl group substituents.” Exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl. Useful alkynyl groups include the lower alkynyl groups.

As used herein, the term “cycloalkyl” refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and/or alkylene. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl. Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

“Heterocyclyl” refers to a nonaromatic 3-8 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). Cxheterocyclyl and Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent. Exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like.

“Aryl” refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms. The aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene and —NRR′, where R and R′ are each independently hydrogen, alkyl, aryl and aralkyl. Exemplary aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl.

As used herein, the term “halogen” or “halo” refers to an atom selected from fluorine, chlorine, bromine and iodine. The term “halogen radioisotope” or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.

A “halogen-substituted moiety” or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.

The term “haloalkyl” as used herein refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different. The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (C1-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, and the like).

As used herein, the term “amino” means —NH2. The term “alkylamino” means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., —NH(alkyl). The term “dialkylamino” means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., —N(alkyl)(alkyl). The term “alkylamino” includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.” The term “arylamino” means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, —NHaryl, and —N(aryl)2. The term “heteroarylamino” means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example —NHheteroaryl, and —N(heteroaryl)2. Optionally, two substituents together with the nitrogen can also form a ring. Unless indicated otherwise, the compounds described herein containing amino moieties can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like. Exemplary alkylamino includes, but is not limited to, NH(C1-C10alkyl), such as —NHCH3, —NHCH2CH3, NHCH2CH2CH3, and —NHCH(CH3)2. Exemplary dialkylamino includes, but is not limited to, —N(C1-C10alkyl)2, such as N(CH3)2, —N(CH2CH3)2, —N(CH2CH2CH3)2, and —N(CH(CH3)2)2.

The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms (—N—) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl. For example, an (C2-C6) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.

The terms “hydroxyl” and “hydroxyl” mean the radical —OH.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto, and can be represented by one of —O-alkyl, —O— alkenyl, and —O-alkynyl. Aroxy can be represented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. The alkoxy and aroxy groups can be substituted as described above for alkyl. Exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n-propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O-hexyl, O-cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.

As used herein, the term “carbonyl” means the radical —C(O)—. It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.

As used herein, the term “oxo” means double bonded oxygen, i.e., ═O.

The term “carboxy” means the radical —C(O)O—. It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes —COOH, i.e., carboxyl group.

The term “ester” refers to a chemical moiety with formula —C(═O)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl.

The term “cyano” means the radical —CN.

The term “nitro” means the radical —NO2.

The term, “heteroatom” refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes a moiety where the atom by which the moiety is attached is not a carbon. Examples of heteroatom moieties include —N═, —NRN—, —N+(O−)═, —O—, —S— or —S(O)2—, —OS(O)2—, and —SS—, wherein RNis H or a further substituent.

The terms “alkylthio” and “thioalkoxy” refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur. In preferred embodiments, the “alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, and —S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups.

The term “sulfinyl” means the radical —SO—. It is noted that the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.

The term “sulfonyl” means the radical —SO2—. It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (—SO3H), sulfonamides, sulfonate esters, sulfones, and the like.

The term “thiocarbonyl” means the radical —C(S)—. It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.

“Acyl” refers to an alkyl-CO— group, wherein alkyl is as previously described. Exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.

“Aroyl” means an aryl-CO— group, wherein aryl is as previously described. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.

“Arylthio” refers to an aryl-S— group, wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.

“Aralkyl” refers to an aryl-alkyl- group, wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.

“Aralkyloxy” refers to an aralkyl-O— group, wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.

“Aralkylthio” refers to an aralkyl-S— group, wherein the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio.

“Aryloxycarbonyl” refers to an aryl-O—CO— group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—CO— group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an H2N—CO— group.

“Alkylcarbamoyl” refers to a R′RN—CO— group, wherein one of R and R′ is hydrogen and the other of R and R′ is alkyl as previously described.

“Dialkylcarbamoyl” refers to R′RN—CO— group, wherein each of R and R′ is independently alkyl as previously described.

“Acyloxy” refers to an acyl-O— group, wherein acyl is as previously described. “Acylamino” refers to an acyl-NH— group, wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH— group, wherein aroyl is as previously described.

The term “optionally substituted” means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified. The term “substituents” refers to a group “substituted” on a substituted group at any atom of the substituted group. Suitable substituents include, without limitation, halogen, hydroxyl, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxylalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido. In some cases, two substituents, together with the carbons to which they are attached to can form a ring.

In some embodiments, an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different.

An “isocyanato” group refers to a NCO group.

A “thiocyanato” group refers to a CNS group.

An “isothiocyanato” group refers to a NCS group.

“Alkoyloxy” refers to a RC(═O)O— group.

“Alkoyl” refers to a RC(═O)— group.

It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., provided herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. The invention is further illustrated by the following example, which should not be construed as further limiting.

The technology may be as described in any one of the following numbered Embodiments:

Embodiment 1: A composition comprising: (a) a polymer; and (b) an antileishmanial compound, optionally, the antileishmanial compound is of Formula (I) or (II):

Embodiment 2: The composition of Embodiment 1, wherein Y is N—R14, e.g., the compound is of Formula (I-A) or (II-A):

Embodiment 7: The composition of Embodiment 6, wherein a is 0, 1, 2, 3 or 4.

Embodiment 10: The composition of Embodiment 9, wherein R15is isopropyl.

Embodiment 11: The composition of Embodiments 1 or 2, wherein R15is a nucleophile.

Embodiment 35: The composition of any one of Embodiments 1-34, wherein the antileishmanial compound has a C Log P from about 3.2 to about 4.3.

Embodiment 36: The composition of any one of the preceding Embodiments, wherein the composition is formulated for topical, intravenous (iv) or oral.

Embodiment 37: The composition of Embodiment 36, wherein the composition is formulated for iv administration.

Embodiment 38: The composition of any one of Embodiments 1-37, wherein the polymer and the antileishmanial compound are comprised in a particle comprising the polymer and the antileishmanial compound.

Embodiment 39: The composition of Embodiment 38, wherein the particle is an expansile particle.

Embodiment 40: The composition of Embodiment 38 or 39, wherein the particle accumulates in the liver of a subject after administering to said subject and releases the antileishmanial compound in the liver.

Embodiment 41: The composition of any one of Embodiments 38-40, wherein the particle is from about 5 nm to about 1500 nm in size.

Embodiment 42: The composition of Embodiment 36, wherein the composition is formulated for topical administration.

Embodiment 43: The composition of Embodiment 42, wherein the composition is in the form of an adhesive.

Embodiment 44: The composition of Embodiment 42 or 43, wherein the composition is in the form of a film, a sheet, a dressing, a cream, a spray, a liquid, a gel, a hydrogel, an emulsion, or a suspension.

Embodiment 45: The composition of any one of Embodiments 1-44, wherein the polymer is selected from the group consisting of polycarbonates, polyesters, polyacrylates, polyamindes, and copolymers and mixtures thereof.

Embodiment 46: The composition of any one of Embodiments 1-45, wherein the polymer comprises one or more monomers of Formula (A), Formula (B), and/or Formula (C):

where each Q is O;each R2is independently selected from the group consisting of hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, arylalkyl, or fluorocarbon chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents;one R3is selected from the group consisting of methoxy, ethoxy, amino, nitro, cycloalkyl, aryl, and olefin;and the remaining R3are each independently selected from the group consisting of hydrogen, methoxy, ethoxy, amino, a straight and branched alkyl, cycloalkyl, aryl, and olefin;R4, R5, and R6are each independently selected from the group consisting of a straight or branched alkyl, cycloalkyl, aryl, and olefin; andR7and R8are each independently selected from the group consisting of hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, alkylaryl, and arylalkyl.

Embodiment 47: The composition of Embodiment 46, wherein the polymer comprises one or more monomers of Formula (A) and/or Formula (B).

Embodiment 48: The composition of Embodiment 47, wherein the polymer comprises one or more monomers of Formula (A).

Embodiment 49: The composition of Embodiment 47 or 48, wherein the polymer comprises one or more monomers of Formula (B).

Embodiment 50: The composition of any one of Embodiments 4649, wherein RAis —OC(O)—RA2, —ORA1, —C(O)RA1, or —C(O)O—RA1.

Embodiment 54: The composition of any one of Embodiments 46-53, wherein the polymer is a poly(glycerol carbonate) or a copolymer thereof.

Embodiment 55: The composition of any one of Embodiments 46-54, wherein the polymer is poly(1,3-glycerol carbonate), poly(1,2-glycerol carbonate), or a copolymer thereof.

Embodiment 57: The composition of Embodiment 56, wherein the copolymer comprises a poly(glycerol carbonate) and polycaprolactone.

Embodiment 58: The composition of Embodiment 57, wherein the copolymer is poly(1,3-glycerol carbonate)-C18-co-poly(ε-caprolactone).

Embodiment 59: The composition of any one of Embodiments 42-58, wherein the composition is an extended or slow release composition.

Embodiment 60: The composition of any one of Embodiments 42-59, wherein the composition further comprises glycerol.

Embodiment 61: The composition of Embodiments 38-40, wherein the particle comprises a first volume at a neutral pH and a second volume at an acidic pH, wherein the second volume is at least 1× greater than the first volume.

Embodiment 62: The composition of Embodiment 61, wherein the antileishmanial compound is released at higher rate at an acidic pH relative to a release at neutral pH.

Embodiment 63: The composition of any one of Embodiments 38-40 or 61-62, wherein the particle is from about 10 nm to about 1,000 nm in size.

Embodiment 64: The composition of Embodiment 63, wherein the particle is from about 50 nm to about 200 nm in size.

Embodiment 65: The composition of any one of Embodiments 38-40 or 61-64, wherein the polymer comprises one or more monomers of Formula (C).

Embodiment 66: The composition of Embodiment 64, wherein R2straight or branched alkyl, hydrogen or cycloalkyl.

Embodiment 68: The composition of Embodiment 66, wherein R2is methyl.

Embodiment 69: The composition of any one of Embodiments 65-68, wherein each R3is independently hydrogen or methoxy.

Embodiment 70: The composition of any one of Embodiments 65-69, wherein RCis

Embodiment 71: The composition of any one of Embodiments 65-70, wherein the monomer of Formula (C) is:

Embodiment 72: The composition of any one of the preceding Embodiments, wherein the composition further comprises a cross-linker.

Embodiment 73: The composition of Embodiment 72, wherein the cross-linker is 1,4-O-methacryloylhydroquinone or 1,4-phenylene-bis(2-methylacrylate).

Embodiment 74: The composition of any one of Embodiments 38-40, wherein the polymer is poly(glycerol carbonate) or a copolymer thereof.

Embodiment 75: The composition of Embodiment 74, wherein the polymer comprises one or more monomers of Formula (A) and/or Formula (B).

Embodiment 76: The composition of Embodiment 75, wherein the polymer comprises one or more monomers of Formula (A).

Embodiment 77: The composition of Embodiment 75 or 76, wherein the polymer comprises one or more monomers of Formula (B).

Embodiment 78: The composition of any one of Embodiments 74-77, wherein RAis —OC(O)—RA2, —ORA1, —C(O)RA1, or —C(O)O—RA1.

Embodiment 82: The composition of any one of Embodiments 74-81, wherein the polymer is a poly(glycerol carbonate) or a copolymer thereof.

Embodiment 83: The composition of any one of Embodiments 74-82, wherein the polymer is poly(1,3-glycerol carbonate), poly(1,2-glycerol carbonate), or a copolymer thereof.

Embodiment 85: The composition of Embodiment 84, wherein the copolymer comprises a poly(glycerol carbonate) and polycaprolactone.

Embodiment 86: The composition of Embodiment 85, wherein the copolymer is poly(1,3-glycerol carbonate)-C18-co-poly(ε-caprolactone).

Embodiment 87: The composition of any one of Embodiments 74-86, wherein the particle is from about 10 nm to about 1,000 nm.

Embodiment 88: The composition of Embodiment 87, wherein the particle is from about 50 nm to about 200 nm.

Embodiment 89: The composition of any one of Embodiments 1-88, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.

Embodiment 90: The composition of any one of Embodiments 1-89, wherein the composition is formulated as a unit dosage formulation.

Embodiment 91: The composition of any one of Embodiments 1-90, wherein the composition further comprises an active agent.

Embodiment 93: The composition of any one of Embodiments 1-92, wherein the composition further comprises one or more of binders, viscosity modifiers, preservatives, humectants, emollients, pH stabilizing agents, chelating agents, gelling agents, thickening agents, emulsifiers, buffers, and carriers.

Embodiment 94: A compound of Formula (I) or (II):

Embodiment 95: The compound of Embodiment 94, wherein Y is N—R14, e.g., the compound is of Formula (I-A) or (II-A):

Embodiment 100: The compound of Embodiment 99, wherein a is 0, 1, 2, 3 or 4.

Embodiment 103: The compound of Embodiment 102, wherein R15is isopropyl.

Embodiment 104: The compound of Embodiment 94 or 95, wherein R15is a nucleophile.

Embodiment 128: The compound of any one of Embodiments 94-127, wherein the antileishmanial compound has a C Log P from about 3.2 to about 4.3.

Embodiment 129: The compound of any one of Embodiments 94-128, wherein the carbon to which the R12group is attached has the S stereochemistry.

Embodiment 130: The compound of any one of Embodiments 94-128, wherein the carbon to which the R12group is attached has the R stereochemistry.

Embodiment 131: A method for treating leishmaniasis or a disease or disorder associated with leishmaniasis, the method comprising: administering a therapeutically effective amount of an antileishmanial compound of Formula (I) or Formula (II) to a subject in need thereof.

Embodiment 134: The method of Embodiment 133, wherein theLeishmaniaparasite is selected from the group consisting ofLeishmania major, Leishmania braziliensis, andLeishmania donovani.

Embodiment 135: The method of any one of Embodiments 131-134, wherein the leishmaniasis is cutaneous leishmaniasis (CL).

Embodiment 136: The method of any one of Embodiments 131-134, wherein the leishmaniasis is visceral leishmaniasis (VL).

Embodiment 137: The method of any one of Embodiments 131-136, wherein the antileishmanial compound is formulated in a composition for administering to the subject.

Embodiment 138: The method of Embodiment 137, wherein the composition is a composition of any one of claims 1-93.

Embodiment 139: A method of reducing, suppressing or inhibiting aLeishmaniaparasite in a vector, the method comprising: administering to the vector an effective amount of Formula (I) or Formula (II).

EXAMPLES

The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

In order to improve topical administration to CL lesions, the inventors have demonstrated an adhesive that can be formulated into a transdermal patch. The structure of the polymer, 1,3-poly(glycerol carbonate) (1,3-PGC), is promising as an adhesive in that it is biodegradable into natural metabolites, tunable in adhesive strength via pendant chain modifications, and structurally similar to poly(acrylate) adhesives. The inventors demonstrated proof-of-concept in drug-loading the adhesive with CMLD011128 through a solvent evaporation method in ethyl acetate with 5% glycerol. Including the glycerol as an additive slowed down the rate of solvent evaporation, ultimately yielding a more uniform adhesive disk (FIG.1).

A new poly(glycerol carbonate) co-polymer nanoparticles was also developed and used for the delivery of active agent to the liver in systemic VL infections. This polymer, 1,3-poly(glycerol carbonate)-C18-co-poly(ε-caprolactone) is formulated into nanoparticles via a oil-in-water nanoemulsion sonication method. This method was optimized to achieve particle sizes of 100-130 nm, which is ideal for cellular uptake and in vivo delivery to the liver. In a preliminary drug release study conducted in sodium phosphate buffer and sodium dodecyl sulfate at pH 7.4, these nanoparticles demonstrate approximately 30% drug release within 7 days (FIG.2).

In a longer-term study, the PGC-co-PCL nanoparticles demonstrated a sustained release of payload, with 65% of the loaded drug released in Week 1 and the remaining 35% released over the next 3-4 weeks (FIG.3). The release media for these experiments was 10 mM phosphate buffer (pH 7.4) containing 0.5% w/v sodium dodecyl sulfate and maintained at 37° C.

Assay Conditions B: THP-1 cells (human acute monocytic leukemia cell line—ATCC TIB202) were grown in RPMI supplemented with 10% Fetal Bovine Serum (FBS) and 50 μM 2-mercaptoethanol at 37° C. in 5% CO2. THP-1 were seeded in microwell plates at 5×105cells/mL density and treated with 0.1 μM phorbol myristate acetate (PMA, Sigma) at 37° C. for 48 h for differentiation into adherent, non-dividing macrophages. After activation by PMA, cells were washed and incubated with complete RPMI medium containing stationary phaseLeishmaniapromastigotes (L. major: strain LV39;L. donovani, strain 1 S/Cl2D) at a 1:15 parasite-cell ratio. Compounds were added and incubated at 37° C. for 72 h. Cells were then washed with phosphate-buffered saline (PBS), fixed for 30 minutes with 4% formaldehyde, rinsed again with PBS, stained for 2 h with 4′,6′-diamidino-2-phenylindole (DAPI 300 nM) and finally washed with PBS.

Assay Conditions C: The intramacrophageLeishmania donovaniactivity assay was performed as described in Antimicrob Agents Chemother (2013) 57(7):2913-22.

Assay Conditions D: B10R cells (CVCL_0155) were seeded at 300 cells/well, andL. donovaniWT promastigotes in stationary phase (7th day after passage) were added at 6,000 parasites/well (ratio of 20 parasites/cell). Both cells and parasites were seeded in DMEM High-Glucose medium (Gibco, cat. no. 11995065) containing 5% Fetal Bovine Serum (Sigma-Aldrich, cat. no. F2442) and 1% Penicillin-Streptomycin (Gibco, cat. no. 15140122). Cells and parasites were incubated in the presence of the compounds for 72 h at 37° C. and 5% CO2. Plates were then fixed with 4% formaldehyde solution for at least 1 h, then washed with 1×PBS and stained with 5 μg/mL DAPI. Plates were read using an ImageXpress microscope (Molecular Devices) and analyzed by MetaXpress software (Molecular Devices) using a custom module optimized for this assay. Compounds that showed relevant antiparasitic activity in the primary screening were retested in serial dilution to obtain a dose-response curve (DRC). Compounds were tested in a 10-point 2-fold serial dilution in 3 technical replicates, and 2 biological replicates. After 72 h, plates were fixed and stained with DAPI as described above. Images were acquired on an ImageXpress microscope, and analyzed using the MetaXpress custom module. The DRCs were plotted, effective concentration inducing 75% activity (EC75) and half-cytotoxic concentration (CC50) were calculated using GraphPad Prism Software, version 6.05 (GraphPad Software, San Diego, CA).

Assay Conditions E: J774 cells (ATCC J774A.1 were grown in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin at 37° C. in 5% CO2. Leishmaniaparasites expressing luciferase and GFP were grown as described in Front. Cell. Infect. Microbiol (2019) 9:237. J774 cells were seeded in microwell plates at 3×105cells/mL density. Cells were washed with PBS and incubated with complete DMEM medium containing stationary phaseLeishmaniapromastigotes (L. major: strain Friedlin;L. braziliensis, strain BA788) at a 1:10 parasite-cell ratio for 24 h. Cells were washed with PBS. Cells and compounds were incubated for 48 h at 37° C. and 5% CO2. Compounds were tested in 8-point 2-fold serial dilution in 4 technical replicates, and 2 biological replicates. After 48 h, OneTiterGlo (Promega) (100 μl) was added to each well. Plates were incubated for 15 min and bioluminescence was measured using a plate reader (Molecular Devices). Effective concentration inducing 75% activity (EC75) was calculated using GraphPad Prism Software (version 9.0).

Assay Conditions F:Leishmaniapromastigotes (L. major: strain LV39;L. donovani, strain 1 S/Cl2D) were maintained as previously described in PloS Negl Trop Dis (2015) 9(3):e0003588 and Pathogens (2021) 10(5):593) at 28° C. in M199 media supplemented with glutamine, adenosine, folic acid, hemin, HEPES, 10% Fetal Bovine Serum (Sigma-Aldrich, cat. no. F2442) and 1% Penicillin-Streptomycin (Gibco, cat. no. 15140122). For the promastigote assay, we followed the method previously described in PloS Negl Trop Dis (2011) 5(7):e1253. Briefly, promastigotes were incubated with the compounds for 72 h at 27° C., then lysed by adding 50 μL of CellTiter-Glo (Promega) and placed on an orbital shaker for 5 min at room temperature. After lysis, the resulting ATP-bioluminescence was measured using the Analyst HT plate reader (Molecular Devices).

Introduction

Leishmaniases are a group of parasitic diseases caused by a variety of species of parasites of the genusLeishmania, which are exist on all continents. About 350 million people are living in high-risk areas. The form and severity of the disease depend on theLeishmaniaspecies and the host's immune status. WhileLeishmania majorcauses most CL infections in North Africa, the Middle East, and Central Asia,L. braziliensisis the leading causative agent of CL in South America, responsible for the majority of the 300,000 total cases. In Brazil, 21,000 new cases are registered per year. CL infection byL. braziliensispresents as several clinical forms, which range from a localized ulcerated lesion (localized cutaneous leishmaniasis, or LCL) to disfiguring lesions in mucosal areas. In Brazil, LCL is caused mainly byL. amazonensisandL. braziliensis. LCL also can produce a large number of skin ulcers on exposed parts of the body, such as the face, legs and arms, which later become permanent scars.L. braziliensiscan also cause disseminated leishmaniasis, characterized by the appearance of dozens to thousands of skin lesions that spread across non-contiguous body segments, also with frequent involvement of mucosal areas.

Treatment alternatives for leishmaniasis are limited to a small number of drugs that, due to the high cost and significant adverse effects, becomes one of the major barriers to cure the disease. In Brazil, the first drug of choice is pentavalent antimonial (Sb v), with two commercial presentations: antimoniate-N-methyl-glucamine (Glucantime®) and sodium stibogluconate (Pentostam®). Pentavalent antimonials interfere with the oxidative metabolism of theLeishmaniaamastigote, causing an inhibition in both glycolysis and oxidation of fatty acids, processes primarily located in the glycosomes. The recommended dose of Glucantime® varies between 10 to 20 mg of Sbv/kg/day/20 days and in the absence of complete healing two weeks after the end of treatment, the regimen is repeated. If the failure persists, the diagnosis should be reassessed in a specialized service to verify the possibility of indicating the drugs of second choice, which in these cases are Amphotericin B and Pentamidine.

Pentavalent antimonials have been used worldwide for the treatment leishmaniasis for more than six decades and, lately, acquired resistance has become a clinical threat. Therefore other alternatives to using of Sbvhave emerged such as Miltefosine, which replaced sodium stibogluconate (Pentostan) in the Indian subcontinent. Amphotericin B is also highly effective, but relatively toxic when injected in its deoxycholate free form. Administration in a liposomal formulation improves the risk of toxicity, although the high cost of this formulation has restricted its use. Lastly, Paromomycin has a relatively narrow range of target species ofLeishmaniawhich makes the range of treatment difficult. In parallel to the search for new therapeutic alternatives for leishmaniasis, another goal is to develop novel forms of therapy besides the intravenous or intramuscular routes used for pentavalent antimonials. Towards this end, the use of bacterial cellulose or biocellulose (BC) membranes was investigated for the topical treatment of LCL. Several microorganisms have the ability to produce BC, however Gram-negative bacteria ofGluconacetobactergenus are capable of producing cellulose in commercial quantities. Cellulose membrane are synthesized at the air/liquid interface of the static culture medium, they highly porous structures constituted of a random microfibrillar 3D-network of cellulose chains aligned in parallel with high permeability to fluids, this being favorable for cell adhesion and proliferation. BC membranes are thus a promising biomaterial for healing wounds, burns and the treatment of tissue implants due to their unique properties such as high crystallinity, high mechanical strength, ultrafine fiber network structure and high water-uptake capability (water content 90%). BC membranes also provide a humid environment to the affected region, promoting exudate absorption and acceleration of wound healing without any toxicity. Efforts to develop new BC-based materials to add new characteristics and therapeutic possibilities have generated a BC-based hydrogel. BC-based-hydrogel has been shown to accelerate the healing processes, suggesting its demonstrating potential for the treatment of dermal lesions, such as those observed in LCL.

A new class of pyrazolopyrrolidinone antileishmanials has recently been exposed and exemplified by early lead CMLD011128 with potent inhibition of both the VL-causative speciesL. donovani, and CL-causative speciesL. major. From this work, two advanced leads CMLD011494 and CMLD010948 showed significant leishmanicidal activity, in micromolar quantities, against the amastigote and promastigote forms, suggesting their therapeutic potential in leishmaniasis.

Given the recent findings describing the use of BC biocuratives formulated with DETC, Diethyldithiocarbamate, a superoxide dismutase 1 inhibitor, herein the leishmanicial potential of CMLD011494 and CMLD010948 was investigated againstL. braziliensis. Experiments conducted in vitro and in vivo, using BC-based hydrogel containing CMLD011494 and CMLD010948 showed that both leads display leishmanicidal activity.

Pyrazolopyrrolidinones Reduce the Parasite Load In Vitro, in a Dose-Dependent Manner

Initially, the leishmanicidal effect of (S)-1 was evaluated and a subset of high-potency advanced leads, including CMLD011494, CMLD0010948, and CMLD010947 against marine macrophages infected withL. braziliensis. It was found that CMLD011494 significantly reduced the percentage of infected macrophages (FIG.4A) and the number of amastigotes (FIG.4B) in a dose dependent manner. To confirm that CMLD011494 compromised parasite viability, intracellular parasite survival was quantified by transformation of amastigotes into proliferating promastigotes in Schneider's medium, as described.L. braziliensispromastigotes were also significantly reduced following exposure of infected BMDM to CMLD011494 (FIG.4C).

In similar experiments, compound CMLD010948 also induced a significant decrease in the percentage of infected cells (FIG.5A) and of intracellular amastigotes (FIG.5B). These results again confirm that these small molecules are targeting the intracellular amastigote, leading to parasite killing. Of note, however, is that compounds CMLD010947 and CMLD011128, both potent inhibitors ofL. donovani, and the latter an inhibitor ofL. major, did not show leishmanicidal effects when probed againstL. braziliensisinfection. The percentage of infected macrophages did not decrease upon treatment with CMLD011128 (FIGS.8A and8B). However, killing effect was seen with for CMLD010947, at the two lowest concentrations tested (50 and 100 nM) (FIGS.8C and8D) but not at the higher concentration range (500 and 1000 nM).

Given the dose dependent effect of compounds CMLD011494 (FIG.5A) and CMLD010948 (FIG.5B), the investigation was extended toL. majorsince this is the main etiological agent of LCL in the Middle East. Differently fromL. braziliensis, L. majordoes not cause disfigurative disease such as mucosal leishmaniasis. The results show that exposure ofL. major-infected murine macrophages to both CMLD011494 and CMLD010948 also leads to a significant decreased percentage of infected cells (FIG.6AandFIG.6C, respectively) and number of intracellular amastigotes (FIG.6BandFIG.6D, respectively). However, these effects were only observed at the highest dose (1 μM). Exposure of macrophages to CMLD011494 and CMLD010948 at concentrations ranging from 500-1000 nM did not change cell viability (FIGS.9A and9B). The EC50of CMLD011494 againstL. braziliensis-infected macrophages was 1.64 μM and for CMLD010948 was >5 μM.

The in vitro data collectively show that CMLD011494 and CMLD010948 display a leishmanicidal potential againstL. braziliensisandL. major, as seen by the reduction in the percentage of infected cells as well the number of intracellular amastigotes.

Topically-Applied BC-Hydrogel Containing Pyrazolopyrrolidinones Reduces the Lesion Size and Parasite Load In Vivo

Next, the effect of the compounds was tested using an in vivo experimental model of CL. Mice were inoculated withL. braziliensisand three weeks later, treated with BC-hydrogel containing CMLD011494 at 10 μg. The hydrogel was applied to cutaneous lesions, with treatment applied three times a week, for eight weeks. BC-hydrogel alone (control) did not interfere with lesion development; however, the BC-hydrogel containing CMLD011494 significantly impaired lesion development (FIG.7A). Of note,L. braziliensistreatment with BC-Hydrogel alone did not change lesion development in comparison to untreated mice (FIG.10), indicating that BC-hydrogel containing CMLD011494 or CMLD010948 exert therapeutic effects in vivo. Six weeks after parasite inoculation, parasite load was determined at the infection site and in draining lymph nodes. As shown inFIG.7, treatment with BC-hydrogel containing CMLD011494 significantly reduced parasite load at the ear (FIG.7B), in levels compared to pentavalent antimony (the current standard care treatment for LCL caused byL. braziliensis, in Brazil). In comparison, the parasite load was similar amongst the four groups (FIG.7C) in the draining lymph nodes, indicating that the local topical exposure to CMLD011494 does not impact distal sites.

Experimental Methods

Ethics Statements

Female BALB/c mice, 6-8 weeks of age, were obtained from the IGM-FIOCRUZ animal facility where they were maintained under pathogen-free conditions. All animal experimentation was conducted in accordance with the Guidelines for Animal Experimentation established by the Brazilian Council for Animal Experimentation Control (CONCEA). All procedures involving animals were approved by the local Institutional Review Board for Animal Care and Experimentation (CEUA-IGM-FIOCRUZ-002/2019-2068.

Cells

BMDM were obtained as described above. Cells were resuspended in DMEM medium supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated Fetal Bovine Serum (all from Invitrogen) and seeded at a density of 3×105cells per well in 24-well culture plates. Monolayers received 3×106cellsL. braziliensispromastigotes and were incubated at 37° C. in supplemented DMEM medium for 24 h. Infected macrophages were then washed to remove non-internalized parasites. Compounds CMLD 011128, CMLD010947, CMLD010498, and CMLD011494 were added at different concentrations. After 24 hours, cells were extensively washed, fixed and stained with hematoxylin and eosin (H&E). The number of infected cells and of intracellular amastigotes were counted by optical microscopy in 200 macrophages. Positive controls consisted of cultures treated with Amphotericin B (0.25 μg/mL, Invitrogen). Cultures were performed in quintuplicate. Alternatively, infected macrophages were treated as described, monolayers were extensively washed and the medium was replaced by 0.5 ml of supplemented Schneider medium. Cells were cultured for seven additional days at 26° C., when number of viable promastigotes was determined using hemocytometer.

To calculate the half-maximal effective concentration (EC50),L. braziliensis-infected macrophages were exposed to different concentrations of CMLD011494 (10, 25, 50, 100, 250, 500 and 1000 nM) After 24 hours, the percentage of infected macrophages was determined by optical microscopy. Results were expressed as the mean percentage reduction of infected cells compared with untreated control wells. Half-maximal effective concentrations (EC50) were determined by sigmoidal regression curves using Prism 7.0 software (GraphPad Software Inc.)

Cellular Viability

Briefly, macrophages were obtained from bone marrow and seeded at a density of 1×106cells per well in 24-well culture plates. After 2 hours, cells were treated with compounds at two concentrations (500 and 1000 nm) for 24 hours. Cell viability was determined using Cell Titer Glo (Promega), according to manufacturer's instructions. For positive control, pararosaniline chloride was used.

Superoxide and Cytokine Quantification in Culture Supernatants.

BMDM were seeded at a density of 1×106cells per well in 24-well culture plates and cells were infected withL. braziliensisas described above. To determine superoxide production, BC-DETC or empty BC were placed within wells containing infected cells for 48 h in presence of 0.5 mM hydroxylamine hydrochloride (Acros Organics). Superoxide was quantified in culture supernatants using Griess reagent.

Cytokine levels were determined in culture supernatants using commercial ELISA kits, following manufacturer's instructions.

BALB/c mice were inoculated intradermally withL. braziliensispromastigotes (105parasites in 10 μl of saline) using a 27.5-gauge needle18. Ear thickness (as a surrogate for lesion development) was recorded weekly using a digital caliper (Thomas Scientific). Three weeks after parasite inoculation mice were randomly assigned into three groups: one group was topically treated with BC-11494 or BC-10948. BC-based hydrogel was kindly provided by Seven Industria de Produtos Biotecnologicos Ltda. (Ibipord, PR, Brazil). BC-based hydrogel (Nexfill® Hydrogel) composition was obtained according to the PI 0601330-9 A2 patent. CMLD011494 or CMLD010498 solution (10 mM) were mixed to BC-hydrogel (20 uL) and placed on the lesions of infected mice, three times a week, for three consecutive weeks. Ear thickness continued to be recorded weekly. Controls consisted of mice treated with BC-hydrogel alone or with Sbv(50 mg/kg), following the same regime described for BC-hydrogel. Six weeks after infection, parasite load was determined using by limiting-dilution analysis, as described previously.

REFERENCES

Leishmaniasis is a disease caused by theLeishmaniagenus of parasites that affects approximately 2 million people worldwide, with 700,000-1 million new cases and as many as 50 thousand deaths annually. It is the second deadliest parasitic disease after Malaria. Leishmaniasis has different clinical manifestations depending on the leishmanial species and patient immune system. Visceral leishmaniasis (VL) is a febrile condition affecting internal organs that can lead to death if left untreated. Current first line treatment for VL, predominantly caused by the speciesL. donovaniandL. infantum/L. chagasi, is based on antimonials, a drug formulation using the toxic metal antimony. Second line treatments include IV-administered liposomal amphotericin B (AmBisome), and miltefosine as an orally administered pill. AmBisome is the most effective but prohibitively expensive for the disease population most affected by leishmanial infections. Availability and supply is often a challenge, with the additional requirement that it must be administered in a clinical setting. Miltefosine is teratogenic, toxic to the kidneys and causes gastrointestinal discomfort at the doses necessary to treat the disease, leading to poor compliance in completing a full treatment regimen. Resistance has already become an issue with miltefosine, and there are supply challenges due to the public-private partnership model.

Cutaneous leishmaniasis (CL) is a generally non-fatal skin condition that produces lesions ultimately leading to permanent scarring and disfigurement.Leishmania majorcauses most CL infections in North Africa, the Middle East, and Central Asia, whileL. braziliensisandL. amazonensisare the leading causative agents of CL in South America. In total, CL infects 1.5 million people worldwide, and the current first line treatment is a pentavalent antimony compound that is delivered by painful intralesional needle injection. Additional challenges in supply, administration, toxicity, and resistance also make this treatment less than ideal. Advances have been made using topically-administered miltefosine, however, there have already been documented failures in this approach due to the rate of parasite mutation.

All current approved small-molecule treatments for leishmaniasis are “repurposed” drugs that were developed for other diseases, especially cancer. The current pipeline is underdeveloped. Drugs for Neglected Diseases (DNDi: dndi.org) lists five new compound classes in their clinical antileishmanial portfolio; none have yet progressed beyond Phase I. GlaxoSmithKline's lead CRK12 inhibitor GSK3186899 (VL only) completed a Phase I single ascending dose study in 2019, but further clinical evaluation of this compound has been paused following the emergence of non-clinical data for a non-GSK asset with a similar mode-of-action. Oxaborole DNDI-6148 (CL/VL) and nitroimidazole DNDI-0690 (CL/VL), have both completed Phase I single ascending dose studies with multiple ascending dose trials underway. Oligo-deoxynucleotide CpG-D35 (CL only) and GSK's recently-reported proteasome inhibitor GSK3494245 are also both slated for Phase I study. There remains an unmet clinical need to develop new treatments against leishmaniasis that are ideally inexpensive, readily produced, and orally available as a short course of chemotherapy. Herein, the discovery of a novel antileishmanial compound class is described, with potent activity against the intracellular stage of the parasite (the most relevant for human disease) in multipleLeishmaniaspecies.

Methods

Chemistry

All pyrazolopyrrolidinones were synthesized via a two-step sequence in which pyrrolidinones 4 were first synthesized via Mannich condensation/cyclization of a/y diketo esters 5 with either pre-isolated or in-situ-generated imines 6, followed by Knorr pyrazole condensation with a requisite hydrazine 3 (Scheme 1) produced the desired pyrazolopyrrolidinones. All compounds tested had a purity of >90% as measured by UPLC-MS-ELSD. Full details for compound synthesis and characterization for select pyrazolopyrrolidinones are provided in the Supplementary Information.

High-Throughput Screen for Antileishmanial Compounds at UCSD

Compounds were obtained as 0.2 μmoles of dried film for primary single point screening. Each compound was diluted in DMSO to 10 μM final testing concentration. These compounds were tested in 2 biological replicates. The compounds were pre-spotted onto 384-well assay plates in single concentration. B10R cells (CVCL_0155) were seeded at 300 cells/well, andL. donovaniWT promastigotes in stationary phase (7th day after passage) were added at 6,000 parasites/well (ratio of 20 parasites/cell). Both cells and parasites were seeded in DMEM High-Glucose medium (Gibco, cat. no. 11995065) containing 5% Fetal Bovine Serum (Sigma-Aldrich, cat. no. F2442) and 1% Penicillin-Streptomycin (Gibco, cat. no. 15140122). Cells and parasites were incubated in the presence of the compounds for 72 h at 37° C. and 5% CO2. Plates were then fixed with 4% formaldehyde solution for at least 1 h, then washed with 1×PBS and stained with 5 μg/mL DAPI. Plates were read using an ImageXpress microscope (Molecular Devices) and analyzed by MetaXpress software (Molecular Devices) using a custom module optimized for this assay. Compounds that showed relevant antiparasitic activity in the primary screening were retested in serial dilution to obtain a dose-response curve (DRC). Compounds were tested in a 10-point 2-fold serial dilution in 3 technical replicates, and 2 biological replicates. After 72 h, plates were fixed and stained with DAPI as described above. Images were acquired on an ImageXpress microscope, and analyzed using the MetaXpress custom module. The DRCs were plotted, half-effective concentration (EC50) and half-cytotoxic concentration (CC50) were calculated using GraphPad Prism Software, version 6.05 (GraphPad Software, San Diego, CA).

The intramacrophageLeishmania donovaniactivity assay (LD AMMAC) at GlaxoSmithKline was performed as described.

Solubility Assays

Solubility of compounds using ChemiLuminescent Nitrogen Detection (CLND) was measured as described. Solubility of compounds using Charged Aerosol Detection (CAD) was measured as described. Solubility of solid compounds in Fasted Simulated Intestinal Fluid (FaSSIF) was measured as described.

Artificial Membrane Permeability (AMP) Assays

Passive permeability of compounds via rate of permeation through an artificial phospholipid membrane at pH 7.4 was measured in a high-throughput format, in duplicate. A solution of 1.8% phosphatidylcholine in 1% decane was added to a 96-well Millicell filter plate along with 250 μL of 50 mM phosphate buffer, pH 7.4 on the donor side, and 100 μL of the same buffer solution on the receiver side. The assay plate was shaken for 45 minutes before adding test compounds. Test compounds were then added to the filter plate and then incubated at room temperature with shaking for three hours. The donor and receiver solutions were next transferred to a 384-well plate for analysis by LC/MS.

Microsomal Stability Assays

Mouse microsomal stability assays were performed as described. Test compound (0.5 μM) was incubated with female CD 1 mouse (Xenotech) liver microsomes and their action started with addition of excess NADPH (8 mg/mL 50 mM potassium phosphate buffer, pH 7.4). Aliquots (50 μL) of the incubation mixture were removed immediately (at time 0) and at 3, 6, 9, 15, and 30 min and mixed with acetonitrile (100 μL) to stop the reaction. Internal standard was added to all samples, the samples were centrifuged to sediment precipitated protein, and the plates were then sealed prior to UPLC-MS/MS analysis using a Quattro Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of the peak area of test compound to internal standard at each time point. The rate of intrinsic clearance (Cli) of each test compound was then calculated using the equation Cli(mL/min/g liver)=k×V×microsomal protein yield, where V (mL/mg protein) is the incubation volume/mg protein added and microsomal protein yield is taken as 52.5 mg protein/g liver. Verapamil (0.5 μM) was used as a positive control to confirm acceptable assay performance.

Human Serum Albumin (HSA) Assay

The Percentage of Compound Bound to Human Serum Albumin was Measured Using a chromatographic method as described. Briefly, each compound was assayed on an immobilized HSA column and gradient retention times measured, with chromatographic peak detection by UV. Each retention time was then converted to a % HSA bound value using a calibration set of compounds with a known % HSA binding.

Plasma Protein Binding (PPB) Assay

The unbound fraction of compound 1 in plasma was measured using a commercial RED (Rapid Equilibrium Dialysis) plate with inserts (Thermo) with a molecular weight membrane cut off of 8K. The relevant volume of spiked sample matrix was added into the corresponding sample chambers of the RED insert. Three volume equivalents of dialysis buffer were added to the buffer chamber. The dialysis plate was sealed and incubated at 37° C. on a plate shaker for approximately 4 h at 100 rpm. An equivalent volume was removed from each of the three buffer sample chambers and placed into its own well in a clean plate. A specific volume of control matrix was added to each buffer sample for matrix matching. Next, >3× volume of precipitation solvent (acetonitrile+internal standard) was added and the plate was centrifuged. A measured volume of the resulting supernatants was transferred into a clean plate and a specific volume of analytical grade water was added to all samples. Samples were analyzed using a compound-specific LC-MS/MS method to generate analyte peak area ratios which are representative of bound and free drug.

Chiral Chromatographic Resolution of 1

The enantiomers of compound 1 were resolved using semi-preparative chiral HPLC on a Chiralpak IC column (0.46×25 cm) using an isocratic mobile phase of 70:30 heptane:ethanol with a 1 mL/min flowrate for 30 minutes. The first- and second-eluding enantiomers of 1 had retention times of 13.9 minutes, and 22.6 minutes, respectively (FIG.12A-12B). Independent biological testing of each enantiomer in the LD AMMAC assay indicated that the first-eluting enantiomer (1a, TR=13.9 min) had an EC50of 0.398 μM, and the second-eluting enantiomer (1b, TR=22.6 min) had an EC50of ˜10 μM. The separated enantiomers were next subjected to VCD analysis for absolute stereochemistry assignment as described below.

VCD Analysis

A VCD spectrum for each of the separated enantiomers of 1 was obtained in deuterated acetonitrile (˜9.8 mg/175 μL concentration) on a BioTools ChirallR-2× FT-VCD spectrometer operated at 4 cm−1. VCD frequency range was measured from 2400-800 cm1with PEM calibrated at 1400 cm−1and PEM retardation applied. The first-eluting enantiomer (1a) was analyzed using a single two-hour block scan (6240 total scans) and the second-eluting enantiomer (1b) was analyzed using the average of six two-hour block scans (37,440 total scans). These experimentally-obtained VCD spectra were utilized in the computational enantiomer assignment as described below.

Computational Methods and Enantiomer Determination

Predicted VCD and IR spectra for the (R) enantiomer of compound 1 were generated according to the following computational workflow: first, a conformational search was performed using MOE LowMode algorithm and Amber12:EHT force field with a generalized Born implicit solvent model (dielectric constant=1). Each unique conformer was then subjected to DFT optimization (B3LYP/DGDZVP2) with VCD vibrational frequency calculation using a polarizable continuum solvent model for acetonitrile. A VCD spectrum was then predicted with fractional populations of each conformer estimated using Boltzmann statistics with a Lorentzian band width of 8 cm1and a frequency scale factor of 9.9825. This computationally-predicted spectrum was compared to the experimentally obtained spectra using CompareVOA software (BioTools, Inc.) (FIG.12A-12B). Inspection of the VCD data in the analysis range indicated that the (R) model spectrum was largely coincident with that measured on the second-eluting enantiomer 1b, and was the mirror image of that obtained for the first-eluting enantiomer 1a. Based on these findings, the bioactive enantiomer 1a was assigned with (S) absolute configuration((S)-1), and enantiomer 1b was assigned with (R) absolute configuration ((R)-1). The confidence limit for these assignments was determined from the absolute values of two parameters in the CompareVOA software: total neighborhood similarity (TNS (VCD)) and the enantiomeric similarity index (ESI). The thresholds for “high” reliability (CL of >99%) are TNS (VCD)≥70 and ESI≥60. In this study, the TNS (VCD) and ESI values were 81.0, and 77.5, respectively, providing an estimated confidence limit of >>99% (very high reliability).

Results and Discussion

In a collaborative effort to identify new antileishmanial chemotypes with minimal host cell cytotoxicity, compounds from the Boston University Center for Molecular Discovery (BUCMD) screening collection were assessed in a phenotypic, high content primary screen at the University of California's Center for Discovery and Innovation in Parasitic Diseases (CDIPD) for the ability to inhibit growth ofL. donovaniintracellular amastigotes infecting THP-1 cells. From this screen, two pyrazolopyrrolidinones were identified (1 and 2, Table 1) which exhibited >99% inhibition of parasite growth with minimal cytotoxicity to the host THP-1 cells (<13% GI). Dose-response testing inL. donovani(both intracellular amastigotes and promastigotes) confirmed concentration-dependent growth inhibition of both morphologies of the parasite at low micromolar EC50values for both compounds (Table 1). Similar activity was subsequently confirmed against both morphologies the cutaneous leishmaniasis-causative speciesL. major, suggestive of broad spectrum antileishmanial activity. Notably, these initial hits had potencies comparable to all existing non-antimonial treatments for the disease (Table 1), as well as to GlaxoSmithKline's current Phase I VL candidates GSK3186899 (intramacrophage EC50=1.4 μM) and GSK3494245 (intramacrophage EC50=1.6 μM), which both were chosen for advancement over more potent analogues due to favorable drug properties (e.g. safety, solubility).

Compounds 1 and 2 were generated as part of a larger combinatorial library of pyrazolopyrrolidinones (Scheme 1, 3), obtained via Knorr pyrazole condensation of 4-acylated 3-hydroxydihydropyrrol-2-ones 4 with hydrazine hydrate. Precursors 4 are easily produced from a Mannich reaction/intramolecular cyclization between α/γ-diketo esters 5 and pre-formed or in situ-generated imines 6. The lack of activity for several near-neighbor analogues in the primary screen provided some nascent SAR (FIG.13), hinting at the importance of the para-methoxyphenyl moiety at R1(vs. phenyl), and the isobutyl group at R3(vs. methyl, isopropyl and phenyl).

At the outset of the project, compound 1 was evaluated against GSK's established criteria for antileishmanial compound advancement (Table 2). Some of these assessments were performed on racemic 1, while chiral separation was also pursued of the 1 racemate to determine the active enantiomer. Preparative chiral-SFC was used to separate enantioenriched 1 on a multigram scale, and vibrational circular dichroism (VCD) analysis confirmed the absolute (S)-stereochemistry of the active enantiomer (FIG.11,FIGS.12A-12B), which had an improved EC50of 0.8 μM. As shown in

Table 2, compound 1 performed well against most of GSK's lead selection criteria, and met minimum standards toward advancement as a lead compound, human serum albumin binding and property forecast index (PFI), a hydrophobicity metric developed at GSK which considers lipophilicity and aromatic ring count and is predictive of downstream developability. Based on this promising profile, it progressed into medicinal chemistry optimization to better understand structure-activity relationships (SAR) toward improved potency, as well as structure-property relationships (SPR) with an eye toward reducing PFI and plasma protein binding.

The pyrazolopyrrolidinone chemotype is well-described in the research and patent literature, with a rich array of reported biological activities, the most prominent of which are p53/MDM2 interaction inhibition, phosphodiesterase inhibition, and GPR55 modulation. In addition, there are examples of pyrazolopyrrolidinones exhibiting P2X3 antagonism, GPR68 agonism, 5-HT1A receptor binding, BET inhibition, 14-3-3-PMA2 interaction stabilization, P-glycoprotein inhibition, antitumor activity, and antimicrobial activity against various parasitic, viral and bacterial species includingT cruzi, HIV, flaviviruses,M. tuberculosis, P. falciparum, andV. cholerae. Interestingly, most of the aforementioned activities are relegated to pyrazolopyrrolidinones wherein R3is an aryl substituent. This phenomenon may, however, be attributable to the ease of synthesis of such compounds and their precursors. An important exception to the R3arylation trend is observed among select inhibitors of the p53/MDM2 interaction. In all of these inhibitors, the R1/R2diarylated motif has been shown crystallographically to be a critical binding element at the Leu26 and Trp23 subpockets of MDM2, a similar pharmacophore and binding mode to that exhibited by other diarylated p53/MDM2 inhibitors such as nutlin. Among these inhibitors, non-aryl R3substitutions such as methyl, isopropyl, and tert-butyl have all been shown to confer some degree of inhibition. Other scattered exceptions include a class of purinoreceptor antagonists with similarly broad tolerance for R3substitution, and two examples of R3-methyl substituted inhibitor chemotypes: EPX-107979, annotated as a folding corrector of F508del-CFTR and 11β-hydroxysteroid dehydrogenase inhibitors ZINC01292412 and ZINC01260941. Importantly, however, there are no reported examples to-date of pyrazolopyrrolidinones bearing the R3=iBu substitution, which from the primary screen SAR (FIG.13) appeared to be critical for antileishmanial activity in the absence of host toxicity. While the target of antileishmanial pyrazolopyrrolidinones has yet to be defined, and it cannot conclusively rule out any of the aforementioned targets as being implicated in this activity, the consistent lack of antileishmanial activity among the many R3phenyl-, isopropyl- and methyl-substituted pyrazolopyrrolidiones tested in the primary screen is suggestive of a target for the R3isobutylated compounds which is orthogonal to those already appearing in the vast pyrazolopyrrolidinone literature.

Concurrent with the evaluation of screening hit 1 against GSK TCOLF's lead advancement criteria (Table 2), a preliminary medicinal chemistry campaign was executed to improve the understanding of structure-activity relationships (SAR) for this series, in order to target compounds with improved potencies and physicochemical properties to potentially supersede compound 1 as an advanced lead.

Given the literature precedents described above and the apparent narrow tolerance for R3substitutions observed in the primary screen compounds, a thorough and methodical assessment was undertaken of tolerated groups at the three points of diversity (R1/R2/R3) for the core. At this stage of the project, all analogues were assessed using a battery of assays performed in-house at GlaxoSmithKline. For antileishmanial activity, GSK's inMac assay was utilized. This assay provides two readouts of compound potency: average number of intracellular amastigotes per infected cell (AMMAC) EC50, percentage of infected cells per well (INFCELL) EC50, as well as a toxicity output derived from the number of host cells (MAC EC50). In addition, compounds were assessed for toxicity against HepG2 cells (HEPG2 EC50). Here, AMMAC EC50 values were focused on for relative potency assessments. Using this data, a selectivity index (SI) was calculated for each compound, described here as a macrophage SI (MAC SI), using the equation MAC SI=(MAC EC50)/(AMMAC EC50). It should be noted that for all compounds assessed in this project, the measured toxicity against THP-1 macrophages either equaled or exceeded that of HepG2 hepatocytes, therefore the MAC SI is used here as the more conservative estimate of therapeutic index.

Revisiting the initial profile of compound 1 against GSK's lead selection criteria, a number of properties were identified requiring improvement, including PFI, plasma protein binding, and a larger SI relative to THP-1 and HepG2 cells. While the CLND solubility fell below the ideal range, good FaSSIF solubility suggested viability as an orally available drug. The potency and physiochemical data was used for 1 as a benchmark for guidance as investigation began into the SAR to identify an improved lead compound for series progression and advancement to animal studies. In these studies, human serum albumin (HSA) binding was employed as a surrogate for plasma protein binding.

Starting first at R3, a variety of aliphatic substitutions were explored, determining that some branched aliphatics of similar size to the parent isobutyl (e.g. isopentyl/neopentyl, Table 3, compounds 9-10) exhibited comparable potencies and low host cell toxicities, whereas the linear n-butyl (compound 11) showed a significant increase in potency (˜300 nM) that was accompanied by a toxicity increase to the low micromolar range (2.5 μM). Similar effects were observed with n-but-1-ene and 2-methyl-n-but-1-ene substitutions (compounds 12-13). Finally, surveys of additional branched aliphatic (13) and aromatic (compounds 14-17) substituents at R3failed to produce more potent compounds than 1, and often showed significant decreases in selectivity index. While the isopentyl/neopentyl analogues 9 and 10 showed marginal improvements over 1 with respect to their macrophage toxicity, these improvements were offset by equivalently small increases in HepG2 toxicity and significant reductions in solubility/permeability; as such it was opted to retain the R3isobutyl substituent in all future analogues.

TABLE 3Surveying the effects of variations at R3. Values bolded andunderlined are considered improved in comparison to initial leadcompound rac-1.LDLDAMMACMACHEPG2HSAfEC50EC50SIEC50SolubilitydAMPeBindingCpdR3(μM)a(μM)bMACc(μM)(μM)(nm/sec)(%)PFIgrac-1isobutyl2.515.86.363.110734596.48.49isopentyl2.531.612.679.41510098.09.110neopentyl2.525.110.039.83617097.49.011n-butyl0.12.525.050.14937096.78.4121.63.22.050.113541097.58.1130.51.63.250.15237096.88.5146.320.03.250.11727097.98.9156.325.14.050.1636098.09.7165.07.91.625.11713097.810.6174.07.92.015.8<134098.011.4aEC50for growth inhibition ofL. donovaniintracellular amastigotes infecting THP-1 macrophages;bEC50for cytotoxicity against host THP-1 macrophages;cSI MAC = selectivity index in macrophages, calculated as SI MAC = (LD MAC EC50)/(LD AMMAC EC50);dkinetic aqueous solubility as determined by high-throughput CLND (chemoluminescent nitrogen detection);eartificial membrane permeability;fhuman serum albumin binding;gPFI = ChromLogD7.4+ Aromatic rings

The effects of modifying the R1para-methoxyphenyl substituent were examined (Table 4). Direct conversion of the methyl ether to phenol (compound 18) suppressed both antileishmanial activity and toxicity. The ethyl ether analogue 19 exhibited modest improvements in both activity and toxicity as compared to the parent methyl, while the trifluoromethoxy ether (20) ablated antileishmanial activity to levels below that of the inherent THP1-cell toxicity. The dimethylamino analogue 21 showed significantly improved potency and selectivity index, while the ethyl-, fluoro-, bromo-, tert-butyl- and methyl ester-substituted analogues (compounds 22-25) had comparable activities and therapeutic indices to 1. In contrast to methyl ester 25, hydrolyzed carboxylic acid 26 was inactive. Lastly, replacement of the para-methoxy with an N-linked imidazole (compound 27) afforded an equipotent compound with reduced cytotoxicity, leading to an improved SI. However, all improvements in potency (24, 26) or host cell toxicity (26, 28) leading to improved selectivity index were accompanied by significant reductions CLND solubility.

TABLE 4Surveying effects of various p-substituted aromatics at R1.Values in bold and underlined are considered improved in comparison toinitial lead compound 1.LDLDAMMACMACHEPG2HSAfCom-EC50EC50SIEC50SolubilitydAMPeBindingpoundX(μM)a(μM)bMACc(μM)(μM)(nm/sec)(%)PFIg1—OCH32.515.86.363.110734596.48.418—OH5.039.88.0>100≥43028595.67.119—OEt1.620.012.550.13825097.58.920—OCF33.27.92.525.1529098.19.821—N(CH3)21.039.839.850.13316096.19.022—Et4.020.05.050.19<1097.49.723—F6.325.14.050.16112097.78.824—Br1.315.812.239.8932097.99.725—t-Bu10.0>50>5.050.1<113097.910.526—CO2CH31.631.620.039.82641097.58.527—CO2H>50>50n/a>100≥389<395.24.9282.5>50>20.0>1007<396.38.1aEC50for growth inhibition ofL. donovaniintracellular amastigotes infecting THP-1 macrophages;bEC50for cytotoxicity against host THP-1 macrophages;cSI MAC = selectivity index in macrophages, calculated as SI MAC = (LD MAC EC50)/(LD AMMAC EC50);dkinetic aqueous solubility as determined by high-throughput CLND (chemoluminescent nitrogen detection);eartificial membrane permeability;fhuman serum albumin binding;gPFI = ChromLogD7.4+ Aromatic rings

Next, alternate substitution patterns were examined on the R1aryl ring (Table 5). Movement of the methoxy group from para- to the ortho- (29) or meta-positions (30) ablated activity, as did nitrogenation of the ring in the presence (31) or absence (32-33) of the para-methoxy group. Additional unsuccessful modifications explored included homologation of the para-methoxyphenyl moiety to a para-methoxybenzyl (34), and additional furyl (35) and non-aromatic substituents (36-41); although several of these modifications led to significant improvements in key properties such as reduced host cell toxicity, and improved solubility, permeability, HSA binding, and PFI, none were able to achieve inhibition of parasite replication below 10 μM EC50values.

TABLE 5Probing expanded diversity at R1. Values in bold and underlined areconsidered improved in comparison to initial lead compound 1.LDLDAMMACMACHEPG2HSAfEC50EC50SIEC50SolubilitydAMPeBindingCpdR1(μM)a(μM)bMACc(μM)(μM)(nm/sec)(%)PFIg12.515.86.363.110734596.48.42920.0>50>2.579.424059097.58.53012.631.62.563.16437097.38.43110.0>50>5.0>10019426095.58.03225.1>50>2.0>10018247093.47.23320.0>50>2.5>10021920096.68.53412.631.6>2.550.14555098.29.03515.839.8>2.550.11937097.28.93625.1>50>2.0>100≥45057092.17.33725.1>50>2.0>100≥44613081.14.63831.6>50>1.6>100≥38149090.26.53915.8>50>3.2>100≥43323076.75.340>50>50>1.0>100≥351<1077.54.841>50>50>1.0>100≥42152090.26.7aEC50for growth inhibition ofL. donovaniintracellular amastigotes infecting THP-1 macrophages;bEC50for cytotoxicity against host THP-1 macrophages;cSI MAC = selectivity index in macrophages, calculated as SI MAC = (LD MAC EC50)/(LD AMMAC EC50);dkinetic aqueous solubility as determined by high-throughput CLND (chemoluminescent nitrogen detection);eartificial membrane permeability;fhuman serum albumin binding;gPFI = ChromLogD7.4+ Aromatic rings.

There was more success in replacing the para-methoxyphenyl group with disubstituted benzene and bicyclic heteroaromatic substituents (Table 6). For example, meta-fluorination of 1 (42) led to modest increases in both potency and selectivity, albeit with the reduction in solubility as would be expected due to the increased lipophilicity. In contrast, addition of an ortho-methoxy substituent to 1 (43) improved solubility, again at the expense of activity. The replacement of the methoxy moiety with various 3,4-fused heterocycles (methylenedioxy 44, ethylenedioxy 45, and triazolopyridine 46) all led to modest improvements in selectivity via reduced host cell toxicity. However, none of these analogues showed improved solubility relative to 1 despite the presence of additional heteroatoms, which was apparently offset by the increased planarity imparted by the bicyclic systems.

TABLE 6Surveying effects of di- and tri- substitutions at R1. Values in boldand underlined are considered improved in comparison to initial leadcompound 1.LDLDAMMACMACHEPG2HSAfEC50EC50SIEC50SolubilitydAMPeBindingCpdR1(μM)a(μM)bMACc(μM)(μM)(nm/sec)(%)PFIg12.515.86.363.110734596.48.4421.325.119.350.13929096.98.64320.031.61.663.113817096.28.5440.625.141.863.16339096.28.4453.225.17.9>505051096.28.4465.0>50>10>504992091.87.8aEC50for growth inhibition ofL. donovaniintracellular amastigotes infecting THP-1 macrophages;bEC50for cytotoxicity against host THP-1 macrophages;cSI MAC = selectivity index in macrophages, calculated as SI MAC = (LD MAC EC50)/(LD AMMAC EC50);dkinetic aqueous solubility as determined by high-throughput CLND (chemoluminescent nitrogen detection);eartificial membrane permeability;fhuman serum albumin binding;gPFI = ChromLogD7.4+ Aromatic rings

With an improved understanding of R1and R3SAR, next was advancing to modifications of R2(Table 7), where the initial screening SAR indicated that deletion of the para-fluoro substituent (compound 2) afforded a similarly potent compound to 1, whereas replacement of the fluorine with a methyl group resulted in 0% inhibition at 10 μM (CMLD007430,FIG.13). Consistent with this, the efforts to replace the fluorine with other halogens (47-48), trifluoromethyl (49), carboxylate (50) and methyl carboxylate (51) substituents all reduced potency, as did replacement of the phenyl ring with cyclohexyl and cyclopentyl moieties (compounds 52-53). Interestingly, improved potencies and selectivity indexes were achieved with several types of ortho-substituents, including halogens (53-56) and a methyl ether (57), whereas none such improvements were observed with the equivalent meta-substituents (59-63). Consistent with this trend, addition of ortho-substituents to the para-fluorinated 1 (64-65) led to improved potency, whereas addition of a meta-fluoro to the same scaffold did not (66). Lastly, 2,6-dichloro substitution of the R2phenyl ring (compound 67) led to improved potency but with a considerable increase in host cell toxicity.

TABLE 7Surveying effects of simple aliphatic and aromatic R2.Values in bold and underlined are considered improved incomparison to initial lead compound 1.LDLDAMMACMACHEPG2HSAfEC50EC50SIEC50SolubilitydAMPeBindingCpdR2(μM)a(μM)bMACc(μM)(μM)(nm/sec)(%)PFIg12.515.86.363.110734596.48.42Ph—3.231.69.979.49037095.88.4474.015.84.050.12233096.99.2486.315.82.550.1942097.29.3498.015.82.050.11221096.99.350>50>501.0>100≥470<390.74.55125.139.81.663.144380958.2527.925.13.250.12933097.68.55312.620.01.6>10011959096.38.1543.231.69.931.67523097.38.6551.320.015.331.63033097.88.8560.525.150.239.81230097.19.2570.525.150.26.35735095.78.6585.012.62.550.19137096.98.3597.939.85.039.81928096.89.0607.925.13.239.810370979.1614.020.05.063.163290958.4626.315.82.550.12022097.88.9632.025.112.663.15214096.18.8640.525.150.250.14311097.28.8655.025.15.050.14414096.28.7661.02.52.56.3633097.19.1aEC50for growth inhibition ofL. donovaniintracellular amastigotes infecting THP-1 macrophages;bEC50for cytotoxicity against host THP-1 macrophages;cSI MAC = selectivity index in macrophages, calculated as SI MAC = (LD MAC EC50)/(LD AMMAC EC50);dkinetic aqueous solubility as determined by high-throughput CLND (chemoluminescent nitrogen detection);eartificial membrane permeability;fhuman serum albumin binding;gPFI = ChromLogD7.4+ Aromatic rings

In an effort to improve solubility via R2modifications, a diverse array of substituted and unsubstituted heteroaromatic groups were also surveyed at this position (Table 9). While several of these compounds exhibited the expected improvements in CLND solubility and reduction in HSA binding, potency was also significantly compromised for this set.

With the scope and limitations of R1/R2substitutions mapped with respect to potency and property improvements, it was next attempted to pair promising groups at each site to arrive at optimized new inhibitors. Based on the trends observed in the initial series, it was clear that improvements in solubility and reduced human serum albumin binding would require reduced lipophilicity (C Log P), a modification which generally also correlated with reduced potency in the initial analogues. To offset this, global Log P were reduced via modifications to R1(where increased polarity appeared to be more tolerable), in combination with the apparent potency-enhancing ortho-substituents at R2. Table 8 depicts the most successful of these pairings with respect to potency, selectivity, HSA binding, and solubility. Of note, at this later stage in the project solubility was measured using charged aerosol detection (CAD), due to a change in standard in vitro ADME methods employed at GSK. In addition, infection EC50and host cell CC50measurements were obtained in a comparableL. donovaniinfection model performed at the University of California, San Diego (see Methods). In order to benchmark compound performance across the two assays, a random sampling of compounds was selected for re-assessment in the UCSD infection assay (Table 10). Most compounds showed slightly improved potency in the UCSD assay than was observed in the LD AMMAC assay run at GSK; as a representative example, the UCSD potency for racemic 1 was found to be 0.82 μM (Table 8, entry 1), compared to 2.5 μM in the GSK LD AMMAC assay. Despite the change in absolute potency values, the two assays were well-correlated with respect to relative potencies, with a Pearson's correlation coefficient of 0.74. (Table 10 andFIG.14).

From this compound series, the ortho-substituted R2groups (B1-B4) significantly improved potency and selectivity, even when paired with groups at R1which had conferred reduced potency when paired with the R2para-fluorophenyl moiety (e.g. A4/A5, Compounds 78-87). Several inhibitors in this series, 69, 86 and 87, exhibited the desired improvements across all key physicochemical properties, in addition to improved potency and selectivity. However, for a large proportion of these compounds the most significant gains in potency were offset by an increase in toxicity, PFI and HSA binding, and a reduction in solubility owing to increased lipophilicity. Efforts to optimize from 69, 86 and 87 toward further improved analogues are ongoing in the laboratory.

TABLE 8R1A1A2A3A4A5A6R2B1B2B3B4B5Pairing of promising R1/R2moieties toward improved inhibitors. Values inbold and underlined are considered improved in comparison to initial lead compound 1.L. donovaniHSAeEC50CC50SolubilitydbindingCpdR1R2(μM)a(μM)bSIc(μM)(%)PFIg(rac)-1——0.82>20.00>24.489f96.48.467A1B10.1612.2276.43598.08.768A1B20.29>20.00>69.02797.08.869A1B30.27>20.00>74.16695.38.270A1B40.1012.08120.84697.68.671A2B20.423.047.22096.99.272A2B30.380.260.74395.98.573A2B40.1610.8968.13596.38.874A3B20.394.9812.8<198.010.475A3B30.241.144.8<197.59.676A3B40.123.0425.3<197.79.877A3B50.124.0433.7<198.59.778A4B10.267.3228.22196.79.479A4B20.085.7271.5<196.99.480A4B30.073.4549.36695.78.681A4B40.117.5168.32796.59.182A4B50.048.50212.54597.18.883A5B11.119.938.97797.08.784A5B20.98>20.00>54.16396.68.885A5B31.11>20.00>76.917693.98.086A5B40.37>20.00>36.411994.78.387A5B50.26>20.00>23.014594.18.188A6B10.55>20.00>55.6<194.18.089A6B40.87>20.00>24.4<193.28.1aEC50for growth inhibition ofL. donovaniintracellular amastigotes infecting THP-1 macrophages (UCSD assay), average of two biological replicates;bEC50for cytotoxicity against host THP-1 macrophages (UCSD assay);cSI = selectivity index, calculated as SI = (L. donovaniinfection EC50)/(CC50);dkinetic aqueous solubility as determined by high-throughput CAD (charged aerosol detection);ehuman serum albumin binding.fObtained on the single enantiomer (S)-1;gPFI = ChromLogD7.4+ Aromatic rings

CONCLUSION

In this study, it was discovered a novel antileishmanial pyrazolopyrrolidinone chemotype that is effective against the intracellular amastigote parasite morphology in multipleLeishmaniaspecies with minimal host cytotoxicity. Compared to all of the advanced leads in the current antileishmanial pipeline, pyrazolopyrrolidinones are extremely facile to produce, without the need for sophisticated reaction apparatus in two synthetic steps from low-cost commodity starting materials—an ideal attribute for a therapeutic targeting a neglected tropical disease. Subsequent medicinal chemistry optimization has produced multiple advanced leads with significantly improved potency and ADME parameters relative to the initial hit, and support further preclinical optimization of the series. Work to advance these and similar candidates into in vivo pharmacokinetic and efficacy assessments is ongoing.

TABLE 10Comparative antileishmanial potencies for select compoundsin the UCSD vs. GSK infection assays. Pearson correlationr = .7429, n = 34, p < 0.10. A single outlier compound(70) did not demonstrate a measurable IC50value in theGSK LD AMMAC assay and was omitted from this analysis.UCSDL. donovaniGSK LD AMMACCompoundinfection IC50(μM)IC50(μM)(S)-10.40.79rac-10.82.521.13.2210.21.03910.415.9420.61.3440.30.6450.93.2461.75.0550.21.3670.20.5680.30.0690.30.4700.1>100710.41.0720.41.3730.23.2740.410.0750.20.5760.10.6770.10.1780.30.8790.10.0800.10.2810.10.1820.04.0831.14.0841.01.0851.17.9860.410.0870.30.8880.61.6890.91.6S34.06.3S103.410.0
Supplementary Methods: Chemical Synthesis
General Methods.

All1H NMR spectra were obtained at 400 or 500 MHz and referenced to the CHCl3singlet at 7.26 ppm, or the center peak of the quintet from the residual1H resonance of DMSO-d6at 2.50 ppm.13C NMR spectra were obtained at 125 or 100 MHz, and referenced to the center peak of the CDCl3triplet at 77.16 ppm, or the center peak of the DMSO-d6heptet at 39.52 ppm. Chemical shifts are reported in parts per million as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, h=heptet m=multiplet, br=broad), coupling constant, and integration. Optical rotations were recorded on a Rudolph AUTOPOL II digital polarimeter at 589 nm, and were reported as [α]D(concentration in grams/100 mL solvent). Analytical thin layer chromatography was performed using EMD 0.25 mm silica gel 60-F plates. Flash column chromatography was performed on Sorbent Technologies 60 Å silica gel. Chiral HPLC analysis was performed using an Agilent 1100 series HPLC with a multiple wavelength detector. Chiral columns include Chiralcel®OD (Chiral Technologies Inc., 25 cm×4.6 mm I.D.), Chiralpak®IA (Chiral Technologies Inc., 25 cm×4.6 mm I.D.). High resolution mass spectrometry data was obtained on a Waters Qtof (hybrid quadrupolar/time-of-flight) API US system by electrospray (ESI) in the positive mode. Mass correction was done by an external reference using a Waters Lockspray accessory. Mobile phases were water and acetonitrile with 0.1% formic acid. The MS settings were: capillary voltage=3 kV, cone voltage=35, source temperature=120° C. and desolvation temperature=350° C. UPLC-MS analysis was performed on a XBridge C18 column (1.7 mm, 2.1×50 mm) with CH3CN:H2O gradient as eluent with UV, ELSD and electrospray ionization (ESI) positive ion detection. Purity analysis was performed by HPLC and quantified by UV peak area at the indicated wavelength.

Detailed Synthetic Methods.

Methyl keto-enol esters (5) were prepared analogously to literature precedents (1) via the following representative procedure: An oven dried 250 mL round-bottom flask equipped with a stir bar was charged with methanol (75 mL) and cooled to 0° C. in an ice-water bath. Sodium metal (5.75 g, 250 mmol, 1.00 equiv.) was added portion-wise, and the flask was stirred and allowed to warm to room temperature. A separate flame-dried 100 mL flask was charged with dimethyl oxalate (29.5 g, 250 mmol, 1.00 equiv.) and 4-methylpentan-2-one (31.3 mL, 250 mmol, 1.00 equiv) and methanol (75 mL). After the sodium metal had completely dissolved, the contents of the 100 mL flask were transferred by cannula to the freshly prepared sodium methoxide solution. The 250 mL flask was fitted with an Ar balloon, and the reaction was stirred at 22° C. for 16 h, at which time the reaction was cooled to 0° C. in an ice-water bath, and quenched by the addition of 80 mL 4 M aq. sulfuric acid. The solution was poured into a 1 L separatory funnel, diluted with 250 mL water, and extracted 3×250 mL dichloromethane. The combined organics were dried over anhydrous Na2SO4, and concentrated by rotary evaporation. The crude oil was distilled under reduced pressure (8 mbar, 105° C.) to yield methyl (Z)-2-hydroxy-6-methyl-4-oxohept-2-enoate as a light yellow oil. Yield: 27.2 g, 59.5%.1H NMR: (400 MHz, CDCl3) δ 6.34 (s, 1H), 3.89 (s, 3H), 2.34 (d, J=7.1 Hz, 2H), 2.14 (dh, J=7.1, 6.6 Hz, 1H), 0.96 (d, J=6.6 Hz, 7H).

Pyrazolopyrrolidinones (1, 9-89) were synthesized on a ˜60 mg scale, as a modification of a literature precedent, according to the following representative procedure:

REFERENCES

Formulations of Pyrazolopyrrolidinones or Other Anti-Leishmanial Compounds Loaded into Biocompatible Expansile Particles (eNPs) for Systemic Delivery

Expansile particles (eNPs) that possess either a 2,4,6-trimethoxyaldene actetal or a benzaldehyde acetal protecting group (control). The specific monomer unit is (5-methyl-2-(2,4,6-trimethoxyphenyl)-1,3-dioxan-5-yl)methyl methacrylate, prepared from the 1,1,1-tris(hydroxymethyl)ethane acetal of the aldehyde reacted with methacryloyl chloride.Particle size and monodispersity controlled by varying the crosslinker, surfactant, and sonication parameters during preparation. For example, 1,4-O-methacryloylhydroquinone and 1,4-phenylene bis(2-methylacrylate) can be used as crosslinkers.Expansile particles are stable at neutral pH, but hydrolyze in the mildly acidic pH (˜5) of the cellular endosome whereLeishmaniaparasites are intracellularly compartmentalized. SeeFIG.16.Expansile particles have been demonstrated to show significant uptake in the liver (˜40%) and spleen (˜2%) following IV injection;Leishmaniaparasites predominantly infect monocytes in the reticulo-endothelial system.

Formulations of Pyrazolopyrrolidinones or Other Anti-Leishmanial Compounds Loaded into Biocompatible, Biodegradable Glycerol Carbonate Polymer and Polycaprolactone Particles for Systemic Delivery

Poly(glycerol carbonate) is prepared from the tin-catalyzed ring opening polymerization of various trimethylene carbonates substituted at the 5-position with hydroxyl, benzyl, ester, or other similar functionality. Poly(glycerol carbonate is additionally prepared through the copolymerization of CO2with various oxiranyl monomers. Additional copolymers are prepared with poly(glycerol carbonate) and polycaprolactone through a similarly catalyzed ring opening polymerization reaction.

Particles are formulated through water-in-oil ultrasonication methods and purified via dialysis. Particle size and monodispersity is controlled through sonication time, sonication pulse parameters, and polymer-to-surfactant ratio.Polymer nanoparticles of similar size and composition to the aforementioned systems have been shown to demonstrate significant uptake in the liver following IV injection;Leishmaniaparasites predominantly infect monocytes in the reticulo-endothelial system.

Formulations of Pyrazolopyrrolidinones or Other Anti-Leishmanial Compounds Loaded into Biocompatible, Biodegradable Glycidyl Carbonate Polymeric Adhesive for Topical Delivery

Poly(Alkyl Glycidate Carbonate)s are prepared from copolymerization of CO2and the various oxiranyl glycidyl monomers as well as those polymerized from a carbonate monomer. Monomer units include 1,2 and 1,3 polyglycerol carbonates. SeeFIG.17.

Another variation of this carbonate polymer hydrogel can be cross-linked hydrogels using PEG-diaziridine. SeeFIG.18.

Pyrazolopyrrolidinones or other antileishmanial compounds can be loaded into these hydrogels through solvent evaporation or similar methods and applied to cutaneous leishmaniasis skin lesions with a brush or as a patch. Additives, tackifiers, or plasticizers may be included to improve adhesive performance.

Electrospun, porous polymer meshes are generated as a durable and moisture permeable backing to the adhesive patch. These meshes are flexible and conformable to the site of intended application. Polymer or hydrogel adhesives are coated onto the backing through dip coating, extrusion, gravure, or other similar methods. SeeFIG.19.

Liners are included to protect the adhesive's integrity until the time of application. Liner composition may include silicone coated paper, polymer, or similar material.

In order to improve topical administration to CL lesions, development begun of an adhesive that can be formulated into a transdermal patch. The structure of the polymer, 1,3-poly(glycerol carbonate) (1,3-PGC), is promising as an adhesive in that it is biodegradable into natural metabolites, tunable in adhesive strength via pendant chain modifications, and structurally similar to poly(acrylate) adhesives. Proof-of-concept was demonstrated in drug-loading the adhesive through a solvent evaporation method in ethyl acetate with 5% glycerol. Including the glycerol as an additive slowed down the rate of solvent evaporation, ultimately yielding a more uniform adhesive disk.

New poly(glycerol carbonate) co-polymer nanoparticles were developed and used for the delivery of active agent to the liver in systemic VL infections. This polymer, 1,3-poly(glycerol carbonate)-C18-co-poly(ε-caprolactone) is formulated into nanoparticles via a oil-in-water nanoemulsion sonication method. This method was optimized to achieve particle sizes of 100-130 nm, which is ideal for cellular uptake and in vivo delivery to the liver. Drug release in physiologically relevant buffer demonstrates sustained release over three weeks.

For CL, the pendant chain character of the polymer was modified to achieve optimal adhesion to human skin. The adhesive layer was developed into an easy-to-apply patch using an electrospun poly(lactic acid) backing. Drug release from the adhesive will be optimized once fully formulated.

For VL a more comprehensive drug release study will be performed with more replicates over a longer duration of time to obtain a formulation with optimal release profile.