Hybrid amide derivatives of amphotericin B

Abstract

C16 amide derivatives of C2'-epi-amphotericin B (C2'epiAmB) and salts thereof are disclosed, characterized by improved clinical efficacy with reduced toxicity compared to AmB. Pharmaceutical compositions comprising any of the disclosed compounds or salts thereof, as well as methods of treatment and therapeutic uses thereof, are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 063,655, filed August 10, 2020, and U.S. Provisional Patent Application No. 63 / 175,771, filed April 16, 2021. GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant AI135812 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Morbidity and mortality from invasive fungal infections are significant and are primarily caused by two genera of fungal pathogens: Candida and Aspergillus. Candida species are the fourth most common pathogen in all bloodstream infections by themselves. Treatment of invasive candidiasis has limited success (50-70%), usually only in the healthiest patients. Mortality from invasive candidiasis is substantial (20-30%). The incidence of invasive aspergillosis due to A. fumigatus has increased threefold in the past decade, and its mortality rate has risen by more than 300%. Furthermore, current treatments for invasive aspergillosis have a low treatment success rate of 40-50%. Invasive aspergillosis is consistently the leading cause of death in immunocompromised patients. Furthermore, invasive mold infections (fusariosis, scedosporosis, and macromycosis) have even higher mortality rates and lack effective treatment options. The current guideline-recommended first-line treatment for invasive aspergillosis and most other invasive mold infections is the triazole antifungal voriconazole. However, pan-triazole resistance in Aspergillus is as high as 30% in some geographic regions and in certain high-risk patient populations. Recognizing this lack of effective treatments, the Infectious Diseases Society of America has highlighted A. fumigatus as one of only six pathogens for which "substantial breakthroughs are urgently needed."

[0004] Amphotericin B (AmB) is a very promising starting point because this drug has potent, dose-dependent bactericidal activity against a broad range of fungal pathogens and has evaded resistance for over half a century. The fungicidal, as opposed to fungistatic, activity of AmB is essential for immunocompromised patients who lack a strong immune system to help clear infections. Broad antifungal activity is particularly important in critically ill patients where the identity of the pathogen is unknown and immediate empirical treatment is required. An international expert committee recently mandated that a new therapeutic approach centered on AmB, without resistance issues, is needed. The problem is that AmB is so toxic that its use is limited to low-dose protocols that often fail to eradicate the disease.

[0005] A new, paradigm-shifting mechanistic understanding of AmB that eluded the field for half a century has been achieved. Previous studies have reported binding of AmB to sterols, which was thought to primarily promote the formation of membrane permeable pores to kill both fungal and human cells. After a decade of intensive synthesis-enabled atomistic investigations of this natural product and frontier SSNMR experiments, AmB was discovered to primarily kill both fungal and human cells by forming a cell-destructive extramembrane sterol sponge. This large aggregate resides at the surface of the lipid bilayer and rapidly extracts membrane sterols leading to cell death; without the need for membrane permeabilization. Based on this new mechanism and increasingly refined structural information, it is proposed that a small-molecule-based ligand-selective allosteric effect may enable selective binding of ergosterol over cholesterol. Guided by this model, elimination of cholesterol binding and therefore mammalian toxicity in the form of a new derivative, C2'epiAmB, has been achieved.

[0006] However, a limitation of C2'epiAmB is its relative lack of efficacy against many clinically relevant yeasts and molds. AmB derivatives that retain potent, broad-spectrum resistance-evading bactericidal activity but lack dose-limiting toxicity could enable new high-dose treatment paradigms with improved clinical efficacy. Summary of the Invention [Means for solving the problem]

[0007] In certain aspects, provided herein is a compound selected from the group consisting of: [ka] and [ka] Or a pharma- ceutically acceptable salt thereof.

[0008] In another aspect, the present invention provides a pharmaceutical composition comprising one of the disclosed compounds or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0009] In yet another aspect, provided herein is a method of treating a fungal infection, the method comprising administering a therapeutically effective amount of a disclosed compound, or a pharma- ceutically acceptable salt thereof, to a subject in need thereof, thereby treating the fungal infection.

[0010] In yet another aspect, provided herein is the use of a disclosed compound, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a fungal infection.

[0011] In certain aspects, provided herein are compounds and pharmaceutical compositions for use as medicines, and for use in treating fungal infections. [Brief description of the drawings]

[0012] [Figure 1A] 1 shows the results of an in vitro safety test using Compound 1. [Figure 1B] 1 shows the results of an in vitro safety test using Compound 1. [Diagram 2] 1 shows in vivo mouse pharmacokinetic data for Compound 1. [Diagram 3]1 shows serum concentrations of Compound 1 after high dose administration. [Figure 4A] Characterization data for compound 1 are shown below. 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1). [Figure 4B] Characterization data for compound 1 are shown below. 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1). [Figure 4C] Characterization data for compound 1 are shown below. 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1). [Figure 5A] Characterization data for compound 2 is shown. [Figure 5B] Characterization data for compound 2 is shown. [Figure 6] 1 shows the results of a killing kinetics assay using DMSO, AmB, EpiAmB (C2′epiAmB), and compound 1 with Candida albicans. [Figure 7] Figure 1 shows the difference in CFU of C.albicans in mice treated with different doses of Compound 1 compared to saline and AmB. *=p<0.05; ****=p<0.001. [Figure 8] Figure 1 shows the difference in CFU of C.albican in mice treated with different doses of compound 3 compared to saline and AmB. NS = not significant (p>0.05); **** = p<0.001. [Figure 9] Differences in CFU of invasive candidiasis in mice treated with different doses of compound 3 compared to saline and AmB. Direct Kruskal-Wallis test vs. vehicle_24h unless otherwise stated: NS = not significant (p>0.05); **p<0.01***p<0.005, ****p<0.001. [Figure 10] Figure 1 shows the difference in CFU of C. auris in mice treated with different doses of compound 3 compared to saline and AmB. NS=not significant (p>0.05); **=p<0.01; ***=p<0.005. [Figure 11]Figure 1 shows the difference in CFU of Aspergillus fumigatus in mice treated with different doses of compound 3 compared to saline and AmB. Direct Kruskal-Wallis test vs. vehicle_24h unless otherwise stated: NS = not significant (p>0.05); **p<0.01***p<0.005, ****p<0.001. [Figure 12A] 1 shows the plasma concentration of Compound 3 in mice. [Figure 12B] 1 shows the plasma concentration of Compound 3 in rats. [Figure 12C] 1 shows the plasma concentrations of Compound 3 in dogs. [Figure 12D] 1 shows plasma concentrations of Compound 3 in primates. [Figure 13] 1 shows Wajima overlay for predicting human concentration-time profile of compound 3. [Figure 14] The percent hemolysis for Compound 1 and Compound 3 is shown. [Figure 15A] 4 shows BUN levels following administration of Compound 3. [Figure 15B] 4 shows cholesterol levels after administration of Compound 3. [Figure 15C] 2 shows creatinine levels following administration of Compound 3. [Figure 16] 1 shows changes in gene expression following administration of Compound 3. [Figure 17A] Group mean daily body weights of mice inoculated with Aspergillus fumigatus and treated with saline, Compound 3, or AmB are shown. [Figure 17B] Group mean daily body weights compared to the day of infection for mice inoculated with Aspergillus fumigatus and treated with saline, Compound 3, or AmB are shown. [Figure 18A] Group mean daily body weights of mice inoculated with C. albicans and treated with saline, Compound 3, or AmB are shown. [Figure 18B] Group mean daily body weights compared to the day of infection for mice inoculated with C. albicans and treated with saline, compound 3, or AmB are shown. [Figure 19A] Characterization data for compound 3 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). [Figure 19B] Characterization data for compound 3 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). [Figure 19C] Characterization data for compound 3 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). [Figure 19D] Characterization data for compound 3 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). [Figure 20A] Figure 20 shows the results of an in vitro tolerance experiment using compound 3. Figure 20A: Neurotoxicity. [Figure 20B] Shown are the results of an in vitro tolerance experiment using compound 3. Figure 20B: Hepatotoxicity. [Figure 20C] Shown are the results of an in vitro tolerance experiment using compound 3. Figure 20C: Cardiotoxicity. [Figure 20D] Shown are the results of an in vitro tolerance experiment using compound 3. Figure 20D: Hematological toxicity. [Figure 21A] 1 shows an in vivo tolerance study of compound 3 in mice. [Figure 21B] 1 shows an in vivo tolerance study of compound 3 in mice. [Figure 21C] 1 shows an in vivo tolerance study of compound 3 in mice. [Figure 21D] 1 shows an in vivo tolerance study of compound 3 in mice. [Figure 21E] 1 shows an in vivo tolerance study of compound 3 in mice. [Figure 21F]1 shows an in vivo tolerance study of compound 3 in mice. [Figure 21G] 1 shows an in vivo tolerance study of compound 3 in mice. [Figure 22] 1 shows the in vivo efficacy of Compound 3 against pulmonary aspergillosis in mice. [Diagram 23] FIG. 1 shows the in vivo resistance of compound 3 to pulmonary aspergillosis in mice. [Figure 24A] Characterization data for compound 4 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). [Figure 24B] Characterization data for compound 4 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). [Figure 24C] Characterization data for compound 4 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). [Figure 24D] Characterization data for compound 4 is shown; 1H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Amphotericin B (AmB) is a polyene macrolide with a mycosamine appendage, the compound having the following structure: [ka]

[0014] AmB is generally obtained from strains of Streptomyces nodosus. It is currently approved for clinical use in the United States for the treatment of progressive and potentially life-threatening fungal infections, including, among others, systemic or deep tissue candidiasis, aspergillosis, cryptococcosis, blastomycosis, coccidioidomycosis, histoplasmosis, mucormycosis, and other infections. It is usually formulated for intravenous injection. Amphotericin B is commercially available, for example, as Fungizone® (Squibb), Amphocin® (Pfizer), Abelcet® (Enzon), and Ambisome® (Astellas). Due to its undesirable toxic side effects, the dosage is generally limited to a maximum of about 1.0 mg / kg / day, with a total cumulative dosage not to exceed about 3 g in humans.

[0015] AmB kills both fungal and human cells by forming a cytocidal extramembrane sterol sponge. Anderson, TM et al., Nat Chem Biol 2014, 10(5), 400-6. These large aggregates reside at the surface of the lipid bilayer and rapidly extract membrane sterols leading to cell death. No membrane permeabilization is required. Based on this mechanism, a small molecule-based ligand-selective allosteric effect allows selective binding of ergosterol over cholesterol, eliminating the mammalian toxicity of AmB (in the form C2'epiAmB). See Wilcock, BC et al., J Am Chem Soc 2013, 135(23), 8488-91. The present invention provides a novel method for determining the K of binding of both ergosterol and cholesterol to the AmB sterol sponge. D We disclose quantitative and mechanistically based biophysical parameters to guide the rational optimization of the therapeutic index of this clinically important natural product.

[0016] The present invention relates, at least in part, to the inventors' discovery of further derivatives of AmB that feature improved therapeutic indices compared to AmB. The various derivatives, i.e., the compounds of the present invention, can be semi-synthetic or fully synthetic. One aspect of the present invention is the development of novel synthetic derivatives of AmB that retain the strong binding of ergosterol but do not exhibit detectable binding of cholesterol. The derivatives retain bactericidal activity against many yeasts and molds, but have no detectable toxicity to mammals. This indicates that a different binding of ergosterol to cholesterol is possible, providing a non-toxic variant of AmB that retains the desirable antifungal properties. The compounds of the present invention enable new high-dose therapeutic strategies to eradicate life-threatening invasive fungal infections with significantly improved safety profiles.

[0017] The compounds of the present invention and pharmaceutical compositions of the present invention are useful for inhibiting fungal growth. In one embodiment, an effective amount of a compound of the present invention is contacted with a fungus, thereby inhibiting fungal growth. In one embodiment, the compound of the present invention or a pharma- ceutically acceptable salt thereof is added to or included in tissue culture media.

[0018] The compounds of the present invention and pharmaceutical compositions of the present invention are useful for treating a fungal infection in a subject. In one embodiment, a therapeutically effective amount of a compound of the present invention or a pharma- ceutical acceptable salt thereof is administered to a subject in need thereof, thereby treating a fungal infection.

[0019] Yeasts are eukaryotic organisms classified in the kingdom Fungi. Fungi include larger organisms including yeasts, molds, and mushrooms. Yeasts and molds are clinically relevant as infectious agents. Yeasts are usually described as budding forms of fungi. Of particular importance in the context of the present invention are yeast species that are capable of causing infections in mammalian hosts. Such infections occur most commonly in immunocompromised hosts, including those with compromised barriers to infection (such as burn victims) and those with compromised immune systems (such as those undergoing chemotherapy or immunosuppressive therapy, those infected with HIV, etc.). Pathogenic yeasts include, but are not limited to, various species of the genera Candida and Cryptococcus. Of particular note among pathogenic yeasts in the genus Candida are C. albicans, C. tropicalis, C. stellatoidea, C. glabrata, C. krusei, C. parapsilosis, C. guilliermondii, C. viswanathii, and C. lusitaniae. The genus Cryptococcus includes, inter alia, Cryptococcus neoformans. Yeasts can cause infections of mucous membranes, such as infections of the oral cavity, esophagus, and vagina in humans, as well as infections of bone, blood, urogenital tract, and central nervous system. This list is illustrative and in no way limiting.

[0020] Many fungi (except yeasts) can cause infections in mammalian hosts. Such infections occur most commonly in immunocompromised hosts, including those with compromised barriers to infection (such as burn victims) and those with compromised immune systems (such as those undergoing chemotherapy or immunosuppressive therapy, HIV-infected hosts). Pathogenic fungi (except yeasts) include, but are not limited to, species of Aspergillus, Rhizopus, Mucor, Histoplasma, Coccidioides, Blastomyces, Trichophyton, Microsporum, and Epidermophyton. Of particular note among the foregoing are A. fumigatus, A. flavus, A. niger, H. capsulatum, C. immitis, and B. dermatitidis. Fungi can cause systemic and deep tissue infections in the lungs, bones, blood, urogenital tract, and central nervous system, to name a few. Some fungi are responsible for skin and nail infections.

[0021] definition Definitions of specific functional groups and chemical terms are explained in more detail below. Chemical elements are listed in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., inside cover, and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in detail in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 thEdition, John Wiley &Sons, Inc., NewYork, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0022] The compounds described herein may contain one or more asymmetric centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be isolated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPFC) and the formation and crystallization of chiral salts, or preferred isomers may be prepared by asymmetric synthesis. See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962) and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.F. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0023] The present invention further includes the compounds described herein as individual isomers substantially free of other isomers, or alternatively, as mixtures of various isomers.

[0024] The following terms are intended to have the meanings indicated below and are helpful in understanding the description and intended scope of the present invention. When describing the present invention, which may include compounds, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions, the following terms, when present, have the following meanings unless otherwise indicated. It should also be understood that, as described herein, any of the moieties defined below may be substituted with various substituents, and each definition is intended to include such substituted moieties within the scope set forth below. Unless otherwise indicated, the term "substituted" is defined as set forth below. It should further be understood that the terms "group" and "radical" can be considered interchangeable when used herein. The articles "a" and "an" may be used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an analog" means one analog or more than one analog.

[0025] The term "pharmaceutical acceptable" refers to a substance that is approved or approvable by a regulatory authority of the federal or state government, or the equivalent in a country other than the United States, or that is listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals or, especially, in humans.

[0026] "Pharmaceutically acceptable salt" of a compound refers to a salt of a compound of the present invention that is pharma-ceutically acceptable and has the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and can be inorganic or organic acid addition salts and base addition salts. In particular, such salts include: (1) salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or salts formed with acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo- [2.2.2] Included are acid addition salts formed with organic acids such as oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is either replaced with a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinated to an organic base, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and when the compound contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like.

[0027] "Pharmaceutically acceptable cation" refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e.g., Berge, et al., J. Pharm. Sci. 66(1):1-79 (January 77)).

[0028] "Pharmaceutically acceptable vehicle" means a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered.

[0029] A "subject" to which administration is contemplated includes, but is not limited to, a human (i.e., male or female of any age, e.g., a pediatric subject (e.g., infant, child, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult)) and / or a non-human animal, e.g., a mammal such as a primate (e.g., cynomolgus monkey, rhesus monkey), cow, pig, horse, sheep, goat, rodent, cat, and / or dog. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms "human," "patient," and "control" are used interchangeably herein.

[0030] An "effective amount" refers to an amount of a compound that, when administered to a patient for treating or preventing a disease, is sufficient to have an effect on such treatment or prevention. An "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the patient being treated. A "therapeutically effective amount" refers to an amount effective for therapeutic treatment. A "prophylactically effective amount" refers to an amount effective for prophylactic treatment.

[0031] "Prevent" or "prevention" or "prophylactic treatment" refers to reducing the risk of suffering from or developing a disease or disorder (i.e., preventing at least one clinical symptom of the disease from developing in a patient who has not yet been exposed to a pathogenic agent or who is susceptible to the disease prior to the onset of the disease).

[0032] "Prophylaxis" is related to "prevention" and refers to a measure or procedure that aims to prevent, rather than treat or cure, a disease. Non-limiting examples of preventive measures may include administration of a vaccine; administration of low molecular weight heparin to hospitalized patients who are at risk of thrombosis due to reduced mobility; and administration of antimalarial drugs, such as cyclosporine and chloroquine, prior to visiting geographic areas where malaria is prevalent or there is an increased risk of contracting malaria.

[0033] "Treating" or "treatment" or "therapeutic treatment" of any disease or disorder, in one embodiment, refers to ameliorating the disease or disorder (i.e., arresting the disease or reducing the signs, extent, severity of at least one clinical symptom thereof). In other embodiments, "treating" or "treatment" refers to improving at least one physical parameter that is not discernible by the subject. In yet other embodiments, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physiological parameter), or both. In further embodiments, "treating" or "treatment" relates to slowing the progression of the disease.

[0034] As used herein, the term "isotopic variant" refers to a compound that contains an unnatural proportion of an isotope at one or more of the atoms that constitute such compound. For example, an "isotopic variant" of a compound can contain, for example, deuterium ( 2 H or D), carbon-13 ( 13 C), nitrogen-15( 15 In compounds where such isotopic substitutions are made, the following atoms may be altered, if present: any hydrogen, 2 H / D, any carbon 13 C or any nitrogen 15N, and the presence and configuration of such atoms can be determined within the skill of the art. Similarly, the present invention can include the preparation of isotopic variants using radioactive isotopes, for example, where the resulting compounds can be used in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H and carbon-14, i.e. 14 C are particularly useful for this purpose given their ease of incorporation and ready means of detection. 11 C. 18 F, 15 O and 13 The compounds may be substituted with positron emitting isotopes, such as N, which may be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. All isotopic variations of the compounds provided herein, whether radioactive or not, are intended to be encompassed within the scope of the invention.

[0035] "Tautomers" refer to compounds that are interchangeable forms of a particular compound structure, differing in the replacement of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the transfer of electrons and atoms (usually H). For example, enols and ketones are tautomers because they rapidly interconvert when treated with either acid or base. Another example of a tautomer is the acid and nitro forms of phenylnitromethane, which are similarly formed by treatment with acid or base. Tautomers can be relevant to achieving optimal chemical reactivity and biological activity of a compound of interest.

[0036] As used herein, a pure enantiomer compound is substantially free of the other enantiomer or stereoisomer of the compound (i.e., enantiomeric excess). In other words, the "S" form of the compound is substantially free of the "R" form of the compound and is therefore in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" means that the compound contains more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight, more than 99.9% by weight of the enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0037] As used herein, unless otherwise indicated, the term "enantiomerically pure R compounds" refers to at least about 95% by weight R compounds and up to about 5% by weight S compounds, at least about 99% by weight R compounds and up to about 1% by weight S compounds, or at least about 99.9% by weight R compounds and up to about 0.1% by weight S compounds. In certain embodiments, the weights are based on the total weight of the compounds.

[0038] As used herein, unless otherwise specified, the term "enantiomerically pure S compounds" or "S compounds" refers to at least about 95% by weight S compounds and up to about 5% by weight R compounds, at least about 99% by weight S compounds and up to about 1% by weight R compounds, or at least about 99.9% by weight S compounds and up to about 0.1% by weight R compounds. In certain embodiments, the weights are based on the total weight of the compounds.

[0039] In the compositions provided herein, the enantiomerically pure compound or its pharma- ceutically acceptable salt can be present together with other active or inactive ingredients. For example, a pharmaceutical composition comprising an enantiomerically pure R compound can comprise, for example, about 90% of excipients and about 10% of the enantiomerically pure R compound. In certain embodiments, the enantiomerically pure R compound in such a composition can comprise, for example, at least about 95% by weight of the R compound and up to about 5% by weight of the S compound, based on the total weight of the compound. For example, a pharmaceutical composition comprising an enantiomerically pure S compound can comprise, for example, about 90% of excipients and about 10% of the enantiomerically pure S compound. In certain embodiments, the enantiomerically pure S compound in such a composition can comprise, for example, at least about 95% by weight of the S compound and up to about 5% by weight of the R compound, based on the total weight of the compound. In certain embodiments, the active ingredient can be formulated with little or no excipients or carriers.

[0040] Compounds of the Invention In certain aspects, provided herein is a compound selected from the group consisting of: [ka] and [ka] Or a pharma- ceutically acceptable salt thereof.

[0041] In certain embodiments, the compound is [ka] Or a pharma- ceutically acceptable salt thereof.

[0042] In another embodiment, the compound is [ka] It is.

[0043] In a further embodiment, the compound is [ka] It is.

[0044] In yet other embodiments, the compound is [ka] Or a pharma- ceutically acceptable salt thereof.

[0045] In still yet other embodiments, the compound is [ka]

[0046] In a further embodiment, the compound is [ka] It is.

[0047] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is an intravenous dosage form. In other embodiments, the pharmaceutical composition is an oral dosage form.

[0048] In yet another aspect, provided herein is a method of treating a fungal infection, comprising administering a therapeutically effective amount of a disclosed compound or a pharma- ceutically acceptable salt thereof to a subject in need thereof, thereby treating the fungal infection. In certain embodiments, the compound is administered intravenously. In other embodiments, the compound is administered orally.

[0049] In yet another aspect, provided herein is the use of a disclosed compound, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a fungal infection.

[0050] In certain aspects, provided herein are compounds and pharma- ceutically acceptable salts thereof for use as pharmaceuticals, and for use in the treatment of fungal infections.

[0051] Pharmaceutical Compositions The invention also provides pharmaceutical compositions of the disclosed compounds or salts thereof, and methods for preparing such compositions.

[0052] One aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention and a pharma- ceutically acceptable carrier. In a particular embodiment, the present invention is a pharmaceutical composition comprising a compound of the present invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. The term "pharma- ceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic component with which an active component is combined to facilitate application. The components of the pharmaceutical composition can also be mixed with the compound of the present invention and with each other such that there is no interaction that would substantially impair the desired medicinal effect.

[0053] In certain embodiments, the pharmaceutical composition is in an intravenous dosage form.

[0054] In certain embodiments, the pharmaceutical composition is an oral dosage form.

[0055] In certain embodiments, the pharmaceutical composition is a lyophilized preparation of the liposome-intercalated or liposome-encapsulated active compound.

[0056] In certain embodiments, the pharmaceutical composition is a lipid complex of the compound in aqueous suspension.

[0057] The foregoing embodiments of pharmaceutical compositions of the present invention are meant to be illustrative and not limiting.

[0058] Also provided is a process for preparing such a pharmaceutical composition, the process comprising combining a compound of the invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0059] Methods of the Invention The compounds of the present invention are useful for inhibiting the growth of fungi and yeasts, particularly including fungi and yeasts that are clinically important as pathogens. Advantageously, the compounds of the present invention have an improved therapeutic index compared to AmB, thereby providing a drug with improved efficacy and reduced toxicity compared to AmB. The compounds of the present invention are useful in methods for treating fungal and yeast infections, particularly including systemic fungal and yeast infections. The compounds of the present invention are also useful in the manufacture of medicaments for treating fungal and yeast infections, particularly including systemic fungal and yeast infections. The present invention further provides the use of the compounds of the present invention for the treatment of fungal and yeast infections, particularly including systemic fungal and yeast infections.

[0060] An aspect of the invention is a method of treating a fungal infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention, or a pharma-ceutically acceptable salt thereof, thereby treating the fungal infection.

[0061] As used herein, "inhibit" or "inhibiting" refers to a decrease in an objectively measurable amount or extent compared to a control. In one embodiment, inhibit or inhibiting refers to a decrease by at least a statistically significant amount compared to a control. In one embodiment, inhibit or inhibiting refers to a decrease of at least 5 percent compared to a control. In various individual embodiments, inhibit or inhibiting refers to a decrease of at least 10, 15, 20, 25, 30, 33, 40, 50, 60, 67, 70, 75, 80, 90, or 95 percent (%) compared to a control.

[0062] As used herein, the terms "treatment" and "treating" refer to (a) preventing a condition or disease from occurring in a subject at risk of developing the condition or disease but not yet diagnosed as having it, (b) inhibiting the condition or disease, e.g., slowing or stopping its onset, or (c) alleviating or ameliorating the condition or disease, e.g., causing regression of the condition or disease. In one embodiment, the terms "treating" and "treatment" refer to (a) inhibiting the condition or disease, e.g., slowing or stopping its onset, or (b) alleviating or ameliorating the condition or disease, e.g., causing regression of the condition or disease. In one embodiment, the terms "treating" and "treatment" refer to (a) inhibiting the fungal infection, e.g., slowing or stopping its onset, or (b) alleviating or ameliorating the fungal infection, e.g., causing regression of the fungal infection.

[0063] As used herein, "fungal infection" refers to an infection in or of a subject with a fungus as defined herein. In one embodiment, the term "fungal infection" includes a yeast infection. As used herein, "yeast infection" refers to an infection in or of a subject with a yeast as defined herein.

[0064] As used herein, the term "subject" refers to a living mammal. In various embodiments, the subject is, but is not limited to, a mouse, a rat, a hamster, a guinea pig, a rabbit, a sheep, a goat, a cat, a dog, a pig, a horse, a cow, or a non-human primate. In one embodiment, the subject is a human.

[0065] As used herein, "subject suffering from fungal infection" refers to a subject that shows at least one objective symptom of fungal infection. In one embodiment, a subject suffering from fungal infection is a subject that has been diagnosed as suffering from fungal infection and is in need of treatment. Methods for diagnosing fungal infection are well known and do not need to be described in detail here.

[0066] As used herein, "subject suffering from yeast infection" refers to a subject exhibiting at least one objective symptom of yeast infection. In one embodiment, a subject suffering from yeast infection is a subject diagnosed as suffering from yeast infection and in need of treatment. Methods for diagnosing yeast infection are well known and need not be described in detail here.

[0067] In certain embodiments, the compound is administered intravenously.

[0068] In certain embodiments, the compound is administered orally.

[0069] In certain embodiments, the compound is administered systemically.

[0070] In certain embodiments, the compound is administered parenterally.

[0071] In certain embodiments, the compound is administered intraperitoneally.

[0072] In certain embodiments, the compound is administered enterally.

[0073] In certain embodiments, the compound is administered intraocularly.

[0074] In certain embodiments, the compound is administered topically.

[0075] Additional routes of administration of the compounds of the present invention are contemplated by the present invention, including, but not limited to, intravesical (bladder), pulmonary, and intrathecal.

[0076] As used herein, the term "effective amount" means any amount sufficient to achieve a desired biological effect.

[0077] As used herein, the term "therapeutically effective amount" means an amount sufficient to achieve the desired therapeutic effect, e.g., to treat a fungal or yeast infection.

[0078] For any compound described herein, a therapeutically effective amount can generally be initially determined from in vitro studies, animal models, or both in vitro studies and animal models. In vitro methods are well known and include methods such as the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MFC), the concentration at which growth is inhibited by 50% (IC 50 ), and the concentration at which proliferation is inhibited by 90% (IC 90 ) and the like. Therapeutically effective amounts can also be determined from human data of compounds of the invention tested in humans and compounds known to exhibit similar pharmacological activity, such as other related active agents (e.g., AmB). Higher doses may be required for parenteral administration. The applied doses can be adjusted based on the relative bioavailability and potency of the administered compounds. Adjusting the dose to achieve maximum efficacy based on the methods described herein and other methods known to those of skill in the art is well within the capabilities of one of skill in the art.

[0079] For any compound described herein, the therapeutically effective amount for use in human subjects can be initially determined from in vitro studies, animal models, or both in vitro studies and animal models. The therapeutically effective amount for use in human subjects can also be determined from human data of the compounds of the present invention that have been tested in humans, and of compounds known to exhibit similar pharmacological activity, such as other related active agents (e.g., AmB). Higher doses may be required for parenteral administration. The applied doses can be adjusted based on the relative bioavailability and potency of the compounds administered. Adjusting the dose to achieve maximum efficacy based on the methods described above and other methods known to those skilled in the art is well within the capabilities of those skilled in the art.

[0080] Dosage and Formulation The compound of the present invention can be combined with other therapeutic agents. The compound of the present invention and other therapeutic agents can be administered simultaneously or sequentially. When other therapeutic agents are administered simultaneously, they can be administered in the same or separate formulations, but they are administered substantially simultaneously. When the administration of other therapeutic agents and the compound of the present invention is separated in time, the other therapeutic agents are administered sequentially with each other and with the compound of the present invention. The interval between the administration of these compounds can be several minutes or longer.

[0081] Examples of other therapeutic agents include other antifungal agents, including AmB, as well as other antibiotics, antivirals, anti-inflammatory agents, immunosuppressants, and anti-cancer agents.

[0082] As stated above, the term "effective amount" means any amount sufficient to achieve a desired biological effect. By selecting among various active compounds and weighting factors such as potency, relative bioavailability, patient weight, severity of adverse side effects and preferred mode of administration, effective prevention or treatment, in combination with the teachings provided herein, one can design a treatment regimen that is effective to treat a particular subject without causing substantial undesired toxicity. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular compound of the invention being administered, the size of the subject, or the severity of the disease or condition. Those of skill in the art can empirically determine the effective amount of a particular compound of the invention and / or other therapeutic agent without necessitating undue experimentation. In general, it is preferred to use a maximum dose, i.e., the highest safe dose according to any medical judgment. Multiple administrations per day may be contemplated to achieve an appropriate systemic level of the compound. An appropriate systemic level may be determined, for example, by measuring the patient's peak or sustained plasma levels of the drug. "Dose" and "administration" are used interchangeably herein.

[0083] In general, the daily oral dosage of the active compound is about 0.01 milligrams / kg per day to 1000 milligrams / kg per day for human subjects. Oral dosages ranging from 0.5 to 50 milligrams / kg are expected to produce the desired results with one or several administrations per day. The dosage may be appropriately adjusted to achieve the desired drug levels locally or systemically, depending on the mode of administration. For example, intravenous administration is expected to be one to several orders of magnitude lower per day. If the response in the subject is insufficient at such dosages, higher dosages (or higher effective dosages by another, more localized delivery route) may be used, as long as the patient's tolerance allows. Multiple administrations per day may be contemplated to achieve adequate systemic levels of the compound.

[0084] In one embodiment, intravenous administration of the compound of the present invention can typically be 0.1 mg / kg / day to 20 mg / kg / day. Thus, intravenous administration can be similar to or advantageously exceed the maximum tolerated dose of AmB. Intravenous administration can also be similar to or advantageously exceed the maximum tolerated daily dose of AmB. Intravenous administration can also be similar to or advantageously exceed the maximum tolerated cumulative dose of AmB.

[0085] Intravenous administration may also be similar to or advantageously exceed the maximum recommended dose of AmB. Intravenous administration may also be similar to or advantageously exceed the maximum recommended daily dose of AmB. Intravenous administration may also be similar to or advantageously exceed the maximum recommended cumulative dose of AmB.

[0086] For any compound described herein, the therapeutically effective amount can be determined first from an animal model. The therapeutically effective amount can also be determined from human data of the compounds of the present invention tested in humans and compounds known to exhibit similar pharmacological activity, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximum efficacy based on the above methods and other methods known to those skilled in the art is well within the capabilities of those skilled in the art.

[0087] The formulations of the present invention will be administered in pharma- ceutically acceptable solutions which may ordinarily contain pharma- ceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0088] Amphotericin B is commercially available in many formulations, including deoxycholate-based (sometimes referred to as deoxycholate-based) and lipid-based (including liposomal) formulations. The amphotericin B derivative compounds of the present invention can be similarly formulated, for example, but not limited to, as deoxycholate-based and lipid-based (including liposomal) formulations.

[0089] For use in treatment, an effective amount of the compound of the present invention can be administered to a subject by any method that delivers the compound of the present invention to the desired surface.The administration of the pharmaceutical composition of the present invention can be achieved by any means known to those skilled in the art.Routes of administration include, but are not limited to, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, direct injection (e.g., into tumor or abscess), mucosal, pulmonary (e.g., inhalation), and topical.

[0090] For intravenous and other parenteral administration routes, the compounds of the present invention can generally be formulated in the same manner as AmB.For example, the compounds of the present invention can be formulated as lyophilized preparations with deoxycholic acid, as lyophilized preparations of liposome-inserted or encapsulated active compounds, as lipid complexes in aqueous suspension, or as cholesteryl sulfate complexes.Lyophilized preparations are generally reconstituted with suitable aqueous solutions, such as sterile water or saline, immediately before administration.

[0091] For oral administration, the compounds (i.e., the compounds of the present invention and other therapeutic agents) can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like for oral ingestion by the subject. Formulations for oral use can be obtained as solid excipients, optionally, after adding suitable auxiliary agents, if desired, the resulting mixture is milled, and the granular mixture is processed to obtain tablets or dragee cores. Suitable excipients are, in particular, sugars such as lactose, sucrose, mannitol, or sorbitol; for example, corn starch, wheat starch, rice starch, or potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or cellulose preparations such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, e.g., sodium alginate. Optionally, oral preparations can be formulated with saline or buffers, e.g., EDTA for neutralizing acidic conditions in the body, or can be administered without a carrier.

[0092] Oral dosage forms of the one or more components are also specifically contemplated. One or more components can be chemically modified so that oral delivery of the derivative is effective. In general, the contemplated chemical modification is the attachment of at least one moiety to the component molecule itself, said moiety allowing for (a) acid hydrolysis and (b) uptake into the bloodstream from the stomach or intestine. Also desired is an increase in the overall stability of the one or more components and an increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts", In:Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383 (1981); Newmark et al., J Appl Biochem 4: 185-9 (1982). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane.Preferred for pharmaceutical applications are the polyethylene glycol moieties, as indicated above.

[0093] The location of release of the component (or derivative) may be the stomach, the small intestine (the duodenum, jejunum, or ileum), or the large intestine. Those skilled in the art have available formulations that will not dissolve in the stomach but will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the compound (or derivative) of the invention or by release of the bioactive material beyond the stomach environment, such as the intestine.

[0094] To ensure full gastric resistance, a coating that is impermeable to at least pH 5.0 is essential. Examples of the more common inactive ingredients used as enteric coatings include cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

[0095] A coating or mixture of coatings may also be used on tablets that are not intended for protection against the stomach. This may include sugar coatings, or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutics (e.g., powders), and for liquids, a soft gelatin shell may be used. The shell material for cachets may be thick starch or other edible paper. For pills, troches, molded tablets, or tablet triturates, wet mashing techniques may be used.

[0096] The therapeutic agent may be included in the formulation as fine multiparticulates in the form of granules or pellets with a particle size of about 1 mm. Formulations of the material for capsule administration may also be as a powder, lightly compressed plugs, or tablets. The therapeutic agent may be prepared by compression.

[0097] Colorants and flavoring agents may all be included. For example, the compounds (or derivatives) of the present invention may be formulated (such as by liposome or microsphere encapsulation) and then further included within an edible product, such as a refrigerated beverage, that contains colorants and flavoring agents.

[0098] The amount of therapeutic agent can be diluted or increased with inert substances. These diluents can include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, and starch. Certain inorganic salts can also be used as fillers, including calcium triphosphate, magnesium carbonate, and sodium chloride. Commercially available diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress, and Avicell.

[0099] Disintegrants may be included in the formulation of therapeutic agents into solid dosage forms. Materials used as disintegrants include, but are not limited to, starch, including the commercially available disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge and bentonite can all be used. Another form of disintegrant is the insoluble cation exchange resin. Powdered gums can be used as disintegrants and binders, and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0100] Binders can be used to hold the therapeutic agent together to form a hard tablet and can include materials from natural sources such as acacia, tragacanth, starch, and gelatin. Others include methylcellulose (MC), ethylcellulose (EC), carboxymethylcellulose (CMC), etc. Both polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can be used in alcoholic solutions to granulate the therapeutic agent.

[0101] Antifriction agents can be included in the formulation of the therapeutic agent to prevent sticking during the formulation process. Lubricants can be used as a layer between the therapeutic agent and the mold wall, and these can include, but are not limited to, stearic acid with magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000 can also be used.

[0102] Glidants may be added which may improve the flow properties of the drug in the formulation and aid in rearrangement during compression. Glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

[0103] Surfactants may be added as wetting agents to aid in dissolving the therapeutic agent in the aqueous environment. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents may be used and include benzalkonium chloride and benzethonium chloride. Potential nonionic surfactants that may be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants may be present alone or as a mixture in different ratios in the formulation of the compound or derivative of the present invention.

[0104] Preparation of pharmaceutical preparations that can be used orally include tablets, push-fit capsules made of gelatin, and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient in a mixture with an injecting agent such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are clearly described in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0105] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0106] For administration by inhalation, the compound for use according to the present invention can be conveniently delivered in the form of aerosol spray from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.For the pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0107] Pulmonary delivery of the compounds of the invention (or derivatives thereof) is also contemplated herein. The compounds of the invention (or derivatives) are delivered to the lungs of a mammal during inhalation and traverse the lung epithelial lining to the bloodstream. Other reports of inhaled molecules include Adjei et al., Pharm Res7:565-569(1990); Adjei et al., Int J Pharmaceutics63:135-144(1990)(leuprolide acetate); Braquet et al., J Cardiovasc. Pharmacol13(suppl.5):143-146(1989)(endothelin-1);Hubbard et al.,Annal Int Med3:206-212(1989)(α1-antitrypsin);Smith et al.,1989,J Clin Invest84:1145-1146(a-1-proteinase);Oswein et al. al., 1990, “Aerosolization of Proteins”, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., US Pat. No. 5,284,656 (granulocyte colony stimulating factor). Methods and compositions for pulmonary delivery of drugs for systemic effect are described in U.S. Patent No. 5,451,569, issued September 19, 1995 to Wong et al.

[0108] Contemplated for use in the practice of the present invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers, and dry powder inhalers, all of which are well known to those skilled in the art.

[0109] Some specific examples of commercially available devices suitable for the practice of the invention include the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina, and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

[0110] All such devices require the use of a formulation suitable for dispensing the compound (or derivative) of the present invention. Typically, each formulation is specific to the type of device used and may involve the use of a suitable propellant in addition to the usual diluents, adjuvants and / or carriers useful in therapy. Also contemplated is the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers. The chemically modified compounds of the present invention may also be prepared in different formulations depending on the type of chemical modification or the type of device used.

[0111] Formulations suitable for use in either jet or ultrasonic nebulizers typically contain a compound (or derivative) of the invention dissolved in water at a concentration of about 0.1-25 mg of bioactive compound of the invention per mL of solution. The formulation may also contain a buffer and a simple sugar (e.g., for stabilization of the compound of the invention and regulation of osmotic pressure). Nebulizer formulations may also contain a surfactant to reduce or prevent surface-induced aggregation of the compound of the invention caused by atomization of the solution in forming the aerosol.

[0112] Formulations for use in metered dose inhaler devices will generally comprise a fine powder containing the compound (or derivative) of the present invention suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soybean lecithin. Oleic acid is also useful as a surfactant.

[0113] Formulations for dispensing from powder inhaler devices comprise a finely divided dry powder containing the compound (or derivative) of the invention and may also contain a bulking agent such as lactose, sorbitol, sucrose, or mannitol in an amount to facilitate dispersion of the powder from the device, e.g., 50-90% by weight of the formulation. The compound (or derivative) of the invention should advantageously be prepared in particulate form with a mean particle size of less than 10 micrometers (μm), most preferably 0.5-5 μm, for most effective delivery to the deep lung.

[0114] Nasal delivery of the pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the pharmaceutical composition of the present invention to pass into the bloodstream immediately after administration of the therapeutic product to the nose, without the need for the product to be deposited in the lungs. Formulations for nasal delivery include those containing dextran or cyclodextran.

[0115] For nasal administration, a small, hard bottle with a metered sprayer attached is a convenient device.In one embodiment, a metered dose is delivered by drawing a solution of the pharmaceutical composition of the present invention into a chamber of a specified volume, and the chamber has an opening that is dimensioned to aerosolize and aerosolize by forming a spray when the liquid in the chamber is compressed.The chamber is compressed to administer the pharmaceutical composition of the present invention.In a particular embodiment, the chamber is a piston arrangement.Such devices are commercially available.

[0116] Alternatively, a plastic squeeze bottle is used with an aperture or opening dimensioned to aerosolize the aerosol formulation by forming a spray when squeezed. The opening is usually at the top of the bottle, which is generally tapered to partially fit into the nasal cavity for efficient administration of the aerosol formulation. Preferably, the nasal inhaler provides a metered amount of the aerosol formulation to administer a measured dose of the drug.

[0117] Compounds can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion, when it is desired to deliver them systemically.Injectable preparations can be provided, for example, in ampoules or multi-dose containers with added preservatives.Compounds can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0118] Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compound in water-soluble form.Furthermore, suspensions of the active compound can be prepared as suitable oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound, allowing the preparation of highly concentrated solutions.

[0119] Alternatively, the active compound may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

[0120] The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0121] In addition to the formulations described above, the compounds can be formulated as depot preparations. Such long-acting compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0122] The pharmaceutical compositions may also include suitable solid- or gel-phase carriers or excipients, examples of which include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0123] Suitable liquid or solid pharmaceutical formulation forms are, for example, aqueous solutions or saline solutions for inhalation, microencapsulation, encapsulation, coating on fine gold particles, inclusion in liposomes, nebulization, aerosolization, pellets for skin implantation, or drying on sharp objects for skin wounding. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations that release active compounds in a sustained manner, in which excipients and additives and / or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers are conventionally used as described above. Pharmaceutical compositions are suitable for use in various drug delivery systems. For a brief review of drug delivery methods, see Langer R, Science 249:1527-33 (1990), which is incorporated herein by reference.

[0124] The compounds of the present invention and optionally other therapeutic agents may be administered per se (neat) or in the form of a pharma- ceutically acceptable salt. When used in medicine, the salt must be pharma- ceutically acceptable, but pharma-ceutically unacceptable salts may conveniently be used to prepare their pharma- ceutically acceptable salts. Such salts include, but are not limited to, those prepared from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts may also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts of the carboxylic acid group.

[0125] Suitable buffering agents include acetic acid and salts (1-2% w / v), citric acid and salts (1-3% w / v), boric acid and salts (0.5-2.5% w / v), and phosphoric acid and salts (0.8-2% w / v).Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v), chlorobutanol (0.3-0.9% w / v), parabens (0.01-0.25% w / v), and thimerosal (0.004-0.02% w / v).

[0126] Pharmaceutical compositions of the present invention comprise an effective amount of a compound of the present invention and optionally at least one additional therapeutic agent in a pharma- ceutically acceptable carrier.

[0127] Specifically, therapeutic agents, including but not limited to the compounds of the present invention, may be provided in particles. Particles, as used herein, refer to nanoparticles or microparticles (or possibly larger particles) that may consist in whole or in part of the compounds of the present invention or other therapeutic agent(s) described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including but not limited to an enteric coating. The therapeutic agent(s) may also be dispersed throughout the particle. The therapeutic agent(s) may also be adsorbed to the particle. The particles may be of any order of release kinetics, including zero-order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof. The particles may contain, in addition to the therapeutic agent(s), any of the materials routinely used in the pharmaceutical and medical fields, including but not limited to erodible, non-erodible, biodegradable, or non-biodegradable materials, or combinations thereof. The particles may be microcapsules that contain the compounds of the present invention in solution or in a semi-solid state. The particles may be of virtually any shape.

[0128] In the manufacture of particles for delivering therapeutic agent(s), both non-biodegradable and biodegradable polymeric materials can be used. Such polymers can be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include the biodegradable hydrogels described in Sawhney HS et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acid, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginic acid, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0129] The therapeutic agent(s) may be included in a controlled release system. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation is controlled. It refers to immediate and non-immediate release formulations, including non-immediate release formulations, including, but not limited to, sustained release formulations and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides sustained release of drug over an extended period of time, and preferably, but not necessarily, results in substantially constant blood drug levels over an extended period of time. The term "delayed release" is used in its conventional sense to refer to a formulation in which there is a time delay between administration of the formulation and the release of drug therefrom. "Delayed release" may or may not include gradual release of drug over an extended period of time, and therefore may or may not be "sustained release".

[0130] The use of long-term sustained release implants may be particularly suitable for treating chronic diseases. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic concentrations of the active ingredient for at least 7 days, and preferably 30 to 60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

[0131] Incorporation by Reference All U.S. patent application publications and U.S. patents mentioned herein are herein incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0132] Other embodiments In the claims, articles such as "a," "an," and "the" may mean one or more, unless specifically indicated to the contrary or otherwise clear from the context. A claim or description containing "or" between one or more elements of a group is considered to be satisfied if one, more than one, or all of the group elements are present in, employed in, or otherwise relevant to a given product or process, unless the contrary is indicated or otherwise clear from the context. The invention includes embodiments in which exactly one element of a group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group elements are present in, employed in, or otherwise relevant to a given product or process.

[0133] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim may be amended to include one or more limitations found in any other claim that is dependent on the same base claim. When elements are presented as a list, e.g., in Markush group format, each subgroup of elements is also disclosed, and any element(s) may be removed from the group. In general, when the invention, or aspects of the invention, are described as including certain elements and / or features, etc., it should be understood that the particular embodiment of the invention or aspect of the invention consists of or consists essentially of such elements and / or features. For simplicity, these embodiments have not been specifically described verbatim herein. It should be noted that the terms "comprise" and "contain" are open-ended and are intended to permit the inclusion of additional elements or steps. When ranges are given, the endpoints are included. Further, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges take any particular value or subrange within the range of conditions in different embodiments of the invention, to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0134] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any inconsistency between the incorporated references and this specification, this specification shall control. In addition, any particular embodiment of the present invention within the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed known to those skilled in the art. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0135] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. It will be apparent to those skilled in the art that various changes and modifications can be made to this description without departing from the spirit or scope of the invention, as defined in the following claims. EXAMPLES

[0136] In order that the invention described herein may be fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the compounds, pharmaceutical compositions, and methods provided herein, and should not be construed as limiting the scope thereof in any way.

[0137] Example 1. Synthetic Procedures and HPLC Methods for the Disclosed Compounds General synthetic procedure for amides of AmB: [ka] Freshly distilled Et 3N was added dropwise to a solution of amphotericin B (10 mg; 0.01 mmol) and amine (3 equiv.) in DMF (500 μL) until pH=9 (by pH paper) was reached. The reaction mixture was stirred at room temperature for 15 min. Solid PyBOP (1.5 equiv.; 8.4 mg) was added under nitrogen atmosphere and the sealed vial was stirred overnight at room temperature. The progress of the reaction was monitored by analytical HPLC trace.

[0138] Upon completion, the product was precipitated and washed with anhydrous diethyl ether (10 mL). The suspension was centrifuged at 3000 g for 5 min. The solvent was decanted and the pellet was dissolved in DMSO and filtered through a 0.2 micron syringe filter for purification on a C18Prep HPLC system. The pure product was dried in a lyophilizer as a yellowish powder and stored at -80°C under nitrogen atmosphere.

[0139] This method is suitable for making the disclosed amides of C2'-epi-AmB starting from C2'-epi-AmB.

[0140] HPLC method: JPEG0007656359000013.jpg84164JPEG0007656359000014.jpg86164

[0141] Synthesis of compound 1 [ka] New Et 3 N (direct from the bottle) (12 μL) was added to C2'epiAmB (10 mg; 0.01 mmol) and 2-amino-1,3-propanediol (3 eq; 3 mg) in DMF (400 μL). The reaction mixture was stirred at room temperature for 15 min. Solid PyBOP (1.5 eq; 8.4 mg) was added to the mixture and the sealed vial was stirred at room temperature for 2 h. The progress of the reaction was monitored by analytical HPLC trace.

[0142] Upon completion, the reaction was diluted with DMSO to form a clear solution and filtered through a 0.2 micron syringe filter before being purified on a C18Prep HPLC system. The pure product was dried in a lyophilizer as a yellowish powder and stored at -80°C under inert atmosphere. Yield: 5.6mg (52%) Product moles, weight: 997.1860

[0143] Synthesis of compound 3 from compound 1 [ka] To a clean, oven-dried, 40-mL glass vial containing 50 mg (X mg) of HPLC-purified compound 1, add 2.5 mL (0.05X mL) of Milli-Q water, followed by 200 mL (4X mL) of 0.5 (M) acetic acid. After the acid has been added, the solution will begin to become clear. Vortex the solution as needed to completely dissolve the solute. After sonicating the solution for 1-2 minutes at room temperature, add clean stir beads to the solution and stir at 800 rpm for 15 minutes. Check for any residue stuck to the top of the glass vial. After stirring is complete, visually inspect the solution for any signs of undissolved parts or suspension. Then remove the stir beads from the solution, freeze them in liquid nitrogen, and place in a freeze dryer to dry overnight.

[0144] Synthesis of compound 4 [ka] To a clean, oven-dried, 40-mL glass vial containing 50 mg (X mg) of HPLC-purified compound A, add 2.5 mL (0.05X mL) of Milli-Q water, followed by 200 mL (4X mL) of 0.5 (M) acetic acid. After the acid has been added, the solution will begin to become clear. Vortex the solution as needed to completely dissolve the solute. After sonicating the solution at room temperature for 1-2 minutes, add clean stirring beads to the solution and stir at 800 rpm for 15 minutes. Check for any residue stuck to the top of the glass vial. After stirring is complete, visually inspect the solution for any signs of undissolved parts or suspension. Then remove the stirring beads from the solution, freeze them in liquid nitrogen, and place in a freeze dryer to dry overnight.

[0145] Example 2. Characterization data for the disclosed compounds compound 1 [ka] The NMR spectrum of compound 1 is shown in FIG. 4C. LCMS: [M+H] + =998.1935. The chromatograph and mass spectrum of compound 1 are shown in Figure 4A and Figure 4B.

[0146] Synthesis of compound 4 [ka] To a clean, oven-dried, 40-mL glass vial containing 50 mg (X mg) of HPLC-purified compound A, add 2.5 mL (0.05X mL) of Milli-Q water, followed by 200 mL (4X mL) of 0.5 (M) acetic acid. After the acid has been added, the solution will begin to become clear. Vortex the solution as needed to completely dissolve the solute. After sonicating the solution at room temperature for 1-2 minutes, add clean stirring beads to the solution and stir at 800 rpm for 15 minutes. Check for any residue stuck to the top of the glass vial. After stirring is complete, visually inspect the solution for any signs of undissolved parts or suspension. Then remove the stirring beads from the solution, freeze them in liquid nitrogen, and place in a freeze dryer to dry overnight.

[0147] Example 2. Characterization data for the disclosed compounds compound 1 [ka] The NMR spectrum of compound 1 is shown in FIG. 4C. LCMS: [M+H] + =998.1935. The chromatograph and mass spectrum of compound 1 are shown in Figure 4A and Figure 4B.

[0148] compound 2 [ka] LCMS: [M+H] + =1028.2195. The chromatograph and mass spectrum of compound 2 are shown in Figure 5A and Figure 5B.

[0149] compound 3 [ka] Compound 3 1 The HNMR spectrum is shown in Figure 19A. Compound 3 13 The C NMR spectrum is shown in Figure 19B. LCMS: [M+H] + =998.0192. The chromatograph and mass spectrum of compound 3 are shown in Figures 19C and 19D.

[0150] compound 4 [ka] Compound 3 1 The HNMR spectrum is shown in Figure 24A. Compound 3 13 The C NMR spectrum is shown in Figure 24B. LCMS: [M+H] + =997.4949. The chromatograph and mass spectrum of compound 4 are shown in Figures 24C and 24D.

[0151] Example 3. The disclosed compounds exhibit comparable efficacy and DMPK to AmB, but with improved safety. [Table 1]

[0152] Example 4. Antifungal efficacy of the disclosed compounds and AmB Fungal or mold colonies from SDA plates were suspended in RPMI medium at an inoculation density of 10 5 CFU / mL. A 990 μL aliquot of the diluted cell suspension was added to a sterile 1.7 mL Eppendorf tube, followed by 10 μL of a 400 μM solution of compound (in DMSO). The DMSO concentration in each Eppendorf tube was 1%, and a control sample was also run to check viability using only 1% DMSO. At predetermined time points (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 24 hours), 10 μL samples were removed from each tube and serially diluted 10-fold in RPMI, and 10 μL aliquots were plated onto SDA plates to determine colony counts. If colony counts were expected to be less than 1,000 CFU / mL, a 50 μL aliquot was taken directly from the test solution and plated undiluted onto SDA plates. Plates were incubated at 37°C for 24 hours before testing. All experiments were performed in duplicate.

[0153] [Table 2-1] [Table 2-2]

[0154] [Table 2A]

[0155] [Table 3]

[0156] [Table 3A]

[0157] [Table 4]

[0158] Example 5. In vitro and in vivo safety of the disclosed compounds UV-Vis binding assay (Figure 1A): The protocol for sterol binding assay (UV-Vis) was developed in our laboratory. Compounds were dissolved in DMSO at a final concentration of 1 mM. Sterols were first dissolved in CHCl3 (>200 mM) and then diluted to 1 mM concentration in DMSO. To synthesize the complexes, 1 ul of compound solution was taken in a clean Eppendorf tube (2 ml) and sterol solution (volume depends on stoichiometry) was added to it and the volume was made up to 20 ul with DMSO. 0.98 ml of PBS buffer was added to the Eppendorf tube and mixed properly. The absorbance of the solution was measured after 30 min of incubation.

[0159] MHC (Figure 1B and Figure 10): The protocol for the hemolysis assay was adapted from the report by Paquet and coworkers (Chem. Eur. J. 2008, 14, 2465-2481). Whole human blood (sodium heparin) was purchased from Bioreclamation LLC (Westbury, NY), stored at 4 °C, and used within 2 days of receipt. 1 mL of whole human blood was added to a 2.0 mL Appendorff tube and centrifuged at 10,000 g for 2 min. The supernatant was removed and the red blood cell pellet was washed with 1 mL of sterile saline and centrifuged at 10,000 g for 2 min. The saline wash was repeated a total of three times. The red blood cell pellet was suspended in 1 mL of RBC buffer (10 mM NaH2PO4, 150 mM NaCl, 1 mM MgCl2, pH 7.4) to form a red blood cell stock suspension.

[0160] Compounds were prepared as stock solutions at >15 mM in DMSO and serially diluted in DMSO to the following concentrations: 7689, 5126, 2563, 2050, 1538, 1025, 769, 513, 384, 256, 205, 154, 103, 77, 51, 26 μM. 24 μL of RBC buffer and 1 μL of compound stock solution were added to 0.2 mL PCR tubes to give final concentrations of 500, 300, 200, 100, 80, 60, 40, 30, 20, 15, 10, 8, 6, 4, 3, 2, 1 μM. Positive and negative controls were prepared in 0.2 mL PCR tubes by adding 1 μL of DMSO to MilliQ water or RBC buffer, respectively. 0.63 μL of red blood cell stock suspension was added to each PCR tube and mixed by inversion. Samples were incubated at 37°C for 2 hours. Samples were mixed by inversion and centrifuged at 10,000g for 2 minutes. 15 μL of supernatant from each sample was added to a 384-well plate. Absorbance was read at 540 nm using a Biotek H1 Synergy Hybrid Reader (Winooski, VT). Experiments were performed in triplicate and reported MHCs represent the average of three experiments.

[0161] Example 6. In vivo mouse pharmacokinetic studies of the disclosed compounds Mouse PK (Figure 2): Experiments were performed with compounds synthesized in the laboratory and purified by preparative HPLC (>91%). All compounds were dissolved in D5W (5% dextrose in water) for IV injection. Female CD-1 mice (3 per group, each weighing approximately 30 g) were injected with compounds (according to the planned dose), blood samples were collected at different time points, and compound content was analyzed using the following procedure: Instrument: TripleQuad6500+ Matrix: Male CD-1 mouse plasma (EDTA-K2) Analyte: Compound 1 Internal standard: 100ng / mL labetalol & 100ng / mL tolbutamide in ACN MS conditions ESI: positive SRM detection Compound 1:[M+H]+m / z979.8>798.4 Labelatol(IS):[M+H]+m / z329.2>162.1 UPLC conditions Mobile phase A: 0.1% FA in water Mobile phase B: 0.1% FA in ACN Time (min) Mobile phase B (%) 1.10 98 1.50 98 1.51 15 2.10 Stop Column: Waters ACQUITY UPLC HSST 3 1.8 μm 2.1 × 50 mm Flow rate: 0.6000mL / min Retention time: Labelatol (IS): 0.998 minutes Sample preparation: An aliquot of 24 μL of sample was protein precipitated with 120 μL of IS solution (100 ng / mL labetalol & 100 ng / mL tolbutamide in ACN), the mixture was vortexed well and centrifuged at 3900 rpm for 10 min at 4°C. An aliquot of 90 μL of supernatant was transferred to a sample plate and mixed with 60 μL of water, then the plate was shaken at 800 rpm for 10 min. 15.0 μL of supernatant was injected for LC-MS / MS analysis. Calibration curve: Compound 1 in female CD-1 mouse plasma (EDTA-K2) at 1.00-3000 ng / mL

[0162] Additional mouse PK experiments (Figure 3): After 24 hours, blood samples were collected from mice treated with compounds for in vivo toxicity experiments (see above). To 50 μL of mouse serum, 300 μL of HPLC grade methanol was added. The mixture was mixed well by vortexing for 30 seconds. The mixture was allowed to stand at room temperature for 0.5 hours to allow complete extraction of AmB (or derivatives) and precipitation of proteins. The mixture was then centrifuged at 16000g for 10 minutes and the unfiltered supernatant was used for HPLC analysis. (Note - AmB was calculated from a previously established standard curve for the concentration of AmB (or derivatives) binding to the prefilter.

[0163] The standard curve was generated as follows. 1: A series of different concentrations (10, 20, 40, 80100, 200, 400, 1000 μM) in methanol, measured by UV-vis, extinction coefficient at 406 nm: 164 mM -1 ) to prepare a solution of AmB (or derivative). 2: Inject 10 μL of the standard solution into the HPLC and create a standard curve using the area under the curve and its corresponding concentration.

[0164] HPLC method: C18SiO2 column: Sunfire column, 5μM, 15cm. Aqueous buffer (A): 0.1% formic acid filtered in MQ water Organic solvent (B): filtered HPLC grade acetonitrile Flow rate: 1 mL / min, DAD: 406.4 nm, Ionization method: AJSESI, positive mode Gradient change: 95:5 (0.1% formic acid in water:MeCN) to 5:95 (0.1% formic acid in water:MeCN) in 8 min, gradient change time schedule is as follows: 0 min 95%A, 5%B 0.5 min 95%A, 5%B 8.5 minutes 5%A, 95%B 9.5 minutes 5%A, 95%B 10.5 minutes 95%A, 5%B

[0165] Example 7. Solubility of the disclosed compounds Test substance 1: Synthesized in the laboratory (purity >95%) D5W: BraunUSA (product number: L5101) Instrument details: Sonicator: Branson Ultrasonics 2800; Vortex-Genie 2 Lab Mixer UV-Vis:Thermo Fisher NanodroponeC procedure:* Take 6 mg of sample (measured by UV-Vis) in a clean, oven-dried 7 mL vial Add 1.65 mL of sterile D5W (at room temperature) Vortex for 2 minutes Water bath sonication: 2 min x 2 Repeat steps 3 and 4 until the solution is clear. Transfer to a 2mL Eppendorf tube and centrifuge (3000gX2min) to completely dissolve the compound and ensure that there are no insoluble parts (optional step). Concentration measured by UV (absolute value at 406 nm) (any step) *Amounts are based on solutions prepared for in vivo toxicity studies at a dose of 50 mg / kg

[0166] [Table 5]

[0167] Example 8. Killing kinetics assay with Candida albicans: (Figure 6) The protocol for the kill kinetics assay was adapted from the CLSI protocol for MIC determination. From the backstock, fungal colonies were suspended in 0.9% sterile saline and diluted to an inoculum density of approximately 103. Aliquots of 10ul were plated onto Sabouraud Dextrose Agar (SDA) plates using an L spreader and incubated at 35°C for 24-48 hours, and colony growth was monitored. During the experiment, 2-3 single colonies were suspended in 10mL RPMI1640 medium (prepared according to the CLSI protocol) and cell density was measured using a hemocytometer. The inoculum density was then adjusted to approximately 105 cfu / mL. In a sterile Eppendorf tube (1.7mL-2mL), a 990µL inoculum aliquot was mixed with 10µL of a 100X solution of the compound (the solution was prepared in DMSO, and the DMSO content of each test solution is 1%). Control samples were prepared to confirm viability using only 1% DMSO in RPMI1640 medium. At the designated time points (0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8, 10, 16, and 24 h), 10 μL samples were removed from each tube, serially diluted 10-fold in RPMI1640 medium, and 10 μL aliquots were plated onto SDA plates to determine colony counts. If colony counts were expected to be less than 1,000 CFU / mL, 50 μL aliquots were taken, diluted in half with RPMI1640 medium, and 50 μL were plated onto SDA plates. Plates were incubated at 35 °C for 24–48 h before testing. All experiments were performed in triplicate.

[0168] Example 9. In vivo efficacy in a mouse candidiasis model (C. albicans SN250): (Figures 7 and 8) FIG. 7: Female CD-1 mice (average body weight = 27 g) were divided into groups of four based on different subject numbers and different doses.

[0169] Prior to treatment, all groups were inoculated via the tail vein with C.albicans SN250 (approximately 105 CFU / mL). Two hours after inoculation, the groups were treated with a single-dose IV formulation of the test article (in saline). After 24 hours of incubation, the animals were euthanized and the kidneys were harvested and immediately homogenized. To measure the fungal load, serum 24 hours after treatment was plated and the colony density (CFU / mL) was calculated. In this experiment, AmBisome and C2'epiAmB were used as controls, and the nipple article compound 1 was administered at a dose of 5 mg / kg as the control.

[0170] Figure 8: To test the efficacy of high doses of compound 1, we developed an acetate version of the drug which was found to be highly soluble in aqueous solutions, specifically D5W and saline vehicles. Therefore, we went ahead and repeated the aforementioned in vivo study, testing the efficacy of drug candidate compound 3 at four different single doses of 1.5 mg / kg, 5 mg / kg, 15 mg / kg and 45 mg / kg. Following the experiment, the fungal load was measured using the procedure described above. In this study, we were able to show that high dose eradication of fungal infections is possible using the non-toxic compound 3.

[0171] Example 10. In vivo efficacy in a mouse candidiasis model (C. albicans SC5314): (Figure 9) In this study, the efficacy and tolerability of compound 3 in a multiple-dose regimen was investigated against C.albicans SC5314 in male ICR mice. Three different doses of compound 3 were tested, 1 mg / kg, 5 mg / kg, and 15 mg / kg, administered once daily for 7 days. Body weights were also recorded daily. Two different doses of Ambisome were used as controls. Each group consisted of 6 mice. Before treatment, all groups were inoculated with C.albicans SC5314 via the tail vein (approximately 1.9x105 CFU / mL). Five hours after inoculation, the groups received their first IV dose (in D5W). After completing the 7-day treatment, all mice were sacrificed and the fungal burden in the kidneys was measured using plating techniques. The weights of the boys are shown in Figures 22A and 22B.

[0172] Example 11. In vivo efficacy in a mouse candidiasis model (C. auris): (Figure 10) Female CD-1 mice (average body weight = 27 g) were divided into groups of four based on different subject numbers and different doses.

[0173] Prior to treatment, all groups were inoculated with C. auris via the tail vein (approximately 105 CFU / mL). Two hours after inoculation, the groups received the first IP dose of compound 3 (in saline). Over the next four days, multiple doses were administered at 24, 48, and 72 hours after inoculation. Four days after treatment, all animals were euthanized, kidneys were harvested, and immediately homogenized. To measure fungal load, serum 24 hours after treatment was plated and colony density (CFU / mL) was calculated. In this experiment, two different doses of AmBisome were used as controls, and the test article compound 3 was administered at doses of 1.5, 5, 15, and 45 mg / kg.

[0174] Example 12. In vivo efficacy in a mouse aspergillosis model: (Figure 11) In this study, the efficacy and tolerance of compound 3 in a multiple-dose regimen was investigated against Aspergillus fumigatus 1163 in male ICR mice. Two different doses of compound 3, 5 mg / kg and 15 mg / kg, were tested, administered once daily for four days. Body weights were also recorded daily. Ambisome (10 mg / kg) was used as a control in this experiment. Each group consisted of six mice. Three days prior to the study, all mice were treated with cyclophosphamide (200 mg / kg) as an immunosuppressant. On the day of the study, all groups were inoculated with Aspergillus fumigatus 1163 via the tail vein (approximately 2.9x105 CFU / mL). Five hours after inoculation, the groups were given their first IV dose (in D5W). After the completion of the four-day treatment, all mice were sacrificed and the fungal burden in the kidneys was measured using the plating technique. Body weights are listed on slide 17.

[0175] Example 13. Wajima Overlay: (Figures 12 and 13) Prediction of human DMPK using Wajima superposition was performed using standard protocols.

[0176] [Table 6]

[0177] [Table 7]

[0178] [Table 8] *Antimicrobial Agents Chemother.2002,46(3):828-833 **Dose proportionality assumed

[0179] [Table 9]

[0180] Example 14. In vivo toxicity in mouse models: (Figures 15 and 16) In this study, the expression of nephrotoxicity biomarker genes was used to study the in vivo toxicity of compound 3. Female CD-1 mice (average body weight = 27 g) were divided into groups of four mice each based on different subject numbers and different doses. On the day of the experiment, different doses of compound 3 were administered intravenously. Ambisome and C2'epiAmB treated groups were used as the reference. After 24 hours, all mice were sacrificed and kidney fungal burdens were collected and stored in RNAlater at -80°C. Then, kidneys were homogenized and RNA was extracted. Expression of KIM1, LCN2, TIMP1, and SPP1 genes was measured relative to GAPDH using RT-PCR technique.

[0181] [Table 10]

[0182] Example 15. Tolerance of Compound 3 and changes in renal biomarkers and gene expression following administration of Compounds 1 or 3 (Figures 11A-11C and 12) hERG inhibition, plasma compatibility, and Ames assays were performed using industry standard protocols.

[0183] hERG inhibition studies: Did not inhibit hERG potassium currents up to 100 uM, the highest concentration tested.

[0184] Plasma compatibility: ppts observed at all three concentrations at 1:1 dilution. Observed only at 5mg / mL at 1:0.1 dilution. Formulation optimization ongoing.

[0185] Ames test: No genotoxic responses were observed at any dose level tested

[0186] Example 16. In vitro resistance of compound 3 (Figures 20A-20D) Procedure: Four different cell lines were obtained from ATCC and the resistance of compound 3 was tested together with AmB, C2'epiAmB and compound 4 according to the ATCC recommendations.

[0187] Neurovirulence: SH-SY5Y (CRL-2266; human neuroblastoma; Fig. 20A). Cells were grown in complete MEM medium and resistance was tested in triplicate 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The inoculation density was 10 4 Cells / well (5×10 5 All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was maintained at 1%. After compound addition to the cultures, the cells were incubated at 5% CO 2 After incubation at 35° C. under atmospheric conditions for 24 hours, cell viability was measured using Alamar blue fluorescent dye (excitation 555 nm and emission 585 nm).

[0188] Hepatotoxicity: Hep-G2 (HB-8065; human hepatocytes; FIG. 20B). Cells were grown in complete MEM medium and resistance was tested in triplicate 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The inoculation density was 10 4 Cells / well (5×10 5 All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was maintained at 1%. After compound addition to the cultures, the cells were incubated at 5% CO 2 After incubation at 35° C. under atmospheric conditions for 24 hours, cell viability was measured using Alamar blue fluorescent dye (excitation 555 nm and emission 585 nm).

[0189] Cardiotoxicity: H9C2 (CRL-1446; rat cardiomyocytes; FIG. 20C). Cells were grown in complete DMEM medium and resistance was tested in triplicate 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The inoculation density was 10 4 Cells / well (5×10 5 All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was maintained at 1%. After compound addition to the cultures, the cells were incubated at 5% CO 2 After incubation at 35° C. under atmospheric conditions for 24 hours, cell viability was measured using Alamar blue fluorescent dye (excitation 555 nm and emission 585 nm).

[0190] Hematotoxicity: K562 (CCL-243; human lymphoblasts; Fig. 20D). Cells were grown in complete IMDM medium and resistance was tested in triplicate 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The inoculation density was 10 5 Cells / well (5×10 6 All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was maintained at 1%. After compound addition to the cultures, the cells were incubated at 5% CO 2After incubation at 35° C. under atmospheric conditions for 24 hours, cell viability was measured using Alamar blue fluorescent dye (excitation 555 nm and emission 585 nm).

[0191] Example 17. In vivo tolerance of compound 3 in mice (Figures 21A-21G) Healthy female CD-1 mice were divided into groups of 4 mice. Test compounds were dissolved in sterile, clinically approved D5W (5% dextrose) and injected via the tail vein during the study. Compound concentrations were adjusted accordingly to obtain the target dose in a 0.3 mL injection. After 24 h of incubation, animals were euthanized, blood samples were taken for quantitative analysis of clinical specimens, and organs were harvested for histopathological analysis.

[0192] [Table 11]

[0193] 0=histologic unremarkable; 1=minimal; 2=mild; 3=moderate; 4=severe; 5=severe D=diffuse; F=focal; M=multifocal; P=current [Table 12] [Table 13] [Table 14] [Table 15]

[0194] Example 18: In vitro efficacy of compound 3 against azole- and echinocandin-resistant strains The experiments were performed according to standard CLSI protocols. [Table 16]

[0195] Example 19: Protocol development for efficacy study of Compound 3 against pulmonary aspergillosis in mice (Figures 22 and 23).

[0196] Efficacy study (Figure 22): Healthy mice were divided into 10 mice / group. All animals were treated with cyclophosphamide (150 mg / kg) on ​​days -2 and +3 and cortisone acetate (250 mg / kg) on ​​days -1 and +6 to convert to neutropenia. Animals were infected with A. fumigatus (CEA10 strain) (intranasally) on day 0. All animals were administered Ambisome or Compound 3 at 15 mg / kg / day (IP) for 7 days and monitored until day 14 post-infection. Survival of the animals was monitored over the course of the study. Objective: Testing compound 3 and AmB against CEA10 (wild type) IA 1e8 spores / mL (40uL) intranasal CEA10 n=10 mice / group Cyclophosphamide: 150mg / kg on days -2 and +3 Cortisone acetate: 250mg / kg on day -1 and +6 Survival rate: D5W only: 10% Compound 315mg / kgQD: 80% Amino Acid 15mg / kg QD: 80% Log-rank statistics: D5W vs Compound 315mg / kgQD:p=0.0005 D5W vs AmB 15 mg / kg QD: p=0.0044 Points to note: Compound 3 and AmB IP at 15 mg / kg given daily for 7 days post-infection are equally protective against CEA10IA infection after 14 days. Amb IP dose of 15 mg / kg is estimated to be at least 5 mg / kg IV. Because IP dosing of Compound 3 and AmB is new, we would like to understand the tolerability of both in immunosuppressed mouse models.

[0197] Tolerance study (Figure 23): Healthy mice were divided into groups of 3 mice. All animals were converted to neutropenia with cyclophosphamide (150 mg / kg) on ​​days -2 and +3 and cortisone acetate (250 mg / kg) on ​​days -1 and +6. Target doses of Compound 3 and Ambisome were administered IP daily for 7 days and survival was monitored for 14 days. Objective: Tolerance of Compound 3 and AmB-dosed IP in immunodeficiency models No infections, only tolerability tests n=3 mice / group Cyclophosphamide: 150mg / kg on days -2 and +3 Cortisone acetate: 250 mg / kg daily -1 and +6 Each drug was administered IP for 7 days. The mice were observed for 14 days. Survival rate: Compound 315mg / kgQD:66% Compound 325mg / kgQD:100% Compound 335mg / kgQD:100% Compound 345mg / kgQD:66% Amino Acid 25mg / kg QD: 66% Points to note: Clinically, Compound 3 at 45 mg / kg and AmBIP at 25 mg / kg showed similar signs of lethargy and rough fur starting on D3-D12. Compound 3 at 25-35 mg / kg did not affect the mice's ability to feed, keep clean, and activity throughout the experiment, but 45 mg / kg did. Compound 3 administered IP at 35 mg / kg for 7 days can be used in the infection model. Finally, preferred embodiments of the present invention are described in sections. [Embodiment 1] [ka] or a pharma- ceutically acceptable salt thereof. [Embodiment 2] [ka] or a pharma- ceutically acceptable salt thereof. [Embodiment 3]

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Claims

【Request 1】 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof. 【Request 2】 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof. 【Request 3】 【Chemistry 3】 2. The compound of claim 1, 【Request 4】 【Chemical 4】 2. The compound of claim 1, 【Request 5】 【Chemistry 5】 or a pharma- ceutically acceptable salt thereof. 【Request 6】 【Chemical 6】 2. The compound of claim 1, 【Request 7】 【Chemical 7】 2. The compound of claim 1,

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

9. The pharmaceutical composition of claim 8 in an intravenous dosage form.

10. The pharmaceutical composition of claim 8 in an oral dosage form.

11. A composition for treating a fungal infection comprising a compound according to any one of claims 1 to 7.

12. The composition of claim 11 , which is administered intravenously.

13. The composition of claim 11 , which is administered orally.

14. 10. Use of a compound according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment of a fungal infection.

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