Hybrid amide derivatives of amphotericin B

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 063,655 filed on August 10, 2020 and U.S. Provisional Patent Application No. 63 / 175,771 filed on April 16, 2021.

[0003] Government support

[0004] This invention was made with government support under Grant No. AI 135812 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the Invention

[0006] The morbidity and mortality of invasive fungal infections are significant and are mainly caused by two genera of fungal pathogens: Candida and Aspergillus. Candida species are the 4th most common pathogens isolated in all bloodstream infections. Treatment for invasive candidiasis has limited success rates (50%-70%), and this is usually only in the healthiest patients. The attributable mortality rate of invasive candidiasis is significant (20%-30%). The incidence of invasive aspergillosis caused by Aspergillus fumigatus has increased threefold in the past decade, and its mortality rate has increased by more than 300%. In addition, the current treatment for invasive aspergillosis has a low (40%-50%) treatment success rate. Invasive aspergillosis has always been the main killer of immunodeficient patients. In addition, invasive mold infections (fusarium, sedosporium and mucormycosis) have even higher mortality rates and no effective treatment options. Current guideline-recommended first-line treatment for invasive aspergillosis, as well as most other invasive mold infections, is the triazole antifungal voriconazole. However, resistance to pantriazoles among Aspergillus species is as high as 30% in some regions and certain high-risk patient groups. 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.”

[0007] Amphotericin B (AmB) is a very promising starting point because this drug has potent and dose-dependent fungicidal activity against a broad range of fungal pathogens and resistance has not emerged in more than half a century. In contrast to its fungistatic activity, the fungicidal activity of AmB is critical for immunocompromised patients who lack a robust immune system to help clear infection. Broad antifungal activity is particularly important in critically ill patients when the pathogen identity is unknown and immediate empirical therapy is required. An international panel of experts recently authorized the need for new treatment approaches centered around AmB that do not have resistance issues. The problem is that AmB is extremely toxic, which limits its use to low-dose regimens that generally fail to eradicate the disease.

[0008] For half a century, the field has not achieved a paradigm-shifting mechanistic understanding of AmB. Previous studies reported the binding of AmB to sterols, which was thought to primarily drive the formation of membrane permeabilization pores to kill both fungi and human cells. After 10 years of intensive synthesis, atomic interrogation of this natural product was enabled, and cutting-edge SSNMR experiments were performed, and it was also found that AmB kills both fungi and human cells primarily by forming a cell-killing extracellular sterol sponge. This large aggregate sits on the surface of the lipid bilayer and rapidly extracts membrane sterols, leading to cell death. No membrane permeabilization is required. Based on this new mechanism and increasingly refined structural information, it has been proposed that small molecule-based ligand-selective allosteric effects can be superior to cholesterol in selectively binding to ergosterol. Guided by the model, cholesterol binding was eliminated in the form of a new derivative, C2'epiAmB, and elimination of mammalian toxicity was achieved.

[0009] However, one limitation of C2'epiAmB is its relative lack of potency against many clinically relevant yeasts and molds. An AmB derivative that retains potent, broad-spectrum, and resistance-evading fungicidal activity but lacks dose-limiting toxicity would enable new high-dose treatment paradigms and improve clinical efficacy. SUMMARY OF THE INVENTION

[0011] In certain aspects, provided herein is a compound selected from the group consisting of:

[0012]

[0013] or a pharmaceutically acceptable salt thereof.

[0014] In other aspects, provided herein is a pharmaceutical composition comprising one of the disclosed compounds or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0015] In yet other aspects, provided herein are methods of treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof, thereby treating the fungal infection.

[0016] In still other aspects, provided herein is the use of a disclosed compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a fungal infection.

[0017] In certain aspects, provided herein are compounds and pharmaceutical compositions for use as medicaments and for treating fungal infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A-1B Results of in vitro safety studies using Compound 1 are depicted.

[0020] Figure 2 In vivo mouse pharmacokinetic data for Compound 1 are depicted.

[0021] Figure 3 Serum concentrations of Compound 1 after high-dose administration are depicted.

[0022] Figures 4A-4C Characterization data of compound 1 are depicted; 1 H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1).

[0023] Figure 5A-5B Characterization data for Compound 2 are depicted.

[0024] Figure 6 Depicted are the results of a kinetic killing assay against Candida albican using DMSO, AmB, EpiAmB (C2'epiAmB), and Compound 1.

[0025] Figure 7 Depicted are the differences 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.

[0026] Figure 8 Depicted are the differences in CFU of C. albicans in mice treated with different doses of Compound 3 compared to saline and AmB; NS = not significant (p>0.05); **** = p<0.001.

[0027] Fig. 9Depicted are the differences in CFU of invasive candidiasis in mice treated with different doses of compound 3 compared to saline and AmB; Kruskal-Wallis test performed directly versus vehicle at 24h unless otherwise stated: NS = not significant (p>0.05); **p<0.01 ***p<0.005, ****p<0.001.

[0028] Fig.10 Depicted are the differences in C. auris CFU 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.

[0029] Fig.11 Depicted are the differences in A. fumigatus CFU of mice treated with different doses of Compound 3 compared to saline and AmB; Kruskal-Wallis test performed directly versus vehicle at 24 h unless otherwise stated: NS = not significant (p>0.05); **p<0.01 ***p<0.005, ****p<0.001.

[0030] Fig. 12A Plasma concentrations of compound 3 in mice are depicted.

[0031] Fig. 12B Plasma concentrations of Compound 3 in rats are depicted.

[0032] Fig. 12C Plasma concentrations of Compound 3 in dogs are depicted.

[0033] Fig.12D Plasma concentrations of Compound 3 in primates are depicted.

[0034] Fig.13 Wajima superposition was depicted to predict the human concentration-time profile of compound 3.

[0035] Fig.14 The percentage of hemolysis of Compound 1 and Compound 3 is depicted.

[0036] Fig.15A BUN levels after administration of Compound 3 are depicted.

[0037] Fig. 15B Cholesterol levels after administration of Compound 3 are depicted.

[0038] Fig. 15C Creatinine levels after administration of Compound 3 are depicted.

[0039] Fig.16 Gene expression changes after administration of Compound 3 are depicted.

[0040] Fig.17A Depicted are group mean daily body weights of mice inoculated with A. fumigatus and treated with saline, Compound 3, or AmB.

[0041] Fig. 17B Depicted are group mean daily body weights of mice inoculated with A. fumigatus and treated with saline, Compound 3, or AmB relative to the day of infection.

[0042] Fig.18A Depicted are group mean daily body weights of mice inoculated with C. albicans and treated with saline, Compound 3, or AmB.

[0043] Fig.18B Depicted are group mean daily body weights of mice inoculated with C. albicans and treated with saline, Compound 3, or AmB relative to the day of infection.

[0044] Figures 19A-19D depicts the characterization data of compound 3; 1 H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13 C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1).

[0045] Figures 20A-20D The results of in vitro tolerability studies using compound 3 are depicted. Fig. 20A : Neurotoxicity; Fig. 20B : Hepatotoxicity; Fig. 20C : Cardiotoxicity; and Fig.20D :Hematotoxicity.

[0046] Figures 21A-21G Depicted is an in vivo tolerability study of compound 3 in mice.

[0047] Fig. 22 The in vivo efficacy of compound 3 against pulmonary aspergillosis in mice is depicted.

[0048] Fig.23 The in vivo tolerability of compound 3 against pulmonary aspergillosis in mice is depicted.

[0049] Figures 24A-24D depicts the characterization data of compound 4; 1 H NMR parameters: 500 MHz, pyridine-d5:CD3OD (1:1); 13 C NMR parameters: 126 MHz, pyridine-d5:CD3OD (1:1). DETAILED DESCRIPTION OF THE INVENTION

[0051] Amphotericin B (AmB) is a polyene macrolide with a trehalose amine appendage having the following structure:

[0052]

[0053] AmB is typically obtained from strains of Streptomyces nodosus. AmB is currently approved for clinical use in the United States for the treatment of progressive, potentially life-threatening fungal infections, including infections such as systemic or deep tissue candidiasis, aspergillosis, cryptococcosis, blastomycosis, coccidioidomycosis, histoplasmosis, and mucormycosis. AmB is typically formulated for intravenous injection. Amphotericin B is commercially available, for example, as (Squibb), (Pfizer), (Enzon) and (Astellas) is commercially available. Due to its adverse toxic side effects, the dosage is usually limited to a maximum of about 1.0 mg / kg / day, and the total cumulative dose in humans does not exceed about 3 g.

[0054] AmB kills fungi and human cells by forming a cell-killing extracellular sterol sponge. Anderson, TM et al., Nat Chem Biol 2014, 10 (5), 400-6. This large aggregate is located on the surface of the lipid bilayer and rapidly extracts membrane sterols, leading to cell death. No membrane permeabilization is required. Based on this mechanism, the selective allosteric effect of small molecule ligands will allow ergosterol to selectively bind cholesterol and eliminate the toxicity of AmB (in the form of C2'epiAmB) to mammals. See Wilcock, BC et al., J Am Chem Soc 2013, 135 (23), 8488-91. The present invention discloses a K-type binding of ergosterol and cholesterol to AmB sterol sponge. D , which provides quantitative and mechanistically based biophysical parameters to guide the rational optimization of the therapeutic index of this clinically relevant natural product.

[0055] The present invention relates at least in part to other derivatives of AmB discovered by the inventors, which are also characterized by an improved therapeutic index compared to AmB. Various derivatives, i.e., compounds of the present invention, can be semi-synthetic or fully synthetic. One aspect of the present invention is to develop a new synthetic derivative of AmB that retains effective binding to ergosterol but does not show detectable cholesterol binding. This derivative retains fungicidal efficacy against many yeasts and molds but does not show detectable mammalian toxicity. This shows that differential binding of ergosterol to cholesterol is possible and provides a non-toxic AmB variant that retains desirable antifungal properties. The compounds of the present invention make new high-dose treatment strategies possible to eradicate life-threatening invasive fungal infections and significantly improve safety.

[0056] The compounds of the present invention and the pharmaceutical compositions of the present invention can be used to inhibit fungal growth. In one embodiment, an effective amount of the compounds of the present invention is contacted with fungi to inhibit fungal growth. In one embodiment, the compounds of the present invention or pharmaceutically acceptable salts thereof are added to or included in tissue culture medium.

[0057] The compounds of the present invention and the pharmaceutical compositions of the present invention can be used to treat fungal infections in subjects. In one embodiment, a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof, thereby treating fungal infections.

[0058] Yeasts are eukaryotic organisms belonging to the kingdom Fungi. Fungi include yeasts, molds, and larger organisms, including mushrooms. Yeasts and molds are of clinical relevance as pathogens. Yeasts are generally described as budding forms of fungi. Of particular importance in connection with the present invention are yeast species that can cause infections in mammalian hosts. Such infections most often occur in immunodeficient hosts, including hosts with impaired infection barriers (e.g., burn victims) and hosts with compromised immune systems (e.g., hosts undergoing chemotherapy or immunosuppressive therapy, and hosts infected with HIV). Pathogenic yeasts include, but are not limited to, various species of Candida and Cryptococcus. Of particular note among the pathogenic yeasts of 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 specifically includes Cryptococcus neoformans. Yeasts can cause mucosal infections, such as oral, esophageal and vaginal infections in humans, as well as infections of the bones, blood, urogenital tract and central nervous system. The list is exemplary and not limiting in any way.

[0059] Many fungi (except yeast) can cause infections in mammalian hosts. Such infections most often occur in immunodeficient hosts, including hosts with impaired infection barriers (e.g., burn victims) and hosts with compromised immune systems (e.g., hosts undergoing chemotherapy or immunosuppressive therapy, and hosts infected with HIV). Pathogenic fungi (except yeast) include, but are not limited to, species of Aspergillus, Rhizopus, Mucor, Histoplasma, Coccidioides, Blastomyces, Trichophyton, Microsporum, and Epidermophyton. Of particular note among the above are Aspergillus fumigatus, A. flavus, A. niger, H. capsulatum, C. immitis, and B. dermatitidis. Fungi can cause systemic and deep tissue infections of the lungs, bones, blood, urogenital tract, and central nervous system (to name a few). Some fungi can cause skin and nail infections.

[0060] definition

[0061] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the CAS version of the periodic table, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry, as well as specific functional moieties and reactivity are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd edition, Cambridge University Press, Cambridge, 1987.

[0062] The compounds described herein may contain one or more asymmetric centers and may therefore exist in various isomeric forms (e.g., 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 stereoisomer mixtures, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated 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).

[0063] The present invention additionally encompasses the compounds described herein as individual isomers substantially free of other isomers and, alternatively, as mixtures of various isomers.

[0064] The following terms are intended to have the following meanings presented and to help understand the description and intended scope of the present invention. When describing the present invention that may include compounds, pharmaceutical compositions containing such compounds, and methods using such compounds and compositions, unless otherwise specified, the following terms (if present) have the following meanings. It should also be understood that when described in this article, any of the parts defined below may be substituted by a variety of substituents, and the respective definitions are intended to include such substituted parts within their scope as listed below. Unless otherwise specified, the term "substituted" is defined as follows. It should also be understood that the terms "group" and "free radical" can be considered interchangeable when used in this article. The article "a (kind) (a / an)" can be used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an analog" means an analog or more than one analog.

[0065] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or a state government or equivalent in a country outside the United States, or listed in the US Pharmacopoeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans.

[0066] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that are pharmaceutically acceptable and have the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic salts or organic acid addition salts and base addition salts. In particular, such salts include: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as 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]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; or (2) salts formed when the acidic protons present in the parent compound are replaced by metal ions (e.g., alkali metal ions, alkaline earth ions or aluminum ions); or salts formed by complexation with organic bases (ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc.). Salts also include, by way of example only, sodium potassium salts, calcium salts, magnesium salts, ammonium salts, tetraalkylammonium salts, etc.; when the compound contains a basic functional group, non-toxic organic or inorganic acid salts such as hydrochlorides, hydrobromides, tartrates, methanesulfonates, acetates, maleates, oxalates, etc.

[0067] "Pharmaceutically acceptable cation" refers to an acceptable cationic counterion of an acidic functional group. Examples of such cations include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e.g., Berge et al., J. Pharm. Sci. 66(1): 1-79 (Jan. 77)).

[0068] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered.

[0069] "Subjects" contemplated for administration include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults) and / or non-human animals, e.g., mammals, such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0070] "Effective amount" means the amount of a compound that, when administered to a subject to treat or prevent a disease, is sufficient to achieve such treatment or prevention. "Effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. "Therapeutically effective amount" means an effective amount for therapeutic treatment. "Prophylactic effective amount" means an effective amount for prophylactic treatment.

[0071] "Preventing" or "preventive treatment" refers to reducing the risk of acquiring or developing a disease or condition (i.e., causing at least one clinical symptom of a disease not to occur in a subject not yet exposed to the pathogen or predisposed to the disease).

[0072] "Prophylaxis" is related to "prevention" and refers to measures or procedures aimed at preventing rather than treating or curing a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients who are at risk of thrombosis, for example, due to immobility, and the administration of antimalarial agents such as chloroquine before visiting geographic areas where malaria is prevalent or the risk of contracting malaria is high.

[0073] In one embodiment, "treating / treatment" or "therapeutic treatment" of any disease or condition refers to ameliorating the disease or condition (i.e., curbing the disease or reducing the manifestation, extent, or severity of at least one clinical symptom thereof). In another embodiment, "treating / treatment" refers to improving at least one physical parameter, which may not be perceived by the subject. In yet another embodiment, "treating / treatment" refers to regulating the disease or condition physically (e.g., stabilization of perceptible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In another embodiment, "treating / treatment" refers to slowing the progression of the disease.

[0074] As used herein, the term "isotopic variant" refers to a compound that contains unnatural proportions of isotopes at one or more atoms that constitute the compound. For example, an "isotopic variant" of a compound may contain one or more non-radioactive isotopes, such as deuterium ( 2 H or D), carbon-13 ( 13 C), nitrogen-15 ( 15 It will be appreciated that in compounds subject to such isotopic substitution, the following atoms (if present) may vary, so that, for example, any hydrogen may be 2 H / D, any carbon can be 13 C, or any nitrogen can be 15N , and the presence and position of these atoms can be determined within the skill of the art. Likewise, the invention may include the use of radioactive isotopes to prepare isotopic variants, in which case, for example, the resulting compounds may be useful 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 suitable for this purpose in view of their ease of incorporation and readily available means of detection. 11 C. 18 F. 15 O and 13 N) substituted compounds and can be used 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 present invention.

[0075] "Tautomers" refer to compounds that are interchangeable forms of a particular compound structure and differ in the replacement of hydrogen atoms and electrons. Thus, the two structures may be in equilibrium through the movement of electrons and atoms (usually H). For example, enols and ketones are tautomers because they rapidly interconvert upon treatment with acid or base. Another example of tautomerism is the acid and nitro forms of phenylnitromethane, which are also formed by treatment with acid or base. Tautomeric forms may be relevant to the achievement of optimal chemical reactivity and biological activity of the target compound.

[0076] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., enantiomeric excess). In other words, the "S" form of a compound is substantially free of the "R" form of the compound, and is therefore an 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, or 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.

[0077] As used herein and unless otherwise indicated, the term "enantiomerically pure R-compound" refers to at least about 95% by weight of the R-compound and at most about 5% by weight of the S-compound, at least about 99% by weight of the R-compound and at most about 1% by weight of the S-compound, or at least about 99.9% by weight of the R-compound and at most about 0.1% by weight of the S-compound. In certain embodiments, the weight is based on the total weight of the compound.

[0078] As used herein and unless otherwise indicated, the term "enantiomerically pure S-compound" or "S-compound" refers to at least about 95% by weight of the S-compound and up to about 5% by weight of the R-compound, at least about 99% by weight of the S-compound and up to about 1% by weight of the R-compound, or at least about 99.9% by weight of the S-compound and up to about 0.1% by weight of the R-compound. In certain embodiments, the weight is based on the total weight of the compound.

[0079] In the compositions provided herein, enantiomerically pure compounds or pharmaceutically acceptable salts thereof may be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising an enantiomerically pure R-compound may comprise, for example, about 90% of an excipient and about 10% of an enantiomerically pure R-compound. In certain embodiments, the enantiomerically pure R-compound in such compositions may, for example, comprise at least about 95% by weight of an R-compound and up to about 5% by weight of an S-compound by the total weight of the compound. For example, a pharmaceutical composition comprising an enantiomerically pure S-compound may comprise, for example, about 90% of an excipient and about 10% of an enantiomerically pure S-compound. In certain embodiments, the enantiomerically pure S-compound in such compositions may, for example, comprise at least about 95% by weight of an S-compound and up to about 5% by weight of an R-compound by the total weight of the compound. In certain embodiments, the active ingredient may be formulated with very little or no excipient or carrier.

[0080] Compounds of the present invention

[0081] In certain aspects, provided herein is a compound selected from the group consisting of:

[0082]

[0083] or a pharmaceutically acceptable salt thereof.

[0084] In certain embodiments, the compound is:

[0085]

[0086] or a pharmaceutically acceptable salt thereof.

[0087] In other embodiments, the compound is:

[0088]

[0089] In other embodiments, the compound is:

[0090]

[0091] In yet other embodiments, the compound is:

[0092]

[0093] or a pharmaceutically acceptable salt thereof.

[0094] In yet other embodiments, the compound is:

[0095]

[0096] In other embodiments, the compound is:

[0097]

[0098] In other aspects, provided herein are pharmaceutical compositions comprising a disclosed compound or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is an intravenous dosage form. In other embodiments, the pharmaceutical composition is an oral dosage form.

[0099] In yet other aspects, there is provided herein a method for treating a fungal infection, the method comprising administering a therapeutically effective amount of a disclosed compound or a pharmaceutically acceptable salt thereof to a subject in need thereof, thereby treating a fungal infection. In certain embodiments, the compound is administered intravenously. In other embodiments, the compound is administered orally.

[0100] In still other aspects, provided herein is the use of a disclosed compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a fungal infection.

[0101] In certain aspects, provided herein are compounds and pharmaceutically acceptable salts thereof for use as medicaments and for treating fungal infections.

[0102] Pharmaceutical composition

[0103] The present invention also provides pharmaceutical compositions of the disclosed compounds or salts thereof, and methods for preparing such compositions.

[0104] One aspect of the present invention is a pharmaceutical composition comprising a compound of the present invention; and a pharmaceutically acceptable carrier. In certain embodiments, the present invention is a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents, or encapsulating materials that are suitable for administration to humans or other vertebrates. The term "carrier" means a natural or synthetic organic or inorganic ingredient to which the active ingredient is combined to facilitate application. The components of the pharmaceutical composition can also be mixed with the compounds of the present invention and with each other in a manner such that there is no interaction that would substantially impair the desired drug effect.

[0105] In certain embodiments, the pharmaceutical composition is an intravenous dosage form.

[0106] In certain embodiments, the pharmaceutical composition is an oral dosage form.

[0107] In certain embodiments, the pharmaceutical composition is a lyophilized formulation of liposome-embedded or liposome-encapsulated active compound.

[0108] In certain embodiments, the pharmaceutical composition is a lipid complex of the compound in aqueous suspension.

[0109] The foregoing embodiments of the pharmaceutical compositions of the present invention are exemplary and not limiting.

[0110] Also provided is a method for preparing such a pharmaceutical composition, comprising mixing the compound of the present invention or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier.

[0111] Method of the present invention

[0112] The compounds of the present invention can be used to inhibit the growth of fungi and yeasts, particularly including fungi and yeasts of clinical significance as pathogens. Advantageously, the compounds of the present invention have an improved therapeutic index compared to AmB, thereby providing an agent with improved efficacy and reduced toxicity compared to AmB. The compounds of the present invention can be used to treat fungal and yeast infections, particularly including methods of systemic fungal and yeast infections. The compounds of the present invention can also be used to manufacture medicaments for treating fungal and yeast infections, particularly including systemic fungal and yeast infections. The present invention also provides the use of the compounds of the present invention for treating fungal and yeast infections, particularly including systemic fungal and yeast infections.

[0113] One aspect of the present invention is a method for treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention or a salt thereof, thereby treating the fungal infection.

[0114] As used herein, "inhibit / inhibiting" means to reduce an objectively measurable amount or degree compared to a control. In one embodiment, inhibition / inhibiting means to reduce at least a statistically significant amount compared to a control. In one embodiment, inhibition / inhibiting means to reduce at least 5% compared to a control. In various individual embodiments, inhibition / inhibiting means to reduce at least 10%, 15%, 20%, 25%, 30%, 33%, 40%, 50%, 60%, 67%, 70%, 75%, 80%, 90% or 95% compared to a control.

[0115] As used herein, the term "treat" or "treating" refers to an intervention that results in (a) preventing the occurrence of a condition or disease in a subject who may be at risk of or prone to a condition or disease but has not yet been diagnosed as having the condition or disease; (b) inhibiting the condition or disease, such as slowing or curbing the development of the condition or disease; or (c) alleviating or ameliorating the condition or disease, such as causing regression of the condition or disease. In one embodiment, the term "treat" or "treating" refers to an intervention that results in (a) inhibiting the condition or disease, such as slowing or curbing the development of the condition or disease; or (b) alleviating or ameliorating the condition or disease, such as causing regression of the condition or disease. For example, in one embodiment, the term "treat" or "treating" refers to an intervention that results in (a) inhibiting a fungal infection, such as slowing or curbing the development of the fungal infection; or (b) alleviating or ameliorating a fungal infection, such as causing regression of the fungal infection.

[0116] As used herein, "fungal infection" refers to an infection in a subject or in 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 a subject or in a subject with a yeast as defined herein.

[0117] As used herein, "subject" refers to a living mammal. In various embodiments, the subject is a non-human mammal, including but not limited to mice, rats, hamsters, guinea pigs, rabbits, sheep, goats, cats, dogs, pigs, horses, cattle, or non-human primates. In one embodiment, the subject is a human.

[0118] As used herein, a "subject with a fungal infection" refers to a subject that exhibits at least one objective manifestation of a fungal infection. In one embodiment, a subject with a fungal infection is a subject that has been diagnosed as having a fungal infection and is in need of treatment thereof. Methods for diagnosing fungal infections are well known and need not be described in detail herein.

[0119] As used herein, a "subject with a yeast infection" refers to a subject that exhibits at least one objective manifestation of a yeast infection. In one embodiment, a subject with a yeast infection is a subject that has been diagnosed as having a yeast infection and is in need of treatment. Methods for diagnosing yeast infections are well known and need not be described in detail here.

[0120] In certain embodiments, the compound is administered intravenously.

[0121] In certain embodiments, the compound is administered orally.

[0122] In certain embodiments, the compound is administered systemically.

[0123] In certain embodiments, the compound is administered parenterally.

[0124] In certain embodiments, the compound is administered intraperitoneally.

[0125] In certain embodiments, the compound is administered enterally.

[0126] In certain embodiments, the compound is administered intraocularly.

[0127] In certain embodiments, the compound is administered topically.

[0128] The present invention contemplates other routes of administration of the compounds of the invention including, but not limited to, intravesical (bladder), pulmonary, and intrathecal.

[0129] As used herein, the phrase "effective amount" refers to any amount sufficient to achieve a desired biological effect.

[0130] As used herein, the phrase "therapeutically effective amount" refers to an amount sufficient to achieve the desired therapeutic effect, such as treating a fungal or yeast infection.

[0131] For any compound described herein, a therapeutically effective amount can generally be initially determined by in vitro studies, animal models, or both. In vitro methods are well known and may include determining the minimum inhibitory concentration (MIC), the minimum fungicidal concentration (MFC), the concentration at which growth is inhibited by 50% (IC 50 ), the concentration at which growth is inhibited by 90% (IC 90 ) and the like. The therapeutically effective amount can also be determined based on human data of compounds of the present invention and compounds known to exhibit similar pharmacological activity (such as other related active agents (e.g. AmB)) that have been tested in humans. Higher doses may be required for parenteral administration. The dose applied can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is entirely within the capabilities of those of ordinary skill in the art to adjust the dose to achieve maximum efficacy based on the methods described herein and other methods known in the art.

[0132] For any compound described herein, the therapeutically effective amount for human subjects can be initially determined by in vitro studies, animal models, or both in vitro studies and animal models. The therapeutically effective amount for human subjects can also be determined based on human data of the compounds of the present invention tested in humans and known compounds that exhibit similar pharmacological activity, such as other related activators (e.g., AmB). Parenteral administration may require higher dosages. The dosage applied can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is completely within the capabilities of those of ordinary skill in the art to adjust the dosage to achieve maximum efficacy based on the methods described above and other methods known in the art.

[0133] Dosage and formulation

[0134] The compounds of the present invention may be combined with other therapeutic agents. The compounds of the present invention and other therapeutic agents may be administered simultaneously or sequentially. When other therapeutic agents are administered simultaneously, they may be administered in the same or separate formulations, but they are administered substantially simultaneously. When the administration of other therapeutic agents and the compounds of the present invention is separated in time, the other therapeutic agent is administered sequentially with another therapeutic agent and with the compounds of the present invention. The time interval between the administration of these compounds may be only a few minutes or may be longer.

[0135] Examples of other therapeutic agents include other antifungal agents, including AmB, and other antibiotics, antivirals, anti-inflammatory agents, immunosuppressants, and anticancer agents.

[0136] As mentioned above, "effective amount" refers to any amount sufficient to achieve the desired biological effect. In conjunction with the teachings provided herein, by selecting between various active compounds and trade-offs (e.g., efficacy, relative bioavailability, patient weight, severity of adverse side effects, and preferred modes of administration), an effective preventive or therapeutic treatment regimen can be planned that does not cause substantial undesirable toxicity, but can still effectively treat a specific subject. The effective amount of any particular application may vary depending on factors such as the disease or condition being treated, the specific compound of the present invention being administered, the size of the subject, or the severity of the disease or condition. A person of ordinary skill in the art can empirically determine the effective amount of a specific compound of the present invention and / or other therapeutic agent without excessive experimentation. It is generally preferred to use the maximum dose, i.e., the highest safe dose according to some medical diagnosis. Multiple doses per day may be considered to achieve appropriate systemic levels of the compound. Appropriate systemic levels may be determined, for example, by measuring the peak or sustained plasma levels of the drug in the patient. "Dose / dosage" is used interchangeably herein.

[0137] Typically, for human subjects, the daily oral dose of the active compound will be about 0.01 mg / kg to 1000 mg / kg per day. It is expected that the oral dose will be in the range of 0.5 to 50 mg / kg, once or multiple times per day, and the desired results will be produced. The dosage can be appropriately adjusted to reach the desired local or systemic drug level, depending on the mode of administration. For example, it is expected that the daily dose of intravenous administration will be reduced by an order of magnitude to several orders of magnitude. If the subject's response at this dosage is insufficient, a higher dose (or an effective higher dose by a different, more localized delivery route) can be used within the range allowed by the patient's tolerance. Consider multiple doses per day to reach the appropriate systemic level of the compound.

[0138] In one embodiment, the intravenous administration of the compound of the invention may typically be 0.1 mg / kg / day to 20 mg / kg / day. Therefore, intravenous administration may be similar to or advantageously may exceed the maximum tolerated dose of AmB. Intravenous administration may also be similar to or advantageously may exceed the maximum tolerated daily dose of AmB. Intravenous administration may also be similar to or advantageously may exceed the maximum tolerated cumulative dose of AmB.

[0139] Intravenous administration may also be similar to or advantageously may exceed the maximum recommended dose of AmB. Intravenous administration may also be similar to or advantageously may exceed the maximum recommended daily dose of AmB. Intravenous administration may also be similar to or advantageously may exceed the maximum recommended cumulative dose of AmB.

[0140] For any compound described herein, the therapeutically effective amount can be initially determined by an animal model. The therapeutically effective dose can also be determined based on human data of the compounds of the present invention and compounds known to exhibit similar pharmacological activity (such as other related active agents) that have been tested in humans. Higher doses may be required for parenteral administration. The dose applied can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is entirely within the capabilities of those of ordinary skill in the art to adjust the dose to achieve maximum efficacy based on the methods described above and other methods known in the art.

[0141] The formulations of the invention are administered in the form of pharmaceutically acceptable solutions which may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

[0142] Amphotericin B is commercially available in a variety of formulations, including deoxycholate-based (sometimes referred to as deoxycholate-based) formulations and lipid-based (including liposome) formulations. The amphotericin B derivative compounds of the present invention can be similarly formulated, for example but not limited to, deoxycholate-based formulations and lipid-based (including liposome) formulations.

[0143] For use in treatment, an effective amount of the compounds of this invention can be administered to a subject by any mode of delivering the compounds of this invention to the desired surface. The use of the pharmaceutical composition of the present invention can be completed 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 a tumor or abscess), mucosa, lung (e.g., inhalation), and topical.

[0144] For intravenous and other parenteral administration routes, the compounds of the present invention can generally be formulated similarly to AmB. For example, the compounds of the present invention can be formulated as lyophilized preparations with deoxycholic acid, lyophilized preparations of active compounds embedded or encapsulated by liposomes, lipid complexes in aqueous suspensions, or cholesterol sulfate complexes. The lyophilized preparations are generally reconstituted in a suitable aqueous solution, such as sterile water or saline, shortly before administration.

[0145] For oral administration, the compound (i.e., the compound of the present invention and other therapeutic agents) can be easily formulated by combining the active compound with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the compound of the present invention to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject to be treated. Oral pharmaceutical preparations can be obtained as solid excipients, the resulting mixture is optionally ground, and if necessary, the granular mixture is processed after adding a suitable auxiliary agent to obtain a tablet or dragee core. Suitable excipients are especially fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methyl-cellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, a disintegrant, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, can be added. Optionally, oral formulations may also be formulated in saline or buffer (such as EDTA for neutralization of internal acidic conditions), or may be administered without any carriers.

[0146] Also particularly contemplated are oral dosage forms of one or more of the above components. One or more of the components may be chemically modified to allow for effective oral delivery of the derivative. Typically, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, wherein the moiety allows (a) inhibition of acid hydrolysis; (b) absorption from the stomach or intestine into the bloodstream. It is also desirable to increase the overall stability of one or more components and to increase the in vivo circulation time. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, 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 may be used are poly-1,3-dioxolane and poly-1,3,6-trioxoctane. As mentioned above, preferred for pharmaceutical use are polyethylene glycol moieties.

[0147] For the ingredients (or derivatives), the release site can be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. Formulations that will not dissolve in the stomach but will release the substance in the duodenum or elsewhere in the intestine are available to those skilled in the art. Preferably, the release will avoid the deleterious effects of the gastric environment by protecting the compound (or derivative) of the invention or by releasing the biologically active substance outside the gastric environment, such as in the intestine.

[0148] To ensure complete gastric resistance, a coating that is impermeable to at least pH 5.0 is essential. Examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and shellac. These coatings can be used as mixed films.

[0149] Coatings or coating mixtures may also be used on tablets whose purpose is not to protect the stomach. Such coatings 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 therapeutic agents (such as powders); for liquid forms, a soft gelatin shell may be used. The shell material for cachets may be thick starch or other edible paper. For pills, lozenges, molded tablets, or stamped tablets, moist massing techniques may be used.

[0150] The therapeutic agent may be included in the formulation as fine multiparticulates in the form of granules or pellets having a particle size of about 1 mm. The formulation of the material for capsule administration may also be a powder, a lightly compressed plug or even a tablet. The therapeutic agent may be prepared by compression.

[0151] Both coloring agents and flavoring agents may be included.For example, a compound (or derivative) of the invention may be formulated (such as by liposome or microsphere encapsulation) and then further included in an edible product such as a refrigerated beverage containing coloring agents and flavoring agents.

[0152] Inert materials may be used to dilute or increase the volume of the therapeutic agent. These diluents may include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts may also be used as fillers including tricalcium phosphate, magnesium carbonate, and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress, and Avicell.

[0153] Disintegrants may be included in the formulation of the therapeutic agent to form a solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including the commercial disintegrant Explotab based on starch. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, super branched starch, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge, and bentonite can all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered glue can be used as a disintegrant and a binder, and these disintegrants and binders may include powdered glue, such as agar, gum karaya, or gum tragacanth. Alginic acid and its sodium salt may also be used as a disintegrant.

[0154] Binders can be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as gum arabic, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can both be used to granulate the therapeutic agent in an alcohol solution.

[0155] Anti-friction agents may be included in the formulation of the therapeutic agent to prevent sticking during formulation. Lubricants may be used as a layer between the wall of the therapeutic agent and the wall of the mold, and these lubricants may include, but are not limited to, stearic acid, including its 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 may also be used.

[0156] Glidants may be added which may improve the flow characteristics of the drug during formulation and aid in rearrangement during compression. Glidants may include starch, talc, fumed silica, and hydrated aluminosilicates.

[0157] In order to help the therapeutic agent dissolve in the aqueous environment, a surfactant can be added as a wetting agent. The surfactant can include anionic detergents, such as sodium lauryl sulfate, sodium sulfosuccinate dioctyl ester and dioctyl sodium sulfonate. Available cationic detergents can include benzalkonium chloride and benzethonium chloride. Potential nonionic detergents that can be included in the formulation as a surfactant include lauromacrogol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10,50 and 60, glyceryl monostearate, polysorbate 40,60,65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. These surfactants can be present in the formulation of the compound of the present invention or derivatives alone or as a mixture of different proportions.

[0158] Orally available pharmaceutical preparations include push-in capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-in capsules may contain active ingredients mixed with fillers such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate, and optional stabilizers. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in a dosage suitable for such administration.

[0159] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0160] For administration by inhalation, the compounds for use according to the invention may be conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer, with the use of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may 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 may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0161] 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 upon inhalation and cross the lung epithelial lining into the bloodstream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(Suppl 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (α1-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (a-1-proteinase); Oswein et 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., U.S. 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. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong et al.

[0162] 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 powder inhalers, all of which are familiar to those skilled in the art.

[0163] Some specific examples of commercially available devices suitable for practicing the present invention are 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.

[0164] All such devices require the use of formulations suitable for dispensing the compounds of the invention (or derivatives). Typically, each formulation is related to the type of device used and may involve the use of appropriate propellant materials in addition to conventional diluents, adjuvants and / or carriers for treatment. In addition, the use of liposomes, microcapsules or microspheres, inclusion compounds or other types of carriers is contemplated. The chemically modified compounds of the invention may also be prepared in different formulation forms depending on the type of chemical modification or the type of device used.

[0165] The formulation suitable for a jet or ultrasonic nebulizer generally comprises a compound of the invention (or derivative) dissolved in water at a concentration of about 0.1 mg to 25 mg of the biologically active compound of the invention per milliliter of solution. The formulation may also include a buffer and a monosaccharide (e.g., a compound of the invention for stabilizing and regulating osmotic pressure). The nebulizer formulation may also contain a surfactant to reduce or prevent surface-induced aggregation of the compound of the invention caused by atomization of the solution when forming an aerosol.

[0166] The formulation used with a metered dose inhaler device typically comprises a fine powder containing a compound of the invention (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, or a combination thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be used as a surfactant.

[0167] The formulation for dispensing from a powder inhaler device will contain a fine dry powder containing the compound (or derivative) of the present invention, and may also include a bulking agent, such as lactose, sorbitol, sucrose or mannitol, in an amount that facilitates the dispersion of the powder from the device (e.g., 50% to 90% by weight of the formulation). The compound (or derivative) of the present invention should advantageously be prepared in a particle form having an average particle size of less than 10 microns (μm), most preferably 0.5 to 5 μm, to be most effectively delivered to the deep lung.

[0168] Nasal delivery of the pharmaceutical compositions of the present invention is also contemplated. Nasal delivery allows the pharmaceutical compositions of the present invention to enter the bloodstream directly after administration of the therapeutic product to the nose, without the need for product deposition in the lungs. Formulations for nasal delivery include those with dextran or cyclodextrin.

[0169] For nasal administration, a useful device is a small, hard bottle with a metered sprayer mounted on it. In one embodiment, a metered dose is delivered by inhaling a solution of the pharmaceutical composition of the present invention into a chamber of defined volume, the chamber having a hole sized to atomize and aerosolize the formulation 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 specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

[0170] Alternatively, a plastic squeeze bottle with a hole or opening is used, sized to atomize the aerosol formulation by forming a spray when squeezed. The opening is usually located at the top of the bottle, and the top is usually tapered to partially fit into the nasal passages to effectively administer the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation for administering a measured dose of the drug.

[0171] When systemic delivery is desired, the compound may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with added preservatives. The composition may take the form of a suspension, solution, or emulsion, such as in an oily or aqueous vehicle, and may contain preparatants such as suspending agents, stabilizers, and / or dispersing agents.

[0172] Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds in water-soluble form. In addition, suspensions of active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain a suitable stabilizer, or increase the solubility of the compound to allow the preparation of a highly concentrated solution.

[0173] Alternatively, the active compound may be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

[0174] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0175] In addition to the formulations described above, the compounds may also be formulated as a depot preparation. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0176] The pharmaceutical composition may also contain a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycol.

[0177] Suitable liquid or solid pharmaceutical preparation forms are aqueous solutions or saline solutions for example for suction, which are microencapsulated, embedded, coated on tiny gold particles, contained in liposomes, atomized, for implanting aerosols, pellets or drying on sharp objects for skin to scrape into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations of sustained-release active compounds, and in the preparation of the pharmaceutical compositions, excipients and additives and / or adjuvants are usually used as described above, such as disintegrants, adhesives, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers. Pharmaceutical compositions are applicable to multiple drug delivery systems. For a brief review of the method for drug delivery, see Langer R, Science 249: 1527-33 (1990), which is incorporated herein by reference.

[0178] The compounds of the present invention and optional other therapeutic agents can be administered per se (pure) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, salts prepared from the following acids: 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. In addition, such salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts of carboxylic acid groups.

[0179] Suitable buffers include: acetic acid and salt (1-2% w / v); citric acid and salt (1-3% w / v); boric acid and salt (0.5-2.5% w / v); and phosphoric acid and salt (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).

[0180] The pharmaceutical compositions of the present invention contain an effective amount of a compound of the present invention and, optionally, at least one additional therapeutic agent in a pharmaceutically acceptable carrier.

[0181] The therapeutic agent, specifically including but not limited to the compounds of the present invention, can be provided in the form of particles. The particles used herein mean nanoparticles or microparticles (or larger particles in some cases), which can be composed of the compounds of the present invention or other therapeutic agents as described herein in whole or in part. The particles can contain the therapeutic agent in a core surrounded by a coating (including but not limited to an enteric coating). The therapeutic agent can also be dispersed throughout the particles. The therapeutic agent can also be adsorbed into the particles. The particles can have 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. In addition to the therapeutic agent, the particles can include any of those materials (including but not limited to corrodible, non-corrodible, biodegradable or non-biodegradable materials or combinations thereof) conventionally used in the pharmaceutical and medical fields. The particles can be microcapsules containing the compounds of the present invention in a solution or semi-solid state. The particles can be in practically any shape.

[0182] Non-biodegradable and biodegradable polymeric materials can be used to manufacture particles for delivering therapeutic agents. Such polymers can be natural polymers or synthetic polymers. Polymers are selected based on the time period of required release. Particularly interesting bioadhesive polymers include the bioerodible hydrogels described in Sawhney HS et al. (1993) Macromolecules 26:581-7, and the teaching of the document is incorporated herein. These polymers include polyhyaluronic acid, casein, gelatin, gelatin, polyanhydrides, polyacrylic acid, alginate, 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).

[0183] The therapeutic agent may be included in a controlled release system. The term "controlled release" means any drug-containing formulation in which the manner and distribution of the drug released from the formulation is controlled. This refers to both immediate and non-immediate release formulations, wherein non-immediate release formulations include, but are not limited to, sustained release 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 for gradual release of the drug over an extended period of time, and preferably, but not necessarily, results in a substantially constant blood level of the drug over an extended period of time. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between the administration of the formulation and the release of the drug therefrom. "Delayed release" may or may not involve the gradual release of the drug over an extended period of time, and therefore may or may not be "sustained release".

[0184] The use of long-term sustained release implants may be particularly useful for treating chronic conditions. As used herein, "long-term" release refers to implants that are constructed and arranged to deliver therapeutic levels of active ingredients for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.

[0185] Incorporated by Reference

[0186] All U.S. patent application publications and U.S. patents mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0187] Other Implementations

[0188] In the claims, articles such as "a," "an," and "the" may mean one or more unless indicated to the contrary or clear from the context. Statements or descriptions including "or" between one or more members of the group are deemed to satisfy if one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless otherwise indicated to the contrary or clear from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, used in, or otherwise relevant to a given product or process.

[0189] In addition, the present invention encompasses all variations, combinations and permutations, wherein one or more limitations, elements, clauses and descriptive terms in one or more listed claims are introduced into another claim. For example, any claim that is subordinate to another claim may be modified to include one or more limitations found in any other claim that is subordinate to the same base claim. In the case where the elements are presented in a list form, such as in a Markush group format, each subgroup of the elements is also disclosed, and any element may be removed from the group. It should be understood that, in general, in the case where the present invention or aspects of the present invention are referred to as including specific elements and / or features, certain embodiments of the present invention or aspects of the present invention are composed of such elements and / or features or are substantially composed of such elements and / or features. For simplicity, those embodiments are not specifically described herein. It should also be noted that the terms "comprising" and "containing" are intended to be open and allow the inclusion of additional elements or steps. In the case of a given range, the endpoints are also included. In addition, unless otherwise specified or it is obvious from the context and the understanding of a person of ordinary skill in the art, the values ​​expressed as ranges can adopt any specific value or subrange within the range stated in different embodiments of the present invention, to one tenth of the lower limit unit of the range, unless the context clearly specifies otherwise.

[0190] This application refers to various issued patents, published patent applications, magazine articles and other publications, all of which are incorporated herein by reference in their entirety. If there is a conflict between any incorporated reference and this specification, this specification shall prevail. In addition, any specific embodiment of the present invention that falls within the scope of the prior art may be explicitly excluded from any one or more claims. Because such embodiments are considered to be known to those of ordinary skill in the art, even if the exclusion is not explicitly set forth in this article, they may also be excluded. For any reason, whether or not it is relevant to the existence of the prior art, any specific embodiment of the present invention may be excluded from any claim.

[0191] Those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments of the invention described herein using only routine experimentation. The scope of the embodiments described herein is not intended to be limited to the above description, but as set forth in the appended claims. It will be appreciated by those of ordinary skill in the art that various changes and modifications may be made to this description without departing from the spirit or scope of the invention as defined in the following claims. Example

[0192] In order that the invention described herein may be more 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 in any way as limiting the scope thereof.

[0193] Example 1. Synthetic procedures and HPLC methods of disclosed compounds

[0194] General synthetic procedure for the amides of AmB:

[0195]

[0196] Freshly distilled Et3N was added dropwise to a solution of amphotericin B (10 mg; 0.01 mmol) and amine (3 eq.) in DMF (500 μL) until pH = 9 (by pH paper) was reached. The reaction mixture was stirred at room temperature for 15 minutes. Solid PyBOP (1.5 eq.; 8.4 mg) was added under nitrogen atmosphere, and the sealed vial was stirred at room temperature overnight. The progress of the reaction was monitored by analytical HPLC traces.

[0197] Once completed, the product was precipitated and washed with anhydrous diethyl ether (10 mL). The suspension was centrifuged at 3000 g for 5 minutes. The solvent was decanted and the pellet was dissolved in DMSO and filtered through a 0.2 micron syringe filter to purify on a C18 preparative HPLC system. The pure product was dried to a pale yellow powder on a freeze dryer and stored at -80 ° C in a nitrogen atmosphere.

[0198] The method is applicable to the preparation of the disclosed amides of C2'-epi-AmB starting from C2'-epi-AmB.

[0199] HPLC method:

[0200] Analytical column: C18 Agilent column (Catalog number: 993967-902)

[0201]

[0202] Preparative column: C18 Agilent column (Catalog number: 410910-502)

[0203] Time (min) Acetonitrile <![CDATA[10 mM NH4OAc buffer]]> Flow rate (mL / min) 0 5 95 30 1 5 95 50 15 95 5 50 16 95 5 50 17 5 95 50 18 5 95 30

[0204] Synthesis of compound 1:

[0205]

[0206] Fresh Et3N (directly from the vial) (12 μL) was added to a solution of 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 minutes. Solid PyBOP (1.5 eq; 8.4 mg) was added to the mixture and the sealed vial was stirred at room temperature for 2 hours. The progress of the reaction was monitored by analytical HPLC traces.

[0207] Once complete, the reaction was diluted with DMSO to form a clear solution and filtered through a 0.2 micron syringe filter and then purified on a C18 preparative HPLC system. The pure product was dried on a lyophilizer to a light yellow powder and stored under an inert atmosphere at -80°C.

[0208] Yield 5.6 mg (52%)

[0209] Product molecular weight: 997.1860

[0210] Compound 3 was synthesized from compound 1:

[0211]

[0212] In a clean oven-dried 40 mL glass vial containing 50 mg (X mg) of HPLC-purified compound 1, 2.5 mL (0.05X mL) of ultrapure water was added, followed by 200 mL (4X mL) of 0.5 (M) acetic acid. After the addition of acid, the solution began to clarify. The solution was vortexed as needed to completely dissolve the solute. After the solution was ultrasonically treated for 1-2 minutes at room temperature, clean stirring beads were added to the solution and stirred at 800 rpm for 15 minutes. Check if there are any solid residues adhering to the top of the glass vial. After stirring is complete, visually inspect the solution for any signs of insoluble parts or suspensions. Now remove the stirring beads from the solution, freeze them in liquid nitrogen and place them on a freeze dryer to dry overnight.

[0213] Synthesis of compound 4:

[0214]

[0215] In a clean oven-dried 40 mL glass vial containing 50 mg (X mg) of HPLC-purified Compound A, 2.5 mL (0.05X mL) of ultrapure water was added, followed by 200 mL (4X mL) of 0.5 (M) acetic acid. After the addition of acid, the solution began to clarify. The solution was vortexed as necessary to completely dissolve the solute. After the solution was ultrasonically treated for 1-2 minutes at room temperature, clean stirring beads were added to the solution and stirred at 800 rpm for 15 minutes. Check if there are any solid residues adhering to the top of the glass vial. After stirring is complete, visually inspect the solution for any signs of insoluble parts or suspensions. Now remove the stirring beads from the solution, freeze them in liquid nitrogen and place them on a freeze dryer to dry overnight.

[0216] Example 2. Characterization data of disclosed compounds

[0217] Compound 1

[0218]

[0219] The NMR spectrum of compound 1 showed Figure 4C middle.

[0220] LCMS: [M+H] + =998.1935. The chromatogram and mass spectrum of compound 1 are shown in Figure 4A and Figure 4B middle.

[0221] Synthesis of compound 4:

[0222]

[0223] In a clean oven-dried 40 mL glass vial containing 50 mg (X mg) of HPLC-purified Compound A, 2.5 mL (0.05X mL) of ultrapure water was added, followed by 200 mL (4X mL) of 0.5 (M) acetic acid. After the addition of acid, the solution began to clarify. The solution was vortexed as necessary to completely dissolve the solute. After the solution was ultrasonically treated for 1-2 minutes at room temperature, clean stirring beads were added to the solution and stirred at 800 rpm for 15 minutes. Check if there are any solid residues adhering to the top of the glass vial. After stirring is complete, visually inspect the solution for any signs of insoluble parts or suspensions. Now remove the stirring beads from the solution, freeze them in liquid nitrogen and place them on a freeze dryer to dry overnight.

[0224] Example 2. Characterization data of disclosed compounds

[0225] Compound 1

[0226]

[0227] The NMR spectrum of compound 1 showed Figure 4C middle.

[0228] LCMS: [M+H] + =998.1935. The chromatogram and mass spectrum of compound 1 are shown in Figure 4A and Figure 4B middle.

[0229] Compound 2

[0230]

[0231] LCMS: [M+H] + =1028.2195. The chromatogram and mass spectrum of compound 2 are shown in Figure 5A and Figure 5B middle.

[0232] Compound 3

[0233]

[0234] Compound 3 1 H NMR spectrum shows Fig.19A middle.

[0235] Compound 3 13 C NMR spectrum shows Fig.19B middle.

[0236] LCMS: [M+H] + =998.0192. The chromatogram and mass spectrum of compound 3 are shown in Fig.19C and Fig.19D middle.

[0237] Compound 4

[0238]

[0239] Compound 3 1 H NMR spectrum shows Fig.24A middle.

[0240] Compound 3 13 C NMR spectrum shows Fig. 24B middle.

[0241] LCMS: [M+H] + =997.4949. The chromatogram and mass spectrum of compound 4 are shown in Fig.24C and Fig.24D middle.

[0242] Example 3. The disclosed compounds exhibit comparable efficacy and DMPK to AmB, but with an improved safety profile.

[0243] Table 1. Efficacy, safety and DMPK data of disclosed compounds and AmB.

[0244]

[0245]

[0246] Example 4. Antifungal efficacy of disclosed compounds and AmB

[0247] Suspend the fungal or mold colonies from the SDA plate in RPMI medium and keep the inoculum density at 10 5 CFU / mL. 990 μL aliquots of the diluted cell suspension were added to sterile 1.7mL eppendorf tubes, followed by 10uL of 400uM compound solution (in DMSO). The DMSO concentration in each eppendorf tube was 1%, and a control sample was also performed to confirm viability using only 1% DMSO. At predetermined time points (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 24h), 10 μL samples were taken out from each tube and diluted 10 times with RPMI, and 10 μL aliquots were spread on SDA plates for colony count determination. When the expected colony count was less than 1,000 CFU / mL, 50 μL aliquots were taken directly from the test solution and spread on the SDA plates without dilution. The plates were incubated at 37°C for 24h before inspection. All experiments were performed in duplicate.

[0248] Table 2. Minimum inhibitory concentrations of AmB (Anbisu) and disclosed compounds against different molds.

[0249]

[0250]

[0251] Table 2A. Minimum inhibitory concentrations of AmB (AmB) and disclosed compounds against different molds.

[0252]

[0253] Table 3. Minimum inhibitory concentrations of AmB (Anbisu) and disclosed compounds against different yeasts.

[0254]

[0255] Table 3A. Minimum inhibitory concentrations of AmB (Anbisu) and disclosed compounds against different yeasts.

[0256]

[0257] Table 4. Minimum inhibitory concentrations of AmB (Anbisu) and disclosed compounds against different rare molds resistant to AmB.

[0258]

[0259] Example 5. In vitro and in vivo safety of disclosed compounds

[0260] 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 with DMSO. To synthesize the complex, 1 ul of compound solution was placed in a clean eppendorf tube (2 ml) and sterol solution was added thereto (volume depends on stoichiometry) 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 appropriately. The absorbance of the solution was measured after incubation for 30 minutes.

[0261] MHC( Figure 1B and Fig.10 ): The protocol for hemolysis assay was adapted from a report by Paquet and colleagues (Chem. Eur. J. 2008, 14, 2465-2481). Whole human blood (sodium heparin) was purchased from Bioreclamation LLC (Westbury, NY) and stored at 4°C and used within two days of receipt. 1 mL of whole human blood was added to a 2.0 mL appendorf tube and centrifuged at 10,000 g for 2 minutes. 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 minutes. 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.

[0262] Compounds were prepared as >15 mM stock solutions in DMSO and serially diluted with 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 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 by adding 1 μL of DMSO to ultrapure water or RBC buffer, respectively, to 0.2 mL PCR tubes. 0.63 μL of the red blood cell stock suspension was added to each PCR tube and mixed by inversion. Samples were incubated at 37°C for 2 hours. The samples were mixed by inversion and centrifuged at 10,000 g for 2 minutes. 15 μL of supernatant from each sample was added to the 384-well position. The absorbance was read at 540 nm using a Biotek H1 Synergy Hybrid Reader (Winooski, VT). The experiments were performed in triplicate, and the reported MHC represents the average of three experiments.

[0263] Example 6. In vivo pharmacokinetic study of the disclosed compounds in mice

[0264] Mouse PK ( Figure 2 ): Experiments were performed using compounds synthesized in the laboratory and purified by preparative HPLC (>91%). All compounds were dissolved in D5W (5% glucose in water) for IV injection. Female CD-1 mice (3 per group; each weighing approximately 30 g) were injected with compounds (as per the planned dose), and blood samples were collected at different time points and analyzed for compound content using the following procedure.

[0265] Instrument: Triple Quad 6500+

[0266] Matrix Male CD-1 Mouse Plasma (EDTA-K2)

[0267] Analyte: Compound 1

[0268] Internal standards: 100 ng / mL labetalol and 100 ng / mL tolbutamide in ACN

[0269] MS condition ESI: Positive

[0270] SRM detection

[0271] Compound 1: [M+H]+m / z 979.8>798.4

[0272] Labetalol (IS): [M+H]+m / z 329.2>162.1

[0273] UPLC conditions

[0274] Mobile phase A: 0.1% FA in water

[0275] Mobile phase B: 0.1% FA in ACN

[0276]

[0277] Column: Waters ACQUITY UPLC HSS T3 1.8μm 2.1×50mm

[0278] Flow rate: 0.6000mL / min

[0279] Retention time: Labetalol (IS): 0.998min

[0280] Sample preparation:

[0281] A 24 μL sample aliquot was protein precipitated with 120 μL IS solution (100 ng / mL labetalol and 100 ng / mL tolbutamide in ACN), the mixture was vortexed thoroughly and centrifuged at 3900 rpm for 10 minutes at 4°C. A 90 μL aliquot of the supernatant was transferred to a sample plate and mixed with 60 μL water, and the plate was then shaken at 800 rpm for 10 minutes. 15.0 μL of the supernatant was injected for LC-MS / MS analysis.

[0282] Calibration curve:

[0283] Compound 1 in female CD-1 mouse plasma (EDTA-K2) was 1.00-3000 ng / mL

[0284] Additional mouse PK studies ( Figure 3 ): After 24 h, blood samples were collected from mice treated with the compound for in vivo toxicity experiments (see above). 300 μL of HPLC grade methanol was added to 50 μL of mouse serum. The mixture was thoroughly mixed by vortexing for 30 s. To allow complete extraction of AmB (or derivatives) and precipitation of proteins, the mixture was allowed to stand at room temperature for 0.5 h. The mixture was then centrifuged at 16000 g for 10 minutes, and the unfiltered supernatant was used for HPLC analysis. (Note - AmB is bound to the pre-filter). The concentration of AmB (or derivative) was calculated by a pre-established standard curve.

[0285] The standard curve was prepared as follows:

[0286] 1: Prepare a series of different concentrations (10, 20, 40, 80, 100, 200, 400, 1000 μM) in methanol, measured by UV-vis, extinction coefficient at 406 nm: 164 mM -1 ) of AmB (or derivative) solution,

[0287] 2: Take 10 μL of standard solution and inject it into HPLC, and use the area under the curve and its corresponding concentration to establish a standard curve.

[0288] HPLC method:

[0289] C18 SiO2 column: Sunfire column, 5μM, 15cm;

[0290] Aqueous buffer (A): 0.1% formic acid filtered in MQ water

[0291] Organic solvent (B): filtered HPLC grade acetonitrile

[0292] Flow rate: 1 mL / min, DAD: 406.4 nm, ionization method: AJS ESI, positive mode

[0293] Gradient change: 95:5 (0.1% formic acid in water: MeCN) to 5:95 (0.1% formic acid in water: MeCN) in 8 minutes, the gradient change schedule is as follows

[0294] 0min 95% A, 5% B

[0295] 0.5min 95% A, 5% B

[0296] 8.5min 5% A, 95% B

[0297] 9.5min 5% A, 95% B

[0298] 10.5min 95% A, 5% B

[0299] Example 7. Solubility of Disclosed Compounds

[0300] Test 1: Synthesized in the laboratory (purity>95%)

[0301] D5W: Braun USA (product number L5101)

[0302] Instrument Details:

[0303] Ultrasonicator: Branson Ultrasonics 2800; Vortex-Genie 2 laboratory mixer

[0304] UV-Vis: Thermo Fisher Nanodrop oneC

[0305] step:*

[0306] ■ Place 6 mg of sample (measured by UV-Vis) in a clean, oven-dried 7 mL vial

[0307] ■Add 1.65mL sterile D5W (at room temperature)

[0308] ■ Vortex it for 2 minutes.

[0309] ■Water bath ultrasonic treatment: 2min x 2

[0310] ■ Repeat steps 3 and 4 until the solution is clear

[0311] ■ Transfer to a 2 mL Eppendorf tube and centrifuge (3000 g x 2 min) to ensure that the compound is completely dissolved and there is no insoluble part. (Optional step)

[0312] ■Concentration measured by UV (absolute value at 406 nm) (optional step)

[0313] *Amounts based on solutions prepared for in vivo toxicity studies at a dose of 50 mg / kg

[0314] Table 5. Solubility of disclosed compounds and AmB in D5W.

[0315] AB Compound 1 Compound 2 Solubility (mM) 0.023 >7.22 0.366 The increase relative to AmB -- >314 15.9

[0316] Example 8. Determination of killing kinetics of Candida albicans: ( Figure 6 )

[0317] The scheme of killing kinetics determination is adapted from the CLSI scheme for MIC measurement. From the stock, the fungal colony is suspended in 0.9% sterile saline solution and diluted to ≈103 inoculation density. 10ul aliquots are spread on Sabouraud dextrose agar (SDA) plates using L-applicator, and incubated at 35°C for 24-48h, and colony growth is monitored. During the experiment, 2-3 single colonies are suspended in 10mL RPMI 1640 culture medium (prepared according to CLSI scheme), and cell density is measured using a hemocytometer. The inoculation density is then adjusted to ≈105cfu / mL. In a sterile Eppendorf tube (1.7mL-2mL), 990μL inoculation aliquots are mixed with 10μL 100X compound solutions (solutions are prepared in DMSO, and DMSO content is 1% in each test solution). Control samples are prepared using only 1% DMSO in RPMI 1640 culture medium to confirm viability. At predetermined time points (0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8, 10, 16 and 24 h), 10 μL of sample was taken from each tube and serially diluted 10 times with RPMI 1640 medium, and 10 μL aliquots were plated on SDA plates for colony count determination. When the expected colony count was less than 1,000 CFU / mL, 50 μL aliquots were taken and half-diluted in RPMI 1640 medium, and 50 μL were plated on SDA plates. The plates were incubated at 35°C for 24 to 48 hours before inspection. All experiments were performed in triplicate.

[0318] Example 9. In vivo efficacy in a murine candidiasis model (Candida albicans SN250): Figure 7 and 8 )

[0319] Figure 7 Female CD-1 mice (average body weight = 27 g) were divided into groups based on the number of different test candidates and different doses, with each group consisting of 4 mice.

[0320] All groups were inoculated with Candida albicans SN250 (≈105 CFU / mL) via the tail vein before treatment. Two hours after inoculation, each group was 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 collected and immediately homogenized. To measure the fungal load 24 hours after treatment, serum was spread and colony density (CFU / mL) was calculated. In this experiment, Anbisu and C2'epiAmB were used as controls, and similar to the control, the test article compound 1 was administered at a dose of 5 mg / kg.

[0321] Figure 8: To test the efficacy of higher doses of compound 1, we developed an acetate version of the drug and found it to be highly soluble in aqueous solutions, especially in D5W saline vehicles. Therefore, we continued and repeated the above in vivo studies and tested the efficacy of drug candidate compound 3 at four different single doses: 1.5 mg / kg; 5 mg / kg; 15 mg / kg and 45 mg / kg. Fungal load was measured after the experiment using the procedure mentioned above. In this study, we were able to demonstrate that fungal infections can be eradicated at high doses using non-toxic compound 3.

[0322] Example 10. In vivo efficacy in a murine candidiasis model (Candida albicans SC5314): Fig. 9 )

[0323] In this study, the efficacy and tolerability of compound 3 against Candida albicans SC5314 in male ICR mice in a multiple dose regimen were studied. Three different doses of compound 3 were tested as a single daily dose over 7 days: 1 mg / kg; 5 mg / kg and 15 mg / kg. Body weight was also recorded every day. Two different doses of Anbisu were used as a control. Each group consisted of 6 mice. All groups were inoculated with Candida albicans SC5314 (≈1.9x105 CFU / mL) via the tail vein before treatment. Five hours after inoculation, each group was given the first IV dose (in D5W). After completing 7 days of treatment, all mice were killed and the fungal load in the kidneys was measured using a spreading technique. Male body weights are shown in Fig. 22 A and 22B.

[0324] Example 11. In vivo efficacy in a murine candidiasis model (Candida auris): Fig.10 )

[0325] Based on the number of different test candidates and different dosages, female CD-1 mice (average weight = 27g) were grouped, with each group consisting of 4 mice. All groups were inoculated with Candida auris (≈105CFU / mL) via the tail vein before treatment. After 2 hours of inoculation, each group of compound 3 was given its first IP dose (in saline). In the following four days, multiple doses were given 24h, 48h and 72h after inoculation. After 4 days of treatment, all animals were euthanized, and kidneys were collected and homogenized immediately. In order to measure the fungal load 24h after treatment, serum was spread and colony density (CFU / mL) was calculated. In this experiment, two different doses of Anbisu were used as controls, and the test compound 3 was administered at a dosage of 1.5, 5, 15 and 45mg / kg.

[0326] Example 12. In vivo efficacy in a murine aspergillosis model: ( Fig.11 )

[0327] In this study, the efficacy and tolerability of compound 3 against Aspergillus fumigatus 1163 in male ICR mice in a multiple dose regimen were studied. Two different doses of compound 3 as a single daily dose were tested within 4 days: 5mg / kg and 15mg / kg. Body weight was also recorded every day. Anbisu (10mg / kg) was used as a control in this experiment. Each group consisted of 6 mice. Three days before the study, all mice were treated with cyclophosphamide (200mg / kg) as an immunosuppressant. On the day of the study, all groups were inoculated with Aspergillus fumigatus 1163 (≈2.9x105 CFU / mL) via the tail vein. After 5 hours of inoculation, each group was given the first IV dose (in D5W). After completing 4 days of treatment, all mice were killed and the fungal load in the kidney was measured using a spreading technique. Body weight can be found on slide 17.

[0328] Example 13. Wajima superposition: (Figures 12 and 13)

[0329] Prediction of human DMPK using the Wajima superposition was done using a standard protocol.

[0330] PK parameters Rat dog monkey people <![CDATA[CL p (mL / min / kg)]]> 1.56 0.365 0.169 NA <![CDATA[V ss (L / kg)]]> 0.873 0.615 0.249 NA <![CDATA[Plasma protein binding (f u,p )]]> 0.0406 0.0364 0.0273 0.0445 Blood: Plasma 0.757 0.753 0.751 0.597

[0331] NCA parameters value <![CDATA[CL p (mL / min / kg)]]> 0.11 <![CDATA[V ss (L / kg)]]> 0.39 <![CDATA[t 1 / 2 (a)(h)]]> 3.8 <![CDATA[t 1 / 2 (b)(h)]]> 44 <![CDATA[AUC 0-t (μg h / mL)]]> 139.3 <![CDATA[AUC 0-24 (μg h / mL)]]> 60.8 <![CDATA[C0(μg / mL)]]> 3.85

[0332] Human PK:

[0333]

[0334]

[0335] *From Antimicrobial Agents Chemother. 2002, 46(3): 828-833

[0336] **Assuming dose proportionality

[0337] Mouse PK, IV bolus (female CD-1):

[0338]

[0339] Example 14. In vivo toxicity in a murine model: (Figures 15 and 16)

[0340] In this study, the in vivo toxicity of compound 3 was studied using the expression of nephrotoxicity biomarker genes. Based on the number of different test candidates and different doses, female CD-1 mice (average weight = 27g) were grouped, with each group consisting of 4 mice. On the day of the experiment, different doses of compound 3 were administered by IV. The group treated with Anbisu and C2'epi AmB was used as a reference. After 24h, all mice were killed and the fungal load in the kidneys was harvested and stored in RNAlater at -80°C. The kidneys were then homogenized and RNA was extracted. The expression of KIM1, LCN2, TIMP1 and SPP1 genes was measured with reference to GAPDH using RT-PCR technology.

[0341]

[0342]

[0343] Example 15. Tolerance of Compound 3 and Changes in Renal Biomarkers and Gene Expression after Administration of Compound 1 or 3 ( Fig.11 A-11C and 12)

[0344] hERG inhibition, plasma compatibility, and Ames assays were performed using industry standard protocols.

[0345] hERG inhibition study: There was no inhibition of hERG potassium current at 100 uM (the highest concentration tested).

[0346] Plasma compatibility: ppt observed at all 3 concentrations at 1:1 dilution; only observed at 5 mg / mL at 1:0.1 dilution. Formulation optimization is ongoing.

[0347] Ames study: No genotoxic effects observed at any dose level tested

[0348] Example 16. In vitro tolerance of compound 3 ( Figures 20A-20D )

[0349] Procedure: Four different cell lines were obtained from ATCC and the tolerance of compound 3 was tested together with AmB, C2'epiAmB and compound 4 as recommended by ATCC.

[0350] Neurotoxicity: SH-SY5Y (CRL-2266; human neuroblastoma; Fig. 20A ). Cells were grown in complete MEM medium and tested for tolerance in triplicate in 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The seeding density was maintained at 10 4 cells / well (5x 10 5CFU / mL). All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was kept at 1%. After the compounds were added to the culture, they were incubated for 24 hours at 35°C in a 5% CO2 atmosphere, and cell viability was measured using Alamar Blue fluorescent dye (excitation 555nm and fluorescence 585nm).

[0351] Hepatotoxicity: Hep-G2 (HB-8065; human hepatocytes; Fig. 20B ). Cells were grown in complete MEM medium and tested for tolerance in triplicate in 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The seeding density was maintained at 10 4 cells / well (5x 10 5 CFU / mL). All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was kept at 1%. After the compounds were added to the culture, they were incubated for 24 h at 35°C in a 5% CO2 atmosphere, and cell viability was measured using Alamar Blue fluorescent dye (excitation 555 nm and fluorescence 585 nm).

[0352] Cardiotoxicity: H9C2 (CRL-1446; rat cardiomyocytes; Fig. 20C ). Cells were grown in complete DMEM medium and tested for tolerance in triplicate in 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The seeding density was maintained at 10 4 cells / well (5x 10 5 CFU / mL). All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was kept at 1%. After the compounds were added to the culture, they were incubated at 35°C for 24 hours under a 5% CO2 atmosphere, and cell viability was measured using Alamar blue fluorescent dye (excitation 555nm and fluorescence 585nm).

[0353] Hematologic toxicity: K562 (CCL-243; human lymphoblastoid cells; Fig.20D ). Cells were grown in complete IMDM medium and tested for tolerance in triplicate in 96-well plates. DMSO and puromycin (100 μM) were used as positive and negative controls. The seeding density was maintained at 10 5 cells / well (5x 10 6CFU / mL). All test compound stock solutions were prepared in DMSO, and the DMSO concentration in the final culture was kept at 1%. After the compounds were added to the culture, they were incubated at 35°C for 24 hours under a 5% CO2 atmosphere, and cell viability was measured using Alamar blue fluorescent dye (excitation 555nm and fluorescence 585nm).

[0354] Example 17. In vivo tolerance of compound 3 in mice ( Figures 21A-21G )

[0355] Healthy female CD-1 mice were divided into 4 / group. The test compound was dissolved in clinically approved sterile D5W (5% glucose) and infused through the tail vein during the study. The concentration of the compound was adjusted accordingly to obtain a target dose of 0.3 mL injection. After 24 hours of incubation, the animals were euthanized, blood samples were collected for quantitative analysis of clinical analytes, and organs were collected for histopathological analysis.

[0356]

[0357]

[0358] 0 = not evident histologically; 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe

[0359] D = diffuse; F = focal; M = multifocal: P = present

[0360]

[0361]

[0362]

[0363] Example 18: In vitro efficacy of compound 3 against azole and echinocandin-resistant fungal strains

[0364] This experiment was performed according to standard CLSI protocols.

[0365]

[0366]

[0367] Example 19: Development of a protocol for studying the efficacy of compound 3 on pulmonary aspergillosis in mice ( Fig. 22 and 23 ).

[0368] Efficacy studies ( Fig. 22): Healthy mice were divided into 10 mice / group. All animals were treated with cyclophosphamide (150 mg / kg) on ​​days -2 and +3, and with cortisone acetate (250 mg / kg) on ​​days -1 and +6 to convert them into neutrophil deficiency. Animals were infected with Aspergillus fumigatus (CEA10 strain) (intranasally) on day 0. All animals were given 15 mg / kg / day (IP) of Anbisu or compound 3 for 7 days and monitored until the 14th day after infection. The survivability of animals was monitored during the study process.

[0369] Purpose: Testing of Compound 3 and AmB against CEA10 (wild type) IA

[0370] 1e8 spores / mL (40uL) intranasal CEA10

[0371] n = 10 mice / group

[0372] Cyclophosphamide: 150 mg / kg on days -2 and +3

[0373] Cortisone acetate: 250 mg / kg on days -1 and +6

[0374] Survival:

[0375] D5W only: 10%

[0376] Compound 3 15 mg / kg QD: 80%

[0377] AmB 15mg / kg QD: 80%

[0378] Log-rank statistics:

[0379] D5W vs. Compound 3 15 mg / kg QD: p=0.0005

[0380] D5W vs. AmB 15 mg / kg QD: p = 0.0044

[0381] Notes:

[0382] Compound 3 and AmB were given at 15 mg / kg daily for 7 days, IP post infection and similarly protected against CEA10 IA infection 14 days later. The Amb IP dose was 15 mg / kg, which was estimated to be at least up to 5 mg / kg IV. Since IP dosing was new for compound 3 and AmB, we wanted to understand the tolerability of both in an immunosuppressed mouse model.

[0383] Tolerance studies ( Fig.23): Healthy mice were divided into 3 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 them into neutrophil deficiency. The target dose of compound 3 and Anbisu was administered IP daily for 7 days, and the survival rate was monitored for 14 days.

[0384] Purpose: Tolerability of compound 3 and AmB IP administration in an immunodeficiency model

[0385] No infection, only tolerance study

[0386] n = 3 mice / group

[0387] Cyclophosphamide: 150 mg / kg on days -2 and +3

[0388] Cortisone acetate: 250 mg / kg on days -1 and +6

[0389] IP administration of the corresponding drugs continued for 7 days

[0390] Observe the mice for 14 days

[0391] Survival:

[0392] Compound 3 15 mg / kg QD: 66%

[0393] Compound 3 25 mg / kg QD: 100%

[0394] Compound 3 35 mg / kg QD: 100%

[0395] Compound 3 45 mg / kg QD: 66%

[0396] AmB 25mg / kg QD: 66%

[0397] Notes:

[0398] Clinically, 45mg / kg compound 3 and 25mg / kg AmB IP showed similar signs of lethargy and ruffled fur from D3 to D12. Compound 3 at 25mg / kg to 35mg / kg did not affect the mice's ability to eat, stay clean, or their activities throughout the experiment, but 45mg / kg did. We can use 35mg / kg compound 3 IP administration for 7 days in the infection model.

Claims

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

2. The compound according to claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein the compound is:

4. The compound according to claim 1, wherein the compound is:

5. The compound according to claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 1, wherein the compound is:

7. The compound according to claim 1, wherein the compound is:

8. A pharmaceutical composition comprising the compound according to any one of claims 1 to 7; and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is in an intravenous injection dosage form.

10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is an oral dosage form.

11. Use of a compound according to any one of claims 1 to 7 in the manufacture of a medicament for treating a fungal infection, the treatment comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 7 to a subject in need thereof.

12. The use according to claim 11, wherein the compound is administered intravenously.

13. The use according to claim 11, wherein the compound is administered orally.

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