Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
  • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
  • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
  • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration

Definitions

• Amphotericin B has potent and dose-dependent fungicidal activity against a broad range of fungal pathogens and has evaded resistance for over half a century.
• the fungicidal, as opposed to fungistatic, activity of AmB is essential in immunocompromised patients which lack a robust immune system to help clear an infection. Broad antifungal activity is especially important in critically ill patients when the identity of the pathogen is unknown and immediate empirical therapy is required.
• AmB is exceptionally toxic, which limits its use to low-dose protocols that often fail to eradicate disease.
• An AmB derivative that retains potent, broad spectrum, and resistance-evasive fungicidal activity but lacks dose-limiting toxicities would enable a new high-dose treatment paradigm with improved clinical efficacy.
• An aspect of the invention is a compound represented by Formula (I) or a
• X is -N(R 2 )-;
• R 1 is a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl; or R 1 and R 2 , together with the nitrogen to which they are attached, may form a substituted or unsubstituted 3- to lO-membered heterocyclic ring, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic;
• R 2 is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl;
• R 4 is selected from the group consisting of secondary amino, tertiary amino, amido, azido, isonitrile, nitro, urea, isocyanate, carbamate, and guanidinyl;
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl.
• An aspect of the invention is a compound represented by Formula (IV) or a
• X is -N(R 2 )-;
• R 1 is a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl; or R 1 and R 2 , together with the nitrogen to which they are attached, may form a substituted or unsubstituted 3- to lO-membered heterocyclic ring, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic;
• R 2 is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl;
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl
• R 6 is C(0)0R f ;
• R f is selected from the group consisting of 2-alken-l-yl, tert- butyl, benzyl and fluorenylmethyl.
• An aspect of the invention is a compound represented by Formula (V) or a
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl
• -XR 1 is selected from the group consisting of
• An aspect of the invention is a compound represented by Formula (II) or a pharmaceutically acceptable salt thereof:
• R 4 is selected from the group consisting of primary amino, secondary amino, tertiary amino, amido, azido, isonitrile, nitro, urea, isocyanate, carbamate, and guanidinyl; and R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl.
• An aspect of the invention is a compound represented by Formula (III) or a pharmaceutically acceptable salt thereof:
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl
• R 6 is -C(0)0R f ;
• R f is selected from the group consisting of 2-alken-l-yl, tert- butyl, benzyl and fluorenylmethyl.
• An aspect of the invention is a pharmaceutical composition, comprising a compound of the invention and a pharmaceutically acceptable carrier.
• An aspect of the invention is a method of treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention, thereby treating the fungal infection.
• An aspect of the invention is a method of making a Cl 6 urea derivative of C2’epi- Amphotericin B according to any one of the four transformations shown in Scheme 1 :
• each instance of R is independently selected from the group consisting of hydrogen, halogen, straight- and branched-chain alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl, hydroxyl, sulfhydryl, carboxyl, amino, amido, azido, nitro, cyano, aminoalkyl, and alkoxyl.
• Figure 1A represents chemical structures of amphotericin B, the primary fungal sterol - ergosterol, and the primary human sterol - cholesterol.
• Figure IB depicts a two-step“Sterol Sponge” model for the cytocidal action of AmB.
• Figure 2A represents chemical structures and biophysical activities of AmB, AmdeB,
• Figure 2B represents biophysical activities of AmB, AmdeB, C2’deOAmB, and
• Figure 2C represents ergosterol and cholesterol activities of AmB, AmdeB,
• Figure 3A is an X-ray crystal structure of A'-iodoacyl AmB.
• Figure 3B depicts a proposed structural model for AmB-Erg complex. A similar model is proposed for cholesterol.
• Figure 4 represents the 11 -step synthesis of C2’epiAmB from AmB.
• Figure 5A depicts sterol binding.
• Sterol sponges formed in vitro from AmB were titrated with ergosterol and analyzed by UV-Vis spectroscopy.
• Figure 5B depicts sterol binding. Sterol sponges formed in vitro from AmB were titrated with cholesterol and analyzed by UV-Vis spectroscopy.
• Figure 5C depicts sterol binding.
• Sterol sponges formed in vitro from C2’epiAmB were titrated with ergosterol and analyzed by UV-Vis spectroscopy.
• Figure 5D depicts sterol binding.
• Sterol sponges formed in vitro from C2’epiAmB were titrated with cholesterol and analyzed by UV-Vis spectroscopy.
• Figure 6 represents toxicity data of AmB-deoxycholate and C2’epiAmB-doxycholate in mice.
• Figure 7 represents toxicity data of AmBisome® compared directly with C2’epiAmB, as judged by renal genotoxicity biomarkers.
• Figure 8A depicts in vitro antifungal activity of AmB and C2’epiAmB against a broad range of fungal pathogens in a panel of Candida and Aspergillus isolates.
• Figure 8B depicts in vitro antifungal activity of AmB and C2’epiAmB against a broad range of fungal pathogens in a panel of Aspergillus isolates.
• Figure 8C depicts in vitro antifungal activity of AmB and C2’epiAmB against a broad range of fungal pathogens in a panel of clinically relevant invasive molds.
• Figure 9 depicts the MICs of AmB and C2'epiAmB against C. albicans with and without pre-complexation with ergosterol.
• Figure 10 represents the efficacy of AmB and C2’epiAmB in a mouse model of invasive candidiasis.
• Figure 11A represents a practical three-step synthesis of AmBUreas from AmB.
• Figure 11B depicts in vitro antifungal activity of several derivatives against a panel of clinical isolates.
• Figure 11C depicts in vitro antifungal activity of several derivatives against a wide range of clinically relevant pathogens.
• Figure 11D depicts in vitro antifungal activity of AmB, AmBAU and AmBTACBU against clinically relevant Candida species and challenging strains of A. fumigatus.
• Figure 12 represents the efficacy of AmB, AmBMU and AmBAU in a mouse model of invasive candidiasis.
• Figure 13 represents the efficacy of AmBCBU, AmBMEU, AmBAU, Fungizone®, and AmBisome® in a candidiasis mouse model.
• Figure 14 depicts the PK properties of AmB and AmBAU in mice, rats and dogs.
• Figure 15 depicts the binding of AmB or derivatives to ergosterol and cholesterol, and shows that AmBMU retains the capacity to bind cholesterol, which is consistent with the retained mammalian toxicity of AmBUreas.
• Figure 16 represents a hybrid AmB derivative, C2’epiAmBAU, with exceptional potency and minimal toxicity.
• Figure 17 represents hybrid C2’epiAmBUreas targeted for synthesis.
• Figure 18 depicts the comparison of in vitro antifungal activity of C2’epiAmBAU hybrid to AmB, C2’epiAmB and AmBAU.
• Figure 19 shows a clinically oriented screening funnel to identify the most promising C2'epiAmBUreas.
• Figure 20 depicts a systematic efficacy evaluation of high-dose C2’epiAmBUreas.
• Amphotericin B is a polyene macrolide with a mycosamine appendage, the complete compound having the following structure:
• AmB is generally obtained from a strain of Streptomyces nodosus. It is currently approved for clinical use in the United States for the treatment of progressive, potentially life- threatening fungal infections, including such infections as systemic or deep tissue candidiasis, aspergillosis, cryptococcosis, blastomycosis, coccidioidomycosis, histoplasmosis, and mucormycosis, among others. It is generally formulated for intravenous injection.
• Amphotericin B is commercially available, for example, as Fungizone® (Squibb), Amphocin® (Pfizer), Abelcet® (Enzon), and Ambisome® (Astellas). Due to its undesirable toxic side effects, dosing is generally limited to a maximum of about 1.0 mg/kg/day and total cumulative doses not to exceed about 3 g in humans.
• This large aggregate sits on the surface of lipid bilayers and rapidly extracts membrane sterols, which leads to cell death.
• Membrane permeabilization is not required. Based on this mechanism, a small molecule-based ligand-selective allosteric effect would enable selective binding of ergosterol over cholesterol and would eliminate the mammalian toxicity of AmB (in the form of C2’epiAmB). See
• the present invention discloses the KDS for the binding of both ergosterol and cholesterol to the AmB sterol sponge, which provides a quantitative and mechanistically-grounded biophysical parameter to guide rational optimization of the therapeutic index of this clinically significant natural product.
• the present invention relates, at least in part, to the discovery by the inventors of further derivatives of AmB which also are characterized by improved therapeutic index compared to AmB.
• the various derivatives, i.e., compounds of the invention can be semi-synthetic or fully synthetic.
• An aspect of the invention is the development of a new synthetic derivative of AmB that retains potent binding of ergosterol but shows no detectable binding of cholesterol.
• This derivative retains fungicidal potency against many yeasts and molds but shows zero detectable mammalian toxicity.
• This demonstrates that differential binding of ergosterol over cholesterol is possible and provides a non-toxic variant of AmB that preserves desirable antifungal properties.
• Compounds of the invention enable a new high-dose treatment strategy to eradicate life- threatening invasive fungal infections with a significantly improved safety profile.
• Compounds of the invention and pharmaceutical compositions of the invention are useful for inhibiting the growth of a fungus.
• an effective amount of a compound of the invention is contacted with a fungus, thereby inhibiting growth of the fungus.
• a compound of the invention, or a pharmaceutically acceptable salt thereof is added to or included in tissue culture medium.
• Compounds of the invention and pharmaceutical compositions of the invention are useful for the treatment of fungal infections in a subject.
• a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof, thereby treating the fungal infection.
• Yeasts are eukaryotic organisms classified in the kingdom Fungi. Fungi include yeasts, molds, and larger organisms including mushrooms. Yeasts and molds are of clinical relevance as infectious agents. Yeasts are typically described as budding forms of fungi. Of particular importance in connection with the invention are species of yeast that can cause infections in mammalian hosts. Such infections most commonly occur in immunocompromised hosts, including hosts with compromised barriers to infection (e.g., burn victims) and hosts with compromised immune systems (e.g., hosts receiving chemotherapy or immune suppressive therapy, and hosts infected with HIV). Pathogenic yeasts include, without limitation, various species of the genus Candida, as well as of Cryptococcus. Of particular note among pathogenic yeasts of the genus Candida are C.
• Cryptococcus specifically includes Cryptococcus neoformans.
• Yeast can cause infections of mucosal membranes, for example oral, esophageal, and vaginal infections in humans, as well as infections of bone, blood, urogenital tract, and central nervous system. This list is exemplary and is not limiting in any way.
• a number of fungi can cause infections in mammalian hosts. Such infections most commonly occur in immunocompromised hosts, including hosts with
• Pathogenic fungi include, without limitation, species of
• Microsporum and Epidermophyton.
• A. fumigatus A. flavus, A. niger, H. capsulatum, C. immitis, and B. dermatitidis.
• Fungi can cause systemic and deep tissue infections in lung, bone, blood, urogenital tract, and central nervous system, to name a few. Some fungi are responsible for infections of the skin and nails.
• Amides (RC(0)NR 2 ) and esters (RC(O)OR’) are classes of acyl compounds, as are ketones (RC(O)R) and aldehydes (RC(O)H).
• Non-limiting examples of acyl groups include formyl, acetyl, propionyl, and benzyl.
• alkenyl and“alkynyl” are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described herein, but that contain at least one double or triple bond, respectively.
• alkoxy means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, /7-butoxy, pentyloxy, and hexyloxy.
• Representative examples of alkoxycarbonyl include, but are not limited to, methoxy carbonyl, ethoxycarbonyl, and /7-butoxycarbonyl .
• alkyl means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms.
• Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n- hexyl.
• alkyl is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, and cycloalkyl (alicyclic) groups.
• a straight-chain or branched-chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and alternatively, about 20 or fewer.
• a straight-chain or branched-chain alkyl has about 10 or fewer carbon atoms in its backbone.
• a straight- chain alkyl has 1 to 6 carbon atoms in its backbone.
• a branched-chain alkyl has 3 to 8 carbon atoms in its backbone.
• Representative examples of linear and branched- chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, /.v -propyl, n-butyl, sec-butyl, /.vo- butyl, c/7-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl.
• Cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure. In certain embodiments, cycloalkyls have 3, 4, 5, 6, or 7 carbons in the ring structure.
• Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
• alkylcarbonyl means an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein.
• alkylcarbonyl include, but are not limited to, acetyl, 1 -oxopropyl, 2,2-dimethyl- l-oxopropyl, l-oxobutyl, and l-oxopentyl.
• alkylcarbonyloxy means an alkylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• alkylcarbonyloxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, and /tw-butylcarbonyloxy.
• alkylthio means an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfur atom.
• alkylthio include, but are not limited, methylthio, ethylthio, /er/-butylthio, and hexylthio.
• arylthio “alkenylthio”, and“arylalkylthio,” for example, are likewise defined in a
• ami do refers to a moiety that may be represented by the general formula:
• R 10 and R 11 each independently represent hydrogen or a substituted or unsubstituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, alkenyl, cycloalkenyl, aminoalkyl, aryl, heteroaryl, aralkyl, and heteroaralkyl.
• amido include those for which R 10 is hydrogen, and R 11 is selected from methyl, ethyl, propyl, isopropyl,
• amido examples include those for which R 10 is hydrogen, and R 11 is selected from -CH2NH2, -CH2N(CH3)2, and -CH(NH2)(CH2)nNH2, where n is an integer 1-6.
• R 10 is hydrogen
• R 11 is selected from
• amino and“amine” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:
• R 20 , R 21 , and R 22 each independently represent a hydrogen, an alkyl, an alkenyl, - (CH 2 ) 111- R 61 ; or R 20 and R 21 , taken together with the N atom to which they are attached, complete a heterocycle having from 4 to 10 atoms in the ring structure, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic; R 61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8.
• R 20 and R 21 each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)m-R 61 .
• the term“alkylamine” includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R 20 and R 21 is an alkyl group.
• Nonlimiting examples of amino groups include -NH 2 , -N(H)CH3,
• amino is -N(H)CH3.
• aminoalkyl as used herein, means an amino group, as defined herein, appended to the parent molecular moiety through an alkyl group, also as defined herein.
• aromatic refers to a planar monocyclic or polycyclic structure characterized by a cyclically conjugated molecular moiety containing 4n+2 electrons, wherein n is the absolute value of an integer.
• Aromatic groups comprising only carbon atoms in their ring structure are termed“aryl” groups.
• Aromatic groups comprising one or more heteroatoms in their ring structure are termed“heteroaryl” or“heteroaromatic” groups.
• Aromatic groups containing fused, or joined, rings also are referred to as polycyclic aromatic groups.
• bicyclic aromatic groups containing heteroatoms in a hydrocarbon ring structure are referred to as bicyclic heteroaryl groups.
• Examples of 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms include, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
• Non-limiting examples of polycyclic aromatic and heteroaromatic groups include quinoline, isoquinoline, carbazole, naphthalene, anthracene, and pyrene.
• aryl groups of the invention can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkenyl, alkoxy,
• aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are“fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
• arylcarbonyloxy means an arylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• Representative examples of arylcarbonyloxy include, but are not limited to, phenylcarbonyloxy.
• arylene is art-recognized, and, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms of an aryl ring, as defined above.
• arylalkyl or“aralkyl”, as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
• arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3- phenylpropyl, and 2-naphth-2-ylethyl.
• R 30 and R 31 each independently represent hydrogen or a substituted or unsubstituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, alkenyl, cycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl.
• Nonlimiting examples of carbamate include those for which R 30 is hydrogen, and R 31 is selected from methyl, ethyl, propyl,
• cyano means a -CN group.
• cycloalkylalkyl refers to a cycloalkyl group, as defined herein, appended to the parent molecular moiety through an alkyl group, also as defined herein.
• guanidinyl refers to a moiety that may be represented by the general formula:
• R 40 , R 41 , R 42 , and R 43 each independently represent hydrogen or a substituted or unsubstituted group selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, alkenyl, cycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl.
• R 40 , R 41 , R 42 , and R 43 each represent hydrogen.
• halo or“halogen” means -F, -Cl, -Br, or -I.
• haloalkyl means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
• Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.
• heteroaryl means a heteroaryl, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
• Representative examples of heteroarylalkyl include, but are not limited to, pyri din-3 -ylmethyl and 2-(thien-2- yl)ethyl.
• heteroaryl includes aromatic ring systems, including, but not limited to, monocyclic, bicyclic, and tricyclic rings, and have 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur.
• heteroaryl azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3- d]pyrimidinyl, pyrazolo[3,4-d]
• alkylcarbonyl alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl, amido, amino, carboxy, cyano, formyl, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, phosphinyl, silyl and silyloxy.
• heteroatom is art-recognized and refers to an atom of any element other than carbon or hydrogen.
• Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur, and selenium.
• heterocyclyl refers to non-aromatic ring systems, including, but not limited to, monocyclic, bicyclic, tricyclic and spirocyclic rings, which can be completely saturated or which can contain one or more units of unsaturation (for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system) and have 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur.
• heterocyclic rings azepines, azetidinyl, morpholinyl, oxopiperidinyl, oxopyrrolidinyl, piperazinyl, piperidinyl, pyrrolidinyl, quinicludinyl, thiomorpholinyl, tetrahydropyranyl and tetrahydrofuranyl.
• the heterocyclyl groups may be substituted with 0, 1, 2, 3, 4 or 5 substituents independently selected from alkenyl, alkoxy, alkoxy carbonyl, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl, amido, amino, carboxy, cyano, formyl, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, phosphinyl, silyl and silyloxy.
• hydroxyl means an -OH group.
• hydroxyalkyl means at least one hydroxy group, as defined herein, is appended to the parent molecular moiety through an alkyl group, as defined herein.
• Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2- hydroxy ethyl, 3-hydroxypropyl, 2,3-dihydroxypentyl, and 2-ethyl-4-hydroxyheptyl.
• nitro means a -NO2 group.
• silyl H3S1-
• hydrocarbyl hydrocarbyl derivatives of the silyl (H3S1-) group (i.e., (hydrocarbyl)3Si-), wherein a hydrocarbyl groups are univalent groups formed by removing a hydrogen atom from a hydrocarbon, e.g., ethyl, phenyl.
• the hydrocarbyl groups can be combinations of differing groups which can be varied in order to provide a number of silyl groups, such as trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]methyl (SEM).
• TMS trimethylsilyl
• TDPS tert-butyldiphenylsilyl
• TIPS triisopropylsilyl
• SEM [2-(trimethylsilyl)ethoxy]methyl
• silyloxy means a silyl group, as defined herein, is appended to the parent molecule through an oxygen atom.
• sulfhydryl means a -SH group.
• sulfonyl is art-recognized and refers to -SO2 .
• urea means a moiety that may be represented by the general formula:
• R 1 is a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl; or -N(R 1 )(R 2 ) may represent a substituted or unsubstituted 3- to lO-membered heterocyclic ring, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic;
• R 2 is independently hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl.
• triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to
• triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, /Moluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
• Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, /Moluenesulfonyl and methanesulfonyl, respectively.
• a more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry ; this list is typically presented in a table entitled Standard List of Abbreviations.
• compositions of the invention may exist in particular geometric or stereoisomeric forms.
• polymers of the invention may also be optically active.
• the invention contemplates all such compounds, including cis- and trans-isomers, R- and L-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
• Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
• a particular enantiomer of compound of the invention may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
• the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
• substitution or“substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
• the term“substituted” is also contemplated to include all permissible substituents of organic compounds.
• the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
• Illustrative substituents include, for example, alkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl, (heterocyclyl)alkyl, (cycloalkyl)alkyl, alkoxy, aryloxy,
• arylsulfonyloxy alkylthio, arylthio, amido, amino, carboxy, cyano, formyl, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, phosphinyl, acyl, acyloxy, silyl and silyloxy.
• the permissible substituents may be one or more and the same or different for appropriate organic compounds.
• the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
• protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
• protecting groups examples include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
• the field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M Protective Groups in Organic Synthesis, 2 nd ed.; Wiley: New York, 1991). Protected forms of the inventive compounds are included within the scope of this invention.
• the invention provides a number of derivatives of AmB, including derivatives characterized by (i) certain modifications at Cl 3; (ii) certain N modifications at C3’; (iii) certain urea derivatives at C16; and (iv) the combination of certain urea derivatives at C16 and
• the invention provides a number of derivatives of AmB, including derivatives characterized by (i) certain modifications at Cl 3; (ii) certain N modifications at C3’; (iii) certain urea derivatives at Cl 6; and (iv) the combination of certain urea derivatives at Cl 6 and C2’epiAmB.
• An aspect of the invention is a compound represented by Formula (I) or a
• X is -N(R 2 )-;
• R 1 is a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl; or R 1 and R 2 , together with the nitrogen to which they are attached, may form a substituted or unsubstituted 3- to lO-membered heterocyclic ring, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic;
• R 2 is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl;
• R 4 is selected from the group consisting of secondary amino, tertiary amino, amido, azido, isonitrile, nitro, urea, isocyanate, carbamate, and guanidinyl;
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl.
• R 2 is hydrogen
• -XR 1 is selected from the group consisting of -NHCH2CH3, - NHCH2CH2CH3, -NHCH(CH 3 )2, -NH(2-butyl), -NHcyclopropyl, -NHcyclobutyl, -
• R a is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl;
• R b is hydrogen, halogen, hydroxyl, sulfhydryl, nitro, cyano, or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, carboxyl, acyl, acyloxy, amino, amido, azido, aminoalkyl, and alkoxyl;
• R c is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, and aminoalkyl; and
• R d is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl,
• heteroaralkyl acyl, amino, amido, aminoalkyl, and alkoxyl; or, when -
• R a and R d together with the nitrogen to which they are attached, may form a substituted or unsubstituted 3- to lO-membered heterocyclic ring, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic.
• -XR 1 is selected from the group consisting of
• -XR 1 is selected from the group consisting of *
• -XR 1 is selected from the group consisting of -NHCH2CH3, -
• -XR 1 is selected from the group consisting of
• R 4 is secondary amino
• R 4 is tertiary amino
• R 4 is amido
• R 4 is azido
• R 4 is isonitrile
• R 4 is nitro
• R 4 is urea
• R 4 is isocyanate
• R 4 is carbamate
• R 4 is guanidinyl
• R 4 is selected from the group consisting of
• R e is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl.
• R 5 is hydrogen
• R 5 is alkyl
• R 5 is haloalkyl
• An aspect of the invention is a compound represented by Formula (IV) or a
• X is -N(R 2 )-;
• R 1 is a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl; or R 1 and R 2 , together with the nitrogen to which they are attached, may form a substituted or unsubstituted 3- to lO-membered heterocyclic ring, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic;
• R 2 is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl;
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl
• R 6 is C(0)0R f ;
• R f is selected from the group consisting of 2-alken-l-yl, tert- butyl, benzyl and fluorenylmethyl.
• R 2 is hydrogen
• -XR 1 is selected from the group consisting of -NHCH2CH3, - NHCH2CH2CH3, -NHCH(CH 3 )2, -NH(2-butyl), -NHcyclopropyl, -NHcyclobutyl, -
• R a is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl;
• R b is hydrogen, halogen, hydroxyl, sulfhydryl, nitro, cyano, or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, carboxyl, acyl, acyloxy, amino, amido, azido, aminoalkyl, and alkoxyl;
• R c is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, and aminoalkyl; and
• R d is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl,
• heteroaralkyl acyl, amino, amido, aminoalkyl, and alkoxyl; or, when -
• R a and R d together with the nitrogen to which they are attached, may form a substituted or unsubstituted 3- to lO-membered heterocyclic ring, wherein said ring is monocyclic, bicyclic, tricyclic, or spirocyclic.
• -XR 1 is selected from the group consisting of
• -XR 1 is selected from the group consisting of
• -XR 1 is selected from the group consisting of
• -XR 1 is selected from the group consisting of
• R 5 is hydrogen
• R 5 is alkyl
• R 5 is haloalkyl
• R f is 2-alken-l-yl.
• R f is /er/-butyl.
• R f is benzyl
• R f is fluorenylmethyl
• An aspect of the invention is a compound represented by Formula (V) or a pharmaceutically acceptable salt thereof:
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl; -XR 1 is selected from the group consisting of
• R 5 is hydrogen
• R 5 is alkyl
• R 5 is haloalkyl
• R 5 is hydrogen; and -XR 1 is H
• An aspect of the invention is a compound represented by Formula (II) or a pharmaceutically acceptable salt thereof:
• R 4 is selected from the group consisting of primary amino, secondary amino, tertiary amino, amido, azido, isonitrile, nitro, urea, isocyanate, carbamate, and guanidinyl; and R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl.
• R 4 is primary amino
• R 4 is secondary amino. In certain embodiments, R 4 is tertiary amino.
• R 4 is amido
• R 4 is azido
• R 4 is isonitrile
• R 4 is nitro
• R 4 is urea
• R 4 is isocyanate
• R 4 is carbamate
• R 4 is guanidinyl
• R 4 is selected from the group consisting of
• R e is hydrogen or a substituted or unsubstituted group selected from the group consisting of alkyl, cycloalkyl, (cycloalkyl)alkyl, heterocyclyl, (heterocyclyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, amino, amido, aminoalkyl, and alkoxyl.
• R 5 is hydrogen
• R 5 is alkyl
• R 5 is haloalkyl
• An aspect of the invention is a compound represented by Formula (III) or a
• R 5 is selected from the group consisting of hydrogen, alkyl, and haloalkyl
• R 6 is -C(0)0R f ;
• R f is selected from the group consisting of 2-alken-l-yl, tert- butyl, benzyl and fluorenylmethyl.
• R 5 is hydrogen
• R 5 is alkyl
• R 5 is haloalkyl
• R f is 2-alken-l-yl.
• R f is tert- butyl
• R f is benzyl
• R f is fluorenylmethyl
• the invention also provides pharmaceutical compositions and methods for making same.
• An aspect of the invention is a pharmaceutical composition comprising a compound of the invention; and a pharmaceutically acceptable carrier.
• the invention is a pharmaceutical composition, comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier means one or more compatible solid or liquid filler, diluent, or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
• carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
• the components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
• the pharmaceutical composition is an intravenous dosage form.
• the pharmaceutical composition is an oral dosage form.
• the pharmaceutical composition is a lyophilized preparation of a liposome-intercalated or liposome -encapsulated active compound.
• the pharmaceutical composition is a lipid complex of the compound in aqueous suspension.
• compositions of the invention are meant to be exemplary and are not limiting.
• the method comprises placing a compound of the invention, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.
• Compounds of the invention are useful for inhibiting growth of fungi and yeast, including, in particular, fungi and yeast of clinical significance as pathogens.
• the compounds of the invention have improved therapeutic indices compared to AmB, thereby providing agents with improved efficacy and reduced toxicity as compared to AmB.
• Compounds of the invention are useful in methods of treating fungal and yeast infections, including, in particular, systemic fungal and yeast infections.
• Compounds of the invention are also useful in the manufacture of medicaments for treating fungal and yeast infections, including, in particular, systemic fungal and yeast infections.
• the invention further provides the use of compounds of the invention for the treatment of fungal and yeast infections, including, in particular, systemic fungal and yeast infections.
• An aspect of the invention is a method of treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention, thereby treating the fungal infection.
• inhibit or inhibiting means reduce by an objectively measureable amount or degree compared to control. In one embodiment, inhibit or inhibiting means reduce by at least a statistically significant amount compared to control. In one embodiment, inhibit or inhibiting means reduce by at least 5 percent compared to control. In various individual embodiments, inhibit or inhibiting means reduce by at least 10, 15, 20, 25, 30, 33, 40, 50, 60, 67, 70, 75, 80, 90, or 95 percent (%) compared to control.
• the terms“treat” and“treating” refer to performing an intervention that results in (a) preventing a condition or disease from occurring in a subject that may be at risk of developing or predisposed to having the condition or disease but has not yet been diagnosed as having it; (b) inhibiting a condition or disease, e.g., slowing or arresting its development; or (c) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease.
• the terms“treating” and“treat” refer to performing an intervention that results in (a) inhibiting a condition or disease, e.g., slowing or arresting its development; or (b) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease.
• the terms“treating” and“treat” refer to performing an intervention that results in (a) inhibiting a fungal infection, e.g., slowing or arresting its development; or (b) relieving or ameliorating a fungal infection, e.g., causing regression of the fungal infection.
• A“fungal infection” as used herein refers to an infection in or of a subject with a fungus as defined herein.
• the term“fungal infection” includes a yeast infection.
• a “yeast infection” as used herein refers to an infection in or of a subject with a yeast as defined herein.
• a“subject” refers to a living mammal.
• a subject is a non-human mammal, including, without limitation, a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, cat, dog, pig, horse, cow, or non-human primate.
• a subject is a human.
• a“subject having a fungal infection” refers to a subject that exhibits at least one objective manifestation of a fungal infection.
• a subject having a fungal infection is a subject that has been diagnosed as having a fungal infection and is in need of treatment thereof. Methods of diagnosing a fungal infection are well known and need not be described here in any detail.
• a“subject having a yeast infection” refers to a subject that exhibits at least one objective manifestation of a yeast infection.
• a subject having a yeast infection is a subject that has been diagnosed as having a yeast infection and is in need of treatment thereof. Methods of diagnosing a yeast infection are well known and need not be described here in any detail.
• the compound is administered intravenously.
• the compound is administered orally.
• the compound is administered systemically.
• the compound is administered parenterally.
• the compound is administered intraperitoneally.
• the compound is administered enterally.
• the compound is administered intraocularly.
• the compound is administered topically.
• Additional routes of administration of compounds of the invention are contemplated by the invention, including, without limitation, intravesicularly (urinary bladder), pulmonary, and intrathecally.
• the phrase“effective amount” refers to any amount that is sufficient to achieve a desired biological effect.
• the phrase“therapeutically effective amount” refers to an amount that is sufficient to achieve a desired therapeutic effect, e.g., to treat a fungal or yeast infection.
• a therapeutically effective amount can, in general, be initially determined from in vitro studies, animal models, or both in vitro studies and animal models.
• In vitro methods are well known and can include determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), concentration at which growth is inhibited by 50 percent (ICso), concentration at which growth is inhibited by 90 percent (IC90), and the like.
• a therapeutically effective amount can also be determined from human data for compounds of the invention which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents (e.g., AmB). Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound.
• a therapeutically effective amount for use in human subjects can be initially determined from in vitro studies, animal models, or both in vitro studies and animal models.
• a therapeutically effective amount for use in human subjects can also be determined from human data for compounds of the invention which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents (e.g., AmB). Higher doses may be required for parenteral administration.
• the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
• Compounds of the invention can be combined with other therapeutic agents.
• the compound of the invention and other therapeutic agent may be administered simultaneously or sequentially.
• the other therapeutic agents When the other therapeutic agents are administered simultaneously, they can be administered in the same or separate formulations, but they are administered substantially at the same time.
• the other therapeutic agents are administered sequentially with one another and with compound of the invention, when the administration of the other therapeutic agents and the compound of the invention is temporally separated. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
• Examples of other therapeutic agents include other antifungal agents, including AmB, as well as other antibiotics, anti-viral agents, anti-inflammatory agents, immunosuppressive agents, and anti-cancer agents.
• an“effective amount” refers to any amount that is sufficient to achieve a desired biological effect.
• an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject.
• the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound of the invention being administered, the size of the subject, or the severity of the disease or condition.
• One of ordinary skill in the art can empirically determine the effective amount of a particular compound of the invention and/or other therapeutic agent without necessitating undue experimentation.
• a maximum dose that is, the highest safe dose according to some medical judgment. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. “Dose” and“dosage” are used interchangeably herein.
• daily oral doses of active compounds will be, for human subjects, from about
• intravenous administration of a compound of the invention may typically be from 0.1 mg/kg/day to 20 mg/kg/day. Intravenous dosing thus may be similar to, or advantageously, may exceed maximal tolerated doses of AmB. Intravenous dosing also may be similar to, or advantageously, may exceed maximal tolerated daily doses of AmB. Intravenous dosing also may be similar to, or advantageously, may exceed maximal tolerated cumulative doses of AmB. Intravenous dosing also may be similar to, or advantageously, may exceed maximal recommended doses of AmB. Intravenous dosing also may be similar to, or advantageously, may exceed maximal recommended daily doses of AmB. Intravenous dosing also may be similar to, or advantageously, may exceed maximal recommended cumulative doses of AmB. Intravenous dosing also may be similar to, or advantageously, may exceed maximal recommended cumulative doses of AmB.
• the therapeutically effective amount can be initially determined from animal models.
• a therapeutically effective dose can also be determined from human data for compounds of the invention which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration.
• the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound.
• compositions of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
• Amphotericin B is commercially available in a number of formulations, including deoxycholate-based (sometimes referred to as desoxycholate-based) formulations and lipid-based (including liposomal) formulations.
• Amphotericin B derivative compounds of the invention similarly may be formulated, for example, and without limitation, as deoxycholate-based formulations and lipid-based (including liposomal) formulations.
• an effective amount of the compound of the invention can be administered to a subject by any mode that delivers the compound of the invention to the desired surface.
• Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, intravenous, intramuscular, intraperitoneal, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal, pulmonary (e.g., inhalation), and topical.
• the compounds of the invention generally may be formulated similarly to AmB.
• a compound of the invention can be formulated as a lyophilized preparation with deoxycholic acid, as a lyophilized preparation of liposome- intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a cholesteryl sulfate complex.
• Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.
• the compounds i.e., compounds of the invention, and other therapeutic agents
• the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
• Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions or may be administered without any carriers.
• oral dosage forms of the above component or components may be chemically modified so that oral delivery of the derivative is efficacious.
• the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine.
• the increase in overall stability of the component or components and increase in circulation time in the body examples include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
• the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
• the stomach the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
• One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine.
• the release will avoid the deleterious effects of the stomach environment, either by protection of the compound of the invention (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.
• a coating impermeable to at least pH 5.0 is essential.
• cellulose acetate trimellitate hydroxypropylmethylcellulose phthalate
• HPMCP 50 HPMCP 55
• PVAP polyvinyl acetate phthalate
• Eudragit L30D Aquateric
• CAP cellulose acetate phthalate
• Eudragit S Eudragit S, and shellac.
• a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
• Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell may be used.
• the shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.
• the therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm.
• the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
• the therapeutic could be prepared by compression.
• Colorants and flavoring agents may all be included.
• the compound of the invention (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
• diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
• Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
• Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
• Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
• Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid
• carboxymethyl cellulose, natural sponge and bentonite may all be used.
• Another form of the disintegrants are the insoluble cationic exchange resins.
• Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
• Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).
• MC methyl cellulose
• EC ethyl cellulose
• CMC carboxymethyl cellulose
• Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
• Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
• the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
• Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
• anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
• Cationic detergents which can be used and can include benzalkonium chloride and benzethonium chloride.
• Non- ionic detergents that could be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound of the invention or derivative either alone or as a mixture in different ratios.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added.
• Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• pulmonary delivery of the compounds of the invention is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
• inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., IntJ Pharmaceutics 63: 135-144 (1990) (leuprolide acetate); Braquet et al J Cardiovasc Pharmacol l3(suppl. 5): 143-146 (1989) (endothelin-l); Hubbard et al , AhhaI IntMed 3:206-212 (1989)
• Contemplated for use in the practice of this 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.
• Some specific examples of commercially available devices suitable for the practice of this 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.
• each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
• Chemically modified compound of the invention may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.
• Formulations suitable for use with a nebulizer will typically comprise compound of the invention (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active compound of the invention per mL of solution.
• the formulation may also include a buffer and a simple sugar (e.g., for compound of the invention stabilization and regulation of 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 in forming the aerosol.
• Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compound of the invention (or derivative) suspended in a propellant with the aid of a surfactant.
• the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,
• Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
• Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing compound of the invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
• the compound of the invention (or derivative) should advantageously be prepared in particulate form with an average particle size of less than 10 micrometers (pm), most preferably 0.5 to 5 pm, for most effective delivery to the deep lung.
• Nasal delivery of a pharmaceutical composition of the present invention is also contemplated.
• Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
• Formulations for nasal delivery include those with dextran or cyclodextran.
• a useful device is a small, hard botle to which a metered dose sprayer is attached.
• the metered dose is delivered by drawing the
• composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed.
• the chamber is compressed to administer the pharmaceutical composition of the present invention.
• the chamber is a piston arrangement. Such devices are commercially available.
• a plastic squeeze botle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used.
• the opening is usually found in the top of the botle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation.
• the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.
• the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may 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 which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• a suitable vehicle e.g., sterile pyrogen-free water
• the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• 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.
• compositions also may comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
• Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin.
• the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as
• compositions are suitable for use in a variety of drug delivery systems.
• Langer R Science 249: 1527-33 (1990), which is incorporated herein by reference.
• the compounds of the invention and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
• the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
• Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic.
• such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
• Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a 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).
• the therapeutic agent(s), including specifically but not limited to the compound of the invention, may be provided in particles.
• Particles as used herein means nanoparticles or microparticles (or in some instances larger particles) which can consist in whole or in part of the compound of the invention or the other therapeutic agent(s) as described herein.
• the particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating.
• the therapeutic agent(s) also may be dispersed throughout the particles.
• the therapeutic agent(s) also may be adsorbed into the particles.
• the particles may be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc.
• the particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof.
• the particles may be microcapsules which contain the compound of the invention in a solution or in a semi-solid state.
• the particles may be of virtually any shape.
• Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s).
• Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired.
• Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney H S et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),
• the therapeutic agent(s) may be contained in controlled release systems.
• controlled release is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations.
• 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 a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period.
• “delayed release” is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be“sustained release.”
• Long-term sustained release implant may be particularly suitable for treatment of chronic conditions.
• Long-term release as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient 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.
• the invention provides a number of derivatives of AmB, including derivatives characterized by (i) certain modifications at Cl 3; (ii) certain N modifications at C3’; (iii) certain urea derivatives at C16; and (iv) the combination of certain urea derivatives at C16 and
• the invention describes a synthesis platform to make atomistic modifications of AmB, which led to the discovery that sterol binding, rather than membrane permeabilization, primarily drives cytocidal action.
• a new method for site-selective modification of AmB involves electronic tuning of acylation reagents to achieve site-discriminating transition states for acyl transfer which achieved site-selective acylations of the 10 hydroxyl groups appended to AmB. See Wilcock, B. C. et al., Nat Chem 2012, 4 (12), 996-1003, the teachings of which are incorporated herein by reference.
• the highly complex macrolide skeleton of AmB is amenable to a tandem sequence involving Curtius rearrangement at Cl 6 and trapping the resulting isocyanate by the C15-OH. This generates an isolable but conformationally strained and thus“spring-loaded” oxazolidinone intermediate poised for one-step late-stage
• An aspect of the invention is a method of making a Cl 6 urea derivative of C2’epi- Amphotericin B according to any one of the four transformations shown in Scheme 1 :
• each instance of R is independently selected from the group consisting of hydrogen, halogen, straight- and branched-chain alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl, hydroxyl, sulfhydryl, carboxyl, amino, amido, azido, nitro, cyano, aminoalkyl, and alkoxyl.
• C2’deOAmB The resulting derivative, C2’epiAmB (FIG. 2A), selectively binds ergosterol and exerts cytocidal action against fungal but not human cells. Notably, C2’epiAmB differs from AmB only in the stereochemistry at a single atom.
• C2’epiAmB is significantly less toxic than AmBisome ® in mice.
• C2’epiAmB is non-toxic to human red blood cells, primary hREC, mice, and rats up to the highest dose tested.
• C2’epiAmB In vitro antifungal activity of C2’epiAmB was compared with that of AmB against an extensive series of Candida and Aspergillus clinical isolates (FIG. 8A) at Evotec (Oxfordshire, UK). C2’epiAmB showed good activity against many Candida and several Aspergillus strains. However, there were several strains of A. fumigatus (AF293, A1163, and ATC204305), for which C2’epiAmB was 4-fold less potent than AmB, and in one strain (AF91) C2’epiAmB was >32 times less potent.
• C2’epiAmB was also sent to the US national Fungus Testing Laboratory at UT-San Antonio for antifungal testing against an extended panel of especially challenging 40 Aspergillus clinical isolates, including azole- resistant A. fumigatus, A. flavus, and A. terreus (FIG. 8B).
• C2’epiAmB was found to be 2-16 times less potent than AmB (average 5.6-fold less potent across all 40 strains).
• Steinbach and Burke directly compared the activity of AmB, AmBisome ® , caspofungin, voriconazole, and C2’epiAmB against an even broader panel of clinically relevant invasive molds (FIG. 8C). These studies again showed good antifungal potency for C2’epiAmB against many strains, including a pan-azole resistant strain (F14196), but also important opportunities for improved activity against Aspergillus.
• C2’epiAmB primarily kills cells via the same sterol sponge mechanism
• the C2’epiAmB sponge was similarly pre- complexed with ergosterol (FIG. 9).
• the same reduction in potency for AmB and C2’epiAmB upon ergosterol pre-complexation was observed.
• C2’epiAmB similarly kills yeast primarily via sterol binding, and, by extension, the new compounds targeted in this application are expected to have a similar barrier to fungal resistance that has been observed for the past 50+ years with AmB.
• Example 5 Non-toxic dose-dependent efficacy in murine invasive candidiasis
• C2’epiAmB is a unique antifungal agent with potent fungicidal activity against several Candida and Aspergillus strains and no detectable mammalian toxicity, a first for an amphotericin derivative.
• C2’epiAmB also has some important limitations with respect to potency and pathogen scope.
• the next plan is to develop a new series of “hybrid” derivatives designed to improve the antifungal potency and pathogen scope of
• AmB urea derivatives modified at Cl 6 have shown to substantially increase antifungal activity in vitro and in vivo relative to AmB. These compounds are orders of magnitude more water soluble than AmB, which may in part account for their improved potency. These urea derivatives evaded pathogen resistance and also displayed excellent PK/PD properties in mice, rats, and dogs. However, these derivatives had unacceptable toxicities. Thus, the toxicity- eliminating modification found in C2’epiAmB was combined with the efficacy-promoting modifications at Cl 6 to develop a new class of hybrid polyene fungicidal agents that are both non-toxic and exceptionally effective in eradicating invasive fungal infections.
• AmBAU AmBAU, AmBMU, and AmBCU
• AmBisome ® a series of AmBUreas
• C2’epiAmB a series of AmBUreas
• caspofungin a series of AmBUreas
• voriconazole against a wide range of clinically relevant pathogens, including AmB-resistant Scedosporium strains (FIG. 11C).
• AmBAU showed excellent potency, equal if not better than that shown by AmB across a wide range of pathogens, importantly it was active against the recalcitrant strain Scedosporium prolificans.
• AmBAU proved to be exceptionally effective when administered intraperitoneally in a murine model of invasive candidiasis (FIG. 12).
• AmB was delivered as a non-deoxycholate complex. The lack of solubility likely accounts for the atypical lack of dose-response observed for AmB in these experiments.
• These AmBUreas were also tested via intravenous administration in a similar model at EvoTec (Oxfordshire, UK), and their activities were compared directly with IV AmB- deoxycholate (Fungizone ® ) and liposomal AmB (AmBisome ® ) (FIG. 13).
• AmBAU This was equal to the activity of IV Fungizone ® delivered at its MTD (1.5 mg/kg), and superior to IV AmBisome ® (2.5 mg/kg). Most importantly, complete sterilization was achieved with AmBAU at 16 mg/kg. AmBAU demonstrated favorable PK/PD properties in mice, rats, dogs (FIG. 14), and had a similar capacity to evade pathogen resistance as AmB.
• AmBUreas were also less toxic than AmB in vitro and in vivo, but were less than the complete elimination of toxicity observed with C2’epiAmB (FIGs. 6 and 7).
• the minimum toxic concentrations (MTC) against primary hRECs are 2.4 mM for AmB, 11.3 mM for AmBAU, 44.4 mM for AmBMU, and >80 mM for C2’epiAmB.
• MTC minimum toxic concentrations
• In IV-injected mice death was observed at 32 mg/kg for AmBAU, whereas all mice treated with C2’epiAmB at 128 mg/kg survived.
• both AmBMU and AmBAU caused significant toxicity at 6 mg/kg, precluding further development. As described above, no such toxicity was observed in the same rats at the highest tested dose of C2’epiAmB (17.5 mg/kg) (FIG. 6).
• C2’epiAmB showed retained binding to ergosterol but no detectable binding to cholesterol and no mammalian toxicity. It was reasoned that the lack of cholesterol binding in C2’epiAmB to a ligand-selective allosteric effect was caused by epimerization of the C2’ stereocenter (see Example 1), and thus predict that the biophysical effects associated with C2’ -epimerization should be transposable to other AmB derivatives.
• Example 7 Synthesis of a new series of AmB derivatives that hybridize the toxicity- eliminating epimerization at C2’ with efficacy-promoting aminoalkylurea modifications at C16
• This oxazolidinone intermediate will then be subdivided into 20 mg batches, and condensed with a collection of small alkyl diamines (obtained from commercial sources or synthesized using established methods), to yield new targeted hybrid C2’epi AmBUreas (representative examples in FIG. 17) This places the diversification step last in the sequence and employ a scalable, accessible and stable oxazolidinone intermediate, substantially maximizing the overall efficiency of this discovery program.
• Each derivative will be purified by reverse-phase HPLC, using the same methods that we previously employed to purify the corresponding AmB ureas. Davis, S. A., et al, Nat Chem Biol 2015, 11 (7), 481-7. The structure of each product will be unambiguously confirmed via a standard suite of one- and two-dimensional 'H and 13 C NMR techniques (COSY, HMBC, HMQC, NOESY) as well as high resolution mass spectroscopy, as previously done with the AmBEheas. Purity of each product will be judged by analytical HLPC at three different wavelengths (406, 383, 254nm), with a cut-off of 95% purity in each case. Compounds will be stored as dry powders under inert atmospheres in foil-wrapped vials, and shipped on dry ice to the Steinbach and Andes labs.
• FIG. 20 depicts a systematic efficacy evaluation of high-dose C2’epiAmBUreas.
• C2’epiAmB is non- toxic in animals at the highest doses tested.
• two assays will be applied that have mechanistically supported C2’epiAmB’s lack of specificity in vitro toxicity against primary hREC and ETV-Vis sterol binding.
• the corresponding KDS will be determined for each of the new C2’epiAmBEireas and prioritize advancement of those derivatives that similarly show retained binding to ergosterol (Ko.crg ⁇ 200 nM) and little or no binding to cholesterol (Ko.ciioi > 2000 nM).
• this step will 1) test for a retained AmB-like pattern of MICs against an established panel of C. albicans erg mutant strains, and 2) perform gradual resistance-selection protocol in liquid culture, with serial twofold increases in C2’epiAmBUrea concentration to identify any mutants that exhibit a greater than or equal to four-fold increase in MIC. The next step will then test whether any such mutants can 3) elude the marked fitness defects previously demonstrated for AmB-resistant strains, including sensitivity to oxidative stress heightened dependence on Hsp90, 4) retain the capacity for filamentation upon stimulation with fetal bovine serum, and/or 5) retain the capacity to cause lethal infection in mice.
• C2’epiAmBUreas that are verified to primarily operate via the sterol sponge mechanism and possess AmB-like capacity to evade resistance, will be advanced to secondary in vitro screening (see C.3.5.).
• Remaining C2’epiAmBUreas will next be evaluated for their broad-spectrum efficacy in an extended panel of climcaliy-relevant pathogens.
• the Steinbach lab will determine the activity of these compounds, tested in triplicate against azole-resistant C. albicans, echinocandin-resistant C. glabrata, Cryptococcus neoformans, A. calidoustus, A. lentulus, azole- resistant A. fumigatus, echinocandin-resistant A. fumigatus, Scedosporium prolificans,
• neutropenic ICR/Swiss mice (4 per group) will be injected IP QD for 7 days.
• One kidney will be analyzed for renal genotoxicity markers Kiml, Fcn2, Timpl, and Sppl via RT-PCR, and the other kidney will be analyzed for renal pathology via osteopontin and H&E staining.
• MTD for the 7 day QD treatment protocol will be defined as the dose of each compound that causes no deaths and only mild changes ( ⁇ 20% increase) in BUN/Cr, renal genotoxicity markers, and renal pathology metrics.
• the study will next determine the PK profiles of the MTD of AmB, AmBisome ® , voriconazole, caspofungin, C2’epiAmB, and the top 5 C2’epiAmBUreas following QD multi dose treatments for 7 days. Specifically, neutropenic ICR/Swiss mice will be injected IP with the MTD of each compound (as determined in study C.3.7) QD for 7 days, and a 13 point PK curve will be generated as detailed in C.3.6.
• Animals will be treated QD with the MTD of each compound or vehicle control for 7 days. Animals will be monitored daily for adverse events and 24 h after the last injection all surviving animals will be sacrificed and both kidneys removed, homogenized, and plated for viable fungal colony counts.
• mice cyclophosphamide 150 mg/kg (days -2, +3) and triamcinolone 40 mg/kg (days -1, +6)] and exposed to an aerosol of the strain (day 0) to develop pulmonary invasive aspergillosis.
• Each arm will contain 10 mice for adequate statistical power. Survival will be plotted on a Kaplan- Meier curve with log rank pair-wise comparison. Fungal burden with galactomannan assay at a pre-determined time point (day +5 after infection) will be analyzed with the Kruskal-Wallis test with Dunn’s post-test.
• C2’epiAmBAU strongly support the prediction that Cl 6 modifications will show improved potency compared to C2’epiAmB.
• C2’epiAmBAU is also substantially less toxic than AmB, but this study did observe low but measurable toxicity to hRECs.
• AmBAU was one of the most toxic of the earlier series of AmBUreas, and many other AmBUreas were much less toxic that AmBAU yet still demonstrated excellent solubilities and antifungal potencies.
• hybridizations of C2’epiAmB with other urea side chains will yield similar increases in potency without any mammalian toxicity.
• C2’epiAmB C16 methyl ester C2’epiAmB ME
• C2’epiAmBME C2’epiAmB C16 methyl ester
• MIC >64 mM for C2’epiAmB and 4 mM for C2’epiAmBME
• the two top C2’epi AmBUreas selected from C.3.7 will be administered IV at 1, 10, 20, 40 and 80 mg/kg to Sprague Dawley rats (3 male/ 3 female, again to account for sex as a biological variable) to evaluate toxicity and pharmacokinetic properties (as described for mice in C.3.6). Rats will be evaluated for weight loss, death, and elevations in BUN, Creatinine, and
• the top performing C2’epiAmBUrea will be further characterized in healthy beagle dogs, a large mammalian, non-rodent species. Extensive preclinical toxicity data of AmB- deoxycholate exists in dogs, identifying 0.625 mg/kg IV daily for 30 consecutive days as the MTD associated with reproducible renal pathology. With the expectation that the best performing C2’epiAmBUrea will afford at least a 10-fold increase in biologic tolerability in comparison with AmB-deoxycholate while retaining potent antifungal activities, 6 sexually- intact beagle dogs (3 male/3 female) will be treated daily for 14 consecutive days (a clinically relevant exposure duration for managing invasive fungal infections in humans) with the top
• C2’epiAmBUrea at 6.25 mg/kg as a 10-minute slow IV bolus.
• the study will then determine the corresponding drug concentrations in serum at 0, lOm, 20m, 30m, 40m, lh, 2h, 4h, 6h, l2h, and 24h via HPLC on Day 1 (initial) and Day 14 (final) of C2’epiAmBUrea administration.
• Serial complete blood counts, chemistry panels, and urinalyses will be assessed pre-treatment (Day 0), and on Days 7 and 14 of drug administration for the detection of associated hematologic, non- hematologic, renal tubular toxicities.
• Beagle dogs will be observed daily for clinical symptoms associated with toxicity including lethargy, inappetence, vomiting, and diarrhea.
• beagle dogs will be humanely sacrificed, and a warm necropsy performed with detailed weighing and histologic assessment of the following internal organs [brain, lung, heart, thymus, thyroid gland, liver, spleen, lymph node, stomach, kidney, adrenal gland, small intestine, large intestine, bladder, gonadal organs (ovaries or testes) and bone marrow]
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the reaction mixture was then poured into diethyl ether (0.5 L), and the resulting yellow precipitate was isolated via Biichner filtration using Whatman #50 filter paper to afford a yellow solid.
• the solid was dissolved in DMSO (-100 mg/mL) and purified by a single prep-HPLC purification (Cl 8, 5-pm, 50 x 250 mm, 75 mL/min, 80:20 to 59:41 0.3% HC02H (aq):MeCN over 9 minutes), Following HPLC purification, the solvent was removed in vacuo at 40 °C. Upon complete solvent removal, residual formic acid was removed via azeotroping with milliQ water (10 mL) and toluene (50 mL).
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the reaction mixture was then poured into diethyl ether (0.5 L), and the resulting yellow precipitate was isolated via Biichner filtration using Whatman #50 filter paper to afford a yellow solid.
• the solid was dissolved in DMSO (-100 mg/mL) and purified by a single prep-HPLC purification (Cl 8, 5-pm, 50 x 250 mm, 75 mL/min, 80:20 to 59:41 0.3% HC02H (aq):MeCN over 9 minutes), Following HPLC purification, the solvent was removed in vacuo at 40 °C. Upon complete solvent removal, residual formic acid was removed via azeotroping with milliQ water (10 mL) and toluene (50 mL).
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the reaction mixture was then poured into diethyl ether (0.5 L), and the resulting yellow precipitate was isolated via Biichner filtration using Whatman #50 filter paper to afford a yellow solid.
• the solid was dissolved in DMSO (-100 mg/mL) and purified by a single prep-HPLC purification (Cl 8, 5-pm, 50 x 250 mm, 75 mL/min, 80:20 to 59:41 0.3% HC02H (aq):MeCN over 9 minutes), Following HPLC purification, the solvent was removed in vacuo at 40 °C. Upon complete solvent removal, residual formic acid was removed via azeotroping with milliQ water (10 mL) and toluene (50 mL).
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.
• the resulting powder was dissolved in 1 : 1 THF:MeOH (18 mL) and cooled to 0°C. To this solution was added camphorsulfonic acid (69 mg, 0.30 mmol, 0.55 eq) and the resulting mixture was stirred for 1 hour at 0°C. The reaction was then quenched at 0°C with triethylamine (0.07 mL, 0.30 mmol, 0.55 eq). The reaction was concentrated in vacuo removing approximately half of the solvent.

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• 2019
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  • 2019-09-06 WO PCT/US2019/049971 patent/WO2020051465A1/en not_active Ceased
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