Synthesis of echinocandin class antifungal agents

Abstract

The present invention relates to echinocandin cyclic peptides and to processes for preparing echinocandin cyclic peptides.

Description

BACKGROUND

[0001] The present invention features methods for synthesizing compounds useful in the treatment of fungal infections and related conditions thereof.

[0002] Fungal infections, such as those caused by Candida and Aspergillus, can be serious and life-threatening infections that represent a major public health problem, particularly in highly susceptible populations, including the elderly, post-operative patients, critically ill patients, and other hospitalized patients with serious medical conditions. Due to increasing resistance to existing antifungal drugs, there is an urgent need to develop new and more effective antifungal agents to treat these serious infections. The echinocandins are members of the major class of antifungal agents used to treat fungal infections. These compounds target the cell wall by preventing the production of 1,3-beta-D-glucan through inhibition of the catalytic subunit of the 1,3-beta-D-glucan synthase complex.

[0003] While nature can provide important parts of the complex chemical structure of semisynthetic cyclic peptides with all chiral centers in the desired configuration in many cases, subsequent chemical transformations to therapeutically active derivatives often still require unprecedented methods. Often, the structures in question are chemically unstable and / or prone to racemization, and simply do not allow for additional obvious synthetic operations taught in synthetic organic chemistry textbooks. This chemical instability is more pronounced in anidulafungin, caspofungin, and micafungin due to the presence of a well-known fragile hemiaminal or acetal amine moiety. The production of pharmaceutical grade echinocandins is complicated by the difficulty and expense of relying on chromatographic methods to remove structurally similar impurities generated during commercial scale production processes.

[0004] There is a need for convenient synthetic alternatives that allow for the commercial scale production of semisynthetic echinocandins. These methods can serve as useful alternatives to existing synthetic methods and can achieve higher yields, higher isomer purity, elimination of mutagenic impurities, reduced waste streams, or any combination of the above. SUMMARY

[0005] The present invention features a method of synthesizing Compound 1, the method comprising the steps of: (a) providing a first composition comprising a boronate ester of anidulafungin; (b) providing a second composition comprising a salt of choline; (c) combining the first composition, the second composition, and an acid to form a mixture, wherein the solvent system is selected to form a precipitate of the reaction product having Formula (I):

[0006]

[0007] wherein X – is an anion; and R is C1-C6alkyl, C3-C 10 carbocyclyl, C2-C6alkenyl, C6-C 10 aryl, or C2-C9heteroaryl; and (d) hydrolyzing the compound of Formula (I) to form Compound 1, or a salt or neutral form thereof.

[0008] In some embodiments, R is C1-C6alkyl or C6-C 10 aryl. In some embodiments, R is C6-C 10 aryl. In some embodiments, R is substituted or unsubstituted C6aryl.

[0009] In some embodiments, the concentration of the mixture is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 moles / liter (e.g., 0.01 to 0.03 moles / liter, 0.03 to 0.05 moles / liter, 0.05 to 0.1 moles / liter, or 0.1 to 0.2 moles / liter) relative to the compound of Formula (I). In some embodiments, the concentration of the mixture is at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 moles / liter (e.g., 0.1 to 0.3 moles / liter, 0.2 to 0.4 moles / liter, 0.3 to 0.5 moles / liter, 0.4 to 0.6 moles / liter, or 0.5 to 0.7 moles / liter) relative to the compound of Formula (I). In some embodiments, the concentration of the mixture is at least 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 moles / liter (e.g., 0.5 to 0.8 moles / liter, 0.6 to 0.9 moles / liter, 0.7 to 1.0 moles / liter, 1.0 to 1.3 moles / liter, 1.0 to 1.5 moles / liter, or 1.5 to 2.0 moles / liter) relative to the compound of Formula (I).

[0010] In some embodiments, step (c) comprises a solvent system comprising acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, cyclopentyl methyl ether, or t-butyl methyl ether, or a mixture thereof. In some embodiments, step (c) comprises a solvent system comprising a mixture of tetrahydrofuran and acetonitrile. In some embodiments, step (c) comprises a solvent system comprising a mixture of 2-methyltetrahydrofuran and acetonitrile. Optionally, the solvent system further comprises trifluoroacetic anhydride. In some embodiments, the solvent system contains 0.1% to 5% (w / w) water. In some embodiments, the solvent system is anhydrous.

[0011] In some embodiments, step (c) comprises combining at least 10, 15, 20, 25, 30, 35, or 40 molar equivalents (e.g., 10 to 80, 10 to 40, 20 to 60, or 20 to 40 equivalents) of a salt of choline and at least 1 molar equivalent of anisulpride as its borate ester.

[0012] In particular embodiments, step (c) is performed at a temperature of less than 40 °C, 35 °C, 30 °C, 25 °C, 20 °C, 18 °C, 15 °C, 12 °C, or 10 °C (e.g., 2 to 40 °C, 5 to 40 °C, 8 to 20 °C, 8 to 18 °C, or 8 to 12 °C).

[0013] In some embodiments, step (c) comprises a step of forming a mixture in which at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 99%, or 98% to 99%) of the compound of formula (I) is precipitated relative to the final amount of the compound of formula (I) produced.

[0014] In some embodiments, step (c) comprises precipitating at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 99%, or 98% to 99%) of the compound of formula (I) relative to the final amount of the compound of formula (I) produced.

[0015] In some embodiments, the second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and trifluoroacetic acid (TFA), optionally with one or more additional organic acids. For example, an additional organic acid that can be used in combination with acetonitrile and TFA can be methanesulfonic acid or acetic acid. In some embodiments, the second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and acetic acid. In some embodiments, the second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and methanesulfonic acid. In some embodiments, the second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and trifluoromethanesulfonic acid. In particular embodiments, the second composition further comprises trifluoroacetic anhydride. In some embodiments, the second composition contains 0.1% to 5% (w / w) water. In some embodiments, the second composition is an anhydrous solution, or the second composition is a mixture comprising one or more anhydrous solvents.

[0016] The present invention features a method of synthesizing Compound 1, the method comprising the steps of: (a) providing a first composition comprising an aryl borate ester of anidulafungin; (b) providing a second composition comprising a salt of choline; (c) combining the first composition, the second composition, and an acid to form a mixture comprising a compound of Formula (II):

[0017]

[0018] wherein X – is an anion; and Ar is a substituted or unsubstituted C6 aryl group; and (d) hydrolyzing the compound of Formula (II) to form Compound 1, or a salt or neutral form thereof.

[0019] In some embodiments, Ar is phenyl, 3,4-dimethoxyphenyl, 4-trifluoromethylphenyl, or 2,6-dimethylphenyl. In some embodiments, Ar is phenyl. In some embodiments, Ar is 3,4-dimethoxyphenyl. In other embodiments, Ar is 4-trifluoromethylphenyl. In other embodiments, Ar is 2,6-dimethylphenyl.

[0020] In certain embodiments of the method, step (c) comprises combining at least 10, 15, 20, 25, 30, 35, or 40 molar equivalents (e.g., 10 to 80, 10 to 40, 20 to 60, or 20 to 40 equivalents) of the salt of choline with 1 molar equivalent of the 3,4-dimethoxyphenyl borate ester of anidulafungin. In certain embodiments of the method, step (c) comprises combining at least 10, 15, 20, 25, 30, 35, or 40 molar equivalents (e.g., 10 to 80, 10 to 40, 20 to 60, or 20 to 40 equivalents) of the salt of choline with 1 molar equivalent of the 4-trifluoromethylphenyl borate ester of anidulafungin. In certain embodiments of the method, step (c) comprises combining at least 10, 15, 20, 25, 30, 35, or 40 molar equivalents (e.g., 10 to 80, 10 to 40, 20 to 60, or 20 to 40 equivalents) of the salt of choline with 1 molar equivalent of the 2,6-dimethylphenyl borate ester of anidulafungin.

[0021] In some embodiments, the first composition comprises a solution of 3,4-dimethoxyphenyl boronate of anidulafungin dissolved in an organic solvent selected from the group consisting of acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, cyclopentyl methyl ether, t-butyl methyl ether, or a mixture thereof. In some embodiments, the first composition comprises a solution of 4-trifluoromethylphenyl boronate of anidulafungin dissolved in an organic solvent selected from the group consisting of acetonitrile, butyronitrile, tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, cyclopentyl methyl ether, t-butyl methyl ether, or a mixture thereof. In some embodiments, the first composition comprises a solution of 2,6-dimethylphenyl boronate of anidulafungin dissolved in an organic solvent selected from the group consisting of acetonitrile, butyronitrile, tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, cyclopentyl methyl ether, t-butyl methyl ether, or a mixture thereof. In some embodiments, the organic solvent contains 0.1% to 5% (w / w) water. In some embodiments, the organic solvent is anhydrous.

[0022] In some embodiments, the second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and trifluoroacetic acid (TFA). In particular embodiments, the second composition comprises a solution of a salt of choline dissolved in a mixture of acetonitrile, trifluoroacetic acid, and trifluoroacetic anhydride. The second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and acetic acid. In particular embodiments, the second composition comprises a solution of a salt of choline dissolved in a mixture of acetonitrile, trifluoroacetic acid, and acetic acid. The second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and methanesulfonic acid.

[0023] The second composition can comprise a solution of a salt of choline dissolved in a mixture of acetonitrile and trifluoromethanesulfonic acid. In particular embodiments, the second composition further comprises trifluoroacetic anhydride. In some embodiments, the second composition contains 0.1% to 5% (w / w) water. In some embodiments, the second composition is anhydrous, or the second composition is a mixture of one or more anhydrous solvents.

[0024] In particular embodiments of any of the above methods, step (c) further comprises the step of adding acetonitrile to the mixture to reduce the level of the compound 1 β- diastereomer.

[0025] In some embodiments of any of the above methods, the mixture is formed at a temperature of less than 40 °C, 35 °C, 30 °C, 25 °C, 20 °C, 18 °C, 15 °C, 12 °C, or 10 °C (e.g., 2 to 40 °C, 5 to 40 °C, 8 to 20 °C, 8 to 18 °C, or 8 to 12 °C).

[0026] In some embodiments, step (c) further comprises dilution with at least 5, 6, 7, 8, 9, or 10 volumes of water or a mixture of water and acetonitrile relative to anidulafungin. In some embodiments, step (c) further comprises dilution with at least 10, 11, 12, 13, 14, or 15 volumes of water or a mixture of water and acetonitrile relative to anidulafungin. In some embodiments, step (c) further comprises dilution with at least 15, 20, 25, 30, 35, 40, 45, or 50 volumes of water or a mixture of water and acetonitrile relative to anidulafungin.

[0027] In some embodiments, the mixture of water and acetonitrile comprises at least 5%, 10%, 15%, 20%, 25%, or 30% water. In some embodiments, the mixture of water and acetonitrile comprises at least 30%, 35%, 40%, 45%, or 50% water. In some embodiments, the mixture of water and acetonitrile comprises at least 50%, 55%, 60%, 65%, or 70% water. In some embodiments, the mixture of water and acetonitrile comprises at least 70%, 75%, 80%, 85%, 90%, or 95% water.

[0028] In some embodiments, step (d) further comprises adding a base to adjust the pH to at least 2 (e.g., 2 to 3, 2 to 4, or 2 to 5). In particular embodiments, step (d) comprises dilution with at least 5 volumes of about 80:20 to 50:50 water:acetonitrile mixture relative to anidulafungin, and adjusting the pH to pH 2 to 5 with a base.

[0029] In some embodiments, the base is ammonium acetate, ammonium hydroxide, or ammonium carbonate. In some embodiments, the base is ammonium acetate. In some embodiments, the base is ammonium hydroxide. In some embodiments, the base is ammonium carbonate.

[0030] In particular embodiments of any of the above methods, step (d) comprises forming a reaction product comprising greater than 70%, 75%, 80%, 85%, 88%, or 90% (e.g., 70% to 95%, 75% to 90%, 80% to 90%, or 85% to 90%) of Compound 1 (as measured by HPLC) and less than 5%, 4%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, or 1.0% (e.g., 0% to 1%, 0% to 2%, 0% to 3%, 1.0% to 4.0%, 1.0% to 3.0%, 1.5% to 3.5%, or 2.0% to 3.0%) of Compound 1 β- diastereomer (as measured by HPLC). For example, step (d) can comprise forming a reaction product comprising greater than 75% of Compound 1 (as measured by HPLC), less than 2% of Compound 1 β-diastereomer (as measured by HPLC), and optionally less than 1% (e.g., 0% to 0.5%, 0.5% to 1.0%, or 0.7% to 1.0%) of Compound 1 epimer (as measured by HPLC). In some embodiments, step (d) comprises forming a reaction product comprising 75% to 90% of Compound 1 (as measured by HPLC) and 1.5% to 3.5% of Compound 1 β-diastereomer (as measured by HPLC). In some embodiments, step (d) comprises forming a reaction product comprising 75% to 90% of Compound 1 and 0.5% to 2.5% of Compound 1 β-diastereomer. In other embodiments, step (d) comprises forming a reaction product comprising 75% to 90% of Compound 1 (as measured by HPLC), 1.5% to 3.5% of Compound 1 β-diastereomer (as measured by HPLC), and 0.5% to 1.0% of Compound 1 epimer (as measured by HPLC). In other embodiments, step (d) comprises forming a reaction product comprising 75% to 90% of Compound 1, 0.5% to 2.5% of Compound 1 β-diastereomer, and 0.1% to 1.0% of Compound 1 epimer. In related aspects, the application features a method of synthesizing Compound 1, the method comprising: hydrolyzing a compound of any of Formula (I), (II), (IIa), (IIb), or (IIc):

[0031]

[0032] wherein X - is an anion, to form Compound 1 or a salt or neutral form thereof.

[0033] In any of the above methods, the hydrolyzing can comprise contacting a compound of any of Formula (I), (II), (IIa), (IIb), or (IIc) with a base.

[0034] In some embodiments, the base is an aqueous base. In particular embodiments, the hydrolysis is carried out at a temperature of less than 15 °C, 12 °C, 10 °C, or 8 °C (e.g., 2 °C to 15 °C, 5 °C to 15 °C, 5 °C to 12 °C, 5 °C to 10 °C, or 2 °C to 10 °C).

[0035] In particular embodiments, the hydrolysis comprises dilution with at least 5 volumes of about 80:20 to 50:50 water:acetonitrile mixture relative to anidulafungin, and adjustment of the pH to pH 2 to 5 with a base.

[0036] In any of the above methods, after hydrolyzing the compound of Formula (IIa) to form Compound 1, or a salt or neutral form thereof, Compound 1, or a salt or neutral form thereof, can be separated from 3,4-dimethoxyphenylboronic acid by passing through an ion exchange column or by preparative HPLC.

[0037] In any of the above methods, after hydrolyzing the compound of Formula (IIb) to form Compound 1, or a salt or neutral form thereof, Compound 1, or a salt or neutral form thereof, can be separated from 4-trifluoromethylphenylboronic acid by passing through an ion exchange column or by preparative HPLC.

[0038] In any of the above methods, after hydrolyzing the compound of Formula (IIc) to form Compound 1, or a salt or neutral form thereof, Compound 1, or a salt or neutral form thereof, can be separated from 2,6-dimethylphenylboronic acid by passing through an ion exchange column or by preparative HPLC.

[0039] In other embodiments, the hydrolysis to form Compound 1 is carried out at a scale that produces 100 grams to 50 kilograms (e.g., 100 grams to 200 grams, 200 grams to 500 grams, 500 grams to 1000 grams, 1 kilogram to 5 kilograms, 5 kilograms to 10 kilograms, 10 kilograms to 20 kilograms, 20 kilograms to 40 kilograms, or 30 kilograms to 50 kilograms) of Compound 1.

[0040] In any of the above methods, the method can further comprise producing a pharmaceutical composition by combining Compound 1, or a salt or neutral form thereof, with a pharmaceutically acceptable excipient (e.g., any of the excipients described herein). For example, the pharmaceutical composition can be formulated for topical or parenteral administration or any of the forms of administration described herein.

[0041] In a related aspect, the application features a compound of Formula (IIa):

[0042]

[0043] wherein X - is an anion.

[0044] In another aspect, the application features a compound of Formula (IIb):

[0045]

[0046] wherein X - is an anion.

[0047] In another aspect, the application features a compound of formula (IIc):

[0048]

[0049] wherein X - is an anion.

[0050] In another aspect, the application features a pharmaceutical composition comprising Compound 1 or a salt or neutral form thereof and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises less than 5%, 4%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, or 1.0% (e.g., 0% to 2.0%, 0.1% to 4.0%, 0.75% to 3.0%, 0.5% to 3.5%, or 1.0% to 3.0%) (w / w) of Compound 1 β- diastereomer. In particular embodiments, the pharmaceutical composition further comprises less than 1% (e.g., 0.5% to 1.0% or 0.7% to 1.0%) (w / w) of Compound 1 epimer relative to the weight of Compound 1 or a salt or neutral form thereof in the pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises 1.5% to 3.5% (w / w) of Compound 1 β- diastereomer and 0.5% to 1.0% (w / w) of Compound 1 epimer relative to the weight of Compound 1 or a salt or neutral form thereof in the pharmaceutical composition.

[0051] Definitions

[0052] As used herein, the term “anhydrous solvent system” or “the solvent system is anhydrous” refers to a solvent system that is dried prior to use in a reaction and / or contains less than 0.1% water. For example, “anhydrous acetonitrile” or “the acetonitrile is anhydrous” refers to acetonitrile that is dried prior to use in a reaction and / or contains less than 0.1% water.

[0053] As used herein, the term “Compound 1” refers to a compound having the structure shown below. The term “salt form of Compound 1” or “salt of Compound 1” refers to Compound 1 when the positive charge of the tertiary ammonium ion is balanced by a negative counterion (e.g., acetate).

[0054]

[0055] As used herein, the term “neutral form” includes the zwitterionic form of Compound 1, wherein Compound 1 does not have a net positive or negative charge. The zwitterion exists in higher proportion in basic media (e.g., between pH 7 and 8, between 8 and 9, or between 9 and 10) relative to Compound 1 or a salt of Compound 1. In some embodiments, the zwitterion can also exist in its salt form.

[0056] As used herein, the term “Compound 1 beta-enantiomer” or “beta-enantiomer” refers to the compound having the structure shown below, and salts thereof.

[0057]

[0058] As used herein, the term “Compound 1 epimer” or “epimer” refers to the compound having the structure shown below, and salts thereof.

[0059]

[0060] As used herein, the term “Anidulafungin arylboronic acid” refers to the compound having the structure shown below, and salts thereof.

[0061]

[0062] wherein Ar is a substituted or unsubstituted C6aryl group.

[0063] As used herein, the term “containing echinocandin” refers to Compound 1, Compound 1 beta-enantiomer, and / or Compound 1 epimer. For example, “a reaction product containing echinocandin” can refer to a reaction product comprising Compound 1, Compound 1 beta-enantiomer, and / or Compound 1 epimer.

[0064] As used herein, the term “about” refers to a range of values ±10% of the specified value. For example, “about 150 mg” includes ±10% of 150 mg, or 135 mg to 165 mg. Such a range performs the desired function or achieves the desired result. For example, “about” can refer to an amount that is within less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the stated amount.

[0065] As used herein, the term “between” refers to any numerical value within the indicated range and including each endpoint of the indicated range. For example, between 5 and 7 pH refers to any numerical value between 5 and 7, as well as a pH of 5 and a pH of 7.

[0066] As used herein, the term "infection" or "fungal infection" refers to a microbial ecological imbalance characterized by the overgrowth or colonization of one or more fungi (e.g., fungal pathogens or opportunistic pathogens) in any part of a human subject's body, the reduction of which may provide a benefit to the host. For example, an infection may include the overgrowth or colonization of fungal species normally present in or on a human subject's body, or it may include the colonization of fungal species not normally present in or on a human subject's body. In some cases, an infection may include the colonization of some parts of the body by fungi that are inherent to certain parts of the body (e.g., the GI tract) but harmful when found in other parts of the body (e.g., tissues outside the GI tract). More generally, an infection can be any situation where the presence of one or more microbial populations is harming the host's body.

[0067] As used herein, the term “C6 aryl” refers to an unsubstituted or substituted aromatic group having six carbon atoms. Substituents may include halogens, methyl, ethyl, ethoxy, methoxy, fluoromethyl, difluoromethyl, and trifluoromethyl. C6 aryl groups include, but are not limited to, phenyl, 3,4-dimethoxyphenyl, 4-trifluoromethylphenyl, and 2,6-dimethylphenyl. In some embodiments, the C6 aryl group is substituted by one, two, three, four, or five substituents independently selected from the group consisting of: (1) a halogroup; (2) a C1-C6 alkoxy group; (3) a C1-C6 alkyl group (e.g., a C1-C6 perfluoroalkyl group); and (4) a C6-C6 alkyl group. 10 Aryl groups. In some embodiments, these groups may each be further substituted as described herein.

[0068] As used herein, the term "salt" refers to any salt form commonly used in the pharmaceutical industry. Acid addition salts include organic acids such as acetic acid, formic acid, lactic acid, palmitic acid, maleic acid, citric acid, cholic acid, capric acid, caprylic acid, lauric acid, glutaric acid, glucuronic acid, glyceric acid, glycocholic acid, glyoxylic acid, isocitrate, isovaleric acid, lactic acid, malic acid, oxaloacetic acid, oxalosuccinic acid, propionic acid, pyruvic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, succinic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, and trifluoroacetic acid, as well as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts.

[0069] Throughout this specification, substituents of the compounds disclosed herein are disclosed in groups or ranges. This disclosure is expressly intended to include individual and each individual sub-combination of members of such groups and ranges. For example, the term "C1-C6 alkyl" is expressly intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl. Furthermore, when a compound contains multiple positions of substituents disclosed in groups or ranges, unless otherwise specified, this disclosure is intended to cover individual compounds and groups of compounds (e.g., genera and subgenera) containing individual and each individual member sub-combination at each position.

[0070] As used herein, the term "alkyl" refers to a saturated hydrocarbon group containing 1 to 20 carbons (e.g., 1 to 10 or 1 to 6). In some embodiments, the alkyl group is unbranched (i.e., linear); in some embodiments, the alkyl group is branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl and isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl, and neopentyl. In some embodiments, the alkyl group is unsubstituted. In some embodiments, the alkyl group is substituted by one, two, three, four or five substituents independently selected from the group consisting of: (1) halogroup; (2) C1-C6 alkoxy; (3) C1-C6 perfluoroalkyl; and (4) C6-C6 perfluoroalkyl. 10 Aryl groups. In some embodiments, these groups may each be further substituted as described herein.

[0071] As used herein, the term "alkenyl" means, unless otherwise specified, a monovalent straight-chain or branched group having 2 to 10 carbons (e.g., 2 to 4 or 2 to 6 carbons) and containing one or more carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl. In some embodiments, the alkenyl group is unsubstituted. In some embodiments, the alkenyl group is substituted by one, two, three, four, or five substituents independently selected from the group consisting of: (1) a halogroup; (2) a C1-C6 alkoxy group; (3) a C1-C6 alkyl group (e.g., a C1-C6 perfluoroalkyl group); and (4) a C6-C6 alkyl group. 10 Aryl groups. In some embodiments, these groups may each be further substituted as described herein.

[0072] As used herein, the term "aryl" denotes a monocyclic, bicyclic, or polycyclic carbocyclic ring system having one or two aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthryl, phenanthryl, fluorenyl, indanyl, and indenyl. In some embodiments, aryl is unsubstituted. In some embodiments, aryl is substituted with one, two, three, four, or five substituents independently selected from the group consisting of: (1) halo; (2) C1-C6alkoxy; (3) C1-C6alkyl (e.g., C1-C6perfluoroalkyl); and (4) C6-C 10 aryl. In some embodiments, each of these groups can be further substituted as described herein. Examples of substituents include, but are not limited to, halo, methyl, ethyl, ethoxy, methoxy, fluoromethyl, difluoromethyl, and trifluoromethyl.

[0073] As used herein, the term "carbocyclyl" refers to a monocyclic, bicyclic, or tricyclic nonaromatic ring structure formed from carbon atoms. Examples of carbocyclyl groups include, but are not limited to, cycloalkyl and cycloalkenyl. In some embodiments, carbocyclyl is unsubstituted. In some embodiments, carbocyclyl is substituted with one, two, three, four, or five substituents independently selected from the group consisting of: (1) halo; (2) C1-C6alkoxy; (3) C1-C6alkyl (e.g., C1-C6perfluoroalkyl); and (4) C6-C 10 aryl. In some embodiments, each of these groups can be further substituted as described herein.

[0074] As used herein, the term "halo" or "halogen" refers to a fluoro (fluorinated), chloro (chlorinated), bromo (brominated), or iodo (iodinated) group.

[0075] As used herein, the term "heteroaryl" denotes a subset of heterocyclyl groups as defined herein that are aromatic: that is, they contain 4n+2 π electrons within a monocyclic or polycyclic ring system. Exemplary unsubstituted heteroaryls have 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In some embodiments, heteroaryl is unsubstituted. In some embodiments, heteroaryl is substituted with one, two, three, four, or five substituents independently selected from the group consisting of: (1) halo; (2) C1-C6alkoxy; (3) C1-C6alkyl (e.g., C1-C6perfluoroalkyl); and (4) C6-C 10 aryl. In some embodiments, each of these groups can be further substituted as described herein.

[0076] As used herein, the term "heterocyclyl" denotes a 5-, 6-, or 7-membered ring, which ring contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, unless otherwise indicated. In some embodiments, the heterocyclyl group is unsubstituted. In some embodiments, the heterocyclyl group is substituted with one, two, three, four, or five substituents independently selected from the group consisting of: (1) halo; (2) Ci-C6alkoxy; (3) Ci-C6alkyl (e.g., Ci-C6perfluoroalkyl); and (4) C6-Ci0aryl. 10 aryl. In some embodiments, these groups can each be further substituted as described herein.

[0077] Other features and advantages of the present application will be apparent from the following detailed description and from the claims. DETAILED DESCRIPTION

[0078] Provided herein are synthetic methods and intermediates for preparing the echinocandin antifungal agent Compound 1, or a salt or neutral form thereof. The methods and intermediates can be used to achieve higher yield, higher chemical purity, and / or higher diastereomeric purity, as well as lower cost for preparing Compound 1. Further synthetic details are provided in the Examples.

[0079] The present application features a method for synthesizing Compound 1 acetate from anidulafungin using arylboronic acids as an in situ protecting group, which developed as follows. In one embodiment, the first step involves slurry of choline chloride in 2-methyltetrahydrofuran, which is then distilled off. The resulting solid is further dried in a vacuum oven at high temperature. The second step involves converting the anidulafungin starting material to a 3,4-dimethoxyphenyl boronic ester by reacting it with 1.3 equivalents of 3,4-dimethoxyphenyl boronic acid in tetrahydrofuran, thereby protecting the anidulafungin starting material. Evaporation of the solvent under reduced vacuum gives the protected intermediate as a solid, which is further dried by repeated azeodrying cycles with 2-methyltetrahydrofuran. Alternatively, other water removal methods such as addition of activated molecular sieves, continuous distillation, or addition of a dehydrating agent can be employed. In the third and final step of the crude material, the azeodried choline chloride is dissolved in a mixture of TFA and acetonitrile, and it is conjugated to the protected anidulafungin backbone to give Compound 1 in its TFA / chloride form. The reactants are then quenched by the addition of a water:acetonitrile mixture, and the pH is adjusted to give a fairly stable crude mixture, which is prepared for the purification process.

[0080] The present invention features a method for the synthesis of Compound 1 acetate from anidulafungin that requires the use of 3,4-dimethoxyphenylboronic acid as an in situ protecting group, wherein additional acetonitrile (20 to 50 volumes relative to anidulafungin) is added when the conjugation reaction is complete. This causes Compound 1 to precipitate. Since the equilibrium between Compound 1 and the beta isomer of 1 (about 95:5) in solution is maintained under acidic conditions, the precipitation of Compound 1 from solution results in driving the formation of Compound 1 and reducing the amount of the beta-isomer. Under these conditions, the beta isomer at the end of the reaction can be controlled to no more than 2.0%.

[0081] The present invention features a method for the synthesis of Compound 1 acetate from anidulafungin that uses 3,4-dimethoxyphenylboronic acid as an in situ protecting group, which involves a conjugation reaction with 12 to 18 equivalents of choline chloride under more concentrated conditions. As the reaction proceeds, this causes Compound 1 to precipitate. Since the equilibrium between Compound 1 and the beta isomer of 1 (about 95:5) in solution is maintained under acidic conditions, the precipitation of Compound 1 from solution results in driving the formation of Compound 1 and reducing the amount of the beta-isomer. Under these conditions, the beta isomer at the end of the reaction can be controlled to less than 2.0%. The present invention also features a method for the synthesis of Compound 1 acetate from anidulafungin that uses 2,6-dimethylphenylboronic acid as an in situ protecting agent.

[0082] The present invention also features a method for the synthesis of Compound 1 acetate from anidulafungin that uses 4-trifluoromethylphenylboronic acid as an in situ protecting agent.

[0083] The present invention features a purification method in which the crude reaction is purified by reverse phase preparative high performance liquid chromatography (RP-HPLC) or reverse phase preparative medium pressure liquid chromatography (RP-MPLC). The final product can be isolated by lyophilization.

[0084] Advantages of the present invention include a significant improvement in diastereomeric purity, which allows for a more straightforward purification method and a product that is overall more pure. Surprisingly, it was found that the nature of the group on the arylboronic acid had a significant impact on the diastereoselectivity in the conjugation reaction, despite the boronic acid group being distal to the reaction center. In particular, the use of anidulafungin 3,4-dimethoxyphenylboronate reduced the amount of Compound 1 beta-diastereomer formed relative to other boronates, resulting in a simpler purification method and a more pure Compound 1.

[0085] Compound 1 can be used to treat, ameliorate, or prevent a fungal infection or associated condition thereof in a human subject in need thereof.

[0086] Compound 1 can be prepared in the form of a pharmaceutical composition. The pharmaceutical composition can include a salt or the neutral form of Compound 1, as well as pharmaceutically acceptable carriers and excipients. The pharmaceutical composition can be formulated for subcutaneous injection or intravenous infusion. Depending on the mode of administration (e.g., subcutaneous or intravenous) and the dosage, Compound 1 can be formulated into suitable pharmaceutical compositions to allow for easy delivery. An overview of such techniques is found in Remington: The Science and Practice of Pharmacy, 22ndEd., Lippincott Williams & Wilkins, (2012); and Encyclopedia of Pharmaceutical Technology, Eds. J. Swarbrick and J. C. Boylan, 2006, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0087] For subcutaneous administration, Compound 1 can be formulated into an aqueous pharmaceutical composition. In some embodiments, a pharmaceutical composition containing Compound 1 formulated for subcutaneous administration can be free of a buffering agent. In some embodiments, a pharmaceutical composition formulated for subcutaneous administration can contain a weak buffering agent. Examples of weak buffering agents that can be used in the pharmaceutical composition include, but are not limited to, acetate, lactate, histidine, glycine, and formate.

[0088] A pharmaceutical composition comprising Compound 1 in a salt or neutral form can optionally contain an amount of a solubilizing agent. Examples of solubilizing agents include, but are not limited to, polysorbate 20 (Tween 20; polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (Tween 40; polyoxyethylene (40) sorbitan monopalmitate), polysorbate 60 (Tween 60; polyoxyethylene (60) sorbitan monostearate), polysorbate 80 (Tween 80; polyoxyethylene (80) sorbitan monooleate), beta-cyclodextrin, polyoxyethylene 35 castor oil (Cremophor EL), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), polyoxyethylene 60 hydrogenated castor oil (Cremophor RH 60), D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), sorbitan monostearate (Span 20), polyoxyethylene 8 stearate (PEG 400 monostearate), polyoxyethylene 40 stearate (PEG 1750 monostearate), PEG 400 caprylic / capric glyceride (Labrasol), PEG 300 oleic glyceride (Labrafil M-1944CS), phosphatidylcholine (lecithin), alkylglycoside, sucrose monolaurate, sucrose monooleate, and polyoxyethylene-polyoxypropylene block copolymer (Poloxamer).

[0089] In addition, a pharmaceutical composition comprising Compound 1 in a salt or neutral form can contain 0.5% to 3% (w / w) of a sugar. Examples of the sugar that can be included in a pharmaceutical composition comprising Compound 1 in a salt or neutral form used in the methods of the present application include, but are not limited to, mannitol, sucrose, trehalose, fructose, glucose, dextrose, dextran, lactose, and sorbitol.

[0090] A pharmaceutical composition comprising Compound 1 in a salt or neutral form can be formulated as a lyophilized composition. In addition, the pH of a lyophilized composition comprising Compound 1 can be between 5 and 6.5 (e.g., about 5, about 5.3, about 5.6, about 5.9, about 6.2, or about 6.5) when reconstituted with water for injection. In some embodiments, Compound 1 in a salt form can be Compound 1 acetate.

[0091] The pharmaceutical compositions used in the methods of the present application can be formulated in a liquid solution or suspension or in a lyophilized cake and administered by parenteral routes (e.g., subcutaneously or intravenously). Pharmaceutical compositions for parenteral administration can be formulated using sterile solutions or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, or cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), a-Modified Eagle Medium (a-MEM), F-12 medium). Formulation methods are known in the art, see, e.g., Gibson (ed.) Pharmaceutical Preformulation and Formulation (2ndEdition) Taylor & Francis Group, CRC Press (2009).

[0092] In addition, the acceptable carriers and excipients in the pharmaceutical compositions used in the methods of the present application are nontoxic to the recipient at the dosages and concentrations employed. Acceptable carriers and excipients can include buffers such as phosphates, citrates, histidine, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, and lysine; and carbohydrates such as dextrose, mannose, sucrose, and sorbitol. The compositions can be formulated according to conventional pharmaceutical practices. The concentration of the compound in the formulation will vary depending on many factors, including the dose of the drug to be administered and the route of administration.

[0093] The pharmaceutical compositions of the present application can be administered to a human subject in a therapeutically effective amount. The preferred dose of the drug to be administered can depend on variables such as the type and extent of the disorder, the overall health of the particular human subject, the particular compound being administered, the excipients used to formulate the compound, and the route of administration thereof.

[0094] The timing of administration of the pharmaceutical compositions containing Compound 1 in salt or neutral form depends on the medical and health status of the human subject. In some cases, the human subject is at risk of developing a fungal infection or related condition and receives one or more doses of Compound 1 prior to developing symptoms or signs of the fungal infection. In some cases, the human subject has developed a fungal infection or related condition and receives one or more doses of Compound 1. The physician can optimize the timing of administration of one or more doses of Compound 1 to reduce the risk of or treat the fungal infection in the human subject.

[0095] The following examples, described below, are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the methods and compounds claimed herein, and are intended to be purely exemplary of the application and are not intended to be limiting to the scope of the inventors' claimed invention.

[0096] Example

[0097] Example 1. Synthesis of Compound 1 from the phenylboronic ester of anidulafungin.

[0098] Phenylboronic ester of anidulafungin:

[0099] To a solution of anidulafungin (5 g) in tetrahydrofuran (70 mL) was added a solution of phenylboronic acid (0.7 g) in tetrahydrofuran (30 mL). The reaction mixture was stirred at room temperature for 90 minutes. The reaction mixture was concentrated by rotary evaporation. The resulting solid was dissolved in tetrahydrofuran (60 mL) and concentrated by rotary evaporation. The resulting solid was again dissolved in tetrahydrofuran (60 mL) and concentrated by rotary evaporation. The resulting solid mixture was re-dissolved in acetonitrile / tetrahydrofuran (30 mL / 15 mL) and concentrated by rotary evaporation. The resulting anidulafungin phenylboronic ester solid was dried in vacuo overnight.

[0100] Choline chloride drying:

[0101] In a round bottom flask, choline chloride (18.6 g) was suspended in acetonitrile (150 mL) and stirred for 4 hours. The suspension was concentrated by rotary evaporation. The choline chloride was suspended in acetonitrile (150 mL) and concentrated by rotary evaporation, and this step was repeated once more. The resulting solid was dried in vacuo overnight.

[0102] Conjugation:

[0103] In a round bottom flask, the dried choline chloride was dissolved in acetonitrile (50 mL) and trifluoroacetic acid (TFA) (12.5 mL). The resulting choline chloride solution was added to the dried anidulafungin phenylboronic ester. The resulting reaction mixture was stirred at room temperature for 2.5 hours. The reaction was quenched by the addition of water (125 mL) and basified to pH ~2 with NH4OH (2N, ~40 mL). A white material formed and was dissolved with acetonitrile (300 mL). The material contained 4.55% Compound 1 β-diastereomer (average of two runs).

[0104] Purification:

[0105] The material was purified by preparative reverse phase HPLC using a C18 silica media using Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in 50% acetonitrile / 50% water). The product was eluted using a 90 minute gradient starting at 70% B / 30% A to 100% B. The resulting pool from the final purification was lyophilized to obtain the dry final drug substance (2.9 g of isolated Compound 1).

[0106] Example 2. Synthesis of Compound 1 from the 4-(trifluoromethyl)phenyl boronic acid ester of anidulafungin.

[0107] The reaction was performed at a similar 200 mg scale as the method of Example 1 except the boronic acid was changed to 4-(trifluoromethyl)phenyl boronic acid and the reaction time was 24 hours. Results: 63% Compound 1; 7.0% Compound 1 β- diastereomer.

[0108] A second conjugation experiment was performed in which the 4-(trifluoromethyl)phenyl boronic acid ester was first dissolved in an acetonitrile:TFA mixture and then a dry choline chloride solution was added to it. After 2.5 h, the reaction mixture was diluted with water:acetonitrile (70:30) and the pH was adjusted to 2.0 by the addition of ammonium hydroxide. Results: 75% Compound 1; 4.8% Compound 1 β- diastereomer.

[0109] Example 3. Synthesis of Compound 1 from the 2,6-dimethylphenyl boronic acid ester of anidulafungin.

[0110] The reaction was performed at a similar 200 mg scale as the method of Example 1 except the boronic acid was changed to 2,6-dimethylphenyl boronic acid. Results: 55% Compound 1; 7.4% Compound 1 β- diastereomer.

[0111] Example 4. Synthesis of Compound 1 from the 3,4-dimethoxyphenyl boronic acid ester of anidulafungin.

[0112] The reaction was performed at a similar 200 mg scale as the method of Example 1 except the boronic acid was replaced with 3,4-dimethoxyphenyl boronic acid. One to three runs were performed to establish reproducibility of the method. The fourth run was performed with 10 equivalents of choline chloride (instead of the 30 equivalents used in Example 1). The fifth run was performed at 40 °C (instead of room temperature as described in Example 1). Results are provided in Table 1 below.

[0113] Table 1.

[0114] Run No. Compound 1 β-diastereoisomer 1 88.8％ 2.6％ 2 86.4％ 3.7％ 3 86.4％ 3.0％ 4 78.8％ 3.4％ 5 88.4％ 4.0％

[0115] The use of the 3,4-dimethoxyphenyl boronic acid ester of anidulafungin reduced the amount of Compound 1 β-diastereomer formed relative to other boronic acid esters.

[0116] Example 5. Compound 1 synthesis from stoichiometric 3,4-dimethoxyphenylboronic acid anidulafungin 3,4-dimethoxyphenylboronate.

[0117] The effect of using stoichiometric (1.05 equivalents) 3,4-dimethoxyphenylboronic acid in the conjugation step was investigated. The reaction was performed on a 500 mg scale of anidulafungin and was performed as previously described in Example 1 except that the boronic acid was replaced with 3,4-dimethoxyphenylboronic acid. The results are provided in Table 2 below.

[0118] Table 2

[0119]

[0120] From these data it can be concluded that while the use of stoichiometric boronic acid in the process can result in a further reduction in the fraction of the compound 1 beta- diastereoisomer produced, on the other hand, the amount of compound 1 epimer byproduct is significantly increased.

[0121] Example 6. Compound 1 synthesis from anidulafungin 3,4-dimethoxyphenylboronate.

[0122] Choline chloride was dried:

[0123] Choline chloride (185 g) was suspended in 2-methyltetrahydrofuran (500 ml) and stirred at room temperature for 1 hour. The solvent was removed under vacuum to near dryness and then dried under vacuum at 70-75 °C for 1 hour.

[0124] Preparation of anidulafungin boronate:

[0125] Anidulafungin (50 g), 3,4-dimethoxyphenylboronic acid (10.37 g) and tetrahydrofuran (250 ml) were charged into a 1000 mL round bottom flask. The suspension was stirred at room temperature for 1.5 hours. The solvent was removed under vacuum. The resulting solid was dissolved in 2-methyltetrahydrofuran (400 mL) and the solvent was evaporated under vacuum. This process was repeated once more.

[0126] Conjugation:

[0127] Dry choline chloride (73.6 g), acetonitrile (200 mL), and trifluoroacetic acid (48 mL) were combined. The suspension was stirred for 10 min. In a second reactor, dry anidulafungin boronate ester (25.6 g) and anhydrous tetrahydrofuran (150 mL) were combined and stirred at room temperature until the material was completely dissolved (30 minutes). The acidic solution of choline chloride was added to the stirred boronate ester solution over 30 minutes. The resulting suspension was stirred at room temperature for 3 hours, then cooled to <10 °C, and quenched by the addition of a 70 / 30 water:acetonitrile mixture (560 mL). The pH of the crude reaction mixture was adjusted to be in the range of 2.0-2.2 by the slow addition of a chilled, half-diluted ammonium hydroxide solution (typically 80-82 mL). The crude solution was diluted to a final volume of 2000 mL with a 70 / 30 water:acetonitrile solution. The crude solution had a compound 1 beta- diastereomer content of 3.7% and a compound 1 epimer content of 0.43%.

[0128] After synthesis of the crude mixture, compound 1 was purified using reverse phase C18 silica media, where a water acetonitrile gradient was used to elute the product from the column. Formal acetate exchange and boronic acid removal were performed in the same process. The final pooled solution of appropriate purity was brought up to column concentration using the same media to generate a concentrated solution. After concentration, the compound 1 solution was concentrated by removing acetonitrile under reduced pressure; the concentrated solution was filtered through a 0.2 pm filter and freeze-dried to yield compound 1 acetate as a white solid with a purity of 97.7%, 1.6% compound 1 beta-diastereomer, and 0.43% compound 1 epimer.

[0129] Example 7. Synthesis of compound 1 from 3,4-dimethoxyphenyl boronic ester of anidulafungin - effect of dilution with acetonitrile.

[0130] The boronic ester was prepared using the conditions reported in Example 6 and coupled with choline chloride in acetonitrile. The reaction was allowed to complete in 2-3 hours and formed a mixture of compound 1 : compound 1 beta-diastereomer of about 96:4. By diluting the reaction mixture with additional acetonitrile (20 to 50 volumes relative to anidulafungin) at the end of the reaction, this ratio was increased to >98:2, which precipitated the alpha isomer and caused the beta isomer to convert to the alpha isomer. The reaction was then quenched to pH 4 with ammonia water / ammonium acetate. The crude yield of compound 1 trifluoroacetate was 75-80%.

[0131] Example 8. Synthesis of compound 1 from 3,4-dimethoxyphenyl boronic ester of anidulafungin - combination of TFAA and dilution with acetonitrile.

[0132] Synthesis of boronic ester slurry:

[0133] A 1000 mL reactor was charged with the following: tetrahydrofuran (250 mL), anidulafungin (25 g), 3,4-dimethoxyphenylboronic acid (5.25 g). The suspension was stirred at room temperature for 1 h. The jacket temperature was set to 30-35 °C, vacuum was applied, and tetrahydrofuran distillation was started. Tetrahydrofuran was added in portions (62.5 mL) to maintain a constant volume in the reactor while distilling. A total of 1250 mL of tetrahydrofuran was distilled. Then, acetonitrile (500 mL) was charged and distillation was restarted. Approximately 600 mL of tetrahydrofuran / acetonitrile mixture was distilled. Additional acetonitrile (250 mL) was charged and 250 mL of acetonitrile / tetrahydrofuran mixture was distilled under vacuum. The reactor contents were cooled to 18-22 °C.

[0134] Composition of acidic choline chloride solution:

[0135] Acetonitrile (57.5 mL), choline chloride (52.5 g), trifluoroacetic acid (32.5 mL), and trifluoroacetic anhydride (2.0 mL) were charged into a 250 mL round bottom flask. The mixture was stirred at 18-22 °C for one hour.

[0136] Conjugation:

[0137] The acidic choline chloride solution was transferred to the reactor containing the boronate slurry. After 1.75 to 2.00 hours of mixing, acetonitrile (285 mL) was added to the reaction mixture and stirred at 10-15 °C for 1 hour. Then additional acetonitrile (285 mL) was added. If Compound 1 beta- diastereomer % > 2.0%, additional acetonitrile (142 mL) was added. After 0.5 hours, the reaction was quenched by the addition of a chilled ammonium acetate solution (143 mL) followed by the slow addition of a chilled solution of 9 M aqueous ammonium hydroxide (28.7 mL) to maintain the temperature < 15 °C and to keep the pH in the range of 4.0-4.7. The crude yield of Compound 1 trifluoroacetate salt was 75-80% with less than 2% Compound 1 beta-diastereomer.

[0138] Example 9. Synthesis of Compound 1 from 3,4-dimethoxyphenyl boronate ester of anidulafungin - coupling in the presence of TFAA.

[0139] A 1 L reactor was charged with tetrahydrofuran (700 mL) and anidulafungin (108.44 g). Then 3,4-dimethoxyphenylboronic acid (21.0 g) was charged and the mixture was stirred at 18-22 °C. The reaction mixture was azeotropically dried by distilling tetrahydrofuran while simultaneously adding fresh tetrahydrofuran (7.0 L). The constant volume solvent exchange into acetonitrile was performed by adding acetonitrile (2.1 L) while simultaneously performing a vacuum distillation. After complete conversion to acetonitrile, further distillation was performed to reduce the volume to 420 mL.

[0140] In a separate vessel, the following were combined with stirring: choline chloride (172 g), acetonitrile (217 mL), trifluoroacetic acid (142 mL), and trifluoroacetic anhydride (8.6 mL). This solution was then added to the slurry containing the anidulafungin borate ester and the resulting mixture was stirred at 15 °C for 8 hours. The reaction was quenched by charging the reactor with a cooled (T < 10 °C) ammonium acetate solution (4.2 M, 221 mL) followed immediately by a chilled (T < 10 °C) water (221 mL). Then, a cooled (10 °C) ammonium hydroxide solution (9.0 M, 126.4 mL) was added. The final pH was adjusted to pH 4.0-4.6 by the addition of ammonium hydroxide. The crude reaction mixture was diluted with water:acetonitrile (3:1, 6 L) and stored at -20 °C.

[0141] Results: Compound 1, 76.8%; Compound 1 β- diastereomer, 0.8%.

[0142] The reduction in Compound 1 β-diastereomer levels has allowed the replacement of HPLC purification with medium pressure chromatography (MPLC) using a coarser grade of C18 silica (25-50 μm). The 3,4-dimethoxyphenylboronic acid can be isolated by ion exchange capture, eluted with 100 mM ammonium acetate in water:acetonitrile 50:50 v:v (pH 4.5), providing a salt exchange from trifluoroacetate to acetate.

[0143] Following chromatography, the Compound 1 acetate solution was concentrated by vacuum distillation to remove most of the acetonitrile. The concentrated solution was filtered through a 0.2 μm filter and freeze-dried to yield Compound 1 acetate. Purity following MPLC and following ion exchange and lyophilization is provided in Table 3 below.

[0144] Table 3

[0145]

[0146] Other Embodiments

[0147] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0148] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure that come within known or customary practice within the art to which the disclosure pertains and fall within the scope of the appended claims. Other embodiments are within the claims.

Claims

1. A method for synthesizing compound 1: The method includes: (a) Providing a first composition comprising an arylboronic acid ester of anidoxane; (b) A second composition comprising a salt of choline, acetonitrile, trifluoroacetic acid, and trifluoroacetic anhydride; (c) Combining the first composition and the second composition to form a mixture comprising a precipitate having the reaction product of formula (I): in X – It is an anion; and R represents the replacement of C6-C. 10 Aryl; and (d) Hydrolyze the compound of formula (I) to form compound 1 or its salt or neutral form.

2. The method of claim 1, wherein the concentration of the mixture is at least 0.01 mol / L relative to the compound of formula (I).

3. The method of claim 1 or 2, wherein step (c) comprises forming a mixture comprising at least 10 molar equivalents of the salt of said choline and at least 1 molar equivalent of the arylboronic acid ester of said anifenidine.

4. The method of claim 1 or 2, wherein step (c) is performed at a temperature less than 40°C.

5. The method of claim 1 or 2, wherein step (c) comprises forming a mixture in which at least 50% of the compound of formula (I) is precipitated.

6. The method of claim 1 or 2, wherein R is a substituted C6 aryl group.

7. A method for synthesizing compound 1: The method includes: (a) Providing a first composition comprising an arylboronic acid ester of anidoxane; (b) A second composition comprising a salt of choline, acetonitrile, and trifluoroacetic acid; (c) Combining the first composition and the second composition to form a mixture comprising a compound of formula (II): in X – It is an anion; and Ar is selected from 3,4-dimethoxyphenyl and 4-trifluoromethylphenyl; and (d) Hydrolyze the compound of formula (II) to form compound 1 or its salt or neutral form.

8. The method of claim 7, wherein Ar is 3,4-dimethoxyphenyl.

9. The method of claim 7, wherein Ar is 4-trifluoromethylphenyl.

10. The method of claim 7, wherein step (c) comprises combining at least 10 molar equivalents of the salt of choline with at least 1 molar equivalent of the arylboronic acid ester of anidoxane.

11. The method of any one of claims 1, 2 and 7, wherein the first composition comprises a solution of the aryl borate ester of anisfenol dissolved in an organic solvent selected from acetonitrile, butyronitrile, tetrahydrofuran or 2-methyltetrahydrofuran, or mixtures thereof.

12. The method of claim 7, wherein the second composition comprises a solution of the choline salt dissolved in a mixture of acetonitrile, trifluoroacetic acid, and trifluoroacetic anhydride.

13. The method of any one of claims 1, 2 and 7, wherein step (c) further comprises adding acetonitrile to the mixture to reduce the level of β-diastereomer.

14. The method of any one of claims 1, 2 and 7, wherein the mixture is formed at a temperature of less than 40°C.

15. The method of any one of claims 1, 2 and 7, wherein step (d) further comprises diluting with a water:acetonitrile mixture of about 80:20 to 50:50 relative to at least 5 volumes of anisfenol, and adjusting the pH to pH 2 to 5 with an alkali.

16. The method of any one of claims 1, 2 and 7, wherein step (d) comprises forming a reaction product comprising more than 70% of compound 1 and less than 4% of the β-diastere of compound 1.

17. The method of any one of claims 1, 2 and 7, wherein step (d) comprises forming a reaction product comprising more than 70% of compound 1, less than 2% of compound 1 β-diastere and less than 1% of compound 1 epistere.

18. A method for synthesizing compound 1: The method includes hydrolyzing the compound of formula (IIa): Where X - It is an anion, to form compound 1 or its salt or neutral form.

19. The method of claim 18, wherein the hydrolysis comprises contacting the compound of formula (IIa) with an aqueous alkaline solution.

20. The method of claim 19, wherein the hydrolysis comprises diluting with a water:acetonitrile mixture of about 80:20 to 50:50 relative to at least 5 volumes of anisfenol, and adjusting the pH to pH 2 to 5 with an alkali.

21. The method of claim 19, wherein the hydrolysis is carried out at a temperature of less than 15°C.

22. The method of any one of claims 18 to 21, wherein after hydrolyzing the compound of formula (IIa) to form compound 1, compound 1 is separated from 3,4-dimethoxyphenylboronic acid by passing it through an ion exchange column.

23. The method according to any one of claims 18 to 21, wherein the compound of formula (IIa) is hydrolyzed to form compound 1 on a scale of producing 100 grams to 50 kilograms of compound 1.

24. The method of any one of claims 1, 2, 7 and 18 to 21, wherein the method further comprises producing a pharmaceutical composition by combining the compound 1 with a pharmaceutically acceptable excipient.

25. The method of claim 24, wherein the pharmaceutical composition is formulated for topical or parenteral application.

26. A compound of formula (IIa): Where X - It is an anion.

27. A compound of formula (IIb): Where X - It is an anion.

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