Synthesis of echinocandin antifungal agent

Abstract

The present invention relates to echinocandin cyclopeptides and to methods for preparing echinocandin cyclopeptides.

Claims

1. A method of synthesizing compound 1: ##STR00011## said method comprising: (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 trifluoroacetic acid in a solvent system that comprises acetonitrile and trifluoroacetic anhydride to form a precipitate of a reaction product having formula (I): ##STR00012## wherein X.sup.− is an anion; and R is C.sub.1-C.sub.6 alkyl or C.sub.6-C.sub.10 aryl; and; (d) hydrolyzing the compound of formula (I) to form compound 1, or a salt or neutral form thereof.

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

3. The method of claim 1, wherein step (c) comprises forming a mixture in a solvent system comprising a mixture of tetrahydrofuran and acetonitrile.

4. The method of claim 1, wherein step (c) comprises forming a mixture comprising at least 10 molar equivalents of the salt of choline and at least 1 molar equivalents of the boronate ester of anidulafungin.

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

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

7. The method of claim 1, wherein R is C.sub.6-C.sub.10 aryl.

8. The method of claim 1, wherein R is C.sub.1-C.sub.6 alkyl.

9. A method of synthesizing compound 1: ##STR00013## said method comprising: (a) providing a first composition comprising an aryl 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 comprising a compound of formula (II): ##STR00014## wherein X.sup.− is an anion; and Ar is substituted C.sub.6 aryl; and (d) hydrolyzing the compound of formula (II) to form compound 1, or salt or neutral form thereof.

10. The method of claim 9, wherein Ar is selected from 3,4-dimethoxyphenyl, 2,6-dimethylphenyl, and 4-trifluoromethylphenyl.

11. The method of claim 9, wherein step (c) comprises combining at least 10 molar equivalents of the salt of choline with at least 1 molar equivalents of the boronate ester of anidulafungin.

12. The method of claim 9, wherein the first composition comprises a solution of the boronate ester of anidulafungin dissolved in an organic solvent selected from acetonitrile, butyronitrile, tetrahydrofuran, or 2-methyltetrahydrofuran, or a mixture thereof.

13. The method of claim 9, wherein the second composition comprises a solution of the salt of choline dissolved in a mixture of acetonitrile and trifluoroacetic acid.

14. The method of claim 9, wherein the second composition comprises a solution of the salt of choline dissolved in a mixture of acetonitrile, trifluoroacetic acid, and trifluoroacetic anhydride.

15. The method of claim 9, wherein step (c) further comprises adding acetonitrile to the mixture to reduce the level of beta-diastereomer.

16. The method of claim 9, wherein step (d) further comprises diluting with at least 5 volumes relative to anidulafungin of water:acetonitrile mixture of about 80:20 to 50:50 and adjusting the pH with base to a pH of from 2 to 5.

17. The method of claim 9, wherein step (d) comprises forming a reaction product comprising greater than 70% compound 1 and less than 4% compound 1 beta-diastereomer.

18. The method of claim 9, wherein the hydrolyzing is performed at a temperature of less than 15° C.

19. The method of claim 9, wherein the method further comprises producing a pharmaceutical composition by combining the compound 1 with a pharmaceutically acceptable excipient.

20. The method of claim 9, wherein the acid of step (c) is trifluoroacetic acid.

21. The method of claim 9, wherein the solvent system of step (c) comprises acetonitrile and trifluoroacetic anhydride.

22. The method of claim 9, wherein the method further comprises the step (e) purifying compound 1, or a salt or neutral form thereof, by reverse phase preparative high performance liquid chromatography or reverse phase preparative medium pressure liquid chromatography.

23. The method of claim 1, wherein the method further comprises step (e) purifying compound 1, or a salt or neutral form thereof, by reverse phase preparative high performance liquid chromatography or reverse phase preparative medium pressure liquid chromatography.

Description

DETAILED DESCRIPTION

(1) Provided herein are synthetic methods and intermediates for making the echinocandin antifungal agent compound 1, or a salt or neutral form thereof. The methods and intermediates can be useful for achieving a higher yield, a higher chemical purity, and/or a higher diastereomeric purity, and a lower cost for the preparation of compound 1. Further synthetic details are provided in the Examples.

(2) The invention features a process for synthesis of compound 1 acetate from anidulafungin using aryl boronic acids as an in situ protecting group, which was developed as follows. In one embodiment, the first step involves slurrying of choline chloride in 2-methyltetrahydrofuran, which is then distilled off. The resulting solid is further dried in a vacuum oven at elevated temperature. The second step involves protecting the anidulafungin starting material by converting it into its 3,4-dimethoxyphenylboronate ester by reacting with 1.3 equivalents of 3,4-dimethoxyphenylboronic acid in tetrahydrofuran. Evaporation of the solvent under reduced vacuum affords the protected intermediate as a solid which is further dried by repeated azeodrying cycles with 2-methyltetrahydrofuran. Alternatively, other methods of water removal can be employed, such as addition of activated molecular sieves, continuous distillation, or addition of dehydrating agents. In the third and final step to the crude material, the azeodried choline chloride is dissolved in a mixture of TFA and acetonitrile and conjugated to the protected anidulafungin backbone to afford the compound 1 as its TFA/chloride form. The reaction is then quenched by addition of a water:acetonitrile mixture and the pH is adjusted to afford a reasonably stable crude mixture that is ready to be fed into the purification process.

(3) The invention features a process for the synthesis of compound 1 acetate from anidulafungin, which entails using 3,4-dimethoxyphenyl boronic acid as an in situ protecting group, wherein, when the conjugation reaction is complete, additional acetonitrile (20 to 50 volumes relative to anidulafungin) is added. This causes precipitation of compound 1. Since the equilibrium between compound 1 and the beta-isomer of 1 (approximately 95:5) in solution is maintained under the acidic conditions, the precipitation of compound 1 from solution results in driving formation of compound 1 and lowering the beta-isomer amount. The beta isomer at the end of reaction can be controlled to no more than 2.0% under these conditions.

(4) The invention features a process for the synthesis of compound 1 acetate from anidulafungin using 3,4-dimethoxyphenyl boronic acid as an in situ protecting group involves conducting the conjugation reaction with 12-18 equivalents of choline chloride under more concentrated conditions. This causes precipitation of compound 1 as the reaction proceeds. Since the equilibrium between compound 1 and the beta-isomer of 1 (approximately 95:5) is solution is maintained under the acidic conditions, the precipitation of compound 1 from solution results in driving formation of compound 1 and lowering the beta-isomer amount. The beta isomer at the end of reaction can be controlled to less than 2.0% under these conditions. The invention also features a process for synthesis of compound 1 acetate from anidulafungin using 2,6-dimethylphenyl boronic acid as an in situ protecting agent.

(5) The invention also features a process for synthesis of compound 1 acetate from anidulafungin using 4-trifluromethylphenyl boronic acid as an in situ protecting agent.

(6) The invention features a purification process where the crude reaction is purified either 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.

(7) The advantages of the invention include a significant improvement in diastereomeric purity, which allows for a more straightforward purification process and an overall higher purity product. Although the boronic acid group is far from the reacting center, it was surprisingly found that the nature of the groups on the aryl boronic acid had a significant impact on the diastereoselectivity in the conjugation reaction. In particular, the use of the 3,4-dimethoxyphenylboronate ester of anidulafungin reduced the amount of compound 1 beta-diastereomer formed relative to other boronate esters, resulting in a simpler purification method and higher purity of compound 1.

(8) Compound 1 can be useful for treating, mitigating, or preventing a fungal infection or related conditions thereto in a human subject in need thereof.

(9) Compound 1 may be prepared in a pharmaceutical composition. The pharmaceutical composition can include a salt of compound 1, or a neutral form thereof, and 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., subcutaneously or intravenously) and the dosage, compound 1 may be formulated into suitable pharmaceutical compositions to permit facile delivery. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 22nd Edition, 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.

(10) For subcutaneous administration, compound 1 may be formulated as an aqueous pharmaceutical composition. In some embodiments, the pharmaceutical composition containing compound 1 formulated for subcutaneous administration may not contain a buffer. In some embodiments, the pharmaceutical composition formulated for subcutaneous administration may contain a weak buffer. Examples of a weak buffer that may be used in the pharmaceutical composition include, but are not limited to, acetate, lactate, histidine, glycine, and formate.

(11) A pharmaceutical composition including compound 1 in salt or neutral form may optionally contain an amount of a solubilizing agent. Examples of a solubilizing agent 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), β-cyclodextrin, polyoxyl 35 castor oil (Cremophor EL), polyoxyl 40 hydrogenated castor oil (Cremophor RH 40), polyoxyl 60 hydrogenated castor oil (Cremophor RH 60), D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), sorbitan monooleate (Span 20), polyoxyl 8 stearate (PEG 400 monosterate), polyoxyl 40 stearate (PEG 1750 monosterate), PEG 400 caprylic/capric glycerides (Labrasol), PEG 300 oleic glycerides (Labrafil M-1944CS), phosphatidylcholine (lecithin), alkylglucoside, sucrose monolaurate, sucrose monooleate, and polyoxyethylene-polyoxypropylene block copolymer (Poloxamer).

(12) Furthermore, a pharmaceutical composition including compound 1 in salt or neutral form may contain between 0.5% to 3% (w/w) of a saccharide. Examples of a saccharide that may be included in the pharmaceutical composition including compound 1 in salt or neutral form used in the methods of the invention include, but are not limited to, mannitol, sucrose, trehalose, fructose, glucose, dextrose, dextran, lactose, and sorbital.

(13) A pharmaceutical composition including compound 1 in salt or neutral form may be formulated as a lyophilized composition. Moreover, the lyophilized composition including compound 1, when re-constituted in water for injection, may have a pH of 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). In some embodiments, compound 1 in salt form may be compound 1 acetate.

(14) The pharmaceutical compositions used in methods of the invention may be formulated in the form of liquid solutions or suspensions or lyophilized cakes and administered by a parenteral route (e.g., subcutaneous or intravenous). Pharmaceutical compositions for parenteral administration can be formulated using a sterile solution 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), α-Modified Eagles Medium (α-MEM), F-12 medium). Formulation methods are known in the art, see e.g., Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd ed.) Taylor & Francis Group, CRC Press (2009).

(15) Furthermore, acceptable carriers and excipients in the pharmaceutical composition used in methods of the invention are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, 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 glucose, mannose, sucrose, and sorbitol. The compositions may be formulated according to conventional pharmaceutical practice. The concentration of the compound in the formulation will vary depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

(16) The pharmaceutical compositions of the invention can be administered to human subjects in therapeutically effective amounts. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular human subject, the specific compound being administered, the excipients used to formulate the compound, and its route of administration.

(17) The timing of the administration of the pharmaceutical composition containing compound 1 in salt or neutral form depends on the medical and health status of the human subject. In some instances, the human subject is at risk for developing a fungal infection or a related condition and receives one or more doses treatment with compound 1 before developing symptoms or signs of a fungal infection. In some instances, the human subject has already developed a fungal infection or a related condition and receives one or more doses treatment with compound 1. The timing of the administration of the dose(s) of compound 1 may be optimized by a physician to reduce the risk of or to treat a fungal infection in a human subject.

(18) The following examples, as set forth below, are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1. Synthesis of Compound 1 from the phenylboronate Ester of Anidulafungin

(19) Anidulafungin phenylboronate ester:

(20) 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 phenylboronate ester solid was dried in vacuum overnight.

(21) Choline Chloride Drying:

(22) 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 one more time. The resulting solid was dried overnight in vacuum.

(23) Conjugation:

(24) 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 phenylboronate 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 was basified with NH.sub.4OH (2N, ˜40 mL) to pH ˜2. A white material was formed and was dissolved with acetonitrile (300 mL). The material contained 4.55% compound 1 beta-diastereomer (average of two runs).

(25) Purification:

(26) The material was purified by preparative reversed-phase HPLC with 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 with 70% B/30% A to 100% B. Pools resulting from the final purification were lyophilized to obtain the dried final bulk drug substance (2.9 g isolated compound 1).

Example 2. Synthesis of Compound 1 from the 4-(trifluoromethyl)phenylboronate Ester of Anidulafungin

(27) The reaction was carried out on 200 mg scale similar to the process of Example 1 except for the change in boronic acid to 4-(trifluoromethy)phenylboronic acid and a reaction time of 24 hours. Results: 63% compound 1; 7.0% compound 1 beta-diastereomer.

(28) A second conjugation experiment was performed where the 4-(trifluoromethyl)phenylboronate ester was solubilized in acetonitrile:TFA mixture initially, and then the dried 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 addition of ammonium hydroxide. Results: 75% compound 1; 4.8% compound 1 beta-diastereomer.

Example 3. Synthesis of Compound 1 from the 2,6-dimethylphenylboronate Ester of Anidulafungin

(29) The reaction was carried out on 200 mg scale similar to the process of Example 1 except for the change to the boronic acid to 2,6-dimethylphenylboronic acid. Results: 55% compound 1; 7.4% compound 1 beta-diastereomer.

Example 4. Synthesis of Compound 1 from the 3,4-dimethoxyphenylboronate Ester of Anidulafungin

(30) The reactions were carried out on 200 mg scale similar to the process of Example 1 except for the replacing the boronic acid to 3,4-dimethoxyphenylboronic acid. Runs 1-3 were carried out to establish reproducibility of the process. Run 4 was performed with 10 equivalents of choline chloride (rather than the 30 equivalents used in Example 1). Run 5 was performed at 40° C. (rather than at room temperature as described in Example 1). Results are provided in Table 1 below.

(31) TABLE-US-00001 TABLE 1 Run No. Compound 1 Beta-diastereomer 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%

(32) The use of the 3,4-dimethoxyphenylboronate ester of anidulafungin reduced the amount of compound 1 beta-diastereomer formed relative to other boronate esters.

Example 5. Synthesis of Compound 1 from the 3,4-dimethoxyphenylboronate Ester of Anidulafungin Made from a Stoichiometric Amount of 3,4-dimethoxyphenylboronic Acid

(33) The effect of using a stoichiometric amount (1.05 eq) of 3,4-dimethoxyphenylboronic acid in the conjugation step was investigated. The reaction was performed on a 500 mg anidulafungin input and performed as previously described in Example 1 except for the replacing the boronic acid to 3,4-dimethoxyphenylboronic acid. Results are provided in Table 2 below.

(34) TABLE-US-00002 TABLE 2 Boronic compound compound 1 beta- unreacted compound 1 acid (eq) 1 diastereomer anidulafungin epimer 1.3 89.5% 2.5% 5.7% 0.5% 1.05 89.4% 2.0% 4.2% 1.3%

(35) We can conclude from these data that while using a stoichiometric amount of boronic acid in the process may lead to a further reduction in the fraction of compound 1 beta-diastereomer that is generated, the amount of compound 1 epimer byproduct, on the other hand, is significantly increased.

Example 6. Synthesis of Compound 1 from the 3,4-dimethoxyphenylboronate Ester of Anidulafungin

(36) Choline chloride drying:

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

(38) Anidulafungin boronate ester preparation:

(39) Anidulafungin (50 g), 3,4-dimethoxyphenylboronic acid (10.37 g), and tetrahydrofuran (250 ml) were charged in 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 solubilized in 2-methyltetrahydrofuran (400 mL) and the solvent was evaporated under vacuum. This process was repeated one more time.

(40) Conjugation:

(41) Dried 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, dried anidulafungin boronate ester (25.6 g) and dry tetrahydrofuran (150 mL) were combined and stirred at room temperature until the material was completely solubilized (30 minutes). The acidic solution of choline chloride was added to the stirred boronate ester solution over 30 minutes. The resulting suspension was stirred for 3 hours at room temperature then cooled to ≤10° C. and quenched by addition of 70/30 water:acetonitrile mixture (560 mL). The pH of the crude reaction mixture was adjusted within the 2.0-2.2 range by slow addition of chilled half-dilute ammonium hydroxide solution (typically 80-82 mL). The crude solution was diluted to a final volume of 2000 mL with 70/30 water:acetonitrile solution. The compound 1 beta-diastereomer content of the crude solution was 3.7% and the compound 1 epimer content was 0.43%.

(42) After synthesis of the crude mixture, compound 1 was purified using a reversed phase C18 silica media, with the product eluted from the column using an aqueous acetonitrile gradient. A formal acetate exchange and removal of boronic acid was performed in the same process. Final pools of the appropriate purity were brought forward to an on-column concentration using the same media to generate a concentrated solution. Post concentration, compound 1 solution was concentrated via acetonitrile removal under reduced pressure; the concentrated solution was filtered through a 0.2 μm filter and freeze-dried to produce compound 1 acetate as a white solid with 97.7% purity, 1.6% compound 1 beta-diastereomer, and 0.43% compound 1 epimer.

Example 7. Synthesis of Compound 1 from the 3,4-dimethoxyphenylboronate Ester of Anidulafungin—Effect of Dilution with Acetonitrile

(43) The boronate ester was prepared and coupled with choline chloride in acetonitrile using the conditions reported in example 6. The reaction was complete in 2-3 hours and formed a approx. 96:4 mixture of compound 1:compound 1 beta-diastereomer. This ratio was improved to >98:2 by dilution of the reaction mixture with additional acetonitrile (20-50 volumes relative to anidulafungin) at the end of the reaction, which precipitates the alpha isomer and results in conversion of beta to alpha isomer. The reaction was then quenched with aqueous ammonia/ammonium acetate to pH 4. The crude yield of compound 1 trifluoroacetate was 75-80%.

Example 8. Synthesis of Compound 1 from the 3,4-dimethoxyphenylboronate Ester of Anidulafungin—Combination of TFAA and Dilution with Acetonitrile

(44) Boronate ester slurry synthesis:

(45) To a 1000 mL reactor the following were charged: tetrahydrofuran (250 mL), anidulafungin (25 g), 3,4-dimethoxyphenylboronic acid (5.25 g). The suspension was stirred for 1 h at room temperature. The jacket temperature was set to 30-35° C., a vacuum applied, and tetrahydrofuran distillation was initiated. Portion wise (62.5 mL) additions of tetrahydrofuran were made 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 distilling re-initiated. Approximately 600 mL of tetrahydrofuran/acetonitrile mixture were distilled. Additional acetonitrile (250 mL) was charged and 250 mL of acetonitrile/tetrahydrofuran mixture distilled under vacuum. The reactor contents were cooled to 18-22° C.

(46) Acidic choline chloride solution makeup:

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

(48) Conjugation:

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

Example 9. Synthesis of Compound 1 from the 3,4-dimethoxyphenylboronate Ester of Anidulafungin—Coupling in the Presence of TFAA

(50) Tetrahydrofuran (700 mL) and anidulafungin (108.44 g) were charged to a 1 L reactor. 3,4-Dimethoxyphenylboronic acid (21.0 g) was then charged and the mixture was stirred at 18-22° C. The reaction mixture was azeodried by distillation of tetrahydrofuran and simultaneous addition of fresh tetrahydrofuran (7.0 L). A constant volume solvent swap to acetonitrile was carried out by addition of acetonitrile (2.1 L) and simultaneous vacuum distillation. After complete turnover to acetonitrile, further distillation was carried out to reduce the volume to 420 mL.

(51) 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 boronate ester and the resulting mixture was stirred at 15° C. for 8 hours. The reaction was quenched by charging cooled (T<10° C.) solution of ammonium acetate (4.2 M, 221 mL) to the reactor at once followed by addition of chilled (T<10° C.)) water (221 mL). Then, a cooled (10° C.) solution of ammonium hydroxide (9.0 M, 126.4 mL) was added. The final pH was adjusted to pH 4.0-4.6 by addition of ammonium hydroxide. The crude reaction mixture was diluted with water:acetonitrile (3:1, 6 L) and stored at −20° C.

(52) Results: compound 1, 76.8%, compound 1 beta-diastereomer, 0.8%.

(53) A reduction in the level of compound 1 beta-diastereomer has allowed for replacement of the HPLC purification with medium pressure chromatography (MPLC) using a coarser grade of C18 silica (25 to 50 μm). The 3,4-dimethoxyphenyl boronic acid can be separated by ion-exchange capture, eluting with 100 mM ammonium acetate (pH 4.5) in water:acetonitrile 50:50 v:v, which affords salt exchange from trifluoroacetate to acetate.

(54) Post chromatography, the compound 1 acetate solution was concentrated by vacuum distillation to remove the majority of acetonitrile. The concentrated solution was filtered through a 0.2 μm filter and freeze-dried to produce compound 1 acetate. The purity after MPLC and after ion exchange and lyophilization is provided in Table 3 below.

(55) TABLE-US-00003 TABLE 3 compound 1 beta- compound 1 Stage compound 1 diastereomer epimer after MPLC 98.47% 0.77% 0.47% after ion exchange and 98.49% 0.77% 0.51% lyophilization

Other Embodiments

(56) 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.

(57) 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 may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.