A new method for the preparation of amorphous voclosporin.

Abstract

The present invention relates to a new process for the preparation of amorphous forms of voclosporin.

Description

[Technical Field]

[0001] The present invention relates to a new process for the preparation of amorphous voclosporin. [Background technology]

[0002] Voclosporin is a compound of formula (I) and is an analogue of cyclosporin A. [ka] It is a calcineurin inhibitor and is used as an active ingredient in medicines, particularly with immunosuppressive effects, in particular in the treatment of certain types of lupus. Although the compound itself has been known for some time, the applicant has found that the currently known methods for synthesizing voclosporin have technical limitations regarding their industrial applicability and scalability, in particular with regard to the possibility of obtaining a highly pure product in high yield and at low cost.

[0003] The applicant has particularly noted that the synthetic methods known to date often start with cyclosporin A, which is derived from natural products and has immunosuppressive properties, and subject said cyclosporin to a series of reactions involving the production of various intermediates, including acetylcyclosporin A aldehyde and acetylcyclosporin (the two compounds of formulae (III) and (IV), respectively). [ka] [ka] In this regard, the applicant has pointed out that the synthetic steps relating to these intermediate products and the form and ease with which the final voclosporin is obtained are particularly important in order to provide a synthetic method that can achieve high yields at low cost and provide a highly pure product. Summary of the Invention

[0004] It is therefore an object of the present invention to provide a new process for the synthesis of voclosporin that overcomes current difficulties and limitations, thereby achieving high yields at low cost and providing a product of high purity. In accordance with the present invention, the applicant has surprisingly found that it is possible to achieve the above-mentioned objectives by employing special reaction conditions and ingenuity in the steps involved in producing voclosporin from cyclosporin A, obtaining acetyl cyclosporin A, acetyl cyclosporin A aldehyde and acetyl voclosporin.

[0005] In particular, the applicant has discovered the possibility of obtaining the intermediates simply and in high yield, and also of obtaining the final product, voclosporin, in amorphous form, which has been found to be particularly easy to handle and to be a stable solid form over time. Thus, the process according to the present invention is competitive with existing methods for synthesizing voclosporin and offers an industrially scalable option for obtaining voclosporin in high yield and in a form that is easy to handle and stable over time.

[0006] Thus, in a first aspect, the present invention relates to a process for preparing voclosporin of formula (I) in amorphous form, comprising: [ka] a) acetylating cyclosporin A with acetic anhydride in a stoichiometric amount relative to cyclosporin A in the presence of methyl tert-butyl ether (MTBE), thereby obtaining acetyl cyclosporin A of formula (II); [ka] b) oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), the base being in an amount such that a pH value ranging from 6 to 8 is achieved, and in the presence of a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites, thereby obtaining acetylcyclosporin A aldehyde of formula (III), [ka] c) converting acetylcyclosporin A aldehyde of formula (III) to acetylcyclosporin A of formula (IV); [ka] d) crystallizing acetylvoclosporin of formula (IV) using a solvent system selected from the group consisting of THF / n-heptane and MTBE, thereby obtaining acetylvoclosporin in crystalline form; e) hydrolyzing acetylvoclosporin of formula (IV) to obtain voclosporin of formula (I); and f) isolating the voclosporin of formula (I) obtained in step e) to obtain amorphous voclosporin of formula (I), wherein the isolation is carried out by precipitation using a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol, and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane, and pentane.

[0007] Indeed, it has surprisingly been found that by using stoichiometric amounts of acetic anhydride and MTBE in step a), it is possible to avoid the consumption of large amounts of acetic anhydride, a toxic reagent with a high environmental impact, to increase the reaction yield and improve the purity with respect to the methods applied in the prior art.

[0008] Furthermore, advantageously and surprisingly, the oxidation reaction of acetylcyclosporin A to obtain acetylcyclosporin A aldehyde by using osmates as the oxidizing agent in step b) and a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites was particularly advantageous in terms of yield and ease of operation. The presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine) made it possible to advantageously suppress carboxylic acid impurities, as will become clear from the experimental section.

[0009] It should also be noted that the intermediate product could be advantageously obtained in crystalline form by using a solvent system selected from the group consisting of THF / n-heptane and MTBE for the isolation of acetylvoclosporin. The crystalline acetylvoclosporin of the present invention exhibits an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2, and 20.9.

[0010] Furthermore, the applicant has surprisingly found that by precipitating voclosporin in a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone ethanol and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane and pentane, it is possible to obtain an amorphous product characterized by high long-term stability and easy handling due to its powdery, non-sticky form.

[0011] Due to the various improvements made in the various synthetic steps starting from cyclosporin A and leading to the production of voclosporin, the present invention also has several additional aspects. In a further aspect, the present invention specifically relates to a process for the preparation of acetyl cyclosporin A aldehyde of formula (III): [ka] A) acetylating cyclosporin A with acetic anhydride in a stoichiometric amount relative to cyclosporin A in the presence of methyl tert-butyl ether (MTBE), thereby obtaining acetyl cyclosporin A of formula (II); and [ka] B) oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), wherein the base is in an amount such that a pH value in the range of 6 to 8 is achieved, and in the presence of a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites, thereby obtaining acetylcyclosporin A aldehyde of formula (III).

[0012] Advantageously, acetylcyclosporin A of formula (II) is obtained in crystalline form using MTBE / n-heptane solvent system and exhibits an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 8.3, 9.1, 11.1, 11.3, 13.0, 15.0, 17.0, 17.6, 17.8, 19.1 and 19.6. Advantageously, acetylcyclosporin A of formula (II) is obtained in crystalline form using MTBE / cyclohexane solvent system and exhibits an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.1, 5.4, 5.5, 9.0, 9.5, 10.2, 15.5, 17.4, 20.2 and 20.9.

[0013] The advantages in terms of yield and ease of operation for the preparation of the intermediate product acetyl cyclosporin A, advantageously in the crystalline form described, and the product acetyl cyclosporin aldehyde resulting from a) the process and b) the process characteristics, make a substantial and decisive contribution to obtaining the desired product in high yield under industrially scalable conditions, and are therefore very advantageous for the production of voclosporin.The applicant has therefore found that this further aspect, included in the first and main aspect of the present invention, represents an independently important innovative and advantageous aspect of the process for obtaining voclosporin.

[0014] In a further aspect thereof, the present invention also relates to a process for obtaining acetylvoclosporin of formula (IV) in crystalline form, and to acetylvoclosporin in crystalline form thus obtained. Therefore, the present invention also relates to a process for obtaining acetylvoclosporin of formula (IV) in crystalline form, [ka] - preparing acetylvoclosporin of formula (IV), for example, but not limited to, according to steps a) to c) of the process according to the first aspect of the present invention, - crystallizing said acetylvoclosporin of formula (IV) using a solvent system selected from the group consisting of THF / n-heptane and MTBE, thus obtaining acetylvoclosporin in crystalline form.

[0015] Furthermore, the present invention relates to a novel crystalline form of acetylvoclosporin thus obtained, and in particular to a crystalline form of acetylvoclosporin of the present invention having an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2 and 20.9. The possibility of obtaining acetylvoclosporin in crystalline form not only represents an advantage within the process according to the first and main aspect of the present invention, but also represents an important innovative and advantageous aspect of the process for obtaining voclosporin independently. Indeed, obtaining a key intermediate such as acetylvoclosporin in crystalline form allows for more efficient purification, benefiting the overall process.

[0016] In a further additional aspect, the present invention also relates to a process for obtaining amorphous voclosporin of formula (I), comprising: [ka] - preparing a solution of voclosporin of formula (I) in a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane and pentane; and - recovering the amorphous voclosporin by precipitation from the solution thereby produced. Indeed, the precipitation of voclosporin according to the invention makes it possible to obtain an amorphous product which is characterized by a high stability over time and which is easy to handle since it is powdery and non-sticky. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the XRPD spectrum of crystalline acetylcyclosporin A of formula (II) obtained according to Example 2. [Figure 2] FIG. 2 shows the chromatogram obtained in Example 3. [Figure 3] FIG. 3 shows the XRPD spectrum of crystalline acetylcyclosporin A of formula (II) obtained according to Example 3. [Figure 4] FIG. 4 shows a chromatogram obtained in Example 4 when the reaction was carried out in the presence of a base selected from dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine). [Figure 5] FIG. 5 shows a chromatogram obtained when the reaction in Example 4 was carried out in the absence of a base selected from dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine). [Figure 6] FIG. 6 shows the XRPD spectrum of the crystalline acetylvoclosporin obtained according to Examples 12 and 13. [Figure 7] FIG. 7 shows the XRPD spectrum of amorphous voclosporin obtained according to Examples 17 and 18. [Figure 8] FIG. 8 shows a comparison of XRPD spectra of amorphous voclosporin samples at the end of the stability study according to Example 19. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention In a first aspect, the present invention relates to a process for preparing voclosporin of formula (I) in amorphous form, comprising: [ka] a) acetylating cyclosporin A with acetic anhydride in a stoichiometric amount relative to cyclosporin A in the presence of methyl tert-butyl ether (MTBE), thereby obtaining acetyl cyclosporin A of formula (II); [ka] b) oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), the base being in an amount such that a pH value ranging from 6 to 8 is achieved, and in the presence of a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites, thus obtaining acetylcyclosporin A aldehyde of formula (III), [ka] c) converting acetylcyclosporin A aldehyde of formula (III) to acetylcyclosporin A of formula (IV); [ka] d) crystallizing acetylvoclosporin of formula (IV) using a solvent system selected from the group consisting of THF / n-heptane and MTBE, thereby obtaining acetylvoclosporin in crystalline form; e) hydrolyzing acetylvoclosporin of formula (IV) to obtain voclosporin of formula (I); and f) isolating the voclosporin of formula (I) obtained in step e) to obtain said voclosporin of formula (I) in amorphous form, said isolation being carried out by precipitation with a solvent / antisolvent system, said solvent being selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol and cyclopentyl methyl ether, and said antisolvent being a hydrocarbon selected from the group consisting of normal heptane, cyclohexane and pentane. Indeed, it has surprisingly been found that the use of stoichiometric amounts of acetic anhydride and MTBE in step a) makes it possible to avoid the consumption of large amounts of acetic anhydride, a toxic reagent with a high environmental impact, and improves the reaction yield and purity compared to the methods applied in the prior art.

[0019] Furthermore, advantageously and surprisingly, the oxidation reaction of acetylcyclosporin A to obtain acetylcyclosporin A aldehyde by using osmates as the oxidizing agent in step b) and a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites was particularly advantageous in terms of yield and ease of operation. The presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine) made it possible to advantageously suppress carboxylic acid impurities, as will become clear from the experimental section.

[0020] It should also be noted that the intermediate product could be advantageously obtained in crystalline form by using a solvent system selected from the group consisting of THF / n-heptane and MTBE for the isolation of acetylvoclosporin. The crystalline acetylvoclosporin of the present invention exhibits an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2, and 20.9.

[0021] Furthermore, the applicant has surprisingly found that by precipitating voclosporin in a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane and pentane, it is possible to obtain an amorphous product characterized by high long-term stability and easy handling due to its powdery, non-sticky form.

[0022] In this specification and the following claims, all numerical magnitudes expressing quantities, parameters, percentages, and the like, unless otherwise indicated, are intended to be preceded in all circumstances by the term "about." Moreover, all ranges of numerical magnitudes include all possible combinations of maximum and minimum numerical values, and all possible intermediate ranges, as set forth below.

[0023] In the present invention, when dimethylpyridine (or lutidine) is mentioned, it means a 2,4-dimethylpyridine compound or a 2,6-dimethylpyridine compound, preferably 2,6-dimethylpyridine. The present invention can be represented by one or more of its aspects or one or more of the preferred features reported below, which can be combined with each other according to the application requirements.

[0024] The process according to the present invention comprises the step a) of acetylating cyclosporin A with acetic anhydride in a stoichiometric amount relative to cyclosporin A in the presence of methyl tert-butyl ether (MTBE), thus obtaining acetyl cyclosporin A of formula (II). [ka]

[0025] Advantageously, the use of stoichiometric amounts of MTBE and acetic anhydride made it possible to avoid the consumption of large amounts of acetic anhydride. In fact, acetic anhydride is known to be a toxic reagent with significant environmental impact. The use of stoichiometric amounts of acetic anhydride in the presence of MTBE was indeed surprising, as it not only had a low environmental impact, but also allowed for improved reaction and purity compared to methods applied in the prior art.

[0026] The acetylation reaction is preferably carried out at a temperature in the range of 10 to 50°C, preferably 25 to 40°C, advantageously using one or more solvents and one or more reagents capable of forming acetate groups from hydroxyl groups. The acetylation of cyclosporin in step a) can be advantageously carried out using acetic anhydride and dimethylaminopyridine; acetic anhydride and pyridine; acetic anhydride, pyridine and dimethylaminopyridine; acetic anhydride and sodium acetate; acetic anhydride and p-toluenesulfonic acid; acetyl chloride, pyridine and dimethylaminopyridine; and ketene. At the end of the acetylation reaction, acetyl cyclosporin A is advantageously recovered from the reaction mixture by filtration, for example through a Buchner filter, and advantageously purified by washing with a suitable solvent.

[0027] Advantageously, the acetylcyclosporin A of formula (II) was obtained in crystalline form. Thus, the present invention provides step a) followed by step a1), which consists of crystallizing acetylcyclosporin A from a solvent system selected from the group consisting of MTBE / n-heptane and MTBE / cyclohexane.

[0028] Acetylcyclosporin A can be reproducibly obtained in crystalline form using an MTBE / n-heptane solvent system and has an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 8.3, 9.1, 11.1, 11.3, 13.0, 15.0, 17.0, 17.6, 17.8, 19.1, and 19.6. Thus, in a further aspect, the present invention relates to a crystalline form of acetyl cyclosporin A of formula (II) having an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 8.3, 9.1, 11.1, 11.3, 13.0, 15.0, 17.0, 17.6, 17.8, 19.1 and 19.6. The powder X-ray diffraction spectrum (XRPD) of the crystalline form of acetyl cyclosporin A obtained using an MTBE / n-heptane solvent system is shown in Figure 1.

[0029] Crystalline acetylcyclosporine A of formula (II) was also obtained in a reproducible crystalline form using the MTBE / cyclohexane solvent system, and exhibited a powder X-ray diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.1, 5.4, 5.5, 9.0, 9.5, 10.2, 15.5, 17.4, 20.2, and 20.9. The powder X-ray diffraction spectrum (XRPD) of the crystalline acetylcyclosporine A obtained using the MTBE / cyclohexane solvent system is shown in Figure 3.

[0030] The process according to the invention comprises step b) of oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), the base being in an amount such that a pH value ranging from 6 to 8 is achieved, and in the presence of a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites, thus obtaining acetylcyclosporin A aldehyde of formula (III). [ka]

[0031] As is evident from the experimental part, the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine) in step b) in an amount such that a pH value ranging from 6 to 8, preferably from 6.5 to 7.5, is achieved, advantageously suppressing the impurity carboxylic acids of the following formula: [ka] The base is preferably dimethylpyridine (or lutidine) in an amount such that the pH value is in the range of 6 to 8, preferably 6.5 to 7.5.

[0032] The reaction of step b) of the process according to the invention is preferably carried out at a temperature in the range of from 0 to 40°C, preferably from 15 to 35°C. The reaction is also preferably carried out in the presence of at least one suitable solvent or solvent mixture, which is preferably selected from the group consisting of water and acetonitrile (CH3CN). Preferably, in said solvent mixture, the volume ratio of H2O:CH3CN ranges from 1:1 to 1:5. In a more preferred embodiment, the solvent mixture is a H2O:CH3CN mixture, preferably in a volume ratio ranging from 1:1 to 1:3, more preferably about 1:1, 1:2 or 1:3, even more preferably about 1:3.

[0033] The at least one oxidizing agent in step b) is preferably osmate, used in an amount ranging from 0.01 to 0.1 equivalents per equivalent of acetylcyclosporin A, preferably from 0.02 to 0.08 equivalents per equivalent of acetylcyclosporin A, for example, about 0.03 equivalents per equivalent of acetylcyclosporin A. Preferably, the osmate is an alkali or alkaline earth metal osmate, more preferably selected from the group consisting of potassium osmate (K2OsO4) and sodium osmate (Na2OsO4). In a particularly preferred embodiment, the at least one oxidizing agent is potassium osmate (K2OsO4).

[0034] The reaction is carried out in the presence of at least one co-oxidant selected from the group consisting of alkali or alkaline earth metal periodates and alkali or alkaline earth metal hypochlorites, the alkali or alkaline earth metal preferably being selected from the group consisting of sodium and potassium. In a particularly preferred embodiment, the at least one co-oxidant is sodium periodate (NaIO4).

[0035] Preferably, the co-oxidant is used in an amount within the range of 1 to 5 equivalents per equivalent of acetylcyclosporin A, preferably 1.8 to 4.5 equivalents per equivalent of acetylcyclosporin A, for example, about 2.1 equivalents per equivalent of acetylcyclosporin A. In a particularly preferred embodiment, the co-oxidant is sodium periodate, preferably used in an amount of about 2.1 equivalents per equivalent of acetylcyclosporin A.

[0036] The oxidant and / or co-oxidant may be added to the reaction mixture in any manner known to those skilled in the art, such as in a single aliquot at the beginning of the reaction or in several equal or different aliquots added over a period of time. Advantageously, at the end of the reaction, the acetyl cyclosporin A aldehyde of formula (III) is separated from the reaction mixture and purified, advantageously by separating it from the reaction mixture, for example by filtration, and then redissolving it in a suitable solvent, for example Me-THF.

[0037] The process according to the invention therefore comprises a step c) of converting acetylcyclosporin A aldehyde of formula (III) into acetylcyclosporin A of formula (IV). [ka] Preferably, the reaction for converting acetylcyclosporin A aldehyde of formula (III) to acetylcyclosporin A of formula (IV) is advantageously carried out by the Peterson reaction (also known as Peterson olefination) or the Wittig reaction. Both the Peterson reaction and the Wittig reaction are well known to those skilled in the art and are commonly used in the field of organic synthesis. In a preferred embodiment, step c) is carried out by a Peterson reaction in the presence of at least one α-silyl-carbanion to provide the corresponding hydroxysilane. Preferably, the α-silyl-carbanion is selected from the group consisting of allyltrimethylsilane and compounds of formula (V). [ka]

[0038] Preferably, the α-silyl-carbanion is used in an amount ranging from 1 to 1.5 equivalents per equivalent of acetyl cyclosporin A aldehyde, more preferably from 1.1 to 1.4 equivalents per equivalent of acetyl cyclosporin A aldehyde, and even more preferably about 1.2 equivalents per equivalent of acetyl cyclosporin A aldehyde. Preferably, in step c), the Peterson reaction is carried out in the presence of an organolithium compound.

[0039] Preferably, the organolithium compound is an alkyllithium, preferably selected from the group consisting of n-butyllithium, s-butyllithium, tert-butyllithium, n-hexyllithium, and mixtures thereof. In a preferred embodiment of the invention, the organolithium compound is n-butyllithium.

[0040] The organolithium compound can be added to the mixture in step c) in any form known to those skilled in the art, such as a solution in a hydrocarbon solvent. In one embodiment, for example, the organolithium compound is added as a 15 wt % solution of the organolithium compound in hexane. The organolithium compound can also be added to the mixture of step c) in any manner known to one skilled in the art, either in a single addition aliquot or divided into several equal or different aliquots over a period of time.

[0041] Preferably, in step c) of the Peterson reaction, the hydroxysilane formed by addition of the α-silyl carbanion is hydrolyzed under acid hydrolysis conditions, preferably using an acid selected from the group consisting of sulfuric acid (HSO) and hydrochloric acid (HCl), at a pH preferably comprised between 0 and 3. Preferably, said step c) is carried out in the presence of a suitable solvent, more preferably a solvent selected from the group consisting of dichloromethane (DCM), water, THF and mixtures thereof.

[0042] In one embodiment, step c) is carried out in the presence of a solvent consisting of a DCM:water mixture, preferably in a volume ratio of 2:1. In a further embodiment, step c) is carried out in the presence of THF as a solvent or in the presence of a mixture of THF:water, preferably in a volume ratio of 1:1. Advantageously, at the end of the reaction, the acetylvoclosporin of formula (IV) is separated from the reaction mixture and purified, which is advantageously carried out by separating the product from the reaction mixture, for example by filtration, and then recombining it with a suitable solvent.

[0043] Advantageously, the process according to the invention comprises an optional step d) of crystallizing acetylvoclosporin of formula (IV) using a solvent system selected from the group consisting of THF / n-heptane and MTBE, thereby obtaining acetylvoclosporin in crystalline form. Thus, in a preferred advantageous embodiment, the use of a solvent system selected from the group consisting of THF / n-heptane and MTBE for the isolation of acetylvoclosporin advantageously made it possible to always obtain said intermediate product in a reproducible crystalline form.

[0044] Said crystalline form can be characterized by powder X-ray diffraction spectroscopy (XRPD) using CuKa radiation, with peaks at characteristic angle values ​​2θ (±0.2) of 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2 and 20.9. Therefore, in a further advantageous embodiment, the present invention relates to crystalline acetylvoclosporin having an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2 and 20.9.

[0045] Preferably, in embodiments using a THF / n-heptane solvent system, the volume ratio of THF to n-heptane ranges from 3:8 to 5:8, more preferably about 4:8. Preferably, in a further embodiment, for an MTBE solvent system, the amount used is in the range of 30-40 volumes, preferably about 35 volumes.

[0046] d) At the end of the process, acetylvoclosporin in crystalline form is obtained. The X-ray powder diffraction spectrum (XRPD) of the crystalline form of acetylvoclosporin obtained according to the present invention is illustrated in FIG. The process according to the invention comprises a step e) of hydrolyzing acetylvoclosporin of formula IV, thus obtaining voclosporin. The hydrolysis of acetylvoclosporin in step e) of the process according to the invention can be carried out according to any method known to those skilled in the art. The hydrolysis reaction is preferably carried out at a temperature in the range of 0 to 30°C, more preferably 10 to 20°C, and more advantageously using one or more solvents and one or more bases capable of hydrolyzing the acetate group of acetylvoclosporin.

[0047] The hydrolysis in step e) can advantageously be carried out in a solvent selected from the group consisting of methanol (MeOH), water, ethanol, isopropanol, normal-propanol and mixtures thereof, and is preferably carried out in methanol or with a methanol:water mixture in which the volume ratio of MeOH:water is in the range of 10:1 to 1:1, more preferably about 3:1. Preferably, at least one base is added in step e), which can be any base known to those skilled in the art for the hydrolysis of acetate groups. Preferably, the base is selected from the group consisting of alkali metal or alkaline earth metal carbonates. In one embodiment, the base is potassium carbonate (K2CO3).

[0048] Thus, the process according to the present invention comprises a step f) of isolating the voclosporin of formula (I) obtained in step e) to obtain the voclosporin of formula (I) in amorphous form, wherein the isolation is carried out by precipitation using a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol, and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane, and pentane. Preferably, when step f) involves the use of a solvent / antisolvent system, the volume ratio of solvent:antisolvent is in the range of 1:1 to 1:15, more preferably 1:5 to 1:15, and even more preferably 1:6 to 1:10.

[0049] In one embodiment, step f) comprises: (fi-1) dissolving voclosporin in MTBE; and (fi-2) precipitating voclosporin from the solution at room temperature by removing MTBE. In a further embodiment, step f) comprises: (f-ii-1) dissolving voclosporin in a solvent, preferably MTBE; (f-ii-2) adding an antisolvent, preferably n-heptane, at a solvent volume ratio, preferably MTBE to antisolvent volume ratio, in the range of 1:1 to 1:10, preferably about 1:6, until a suspension is formed; and (f-ii-3) isolating the voclosporin thus precipitated. Preferably, in said step (f-ii-2), said antisolvent is added in at least two equal or different aliquots at predetermined time intervals.

[0050] In a further embodiment, step f) comprises: (f-iii-1) dissolving voclosporin in a solvent, preferably MTBE; (f-iii-2) adding an antisolvent, preferably cyclohexane, at a solvent volume ratio, preferably MTBE to antisolvent volume ratio of 1:6 to 1:15, preferably about 1:10, until a suspension is formed; and (f-iii-3) separating the voclosporin thus precipitated.

[0051] In a further embodiment, step f) comprises steps (f-iv-1) of dissolving voclosporin in a solvent, preferably MTBE, THF, acetonitrile, acetone, or ethanol, to obtain a voclosporin solution, and (f-iv-2) of adding the voclosporin solution obtained in step (f-iv-1) dropwise to an antisolvent selected from the group consisting of normal heptane, cyclohexane, and pentane. Preferably, in this further embodiment, the ratio of solvent to antisolvent is in the range of 1:6 to 1:15, more preferably 1:10.

[0052] f) At the end of the process, voclosporin in amorphous form is obtained. The powder X-ray diffraction spectrum (XRPD) of the amorphous form of voclosporin obtained according to the present invention is illustrated in FIG. Due to the various improvements made in the various synthetic steps starting from cyclosporin A and leading to the production of voclosporin, the present invention also has several additional aspects. In a further aspect thereof, the present invention relates to a process for the preparation of acetyl cyclosporin A aldehyde of formula (III), [ka] A) Acetylation of cyclosporin A with acetic anhydride in a stoichiometric amount relative to cyclosporin A in the presence of methyl tert-butyl ether (MTBE), thus obtaining acetyl cyclosporin A of formula (II): [ka] B) oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), the base being in an amount such that a pH value ranging from 6 to 8 is achieved, and in the presence of a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites, thus obtaining acetylcyclosporin A aldehyde of formula III.

[0053] As advantageously shown above, the use of stoichiometric amounts of MTBE and acetic anhydride makes it possible to avoid the consumption of large amounts of acetic anhydride. According to the present invention, the use of stoichiometric amounts of acetic anhydride in the presence of MTBE not only reduces the environmental impact, but also improves the reaction and purity compared to the methods applied in the prior art. The present invention provides step A1) following step A), which comprises crystallizing acetylcyclosporin A from a solvent system selected from the group consisting of MTBE / n-heptane and MTBE / cyclohexane.

[0054] A1) At the end of step A1), acetylcyclosporin A is in a crystalline form, which can be reproducibly obtained using an MTBE / n-heptane solvent system and which exhibits an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 8.3, 9.1, 11.1, 11.3, 13.0, 15.0, 17.0, 17.6, 17.8, 19.1, and 19.6. Thus, in a further aspect, the present invention relates to a crystalline form of acetyl cyclosporin A of formula II having an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 8.3, 9.1, 11.1, 11.3, 13.0, 15.0, 17.0, 17.6, 17.8, 19.1, and 19.6. The powder X-ray diffraction spectrum (XRPD) of the crystalline form of acetyl cyclosporin A obtained using an MTBE / n-heptane solvent system is shown in Figure 1.

[0055] Crystalline acetylcyclosporine A of formula II has also been obtained reproducibly using the MTBE / cyclohexane solvent system and exhibits an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.1, 5.4, 5.5, 9.0, 9.5, 10.2, and 15.56. The X-ray powder diffraction spectrum (XRPD) of the crystalline acetylcyclosporine A obtained using the MTBE / cyclohexane solvent system is shown in Figure 2.

[0056] The process according to the present invention comprises step B) of oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), thereby obtaining acetylcyclosporin A aldehyde of formula (III). Other advantages and preferred conditions for carrying out steps A), A1) and B) of this further aspect of the invention have already been described with reference to steps a), a1) and b) of the first aspect of the invention and therefore will not be repeated here.

[0057] In fact, as is evident from the experimental part, in step B), which corresponds to step b) of the first embodiment, the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine) advantageously made it possible to suppress the impurity carboxylic acid of the formula: [ka] The advantages in yield and ease of handling of the production of the intermediate acetylcyclosporin A aldehyde resulting from the conditions of steps A) and B), preferably steps A), A1), and B), are a substantial advantage for the production of voclosporin, since they contribute substantially, even decisively, to obtaining the desired product in high yield under industrially scalable conditions. The applicant has therefore found that this subsidiary aspect of the invention, included in its first and main aspects, independently represents an important innovative and advantageous aspect in the context of a process for obtaining voclosporin.

[0058] In a further aspect thereof, the present invention also relates to a process for obtaining acetylvoclosporin of formula (IV) in crystalline form, and to acetylvoclosporin in crystalline form thus obtained. In particular, the present invention also relates to a process for obtaining acetylvoclosporin of formula (IV) in crystalline form, [ka] - a process for producing acetylvoclosporin, for example, but not limited to, according to steps a) to c) of the process according to the first aspect of the present invention, - crystallizing acetylvoclosporin in a solvent system selected from the group consisting of THF / n-heptane and MTBE to obtain acetylvoclosporin IV in crystalline form.

[0059] Furthermore, the present invention relates to a novel crystalline form of acetylvoclosporin thus obtained, i.e., a crystalline form of acetylvoclosporin which exhibits an X-ray powder diffraction spectrum (XRPD) with peaks at characteristic angle values ​​2θ (±0.2) of 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2 and 20.9.

[0060] The possibility of obtaining acetylvoclosporin in crystalline form also represents an advantage within the process according to the first and main aspects of the invention, but independently constitutes an important innovative and advantageous aspect of the process for obtaining voclosporin. Indeed, obtaining a key intermediate such as acetylvoclosporin in crystalline form allows for more efficient purification, benefiting the overall process.

[0061] The X-ray powder diffraction spectrum (XRPD) of the crystalline form of acetylvoclosporin obtained according to the present invention is illustrated in FIG. Other advantages and preferred conditions for carrying out the steps of this further aspect of the invention have already been described with reference to steps a) to c) and d) of the first aspect of the invention and will not be repeated here. In yet another aspect thereof, the present invention relates to a process for obtaining amorphous voclosporin of formula I: [ka] - preparing a solution of voclosporin in a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol, and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane, and pentane; - recovering said voclosporin in amorphous form by precipitation from the solution thus produced. Indeed, the precipitation of voclosporin according to the invention makes it possible to obtain an amorphous product which is characterized by a high stability over time and which is easy to handle since it is powdery and non-sticky. Other advantages and preferred conditions for carrying out this further aspect of the invention have already been described with reference to step f) of the first aspect of the invention and will not be repeated here. The invention will now be described by means of some examples which are to be understood for illustrative and non-limiting purposes. [Example]

[0062] method XRPD analysis: X-ray diffraction analysis of the samples was performed using an X-ray diffractometer (voltage 45 kV, current 40 mA, scan rate 0.025710° / s, CuKα light source, θ angle range 3.0° to 49.992°). HPLC analysis: To perform the analysis, the samples were dissolved in methanol and the following equipment and conditions were used: Detector: UV 210nm Column: Zorbax SB-C18 250mm x 4.6mm, 5μm Temperature: 65℃ Sampler temperature 15℃ Mobile phase A: 0.1% formic acid and a mixture of 55.2% acetonitrile / 42.3% water / 2.5% MTBE Mobile phase B: 0.1% formic acid in acetonitrile Injection volume: 10μL Flow: 1.5mL / min Run Time: 28 minutes Elution gradient:

[0063] [Table 1]

[0064] Example Example 1 - Step a) and step a1) of the present invention In a three-necked round-bottom flask, 20 g (16.63 mmol) of cyclosporin A was dissolved in 200 mL (10 V) of MTBE. The white suspension was left under stirring until a clear solution appeared. 10.386 g (99.78 mmol, 6 eq) of acetic anhydride was added dropwise to the solution. 4.63 g (33.26 mmol, 2 eq) of 4-dimethylaminopyridine was added portionwise to the solution, maintaining the temperature at 25 ± 5 °C. After 1 h, the product began to precipitate, resulting in a white suspension. This suspension was left under stirring for 48 h. To quench the reaction, 50 mL (2.5 V) of water was added dropwise. The temperature was maintained at 25 ± 5 °C using an ice bath. A pale two-phase solution formed. The organic phase was separated, and 50 mL (1 V) of water was added, followed by an aqueous solution of NaHCO3 (8% w / w) until the pH reached 6 to 6.5. The resulting biphasic solution was separated, and the organic phase was washed with brine. The organic phase was distilled under vacuum until a 7-volume residue was obtained. 100 mL of n-heptane (5V) was added dropwise to the shaken solution at 25±5°C. Precipitation occurred. The white suspension was left under stirring at 25±5°C for 16 hours. The suspension was cooled to -15°C for 2 hours and filtered. The solid was washed twice with 10 mL of a 1:1 MTBE / n-heptane solution. The solid was dried at 45°C overnight. Acetyl cyclosporine solid was recovered as a dry powder. 18.21 g (88% yield).

[0065] Example 2 - Steps a) and a1) of the present invention In a three-necked round-bottom flask, 20 g (16.63 mmol) of cyclosporin A was dissolved in 200 mL (10 V) of MTBE. The white suspension was left stirring until a clear solution appeared. 10.386 g (99.78 mmol, 6 eq) of acetic anhydride was added dropwise to the solution. The MTBE solution was heated to 45 ± 5 °C. 4.63 g (33.26 mmol, 2 eq) of 4-dimethylaminopyridine was added portionwise to the solution while maintaining the temperature at 45 ± 5 °C. The solution was left stirring for 6 h. The reaction was cooled to 25 ± 5 °C and quenched by the dropwise addition of 50 mL (2.5 V) of water. The temperature was maintained at 25 ± 5 °C using an ice bath. A pale two-phase solution formed. The organic phase was separated, and 50 mL (1 V) of water was added, followed by an aqueous solution of NaHCO3 (8% w / w) until the pH reached 6–6.5. The resulting biphasic solution was separated, and the organic phase was washed with brine. The organic phase was distilled under vacuum until a 7-volume residue was obtained. 100 ml of n-heptane (5V) was added dropwise to the shaken solution at 25±5°C. Precipitation occurred. The white suspension was left under stirring at 25±5°C for 16 hours. The suspension was cooled to -15°C for 2 hours and filtered. The solid was washed twice with 10 mL of a 1:1 MTBE / n-heptane solution. The solid was dried at 45°C overnight. Acetyl cyclosporine solid was recovered as a dry powder. 17.93 g (87% yield).

[0066] The solids thus obtained in Examples 1 and 2 were analyzed to obtain the powder X-ray diffraction spectrum (XRPD) of the crystalline form of acetylcyclosporin A shown in Figure 1. The characteristic peaks of the crystalline form at angles 2θ (±0.2) were 8.3, 9.1, 11.1, 11.3, 13.0, 15.0, 17.0, 17.6, 17.8, 19.1 and 19.6.

[0067] Example 3 - Steps a) and a1) of the present invention 2.0 kg of cyclosporin A (1.66 mol, 1.0 equiv.) was dissolved in 20 L of MTBE. 1.02 kg of acetic anhydride (9.96 mol, 6.0 equiv.) and 415.4 g of DMAP (3.32 mol, 2.0 equiv.) were added under stirring. The final mixture was heated to 45°C and maintained under stirring for 16 hours. The reaction mixture was cooled to 20°C and quenched with 5.0 kg of water. The two phases were separated, and the organic phase was washed twice with 1 M hydrochloric acid (4.0 L x 2) and once with an aqueous mixture of 8% NaHCO3:15% brine (2.0 L + 3.0 L). The final organic layer was concentrated under reduced pressure to 8.0 L and heated to 55°C. 10.0 L of cyclohexane was added, and the suspension was cooled to 0°C. The suspension was filtered and the solid was washed once with MTBE:cyclohexane (0.8 L + 1.0 L). The solid was dried under vacuum at 50°C for 16 hours to give a white solid (1.86 kg, yield = 90.0%, typical A% HPLC purity ≥ 98%).

[0068] Figure 2 shows the chromatogram obtained from the reaction of step (a) (Step (A)), demonstrating the high purity of crystalline acetylcyclosporin A obtained by using a stoichiometric amount of acetic anhydride in the presence of MTBE. The results are shown in the table below.

[0069] [Table 2]

[0070] The solid thus obtained in Example 3 was analyzed to obtain the X-ray powder diffraction spectrum (XRPD) of the crystalline form of acetylcyclosporin A shown in Figure 3. The characteristic peaks of said crystalline form at angles 2θ (±0.2) were 5.1, 5.4, 5.5, 9.0, 9.5, 10.2, 15.5, 17.4, 20.2 and 20.9.

[0071] Example 4-b) Process or B) Process 11.2 g (9.00 mmol, 1.0 eq) of acetylcyclosporin A from Example 1 was dissolved in 168 ml CH3CN (15 V) to obtain a clear solution. The solution was concentrated to 8 volumes under vacuum, and 15 V of CH3CN was added. 56 mL of water (5 V) was added. To the resulting solution, 1.9 g of 2,6-lutidine (18 mmol, 2 eq) was added, and the solution was adjusted to pH 7. The suspension was stirred at 25 ± 5 °C for 30 min. 0.050 g (0.136 mmol, 0.015 eq) of potassium osmate was added in one portion. After dissolution of the osmate, the solution acquired a brownish color. After 15 min, 3.850 g of NaIO4 (18 mmol, 2 eq.) was added in five portions (0.770 g, 3.6 mmol, 0.4 eq. each), spaced 1 h apart. During the addition, a brown suspension formed, and the salts formed were dissolved by adding 50 mL of water. Next, 62 mL of water was added, reaching 15 V. The suspension was left stirring for 16 h. 44.8 mL of IPAC (4 V) was added, followed by 10 mL of water, and the resulting biphasic solution was left stirring for 30 min. The organic phase was separated, and the aqueous phase was extracted twice with 56 mL of IPAC (5 V). The oxidant in each organic and aqueous phase was monitored with a Quantofix Peroxide 100® analyzer, which indicated no oxidant was present. The organic phases were combined and concentrated to 5 V. The concentrated organic phase was washed with 56 mL (5 V) of brine. The organic phase was concentrated and solvent exchanged with THF (5 volumes of final solution). 200 mL of n-heptane (4 V based on total residue) was added dropwise over 1.5 h. The resulting mixture was stirred at room temperature for 16 h. Filtration yielded a white solid, which was dried overnight at 40 °C. 10.4 g of acetylcyclosporin A aldehyde was obtained with a yield of 94%. This solid was subjected to HPLC analysis, the graph of which is shown in FIG. 4, and the results are shown in the table below.

[0072] [Table 3]

[0073] As can be seen from the table, the impurities were very low, with only 1.35% being a carboxylic acid impurity (+16 impurity). To assess the critical importance of the presence of a base in step b) (or step B), step b) (or step B)) was repeated without the presence of a base.

[0074] 5.580 g (4.48 mmol, 1.0 eq) of acetylcyclosporin A was dissolved in 55 mL (30 V) of CH3CN to obtain a clear solution. 55 mL of water (10 V) was added. The suspension was stirred at 25 ± 5 °C for 30 min. 0.050 g (0.136 mmol, 0.03 eq) of potassium osmate was added in one portion. The resulting suspension acquired a brownish color after the osmate dissolved. After 15 min, 2.012 g of NaIO4 (9.408 mmol, 2.1 eq.) was added in three portions (0.670 g; 3.136 mmol, 0.7 eq. each) at 30 min intervals. A brown suspension formed during the addition. The suspension was left under stirring at 25 ± 5 °C for 16 h. 10 ml of a saturated solution of 7.083 g (44.8 mmol, 10.0 eq) of NaSO was added dropwise to the reaction mixture. After 30 minutes of stirring, the oxidant was monitored with Quantofix Peroxide 100®. 5 ml of water (1 V) was added to the reaction mixture until the salt dissolved and a homogeneous, clear solution appeared. The solution was extracted three times with 100 ml of Me-THF (20 V). The organic phases were collected and dried under vacuum. 5.33 g of a white solid was obtained, with a yield of 96%. The resulting product was subjected to HPLC analysis as described above, yielding the results shown in the graph of Figure 5 and the table below.

[0075] [Table 4]

[0076] As can be seen from the table above, the spectrum showed the presence of a large amount of the impurity carboxylic acid (12.56%) of the formula: [ka] Therefore, when carrying out step b) (or step B)), it is highly advantageous to have a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine) present in an amount such that the pH value is in the range of 6 to 8, since this allows for a drastic reduction of undesired carboxylic acid impurities.

[0077] Example 5 - Preparation of α-silyl-carbanions of formula (V) [ka] 2.0 g (2.78 ml, 17.53 mmol) of allyltrimethylsilane was placed in a three-neck round-bottom flask under a nitrogen atmosphere. 6 ml of dry THF (3 vol.) was added, and the formation of a clear solution was observed. Next, 11.5 ml of a 1.6 M n-butyllithium solution in hexane (18.40 mmol, 1.05 eq) was added dropwise over 30 minutes at 20 °C until the solution turned yellow-orange. An ice bath was used to monitor the exotherm of the addition. The solution was left stirring for 1 hour. The solution was then cooled to -78 °C in a dry ice bath in acetone. 3.460 g (4.24 ml, 18.40 mmol, 1.05 eq) of isopropyl borate was added dropwise. The reaction mixture was left stirring for 1 hour until the color changed from yellow to white. Next, the reaction mixture was poured into 1 M aqueous hydrochloric acid (10 ml) until the pH reached 4-5. The aqueous phase was discarded, and the organic phase was collected. A solution of diethanolamine (1.750 g, 16.65 mmol, 0.95 eq) in isopropyl alcohol (iPrOH, 4 ml) was prepared separately and added dropwise to the organic phase containing the boronic acid at 20 °C. The resulting solution was left under stirring for at least 1 hour. 30 ml of n-heptane (15 vol.) was then added, resulting in a two-phase emulsion. The emulsion was concentrated until a white solid began to precipitate. 30 ml of n-heptane was added and the mixture was concentrated to the original volume to remove traces of THF. The suspension was filtered to obtain a white solid. This solid contained traces of diethanolamine, so it was suspended in THF (4.4 vol, 8.8 ml) and filtered to obtain a white solid.

[0078] Example 6 - Preparation of α-silyl-carbanions of formula (V) [ka] 2.0 g (2.78 ml, 17.53 mmol) of allyltrimethylsilane was placed in a three-neck round-bottom flask under a nitrogen atmosphere. 6 ml of dry THF (3 vol.) was added, and the formation of a clear solution was observed. Next, 11.5 ml of a 1.6 M n-butyllithium solution in hexane (18.40 mmol, 1.05 eq) was added dropwise over 30 min at 20 °C until the solution turned yellow-orange. An ice bath was used to monitor the exotherm of the addition. The solution was left stirring for 1 h. The solution was then cooled to -30 °C in a dry ice bath in acetone. 3.460 g (4.24 ml, 18.40 mmol, 1.05 eq) of isopropyl borate was added dropwise. The reaction mixture was left stirring for 1 h until the color changed from yellow to white. Next, the reaction mixture was poured into 1 M aqueous hydrochloric acid (10 ml) until the pH reached 4-5. The aqueous phase was discarded, and the organic phase was collected. A solution of diethanolamine (1.750 g, 16.65 mmol, 0.95 eq) in isopropyl alcohol (4 ml) was prepared separately and added dropwise to the organic phase containing the boronic acid at 20°C. The solution was left under stirring for 1 hour. 30 ml of n-heptane (15 vol.) was added until a two-phase emulsion appeared. The emulsion was concentrated until a white precipitate appeared. 30 ml of n-heptane was added and the mixture was concentrated to the original volume to remove traces of THF. The suspension was filtered to give a white solid. This solid still contained traces of diethanolamine, so it was suspended in THF (4.4 vol.) (8.8 ml) and filtered to give a white solid.

[0079] Example 7-c) Step 7.0 g (5.679 mmol) of acetylcyclosporin A aldehyde obtained according to Example 4 (2,6-lutidine in the presence of base) and 1.740 g (7.659 mmol, 1.2 eq, 90% purity) of the compound of formula (V) obtained according to Example 5 or 6 were dissolved in 63 ml of a 2:1 v / v (9 vol.) mixture of DCM and HO to obtain a heterogeneous biphasic mixture. 1.022 mg (0.973 ml, 17.037 mmol, 3 eq.) of concentrated acetic acid was added dropwise to the reaction mixture over 30 minutes. The pH was monitored at this stage and was approximately 4.5. The reaction mixture was left under stirring overnight. The organic phase was then separated, and the aqueous phase was extracted three times with 20 ml of dichloromethane (3 vol.). The organic extracts were pooled, and the solvent was replaced with THF (40 ml, 6 vol.). Briefly, the crude extract was evaporated to dryness and subjected to vacuum dissolution-concentration cycles with THF alone to remove residual dichloromethane. The reaction mixture was cooled to 0°C, and 1.113 g (0.562 ml, 11.358 mmol, 2 eq) of concentrated sulfuric acid was added dropwise. The temperature was raised to 20°C. The pH was monitored and maintained at approximately 1.5. The reaction mixture was left stirring overnight at 20°C. An additional 1.113 g (0.562 ml, 2 eq) of concentrated sulfuric acid was added dropwise at 0°C. After the reaction was complete, 20 mL (3 vol) of water was added dropwise to the reaction mixture. 2 M NaOH solution was then added dropwise to the reaction mixture until the pH reached approximately 4.5. This solution was extracted three times with 35 mL aliquots of MTBE (5 vol.). The combined organic phases were concentrated under reduced pressure. The resulting white solid was dissolved in 100 mL of MTBE (14 vol.) until crystallization began. The suspension was left stirring overnight at 20°C. Acetylvoclosporin (white solid, 5.35 g, 75% yield) was then obtained by filtration.

[0080] Example 8-c) Step 7.0 g (5.679 mmol) of acetyl cyclosporin A aldehyde obtained according to Example 4 (2,6-lutidine in the presence of base) and 1.740 g (7.659 mmol, 1.2 eq., 90% purity) of the compound of formula V obtained according to Example 5 or 6 were dissolved in 63 ml (9 vol.) of DCM:HO 2:1 v / v, resulting in a heterogeneous biphasic mixture. 1.022 mg / 0.973 ml (17.037 mmol, 3 eq.) of concentrated acetic acid was added dropwise to the reaction mixture over 30 minutes. The pH was checked and found to be approximately 4.5. The reaction mixture was left under stirring overnight. The organic phase was separated, and the aqueous phase was extracted three times with 20 ml aliquots of dichloromethane (3 volumes). The organic phases were pooled, and the solvent was changed to THF (40 ml, 6 volumes). Briefly, the crude extract was evaporated to dryness and subjected to vacuum dissolution-concentration cycles with THF alone to remove residual dichloromethane. The reaction mixture was cooled to 0°C, and 1.119 g / 0.940 ml (11.358 mmol, 2 eq) of concentrated hydrochloric acid (37% HCl solution) was added dropwise. The temperature was raised to 20°C. The pH was checked and found to be approximately 1.5. The reaction mixture was left stirring overnight at room temperature. An additional 1.119 g / 0.940 ml (2 eq) of concentrated HCl (37% HCl solution) was added dropwise at 0°C. After the reaction was complete, 20 ml (3 vol) of water was added dropwise to the reaction mixture. Next, 2 M NaOH solution was added dropwise to the reaction mixture until the pH reached approximately 4.5. This solution was extracted three times with 35 ml aliquots of MTBE (5 vol.). The combined organic phases were concentrated under reduced pressure. The resulting white solid was suspended in 100 ml (14 vol) of MTBE and allowed to crystallize. The suspension was left under stirring at room temperature overnight and then filtered to give a white solid (acetylvoclosporin, 5.35 g, 75% yield).

[0081] Example 9-c) Step 0.1 g (0.081 mmol) of acetylcyclosporin A aldehyde obtained according to Example 4 (2,6-lutidine in the presence of base) and 0.025 g (0.097 mmol, 1.2 eq, 90% purity) of the compound of formula V obtained according to Example 5 or 6 were dissolved in THF (0.6 ml, 6 vol.). 0.015 mg / 0.014 ml (0.243 mmol, 3 eq) of concentrated acetic acid was added dropwise to the reaction mixture over 30 minutes. The pH was checked and found to be approximately 4.5. 0.6 ml of HO (6 vol.) was then added to the reaction mixture, resulting in a two-phase mixture (THF:HO 1:1 v / v). The reaction mixture was left under stirring overnight. 0.015 mg / 0.014 ml (0.243 mmol, 3 eq) of acetic acid was added dropwise. The organic phase was separated and the aqueous phase was extracted three times with 3 ml (30 vol.) aliquots of DCM. The organic phases were combined and the solvent was changed to THF (0.6 ml, 6 vol.). The reaction mixture was cooled to 0°C, and 0.016 g / 0.009 ml (0.162 mmol, 2 eq) of concentrated sulfuric acid was added dropwise. The temperature was raised to 20°C. The pH was checked and found to be approximately 1.5. The reaction mixture was left stirring overnight at 20°C. An additional 0.016 g / 0.009 ml (0.162 mmol, 2 eq) of 98% H2SO4 was added dropwise at 0°C. After the reaction was complete, 1 mL (10 vol.) of water was added dropwise to the reaction mixture. 2 M NaOH solution was also added dropwise to the reaction mixture until the pH reached approximately 4.5. This solution was extracted three times with 5 ml aliquots of MTBE (50 vol.). The combined organic phases were concentrated under reduced pressure to give a white solid (acetylvoclosporin, 88.16 mg, 88.3% yield).

[0082] Example 10-c) Step 1.0 g (8.752 mmol) of allyltrimethylsilane was placed in a three-necked round-bottom flask under a nitrogen atmosphere. 7.4 ml (7.4 vol.) of THF was added, and the formation of a clear solution was observed. 1.6 ml of n-butyllithium (8.8 mmol, 1.05 eq) in 5.5 ml of hexane was added dropwise over 30 minutes until the solution turned from orange to yellow. The solution was then cooled to -78 °C using a dry ice bath in acetone. 1.646 g (2.02 ml, 8.752 mmol, 1 eq) of isopropyl borate was added. The reaction mixture was left stirring for 2 hours until the color changed from yellow to white. The reaction mixture was then poured into 1 M aqueous hydrochloric acid (8 ml) until the pH reached approximately 4. The aqueous phase was then extracted three times with 10 ml of DCM. The DCM solution was concentrated until the boronic acid solution was 10 wt%. In a separate flask, 0.719 g (0.583 mmol, 1 eq) of acetylcyclosporin A aldehyde obtained according to Example 4 was dissolved in 4 ml of DCM (5 vol.) and then added to the previously prepared trimethylsilane allylboronic acid solution. The reaction mixture was left overnight at 20 °C. 10 ml of THF was then added, and the solution was brought to 0 °C. At this point, 228 mg (0.125 ml, 2 eq.) of 98% H2SO4 was added dropwise, and the temperature was adjusted to approximately 20 °C. An additional 228 mg (0.125 ml, 2 eq.) of 98% H2SO4 was added dropwise at 0 °C. Upon completion of the reaction, 2 M NaOH solution was added dropwise to the reaction mixture until the pH reached approximately 4.5. This solution was extracted three times with 35 ml aliquots of MTBE (5 vol.). The combined organic phase was concentrated under reduced pressure. The product was obtained as a white solid (acetylvoclosporin, 685 mg, 93.5% yield).

[0083] Example 11-d) Step, Purification in THF / n-heptane 127.0 g (0.101 mol, 1.0 eq) of acetylvoclosporin was dissolved in 500 mL (4 V) of THF. 1 L (8 V) of n-heptane was added to the clear solution over 1 hour and 30 minutes. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0-5 °C for 2 hours and filtered to give 110 g of the desired product. The resulting solid was dried under vacuum at 40 °C for 16 hours to give 100 g in 80% yield. The resulting solid was analyzed to obtain the powder X-ray diffraction spectrum (XRPD) shown in Figure 6. The characteristic peaks of the crystalline form at angles 2θ (±0.2) were 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2, and 20.9.

[0084] Example 12-d) Step, Purification of Acetylvoclosporin in MTBE Acetylvoclosporin (0.5 g) was suspended in 17.5 mL (35 V) of MTBE. The resulting mixture was heated until completely dissolved and then cooled to room temperature. The resulting solid was collected by filtration and dried under vacuum at 40°C for 48 hours. The resulting solid was analyzed to obtain the powder X-ray diffraction spectrum (XRPD) shown in Figure 6. The characteristic peaks of the crystalline form at angles 2θ (±0.2) were 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2, and 20.9. Crystalline forms of acetylvoclosporin were reproducibly obtained in solvent systems selected from the group consisting of THF / n-heptane and MTBE.

[0085] Example 13-e) Step 2 g (1.590 mmol) of acetyl voclosporin obtained according to Example 11 was dissolved in 40 ml (20 vol.) of methanol. 1.099 g (7.95 mmol, 5 eq) of potassium carbonate was added at 20°C, resulting in the formation of a white suspension. After stirring at 20°C for 5 hours, the reaction mixture was diluted with 15 ml (7.5 vol.) of water to obtain a clear solution (added dropwise over 30 minutes while maintaining the temperature below 25°C). Most of the methanol was evaporated under reduced pressure until an aqueous suspension was formed, and then 30 ml (15 vol.) of MTBE was added with stirring. The aqueous layer was separated and extracted with 2 x 30 ml (15 vol.) of MTBE. The combined organic phases were washed with brine and dried under reduced pressure at 45°C. A white solid was obtained (voclosporin, 1.91 g, 98.9% yield).

[0086] Example 14-e) Step 0.100 g (0.08 mmol) of acetyl voclosporin obtained according to Example 11 was dissolved in 10.6 ml (106 vol.) of methanol. 0.264 g (1.911 mmol, 24 eq.) of potassium carbonate was added at 20°C, resulting in the formation of a white suspension. After stirring overnight at room temperature, a clear solution was formed. This solution was diluted with 1 ml (10 vol.) of water (HO). Most of the methanol was evaporated under reduced pressure until an aqueous suspension was formed, after which 1.5 ml (15 vol.) of MTBE was added with stirring. The aqueous layer was separated and extracted with 2 x 1.5 ml (15 vol.) of MTBE. The combined organic phases were washed with brine and dried under reduced pressure at 45°C. A white solid was observed to be formed (voclosporin, 92.6 mg, 95.3% yield).

[0087] Example 15-e) Step 500 mg (0.398 mmol) of acetyl voclosporin obtained according to Example 11 was dissolved in 53 ml (106 vol.) of methanol. 1.321 g (9.55 mmol) (24 eq.) of potassium carbonate was added to the reaction mixture at 20°C, forming a white suspension. This suspension was left overnight at 20°C. Water was then added until the salt was completely dissolved. The solution was concentrated on a rotary evaporator until solid precipitation was observed. The suspension was extracted three times with MTBE (20 ml, 40 vol.). The combined organic phases were concentrated under reduced pressure to obtain solid voclosporin.

[0088] Example 16-e) Step 0.250 g (0.199 mmol) of acetyl voclosporin obtained according to Example 11 was dissolved in 5 ml of a 3:1 v / v (20 vol.) mixture of MeOH and water. 0.138 g (0.995 mmol, 5 eq.) of potassium carbonate was added at 20°C. After stirring at room temperature for 30 hours, the mixture was concentrated under reduced pressure, evaporating most of the methanol until an aqueous suspension was formed. The residue was redissolved in 3.75 ml (15 vol.) of MTBE. The aqueous layer was separated and extracted with 2 x 3.75 ml (15 vol.) of MTBE. The combined organic phases were washed with brine and dried under reduced pressure at 45°C. A white solid was obtained (voclosporin, 194.34 mg, 80.7% yield).

[0089] Example 17-f) Step 250 mg (206 mmol) of solid voclosporin obtained according to Example 11 was dissolved in 1 ml of MTBE (4 vol.). 1.0 ml of n-heptane (1.2 ml) was added to form a white precipitate. The suspension was left under stirring for 10 minutes until the solution became clear. 1.4 ml of n-heptane was then added to form a cloudy suspension, and an additional 3.3 ml of n-heptane was added, and the suspension was left under stirring. The suspension was filtered through a Buchner funnel to obtain a solid (90.9 mg). This solid was subjected to powder X-ray diffraction spectroscopy (XRPD) according to the method reported in the "Methods" section above, resulting in the spectrum shown in Figure 7, which revealed that an amorphous form of voclosporin was obtained.

[0090] Example 18-f) Step 250 mg (0.206 mmol) of solid voclosporin obtained according to Example 11 was dissolved in 1 ml (4 vol.) of MTBE. After adding 10 ml (40 vol.) of cyclohexane, a white precipitate formed. The solid was filtered through a Buchner funnel to obtain 25 mg of solid precipitate (10% yield). This solid was subjected to powder X-ray diffraction spectroscopy (XRPD) according to the method reported in the "Methods" section above, which gave a spectrum completely similar to that shown in Figure 7, indicating that amorphous voclosporin was obtained.

[0091] Example 19 - Stability testing of amorphous voclosporin obtained according to the method of the present invention Five 500 mg samples of amorphous voclosporin obtained according to Example 17 were prepared. The first reference sample was subjected to X-ray powder diffraction spectroscopy (XRPD) according to the method reported in the Methods section above, which gave a spectrum completely similar to that shown in Figure 7, confirming that an amorphous form of voclosporin was obtained. The remaining four samples were tested for stability over time under different conditions of temperature, humidity, lighting, and time, as detailed below. Test 1: A voclosporin sample was placed in a sealed vial and placed in a sealed oven at a constant temperature of 70°C for 92 hours. Test 2: Voclosporin samples were placed in sealed vials and placed in a sealed oven at a constant temperature of 70°C for 14 days. Test 3: Voclosporin samples were placed in clear vials and left in direct sunlight for 7 days. Test 4: Voclosporin samples were placed in open vials and placed in an isolated environment with saturated aqueous NaCl. The relative humidity reached 70% and the samples were left in these conditions for 7 days. After the study was completed, each sample was subjected to X-ray powder diffraction (XRPD) according to the method reported in the Methods section above, which again yielded spectra completely similar to those shown in Figure 7, highlighting the maintenance of the amorphous form of voclosporin. Figure 8 shows a comparison of the amorphous forms obtained from the analysis at the end of the stability study.

Claims

1. 1. A process for preparing voclosporin of formula (I) in amorphous form, comprising: 【Chemistry 1】 a) acetylating cyclosporin A with acetic anhydride in a stoichiometric amount relative to cyclosporin A in the presence of methyl tert-butyl ether (MTBE), thus obtaining acetyl cyclosporin A of formula (II); 【Chemistry 2】 b) oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), the base being in an amount such that a pH value ranging from 6 to 8 is achieved, and in the presence of a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites, thus obtaining acetylcyclosporin A aldehyde of formula (III), 【Transformation 3】 c) converting acetylcyclosporin A aldehyde of formula (III) to acetylcyclosporin A of formula (IV); 【Chemistry 4】 d) crystallizing acetylvoclosporin of formula (IV) using a solvent system selected from the group consisting of THF / n-heptane and MTBE, thereby obtaining acetylvoclosporin in crystalline form; e) hydrolyzing acetylvoclosporin of formula (IV) to obtain voclosporin of formula (I); and f) isolating the voclosporin of formula (I) obtained in step e) to obtain amorphous voclosporin of formula (I), wherein the isolation is carried out by precipitation using a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol, and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane, and pentane.

2. 2. The process according to claim 1, further comprising, after step a), a step a1) of crystallizing acetylcyclosporin A from a solvent system selected from the group consisting of MTBE / n-heptane and MTBE / cyclohexane.

3. 3. The method according to claim 1, wherein the base in step b) is dimethylpyridine (or lutidine).

4. The osmate is an osmate of an alkali metal or alkaline earth metal, preferably potassium osmate (K ２ OsO ４ 4. The method according to claim 1, wherein the

5. The step b) is carried out by adding sodium periodate (NaIO) as a co-oxidant. ４ The process according to any one of claims 1 to 4, wherein the process is carried out in the presence of

6. The process according to any one of claims 1 to 5, wherein in step d), the solvent system is THF / n-heptane, preferably in a volume ratio ranging from 3:8 to 5:8, more preferably about 4:

8.

7. 6. The process according to claim 1, wherein in step d), the solvent system is MTBE, preferably used in an amount ranging from 30 to 40 volumes, more preferably about 35 volumes.

8. A process for preparing acetyl cyclosporin A aldehyde of formula (III), comprising: 【Transformation 5】 A) acetylating cyclosporin A with acetic anhydride in a stoichiometric amount relative to cyclosporin A in the presence of methyl tert-butyl ether (MTBE), thus obtaining acetyl cyclosporin A of formula (II); and 【Transformation 6】 B) oxidizing acetylcyclosporin A of formula (II) with at least one oxidizing agent which is an osmate in the presence of a base selected from the group consisting of dimethylpyridine (or lutidine) and 2,4,6-trimethylpyridine (or sym-collidine), wherein the base is in an amount such that a pH value in the range of 6 to 8 is achieved, and in the presence of a co-oxidizing agent selected from the group consisting of alkali metal or alkaline earth metal periodates and alkali metal or alkaline earth metal hypochlorites, thereby obtaining acetylcyclosporin A aldehyde of formula (III).

9. 9. The process according to claim 8, further comprising a step A1) following step A) of crystallizing acetylcyclosporin A from a solvent system selected from the group consisting of MTBE / n-heptane and MTBE / cyclohexane.

10. A process for obtaining acetylvoclosporin of formula (IV) in crystalline form, comprising: 【Transformation 7】 - preparing acetylvoclosporin of formula (IV), - crystallizing said acetylvoclosporin using a solvent system selected from the group consisting of THF / n-heptane and MTBE, thus obtaining acetylvoclosporin (IV).

11. A crystalline form of acetylvoclosporin of formula (IV): 【Transformation 8】 The crystalline form of acetylvoclosporin is characterized in that the crystalline form exhibits an X-ray powder diffraction spectrum (XRPD) having peaks at characteristic angle values ​​2θ (±0.2) of 5.0, 5.5, 7.0, 8.5, 8.8, 10.2, 12.4, 16.5, 17.2 and 20.

9.

12. A process for obtaining amorphous voclosporin of formula (I), comprising the steps of: 【Chemistry 9】 - preparing a solution of voclosporin of formula I in a solvent / antisolvent system, wherein the solvent is selected from the group consisting of MTBE, THF, 2-methyl THF, diisopropyl ether, acetonitrile, acetone, ethanol and cyclopentyl methyl ether, and the antisolvent is a hydrocarbon selected from the group consisting of normal heptane, cyclohexane and pentane; - recovering the amorphous form of voclosporin by precipitation from the solution thus prepared.

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