Process for the preparation of voclosporin

Abstract

Disclosed is a process for the preparation of a mixture of (E)-voclosporin and (Z)-voclosporin with a low Z isomer content. The process is characterised in that it includes a step of crystallisation of the precursor acetyl voclosporin (5a, 5b) from organic solvent or organic solvent and water in specific ratios.

Description

[0001] PROCESS FOR THE PREPARATION OF VOCLOSPORIN

[0002] The invention relates to a process for the preparation of a mixture of (E)-voclosporin and (Z)-voclosporin, a calcineurin-inhibitor immunosuppressant active ingredient.

[0003] Background of the invention

[0004] Voclosporin, in particular the mixture of the (E) and (Z) isomers thereof (Formulas la and lb respectively), is a calcineurin-inhibitor immunosuppressant active ingredient obtained by derivatisation of cyclosporin (Formula 2); as a result of said derivatisation, the calcineurin inhibition capacity and metabolic stability of voclosporin exceed those of cyclosporin.

[0005] Approved in Europe and the USA under the trademark Lupkynis™, voclosporin is used in combination with mycophenolate mofetil in the treatment of glomerulonephritis caused by systemic lupus erythematosus, called lupus nephritis; voclosporin is also used as a candidate in clinical trials focusing on disorders such as focal segmental glomerulosclerosis, psoriasis, dry eye syndrome, non-infectious uveitis and keratoconjunctivitis sicca, or on kidney transplant patients.

[0006] Although the (E) isomer is the active one, it must be administered in combination with a minimal amount of the Z isomer to reduce its toxicity; in the commercially available products, the E / Z ratio can range from 90: 10 to 95:5.

[0007] (E)-Voclosporin la (Z)-Voclosporin lb Cyclosporin 2

[0008] Processes for the preparation of mixtures of voclosporin with an (E) / (Z) ratio ranging between 1 :9 and 9:1 are disclosed in WO 2003 / 033526 (Isotechnika Inc. and Hoffman-La Roche Ltd.); said mixtures are prepared by stereoselective and non-stereoselective synthesis processes. The drawback of said methodologies is that in order to obtain a given (E) / (Z) ratio a stereoselective procedure must be used, with no possibility of modulating the ratio of the isomers in mixtures obtained by non-stereoselective processes, such as Wittig olefination.

[0009] For the sake of clarity, in the present invention the term “stereoselective process” means a process that produces a single isomer; in this specific case, a stereoselective process would produce the (E) isomer or the (Z) isomer.

[0010] A process for the preparation of a mixture of voclosporin with an (E) / (Z) ratio ranging between 9: 1 and 95:5 is disclosed in WO 2023 / 118045 (Curia Spain S.A.U); in said application, the ratio between isomers la and lb is modulated by conducting two sequential olefinations, in particular a first olefination (the Wittig reaction) on aldehyde 3, followed by a second (Petersen- like) olefination on the residual aldehyde 3, unreacted in the first olefination.

[0011] Aldehyde 3

[0012] By adopting specific conditions of elimination of the P-hydroxy-silane formed as intermediate of the second olefination, compound la or compound lb can be selectively obtained, thus modulating the ratio between the two isomers in the final mixture. Said procedure is disadvantageous because although it enables the (E) / (Z) ratio to be modulated, unlike the classic methodologies for preparing voclosporin (such as those described in WO 2003 / 033526, WO 2003 / 033527 and WO 2004 / 89960), it involves not one but two olefination steps, making the synthesis more complex, especially when conducted on an industrial scale.

[0013] The voclosporin preparation processes disclosed in WO 2003 / 033526, WO 2003 / 033527, WO 2004 / 89960 and WO 2023 / 118045 involve at least one olefination step using the Wittig reaction, Peterson olefination, or phospho- or titano-allyls.

[0014] The aldehyde of Formula 3 represents a key intermediate in said processes, and is obtained by ozonolysis of the compound of Formula 4.

[0015]

[0016] Compound 4

[0017] In view of the usefulness of and interest in voclosporin, there is a need to develop alternative methods of obtaining voclosporin wherein the ratio between the (E) / (Z) isomers and the impurity content are in line with those required for the commercial product.

[0018] Description of the invention

[0019] The Applicant has developed a process for the production of a mixture of (E) / (Z) voclosporin with a low Z isomer content, characterised in that it includes a step of crystallisation of the precursor acetyl voclosporin (5a, 5b) from organic solvent or organic solvent and water at the volume ratio of at least 2.2: 1; preferably, the reduction in Z isomer content compared with the starting acetyl voclosporin (5a, 5b) is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%; more preferably, the reduction in Z isomer content ranges between 1% and 10%; even more preferably, the Z isomer content is reduced until a mixture of (E) / (Z) acetyl voclosporin with an (E) / (Z) ratio ranging between 9: 1 and 95:5 is obtained.

[0020] (E)-Acetyl voclosporin 5a (Z)- Acetyl voclosporin 5b

[0021] WO 2003 / 033526 and WO 2023 / 118045 describe methods of preparation of (E) / (Z) mixtures of acetyl voclosporin by stereoselective olefination of aldehyde 3 (with R= acetyl); in said methodologies, the use of organometallic reagents comprising elements such as boron, titanium, sulphur, phosphorus and lithium or the use of reagents with a particular steric bulk, elements which affect the geometry of the state of transition of the reaction, allows access to (E) / (Z) mixtures with a given ratio.

[0022] The process according to the present invention, which involves a step of crystallisation of an (E) / (Z) mixture of acetyl voclosporin, is particularly advantageous, because whatever the preparation method of said mixture, it enables the ratio between the isomers to be modulated; said crystallisation therefore enables the desired isomeric ratio to be obtained without the use of stereoselective olefination methods.

[0023] The (E) / (Z) mixture of acetyl voclosporin obtained by crystallisation is then converted to an (E) / (Z) mixture of voclosporin by deacetylation and isolation.

[0024] Crystallisation of (E) / (Z) acetyl voclosporin

[0025] In a first embodiment, the process according to the present invention comprises

[0026] (i) dissolving or suspending a mixture of (E) / (Z) acetyl voclosporin in an organic solvent to give a solution Soil or a suspension Sosl,

[0027] (ii) heating solution Soil or suspension Sosl to give a solution Sol2 or a suspension Sos2,

[0028] (iii) cooling solution Sol2 or suspension Sos2, and

[0029] (iv) isolating the (E) / (Z) mixture of acetyl voclosporin formed.

[0030] Typically, steps (ii) and (iii) and, for clarity, the final step (iv), can be repeated two or more times, preferably twice; in other words, after isolation of the (E) / (Z) mixture of acetyl voclosporin performed in step (iv), the solution Soil or suspension Sosl can again be subjected to steps (ii) and (iii’) to isolate more (E) / (Z) acetyl voclosporin with the desired ratio between the isomers.

[0031] In a preferred alternative embodiment, the process according to the present invention involves a crystallisation from organic solvent and water at a volume ratio of at least 2.2: 1. In the present invention, references to the use of organic solvent and water mean either that the organic solvent and water can come into contact with acetyl voclosporin at the same time, i.e. be mixed and then added, or that the organic solvent and water can come into contact with acetyl voclosporin at two sequential times, first the organic solvent and then the water or vice versa; preferably, the (E) / (Z) mixture of acetyl voclosporin comes into contact with the organic solvent and then with the water.

[0032] In said alternative embodiment, the process according to the present invention comprises (i) dissolving or suspending a mixture of (E) / (Z) acetyl voclosporin in an organic solvent to give a solution Soil or a suspension Sosl,

[0033] (ii) heating solution Soil or suspension Sosl to give a solution Sol2 or a suspension Sos2,

[0034] (ii’) adding water to solution Sol2 or suspension Sos2 to give a solution Sol3 or a suspension Sos3, with an organic solvent: water ratio of at least 2.2: 1,

[0035] (iii’) cooling solution Sol3 or suspension Sos3, and

[0036] (iv) isolating the (E) / (Z) mixture of acetyl voclosporin formed.

[0037] Typically, steps (ii), (ii’) and (iii’) and, for clarity, final step (iv), can be repeated two or more times; preferably, twice; in other words, after isolation of the (E) / (Z) mixture of acetyl voclosporin performed in step (iv), the solution Soil or suspension Sosl can again be subjected to steps (ii), (ii’) and (iii’) to isolate more (E) / (Z) acetyl voclosporin with the desired ratio between the isomers.

[0038] Typically, the organic solvent is an organic solvent other than tert-butanol; it is preferably selected from methanol, ethanol, propanol, isopropanol, butanol, acetonitrile, acetone, dichloromethane, dimethylformamide, dimethyl sulphoxide, tetrahydrofuran, methyltetrahydrofuran, methyl tert-butyl ether, ethyl acetate and isopropyl acetate; more preferably, it is selected from methanol, isopropanol, acetonitrile, methyltetrahydrofuran and methyl tert-butyl ether. In the first embodiment, the preferred organic solvent is acetonitrile. In the alternative embodiment, methanol and isopropanol are preferred, methanol being more preferred.

[0039] Typically, in the second embodiment, the ratio between organic solvent and water is at least 2.2: 1; preferably between 4.5: 1 and 2.2: 1. The ratio between the organic solvent and water is an essential parameter to ensure that the recrystallisation actually modulates the ratio between the (E) / (Z) isomers; in fact, WO 2003 / 033526 describes a procedure for preparation of an (E) / (Z) mixture of acetyl voclosporin by reacting aldehyde 3 with allyltributyl -phosphonium bromide wherein, at the end of the reaction, the reaction mixture is concentrated, dissolved in methanol and precipitated by adding one volume of water relative to the volume of methanol (methanol: water ratio 1 : 1). The resulting (E) / (Z) mixture of acetyl voclosporin has an (E) / (Z) ratio of 91 :9; said ratio derives from the use of allyltributyl -phosphonium bromide which, with its particular steric bulk, mainly leads to the formation of the (E) isomer. The crystallisation step with a 1 : 1 methanol: water mixture does not alter the ratio between the isomers, as confirmed by the tests illustrated in Table 1. Table 1

[0040] Crystallisation solvent % yield E / Z ratio (%)

[0041] 14 (before crystallisation) Acetonitrile 50 8

[0042] Methyl tert-butyl ether 80 12

[0043] Tert-butanol 50 14

[0044] Methyltetrahydrofuran 68 13

[0045] Isopropanol: water (3:2) 50 9

[0046] Methanol: water 1.1 90 14

[0047] Methanol : water 2: 1 87 14

[0048] Methanol : water 2.2: 1 89 13

[0049] Methanol: water 2.7: 1 82 12

[0050] Methanol : water 3.1 : 1 82 11

[0051] Methanol: water 3.6: 1 79 10

[0052] Methanol : water 3.1 : 1 (double crystallisation) 71 8

[0053] Typically, the weight / volume ratio (g / mL) between the (E) / (Z) mixture of acetyl voclosporin and organic solvent ranges between 1 : 1 and 1 : 10; preferably between 1 :3 and 1 :5; and more preferably, 1 :4.

[0054] Typically, the weight / volume ratio (g / mL) between the (E) / (Z) mixture of acetyl voclosporin and water ranges between 1 :0.2 and 1 :5; preferably between 1 :0.5 and 1 :3; more preferably, between 1 : 1 and 1 :2.

[0055] Typically, solution Soil or suspension Sosl is heated to a temperature ranging between 40 and 90°C; preferably, 50°C.

[0056] Typically, solutions Sol2 and Sol3 or suspensions Sos2 and Sos3 are cooled to temperatures ranging between 30 and -10°C; preferably between 20 and -5°C.

[0057] Typically, the mixture of (E) / (Z) acetyl voclosporin is isolated by filtration, centrifugation or decanting of the solid and removal of the supernatant.

[0058] A preferred aspect of the second embodiment of the invention involves the selection of methanol as solvent, a methanol -to-water ratio of at least 2.1 : 1, more preferably at least 3.1 : 1, and repetition of steps (ii), (ii’), (iii ’) and (iv) (double crystallisation).

[0059] Preparation method of (E) / (Z) voclosporin by crystallisation and deacetylation of (E) / (Z) acetyl voclosporin

[0060] A second aspect of the present invention relates to a voclosporin preparation method comprising the following steps:

[0061] A. crystallisation of the (E) / (Z) mixture according to the first aspect of the invention;

[0062] B. deacetylation of the mixture used in step A to give an (E) / (Z) mixture of voclosporin; Step B

[0063] The (E) / (Z) mixture of acetyl voclosporin, after being crystallised according to the first aspect of the invention, is converted to the (E) / (Z) mixture of voclosporin by removing protecting group R. Said group can be removed by using the removal conditions known to the skilled person. In the case of R= acetyl, the removal is conducted by basic hydrolysis.

[0064] Typically, the (E) / (Z) mixture of acetyl voclosporin is dissolved in a solvent selected from methanol, ethanol, isopropanol, tetrahydrofuran and 2-methyl tetrahydrofuran, preferably methanol, obtaining a solution Sol4 to which an inorganic base selected from K2CO3, CS2CO3, ISfeCCh, NaHCCh, KHCO3, KOH, LiOH and NaOH is added; preferably K2CO3, dissolved in water. Typically, the ratio between the equivalents of the (E) isomer and the base ranges between 1 :0.8 and 1 :2, preferably, 1 :2.

[0065] The mixture is first left to react for 8-24 hours, preferably 12-16 hours, at room temperature, and then quenched by adding an acid aqueous solution, preferably IM HC1.

[0066] The mixture is concentrated to a volume of a fifth, a quarter, a third or half of the starting volume, preferably half.

[0067] Typically, a 2:1 mixture of methyl tert-butyl ether: water, wherein the volume / volume ratio between the concentrated solution and MTBE ranges between 3: 1 and 1 :3; preferably 1- 1, is added to the concentrated solution. After phase separation, the organic phases are washed two or more times with water. The washed organic phases are concentrated by removing the solvent under vacuum until a residue is obtained, namely the (E) / (Z) mixture of crude voclosporin.

[0068] A preparation method of an (E) / (Z) mixture of voclosporin is described in WO 2003 / 033256; in said prior art document, removal of the protecting group is followed by a chromatographic purification, wherein a 3:2 mixture of acetone: hexane is used as eluent. The Applicant has established that when the method of WO 2003 / 033256 is applied to the (E) / (Z) mixture of crude voclosporin obtained from the deacetylation reaction, it is impossible to obtain fractions wherein the amount of the single impurities is less than 0.10%, as will be seen from Table 2. Table 2

[0069] The impurity content is expressed as Area% and determined by applying the HPLC method reported in the experimental section.

[0070] The Applicant has found that by using methyl tert-butyl ether, methanol or mixtures thereof, preferably methyl tert-butyl ether, as eluent, the efficiency of the chromatography can be considerably improved, improving the separation of the impurities and increasing the purity of the end product.

[0071] As will be seen from Table 3, by using the Applicant’s chromatography method with methyl tert-butyl ether as eluent, fractions can be obtained wherein the content of the single impurities is lower than or equal to 0.10% (fractions 6-11). Table 3

[0072] Typically, the fractions wherein the content of the single impurities is lower than or equal to 0.10% are combined at the end of the chromatographic purification. The combined fractions can be directly dropped into heptane; alternatively, the solvent can be replaced with methanol or acetone, and then dropped into water. Said step allows the isolation of the (E) / (Z) mixture of voclosporin in amorphous form.

[0073] (E) / (Z) voclosporin preparation method

[0074] A third aspect of the present invention relates to a process for preparation of (E) / (Z) voclosporin wherein the process according to the second aspect of the invention is preceded by preparation of (E) / (Z) acetyl voclosporin by olefination of the aldehyde of Formula 3. The aldehyde of Formula 3, with R=acetyl, is converted to an (E) / (Z) mixture of acetyl voclosporin using one of the olefination methods described in the prior art; preferably, the olefination is conducted under Wittig conditions, by reacting the aldehyde with an allyl phosphonium salt in the presence of a base.

[0075] Typically, the ratio between the aldehyde equivalents of Formula 3 and the allyl bromide equivalents ranges between 1 : 1 and 1 :3; preferably 1-1.6.

[0076] Typically, the allyl phosphonium salt is prepared by reacting an allyl halide, selected from the group consisting of allyl chloride, allyl bromide and allyl iodide, with a phosphine selected from the group consisting of triphenylphosphine, trimethylphosphine, triethylphosphine and tributylphosphine; preferably allyl bromide and tributylphosphine. The ratio between the allyl halide and phosphine equivalents ranges between 1 : 1 and 2: 1, preferably 1 : 1.

[0077] Typically, the solvent used to prepare the allyl phosphonium salt is selected from diethyl ether, diisopropyl ether, methyl tert-butyl ether, dichloromethane, tetrahydrofuran, 2-methyl- tetrahydrofuran and toluene; preferably methyl tert-butyl ether.

[0078] Typically, the preparation reaction of the allyl phosphonium salt is conducted for 2-30 hours at a temperature ranging between 10 and 50°C; preferably for 16 hours at 20-25°C.

[0079] Typically, the base used in the olefination is selected from lithium diisopropylamide, potassium tert-butoxide and hexamethyldisilazane; preferably potassium tert-butoxide.

[0080] Typically, the olefination reaction is conducted at a temperature ranging between -78°C and -20°C for a time ranging between 10 and 180 min.; preferably at -45°C for 1 hour. By conducting the reaction in the temperature range indicated, an (E) / (Z) mixture wherein the amount of E isomer is greater than that of the Z isomer can be obtained.

[0081] Typically, the reaction is quenched by adding an inorganic acid selected from hydrochloric, phosphoric and sulphuric acid; preferably phosphoric acid.

[0082] After quenching, the reaction mixture is brought to room temperature and diluted with methyl tert-butyl ether (MTBE) and water, with a volume / volume ratio between MTBE and water ranging between 2:0.5 and 0.5:2; preferably, the mixture is diluted with MTBE and water at the ratio of 1 : 1.

[0083] After phase separation, the organic phase is concentrated to residue by vacuum distillation and diluted with methanol, heated to a temperature ranging between 40 and 80°C, and then diluted with water; the volume / volume ratio between organic solvent and water ranges between 2: 1 and 1 : 1; preferably, the residue is diluted with methanol, heated to 50°C and further diluted with water, with an organic solvent : water ratio of 2:1. After the addition of water and cooling to room temperature, an (E) / (Z) mixture of acetyl voclosporin precipitates as a solid. Said mixture is isolated by filtration, dried under vacuum and crystallised as in the first aspect of the invention.

[0084] Experimental section

[0085] The HPLC analyses were conducted with an HPLC apparatus consisting of a quaternary pump, a thermostated autosampler, a column compartment and a UV / VIS detector, using a Zorbax RRHD SB-C18 column (1=100 mm, i.d. = 2.1 mm, particle size = 1.8 pm), and solvents A (water + 0.01% phosphoric acid) and B (acetonitrile + 0.01% phosphoric acid) as mobile phase. The elution gradient used is as follows:

[0086] Table 4

[0087] The 'H-NMR analyses were conducted with a Bruker 400 MHz NMR instrument; spectra recorded in CDCL at the temperature of 30°C.

[0088] Example 1 - Synthesis of acetyl voclosporin

[0089] 18 g of tributylphosphine (89.1 mmol, 2.2 equiv.) was diluted with 250 mL of methyl tert-butyl ether at 20±5°C and maintained under stirring for 30 min, until a clear solution was obtained; 11 g of allyl bromide (89.1 mmols, 2.2 equiv.) was dropped into said solution, and the mixture was left under stirring for 16 h, at the end of which the phosphonium salt formed was filtered and washed with methyl tert-butyl ether (23.2 g, yield= 80%).

[0090] 21 g of wet phosphonium salt (6.48 mmols, 1.6 equiv.) and 50 g of aldehyde (40.5 mmols) were dissolved in 350 mL of tetrahydrofuran; the resulting solution was cooled to -45±5°C. 6.36 g of potassium tert-butoxide (56.0 mmols, 1.4 equiv.) in 100 mL of tetrahydrofuran was added drop by drop to the solution containing the phosphonium salt and the aldehyde, maintaining the temperature constant at -45±5°C. The reaction mixture was first maintained under stirring for 1 h, then quenched by adding 6 g of phosphoric acid (61.2 mmol), heated to 25±5°C, and finally diluted with 100 mL of MTBE and 100 mL of water. After phase separation, the organic phase was washed with a further 100 mL of water and concentrated under vacuum until a residue was obtained; said residue was diluted with 150 mL of methanol until a solution / suspension was obtained, then heated to 50±5°C and diluted with 100 mL of water. The suspension was cooled to 20±5°C, and the solid formed, acetyl voclosporin, was then filtered and dried at 40°C under vacuum. (45.7 g, yield=90%,

[0091] Example 2 - Crystallisation of acetyl voclosporin in acetonitrile

[0092] 10 g of acetyl voclosporin NMR Z=14%) was suspended in 40 mL of acetonitrile. The suspension was first heated to 50°C, then cooled to 20±5°C. The suspended solid was filtered and dried under vacuum at 40°C. (yield 50%,

[0093] Example 3 - Crystallisation of acetyl voclosporin in methanol water

[0094] 10 g of acetyl voclosporin NMR Z=14%) was dissolved in 40 mL of methanol, and the resulting solution was heated to 50°C. 11 mL of water was added to the solution, which was then cooled to 0±5°C, with formation of a solid. The solid was filtered and dried under vacuum at 40°C. (yield 79%,1H NMR Z=10%)

[0095] Example 4 - Double crystallisation of acetyl voclosporin in methanol rwater

[0096] 20 g of acetyl voclosporin NMR Z=14%) was dissolved in 80 mL of methanol, and the resulting solution was heated to 50°C. 26 mL of water was then added to the solution, which was then cooled to 0±5°C. The solid formed was filtered and again dissolved in 80 mL of methanol. The solution was heated to 50°C, 33 mL of water was then added, and finally, the solution was cooled to 0±5°C. The solid was dried under vacuum at 40°C. (yield=76%, NMR Z=8%)

[0097] Example 5 - Crystallisation of acetyl voclosporin in isopropanohwater

[0098] 10 g of acetyl voclosporin NMR Z=14%) was dissolved in 30 mL of isopropanol, and the solution was heated to 50°C. 20 mL of water was added to the solution. The solution was cooled to 20±5°C. The solid was filtered and dried under vacuum at 40°C. (yield 50%, NMR Z=9%)

[0099] Example 6 - Synthesis of voclosporin

[0100] 25 g of acetyl voclosporin (19.90 mmol, 1.0 equiv.) was dissolved in 250 mL of methanol at 25±5°C, to give a methanol solution. 5.55 g of K2CO3 (35.8 mmol, 2.0 equiv.) was dissolved in 13 mL of water, to give an aqueous solution. Said solution was dropped into the methanol solution at 25±5°C and left under stirring for 16h. The reaction mixture was quenched with 50 mL of IM HC1 and concentrated to 125 mL. The concentrated mixture was diluted with 125 mL of methyl tert-butyl ether and 63 mL of water. After phase separation, the organic phase was washed twice with 125 mL of water. Finally, the final organic phase was purified by silica gel chromatography, using methyl tert-butyl ether as eluent. The column fractions containing impurities in amounts lower than or equal to 0.10% were combined, and subjected to solvent substitution from methyl tert-butyl ether to methanol three times. The final methanol solution was dropped into 300 mL of water and left under stirring for Ih. The solid was filtered and dried at 30°C under nitrogen flow. An amorphous white solid was obtained with a yield of 60% and HPLC purity of 99.70 % (each impurity was under 0.10%). Optionally, the precipitation can be conducted by:

[0101] • combining the fractions at the end of the column, replacing the solvent with acetone and dropping into 300 mL of water

[0102] • combining the fractions at the end of the column and dropping into 300 mL of heptane.

[0103] All said precipitations produce an amorphous voclosporin.

Claims

CLAIMS1. A process for obtaining a mixture of (E) / (Z) voclosporin (la and lb) with a ratio between the (E) isomer (la) and the (Z) isomer (lb) ranging between 9: 1 and 95:5,(E)-voclosporin la (Z)-voclosporin lb characterised in that it involves a crystallisation step of the precursor acetyl voclosporin (5a, 5b) from organic solvent or from organic solvent and water in a volume ratio of at least 2.2: 1(E)-acetyl voclosporin 5a (Z)-acetyl voclosporin 5b2. The process according to claim 1, comprising (i) dissolving or suspending a mixture of (E) / (Z) acetyl voclosporin in an organic solvent to give a solution Soil or a suspension Sosl, (ii) heating solution Soil or suspension Sosl to give a solution Sol2 or a suspension Sos2, (ii’) adding water to solution Sol2 or suspension Sos2 to give a solution Sol3 or a suspension Sos3, with an organic solvent : water ratio of at least 2.2: 1; (iii’) cooling solution Sol3 or the suspension Sos3, and (iv) isolating the (E) / (Z) mixture of acetyl voclosporin formed.

3. The process according to claim 2 wherein steps (ii), (ii’) and (iii’) are repeated two or more times.

4. The process according to claims 1 to 3 wherein the ratio of organic solvent to water ranges between 4.5: 1 and 2.2: 1.

5. The process according to claims 1 to 4 wherein the weight / volume ratio (g / mL) between mixture (E) / (Z) of acetyl voclosporin and water ranges between 1 :0.2 and 1 :5.

6. The process according to any one of the preceding claims wherein the organic solvent is an organic solvent other than tert-butanol.

7. The process according to any one of the preceding claims wherein the organic solvent is selected from methanol, ethanol, propanol, isopropanol, butanol, acetonitrile, acetone, dichloromethane, dimethylformamide, dimethyl sulphoxide, tetrahydrofuran, methyltetrahydrofuran, methyl tert-butyl ether, ethyl acetate and isopropyl acetate.

8. The process according to any one of the preceding claims wherein the weight / volume (g / mL) ratio between the (E) / (Z) mixture of acetyl voclosporin and organic solvent ranges between 1 : 1 and 1 :

10.

9. The process according to any one of the preceding claims wherein the organic solvent is methanol or isopropanol.

10. The process according to claim 9 wherein the solvent is methanol.

11. The process according to claim 10 wherein the methanol : water ratio is at least 2.1 : 1, and steps (ii), (ii’), (iii’) and (iv) are repeated twice.

12. The process according to any one of the preceding claims wherein solution Soil or suspension Sosl is heated to a temperature ranging between 40 and 90°C.

13. The process according to any one of the preceding claims wherein solutions Sol2 and Sol3 or suspensions Sos2 and Sos3 are cooled to temperatures ranging between 30 and -10°C.

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