Process for preparing voclosporin

Abstract

The present invention provides an industrially applicable process for preparing voclosporin (compound of formula (I)). This process provides voclosporin (compound of formula (I)) with the desired mixture of E isomer (from 90% to 95%) and Z isomer (from 5% to 10%).

Description

[0001] PROCESS FOR PREPARING VOCLOSPORIN

[0002] This application claims the benefit of European Patent Application EP23383258 filed 5 December 2023.

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to an industrially applicable process for preparing voclosporin.

[0005] BACKGROUND OF THE INVENTION

[0006] Voclosporin (compound of formula I) is a mixture of cis and trans isomers of cyclosporin diene analogue, which is chemically described as cyclo{{(2S,3R,4R)-3-hydroxy-4- methyl-2-(methylamino)-6,8-nonadienoyl}-L-2-aminobutyryl-N-methyl-glycyl-N-methyl- L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L- leucyl-N-methyl-L-valyl}. It has empirical formula of C63H111N11O12 and its molecular weight is 1214.65 g / mol.

[0007] I

[0008] Voclosporin (compound of formula I), which is a synthetic derivative of Cyclosporin A where an additional carbon connected by a double bond is introduced, is an immunosuppressant drug being developed by Aurinia Pharmaceuticals and it is approved in the USA and in Europe for the treatment of lupus nephritis (LN).

[0009] The commercial pharmaceutical product Lupkynis® which contains voclosporin (compound of formula I), is defined as an enriched trans / (E) compound with the cis / (Z) isomer being present in small percentage. Commercial voclosporin (compound of formula I) is a mixture of the E and Z isomers being the E isomer prevalent (90-95%).

[0010] EP0991660B1 describes the preparation of deuterated and undeuterated cyclosporin analogues, which can be used as immunomodulating agents. The synthetic route starting from cyclosporin A (compound of formula VII) involves the following steps which involved the use of Wittig reagents (see scheme 1).

[0011] Scheme 1

[0012] CsA represents hereinafter the cyclosporine A ring as shown above. The present nomenclature provides a shorthand means of displaying the region where the synthetic reactions described take place, without having to re-draw the remainder of the molecule each time a reaction is described. No specific example is given in EP0991660B1 for the preparation of voclosporin (compound of formula I, i.e., when R is -CH=CH2).

[0013] WO03033526A2, WO03033527A2, W02004089960A2 and WO2024052941A1 disclose several processes for preparing isomeric mixtures of cyclosporin derivatives, such as voclosporin (compound of formula I). In these documents the compound of formula IV, in particular the compound of formula IV, wherein Pg is acetyl, is a key intermediate which can react with Wittig reagents, phosphonium ylides, boranes, boronate esters, organo-titanium reagents or organo-aluminum as sources to provide the additional -CH=CH2 fragment.

[0014] Wittig reactions have as main drawback the formation of phosphine oxide side-products which are very difficult to remove, making this process in general not suitable at industrial scale. Also, Wittig reactions involve the use of a strong base (such as alkali metal alkoxides or alkali hydrides) which, at the case at hand, in view of the amido groups present in the cyclosporin derivatives can generate additional by-products by hydrolysis and / or a-epimerization at any of the chiral aminoacids. On the top of it, most of the Wittig reactions disclosed in WO03033526A2, WO03033527A2 and W02004089960A2 gives voclosporin (compound of formula I) with an undesired ratio of E / Z isomers.

[0015] More convenient and industrially applicable process disclosed in WO03033526A2, WO03033527A2, W02004089960A2, CN117886892A, Org. Process Res. Dev. 2024, 28, 4, 1151-1158, and WO2024157186A1 is the one depicted in scheme 2:

[0016] Si Meg Peterson eli Peterson elimination and deprote

[0017] Pg is acetyl or trimethylsily

[0018] Scheme 2

[0019] In the process of scheme 2, the compound of formula I reacts with a (E)-y- (trimethylsilylallyl) boronate ester or a (E)-y-(trimethylsilylallyl) dialkylborane to form the mixture of the anti-isomers of the p-trimethylsilyl alcohol. Peterson elimination of the mixture of the anti-isomers of the p-trimethylsilyl alcohol and simultaneous or subsequent deprotection reaction gives voclosporin (compound of formula I) showing variable ratios of the E isomer and the Z isomer depending on the conditions used, but most of them not being the desirable ratios.

[0020] WO03033526A2, WO03033527A2, W02004089960A2, CN117886892A, Org. Process Res. Dev. 2024, 28, 4, 1151-1158, and WO2024157186A1 teach that when Peterson elimination of the mixture of the anti-isomers of the p-trimethylsilyl alcohol is performed under acid conditions, substantially pure E isomers of the compounds of formula II’ and I are obtained. On the other hand, when Peterson elimination of the mixture of the antiisomers of the p-trimethylsilyl alcohol is performed under basic conditions, compounds of formula II’ and I enriched in the Z isomer are obtained, although some substantial amounts of the E isomer are also obtained. The bases disclosed in WO03033526A2 and WO03033527A2 to perform the Peterson elimination are strong bases such as potassium tert-butoxide or potassium hydride. As previously mentioned, the use of strong bases is not advisable in the present case in view of the presence of the amido groups in the cyclosporin derivatives which can be hydrolysable and / or epimerizable under strong basic conditions. All the examples provided by W02004089960A2, CN117886892A, Org. Process Res. Dev. 2024, 28, 4, 1151-1158 and WO2024157186A1 disclose the preparation of the substantially pure E isomer of the compounds of formula II’ and I.

[0021] Nearly none of the examples of WO03033526A2 and WO03033527A2 related to the process of scheme 2, provide voclosporin (compound of formula I) with the desired mixtures of isomers E and Z. Only Example 34 of WO03033526A2 and WO03033527A2 discloses a process wherein the mixture of the anti-isomers of the p-trimethylsilyl alcohol, wherein Pg is trimethylsilyl, is contacted with the Lewis acid boron trifluoride diethylether in dichloromethane to directly obtain voclosporin (compound of formula I) containing 91% of the E isomer and 9% of the Z isomer as measured by NMR, which this would be, although in the limit, within the desired isomers ratio. However, this process has the disadvantage of the use of dichloromethane which is not recommended at industrial scale. Dichloromethane has a low boiling point, and it is classified as human carcinogen. Consequently, dichloromethane has a very low emission limit, so that it brings a lot of inconvenience when used at large scale, i.e., at industrial scale. Dichloromethane is a substance that should be handled under controlled conditions. In conclusion, dichloromethane is a solvent which must be replaced by other solvents when scaling up processes. Also, boron trifluoride is a gas compound that is corrosive and fatal if inhaled, causing severe damage to kidneys, lungs and teeth through prolonged or repeated exposure, and therefore it is not convenient to use boron trifluoride derivatives such as boron trifluoride diethylether at industrial scale.

[0022] W02006014872A2 discloses a four-step synthesis of voclosporin (compound of formula I) wherein the diene moiety is introduced through silver-catalyzed reaction of the corresponding aldehyde (compound of formula IV) with an organo-zirconium reagent and deprotection. However, ten equivalents of the organometallic reagent are needed and only a moderate 47% product yield is achieved. Voclosporin (compound of formula I) is obtained as the E isomer, so not the desirable mixture of isomers is obtained either.

[0023] In order to obtain voclosporin (compound of formula I) with the desired mixture of isomers E and Z, WO2023118045A1 teaches a process which comprises a first step wherein the compound of formula IV reacts with less than one equivalent of the Wittig reagent allyl triphenylphosphonium bromide to obtain a mixture comprising the compound of formula II, being mostly the E isomer but also containing some amounts of the Z isomer, and the unreacted compound of formula IV. In a second step, and without any isolation, the boron complex, tetrahydro-2-[(2E)-3-(trimethylsilyl)-2-propen-1-yl]-4H-1 ,3,6,2-dioxazaborocine is added over the mixture obtained in the first step, so that the unreacted compound of formula IV forms the corresponding p-trimethylsilyl alcohol. An acid, e.g, acetic acid, sulfuric acid, methanesulfonic acid and / or formic acid, is added either after the preparation of the silylated intermediate or simultaneously with the formation of the silylated intermediate, obtaining the E isomer of the compound of formula II. In this way, combining Wittig reaction (using less than one equivalent of the Wittig reagent) and trans-boron compound addition followed by a Peterson elimination, the desired mixtures of isomers E and Z are achieved. However, several drawbacks of this process can be pointed out: the process needs a Wittig reaction which is a process to be avoided at industrial scale as previously mentioned and involves the use of a strong base, concretely an alkali metal alkoxide, which in the present case can generate additional by-products. Yields and chemical purities are not provided in WO2023118045A1.

[0024] Therefore, prior art does not provide a suitable and industrially scalable process to provide with the desired E and Z isomer ratios of voclosporin (compound of formula I). Prior art does not provide any specific conditions to carry out the process of scheme 2, i.e., the process which involves Peterson elimination of the anti-isomers of the - trialkylsilyl alcohol intermediate, in order to obtain substantially pure Z isomer of the compounds of formula II’ and I, either. Therefore, there is still the need of an effective, robust, and also industrially applicable process for preparing voclosporin (compound of formula I) having the specific ratio E (trans) and Z (cis) isomers of the commercial product: 90%-95% of the E (trans) isomer and 5%-10% of the Z (cis) isomer.

[0025] BRIEF SUMMARY OF THE INVENTION

[0026] The present invention relates to an improved process for the preparation of voclosporin (compound of formula I) having the desired mixture of E and Z isomers which is suitable at industrial scale.

[0027] BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 X-Ray Powder Diffractogram (XRPD) of the substantially pure Z isomer of the compound of formula II as obtained in Example 3.

[0029] Figure 2 X-Ray Powder Diffractogram (XRPD) of the substantially pure E isomer of the compound of formula II as obtained in Example 4.

[0030] Figure 3 X-Ray Powder Diffractogram (XRPD) of voclosporin (compound of formula I) as obtained in Example 5.

[0031] Figure 4 X-ray Powder Diffraction (XRPD) of voclosporin (compound of formula I) as obtained in Example 6.

[0032] Figure 5 X-Ray Powder Diffraction (XRPD) of compound of formula VI as obtained in Example 1 .

[0033] Figure 6 X-Ray Powder Diffraction (XRPD) of compound of formula IV as obtained in Example 2.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] A first aspect of the present invention provides a process for preparing voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer, which comprises: a) Preparing substantially pure Z isomer of the compound of formula II’ substantially pure Z isomer of II' wherein Pg is a hydroxyl protecting group, and optionally deprotecting substantially pure Z isomer of the compound of formula II’ to obtain substantially pure Z isomer of voclosporin (compound of formula I); substantially pure Z isomer of I b) Preparing substantially pure E isomer of the compound of formula II’ substantially pure E isomer of II’ wherein Pg is a hydroxyl protecting group, and optionally deprotecting substantially pure E isomer of the compound of formula II’ to obtain substantially pure E isomer of voclosporin (compound of formula I); substantially pure E isomer of I and, either c-1) Mixing the substantially pure Z isomer of the compound of formula II’ of step a) with the substantially pure E isomer of the compound of formula II’ of step b), in order to obtain compound of formula II’ having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer; and c-2) Deprotecting the compound of formula II’ obtained in step c-1) to obtain voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer; or d) In case that the steps a) and b) above include the deprotection step, mixing the substantially pure Z isomer of voclosporin (compound of formula I) of step a) with the substantially pure E isomer of voclosporin (compound of formula I) of step b), in order to obtain voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer.

[0036] The term “voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer” means that the sum of the E isomer of voclosporin (compound of formula I) and of the Z isomer of voclosporin (compound of formula I) is the 100% of the weight of voclosporin (compound of formula I).

[0037] The term “compound of formula II’” having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer” means that the sum of the E isomer of the compound of formula II’ and of the Z isomer of the compound of formula II’ is the 100% of the weight of the compound of formula II’.

[0038] The term “substantially pure E or Z isomer of the compound of formula II’” as used herein means a compound of formula II’ having an amount equal to or more than 95% by weight, preferably an amount equal to or more than 96% by weight, more preferably an amount equal to or more than 97% by weight, even more preferably an amount equal to or more than 98% by weight, of the E or Z isomer with respect to the total weight of the E and Z isomers of the compound of formula II’.

[0039] The term “substantially pure E or Z isomer of voclosporin (compound of formula I)” as used herein means voclosporin (compound of formula I) having an amount equal to or more than 95% by weight, preferably an amount equal to or more than 96% by weight, more preferably an amount equal to or more than 97%, even more preferably an amount equal to or more than 98%, of the E or Z isomer with respect to the total weight of the E and Z isomers of voclosporin (compound of formula I).

[0040] The step a) of the process of the present invention, i.e., the preparation of the substantially pure Z isomer of the compound of formula II’, comprises the reaction of the compound of formula III’ with a weak base to obtain the substantially pure isomer Z of the compound of formula II’ (see scheme 3): l , substantially pure Z isomer of II' wherein Pg is a hydroxyl protecting group and R is Ci-Ce alkyl.

[0041] Scheme 3

[0042] The compound of formula III’ involved in the process of the present invention is a mixture of the two anti-isomers of the p-trialkylsilyl alcohol as shown above.

[0043] The term “hydroxyl protecting group” refers to a group blocking the OH function for subsequent reactions that can be removed under controlled conditions. Hydroxyl protecting groups are well known in the art. Illustrative examples of hydroxyl protecting groups have been described by Green TW et al. in “Protective Groups in Organic Synthesis”, 3rd Edition (1999), Ed. John Wiley & Sons. Non-limiting examples of hydroxyl protecting groups include silyl ethers such as trimethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether or triisopropylsilyl ether; ethers such as methyl ether, tert-butyl ether, benzyl ether, trityl ether, p-methoxybenzyl ether, allyl ether, methoxymethyl ether, 2-methoxyethoxymethyl ether or tetrahydropyranyl and related ethers; esters such as acetate ester, propionate ester, butyrate ester, isobutyrate ester, valerate ester, benzoate ester, pivalate ester, levulinate ester; and carbonates such as benzyl carbonate, tert-butyl carbonate, 2-(trimethylsilyl)ethyl carbonate, allyl carbonate. The term “Ci-Ce alkyl” or “C1-C18 alkyl” as used herein refers to a linear or branched alkane derivative containing from 1 to 6 or from 1 to 18 carbon atoms, and which is bound to the rest of the molecule through a single bond. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, pentyl, hexyl, octyl, decyl.

[0044] Preferably, in the process of scheme 3, Pg is acetyl and R is methyl (see scheme 3’): substantially pure Z Isomer of II

[0045] Scheme 3’

[0046] The term “weak base” as used herein means a base that is milder than hydroxyl anion, e.g., any base having pKa lower than 14. Non-limiting examples of weak bases that can be used in the processes of schemes 3 and 3’ are sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, 1 ,8-diazabicyclo[5.4.0]undec-7- ene (DBU), triethylenediamine (DABCO), triethylamine, methylamine, diisopropylethylamine (DI PEA), pyridine, / V, / V-dimethyl-4-aminopyridine (DMAP), ammonia, etc. In a preferred embodiment, the weak base used in the processes of schemes 3 and 3’ is 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylenediamine (DABCO), triethylamine, methylamine, diisopropylethylamine (DI PEA), pyridine, / V, / V- dimethyl-4-aminopyridine (DMAP), more preferably DBU.

[0047] The authors of the present invention have surprisingly found that by using a weak base, for example DBU, in the Peterson elimination of the compound of formula III’, preferably compound of formula III, is possible to obtain substantially pure Z isomer of the compound of formula II’, preferably the compound of formula II, in contrast to the Peterson elimination disclosed in the prior art which involves the use of strong bases, where undesirable mixtures of Z and E isomers of the compounds of formula II’ or II are obtained.

[0048] Therefore, the present invention provides a process for preparing voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer, which comprises the preparation of substantially pure Z isomer of the compound of formula II’, preferably compound of formula II, by reacting a mixture of the two anti-isomers of the p-trialkylsilyl alcohol of formula III’, preferably a mixture of the two anti-isomers of the p-methylsilyl alcohol of formula III, with a weak base (see schemes 3 and 3’), preferably 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylenediamine (DABCO), triethylamine, methylamine, diisopropylethylamine (DIPEA), pyridine, / V, / V-dimethyl-4-aminopyridine (DMAP), more preferably DBU.

[0049] Non-limiting examples of suitable solvents which can be used in the processes of schemes 3 and 3’ are: ethers such as tetrahydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; halogenated solvents such chlorobenzene or 1 ,2-dichlorobenzene; polar aprotic solvents such as / V, / V-dimethylformamide, acetonitrile, / V, / V-dimethylacetamide, / V-methyl-2-pyrrolidone or dimethylsulfoxide; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; hydrocarbon aromatic solvents such as toluene, benzene, o-xylene, m-xylene or p-xylene; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate or mixtures of two or more of the solvents listed. Particularly preferred solvents are esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate or mixtures thereof, more preferably isopropyl acetate.

[0050] The substantially pure Z isomer of the compound of formula II’, preferably the substantially pure Z isomer of the compound of formula II, obtained according to the process of the present invention can be purified by means of recrystallization or slurry in a suitable solvent. Non-limiting examples of suitable solvents which can be used are: alcohols such as methanol, ethanol, propanol, isopropanol, tert-butanol; ethers such as tetra hydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; halogenated solvents such as chlorobenzene or 1 ,2- dichlorobenzene; polar aprotic solvents such as / V, / V-dimethylformamide, acetonitrile, / V, / V-dimethylacetamide, / V-methyl-2-pyrrolidone or dimethylsulfoxide; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; hydrocarbon aromatic solvents such as toluene, benzene, o-xylene, m-xylene or p- xylene; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate; water or mixtures of two or more of the solvents listed. In a preferred embodiment, the substantially pure Z isomer of the compound of formula II’, preferably the substantially pure Z isomer of the compound of formula II, obtained according to the process of the present invention, is purified by means of slurry in a hydrocarbon aliphatic solvent, preferably heptane, and / or by means of a recrystallization in a mixture of a ketone solvent and water, preferably in a mixture of acetone and water. The substantially pure Z isomer of the compound of formula II obtained according to the process of the present invention can be amorphous, crystalline of a mixture of crystalline and amorphous forms. More specifically, the present invention provides a novel crystalline form of the substantially pure Z isomer of the compound of formula II which shows and X-Ray Powder Diffractogram (XRPD) that comprises characteristic peaks at an angle of refraction 2 theta (20) of 6.85, 6.99, 7.86, 9.75, 10.22 and 15.93 (± 0.2), preferably at an angle of refraction of 2 theta (20) of 6.85, 6.99, 7.86, 9.75, 10.22, 12.02, 13.90, 14.54, 15.93, 18.99 and 22.08 (± 0.2), more preferably at an angle of refraction of 2 theta (20) of 6.85, 6.99, 7.86, 8.53, 9.75, 10.22, 11.04, 12.02, 13.90, 14.54, 15.93, 16.16, 17.83, 18.99, 19.43, 21.35 and 22.08 (± 0.2), even more preferably an X-Ray Powder Diffraction (XRPD) as shown in Figure 1 , as measured in an X-ray diffractometer with Cu Ka radiation (1.54056 A).

[0051] The substantially pure isomer E of the compound of formula II’ used in the process of the present invention can be obtained by any of the processes disclosed in the prior art.

[0052] Preferably, step b) of the process of the present invention, i.e., the preparation of the substantially pure E isomer of the compound of formula II’, comprises the reaction of the compound of formula III’ with an acid to obtain the substantially pure isomer E of the compound of formula II’ (see scheme 4): of I* wherein Pg is a hydroxyl protecting group and R is Ci-Ce alkyl.

[0053] Scheme 4

[0054] Preferably, in the process of scheme 4, Pg is acetyl and R is methyl (see scheme 4’): substantially pure E isomer of II'

[0055] Scheme 4’

[0056] Non-limiting examples of acids which can be used in the process of schemes 4 and 4’ above are sulfuric acid, hydrochloric acid, perchloric acid, formic acid, acetic acid, trifluoroacetic acid, tetrafluoroboric acid, methanesulfonic acid, p-toluensulfonic acid, triflic acid and mixtures thereof. In a preferred embodiment of the present invention the acid used in the process of schemes 4 and 4’ is formic acid or a mixture of acetic acid and formic acid.

[0057] The processes of schemes 4 and 4’ can be performed in the same list of solvents mentioned above for the processes of schemes 3 and 3’. Particularly preferred solvents are again esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate or mixtures thereof, more preferably isopropyl acetate.

[0058] The substantially pure E isomer of the compound of formula II’, preferably the substantially pure E isomer of the compound of formula II, obtained according to the process of the present invention can be purified by means of recrystallization or slurry in a suitable solvent. Non-limiting examples of suitable solvents which can be used are the same as those disclosed for the purification of the pure Z isomer of the compound of formula II’, preferably substantially pure Z isomer of the compound of formula II. In a preferred embodiment, the substantially pure E isomer of the compound of formula II’, preferably the substantially pure E isomer of the compound of formula II, obtained according to the process of the present invention, is purified by means of a recrystallization in a mixture of a ketone solvent and water, preferably in a mixture of acetone and water.

[0059] The substantially pure E isomer of the compound of formula II obtained according to the process of the present invention can be amorphous, crystalline of a mixture of crystalline and amorphous forms. More specifically, the present invention provides a novel crystalline form of the substantially pure E isomer of the compound of formula II which shows and X-Ray Powder Diffractogram (XRPD) that comprises characteristic peaks at an angle of refraction 2 theta (20) of 7.83, 8.82, 14.08, 14.58, 17.46 and 17.85 (± 0.2), preferably at an angle of refraction of 2 theta (20) of 6.98, 7.83, 8.82, 10.36, 11.38, 12.32, 12.72, 14.08, 14.58, 15.01 , 16.35, 17.46, 17.85 and 20.71 (± 0.2), more preferably at an angle of refraction of 2 theta (20) of 6.24, 6.98, 7.83, 8.82, 10.36, 11.16, 11.38, 12.32, 12.72, 14.08, 14.58, 15.01 , 15.72, 16.35, 17.46, 17.85, 19.35, 20.71 , 21.87 and 22.22 (± 0.2), even more preferably an X-Ray Powder Diffraction (XRPD) as shown in Figure 2, as measured in an X-ray diffractometer with Cu Ka radiation (1.54056 A).

[0060] Once substantially pure Z isomer of the compound of formula II’, preferably substantially pure Z isomer of the compound of formula II, and pure E isomer of the compound of formula II’, preferably substantially pure E isomer of the compound of formula II, are provided, both are mixed so that the compound of formula II’, preferably the compound of formula II, with the desirable mixture of Z and E isomers, is obtained (step c-1). The mixing process of the step c-1) consists of weighting suitable amounts of the substantially pure Z isomer of the compound of formula II’, preferably substantially pure Z isomer of the compound of formula II, and of the substantially pure E isomer of the compound of formula II’, preferably substantially pure E isomer of the compound of formula II, and put these amounts in the same reactor or mixer, optionally with stirring, in order to obtain the compound of formula II’, preferably the compound of formula II, having from 90% to 95% of the E isomer and from 5% to 10% of the Z isomer.

[0061] The compound of formula II’, preferably the compound of formula II, having from 90% to 95% of the E isomer and from 5% to 10% of the Z isomer, is deprotected to obtain voclosporin (compound of formula I) having from 90% to 95% of the E isomer and from 5% to 10% of the Z isomer (step c-2). The deprotection of the step c-2) takes place by the reaction of the compound of formula II’, preferably the compound of formula II, having from 90% to 95% of the E isomer and from 5% to 10% of the Z isomer, with a suitable base in a suitable solvent.

[0062] Preferably, the bases used in the deprotection of the step c-2) of the process of the present invention are ammonium salts hydroxides represented by the general formula RIR2R3R4N+OH-, wherein Ri, R2, R3and R4 can be a C1-C18 alkyl or a phenyl or benzyl, for example tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetraisopropylammonium hydroxide, tetrabutylammonium hydroxide, tetradecylammonium hydroxide or benzyltrimethylammonium hydroxide. In a preferred embodiment of the present invention the base used in the deprotection of the step c-2) is tetramethylammonium hydroxide.

[0063] Non-limiting examples of suitable solvents which can be used in the deprotection reaction of step c-2) of the process of the present invention are alcohols such as methanol, ethanol, propanol, isopropanol or tert-butanol; ethers such as tetrahydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; halogenated solvents such as chlorobenzene or 1 ,2- dichlorobenzene; polar aprotic solvents such as / V, / V-dimethylformamide, acetonitrile, / V, / V-dimethylacetamide, / V-methyl-2-pyrrolidone or dimethylsulfoxide; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; hydrocarbon aromatic solvents such as toluene, benzene, o-xylene, m-xylene or p- xylene or mixtures of two or more of the solvents listed. Particularly preferred solvents are alcohols such as methanol, ethanol, propanol, isopropanol or tert-butanol, more preferably methanol.

[0064] When steps a) and b) of the process of the present invention also includes the deprotection, respectively of the substantially pure isomer Z of the compound of formula II’, preferably the substantially pure isomer Z of the compound of formula II, as well as the deprotection of the substantially pure isomer E of the compound of formula II’, preferably the substantially pure isomer E of the compound of formula II, then the deprotection reactions are performed using the same conditions, i.e,. the same bases and solvents as those ones mentioned for the step c-2).

[0065] The authors of the present invention have found that the use of ammonium salts hydroxides of the general formula RIR2R3R4N+OH wherein Ri, R2, R3 and R4 can be a C1-C18 alkyl or a phenyl or benzyl, preferably tetramethylammonium hydroxide, in the deprotection reactions allows to decrease the amount of dehydration impurity. Therefore, this is an advantageous deprotecting process compared with other process processes disclosed in the prior art, e.g., involving the use of potassium carbonate, which leads to higher amounts of the dehydration impurity.

[0066] Dehydration impurity

[0067] The cleavage of the hydroxyl protecting group, preferably acetyl, does not affect the (E) / (Z) ratio. Therefore, the same (E) / (Z) ratio of the mixture obtained in step c-1) is maintained after deprotection of the protecting group of step c-2).

[0068] In case that steps a) and b) also comprise the conversion of the substantially pure Z and E isomers of the compound of formula II’, preferably of the compound of formula II, to respectively substantially pure Z and E isomers of voclosporin (compound of formula I), then in step d) the substantially pure Z and E isomers of voclosporin (compound of formula I), are mixed in order to obtain voclosporin (compound of formula I) having from 90% to 95% of the E isomer and from 5% to 10% of the Z isomer analogously as disclosed above for step c-1).

[0069] The obtained voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer can be purified by means of crystallization or slurry in an organic solvent, mixtures of organic solvents or mixtures of one or more organic solvents and water, for example in mixtures of an alcohol, for example methanol, and water or in mixtures of a ketone, for example acetone, and water.

[0070] Voclosporin (compound of formula I) obtained according to the process of the present invention can be amorphous, crystalline or a mixture of crystalline and amorphous forms. More specifically, the present invention provides a novel crystalline form of the voclosporin (compound of formula I) which shows and X-Ray Powder Diffractogram (XRPD) that comprises characteristic peaks at an angle of refraction 2 theta (20) of 7.04, 7.48 and 8.33 (± 0.2), preferably at an angle of refraction of 2 theta (20) of 6.03, 7.04, 7.48, 8.33, 9.84, 12.48, 14.20 and 19.12 (± 0.2), more preferably at an angle of refraction of 2 theta (20) of 6.03, 7.04, 7.48, 8.33, 9.84, 11.09, 11.32, 12.48, 12.67, 12.87, 14.20, 14.83, 16.70, 17.34 and 19.12 (± 0.2), even more preferably an X-Ray Powder Diffraction (XRPD) as shown in Figure 4, as measured in an X-ray diffractometer with Cu Ka radiation (1.54056 A).

[0071] Alternatively, the voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer obtained according to the present invention can be purified by chromatographic methods.

[0072] Voclosporin (compound of formula I) obtained according to the process of the present invention may have a particle size distribution (PSD), defined as the equivalent circular diameter (ECD), characterized by: (i) a D10 value of less than about 10 pm, preferably of less than 8 pm, more preferably of less than 5 pm; (ii) a D50 value of less than 50 pm, preferably of less than 30 pm, more preferably of less than 20 pm; or (iii) a D90 value of less than 100 pm, preferably of less than 90 pm, more preferably of less than 75 pm.

[0073] The particle size of the voclosporiin (compound of formula I) can be measured by any of the methods known by the skilled person, The skilled person knows that the results of PSD determination by one technique can be correlated with results from another technique on an empirical basis by routine experimentation. Voclosporin (compound of formula I) with this particle size distribution can be obtained directly from the purification processes of the present invention or applying a subsequent step or reducing the particle size by any know process disclosed in the prior art such as pin milling, ball milling or jet milling.

[0074] The percentage of one isomer or another in the compound of formula II’, preferably the compound of formula II, or in voclosporin (compound of formula I) can be verified using nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR), high performance liquid chromatography (HPLC) or other techniques disclosed in the art for the determination of the percentages of the E and Z isomers. In a particular embodiment, the percentage of each isomer is determined by NMR, such as1H-NMR.

[0075] The compound of formula III’ used in the process of the present invention can be obtained according to any of the processes disclosed in the prior art (e.g., EP0991660B1 , W02003033527A2, W02004089960A2).

[0076] Preferably, the compound of formula III’ used in the process of the present invention is obtained by a process comprising the following steps (see scheme 5):

[0077] Step 1) Reacting cyclosporin A (compound of formula VII) with a suitable protecting agent to obtain the compound of formula VI’;

[0078] Step 2) Reacting the compound of formula VI’ with a suitable oxidizing agent to obtain the compound of formula V’;

[0079] Step 3) Reacting the compound of formula V’ with a suitable oxidizing agent to obtain the compound of formula IV’; and

[0080] Step 4) Reacting the compound of formula IV’ with a borane compound of formula A to obtain the mixture of anti-isomers of the p-trialkylsilyl alcohol (compound of formula III’). wherein Pg is a hydroxyl protecting group, R is Ci-Ce alkyl and R’ and R” are independently selected from the group consisting of: Ci-Ce alkyl, C3-C7 cycloalkyl, together they form a C5-C9 cycloalkyl group, the -BR’R” group is -BF3K, or -OR”, wherein each R’” is independently selected from the group consisting of: H, Ci-Ce alkyl, C3-C7 cycloalkyl, or together the two R’” groups form a group selected from: C3-C7 cycloalkyl, 5- to 7-membered heterocycloalkyl, 5- to 7-membered heteroaryl, Ce-C aryl and C2-5 alkylene optionally substituted by Ci-Ce alkyl or -COO(Ci-Ce alkyl) wherein one of the carbon atoms in the C2-C5 alkylene group is optionally replaced by O, S or NR*, wherein R* is selected from H and C1-6 alkyl.

[0081] Scheme 5

[0082] Preferably, in the process of scheme 5, Pg is acetyl, R is methyl and R’ and R” have the meaning given above in scheme 5 (see scheme 5’):

[0083] Scheme 5’

[0084] The cyclosporin A (compound of formula VII) used as starting material can be obtained by any of the processes disclosed in the prior art.

[0085] Step 1) can be performed by conventional methods known by those skilled in the art (e.g., Green TW et al. in “Protective Groups in Organic Synthesis”, 3rd Edition (1999), Ed. John Wiley & Sons (ISBN 0-471-16019-9).

[0086] In the preferred embodiment wherein cyclosporin A (compound of formula VII) is protected with an acetyl to form the compound of formula VI, cyclosporin A (compound of formula VII) reacts with acetylating agents such as acetic anhydride or acetyl chloride, preferably acetic anhydride, in the presence of a base in a suitable solvent. Non-limiting examples of suitable solvents which can be used in the reaction between cyclosporin A (compound of formula VII) and the acetylating agent, preferably acetic anhydride, of the step 1) are ethers such as tetrahydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; halogenated solvents such as chlorobenzene or 1 ,2-dichlorobenzene; polar aprotic solvents such as / V, / V-dimethylformamide, acetonitrile, / V, / V-dimethylacetamide, / V-methyl-2-pyrrolidone or dimethylsulfoxide; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; hydrocarbon aromatic solvents such as toluene, benzene, o-xylene, m-xylene or p-xylene; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate or mixtures of two or more of the solvents listed. Particularly preferred solvents are esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate, preferably isopropyl acetate.

[0087] Non-limiting examples of suitable bases which can be used in the reaction between cyclosporin A (compound of formula VII) and the acetylating agent, preferably acetic anhydride, of the step 1) are inorganic or organic bases, preferably organic bases such as / V, / V-diisopropylethylamine (DI PEA), trimethylamine, tripropylamine, / V- methylpiperidine, / V, / V-dimethylaminopyridine (DMAP), / V-methylpyrrolidine and 1 ,4- diazabicyclo[2.2.2]octane (DABCO), or amidine bases selected from the group of 1 ,5- diazabicyclo[4.3.0]non-5-ene (DBN) and 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or mixtures thereof, and more preferably / V, / V-diisopropylethylamine (DI PEA).

[0088] The compound of formula VI obtained according to the process of the present invention can be amorphous, crystalline or a mixture of crystalline and amorphous forms. More specifically, the present invention provides a novel crystalline form of the compound of formula VI which shows and X-Ray Powder Diffractogram (XRPD) that comprises characteristic peaks at an angle of refraction 2 theta (20) of 7.68, 8.62, 9.47, 10.29, 11 .58 and 15.33 (± 0.2), preferably at an angle of refraction of 2 theta (20) of 7.68, 8.62, 9.47, 10.29, 11.58, 14.86, 15.33, 17.42, 18.17 and 20.04 (± 0.2), more preferably at an angle of refraction of 2 theta (20) of 7.68, 8.62, 9.47, 10.29, 11.58, 13.43, 14.14, 14.86, 15.33, 16.73, 17.42, 18.17, 18.62, 19.48, 20.04, 20.38, 21.80 and 22.89 (± 0.2), even more preferably an X-Ray Powder Diffraction (XRPD) as shown in Figure 5, as measured in an X-ray diffractometer with Cu K a radiation (1 .54056 A).

[0089] The epoxidation of the double bond of the step 2) of schemes 5 and 5’ can be performed with Oxone®, a peroxide, a peroxyacid or a persulfate, preferably with Oxone®, preferably in the presence of a base, preferably an inorganic base, for example sodium bicarbonate, in a suitable solvent. Oxone® is potassium peroxymonosulfate (KHSOs O.SKHSOr 0.5K2SO4).

[0090] Non-limiting examples of suitable solvents which can be used in the epoxidation of the double bond of the step 2) of schemes 5 and 5’, are alcohols such as methanol, ethanol, propanol, isopropanol or tert-butanol; ethers such as tetra hydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; halogenated solvents such as chlorobenzene or 1 ,2-dichlorobenzene; polar aprotic solvents such as / V, / V-dimethylformamide, acetonitrile, / V, / V-dimethylacetamide or / V-methyl-2-pyrrolidone; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; hydrocarbon aromatic solvents such as toluene, benzene, o-xylene, m-xylene or p-xylene; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate; water or mixtures of two or more of the solvents listed. Particularly preferred solvents are ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate; water or mixtures thereof, preferably a mixture of acetone, ethyl acetate and water.

[0091] The oxidative cleavage of the epoxide of formula V’, preferably compound of formula V, of step 3) can be performed without being previously isolated. In a preferred embodiment of the present invention, the compound of formula V’, preferably compound of formula V, obtained in step 2) is subjected to the oxidation of step 3) without being previously isolated.

[0092] The oxidation step 3) of the process of the schemes 5 and 5’ is performed with an oxidizing agent such as periodic acid or periodate salt, preferably sodium periodate, in the presence of a suitable acid, for example sulfuric acid, in a suitable solvent.

[0093] Non-limiting examples of suitable solvents which can be used in step 3) of the process of the schemes 5 and 5’ are alcohols such as methanol, ethanol, propanol, isopropanol or tert-butanol; ethers such as tetra hydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; halogenated solvents such as chlorobenzene or 1 ,2-dichlorobenzene; polar aprotic solvents such as / V, / V- dimethylformamide, acetonitrile, / V, / V-dimethylacetamide, / V-methyl-2-pyrrolidone or dimethylsulfoxide; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; hydrocarbon aromatic solvents such as toluene, benzene, o-xylene, m-xylene or p-xylene; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate, water or mixtures of two or more of the solvents listed. Particularly preferred solvents are polar aprotic solvents such as / V, / V-dimethylformamide, acetonitrile, / V, / V-dimethylacetamide, / V- methyl-2-pyrrolidone or dimethylsulfoxide; water or mixtures thereof, preferably mixtures of acetonitrile and water.

[0094] The obtained compound of formula IV’, preferably the compound of formula IV, can be purified by means of recrystallization or slurry in an organic solvent or mixtures of organic solvents. Non-limiting examples of suitable solvents which can be used are: alcohols such as methanol, ethanol, propanol, isopropanol, tert-butanol; ethers such as tetra hydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones such as methyl ethyl ketone, methyl isobutyl ketone or acetone; halogenated solvents such as chlorobenzene or 1 ,2- dichlorobenzene; polar aprotic solvents such as / V, / V-dimethylformamide, acetonitrile, / V, / V-dimethylacetamide, / V-methyl-2-pyrrolidone or dimethylsulfoxide; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; hydrocarbon aromatic solvents such as toluene, benzene, o-xylene, m-xylene or p- xylene; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate; water or mixtures of two or more of the solvents listed.

[0095] In an embodiment of the present invention, the obtained compound of formula IV’, preferably the compound of formula IV, is purified by means of a crystallization process which comprises first preparing a solution of the compound of formula IV’, preferably the compound of formula IV, in a ketone such as methyl ethyl ketone, methyl isobutyl ketone or acetone, preferably acetone, and then contacting this solution with an anti-solvent, preferably water. This purification process can be repeated until the desired purity is achieved.

[0096] The compound of formula IV obtained according to the process of the present invention can be amorphous, crystalline or a mixture of crystalline and amorphous forms. More specifically, the present invention provides a novel crystalline form of the compound of formula IV which shows and X-Ray Powder Diffractogram (XRPD) that comprises characteristic peaks at an angle of refraction 2 theta (20) of 8.77, 11.56, 16.24, 16.56, 18.38 (± 0.2), preferably at an angle of refraction of 2 theta (20) of 8.77, 11.56, 13.02, 14.66, 16.24, 16.56, 17.32, 18.38, 19.04, 19.96 and 20.25 (± 0.2), more preferably at an angle of refraction of 2 theta (20) of 6.18, 8.77, 11.56, 11.91 , 13.02, 14.66, 16.24, 16.56, 17.32, 18.38, 19.04, 19.96, 20.25, 21.85, 22.77, 23.21 and 25.78 (± 0.2), even more preferably an X-Ray Powder Diffraction (XRPD) as shown in Figure 6, as measured in an X-ray diffractometer with Cu K a radiation (1 .54056 A).

[0097] The borane compound of formula A used in the step 4) of the processes of schemes 5, 5’ has E configuration, thus yielding the mixture of the two anti-isomers of the p- trialkylsilyl alcohols of formula III’, preferably of formula III. In a preferred embodiment of the present invention, the borane compound of formula A used is the step 4) of the processes of schemes 5 and 5’ is (E)-trimethyl(3-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)allyl)silane.

[0098] The obtained compound of formula III’, preferably compound of formula III, can be used without isolation in the subsequent step, i.e. , the Peterson elimination (see schemes 3, 3’, 4 and 4’). In case that the Peterson elimination is performed with a weak base (schemes 3 and 3’), the acid used in the step 4) for the preparation of the compound of formula III’, preferably the compound of formula III, must be previously neutralized before the addition of the weak base. Thus, the reaction mixture obtained in step 4) of the schemes 5 and 5’ can be washed with an aqueous solution of a base, for example an aqueous solution of sodium hydroxide and subsequently buffered, for example, with an aqueous solution of ammonium formate, in order to neutralize the acid used in the process of step 4). However, in case that the Peterson elimination is performed with an acid (schemes 4 and 4’), such acid can be directly added to the reaction mixture obtained after the step 4) of schemes 5 and 5’. The preparation of the compound of formula III’, preferably compound of formula III, and the subsequent Peterson elimination can be then performed in the same solvent or mixture of solvents as those listed before. In a preferred embodiment, the preparation of the compound of formula III’, preferably compound of formula III, is performed in an ester such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate, preferably isopropyl acetate.

[0099] Step 4) of the processes of schemes 5 and 5’ is preferably performed in the presence of an acid, for example sulfuric acid, phosphoric acid, hydrochloric acid, formic acid, acetic acid, methanesulfonic acid, trifuloroacetic acid, preferably acetic acid.

[0100] Another aspect of the present invention provides a process for preparing pharmaceutical compositions comprising voclosporin (compound of formula I) by mixing voclosporin (compound of formula I) having from 90% to 95% of the E isomer and from 5% to 10% of the Z isomer obtained according to the process of the present invention and one or more pharmaceutically acceptable excipient. As used herein, the term "pharmaceutical compositions" or "pharmaceutical formulations" include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, or injection preparations, preferably tablets or capsules, in particular soft capsules.

[0101] The pharmaceutically acceptable excipient can be one or more substances which may also act as diluent, flavouring agent, solubiliser, lubricant, suspending agent, binder, glidant, or disintegrant.

[0102] Voclosporin (compound of formula I) having from 90% to 95% of the E isomer and from 5% to 10% of the Z isomer obtained according to the process of the present invention or pharmaceutical compositions thereof may be used to treat alleviate an autoimmune disease or condition or an inflammatory disease or condition, for example, systemic lupus erythematosus, which can cause lupus nephritis.

[0103] The term “about” when used in the present invention preceding a number and referring to it, is meant to designate any value which lies within the range defined by the number ±10% of its value, preferably a range defined by the number ±5%, more preferably a range defined by the number ±2%, still more preferably a range defined by the number 10 ±1%. For example, “about 10” should be construed as meaning within the range of 9 to 11 , preferably within the range of 9.5 to 10.5, more preferably within the range of 9.8 to 10.2, and still more preferably within the range of 9.9 to 10.1.

[0104] Examples

[0105] XRPD Method of Analysis:

[0106] The XRPD pattern was recorded on a Bruker D2 Phaser diffractometer equipped with a vertical goniometer under Bragg-Brentano geometry and a SSD160-2 position-sensitive detector with a 6-position sample changer and a horizontal sample holder rotating at 15 rpm. Cu Ka radiation (1 = 1.54056 A) was obtained from a copper X-ray tube operated at 30 kV and 10 mA. The diffraction pattern was recorded including values of 20 that range from 3 to 50° with a sampling rate of 0.02° per second and a step time of 0.7 seconds per step. The powdered sample was placed on a low-background silicon sample holder and covered with Kapton foil. DIFFRAC MEASUREMENT CENTER software with EVA evaluation software (Bruker) was used to record the data and for a primary analysis of the diffraction pattern. The equipment was periodically calibrated using a corundum (AI2O3) reference sample.

[0107] The process described in the present invention is illustrated in examples below. These examples are provided as illustration only and therefore should not be construed as limitation of the scope of the invention: Example 1 : Preparation of the compound of formula VI from cyclosporin (compound of formula VII)

[0108] 75 g of Cyclosporin A (compound of formula VII) and 5.71 g of A / . / V- dimethylaminopyridine were combined with 375 mL of isopropyl acetate and the mixture was stirred until complete dissolution at 20-25°C. 2.82 g of / V, / V-diisopropylethylamine and 31.8 g of acetic anhydride were added onto the previous solution. The mixture was heated up to 39-43°C and stirred at this temperature for about 72 hours. The mixture was cooled down to 20-25°C and 225 mL of deionized water were added. The biphasic system was stirred for 15 minutes, then the phases were allowed to settle, and the aqueous phase was separated. The organic phase was mixed with 188 mL of deionized water, followed by the addition of 44.3 g of concentrated hydrochloric acid, while keeping the temperature below 25°C. The biphasic mixture was stirred for 15 minutes, the phases were allowed to settle, and the aqueous phase was separated. The organic phase was mixed with 225 mL of deionized water. The biphasic mixture was stirred for 15 minutes, the phases were allowed to settle, and the aqueous phase was separated. The organic phase was submitted to distillation under vacuum to almost dryness at a temperature below 45°C. 50 mL of acetone were loaded onto the residue, the mixture was stirred for 30 minutes and then it was submitted to distillation to almost dryness at a temperature below 45°C. This last operation was repeated one more time. 210 mL of acetone were loaded onto the residue at 20-25°C, the mixture was stirred for 30 minutes and then 373 mL of deionized water were added. The resulting suspension was stirred at that temperature for 1 hour and was filtered, washing the cake with deionized water (2 x 40 mL). The solid, corresponding to the compound of formula VI, was used without drying for the next step. X-Ray Powder Diffraction as shown in Figure 5. Yield: 90%, purity by HPLC (% area): 99.1%.

[0109] Example 2: Preparation of the compound of formula IV 60 g of the compound of formula VI were combined with 480 mL of ethyl acetate and the mixture was stirred at 20-25°C until complete dissolution. Then, 300 mL of deionized water, 16.2 g of sodium bicarbonate and 56 g of acetone were added onto the solution. On the other hand, 88.9 g of Oxone® were dissolved in 432 mL of deionized water and this solution was added in 1-1.5 hours onto the initial solution. The final mixture was stirred at 20-25°C for 1 hour and then a second load of 4.05 g of sodium bicarbonate were added and the mixture was stirred for 1 hour. The same operation was repeated 3 more times. After the fifth load of sodium bicarbonate, the biphasic mixture was stirred for 24 hours at 20-25°C. After that time, stirring was stopped, the phases were allowed to settle for 15-30 minutes, and the aqueous phase was separated. The organic phase was treated with an aqueous solution of 25 g of sodium chloride in 110 mL of deionized water for 15 minutes, the phases were allowed to settle for 15-30 minutes and the aqueous phase was separated. The organic phase was submitted to distillation under vacuum at a temperature below 45°C until a residual volume of about 180 mL. 300 mL of acetonitrile were loaded onto the residue at 20-25°C and the mixture was submitted to distillation under vacuum at a temperature below 45°C until a residual volume of about 180 mL. The solution, containing compound of formula V, was used as such for the next step.

[0110] 835 mL of acetonitrile and 820 mL of deionized water were loaded onto the previous solution. Then, 15.47 g of sodium periodate and 4.82 g of 98% concentrated sulfuric acid were added. The mixture was heated up to 30±3°C and stirred at this temperature for 9 hours. The reaction was then cooled down to 20-25°C and was submitted to distillation under vacuum at a temperature below 30-35°C to remove almost all the acetonitrile. 304 mL of methyl t-butyl ether were loaded at 20-25°C and the mixture was stirred for 15 minutes. The phases were allowed to settle for 30 minutes, and the organic phase was separated. The aqueous phase was treated with 304 mL of methyl t-butyl ether and the mixture was stirred for 15 minutes. The phases were allowed to settle for 30 minutes, and the aqueous phase was separated. The two organic phases were combined and were treated with an aqueous solution of 88 g of sodium chloride in 275 mL of deionized water for 15 minutes. The phases were allowed to settle for 30 minutes and the aqueous phase was separated. The organic phase was submitted to distillation under vacuum at a temperature below 40°C almost until dryness. 267 mL of dimethyl sulfoxide were loaded onto the residue and the mixture was submitted to distillation under vacuum at a temperature below 40°C to remove residual methyl t-butyl ether. Keeping the temperature at 37-43°C, 89 mL of deionized water were slowly added during about 1 hour, then the mixture was cooled down slowly to 20-25°C and stirred for 1 hour. The resulting suspension was filtered, washing the cake with deionized water (3 x 50 mL). Wet crude compound of formula IV was obtained (42.6 g dry equivalent).

[0111] Purification of the compound of formula IV

[0112] Wet compound of formula IV (14 g dry equivalent) was combined with 56 mL of acetone. The mixture was stirred until complete dissolution and then 70 mL of deionized water were added. The obtained suspension was heated to 40°C and then cooled down slowly to 20-25°C and stirred at this temperature for 4 hours. The suspension was filtered, washing the cake with deionized water, obtaining 14.72 g of wet compound of formula IV. 13.7 g of this second crude (10.5 g dry equivalent) were dissolved with 42 mL of acetone at 20-25°C followed by the addition of 52 mL of deionized water. The suspension was stirred at this temperature for 4 hours and was filtered, washing the cake with deionized water. The solid was dried and a third crude compound of formula IV was obtained (9.95 g). 5 g of this third crude were dissolved in 20 mL of acetone at 20-25°C and 25 mL of deionized water were added. The suspension was stirred at this temperature for 4 hours and filtered, washing the cake with deionized water. The solid was dried and 4.67 g of final compound of formula IV were obtained. X-Ray Powder Diffraction as shown in Figure 6. Purity by HPLC (% area): 98.1 %.

[0113] Example 3: Preparation of substantially pure Z isomer of the compound of formula II Z isomer of II

[0114] 25.0 g of the compound of formula IV were combined with 50 mL of isopropyl acetate and the mixture was stirred at 20-25°C until complete dissolution. Then, 31 mL of acetic acid and 7.3 g of (E)-trimethyl(3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)allyl)silane were added onto the solution. The resulting mixture was stirred for 24 h at 40°C. Over this mixture, 175 mL of isopropyl acetate and 125 mL of water were added. Layers were separated and the organic layer was washed twice with NaOH 2N and finally with ammonium formate 5% aqueous solution. Then, 9.3 g of DBU were added to the organic phase and the resulting solution was stirred 20 hours at reflux. Then, the mixture was cooled to 20-25°C and over this mixture, 125 mL of HCI 1 N were added. Layers were separated and the organic layer was washed with ammonium formate 5% aqueous solution. The organic layer was concentrated almost to dryness and 300 mL of heptane were added. The resulting suspension was stirred 2 hours at 15°C, filtered and washed with heptane. 22.41 g of dry equivalent substantially pure Z isomer of compound of formula II were obtained.

[0115] The obtained wet solid was stirred with 170 mL of acetone at reflux. Over this solution 145 mL of water were slowly added. The resulting solution was cooled down to 10-15 °C and it was stirred for at least 2 hours at 10-15°C. The resulting solid was filtered, washed with water and dried under vacuum at 50 °C. 20.19 g of compound of formula II having 97% by weight of the Z isomer and 3% by weight of the E isomer (as determined by1H-NMR). X-Ray Powder Diffraction as shown in Figure 1. Purity by HPLC (% area): 99.5% (this includes Z and E isomers of the compound of formula II).

[0116] Example 4: Preparation of substantially pure E isomer of the compound of formula II

[0117] 17.0 g of compound of formula IV were combined with 34 mL of isopropyl acetate and the mixture was stirred at 20-25°C until complete dissolution. Then, 21 mL of acetic acid and 5.0 g of (E)-trimethyl(3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)allyl)silane were added onto the solution. After stirring 10 minutes at room temperature, 21 mL of formic acid were added, and the resulting mixture was heated to 40°C. The reaction crude was stirred for 24 h at 40°C and then 120 mL of isopropyl acetate and 85 mL of water were added. Layers were separated and the organic layer was washed twice with NaOH 2N and finally with ammonium formate 5% aqueous solution. The organic layer was concentrated almost to dryness and 100 mL of heptane were added. The resulting suspension was stirred 2 hours at 15°C, filtered and washed with heptane. 15.43 g of dry compound of formula II were obtained.

[0118] Wet solid was stirred with 100 mL of acetone at reflux. Over this solution 80 mL of water were slowly added. The resulting solution was cooled down to 10-15 °C and it was stirred for at least 2 hours at 10-15°C. The resulting solid was filtered, washed with water and dried under vacuum at 50°C. 13.27 g of the compound of formula II having 98% by weight of the E isomer and 2% by weight of the Z isomer (as determined by1H-NMR). X-Ray Powder Diffraction as shown in Figure 2. Purity by HPLC (% area): 99.5% (this includes E and Z isomers of the compound of formula II). Example 5: Preparation of Voclosporin (compound of formula I) substantially pure substantially pure Z isomer of II E isomer of II

[0119] A mixture of 55.0 g of substantially pure E isomer of the compound of formula II (as a mixture of isomers Z / E of 2% / 98% as determined by1H-NMR), 3.6 g of substantially pure Z isomer of the compound of formula II (as a mixture of isomers Z / E of 97% / 3% as determined by1H-NMR) and 880 mL of methanol were cooled to 10-15°C. Over this solution, 54.38 g of tetramethylammonium hydroxide 25% aqueous solution were added and the mixture was stirred for 4 days at 10-15°C. Over this mixture, 125 mL of citric acid 10% aqueous solution were added maintaining the temperature below 15°C. Solvent was partially distilled under pressure and then 600 mL of isopropyl acetate and 350 mL of sodium chloride 5% aqueous solution were added. Layers were separated and the organic layer was concentrated under pressure at temperature below 40 °C to give crude Voclosporin (compound of formula I) having 93% by weight of E isomer and 7% by weight of Z isomer (as determined by1H-NMR).

[0120] 38.5 g (dry equivalent weight) of Voclosporin (compound of formula I) were solved in a mixture of 80 mL of MTBE and 35 mL of n-heptane at 45-50 °C. The resulting solution was added slowly over 700 mL of n-heptane at room temperature. The mixture was stirred for 2 hours at room temperature and the obtained solid was filtered and washed with 70 mL of n-heptane. The solid was dried at 60 °C under vacuum. 33.5 g of Voclosporin (compound of formula I) were obtained as a white solid having 93% by weight of E isomer and 7% by weight of Z isomer (as determined by1H-NMR). X-Ray Powder Diffraction as shown in Figure 3. Purity by HPLC (% area): 99.0% (this includes E and Z isomers of the compound of formula I).

[0121] Example 6: Preparation of Voclosporin Form M:

[0122] A mixture of 5.0 g of substantially pure E isomer of the compound of formula II (as a mixture of isomers Z / E of 2% / 98% as determined by1H-NMR), 325 mg of substantially pure Z isomer of the compound of formula II (as a mixture of isomers Z / E of 97% / 3% as determined by1H-NMR) and 80 mL of methanol were cooled below 20°C. Over this solution, 5.57 g of tetramethylammomum hydroxide 25% aqueous solution were added and the mixture was stirred for 3 days at 20°C. Over this mixture, 0.70 mL of formic acid were added maintaining the temperature below 20 °C. Solvent was partially distilled under pressure and then 50 mL of isopropyl acetate and 50 mL of sodium chloride 5% aqueous solution were added. Layers were separated and the organic layer was concentrated under pressure at temperature below 40 °C to give crude voclosporin

[0123] (compound of formula I) as a mixture having 93% by weigh of E isomer and 7% by weight of Z isomer (as determined by1H-NMR).

[0124] The obtained wet solid was stirred with 35 mL of methanol and 15 mL of water at 25°C for 2 days. The resulting solid was filtered and washed with water. Crystalline Form M of voclosporin (compound of formula I) was obtained. X-Ray Powder Diffraction as shown in Figure 4.

Claims

CLAIMS1 . A process for preparing voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer,which comprises: a) Preparing substantially pure Z isomer of the compound of formula II’substantially pure Z isomer of II' wherein Pg is a hydroxyl protecting group, and optionally deprotecting substantially pure Z isomer of the compound of formula II’ to obtain substantially pure Z isomer of voclosporin (compound of formula I);substantially pure Z isomer of Ib) Preparing substantially pure E isomer of the compound of formula II’substantially pure E isomer of II' wherein Pg is a hydroxyl protecting group, and optionally deprotecting substantially pure E isomer of the compound of formula II’ to obtain substantially pure E isomer of voclosporin (compound of formula I);substantially pure E isomer of I and, either c-1) Mixing the substantially pure Z isomer of the compound of formula II’ of step a) with the substantially pure E isomer of the compound of formula II’ of step b), in such a way that compound of formula II’ having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer is obtained; and c-2) Deprotecting the compound of formula II’ obtained in step c-1) to obtain voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer; or d) In case that the steps a) and b) above include the deprotection step, mixing the substantially pure Z isomer of voclosporin (compound of formula I) of step a) with the substantially pure E isomer of voclosporin (compound of formula I) of step b), in such a way that voclosporin (compound of formula I) having from 90% to 95% of weight of the E isomer and from 5% to 10% by weight of the Z isomer is obtained; wherein,the term substantially pure Z isomer of the compound of formula II’ means a compound of formula II’ having an amount equal to or more than 95% by weight of the Z isomer with respect to the total weight of the E and Z isomers of the compound of formula II’; the term substantially pure Z isomer of voclosporin (compound of formula I) means voclosporin (compound of formula I) having an amount equal to or more than 95% by weight of the Z isomer with respect to the total weight of the E and Z isomers of voclosporin (compound of formula I); the term substantially pure E isomer of the compound of formula II’ means a compound of formula II’ having an amount equal to or more than 95% by weight of the E isomer with respect to the total weight of the E and Z isomers of the compound of formula II’; and the term substantially pure E isomer of voclosporin (compound of formula I) means voclosporin (compound of formula I) having an amount equal to or more than 95% by weight of the E isomer with respect to the total weight of the E and Z isomers of voclosporin (compound of formula I).

2. The process according to claim 1 , wherein step a) comprises the reaction of the compound of formula III’ with a weak base to obtain the substantially pure isomer Z of the compound of formula II’:substantially pure Z isomer of II'wherein Pg is a hydroxyl protecting group and R is Ci-Ce alkyl.

3. The process according to the claim 2 wherein the weak base is selected from the group consisting of 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylenediamine (DABCO), triethylamine, methylamine, diisopropylethylamine (DI PEA), pyridine and / V, / V- dimethyl-4-aminopyridine (DMAP), preferably DBU.

4. The process according to any one of claims 1 to 3, wherein step b) comprises the reaction of the compound of formula III’ with an acid to obtain the substantially pure isomer E of the compound of formula II’.substantially pure E isomer of II' wherein Pg is a hydroxyl protecting group and R is Ci-Ce alkyl.

5. The process according to claim 4 wherein the acid used is selected from the group consisting of sulfuric acid, hydrochloric acid, perchloric acid, formic acid, acetic acid, trifluoroacetic acid, tetrafluoroboric acid, methanesulfonic acid, p-toluensulfonic acid, triflic acid, Lewis acid (such as boron trifluoride diethyletherate) and mixtures thereof, preferably formic acid or a mixture of acetic acid and formic acid.

6. The process according to any one of claims 1 to 5, wherein the deprotection reaction of steps a), b) or c-2) is performed by using an ammonium salt hydroxide represented by the general formula RIR2R3R4N+OH wherein R1, R2, R3 and R4 can be a C1-C18 alkyl or a phenyl or benzyl, preferably tetramethylammonium hydroxide.

7. The process according to any one of claims 2 to 6, which comprises the following previous steps of:Step 1) Reacting cyclosporin A (compound of formula VII) with a suitable protecting agent to obtain the compound of formula VI’;Step 2) Reacting the compound of formula VI’ with a suitable oxidizing agent to obtain the compound of formula V’;Step 3) Reacting the compound of formula V’ with the suitable oxidizing agent to obtain the compound of formula IV’; andStep 4) Reacting the compound of formula IV’ with a borane compound of formula A to obtain the mixture of anti-isomers of the p-trialkylsilyl alcohol (compound of formula III’).wherein Pg is a hydroxyl protecting group, R is Ci-Ce alkyl and R’ and R” are independently selected from the group consisting of: Ci-Ce alkyl, C3-C7 cycloalkyl, together they form a C5-C9 cycloalkyl group, the -BR’R” group is -BF3K, or -OR”, wherein each R’” is independently selected from the group consisting of: H, Ci-Ce alkyl, C3-C7 cycloalkyl, or together the two R’” groups form a group selected from: C3-C7 cycloalkyl, 5- to 7-membered heterocycloalkyl, 5- to 7-membered heteroaryl, Ce-C aryl and C2-5 alkylene optionally substituted by Ci-Ce alkyl or -COO(Ci-Ce alkyl) wherein one of the carbon atoms in the C2-C5 alkylene group is optionally replaced by O, S or NR*, wherein R* is selected from H and C1-6 alkyl.

8. The process according to any one of claims 1 to 7, wherein Pg is acetyl.

9. The process according to any one of claims 2 to 8, wherein R is methyl.

10. The process according to any one of claims 7 to 9, whereien the borane compound of formula A is (E)-trimethyl(3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)allyl)silane.

11. The process according to any one of claims 7 to 10, wherein step 4) is performed in the presence of an acid selected from the group consisting of sulfuric acid, phosphoric acid, hydrochloric acid, formic acid, acetic acid, methanesulfonic acid, trifuloroacetic acid, preferably acetic acid.

12. The process according to any one of claims 2 to 11 , wherein the compound of formula III’ is not isolated before its conversion to the substantially pure Z or E isomer of the compound of formula II’.

13. The process according to any one of claims 7 to 12, wherein the compound of formula V’ obtained in step 2) is subjected to the oxidation of step 3) without being previously isolated.

14. A process according to any one of claims 1 to 13, further comprising mixing the voclosporin thus obtained (compound of formula I) with one or more pharmaceutically acceptable excipients to form a pharmaceutical formulation.

15. A pharmaceutical formulation obtained by the process of claim 14 for use in treatment of lupus nephritis.

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