MX2007014278A - Process for purifying tacrolimus - Google Patents

Abstract

The invention provides a process for the chromatographic purification of Tacrolimus, using a silver modified sorbent, selected from the group consisting of silver modified aluminum oxide, zirconium oxide, styrene divinylbenzene copolymer, adsorption resin, cation exchange resin, anion exchange resin, reverse phase silica gel, and cyano silica-gel, and separating the Tacrolimus chromatographically with the silver modified sorbent as a stationary phase.

Description

PROCESS TO PURIFY TACROLIMUS FIELD OF THE INVENTION The present invention relates to a process for the purification of Tacrolimus.

Background of the Invention Tacrolimus, [3S- [3R [E (1S, 3S, AS)], AS, SR, 8S, 9E, 12R, 1AR, 15S, 16R, 185, 19S, 26aR]] - 5, 6, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 24, 25, 26, 26a-hexadecahydro-5,19-dihydroxy-3- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylethhenyl] -14, 16-dimethoxy-4, 10, 12, 18-tetramethyl-8- (2-propenyl) -15, 19-epoxy-3H-pyrido [2, 1-c] [1,4 ] oxaazacyclyltricosin-1, 7,20,21 (4H, 23H) -tetron, monohydrate (formerly known as FK506), has a molecular weight of 822.05, the formula C44H69 O12H2O, and the structural formula Tacrolimus is a naturally occurring macrolide immunosuppressant produced by Streptomyces tsukubaensis. It is found on the market under the name PROGRAF® from Fujisawa, and is available for oral administration as capsules (tacrolimus capsules). Tacrolimus prolongs the survival of the host and transplanted graft in models of transplants in animals of liver, kidney, heart, spinal cord, small intestine and pancreas, lung and trachea, skin, cornea and limbs inhibiting the activation of T-lymphocyte. In animals, tacrolimus has been shown to suppress humoral immunity and, to some extent, cell-mediated reactions, such as allograft, delayed-type hypersensitivity, collagen-induced arthritis, experimental allergic encephalomyelitis, and graft versus disease. Guest.

Tacrolimus was first described in U.S. Patent No. 4,894,366 and European Patent No. EP 0 184 162. When Tacrolimus is prepared, two Tacrolumus analogues, which are impurities, are also obtained: Ascomycin, which has the structural formula and Dihydrotacrolimus, which has the structural formula.

The separation of tacrolimus from these impurities by conventional methods, such as crystallization, is difficult because of the structural similarity. In addition, purification is required by column chromatography. The efficiency of the purification can be improved by using silver ions in column chromatography. Silver ions interact with the double bond of the allyl side chain found in tacrolimus but which is absent in ascomycin and dihydrotacrolimus in which the side chain is an alkyl group. In ascomycin, the side chain is an ethyl group, and Dihydrotacrolimus, the side chain is a propyl group. As a result, the bond between the tacrolimus and the stationary phase or the mobile phase is fortified, and thus, the difference in tacrolimus retention time compared with the retention time of its analogs increases.

U.S. Patent No. 6,492,513 discloses separating tacrolimus from impurities using a sulfuric acid group containing a strong cation exchange resin pre-treated as silver ions, and an eluent of acetone or ethyl acetate and methanol . As tacrolimus is sensitive to the revealed exchange resin, tacrolimus contamination should be expected.

U.S. Patent Nos. 6,576,135 and 6,881,341 disclose a two-step column chromatography process for the removal of tacrolimus from impurities, particularly analogue tacrolimus-dimethyl. One disclosed step includes absorbing a mixture containing tacrolimus with a non-anionic adsorption resin, and eluting with an aqueous solvent containing silver ions. The other disclosed step includes absorbing the mixture with a basic active alumina and eluting with an organic solvent to perform the separation. The use of water in the eluent results in the formation of tacrolimus isomers, thus contaminating tacrolimus. In addition, the use of an eluent containing free silver ions requires the separation of silver ions from the eluent from the tacrolimus followed by elution from the column.

International Patent Application Publication No. WO 05/054253 discloses the chromatographic separation of tacrolimus using silica gel, optionally, either in reverse phase chromatography or pre-treated with silver, followed by treatment with ammonia gas to remove the impurities The disclosed eluent was a mixture of n-butanol, acetonitrile, and a buffer for reverse phase separation, and a mixture of ethyl acetate and hexane for removal of silver-treated silica gel.

International Patent Application Publication No. WO 05/098011 discloses a separation of tacrolimus from impurities by silica gel chromatography, where the silica gel is optionally pretreated with silver. Optionally, tacrolimus is also purified by reverse phase chromatography using previously treated silica. A C-8 column is exemplified for reverse phase chromatography. Eluent exemplified include ethyl acetate in hexane for the silver-treated column and a mixture of acetonitrile, n-butanol, and buffer for reverse phase separation.

International Patent Application Publication No. 0 05/010015 discloses the separation using a non-ionic adsorption resin without silver and with an eluent containing THF or acetonitrile, water, and, optionally, an additional organic solvent.

There is a need in the art for a method to purify tacrolimus, particularly Ascomycin and Dihydrotacrolimus, which would be suitable for use on an industrial scale. The present invention provides said method.

Extract of the Invention In one embodiment the present invention provides a process for separating tacrolimus from impurities comprising: a) charging a mixture comprised of tacrolimus and impurities in a bed of a sorbent pre-treated with silver ions, characterized in that the sorbent is selected from the group formed by silver modified aluminum oxide, zirconium oxide, styrene divinylbenzene copolymer, adsorption resin, cation exchange resin, anion exchange resin, reversed phase silica gel, and cyano silica gel; and b) bypassing the mixture from the bed of the sorbent resin to remove tacrolimus from the impurities present in the mixture.

Brief description of the figures Figure 1 shows HPLC analysis of chromatographic fractions (mg / 1) according to example 2.

Figure 2 shows HPLC analysis of chromatographic fractions (mg / 1) according to example 5.

Figure 3 shows HPLC analysis of chromatographic fractions (mg / 1) according to example 21.

Figure 4 shows HPLC analysis of chromatographic fractions (mg / 1) according to example 25.

Figure 5 shows HPLC analysis of chromatographic fractions (mg / ml) according to comparative example 23.

Detailed description of the invention The present invention is directed to a process for the chromatographic purification of tacrolimus using a sorbent pretreated with silver ions for the stationary phase ("silver modified sorbent"), where the sorbent is one of aluminum oxide, zirconium oxide, styrene divinylbenzene copolymer, adsorption resin, cation exchange resin, anion exchange resin, reverse phase silica gel, and cyano silica gel. The process of the invention is particularly effective for the separation of tacrolimus from ascomycin and dihydrotacrolimus.

The process of the invention is suitable for industrial use. It can be carried out with an eluent that does not contain water and / or silver ions, and provides an effective separation of tacrolimus from dihydrotacrolimus and ascomycin. An eluent that does not contain water prevents tacrolimus contamination facilitating the formation of its isomers. Furthermore, by avoiding the use of silver ions in the eluent, the contamination of the final product with silver ions is substantially reduced. The use of a strong acid resin, to which tacrolimus is sensitive, and the use of two-step chromatography can also be avoided in the process of the present invention.

Tacrolimus, which is present in a mixture with impurities, is charged in the silver-modified sorbent. The loading can be carried out, for example, by dissolving the mixture in a solvent, preferably a minimum amount of the solvent, which allows the dissolution, and charging the solution in the silver-modified sorbent as a concentrated solution. A minimum amount of the solvent may be approximately twice the volume of the solvent mixture calculated to start the tacrolimus mass. The solvent in which the mixture dissolves may be the same as that used during the elution. Examples of said solvents are given below. The mixture can be charged in other ways, including as an oil or as a solid residue obtained from the evaporation of a non-concentrated solution of the mixture.

The mixture formed by tacrolimus is then eluted with a solvent or solvents from the silver-modified sorbent. Preferably the solvent is substantially anhydrous. In one embodiment, a non-aqueous eluent is used which contains either polar organic solvent from the group consisting of C3-9 linear or branched ketone, C3-7 linear or branched ester, C1-7 linear or branched alcohol, C2-B linear or branched cyclic ether, C2-s nitrile, and mixtures thereof, or a mixture of a polar organic solvent and an apolar organic solvent selected from the group consisting of C5-8 linear or branched cyclic hydrocarbon, C6-io aromatic hydrocarbon and mixtures thereof. The eluents are preferably anhydrous, ie free of water, or have a water content that is low enough to avoid the formation of tacrolimus isomers. As used herein, the term "anhydrous" refers to eluents that have no water, except, possibly, as an impurity trace that is insufficient to result in the formation of a detectable amount of tacrolimus isomers. Preferably, the eluent contains less than 2% by weight, more preferably, less than 1% by weight, even more preferably, less than 0.05% by weight of water.

Preferably, C3-9 linear or branched ketone is acetone, ethylmethyl ketone, or isobutylmethyl ketone. Preferred linear or branched C3-7 esters include ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, and ethyl propionate. Ci-7 linear or branched alcohols include methanol, ethanol, isopropanol, n-propanol, n-butanol, and isobutanol. Preferred linear, branched or cyclic C2-B ethers include tert-butylmethyl ether, diethyl ether, diisopropyl ether, and tetrahydrofuran. A preferred C2-5 nitrile is acetonitrile. Preferably, C5-8 linear, branched or cyclic hydrocarbon is hexane, heptane, octane, cyclohexane, or cycloheptane. Preferably, the C6-io aromatic hydrocarbon is toluene. The preferred polar organic solvent is acetone. Preferably, the eluent contains acetone as a single solvent or a mixture with another solvent, and more preferably, the eluent contains acetone and hexane or ethyl acetate.

When a mixture of acetone and hexane is used, at least one of the following proportions of the solvent may be used: about 1: 1, about 2: 3, about 1: 4 or about 1: 9, respectively, depending on the sorbent being used . When a mixture of ethyl acetate and acetone is used, the proportion of the solvent is preferably about 50-80% acetone by volume, more preferably about 30:70 ethyl acetate to acetone by volume. Optionally, the elution of the tacrolimus comprises a gradient elution process, starting with the elution with a mixture of solvents, preferably acetone and hexane, preferably in a ratio of approximately 3: 2, followed by an elution with approximately 100 percent of a simple solvent, preferably acetone.

Preferably, the type of elusion process is chosen according to the length of the column. Preferably, with a short column, for example a column of approximately 10 cm in length, the elution gradient is preferred, and with a longer column, for example a column of approximately 1 m in length, no elution gradient is used.

In a preferred process of the invention, a column is packaged with a silver-modified sorbent which is then washed with eluent. A solution of tacrolimus in the eluent is then loaded, followed by elution of tacrolimus, ascomycin, and dihydrotacrolimus from the column, and recovery and analysis of the obtained samples. The eluent is preferably anhydrous.

Tacrolimus of any purity can be purified by the process of the invention and can contain at least one of the following impurities: ascomycin, commonly in an amount of about 0 to about 10 percent per area by HPLC, and / or dihydrotacrolimus, commonly in an amount of about 6 percent to about 0 percent per area by HPLC. Crude tacrolimus containing approximately 7.9 percent per area by HPLC of Ascomycin and / or approximately 3.8 percent per area by HPLC of dihydrotacrolimus has been purified by the process of the invention.

The order of circumvention depends on the nature of the sorbent and the eluent. Commonly, ascomycin and dihydrotacrolimus elute before tacrolimus, since said tacrolimus has greater affinity than the silver-modified sorbent.

Preferably, the aluminum oxide used for the preparation of the silver-modified sorbent is active, aluminum oxide acid, neutral active, or basic active or aluminum oxide standardized according to Brockmann, activity II-III. More preferably, aluminum oxide 90 standardized according to Brockmann or neutral active of aluminum oxide or acidic active are used.

A styrene divinylbenzene copolymer is nonionic divinylbenzene copolymer or anionic styrene divinibenze copolymer.

The cyano silica gel used for the preparation of silver-modified silica gel can be a commercial sorbent or can be prepared by treatment of silica gel with trichloro-3-cyanopropyl silane, resulting in silica gel covalently bound to its surface. a cyanoalkyl group. The most preferred sorbent for tacrolimus purification is silver modified aluminum oxide or silver modified cyano silica gel.

The sorbent adsorption resin that is preferably used has a small molecule size. The particle size can be 75 μp? at approximately 35 μta.

Preferably, for effective purification, the sorbent has a specific surface area of at least about 50 μl / more preferably about 50 about 600 m2 / g.

Preferably, the silver-modified sorbent is prepared by mixing the sorbent, a silver cation source, and a solvent selected from the group consisting of Ci_4 alcohol, water, and a mixture of water and a water-miscible solvent, and drying the resulting mixture . Preferably, when the solvent is a C1-4 alcohol, the silver cation source and Ci-4 alcohol are initially combined to obtain a solution, and the sorbent is then added to obtain a suspension. The resulting suspension is then dried, preferably, on a rotary evaporator, followed by oven drying in vacuum for about 15 minutes to about 5 hours.

The C1-4 alcohol preferably comprises a simple alcohol or a mixture of alcohols. The preferred C1-4 alcohol is methanol. Preferably, when the solvent is Ci-4 alcohol, the source of the silver ion and the C-alcohol are heated to about reflux temperature of the solvent, until a solution is obtained.

When the solvent is water or a mixture of water and a water-miscible solvent, and the sorbent is a reversed-phase silica gel, the silver ion source is preferably dissolved at room temperature, and, after dissolution, but before drying, a solution of ammonia is added. The treated solution is then dried, preferably, in an oven and, more preferably, at a temperature of about 70 ° C and at a pressure of about 30 mbar.

The inorganic silver salt or complex salt of inorganic silver can be used. The salt or complex can be contacted with the sorbent as a solution. Preferred silver salts include silver acetate, silver sulfate and silver nitrides. Preferred silver complexes include a palmetto amino complex, a cyano silver complex and a silver thiosulfate complex and silver acetate. More preferably, the silver source is silver nitrate.

The sorbent may comprise a different amount of silver ions. The silver-modified sorbent obtained after drying preferably contains about 0.1 percent to about 15 percent w / w silver salt and, more preferably, about 3 percent to about 13 percent w / w silver salt .

Preferably, the eluted fractions are recovered by collecting groups of fractions, and evaporating the solvent, thus, providing a sample. The recovered sample is then analyzed, preferably by HPLC, providing the content of tacrolimus, ascomycin, and dihydrotacrolimus in the sample. Commonly, when the elusion process progresses, the level of ascomycin and dihydrotacrolimus decreases while the level of tacrolimus increases. Preferably, the process is repeated, so that the final sample contains substantially pure tacrolimus. Preferably, the final sample contains at least about 93 percent by HPLC area of tacrolimus, more preferably, at least about 95 percent by HPLC area of tacrolimus, and, even more preferably, at least about 99 percent by area of HPLC.

Preferably, the final sample also contains less than about 0.20 percent by HPLC area of ascomycin, more preferably, less than about 0.09 percent by HPLC area, and, even more preferably, less than about 0.06. percent per HPLC area.

Preferably, the final sample also contains less than about 0.04 percent by HPLC area of dihydrotacrolimus, and, more preferably, less than about 0.03 percent by HPLC area of dihydrotacrolimus.

The column can be regenerated and used for further purification process.

The eluted tacrolimus may contain silver ions leaving the resin; the amount of said silver ions are substantially lower if an eluent containing silver ions is used. The eluted tacrolimus can be separated from the silver ions by evaporation of the eluent to obtain a residue; dissolving the residue in an organic solvent; mixing with a solution comprising a reagent that precipitates silver ions; filtering the precipitated silver salt. The reagent may be a chloride salt, such as, for example, sodium or ammonium chloride, the addition of which would result in precipitation of silver chloride. Another method for extracting silver ions is to pass the eluate through a column filled with gel modified by silica gel. Silica gel modified by sodium chloride can easily be prepared by mixing silica gel with an aqueous solution of sodium chloride.

Commonly, the organic solvent is a solvent in which the silver salts are immiscible. Preferably, the organic solvent is selected from the group consisting of: ethyl acetate, toluene, isopropyl ethyl acetate or normal, n-butyl acetate, isobutyl acetate, propionate, formate, acetone, ethyl methyl ketone, isobutyl methyl ketone, ethanol, propanol, n-butanol, iso-butanol, mixtures of ethyl acetate and acetone, mixtures of toluene and acetone, and mixtures thereof with water. Preferably, the solvent is ethyl acetate.

Preferably, the organic solvent is in an amount of about 2 volumes per gram of residue at about 30 volumes per gram of the residue, more preferably, from about 10 to about 30.

Commonly, reagents that precipitate silver ions form a silver salt that has a low solubility in water and in organic solvents. Preferably, the reagent includes a counting ion selected from the group consisting of acetate, sulfate, nitride, bromate, salicylate, iodate, chromate, carbonate, citrate, phosphate, chloride, stearate, sulfur, bromide, iodide, cyanide, benzoate, oxalate, sulfite, and thiocyanate. More preferably, the counting ion is carbonate, citrate, phosphate, chloride, stearate, sulfur, bromide, iodide, cyanide, benzoate, oxalate, sulfide, or thiocyanate. Even more preferably, the counting ion is bromide, iodide, stearate, sulfur, or cyanide. Preferably, the precipitating reagent is a salt comprising a counting ion. Even more preferably, the precipitating reagent is NH 4 Cl.

Preferably, the precipitation reagent is added in an amount of about 5 equivalent molecules per silver molecule, and more preferably, in an amount of about 1.2 to about 4 equivalent molecules per silver equivalent molecule. Preferably, the silver ion solution of reagent precipitation is an aqueous solution. Preferably, the aqueous solution is added to the solution of the residue in the organic solvent. The addition of the aqueous solution comprising a reagent to precipitate precipitation silver provides a system having at least two phases, including an organic phase, comprising the organic solvent, an aqueous phase and a silver salt precipitate. Preferably, the silver salt is silver chloride. The salt is then filtered, and the filtrate is passed through phase separation, yielding an organic phase comprising the eluted tacrolimus, substantially free of silver ions. Commonly, the term "free from" as used in reference to tacrolimus refers to tacrolimus containing less than 20 ppm silver ions. The level of the silver ions can be determined by any method known to an expert, for example, by the method described in pharmacopoeias of the United States of America.

The tacrolimus obtained directly from the column or followed by separation of silver ions can further be purified by a crystallization process, preferably comprising dissolving the final sample in a mixture of 2-propanol and n-heptane, followed by filtration. The filtrate is then preferably combined with a mixture of water and n-heptane, followed by cooling to obtain a precipitate. The precipitate is then preferably recovered by filtration and washing with water and then with n-heptane, followed by drying.

Preferably, before recovering the precipitate, the filtrate combined with the solvent mixture is maintained for about 24 hours.

The still more purified sample preferably contains tacrolimus which has a purity higher than that obtained with the chromatographic process. Commonly, after crystallization, the levels of any remaining ascomycin and dihydrotacrolimus decrease further, and tacrolimus levels decrease. Preferably, the purified sample contains at least 97 percent by HPLC area of tacrolimus, more preferably, at least about 99 percent by HPLC area or at least 99.5% and even more preferably, at least 99.9 percent per HPLC area.

Having thus described the invention with reference to particular preferred embodiments and illustrative examples, those skilled in the art will appreciate modifications to the invention as described and illustrated, which do not depart from the spirit or scope of the invention as disclosed in descriptive memory. The examples are set forth to help understand the invention but are not intended to limit and should not be considered as a limit to such scope in any way.

Examples HPLC Columns aters Symmetry C18 4,6 * 150 mm 3,5 μta Mobile phase; TO; Weigh 200 ml of acetonitrile in a jar volumetric of 2000 ml, fill the flask with distilled water, and then add 100 μ? 50 percent CH3COOH (pH = 4.0) B: Add 100 μ? 50% -os CH3COOH to 2000 ml of acetonitrile.

Gradient Table Flow: 2.2 ml / min Detection wavelength 200 nm Injection volume: 20 μ? Diluent: Acetonitrile Sample temperature 20 ° C Column temperature: 60 ° C Execution time: 47 minutes Typical retention time: Tacrolimus 24.5 min Tac. I. (Ascomycin) trt = 0.91 Tac. II. (Dihydro tacrolimus) trt = l, 35 Example 1; Preparation of alumina modified by silver nitrate Silver nitrate (10.0 g) was dissolved in hot methanol (500 ml), then adding alumina (basic active aluminum oxide 90, 63-200 / m, activity II-III, manufactured by Merck, 100 g) to get a suspension. The suspension was evaporated to dryness in a rotary evaporator. The residue was dried under vacuum (30 mbar) at 70 ° C for 5 hours. The silver content: 10 percent p / p.

Example 2% Purification of crude tacrolimus through alumina modified by silver, isocratic elution.

A glass column (diameter 22 mm, bed height 100 mm, dry fill) was filled with alumina modified with silver nitrate (40 g) prepared according to example 1, and the column was washed with the mobile phase (approximately 200 ml), which contains 50 percent (v / v) acetone and 50 percent (v / v) n-hexane. Raw tacrolimus (306 mg) containing, according to HPLC analysis, 79.7 percent tacrolimus, 7.9 percent ascomycin and 3.8 percent dihydrotacrolimus, was dissolved in the mobile phase (10 ml) , and the solution was loaded into the column. The column was eluted by the mobile phase (approximately 20 ml / min), and 30 ml of fractions were collected. The fractions were analyzed by HPLC, and the reconstructed chromatogram was presented in Figure 1. The first four fractions, representing separated ascomycin and dihydrotacrolimus, were concentrated to dryness. The residue (52 mg) contained 2.1 percent tacrolimus, 31.7 percent dihydrotacrolimus, and 63.0 percent ascomycin, according to HPLC analysis. The next three Inter-fractions were concentrated to dryness (15 mg), and according to HPLC analysis, they contained 37.5 percent tacrolimus. The following fractions, containing purified tacrolimus (15 fractions), were concentrated providing 223 mg of residue that was analyzed by HPLC, and showed to contain 95.54 percent tacrolimus, 0.20 percent ascomycin, and 0.04 percent one hundred dihydrotacrolimus.

Example 3; Purification of crude tacrolimus by silver-modified alumina, gradient elution The column after Example 2 was washed with a mobile phase consisting of 40 percent (v / v) acetone and 60 percent (v / v) n-hexane . Raw tacrolimus (321 mg, 79.7 percent tacrolimus, 7.9 percent ascomycin, and 3.8 percent dihydrotacrolimus) was dissolved in the mobile phase (10 ml) and then loaded onto the column. The elution was carried out with the mobile phase (350 ml) containing 40 percent acetone and 60 percent n-hexane (v / v), and the eluate was concentrated to dryness, yielding 59 mg of residue, which contains, according to HPLC analysis, 3.3 percent of tacrolimus, 34.5 percent of dihydrotacrolimus and 67.0 percent of ascomycin. The column was then eluted with acetone (250 ml), and the eluate was concentrated to provide 218 mg of dry residue, which contains 95.7 percent tacrolimus, 0.09 percent ascomycin, and 0.03 percent dihydrotacrolimus.

Example 4; Purification of crude tacrolimus by substitution chromatography of tacrolimus with modified alumina and eluent of acetone Tacrolimus (3.8 g) was dissolved in acetone (15.6 ml). The solution was passed through a bed of alumina sorbent modified by silver (containing about 10 percent silver w / w), which has a height of 85 cm that was placed on a glass column, which has a diameter of 3. , 2 cm. The column was then eluted with acetone. The average elution was 30 ml / hour. One fraction contained 0.3 percent dihydrotacrolimus, 0.45 percent ascomycin, and 93.0 percent tacrolimus.

Example 5s Purification of crude tacrolimus by substitution chromatography of tacrolimus with modified alumina and eluent of acetone Raw tacrolimus (2 g) containing, according to the HPLC analysis, 1.2 percent of ascomycin and 1.8 percent of dihydrotacrolimus, was dissolved in acetone (20 ml). The solution was passed through a bed of alumina sorbent modified by silver (200 g, containing about 10 percent silver w / w). The sorbent bed was then eluted with acetone, and fractions of 50 ml were collected and analyzed by HPLC. The reconstructed chromatogram was presented in figure 2. The first three fractions containing macrolides were concentrated, providing 0.41 g of dry residue, which contains, according to HPLC analysis, 6.1 percent ascomycin and 9.4 percent of dihydrotacrolimus. The following seven fractions were concentrated, yielding 1.53 g of dry residue, which contains, according to HPLC analysis, 0.18 percent ascomycin and 0.06 percent dihydrotacrolimus.

Example 6; Purification of crude tacrolimus by substitution chromatography of tacrolimus with modified alumina and acetone mixture of hexane eluent. Tacrolimus (3.8 g) was dissolved in acetone (15.6 ml). The solution was passed through a bed of neutral active alumina sorbent modified by silver (containing approximately 10 percent silver w / w), which has a height of 85 cm that was placed on a glass column of 3.2 cm diameter. The sorbent bed was then eluted with acetone and ethyl acetate in an acetone: ethyl acetate ratio of 70:30. The average elution was 90 ml / hour. One fraction contained 0.07 percent dihydrotacrolimus, 0.39 percent ascomycin, and 94.7 percent tacrolimus.

Example 7; Preparation of active acid alumina modified by silver nitrate Silver nitrate (75.0 g) was dissolved in hot methanol (1400 ml), followed by the addition of alumina (active acid aluminum oxide 90, 63 to 200 μt, activity I, manufactured by Merck, 750 g) to obtain a suspension. The suspension was evaporated to dryness in a rotary evaporator. The residue was dried under vacuum. The residue had a silver content of 10 percent w / w. Example 8: Purification of Tacrolimus by chromatography with active acidic alumina modified by silver Tacrolimus (6.0 g) containing, according to the HPLC analysis, 1.03 percent ascomycin and 1.85 percent dihydrotacrolimus, was dissolved in a solvent mixture of acetone and ethyl acetate (24 ml) in a ratio of acetone: ethyl acetate of 70:30. The solution was passed through a bed of activated acid alumina sorbent modified by silver (containing approximately 10 percent silver w / w), which has a height of 85 cm which was placed on a 3.2 cm glass column diameter. The column was eluted at -5 ° C with acetone and ethyl acetate in a ratio of acetone: ethyl acetate of 70:30. The average elution was 90 ml / hour. The fractions were collected and analyzed by HPLC. The main fraction chosen evaporated and crystallized. According to the HPLC analysis, the product contained 96.25 percent tacrolimus, 0.22 percent ascomycin, and 0.04 percent dihydrotacrolimus.

Example 9s Crystallization of Tacrolimus The residue obtained by evaporation of the tacrolimus fractions after chromatography on a silver-modified alumina (55.3 g) was dissolved in a mixture of 2-propanol (150 ml) and n-heptane (150 ml). ), followed by filtration of the solution. The filtrate was then diluted with water (250 ml) and n-heptane (250 ml), and the resulting mixture was cooled and stirred for 24 hours. The crystalline precipitated product was filtered, washed with water and n-heptane, and dried. 47.8 g crystalline tacrolimus was obtained. According to the HPLC analysis, the product contained 99.1 percent tacrolimus, 0.33 percent tacrolimus tautomer II, 0.18 percent tacrolimus tautomer I, 0.11 percent ascomycin, and 0.04 percent percent of dihydrotacrolimus.

Example 10: Modification of active alumina Silver nitrate (20 g) was dissolved in heated methanol (650 ml). Aluminum oxide (750 g) (active neutral, Merck, 0.063-0.200 mm, stage I activity) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness. The silver content of the dry suspension was 2.5 percent w / w.

Example 11: Modification of Reverse Phase Silica Gel Silver nitrate (20 g) was dissolved in heated methanol (650 ml). Reverse phase silica gel (330 g) (LiChroprep RP-18, Merck) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 12: Modification of resin adsorption Silver nitrate (30 g) was dissolved in heated methanol (650 ml). The adsorption resin XAD 1180 (650 g) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 13: Modification of adsorption resin Silver nitrate (30 g) was dissolved in heated methanol (650 ml) Adsorption resin SP 207 (650 g) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 14: Modification of the adsorption resin having small particle size Silver nitrate (30 g) was dissolved in heated methanol (650 ml). The Amberchrom CG 300 M adsorption resin (650 g) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 15s Modification of the adsorption resin having small particle size Silver nitrate (30 g) was dissolved in methanol (650 ml) by heating to reflux temperature. The adsorption resin Amberchrom CG 300 S (650 g) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 16: Modification of cation exchange resin. Silver nitrate (30 g) was dissolved in heated methanol (650 ml). The cation exchange resin SK 104 (650 g) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 17: Modification of anion exchange resin. Silver nitrate (30 g) was dissolved in heated methanol (650 ml). The anion exchange resin IRA 68 (650 g) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 18s Modification of Reverse Phase Silica Gel Silver nitrate (20 g) was dissolved in water (500 ml) at room temperature. The 40 ml concentrated ammonia solution was added to the silver nitrate solution in several portions in the presence of 330 g reverse phase silica gel (LiChroprep RP-18, Merck). The solution was carefully concentrated under high vacuum to dryness.

Example 19; Modification of anion exchange resin Silver nitrate (30 g) was dissolved in heated methanol (650 ml). Anion exchange resin IRA 900 (650 g) was added to the solution to obtain a suspension. The suspension was concentrated under reduced pressure to dryness.

Example 20: Cyano silica gel modified by silver nitrate Silver nitrate (10.0 g) was dissolved in hot methanol (500 ml) and silica cyano gel (100 CN silica gel, 15-35 μ, manufactured by Fluka, 100 g) was added to the solution, to obtain a suspension. The suspension was evaporated to dryness in a rotary evaporator. The residue was dried under vacuum (30 mbar) at 70 ° C for 5 hours.

Example 21: Purification of crude gel tacrolimus by silver modified silica cyano A chromatographic column (diameter 25 mm (250 mm in length) was filled with the sorbent prepared in Example 18 (54.4 g), and the column was washed with the mobile phase (mixture of acetone and n-hexane 25:75, 1000 ml), then raw tacrolimus (510 mg) (79.7 percent tacrolimus, 7.9 percent ascomycin and 3.8 percent of dihydrotacrolimus) was dissolved in the mobile phase (25 ml), loaded onto the column, and the column was eluted with the mobile phase.Fractions of 100 ml each were taken and analyzed by HPLC.The reconstructed chromatogram is shown in Figure 3 Example 22: Silica gel modified by silver nitrate Silver nitrate (10.0 g) was dissolved in hot methanol (500 ml) and silica gel (silica gel 60 15-40 μp ?, manufactured by Merck, 100 g) was added to the solution, to obtain a suspension . The suspension was evaporated to dryness in a rotary evaporator. The residue was dried under vacuum (30 mbar) at 70 ° C for 8 hours.

Comparative Example 23: Purification of crude tacrolimus by silver-modified silica gel A chromatographic column (diameter 25 mm, 250 mm in length) was filled with the sorbent prepared in Example 26 (62.6 g), and the column was washed with the mobile phase (mixture of acetone and n-hexane 20:80, 1000 ml). Then the crude tacrolimus (500 mg) (79.7 percent tacrolimus, 7.9 percent ascomycin and 3.8 percent dihydrotacrolimus) was dissolved in the mobile phase (25 ml), loaded into the column, and the column was eluted with the mobile phase. Fractions of 100 ml each were taken and analyzed by HPLC. The reconstructed chromatogram is shown in Figure 5.

Example 24; Zirconium oxide modified by silver nitrate Silver nitrate (20.0 g) was dissolved in hot methanol (1000 ml), and zirconium oxide (manufactured by Merck, 200 g) was added to the solution to obtain a suspension. The suspension was evaporated to dryness in a rotary evaporator. The residue was dried under vacuum (30 mbar) at 70 ° C for 5 hours.

Example 25s Purification of crude tacrolimus oxide by silver-modified zirconium A chromatographic column (diameter 25 mm, 250 mm in length) was filled with the sorbent prepared in Example 20 (345 g) (the sorbent preparation was repeated), and the column was washed with the mobile phase (approximately 1000 ml) (mixture of acetone and n-hexane 10: 90). Then, crude tacrolimus (210 mg) (79.7 percent tacrolimus, 7.9 percent ascomycin, and 3.8 percent dihydrotacrolimus) was dissolved in the mobile phase (25 ml), and loaded into the column, and the column was eluted with the mobile phase. Fractions of 100 ml each were collected and analyzed by HPLC. The reconstructed chromatogram is shown in Figure 4.

Example 26: Separation of Tacrolimus from silver ions The main fraction chosen according to Examples 6 and 8 was evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (20 times the volume of the residue). NH4C1 (1.3 times the mass of the residue) was dissolved in water (5 times the volume of the residue), and the salt solution was added to the ethyl acetate solution. After 30 minutes of stirring, the precipitated silver chloride was filtered, and the phases separated. The macrolide was crystallized from the ethyl acetate phase.

Example 27 Separation of Tacrolimus from silver ions The eluate from the column was found to contain approximately 80 mg / 1 silver. The eluate was passed through a column filled with silica gel modified by sodium chloride. The silver was completely retained in the column. The silver content was less than 10 ppm. The modified silica gel was prepared by adding an aqueous solution of sodium chloride to a silica gel followed by homogenization.

Claims (54)

REIVI DICACIONES

1. A process for separating Tacrolimus from impurities comprising: a) charging a mixture comprising tacrolimus and impurities in a sorbent bed pre-treated with silver ions, characterized in that the sorbent is selected from the group consisting of aluminum oxide modified by silver, zirconium oxide, styrene-divinylbenzene copolymer, resin of adsorption, cation exchange resin, anion exchange resin, reverse phase silica gel and cyano silica gel; Y b) bypass the mixture from the bed of the sorbent resin to separate the Tacrilomus from the impurities present in the mixture.

2. The process according to claim 1, characterized in that the mixture is eluted with an anhydrous eluent.

3. The process according to any of claims 1-2, characterized in that the impurities are Ascomycin and dihydrotacrolimus.

4. The process according to any of claims 1-3, characterized in that the anhydrous eluent is selected from the group consisting of polar organic solvent and mixtures of the polar organic solvents and apolar organic solvents.

5. The process according to claim 4, characterized in that the polar organic solvent is selected from the group consisting of C3-9 linear or branched ketones, C3-7 linear or branched esters, Ci-7 linear or branched alcohols, C2-8 linear, branched or cyclic ethers, C2-5 nitriles, and mixtures thereof.

6. The process according to claim 4, characterized in that the apolar organic solvent is selected from the group consisting of C5-8 linear, branched or cyclic hydrocarbon, C6-io aromatic hydrocarbon and mixtures thereof.

7. The process according to any of claims 2-6, characterized in that the anhydrous eluent has less than about 2% water by volume.

8. The process according to any of claims 2-7, characterized in that the anhydrous eluent is selected from the group consisting of acetone, ethyl-methyl ketone, iso-butyl methyl ketone, ethyl acetate, n-propyl acetate, isopropyl, n-butyl acetate, ethylpropionate, methanol, ethanol, isopropanol, n-propanol, n-butanol, iso-butanol, tert-butyl methyl ether, diethyl ether, diisopropyl ether, tetrahydrofuran, acetonitrile, toluene, and linear, branched or cyclic hexane , heptane, octane, cyclohexane, and cycloheptane.

9. The process according to claim 8, characterized in that the anhydrous eluent comprises acetone.

10. The process according to claim 8, characterized in that the anhydrous eluent comprises hexane or ethyl acetate.

11. The process according to claim 10, characterized in that the anhydrous eluent comprises acetone and hexane, characterized in that acetone and hexane are in an acetone: hexane ratio of about 1: 1, about 2: 3, about 1: 4, or about 1: 9.

12. The process according to claim 9, characterized in that the eluent is a mixture of acetone and ethyl acetate, and acetone and ethyl acetate are present in about 50-80% acetone by volume as compared to ethyl acetate.

13. The process according to claim 2, characterized in that the anhydrous eluent is acetone.

14. The process according to any of claims 2-13, characterized in that Tacrolimus is eluted with an elution gradient, starting with elution with a mixture of solvents followed by elution with approximately 100 percent of a simple solvent.

15. The process according to claim 14, characterized in that the elution of the gradient comprises starting the elution with a mixture comprising acetone and hexane, followed by elution with acetone.

16. The process according to claim 15, characterized in that acetone and hexane are in an acetone: hexane ratio of about 3: 2 v / v.

17. The process according to any of claims 1-16, characterized in that the mixture elutes with a gradient elution with a column of approximately 10 cm or less in length.

18. The process according to any of claims 1-16, characterized in that the mixture is eluted without gradient elution with a column of at least about 1 m in length.

19. The process according to any of claims 1-18, characterized in that the process comprises filling a column with silver-modified sorbent, washing the silver-modified sorbent with an eluent, charging a tacrolimus solution into the eluent in the column, and elude tacrolimus, Ascomycin and Dihydrotacrolimus from the Column.

20. The process according to any of claims 1-19, characterized in that at least one of Ascomycin and Dihydrotacrolimus are eluted from the column before Tacrolimus.

21. The process according to any of claims 1-19, characterized in that the Tacrolimus before separation, comprises up to about 10 percent by HPLC area of Ascomycin and / or up to about 6 percent by HPLC area of Dihydrotacrolimus.

The process according to any of the claims, characterized in that the sorbent is aluminum oxide.

23. The process according to claim 22, characterized in that the aluminum oxide sorbent is an active form, selected from the group consisting of acid, neutral and basic aluminum oxide.

24. The process according to claim 23, characterized in that the aluminum oxide sorbent is neutral active aluminum oxide or acid active aluminum oxide.

25. The process according to any of claims 1-21, characterized in that the sorbent is a copolymer of styrene divinylbenzene, selected from the group consisting of copolymer of non-anionic styrene divinylbenzene and anionic styrene divinylbenzene copolymer.

26. The process according to any of claims 1-25, characterized in that the sorbent is an adsorption resin having a particle size of about 35 μp? or about 75 μ? .

27. The process according to any of claims 1-26, characterized in that the sorbent has a specific surface area of at least about 50 m2 / g.

28. The process according to any of claims 1-27, further comprises preparing the modified sorbent for silver in a process comprising mixing the sorbent, a silver cation source and a solvent selected from the group consisting of one or more Ci-4 alcohols, water, and a mixture of water and a solvent miscible with water, and dry.

29. The process according to any of claims 1-21, characterized in that the sorbent is cyano silica gel.

30. The process according to claim 29, characterized in that the cyano silica gel comprises silica gel covalently bonded on the surface to the cyanoalkyl group.

31. The process according to claim 28, characterized in that the solvent is Ci-4 alcohol, and the cation source of silver and Ci-4 alcohol combine to form a solution, and the sorbent is then added to form a suspension.

32. The process according to claim 31, characterized in that the C1-alcohol is methanol.

33. The process according to claim 30, characterized in that the solvent is Ci-4 alcohol, and the source of the silver ion and Ci-4 alcohol are heated to approximately reflux temperature of the solvent to obtain a solution.

34. The process according to claim 28, characterized in that the solvent is water or a mixture of water and a water miscible solvent, the sorbent is a reverse phase silica gel, the silver ion source is dissolved at room temperature, and before drying, an ammonia solution is added to the solvent solution and the silver cation source.

35. The process according to claim 28, characterized in that the source of silver cations is a silver salt solution or a silver complex.

36. The process according to claim 28, characterized in that the source of silver cations is selected from the group consisting of silver nitrate, silver acetate, silver sulfate, a cyano silver complex and a silver thiosulfate complex.

37. The process according to claim 35, characterized in that the source of silver cations comprises at least one silver nitrate and one silver acetate.

38. The process according to claim 35, characterized in that the source of silver cations comprises a complex of silver cyano.

39. The process according to claim 28, characterized in that the silver-modified sorbent, after drying, comprises about 0.1 to about 15 percent w / w silver salt.

40. The process according to any of claims 1-39, characterized in that the separated Tacrolimus comprises at least about 93 percent by HPLC area of Tacrolimus.

41. The process according to any of claims 1-39, characterized in that the separated Tacrolimus comprises less than about 0.20 percent by HPLC area of Ascomycin

42. The process according to any of claims 1-39, characterized in that the separated tacrolimus comprises less than about 0.04 percent per HPLC area of Dihydrotacrolimus.

43. The process according to any of claims 1-39, further comprises separating any silver ion eluded with tacrolimus from Tacrolimus.

44. The process according to any of claims 1-43, which comprises evaporating the eluent to obtain a residue, dissolving the residue in an organic solvent, mixing the resulting solution with a solution comprising a reagent that precipitates silver ions, and filtering the precipitated silver salt.

45. The process according to claim 44, characterized in that the organic solvent is selected from the group consisting of: ethyl acetate, toluene, isopropyl ethyl acetate or normal, n-butyl acetate, isobutyl acetate, propionate, formate, acetone, ethyl methyl ketone, isobutyl methyl ketone, ethanol, propanol, n-butanol, iso-butanol, mixtures of ethyl acetate and acetone, mixtures of toluene and acetone, and mixtures thereof with water.

46. The process according to claim 45, characterized in that the organic solvent is ethyl acetate.

47. The process according to claim 44, characterized in that the reagent comprises an ion count selected from the group consisting of acetate, sulfate, nitride, bromate, salicylate, iodate, chromate, carbonate, citrate, phosphate, chloride, stearate, sulfur, bromide , iodide, cyanide, benzoate, oxalate, sulfite, and thiocyanate.

48. The process according to claim 47, characterized in that the precipitating reagent is NH 4 Cl.

49. The process according to claim 44, further comprises adding the precipitating reagent in an amount of 1 to about 5 equivalent molecules per equivalent molecule of silver.

50. The process according to claim 44, characterized in that the solution comprising a reagent that precipitates the silver ions is an aqueous solution.

51. The process according to claim 44 comprises passing the eluate through a column filled with a mixture of silica gel and a precipitating reagent.

52. The process according to claim 44, characterized in that the precipitating reagent is sodium chloride or ammonium chloride.

53. The process according to any of claims 1-52, further comprises collecting at least one effluent fraction having tacrolimus.

54. The process according to claim 53, further comprises crystallizing the separated tacrolimus.