CN101068818A - Process for the preparation of pimecrolimus - Google Patents

Abstract

The present invention provides a method for preparing pimecrolimus from ascomycin, wherein ascomycin is reacted with a conversion reagent to convert it into an activated C-32 derivative. The activated ascomycin is then reacted with chloride ion. The method of the present invention requires fewer steps than prior art methods and does not require protection of the C-24 hydroxyl group of the ascomycin or purification of the activated ascomycin derivative.

Description

Process for the preparation of pimecrolimus

related application

[0001] This application claims the benefit of the following U.S. provisional patent applications: 60/632,372 filed December 1, 2004, 60/633,926 filed December 6, 2004, 60/641,697 filed January 5, 2005 , 60/641,868 filed January 5, 2005, 60/641,869 filed January 5, 2005, 60/662,440 filed March 16, 2005, 60/705,681 filed August 3, 2005, and 2005 60/709,160, filed August 17, 1999, the entire contents of which are hereby incorporated by reference in their entirety.

field of invention

[0002] The present invention relates to a process for the preparation of the anti-inflammatory compound pimecrolimus and to pure pimecrolimus.

Background of the invention

[0003] Pimecrolimus is an anti-inflammatory compound derived from the macrolide natural product ascomycin produced by certain strains of Streptomyces. Pimecrolimus is sold in the United States under the trade name ELTJDEL(R) and is approved for the treatment of atopic dermatitis. The systematic designation of pimecrolimus is (1R, 9S, 12S, 13R, 14S, 17R, 18E, 21S, 23S, 24R, 25S, 27R)-12-[(1E)-2-{(1R, 3R, 4S )-4-chloro-3-methoxycyclohexyl}-1-methylvinyl]-17-ethyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21, 27-Tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.04,9]octacos-18-ene (octacos-18-ene)-2,3,10, 16-tetraketone. Pimecrolimus is a 32-epichloro derivative of ascomycin. Its empirical formula is C ₄₃ H ₆₈ ClNO ₁₁ , and its molecular weight is 810.47.

[0004] European Patent EP 427 680 B1 discloses a method for the synthesis of pimecrolimus in the form of a colorless foamy resin. The disclosed method comprises the following four reaction steps:

(a) two hydroxyl groups of C-24 and C-32 are protected with tert-butyldimethylsilyl ether;

(b) deprotecting the C-32 hydroxyl group protected by the silyl group, and the hydroxyl group at the C-24 position remains protected;

(c) chlorine substitution of the free hydroxyl group at C-32, with a configurational shift; and

(d) deprotecting the silyl-protected C-24 hydroxyl group.

[0005] EP 427 680 does not disclose the yield of each step in the synthesis, but discloses that the product of this step is purified by chromatography after each step. Therefore, the overall yield of the process disclosed in EP 427 680 may be expected to be low from the number of reaction steps and the number of chromatographic purifications required.

[0006] Accordingly, a process for the preparation of pimecrolimus with fewer steps, particularly chromatographic steps, would be advantageous. The present invention provides such a method.

Brief description of the invention

The present invention provides the method for synthesizing pimecrolimus, described method comprises that ascomycin is dissolved in organic solvent, ascomycin solution is mixed with alkali, obtains reaction mixture, reaction mixture and transformation reagent (conversion reagent) are mixed to obtain activated ascomycin derivatives, and the activated ascomycin derivatives are mixed with a chloride ion source until pimecrolimus is obtained, and the pimecrolimus is recovered.

[0008] Preferably, the pimecrolimus obtained by the above method is purified by column chromatography.

[0009] Preferably, the purified pimecrolimus is at least about 95% pure by HPLC peak area, more preferably at least about 98% pure by HPLC peak area.

[00010] The present invention also provides pimecrolimus having a purity of at least about 95% by HPLC peak area, more preferably at least about 98% pure by HPLC peak area.

[0001] In another embodiment of the present invention, there is provided a pharmaceutical preparation comprising a therapeutically effective amount of the above pure pimecrolimus.

[0002] In another embodiment of the present invention, there is provided a method of treating a patient suffering from atopic dermatitis, said method comprising the step of administering to the patient the aforementioned pharmaceutical formulation.

Brief description of the drawings:

Figure 1: Chromatogram of pimecrolimus.

Detailed description of the invention

The present invention provides the method for synthesizing pimecrolimus, described method comprises that ascomycin is dissolved in organic solvent, ascomycin solution is mixed with alkali, obtains reaction mixture, and reaction mixture is mixed with conversion reagent to obtain Activated ascomycin derivatives, the activated ascomycin derivatives are mixed with a chloride ion source until pimecrolimus is obtained, and crude pimecrolimus is recovered. Optionally, the hydroxyl group at the C-24 position of ascomycin is not protected during the process. Unlike the method described in EP 427 680, selective protection of the C-24 hydroxyl group of ascomycin is not necessary because the C-32 hydroxyl group is position-selectively active (the C-24 hydroxyl group remains intact). The transformation reagent converts the ascomycin to an active ascomycin derivative at the C-32 position.

[0004] Preferably, the activated ascomycin derivatives are sulfonate, more preferably tosylate or mesylate, most preferably trifluoromethylsulfonate.

Preferably, described organic solvent is selected from: methylene chloride, chloroform, ether, diisopropyl ether, methyl tert-butyl ether, toluene, ethyl acetate, isobutyl acetate, acetone, methyl ethyl ketone , acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and mixtures thereof. More preferably, the organic solvent is toluene or acetonitrile. Preferably, the ascomycin is dissolved in the organic solvent at a temperature above about 25°C. The resulting ascomycin solution is then stirred under an inert gas such as nitrogen at a low temperature below about 25°C, preferably below about 0°C, more preferably below about -20°C. Low temperature is necessary to react selectively and obtain less by-products. Stirring is preferably continued sufficiently to substantially dissolve all of the ascomycin.

[0006] Preferably, the amount of the base is from about 1 to about 4 equivalents. The base can be added dropwise, batchwise or all at once. The base may be an organic or inorganic base. Preferably, the base is selected from triethylamine, diisopropylethylamine (EDIPA), N-methyl-morpholine, N,N-dimethylaniline, pyridine and substituted pyridine derivatives such as 2 , 6-lutidine, 2,4,6-collidine and 4-dimethylaminopyridine. More preferably, the base is selected from the group consisting of: diisopropylethylamine (EDIPA), 2,4,6-collidine and 2,6-lutidine. The base can also be added to the ascomycin solution in the next step along with the activated transformation reagent, as described below. The added base can be in the form of a solution, wherein the solvent is preferably selected from the group consisting of dichloromethane, chloroform, ether, diisopropyl ether, methyl tert-butyl ether, toluene, ethyl acetate, isobutyl acetate, acetone, methyl Ethyl ketone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, n-hexane, n-heptane, cyclohexane and mixtures thereof. More preferably, the solvent is toluene. Preferably, the base is added directly (under neat conditions).

The addition of a base is preferred to prevent an increase in the acidity of the reaction mixture since, during the course of the reaction, the activator will form an acid which, in the absence of base, will acidify the reaction mixture. For example, when the activator is trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid is formed during the reaction, and when the activator is an acid chloride such as trifluoromethanesulfonyl chloride, HCl is formed during the reaction. If no base is added, the formation of acid in the reaction mixture will increase the acidity of the reaction process, slow down the reaction rate and degrade the macrocycle. Therefore, it is preferred to add a base to the reaction mixture to neutralize the acid formed during the reaction.

[0007] After adding the base dropwise, batchwise or all at once or simultaneously, the reaction mixture is mixed with the activated transformation reagent. Preferably, the progress of the reaction is monitored, eg by thin layer chromatography (TLC), until the reaction is complete or nearly complete.

Preferably, described transformation reagent is selected from: fluorosulfonic anhydride, fluorosulfonyl chloride, trifluoromethanesulfonic anhydride, trifluoromethanesulfonyl chloride, methanesulfonic anhydride, methanesulfonyl chloride, phenylmethanesulfonic anhydride, phenylmethanesulfonic anhydride Sulfonyl chloride, p-toluenesulfonic anhydride, p-toluenesulfonyl chloride, benzenesulfonic anhydride, and benzenesulfonyl chloride. More preferably, the transformation reagent is selected from: trifluoromethanesulfonic anhydride and trifluoromethanesulfonyl chloride. The transformation reagent added may be in solution, wherein the solvent is selected from the same solvents used to form the base solution, as described above. Preferably, the solvent is selected from the group consisting of toluene, n-hexane, n-heptane and cyclohexane.

The time required for complete or almost complete conversion of ascomycin to the corresponding activated ascomycin such as ascomycin 32-triflate may vary depending on reaction conditions such as temperature, solvent , base and activator used. Those skilled in the art will know how to monitor the reaction, eg by TLC at appropriate time intervals depending on the conditions chosen. As a non-limiting example, when trifluoromethanesulfonic anhydride is used as the activator, the reaction occurs almost instantaneously, even at -40°C, provided that there is a sufficient amount of base in the reaction mixture, i.e., about 3 to about 4 equivalent. In contrast, for example, when p-toluenesulfonyl chloride is used as an activator, 3-4 equivalents of base are required for 1-3 hours at 0°C, and at least one day is required for 1 equivalent of base.

[00010] After adding the activated transformation reagent, the reaction mixture is mixed with a solution of the chloride ion source. Preferably, the chloride ion source solution is added to the reaction mixture. Effective sources of chloride ions include, but are not limited to, lithium chloride, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, aluminum chloride, iron(II) chloride, iron(III) chloride, ammonium chloride, organic Hydrochlorides of bases, quaternary ammonium chlorides, quaternaryphosphonium chlorides, tetrabutylammonium chloride, benzyl-triethylammonium chloride and similar quaternary ammonium chlorides and Hydrochlorides of the bases discussed above. Preferably, the source of chloride ions is benzyltriethylammonium chloride. Solvents for the source of chloride ions include those discussed above for the base and activator. Alternatively, the source of chloride ions is neatly added to the reaction mixture.

[00011] After adding the source of chloride ions, the reaction is stirred at a temperature above about 0°C but below the reflux temperature of the solvent or solvent mixture used in the reaction mixture. Preferably, the temperature is at least about 25°C. The progress of the reaction is again monitored to confirm completion of the reaction, eg, by TLC.

[00012] The time of disappearance of intermediate activated ascomycin derivatives such as 32-trifluoromethylsulfonate can vary depending on the precise conditions used, mainly depending on the reaction temperature and the selected chloride ion source. Solubility. Typically, the time is from about 1 hour to 1 day at room temperature. At lower temperatures and/or with poor solubility of the chloride ion source, the reaction is significantly slower. Longer reaction times are not beneficial because they increase the possibility of formation of unwanted by-products.

[00013] The pimecrolimus recovery step comprises: adding water together with a water-insoluble organic solvent to obtain a two-phase system; separating the two-phase system; using KHSO _aqueous solution, _NaHCO solution and brine to extract the organic phase, and concentrate the organic phase; and dry.

[00014] Or the recovery step comprises: adding water to the reaction mixture to obtain a biphasic system; separating the biphasic system; and concentrating the organic phase.

[00015] High vacuum was used in the final stage of concentration to obtain the crude product as an amorphous solid.

[00016] Preferably, the crude product is purified by column chromatography prior to the drying step. The solution containing the crude product was concentrated and placed on a chromatography column. After chromatographic elution the pure fractions were combined and concentrated to give an amorphous solid.

[00017] Preferably, the resulting pimecrolimus after the purification step is at least about 95% pure by HPLC peak area, more preferably at least about 98% pure by HPLC peak area.

[00018] In one embodiment, the invention comprises pimecrolimus having a purity of at least about 95% by HPLC peak area, more preferably at least about 98% pure by HPLC peak area.

[00019] Another embodiment of the present invention is a pharmaceutical formulation comprising a therapeutically effective amount of the above pure pimecrolimus and an amount of a pharmaceutically acceptable excipient.

[00020] "Therapeutically effective amount" means that amount of pure pimecrolimus that, when administered to a patient for treating a disease or other undesired medical condition, has a sufficient effective effect on the disease or condition. The "therapeutically effective amount" varies depending on the purity, the disease or condition being treated and its severity, the age, weight, etc. of the patient being treated. Determining a therapeutically effective amount of a given pure pimecrolimus is within the purview of one of ordinary skill in the art without further undue experimentation.

[00021] Another embodiment of the present invention is a method of treating a patient suffering from atopic dermatitis, said method comprising the step of administering to said patient a pharmaceutical formulation comprising a therapeutically effective amount of pure pimecrolimus produced by the present invention . A further embodiment of the present invention is a method of providing immunosuppression to a patient in need thereof, said method comprising the step of administering to said patient a pharmaceutical formulation comprising a therapeutically effective amount of pure pimecrolimus produced by the present invention.

[00022] The pharmaceutical formulation of the invention comprises pure pimecrolimus produced by the method of the invention. The pharmaceutical formulations of the present invention may contain, in addition to the active ingredient, one or more excipients. Excipients are added to formulations for various purposes.

[00023] Diluents may be added to the formulations of the invention. Diluents increase the bulk of the solid pharmaceutical composition and facilitate the administration of pharmaceutical dosage forms comprising the composition by patients and caregivers. Diluents for solid compositions include, for example, microcrystalline cellulose (such as AVICEL(R), microfine cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sucrose, dextrates , dextrin, dextrose, calcium hydrogen phosphate dihydrate, tricalcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylate (such as EUDRAGIT®), potassium chloride, powdered fiber Sodium Chloride, Sorbitol and Talc.

[00024] Solid pharmaceutical compositions that are compressed into dosage forms such as tablets may include excipients to help hold the active ingredient and other excipients together after compression. Binders for solid pharmaceutical compositions include gum arabic, alginic acid, carbomers (such as carbopol), sodium carboxymethylcellulose, dextrin, ethylcellulose, gelatin, guar gum, hydrogenated Vegetable oil, hydroxyethylcellulose, hydroxypropylcellulose (such as KLUCEL(R), hydroxypropylmethylcellulose (such as METHOCEL(R), liquid dextrose, magnesium aluminum silicate, maltodextrin, methylcellulose, poly Methacrylates, povidone (eg KOLLIDON(R), PLASDONE(R), pregelatinized starch, sodium alginate and starch.

[00025] A disintegrant can be added to the composition to increase the dissolution rate of the compressed solid pharmaceutical composition in the patient's stomach. Disintegrants include alginic acid, calcium carboxymethylcellulose, sodium carboxymethylcellulose (such as AC-DI-SOL(R), PRMELLOSE(R), colloidal silicon dioxide, croscarmellose sodium, cross-linked polymer Vitone (eg, KOLLIDON®, POLYPLASDONE®), guar gum, magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, pocridine potassium, powdered cellulose, pregelatinized starch, sodium alginate, Sodium starch glycolate (eg EXPLOT AB(R)) and starch.

[00026] Glidants can be added to improve the flowability of non-compressed solid compositions and to increase the accuracy of dosage. Excipients that can be used as glidants include colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc and tricalcium phosphate.

[00027] When dosage forms such as tablets are made from compressed powder compositions, the compositions are subjected to the pressure of punches and dies. Some excipients and active ingredients can adhere to the surface of punches and dies, which can cause products to have pitting and other surface irregularities. Lubricants may be added to the composition to reduce sticking and to facilitate release of the product from the die. Lubricants include magnesium stearate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, Sodium stearyl fumarate, stearic acid, talc and zinc stearate.

[00028] Flavoring and flavor enhancers make the dosage form more palatable to the patient. Flavoring and flavoring agents commonly used in pharmaceutical products that may be included in the compositions of the present invention include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid .

[00029] Solid and liquid compositions may also be dyed with any pharmaceutically acceptable colorant to improve their appearance, and/or to facilitate patient identification of product and unit dosage levels.

[00030] In liquid pharmaceutical compositions prepared from pure pimecrolimus produced by the method of the present invention, pimecrolimus and any other solid excipients are dissolved or suspended in a liquid carrier such as water, vegetable oil, alcohol, in polyethylene glycol, propylene glycol or glycerin.

[00031] Liquid pharmaceutical compositions can contain emulsifying agents to disperse uniformly throughout the composition active ingredients or other excipients that are insoluble in the liquid carrier. Emulsifiers useful in the liquid compositions of the present invention include, for example, gelatin, egg yolk, casein, cholesterol, acacia, tragacanth, carrageenan, pectin, methylcellulose, carbomer, stearyl alcohol Cetyl Alcohol Mixture and Cetyl Alcohol.

[00032] Liquid pharmaceutical compositions may also contain viscosity-increasing agents to improve mouthfeel and/or coat the lining of the gastrointestinal tract. Such agents include gum arabic, alginic acid, bentonite, carbomer, calcium or sodium carboxymethylcellulose, cetostearyl alcohol, methylcellulose, ethylcellulose, gelatin, guar gum, hydroxy Ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polyvinyl alcohol, povidone, propylene carbonate, propylene glycol alginate, sodium alginate, sodium starch glycolate, starch , Tragacanth and Xanthan Gum.

[00033] Sweeteners such as sorbitol, saccharin, sodium saccharin, sucrose, aspartame, fructose, mannitol and invert sugar may also be added to improve taste.

[00034] Preservatives and chelating agents such as alcohol, sodium benzoate, butylated hydroxytoluene, butylated hydroxyanisole, and ethylenediaminetetraacetic acid may be added at levels safe for ingestion to enhance storage stability.

[00035] The liquid composition may also comprise a buffering agent such as guconic acid, lactic acid, citric acid or acetic acid, sodium guconate, sodium lactate, sodium citrate or sodium acetate. The choice of excipients and the amounts to be used can readily be determined by a formulation scientist empirically and by consideration of standard procedures and references in the art.

[00036] The solid compositions of the present invention include powders, granules, aggregates and compact compositions. Dosages include those suitable for oral, buccal, rectal, parenteral (including subcutaneous, intramuscular and intravenous), inhalation and ophthalmic administration. The most preferred route according to the invention is oral, although the most suitable mode of administration in any event will depend on the nature and severity of the condition being treated. The dosage may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

[00037] Dosage forms include solid dosage forms such as tablets, powders, capsules, suppositories, sachets, troches and lozenges as well as liquid syrups, suspensions and elixirs.

[00038] Oral dosage forms of the present invention are preferably in the form of oral capsules in dosages from about 10 mg to about 160 mg, more preferably from about 20 mg to about 80 mg, most preferably 20, 40, 60 and 80 mg. The daily dose may consist of 1, 2 or more capsules per day.

[00039] The dosage form of the present invention may be a capsule comprising a composition, preferably a powdered or granular solid composition of the present invention, in a hard or soft shell. The shell can be prepared from gelatin and optionally contain plasticizers, such as glycerol and sorbitol, and opacifiers or coloring agents.

[00040] Compositions for tablet compression or capsule filling may be prepared by wet granulation. In wet granulation, some or all of the active ingredient in powder form is mixed with excipients and then mixed in the presence of a liquid, usually water, to agglomerate the powder into granules. The granules are sieved and/or ground, dried, and then sieved and/or ground to the desired particle size. The granules may then be compressed, or other excipients such as glidants and/or lubricants may be added prior to compression.

[00041] Tabletting compositions can be conventionally prepared by dry blending. For example, the mixed composition of active ingredient and excipients can be compressed into pre-compressed tablets or sheets and then comminuted into tightly bound granules. The tightly bound granules are then compressed into tablets.

[00042] As an alternative to dry granulation, the blended composition can be compressed directly into dosage form using direct compression technology. Direct compression yields a more uniform tablet without granules. Excipients particularly suitable for direct compression include microcrystalline cellulose, spray-dried lactose, dicalcium phosphate dihydrate and colloidal silicon. The appropriate use of these and other excipients in direct compression is known to those skilled in the art with experience and skill in the particular formulation challenges of direct compression.

[00043] The filled capsules of the present invention may contain any of the above mixtures and granules mentioned in the tabletting, however, they are not subjected to the final tabletting step.

[00044] Active ingredients and excipients can be formulated into compositions and dosage forms according to methods known in the art.

[00045] Having described the invention in terms of certain preferred embodiments, other embodiments will be apparent to those skilled in the art from the description. The invention is also defined by reference to the following examples which describe in detail the preparation of the compositions and methods of use of the invention. Those skilled in the art will appreciate that many modifications in materials and methods can be made without departing from the scope of the invention.

instrument

Chromatographic method for determination of purity:

Eluent: n-hexane: acetone: acetonitrile 20:2:1 (by volume)

Flow rate: 20-40mL/min

Detection: TLC (UV, 254nm)

Sample concentration: 400-500g/L

Sample volume: 60-70mL

Column temperature: 25°C

Detection limit: not determined

Example

[00046] The following non-limiting examples are intended only to illustrate preferred embodiments of the invention and should not be construed as limiting the invention, the scope of which is defined by the appended claims.

Example 1

[00047] A 3.0 g sample of crude ascomycin was purified by a short silica gel column. The resulting 3.0 g syrup was dissolved in 25 ml dry dichloromethane. A stream of dry nitrogen was passed slowly through the solution, which was cooled to -15°C.

[00048] A 5% by weight solution of trifluoromethanesulfonic anhydride in anhydrous dichloromethane was added to the above solution in 6 ml portions together with 0.3 g 2,6-lutidine in each portion. After adding a total of 24 ml of trifluoromethanesulfonic anhydride solution, 28 g of a 10% by weight lithium chloride solution were added to the reaction mixture, and the reaction mixture was raised to room temperature, i.e. about 21°C. The reaction mixture was then stirred for 4 days.

[00049] The reaction mixture was diluted with a mixture of 200ml ethyl acetate and 25ml water, poured into a separatory funnel, and shaken. After extraction, 25 ml of 10% by weight aqueous _KHSO4 solution was added. After further shaking, the aqueous layer was removed and the organic phase was washed three times with 25 ml of 10% by weight aqueous KHSO ₄ , twice with 25 ml of saturated aqueous sodium bicarbonate and twice with 25 ml of brine. Dry the organic phase with anhydrous _MgSO4 . After filtration, concentration in vacuo and finally complete removal of solvent under high vacuum. The yield of crude amorphous pimecrolimus was 2.94 g.

[00050] The resulting product was purified by flash chromatography using n-hexane/acetone (2:1, V/V) as the eluent. The yield of amorphous pimecrolimus was 2.54 g.

Example 2

[00051] A 31.4g ascomycin (assayed at 92.7percent) sample was dissolved in 200ml toluene. The solution was concentrated to dryness (slurry) at 40°C. Anhydrous toluene was added to obtain 230 g of solution, and then 275 ml of anhydrous acetonitrile was added. The solution was cooled to a jacket temperature of -15°C with a stream of dry nitrogen slowly passed over the surface of the solution. Simultaneously, 315 ml of anhydrous toluene were cooled to a jacket temperature of -15° C. in a small reactor, also with a stream of dry nitrogen slowly passing over the surface of the liquid. When the stabilization of the toluene reached about -12.5°C, 13.85 g of triflic anhydride was added dropwise. At about the same time, 11.33 g of ethyldiisopropylamine (EDIPA) were added dropwise to the ascomycin solution. After a few minutes, the triflic anhydride solution was transferred to the ascomycin solution. After the addition was complete, the temperature of the jacket was set to 26°C. When the temperature of the reaction mass reached about 0°C, 300 g of a 12.5% by weight solution of benzyl-triethylammonium chloride (" _BnEt3NCl ") in anhydrous acetonitrile was added. 200 ml of water were added 45 minutes after the temperature of the reaction mass had risen to about 24°C to about 25°C. After a period of vigorous stirring, the water phase was removed, 200 ml of water was added, and the water phase was removed again after stirring. The organic phase was concentrated at 40°C until almost all acetonitrile was removed. The solution was diluted with an equal volume of toluene with stirring. The precipitated solid was filtered and washed with toluene. The filtrate was concentrated to dryness at 40 °C. Crude amorphous pimecrolimus was obtained as a brown foam in a yield of 33 g.

Example 3

[00052] According to embodiment 2, 30g ascomycin is converted into crude pimecrolimus. The resulting concentrated solution of the crude product in toluene was placed on a 600 g column of silica gel 60 (0,040-0,063 mm). Elution was carried out with a mixture of n-hexane-acetone-acetonitrile (20:2:1).

[00053] Fractions containing pimecrolimus of sufficient purity by HPLC analysis were pooled and extracted with 10 V/V% acetonitrile. The lower layer containing pimecrolimus in a mixture of acetonitrile and acetone (with some n-hexane) was removed and the upper layer (mainly n-hexane) was extracted twice with 5 V/V% acetonitrile. The lower layers (acetonitrile-acetone) were combined and concentrated at 40°C to give a colorless resin. It was then dissolved in 217 ml of acetone at 40°C and concentrated. Residue: 38,76 g. The residue was diluted with 6 ml of distilled water with stirring. Finally 1 ml of acetone was added. The solution was slowly added to 2L of pre-cooled distilled water and stirred thoroughly. After the addition was complete, the suspension was stirred at 0°C for 20 minutes. The solid was then filtered off and dried under vacuum at 45°C overnight. Product: 15,65 g pale yellow solid. Amorphous (XRD, DSC). Purity: The purity determined by HPLC peak area method is 95.75%.

[00054] While the invention disclosed herein well accomplishes the foregoing objectives, it is to be understood that numerous modifications and embodiments can be devised by those skilled in the art. Accordingly, the appended claims cover all such modifications and embodiments as fall within the true spirit and scope of this invention.

Claims (31)

1. A method for preparing pimecrolimus, the method comprising: a) dissolving ascomycin in an organic solvent; b) mixing Ascomycin with a base and a transformation reagent to obtain an activated Ascomycin derivative; c) reacting an activated derivative of ascomycin with a source of chloride ions to obtain pimecrolimus; and d) recovering the resulting pimecrolimus. 2. The method of claim 1, wherein said organic solvent is selected from the group consisting of: dichloromethane, chloroform, ether, diisopropyl ether, methyl tert-butyl ether, toluene, ethyl acetate, isobutyl acetate, acetone, methyl Ethyl ketone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide and mixtures thereof. 3. The method of claim 2, wherein the organic solvent is toluene, acetonitrile or a mixture thereof. 4. The method of claim 1, wherein the ascomycin is dissolved in the organic solvent at a temperature of at least about 25°C. 5. The method of claim 1, wherein the resulting ascomycin solution is stirred at a temperature below about 25°C. 6. The method of claim 5, wherein the resulting ascomycin solution is stirred at a temperature below 0°C. 7. The method of claim 6, wherein the resulting ascomycin solution is stirred at a temperature below -20°C. 8. The method of claim 1, wherein the amount of the base is about 1 to about 4 equivalents. 9. The method of claim 1, wherein the alkali in the step (b) is added dropwise, batchwise or all at once. 10. The method of claim 1, wherein said base is selected from the group consisting of: triethylamine, diisopropylethylamine (EDIPA), N-methyl-morpholine, N,N-dimethylaniline, pyridine and Substituted pyridine derivatives such as 2,6-lutidine, 2,4,6-collidine and 4-dimethylaminopyridine. 11. The method of claim 10, wherein the base is selected from the group consisting of: diisopropylethylamine (EDIPA), 2,4,6-Collidine and 2,6-Lutidine. 12. The method of claim 1, wherein said base is added in the form of a solution comprising a solvent selected from the group consisting of dichloromethane, chloroform, diethyl ether, diisopropyl ether, methyl tert-butyl ether, toluene, Ethyl acetate, isobutyl acetate, acetone, methyl ethyl ketone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, n-hexane, n-heptyl alkanes, cyclohexanes and mixtures thereof. 13. The method of claim 12, wherein the base is added as a solution, the solution comprising toluene. 14. The method of claim 1, wherein said activated ascomycin derivative is selected from the group consisting of sulfonate, tosylate or mesylate and triflate. 15. The method of claim 1, wherein the transformation reagent in the step (a) is selected from the group consisting of: fluorosulfonic anhydride, fluorosulfonyl chloride, trifluoromethanesulfonic anhydride, trifluoromethanesulfonyl chloride, methanesulfonic anhydride, methanesulfonyl chloride, Phenylmethanesulfonic anhydride, phenylmethanesulfonyl chloride, p-toluenesulfonic anhydride, p-toluenesulfonyl chloride, benzenesulfonic anhydride, and benzenesulfonyl chloride. 16. The method of claim 15, wherein the transforming reagent is selected from the group consisting of trifluoromethanesulfonic anhydride and trifluoromethanesulfonyl chloride. 17. The method of claim 1, wherein said transformation reagent is added in the form of a solution comprising a solvent selected from the group consisting of dichloromethane, chloroform, diethyl ether, diisopropyl ether, methyl tert-butyl ether, toluene , ethyl acetate, isobutyl acetate, acetone, methyl ethyl ketone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, n-hexane, n- Heptane, cyclohexane and mixtures thereof. 18. The method of claim 17, wherein the transformation reagent is added as a solution comprising a solvent selected from the group consisting of toluene, n-hexane, n-heptane and cyclohexane. 19. The method of claim 1, wherein the source of chloride ions in the step (c) is selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, aluminum chloride, iron(II) chloride ), iron(III) chloride, ammonium chloride, hydrochlorides of organic bases, quaternary ammonium chlorides, quaternary phosphonium chlorides, tetrabutylammonium chloride, benzyl-triethylammonium chloride and similar Quaternary ammonium chlorides, and hydrochlorides of bases selected from the group consisting of triethylamine, diisopropylethylamine (EDIPA), N-methyl-morpholine, N,N-dimethylaniline, pyridine and substituted pyridine derivatives such as 2,6-lutidine, 2,4,6-collidine and 4-dimethylaminopyridine. 20. The method of claim 19, wherein the source of chloride ions is benzyltriethylammonium chloride. 21. The method of claim 1, wherein said source of chloride ions is added as a solution comprising a solvent selected from the group consisting of dichloromethane, chloroform, diethyl ether, diisopropyl ether, methyl tert-butyl ether, Toluene, ethyl acetate, isobutyl acetate, acetone, methyl ethyl ketone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, n-hexane, n-heptane, cyclohexane and mixtures thereof. 22. The method of claim 1, further comprising stirring the reaction mixture prior to step d). 23. The method of claim 1, wherein said recovering step comprises: adding water together with a water-insoluble organic solvent to obtain a biphasic system; separating said biphasic system; extracting the organic phase with aqueous KHSO ₄ , aqueous NaHCO ₃ and brine , the organic phase was concentrated; and dried. 24. The method of claim 1, wherein said recovering step comprises: adding water to the reaction mixture to obtain a biphasic system; separating said biphasic system; and concentrating the organic phase. 25. The method of claim 1, further comprising purifying the obtained pimecrolimus by column chromatography to obtain purified pimecrolimus. 26. The method of claim 25, wherein said purified pimecrolimus has a purity of at least about 95% as determined by HPLC peak area. 27. The method of claim 26, wherein said purified pimecrolimus has a purity of at least about 98% as determined by HPLC peak area. 28. Pimecrolimus having a purity of at least about 95% as determined by HPLC peak area method. 29. The pimecrolimus of claim 28 having a purity of at least about 98% as determined by HPLC peak area method. 30. A pharmaceutical formulation comprising a therapeutically effective amount of pimecrolimus according to claim 28. 31. A method of treating a patient suffering from atopic dermatitis, said method comprising the step of administering to said patient a pharmaceutical formulation according to claim 30.

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