CN106854228B

CN106854228B - Preparation method of pimecrolimus

Info

Publication number: CN106854228B
Application number: CN201610384697.6A
Authority: CN
Inventors: 袁建栋; 黄仰青; 池建文; 马萨·康德瑞斯; 乔迪·普施
Original assignee: Modicon; Borui Biomedical Suzhou Co Ltd
Current assignee: Modicon; Borui Biomedical Suzhou Co Ltd
Priority date: 2015-12-08
Filing date: 2016-06-02
Publication date: 2020-05-29
Anticipated expiration: 2036-06-02
Also published as: CN106854228A

Abstract

The invention relates to a method for preparing pimecrolimus from ascomycin, which is suitable for industrial application. The method provided by the invention has the advantages that the yield is obviously improved, and the obtained pimecrolimus can be purified by a crystallization method without column chromatography.

Description

Preparation method of pimecrolimus

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a preparation method of pimecrolimus.

Background

Pimecrolimus is an anti-inflammatory compound obtained by structural modification of the macrolide natural product ascomycin produced by a particular streptomyces strain. Pimecrolimus is sold in the United states under the trade name ELIDEL^®Sold, approved for the treatment of atopic dermatitis. The systemic name of pimecrolimus is (1R, 9S,12S,13R,14S,17R,18E,21S,23S,24R,25S, 27R) -12- [ (1E) -2- { (1R,3, R,4S) -4-chloro-3-methoxycyclohexyl } -1-methylethenyl]-17-ethyl-1, 14, dihydroxy-23, 25-dimethoxy-13, 19,21, 27-tetramethyl-11, 28-dioxa-4-aza-tricyclo [22.3.1.0 [^4,9]Dioctadecyl-18-ene-2, 3,10, 16-tetraone. Pimecrolimus is a 32-epichloro derivative of ascomycin. It has an empirical formula of C₄₃H₆₈ClNO₁₁The molecular weight is 810.47, and the structural formula is as follows:

，

european patent EP0427680B1 discloses pimecrolimus for the first time and discloses a process for the synthesis of pimecrolimus in the form of a colourless foamy resin. The disclosed process comprises the following four reaction steps:

(a) protecting two hydroxyl groups at the C-24 position and the C-32 position of the ascomycin by using tert-butyldimethylsilyl ether;

(b) deprotection of the silyl protected hydroxyl group at the C-32 position, the hydroxyl group at the C-24 position remaining protected;

(c) chlorine substitution is carried out on the free hydroxyl at the C-32 position, and configuration transformation is carried out; and

(d) deprotection of the silyl protected hydroxyl group at the C-24 position is carried out.

EP0427680B1 does not disclose the yield of each step, but discloses that the product of each step is purified by chromatography. Thus, the overall yield of the process disclosed in EP0427680B1 can be expected to be low from the number of reaction steps and the number of chromatographic purifications required.

EP1802635B1 discloses a process for the preparation of pimecrolimus from ascomycin by a one-step process comprising reacting ascomycin with a suitable source of chloride ions in an organic solvent, preferably in the presence of a base. As can be seen from the specification examples 1-3, the crude product was purified by silica gel chromatography to obtain pimecrolimus in yields of 51.4%, 45%, and 48.1%, respectively. Example 4 further discloses that pimecrolimus obtained in examples 1 to 3 was purified by ethanol/water crystallization to obtain pimecrolimus with a yield of 87% and a purity of 98%. It can be seen that the total yield of pimecrolimus prepared by the method with the purity of 98 percent is not higher than 45 percent.

EP1817317B1 discloses a two-step process for the preparation of pimecrolimus comprising dissolving ascomycin in an organic solvent, mixing the organic solvent of ascomycin with a base to obtain a mixture, mixing the mixture with an activator mixture to obtain an activated ascomycin derivative, combining the activated ascomycin derivative with a source of chloride ions to obtain pimecrolimus, and recovering the pimecrolimus thus obtained. The use of lithium chloride and benzyltriethylammonium chloride as a source of chloride ions is mentioned in examples 1 and 2 of EP1817317B 1. In example 1, pimecrolimus obtained was purified by flash chromatography to yield pimecrolimus in a yield of 82.6%, but no purity data are given. Example 3 discloses purification of pimecrolimus obtained from example 2 by column chromatography to yield purified pimecrolimus in 51% yield and 95.75% purity (HPLC peak area method).

The disclosure of EP1817317B1 is based on the use of a single chloride source, and the inventors have found that the use of a single chloride source according to the method disclosed in EP1817317B1 does not completely convert the activated ascomycin into pimecrolimus, the remaining activated ascomycin is converted back to ascomycin during the post-treatment process, resulting in a decrease in yield, and the presence of ascomycin increases the difficulty and cost of post-treatment purification, and the subsequent crystallization purification process is difficult to remove ascomycin. The crude product obtained contains both unreacted starting material and other introduced reagents and is also of very low HPLC purity. EP2432791B1 discloses a four-step and five-step process for the preparation of pimecrolimus comprising enzymatic acylation and enzymatic alcoholysis of ascomycin with canadian lipase. The total yield after the four-step reaction is about 30 percent, and the total yield after the five-step reaction is about 13 percent. And both methods involve purification by column chromatography.

Therefore, a simple method for preparing pimecrolimus with high purity, less reaction steps and high yield, which is suitable for industrial production, is still needed.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for preparing pimecrolimus, comprising: a) dissolving ascomycin in an organic solvent; b) activating C-32 site of ascomycin by using organic base or inorganic base and an activating agent to obtain an activated ascomycin derivative; c) reacting the activated ascomycin derivative with two chloride ion sources A and B to obtain pimecrolimus; and, d) recovering the pimecrolimus obtained; the method is characterized in that the chloride ion source A is selected from: lithium chloride, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, aluminum chloride, ferrous chloride, ferric chloride, ammonium chloride or mixtures thereof; and, the source of chloride ions B is selected from: quaternary phosphonium chlorides, quaternary ammonium chlorides, such as tert-butylammonium chloride, benzyltriethylammonium chloride and hydrochlorides of organic bases selected from: triethylamine, Diisopropylethylamine (DIPEA), N-methylmorpholine, N-dimethylaniline, pyridine and substituted pyridine derivatives, such as 2, 6-lutidine, 2,4, 6-collidine and 4-dimethylaminopyridine and mixtures thereof.

The inventors surprisingly found that the combined use of two chloride ion sources (i.e. the chloride ion source a and the chloride ion source B according to the invention) significantly increases the reaction yield compared to the use of a single chloride ion source in the reaction of an activated ascomycin derivative with a chloride ion source, and that the pimecrolimus yield is significantly increased when the molar ratio of the total amount of chloride ion sources to ascomycin is 4-20: 1, preferably 10-20: 1, more preferably 15:1, and the chloride ion sources a and B replace the single chloride ion source (a or B). The yield of pimecrolimus is increased, and the purity of the crude pimecrolimus prepared is higher, so that the purification method of the crude pimecrolimus is simpler, for example, the purification by a chromatographic column is not needed, or the purification steps are reduced.

In a preferred embodiment of the present invention, the organic solvent for dissolving the ascomycin is selected from the group consisting of dichloromethane, chloroform, diethyl ether, diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, toluene, xylene, hexane, heptane, cyclohexane, methylcyclohexane, acetonitrile and a mixture thereof; preferably, dichloromethane, acetonitrile toluene or mixtures thereof.

Further, preferably, the organic solvent for dissolving the ascomycin is anhydrous. Alternatively, the resulting ascomycin solution may be subjected to anhydrous treatment, for example by removing water by molecular sieves.

In another preferred embodiment of the invention, the temperature at which the ascomycin is reacted with the activator to produce the activated ascomycin derivative is below 0 ℃, preferably below-20 ℃, more preferably-35 ℃ to-30 ℃. The method for preparing pimecrolimus provided by the invention has obvious advantages, and particularly has higher yield while improving the purity of the product.

In the method of the present invention, the organic base or inorganic base used in step b) may be slowly added dropwise in batches or at a time.

In a preferred embodiment of the present invention, the base used in step b) is an organic base selected from the group consisting of: triethylamine, Diisopropylethylamine (DIPEA), N-methylmorpholine, N-dimethylaniline, pyridine, and substituted pyridine derivatives such as 2, 6-lutidine, 2,4, 6-trinitrotoluene and 4-dimethylaminopyridine, preferably Diisopropylethylamine (DIPEA), 2, 6-lutidine, 2,4, 6-trinitrotoluene and mixtures thereof.

In the method of the present invention, the organic base or inorganic base used in step b) may be added to the reactor before the addition of the activator, or may be added to the reactor together with the activator; preferably, the organic or inorganic base is added to the reactor prior to the addition of the activator.

The organic or inorganic base used in step b) of the process of the present invention may be added alone or dissolved in a solvent, preferably dichloromethane, chloroform, diethyl ether, diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, toluene, xylene, hexane, heptane, cyclohexane, methylcyclohexane and mixtures thereof, and added as a solution.

Preferably, the amount of the organic base or inorganic base used in step b) of the method of the present invention is 1 to 6 molar equivalents of the ascomycin, preferably 3 to 4 molar equivalents of the ascomycin.

In another preferred embodiment of the invention, the reaction mixture is mixed with the activator after or simultaneously with the dropwise, batchwise or one-time addition of the base. Preferably, the reaction solution is stirred at a temperature of less than 0 ℃, preferably less than-20 ℃, more preferably-35 ℃ to-30 ℃. Preferably, the progress of the reaction is monitored, for example using Thin Layer Chromatography (TLC) until the reaction is complete or near completion.

In another preferred embodiment of the invention, the activated ascomycin derivative is a sulphonate selected from: tosylate, mesylate and triflate, preferably triflate.

In another preferred embodiment of the invention, the activator 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, preferably the activating agent is trifluoromethanesulfonic anhydride.

The activator may be added alone or in solution, wherein the solvent is selected from the same solvents as used for the solution formed with the base, as described above.

The time for complete or near complete conversion of the ascomycin to the corresponding activated ascomycin derivative may vary depending on the reaction conditions, such as temperature, solvent, base and activator used. One of ordinary skill in the art would know how to monitor the reaction, such as by TLC at appropriate time intervals depending on the conditions selected.

Once the activated ascomycin derivative is obtained, the reaction mixture is mixed with two chloride ion sources a and B. The two sources of chloride ions A and B can be added to the reaction mixture simultaneously or sequentially, separately or in solution. Alternative solvents for dissolving the chloride ion source include those described above for dissolving the organic or inorganic base and the activator.

During the addition of the chloride ion sources A and B, the temperature of the reaction mixture is controlled to be less than 20 deg.C, preferably not more than 10 deg.C, more preferably-5 deg.C.

The source of chloride ions A is selected from: lithium chloride, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, aluminum chloride, ferrous chloride, ferric chloride, ammonium chloride or mixtures thereof; and, the source of chloride ions B is selected from the group of: quaternary phosphonium chlorides, quaternary ammonium chlorides, such as tert-butylammonium chloride, benzyltriethylammonium chloride and hydrochlorides of organic bases selected from: triethylamine, Diisopropylethylamine (DIPEA), N-methylmorpholine, N-dimethylaniline, pyridine and substituted pyridine derivatives, such as 2, 6-lutidine, 2,4, 6-collidine and 4-dimethylaminopyridine and mixtures thereof.

In a preferred embodiment of the invention, said source of chloride ions a is selected from lithium chloride and said source of chloride ions B is selected from benzyltriethylammonium chloride.

In another embodiment of the present invention, the molar ratio of the source of chloride ions A to the source of chloride ions B is from about 5:1 to about 1:5, preferably from 2:1 to about 1:2, more preferably about 1.8: 1. Under the condition, the purity of pimecrolimus is improved, and the yield is improved.

Preferably, the molar ratio of the total amount of chloride ion source a and chloride ion source B to the corresponding ascomycin is higher than 1, more preferably higher than 4, even more preferably higher than 20.

After the chloride ion sources A and B are added, the reaction mixture is stirred at 20 ℃ to 35 ℃, preferably 20 ℃ to 30 ℃. Also, the reaction progress is monitored to determine completion, e.g., by TLC.

The disappearance of the intermediate activated ascomycin derivative may vary depending on the precise conditions used. One of ordinary skill in the art would know how to monitor the reaction, such as by TLC at appropriate time intervals depending on the conditions selected.

The method for recovering pimecrolimus comprises the following steps: adding water to the reaction mixture to obtain a two-phase system; the two phases were separated and the organic phase was concentrated to give a first residue. The first residue is preferably an amorphous solid. The recovering step of pimecrolimus may further comprise mixing the first residue with a water-insoluble organic solvent and washing the resulting mixture with an acidic solution and water; the organic phase is recovered and then concentrated to give a second residue.

Without limitation, the water-insoluble organic solvent added to the first residue is diisopropyl ether, diethyl ether, 2-methyltetrahydrofuran, cyclopentyl methyl ether, methyl tert-butyl ether, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, or dichlorobenzene; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; a hydrocarbon aromatic solvent such as toluene, benzene, o-xylene, m-xylene, p-xylene or a mixture of two or more thereof. Preferably, the water-insoluble organic solvent is dichloromethane, heptane, or a mixture thereof.

In a preferred embodiment, pimecrolimus may be purified by a recrystallization process. The purified pimecrolimus obtained is preferably in crystalline form, more preferably the purified pimecrolimus obtained is form a or form B as described in WO9901458a 1.

Without limitation, suitable recrystallization solvents are alcoholic solvents such as methanol, ethanol, isopropanol, butanol; ethers such as tetrahydrofuran, dioxane, isopropyl ether, diethyl ether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; ketones, such as methyl ethyl ketone, methyl isobutyl ketone or acetone; a halogenating agent such as dichloromethane, chloroform, tetrahydrofuran, dichloroethane, chlorobenzene or dichlorobenzene; polar aprotic solvents such as N, N-dimethylformamide, acetonitrile, N-dimethylacetamide, N-methyl-2-pyridone, or dimethylsulfoxide; hydrocarbon aliphatic solvents such as methylcyclohexane, cyclohexane, heptane or hexane; a hydrocarbon aromatic solvent such as toluene, benzene, o-xylene, m-xylene, p-xylene or water or a mixture of two or more thereof. Particularly preferably, the solvent is an alcohol solvent, a ketone solvent, a hydrocarbon aliphatic solvent and water or a mixture thereof. More preferably, the solvent is a mixture of an alcohol and a hydrocarbon aliphatic solvent.

Alternatively, crude pimecrolimus may be purified by chromatography.

Preferably, the purified pimecrolimus has a purity of greater than 97% by HPLC peak area method, preferably the purified pimecrolimus has a purity of greater than 98% by HPLC peak area method, and more preferably the purified pimecrolimus has a purity of greater than 99% by HPLC peak area method.

The term "about" used before a number means any number within a range of error of 10% of the number, preferably any number within a range of error of 5% of the number, more preferably any number within a range of error of 2% of the number, and still more preferably any number within a range of error of 1% of the number. For example, "about 10" can be interpreted as 9 to 11, preferably 9.5 to 10.5, more preferably 9.8 to 10.2, and further preferably 9.9 to 10.1.

The term "anhydrous" means that the organic solvent contains no more than 1% (w/w) of water, preferably no more than 0.5% (w/w), more preferably no more than 0.1% (w/w).

The term "crystalline" means that at least 20% (w/w) of the pimecrolimus prepared by the process of the invention is in crystalline form, preferably more than 50% (w/w) of the pimecrolimus prepared by the process of the invention is in crystalline form, more preferably more than 70% (w/w) of the pimecrolimus prepared by the process of the invention is in crystalline form; further preferably, greater than 90% (w/w) of the pimecrolimus prepared by the process of the present invention is in crystalline form.

The advantages of the present invention using a mixed chloride ion source are: can completely convert activated ascomycin into pimecrolimus, and the conversion rate is more than 99 percent. Thereby greatly improving the reaction yield, simultaneously improving the conversion rate and the yield, greatly simplifying the post-treatment purification method, and obtaining the pimecrolimus with higher purity through the crystallization purification process.

Drawings

FIG. 1 shows the purity of pimecrolimus prepared by the method of example 1, wherein the retention time of pimecrolimus: 37.700min,% peak area: 59.567% with a retention time of the tautomer of 35.663min peak area%: 1.12%, i.e.% main component peak area: 60.687 percent.

FIG. 2 shows the purity of pimecrolimus prepared by the method of example 2, wherein the retention time of pimecrolimus: 37.31min,% peak area: 63.386%, retention time of its tautomer: 35.564min,% peak area: 1.005%, i.e.% of main component peak area: 64.39 percent.

FIG. 3 shows the purity of pimecrolimus prepared by the method of example 3, wherein the retention time of pimecrolimus: 37.269min,% peak area: 78.502%, retention time of its tautomer: 35.566min,% peak area: 1.197%, main component peak area%: 79.699 percent.

FIG. 4 shows the purity of pimecrolimus prepared by the method of example 5, wherein the retention time of pimecrolimus: 35.543min,% peak area: 96.787%, retention time of its tautomer: 33.576min,% peak area: 2.231%, i.e.% of main component peak area: 99.018 percent.

FIG. 5 shows the purity of pimecrolimus prepared by the method of example 7, wherein the retention time of pimecrolimus: 35.588min,% peak area: 97.150%, retention time of its tautomer: 33.590min,% peak area: 1.863%, i.e.% of main component peak area: 99.013 percent.

FIG. 6 shows the purity of crude pimecrolimus prepared by the method of example 9, wherein the retention time of pimecrolimus: 35.505min,% peak area: 97.043%, retention time of its tautomer: 33.571min,% peak area: 2.383%, i.e.% of main component peak area: 99.426 percent.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following specific examples further illustrate the present disclosure, but it should not be construed as in any way limiting its scope.

The HPLC purity detection method of the pimecrolimus crude product comprises the following steps:

the chromatographic separation method adopts Poroshell 120 EC-18, 2.7 mm (4.6 x 150 mm) column, and the temperature is 60 ℃;

mobile phase A: 0.1% (V/V) phosphoric acid aqueous solution;

mobile phase B: methanol;

gradient elution conditions: initial 0-10min, isocratic 35% mobile phase A elution. 10-30min, linear elution to 25% mobile phase A. 30-60min, and eluting with 25% of mobile phase A at equal degree. 60-63min, linear elution to 35% mobile phase A. 63-70min, isocratic 35% mobile phase A elution.

A detector: a 210 nm UV detector. Flow rate: 1.0 ml/min;

sample preparation: about 40mg of the sample was dissolved in 10.0ml of a diluent (acetonitrile) in an amount of 10 μ l.

The ascomycin used in the invention is purchased from Chongqing Qiantai biological medicine, and the purity is not lower than 98%.

Example 1: comparative example based on example 1 in EP1817317B1 (using only chloride ion source A)

A solution of 3.0g (3.79 mmol) of the ascomycin in 25ml of anhydrous dichloromethane was slowly bubbled through the solution using a stream of dry nitrogen and cooled to-15 ℃. 24ml of a 5% by weight solution of trifluoromethanesulfonic anhydride in dry dichloromethane are then added to the above solution together with 1.8g of 2, 6-lutidine. After the addition was complete, 28g of a 10% by weight solution of lithium chloride (66.05 mmol, corresponding to 17.4 molar equivalents of ascomycin) in dichloromethane were added to the reaction mixture. The reaction mixture was warmed to about 21 ℃ and stirred at this temperature for 4 days. The reaction mixture was diluted with a mixture of 200ml of ethyl acetate and 25ml of water, the organic layer was extracted, and then the solvent was distilled off to obtain 3.4g of crude pimecrolimus in yield (110.8%), HPLC purity (59.57%) and HPLC profile as in fig. 1.

Example 2: comparative example based on example 2 in EP1817317B1 (using only chloride ion source B)

A solution of 2.9g (3.66mmol) of ascomycin in 20ml of toluene is added. The solution was concentrated to dryness at 40 ℃. The residue was dissolved in 23ml of anhydrous toluene under nitrogen protection, and 28ml of anhydrous acetonitrile was added. The resulting solution was cooled to-15 ℃ and 1.13g of diisopropylethylamine was added to the reaction mixture. 32ml of an anhydrous toluene solution of 1.39g of trifluoromethanesulfonic anhydride, previously cooled to-15 ℃ was added to the above reaction mixture. After the addition was complete, the reaction temperature was raised to 0 ℃ and then 30g of a 12.5% by weight solution of benzyltriethylammonium chloride (16.46 mmol, corresponding to 4.5 molar equivalents of ascomycin) in anhydrous acetonitrile was added. The reaction temperature was then raised to about 25 ℃, stirred at 24-25 ℃ for 45min, and then 20ml water was added. After sufficient stirring, 20ml of water was added, the organic layer was extracted, and the solvent was distilled off to give 6.6g of crude pimecrolimus in yield (222%) with HPLC purity (63.39%) containing a large amount of benzyltriethylammonium chloride and unreacted ascomycin as shown in fig. 2.

Example 3: preparation of pimecrolimus according to the Process of the invention

2.9g (3.66mmol) of ascomycin are dissolved in 20ml of toluene and the solution is concentrated to dryness at 40 ℃. The residue was dissolved in 23ml of anhydrous toluene under nitrogen protection, and 28ml of anhydrous acetonitrile was added. The resulting solution was cooled to-15 ℃ and 1.13g of diisopropylethylamine was added to the reaction mixture. 32ml of an anhydrous toluene solution of 1.39g of trifluoromethanesulfonic anhydride, previously cooled to-15 ℃ was added to the above reaction mixture. After the addition was complete, the reaction temperature was raised to 0 ℃ and then a solution of 1.90g (8.34 mmol) benzyltriethylammonium chloride and 0.34g (8.02 mmol) LiCl (total chloride ion 16.36mmol, corresponding to 4.5 molar equivalents of ascomycin) in anhydrous acetonitrile was added. The reaction temperature was then raised to about 25 ℃, stirred at 24-25 ℃ for 45min, and then 20ml water was added. After sufficient stirring, 20ml of water was added, the organic layer was extracted, and the solvent was distilled off to obtain 3.3g of crude pimecrolimus, yield (112%), HPLC purity: 78.50%, see FIG. 3.

Example 4: preparation of pimecrolimus according to the Process of the invention

100g (0.126 mol) of ascomycin is dissolved in 600ml of dichloromethane at the temperature of 15-20 ℃, 17.9g of molecular sieve is added into the reaction liquid, the mixture is stirred for 2 hours at the temperature of 20-25 ℃, then the mixture is filtered, the solid is washed by 75ml of dichloromethane, the filtrate is collected and cooled to the temperature of-35 ℃ to-30 ℃, and 46.9g of 2, 6-dimethylpyridine and 39.3g of trifluoromethanesulfonic anhydride are sequentially added. The reaction mixture was stirred at-35 ℃ to-30 ℃ for about 30 min. Then, 52.2g (1.23 mol) of lithium chloride and 287.9g (1.26 mol) of benzyltriethylammonium chloride were added to the reaction solution while controlling the reaction temperature at-10 ℃. The reaction mixture is then heated to-5 ℃ to 0 ℃ and stirred at this temperature for 2 hours. The reaction mixture was then heated to 15-20 ℃ and stirred at this temperature for 30 minutes. Then cooled to 0 ℃, 400ml of water was added to the resulting mixture and stirred at 0 ℃ to 5 ℃ for 30 minutes. The organic layer was extracted and concentrated to dryness. Then, 200ml of methylene chloride and 400ml of n-heptane were added to the obtained residue, and stirred at 20 ℃ to 25 ℃ for 1 hour. The resulting suspension was filtered and the solid was washed with a mixture of 200ml dichloromethane and 400ml heptane. The filtrate was collected, washed three times with 300ml of 0.5M HCl, three times with 300ml of water, the organic phase was collected and concentrated to dryness to give 120g of crude pimecrolimus product with a yield of 117% and an HPLC purity: 82.6 percent.

Example 5: purification of pimecrolimus

390ml of isopropanol was added to the crude pimecrolimus prepared in example 4, and then heated to 30-35 ℃ until completely dissolved. 200ml of water are added dropwise at 25 ℃ to 30 ℃ and stirred at this temperature for about 2 hours. The reaction solution was then cooled to 0-5 ℃ and stirred at this temperature for 4 hours. Then filtered and the solid washed with a mixture of 120ml isopropanol and 80ml water and then with 140ml heptane. The solid obtained was dried under vacuum at 35 ℃. + -. 5 ℃ to give 84.8g of pimecrolimus (starting from ascomycin, overall yield 82.9%), purity: 96.79% (HPLC peak area method) as shown in fig. 4.

Example 6: preparation of pimecrolimus according to the Process of the invention

29g (36.6mmol) of ascomycin are dissolved in 250ml of anhydrous dichloromethane and stirred at 20 ℃ to 25 ℃ for 2 hours. The resulting solution was cooled to 0 ℃ and 1.13g of diisopropylethylamine was added to the reaction mixture. To the above reaction mixture was added 320ml of an anhydrous dichloromethane solution of 13.9g of trifluoromethanesulfonic anhydride previously cooled to 0 ℃. After the addition was complete, the reaction temperature was controlled at 0 ℃ and then a solution of 111.2g (488 mmol) benzyltriethylammonium chloride and 10.3g (244 mmol) LiCl (total chloride ion: 732mmol, corresponding to 20 molar equivalents of ascomycin) in dry dichloromethane was added. Then the reaction temperature is raised to about 20 ℃, stirred for 45min at 20-21 ℃, and then 200ml of water is added. After sufficient stirring, 200ml of water was added, the organic layer was extracted, and the solvent was distilled off to obtain 135g of crude pimecrolimus having a purity of about 79%.

750ml of ethanol was added to the crude pimecrolimus prepared in example 6, and then heated to 30-35 ℃ until completely dissolved. 250ml of water are added dropwise at 25 ℃ to 30 ℃ and stirred at this temperature for about 2 hours. The reaction solution was then cooled to 0-5 ℃ and stirred at this temperature for 4 hours. Then filtered and the solid washed with a mixture of 90ml ethanol and 30ml water and then 100ml heptane. The obtained solid was dried under vacuum at 35 ℃. + -. 5 ℃ to give 24.3g of pimecrolimus (starting from ascomycin, total yield 82.3%), purity: 99.62% (HPLC peak area method).

Example 7: preparation of pimecrolimus according to the Process of the invention

3.0kg of ascomycin was dissolved in 18L of anhydrous dichloromethane. The resulting solution was cooled to-20 ℃ and 1.625Kg of 2, 6-lutidine solution was added to the reaction mixture, and the temperature of the reaction mixture was controlled not to exceed-20 ℃ during the dropwise addition. Then 1.39Kg of trifluoromethanesulfonic anhydride solution was added dropwise to the reaction solution, and the temperature of the reaction solution was controlled not to exceed-20 ℃ during the dropwise addition. After the dropwise addition, stirring for 1 hour at minus 20 ℃ (-2 ℃). TLC monitored the reaction until the starting material was completely reacted.

Heating the reaction solution to 5 ℃ (± 2 ℃), adding 4.315Kg triethyl benzyl ammonium chloride and 1.445Kg anhydrous lithium chloride in turn, heating to 28 ℃ (± 2 ℃), and reacting for 2 hours. TLC monitored until the reaction was complete. Then 20L of water was added. After stirring well, 20L of water was added, the organic layer was extracted, and the solvent was distilled off to give 3000g of crude pimecrolimus in yield (98%) and HPLC purity (82.1%).

Adding 12L of ethanol into the prepared pimecrolimus crude product, and heating to 30-35 ℃ until the pimecrolimus crude product is completely dissolved. 4L of water was added dropwise at 25 ℃ to 30 ℃ and stirred at this temperature for about 2 hours. The reaction solution was then cooled to 0-5 ℃ and stirred at this temperature for 4 hours. Then filtered and the solid washed with a mixture of 1.5L ethanol and 0.5L water, followed by 1.5L heptane. The resulting solid was dried under vacuum at 35 ℃. + -. 5 ℃ to give 2610g of pimecrolimus (ascomycin as starting material, total yield: 87%), HPLC: 97.15% (HPLC peak area method), as shown in FIG. 5.

Example 8: preparation of pimecrolimus according to the Process of the invention

Heating the reaction solution to 5 ℃ (± 2 ℃), adding 4.315Kg triethyl benzyl ammonium chloride and 1.445Kg anhydrous lithium chloride in turn, heating to 28 ℃ (± 2 ℃), and reacting for 2 hours. TLC monitored until the reaction was complete. Then 20L of water was added. After stirring sufficiently, 20L of water was added, the organic layer was extracted, and the solvent was distilled off to obtain crude pimecrolimus.

Adding 16L of isopropanol into the prepared pimecrolimus crude product, and heating to 30-35 ℃ until the pimecrolimus crude product is completely dissolved. 1.5L of water was added dropwise at 25 ℃ to 30 ℃ and stirred at this temperature for about 2 hours. The reaction solution was then cooled to 0-5 ℃ and stirred at this temperature for 4 hours. Then filtered and the solid washed with a mixture of 1.5L isopropanol and 0.5L water, followed by 1.5L heptane. The resulting solid was dried under vacuum at 35 ℃ ± 5 ℃ to give pimecrolimus (starting material ascomycin, total yield 89.1%), HPLC: 99.89% (HPLC peak area method).

Example 9: preparation of pimecrolimus according to the Process of the invention

3.0kg (3.79 mol) of ascomycin was dissolved in 18L of anhydrous tetrahydrofuran. The resulting solution was cooled to-20 ℃ and 2.76Kg (22.74 mol) of N, N-dimethylaniline was added to the reaction mixture, and the temperature of the reaction mixture was controlled not to exceed-20 ℃ during the dropwise addition. Then 0.65Kg of methanesulfonyl chloride is added into the reaction solution dropwise, and the temperature of the reaction solution is controlled not to exceed minus 20 ℃ in the dropwise adding process. After the dropwise addition, stirring for 1 hour at minus 20 ℃ (-2 ℃). TLC monitored the reaction until the starting material was completely reacted.

The temperature of the reaction solution is raised to 18 ℃ (± 2 ℃), 8.78Kg (31.6mol) tetrabutylammonium chloride and 0.6Kg (6.3 mol) anhydrous magnesium chloride are added in sequence, then the temperature is raised to 25 ℃ (± 2 ℃), and the reaction is kept for 2 hours. TLC monitored until the reaction was complete. Then 20L of water was added. After stirring well, 20L of water was added, the organic layer was extracted, and the solvent was distilled off to give 2800g of crude pimecrolimus in yield (91.2%) and purity (99.87%).

Adding 16.8L of isopropanol into the prepared pimecrolimus crude product, and heating to 30-35 ℃ until the pimecrolimus crude product is completely dissolved. 5.6L of water was added dropwise at 25 ℃ to 30 ℃ and stirred at this temperature for about 2 hours. The reaction solution was then cooled to 0-5 ℃ and stirred at this temperature for 4 hours. Then filtered and the solid washed with a mixture of 1.5L isopropanol and 0.5L water, followed by 15L heptane. The obtained solid was dried under vacuum at 35 ℃. + -. 5 ℃ to give 2530g of pimecrolimus (starting material ascomycin, total yield 82.4%), purity: 97.04% (HPLC peak area method) is shown in FIG. 6.

Claims

1. A process for preparing pimecrolimus, comprising:

a) dissolving ascomycin in an organic solvent;

b) activating C-32 site of ascomycin by using organic base or inorganic base and an activating agent to obtain an activated ascomycin derivative;

c) reacting the activated ascomycin derivative with two chloride ion sources A and B to obtain pimecrolimus;

d) recovering the pimecrolimus thus obtained;

characterized in that the source of chloride ions A is selected from: lithium chloride, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, aluminum chloride, ferrous chloride, ferric chloride, ammonium chloride or mixtures thereof; and, the source of chloride ions B is selected from: hydrochloride salts of quaternary phosphonium chlorides, quaternary ammonium chlorides and organic bases selected from: triethylamine, Diisopropylethylamine (DIPEA), N-methylmorpholine, N-dimethylaniline, pyridine and substituted pyridine derivatives and mixtures thereof; the activated ascomycin derivative is a sulfonate; the molar ratio of the chloride ion source A to the chloride ion source B is 5: 1-1: 5; controlling the temperature of the reaction mixture to be lower than 20 ℃ when two chloride ion sources are added; the molar ratio of the total amount of the two chloride ion sources to the ascomycin is 4-20: 1.

2. The method of claim 1, wherein the quaternary ammonium chloride is tetrabutylammonium chloride or benzyltriethylammonium chloride; the substituted pyridine derivative is 2, 6-lutidine, 2,4, 6-collidine or 4-dimethylaminopyridine.

3. The method according to claim 1, wherein the organic solvent is selected from the group consisting of: dichloromethane, chloroform, diethyl ether, diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, toluene, xylene, hexane, heptane, cyclohexane, methylcyclohexane, acetonitrile and mixtures thereof.

4. The process according to claim 3, wherein the organic solvent is selected from dichloromethane, acetonitrile, toluene or mixtures thereof.

5. A process according to any one of claims 1 to 3, wherein the temperature at which the ascomycin is reacted with the activating agent to produce the activated ascomycin derivative is less than 0 ℃.

6. The method of claim 5, wherein the temperature at which the ascomycin is reacted with the activating agent to produce the activated ascomycin derivative is less than-20 ℃.

7. The method of claim 6, wherein the temperature for reacting the ascomycin with the activating agent to prepare the activated ascomycin derivative is from-35 ℃ to-30 ℃.

8. A process according to any one of claims 1 to 3, wherein the organic base of step b) is selected from: triethylamine, Diisopropylethylamine (DIPEA), N-methylmorpholine, N, N-dimethylaniline, pyridine, 2, 6-lutidine, 2,4, 6-collidine or 4-dimethylaminopyridine.

9. The process according to any one of claims 1 to 3, wherein the organic base in step b) is selected from Diisopropylethylamine (DIPEA), 2, 6-dimethylpyridine, 2,4, 6-collidine and mixtures thereof.

10. A process according to any one of claims 1 to 3, wherein the sulfonate is selected from the group consisting of tosylate, mesylate and triflate.

11. A process according to any one of claims 1 to 3, wherein the activator 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.

12. The method according to any one of claims 1 to 3, wherein the source A of chloride ions is lithium chloride and the source B of chloride ions is benzyltriethylammonium chloride.

13. The method according to any one of claims 1 to 3, wherein the molar ratio of the source of chloride ions A to the source of chloride ions B is from 2:1 to 1: 2.

14. A process according to any one of claims 1 to 3, wherein the temperature of the reaction mixture is controlled to be less than 10 ℃ when two chloride ion sources are added.

15. The method of claim 14, wherein the molar ratio of the total amount of the two chloride ion sources to the ascomycin is 10 to 20: 1.

16. The process according to any one of claims 1 to 3, further comprising purifying the pimecrolimus obtained by crystallization.