Preparation method of tacrolimus position isomer

Abstract

The invention relates to the technical field of medical engineering, in particular to a preparation method of a tacrolimus position isomer. By introducing the catalyst tetra (triphenylphosphine) palladium in the reaction process, the tacrolimus position isomer with higher purity can be prepared on the basis of reducing the reaction temperature, and compared with the prior art, the method has the technical advantage of low-temperature reaction, and has the technical effects of reducing the side reaction of the whole reaction, reducing the purification difficulty and improving the product yield.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical engineering technology, and in particular to a method for preparing a tacrolimus positional isomer. Background Art

[0002] Tacrolimus may isomerize under high temperature conditions to form a positional isomer (tacrolimusregioisomer, CAS 131944-48-4), which has been included in the quality standard of tacrolimus capsules by the United States Pharmacopoeia USP-NF2024.

[0003] The preparation methods of this isomer reported in the literature generally adopt high temperature reactions, which have the problems of many side reactions, difficult purification, cumbersome operations, and low product yields. Summary of the invention

[0004] In order to solve the above problems, the embodiments of the present application provide a method for preparing positional isomers of tacrolimus, which can reduce the overall reaction temperature by introducing a catalyst, thereby reducing side reactions and greatly improving the yield.

[0005] In order to achieve the above purpose, the technical solution adopted in the embodiment of the present application is as follows: In a first aspect, an embodiment of the present application provides a method for preparing a tacrolimus positional isomer, wherein the tacrolimus positional isomer is a (E / Z)-FK-506 26,28-allyl ester rearrangement impurity with a CAS number of 131944-48-4. The method comprises: adding tetrakis(triphenylphosphine)palladium to a solvent in which tacrolimus is dissolved and performing a temperature reaction, monitoring the reaction process every 30 minutes by liquid chromatography until the tacrolimus content is less than 2%, filtering and distilling to obtain a crude product of the tacrolimus positional isomer, and recrystallizing the crude product of the tacrolimus positional isomer to obtain the tacrolimus positional isomer.

[0006] Furthermore, the crude tacrolimus position isomer product is recrystallized by adding two different solvents in sequence.

[0007] Furthermore, the solvent includes any one of ethanol, methanol, tetrahydrofuran, chloroform, toluene, xylene, acetone, butanone, and ethyl acetate.

[0008] Furthermore, the volume ratio of the solvent to the tacrolimus is 2-30:1.

[0009] Furthermore, the volume ratio of the solvent to the tacrolimus is 5-10:1.

[0010] Furthermore, the amount of tetrakis(triphenylphosphine)palladium used is 0.1% to 5% of the amount of the solvent.

[0011] Furthermore, the amount of tetrakis(triphenylphosphine)palladium used is 1-2% of the amount of the solvent.

[0012] Furthermore, the reaction temperature of the temperature-raising reaction is 50-100°C.

[0013] Furthermore, the first solvent of the two different solvents includes any one of ethyl acetate, isopropyl acetate, butyl acetate, toluene and xylene, and the second solvent includes any one of n-hexane, n-heptane, n-pentane, cyclohexane, isooctane and petroleum ether.

[0014] Furthermore, the second solvent is added at a temperature of 10-30°C.

[0015] In the technical solution provided in the embodiments of the present application, by introducing the catalyst tetrakis(triphenylphosphine)palladium during the reaction process, it is possible to prepare tacrolimus positional isomers with higher purity on the basis of lowering the reaction temperature. Compared with the prior art, it has the technical advantage of low-temperature reaction, which reduces the overall reaction side reactions, reduces the difficulty of purification, and improves the product yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 Schematic diagram of the structure of tacrolimus and its positional isomers.

[0018] Figure 2 It is a flow chart of the method for preparing tacrolimus positional isomers provided in the examples of the present application.

[0019] Figure 3 It is a schematic diagram of the HPLC detection spectrum of the tacrolimus positional isomers prepared in Example 1.

[0020] Figure 4 is the hydrogen spectrum of the tacrolimus positional isomer prepared in Example 1.

[0021] Figure 5 is the carbon spectrum of the tacrolimus positional isomer prepared in Example 1. DETAILED DESCRIPTION

[0022] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. The embodiments described below are some embodiments of the present invention, rather than all embodiments. In conjunction with the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention.

[0023] The present application embodiment provides a method for preparing a tacrolimus positional isomer, wherein tacrolimus is a macrolide compound isolated from the genus Streptomyces and is a potent immunosuppressant. Its mechanism of action is mainly to inhibit the release of interleukin-2 (IL-2) and comprehensively inhibit the activity of T lymphocytes. At the molecular level, tacrolimus binds to the FKBP12 protein in the cell to form a complex and accumulate in the cell, thereby inhibiting the activation and proliferation of T cells, while inhibiting the production of lymphokines and the expression of interleukin-2 receptors. Compared with cyclosporine (CsA), the immunosuppressive effect of tacrolimus is about 100 times stronger. In addition, tacrolimus has the advantages of low toxicity and side effects, high transplant survival rate, and low dependence. Therefore, it is widely used in the treatment of various autoimmune diseases in clinical practice, such as dermatitis, psoriasis, lupus erythematosus, lichen planus, vitiligo, and Netherton syndrome. At the same time, it is also used to prevent rejection reactions after organ transplantation such as liver, kidney, heart, lung, and pancreas.

[0024] The positional isomer of tacrolimus is produced by isomerization of tacrolimus at high temperature. For the structure of this isomer, please refer to Figure 1 As shown, in Figure 1The specific structures of tacrolimus and its positional isomers are shown in Fig. 1. The preparation of this positional isomer is currently mainly achieved by high-temperature reaction. For example, in the literature (Eur J Pharm Sci, 2013, 48, 514–522), o-xylene (boiling point 143~145 ℃) was refluxed for 24 hours, and the yield was only 19% after separation and purification by preparative chromatography. In Chinese patent CN201210483603.2, tacrolimus was heated at 110~150℃, and a pure product was obtained after silica gel column chromatography and recrystallization, with a yield of 17~18%. In addition, since the solid-phase reaction is highly sensitive to the particle size and dispersion of the raw material particles, the reaction mass transfer is uneven, the by-products are complex, and the batch reproducibility is poor; and since the particle size, morphology and other parameters of the solid in the solid-phase reaction have a great influence on the heat and mass transfer during the reaction, the reaction process and reaction results are difficult to control and even lead to poor process reproducibility. In Chinese patent CN201410057153.X, a tacrolimus solution is isomerized at 130-140°C, and a pure product is obtained after column chromatography and recrystallization several times with a yield of 14.5%.

[0025] In order to solve the above problems in the prior art accordingly, a method for preparing a tacrolimus positional isomer is provided in this embodiment. By adopting a reaction catalyst different from that in the prior art, the reaction temperature can be lowered to reduce side reactions and directly increase the yield of the tacrolimus positional isomer.

[0026] The process flow for this method can be found in Figure 2 , a method for preparing a tacrolimus positional isomer comprises the following steps: Step S21. Adding tetrakis(triphenylphosphine)palladium to the solvent in which tacrolimus is dissolved and heating the solvent to react until the tacrolimus content is less than 2%.

[0027] In this embodiment, the content of tacrolimus is determined by liquid chromatography, specifically, the reaction process is monitored every 30 minutes by liquid chromatography until the content of tacrolimus is less than 2%. The solvent for dissolving tacrolimus is any one of ethanol, methanol, tetrahydrofuran, chloroform, toluene, xylene, acetone, butanone, and ethyl acetate, preferably tetrahydrofuran or ethyl acetate.

[0028] The volume ratio of the solvent to tacrolimus is 2 to 30:1, with a 5 to 10-fold volume ratio being preferred.

[0029] Furthermore, tetrakis(triphenylphosphine)palladium is used as a catalyst in this embodiment, and its usage accounts for 0.1% to 5% of the solvent in which tacrolimus is dissolved, preferably 1% to 2%.

[0030] In this embodiment, a temperature-raising reaction is adopted for the reaction process, wherein the reaction temperature is selected to be in the range of 50-100° C., preferably 60-70° C. Compared with the reaction temperature of 140° C. adopted in the prior art, the reaction temperature in this embodiment is lower, thus avoiding the problems of many side reactions, difficult passivation, complicated operation and low product yield under high temperature reaction conditions in the prior art.

[0031] Step S22: filtering and distilling the solvent to obtain a crude product of tacrolimus isomers.

[0032] In this embodiment, filtration is used to remove the catalyst, i.e., tetrakis(triphenylphosphine)palladium, in the solvent, and distillation is used to remove the solvent, thereby obtaining an intermediate product containing only a crude product of tacrolimus isomers. The filtration and distillation treatments can be implemented by methods in the prior art, which will not be described in detail in this embodiment.

[0033] Step S23: Recrystallizing the crude product of tacrolimus position isomer to obtain tacrolimus position isomer.

[0034] In this embodiment, the recrystallization process is implemented by a dual solvent method, specifically, by sequentially adding two different solvents to the crude tacrolimus isomer product. First, the crude tacrolimus isomer position isomer product is dissolved in a first solvent, and then the second solvent is slowly added based on the degree of solubility.

[0035] In this embodiment, the second solvent is added when the reaction temperature reaches 10-30°C.

[0036] Furthermore, in this embodiment, the first solvent is any one of ethyl acetate, isopropyl acetate, butyl acetate, toluene and xylene, and the second solvent is any one of n-hexane, n-heptane, n-pentane, cyclohexane, isooctane and petroleum ether.

[0037] Finally, the product after the above reaction is filtered and dried to obtain the tacrolimus positional isomer. The filtering and drying processes can be carried out by methods in the prior art, which will not be described in detail in this embodiment.

[0038] The following specific examples are used to further illustrate the method for preparing the tacrolimus positional isomers of the present invention.

[0039] Example 1 5g of tacrolimus was dissolved in 40ml of tetrahydrofuran solvent, 0.05g of 1% w / w tetrakis(triphenylphosphine)palladium was added, and the temperature was maintained at 60-65°C for 3h. During this process, the reaction process was monitored by liquid chromatography every 30min. At this time, the tacrolimus residue was 0.13%, and the tacrolimus position isomer content was 82.11%. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. 15ml of ethyl acetate was added to the residue to dissolve it, and then 30ml of n-hexane was slowly added dropwise at 25°C. After filtering and drying, 3.32g of tacrolimus position isomer was obtained in the form of white powder.

[0040] Example 2 2g of tacrolimus was dissolved in 20ml of ethyl acetate solvent, 0.04g of 2% w / w tetrakis(triphenylphosphine)palladium was added, and the temperature was maintained at 65-70°C for 2.5h. During this process, the reaction process was monitored by liquid chromatography every 30min. At this time, the tacrolimus residue was 0.08%, and the tacrolimus position isomer content was 78.63%. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. 6ml of ethyl acetate was added to the residue to dissolve, and 12ml of n-heptane was slowly added dropwise at 30°C. After filtering and drying, 1.24g of tacrolimus position isomer was obtained.

[0041] Example 3 2g of tacrolimus was dissolved in 15ml of acetone solvent, and 0.04g of 2% w / w tetrakis(triphenylphosphine)palladium was added, and the reaction was refluxed until the residual amount of tacrolimus was 0.20%. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. 6ml of propyl acetate was added to the residue to dissolve it, and then 12ml of cyclohexane was slowly added dropwise at 30°C. After filtering and drying, 1.36g of tacrolimus positional isomer was obtained.

[0042] Example 4 2g of tacrolimus was dissolved in 60ml of toluene, 0.002g of 0.1% w / w tetrakis(triphenylphosphine)palladium was added, the temperature was raised to 80°C for 4h, and the reaction process was monitored every 30min by liquid chromatography during the process. At this time, the tacrolimus residue was 0.75%, and the tacrolimus positional isomer content was 71.42%. The reaction solution was filtered, the filtrate was concentrated to dryness under reduced pressure, 5ml of ethyl acetate was added to dissolve, 15ml of isooctane was added dropwise at 10°C, and 1.21g of the product was obtained after filtration and drying.

[0043] Example 5 2g of tacrolimus was dissolved in 4ml of methanol, 0.1g of 5% w / w tetrakis(triphenylphosphine)palladium was added, the temperature was raised to 50°C for 4h, and the reaction process was monitored every 30min by liquid chromatography during the process. At this time, the tacrolimus residual content was 1.68%, and the tacrolimus position isomer content was 81.33%. The reaction solution was filtered, the filtrate was concentrated to dryness under reduced pressure, 12ml of butyl acetate was added to dissolve, 24ml of n-hexane was added dropwise at 25°C, and 1.18g of the product was obtained by filtration and drying.

[0044] Example 6 2g of tacrolimus was dissolved in 60ml of tetrahydrofuran, 0.04g of 2% w / w tetrakis(triphenylphosphine)palladium was added, the temperature was raised to 60°C for 6h, and the reaction process was monitored every 30min by liquid chromatography during the process. At this time, the tacrolimus residue was 0.11%, and the tacrolimus position isomer content was 80.15%. The reaction solution was evaporated under reduced pressure to remove the solvent, 6ml of ethyl acetate was added to the residue to dissolve, and then 12ml of n-hexane was slowly added dropwise at 30°C, and 1.29g of the product was obtained by filtering, mixing and drying.

[0045] Experimental Example 1 The tacrolimus positional isomers of Examples 1 to 6 were sequentially subjected to HPLC detection, wherein the parameters of the HPLC detection were: two SUPELCOSTLC- DIOL columns with a size of 4.6 mm * 250 mm were used as chromatographic columns, the mobile phase was n-hexane-chlorobutane-acetonitrile, the ratio was 7:2:1, the flow rate was 1.3 ml / min, the column temperature was 30 ° C, the detection wavelength was 225 nm, the injection volume was 20 ul, and the injection concentration was 2 mg / ml.

[0046] The purity results of the tacrolimus positional isomers in Examples 1 to 6 can be determined by HPLC detection. The results can be found in Table 1, and Table 1 also records the yields of the tacrolimus positional isomers in Examples 1 to 6.

[0047] Table 1. Results of tacrolimus positional isomers

[0048] As shown in Table 1, the preparation methods disclosed in Examples 1 to 6 can effectively obtain high-purity tacrolimus positional isomers with a relatively stable yield. Moreover, among Examples 1 to 6, Example 1 can be used as the optimal example in terms of purity and yield. Moreover, the HPLC detection spectrum of the tacrolimus positional isomers prepared in Example 1 can be found in Figure 3 .

[0049] Experimental Example 2 The tacrolimus positional isomers obtained in Example 1 were detected by nuclear magnetic resonance hydrogen spectroscopy (400M, CDCl3) to obtain a spectrum for indicating the structural characteristics of the isomers. For the nuclear magnetic resonance spectrum, please refer to Figure 4 shown.

[0050] Experimental Example 3 The tacrolimus positional isomers obtained in Example 1 were detected by carbon nuclear magnetic resonance spectroscopy (100M, CDCl3) to obtain a spectrum for indicating the structural characteristics of the isomers. For the nuclear magnetic resonance spectrum, please refer to Figure 5 shown.

[0051] Comparative Example 1 In this example, 0.5 g of tacrolimus was placed in a culture dish and heated in an oven at 130° C. for 5 minutes. Samples were taken for HPLC detection. The test results showed that tacrolimus had been completely degraded, but no tacrolimus positional isomers were detected in the degradation products. The product was heated at 130° C. for 1 hour, and samples were taken for HPLC detection. No tacrolimus positional isomers were detected in the product.

[0052] Comparative Example 2 This embodiment is a method for preparing a tacrolimus positional isomer provided in the document (CN201210483603.2, 2012). The document records that tacrolimus is heated at 110-150° C. for 2-25 minutes to obtain a crude tacrolimus positional isomer; the crude tacrolimus positional isomer is passed through a silica gel column with a particle size of 100-400 mesh, and the eluate with a purity of ≥75% is collected and concentrated under reduced pressure; and the pure tacrolimus positional isomer is obtained after recrystallization with a yield of 17-18% and a purity of 97.8-98.6%.

[0053] Comparative Example 3 This embodiment is another method for preparing positional isomers of tacrolimus provided in the document (201410057153.X, 2014), which records that tacrolimus is isomerized at 130-140°C; purified by column chromatography; and recrystallized several times to obtain pure positional isomers of tacrolimus with a yield of 14.5% and a purity of 98.54%.

[0054] Comparative Example 4 This example is a document (European Journal of Pharmaceutical Sciences, Vol. 48, pp514-522, 2013), which records that tacrolimus was refluxed in o-xylene for 24 hours, concentrated under reduced pressure, and then purified by preparative chromatography to obtain pure tacrolimus positional isomers with a yield of 19%.

[0055] By comparing Example 4, it can be seen that the tacrolimus positional isomer can also be obtained without high temperature reaction conditions, but the yield of the above method is much lower than the yield corresponding to Examples 1 to 6. By using Comparative Examples 2 and 3, the tacrolimus positional isomer with higher purity can be obtained by preparing under high temperature reaction conditions, but the yield is also lower than the yield corresponding to Examples 1 to 6.

[0056] It can be seen from Examples 1 to 6, Experimental Examples 1 to 3 and Comparative Examples that in the preparation method provided in the embodiments of the present application, by introducing the catalyst tetrakis(triphenylphosphine)palladium during the reaction process, it is possible to prepare tacrolimus positional isomers with higher purity on the basis of lowering the reaction temperature. Compared with the prior art, it has the technical advantage of low-temperature reaction, which reduces the overall reaction side reactions, reduces the difficulty of purification, and improves the product yield.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a tacrolimus positional isomer, characterized in that: The tacrolimus positional isomer is a (E / Z)-FK-506 26,28-allyl ester rearrangement impurity with a CAS number of 131944-48-4. The method comprises: adding tetrakis(triphenylphosphine)palladium to a solvent in which tacrolimus is dissolved and performing a temperature-raising reaction, monitoring the reaction process every 30 minutes by liquid chromatography until the tacrolimus content is less than 2%, filtering and distilling to obtain a crude product of the tacrolimus positional isomer, and recrystallizing the crude product of the tacrolimus positional isomer to obtain the tacrolimus positional isomer.

2. The method for preparing a tacrolimus positional isomer according to claim 1, characterized in that: The crude tacrolimus positional isomers were recrystallized by sequentially adding two different solvents.

3. The method for preparing a tacrolimus positional isomer according to claim 1, characterized in that: The solvent includes any one of ethanol, methanol, tetrahydrofuran, chloroform, toluene, xylene, acetone, butanone, and ethyl acetate.

4. The method for preparing a tacrolimus positional isomer according to claim 3, characterized in that: The volume ratio of the solvent to the tacrolimus is 2-30:

1.

5. The method for preparing a tacrolimus positional isomer according to claim 4, characterized in that: The volume ratio of the solvent to the tacrolimus is 5-10:

1.

6. The method for preparing a tacrolimus positional isomer according to claim 1, characterized in that: The amount of the tetrakis(triphenylphosphine)palladium used is 0.1% to 5% of the amount of the solvent.

7. The method for preparing a tacrolimus positional isomer according to claim 6, characterized in that: The amount of the tetrakis(triphenylphosphine)palladium used is 1-2% of the amount of the solvent.

8. The method for preparing a tacrolimus positional isomer according to claim 1, characterized in that: The reaction temperature of the temperature-raising reaction is 50-100°C.

9. The method for preparing a tacrolimus positional isomer according to claim 2, characterized in that: The first solvent of the two different solvents includes any one of ethyl acetate, isopropyl acetate, butyl acetate, toluene and xylene, and the second solvent includes any one of n-hexane, n-heptane, n-pentane, cyclohexane, isooctane and petroleum ether.

10. The method for preparing a tacrolimus positional isomer according to claim 9, characterized in that: The second solvent is added at a temperature of 10-30°C.

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