A method for preparing a tacrolimus positional isomer

Abstract

The present invention relates to the technical field of pharmaceutical engineering, and specifically relates to a preparation method of tacrolimus position isomers. By introducing the catalyst tetrakis(triphenylphosphine)palladium during the reaction process, it is possible to prepare tacrolimus position isomers with relatively high purity on the basis of reducing the reaction temperature. Compared with the prior art, it has the technical advantage of low-temperature reaction, resulting in the technical effects of reducing side reactions in the overall reaction, lowering the purification difficulty, and increasing the product yield.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical engineering, and particularly relates to a preparation method of tacrolimus regioisomer. Background Art

[0002] Tacrolimus may undergo isomerization under high temperature conditions to generate a regioisomer (tacrolimus regioisomer, CAS 131944-48-4), which has been included in the quality standard of tacrolimus capsules in the United States Pharmacopeia USP-NF2024.

[0003] Currently, the preparation methods of this isomer reported in the literature generally use high-temperature reactions, thus having problems such as many side reactions, difficult purification, cumbersome operation, and low product yield. Summary of the Invention

[0004] To solve the above problems, the embodiments of the present application provide a preparation method of tacrolimus regioisomer. By introducing a catalyst, the overall reaction temperature can be reduced, thereby reducing side reactions and greatly improving the yield.

[0005] To achieve the above object, the technical solutions adopted in the embodiments of the present application are as follows:

[0006] In a first aspect, the embodiments of the present application provide a preparation method of tacrolimus regioisomer. The tacrolimus regioisomer is (E / Z)-FK-506 26,28-allyl ester rearrangement impurity, with a CAS number of 131944-48-4. The method includes: adding tetrakis(triphenylphosphine)palladium to a solvent in which tacrolimus is dissolved and carrying out a temperature-raising reaction. The reaction process is monitored by liquid chromatography every 30 minutes until the tacrolimus content is less than 2%. After filtration and distillation treatment, a crude product of tacrolimus regioisomer is obtained, and the crude product of tacrolimus regioisomer is recrystallized to obtain tacrolimus regioisomer.

[0007] Further, the crude product of tacrolimus regioisomer is recrystallized by sequentially adding two different solvents.

[0008] Further, the solvent includes any one of ethanol, methanol, tetrahydrofuran, chloroform, toluene, xylene, acetone, methyl ethyl ketone, and ethyl acetate.

[0009] Further, the volume ratio of the solvent to tacrolimus is 2 to 30:1.

[0010] Further, the volume ratio of the solvent to tacrolimus is 5 to 10:1.

[0011] Further, the dosage of tetrakis(triphenylphosphine)palladium accounts for 0.1% to 5% of the solvent amount.

[0012] Further, the dosage of tetrakis(triphenylphosphine)palladium accounts for 1-2% of the solvent amount.

[0013] Further, the reaction temperature of the temperature-raising reaction is 50-100 °C.

[0014] Further, the first solvent in 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.

[0015] Further, the second solvent is added at a temperature of 10-30 °C.

[0016] In the technical solution provided by the embodiment of the present application, by introducing the catalyst tetrakis(triphenylphosphine)palladium during the reaction process, it is possible to prepare a relatively high-purity tacrolimus positional isomer on the basis of reducing the reaction temperature. Compared with the prior art, it has the technical advantage of low-temperature reaction, resulting in the technical effects of reducing side reactions in the overall reaction, reducing the purification difficulty, and increasing the product yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is a schematic structural diagram of tacrolimus and its positional isomers.

[0019] Figure 2 It is a flowchart of the method for preparing tacrolimus positional isomers provided by the embodiment of the present application.

[0020] Figure 3 It is a schematic diagram of the HPLC detection spectrum of the tacrolimus positional isomer prepared in Example 1.

[0021] Figure 4 It is the hydrogen spectrum of the tacrolimus positional isomer prepared in Example 1.

[0022] Figure 5 It is the carbon spectrum of the tacrolimus positional isomer prepared in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated with the manufacturer are all conventional products that can be obtained through commercial purchase. The embodiments described below are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts in combination with the embodiments of the present invention belong to the scope of protection of the present invention.

[0024] The embodiments of the present application provide a method for preparing the positional isomer of tacrolimus. Tacrolimus is a macrolide compound isolated from Streptomyces and belongs to 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 intracellular FKBP12 protein, forms a complex and accumulates in the cell, thereby inhibiting the activation and proliferation of T cells, and simultaneously 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 advantages such as low toxicity and side effects, high transplantation survival rate, and low dependence. Therefore, it is widely used clinically in the treatment of various autoimmune diseases, 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 of the liver, kidney, heart, lung, pancreas, etc.

[0025] The positional isomer of tacrolimus is a positional isomer generated by the 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. For the preparation of this positional isomer, high-temperature reactions are currently mainly used. For example, in the literature (Eur J Pharm Sci, 2013, 48, 514–522), o-xylene (boiling point 143~145 °C) was refluxed for 24 hours, and after purification by preparative chromatography, the yield was only 19%. In Chinese Patent CN201210483603.2, tacrolimus was heated at 110~150 °C, and after silica gel column chromatography and recrystallization, the pure product was obtained with a yield of 17~18%. Moreover, due to the high sensitivity of the solid-phase reaction to the particle size and dispersion degree of the raw materials, the reaction mass transfer was uneven and the by-products were complex, resulting in poor batch-to-batch reproducibility. And because parameters such as the particle size and morphology 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 the process reproducibility is poor. In Chinese Patent CN201410057153.X, the tacrolimus solution was isomerized at 130~140 °C, and after several times of column chromatography and recrystallization, the pure product was obtained with a yield of 14.5%.

[0026] Aiming at the problems in the above prior art, in order to solve them correspondingly, in this embodiment, a preparation method of tacrolimus positional isomer is provided. By using a reaction catalyst different from that in the prior art, the reaction temperature can be reduced, thereby reducing side reactions and directly increasing the yield of tacrolimus positional isomer.

[0027] For the process flow of this method, reference can be made to Figure 2 Regarding the preparation method of tacrolimus positional isomer, it includes the following steps:

[0028] Step S21. Add palladium tetrakis(triphenylphosphine) to the solvent in which tacrolimus is dissolved and heat up the reaction until the tacrolimus content is less than 2%.

[0029] In this embodiment, the determination of the tacrolimus content is determined by liquid chromatography. Specifically, the reaction process is monitored every 30 min by liquid chromatograph until the tacrolimus content is less than 2%. Among them, the solvent for dissolving tacrolimus is any one of ethanol, methanol, tetrahydrofuran, chloroform, toluene, xylene, acetone, butanone, and ethyl acetate, and tetrahydrofuran or ethyl acetate is preferably selected.

[0030] Among them, the volume ratio of the solvent to tacrolimus is 2~30:1, and a volume ratio of 5~10 times is preferably selected.

[0031] Furthermore, palladium tetrakis(triphenylphosphine) is used as a catalyst in this embodiment. Regarding its dosage, it accounts for 0.1%~5% of the solvent in which tacrolimus is dissolved, and 1~2% is preferably selected.

[0032] In this embodiment, a temperature-raising reaction is adopted for this reaction process, where the reaction temperature is selected in the range of 50-100°C for the temperature-raising range, and 60-70°C is preferably selected. Compared with the reaction temperature of 140°C in the prior art, the reaction temperature in this embodiment is lower, avoiding the problems of many side reactions, difficult passivation, cumbersome operation, and low product yield under high-temperature reaction conditions in the prior art.

[0033] Step S22. Filter and distill the solvent to obtain a crude product of tacrolimus isomers.

[0034] In this embodiment, the filtration is used to remove the catalyst, i.e., tetrakis(triphenylphosphine)palladium, in the solvent, and the distillation is used to remove the solvent, thereby obtaining an intermediate product containing only the crude product of tacrolimus isomers. Among them, the filtration and distillation processes can be implemented by the methods in the prior art and will not be elaborated in this embodiment.

[0035] Step S23. Recrystallize the crude product of tacrolimus position isomers to obtain tacrolimus position isomers.

[0036] In this embodiment, the recrystallization process is implemented by the double-solvent method. Specifically, two different solvents are sequentially added to the crude product of tacrolimus isomers. First, the crude product of tacrolimus position isomers is dissolved in the first solvent, and then the second solvent is slowly added based on the dissolution degree.

[0037] Among them, the time node for adding the second solvent in this embodiment is when the reaction temperature reaches 10-30°C.

[0038] 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.

[0039] Finally, filter and dry the product after the above reaction to obtain tacrolimus position isomers. Among them, the filtration and drying processes can be implemented by the methods in the prior art and will not be elaborated in this embodiment.

[0040] The preparation method of the tacrolimus position isomers described in the present invention will be further illustrated below through specific examples.

[0041] Example 1

[0042] Dissolve 5 g of tacrolimus in 40 ml of tetrahydrofuran solvent, add 0.05 g of tetrakis(triphenylphosphine)palladium with a concentration of 1% w / w, and react at a temperature of 60 - 65 °C for 3 h. During this process, monitor the reaction process every 30 min by liquid chromatography. At this time, the residual amount of tacrolimus is 0.13%, and the content of tacrolimus positional isomers is 82.11%. Filter the reaction solution, concentrate the filtrate under reduced pressure to dryness, dissolve the residue in 15 ml of ethyl acetate, and slowly add 30 ml of n-hexane dropwise while maintaining a temperature of 25 °C. After filtration and drying, 3.32 g of tacrolimus positional isomers are obtained, which are in the form of white powder.

[0043] Example 2

[0044] Dissolve 2 g of tacrolimus in 20 ml of ethyl acetate solvent, add 0.04 g of tetrakis(triphenylphosphine)palladium with a concentration of 2% w / w, and react at a temperature of 65 - 70 °C for 2.5 h. During this process, monitor the reaction process every 30 min by liquid chromatography. At this time, the residual amount of tacrolimus is 0.08%, and the content of tacrolimus positional isomers is 78.63%. Filter the reaction solution, concentrate the filtrate under reduced pressure to dryness, dissolve the residue in 6 ml of ethyl acetate, and slowly add 12 ml of n-heptane dropwise while maintaining a temperature of 30 °C. After filtration and drying, 1.24 g of tacrolimus positional isomers are obtained.

[0045] Example 3

[0046] Dissolve 2 g of tacrolimus in 15 ml of acetone solvent, add 0.04 g of tetrakis(triphenylphosphine)palladium with a concentration of 2% w / w, and reflux the reaction until the residual amount of tacrolimus is 0.20%. Filter the reaction solution, concentrate the filtrate under reduced pressure to dryness, dissolve the residue in 6 ml of propyl acetate, and slowly add 12 ml of cyclohexane dropwise while maintaining a temperature of 30 °C. After filtration and drying, 1.36 g of tacrolimus positional isomers are obtained.

[0047] Example 4

[0048] Dissolve 2 g of tacrolimus in 60 ml of toluene, add 0.002 g of tetrakis(triphenylphosphine)palladium with a concentration of 0.1% w / w, heat up to 80 °C and react for 4 h. During this process, monitor the reaction process every 30 min by liquid chromatography. At this time, the residual amount of tacrolimus is 0.75%, and the content of tacrolimus positional isomers is 71.42%. Filter the reaction solution, concentrate the filtrate under reduced pressure to dryness, dissolve it in 5 ml of ethyl acetate, and add 15 ml of isooctane dropwise at 10 °C. After filtration and drying, 1.21 g of the product is obtained.

[0049] Example 5

[0050] Dissolve 2 g of tacrolimus in 4 ml of methanol, add 0.1 g of tetrakis(triphenylphosphine)palladium with a concentration of 5% w / w, heat up to 50 °C and react for 4 h. During this process, monitor the reaction process by liquid chromatography every 30 min. At this time, the residual amount of tacrolimus is 1.68%, and the content of the tacrolimus positional isomer is 81.33%. Filter the reaction solution, concentrate the filtrate under reduced pressure to dryness, dissolve it in 12 ml of butyl acetate, and slowly add 24 ml of n-hexane dropwise at 25 °C. Obtain 1.18 g of the product through filtration and drying.

[0051] Example 6

[0052] Dissolve 2 g of tacrolimus in 60 ml of tetrahydrofuran, add 0.04 g of tetrakis(triphenylphosphine)palladium with a concentration of 2% w / w, heat up to 60 °C and react for 6 h. During this process, monitor the reaction process by liquid chromatography every 30 min. At this time, the residual amount of tacrolimus is 0.11%, and the content of the tacrolimus positional isomer is 80.15%. Evaporate the solvent from the reaction solution under reduced pressure, dissolve the residue in 6 ml of ethyl acetate, and slowly add 12 ml of n-hexane dropwise while maintaining the temperature at 30 °C. Obtain 1.29 g of the product through filtration, mixing, and drying.

[0053] Experimental Example 1

[0054] Perform HPLC detection on the tacrolimus positional isomers of Examples 1 - 6 in sequence. The parameters for HPLC detection are as follows: Use two SUPELCOSTLC-DIOL columns with dimensions of 4.6 mm * 250 mm as the chromatographic columns, the mobile phase is n-hexane - chloro-butane - acetonitrile, with a ratio of 7:2:1, the flow rate is 1.3 ml / min, the column temperature is 30 °C, the detection wavelength is 225 nm, the injection volume is 20 μl, and the injection concentration is 2 mg / ml.

[0055] Through HPLC detection, the purity results of the tacrolimus positional isomers in Examples 1 - 6 can be determined. For this result, refer to Table 1, and Table 1 also records the yields of the tacrolimus positional isomers in Examples 1 - 6.

[0056] Table 1. Results Table of Tacrolimus Positional Isomers

[0057]

[0058] As can be seen from Table 1, through the preparation methods disclosed in Examples 1 - 6, high-purity tacrolimus positional isomers can be effectively obtained, and the yields are relatively stable. Moreover, among Examples 1 - 6, Example 1 can be used as the optimal example considering both purity and yield. And for the HPLC detection spectrum of the tacrolimus positional isomer prepared in Example 1, refer to Figure 3 .

[0059] Experimental Example 2

[0060] The tacrolimus position isomers obtained in Example 1 were detected by 1H NMR (400M, CDCl3), and the obtained spectrum was used to show the structural characteristics of the isomers. For the NMR spectrum, refer to Figure 4 as shown.

[0061] Experimental Example 3

[0062] The tacrolimus position isomers obtained in Example 1 were detected by 13C NMR (100M, CDCl3), and the obtained spectrum was used to show the structural characteristics of the isomers. For the NMR spectrum, refer to Figure 5 as shown.

[0063] Comparative Example 1

[0064] In this example, 0.5 g of tacrolimus was placed in a petri dish and heated in an oven at 130 °C for 5 minutes. The sample was taken for HPLC detection. The detection results showed that tacrolimus had been completely degraded, but no tacrolimus position isomers were detected in the degradation products. Heating was continued at 130 °C for 1 h, and the sample was taken for HPLC detection. No tacrolimus position isomers were still detected in the product.

[0065] Comparative Example 2

[0066] This example was a method for preparing tacrolimus position isomers provided in the literature (CN201210483603.2, 2012). It was recorded in this literature that tacrolimus was heated at 110 - 150 °C for 2 - 25 minutes to obtain a crude product of tacrolimus position isomers; silica gel with a particle size of 100 - 400 mesh was used for column chromatography, and the eluate with a purity ≥ 75% was collected and concentrated under reduced pressure; after recrystallization, a pure product of tacrolimus position isomers was obtained, with a yield of 17 - 18% and a purity of 97.8 - 98.6%.

[0067] Comparative Example 3

[0068] This example was another method for preparing tacrolimus position isomers provided in the literature (201410057153.X, 2014). It was recorded in this literature that tacrolimus was isomerized at 130 - 140 °C; purified by column chromatography; after several recrystallizations, a pure product of tacrolimus position isomers was obtained, with a yield of 14.5% and a purity of 98.54%.

[0069] Comparative Example 4

[0070] This example is from the literature (European Journal of Pharmaceutical Sciences, Vol. 48, pp514–522, 2013). In this literature, it is recorded that tacrolimus was refluxed in o-xylene for 24 h, concentrated under reduced pressure, and then separated and purified by preparative chromatography to obtain pure tacrolimus position isomers with a yield of 19%.

[0071] It can be seen from Comparative Example 4 that tacrolimus position isomers can also be obtained without using high-temperature reaction conditions. However, the yield of the above method is much lower than that of Examples 1 - 6. By using Comparative Example 2 and Comparative Example 3, tacrolimus position isomers with higher purity can be obtained by high-temperature reaction conditions, but their yields are also lower than those of Examples 1 - 6.

[0072] It can be seen from Examples 1 - 6, Experimental Examples 1 - 3, and Comparative Examples that in the preparation method provided in the examples of the present application, by introducing the catalyst tetrakis(triphenylphosphine)palladium during the reaction process, tacrolimus position isomers with higher purity can be prepared on the basis of reducing the reaction temperature. Compared with the prior art, it has the technical advantage of low-temperature reaction, resulting in the technical effects of reducing side reactions in the overall reaction, reducing the difficulty of purification, and increasing the product yield.

[0073] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the principles of the present invention shall be included within 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 containing tacrolimus 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; and the reaction temperature of the temperature-raising reaction is 50 to 100°C.

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 to 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 to 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 accounts for 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 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.

9. The method for preparing a tacrolimus positional isomer according to claim 8, characterized in that: The second solvent is added at a temperature of 10 to 30°C.

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