Method for preparing salts of isocyclosporin A

Abstract

The present invention belongs to the technical field of drug synthesis. In particular, the present invention relates to a method for preparing salts of isocyclosporin A, in particular a method for preparing salts of isocyclosporin A by transesterification of cyclosporin A.

Description

[Technical field]

[0001] The present invention belongs to the technical field of drug synthesis. In particular, the present invention relates to a method for preparing salts of isocyclosporin A, in particular a method for preparing salts of isocyclosporin A by transesterification of cyclosporin A. [Background technology]

[0002] Cyclosporine is an oligopeptide with a cyclic structure that has antifungal properties and immunosuppressive effects and is used to regulate the body's immune response in organ transplants to prevent rejection.

[0003] Since the original discovery of cyclosporin, several natural cyclosporins have been isolated and identified, while non-natural cyclosporins have been obtained through semi-synthetic or culture techniques. Cyclosporin A is the cyclosporin primarily used as a drug.

[0004] Cyclosporine A is used as monotherapy or in combination with other immunosuppressants, but its main indication is the prevention of rejection in organ transplants, particularly kidney, pancreas, liver and heart transplants. Cyclosporin A can also be used to treat autoimmune diseases such as uveitis, rheumatoid arthritis, psoriasis and ulcerative colitis.

[0005] Cyclosporine has a complex chemical structure, since it is formed by 11 peptides and contains several N-methylated amino acids. As a result, the synthesis using peptide condensation reagents is quite complicated and time-consuming. Therefore, the method mainly used for the synthesis of cyclosporine at present is the fermentation method using two fungi, namely Trichoderma polysporum and Cylindrocarpon lucidum (Survase, SA, Kagliwal, LD, Annapure, US & Singhal, RS Cyclosporin A - a review on fermentative production, downstream processing and pharmacological applications. Biotech. Advances. 29, 418-435 (2011)). However, this synthesis method does not provide a high yield of cyclosporine.

[0006] In 2010, a research group led by chemist Danishevsky attempted to synthesize cyclosporine A by isonitrile condensation in liquid phase. However, this synthesis method requires the use of many condensation reagents and is therefore complicated. Therefore, solid-phase synthesis is currently not feasible except through a method that requires many difficulties and a long time.

[0007] Furthermore, the use of cyclosporine A is limited due to its low bioavailability and high toxicity, especially nephrotoxicity. Indeed, after oral administration of cyclosporine, the blood concentration level reaches a high peak and then declines rapidly. As a result, oral administration of an effective dose of cyclosporine leads to transient but dangerously high concentrations of cyclosporine in the blood at the peak blood concentration level, which leads to several side effects, especially kidney and liver damage.

[0008] Recently, it has been observed that some isocyclosporines have improved pharmacokinetic profiles compared to cyclosporine, particularly isocyclosporines A, B, D, and G. Advantageously, isocyclosporines, i.e., isomers of cyclosporine, are absorbed in the intestine in relatively inactive, non-toxic isoforms that are subsequently converted to the pharmacologically active cyclosporine form, thereby reducing peak blood concentrations following administration.

[0009] It is an object of the present invention to provide a process for the preparation of salts of isocyclosporin A to overcome the problems encountered in existing cyclosporin synthesis processes outlined above. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Survase, SA, Kagliwal, LD, Annapure, US & Singhal, RS Biotech. Advances. 29, 418-435 (2011) Summary of the Invention

[0011] The applicant has developed a process for the preparation of salts of isocyclosporine A by direct conversion of cyclosporine A. The isocyclosporine obtained by the process according to the invention has a good pharmacokinetic profile and can therefore be used as a drug instead of cyclosporine.

[0012] A first aspect of the present invention relates to a process for the preparation of a salt of isocyclosporine A by transesterification of cyclosporine A to a salt of isocyclosporine A, the process comprising: a) dissolving the cyclosporin A in anhydrous methanol and adding trifluoroacetic acid; b) heating the solution obtained by step a) to a temperature ranging from 50° C. to the reflux temperature of the reaction mixture for a period ranging from 30 to 60 hours; c) removing the methanol and excess of the trifluoroacetic acid; d) Recovering the salt of isocyclosporin A with trifluoroacetic acid. The molar ratio of the trifluoroacetic acid to the methanol in the solution obtained by step a) is 1:3.

[0013] The applicants have observed that with an optimum molar ratio of 1:3 between the acid compounds, particularly trifluoroacetic acid and methanol, a 53% conversion of cyclosporine A to isocyclosporine A is obtained without by-products, whereas a molar ratio of 1:1 or 1:4 between the acid compounds and methanol results in a low conversion rate of cyclosporine A to isocyclosporine A (30% or 20%) (see Examples 1 and 2). On the other hand, a molar ratio of 3:1 between the acid compounds and methanol results in a high conversion rate of cyclosporine A to isocyclosporine A (75%) but with numerous by-products (Example 2).

[0014] With this in mind, a molar ratio of 1:3 resulted to be the best ratio since 53% of cyclosporine was converted to the isoforms without by-products and the unconverted residue could be recycled.

[0015] In a second embodiment of the invention, the solution of cyclosporin A and methanol according to step a) is heated by microwaves. According to said second aspect, a process for the preparation of a salt of isocyclosporine A by transesterification of cyclosporine A to a salt of isocyclosporine A comprises the steps of: a) dissolving the cyclosporin A in anhydrous methanol and adding trifluoroacetic acid; b) heating the solution obtained by step a) in a microwave oven; c) removing the methanol and excess of the trifluoroacetic acid; d) Recovering the salt of isocyclosporin A with trifluoroacetic acid. The process includes:

[0016] In particular, the microwave heating according to process step b) is carried out at a temperature ranging from 55° C. to 65° C. for a time ranging from 10 to 20 hours, preferably about 15 hours. In a particularly preferred embodiment, step b) is carried out at 60° C. for 15 hours.

[0017] In fact, microwave heating at 60° C. for 15 hours makes it possible to obtain a yield of isocyclosporin A or its salts of 100%. A second aspect of the second embodiment of the present invention is a continuous flow microwave system for producing salts of isocyclosporine A according to the method of the present invention.

[0018] The continuous flow microwave system includes one or more distribution units for starting reagents, one or more microwave reactors, and one or more product collectors. In particular, the starting reagents are fed to one or more microwave reactors using one or more pumps, preferably one or more HPLC pumps or syringe pumps.

[0019] A system according to the present invention also includes one or more coolers and one or more backpressure regulators. In a preferred embodiment, the continuous flow microwave system comprises multiple microwave reactors in parallel.

[0020] The combination of microwave heating and continuous flow technology allows increased yields of isocyclosporin A to be advantageously obtained. [Brief description of the drawings]

[0021] [Figure 1] 1 shows the results of liquid chromatography of a salt of isocyclosporine A obtained by using a molar ratio of trifluoroacetic acid and methanol of 1:3. In FIG. 1, "IsoCsA" represents isocyclosporine A, and "CsA" represents cyclosporine A. [Diagram 2]2 shows the results of liquid chromatography of a salt of isocyclosporine A obtained by using a molar ratio of trifluoroacetic acid to methanol of 1:4 (see Example 2). In FIG. 2, "IsoCsA" represents isocyclosporine A, and "CsA" represents cyclosporine A. [Diagram 3] Figure 3 shows the liquid chromatography results of the salt of isocyclosporine A obtained by carrying out heating of the reaction mixture in a microwave. In Figure 3, "IsoCsA" stands for isocyclosporine A. [Figure 4] Figure 4 is a diagrammatic representation of a continuous flow microwave system in accordance with the present invention. In particular, the continuous flow microwave system shown in Figure 4 includes a distribution unit (1), a microwave reactor (2), and a product collector (3). Also shown in Figure 4 is a pump (4) that conveys starting reagents from the distribution unit (1) to the microwave reactor (2), a cooler (5), and a back pressure regulator (6). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In order to reduce the side effects caused by high blood cyclosporine concentrations after oral administration, the applicant has devised a method for producing a salt of isocyclosporine A, which is an isomer of cyclosporine A. This is achieved by producing a salt of isocyclosporine by transesterification of cyclosporine A. This method makes it possible to overcome the problems encountered in the production of salts of isocyclosporine A due to its complex chemical structure.

[0023] In fact, cyclosporine has the following formula I:

[0024] [ka]

[0025] It is a hydrophobic cyclic undecapeptide having the formula: Isocyclosporin A, an isomer of cyclosporin A, can alternatively be represented by the following formula (II):

[0026] [ka]

[0027] has. The structural differences between cyclosporine A and its isomers are shown in Scheme 1 below:

[0028] [ka]

[0029] This is represented in the figure. Isocyclosporine A is absorbed into the intestine in a relatively inactive and non-toxic isoform, which is then converted to the pharmacologically active form of cyclosporine, resulting in a lower peak blood concentration after administration. Therefore, isocyclosporine A can be used instead of cyclosporine A, since it has the same pharmacological effects as cyclosporine A but is less toxic.

[0030] The object of a first aspect of the present invention is a process for the preparation of a salt of isocyclosporine A by transesterification of cyclosporine A to a salt of isocyclosporine A, the process comprising the steps of: a) dissolving the cyclosporin A in anhydrous methanol and adding trifluoroacetic acid; b) heating the solution obtained by step a) to a temperature ranging from 50° C. to the reflux temperature of the reaction mixture for a period ranging from 30 to 60 hours; c) removing the methanol and excess of the trifluoroacetic acid; d) Recovering the salt of isocyclosporin A with trifluoroacetic acid. The molar ratio of the trifluoroacetic acid to the methanol in the solution obtained by step a) is 1:3.

[0031] Applicants have advantageously observed that a molar ratio between trifluoroacetic acid and methanol in the solution (comprising cyclosporine A, trifluoroacetic acid and methanol) formed in step a) equal to 1:3 results in a yield of 80% of the salt of isocyclosporine A. In one embodiment, the solution according to step a) comprises about 2 mmol of cyclosporine A and 60 mmol of methanol (see Table 1 in the Examples).

[0032] In particular, in the process according to the invention, step b) is carried out at a temperature ranging from 50° C. to the reflux temperature of the reaction mixture, preferably at a temperature of 60° C. In a particularly preferred embodiment, the solution according to step a), i.e. the solution containing cyclosporin A dissolved in methanol, is heated for 48 hours, preferably at a temperature of 60°C.

[0033] The reaction scheme according to the method of the present invention is shown below.

[0034] [ka]

[0035] The excess trifluoroacetic acid in step c) can be removed by stripping with diethyl ether under vacuum. As can be seen from Scheme 2 above, the starting cyclosporine can be removed during the salification step (step d') by adding DCM / NaHCO3 to the resulting salt of isocyclosporine A with trifluoroacetic acid.

[0036] After recovering the salt of isocyclosporin A with trifluoroacetic acid, the process finally comprises dissolving the salt of isocyclosporin A with trifluoroacetic acid obtained in step d) in a solution comprising an acid compound selected from citric acid and lactic acid and methanol.

[0037] In particular, the process according to the invention comprises downstream of step d) the following step: e) dissolving an acid compound selected from citric acid and lactic acid in methanol; f) dissolving said salt of isocyclosporine A with trifluoroacetic acid in the solution obtained in step e) while stirring the resulting solution for a time period ranging from 0.5 to 2 hours; g) removing the methanol and the trifluoroacetic acid; and h) recovering the salt of isocyclosporin A with the acid compound selected from citric acid and lactic acid. The method may include the steps of:

[0038] Examples of the preparation of salts of isocyclosporin A with acids selected from citric acid and lactic acid are given in the experimental section (Examples 3 and 4). The applicants have observed that an optimal molar ratio of 1:3 between trifluoroacetic acid and methanol results in 53% conversion of cyclosporine A to isocyclosporine A without any by-products, whereas a molar ratio of 1:1 or 1:4 between the acid compound and methanol results in lower conversion of cyclosporine A to isocyclosporine A (30% or 20%) (see Examples 1 and 2).

[0039] A molar ratio of acid to methanol of 3:1 results in a high conversion of cyclosporin A to isocyclosporin A (75%), but with numerous by-products. The overall yield of isocyclosporin A obtained by the above process is 80%.

[0040] According to a second aspect of the present invention, a process for the preparation of a salt of isocyclosporine A by transesterification of cyclosporine A to a salt of isocyclosporine A comprises the steps of: a) dissolving the cyclosporin A in anhydrous methanol and adding trifluoroacetic acid; b) heating the solution obtained by step a) in a microwave oven; c) removing the methanol and excess of the trifluoroacetic acid; and d) Recovering the salt of isocyclosporin A with trifluoroacetic acid. The process includes:

[0041] In particular, in step b) of the process, the solution obtained according to step a) is heated in a microwave at a temperature ranging from 55°C to 65°C, preferably at a temperature of 60°C. In particular, step b) is carried out for a time ranging from 10 to 20 hours, preferably for about 15 hours.

[0042] In a particularly preferred embodiment, the microwave heating according to process step b) is carried out at 60° C. for 15 hours. In fact, under these conditions it is possible to obtain a 100% yield of the salt of isocyclosporin A.

[0043] Scheme 3 below shows a reaction scheme according to the method of the present invention, in which a reaction solution between a compound (trifluoroacetic acid) and methanol is heated in a microwave (MW).

[0044] [ka]

[0045] In step c) of the process according to the invention, the excess acid compound is removed by stripping with diethyl ether under vacuum. After recovering the salt of isocyclosporin A with trifluoroacetic acid (step d), the method finally comprises dissolving said salt of isocyclosporin A with trifluoroacetic acid in a solution comprising an acid compound selected from citric acid and lactic acid and methanol.

[0046] In particular, the process according to the invention comprises downstream of step d) the following step: e) dissolving an acid compound selected from citric acid and lactic acid in methanol; f) dissolving said salt of isocyclosporine A with trifluoroacetic acid in the solution obtained in step e) while stirring the resulting solution for a time period ranging from 0.5 to 2 hours; g) removing the methanol and the trifluoroacetic acid; and h) recovering the salt of isocyclosporin A with the acid compound selected from citric acid and lactic acid. The method may include the steps of:

[0047] Examples of the preparation of salts of isocyclosporin A with acids selected from citric acid and lactic acid are given in the experimental section (Examples 3 and 4). The process for the preparation of salts of isocyclosporine A comprising microwave heating can be carried out by a continuous flow system comprising one or more microwave reactors. In particular, the applicant has observed that by combining microwave heating with continuous flow technology, high yields of isocyclosporine A can be obtained. Thus, a second aspect of the second embodiment of the present invention is a continuous flow microwave system for the preparation of salts of isocyclosporine A comprising heating the solution according to step a) in a microwave oven. In particular, the continuous flow microwave system for the preparation of salts of isocyclosporine A comprises one or more distribution units for the starting reagents represented by the solutions obtained in step a) of the process according to the present invention, one or more microwave reactors and one or more product collectors.

[0048] In particular, in said system the starting reagents are transported from a distribution unit to the microwave reactor by a pump, preferably an HPLC pump or a syringe pump. The system may also include one or more coolers and one or more backpressure regulators for monitoring pressure.

[0049] Additionally, there may be a sensor, such as a fiber optic sensor, to monitor the reaction temperature. In one embodiment, the system includes a single distribution unit for starting reagents, a single microwave reactor, and a single product collector (as shown in Figure 4). The reactor is also equipped with a pump, a cooler, and a back pressure cooler.

[0050] Preferably, the continuous flow microwave system includes two or more distribution units for starting reagents, two or more microwave reactors, and two or more product collectors, and may also include two or more pumps, two or more coolers, and two or more back pressure coolers.

[0051] More preferably, the continuous flow microwave system comprises multiple microwave reactors in parallel. EXAMPLES

[0052] Example 1: Preparation of isocyclosporin A salt with trifluoroacetic acid by transesterification of cyclosporin A - TFA:methanol molar ratio 1:3 Cyclosporine A (2.5 g, 2.08 mol) was dissolved in anhydrous methanol (2.45 ml). Trifluoroacetic acid (TFA) (1.5 ml) was added and the reaction was stirred at reflux at 60° C. for 48 hours.

[0053] The solvent was removed under reduced pressure and excess residual TFA was removed by stripping under vacuum with diethyl ether (2 x 15 ml). The salt of isocyclosporine A with dry TFA (1.33 g) appeared as a white powder. Approximately 53% conversion of the starting material and quantitative yield of Iso-CsA was obtained. No by-products were observed in the final reaction. The remaining starting material (CsA) was removed by adding NaHCO3 during the salification step.

[0054] As can be seen from Table 1, the molar ratio between trifluoroacetic acid and methanol is 1:3.

[0055] [Table 1]

[0056] The product was characterized by liquid chromatography (see Figure 1). Applicants have observed that extending the reaction time beyond 60 hours, e.g., to 72 hours, does not result in a significant increase in reaction yield and results in the formation of impurities. It is believed that extending the reaction time beyond 60 hours is not beneficial.

[0057] Therefore, the applicants concluded that the optimal reaction conditions are a molar ratio between trifluoroacetic acid and methanol of 1:3 with reaction times up to 60 hours. Example 2: Preparation of isocyclosporine A salts by transesterification of cyclosporine A - TFA:methanol molar ratio 1:1, 1:4, 3:1 Applicants conducted separate experiments varying the molar ratio between trifluoroacetic acid and methanol, as shown in Table 2.

[0058] [Table 2]

[0059] As can be seen from Table 2, a molar ratio between trifluoroacetic acid compound and methanol of 1:1 or 1:4 results in a low conversion rate of cyclosporine A to isocyclosporine A (30% or 20%), whereas a molar ratio between acid compound and methanol of 3:1 results in a high conversion rate of cyclosporine A to isocyclosporine A (75%), but with many by-products.

[0060] The product was characterized by liquid chromatography (see Figure 2). The isomerization yield obtained with a molar ratio of trifluoroacetic acid to methanol of 3:1 (reagent c) in Table 2) was measured after 12 and 24 hours. In particular, a conversion of 55% was observed after 12 hours of reaction and 70% after 24 hours, without any noticeable improvement in the amount of by-products detected.

[0061] The Applicant further observed that by operating under the same conditions as shown for reagent c) in Table 2, but at an increased reaction temperature from 60° C. to 65° C., a conversion rate of 77% was achieved, again without any noticeable improvement in the amount of by-products detected.

[0062] Example 3: Preparation of the salt of isocyclosporin A with citric acid Isocyclosporin A trifluoroacetic acid salt (1 mmol) was dissolved in a solution of MeOH and citric acid (1 mmol). After the solution was kept under stirring for 1 hour, the solvent was removed under reduced pressure and excess residual trifluoroacetic acid was removed by stripping with diethyl ether (2×15 ml) under vacuum. A 90% yield of Isocyclosporin A citric acid salt was obtained.

[0063] Example 4: Preparation of the salt of isocyclosporin A with lactic acid Isocyclosporine A trifluoroacetic acid salt (1 mmol) was dissolved in a solution of MeOH and lactic acid (1 mmol). After the solution was kept under stirring for 1 hour, the solvent was reduced under reduced pressure and excess residual trifluoroacetic acid was removed by stripping with diethyl ether (2×15 ml) under vacuum. A 91% yield of isocyclosporine A lactic acid salt was obtained.

[0064] Example 5: Preparation of salts of isocyclosporine A by transesterification of cyclosporine A - microwave heating Cyclosporin A (2.5 g, 2.08 mol) was dissolved in anhydrous methanol, then trifluoroacetic acid was added (5 ml) and the reaction vial was heated at 60° C. in a microwave for 15 hours by using a Biotage MW reactor.

[0065] The solvent was reduced under reduced pressure and excess residual trifluoroacetic acid was removed by stripping under vacuum with diethyl ether (2 x 15 ml). The dry TFA salt of isocyclosporin A (3.386 g) appeared as a white powder. The product was characterized by liquid chromatography (see FIG. 3). [Explanation of symbols]

[0066] 1 Distribution Unit 2. Microwave Reactor 3. Product collector 4. Pump 5. Cooler 6 Back pressure regulator

Claims

1. 1. A process for preparing a salt of isocyclosporin A by transesterification of cyclosporin A to a salt of isocyclosporin A, comprising the steps of: a) dissolving the cyclosporin A in anhydrous methanol and adding trifluoroacetic acid; b) heating the solution obtained from step (a) to a temperature ranging from 50°C to the reflux temperature of the reaction mixture for a time ranging from 30 to 60 hours; c) removing the methanol and excess trifluoroacetic acid; d) Recovering the salt of isocyclosporin A with trifluoroacetic acid. wherein the molar ratio of trifluoroacetic acid to methanol in the solution obtained by step a) is 1:

3.

2. 10. The method of claim 1, wherein step b) is carried out at a temperature of 60°C.

3. 10. The method of claim 1, wherein step b) is carried out for about 48 hours.

4. The method of any one of claims 1 to 3, wherein the excess trifluoroacetic acid in step c) is removed by stripping with diethyl ether under vacuum.

5. downstream of step d) the following step: e) dissolving an acid compound selected from citric acid and lactic acid in methanol; f) dissolving the salt of isocyclosporin A with trifluoroacetic acid in the solution obtained in step e), while stirring the resulting solution for a time ranging from 0.5 to 2 hours; g) removing the methanol and the trifluoroacetic acid; and h) recovering the salt of isocyclosporin A with the acid compound selected from citric acid and lactic acid. The method according to any one of claims 1 to 3, further comprising the step of:

6. 4. The method of any one of claims 1 to 3, wherein step a) comprises dissolving about 2 mmol of cyclosporin A in 60 mmol of methanol.

7. 4. The process according to any one of claims 1 to 3, wherein the overall yield of isocyclosporin A is 80%.

8. 4. The process according to any one of claims 1 to 3, wherein the conversion percentage of the starting cyclosporin A is 53%.

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