Method of production of cladribine

Abstract

The invention relates to a method of producing cladribine with a purity greater than 99.9 wt.% from 2-deoxyuridine and 2-chloroadenine by using the catalytic action of high concentrations (at least 20.000 U / L) of freely dissolved transglycosidases UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) in an enzymatic synthesis in an aqueous environment without the presence of any organic solvent.

Description

[0001] Method of production of cladribine

[0002] Area of technology

[0003] The invention relates to a method of producing cladribine through enzymatic synthesis in an aqueous environment without the use of organic solvents, utilizing high concentrations of freely dissolved transglycosidases to catalyze the reaction.

[0004] State of the art

[0005] Cladribine (2-chlorodeoxyadenosine, 2-CdA) is a drug that was developed in the late 1970s and is used in the treatment of mainly haemato-oncological diseases. The first registration of a parenteral formulation of cladribine was granted in 1993 for the treatment of haemato- oncological diseases.

[0006] It is a synthetic purine nucleoside analogue of deoxyadenosine substituted with chlorine at position 2 of the purine ring with the following structure:

[0007] Several synthetic methods of cladribine production are described in the literature. The first publicly known chemical synthesis method was described by Chen et al., 2002, which describes a seven-step synthesis based on guanosine. Also known is US 6,252,061, which describes a chemical synthesis based on the halogenation of 2,6-diaminopurine deoxyribose in a mixture of protic and aprotic solvents. Similarly based is the invention of WO 2004 / 028462, which describes the synthesis of cladribine by direct halogenation of 2'-deoxyguanosine. The invention published in document US 2004 / 0039190 mentions a synthesis based on chloradenine and deoxyribose.

[0008] It is well known that the chemical synthesis of cladribine, which remains the only commercially available method, has significant limitations. These chemical processes typically involve multiple reaction steps, including protective and deprotective reactions, and often require purification of intermediates after each step.

[0009] Additionally, chemical reactions are often non-stereospecific, producing both a and P conformations of the final product. This increases purification costs and reduces the overall yield of the synthesis.

[0010] Furthermore, from an economic standpoint, the purification process following chemical synthesis is complex and involves the use of many organic solvents. This not only increases production costs but also significantly contributes to environmental pollution and ecological impact.

[0011] An alternative to chemical synthesis is the use of chemoenzymatic methods for producing nucleoside analogues, employing recombinant enzymes. Although cladribine was first synthesized experimentally in 1980 using semi -purified deoxyribosyltransf erase, its biosynthesis under industrial or field conditions has not yet been successfully achieved. A primary obstacle is the high cost of enzymes. Consequently, researchers are exploring solutions such as replacing purified enzymes with bacterial cell suspensions, reducing the number of enzymes required, or enabling enzyme reuse through immobilization on suitable carriers.

[0012] All of the aforementioned procedures have been experimentally tested in the biosynthesis of cladribine. While they help reduce enzyme costs, they are associated with several technical challenges. For instance, living cells — whether free in suspension or immobilized on carriers — produce various side metabolites alongside the target enzymes, which can interfere with the reaction process or complicate the isolation of the desired product. Additionally, low enzyme concentrations often necessitate prolonged reaction times and typically require the use of organic solvents in the reaction mixture. Immobilization of enzymes on suitable carriers, as proposed by some scholars experts, appears to be a very promising approach. However, it also has several limitations, including the cost of acquiring carriers, the complexity of immobilization methods, the expenses and challenges associated with disposing of immobilized biocatalysts, and the potential reduction in enzymatic activity compared to free enzymes. Additionally, enzyme inactivation can occur due to immobilization procedures, and some degree of empiricism remains involved in optimizing immobilization methodologies.

[0013] On the other hand, despite the notable limitations mentioned above, the process described in EP 1 932 918 most closely aligns with the operating conditions known to the inventors of the claimed invention.

[0014] However, in a detailed study of the above invention, several technical inconsistencies cannot be overlooked. The invention discloses the use of both aqueous reaction media and media containing apolar organic solvents, specifically 20% DMSO or DMF. In doing so, the proposed isolation process disclosed in the patent can be understood, based on the information provided, to be universal, i.e. applicable to both aqueous solutions and solutions containing organic solvents.

[0015] From a knowledge of the physicochemical properties of cladribine, it is obvious to one skilled in the art that the solubility of cladribine will vary substantially with the concentration of the aprotic solvents and that it can hardly be expected to precipitate under the same conditions from aqueous solutions as from solutions containing organic solvents.

[0016] Based on verification experiments by the inventors of the claimed invention, the process proposed in EP 1 932918 is not applicable to solutions containing the above-mentioned organic solvents.

[0017] Moreover, for aqueous solutions alone, no example of implementation is given in the cited document. Verification experiments have shown that even after numerous repetitions the isolation procedure according to the cited document EP 1 932 918 could not be repeated. Moreover, the preparation of the 2-chloroadenine solution in KOH referred to in the document is a known procedure. According to the published description of the reaction, the authors used a total enzymatic activity of 187.5 U / l medium. Unfortunately, a detailed description of the reaction time is missing from the text of the invention, since at such a low enzyme concentration, a very low cladribine production of somewhere in the range of 0.10 g / L / hr can be expected, which, at the presented production of 4.0 to 5.0 g / L, represents a reaction time in the order of days.

[0018] This implies further disadvantages of the known invention, as long-term synthesis brings with it several problems and limitations, including increased requirements for the sterility of the synthetic process. With respect to working with immobilized cells, the production of other enzymes (metabolites) can be expected, which may substantially affect the purity and quality of the cladribine produced.

[0019] In view of the foregoing, the inventors of the claimed invention have decided to develop a new method of synthesizing cladribine which would eliminate the shortcomings of the above-quoted invention of EP 1932 918 and at the same time provide a rapid, economically advantageous and environmentally more environmentally friendly synthesis of cladribine in a quality corresponding to the requirements of the EP and USP.

[0020] The essence of the invention

[0021] The problem is solved by a method of producing cladribine according to the invention below.

[0022] The invention offers an alternative method for the production of cladribine, wherein the synthesis is carried out using a high concentration of freely dissolved transglycosidases in an aqueous solution, thereby eliminating the need for organic solvents.

[0023] The invention is further supported by the fact that, to the best of the inventors' knowledge and based on the prior art, there has been no disclosure in the literature of an enzymatic synthesis of cladribine utilizing high concentrations of freely dissolved transglycosidases in aqueous solutions, despite diligent efforts. The technical objective of the invention is to develop a novel and entirely new method for the production of cladribine. The inventive method enables the production of cladribine with a yield of 5 or more grams per liter per hour, allows for easy isolation from the aqueous environment, ensures high purity of the final product, and offers advantages such as low production costs and minimal ecological impact.

[0024] The essence of the invented method of producing cladribine is that the production method comprises the following production steps: a) a reaction solution is formed by dissolving the transglycosidase enzymes at a concentration of at least 20 000 U / l in aqueous phosphate buffer containing 2'- deoxyuridine, b) 2-chloroadenine is gradually added to the reaction solution to prevent its precipitation, while cladribine is synthesized in the reaction solution, c) transglycosidases are heat inactivated and allowed to precipitate from the reaction solution at room temperature, d) the solid portion is removed from the reaction solution, e) the filtrate is concentrated and alkalinised to a pH between 11.0 and 12.0, f) The cladribine is allowed to precipitate freely in the filtrate at room temperature, the precipitate containing at least 98 % by weight of cladribine, g) the precipitate containing at least 98 % w / w cladribine is separated by filtration and dried, h) cladribine recrystallizes under reflux, i) cladribine is isolated by filtration from the mother liquor, j) the recrystallized cladribine is dried.

[0025] Transglycosidases are present in the reaction medium in the form of free enzymes and may be in either semi-purified (e.g., ammonium sulfate precipitate) or purified form. The semi-purified form refers to a crude protein precipitate obtained through a combination of thermal precipitation followed by salting out with, for example, ammonium sulfate. The purified form denotes an enzyme that has been purified using chromatographic methods.

[0026] Unlike published processes that employ immobilized cells or enzymes, the present invention pertains to a technology for synthesizing cladribine using freely dissolved enzymes. Surprisingly, it has been discovered that a single application of high doses of freely dissolved enzymes — equivalent to tens of thousands of enzyme units per liter of buffered aqueous reaction medium — can produce cladribine at a rate of 5 g / L per hour, even without the use of organic solvents in the reaction medium.

[0027] Moreover, it is advantageous that after the reaction is complete, the enzymes can be easily removed from the reaction solution through thermal inactivation followed by filtration. The resulting cladribine can then be isolated simply by precipitation from the concentrated medium.

[0028] Furthermore, it was surprisingly found that the disadvantages of previously known enzymatic synthesis methods could be circumvented, allowing for the production of pure cladribine with yields exceeding 60 wt.% based on the amount of starting material.

[0029] The advantage of the inventive method is that the reaction takes place only in the presence of an aqueous buffered medium, in the presence of free recombinant enzymes - transglycosidases and the isolation of the synthesized cladribine does not require the use of chromatographic methods or buffers containing organic solvents, since the precipitation from the concentrated aqueous medium is sufficient.

[0030] This means that, in addition to being environmentally friendly, the above-invented process enables a significantly faster and cheaper way of producing cladribine in pharmacopoeial purity.

[0031] The enzymatic method for producing cladribine according to the invention is rapid, cost- effective in terms of technology and energy, and environmentally friendly. It does not require the use of organic solvents or chromatographic purification of pure cladribine, making it readily applicable to both laboratory-scale and industrial production settings.

[0032] Thus, among the advantages of the invention are its rapid, environmentally friendly, and cost- effective synthesis of cladribine from aqueous solutions, conducted without the use of organic solvents, and its favorable technological and energetic profile. In another aspect, the present invention pertains to a method for in vitro enzymatic synthesis of cladribine in an aqueous buffered medium. The reaction is catalyzed by high concentrations of freely soluble transglycosidases, preferably purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UP), amounting to tens of thousands of units per liter of medium.

[0033] In another aspect, the invention pertains to the enzymatic synthesis of cladribine, wherein the deoxyribose moiety of cladribine is derived from 2'-deoxyuridine, and the nucleobase is derived from 2'-chloroadenine.

[0034] In a further aspect, it is advantageous to purify the product by precipitating it from the aqueous reaction solution through pH adjustment, resulting in a crude product with a purity greater than 98 wt%. After purification under reflux, the resulting cladribine exhibits a purity with less than 0.1 wt% of unidentified impurities.

[0035] Last but not least, the invention does not require the use of organic solvents nor the chromatographic purification of pure cladribine.

[0036] Example of the implementation of an invention

[0037] It is understood that the specific embodiments of the invention described and illustrated below are presented for purposes of illustration and not as a limitation of the invention to the examples provided. Those skilled in the art will find or be able to provide, using routine experimentation, a greater or lesser number of equivalents to the specific embodiments of the invention described herein.

[0038] Example 1 : Cladribine production process

[0039] Dissolve 2 '-deoxyuridine in 1 L of phosphate buffer and heat the solution to 55 °C. The enzymes UP and PNP are added to the heated solution with stirring at a minimum concentration of 20 000 U / l. A solution of 2-chloroadenine in 3% KOH is gradually added. The pH of the reaction is maintained at 7.5. The reaction is terminated after 60 minutes when the solution is heated to 75 °C / 15 minutes. The resulting precipitate from the reaction solution is filtered and concentrated on a vacuum evaporator approximately ten times. Allow the filtrate to precipitate freely at room temperature. Precipitation of the product from the solution occurs at pH 11.5. The product is filtered off and dried at 60 °C / 3 hours. The purity of the crude precipitate is 98, 1 % w / w. To increase the purity, the product is dissolved in a 1 : 12.5 hydroalcoholic solution and recrystallised under reflux. The concentration of cladribine produced is around 5 g / L.

[0040] Industrial applicability

[0041] The method of producing cladribine according to the invention is readily applicable to both laboratory operating conditions and industrial production operating conditions.

Claims

PATENT CLAIMS1. Method of producing cladribine, characterized by comprising the following process steps: a) Forming a reaction solution by dissolving transglycosidase enzymes at a concentration of at least 20,000 U / l in aqueous phosphate buffer containing 2'- deoxyuridine, b) Gradually adding 2-chloroadenine to the reaction solution to prevent its precipitation, while allowing cladribine to be synthesized in the reaction solution, c) Heat inactivating the transglycosidases and permitting them to precipitate from the reaction solution at room temperature, d) Removing the solid portion from the reaction solution, e) Concentrating and alkalinizing the filtrate to a pH between 11.0 and 12.0, f) Allowing cladribine to precipitate freely in the filtrate at room temperature, with the precipitate containing at least 98% by weight of cladribine, g) Separating the precipitate containing at least 98% by weight of cladribine by filtration and drying, h) Recrystallizing cladribine under reflux, i) Isolating cladribine by filtration from the parent solution, j) Drying the recrystallized cladribine.

2. The method of claim 1, characterized in that the reaction in process step (a) is carried out in an aqueous phosphate medium with a pH range of 6.5 to 8.5 and a temperature range of 40°C to 55°C.

3. The method according to claim 1 or 2, characterized in that in process step (e), the volume of the filtrate is concentrated tenfold.

4. The method according to any one of claims 1 to 3, characterized in that in process step (g), the precipitate containing more than 98% w / w cladribine is separated by filtration, washed with distilled water, and dried at 60°C.

5. The method according to any one of claims 1 to 4, characterized in that in process step (h), the cladribine is recrystallized under reflux from a hydroalcoholic solution at a ratio of 1 : 12.5.

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