Methods for the production of nucleosides

Abstract

The invention relates to a method for the production of nucleosides or corresponding derivatives. According to the invention, thymidine or a corresponding derivative thereof is converted into the corresponding nucleoside using a mixture of phosphorylase 1 and phosphorylase 2 in the presence of a nucleobase other than thymine, with the thymine acting as a leaving group. The reaction according to the invention can be carried out in one step. To purify the resulting product, it can be extracted, filtered, and washed. The residues present in the crude product can be readily removed, achieving a purity of at least 99.5%. Surprisingly, the reaction has been found to exhibit remarkable substrate variability.

Description

[0001] The invention relates to a process for the preparation of nucleosides or corresponding derivatives.

[0002] Nucleosides (also called nucleosides) are organic molecules consisting of a nucleic base and a ribose, with the nucleic base and ribose being linked by a covalent bond. Nucleic bases (also called nucleobases) include adenine, guanine, cytosine, thymine, and uracil, as well as hypoxanthine and xanthine. Riboses are ring-shaped sugar molecules with a five-membered ring. When nucleosides are coupled with a phosphate residue, nucleotides are formed. Monophosphates, diphosphates, and triphosphates, so-called dNTPs (deoxynucleoside triphosphate) or NTPs (nucleoside triphosphate), are frequently used building blocks in biochemistry. The polymerization of nucleotides by linking different nucleotides ultimately leads to nucleic acid, a basic building block of cells in which genetic information about cell structure is stored. Nucleosides therefore represent the basic structure for the production of synthetic nucleic acids.Synthetic nucleic acids are required for a variety of medical and analytical applications. One medical application of nucleotides is the production of specific mRNA, which can be used as a vaccine. In addition to the production of nucleic acids as polymers from nucleotides, simpler structures that act as cosubstrates are also known, such as NADH (nicotinamide adenine dinucleotide hydride). This molecule, which looks like a simple dimer of two different nucleotides, has a nicotinamide residue in one of the nucleosides instead of the typical nucleic bases. Nucleotides are required in large quantities for a variety of analytical and forensic procedures, such as the now well-known PCR diagnostics. Eukaryotic cells contain various nucleosides that differ in their base or sugar content.There are a variety of pharmaceutical approaches for using nucleosides as drugs, possibly with derivatization of the base. The number of different applications of nucleosides in pharmacy, biochemistry, chemical analysis, forensics, and even as a base material is very large.

[0003] The synthesis of nucleotides or their derivatives requires multi-step reactions. These steps also include steps for protecting group chemistry. A further complexity in the production of nucleosides is the fact that multiple chiral centers exist on the ribose. Reactions involving chiral centers can produce racemates. If two or more chiral centers are involved, diastereomeric mixtures can also form, which are difficult to purify and reduce the selective yield during production.

[0004] European patent application EP 1 932 918 A1 presents a process for the synthesis of cladribine (2-chloro-2'-deoxyadenosine). Starting from uracil in a single step, using enzymes in bacterial cells or on solid supports as a biocatalyst, the cladribine is produced by transglycosylation. The biocatalyst itself is produced using genetically modified strains of Escherichia coli capable of overexpressing phosphorylase activities due to the enzymes uridine phosphorylase and purine nucleoside phosphorylase. Immobilization of cell suspensions containing the enzymatic activity of UPase and PNPase is prepared from a mixture of cell suspensions in order to achieve a ratio between the enzymatic activity of the UPase enzyme and the enzymatic activity of the PNPase enzyme in the range of 1:1 to 3:1.

[0005] European patent application EP 0 173 059 A2 presents a process for the preparation of 2'-deoxyadenosine compounds and related analogues. The process involves the glycosylation of the sodium salt of 2,6-dichloropurine or 6-chloropurine and the ammonolysis of the glycosylate to obtain the corresponding 2-chloro-deoxyadenosine or deoxyadenosine. For this purpose, the purine base and a suitably protected deoxyribose are condensed.

[0006] US patent application US 2002 / 0052491 A1 presents a process for the preparation of cladribine (2-chloro-2'-deoxyadenosine), which involves the direct glycosylation of 2-chloro-6-aminopurine with 1-chloro-2-deoxy-3,5-di-Op-toluoyl-alpha-D-erythropentofuranose. The process is carried out by first forming the sodium salt of 2-chloro-6-aminopurine and reacting the sodium salt with 1-chloro-2-deoxy-3,5-di-Op-toluoyl-alpha-D-erythropentofuranose in the presence of a moderately polar solvent such as acetone. The final product, cladribine, is formed by removal of the p-toluoyl groups by the action of methanolic ammonia or methanolic sodium methoxide. This process offers improved yields compared to the process described in the aforementioned EP 173 059 and the column purification step is not necessary.

[0007] European patent EP 2467 391 B1 presents a multi-step and complex process for the synthesis of cladribine, in which hydroxy-protecting group chemistry is used and in which exclusively the β-anomer is formed.

[0008] US patent application US 2006 / 0094869 A1 describes a reaction between chloradenine and deoxyribose 1-phosphate in the presence of the purified purine nucleoside phosphorylase (PNP) enzyme. A desired isomer is selectively produced by phosphorylation and isomerization of an anomer mixture of a 1-phosphorylated saccharide derivative, while simultaneously crystallizing one of the isomers to shift the equilibrium. Furthermore, using the action of a nucleoside phosphorylase, a nucleoside is produced from the resulting 1-phosphorylated saccharide derivative and a base with improved stereoselectivity and higher yield. This process is an anomer-selective process for producing a 1-phosphorylated saccharide derivative and a nucleoside.

[0009] The processes described above are complex, therefore expensive and do not offer the exclusive possibility of producing the β-anomer which is of interest for pharmaceutical purposes.

[0010] The object of the invention is therefore to provide a simple and selective process for the production of nucleosides.

[0011] The object of the invention is achieved by a method having the features of claim 1. Further advantageous embodiments are specified in the subclaims to claim 1.

[0012] The invention is based on the observation that thymidine (deoxythymidine, 1-(2-deoxy-β-D-ribofuranosyl)-thymine), uridine (1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2,4-dioxo-3H-pyrimidine), cytidine (1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4-amino-pyrimidin-2-one) or guanosine (9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-amino-1H-purin-6-one) or a derivative of the previous ones according to the following structures: Thymidine Uridine Cytide Guanosin where R0 as ribose (R0) or as ribose derivative (R0) has the following structure with R1 is the previously shown pyrimidine residue, R2, R3, R4, R5 selected from the group consisting of: -H, -OH, -NH2, -CH2OH, -OPO3H, -F, -Cl, -Br, -I, -CF3, - NH(alkyl), -NHCO2-alkyl, and -X1 selected from the group consisting of: O, S, Se, NH, CH2, N-alkyl, CH-alkyl can be converted into another nucleoside in a reaction requiring only one step using a mixture of phosphorylase 1 and phosphorylase 2.

[0013] In the reaction with phosphorylase 1 and phosphorylase 2, another nucleic base can take the place of the thymine residue. The following structures with R0 = H are possible nucleic bases, where R0 indicates the coupling site. As a nucleoside, R0 is equal to ribose (R0) or the ribose derivative (R0), and X1, X2, X3, X4 are selected from the group consisting of: O, S, Se, NH, CH2, N-alkyl, CH-alkyl, R2 is selected from the group consisting of: H, OH, NH2, CH2OH, OPO3H, F, Cl, Br, I, CF3, NH(alkyl), NHCO2-alkyl, and Y1, Y2, Y3 are selected from the group consisting of: N, CH, C-alkyl, CCF3, CF, CCl, CBr, Cl. The reference to alkyl includes a C1, C2, C3 or iso-C3, C4 or iso-C4, C5 or iso-C5, or C6 or iso-C6 alkyl radical.

[0014] The process according to the invention comprises initially charging thymidine (deoxythymidine, 1-(2-deoxy-β-D-ribofuranosyl)-thymine) or a derivative thereof according to the following structure: where R0 is the previously described ribose (R0) or the ribose derivative (R0), in excess, and a previously described nucleic base (R1) in a mixture of water and dimethylacetamide (C4H9NO), adjusting the pH of the initial charge to approximately pH 8.5 to 9.5, heating the initial charge to at least 70°C, and adding phosphorylase 1 and phosphorylase 2, filtering off the solid, extracting the aqueous phase with THF (tetrahydrofuran, C4H8O) under basic conditions (pH>12), and distilling off the THF under reduced pressure to leave a residue. The template can be buffered with K2HPO4·3 H2O.The ideal phosphorylase ratio is a mass ratio of phosphorylase 1 to phosphorylase 2 between 3:4 and 5:6, preferably 2:3 with a tolerance of 0.5. The mass ratio of phosphorylase 1 and phosphorylase 2 to the nucleobase or its derivative can be between 1:1,000 and 1:3,000, preferably 1:1,700 with a tolerance of 200. The reaction temperature can be 90°C with a tolerance of 7°C. Finally, the reaction time on a laboratory scale can be between 12 and 24 hours, preferably between 14 and 18 hours, particularly preferably 16 hours with a tolerance of 1 hour. To carry out the reaction, the stoichiometric excess of the thymidine or the derivative thereof can be between 3:1 and 5:1, preferably 4:1 with a tolerance of 0.5 parts relative to the nucleic base (R1) or the nucleic base derivative (R1).

[0015] The surprising effect of the process according to the invention is that the mixture of phosphorylase 1 and phosphorylase 2 exhibits unforeseen substrate variability, allowing for a variety of derivatives as reactants. The common pathway of the reaction is the conversion of thymidine or a derivative thereof into another nucleoside, with the thymine residue in the thymidine acting as the leaving group. By converting thymidine or its corresponding derivative into the desired nucleoside, exclusively the β-anomer, which is of pharmacological interest, is obtained. After distillation, it is only necessary to purify the resulting product. A feasible approach has been shown to be to redissolve the residue in THF after distilling off the solvent and precipitate it from methyl tert-butyl ether. The precipitate can be recrystallized from aqueous ethanol (EtOH / water 1:1).

[0016] The method according to the invention is explained using a concrete example: Example 1:

[0017] Dimethylacetamide (200 mL) and deionized water (1000 mL) were stirred in a 3 L, 4-neck flask. Thymidine (193.86 g, 800 mmol, 4 eq.), K2HPO4·3H2O (0.91 g, 4.0 mmol, 0.02 eq.), and 2-chloroadenine (33.92 g, 200 mmol, 1.0 eq.) were added successively. The mixture was rinsed with deionized water (800 mL) and adjusted to pH 9 with aq. NaOH (40%, 4.3 mL). The suspension was heated to 90°C in an oil bath. As soon as the internal temperature exceeded 70 °C, the enzymes phosphorylase 1 (20 mg in 2.75 mL aq. 10 mM K2HPO4 solution) and phosphorylase 2 (30 mg in 3.19 mL aq. 10 mM K2HPO4 solution) were added.

[0018] After 16 h, the oil bath was removed, NaCl (620 g) was added and the reaction mixture was heated to T InnenCooled to < 30 °C. The suspension was filtered through a Büchner funnel with water-moistened filter paper. Filtering was continued until the filtrate was no longer turbid. The resulting filtrate was filtered again through the same filter, and the remaining reaction mixture was also filtered. The filter cake was rinsed with deionized water (50 mL), and the filtrate was adjusted to pH ≥ 12.8 with aq. NaOH (40%, 94 mL). The filtrate was transferred to a 4 L separatory funnel, and THF (1 L) was added. After shaking in the separatory funnel, the mixture was filtered again through a Büchner funnel with water-moistened filter paper. The filter cake was rinsed with THF (200 mL). The filtrate was transferred to a 4 L separatory funnel and shaken. After phase separation, the aqueous phase was extracted with THF (3 × 1 L). The combined organic phases were washed with aqueous saturated NaCl (250 mL), which had previously been adjusted to pH 13 with aqueous NaOH (40%).After phase separation, the organic phase was filtered through Na2SO4 (252 g). The solvent was removed under reduced pressure at 45 °C. The residue was transferred to a 500 mL flask with distillate (100 mL). The solvent was removed under reduced pressure at 45 °C.

[0019] THF (65 mL) was added to the crude product (88.39 g) and stirred at RT. After a cloudy phase was obtained, methyl tert-butyl ether (400 mL) was added, and a colorless precipitate formed. After stirring at RT for 20 min, the resulting solid was filtered off, and the remaining solid was also transferred using the filtrate. The residue was washed with methyl tert-butyl ether (2 × 50 mL) and filtered. The colorless precipitate was dried under reduced pressure at 45 °C for 2 h.

[0020] The precipitate (32.7 g) was suspended in EtOH:water (1:1, 134 mL) and heated to 90 °C in an oil bath. EtOH:water (1:1, 154 mL) was added under reflux until a clear, slightly brownish solution was obtained. After cooling to RT, the resulting suspension was stored in an icebox overnight. The resulting precipitate was filtered through a frit, and the residue was resuspended with ice-cold EtOH:water (1:1, 20 mL). After filtration, the residue was resuspended in MTBE (2 × 100 mL) and filtered. The precipitate was dried under reduced pressure at 45 °C for 6 h. A colorless, powdery solid (25.39 g, 88.7 mmol, 44.4%) was obtained. The purity of the product was > 99.5% (HPLC).

[0021] The method presented here is highly selective, as only the β-anomer is formed. In contrast to chemically produced nucleotides or nucleotide analogues, this process is much shorter, more cost-effective, and more resource-efficient, as it is carried out in a single reaction vessel. When a naturally occurring nucleotide is used as the starting material, only a naturally occurring nucleic base is formed as a byproduct. LIST OF REFERENCE SYMBOLS

[0022] - no reference symbols - QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] EP 1 932 918 A1

[0004] EP 0 173 059 A2

[0005] US 2002 / 0052491 A1

[0006] EP 173 059

[0006] EP 2467 391 B1

[0007] US 2006 / 0094869 A1

[0008]

Claims

[1] A process for the preparation of a nucleoside (R0-R1) or its derivative, wherein the nucleoside (R0-R1) - a first residue of a ribose (R0) or a ribose derivative (R0), and - a second residue of a nucleic base (R1) or a nucleic base derivative (R1), wherein the two residues (R0, R1) are covalently bonded to one another, and wherein - the ribose (R0) or the ribose derivative (R0) has the following structure with R1 equal to the nucleic base (R1) or the nucleic base derivative (R1) - and the nucleic base (R1) or the nucleic base derivative (R1) is selected from the group consisting of and with R0 = H where R0 in the nucleoside is the ribose (R0) or the ribose derivative (R0), and X1, X2, X3, X4 are selected from the group consisting of: O, S, Se, NH, CH2, N-alkyl, CH-alkyl, R2, R3, R4, R5 is selected from the group consisting of: -H, -OH, -NH2, -CH2OH, -OPO3, -F, -Cl, -Br, -I, -CF3, -NH(Alkyl), - NHCO2-Alkyl, and Y1, Y2, Y3 is selected from the group consisting of: N, CH, C-alkyl, CCF3, CF, CCl, CBr, Cl comprising the following steps - Presentation of - Thymidine (deoxythymidine, 1-(2-deoxy-β-D-ribofuranosyl)-thymine), uridine (1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2,4-dioxo-3H-pyrimidine), cytidine (1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4-amino-pyrimidin-2-one) or guanosine (9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-amino-1H-purin-6-one) or a derivative the previous according to the following structures: Thymidine Uridine Cytidine Guanosine where R0 is the previously described ribose (R0) or the ribose derivative (R0), in excess and - a previously described nucleic base (R1) in a mixture of water and dimethylacetamide (C4H9NO), - Setting a basic pH value of the sample at approximately pH 8.5 to 9.5, - Heating the template to at least 70°C, and - Addition of phosphorylase 1 and phosphorylase 2, - Filtering off the solid, - Extracting the aqueous phase with THF (tetrahydrofuran, C4H8O) under basic conditions (pH > 12), and - Distill off the THF under reduced pressure, leaving a residue. [2] Method according to claim 1, characterized by - Take up the residue with THF and precipitate from methyl tert-butyl ether. [3] Method according to claim 2, characterized by - Recrystallize the precipitate with EtOH / water. [4] Method according to one of claims 1 to 3, characterized bythat the stoichiometric excess of the thymidine or the derivative thereof is between 3:1 and 5:1, preferably 4:1 with a tolerance of 0.5 parts relative to the nucleic base (R1) or to the nucleic base derivative (R1). [5] Method according to one of claims 1 to 4, characterized by that the template is buffered with K2HPO4·3 H2O. [6] Method according to one of claims 1 to 5, characterized by that the mass ratio of phosphorylase 1 to phosphorylase 2 is between 3:4 and 5:6, preferably 2:3 with a tolerance of 0.

5. [7] Method according to one of claims 1 to 6, characterized by that the mass ratio of phosphorylase 1 and phosphorylase 2 to the nucleic base or its derivatives is between 1:1,000 and 1:3,000, preferably 1:1,700 with a tolerance of 200. [8] Method according to one of claims 1 to 7, characterized by that the reaction temperature is 90°C with a tolerance of 7°C. [9] Method according to one of claims 1 to 8, characterized by that the reaction time is between 12 and 24 hours, preferably between 14 and 18 hours, particularly preferably 16 hours with a tolerance of 1 hour.