FI90552B - Method for preparing nucleoside derivatives - Google Patents

Description

90552

METHOD FOR THE PREPARATION OF NUCLEOSIDE DERIVATIVES

The invention describes a chemical synthesis process by which deoxyribose derivatives of nucleosides, in particular thymine, can be prepared more easily.

The present invention therefore relates to a process for the preparation of chemical derivatives of compounds between nucleic acid bases and ribose sugars of the formula HOCHjq b 0 N, in which B represents a keto group-containing nucleic acid base group in the 2-position, in particular

1 X

thyrine group to '' "^ Ύ 0.3 uracil group ^ | - O ^ | 25 N | H2 or cytosine group N / \ 30 especially thymine.

Such nucleoside analogs play a commercially important role as agents for the prevention or treatment of bacterial, fungal and viral diseases, as well as anticancer drugs, especially in the pharmaceutical industry.

A very large number of small molecule nucleoside and nucleotide analogs have been described in the literature. Of particular interest are these substances because they often inhibit the growth of bacteria, fungi, viruses, and cancer cells. The most commercially important nucleoside analog is 3'-azido-3'-deoxythymidine, trivially named azidoty-5 mididine, AZT, and trade name Retrovir. AZT is widely accepted for the treatment of autoimmune disease (AIDS) as well as for the prevention of the onset of the disease. Although AZT has been a known substance for a long time, it is still the best of a wide range of nucleotide and nucleoside analogs tested. AIDS is considered one of the 10 most threatening diseases of humankind and there are no clear solutions in sight to overcome it.

AZT is mainly manufactured by US Burroughs Wellcome Co. (Pharmaceutical Business News 27, April, pp. 13-16, 1990). 15 Despite its efforts to increase its production volume, there is a continuing need for AZT on the world market as the number of people living with and infected with AIDS grows faster. This, as well as the relatively old AZT synthesis route, causes the price of the drug raw material 20 to be high. In particular, the high number of people living with AIDS in poor developing countries makes it necessary to develop cheaper medicines. Although new nucleoside and nucleotide analogs are constantly being synthesized and tested in the treatment of AIDS, the relatively minor side effects of AZT make it the best known AIDS drug. Some of the side effects of AZT have been exaggerated due to overdosing and residual impurities produced by the synthetic method used.

The described methods for the synthesis of AZT and similar types of compounds use two chemical pathways. The first widely used method involves, as a first step, the protection of the 5'-hydroxyl group of a nucleoside with a trityl group ("Drugs for Future" vol. 11, pp. 1017-1019, 35 1986), which is sensitive to acid hydrolysis and can thus be removed in acidic solution. The acid-labile protecting group is seemingly preferred for AZT because the acidification reaction to the 3'-hydroxyl group occurs in a slightly basic solution. However, a very great difficulty is the removal of solid tritylcarbinol formed in acid hydrolysis from the final product. If the final product contains trityl-5 carbinol, it is assumed that significant adverse side effects will be observed with the drug.

Under the chemistry of sugars, it is known to use large protecting groups, such as a pivaloyl group, to protect the 5-hydroxyl group. This reaction has been exploited in nucleoside synthesis ("Nucleosides and Nucleotides" vol. 9, pp. 245-258, 1990), in which said protecting group has been used to form a double bond in the ribose ring by m-chloroperbenzoic acid treatment. The protecting group is removed by 15 amine treatments.

Another described method for the synthesis of AZT uses 2-chloro-1,2-difluorotriethylamine as a cyclizing agent (DE 3608606). Said reagent is not available in mark-20 cinemas. In addition, it is difficult to remove 2,3'-O-anhydrothymidine from the post-acidification mixture.

The disadvantages of the AZT synthesis methods currently in use are the complexity of the synthesis processes and the high cost of the starting materials required. These cause the price of AZT drug raw material to be high and not be able to be produced in sufficient quantities. In addition, currently used methods leave residues of harmful impurities in the final product and must be purified by chromatographic methods which are uneconomical.

The method according to the invention provides a decisive improvement in the above-mentioned drawbacks. To accomplish this, the method of the invention is characterized in that the 5'-O-derivative of the 2,3'-anhydronucleoside has the formula 4 90552 / "N".

5 Ο "^ u'J

X ° H, C (II) w 10 wherein X is a stable protecting group in a slightly basic solution, especially a pivaloyl or 1-adamantoyl or other similar sterically hindered protecting group, and the dashed line together with the associated nitrogen atom forms a corresponding nucleic base B as defined above. ring, is reacted in a slightly basic solution with sodium azide and lithium chloride in the presence of a buffering salt, then the protecting group X is removed by treatment with a strong base and the desired final product of formula I is recovered.

According to the invention, a 2,3'-anhydro derivative of formula (II) is preferably prepared by reacting a compound of formula HOC Hj 0 b

O

I1 (III)

HO

: Wherein B is as defined above, to react with a halide of formula XHal, wherein X is as defined above and Hal is halogen, in a basic organic solvent to give a 5'-35 XO derivative of the nucleoside, followed by the addition of a halide of formula YHal, wherein Y represents an alkyl or arylsulfonyl group, and Hal is the same as above, to prepare a compound of formula

5 X OC H j o B

O * 1 I (V) Y, 10 10 which compound is then converted into a 2,3'-anhydro-derivative of formula (II) by a base-catalyzed cyclization reaction.

In the above formulas, X is preferably a pivaloyl-15 group and Y is a methanesulfonyl group. Ammonium chloride can be preferably used as the buffering salt, but other buffering salts known to the person skilled in the art can also be used, e.g. ammonium acetate.

Compounds of formula II in which the dashed line has the same meaning as above and X is pivaloyl or 1-adamantoyl are also the subject of the present invention as novel compounds.

The invention provides a much simpler and more efficient method of preparation for nucleoside analogs, especially when the cyclization and acidification reaction is performed in a slightly alkaline solution with a stable protecting group, especially below pH about 11, however, the protecting group can be removed by concentrated base treatment, preferably above pH. about 14, without degradation of the product.

A significant advantage is also that the formation of the azido group in the final product is carried out without separate synthesis of lithium azide. Namely, the acidification step is generally performed using lithium azide which is highly soluble in dimethylformamide but which is not a readily commercially available product. Thus, hitherto known methods have included the preparation of an additional step - lithium azide. According to the invention, this additional step is avoided simply by using a commercially available mixture of lithium chloride and sodium azide, using a buffering salt to neutralize the lithium and sodium ions formed during the acidification reaction, which is important for the stability of the 5 'protecting group. The persistence of these 10 groups, on the other hand, is very important for the quantitative isolation and separation of the product from the salts. Purification of the final product is simpler and does not involve chromatographic steps.

Starting from a nucleoside of formula (III), it is dissolved in a basic organic solvent such as pyridine, pyrrolidone, dimethylformamide, imidazole or the like, or a mixture thereof, while stirring and / or heating. The organic base can also be replaced in industrial use by a less cumbersome tetrahydrofuran to which a small amount of a suitable organic base, e.g. 1-methylimidazole, is added. To this solution is added a specific reagent for 5 'protection, such as adamantoyl or pivaloyl halide, such as chloride, at a temperature of 0-40 ° C. The reaction is allowed to proceed at 5-30 ° C until all the nucleoside has reacted. An alkyl or arylsulfonyl halide such as methanesulfonyl, p-toluenesulfonyl or trifluoromethanesulfonyl chloride is then added to the reaction mixture and the reaction is allowed to proceed at 10-30 ° C with stirring for 2-20 hours. The 5 ', 3' protected nucleoside analog of formula V is then isolated by standard methods used in organic chemistry.

35 In the next reaction step, a base-catalytic intramolecular cyclization reaction takes place. In this case, a protected anhydro derivative is formed in high yield from the 5 'group.

U

7 90552 female (II). The base used in this step may be, for example, an alkali or alkaline earth metal hydroxide or an organic nitrogen-containing base such as an alkyl or arylamine. Although the 5 'protecting group used is a base-5-bile, it will withstand this step as well as the mild basic conditions of the next reaction step.

The next step specifically involves a new reaction in which the cyclic anhydro derivative (II) is converted in high yield into the 3'-deoxy-3'-azido-deoxyribose derivative (I). In this reaction, sodium azide is converted in situ to lithium azide in the presence of a buffering salt. In addition to the above-mentioned advantages associated with this step, the process according to the invention substantially reduces the inherent risk of explosion in the treatment of azides 15, which is of particular importance in the synthesis of large quantities of substances.

EXAMPLE

Thymidine (0.5 kg) is dissolved in 5 liters of pyridine. Pivalo-yl chloride (0.3 liters) is added with stirring to a solution of thymidine at 20 ° C. After all the thymidine has reacted, methanesulfonyl chloride (0.24 liters) is added and the mixture is stirred at 20 ° C for 5-10 hours. The precipitated material is filtered off and 0.4 kg of precipitate of rose is added to the filtrate. The mixture is stirred for 2 hours at 20 ° C and the pyridine is evaporated off in vacuo. Ethyl acetate and water are added to the residue, and after stirring, the organic layer is recovered.

30

Alternatively, thymidine (0.3 kg) can be dissolved in a mixture of 1-methylimidazole (0.5 L) and tetrahydrofuran (2 L). The mixture is cooled in an ice-salt bath and a solution of pivaloyl chloride (0.19 L) in tetrahydrofuran (0.8 35 L) is added dropwise with stirring. The mixture is stirred for two h in an ice-salt bath and then for 6 h at room temperature. The mixture is cooled again in an ice-salt bath and a mixture of methanesulfonyl chloride (0.25 L) and tetrahydrofuran is added with stirring. The solution is stirred for 4 h at room temperature and re-cooled in an ice-salt bath. Water (1.5 L) is added dropwise with stirring. The mixture is stirred for 2 h at room temperature. The lower layer is separated, extracted with ethyl acetate (2 x 21). The organic layers are combined.

The organic layer obtained in either of the above ways is washed with sodium hydrogen carbonate solution. The ethyl acetate solution is dried over sodium sulfate and the ethyl acetate solution is evaporated to dryness. The residue is crystallized from toluene. The yield of 5'-O-pivaloyl-3'-O-methanesulfonylthymidine is 75-85% of the calculated theoretical yield of thymidine. The white crystals have a melting point of 140 ° C (decomposes). The product gives an Rf value of 0.8 on silica gel thin layer chromatography (chloroform / methanol 9: 1 v / v). The absorption maximum in the ultraviolet range is 265 nm. The proton NMR spectrum (CDCl 3) shows the following typical resonances (tetramethylsilane resonance = 0 ppm): 9.13 ppm (1H, singlet, 3-NH); 7.21 ppm (1H, doublet, 6-H); 6.26 ppm (1H, doublet of doublets, 1'-H); 5.26 ppm (1H, multiplet, 3'-H); 4.46 ppm (1H, multiplet, 4'-H); 4.36 ppm (2H, multiplet, 5a, b-H); 3.10 ppm (3H, singlet, methanesulfonyl group); 2.69 (1H, multiplet, 2'a-H); 2.25 (1H, multiplet, 2'b-H9); 1.94 (3H, doublet, 5-CH 3); 1.24 (9H, singlet, pivaloyl).

The resulting compound (0.7 kg) is dissolved in dioxane or methylene chloride (4 liters) and 10% aqueous sodium hydroxide solution is added with stirring at 30-50 ° C until the reaction no longer proceeds and the pH remains at 6-7.5, pH measured on indicator paper. The reaction mixture is kept overnight at + 4 ° C and the crystals are filtered off. The filtrate is evaporated to dryness, after which chloroform is added to the residue, and the mixture is stirred for 30 minutes at 20 ° C, after which it is filtered. The filtrate is concentrated to a volume of about 9 liters 9 90552 and left at + 4 ° C. When crystals appear in abundance, they are filtered off. The crystals are dried in vacuo at 50-60 ° C to give 0.49 kg of 5'-O-pivaloyl-2,3'-anhydrothymidine. The melting point of the crystals is 216-217 ° C 5 (decomposes). On silica gel thin layer chromatography (chloroform / methanol 9: 1) the product gives an RF value of 0.3. The UV absorption maximum in methanol is 245 nm. The following proton NMR spectral resonances are observed (CDCl 3; tetramethylsilane resonance is 0 ppm): 7.18 ppm (1H, doublet, 6-H); 5.92 ppm (1H, doublet, 1'-H); 5.21 ppm (1H, multiplet, 3'-H); 4.41 ppm (1H, multiplet, 4'-H); 4.24 ppm (2H, multiplet, 5 'ab-H); 2.84 ppm (1H, multiplet, 2'a-H); 1.91 ppm (3H, doublet, 5-CH 3); 1.20 ppm (9H, singlet, pivaloyl).

15

A mixture of 0.53 kg of sodium azide, 0.2 kg of lithium chloride monohydrate in 6.9 liters of N, N'-dimethylformamide is heated at reflux for 1 hour with stirring. The mixture is cooled to 100-102 ° C. Ammonium chloride (0.174 kg) and 0.494 kg of 5'-O-pivaloyl-2,3'-anhydrothymidine are added. The mixture is stirred for 18-20 hours at 100-105 ° C and cooled to 20 ° C. The solid is filtered off and the filtrate is evaporated to dryness in vacuo at 50 ° C. The residue is treated with 2-5 liters of ethyl acetate-25 and 1-3 liters of water. The organic layer is separated, washed with water and finally the organic layer is evaporated to dryness under reduced pressure at 40-45 ° C. The residue is dissolved in 5.7 liters of dioxane and about 1.25 liters of 5M aqueous sodium hydroxide solution are added to the solution. The mixture is stirred for 2 hours at 20 ° C after which the solution is neutralized with a calculated equivalent amount of strong cation exchange resin (Dowex 50Wx8, 50-100 mesh, in H + form, about 2.4 liters) to pH 6.5-7. The resin is filtered off and washed as a filter with a mixture of 50% (v / v) dioxane and water. The combined filtrates are evaporated to dryness under reduced pressure. Add water to the residue and evaporate to dryness. This process is repeated 5-6 times. Dissolve the residue in about 10 liters of water and treat the solution with activated carbon for 30 minutes at 20 ° C. The activated carbon is filtered off and the filtrate is concentrated to 1.5 liters and left at + 4 ° C for 24 hours. The crystals 5 are filtered off and dried in vacuo at 50 ° C to constant weight. The yield of azidothymidine is 0.365 kg, 85%. Melting point 121-122 ° C. Silica gel thin layer chromatography (chloroform / methanol 9: 1) has an Rf value of 0.8. The optical rotation at 20 ° C at a wavelength of 10 ° C in a 0.8% solution of deuterium lamp is + 59 °. Elemental analysis for C 10 H 13 N 5 O 4 (m ° C weight 267.24): Calculated: C 44.94% H 4.90% N 26.21% Found: C 44.91% H 4.93% N 26.14% UV spectrum in methanol shows a wavelength maximum at 2 66 nm (molar absorbance 9877 measured in a 1 cm cuvette). Infrared spectrum (KBr tablet) vmax 2110 cm-1 (-N3). Proton NMR spectrum (in CDCl 3; tetrame-20-silane 0 ppm): 8.21 ppm (1H, broad singlet, 3-NH); 7.37 ppm (1H, doublet, 6-H); 6.06 ppm (1H, triplet, 1'-H); 4.41 ppm (1H, multiplet, 3'-H); 4.01 ppm (1H, multiplet, 5'a-H); 3.98 ppm (1H, multiplet, 4'-H); 3.83 (1H, multiplet, 5'b-H); 2.57 ppm (1H, multiplet, 2'a-25 H); 2.41 ppm (1H, multiplet, 2'b-H); 2.37 ppm (1H, multiplet, 5'-OH); 1.94 (3H, doublet, 5-CH 3).

Tue

Claims (13)

A process for the preparation of 3'-azido-31-deoxynucleosides of formula 5 HOCH₂Ob ON, wherein B represents a nucleotide group having a keto group in the 2-position, especially a tyrnine, uracil or cytosine group , characterized in that a 5'-O derivative of a 2,3'-anhydronucleoside of formula I 0 H '' (II) 2. XOH, C in which X is a weakly basic protecting group, especially a pivaloyl or 1-admantoyl group or a corresponding sterically hindered protecting group, and the dashed line with the corresponding nitrogen atom forms a ring corresponding to the the above-mentioned nucleic base B, is reacted in a weakly basic solution with sodium azide and lithium chloride in the presence of a buffering site, after which the protecting group X is removed by treatment with a strong base, and the desired final product is recovered. A method according to claim 1, characterized in that B is a thymine base. 3. A method according to claim 1 or 2, characterized in that ice 90552 is characterized in that the protecting group, which is stable in weak basic solution, is stable at a pH below about 11. Method according to any of the preceding claims, characterized in that X is a pivaloyl group. Process according to any of the preceding claims, characterized in that the buffer used in the azidation reaction is an ammonium salt. 10 Process according to any of the preceding claims, characterized in that the protecting group at the 5 'position is removed at a pH above about 14, by treatment with an alkali or alkaline earth metal hydroxide, or an organic nitrogen-containing base, such as alkyl or arylamine, preferably with 1-10 N alkali metal hydroxide. Process according to any of the preceding claims, characterized in that the 2,31-anhydro derivative used as a starting material is prepared by reacting a compound of the formula HOC 0 b: „Ό:: HO which B is the same as above, with a halide - of formula XHal, in which X represents the same as above and Hai represents halogen, in a basic organic solvent, whereby the 51-XO derivative of nucleoside is obtained, then a halide of formula YHal is added in which Y represents a alkyl or arylsulfonyl group, and Hai represents the same as above, to prepare a compound of formula 16 90552 X OC H; q B Ό1 5 γ0 (V) which compound is then transferred in a base catalyzed cyclization reaction to 2.3 ' the anhydrous derivative of formula II. 8. A process according to claim 7, characterized in that pyridine, a mixture of tetrahydrofuran and 1-methylimidazole or a mixture of N, N'-dimethylformamide and as a organic solvent is used in the step of protecting the 5 'position. imidazole. 9. A process according to claim 7, characterized in that Y is a methanesulfonyl group. 20 10. Compounds of Formula II 1 N U'J XOH, C (II); which X is a pivaloyl or a 1-adamantoyl group, and the dotted line means the same as in claim 1.