WO1993007162A1

WO1993007162A1 - Process for the preparation of nucleoside derivatives

Info

Publication number: WO1993007162A1
Application number: PCT/FI1992/000269
Authority: WO
Inventors: Alex V. Azhayev; Timo Korpela
Original assignee: Leiras Oy
Priority date: 1991-10-09
Filing date: 1992-10-07
Publication date: 1993-04-15
Also published as: JPH07500100A; FI90552C; FI914767A; CA2119914A1; FI90552B; ZA927464B; FI914767A0; EE9400304A

Abstract

The invention concerns a process for the preparation of nucleoside derivatives espcecially of AZT. As the starting material a 2,3'-anhydroderivative of the said nucleoside is used, which in its 5'-position carries a largish protecting group, which is stable under weakly alkaline, but which is split off in strongly alkaline conditions, especially pivaloyl, or adamantoyl. This compound is reacted with sodium or potassium azide and lithium halide in the presence of a buffering salt, whereafter the 5'-protecting group is split off from the obtained 3'-azido-nucleoside by treating with a base.

Description

PROCESS FOR THE PREPARATION OF NUCLEOSIDE DERIVATIVES

The invention describes a chemical synthesis method by means of which it is possible to prepare, in a manner easier than before, deoxyribose derivatives of nucleosides, especially thymine.

The object of the present invention is therefore a process for the preparation of chemical derivatives of compounds formed between nucleic acid bases and ribose sugars having the formula

wherein B has the meaning of a nucleobase group containing a eto group in the 2-position, especially

a thymine group

or a cytosine group

especially thymine.

Such nucleoside analogues are commercially important as agents inhibiting or curing bacterial, fungal and viral diseases, as well as as cancer agents, especially in the pharmaceutical industry. A very large number of low molecular nucleoside and nucleotide analogues are described in literature. These agents are subject to special interest as they often prevent the growth of bacteria, fungi, viruses and cancer cells. The commercially most important nucleoside analogue is 3'-azido-3'-deoxythymidine, of the trivial name azido- thymidine, AZT, and having the commercial name of retrovir. AZT is widely accepted for the treatment of the autoimmune disease (AIDS) as well as for the treatment aiming at preventing the disease from developing. Although AZT has been a known agent already for a long time, it is still the best in the large group of tested nucleotide and nucleoside analogues. AIDS is considered to be one of the diseases most threatening to mankind, and there are no clear solu¬ tions in sight for the conquer thereof.

AZT is mainly manufactured by US Burroughs Wellcome Co. (Pharmaceutical Business News 27, April, pp. 13-16, 1990) . Irrespective of its efforts to increase its production volume, there is a continuous additional need for AZT on the world market as the number of people taken sick with AIDS and infected by AIDS grows faster. This as well as the relatively old synthesis route for AZT results in that the price of the drug raw material is high. The large number of people with AIDS in the poor developing countries makes it especially necessary to develop cheaper drugs. Although new nucleoside and nucleotide analogues are constantly synt¬ hesized and tested in the treatment of AIDS, the relatively few side effects of AZT make it the best known AIDS drug. Some of the side effects of AZT have been exaggerated due to over-dosing and to residues of contaminants produced by the synthesis method used.

The synthesis methods described for AZT and corresponding compounds use two chemical routes. The first method widely in use comprises as the first step the protection of the 5'-hydroxyl group in the nucleoside with a trityl group

("Drugs for Future", vol. 13, pp 1017-1019, 1986) , which is sensitive to acid hydrolysis and may thus be removed in an acidic solution. An acid sensitive protecting group is seemingly advantageous in connection with AZT as the azide reaction on the 3'-hydroxyl group takes place in a mildly alkaline solution. However, a very big problem is the removal from the end product of solid trityl carbinol formed in the acid hydrolysis. If the end product contains trityl carbinol, it can be assumed that considerable unfavourable side effects can be observed in the drug.

Large protecting groups, such as the pivaloyl group, are known to be used -in the field of sugar chemistry for the protection of the hydroxyl group in the 5-position. This reaction has been applied to nucleoside synthesis ("Nucleo- sides and Nucleotides" , vol. 9, pp. 245-258, 1990) , wherein the said protecting group has been used for the introduc¬ tion of a double bond in the ribose ring with m-chloroper- benzoic acid treatment. The protecting group is removed with amine treatment.

Another disclosed AZT synthesis method uses 2-chloro-l, 2- difluorotriethylamine as the cyclization agent (DE 3608606) . The said reagent is not available on the market. In addition, it is difficult to remove it from the mixture remaining after forming the azide of 2 , 3 '-O- anhydrothymidine.

Disadvantages of the AZT synthesis methods presently in use are the complexicity of the process of synthesis and the high price of the required starting materials. As a con¬ sequence the price of the AZT drug raw material is high and it is not possible to manufacture sufficient amounts there- of. In addition, in the methods presently in use, residues of harmful contaminants remain in the end product and they have to be purified by chromatographic methods, which are uneconomical-

According to the process of the invention, a decisive improvement to the above mentioned disadvantages is obtained. To this end the process according to the inventi¬ on is characterized in that a 5'-O-derivative of a 2,3'- anhydronucleoside having the formula

wherein X is a protecting group stable in a weakly alkaline solution, especially a pivaloyl or l-adaman oyl, or other corresponding, sterically hindered protecting group, and the dotted line together with the nitrogen atom connected thereto forms a ring corresponding to the nucleobase B defined above, is reacted in a weakly alkaline solution with sodium or potassium azide and a lithium halide, LiHal, wherein Hal means halogen, in the presence of a buffering salt, whereafter the protecting group X is removed by treating with a strong base, and recovering the desired end product of the formula I.

According to the invention the 2,3'-anhydroderivative of the formula (II) is advantageously prepared by reacting a compound having the formula

(III)

wherein B has the meaning given above, with a halide of the formula XHal, wherein X has the meaning given above, and Hal means halogen, in an alkaline organic solvent, whereby the 5'-XO-derivative of the nucleoside is obtained, there- after adding a halide of the formula YHal, wherein Y has the meaning of an alkyl- or arylsulphonyl group, and Hal has the meaning given above, to prepare a compound having the formula

YO

which compound thereafter in a base catalyzed cyclization reaction is converted to the 2,3'-anhydroderivative of the formula (II) .

In the above mentioned formulas X is preferably the pivaloyl group and Y is the ethanesulphonyl group. Halogen means chlorine, bromine and fluorine. The azide is prefera¬ bly sodium azide and the lithium halide is preferably lithium chloride. As the buffering salt advantageously ammonium chloride may be used, but also other buffering salts known to one skilled in the art can come into question, e.g. ammonium acetate.

The compounds of the formula II, wherein the dotted line has the meaning given above and X is pivaloyl or ^' 1- adamantoyl, are, as new compounds, also an object of this invention.

According to the invention a substantially simpler and more effective process of preparation of nucleoside analogues is obtained, especially as the cyclization and azide reaction is carried out in a weakly alkaline solution, where the protecting group is stable, especially at a pH value of less than appr. 11, whereby, however, the protecting group may be removed by strong alkaline treatment, preferably at a pH value over appr. 14, without degrading the product.

A considerable advantage is also the fact that the forma¬ tion of the azido group in the end product is carried out without separate synthesis of lithium azide. The azide step namely has conventionally been carried out by using lithium azide which dissolves well in dimethylformamide but which is not a product easily available commercially. Thus the methods known so far have contained an additional step - the prepararion of lithium azide. According tc the invention, this additional step is avoided by simply using as a mixrure e.g. commercially available lithium chloride and sodium azide, whereby a buffering salt is present in order to neutralize the lithium and sodium ions formed during the azide reaction, which is important from the point of view of the stability of the protecting group in the 5'-position. The stability of this group again is very important from the point of view of quantitative isolation of the product and its separation f om the salts. The purification of the end product is simpler and does not contain chromatographic steps.

When starting from a nucleoside of the formula (III) this is dissolved in an alkaline organic solvent, such as pyridine, pyrrolidone, dimethylformamide, imidazole or a corresponding solvent, or in a mixture thereof, while stirring and/or heating. The organic base can also be substituted by tetrahydrofuran, which is less difficult in industrial use, and to which a small amount of a suitable base, e.g. 1-methyl-imidazole, is added. To this solution the specific reagent for use as the 5'-protection, such as a adamantoyl or pivaloyl halide, such as chloride, is added at a temperature of 0 to 40 °C. The reaction is allowed to proceed at a temperature of 5 to 30 °C until all the nucleoside has reacted. To the reaction mixture is thereaf¬ ter added alkyl- or arylsulphonyl halide, such as ethane- sulphonyl-, p-toluenesulphonyl- or trifluoromethanesul- phonylchloride and the reaction is allowed to proceed at a temperature of 10 to 30 °C while stirring for 2 to 20 hours. The 5'-, 3'-protected nucleoside analogue of the formula V is therafter isolated with conventional methods used in organic chemistry.

In the next reaction step the base catalyzed intramolecular cyclization reaction is carried out. Hereby the anhydrode- rivative protected at the 5'-group (II) is formed with a large yield. The base used in this step can be, for example, an alkali metal or alkaline earth metal hydroxide or an organic nitrogen containing base, such as an alkyl- or aryl amine. Although the protecting group of the 5'- position is base sensitive, it withstands the weak alkaline conditions of this step as well as of the next reaction step.

The next step comprises specifically a new reaction wherein the cyclic anhydroderivative (II) is converted with a high yield to 3 '-deoxy-3 '-azido-deoxyribose derivative (I) . In this reaction sodium or potassium azide is converted in situ to lithium azide in the presence of a buffering salt in a weakly alkaline solution, e.g. in dimethylformamide. In addition to the above mentioned advantages relating to this step the method according to the invention substan¬ tially reduces . the danger of explosion relating to the handling of azides, which is of great importance especially in the synthesis of large amounts of material.

In the final step the protecting group in the 5'-position is removed with a strong base, such as by treatment with an alkali or alkaline earth metal hydroxide, or an organic nitrogen containing base, such as an alkyl or aryl amine, especially with 1 to 10 N alkalimetal hydroxide. EXAMPLE

Thymidine (0.5 kg) is dissolved in 5 litres of pyridine.

Pivaloyl chloride (0.3 litres) is added while stirring to the thymidine solution at a temperature of 20 °C. When all thymidine has reacted, methanesulphonylchloride (0.24 litres) is added and the mixture stirred at a temperature of 20 °C for 5 to 10 hours. The precipitated material is filtered off and to the filtrate, 0.4 kg of sucrose is added. The mixture is stirred for 2 hours at a temperature of 20 °C and the pyridine is evaporated in vacuo. To the residue, ethyl acetate and water is added, and after mixing the organic layer is recovered.

Alternatively thymidine (0.3 kg) may be dissolved in a mixture of 1-methyl-imidazole (0.5 1) and tetrahydrofuran (2 1) . The mixture is cooled on a ice-salt-bath and a solu¬ tion of pivaloyl chloride (0.19 1) in tetrahydrofuran (0.8 1) is added dropwise while stirring. The mixture is stirred for 2 h on the ice-salt-bath and thereafter for 6 h at room temperature. The mixture is again cooled on the ice-εalt- bath and a mixture of methanesulphonylchloride (0.25 1) and tetrahydrofuran is added while stirring. The solution is stirred for 4 h at room temperature and cooled again on the ice-salt-bath. Water (1.51) is added dropwise while stirr¬ ing. The mixture is stirred for 2 h at room temperature. The lower layer is separated, extracted with ethyl acetate (2 x 2 1) . The organic layers are combined.

The organic layer obtained by either one of the above mentioned methods is washed with a sodium bicarbonate solution. The ethyl acetate solution is dried on sodium sulphate and the ethyl acetate solution is evaporated to dryness. The residue is crsytallized from toluene. The yield of 5'-0-pivaloyl-3'-0-methanesulphonylthymidine is 75 - 85 % of the theoretical yield calculated on the amount of thymidine. The melting point of the white crystals is 140 °C (dec.) The product gives a Rf-value of 0.8 in 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 (CDC1₃) gives the following typical resonances (tetramethylsilane resonance = 0 ppm) : 9.13 pp (1 H, singlet, 3-NH) ; 7.21 ppm (1H, dublet, 6-H) ; 6.26 ppm (1H, twin dublet, l'-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, methanesulphonyl group) ; 2.69 (1H, multiplet, 2' a-H) ; 2.25 (1H, multiplet, 2'b- H9) ; 1.94 (3H, dublet, 5-CH3) ; 1.24 (9H, singlet, pivalo¬ yl) .

The compound formed (0.7 kg) is dissolved in dioxane cr methylene chloride (4 litres) and therein an aqueous 10 % sodium hydroxide solution is added while stirring at a temperature of 30 to 50 °C until the reaction does not proceed any longer and the pH remains at 6 to 7.5, measured with pH-reactive paper. The reaction mixture is kept over night at +4 °C and the crystals are filtered off. The filtrate is evaporated to dryness, whereafter to the residue chloroform is added and the mixture stirred for 30 minutes at +20 °C, wherafter it is filtered. The filtrate is concentrated to a volume of about 1 liter and kept at +4 °C. When an abundance of crystals appear, these are filtered off. The crystals are dried in vacuo at 50 to 60 °C, whereby 0.49 kg of 5'-0-pivaloyl-2 , 3 '-anhydrothymidine is obtained. The melting point of the crystals is 216 to 217 °C (dec) . In silica gel thin layer chromatography (chloroform/methanol 9:1) the product gives a Rf value of 0.3. The UV absorption maximum in methanol is 245 nm. The following proton-NMR-spectrum resonances are observed (CDC1₃; tetramethylsilane resonance is 0 ppm) : 7.18 ppm (1H, dublet, 6-H) ; 5.92 ppm (1H, dublet, l'-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, dublet, 5-CH₃) ; 1.20 ppm (9H, singlet- , pivaloyl )

A mixture that contains 0.53 kg of sodium azide, 0.2 kg of lithium chloride monohydrate in 6.9 litres of N,N'-dime- thylformamide is heated at reflux temperature for 1 hour while stirring. The mixture is cooled to 100 to 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 to 20 hours at 100 to 105 °C and cooled to 20 °C. The solid material is filtered off and the filtrate evaporated to dryness in vacuo at 50 °C. The residue is treated with 2 to 5 litres of ethyl acetate and 1 to 3 litres of water. The organic layer is separated, washed with water and finally the organic layer is evaporated to dryness at reduced pressure at a temperature of 40 to 45 °C. The residue is dissolved in 5.7 litres of dioxane and appr. 1.25 litres of an aqueous 5M sodium hydroxide solution is added to the solution. The mixture is stirred for 2 hours at a temperature of 20 °C, whereafter the solution is neutralized with the calculated equivalent amount of strong cation exchange resin (Dowex 50Wx8, 50 to 100 mesh, H÷ form, about 2.4 litres) to a pH value of 6.5 to 7. The resin is filtered off and washed on a filter with 50% (v/v) mixture of dioxane and water. The combined filtrates are evaporated to dryness under reduced pressure. Water is added to the residue and it is evaporated to dryness. This procedure is repeated 5 to 6 times. The residue is dis¬ solved into about 10 litres of water and the solution treated with active carbon for 30 minutes at 20 °C. The active carbon is filtered off and the filtrate concentrated to 1.5 litres and kept for one day at +4 °C. The crystals are filtered off and dried in vacuo at 50 °C to constant weight. The yield of azidothymidine is 0.365 kg, 85 %. The melting point is 121-122 °C. In silica gel thin layer chromatography (chloroform/methanol 9:1) the Rf-value of the product is 0.8. The optical rotation is +59° at 20 °C dissolved in methanol as a 0.8 % solution, at the wave length of a deuterium lamp. Elementary analysis for the formula ^c ₁₀ ^H _i3 ^Ns°₄ (molecular weight 267.24) :

calculated: C 44.94% H 4.90% N 26.21% found: C 44.91% H 4.93% N 26.14%

The UV-spectrum in methanol shows a wave length maximum at 266 nm (molar absorptivity 9877 measured in a 1 cm cuvet¬ te) . The infrared spectrum (KBr-tablet) v 2110 cm^-1 (- N₃) . The proton-NMR-spectrum (CDC1₃; tetramethyl silane 0 ppm) : 8.21 ppm (IH, broad singlet, 3-NH) ; 7.37 ppm (IH, dublet, 6-H) ; 6.06 ppm (IH, triplet, l'-H) ; 4.41 ppm (IH, multiplet, 3'-H) ; 4.01 ppm (IH, multiplet, 5' a-H) ; 3.9S ppm (IH, multiplet, 4'-H) ; 3.83 (IH, multiplet, 5' b-H) ; 2.57 ppm (IH, multiplet, 2' a-H) ; 2.41 ppm (IH, multiplet, 2' b-H) ; 2.37 ppm (IH, multiplet, 5'-OH) ; 1.94 (3H, dublet, 5-CH₃) .

Claims

1. Process for the preparation of a 3'-azido-3'-deoxynucle- oside having the formula

wherein B has the meaning of a nucleobase group containing a keto group in the 2-position, especially a thymine, uracil or cytosine group, characterized in that a 5'-θ- derivative of a 2,3'-anhydronucleoside of the formula

wherein X is a protecting group stable under weakly alkaline conditions, especially a pivaloyl or l-adamantoyl or other corresponding sterically hindered protecting group, and the dotted line together with the nitrogen atom connected thereto forms a ring corresponding to the nucleobase B defined above, is reacted in a weakly alkaline solution with sodium or potassium azide and LiHal, wherein Hal means halogen, in the presence of a buffering salt, whereafter the protecting group X is removed by treating with a strong base, and the desired end product is recove- red.

2. Process according to claim 1, characterized in that the azide is sodium azide and the lithium halide is lithium chloride.

3. Process according to claim 1 or 2, characterized in that B is a thymine base.

4. Process according to any one of claim 1 to 3, characte¬ rized in that the protecting group stable in a weakly alkaline solution is -stable at pH-values less than about 11.

5. Process according to any one of the preceeding claims, characterized in that X is the pivaloyl group.

6. Process according to any one of the preceeding claims, characterized in that the buffer used in the azide re¬ action is an ammonium salt.

7. Process according to any one of the preceeding claims, characterized in that the protecting group in the 5'- position is removed at a pH of over appr. 14, by treating with an alkalimetal or earth alkaline metal hydroxide, or an organic nitrogen base, such as an alkyl- or arylamine, especially with 1 to 10 N alkalimetal hydroxide.

8. Process according to any one of the preceeding claims, characterized in that the 2,3'-anhydroderivative used as a starting material is prepared by reacting a compound of the formula

wherein B has the meaning given above, with a halide of the formula XHal, wherein X has the meaning given above, and Hal means halogen, in an alkaline organic solvent, whereby the 5'-XO-derivative of the nucleoside is obtained, there¬ after adding a halide of the formula YHal, wherein Y has the meaning of an alkyl- or arylsulphonyl group, and Hal has the meaning given above, to prepare a compound having the formula

which compound thereafter in a base catalyzed cyclization reaction is converted to the 2,3'-anhydro derivative of the formula (II) .

9. Process according to claim 8, characterized in that in the step for protecting the 5'-position of the nucleoside, pyridine, a mixture of tetrahydrofuran and l-methyl- imidazole or a mixture of N,N'-dimethylformamide and imidazole is used.

10. Process according to claim 8, characterized in that Y is the methanesulphonyl group.

11. Compounds of the formula II

wherein X is the pivaloyl or 1-adamantoyl group, and the dotted line has the same meaning as in the claim 1.