CS220430B1 - Method of preparing 2-deoxy-5-azacytidine - Google Patents

Description

The invention relates to a process for the preparation of 2'-deoxy-5-azacytidines of the general formula I

R ² R ^{* 1}

Ί /

N ^ N

BOY

Wherein R 1 and R 4 are the same or different and denote hydrogen or C 1 -C 4 alkyl.

The most prominent of this type is the pyrimidine antimetabolite 2'-deoxy-5-azacytidine [4-amino-1- (2-deoxy-beta-D-erythro-pentofuranosyl) -α 'triazin-2 (1H) -one, which, like 5-Azacytidine inhibits bacterial growth even at low concentrations and has pronounced canceroatatic effects * The lymphatic system has the highest affinity for this substance, so its clinical use in lymphatic tumor tumors is offered. Biological activity, although to a lesser extent, also has N-alkyl 5-azacytidine derivatives, and the same can be expected for the corresponding deoxyanalogs. The compounds of formula I can furthermore be used as tools for biochemical, pharmacological and molecular biological research.

2'-Deoxy-5-azacytidine can be prepared according to the Czechoslovak patents S. 116 297 et al. 139 542. The synthesis of this substance has long been the subject of a number of publications [Pliml, Šorm: Collect.

Czech. Chem. Commun. 22 »2576 (1964); Winkley, Robins, J. Org. Chem. £ 491 (1970) 1

Niedballa, Vorbrflgen, J. Org. Chem. £ g, 3,672 (1974), Pískala et al., Nucleic Acids Res.,

Special Publication No 4, s 109 (1978) and Pískala, Šorm: Nucleic Acid Chemistry (L. B. Townsend,

R. S. Tipson, eds., Part 1, 443 (1978)]. In this work, different variants of two fundamentally different general approaches with which 5-azacytidine was previously prepared are applied.

The first consists in a multi-step synthesis starting from 3,5-di-O-p-toluoyl-2-deoxy-D-erythro-pentosyl isocyanate and the second in direct deoxyribosylation of silylated 5-azacytosine.

Since the steric course of deoxyribosylation is not controlled as in the case of ribosylation by the adjacent acyloxy group, the aforementioned procedures result in a mixture of alpha and beta-anomers. In the isocyanate process, the desired beta-anomer is greatly predominated in the mixture, but production is lengthy. Shorter and preparatively preferred is direct deoxyribosylation catalysed by Lewis acids, which results in a high yield of an anomeric mixture in which the 3-anomer predominates slightly. The preparative yields of 2'-deoxy-5-azacyclidine in this final step are in the range of 25-30%. Furthermore, the treatment of the mother liquors yields, in 45-50% yields, a crude crystalline alpha-anomer, which is biologically poorly active and is therefore essentially worthless waste.

By studying the hydrolysis of 2'-deoxy-5-azacytidine and its alpha-anomer, it has now become very surprising to find that reaction with dilute ammonia at room temperature gives both anomers virtually quantitatively the same mixture of N-amidino-N '- (2-deoxy) formates (D-erythro-pento-1-furanosyl) urea in which the beta-anomer is predominant. Furthermore, it has been found that these formates can be cyclocondensed with dimethylformamide dimethylacetal in good yield to 2'-deoxy-5-azacytidine directly, without releasing said amidine ureas. The byproduct is again an alpha-isomer which, without isolation, can be used again in the next cycle to prepare 2'-deoxy-5-azacytidine. By repeating this cycle twice, up to 60% conversion of the alpha-anomer to 2'-deoxy-5-azacytidine can be achieved. This virtually doubles the total yield of this compound calculated on 2-deoxyribos, which is the most expensive starting material. The procedure is simple, short and does not require any special equipment. Due to the high cost of 2'-deoxy-5-azacytidine, the use of this process results in a significant increase in the efficiency of production. Not the same principle can also be prepared by N? -Alkyl derivatives 2 ^{<-deoxy-5-azacytidihu.}

The present invention provides a process for the preparation of the 2 * -deoxy-5-azacydidines of formula (I) by hydrolyzing the alpha-anomers of the 2 * -deoxy-5-azacytidines of formula (II),

wherein R and R ^{2 are the} same as in Formula I, to the N-emidino-N '- (2-deoxy-beta-D-erythro-pentofuranose) ureas of Formula III r 1

Y

HOCH 3 _λ NH-CO-N = C-NH, HCOOH

O

Wherein R ¹ and R ^{2 are the} same as in Formula I, which are then cyclocondensed with dimethylformamide dialkyl acetal in C 1-4 alkamol.

(III)

220430 '

The hydrolysis of the alpha-anomers of the formula 11 to the amidine-urea formates of the formula III takes place very rapidly already at room temperature in an alkaline medium. From a preparative point of view, it is suitable to use dilute solutions of volatile bases. Dilute ammonia solution has proven to work well. Since the formic acid formed during the evaporation partially escapes from the hydrolysis, it is advisable to add 10-20% of the theoretical amount of formic acid to the mixture before crystallization to achieve high preparative yields. The yields of the amidine-urea formates III are in the range of 92-98 Si. These intermediates are not sterically uniform and contain minor amounts of the corresponding alpha-anomers. However, they can be used directly in the next stage of the synthesis without further purification.

The cyclo-condensation of the amidine ureas of the formula III is carried out in a C1 -C4 alkanol medium and dimethylformamide dialkyl acetals having a C1 -C4 alkyl radical are used. Preferably, this reaction is carried out with dimethylformamide dimethyl acetal in methanol. Good results can be obtained by using 1.5-2.5 mol equivalents of dimethylformamide dialkyl acetal. The cyclo-condensation may be carried out in a wide temperature range from 0 ° C to the boiling point of the corresponding alkanol. The reaction at room temperature has proven to work very well. Since the amidineureas of the formula III during the reaction before the cyclization change the anomeric configuration, some of the starting alpha-enomers of the formula II are formed by this process, but can be used again for the preparation of 2-deoxy-5-azacytidine of the formula I without further purification. By repeating the production cycle, good yields of the desired beta-anomers can be achieved.

In the following, the invention is illustrated in more detail by way of non-limiting examples.

He did

Preparation of N-amldino-N * - (2-deoxy-beta-D-erythro-pentofuranosyl) urea formates

A solution of 2.28 g of pure alpha-anomer of 2 ' -deoxy-5-azacytidine, m.p. 82 DEG-183 DEG C. (decomposition) in 200 ml of aqueous 1M ammonia was allowed to stand at room temperature for 1 h and then evaporated in vacuo. The residue is thoroughly stirred with 10 ml of ethanol and the crystalline product is filtered off with suction. The mother liquor is concentrated to a small volume, 0.1 ml formic acid is added and the mixture is left in the refrigerator overnight. The second crop was aspirated and washed with ice-cold ethanol. After air-drying, a total of 2.55 g, i.e. 96% of N-amidino-N * - (2-deoxy-beta-D-erythropentofuranosyl) urea formates, m.p. 153-156 ° C (decomposition) is obtained. This product can be used without further purification for the subsequent preparation of 2'-deoxy-5-azacytidine. Recrystallization from methanol gave the pure product, m.p. 160-162 ° C (dec.).

Example 2

A solution of 2.28 g of the crude alpha-anomer of 2'-deoxy-5-azaoytidine [t. 165-176 [deg.] C. (decomposition)] is hydrolyzed in the same manner as in Example 1. 2.50 g, i.e. 94% of N-amidino-N '- (2-deffxy-beta-D-erythro) formates, are obtained. m.p. 152-156 ° C (decomposition).

Example 3

A solution of 2.28 g of the mother liquor residue after preparation of 2 ^, -deoxy-5-azacytidine (see Example 4) is hydrolyzed as described in Example 1. 1.54 g (58%) of N-amidino-N '- (-) formates are obtained. 2-deoxy-beta-D-erythro-pentofuranosyl) urea; mp 150-155 ° C (dec.).

Example 4

Preparation of 2'-deoxy-5-azacytidine

Mix 2.64 g of N-amidino-K '- (J-deoxy-beta-D-erythro-pentofuranosyl) urea formate, ml of methanol and 2.6 ml of dimethyl acetamide dimethylacetate were magnetically stirred in a sealed flask at room temperature for 24 hours. The starting material initially goes into solution and after about 1 hour the reaction product begins to precipitate. The mixture was cooled after this time, the product was filtered off with suction, washed with ice-cold methanol and dried in vacuo. 0.91 g, i.e. 40% of 2'-deoxy-5-azacytidinate, m.p. 202-204 ° C (decomposition), is obtained. The mother liquor residue ee is used to recover the starting amine urea (see Example 3).

Claims (4)

OBJECT OF THE INVENTION A process for the preparation of 2'-deoxy-5-azacytidines of the general formula I (I) wherein r 1 and R ² are either the same or different and denote hydrogen or alkyl characterized in that they are alpha-anomers of 2'-deoxy-5-azacytidines common 1-4 carbon atoms, of formula II (II) wherein R ¹ and R ^{2 are the} same as in formula I, hydrolyze to formates of N-amidino-N * - (2-deoxy-bete-D-erythro-pententouranosyl) ureas of formula III HOCH 2 _nH - _C ON = C-NH ₂ .HCOOH (III) Wherein R 1 and R ^{2 are the} same as in Formula I, which are then cyclo-condensed with a dialkyl acetal of dimethylformamide having an alkyl radical of 1 to 4 carbon atoms, in an alkanol of 1 to 4 carbon atoms. 2. A process according to claim 1 wherein the hydrolysis is carried out with a dilute solution of the volatile base at room temperature. 3. A process according to claim 2 wherein the hydrolysis is carried out with dilute ammonia. 4. The method of claim 1 wherein the cyclo-condensation is performed with dimethyl acetamide dimethyl acetal at room temperature in methanol.