DE2146733A1 - 1-(beta-d-arabinofuranosyl)-cytosine prepn - from tri-o-acyl 1-(beta-d-arabinofuranosyl)-uracil - Google Patents

Abstract

1-(beta-D-arabinofluranoxyl)cytosine, which is active against acute and chronic leukaemia, is prepd. by halogenating (II) to give (III) (where R1, R2 and R3 are acyl, and X is halogen) aminating (III) and deacylating. Preferred halogenating agents are PX5 and POX5, used in the presence of base and amination is pref. effected with NH3 which may also be used for deacylation.

Description

Process for the preparation of 1- (β-D-arabinofuranosyl) -cytosine.

The invention relates to an improvement in manufacture of 1- (β-D-arabinofuranosyl) -cytosine.

In particular, it relates to a new and improved process for the preparation of 1- (ß-D-arabinofuranosyl) -cytosine represented by the following formula (I) from the corresponding Gri-O-acylarabinofuranosyluracil derivative: 1- (ß-D-arabinofuranosyl) -cytosine is a known substance which exerts convincing therapeutic activities against acute or chronic leukemia, and so can be used as a medicament. For example, the publication on page 561 of "Drugs of choice 1970-1971" edited by Walter Modell? 1970, The CV Mosby Company, USA reports that the usual dose of 1- (β-D-arabinofuranosyl) -cytosine administered intravenously is 1 to 3 mg / kg daily until ringing in the ears due to toxicity occurs.

Ji; s are numerous methods of manufacture so far in the art of 1- (ß-D-irabinofuranosyl) -cytosine has been suggested. Under these methods a method was considered satisfactory, in which the price-worthy and economical most conveniently available starting material, uridine, by rearrangement of the sugar part of the above-mentioned starting material is converted into 1- (β-D-arabinofuranosyl) -uracil and the uracil product thus obtained was converted into 1 - (ß-D) -arabinofuranosyl) -cytosine by substituting the substituent in the base part of the product. In this favorable method, the first stage of the procedure was acceptable because of the few steps, simple process management and a satisfactory one high yield (Biochemistry, 5, (1966), 2076), the second process section however, it contains some peculiarities to be improved, namely a number more complicated Working iron and an economically insufficient loot £ U. $. Patent No. 3 116 282). It has therefore become known in the art to make improvements in the above attention paid to the second stage of the process, wherein 1- (ß-D-arabinofuranosyl) -uracil was converted into the corresponding cytosine derivative to 1- (ß-D-arabinofuranosyl) -cytosine much cheaper and more convenient to obtain.

r; s is therefore a primary object of the invention, an improved and economically advantageous process for the preparation of 1- (β-D-arabinofuranosyl) -cytosine from the corresponding uracil derivative.

This and other features of the present invention result to those skilled in the art from the following more specific description.

As a result of extensive studies on the production of 1- (BD-arabinofuranosyl) -cytosine, it has now been found that the above objects can be achieved by a process which comprises the steps of halogenating a ri-O-acyl-arabinofuranosyluracil with the formula wherein R1, R2 and R3 can be the same or different and each represent an acyl group such as an aliphatic acyl group such as acetyl, propionyl or butyryl, or an aromatic acyl group such as benzoyl or p-nitrobenzoyl, around a compound of the formula to obtain in which R1, R2 and R3 have the meaning described above and X represents a halogen atom, such as chlorine or bromine; the amination of the latter product thus obtained; and the subsequent deacylation or removal of the acyl groups.

As set forth above, the improved lytic method of the present invention includes mainly three levels. However, it was also pointed out that the deacylation at the same time with the amination can take place depending on the type or Types of acyl groups represented by k1, R2 and R3; in other words, the method according to the invention can in some cases be effected in two stages, their specific implementation within the scope of the present invention is considered to be falling, particularly when any aliphatic acyl group is present is, the deacylation can take place simultaneously with the amination by the Exposure to ammonia while if any aromatic acyl group is present is, the deacylation is advantageously carried out separately after the amination, because the aromatic acyl group often has far less deacylation from exposure may be subject to ammonia.

When carrying out the method according to the invention, the first Stage, namely the halogenation stage, can be conveniently carried out by using the Bringing starting material (II) into contact with a halogenating agent, preferably under moisture-free conditions.

The reaction can be effected in the presence or absence of one Reaction solvent, however, it is convenient to carry out the reaction in the presence of a suitable solvent, because the reaction then proceeds more smoothly. The reaction solvent which can be used in this step can be be any such solvent that does not adversely affect the reaction. Examples of the reaction solvents include lower aliphatic carboxylic acid esters, such as ethyl acetate or butyl acetate; halogenated hydrocarbons such as dichloromethane or chloroform, and the like. Lower aliphatic carboxylic acid esters are preferred, and ethyl acetate is particularly preferred. The halogenating agent used in this Stage can be used includes phosphorus halides, for example phosphorus oxyhalides, such as phosphorus oxychloride, phosphorus oxybromide; Phosphorus pentahalides, such as phosphorus pentachloride, Phosphorus pentabromide, and the like. Phosphorus oxyhalides, and Phosphorus oxychloride is particularly preferred.

The reaction in this stage can be accelerated even if the yield is higher are made by using a base with a tertiary nitrogen atom or a Acid addition salt thereof as a catalyst. Examples of the catalyst shown in can be used at this stage include bases such as pyridine, picoline, Quinoline, lutidine, triethylamine, tri-n-butylamine, dimethylaniline, diethylaniline, and acid addition salts thereof, such as hydrochloride, hydrobromide, sulfate, acetate, Oxalate.

The reaction temperature is not a critical feature of this stage, However, it is advisable to carry out the reaction at approximately the reflux temperature of the used To carry out reaction solution means or halation means, there the reaction can be accelerated using an elevated temperature. The reaction time is also not critical and can be varied on special request mainly depending on the reaction temperature, the type of catalyst, the presence or absence of a reaction solvent and other factors; in general, however, the reaction is complete in about 1 to 10 hours.

After completion of the reaction, the intermediate (III) from the Reaction mixture can be obtained by one of the usual methods. For example the solvent is distilled off from the reaction mixture and the residue purified by any of the usual methods such as chromatography. Alternatively can the product (III) without further purification in the following step for the preparation of the desired product (I) can be used.

The second stage, namely the amination stage, can easily be effected by adding »*. intermediate (III) obtained in the previous stage with ammonia. The reaction is usually caused by the use of gaseous ammonia in the presence of a reaction solvent, however, can the reaction can alternatively be carried out in the presence of liquid ammonia in much larger amount than the stoichiometric, because ammonia both as Reagent as well as reaction solvent can serve. The reaction solvent if used, it can be any of those solvents which do not adversely affect the response. examples of reaction solvent, which can be used at this stage include e iii h logenie rte Koh hydrogen substances such as methylene chloride or chloroform; cyclic ethers, such as l'etraNydrofuran or dioxane; and the same. halogenated hydrocarbons are preferred most preferred is chloroform. The reaction temperature is not critical at this stage, and the reaction will generally be at room temperature executed. However, higher or lower temperatures can also be used favorably. The reaction time is also not critical. However, the reaction is general finished in about two to 16 hours.

After the reaction has ended, the aminated product can be removed from the reaction mixture can be obtained by one of the usual methods. For example, that will be the excess Ammonia and, if used, the reaction solvent from the reaction mixture distilled off, whereby the end product is obtained at this stage. In the case, that the deacylation can take place at the same time, is the end product thus obtained - that desired product (I).

If not deacylated, the end product aminated in this stage can be used can be used in the following step without further purification.

The third stage, namely the deacylation stage, can easily be carried out by bringing together the aminated product obtained in the previous step with a base in the presence of a solvent. The base, which in this stage can be used can be any of such bases generally disclosed in US Pat Technique of saponification can be applied. Examples of the base include alkali metal alkoxides, such as sodium methoxide or sodium ethoxide; Alkali metal hydroxides such as sodium hydroxide or potassium hydroxide; Ammonia; strongly basic ion exchange resins, such as resins from Type of quaternary ammonium compounds; etc. The alkoxides are preferred for such Acyl groups, which are not so much amenable to deacylation, and ammonia for those acyl groups that are easily accessible. The reaction solvent, which Any of those solvents can be used at this stage which are commonly used in the art for deacylation. Examples the reaction solvent include water; Alcohols such as methanol or ethanol; aqueous Alcohols such as aqueous methanol or aqueous ethanol; and the same. The reaction temperature is not critical, but the reaction can generally take place at room temperature or a reflux temperature of the reaction solvent used. However, a higher or lower temperature can also be used, if desired will. The reaction time is also not critical and can vary as a function the type or nature of the acyl group and the base concerned and the reaction temperature, However, the reaction is generally in 2 minutes to about 16 hours completed.

After the reaction has ended, the desired product (I) can be selected from the reaction mixture can be obtained by an ordinary method. For example the solvent is distilled off from the reaction mixture if ammonia is used or the same procedure is followed after neutralizing the reaction mixture applied with an appropriate acid when an alkali metal compound is used and the residue can be further processed by an ordinary method such as chromatography are purified to obtain the desired product (I) in a pure form.

As described in detail above, the inventive improved process several advantages and results in an unexpected technical Progress over the known methods, especially in the respect that a inexpensive and readily commercially available starting material and simple procedures can be used to obtain a yield of 1- (ß-D-arabinofuranosyl) -cytosine in commercial scale.

The following examples are provided for the purpose of explaining the Invention given without limiting it.

Example 1 (A) In 3 ml of dry ethyl acetate were added with heating 550 mg of diethylaniline hydrochloride and 5 ml of phosphorus oxychloride dissolved. To the received 556 mg of tri-O-benzoyl-1- (ß-D-arabinofuranosyl) uracil were added to the solution, and the resulting mixture was refluxed for 7 hours while moisture-free Conditions were maintained. Thereafter, the solvent was under a reduced pressure at a bath temperature below 70 from distilled off. The residue was then mixed well with 10 ml of dry ethyl acetate and the solvent became again distilled off under reduced pressure. The residue was dried in vacuo, whereby a yellow caramelized substance was obtained.

(B) The substance thus obtained was dissolved in 10 ml of dry chloroform, and then dry gaseous ammonia was bubbled into the solution obtained, while cooling with a dry sis-acetone cooling mixture. The gushing was interrupted at a time when the volume of the reaction mixture was increasing doubled in size. Then the reaction container was closed and Left to stand at room temperature for 16 hours. After the ammonia from the reaction mixture was completely removed, the in situ deposited crystalline substance was through Filtration recovered and washed with 10 ml of chloroform. The filtrate was with the Washes were combined and the resulting mixture became a viscous substance distilled off.

(C) In 10 ml of a 0.1 N methanolic solution of sodium methoxide the viscous substance obtained in the above manner was dissolved, and the resulting solution was refluxed for 10 minutes. After cooling it was the The reaction mixture was neutralized with 0.1 N hydrochloric acid.

Then the solvent was distilled off under reduced pressure. To the residue were added 20 ml of water and 10 ml of chloroform, and the obtained Mixture was shaken thoroughly and an organic layer from an aqueous Layer separated. The aqueous layer thus deposited was again made up of 10 ml Chloroform, and then 10 ml of ethanol was added.

The resulting mixture was reduced to a volume of 5 ml Pressure restricted. The residue was adsorbed on a column (2 cm diameter and 7 cm length) of "Dowex 1 x 4" (OH-Form), which then with 300 ml of water and then was eluted with a 50 point aqueous methanol. From the aqueous methanolic Fractions were collected as those showing the same ultraviolet spectrum such as cytidine, and then the solvent was distilled off under reduced pressure. The residue was concentrated to dryness and recrystallized from 95% ethanol, 167 mg of 1- (ß-D-arabinofuranosyl) -cytosine were obtained as colorless plates, which melt at 212 to 213 0C.

Example 2 (A) In 5 ml of dry ethyl acetate were added with heating 550 mg of diethylaniline hydrochloride and 5 ml of phosphorus oxychloride dissolved. To the received 370 mg of tri-O-acetyl-1- (ß-D-arabinofuranosyl) -uracil were added to the solution, and the resulting mixture was refluxed for 7 hours while moisture-free Conditions were maintained. Thereafter, the solvent was reduced under Pressure distilled off at a bath temperature below 70 ° C. The residue was then mixed well with 10 ml of dry ethyl acetate and the solvent was again distilled under reduced pressure. The residue was dried in vacuo.

(B) and (C) The residue was dissolved in 10 ml of dry chloroform, and dry ammonia gas was bubbled into the resulting solution while was cooled with a dry ice-acetone cooling mixture. The gushing turned to interrupted a time when the volume of the reaction mixture doubled was enlarged. The reaction vessel was then sealed and held for 16 hours Let stand room temperature. 10 ml of ethanol were added to the reaction mixture, and the resulting mixture was left to stand for an additional 4 hours.

Then 5 ml of water was added to the reaction mixture to reduce the to dissolve crystalline substance, which had deposited in situ, and that whole mixture was concentrated to dryness. The residue was then dissolved in 5 ml of water dissolved, and the resulting solution was on a column (2 cm diameter and 10 cm length) of "Dowex 1 x 4" (OH form) adsorbed, which with 500 ml of water and then was eluted with 50% aqueous methanol.

Of the aqueous methanolic fractions these were nigen, which have the same ultraviolet spectrum as cytidine, collected and under reduced Pressure concentrated to dryness. The residue was recrystallized from the above ethanol, 172 mg of 1- (B-D-arabinofuranosyl) cytosine were obtained.

Example 3 Essentially the same procedure as in the above Example 1 described was repeated with the exception that 2 ml of dry Pyridine was used instead of diethylamine hydrochloride and the reaction became effected in stage (A) in 1.5 hours, 110 mg of 1- (ß-D-arabinofuranosyl) -cytosine received.

Example 4 Essentially the same procedure as in the above Example 2 is described, was repeated with the exception that 2 ml of dry Pyridine were used instead of diethylaniline hydrochloride and that the reaction in the step (A) was effected in 1.5 hours, giving 120 mg of 1- (β-D-arabinofuranosyl) -cytosine received.

Example 5 Essentially the same procedure as described in the above Example 1 is described, was repeated with the exception that 4-chloro-tri-O-benzoyl-1- (B-D-arabinofuranosyl) -cytosine, which was prepared in the manner described below was used instead the yellow caramelized substance, 1- (ß-D-arabinofuranosyl) -cytosine received.

550 mg of diethylaniline hydrochloride were added to 5 ml of dry ethyl acetate with heating and 5 ml of phosphorus oxychloride dissolved. 556 mg of tri-O-benzoyl-1- (β-D-arabinofuranosyl) uracil were added to the resulting solution and the resulting mixture was refluxed for 7 hours while moisture-free conditions were maintained. Then the solvent became distilled off under reduced pressure, and the residue was dried with 5 ml ethyl acetate mixed well. After removing the solvent by distillation the residue was mixed well with 5 ml of dry benzene, and that benzene was removed by distillation. The cleaning procedures outlined above were used repeated three times.

Thereafter, the residue was dissolved in 3 ml of chloroform and on a Adsorbed silica gel thin layer chromatography plate with a thickness of 1.8mm, which was then developed with a mixture of benzene with acetone (22: 3).

The silica gel fractions, which can be detected by an ultraviolet lamp were scraped together and extracted with acetone while still hot. From the extract the solvent was distilled off, 250 mg of 4-chlorotri-0-benzoyl-1- (β-D-arabinofuranosyl) cytosine being added received.

UV spectrum JA CHCl3 mµ: 278 (Sh.), 284, 308.

max IR spectrum 3 a = o (cm-1): 1725 (a carbonyl group of a benzoyl group).

z an absorption band at 1735 cm 1, which is a carbonyl group in the 4-position was not observed.

Analysis values for 030H23O8N2Cl Calculated C = 62.66%; H = 4.03%; N = 4.87 %; C1 = 6.17 ° / 0; Found: C562.25%; H = 4.09%; N = 4.67 / 0; C1 = 6.28 Vo.

Claims (8)

Patent claims: 1. A process for the preparation of 1- (ß-D-arabinofuranosyl) -cytosine, characterized by the steps of halogenation of a compound of the formula in which R1, R2 and R3 can be identical or different and each represent an acyl group, for the preparation of a compound of the formula wherein R1, R2 and R3 have the meaning described above and X represents a halogen atom; the amination of the latter product (III); and the subsequent deacylation. 2. The method according to claim 1, characterized in that each of the Substituents R1, R2 and R3 are acetyl or benzoyl and that X is chlorine. 3. The method according to claim 1, characterized in that the halogenation is effected by the use of a halogenating agent selected from Group consisting of phosphorus oxyhalides and phosphorus pentahalides. 4. The method according to claim 1, characterized in that the halogenation is effected in the presence of a catalyst selected from the group consisting of from bases having a tertiary nitrogen atom and acid addition salts thereof. 5. The method according to claim 1, characterized in that the amination is effected using ammonia. 6. The method according to claim 1, characterized in that the deacylation is effected by using a base. 7. The method of claim 1, wherein each of the substituents R1, R2 and R3 is acetyl or benzoyl and X is chlorine, characterized in that the halogenation is effected using phosphorus oxychloride in the presence of diethylaniline or pyridine as a catalyst and of ethyl acetate as the reaction solvent that the amination is effected using gaseous ammonia, and that the Deacylation is effected using sodium methoxide. 8. The method according to claim 1, characterized in that each of the Substituents R1, R2 and R3 is acetyl, and that the deacylation is simultaneous with the amination is effected using ammonia.