2,2' -O-CYCLOCYTIDINE

FIELD OF THE INVENTION

The present invention refers to a synthetic process for the preparation of 2,2'-anhydro-l-P- D-arabinofuranosylcytosine hydrochloride as well as Ι-β-D-arabinofuranosylcytosine or cytarabine or Ara-C starting from cytidine, which is obtained in two steps.

BACKGROUND OF THE INVENTION

Ι-β-D-arabinofuranosyl cytosines are known to be pharmaceutically useful for their antiviral, cytotoxic and antineoplastic activities. Cytarabine which is also chemically known as 4-amino-l-[(2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxol an-2-yl]pyrim- idin-2- one and cytosine arabinoside, is a chemotherapy agent used mainly in the treatment of cancers of white blood cells such as acute myeloid leukemia (AML) and non-Hodgkin lymphoma. It kills cancer cells by interfering with DNA synthesis. Cytarabine has the following chemical structure:

There have theretofore been many processes reported for the synthesis of Ι-β-D- arabinofuranosylcytosine in the literature.

Hoffer (U.S. Pat. No. 3.721.664) explains a process for the preparation of cytosine nucleoside by a glycolysation reaction between silylated cytosine and acylated sugar halide. The synthesis is accomplished at a temperature of about 120-170 °C and utilizing at least 2 moles of the silylated cytosine in which process two different isomers (α+β) of Ara- C are formed. Beranek et al (Nucleic Acid Chemistry, Vol. 1, 249, Edited by Townsend and Tipson, Wiley, N.Y.) teach the production of cytarabine from cytidine. Specifically, cytidine is reacted with incremental amounts of diphenyl carbonate in the presence of DMF and water at 120 °C.

Roberts et al (J. Org. Chem. 32, 816 (1967)) teach the production of cytarabine from cytidine (or from 2'(3')-cytidylic acid). Specifically, cytidine is reacted with phosphoric acid at 80 °C for a period of 30 hours to produce a 2,2'-0-cyclocytidine analogue intermediate. This intermediate is then hydrolyzed at a pH of 9 utilizing lithium hydroxide to produce the 3',5'-diphosphate of cytarabine. The diphosphate is then treated with magnesium chloride, ammonium chloride and concentrated ammonium hydroxide, and thereafter purified by column chromatography to yield pure cytarabine. The overall yield is 53% based on the uncovered portion of starting cytidine. However, it is impossible to put this process into commercial practice since the process has numerous shortcomings that make it very troublesome such as purification of the product utilizing column chromatography .

Kanai et al (U.S. Pat. No. 3.755.296) describe preparation of arabinofuranosylcytosine. The process comprises of reacting cytidine with a Vilsmeier-Haack reagent formed from an acid chloride such as thionyl chloride, phosphorus oxychloride or phosgene in the presence of DMF. Sulfurous acid was removed and then the pH was basified to 9 with ammonia. The solution was then acidified to pH 2 with hydrochloric acid. After treating with active charcoal and washing with water and elution with 50 percent ethanol containing 2 percent ammonia, the eluent was concentrated to dryness and treated with a column of ion exchange resin. In the end, the crystals of Ι-β-D-arabinofuranosylcytosine were obtained from ethanol to yield 70% of the titled compound. Inventors use a complicat

-ed and time consuming process to isolate the product as well as utilize column of ion exchange resin to purify the product which is difficult to handle for large quantity of the product, making it an expensive process to obtain cytarabine.

Kikugawa et al (J. Org. Chem., 37, 284-288 (1972)) teach the conversion of 2,2'-0- cyclocytidine hydrochloride to cytarabine. Specifically, ammonia is added to an aqueous solution of 2,2'-0-cyclocytidine thereby raising the pH to 9. The solution is thereafter acidified with hydrochloric acid and run through an ion exchange column. Thereafter, cytar-abine is crystallized from ethanol in a ¾ield of 90% (last step). Ishido et al (U.S. Pat. No. 3.856.777) disclose the production of cytarabine from cytidine. Cytidine hydrochloride is reacted with ethylene carbonate in the presence incremental amounts of ethylene carbonate at 150 °C for a period of 50 minutes. The resulting mixture was then dissolved in water and then the solution was passed through a column packed with active charcoal. Thereafter, the eluate was evaporated to dryness and the residue was recrystallized from water: ethanol mixture to obtain 2,2'-0-cyclocytidine hydrochloride. Thereafter, sodium hydroxide is added to an aqueous solution of 2,2'-0-cyclocytidine hydrochloride and followed by extraction with methanol and crystallization from water to obtain cytosine nucleoside with the overall yield of 35%. Use of a column packed with active charcoal to purify the product, as well as large quantity of water used as eluent (which is difficult to evaporate) are cumbersome, expensive and difficult to carry out on commercial scale.

Sowa et al (Bull. Chem. Soc. Jap., 48, 505-507 (1975) teach the production of cytarabine from 2,2'-0-cyclocytidine. Specifically, sodium hydroxide is added to an aqueous solution of 2,2'-0-cyclocytidine hydrochloride thereby raising the pH of the solution to 10. Thereafter, the solution is run through an acid ionic exchange resin followed by recrystallization of pure cytarabine from ethanol.

Inoue et al (U.S. Pat. No. 3.950.325) explains a process for producing cytarabine. The process comprises reacting cytidine with a functional derivative of silicon such as silicon tetrachloride in acetic acid under reflux condition. After the reaction is complete, the mixture is concentrated under reduced pressure and the reside is dissolved in ice-water and pH raised to 12.5 utilizing aqueous sodium hydroxide. Thereafter, the solution passed through a column of an acidic ion exchange and eluted with aqueous ammonia. Finally, the eluate is concentrated under reduced pressure and the residue is crystallized from 50% ethanol to obtain cytarabine with the yield of 55%. Although the titled compound is obtained with acceptable yield, an obstacle to such synthesis, from the point of view of the industrial insight, derives from the fact that the aforementioned technique for the preparation of Ι-β-D-arabinofuranosylcytosine is deficient in that the process utilizes a large quantity of water as well as aqueous ammonia for producing small amount of the tiled compound. Moreover, the process comprises concentration of large amount of aqueous solution as well as requiring the use of acidic ion exchange. Karimian (U.S. Patent No. 5.610.292) teaches a process for producing cytarabine wherein cytidine is reacted with dibutyl tin oxide in the presence of triethylamine followed by slow addition of p-toluenesulfonyl chloride in methanol to produce 2,2' -O-cyclocytidine. Thereafter, 2,2' -O-cyclocytidine is converted to cytarabine utilizing t-butylamine in water at 80°C with the overall yield of 24%. In spite of the novelty of the foresaid process, it faces the obstacle of using dibutyl tin oxide which is both toxic and expensive reagent. Additionally, utilizing water in the production of 2,2'-0-cyclocytidineas as well as cytarabine and furthermore, the need of evaporating large quantity of water is another shortcoming of the invention.

Meglio (E.P. Pat. No. 0.757.056 Al) teaches a process for the preparation of cytarabine from arabinofuranosyluracil. The process comprises the reaction of Ι-β-D- arabinofuranosyluracil (Ara-U) under a pressure comprised between 8-18 bar and a temperature 130-140°C with hexamethyldisilazane and a C1-C3 acylamide, subsequently dry evaporating the resulting mixture and then treating the oily residue with methyl alcohol and aqueous ammonia, further dry evaporating and purifying said residual by percolating on an acidic resin, washing with water and subsequently eluting with ammonia, finally after evaporating a large volume of diluted ammonia, the oily product is made crystalline with methanol to obtained the titled compound with 77% yield. Even though the product is obtained in good yield, the process suffers from reduction of a large volume of water, utilizing an acidic resin which is not easy to handle for large scale production as well as the reaction condition which is accomplished under high pressure.

Kanai et al (U.S. Patent No. 3.595.853) teach the production of arabinofuranosylcytosine wherein cytidine is reacted with fuming nitric acid at -20 °C in the presence of phosphorous

-s pentoxide to give l-(3',5'-tri-0-nitro-P-D-ribinofuranosyl)cytosine in three steps. Thereafter, the titled compound was dissolved in 70% methanol and adjusted to pH 2 with hydrochloric acid and treated with 5% Pd on BaS0 ₄ under atmospheric pressure in a stream of hydrogen to obtain cytarbine with the overall yield of 36%. It goes without saying that difficult and time consuming isolation and purification process as well as utilizing Pd which is an expensive reagent and dangerous to apply hydrogen in industrial scale production, makes the invention less favored. Further, the production of cyclonucleosides is known. For example, Walwick et al (Proc. Chem. Soc, 84 (1959)) teach the production of 2,2'-0-cyclocytidine hydrochloride from cytidine. The process involved heating cytidine with polyphosphoric acid followed by dephosphorylation of one of the reaction products, 2,2'-0-cyclocytidine-3',5'-diphosphate.

Doerr et al (J. Org. Chem., 32, 1462 (1967)) teach the production of 2,2'-0-cyclocytidine chloride from uridine using a process comprising six steps. It is interesting to note that in the final step, 2,2'-0-cyclotidine hydrochloride was obtained only in a 57% yield. Taking into account the fact that each step is not quantitative, the overall yield of 2,2'-0- cyclocytidine hydrochloride from uridine can be expected to be on the order of from 10% to 20%.

The above-noted processes, however, could not be operated easily, since they require multistage operation for the synthesis with inherent loss of yield. Additionally, they require silica/resin columns for isolation and purification. Furthermore, the prior art processes for the production of cytarabine and its analogues are deficient in that the purified product is obtained in a relatively low yield and/or the process is complicated requiring a series of steps including the use of ion exchange resins as well as requiring evaporating large volumes of water which is not easily feasible in the industry.

It would be desirable to have a relatively simple process for producing 2,2'-0- cyclonucleosides in acceptable and/or comparable yields. Furthermore, it would be desirable to have a process for the production of 2,2'-0-cyclonucleosides such as cytarabine and pharmaceutically acceptable salts thereof in relatively high yields and by a relatively simple process.

BRIEF DESCRIPTION OF THE INVENTION

2,2' -O-cyclocytidine hydrochloride is obtained by reacting cytidine hydrochloride with ethylene carbonate at 130°C by precipitation of the pure product from the resulting mixture from a highly polar solvent such as alcohol, DMF, etc. at 0-20°C via a simple and inexpensive isolation technique with an acceptable yield. Resulting 2,2' -O-cyclocytidine hydrochloride was converted to cytarabine utilizing lithium hydroxide in a protic solvent such as water, methanol, ethanol and a mixture of water and at least one other solvent miscible therewith and preferably methanol. The crude cytarabine was purified using methanol to obtain pharmaceutical grade product. SUMMARY OF THE INVENTION

It is an object of the present invention to provide an efficient process for producing Ι-β-D- arabinofuranosylcytosine and pharmaceutically acceptable salts thereof, which is simple and inexpensive.

It is a further object of the present invention to provide a process which avoids the troublesome heretofore encountered in the prior art for the production of 2,2'-anhydro-l-P- D-arabinofuranosyl cytosine.

Accordingly, the present invention provides a process for preparing a compound of Formula I, or a pharmaceutically acceptable salt thereof:

Starting from a compound of Formula II, or a pharmaceutically acceptable salt thereof:

(ID Which comprises the step of reacting with cyclic carbonates such as ethylene carbonate to produce a compound of Formula III or a pharmaceutically acceptable salt thereof:

wherein Ri is selected from the group comprising hydrogen, trityl, methoxytrityl, dimethoxytrityl, acetyl, a C ₂ -C ₆ alkylacyl group, a C ₆ -C9 arylacyl group, allyl, 2,2,2- trichloroethyl, phosphates and salts thereof, tosyl and mesyl and the like; W is selected from the group comprising -NH-CO- and -NH-C(NH ₂ ) -; Z is selected from the group comprising hydrogen, methyl and halide; and Y is selected from the group comprising - N(H)- or O.

DETAILED DESCRIPTION OF THE INVENTION

The process of the invention is schematically represented as follows:

Preferably, the process for producing a compound of Formula III can be used to produce 2,2'-0-cycloribonucleosides such as 2,2'-0-cyclocytidine, 2,2'-0-cyclouridine, 2,2'-0- cyclothymidine, or pharmaceutically acceptable salts thereof. Generally, 2,2'-0- cycloribonucleosides may be prepared by reacting the appropriate nucleoside with ethylene carbonate. More preferably, this process is used to produce 2,2'-0-cyclocytidine by reacting cytidine and its salts thereof with at least 5 eq moles of ethylene carbonate.

Typically, the above-noted reaction can be conducted at a temperature comprised between 110 to 140°C and preferably at 130°C.

The most preferred starting material of Formula III for the process of producing a compound of Formula I is 2,2'-0-cyclocytidine in which Ri of Formula III is hydrogen. In this embodiment, the product of Formula I is cytarabine. It will of course be understood that the manner in which starting compound of Formula III is made is not particularly restricted as regards the process of making Formula I.

The base suitable for use in the process of producing a compound of Formula I is selected from the group comprising lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, cesium hydroxide and barium hydroxide.

The crude 2,2'-0-cyclonucleoside precursor product, preferably, 2,2'-0-cyclocytidine, as well as the crude final products, preferably, Cytarabine and its analogues or pharmaceutically acceptable salts thereof, may be separated from the reaction mixture and purified using conventional techniques within the purview of a person skilled in the art. For example, after the reaction is complete, crude 2,2'-0-cyclonueleoside may be suspended in a suitable polar solvent. Examples of suitable polar solvents include alcohols, ketones and amides. The most preferred solvent is selected from amides and preferably dimethy If oramide .

Preferably, the process of producing a compound of Formula I is conducted in the presence of a protic solvent. Examples of suitable protic solvents include water, methanol, ethanol and a mixture of water and at least one other solvent miscible therewith. The most preferred solvent for use in this process is methanol.

The resulting final cytarabine solid may be suspended and agitated in a suitable medium to produce a purified product. Examples of such media include alcohol and mixtures containing alcohol and water. The preferred alcohol for use is methanol.

Aspects of the present invention will be described with reference to the following examples which should not be considered to limit the scope of the invention.

EXAMPLE 1

Preparation of 2,2'-0-cyclocytidine hydrochloride

A 500 ml three-necked flask was equipped with a mechanical stirrer and condenser and then charged with 157,52 g (1.79 mol) of ethylene carbonate and heated to 130°C with slow stirring. 100 g (0.358 mol) cytidine hydrochloride was added into the flask in a period of 50 minutes. The resulting suspension was stirred at 130°C for four hours and then the mixture was cooled to room temperature. 250 ml dimethylformamide was cooled to 10°C and added to the resulting brown gummy precipitate and the resulting mixture was stirred at 10°C for two hours. The creamy precipitate was filtered and washed with dimethylformamide and then with acetone and dried to yield 50 g of 2,2'-0-cyclocytidine hydrochloride (53% yield). The product was characterized by comparison of its melting point, UV and IR spectra with those previously reported for 2,2'-G-cyclocytidme hydrochloride. The product was used without further purification for the next step.

EXAMPLE 2

Preparation of Ι-β-D-arabinofuranosylcytosine

2,2'-0-cyclocytidine hydrochloride (50 g, 0.191 mol) was suspended in 500 ml methanol and stirred a reflux temperature. Then, 8.026 g (0.191 mol) lithium hydroxide monohydrate was added to the resulting suspension. Thereafter, the resulting yellow solution was stirred at reflux for 75 minutes. Then the reaction mixture was adjusted to pH 7-8 using concentrated hydrochloric acid. The resulting solution treated with active charcoal and the yellow filtrate was evaporated under reduced pressure to about 250 ml. Thereafter, the resulting solution was stirred at room temperature for an hour and then the precipitate was filtered and dried to yield 37 g of crude cytarabine (79% yield). A 2000 ml three-necked flask was equipped with a mechanical stirrer and then charged with 37 g of crude cytarabine and 1221 ml methanol and then heated to reflux until a homogenous solution was obtained. Thereafter, the resulting solution was treated with 1.85 g active charcoal and stirred at reflux for an hour. The boiling reaction mixture was filtered on Celite and the filtrate was evaporated under reduced pressure until about 370 ml of solvent remained. The resulting solution was stirred at room temperature for one hour and then the precipitate was filtered and dried to yield 32.88 g of pure cytarabine (88.9% yield). The product was characterized by comparison of its HPLC, melting point, UV, NMR and IR spectra with those previously reported for cytarabine.