Method of manufacturing 1 -(2-deoxy-alpha-D-eryt/zro-pentofuranosyl)-5-azacytosine

Technical Field

The invention relates to a method of manufacturing l-(2-deoxy-alpha-D-eryt/zro- pentofuranosyl)-5-azacytosine, which is efficacious as an agent inducing DNA hypomethylation.

Background Art

The most considerable compound of the group of deoxynucleosides of 5-azacytosine is 5-aza-2'-deoxycytidine (decitabine, 1 -(2-deoxy-beta-D-eryf/zro-pentofuranosyl)-5- azacytosine), which is a potent chemotherapeutic effective against a broad variety of malignant tumor diseases (Momparler, R. L.: Semin. Hematol. 2005, 42, (Suppl. 2), S9- 16). In 2006 this nucleoside was approved for the treatment of all types of myelodisplastic syndromes (Hennessy, B. T.; Garcia-Manero, G.; Kantarjian, H. M.; Giles, F. J.: Expert. Opin. Investig. Drugs, 2003, 12, 1985-1993). The efficacy of 5-aza- 2'-deoxycytidine is based on its ability to inhibit DNA methyltransferase and thus decrease the level of DNA methylation, which is one of the epigenetic mechanisms playing a crucial role in cancerogenesis and tumor development. DNA hypomethylation leads to reactivation of the genes being essential for the normal cell function, which were silenced because of aberrant methylation during tumorogenesis. Thus, the malignant cell population is reverted to a more normal stage, which is the principle of the epigenetic therapy of cancer diseases (Yoo, C. B.; Jones, P. A.: Nature Rev. 2006, 5, 37-50). 5-Aza-2'-deoxycytidine is hitherto the most potent hypomethylating agent from the compounds studied so far. Screening of new drugs targeting the specific enzymes involved in epigenetic regulation of gene expression represents an efficient and valuable approach to the chemotherapy of cancer.

It is known that the above mentioned l-(2-deoxy-alpha-D-ery//zrø-pentofuranosyl)-5- azacytosine (compound of the formula I), which is an alpha anomer of 5-aza-2'- deoxycytidine, exhibits also a potent antileukemic activity. I ts efficacy is lower in

comparison to 5-aza-2'-deoxycytidine, but the lack of activity is compensated by much lower toxicity (Vesely, J.; Piskala, A.: Cancer Res. 1984, 44, 5165-5168). Recently we found that l-(2-deoxy-alpha-D-eryt/zro-pentofuranosyl)-5-azacytosine possesses also an ability to induce DNA hypomethylation to a comparable degree as the clinically used 5- aza-2'-deoxycytidine without incorporating into DNA and without being deaminated by cytidine deaminase (Fojtova, M.; Piskala, A., Votruba, L; Otmar, M.; Bartova, E.; Kovarik, A.: Pharmacol. Res. 2007 , 55, 16-22; Holy, A.; Otmar, M.; Piskala, A.; Votruba, I.; Kovarik, A.; Fojtova, M.; Bartova, E.: CZ patent application PV 2007-18). With regard to the lower toxicity, higher stability in aqueous solution, and easier manufacturing compared to 5-aza-2'-deoxycytidine, l-(2-deoxy-alpha-D-erytλro- pentofuranosyl)-5-azacytosine is applicable to epigenetic therapy of cancer.

The exact mechanism of action of l-(2-deoxy-alpha-D-eryt/zro-pentofuranosyl)-5- azacytosine was never investigated in detail, however, it is supposed that its biological activity is based on its successive anomerization to the highly efficient beta anomer - 5- aza-2'-deoxy cytidine, which was observed in aqueous solution in vitro. The anomerization consists in a reversible hydrolytic cleavage of the 5-azacytidine ring to 2- deoxy-alpha-D-erytλro-pentofuranosyl derivative of formylcarbamoylguanidine followed by epimerization at the anomeric carbon of the sugar moiety and recyclization to 5-aza-2'-deoxycytidine.

l-(2-Deoxy-alpha-D-erytλro-pentofuranosyl)-5-azacytosine was usually obtained as a side product at the preparation of 5-aza-2'-deoxycytidine. 5-Aza-2'-deoxycytidine was prepared for the first time in 1963 using various modifications of a general procedure consisting in a successive construction of azacytosine ring on peracylglycosyl isocyanates (Soπn, F.; Piskala A.: CS patent 139 542 (Neth. 6 414 959)). Another variant of the general procedure, which was used originally to prepare 5-azacytidine (Sorm, F.; Piskala, A.: CS patent 116 297; Piskala, A.; Sorm, F.: Collect. Czech. Chem. Commun. 1964, 29, 2060-2075), was also employed to the synthesis of 5-aza-2'- deoxy cytidine, but without assigning the anomeric configuration of the product (Pliml, J.; Sorm, F.: Collect. Czech. Chem. Commun. 1964, 29, 2576-2578). Later, by further optimization of this procedure, both 5 -aza-2' -deoxy cytidine as well as its alpha-anomer - l-(2-deoxy-alpha-D-ery/λro-pentofuranosyl)-5-azacytosine were prepared in good

yields (Piskala, A.; Sorm, F.: Nucleic Acid Chem. 1978, 1, 443-449 (Townsend, L. B.; Tipson, R. S.; Eds), Wiley, New York).

Another method for the preparation 5-aza-2'-deoxycytidine and its alpha-anomer - l-(2- deoxy-alpha-D-eryt/?ro-pentofuranosyl)-5-azacytosine consists in a reaction of protected 2-deoxy-D-erjtfλrø-pentofuranosyl halides with C^ _j A^-bis-^rimethylsily^-S-azacytosine (Winkley, M. W.; Robins, R. K. J Org. Chem. 1970, 35, 491-495; Piskala, A.; Synackova, M.; Tomankova, H.; Fiedler, P., Zizkovsky, V. Nucleic Acid Res., Spec. Publ, 1978, 4, 109-113; Ben-Hattar, J.; Jiricny, J. J. Org. Chem. 1986, 51, 3211-3213). Thus, treatment of 2-deoxy-3,5-di-O-j9-toluoyl-alpha-D-eryt/?ro-pentofuranosyl chloride with O^λ^-bis-^rimethylsilyrj-S-azacytosine in the presence of tin(IV) chloride afforded a product in lower yield, which was declared as 2 '-deoxy-3 ',5 '-di-O-p-toluoyl- 5-azacytidine (Niedballa, U.; Vorbriiggen, H.: J. Org. Chem. 1974, 39, 3672-3674). However, according to the presented ¹ H NMR spectra it is clear, that the product is alpha-anomer and not beta-anomer, as was erroneously assumed by the authors. Preparation of the corresponding unprotected nucleoside was not reported in the mentioned publication. The free nucleoside, obtained by removal of the protecting groups from the 3,5-di-O-/7-toluoyl derivative prepared according to Niedballa and Vorbruggen, was described later (Benjamin, E. J.: Ph.D. Thesis, 1979, University of Kansas; Diss. Abstr. 1980, 41, 208-B); however, it was still erroneously interpreted as beta-anomer, although it clearly shows the ¹ H NMR spectrum as well as the melting point of the alpha-anomer. Additionally, the kinetics of hydrolysis of the free nucleoside showed a higher stability, than was found later for a true beta-anomer (Lin, K.-T.; Momparler, R. L.; Rivard, G. E.: J. Pharm. Sci. 1981, 70, 1228-1232). A comparison of the hydrolytic stability stability of both anomers is also described in the paper mentioned above (Vesely J., Piskala A.: Cancer Res. 1984, 44, 5165-5168).

A further published method of preparation of 5-aza-2'-deoxycytidine employs a partial anomerization, which takes place during the hydrolysis of the alpha-anomer.

Recyclization of the mixture of hydrolytic products, which were formed by a treatment of the alpha-anomer with aqueous ammonia, by dimethylformamide dimethylacetal afforded 5-aza-2'-deoxycytidine in lower yield (Piskala, A.; Vesely, J.: CS AO

220430). In the mentioned patent, a considerable transformation of alpha-anomer to beta-anomer was supposed, which was later revised on the base of a rigorous analysis of the NMR spectra, showing that under the given conditions a mixture of anomers of furanosyl and in a lower rate of pyranosyl derivatives is formed (Piskala, A.; Hanna, N. B.; Masojidkova, M.; Otmar, M.; Fiedler, P.; Ubik, K. Collect. Czech. Chem. Commun. 2003, 68, 711-743). That also explains the mentioned lower yield of 5-aza-2'- deoxycytidine.

The above mentioned synthetic procedures afford only mixtures of both anomers, whose separation is difficult and requires repetitive chromatography or a highly lossy fractional crystallization, which considerably complicates a large scale preparation. These disadvantages are eliminated by the method of manufacturing according to the presented invention consisting in a reaction of protected 2-deoxy-D-erythro- pentofuranoside with silylated 5-azacytosine in the presence of Lewis acid and subsequent removal of the protecting groups.

Description of the Invention

Object of the present invention is a method of manufacturing of hypomethylating agent l-(2-deoxy-alpha-D-eryt/z/O-pentofuranosyl)-5-azacytosine of the formula I,

which consists in the reaction of protected 2-deoxy-D-eryt/zrø-pentofuranoside of the general formula II,

(H)

wherein R ¹ represents an alkyl group having 1 to 6 carbon atoms or aralkyl group having 7 to 10 carbons, being eventually substituted on aromatic ring with an alkyl group having 1 to 3 carbon atoms, halogen, methoxy group or nitro group, and R ² represents an alkanoyl group having 1 to 6 carbon atoms or aralkanoyl group having 7 to 11 carbon atoms, being eventually substituted on aromatic ring with an alkyl group having 1 to 3 carbons, halogen, methoxy group or nitro group, or aroyl having 7 to 11 carbon atoms, being eventually substituted on aromatic ring with an alkyl group having 1 to 3 carbon atoms, halogen, methoxy group or nitro group, with (fj^-bis- (trialkylsilyl)-5-azacytosine of the general formula III,

NHSi(R ³ ) ₃

^" N ^' ~OSi(R ³ ) ₃ (III)

wherein R ³ represents an alkyl group having 1 to 4 carbon atoms, wherein the substituents R ³ can be identical or different, in an inert organic solvent in the presence of a Lewis acid under formation of protected l-(2-deoxy-alpha-D-er)tf/jrø- pentofuranosyl)-5-azacytosine of the general formula IV,

(IV)

wherein R ² represents the same substituents as in formula II, and subsequently the alkanoyl, aralkanoyl or aroyl protecting groups are removed by an excess of alkali alkoxide having 1 to 4 carbon atoms or by ammonia in alcohol having 1 to 4 carbon atoms.

The preferred protecting groups R ¹ are according to the present invention methyl and benzyl group, R ² is preferably selected from the group consisting of the acetyl, benzoyl and/?-toluoyl group, and R ³ is preferably methyl.

It is an aspect of the method according to the present invention that the suitable Lewis acid is preferably tin(IV) chloride.

The suitable inert organic solvent for the method of the present invention is a solvent selected from the group consisting of halogenated hydrocarbons and acetonitrile.

It is a further aspect of the method of the present invention that the reaction is carried out with an anomeric mixture of methyl 2-deoxy-3,5-di-O-p-toluoyl-D-eryt/zrø- pentofuranosides and ^^-bis^trimethylsily^-S-azacytosine.

It is a further aspect of the method according the present invention that the reaction of protected 2-deoxy-D-eryt/jro-pentofuranoside of the general formula II and C^^-bis- (trimethylsilyl)-5-azacytosine of the general formula III is carried out in acetonitrile in the presence of excess of tin(IV) chloride at room temperature.

It is an aspect of the method of the present invention that the removal of alkanoyl, aralkanoyl or aroyl protecting groups R ² of the compound of the general formula IV is carried out with excess sodium methoxide in methanol at room temperature.

Particularly preferable for carrying out the method according to the present invention is the use of acetyl, benzoyl, and/7-toluoyl groups for the protection of hydroxy Is (R ² ), and methyl and benzyl as alkyl groups R ¹ in compounds of the general formula II, and methyl group as R ³ in compounds of the general formula III. The reaction of compounds of the general formula II with silylated bases of the general formula III is carried out in inert organic solvents as, e.g. halogenated hydrocarbons - dichloromethane, dichloroethane or chloroform; acetonitrile is particularly preferable. The preferred Lewis acid is tin(IV) chloride. It is preferable to carry out the reaction in inert atmosphere not only because of the removal of air moisture, but also to prevent an oxidation of the nucleobase in position 6 by air oxygen, which partially takes place

under the conditions of condensation and leads to its transformation to 6-oxo-5- azacytosine (ammelide).

The present method of manufacturing is thus based on the reaction of C^^-bis- (trimethysilyl)-5-azacytosine with a mixture of alpha- and beta-anomers of methyl 2- deoxy-3,5-di-O-p-toluoyl-D-eryt/zro-pentofuranoside in acetonitrile in the presence of tin(IV) chloride, in which l-(2-deoxy-3,5-di-O-/?-toluoyl-alpha-D-erμt/zro- pentofuranosyl)-5-azacytosine is formed in a very high selectivity. With regard to a lower solubility of the compound in comparison to its beta-anomer, which can be present in a small amount in the crude product, it is possible easily to obtain a quite pure product in high yield by crystallization. After removal of the protecting groups by treatment with sodium methoxide in methanol, pure l-(2-deoxy-alpha-D-eryt/zro- pentofuranosyl)-5-azacytosine of the formula I is obtained in high yield.

The mentioned procedure affords the required product selectively in high yield and purity. The availability of the starting compound - mixture of alpha and beta anomers of methyl 2-deoxy-3,5-di-O-p-toluoyl-D-erytλro-pentofuranoside is markedly better than of the starting compounds employed in the previously described methods. Moreover, this starting compound is quite stable in contrast to the unstable peracylglycosyl isocyanates or protected 2-deoxy-D-erytλro-pentofuranosyl halides, which are precursors in the previous syntheses. Additionally, in contrary to them, isolation of the product is much easier - the tedious chromatographic separation of anomers and lossy fraction crystallization is not necessary.

The following examples illustrate the details of this invention, without thereby limiting it in any manner.

Examples

Example 1

Preparation of 1 -(2-deoxy-3,5-di-0-p-toluoyl-alpha-D-erytλro-pentofuranosyl )-5- azacytosine

A solution of tin(IV) chloride (1.8 ml) in dry acetonitrile (20 ml) is added dropwise to a stirred mixture of anomeric methyl 2-deoxy-3,5-di-O-p-toluoyl-D-eryt/zro- pentofuranosides (3.8 g) and C^^-bis-^rimethylsilyO-S-azacytosine (2.6 g) in dry acetonitrile (20 ml) at 0 °C under argon atmosphere and the mixture is kept at room temperature overnight. The proceeding of the reaction is monitored by TLC. If on the next day the starting mixture of the protected methyl furanosides is still present, another portion of the solution of tin(IV) chloride (0.9 ml) in dry acetonitrile (10 ml) is added dropwise under stirring at 0 °C and the reaction time is prolonged by one day. Than the reaction mixture is poured under vigorous stirring into an ice chilled mixture of saturated sodium hydrogen carbonate (250 ml) and chloroform (100 ml). The mixture is still vigorously stirred for 15 min, then diatomaceous earth (Celite) (10 g) is admixed and the mixture is filtered through a thick pad of diatomaceous earth (Celite). The filtration is accelerated by scratching the surface of the filtration pad by spatula. The material on the filtration pad is washed with chloroform (5 x 10 ml), the organic layer of a clear filtrate is, after drying with MgSO ₄ , evaporated in vacuo at 40 ⁰ C in water bath to thick syrup, which is then refluxed in benzene (30 ml) until a crystalline product is formed. The mixture is left at room temperature overnight then the solid is filtered off and washed with benzene. The crude product is dissolved under reflux in 1,2- dichloroethane (30 ml) and petroleum ether is added by portions until a slight opalescence appears. The solution is left to crystallize overnight, the crystalline product is filtered off and washed with a mixture 1,2-dichloroethane-petroleum ether (2:1). After working up the mother liquor, the overall yield of l-(2-deoxy-3,5-di-O-p-toluoyl- alpha-D-erytλro-pentofuranosyl)-5-azacytosine, mp 221-223 °C (dec), is 3.3 g (70%).

Example 2

Preparation of 1 -(2-deoxy-alpha-D-eryt/zro-pentofuranosyl)-5-azacytosine

A mixture of l-(2-deoxy-3,5-di-0-/7-toluoyl-alpha-D-eryt/zro-pentofuranos yl)-5- azacytosine (2.32 g), methanol (14 ml), ethyl acetate (6 ml) and a methanolic solution of sodium methoxide (IM, 5 ml) is stirred at room temperature for 24 h. The resulted crystalline product is filtered off, washed with a mixture of methanol-ethyl acetate (1:1,

6 ml) and dried in vacuo. The yield of pure l-(2-deoxy-alpha-D-eryt/zro- pentofuranosyl)-5-azacytosine, mp 175-177 °C (dec), is 0.91 g (80%). By working up the mother liquor, a further quantity (10%) of the product is obtained. By recrystallization from methanol, the melting point increases to 182-184 ⁰ C (dec). ¹ H NMR (500 MHz, D ₂ O): 8.47 (s, H-6), 6.12 (dd, J ₁ ^ ₅ = 1.7, J _r>2 ' _a = 6.9, H-I'), 2.70 (ddd, J ₂ ' _a , ₃ - = 5.7, Jra,r = 6.9, J _g =14.9, H-2'a), 2.27 (dt, J _rb , _r = Jr _b ,y = 1.7, Jg =14.9, H- 2'b), 4.45 (dt, J ₃ . _,rb = J ₃ 7 _T = 1.7, J ₃ .,r _a = 5.7, H-3 '), 4.52 (ddd, J ₄ ^ ₃ - = 1.7, J ₄ ^ ₃ = 4.2, J ₄ -, ₅ ' _b = 5.5, H-4'), 3.73 (dd, J _5%4 - = 4.2, J _g = 12.2, H-5'a), 3.65 (dd, J ₅ - _b>4 - = 5.5, J _g = 12.2, H-5'b), [α] _D -45.4 (c 0.50, water).

Industrial application

l-(2-Deoxy-alpha-D-eryt/2ro-pentofuranosyl)-5-azacytosine of the formula I as an agent inducing DNA hypomethylation is suitable for manufacturing of a pharmaceutical composition for epigenetic therapy of cancer and other epigenetically determined diseases.