"2',3'-Di-O-acyl-5-fluoronucleosides"

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FIELD OF THE INVENTION

The present invention concerns new 2',3'-di-O-acyl-5-fluoronucleosides useful as intermediates in the preparation of 5'-deoxy-2',3'-di-O-acyl-5-fluoronucleosides which, by simple deacetylation in position 2' and 3', provide important medicaments such as for example capecitabine.

More particularly, the invention refers to 2',3'-di-O-acetyl-5-fluorocytidine derivatives and to a process for their preparation by enzymatic hydrolysis of only the acetyl group in position 5' of the corresponding 2',3',5'-tri-O-acetyl-5- fluorocytidine.

BACKGROUND OF THE INVENTION

Capecitabine, International Non-proprietary Name of 5'-deoxy-N4- pentyloxycarbonyl-5-fluorocytidine or pentyl l-(5-deoxy-β-D-ribofuranosyl)-5- fluoro- 1 ,2-dihydro-2-oxo-4-p yrimidine carbamate of formula A

is a chemotherapeutical agent used for the treatment of some tumoral forms, among which the advanced stage colon cancer, breast cancer, ovary cancer and tested in lung, bladder and pancreas carcinomas. PRIOR ART

According to literature, capecitabine is prepared by reaction of 5-fluorocytosine with 5-deoxy-tri-O-acetylribose in the presence of a silylating agent, in particular of hexamethyldisilazane, subsequent - reaction of the 5'-deoxy-2',3'-di-O-acetyl-5- fluorocytidine thus obtained with pentanoyloxycarbonyl chloride and final deacetylation, as described in Bioorg. Med. Chem. 2000, 8, 1697-1706. Other methods described in the literature, for example in US 4,966,891, US 5,472,949 and

US 5,476,932, always involve a reaction with a 5-deoxyribose having its secondary hydroxyls differently protected.

All the method disclosed in the literature present the problem of the preparation of 5- deoxyribose which involves the selective protection of the two hydroxyls in the positions 2 and 3. An example is described in Bull. Korean Chem. Soc. 2005, 26(11), 1865-1868, where the 5-deoxy-tri-O-acetylribose is prepared starting from ribose by five steps: (i) methylation of the anomeric hydroxyl; (ii) protection of the secondary hydroxyls in form of acetonide; (iii) tosylation of the primary hydroxyl, (iv) reduction with LiAlH ₄ ; and (v) acetylation to give the 5-deoxy-tri-O-acetylribose. SUMMARY OF THE INVENTION

The present invention concers new 2',3'-di-O-acyl-5-fiuoronucleosides, in particular the 2',3'-di-O-acetyl-5-fluorocytidine and its N ⁴ -derivatives (globally designated "2',3'-di-O-acetyl-5-fluorocytidine-derivatives", useful as intermediates in the preparation of capecitabine. hi fact, it has been found that, by treating a 2',3',5'-tri-O-acetyl-5-fluorocytidine- derivative with an immobilized lipase in an aqueous medium buffered at a pH of from 4.0 to 9.0, a selective hydrolysis of the acetoxy group in the position 5' occurs, whereby the 2',3',5'-tri-O-acetyl-5-fiuorocytidine-derivative is transformed, in a yield of 75-95%, into the corresponding 2',3'-di-O-acetyl-5-fluorocytidine-derivative which may be easily converted to the corresponding 5'-deoxy-2',3'-di-O-acetyl-5- fluorocytidine-derivative and in capecitabine. DETAILED DESCRIPTION

Thus, it is an object of the present invention to provide a 2',3'-di-O-acyl-5- fluorocytidine-derivative of formula I

wherein Ac represents an acyl group, X represents hydrogen or a n- pentyloxycarbonyl group and Y represents a hydroxyl group.

The term "acyl", in the present context, designates an acyl containing from 2 to 9 carbon atoms such as acetyl, propionyl, butyroyl, pivaloyl, benzoyl, p-toluoyl, phenylacetyl, p-toluoylacetyl, the acetyl group being preferred. Said 2',3'-di-O-acyl-5-fluorocytidine-derivative of formula I is prepared by a process which comprises submitting the corresponding 2',3',5'-tri-O-acyl-5-fluorocytidine- derivative of formula II

wherein Ac and X have the above defined meaning, to a selective hydrolysis of the acyloxy group in the position 5' in a buffer at a pH of from 4.0 to 9.0 in the presence of an immobilized lipase or esterase.

The starting 2',3',5'-tri-O-acyl-5-fluoronucleosides of formula II are known products or can easily be prepared by reaction of the corresponding tetra-O-acylribose with cytosine. In particular, the 2',3',5'-tri-O-acetyl-5-fluorocytidine (formula II, X = H) may be easily prepared by reaction of tetra-O-acetylribose with cytosine. Also the 2',3\5'-tri-O-acetyl-N ⁴ -n-pentyloxycarbonylcytidine (formula II, X = n- pentyloycarbonyl) is known in the literature (Biorg. Med. Chem. Lett., EN, 12, 3, 2002, 483-486).

The selective hydrolysis is carried out in aqueous medium, buffered for example with TRIS buffer or, preferably, with phosphate buffer (by "phosphate buffer" is meant a KH ₂ PO ₄ buffer at a concentration which may vary between 10 and 100 mM and which will preferably be of about 25 mM), at a pH value from 4.0 to 9, advantageously from 6.5 to 7.5, optionally in the presence of an organic co-solvent at a concentration up to 50%, preferably at 10-30% of acetonitrile or acetone, at a temperature of from 0°C to 25°C. As catalysts, optionally immobilized hydrolases such as lipases of esterases may be used.

The lipase used as a catalyst for the selective hydrolysis is generally obtainable from

a wild or recombinant micro-organism, for example of the genus Rhizomucor, Candida or Pseudomonas. Advantageous microbial lipases are those obtainable from micro-organisms of the Candida and Pseudomonas genus.

The lipase from Candida may be obtained from wild or recombinant Candida rugosa, Candida antarctica, Candida lipolytica, that from Candida rugosa, preferibly immobilized as described in WO 03/057894, being preferred. An advantageous recombinant lipase is the isoform encoded by the gene lipl synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast, for example in Saccharomices cervisiae or in Pichia -pastoris as described by S. Brocca et al. in Protein Science, 1998, VoI 7, Issue 6, 1415-1422.

The lipase from Pseudomonas may be obtained from Pseudomonas putida, Pseudomonas cepacia or, advantageously, from Pseudomonas βuorescens, preferably immobilized as described in WO 03/057894. The immobilization of the lipase is normally made on solid hydrophobic supports. Advantageously, the immobilization of lipase may be made on a silicon matrix consisting of an organosilicon compound, namely of a compound containing at least a Si-C bond (US 6,080,402). More advantageously, the immobilization may be made on a octyl agarose gel such as Octyl Sepharose ^® CL-4B, or on polymetacrylate resins and a butyl character such as Sapebeads FP-BU or a decaoctyl character such as Sapebeads FP-RPOD (RP-OD) which are already totally derivatized with hydrophobic groups, i.e. butyl and, decaoctyl chains, respectively. Preferably, the hydrophobic immobilizing support is octyl-agarose or decaoctyl-Sapebeads RP-OD. Alternatively, the immobilization may be made on a macroporous matrix of silica or silicates (EP 444092), on a matrix consisting of adsorbing, optionally reticulated acrylic-type resins such as Amberlite ^® XAD-8 or Lewatit ^® E 2001/85 (EP 529 424), of an amphiphilic support containing lipophilic chains (US 5,182,201), on a styrene and divinylbenzene matrix optionally containing epoxy groups such as Lewatit ^® R 259 K or R 260 K or Diaion ^® HP-40, on a polyacrylic resin containing epoxy groups such as FP 4000, on a polymethacrylic resin containing epoxy groups such as Sepabeads ^® FP-EP or Eupergit ^® C, suitably derivatized with hydrophobic groups.

As esterases, enzymes of animal origin such as pig pancreas esterases or of fungi

origin such as Aspergillus niger esterase immobilized on Eupergit C may be used. Advantageously, the solution containing the 2',3',5'-tri-O-acetyl-5-fluoronucleoside of formula II (Ac = acetyl) and the immobilized enzyme is brought to a pH from 4.0 to 9.0, advantageously from 6.5 to 7.5 and maintained at the chosen pH and at a temperature from 0°C to 25°C for 12-120 hours.

Preferably, according to the process of the present invention, the 2',3',5'-tri-O- acetyl-5-fluoronucleoside is dissolved in a buffer at the chosen pH, for example in a 25 mM phosphate buffer containing 20-30% acetonitrile or acetone kept at the desired pH value. The solution thus obtained is treated with a preferably immobilized lipase from Candida rugosa or from Pseudomonas fluorescens or with an esterase from pig pancreas or from Aspergillus niger and left to incubate for a period of time from 12 to 120 hours, by controlling the hydrolysis reaction by HPLC. The immobilization, for example on octyl agarose, may be made as described in WO 03/57894. The 2',3'-di-O-acetyl-5-fluorocytidine-derivative of formula I (Ac = acetyl) thus obtained is isolated according to conventional methods, optionally by eliminating the organic solvent, extracting the final product from the aqueous solution, for example with ethyl acetate, and isolating the 2',3'-di-O-acetyl-5-fluorocytidine-derivative of formula I (Ac = acetyl) for example by column chromatography. The selective enzymatic hydrolysis of the acyloxy group in the position 5', according to the present invention, showed to be a simple method characterized by mild experimental conditions, reduced percent of organic solvent in the aqueous solution and low temperature, for obtaining high yields in 2',3'-di-O-acyl-5-fluorocytidine- derivatives of formula I characterized by a free hydroxyl in position 5'. The free hydroxyl in position 5' allows the conversion of the 2',3'-di-O-acyl-5- fluorocytidine-derivatives of formula I into the corresponding 5'-deoxy-2',3'-di-O- acyl-5-fluorocytidine-derivatives, for example via the corresponding sulfonic esters and iodo or bromo derivatives and subsequent replacement of the halogen by a hydrogen atom with consequent obtaining of the corresponding 5'-deoxy-2',3'-di-O- acyl-5-fluorocytidine-derivatives. According to a preferred embodiment, a 2',3'-di- O-acetyl-5-fluorocytidine-derivative of formula I is treated with an alkyl, aralkyl or

aryl sulfonyl chloride, for example with methane sulfonyl chloride or with /(-toluene sulfonyl chloride in an organic solvent such as pyridine to obtain the corresponding, new 5'-O-(alkyl, aryl or aryl sulfonyl)-2',3'-di-O-acetyl-5-fluorocytidine-derivative which, by reaction with a brominating or iodinating agent, for example with a quaternary ammonium bromide such as tetra(«-butyl)ammonium bromide or sodium iodide in a polar aprotic solvent such as dimethylacetamide, dimethylformamide or dimethyl sulfoxide to give the corresponding, new 5'-bromo- or 5'-iodo-2,3-di-O- acetyl-5-fiuorocytidine-derivative which is submitted to a reduction with NaBH ₄ , with LiAlH ₄ , with (W-C ₄ Hg) ₃ SnH or with hydrogen in the presence of a catalyst such as palladium, to obtain the 5'-deoxy-2,3-di-O-acetyl-5-fiuorocytidine-derivative, known immediate precursors of capecitabine which can be converted into capecitabine according to literature methods.

Thus, it is a further object of the present invention to provide the use of the compounds of formula I above for the preparation of capecitabine. The following examples illustrate the invention. The control of the pH during the hydrolysis was made with an automatic pH-Stat, 718 Stat Tritino by Metrohm (Herisau, Switzrland). The HPLC analyses were carried out by using a HPLC Merck Hitachi L-7100 apparatus (E.Merck, Darmstadt, Germany), equipped with UV L- 7400 detector and with an injection valve with a 20 μL loop. A column Shiseido Capcell Pak RP Ci ₈ (250 x 4.6 mm; 5 μm) was used. The analyses were carried out at room temperature at a wave length of 260 nm. In monitoring the hydrolysis reactions the mobile phases were of 10% acetonitrile in 1OmM KH2PO4 buffer at spontaneous pH and 10% H2O in acetonitrile; the mobile phases were filtered and de-gased before the use; the flux was of 1 mL/minute. Monitoring was made using TLC on silica gel 60 (0.25 mm, E. Merck, Darmstadt, Germany).

The 2',3'-di-O-acetyl-5-fluoronucleosides were identified by ¹ H-NMR and COSY 2D NMR analyses registered in DMSO-d6 (δ=ppm) using an instrument Bruker AMX 400. PREPARATION I

General method for the immobilization of lipase

The immobilization process is based on the principle of the interface absorption on strongly hydrophobic supports, said absorption being performed at a low ionic strength. The process consists of dissolving the calculated amount of enzymatic extract in water buffered at neutral pH with 25 mM phosphate buffer under stirring and continuing stirring (oscillating mixer) for 30 minutes. The suspension is added stirring the immobilizing support, previously washed and conditioned with the same buffer and stirring is continued for 2 hours. The suspension is filtered under reduced pressure and the enzymatic material is washed with distilled water. The amount of immobilized enzyme is calculated by spectrophotometry by determining the concentration of the residual supernatant enzyme in the immobilization suspension according to the Bredford method based on the absorbance of the enzyme/Bradford reagent complex at 595 nm (Bradford M: A rapid, sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem, 1976, 72:248-254). PREPARATION II

Immobilization of the lipase from Candida rugosa

A solution of 7.4 mL of lipase from Candida rugosa lip 1 wild-type (containing 16.1 U/mL of solution) in 4.6 ml of phosphate buffer 25 mM at pH 7 is let under stirring for about 30 minutes at room temperature, then 600 mg of gel octyl agarose (Octyl Sepharose® CL-4B, Pharmacia Biotech) or decaoctyl sepabeads (Sepabeads® FP- RPOD, Resindion) previously washed first with water and, then, with the immobilizing buffer (phosphate buffer 25 mM at pH 7), is added thereto. The mixture is let under stirring for about two hours at room temperature then filtered. The derivative thus obtained, consisting of the resin whereon the enzyme is immobilized, is washed with the minimal amount of water. Thus, the lipase from Candida rugosa lipl immobilized on supports, containing 9.8 mg of protein per gram of gel is obtained in an immobilization yield, calculated as expressed activity, equal to about 100% or 49% respectively. PREPARATION III Immobilization of the protease N from Bacillus Subtilis

A solution of 500 mg of protease N from Bacillus Subtilis (Amano Pharmaceuticals

Co.), containing 194 mg of protein/g of powder, equal to 150 U/g powder), in 12 ml of phosphate 1 M buffer at pH 7 is let to stand under stirring for about 30 minutes at room temperature, then 500 mg of gel eupergit C (Eupergit C®, Rohm, Degussa-hiils gruppe) previously washed first with water and, then, with the immobilizing buffer (I M phosphate buffer at pH 7) is added thereto. The mixture is let under stirring for about 24 hours at room temperature then filtered. The derivative thus obtained, consisting of the resin whereon the enzyme is immobilized, is washed with the minimal amount of water. Thus, the protease N from Bacillus subtilis immobilized on Eupergit ^® C, containing 123 U per gram of gel is obtained in an immobilization yield, calculated as amount of immobilized enzyme, equal to 82% (Bradford assay). Example 1

2 ',3 '-Di-O-acetylS-fluorocytidine

To a solution of 100 mM 2',3',5'-tri-O-acetyl-5-fluorocytidine in 25 mM phosphate buffer containing 10% acetoniirile at a pH kept constant at the value of 7,0, 4 g of immobilized Candida rugosa lipase were added. The solution was left to stand under mechanical stirring, at room temperature, under the control of its course by HPLC and the pH was kept constant by automatic titration. After a 3-day incubation 90% conversion of the substrate was observed. The enzyme was removed by filtration, the 2',3'-di-O-acetyl-5-fluorocytidine thus obtained was isolated by removing the possibly present organic solvent and extracting the product from the aqueous solution by ethyl acetate. After evaporation of the collected extracts under reduced pressure, the residue was purified using a chromatographic silica gel column by using a (CH2C12 100- CH ₂ Cl ₂ -MeOH 97:3) mixture as an eluent, thus obtaining the 2',3'-di- O-acetyl-5-fluorocytidine. Global yield 85%. The purified product was identified by analysis ¹ H-NMR and COSY 2D NMR registered in DMSO-d6 (δ=ppm) using an instrument Bruker AMX 400. ¹ H NMR (400 MHz, DMSO-d ₆ , 25 ⁰ C): δ = 8.20 (d, 1 H, 6-H, J 7.8 Hz), 7.90 (s, 2 H, NH ₂ ), 5.97 (d, 1 H, 1 '-H, J4.8 Hz), 5.43 (t, 5'-OH, J ₁₂ 5.9 Hz, J ₁₃ 9.8 Hz ), 5.30-5.26 (m, 2 H, 2'-H,3'-H), 4.08-4.10 (m, 1 H, 4'-H), 3.68-3.58 (m, 2 H, 5'-H), 2.50 (s, 3 H, OAc), 2.06 (s, 3 H, OAc). Example 2

2 ',3 '-Di-O-acetyl-N^n-pentyloxycarbonytyS-fluorocytidine

To a solution of 20 mM of 2',3',5'-tri-O-acetil-N ⁴ -(«-pentyloxycarbonyl)-5- fluorocytidine in 25 mM phosphate buffer containing 30% acetonitrile at a pH kept constant at a value of 7, 0.7 g of immobilized Pseudomonas fluorescens lipase was added. The solution was left to stand under mechanical stirring, at room temperature, under the control of its course by HPLC and the pH was kept constant at a value of 7 by automatic titration. After a 6-hour incubation a 82% conversion of the substrate was observed. The enzyme was removed by filtration, the 2',3'-di-O-acetyl-N ⁴ («- pentyloxycarbonyl)-5-fluorocytidine thus obtained was isolated by removing the possibly present organic solvent and extracting the product from the aqueous solution with ethyl acetate. After evaporation of the collected extracts under reduced pressure, the 2',3'-di-O-acetil-N ⁴ («-pentyloxycarbonyl)-5-fluorocytidine was obtained. The purified product was identified by analysis ¹ H-NMR and COSY 2D NMR registered in DMSO-d ₆ (δ=ppm) using an instrument Bruker AMX 400. HPLC: Rt 14.90 minutes (A: 10 mM buffer K ₂ HPO ₄ 90%/CH ₃ CN 10%, B: CH ₃ CN 90%/H ₂ O 10%, spontaneous pH; Method: 0-3 minutes 75% A-25% B, 3-10 minutes 60% A-40% B, 10-11 minutes 60% A-40% B, 11-12 minutes 50% A-50% B, 12-18 minutes 50% A-50% B,18-23 minutes 75% A-25% B, Flux: 1 mL/ninute, λ: 240 nm, column: RP- 18 Shiseido Capcell Pak). ¹ H NMR (400 MHz, DMSO U ₆ , 25°C): δ = 8.10 (s, 1 H, N-H), 7.80 (d, 1 H, 6-H, J 7.6 Hz), 6.02 (d, 1 H, l'-H, J4.6 Hz), 5.40 (t, 5'-OH, , J ₁₂ 5.8 Hz, J ₁₃ 9.6 Hz ), 5.20- 5.10 (m, 2 H, 2'-H,3'-H), 4.05-4.10 (m, 1 H, 4'-H), 4.13-4.20 (m, 2H), 3.60-3.50 (m, 2 H, 5'-H), 2.40 (s, 3 H, OAc), 2.10 (s, 3 H, OAc), 1.28-1.60 (m, 6 H), 0.91-1.00 (d, 3H) ppm. Examples 3-7

A solution of 2',3',5'-tri-O-acetyl-5-fluorocytidine (10 mM) in a buffer consisting of KH ₂ PO ₄ 25 mM 90% and acetonitrile 10% at pH 7 is added with commercial Candida rugosa lipase (CRL) immobilized on Octyl-Sepharose (Example 3); or commercial Candida rugosa lipase (CRL) immobilized on Decaoctyl-Sepabeads RP- OD (Example 4); or recombinant Candida rugosa lipl lipase (CRL lipl) immobilized on Octyl-Sepharose (Example 5); or recombinant Candida rugosa lipl

lipase (CRL lipl) immobilized on Decaoctyl-Sepabeads RP-OD (Example 6), or Protease N (Amano Pharmaceutical Co.) immobilized on decaoctyl-Sepabeads (Seapabeads RP-OD), respectively. By operating as described in Example 1, a 2',3'- di-O-acetyl-5-fluorocytidine identical to the product of Example 1 is obtained as summarized in Table 1 below.

Table 1

Example 7-10 A solution of 2',3',5'-tri-O-acetil-N ⁴ -(n-pentyloxycarbonyl)-5-fluorocytidine (10 mM) in a buffer consisting of KH ₂ PO ₄ 25 mM 90% and acetonitrile 10% at pH 7 is added with commercial Candida rugosa lipase (CRL) immobilized on Octyl- Sepharose (Example 7), or recombinant Candida rugosa lipl lipase (CRL lipl) immobilized on Decaoctyl-Seapabeads (Example 8), or commercial Pseudomonas fluorescens lipase (PFL) immobilized on Octyl-Sepharose (Example 9); or Protease N (EC 3.4.21.66, Amano Pharmaceutical Co.) immobilized on decaoctyl-Sepabeads (Sepabeads RP-OD) (Example 10), respectively. By operating as described in Example 2, a 2',3'-di-O-acetil-N ⁴ («-pentyloxycarbonyl)-5-fluorocytidine identical to the product of Example 2 is obtained as summarized in Table 2 below.

Table 2