RU2368662C1 - METHOD OF OBTAINING 9-(β-D-ARABINOFURANOSYL)-2-FLUOROADENINE - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of obtaining 9-(β-D-arabinofuranosyl)-2-fluoroadenine (fludarabine), involving reacting 2-fluoroadenosine and 1-β-D- arabinofuranosyluracil in molar ratio 1:2 respectively, in a potassium phosphate buffer at neutral pH, temperature 48-52°C in the presence of purine nucleoside phosphorylase in amount of 62.5 points act. per 1 mmol 2-fluoroadenosine and uridine phosphorylase in amount of 62.5 points act.per 1 mmol 1-β-D-arabinofuranosyluracil. Fludarabine is a precursor of pharmaceutical preparation 5'-fludarabine phosphate.

EFFECT: wide use in medicine as anticancer drug.

1 cl, 1 tbl, 5 dwg, 3 ex

Description

The invention relates to biotechnology, in particular to the production of antitumor compounds, and can be used to obtain an antitumor drug of nucleoside nature - 9- (β-D-arabinofuranosyl) -2-fluoroadenine (fludarabine).

Fludarabine is a precursor to the pharmaceutical preparation of fludarabine 5'-phosphate, widely used in medicine as an antitumor drug used in the treatment of chronic B-cell lymphocytic leukemia and non-Hodgkin lymphomas of low malignancy (Fludarabine-induced apoptosis in CD19 + / CD5 + B-CLL cells is a direct and nurse-like-cell independent effect Martinez-Lostao L., Briones J., Martinez-Gallo M. et al. Leukemia & lymphoma. 2004, V. 45, no. 11, p. 2307-14).

According to the chemical structure, fludarabine is a modified nucleoside - a fluorinated analogue of 9-β-D-arabinofuranosyladenine (Fig. 1).

The most effective currently recognized biotechnological method for producing modified nucleosides using genetically engineered enzymes nucleoside phosphorylases (NF) is the enzymatic transglycosylation reaction, during which the sugar residue is transferred from a natural purine or pyrimidine nucleoside to a modified heterocyclic base (Purine nucleoside phosphorylases: properties , functions, and clinical aspects.Bzowska A., Kulikowska E., Shugar D. Pharmacology & Therapeutics. - 2000. - V.88. P. - 349-425). A schematic diagram of the process is presented in figure 2.

A known method for the enzymatic synthesis of fludarabine by the transglycosylation reaction of the starting heterocyclic base of 2-fluoroadenine with 1-β-D-arabinofuranosiluracil at a temperature of 60 ° C in the presence of Enterobacter aerogenes cell suspension at 60 ° C (EP Patent No. 1464708, IPC ⁷ CL 12/1932 published on April 3, 2003). The disadvantages of this method include the following:

1) the process is carried out in buffer solutions with dimethyl sulfoxide (up to 40% vol) added to the reaction mixture, which affects the partial loss of activity of enzyme preparations and makes it difficult to isolate the product from the reaction mixture,

2) fludarabine is produced in a culture medium containing proliferating microorganisms during the growth of microorganisms,

3) cell enzymes produce not only fludarabine, but also the entire spectrum of natural nucleosides, their metabolites, which complicates the process of isolating the target product.

Known closest to the claimed method for producing fludarabine by the transglycosylation reaction of the starting base of 2-fluoroadenine 1-β-D-arabinofuranosyluracil using the enzymes purinucleoside phosphorylase (EC 2.4.2.1.) And uridine phosphorylase (EC 2.4.2.3.) From a suspension of Enterobacter cells isolated and immobilized on polymer carriers (patent EP No. 1439220, IPC ⁷ cells C12N 19/10, published. 16.01.2003). The disadvantages of this method include the following:

1) the initial 2-fluoroadenine is poorly soluble in water, as a result of which the process is carried out in buffer solutions with dimethyl sulfoxide (up to 40% vol.) Or ethyl alcohol (10% vol.) Added to the reaction mixture, which affects the partial loss of activity of enzyme preparations,

2) at the end of the process, the product crystallizes together with the starting base due to its low solubility, which leads to the need for additional recrystallization of fludarabine.

The invention solves the problem of simplifying the technology of obtaining fludarabine and increasing its yield.

The problem is solved due to the fact that fludarabine is obtained by the interaction of 2-fluoroadenosine and 1-β-D-arabinofuranosiluracil at a molar ratio of 1: 2, respectively, in potassium phosphate buffer at a neutral pH value, temperature 48-52 ° С the presence of purine nucleoside phosphorylase in the amount of 62.5 units. Act. per 1 mmol of 2-fluoroadenosine and uridine phosphorylase in an amount of 62.5 units. Act. per 1 mmol of 1-β-D-arabinofuranosyluracil.

Using 2-fluoroadenosine allows the process of obtaining fludarabine at high concentrations of the starting nucleosides without adding dimethyl sulfoxide to the reaction mixture, which simplifies the process of isolating the target product from the reaction mixture and leads to a significant increase in its yield.

The invention is as follows.

The starting 2-fluoroadenosine is obtained by treating with ammonia in methanol 2 ′, 3 ′, 5′-tri-O-acetyl-2-fluoroadenosine (RF patent No. 2325395, 02/05/2007). The synthesis scheme is presented in figure 3.

In parallel, the synthesis of the second starting nucleoside 1-β-D-arabinofuranosiluracil (compound VI, Fig. 4) is carried out, the preparation of which is based on the acid-catalyzed uridine acetylation reaction with the formation of 2,2'-anhydro-1- (3 ', 5' -di-O-acetyl-β-D-arabinofuranosyl) uracil (Kondo K. and Inoue IJOrg. Chem. 1977. V.42. N.17. P.2809-2812), saponification of which with aqueous alkali at room temperature and leads to the formation of 1-β-D-arabinofuranosiluracil.

Purine nucleoside phosphorylase (PNP, KF 2.4.2.1) and uridine phosphorylase (UV, KF 2.4.2.3) are used as genetic engineering enzymes.

The synthesis of fludarabine (I, Fig. 5) from 2-fluoroadenosine is carried out by the transglycosylation reaction at a molar ratio of 2-fluoroadenosine to 1-β-D-arabinofuranosiluracil - 1: 2 at 48-52 ° С to the content of the initial 2-fluoroadenosine 0.5 -one%. At this temperature, fludarabine gradually crystallizes, shifting the equilibrium of the enzymatic reaction in the direction of product formation. Process control is carried out using HPLC. At the end of the reaction, the mixture was kept at 8-15 ° C for 20-24 hours, the precipitated fludarabine was filtered off. The process of obtaining fludarabine in the described manner allows to obtain a product with a yield of 92% and a purity of at least 99%. Data confirming the achievement of the technical result is presented in the table.

Table. The proposed method EP 1,439,220 Temperature (° C) 50 ± 2 60 Ratio of components 1: 2 1: 1.5 Reaction time, h 110-120 four The presence of DMSO in the reaction mixture,% - 40 Enzyme source Purified genetically engineered enzymes from E. coli Immobilized Enterobacter aerogenes Enzymes The need for additional crystallization of the product - + The yield of fludarabine,% 92 70

The invention is illustrated by the drawings:

Figure 1. - structural formula of fludarabine (9- (β-D-arabinofuranosyl) -2-fluoroadenine).

Figure 2 is a schematic diagram of a transglycosylation reaction.

Figure 3 - synthesis scheme of 2-fluoroadenosine.

4 is a diagram of the synthesis of 1-β-D-arabinofuranosyluracil.

Figure 5. - Scheme of enzymatic synthesis of 9- (β-D-arabinofuranosyl) -2-fluoroadenine (fludarabine).

The invention is illustrated by examples.

Example 1

Obtaining 2-fluoroadenosine (compound III, figure 3).

A suspension of 33.35 g (117 mmol) of 2 ', 3', 5'-tri-O-acetyl-2-fluoroadenosine in 1.25 L of methanol is stirred and saturated with dried ammonia at 0-2 ° C for 6 hours, the resulting solution is kept closed the flask at the indicated temperature for 5 days, and then 4 hours at 20 ° C. The precipitate formed is filtered off and 11.96 g of 2-fluoroadenosine, mp. 249-251 ° C. Mass spectrum: m / z 308 (M + Na) ⁺ , 286 (M + H) ⁺ , 154 (B + H) ⁺ , m.v. 285.237. ¹ H-NMR spectrum (DMSO-d ₆ ), (δ ppm): 8.38 (s, 1H, 8-H), 7.9 (s, 2H, NH ₂ ), 5.81 (d, 1H, J ₁ = 5.1 Hz, 1'-H), 4.54 (m, 1H, 2'-H), 4.15 (m, 1H, 3'-H), 3.96 (m, 1H, 4'-H), 3.50-3.75 (m , 2H, 5'-H ₂ ).

The mother liquor is concentrated in vacuo (15 mmHg), the residue is boiled for 15 minutes in 300 ml of methanol containing 5% ammonia, the reaction mixture is cooled to 20 ° C and maintained at this temperature for 2 hours, the precipitate formed is filtered off and an additional 9.21 is obtained. g 2-fluoroadenosine (compound III, figure 3), 98% purity, so pl. 244-246 ° C. The total yield of 2-fluoroadenosine is 21.17 g (91.56%).

Example 2

Obtaining 1-β-D-arabinofuranosiluracil (compound VI, figure 4). A solution of acetic anhydride (210 g, 200 ml) was added dropwise to a stirred boiling suspension of uridine (compound IV, Figure 4) (100 g, 0.4 mol) with boron trifluoride etherate (160 ml, 1.28 mol) in 1 liter of toluene-acetonitrile mixture , 1: 1. After 2 hours, the reaction mixture was cooled with ice and neutralized with sodium carbonate.

(Na ₂ CO ₃ ) to a pH of 8-8.5. The precipitate is removed, washed with acetonitrile. The supernatant was concentrated in vacuo. The residue of acetic acid is removed by evaporation with toluene. The residue is dissolved in ethanol, the product is precipitated from the solution by the addition of toluene. The precipitated crystals are filtered off, washed with ethanol (2 × 100 ml), dried in vacuo (15 mmHg). Yield of 2-2'-anhydro-1- (3 ', 5'-di-O-acetyl-β-D-arabinofuranosyl) uracil (compound V, Fig. 4) 72.8 g (58.5%); t. pl. 185 ° C; λ _max , nm: 246.2; m / z: 311.3 [M] ⁺ , 333.4 [M + Na] ⁺ .

2-2'-Anhydro-1- (3 ', 5'-di-O-acetyl-β-D-arabinofuranosyl) uracil (compound V, Fig. 4) (72.8 g) was dissolved in 1 L of H ₂ O, 140 ml of 5M KOH to pH 12. After 5 hours, the reaction mixture was neutralized with Dowex 50 cation exchanger (H ⁺ ) to pH 7. The resin was filtered off, washed with water (3 × 100 ml). The solvent was removed in vacuo (15 mmHg). The target product is crystallized from 600 ml of ethanol, washed with chilled ethanol (2 × 100 ml). Yield 30.4 g (30%); t. pl. 238 ° C; λ _max , nm: 262.6; m / z: 245.3 [M + H] ⁺ , 267.2 [M + Na] ⁺ . ¹ H-NMR (D ₂ O) (δ, ppm): 7.83 (1H, d, J _6.5 8.09, H ₆ ), 6.15 (1H, d, J _{1 ', 2'} 4.98, H _{1 '} ), 5.85 (1H, d, J _5.6 8.09, H ₅ ), 4.43 (1H, t, J _{2 ', 1'} . 4.8, H ₂ ), 4.11 (1H, t, J _{3 ', 2'} 5.14, H _{3 '} ), 3.97 (1H, m, H _4' ), 3.9 (1H, dd, J _{5'a, 5'b} 12.45, J _{5'a, 4 '} 3.11, H _5'a ), 3.81 (1H, dd, J _{5'b, 5'a} 12.45, J _{5'b, 4 '} 5.61, H _5'b ).

Example 3

Getting fludarabine (compound I, figure 5).

A mixture of 17.116 g (0.06 mol) of 2-fluoroadenosine (compound III, FIG. 5) and 29.3 g (0.12 mol) of 1-β-D-arabinofuranosiluracil (compound VI, FIG. 5) in 2 L of 60 mM potassium phosphate buffer ( pH 7.0) thermostat for 120 hours at 50 ± 2 ° C in the presence of 15 ml of the enzyme preparation PNP (10 mg / ml, 25 units of act./ mg of protein or 62.5 units of act. on 1 mmol of 2-fluoroadenosine) and 15 ml of UV enzyme preparation (10 mg / ml, 50 units of act./ mg of protein or 62.5 units of act. per 1 mmol of 1-β-D-arabinofuranosyluracil). Monitoring the completeness of the process is carried out using HPLC. The process is considered complete if the content of the starting 2-fluoroadenosine (compound III, FIG. 5) in the reaction mixture is 1%.

The reaction mixture is cooled to a temperature of 8 ° C. The precipitated target product is filtered off, washed with water (3 × 20 ml) and ethyl alcohol (1 × 20 ml), then dried in a desiccator to constant weight. Yield: 15.75 g (92%). Product purity according to HPLC 99-99.4%. T. pl. 260-262 ° C. λ _max , nm (ε, M ^-1 cm ^-1 ): 262 (14800). ¹ H-NMR spectrum (DMSO-d ₆ ), (δ ppm): 8.17 (s, 1H, H-8), 7.76 (s, 2H, NH ₂ ), 6.11 (d, 1H, H _{1 '} ), 5.61 (d, OH _{2 ′} ), 5.51 (d, OH _{5 '} ), 5.05 (t, OH _3' ), 4.14 (m, 1H, H _{2 '} ), 3.77 (m, 1H, H _3' ) , 3.65 (m, 1H, H _{4 '} ), 3.35 (s, 2H, H _5' )

Claims (1)

The method of obtaining 9- (β-D-arabinofuranosyl) -2-fluoroadenine, comprising the interaction of 2-fluoroadenosine and 1-β-D-arabinofuranosyl uracil at a molar ratio of 1: 2, respectively, in potassium phosphate buffer at a neutral pH value, temperature 48- 52 ° C in the presence of purine nucleoside phosphorylase in an amount of 62.5 units. Act. per 1 mmol of 2-fluoroadenosine and uridine phosphorylase in an amount of 62.5 units. Act. per 1 mmol of 1-β-D-arabinofuranosyluracil.

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