RU2624023C2 - METHOD FOR PRODUCTION OF PURINE NUCLEOSIDES OF β-D-ARABINOFURANOSE SERIES - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for production of modified purine nucleosides of β-D-arabinofuranose series. The method is implemented by reacting a purine ribonucleoside with 1-β-D-arabinofuranosyluracil or 1-α-phosphate of D-arabinose in a potassium phosphate buffer in the presence of purine nucleoside phosphorylase or a mixture of purine and pyrimidine nucleoside phosphorylases in the presence of ortho-arsenic acid salts.

EFFECT: simplification of the technique of β-D-arabinofuranose series modified purine nucleosides production from natural or chemically modified β-D-ribofuranose nucleosidesβ.

1 tbl, 16 dwg, 11 ex

Description

The invention relates to biotechnology, in particular the production of modified purine nucleosides of a number of β-D-arabinofuranose used in medical practice for the treatment of tumor diseases (fludarabine, non-narabin, etc.) and viral infections (vidarabine).

The invention solves the problem of developing an effective method for the synthesis of modified purine nucleosides of a series of β-D-arabinofuranose through the use of a chemical-enzymatic approach (Fig. 1, 2), combining two processes: phosphorolysis and arsenolysis of nucleosides in the active center of nucleoside phosphorylase enzymes.

The most effective currently recognized biotechnological method for producing modified nucleosides using enzymes nucleoside phosphorylases (NF) - an enzymatic transglycosylation reaction - during which the sugar residue is transferred from a natural purine or pyrimidine nucleoside to a modified heterocyclic base (Mikhaylopulo I.A., Miroshnikov A. I. Current trends in biotechnology of nucleosides. // Acta Naturae. 2010; 2 (2), pp. 38-61.). A schematic diagram of the process is shown in Figure 1.

The enzymatic transglycosylation reaction is usually reversible, which limits its use in industrial biotechnological processes. To shift the equilibrium, a number of approaches are used. These include:

1) the use of an excess of one of the components of the reaction (Purine nucleoside phosphorylases: properties, functions and clinical aspects. Bzowska A., Kulikowska E., Shugar D. Pharmacology & Therapeutics. - 2000. - V. 88. P. - 349-425) ,

2) the use of pentose-1-phosphate barium salts as pentose donors (The use of pentose-1-phosphoric acid barium salts in the enzymatic synthesis of ribotimidine and bromovinyldeoxyuridine. Bokut SB, Baray VN, Zinchenko AI Prikl. Biochem. Microbiol. 1995.Vol. 31. Issue 3. S. 308-310). In this case, barium phosphate formed during the reaction precipitates and is removed from the reaction.

3) There is a method of conducting the phosphorolysis reaction of natural nucleosides using instead of inorganic phosphates salts of ortho-arsenic acid (H ₃ AsO ₄ , arsenolysis) to shift the equilibrium of the enzymatic reaction towards the formation of a heterocyclic base. The α-D-ribose-1-arsenate formed during the reaction is unstable in solution and is excreted from the enzymatic reaction (Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction. Kline, PC, & Schramm, VL Biochemistry 1993. V 32. P. 13212-13219).

The disadvantages of these methods include the following:

1) the use of large excesses of one of the components of the reaction — a nucleoside or a base — is a rather costly process, requiring the completion of chromatographic separation of the components of the reaction mixtures and returning them to the reaction by recycling.

2) the use of barium salts is impossible if the target product is poorly soluble and crystallizes during the reaction simultaneously with barium phosphate.

3) The disadvantages of arsenolysis include the following: salts of ortho-arsenic acid are used only in the hydrolysis of nucleosides (ribosides and 2'-deoxyribosides) to heterocyclic bases.

A known method for the synthesis of nucleosides by the transglycosylation reaction using salts of ortho-arsenic acid in the synthesis of 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) (Konstantinova I.D., Fateev I.V., Miroshnikov AI “Method for the production of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide." RF patent No. 2480218 from 04/27/2013, Priority dated 12/06/2011), characterized in that that as a result of the reaction, 1,2,4-triazole-3-carboxamide riboside is synthesized and sodium arsenate is added to the reaction mixture after completion of the synthesis of ribavirin to remove I have unreacted guanosine residues. However, this method of synthesis of the target nucleoside 1-β-D-ribofuranose is unique and it is practically impossible to extend it to the synthesis of other ribosides in the active center of the enzyme (I.D. Konstantinova, I.V. Fateev, G.A. Galegov, P.G. Deryabin, A.G. Botikov, I.S.Music, D.K. Lvov, A.I. Miroshnikov, Arsenolysis in the biotechnological method for producing ribavirin, Inhibition of the reproduction of influenza A virus in vitro and in vivo using a combination ribavirin and oseltamivir. // Bioorganic chemistry. 2013.V. 39., No. 5. P. 594-603).

The use of salts of ortho-arsenic acid in the synthesis of nucleosides of a number of β-D-arabinofuranose is not known in the literature.

The invention solves the problem of simplifying the technology for producing modified purine nucleosides of a number of β-D-arabinofuranose from natural or chemically modified nucleosides of β-D-ribofuranose. The use of ribosides in the transglycosylation reactions in the development of modified nucleoside production technologies is motivated by higher solubility compared to the corresponding heterocyclic bases.

The problem is solved due to the fact that the method for producing modified purine nucleosides of a series of β-D-arabinofuranose involves the interaction of purine riboside with 1-β-D-arabinofuranosyl uracil or 1-α-phosphate D-arabinose in potassium phosphate buffer using nucleoside phosphorylase enzymes in the presence of catalytic amounts of salts of ortho-arsenic acid (sodium or potassium arsenate in an amount of from 0.1% per mole).

The method is as follows.

The transglycosylation reaction is introduced:

1) purine base source: natural or chemically modified ribonucleoside (compound II, figure 2),

2) the source of β-D-arabinose: 1-β-D-arabinofuranosylluracil (compound I, Figure 2) or 1-α-phosphate-D-arabinose (compound III, Figure 2),

3) nucleoside phosphorylase enzymes,

4) sodium arsenate (NaH ₂ AsO ₄ ) in a catalytic amount from 0.1% to 10% (moles per mole of purine ribonucleoside).

A schematic diagram of the process is shown in Figure 2.

The reaction is carried out in K-phosphate buffer solution.

In the active center of the enzyme, 1-α-D-ribofuranose arsenate is formed very easily, while 1-α-D-arabinofuranose arsenate is practically not synthesized. The arsenate of 1-α-D-ribofuranose is extremely unstable and hydrolyzes to ribose, which is derived from the enzymatic reaction.

The use of catalytic amounts of sodium arsenate in transglycosylation reactions significantly accelerates the enzymatic process, reduces the reaction time, simplifies the composition of reaction mixtures and facilitates the process of separation of reaction components. Figures 11-16 show the HPLC data of the reaction mixtures of the enzymatic preparation of nucleosides of the β-D-arabinofuranose series without sodium arsenate (Figures 11, 13, 15) and with its use (Figures 12, 14, 16).

The invention is as follows.

The source of purine base is any natural or chemically modified ribo-nucleoside (compound II, Figure 2) (e.g. adenosine, inosine, guanosine, 2-fluoroadenosine, 2-methoxyadenosine, 2-chloradenosine, 6-O-methylguanosine, etc.).

As a source of 1-α-phosphate-D-arabinose, 1-β-D-arabinofuranosylluracil (compound I, Figure 2) (CAS Number: 3083-77-0) or 1-α-phosphate-D-arabinose (compound III) is used Figures 2, 6-8) (ID Konstantinova, KV Antonov, IV Fateev, AI Miroshnikov, VA Stepchenko, AV Baranovsky, IA Mikhailopulo. A Chemo-Enzymatic Synthesis of β-D-Arabinofuranosyl Purine Nucleosides. // Synthesis, 2011. N. 10. P. 1555-1560. DOI: 10.1055 / s-0030-1260010).

When preparing a phosphate buffer solution, a catalytic amount of sodium or potassium arsenate is added (from 0.1% to 10% by mole).

The temperature and pH of the solution are determined by the requirements of nucleoside phosphorylase enzymes.

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

The process of obtaining any modified nucleosides - derivatives of β-D-arabinose in the described manner allows to obtain nucleosides with a yield of more than 90%. Data confirming the achievement of the technical result is presented in the table.

The invention is further illustrated by the table and the following graphic materials:

Table. Comparative data on the production of nucleosides of a number of D-arabinose without the use of sodium arsenate and with it. The composition of the reaction mixtures, the synthesis time (h) and the conversion of purine β-D-riboside to β-D-arabinoside are presented. The detailed procedures are given in examples 1-11.

Figure 1. Schematic diagram of the transglycosylation reaction.

Figure 2. Scheme of the transglycosylation reaction using sodium arsenate.

Figure 3. Scheme of enzymatic synthesis of 9- (β-D-arabinofuranosyl) -2-fluoroadenine (fludarabine) using two enzymes PNP and UV.

Figure 4. Scheme of enzymatic synthesis of 9- (β-D-arabinofuranosyl) adenine (Ara-A) using two enzymes PNP and UV.

Figure 5. Scheme of enzymatic synthesis of 9- (β-D-arabinofuranosyl) -6-O-methylguanine (nonlarabin) using two enzymes PNP and UV.

Figure 6. Scheme of enzymatic synthesis of 9- (β-D-arabinofuranosyl) -2-fluoroadenine (fludarabine) from 2-fluoroadenosine and 1-α-phosphate D-arabinose using a single PNP enzyme.

Figure 7. Scheme of enzymatic synthesis of 9- (β-D-arabinofuranosyl) adenine (Ara-A) from adenosine and 1-α-phosphate of D-arabinose using a single PNP enzyme.

Figure 8. Scheme of enzymatic synthesis of 9- (β-D-arabinofuranosyl) -6-O-methylguanine from 9- (β-D-ribofuranosyl) -6-O-methylguanine and 1-α-phosphate D-arabinose using one enzyme PNF.

Figure 9. Diagram of the enzymatic synthesis of 9- (β-D-arabinofuranosyl) hypoxanthine (Ara-Hyp) using two enzymes PNP and UV.

Figure 10. Scheme of enzymatic synthesis of 2-chloro-9- (β-D-arabinofuranosyl) adenine (Cl-AraA) using two enzymes PNP and UV.

Figure 11. HPLC data of a fludarabine synthesis reaction mixture without sodium arsenate using two enzymes PNP and UV.

Figure 12. HPLC data of a reaction mixture for the synthesis of fludarabine with sodium arsenate using two enzymes PNP and UV.

Figure 13. HPLC data of a reaction mixture for the synthesis of vidarabine without sodium arsenate using two enzymes PNP and UV.

Figure 14. HPLC data of a reaction mixture for the synthesis of vidarabine with sodium arsenate using two enzymes PNP and UV.

Figure 15. HPLC data of a nonlarabin synthesis reaction mixture without sodium arsenate using two enzymes PNP and UV.

Figure 16. HPLC data of a reaction mixture for the synthesis of non-narabine with sodium arsenate using two enzymes PNP and UV.

The invention is illustrated by examples.

Example 1

Preparation of 9- (β-D-arabinofuranosyl) -2-fluoroadenine (fludarabine, compound IX, Figure 3).

A mixture of 17.116 g (0.06 mol) of 2-fluoroadenosine (CAS Number: 146-78-1, compound VII, Fig. 3), 22.0 g (0.09 mol) of 1-β-D-arabinofuranosyluracil (compound VIII, Fig. 3) and 0.882 g (0.003 mol) of NaH ₂ AsO ₄ in 2 L of 60 mM potassium phosphate buffer (pH 7.0) was incubated for 72 hours at 48 ° C in the presence of 3750 units. Act. PNP enzyme preparation (62.5 units of act. per 1 mmol of 2-fluoroadenosine) and 5625 units. Act. UV enzyme preparation (62.5 units of act. per 1 mmol of 1-β-D-arabinofuranosiluracil). The control of the completeness of the process is carried out using HPLC. The process is considered complete if the content of the starting 2-fluoroadenosine (compound VII, Fig. 3) in the reaction mixture is <0.2%.

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% (method from 0 to 20% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, Nova-Pak C18 column, 4.6 × 150 mm, 4 μm). Mp 260-262 ° C. λ _max , nm (ε, M ^-1 cm ^-1 ): 262 (14800). Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.17 (s, 1H, H-8), 7.76 (s, 2H, NH ₂ ), 6.11 (d, J = 5.0 Hz, 1H, H _{1 '} ), 5.61 (d, J = 5.3 Hz, 1H, OH _2' ), 5.51 (d, J = 4.7 Hz, 1H, OH _{3 '} ), 5.05 (t, J = 4.3 Hz, 1H, OH _{5 '} ), 4.14 (m, 1H, H _2' ), 4.13 (m, 1H, H _{3 '} ), 3.77 (m, 1H, H _4' ), 3.66 (m, 2H, H _{5 '} ).

Example 2

Preparation of 9- (β-D-arabinofuranosyl) adenine (Ara-A, compound XII, Fig. 4).

A mixture of 2.84 g (0.01 mol) of adenosine (compound XI, Fig. 4), 3.66 g (0.015 mol) of 1-β-D-arabinofuranosyluracil (compound VIII, Fig. 4) and 0.294 g (0.001 mol) of NaH ₂ AsO ₄ 0.2 l of 50 mm potassium phosphate buffer (pH 7.0) thermostat for 16 hours at 52 ° C in the presence of 300 units. Act. PNP enzyme preparation (30 units of act. per 1 mmol of adenosine) and 495 units. Act. UV enzyme preparation (33 units of act. per 1 mmol of 1-β-D-arabinofuranosiluracil). The control of the completeness of the process is carried out using HPLC. The process is considered complete if the content of the starting adenosine (compound XI, Fig. 4) in the reaction mixture is <1%.

The reaction mixture was concentrated in vacuo (15 mmHg) to 0.1 L, 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: 2.65 g (93%). Product purity according to HPLC 99-99.4% (RT = 10.2 min, method from 0 to 10% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, column Nova-Pak C18, 4.6 × 150 mm, 4 μm). T. pl. 260-262 ° C. λ _max , nm (ε, M ^-1 cm ^-1 ): 262 (14800). Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.17 (s, 1H, H-8), 8.12 (s, 1H, H-2), 7.19 (s, 2H, NH ₂ ), 6.25 (d, J = 4.3 Hz, 1H, H _{1 '} ), 5.60 (br.s, 1H, OH _2' ), 5.49 (d, J = 3.3 Hz, 1H, OH _{3 '} ), 5.05 (br t, 1H, OH _{5 '} ), 4.13 (m, 2H, H _2' and H _{3 '} ), 3.78 (m, 1H, H _4' ), 3.68 and 3.63 (2m, 2H, H _{5 '} ).

Example 3

Preparation of 9-β-D-arabinofuranosyl-6-O-methylguanine (nonlarabine, compound XIV, Fig. 5).

3.50 g (11.75 mmol) of 9- (β-D-ribofuranosyl) -6-O-methylguanine (CAS Number: 7803-88-5), 4.3 g (17.6 mmol) 1- (β) are dissolved in 590 ml of distilled water when heated. -D-arabinofuranosyl) uracil, 0.294 g (0.001 mol) NaH ₂ AsO ₄ and 1.605 g (11.8 mmol) potassium dihydroorthophosphate. The pH of the reaction mixture was adjusted to 7.0. 352 units were added to the reaction mixture. purinnucleoside phosphorylase (30 units of act. per 1 mmol of 9- (β-D-ribofuranosyl) -6-O-methylguanine) and 616 units. uridine phosphorylase (35 units of act. per 1 mmol of 1-β-D-arabinofuranosyluracil). The solution was thermostated at 52 ° C for 7 days. At the end of the process, the reaction mixture was concentrated in vacuo (15 mmHg) to a volume of 50 ml and chromatographed on a Octadecyl = Si 100polyol reverse phase sorbent (0.03 mm), column size: 25 × 400 mm The target compound was eluted with 2% acetonitrile in water. The fractions containing the product are combined, the solvent is removed in vacuo (15 mmHg). The product is dried in vacuum (5 mm RT. Art.) Over phosphorus pentoxide. Yield: 3.33 g (95%). Product purity (according to HPLC) 99.25% (RT = 9.6 min, method from 0 to 20% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, column Nova-Pak C18, 4.6 × 150 mm, 4 μm). λ _max 286, 244, 214 nm. Mass spectrum, m / z: 298.1208 [M + H] ⁺ , 166.0777 [base + H] ⁺ . Calc. 298.2765 [M + H] ⁺ , 166.1613 [base + H] ⁺ .

¹ H-NMR (DMSO-d6, δ, ppm, J, Hz): 7.92 (s, 1H, H-8), 6.41 (s, 2H, NH ₂ ), 6.12 (d, J = 4.7, 1H, H _{1 '} ), 5.60 (broad s, 1H, OH _5' ), 5.48 (broad s, 1H, OH _{3 '} ), 5.04 (broad s, 1H, OH _2' ), 4.09 (dd, J = 3.9 and 4.1, 1H, H _{3 '} ), 4.06 (dd, J = 4.2 and 4.4, 1H, H _2' ), 3.75 (m, 1H, H _{4 '} ), 3.65 (m, 1H, H _5a' ), 3.60 (m, 1H, H _{5b '} ).

¹³ C-NMR (DMSO-d6, δ, ppm): 160.98 (C6), 154.44 (C4), 139.50 (C8), 113.62 (C5), 84.69 (C-4 '), 83.82 (C-1'), 75.96 (C-2 '), 75.81 (C3'), 61.44 (C-5 ').

¹⁵³ N-NMR (DMSO-d6, δ, ppm): 239.56 (N7), 205.19 (N1), 188.81 (N3), 163.61 (N9).

Example 4

Preparation of 9- (β-D-arabinofuranosyl) -2-fluoroadenine (fludarabine, compound IX, Figure 6).

A mixture of 1.70 g (0.006 mol) of 2-fluoroadenosine (CAS Number: 146-78-1, compound VII, Fig. 6), 5.8 g (0.024 mol) of 1-α-phosphate-D-arabinose (compound III, Fig. 6 ) and 0.09 g (0.3 mmol) of NaH ₂ AsO ₄ in 250 ml of 60 mM potassium phosphate buffer (pH 7.0) were incubated for 24 hours at 50 ° C in the presence of 375 units. Act. PNP enzyme preparation (62.5 units of act. per 1 mmol of 2-fluoroadenosine). The control of the completeness of the process is carried out using HPLC. The process is considered complete if the content of the starting 2-fluoroadenosine (compound VII, Fig. 6) in the reaction mixture is <0.2%.

The reaction mixture is cooled to a temperature of 8 ° C. The precipitated target product is filtered off, washed with water (3 × 10 ml) and ethyl alcohol (1 × 10 ml), then dried in a desiccator to constant weight. Yield: 1.67 g (98%). Product purity according to HPLC 99-99.4% (method from 0 to 20% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, Nova-Pak C18 column, 4.6 × 150 mm, 4 μm). T. pl. 260-262 ° C. λ _max , nm (ε, M ^-1 cm ^-1 ): 262 (14800). Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.17 (s, 1H, H-8), 7.76 (s, 2H, NH ₂ ), 6.11 (d, J = 5.0 Hz, 1H, H _{1 '} ), 5.61 (d, J = 5.3 Hz, 1H, OH _2' ), 5.51 (d, J = 4.7 Hz, 1H, OH _{3 '} ), 5.05 (t, J = 4.3 Hz, 1H, OH _{5 '} ), 4.14 (m, 1H, H _2' ), 4.13 (m, 1H, H _{3 '} ), 3.77 (m, 1H, H _4' ), 3.66 (m, 2H, H _{5 '} ).

Example 5

Preparation of 9- (β-D-arabinofuranosyl) adenine (Ara-A, compound XII, Fig. 7).

A mixture of 2.84 g (0.01 mol) of adenosine (compound XI, Fig. 7), 9.68 g (0.04 mol) of 1-α-phosphate-D-arabinose (compound III, Fig. 7) and 0.294 g (0.001 mol) of NaH ₂ AsO ₄ in 0.2 l of 50 mm potassium phosphate buffer (pH 7.0) thermostat for 4 hours at 52 ° C in the presence of 300 units. Act. PNP enzyme preparation (30 units of act. per 1 mmol of 2'-deoxyadenosine). The control of the completeness of the process is carried out using HPLC. The process is considered complete if the content of the starting adenosine (compound XI, Fig. 7) in the reaction mixture is <1%.

The reaction mixture was concentrated in vacuo (15 mmHg) to 0.1 L, 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: 2.60 g (92%). Product purity according to HPLC 99.0-99.4% (RT = 10.2 min, method from 0 to 10% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, column Nova-Pak C18, 4.6 × 150 mm, 4 μm). T. pl. 260-262 ° C. λ _max , nm (ε, M ^-1 cm ^-1 ): 262 (14800). Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.17 (s, 1H, H-8), 8.12 (s, 1H, H-2), 7.19 (s, 2H, NH ₂ ), 6.25 (d, J = 4.3 Hz, 1H, H _{1 '} ), 5.60 (br.s, OH _2' ), 5.49 (d, J = 3.3 Hz, OH _{3 '} ), 5.05 (br, OH _{5 '} ), 4.13 (m, 2H, H _2' and H _{3 '} ), 3.78 (m, 1H, H _4' ), 3.68 and 3.63 (2m, 2H, H _{5 '} ).

Example 6

Preparation of 9-β-D-arabinofuranosyl-6-O-methylguanine (non-larabine, compound XIV, Fig. 8).

3.50 g (11.75 mmol) of 9- (β-D-ribofuranosyl) -6-O-methylguanine (CAS Number: 7803-88-5), 11.4 g (47.0 mmol) 1-α- are dissolved in 590 ml of distilled water when heated. phosphate-D-arabinose, 0.294 g (0.001 mol) of NaH ₂ AsO ₄ and 1.605 g (11.8 mmol) of potassium dihydroorthophosphate. The pH of the reaction mixture was adjusted to 7.0. 352 units were added to the reaction mixture. purinucleoside phosphorylase (30 units of act. per 1 mmol of 9- (β-D-ribofuranosyl) -6-O-methylguanine). The solution was thermostated at 52 ° C for 7 days. At the end of the process, the reaction mixture was concentrated in vacuo (15 mmHg) to a volume of 50 ml and chromatographed on a Octadecyl = Si 100polyol reverse phase sorbent (0.03 mm), column size: 25 × 400 mm The target compound was eluted with 2% acetonitrile in water. The fractions containing the product are combined, the solvent is removed in vacuo (15 mmHg). The product is dried in vacuum (5 mm RT. Art.) Over phosphorus pentoxide. Yield: 3.20 g (91%). Product purity (according to HPLC) 99.0-99.25% (RT = 9.6 min, method from 0 to 20% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA column Nova-Pak C18, 4.6 × 150 mm, 4 μm). λ _max 286, 244, 214 nm. Mass spectrum, m / z: 298.1208 [M + H] ⁺ , 166.0777 [base + H] ⁺ . Calc. 298.2765 [M + H] ⁺ , 166.1613 [base + H] ⁺ .

¹ H-NMR (DMSO-d6, δ, ppm, J, Hz): 7.92 (s, 1H, H-8), 6.41 (s, 2H, NH ₂ ), 6.12 (d, J = 4.7, 1H, H _{1 '} ), 5.60 (broad s, 1H, OH _5' ), 5.48 (broad s, 1H, OH _{3 '} ), 5.04 (broad s, 1H, OH _2' ), 4.09 (dd, J = 3.9 and 4.1, 1H, H _{3 '} ), 4.06 (dd, J = 4.2 and 4.4, 1H, H _2' ), 3.75 (m, 1H, H _{4 '} ), 3.65 (m, 1H, H _5a' ), 3.60 (m, 1H, H _{5b '} ).

¹³ C-NMR (DMSO-d6, δ, ppm): 160.98 (C6), 154.44 (C4), 139.50 (C8), 113.62 (C5), 84.69 (C-4 '), 83.82 (C-1'), 75.96 (C-2 '), 75.81 (C3'), 61.44 (C-5 ').

¹⁵³ N-NMR (DMSO-d6, δ, ppm): 239.56 (N7), 205.19 (N1), 188.81 (N3), 163.61 (N9).

Example 7

Obtaining 9- (β-D-arabinofuranosyl) -2-fluoroadenine (fludarabine, compound IX) without sodium arsenate.

A mixture of 1.7 g (0.006 mol) of 2-fluoroadenosine (CAS Number: 146-78-1, compound VII), 2.2 g (0.009 mol) of 1-β-D-arabinofuranosilouracil (compound VIII) and 0.882 g (0.003 mol) in 0.2 l 60 mm potassium phosphate buffer (pH 7.0) thermostat for 120 hours at 48 ° C in the presence of 375 units. Act. PNP enzyme preparation (62.5 units of act. per 1 mmol of 2-fluoroadenosine) and 563 units. Act. UV enzyme preparation (62.5 units of act. per 1 mmol of 1-β-D-arabinofuranosiluracil). The control of the completeness of the process is carried out using HPLC. The process is considered complete if, according to HPLC, the content of the starting 2-fluoroadenosine (compound VII, Fig. 3) and the product of fludarabine (compound IX) does not change.

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: 1.2 g (70%). Product purity according to HPLC 99-99.4% (method from 0 to 20% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, Nova-Pak C18 column, 4.6 × 150 mm, 4 μm). T. pl. 260-262 ° C. λ _max , nm (ε, M ^-1 cm ^-1 ): 262 (14800). Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.17 (s, 1H, H-8), 7.76 (s, 2H, NH ₂ ), 6.11 (d, J = 5.0 Hz, 1H, H _{1 '} ), 5.61 (d, J = 5.3 Hz, 1H, OH _2' ), 5.51 (d, J = 4.7 Hz, 1H, OH _{3 '} ), 5.05 (t, J = 4.3 Hz, 1H, OH _{5 '} ), 4.14 (m, 1H, H _2' ), 4.13 (m, 1H, H _{3 '} ), 3.77 (m, 1H, H _4' ), 3.66 (m, 2H, H _{5 '} ).

Example 8

Preparation of 9- (β-D-arabinofuranosyl) adenine (Ara-A, compound XII) without sodium arsenate.

A mixture of 1.4 g (0.005 mol) of adenosine (compound XI), 1.8 g (0.0075 mol) of 1-β-D-arabinofuranosyluracil (compound VIII) in 0.1 L of 50 mM potassium phosphate buffer (pH 7.0) was incubated for 24 hours at 52 ° C in the presence of 150 units Act. PNP enzyme preparation (30 units of act. on 1 mmol of 2'-deoxyadenosine) and 250 units. Act. UV enzyme preparation (33 units of act. per 1 mmol of 1-β-D-arabinofuranosiluracil). The control of the completeness of the process is carried out using HPLC. The process is considered complete if the residual content of the starting adenosine (compound XI) in the reaction mixture remains unchanged.

The reaction mixture was concentrated in vacuo (15 mmHg) to 0.05 L, cooled to a temperature of 8 ° C. The precipitated target product is filtered off, washed with water (3 × 5 ml) and ethyl alcohol (1 × 5 ml), then dried in a desiccator to a constant weight. Yield: 0.65 g (45%). Product purity according to HPLC 99-99.4% (RT = 10.2 min, method from 0 to 10% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, column Nova-Pak C18, 4.6 × 150 mm, 4 μm). T. pl. 260-262 ° C. λ _max , nm (ε, M ^-1 cm ^-1 ): 262 (14800). Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.17 (s, 1H, H-8), 8.12 (s, 1H, H-2), 7.19 (s, 2H, NH ₂ ), 6.25 (d, J = 4.3 Hz, 1H, H _{1 '} ), 5.60 (br.s, OH _2' ), 5.49 (d, J = 3.3 Hz, OH _{3 '} ), 5.05 (br., OH _{5 '} ), 4.13 (m, 2H, H _2' and H _{3 '} ), 3.78 (m, 1H, H _4' ), 3.68 and 3.63 (2m, 2H, H _{5 '} ).

Example 9

Preparation of 9-β-D-arabinofuranosyl-6-O-methylguanine (nonlarabine, compound XIV) without sodium arsenate.

In 200 ml of distilled water, 1.17 g (4.0 mmol) of 9- (β-D-ribofuranosyl) -6-O-methylguanine (CAS Number: 7803-88-5), 1.46 g (6.0 mmol) 1- (β -D-arabinofuranosyl) uracil and 1.605 g (11.8 mmol) of potassium dihydroorthophosphate. The pH of the reaction mixture was adjusted to 7.0. 120 units were added to the reaction mixture. purinnucleoside phosphorylase (30 units of act. per 1 mmol of 9- (β-D-ribofuranosyl) -6-O-methylguanine) and 210 units. uridine phosphorylase (35 units of act. per 1 mmol of 1-β-D-arabinofuranosyluracil). The solution is thermostated at 52 ° C for 7-8 days. At the end of the process, the reaction mixture was concentrated in vacuo (15 mmHg) to a volume of 10 ml and chromatographed on a Octadecyl = Si 100polyol reverse phase sorbent (0.03 mm), column size: 20 × 300 mm The target compound was eluted with 2% acetonitrile in water. The fractions containing the product are combined, the solvent is removed in vacuo (15 mmHg). The product is dried in vacuum (5 mm RT. Art.) Over phosphorus pentoxide. Yield: 0.78 g (67%). Product purity (according to HPLC) 90-91% (RT = 9.6 min, method from 0 to 20% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA Column Nova-Pak C18, 4.6 × 150 mm, 4 μm). λ _max 286, 244, 214 nm. Mass spectrum, m / z: 298.1208 [M + H] ⁺ , 166.0777 [base + H] ⁺ . Calc. 298.2765 [M + H] ⁺ , 166.1613 [base + H] ⁺ .

¹ H-NMR (DMSO-d6, δ, ppm, J, Hz): 7.92 (s, 1H, H-8), 6.41 (s, 2H, NH ₂ ), 6.12 (d, J = 4.7, 1H, H _{1 '} ), 5.60 (broad s, 1H, OH _5' ), 5.48 (broad s, 1H, OH _{3 '} ), 5.04 (broad s, 1H, OH _2' ), 4.09 (dd, J = 3.9 and 4.1, 1H, H _{3 '} ), 4.06 (dd, J = 4.2 and 4.4, 1H, H _2' ), 3.75 (m, 1H, H _{4 '} ), 3.65 (m, 1H, H _5a' ), 3.60 (m, 1H, H _{5b '} ).

¹³ C-NMR (DMSO-d6, δ, ppm): 160.98 (C6), 154.44 (C4), 139.50 (C8), 113.62 (C5), 84.69 (C-4 '), 83.82 (C-1'), 75.96 (C-2 '), 75.81 (C3'), 61.44 (C-5 ').

¹⁵³ N-NMR (DMSO-d6, δ, ppm): 239.56 (N7), 205.19 (N1), 188.81 (N3), 163.61 (N9).

Example 10

Preparation of 9- (β-D-arabinofuranosyl) hypoxanthine (Ara-Hyp, compound XVI, Fig. 9).

A mixture of 0.27 g (1.0 mmol) of inosine (CAS Number: 58-63-9) (compound XV, Fig. 9), 0.314 g (1.3 mmol) of 1-β-D-arabinofuranosiluracil (compound VIII, Fig. 9) and 0.031 g (0.1 mmol) NaH ₂ AsO ₄ in 50.0 ml of 50 mm potassium phosphate buffer (pH 7.0) thermostat for 48 hours at 52 ° C in the presence of 30 units Act. PNP enzyme preparation (30 units of act. per 1 mmol of adenosine) and 45 units. Act. UV enzyme preparation (35 units of act. per 1 mmol of 1-β-D-arabinofuranosiluracil). The control of the completeness of the process is carried out using HPLC. The process is considered complete if the initial inosine content (compound XV, Fig. 9) in the reaction mixture is <1%.

The reaction mixture was concentrated in vacuo (15 mmHg) to 5 ml and chromatographed on a Octadecyl = Si 100polyol reverse phase sorbent (0.03 mm), column size: 15 × 150 mm The target compound was eluted with water. The fractions containing the product are combined, the solvent is removed in vacuo (15 mmHg). The product is dried in vacuum (5 mm RT. Art.) Over phosphorus pentoxide. Yield: 0.26 g (98%). Product purity according to HPLC 98.2% (RT = 8.34, method from 0 to 10% eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, Nova-Pak column C18, 4.6 × 150 mm, 4 μm). Mass spectrum, m / z: 268.0800 [M + H] ⁺ , 136.0400 [base + H] ⁺ . Calc. 268.0808 [M + H] ⁺ , 136.0385 [base + H] ⁺ . Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.08 (s, 1H, H-8), 8.00 (s, 1H, H-2), 6.20 (d, J = 4.9 Hz , 1H, H _{1 '} ), 4.15 (m, 1H, H _3' ), 4.13 (m, 1H, H _{3 '} ), 3.77 (m, 1H, H _4' ), 3.68 (m, 1H, H _{5 '} ), 3.63 (m, 1H, H _{5 '} ).

Example 11

Preparation of 2-chloro-9- (β-D-arabinofuranosyl) adenine (Cl-AraA), compound XVIII, Fig. 10).

A mixture of 0.30 g (1.0 mmol) of 2-chloro-adenosine (CAS Number: 146-77-0) (compound XVII, Fig. 10), 0.29 g (1.2 mmol) of 1-β-D-arabinofuranosyluracil (compound VIII, Fig. 10) and 0.003 g (0.01 mmol) of NaH ₂ AsO ₄ in 50.0 ml of 50 mM potassium phosphate buffer (pH 7.0) are thermostated for 48 hours at 52 ° C in the presence of 60 units. Act. PNP enzyme preparation (60 units of act. per 1 mmol of adenosine) and 78 units. Act. UV enzyme preparation (65 units of act. per 1 mmol of 1-β-D-arabinofuranosiluracil). The control of the completeness of the process is carried out using HPLC. The process is considered complete if the content of the starting 2-chloro-adenosine (compound XVII, Fig. 10) in the reaction mixture is <2%.

The reaction mixture was concentrated in vacuo (15 mmHg) to 10 ml and chromatographed on a Octadecyl = Si 100polyol reverse phase sorbent (0.03 mm), column size: 15 × 250 mm The target compound was eluted in a gradient of 0-20% acetonitrile in water. The fractions containing the product are combined, the solvent is removed in vacuo (15 mmHg). The product is dried in vacuum (5 mm RT. Art.) Over phosphorus pentoxide. Yield: 0.297 g (99%). Product purity according to HPLC 99.8% (RT = 9.42 min, isocratic method 7% of eluent B in 20 minutes, eluent A - water with 0.1% TFA, eluent B - 70% acetonitrile in water with 0.1% TFA, Nova-Pak C18 column , 4.6 × 150 mm, 4 μm). λ _max 262, 210 nm. Spectrum ¹ H-NMR (DMSO-d ₆ ), (δ ppm): 8.20 (s, 1H, H-8), 7.44 (s, 2H, NH ₂ ), 6.14 (d, J = 5.15 Hz, 1H, H _{1 '} ), 5.63 (1H, br.s, OH _2' ), 5.54 (1H, br.s, OH _{3 '} ), 5.07 (1H, br.s, OH _5' ), 4.16 (m, 1H, H _{2 '} ), 4.11 (m, 1H, H _3' ), 3.77 (m, 1H, H _{4 '} ), 3.67 and 3.63 (2m, 2H, H _5' ).

Claims (1)

A method for producing modified β-D-arabinofuranose series purine nucleosides by reacting a purine ribonucleoside with 1-β-D-arabinofuranosiluracil or 1-α-phosphate of D-arabinose in potassium phosphate buffer in the presence of purine nucleoside phosphorylase in the presence of purine and pyrimidosulfuric acid nucleoside .

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