Preparation method of 2 '-alkoxy uridine derivative

Abstract

The invention discloses a preparation method of a 2 '-alkoxy uridine derivative, which comprises the following steps: by taking cytidine as an initial raw material, firstly substituting 2'-OH of the cytidine with long alkyl to obtain 2 '-alkoxy cytidine, then removing amino on the 2'-alkoxy cytidine to obtain a compound 3, then protecting 5 '-OH in the compound 3 with DMTr to obtain a compound 4, and finally, preparing the 2'-alkoxy uridine derivative. Finally, a phosphorus reagent reacts with 3 '-OH in the compound 4, and the target 2'-alkoxy uridine derivative is obtained.The method is mild in reaction condition, high in synthesis efficiency, environmentally friendly and suitable for large-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic chemical synthesis, and particularly relates to a preparation method of 2'-alkoxyuridine derivatives. Background Art

[0002] The ability to effectively and completely deliver nucleoside drugs to the targeted position is one of the main limiting factors in their in vivo experiments and clinical applications.

[0003] Introducing groups with different sizes and polarities at the 2'-position of oligonucleosides will cause different changes in the properties of nucleosides. For example, methoxy modification can enhance the binding of drugs to target mRNA and inhibit the hydrolysis of nucleases; fluorine substitution can improve the stability of drugs in serum; while modifying the 2'-position with long-chain aliphatic hydrocarbons can increase the liposolubility of the nucleoside structure, which is more conducive to efficient delivery into targeted cells.

[0004] WO 2020 / 257194A1 discloses a method for synthesizing 2'-hexadecoxyuridine derivatives. This method uses cyclic uridine as a raw material, the 5'-OH is protected by TBS or DMTr, and then the long-chain alkane is activated under the action of methylaluminum, and further ring-opening generates 2'-alkoxyuridine. This method is difficult to react with alkanes with longer carbon chains, and the greatly excessive fatty carbon chains are very difficult to dissolve, often requiring a reaction temperature above 130°C, with a long reaction time and low conversion rate. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of 2'-alkoxyuridine derivatives, which has mild reaction conditions, high synthesis efficiency, is environmentally friendly, and is more suitable for large-scale production.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A preparation method of 2'-alkoxyuridine derivatives, the preparation method comprising the following steps:

[0008] (1) React cytidine with C n H 2n+1 X under the catalysis of an inorganic base to generate 2'-alkoxycytidine; wherein, n is 16-22, and X is a halogen;

[0009] (2) 2'-alkoxycytidine reacts with sodium nitrite under acidic conditions to deaminate and generate compound 3;

[0010] (3) Compound 3 reacts with 4,4'-dimethoxytriphenylmethyl chloride under the action of a basic catalyst to generate compound 4;

[0011] (4) Compound 4 reacts with a phosphorus reagent under the action of a catalyst to generate 2'-alkoxyuridine derivatives;

[0012] The structural formula of the said compound 3 is:

[0013] The structural formula of the said compound 4 is:

[0014] The structural formula of the said 2'-alkoxyuridine derivative is:

[0015] Furthermore, in step (1), the reaction solvent is selected from one or more of DMSO, DMF, THF, and DMAc.

[0016] In step (1), the said inorganic base is selected from one or more of sodium hydride, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0017] In step (1), the molar ratio of cytidine, C n H 2n+1 X, and the inorganic base is 1:1 - 1.7:1 - 3.

[0018] In step (1), the reaction temperature is 10 - 60 °C and the time is 6 - 14 h.

[0019] In step (1), the post-treatment method of the reaction is: after the reaction ends, the solvent is concentrated and removed, and 2'-alkoxycytidine is obtained by column chromatography separation. The eluent used for column chromatography is a mixed solution composed of dichloromethane and methanol in a volume ratio of 10 - 25:1.

[0020] In step (2), the molar ratio of 2'-alkoxycytidine to sodium nitrite is 1:20 - 40.

[0021] In step (2), the reaction solvent is selected from a mixed solvent composed of an organic acid and water in a volume ratio of 1:3 - 6; the said organic acid is one or more of formic acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid.

[0022] In steps (2) and (4), the reaction conditions are both reacting at 20 - 60 °C for 6 - 14 h.

[0023] In step (2), the post-treatment method of the reaction is: after the reaction is completed, the pH of the system is adjusted to 7 with a 2 - 4M sodium hydroxide solution, filtered, the obtained solid is redissolved in dichloromethane, dried over anhydrous sodium sulfate and then concentrated, and compound 3 is obtained by column chromatography separation. The eluent used for column chromatography is a mixed solution composed of dichloromethane and methanol in a volume ratio of 35 - 40:1.

[0024] In step (3), the reaction solvent is selected from one or more of acetonitrile, DCM, pyridine, and THF.

[0025] In step (3), the basic catalyst is selected from one or more of pyridine, 2,6-dimethylpyridine, DMAP, imidazole, DIPEA, and N-methylmorpholine.

[0026] In step (3), the mass ratio of compound 3, 4,4'-dimethoxytriphenylmethyl chloride, and the basic catalyst is 1:0.5 - 1.0:0.5 - 1.0.

[0027] In step (3), the reaction temperature is 10 - 60 °C, and the time is 1.5 - 2.0 h.

[0028] In step (3), the post-treatment method of the reaction is as follows: after the reaction is completed, it is washed successively with saturated sodium bicarbonate solution, 2% citric acid solution, saturated sodium bicarbonate solution, and saturated NaCl solution, concentrated, and then separated by column chromatography to obtain compound 4. The eluent used for column chromatography is a mixed solution composed of dichloromethane and ethyl acetate, or hexane and ethyl acetate in a volume ratio of 2 - 5:1.

[0029] In step (4), the catalyst is selected from one or more of diisopropylammonium salt tetrazole, tetrazole, diisopropylethylamine tetrazole, 5-ethylthiotetrazole, DCI, pyridinium trifluoroacetate, DIPEA, and N-methylmorpholine.

[0030] In step (4), the reaction solvent is selected from one or more of DCM, acetonitrile, THF, and 2-methyltetrahydrofuran.

[0031] In step (4), the molar ratio of compound 4, phosphorus reagent, and catalyst is 1:1.0 - 1.5:0.7 - 1.0.

[0032] In step (4), the post-treatment method of the reaction is as follows: add sodium bicarbonate solution to quench the reaction, extract with dichloromethane, wash the organic phase three times with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, and then separate by column chromatography to obtain the 2'-alkoxyuridine derivative. The eluent used for column chromatography is a mixed solution composed of hexane and ethyl acetate in a volume ratio of 3 - 5:1.

[0033] The preparation method of the 2'-alkoxyuridine derivative provided by the present invention uses cytidine as the starting material, and reacts cytidine with C n H 2n+1X reacts under the catalysis of an inorganic base. The 2'-hydroxyl group on the ribose ring of cytidine has the strongest acidity and will be preferentially activated by the base, and then substituted by a long alkyl group to obtain 2'-alkoxycytidine. Then, the amino group on 2'-alkoxycytidine is removed to obtain compound 3. Then, the 5'-OH in compound 3 is protected with DMTr to obtain compound 4. Finally, a phosphorus reagent (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite) reacts with the 3'-OH in compound 4 to generate the target 2'-alkoxylated uridine derivative. The synthesis route is as follows:

[0034]

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] Based on the limitations of the traditional preparation of 2'-long-chain alkane substitution reactions, the present invention uses cytidine as the starting material and develops a method for converting cytidine into uridine derivatives by deamination, further overcoming the defects of slow reaction time, high reaction temperature, low conversion rate, and unfavorable for large-scale production in the prior art, and providing a new method for selectively introducing a long fatty chain at the 2'-position of uridine. Description of the Drawings

[0037] Figure 1 is the 1H NMR spectrum of compound 4a;

[0038] Figure 2 is the 1H NMR spectrum of compound 5a;

[0039] Figure 3 is the 31P NMR spectrum of compound 5a;

[0040] Figure 4 is the 1H NMR spectrum of compound 4b

[0041] Figure 5 is the 1H NMR spectrum of compound 5b;

[0042] Figure 6 is the 31P NMR spectrum of compound 5b;

[0043] Figure 7 is the synthesis route diagram of the 2'-alkoxylated uridine derivative. Detailed Embodiments

[0044] The present invention will be described in detail below with reference to the embodiments.

[0045] Example 1

[0046] A preparation method of a 2'-alkoxylated uridine derivative, and the preparation route is as follows:

[0047]

[0048] Among them, n = 16 and X = Br.

[0049] The preparation method includes the following steps:

[0050] (1) Add 50 g of cytidine and 750 mL of DMF to the reaction flask in sequence, heat to 100 °C until dissolved, quickly cool to room temperature, then add 7.4 g (308.33 mmol) of NaH, react at room temperature for 2 h, and dropwise add 100.49 g (329.09 mmol) of C 16 H 33 Br at room temperature, heat to 40 °C and react overnight, concentrate to remove the solvent, and then separate by column chromatography. The eluent is a mixed solution composed of dichloromethane and methanol in a volume ratio of 15:1. The collected product is concentrated to obtain compound 2a;

[0051] (2) Add 23 g (49.21 mmol) of compound 2a, 460 ml of deionized water, 102 g (1.48 mol) of sodium nitrite, and 115 mL of acetic acid, stir overnight at 40 °C, adjust the pH to 7 with 2 M sodium hydroxide solution, filter to obtain a solid, dissolve it with dichloromethane, dry it with anhydrous sodium sulfate, concentrate, and then separate by column chromatography. The eluent is a mixed solution composed of dichloromethane and methanol in a volume ratio of 35:1. The collected product is concentrated and dried to obtain 16.6 g of white solid compound 3a;

[0052] (3) Add 16.6 g (35.45 mmol) of compound 3a, 166 mL of dichloromethane, and 16.6 mL of pyridine to the flask, then add 12.61 g (37.22 mmol) of DMTrCl. DMTrCl is added in 3 portions at half-hour intervals. After the addition is completed, stir at room temperature for 2 h. Wash the reaction solution twice with 7% mass concentration of NaHCO3 solution, then wash twice with 10% mass concentration of citric acid, wash once with 7% mass concentration of NaHCO3 solution, wash once with saturated NaCl solution, dry with anhydrous Na2SO4, concentrate, and then separate by column chromatography. The eluent is a mixed solution composed of dichloromethane and ethyl acetate in a volume ratio of 3:1. The collected product is concentrated and dried to obtain 10 g of solid compound 4a.

[0053] Its 1H NMR (400 MHz, DMSO): δ 11.38 (s, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.41–7.21 (m, 10H), 6.90 (d, J = 8.9 Hz, 4H), 5.80 (d, J = 3.8 Hz, 1H), 5.28 (d, J = 8.1 Hz, 1H), 5.13 (d, J = 6.6 Hz, 1H), 4.18 (dd, J = 11.6, 6.1 Hz, 1H), 3.97 (dd, J = 8.7, 4.1 Hz, 1H), 3.92–3.88 (m, 1H), 3.74 (s, 6H), 3.56 (dtd, J = 16.3, 9.7, 6.5 Hz, 2H), 3.31–3.20 (m, 2H), 1.49 (dd, J = 13.1, 6.4 Hz, 2H), 1.22 (s, 25H), 0.84 (t, J = 6.8 Hz, 3H).

[0054] (4) Add 8 g (10.38 mmol) of compound 4a, 80 mL of dichloromethane, 1.42 g (8.30 mmol) of diisopropylethylamine salt of tetrazole, and 4.07 g (13.5 mmol) of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite into a flask. Under nitrogen protection, stir overnight at 30 °C. Quench the reaction with sodium bicarbonate solution, extract with dichloromethane. Wash the organic phase three times with saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate, and separate by column chromatography. The eluent is a mixed solution composed of hexane and ethyl acetate in a volume ratio of 4:1. The collected product is concentrated and dried to obtain 5.3 g of compound 5a with a purity of 96.88%.

[0055] Its 1H NMR (400 MHz, DMSO): δ 11.38 (s, 1H), 7.79 (dd, J = 13.6, 8.1 Hz, 1H), 7.41–7.22 (m, 10H), 6.92–6.86 (m, 4H), 5.80 (t, J = 3.5 Hz, 1H), 5.26 (dd, J = 14.1, 8.1 Hz, 1H), 4.46–4.26 (m, 1H), 4.09 (ddd, J = 15.6, 8.5, 3.7 Hz, 2H), 3.73 (d, J = 2.6 Hz, 6H), 3.56 (ddd, J = 13.5, 9.8, 6.8 Hz, 5H), 2.76 (t, J = 5.9 Hz, 1H), 2.60 (dd, J = 9.8, 5.5 Hz, 1H), 1.54–1.45 (m, 2H), 1.21 (s, 30H), 1.11 (dd, J = 11.8, 6.7 Hz, 10H), 0.98 (d, J = 6.7 Hz, 2H), 0.84 (t, J = 6.7 Hz, 3H).

[0056] Its 1P NMR (162 MHz, DMSO) δ 149.15, 148.62.

[0057] Example 2

[0058] A preparation method of a 2'-alkoxyuridine derivative, the preparation route is as follows:

[0059]

[0060] Wherein, n = 22, X = Br.

[0061] The said preparation method comprises the following steps:

[0062] (1) Add 30 g (123.35 mmol) of cytidine and 300 mL of DMF into a flask, heat up to 100 °C until dissolved, quickly cool down to 60 °C, add 6.79 g (283.71 mmol) of lithium hydroxide, stir at 60 °C for 1 hour, add 72.07 g (185.03 mmol) of 1-bromodocosane, stir at 60 °C overnight, pour it into stirred water, filter to obtain a solid, and then separate by column chromatography, the eluent is a mixed solution composed of dichloromethane and methanol in a volume ratio of 20:1, to obtain 21 g of a white solid compound 2b.

[0063] (2) Add 21 g (38.08 mmol) of compound 2b, 420 mL of water, 78.84 g (1.14 mol) of sodium nitrite, and 105 mL of acetic acid into a flask, stir at 40 °C overnight, adjust the pH to 7 with 4 M sodium hydroxide solution, filter to obtain a solid, redissolve it with dichloromethane, dry with anhydrous sodium sulfate, concentrate and then separate by column chromatography, the eluent is a mixed solution composed of dichloromethane and methanol in a volume ratio of 35:1, the collected product is concentrated and dried to obtain 9.5 g of compound 3b.

[0064] (3) Add 9.5 g (17.20 mmol) of compound 3b, 95 mL of dichloromethane, 9 mL of pyridine, and 6.17 g (18.06 mmol) of DMTrCl into a flask, stir at room temperature for 2 h, wash twice with a 7% mass concentration of NaHCO3 solution, then wash twice with a 10% mass concentration of citric acid, wash once with a 7% mass concentration of NaHCO3, wash once with saturated NaCl solution, dry with anhydrous Na2SO4, concentrate and then separate by column chromatography, the eluent is a mixed solution composed of hexane and ethyl acetate in a volume ratio of 3:1, the collected product is concentrated and dried to obtain 11 g of a white solid compound 4b.

[0065] Its 1H NMR (400 MHz, DMSO): δ 11.38 (s, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.40–7.22 (m, 10H), 6.89 (d, J = 8.8 Hz, 4H), 5.80 (d, J = 3.7 Hz, 1H), 5.27 (d, J = 8.1 Hz, 1H), 5.11 (d, J = 6.6 Hz, 1H), 4.18 (dd, J = 11.6, 6.1 Hz, 1H), 3.96 (dd, J = 8.7, 3.9 Hz, 1H), 3.91–3.87 (m, 1H), 3.73 (s, 6H), 3.63–3.49 (m, 2H), 3.26 (dd, J = 25.7, 6.2 Hz, 2H), 1.54–1.46 (m, 2H), 1.21 (s, 37H), 0.83 (t, J = 6.7 Hz, 3H).

[0066] (4) 3.8 g (4.45 mmol) of compound 4b, 609 mg (3.56 mmol) of diisopropylethylamine salt of tetrazole, 1.74 g (5.78 mmol) of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite and 38 mL of dichloromethane were added to a reaction flask, stirred overnight under a nitrogen atmosphere, quenched with sodium bicarbonate solution, extracted with dichloromethane, the organic phase was washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated and separated by column chromatography. The eluent was a mixed solution composed of hexane and ethyl acetate in a volume ratio of 4:1. The collected product was concentrated and dried to obtain 3.9 g of white solid compound 5b, with a yield of 83%. The purity was 97.71%, and the PNMR was 99.23%. Its 1H NMR (400 MHz, DMSO): δ 11.37 (s, 1H), 7.79 (dd, J = 13.2, 8.1 Hz, 1H), 7.42–7.20 (m, 10H), 6.88 (t, J = 6.5 Hz, 4H), 5.79 (t, J = 3.3 Hz, 1H), 5.24 (dd, J = 13.5, 8.1 Hz, 1H), 4.38 (ddd, J = 18.7, 10.3, 5.3 Hz, 1H), 4.14–4.01 (m, 2H), 3.74–3.69 (m, 7H), 3.56 (dt, J = 15.6, 6.4 Hz, 5H), 2.76 (t, J = 5.9 Hz, 1H), 2.60 (dd, J = 10.1, 5.4 Hz, 1H), 1.54–1.44 (m, 2H), 1.21 (s, 38H), 1.11 (dd, J = 12.6, 6.7 Hz, 11H), 0.97 (d, J = 6.7 Hz, 2H), 0.83 (t, J = 6.5 Hz, 3H).

[0067] Its 1P NMR (162 MHz, DMSO) δ 149.13, 148.58.

[0068] The detailed description of the preparation method of a 2'-alkoxyuridine derivative in the above reference examples is illustrative rather than restrictive. Several embodiments can be enumerated within the defined scope. Therefore, changes and modifications without departing from the general concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a 2'-alkoxyuridine derivative, characterized in that: The preparation method comprises the following steps: (1) Cytidine and C n H 2n+1 X reacts under the catalysis of an inorganic base to generate 2'-alkoxycytidine; wherein n is 16 to 22, and X is a halogen; (2) 2'-alkoxycytidine reacts with sodium nitrite under acidic conditions to generate compound 3; (3) Compound 3 reacts with 4,4'-dimethoxytrityl chloride in the presence of a basic catalyst to generate compound 4; (4) Compound 4 reacts with a phosphorus reagent under the action of a catalyst to generate a 2'-alkoxyuridine derivative; The structural formula of the compound 3 is: The structural formula of the compound 4 is: The structural formula of the 2'-alkoxyuridine derivative is:

2. The preparation method according to claim 1, characterized in that: In step (1), the reaction solvent is selected from one or more of DMSO, DMF, THF, and DMAc; and the inorganic base is selected from one or more of sodium hydrogen sulfide, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

3. The preparation method according to claim 1, characterized in that: In step (1), cytidine, C n H 2n+1 The molar ratio of X and inorganic base is 1:1-1.7:1-3.

4. The preparation method according to claim 1, characterized in that: In step (1), the reaction temperature is 10 to 60° C. and the reaction time is 6 to 14 hours.

5. The preparation method according to claim 1, characterized in that: In step (2), the molar ratio of 2'-alkoxycytidine to sodium nitrite is 1:20-40.

6. The preparation method according to claim 1, characterized in that: In step (2), the reaction solvent is selected from a mixed solvent composed of an organic acid and water in a volume ratio of 1:3 to 6; the organic acid is one or more of formic acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid.

7. The preparation method according to claim 1, characterized in that: In step (2) and step (4), the reaction conditions are both 20-60° C. for 6-14 hours.

8. The preparation method according to claim 1, characterized in that: In step (3), the reaction solvent is selected from one or more of acetonitrile, DCM, pyridine, and THF; the basic catalyst is selected from one or more of pyridine, 2,6-lutidine, DMAP, imidazole, DIPEA, and N-methylmorpholine.

9. The preparation method according to claim 1, characterized in that: In step (4), the catalyst is selected from one or more of diisopropylammonium tetrazolium, tetrazole, diisopropylethylamine tetrazolium, 5-ethylthiotetrazolium, DCI, pyridinium trifluoroacetate, DIPEA, and N-methylmorpholine.

10. The preparation method according to any one of claims 2 to 9, characterized in that: In step (4), the molar ratio of compound 4, phosphorus reagent and catalyst is 1:1.0-1.5:0.7-1.0.

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