A method for the synthesis of cytosine nucleosides and derivatives thereof

Abstract

This invention discloses a method for synthesizing cytosine nucleosides and their derivatives, belonging to the field of phosphorus amide and small nucleic acid drug preparation technology. Using uracil nucleosides and their derivatives represented by Formula I as raw materials, this invention involves a one-step direct ammoniation reaction with an ammonia source under the action of an ammonium salt catalyst. This eliminates the need for pre-protection and subsequent deprotection of the 3'- and 5'-hydroxyl groups in the raw materials, thus obtaining cytosine nucleosides and their derivatives represented by Formula II. The reaction conditions of this invention are mild, avoiding the large-scale use of phosphorus oxychloride, strong acids and bases, and highly toxic phosphorus / chlorine-containing reagents, resulting in low emissions of waste. The obtained product can achieve a purity of over 99.5% through simple isopropanol crystallization, with a total yield of 75%–82%. This method solves the problems of long synthetic routes, low atom utilization, difficult waste treatment, and high production costs associated with existing technologies, making it suitable for large-scale industrial production and providing a novel green approach for the preparation of key intermediates for small nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of phosphorus amide and small nucleic acid drug preparation technology, specifically relating to a method for synthesizing cytosine nucleosides and their derivatives. Background Technology

[0002] Cytosine nucleosides are one of the basic building blocks of RNA. Their base moiety, cytosine, participates in the specific binding of small nucleic acid drugs to target mRNA through complementary base pairing. In drugs such as antisense oligonucleotides (ASO) and small interfering RNA (siRNA), cytosine residues help improve the accuracy of sequence recognition and binding stability. In small nucleic acid drug development, the 2'-OH position of cytosine nucleosides is often chemically modified to enhance drug performance, for example, with 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), and 2'-O-methoxyethyl (2'-MOE). These modifications can improve the drug's serum stability, target affinity, and reduce immunogenicity. For example, in a patent for siRNA targeting hepatitis B (CN102140461B), both the sense and antisense strands of cytosine nucleosides were modified with 2'-OMe or 2'-F to optimize pharmacokinetic properties.

[0003] Currently, the main routes for synthesizing cytosine nucleosides and their derivatives from uracil nucleosides and their derivatives are as follows.

[0004] Route 1:

[0005] This route reacts the protected uridine and its derivatives with phosphorus oxychloride (POCl3) to convert the carbonyl group at the C4 position into a chlorinated group, generating structural formula 3. The chlorinated product reacts in liquid ammonia or an alcoholic solution of ammonia, where the chlorine is replaced by an amino group, forming the target product II.

[0006] Route 2:

[0007] This route requires first enolizing the carbonyl group on uracil nucleosides and their derivatives to obtain structural formula 2, where group R4 is typically tetrazolium, p-toluenesulfonyl, or 2,4,6-triisopropylbenzenesulfonyl. Substituting structural formula 2 with ammonia yields structural formula II.

[0008] Both routes require protecting the exposed hydroxyl groups before removing them, significantly increasing material costs and reaction steps, and reducing the overall reaction yield. Enolization of carbonyl groups requires strong acids and bases, as well as phosphorus- or chlorine-containing reagents, greatly increasing the difficulty of waste treatment. Strict temperature control is required during the reaction, demanding advanced production equipment and increasing energy consumption. Furthermore, the R4 group is only used as a transition state, resulting in low atom utilization and making overall route approval difficult.

[0009] In summary, existing techniques for synthesizing cytosine nucleosides and their derivatives still have considerable room for improvement. Therefore, this invention provides a novel route for the synthesis of cytosine nucleosides and their derivatives. Summary of the Invention

[0010] The purpose of this invention is to provide a method for synthesizing cytosine nucleosides and their derivatives, which achieves one-step amination under hydroxyl-free protection conditions. This method is not only applicable to the synthesis of natural cytosine, but can also be widely used to prepare various 2'-modified cytosine derivatives. At the same time, it solves the problems of long technical routes, large emissions of waste, and high production costs in the existing technology.

[0011] The objective of this invention can be achieved through the following technical solutions: A method for synthesizing cytosine nucleosides and their derivatives without prior protection of the 3'- and 5'-hydroxyl groups in the raw materials includes the following steps: mixing uracil nucleosides and their derivatives as shown in Formula I, an ammonium salt catalyst, and a solvent; heating the mixture to 60-80°C under normal pressure; introducing an ammonia source to carry out a direct amination reaction of the carbonyl group at the C4 position; and after the reaction is completed, post-treatment is performed to obtain cytosine nucleosides and their derivatives as shown in Formula II. ; R1 is selected from one of -H, -OH, -OMe (methoxy), -F, and -MOE (methoxyethyl); R2 and R3 are each independently selected from one of -H, -TES (triethylsilyl), -TBDMS (tert-butyldimethylsilyl), -BOC (tert-butyloxycarbonyl), -TBDPS (tert-butyldiphenylsilyl), -Bn (benzyl), -Ac (acetyl), -Bz (benzoyl), and -DMTr (4,4'-dimethoxytriphenylmethyl).

[0012] In the above synthetic method, the direct amination of the carbonyl group at the C4 position of uracil nucleoside under ammonium salt catalysis has the following reaction mechanism: ① Carbonyl activation: Ammonium methanesulfonate transfers a proton to the carbonyl oxygen atom at the C4 position, enhancing the positive charge of the carbonyl carbon atom and increasing its electrophilic activity; ② Nucleophilic addition: Ammonia molecules attack the activated carbonyl carbon atom to form a tetrahedral intermediate. Due to the mild reaction conditions, the 3' and 5'-hydroxyl groups do not participate in the reaction, so no protection is required. ③ Dehydration elimination: The tetrahedral intermediate is dehydrated under the catalysis of ammonium salt to generate cytosine nucleoside derivatives, while regenerating the ammonium methanesulfonate catalyst.

[0013] This mechanism fundamentally breaks through the traditional paradigm that requires carbonyl activation followed by ammonolysis, omitting the hydroxyl protection-deprotection step and significantly shortening the synthetic route.

[0014] Furthermore, the ammonium salt catalyst is selected from one or more of ammonium trifluoroacetate, ammonium trifluoromethanesulfonate, and ammonium methanesulfonate, preferably ammonium methanesulfonate. Among them, ammonium methanesulfonate, as a bifunctional catalyst, can both activate the carbonyl group at the C4 position of the uracil ring through protonation and promote the nucleophilic attack of ammonia through ammonium ions, exhibiting the highest catalytic activity and low cost.

[0015] Furthermore, the amount of the ammonium salt catalyst is 0.005 to 0.05 times the molar amount of the compound of Formula I, preferably 0.01 to 0.02 times. Too little catalyst will result in a slow reaction rate, while too much catalyst may trigger a ring-opening side reaction of the pyrimidine ring.

[0016] Furthermore, the ammonia source is preferably a combination of ammonia and hexamethyldisilamine, wherein the amount of ammonia is 2.0 to 3.0 times the molar amount of the compound of Formula I, and the amount of hexamethyldisilamine is 1.5 to 2.5 times the molar amount of the compound of Formula I. Ammonia is the main amination reagent, and hexamethyldisilamine acts as an auxiliary base and dehydrating agent, absorbing the water generated in the reaction and promoting a forward shift of equilibrium.

[0017] Furthermore, the ammonia source is selected from ammonia gas, a tetrahydrofuran solution of ammonia, or a 2-methyltetrahydrofuran solution of ammonia, and the amount used is 3.0 to 4.0 times the molar amount of the compound of Formula I.

[0018] Furthermore, the solvent is selected from one or more of toluene, tetrahydrofuran, 2-methyltetrahydrofuran, and N,N-dimethylformamide. The solvent is preferably tetrahydrofuran, which has good solubility for both the raw materials and the product, a moderate boiling point (66°C), facilitates reaction temperature control and solvent recovery, and achieves a recovery rate of over 92%.

[0019] Furthermore, the mass of the solvent is 4 to 10 times the mass of the compound of Formula I.

[0020] Furthermore, the amination reaction is carried out at a temperature of 60-65°C for a reaction time of 24-30 hours. This temperature range ensures both the reaction rate and effectively suppresses side reactions.

[0021] Furthermore, the post-processing steps include: concentrating the reaction solution under reduced pressure to dryness at 50-55°C and 20-50 Pa, adding methanol and refluxing for 5-8 hours to decompose the silicon-based byproducts, concentrating under reduced pressure to dryness again, then crystallizing with isopropanol, filtering, and drying to obtain compound II. The purity of the product can reach more than 99.5% with one crystallization.

[0022] Furthermore, when R2 and / or R3 are not -H, a deprotection step is included after the amination reaction is completed; the deprotection method is one of acid hydrolysis, alkaline hydrolysis or fluoride ion hydrolysis, depending on the type of protecting group.

[0023] The beneficial effects of this invention are: (1) The present invention uses uracil nucleoside and its derivatives as shown in Formula I as raw materials. There is no need to pre-protect the 3'- and 5'-hydroxyl groups in the raw materials. Under the action of ammonium salt catalyst, the cytosine nucleoside and its derivatives shown in Formula II are directly aminated with the C4 carbonyl group in a one-step process. The synthesis steps are shortened from 4 to 6 steps in the prior art to 1 step, and the total reaction time is shortened from 48 to 72 hours to 30 to 36 hours, and the production efficiency is increased by more than 100%. At the same time, there is no need to use protecting groups and leaving groups, and the atom utilization rate is increased from about 30% to more than 70%, which significantly reduces the consumption of raw materials.

[0024] (2) The ammoniation reaction of the present invention is carried out under mild conditions of normal pressure and 60~80℃, without the need for high temperature and high pressure equipment, and without the need for highly toxic or corrosive reagents such as phosphorus oxychloride, strong acids and bases and heavy metal Lewis acids, which greatly improves the safety of operation and reduces equipment investment by more than 50%. By selecting inexpensive and readily available ammonium salts such as ammonium methanesulfonate as bifunctional catalysts, the catalytic activity is high and the amount used is only 0.005~0.05 times the molar amount of raw materials, which further reduces the production cost.

[0025] (3) This invention optimizes the ammonia source to be a combination of ammonia and hexamethyldisilamine. Hexamethyldisilamine absorbs the water generated in the reaction, promotes the forward shift of the reaction equilibrium, and avoids product decomposition caused by excessively high pH in the reaction system, so that the product yield is stable at 75%~82%. The three wastes generated in the reaction process are mainly recyclable solvents and a small amount of inorganic salts. The solvent recovery rate can reach more than 92%, and the cost of waste treatment is reduced by more than 60%, which meets the requirements of the national green chemical development.

[0026] (4) This invention is not only applicable to the synthesis of natural cytidine, but can also be widely applied to the preparation of key modification intermediates for various small nucleic acid drugs such as 2'-deoxycytidine, 2'-O-methylcytidine, 2'-deoxy-2'-fluorocytidine, and 2'-O-methoxyethylcytidine. It solves the technical problem that existing de novo synthesis methods cannot synthesize 2'-modified cytidine derivatives. The obtained product can achieve a purity of more than 99.5% by simple isopropanol crystallization, with a single maximum impurity content of ≤0.1%. The residual solvent meets the ICHQ3C standard, and no complicated column chromatography purification is required, making it suitable for large-scale industrial production. Attached Figure Description

[0027] The invention will now be further described with reference to the accompanying drawings.

[0028] Figure 1 The LC-MS positive ion spectrum of 2'-deoxy-2'-fluorocytidine prepared in Example 1 of this invention; Figure 2 The LC-MS negative ion spectrum of 2'-deoxy-2'-fluorocytidine prepared in Example 1 of this invention; Figure 3 The 2'-deoxy-2'-fluorocytidine prepared in Example 1 of this invention 1 HNMR spectrum; Figure 4 The 2'-deoxy-2'-fluorocytidine prepared in Example 1 of this invention 13 CNMR spectrum. Detailed Implementation

[0029] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0030] Example 1: Preparation of 2'-deoxy-2'-fluorocytidine In a 250 mL three-necked flask, add 2'-deoxy-2'-fluorouridine (20.0 g, 0.08124 mol), tetrahydrofuran (100.0 g), and hexamethyldisilazane (26.2 g, 0.1625 mol) sequentially, and stir at 25 °C until dissolved. Add ammonium methanesulfonate (1.0 g, 0.008124 mol), and slowly heat to 60–65 °C. Slowly bubble ammonia gas (5.5 g, 0.3250 mol) through the flask for approximately 6 hours.

[0031] The reaction was maintained at this temperature for 24 hours, and the conversion rate of the starting material was 99.8% as determined by HPLC. The reaction solution was concentrated to dryness under reduced pressure at 50-55℃ and 20-50 Pa. Methanol (50g) was added and refluxed for 6 hours to decompose the silicon-based byproducts, and the solution was concentrated to dryness again. Isopropanol (80g) was added and refluxed to dissolve the product. The solution was then slowly cooled to 0-5℃ to crystallize for 12 hours. After filtration and washing with cold isopropanol, the solution was dried under vacuum at 40℃ for 8 hours to obtain 15.8g of 2'-deoxy-2'-fluorocytidine, with a yield of 79.31%, an HPLC purity of 99.52%, and a maximum single impurity of 0.08%.

[0032] Product characterization: The LC-MS positive ion spectrum of the 2'-deoxy-2'-fluorocytidine prepared above is shown below. Figure 1 As shown; LC-MS negative ion spectrum as shown Figure 2 As shown; 1 HNMR spectrum as follows Figure 3 As shown; 13 CNMR spectrum as shown Figure 4 As shown.

[0033] LCMS: [M+1]=246[2M+1]=491[M+46-1]=290; 1 HNMR(400MHz,DMSO)δ7.89(d,J=7.4Hz,2H),7.22(d,J=12.1Hz,4H),5.88(dd,J= 18.1,1.7Hz,2H),5.72(d,J=7.4Hz,2H),5.54(d,J=6.3Hz,2H),5.15(t,J=5.1Hz, 2H),4.94(dd,J=4.3,1.7Hz,1H),4.81(dd,J=4.3,1.7Hz,1H),4.11(dddd,J=22. 5,8.0,6.3,4.3Hz,2H),3.90-3.73(m,4H),3.66-3.58(m,1H),3.62-3.54(m,1H).

[0034] 13 CNMR(101MHz,DMSO)δ166.3,155.3,141.6,95.4,94.4,93.6,88.7,88.36,83.2,67.9,67.7,59.8.

[0035] Example 2: Preparation of 2'-O-methylcytidine In a 250 mL three-necked flask, 2'-O-methyluridine (21.2 g, 0.08124 mol), tetrahydrofuran (100.0 g), and hexamethyldisilazane (26.2 g, 0.1625 mol) were added sequentially, and the mixture was stirred at 25 °C until the solution became clear. Ammonium methanesulfonate (1.0 g, 0.008124 mol) was added, and the mixture was slowly heated to 60–65 °C. Ammonia gas (5.5 g, 0.3250 mol) was slowly bubbled into the reaction flask over approximately 6 hours.

[0036] The reaction was continued at 60-65℃ for 24 hours, and samples were taken for HPLC analysis, showing a raw material conversion rate of 99.5%. The reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure at 50-55℃ and 20-50 Pa. Methanol (50g) was added, and the mixture was heated under reflux for 6 hours to decompose the silicon-based byproducts, followed by further concentration to dryness under reduced pressure.

[0037] Isopropanol (80 g) was added to the residue, and the mixture was heated to reflux to dissolve the solid. The mixture was then slowly cooled to 0–5 °C and stirred to induce crystallization for 12 hours. The residue was filtered, and the filter cake was washed with cold isopropanol (20 g) and dried under vacuum at 40 °C for 8 hours to obtain 16.4 g of a white solid product, with a yield of 77.73%, an HPLC purity of 99.48%, and a maximum single impurity content of 0.10%.

[0038] Example 3: Preparation of 2'-deoxy-2'-fluorocytidine catalyzed by ammonium trifluoroacetate In a 250 mL three-necked flask, 2'-deoxy-2'-fluorouridine (20.0 g, 0.08124 mol), tetrahydrofuran (100.0 g), and hexamethyldisilazane (26.2 g, 0.1625 mol) were added sequentially, and the mixture was stirred at 25 °C until the solution became clear. Ammonium trifluoroacetate (1.1 g, 0.008124 mol) was added, and the mixture was slowly heated to 60–65 °C. Ammonia gas (5.5 g, 0.3250 mol) was slowly bubbled into the reaction flask over approximately 6 hours.

[0039] The reaction was continued at 60-65℃ for 24 hours, and samples were taken for HPLC analysis, showing a raw material conversion rate of 98.5%. The reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure at 50-55℃ and 20-50 Pa. Methanol (50g) was added, and the mixture was heated under reflux for 6 hours to decompose the silicon-based byproducts, followed by further concentration to dryness under reduced pressure.

[0040] Isopropanol (80 g) was added to the residue, and the mixture was heated to reflux to dissolve the solid. The mixture was then slowly cooled to 0–5 °C and stirred to induce crystallization for 12 hours. The residue was filtered, and the filter cake was washed with cold isopropanol (20 g) and dried under vacuum at 40 °C for 8 hours to obtain 15.1 g of a white solid product, with a yield of 75.62%, an HPLC purity of 99.38%, and a maximum single impurity content of 0.12%.

[0041] Example 4: Preparation of 2'-deoxy-2'-fluorocytidine using ammonia in tetrahydrofuran solution as the ammonia source In a 250 mL three-necked flask, add 2'-deoxy-2'-fluorouridine (20.0 g, 0.08124 mol) and tetrahydrofuran (100.0 g) sequentially, and stir at 25 °C until the solution is clear. Add ammonium methanesulfonate (1.0 g, 0.008124 mol), and slowly heat to 60–65 °C. Slowly add a tetrahydrofuran solution of ammonia (162.5 mL, 2 mol / L, 0.3250 mol) over approximately 6 hours.

[0042] The reaction was continued at 60-65℃ for 24 hours, and samples were taken for HPLC analysis, showing a raw material conversion rate of 99.0%. The reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure at 50-55℃ and 20-50 Pa. Methanol (50g) was added, and the mixture was heated under reflux for 6 hours, then concentrated to dryness again under reduced pressure.

[0043] Isopropanol (80 g) was added to the residue, and the mixture was heated to reflux to dissolve the solid. The mixture was then slowly cooled to 0–5 °C and stirred to induce crystallization for 12 hours. The residue was filtered, and the filter cake was washed with cold isopropanol (20 g) and dried under vacuum at 40 °C for 8 hours to obtain 15.3 g of a white solid product, with a yield of 76.89%, an HPLC purity of 99.42%, and a maximum single impurity content of 0.11%.

[0044] Example 5: Preparation of 2'-deoxy-2'-fluorocytidine using 2-methyltetrahydrofuran as solvent In a 250 mL three-necked flask, add 2'-deoxy-2'-fluorouridine (20.0 g, 0.08124 mol), 2-methyltetrahydrofuran (100.0 g), and hexamethyldisilazane (26.2 g, 0.1625 mol) sequentially, and stir at 25 °C until the solution is clear. Add ammonium methanesulfonate (1.0 g, 0.008124 mol), and slowly heat to 60–65 °C. Slowly bubble ammonia gas (5.5 g, 0.3250 mol) into the reaction flask for approximately 6 hours.

[0045] The reaction was continued at 60-65℃ for 24 hours, and samples were taken for HPLC analysis, showing a raw material conversion rate of 98.7%. The reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure at 50-55℃ and 20-50 Pa. Methanol (50g) was added, and the mixture was heated under reflux for 6 hours to decompose the silicon-based byproducts, followed by further concentration to dryness under reduced pressure.

[0046] Isopropanol (80 g) was added to the residue, and the mixture was heated to reflux to dissolve the solid. The mixture was then slowly cooled to 0–5 °C and stirred to induce crystallization for 12 hours. The residue was filtered, and the filter cake was washed with cold isopropanol (20 g) and dried under vacuum at 40 °C for 8 hours to obtain 15.2 g of a white solid product, with a yield of 76.23%, an HPLC purity of 99.41%, and a maximum single impurity content of 0.11%.

[0047] Example 6 In a 250 mL three-necked flask, add 5'-O-DMTr-2'-deoxy-2'-fluorouridine (50.0 g, 0.08124 mol), tetrahydrofuran (250.0 g), and hexamethyldisilazane (26.2 g, 0.1625 mol) sequentially, and stir at 25 °C until the solution is clear. Add ammonium methanesulfonate (1.0 g, 0.008124 mol) and slowly heat to 60–65 °C. Slowly bubble ammonia gas (5.5 g, 0.3250 mol) into the reaction flask for approximately 6 hours.

[0048] The reaction was continued at 60-65℃ for 26 hours, and samples were taken for HPLC analysis, showing a raw material conversion rate of 99.6%. The reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure at 50-55℃ and 20-50 Pa. Methanol (100g) was added, and the mixture was heated under reflux for 6 hours to decompose the silicon-based byproducts. The solution was then concentrated to dryness again under reduced pressure. The residue was dissolved in dichloromethane (200mL), washed with 5% sodium bicarbonate solution (100mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 46.8g of crude 5'-O-DMTr-2'-deoxy-2'-fluorocytidine, with a yield of 93.8%.

[0049] The crude product was dissolved in dichloromethane (100 mL), and 5% trifluoroacetic acid dichloromethane solution (50 mL) was added. The mixture was stirred at 25 °C for 1 hour to remove the DMTr protecting group. After the reaction was complete, the solution was neutralized to pH 7 with saturated sodium bicarbonate solution. The mixture was separated, and the organic phase was concentrated to dryness. Isopropanol (80 g) was added and refluxed to dissolve the product. The mixture was cooled to 0–5 °C for 12 hours to crystallize. After filtration and drying, 14.9 g of 2'-deoxy-2'-fluorocytidine was obtained, with a total yield of 74.8% and an HPLC purity of 99.45%.

[0050] Comparative Example 1: Preparation of 2'-deoxy-2'-fluorocytidine via the existing chlorination-ammonolysis route Hydroxyl protection: 2'-deoxy-2'-fluorouridine (20.0 g, 0.08124 mol) was dissolved in pyridine (100 mL), and benzoyl chloride (22.7 g, 0.1625 mol) was added dropwise at 0 °C. The reaction was carried out for 6 hours, and the result was post-processed to obtain 33.2 g of 3',5'-di-O-benzoyl-2'-deoxy-2'-fluorouridine, with a yield of 92.3%.

[0051] Chlorination reaction: The above product was dissolved in phosphorus oxychloride (50 mL), N,N-dimethylaniline (10 mL) was added, and the reaction was carried out at 80 °C for 6 hours. After post-treatment, 31.5 g of 3',5'-di-O-benzoyl-4-chloro-2'-deoxy-2'-fluorouridine was obtained, with a yield of 91.2%.

[0052] High-pressure ammonolysis: The above product was added to a high-pressure reactor, ammonia gas was introduced to 0.5 MPa, and the reaction was carried out at 50 °C for 12 hours. After post-treatment, 28.7 g of 3',5'-di-O-benzoyl-2'-deoxy-2'-fluorocytidine was obtained, with a yield of 95.8%.

[0053] Deprotection: The above product was dissolved in sodium methoxide methanol solution and reacted at 25°C for 4 hours. After neutralization, concentration, and isopropanol crystallization, 10.2 g of product was obtained, with a total yield of 51.2%, HPLC purity of 98.7%, and a single maximum impurity of 0.52%.

[0054] Comparative Example 2: Preparation of 2'-deoxy-2'-fluorocytidine via existing enolization-substitution route Hydroxyl protection: 2'-deoxy-2'-fluorouridine (20.0 g, 0.08124 mol) was dissolved in DMF (100 mL), and imidazole (16.6 g) and tert-butyldimethylchlorosilane (24.5 g) were added. The mixture was reacted at 25 °C for 12 hours. Post-treatment yielded 36.8 g of 3',5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluorouridine, with a yield of 94.5%.

[0055] Enolization-sulfonation: The above product was dissolved in dichloromethane (150 mL), triethylamine (15.5 g) was added, and 2,4,6-triisopropylbenzenesulfonyl chloride (23.2 g) was added dropwise at 0 °C. The reaction was carried out for 6 hours, and the 4-sulfonyloxy intermediate was obtained by post-treatment with a yield of 90.1%.

[0056] Ammonolysis reaction: The above product was dissolved in a dioxane solution of ammonia and reacted at 25°C for 24 hours. After post-treatment, 33.6 g of 3',5'-di-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluorocytidine was obtained, with a yield of 92.3%.

[0057] Deprotection: The above product was dissolved in THF, tetrabutylammonium fluoride was added, and the reaction was carried out at 25°C for 4 hours. The product was concentrated and crystallized with isopropanol to obtain 9.8 g of product, with a total yield of 49.2%, HPLC purity of 98.5%, and a single maximum impurity of 0.61%.

[0058] Data Analysis: Data from Example 3 shows that when ammonium trifluoroacetate is used as a catalyst, both the feed conversion rate and product yield are lower than those of ammonium methanesulfonate. This is because ammonium methanesulfonate has moderate acidity, which can effectively protonate and activate the carbonyl group at the C4 position without causing ring-opening of the pyrimidine ring or side reactions of the hydroxyl group due to excessive acidity, thus verifying the rationality of using ammonium methanesulfonate as the preferred catalyst in claim 2.

[0059] Data from Example 4 shows that when a tetrahydrofuran solution of ammonia is used alone as the ammonia source, the product yield is 2.42% lower than that of the combination of ammonia and hexamethyldisilamine. This indicates that HMDS mainly acts as a dehydrating agent to absorb the water generated in the reaction, promoting a positive shift in equilibrium, while ammonia is the main ammonifying agent. The synergistic effect of the two achieves the best reaction effect, corresponding to the technical feature of optimized ammonia source in the beneficial effect.

[0060] As can be seen from the data in Example 5, high yields and purity can also be obtained when 2-methyltetrahydrofuran is used as a solvent, proving that the solvent selection range of the present invention is wide and can be flexibly adjusted according to actual production, further enhancing the industrial applicability of the process.

[0061] This invention is superior to existing uracil nucleoside conversion routes in terms of reaction steps, yield, purity, waste generation, and production cost. More importantly, this invention eliminates the need to protect hydroxyl groups for the amination reaction, enabling one-step synthesis of 2'-modified cytidine derivatives, thus overcoming the core deficiency of existing technologies (Comparative Examples 1-2) in their initio synthesis methods that cannot prepare modified intermediates.

[0062] Even when using pre-protected raw materials, this invention still offers significant advantages over existing technologies. In Example 6, when synthesizing 2'-deoxy-2'-fluorocytidine from 5'-O-DMTr-2'-deoxy-2'-fluorouridine, this invention requires only two steps: "ammoniation → deprotection," achieving a total yield of approximately 74.8%. In contrast, the existing chlorination-ammonolysis route requires four steps: "protection → chlorination → ammonolysis → deprotection," with a total yield of only about 42%. This is because the ammoniation reaction of this invention does not require the introduction of an additional protecting group, avoiding the yield loss and raw material waste caused by multiple protection-deprotection steps in existing technologies.

[0063] The above-disclosed embodiments are merely a few specific examples of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A method of synthesizing cytosine nucleosides and derivatives thereof, characterized by, No pre-protection of the 3'- and 5'-hydroxyl groups in the raw materials is required. The process includes the following steps: mixing the uracil nucleoside and its derivatives shown in Formula I, an ammonium salt catalyst, and a solvent, heating to 60-80°C under normal pressure, introducing an ammonia source to carry out a direct amination reaction of the carbonyl group at the C4 position, and obtaining the cytosine nucleoside and its derivatives shown in Formula II after post-treatment after the reaction is completed. ； R1 is selected from one of -H, -OH, -OMe, -F, and -MOE; R2 and R3 are each independently selected from one of -H, -TES, -TBDMS, -BOC, -TBDPS, -Bn, -Ac, -Bz, and -DMTr.

2. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The ammonium salt catalyst is selected from one or more of ammonium trifluoroacetate, ammonium trifluoromethanesulfonate, and ammonium methanesulfonate.

3. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The amount of the ammonium salt catalyst used is 0.005 to 0.05 times the molar amount of the compound of Formula I.

4. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The ammonia source is a combination of ammonia and hexamethyldisilazane, wherein the amount of ammonia is 2.0 to 3.0 times the molar amount of the compound of Formula I, and the amount of hexamethyldisilazane is 1.5 to 2.5 times the molar amount of the compound of Formula I.

5. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The ammonia source is selected from ammonia gas, a tetrahydrofuran solution of ammonia, or a 2-methyltetrahydrofuran solution of ammonia, and the amount used is 3.0 to 4.0 times the molar amount of the compound of Formula I.

6. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The solvent is selected from one or more of toluene, tetrahydrofuran, 2-methyltetrahydrofuran, and N,N-dimethylformamide.

7. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The mass of the solvent is 4 to 10 times the mass of the compound of Formula I.

8. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The amination reaction is carried out at a temperature of 60-65°C for 24-30 hours.

9. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, The post-processing steps include: concentrating the reaction solution under reduced pressure to dryness at 50-55℃ and 20-50Pa, adding methanol and refluxing for 5-8 hours, concentrating again under reduced pressure to dryness, then crystallizing with isopropanol, filtering and drying to obtain compound II.

10. The method for synthesizing cytosine nucleosides and their derivatives according to claim 1, characterized in that, When R2 and / or R3 are not -H, a deprotection step is included after the amination reaction is completed; the deprotection method is one of acid hydrolysis, alkaline hydrolysis or fluoride ion hydrolysis, depending on the type of protecting group.

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