Synthesis method of cytidine analogue
By simplifying the synthesis route of cytidine analogs, controlling the reaction conditions and selecting suitable solvents and catalysts, the problems of low yield and difficulty in improving purity in the existing technology are solved, and the synthesis of cytidine analogs with high yield and high purity is achieved.
Patent Information
- Application Number
- CN202511090311.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-05
- Publication Date
- 2025-10-17
AI Technical Summary
Existing methods for synthesizing cytidine analogs suffer from problems such as numerous reaction steps, low yield, many side reactions, high excipient costs, and difficulty in improving purity.
A simplified synthetic route is adopted, reaction conditions are controlled, suitable solvents and catalysts are selected, reaction steps and post-processing steps are reduced, and non-explosive compounds are used as auxiliary materials to improve product yield and purity.
It significantly improved the product yield and purity of cytidine analogs, reduced side reactions, and lowered excipient costs.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of cytidine analog synthesis, and particularly relates to a synthesis method of cytidine analog. BACKGROUND
[0002] Cytidine analogs are a class of compounds designed by chemically modifying the structure of natural cytidine. They have diverse mechanisms of action, interfere with DNA / RNA synthesis or related metabolic processes by mimicking the structure of natural cytidine, and are widely used in the fields of antiviral, antitumor and epigenetic regulation. In the field of genetic regulation, as DNA methyltransferase inhibitors (such as decitabine and azacitidine), they can reverse the hypermethylation silencing state of tumor suppressor genes, become key tools for studying epigenetic regulation, and reveal the dynamic correlation between DNA methylation and gene expression by depleting DNMT enzyme activity at low doses. They induce tumor cell redifferentiation and are used to construct in vitro research models of diseases such as myelodysplastic syndrome (MDS). In the field of antitumor mechanisms and drug development, cytidine analogs (such as azacitidine and cytarabine) competitively incorporate DNA or RNA by mimicking the structure of natural cytidine, block nucleic acid chain extension, and interfere with tumor cell replication. Low doses of certain cytidine analogs can selectively inhibit DNA methyltransferase (DNMT), reverse the abnormally high methylation of tumor suppressor genes, and restore the expression of tumor suppressor genes (such as the treatment of MDS and AML). In today's society, small nucleic acid drugs have developed rapidly due to their unique molecular structure and therapeutic mechanism. Among them, cytidine analogs, as key representatives of synthetic small nucleic acid drugs, play a crucial role in the construction of small nucleic acid drugs. Cytidine analogs can be divided into base modification type and ribose modification type according to their structure, and we mainly introduce the synthesis method of ribose modification type cytidine here.
[0003] The existing synthesis route is as follows:
[0004] Z=CH3,H;X=H,F,OCH3,OMOE ;R=H, NO2; Reference: Helvetica Chimica Acta, 1997, vol. 80, # 6, p. 1952 - 1971 Nucleosides and Nucleotides, 1997, vol. 16, # 1-2, p. 53 - 65
[0005] Z=CH3,H;X=H,F,OCH3,OMOE ;R=H, NO2; The prior art has long reaction time, many intermediates, low overall yield and low efficiency. The auxiliary material triazole compound is an explosive compound and has high cost. The overall reaction has many by-products, impurities are difficult to remove, and the purity cannot be improved. SUMMARY
[0006] In view of the above technical problems, the present application provides a synthesis method of a cytidine analogue, aiming to reduce reaction steps and post-processing steps, improve overall yield of products, reduce auxiliary material cost, reduce side reactions, and improve product purity.
[0007] To solve the above technical problems, the present application adopts the following technical solutions: the present application provides a synthesis method of a cytidine analogue, comprising: The present application has the advantages that the present application reduces reaction steps and post-processing steps, has less side reactions, and has high purity of the obtained product, greatly improving the yield. DETAILED DESCRIPTION
[0008] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.
[0009] The abbreviations and key terms involved in the present embodiment are defined as follows: DMF: N,N-dimethylformamide; DIPEA: N,N-diisopropyl ethylamine; TMSCl: trimethylchlorosilane; TEA: triethylamine.
[0010] Embodiment 1 A synthesis method of a cytidine analogue comprises the following steps:
[0011] Z= H, X= F; A1, the raw material (50g, 1.0eq) is dissolved in 10v anhydrous dichloromethane, TEA (2.5eq) and acetic anhydride (2.5eq) are added to the reaction solution after nitrogen replacement for three times, the mixture is reacted at 25℃ for 16h, TLC point plate reaction is complete; the reaction solution is poured into water, then extracted with ethyl acetate, the organic phase is separated and dried with anhydrous sodium sulfate, filtered and concentrated to obtain the intermediate as shown in formula 1, the molar yield is 100%, and the purity is >95%.
[0012] A2, the intermediate as shown in formula 1 is dissolved in 10v anhydrous dichloromethane, after nitrogen replacement for three times, DMF (1.0 eq) and TEA (2 eq) are added to the reaction solution, and the temperature is controlled at 5°C to drop phosphorus oxychloride (1.5 eq), 30 min drop is completed, the temperature is controlled at 35°C for 1h; reaction is finished, the reaction solution is reduced to oil, 10v methanol and 2v ammonia water are added, 15°C reaction is carried out for 24h; reaction is finished, the reaction solution is reduced to oil, 5v methanol is added, and the temperature is increased to 40°C-60°C to dissolve, cooling crystallization, suction filtration is carried out to obtain the intermediate as shown in formula 2, the molar yield is 90%, and the purity is >98%. Example
[0013] A method for synthesizing a cytidine analogue, comprising the following steps:
[0014] Z=CH3 A1, the raw material (50g, 1.0eq) is dissolved in 6v anhydrous DMF, after nitrogen replacement for three times, pyridine (3.0eq) and acetic anhydride (3.0eq) are added to the reaction solution, the mixture is reacted at 40°C for 12h, TLC point plate reaction is completed, the reaction solution is poured into water, then extracted with ethyl acetate, the organic phase is separated, and then washed with water once. The organic phase is dried with anhydrous sodium sulfate, suction filtration, and the filtrate is concentrated to obtain the intermediate as shown in formula 1, the molar yield is 100%, and the purity is >95%.
[0015] A2, the intermediate as shown in formula 1 is dissolved in 15v anhydrous dioxane, after nitrogen replacement for three times, DMF (0.1 eq) and DIPEA (6eq) are added to the reaction solution, and the temperature is controlled at 5°C to drop thionyl chloride (4eq), 30 min drop is completed, the temperature is controlled at 70°C for 1h; reaction is finished, the reaction solution is reduced to oil, 15v methanol and 5v ammonia water are added, 50°C reaction is carried out for 12h; reaction is finished, the reaction solution is reduced to oil, 5v methanol is added, and the temperature is increased to 40°C-60°C to dissolve, cooling crystallization, suction filtration is carried out to obtain the intermediate as shown in formula 2, the molar yield is 85%, and the purity is >98%. Example
[0016] A method for synthesizing a cytidine analogue, comprising the following steps:
[0017] Z=H A1, the raw material (50 g, 1.0 eq) was dissolved in 12 v of anhydrous dichloromethane, and after three times of nitrogen replacement, DIPEA (3.0 eq) and acetic anhydride (3.0 eq) were added to the reaction solution, and the mixture was reacted at 20°C for 24 h. After TLC point plate reaction was completed, the reaction solution was poured into water, and then extracted with ethyl acetate. The organic phase was separated and washed once with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain an intermediate shown in formula 1, with a molar yield of 100% and a purity of >95%.
[0018] A2, the intermediate shown in formula 1 was dissolved in 5 v of anhydrous 1,2-dichloroethane, and after three times of nitrogen replacement, DMF (3 eq) and DBU (4 eq) were added to the reaction solution. Sulfurous chloride (3 eq) was added dropwise at 5°C, and the reaction was completed after 30 min. The reaction solution was concentrated under reduced pressure to an oil, 10 v of isopropyl alcohol and 2 v of ammonia water were added, and the reaction was carried out at 20°C for 24 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure to an oil, 5 v of isopropyl alcohol was added, and the solution was dissolved by heating to 40°C-60°C. After cooling and crystallization, an intermediate shown in formula 2 was obtained by filtration, with a molar yield of 90% and a purity of >98%. Example
[0019] A method for synthesizing a cytidine analogue, comprising the following steps:
[0020] Z = CH3 A1, the raw material (50 g, 1.0 eq) was dissolved in 12 v of anhydrous dichloromethane, and after three times of nitrogen replacement, DIPEA (3.0 eq) and acetic anhydride (3.0 eq) were added to the reaction solution, and the mixture was reacted at 20°C for 24 h. After TLC point plate reaction was completed, the reaction solution was poured into water, and then extracted with ethyl acetate. The organic phase was separated and washed once with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain an intermediate shown in formula 1, with a molar yield of 100% and a purity of >95%.
[0021] A2, the intermediate shown in formula 1 was dissolved in 5 v of anhydrous 1,2-dichloroethane, and after three times of nitrogen replacement, DMF (3 eq) and DBU (4 eq) were added to the reaction solution. Sulfurous chloride (3 eq) was added dropwise at 5°C, and the reaction was completed after 30 min. The reaction solution was concentrated under reduced pressure to an oil, 10 v of isopropyl alcohol and 2 v of ammonia water were added, and the reaction was carried out at 20°C for 24 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure to an oil, 5 v of isopropyl alcohol was added, and the solution was dissolved by heating to 40°C-60°C. After cooling and crystallization, an intermediate shown in formula 2 was obtained by filtration, with a molar yield of 90% and a purity of >98%. Example
[0022] A process for the synthesis of a cytidine analogue, comprising the steps of:
[0023] Z = H; A1, dissolving the raw material (50 g, 1.0 eq) in 10 v of anhydrous dichloromethane, replacing with nitrogen for three times, then adding pyridine (2.5 eq) and acetic anhydride (2.5 eq) into the reaction solution, reacting the mixture at 40 °C for 12 h, TLC spotting plate reaction is complete, pouring the reaction solution into water, then extracting with ethyl acetate, separating the organic phase, washing once with water, drying the organic phase with anhydrous sodium sulfate, suction filtration, and concentrating the filtrate to obtain the intermediate shown as Formula 1, molar yield: 100%, purity > 95%.
[0024] A2, dissolving the intermediate shown as Formula 1 in 15 v of anhydrous acetonitrile, replacing with nitrogen for three times, then adding DMF (2.0 eq) and TEA (4 eq) into the reaction solution, adding phosphorus oxychloride (3 eq) dropwise while controlling the temperature at 5 °C, dropwise adding for 30 min, and reacting at 50 °C for 2 h; after the reaction is completed, reducing pressure to remove the solvent to obtain an oil, adding 10 v of ethanol and 3 v of ammonia water, reacting at 25 °C for 24 h; after the reaction is completed, reducing pressure to remove the solvent to obtain an oil, adding 5 v of ethanol, dissolving at 40 °C-60 °C, cooling to crystallize, and suction filtering to obtain the intermediate shown as Formula 2, molar yield: 90%, purity > 98%. Example
[0025] A process for the synthesis of a cytidine analogue, comprising the steps of:
[0026] Z = CH3, X = OMOE 5' position is DMTr protected, then TMS transient protection is required.
[0027] B1, dissolving the raw material (50 g, 1.0 eq) in 10 v of anhydrous dichloromethane, replacing with nitrogen for three times, then adding TEA (5.0 eq) and trimethylsilyl chloride (3.0 eq) dropwise into the reaction solution, reacting the mixture at 10 °C for 3 h, TLC spotting plate reaction is complete, pouring the reaction solution into water, then extracting with ethyl acetate, separating the organic phase, washing once with water, drying the organic phase with anhydrous sodium sulfate, suction filtering, and concentrating the filtrate to obtain the intermediate shown as Formula 1, molar yield: 99%, purity > 95%.
[0028] B2, the intermediate as shown in formula 1 is dissolved in 10v anhydrous dichloromethane, after nitrogen replacement for three times, DMF (1.0 eq) and TEA (4 eq) are added to the reaction solution, and temperature control is performed at 5°C, and phosphorus oxychloride (3 eq) is added dropwise, 30 min dropwise completion, temperature control at 10°C reaction 4h; reaction is completed, the reaction solution is reduced to oil, 10v methanol and 3v ammonia water are added at 25°C for 24h; reaction is completed, the reaction solution is reduced to oil, 5v methanol is added to dissolve at 40°C-60°C, cooling crystallization, suction filtration to obtain the intermediate as shown in formula 2, the molar yield is 85%, and the purity is >98%. Example
[0029] A synthesis method of a cytidine analogue, comprising the following steps:
[0030] Z=CH3; B1, the raw material (50g, 1.0eq) is dissolved in 15v anhydrous DMF, after nitrogen replacement for three times, TEA (4.0eq) and trimethylchlorosilane (2.0eq) are added dropwise to the reaction solution, the mixture is reacted at 0°C for 5h, TLC point plate reaction is completed, the reaction solution is poured into water, then extracted with ethyl acetate, the organic phase is separated, washed with water once, the organic phase is dried with anhydrous sodium sulfate, suction filtered, and the filtrate is concentrated to obtain the intermediate as shown in formula 1, the molar yield is 99%, and the purity is >95%.
[0031] B2, the intermediate as shown in formula 1 is dissolved in 15v anhydrous acetonitrile, after nitrogen replacement for three times, DMF (5.0 eq) and DIPEA (4.0 eq) are added to the reaction solution, and temperature control is performed at 0°C, and phosphorus oxychloride (3.0 eq) is added dropwise, 30 min dropwise completion, temperature control at 0°C reaction 5h; reaction is completed, the reaction solution is reduced to oil, 10v methanol and 3v ammonia water are added at 25°C for 24h; reaction is completed, the reaction solution is reduced to oil, 5v methanol is added to dissolve at 40°C-60°C, cooling crystallization, suction filtration to obtain the intermediate as shown in formula 2, the molar yield is 85%, and the purity is >98%. Example
[0032] A synthesis method of a cytidine analogue, comprising the following steps:
[0033] Z=H; B1, the raw material (50 g, 1.0 eq) is dissolved in 10v anhydrous dichloromethane, and TEA (3.0 eq) and trimethylchlorosilane (1.5 eq) are added dropwise into the reaction solution after being replaced by nitrogen for three times, and the mixture is reacted at 5℃ for 5h, TLC point plate reaction is complete, the reaction solution is poured into water, then extracted with ethyl acetate, the organic phase is separated, washed with water once, the organic phase is dried with anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain an intermediate shown as formula 1, the molar yield is 98%, and the purity is >95%.
[0034] B2, the intermediate shown as formula 1 is dissolved in 10v anhydrous 1,2-dichloroethane, and DMF (0.5 eq) and DBU (4 eq) are added into the reaction solution after being replaced by nitrogen for three times, and the temperature is controlled at 5℃, and thionyl chloride (3 eq) is added dropwise, and the dropwise addition is completed after 30 min, and the reaction is carried out at 5℃ for 5h; after the reaction is completed, the reaction solution is reduced to an oil, 10v ethanol and 5v ammonia water are added, and the reaction is carried out at 35℃ for 12h; after the reaction is completed, the reaction solution is reduced to an oil, 5v ethanol is added, and the temperature is raised to 40℃-60℃ to dissolve, and the product is cooled and crystallized, and filtered to obtain an intermediate shown as formula 2, the molar yield is 85%, and the purity is >98%. Example
[0035] A method for synthesizing a cytidine analogue, comprising the following steps:
[0036] Z=H B1, the raw material (50 g, 1.0 eq) is dissolved in 10v anhydrous DMF, and TEA (4.0 eq) and trimethylchlorosilane (2.0 eq) are added dropwise into the reaction solution after being replaced by nitrogen for three times, and the mixture is reacted at 10℃ for 3h, TLC point plate reaction is complete, the reaction solution is poured into water, then extracted with ethyl acetate, the organic phase is separated, washed with water once, the organic phase is dried with anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain an intermediate shown as formula 1, the molar yield is 98%, and the purity is >95%.
[0037] B2, the intermediate shown as formula 1 is dissolved in 10v anhydrous dioxane, and DMF (0.5 eq) and DCBAO (4 eq) are added into the reaction solution after being replaced by nitrogen for three times, and the temperature is controlled at 5℃, and phosphorus oxychloride (3 eq) is added dropwise, and the dropwise addition is completed after 30 min, and the reaction is carried out at 5℃ for 5h; after the reaction is completed, the reaction solution is reduced to an oil, 10v ethanol and 3v ammonia water are added, and the reaction is carried out at 25℃ for 16h; after the reaction is completed, the reaction solution is reduced to an oil, 5v ethanol is added, and the temperature is raised to 40℃-60℃ to dissolve, and the product is cooled and crystallized, and filtered to obtain an intermediate shown as formula 2, the molar yield is 85%, and the purity is >98%. Example
[0038] A method for synthesizing a cytidine analogue comprises the following steps:
[0039] Z=H; B1. Dissolve the raw material (50 g, 1.0 eq) in 10 v of anhydrous dichloromethane. After nitrogen replacement three times, TEA (4.0 eq) and trimethylsilyl chloride (2.0 eq) were added dropwise to the reaction solution. The mixture was reacted at 10°C for 3 h. The reaction was complete on TLC. The reaction solution was poured into water and then extracted with ethyl acetate. The organic phase was separated and washed once with water. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to obtain the intermediate shown in Formula 1 with a molar yield of 98% and a purity of >95%.
[0040] B2. The intermediate shown in Formula 1 was dissolved in 10 v of anhydrous chloroform. After nitrogen replacement three times, DMF (0.5 eq) and TEA (4 eq) were added to the reaction solution. Thionyl chloride (3 eq) was added dropwise at 5°C for 30 minutes, and the temperature was controlled at 5°C for 5 hours. After the reaction was completed, the reaction solution was decompressed and desolvated to an oily substance. 10 v of ethanol and 3 v of ammonia water were added and reacted at 25°C for 16 hours. After the reaction was completed, the reaction solution was decompressed and desolvated to an oily substance. 5 v of ethanol was added and the temperature was raised to 40°C-60°C for dissolution. The mixture was cooled and crystallized. The intermediate shown in Formula 2 was obtained by filtration with a molar yield of 85% and a purity of >98%.
[0041] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A method for synthesizing a cytidine analog, characterized in that: The following steps are involved: ； Wherein, Z = CH3 or H; X = H, F, OCH3 or OMOE; A1. Dissolve the raw materials in a first organic solvent, replace the solvent with nitrogen three times, and then add a base and acetic anhydride to the reaction solution to obtain a mixture; control the temperature of the mixture to react, and the reaction is complete on a TLC plate. Pour the reaction solution into water, and then extract with ethyl acetate. Separate the organic phase, dry it with anhydrous sodium sulfate, filter it with suction, and concentrate the filtrate to obtain the intermediate shown in Formula 1; A2. Dissolve the intermediate shown in Formula 1 in a second organic solvent, replace the atmosphere with nitrogen three times, add DMF and a base to the reaction solution, add the chlorination reagent dropwise under temperature control, and control the temperature to react after the addition is complete. After the reaction is completed, decompress the reaction solution to desolventize to an oily substance, add an ammonolysis reaction solvent and aqueous ammonia to control the temperature to react. After the reaction is completed, decompress the reaction solution to desolventize to an oily substance, add an ammonolysis reaction solvent, heat and dissolve, cool and crystallize, and filter to obtain the intermediate shown in Formula 2.
2. The method for synthesizing a cytidine analog according to claim 1, wherein The following steps are also included: ； Wherein, Z = CH3 or H; X = H, F, OCH3 or OMOE; B1. Dissolve the raw materials in a first organic solvent, replace the atmosphere with nitrogen three times, and then add TEA and trimethylsilyl chloride to the reaction solution to obtain a mixture; control the temperature of the mixture to react, and the reaction is complete when the TLC plate is spotted. Pour the reaction solution into water, and then extract with ethyl acetate. Separate the obtained organic phase, dry it with anhydrous sodium sulfate, filter it, and concentrate the filtrate to obtain the intermediate shown in Formula 1; B2. Dissolve the intermediate shown in Formula 1 in a second organic solvent, replace the nitrogen three times, add DMF and alkali to the reaction solution, control the temperature and add the chlorination reagent dropwise. After the addition is completed, control the temperature to react. After the reaction is completed, reduce the pressure on the reaction solution to desolventize to an oily substance, add an ammonolysis reaction solvent and ammonia water to control the temperature to react. After the reaction is completed, reduce the pressure on the reaction solution to desolventize to an oily substance, add an ammonolysis reaction solvent, raise the temperature to dissolve, cool and crystallize, and filter to obtain the intermediate shown in Formula 2.
3. The method for synthesizing a cytidine analog according to any one of claims 1 or 2, wherein: The first organic solvent is any one of dichloromethane and DMF; the volume of the first organic solvent is 6v~12v.
4. The method for synthesizing a cytidine analog according to any one of claims 1 or 2, wherein: The base in step A1 is any one of TEA, pyridine, and DIPEA, and the base equivalent in step A1 is 2.5eq~3.0eq; The base in step B1 is TEA, and the base equivalent in step B1 is 3.0eq-6.0eq.
5. The method for synthesizing a cytidine analog according to claim 1, wherein The acetic anhydride equivalent is 2.5eq~3.0eq.
6. The method for synthesizing a cytidine analog according to any one of claims 1 or 2, wherein: The second organic solvent is any one of dichloromethane, dioxane, 1,2-dichloroethane, chloroform, and acetonitrile; and the volume of the second organic solvent is 5v~15v.
7. The method for synthesizing a cytidine analog according to any one of claims 1 or 2, wherein: The DMF equivalent is 0.1eq~5.0eq.
8. The method for synthesizing a cytidine analog according to any one of claims 1 or 2, wherein: The base in step A2 and step B2 is any one of TEA, DIPEA, DBU, and DABCO, and the base equivalent is 2eq~6eq; The equivalent of the chlorination reagent is 1.5eq~4.0eq; Here, the equivalent of the base exceeds the equivalent of the chlorinating agent.
9. The method for synthesizing a cytidine analog according to any one of claims 1 or 2, wherein: The chlorination agent is any one of thionyl chloride and phosphorus oxychloride; The aminolysis reaction solvent is any one of methanol, ethanol and isopropanol.
10. The method for synthesizing a cytidine analog according to claim 2, wherein: The trimethylchlorosilane equivalent is 1.5eq~3.0eq.