Preparation method and application of cytidine vinyl phosphate compound

Abstract

The invention relates to a divisional application of which the application number is 202411350349.8. The invention relates to a preparation method of a cytidine vinyl phosphate compound, which comprises the step of contacting a compound shown in a formula (I) with an ammonia source compound in an organic solvent under an ammonolysis reaction condition, in the compound as shown in the formula (I), R1, R2, R5, R6, R8 and R9 are respectively and independently hydrogen, methyl or ethyl; r3 and R4 are respectively and independently one of hydrogen, halogen, hydroxyl, protected hydroxyl, alkyl with 1 to 3 carbon atoms, alkoxy with 1 to 3 carbon atoms, substituted alkyl with 1 to 3 carbon atoms and substituted alkoxy with 1 to 3 carbon atoms; each R7 is independently one of a hydroxyl group, a protected hydroxyl group, a C1-C3 alkyl group, a C1-C3 alkoxy group, a substituted C1-C3 alkyl group, and a substituted C1-C3 alkoxy group; and R10 is hydrogen or methyl. The invention also relates to application of the cytidine vinyl phosphate compound prepared by the method.

Description

[0001] This application is a divisional application. The parent application's application number is 202411350349.8, the application date is September 26, 2024, and the invention title is "Preparation method of cytidine vinyl phosphate compound and its application". Technical Field

[0002] This disclosure relates to a method for preparing a cytidine vinyl phosphate compound. This disclosure also relates to the method and the use of the cytidine vinyl phosphate compound obtained therefrom. Background Technology

[0003] In recent years, small nucleic acid drugs have become a research hotspot in the biopharmaceutical field due to their advantages such as high specificity, simple design, short development cycle, and abundant targets. 5'-vinyl phosphate-terminated oligonucleotides have shown increasing attention in the field due to their beneficial effects, including stability against exonucleases, significantly increased oligonucleotide pharmacodynamic activity, and / or long-lasting effects. Vinyl phosphate phosphoramide nucleoside monomers are important intermediates commonly used in solid-phase nucleic acid synthesis methods for introducing vinyl phosphates at the 5'-end of oligonucleotides. Among existing technologies, uridine / thymidine vinyl phosphates are the most widely used due to their ease of synthesis, fewer byproducts, and lower cost. However, the application of nucleoside vinyl phosphates based on other bases, such as cytidine vinyl phosphate, in small nucleic acid drugs is also gradually being developed.

[0004] The preparation of nucleoside vinyl phosphates is typically carried out via the phosphorus ylide reaction. This reaction requires relatively harsh reaction conditions, and the starting compounds usually use nucleoside monomers with different bases. Non-patent literature (Hironori Komatsu, Kunihiko Morizane, Toshiyuki Kohno, etc., An Efficient Amination Method for Manufacturing Cytidines, Organic Process Research & Development 2004, 8, 564-567) reports a novel amination method for uracil derivatives and its application in the synthesis of cytosine. This literature reports an innovative amination synthesis method for uracil derivatives involving an activation step at the C4 position of the uracil base using 1-methylpiperidine, thereby achieving the large-scale synthesis of 2'-deoxycytosine. However, this literature does not address uracil derivatives with a vinyl phosphate group at the 5' position, which may be a limitation in certain chemical synthesis and biochemical applications. Summary of the Invention

[0005] The first aspect of this disclosure provides a method for preparing a cytidine vinyl phosphate compound, the method comprising contacting a compound of formula (I) with an ammonia source compound in an organic solvent under ammonolysis reaction conditions:

[0006]

[0007] Among them, R1, R2, R5, R6, R8 and R9 are each independently hydrogen, methyl or ethyl;

[0008] R3 and R4 are each independently one of hydrogen, halogen, hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, or substituted C1-C3 alkoxy.

[0009] Each R7 is independently one of hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, or substituted C1-C3 alkoxy.

[0010] R 10 It can be hydrogen or methyl.

[0011] A second aspect of this disclosure provides a method for preparing an amino-protected cytidine vinyl phosphate compound, the method comprising contacting the cytidine vinyl phosphate compound with an amino-protecting agent in an aprotic organic solvent under acylation reaction conditions, wherein the cytidine vinyl phosphate compound is prepared by the method provided in this disclosure described above.

[0012] The third aspect of this disclosure provides a method for preparing a hydroxyl-containing amino-protected cytosine vinyl phosphate compound, the method comprising contacting the amino-protected cytosine vinyl phosphate compound with a deprotecting agent in an aprotic organic solvent under deprotection reaction conditions.

[0013] The fourth aspect of this disclosure provides a method for preparing an amino-protected cytidine vinyl phosphate phosphoramide compound, the method comprising contacting the hydroxyl-containing amino-protected cytidine vinyl phosphate compound with a phosphorylating agent in the presence of a catalyst under phosphorylation reaction conditions in an aprotic organic solvent.

[0014] Beneficial effects

[0015] The inventors discovered in their experiments that it is difficult to obtain cytidine vinyl phosphate compounds using the phosphorus ylide reaction process commonly used in the field. In contrast, the method disclosed in this paper avoids the side reaction of deprotection of amino protecting groups that may occur during the direct synthesis of VPCm, and can rapidly and efficiently synthesize the target cytidine vinyl phosphate compound, making it suitable for industrial production.

[0016] Incorporate by reference

[0017] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually incorporated herein by reference. Detailed Implementation

[0018] In one aspect, this disclosure provides a method for preparing a cytidine vinyl phosphate compound, the method comprising contacting a compound of formula (I) with an ammonia source compound in an organic solvent under ammonolysis reaction conditions:

[0019]

[0020] Among them, R1, R2, R5, R6, R8 and R9 are each independently hydrogen, methyl or ethyl;

[0021] R3 and R4 are each independently one of hydrogen, halogen, hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, or substituted C1-C3 alkoxy.

[0022] Each R7 is independently one of hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, or substituted C1-C3 alkoxy.

[0023] R 10 It can be hydrogen or methyl.

[0024] In some implementations, R1, R2, R5, R6, R8, and R9 are each independently hydrogen;

[0025] R3 is a C1-C3 alkoxy group;

[0026] R4 is a protected hydroxyl group;

[0027] Each R7 is independently a C1-C3 alkoxy group;

[0028] R 10 It is hydrogen.

[0029] As used herein, “alkyl” refers to a straight-chain and branched saturated hydrocarbon group having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, or from 1 to 6 or 1 to 3 carbon atoms. For example, C1-C3 alkyl groups comprise straight-chain and branched alkyl groups with 1 to 3 carbon atoms. When referring to residues having a specific number of alkyl groups, it is intended to cover all branched and straight-chain forms having that number of carbon atoms; thus, for example, “propyl” includes n-propyl and isopropyl. Alkyl groups as used herein may optionally contain one or more further substituents.

[0030] As used herein, "alkoxy" refers to an alkyl group with a specified number of carbon atoms attached by an oxygen bridge, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentooxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms attached by an oxygen bridge. In some embodiments, the alkoxy group is methoxy, ethoxy, propoxy, or isopropoxy. Alkoxy groups as used herein may optionally contain one or more further substituents.

[0031] As used herein, the term "protecting group" refers to an unstable chemical moiety known in the art for protecting reactive groups (including, but not limited to, hydroxyl and amino groups) from undesirable reactions during synthesis. Protecting groups are typically used selectively and / orthogonally to protect certain sites during reactions at other reactive sites, and can then be removed to release the unprotected group as is or in a manner suitable for further reactions. Protecting groups known in the art are generally described in "Greene's Protective Groups in Organic Synthesis," 4th edition, John Wiley & Sons, New York, 2007.

[0032] As used herein, the term "protected hydroxyl group" refers to a 2'-hydroxyl functional group protected by a suitable protecting group, such as an acidically active acetal protecting group, particularly 1-(aryl)-4-alkoxypiperidin-4-yl, such as 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) or 1-(2-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep). Alternatively, the 2'-O hydroxyl group of the nucleoside intermediate can be a 2'-O-alkyl, 2'-O-CH2CN, 2'-O-alkoxyalkyl, or 2'-O-alkenyl derivative, typically a C1-C4 alkyl, C1-C4 alkoxyC1-C4 alkyl, or alkenyl derivative. In this case, no further protection at the 2' position is required. Alternatively, the nucleoside intermediate may also include nucleosides substituted with 2'-amino, 2'-alkyl, or 2'-carbon-alkenyl groups.

[0033]

[0034] In this disclosure, an amino-protected cytosine vinyl phosphate refers to a cytosine vinyl phosphate in which an amino protecting group is attached to the cytosine amino group. Examples of an "amino protecting group" include, but are not limited to, urethane protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups such as 2-nitrobenzenesulfonyl; and imine and cyclic imine protecting groups such as phthalimide and dithiasuccinoyl.

[0035] Those skilled in the art are familiar with methods for preparing compounds represented by formula (I), for example, methods for preparing various compounds represented by formula (I) are disclosed in Examples 13, 16, 21, 22, 26, and 35a of CN103154014B. In one embodiment, the compound represented by formula (I) is prepared according to the method of Preparation Examples 14-1 to 14-2 of CN110959011A.

[0036] In some embodiments, the organic solvent is an aprotic polar solvent, such as one or more nitrile or ether solvents. In some embodiments, the organic solvent is at least one of acetonitrile, tetrahydrofuran, or 1,4-dioxane.

[0037] In some embodiments, the amount of the organic solvent used is 1.5-5.5 L / mol, for example 2.0-5.0 L / mol, relative to the compound shown in formula (I).

[0038] In some embodiments, the ammonia source compound is selected from at least one of ammonia, urea, or ammonium salts. Considering the availability and cost of raw materials, in some embodiments, the source compound is ammonia. Ammonia can be provided in the form of gaseous ammonia or in the form of ammonia solution. The concentration of the ammonia solution can be 20-30 wt%. In some embodiments, the molar ratio of the ammonia source compound to the compound shown in formula (I) is 2.0-4.0:1, for example, 2.5-3.5:1.

[0039] Those skilled in the art are familiar with various ammonolysis reaction conditions. Any ammonolysis reaction condition that allows the compound of formula (I) to be contacted with an ammonia source compound to obtain the above-mentioned cytidine vinyl phosphate compound can be used in this disclosure. In some embodiments, the ammonolysis reaction is carried out in the presence of an activator. The presence of the activator can promote the activation of reactant molecules, thereby lowering the activation energy of the reaction and accelerating the reaction rate. In some embodiments, the molar ratio of the activator to the compound shown in formula (I) is 1:0.20-0.40. In some embodiments, the ammonolysis reaction conditions include one or more of the following:

[0040] The reaction temperature is: -10℃ to -10℃;

[0041] The reaction time is 5-20 hours.

[0042] The pH value of the reaction is 7.5-12.5;

[0043] The activator of the reaction is a sulfonyl halide and / or a nitrogen-containing heterocyclic compound;

[0044] The molar ratio of the activator to the compound shown in formula (Ⅰ) is 1:0.25-0.35.

[0045] In some embodiments, the ammonolysis reaction conditions include one or more of the following conditions:

[0046] The reaction temperature is: -5℃ to -5℃;

[0047] The reaction time is 9-15 hours.

[0048] The pH value of the reaction is 8-12;

[0049] The activator of the reaction is at least one of p-toluenesulfonyl chloride, N-methylpiperidine, or piperidine.

[0050] In some embodiments, the ammonolysis reaction is carried out in the presence of a basic compound selected from at least one of organic amines, multidentate chelating agents, or nitrogen-containing heterocyclic compounds. Considering cost and stability under basic conditions, in some embodiments, the basic compound is at least one of triethylamine, ethylenediaminetetraacetic acid, 2,6-methylpyridine, or pyridine.

[0051] In some embodiments, the weight ratio of the compound represented by formula (I) to the basic compound is 2.0-2.8:1. In some embodiments, the weight ratio of the compound represented by formula (I) to the basic compound is 2.2-2.6:1.

[0052] In some embodiments, the compound represented by formula (I) is the compound represented by formula (100):

[0053]

[0054] In some embodiments, the cytidine vinyl phosphate compound is a compound represented by formula (II):

[0055]

[0056] In some embodiments, the cytidine vinyl phosphate compound is a compound of formula (101):

[0057]

[0058] In some embodiments, the method further includes a separation and purification step. In some embodiments, the separation and purification step includes one or more of concentration, extraction, filtration, and separation. In some embodiments, the separation and purification step includes adding water and an extraction solvent to the reaction mixture for extraction; washing the extracted organic phase with a detergent; separating the aqueous and organic phases; adding a desiccant to the collected organic phase; filtering and concentrating to obtain a crude product; and further purifying the crude product to obtain a cytidine vinyl phosphate compound. In some embodiments, examples of extraction solvents include, but are not limited to, ethyl acetate. In some embodiments, examples of desiccant include, but are not limited to, anhydrous sodium sulfate solid. In some embodiments, examples of detergent include, but are not limited to, saturated sodium chloride solution. In some embodiments, the separation and purification is carried out in a chromatography column. In some embodiments, the stationary phase of the chromatography column can be silica gel. Examples of eluents used in the chromatography column include, but are not limited to, petroleum ether, ethyl acetate, and ethanol. In some embodiments, the eluent can be a mixed solvent of petroleum ether:ethyl acetate:ethanol, where the volume ratio of petroleum ether to ethyl acetate is 1:1-1:5; and the volume ratio of petroleum ether to ethanol is 1:0.1-1:0.3.

[0059] Application of the method disclosed herein

[0060] The methods disclosed herein and the cytidine vinyl phosphate compounds obtained therefrom can be used for a variety of purposes, such as the preparation of cytidine vinyl phosphate derivatives. In some embodiments, the cytidine vinyl phosphate compounds prepared by the methods of this disclosure can be used to prepare amino-protected cytidine vinyl phosphate phosphorimide monomers.

[0061] In another aspect, this disclosure also provides a method for preparing an amino-protected cytidine vinyl phosphate compound, the method comprising contacting the cytidine vinyl phosphate compound with an amino protecting agent in an aprotic organic solvent under acylation reaction conditions, wherein the cytidine vinyl phosphate compound is prepared by the method provided in this disclosure.

[0062] Those skilled in the art are familiar with various acylation reaction conditions. Any acylation reaction condition that enables the cytidine vinyl phosphate compound provided in this disclosure to contact with an amino protecting agent to obtain the compound containing the protected amino group provided in this disclosure can be used in this disclosure. In some embodiments, the acylation reaction conditions include: a reaction temperature of -10℃ to 35℃ and a reaction time of 5 to 20 hours; in other embodiments, the acylation reaction conditions include: a reaction temperature of 10℃ to 25℃ and a reaction time of 8 to 16 hours.

[0063] In some embodiments, the aprotic organic solvent is selected from amides, nitriles, esters, or ethers. Examples of aprotic organic solvents in some embodiments include, but are not limited to, N,N-dimethylformamide, acetonitrile, ethyl acetate, tetrahydrofuran, or dioxane.

[0064] Depending on the type of amino protecting agent used, the amino protecting group in the obtained amino-protected cytosine vinyl phosphate compound will vary. In some embodiments, examples of amino protecting agents include, but are not limited to, benzoic anhydride, acyl chloride, Boc-Cl, Fmoc-Cl, Cbz-Cl, and activated esters; in other embodiments, benzoic anhydride or acetyl chloride is used. In some embodiments, the molar ratio of the cytosine vinyl phosphate compound to the amino protecting agent can be 1-1.0-2.5. In some embodiments, the molar ratio of the cytosine vinyl phosphate compound to the amino protecting agent can be 1-1.2-2.0.

[0065] In some embodiments, the compound containing the protected amino group is a compound represented by formula (III):

[0066]

[0067] Among them, R 11 The amino protecting group is preferably selected from benzoyl, C1-C8 ketone carbonyl, C1-C8 alkyl, Boc, Cbz, Fmoc, and more preferably benzoyl or acetyl.

[0068] R4 is a protected hydroxyl group; preferably, R4 is tert-butyldimethylsilyl (TBDMS) or tert-butyldiphenylsilyl (TBDPS).

[0069] In some embodiments, the compound containing the protected amino group is a compound of formula (102):

[0070]

[0071] In another aspect, this disclosure also provides a method for preparing a hydroxyl-containing amino-protected cytosine vinyl phosphate compound, the method comprising contacting the compound containing the protected amino group with a deprotecting agent in an aprotic organic solvent under deprotection reaction conditions, wherein the amino-protected cytosine vinyl phosphate compound is prepared by the method provided by this disclosure, and the compound containing the protected amino group also contains a protected hydroxyl group.

[0072] Those skilled in the art are familiar with various deprotection reaction conditions. Any deprotection reaction condition that enables the compound containing the protected amino group provided in this disclosure to be contacted with the deprotecting agent to obtain the compound containing the hydroxyl group provided in this disclosure can be used in this disclosure. In some embodiments, the deprotection reaction conditions include: a reaction temperature of -10℃ to 35℃ and a reaction time of 5 to 20 hours; in some embodiments, the deprotection reaction conditions include: a reaction temperature of 10℃ to 25℃ and a reaction time of 8 to 16 hours.

[0073] In some embodiments, examples of deprotecting agents include, but are not limited to, various fluoride-containing compounds, such as triethylamine hydrofluoride and tetrabutylammonium fluoride. In some embodiments, the molar ratio of the compound containing the protected amino group to the deprotecting agent may be 1-4-6; in some embodiments, the molar ratio of the compound containing the protected amino group to the deprotecting agent may be 1-4.5-5.5.

[0074] In some embodiments, the hydroxyl-containing compound is a compound represented by formula (Ⅳ):

[0075]

[0076] In some embodiments, the hydroxyl-containing compound is a compound represented by formula (103):

[0077]

[0078] In another aspect, this disclosure also provides a method for preparing an amino-protected cytidine vinyl phosphate phosphoramide monomer, the method comprising contacting the above-mentioned hydroxyl-containing amino-protected cytidine vinyl phosphate compound with a phosphorylating agent in the presence of a catalyst under phosphorylation reaction conditions in an aprotic organic solvent.

[0079] Those skilled in the art are familiar with various phosphorylation reaction conditions. Any phosphorylation reaction condition that enables the amino-protected cytidine vinyl phosphate compound provided in this disclosure to be contacted with a phosphorylating agent to obtain an amino-protected cytidine vinyl phosphate phosphorimide monomer can be used in this disclosure. In some embodiments, the phosphorylation reaction conditions include: a reaction temperature of -10℃ to 35℃ and a reaction time of 5 to 20 hours; in other embodiments, the phosphorylation reaction conditions include: a reaction temperature of 10℃ to 25℃ and a reaction time of 8 to 16 hours.

[0080] In some embodiments, examples of phosphorylating agents include, but are not limited to, phosphorus reagents (bis(diisopropylamino)(2-cyanoethoxy)phosphine). In some embodiments, the molar ratio of the hydroxyl-containing amino-protected cytidine vinyl phosphate compound to the phosphorylating agent is 1:1-2. In some embodiments, the molar ratio of the compound containing the protected amino group to the phosphorylating agent is 1:1.2-1.5.

[0081] In some embodiments, examples of catalysts include, but are not limited to, 4,5-dicyanimidazole. In some embodiments, the molar ratio of the hydroxyl-containing amino-protected cytosine vinyl phosphate compound to the catalyst is 1:1-2. In some embodiments, the molar ratio of the hydroxyl-containing amino-protected cytosine vinyl phosphate compound to the catalyst is 1:1.2-1.5.

[0082] In this disclosure, after preparing amino-protected cytidine vinyl phosphate compounds, hydroxyl-containing amino-protected cytidine vinyl phosphate compounds, and amino-protected cytidine vinyl phosphate phosphoramide monomers, in some embodiments, the method may further include a separation and purification step, which includes one or more of the steps of extraction, drying, washing, filtration, concentration, and separation. In the method of this disclosure, the method includes adding an impurity remover and an extractant to the reaction mixture for extraction; washing the extracted organic phase with a detergent, separating the aqueous phase and the organic phase, adding a desiccant to the collected organic phase, filtering and concentrating to obtain a crude product; and separating and purifying the crude product to obtain a hydroxyl-containing compound. In some embodiments, examples of impurity removers include, but are not limited to, saturated sodium bicarbonate aqueous solution. In some embodiments, examples of extractants include, but are not limited to, ethyl acetate. In some embodiments, examples of desiccant include, but are not limited to, sodium sulfate. In some embodiments, examples of detergents include, but are not limited to, saturated sodium chloride solution. In some embodiments, the separation and purification are carried out in a chromatography column. In some embodiments, the stationary phase of the chromatography column may be silica gel. Examples of eluents used in the chromatography column include, but are not limited to, petroleum ether and ethyl acetate. In the process of separation and purification by chromatography after the preparation of compounds containing protected amino groups, compounds containing hydroxyl groups, and amino-protected cytidine vinyl phosphate phosphoramide monomers, the eluent of the chromatography column is a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate can be 1:1-1:3, 1:1-1:10, and 1:1-1:3, respectively.

[0083] In some embodiments, the amino-protected cytidine vinyl phosphate phosphoramide monomer is a compound represented by formula (V):

[0084]

[0085] Among them, R 12 It is a hydroxyl protecting group.

[0086] R 13 R 14 They may be the same or different, each independently selected from C1-C8 alkyl and / or substituted C1-C8 alkyl groups.

[0087] In some embodiments, the amino-protected cytidine vinyl phosphate phosphoramide monomer is a compound of formula (104):

[0088]

[0089] Example

[0090] The present disclosure and its beneficial effects will be described in detail below with reference to specific embodiments.

[0091] Example 1: Synthesis of Compound 6

[0092]

[0093] Compound 5 (72 g, synthesized according to Preparation Examples 14-1 and 14-2 in CN110959011A, the only difference being the use of tert-butyldimethylchlorosilane (TBDMS) instead of tert-butyldiphenylchlorosilane (TBDPS)), triethylamine (30 g, 0.30 mol), and N-methylpiperidine (17 g, 0.17 mol) were added to a solution of acetonitrile in 360 mL. A solution of 56 g TsCl (0.29 mol) in 170 mL of acetonitrile was added, and the reaction was allowed to proceed for 3 hours. Then, 75 mL of 25 wt% ammonia was added, and the reaction was allowed to proceed for 12 hours. The entire reaction system was concentrated to an oily state, and then 400 mL of water was added. Extraction was performed with 150 mL of ethyl acetate to separate the organic and aqueous phases. The aqueous phase was then extracted twice more with 150 mL of ethyl acetate. The organic phases were combined and washed with 50 mL of saturated brine. The organic and aqueous phases were separated. 100 g of anhydrous sodium sulfate was added to the organic phase for drying. The mixture was filtered and concentrated to obtain 90 g of crude oil. The crude product was purified by column chromatography using 150-mesh normal-phase silica gel as the stationary phase. The eluent was a mixture of petroleum ether, ethyl acetate, and EtOH in a volume ratio of 1:1:0.1. The eluent containing the product was collected, and the solvent was removed by vacuum distillation, finally yielding 50 g of compound 6. The purity was 80% as determined by HPLC.

[0094] Example 2 Synthesis of Compound 7

[0095]

[0096] 34 g of benzoic anhydride (0.15 mol) was added to a solution of 50 g of compound 6 (0.1 mol) prepared in Example 1 in 150 mL of N,N-dimethylformamide, and the mixture was stirred overnight at room temperature to obtain a reaction mixture. The reaction mixture was added to 300 mL of a 10 wt.% saturated sodium bicarbonate aqueous solution, and then extracted three times with 100 mL of ethyl acetate. The organic phases were combined, washed with 100 mL of saturated brine, and the organic phase was collected. 20 g of anhydrous sodium sulfate was added for drying, the mixture was filtered, and the filtrate was concentrated to remove the solvent, yielding 60 g of a yellow oily substance. The crude product was purified by column chromatography using 150-mesh normal-phase silica gel as the stationary phase and a mixture of petroleum ether and ethyl acetate (volume ratio of petroleum ether to ethyl acetate 1:1) as the eluent to obtain 42 g of compound 7. HPLC analysis showed that the purity of compound 7 was 83%.

[0097] In the above process, the process of the present invention is used, which avoids the side reaction of deprotection of amino protecting groups that may occur during the direct synthesis of VPCm, and the compound 7 is synthesized quickly and efficiently.

[0098] Example 3 Synthesis of Compound 8

[0099]

[0100] 30g of compound 7 (0.05mol) prepared in Example 2 above was added to 210mL of anhydrous tetrahydrofuran solution, and 40g of triethylamine hydrofluoric acid (0.25mol) was added. The mixture was stirred overnight at room temperature to obtain a reaction mixture. 1000mL of 10wt.% saturated sodium bicarbonate aqueous solution was added to the reaction mixture to neutralize the reaction solution, followed by the addition of 500mL of ethyl acetate and thorough stirring for extraction. The aqueous phase was then extracted twice more with ethyl acetate, 500mL each time. All organic phases were combined and washed once with 500mL of saturated brine. After separating the organic and aqueous phases, 200g of anhydrous sodium sulfate was added for drying. The mixture was filtered and the filtrate was concentrated to remove the solvent, yielding 60g of crude product. The crude product was purified by column chromatography using 150-mesh normal-phase silica gel as the stationary phase and a mixed solvent of petroleum ether and ethyl acetate (volume ratio of petroleum ether to ethyl acetate 1:1) as the eluent. Finally, 15g of compound 8 was obtained, and the purity of compound 8 was 89% as determined by HPLC.

[0101] Example 4 Synthesis of Compound 9

[0102]

[0103] 15 g of compound 8 (0.03 mol) was added to 100 mL of DMF solution with 3.6 g of 4,5-dicyanimidazolium (0.03 mol) and 12 g of phosphorus reagent (bis(diisopropylamino)(2-cyanoethoxy)phosphine) (0.4 mol). The mixture was stirred at room temperature for three hours to obtain a reaction mixture. 100 mL of ice water was added to the reaction mixture, followed by extraction three times with 80 mL of ethyl acetate each time, and the organic phase was collected. The combined organic phases were washed twice with 100 mL of ice-cold DMF aqueous solution (DMF:H2O = 1:1 v / v), and the organic phase was collected. The mixture was then washed once with 100 mL of semi-saturated brine and once with 100 mL of saturated brine. The organic phase was collected and 50 g of anhydrous sodium sulfate was added. The mixture was filtered, and the filtrate was concentrated to remove the solvent, yielding 21 g of crude compound 9. The crude product was purified by column chromatography using 100-200 mesh silica gel as the stationary phase and a mixed solvent of petroleum ether and ethyl acetate (volume ratio of petroleum ether to ethyl acetate 1:1) as the eluent. 15g of compound 9 was finally obtained, and HPLC analysis showed that the purity of compound 9 was 95%.

[0104] Preparation of protected cytidine vinyl phosphates in Comparative Examples 1-4

[0105] Compound 7 was synthesized according to the methods of Preparation Examples 14-1 and 14-2 in CN110959011A, except that the starting compound was replaced by 2-methoxycytosine (compound of formula 1') with 5'-hydroxyl protected by DMTr and amino group on cytosine base protected by benzoyl group instead of 2'-OMe-U in Preparation Example 14-1.

[0106]

[0107] Furthermore, corresponding to the steps of VP-U-3 to VP-U-4 in Preparation Example 14-2 of CN110959011A:

[0108] In Comparative Example 1, the solvent (tetrahydrofuran) and the organic base (potassium tert-butoxide (t-BuOK)) were not substituted;

[0109] In Comparative Example 2, the solvent was not replaced, but sodium hydride was used instead of potassium tert-butoxide as the organic base for the reaction.

[0110] In Comparative Example 3, the organic base was not replaced, and dichloromethane was used instead of tetrahydrofuran as the solvent for the reaction.

[0111] In Comparative Example 4, dichloromethane was used instead of tetrahydrofuran as the solvent, and sodium hydride was used instead of potassium tert-butoxide as the organic base for the reaction.

[0112] As a result, no compound 7 product was obtained in Comparative Examples 1-4. These results indicate that it is difficult to obtain cytidine vinyl phosphate compounds using existing techniques and simply by replacing the starting material compounds.

[0113] The above results indicate that it is difficult to obtain cytidine vinyl phosphate compounds using the phosphorus ylide reaction process commonly used in the art. In contrast, the method disclosed herein avoids the side reaction of amino protecting group deprotection that may occur during the direct synthesis of VPCm, and can rapidly and efficiently synthesize the target cytidine vinyl phosphate compound, making it suitable for industrial production.

Claims

1. A method for preparing a cytidine vinyl phosphate compound, the method comprising contacting the compound represented by formula (I) with an ammonia source compound in an organic solvent under ammonolysis reaction conditions: in, R1, R2, R5, R6, R8, and R9 are each independently hydrogen, methyl, or ethyl; R3 and R4 are each independently one of hydrogen, halogen, hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, or substituted C1-C3 alkoxy. Each R7 is independently one of hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, or substituted C1-C3 alkoxy. R 10 It can be hydrogen or methyl.

2. The method according to claim 1, wherein, R1, R2, R5, R6, R8, and R9 are each independently hydrogen; R3 is one of halogen, C1-C3 alkoxy, or substituted C1-C3 alkoxy; optionally, R3 is one of fluorine, methoxy, or -O-2-methoxyethyl. R4 is a protected hydroxyl group; Each R7 is independently a C1-C3 alkoxy group; R 10 It is hydrogen.

3. The method according to claim 1, wherein, The organic solvent is a nonprotic polar solvent; and / or The amount of organic solvent used is 1.5-5.5 L / mol relative to the compound shown in formula (I).

4. The method according to claim 3, wherein, The organic solvent is one or more of nitrile or ether solvents.

5. The method according to claim 4, wherein, The organic solvent is at least one of acetonitrile, tetrahydrofuran, or 1,4-dioxane.

6. The method according to claim 1, wherein, The ammonia source compound is selected from at least one of ammonia, urea, or ammonium salts; and / or The molar ratio of the ammonia source compound to the compound shown in formula (Ⅰ) is 2.0-4.0:

1.

7. The method according to claim 1, wherein, The ammonolysis reaction conditions include one or more of the following conditions: The reaction temperature is: -10℃ to -10℃; The reaction time is 5-20 hours. The pH value of the reaction is 7.5-12.5; The reaction is carried out in the presence of an activator, which is a sulfonyl halide and / or a nitrogen-containing heterocyclic compound; the molar ratio of the activator to the compound shown in formula (I) is 1:0.20-0.

40.

8. The method according to claim 7, wherein, The ammonolysis reaction conditions include one or more of the following conditions: The reaction temperature is: -5℃ to -5℃; The reaction time is 9-15 hours. The pH value of the reaction is 8-12; The activator of the reaction is at least one of p-toluenesulfonyl chloride, N-methylpiperidine, or piperidine; the molar ratio of the activator to the compound shown in formula (I) is 1:0.25-0.

35.

9. The method according to claim 1, wherein, The ammonolysis reaction is carried out in the presence of a basic compound selected from at least one of organic amine compounds, multidentate chelating agents, or nitrogen-containing heterocyclic compounds; and / or The weight ratio of the compound represented by formula (Ⅰ) to the basic compound is 2.0-2.8:

1.

10. The method according to claim 9, wherein, The basic compound is at least one of triethylamine, ethylenediaminetetraacetic acid, 2,6-methylpyridine, or pyridine.

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