Preparation method and application of cytidine vinyl phosphate compound

Abstract

The present disclosure relates to a method for preparing a cytidine vinyl phosphate compound, comprising contacting a compound represented by formula (I) with an ammonia source compound in an organic solvent under aminolysis reaction conditions; in the compound represented by formula (I), 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, substituted C1-C3 alkoxy; each R7 is independently one of hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, substituted C1-C3 alkoxy; R 10 The present disclosure also relates to the use of the cytidine vinyl phosphate compound prepared by the method.

Description

Technical Field

[0001] The present disclosure relates to a method for preparing a cytidine vinyl phosphate compound and also relates to uses of the cytidine vinyl phosphate compound prepared by the method. Background Art

[0002] In recent years, small nucleic acid drugs have become a hot topic in the current biomedical research field due to their advantages such as strong specificity, simple design, short R&D cycle, and rich targets. 5'-vinyl phosphate-terminated oligonucleotides have beneficial effects such as stability to phosphoexonucleases, significant increase in oligonucleotide pharmacodynamic activity and / or long-term effectiveness, and are receiving increasing attention in the field. Vinyl phosphate phosphoramidite nucleoside monomers are important intermediates commonly used to introduce vinyl phosphate at the 5'-end of oligonucleotides in solid-phase nucleic acid synthesis methods. In the prior art, uridine / thymidine vinyl phosphate is the most widely used due to its easy synthesis, fewer by-products, 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.

[0003] The preparation of nucleoside vinyl phosphate is usually carried out by phosphorus ylide reaction. This reaction requires relatively harsh reaction conditions, and the starting compound usually uses nucleoside monomers of 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 new uracil derivative amination method, and applies it to the synthesis of cytosine. This document reports a kind of innovative uracil derivative amination synthesis method, which involves using 1-methyl piperidine to carry out activation steps at the C4 position of uracil base, thereby realizing the large-scale synthesis of 2'-deoxycytosine. However, this document does not involve uracil derivatives whose 5' position is a vinyl phosphate group, which may be a limitation in some specific chemical synthesis and biochemical applications. Summary of the invention

[0004] The first aspect of the present disclosure provides a method for preparing a cytidine vinyl phosphate compound, which comprises contacting a compound represented by formula (I) with an ammonia source compound in an organic solvent under aminolysis reaction conditions:

[0005] Formula (I),

[0006] wherein R1, R2, R5, R6, R8 and R9 are each independently hydrogen, methyl or ethyl;

[0007] R3 and R4 are each independently one of hydrogen, halogen, hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, substituted C1-C3 alkoxy;

[0008] Each R7 is independently one of hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, substituted C1-C3 alkoxy;

[0009] R 10 It is hydrogen or methyl.

[0010] A second aspect of the present disclosure provides a method for preparing an amino-protected cytidine vinyl phosphate compound, the method comprising contacting a cytidine vinyl phosphate compound with an amino-protecting agent in a non-protonic organic solvent under acylation reaction conditions, wherein the cytidine vinyl phosphate compound is prepared by the method provided by the present disclosure above.

[0011] The third aspect of the present disclosure provides a method for preparing a hydroxyl-containing amino-protected cytidine vinyl phosphate compound, the method comprising contacting the amino-protected cytidine vinyl phosphate compound with a deprotecting agent in a non-protonic organic solvent under deprotection reaction conditions.

[0012] The fourth aspect of the present disclosure provides a method for preparing an amino-protected cytidine vinyl phosphate phosphoramidite compound, which comprises contacting the above-mentioned hydroxyl-containing amino-protected cytidine vinyl phosphate compound with a phosphating agent in a non-protonic organic solvent under phosphorylation reaction conditions and in the presence of a catalyst.

[0013] Beneficial Effects

[0014] The inventors found in experiments 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 deprotection of the amino protecting group that may be produced when directly synthesizing VPCm, and can quickly and efficiently synthesize the target cytidine vinyl phosphate compound, which is suitable for industrial production.

[0015] Incorporated by Reference

[0016] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into the present disclosure to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference into the present disclosure. DETAILED DESCRIPTION

[0017] In one aspect, the present disclosure provides a method for preparing a cytidine vinyl phosphate compound, the method comprising contacting a compound represented by formula (I) with an ammonia source compound in an organic solvent under aminolysis reaction conditions:

[0018] Formula (I),

[0019] wherein R1, R2, R5, R6, R8 and R9 are each independently hydrogen, methyl or ethyl;

[0020] R3 and R4 are each independently one of hydrogen, halogen, hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, substituted C1-C3 alkoxy;

[0021] Each R7 is independently one of hydroxyl, protected hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, substituted C1-C3 alkyl, substituted C1-C3 alkoxy;

[0022] R 10 It is hydrogen or methyl.

[0023] In some embodiments, R1, R2, R5, R6, R8 and R9 are each independently hydrogen;

[0024] R3 is a C1-C3 alkoxy group;

[0025] R4 is a protected hydroxyl group;

[0026] Each R7 is independently a C1-C3 alkoxy group;

[0027] R 10 For hydrogen.

[0028] As used herein, "alkyl" refers to straight and branched saturated hydrocarbon groups having a specified number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 6 or 1 to 3 carbon atoms. For example, C1-C3 alkyl includes straight and branched alkyl groups of 1 to 3 carbon atoms. When referring to a residue having a specific number of alkyl groups, it is intended to cover all branched and straight chain forms having that number of carbons; thus, for example, "propyl" includes n-propyl and isopropyl. Alkyl groups used herein may optionally contain one or more further substituted groups.

[0029] As used herein, "alkoxy" refers to an alkyl group of a specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 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 through an oxygen bridge. In some embodiments, the alkoxy group is methoxy, ethoxy, propoxy, or isopropoxy. The alkoxy group used herein may optionally contain one or more further substituent groups.

[0030] As used herein, term " blocking group " refers to the unstable chemical part that is used to protect reactive groups (including but not limited to hydroxyl and amino) and avoids undesirable reaction in building-up process known in the art. Blocking group is usually used to protect some sites selectively and / or orthogonally in the reaction process of other reactive sites, and can be removed to discharge unprotected group as is or can be used for further reaction mode then. As known in the art, blocking group is generally described in " Greene ' s Protective Groups in Organic Synthesis ", the 4th edition, John Wiley & Sons, New York, 2007.

[0031] As used herein, the term "protected hydroxyl" refers to a 2'-hydroxyl functional group protected by a suitable protecting group, such as an acid-active acetal protecting group, particularly a 1-(aryl)-4-alkoxypiperidin-4-yl, such as a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) or a 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 alkoxy C1-C4 alkyl or alkenyl derivative. In this case, the 2' position does not require further protection. Alternatively, the nucleoside intermediate also includes 2'-amino, 2'-alkyl or 2'-carbon-alkenyl substituted nucleosides.

[0032] .

[0033] In the present disclosure, amino protected cytidine vinyl phosphate refers to cytosine amino group in cytidine vinyl phosphate connected with amino protecting group. Examples of "amino protecting group" include but are not limited to carbamate 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 imide and cyclic imide protecting groups, such as phthalimido and dithiosuccinoyl.

[0034] Those skilled in the art know the preparation method of the compound represented by formula (I), for example, Examples 13, 16, 21, 22, 26, and 35a in CN103154014B disclose the preparation methods of various compounds represented by formula (I). In one embodiment, the compound represented by formula (I) is prepared according to the method of Preparation Examples 14-1 to 14-2 in CN110959011A.

[0035] 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.

[0036] 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 represented by formula (I).

[0037] In some embodiments, the ammonia source compound is selected from at least one of ammonia gas, urea or ammonium salt. Considering the availability and cost of raw materials, in some embodiments, the ammonia source compound is ammonia. Ammonia can be provided in the form of ammonia gas or in the form of ammonia water. The concentration of ammonia water can be 20-30wt%. In some embodiments, the molar ratio of the ammonia source compound to the compound represented by formula (I) is 2.0-4.0:1, for example, 2.5-3.5:1.

[0038] Those skilled in the art are aware of various aminolysis reaction conditions. As long as the aminolysis reaction conditions can obtain the above-mentioned cytidine vinyl phosphate compound after the compound of formula (I) is contacted with the ammonia source compound, they can be used in the present disclosure. In some embodiments, the aminolysis reaction is carried out in the presence of an activator. The presence of the activator can promote the activation of the reactant molecules, thereby reducing the activation energy of the reaction and accelerating the reaction rate. In some embodiments, the molar ratio of the activator to the compound represented by formula (I) is 1:0.20-0.40. In some embodiments, in some embodiments, the aminolysis reaction conditions include one or more of the following conditions:

[0039] Reaction temperature: -10℃-10℃;

[0040] Reaction time: 5-20 hours;

[0041] The pH of the reaction is 7.5-12.5;

[0042] The activator of the reaction is a sulfonyl halide and / or a nitrogen-containing heterocyclic compound;

[0043] The molar ratio of the activator to the compound represented by formula (I) is 1:0.25-0.35.

[0044] In some embodiments, the aminolysis reaction conditions include one or more of the following conditions:

[0045] Reaction temperature: -5℃-5℃;

[0046] Reaction time: 9-15 hours;

[0047] The pH of the reaction is 8-12;

[0048] The activating agent of the reaction is at least one of p-toluenesulfonyl chloride, N-methylpiperidine or piperidine.

[0049] In some embodiments, the aminolysis reaction is carried out in the presence of a basic compound, and the basic compound is selected from at least one of an organic amine compound, a multidentate chelating agent, or a nitrogen-containing heterocyclic compound. Considering the cost and stability of the basic conditions, in some embodiments, the basic compound is at least one of triethylamine, ethylenediaminetetraacetate, 2,6-methylpyridine, or pyridine.

[0050] 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.

[0051] In some embodiments, the compound represented by formula (I) is a compound represented by formula (100):

[0052] Formula (100).

[0053] In some embodiments, the cytidine vinyl phosphate compound is a compound represented by formula (II):

[0054] (II).

[0055] In some embodiments, the cytidine vinyl phosphate compound is a compound represented by formula (101):

[0056] Formula (101).

[0057] In some embodiments, the method further comprises a separation and purification step. In some embodiments, the separation and purification step comprises one or more of concentration, extraction, filtration, and separation. In some embodiments, the separation and purification step comprises adding water and an extraction solvent to the reaction mixture for extraction; washing the organic phase obtained by extraction 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; separating and 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 detergents include but are not limited to saturated sodium chloride solutions. In some embodiments, the separation and purification is performed 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 use a mixed solvent of petroleum ether: ethyl acetate: ethanol, and the volume ratio of petroleum ether to ethyl acetate can be 1: 1-1: 5; the volume ratio of petroleum ether to ethanol can be 1: 0.1-1: 0.3.

[0058] Application of the disclosed method

[0059] The method disclosed herein and the cytidine vinyl phosphate compound prepared therefrom can be used for a variety of purposes, such as preparing cytidine vinyl phosphate derivatives. In some embodiments, the cytidine vinyl phosphate compound prepared by the method disclosed herein can be used to prepare amino-protected cytidine vinyl phosphate phosphoramidite monomers.

[0060] On the other hand, the present disclosure also provides a method for preparing an amino-protected cytidine vinyl phosphate compound, the method comprising contacting a cytidine vinyl phosphate compound with an amino-protecting agent in a non-protonic organic solvent under acylation reaction conditions, wherein the cytidine vinyl phosphate compound is prepared by the method provided by the present disclosure.

[0061] Those skilled in the art are aware of various acylation reaction conditions, and any acylation reaction conditions that can obtain the protected amino-containing compound provided by the present disclosure after the cytidine vinyl phosphate compound provided by the present disclosure is contacted with the amino protecting agent can be used in the present disclosure. In some embodiments, the acylation reaction conditions include: reaction temperature: -10°C-35°C; reaction time: 5-20 hours; In some embodiments, the acylation reaction conditions include: reaction temperature: 10°C-25°C; reaction time: 8-16 hours.

[0062] In some embodiments, the aprotic organic solvent is selected from amides, nitriles, esters or ethers. In some embodiments, examples of aprotic organic solvents include, but are not limited to, N,N-dimethylformamide, acetonitrile, ethyl acetate, tetrahydrofuran or dioxane.

[0063] According to the different types of amino protecting agents used, the amino protecting groups in the obtained amino protected cytidine vinyl phosphate compounds are also different. In some embodiments, examples of amino protecting agents include but are not limited to benzoic anhydride, acyl chlorides, Boc-Cl, Fmoc-Cl, Cbz-Cl, activated esters, and in some embodiments benzoic anhydride or acetyl chloride. In some embodiments, the molar ratio of the cytidine vinyl phosphate compound to the amino protecting agent can be 1-1.0-2.5. In some embodiments, the molar ratio of the cytidine vinyl phosphate compound to the amino protecting agent can be 1-1.2-2.0.

[0064] In some embodiments, the compound containing a protected amino group is a compound represented by formula (III):

[0065] Formula (III),

[0066] Among them, R 11 is an amino protecting group, preferably selected from benzoyl, C1-C8 ketocarbonyl, C1-C8 alkyl, Boc, Cbz, Fmoc, and more preferably benzoyl or acetyl;

[0067] R4 is a protected hydroxyl group; preferably, R4 is tert-butyldimethylsilyl (TBDMS) or tert-butyldiphenylsilyl (TBDPS).

[0068] In some embodiments, the compound containing a protected amino group is a compound represented by formula (102):

[0069] Formula (102).

[0070] On the other hand, the present disclosure also provides a method for preparing a hydroxyl-containing amino-protected cytidine vinyl phosphate compound, the method comprising contacting a protected amino-containing compound with a deprotecting agent in a non-protonic organic solvent under deprotection reaction conditions, wherein the amino-protected cytidine vinyl phosphate compound is prepared by the method provided by the present disclosure, and the protected amino-containing compound also contains a protected hydroxyl group.

[0071] Those skilled in the art are aware of various deprotection reaction conditions, and any deprotection reaction conditions that can obtain a hydroxyl-containing compound provided by the present disclosure after the protected amino-containing compound provided by the present disclosure is contacted with a deprotecting agent can be used in the present disclosure. In some embodiments, the deprotection reaction conditions include: a reaction temperature of -10°C to 35°C; a reaction time of 5 to 20 hours; in some embodiments, the deprotection reaction conditions include: a reaction temperature of 10°C to 25°C; a reaction time of 8 to 16 hours.

[0072] In some embodiments, examples of deprotecting agents include, but are not limited to, various fluoride-containing compounds, such as triethylamine hydrofluoride, tetrabutylammonium fluoride. In some embodiments, the molar ratio of the compound containing a protected amino group to the deprotecting agent may be 1-4-6; in some embodiments, the molar ratio of the compound containing a protected amino group to the deprotecting agent may be 1-4.5-5.5.

[0073] In some embodiments, the hydroxyl-containing compound is a compound represented by formula (IV):

[0074] Formula (IV).

[0075] In some embodiments, the hydroxyl-containing compound is a compound represented by formula (103):

[0076] Formula (103).

[0077] On the other hand, the present disclosure also provides a method for preparing an amino-protected cytidine vinyl phosphate phosphoramidite monomer, which comprises contacting the above-mentioned hydroxyl-containing amino-protected cytidine vinyl phosphate compound with a phosphating agent in a non-protonic organic solvent under phosphitylation reaction conditions in the presence of a catalyst.

[0078] Those skilled in the art are aware of various phosphorylation reaction conditions, and any phosphorylation reaction conditions that can obtain amino-protected cytidine vinyl phosphate phosphoramidite monomers after contacting the amino-protected cytidine vinyl phosphate compound provided by the present disclosure with a phosphorylation agent can be used in the present disclosure. In some embodiments, the phosphorylation reaction conditions include: reaction temperature: -10°C-35°C; reaction time: 5-20 hours; In some embodiments, the phosphorylation reaction conditions include: reaction temperature: 10°C-25°C; reaction time: 8-16 hours.

[0079] In some embodiments, examples of the phosphorylating agent include, but are not limited to, phosphorus reagent (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 protected amino-containing compound to the phosphorylating agent is 1:1.2-1.5.

[0080] In some embodiments, examples of the catalyst include, but are not limited to, 4,5-dicyanoimidazole. In some embodiments, the molar ratio of the hydroxyl-containing amino-protected cytidine vinyl phosphate compound to the catalyst is 1:1-2. In some embodiments, the molar ratio of the hydroxyl-containing amino-protected cytidine vinyl phosphate compound to the catalyst is 1:1.2-1.5.

[0081] In the present disclosure, after preparing the amino-protected cytidine vinyl phosphate compound, the hydroxyl-containing amino-protected cytidine vinyl phosphate compound and the amino-protected cytidine vinyl phosphate phosphoramidite monomer, in some embodiments, the method can further include the step of separation and purification, and the separation and purification includes one or more of extraction, drying, washing, filtering, concentration, and separation steps. In the method of the present disclosure, the method includes adding an impurity remover and an extractant to the reaction mixture for extraction; washing the organic phase obtained by extraction 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; 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 solutions. 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 and ethyl acetate. In the process of separation and purification by chromatography column after the preparation of protected amino-containing compounds, hydroxyl-containing compounds and amino-protected cytidine vinyl phosphate phosphoramidite 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.

[0082] In some embodiments, the amino-protected cytidine vinyl phosphate phosphoramidite monomer is a compound represented by formula (V):

[0083] Formula (V),

[0084] Among them, R 12 is a hydroxyl protecting group,

[0085] R 13 , R 14 The same or different, each independently selected from C1-C8 alkyl and / or substituted C1-C8 alkyl.

[0086] In some embodiments, the amino-protected cytidine vinyl phosphate phosphoramidite monomer is a compound represented by formula (104):

[0087] Formula (104).

[0088] Example

[0089] The present disclosure and its beneficial effects are described in detail below in conjunction with specific embodiments.

[0090] Example 1 Synthesis of Compound 6

[0091]

[0092] To a solution of compound 5 (72 g, synthesized according to the method of Preparation Examples 14-1 and 14-2 in CN110959011A, the only difference is that tert-butyldimethylsilyl chloride (TBDMS) is used instead of tert-butyldiphenylsilyl chloride (TBDPS) for preparation), triethylamine (30 g, 0.30 mol), and N-methylpiperidine (17 g, 0.17 mol) in 360 mL of acetonitrile, a solution of 56 g of TsCl (0.29 mol) in 170 mL of acetonitrile was added, and the reaction was carried out for 3 hours, followed by the addition of 75 mL of 25 wt% aqueous ammonia and the reaction was carried out for 12 hours. The reaction system was concentrated to an oily state, followed by the addition of 400 mL of water, extraction with 150 mL of ethyl acetate, separation of the organic phase and the aqueous phase, and the aqueous phase was extracted twice with 150 mL of ethyl acetate. The organic phases were combined, washed with 50 mL of saturated brine, separated from the organic phase and the aqueous phase, 100 g of anhydrous sodium sulfate was added to the organic phase for drying, filtered and concentrated to obtain 90 g of an oily crude product. The crude product was separated and purified by a chromatography column, the stationary phase of the chromatography column was 150 mesh normal phase silica gel, the eluent was a mixed solvent of petroleum ether, ethyl acetate and EtOH, and the volume ratio of the three was 1: 1: 0.1, the eluate containing the product was collected, and the solvent was evaporated under reduced pressure to finally obtain 50 g of compound 6, which was 80% pure by HPLC.

[0093] Example 2 Synthesis of Compound 7

[0094]

[0095] To a solution of 50 g of compound 6 (0.1 mol) prepared in Example 1 above in 150 mL of N, N-dimethylformamide, 34 g of benzoic anhydride (0.15 mol) was added, and the mixture was stirred at room temperature overnight to obtain a reaction mixture. The reaction mixture was added to 300 mL of a saturated sodium bicarbonate aqueous solution having a concentration of 10 wt.%, and then 100 mL of ethyl acetate was added for extraction three times. The organic phases were combined, washed with 100 mL of saturated brine, and the organic phases were collected. 20 g of anhydrous sodium sulfate was added for drying, the filtrate was filtered and concentrated to remove the solvent, and 60 g of a yellow oil was obtained. The crude product was separated and purified by a chromatography column, the stationary phase of the chromatography column was 150 mesh normal phase silica gel, and the eluent of the chromatography column was a mixed solvent of petroleum ether and ethyl acetate (the volume ratio of petroleum ether to ethyl acetate was 1:1) to obtain 42 g of the compound, and the purity of compound 7 was 83% as determined by HPLC.

[0096] In the above process, due to the use of the process of the present invention, the side reaction of deprotection of the amino protecting group that may be generated when directly synthesizing VPCm is avoided, and compound 7 is synthesized quickly and efficiently.

[0097] Example 3 Synthesis of Compound 8

[0098]

[0099] 40 g of triethylamine hydrofluoride (0.25 mol) was added to a solution of 30 g of compound 7 (0.05 mol) prepared in Example 2 above in 210 mL of anhydrous tetrahydrofuran, and stirred overnight at room temperature to obtain a reaction mixture. 1000 mL of 10 wt.% saturated sodium bicarbonate aqueous solution was added to the reaction mixture to neutralize the reaction solution, followed by adding 500 mL of ethyl acetate to fully stir and extract, and the aqueous phase was extracted twice with ethyl acetate, 500 mL each time. All organic phases were combined, the organic phase was washed once with 500 mL of saturated brine, the organic phase and the aqueous phase were separated, 200 g of anhydrous sodium sulfate was added to dry, the filtrate was filtered and concentrated to remove the solvent, and 60 g of crude product was obtained. The crude product was separated and purified by a chromatography column, the stationary phase of the chromatography column was 150 mesh normal phase silica gel, and the eluent of the chromatography column was a mixed solvent of petroleum ether and ethyl acetate ((volume ratio of petroleum ether and ethyl acetate 1:1)) to finally obtain 15 g of compound 8, and the purity of compound 8 was 89% by HPLC.

[0100] Example 4 Synthesis of Compound 9

[0101]

[0102] 3.6 g 4,5-dicyanoimidazole (0.03 mol) and 12 g phosphorus reagent (bis(diisopropylamino)(2-cyanoethoxy)phosphine) (0.4 mol) were added to a solution of 15 g of compound 8 (0.03 mol) in 100 mL of DMF, and 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 with ethyl acetate three times, 80 mL each time, and the organic phase was collected. After the organic phases were combined, they were washed twice with an icy DMF aqueous solution (DMF:H2O=1:1 v / v), 100 mL each time, and the organic phase was collected. The organic phase was continued to be washed once with 100 mL of semi-saturated brine and once with 100 mL of saturated brine, and the organic phase was collected and 50 g of anhydrous sodium sulfate was added. After filtering, the filtrate was concentrated to remove the solvent to obtain 21 g of crude compound 9. The crude product was separated and purified by chromatography column, the stationary phase was 100-200 mesh silica gel, and the eluent of the chromatography column was a mixed solvent of petroleum ether and ethyl acetate (volume ratio of petroleum ether and ethyl acetate was 1:1). Finally, 15 g of compound 9 was obtained, and the purity of compound 9 was 95% as determined by HPLC.

[0103] Comparative Example 1-4 Preparation of Protected Cytidine Vinyl Phosphate

[0104] Compound 7 was synthesized according to the method of Preparation Examples 14-1 and 14-2 in CN110959011A, except that 2-methoxycytosine (compound of formula 1') in which the 5'-hydroxyl group was protected by DMTr and the amino group on the cytosine base was protected by benzoyl was used as the starting compound instead of 2'-OMe-U in Preparation Example 14-1;

[0105] Formula (1');

[0106] And, corresponding to the steps of VP-U-3 to VP-U-4 in Preparation Example 14-2 in CN110959011A:

[0107] In Comparative Example 1, the solvent (tetrahydrofuran) and the organic base (potassium tert-butoxide (t-BuOK)) were not replaced;

[0108] In Comparative Example 2, the solvent was not replaced, and sodium hydride was used to replace potassium tert-butoxide as the organic base for the reaction;

[0109] In Comparative Example 3, the organic base was not replaced, and dichloromethane was used instead of tetrahydrofuran as the solvent for the reaction;

[0110] In Comparative Example 4, dichloromethane was used as the solvent instead of tetrahydrofuran, and sodium hydride was used as the organic base instead of potassium tert-butoxide to carry out the reaction.

[0111] As a result, no compound 7 product was obtained in Comparative Examples 1 to 4. The above results show that it is difficult to obtain a cytidine vinyl phosphate compound by using the prior art method and only by replacing the raw material compound.

[0112] The above results show 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 deprotection of the amino protecting group that may occur when directly synthesizing VPCm, and can quickly and efficiently synthesize the target cytidine vinyl phosphate compound, which is suitable for industrial production.

Claims

1. A method for preparing an amino-protected cytidine vinyl phosphate phosphoramidite monomer, the method comprising the following steps: 1) In an organic solvent, under the conditions of ammonolysis reaction, the compound represented by formula (100) is contacted with an ammonia source compound: Formula (100), The cytidine vinyl phosphate compound represented by formula (101) is obtained: Formula (101); 2) Under acylation reaction conditions, in a non-protonic organic solvent, the cytidine vinyl phosphate compound represented by formula (101) is contacted with an amino protecting agent to obtain an amino protected cytidine vinyl phosphate compound represented by formula (102): Formula (102); 3) Under deprotection reaction conditions, in an aprotic organic solvent, the amino-protected cytidine vinyl phosphate compound represented by formula (102) is contacted with a deprotecting agent to obtain a hydroxyl-containing amino-protected cytidine vinyl phosphate compound represented by formula (103): Formula (103); 4) under phosphitylation reaction conditions, in the presence of a catalyst, in an aprotic organic solvent, contacting the amino-protected cytidine vinyl phosphate compound of formula (103) with a phosphitylation agent to obtain an amino-protected cytidine vinyl phosphate phosphoramidite monomer of formula (104): Formula (104).

2. The method according to claim 1, wherein: The organic solvent is an aprotic polar solvent; and / or The amount of the organic solvent used is 1.5-5.5 L / mol relative to the compound represented by formula (100).

3. The method according to claim 2, wherein: The organic solvent is one or more nitrile or ether solvents.

4. The method according to claim 3, wherein: The organic solvent is at least one of acetonitrile, tetrahydrofuran or 1,4-dioxane.

5. The method according to claim 1, wherein: The ammonia source compound is selected from at least one of ammonia, urea or ammonium salt; and / or The molar ratio of the ammonia source compound to the compound represented by formula (100) is 2.0-4.0:

1.

6. The method according to claim 1, wherein: The aminolysis reaction conditions include one or more of the following conditions: Reaction temperature: -10℃-10℃; Reaction time: 5-20 hours; The pH 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 represented by formula (100) is 1:0.20-0.

40.

7. The method according to claim 6, wherein: The aminolysis reaction conditions include one or more of the following conditions: Reaction temperature: -5℃-5℃; Reaction time: 9-15 hours; The pH of the reaction is 8-12; The activating agent of the reaction is at least one of p-toluenesulfonyl chloride, N-methylpiperidine or piperidine; the molar ratio of the activating agent to the compound represented by formula (100) is 1:0.25-0.

35.

8. The method according to claim 1, wherein: The aminolysis reaction is carried out in the presence of an alkaline compound, wherein the alkaline compound is selected from at least one of an organic amine compound, a multidentate chelating agent or a nitrogen-containing heterocyclic compound; and / or The weight ratio of the compound represented by formula (100) to the basic compound is 2.0-2.8:

1.

9. The method according to claim 8, wherein: The alkaline compound is at least one of triethylamine, ethylenediaminetetraacetic acid, 2,6-methylpyridine or pyridine.

10. The method according to claim 1, wherein: The method further comprises a separation and purification step.

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