Preparation method of nucleotide monomer

Abstract

The invention discloses a preparation method of a nucleotide monomer, which comprises the following steps: taking methyl-D-ribofuranoside as an initial raw material, and finally realizing the synthesis of a compound monomer as shown in a formula P through the steps of selective protection, selective deprotection, C-C bond construction, acylating chlorination reaction, basic group introduction through glycosylation, deprotection, hydroboration oxidation and ring closing in sequence. By providing a brand new synthesis method, the reaction yield is improved, the cost of the whole synthesis molecule is reduced, the binding affinity of the prepared target nucleoside monomer serving as a reported drug applied to HCV resistance to RNA can be improved so as to further improve nuclease resistance, and the method has great significance for subsequent nucleic acid drug research.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology and relates to a method for preparing nucleotide monomers. Background Technology

[0002] In 1978, Zamecnik and colleagues at Harvard University discovered the inhibitory effect of complementary nucleotide chains on RSV virus, thus proposing the concept of ASO (Acute Oxygen Optimizer). However, the first ASO drug, Vitravene, was not approved for marketing until 20 years later in 1998. In 1990, Craig Tuerk and Larry Gold published an article in Science, screening RNA chains (RNA aptamers) with strong affinity for target protein molecules in vitro. The first RNA aptamer drug, pegatatanib, was approved for marketing in 2004. In 1998, Andrew Fire and Craig Mello first revealed the mechanism of RNAi in nematodes, and were awarded the 2006 Nobel Prize for their work. The first siRNA drug based on this mechanism, Patisiran, was approved in 2018.

[0003] Unlike small molecule or antibody drugs, nucleic acid drugs can selectively target untreatable human and viral genomes to inhibit gene expression, alter mRNA splicing, target non-coding RNAs involved in transcription and epigenetic regulation, upregulate target genes, and edit the genome. This provides a novel approach for treating genetic diseases, rare diseases, tumors, and chronic diseases, and is expected to become the third major class of drugs after small molecule chemical drugs and antibody drugs.

[0004] The numerous advantages of small nucleic acid drugs have shown their potential for addressing undrugable targets and providing both symptomatic relief and fundamental cure. However, many aspects still warrant optimization in practical applications. For example: In vivo stability: Naked nucleic acids are not stable in the human body and are easily degraded by nucleases in the blood and tissues. They are also easily cleared by the reticuloendothelial system of the liver and kidneys.

[0005] Immunogenicity: Exogenous nucleic acids are immunogenic and can easily trigger intracellular and extracellular immune responses.

[0006] Cellular uptake efficiency: The phospholipid bilayer of the cell membrane only allows neutral microhydrophobic molecules smaller than 1000 Da to enter the cell through passive diffusion, while small nucleic acid drugs (ASO: 4-10 kDa; siRNA: ~14 kDa) often cannot diffuse into the cell due to their large molecular weight, charged nature, etc.

[0007] Endosome escape: Small nucleic acid drugs are difficult to escape from endosomes after being endocytosed, resulting in insufficient effective drug concentration.

[0008] Chemical modifications to nucleotides can improve substrate specificity, enhance stability against nucleases, and reduce immunogenicity. Modification methods include improvements to ribose, phosphate backbone, bases, and nucleic acid chain ends.

[0009] In summary, there is an urgent need in the field for a method to prepare modified nucleotide monomers that can improve the binding affinity to RNA and thus further enhance nuclease resistance. Summary of the Invention

[0010] To address the above technical problems, the present invention aims to provide a method for synthesizing monomers based on modifications at the 2' position of the ribose. This method is simple to prepare, has a high yield, and achieves efficient synthesis of monomers. These monomers can improve their binding affinity to RNA, thereby further enhancing nuclease resistance.

[0011] To achieve the above objectives, this invention provides a method for preparing nucleotide monomers, and the technical solution adopted is as follows: A method for preparing a nucleotide monomer includes the following steps:

[0012] Starting with methyl-D-furanoside, the monomer of the compound shown in formula P was finally synthesized through a series of steps including selective protection, selective deprotection, C / C bond construction, acyl chloride reaction, glycosylation to introduce a base, deprotection, hydroboration oxidation and cyclization. In the formula: R1 represents any one of the following groups I to IV:

[0013] R2 represents a H atom or a DMTr group, and Z represents an O atom.

[0014] In some embodiments, methyl-D-furanoside is used as the starting material. All hydroxyl groups are selectively protected with hydroxyl protecting groups, and then the protection at the target position is selectively removed. After oxidation to ketone, it reacts with an olefin Grignard reagent, and after protecting the hydroxyl group, it is glycosylated to attach a base. The hydroxyl protection at the target position is selectively removed, and after hydroboration oxidation, an acylation reagent is introduced to close the five-membered oxygen ring. Finally, the protection is removed to obtain the target product.

[0015] In some embodiments, the preparation method includes the following steps: Starting with S1 and methyl-D-furanoside, protecting groups were introduced to the 2', 3' and 5' hydroxyl groups to obtain compound 2; S2 and compound 2 selectively remove the protecting group on the 2' hydroxyl group to generate compound 3; S3 and compound 3 undergo an oxidation reaction to oxidize the hydroxyl group at the 2' position to a ketone to obtain compound 4; S4, compound 4, and olefin Grignard reagent react to give compound 5 with an olefin group and a hydroxyl group at the 2' position; S5 and compound 5 continue to undergo acylation of the hydroxyl group at the 2' position to give compound 6; S6, Compound 6 undergoes a glycosylation reaction with a pyrimidine or purine base at the 1' position to give Compound 7; S7 and compound 7 are deprotected at the 2' position to obtain compound 8; S8 and compound 8 were given compound 9 via a hydroboration oxidation reaction; S9 and compound 9 react with an acylation reagent at the 2' position to give compound 10; S10 and compound 10 undergo intramolecular cyclization at the 1' and 2' positions under basic conditions to yield compound 11; S11 removes the benzyl hydroxyl protecting group from compound 11 to generate the corresponding target molecule.

[0016] In some embodiments, in step S1, the hydroxyl protecting group is benzylBn, and NaH is used as a base to introduce protecting groups at the 2', 3', and 5' hydroxyl positions, resulting in compound 2: ; In step S2, compound 2 selectively loses its protecting group at the 2' hydroxyl group under the action of SnCl4, yielding compound 3: .

[0017] In some embodiments, in step S3, the hydroxyl group at the 2' position of the compound is oxidized to a ketone by a Desmond-Martin oxidant, resulting in compound 4: ; The olefin-based Grignard reagent used in step S4 is a vinyl or propylene Grignard reagent. When a propylene Grignard reagent is used, the temperature is -78°C. o Compound 5, obtained by reacting C with an olefinic Grignard reagent, is: .

[0018] In some embodiments, the acylation reagent in step S5 is BzCl, and compound 5 reacts with BzCl under DMAP-based conditions to give compound 6 as follows: ; In step S6, compound 6 undergoes a glycosylation reaction with uracil, cytosine, adenine, or guanine to yield compound 7; wherein, when compound 6 reacts with uracil, compound 7 is formed as follows: ; In step S7, compound 7 undergoes deprotection of the Bz protecting group under the action of sodium methoxide to obtain compound 8; when compound 6 reacts with uracil, compound 8 is formed as follows: .

[0019] In some embodiments, in step S8, compound 8 undergoes a borohydride oxidation reaction under the conditions of 9-BBN and hydrogen peroxide to yield compound 9; when compound 6 reacts with uracil, compound 9 is formed as follows: ; The acylation reagent in step S9 is MsCl; compound 9 reacts with MsCl to give compound 10. When compound 6 reacts with uracil, compound 10 is formed as follows: ; In step S10, compound 10 undergoes intramolecular cyclization under the action of NaH to yield compound 11; when compound 6 reacts with uracil, compound 11 is formed as follows: ; In step S11, the target molecule is generated by removing the benzyl hydroxyl protecting group using BCl3.

[0020] In some embodiments, the molar ratio of the methyl-D-furanoside to the NaH base is 1:(3-5), and the molar ratio of the methyl-D-furanoside to the protecting benzyl group is 1:(3-4).

[0021] In some embodiments, in step S3, the molar ratio of compound 3 to the Des Martin oxidant is 1:(2-5).

[0022] In some embodiments, in step S4, the molar ratio of compound 4 to the propylene Grignard reagent is 1:(2-5).

[0023] In some embodiments, in step S5, the molar ratio of compound 5 to DMAP and benzoyl chloride is 1:(0.2-1):(2-5).

[0024] In some embodiments, during the glycosylation reaction in step S6, the molar ratio of compound 6 to uracil or cytosine or adenine or guanine and tin tetrachloride is controlled to be 1:(2-5):(3-7).

[0025] In some embodiments, the alkaline condition in step S10 is NaH or potassium tert-butoxide, and the molar ratio of compound 10 to NaH or potassium tert-butoxide is 1:(2-5).

[0026] Compared with traditional technologies, the beneficial effects of this invention are: 1) The purpose of this invention is to provide a method for preparing a nucleotide monomer, using methyl-D-furanoside as the starting material, and through key steps such as selective protection / deprotection, glycosylation to introduce bases, hydroboration oxidation and cyclization, the monomer is finally synthesized efficiently.

[0027] 2) In the synthetic route of this invention, after selective deprotection of methyl-D-furanoside protected by Bn and subsequent oxidation to a ketone, the product protected by benzyl Bz undergoes a high glycosylation reaction with Grignard reagent, requiring no purification. The crude product is purified after deprotection of benzyl Bz to obtain a two-step yield of up to 79.5%. Furthermore, by using a benzyl-protected compound as the starting material, this invention effectively avoids the cumbersome steps of deprotection followed by silicon protection required in similar reactions in existing literature.

[0028] 3) The raw materials used in the preparation method of this invention are simple and readily available, the reaction conditions are mild, the operation is simple, there are no selectivity issues, the yield is high, and it is universal for this series of compounds.

[0029] In summary, this invention provides a novel synthetic method that improves reaction yield while reducing the overall cost of the synthesized molecule. The prepared target nucleoside monomer, as a reported drug used in anti-HCV therapy, can enhance its binding affinity to RNA, thereby further improving nuclease resistance. This is of great significance for subsequent nucleic acid drug research. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] Throughout the invention, when a composition is described as having, containing, or including specific ingredients, or a method is described as having, containing, or including specific process steps, it should be understood that the compositions of the invention are also substantially composed of or consisting of the mentioned ingredients, and the methods of the invention are also substantially composed of or consisting of the mentioned process steps.

[0032] In this invention, when an element or component is referred to as being included in and / or selected from the list of mentioned elements or components, it should be understood that the element or component may be any one of the mentioned elements or components, or the element or component may be selected from the group consisting of two or more mentioned elements or components. Furthermore, it should be understood that the elements or features of the compositions, apparatus, or methods described herein, whether expressly or implicitly stated, can be combined in any manner without departing from the subject matter and scope of the invention.

[0033] It should be understood that the order of steps or the sequence of actions is not important as long as the teachings of this invention are operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs.

[0035] Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. In the examples of this application, NMR: all NMR spectra were detected by a Bruker AV 400 MHz NMR spectrometer, with TMS as an internal standard or without an internal standard. MS: all mass spectra were detected by a Shimadzu LCMS 2020.

[0036] According to a preferred embodiment of the present invention, a method for preparing a nucleotide monomer includes the following steps:

[0037] Starting with methyl-D-furanoside, the monomer of the compound shown in formula P was synthesized through a series of steps including selective protection, selective deprotection, C / C bond construction, acylation reaction, glycosylation to introduce a base, deprotection, hydroboration oxidation and cyclization.

[0038] In the formula: R1 represents any one of the following groups I to IV:

[0039] R2 represents a H atom or a DMTr group, and Z represents an O atom.

[0040] In some embodiments, the preparation method uses methyl-D-furanoside as the starting material, selectively protects all hydroxyl groups with a hydroxyl protecting group, then selectively removes the protection at the target position, oxidizes to a ketone, reacts with an olefin Grignard reagent, protects the hydroxyl group, then glycosylates and attaches a base, selectively removes the hydroxyl protection at the target position, oxidizes with borohydride, introduces an acylation reagent, closes the five-membered oxygen ring, and finally removes the protection to obtain the target product.

[0041] As used herein, "olefinic group" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond obtained by removing a hydrogen molecule from an adjacent carbon atom of the parent alkyl group. The group can be in either the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl groups; propenyl groups, etc.

[0042] Various hydroxyl protecting groups may be used in this paper. Generally, protecting groups make chemical functional groups insensitive to specific reaction conditions and can be added to and removed from the functional group in the molecule without substantially impairing the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2ded, John Wiley & Sons, New York, 1991, which are incorporated herein by reference in their entirety.

[0043] In some embodiments, the preparation method includes the following specific steps: Starting with S1 and methyl-D-furanoside, using NaH as a base, protecting groups were introduced to the 2', 3' and 5' hydroxyl groups to obtain compound 2; S2 and compound 2 selectively remove the protecting group on the 2' hydroxyl group to generate compound 3; S3 and compound 3 undergo an oxidation reaction to oxidize the hydroxyl group at the 2' position to a ketone to obtain compound 4; S4, compound 4, and olefin Grignard reagent react to give compound 5 with an olefin group and a hydroxyl group at the 2' position; S5 and compound 5 continue to undergo acylation of the hydroxyl group at the 2' position to give compound 6; S6, Compound 6 undergoes a glycosylation reaction with a pyrimidine or purine base at the 1' position to give Compound 7; S7 and compound 7 are deprotected at the 2' position to obtain compound 8; S8 and compound 8 undergo a borohydride oxidation reaction under the conditions of 9-BBN and hydrogen peroxide to give compound 9; S9 and compound 9 react with an acylation reagent at the 2' position to give compound 10; S10 and compound 10 undergo intramolecular cyclization at the 1' and 2' positions under basic conditions to yield compound 11; S11 removes the benzyl hydroxyl protecting group from compound 11 to generate the corresponding target molecule.

[0044] In some implementations, the steps may be performed as follows: In step S1, the hydroxyl protecting group is benzylBn, resulting in compound 2: ; In step S2, compound 2 selectively removes the protecting group at the 2' hydroxyl position under the action of SnCl4 to generate compound 3: ; In step S3, the hydroxyl group at the 2' position of the compound is oxidized to a ketone by the Des Martin oxidant, resulting in compound 4: ; The olefin-based Grignard reagent used in step S4 is a vinyl or propylene Grignard reagent. When a propylene Grignard reagent is used, the temperature is -78°C. o Compound 5, obtained by reacting C with an olefinic Grignard reagent, is: ; The acylation reagent in step S5 is BzCl. Compound 5 reacts with BzCl under DMAP as a base to give compound 6: ; In step S6, compound 6 undergoes a glycosylation reaction with uracil, cytosine, adenine, or guanine to yield compound 7; wherein, when compound 6 reacts with uracil, compound 7 is formed as follows: ; In step S7, compound 7 undergoes deprotection of the Bz protecting group under the action of sodium methoxide to obtain compound 8; when compound 6 reacts with uracil, compound 8 is formed as follows: ; In step S8, compound 8 undergoes a borohydride oxidation reaction under the conditions of 9-BBN and hydrogen peroxide to yield compound 9; when compound 6 reacts with uracil, compound 9 is formed as follows: ; The acylation reagent in step S9 is MsCl; compound 9 reacts with MsCl to give compound 10. When compound 6 reacts with uracil, compound 10 is formed as follows: ; In step S10, compound 10 undergoes intramolecular cyclization under the action of NaH or potassium tert-butoxide to yield compound 11; when compound 6 reacts with uracil, compound 11 is formed as follows: ; In step S11, the target molecule is generated by removing the benzyl hydroxyl protecting group using BCl3.

[0045] In the above embodiments, the structural formula of the obtained compound 7-11 is adjusted accordingly based on the change of the selected R1 group.

[0046] In some embodiments, in step S1, the molar ratio of methyl-D-furanoside to NaH base is 1:(3-5), and the molar ratio of methyl-D-furanoside to benzyl bromide (a Bn-protecting source) is 1:(3-4). In step S3, the molar ratio of compound 3 to the Desmond-Martin oxidant is 1:(2-5). In step S4, the molar ratio of compound 4 to the propenyl Grignard reagent is 1:(2-5). In step S5, the molar ratio of compound 5 to DMAP and benzoyl chloride is 1:(0.2-1):(2-5). In the glycosylation reaction of step S6, the molar ratio of compound 6 to uracil or cytosine or adenine or guanine and tin tetrachloride is controlled to be 1:(2-5):(3-7). In step S10, the molar ratio of compound 10 to NaH or potassium tert-butoxide is 1:(2-5). This further improves the synthesis efficiency of the reaction.

[0047] To fully understand the above scheme and clarify its application value, this application provides the following specific example using uracil. It should be noted that if uracil is replaced with cytosine, adenine, or guanine, the corresponding nucleotide monomer synthesis will also be obtained.

[0048]

[0049] Example 1 Synthesis of S1. (2R,3R,4R,5S)-3,4-bis(benzyloxy)-2-(benzyloxymethyl)-5-methoxytetrahydrofuran:

[0050] (2R,3S,4R,5S)-2-(hydroxymethyl)-5-methoxytetrahydrofuran-3,4-diol (50 g, 305 mmol) was dissolved in dimethylformamide (1 L), and sodium hydride (48.7 g, 1218 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 1 hour, and then tetrabutylammonium iodide (11.3 g, 30.5 mmol) and benzyl bromide (116 mL, 975 mmol) were slowly added at 0 °C. The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated aqueous solution of ammonium chloride (500 mL) and extracted three times with ethyl acetate (250 mL). The combined organic phase was washed with brine and dried over anhydrous sodium sulfate, then concentrated and evaporated to dryness. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 1 / 1) to give a yellow oily compound (2R,3R,4R,5S)-3,4-bis(benzyloxy)-2-(benzyloxymethyl)-5-methoxytetrahydrofuran (100 g, yield 75.6%).

[0051] LC-MS (ESI): m / z 435.53 [M+1] + . S2. Synthesis of (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol

[0052] (2R,3R,4R,5S)-3,4-bis(benzyloxy)-2-(benzyloxymethyl)-5-methoxytetrahydrofuran (25 g, 57.5 mmol) was dissolved in dichloromethane (420 mL), and tin tetrachloride (7 mL) was slowly added at 0 °C. The mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated aqueous solution of ammonium chloride (500 mL) and extracted three times with dichloromethane (150 mL). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, concentrated, and evaporated to dryness. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 1 / 1) to give a yellow oily compound (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol (15 g, 75.7% yield).

[0053] LC / MS (ESI): m / z 345.41 [M+1] + . S3. Synthesis of (2S,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxydihydrofuran-3(2H)-one

[0054] (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol (15 g, 43.6 mmol) was dissolved in dichloromethane (210 mL), and Dys-Martin oxidant (55.4 g, 131 mmol) was slowly added at 0 °C. The mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated, filtered, and poured into a saturated aqueous solution of sodium bicarbonate (250 mL). It was extracted three times with ethyl acetate (150 mL). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, concentrated, and evaporated to dryness to give a crude yellow oily compound (2S,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxydihydrofuran-3(2H)-one (16 g, 100% yield).

[0055] LC / MS (ESI): m / z 343.39 [M+1] + . Synthesis of S4. (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol

[0056] (2S,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxydihydrofuran-3(2H)-one (16 g, 46.7 mmol) was dissolved in tetrahydrofuran (200 mL) and heated at -78°C. o Allyl magnesium bromide (33.9 g, 234 mmol) was slowly added dropwise at C. After the addition was complete, the mixture was brought to room temperature and stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated aqueous solution of ammonium chloride (100 mL) and extracted three times with ethyl acetate (100 mL). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, concentrated, and evaporated to dryness. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 1 / 1) to give a yellow oily compound (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol (17 g, 94.6% yield).

[0057] LC / MS (ESI): m / z 385.47 [M+1]+ 。

[0058] Synthesis of S5. (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ylbenzoate

[0059] (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol (17 g, 44.2 mmol), 4-dimethylaminopyridine (2.70 g, 22.1 mmol), and triethylamine (122.7 mL, 884 mmol) were dissolved in dichloromethane (250 mL), and benzoyl chloride (18.6 g, 132.6 mmol) was slowly added at 0 °C. The mixture was stirred at room temperature for 72 hours. The reaction mixture was poured into a saturated aqueous solution of sodium bicarbonate (250 mL) and extracted three times with dichloromethane (150 mL). After the organic phases were combined, the product was washed with brine and dried over anhydrous sodium sulfate. The product was then concentrated and evaporated to dryness. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 1 / 1) to give a yellow oily compound (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ylbenzoate (21 g, yield 97.2%).

[0060] LC / MS (ESI): m / z 489.58 [M+1] + 。

[0061] Synthesis of S6. (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrido[2,4-b]pyrimidin-1(2H)yl)tetrahydrofuran-3-ylbenzoate

[0062] (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ylbenzoate (21 g, 43.6 mmol) and uracil (11.3 g, 100.28 mmol) were dissolved in anhydrous acetonitrile (300 mL). The mixture was refluxed for 1 hour and then cooled to room temperature. Tin tetrachloride (48.8 g, 187.48 mmol) was slowly added dropwise to the mixture while stirring at room temperature for another 1 hour. The mixture was refluxed for 1.5 hours and then cooled to room temperature. Ethyl acetate (250 mL) and a saturated sodium bicarbonate solution (250 mL) were added to the mixture. After separation, the organic phase was washed with brine and dried over anhydrous sodium sulfate, then concentrated and evaporated to dryness to obtain a yellow oily compound (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrido[1,2-b]pyrimidin-1(2H)-yl)tetrahydrofuran-3-ylbenzoate (20 g, crude product), which was directly used in the next step of the reaction.

[0063] LC / MS (ESI): m / z 569.2 [M+1] + 。

[0064] S7. Synthesis of 1-((2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0065] (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrido[1,2-b]pyrimidin-1(2H)-yl)tetrahydrofuran-3-ylbenzoate (20 g, 35.2 mmol) was dissolved in methanol (200 mL), and sodium methoxide (11.8 g, 218 mmol) was added at room temperature. The mixture was stirred overnight at room temperature. 30 mL of acetic acid was slowly added to the mixture. After concentration and drying, the crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 1 / 1) to give a yellow solid compound 1-((2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (13 g, yield 79.5%).

[0066] LC / MS (ESI): m / z 465.0 [M+1] + 。

[0067] 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.29 (s, 1H), 7.74 (d, J = 8.1 Hz, 1H),7.41 – 7.28 (m, 10H), 5.85 (s, 1H), 5.78 – 5.65 (m, 1H), 5.33 (s, 1H), 5.25(d, J = 8.1 Hz, 1H), 4.94 – 4.84 (m, 2H), 4.67 (d, J = 11.7 Hz, 1H), 4.54 (d, J = 11.7 Hz, 1H), 4.52 – 4.44 (m, 2H), 4.12 – 4.04 (m, 1H), 3.96 (d, J = 8.9Hz, 1H), 3.84 – 3.77 (m, 1H), 3.63 (dd, J = 11.3, 3.1 Hz, 1H), 2.38 (dd, J =15.0, 6.9 Hz, 1H), 2.18 (dd, J = 15.0, 6.8 Hz, 1H). S8. Synthesis of 1-((2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-3-(3-hydroxypropyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0068] 1-((2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione was dissolved in tetrahydrofuran (150 mL) and then... o C. Slowly add 9-boronbicyclo[3.3.1]nonane (336 mL, 168 mmol, 0.5 M tetrahydrofuran solution). Stir the mixture at room temperature for 3 hours, then cool to 0. oC, and slowly add 70 mL of saturated aqueous solution of NaOH (140 mmol) and 50 mL of 30% hydrogen peroxide (420 mmol). The mixture is stirred at room temperature for 2 hours. Add 500 mL of ethyl acetate and 500 mL of water to the mixture. After separation, the aqueous phase is extracted three times with 150 mL of ethyl acetate. Combine the organic phases, wash with brine, dry over anhydrous sodium sulfate, concentrate and evaporate to dryness to give a yellow oily compound 1-((2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-3-(3-hydroxypropyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (12 g, yield 88.9%), which is used directly in the next step of the reaction.

[0069] LC / MS (ESI): m / z 483.2 [M+1] + 。

[0070] Synthesis of S9. 3-((2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrido[1,2-b]pyrimidin-1(2H)yl)-3-hydroxytetrahydrofuran-3-yl)propyl methanesulfonate

[0071] 1-((2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-3-(3-hydroxypropyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (12 g, 24.9 mmol) was dissolved in anhydrous pyridine (150 mL) and dichloromethane (150 mL), and methanesulfonyl chloride (7.13 g, 62.25 mmol) was slowly added at 0 °C. The mixture was stirred at room temperature for 16 hours. After concentration and drying, the crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 1 / 1) to give a yellow liquid compound 3-((2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrido[1,2-b]pyrimidin-1(2H)yl)-3-hydroxytetrahydrofuran-3-yl)propyl methanesulfonate (11.8 g, yield 84.3%).

[0072] LC / MS (ESI): m / z 561.1 [M+1] + . S10. Synthesis of 1-((5R,6S,8R,9R)-9-(benzyloxy)-8-((benzyloxy)methyl)-1,7-dioxo-spiro[4.4]non-6-yl)pyrimidine-2,4(1H,3H)-dione

[0073] 3-((2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrido[1,2-b]pyrimidin-1(2H)yl)-3-hydroxytetrahydrofuran-3-yl)propyl methanesulfonate (11.8 g, 21 mmol) was dissolved in tetrahydrofuran (500 mL) and at 0 o C. Sodium hydroxide (4.2 g, 105 mmol) was slowly added, and the mixture was stirred at room temperature for 2 hours. After quenching the reaction with water, the solvent was evaporated to dryness. The mixture was separated using ethyl acetate (250 mL) and water (150 mL). The organic phase was washed with brine and dried over anhydrous sodium sulfate, then concentrated and evaporated to dryness. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (5 / 1 to 1 / 1) to give a yellow liquid compound 1-((5R,6S,8R,9R)-9-(benzyloxy)-8-((benzyloxy)methyl)-1,7-dioxo-spiro[4.4]non-6-yl)pyrimidine-2,4(1H,3H)-dione (8.1 g, yield 82.8%).

[0074] LC / MS (ESI): m / z 465.0 [M+1] + 。

[0075] 1 H NMR (400 MHz, DMSO) δ 11.40 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H),7.42 – 7.29 (m, 10H), 5.74 (s, 1H), 5.20 (d, J = 8.1 Hz, 1H), 4.62 (s, 2H), 4.55 – 4.48 (m, 2H), 4.02 (s, 2H), 3.85 (t, J = 9.4 Hz, 3H), 3.69 (d, J =11.3 Hz, 1H), 1.90 – 1.77 (m, 3H), 1.63 – 1.53 (m, 1H). S11. Synthesis of 1-((5R,6S,8R,9R)-9-hydroxy-8-(hydroxymethyl)-1,7-dioxo-spiro[4.4]nonane-6-yl)pyrimidine-2,4(1H,3H)-dione

[0076] 1-((5R,6S,8R,9R)-9-(benzyloxy)-8-((benzyloxy)methyl)-1,7-dioxo-spiro[4.4]non-6-yl)pyrimidine-2,4(1H,3H)-dione (8.1 g, 17.4 mmol) was dissolved in dichloromethane (100 mL) and heated at -70°C. o BCl3 (87 mL, 1 M in DCM) was slowly added dropwise, and the mixture was stirred at -70 °C for 2 hours. After the reaction was completed, methanol was slowly added to quench the reaction. The mixture was concentrated and evaporated to dryness, and the crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (5 / 1 to 1 / 1) to give a white solid compound 1-((5R,6S,8R,9R)-9-hydroxy-8-(hydroxymethyl)-1,7-dioxo-spiro[4.4]nonane-6-yl)pyrimidine-2,4(1H,3H)-dione, which is the compound shown in Formula I (4.11 g, yield 82.8%).

[0077] LC / MS (ESI): m / z 285.0 [M+1] + 。

[0078] 1 H NMR (400 MHz, DMSO) δ 11.39 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 5.76 (d, J = 4.4 Hz, 1H), 5.63 (d, J = 8.1 Hz, 1H), 5.19 (t, J = 4.6 Hz, 1H), 5.05 (d, J = 8.2 Hz, 1H), 3.87 (t, J = 6.4 Hz, 2H), 3.83 – 3.76 (m, 2H), 3.72(dt, J = 9.2, 2.1 Hz, 1H), 3.64 – 3.56 (m, 1H), 1.90 – 1.76 (m, 3H), 1.62 –1.52 (m, 1H). Example 2

[0079] This embodiment is basically the same as Embodiment 1, with the only difference being: The hydroxyl protecting agent in steps S1-S6 is replaced with benzoyl Bz; Step S7 is replaced by the following deprotection and silicon protection: S71. Synthesis of 1-((2R,3R,4R,5R)-3-allyl-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0080] (2R,3R,4R,5R)-3-allyl-5-(benzoyloxymethyl)-2-(2,4-dioxo-3,4-dihydropyrido[1,2-b]pyrimidin-1(2H)-yl)tetrahydrofuran-3,4-diol diphenyl ester (5 g, 8.38 mmol) was dissolved in methanol (50 mL), and sodium methoxide (2.76 g, 51.1 mmol) was added at room temperature. The mixture was stirred overnight at room temperature. After concentration and drying, the crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 1 / 1) to give a yellow solid compound 1-((2R,3R,4R,5R)-3-allyl-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (800 mg, 34% yield).

[0081] LC / MS (ESI): m / z 285.0 [M+1] + 。

[0082] S72. Synthesis of 1-((6aR,8R,9R,9aR)-9-allyl-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxodihydro-8-yl)pyrimidine-2,4(1H,3H)-dione

[0083] 1-((2R,3R,4R,5R)-3-allyl-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (900 mg, 3.17 mmol) was dissolved in anhydrous pyridine (50 mL), and triisopropylchlorosilane (672 mg, 3.49 mmol) was slowly added. The mixture was stirred at room temperature for 16 hours. After concentration and drying, the crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent (10 / 1 to 5 / 1) to give a yellow solid compound 1-((6aR,8R,9R,9aR)-9-allyl-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxodihydro-8-yl)pyrimidine-2,4(1H,3H)-dione (1.2 g, yield 71.9%).

[0084] LC / MS (ESI): m / z 527.1 [M+1] + . The subsequent steps included hydroboration oxidation (S8), acylation (S9), and intramolecular cyclization (S10), followed by removal of the Si protecting group in step S11 to obtain the same molecular product. It can be seen that due to the very low yield in step S7, even if the target product is eventually synthesized, there is a significant disadvantage.

[0085] Example 3 This embodiment is basically the same as Embodiment 1, with the only difference being: In step S4, the amount of propylene Grignard reagent added was 13.5 g, 93.4 mmol. A yellow oily compound (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol (16.5 g, 92% yield) was obtained.

[0086] LC / MS (ESI): m / z 385.47 [M+1] + 。

[0087] Example 4 This embodiment is basically the same as Embodiment 1, with the only difference being: In step S3, the amount of Desmond-Martin oxidant added was 36.9 g, 87.2 mmol. The same product was obtained, which was compound 4 (15.7 g, yield 98%).

[0088] Example 5 This embodiment is basically the same as Embodiment 1, with the only difference being: In step S5, 1.1 g of 4-dimethylaminopyridine (8.84 mmol) and 12.4 g of benzoyl chloride (88.4 mmol) were added. The same product, compound 6 (20 g, 92.6% yield), was obtained.

[0089] Example 6 This embodiment is basically the same as Embodiment 1, with the only difference being: In step S5, 5.4 g of 4-dimethylaminopyridine (44.2 mmol) and 31 g of benzoyl chloride (221 mmol) were added. The same product was obtained, which is compound 6 (21.3 g, yield 98.5%).

[0090] Example 7 This embodiment is basically the same as Embodiment 1, with the only difference being: In the glycosylation reaction of step S6, 9.8 g of uracil (87.2 mmol) and 34 g of tin tetrachloride (130.8 mmol) were added. The same product, compound 7 (19.8 g, crude), was obtained and directly used in the next step of the reaction. In step S7, compound 8 (12.8 g, yield 78.3%) was obtained.

[0091] Example 8 This embodiment is basically the same as Embodiment 1, with the only difference being: In the glycosylation reaction of step S6, 24.6 g of uracil (218 mmol) and 79.4 g of tin tetrachloride (305.2 mmol) were added. The same product, compound 7 (22 g, crude), was obtained and directly used in the next step of the reaction. In step S7, compound 8 (13.8 g, yield 84.4%) was obtained.

[0092] Example 9 This embodiment is basically the same as Embodiment 1, with the only difference being: In step S10, the amount of sodium hydrogen added was (1.7 g, 42 mmol). The same product was obtained, which was compound 11 (7.8 g, yield 80%).

[0093] Example 10 This embodiment is basically the same as Embodiment 1, with the only difference being: In step 10, potassium tert-butoxide was used instead of NaH under alkaline conditions. A yellow liquid compound, 1-((5R,6S,8R,9R)-9-(benzyloxy)-8-((benzyloxy)methyl)-1,7-dioxo-spiro[4.4]non-6-yl)pyrimidine-2,4(1H,3H)-dione (8.3 g, 85% yield), was obtained.

[0094] Based on the above-described preferred embodiments of the present invention, those skilled in the art can make various changes and modifications without departing from the inventive concept, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing a nucleotide monomer, characterized in that, Includes the following steps: ； Starting with methyl-D-furanoside, the monomer of the compound shown in formula P was synthesized by sequentially performing selective protection, selective deprotection, C / C bond construction, acyl chloride reaction, glycosylation to introduce a base, deprotection, hydroboration oxidation and cyclization steps. In the formula: R1 represents any one of the following groups I to IV: ； R2 represents a H atom or a DMTr group, and Z represents an O atom.

2. The preparation method according to claim 1, characterized in that: This preparation method uses methyl-D-furanoside as the starting material, selectively protects all hydroxyl groups with a hydroxyl protecting group, then selectively removes the protection at the target position, oxidizes to a ketone, reacts with an olefin Grignard reagent, protects the hydroxyl group, then glycosylates and attaches a base, selectively removes the hydroxyl protection at the target position, oxidizes with borohydride, introduces an acylation reagent, closes the five-membered oxygen ring, and finally removes the protection to obtain the target product.

3. The preparation method according to claim 2, characterized in that, The preparation method includes the following steps: Starting with S1 and methyl-D-furanoside, protecting groups were introduced to the 2', 3' and 5' hydroxyl groups to obtain compound 2; S2 and compound 2 selectively remove the protecting group on the 2' hydroxyl group to generate compound 3; S3 and compound 3 undergo an oxidation reaction to oxidize the hydroxyl group at the 2' position to a ketone to obtain compound 4; S4, compound 4, and olefin Grignard reagent react to give compound 5 with an olefin group and a hydroxyl group at the 2' position; S5 and compound 5 continue to undergo acylation of the hydroxyl group at the 2' position to give compound 6; S6, Compound 6 undergoes a glycosylation reaction with a pyrimidine or purine base at the 1' position to give Compound 7; S7 and compound 7 are deprotected at the 2' position to obtain compound 8; S8 and compound 8 were given compound 9 via a hydroboration oxidation reaction; S9 and compound 9 react with an acylation reagent at the 2' position to give compound 10; S10 and compound 10 undergo intramolecular cyclization at the 1' and 2' positions under basic conditions to yield compound 11; S11 removes the benzyl hydroxyl protecting group from compound 11 to generate the corresponding target molecule.

4. The preparation method according to claim 3, characterized in that: In step S1, benzyl Bn is used as the hydroxyl protecting group, and NaH is used as the base. The compound 2 obtained by introducing protecting groups at the 2', 3', and 5' hydroxyl positions is: ； In step S2, compound 2 undergoes selective removal of the protecting group at the 2' hydroxyl position under the action of SnCl4, resulting in compound 3: 。 5. The preparation method according to claim 3, characterized in that: In step S3, the hydroxyl group at the 2' position of the compound is oxidized to a ketone by the Des Martin oxidant, resulting in compound 4: ； The olefin-based Grignard reagent used in step S4 is a vinyl or propylene Grignard reagent. When a propylene Grignard reagent is used, the temperature is -78°C. o Compound 5, obtained by reacting C with an olefinic Grignard reagent, is: 。 6. The preparation method according to claim 3, characterized in that: The acylation reagent in step S5 is BzCl. Compound 5 reacts with BzCl under DMAP as a base to give compound 6: ； In step S6, compound 6 undergoes a glycosylation reaction with uracil, cytosine, adenine, or guanine to yield compound 7; wherein, when compound 6 reacts with uracil, compound 7 is formed as follows: ； In step S7, compound 7 undergoes deprotection of the Bz protecting group under the action of sodium methoxide to obtain compound 8; when compound 6 reacts with uracil, compound 8 is formed as follows: 。 7. The preparation method according to claim 6, characterized in that: In step S8, compound 8 undergoes a borohydride oxidation reaction under the conditions of 9-BBN and hydrogen peroxide to yield compound 9; when compound 6 reacts with uracil, compound 9 is formed as follows: ； The acylation reagent in step S9 is MsCl; compound 9 reacts with MsCl to give compound 10. When compound 6 reacts with uracil, compound 10 is formed as follows: ； In step S10, compound 10 undergoes intramolecular cyclization under the action of NaH or potassium tert-butoxide to yield compound 11; when compound 6 reacts with uracil, compound 11 is formed as follows: ； In step S11, the target molecule is generated by removing the benzyl hydroxyl protecting group using BCl3.

8. The preparation method according to claim 4, characterized in that: The molar ratio of methyl-D-furanoside to NaH base is 1:(3-5), and the molar ratio of methyl-D-furanoside to the protecting benzyl group is 1:(3-4).

9. The preparation method according to claim 5, characterized in that: In step S3, the molar ratio of compound 3 to the Des Martin oxidant is 1:(2-5); and / or; In step S4, the molar ratio of compound 4 to propylene Grignard reagent is 1:(2-5).

10. The preparation method according to claim 7, characterized in that: In step S5, the molar ratio of compound 5 to DMAP and benzoyl chloride is 1:(0.2-1):(2-5); and / or; In the glycosylation reaction of step S6, the molar ratio of compound 6 to uracil or cytosine or adenine or guanine, and tin tetrachloride is controlled to be 1:(2-5):(3-7); and / or; In step S10, the molar ratio of compound 10 to NaH or potassium tert-butoxide is 1:(2-5).

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