A ribose ring-modified mRNA cap analog, and methods of making and using the same

Abstract

The application provides a ribose ring modified mRNA cap analog and a preparation method and application thereof, wherein hydrogen atoms at R1, R4, R5, R7 and R8 positions connected with carbon atoms on a five-membered sugar ring in the following structure are independently and selectively substituted and modified by OH, alkyl, O-alkyl, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl and halogen; after the modification, the five-membered sugar ring has a molecular conformation advantage, a stronger elF4E protein activity pocket binding capacity, the structure is not easily recognized by a decapping enzyme, the cap structure is more stable, and finally a better mRNA translation effect is shown.

Description

Technical Field

[0001] This invention relates to the fields of chemical and biological engineering technology, specifically to a ribose ring-modified mRNA cap analogue, its preparation method, and its application. Background Technology

[0002] Eukaryotic mRNA possesses a "cap" structure at its 5' end, which is known to play a crucial role in translation. The naturally occurring cap structure consists of 7-methylguanosine, which is linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge, resulting in 7G(5')ppp(5')N, where N is any nucleotide. The mRNA cap plays a vital role in gene expression. The cap structure protects mRNA from degradation by exonucleases, allows RNA to be transported from the nucleus to the cytoplasm, and participates in the assembly of the translation initiation complex. m7G(5')ppp(5')G (mCAP) has been used as a primer for in vitro transcription by T7 or SP6 RNA polymerases to obtain RNA with a cap structure at its 5' end.

[0003] In vitro transcription synthesis of mRNA has become an important tool for introducing foreign genes and expressing proteins, and is widely used in the treatment and prevention of diseases. In vitro transcription synthesis of mRNA enables researchers to prepare RNA molecules that behave appropriately in various biological applications. These applications include research applications and commercial production of peptides, such as producing peptides containing "non-natural" amino acids at specific sites in cell-free translation systems, or producing peptides in cultured cells that require post-translational modifications for their activity or stability. In the latter system, synthesis takes significantly longer and therefore yields more protein.

[0004] The binding affinity of the cap structure to the eukaryotic initiation factor (elF4E) determines the translational expression efficacy of the mRNA within the cell. Existing cap analogs are generally natural cap structures, and their mRNA translation efficacy is unstable, showing significant differences for different sequences or different expression hosts. Summary of the Invention

[0005] This application provides a ribocycle-modified mRNA cap analog, its preparation method, and its application. In this mRNA cap analog structure, the hydrogen atoms attached to the carbon atoms on the five-membered sugar ring are independently substituted with OH, alkyl, O-alkyl, halogen, alkenyl, alkynyl, and cyano groups. The modified five-membered sugar ring has a stronger binding affinity to the active pocket of the elF4E protein, thus promoting the translation of the mRNA. Furthermore, because this structure is non-natural, it cannot be hydrolyzed by ribozymes in vivo and is not easily recognized by uncapping enzymes, making the cap structure more stable and ultimately resulting in better mRNA translation and a lower uncapping rate.

[0006] A ribocyclic modified mRNA cap analog comprising the following structure:

[0007]

[0008] Among them, X1, X2 and X3 are independently O, CH2 or NH;

[0009] Y1, Y2 and Y3 are independently O, S, Se or BH3;

[0010] R1, R2, R3, R4, R5, R8, R9, R 12 R 13 R 14 Independently, they are H, OH, alkyl, O-alkyl, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, and halogen;

[0011] And R2, R 13 Not simultaneously OH, O-alkyl, R3, R 14 They are not both OH or O-alkyl;

[0012] R6, R7, R 10 R 11 Independently, they are H, alkyl, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, and halogen;

[0013] B1 and B2 are either natural, modified, or non-natural nucleoside bases.

[0014] The above X1, X2 and X3 are independently O, CH2 or NH respectively; Y1, Y2 and Y3 are independently O or S respectively.

[0015] The above R1, R2, R3, R4, R5, R8, R9, R 12 R 13 R 14 Independently, it can be H, OH, methyl, ethyl, propyl, or butyl; R6, R7, R 10 R 11 The individual components are H, methyl, ethyl, propyl, or butyl.

[0016] The B1 and B2 mentioned above are adenine, guanine, cytosine, uracil, and thymine, respectively.

[0017] X1, X2, X3, Y1, Y2, and Y3 are all 0; R1, R5, R7, R9, and R 11 All are H; R2, R 13 One is OH, and the other is H or CH3; R3, R 14 One of them is OH and the other is H; R4, R6, R8, R10 R 12 It is H or CH3; B1 is adenine, B2 is guanine; the specific structure is shown below:

[0018]

[0019]

[0020] X1, X2, X3, Y1, Y2, and Y3 are all 0; R1, R5, R7, R9, and R 10 R 11 R 12 All are H; R2, R 13 One is OH, and the other is H or CH3; R3, R 14 One of them is OH and the other is H; R4, R6, and R8 are H or CH3; B1 is adenine and B2 is guanine.

[0021] Furthermore, in order to improve both the capping rate and translation efficiency of capped analogs, this invention provides a ribocycle-modified mRNA capped analog, which is a compound of formula (I), a stereoisomer, or a pharmaceutically acceptable salt:

[0022]

[0023] in,

[0024] X1, X2, and X3 are independently O, CH2, or NH, respectively;

[0025] Y1, Y2 and Y3 are independently O, S, Se or BH3, respectively;

[0026] R1, R4, R5, R7, and R8 are respectively H, OH, halogen, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkenyl, C 1-6 Alkyne or cyano; and at least one of R1, R4, R5, R7, and R8 is OH, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkenyl, C 1-6 Alkyne group, cyano group; the C 1-6 The alkoxy group was not substituted or was replaced by R. 15 Replace, R 15 C 1-6 Alkoxy;

[0027] R2, R3, R 13 R 14 They are H, OH, and C, respectively. 1-6 Alkyl, C1-6 Alkoxy groups, halogens;

[0028] R6, R9, R 10 R 11 R 12 H and C independently 1-6 Alkyl, C 1-6 Alkoxy groups, halogens;

[0029] B1 and B2 are either natural, modified, or non-natural nucleoside bases.

[0030] The ribosome-modified mRNA cap analogs, R2 and R 13 At least one of them is H, R3 and R 14 At least one of them is H.

[0031] The ribosome-modified mRNA cap analogues described herein, wherein only one of R1, R4, R5, R7, or R8 is OH, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkenyl, C 1-6 Alkyne group, cyano group; the C 1-6 The alkoxy group was not substituted or was replaced by R. 15 Replace, R 15 C 1-6 Alkyl group; the other four of R1, R4, R5, R7 or R8 are each independently H.

[0032] The ribosome-modified mRNA cap analogue is a compound of formula (II), a stereoisomer, or a pharmaceutically acceptable salt:

[0033]

[0034] Only one of R1 or R5 is OH, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkenyl, C 1-6 Alkyne group, cyano group; the C 1-6 The alkoxy group was not substituted or was replaced by R. 15 Replace, R 15 C 1-6 alkoxy group; the other of R1 or R5 is H;

[0035] R 13 R 14 They are H, OH, and C, respectively. 1-6 Alkyl, C 1-6 Alkoxy groups, halogens;

[0036] B1 and B2 are either natural, modified, or non-natural nucleoside bases.

[0037] The ribosome-modified mRNA cap analogue, R 13 R 14 Each is an OH group, which can be converted to OH independently.

[0038] The ribosome-modified mRNA cap analogue, wherein R1 is selected from ethynyl, 1-propynyl, 2-propynyl, butynyl, cyano, hydroxyl, methoxy, ethoxy, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, F or Cl; and R5 is H.

[0039] Alternatively, R5 can be selected from ethynyl, 1-propynyl, 2-propynyl, butynyl, cyano, hydroxyl, methoxy, ethoxy, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, F, or Cl; R1 is H.

[0040] The ribocycle-modified mRNA cap analogue is a compound of formula (III), a stereoisomer, or a pharmaceutically acceptable salt:

[0041]

[0042] R1 is OH, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkenyl, C 1-6 Alkyne group, cyano group; the C 1-6 The alkoxy group was not substituted or was replaced by R. 15 Replace, R 15 C 1-6 Alkoxy;

[0043] R 13 R 14 They are H, OH, and C, respectively. 1-6 Alkyl, C 1-6 Alkoxy groups, halogens;

[0044] B1 and B2 are either natural, modified, or non-natural nucleoside bases.

[0045] The ribosome-modified mRNA cap analogue, wherein R1 is ethynyl, 1-propynyl, 2-propynyl, butynyl, cyano, hydroxyl, methoxy, ethoxy, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, F or Cl.

[0046] The ribosome-modified mRNA cap analogue, R 13 R 14Each can be independently H, OH, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, F, or Cl.

[0047] The ribosome-modified mRNA cap analogues described herein are the following compounds, their stereoisomers, or pharmaceutically acceptable salts thereof:

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] The ribocycle-modified mRNA cap analogs described in this application have higher yield, higher capping rate, higher translation efficiency, and lower uncapping rate.

[0055] This application also provides the application of the ribose ring-modified mRNA cap analogue described above in the preparation of an in vitro co-transcribed RNA capping reagent.

[0056] The ribocycle-modified mRNA cap analogue is used to prepare an in vitro co-transcriptional RNA capping reagent. This ribocycle-modified mRNA cap analogue is used for mRNA capping in either the T7 RNA polymerase system or the SP6 RNA polymerase system. T7 RNA polymerase is a DNA-dependent RNA polymerase with high specificity for the bacteriophage T7 promoter sequence. This enzyme inserts into the DNA downstream of the transcription vector from the T7 promoter to synthesize large amounts of RNA. The T7 RNA polymerase-catalyzed IVT (in vitro transcription) reaction system is currently the most mature mRNA preparation system.

[0057] Typically, adding T7 RNA polymerase or SP6 RNA polymerase to the IVT reaction system, along with the capping analogue described in this invention, can yield capped mRNA.

[0058] This application also provides a DNA template of the ribocycle-modified mRNA cap analog, wherein the DNA template includes a promoter region containing a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and B1 is complementary to the nucleoside base at position +1 of the DNA template, and B2 is complementary to the nucleoside base at position +2 of the transcription template on the DNA template.

[0059] This application also provides an RNA molecule comprising the ribocycle-modified mRNA cap analogue described in any of the preceding claims.

[0060] This application also provides a pharmaceutical composition comprising the RNA molecule described above, and a pharmaceutically acceptable carrier.

[0061] This invention provides a ribocycle-modified mRNA cap analog, its preparation method, and its application. The ribocycle-modified mRNA cap analog is suitable for producing mRNA using an in vitro co-transcription method with a DNA sequence as a template. The DNA sequence can be derived from or modified from species such as viruses, animals, and plants. At the same time, the mRNA produced by it has lower immunogenicity, higher protein translation efficiency, and better stability.

[0062] The present invention has the following advantages over the prior art:

[0063] Compared to existing cap-structure analogs like Cleancap, the novel cap structure designed in this invention contains substituents in a five-membered sugar ring, which effectively fixes the spatial structure of the 5' cap and enhances the binding ability of the 5' cap structure of mRNA to the eukaryotic initiation factor (elF4E), thereby improving mRNA translation efficiency. The ribocycle-modified mRNA cap analog of this invention exhibits stronger binding ability to the elF4E protein active pocket due to the conformational advantage of the modified five-membered sugar ring. Simultaneously, this structure is less easily recognized by uncapping enzymes, making the cap structure more stable. Ultimately, it demonstrates better in vitro transcription yield and capping efficiency, better mRNA translation performance, and a lower uncapping rate. Detailed Implementation

[0064] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0065] Terminology Explanation:

[0066] In this invention, "eq." represents equivalent.

[0067] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 6 (e.g., 1, 2, 3, 4, 5, and 6) carbon atoms, and more preferably an alkyl group containing 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted.

[0068] Term "C" 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. C 1-6 Examples of alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, isobutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc.

[0069] Term "C" 1-6 "Alkoxy" refers to the -OR group, where R is defined as "C1-6 alkyl" as mentioned above.

[0070] Term "C" 1-6 "Alkenyl" refers to an alkyl group that contains at least one carbon-carbon double bond and comprises 1 to 6 carbon atoms, as defined above.

[0071] Term "C" 1-6 "Alkyne" refers to a straight or branched alkyl group having one or more carbon-carbon triple bonds and containing 1 to 6 carbon atoms, as defined above.

[0072] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0073] In this application, the terms "base" and "nucleoside base" are used interchangeably and can refer to either natural nucleoside bases or modified nucleoside bases. Natural nucleoside bases include, but are not limited to, any one of adenine (A), guanine (G), cytosine (C), uracil (U), thymine (T), and their derivatives.

[0074] "Modified base" and "modified nucleoside base" can be used interchangeably. They refer to substances obtained by replacing one or more hydrogen atoms of a natural nucleoside base, such as including but not limited to N6-methyladenine. In this embodiment, the modifying group is selected from N6-methyladenine, N1-methyladenine, N6-2'-O-dimethyladenine, pseudouridine, N1-methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N1-methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N1-methylguanine, N1-methylguanine, and isoguanine.

[0075] "Stereoisomers" are compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers.

[0076] The names and sources of the raw materials used in each embodiment are shown in Table 1 below:

[0077] Table 1

[0078]

[0079]

[0080] The reaction route for intermediate A used in the following examples was prepared by the steps shown in equation (1):

[0081]

[0082] 1) Dissolve 20 g of 2,3-O-isopropylidene-β-D-methylfuranoside in 300 mL of acetonitrile. Add 1.8 eq. of 2-iodobenzoic acid to the reaction solution at room temperature. Heat the resulting suspension under reflux for 5 hours and then leave it at room temperature overnight. Filter the suspension, wash with ethyl acetate, and concentrate the filtrate to obtain intermediate A1.

[0083] 2) Dissolve 20g of intermediate A1 and formaldehyde aqueous solution (37%, 76mL) in 380mL of 1,4-dioxane. Add NaOH aqueous solution (2.0M, 188mL) to the reaction solution at 0℃. After stirring at room temperature overnight, neutralize with 10% acetic acid, concentrate and then obtain intermediate A2 by column chromatography.

[0084] 3) Dissolve 20 g of intermediate A2 and 1,4'-bismethoxytriphenylmethyl chloride in pyridine (400 mL) and stir overnight at room temperature. After the reaction is complete, quench the reaction with methanol (40 mL) at 0 °C. After concentration, column chromatography yields intermediate A3.

[0085] 4) Dissolve 20g of intermediate A3 in 200.0mL of anhydrous pyridine. Add benzoyl chloride (1.2 eq.) dropwise to the reaction solution under ice bath conditions. Stir at room temperature for 3 hours until the reaction is complete. Concentrate to obtain crude intermediate A4.

[0086] 5) Dissolve the crude intermediate A4 in an 80% aqueous acetic acid solution and stir overnight at room temperature until the reaction is complete. After concentration, column chromatography yields intermediate A5.

[0087] 6) Dissolve 15g of intermediate A5, DMAP (3.0 eq.), and phenyl thiochloroformate (1.2 eq.) in 150.0 mL of acetonitrile and stir at room temperature for 3 hours until the reaction is complete. After concentration, dissolve in dichloromethane, wash twice with 0.5 M dilute hydrochloric acid, dry the organic phase with anhydrous sodium sulfate, and concentrate to obtain crude intermediate A6.

[0088] 7) Crude intermediate A6 was dissolved in 150.0 mL of toluene. Tris(trimethylsilyl)silane (1.5 eq.) and 1,1'-azo(cyanocyclohexane) (0.25 eq.) were added to the reaction solution, and the temperature was raised to 100 °C. After stirring for 15 hours, the reaction was completed. The reaction solution was concentrated and then subjected to column chromatography to obtain intermediate A7.

[0089] 8) Dissolve 5 g of intermediate A7 in cold trifluoroacetic acid / water (9:1, 50.0 mL), then stir at 0 °C for 1 hour until the reaction is complete. Concentrate at low temperature to remove the solvent, then dissolve in 50.0 mL of glacial acetic acid and acetic anhydride (3.0 eq.), and slowly add concentrated sulfuric acid (0.1 eq.) dropwise under ice bath. Stir overnight at room temperature until the reaction is complete. Pour the reaction solution into ice water and extract with dichloromethane. Dry the organic phase and concentrate, then perform column chromatography to obtain intermediate A.

[0090] The reaction route for intermediate B used in the following examples is prepared by the steps shown in equation (2):

[0091]

[0092] 1) 40 g of Des Martin oxidant was suspended in 200.0 mL of dichloromethane and cooled to an ice bath. 20 g of 1,3,5-tribenzoyl-D-furanose was added to the reaction solution. The mixture was stirred overnight at room temperature until the reaction was complete. The reaction solution was concentrated and suspended in methyl tert-butyl ether and slurried, then filtered. The filtrate was washed with 15% sodium thiosulfate, cold saturated sodium bicarbonate aqueous solution, and saturated brine, and dried over anhydrous sodium sulfate. The solution was then concentrated to obtain intermediate B1.

[0093] 2) Dissolve 17.5 g of intermediate B1 in 175.0 mL of tetrahydrofuran. Add methyl magnesium bromide (3 M in Et₂O, 3.0 eq.) dropwise to the reaction solution at -78 °C. Stir at -78 °C for 2 hours until the reaction is complete. Quench the reaction solution in a saturated ammonium chloride solution, then extract with ethyl acetate. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and concentrate to obtain crude intermediate B2.

[0094] 3) Take 18g of crude intermediate B2, dissolve DMAP (2.0 eq.) and triethylamine (3.0 eq.) in 180.0 mL of dichloromethane, cool to 0℃, and then add isobutyryl chloride (3.0 eq.) dropwise to the reaction solution. Stir overnight at room temperature. Wash the reaction solution with saturated sodium bicarbonate, and extract the aqueous phase with dichloromethane. Combine the organic phases, dry to anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain intermediate B.

[0095] Example 1: Synthesis of ribosome-modified mRNA cap analogs using intermediates C and D as raw materials

[0096] Intermediate C (2 mol) was dissolved in a DMF solution containing MnCl2 (20 mol), and then intermediate D (1.8 mol) was added to the reaction solution. The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB elution buffer. The target product was concentrated, and the reaction route is shown in equation (3):

[0097]

[0098] Compound C is obtained through the following steps:

[0099] 1) BSA (2.0 eq.) was added dropwise to a toluene suspension of N2-isobutyryloylguanine (1.2 eq.) at room temperature. The mixture was heated to reflux and stirred for 1 hour to obtain a clear solution. The solution was then cooled to room temperature, and intermediate A (50 g) and trimethylsilyl trifluoromethanesulfonate (1.5 eq.) were added. The mixture was then heated to reflux and the reaction was continued for 1 hour. After the reaction was complete, the reaction solution was cooled to room temperature, and the pH was adjusted to neutral with saturated sodium bicarbonate aqueous solution. The solution was extracted with ethyl acetate and concentrated. The crude product was purified by column chromatography to obtain intermediate C1.

[0100] 2) Dissolve 30g of intermediate C1 in 300.0mL of ammonia / methanol (1:1) and stir overnight at room temperature. After the reaction is complete, concentrate and purify by reversed-phase chromatography to obtain intermediate C2.

[0101] 3) Dissolve intermediate C2 in 150 ml of trimethyl phosphate, cool the reaction solution to 0 °C, slowly add 1.2 eq. of phosphorus oxychloride, stir for 4 hours in an ice bath, quench the reaction with water, and purify by reverse phase chromatography to obtain intermediate C3.

[0102] 4) Intermediate C3, triphenylphosphine (1.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred thoroughly for 10 hours. After the reaction was completed, 4M sodium perchlorate acetone solution was added to precipitate the mixture. The filter cake was washed thoroughly with acetone to obtain intermediate C4.

[0103] 5) Dissolve 10g of intermediate C4 in DMF, add tributylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.), and stir at room temperature for 5 hours. After the reaction is complete, ion chromatography is used to purify intermediate C5.

[0104] 6) Dissolve intermediate C5 in 20 times its volume of purified water, cool the reaction solution to 4°C, and slowly add dimethyl sulfate dropwise. During the process, adjust the pH to not exceed 5 with 2M sodium hydroxide. Monitor the reaction by HPLC, and after the reaction is complete, ion chromatography is used to purify and obtain intermediate C6.

[0105] 7) Intermediate C6, triphenylphosphine (1.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred thoroughly for 10 hours. After the reaction was completed, 4M sodium perchlorate acetone solution was added to precipitate the intermediate. The filter cake was thoroughly washed with acetone to obtain intermediate C. The reaction route is shown in equation (4):

[0106]

[0107] Compound D is obtained through the following steps:

[0108] 1) Weigh 1 kg of 2'OMe-rA phosphorous amide monomer and dissolve it in dichloromethane (6.0 L). Add 2',3'-acetylguanosine (1.0 eq.) at room temperature. Add tetrazolium (1.0 eq.) under ice bath conditions, then heat to 25 °C and stir for 5 hours. After the reaction is complete as monitored by TLC, add 1.2 eq. of iodopyridine solution to the reaction solution. After the reaction is complete as monitored by TLC, wash the reaction solution with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine, and then concentrate the organic phase. Dissolve the concentrated oil in 4 L of dichloromethane, and slowly add trifluoroacetic acid (4.0 eq.) under ice bath conditions, stirring at room temperature for 2 hours. After the reaction is complete as monitored by TLC, wash the reaction solution with saturated sodium bicarbonate solution and saturated brine, and then concentrate the organic phase. Column chromatography yields intermediate D1.

[0109] 2) Dissolve D1 in 4L of dichloromethane. Add 1.5 eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide and 1.5 eq. of tetrazolium to the reaction solution at room temperature and stir for 3 hours. After the reaction is completed by TLC monitoring, add 1.2 eq. of iodopyridine solution to the reaction solution. After the reaction is completed by TLC monitoring, wash the reaction solution with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine respectively, and then concentrate the organic phase. Dissolve the crude product in 3L of methanol and 3L of concentrated ammonia water and react at room temperature for 14 hours. Monitor the reaction and concentrate after the reaction is completed. Load the crude product onto a DEAE Sephadex column and elute with a linear gradient of 0-1.0M TEAB elution buffer. Concentrate to obtain the target product. The reaction route is shown in equation (5):

[0110]

[0111] Example 2:

[0112] Synthetic method of ribocyclic modified mRNA cap analogs using intermediates D and E as raw materials

[0113] Intermediate E (2 mol) was dissolved in a DMF solution containing MnCl2 (20 mol), and then intermediate D (1.8 mol) was added to the reaction solution. The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB elution buffer. The target product was concentrated, and the reaction route is shown in equation (6):

[0114]

[0115] Compound E is obtained through the following steps:

[0116] 1) BSA (2.0 eq.) was added dropwise to a toluene suspension of N2-isobutyryloylguanine (1.2 eq.) at room temperature. The mixture was heated to reflux and stirred for 1 hour to obtain a clear solution. The solution was then cooled to room temperature, and intermediate B (50 g) and trimethylsilyl trifluoromethanesulfonate (1.5 eq.) were added. The mixture was then heated to reflux and the reaction was continued for 1 hour. After the reaction was complete, the reaction solution was cooled to room temperature, and the pH was adjusted to neutral with saturated sodium bicarbonate aqueous solution. The solution was extracted with ethyl acetate and concentrated. The crude product was purified by column chromatography to obtain intermediate E1.

[0117] 2) Dissolve 30g of intermediate E1 in 300.0mL of ammonia / methanol (1:1) and stir overnight at room temperature. After the reaction is complete, concentrate and purify by reversed-phase chromatography to obtain intermediate E2.

[0118] 3) Dissolve intermediate E2 in 150 ml of trimethyl phosphate, cool the reaction solution to 0°C, slowly add 1.2 eq. of phosphorus oxychloride, stir for 4 hours in an ice bath, quench the reaction with water, and purify by reverse phase chromatography to obtain intermediate E3.

[0119] 4) Intermediate E3, triphenylphosphine (1.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred thoroughly for 10 hours. After the reaction was completed, 4M sodium perchlorate acetone solution was added to precipitate the mixture. The filter cake was washed thoroughly with acetone to obtain intermediate E4.

[0120] 5) Dissolve 10g of intermediate E4 in DMF, add tributylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.), and stir at room temperature for 5 hours. After the reaction is complete, ion chromatography is used to purify intermediate E5.

[0121] 6) Dissolve intermediate E5 in 20 times its volume of purified water, cool the reaction solution to 4°C, and slowly add dimethyl sulfate dropwise. During the process, adjust the pH to not exceed 5 with 2M sodium hydroxide. Monitor the reaction by HPLC, and after the reaction is completed, ion chromatography is used to purify and obtain intermediate E6.

[0122] 7); Intermediate E6, triphenylphosphine (1.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred thoroughly for 10 hours. After the reaction was completed, 4M sodium perchlorate acetone solution was added to precipitate the intermediate. The filter cake was thoroughly washed with acetone to obtain intermediate E. The reaction route is shown in equation (7):

[0123]

[0124] Example 3:

[0125] Synthetic method of ribocyclic modified mRNA cap analogs using intermediates F and G as raw materials

[0126] Intermediate F (2 mol) was dissolved in a DMF solution containing MnCl2 (20 mol), and then intermediate G (1.8 mol) was added to the reaction solution. The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The target product was concentrated. The reaction route is shown in equation (8).

[0127]

[0128] Intermediate F is obtained through the following steps:

[0129] 1) Weigh 5g of guanosine and dissolve it in 70.0ml of trimethyl phosphate. Cool the reaction solution to 0℃ and slowly add phosphorus oxychloride (1.8eq.). Stir for 4 hours in an ice bath, then quench the reaction with water and purify by reverse phase chromatography to obtain intermediate F1.

[0130] 2) Intermediate F1, triphenylphosphine (1.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred thoroughly for 10 hours. After the reaction was completed, 4M sodium perchlorate acetone solution was added to precipitate the product. The filter cake was thoroughly washed with acetone to obtain intermediate F2.

[0131] 3) Dissolve 10g of intermediate F2 in DMF, add tributylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.), and stir at room temperature for 5 hours. After the reaction is complete, ion chromatography is used to purify intermediate F3.

[0132] 4) Dissolve intermediate F3 in 20 times its volume of purified water, cool the reaction solution to 4°C, and slowly add dimethyl sulfate dropwise. During the process, adjust the pH to not exceed 5 with 2M sodium hydroxide. Monitor the reaction by HPLC, and after the reaction is completed, ion chromatography is used to purify and obtain intermediate F4.

[0133] 5) Intermediate F4, triphenylphosphine (1.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred thoroughly for 10 hours. After the reaction was completed, 4M sodium perchlorate acetone solution was added to precipitate the product. The filter cake was thoroughly washed with acetone to obtain intermediate F. The reaction route is shown in equation (10):

[0134]

[0135] Compound G is obtained through the following steps:

[0136] 1) BSA (2.0 eq.) was added dropwise to a toluene suspension of adenine (1.2 eq.) at room temperature. The mixture was heated to reflux and stirred for 1 hour to obtain a clear solution. The solution was then cooled to room temperature, and intermediate A (50 g) and trimethylsilyl trifluoromethanesulfonate (1.5 eq.) were added. The mixture was then heated to reflux and the reaction was continued for 1 hour. After the reaction was complete, the reaction solution was cooled to room temperature, and the pH was adjusted to neutral with saturated sodium bicarbonate aqueous solution. The solution was extracted with ethyl acetate and concentrated. The crude product was purified by column chromatography to obtain intermediate G1.

[0137] 2) Dissolve 30g of intermediate G1 in 300.0mL of ammonia / methanol (1:1) and stir overnight at room temperature. After the reaction is complete, concentrate and purify by reversed-phase chromatography to obtain intermediate G2.

[0138] 3) Dissolve intermediate G2 in 150 ml of DMF. Add sodium hydride (60%, 2.0 eq.) slowly in an ice bath and stir at room temperature for 0.5 hours. Then add iodomethane (1.8 eq.). Stir at room temperature for 2 hours to complete the reaction. Quench the reaction with saturated ammonium chloride solution in an ice bath, concentrate the solution, and purify by column chromatography to obtain intermediate G3.

[0139] 4) Dissolve intermediate G3 in 100.0 mL of pyridine. Add trimethylchlorosilane (5.0 eq.) under ice bath conditions and continue stirring for 5 hours. Add benzoyl chloride (1.5 eq.) dropwise to the reaction solution at 0°C, and heat to room temperature while stirring for 2 hours. Cool to 0°C, and add ammonia water (30%, 6.5 eq.) dropwise to the reaction solution. Stir for 2 hours below 10°C. After concentration, slurry with dichloromethane and water to obtain intermediate G4.

[0140] 5) Dissolve intermediate G4 in pyridine, and add 1.2 eq. of 4,4'-bismethoxytriphenylmethyl chloride to the reaction solution under ice bath conditions. Heat to room temperature and stir for 4 hours. After the reaction is complete, quench the reaction with methanol (5.0 eq.). Concentrate the reaction solution and then perform column chromatography to obtain intermediate G5.

[0141] 6) Intermediate G5 and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.5 eq.) were dissolved in dichloromethane. Tetraazole (1.2 eq.) was added under ice bath conditions, and the mixture was stirred for 2 hours after reaching room temperature. After the reaction was completed, the reaction solution was washed with saturated sodium bicarbonate, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain intermediate G6.

[0142] 7) Intermediate G6 was dissolved in dichloromethane, and 2',3'-acetylguanosine (1.0 eq.) was added at room temperature. Tetraazole (1.0 eq.) was added under ice bath conditions, followed by heating to 25°C and stirring for 5 hours. After the reaction was completed by TLC monitoring, 1.2 eq. of iodopyridine solution was added to the reaction solution. After the reaction was completed by TLC monitoring, the reaction solution was washed with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine, and the organic phase was concentrated. The concentrated oil was dissolved in 4 L of dichloromethane, and trifluoroacetic acid (4.0 eq.) was slowly added under ice bath conditions, followed by stirring at room temperature for 2 hours. After the reaction was completed by TLC monitoring, the reaction solution was washed with saturated sodium bicarbonate solution and saturated brine, and the organic phase was concentrated. Intermediate G7 was obtained by column chromatography.

[0143] 8) Dissolve G7 in 4 L of dichloromethane. Add 1.5 eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide and 1.5 eq. of tetrazolium to the reaction solution at room temperature and stir for 3 hours. After the reaction is completed by TLC monitoring, add 1.2 eq. of iodopyridine solution to the reaction solution. After the reaction is completed by TLC monitoring, wash the reaction solution with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine respectively, and then concentrate the organic phase. Dissolve the crude product in 10 times the volume of methanol / concentrated ammonia (1:1) and react at room temperature for 14 hours. Monitor the reaction and concentrate after the reaction is completed. Load the crude product onto a DEAE Sephadex column and elute with a linear gradient using 0-1.0 M TEAB elution buffer. Concentrate to obtain intermediate G. The reaction route is shown in equation (11):

[0144]

[0145] Example 4:

[0146] Synthetic method of ribocyclic modified mRNA cap analogs using intermediates F and H as raw materials

[0147] Intermediate F (2 mol) was dissolved in a DMF solution containing MnCl2 (20 mol), and then intermediate H (1.8 mol) was added to the reaction solution. The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB elution buffer. The target product was concentrated, and the reaction route is shown in equation (12):

[0148]

[0149] Compound H is obtained through the following steps:

[0150] 1) BSA (2.0 eq.) was added dropwise to a toluene suspension of adenine (1.2 eq.) at room temperature. The mixture was heated to reflux and stirred for 1 hour to obtain a clear solution. The solution was then cooled to room temperature, and intermediate B (50 g) and trimethylsilyl trifluoromethanesulfonate (1.5 eq.) were added. The mixture was then heated to reflux and the reaction was continued for 1 hour. After the reaction was complete, the reaction solution was cooled to room temperature, and the pH was adjusted to neutral with saturated sodium bicarbonate aqueous solution. The solution was extracted with ethyl acetate and concentrated. The crude product was purified by column chromatography to obtain intermediate H1.

[0151] 2) Dissolve 30g of intermediate H1 in 300.0mL of ammonia / methanol (1:1) and stir overnight at room temperature. After the reaction is complete, concentrate and purify by reversed-phase chromatography to obtain intermediate H2.

[0152] 3) Dissolve intermediate H2 in 150 ml of DMF. Add sodium hydride (60%, 2.0 eq.) slowly in an ice bath and stir at room temperature for 0.5 hours. Then add iodomethane (1.8 eq.). Stir at room temperature for 2 hours to complete the reaction. Quench the reaction with saturated ammonium chloride solution in an ice bath, concentrate the solution, and purify by column chromatography to obtain intermediate H3.

[0153] 4) Dissolve intermediate H3 in 100.0 mL of pyridine. Add trimethylchlorosilane (5.0 eq.) under ice bath conditions and continue stirring for 5 hours. Add benzoyl chloride (1.5 eq.) dropwise to the reaction solution at 0°C, and heat to room temperature while stirring for 2 hours. Cool to 0°C, and add ammonia water (30%, 6.5 eq.) dropwise to the reaction solution. Stir for 2 hours below 10°C. After concentration, slurry with dichloromethane and water to obtain intermediate H4.

[0154] 5) Dissolve intermediate H4 in pyridine, and add 1.2 eq. of 4,4'-bismethoxytriphenylmethyl chloride to the reaction solution under ice bath conditions. Heat to room temperature and stir for 4 hours. After the reaction is complete, quench the reaction with methanol (5.0 eq.). Concentrate the reaction solution and then perform column chromatography to obtain intermediate H5.

[0155] 6) Intermediate H5 and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.5 eq.) were dissolved in dichloromethane. Tetraazole (1.2 eq.) was added under ice bath conditions, and the mixture was stirred for 2 hours after reaching room temperature. After the reaction was completed, the reaction solution was washed with saturated sodium bicarbonate, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain intermediate H6.

[0156] 7) Intermediate H6 was dissolved in dichloromethane, and 2',3'-acetylguanosine (1.0 eq.) was added at room temperature. Tetraazole (1.0 eq.) was added under ice bath conditions, followed by heating to 25°C and stirring for 5 hours. After the reaction was completed by TLC monitoring, 1.2 eq. of iodopyridine solution was added to the reaction solution. After the reaction was completed by TLC monitoring, the reaction solution was washed with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine, and the organic phase was concentrated. The concentrated oil was dissolved in 4 L of dichloromethane, and trifluoroacetic acid (4.0 eq.) was slowly added under ice bath conditions, followed by stirring at room temperature for 2 hours. After the reaction was completed by TLC monitoring, the reaction solution was washed with saturated sodium bicarbonate solution and saturated brine, and the organic phase was concentrated. Intermediate H7 was obtained by column chromatography.

[0157] 8) Dissolve H7 in 4L of dichloromethane. Add 1.5 eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide and 1.5 eq. of tetrazolium to the reaction solution at room temperature and stir for 3 hours. After the reaction is completed by TLC monitoring, add 1.2 eq. of iodopyridine solution to the reaction solution. After the reaction is completed by TLC monitoring, wash the reaction solution with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine respectively, and then concentrate the organic phase. Dissolve the crude product in 10 times the volume of methanol / concentrated ammonia (1:1) and react at room temperature for 14 hours. Monitor the reaction and concentrate after the reaction is completed. Load the crude product onto a DEAE Sephadex column and elute with a linear gradient using 0-1.0M TEAB elution buffer. Concentrate to obtain intermediate H. The reaction route is shown in equation (13):

[0158] Example 5:

[0159] Synthetic method of ribocyclic modified mRNA cap analogs using intermediates F and I as raw materials

[0160] Intermediate F (2 mol) was dissolved in a DMF solution containing MnCl2 (20 mol), and then intermediate I (1.8 mol) was added to the reaction solution. The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB elution buffer. The target product was concentrated, and the reaction route is shown in equation (14):

[0161]

[0162] Compound I is obtained through the following steps:

[0163] 1) Dissolve 20g of intermediate C2 in 200.0mL of pyridine, then add acetic anhydride (4.0 eq.) and DMAP (1.0 eq.) to the reaction solution. Heat to 70℃ and stir for 3 hours. After the reaction is complete, concentrate to remove pyridine. The crude product is then subjected to column chromatography to obtain intermediate I1.

[0164] 2) Dissolve 20g of intermediate I1 in 200.0mL of iodine (3.0 eq.) / methanol solution, heat to 67℃ and stir for 30 hours. After the reaction is complete, concentrate the solution. Dissolve the concentrated reaction solution in dichloromethane, wash with 5% sodium thiosulfate aqueous solution and saturated brine, dry with anhydrous sodium sulfate, filter, concentrate, and perform column chromatography to obtain intermediate 12.

[0165] 3) Intermediate I2 was dissolved in dichloromethane, and 1.0 eq. of 2'OMe-rA phosphorous amide monomer was added at room temperature. Tetraazole (1.0 eq.) was added under ice bath conditions, followed by heating to 25°C and stirring for 5 hours. After the reaction was completed by TLC monitoring, 1.2 eq. of iodopyridine solution was added to the reaction solution. After the reaction was completed by TLC monitoring, the reaction solution was washed with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine, and the organic phase was concentrated. The concentrated oil was dissolved in 4 L of dichloromethane, and 4.0 eq. of trifluoroacetic acid was slowly added under ice bath conditions, followed by stirring at room temperature for 2 hours. After the reaction was completed by TLC monitoring, the reaction solution was washed with saturated sodium bicarbonate solution and saturated brine, and the organic phase was concentrated. Intermediate I3 was obtained by column chromatography.

[0166] 4) Dissolve I3 in 4L of dichloromethane. Add bis(2-cyanoethyl)-N,N-diisopropylphosphonamide (1.5 eq.) and tetrazolium (1.5 eq.) to the reaction solution at room temperature and stir for 3 hours. After the reaction is completed by TLC monitoring, add 1.2 eq. of iodopyridine solution to the reaction solution. After the reaction is completed by TLC monitoring, wash the reaction solution with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine respectively, and then concentrate the organic phase. Dissolve the crude product in 10 times the volume of methanol / concentrated ammonia (1:1) and react at room temperature for 14 hours. Monitor the reaction and concentrate after the reaction is completed. Load the crude product onto a DEAE Sephadex column and elute with a linear gradient using 0-1.0M TEAB elution buffer. Concentrate to obtain intermediate I. The reaction route is shown in equation (15):

[0167] Example 6:

[0168] Synthetic method of ribocyclic modified mRNA cap analogs using intermediates F and J as raw materials

[0169] Intermediate F (2 mol) was dissolved in a DMF solution containing MnCl2 (20 mol), and then intermediate J (1.8 mol) was added to the reaction solution. The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB elution buffer. The target product was concentrated. The reaction route is shown in equation (16):

[0170]

[0171] Compound J is obtained through the following steps:

[0172] 1) Dissolve 20g of intermediate E2 in 200.0mL of pyridine, then add acetic anhydride (4.0 eq.) and DMAP (1.0 eq.) to the reaction solution. Heat to 70℃ and stir for 3 hours. After the reaction is complete, concentrate to remove pyridine. The crude product is then subjected to column chromatography to obtain intermediate J1.

[0173] 2) Dissolve 20g of intermediate J1 in 200.0mL of iodine (3.0 eq.) / methanol solution, heat to 67℃ and stir for 30 hours. After the reaction is complete, concentrate the solution. Dissolve the concentrated reaction solution in dichloromethane, wash with 5% sodium thiosulfate aqueous solution and saturated brine, dry with anhydrous sodium sulfate, filter, concentrate, and perform column chromatography to obtain intermediate 12.

[0174] 3) Intermediate J2 was dissolved in dichloromethane, and 1.0 eq. of 2'OMe-rA phosphorous amide monomer was added at room temperature. Tetraazole (1.0 eq.) was added under ice bath conditions, followed by heating to 25°C and stirring for 5 hours. After the reaction was completed by TLC monitoring, 1.2 eq. of iodopyridine solution was added to the reaction solution. After the reaction was completed by TLC monitoring, the reaction solution was washed with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine, and the organic phase was concentrated. The concentrated oil was dissolved in 4 L of dichloromethane, and 4.0 eq. of trifluoroacetic acid was slowly added under ice bath conditions, followed by stirring at room temperature for 2 hours. After the reaction was completed by TLC monitoring, the reaction solution was washed with saturated sodium bicarbonate solution and saturated brine, and the organic phase was concentrated. Intermediate J3 was obtained by column chromatography.

[0175] 4) Dissolve J3 in 4L of dichloromethane. Add 1.5 eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide and 1.5 eq. of tetrazolium to the reaction solution at room temperature and stir for 3 hours. After the reaction is completed by TLC monitoring, add 1.2 eq. of iodopyridine solution to the reaction solution. After the reaction is completed by TLC monitoring, wash the reaction solution with 5% sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine respectively, and then concentrate the organic phase. Dissolve the crude product in 10 times the volume of methanol / concentrated ammonia (1:1) and react at room temperature for 14 hours. Monitor the reaction and concentrate after the reaction is completed. Load the crude product onto a DEAE Sephadex column and elute with a linear gradient using 0-1.0M TEAB elution buffer. Concentrate to obtain intermediate J. The reaction route is shown in equation (17):

[0176] Example 7: Synthetic route for the ammonium salt of compound 7 using intermediates D and K as raw materials

[0177] Using intermediates D and K as raw materials, the ammonium salt of compound 7 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (18):

[0178]

[0179] Compound K was obtained through the following steps: intermediates K2 and K3 were obtained by referring to the synthesis methods of intermediates H1 and H2 in Example 4, respectively. Intermediates K4-K were obtained by referring to the synthesis methods of intermediates F1-F in Example 3.

[0180]

[0181] Example 8: Synthetic route for the ammonium salt of compound 8 using intermediates D and L as raw materials

[0182] Using intermediates D and L as raw materials, the ammonium salt of compound 8 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (20):

[0183]

[0184] Compound L is obtained through the following steps:

[0185] 1) Cool a 3.5 L solution of trimethylsilylacetylene (2.1 mol) in THF to -78 °C. Slowly add 2.3 mol (2.5 M in hexanes) of n-butyllithium to the reaction mixture at -78 °C. After the addition is complete, continue stirring for half an hour. Then add a 0.5 L solution of compound L1 (1.4 mol) in THF to the mixture and continue stirring for 3 hours. Quench the reaction mixture with a saturated ammonium chloride aqueous solution and extract twice with 1 L of ethyl acetate each time. Dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography to obtain intermediate L2.

[0186] 2) Dissolve intermediate L2 (1.0 mol) in DCM (940.0 mL), add TEA (1.4 mol) and DMAP (0.01 mol), and then add acetic anhydride (1.3 mol) dropwise to the reaction solution under ice bath conditions. After returning to room temperature, stir for 5 hours. Wash the reaction solution successively with water and saturated brine, dry with anhydrous sodium sulfate, concentrate the organic phase under reduced pressure, and purify by column chromatography to obtain intermediate L3.

[0187] 3) Intermediate L4 was obtained by referring to the synthesis method of intermediate K2 in Example 7.

[0188] 4) Dissolve intermediate L4 (0.35 mol) in THF (2.0 L). Add glacial acetic acid (1.22 mol) and tetrabutylammonium fluoride (0.7 mol, 1 M in THF) to the reaction solution under ice bath conditions. Stir at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure and purify by column chromatography to obtain intermediate L5.

[0189] 5) Dissolve intermediate L5 (0.25 mol) in 80% formic acid aqueous solution and react overnight at room temperature. After concentration under reduced pressure, the reaction solution is purified by column chromatography to obtain intermediate L6.

[0190] Intermediates L7-L were obtained by referring to the synthesis method of intermediates K4-K in Example 7.

[0191]

[0192] Example 9: Synthetic route for the ammonium salt of compound 13 using intermediates D and M as raw materials

[0193] Using intermediates D and M as raw materials, the ammonium salt of compound 13 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. As shown in equation (22):

[0194]

[0195] Compound M is obtained through the following steps: intermediates M2 and M are obtained respectively by referring to the synthesis method of intermediates L7-L in Example 8.

[0196]

[0197] Example 10: Synthetic route for ammonium salt of compound 14 using intermediates D and N as raw materials

[0198] Using intermediates D and N as raw materials, the ammonium salt of compound 14 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (24):

[0199]

[0200] Compound N is obtained through the following steps: intermediates N2-N are obtained by referring to the synthesis method of intermediate M2-M in Example 9.

[0201]

[0202] Example 11: Synthetic route for ammonium salt of compound 17 using intermediates D and O as raw materials

[0203] Using intermediates D and O as raw materials, the ammonium salt of compound 17 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (26):

[0204]

[0205] Compound O is obtained through the following steps: intermediate O2-O is obtained by referring to the synthesis method of intermediate N2-N in Example 10.

[0206]

[0207] Example 12: Synthetic route for the ammonium salt of compound 18 using intermediates D and P as raw materials

[0208] Using intermediates D and P as raw materials, the ammonium salt of compound 18 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (28):

[0209]

[0210] Compound P was obtained through the following steps: intermediates P2 and P3 were obtained by referring to the synthesis methods of intermediates K2 and K3 in Example 7; intermediates P4 and P were obtained by referring to the synthesis methods of intermediates O2-O in Example 11.

[0211]

[0212] Example 13: Synthetic route for the ammonium salt of compound 19 using intermediates D and Q as raw materials

[0213] Using intermediates D and Q as raw materials, the ammonium salt of compound 19 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (30):

[0214]

[0215] Compound Q is obtained through the following steps: intermediates Q2-Q are obtained by referring to the synthesis method of intermediate P3-P in Example 12.

[0216]

[0217] Example 14: Synthetic route for the ammonium salt of compound 20 using intermediates D and R as raw materials

[0218] Using intermediates D and R as raw materials, the ammonium salt of compound 20 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (32):

[0219]

[0220] Compound R is obtained through the following steps: intermediates R2-R are obtained by referring to the synthesis method of intermediate Q2-Q in Example 13.

[0221]

[0222] Example 15: Synthetic route for the ammonium salt of compound 25 using intermediates F and S as raw materials

[0223] Using intermediates F and S as raw materials, the ammonium salt of compound 25 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (34):

[0224]

[0225] Compound S is obtained through the following steps:

[0226] Weigh out 0.23 mol of S1 and 0.23 mol of N-isobutyryl-2',3'-acetylguanosine and dissolve them in 2.0 L of DCM. Add 0.5 mol of tetrazolium under a nitrogen atmosphere. Stir at room temperature for 2 hours. After the reaction is complete, add 0.69 mol (70%) of tert-butyl hydrogen peroxide aqueous solution to the reaction solution and continue the reaction at room temperature for 1 hour. Add 0.4 L of DCM solution containing 0.69 mol of trichloroacetic acid to the reaction solution and react at room temperature for 2 hours. Wash the reaction solution with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively. After concentrating the organic phase under reduced pressure, purify by column chromatography to obtain intermediate B1.

[0227] Intermediate S2 (0.18 mol) and bis(2-cyanoethyl)-N,N-diisopropylphosphonamide (0.25 mol) were dissolved in DCM (1.7 L). Tetraazole (0.39 mol) was added under a nitrogen atmosphere. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, tert-butyl hydrogen peroxide aqueous solution (0.51 mol, 70%) was added dropwise to the reaction solution, and the reaction was continued at room temperature for 1 hour. The reaction solution was washed with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively. The organic phase was concentrated under reduced pressure and dissolved in methanol (0.5 L) and ammonia (1.0 L), and stirred at room temperature overnight. After removing the solvent by concentration, the solution was diluted with water, loaded onto a DEAE Sephadex, and eluted with a linear gradient of 0-1.0 M TEAB elution buffer. The intermediate S was then concentrated. The reaction route is shown in equation (35):

[0228]

[0229] Example 16: Synthetic route for the ammonium salt of compound 27 using intermediates F and T as raw materials

[0230] Using intermediates F and T as raw materials, the ammonium salt of compound 27 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (36):

[0231]

[0232] Compound T is obtained through the following steps: intermediates T2-T are obtained by referring to the synthesis method of intermediates S2-S in Example 15.

[0233]

[0234] Example 17: Synthetic route for the ammonium salt of compound 28 using intermediates F and U as raw materials

[0235] Using intermediates F and U as raw materials, the ammonium salt of compound 28 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (38):

[0236]

[0237] Compound U is obtained through the following steps: intermediate U2-U is obtained by referring to the synthesis method of intermediate T2-T in Example 16.

[0238]

[0239]

[0240] Example 18: Synthetic route for the ammonium salt of compound 41 using intermediates F and V as raw materials

[0241] Using intermediates F and V as raw materials, the ammonium salt of compound 41 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (40):

[0242]

[0243] Compound V is obtained through the following steps: intermediates V1-V are obtained respectively by referring to the synthesis method of intermediates U2-U in Example 17.

[0244]

[0245] Example 19: Synthetic route for the ammonium salt of compound 41 using intermediates F and W as raw materials

[0246] Using intermediates F and W as raw materials, the ammonium salt of compound 42 was obtained by referring to the synthesis method of compound 1 in the target product of Example 1. The reaction route is shown in equation (42):

[0247]

[0248] Compound W is obtained through the following steps: intermediates W1-W are obtained respectively by referring to the synthesis method of intermediates V1-V in Example 18.

[0249]

[0250] Comparative Example 1: m7 GpppA 2’Ome pG

[0251] m7 GpppA 2’Ome The synthesis method of pG is the same as that in the above embodiments (the raw materials used are the same as those in the embodiments). The reaction route is shown in equation (44):

[0252]

[0253] Test Example 1: Detection of mRNA in vitro transcription yield and capping efficiency

[0254] In vitro synthesis of mRNA using ribocycle-modified mRNA cap analogs: plasmids were first linearized with NotI and digested overnight at 4°C; DNA template was extracted; mRNA was synthesized by in vitro transcription, using ribocycle-modified mRNA cap analogs and comparative example 1 mRNA cap analogs as cap structures.

[0255] The reaction system is shown in Table 3:

[0256] Table 3

[0257] system Dosage T7 RNA polymerase 50U 10X buffer 2μl 100mM ATP 1μl 100mM GTP 1μl 100mM CTP 1μl 100mM N1-Me-pUTP 1μl 100mm cap analogue 1μl Inorganic pyrophosphatase 0.05U nuclease inhibitors 20U sterile enzyme-free water Make up to 20 μl template 1μg

[0258] In the experiment, the required volume of materials for the system was first calculated, and then samples were added. First, sterile, enzyme-free water was added to the system, followed by 10X buffer, NTPs, and cap analogue. After mixing, the mixture was gently centrifuged. Then, nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template were added, mixed thoroughly, and gently centrifuged again. The mixture was incubated at 37°C for 2 hours. After 2 hours, 1U of DNase I was added, and the mixture was incubated at 37°C for another 30 minutes to remove the DNA template. RNA purification was then performed, typically using magnetic beads. The purified mRNA was dissolved in sterile, enzyme-free water and then quantified using Nanodrop One.

[0259] Liquid chromatography-mass spectrometry (LC-MS) was used to detect the IVT capping rate of mRNAs with different initiator cap analogs. First, a labeled DNA probe matching the initiation base of the transcription product mRNA needs to be designed, typically using biotin. Streptavidin-labeled magnetic beads are washed and then incubated with the synthesized DNA probe, mRNA, and 10×RNase H reaction buffer at room temperature for 30 minutes, mixing slowly while incubating. Then, 20 μL of RNase H (5 U / μL) is added and incubated at 37°C for 3 hours, mixing every half hour. After incubation, the magnetic beads are washed, and 100 μL of 75% methanol heated to 80°C is added. The mixture is heated to 80°C on a hot plate for 3 minutes, then the supernatant is aspirated and dried using an evaporative centrifuge at room temperature for 45 minutes to 10 μL. The sample is then resuspended in 50 μL of 100 μM EDTA / 1% MeOH and is ready for LC-MS analysis to determine the RNA capping status during the transcription reaction. Since capped and uncapped bases differ significantly in molecular weight, the capping rate of mRNA transcription initiated by different capping analogs can be determined by using the difference in molecular weight. Specific results are shown in Table 4.

[0260] Table 4

[0261]

[0262]

[0263]

[0264]

[0265] As shown in Table 4, the ribosome-modified cap analogs of this application can all be transcribed into the corresponding target mRNA via IVT. Furthermore, the transcription yield and capping rate of these cap analogs are significantly higher than those of Comparative Example 1, and in some cases, comparable to Comparative Example 1.

[0266] Test Example 2: Translation Efficiency Test

[0267] mRNA translation efficiency was assessed using the eGFP coding sequence as a DNA template, and in vitro transcription was initiated using a ribose loop-modified mRNA cap analog and a cap analog from Comparative Example 1. The resulting different mRNA products were then transfected into 293T cells. The 293T cells were cultured at a concentration of (0.5–1) × 10⁻⁶ cells / cell. 5 Cells were seeded into 24-well plates. A cell density of 60-80% is optimal for transfection. 2 μg of mRNA was transfected into each well. Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) was used as the transfection reagent, and its usage instructions were followed. Transfected cells were incubated at 37°C in a CO2 incubator for 4-6 hours, then replaced with fresh complete culture medium. After incubation at 37°C in a CO2 incubator for 24 hours, the fluorescence intensity of GFP was observed using a fluorescence microscope, and the fluorescence intensity ratio of the ribose ring-modified mRNA cap analog to that of Comparative Example 1 was calculated based on the fluorescence intensity.

[0268] Table 5

[0269]

[0270]

[0271]

[0272]

[0273] As shown in Table 5, the mRNA transcribed from the ribocycle-modified cap analog of this application has a significantly better intracellular translation effect than Comparative Example 1, and is partially comparable to Comparative Example 1.

[0274] Therefore, when the cap analog contains different modifications and substitutions to the five-membered sugar ring, it may improve the binding ability of the cap analog to the elF4E protein and ultimately promote the translation of the target mRNA in the cell.

[0275] Test Example 3: Stability Test

[0276] 30 pmol of RNA purified by polyacrylamide gel electrophoresis (PAGE) was mixed with 50 U mRNA decapping enzyme (New England Biolabs) and 1×MDE buffer, and the mixture was incubated at 37°C for 45 min. The enzymatically synthesized reaction mixture was then subjected to PAGE electrophoresis, stained with SYBR Green II (Lonza), and the gel images were observed on a Typhoon FLA 7000 (GE Healthcare) instrument. The ratio of capped RNA to uncapped RNA band intensity was statistically analyzed using Image Quant (GE Healthcare) software, and the decapping rate of the cap analogue after treatment with the decapping enzyme (DCP2 enzyme) was calculated. Statistical analysis was performed using the Dunnett test in KaleidaGraph (Synergy) software. The decapping rate after decapping enzyme treatment is shown in Table 6.

[0277] Table 6

[0278]

[0279]

[0280]

[0281]

[0282] As shown in Table 6, after treatment with the decapping enzyme (DCP2 enzyme), the decapping rate of the cap analogs of the present invention is significantly lower than that of Comparative Example 1, and the decapping rate of some cap analogs is comparable to that of Comparative Example 1.

[0283] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A ribocycle-modified mRNA cap analog, characterized in that, It is a compound of formula (I) or a pharmaceutically acceptable salt: （I）， in, X1, X2, and X3 are 0; Y1, Y2 and Y3 are 0; Only one of R1 or R5 is OH, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyne group, cyano group; the C 1-6 The alkoxy group was not substituted or was replaced by R. 15 Replace, R 15 C 1-6 alkoxy group; the other of R1 or R5 is H; R4 can be H, OH, halogen, or C. 1-6 alkyl; R7 represents H, OH, halogens, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkynyl, cyano; R8 is H; R2, R3, R 13 R 14 They are H, OH, and C, respectively. 1-6 Alkoxy; R6, R9, R 10 R 11 R 12 H and C independently 1-6 Alkoxy; B1 and B2 are independently natural, modified, or non-natural nucleoside bases; R2 and R 13 At least one of them is H, R3 and R 14 At least one of them is H.

2. The ribosome-modified mRNA cap analog according to claim 1, characterized in that, It is a compound of formula (II) or a pharmaceutically acceptable salt: （Ⅱ）， Only one of R1 or R5 is OH, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyne group, cyano group; the C 1-6 The alkoxy group was not substituted or was replaced by R. 15 Replace, R 15 C 1-6 alkoxy group; the other of R1 or R5 is H; R 13 R 14 They are H, OH, and C, respectively. 1-6 Alkoxy; B1 and B2 are either natural, modified, or non-natural nucleoside bases.

3. The ribosome-modified mRNA cap analog according to claim 2, characterized in that, R 13 R 14 Each is an OH group, which can be converted to OH independently.

4. The ribosome-modified mRNA cap analog according to claim 2, characterized in that, R1 is selected from ethynyl, 1-propynyl, 2-propynyl, butynyl, cyano, hydroxyl, methoxy, ethoxy, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, F or Cl; R5 is H.

5. The ribosome-modified mRNA cap analog according to claim 2, characterized in that, R5 is selected from ethynyl, 1-propynyl, 2-propynyl, butynyl, cyano, hydroxyl, methoxy, ethoxy, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, F or Cl; R1 is H.

6. The ribosome-modified mRNA cap analog according to claim 1, characterized in that, It is a compound of formula (Ⅲ) or a pharmaceutically acceptable salt: （Ⅲ）， R1 is OH, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyne group, cyano group; the C 1-6 The alkoxy group was not substituted or was replaced by R. 15 Replace, R 15 C 1-6 Alkoxy; R 13 R 14 They are H, OH, and C, respectively. 1-6 Alkoxy; B1 and B2 are either natural, modified, or non-natural nucleoside bases.

7. The ribosome-modified mRNA cap analog according to claim 6, characterized in that, R1 is ethynyl, 1-propynyl, 2-propynyl, butynyl, cyano, hydroxyl, methoxy, ethoxy, -O-methylene-O-methyl, -O-ethylidene-O-methyl, -O-methylene-O-ethyl, -O-ethylidene-O-ethyl, F, or Cl.

8. A ribocycle-modified mRNA cap analog, characterized in that, It is the following compounds or their pharmaceutically acceptable salts: Compound 7: , Compound 8: , Compound 9: , Compound 10: , Compound 13: , Compound 14: , Compound 15: , Compound 16: , Compound 19: , Compound 20: , Compound 21: , Compound 22: , Compound 23: , Compound 24: , Compound 25: , Compound 26: , Compound 27: , Compound 28: , Compound 29: , Compound 30: , Compound 31: , Compound 32: , Compound 33: , Compound 34: .

9. The use of the ribose ring-modified mRNA cap analogue according to any one of claims 1-8 in the preparation of an in vitro co-transcribed RNA capping reagent.

10. A complex comprising a DNA template of a ribocycle-modified mRNA cap analog according to any one of claims 1-8, wherein the DNA template includes a promoter region containing a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and B1 is complementary to the nucleoside base at position +1 of the DNA template, and B2 is complementary to the nucleoside base at position +2 of the transcription template on the DNA template.

11. An RNA molecule, characterized in that, mRNA cap analogs comprising the ribocycle-modified form as described in any one of claims 1-8.

12. A pharmaceutical composition, characterized in that, It comprises the RNA molecule of claim 11, and a pharmaceutically acceptable carrier.

Images