Cap analogue, preparation method thereof and mRNA (messenger ribonucleic acid)
By designing cap analogs with multi-ring structures, the problem of poor stability of cap-structured mRNA was solved, and the stability of cap analogs and the effective application of mRNA were achieved.
Patent Information
- Application Number
- CN202411088270.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-08
- Publication Date
- 2026-02-10
AI Technical Summary
mRNAs with caps have poor stability in the medical field, which limits their application.
Design a cap analogue in which the atom adjacent to the base position in the polycyclic structure is not O, and prepare a stable cap analogue through a specific chemical reaction. This includes using guanosine or its analogues as raw materials and synthesizing a structurally stable cap analogue through multiple steps.
It improved the stability of cap analogues, enhanced the stability and translation efficiency of mRNA, and improved the efficacy of drug application.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a cap analogue, its preparation method, and mRNA. Background Technology
[0002] Eukaryotic mRNAs possess a cap structure (7MeGpppN) at one end, which plays a crucial role in mRNA stability and translation. Capped mRNAs hold significant importance in the medical field, but they also exhibit relatively poor stability. Summary of the Invention
[0003] This invention provides a cap analogue, its preparation method, and mRNA, to at least solve some of the technical problems existing in the prior art.
[0004] According to a first aspect of the present invention, a cap analogue is provided, which is a compound represented by structural formula (1) or a pharmaceutically acceptable salt.
[0005]
[0006] Where M is a multi-ring structure, and the atom adjacent to position 1' in the ring is not O, and position 1' is connected to B1;
[0007] C6 is selected from C=O, C=CH2, C=S, C=NR and C(R'R"), where R is selected from H, OH, NH2 and alkyl, and R' and R" are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl and halogen;
[0008] B1 is selected from guanine, modified guanine, guanine analogues, xanthine, hypoxanthine and modified hypoxanthine;
[0009] B2 and Bn are independently selected from natural nucleoside bases, modified nucleoside bases, and non-natural nucleoside bases, respectively.
[0010] X1 to X9 are independently selected from O, S, NH, CH2 and CF2, respectively;
[0011] R1 to R8 are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl, and halogens, respectively.
[0012] n is between 0 and 10.
[0013] In an optional embodiment, the structure of the cap analogue is as shown in formula (2) or formula (3):
[0014]
[0015] Wherein, C1, C2, C3, C4 and C5 are independently selected from C=O, C=CH2, C=S, C=NR or C(R'R"), where R is selected from H, OH, NH2 and alkyl, and R' and R" are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl and halogen;
[0016] Y is selected from O, S, and NR1, and R1 is selected from H and alkyl groups.
[0017] In an optional embodiment, the cap analogue includes:
[0018]
[0019]
[0020]
[0021] In an optional embodiment, n is 0-3.
[0022] In an optional embodiment, the ring consists entirely of single bonds, or the ring contains double bonds.
[0023] According to a second aspect of the present invention, a method for preparing a cap analogue as described in the embodiments of the present invention is provided, comprising:
[0024] by Monophosphate esters were prepared from guanosine or its analogues as shown.
[0025] Diphosphate was prepared from the monophosphate as a raw material.
[0026]
[0027] The first intermediate was prepared by reacting the bisphosphate with dimethyl sulfate.
[0028]
[0029] The bisphosphoester imidazole adduct was prepared using the first intermediate as a raw material.
[0030] With phosphorus amide monomer and protected nucleosides Preparation of raw materials
[0031] by Using raw materials, a second intermediate was prepared.
[0032]
[0033] A dimer was prepared using bis(2-cyanoethyl)-N,N-diisopropylphosphonamide, the second intermediate, and tetrazolium as raw materials.
[0034] Cap analogues were prepared using the bisphosphoimidazole adduct and the dimer as raw materials.
[0035] In an optional embodiment,
[0036] The guanosine or its analogue was dissolved in trimethyl phosphate. The reaction solution was cooled to 0°C, phosphorus oxychloride was added, and the reaction was stirred in an ice bath. The reaction solution was then poured into ice water for hydrolysis. After hydrolysis, the monophosphate was separated by reversed-phase chromatography.
[0037] The monophosphate, imidazole, triphenylphosphine, 2,2'-dithiopyridine and triethylamine were dissolved in DMF and reacted with stirring at room temperature. The reaction solution was then added to a sodium perchlorate acetone solution to precipitate the precipitate. The precipitate was filtered and washed to obtain the monophosphate imidazole adduct.
[0038] The monophosphate adduct and tributylamine phosphate were dissolved in dry DMF, and anhydrous zinc chloride was added. The mixture was stirred at room temperature, and then the reaction was terminated by adding EDTA aqueous solution. The diphosphate was obtained by separation by chromatographic column chromatography.
[0039] The bisphosphate was dissolved in water and added in batches to dimethyl sulfate. The pH was adjusted to 4-5 with sodium acetate, and the first intermediate was obtained by separation by chromatographic column.
[0040] The first intermediate, imidazole, triphenylphosphine, 2,2'-dithiopyridine and triethylamine were dissolved in DMF and reacted with stirring at room temperature. The reaction solution was added to a sodium perchlorate acetone solution to precipitate the precipitate, filtered, and washed to obtain the bisphosphoimidazolium adduct.
[0041] The phosphoramidone monomer and the protected nucleoside were dissolved in anhydrous CH2Cl2, and tetrazolium was added with stirring. The reaction was carried out at room temperature. Iodopyridine solution was added to the reaction solution. The reaction solution was washed with 5% sodium thiosulfate solution and water, respectively. Then trichloroacetic acid was added, and the reaction was stirred at room temperature until the reaction was completed. The reaction solution was washed with saturated sodium bicarbonate solution and water, respectively. The organic phase was concentrated and obtained by column chromatography.
[0042] Bis(2-cyanoethyl)-N,N-diisopropylphosphonamide, the second intermediate, and tetrazolium were dissolved in anhydrous CH2Cl2 and reacted at room temperature. Iodopyridine solution was added to the reaction solution. The reaction solution was washed with 5% sodium thiosulfate solution and water, respectively. The organic phase was concentrated, and the crude product was dissolved in ethanol / concentrated ammonia water. After concentration, the crude product was separated by chromatographic column to obtain the dimer.
[0043] The bisphosphoimidazole adduct and the dimer were dissolved in dry DMF, anhydrous zinc chloride was added, and the reaction was stirred at room temperature. The reaction was terminated by adding 5% EDTA aqueous solution, and the cap analog was obtained by repeated purification by chromatographic column chromatography.
[0044] According to a third aspect of the present invention, an mRNA is provided, comprising the cap analogue described in the embodiments of the present invention.
[0045] According to a fourth aspect of the present invention, a drug is provided, comprising the mRNA described in the embodiments of the present invention.
[0046] One embodiment of the present invention has the following advantages or beneficial effects:
[0047] In the cap analogues of this invention, the atoms adjacent to the base positions in the multi-ring structure are not O, which improves the instability problem caused by nucleotide depurination. Detailed Implementation
[0048] The exemplary embodiments will now be described more fully. However, the exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The same structural designations denote the same or similar structures, and therefore their detailed descriptions will be omitted.
[0049] The terms “a,” “one,” “the,” and “” are used to indicate the existence of one or more elements / components / etc.; the terms “include” and “have” are used to indicate an open-ended meaning of inclusion and that there may be other elements / components / etc. in addition to the listed elements / components / etc.
[0050] In cap analogs containing polycyclic rings, taking 7MeGpppN as an example, when the atom adjacent to the base position in the polycyclic ring is O, depurination is likely to occur, resulting in instability of the cap analog. To address this, the present invention provides a cap analog that improves the instability caused by depurination by ensuring that the atom adjacent to the base position in the polycyclic ring is not O.
[0051] This invention provides a cap analogue, which is a compound represented by structural formula (1) or a pharmaceutically acceptable salt.
[0052]
[0053] Where M is a multi-ring structure, and the atom adjacent to position 1' in the ring is not O, and position 1' is connected to B1;
[0054] C6 is selected from C=O, C=CH2, C=S, C=NR and C(R'R″), where R is selected from H, OH, NH2 and alkyl, and R' and R″ are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl and halogen;
[0055] B1 is selected from guanine, modified guanine, guanine analogues, xanthine, hypoxanthine and modified hypoxanthine;
[0056] B2 and Bn are independently selected from natural nucleoside bases, modified nucleoside bases, and non-natural nucleoside bases, respectively.
[0057] X1 to X9 are independently selected from O, S, NH, CH2 and CF2, respectively;
[0058] R1 to R8 are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl, and halogens, respectively.
[0059] n is between 0 and 10.
[0060] In the cap analogues of this invention, the atom adjacent to the 1' position in the multi-ring structure is not O, which can avoid depurination and thus improve the instability problems of cap analogues and their derivatives and products caused by depurination.
[0061] In the cap analogues of this invention, the number of atoms on the ring of the multi-ring structure can be 3-6. In an exemplary embodiment, the number of atoms on the ring of the multi-ring structure is 4, which is a four-membered ring, or the number of atoms on the ring of the multi-ring structure is 5, which is a five-membered ring.
[0062] In the embodiments of the present invention, n is 0-10. In specific implementations, n can be 0-3, for example, n can be 1, which makes the cap analogue easy to prepare and has better drug application performance.
[0063] In some embodiments, the rings in the multi-ring structure may contain double bonds or all single bonds.
[0064] In some embodiments, the structure of the cap analogue is as shown in formula (2) or formula (3):
[0065]
[0066] Wherein, C1, C2, C3, C4 and C5 are independently selected from C=O, C=CH2, C=S, C=NR and C(R'R"), where R is selected from H and alkyl, and R' and R" are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl and halogen;
[0067] Y is selected from O, S, and NR1, and R1 is selected from H and alkyl groups.
[0068] In the embodiments of the present invention, the alkyl group can be a C1-C6 alkyl group. In specific implementations, the alkyl group in the embodiments of the present invention can be a C1-C4 alkyl group.
[0069] The multi-ring structure in the cap analogue shown in equation (2) is a five-ring structure. The multi-ring structure in the cap analogue shown in equation (3) is a four-ring structure.
[0070] In this embodiment of the invention, the multi-membered ring structure can be a carbon ring or a heterocyclic ring. For example, the four-membered ring in the structure shown in formula (3) is a carbon ring, the five-membered ring in the structure shown in formula (2) is a carbon ring when it is composed of C1-C5, and a heterocyclic ring when it is composed of C1-C4 and Y.
[0071] In an exemplary embodiment, when the number of atoms on the multi-ring structure is less than 5, it can be a carbon ring. When the number of atoms on the multi-ring structure is greater than or equal to 5, it can be either a carbon ring or a heterocycle.
[0072] In some embodiments, C6 is selected from C(R'R"), and C6 in the cap analogue has a stable C(R'R") structure, which is easy to prepare.
[0073] In some embodiments, the cap analogues of the present invention include:
[0074]
[0075]
[0076]
[0077]
[0078] The reaction route of the preparation method in the embodiments of the present invention can be referred to chemical reaction formulas (I), (II) and (III).
[0079]
[0080]
[0081] Referring to chemical reaction formulas (I), (II), and (III), this embodiment of the invention provides a method for preparing a cap analogue, comprising:
[0082] Monophosphate S2 was prepared from guanosine or its analogues as shown in S1.
[0083] Diphosphate S3 was prepared from monophosphate S2 as a raw material;
[0084] The first intermediate S4 was prepared by reacting bisphosphonate S3 with dimethyl sulfate.
[0085] The bisphosphoimidazolium adduct S5 was prepared from the first intermediate S4.
[0086] S11 was prepared using phosphorus amide monomer S10 and protected nucleoside S9 as raw materials;
[0087] Using S11 as raw material, a second intermediate S12 is prepared;
[0088] Dimer S13 was prepared using bis(2-cyanoethyl)-N,N-diisopropylphosphoramide, S12 and tetrazolium as raw materials;
[0089] Cap analogue S15 was prepared using S5 and S13 as raw materials.
[0090] In some embodiments, monophosphate S2 is prepared from guanosine or its analogues as shown in S1, including: dissolving guanosine or its analogues in trimethyl phosphate, cooling the reaction solution to 0°C, adding phosphorus oxychloride, stirring the reaction in an ice bath, pouring the reaction solution into ice water for hydrolysis, and separating the hydrolyzed solution by reversed-phase chromatography to obtain monophosphate S2.
[0091] In the specific implementation, guanosine or its analogue S1 (1Eq) is dissolved in 10-50 times its volume of trimethyl phosphate. The reaction solution is cooled to 0°C, phosphorus oxychloride (2Eq) is added, and the mixture is stirred in an ice bath for 5 hours. The reaction solution is then poured into ice water and hydrolyzed for 1 hour. Phosphate ester S2 is obtained by separation by reversed-phase chromatography.
[0092] In some embodiments, bisphosphate S3 is prepared from monophosphate S2, comprising: dissolving monophosphate, imidazole, triphenylphosphine, 2,2'-dithiopyridine, and triethylamine in DMF; stirring the reaction at room temperature; adding the reaction solution to a sodium perchlorate-acetone solution to precipitate; filtering; and washing to obtain the monophosphate imidazole adduct. The monophosphate adduct and tributylamine phosphate are dissolved in dry DMF; anhydrous zinc chloride is added; the reaction is stirred at room temperature; then an aqueous EDTA solution is added to terminate the reaction; and the bisphosphate S3 is obtained by column chromatography.
[0093] In the specific implementation, the above-mentioned monophosphate S2, imidazole (8.0 Eq), triphenylphosphine (3.0 Eq), 2,2'-dithiopyridine (3.0 Eq), and triethylamine (1.0 Eq) were dissolved in DMF and stirred at room temperature for 12 hours. The reaction solution was then precipitated in 4M sodium perchlorate acetone solution, filtered, and washed with acetone to obtain the monophosphate imidazole adduct. The monophosphate adduct, tributylamine phosphate (3.0 Eq), was dissolved in dry DMF, and anhydrous zinc chloride (8.0 Eq) was added, followed by stirring at room temperature for 12 hours. The reaction was terminated by adding EDTA aqueous solution, and the bisphosphate S3 was obtained by column chromatography.
[0094] In some embodiments, the first intermediate S4 is prepared by reacting bisphosphonate S3 with dimethyl sulfate, including: dissolving bisphosphonate S3 in water, adding it in batches to dimethyl sulfate, adjusting the pH value to 4-5 with sodium acetate, and separating the first intermediate S4 by chromatographic column.
[0095] In the specific implementation, the above-mentioned bisphosphonate S3 was dissolved in water, and dimethyl sulfate (2.0 Eq) was added in batches. The pH value was adjusted with sodium acetate and controlled at 4-5. The first intermediate S4 was obtained by separation by chromatographic column chromatography.
[0096] In some embodiments, the preparation of the bisphosphide imidazole adduct S5 using the first intermediate S4 as a raw material includes: dissolving the first intermediate S4, imidazole, triphenylphosphine, 2,2' dithiodipyridine and triethylamine in DMF, stirring the reaction at room temperature, adding the reaction solution to a sodium perchlorate acetone solution to precipitate, filtering, and washing to obtain the bisphosphide imidazole adduct S5.
[0097] In a specific implementation, the first intermediate S4, imidazole (8.0 Eq), triphenylphosphine (3.0 Eq), 2,2' dithiodipyridine (3.0 Eq) and triethylamine (1.0 Eq) were dissolved in DMF and stirred at room temperature for 12 hours. The reaction solution was then added to a 4M sodium perchlorate acetone solution to precipitate the precipitate. After filtration, the precipitate was washed with acetone to obtain the bisphosphophosphate imidazole adduct S5.
[0098] In some embodiments, S11 is prepared using phosphoramidite monomer S10 and protected nucleoside S9 as raw materials, and the second intermediate S12 is prepared using S11 as a raw material, including:
[0099] Using S11 as a raw material, the second intermediate S12 was prepared by dissolving the phosphoramidite monomer S10H and the protected nucleoside S9 in anhydrous H2Cl2, adding tetrazolium under stirring, reacting at room temperature, adding iodopyridine solution to the reaction solution, washing the reaction solution with 5% sodium thiosulfate solution and water respectively, then adding trichloroacetic acid, stirring at room temperature until the reaction was complete, washing the reaction solution with saturated sodium bicarbonate solution and water respectively, concentrating the organic phase, and obtaining the second intermediate S12 by column chromatography.
[0100] In the specific implementation, phosphoramidite monomer S10 (1.3 Eq) and protected nucleoside S9 (1.0 Eq) were dissolved in anhydrous CH2Cl2. Tetraazole (3.0 Eq) was added with stirring. The reaction was carried out at room temperature for 30 minutes. 1.2 Eq of iodopyridine solution was added to the reaction solution. After 10 minutes, the reaction solution was washed with 5% sodium thiosulfate solution and water, respectively. Then, trichloroacetic acid (4.0 Eq) was slowly added, and the reaction was stirred at room temperature until the reaction was complete. After washing the reaction solution with saturated sodium bicarbonate solution and water, the organic phase was concentrated and obtained by column chromatography to yield intermediate S12.
[0101] In some embodiments, the dimer S13 is prepared using bis(2-cyanoethyl)-N,N-diisopropylphosphamide, S12, and tetrazolium as raw materials, comprising: dissolving bis(2-cyanoethyl)-N,N-diisopropylphosphamide, the second intermediate, and tetrazolium in anhydrous CH2Cl2, reacting at room temperature, adding iodopyridine solution to the reaction solution, washing the reaction solution with 5% sodium thiosulfate solution and water respectively, concentrating the organic phase, dissolving the crude product in ethanol / concentrated ammonia water, and separating the crude product by chromatographic column to obtain the dimer.
[0102] In the specific implementation, bis(2-cyanoethyl)-N,N-diisopropylphosphonamide (1.5 Eq), S12, and tetrazolium (3.0 Eq) were dissolved in anhydrous CH2Cl2 and reacted at room temperature for 30 minutes. A 1.2 Eq solution of 0.05 M iodopyridine was added to the reaction solution. After 10 minutes, the reaction solution was washed with 5% sodium thiosulfate solution and water, respectively. The organic phase was concentrated, and the crude product was dissolved in ethanol / concentrated ammonia water. The concentrated crude product was separated by column chromatography to obtain the dimer S13.
[0103] In some embodiments, cap analog S15 is prepared using S5 and S13 as raw materials, including: dissolving bisphosphonate imidazole adduct S5 and dimer S13 in dry DMF, adding anhydrous zinc chloride, stirring the reaction at room temperature, adding 5% EDTA aqueous solution to terminate the reaction, and repeatedly purifying by chromatographic column to obtain the cap analog.
[0104] In the specific implementation, the above-mentioned bisphosphoimidazole adduct S5 (1.0 Eq) and dimer S13 (1.1 Eq) were dissolved in dry DMF, and anhydrous zinc chloride (8.0 Eq) was added, followed by stirring at room temperature for 12 hours. The reaction was terminated by adding 5% EDTA aqueous solution, and the cap analog S14 was obtained by repeated purification by chromatographic column chromatography.
[0105] Example 1
[0106]
[0107] Referring to the above chemical reaction formula (Ⅳ), entecavir S1 (27.7g) was dissolved in 300ml of trimethyl phosphate. The reaction solution was cooled to 0℃, and phosphorus oxychloride (20g) was added. After stirring in an ice bath for 5 hours, the reaction solution was poured into ice water and hydrolyzed for 1 hour. The entecavir monophosphate S2 was obtained by separation by reversed-phase chromatography.
[0108] Entecavir monophosphate S2, imidazole (8.0 Eq), triphenylphosphine (3.0 Eq), 2,2'-dithiopyridine (3.0 Eq), and triethylamine (1.0 Eq) were dissolved in DMF and stirred at room temperature for 12 hours. The reaction mixture was then precipitated in 4M sodium perchlorate-acetone solution, filtered, and washed with acetone to obtain the entecavir monophosphate imidazole adduct. The entecavir monophosphate adduct was dissolved in dry DMF with tributylamine phosphate (3.0 Eq), and anhydrous zinc chloride (8.0 Eq) was added, followed by stirring at room temperature for 12 hours. The reaction was terminated by adding EDTA aqueous solution, and the mixture was separated by column chromatography to obtain entecavir bisphosphate S3.
[0109] Entecavir bisphosphate S3 was dissolved in water, and dimethyl sulfate (2.0 Eq) was added in portions. The pH was adjusted to 4-5 with sodium acetate. 7-methylentecavir bisphosphate S4 was obtained by column chromatography.
[0110] The above-mentioned 7-methylentecavir bisphosphate S4, imidazole (8.0 Eq), triphenylphosphine (3.0 Eq), 2,2'-dithiopyridine (3.0 Eq), and triethylamine (1.0 Eq) were dissolved in DMF and stirred at room temperature for 12 hours. The reaction solution was added to a 4M sodium perchlorate acetone solution to precipitate the precipitate. The precipitate was filtered and washed with acetone to obtain 7-methylentecavir bisphosphate imidazole adduct S5.
[0111]
[0112] Referring to the above chemical reaction formula (V), the 2'-OMe-rA phosphoramidamide monomer S10 (1.3 Eq) and 2',3',2-triacetylguanosine S9 (1.0 Eq) were dissolved in anhydrous CH2Cl2. Tetraazole (3.0 Eq) was added with stirring. The reaction was allowed to proceed at room temperature for 30 minutes. A 1.2 Eq solution of iodopyridine was added to the reaction solution. After 10 minutes, the reaction solution was washed with 5% sodium thiosulfate solution and water, respectively. Then, trichloroacetic acid (4.0 Eq) was slowly added, and the reaction was stirred at room temperature until the reaction was complete. The reaction solution was washed with saturated sodium bicarbonate solution and water, and the organic phase was concentrated and subjected to column chromatography to obtain intermediate S12.
[0113] Bis(2-cyanoethyl)-N,N-diisopropylphosphonamide (1.5 Eq), S12, and tetrazolium (3.0 Eq) were dissolved in anhydrous CH2Cl2 and reacted at room temperature for 30 minutes. A 1.2 Eq solution of iodopyridine was added to the reaction mixture, and after 10 minutes, the mixture was washed with 5% sodium thiosulfate solution and water, respectively. The organic phase was concentrated, and the crude product was dissolved in ethanol / concentrated ammonia solution. The concentrated crude product was separated by column chromatography to obtain the dimer S13.
[0114]
[0115] Referring to the above chemical reaction formula (VI), the above 7-methylentecavir bisphosphate imidazole adduct S5 (1.0 Eq) and pA(OMe)pG S13 (1.1 Eq) were dissolved in dry DMF, and anhydrous zinc chloride (8.0 Eq) was added and stirred at room temperature for 12 hours. The reaction was terminated by adding 5% EDTA aqueous solution, and the cap analog S14 (7-MeGepppA(OMe)pG) was obtained by repeated purification by chromatographic column chromatography.
[0116] Comparative Example 1
[0117] Guanosine diphosphate S6 was obtained by replacing entecavir with natural guanosine.
[0118]
[0119] 7-Methylentecavir bisphosphate S4 and guanosine bisphosphate S6 obtained in Comparative Example 1 were dissolved in water, and dimethyl sulfate (2.0 Eq) was added in portions to each. The pH was adjusted to 4-5 with sodium acetate. The guanosine bisphosphate reaction product obtained in Comparative Example 1 contained 5-10% depurinated products, which were separated by chromatographic column chromatography to obtain 7-guanosine bisphosphate S7.
[0120] Experiments revealed that no depurination was observed after methylation of the 7-methylentecavir bisphosphate S4 obtained in the embodiments of the present invention. In Comparative Example 1, where natural guanosine was used instead of entecavir, the resulting guanosine bisphosphate S6 exhibited a considerable degree of depurination.
[0121] Example 2
[0122] The cap analogue 7-MeGcpppA(OMe)G can be prepared using the method of Example 1.
[0123]
[0124] The preparation of S5 can be referenced in the following chemical reaction formula (VII).
[0125]
[0126] Example 3
[0127] 4'-Hydroxymethylcyclobutylguanosine was synthesized according to the method described in the literature (J. CHEM. soc. PERKIN TRANS. I 1995, 2081-87).
[0128] Then, cap analog 7-MeGbpppA(OMe)pG was prepared according to Example 1.
[0129]
[0130] Example 4
[0131] IsoG was synthesized according to the literature method (Journal of Medicinal Chemistry (1992), 35(13), 2347-54, J.CHEM.SOC.PERKIN TRANS.1 1992, 1427-36).
[0132] Cap analog 7-MeisoGpppA(OMe)pG was prepared according to Example 1.
[0133]
[0134] Example 5
[0135] mRNA formulations were prepared using 7-MeGepppA(OMe)pG and 7-MeGpppA(OMe)pG (natural guanosine control), respectively. The ROI value was measured 6 hours after intramuscular injection of 5 μg into mice. The ROI value of the cap analog 7-MeGepppA(OMe)pG in this embodiment of the invention is 4.226 × 10⁻⁶. 9 The ROI of the reference standard was 3.688 × 10⁻⁶. 8 The cap analogue of this invention is an order of magnitude higher than the control.
[0136] This invention provides an mRNA including the cap analog described in this invention.
[0137] This invention provides a drug comprising the mRNA described in this invention.
[0138] In this embodiment of the invention, the term "multiple" refers to two or more, unless otherwise explicitly defined. The terms "install," "connect," and "fix" should be interpreted broadly. For example, "connect" can mean a fixed connection, a detachable connection, or an integral connection. Those skilled in the art can understand the specific meaning of the above terms in this embodiment of the invention based on the specific circumstances.
[0139] In the description of the embodiments of the present invention, it should be understood that the terms "upper" and "lower" and other terms indicating the orientation or positional relationship are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or unit referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of the present invention.
[0140] In the description of this specification, the terms "an embodiment," "a preferred embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0141] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. For those skilled in the art, various modifications and variations can be made to the embodiments of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the embodiments of the present invention should be included within the protection scope of the embodiments of the present invention.
Claims
1. A hat-like object, characterized in that, It is a compound represented by structural formula (1) or a pharmaceutically acceptable salt. Where M is a multi-ring structure, and the atom adjacent to position 1' in the ring is not O, and position 1' is connected to B1; C6 is selected from C=O, C=CH2, C=S, C=NR and C(R'R"), where R is selected from H, OH, NH2 and alkyl, and R' and R" are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl and halogen; B1 is selected from guanine, modified guanine, guanine analogues, xanthine, hypoxanthine and modified hypoxanthine; B2 and Bn are independently selected from natural nucleoside bases, modified nucleoside bases, and non-natural nucleoside bases, respectively. X1 to X9 are independently selected from O, S, NH, CH2 and CF2, respectively; R1 to R8 are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl, and halogens, respectively. n is between 0 and 10.
2. The cap analogue according to claim 1, characterized in that, The structure of the cap-like object is shown in formula (2) or formula (3): Wherein, C1, C2, C3, C4 and C5 are independently selected from C=O, C=CH2, C=S, C=NR and C(R'R"), where R is selected from H, OH, NH2 and alkyl, and R' and R" are independently selected from H, OH, N3, vinyl, propynyl, ethynyl, propynyl, alkyl, O-alkyl, S-alkyl, N-alkyl, CH2OH, CH2-O-alkyl, CH2-N-alkyl, CH2-S-alkyl, CH2CH2-O-alkyl, CH2CH2-N-alkyl, CH2CH2-S-alkyl and halogen; Y is selected from O, S and NR1, where R1 is selected from H and alkyl groups.
3. The cap analogue according to claim 3, characterized in that, The cap analogues include:
4. The cap analogue according to claim 1, characterized in that, n is between 0 and 3.
5. The cap analogue according to claim 1, characterized in that, The ring consists entirely of single bonds, or contains double bonds.
6. The method for preparing the cap analogue according to claim 1, characterized in that, include: by Monophosphate esters were prepared from guanosine or its analogues as shown. Diphosphate was prepared from the monophosphate as a raw material. The first intermediate was prepared by reacting the bisphosphate with dimethyl sulfate. The bisphosphoester imidazole adduct was prepared using the first intermediate as a raw material. With phosphorus amide monomer and protected nucleosides Preparation of raw materials by Using raw materials, a second intermediate was prepared. A dimer was prepared using bis(2-cyanoethyl)-N,N-diisopropylphosphonamide, the second intermediate, and tetrazolium as raw materials. Cap analogues were prepared using the bisphosphoimidazole adduct and the dimer as raw materials.
7. The method according to claim 6, characterized in that, The guanosine or its analogue was dissolved in trimethyl phosphate. The reaction solution was cooled to 0°C, phosphorus oxychloride was added, and the reaction was stirred in an ice bath. The reaction solution was then poured into ice water for hydrolysis. After hydrolysis, the monophosphate was separated by reversed-phase chromatography. The monophosphate, imidazole, triphenylphosphine, 2,2'-dithiopyridine and triethylamine were dissolved in DMF and reacted with stirring at room temperature. The reaction solution was then added to a sodium perchlorate acetone solution to precipitate the precipitate. The precipitate was filtered and washed to obtain the monophosphate imidazole adduct. The monophosphate adduct and tributylamine phosphate were dissolved in dry DMF, and anhydrous zinc chloride was added. The mixture was stirred at room temperature, and then the reaction was terminated by adding EDTA aqueous solution. The diphosphate was obtained by separation by chromatographic column chromatography. The bisphosphate was dissolved in water and added in batches to dimethyl sulfate. The pH was adjusted to 4-5 with sodium acetate, and the first intermediate was obtained by separation by chromatographic column. The first intermediate, imidazole, triphenylphosphine, 2,2'-dithiopyridine and triethylamine were dissolved in DMF and reacted with stirring at room temperature. The reaction solution was added to a sodium perchlorate acetone solution to precipitate the precipitate, filtered, and washed to obtain the bisphosphoimidazolium adduct. The phosphoramidone monomer and the protected nucleoside were dissolved in anhydrous CH2Cl2, and tetrazolium was added with stirring. The reaction was carried out at room temperature. Iodopyridine solution was added to the reaction solution. The reaction solution was washed with 5% sodium thiosulfate solution and water, respectively. Then trichloroacetic acid was added, and the reaction was stirred at room temperature until the reaction was completed. The reaction solution was washed with saturated sodium bicarbonate solution and water, respectively. The organic phase was concentrated and obtained by column chromatography. Bis(2-cyanoethyl)-N,N-diisopropylphosphonamide, the second intermediate, and tetrazolium were dissolved in anhydrous CH2Cl2 and reacted at room temperature. Iodopyridine solution was added to the reaction solution. The reaction solution was washed with 5% sodium thiosulfate solution and water, respectively. The organic phase was concentrated, and the crude product was dissolved in ethanol / concentrated ammonia water. After concentration, the crude product was separated by chromatographic column to obtain the dimer. The bisphosphoimidazole adduct and the dimer were dissolved in dry DMF, anhydrous zinc chloride was added, and the reaction was stirred at room temperature. The reaction was terminated by adding 5% EDTA aqueous solution, and the cap analog was obtained by repeated purification by chromatographic column chromatography.
8. An mRNA, characterized in that, Includes the cap analogues as described in any one of claims 1-5.
9. A drug, characterized in that, Includes the mRNA described in claim 8.