Capping analogs for self-replicating mRNA and their applications

Abstract

The present disclosure provides a capping analog for self-replicating mRNA and applications thereof. The capping analog for self-replicating mRNA can improve the stability of the self-replicating mRNA and / or the translation efficiency of the self-replicating mRNA.

Description

Technical Field

[0001] The present disclosure belongs to the field of chemical and bioengineering technology, and particularly relates to capping analogs for self-replicating mRNA and applications thereof. Background Art

[0002] The cap structure is essential for RNA to perform its biological functions in vivo. Its presence helps RNA evade the cellular innate immune response and achieve effective protein translation in vivo, making it a crucial prerequisite for stimulating a response. The chemical nature of the cap structure is a specialized structure at the 5' end of RNA, formed during RNA transcription. This structure, the m7GPPPN structure, is also known as the methylguanosine cap. It is formed through the catalytic action of RNA triphosphatases, guanylyltransferases, RNA (guanine-N7) methyltransferases, and RNA (nucleoside-2') methyltransferases.

[0003] Self-replicating RNA (srRNA), also often called self-amplifying RNA (saRNA), is a specialized RNA molecule with the ability to replicate itself. A key difference between self-replicating mRNA and traditional mRNA is its ability to replicate using its own RNA sequence as a template. This self-replication property enables higher protein expression with lower vaccine dosages, improving vaccine efficacy.

[0004] Similar to conventional mRNA, self-replicating mRNA also requires a cap structure to enhance its intracellular stability and translation efficiency. Self-replicating mRNAs are often designed based on the genomes of positive-sense RNA viruses, such as Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), and Sindbis virus (SIN). The genomes of these positive-sense RNA viruses begin with a 5'-AU sequence, and m7GpppApU-type capping analogs are well-suited to this initiation sequence, thereby better mimicking the natural infection process of the virus.

[0005] Due to differences in sequence length and cap structure type, the structural modification methods used for m7GpppApG capping analogs used in traditional mRNA preparation processes may not be suitable for the development of self-replicating mRNA capping analogs. Therefore, it is crucial to provide capping analogs suitable for self-replicating mRNA. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a capping analog for self-replicating mRNA and its application. The capping analog for self-replicating mRNA described in the present invention can improve the stability of self-replicating mRNA and / or the translation efficiency of self-replicating mRNA.

[0007] The present invention provides a compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0008] ,

[0009] Wherein, n is 0, 1, 2 or 3;

[0010] R5 is -OR 5a 、-SR 5b 、-NR 5c R 5d 、-C(O)R 5e 、 、-NHC(O)-R 5h 、-N(CH3)C(O)-R 5i 、-C(O)NR 5j R 5k 、-C(O)OR 5l 、-OC(O)-R 5m or -S(O)2-R 5n ;

[0011] R 4a 、R 5a 、R 5b 、R 5c 、R 5d 、R 5e 、R 5h 、R 5i 、R 5j 、R 5k 、R 5l 、R 5m and R 5n are independently -H, C1-C3 alkyl or halogenated C1-C3 alkyl;

[0012] R 5f and R 5g are independently -H, halogen, C1-C3 alkyl, -OC1-C3 alkyl, halogenated C1-C3 alkyl or -NR 5-1 C(O)R 5-2 ;

[0013] R 5-1 and R 5-2 are independently -H or C1-C3 alkyl;

[0014] R1, R2, R3, R6 and R7 are each independently -H, C1-C6 alkyl, C 2- C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl or benzyl;

[0015] R4 is -OR 4a , halogen or -H;

[0016] R8 is -H or C1-C3 alkyl.

[0017] In some embodiments, in the compound represented by formula (I), its stereoisomers or pharmaceutically acceptable salts thereof, some of the groups are defined as follows, and the remaining groups are defined as described in any other embodiment (hereinafter referred to as "in some embodiments"):

[0018] n is 0 or 1.

[0019] In some embodiments, R4 is -H, -OC1-C3 alkyl, or halogen.

[0020] In some embodiments, R4 is -H or halogen.

[0021] In some embodiments, R4 is -H, -F, or -OCH3.

[0022] In some embodiments, R5 is -OH, or -C(O)NR 5j R 5k ; R 5f and R 5g are independently halogen, C1-C3 alkyl, -OC1-C3 alkyl or -NR 5-1 C(O)R 5-2 ; R 5j and R 5k are independently C1-C3 alkyl; R 5-1 H; R 5-2 It is a C1-C3 alkyl group.

[0023] In some embodiments, R5 is -OH, or -C(O)NR 5j R 5k ; R 5f is C1-C3 alkyl or halogen; R 5g is halogen, C1-C3 alkyl or -OC1-C3 alkyl; R 5j and R 5k are independently C1-C3 alkyl.

[0024] In some embodiments, R5 is -OH, 、 、 、 or .

[0025] In some embodiments, R5 is -OH, 、 、 or .

[0026] In some embodiments, R1 is -H.

[0027] In some embodiments, R2 is C1-C3 alkyl.

[0028] In some embodiments, R2 is -CH3.

[0029] In some embodiments, R3 is -H.

[0030] In some embodiments, R6 is C1-C3 alkyl.

[0031] In some embodiments, R6 is -CH3.

[0032] In some embodiments, R7 is -H.

[0033] In some embodiments, R8 is -H.

[0034] In some embodiments, n is 0 or 1; R4 is -H or halogen;

[0035] R5 is -OH, or -C(O)NR 5j R 5k ; R 5f and R 5g R is independently halogen, C1-C3 alkyl or -OC1-C3 alkyl; 5j and R 5k are independently C1-C3 alkyl;

[0036] R1 is -H; R2 is C1-C3 alkyl; R3 is -H;

[0037] R6 is C1-C3 alkyl; R7 is -H;

[0038] R8 is -H.

[0039] In some embodiments, the pharmaceutically acceptable salt of the compound represented by formula (I) is any of the following structures:

[0040] 、

[0041] 、

[0042] 、

[0043] 、

[0044] 、

[0045] or

[0046] .

[0047] The present invention provides a use of the compound represented by formula (I) as described above, its stereoisomers or pharmaceutically acceptable salts thereof in preparing an in vitro co-transcribed mRNA capping agent.

[0048] The present invention also provides an RNA molecule comprising the compound represented by formula (I) as described above, its stereoisomers or pharmaceutically acceptable salts thereof as a cap structure or a cap structure fragment.

[0049] The present invention also provides a pharmaceutical composition comprising the RNA molecule described above.

[0050] In some embodiments, the pharmaceutical composition further comprises at least one delivery agent.

[0051] In some embodiments, the at least one delivery agent comprises at least one cationic lipid.

[0052] In some embodiments, the cationic lipid is any one or a combination of at least two of the following:

[0053] (i) A compound represented by formula (II), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-6 Alkylene; G2 is C 2-8 Alkylene; G3 is C 1-3 Alkylene; L1 is C 6-15 Straight chain alkyl; L2 is C 12-25 branched alkyl;

[0054] (II)

[0055] (ii) A compound represented by formula (III), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C 2-8 Alkylene; G2 is C 2-8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6-25 Straight or branched alkyl; R2 is C 6-25Straight or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4-;

[0056] (III)

[0057] (iii) A compound represented by formula (IV), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, wherein: G1 is C 1-6 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-20 Straight or branched alkyl; R2 is C 12-25 Branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-;

[0058] (IV)

[0059] (iv) A compound represented by formula (V), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-8 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-25 Straight or branched alkyl; R2 is C 12-25 Straight or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3 or -CH2CH2OH ;

[0060] (V)

[0061] (v) A compound represented by formula (VI), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G 1 and G 2 Each independently is C6-C 10 Alkylene; G 3 C1-C 12 Alkylene; R 1 and R 2Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 OR 5 、N(R 5 )2, -C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 Hydrocarbon; R 5 is H or a C1-C6 hydrocarbon group;

[0062] (VI)

[0063] (vi) A compound represented by formula (VII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein R4 is -(CH2) n Q; Q is -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocycle; n is 1, 2 or 3; R is independently C 1-8 Alkyl; X is independently H or C 1-8 alkyl;

[0064] (VII)

[0065] (vii) a compound represented by formula (VIII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof,

[0066] (VIII).

[0067] In some embodiments, the cationic lipid is selected from any one or a combination of at least two of the group consisting of YK-009, YK-201, YK-407, YK-305, ALC-0315, SM-102, and DLIN-MC3-DMA:

[0068] 、 、 、 、 、 、 .

[0069] In some embodiments, the cationic lipid is YK-009.

[0070] In some embodiments, the at least one delivery agent further comprises at least one neutral lipid.

[0071] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol.

[0072] In some embodiments, the neutral lipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1,2-di- 2-Dilinoyl-sn-glycero-3-phosphocholine, 1, 2-diamidonoyl-sn-glycero-3-phosphocholine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoyl-sn-glycero-3-phosphoethanolamine, 1, 2-diamidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, or a combination of at least two thereof.

[0073] In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0074] In some embodiments, the at least one delivery agent further comprises a structured lipid.

[0075] In some embodiments, the structured lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids.

[0076] In some embodiments, the structured lipid is cholesterol.

[0077] In some embodiments, the at least one delivery agent further comprises a polymer-conjugated lipid.

[0078] In some embodiments, the polymer-conjugated lipid is selected from any one or a combination of at least two of the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) and methoxypolyethylene glycol ditetradecanoyl acetamide.

[0079] In some embodiments, the polymer-conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000.

[0080] In some embodiments, the at least one delivery agent comprises YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000; the YK-009 is .

[0081] In some embodiments, the molar ratio of YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 is preferably 49:10:39.5:1.5.

[0082] In some embodiments, the pharmaceutical composition further comprises one or at least two cell-penetrating peptides (CPPs).

[0083] The present invention provides a method for synthesizing mRNA molecules for purposes other than disease diagnosis and treatment, comprising the following steps: co-incubating the compound represented by formula (I) as described above, its stereoisomers or pharmaceutically acceptable salts thereof with a polynucleotide template, and transcribing the polynucleotide template.

[0084] The present invention provides a capped mRNA transcription reaction system for non-disease diagnosis and treatment purposes, comprising: (1) a compound as shown in formula (I) as described above, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; and (2) a polynucleotide template, NTPs, and RNA polymerase.

[0085] The present invention provides a kit comprising: (1) a compound represented by formula (I) as described above, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; and (2) a nucleotide triphosphate molecule, and RNA polymerase.

[0086] In some embodiments, the kit further comprises an RNase inhibitor, an inorganic pyrophosphatase, Mg 2+ , a crowding agent and a buffer, or a combination of at least two of the group consisting of.

[0087] The present invention provides a method for improving the stability of RNA, comprising incorporating the compound represented by formula (I) as described above, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof into RNA.

[0088] The present invention provides a method for introducing RNA into a cell, the method comprising contacting the cell with an RNA molecule as described above or a pharmaceutical composition as described above.

[0089] The present invention provides a use of the RNA molecule or the pharmaceutical composition as described above in preparing a vaccine.

[0090] Definition of terms

[0091] All publications and patents mentioned in this disclosure are hereby incorporated into the present disclosure in their entirety by reference. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0092] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0093] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0094] Except in the examples or where otherwise indicated, all numbers stating quantitative properties, such as dosages, in this disclosure are to be understood as being modified in all instances by the term "about." It is also to be understood that any numerical range recited in this disclosure is intended to include all subranges within that range and any combination of the various endpoints of that range or subrange.

[0095] In addition, when referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is within the typical tolerance range of the art, within the experimental variability or the approximate value within the statistical experimental error, and thus the number or numerical range can, for example, vary between 1% and 15% of the number or numerical range. For example, "about" can be understood as about 2 standard deviations of the mean, and when "about" is present before a series of numbers or ranges, it should be understood that "about" can modify each number in the numbers in the series or range.

[0096] Unless otherwise defined, all terms (including technical and scientific terms) used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless explicitly defined in this disclosure.

[0097] In this disclosure, the “ " means that the structural fragment is connected to the rest of the molecule through this bond. For example, It means that through ” is connected to the rest of the molecule.

[0098] As used in this disclosure, the term "C1-C3" refers to a group having any integer value of carbon atoms in the main chain ranging from 1 to 3, such as 1, 2 or 3 carbon atoms. 6-15 " means that the number of carbon atoms in the group is any integer value within the range of 6 to 15, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms. The limitations of other carbon atom ranges are similar and indicate that the number of carbon atoms in the defined group can be any integer value within the defined range.

[0099] As used in the present disclosure, the term "alkyl" refers to a saturated aliphatic hydrocarbon group having a straight or branched chain; non-limiting examples thereof include methyl, ethyl, propyl, isopropyl, and the like.

[0100] In the present invention, the term "alkoxy" refers to a group R Y -O-, R Y The same definition as the term "alkyl". Alkoxy includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like.

[0101] The term "alkenyl" refers to a straight or branched hydrocarbon group having at least one double bond, consisting solely of carbon atoms and hydrogen atoms, having, for example, 2 to 6 (e.g., 2, 3, 4, 5, or 6) carbon atoms, and connected to the rest of the molecule by a single bond. Alkenyl groups include, but are not limited to, vinyl, 、 、 、 wait.

[0102] The term "alkynyl" refers to a straight or branched hydrocarbon group having at least one triple bond, consisting solely of carbon and hydrogen atoms, having, for example, 2 to 6 (e.g., 2, 3, 4, 5, or 6) carbon atoms, and connected to the rest of the molecule by a single bond. Alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, but-1-ynyl, but-2-ynyl, and the like.

[0103] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0104] The term "cycloalkyl" refers to a saturated monocyclic ring group consisting only of carbon atoms, having a specified number of carbon atoms (e.g., C3-C6). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0105] As used in this disclosure, the term "salt" refers to a corresponding salt of the modified nucleoside compound (or nucleotide compound) disclosed herein that can be conveniently or desirably prepared, purified, and / or handled, for example, a pharmaceutically acceptable salt. Unless otherwise indicated, reference to a particular compound in this disclosure also includes its salt form.

[0106] As used herein, "capping analog" refers to a structure at the 5' end of mature RNA formed by post-transcriptional modification in eukaryotes, namely the m7GPPPN structure, also known as a methylguanosine cap. This structure can prevent RNA degradation at the 5' end, help RNA transcripts pass through selective pores in the nuclear membrane and enter the cytoplasm, enhance translation, and help complete the entire splicing process.

[0107] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0108] The term "pharmaceutically acceptable" in this disclosure means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or with humans or mammals for the prevention or treatment of a disease or condition for which it is used.

[0109] The term "polynucleotide template" in this disclosure refers to a polynucleotide chain from which other nucleic acid molecules are synthesized.

[0110] The present disclosure also includes salts of the compounds described herein, particularly pharmaceutically acceptable salts. Compounds of the present disclosure having sufficiently acidic or basic functional groups can react with a variety of bases or acids to form salts. Alternatively, compounds that are inherently charged (e.g., compounds having quaternary nitrogen) can form salts with appropriate counterions (e.g., halides such as bromide, chloride, or fluoride, particularly bromide).

[0111] The pharmaceutically acceptable salts of the present invention may be acid addition salts of the compounds of the present invention having sufficient basicity, such as those formed with the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or with the following organic acids: formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentane Propionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, or thiocyanic acid.

[0112] In addition, another suitable pharmaceutically acceptable salt of the compounds of the present disclosure having sufficient acidity is an alkali metal salt, such as a sodium salt or a potassium salt, an alkaline earth metal salt, such as a calcium salt or a magnesium salt, an ammonium salt (e.g., a salt formed with NH3 or ammonia), or a salt formed with an organic base that provides a physiologically acceptable cation, such as a salt formed with the following substances: triethylamine, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, trishydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. In addition, basic nitrogen-containing groups can be quaternized using reagents such as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides, such as benzyl and phenethyl bromides, etc.

[0113] Those skilled in the art will also recognize that the acid addition salts of the compounds of formula (I) disclosed herein can be prepared by reacting the compounds with a suitable inorganic or organic acid by any of the known methods. Alternatively, the base addition salts of the acidic compounds disclosed herein can be prepared by reacting them with a suitable base by various known methods.

[0114] The present disclosure includes all possible salts of the compounds of formula (I) described herein, which may be a single salt or any mixture of said salts in any ratio.

[0115] Certain compounds disclosed herein may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds of formula (I) disclosed herein also include racemic mixtures, single stereoisomers, and optically active mixtures. It should be understood by those skilled in the art that one stereoisomer may have better efficacy and / or lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution, and the like. Racemates can be chirally resolved by chromatographic resolution or chemical resolution. For example, chiral acid resolution reagents such as chiral tartaric acid and chiral malic acid can be added to the compounds disclosed herein to form salts, and the products can be separated by utilizing the physicochemical properties of the products, such as different solubility.

[0116] In the present disclosure, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

[0117] The description of the present disclosure should be interpreted in accordance with the laws and principles of chemical bonding.In some cases, it may be possible to remove a hydrogen atom in order to accommodate a substituent at a given position.

[0118] It should be understood that the terms "compounds of the present disclosure" and "compounds" used in the present disclosure may refer to: compounds of formula (I), solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, or mixtures thereof, depending on the context.

[0119] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0120] Unless otherwise specified, all reagents and raw materials used in the present disclosure can be obtained through commercial channels.

[0121] The positive progress of the present disclosure is that the compound shown in formula (I) provided by the present disclosure is an mRNA capping analog for self-replication, and has at least one of the following advantages:

[0122] (1) The self-replicating mRNA capping analogs of this application significantly improve the mRNA in vitro transcription yield and integrity;

[0123] (2) The self-replicating mRNA capping analogs of the present application significantly improve the mRNA translation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some exemplary embodiments of the present disclosure, rather than limitations of the present disclosure. For ordinary technicians in this field, various changes made within the spirit and scope of the present disclosure may also be regarded as the scope intended by the drawings of the present disclosure.

[0125] Figure 1 The graph shows the mRNA in vitro transcription yield using YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0126] Figure 2 The graph shows the integrity of mRNA transcription initiated by YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0127] Figure 3 The graph shows the capping efficiency of mRNA transcription initiated by YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0128] Figure 4 The graph shows the translation efficiency of capped mRNA using YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs.

[0129] Figure 5 This figure shows the in vivo expression results of capped mRNA in mice using YK-CAP-201, YK-CAP-202, YK-CAP-203, YK-CAP-204, YK-CAP-205, YK-CAP-206, YK-CAP-207 and N-7114 as capping analogs. DETAILED DESCRIPTION

[0130] The present disclosure is further described below in conjunction with the examples, but the present disclosure is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to the different requirements of specific use. The implementation conditions not specifically specified are conventional conditions in the art. In the specific examples of the present disclosure, the raw materials used can be obtained through commercial channels. Unless otherwise stated, all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present disclosure can be combined with each other as long as they do not conflict with each other.

[0131] The following abbreviations represent the following reagents:

[0132] TEA: triethylamine; DMF: N, N-dimethylformamide; PySSPy: 2, 2'-dipyridyl disulfide; PPh3: triphenylphosphine; TEAB: triethylamine bicarbonate; EDTA: ethylenediaminetetraacetic acid; DSPC: 1, 2-distearoyl-sn-glycero-3-phosphocholine; DMG-PEG 2000: 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000.

[0133] Example 1: Synthesis of capping analogs for self-replicating mRNA

[0134] 1. Synthesis of Intermediate INT-I

[0135]

[0136] pA(2'-OMe)mpU•TEA (3.10 g, 4.64 mmol), imidazole (1.58 g, 23.22 mmol), 2, 2'-dithiodipyridine (2.04 g, 9.28 mmol), triethylamine (0.47 g, 4.64 mmol) and triphenylphosphine (2.43 g, 9.28 mmol) were dissolved in 31 mL of ultra-dry N,N-dimethylformamide and placed in a 25 o After the reaction was completed, the reaction solution was slowly added to a pre-cooled acetone (150 mL) solution containing sodium iodide (5.08 g, 33.89 mmol) and stirred for 25 min. o C crystallize for 30 min, centrifuge, and freeze-dry to obtain a white solid INT-I (2.50 g, 3.28 mmol, yield 75.0%). 23 H 29 N9O 14 P 2， MS(ES): m / z (MH) - 716.1.

[0137] 2. Synthesis of YK-CAP-201

[0138]

[0139] YK-CAP-201-PM1 (synthesized according to the synthetic route of YK-CAP-107-PM11 in patent CN118389495B) (130 mg, 0.26 mmol) and INT-I (513 mg, 0.72 mmol) were dissolved in DMF, ZnCl2 was added, and the mixture was stirred at 37°C under N2 protection for 72 hours. After completion of the reaction, the reaction solution was poured into an aqueous solution of EDTA and stirred for 20 minutes. The pH was adjusted to 6.5 with aqueous ammonia and purified by gel column chromatography (elution with water and 1 M TEAB = 3:1). The target product peak was collected, concentrated, and lyophilized. Further purification by high-performance preparative liquid chromatography yielded the final product, YK-CAP-201 (54 mg, 47 μmol, 18.1%), C 34 H 49 N 12 O 25 P4,MS(ES): m / z (MH) - 1147.2. 1 H NMR (400 MHz, D2O) δ 9.09 (s, 1H), 8.43 (s, 1H), 8.17 (s, 1H), 7.41 (d, J = 2.4 Hz, 1H), 6.03 (d, J = 4.4 Hz, 1H), 5.71 (d, J= 5.6 Hz, 1H), 5.47 (d, J = 2.4 Hz, 1H), 4.90 - 4.83 (m, 1H), 4.67 (t, J =5.4 Hz, 2H), 4.52 (d, J = 5.2 Hz, 1H), 4.47 - 4.43 (m, 2H), 4.42 - 4.38 (m,2H), 4.34 (t, J = 4.8 Hz, 1H), 4.31 - 4.23 (m, 2H), 4.21 - 4.15 (m, 1H), 4.14- 4.11 (m, 2H), 4.10 - 4.06 (m, 1H), 3.93 (s, 3H), 3.66 - 3.55 (m, 1H), 3.42(s, 3H), 3.25 (s, 3H), 2.33 - 2.24 (m, 1H), 1.12 (d, J = 6.0 Hz, 3H). 31P NMR (162 MHz, D2O) δ -0.88, -11.46, -11.60, -22.76 (4P).

[0140] 3. Synthesis of YK-CAP-202

[0141]

[0142] YK-CAP-202-PM1 (synthesized by the synthetic route of YK-CAP-108-PM10 in CN118389495B) (100 mg, 0.19 mmol) and INT-I (179 mg, 0.25 mmol) were used as starting materials. Following the synthetic route of YK-CAP-201, the final product YK-CAP-202 (20 mg, 17 µmol, 9.0%) was obtained. 35 H 49 N 13 O 25 P4, MS(ES): m / z (MH) - 1174.1. 1 H NMR (400 MHz, D2O) δ 9.06 (s, 1H), 8.36 (s, 1H), 8.12 (s, 1H), 7.36 (d, J = 2.4 Hz, 1H), 6.01 (d, J = 4.4 Hz, 1H), 5.68 (d, J = 5.6 Hz, 1H), 5.44(d, J = 2.4 Hz, 1H), 4.90 - 4.80 (m, 1H), 4.64 (t, J = 5.4 Hz, 2H), 4.48 (d,J = 5.2 Hz, 1H), 4.44 - 4.40 (m, 2H), 4.36 - 4.32 (m, 2H), 4.29 (t, J = 4.8Hz, 1H), 4.26 - 4.19 (m, 2H), 4.18 - 4.12 (m, 1H), 4.10 - 4.08 (m, 2H), 4.06- 4.01 (m, 1H), 3.88 (s, 3H), 3.63 - 3.51 (m, 1H), 3.40 (s, 3H), 2.29 - 2.20 (m, 1H), 1.90 (s, 3H), 1.08 (d, J = 6.0 Hz, 3H). 31P NMR (162 MHz, D2O) δ -0.89, -11.47, -11.63, -22.77 (4P).

[0143] 4. Synthesis of YK-CAP-203

[0144]

[0145] YK-CAP-203-PM1 (synthesized by the synthetic route of YK-CAP-109-PM8 in CN118389495B) (100 mg, 0.17 mmol) and INT-I (179 mg, 0.25 mmol) were used as starting materials. Following the synthetic route of YK-CAP-201, the final product YK-CAP-203 (15 mg, 13 µmol, 6.5%) was obtained. 33 H 45 FN 12 O 24 P4, MS(ES): m / z (MH) - 1135.1. 1 HNMR (400 MHz, D2O) δ 9.08 (s, 1H), 8.42 (s, 1H), 8.15 (s, 1H), 7.40 (d, J = 2.4 Hz, 1H), 6.17 (d, J = 4.4 Hz, 1H), 5.65 (d, J = 2.6 Hz ,1H), 5.47 (d, J = 2.4 Hz, 1H), 5.32 - 5.21 (m, 1H), 4.62 - 4.42 (m, 3H), 4.36- 4.33 (m, 4H), 4.22 - 4.15 (m, 3H), 4.11 (s, 2H), 4.03 (s, 1H), 3.91 - 3.82(m, 3H), 3.21 - 3.15 (m, 3H), 3.11 (s, 2H), 2.31 - 2.22 (m, 1H), 1.52 (d, J =6.1 Hz, 3H). 31 PNMR (163MHz, D2O) δ -0.89, -11.21, -11.74, -22.12 (4P).

[0146] 5. Synthesis of YK-CAP-204

[0147]

[0148] YK-CAP-204-PM1 (synthesized by the synthetic route of YK-CAP-110-PM8 in CN118389495B) (180 mg, 0.31 mmol) and INT-I (470 mg, 0.62 mmol) were used as starting materials. Following the synthetic route of YK-CAP-201, the final product YK-CAP-204 (71 mg, 49 μmol, 15.8%) was obtained. 32 H 39 F2N 12 O 24 P4, MS(ES): m / z (MH) - 1138.62. 1 H NMR (400 MHz, D2O) δ 9.01 (s, 1H), 8.33 (s, 1H), 8.06 (s, 1H), 7.35 (d, J = 2.4 Hz, 1H), 6.30 - 5.99 (m, 1H), 5.96 (d, J = 5.2 Hz, 1H), 5.75- 5.71 (m, 2H), 5.40 (d, J = 2.4 Hz, 1H), 4.90 - 4.82 (m, 1H), 4.81 - 4.76(m, 1H), 4.68 - 4.61 (m, 4H), 4.45 - 4.31 (m, 6H), 4.30 - 4.21 (m, 3H), 4.20- 4.10 (m, 4H), 4.07 - 4.00 (m, 2H), 3.93 (s, 4H), 3.38 (s, 4H), 2.97 - 2.85 (m, 1H). 31 P NMR (162 MHz, D2O) δ -0.88, -11.50, -11.64, -22.76 (4P).

[0149] 6. Synthesis of YK-CAP-205

[0150]

[0151] YK-CAP-205-PM1 (synthesized by the synthetic route of YK-CAP-111-PM10 in CN118389495B) (120 mg, 0.22 mmol) and INT-I (477 mg, 0.67 mmol) were used as starting materials. Following the synthetic route of YK-CAP-201, the final product YK-CAP-205 (17 mg, 14 μmol, 6.5%) was obtained. 36 H52 N 13 O 25 P4, MS(ES): m / z (MH) - 1188.2. 1 H NMR (400 MHz, D2O) δ 9.11 (s, 1H), 8.82 (s, 1H), 8.60 (s, 1H), 7.51 (d, J = 2.4 Hz, 1H), 6.09 (d, J = 4.8 Hz, 1H), 5.82 (d, J = 3.6 Hz, 2H), 5.53(d, J = 2.4 Hz, 1H), 4.90 - 4.84 (m, 2H), 4.82 - 4.78 (m, 1H), 4.62 (t, J =4.4 Hz, 1H), 4.52 - 4.36 (m, 5H), 4.34 - 4.09 (m, 6H), 4.08 - 4.00 (m, 1H),3.95 (s, 3H), 3.72 (t, J = 6.4 Hz, 1H), 3.43 (s, 3H), 3.39 - 3.21 (m, 5H),1.05 (dt, J = 21.2, 7.2 Hz, 7H). 31 P NMR (162 MHz, D2O) δ -0.92, 11.48, -11.73, -22.57 (4P).

[0152] 7. Synthesis of YK-CAP-206

[0153]

[0154] YK-CAP-206-PM1 (synthesized by the synthetic route of YK-CAP-118-PM8 in CN118389495B) (200 mg, 0.41 mmol) and INT-I (954 mg, 1.34 mmol) were used as starting materials. Following the synthetic route of YK-CAP-201, the final product YK-CAP-206 (44 mg, 39 μmol, 9.4%) was obtained. 34 H 44 N 12 O 25 P4, MS(ES): m / z (MH) - 1137.2. 1H NMR (400 MHz, D2O) δ 9.01 (s, 1H), 8.36 (s, 1H), 8.09 (s, 1H), 7.35 (d, J = 2.4 Hz, 1H), 5.95 (d, J = 5.2 Hz, 1H), 5.86 (d, J = 5.2 Hz, 1H), 5.73(d, J = 5.6 Hz, 1H), 5.40 (d, J = 2.4 Hz, 1H), 4.87 - 4.81 (m, 1H), 4.68 -4.64 (m, 2H), 4.53 (d, J = 5.6 Hz, 1H), 4.47 - 4.35 (m, 4H), 4.33 (t, J = 5.2Hz, 2H), 4.28 - 4.21 (m, 2H), 4.14 - 4.09 (m, 3H), 3.95 (s, 3H), 3.69 (d, J =10.8 Hz, 1H), 3.59 (d, J = 10.8 Hz, 1H), 3.35 (s, 3H). 31 P NMR (162 MHz, D2O)δ -0.90, -11.41, -11.48, -22.68 (4P).

[0155] 8. Synthesis of YK-CAP-207

[0156]

[0157] YK-CAP-207-PM1 (synthesized by the synthetic route of YK-CAP-117-PM8 in reference patent CN118389495B) (180 mg, 0.35 mmol) and INT-I (514 mg, 0.71 mmol) were used as raw materials and the synthetic route of YK-CAP-201 was followed to obtain the final product YK-CAP-207 (21 mg, 18 µmol, 5.0%). 33 H 47 N 12 O 26 P4, MS(ES): m / z (MH) - 1149.18. 1H NMR (400 MHz, D2O) δ 9.01 (s, 1H), 8.36 (s, 1H), 8.09 (s, 1H), 7.35 (d, J = 2.4 Hz, 1H), 5.95 (d, J = 5.2 Hz, 1H), 5.86 (d, J = 5.2 Hz, 1H), 5.73 (d, J = 5.6 Hz, 1H), 5.40 (d, J = 2.4 Hz, 1H), 4.87 - 4.81 (m, 1H), 4.68- 4.64 (m, 2H), 4.53 (d, J = 5.6 Hz, 1H), 4.44 - 4.39 (m, 2H), 4.33 (t, J =5.2 Hz, 2H), 4.28 - 4.21 (m, 2H), 4.19 - 4.15 (m, 2H), 4.14 - 4.09 (m, 3H), 3.95 (s, 3H), 3.69 (d, J = 10.8 Hz, 1H), 3.59 (d, J = 10.8 Hz, 1H), 3.38 (s,3H), 3.35 (s, 3H). 31 P NMR (162 MHz, D2O) δ -0.92, -11.44, -11.55, -22.74(4P).

[0158] Example 2: mRNA in vitro transcription yield and capping rate

[0159] I. Structural differences between capped analogs

[0160] Table 1 Structures of capped analogs

[0161]

[0162]

[0163]

[0164] As can be seen from Table 1, the chemical structures of compounds YK-CAP-201 to YK-CAP-207 of the present application are similar to each other, as follows:

[0165] 1. The compounds YK-CAP-201 to YK-CAP-207 of the present application differ only in the substituent group at C3 and / or the substituent group at C4 on the first sugar ring.

[0166] 2. The C3 substituents on the first sugar ring of compounds YK-CAP-201 through YK-CAP-205 differ from those of N-7114: the C3 substituent of N-7114 is hydroxyl; the C3 substituents of YK-CAP-201 through YK-CAP-205 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, and N,N-diacetamido, respectively.

[0167] 3. The C4 substituent on the first sugar ring of compounds YK-CAP-206 and YK-CAP-207 of the present application differs from that of N-7114: the C4 substituent of N-7114 is a hydrogen atom; the C4 substituents of YK-CAP-206 and YK-CAP-207 are 1-fluoromethyl and methoxymethyl, respectively.

[0168] II. mRNA in vitro transcription yield and capping rate

[0169] 1. Experimental Methods

[0170] 1.1 Preparation of DNA template

[0171] 1) Luciferase (Luciferase protein CDS) was constructed by EcoRV enzyme digestion and ligation into the pVAX1 vector (Thermo Fisher Scientific);

[0172] 2) Mix the plasmid constructed in the pVAX1 vector from step 1) with 50 μL of competent E. coli Stbl2 cells (Thermo Fisher Scientific). Incubate the mixture on ice for 30 minutes, heat shock at 42°C for 90 seconds, and immediately return to ice for 2 minutes.

[0173] 3) Add 400 μL of LB medium (Thermo Fisher Scientific) and incubate at 30°C with slow shaking for 45-60 minutes.

[0174] 4) Spread 50-100 μL of bacterial suspension onto LB solid medium containing kanamycin (100 μg / mL, Yisheng Biotechnology Co., Ltd.) and incubate inverted at 37°C overnight.

[0175] 5) Sequence the obtained monoclonal colony plate to verify its correctness. Pick the monoclonal colony with the correct sequencing and culture it in a shaker at 30°C overnight;

[0176] 6) Plasmid extraction was performed using the MolPure® Endo-free Plasmid Maxi Kit (Yisheng Biotechnology Co., Ltd., Catalog No. 19036ES10).

[0177] 7) The extracted plasmid is digested with restriction endonucleases into a linearized plasmid for use as a transcription template. For the specific digestion process, see steps ① to ③ below.

[0178] Step 1: Take 1 mg of luciferase circular plasmid and digest it with BspQ I enzyme (purchased from Yisheng Biotechnology Co., Ltd.) at 37°C for 4 hours to obtain a linearized DNA transcription template (see Table 2 for the enzyme digestion reaction system).

[0179] Table 2 Enzyme digestion reaction system

[0180]

[0181] Step ② After the reaction is completed, in the enzyme cleavage reaction product solution, according to V 酶切反应产物 :V 无水乙醇 :V 3M醋酸钠 =1:3:1 volume ratio, anhydrous ethanol and 3 M sodium acetate were added in sequence, and the mixture was allowed to settle at -20 °C for 1 hour, and then centrifuged at 12000 rpm to retain the precipitate;

[0182] Step 3: Wash the precipitate obtained in step 2 twice with 70% ethanol and centrifuge. The centrifuged material is dried at 55°C for 10 minutes, and then dissolved in 1.7 mL of water for injection;

[0183] The concentration of the linearized plasmid in the lysate was 500 ng / µL, the linearization ratio was over 90%, and the purification recovery efficiency was 85%.

[0184] 1.2 In vitro transcription of mRNA

[0185] 1) Co-transcriptional capping reaction:

[0186] YK-CAP-201, YK-CAP-207, and N-7114, synthesized in Example 1, were used as capping analogs, respectively. The Fluc DNA prepared in "1.1 DNA Template Preparation" above was used as a template, and NTP solution (NTPs) was used as the starting material. mRNA was transcribed using T7 RNA polymerase. The specific reaction system is shown in Table 3. The prepared reaction system was placed in a 37°C incubator with shaking for 3 hours.

[0187] Table 3 Co-transcriptional capping reaction system

[0188]

[0189] Note: N-7114 among the capped analogs was purchased from Cangzhou Weikexin Biochemical Technology Co., Ltd., and other capped analogs were synthesized with reference to the aforementioned content of this application; other reagents except the capped analogs were purchased from Yisheng Biotechnology Co., Ltd.

[0190] 2) Digestion of template DNA:

[0191] DNase I (Yisheng Biotechnology Co., Ltd.) was added to the co-transcriptional capping reaction system completed in step 1) above, so that the final concentration of DNase I was equivalent to 1U of DNase I per μg of linearized plasmid in the reaction solution. After mixing, centrifugation was performed and digestion was carried out at 37°C for 1 hour to obtain the co-transcriptional capping product.

[0192] 3) Purification by lithium chloride precipitation:

[0193] The co-transcribed capping product obtained in step 2) above was purified by lithium chloride precipitation as follows:

[0194] Step 1) Adding lithium chloride: Add lithium chloride solution (Thermo Fisher Scientific) to the product of step 2) above to a final concentration of 2.8 M and allow to cool for 2 hours.

[0195] Step ② Precipitation: Centrifuge at 12,000 rpm for 15 minutes and retain the precipitate;

[0196] Step 3: Washing: Wash twice with 75% ethanol and dissolve in water for injection to obtain the mRNA solution. Store the purified mRNA solution at -80°C.

[0197] 1.3 Annealing of the mRNA with the probe

[0198] Annealing in a PCR instrument: 95 o C 5 min; 65 o C 2 min; 55 o C 2 min; 40 o C 2 min; 22 o C2 min.

[0199] 1.4 Magnetic bead pretreatment and probe binding: Place 100 μL of magnetic beads on a magnetic rack for pretreatment. Add 120 μL of sample and magnetic bead solution and incubate at room temperature for 30 min, gently mixing during incubation.

[0200] 1.5 Shearing mRNA and obtaining the 5' single-stranded sequence of mRNA bound to the probe

[0201] Add 20 μL RNaseH (5 U / μL) and incubate at 37 o Incubate at 80°C for 3 h, mixing every half hour. Wash the magnetic beads after incubation, add 100 μL of PBS heated to 80°C to the washed magnetic beads. o The resulting mixture was heated to 80°C on a hot plate. oC, hold for 3 minutes, then place on a magnetic rack, aspirate the supernatant, and dry in an evaporative centrifuge at room temperature for 45 minutes to a volume of 10 μL. The sample is then resuspended in 50 μL of 100 μM EDTA / 1% MeOH and used for LC-MS analysis to determine RNA capping during the transcription reaction. Because capped and uncapped bases have distinct molecular weight differences, this molecular mass difference can be used to determine the capping rate of mRNA transcripts initiated by different capping analogs.

[0202] (6) mRNA integrity was assessed using the Agilent 5200 Fragment Analyzer capillary electrophoresis system.

[0203] 1) Sample preparation: Based on the sample concentration determined by ultra-micro spectrophotometer, each sample was diluted with RNase-free water to 20-100 ng / μL at different times. The sample and the pre-packaged ladder were mixed and placed in a metal bath (MiniH-100D, Hangzhou Youning) at 70 o C for 2 min to denature, then remove from the plate and immediately place on ice.

[0204] 2) Preparation of capillary reagents:

[0205] Table 4 Preparation of capillary reagents

[0206]

[0207] 3) Set the sample operation program and sample loading and determination: set the sample injection voltage to 5 kV, the run time to 4 s, and the separation voltage to 8 kV, the run time to 45 min.

[0208] 4) Data Analysis: The Agilent Fragment Analyzer comes with ProSize analysis software, which allows users to batch calculate the percentage of RNA fragments that meet the requirements or the percentage of degraded RNA through Smear analysis.

[0209] 2. Experimental Results

[0210] The results of the measurement of mRNA in vitro transcription yield, integrity, and capping rate showed that the capping rates were basically consistent and all at very high levels. Compared with the self-replicating mRNA capping analogs used in the prior art, the self-replicating mRNA capping analogs disclosed herein significantly improved the mRNA in vitro transcription yield and integrity, and the capping rates were all at very high levels. Specific mRNA in vitro transcription yields and capping rates are shown in Table 5.

[0211] Table 5 Results of co-transcriptional capping reaction system

[0212]

[0213] As can be seen from Table 5, the self-replicating mRNA capping analogs in this application can all efficiently transcribe mRNA ( Figure 1 Compared with the capping analogs used for self-replicating mRNA in the prior art, the mRNA in vitro transcription yield and integrity are significantly improved.

[0214] The mRNA yield and integrity of N-7114 in vitro transcription ( Figure 2 ) were 175.3 μg and 60.1%, respectively. The in vitro transcription yield and integrity of the mRNA of YK-CAP-206 of the present application were increased by 18.6% and 18.1% compared with N-7114, respectively.

[0215] It can be seen from the in vitro transcription yield and integrity of mRNA that, compared with the prior art N-7114, the in vitro transcription yield and integrity of the capping analogue for self-replicating mRNA of the present application are significantly improved, indicating that the capping analogue of the present application has excellent anti-reverse transcription effect when used for self-replicating mRNA, and can increase the binding ability of the cap structure with the capping enzyme, thereby increasing the capping rate of the transcribed mRNA.

[0216] Example 3: Preparation and characterization of lipid nanoparticles

[0217] 1. Experimental Methods

[0218] Cationic lipid YK-009 (Beijing Yuekang Kechuang Pharmaceutical Technology Co., Ltd.), DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG 2000 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5. mRNA was diluted in 50 mM citrate buffer, pH 4. Using a microfluidic device, the ethanolic lipid solution was mixed with aqueous solutions of FLuc mRNA prepared with different cap structures at a volume ratio of 1:3 at a flow rate of 10 mL / min. LNPs were prepared with a total lipid to mRNA mass ratio of approximately 15:1. The resulting liposomes were diluted to 10 times their original volume with PBS and then ultrafiltered through a 300 kDa ultrafiltration tube to remove the ethanol. The volume was then adjusted to a certain value with PBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain the YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar ratio of 49:10:39.5:1.5) LNP preparation encapsulating self-replicating FLuc-mRNA.

[0219] The structural formula of YK-009 is as follows: .

[0220] The particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer using dynamic light scattering. 25 μL of the liposome solution was diluted to 125 μL with saline and added to the sample cell. Each sample was measured three times. The measurement conditions were: 90 o Scattering angle, 25 o C. The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantification Assay Kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions.

[0221] 2. Experimental Results

[0222] The characterization data of specific lipid nanoparticles are shown in Table 6.

[0223] Table 6 Characterization of lipid nanoparticles

[0224]

[0225] As shown in Table 6, lipid nanoparticles can be prepared from self-replicating FLuc-mRNA transcribed from the capped analogs YK-CAP-201 to YK-CAP-207 described in this application and the capped analog N-7114 disclosed in the prior art. All lipid nanoparticles had particle sizes between 70 and 85 nm, PDI values ​​between 0.031 and 0.096, and encapsulation efficiencies exceeding 90%.

[0226] Example 4: Translation efficiency of luciferase mRNA containing different cap structures

[0227] 1. Experimental Methods

[0228] (1) Incubate in DMEM medium containing 10% FBS and penicillin / streptomycin at 37 o HEK293T cells were cultured under 5% CO2 conditions.

[0229] (2) Digest and count the cells in the culture dish, plate 10,000 cells per well in a 96-well plate, and culture overnight until the cells adhere.

[0230] (3) When the cell density was approximately 80%, transfection was performed by adding 50 ng of mRNA sample and Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) to each well. The transfection steps were performed according to the manufacturer's instructions.

[0231] (4) Place the transfected cells at 37 oAfter incubation for an additional 24 h at 5% CO2, remove the growth medium from the cells to be tested and rinse the cells with PBS. Remove the PBS by centrifugation, add 50 μL of 1x lysis buffer, transfer the cells and all liquid to a microcentrifuge tube, and centrifuge.

[0232] (5) Take 20 μL of sample and add 100 μL of Dual-Lumi™ II Firefly Luciferase Assay Reagent that has been equilibrated to room temperature and mix well.

[0233] (6) Room temperature (about 25 o C) Incubate for 5 min to allow the luminescent signal to stabilize. Use a multifunctional microplate reader with chemiluminescence detection function to perform chemiluminescence detection and record the data (see Table 7 and Figure 4 ).

[0234] 2. Experimental Results

[0235] The relative fluorescence readings of capped mRNA are shown in Table 7. The relative fluorescence intensity is proportional to the translation efficiency of mRNA.

[0236] Table 7 Average fluorescence readings of capped mRNA

[0237]

[0238] It can be seen that the capping analogues for self-replicating mRNA of the present application significantly improve the mRNA translation efficiency compared with the capping analogues for self-replicating mRNA in the prior art.

[0239] The average fluorescence intensity of N-7114 (corresponding to the translation efficiency of mRNA) was 13241. The average fluorescence intensity of YK-CAP-206 was 3.0 times that of N-7114, and the average fluorescence intensity of YK-CAP-203 was 2.1 times that of N-7114.

[0240] The C3 substituents on the first sugar ring of YK-CAP-201 to YK-CAP-205 in this application differ from those in N-7114: N-7114's C3 substituent is a hydroxyl group; the C3 substituents of YK-CAP-201 to YK-CAP-205 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, and N,N-diacetamido, respectively. However, the translation efficiencies of the corresponding mRNAs of YK-CAP-201, YK-CAP-203, YK-CAP-204, and YK-CAP-205 were 1.9-, 2.1-, 2.0-, and 1.6-fold higher than those of N-7114, respectively.

[0241] It can be seen from the translation efficiency of different capped luciferase mRNAs that the capping analogs for self-replicating mRNAs of the present application have significantly improved translation efficiency compared to the prior art capping analog N-7114 for self-replicating mRNAs. This indicates that the use of the capping analogs of the present application to modify ribose can more easily bind to the cap binding protein (EIF4E), thereby improving the translation efficiency of the target mRNA.

[0242] Example 5: Cell viability determination

[0243] 1. Experimental Methods

[0244] The LNP formulation containing 1 μg of self-replicating FLuc-mRNA prepared according to Example 3 was added to the cell culture medium of a 96-well plate. After 24 hours of culture, 10 μL of CCK-8 solution was added to each well. The plate was incubated in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. The results are shown in Table 8.

[0245] The cell survival rate can represent the toxicity of the transcribed self-replicating FLuc-mRNA to the cells. The higher the cell survival rate, the lower the toxicity to the cells.

[0246] 2. Experimental Results

[0247] Table 8 Cell survival rate

[0248]

[0249] It can be seen that the self-replicating FLuc-mRNA transcribed from the capped analogs YK-CAP-201 to YK-CAP-207 of the present application and the capped analog N-7114 of the prior art both have low cytotoxicity and cell survival rates greater than 90%.

[0250] Example 6: Animal Experiment

[0251] 1. Experimental Methods

[0252] LNP formulations containing 5 μg of capped analog-transcribed self-replicating FLuc-mRNA were injected intramuscularly into female BALB / c mice, aged 4-6 weeks and weighing 17-19 g. Fluorescent imaging substrate was injected intraperitoneally at 24, 48, 72, 120, and 192 hours after administration. The mice were allowed to move freely for 5 minutes, and the total radiation intensity (which reflects protein expression) of the protein expressed by the LNP-carried mRNA was measured using an IVIS Spectrum small animal in vivo imager.

[0253] 2. Experimental Results

[0254] The test results are shown in Table 9 and Figure 5 In the mouse in vivo imaging experiment, the total radiation intensity of each group of mice relative to N-7114 is shown in Table 10 (where the total radiation intensity is the value × 10 7 p / s).

[0255] Table 9 Mouse in vivo imaging experimental data

[0256]

[0257] Table 10 Total radiation intensity relative to N-7114 multiples

[0258]

[0259] Compared to capped analogs in the prior art, the total radiation intensity and duration of protein expression in mice from mRNA transcribed from the capped analogs of the present application were significantly improved. For example, the corresponding total radiation intensity of YK-CAP-206 was 2.5 times, 2.1 times, 2.4 times, 2.2 times, and 2.3 times that of N-7114 at 24 hours, 48 ​​hours, 72 hours, 120 hours, and 192 hours, respectively. The total radiation intensity of YK-CAP-205 at the corresponding times was 3.4 times, 2.0 times, 2.0 times, 1.7 times, and 1.7 times that of N-7114, respectively.

[0260] The C3 substituent on the first sugar ring of compounds YK-CAP-201 to 205 in this application differs from that of N-7114. Specifically, the C3 substituent in N-7114 is a hydroxyl group, while the C3 substituents in YK-CAP-201 to 205 are 1-methoxyethyl, 1-acetamidoethyl, 1-fluoroethyl, difluoromethyl, and N,N-diacetamido, respectively. However, the total radioactivity intensities of proteins expressed in mice over a 24-hour period from YK-CAP-201, YK-CAP-203, YK-CAP-204, and YK-CAP-205 were 1.3, 2.4, 1.3, and 3.4 times that of N-7114, and 3.4, 6.3, 3.5, and 8.8 times that of YK-CAP-202, respectively.

[0261] Animal experiments have shown that compared to the prior art capping analog N-7114, the mRNA transcribed from the capping analogs of this application significantly increases protein expression and duration in mice. In vivo experiments further demonstrate that the mRNA transcribed from YK-CAP-201, YK-CAP-203, and YK-CAP-206 in this application can be effectively delivered into the body by LNP delivery vectors and expressed efficiently and continuously.

[0262] In summary, the self-replicating mRNA capping analogs YK-CAP-201, YK-CAP-203, and YK-CAP-206 used in the present application for self-replicating RNA have significantly improved mRNA in vitro transcription yield, integrity, capping rate, mRNA translation efficiency, and protein expression and duration in animals compared to the prior art self-replicating mRNA capping analog N-7114. This indicates that the capping analogs provided by the present disclosure can significantly increase the affinity for binding to the capping enzyme, providing a cap structure that can be used for self-replicating mRNA in vitro transcription with high efficiency.

[0263] 1. Compared with prior art capping analogs for self-replicating mRNA, the capping analogs for self-replicating mRNA in this application significantly improve both the in vitro mRNA transcription yield and integrity. For example, the in vitro mRNA transcription yield and integrity of N-7114 were 175.3 μg and 60.1%, respectively. The in vitro mRNA transcription yield and integrity of YK-CAP-206 in this application were 18.6% and 18.1% higher than those of N-7114, respectively.

[0264] 2. Compared to prior art capping analogs for self-replicating mRNA, the capping analogs for self-replicating mRNA in this application significantly improve mRNA translation efficiency. For example, the translation efficiency of YK-CAP-206 in this application is 3.0 times that of N-7114.

[0265] The above embodiments are intended only to illustrate the technical solutions of the present disclosure and are not intended to limit the present disclosure. Although the present disclosure has been described in detail with reference to the above embodiments, those skilled in the art should understand that they may modify the technical solutions described in the above embodiments or replace some or all of the technical features therein with equivalents. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the claims of the present disclosure.

Claims

1. A compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt of the compound is any of the following structures: 、 、 、 or 。 2. Use of the compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of an in vitro co-transcribed mRNA capping reagent.

3. An RNA molecule, characterized in that It comprises the compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof as a cap structure or a cap structure fragment.

4. A pharmaceutical composition, characterized in that It comprises the RNA molecule according to claim 3.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition further comprises at least one delivery agent.

6. The pharmaceutical composition according to claim 5, characterized in that The at least one delivery agent is selected from any one or a combination of at least two of the group consisting of cationic lipids, neutral lipids, structured lipids and polymer-conjugated lipids.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions (1) to (4): (1) The cationic lipid is any one of the following or a combination of at least two: (i) A compound represented by formula (II), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-6 Alkylene; G2 is C 2-8 Alkylene; G3 is C 1-3 Alkylene; L1 is C 6-15 Straight chain alkyl; L2 is C 12-25 branched alkyl; (II) (ii) A compound represented by formula (III), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C 2-8 Alkylene; G2 is C 2~8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6-25 Straight or branched alkyl; R2 is C 6-25 Straight or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4-; (III) (iii) A compound represented by formula (IV), its stereoisomer, its N-oxide, its solvate or its pharmaceutically acceptable salt, wherein: G1 is C 1-6 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-20 Straight or branched alkyl; R2 is C 12-25 Branched alkyl; G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-; (IV) (iv) A compound represented by formula (V), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G1 is C 1-8 Alkylene; G2 is C 2-8 Alkylene; R1 is C 6-25 Straight or branched alkyl; R2 is C 12-25 Straight or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3, -CH2CH3 or -CH2CH2OH; (V) (v) A compound represented by formula (VI), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein G 1 and G 2 Each independently is C6-C 10 Alkylene; G 3 C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 OR 5 、N(R 5 )2, -C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 Hydrocarbon; R 5 is H or a C1-C6 hydrocarbon group; (WE) (vi) A compound represented by formula (VII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein R4 is -(CH2) n Q; Q is -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)2R or heterocycle; n is 1, 2 or 3; R is independently C 1-8 Alkyl; X is independently H or C 1-8 alkyl; (VII) (vii) a compound represented by formula (VIII), a stereoisomer thereof, an N-oxide thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, (VIII); (2) the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide and sterol; (3) the structural lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids; and (4) The polymer-conjugated lipid is selected from any one of the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycero-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol ditetradecanoyl acetamide, or a combination of at least two of them.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition satisfies any one or a combination of at least two selected from the group consisting of the following conditions (1) to (4): (1) The cationic lipid is selected from any one of the group consisting of YK-009, YK-201, YK-407, YK-305, ALC-0315, SM-102 and DLIN-MC3-DMA, or a combination of at least two of the following: 、 、 、 、 、 、 ； (2) The neutral lipid is selected from 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine, 1, 2-dimyristoyl-sn-glycero-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1, 2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1, 2-Dilinoyl-sn-glycero-3-phosphocholine, 1, 2-diamidonoyl-sn-glycero-3-phosphocholine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoyl-sn-glycero-3-phosphoethanolamine, 1, 2-diamidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine, or a combination of at least two of the group consisting of; (3) the structural lipid is cholesterol; and (4) The polymer conjugated lipid is 1,2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol 2000.

9. The pharmaceutical composition according to claim 8, characterized in that The cationic lipid is YK-009; and / or the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

10. The pharmaceutical composition according to claim 6, wherein The at least one delivery agent comprises YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000; the YK-009 is .

11. The pharmaceutical composition according to claim 10, wherein The molar ratio of YK-009, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 is 49:10:39.5:1.

5.

12. The pharmaceutical composition according to any one of claims 4 to 11, characterized in that The pharmaceutical composition further comprises one or at least two cell penetrating peptides.

13. A method for synthesizing mRNA molecules for purposes other than disease diagnosis and treatment, characterized in that: The following steps are involved: The compound according to claim 1, its stereoisomer, its pharmaceutically acceptable salt or solvate thereof is co-incubated with a polynucleotide template to transcribe the polynucleotide template.

14. A capped mRNA transcription reaction system for non-disease diagnosis and treatment purposes, characterized in that: include: (1) The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof; and (2) polynucleotide template, NTPs, and RNA polymerase.

15. A kit, characterized in that include: (1) The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof; and (2) nucleotide triphosphate molecules, and RNA polymerase.

16. The kit according to claim 15, wherein The kit further comprises RNAse inhibitor, inorganic pyrophosphatase, Mg 2+ , crowding agents and buffers, or a combination of at least two of the group consisting of.

17. A method for improving the stability of RNA, characterized in that: The method comprises incorporating the compound according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof into RNA.

18. A method for introducing RNA into a cell, characterized in that The method comprises contacting the cell with the RNA molecule of claim 3 or the pharmaceutical composition of any one of claims 4-11.

19. Use of the RNA molecule according to claim 3 or the pharmaceutical composition according to any one of claims 4 to 11 in the preparation of a vaccine.

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