Compounds for RNA capping and uses thereof

Abstract

The invention relates to two compounds for RNA capping and application thereof, and belongs to the technical field of genetic engineering. The compound has a structure as shown in a formula (I), can be used as an initial capping oligonucleotide primer, is used for capping an mRNA 5'end, and has good capping efficiency and protein expression quantity; meanwhile, due to the fact that the fluorescent group marker is arranged, the fluorescent group marker can be used for detecting and tracking dynamic changes of mRNA in cells and living bodies, the delivery process, pharmacokinetics and other performance of mRNA in the cells or living bodies can be conveniently researched, and convenience is provided for research of mRNA vaccines / drugs in the cells or living bodies. # imgabs0 #

Description

Compounds for RNA capping and their applications

[0001] This application claims priority to Chinese patent application No. 202310061987.7, filed on January 19, 2023. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the technical field of genetic engineering, in particular to a compound for RNA capping and application thereof. Background Art

[0003] The 5'-prime cap structure (m7GpppN) of messenger RNA (mRNA) was discovered in the 1970s. Its presence confers stability to mRNA and enables efficient translation. There are generally three types of cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), respectively designated as type O (m7G5'ppp5'Np), type I (m7G5'ppp5'NmpNp), and type II (m7G5'ppp5'NmpNmpNp). Type O refers to unmethylated ribose sugars on the terminal nucleotide, type I refers to methylated ribose sugars on one terminal nucleotide, and type II refers to methylated ribose sugars on both terminal nucleotides.

[0004] In eukaryotic cells, in addition to identifying the start of protein synthesis, the 5'-end cap structure also acts as a protective group for cleavage by 5'- to 3'-exonucleases, providing resistance to degradation by 5'-exonucleases. During protein synthesis, the cap structure also serves as a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation, and nuclear export. It also serves as an anchor for recruiting initiation factors, facilitating ribosome recognition and binding to mRNA, enabling the correct initiation of translation.

[0005] The outbreak of the epidemic has greatly promoted the research of mRNA vaccines / drugs, one of the indispensable raw materials of which is the cap structure. Although there have been many studies on the cap structure, there are very few cap raw materials that can be used on a large scale. Therefore, it is necessary to provide more caps with excellent performance for selection to promote the faster development of mRNA vaccines / drugs without being restricted by the cap structure. Therefore, providing a cap structure with a high capping rate and improved stability, expression level, immunogenicity and other properties of the capped mRNA has great application prospects and value.

[0006] In addition, in order to understand the biological processes that exogenous RNA participates in within cells, such as delivery, translation, migration, shearing, and degradation, it is necessary to visualize the life processes that RNA molecules participate in within cells for researchers, and to use imaging technology to track the real-time dynamics of RNA molecules within cells. To achieve this goal, RNA usually needs to be labeled. Currently, in vitro transcribed mRNA is usually labeled with nucleoside triphosphates (NPTs) modified with fluorescent groups. However, nucleoside triphosphates modified with spatially larger fluorescent groups are difficult to be recognized by RNA polymerase and introduced into mRNA, resulting in reduced transcription efficiency and transcription yield; they are also likely to have a certain impact on the stability and translation efficiency of mRNA prepared by in vitro transcription.

[0007] Since the mRNA cap structure is essential for mRNA stability and translation expression, most existing research focuses on the capping rate, immunogenicity, and mRNA expression level of the cap structure. No relevant research has been conducted on the labeling function of the cap. Therefore, a cap structure that retains its original function while also having a labeling function has great application value and is necessary for further research.

[0008] Summary of the Invention

[0009] Based on the importance of the 5'-end cap structure and the huge unmet market demand, the present invention provides a compound for RNA capping. Using this compound for mRNA 5'-end capping not only has good capping efficiency, but also significantly improves the stability and expression level of the capped mRNA. It performs well both in vitro and in animals, and is superior to the positive control.

[0010] In addition, the present invention also provides an RNA-capped compound with a fluorescent group, which not only retains the original excellent performance, such as a good capping rate, but also the capped mRNA has good stability, expression level and other properties; because it is labeled with a fluorescent group, it can also be used for detection and tracking the dynamic changes of mRNA in cells and organisms, facilitating the study of the delivery process of mRNA in cells or organisms, pharmacokinetic and other properties, and providing convenience for the research of mRNA vaccines / drugs.

[0011] One of the objects of the present invention is to provide a compound having a structure of Formula I or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof:

[0012] in:

[0013] R1 is selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, benzyl, R5-substituted C1-C6 alkyl, R5-substituted C2-C6 alkenyl, R5-substituted C2-C6 alkynyl, R5-substituted C3-C6 cycloalkyl, R5-substituted C3-C6 cycloalkenyl or R5-substituted benzyl;

[0014] R2 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, cycloalkenyl, benzyl, aryl, heteroaryl, R5 substituted benzyl, R5 substituted aryl, carbonylalkyl, carbonylalkoxy or sulfonamide;

[0015] R3 is selected from H, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, R5 substituted C1-C6 alkyl, R5 substituted C2-C6 alkenyl, R5 substituted C2-C6 alkynyl, halogen or none;

[0016] R 3a , R 3b Each is independently selected from H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, R5 substituted C1-C6 alkyl, R5 substituted C2-C6 alkenyl or R5 substituted C2-C6 alkynyl;

[0017] R9 is selected from H, halogen, C1-C3 alkyl or R4 substituted C1-C3 alkyl;

[0018] R0 is selected from H, a fluorescent group or a steroidal structural group;

[0019] X0 is selected from O, S, NR4, CH2, CF2, CHF, CCH2 or CCF2;

[0020] W is selected from H, OH, OR4, NR4R4, NR4COR4, F, Cl, N3 or CN;

[0021] W2 is selected from H, OH, halogen, N3, CN, OR4, NR4R4, NR4COR4 or C1-C4 alkyl;

[0022] L1 is selected from O, S, NH, carbonyl or none;

[0023] L2 is selected from NH, carbonyl or none;

[0024] The L3 is selected from -X-(CH2)nNH-, -X-(CH2)nNH(CH2)mNH-, -X-(CH2)nNHCO(CH2)mNH-, -X-(CH2)nCONH(CH2)mNH-, -X-(CH2)n(OC2H4)mNH-, -X-(CH2)n(OC2H4)mOCH2-, -X-(C(O)CHRaNH)n- or none; wherein X is NH, O, a five-membered heteroaryl group, a six-membered heteroaryl group or none; n and m are each independently an integer from 0 to 10; wherein Ra is selected from the side chain groups in amino acids, and when Ra appears each time, it may be the same or different (i.e., in the same substituent, when n≥2, Ra may be the same or different);

[0025] Y a , Y b , Y c , Y d Each independently selected from O, S, CH2, CCl2, CF2 or NH;

[0026] Y 1a , Y 1b , Y 1c Each independently selected from O or S;

[0027] Y 2a , Y 2b , Y 2c Each independently selected from OH, SH or BH3;

[0028] Y3, Y4 are each independently selected from CH2 or O;

[0029] Z1 is selected from O, OH, CH2, S, NR6, CO or SO2;

[0030] Z2, Z3 are each independently selected from O, NR6, CHR7, CHCOOR7, CHCONR7R7, S, CO, SO2, PO(OH), PO(SH), P(O)VCO2H or none; Z2 can be combined with R 3a The connected oxygen atoms form a ring;

[0031] Z4 is selected from O, CH2, S, NR6, CO, SO2 or none;

[0032] B1 and B2 are each independently selected from natural or modified pyrimidine nucleotide bases, and natural or modified purine nucleotide bases;

[0033] R4 is selected from H, C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl;

[0034] R5 is selected from halogen, CN, SO2, NO2, D, N3, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, OR7, SR7, NR7R7, COR7, COOR7, OCOOR7, CONR7R7, NHCOR7, OCONR7R7, aryl or heteroaryl;

[0035] R6 is selected from H, C1-C6 alkyl, COR8 or SO2R8;

[0036] R7 is selected from H, halogen, C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl;

[0037] R8 is selected from H, C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl;

[0038] V is C 1-4 alkyl;

[0039] n0 is an integer selected from 0 to 6;

[0040] m0 is selected from 0, 1 or 2.

[0041] In some embodiments, Ra is selected from side chain groups in common amino acid structures.

[0042] In some embodiments, the compound has a structure of Formula II, or a stereoisomer, a pharmaceutically acceptable salt, or a solvate of the compound of Formula II:

[0043] Among them, R1, R2, R3, R 3a 、R 3b , R4, R5, R6, R7, R8, R9, R0, R a ,L1,L2,L3,X,X0,W,W2,Y a 、Y b 、Y c 、Y d 、Y 1a 、Y 1b 、Y 1c 、Y 2a 、Y 2b 、Y 2c , Y3, Y4, B1, B2, Z1, Z2, Z3, Z4, V, n0, m0, n and m are as described above respectively.

[0044] In some embodiments, when R0 is a fluorescent group, it is a fluorescent group formed by any one of the following dyes: biotin, Cy3, Cy5, Cy7, Cy5.5, DY-776, DY-751, DY-647P1, AF647, AF555, 5-FAM, 6-FAM, 6-TET, 5-SIMA, 6-JCE, ATTO 700, ATTO 680, ATTO 655, Texas Red, DEAC, AMCA, ANT, MANT, DY-480XL, DY-485XL, ATTO 425, ATTO 390, ATTO465, ATTO495, BDP-FL, ATTO 647N, ATTO 633, ATTO Rho14, ATTO Rho13, ATTO Rho12, ATTO Rho11, ATTO Thio12, ATTO 620, ATTO Rho101, ATTO 550, ATTORho6G, ATTO Rho13, ATTO 532, 5 / 6-TARMA, 6-ROX, ATTO 565, ATTO 590, AF594, 5 / 6-RHOX, AF488, AF546.

[0045] When R0 is a fluorescent group, the fluorescent group is any of the following groups:

[0046] In some embodiments, when R0 is a fluorescent group, R0 is biotin, Cy3, Cy5, Cy7, Cy5.5, AF647, AF555, 5-FAM, 6-FAM, 6-TET, 5-SIMA, 6-JCE, ATTO 700, ATTO 680, ATTO 655, ATTO 647N, ATTO 633, ATTO 620, ATTO 590, ATTO 565, ATTO 550, ATTO532, ATTO 495, ATTO 465, ATTO 425, ATTO 390, ATTO Rho14, ATTO Rho13, ATTO Rho12, ATTO Rho11, ATTO Thio12, ATTO Rho101, ATTO Rho6G, DY-776, DY-751, DY-647P1, DY-480XL, DY-485XL.

[0047] In some embodiments, when R0 is a fluorescent group, R0 is biotin, Cy3, Cy5, Cy7, Cy5.5, AF647, AF555, 5-FAM, 6-FAM, 6-TET, 5-SIMA, 6-JCE, Texas Red, DEAC, AMCA, ANT, MANT, BDP-FL, 5 / 6-TARMA, 6-ROX, AF594, 5 / 6-RHOX, AF488, AF546.

[0048] In some embodiments, when R0 is a fluorescent group, R0 is biotin, Cy3, Cy5, Cy7, Cy5.5, AF647, AF555, 5-FAM, 6-FAM, 6-TET, 5-SIMA, 6-JCE, ATTO 700, ATTO 680, ATTO 655, ATTO 647N, ATTO 633, ATTO 620, ATTO 590, ATTO 565, ATTO 550, ATTO 532, ATTO 495, DY-776, DY-751, DY-647P1, DY-480XL, DY-485XL.

[0049] In some embodiments, when R0 is a fluorescent group, R0 is biotin, Cy3, Cy5, Cy7, Cy5.5, AF647, AF555, 5-FAM, 6-FAM, 6-TET, 5-SIMA, 6-JCE, ATTO 655, ATTO 647N, ATTO 590, ATTO 565, ATTO 550, DY-776, DY-751, DY-647P1, DY-485XL. In some embodiments, when R0 is a steroidal structural group, it is a group formed by any of the following steroidal structures: cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, or glycoursodeoxycholic acid.

[0050] The group formed by cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid or ganoursodeoxycholic acid is shown below:

[0051] In some embodiments, L1 and L2 are not NH at the same time; or L1 and L2 are not carbonyl at the same time; and L2 and X are not NH at the same time.

[0052] In some embodiments, when L1 is absent, L2 is selected from NH, carbonyl or absent;

[0053] or, when L1 is NH, L2 is selected from carbonyl or none;

[0054] Alternatively, when L1 is a carbonyl group, L2 is selected from NH or none.

[0055] In some embodiments, X is NH, O, a five-membered nitrogen-containing aryl group, a six-membered nitrogen-containing aryl group, or none; more preferably, NH, O, Or not.

[0056] In some embodiments, L3 is selected from L3 is selected from -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nNHCO(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2)n(OC2H4)mNH-, -O-(CH2)nNH-, -O-(CH2)nNHCO(CH2)mNH-, -O-(CH2)nCONH(CH2)mNH-, -O-(CH2)n(OC2H4)mNH-, -NH-(CH2)nNH-, -NH-(CH2)nNHCO(CH2)mNH-, -NH-(CH2)nCONH(CH2)mNH-, -NH-(CH2)n(OC2H4)mNH-, -(CH2)n(OC2H4)mOCH2-, -O-(CH2)n(OC2H4)mOCH2-, -NH-(CH2)n(OC2H4)mOCH2-, -(C(O)CHRaNH)n- or none;

[0057] wherein Ra is selected from H, CH3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, CH2C8NH6, CH2C6H4OH, CH2COOH, CH2CONH2, (CH2)2COOH, (CH2)4NH2, (CH2)2CONH2, (CH2)2SCH3, CH2OH, CH(CH3)OH, CH2SH, C3H6, CH2C3H3N2 or (CH2)3NHC(NH)NH2; when Ra occurs each time, it may be the same or different;

[0058] wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0059] In a specific embodiment, when n is 0, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0060] When n is 1, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0061] When n is 2, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0062] When n is 3, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0063] When n is 4, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0064] When n is 5, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0065] When n is 6, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0066] When n is 7, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0067] When n is 8, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0068] When n is 9, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0069] When n is 10, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0070] In some embodiments, Ra is selected from H, CH3, C(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, CH2C6H4OH, CH2COOH, (CH2)2COOH, CH2CONH2, (CH2)2CONH2, CH2OH, CH(CH3)OH, C3H6 or (CH2)3NHC(NH)NH2; when Ra appears each time, it may be the same or different.

[0071] In some embodiments, Ra is selected from H, CH3, CH(CH3)2, CH2C6H5, CH2C6H4OH, CH2COOH, CH2CONH2, (CH2)2CONH2, CH2OH or CH(CH3)OH; and each occurrence of Ra may be the same or different.

[0072] In some embodiments, Ra is selected from H, CH3, CH2C6H5, CH2C6H4OH, CH2CONH2 or (CH2)2CONH2; each occurrence of Ra may be the same or different.

[0073] In some embodiments, Ra is selected from H, CH3, CH2C6H5 or (CH2)2CONH2; each occurrence of Ra may be the same or different.

[0074] In some embodiments, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)n(OC2H4)mOCH2-, -NHCO(CH2)n(OC2H4)mOCH2-, -CONH(CH2)n(OC2H4)mOCH2-, -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2) nNHCO(CH2)mNH-, -NHCO(CH2)nNHCO(CH2)mNH-, -NHCONH(CH2)nNH-, -NHCOO(CH2)nNH-, -(CH2)n( OC2H4)mNH-, -NHCO(CH2)n(OC2H4)mNH-, -NHCONH(CH2)n(OC2H4)mNH-, -CONH(CH2)n(OC2H4)mNH-, -NH-(C(O)CHRaNH)n- or none;

[0075] wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different at each occurrence.

[0076] In some embodiments, when R0 is H, the -L1-L2-L3- group is -(CH2)n(OC2H4)mOCH2-, -NHCO(CH2)n(OC2H4)mOCH2-, or -CONH(CH2)n(OC2H4)mOCH2-;

[0077] wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different each time they appear; and n0 is 0, 1, 2, 3 or 4.

[0078] In some embodiments, when R0 is a fluorescent group or a steroidal structural group, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2)nNHCO(CH2)mNH-, -NHCO(CH2)nNHCO(CH2)mNH-, -NHCONH(CH2)nNH-, -NHCOO(CH2)nNH-, -(CH2)n(OC2H4)mNH-, -NHCO(CH2)n(OC2H4)mNH-, -NHCONH(CH2)n(OC2H4)mNH-, -CONH(CH2)n(OC2H4)mNH-, -NH-(C(O)CHRaNH)n- or none;

[0079] wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different each time they appear; and n0 is 0, 1, 2, 3 or 4.

[0080] In a specific embodiment, when n is 0, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0081] When n is 1, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0082] When n is 2, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0083] When n is 3, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0084] When n is 4, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0085] When n is 5, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0086] When n is 6, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0087] When n is 7, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0088] When n is 8, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0089] When n is 9, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0090] When n is 10, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0091] In some embodiments, when R0 is a fluorescent group or a steroidal structural group, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2)nNHCO(CH2)mNH-, -NHCO(CH2)nNHCO(CH2)mNH-, -NHCONH(CH2)nNH-, -NHCOO(CH2)nNH-, -(CH2)n(OC2H4)mNH-, -NHCO(CH2)n(OC2H4)mNH-, -NHCONH(CH2)n(OC2H4)mNH-, Or -NH-(C(O)CHRaNH)n-.

[0092] In some embodiments, when R0 is a fluorescent group or a steroidal structural group, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2)nNHCO(CH2)mNH-, -NHCO(CH2)nNHCO(CH2)mNH-, -(CH2)n(OC2H4)mNH-, -NHCO(CH2)n(OC2H4)mNH-,

[0093] In some embodiments, when R0 is a fluorescent group or a steroidal structural group, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)nNH-, -(CH2)nCONH(CH2)mNH-, -(CH2)nNHCO(CH2)mNH-, -NHCO(CH2)n(OC2H4)mNH-,

[0094] In some embodiments, when R0 is a fluorescent group or a steroidal structural group, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)nNH-, -(CH2)nNHCO(CH2)mNH-, -NHCO(CH2)n(OC2H4)mNH- or

[0095] In some embodiments, R4 is selected from H, C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl;

[0096] R5 is selected from halogen, CN, SO2, NO2, D, N3, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, OR7, SR7, NR7R7, COR7, COOR7, OCOOR7, CONR7R7, NHCOR7, OCONR7R7, aryl or heteroaryl;

[0097] R6 is selected from H, C1-C4 alkyl, COR8 or SO2R8;

[0098] R7 is selected from H, halogen, C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl;

[0099] R8 is selected from H, halogen, C1-C4 alkyl, C2-C4 alkenyl or C2-C5 alkynyl;

[0100] R9 is selected from H, halogen or C1-C3 alkyl.

[0101] In some embodiments, R4 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, ethenyl, propenyl, butenyl, ethynyl, propynyl or butynyl.

[0102] In some embodiments, R4 is selected from H, methyl, ethyl, n-propyl, isopropyl, vinyl, propenyl, ethynyl or propynyl.

[0103] In some embodiments, R4 is selected from H, methyl, ethyl, ethenyl, propenyl, ethynyl or propynyl.

[0104] In some embodiments, R4 is selected from H, methyl, ethyl, vinyl, ethynyl or propynyl.

[0105] In some embodiments, R5 is selected from halogen, CN, D, N3, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, OR7, NR7R7, COR7, pyridine, pyrimidine or morpholine.

[0106] In some embodiments, R5 is selected from halogen, CN, D, N3, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, OR7, pyridine, pyrimidine or morpholine.

[0107] In some embodiments, R6 is selected from H or C1-C4 alkyl.

[0108] In some embodiments, R6 is selected from H, methyl, ethyl, n-propyl, isopropyl or butyl.

[0109] In some embodiments, R6 is selected from H, methyl or ethyl.

[0110] In some embodiments, R7 is selected from H, fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, butyl, ethenyl, propenyl, butenyl, ethynyl, propynyl or butynyl.

[0111] In some embodiments, R7 is selected from H, fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, vinyl, propenyl, ethynyl or propynyl.

[0112] In some embodiments, R7 is selected from H, fluoro, chloro, methyl, ethyl, vinyl, ethynyl or propynyl.

[0113] In some embodiments, R7 is selected from H, fluoro, chloro, methyl, ethyl or vinyl.

[0114] In some embodiments, R8 is selected from H, fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, butyl, ethenyl, propenyl, butenyl, ethynyl, propynyl or butynyl.

[0115] In some embodiments, R8 is selected from H, fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, ethenyl, propenyl, ethynyl or propynyl.

[0116] In some embodiments, R8 is selected from H, fluoro, chloro, methyl, ethyl, vinyl, ethynyl or propynyl.

[0117] In some embodiments, R8 is selected from H, fluoro, chloro, methyl, ethyl or vinyl.

[0118] In some embodiments, Z1 is selected from O, CH2, S or NH;

[0119] Z2, Z3 are each independently selected from O, NH, CHR7, CHCOOR7, CHCONR7R7, S, CO, SO2, PO(OH), PO(SH) or none;

[0120] Z4 is selected from O, CH2, S, NH or none;

[0121] B1 and B2 are each independently selected from: natural or modified cytosine nucleotide base, natural or modified uracil nucleotide base, natural or modified adenine nucleotide base, natural or modified guanine nucleotide base.

[0122] In some more preferred embodiments, Z2 is selected from methylene, ethylene, CO, SO2, PO(OH) or none;

[0123] Z3 is selected from O, CH2 or NH;

[0124] Z4 is selected from CH2 or NH.

[0125] In some embodiments, Y a , Y b , Y c , Y d Simultaneously O or at most one of S, CH2, CCl2, CF2 or NH;

[0126] Y 1a , Y 1b , Y 1c All are O or at most one is S;

[0127] Y 2a , Y 2b , Y 2c Simultaneously OH or at most one SH or BH3;

[0128] Y3, Y4 are independently selected from: CH2.

[0129] In some embodiments, R1 is selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, benzyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl, halogenated C2-C4 alkynyl, halogenated C3-C6 cycloalkyl, halogenated C3-C6 cycloalkenyl or halogenated benzyl;

[0130] R2 is selected from H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl, halogenated C2-C4 alkynyl, halogenated C3-C6 cycloalkyl, halogenated C3-C6 cycloalkenyl, benzyl or halogenated benzyl;

[0131] R3 is selected from H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl, halogenated C2-C4 alkynyl, halogen or none;

[0132] R 3a , R 3b Each is independently selected from H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl or halogenated C2-C4 alkynyl.

[0133] In some embodiments, W is selected from H, OH, OR4, NR4R4, F, Cl or CN;

[0134] R4 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl or butynyl;

[0135] R5 is selected from halogen, CN, D, N3, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, OR7, SR7, NR7R7, COR7, COOR7, pyridine, pyrimidine or morpholine;

[0136] R7 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl or butynyl.

[0137] In some more preferred embodiments, W is selected from OH, F, Cl, methoxy or ethoxy;

[0138] R1 is selected from methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, trifluoropropyl or trifluoroisopropyl;

[0139] R2 is selected from H, methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, trifluoropropyl or trifluoroisopropyl.

[0140] In some preferred embodiments, the compound structure is as shown in Formula III or a stereoisomer, pharmaceutically acceptable salt, or solvate of the compound of Formula III:

[0141] wherein L1 is selected from O, S, NH, carbonyl or none;

[0142] L2 is selected from NH, carbonyl or none;

[0143] L3 is selected from -X-(CH2)nNH-, -X-(CH2)nNH(CH2)mNH-, -X-(CH2)nNHCO(CH2)mNH-, -X-(CH2)nCONH(CH2)mNH-, -X-(CH2)n(OC2H4)mNH-, -X-(CH2)n(OC2H4)mOCH2-, -X-(C(O)CHRaNH)n- or none; wherein X is NH, O, a five-membered heteroaryl group, a six-membered heteroaryl group or none; n and m are each independently an integer from 0 to 10; Ra is selected from the side chain groups in amino acids, and when Ra appears each time, it may be the same or different;

[0144] R0 is selected from H, a fluorescent group or a steroidal structural group;

[0145] n0 is selected from an integer from 0 to 6.

[0146] In some more preferred embodiments, L1 and L2 are not NH at the same time; or L1 and L2 are not carbonyl at the same time; more preferably, L2 and X are not NH at the same time.

[0147] In more preferred embodiments, when L1 is absent, L2 is selected from NH, carbonyl or absent; or, when L1 is NH, L2 is selected from carbonyl or absent; or, when L1 is carbonyl, L2 is selected from NH or absent.

[0148] In some more preferred embodiments, X is NH, O, a five-membered nitrogen-containing aryl group, a six-membered nitrogen-containing aryl group, or none; more preferably, NH, O, Or not.

[0149] In some more preferred embodiments, L3 is selected from -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nNHCO(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2)n(OC2H4)mNH-, -O-(CH2)nNH-, -O-(CH2)nNHCO(CH2)mNH-, -O-(CH2)nCONH(CH2)mNH-, -O-(CH2)n(OC2H4)mNH-, -NH-(CH2)nNH-, -NH-(CH2)nNHCO(CH2)mNH-, -NH-(CH2)nCONH(CH2)mNH-, -NH-(CH2)n(OC2H4)mNH-, -(CH2)n(OC2H4)mOCH2-, -O-(CH2)n(OC2H4)mOCH2-, -NH-(CH2)n(OC2H4)mOCH2-, -(C(O)CHRaNH)n- or none; wherein Ra is selected from H, CH3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, CH2C8NH6, CH2C6H4OH, CH2COOH, CH2CONH2, (CH2)2COOH, (CH2)4NH2, (CH2)2CONH2, (CH2)2SCH3, CH2OH, CH(CH3)OH, CH2SH, C3H6, CH2C3H3N2 or (CH2)3NHC(NH)NH2; when Ra occurs each time, it may be the same or different;

[0150] wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0151] In some embodiments, Ra is selected from H, CH3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, CH2C8NH6, CH2C6H4OH, CH2COOH, CH2CONH2, (CH2)2COOH, (CH2)2CONH2, CH2OH, CH(CH3)OH, C3H6, CH2C3H3N2 or (CH2)3NHC(NH)NH2; when Ra appears each time, it may be the same or different.

[0152] In some embodiments, Ra is selected from H, CH3, CH(CH3)2, CH2CH(CH3)2, CH2C6H5, CH2C8NH6, CH2C6H4OH, CH2COOH, CH2CONH2, CH2OH, CH(CH3)OH, CH2C3H3N2 or (CH2)3NHC(NH)NH2; when Ra appears each time, it may be the same or different.

[0153] In some embodiments, Ra is selected from H, CH3, CH2C6H5, CH2C8NH6, CH2C6H4OH, CH2CONH2, CH2OH, CH2C3H3N2 or (CH2)3NHC(NH)NH2; each occurrence of Ra may be the same or different.

[0154] In some more preferred embodiments, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)n(OC2H4)mOCH2-, -NHCO(CH2)n(OC2H4)mOCH2-, -CONH(CH2)n(OC2H4)mOCH2-, -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2 )nNHCO(CH2)mNH-, -NHCO(CH2)nNHCO(CH2)mNH-, -NHCONH(CH2)nNH-, -NHCOO(CH2)nNH-, -(CH2)n( OC2H4)mNH-, -NHCO(CH2)n(OC2H4)mNH-, -NHCONH(CH2)n(OC2H4)mNH-, -CONH(CH2)n(OC2H4)mNH-, -NH-(C(O)CHRaNH)n- or none;

[0155] wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different at each occurrence.

[0156] In some more preferred embodiments, when R0 is H, the -L1-L2-L3- group is -(CH2)n(OC2H4)mOCH2-, -NHCO(CH2)n(OC2H4)mOCH2-, or -CONH(CH2)n(OC2H4)mOCH2-;

[0157] When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and each occurrence may be the same or different;

[0158] For example, the -L1-L2-L3- group can be any of the following groups (not exhaustive):

[0159] -OC2H4OCH2-, -(OC2H4)2OCH2-, -(OC2H4)3OCH2-, -(OC2H4)4OCH2-, -(OC2H4)5OCH2 -, -(OC2H4)6OCH2-, -(OC2H4)7OCH2-, -(OC2H4)8OCH2-, -(OC2H4)9OCH2-, -(OC2H4) 10 OCH2-;

[0160] -CH2OC2H4OCH2-, -CH2(OC2H4)2OCH2-, -CH2(OC2H4)4OCH2-, -CH2(OC2H4)5OCH2-, -CH2(OC2H4)6OCH2-, -CH2(OC2H4)8OCH2-, -CH2(OC2H4) 10 OCH2-;

[0161] -C3H6OC2H4OCH2-, -C3H6(OC2H4)2OCH2-, -C3H6(OC2H4)4OCH2-, -C3H6(OC2H4)8OCH2-;

[0162] -C4H8(OC2H4)2OCH2-, -C4H8(OC2H4)4OCH2-, -C4H8(OC2H4)5OCH2-, -C4H8(OC2H4)7OCH2-;

[0163] -C5H 10 OC2H4OCH2-,-C5H 10 (OC2H4)2OCH2-,-C5H 10 (OC2H4)4OCH2-,-C5H 10 (OC2H4)5OCH2-,-C5H 10 (OC2H4)6OCH2-,-C5H10 (OC2H4)8OCH2-,-C5H 10 (OC2H4) 10 OCH2-;

[0164] -C6H 12 OC2H4OCH2-,-C6H 12 (OC2H4)5OCH2-,-C7H 14 (OC2H4)9OCH2-,-C8H 16 (OC2H4)6OCH2-;

[0165] -NHCO(OC2H4)2OCH2-,-NHCO(OC2H4)3OCH2-,-NHCO(OC2H4)4OCH2,-NHCO(OC2H4)5OCH2-,-NHCO(OC2H4)6OCH2-,-NHCO(OC2H4)8OCH2-,-NHCO(OC2H4) 10 OCH2-;

[0166] -NHCOCH2(OC2H4)2OCH2-,-NHCOCH2(OC2H4)3OCH2-,-NHCOCH2(OC2H4)5OCH2-,-NHCOCH2(OC2H4)7OCH2-,-NHCOCH2(OC2H4)9OCH2-,-NHCOCH2(OC2H4) 10 OCH2-,;

[0167] -NHCOC2H4(OC2H4)2OCH2-,-NHCOC2H4(OC2H4)3OCH2-,-NHCOC2H4(OC2H4)5OCH2-,-NHCOC2H4(OC2H4)7OCH2-,-NHCOC2H4(OC2H4)9OCH2-,-NHCOC2H4(OC2H4) 10 OCH2-,;

[0168] -NHCOC3H6(OC2H4)2OCH2-,-NHCOC3H6(OC2H4)3OCH2-,-NHCOC3H6(OC2H4)5OCH2-,-NHCOC3H4(OC2H4)7OCH2-,-NHCOC3H6(OC2H4)9OCH2-,-NHCOC3H6(OC2H4) 10 OCH2-,;

[0169] -NHCOC4H8(OC2H4)6OCH2-,-NHCOC4H8(OC2H4)8OCH2-,-NHCOC5H 10(OC2H4)4OCH2-,-NHCOC5H 10 (OC2H4)7OCH2-,-NHCOC7H 14 (OC2H4)9OCH2-,-NHCOC8H 16 (OC2H4) 10 OCH2-,;

[0170] -CONHCH2OC2H4OCH2-,-CONHCH2(OC2H4)3OCH2-,-CONHCH2(OC2H4)5OCH2-,-CONHCH2(OC2H4)6OCH2-,-CONHCH2(OC2H4)8OCH2-,-CONHCH2(OC2H4) 10 OCH2-;

[0171] -CONHC2H4OC2H4OCH2-,-CONHC2H4(OC2H4)2OCH2-,-CONHC2H4(OC2H4)4OCH2-,-CONHC2H4(OC2H4)6OCH2-,-CONHC2H4(OC2H4)8OCH2-,-CONHC2H4(OC2H4) 10 OCH2-,-CONHC3H6OC2H4OCH2-,-CONHC3H6(OC2H4)2OCH2-,-CONHC3H6(OC2H4)4OCH2-,-CONHC3H6(OC2H4)6OCH2-,-CONHC3H6(OC2H4)8OCH2-,-CONHC3H6(OC2H4) 10 OCH2-,-CONHC4H8OC2H4OCH2-,-CONHC4H8(OC2H4)5OCH2-,-CONHC4H8(OC2H4)6OCH2-,-CONHC4H8(OC2H4) 10 OCH2-,-CONHC5H 10 OC2H4OCH2-,-CONHC7H 14 (OC2H4)3OCH2-,-CONHC7H 14 (OC2H4)6OCH2-,-CONHC8H 16 (OC2H4)8OCH2-,-CONHC 10 H 22 OC2H4OCH2-,-CONHC9H 18 (OC2H4)3OCH2-,-CONHC 10 H 22 (OC2H4)4OCH2-,-CONHC9H18 (OC2H4)6OCH2-,-CONHC 10 H 22 (OC2H4)8OCH2-or-CONHC 10 H 22 (OC2H4)9OCH2-.

[0172] In some more preferred embodiments, when R0 is a fluorescent group or a steroidal structural group, the -L1-L2-L3- group is selected from any one of the following groups: -(CH2)nNH-, -(CH2)nNH(CH2)mNH-, -(CH2)nCONH(CH2)mNH-, -(CH2)nNHCO(CH2)mNH-, -NHCO(CH2)nNHCO(CH2)mNH-, -NHCONH(CH2)nNH-, -NHCOO(CH2)nNH-, -(CH2)n(OC2H4)mNH-, -NHCO(CH2)n(OC2H4)mNH-, -NHCONH(CH2)n(OC2H4)mNH-, -CONH(CH2)n(OC2H4)mNH-, -NH-(C(O)CHRaNH)n- or none;

[0173] wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different at each occurrence.

[0174] For example, the -L1-L2-L3- group can be any of the following groups (not exhaustive):

[0175] -NH-, -CH2NH-, -(CH2)2NH-, -(CH2)4NH-, -(CH2)5NH-, -(CH2)6NH-;

[0176] -NH(CH2)2NH-, -NH(CH2)3NH-, -NH(CH2)4NH-, -NH(CH2)5NH-, -NH(CH2)8NH-;

[0177] -CH2NH(CH2)2NH-, -CH2NH(CH2)3NH-, -CH2NH(CH2)5NH-, -CH2NH(CH2)8NH-;

[0178] <h2 style=";text-align:left;direction:ltr">-(CH2)4NH(CH2)2NH-,-(CH2)4NH(CH2)3NH-,-(CH2)4NH(CH2)4NH-,-(CH2)4NH(CH2)5NH-,-(CH2)3NH(CH2)2NH-,-(CH2)3NH(CH2)5NH-,-(CH2)3NH(CH2)6NH-,-(CH2)4NH(CH2)2NH-,-(CH2)4NH(CH2)5NH-,-(CH2)5NH(CH2)3NH-,-(CH2)5NH(CH2)3NH-,-(CH2)5NH(CH2)7NH-,-(CH2)6NH(CH2)4NH(CH2)4NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0179] <h2 style=";text-align:left;direction:ltr"> -CONHCH2NH-,-CONH(CH2)2NH-,-CONH(CH2)3NH-,-CONH(CH2)4NH-,-CONH(CH2)5NH-,-CONH(CH2)6NH-,-CONH(CH2)7NH-,-CONH(CH2)8NH-,-CONH(CH2)9NH-,-CONH(CH2)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0180] <h2 style=";text-align:left;direction:ltr"> -CH2CONHCH2NH-,-CH2CONH(CH2)2NH-,-CH2CONH(CH2)3NH-,-CH2CONH(CH2)4NH-,-CH2CONH(CH2)5NH-,-CH2CONH(CH2)6NH-,-CH2CONH(CH2)7NH-,-CH2CONH(CH2)9NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0181] <h2 style=";text-align:left;direction:ltr"> -(CH2)2CONHCH2NH-,-(CH2)2CONH(CH2)3NH-,-(CH2)2CONH(CH2)5NH-,-(CH2)2CONH(CH2)8NH-,-(CH2)3CONH(CH2)3NH-,-(CH2)3CONH(CH2)6NH-,-(CH2)4CONH(CH2)3NH-,-(CH2)4CONH( CH2)6NH-,-(CH2)5CONH(CH2)2NH-,-(CH2)5CONH(CH2)5NH-,-(CH2)6CONH(CH2)5NH-,-(CH2) 7CONH(CH2)4NH-,-(CH2)8CONH(CH2)4NH-,-(CH2)8CONH(CH2)6NH-,-(CH2)8CONH(CH2)8NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0182] <h2 style=";text-align:left;direction:ltr">-NHCO(CH2)2NH-, -NHCO(CH2)3NH-, -NHCO(CH2)4NH-, -NHCO(CH2)5NH-, -NHCO(CH2)8NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0183] <h2 style=";text-align:left;direction:ltr"> -CH2NHCO(CH2)2NH-, -CH2NHCO(CH2)3NH-, -CH2NHCO(CH2)4NH-, -CH2NHCO(CH2)5NH-, - CH2NHCO(CH2)7NH-,-CH2NHCO(CH2)9NH-,-(CH2)3NHCO(CH2)2NH-,-(CH2)3NHCO(CH2)3NH-,-(CH2)3NHCO(CH2)5NH-,-(CH2)3NHCO(CH2)7NH-,-(CH2)4NHCO(CH2)2NH-,-(CH2)4NHCO(CH2)5NH-,-(CH2)4NHCO(CH2)8NH-,-(CH2)5NHCO(CH2)4NH-,-(CH2)5NHCO(CH2)7NH-,-(CH2)6NHCO(CH2)3NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0184] <h2 style=";text-align:left;direction:ltr"> -NHCOCH2NHCOCH2NH-,-NHCOCH2NHCO(CH2)2NH-,-NHCOCH2NHCO(CH2)3NH-,-NHCOCH2NHCO(CH2)4NH-,-NHCOCH2NHCO(CH2)6NH-,-NHCOCH2NHCO(CH2)8NH-,-NHCO(CH2)2NHCO(CH2)2NHCO(CH2)4NH-,-NHCO(CH2)2NHCO(CH2)6NH-,-NHCO(CH2)2NHCO(CH2)8NH-,-NHCO(CH2)2NHCO(CH2)4NH-,-NHCO(CH2)2NHCO(CH2)6NH-,-NHCO(CH2)2NHCO(CH2)8NH-,-NHCO(CH2)2NHCO(CH2)<h2 style=";text-align:left;direction:ltr"> 10<h2 style=";text-align:left;direction:ltr">NH-,-NHCO(CH2)3NHCO(CH2)2NH-,-NHCO(CH2)3NHCO(CH2)4NH-,-NHCO(CH2)3NHCO(CH2)6NH-,-NH CO(CH2)4NHCO(CH2)2NH-,-NHCO(CH2)4NHCO(CH2)4NH-,-NHCO(CH2)4NHCO(CH2)6NH-,-NHCO(CH2) 4NHCO(CH2)6NH-,-NHCO(CH2)5NHCO(CH2)2NH-,-NHCO(CH2)5NHCO(CH2)6NH-,-NHCO(CH2)5NHCO(C) H2)4NH-,-NHCO(CH2)6NHCO(CH2)6NH-,-NHCO(CH2)8NHCO(CH2)4NH-,-NHCO(CH2)8NHCO(CH2)6NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0185] <h2 style=";text-align:left;direction:ltr"> -NHCONHCOCH2NH-,-NHCONHCO(CH2)2NH-,-NHCONHCO(CH2)3NH-,-NHCONHCO(CH2)4NH-,-NHCONHCO(CH2)5NH-,-NHCONHCO(CH2)6NH-,-NHCONHCO(CH2)7NH-,-NHCONHCO(CH2)8NH-,-NHCONHCO(CH2)9NH-,-NHCONHCO(CH2)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0186] <h2 style=";text-align:left;direction:ltr"> -NHCOOCH2NH-,-NHCOO(CH2)2NH-,-NHCOO(CH2)3NH-,-NHCOO(CH2)4NH-,-NHCOO(CH2)5NH-,-NHCOO(CH2)6NH-,-NHCOO(CH2)7NH-,-NHCOO(CH2)8NH-,-NHCOO(CH2)9NH-,-NHCOO(CH2)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0187] <h2 style=";text-align:left;direction:ltr"> -CH2OC2H4NH-,-CH2(OC2H4)2NH-,-CH2(OC2H4)3NH-,-CH2(OC2H4)4NH-,-CH2(OC2H4)5NH-,-CH2(OC2H4)6NH-,-CH2(OC2H4)7NH-,-CH2(OC2H4)8NH-,-CH2(OC2H4)9NH-,-CH2(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10<h2 style=";text-align:left;direction:ltr">NH-,-(CH2)2OC2H4NH-,-(CH2)2(OC2H4)3NH-,-(CH2)2(OC2H4)5NH-,-(CH2)2(OC2H4)7NH-,-(CH2)2(OC2H4)8NH-,-(CH2)3(OC2H4)2NH-,-(CH2)3(OC2H4)5NH-,-(CH2)3(OC2H4)7NH-,-(CH2)3(OC2H4)8NH-,-(CH2)3(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,-(CH2)4(OC2H4)2NH-,-(CH2)4(OC2H4)5NH-,-(CH2)4(OC2H4)6NH-, -(CH2)4(OC2H4)8NH-,-(CH2)5(OC2H4)2NH-,-(CH2)5(OC2H4)5NH-,-(CH2 )5(OC2H4)7NH-,-(CH2)6(OC2H4)4NH-,-(CH2)6(OC2H4)6NH-,-(CH2)6(O C2H4)8NH-,-(CH2)7(OC2H4)4NH-,-(CH2)7(OC2H4)7NH-,-(CH2)7(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,-(CH2)8(OC2H4)6NH-,-(CH2)8(OC2H4)8NH-,-(CH2)8(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0188] <h2 style=";text-align:left;direction:ltr"> -NHCOCH2OC2H4NH-,-NHCOCH2(OC2H4)2NH-,-NHCOCH2(OC2H4)3NH-,-NHCOCH2(OC2H4)4NH-,-NHCOCH2(OC2H4)5NH-,-NHCOCH2(OC2H4)6NH-,-NHCOCH2(OC2H4)7NH-,-NHCOCH2(OC2H4)8NH-,-NHCOCH2(OC2H4)9NH-,-NHCOCH2(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,-NHCO(CH2)2(OC2H4)2NH-,-NHCO(CH2)2(OC2H4)4NH-,-NHCO(CH2)2(OC2H4)6NH-,-NHCO(CH2)2(OC2H4)7NH-,-NHCO(CH2)2(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10<h2 style=";text-align:left;direction:ltr">NH-,-NHCO(CH2)3(OC2H4)2NH-,-NHCO(CH2)3(OC2H4)4NH-,-NHCO(CH2)3(OC2H4)6NH-,-NHCO(CH2)3(OC2H4) 9NH-,-NHCO(CH2)4(OC2H4)2NH-,-NHCO(CH2)4(OC2H4)6NH-,-NHCO(CH2)4(OC2H4)8NH-,-NHCO(CH2)4(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,-NHCO(CH2)5(OC2H4)4NH-,-NHCO(CH2)5(OC2H4)7NH-,-NHCO(CH2)6(OC2H4)4NH-,-NHCO(CH2)6(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,-NHCO(CH2)7(OC2H4)7NH-,-NHCO(CH2)8(OC2H4)6NH-,-NHCO(CH2)8(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0189] <h2 style=";text-align:left;direction:ltr"> -NHCONH(CH2)2OC2H4NH-,-NHCONH(CH2)2(OC2H4)2NH-,-NHCONH(CH2)2(OC2H4)3NH-,-NHCONH(CH2)2(OC2H4)4NH-,-NHCONH(CH2)2(OC2H4)5NH-,-NHCONH(CH2)2(OC2H4)6NH-,-NHCONH(CH2)2(OC2H4)7NH-,-NHCONH(CH2)2(OC2H4)8NH-,-NHCONH(CH2)2(OC2H4)9NH-,-NHCONH(CH2)2(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0190] <h2 style=";text-align:left;direction:ltr">-NHCONH(CH2)3(OC2H4)2NH-,-NHCONH(CH2)3(OC2H4)4NH-,-NHCONH(CH2)3(OC2H4)5NH-,-NHCONH(CH2)3(OC2H4)7NH -,-NHCONH(CH2)3(OC2H4)8NH-,-NHCONH(CH2)4(OC2H4)4NH-,-NHCONH(CH2)4(OC2H4)6NH-,-NHCONH(CH2)4(OC2H4)4N H-, -NHCONH(CH2)4(OC2H4)7NH-, -NHCONH(CH2)5(OC2H4)4NH-, -NHCONH(CH2)5(OC2H4)8NH-, -NHCONH(CH2)6(OC2H4)3NH-, -NHCONH(CH2)6(OC2H4)6NH-, -NHCONH(CH2)7(OC2H4)4NH-, -NHCONH(CH2)8(OC2H4)4NH-, -NHCONH(CH2)8(OC2H4)8NH<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> NH-,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0191] <h2 style=";text-align:left;direction:ltr"> -CONH(CH2)2OC2H4NH-,-CONH(CH2)2(OC2H4)2NH-,-CONH(CH2)2(OC2H4)3NH-,-CONH(CH2)2(OC2H4)4NH-,-CONH(CH2)2(OC2H4)5NH-,-CONH(CH2)2(OC2H4)6NH-,-CONH(CH2)2(OC2H4)7NH-,-CONH(CH2)2(OC2H4)8NH-,-CONH(CH2)2(OC2H4)9NH-,-CONH(CH2)2(OC2H4)<h2 style=";text-align:left;direction:ltr"> 10NH-, -CONH(CH2)3(OC2H4)2NH-, -CONH(CH2)3(OC2H4)5NH-, -CONH(CH2)4(OC2H4)7NH-, -CONH(CH2)4(OC2H4) 2NH-, -CONH(CH2)4(OC2H4)4NH-, -CONH(CH2)4(OC2H4)7NH-, -CONH(CH2)5(OC2H4)4NH-, -CONH(CH2)5(OC2H4) 8NH-, -CONH(CH2)6(OC2H4)3NH-, -CONH(CH2)6(OC2H4)5NH-, -CONH(CH2)6(OC2H4)8NH-, -CONH(CH2)7(OC2H4) 4NH-, -CONH(CH2)7(OC2H4)8NH-, -CONH(CH2)8(OC2H4)4NH-, -CONH(CH2)8(OC2H4)8NH-, -CONH(CH2)8(OC2H4) 10 NH-;

[0192] In some embodiments, the compound is as shown in any of the following structures, wherein R0 is the fluorescent group or steroidal structure group mentioned above:

[0193] In some more preferred embodiments, R0 is a fluorescent group formed by biotin, Cy3, Cy5, Cy7, 5-FAM or 6-FAM, or a steroidal structural group formed by cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid or glycoursodeoxycholic acid.

[0194] In some more preferred embodiments, the compound is any of the following structures (non-exhaustive):

[0195] The present invention also protects a compound having any of the following structures or its stereoisomers, pharmaceutically acceptable salts, or solvates, wherein the structure is:

[0196] The second object of the present invention is to provide a compound having a structure of Formula IV or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof:

[0197] wherein R1 is selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, benzyl, R5-substituted C1-C6 alkyl, R5-substituted C2-C6 alkenyl, R5-substituted C2-C6 alkynyl, R5-substituted C3-C6 cycloalkyl, R5-substituted C3-C6 cycloalkenyl or R5-substituted benzyl;

[0198] R2 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, cycloalkenyl, benzyl, aryl, heteroaryl, R5 substituted benzyl, R5 substituted aryl, carbonylalkyl, carbonylalkoxy or sulfonamide;

[0199] W is selected from H, OH, OR4, NR4R4, NR4COR4, F, Cl, N3 or CN;

[0200] W2 is selected from H, OH, halogen, N3, CN, OR4, NR4R4, NR4COR4 or C1-C4 alkyl;

[0201] X1 is selected from (CH2) n1 , NR4, or none; n1 is selected from: 1, 2 or 3;

[0202] X2 is selected from O, S, NR4, CO, CO2, CONR4, NR4CO, NR4CO2, NR4CONR4, SO2, SO2NR4, CH2, or none;

[0203] R 20 is selected from H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkyl substituted with R5, C2-C8 alkenyl substituted with R5, C2-C8 alkynyl substituted with R5, aryl, aryl substituted with R5, heteroaryl, heteroaryl substituted with R5, halogen, CN or N3;

[0204] R 12a , R 12b Each is independently selected from H, halogen, alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, R5-substituted C1-C6 alkyl, R5-substituted C2-C6 alkenyl or R5-substituted C2-C6 alkynyl;

[0205] or R 12a , R 12b Each independently selected from OH or R5 substituted C1-C6 alkoxy;

[0206] Ya , Y b , Y c , Y d Each independently selected from O, S, CH2, CCl2, CF2 or NH;

[0207] Y 1a , Y 1b , Y 1c Each independently selected from O or S;

[0208] Y 2a , Y 2b , Y 2c Each independently selected from OH, SH or BH3;

[0209] Y3, Y4 are each independently selected from CH2 or O;

[0210] Z1 is selected from O, OH, CH2, S, NR6, CO or SO2;

[0211] Z2, Z3 are each independently selected from O, NR6, CHR7, CHCOOR7, CHCONR7R7, S, CO, SO2, PO(OH), PO(SH), P(O)VCO2H or none; Z2 can be combined with R 3a The connected oxygen atoms form a ring;

[0212] Z4 is selected from O, CH2, S, NR6, CO, SO2 or none;

[0213] B1 and B2 are each independently selected from natural or modified pyrimidine nucleotide bases, and natural or modified purine nucleotide bases;

[0214] R4 is selected from H, C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl;

[0215] R5 is selected from halogen, CN, SO2, NO2, D, N3, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, OR7, SR7, NR7R7, COR7, COOR7, OCOOR7, CONR7R7, NHCOR7, OCONR7R7, aryl or heteroaryl;

[0216] R6 is selected from H, C1-C6 alkyl, COR8 or SO2R8;

[0217] R7 is selected from H, halogen, C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl;

[0218] R8 is selected from H, C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl;

[0219] V is a C1-4 alkyl group;

[0220] m0 is selected from 0, 1 or 2.

[0221] In some embodiments, R4 is selected from H, C1-C4 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0222] R5 is selected from halogen, CN, SO2, NO2, D, N3, C1-C4 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C1-C4 alkoxy, OR7, SR7, NR7R7, COR7, COOR7, OCOOR7, CONR7R7, NHCOR7, OCONR7R7, aryl or heteroaryl;

[0223] R6 is selected from H, C1-C4 alkyl, COR8 or SO2R8;

[0224] R7 is selected from H, halogen, C1-C4 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0225] R8 is selected from H, C1-C4 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0226] More preferably, R5 is selected from halogen, CN, SO2, NO2, D, N3, C1-C4 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C1-C4 alkoxy, pyridine, pyrimidine or morpholine.

[0227] In some embodiments, W is selected from H, OH, C1-C4 alkoxy, C1-C4 alkylamino, F, Cl, N3 or CN;

[0228] W2 is selected from H, OH, halogen, N3, CN, C1-C4 alkyl, C1-C4 alkoxy or C1-C4 alkylamino;

[0229] X1 is selected from (CH2) n1 , C1-C4 alkyl substituted amino or not; n1 is selected from: 1, 2 or 3;

[0230] X2 is selected from O, S, CO, CO2, CONR4, NR4CO, NR4CO2, NR4CONR4, SO2 or SO2NR4;

[0231] R 12a , R 12b Each independently selected from OH, C1-C6 alkyl, C1-C6 alkoxy, R5-substituted C1-C6 alkyl or R5-substituted C1-C6 alkoxy;

[0232] R 20Selected from H, C1-C4 alkyl, C2-C5 alkenyl, R5 substituted C1-C4 alkyl, R5 substituted aryl, heteroaryl, N3 or none.

[0233] In more preferred embodiments, X1 is selected from CH2, C2H4, C3H6, NH, N(CH3), N(C2H5), N(C3H7), N(C4H9), N(C2H3), N(C3H5), N(C4H7), N(C2H), N(C3H3), N(C4H5), N(C5H7) or none;

[0234] X2 is selected from O, S, CO, CO2, CONH, CON(CH3), CON(C2H5), CON(C3H7), CON(C4H9), CON(C3H5), CON(C4H7), CON(C3H3), CON(C4H5), CON(C5H7),

[0235] N(CH3)CO, N(C2H5)CO, N(C3H7)CO, N(C4H9)CO, N(C2H3)CO, N(C3H5)CO, N(C4H7)CO, N(C2H)CO, N(C3H3)CO, N(C4H5)CO, N(C5H7)CO,

[0236] N(CH3)COO, N(C2H5)COO, N(C3H7)COO, N(C4H9)COO, N(C2H3)COO, N(C3H7)COO, N(C4H9)COO, N(C2H)COO, N(C3H3)COO, N(C4H5)COO, N(C5H7)COO,

[0237] N(CH3)CON(CH3), N(CH3)CON(C2H5), N(CH3)CON(C4H9), N(CH3)CON(C2H3), N(CH3)CON(C 4H7), N(CH3)CON(C3H3), N(C2H5)CON(CH3), N(C2H5)CON(C2H5), N(C2H5)CON(C4H9), N(C 2H5)CON(C2H3), N(C2H5)CON(C4H9), N(C2H5)CON(C3H3), N(C3H7)CON(CH3), N(C3H7)CON (C2H5), N(C3H7)CON(C4H9), N(C3H7)CON(C2H3), N(C3H7)CON(C4H9), N(C3H7)CON(C3H3),

[0238] SO2, SO2NH, SO2N(CH3), SO2N(C2H5), SO2N(C3H7), SO2N(C4H9), SO2N(C2H3), SO2N(C3H5), SO2N(C4H7), SO2N(C2H), SO2N(C3H3), SO2N(C4H5) or SO2N(C5H7).

[0239] In more preferred embodiments, X1 is selected from CH2, C2H4, C3H6, NH, N(CH3), N(C2H5), N(C3H7) or N(C4H9).

[0240] In some embodiments, R 12a , R 12b Each is independently OH, F, Cl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl or C1-C4 haloalkoxy;

[0241] W is OH;

[0242] Y a , Y b , Y c , Y d are each independently selected from O or CH2;

[0243] Y 1a , Y 1b , Y 1c Each independently selected from O or S;

[0244] Y 2a , Y 2b , Y 2c are each independently selected from OH;

[0245] Y3, Y4 are independently selected from CH2 or O;

[0246] Z1 is selected from O, OH, CH2, S or NH;

[0247] Z2, Z3 are each independently selected from O, NH, CHR7, CHCOOR7, CO, SO2 or PO(OH);

[0248] B1 and B2 are each independently selected from natural or modified pyrimidine nucleotide bases, and natural or modified purine nucleotide bases;

[0249] R4 is selected from H or C1-C4 alkyl;

[0250] R5 is selected from halogen, OR7, NR7R7, CONR7R7 or OCONR7R7;

[0251] R6 is selected from H;

[0252] R7 is selected from H or C1-C4 alkyl;

[0253] m0 is selected from 0, 1 or 2.

[0254] In some embodiments, R1 is selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, benzyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl, halogenated C2-C4 alkynyl, halogenated C3-C6 cycloalkyl, halogenated C3-C6 cycloalkenyl or halogenated benzyl;

[0255] R2 is selected from H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl, halogenated C2-C4 alkynyl, halogenated C3-C6 cycloalkyl, halogenated C3-C6 cycloalkenyl, benzyl or halogenated benzyl.

[0256] In some preferred embodiments, the compound structure is shown in any one of Formula IV-2, IV-3, IV-4, IV-5 or IV-6:

[0257] Among them R1, R2, R 20 , R 12a , R 12b , R4, R5, R6, R7, R8, W, W2, Y a , Y b , Y c , Y d , Y 1a , Y 1b , Y 1c , Y 2a , Y 2b , Y 2c , Y3, Y4, B1, B2, Z1, Z2, Z3, B1, B2, V, n1 and m0 are as shown above respectively.

[0258] In some embodiments, R1 is selected from methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl, butynyl, benzyl, halomethyl, haloethyl, halo-n-propyl, haloisopropyl, halobutyl, halovinyl, halopropenyl, halobutenyl, haloethynyl, halopropynyl, halobutynyl or halobenzyl;

[0259] R2 is selected from H, methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, trifluoropropyl or trifluoroisopropyl;

[0260] W is selected from OH, F, Cl, methyl, ethyl, methoxy or ethoxy;

[0261] W2 is selected from H, OH, F, Cl, N3, CN, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, methyl substituted amino or ethyl substituted amino;

[0262] R4 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl or butynyl;

[0263] R 20 Selected from H, C1-C4 alkyl, C2-C5 alkenyl, R5 substituted C1-C4 alkyl, R5 substituted aryl, heteroaryl, N3 or none;

[0264] R5 is selected from halogen, CN, D, N3, C1-C4 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C1-C4 alkoxy, pyridine, pyrimidine or morpholine.

[0265] In some embodiments, R 12a , R 12b Each is independently OH, F, Cl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl or C1-C4 haloalkoxy;

[0266] W is OH;

[0267] Z1 is selected from O, OH, CH2, S or NH;

[0268] Z2, Z3 are each independently selected from O, NH, CH2, CHCOO, CO, SO2 or PO(OH);

[0269] B1, B2 are independently selected from: natural or modified cytosine nucleotide base, natural or modified uracil nucleotide base, natural or modified adenine nucleotide base, natural or modified guanine nucleotide base;

[0270] m0 is selected from 0, 1 or 2.

[0271] In some preferred embodiments, the compound structure is shown in Formula V:

[0272] wherein W2 is selected from H, OH, halogen, N3, CN, C1-C4 alkyl, C1-C4 alkoxy or C1-C4 alkylamino;

[0273] X1 is selected from (CH2) n1 , C1-C4 alkyl substituted amino or not; n1 is selected from: 1, 2 or 3;

[0274] X2 is selected from O, S, CO, CO2, CONR4, NR4CO, NR4CO2, NR4CONR4, SO2 or SO2NR4;

[0275] R 20 Selected from H, C1-C4 alkyl, C2-C5 alkenyl, R5 substituted C1-C4 alkyl, R5 substituted aryl, heteroaryl, N3 or none;

[0276] R4 is selected from H, C1-C4 alkyl, C2-C5 alkenyl or C2-C5 alkynyl;

[0277] R5 is selected from halogen, CN, SO2, NO2, D, N3, C1-C4 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C1-C4 alkoxy, OR7, SR7, NR7R7, COR7, COOR7, OCOOR7, CONR7R7, NHCOR7, OCONR7R7, aryl or heteroaryl;

[0278] R7 is selected from H, halogen, C1-C4 alkyl, C2-C5 alkenyl or C2-C5 alkynyl.

[0279] In some more preferred embodiments, the compound structure is as shown in Formula V-2, V-3, V-4, V-5 or V-6:

[0280] wherein W2 is selected from H, OH, F, Cl, N3, CN, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, methyl substituted amino or ethyl substituted amino;

[0281] R4 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl or butynyl;

[0282] R 20 Selected from H, C1-C4 alkyl, C2-C5 alkenyl, R5 substituted C1-C4 alkyl, R5 substituted aryl, heteroaryl, N3 or none;

[0283] R5 is selected from halogen, CN, D, N3, C1-C4 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C1-C4 alkoxy, pyridine, pyrimidine or morpholine.

[0284] In some embodiments, the compound is represented by any of the following structures (non-exhaustive):

[0285] In some embodiments, the compound is represented by any of the following structures:

[0286] The third object of the present invention is to protect the preparation method of the above-mentioned compound, wherein the preparation route of one type of compound is:

[0287] A0 undergoes a displacement reaction with hydrogen in the presence of a catalyst to obtain B0, and then B0 undergoes a condensation reaction with Ia or Ib to prepare the target compound C0.

[0288] In some embodiments, A0 and B0 can be salt compounds commonly used in chemical reactions, or pharmaceutically acceptable salt compounds; more preferably, A0 and B0 are ammonium salts, sodium salts, potassium salts, Tris salts or phosphates.

[0289] The fourth object of the present invention is to protect the use of the above compound as an in vitro co-transcribed RNA capping agent.

[0290] The fifth object of the present invention is to protect an RNA molecule comprising any one of the above compounds as a cap structure or a cap structure fragment.

[0291] The sixth object of the present invention is to protect a pharmaceutical composition, namely a composition comprising the above-mentioned RNA molecule and a pharmaceutically acceptable carrier.

[0292] A seventh object of the present invention is to protect a method for synthesizing RNA molecules, comprising the following steps:

[0293] Any of the aforementioned compounds is co-incubated with a polynucleotide template to perform template transcription.

[0294] The eighth object of the present invention is to protect a capping RNA transcription reaction system, comprising: a polynucleotide template, any of the aforementioned compounds, NTPs and RNA polymerase system.

[0295] Compared with the prior art, the present invention has the following beneficial effects:

[0296] The present invention provides an RNA-capped compound with a fluorescent group, which not only retains the original excellent performance, such as a good capping rate, but also has good stability, expression level and other properties of the capped mRNA; because it is labeled with a fluorescent group, it can also be used to detect and track the dynamic changes of mRNA in cells and organisms, facilitating the study of the delivery process of mRNA in cells or organisms, pharmacokinetic properties and other properties, and providing convenience for the research of mRNA vaccines / drugs in cells or organisms.

[0297] The present invention provides a compound for RNA capping. The compound is used to cap the 5' end of mRNA, which not only has good capping efficiency, but also significantly improves the performance of the capped mRNA in terms of stability and expression level. It performs well both in vitro and in animals, and is better than the positive control. BRIEF DESCRIPTION OF THE DRAWINGS

[0298] FIG1 shows the fluorescence intensity detection results at different mRNA concentrations in Example 23.

[0299] FIG2 shows the fluorescence detection results at different time points after S10-GFP-mRNA was transfected into cells in Example 25.

[0300] FIG3 shows the fluorescence detection results 20 hours after S10-GFP-mRNA was transfected into cells in Example 25.

[0301] FIG4 shows the in vivo imaging detection results of S10-FLuc-mRNA-LNP injected into mice 1 hour after Example 26.

[0302] FIG5 shows the imaging detection results of the liver 1 hour after the S10-FLuc-mRNA-LNP was injected into mice in Example 26.

[0303] FIG6 shows the imaging detection results of the lungs of mice 1 hour after the injection of S10-FLuc-mRNA-LNP in Example 26.

[0304] FIG7 shows the imaging detection results of the spleen 1 hour after the S10-FLuc-mRNA-LNP was injected into mice in Example 26.

[0305] FIG8 shows the in vivo imaging results of S10-FLuc-mRNA-LNP injected into mice 3 hours after injection in Example 27. DETAILED DESCRIPTION

[0306] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0307] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0308] the term:

[0309] Pyrimidine nucleotide bases include, but are not limited to, uracil (U), thymine (T), cytosine (C), 5-methylcytosine, 5-fluorouracil, 5-fluorocytosine, and the like.

[0310] Purine nucleotide derivatives include, but are not limited to, adenine (A), guanine (G), 6-N-methyladenine (m6A), 6-N,N,-dimethyladenine, 2-N-methylguanine, 2-N,N,-dimethylguanine, and the like.

[0311] "Together are a single bond or a double bond" means that this structure is a chemical bond, and a single bond or a double bond can be selected specifically. For example, "-X4-together are a single bond or a double bond" means that when the optional -X4- is a single bond or a double bond, the ring of the parent core of Formula I is a five-membered ring, and the groups on both sides of X4 are directly connected.

[0312] "Connected to form a ring through chemical bonds" means that two groups are connected through a carbon-carbon bond, carbon-oxygen bond, carbon-nitrogen bond, carbon-sulfur bond, etc. to form a ring structure. If necessary, the corresponding group can reduce 1-2 hydrogen atoms.

[0313] "Stereoisomers" are compounds that have identical chemical constitutions but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0314] Any asymmetric atom (e.g., carbon, etc.) of the compounds disclosed herein can exist in a racemic or enantiomerically enriched form, such as in the (R)-, (S)-, or (R, S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in terms of the (R)- or (S)-configuration.

[0315] The term "substituted" indicates that one or more hydrogen atoms in a given structure are replaced with a specified substituent. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structure is substitutable with one or more substituents selected from a specified group, the substituents may be the same or different at each position.

[0316] The term "independently selected from" should be understood in a broad sense, which can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0317] Throughout this specification, substituents of the compounds disclosed herein are disclosed by group class or range. It is specifically noted that the present invention includes each independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0318] In various sections of the present invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variable listed for that group should be understood to be a linking group. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl", it should be understood that the "alkyl" or "aryl" represents a linking alkylene group or arylene group, respectively.

[0319] As used herein, the term "alkyl" or "alkyl group" refers to a saturated linear or branched monovalent hydrocarbon group, wherein the alkyl group may be optionally substituted with one or more substituents as described herein. The alkyl group may be optionally substituted with one or more substituents as described herein.

[0320] Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -CH(CH3)CH2CH3), u, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1 -butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3 ), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, and the like.

[0321] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon group containing 2 to 30 carbon atoms, wherein there is at least one site of unsaturation, i.e., a carbon-carbon sp2 double bond, including "cis" and "trans" orientations, or "E" and "Z" orientations. Examples of alkenyl groups include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), and the like. The alkenyl group may be optionally substituted with one or more substituents described herein.

[0322] The term "alkynyl" refers to a group having at least one site of unsaturation, i.e., a carbon-carbon sp triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propargyl (-CH2C≡CH), 1-propynyl (-C≡C-CH3), and the like. The alkynyl group may be optionally substituted with one or more substituents described herein.

[0323] The term "cycloalkyl" as used herein, unless otherwise indicated, refers to a monovalent saturated or partially unsaturated (but non-aromatic) monocyclic or polycyclic hydrocarbon. In some embodiments, the cycloalkyl group can be a bridged or non-bridged, spirocyclic or non-spirocyclic, and / or fused or non-fused bicyclic group. In some embodiments, the cycloalkyl group includes 3-10 carbon atoms, i.e., C3 to C10 cycloalkyl. In some embodiments, the cycloalkyl group has 3-15 (C3-15), 3-10 (C3-10), or 3-7 (C3-7) carbon atoms. In some embodiments, the cycloalkyl group is a monocyclic or bicyclic ring. In some embodiments, the cycloalkyl group is a monocyclic ring. In some embodiments, the cycloalkyl group is a bicyclic ring. In some embodiments, the cycloalkyl group is a tricyclic ring. In some embodiments, the cycloalkyl group is fully saturated. In some embodiments, the cycloalkyl group is partially saturated. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decahydronaphthyl, or adamantyl. When the cycloalkyl group is substituted, it can be substituted independently with one or more substituents as described herein on any ring, i.e., on any aromatic or non-aromatic ring contained in the cycloalkyl group.

[0324] The term "haloalkyl" means that at least one H in an alkyl group is replaced by a halogen, wherein the halogen is one or more of fluorine, chlorine, bromine or iodine.

[0325] The term "alkylamino" refers to an amino group in which at least one of the hydrogen atoms is replaced by an alkyl group.

[0326] The terms "heterocyclyl" and "heterocycle" are used interchangeably herein and, unless otherwise indicated, refer to a monovalent monocyclic non-aromatic ring system and / or polycyclic ring system comprising at least one non-aromatic ring; wherein one or more (in certain embodiments, 1, 2, 3, or 4) of the non-aromatic monocyclic atoms are heteroatoms independently selected from O, S(O)0-2 and N, and the remaining ring atoms are carbon atoms; and wherein one or more (in certain embodiments, 1, 2, 3, or 4) of the polycyclic ring atoms are heteroatoms independently selected from O, S(O)0-2 and N, and the remaining ring atoms are carbon atoms. In some embodiments, the heterocycle contains 1 or 2 heteroatoms, both of which are nitrogen atoms. In some embodiments, the heterocyclyl is polycyclic and contains one heteroatom in the non-aromatic ring, one heteroatom in the aromatic ring, two heteroatoms in the aromatic ring, or two heteroatoms, one of which is in the aromatic ring and the other is in the non-aromatic ring. In some embodiments, the heterocyclyl group has 3-20, 3-15, 3-10, 3-8, 4-7, or 5-6 ring atoms. In some embodiments, the heterocyclyl group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system. In some embodiments, the heterocyclyl group can be a bridged or non-bridged, spirocyclic or non-spirocyclic, and / or fused or non-fused bicyclic group. One or more nitrogen atoms and sulfur atoms may be optionally oxidized, one or more nitrogen atoms may be optionally quaternized, and one or more carbon atoms may be optionally quaternized. Replacement. Some rings can be partially or completely saturated, or aromatic, provided that the heterocycle is non-completely aromatic. The monocyclic heterocycle and polycyclic heterocycle can be connected to the main structure on any heteroatom or carbon atom that leads to a stable compound. The polycyclic heterocyclic radical can be connected to the main structure through any ring thereof, including any aromatic ring or non-aromatic ring, regardless of whether the ring contains a heteroatom. In some embodiments, the heterocyclic radical is a "heterocycloalkyl", which is 1) a saturated or partially unsaturated (but non-aromatic) monovalent monocyclic group containing at least one ring heteroatom as described in the present invention, or 2) a saturated or partially unsaturated (but non-aromatic) monovalent bicyclic group or tricyclic group, wherein at least one ring contains at least one heteroatom as described in the present invention. When heterocyclic and heterocycloalkyl are substituted, they can be substituted on any ring, i.e., on any aromatic or non-aromatic ring contained by heterocyclic and heterocycloalkyl. In some embodiments, such heterocyclic groups include, but are not limited to, oxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, 1,3-dioxolane, dithiolanyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thioxanyl, homopiperazinyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepine Base, diazepine Base, thiazolin benzoxanyl, benzodioxanyl, benzodioxolyl, benzofuranonyl, benzopyronyl, benzopyranyl, dihydrobenzofuranyl, chromanthiophene, benzothiopyranyl, benzoxazinyl, β-carbolinyl, chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroquinolinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl, dihydrobenzisothiazinyl, dihydrofuranyl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithiopyranyl, furanonyl, imidazolidinyl, 2,4-dioxo-imidazolidinyl, imidazolinyl, indolinyl, 2-oxo-indolinyl, isochromanyl, isochromanyl, isocoumarinyl, Isoindolinyl, 1-oxo-isoindolinyl, 1,3-dioxo-isoindolinyl, isothiazolidinyl, isoxazolidinyl, 3-oxo-isoxazolidinyl, morpholinyl, 3,5-dioxo-morpholinyl, octahydroindolyl, octahydroisoindolyl, 1-oxo-octahydroisoindolyl, 1,3-dioxo-hexahydroisoindolyl, oxazolidinone, oxazolidinyl, oxiranyl, piperyl Examples of heterocyclic groups in which the -CH2- group is replaced by -C(=O)- include, but are not limited to, 2-oxopyrrolidinyl, oxo-1,3-thiazolidinyl, 2-piperidinyl, 3,5-dioxopiperidinyl, and pyrimidinedione. Examples of heterocyclic groups in which the sulfur atom is oxidized include, but are not limited to, sulfolane and 1,1-dioxothiomorpholinyl. The heterocyclyl group may be optionally substituted with one or more substituents described herein.

[0327] In one embodiment, the heterocyclic group is a 3-8 atom heterocyclic group, which refers to a saturated or partially unsaturated monocyclic ring containing 3-8 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur and oxygen atoms. Unless otherwise indicated, the 3-8 atom heterocyclic group can be a carbon group or a nitrogen group, and the -CH2- group can optionally be replaced by -C(=O)-. The sulfur atom of the ring can optionally be oxidized to an S-oxide. The nitrogen atom of the ring can optionally be oxidized to an N-oxide. Examples of heterocyclic groups of 3-8 atoms include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, 1,3-dioxolane, dithiolanyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thioxanyl, homopiperazinyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepanyl, Base, diazepine Base, thiazolin Examples of heterocyclic groups in which the -CH2- group is replaced by -C(=O)- include, but are not limited to, 2-oxopyrrolidinyl, oxo-1,3-thiazolidinyl, 2-piperidonyl, 3,5-dioxopiperidinyl, and pyrimidinedione. Examples of heterocyclic groups in which the sulfur atom is oxidized include, but are not limited to, sulfolane and 1,1-dioxothiomorpholinyl. The 3-8 atom heterocyclic group may be optionally substituted with one or more substituents described herein.

[0328] In one embodiment, the heterocyclic group is a 3-6 atom heterocyclic group, which refers to a saturated or partially unsaturated monocyclic ring containing 3-6 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur, and oxygen atoms. Unless otherwise indicated, the 3-6 atom heterocyclic group can be a carbon group or a nitrogen group, and the -CH2- group can optionally be replaced by -C(=O)-. The sulfur atom of the ring can optionally be oxidized to an S-oxide. The nitrogen atom of the ring can optionally be oxidized to an N-oxide. The 3-6 atom heterocyclic group can optionally be substituted with one or more substituents described herein.

[0329] In another embodiment, the heterocyclic group is a 5-6 atom heterocyclic group, which refers to a saturated or partially unsaturated monocyclic ring containing 5-6 ring atoms, wherein at least one ring atom is selected from nitrogen, sulfur and oxygen atoms. Unless otherwise indicated, the 5-6 atom heterocyclic group can be a carbon group or a nitrogen group, and the -CH2- group can be optionally replaced by -C(=O)-. The sulfur atom of the ring can be optionally oxidized to S-oxide. The nitrogen atom of the ring can be optionally oxidized to N-oxide. Examples of 5-6 atom heterocyclyl groups include, but are not limited to, pyrrolidinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, 1,3-dioxolane, dithiolanyl, 2-oxopyrrolidinyl, oxo-1,3-thiazolidinyl, sulfolane, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thioxanyl, 2-piperidonyl, 3,5-dioxopiperidinyl, pyrimidinedione, 1,1-dioxothiomorpholinyl. The 5-6 atom heterocyclyl groups may be optionally substituted with one or more substituents described herein.

[0330] As used herein, the term "aryl," unless otherwise indicated, refers to a monovalent C6-C14 carbocyclic ring system comprising at least one aromatic ring, wherein the aromatic ring system is monocyclic, bicyclic, or tricyclic. The aryl group may be attached to the main structure via any of its rings, i.e., any aromatic or non-aromatic ring. In some embodiments, the aryl group is phenyl, naphthyl, bicyclo[4.2.0]octa-1,3,5-trienyl, indanyl, fluorenyl, or tetrahydronaphthyl. When the aryl group is substituted, it may be substituted on any ring, i.e., on any aromatic or non-aromatic ring comprised by the aryl group. In some or any embodiments, the aryl group is phenyl, naphthyl, tetrahydronaphthyl, fluorenyl, or indanyl. The aryl group may be independently and optionally substituted with one or more substituents described herein.

[0331] The term "heteroaryl" as used herein, unless otherwise indicated, refers to a monovalent monocyclic or polycyclic aromatic group, wherein at least one (in certain embodiments, 1, 2, 3 or 4) ring atom is independently selected from the heteroatoms of O, S(O)0-2 and N in the ring. The heteroaryl group is connected to the rest of the molecule by any atom in the ring system, where its valence rules permit. In some embodiments, each ring of the heteroaryl group may contain 1 or 2 O atoms, 1 or 2 S atoms, and / or 1 to 4 N atoms, or a combination thereof, provided that the total number of heteroatoms in each ring is 4 or less, and each ring contains at least 1 carbon atom. In some embodiments, the heteroaryl group has 5-20, 5-15, or 5-10 ring atoms. When a heteroaryl group is substituted, it may be substituted on any ring. In certain embodiments, monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and triazolyl. In certain embodiments, bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridinyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothiophenyl, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridinyl, pyrrolopyridinyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidinyl, and thienopyridinyl. In certain embodiments, tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, furidinyl, phenanthrolinyl, phenanthridinyl, and phenazinyl. In some or any embodiments, heteroaryl is phenylene, naphthylene, pyridinylene, pyrimidinylene, pyrazinylene, pyridazinylene, thiazolylene, benzothiazolyl, benzo[d]isothiazolyl, imidazo[1,2-a]pyridinyl, quinolinyl, 1H-indolyl, pyrrolo[1,2-b]pyridazinyl, benzofuranyl, benzo[b]thiophenyl, 1H-indazolyl, benzo[d]isoxazolyl, quinazolinyl, 1H-pyrrolo[3,2-c]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, or pyrazolo[1,5-a]pyridinyl; each of which is optionally substituted with 1, 2, 3, or 4 groups as defined throughout this specification.

[0332] As used herein, a "solvate" refers to an association formed between one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed when the solvent molecule is water.

[0333] When the solvent is water, the term "hydrate" may be used. In some embodiments, one molecule of the compound of the present invention may be associated with one water molecule, such as a monohydrate; in other embodiments, one molecule of the compound of the present invention may be associated with more than one water molecule, such as a dihydrate; and in still other embodiments, one molecule of the compound of the present invention may be associated with less than one water molecule, such as a hemihydrate. It should be noted that the hydrates of the present invention retain the biological effectiveness of the non-hydrated form of the compound.

[0334] Unless otherwise specified, the compounds used in the following examples are commercially available; the methods used in the following examples are all achievable by conventional methods unless otherwise specified.

[0335] In the compounds of the present invention, when the -L1-L2-L3 substituents are connected to the main skeleton of the compound via NH, the preparation route of the compound is as follows:

[0336] Scheme A

[0337] A0 undergoes a reduction reaction with hydrogen in the presence of a catalyst to produce B0, which then undergoes a condensation reaction with Ia or Ib to produce C0. In C0, when L1 is present, L1 is NH; if L1 is absent and L2 is present, L2 is NH. If both L1 and L2 are absent, the main skeleton of the compound is connected to the NH in the L3 group.

[0338] In the above reaction scheme, A0 and B0 can be salt compounds commonly used in chemical reactions, or pharmaceutically acceptable salt compounds, such as ammonium salts, sodium salts, potassium salts, Tris salts, phosphates, and the like.

[0339] For example, when -L1-L2-L3- are different groups, the preparation route is one of the following (since the process of preparing B0 from A0 is the same, it is not repeated and the reaction route is provided starting from B0). The following examples are only individual examples and are not exhaustive.

[0340] When R0 is a fluorescent group, the preparation route is:

[0341] Scheme A1:

[0342] Scheme A2:

[0343] Example 1 Synthesis of Compound S1

[0344] The ammonium salt of compound 1-1 was synthesized according to the method in patent CN115260264A and used as a raw material to prepare the compound of this example.

[0345] Compound S1-1 (350 mg) was dissolved in water (8 mL) and ethanol (2 mL), palladium carbon (500 mg) was added, and the mixture was replaced with hydrogen and stirred at room temperature. After the reaction, the mixture was purified with anion exchange resin and lyophilized to obtain 320 mg of the ammonium salt of compound S1-2.

[0346] The ammonium salt of compound S1-2 (50 mg) was dissolved in N,N-dimethylformamide (1 mL) and water (1 mL), and compound S1-3 (38 mg, 5 equivalents) was added and stirred at room temperature. After the reaction, the mixture was purified with anion exchange resin and lyophilized to obtain 21 mg of the ammonium salt of compound S1.

[0347] The characterization data of the ammonium salt of compound S1 is: 1382.85[M-1] - . 1 HNMR (400MHz, D2O) δ9.09 (s, 1H), 8.46 (s, 1H), 8.16 (s, 1H), 7.96 (s, 1H), 6.06 (d, J = 5.8Hz, 1H), 5.90 (s, 1H), 5.82 (d, J = 6.0Hz, 1H), 4.96-4.92 (m, 1H), 4.85-4.82 (m, 1H), 4.56 (d, J=4.3Hz, 1H), 4.53-4.46 (m, 4H), 4.43 (t, J=5.0Hz, 1H), 4.38-4.36 (m, 1H), 4 .30-4.28(m, 3H), 4.24-4.21(m, 3H), 4.18-4.14(m, 1H), 4.00(s, 3H), 3.57-3.49(m, 1H), 3.40(s, 3H), 3.23-3.19(m, 1H), 3.03- 3.01(m, 1H), 2.83-2.78(m, 1H), 2.64-2.59(m, 2H), 2.25-2.22(m, 2H), 1.61-1.51(m, 3H), 1.44-1.35(m, 1H), 1.22-1.15(m, 2H); 31 P NMR (162MHz, D2O) δ -0.93 (s, 1H), -11.56 (m, 2P), -22.90 (t, J = 17.6Hz, 1P).

[0348] Example 2 Synthesis of Compound S3

[0349] Following the above reaction route, the ammonium salt of compound S2 was synthesized with reference to the synthesis method of compound S1. Since the reaction from compound S1-1 to compound S1-2 is the same, it is not listed in the preparation route. The following examples are all based on the same situation.

[0350] The characterization data of the ammonium salt of compound S3 is: 1495.83[M-1] - . 1 H NMR (500MHz, D2O) δ9.07 (s, 1H), 8.45 (s, 1H), 8.16 (s, 1H), 7.93 (s, 1H), 6.04 (d, J = 5.3Hz, 1H), 5.82 (s, 1H), 5.76 (d, J = 5.9Hz, 1H), 4.91-4.90 (m, 1H), 4.83-4.75 (m, 1H), 4.56 (d, J=4.2Hz, 1H), 4.54-4.51 (m, 1H), 4.48-4.46 (m, 3H), 4.40 (t, J=4.7Hz, 1H), 4.33-4.27 (m, 4H), 4.24-4.22 (m, 1H), 4.17 (m, 2H), 4.13-4.11 (m, 1H), 3.97 (s, 3H), 3.53-3.48 (m, 1H), 3.40 (s , 3H), 3.24-3.16(m, 2H), 3.06-2.95(m, 2H), 2.91-2.88(m, 1H), 2.70-2.67(m, 1H) , 2.61-2.58(m, 1H), 2.18(t, J=7.4Hz, 2H), 2.14(t, J=7.2Hz, 2H), 1.65-1.57(m, 1H), 1.55-1.44(m, 5H), 1.39-1.33(m, 2H), 1.31-1.23(m, 2H), 1.19-1.15(m, 2H); 31 P NMR (202MHz, D2O) δ -0.99 (s, 1H), -11.60 (m, 2P), -22.87 (t, J = 17.6Hz, 1P).

[0351] Example 3 Synthesis of Compound S17

[0352] According to the above reaction route and referring to the synthesis method of compound S1, the ammonium salt of compound S17 was synthesized.

[0353] The characterization data of the ammonium salt of compound S17 is: 1514.86[M-1] - . 1H NMR (400MHz, d6-DMSO) δ8.48 (s, 1H), 8.41 (s, 1H), 8.31 (d, 1H), 8.10 (s, 1H), 7.94 (s, 1H), 7.31 (d, J=7.8Hz, 1H), 6.70-6.50 (m, 6H), 5.96 (d, J=6 .0Hz, 1H), 5.92 (s, 1H), 5.65 (d, J=6.0Hz, 1H), 4.87 (m, 1H), 4.61-4.53 ( m, 3H), 4.26-4.21 (m, 3H), 4.08-3.90 (m, 9H), 3.68 (m, 2H), 3.26 (m, 4H); 31 P NMR (162MHz, d6-DMSO) δ -1.02 (s, 1H), -11.53 (m, 2P), -22.70 (m, 1P).

[0354] Example 4 Synthesis of Compound S19

[0355] According to the above reaction route and referring to the synthesis method of compound S1, the ammonium salt of compound S19 was synthesized.

[0356] The characterization data of the ammonium salt of compound S19 is: 1627.69[M-1] - . 1 H NMR (500MHz, d6-DMSO) δ8.50 (s, 1H), 8.39 (s, 1H), 8.21 (d, J=8.0Hz, 1H), 8.10 (s, 1H), 7. 96 (s, 1H), 7.31 (d, J = 8.0Hz, 1H), 6.67 (s, 2H), 6.56 (s, 4H), 5.96 (d, J = 6.2Hz, 1H), 5.82 (s , 1H), 5.65 (d, J=6.3Hz, 1H), 4.87 (m, 1H), 4.62-4.56 (m, 2H), 4.38-4.33 (m, 2H), 4.21-3. 89 (m, 12H), 3.32-3.20 (m, 4H), 3.01-3.00 (m, 2H), 2.09 (m, 2H), 1.46 (m, 4H), 1.21 (m, 2H); 31 P NMR (202MHz, d6-DMSO) δ -2.18 (s, 1H), -11.86 (m, 2P), -21.83 (m, 1P).

[0357] Example 5 Synthesis of Compound 5

[0358] Compound 5-1 (77 mg, 5 equivalents) was dissolved in dimethyl sulfoxide (2 mL), and triethylamine (60 μL, 5 equivalents) and HATU (164 mg, 5 equivalents) were added. The mixture was stirred at room temperature for 30 minutes. Compound S1-2 (100 mg) was dissolved in dimethyl sulfoxide (1 mL) and added to the reaction mixture, which was stirred at room temperature. After the reaction, the mixture was purified using anion exchange resin and lyophilized to obtain 17 mg of the ammonium salt of compound 5.

[0359] The characterization data of the ammonium salt of compound 5 is: 1316.85[M-1] - . 1 H NMR (500MHz, D2O) δ8.35 (s, 1H), 8.06 (s, 1H), 7.91 (s, 1H), 5.96 (d, J=5.6Hz, 1H), 5.78 (d, J= 6.0Hz, 1H), 5.73 (s, 1H), 4.90-4.88 (m, 1H), 4.82 (m, 1H), 4.49-4.46 (m, 4H), 4.39 (t, J=5.3Hz , 1H), 4.32-4.20(m, 4H), 4.17(m, 2H), 4.13-4.09(m, 1H), 4.02(s, 2H), 3.98(s, 3H), 3.63-3.5 3(m, 7H), 3.48-3.45(m, 2H), 3.38(s, 3H), 3.27(s, 3H), 3.25-3.21(m, 1H), 2.54-2.50(m, 1H); 31 P NMR (202MHz, D2O) δ -0.98 (s, 1H), -11.68 (m, 2P), -22.94 (m, 1P).

[0360] Example 6 Synthesis of Compound 6

[0361] According to the above reaction route and referring to the synthesis method of compound 5, the ammonium salt of compound 6 was synthesized.

[0362] The characterization data of the ammonium salt of compound 6 is: 1404.88[M-1] - . 1H NMR (500MHz, D2O) δ9.05 (s, 1H), 8.43 (s, 1H), 8.15 (s, 1H), 7.92 (s, 1H), 6.04 (d, J = 4.8Hz, 1H ), 5.77 (s, 1H), 5.75 (d, J = 5.9Hz, 1H), 4.91 (m, 1H), 4.71 (t, J = 5.2Hz, 1H), 4.55 (d, J = 3.8Hz, 1 H), 4.49-4.46 (m, 3H), 4.40 (t, 1H), 4.32-4.28 (m, 3H), 4.24-4.10 (m, 4H), 4.04 (s, 2H), 3.96 (s, 3H), 3.64-3.54 (m, 17H), 3.44 (s, 3H), 3.32 (s, 3H), 3.30-3.27 (m, 1H), 2.60-2.55 (m, 1H); 31 P NMR (202MHz, D2O) δ -1.00 (s, 1H), -11.64 (m, 2P), -22.76 (m, 1P).

[0363] Example 7 Synthesis of Compound S23

[0364] According to the above reaction route and referring to the synthesis method of compound 5, the ammonium salt of compound S23 was synthesized.

[0365] The characterization data of the ammonium salt of compound S23 is: 1747.91[M-1] - . 1 H NMR (500MHz, d6-DMSO) δ8.49 (s, 1H), 8.40 (s, 1H), 8.28 (d, 1H), 8.10 (s, 1H) , 7.95 (s, 1H), 7.31 (d, J = 7.8Hz, 1H), 6.70-6.53 (m, 6H), 5.96 (d, J = 6.0Hz, 1 H), 5.88 (s, 1H), 5.65 (d, J=6.0Hz, 1H), 4.88-4.86 (m, 1H), 4.65-4.55 (m, 3H ), 4.28-4.20(m, 3H), 4.10-3.89(m, 11H), 3.68-3.53(m, 16H), 3.26(m, 6H); 31 P NMR (202MHz, d6-DMSO) δ -1.38 (s, 1H), -11.70 (m, 2P), -22.56 (m, 1P).

[0366] Referring to the synthesis method of compound S1, the compounds of Examples 8-15 were prepared as shown in Table 1.

[0367] Example 16 Preparation of Compound S41

[0368] According to the above reaction route, compound S1-1 was dissolved in DMSO, compound S41-1 and CuI were added, and the mixture was stirred at room temperature. After the reaction, the mixture was purified with an ion exchange resin to synthesize the ammonium salt of compound S41.

[0369] The characterization data of the ammonium salt of compound S41 is: 1464.04[M-1] - . 1 H NMR (400MHz, D2O) δ9.10 (s, 1H), 8.51 (s, 1H), 8.21 (s, 1H), 7.99 (s, 1H), 7.98 (s, 1H), 6.06 (d, J=5.5Hz, 1H), 5.85 (s, 1H), 5.81 (d, J= 5.8Hz, 1H), 4.96 (s, 1H), 4.74-4.72 (m, 1H), 4.69-4.67 (m, 1H), 4.61-4.50 (m, 5H), 4.48-4.45 (m, 3H), 4.42 (s, 1H), 4.36 (m, 2H), 4.32 -4.30(m,2H),4.27-4.25(m,1H),4.21(m,2H),3.99(s,3H),3.95-3.92(m,1H),3.45(s,3H),3.10-3.06(m,1H),3.01-2.96(m,1H),2 .90 (dd, J=13.2, 4.7Hz, 1H), 2.73 (d, J=12.9Hz, 1H), 2.18 (t, J=7.0Hz, 2H), 1.60-1.50 (m, 3H), 1.40-1.33 (m, 1H), 1.15-1.08 (m, 2H); 31 P NMR (202MHz, D2O) δ -0.89 (s, 1P), -11.58 (d, J=17.5Hz, 1P), -11.84 (d, J=18.2Hz, 1P), -22.90 (t, J=17.8Hz, 1P).

[0370] Example 17 Preparation of Compound S170

[0371] The ammonium salt of compound S170-1 was synthesized according to the method in patent CN115260264A, and the ammonium salt of compound S170 was synthesized with reference to the synthesis method of compound S1.

[0372] The characterization data of the ammonium salt of compound S170 is: 1454.02[M-1] - .1 H NMR (400MHz, D2O) δ9.11 (s, 1H), 8.50 (s, 1H), 8.22 (s, 1H), 7.97 (s, 1H), 6.08 (s, 1H), 5.82-5 .81 (m, 2H), 4.94 (s, 1H), 4.56-4.44 (m, 6H), 4.34 (m, 3H), 4.25 (m, 2H), 4.19 (m, 2H), 4.14-4. 12(m, 1H), 4.01(s, 3H), 3.44(s, 3H), 3.29-3.19(m, 5H), 2.93-2.91(m, 1H), 2.73-2.71(m, 1H ), 2.65(m, 1H), 2.48-2.46(m, 2H), 2.16-2.11(m, 2H), 1.58-1.43(m, 4H), 1.27-1.22(m, 3H); 31 P NMR (202MHz, D2O) δ-0.91 (s, 1P), -11.58--11.67 (m, 2P), -22.86 (m, 1P).

[0373] Example 18 Preparation of Compound S171

[0374] The characterization data of the ammonium salt of compound S170 is: 1586.03[M-1] - . 1 H NMR (500MHz, D2O) δ9.03 (s, 1H), 8.41 (s, 1H), 8.09 (br, 2H), 7.88 (s, 1H), 7.61 (br, 1H), 6.82-6.48 (m, 7H), 5.91 (s, 1H), 5.78 (s, 1H), 5.72 (s, 1H), 4.89 (m, 1H ), 4.67-4.64(m, 2H), 4.45(m, 2H), 4.40-4.35(m, 2H), 4.28(s, 2H), 4.22-4.15( m, 4H), 3.90 (s, 3H), 3.50-3.45 (m, 4H), 3.38 (s, 3H), 2.75 (m, 1H), 2.55 (m, 2H); 31 P NMR (202MHz, D2O) δ -0.97 (s, 1P), -11.57 (m, 2P), -22.72 (m, 1P).

[0375] Example 19 Preparation of Compound S168

[0376] According to the above reaction route and referring to the synthesis method of compound S41, the ammonium salt of compound S168 was synthesized.

[0377] The characterization data of compound S168 are: 1667.02[M-1] - . 1 H NMR (500MHz, D2O) δ9.02 (s, 1H), 8.39 (s, 1H), 8.24 (s, 1H), 8.07 (s, 1H), 7.97-7.88 (m, 3H), 7.26 (s, 1H), 6.82 (br, 2H), 6.55(br, 4H), 5.92(s, 1H), 5.72(s, 1H), 5.62(s, 1H), 4.90(m, 1H), 4.68-4.63(m, 3H), 4.47-4.35(m, 6H), 4.28(s , 1H), 4.21-4.14(m, 5H), 4.04(s, 1H), 3.94(s, 3H), 3.65(m, 1H), 3.53(m, 1H), 3.36(s, 3H), 2.59(m, 1H), 2.31(m, 2H); 31 P NMR (202MHz, D2O) δ-0.93 (s, 1P), -11.59--11.67 (m, 2P), -22.81 (m, 1P).

[0378] Example 20 Detecting the efficiency of mRNA capping synthesis

[0379] 1) Linearize the plasmid and purify the DNA template.

[0380] 2) In vitro transcription synthesis of mRNA, using the capping structure prepared in the present invention, the positive control AG (3'OMe) (Trilink, N-7413), negative control (uncapped mRNA) and blank control (DNase / RNase-free water), the reaction system is shown in Table 1:

[0381] Table 1. Reaction system

[0382] Incubate at 37°C for 2-3 hours. Digest with TURBO DNase for 15 minutes. Precipitate the mRNA with LiCl for at least 30 minutes or overnight. Wash the mRNA pellet with 75% ethanol, briefly air dry to evaporate the ethanol, and then reconstitute the mRNA in RNase-free water.

[0383] The reaction systems listed in Table 1 can be adjusted as needed because the solubility of different test compounds varies in different solutions. For ease of operation, the more commonly used concentrations are 10mM and 100mM. If the test compound has good solubility, an initial concentration of 100mM can be used, and the dosage is 0.8μL. If the initial concentration of the test compound is 10mM, the dosage is 8μL. Regardless of the initial concentration, the dosage of different test compounds is the same, and the final concentration is also the same.

[0384] For example, when testing compound S17 and compound S19, their initial concentrations were 10 mM, and the amount added was 8 μL; when testing compounds 5 and 6, their initial concentrations were 100 mM, and the amount added was 0.8 μL.

[0385] 3) The transcription products were purified and the reaction yields were calculated. The test results of some compounds are provided as shown in Table 2.

[0386] Table 2. Final product mass (μg) obtained from a 20 μL mRNA synthesis reaction system

[0387] As can be seen from Table 2, all test groups were able to transcribe mRNA normally with good yields.

[0388] Example 21 Evaluation of the expression efficiency of different capped luciferase mRNAs in HEK293T cells

[0389] 1) HNE293T cells were cultured in DMEM medium containing 10% FBS and penicillin / streptomycin at 37°C and 5% CO2.

[0390] 2) The cultured HEK293T cells were plated in a 96-well plate, with 1.25×10 4 cells.

[0391] 3) After the cells are completely attached, the drug is administered. A mixture of 0.5 μg of mRNA sample and Polyplus jetMESSENGER is added to each well of cells and cultured at 37° C. and 5% CO 2 for 6 hours.

[0392] 4) Remove the growth medium from the cells to be tested and rinse the cells with PBS. Remove the PBS by centrifugation and add 50 μL of 1x lysis buffer. Transfer the cells and all liquid to a microcentrifuge tube and centrifuge at 12,000 x g at 4°C for 2 minutes.

[0393] 5) Transfer the supernatant to a new tube and measure the fluorescence reading using the ONE-Glo Luciferase Assay System Kit.

[0394] All test compounds, N-7413, negative control and blank control were tested under the same conditions. Some experimental results are shown in Table 3.

[0395] Table 3 Relative fluorescence readings of capped mRNA

[0396] Note: The fluorescence reading of N-7413 is set as 1 for calibration. The ratio of the fluorescence reading of other capped mRNA transfected with the fluorescence reading of N-7413 is the relative fluorescence reading.

[0397] The relative fluorescence reading test results (6 hours) after transfection of capped mRNA into 293T cells are shown in Table 3. As can be seen from the table, some compounds of the present invention have higher expression levels of capped mRNA, and some compounds can achieve higher mRNA expression efficiency than N-7413.

[0398] Example 22 Biotin affinity experiment

[0399] 1) preparing S1-30nt-mRNA (mRNA capped with compound S1, with a length of 30nt) and purifying it with LiCl;

[0400] 2) Take 500 μL (2 mg) of magnetic beads with streptavidin, place the test tube on a magnetic rack to collect the beads, remove and discard the supernatant, and wash the beads;

[0401] 3) Resuspend the magnetic beads in 600 μL TES buffer and transfer the S1-30nt-mRNA from 1) to - Add 5.5 μg to the magnetic bead resuspension and mix thoroughly; incubate at room temperature for one hour on a shaker or vortexer;

[0402] 4) Place the sample on a magnetic rack for 5 minutes, wait for the liquid to clear, and remove the supernatant;

[0403] 5) Keep the sample on the magnetic stand and rinse the magnetic beads three times with TES buffer;

[0404] 6) Keep the sample on the magnetic rack and rinse the magnetic beads three times with distilled water;

[0405] 7) Remove deionized water from the beads, add 60 μL of elution buffer heated to 80°C, mix well, heat the mixture to 80°C in a thermostatic metal bath for 3 minutes, then place on a magnetic stand for 3 minutes, and aspirate the supernatant as the eluted product.

[0406] 8) Experimental results

[0407] Amount obtained: 5.5 μg; recovery rate: 100%.

[0408] Compound S3, Compound 169, Compound S41, and Compound S170 prepared in the Examples were tested according to the above test process, and the recovery rates were all above 95%.

[0409] It can be seen from this that cap analogs with biotin groups can be bound by streptavidin magnetic beads. When mRNA is capped with such compounds, the mRNA molecules carry biotin groups and can also be bound by streptavidin magnetic beads, thereby purifying or enriching the desired mRNA molecules. Not only is the obtained mRNA purity high, but the purification or enrichment process is also simple and efficient.

[0410] Cap analogs with biotin groups, such as compounds S1, S3, S169, S41, and S170, are beneficial for improving the expression performance of mRNA. At the same time, during the mRNA production and preparation process, they can be used to purify and enrich the capped target mRNA very conveniently and efficiently, which is very beneficial for the preparation and application of in vitro transcribed mRNA.

[0411] Example 23 Verification of the linear relationship between fluorescently labeled mRNA concentration and fluorescence intensity

[0412] 1) Selecting some compounds to synthesize corresponding mRNAs, for example, using compound S23 (FAM-labeled), compound S166 (MANT-labeled), compound S7 (Cy3-labeled), and compound S10 (Cy5-labeled) as cap analogs, and referring to Example 20, synthesizing FLuc-mRNAs with corresponding cap analogs, and further purifying them using LiCl precipitation and an RNA purification kit. The resulting mRNA stock solutions were designated S23-mRNA, S166-mRNA, S7-mRNA, and S10-mRNA, respectively;

[0413] 2) The mRNA stock solution was serially diluted to concentrations of 200 ng / uL, 100 ng / uL, 50 ng / uL, 25 ng / uL, 12.5 ng / uL, and 6.25 ng / uL, and 20 uL of each was added to a 96-well plate;

[0414] 3) The linear correlation between concentration and fluorescence value was detected by a microplate reader. The excitation / emission wavelengths were 492 / 518 nm for S23-mRNA, 356 / 448 nm for S166-mRNA, 570 / 650 nm for S7-mRNA, and 640 / 675 nm for S10-mRNA.

[0415] The detection results are shown in Figure 1, which shows the fluorescence intensity detection results at different mRNA concentrations. As shown in Figure 1, for mRNA prepared with a cap analog labeled with a fluorescent group, the mRNA concentration and fluorescence intensity are linearly related at the corresponding wavelength.

[0416] Example 24 Preparation of mRNA-LNP

[0417] Preparation process:

[0418] 1) The components of LNP include: cationic lipid (SM102), cholesterol, DSPC and DMG-PEG2000, with molar ratios of 48.5%, 38.9%, 11.1% and 1.5%, respectively.

[0419] 2) Lipid-ethanol solution: The required cationic lipid, cholesterol, DSPC and DMG-PEG2000 were dissolved in anhydrous ethanol at a molar ratio to prepare a lipid-ethanol solution with a total lipid concentration of 8 mM, which was set aside.

[0420] 3) mRNA-acetate buffer: dilute the mRNA stock solution (prepared as described in Example 20) with acetate buffer (200 mM, pH 5.0) to an appropriate concentration for later use.

[0421] 4) Encapsulation: The mRNA-buffer phase and the lipid-ethanol phase were mixed using a microfluidic instrument (Syringe Pump: SPM, Duco Industrial; Hybrid Chip: LNP-B0, FluidicLab) at a nitrogen-phosphorus ratio of 4.8 or more, a flow rate ratio of 3:1 (buffer phase / ethanol phase), and a total flow rate of 3.6 mL / min. The mixed sample was then diluted with 34 volumes of Tris buffer (20.5 mM, pH 7.5, containing 8.95% sucrose) and dialyzed against 25 or 30 kDa MwCO overnight at 4°C to obtain mRNA-encapsulated LNPs.

[0422] Example 25 Cell confocal experiment

[0423] Using compound S10 as the cap analog, the process of Example 20 was followed to prepare compound S10-capped GFP-mRNA, designated S10-GFP-mRNA. Encapsulation was then performed according to the process of Example 24 to obtain encapsulated LNPs, designated S10-GFP-mRNA-LNP. When the cap analog was compound X, the prepared mRNA was designated X-GFP-mRNA, and the encapsulated LNPs were designated X-GFP-mRNA-LNP.

[0424] Experimental methods

[0425] 1) Prepare 35 mm culture dishes for Huh7 cell plating, with approximately 1.0 × 10 cells per plate. 5 The cells were placed in a 37°C, 5% CO2 incubator and cultured for 18 h before LNP transfection.

[0426] 2) Before transfection, take out the pre-encapsulated X-GFP-mRNA-LNP, and use 1.0×10 5Add 1 μg of X-GFP-mRNA to each dish of cells, shake gently and incubate in a 37°C, 5% CO2 incubator;

[0427] 3) After the incubation period, gently aspirate the culture medium from the cell culture dish, rinse twice with PBS, add 300 μL of fixative (4% paraformaldehyde) to the center of the dish to be fixed, and fix for 15 minutes. After fixation, rinse twice with PBS.

[0428] 4) Nuclear staining: Add 100 μL of DAPI staining solution to the fixed culture dish to cover the sample. Incubate at room temperature for 3-5 minutes. Aspirate the DAPI staining solution and wash with PBS 2-3 times, 3-5 minutes each time.

[0429] 5) Before confocal observation, add a small amount of PBS to the fixed or unfixed culture dish to maintain a moist environment.

[0430] 6) Place the culture dish under a confocal microscope and observe the intracellular fluorescence expression and localization of DAPI, GFP, and Compound X using excitation / emission wavelengths of 410 nm / 410-585 nm, 488 nm / 494-572 nm, and the excitation / emission wavelengths of the fluorescent group in Compound X, respectively.

[0431] Taking compound S10 as an example, its structure contains a Cy5 group, and its excitation / emission wavelength is 643 nm / 638-759 nm.

[0432] When the compound carries different fluorescent groups, such as FAM group, MANT group, Cy3 group, Cy5 group, etc., the excitation and emission wavelengths of the corresponding fluorescent groups are selected.

[0433] Taking S10-GFP-mRNA as an example, referring to the above process, its fluorescence expression and distribution in cells were detected, and the measured results are shown in Figures 2 and 3.

[0434] The results of staining and fixation for 20 min, 30 min, and 2 h are shown in FIG2 (imaging results of the same area at the same time point); the results of staining and fixation for 20 h are shown in FIG3 (imaging results of the same area).

[0435] As shown in Figures 2 and 3, 30 minutes after transfection with S10-GFP-mRNA (labeled with a Cy5 group), Cy5 fluorescence distribution in Huh7 cells was observed under a confocal microscope, with clear fluorescence observed 2 hours after transfection. By 20 hours after transfection, significant GFP expression was observed, with Cy5 fluorescence exhibiting intracellular clustering. This demonstrates that Cy5-labeled mRNA can be used as a tracer to observe mRNA distribution at different time points after transfection and that mRNA expression is normal.

[0436] Example 26 Mouse In Vivo Imaging Experiment 1 (Cy5 Fluorescence Detection)

[0437] Compound S10 (with a Cy5 group) was used as a cap analog, and the procedure of Example 20 was followed to prepare compound S10-capped FLuc-mRNA, designated S10-FLuc-mRNA. Encapsulation was then performed according to the procedure of Example 24 to obtain encapsulated LNPs, designated S10-FLuc-mRNA-LNP.

[0438] Experimental methods

[0439] 1) Dilute the S10-FLuc-mRNA-LNP sample to 200 ng / uL for later use;

[0440] 2) Healthy mice were randomly divided into two groups, three in each group; the experimental group received an IV injection of 0.2 mL of the sample (40 μg / mouse) via the tail vein, while the control group received an IV injection of 0.2 mL of normal saline via the tail vein. Live imaging was performed 1 hour later using an excitation wavelength of 640 nm and an emission wavelength of 680 nm.

[0441] 3) After the in vivo imaging test, the liver, lungs, and spleen of the mouse were imaged using an imaging system with an excitation wavelength / emission wavelength of 640 nm / 680 nm.

[0442] Experimental results

[0443] The test results are shown in Figures 4, 5 to 7. Figure 4 shows the in vivo imaging test results of mice 1 hour after injection; Figures 5 to 7 show the imaging test results of the liver, lung, and spleen of mice, respectively.

[0444] As shown in Figure 4 , significant Cy5 fluorescence was observed in living mice 1 hour after IV injection of S10-FLuc-mRNA-LNP. As shown in Figures 5 to 7 , after S10-FLuc-mRNA-LNP injection, it was mainly distributed in the liver, lungs, and spleen of mice.

[0445] Example 27 Mouse In Vivo Imaging Experiment 2 (Fluc Fluorescence Detection)

[0446] The test sample was the same as that in Example 26, namely S10-FLuc-mRNA-LNP.

[0447] Experimental methods

[0448] The S10-FLuc-mRNA-LNP sample was diluted to 50 ng / uL, and the fluorescent substrate (D-Luciferin, which can catalyze FLuc to emit yellow-green fluorescence) was prepared to a concentration of 15 mg / mL and set aside.

[0449] 1) Three healthy mice were injected intravenously (IV) with 0.2 mL of S10-FLuc-mRNA-LNP sample (10 μg / mouse). 3 h later, 0.2 mL of fluorescent substrate was injected intraperitoneally (IP). In vivo abdominal imaging was then performed at excitation / emission wavelengths of 560 / 590 nm.

[0450] The test results are shown in Figure 8 . As can be seen from Figure 8 , 3 hours after IV injection of S10-FLuc-mRNA-LNP encoding the FLuc protein, obvious FLuc fluorescence was observed in mouse live imaging, indicating that after the S10-FLuc-mRNA-LNP was injected into the mouse, the S10-FLuc-mRNA therein could normally express the FLuc protein in the mouse.

[0451] Example 28 Synthesis of Compound D20-1

[0452] At room temperature, compound D20-1A (97.00 g, 375.56 mmol) and CHCl3 (170 mL) were added to anhydrous tetrahydrofuran and cooled to -78°C. Lithium bistrimethylsilylamide (500 mL) was slowly added dropwise to the system, and the reaction was stirred at this temperature for 4 hours. The reaction solution was poured into a saturated aqueous sodium bicarbonate solution (700 mL) at 0°C, separated, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by ethanol slurrying to obtain compound D20-1B as a white solid (73.79 g, 52.3%).

[0453] At room temperature, cesium fluoride (296.20 g, 1.95 mol) was added to a mixed solution of compound D20-1B (73.79 g, 195.4 mmol) in methanol (500 ml) and tert-butanol (500 ml). DBU (117 mL, 781.0 mmol) was then added and stirred for 1 hour. Saturated ammonium chloride solution (150 ml) was added to the reaction system, stirred for 10 minutes, and water (2 L) and ethyl acetate (1 L) were added. The mixture was stirred for 5 minutes and separated. The aqueous phase was extracted with ethyl acetate (500 mL × 2). The organic phases were combined, concentrated under reduced pressure, and purified by ethanol slurrying to obtain compound D20-1C as a white solid (48.50 g, 77.5%).

[0454] At 0°C, sodium borohydride (6.73 g, 177.95 mmol) was added portionwise to a solution of compound D20-1C (28.5 g, 88.98 mmol) in ethanol (290 ml) and stirred at room temperature; saturated ammonium chloride solution (100 ml) was added to the reaction system, the reaction solution was concentrated, and dichloromethane (100 ml) and water (100 ml) were added for extraction. The aqueous phase was extracted with dichloromethane (100 ml × 2), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound D20-1D as a colorless oil (27.13 g, 100%), which was carried out in the next step without purification.

[0455] Methanesulfonyl chloride (7.0 mL, 91.14 mmol) was slowly added dropwise to a solution of compound D20-1D (22.20 g, 75.95 mmol) and triethylamine (21.1 mL, 151.90 mmol) in dichloromethane (220 mL) in an ice bath under nitrogen. After complete addition, the mixture was stirred at room temperature for 1 hour. Water (200 mL) was added to the system, stirred for 2 minutes, and the layers separated. The organic phase was washed with water (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by slurrying with petroleum ether (200 mL) to afford compound D20-1E as a light yellow solid (26.58 g, 94.5%).

[0456] At room temperature, sodium azide (2.19 g, 33.75 mmol) was added to a solution of compound D20-1E (5.00 g, 13.50 mmol) in anhydrous N,N-dimethylformamide (25 mL). The temperature was raised to 120°C and the mixture was stirred overnight. After cooling to room temperature, water (150 mL) was added and extracted with methyl tert-butyl ether (60 mL x 2). The organic phases were combined, washed with water (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound D20-1F as a colorless oil (4.28 g, 100%). This was carried on to the next step without purification.

[0457] At room temperature, acetic acid (35 mL) and water (15 mL) were mixed and added to compound D20-1F (4.28 g, 13.50 mmol). The mixture was heated to 50°C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and saturated aqueous sodium bicarbonate (19 mL) and methanol (19 mL) were added and stirred for 2 minutes. Sodium periodate (3.18 g, 14.85 mmol) was added to the system and stirred for 30 minutes. The filtrate was collected by suction and cooled to 0°C. Sodium borohydride (1.02 g, 27.00 mmol) was added to the system in batches. After the addition was complete, the mixture was stirred for 30 minutes. The reaction was quenched by adding saturated aqueous ammonium chloride (10 mL). The majority of the reaction solution was concentrated under reduced pressure, and water (50 mL) was added. The product was extracted with dichloromethane (60 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford compound D20-1G as a colorless oil (3.34 g, 100%). The product was directly used in the next step without purification.

[0458] Benzoyl chloride (2.35 mL, 20.25 mmol) was slowly added dropwise to a solution of compound D20-1G (3.34 g, 13.50 mmol) and triethylamine (3.75 mL, 27.00 mmol) in dichloromethane (33 mL) in an ice bath under nitrogen. After complete addition, the mixture was stirred at room temperature for 30 minutes. Water (100 mL) was added to the system, the layers were separated, and the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15 / 1) to afford compound D20-1H as a colorless oil (3.64 g, 76.8%).

[0459] At room temperature, concentrated sulfuric acid (83 μL, 1.55 mmol) was diluted with acetic acid (1 mL) and added to a solution of compound D20-1H (3.64 g, 10.36 mmol) in acetic anhydride (2.5 mL, 25.90 mmol) and acetic acid (36 mL). The mixture was heated to 30°C and stirred for 5 hours. The reaction solution was poured into water (200 mL), extracted with ethyl acetate (100 mL), and washed sequentially with saturated aqueous sodium bicarbonate solution (100 mL × 2) and saturated aqueous sodium chloride solution (100 mL). The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to afford compound D20-1I as a colorless oil (3.40 g, 82.9%).

[0460] At room temperature, under nitrogen, N,O-bis(trimethylsilyl)acetamide (12 mL, 44.01 mmol) was added to a suspension of 2-acetamido-9H-purin-6-yldiphenylcarbamate (6.84 g, 17.60 mmol) in 1,2-dichloroethane (70 mL), and the mixture was heated to 70°C and stirred for 2 hours. Trimethylsilyl trifluoromethanesulfonate (3.4 mL, 19.07 mmol) was added to the system, and compound D20-1I (0.50 g, 1.26 mmol) was dissolved in 1,2-dichloroethane (40 mL) and added to the system. The mixture was heated to 80°C and stirred for 2 hours. The mixture was cooled to room temperature, and water (100 mL) was added to the system. The liquid was separated, and the aqueous phase was extracted with dichloromethane (50 mL). The liquid was separated, and the organic phases were combined, washed with saturated aqueous sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 4) to give compound D20-1J as a white solid (2.78 g, 26.2%).

[0461] Aqueous ammonia (6 mL) was added to a solution of compound D20-1J (2.78 g, 3.84 mmol) in methanol (30 mL) at room temperature, and the mixture was heated to 50°C and stirred overnight. The reaction solution was concentrated under reduced pressure and purified by slurrying with acetonitrile (30 mL) to obtain compound D20-1 as a white solid (1.15 g, 88.5%).

[0462] The characterization data of compound D20-1 are: MS (ESI, pos.ion) m / z: 341.15 [M+H]+; 1 H NMR (500MHz, DMSO) δ9.71 (s, 1H), 7.76 (s, 1H), 6.55 (s, 3H), 5.66 (s, 1H), 5.06 (s, 1H), 4.69-4.63(m, 1H), 4.10-4.01(m, 1H), 3.90-3.67(m, 3H), 3.66-3.60(m, 1H).

[0463] The nucleoside intermediates required in the following examples can be synthesized by referring to the method of compound D20-1.

[0464] Synthesis of compounds of Examples 29-38

[0465] Scheme D:

[0466] According to the above synthetic route, nucleoside D0 was dissolved in trimethyl phosphate, and phosphorus oxychloride was added dropwise at 0°C. After the reaction was completed, 1 M TEAB solution was added to quench the reaction and the mixture was directly purified using an ion exchange column. The product was collected, concentrated, and lyophilized to obtain the triethylamine salt of compound D1.

[0467] Compound D1 was dissolved in DMF, iodomethane was added, the reaction was completed, the mixture was concentrated, diluted with water, and purified using an ion exchange column to obtain triethylamine salt of compound D2.

[0468] Compound D3 was suspended in DMF, and CDI was added. After the reaction, an acetone solution of sodium iodide was added for precipitation, and the mixture was filtered. The filter cake was dried in a rotary evaporation machine to obtain compound D4.

[0469] Compound D2, compound D4 and anhydrous magnesium chloride were added to DMSO and stirred at room temperature. After the reaction, the mixture was diluted with water and purified by ion exchange column to obtain product D.

[0470] Examples 29-38 were prepared according to the above synthetic route, wherein D0 was synthesized according to the method of compound D20-1 in Example 28. The compounds prepared in Examples 29-38 and their characterization parameters are shown in the following table.

[0471] Synthesis of Compounds of Examples 46-49

[0472] Example 46 was prepared according to the process of Example 35, and Examples 47-49 were prepared according to the process of Example 32, except that, referring to the synthesis route of Scheme D, compound D3 was replaced with different dinucleoside diphosphate fragments to obtain

[0473] Examples 46-49.

[0474] Example 50 Synthesis of Compound D23

[0475] According to the above synthetic route, the ammonium salt of compound D23 was synthesized with reference to the method in patent CN115260264A. Its characterization data is: 1188.02[M-1] - . 1H NMR (500MHz, D2O) δ9.06 (s, 1H), 8.46 (s, 1H), 8.17 (s, 1H), 7.93 (s, 1H), 6.04 (d, J = 5.2Hz, 1H) , 5.83 (s, 1H), 5.78 (d, J = 5.8Hz, 1H), 4.91-4.87 (m, 1H), 4.76-4.75 (m, 1H), 4.66 (d, J = 12.3Hz, 1H), 4.55 (dt, J=25.5, 5.5Hz, 1H), 4.49-4.46 (m, 2H), 4.38-4.35 (m, 2H), 4.32-4.30 (m, 3H), 4. 21-4.15(m, 3H), 3.96(s, 3H), 3.88-3.80(m, 2H), 3.43(s, 3H), 3.39(s, 3H), 2.53-2.37(m, 2H); 31 P NMR (202MHz, D2O) δ17.39 (d, J=8.3Hz, 1P), 7.62-7.45 (m, 1P), -0.90 (s, 1P), -11.42 (d, J=25.1Hz, 1P).

[0476] Example 51 Synthesis of Compound D28

[0477] According to the above synthetic route, the ammonium salt of compound D28 was synthesized with reference to the method in patent CN115260264A. Its characterization data is: 1270.06 [M-1] - . 1 H NMR (500MHz, D2O) δ8.41 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 5.95 (d, J = 6.1Hz, 1H), 5.87 (s, 1H), 5.79 (d, J=5.8Hz, 1H), 4.92-4.89 (m, 1H), 4.83-4.82 (m, 1H) , 4.66-4.58(m, 2H), 4.50-4.47(m, 2H), 4.41-4.36(m, 3H), 4.32-4.28(m, 2H), 4.23-4.14(m, 3H), 3.98(s, 3H), 3.90-3.82(m, 2H), 3.39(s, 3H), 3.36(s, 3H); 31 P NMR (202MHz, D2O) δ-0.94 (s, 1P), -11.58--11.74 (m, 2P), -22.96--23.05 (m, 2P).

[0478] Example 52 Synthesis of Compound D97

[0479] According to the above synthetic route, the ammonium salt of compound D97 was synthesized with reference to the method in patent CN115260264A. Its characterization data is: 1206.03[M-1] - . 1 H NMR (500MHz, D2O) δ8.84 (s, 1H), 8.49-8.44 (m, 1H), 8.11-8.10 (m, 1H), 7. 91(s, 1H), 6.00-5.97(m, 1H), 5.80-5.78(m, 2H), 4.93(m, 1H), 4.75-4.74( m, 1H), 4.62-4.56 (m, 2H), 4.50 (s, 1H), 4.44-4.42 (m, 2H), 4.34-4.29 (m, 4 H), 4.23-4.15(m, 3H), 3.96(s, 3H), 3.85-3.78(m, 2H), 3.37-3.34(m, 6H); 31 P NMR (202MHz, D2O) δ30.00 (m, 1P), -0.97 (m, 1P), -12.54 (m, 2P).

[0480] Example 53 Detecting the efficiency of mRNA capping synthesis

[0481] The detection was carried out according to the method described in Example 20, and the results of the detection of some compounds are shown in Table 4.

[0482] Table 4. Final product mass (μg) obtained from a 20 μL mRNA synthesis reaction system

[0483] As can be seen from Table 4, all test groups were able to transcribe mRNA normally with good yields.

[0484] Example 54 Evaluation of the expression efficiency of different capped luciferase mRNAs in HEK293T cells

[0485] The test was performed according to the method described in Example 21. All test compounds, N-7413, negative controls and blank controls were tested under the same conditions. Some experimental results are shown in Table 5.

[0486] Table 5 Relative fluorescence readings of capped mRNA

[0487] Note: The fluorescence reading of N-7413 is set as 1 for calibration. The ratio of the fluorescence reading of other capped mRNA transfected with the fluorescence reading of N-7413 is the relative fluorescence reading.

[0488] The relative fluorescence reading results (6 h) after transfection of capped mRNA into 293T cells are shown in Table 5. As can be seen from the table, the mRNA capped by the compounds of the present invention has a high expression level, and some compounds can achieve higher mRNA expression efficiency than N-7413.

[0489] Example 55 PK test of capped Gluc mRNA-LNP in SD rats

[0490] 1) Preparation of mRNA

[0491] Capped Gluc mRNA was prepared according to the method described in Example 20, using different compounds as cap analogs. The compound used was X, and the corresponding target product prepared was recorded as X-Gluc mRNA.

[0492] 2) mRNA encapsulation

[0493] Encapsulation was performed according to the method described in Example 24 to obtain LNPs encapsulating mRNA.

[0494] When the cap analog is compound X, the prepared mRNA is denoted as X-Gluc-mRNA; the encapsulated LNPs are denoted as X-Gluc-mRNA-LNP. Taking compound D1 as an example, the prepared mRNA is denoted as D1-Gluc-mRNA; the encapsulated LNPs are denoted as D1-Gluc-mRNA-LNP.

[0495] by The GlucmRNA prepared with (3'Acm)AG as the cap analogue was used as the control group. The structure of AG (3'Acm) is:

[0496] 3) In vivo testing in rats

[0497] Male SD rats were divided into groups of 3 and the prepared capped X-Gluc-mRNA-LNP was administered intravenously at a dose of 0.05 mg / kg (mRNA). Blood samples were collected at 0, 1, 2, 4, 6, 8, 10, 24, 32, and 48 hours. 0.3 mL of blood was collected at each time point and anticoagulated with K2EDTA / heparin sodium. The samples were placed in an ice bath and centrifuged within 30 minutes to obtain plasma, which was stored at -70°C. The fluorescence intensity was detected using a Gaussian luciferase assay kit, and the AUC was calculated to obtain the experimental test data.

[0498] The experimental detection data of some compounds are shown in Table 6.

[0499] Table 6 PK test data of some test groups in rats

[0500] As can be seen from Table 6, the samples in the test group of the present invention can express the target protein well in rats, and the cumulative expression levels of mRNA samples prepared by using most compounds as cap analogs in rats after 48 hours are higher than those in the control group, indicating that the compounds provided by the present invention as cap analogs are beneficial for increasing the expression level of mRNA in rats.

[0501] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0502] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A compound having a structure of Formula I or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof: in: R 1 Selected from C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkenyl, benzyl, R 5 Substituted C 1 -C 6 Alkyl, R 5 Substituted C 2 -C 6 Alkenyl, R 5 Substituted C 2 -C 6 Alkynyl, R 5 Substituted C 3 -C 6 Cycloalkyl, R 5 Substituted C 3 -C 6 Cycloalkenyl or R 5 substituted benzyl; R 2 Selected from H, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, cycloalkenyl, benzyl, aryl, heteroaryl, R 5 Substituted benzyl, R 5 substituted aryl, carbonylalkyl, carbonylalkoxy or sulfonamide; R 3 Selected from H, OH, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, R 5 Substituted C 1 -C 6 Alkyl, R 5 Substituted C 2 -C 6 Alkenyl, R 5 Substituted C 2 -C 6 Alkynyl, halogen or none; R 3a , R 3b Each independently selected from H, halogen, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, R 5 Substituted C 1 -C 6 Alkyl, R 5 Substituted C 2 -C 6 Alkenyl or R 5 Substituted C 2 -C 6 Alkynyl; R 9 Selected from H, halogen, C 1 -C 3 Alkyl or R 4 Substituted C 1 -C 3 alkyl; R 0 Selected from H, a fluorescent group or a steroidal structural group; X 0 Selected from O, S, NR 4 , CH 2 , C.F. 2 , CHF, CCH 2 or CCF 2 ; W is selected from H, OH, OR 4 , NR 4 R 4 , NR 4 COR 4 , F, Cl, N 3 or CN; W 2 Selected from H, OH, halogen, N 3 , CN, OR 4 , NR 4 R 4 , NR 4 COR 4 or C 1 -C 4 alkyl; L 1 Selected from O, S, NH, carbonyl or none; L 2 Selected from NH, carbonyl or none; L 3 Selected from -X-(CH 2 )nNH-,-X-(CH 2 )nNH(CH 2 )mNH-,-X-(CH 2 )nNHCO(CH 2 )mNH-, -X-(CH 2 )nCONH(CH 2 )mNH-, -X-(CH 2 )n(OC 2 H 4 )mNH-,-X-(CH 2 )n(OC 2 H 4 )mOCH 2 -, -X-(C(O)CHRaNH)n- or none; wherein X is NH, O, a five-membered heteroaryl, a six-membered heteroaryl or none; n and m are each independently an integer from 0 to 10; Ra is selected from the side chain groups in amino acids, and when Ra appears each time, it may be the same or different; Y a , Y b , Y c , Y d Each independently selected from O, S, CH 2 , CCl 2 , C.F. 2 or NH; Y 1a , Y 1b , Y 1c Each independently selected from O or S; Y 2a , Y 2b , Y 2c Each independently selected from OH, SH or BH 3 ; Y 3 , Y 4 Each independently selected from CH 2 or O; Z 1 Selected from O, OH, CH 2 , S, NR 6 , CO or SO 2 ; Z 2 , Z 3 Each independently selected from O, NR 6 , CHR 7 , CHCOOR 7 , CHCONR 7 R 7 , S, CO, SO 2 , PO(OH), PO(SH), P(O)VCO 2 H or None; Z 2 Can be used with R 3a The connected oxygen atoms form a ring; Z 4 Selected from O, CH 2 , S, NR 6 , CO, SO 2 or not; B 1 , B 2 Each independently selected from a natural or modified pyrimidine nucleotide base, a natural or modified purine nucleotide base; R 4 Selected from H, C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl or C 2 -C 8 Alkynyl; R 5 Selected from halogen, CN, SO 2 , NO 2 ,D,N 3 , C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl, C 2 -C 8 Alkynyl, C 1 -C 8 Alkoxy, OR 7 , SR 7 , NR 7 R 7 , COR 7 ,COOR 7 , OCOOR 7 ,CONR 7 R 7 , NHCOR 7 , OCONR 7 R 7 , aryl or heteroaryl; R 6 Selected from H, C 1 -C 6 Alkyl, COR 8 or SO 2 R 8 ; R 7 Selected from H, halogen, C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl or C 2 -C 8 Alkynyl; R 8 Selected from H, C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl or C 2 -C 8 Alkynyl; V = C 1-4 alkyl; n 0 An integer selected from 0 to 6; m 0 Select from 0, 1 or 2.

2. A compound having a structure of formula II or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof: Wherein R 1 , R 2 , R 3 , R 3a , R 3b , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 0 , Ra, L 1 , L 2 , L 3 ,X,X 0 , W, W 2 , Y a , Y b , Y c , Y d , Y 1a , Y 1b , Y 1c , Y 2a , Y 2b , Y 2c , Y 3 , Y 4 , B 1 , B 2 , Z 1 , Z 2 , Z 3 , Z 4 , V, n 0 、m 0 , n and m are as shown in claim 1 respectively.

3. A compound according to claim 1 or 2 or a stereoisomer thereof, a pharmaceutically acceptable salt, or a solvate thereof, Features: When R 0 When it is a fluorescent group, it is a group formed by any of the following dyes: biotin, Cy3, Cy5, Cy7, Cy5.5, DY-776, DY-751, DY-647P1, AF647, AF555, 5-FAM, 6-FAM, 6-TET, 5-SIMA, 6-JCE, ATTO 700, ATTO 680, ATTO 655, Texas Red, DEAC, AMCA, ANT, MANT, DY-480XL, DY-485XL, ATTO 425, ATTO 390, ATTO 465, ATTO495, BDP-FL, ATTO 647N, ATTO 633, ATTO Rho14, ATTO Rho13, ATTO Rho12, ATTO Rho11, ATTO Thio12, ATTO 620, ATTO Rho101, ATTO 550, ATTO Rho6G, ATTO Rho13, ATTO 532, 5 / 6-TARMA, 6-ROX, ATTO 565, ATTO 590, AF594, 5 / 6-RHOX, AF488, AF546; or; when R 0 When it is a steroidal structural group, it is a group formed by any of the following steroidal structures: cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid or glycoursodeoxycholic acid.

4. The compound according to claim 3 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: When the L 1 When L 2 is selected from NH, carbonyl or none; or, when said L 1 When NH, the L 2 is selected as carbonyl or none; or, when the L 1 When L is a carbonyl group, 2 For NH or not.

5. The compound according to claim 4 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The X is NH, O, a five-membered nitrogen-containing aromatic group, a six-membered nitrogen-containing aromatic group or none; more preferably, the X is NH, O, Or not.

6. The compound according to claim 5 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The L 3 Selected from -(CH 2 )nNH-,-(CH 2 )nNH(CH 2 )mNH-,-(CH 2 )nNHCO(CH 2 )mNH-,-(CH 2 )nCONH(CH 2 )mNH-,-(CH 2 )n(OC 2 H 4 )mNH-,-O-(CH 2 )nNH-,-O-(CH 2 )nNHCO(CH 2 )mNH-,-O-(CH 2 )nCONH(CH 2 )mNH-,-O-(CH 2 )n(OC 2 H 4 )mNH-, -NH-(CH 2 )nNH-, -NH-(CH 2 )nNHCO(CH 2 )mNH-, -NH-(CH 2 )nCONH(CH 2 )mNH-, -NH-(CH 2 )n(OC 2 H 4 )mNH-, -(CH 2 )n(OC 2 H 4 )mOCH 2 -,-O-(CH 2 )n(OC 2 H 4 )mOCH 2 -,-NH-(CH 2 )n(OC 2 H 4 )mOCH 2 -, -(C(O)CHRaNH)n- or none; wherein Ra is selected from H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , CH 2 C 6 H 5 , CH 2 C 8 NH 6 , CH 2 C 6 H 4 OH, CH 2 COOH, CH 2 CONH 2 , (CH 2 ) 2 COOH, (CH 2 ) 4 NH 2 , (CH 2 ) 2 CONH 2 , (CH 2 ) 2 SCH 3 , CH 2 OH, CH(CH 3 )OH,CH 2 SH, C 3 H 6 , CH 2 C 3 H 3 N 2 or (CH 2 ) 3 NHC(NH)NH 2 ; When Ra appears each time, it can be the same or different; wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

7. The compound according to claim 6 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: -L 1 -L 2 -L 3 -group is selected from any one of the following groups: -(CH 2 )n(OC 2 H 4 )mOCH 2 -, -NHCO(CH 2 )n(OC 2 H 4 )mOCH 2 -,-CONH(CH 2 )n(OC 2 H 4 )mOCH 2 -,-(CH 2 )nNH-,-(CH 2 )nNH(CH 2 )mNH-,-(CH 2 )nCONH(CH 2 )mNH-,-(CH 2 )nNHCO(CH 2 )mNH-, -NHCO(CH 2 )nNHCO(CH 2 )mNH-, -NHCONH(CH 2 )nNH-, -NHCOO(CH 2 )nNH-,-(CH 2 )n(OC 2 H 4 )mNH-, -NHCO(CH 2 )n(OC 2 H 4 )mNH-, -NHCONH(CH 2 )n(OC 2 H 4 )mNH-,-CONH(CH 2 )n(OC 2 H 4 )mNH-、 -NH-(C(O)CHRaNH)n- or none; wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different each time they appear.

8. The compound according to claim 7 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The R 0 When H, -L 1 -L 2 -L 3 - group is -(CH 2 )n(OC 2 H 4 )mOCH 2 -, -NHCO(CH 2 )n(OC 2 H 4 )mOCH 2 -, or -CONH(CH 2 )n(OC 2 H 4 )mOCH 2 -; wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different each time they appear; n 0 is 0, 1, 2, 3, or 4; The R 0 When it is a fluorescent group or a steroidal structural group, -L 1 -L 2 -L 3 -group is selected from any one of the following groups: -(CH 2 )nNH-,-(CH 2 )nNH(CH 2 )mNH-,-(CH 2 )nCONH(CH 2 )mNH-,-(CH 2 )nNHCO(CH 2 )mNH-, -NHCO(CH 2 )nNHCO(CH 2 )mNH-, -NHCONH(CH 2 )nNH-, -NHCOO(CH 2 )nNH-,-(CH 2 )n(OC 2 H 4 )mNH-, -NHCO(CH 2 )n(OC 2 H 4 )mNH-, -NHCONH(CH 2 )n(OC 2 H 4 )mNH-,-CONH(CH 2 )n(OC 2 H 4 )mNH-、 -NH-(C(O)CHRaNH)n- or none; wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and each time n and m occur, Can be the same or different; n 0 Is 0, 1, 2, 3, or 4.

9. The compound according to claim 6 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The R 4 Selected from H, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl or C 2 -C 4 Alkynyl; The R 5 Selected from halogen, CN, SO 2 , NO 2 ,D,N 3 , C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, C 1 -C 4 Alkoxy, OR 7 , SR 7 , NR 7 R 7 , COR 7 ,COOR 7 , OCOOR 7 ,CONR 7 R 7 , NHCOR 7 , OCONR 7 R 7 , aryl or heteroaryl; The R 6 Selected from H, C 1 -C 4 Alkyl, COR 8 or SO 2 R 8 ; The R 7 Selected from H, halogen, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl or C 2 -C 4 Alkynyl; The R 8 Selected from H, halogen, C 1 -C 4 Alkyl, C 2 -C alkenyl or C 2 -C 5 Alkynyl; The R 9 Selected from H, halogen or C 1 -C 3 alkyl.

10. The compound according to claim 9 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The Z 1 Selected from O, CH 2 , S or NH; The Z 2 , Z 3 Each independently selected from O, NH, CHR 7 , CHCOOR 7 , CHCONR 7 R 7 , S, CO, SO 2 , PO(OH), PO(SH) or none; The Z 4 Selected from O, CH 2 , S, NH or none; B 1 , B 2 Each is independently selected from: a natural or modified cytosine nucleotide base, a natural or modified uracil nucleotide base, a natural or modified adenine nucleotide base, and a natural or modified guanine nucleotide base.

11. The compound according to claim 10 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The Z 2 Selected from methylene, ethylene, CO, SO 2 , PO(OH) or none; The Z 3 Selected from O, CH 2 or NH; The Z 4 Selected from CH 2 or NH.

12. The compound according to claim 9 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The Y a , Y b , Y c , Y d All O or at most one S, CH 2 , CCl 2 , C.F. 2 or NH; The Y 1a , Y 1b , Y 1c All are O or at most one is S; The Y 2a , Y 2b , Y 2c Both are OH or at most one is SH or BH 3 ; The Y 3 , Y 4 Each independently is CH 2 .

13. The compound according to claim 9 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: The R 1 Selected from C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkenyl, benzyl, halogenated C 1 -C 4 Alkyl, haloC 2 -C 4 Alkenyl, haloC 2 -C 4 Alkynyl, haloC 3 -C 6 Cycloalkyl, haloC 3 -C 6 Cycloalkenyl or halogenated benzyl; The R 2 Selected from H, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkenyl, haloC 1 -C 4 Alkyl, haloC 2 -C 4 Alkenyl, haloC 2 -C 4 Alkynyl, haloC 3 -C 6 Cycloalkyl, haloC 3 -C 6 Cycloalkenyl, benzyl or halogenated benzyl; The R 3 Selected from H, OH, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, haloC 1 -C 4 Alkyl, haloC 2 -C 4 Alkenyl, haloC 2 -C 4 Alkynyl, halogen or none; The R 3a , R 3b Each independently selected from H, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, haloC 1 -C 4 Alkyl, haloC 2 -C 4 Alkenyl or halogenated C 2 -C 4 Alkynyl; W is selected from H, OH, OR 4 , NR 4 R 4 , F, Cl or CN; The R 4 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl or butynyl; The R 5 Selected from halogen, CN, D, N 3 , C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, OR 7 , SR 7 , NR 7 R 7 , COR 7 ,COOR 7 , pyridine, pyrimidine or morpholine; The R 7 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, ethenyl, propenyl, butenyl, ethynyl, propynyl or butynyl.

14. The compound according to claim 13 or its stereoisomer, pharmaceutically acceptable salt, or solvate, Features: W is selected from OH, F, Cl, methoxy or ethoxy; The R 1 Selected from methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, trifluoromethyl Fluoropropyl or trifluoroisopropyl; The R 2 is selected from H, methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, trifluoropropyl or trifluoroisopropyl.

15. The compound according to claim 2 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that The compound structure is shown in Formula III: Among them, the L 1 Selected from O, S, NH, carbonyl or none; The L 2 Selected from NH, carbonyl or none; The L 3 Selected from -X-(CH 2 )nNH-,-X-(CH 2 )nNH(CH 2 )mNH-,-X-(CH 2 )nNHCO(CH 2 )mNH-,-X-(CH 2 )nCONH(CH 2 )mNH-,-X-(CH 2 )n(OC 2 H 4 )mNH-,-X-(CH 2 )n(OC 2 H 4 )mOCH 2 -, -X-(C(O)CHRaNH)n- or none; wherein X is NH, O, a five-membered heteroaryl, a six-membered heteroaryl or none; n and m are each independently an integer from 0 to 10; Ra is selected from the side chain groups in amino acids, and when Ra appears each time, it may be the same or different; The R 0 Selected from H, a fluorescent group or a steroidal structural group; n 0 Selected from: integers from 0 to 6.

16. The compound according to claim 15 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that When the L 1 When L 2 is selected from imino, carbonyl or none; or, when said L 1 When L is an imino group, 2 is selected from carbonyl or none; or, when said L 1 When L is a carbonyl group, 2 Selected from imino or none.

17. The compound according to claim 16 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that The X is NH, O, a five-membered nitrogen-containing aromatic group, a six-membered nitrogen-containing aromatic group or none; more preferably, the X is NH, O, Or not.

18. The compound according to claim 17 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that The L 3 Selected from -(CH 2 )nNH-,-(CH 2 )nNH(CH 2 )mNH-,-(CH 2 )nNHCO(CH 2 )mNH-,-(CH 2 )nCONH(CH 2 )mNH-,-(CH 2 )n(OC 2 H 4 )mNH-,-O-(CH 2 )nNH-,-O-(CH 2 )nNHCO(CH 2 )mNH-,-O-(CH 2 )nCONH(CH 2 )mNH-,-O-(CH 2 )n(OC 2 H 4 )mNH-, -NH-(CH 2 )nNH-, -NH-(CH 2 )nNHCO(CH 2 )mNH-, -NH-(CH 2 )nCONH(CH 2 )mNH-, -NH-(CH 2 )n(OC 2 H 4 )mNH-, -(CH 2 )n(OC 2 H 4 )mOCH 2 -,-O-(CH 2 )n(OC 2 H 4 )mOCH 2 -,-NH-(CH 2 )n(OC 2 H 4 )mOCH 2 -, -(C(O)CHRaNH)n- or none; wherein Ra is selected from H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , CH 2 C 6 H 5 , CH 2 C 8 NH 6 , CH 2 C 6 H 4 OH, CH 2 COOH, CH 2 CONH 2 , (CH 2 ) 2 COOH, (CH 2 ) 4 NH 2 , (CH 2 ) 2 CONH 2 , (CH 2 ) 2 SCH 3 , CH 2 OH, CH(CH 3 )OH, CH 2 SH, C 3 H 6 , CH 2 C 3 H 3 N 2 or (CH 2 ) 3 NHC(NH)NH 2 ; When Ra appears each time, it can be the same or different; wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

19. The compound according to claim 18 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that -L 1 -L 2 -L 3 -group is selected from any one of the following groups: -(CH 2 )n(OC 2 H 4 )mOCH 2 -, -NHCO(CH 2 )n(OC 2 H 4 )mOCH 2 -,-CONH(CH 2 )n(OC 2 H 4 )mOCH 2 -,-(CH 2 )nNH-,-(CH 2 )nNH(CH 2 )mNH-,-(CH 2 )nCONH(CH 2 )mNH-,-(CH 2 )nNHCO(CH 2 )mNH-, -NHCO(CH 2 )nNHCO(CH 2 )mNH-, -NHCONH(CH 2 )nNH-, -NHCOO(CH 2 )nNH-,-(CH 2 )n(OC 2 H 4 )mNH-, -NHCO(CH 2 )n(OC 2 H 4 )mNH-, -NHCONH(CH 2 )n(OC 2 H 4 )mNH-,-CONH(CH 2 )n(OC 2 H 4 )mNH-、 -NH-(C(O)CHRaNH)n- or none; wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different each time they appear.

20. The compound according to claim 19 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, Features: The R 0 When H, -L 1 -L 2 -L 3 - group is -(CH 2 )n(OC 2 H 4 )mOCH 2 -, -NHCO(CH 2 )n(OC 2 H 4 )mOCH 2 -, or -CONH(CH 2 )n(OC 2 H 4 )mOCH 2 -; The R 0 When it is a fluorescent group or a steroidal structural group, -L 1 -L 2 -L 3 -group is selected from any one of the following groups: -(CH 2 )nNH-,-(CH 2 )nNH(CH 2 )mNH-,-(CH 2 )nCONH(CH 2 )mNH-,-(CH 2 )nNHCO(CH 2 )mNH-, -NHCO(CH 2 )nNHCO(CH 2 )mNH-, -NHCONH(CH 2 )nNH-, -NHCOO(CH 2 )nNH-,-(CH 2 )n(OC 2 H 4 )mNH-, -NHCO(CH 2 )n(OC 2 H 4 )mNH-, -NHCONH(CH 2 )n(OC 2 H 4 )mNH-,-CONH(CH 2 )n(OC 2 H 4 )mNH-、 -NH-(C(O)CHRaNH)n- or none; wherein n and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n and m may be the same or different each time they appear; n 0 Is 0, 1, 2, 3, or 4.

21. The compound according to claim 20 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that The compound is any of the following structures, wherein R 0 The fluorescent group or steroidal structural group as described in claim 3:

22. The compound according to claim 21 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that Wherein R 0 It is a fluorescent group formed by biotin, Cy3, Cy5, Cy7, 5-FAM or 6-FAM, or a steroidal structural group formed by cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid or glycoursodeoxycholic acid.

23. The compound according to claim 22 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that The compound is any of the following structures:

24. A compound having any of the following structures or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that Its structure is:

25. A compound having a structure of Formula IV or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof: in, R 1 Selected from C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkenyl, benzyl, R 5 Substituted C 1 -C 6 Alkyl, R 5 Substituted C 2 -C 6 Alkenyl, R 5 Substituted C 2 -C 6 Alkynyl, R 5 Substituted C 3 -C 6 Cycloalkyl, R 5 Substituted C 3 -C 6 Cycloalkenyl or R 5 substituted benzyl; R 2 Selected from H, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, cycloalkenyl, benzyl, aryl, hetero Aryl, R 5 Substituted benzyl, R 5 substituted aryl, carbonylalkyl, carbonylalkoxy or sulfonamide; W is selected from H, OH, OR 4 , NR 4 R 4 , NR 4 COR 4 , F, Cl, N 3 or CN; W 2 Selected from H, OH, halogen, N 3 , CN, OR 4 , NR 4 R 4 , NR 4 COR 4 or C 1 -C 4 alkyl; X 1 Selected from (CH 2 ) n1 , NR 4 , or no; n 1 Select from: 1, 2 or 3; X 2 Selected from O, S, NR 4 ,CO,CO 2 ,CONR 4 , NR 4 CO, NR 4 CO 2 , NR 4 CONR 4 , SO 2 , SO 2 NR 4 , CH 2 , or not; R 20 Selected from H, C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl, C 2 -C 8 Alkynyl, R 5 Substituted C 1 -C 8 Alkyl, R 5 Substituted C 2 -C 8 Alkenyl, R 5 Substituted C 2 -C 8 Alkynyl, Aryl, R 5 Substituted aryl, heteroaryl, R 5 substituted heteroaryl, halogen, CN or N3; R 12a , R 12b Each independently selected from H, halogen, alkoxy, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, R 5 Substituted C 1 -C 6 Alkyl, R 5 Substituted C 2 -C 6 Alkenyl or R 5 Substituted C 2 -C 6 Alkynyl; or R 12a , R 12b Each independently selected from OH or R 5 Substituted C 1 -C 6 Alkoxy; Y a , Y b , Y c , Y d Each independently selected from O, S, CH 2 , CCl 2 , C.F. 2 or NH; Y 1a , Y 1b , Y 1c Each independently selected from O or S; Y 2a , Y 2b , Y 2c Each independently selected from OH, SH or BH 3 ; Y 3 , Y 4 Each independently selected from CH 2 or O; Z 1 Selected from O, OH, CH 2 , S, NR 6 , CO or SO 2 ; Z 2 , Z 3 Each independently selected from O, NR 6 , CHR 7 , CHCOOR 7 , CHCONR 7 R 7 , S, CO, SO 2 , PO(OH), PO(SH), P(O)VCO 2 H or None; Z 2 Can be used with R 3a The connected oxygen atoms form a ring; Z 4 Selected from O, CH 2 , S, NR 6 , CO, SO 2 or not; B 1 , B 2 Each independently selected from a natural or modified pyrimidine nucleotide base, a natural or modified purine nucleotide base; R 4 Selected from H, C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl or C 2 -C 8 Alkynyl; R 5 Selected from halogen, CN, SO 2 , NO 2 ,D,N 3 , C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl, C 2 -C 8 Alkynyl, C 1 -C 8 Alkoxy, OR 7 , SR 7 , NR 7 R 7 , COR 7 ,COOR 7 , OCOOR 7 ,CONR 7 R 7 , NHCOR 7 , OCONR 7 R 7 , aryl or heteroaryl; R 6 Selected from H, C 1 -C 6 Alkyl, COR 8 or SO 2 R 8 ; R 7 Selected from H, halogen, C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl or C 2 -C 8 Alkynyl; R 8 Selected from H, C 1 -C 8 Alkyl, C 2 -C 8 Alkenyl or C 2 -C 8 Alkynyl; V = C 1-4 alkyl; m 0 Select from 0, 1 or 2.

26. The compound according to claim 25 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that The R 4 Selected from H, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl or C 2 -C 5 Alkynyl; The R 5 Selected from halogen, CN, SO 2 , NO 2 ,D,N 3 , C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, C 2 -C 5 Alkynyl, C 1 -C 4 Alkoxy, OR 7 , SR 7 , NR 7 R 7 , COR 7 ,COOR 7 , OCOOR 7 ,CONR 7 R 7 , NHCOR 7 , OCONR 7 R 7 , aryl or heteroaryl; The R 6 Selected from H, C 1 -C 4 Alkyl, COR 8 or SO 2 R 8 ; The R 7 Selected from H, halogen, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl or C 2 -C 5 Alkynyl; The R 8 Selected from H, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl or C 2 -C 5 Alkynyl; More preferably, the R 5 Selected from halogen, CN, SO 2 , NO 2 ,D,N 3 , C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, C 2 -C 5 Alkynyl, C 1 -C 4 Alkoxy, pyridine, pyrimidine or morpholine.

27. The compound according to claim 26 or its stereoisomer, pharmaceutically acceptable salt, or solvate, It is characterized in that The W is selected from H, OH, C 1 -C 4 Alkoxy, C 1 -C 4 Alkylamino, F, Cl, N 3 or CN; The W 2 Selected from H, OH, halogen, N 3 , CN, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy or C 1 -C 4 Alkylamino; The X 1 Selected from (CH 2 ) n1 , C 1 -C 4 Alkyl substituted amino or none; n 1 Select from: 1, 2 or 3; The X 2 Selected from O, S, CO, CO 2 ,CONR 4 , NR 4 CO, NR 4 CO 2 , NR 4 CONR 4 , SO 2 or SO 2 NR 4 ; The R 12a , R 12b Each independently selected from OH, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, R 5 Substituted C 1 -C 6 Alkyl or R 5 Substituted C 1 -C 6 Alkoxy; The R 20 Selected from H, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, R 5 Replace C 1 -C 4 Alkyl, R 5 Substituted aryl, heteroaryl, N 3 Or not.

28. The compound according to claim 26 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The R 12a , R 12b Each independently is OH, F, Cl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Haloalkyl or C 1 -C 4 Haloalkoxy; W is OH; The Y a , Y b , Y c , Y d Each independently selected from O or CH 2 ; The Y 1a , Y 1b , Y 1c Each independently selected from O or S; The Y 2a , Y 2b , Y 2c are each independently selected from OH; The Y 3 , Y 4 Independently selected from CH 2 or O; The Z 1 Selected from O, OH, CH 2 , S or NH; The Z 2 , Z 3 Each independently selected from O, NH, CHR 7 , CHCOOR 7 , CO, SO 2 or PO(OH); B 1 , B 2 Each independently selected from a natural or modified pyrimidine nucleotide base, a natural or modified purine nucleotide base; The R 4 Select from H or C 1 -C 4 alkyl; The R 5 Selected from halogen, OR 7 , NR 7 R 7 ,CONR 7 R 7 OCONR 7 R 7 ; The R 6 Selected from H; The R 7 Select from H or C 1 -C 4 alkyl; The m 0 Select from 0, 1 or 2.

29. The compound according to claim 28 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The R 1 Selected from C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkenyl, benzyl, halogenated C 1 -C 4 Alkyl, haloC 2 -C 4 Alkenyl, haloC 2 -C 4 Alkynyl, haloC 3 -C 6 Cycloalkyl, haloC 3 -C 6 Cycloalkenyl or halogenated benzyl; The R 2 Selected from H, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, C 3 -C 6 Cycloalkyl, C 3 -C 6 Cycloalkenyl, haloC 1 -C 4 Alkyl, haloC 2 -C 4 Alkenyl, haloC 2 -C 4 Alkynyl, haloC 3 -C 6 Cycloalkyl, haloC 3 -C 6 Cycloalkenyl, benzyl or halogenated benzyl.

30. The compound according to claim 27 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The compound structure is shown in any one of the general formulas IV-2, IV-3, IV-4, IV-5 or IV-6: Where R 1 , R 2 , R 20 , R 12a , R 12b , R 4 , R 5 , R 6 , R 7 , R 8 , W, W 2 , Y a , Y b , Y c , Y d , Y 1a , Y 1b , Y 1c , Y 2a , Y 2b , Y 2c , Y 3 , Y 4 , B 1 , B 2 , Z 1 , Z 2 , Z 3 , B 1 , B 2 , V, n 1 and m 0 As shown in claim 30 respectively.

31. The compound according to claim 30 or its stereoisomer, pharmaceutically acceptable salt, or solvate, characterized in that R 1 is selected from methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl, butynyl, benzyl, halomethyl, haloethyl, halo-n-propyl, haloisopropyl, halobutyl, halovinyl, halopropenyl, halobutenyl, haloethynyl, halopropynyl, halobutynyl or halobenzyl; The R 2 is selected from H, methyl, ethyl, n-propyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, trifluoropropyl or trifluoroisopropyl; W is selected from OH, F, Cl, methyl, ethyl, methoxy or ethoxy; The W 2 Selected from H, OH, F, Cl, N 3 , CN, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, methyl substituted amino or ethyl substituted amino; The R 4 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl or butynyl; The R 20 Selected from H, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, R 5 Replace C 1 -C 4 Alkyl, R 5 Substituted aryl, heteroaryl, N 3 or not; The R 5 Selected from halogen, CN, D, N 3 , C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, C 2 -C 5 Alkynyl, C 1 -C 4 Alkoxy, pyridine, pyrimidine or morpholine.

32. A compound according to claim 31 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The R 12a , R 12b Each independently is OH, F, Cl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Haloalkyl or C 1 -C 4 Haloalkoxy; W is OH; The Z 1 Selected from O, OH, CH 2 , S or NH; The Z 2 , Z 3 Each independently selected from O, NH, CH 2 , CHCOO, CO, SO 2 or PO(OH); B 1 , B 2 Independently selected from: natural or modified cytosine nucleotide base, natural or modified uracil nucleotide base, natural or modified adenine nucleotide base, natural or modified guanine nucleotide base; The m 0 Select from 0, 1 or 2.

33. The compound according to claim 30 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The compound structure is shown in Formula V: Among them, the W 2 Selected from H, OH, halogen, N 3 , CN, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy or C 1 -C 4 Alkylamino; The X 1 Selected from (CH 2 ) n1 , C 1 -C 4 Alkyl substituted amino or none; n 1 Select from: 1, 2 or 3; The X 2 Selected from O, S, CO, CO 2 ,CONR 4 , NR 4 CO, NR 4 CO 2 , NR 4 CONR 4 , SO 2 or SO 2 NR 4 ; The R 20 Selected from H, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, R 5 Replace C 1 -C 4 Alkyl, R 5 Substituted aryl, heteroaryl, N 3 or not; The R 4 Selected from H, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl or C 2 -C 5 Alkynyl; The R 5 Selected from halogen, CN, SO 2 , NO 2 ,D,N 3 , C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, C 2 -C 5 Alkynyl, C 1 -C 4 Alkoxy, OR 7 , SR 7 , NR 7 R 7 , COR 7 ,COOR 7 , OCOOR 7 ,CONR 7 R 7 , NHCOR 7 , OCONR 7 R 7 , aryl or heteroaryl; The R 7 Selected from H, halogen, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl or C 2 -C 5 Alkynyl.

34. A compound according to claim 33 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The compound structure is shown in Formula V-2, V-3, V-4, V-5 or V-6: Wherein W 2 Selected from H, OH, F, Cl, N 3 , CN, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, methyl substituted amino or ethyl substituted amino; The R 4 is selected from H, methyl, ethyl, n-propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl or butynyl; The R 20 Selected from H, C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, R 5 Replace C 1 -C 4 Alkyl, R 5 Substituted aryl, heteroaryl, N 3 or not; The R 5 Selected from halogen, CN, D, N 3 , C 1 -C 4 Alkyl, C 2 -C 5 Alkenyl, C 2 -C 5 Alkynyl, C 1 -C 4 Alkoxy, pyridine, pyrimidine or morpholine.

35. The compound according to claim 25 or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The compound is represented by any of the following structures:

36. A compound for RNA capping or a stereoisomer, a pharmaceutically acceptable salt, or a solvate thereof, It is characterized in that The compound is represented by any of the following structures:

37. Use of the compound of any one of claims 1-24 or 25-36 as an in vitro co-transcribed RNA capping agent. 38.An RNA molecule, It is characterized in that The method comprises the compound according to any one of claims 1 to 24 or 25 to 36 as a cap structure or a cap structure fragment.

39. A pharmaceutical composition, It is characterized in that Comprising the RNA molecule of claim 38, and a pharmaceutically acceptable carrier.

40. A method for synthesizing RNA molecules, It is characterized in that The following steps are involved: The compound according to any one of claims 1-24 or 25-36 is co-incubated with a polynucleotide template to perform template transcription.

41. A capped RNA transcription reaction system, It is characterized in that include: A polynucleotide template, a compound according to any one of claims 1-24 or 25-36, NTPs and RNA polymerase.

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