Oligonucleotide-modified compound, and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a modified oligonucleotide compound and a preparation method and application thereof. The invention has the following advantages: the invention provides the compound for modifying the oligonucleotide, and the compound can improve the drug effect of the oligonucleotide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a modified oligonucleotide compound, a preparation method and application thereof. Background Art

[0002] Oligonucleotides are deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and are generally composed of 15 to 50 oligonucleotides. Natural oligonucleotides are easily degraded in the body and have low specificity. As a new generation of gene drugs, synthetic oligonucleotides avoid the shortcomings of natural oligonucleotides and can directly regulate gene expression, thereby playing a therapeutic role in diseases.

[0003] Currently available oligonucleotide drugs mainly use chemical modifications to improve the metabolic stability of the oligonucleotide structure and reduce adverse reactions, such as modifications to the phosphodiester bond, ribose, oligonucleotide bases, or ribose-phosphate backbone.

[0004] Oligonucleotide compounds have important therapeutic applications in medicine. Oligonucleotides can be used to silence genes that cause specific diseases. Gene silencing prevents the formation of proteins by inhibiting translation. Importantly, gene silencers are promising alternatives to traditional small organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleotides that prevent protein formation through gene silencing.

[0005] Traditional siRNAs are often less than ideal in efficacy. Many modified siRNA compounds have been developed for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutics for the treatment of various diseases, including central nervous system diseases, inflammatory diseases, metabolic disorders, tumors, infectious diseases, and eye diseases.

[0006] The present disclosure aims to provide a compound for modifying oligonucleotides, a preparation method and application thereof, and the compound can improve the efficacy of oligonucleotides. Summary of the invention

[0007] The technical problem to be solved by the present disclosure is that the efficacy of traditional siRNA in the current market is often not ideal, and a compound of modified oligonucleotides, a preparation method and application thereof are developed, and the compound can improve the efficacy of oligonucleotides.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present disclosure is: In one aspect, the present disclosure provides a modified oligonucleotide compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, which has a structure shown in the following formula (I): (I) in, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3, -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl; R2 is selected from or ; nz is an integer from 0 to 10; R7 and R8 are each independently selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl; R9 is -(CH2) nx CN; nx is an integer from 0 to 10; ny is an integer from 0 to 10.

[0009] In some preferred embodiments of the present disclosure, the aforementioned compound or its pharmaceutically acceptable salt, isomer, solvate or prodrug has a structure shown in the following formula (II): (Ⅱ) Wherein, R1 and R2 are respectively defined as in formula (I).

[0010] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3, -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-10 Alkyl, C 2-10 Alkenyl or C 2-10 Alkynyl.

[0011] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3, -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-5 Alkyl, C 2-5 Alkenyl or C 2-5 Alkynyl.

[0012] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3, -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-3 Alkyl, C 2-3 Alkenyl or C 2-3 Alkynyl.

[0013] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3, -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-10 alkyl.

[0014] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3, -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-5 alkyl.

[0015] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3, -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-3 alkyl.

[0016] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOR6; R6 is C 1-3 alkyl.

[0017] In some preferred embodiments of the present disclosure, R1 is selected from H, F, -OMe, -HNCOCH3.

[0018] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 8.

[0019] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 6.

[0020] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 5.

[0021] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 4.

[0022] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 2.

[0023] In some preferred embodiments of the present disclosure, nz is 1 or 2.

[0024] In some preferred embodiments of the present disclosure, nz is 2.

[0025] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 8.

[0026] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 6.

[0027] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 5.

[0028] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 4.

[0029] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 2.

[0030] In some preferred embodiments of the present disclosure, nx is 1 or 2.

[0031] In some preferred embodiments of the present disclosure, nx is 2.

[0032] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 8.

[0033] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 6.

[0034] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 5.

[0035] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 4.

[0036] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 2.

[0037] In some preferred embodiments of the present disclosure, ny is 1 or 2.

[0038] In some preferred embodiments of the present disclosure, ny is 1.

[0039] In some preferred embodiments of the present disclosure, R7 and R8 are each independently selected from C 1-10 Alkyl, C 2-10 Alkenyl or C 2-10 Alkynyl.

[0040] In some preferred embodiments of the present disclosure, R7 and R8 are each independently selected from C 1-5 Alkyl, C 2-5 Alkenyl or C 2-5 Alkynyl.

[0041] In some preferred embodiments of the present disclosure, R7 and R8 are each independently selected from C 1-3 Alkyl, C 2-3 Alkenyl or C 2-3 Alkynyl.

[0042] In some preferred embodiments of the present disclosure, R7 and R8 are each independently selected from C 1-5 alkyl.

[0043] In some preferred embodiments of the present disclosure, R7 and R8 are each independently selected from C 1-3 alkyl.

[0044] In some preferred embodiments of the present disclosure, R7 and R8 are each independently selected from methyl, ethyl, propyl or isopropyl.

[0045] In some preferred embodiments of the present disclosure, R7 and R8 are both isopropyl.

[0046] In some preferred embodiments of the present disclosure, the compound is denoted as vmap, and the compound is selected from the following compounds: .

[0047] On the other hand, the present disclosure provides a method for preparing the aforementioned compound, wherein the compound has the structural formula of formula (I), which comprises reacting compound M8 with compound M9 to prepare the compound: .

[0048] In some preferred embodiments of the present disclosure, according to the preparation method described in the present disclosure, compound M7 is prepared by reacting compound M5 with compound M6, and compound M8 is prepared by reacting compound M7: .

[0049] In some preferred embodiments of the present disclosure, according to the preparation method described in the present disclosure, compound M5 is prepared by reacting compound M4: .

[0050] In some preferred embodiments of the present disclosure, according to the preparation method described in the present disclosure, compound M3 is prepared by reacting compound M1 with compound M2, and compound M4 is prepared by reacting compound M3: .

[0051] In some preferred embodiments of the present disclosure, according to the preparation method described in the present disclosure, compound M3 is prepared by reacting compound M1 with compound M2, compound M3 is reacted to prepare compound M4, and compound M4 is reacted to prepare compound M5: .

[0052] In another aspect, the present disclosure provides use of the aforementioned compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in preparing an oligonucleotide sequence.

[0053] In another aspect, the present disclosure provides use of the aforementioned compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in the preparation of siRNA or miRNA.

[0054] In another aspect, the present disclosure provides use of the aforementioned compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in preparing siRNA.

[0055] In another aspect, the present disclosure provides an oligonucleotide sequence prepared from the aforementioned compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof.

[0056] In another aspect, the present disclosure provides an oligonucleotide sequence prepared from the aforementioned compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof by an oligonucleotide synthesizer.

[0057] On the other hand, the present disclosure provides an oligonucleotide sequence, which is prepared by an oligonucleotide synthesizer from the above-mentioned compound or its pharmaceutically acceptable salt, isomer, solvate or prodrug; the compound or its pharmaceutically acceptable salt, isomer, solvate or prodrug is located at the 5' end or middle position of the oligonucleotide.

[0058] In some preferred embodiments of the present disclosure, according to the oligonucleotide sequence described in the present disclosure, wherein the aforementioned compound or its pharmaceutically acceptable salt, isomer, solvate or prodrug is located at the 5' end of the oligonucleotide and has a structure shown in formula (III): (III) Wherein, R1 and R2 are respectively defined as in formula (I); X is selected from O, S; The middle wavy lines indicate oligonucleotides; Indicates that O is linked to the oligonucleotide.

[0059] In another aspect, the present disclosure provides an oligonucleotide delivery system, comprising the oligonucleotide sequence as described above.

[0060] In another aspect, the present disclosure provides a siRNA or miRNA delivery system, wherein the siRNA or miRNA delivery system comprises the oligonucleotide sequence as described above.

[0061] In another aspect, the present disclosure provides a siRNA delivery system, comprising the oligonucleotide sequence as described above.

[0062] On the other hand, the present disclosure provides a method for preparing an oligonucleotide delivery system, which comprises the following steps: preparing an oligonucleotide sequence by an oligonucleotide synthesizer using the aforementioned compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof.

[0063] On the other hand, the present disclosure provides a pharmaceutical composition comprising an oligonucleotide sequence prepared by an oligonucleotide synthesizer using the aforementioned compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof and / or the aforementioned oligonucleotide sequence.

[0064] In another aspect, the present disclosure provides an oligonucleotide sequence as described above, an oligonucleotide delivery system as described above, an siRNA or miRNA delivery system as described above, an siRNA delivery system as described above, or a pharmaceutical composition as described above for use in treatment.

[0065] The present disclosure has the following advantages: (1) The present disclosure provides a compound for modifying an oligonucleotide, which can improve the drug efficacy of the oligonucleotide.

[0066] (2) The present disclosure provides nucleotides modified with a phosphoester backbone, which have the effect of enhancing the activity of double-stranded oligonucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. The drawings herein are incorporated into the specification and constitute a part of the specification, showing embodiments that conform to the specification, and are used together with the specification to explain the principles of the specification.

[0068] Figure 1 This is the HNMR spectrum of compound vmap-001.

[0069] Figure 2 This is the PNMR spectrum of compound vmap-001. DETAILED DESCRIPTION

[0070] Definition and Description In order to make it easier to understand the present disclosure, some technical and scientific terms are specifically defined below. In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell and tissue culture, microbiology-related terms and laboratory operation steps used herein are terms and routine steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definition and explanation of the relevant terms are provided below. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in the present application are only for describing specific embodiments and are not intended to be limited.

[0071] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0072] As used herein, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or apparatus comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or apparatuses.

[0073] In the description herein, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0074] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0075] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0076] As used herein, the term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include variants of deuterium and hydrogen, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo group (i.e., =O), it means that two hydrogen atoms are replaced. The term "optionally substituted" or "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of the substituent can be arbitrary on the basis of chemically achievable.

[0077] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and / or variants thereof are permitted only if such combinations result in stable compounds.

[0078] In any embodiment, any or all hydrogens present in the compound, or hydrogens in a specific group or moiety within the compound, may be replaced by deuterium or tritium. From one to the maximum number of hydrogens present in the compound may be replaced by deuterium. From one to the maximum number of hydrogens present in any group in the general compound or in the specific compound may be deuterated. For example, when describing a group as ethyl, the ethyl group may be C2H5 or C2H5 in which x (1 to 5) hydrogens are replaced by deuterium, such as C2D x H 5-x When describing a group as a deuterated ethyl group, the deuterated ethyl group may be C2H5 in which x (1 to 5) hydrogen atoms are replaced by deuterium, for example C2D x H 5-x The stable deuterated derivatives disclosed herein are preferably stable deuterated isotope derivatives obtained by replacing any deuterated hydrogen atom in each formula with 1 to a maximum number (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, etc.) of deuterium atoms.

[0079] The present disclosure mentions the compound of formula (I), which also includes its tautomers, stereoisomers, mixtures of stereoisomers, solvates or derivatives, etc.

[0080] The present disclosure "compounds" also include tautomeric forms. Tautomeric forms are derived from the exchange of a single bond with an adjacent double bond and the migration of a proton. The term "tautomer" or "tautomeric form" means that at room temperature, different functional group isomers are in dynamic equilibrium and can quickly convert to each other. It refers to one of two or more structural isomers that exist in equilibrium and are easily converted from one isomeric form to another isomeric form. This transformation results in the formal migration of hydrogen atoms and is accompanied by the conversion of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomeric groups in solution. In solutions where tautomerism may occur, a chemical equilibrium of tautomers will be reached. The exact ratio of tautomers depends on several factors, including temperature, solvent and pH conditions. The concept of tautomers that can be converted to each other by tautomerization is called tautomerism.

[0081] When the present specification describes a compound that is susceptible to tautomerism, but only one of the tautomers is described, it should be understood that all tautomers are included as part of the chemical meaning described. It should be understood that when a compound has a tautomeric form, it is intended to include all tautomeric forms, and the naming of the compound does not exclude any tautomeric form.

[0082] Of the various types of tautomerism possible, two are commonly observed. In keto-enol tautomerism, both electrons and hydrogen atoms shift simultaneously.

[0083] Common tautomeric pairs are: keto-enol, amide-nitrile, lactam-lactam, amide-imidic acid tautomerism in heterocycles, imine-enamine, and enamine-enamine.

[0084] The term "isomer" refers to different compounds with the same molecular formula but different atomic arrangements and configurations. Depending on their structure, the compounds of the present disclosure may exist in different stereoisomeric forms. These forms include configurational isomers or optical conformers (enantiomers and / or diastereomers, including those of atropisomers). Therefore, the present disclosure includes enantiomers, diastereomers and mixtures thereof. The present disclosure further includes all mixtures of the above stereoisomers, regardless of the ratio, including racemates.

[0085] Depending on their structure, the compounds of the present disclosure may exist in various stable isotopic forms. These forms include those in which one or more hydrogen atoms have been replaced by deuterium atoms, those in which one or more nitrogen atoms have been replaced by 15N atoms, or those in which one or more carbon, fluorine, chlorine, bromine, sulfur or oxygen have been replaced by stable isotopes of the respective original atoms.

[0086] Some compounds and salts according to the present disclosure may exist in different crystalline forms (polymorphs), which are within the scope of the present disclosure.

[0087] In this disclosure, the term “ "and" "Denotes the absolute configuration of a stereocenter." " ” refers to the chemical bond connection.

[0088] When the ring appears ", and the connection position is uncertain, it means that the connection site is located at " "Any atom on the single ring where it is located, as long as the atomic valence permits.

[0089] The term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and can be activated or converted to a more active parent form by enzyme action. The prodrugs disclosed herein include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs that can be converted into more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derived as prodrug forms for use in the present disclosure include, but are not limited to, those chemotherapeutic agents described above.

[0090] The term "alkyl" refers to a chain (straight or branched) saturated aliphatic hydrocarbon group. The term "alkyl" may be a straight or branched chain alkyl group (C 1-10 Alkyl), preferably an alkyl group containing 1 to 6 carbon atoms (C 1-6 Alkyl). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched chain isomers thereof. More preferred are lower alkyl (C 2-4-16-17) groups containing 1 to 3 carbon atoms. 1-3 Alkyl), non-limiting examples include methyl, ethyl, n-propyl, isopropyl, etc. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the groups described in the present application.

[0091] In one embodiment, the substituents are independently selected from oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including linear, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)2alkyl, -C(=O)NH(substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl), -C(=O)N(H or alkyl)2, -OC(=O)N(substituted or unsubstituted alkyl)2, NHC(=O)NH(substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl), -NHC(=O)alkyl, -N(substituted or unsubstituted alkyl)C(=O)(substituted or unsubstituted alkyl), -NHC(=O)(substituted or unsubstituted alkyl), -C(OH)(substituted or unsubstituted alkyl)2 and -C(NH2)(substituted or unsubstituted alkyl)2. In another embodiment, for example, the optional substituents are selected from oxo, fluoro, chloro, bromo, iodo, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, -OCH2CH3, -OCH(CH3)2, -OCF3, -OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, -NHC(=O)NHCH3, -C(=O)CH3, -ON(O)2, and C(=O)OH. In yet another embodiment, the substituents are independently selected from C 1-6 Alkyl, -OH, C 1-6 In yet another embodiment, the substituents are independently selected from C 1-6 Alkyl, C 1-6 Alkoxy, halogen, acetylamino and nitro. As used herein, when the substituent is an alkyl or alkoxy group, the carbon chain may be branched, linear or cyclic.

[0092] The term "alkylene" refers to a divalent group formed by removing another hydrogen of an alkyl group, which may be substituted or unsubstituted. The "alkylene" is preferably a divalent group of a straight or branched chain saturated aliphatic hydrocarbon containing 1 to 8 carbon atoms, more preferably a divalent alkyl group (C 2-6) containing 1 to 6 carbon atoms. 1-6 Examples of alkylene include, but are not limited to, -CH2-, -CH(CH3)-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH2CH2-, or -(CH2)4- and stereoisomers thereof.

[0093] The term "heteroatom" is selected from nitrogen, oxygen or sulfur. Among them, the nitrogen can be optionally substituted; the sulfur can also be optionally substituted, for example, oxo, that is, forming S(O) t3 (where t3 is an integer from 0 to 2).

[0094] The term "aryl" means phenyl or naphthyl, or phenyl or naphthyl substituted by halogen, C 1-8 Alkyl, hydroxyl, nitro, trifluoromethyl, etc. Preferred is phenyl or monosubstituted phenyl; most preferred is phenyl.

[0095] The "solvate" mentioned in the present disclosure refers to a complex formed by the compound of the present disclosure and a solvent. They either react in a solvent or precipitate or crystallize from a solvent. For example, a complex formed with water is called a "hydrate". Solvates of the compound represented by formula (I) of the present disclosure belong to the scope of the present disclosure.

[0096] The present disclosure includes prodrugs of the above compounds. Prodrugs include known amino protecting groups and carboxyl protecting groups, which are hydrolyzed under physiological conditions or released via enzyme reactions to obtain parent compounds. Specific prodrug preparation methods can refer to (Saulnier, MG; Frennesson, DB; Deshpande, MS; Hansel, SB and Vysa, DM Bioorg. Med. Chem Lett. 1994, 4, 1985-1990; and Greenwald, RB; Choe, YH; Conover, CD; Shum, K.; Wu, D.; Royzen, MJ Med. Chem. 2000, 43, 475.).

[0097] As used herein, the term "hydroxy" refers to -OH.

[0098] As used herein, the term "oxo" refers to =0.

[0099] As used herein, the term "carboxy" refers to -C(=0)OH.

[0100] As used herein, the term "acetyl (Ac)" refers to -COCH3.

[0101] As used herein, the term "DMTr" refers to 4,4'-dimethoxytrityl.

[0102] As used herein, the term "Ph" refers to phenyl, ie, C6H5-.

[0103] As used herein, the term "TBSO" refers to tert-butyldimethylsilyloxy, which has the chemical formula (CH3)3C-Si(CH3)2-O-.

[0104] As used herein, the term "t-BuOK" refers to potassium tert-butoxide.

[0105] Example In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. The following is only a further description of the present disclosure, and the protection scope of the present disclosure is not limited thereto.

[0106] Example 1 Preparation of compound vmap-001: Preparation of compound 3: Compound 1 (20 g, 27.9 mmol), compound 2 (8.7 g, 41.8 mmol), and Cs2CO3 (27.2 g, 83.4 mmol) were dissolved in 300 mL (1,4-dioxane / water = 10:1), deoxygenated, and compound Pd(dppf)Cl2 (4.5 g, 5.5 mmol) was added under nitrogen protection. The reaction was carried out at 80°C for 12 hours. TLC (PE:EA=2:1) ​​showed that the reaction was completed. The reaction solution was treated with ethyl acetate and water, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was purified by column chromatography (PE:EA=2:1) ​​to obtain an off-white solid compound 3 (18.2 g, yield 77%).

[0107] 1HNMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 8.65 (m, 1H), 8.58 (d, 1H), 8.33 (d, 1H) , 7.52-7.45 (m, 3H), 7.39-7.31 (m, 5H), 7.28-7.17 (m, 3H), 6.88-6.80 (m, 4H), 6.28-6.20 (m, 1H), 4.55-4.49 (d, 1H), 4.47-4.41 (d, 2H), 4.35(m, 1H), 3.89-3.78 (m, 6H), 3.61-3.58 (m, 1H), 3.55-3.50 (S, 3H), 3.43-3.39(m, 3H), 2.91-2.88 (m, 2H), 2.68-2.61 (m, 2H), 0.91-0.85 (s, 9H), 0.09 (s,6H). Preparation of compound 4: Compound 3 (18 g, 21 mmol) was dissolved in 300 mL of dichloromethane solution containing 3% dichloroacetic acid at 0-5°C, and reacted at 0-5°C for 1 hour. TLC (PE: EA = 1: 1) showed that the reaction was complete. The temperature was controlled, and the pH was adjusted to 8 with a saturated sodium bicarbonate solution. The organic phase was collected, treated with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by column chromatography (PE: EA = 2: 1) to obtain a light yellow solid compound 4 (10.3 g, yield 89%).

[0108] Preparation of compound 5: Compound 4 (15 g, 27.6 mmol) and IBX (15.4 g, 53.8 mmol) were added to 200 mL of acetonitrile and reacted at 80 °C for 1.5 hr. TLC (PE: EA = 2:1) showed that the reaction was completed. The reaction solution was cooled, filtered, and concentrated. The crude product was purified by column chromatography (PE: EA = 2:1) to obtain compound 5 as a light yellow solid (10.4 g, yield 69.6%).

[0109] Preparation of compound 7: Under nitrogen protection, compound 6 (28.5 g, 45.1 mmol) was dissolved in 120 ml of anhydrous THF, the temperature was controlled at 0°C, t-BuOK (5.1 g, 45.1 mmol) was added in batches, and then 80 ml of anhydrous THF solution containing compound 5 (10 g, 18 mmol) was added dropwise to the above reaction solution. After the addition was complete, the reaction was continued for 6 hours. TLC (PE: EA = 1: 1) showed completion. The reaction solution was quenched with saturated ammonium chloride solution, extracted twice with ethyl acetate, and the organic phase was collected. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (PE: EA = 1: 1) to obtain an off-white solid compound 7 (8.3 g, yield 52%).

[0110] Preparation of compound 8: Compound 7 (8 g, 9.1 mmol) was dissolved in 100 ml of HCOOH:H2O=1:1 (V:V) solution and reacted for 56 hours under nitrogen protection. 500 mL of water was added to the reaction system and freeze-dried to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH=10:1) to obtain a light yellow solid compound 8 (3.8 g, yield, yield 55.9%).

[0111] Preparation of compound vmap-001: Under nitrogen protection, compound 8 (3 g, 4 mmol) was dissolved in 30 mL of anhydrous DCM, and compound 9 (1.8 g, 6 mmol) and DCI (0.7 g, 6 mmol) were added. The reaction was continued for 2 hours. TLC (PE:EA=1:2) showed that the reaction was completed. The reaction was also concentrated in vacuo at 20°C. The crude product was purified by rapid column, and then purified by slurrying with anhydrous dichloromethane:n-hexane=1:9, filtered, and the solvent was removed in vacuo to obtain an off-white solid compound vmap-001 (2.3 g, yield 60%).

[0112] 1HNMR (400 MHz, CDCl3) δ 8.95 (s, 1H), 8.62-8.55 (m, 2H), 8.21-8.20(d, 1H), 7.45-7.41 (m, 1H), 7.34-7.31 (m, 1H), 7.12-6.91 (m, 1H), 6.17-6.13(m, 2H), 5.65-5.59 (m, 4H), 4.81-4.67 (m, 2H), 4.61-4.52 (m, 1H), 3.87-3.62(m, 7H), 3.43-3.41 (m, 3H), 3.06-3.02(m, 2H), 2.69-2.64 (m, 4H), 1.21-1.10(m, 30H). 31P NMR (162Mhz, CDCl3) δ 151.86, 151.05, 17.94, 17.67. The HNMR spectrum of compound vmap-001 is shown in Figure 1 .

[0113] The PNMR spectrum of compound vmap-001 is shown in Figure 2 .

[0114] The compound of the present disclosure is prepared by an oligonucleotide synthesizer to produce an oligonucleotide sequence containing the compound of the present disclosure, and the above structure can be located at the 5' end or the middle position of the oligonucleotide, such as coupled to the 5' end or the middle position of the siRNA antisense strand by a phosphate bond or a phosphorothioate bond.

[0115] The abbreviations of nucleotide monomers or modified nucleotide monomers used in the nucleic acid sequence representation shown in Table 1 will be understood that these monomers, when present in an oligonucleotide, are interconnected by 5'-3' phosphodiester bonds unless otherwise specified. It will also be understood that when a nucleotide contains a 2'-fluorine modification, the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2'-fluorine nucleotide).

[0116] Table 1. Nucleotide monomer abbreviations used in nucleic acid sequence representation L96 is a well-known GalNAc delivery vector in the art. It achieves targeted delivery of nucleic acid drugs by binding to the ASGPR (asialoglycoprotein receptor) in the liver, thereby increasing the concentration and efficacy of the drug in the liver. The structure of L96 is as follows: In the absence of specific information on the source of reagents, such reagents can be obtained from any molecular biology reagent supplier at quality / purity standards appropriate for molecular biology.

[0117] Example 2 Preparation of siRNA: The siRNA sequence is synthesized separately on a solid support via a sense strand (SS) and an antisense strand (AS), and is obtained after deprotection, cleavage, purification, annealing, purification, and lyophilization.

[0118] Solid phase synthesis: The sense strand and antisense strand are synthesized separately on a solid support using an oligonucleotide automatic synthesizer using phosphoramidite technology. The synthesizers include AKTA Oligopilot (Cytiva) and Dr.Oligo 192XLc (Kunshan Berleke Precision Instrument Co., Ltd.). Solid phase synthesis starts from the 3' end of the sequence and couples the monomers into the sequence in sequence. Each coupling of a phosphoramidite monomer includes four chemical steps: 1) unblocking or deprotection (dehydroxyl protecting group); 2) coupling; 3) oxidation; 4) end-capping. The phosphoramidite monomers, reagents, and purification consumables used are commercial circulating reagents and consumables, such as various phosphoramidite monomers purchased from Shanghai Zhaowei Technology Development Co., Ltd. (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite), and reaction reagents purchased from Sigma-Aldrich LLC (such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.). The siRNA synthesis and purification methods used in this article are described in US20130178612A1, US2015100197A1, etc., the contents of which are incorporated herein by reference; the synthesis method of the sequence containing the vmap structure is described in J. Med. Chem. 2018, 61, 734-744, the contents of which are incorporated herein by reference.

[0119] (1) Synthesis of the positive chain The solid phase phosphoramidite method is a mature oligonucleotide synthesis method. The reaction is carried out in a stainless steel synthesis column using a computer-controlled synthesizer. In the synthesis of the positive chain, a solid phase support loaded with a targeting ligand (such as L96) is used as the starting point, or the solid phase support is used directly as the starting point. Different raw materials, reagents and solvents are injected in the order of sequence 3' to 5' through different pipelines controlled by the solid phase synthesizer, and phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four cycles of DMT protection group removal reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 19 or 21 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and the synthesized sequence is cut from the solid phase support by aminolysis reaction, filtered, the filter cake is washed with ethanol, the filtrate and washing liquid are collected, and concentrated to obtain the crude positive chain. The crude product is purified by chromatography (SOURCE 15Q) and freeze-dried to obtain the target product positive chain.

[0120] (2) Synthesis of antisense strand The synthesis of the antisense strand is similar to that of the sense strand. Different raw materials, reagents and solvents are injected into different pipelines in the order of 3' to 5' of the sequence through the solid phase synthesizer to connect the phosphoramidite nucleoside monomers one by one. The reaction process includes four cycles of DMT protection group removal reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After the synthesis is completed, the protective group (2-cyanoethyl) is removed on the solid phase synthesis column, and then the synthesized sequence is cut from the solid phase support by aminolysis reaction, filtered, and the filter cake is washed with ethanol. The filtrate and washing liquid are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and dried or freeze-dried to obtain the target product antisense strand siRNA.

[0121] (3) Preparation of double-stranded siRNA Dissolve the sense strand and antisense strand in injection water, mix them in a certain ratio (1.01:1.0~1.2:1.0), incubate at 30-50℃ for 30-90 min, cool to room temperature, and freeze-dry to obtain double-stranded siRNA products.

[0122] Example 3 The effect of the structure described in this case was examined using a sequence having an INHBE gene inhibitory effect as a tool sequence.

[0123] The siRNA sequence was designed based on the mRNA sequence of the INHBE (inhibin subunit beta E) gene. The siRNA modified sequence (as shown in Table 2) was complementary to human INHBE mRNA (Genbank accession number: NM_031479.5) or had one mismatch at the 3' end of the sense strand, or was complementary to cynomolgus monkey INHBE mRNA (Genbank accession number: XM_005571319.4) or had no more than two mismatches. The INHBE-siRNA modified sequence was prepared according to the method of Example 2.

[0124] Table 2. INHBE-siRNA modified sequences In vitro activity assay (1) Cell culture and transfection: Hep3B cells (Wuhan Pronocell Life Science Co., Ltd., Catalog No. CL-0102) were taken and placed in an incubator at 37°C and 5% CO2. DMEM medium (Cellbio (Shanghai) Biotechnology Co., Ltd., Catalog No. iCell-0001) was used, supplemented with 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122) for culture. When the cell confluence reached 90%, they were digested with trypsin-EDTA (Thermo, 25200-072) and counted with a counter (Countstar, IC1000). 190 μl of cell suspension / well was inoculated in a 96-well plate. The inoculation number of Hep3B cells was 2*104 cells / well. Transfection was performed the next day after the cells adhered to the wall.

[0125] LipofectamineTM RNAiMAX (thermofisher, 13778150) was used for transfection. 2.2 μl (100 nM or 20 nM or 10 nM) diluted siRNA (as shown in Table 2), 19.1 μl Opti-MEM (thermofisher, 1105821), and 0.7 μl RNAiMAX were mixed to form a transfection complex. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates for each complex), 10 μl per well, the final concentration of siRNA was 0.5 nM, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0126] (2) RNA extraction and detection (i) Extract total RNA using RNA-Quick Purification Kit (RN001, Yishan Biotechnology): Remove the 12-well plate from the incubator, drain the culture medium, wash once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a new 1.5 ml centrifuge tube. Add 500 μl of anhydrous ethanol to the lysed cells and mix thoroughly (if precipitation occurs, this is normal, just continue the operation). Invert the centrifuge tube several times, or use a pipette to blow and aspirate 10 times to disperse the precipitate, then add the liquid to the centrifuge column, place the centrifuge tube symmetrically in a centrifuge (eppendorf, 5430), and centrifuge at 4000 × g for 1 min. Remove the centrifuge tube and add 500 μl of wash buffer to the column, centrifuge at 12000 × g for 1 min, remove the column after centrifugation, pour out the waste liquid, put the RNA column back into the collection tube, and centrifuge the empty tube once to remove any residual wash buffer. Place the column in a clean 1.5ml centrifuge tube without RNAse, open the cover and let it dry for 2 minutes. Add 30μl of elution buffer to the center of the RNA column membrane, let it stand at room temperature for 2 minutes, centrifuge at 2000 × g for 1min, and place on ice after RNA is eluted. Determine the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0127] (ii) Synthesize cDNA using HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novozymes, R223-01): Prepare a mixture of 4μl 4 × gDNA wiper Mix, 1μg template RNA, and 16μl RNase-free ddH2O in an RNase-free centrifuge tube to remove genomic DNA. Use a pipette to gently mix, and incubate at 42℃ for 2 min. Then add 4μl 5 × HiScript II qRT SuperMix II directly to the reaction tube and mix gently with a pipette. Place in a PCR instrument (Applied Biosystems, 9700) at 50℃ for 15min; 85℃ for 5sec, and maintain at 4℃. The product can be used for qPCR reaction immediately, or stored at -20℃ and used within half a year. For long-term storage, it needs to be stored at -80℃ after aliquoting. Repeated freezing and thawing of cDNA should be avoided.

[0128] (iii) qPCR quantification using ChamQ SYBR qPCR Master Mix (Novozymes, Q311-02): A mixed solution of 10 μl 2 × ChamQ SYBR qPCR Master Mix, 0.5 μl Forword primer (Roboxin), 0.5 μl Reverse primer (Roboxin), 1 μl Template cDNA, and 8 μl ddH2O was prepared to a 20 μl system. Each sample was replicated three times. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed). The following program was performed: pre-denaturation, 95°C, 30 sec; amplification, 95°C, 10 sec, 60°C, 30 sec, 40 cycles; melting curve, 95°C, 15 sec, 60°C, 60 sec, 95°C, 15 sec.

[0129] (3) Data statistical analysis: Export the data to EXCEL format and use CT INHBE -CT GAPDH The control group was normalized. In order to calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method, and the mean and standard deviation of the three parallel repeated data were calculated.

[0130] The results of the INHBE-siRNA activity experiment in Hep3B cells are shown in Table 3.

[0131] As can be seen from Table 3, when the dosage is 1 nM, the inhibition rate of INHBE mRNA by vmap001-modified siRNA can be as high as 80-90%.

[0132] Table 3. INHBE siRNA knockdown levels in Hep3B cells Example 4 A sequence having a DPP4 gene inhibitory effect was used as a tool sequence to examine the effect of the structure described in this case.

[0133] The siRNA sequence was designed based on the mRNA sequence of the DPP4 (Dipeptidyl peptidase-4) gene. The siRNA sequence (as shown in Table 4) was complementary to human DPP4 mRNA (Genbank accession number: NM_001935.4) or had one mismatch at the 3' end of the sense strand, or was complementary to cynomolgus monkey DPP4 mRNA (Genbank accession number: XM_005573318.4) or had no more than two mismatches. The DPP4-siRNA modified sequence was prepared according to the method of Example 2.

[0134] Table 4. INHBE siRNA knockdown levels in Hep3B cells The in vitro activity detection method is the same as in Example 3 Data Statistical Analysis: Export the data to EXCEL format and use CT DPP4 -CT GAPDH The control group was normalized. In order to calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method, and the mean and standard deviation of the three parallel repeated data were calculated.

[0135] The results of the DPP4-siRNA activity experiment in Hep3B cells are shown in Table 5.

[0136] As can be seen from Table 5, when the dosage is 0.5 nM, the inhibition rate of DPP4 mRNA by vmap001-modified siRNA can be as high as 70-90%.

[0137] Table 5. DPP4 siRNA knockdown levels in Hep3B Example 5 DPP4-siRNA activity evaluation experiment in mice The experiment used SPF male C57BL / 6J mice aged 6 to 8 weeks (Beijing Biotechnology Co., Ltd.), which were randomly divided into groups of 6 mice per group. C57Bl / 6 mice were subcutaneously administered 3 mg / kg of DPP4-siRNA reagent with carrier L96 (shown in Table 6) and normal saline (NC, negative control) once. On the 14th day after administration, the mice were euthanized, liver samples were collected, liver mRNA was extracted and the mRNA content was analyzed by RT-PCR method, and reverse transcription, qPCR quantification and data statistical analysis were performed according to the method described in Example 4. During the experiment, no animals showed signs of death or dying. Clinical observation showed no obvious abnormalities in all animals. The experimental data are shown in the table. As can be seen from Table 7, when the dosage is 3 mg / kg, the in vivo inhibition rate of DPP4 mRNA of the modified siRNA reagent can reach or approach 90%.

[0138] Table 6. DPP4-siRNA reagents with carrier L96 Table 7. In vivo inhibition rate of DPP4 siRNA modified sequences with vector The foregoing description of specific exemplary embodiments of the present disclosure is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present disclosure and various different selections and changes. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims

1. A compound for modifying an oligonucleotide or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, which has a structure represented by the following formula (I): (I) in, R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3 or -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl; R2 is selected from or ; nz is an integer from 0 to 10; R7 and R8 are each independently selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl; R9 is -(CH2) nx CN; nx is an integer from 0 to 10; ny is an integer from 0 to 10.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, characterized in that: It has the structure shown in the following formula (II), (Ⅱ) Wherein, R1 and R2 are respectively defined as in formula (I).

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, characterized in that: R1 is selected from H, F, -OMe, -HNCOR6, -OH, -OR3 or -NR4R5; R3, R4, R5, R6 are each independently selected from C 1-5 alkyl.

4. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, characterized in that: nz is an integer from 0 to 5.

5. The compound according to claim 1 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, characterized in that: nx is an integer from 0 to 5.

6. The compound according to claim 1 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, characterized in that: ny is an integer from 0 to 5.

7. The compound according to claim 1 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, characterized in that: R7 and R8 are each independently selected from C 1-5 alkyl.

8. The compound according to claim 1 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof, wherein the compound is denoted as vmap, and the compound is selected from the following compounds: 。 9. A method for preparing the compound according to any one of claims 1 to 8, wherein the compound has the structural formula of formula (I), comprising reacting compound M8 with compound M9 to prepare the compound: 。 10. The preparation method according to claim 9, wherein compound M7 is prepared by reacting compound M5 with compound M6, and compound M8 is prepared by reacting compound M7: 。 11. The preparation method according to claim 10, wherein compound M3 is prepared by reacting compound M1 with compound M2, compound M3 is reacted to prepare compound M4, and compound M4 is reacted to prepare compound M5: 。 12. Use of the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in the preparation of an oligonucleotide sequence.

13. Use of the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in the preparation of siRNA or miRNA.

14. Use of the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in the preparation of siRNA.

15. An oligonucleotide sequence, characterized in that Prepared from the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof.

16. An oligonucleotide sequence, characterized in that The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof is prepared by an oligonucleotide synthesizer.

17. An oligonucleotide sequence, characterized in that The compound or its pharmaceutically acceptable salt, isomer, solvate or prodrug according to any one of claims 1 to 8 is prepared by an oligonucleotide synthesizer; the compound or its pharmaceutically acceptable salt, isomer, solvate or prodrug is located at the 5' end or the middle position of the oligonucleotide.

18. The oligonucleotide sequence according to any one of claims 15 to 17, wherein the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof has a structure represented by formula (III) when located at the 5' end of the oligonucleotide: (Ⅲ) in, R1 and R2 are each defined as in formula (I); X is selected from O, S; The middle wavy lines indicate oligonucleotides; Indicates that O is linked to the oligonucleotide.

19. An oligonucleotide delivery system, characterized in that The oligonucleotide delivery system comprises the oligonucleotide sequence of any one of claims 15 to 18.

20. A siRNA or miRNA delivery system, characterized in that The siRNA or miRNA delivery system comprises the oligonucleotide sequence of any one of claims 15 to 18.

21. A siRNA delivery system, characterized in that The siRNA delivery system comprises the oligonucleotide sequence according to any one of claims 15 to 18.

22. A method for preparing an oligonucleotide delivery system, comprising the following steps: The oligonucleotide sequence is prepared by using an oligonucleotide synthesizer using the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof.

23. A pharmaceutical composition comprising an oligonucleotide sequence prepared by an oligonucleotide synthesizer using a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof and / or an oligonucleotide sequence according to any one of claims 15 to 18.

24. The oligonucleotide sequence of any one of claims 15 to 18, the oligonucleotide delivery system of claim 19, the siRNA or miRNA delivery system of claim 20, the siRNA delivery system of claim 21, or the pharmaceutical composition of claim 23 for use in treatment.

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