Compounds for modifying oligonucleotides, methods for preparing the same, and uses thereof

Abstract

The present disclosure belongs to the field of biomedical technologies, and particularly relates to compounds for modifying oligonucleotides, methods for preparing the same, and applications thereof. The present disclosure has the following advantages: The present disclosure provides compounds for modifying oligonucleotides, and these compounds can improve the pharmaceutical efficacy of oligonucleotides.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedical technologies, and particularly relates to compounds for modifying oligonucleotides, methods for preparing the same, and applications thereof. Background Art

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

[0003] Currently, the marketed oligonucleotide drugs mainly improve the metabolic stability of the oligonucleotide structure and reduce adverse reactions through chemical modification. For example, modification is carried out on phosphodiester bonds, ribose, oligonucleotide bases, or ribose-phosphate backbones.

[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 silencing agents are promising alternatives to traditional small organic compounds that inhibit the functions of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleotides that prevent protein formation through gene silencing.

[0005] The efficacy of traditional siRNA is often not ideal. All along, many modified siRNA compounds have been developed for diagnostic and therapeutic purposes, including SiRNA / RNAi therapeutic agents for treating various diseases, such as central nervous system diseases, inflammatory diseases, metabolic disorders, tumors, infectious diseases, and eye diseases, etc.

[0006] The present disclosure aims to provide compounds for modifying oligonucleotides, methods for preparing the same, and applications thereof, and the compounds 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 to develop compounds for modifying oligonucleotides, methods for preparing the same, and applications thereof, and the compounds can improve the efficacy of oligonucleotides.

[0008] To achieve the above technical objectives, the technical solutions adopted by the present disclosure are as follows:

[0009] On the one hand, the present disclosure provides a compound for modifying oligonucleotides or a pharmaceutically acceptable salt, isomer, solvate, or prodrug thereof, which has a structure shown in the following formula (I),

[0010] (I)

[0011] wherein,

[0012] R 1 is selected from H, F, -OMe, -HNCOR 6 , -OH, -OR 3 , -NR 4 R 5 ; R 3 , R 4 , R 5 , R 6 are each independently selected from C 1-20 alkyl, C 2-20 alkenyl or C 2-20 alkynyl;

[0013] R 2 is selected from or ; nz is an integer from 0 to 10;

[0014] R 7 , R 8 are each independently selected from C 1-20 alkyl, C 2-20 alkenyl or C 2-20 alkynyl;

[0015] R 9 is -(CH 2 ) nx CN; nx is an integer from 0 to 10;

[0016] ny is an integer from 0 to 10.

[0017] In some preferred embodiments of the present disclosure, the compound or its pharmaceutically acceptable salt, isomer, solvate or prodrug as described above has the structure shown in the following formula (Ⅱ),

[0018] (Ⅱ)

[0019] wherein, R 1 , R 2 are each defined as in formula (I).

[0020] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOR 6 , -OH, -OR 3 , -NR 4 R 5 ; R 3 , R 4 , R5 and R 6 are each independently selected from C 1-10 alkyl, C 2-10 alkenyl or C 2-10 alkynyl.

[0021] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOR 6 , -OH, -OR 3 , -NR 4 R 5 ; R 3 , R 4 , R 5 , R 6 are each independently selected from C 1-5 alkyl, C 2-5 alkenyl or C 2-5 alkynyl.

[0022] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOR 6 , -OH, -OR 3 , -NR 4 R 5 ; R 3 , R 4 , R 5 , R 6 are each independently selected from C 1-3 alkyl, C 2-3 alkenyl or C 2-3 alkynyl.

[0023] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOR 6 , -OH, -OR 3 , -NR 4 R 5 ; R 3 , R 4 , R 5 , R 6 are each independently selected from C 1-10 alkyl.

[0024] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOR 6 , -OH, -OR 3 , -NR 4 R 5 ; R 3 , R 4 , R 5 , R6 Each independently selected from C 1-5 alkyl groups.

[0025] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOR 6 , -OH, -OR 3 , -NR 4 R 5 ; R 3 , R 4 , R 5 , R 6 Each independently selected from C 1-3 alkyl groups.

[0026] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOR 6 ; R 6 is C 1-3 alkyl.

[0027] In some preferred embodiments of the present disclosure, R 1 is selected from H, F, -OMe, -HNCOCH 3 .

[0028] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 8.

[0029] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 6.

[0030] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 5.

[0031] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 4.

[0032] In some preferred embodiments of the present disclosure, nz is an integer from 0 to 2.

[0033] In some preferred embodiments of the present disclosure, nz is 1 or 2.

[0034] In some preferred embodiments of the present disclosure, nz is 2.

[0035] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 8.

[0036] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 6.

[0037] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 5.

[0038] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 4.

[0039] In some preferred embodiments of the present disclosure, nx is an integer from 0 to 2.

[0040] In some preferred embodiments of the present disclosure, nx is 1 or 2.

[0041] In some preferred embodiments of the present disclosure, nx is 2.

[0042] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 8.

[0043] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 6.

[0044] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 5.

[0045] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 4.

[0046] In some preferred embodiments of the present disclosure, ny is an integer from 0 to 2.

[0047] In some preferred embodiments of the present disclosure, ny is 1 or 2.

[0048] In some preferred embodiments of the present disclosure, ny is 1.

[0049] In some preferred embodiments of the present disclosure, R 7 、R 8 are each independently selected from C 1-10 alkyl, C 2-10 alkenyl or C 2-10 alkynyl.

[0050] In some preferred embodiments of the present disclosure, R 7 、R 8 are each independently selected from C 1-5 alkyl, C 2-5 alkenyl or C 2-5 alkynyl.

[0051] In some preferred embodiments of the present disclosure, R 7 、R 8 are each independently selected from C 1-3 alkyl, C 2-3 alkenyl or C 2-3 alkynyl.

[0052] In some preferred embodiments of the present disclosure, R 7 、R 8 are each independently selected from C 1-5 alkyl.

[0053] In some preferred embodiments of the present disclosure, R 7 , R 8 are each independently selected from C 1-3 alkyl.

[0054] In some preferred embodiments of the present disclosure, R 7 , R 8 are each independently selected from methyl, ethyl, propyl or isopropyl.

[0055] In some preferred embodiments of the present disclosure, R 7 , R 8 are both isopropyl.

[0056] In some preferred embodiments of the present disclosure, the compound is denoted as vmap, and the compound is selected from the following compounds:

[0057]

[0058] .

[0059] On the other hand, the present disclosure provides a method for preparing the compound as described above, the structural formula of the compound being formula (I), which comprises reacting compound M8 with compound M9 to prepare the compound:

[0060] .

[0061] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, wherein compound M7 is prepared by reacting compound M5 with compound M6, and compound M8 is prepared by reacting compound M7:

[0062] .

[0063] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, wherein compound M5 is prepared by reacting compound M4:

[0064] .

[0065] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, wherein compound M3 is prepared by reacting compound M1 with compound M2, and compound M4 is prepared by reacting compound M3:

[0066] .

[0067] 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 M4 is prepared by reacting compound M3, and compound M5 is prepared by reacting compound M4:

[0068] .

[0069] On the other hand, the present disclosure provides the use of the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in the preparation of an oligonucleotide sequence.

[0070] On the other hand, the present disclosure provides the use of the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in the preparation of siRNA or miRNA.

[0071] On the other hand, the present disclosure provides the use of the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof in the preparation of siRNA.

[0072] On the other hand, the present disclosure provides an oligonucleotide sequence prepared from the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof.

[0073] On the other hand, the present disclosure provides an oligonucleotide sequence prepared from the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof by an oligonucleotide synthesizer.

[0074] On the other hand, the present disclosure provides an oligonucleotide sequence prepared from the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof by an oligonucleotide synthesizer; the compound or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof is located at the 5'-end or the middle position of the oligonucleotide.

[0075] In some preferred embodiments of the present disclosure, according to the oligonucleotide sequence described in the present disclosure, when the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof is located at the 5'-end of the oligonucleotide, it has the structure shown in formula (Ⅲ):

[0076] (Ⅲ)

[0077] Wherein, R 1 , R 2 are each defined as in formula (I);

[0078] X is selected from O, S;

[0079] The wavy line in represents the oligonucleotide; represents the connection of O to the oligonucleotide.

[0080] On the other hand, the present disclosure provides an oligonucleotide delivery system, which includes the oligonucleotide sequence as described above.

[0081] On the other hand, the present disclosure provides an siRNA or miRNA delivery system, which includes the oligonucleotide sequence as described above.

[0082] On the other hand, the present disclosure provides an siRNA delivery system, which includes the oligonucleotide sequence as described above.

[0083] On the other hand, the present disclosure provides a method for preparing an oligonucleotide delivery system, which includes the following steps: preparing an oligonucleotide sequence using the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof by an oligonucleotide synthesizer.

[0084] On the other hand, the present disclosure provides a pharmaceutical composition, which includes the oligonucleotide sequence prepared by an oligonucleotide synthesizer using the compound as described above or a pharmaceutically acceptable salt, isomer, solvate or prodrug thereof and / or the oligonucleotide sequence as described above.

[0085] On the other hand, the present disclosure provides the oligonucleotide sequence as described above, the oligonucleotide delivery system as described above, the siRNA or miRNA delivery system as described above, the siRNA delivery system as described above, or the pharmaceutical composition as described above, which is used for treatment.

[0086] The present disclosure has the following advantages:

[0087] (1) The present disclosure provides a compound for modifying an oligonucleotide, and this compound can improve the efficacy of the oligonucleotide.

[0088] (2) The present disclosure provides a nucleotide modified with a phosphor backbone, which has the effect of enhancing the activity of a double-stranded oligonucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings required to be used in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. The drawings here are incorporated into the specification and constitute a part of this specification, showing the embodiments in line with this specification, and are used together with the specification to explain the principles of this specification.

[0090] Figure 11H NMR spectrum of compound vmap-001.

[0091] Figure 2 31P NMR spectrum of compound vmap-001. Detailed implementation manners

[0092] Definitions and explanations

[0093] To facilitate a better understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the terms related to cell and tissue culture, microbiology, and laboratory operation procedures used herein are all widely used terms and conventional procedures in the relevant fields. Meanwhile, to better understand the present disclosure, the definitions and explanations of 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, changes can be made thereto. It should also be understood that the terms used in this application are only for describing specific implementation manners and are not intended to be limiting.

[0094] Unless the content is otherwise clearly stated, the singular forms "a", "an", and "the" used in this specification and the appended claims include plural referents.

[0095] As used herein, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products, or devices.

[0096] In the description herein, reference is made to "some embodiments", "some implementation manners", or "some implementation schemes", which describe subsets of all possible embodiments. However, it can be understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0097] As used herein and unless otherwise stated, the terms "comprising", "including", "having", "containing", including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, for example, not excluding other unlisted elements or steps.

[0098] When listing a numerical range, each value and sub-ranges within the stated range are included. For example, "C 1-6 alkyl" includes C 1 、C 2 、C 3 、C 4 、C5 , C 6 , 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 groups.

[0099] 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 deuterium and variants of hydrogen, provided that the valence of the particular atom is normal and the resulting 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 with" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary based on what is chemically feasible.

[0100] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 - 2 R's, the group may optionally be substituted with up to two R's, and each R has independent options in each case. In addition, combinations of substituents and / or their variants are permitted only if such combinations result in a stable compound.

[0101] In any embodiment, any or all of the hydrogens present in a compound, or the hydrogens within a particular group or moiety within the compound, may be replaced by deuterium or tritium. One to the maximum number of hydrogens present in the compound may be replaced by deuterium. One to the maximum number of hydrogens present in any group of a general formula compound or a specific compound may be deuterated. For example, when a group is described as an ethyl group, the ethyl group may be C 2 H 5 or C 2 H 5 where x (1 to 5) of the hydrogens are replaced by deuterium, e.g., C 2 D x H 5-x . When a group is described as a deuterated ethyl group, the deuterated ethyl group may be C 2 H 5 where x (1 to 5) of the hydrogens are replaced by deuterium, e.g., C 2 D x H5-x The stable deuterated derivatives described in the present disclosure are preferably stable deuterated isotope derivatives obtained by substituting any hydrogen atoms that can be deuterated in the various formulas with 1 to the maximum number (such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, etc.) of deuterium atoms.

[0102] The present disclosure mentions the compounds of formula (I), and also includes their tautomers, stereoisomers, mixtures of stereoisomers, solvates, or derivatives, etc.

[0103] The "compounds" in the present disclosure also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond and are accompanied by the migration of a proton. The term "tautomer" or "tautomeric form" refers to different functional group isomers that are in dynamic equilibrium at room temperature and can rapidly interconvert. It refers to one of two or more structural isomers in which the equilibrium exists and it is easy to convert from one isomer form to another. This transformation results in the formal migration of a hydrogen atom and is accompanied by the conversion of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomeric groups in solution. In a solution where tautomerization can 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 interconverted by tautomerization is called tautomerism.

[0104] When the present specification describes compounds that are prone to tautomerization but only describes one of the tautomers, it should be understood that all tautomers are included as part of the described chemical meaning. It should be understood that when a compound has tautomeric forms, all tautomeric forms are intended to be included, and the naming of the compound does not exclude any tautomeric form.

[0105] Among the various possible types of tautomerism, two are usually observed. In keto-enol tautomerism, electrons and hydrogen atoms move simultaneously.

[0106] Common tautomeric pairs are: keto-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocycles, imine-enamine, and enamine-enamine.

[0107] The term "isomer" refers to different compounds that have the same molecular formula but different atomic arrangements and configurations. According to their structures, the compounds of the present disclosure can exist in different stereoisomeric forms. These forms include configurational isomers or optical conformational isomers (enantiomers and / or diastereomers, including those of atropisomers). Therefore, the present disclosure includes enantiomers, diastereomers, and their mixtures. The present disclosure further includes all mixtures of the above stereoisomers, regardless of the ratio, including racemates.

[0108] Depending on their structure, the compounds of the present disclosure may exist in various stable isotope 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 their respective original atoms.

[0109] 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.

[0110] In the present disclosure, " " and " " are used to represent the absolute configuration of a stereocenter. The " " in " " refers to the bond connection site.

[0111] When " " appears in a ring and the connection position is uncertain, it means that the connection site is at any atom on the single ring where " " is located, as long as the atomic valence permits.

[0112] The term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance, which has lower cytotoxicity to tumor cells compared to the parent drug and can be activated or transformed into a more active parent form through enzymatic action. The prodrugs of the present disclosure include, but are not limited to, phosphate (ester) prodrugs, thiophosphate (ester) prodrugs, sulfate (ester) prodrugs, peptide prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, prodrugs containing optionally substituted phenoxyacetamide or phenylacetamide, 5-fluorocytosine, and other 5-fluorouridine prodrugs that can be converted into more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present disclosure include, but are not limited to, the above-mentioned chemotherapeutic agents.

[0113] The term "alkyl" refers to a chain-like (straight-chain or branched-chain) saturated aliphatic hydrocarbon group. The term "alkyl" can be a straight-chain or branched-chain alkyl containing 1 to 10 carbon atoms (C 1-10 alkyl), preferably an alkyl containing 1 to 6 carbon atoms (C 1-6alkyl). 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 isomers thereof, etc. More preferably, it is a lower alkyl containing 1 to 3 carbon atoms (C 1-3 alkyl), non-limiting examples include methyl, ethyl, n-propyl, isopropyl, etc. The alkyl can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the groups described in this application.

[0114] In one embodiment, the substituents are independently selected from oxo, halogen, -CN, -NH 2 , -OH, -NH(CH 3 ), -N(CH 3 ) 2 , alkyl (including straight-chain, branched-chain) 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) 2 alkyl, -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(NH 2 )(substituted or unsubstituted alkyl) 2 . In another embodiment, for example, the optional substituents are selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH 2 , -OH, -NH(CH 3 ), -N(CH 3 ) 2 , -CH 3 , -CH 2 CH 3 , -CH(CH 3 ) 2, -CF 3 , -CH 2 CF 3 , -OCH 3 , -OCH 2 CH 3 , -OCH(CH 3 ) 2 , -OCF 3 , -OCH 2 CF 3 , -S(=O) 2 -CH 3 , -C(=O)NH 2 , -C(=O)-NHCH 3 , -NHC(=O)NHCH 3 , -C(=O)CH 3 , -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 alkoxy, halogen, amino, acetamido, oxo, and nitro. In yet another embodiment, the substituents are independently selected from C 1-6 alkyl, C 1-6 alkoxy, halogen, acetamido, and nitro. As used herein, when the substituent is alkyl or alkoxy, the carbon chain may be branched, straight-chain, or cyclic.

[0115] The term "alkylene" refers to a divalent group formed by removing another hydrogen from an alkyl group, which may be substituted or unsubstituted. The "alkylene" is preferably a divalent group of a straight-chain or branched-chain saturated aliphatic hydrocarbon containing 1 to 8 carbon atoms, more preferably a divalent alkyl group (C 1-6 alkylene) containing 1 to 6 carbon atoms. Examples of alkylene include, but are not limited to, -CH 2 -, -CH(CH 3 )-, -CH 2 CH 2 -, -CH(CH 3 )CH 2 -, -CH 2 CH 2 CH 2 -, or -(CH 2 ) 4 - and its stereoisomers.

[0116] The term "heteroatom" is selected from nitrogen, oxygen, or sulfur. Among them, nitrogen may be optionally substituted; sulfur may also be optionally substituted, such as oxo, i.e., forming S(O) t3 (where t3 is an integer from 0 to 2).

[0117] The term "aryl" means phenyl or naphthyl, or phenyl or naphthyl substituted by the following groups: halogen, C 1-8 alkyl, hydroxy, nitro, trifluoromethyl, etc. Preferred is phenyl or monosubstituted phenyl; most preferred is phenyl.

[0118] As used herein, the term "solvate" refers to a complex formed by a compound of the present disclosure and a solvent. They either react in a solvent or precipitate or crystallize out from a solvent. For example, a complex formed with water is called a "hydrate". The solvates of the compounds represented by formula (I) of the present disclosure are within the scope of the present disclosure.

[0119] The present disclosure includes prodrugs of the above compounds. Prodrugs include known amino protecting groups and carboxyl protecting groups, which are hydrolyzed or released via an enzymatic reaction under physiological conditions to obtain the parent compound. Specific methods for preparing prodrugs can be referred to (Saulnier, M.G.; Frennesson, D.B.; Deshpande, M.S.; Hansel, S.B and Vysa, D.M. Bioorg. Med.Chem Lett. 1994, 4, 1985 - 1990; and Greenwald, R.B.; Choe, Y.H.; Conover, C.D.; Shum, K.; Wu, D.; Royzen, M. J. Med. Chem. 2000, 43, 475.).

[0120] As used herein, the term "hydroxy" means -OH.

[0121] As used herein, the term "oxo group" means =O.

[0122] As used herein, the term "carboxyl" means -C(=O)OH.

[0123] As used herein, the term "acetyl (Ac)" means -COCH 3 .

[0124] As used herein, the term "DMTr" means 4,4'-dimethoxytrityl.

[0125] As used herein, the term "Ph" means phenyl, i.e., C 6 H 5 -.

[0126] As used herein, the term "TBSO" refers to tert-butyldimethylsilyloxy, and its chemical structural formula is (CH 3 ) 3 C-Si(CH 3 ) 2 -O-.

[0127] As used herein, the term "t-BuOK" refers to potassium tert-butoxide.

[0128] Examples

[0129] To make the objectives, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present disclosure. Apparently, the described embodiments are some, but not all, of the embodiments of the present disclosure. The following is only a further description of the present disclosure, and the protection scope of the present disclosure is not limited thereto.

[0130] Example 1

[0131] Preparation of Compound vmap-001:

[0132]

[0133] Preparation of Compound 3:

[0134] Dissolve Compound 1 (20 g, 27.9 mmol), Compound 2 (8.7 g, 41.8 mmol), and Cs2CO3 (27.2 g, 83.4 mmol) in 300 mL (1,4-dioxane / water = 10:1), deoxygenate, add Compound Pd(dppf)Cl2 (4.5 g, 5.5 mmol) under nitrogen protection, react at 80 °C for 12 hours. TLC (PE:EA = 2:1) shows that the reaction is completed. Treat the reaction solution with ethyl acetate and water, collect the organic phase, dry the organic phase with anhydrous sodium sulfate, concentrate to obtain the crude product, and purify the crude product by column chromatography (PE:EA = 2:1) to obtain off-white solid Compound 3 (18.2 g, yield 77%).

[0135] 1H NMR (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).

[0136] Preparation of Compound 4:

[0137] Dissolve Compound 3 (18 g, 21 mmol) in 300 mL of dichloromethane solution containing 3% dichloroacetic acid at 0 - 5 °C, react for hours at 0 - 5 °C. TLC (PE:EA = 1:1) shows the reaction is complete. Control the temperature, adjust the pH to 8 with saturated sodium bicarbonate solution, collect the organic phase, treat the organic phase with saturated brine, dry with anhydrous sodium sulfate, concentrate to obtain the crude product, and purify the crude product by column chromatography (PE:EA = 2:1) to obtain pale yellow solid Compound 4 (10.3 g, yield 89%).

[0138] Preparation of Compound 5:

[0139] Add Compound 4 (15 g, 27.6 mmol) and IBX (15.4 g, 53.8 mmol) to 200 mL of acetonitrile, react at 80 °C for 1.5 hr. TLC (PE:EA = 2:1) shows the reaction is over. Cool the reaction solution, filter, concentrate, and purify the crude product by column chromatography (PE:EA = 2:1) to obtain pale yellow solid Compound 5 (10.4 g, yield 69.6%).

[0140] Preparation of Compound 7:

[0141] Under nitrogen protection, compound 6 (28.5 g, 45.1 mmol) was dissolved in 120 ml of anhydrous THF. While controlling the temperature at 0 °C, t-BuOK (5.1 g, 45.1 mmol) was added portionwise. Subsequently, an 80-ml 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 h. TLC (PE:EA = 1:1) indicated the reaction was complete. The reaction solution was quenched with saturated ammonium chloride solution and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by column chromatography (PE:EA = 1:1) to obtain 8.3 g of off-white solid compound 7 (yield 52%).

[0142] Preparation of compound 8:

[0143] Compound 7 (8 g, 9.1 mmol) was dissolved in 100 ml of HCOOH:H2O = 1:1 (V:V) solution. Under nitrogen protection, the reaction was carried out for 56 h. 500 mL of water was added to the reaction system, and after freeze-drying, the crude product was obtained. The crude product was purified by column chromatography (DCM:MeOH = 10:1) to obtain 3.8 g of pale yellow solid compound 8 (yield 55.9%).

[0144] Preparation of compound vmap-001:

[0145] Under nitrogen protection, compound 8 (3 g, 4 mmol) was dissolved in 30 mL of anhydrous DCM. Compound 9 (1.8 g, 6 mmol) and DCI (0.7 g, 6 mmol) were added, and the reaction was carried out for 2 h. TLC (PE:EA = 1:2) indicated the reaction was complete. The reaction was concentrated in vacuo at 20 °C. The crude product was purified by flash column chromatography, and then slurried and purified with anhydrous dichloromethane:n-hexane = 1:9, filtered, and the solvent was removed in vacuo to obtain 2.3 g of off-white solid compound vmap-001 (yield 60%).

[0146] 1H NMR (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).

[0147] 31P NMR (162 MHz, CDCl3) δ 151.86, 151.05, 17.94, 17.67.

[0148] The 1H NMR spectrum of compound vmap - 001 is shown in Figure 1 .

[0149] The 31P NMR spectrum of compound vmap - 001 is shown in Figure 2 .

[0150] The oligonucleotide sequence containing the compound described in the present disclosure is prepared by an oligonucleotide synthesizer, and the above structure can be located at the 5'-end or the middle position of the oligonucleotide. For example, it is coupled at the 5'-end or the middle position of the antisense strand of siRNA by a phosphodiester bond or a phosphorothioate bond.

[0151] The abbreviations of nucleotide monomers or modified nucleotide monomers used in the nucleic acid sequence representation shown in Table 1 are understood. When these monomers are present in an oligonucleotide, unless otherwise specified, they are connected to each other by 5'-3' phosphodiester bonds. And it should be understood that when a nucleotide contains a 2'-fluoro modification, the fluorine replaces the hydroxyl group at that position in the parental nucleotide (i.e., it is a 2'-fluoro nucleotide).

[0152] Table 1. Abbreviations of nucleotide monomers for nucleic acid sequence representation

[0153]

[0154] L96 is a well-known GalNAc delivery vector in the art. It binds to ASGPR (asialoglycoprotein receptor) in the liver to achieve targeted delivery of nucleic acid drugs and improve the concentration and efficacy of drugs in the liver. The structure of L96 is as follows:

[0155]

[0156] In the case where the source of the reagent is not specifically given in this article, such reagents can be obtained from any molecular biology reagent supplier, and their quality / purity standards are applicable to molecular biology.

[0157] Example 2

[0158] Preparation of siRNA:

[0159] The siRNA sequence was separately synthesized on a solid support via the sense strand (SS) and the antisense strand (AS), and obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0160] Solid-phase synthesis: The sense strand and the antisense strand were separately synthesized on a solid support using the phosphoramidite technique with an oligonucleotide synthesizer. Such synthesizers include AKTA Oligopilot (Cytiva) and Dr. Oligo 192XLc (Kunshan Berlic Precision Instruments Co., Ltd.). The solid-phase synthesis starts from the 3'-end of the sequence, and the monomers are sequentially coupled into the sequence in order. For each coupling of a phosphoramidite monomer, it includes four chemical steps: 1) deblocking or deprotection (removing the hydroxyl protecting group); 2) coupling; 3) oxidation; 4) capping. The phosphoramidite monomers, reagents, and purification consumables used are all commercially available reagents and consumables. For example, various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) were purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40 wt% aqueous methylamine solution, 28 wt% aqueous ammonium hydroxide solution, etc.) were purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this article are as 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 as described in J. Med. Chem. 2018, 61, 734−744, the content of which is incorporated herein by reference.

[0161] (1) Synthesis of the sense strand

[0162] The solid-phase phosphoramidite method is a well-established method for synthesizing oligonucleic acids. Using a computer-controlled synthesizer, the reaction is carried out in a stainless-steel synthesis column. In the synthesis of the sense strand, it starts with a solid support loaded with a targeting ligand (such as L96), or directly with a solid support. Through the solid-phase synthesizer, different raw materials, reagents, and solvents are injected into different pipelines in the order of 3' to 5' of the sequence, and phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four-step cycles of DMT protecting group removal reaction, condensation reaction, oxidation or thiolation reaction, and capping reaction. Each cycle connects one nucleotide unit, and an oligonucleic acid sequence of 19 or 21 nucleotide units is obtained. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and then the synthesized sequence is cleaved from the solid support by an ammonolysis reaction, filtered, the filter cake is washed with ethanol, the filtrate and washing solution are collected, and concentrated to obtain the crude sense strand. The crude product is purified by chromatography (SOURCE 15Q) and freeze-dried to obtain the target product sense strand.

[0163] (2)Synthesis of antisense strand

[0164] The synthesis of the antisense strand is similar to that of the sense strand. Through the solid-phase synthesizer, different raw materials, reagents, and solvents are injected into different pipelines in the order of 3' to 5' of the sequence, and phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four-step cycles of DMT protecting group removal reaction, condensation reaction, oxidation or thiolation reaction, and capping reaction. Each cycle connects one nucleotide unit, and an oligonucleic acid sequence of 21 or 23 nucleotide units is obtained. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and then the synthesized sequence is cleaved from the solid support by an ammonolysis reaction, filtered, the filter cake is washed with ethanol, the filtrate and washing solution are collected, and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, dried by suction or freeze-dried to obtain the target product antisense strand siRNA.

[0165] (3)Preparation of double-stranded siRNA

[0166] The sense strand and the antisense strand are respectively dissolved in injection water, mixed in a certain ratio (1.01:1.0~1.2:1.0), incubated at 30 - 50 °C for 30 - 90 min, and cooled to room temperature. After freeze-drying, the double-stranded siRNA product is obtained.

[0167] Example 3

[0168] Use the sequence with the inhibitory effect on the INHBE gene as a tool sequence to investigate the effect produced by the structure described in this case.

[0169] Design siRNA sequences based on the mRNA sequence of the INHBE (Inhibin Subunit Beta E) gene. The siRNA modification sequences (specifically shown in Table 2) are complementary to human INHBE mRNA (Genbank accession number: NM_031479.5) or have 1 mismatch at the 3' end of the sense strand, or are also complementary to cynomolgus monkey INHBE mRNA (Genbank accession number: XM_005571319.4) with no more than 2 mismatches. Prepare the INHBE-siRNA modification sequences according to the method of Example 2.

[0170] Table 2. INHBE-siRNA modification sequences

[0171]

[0172] In vitro activity detection

[0173] (1) Cell culture and transfection:

[0174] Take Hep3B cells (Wuhan Punosai Life Science Co., Ltd., product number CL-0102), place them in an incubator at 37°C and 5% CO2, use DMEM medium (Saibakang (Shanghai) Biotechnology Co., Ltd., product number iCell-0001), add 10% FBS (GIBCO, 12483020), 1% penicillin-streptomycin (GIBCO, 15140-122) for culture. When the cell confluence reaches 90%, digest with trypsin-EDTA (Thermo, 25200-072), count with a cell counter (Countstar, IC1000), inoculate 190 μl of cell suspension per well into a 96-well plate, and the inoculation number of Hep3B cells is 2×104 cells / well. Wait for the cells to adhere the next day for transfection.

[0175] Use LipofectamineTM RNAiMAX (thermofisher, 13778150) for transfection. Mix 2.2 μl (100 nM or 20 nM or 10 nM) of diluted siRNA (shown in Table 2), 19.1 μl of Opti-MEM (thermofisher, 1105821), and 0.7 μl of RNAiMAX to form a transfection complex. After incubating for 5 minutes, add the transfection complex to the cells (two technical replicates for each complex), 10 μl per well, and the final concentration of siRNA is 0.5 nM. Culture in an incubator at 37°C and 5% CO 2 for 24 hours.

[0176] (2) RNA extraction and detection

[0177] (i)Total RNA extraction using RNA-Quick Purification Kit (Yishan Biotech, RN001): Take out the 12-well plate from the incubator, aspirate 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 absolute ethanol to the lysed cells and mix well (if precipitation occurs, this is normal and continue with the operation). Invert the centrifuge tube several times or pipette forcefully 10 times to disperse the resulting precipitate, then add the liquid to the spin column. Place the centrifuge tubes symmetrically in a centrifuge (eppendorf, 5430) and centrifuge at 4000 × g for 1 min. Take out the centrifuge tube, add 500 μl of wash buffer to the column, centrifuge at 12000 × g for 1 min. After centrifugation, take out the column, discard the waste liquid, reinstall the RNA column into the collection tube, and centrifuge the empty tube once to remove any possible residual wash buffer. Place the column on a clean RNase-free 1.5 ml centrifuge tube, open the lid and air-dry for 2 minutes. Add 30 μl of elution buffer to the center of the membrane of the RNA column, let stand at room temperature for 2 minutes, centrifuge at 2000 × g for 1 min. After the RNA is eluted, place it on ice. Measure the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80 °C for later use.

[0178] (ii)Synthesis of cDNA using HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme, R223-01):

[0179] Prepare a mixture in a RNase-free centrifuge tube: 4 μl of 4 × gDNA wiper Mix, 1 μg of template RNA, add RNase-free ddH2O to 16 μl to remove genomic DNA, gently pipette to mix well, and incubate at 42 °C for 2 min. Then directly add 4 μl of 5 × HiScript II qRT SuperMix II to the reaction tube, gently pipette to mix well, place in a PCR instrument (Applied Biosystems, 9700) at 50 °C for 15 min; 85 °C for 5 sec, and hold at 4 °C. The product can be used immediately for qPCR reaction or stored at -20 °C and used within half a year. For long-term storage, it needs to be aliquoted and stored at -80 °C. cDNA should be avoided from repeated freezing and thawing.

[0180] (iii)qPCR quantification using ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02):

[0181] Prepare a 20 μl reaction system by adding 10 μl of 2 × ChamQ SYBR qPCR Master Mix, 0.5 μl of Forward primer (Ruibo Xingke), 0.5 μl of Reverse primer (Ruibo Xingke), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample has 3 replicates. Place the 96-well plate into a qPCR instrument (ROCGENE, Archimed) and execute the following program: pre-denaturation at 95°C for 30 sec; amplification at 95°C for 10 sec, 60°C for 30 sec, for 40 cycles; melting curve at 95°C for 15 sec, 60°C for 60 sec, 95°C for 15 sec.

[0182] (3)Data statistical analysis:

[0183] Export the data in EXCEL format and use CT INHBE -CT GAPDH , normalize the control group. To calculate the fold change of relative silencing efficiency, the data is analyzed using the ΔΔCT method, and the average value and standard deviation are calculated from the three parallel replicated data obtained.

[0184] The experimental results of INHBE-siRNA activity in Hep3B cells are shown in Table 3.

[0185] 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%.

[0186] Table 3. INHBE siRNA knockdown levels in Hep3B cells

[0187]

[0188] Example 4

[0189] Use the sequence with DPP4 gene inhibitory effect as a tool sequence to investigate the effect produced by the structure described in this case.

[0190] Design siRNA sequences based on the mRNA sequence of DPP4 (Dipeptidyl peptidase-4) gene. The siRNA sequences (specifically shown in Table 4) are complementary to human DPP4 mRNA (Genbank accession number: NM_001935.4) or have 1 mismatch at the 3' end of the sense strand, or are also complementary to cynomolgus monkey DPP4 mRNA (Genbank accession number: XM_005573318.4) with no more than 2 mismatches. Prepare the DPP4-siRNA modified sequences according to the method of Example 2.

[0191] Table 4. INHBE siRNA knockdown levels in Hep3B cells

[0192]

[0193] The in vitro activity detection method was the same as that in Example 3

[0194] Data statistical analysis:

[0195] Export the data in EXCEL format and use CT DPP4 -CT GAPDH , normalize the control group. To calculate the fold change of the relative silencing efficiency, the data was analyzed using the △△CT method, and the average value and standard deviation were calculated for the three parallel replicated data obtained

[0196] The experimental results of DPP4-siRNA activity in Hep3B cells are shown in Table 5

[0197] As can be seen from Table 5, when the dosage was 0.5 nM, the inhibition rate of DPP4 mRNA of vmap001-modified siRNA could be as high as 70 - 90%

[0198] Table 5. DPP4 siRNA knockdown levels in Hep3B

[0199]

[0200] Example 5

[0201] In vivo activity evaluation experiment of DPP4-siRNA

[0202] SPF-grade male C57BL / 6J mice aged 6 - 8 weeks (Beijing Specif Biological Technology Co., Ltd.) were used in the experiment. They were randomly grouped with 6 mice in each group. C57Bl / 6 mice were subcutaneously administered with 3 mg / kg of DPP4-siRNA reagent with carrier L96 (shown in Table 6), 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. Specifically, reverse transcription, qPCR quantification and data statistical analysis were carried out according to the methods described in Example 4. During the experiment, no death or near-death symptoms were observed in all animals. No obvious abnormalities were observed in all animals during clinical observation. The experimental data is shown in the table. As can be seen from Table 7, when the dosage was 3 mg / kg, the in vivo inhibition rate of DPP4 mRNA of the modified siRNA reagent could reach or approach 90%

[0203] Table 6. DPP4-siRNA reagent with carrier L96

[0204]

[0205] Table 7. In vivo inhibition rate of DPP4 siRNA modified sequences with carriers

[0206]

[0207] The foregoing description of specific exemplary embodiments of the present disclosure is for purposes of illustration and exemplification. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it is apparent that many changes and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present disclosure, as well as 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 thereof, the compound being denoted as vmap, and the compound being selected from the following compounds: 、 。 2. A method for preparing the compound according to claim 1, wherein the compound is vmap-001, and the preparation method comprises the following steps: 。 3. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in preparing an oligonucleotide sequence.

4. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of siRNA or miRNA.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of siRNA.

6. An oligonucleotide sequence, characterized in that The method is prepared from the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

7. An oligonucleotide sequence, characterized in that The compound according to claim 1 or a pharmaceutically acceptable salt thereof is prepared by an oligonucleotide synthesizer.

8. An oligonucleotide sequence, characterized in that The compound or a pharmaceutically acceptable salt thereof according to claim 1 is prepared by an oligonucleotide synthesizer; the compound or a pharmaceutically acceptable salt thereof is located at the 5' end or the middle position of the oligonucleotide.

9. An oligonucleotide delivery system, characterized in that The oligonucleotide delivery system comprises the oligonucleotide sequence of any one of claims 6 to 8.

10. A siRNA or miRNA delivery system, characterized in that: The siRNA or miRNA delivery system comprises the oligonucleotide sequence of any one of claims 6 to 8.

11. A siRNA delivery system, characterized in that: The siRNA delivery system comprises the oligonucleotide sequence according to any one of claims 6 to 8.

12. A method for preparing an oligonucleotide delivery system, comprising the following steps: The compound according to claim 1 or a pharmaceutically acceptable salt thereof is used to prepare an oligonucleotide sequence by an oligonucleotide synthesizer.

13. A pharmaceutical composition comprising an oligonucleotide sequence prepared by an oligonucleotide synthesizer using the compound according to claim 1 or a pharmaceutically acceptable salt thereof and / or an oligonucleotide sequence according to any one of claims 6 to 8.

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