Oligonucleotide coupled compound as well as preparation method and application thereof

Abstract

The invention particularly discloses an oligonucleotide coupled compound modified by cholic acid or a derivative thereof with a structure (I), a preparation method and application. Compared with unmodified oligonucleotide, the oligonucleotide coupled compound shown in the structure (I) has better biological activity and safety.

Description

Technical Field

[0001] The present invention relates to the field of biologically active nucleic acid compounds, and more specifically, to oligonucleotide conjugates modified with bile acid or its derivatives, preparation methods and uses thereof. Background Art

[0002] Nucleic acid drugs refer to artificially designed DNA or RNA with disease prevention or treatment functions. They act on pathogenic target genes or target mRNAs to regulate the expression of pathogenic genes from the root to achieve the purpose of disease prevention or treatment. Nucleic acid drugs mainly include antisense oligonucleotide (ASO), small interfering RNA (siRNA), micro RNA (miRNA), messenger RNA (mRNA), etc. As of the end of 2022, the US FDA has approved more than 10 nucleic acid drugs, and many candidate drugs are in clinical trials or preclinical trials.

[0003] The delivery of therapeutic nucleic acids into cells remains a challenging area of ​​research. Thus, there is a need for improved nucleic acid compounds and strategies for introducing such compounds into cells. Summary of the invention

[0004] The present invention provides a compound having the following structure or a nucleic acid compound modified with bile acid or its derivatives:

[0005]

[0006] in

[0007] A is an oligonucleotide, which includes small interfering nucleic acid (siRNA) and antisense oligonucleotide (ASO);

[0008] S1 is linked to the 3' carbon, 5' carbon or nucleobase of a double-stranded oligonucleotide, or to the 3' carbon, 5' carbon or nucleobase of a single-stranded oligonucleotide;

[0009] The specific structure of S1 is X represents O or S;

[0010] L1, L2, and L3 are each independently absent, -(CH2) h -、-(CH2) h -A group in which any one or more alkylene groups are replaced by a B group; wherein h is 0 to 15; the B group includes

[0011] -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -CH(RC)-, -C(R')(R”)-, -NH-, -N(RN)-, -SS-, -C(R')=C(R”)-, -C≡C-, one or more of; RC, RN, R', R" in the B group indicates that any one or more hydrogen atoms on the designated atom of the group are replaced by a D group, and the conditions for replacement are: the valence of the designated atom does not exceed its normal valence and the compound can generate a stable compound after the hydrogen atom is replaced; the designated atom includes a carbon atom or a nitrogen atom; the D group includes a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, a cyano group, a hydroxyl group, an oxo group, a carboxyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclic group, a heteroaryl group, an aryl group, a ketone, an alkoxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group or a halogen; the halogen includes F, Cl, Br or I; (O) in the B group represents a carbonyl oxygen atom;

[0012] T1, T2, T3, T4, and T5 are linkers for conjugating an oligonucleotide to a moiety containing cholic acid or its derivatives, and T1, T2, T3, T4, and T5 are each independently absent, -O-, -S-, -C(O)-, -NH-, -CH2-, -C(O)NH-, -NHC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-,

[0013]

[0014] One or more of; wherein (O) represents a carbonyl oxygen atom; is the connection site;

[0015] k is 1 or 2;

[0016] t is an integer from 1 to 5;

[0017] j, m, and n are each independently 1 to 15;

[0018] R1 is bile acid or its derivatives;

[0019] R2 is absent, cholic acid or its derivatives, cholesterol or its derivatives, unsubstituted C1-C 22 Alkyl, linear or branched C1-C 22 Alkyl, saturated or unsaturated C1-C 22 Alkyl, etc.

[0020] As a preferred embodiment of the present invention, t is preferably 1, 2 or 3.

[0021] As a preferred embodiment of the present invention, R1 is cholic acid or a derivative thereof, and cholic acid is preferably ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid, deoxycholic acid or others. Furthermore, "cholic acid derivative" refers to a compound derived from cholic acid, for example, by substitution, addition or removal of a substituent.

[0022] As a preferred embodiment of the present invention, R2 is absent, cholic acid or its derivatives, cholesterol or its derivatives, unsubstituted C1-C22 alkyl, linear or branched C1-C22 alkyl, saturated or unsaturated C1-C22 alkyl, etc.; cholic acid is preferably ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid or deoxycholic acid, etc. Further, "cholic acid derivatives" refer to compounds derived from cholic acid, for example, by substitution, addition or removal of substituents. Further, "cholesterol derivatives" refer to compounds derived from cholesterol, for example, by substitution, addition or removal of substituents.

[0023] As a preferred embodiment of the present invention, the oligonucleotide is modified.

[0024] As a preferred embodiment of the present invention, the oligonucleotide comprises a nucleotide analog.

[0025] As a preferred embodiment of the present invention, the oligonucleotide includes locked nucleic acid (LNA) residues, bicyclic nucleic acid (BNA) residues, constrained ethyl (cEt) residues, unlocked nucleic acid (UNA) residues, phosphorodiamidate morpholino oligomers (PMO) monomers, peptide nucleic acid (PNA) monomers, 2'-O-methyl (2'-OMe) residues, 2'-O-methyloxyethyl residues, 2'-deoxy-2'-fluoro residues, 2'-O-methoxyethyl / thioate residues, phosphoramidates, phosphorodiamidates, thiophosphates, dithiophosphates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methylphosphonates, borophosphonates or O-methylphosphoramidates.

[0026] As a preferred embodiment of the present invention, the above compound is any one of compound numbers 1-11; the specific structures of compound numbers 1-11 are shown in the following table:

[0027]

[0028]

[0029]

[0030] Further preferably, the compound also includes but is not limited to the optical isomers, racemic compounds or other types of mixtures of compound numbers 1-11.

[0031] In one aspect, the present invention provides a compound conjugated with cholic acid or a derivative thereof having a structure of formula II,

[0032]

[0033] or a pharmaceutically acceptable salt thereof, wherein:

[0034] A is an oligonucleotide, which includes small interfering nucleic acid (siRNA) and antisense oligonucleotide (ASO);

[0035] L1, L2, and L3 are each independently absent, -(CH2) h -、-(CH2) h -A group in which any one or more alkylene groups are replaced by a B group; wherein h is 0 to 15; the B group includes

[0036] -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -CH(RC)-, -C(R')(R”)-, -NH-, -N(RN)-, -SS-, -C(R')=C(R”)-, -C≡C-, one or more of; RC, RN, R', R" in the B group indicates that any one or more hydrogen atoms on the designated atom of the group are replaced by a D group, and the conditions for replacement are: the valence of the designated atom does not exceed its normal valence and the compound can generate a stable compound after the hydrogen atom is replaced; the designated atom includes a carbon atom or a nitrogen atom; the D group includes a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, a cyano group, a hydroxyl group, an oxo group, a carboxyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclic group, a heteroaryl group, an aryl group, a ketone, an alkoxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group or a halogen; the halogen includes F, Cl, Br or I; (O) in the B group represents a carbonyl oxygen atom;

[0037] R1 is bile acid or its derivatives;

[0038] R2 is absent, cholic acid or its derivatives, cholesterol or its derivatives, unsubstituted C1-C 22 Alkyl, unbranched C1-C 22 Alkyl, saturated or unsaturated C1-C 22 alkyl, etc. Further, bile acid is preferably ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid or deoxycholic acid. Further, "cholic acid derivative" refers to a compound derived from bile acid, for example, by substitution, addition or removal of a substituent; further, "cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition or removal of a substituent.

[0039] As a preferred embodiment of the present invention, R1 is cholic acid or a derivative thereof, and cholic acid is preferably ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid, deoxycholic acid or others. Furthermore, "cholic acid derivative" refers to a compound derived from cholic acid, for example, by substitution, addition or removal of a substituent.

[0040] As a preferred embodiment of the present invention, A is a modified double-stranded oligonucleotide or a modified single-stranded oligonucleotide, wherein the modified double-stranded oligonucleotide is conjugated with a portion containing bile acid or its derivatives at the 3' end of one strand of the modified double-stranded oligonucleotide, or the modified single-stranded oligonucleotide is conjugated with a portion containing bile acid or its derivatives at the 3' end of the modified single-stranded oligonucleotide.

[0041] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide contains at least one phosphorothioate bond.

[0042] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide contains at least one 2'-O-methyl residue.

[0043] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide contains at least one 2'-deoxy-2'-fluoro residue.

[0044] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO) monomer.

[0045] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or modified single-stranded oligonucleotide comprises a bicyclic nucleic acid (BNA) residue. Further preferably, the bicyclic nucleic acid residue of the modified double-stranded oligonucleotide or modified single-stranded oligonucleotide is a locked nucleic acid (LNA) residue or a restricted ethyl (cEt) residue.

[0046] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide is siRNA or microRNA.

[0047] Further preferably, cholic acid or a derivative thereof is linked to the 3' end of the sense strand of the siRNA or microRNA.

[0048] As a preferred embodiment of the present invention, A is an antisense oligonucleotide.

[0049] In one aspect, the present invention provides a compound conjugated with cholic acid or a derivative thereof having a structure of formula III,

[0050]

[0051] or a pharmaceutically acceptable salt thereof, wherein A is a modified double-stranded oligonucleotide or a modified single-stranded oligonucleotide;

[0052] wherein the modified double-stranded oligonucleotide is conjugated to Z1 at the 3' end of one strand of the modified double-stranded oligonucleotide, or the modified single-stranded oligonucleotide is conjugated to Z1 at the 3' end of the modified single-stranded oligonucleotide, wherein Z1 is

[0053]

[0054] wherein the modified double-stranded oligonucleotide is conjugated to Z2 at the 5' end of one strand of the modified double-stranded oligonucleotide, or the modified single-stranded oligonucleotide is conjugated to Z2 at the 5' end, wherein Z2 is

[0055]

[0056] Wherein: L1, L2, L3, R1 and R2 include any corresponding values ​​described in the present invention.

[0057] Specifically, L1, L2, and L3 are each independently absent, -(CH2) h -、-(CH2) h - is a group formed by replacing any one or more alkylene groups in the group by a B group; wherein h is 0 to 15; the B group includes -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -CH(RC)-, -C(R')(R")-, -NH-, -N(RN)-, -SS-, -C(R')=C(R")-, -C≡C-, one or more of; RC, RN, R', R" in the B group indicates that any one or more hydrogen atoms on the designated atom of the group are replaced by a D group, and the conditions for replacement are: the valence of the designated atom does not exceed its normal valence and the compound can generate a stable compound after the hydrogen atom is replaced; the designated atom includes a carbon atom or a nitrogen atom; the D group includes a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, a cyano group, a hydroxyl group, an oxo group, a carboxyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclic group, a heteroaryl group, an aryl group, a ketone, an alkoxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group or a halogen; the halogen includes F, Cl, Br or I; (O) in the B group represents a carbonyl oxygen atom;

[0058] R1 is bile acid or its derivatives;

[0059] R2 is absent, cholic acid or its derivatives, cholesterol or its derivatives, unsubstituted C1-C 22 Alkyl, linear or branched C1-C 22 Alkyl, saturated or unsaturated C1-C 22alkyl, etc. Further, bile acid is preferably ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid or deoxycholic acid. Further, "cholic acid derivative" refers to a compound derived from bile acid, for example, by substitution, addition or removal of a substituent; "cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition or removal of a substituent.

[0060] As a preferred embodiment of the present invention, R1 is cholic acid or a derivative thereof, and cholic acid is preferably ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid, deoxycholic acid or others. Furthermore, "cholic acid derivative" refers to a compound derived from cholic acid, for example, by substitution, addition or removal of a substituent.

[0061] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide contains at least one phosphorothioate bond.

[0062] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide contains at least one 2'-O-methyl residue.

[0063] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide contains at least one 2'-deoxy-2'-fluoro residue.

[0064] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO) monomer.

[0065] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide or modified single-stranded oligonucleotide comprises a bicyclic nucleic acid (BNA) residue. Further preferably, the bicyclic nucleic acid residue of the modified double-stranded oligonucleotide or modified single-stranded oligonucleotide is a locked nucleic acid (LNA) residue or a restricted ethyl (cEt) residue.

[0066] As a preferred embodiment of the present invention, the modified double-stranded oligonucleotide is siRNA or microRNA. Further preferably, cholic acid or its derivative is linked to the 3' end of the sense strand of the siRNA or microRNA.

[0067] As a preferred embodiment of the present invention, A is an antisense oligonucleotide.

[0068] In one aspect, the present invention provides a cell containing the compound of the above technical solution.

[0069] As a preferred embodiment of the present invention, the cells are hepatocytes, fibroblasts, endothelial cells, kidney cells, human umbilical vein endothelial cells (HUVEC), adipocytes, macrophages, neuronal cells, muscle cells, differentiated primary human skeletal muscle cells or tumor cells.

[0070] In one aspect, the present invention provides a method for introducing the compound of the above technical solution into cells in vitro, comprising contacting the cells with the compound under free uptake conditions.

[0071] As a preferred embodiment of the present invention, the contacting is ex vivo contacting.

[0072] As a preferred embodiment of the present invention, the contacting occurs in vivo.

[0073] As a preferred embodiment of the present invention, A in the compound is a modified double-stranded oligonucleotide.

[0074] A method of introducing a compound into cells in a subject, comprising administering the compound to the subject, wherein the subject suffers from a disease or disorder of the eye, liver, kidney, heart, adipose tissue, lung, muscle or spleen, and the like.

[0075] In one aspect, the present invention provides a method for introducing a modified double-stranded oligonucleotide into a cell, comprising contacting the cell with a compound of the above technical solution under free uptake conditions.

[0076] In the prior art, the siRNA ends are modified by a generally adopted synthesis method of first synthesizing a single strand or a modified single strand and then annealing. The preparation process is as follows:

[0077]

[0078] represents the solid phase carrier CPG, and DMTr represents 4,4'-dimethoxytrityl.

[0079] The preparation method adopted by the present invention is to first synthesize single-stranded RNA, then anneal it, and finally modify the siRNA. The preparation process is as follows:

[0080]

[0081] R represents a lipid moiety modified with bile acid or its derivatives.

[0082] The specific preparation method includes:

[0083] a) introducing a reactive active site at the 5' end of the sense strand of a double-stranded oligonucleotide siRNA or microRNA, or the 3' end of the sense strand of a double-stranded oligonucleotide siRNA or microRNA, or the 5' end of an antisense oligonucleotide, or the 3' end of an antisense oligonucleotide;

[0084] b) preparing cholic acid or its derivatives into activated esters;

[0085] c) mixing the two substances obtained in steps a) and b) together under the condition that the aqueous phase and the organic solvent are mixed at pH=3-12, and shaking overnight;

[0086] d) Purification, desalting, and removal of solvent to obtain the product.

[0087] As a preferred embodiment of the present invention, the active site is preferably an amino group.

[0088] As a preferred embodiment of the present invention, the reaction conditions of step c) include a reaction temperature of 0-100° C., a reaction time of 0.1-24 hours, and a reaction pH of 3-12.

[0089] More preferably, the reaction temperature is 10-40° C., the reaction time is 0.5-16 hours, and the reaction pH is 5-10.

[0090] As a preferred embodiment of the present invention, in step b), the activated lipid prepared from cholic acid or its derivatives is an activated carboxylic acid ester. Further preferably, the activated carboxylic acid ester is preferably NHS ester or pentafluorophenyl ester.

[0091] As a preferred embodiment of the present invention, in step c), the organic solvent includes but is not limited to one or more of acetonitrile, acetone, alcohol solvent, epoxy solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. Further preferably, the alcohol solvent is one or more of methanol, ethanol and propanol. Further preferably, the epoxy solvent is dioxane and / or tetrahydrofuran.

[0092] In one aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound described in the above technical solution.

[0093] As a preferred embodiment of the present invention, the compound is at least one pharmacologically effective amount of a therapeutic compound.

[0094] As a preferred embodiment of the present invention, the excipient is one or more pharmaceutically acceptable excipients.

[0095] As a preferred embodiment of the present invention, the excipients include but are not limited to absorption enhancers, anti-adherents, defoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavoring agents, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents or wetting agents.

[0096] In one aspect, the present invention provides a compound described in the above technical solution for use in preparing a drug.

[0097] On the one hand, the present invention provides administration methods of the compound described in the above technical scheme, including but not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumor injection, ocular administration, nasal administration, oral administration, anal administration or vaginal administration, etc.

[0098] On the one hand, the present invention provides that the subjects for administration of the compound described in the above technical scheme include but are not limited to mammals, poultry, fish, non-human primates or humans; wherein mammals include but are not limited to cattle, horses, mules, donkeys, camels, pigs, sheep, dogs, foxes or rabbits; poultry include but are not limited to chickens, ducks, geese or pigeons.

[0099] The preparation process is suitable for performing different modifications on siRNA and improves work efficiency.

[0100] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 . A structural diagram of a compound of structure (I);

[0102] Figure 2 .H NMR spectrum of intermediate 1-2;

[0103] Figure 3 .Mass spectrum of siRNA conjugate 1;

[0104] Figure 4 .H NMR spectrum of intermediate 2-2;

[0105] Figure 5 .Mass spectrum of siRNA conjugate 2;

[0106] Figure 6 .H NMR spectrum of intermediate 4-5;

[0107] Figure 7 .Mass spectrum of siRNA conjugate 4;

[0108] Figure 8 .H NMR spectrum of intermediate 8-7;

[0109] Fig. 9 .Mass spectrum of siRNA conjugate 8;

[0110] Fig.10 .Knockdown effect of different Luciferase siRNA conjugates on target protein. DETAILED DESCRIPTION

[0111] The present invention is illustrated by the following examples, but it is not intended to limit the present invention in any way. The present invention has been described in detail herein, and its specific embodiments are also disclosed. It will be apparent to those skilled in the art that various changes and modifications will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. It should be noted that compounds and oligonucleotide conjugates herein refer to compounds coupled to oligonucleotides.

[0112] Example 1: Synthesis of siRNA Conjugate 1

[0113]

[0114] Step 1: Preparation of intermediate 1-2

[0115] The intermediate 1-1 (500 mg, 1.27 mmol) was dissolved in DCM (dichloromethane) (15 mL), and the intermediate NHS (N-hydroxysuccinimide) (176 mg, 1.53 mmol), EDCI (carbodiimide hydrochloride) (293 mg, 1.53 mmol), DMAP (4-dimethylaminopyridine) (31 mg, 0.25 mmol) were added in sequence at room temperature, and the mixture was stirred at room temperature overnight under nitrogen protection. TLC (thin layer chromatography) detection was performed. After the reaction was completed, an appropriate amount of water was added, and the mixture was extracted with ethyl acetate for 3 times. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain a crude product. Silica gel column chromatography was performed, and PE / EA (petroleum ether / ethyl acetate) (1:2) was used as the eluent to obtain a white solid 1-2 (514 mg, 82.4%).

[0116] The white solid 1-2 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, CDCl3) δ3.63-3.54 (m, 2H), 2.83 (s, 4H), 2.70-2.60 (m, 1H), 2.58-2.48 (m, 1H), 2.03-1.97 (m, 1H), 0.99-0.94 (m, 6H), 0.69 (s, 3H). The specific hydrogen nuclear magnetic resonance image of the white solid 1-2 is shown in Figure 2 .

[0117] Step 2: Preparation of siRNA conjugate 1

[0118] The double-stranded oligonucleotide siRNA targeting the Luc gene used in this example was purchased from Suzhou Ouli Biopharmaceutical Technology Co., Ltd. We introduced C7-NH2 modification at the 3' end of the sense strand of the double-stranded oligonucleotide siRNA so that it can be efficiently coupled with bile acid or its derivatives to prepare activated carboxylic acid esters through amidation reaction. The modified structure is shown below:

[0119]

[0120]

[0121] Here, lowercase letters represent 2'-O-methyl residues.

[0122] The intermediate Luc-C7-NH2 (200ug, 1eq) was dissolved in PBS phosphate buffer (150uL) at pH = 7.4, and a solution of the intermediate 1-2 (72ug, 10eq) in DMSO (dimethyl sulfoxide) (150uL) was added in batches at room temperature, and shaken overnight at room temperature. RP-HPLC (reverse phase high performance liquid chromatography) purification (Phase A 0.1M TEAA triethylamine acetic acid, Phase B ACN propionitrile), desalting, and removing the solvent to obtain a white powder 1 (123.76ug, 61.9%).

[0123] Characterization by MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry): siRNA conjugate 1 positive strand [M+H] + 7388.6,Found 7390.1; siRNA conjugate 1 guide strand [M+H] + 6635.1,Found6636.1. The mass spectrum of siRNA conjugate 1 is shown in Figure 3 . HPLC (high performance liquid chromatography) purity: 95%.

[0124] Example 2: Synthesis of siRNA Conjugate 2

[0125]

[0126] Step 1: Preparation of intermediate 2-2

[0127] The intermediate 2-1 (500 mg, 1.19 mmol) was dissolved in DCM (15 mL), and the intermediate NHS (164 mg, 1.43 mmol), EDCI (273 mg, 1.43 mmol), and DMAP (29 mg, 0.24 mmol) were added in sequence at room temperature. The mixture was protected by nitrogen and stirred at room temperature overnight. After TLC detection, an appropriate amount of water was added after the reaction was completed, and the mixture was extracted with ethyl acetate for 3 times. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain a crude product. Silica gel column chromatography was performed and eluted with PE / EA (1:2) to obtain a white solid 2-2 (505 mg, 82.1%).

[0128] The white solid 2-2 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, CDCl3) δ3.70 (s, 1H), 3.47-3.32 (m, 1H), 2.83 (s, 4H), 2.70-2.60 (m, 1H), 2.60-2.48 (m, 1H), 1.99-1.95 (m, 1H), 0.97 (d, J = 6.3Hz, 3H), 0.92-0.88 (m, 6H), 0.68 (s, 3H). The hydrogen nuclear magnetic resonance image of the white solid 2-2 is shown in detail. Figure 4 .

[0129] Step 2: Preparation of siRNA conjugate 2

[0130] The intermediate Luc-C7-NH2 (200ug, 1eq) was dissolved in a pH = 7.4 PBS buffer (150uL), and a solution of the intermediate 2-2 (76ug, 10eq) in DMSO (150uL) was added in batches at room temperature, and shaken overnight at room temperature. RP-HPLC purification (0.1M TEAA in phase A, ACN in phase B), desalting, and removal of the solvent gave a white powder 2 (118.30ug, 59.2%).

[0131] Characterization by MALDI-TOF MS: siRNA conjugate 2 sense strand [M+H] + 7416.6,Found 7418.1; siRNA conjugate 2 guide strand [M+H] + 6635.1,Found 6636.1. The mass spectrum of siRNA conjugate 2 is shown in Figure 5 . HPLC purity: 95%.

[0132] Example 3: Synthesis of siRNA Conjugate 4

[0133]

[0134] Step 1: Preparation of intermediate 4-3

[0135] Dissolve the intermediate 1-1 (500 mg, 1.27 mmol) in DMF (n,n-dimethylformamide) (15 mL), add the intermediate 4-2 (653 mg, 1.66 mmol), K2CO3 (528 mg, 3.82 mmol) in turn at room temperature, protect with nitrogen, and stir at 60 ° C overnight. TLC detection, after the reaction is completed, add appropriate amount of water, extract with ethyl acetate 3 times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with PE / EA (1:2) to obtain a colorless oil 4-3 (664 mg, 74.2%).

[0136] The colorless oil 4-3 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, CDCl3) δ4.05 (t, J = 6.7Hz, 2H), 3.64-3.54 (m, 2H), 2.39-2.29 (m, 1H), 2.26-2.16(m,3H),2.02-1.97(m,1H),1.44(s,9H),0.96-0.91(m,6H),0.67(s,3H).

[0137] Step 2: Preparation of intermediate 4-4

[0138] Dissolve the intermediate 4-3 (780 mg, 1.11 mmol) in 4 mol / L HCl-Dioxane solution (10 mL) and stir the mixture at room temperature overnight. After the reaction is complete, concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM / MeOH (dichloromethane / methanol) (10:1) to obtain a colorless oil 4-4 (390 mg, 56.5%).

[0139] The colorless oil 4-4 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR(400MHz, CDCl3)δ4.06(t,J=6.6Hz,2H),3.66-3.55(m,2H),2.38-2.31( m,3H),2.25-2.21(m,1H),2.01-1.98(m,1H),0.96-0.90(m,6H),0.68(s,3H).

[0140] Step 3: Preparation of intermediate 4-5

[0141] Dissolve the intermediate 4-4 (150 mg, 0.23 mmol) in DCM (4 mL), add the intermediate NHS (40 mg, 0.35 mmol), EDCI (67 mg, 0.35 mmol), DMAP (6 mg, 0.05 mmol) in turn at room temperature, protect with nitrogen, stir at room temperature and react overnight. TLC detection, after the reaction is completed, add appropriate amount of water, extract with ethyl acetate 3 times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluted with DCM / MeOH (10:1), to obtain a colorless oil 4-5 (84 mg, 48.5%).

[0142] The colorless oil 4-5 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, CDCl3) δ4.05 (t, J = 6.7Hz, 2H), 3.64-3.52 (m, 2H), 2.83 (s, 4H), 2.60 (t, J = 7.5Hz, 2H), 2.39-2.29 (m, 1H), 2.25-2.17 (m, 1H), 2.02-1.97 (m, 1H), 0.95-0.91 (m, 6H), 0.67 (s, 3H). The specific H NMR of the colorless oil 4-5 is shown in Figure 6 .

[0143] Step 4: Preparation of siRNA conjugates

[0144] The intermediate Luc-C7-NH2 (200ug, 1eq) was dissolved in a sodium carbonate / sodium bicarbonate buffer (100uL) at pH = 8.5, and a solution of the intermediate 4-5 (2.18mg, 200eq) in DMSO (200uL) was added in batches at room temperature, and shaken overnight at room temperature. RP-HPLC purification (0.1M TEAA in phase A, ACN in phase B), desalting, and removal of the solvent gave a white powder 4 (50.31ug, 25.2%).

[0145] Characterization by MALDI-TOF MS: siRNA conjugate 4 sense strand [M+H] + 7642.8,Found 7644.0; siRNA conjugate 4 guide strand [M+H] + 6635.1,Found 6636.1. The mass spectrum of siRNA conjugate 4 is shown in Figure 7 . HPLC purity: 95%.

[0146] Example 4: Synthesis of siRNA Conjugate 8

[0147]

[0148]

[0149] Step 1: Preparation of intermediate 8-3

[0150] Dissolve the intermediate 8-1 (2.0 g, 2.96 mmol) in DMF (40 mL), add the intermediate 8-2 (2.36 g, 3.85 mmol), K2CO3 (3.28 g, 11.84 mmol) in turn at room temperature, protect with nitrogen, and stir at 60 ° C overnight. TLC detection, after the reaction is completed, add appropriate amount of water, extract with ethyl acetate 3 times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluted with PE / EA (5:1), to obtain a white solid 8-3 (1.38 g, 41.6%).

[0151] The white solid 8-3 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, CDCl3) δ7.39-7.30(m,5H),5.15-5.06(m,3H),4.37-4.27(m,1H),4.11(t,J=6.8Hz,2H),2.54-2.38(m, 2H),2.26-2.24(m,1H),2.01-1.90(m,1H),1.63-1.58(m,2H),1.43(s,9H),1.28-1.23(m,26H),0.88(t,J=6.8Hz,3H).

[0152] Step 2: Preparation of intermediate 8-4

[0153] The intermediate 8-3 (1.38 g, 2.46 mmol) was dissolved in 2 mol / L HCl-EA solution (hydrochloric acid-ethyl acetate solution) (25 mL), and stirred at room temperature overnight. After TLC detection, the filter cake was filtered and dried to obtain a white solid 8-4 (1.20 g, 98.4%).

[0154] The white solid 8-4 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, DMSO) δ8.61 (s, 2H), 7.42-7.29 (m, 5H), 5.10 (s, 2H), 4.12 (t, J = 6.5Hz, 2H), 4.05 (t, J = 6.6Hz, 1H), 2.69 -2.59(m,1H),2.58-2.51(m,1H),2.08(q,J=7.3Hz,2H),1.62-1.53(m,2H),1.26-1.21(m,26H),0.85(t,J=6.8Hz,3H).

[0155] Step 3: Preparation of intermediate 8-5

[0156] The intermediate ursodeoxycholic acid (UDCA) (450 mg, 1.15 mmol) was dissolved in DMF (20 mL), and the intermediate 8-4 (608 mg, 1.38 mmol), DIPEA (N, N-diisopropylethylamine) (741 mg, 5.73 mmol), HBTU (o-benzotriazole-N, N, N, N-tetramethyluronium tetrafluoroborate) (522 mg, 1.38 mmol) were added in sequence at room temperature, and nitrogen was protected. The reaction was stirred at room temperature overnight. TLC detection, after the reaction was completed, an appropriate amount of water was added, and the mixture was extracted with ethyl acetate for 3 times. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain a crude product. Silica gel column chromatography, eluted with PE / EA (1:2), to obtain a yellow oil 8-5 (902 mg, 94.1%).

[0157] The yellow oil 8-5 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, CDCl3) δ7.39-7.31(m,5H),6.13(d,J=7.8Hz,1H),5.11(s,2H),4.65-4.58(m,1H),4.15-4.08(m,2H),3.63-3 .54(m,2H),2.53-2.34(m,2H),2.30-2.18(m,2H),2.12-1.97(m,3H),0.95-0.91(m,6H),0.88(t,J=6.8Hz,3H),0.66(s,3H).

[0158] Step 4: Preparation of intermediate 8-6

[0159] The intermediate 8-5 (890 mg, 1.06 mmol) was dissolved in MeOH (10 mL), 10% Pd / C (134 mg, 15% wt) was added, and hydrogen was replaced 3 times. The reaction was stirred at 35°C for 4 h. After TLC detection, the filtrate was filtered and the filter cake was washed with MeOH twice. The filtrate was combined and the solvent was concentrated to obtain a crude product. Silica gel column chromatography was performed and eluted with DCM / MeOH (10:1) to obtain a yellow solid 8-6 (582 mg, 65.2%).

[0160] The yellow solid 8-6 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1HNMR (400MHz, CDCl3) δ6.37(d,J=7.8Hz,1H),4.64(td,J=8.1,5.0Hz,1H),4.13(t,J=6.7Hz,2H),3.65-3.55(m,2H ),2.46-2.37(m,2H),2.36-2.07(m,4H),2.04-2.00(m,1H),0.96-0.93(m,6H),0.88(t,J=6.8Hz,3H),0.67(s,3H).

[0161] Step 5: Preparation of Intermediate 8-7

[0162] Dissolve the intermediate 8-6 (150 mg, 0.20 mmol) in DCM (4 mL), add the intermediate NHS (35 mg, 0.30 mmol), EDCI (58 mg, 0.30 mmol), DMAP (5 mg, 0.04 mmol) in turn at room temperature, protect with nitrogen, stir at room temperature and react overnight. TLC detection, after the reaction is completed, add appropriate amount of water, extract with ethyl acetate 3 times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluted with DCM / MeOH (20:1), to obtain a light yellow solid 8-7 (64 mg, 37.9%).

[0163] The light yellow solid 8-7 was detected by hydrogen nuclear magnetic resonance to obtain the following data: 1 HNMR (400MHz, CDCl3) δ6.22 (d, J = 8.0Hz, 1H), 4.70 (td, J = 7.9, 5.0Hz, 1H), 4.21-4.11 (m, 2H), 3.62-3.53 (m, 2H), 2.84 (s, 4H), 2.80-2.69 (m, 1H), 2.68-2.58 (m, 1H), 2.39-2.26 (m, 2H), 2.18-2.09 (m, 2H), 2.04-1.98 (m, 1H), 0.95-0.92 (m, 6H), 0.88 (t, J = 6.8Hz, 3H), 0.68 (s, 3H). The specific hydrogen nuclear magnetic resonance image of the light yellow solid 8-7 is shown in Figure 8 .

[0164] Step 6: Preparation of siRNA conjugates 8

[0165] The intermediate Luc-C7-NH2 (200ug, 1eq) was dissolved in a sodium carbonate / sodium bicarbonate buffer (100uL) at pH = 8.5, and a solution of the intermediate 8-7 (2.47mg, 200eq) in DMSO (200uL) was added in batches at room temperature, and shaken overnight at room temperature. RP-HPLC purification (0.1M TEAA in phase A, ACN in phase B), desalting, and removal of the solvent gave a white powder 8 (78.52ug, 39.2%).

[0166] Characterization by MALDI-TOF MS: siRNA conjugate 8 sense strand [M+H] + 7741.9,Found 7743.7; siRNA conjugate 8 guide strand [M+H] + 6635.1,Found 6636.7,The mass spectrum of siRNA conjugate 8 is shown in Fig. 9 . HPLC purity: 95%.

[0167] Example 5 Cell evaluation test

[0168] HEK-293T-Luc is derived from HEK-293T cells infected with CMV-Luc-Puro Lentivirus (purchased from Yoshiman Biotechnology). 5 HEK-293T cells were inoculated in 6-well plates. 4 hours after inoculation, 2 μl of virus solution was added. After 24 hours of culture, the culture medium was changed to selection medium for 5 days. 24 hours before transfection, HEK-293-Luc cells were inoculated into white 96-well plates at 30,000 cells / well in 100 μL culture medium. After 24 hours of culture, the culture medium was changed to 90 μL Opti-MEM reduced serum medium. The siRNA conjugate was diluted 1:16.7 in Opti-MEM reduced serum medium, and Lipofectamine 2000 (transfection reagent) was diluted 1:10 in Opti-MEM reduced serum medium. After incubation for 15 minutes, 10 μL of siRNA conjugate: Lipofectamine 2000 complex was added to the cells in reduced serum medium. Luciferase luminescence value was detected using Bright-Lite Luciferase Assay System (firefly luciferase reporter gene detection kit) 24 hours after transfection. The siRNA conjugate preparation was diluted at different concentrations in Opti-MEM reduced serum medium, and 50 μL of the siRNA conjugate preparation dilutions of different concentrations were added to the cells containing 50 μL reduced serum medium. After 24 hours of culture, the luciferase luminescence value was detected using the Bright-Lite Luciferase Assay System.

[0169] like Fig.10 As shown, siRNA conjugates 1, 4 and Luciferase siRNA conjugated cholesterol (i.e., compound 3) were incubated on HEK-293-Luc cells in Opti-MEM medium for 24 hours. The Luciferase luminescence value was detected using the Bright-Lite Luciferase Assay System, and the knockdown effect of siRNA on the target protein was calculated. The results showed that under free uptake conditions, at a concentration of 1000nM, siRNA conjugate 4 could significantly inhibit Luciferase protein expression, while compound 3 had almost no effect.

[0170] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify and vary the above embodiments within the scope of the present invention.

Claims

1. A compound having the structure: in A is an oligonucleotide, which includes small interfering nucleic acid (siRNA) and antisense oligonucleotide (ASO); S1 is linked to the 3' carbon, 5' carbon or nucleobase of a double-stranded oligonucleotide, or to the 3' carbon, 5' carbon or nucleobase of a single-stranded oligonucleotide; The specific structure of S1 is X represents O or S; L1, L2, and L3 are each independently non-existent, -(CH2)h-, or any one or more alkylene groups in -(CH2)h- replaced by a B group; wherein, h is 0 to 15; the B group includes -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -CH(RC)-, -C(R')(R”)-, -NH-, -N(RN)-, -SS-, -C(R')=C(R”)-, -C≡C-, one or more of; RC, RN, R', R" in the B group indicates that any one or more hydrogen atoms on the designated atom of the group are replaced by a D group, and the conditions for replacement are: the valence of the designated atom does not exceed its normal valence and the compound can generate a stable compound after the hydrogen atom is replaced; the designated atom includes a carbon atom or a nitrogen atom; the D group includes a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, a cyano group, a hydroxyl group, an oxo group, a carboxyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclic group, a heteroaryl group, an aryl group, a ketone, an alkoxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group or a halogen; the halogen includes F, Cl, Br or I; (O) in the B group represents a carbonyl oxygen atom; T1, T2, T3, T4, and T5 are each independently absent, -O-, -S-, -C(O)-, -NH-, -CH2-, -C(O)NH-, -NHC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, One or more of; (O) in the T1, T2, T3, T4, T5 groups represents a carbonyl oxygen atom; is the connection site; k is 1 or 2; t is an integer from 1 to 5; j, m, and n are each independently 1 to 15; R1 is bile acid or its derivatives, bile acid includes ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid or deoxycholic acid; R2 is absent, cholic acid or its derivatives, cholesterol or its derivatives, unsubstituted C1-C 22 Alkyl, linear or branched C1-C 22 Alkyl, saturated or unsaturated C1-C 22 Alkyl; bile acids include ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid or deoxycholic acid.

2. The compound according to claim 1, wherein the oligonucleotide is modified.

3. The compound of claim 1, wherein the oligonucleotide comprises a nucleotide analog.

4. The compound of claim 1, wherein the oligonucleotide comprises a locked nucleic acid (LNA) residue, a bicyclic nucleic acid (BNA) residue, a constrained ethyl (cEt) residue, an unlocked nucleic acid (UNA) residue, a phosphorodiamidate morpholino oligomer (PMO) monomer, a peptide nucleic acid (PNA) monomer, a 2'-O-methyl (2'-OMe) residue, a 2'-O-methyloxyethyl residue, a 2'-deoxy-2'-fluoro residue, a 2'-O-methoxyethyl / phosphorothioate residue, a phosphoramidate, a phosphorodiamidate, a phosphorothioate, a phosphorodithioate, a phosphonocarboxylic acid, a phosphonocarboxylate, a phosphonoacetic acid, a phosphonoformic acid, a methylphosphonate, a borophosphonate or an O-methylphosphoramidite.

5. The compound according to claim 1, which is a compound conjugated with cholic acid or its derivative having a structure of Formula II, or a pharmaceutically acceptable salt of the compound, wherein the structure of Formula II is as follows: in: A is a modified double-stranded oligonucleotide or a modified single-stranded oligonucleotide, wherein the modified double-stranded oligonucleotide is conjugated with a moiety containing cholic acid or its derivative at the 3' end of one strand of the modified double-stranded oligonucleotide, or the modified single-stranded oligonucleotide is conjugated with a moiety containing cholic acid or its derivative at the 3' end of the modified single-stranded oligonucleotide; L1, L2, and L3 are each independently non-existent, -(CH2)h-, or any one or more alkylene groups in -(CH2)h- replaced by a B group; wherein h is 0 to 15; and the B group includes: -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -CH(RC)-, -C(R')(R”)-, -NH-, -N(RN)-, -SS-, -C(R')=C(R”)-, -C≡C-, one or more of; RC, RN, R', R" in the B group indicates that any one or more hydrogen atoms on the designated atom of the group are replaced by a D group, and the conditions for replacement are: the valence of the designated atom does not exceed its normal valence and the compound can generate a stable compound after the hydrogen atom is replaced; the designated atom includes a carbon atom or a nitrogen atom; the D group includes a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, a cyano group, a hydroxyl group, an oxo group, a carboxyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclic group, a heteroaryl group, an aryl group, a ketone, an alkoxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group or a halogen; the halogen includes F, Cl, Br or I; (O) in the B group represents a carbonyl oxygen atom; R1 is bile acid or its derivatives, bile acid includes ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid or deoxycholic acid; R2 is absent, bile acid or its derivatives, cholesterol or its derivatives, unsubstituted C1-C 22 Alkyl, unbranched C1-C 22 Alkyl, saturated or unsaturated C1-C 22 Alkyl, bile acids include ursodeoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, dehydrocholic acid or deoxycholic acid.

6. The compound according to claim 5, wherein the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide contains at least one phosphorothioate bond; and / or The modified double-stranded oligonucleotide or modified single-stranded oligonucleotide contains at least one 2'-O-methyl residue; and / or The modified double-stranded oligonucleotide or modified single-stranded oligonucleotide contains at least one 2'-deoxy-2'-fluoro residue; and / or The modified double-stranded oligonucleotide or modified single-stranded oligonucleotide includes a phosphorodiamidate morpholino oligomer (PMO) monomer.

7. The compound according to claim 5, wherein the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide comprises a bicyclic nucleic acid (BNA) residue. 8 . The compound according to claim 7 , wherein the bicyclic nucleic acid residue of the modified double-stranded oligonucleotide or the modified single-stranded oligonucleotide is a locked nucleic acid (LNA) residue or a constrained ethyl (cEt) residue.

9. The compound according to claim 5, wherein the modified double-stranded oligonucleotide is siRNA or microRNA. 10 . The compound according to claim 9 , wherein cholic acid or a derivative thereof is linked to the 3′ end of the sense strand of the siRNA or microRNA. The compound according to claim 5 , wherein A is an antisense oligonucleotide.

12. The compound according to claim 1, wherein the compound is any one of compound numbers 1 to 11, The optical isomers, racemic compounds or mixtures of Compound Nos. 1 to 11 are also included.

13. A method for preparing the compound according to any one of claims 5 to 12: a) introducing a reactive active site at the 5' end of the sense strand of a double-stranded oligonucleotide siRNA or microRNA, or the 3' end of the sense strand of a double-stranded oligonucleotide siRNA or microRNA, or the 5' end of an antisense oligonucleotide, or the 3' end of an antisense oligonucleotide; b) preparing cholic acid or its derivatives into activated esters; c) mixing the two substances obtained in the above steps a) and b) together under the condition that the aqueous phase and the organic solvent are mixed at pH=3-12, and shaking overnight; d) Purification, desalting, and removal of solvent to obtain the product.

14. A cell containing the compound of claim 1, wherein the cell is a hepatocyte, a fibroblast, an endothelial cell, a kidney cell, a human umbilical vein endothelial cell (HUVEC), an adipocyte, a macrophage, a neuronal cell, a muscle cell, a differentiated primary human skeletal muscle cell or a tumor cell.

15. A method of introducing a compound according to any one of claims 1 to 12 into a cell, the method comprising contacting the cell with the compound, wherein the contact occurs in vitro or in vivo.

16. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound according to any one of claims 1 to 12, wherein the compound is at least one pharmacologically effective amount of a therapeutic compound, and the excipient is one or more pharmaceutically acceptable excipients.

17. A method of introducing a compound according to any one of claims 1 to 12 into cells in a subject, comprising administering the compound to the subject, wherein the subject suffers from a disease or disorder of the eye, liver, kidney, heart, adipose tissue, lung, muscle or spleen.

18. Use of a compound according to any one of claims 1 to 12 for the preparation of a medicament.

19. The compound according to claim 1, wherein the administration method comprises intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumor injection, ocular administration, nasal administration, oral administration, anal administration or vaginal administration.

20. The compound according to claim 1, wherein the subject to be administered includes but is not limited to mammals, birds, fish, non-human primates or humans; wherein, Mammals include cattle, horses, mules, donkeys, camels, pigs, sheep, dogs, foxes or rabbits; poultry include chickens, ducks, geese or pigeons.

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