Targeting ligands and their uses

Abstract

The present invention is applied to the technical field of nucleic acid drug delivery, and specifically provides a nucleic acid delivery compound, a liver-targeting nucleic acid ligand and its use.The present invention provides a compound represented by formula (Z-1) and a compound represented by formula (I) and formula (II), and the oligonucleotide conjugate according to the embodiment of the present invention can improve the binding efficiency of the targeting moiety to cells or cell receptors, and increase the effect of RNA interference.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to a Chinese patent application filed on January 20, 2022, bearing application number 202210068123.3 and entitled "siRNA Conjugate and Use Thereof," the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the technical field of nucleic acid drug delivery, and specifically to a nucleic acid delivery compound, a liver-targeting nucleic acid ligand and uses thereof. [Background technology]

[0003] RNA interference (RNAi) is widespread in nature and has now become a mature technological tool in molecular biology and medicine.

[0004] The targeting ligand is a very important part in the RNAi process, helping to guide the delivery of the therapeutic agent linked thereto to the desired target location, and the targeting portion of the targeting ligand consists of one or more targeting groups or targeting moieties. In some cases, the targeting moiety can bind to a cell or a cell receptor and initiate endocytosis to facilitate the entry of the therapeutic agent into the cell.

[0005] The liver is an important organ of metabolism in the human body, and the most major cell type performing liver functions in the liver is the hepatocyte, which has abundant asialoglycoprotein receptors (ASGPR) on its surface. It is known that targeting moieties that bind to the asialoglycoprotein receptor (ASGPR) can specifically deliver oligonucleotide compounds (such as siRNA) to the liver. Targeting moieties that target the asialoglycoprotein receptor include galactose or galactose derivatives. In the process of use, the N-acetyl-galactosamine (GalNAc) clusters coupled to the oligomeric compounds can guide the composition to the liver, and the N-acetyl-galactosamine can promote the entry of the compound into the cell interior by binding to the ASGPR receptor on the surface of the liver cells. Therefore, the improvement of this targeting ligand can improve the working efficiency of RNAi.

[0006] In addition, because nucleotide molecules usually have a high negative charge, the complex system of cell membranes restricts nucleic acid molecules or other compounds that are difficult to permeate through the cell membrane from penetrating the cell membrane and directly diffusing into living cells. Therefore, the main obstacle to the delivery of nucleic acid molecules is how to deliver nucleic acid molecules to the cytoplasm or cell nucleus. Currently, known solutions include the use of nanoliposomes, viral vectors, conjugated coupling delivery molecules, etc. Conjugated coupling delivery molecules, such as coupling GalNAc molecules, polypeptide molecules, antibody molecules, etc., function as guides to guide siRNA to target cells. Therefore, it is necessary to develop a cleavable linker to achieve this function, as to how the coupled delivery molecule can be separated from the siRNA molecule after completing its guide role. Summary of the Invention

[0007] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides compounds that can be used for nucleic acid coupling delivery ligands, liver targeting nucleic acid ligands and their use in nucleic acid molecule delivery, as well as applications in the field of disease treatment.

[0008] A first aspect of the present invention provides a cleavable compound represented by formula (Z-1) or a pharma- ceutically acceptable salt thereof: [ka] In the formula, R1 is O, S, NR3, or CR3R4, and R3 and R4 are each independently hydrogen, halogen, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocycle, or a substituted or unsubstituted cycloalkyl group; R2 may be -O-, -S-, -NH-, -CH2-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2NH-, -CHO-, -NH-C(O)-CH2-, -C(O)-CH2-NH-, or -NH(CO)NH-, wherein the -CH2- may be further optionally substituted with a substituent selected from a halogen, an alkyl group (which may be optionally substituted with a substituent selected from a hydroxy group, an amino group, and a halogen), an alkoxy group, and an alkylamino group; The a and b are the same or different and each is an integer selected from 0 to 20, preferably 1 to 10, and more preferably 1 to 5.

[0009] In one preferred embodiment, the general formula of compound Z is represented by the following formula (Z-2): [ka] Preferably, R2 is -NH-, and then compound Z is [ka] and In another embodiment, R2 is preferably -C(O)-, and the compound Z is [ka] It is.

[0010] The target ligand compound or a pharma- ceutically acceptable salt thereof according to the second aspect of the present invention includes the above-mentioned cleavable compound, and its general formula is represented by the following formula (I): [ka] (I), In the formula, Z is a cleavable compound represented by the above general formula (Z-1), Alternatively, the compound comprises a structure of general formula (II): [ka] (II), In the formula, L3 includes a cleavable compound represented by the above formula (Z-1).

[0011] Specifically, in the general formula (I), a1 is an integer selected from 1 to 10, preferably 1 to 5; Z is a first linker portion of a nucleotide sequence (the above-mentioned cleavable compound Z-1), which is linked to a nucleotide sequence X, which may be any nucleotide sequence, preferably an oligonucleotide sequence, which may be a sense strand or an antisense strand of an siRNA; Z and X may be linked directly or via a chemical group; and the general formula of Z is as shown in the above-mentioned (Z-1), one end of the O group is linked to the nucleotide sequence; [ka] Preferably, said nucleotide sequence X comprises 30 or fewer nucleotides, more preferably 23 or fewer nucleotides.

[0012] Specifically, in formula (I), L1 is a second linker moiety, E is a branch point group, and the two are directly linked.

[0013] Preferably, the branch point group E is linked to a1 targeting complexes, a1 being an integer selected from 0 to 10, preferably 1 to 5, and the targeting complexes comprise a 1:1 ratio of tethering moiety L2 to targeting moiety T.

[0014] When the above formula (II) binds to a nucleotide sequence, it may bind to the 5' end of the nucleotide sequence, or to the 3' end of the nucleotide sequence, or to the 5' end and the 3' end simultaneously, or it may bind to a nucleotide sequence X according to the following formula (III), in which L4 and L3 are the same or different and are both targeting moieties, Y is O, S or N, and b, c, d and e are integers selected from 0 to 10. [ka] (III)

[0015] Specifically, in general formulas (II) and (III), L3 comprises a third linker moiety, and L4 in general formula (III) is a targeting moiety. The targeting moiety T in general formula (I) and L3 and L4 in general formula (III) may be the same or different, and preferably, L3 and L4 are, respectively: [ka] (II-aI), [ka] (II-a-II), [ka] (II-a-III), [ka] (II-a-III) In the formula, R2 may be -O-, -S-, -NH-, -CH2-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2NH-, -CHO-, -NH-C(O)-CH2-, -C(O)-CH2-NH-, or -NH(CO)NH-, wherein the -CH2- may be optionally substituted with a substituent selected from a halogen and an alkyl group (which may be optionally substituted with a substituent selected from a hydroxy group, an amino group, a halogen, an alkoxy group, and an alkylamino group); The above p, q, r, s, t and u are each independently an integer selected from 0 to 20, preferably 1 to 10, and more preferably 1 to 5.

[0016] In the above formula (III), X is a nucleotide sequence, which may be a sense strand or an antisense strand; Y is O or S; b, c, d and e are each an integer selected from 0 to 10, preferably 0 to 5, and b and e are not simultaneously 0. In one embodiment, b is 0, and e is an integer from 3 to 6, and in one preferred embodiment, b=0, d=2, and e=1.

[0017] Specifically, in the general formula (I), the second linker portion L1 is [ka] [ka] [ka] or [ka] It has the following structure: In the formula, f, g, h and i each represent an integer of 1 to 20, preferably an integer of 1 to 10, and more preferably an integer of 1 to 5.

[0018] Specifically, the structural formula of the compound L2 in the general formula (I) is [ka] [ka] [ka] or [ka] and In the formula, j, k, l, m, n and o each represent an integer of 1 to 20, preferably an integer of 1 to 10, and more preferably an integer of 1 to 5.

[0019] Further, the targeting moiety T is selected from a tissue specific targeting ligand, for example, the targeting moiety T is a liver specific targeting ligand or other tissue specific targeting ligand, preferably, the targeting moiety T has a structure that enhances uptake of the oligomeric compound into a target cell, and in one preferred embodiment, the target cell is a liver cell.

[0020] The oligonucleotide conjugates according to the embodiments of the present invention can improve the binding efficiency of the targeting moiety to cells or cell receptors, and improve the RNA interference effect.

[0021] The present invention further provides the use of the compounds of formula (Z-1), formula (I) and formula (II) and their pharma- ceutically acceptable salts in a delivery nucleic acid molecule, which can deliver a nucleic acid molecule to a specific cell in the body.

[0022] Furthermore, the compound of formula (Z-1) or a pharma- ceutically acceptable salt thereof is cleaved at the R2 site after delivering the nucleic acid molecule to a target cell.

[0023] Additionally, the compounds of formula (I) and formula (II) and their pharma- ceutically acceptable salts are used for targeting and delivering nucleic acids to human or mammalian liver cells.

[0024] Further, the liver cells are hepatocytes.

[0025] The compounds represented by formula (Z-1), formula (I) and formula (II) and pharma- ceutically acceptable salts thereof according to the embodiments of the present invention are used as a nucleic acid coupling delivery ligand or a part of the structure thereof, and the delivery ligand can deliver a target nucleic acid to a specified cell with high delivery efficiency. [Brief description of the drawings]

[0026] [Figure 1] 1A and 1B are graphs showing the results of detection 28 and 35 days after the compound according to an embodiment of the present application suppressed the expression of ANGPTL3. [Diagram 2] FIG. 1 shows the results of detecting LPA siRNA concentrations in the liver and kidney of a mouse in one example of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In order to clarify the technical problems to be solved, the technical solutions and the beneficial effects of the present invention, the present invention will be described in more detail below with reference to the embodiments and chemical reaction formulas. Note that the specific embodiments described herein are merely for interpreting the present invention and are not intended to limit the present invention.

[0028] Definition of Terms In this application, the term "oligonucleotide" generally refers to a polymer consisting of multiple nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogs thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogs thereof). Thus, the term "oligonucleotide" generally refers to a nucleotide polymer in which the nucleotide residues and the linkages between them are naturally occurring, but it is understood that the term also includes within its scope various analogs, including, but not limited to, peptide nucleic acid (PNA), phosphoramidate, phosphorothioate, methylphosphonate, 2-O-methyl ribonucleic acid, and the like. The appropriate size of the molecule depends on the specific application. Oligonucleotides are generally short in length, usually about 10-30 nucleotide residues, although the term may refer to molecules of any length. The terms "polynucleotide" or "nucleic acid" are generally used for larger oligonucleotides.

[0029] In some embodiments, an oligonucleotide comprises one or more unmodified nucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides. The term "modified oligonucleotide" generally refers to an oligonucleotide that contains at least one modified nucleotide and / or at least one modified internucleotide linkage.

[0030] As used herein, the term "modified nucleotide" refers to a nucleotide that contains at least one chemical modification compared to a naturally occurring RNA or DNA nucleotide. Modified nucleotides include modified sugar moieties and / or modified nucleobases.

[0031] In this application, the term "nucleobase" generally refers to a heterocyclic pyrimidine or purine compound, which is a component of all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Nucleotides may include modified nucleotides or nucleotide analogs, abasic sites (Ab or X) or alternative moieties. As used herein, "nucleobase sequence" generally refers to the order of consecutive nucleobases, independent of any sugar, linkage or nucleobase modification. The term "unmodified nucleobase" or "naturally occurring nucleobase" generally refers to the naturally occurring heterocyclic nucleobases of RNA or DNA, i.e., the purine bases (adenine (A) and guanine (G)) and the pyrimidine bases (thymine (T), cytosine (C) (including 5-methyl C) and uracil (U)). "Modified nucleobase" generally refers to any nucleobase that is not a naturally occurring nucleobase.

[0032] In this application, the term "ligand" generally refers to any compound or molecule that can be covalently or otherwise chemically bound to a biologically active substance (e.g., an oligonucleotide). In some embodiments, a ligand can directly or indirectly interact with another compound, such as a receptor, which may be present on the cell surface or, alternatively, may be an intracellular and / or intercellular receptor, and the interaction between the ligand and the receptor may result in a biochemical reaction or may be a physical interaction or binding.

[0033] An embodiment of the present invention provides a cleavable compound represented by formula (Z-1): [ka] In the formula, R1 is O, S, NR3, or CR3R4, and R3 and R4 are each independently hydrogen, halogen, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocycle, or a substituted or unsubstituted cycloalkyl group; R2 may be -O-, -S-, -NH-, -CH2-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2NH-, -CHO-, -NH-C(O)-CH2-, -C(O)-CH2-NH-, or -NH(CO)NH-, wherein the -CH2- may be further optionally substituted with a substituent selected from a halogen, an alkyl group (which may be optionally substituted with a substituent selected from a hydroxy group, an amino group, and a halogen), an alkoxy group, and an alkylamino group, and a and b are the same or different and each is an integer selected from 0 to 20, preferably 1 to 10, and more preferably 1 to 5.

[0034] In one preferred embodiment, compound Z is [ka] or [ka] or [ka] It is.

[0035] The compound O group binds to the nucleotide sequence and is cleaved at the left R2 position after delivering the nucleic acid sequence to the target location.

[0036]

[0013] The present embodiment further provides an oligonucleotide liver targeting ligand, the oligonucleotide liver targeting ligand having the general formula: [ka] (I), or [ka] (II), or after binding to nucleotide sequence X, [ka] It has the structure (III).

[0037] Specifically, in the general formula (I), X is a sense or antisense strand of a nucleotide, Z is a linker moiety of the sense or antisense strand, E is a branch point group, L1 is a linking moiety between the linker moiety of the sense or antisense strand and the branch point group E, T is a targeting moiety, L2 is a tethering moiety between the targeting moiety and the branch point group E, and a1 is an integer selected from 1 to 10, preferably 1 to 5, and more preferably 1 or 3.

[0038] In the general formulas (II) and (III), L3, L4 are targeting moieties, X is a sense strand or an antisense strand, Y is O, S or N, and b, c, d and e are integers selected from 0-10, preferably 1-5.

[0039] Specifically, the above T, L3, and L4 may be the same or different.

[0040] In one embodiment, in formula (I), the targeting ligand L1 is [ka] [ka] [ka] or [ka] It has the general formula:

[0041] In the formula, f, g, h and i each represent an integer of 1 to 20, preferably an integer of 1 to 10, and more preferably an integer of 1 to 5.

[0042] Preferably, L1 is [ka] [ka] [ka] [ka] It has the following structure.

[0043] In one embodiment, in general formula (I), the compound E is [ka] is selected from.

[0044] In one embodiment, the compound L2 in the general formula (I) is [ka] [ka] [ka] or [ka] and In the formula, j, k, l, m, n, and o each represent an integer of 1 to 20, preferably an integer of 1 to 10, and more preferably an integer of 1 to 5. Preferably, L2 is [ka] [ka] [ka] [ka] [ka] [ka] or [ka] It has the structural formula:

[0045] In a preferred embodiment, the general formula (I) is [ka] (II) (wherein a1=3), [ka] (I-II) (wherein a1=3), [ka] (I-III) (wherein a1=3), [ka] (I-IV) (wherein a1=3), [ka] (IV) (wherein a1=3), [ka] (I-VI) (wherein a1=3), [ka] (I-VII) (wherein a1=3), or [ka] (I-VIII) (wherein a1=3).

[0046] In one embodiment, T of the targeting ligand is one selected from N-acetyl-galactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine and N-isobutyryl-galactosamine, preferably N-acetyl-galactosamine, having the structural formula: [ka] or [ka] It is.

[0047] In one preferred embodiment, the targeting ligand represented by general formula (I) is linked to the siRNA terminus via a phosphate, thiophosphate, or phosphonate group.

[0048] In one preferred embodiment, the targeting ligand is [ka] (I-1), [ka] (I-2), [ka] (I-3), [ka] (I-4), [ka] (I-5), [ka] (I-6), [ka] (I-7), [ka] (I-8), [ka] (I-9), [ka] (I-10), [ka] (I-11), [ka] (I-12), [ka] (I-13), [ka] (I-14), [ka] (I-15), or [ka] It has the structure (I-16).

[0049] In one embodiment, the target ligand compound is [ka] (II-aI), [ka] (II-a-II), [ka] (II-a-III), or [ka] (II-a-III) In the formula, R2 is -O-, -S-, -NH-, -CH2-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2NH-, -CHO-, -NH-C(O)-CH2-, -C(O)-CH2-NH-, or -NH(CO)NH-, wherein the -CH2- is optionally substituted with a substituent selected from a halogen, an alkyl group (which may be further optionally substituted with a substituent selected from a hydroxy group, an amino group, a halogen, an alkoxy group, and an alkylamino group), an alkoxy group, and an alkylamino group; The above p, q, r, s, t and u are each independently an integer selected from 0 to 20, preferably 1 to 10, and more preferably 1 to 5.

[0050] In one preferred embodiment, the target ligand compound is [ka] (II-a-1), [ka] (II-a-2), [ka] (II-a-3), [ka] (II-a-4), [ka] (II-a-5), or [ka] It contains the structure (II-a-6).

[0051] In another preferred embodiment, the target ligand compound comprises [ka] (II-a-IV), [ka] (II-aV), [ka] (II-a-VI), or [ka] It contains the structure (II-a-VII).

[0052] In the formula, R2 is -O-, -S-, -NH-, -CH2-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2NH-, -CHO-, -NH-C(O)-CH2-, -C(O)-CH2-NH-, or -NH(CO)NH-, wherein the -CH2- is optionally substituted with a substituent selected from a halogen, an alkyl group (which may be further optionally substituted with a substituent selected from a hydroxy group, an amino group, a halogen, an alkoxy group, and an alkylamino group), an alkoxy group, and an alkylamino group; The p, q, r, s, t and u are each independently an integer selected from 0 to 20, preferably 1 to 10, and more preferably 1 to 5. In one preferred embodiment, the target ligand compound is [ka] (II-a-7), [ka] (II-a-8), [ka] (II-a-9), [ka] (II-a-10), [ka] (II-a-11), or [ka] It contains the structure (II-a-12).

[0053] In one preferred embodiment, the general formula (III) is [ka] (II-1), [ka] (II-2), [ka] (II-3), [ka] (II-4), [ka] (II-5), [ka] (II-6), [ka] (II-7), [ka] (II-8), [ka] (II-9), or [ka] It has the structure (II-10).

[0054] In the formula, Y is O or S.

[0055] In another preferred embodiment, the targeting ligand compound which binds to the nucleotide X has the formula (III): [ka] (II-11), [ka] (II-12), [ka] (II-13), [ka] (II-14), [ka] (II-15), [ka] (II-16), [ka] (II-17), [ka] (II-18), [ka] (II-19), or [ka] It has the structure (II-20), In the formula, Y is O, S or N.

[0056] In some embodiments, the sense strand is 5'-NmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm-3', 5'-NmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNms-3, 5'-NmNmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm-3', 5'-sNmNmNmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm-3', or The nucleotide sequence is selected from the nucleotide sequence represented by the formula: 5'-sNmNmNmNmNmNmNmNmNfNfNmNmNmNmNmNmNmNmNmNms-3'.

[0057] where Nm represents any methoxy-modified nucleotide, such as methoxy-modified cytosine (C), guanine (G), uracil (U), adenine (A), thymine (T), and Nf represents any fluoro-modified nucleotide, such as fluoro-modified C, G, U, A, T; When a lowercase letter s is in the center of a capital letter, it indicates that the two adjacent nucleotides to the left and right of the letter s are linked by a thiophosphate group; when the lowercase letter s is in the first position at the 3' end, it indicates that the one nucleotide terminal adjacent to the left of the letter s is a thiophosphate group; and when the lowercase letter s is in the first position at the 5' end, it indicates that the one nucleotide terminal adjacent to the right of the letter s is a thiophosphate group.

[0058] In some embodiments, the antisense strand is It has a nucleotide sequence represented by the formula: 5'-Nms'Nfs'Nm'Nm'Nm'Nf'Nm'Nm'Nm'Nm'Nm'Nm'Nm'Nf'Nm'Nm'Nm'Nm'Nf'Nm'Nm'Nm'Nm'Nm'Nms'Nms'Nm-3'.

[0059] where Nm' represents any methoxy modified nucleotide, e.g., methoxy modified C, G, U, A, T; Nf' represents any fluoro modified nucleotide, e.g., fluoro modified C, G, U, A, T; When a lowercase s is placed in the middle of an uppercase letter, it indicates that the two adjacent nucleotides on the left and right of the s are linked by a phosphorothioate group.

[0060] The synthetic route of the target ligand of the present invention will be described below with reference to specific examples.

[0061] Example 1 Synthesis of the aminogalactose compound GENO-Gal-5 linked to a solid-phase vector (1) Synthesis Route [ka] [ka] [ka] (2) Specific synthesis process 1) Preparation of compound Int-11-2 [ka] Int-11-1 (10 g) was dissolved in (140 mL) DCM (dichloromethane) and cooled to 0 ° C., and TMSCN (trimethylsilyl cyanide, trimethylsilyl cyanide (TMSCN)) (4.01 g) and BF3Et2O (2.83 mL) were added dropwise and reacted for 10 min. The reaction was monitored on a TLC plate until the reaction of the raw material was completed (Hexane: EtOAc = 5: 1, KMnO4 color development, raw material Rf = 0.3, α configuration Rf = 0.28, β configuration Rf = 0.27). After the reaction was completed, 100mL of saturated NaHCO3 aqueous solution was added to the reaction solution, 100ml of DCM was added to separate the organic phase, the organic phase was washed once with saturated saline, the organic phase was concentrated, and then dissolved in EtOAc (200mL), washed once with NaHCO3 aqueous solution (100mL), washed once with saturated saline, the organic phase was dried over anhydrous sodium sulfate and concentrated. Then, it was purified with a normal phase silica gel column, and the polarity was gradually increased, and when Hexane / EtOAc = 20%, α configuration was shown, and when it was 25%, β configuration was shown, a white solid (5.3g) was obtained in the α configuration, and a colorless oil (3.7g) was obtained in the β configuration. 1 HNMR(400MHz,DMSO)δ6.03~6.06(dt,1H),5.91~5.94(dt,1H),5.35(dq,1H), 5.12(m,1H),4.30(dd,1H),4.30(dd,1H),3.82(ddd,1H),2.10~2.12(2s,6H).

[0062] 2) Preparation of compound Int-11-3 [ka] Aqueous HCl (1M, 17.2mL) was added to a suspension of Int-11-2 (3.7g, β-configuration, colorless oil) and 10% Pd / C (377mg), and a mixture of ethyl acetate / 2-propanol / ethanol (2:1:1, total 90mL) was added and the reaction was stirred under hydrogen (40Psi) for 48 hours. The reaction was monitored by TLC, filtered through diatomaceous earth to remove the catalyst, and the solvent was concentrated under reduced pressure. The obtained crude product was washed twice with toluene, dissolved in methanol (10mL), added with 28% NH3H2O ​​(30mL), stirred at room temperature for 16 hours, concentrated the reaction solution, and then washed three times with a 1:1 toluene-acetonitrile mixture (50mL). 1g of the above crude product was dissolved in H2O (40mL) and cooled to 0°C, NaHCO3 (1.4g) and Na2CO3 (0.88g) were added, FmocOSu (2.13g) was dissolved in dioxane (40mL) solvent, and then added dropwise to the above aqueous solution and reacted at room temperature for 1 hour. The reaction was monitored by TLC (DCM:MeOH=10:1, 254nm, Rf=0.5), and purified with a normal phase column to obtain the product Int-11-3 (340mg) as a colorless solid with MeOH / DCM=5%. 1 HNMR(CDCl3):δ7.76-7.78(d,2H),7.58-7.60(d,2H),7.39-7.42(t,2H),7.30-7.34(t,2H),5.12(t,1H),4.42-4.52( m,2H),4.22(t,1H),3.84(br.s,2H).3.47-3.57(m,3H),3.11-3.24(m,2H),1.30-1.56,1.67-1.72,2.05-2.22(m,4H).

[0063] 3) Preparation of compound Int-11-4 [ka] Int-11-3 (340 mg) was dissolved in 2 ml of pyridine and cooled to 0°C. DMTrCl (450 mg) was dissolved in 2 ml of pyridine and then added dropwise to the above solution. The reaction was monitored by TLC until the reaction of the raw material was completed. The reaction was quenched by adding 5 ml of aqueous solution, and the product int-11-4 (290 mg) was obtained by purification with reverse phase column C18 and MeCN / H2O=80%.

[0064] 4) Preparation of compound GENO-Int-11 [ka] Int-11-4 (700 mg, 1.02 mmol) was dissolved in 2 mL of dichloromethane, and then DBU (310 mg, 2.04 mmol) was added to the above solution, and the reaction was monitored by TLC until the reaction of the raw material was completed. The reaction was quenched by adding 5 mL of saturated Na2CO3 solution, extracted with 20 mL of dichloromethane, and the organic phase was concentrated with DCM:MeOH=10:1 (254 nm, Rf=0.5). The product was purified by normal phase column with MeOH / DCM=5% to obtain the product GENO-Int-11 (550 mg) as a yellow solid.

[0065] 5) Preparation of Compound Gal-5-1 [ka] 11-Benzyloxycarbonylundecanoic acid (387 mg, 1.2 mmol) was dissolved in anhydrous DMF (N,N-Dimethylformamide) (10 mL) solvent, then HBTU (O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate) (546 mg, 1.44 mmol), DIEA (N,N-Diisopropylethylamine) (0.65 mL, 3.6 mmol), HOBT (1-Hydroxybenzotriazole) (324 mg, 2.4 mmol) and GENO-Int-11 (560 mg, 1.2 mmol) were added, and the mixture was stirred overnight at room temperature under argon protection. The reaction solution was purified with a reverse phase column C18, and the product Gal-5-1 (200 mg) was obtained by MeCN / H2O = 70%. 1 HNMR(400MHz,DMSO)δ7.68(t,1H),7.49-7.12(m,14H),6.86(d,4H),5.07(s,2H),4.58(d,1H),3.73(s,6H),3.38(dd,1H),3.28-3.16 (m,3H),3.22-2.85(m,3H),2.41-2.25(m,2H),2.11-2.00(m,2H),1.92(dd,1H),1.64(d,1H),1.57-1.41(m,4H),1.27-1.06(m,14H).

[0066] 6) Preparation of Compound Gal-5-2 [ka] Gal-5-1 (200 mg, 0.26 mmol) was dissolved in 5 mL of ethyl acetate, 50 mg of palladium on carbon and triethylamine (0.11 mL, 0.78 mmol) were added, and the reaction was stirred under hydrogen (15 psi) at room temperature for 16 h. The reaction was filtered and concentrated to give Gal-5-2 (150 mg).

[0067] 7) Preparation of Compound Gal-5-3 [ka] Gal-5-2 (150 mg, 0.22 mmol) was dissolved in 5 mL of anhydrous DMF, and HBTU (101 mg, 0.27 mmol), DIEA (0.16 mL), 3A molecular sieves (1 g) and Gal-3-4C (441 mg, 0.22 mmol) were added to the solution. The mixture was then stirred at room temperature for 16 hours, and the reaction solution was purified by reverse phase column C18 and MeCN / H2O=60% to obtain the product Gal-5-3 (380 mg). 1 HNMR(400MHz,DMSO)δ7.91-7.67(m,10H),7.42(d,2H),7.34-7.24(m,6H),7.20(d,1H),6.99(s,1H), 6.87(d,4H),5.21(d,3H),4.97(dd,3H),4.60(s,1H),4.48(d,3H),4.09-3.95(m,10H),3.93-3.82(m ,3H),3.77-3.66(m,9H),3.61-3.48(m,12H),3.45-3.20(m,16H),3.09-2.95(m,15H),2.27(t,6H),2 .10(s,9H),2.05(dd,10H),1.99(s,9H),1.89(s,9H),1.76(d,9H),1.63-1.38(m,24H),1.16(s,14H).

[0068] 8) Preparation of Compound Gal-5-4 [ka] Gal-5-3 (370 mg, 0.15 mmol), succinic anhydride (75 mg, 0.76 mmol), 3A molecular sieves (0.5 g) and DMAP (4-Dimethylaminopyridine) (46 mg, 0.38 mmol) were dissolved in 5 mL of THF (Tetrahydrofuran), and the reaction solution was stirred at 40° C. overnight. The reaction solution was purified on a reversed-phase C18 column and the product Gal-5-4 (220 mg) was obtained using MeCN / H2O=40%.

[0069] 9) Synthesis of the aminogalactose compound GENO-Gal-5 linked to a solid-phase vector [ka] Gal-5-4 (220 mg, 0.086 mmol) was suspended in 4 mL of CH3CN and 2 mL of DMF, DIEA (0.035 mL, 0.216 mmol) and HBTU (49.07 mg, 0.129 mmol) were added dropwise, and the mixture was stirred at room temperature for 5 minutes. Aminomethyl resin (99.51 mg, 100-200 mesh, amino loading amount 250 umol / g) was added to the reaction solution, and the reaction was carried out on a shaker at 25°C with a rotation speed of 220 rpm. After reacting for 16 h, it was filtered, the filter cake was rinsed 3 times with DCM at 30 ml each time, rinsed 3 times with acetonitrile at 30 ml each time, rinsed 3 times with 30 ml of n-hexane, dried with a vacuum oil pump for 2 h, and then the mixed reagent (CapB1, 4-dimethylaminopyridine, N-methylimidazole, acetonitrile, 11.2 mL / 12.4 mg / 0.50 mL / 4.32 mL) was added to perform the capping reaction. It was placed on a shaker at 25 ° C, the rotation speed was set to 220 rpm, and reacted for 16 h, the reaction solution was filtered, the filter cake was rinsed 3 times with acetonitrile at 30 ml each time, suction filtered until dry, and dried overnight under reduced pressure with a vacuum oil pump to obtain the target product GENO-Gal-5 compound (160 mg).

[0070] 10) Preparation of Compound Int-6-1 [ka] Gal-3-5B (1.45 g, 3.24 mmol) was dissolved in anhydrous DMF (10 mL) solvent, then HATU (1.64 g, 4.32 mmol), 3A molecular sieves (1 g) and DIEA (1.07 mL, 6.47 mmol) were added, and the reaction solution was stirred at room temperature for 30 min. GENO-int-11 (1 g, 2.16 mmol) was dissolved in DMF (10 mL) and added to the reaction solution, and the reaction solution was stirred at room temperature overnight under argon protection. The reaction solution was filtered and purified with reverse phase column C18, and the product Int-6-1 (1.8 g) was obtained by MeCN / H2O=60%. 1HNMR:(400MHz,DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.71-7.73(m,1H),7.39-7.41(m,2H),7.24-7.30(m,6H), 7.18-7.21(m,2H),6.87(d,J=8.8Hz,4H),5.21(d,J=3.6Hz,1H),4.95-4.98(m,1H),4.59(d,J=6.4Hz,1H), 4.47(d,J=8.4Hz,1H),4.00-4.05(m,2H),3.83-3.90(m,1H),3.64-3.73(m,7H),3.23-3.38(m,8H),2.97-3 .14(m,3H),2.04-2.14(m,5H),1.98(s,3H),1.89(s,3H),1.77(s,3H),1.64-4.66(m,1H)1.42-1.50(m,4H).

[0071] [ka] Int-6-1 (2.1g, 2.35mmol) was dissolved in DCM (30mL), tetrazole (33mg, 0.47mmol), NMI (77.2mg, 0.94mmol) and 2g of molecular sieves were added, the reaction solution was replaced with argon three times, and stirred at room temperature for 20min. After that, phosphorus reagent (920.81mg, 3.06mmol) was dissolved in a small amount of dichloromethane and added to the reaction solution, and stirred at room temperature for 1h. The reaction solution was washed twice with saturated NaHCO3 aqueous solution, once with water, once with saline, concentrated at room temperature, and purified with reverse phase column C18 and MeCN / H2O=65% to obtain the product GENO-Gal-6 (1.5g). 1HNMR:(400MHz,CD3CN)δ7.48-7.50(m,2H),7.20-7.36(m,7H),6.83-6.87(m,4H),6.45-6.51(m ,1H),5.28(d,J=3.2Hz,1H),4.98-5.02(m,1H),4.49(d,J=8.4Hz,1H),3.90-4.12(m,4H),3.24 -3.76(m,20H),3.02-3.11(m,1H),2.49-2.59(m,1H),2.36-2.39(m,1H),2.08-2.25(m,9H),1. 97(s,3H),1.91(s,3H),1.83(s,3H),1.71-1.74(m,1H),1.46-1.63(m,5H),0.85-1.39(m,20H).

[0072] Example 2 Synthesis of compound GENO-Gal-7 (1) Synthesis Route [ka] [ka] 1) Preparation of compound Gal-7-1 [ka] GENO-Int-5A (5.91 g, 15.53 mmol) was dissolved in anhydrous DMF (40 mL) solvent, then HATU (5.91 g, 15.53 mmol), 3A molecular sieves (3 g) and DIEA (6.42 mL, 38.83 mmol) were added, and the reaction solution was stirred at room temperature for 30 min. GENO-int-11 (3.6 g, 7.77 mmol) was dissolved in DMF (10 mL) and added to the reaction solution, and the reaction solution was stirred at room temperature overnight under argon protection. The reaction solution was filtered and purified with reverse phase column C18 and MeCN / H2O=60% to obtain product Gal-7-1 (4.7 g).

[0073] 2) Preparation of compound Gal-7-2 [ka] Gal-7-1 (800 mg, 0.91 mmol) was dissolved in 10 mL of dichloromethane, and then DBU (0.27 mL, 1.83 mmol) was added to the above solution and stirred at room temperature for 1 h. The reaction was monitored by TLC until the reaction of the raw material was completed. The reaction was quenched by adding 5 mL of saturated Na2CO3 solution, extracted with 30 mL of dichloromethane, and the organic phase was concentrated and purified by reverse phase column C18 to obtain the product (330 mg) with MeCN / H2O=35%. (The product was dissolved in CH3CN / H 20 (=20 / 1 silica gel thin layer chromatography.) 3) Preparation of compound Gal-7-3 [ka] Gal-3-5B (318.72 mg, 0.712 mmol) was dissolved in anhydrous DMF (4 mL) solvent, then HATU (361.14 mg, 0.95 mmol), 3A molecular sieves (1 g) and DIEA (0.24 mL, 1.43 mmol) were added, and the reaction solution was stirred at room temperature for 30 min. Gal-7-2 (310 mg, 0.48 mmol) was dissolved in DMF (3 mL) and added to the reaction solution, and the reaction solution was stirred at room temperature overnight under argon protection. The reaction solution was filtered and purified with reverse phase column C18, and the product Gal-7-3 (370 mg) was obtained by MeCN / H2O=50%.

[0074] 4) Preparation of compound GENO-Gal-7 [ka] Gal-7-3 (3.0 g, 2.77 mmol) was dissolved in DCM (30 mL), 7-B (1.34 g, 4.44 mmol), DCI (491 mg, 4.16 mmol) and 2 g of molecular sieves were added, the reaction mixture was purged with argon three times, stirred at room temperature for 20 min, and stirred at room temperature for 1 h. The reaction mixture was washed twice with saturated aqueous NaHCO3, once with water, once with brine, concentrated at room temperature, and purified by reverse phase column C18 with MeCN / H2O=65% to give the product GENO-Gal-7 (1.5 g).

[0075] 1 HNMR:δ7.46-7.44(d,J=7.7Hz2H),7.32-7.25(m,6H),7.18-7.16(m,2H),6 .84-6.80(m,4H),6.63-6.59(m,2H),5.25-5.24(d,J=3.2Hz1H),5.00-4.9 6(dd,J=11.2Hz,3.3Hz1H),4.51-4.49(d,J=8.5Hz1H),4.08-4.01(m,3H), 3.99-3.88(m,4H),3.78-3.72(m,7H),3.48-3.32(m,24H),3.8-3.01(m,1H ),2.78-2.76(m,1H),2.56-2.53(m,1H),2.36-2.33(m,1H),2.21-2.18(m, 1H),2.08-2.06(m,2H),1.94(m,4H),1.91-1.87(m,4H),1.80(s,3H),1.70 -1.67(d,J=13.1Hz1H),1.59-1.45(m,5H),1.39-1.29(m,1H),1.25-1.22( m,1H),1.18-1.10(m,2H),1.06-0.99(m,10H),0.83-0.82(d,J=6.7Hz,2H), 31 PNMR:ET51671-213-P1O1(162MHz, CD3CN), δ146.92,146.03.

[0076] 5) Preparation of Compound Gal-7-3a [ka] Gal-7-3 (3.0 g, 2.77 mmol), succinic anhydride (2.22 g, 22.1 mmol), 3A molecular sieves (0.5 g) and triethylamine (2 mL, 3.8 mmol) were dissolved in DCM (6 mL), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered and washed with 5% NaCl solution, and the reaction mixture was concentrated at room temperature and purified by reverse phase column C18 with MeCN / HO=40% to obtain product Gal-7-3a (1.4 g).

[0077] 6) Preparation of Compound Gal-7-3a [ka] Gal-7-3a (0.7 g, 0.6 mmol) was suspended in 8 mL of acetonitrile and 4 mL of N,N-dimethylformamide, DIEA (0.83 mL, 4.74 mmol) and HBTU (1.12 g, 2.96 mmol) were added dropwise, and the mixture was shaken at room temperature for 5 minutes. Aminomethyl resin (409.43 mg, 100-200 mesh, amino loading amount 250 μmol / g) was added to the reaction solution, and the reaction was carried out on a shaker at 25 °C and rotated. The rotation speed was set to 220 rpm, and the mixture was reacted for 16 h, then filtered, the filter cake was rinsed with DCM 3 times at 30 ml per time, rinsed with acetonitrile 3 times at 30 ml per time, rinsed with 30 ml of n-hexane 3 times, dried with a vacuum oil pump for 2 h, and then the raw materials (CapB1, 4-dimethylaminopyridine, N-methylimidazole and acetonitrile) were added in the raw material mixing ratio shown in Table 2 to perform the capping reaction. The mixture was placed on a shaker at 25 ° C., the rotation speed was set to 220 rpm, and the mixture was reacted for 16 h, the reaction liquid was filtered, the filter cake was rinsed with acetonitrile 3 times at 30 ml per time, suction filtered until dry, and dried overnight under reduced pressure with a vacuum oil pump to obtain the GENO-Gal-7A compound (4.9 g).

[0078] Example 3 Synthesis of oligonucleotides conjugated to aminogalactose molecular clusters The synthesis of siRNA is basically the same as that of the conventional phosphoramidite solid-phase synthesis method, but differs in that, when synthesizing the SS strand of siRNA, the CPG vector to which the aminogalactose cluster is linked as above is used instead of the conventional Universal-CPG vector. The synthesis process of siRNA (specific sequences are shown in Table 1) is briefly described as follows. Starting from the Universal-CPG vector or the CPG vector to which the aminogalactose cluster is linked, nucleoside phosphoramidite monomers are linked in sequence according to the synthesis procedure on the Dr.Oligo48 synthesizer (Biolytic), and nucleoside phosphoramidite monomer raw materials such as 2'-FRNA, 2'-O-methyl RNA, etc. are purchased from Wuhu Huaren and Shanghai Zhaowei. 5-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M solution in acetonitrile), 0.22 M PADS was dissolved in a mixed solvent of acetonitrile and trimethylpyridine (Shanghai Lingjiang) with a volume ratio of 1:1, and the resulting solution was used as the sulfurizing reagent, and iodopyridine / water solution (Shanghai Lingjiang) was used as the oxidizing agent.

[0079] After the solid-phase synthesis was completed, the oligoribonucleotide was decomposed from the solid support and soaked in 28% aqueous ammonia at 50°C for 16 hours. Then, it was centrifuged, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, and then purified by C18 reverse phase chromatography, with the mobile phase being 0.1M TEAA and acetonitrile. After collecting the target oligonucleotide, it was repeatedly freeze-dried, identified as the target product by LC-MS, and quantified by UV (260nm).

[0080] The resulting single-stranded oligonucleotide was annealed with the AS strand by forming a complementary pair in an equimolar ratio, and finally the resulting double-stranded siRNA was dissolved in 1x PBS and adjusted to the concentration required for the experiment.

[0081] [Table 1]

[0082] [Table 2]

[0083] L96 structural formula: [ka] Gal-3 structural formula: [ka] Gal-5: [ka] Gal-6: [ka] [ST23sST23sST23sC6XLT]Structural formula: [ka]

[0084] In the sequences of Tables 1 and 2, each symbol represents the following modified nucleotides (esters):

[0085] A = adenosine 3'-phosphate C=Cytidine-3'-phosphate G = guanyl-3'-phosphate U = Uridyl-3'-phosphate Am = 2'-O-methyladenosine-3'-phosphate Ams = 2'-O-methyl adenosine-3'-thiophosphate Cm = 2'-O-methylcytidine-3'-phosphate Cms = 2'-O-methylcytidine-3'-thiophosphate Gm = 2'-O-methylguanyl-3'-phosphate Gms = 2'-O-methylguanyl-3'-thiophosphate Um = 2'-O-methyluridyl-3'-phosphate ester Ums = 2'-O-methyluridyl-3'-thiophosphate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-thiophosphate Cf = 2'-fluorocytidine-3'-phosphate ester Cfs = 2'-fluorocytidine-3'-thiophosphate Gf = 2'-fluoroguanyl-3'-phosphate Gfs = 2'-fluoroguanyl-3'-thiophosphate Uf = 2'-fluorouridyl-3'-phosphate ester Ufs = 2'-fluorouridyl-3'-thiophosphate A lower case m indicates that the one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, and a lower case f indicates that the one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide; When a lowercase s is placed in the middle of an uppercase letter, it indicates that the two adjacent nucleotides on the left and right sides of the s are linked by a phosphorothioate group; When the lowercase letter s is at the first position of the 3' terminus, it indicates that the adjacent nucleotide terminus to the left of the letter s is a thiophosphate group; In oligonucleotides, the nucleotide monomers are linked to each other by 5'-3'-phosphodiester bonds, including phosphorothioate and phosphodiester bonds.

[0086] Example 4 Suppression of Angptl3 mRNA expression in mice by ANGPTL3 RNAi agents To evaluate the in vivo activity of ANGPTL3RNAi, male C57BL / 6 mice were used. After adapting to the environmental facility, the mice were divided equally according to body weight, with 3 mice per group. On the day of administration (day 0), the mice were subcutaneously injected with ANGPTL3RNAi solution, and the control group mice were administered with PBS solution. All mice were subcutaneously injected once in the neck, with a dose of 1 mg / Kg and a volume of 5 ml / Kg. Blood was collected from the orbital venous plexus (after isoflurane anesthesia) for all mice 3 days before administration, and on days 5, 14, 21, and 28 after administration to collect serum, and blood was continued to be collected on days 35 and 42 for some mice in the administration group. After blood collection, the animals were euthanized.

[0087] The mANGPTL3 expression knockdown status was evaluated by detecting the mANGPTL3 protein level in the serum of mice using ELISA (mANGPTL3, R&D). For normalization, the mANGPTL3 level of each animal at a given time point was divided by the pretreatment level for that animal (Day-3) to determine the expression ratio "normalized to pretreatment", and then the expression at a particular time point was normalized to the control group by dividing the "normalized to pretreatment" ratio of each animal by the average value of the "normalized to pretreatment" ratio of all mice in the control group. This normalizes the expression at each time point to the expression of the control group, and the specific detection results after normalization are shown in Figure 1.

[0088] ANGPTL3 RNAi agents (containing the oligonucleotide sequences listed in Table 1) were administered to C57BL / 6 mice as described above. Each mouse was administered a single subcutaneous injection (SC) dose of 1 mg / Kg of ANGPTL3 RNAi solution, and serum mANGPTL3 protein levels were monitored for 42 days. The knockdown levels and response times are shown in Table 3. As can be seen from Table 3, at day 5 after administration, 11 mice administered 1 mg / Kg of ANGPTL3 RNAi agent showed greater than 80% knockdown, 6 mice administered 1 mg / Kg of ANGPTL3 RNAi agent showed greater than 90% knockdown, at day 14 after administration, 8 mice administered 1 mg / Kg of ANGPTL3 RNAi agent showed greater than 70% knockdown, 5 mice administered 1 mg / Kg of ANGPTL3 RNAi agent showed greater than 80% knockdown, and at day 21 after administration, 10 mice administered 1 mg / Kg of ANGPTL3 RNAi agent showed greater than 90% knockdown. Eight mice administered 1 mg / Kg of ANGPTL3 RNAi agent showed greater than 50% knockdown, three mice administered 1 mg / Kg of ANGPTL3 RNAi agent showed greater than 70% knockdown, and at 28 days post-treatment, five mice administered ANGPTL3 RNAi agent still maintained greater than 50% knockdown in mANGPTL3 protein levels, and at 35 days post-treatment, two mice administered ANGPTL3 RNAi agent still maintained greater than 45% knockdown in mANGPTL3 protein levels.

[0089] Angiopoietin-like 3 (ANGPTL3) is an angiopoietin protein encoded by the human angiopoietin-like 3 gene, which is mainly expressed in the liver and is used to regulate lipid metabolism. Effective therapeutic agents targeting ANGPTL3 can be used for the treatment (including preventive treatment) of metabolic diseases such as hypertriglyceridemia. As can be seen from the experimental results in Table 3, the targeting ligand of the present application can efficiently deliver oligonucleotide sequences to liver cells and inhibit the expression of ANGPTL3.

[0090] [Table 3]

[0091] Example 5 Distribution ratio of conjugated LPA RNAi agents in the liver and kidney of wild-type mice after subcutaneous administration Thirty C57BL6 / J mice were divided into 10 groups, and the designated 5 groups of LPA RNAi agents (containing the oligonucleotide sequences shown in Table 2) were subcutaneously injected into the mice in each group on day 1 at a dose of 10 mg / kg. For the mice in 5 groups, tissues were collected 1 hour after administration, and for the mice in the other 5 groups, tissues were collected 24 hours after administration. The collected tissue samples were flash frozen and transferred to -80°C for storage. After homogenizing the tissues, the concentrations of the corresponding LPA RNAi agents in the liver tissue homogenates and kidney tissue homogenates were measured by hybridization fluorescent probe-enzyme-linked immunosorbent assay, and the liver-kidney ratio was calculated and evaluated by dividing the concentration in the liver tissue homogenates by the concentration in the kidney tissue homogenates. The results are shown in Table 4 and Figure 2.

[0092] FIG. 2 shows the ratio of compound concentration in the liver to compound concentration in the kidney of wild-type mice after SC administration of 10 mg / kg LPA RNAi agent. As can be seen from the results, the concentrations in the liver, which is the target organ, of Geno-1-105M to Geno-1-108M are all much greater than the concentrations in the kidney, which is the non-target organ, at different time points, and the ratio of the drug concentration in the liver to the drug concentration in the kidney is much greater than 1. And, at the two test time points, the concentrations in the kidney, which is the non-target organ, of Geno-1-1004M are all greater than the concentrations in the liver, and the ratio of the drug concentration in the liver to the drug concentration in the kidney is less than 1.

[0093] [Table 4]

[0094] Lipoprotein(a) [Lp(a)] is a heterogeneous low-density lipoprotein (LDL)-like particle, and the LPA (Apo(a)) gene is mainly expressed in the liver. High Lp(a) levels are an independent risk factor for cardiovascular disease, stroke, and other related diseases, including atherosclerotic stenosis. The above examples demonstrate that the targeting ligand of the present application can efficiently deliver the target oligonucleotide sequence to liver cells. In Table 4, compared with the control, Geno-1-1004M caused more target substance to accumulate in the kidney, while the other ligands mainly delivered the target substance to the liver.

[0095] The above is merely a preferred embodiment of the present invention, and does not limit the present invention. All modifications, equivalent replacements, improvements, etc. made within the principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A cleavable compound represented by formula (Z-1) or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 (In the formula, R １ is O, S, NR ３ or CR ３ R ４ and R ３ and R ４ each independently represents hydrogen, halogen, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocycle, or a substituted or unsubstituted cycloalkyl group; R ２ is -O-, -S-, -NH-, -CH ２ -, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH ２ NH-, -CH ２ O-, -NH-C(O)-CH ２ -, -C(O)-CH ２ -NH- or -NH(CO)NH-, ２ - is optionally substituted with a substituent selected from halogen, an alkyl group, an alkoxy group, and an alkylamino group, and a and b are the same or different and each represents an integer selected from 0 to 20, preferably 1 to 10, and more preferably 1 to 5.

2. The structural formula of the compound is: 【Chemistry 2】 【Transformation 3】 or 【Chemistry 4】 2. The cleavable compound of claim 1, wherein:

3. 10. An oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof, comprising the cleavable compound of claim 1 or a pharmaceutically acceptable salt thereof, said oligonucleotide ligand compound comprising: It is of general formula (I) or contains a structure of general formula (II), 【Transformation 5】 (I) 【Transformation 6】 (II) In general formula (I), Z is a cleavable compound represented by formula (Z-1) linked to the nucleotide sequence X, and １ is a second linker moiety, E is a branch point group, said branch point group E is linked to a1 targeting complexes, said a1 being an integer selected from 0 to 10, preferably 1 to 5, said targeting complexes being linked to a 1:1 ratio of tethering moieties L ２ and a targeting moiety T, In the general formula (II), Y is O, S or N, and L ３ teeth, 【Transformation 7】 (II-a-I), 【Transformation 8】 (II-a-II), 【Chemistry 9】 (II-a-III), 【Chemistry 10】 (II-a-III) In the formula, R ２ is -O-, -S-, -NH-, -CH ２ -, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH ２ NH-, -CH ２ O-, -NH-C(O)-CH ２ -, -C(O)-CH ２ -NH- or -NH(CO)NH-, ２ - is optionally substituted with a substituent selected from a halogen and an alkyl group, and the alkyl group is further optionally substituted with a substituent selected from a hydroxy group, an amino group, a halogen, an alkoxy group, and an alkylamino group; An oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof, wherein p, q, r, s, t, and u are integers selected from 0 to 20, preferably 1 to 10.

4. 4. The oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein the general formula (II) is linked to a nucleotide sequence.

5. In the general formula (I), the second linker moiety L １ teeth, 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 or 【Chemistry 14】 It has the structure The oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, in the formula, f, g, h and i are each an integer of 1 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.

6. In the general formula (I), compound L ２ The structural formula is 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 or [Chemistry 18] and The oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, in the formula, j, k, l, m, n, and o each represent an integer of 1 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.

7. 4. The oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein the targeting moiety T is selected from tissue-specific targeting ligands, preferably the targeting moiety T is a liver-specific targeting ligand, and more preferably the targeting moiety T has a structure that enhances uptake of the oligomeric compound into liver cells.

8. The target portion T is L ３ 4. The oligonucleotide ligand compound of claim 3, wherein the oligonucleotide ligand compound has the same or different structure as:

9. The ligand compound is 【Chemistry 19】 (I-1), 【Chemistry 20】 (I-2), 【Chemistry 21】 (I-3), 【Chemistry 22】 (I-4), 【Chemistry 23】 (I-5), 【Chemistry 24】 (I-6), 【Chemistry 25】 (I-7), 【Chemistry 26】 (I-8), 【Chemistry 27】 (I-9), 【Chemistry 28】 (I-10), 【Chemistry 29】 (I-11), 【Transformation 30】 (I-12), 【Chemistry 31】 (I-13), 【Chemistry 32】 (I-14), 【Transformation 33】 (I-15), or 【Transformation 34】 9. The oligonucleotide ligand compound according to claim 3, wherein the compound has the structure (I-16) or a pharmaceutically acceptable salt thereof.

10. The ligand compound is 【Chemistry 35】 (II-a-1), 【Transformation 36】 (II-a-2), 【Chemistry 37】 (II-a-3), 【Transformation 38】 (II-a-4), 【Chemistry 39】 (II-a-5), 【Chemistry 40】 (II-a-6), 【Chemistry 41】 (II-a-IV), 【Chemistry 42】 (II-a-V), 【Chemistry 43】 (II-a-VI), 【Chemistry 44】 (II-a-VII), 【Chemistry 45】 (II-a-7), 【Chemistry 46】 (II-a-8), 【Chemistry 47】 (II-a-9), 【Chemistry 48】 (II-a-10), 【Chemistry 49】 (II-a-11), or [Transformation 50] 9. The oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 8, which comprises the structure (II-a-12).

11. After binding to the nucleotide sequence X, the ligand compound 【Chemistry 51】 (II-1), 【Chemistry 52】 (II-2), 【Chemistry 53】 (II-3), 【Chemistry 54】 (II-4), 【Transformation 55】 (II-5), 【Transformation 56】 (II-6), 【Chemistry 57】 (II-7), 【Chemistry 58】 (II-8), 【Chemistry 59】 (II-9), 【Transformation 60】 (II-10), 【Chemistry 61】 (II-11), 【Transformation 62】 (II-12), 【Transformation 63】 (II-13), 【Chemistry 64】 (II-14), 【Transformation 65】 (II-15), 【Chemical Formula 66】 (II-16), 【Transformation 67】 (II-17), 【Transformation 68】 (II-18), 【Transformation 69】 (II-19), or 【Transformation 70】 (II-20) 9. The oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 8, wherein Y is O or S.

12. An RNA interference agent comprising a sense strand and / or an antisense strand and the oligonucleotide ligand compound according to any one of claims 3 to 8 or a pharmaceutically acceptable salt thereof.

13. The RNA interference agent of claim 12, comprising an antisense oligonucleotide, siRNA, or miRNA.

14. The RNA interference agent of claim 12, comprising one or more modified nucleotides.

15. The RNA interference agent according to claim 12, wherein the oligonucleotide ligand compound or a pharmaceutically acceptable salt thereof is coupled to the sense strand and / or the antisense strand.

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