WO2023138664A1 - 靶向配体及其用途 - Google Patents

靶向配体及其用途 Download PDF

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WO2023138664A1
WO2023138664A1 PCT/CN2023/073193 CN2023073193W WO2023138664A1 WO 2023138664 A1 WO2023138664 A1 WO 2023138664A1 CN 2023073193 W CN2023073193 W CN 2023073193W WO 2023138664 A1 WO2023138664 A1 WO 2023138664A1
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pharmaceutically acceptable
acceptable salt
rule
compound
ligand compound
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PCT/CN2023/073193
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English (en)
French (fr)
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李冲
尹科
李倩
马海萍
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上海金中锘美生物医药科技有限公司
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Priority to CN202380012476.4A priority Critical patent/CN117561268A/zh
Priority to AU2023209536A priority patent/AU2023209536A1/en
Publication of WO2023138664A1 publication Critical patent/WO2023138664A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the invention belongs to the technical field of nucleic acid drug delivery, and in particular relates to a nucleic acid delivery compound, a liver-targeting nucleic acid ligand and uses thereof.
  • RNA interference widely exists in nature, and has developed into a mature technical means in the field of molecular biology and medical treatment.
  • the targeting ligand is a very important part in the RNAi process, and the targeting ligand assists in guiding the delivery of the attached therapeutic agent to the desired target position, wherein the targeting part of the targeting ligand is composed of one or more targeting groups or targeting moieties.
  • a targeting moiety can bind a cell or a cell receptor and initiate endocytosis to facilitate entry of a therapeutic agent into the cell.
  • the liver is an important organ of human metabolism.
  • the most important cell type in the liver that performs liver functions is hepatic parenchymal cells, and there are abundant asialoglycoprotein receptors (ASGPR) on the surface of hepatic parenchymal cells.
  • Targeting moieties known to bind the asialoglycoprotein receptor (ASGPR) are particularly useful for delivering oligonucleotide compounds (siRNA, etc.) to the liver.
  • Targeting moieties targeting asialoglycoprotein receptors include galactose or galactose derivatives.
  • N-acetyl-galactosamine (GalNAc) clusters coupled to oligomeric compounds are used to direct the composition to the liver, where N-acetyl-galactosamine can bind to the ASGPR receptor on the surface of liver cells, thereby facilitating the entry of the compound into the cell interior. Therefore, the improvement of this targeting ligand can improve the working efficiency of RNAi.
  • nucleoside molecules since nucleoside molecules usually have a relatively high negative charge, the complex system of the cell membrane limits the direct diffusion of nucleic acid molecules or other compounds that do not easily pass through the cell membrane through the cell membrane into living cells. Therefore, the main obstacle in the delivery of nucleic acid molecules is how to deliver nucleic acid molecules into the cytoplasm or nucleus.
  • the published solutions include nanoliposomes, viral vectors, conjugated delivery molecules, etc.
  • Conjugated delivery molecules such as coupled GalNAc molecules, polypeptide molecules, antibody molecules, etc., serve as guides to direct siRNA to specific target cells. Therefore, how to separate the coupled delivery molecule from the siRNA molecule after completing the guidance function requires the development of a cleavable linker to achieve this function.
  • the embodiment of the present invention provides a compound that can be used for nucleic acid coupling delivery ligand, a liver-targeting nucleic acid ligand and its use for delivering nucleic acid molecules, as well as its application in the field of disease treatment.
  • the first aspect of the present invention provides a cleavable compound represented by formula (Z-1) or a pharmaceutically acceptable salt thereof,
  • R 1 is O, S, NR 3 or CR 3 R 4 , wherein R 3 and R 4 are each independently hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocycle or substituted or unsubstituted cycloalkyl group;
  • R 2 Can be -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-, wherein, the -CH 2 -can also be optionally substituted by a substituent selected from halogen, alkyl (optionally, substituted by a substituent selected from hydroxyl, amino, halogen), alkoxy, and alkylamino; the a and b are the same or different, and are respectively selected from an integer of 0 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
  • R 2 is -NH-, at this time compound Z is:
  • R 2 is preferably -C(O)-, at this time compound Z is
  • the above compound delivers the nucleic acid sequence to the target position, it breaks at the left R2 , and breaks only at this position, which makes the target product after delivery predictable and avoids the negative impact of irregular breaks.
  • the second aspect of the present invention provides a targeting ligand compound or a pharmaceutically acceptable salt thereof, which includes the above-mentioned cleavable compound, and its general formula is shown in the following formula (I): (I), wherein Z is a cleavable compound represented by the above general formula (Z-1); Or a structure comprising general formula (II): (II), wherein L3 includes the cleavable compound represented by the above formula (Z-1).
  • a1 is an integer selected from 1 to 10, preferably an integer from 1 to 5
  • Z is the first linker part of the nucleotide sequence (the above-mentioned cleavable compound Z-1), and is used to connect to the nucleotide sequence X.
  • the nucleotide sequence can be any nucleotide sequence, preferably an oligonucleotide sequence, wherein the nucleotide sequence can be the sense strand or the antisense strand of siRNA, wherein Z and X can be directly connected or connected through a chemical group, and the general formula of Z is as shown in (Z-1) above, wherein one end of the O group is used to connect to the nucleotide sequence:
  • the nucleotide sequence X includes no more than 30 nucleotides, more preferably, no more than 23 nucleotides.
  • said L 1 is the second linker moiety
  • E is a branch point group, and the two are directly connected.
  • the branch point group E is connected to a1 targeting combinations, where a1 is an integer selected from 0 to 10, preferably an integer from 1 to 5; wherein the targeting combination includes a tethering moiety L2 and a targeting moiety T in a ratio of 1:1.
  • the above formula (II) binds to the nucleotide sequence, it can bind to the 5' end of the nucleotide sequence, or it can bind to the 3' end of the nucleotide sequence, and it can also bind to the 5' end and the 3' end at the same time. It can be combined with the nucleotide sequence X according to the following formula (III), wherein L4 and L3 are the same or different, and both are targeting moieties, and Y is O, S or N; wherein b, c, d and e are selected from integers from 0 to 10.
  • L3 includes a third linker moiety
  • L4 in general formula (III) is a targeting moiety
  • targeting moiety T in general formula (I) and L3 and L4 in general formula (III) can be the same or different, preferably, L3 and L4 are the following structures respectively:
  • R2 can be -O-, -S-, -NH-, -CH2- , -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2NH-, -CH2O- , -NH -C(O) -CH2- , -C(O) -CH2 -NH-, or -NH(CO)NH-, wherein said -CH 2 - can also be optionally substituted by a substituent selected from halogen, alkyl (optionally further substituted by a substituent selected from hydroxy, amino, halogen, alkoxy and alkylamino);
  • the p, q, r, s, t and u are independently selected from an integer of 0 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
  • X is a nucleotide sequence, which can be a sense strand or an antisense strand
  • Y is O or S
  • b, c, d and e are respectively selected from an integer of 0 to 10, preferably an integer of 0 to 5, and b and e are not 0 at the same time.
  • the second linker part L has the following structure:
  • f, g, h and i are respectively an integer from 1 to 20, preferably an integer from 1 to 10, more preferably an integer from 1 to 5.
  • j, k, l, m, n and o are respectively integers from 1 to 20, preferably integers from 1 to 10, more preferably 1 Integers up to 5.
  • the targeting moiety T is selected from tissue-specific targeting ligands, for example, the targeting moiety T is a liver-specific targeting ligand or other tissue-specific targeting ligands; preferably, the targeting moiety T has a structure for enhancing the uptake of oligomeric compounds by target cells; in a preferred embodiment, the target cells are hepatocytes.
  • the oligonucleotide conjugates provided in the embodiments of the present invention can improve the efficiency of the targeting moiety in binding to cells or cell receptors, and increase the effect of RNA interference.
  • the embodiment of the present invention also provides the use of the above-mentioned compounds of formula (Z-1), formula (I) and formula (II) and pharmaceutically acceptable salts thereof for the delivery of nucleic acid molecules.
  • the delivery of the nucleic acid molecule can deliver the nucleic acid molecule to a specific in vivo cell.
  • the compound of the above formula (Z-1) or a pharmaceutically acceptable salt thereof undergoes cleavage at R 2 after delivering the nucleic acid molecule to the target cell.
  • the compounds of formula (I) and formula (II) and their pharmaceutically acceptable salts are used for targeted binding to nucleic acids and delivery to human or mammalian liver cells.
  • liver cells are hepatic parenchymal cells.
  • the compounds represented by formula (Z-1), formula (I) and formula (II) and their pharmaceutically acceptable salts provided in the embodiments of the present invention are used as nucleic acid coupling delivery ligands or part of their structures.
  • the delivery ligands can deliver target nucleic acids to designated cells with high delivery efficiency.
  • Figure 1A is a graph of the detection results after 28 days of inhibition of ANGPTL3 expression by a compound provided by an embodiment of the present application
  • Figure 1B is a detection result graph of 35 days after inhibition of ANGPTL3 expression
  • Figure 2 is a graph showing the concentration detection results of LPA siRNA in mouse liver and kidney in one embodiment of the present application.
  • oligonucleotide generally refers to a polymer consisting of multiple nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogs thereof) linked by phosphodiester bonds (or related structural variants or synthetic analogs thereof).
  • oligonucleotide generally refers to polymers of nucleotides in which nucleotide residues and linkages between them are naturally occurring, it is understood that the scope of the term also includes a variety of analogs, including but not limited to: peptide nucleic acid (PNA), phosphoramidate, phosphorothioate, methylphosphonate, 2-O-methyl ribonucleic acid, and the like. The exact size of the molecule can depend on the particular application.
  • PNA peptide nucleic acid
  • phosphoramidate phosphoramidate
  • phosphorothioate phosphorothioate
  • methylphosphonate 2-O-methyl ribonucleic acid
  • Oligonucleotides are generally short in length, usually about 10-30 nucleotide residues, but the term Molecules of any length may also be referred to, although the terms "polynucleotide” or “nucleic acid” are generally used for larger oligonucleotides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • modified oligonucleotide generally means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • modified nucleoside generally means a nucleoside comprising at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside.
  • Modified nucleosides comprise modified sugar moieties and/or modified nucleobases.
  • nucleobase generally means 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 mimetics, abasic sites (Ab or X), or surrogate replacement moieties. As used herein, “nucleobase sequence” generally means the sequence of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
  • unmodified nucleobase or “naturally occurring nucleobase” generally refers to the naturally occurring heterocyclic nucleobases of RNA or DNA: 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 means any nucleobase that is not a naturally occurring nucleobase.
  • ligand generally refers to any compound or molecule capable of covalently or otherwise chemically binding to a biologically active substance such as an oligonucleotide.
  • a ligand is capable of interacting directly or indirectly with another compound, such as a receptor, the receptor with which the ligand interacts may be present on the cell surface, or alternatively may be an intracellular and/or intercellular receptor, the interaction of the ligand with the receptor may result in a biochemical reaction, or may be merely a physical interaction or binding.
  • the embodiment of the present invention provides a cleavable compound represented by formula (Z-1),
  • R 1 is O, S, NR 3 or CR 3 R 4 , wherein R 3 and R 4 are each independently hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocycle or substituted or unsubstituted cycloalkyl group;
  • R 2 can be -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-, wherein, the - CH2- can also optionally be selected from halogen, alkyl (optionally, substituted with substituents selected from hydroxy, amino, halogen), alkoxy, Alkylamino substituents are substituted; said a and b are the same or different, and are respectively selected from an integer of 0 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
  • compound Z is:
  • the O group of the above compound binds to the nucleotide sequence, and after the nucleic acid sequence is delivered to the target position, a break occurs at the left R2 .
  • the embodiment of the present application also provides an oligonucleotide liver-targeting ligand, the general formula of which is as follows, Or have the following structure after combining the nucleotide sequence X:
  • X is the sense strand or antisense strand of nucleotides
  • Z is the linker part of the sense strand or antisense strand
  • E is the branch point group
  • L 1 is the connection part between the linker part of the sense strand or the antisense strand and the branch point group E
  • T is the targeting part
  • L2 is the tethering part between the targeting part and the branch point group E, wherein a1 is selected from an integer of 1 to 10, preferably an integer of 1 to 5, more preferably 1 or 3.
  • L 3 and L 4 are targeting moieties
  • X is sense strand or antisense strand
  • Y is O or S or N
  • b, c, d and e are selected from integers from 0 to 10, preferably integers from 1 to 5.
  • T, L 3 , and L 4 may be the same or different from each other.
  • L in the targeting ligand described in formula (I) has the following general formula:
  • f, g, h and i are respectively an integer from 1 to 20, preferably an integer from 1 to 10, more preferably an integer from 1 to 5;
  • L1 is the following structure:
  • said compound E is selected from:
  • compound L in general formula (I) 2 is
  • j, k, l, m, n and o are respectively integers from 1 to 20, preferably integers from 1 to 10, more preferably integers from 1 to 5;
  • L2 has the following structural formula:
  • T in the targeting ligand is selected from one of N-acetyl-galactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine, preferably N-acetyl-galactosamine, the structural formula is as follows:
  • the targeting ligand represented by the general formula (I) is connected to the end of the siRNA through a phosphate group, a phosphorothioate group or a phosphonic acid group.
  • the targeting ligand has the following structure:
  • the targeting ligand compound includes the following structure:
  • 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-, wherein said -CH 2 - can optionally be selected from halogen, alkyl (optionally further substituted by substituents selected from hydroxy, amino, halogen, alkoxy and alkylamino) Substituent), alkoxy, alkylamino substituent substitution;
  • the p, q, r, s, t and u are independently selected from an integer of 0 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
  • the targeting ligand compound includes the following structure:
  • the targeting ligand compound includes the following structure:
  • 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-, the -CH 2 - Can be optionally substituted by a substituent selected from halogen, alkyl (optionally further substituted by a substituent selected from hydroxyl, amino, halogen, alkoxy and alkylamino), alkoxy, alkylamino;
  • the p, q, r, s, t and u are independently selected from an integer of 0 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
  • the targeting ligand compound includes the following structure:
  • general formula (III) has the following structure:
  • Y is O or S.
  • the targeting ligand compound that binds to nucleotide X shown in formula (III) has the following structure:
  • Y is O or S or N.
  • the sense strand is selected from a nucleotide sequence represented by the following formula:
  • Nm represents any nucleotide modified by methoxy, such as cytosine (C), guanine (G), uracil (U), adenine (A), thymine (T) modified by methoxy;
  • Nf represents any nucleotide modified by fluorine, such as C, G, U, A, T modified by fluorine;
  • the lowercase letter s When the lowercase letter s is in the middle of the uppercase letter, it means that there is a phosphorothioate group connection between the two nucleotides adjacent to the left and right of the letter s.
  • the lowercase letter s When the lowercase letter s is the first at the 3' end, it means that the nucleotide end adjacent to the left side of the letter s is a phosphorothioate group.
  • the lowercase letter s When the lowercase letter s is the first at the 5' end, it means that the nucleotide end adjacent to the right side of the letter s is a phosphorothioate group.
  • the antisense strand has a nucleotide sequence represented by the following formula:
  • Nm' represents any nucleotide modified by methoxy, such as C, G, U, A, T modified by methoxy
  • Nf' represents any nucleotide modified by fluorine, such as C, G, U, A, T modified by fluorine
  • Embodiment 1 is connected to the galactosamine compound GENO-Gal-5 synthesis on the solid phase carrier
  • Int-11-1 (10g) was dissolved in (140mL) DCM (Dichloromethane) and cooled to 0°C, TMSCN (Trimethylsilyl cyanide, trimethylnitrile silane (TMSCN)) (4.01g) and BF 3 .Et 2 O (2.83mL) were added dropwise, and reacted for 10min.
  • Gal-5-1 (200 mg, 0.26 mmol) was dissolved in 5 mL of ethyl acetate, 50 mg of palladium carbon and triethylamine (0.11 mL, 0.78 mmol) were added, the reaction solution was stirred under hydrogen (15 psi) at room temperature for 16 hours, and the reaction solution was filtered and concentrated to obtain Gal-5-2 (150 mg).
  • THF Tetrahydrofuran
  • Gal-5-4 (220mg, 0.086mmol) was suspended in 4ml CH 3 Add DIEA (0.035mL, 0.216mmol) and HBTU (49.07mg, 0.129mmol) dropwise to CN and 2mL of DMF, stir at room temperature for 5 minutes, add aminomethyl resin (99.51mg, 100-200 mesh, amino load 250umol/g, react on a shaking table at 25°C, rotate at 220 rpm, react for 16h, and filter the filter cake with DCM.
  • DIEA 0.035mL, 0.216mmol
  • HBTU 49.07mg, 0.129mmol
  • Int-6-1 (2.1g, 2.35mmol) was dissolved in DCM (30mL), Tetrazole (33mg, 0.47mmol), NMI (77.2mg, 0.94mmol) and 2g molecular sieves were added, the reaction solution was replaced with argon three times, stirred at room temperature for 20min, then phosphorus reagent (920.81mg, 3.06mmol) was dissolved in a small amount of dichloromethane and added to the reaction solution, at room temperature Stir for 1 hour.
  • Embodiment 2 compound GENO-Gal-7 is synthesized
  • siRNA The synthesis of siRNA is basically the same as the usual phosphoramidite solid-phase synthesis method, except that when synthesizing the SS chain of siRNA, the CPG carrier linked with galactosamine clusters synthesized above is used instead of the usual Universal-CPG carrier.
  • the synthesis process of siRNA (see Table 1 for the specific sequence) is briefly described as follows: On Dr. Oligo 48 synthesizer (Biolytic), starting with Universal CPG carrier or CPG carrier linked with galactosamine cluster, the nucleoside phosphoramidite monomers were connected one by one according to the synthesis procedure.
  • nucleoside phosphoramidite monomers such as 2’-F RNA and 2’-O-methyl RNA were purchased from Wuhu Huaren and Shanghai Zhaowei.
  • 5-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution)
  • 0.22M PADS was dissolved in a mixed solvent of acetonitrile and collidine (Shanghai Lingjiang) with a volume ratio of 1:1, and the resulting solution was used as a vulcanizing agent
  • iodopyridine/water solution (Shanghai Lingjiang) was used as an oxidant.
  • the oligoribonucleotide is cleaved from the solid support, and soaked in 28% ammonia water at 50° C. for 16 hours. Then centrifuge, transfer the supernatant to another centrifuge tube, concentrate and evaporate to dryness, and then use C18 reverse chromatography to purify, the mobile phase is 0.1M TEAA and acetonitrile.
  • the target oligonucleotides were collected and freeze-dried repeatedly, identified as target products by LC-MS, and then quantified by UV (260nm).
  • the obtained single-stranded oligonucleotides were annealed with the AS strand according to the equimolar ratio and complementary pairing, and finally the obtained double-stranded siRNA was dissolved in 1X PBS and adjusted to the concentration required for the experiment.
  • the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide
  • the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide
  • connection between the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate group connection
  • nucleotide terminal adjacent to the left side of the letter s is a phosphorothioate group
  • nucleotide monomers are linked to each other by 5'-3'-phosphodiester linkages, including phosphorothioate linkages and phosphodiester linkages.
  • Example 4 ANGPTL3 RNAi agent inhibits Angptl3 mRNA expression in mice
  • mice Male C57BL/6 mice were used. After adapting to the environment and facilities, the mice were divided into equal groups according to body weight, with 3 mice in each group. On the day of administration (day 0), mice were subcutaneously injected with ANGPTL3 RNAi solution, mice in the control group were given PBS solution, and all mice were subcutaneously injected once at the back of the neck, with a dosage of 1mg/Kg and an administration volume of 5ml/Kg.
  • the knockdown of mANGPTL3 expression was evaluated by detecting the protein level of mANGPTL3 in mouse serum by ELISA method (mANGPTL3, R&D).
  • mANGPTL3, R&D ELISA method
  • the mANGPTL3 level for each animal at a given time point was divided by the pretreatment level in this animal (Day-3) to determine the ratio of "normalized to pretreatment” expression; expression at a particular time point was then normalized to the control group by dividing the "normalized to pretreatment” ratio for each animal by the average “normalized to pretreatment” ratio for 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 FIG. 1 .
  • ANGPTL3 RNAi agents (comprising the oligonucleotide sequences described in Table 1) were administered to C57 BL/6 mice as described above. Each mouse received a single subcutaneous (SC) dose of 1 mg/Kg ANGPTL3 RNAi agent solution, and mANGPTL3 protein levels in serum were monitored for up to 42 days. Knockdown levels and duration of response are shown in Table 3.
  • Angiopoietin-like 3 (ANGPTL3) is an angiopoietin protein encoded by the human angiopoietin-like 3 gene, mainly expressed in the liver and used to regulate lipid metabolism. Effective therapeutics targeting ANGPTL3 are useful in the treatment, including prophylactic treatment, of metabolic diseases such as hypertriglyceridemia.
  • the experimental results in Table 3 show that the targeting ligand of the present application can efficiently deliver the oligonucleotide sequence to liver cells and inhibit the expression of ANGPTL3.
  • Example 5 Conjugated with LPA RNAi agent after subcutaneous administration in the distribution ratio of wild-type mice liver and kidney
  • mice Thirty C57BL6/J mice were divided into 10 groups, and the designated 5 groups of LPA RNAi agents (including the oligonucleotide sequences shown in Table 2) were subcutaneously injected into each group of mice at a dose of 10 mg/kg on the first day. 5 groups of mice were collected 1 hour after administration, and the other 5 groups of mice were collected 24 hours after administration. The collected tissue samples were quickly frozen and transferred to -80°C for storage.
  • LPA RNAi agents including the oligonucleotide sequences shown in Table 2
  • the concentration of the corresponding LPA RNAi agent in liver tissue homogenate and kidney tissue homogenate was measured by hybridization fluorescent probe-enzyme-linked immunosorbent assay, and the liver-kidney ratio was calculated by dividing the concentration of liver tissue homogenate by the concentration of kidney tissue homogenate. The results are shown in Table 4 and Figure 2.
  • Figure 2 shows the ratio of compound concentration in liver to kidney of wild-type mice after SC administration of 10 mg/kg LPA RNAi agent.
  • the results showed that the concentration of Geno-1-105M ⁇ Geno-1-108M in the target organ liver was much higher than that in the non-target organ kidney at different time points, and the drug concentration ratio of liver to kidney was much greater than 1.
  • Geno-1-1004M is testing two At each time point, the concentration in the non-target organ kidney was greater than that in the liver, and the ratio of the drug concentration in the liver to the kidney was less than 1.
  • Lipoprotein(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 conditions, including atherosclerotic stenosis.
  • the above examples prove that the targeting ligand of the present application can efficiently deliver the target oligonucleotide sequence to liver cells.
  • Table 4 compared with the control Geno-1-1004M, more target substances were accumulated in the kidney, while other ligands mainly delivered the target substances to the liver.

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Abstract

本发明适用于核酸药物递送技术领域，具体提供一种核酸递送化合物，肝脏靶向核酸配体及其用途。本发明提供了如式(Z-1)所示的化合物，以及式(I)和式(II)所示的化合物：本发明实施例提供的寡核苷酸缀合物可以提高靶向部分结合细胞或细胞受体的效率，增加RNA干扰的作用效果。

Description

靶向配体及其用途
交叉引用
本申请要求2022年01月20日递交的，申请号为202210068123.3，名称为siRNA缀合物及其用途的中国专利申请，其通过引用全文结合至本申请中。
技术领域
本发明属于核酸药物递送技术领域，具体涉及一种核酸递送化合物，肝脏靶向核酸配体及其用途。
背景技术
RNA干扰(RNAi)广泛存在于自然界中，目前在分子生物学和医疗领域已经发展成为成熟的技术手段。
靶向配体是RNAi过程中非常重要的部分，靶向配体协助引导将与其连接的治疗性试剂递送至所需靶位置，其中靶向配体的靶向部分是由一个或多个靶向基团或靶向部分组成。在一些情况中，靶向部分可以结合细胞或细胞受体，并且启动内吞作用以促进治疗性试剂进入细胞。
肝脏是人体新陈代谢的重要器官，肝脏中执行肝脏功能的最主要的细胞类型是肝实质细胞，并且在肝实质细胞表面有着丰富的去唾液酸糖蛋白受体(Asialoglycoprotein Receptor，ASGPR)。已知结合去唾液酸糖蛋白受体(ASGPR)的靶向部分可特别用于将寡聚核苷酸化合物(siRNA等)递送至肝脏。靶向去唾液酸糖蛋白受体的靶向部分包括半乳糖或者半乳糖衍生物。使用过程中，偶联寡聚化合物的N-乙酰基-半乳糖胺(GalNAc)簇用于引导组合物至肝脏，在这里N-乙酰基-半乳糖胺能够结合肝脏细胞表面的ASGPR受体，从而促进化合物进入细胞内部。因此对于此靶向配体的改进，可以提高RNAi的工作效率。
另一方面，由于核苷分子通常带有较高负电荷，细胞膜复杂的系统限制了核酸分子或其他不易穿过细胞膜的化合物直接扩散穿过细胞膜至活细胞内。因此，核酸分子递送的主要障碍在于如何将核酸分子递送至细胞质或细胞核中。目前，已发表的解决方法有借助纳米脂质体、病毒载体、共轭偶联递送分子等。对于共轭偶联递送分子，诸如偶联GalNAc分子、多肽分子、抗体分子等，其作为向导指引siRNA到特定的靶向细胞中。故偶联的递送分子在完成向导作用后，如何与siRNA分子分开，需要开发可断裂的连接子来实现这一功能。
发明内容
本发明实施例提供一种可用于核酸偶联递送配体的化合物，肝脏靶向核酸配体及其用于递送核酸分子的用途，以及在疾病治疗领域中的应用。
本发明第一方面提供了如式(Z-1)所示的可断裂化合物或其药学上可接受的盐，
其中R1是O、S、NR3或者CR3R4,其中R3和R4各自独立地是氢、卤素、取代或未取代的脂族基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的杂环或取代或未取代的环烷基；
其中R2可以为-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或者-NH(CO)NH-，其中，所述-CH2-还可以任选被选自卤素，烷基(任选地，被选自羟基、氨基、卤素的取代基取代)，烷氧基，烷氨基的取代基取代；所述a和b相同或者不相同，并且分别选自0到20的整数，优选为1到10的整数，进一步优选1到5的整数。
一个优选实施方案中，化合物Z的通式如下(Z-2)所示：
优选地R2为-NH-，此时化合物Z为：
另一个实施方案中，R2优选地为-C(O)-，此时化合物Z为
上述化合物在将核酸序列递送至目的位置后，在左侧R2处发生断裂，并且只在该位置发生断裂，这使得递送后的目标产物具有可预测性，避免了不规则断裂产生的负面影响。
本发明第二方面提供了一种靶向配体化合物或其药学上可接受的盐，其包括上述可断裂化合物，且其通式如下式(I)所示：

(I)，其中Z为上述通式(Z-1)所示的可断裂化合物；
或者包括通式(II)的结构：

(II)，其中L3中包括上述式(Z-1)所示的可断裂化合物。
具体地，通式(I)中：
a1选自1到10的整数，优选1到5的整数，Z为核苷酸序列的第一接头部分(上述可断裂化合物Z-1)，用于与核苷酸序列X连接，该核苷酸序列可以为任意核苷酸序列，优选为寡核苷酸序列，其中该核苷酸序列可以为siRNA的正义链或反义链，其中Z与X可以直接连接，也可以通过化学基团连接，且Z的通式如上面(Z-1)所示，其中O基一端用于与核苷酸序列连接：
优选地，所述核苷酸序列X包括不超过30个核苷酸，进一步优选地，包括不超过23个核苷酸。
具体地，在式(I)中，所述L1为第二接头部分，E为分支点基团，两者直接连接。
优选地，所述分支点基团E与a1个靶向组合体连接，所述a1为选自0到10的整数，优选1到5的整数；其中所述靶向组合体包括1：1比例的栓系部分L2和靶向部分T。
其中上式(II)在结合核苷酸序列时，可以结合至核苷酸序列的5’端，也可以结合至核苷酸序列的3’端，并且也可以同时结合至5’端和3’端，可以按照下式(III)的方式与核苷酸序列X结合，其中L4与L3相同或不同，均为为靶向部分，Y是O或S或N；其中b,c,d和e选自0到10的整数。
具体地，通式(II)和(III)中，L3包括第三接头部分，通式(III)中的L4为靶向部分，通式(I)中的靶向部分T和通式(III)中的L3和L4可以相同或者不同，优选地，L3和L4分别为以下结构：

其中R2可以是-O-，-S-，-NH-，-CH 2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或-NH(CO)NH-，其中所述-CH2-还可以任选被选自卤素，烷基(任选进一步被选自羟基、氨基、卤素、烷氧基和烷氨基的取代基取代)的取代基取代；
所述p,q,r,s,t和u分别独立地选自0到20的整数，优选为1到10的整数，进一步优选为1到5的整数。
上述式(III)中，X为核苷酸序列，可以为正义链或反义链；
Y是O或S，
其中b,c,d和e分别选自0到10的整数，优选为0到5的整数，且b和e不同时为0。在一个实施方案中，b为0，e为3至6的整数，一个优选实施例中，b＝0,d＝2,e＝1。
具体地，在通式(I)中，所述第二接头部分L1具有以下结构：
其中f,g，h和i分别是从1至20的整数，优选为1至10的整数，进一步优选为1至5的整数。
具体地，通式(I)中，化合物L2的结构式为：
其中j,k,l,m,n和o分别是从1至20的整数，优选为1至10的整数，进一步优选为1 至5的整数。
进一步地，所述靶向部分T选自组织的特异性靶向配体，例如，所述靶向部分T为肝特异性靶向配体或其他组织的特异性靶向配体；优选地，所述靶向部分T具有用于增强目标细胞对寡聚化合物的摄取的结构；一个优选实施方案中，所述目标细胞为肝细胞。
本发明实施例提供的寡核苷酸缀合物可以提高靶向部分结合细胞或细胞受体的效率，增加RNA干扰的作用效果。
本发明实施例还提供上述式(Z-1),式(I)及式(II)化合物及其药学上可接受的盐用于递送核酸分子的用途。具体地，所述递送核酸分子可以将核酸分子递送至特定体内细胞中。
进一步地，上述式(Z-1)的化合物或其药学上可接受的盐在将核酸分子递送至目标细胞后在R2处发生断裂。
进一步地，式(I)及式(II)化合物及其药学上可接受的盐用于靶向结合核酸并将其递送至人或哺乳动物的肝脏细胞。
进一步地，所述肝脏细胞为肝实质细胞。
本发明实施例提供的式(Z-1),式(I)及式(II)所示的化合物及其药学上可接受的盐，用做核酸偶联递送配体或其一部分结构，该递送配体可以将目标核酸递送至指定细胞中，递送效率高。
附图说明
图1A为本申请一个实施例提供的化合物抑制ANGPTL3表达的28天后的检测结果图，图1B为抑制ANGPTL3表达的35天后的检测结果图；
图2为本申请一个实施例中小鼠肝肾中LPA siRNA的浓度检测结果图。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例及化学反应式，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
术语定义
在本申请中，术语“寡核苷酸”通常是指由多个核苷酸残基(脱氧核糖核苷酸或核糖核苷酸，或其相关的结构变体或合成类似物)通过磷酸二酯键(或其相关的结构变体或合成类似物)连接组成的聚合物。因此，虽然术语“寡核苷酸”一般指其中核苷酸残基和它们之间的连接是天然产生的核苷酸聚合物，但应理解，该术语的范围也包括各种类似物，包括但不限于：肽核酸(PNA)、氨基磷酸酯、硫代磷酸酯、甲基膦酸酯、2-O-甲基核糖核酸等。该分子的确切大小可取决于具体应用。寡核苷酸一般长度较短，通常约有10-30个核苷酸残基，但该术语 也可指任何长度的分子，尽管术语“多核苷酸”或“核酸”一般用于较大的寡核苷酸。
在某些实施方案中，寡核苷酸包含一个或多个未修饰的核糖核苷(RNA)和/或未修饰的脱氧核糖核苷(DNA)和/或一个或多个修饰核苷。术语“修饰寡核苷酸”通常意指包含至少一个修饰核苷和/或至少一个修饰的核苷间键联的寡核苷酸。
在本申请中，术语“修饰的核苷”通常意指与天然存在的RNA或DNA核苷相比包含至少一个化学修饰的核苷。修饰的核苷包含修饰的糖部分和/或修饰的核碱基。
在本申请中，术语“核碱基”通常意指杂环嘧啶或嘌呤化合物，它是所有核酸的组分且包括腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、胸腺嘧啶(T)和尿嘧啶(U)。核苷酸可包括经修饰的核苷酸或核苷酸模拟物、无碱基位点(Ab或X)或替代物替代部分。如本申请所使用，“核碱基序列”通常意指不依赖于任何糖、键联或核碱基修饰的连续核碱基的顺序。术语“未修饰的核碱基”或“天然存在的核碱基”通常意指RNA或DNA的天然存在的杂环核碱基：嘌呤碱基腺嘌呤(A)和鸟嘌呤(G)；以及嘧啶碱基胸腺嘧啶(T)、胞嘧啶(C)(包括5-甲基C)和尿嘧啶(U)。“修饰的核碱基”通常意指并非天然存在的核碱基的任何核碱基。
在本申请中，术语“配体”通常是指能够共价地或以其它化学方式与生物活性物质(如寡核苷酸)结合的任何化合物或分子。在某些实施方案中，配体能够与另一种化合物例如受体直接或间接地相互作用，与配体相互作用的受体可以存在于细胞表面上，或可替代地可以是细胞内和/或细胞间受体，配体与受体的相互作用可以导致生化反应，或可以仅仅是物理相互作用或结合。
本发明实施例提供了如式(Z-1)所示的可断裂化合物，
其中R1是O、S、NR3或者CR3R4,其中R3和R4各自独立地是氢、卤素、取代或未取代的脂族基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的杂环或取代或未取代的环烷基；
其中R2可以为-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或者-NH(CO)NH-，其中，所述-CH2-还可以任选被选自卤素，烷基(任选地，被选自羟基、氨基、卤素的取代基取代)，烷氧基， 烷氨基的取代基取代；所述a和b相同或者不相同，并且分别选自0到20的整数，优选为1到10的整数，进一步优选1到5的整数。
一个优选实施方案中，化合物Z为：
上述化合物O基结合核苷酸序列，并在将核酸序列递送至目的位置后，在左侧R2处发生断裂。
本申请实施例还提供一种寡聚核苷酸的肝脏靶向配体，其通式如下所示，

或者在结合核苷酸序列X后具有如下结构：
具体地，通式(I)中：
X为核苷酸的正义链或反义链,Z为正义链或反义链的接头部分，E为分支点基团，L1 为正义链或反义链的接头部分与分支点基团E间的连接部分，T为靶向部分，L2为靶向部分与分支点基团E间的栓系部分，其中a1选自1到10的整数，优选1到5的整数，进一步优选为1或者3。
通式(II)和(III)中：
L3，L4为靶向部分，X为正义链或反义链，Y是O或S或N，其中b,c,d和e选自0到10的整数，优选为1到5的整数。
具体地，上述T，L3，L4可以彼此相同或者不同。
一个实施方案中，式(I)中所述靶向配体中L1具有以下通式：
其中f,g，h和i分别是从1至20的整数，优选为1至10的整数，更优选为1至5的整数；
优选地，L1为以下结构：
一个实施方案中，在通式(I)中，所述化合物E选自：
一个实施方案中，通式(I)中的化合物L2为
其中j,k,l,m,n和o分别是从1至20的整数，优选为1至10的整数，更优选为1至5的整数；
优选地，L2具有以下结构式：
优选实施方案中，通式(I)具有以下结构：

(I-I)，其中a1＝3，

(I-II)，其中a1＝3,

(I-III)，其中a1＝3,

(I-IV)，其中a1＝3,

(I-V)，其中a1＝3,

(I-VI)，其中a1＝3,

(I-VII)，其中a1＝3,或者

(I-VIII)，其中a1＝3。
一个实施方案中，所述靶向配体中T选自N-乙酰基-半乳糖胺、半乳糖、半乳糖胺、N-甲酰基-半乳糖胺、N-丙酰基-半乳糖胺、N-正丁酰基半乳糖胺和N-异丁酰基-半乳糖胺中的一种，优选为N-乙酰基-半乳糖胺，结构式如下：
一个优选实施例中，通式(I)显示的靶向配体通过磷酸酯基团或硫代磷酸酯基团或膦酸基团与siRNA末端连接。
一个优选实施方案中,所述靶向配体具有以下结构:





一个实施方案中，所述靶向配体化合物包括如下结构：
其中R2是-O-，-S-，-NH-，-CH 2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-或-NH(CO)NH-，其中所述-CH2-可以任选被选自卤素，烷基(任选进一步被选自羟基、氨基、卤素、烷氧基和烷氨基的取代基取 代)，烷氧基，烷氨基的取代基取代；
所述p,q,r,s,t和u分别独立选自0到20的整数，优选为1到10的整数，进一步优选为1至5的整数。
在一个优选的实施方案中，所述靶向配体化合物包括以下结构：

在另一优选实施方案中，所述靶向配体化合物包括如下结构：
其中R2是-O-，-S-，-NH-，-CH 2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-或-NH(CO)NH-，所述-CH2-可以任选被选自卤素，烷基(任选进一步被选自羟基、氨基、卤素、烷氧基和烷氨基的取代基取代)，烷氧基，烷氨基的取代基取代；
所述p,q,r,s,t和u分别独立选自0到20的整数，优选为1到10的整数，进一步优选为1至5的整数。在一个优选实施方案中，所述靶向配体化合物包括如下结构：
一个优选实施方案中，通式(III)具有以下结构：



其中Y为O或者S。
另一优选实施方案中，式(III)显示的结合核苷酸X的所述靶向配体化合物具有以下结构：




其中Y为O或者S或者N。
在一些实施方案中，正义链选自如下式所示的核苷酸序列：
5’-NmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，或，
5’-NmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNms-3，或，
5’-NmNmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，或，
5’-sNmNmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’或，
5’-sNmNmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNms-3’，
其中，Nm表示甲氧基修饰的任意核苷酸，例如甲氧基修饰的胞嘧啶(C)、鸟嘌呤(G)、尿嘧啶(U)、腺嘌呤(A)、胸腺嘧啶(T)；Nf表示氟代修饰的任意核苷酸，例如氟代修饰的C、G、U、A、T；
小写字母s在大写字母中间时表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接，小写字母s在3’端第一个时表示与该字母s左侧相邻的一个核苷酸末端为硫代磷酸酯基,小写字母s在5’端第一个时表示与该字母s右侧相邻的一个核苷酸末端为硫代磷酸酯基,。
在一些实施方案中，反义链具有如下式所示的核苷酸序列：
5’-Nms’Nfs’Nm’Nm’Nm’Nf’Nm’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nm’Nm’Nm’Nms’Nms’Nm-3’，
其中，Nm’表示甲氧基修饰的任意核苷酸，例如甲氧基修饰的C、G、U、A、T；Nf’表示氟代修饰的任意核苷酸，例如氟代修饰的C、G、U、A、T；
小写字母s在大写字母中间时表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接。
以下通过具体实施例描述本申请的靶向配体的合成路线。
实施例1连接到固相载体上的氨基半乳糖化合物GENO-Gal-5合成
(1)合成路线
(2)具体合成步骤
1)化合物Int-11-2的制备
将Int-11-1(10g)溶于(140mL)DCM(Dichloromethane)并降温至0℃,滴加TMSCN(Trimethylsilyl cyanide，三甲基腈硅烷(TMSCN))(4.01g)和BF3.Et2O(2.83mL),反应10min.TLC板监测反应(使用Hexane:EtOAc＝5:1,KMnO4显色，原料Rf＝0.3,α构型Rf＝0.28，β构型Rf＝0.27)原料反应完全。反应完全后，将100mL饱和NaHCO3水溶液加入到反应液中，另加入100ml DCM分出有机相，将有机相用饱和食盐水洗一次，使有机相浓缩，然后溶于EtOAc(200mL)，用NaHCO3水溶液(100mL)洗一次，饱和食盐水洗一次，有机相使用无水硫酸钠干燥，浓缩。然后用正向硅胶柱纯化，缓慢增加极性，其中Hexane/EtOAc＝20％时出α构型，25％时出β构型，α构型得到(5.3g)白色固体,β构型得到(3.7g)无色油状。1H NMR(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).2)化合物Int-11-3的制备:
将HCl水溶液(1M,17.2mL)加入到Int-11-2(3.7g，β构型，无色油状)和10％Pd/C(377mg)的悬浮液中,加入乙酸乙酯/2-丙醇/乙醇(2:1:1,共90mL)的混合物中，在氢气(40Psi)下搅拌反应48小时。利用TLC监测反应，通过硅藻土过滤去除催化剂，减压浓缩溶剂。将得到的粗品用甲苯带两遍，然后溶于(10mL甲醇中，加入28％NH3.H2O(30mL),室温搅拌16小时，反应液浓缩后，用甲苯乙腈1:1混合溶液(50mL)带3次。将上述粗品取1g溶于(40mL)H2O降温至0℃,加入NaHCO3(1.4g),Na2CO3(0.88g),将FmocOSu(2.13g)溶于二氧六环(dioxane)(40mL)溶剂，然后滴加到上述水溶液中，室温反应1小时。TLC监测反应(DCM:MeOH＝10:1,254nm,Rf＝0.5).正向柱纯化，MeOH/DCM＝5％出产物，得Int-11-3(340mg)无色固体。1H NMR(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)。
3)化合物Int-11-4的制备
将Int-11-3(340mg)溶于2ml吡啶，降温至0℃,将DMTrCl(450mg)溶于2ml吡啶后逐滴滴加到上述溶液中，TLC监测，原料反应完全。加5ml水溶液淬灭反应，反向柱C18纯化MeCN/H2O＝80％出产物int-11-4(290mg)。
4)化合物GENO-Int-11的制备
将Int-11-4(700mg,1.02mmol)溶于2mL二氯甲烷，然后将DBU(310mg,2.04mmol)加到上述溶液中，TLC监测，原料反应完全。加5ml饱和Na2CO3溶液淬灭反应，用二氯甲烷20mL萃取，有机相浓缩用DCM:MeOH＝10:1,(254nm,Rf＝0.5)。正向柱纯化，MeOH/DCM＝5％出产物，得GENO-Int-11(550mg)黄色固体。
5)化合物Gal-5-1的制备
将1,12-十二烷二酸单苄酯(387mg，1.2mmol)溶于无水DMF(N,N-Dimethylformamide)(10mL)溶剂中，然后加入HBTU(O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate)(546mg，1.44mmol),DIEA(N,N-Diisopropylethylamine)(0.65mL，3.6mmol),HOBT(1-Hydroxybenzotriazole)(324mg，2.4mmol)和GENO-Int-11(560mg，1.2mmol，然后在氩气保护下室温搅拌过夜。反应液通过反向柱C18纯化MeCN/H2O＝70％出产物得到Gal-5-1(200mg)。1H NMR(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)。
6)化合物Gal-5-2的制备
将Gal-5-1(200mg，0.26mmol)溶于5mL乙酸乙酯中，加入50毫克钯炭和三乙胺(0.11mL，0.78mmol)，反应液在氢气(15psi))，室温下搅拌16小时，反应液过滤浓缩得Gal-5-2(150mg)。
7)化合物Gal-5-3的制备
将Gal-5-2(150mg，0.22mmol)溶于5mL无水DMF中，向此溶液中加HBTU(101mg，0.27mmol),DIEA(0.16mL),3A分子筛(1g)和Gal-3-4C(441mg.0.22mmol)。然后上述混合物于室温下搅拌16小时，反应液通过反向柱C18纯化MeCN/H2O＝60％出产物得到Gal-5-3(380mg)。1H NMR(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)。
8)化合物Gal-5-4的制备
将Gal-5-3(370mg，0.15mmol)，丁二酸酐(75mg，0.76mmol),3A分子筛(0.5g)和DMAP(4-Dimethylaminopyridine)(46mg，0.38mmol)溶于5mL THF(Tetrahydrofuran)中，然后反应液40℃搅拌过夜，反应液通过反向柱C18纯化MeCN/H2O＝40％出产物得到 Gal-5-4(220mg)。
9)连接到固相载体上的氨基半乳糖化合物GENO-Gal-5合成
将Gal-5-4(220mg，0.086mmol)悬浮于4ml CH3CN和2mL DMF中，滴加DIEA(0.035mL，0.216mmol)和HBTU(49.07mg，0.129mmol)，室温搅拌5分钟，向反应液中加入氨甲基树脂(99.51mg，100-200目，氨基载量250umol/g，25℃下进行摇床反应，转速220转/分钟，反应16h后过滤，滤饼以DCM淋洗3次，每次30ml，乙腈淋洗3次，每次30ml，30ml正己烷淋洗3次，真空油泵干燥2h，随后加入混合试剂(CapB1,4-二甲氨基吡啶,N-甲基咪唑和乙腈，11.2mL/12.4mg/0.50mL/4.32mL)进行盖帽反应。25℃下置于摇床上，转速220转/分钟，反应16h，反应液过滤，滤饼用乙腈淋洗3次，每次30ml，抽滤至干，真空油泵减压下干燥过夜，得目标产物，GNEO-Gal-5化合物160mg。
10)化合物Int-6-1的制备
将Gal-3-5B(1.45g，3.24mmol)溶于无水DMF(10mL)溶剂中，然后加入HATU(1.64g，4.32mmol),3A分子筛(1g)和DIEA(1.07mL，6.47mmol),反应液室温下搅拌30min,然后把GENO-int-11(1g，2.16mmol)溶于DMF(10mL)加入到上述反应液中，反应液在氩气保护下室温搅拌过夜。反应液过滤，通过反向柱C18纯化MeCN/H2O＝60％出产物得到Int-6-1(1.8g)。1H NMR:(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＝3.6 Hz,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).
将Int-6-1(2.1g，2.35mmol)溶于DCM(30mL)，加入Tetrazole(33mg,0.47mmol),NMI(77.2mg，0.94mmol)和2g分子筛，反应液用氩气置换三次，在室温下搅拌20min，然后把磷试剂(920.81mg，3.06mmol)溶于少量二氯甲烷加入到反应液中，室温下搅拌1小时。反应液用饱和NaHCO3水溶液洗两次，水洗一次，食盐水洗一次，室温浓缩，通过反向柱C18纯化MeCN/H2O＝65％出产物得到GENO-Gal-6(1.5g)。1H NMR:(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).
实施例2化合物GENO-Gal-7合成
(1)合成路线

1)化合物Gal-7-1的制备
将GENO-Int-5A(5.91g，15.53mmol)溶于无水DMF(40mL)溶剂中，然后加入HATU(5.91g，15.53mmol),3A分子筛(3g)和DIEA(6.42mL，38.83mmol),反应液室温下搅拌30min,然后把GENO-int-11(3.6g，7.77mmol)溶于DMF(10mL)加入到上述反应液中，反应液在氩气保护下室温搅拌过夜。反应液过滤，通过反向柱C18纯化MeCN/H2O＝60％出产物得到Gal-7-1(4.7g)。
2)化合物Gal-7-2的制备
将Gal-7-1(800mg,0.91mmol)溶于10mL二氯甲烷，然后将DBU(0.27mL,1.83mmol)加到上述溶液中，室温搅拌1h,TLC监测，原料反应完全。加5ml饱和Na2CO3溶液淬灭反应，用二氯甲烷30mL萃取，有机相浓缩，用反相柱C18纯化MeCN/H2O＝35％出产物得330mg。(产物用CH3CN/H20＝20/1硅胶薄层层析处理)。
3)化合物Gal-7-3的制备
将Gal-3-5B(318.72mg，0.712mmol)溶于无水DMF(4mL)溶剂中，然后加入HATU(361.14mg，0.95mmol),3A分子筛(1g)和DIEA(0.24mL，1.43mmol),反应液室温下搅拌30min,然后把Gal-7-2(310mg，0.48mmol)溶于DMF(3mL)加入到上述反应液中，反 应液在氩气保护下室温搅拌过夜。反应液过滤，通过反向柱C18纯化MeCN/H2O＝50％出产物得到Gal-7-3(370mg)。
4)化合物GENO-Gal-7的制备
将Gal-7-3(3.0g，2.77mmol)溶于DCM(30mL)，加入7-B(1.34g,4.44mmol),DCI(491mg,4.16mmol)和2g分子筛，反应液用氩气置换三次，在室温下搅拌20min，，室温下搅拌1小时。反应液用饱和NaHCO3水溶液洗两次，水洗一次，食盐水洗一次，室温浓缩，通过反向柱C18纯化MeCN/H2O＝65％出产物得到GENO-Gal-6(1.5g)；
1H NMR:δ7.46-7.44(d,J＝7.7Hz 2H),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＝3.2Hz 1H),5.00-4.96(dd,J＝11.2Hz,3.3Hz 1H),4.51-4.49(d,J＝8.5Hz 1H),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)；
31PNMR:ET51671-213-P1O1(162MHz,CD3CN)；
δ146.92,146.03.
5)化合物Gal-7-3a的制备
将Gal-7-3(3.0g，2.77mmol)，丁二酸酐(2.22g，22.1mmol),3A分子筛(0.5g)和三乙胺(2mL，3.8mmol)溶于DCM(6mL)中，然后反应液室温下搅拌过夜，反应液过滤，用5％NaCl溶液洗涤，反应液室温浓缩，通过反向柱C18纯化MeCN/H2O＝40％出产物得到Gal-7-3a(1.4g)。
6)化合物Gal-7-3a的制备
将Gal-7-3a(0.7g，0.6mmol)悬浮于8mL乙腈和4mL N,N-二甲基甲酰胺中，滴加DIEA(0.83mL，4.74mmol)和HBTU(1.12g，2.96mmol)，室温摇晃5分钟，向反应液中加入氨甲基树脂(409.43mg，100-200目，氨基载量250umol/g，25℃下进行摇床反应，转速220转/分钟，反应16h后过滤，滤饼以DCM淋洗3次，每次30ml，乙腈淋洗3次，每次30ml，30ml正己烷淋洗3次，真空油泵干燥2h，随后再按照表2中示出的投料配比加入原料(CapB1,4-二甲氨基吡啶,N-甲基咪唑和乙腈)进行盖帽反应。25℃下置于摇床上，转速220转/分钟，反应16h，反应液过滤，滤饼用乙腈淋洗3次，每次30ml，抽滤至干，真空油泵减压下干燥过夜，得GNEO-Gal-7A化合物4.9g.
实施例3合成氨基半乳糖分子簇缀合的寡核苷酸
siRNA的合成与通常的亚磷酰胺固相合成法基本相同，所不同的是在合成siRNA的SS链时，使用上述所合成的连接有氨基半乳糖簇的CPG载体代替通常的Universal-CPG载体。siRNA的合成(具体序列见表1)过程简要描述如下：于Dr.Oligo 48合成器(Biolytic)上，以Universal CPG载体或连接有氨基半乳糖簇的CPG载体为起始，根据合成程序逐个连接核苷亚磷酰胺单体，其中2’-F RNA、2’-O-甲基RNA等核苷亚磷酰胺单体原料购自芜湖华仁和上海兆维。采用5-乙基硫-1H-四唑(ETT)作为活化剂(0.6M乙腈溶液)，使用0.22M的PADS溶于1:1体积比的乙腈和三甲基吡啶(上海凌江)混合溶剂中，得到的溶液作为硫化试剂，使用碘吡啶/水溶液(上海凌江)作为氧化剂。
固相合成完成后，寡核糖核苷酸自该固体支撑物裂解，采用28％氨水在50℃条件下浸泡16小时。然后离心，将上清液转移到另一个离心管中，浓缩蒸发干后，使用C18反向色谱纯化，流动相为0.1M TEAA和乙腈。目标寡核苷酸收集后反复冻干，并经LC-MS鉴定为目标产物，再经过UV(260nm)定量。
所得到的单链寡核苷酸，根据等摩尔比，按照互补配对，与AS链退火，最后所得到的双链siRNA溶于1X PBS中，并调整至实验所需浓度。
表1本申请实施例中用于抑制ANGPTL3表达的寡核苷酸序列(被递送的序列)

表2本申请实施例中用于检测LPA siRNA在肝脏中分布的寡核苷酸序列(被递送的序列)
L96结构式：
Gal-3结构式：
[ST23sST23sST23sC6XLT]结构式:
表1和表2的序列中，各符号分别用于表示如下修饰的核苷酸(酯)：
A＝腺苷-3’-磷酸酯
C＝胞苷-3’-磷酸酯
G＝鸟苷-3’-磷酸酯
U＝尿苷-3’-磷酸酯
Am＝2’-O-甲基腺苷-3’-磷酸酯
Ams＝2’-O-甲基腺苷-3’-硫代磷酸酯
Cm＝2’-O-甲基胞苷-3’-磷酸酯
Cms＝2’-O-甲基胞苷-3’-硫代磷酸酯
Gm＝2’-O-甲基鸟苷-3’-磷酸酯
Gms＝2’-O-甲基鸟苷-3’-硫代磷酸酯
Um＝2’-O-甲基尿苷-3’-磷酸酯
Ums＝2’-O-甲基尿苷-3’-硫代磷酸酯
Af＝2’-氟腺苷-3’-磷酸酯
Afs＝2’-氟腺苷-3’-硫代磷酸酯
Cf＝2’-氟胞苷-3’-磷酸酯
Cfs＝2’-氟胞苷-3’-硫代磷酸酯
Gf＝2’-氟鸟苷-3’-磷酸酯
Gfs＝2’-氟鸟苷-3’-硫代磷酸酯
Uf＝2’-氟尿苷-3’-磷酸酯
Ufs＝2’-氟尿苷-3’-硫代磷酸酯
其中：小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；
小写字母s在大写字母中间时表示与该字母s左右相邻的两个核苷酸之间的连接为硫代磷酸酯基连接；
小写字母s在3’端第一个时表示与该字母s左侧相邻的一个核苷酸末端为硫代磷酸酯基；
在寡核苷酸中，核苷酸单体通过5’-3’-磷酸二酯键相互连接，包含硫代磷酸酯连键和磷酸二酯连接。
实施例4 ANGPTL3 RNAi剂在小鼠体内抑制Angptl3 mRNA表达
为了评价ANGPTL3 RNAi的体内活性，使用雄性C57BL/6小鼠。适应环境设施后，根据体重，将小鼠平均分组，每组3只。在给药当天(第0天)，给予小鼠皮下注射ANGPTL3 RNAi溶液，对照组小鼠给予PBS溶液，所有小鼠在颈背部皮下注射给药一次，给药剂量为1mg/Kg，给药体积5ml/Kg。给药前3天、给药后第5天、第14天、第21天和第28天，所有小鼠通过眼眶静脉丛(异氟烷麻醉后)采血用于收集血清，部分给药组小鼠在第35天和42天继续采血。采血结束后，将动物安乐死。
通过使用ELISA方法(mANGPTL3,R&D)检测小鼠血清中mANGPTL3蛋白水平来评价mANGPTL3表达敲低情况。为了归一化，将给定时间点的每只动物的mANGPTL3水平除以在此动物中的预处理水平(Day-3)以确定“归一化至预处理”的表达的比率；然后通过将各只动物的“归一化至预处理”比率除以对照组中所有小鼠的平均“归一化至预处理”比率，将特定时间点的表达归一化至对照组。这就使每个时间点的表达归一化至对照组的表达，归一化之后的具体检测结果如图1所示。
将ANGPTL3 RNAi剂(包括表1所述的寡核苷酸序列)如上所述施用给C57 BL/6小鼠。每只小鼠接受单次皮下注射(SC)剂量1mg/Kg ANGPTL3 RNAi剂溶液，监测血清中的mANGPTL3蛋白水平长达42天。敲低水平和响应持续时间示于表3中。从表3可看出，施用第5天时，11个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于80％的敲低，其中有6个 施用的ANGPTL3 RNAi剂1mg/Kg显示出大于90％的敲低；施用第14天时，有8个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于70％的敲低，其中有5个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于80％的敲低；施用第21天时，有8个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于50％的敲低，其中有3个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于70％的敲低；施用第28天时，有5个施用的ANGPTL3 RNAi剂对mANGPTL3蛋白水平敲低仍保持大于50％的敲低；施用第35天时，有2个施用的ANGPTL3 RNAi剂对mANGPTL3蛋白水平敲低仍保持大于45％的敲低。
血管生成素样3(ANGPTL3)是由人类血管生成素-样3基因编码的血管生成素蛋白，主要在肝脏中表达并用于调节脂质新陈代谢。靶向ANGPTL3的有效治疗剂可用于治疗(包括预防性治疗)代谢类疾病，例如高甘油三酯血症。表3的实验结果表明，本申请的靶向配体，可以将寡核苷酸序列高效地递送到肝脏细胞中并抑制ANGPTL3的表达。
表3.皮下施用1mg/Kg ANGPTL3 RNAi剂后小鼠血清中的相对mANGPTL3水平
实施例5缀合LPA RNAi剂皮下给药后在野生型小鼠肝脏和肾脏中的分布比例
将30只C57BL6/J小鼠分为10组，将指定的5组LPA RNAi剂(包括如表2所示的寡核苷酸序列)在第一天以10mg/kg的剂量对每组小鼠进行皮下注射。5组小鼠在给药后1小时进行组织收集，另外5组小鼠在给药后24小时进行组织收集。将收集的组织样本清速冻，并转移至-80℃保存。组织匀浆后，通过杂交荧光探针-酶联免疫吸附测定法测量肝组织匀浆液与肾组织匀浆液中对应LPA RNAi剂的浓度，以肝组织匀浆液浓度除以肾组织匀浆液浓度，计算肝肾比进行评价。结果如表4和图2所示。
图2显示了SC给药10mg/kg LPA RNAi剂后，野生型小鼠肝脏与肾脏化合物浓度比值。结果显示Geno-1-105M～Geno-1-108M在靶向器官肝脏中的浓度在不同时间点均远高于在非靶向器官肾脏中的浓度，肝脏比肾脏的药物浓度比值远大于1。同时Geno-1-1004M在测试的两 个时间点中，非靶向器官肾脏中的浓度均大于肝脏，肝脏比肾脏的药物浓度比值小于1。
表4 SC给药10mg/kg LPA RNAi剂后，野生型小鼠肝脏与肾脏化合物浓度比值
脂蛋白(a)[Lp(a)]是一种异质型低密度脂蛋白(LDL)-样颗粒，LPA(Apo(a))基因主要在肝脏中表达。高Lp(a)水平是心血管疾病、中风和其它相关病症(包括动脉粥样硬化性狭窄)的独立风险因子。上述实施例证明本申请的靶向配体，能高效地将目标寡核苷酸序列递送至肝脏细胞。表4中相比对照Geno-1-1004M使更多的目标物累积至肾脏中，其他配体则主要是将目标物递送至肝脏。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (23)

  1. [细则91条20.02.2023]一种具有式(Z-1)所示的可断裂化合物或其药学上可接受的盐，
    其中R1是O、S、NR3或者CR3R4,其中R3和R4各自独立地是氢、卤素、取代或未取代的脂族基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的杂环或取代或未取代的环烷基；
    其中R2为-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或者-NH(CO)NH-，其中，所述-CH2-任选被选自卤素，烷基，烷氧基，烷氨基的取代基取代；所述a和b相同或者不相同，并且分别选自0到20的整数，优选为1到10的整数，进一步优选为1到5的整数。
  2. [细则91条20.02.2023]根据权利要求1所述的可断裂化合物或其药学上可接受的盐，其特征在于，所述化合物结构式为：
  3. [细则91条20.02.2023]一种寡核苷酸配体化合物或其药学上可接受的盐，包括根据权利要求1或2所述的可断裂化合物或其药学上可接受的盐，所述寡核苷酸配体化合物通式为：
    (I)或者包括通式(II)的结构:
    其中通式(I)中：
    Z为所述可断裂化合物，用于与核苷酸序列X连接，所述L1为第二接头部分，E为分支点基团；所述分支点基团E与a1个靶向组合体连接，所述a1为选自0到10的整数，优选1到5的整数；其中所述靶向组合体包括1：1比例的栓系部分L2和靶向部分T；
    通式(II)中，Y是O或S或N，且L3具有以下结构：
    其中R2是-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或-NH(CO)NH-，其中所述-CH2-任选被选自卤素，烷基的取代基取代，且所述烷基任选进一步被选自羟基、氨基、卤素、烷氧基和烷氨基的取代基取代；
    所述p,q,r,s,t和u选自0到20的整数，优选为1到10的整数。
  4. [细则91条20.02.2023]根据权利要求3所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述通式(II)用于与核苷酸序列连接。
  5. [细则91条20.02.2023]根据权利要求3所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述通式(I)中，所述第二接头部分L1具有以下结构： 或者
    其中f,g，h和i分别是从1至20的整数，优选为1至10的整数，进一步优选为1至5的整数。
  6. [细则91条20.02.2023]根据权利要求3所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述通式(I)中，化合物L2的结构式为：
    其中j,k,l,m,n和o分别是从1至20的整数，优选为1至10的整数，进一步优选为1至5的整数。
  7. [细则91条20.02.2023]根据权利要求3所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述靶向部分T选自组织的特异性靶向配体，优选地，所述靶向部分T为肝特异性靶向配体；进一步优选地，所述靶向部分T具有用于增强肝细胞对寡聚化合物的摄取的结构。
  8. [细则91条20.02.2023]根据权利要求3所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述靶向部分T与L3具有相同或不同的结构。
  9. [细则91条20.02.2023]根据权利要求3-8所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述配体化合物具有以下结构：
  10. [细则91条20.02.2023]根据权利要求3-8所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述配体化合物包括以下结构：
  11. [细则91条20.02.2023]根据权利要求3-8所述的寡核苷酸配体化合物或其药学上可接受的盐，其特征在于，所述配体化合物结合核苷酸序列X后具有以下结构：
    其中Y为O或者S。
  12. [细则91条20.02.2023]一种RNA干扰剂，包括：正义链和/或反义链，以及根据权利要求3-11中任一项所述的寡核苷酸配体化合物或其药学上可接受的盐。
  13. [细则91条20.02.2023]根据权利要求12所述的RNA干扰剂，其特征在于，包括反义寡核苷酸，siRNA或miRNA。
  14. [细则91条20.02.2023]根据权利要求12所述的RNA干扰剂，其特征在于，包括一个或多个修饰的核苷酸。
  15. [细则91条20.02.2023]根据权利要求12所述的RNA干扰剂，其特征在于，所述正义链和/或反义链上的一个或多个核苷酸被修饰以形成修饰的核苷酸。
  16. [细则91条20.02.2023]根据权利要求12所述的RNA干扰剂，经由磷酸酯基团、硫代磷酸酯基团或膦酸酯基团与寡核苷酸配体化合物或其药学上可接受的盐连接。
  17. [细则91条20.02.2023]根据权利要求12所述的RNA干扰剂，其特征在于，所述寡核苷酸配体化合物或其药学上可接受的盐偶联于正义链和/或反义链。
  18. [细则91条20.02.2023]根据权利要求12所述的RNA干扰剂，其特征在于，所述寡核苷酸配体化合物或其药学上可接受的盐偶联于反义链的5’末端和/或3’末端，或者偶联于正义链的5’末端和/或3’末端。
  19. [细则91条20.02.2023]一种药物组合物，其包含根据权利要求1或2所述的可断裂化合物或其药学上可接受的盐，或者包含根据权利要求3-11中任一项所述的寡核苷酸配体化合物或其药学上可接受的盐。
  20. [细则91条20.02.2023]根据权利要求1或2所述的可断裂化合物或其药学上可接受的盐，或者根据权利要求3-11中任一项所述的寡核苷酸配体化合物或其药学上可接受的盐在制备用于预防和/或治疗由肝细胞中特定基因的表达而引起的生理状况或疾病的药物，或者肝细胞功能异常而引起的生理状况或疾病的药物中的用途。
  21. [细则91条20.02.2023]根据权利要求20所述的用途，其特征在于，所述用途包括将根据权利要求1或2所述的可断裂化合物或其药学上可接受的盐，或者根据权利要求3-11中任一项所述的寡核苷酸配体化合物或其药学上可接受的盐与肝细胞进行接触。
  22. [细则91条20.02.2023]根据权利要求20所述的用途，其特征在于，所述可断裂化合物在将核酸分子递送至目标细胞后在R2处发生断裂。
  23. [细则91条20.02.2023]根据权利要求1或2所述的可断裂化合物或其药学上可接受的盐，或者根据权利要求3-11中任一项所述的寡核苷酸配体化合物或其药学上可接受的盐用于靶向结合核酸并将其递送至人或哺乳动物的肝脏细胞的用途。
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