Drug delivery compound, oligonucleotide conjugate and application thereof

Abstract

The invention relates to a transferrin receptor-targeting drug delivery compound as shown in a formula I, an oligonucleotide conjugate and application of the transferrin receptor-targeting drug delivery compound and the oligonucleotide conjugate. The compounds of the present invention are capable of efficiently delivering oligonucleotides to target organs or target tissues. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and particularly relates to a drug delivery compound, an oligonucleotide conjugate and their uses. Background Art

[0002] Oligonucleotides include, but are not limited to, small interfering RNA (siRNA), small activating RNA (saRNA) and antisense nucleotides. In recent years, oligonucleotides, especially siRNA, have made considerable progress in drug development. siRNA as a drug active ingredient is known to the public, and the RNA interference mechanism corresponding to siRNA is a type of RNA interference mediated by an endonuclease complex. At the gene level, almost all diseases, including tumors, infectious diseases, genetic diseases, etc., can be treated by siRNA. Therefore, siRNA has become the most popular biomedical research field.

[0003] However, siRNA therapy has also encountered unique challenges in development. The targeted delivery, potential off-target effects and in vivo stability of oligonucleic acid substances have been the main challenges in the development of siRNA drugs. Among them, achieving efficient siRNA drug delivery has become the primary limiting factor for the development of such drugs into clinical applications. Many siRNAs showing excellent pharmaceutical activities in preclinical pharmaceutical research centers are difficult to effectively reach specific target organs or tissues due to the lack of effective delivery vectors, and thus are difficult to be used in actual drug research and development, especially for some neurological diseases, including central nervous system diseases and peripheral nervous system diseases, muscle diseases. Therefore, there is still a significant practical need in this field for oligonucleotide drugs that can be effectively delivered to specific target organs or tissues, especially such as the brain, spinal cord, optic nerve tissue, olfactory nerve tissue, auditory nerve and other peripheral nerve tissues, neuromuscular junctions or muscle tissues, and tumor tissues.

[0004] To solve the above problems, the present invention provides a drug delivery compound targeting the transferrin receptor, and its conjugate with an oligonucleotide, which can effectively deliver the oligonucleotide to the target organ or tissue. Summary of the Invention

[0005] The present invention first provides a compound represented by Formula I, or its stereoisomer, or its deuterated compound, or its fluorinated compound, or its pharmaceutically acceptable salt:

[0006]

[0007] Wherein,

[0008] n is 1, 2, 3 or 4;

[0009] X 1 、X 2, X 3 , X 4 are each independently selected from N or CR X ;

[0010] Each R X is independently selected from hydrogen, halogen, cyano, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, -OH, -NH2, -O(C 1～6 alkyl), -O(halogen-substituted C 1～6 alkyl), -NH(C 1～6 alkyl), -N(C 1～6 alkyl)(C 1～6 alkyl), 3- to 6-membered carbocyclic group;

[0011] R 1 , R 2 are each independently selected from hydrogen, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, 3- to 6-membered carbocyclic group.

[0012] Furthermore,

[0013] each R X is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl, trifluoromethyl, -OH, -NH2, -O(methyl), -O(ethyl), -NH(methyl), -NH(ethyl), -N(methyl)(methyl), cyclopropyl;

[0014] R 1 , R 2 are each independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl.

[0015] More specifically,

[0016]

[0017] The present invention also provides the use of any one of the above compounds as a delivery ligand targeting the transferrin receptor in the preparation of a drug.

[0018] The present invention also provides the use of any one of the above compounds as an intermediate in the preparation of a drug.

[0019] Furthermore, the drug is an oligonucleotide drug; preferably, the oligonucleotide drug is an antisense nucleotide, siRNA or saRNA.

[0020] The present invention also provides an oligonucleotide conjugate represented by Formula II, or a pharmaceutically acceptable salt thereof:

[0021]

[0022] wherein,

[0023] n is 1, 2, 3 or 4;

[0024] X 1 、X 2 、X 3 、X 4 are each independently selected from N or CR X ;

[0025] Each R X is independently selected from hydrogen, halogen, cyano, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, -OH, -NH2, -O(C 1～6 alkyl), -O(halogen-substituted C 1～6 alkyl), -NH(C 1～6 alkyl), -N(C 1～6 alkyl)(C 1～6 alkyl), 3- to 6-membered carbocyclic group;

[0026] R 1 、R 2 are each independently selected from hydrogen, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, 3- to 6-membered carbocyclic group;

[0027] m is 1, 2, 3, 4, 5 or 6;

[0028] L 1 、L 2 are each independently a linking group;

[0029] Nu is an oligonucleotide moiety.

[0030] Furthermore,

[0031] Each R X is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl, trifluoromethyl, -OH, -NH2, -O(methyl), -O(ethyl), -NH(methyl), -NH(ethyl), -N(methyl)(methyl), cyclopropyl;

[0032] R 1 、R2 Independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, and cyclopropyl, respectively.

[0033] More specifically, the oligonucleotide conjugate shown in Formula II is specifically:

[0034]

[0035] Wherein,

[0036] m is 1, 2, 3, 4, 5, or 6;

[0037] L 1 and L 2 Are independently linking groups;

[0038] Nu is an oligonucleotide moiety.

[0039] In some embodiments of the present invention, the Nu is a single-stranded oligonucleotide moiety or a double-stranded oligonucleotide moiety.

[0040] In some embodiments of the present invention, the double-stranded oligonucleotide moiety comprises a sense strand and an antisense strand, and the sense strand and the antisense strand each comprise 15 to 30 nucleotides, wherein each of the nucleotides is independently a modified or unmodified nucleotide.

[0041] In some embodiments of the present invention, the double-stranded oligonucleotide moiety is linked to L 2 via the sense strand, or the double-stranded oligonucleotide moiety is linked to L 2 via the antisense strand.

[0042] In some embodiments of the present invention, at least one of the 5'-end or 3'-end of the sense strand of the double-stranded oligonucleotide moiety is linked to L 2

[0043] In some embodiments of the present invention, the sense strand and the antisense strand at least partially complement each other to form a double-stranded region, and the antisense strand is at least partially reverse complementary to the target mRNA, and the target mRNA is the mRNA expressed by the target gene in the target cells in the target tissue or target organ, and the target cells are the cells with transferrin receptors on the cell surface.

[0044] In some embodiments of the present invention, at least one of the nucleotides in the sense strand and the antisense strand is a modified nucleotide.

[0045] In some embodiments of the present invention, the double-stranded oligonucleotide in the double-stranded oligonucleotide moiety is siRNA or saRNA.

[0046] In some embodiments of the present invention, further,

[0047] Each L​1 independently selected from linking groups having 1 to 30 carbon atoms in length, wherein one or more methylene groups may optionally be replaced by one or more of the following groups: -C(O)-, -NH-, O, S, -S(O)-, -S(O)2-, -P(O)(OH)-, -P(S)(OH)-, -CH=CH-, -C≡C-, -CH=N-, a carbocyclic group, a heterocyclic group; and may each optionally be substituted by one or more of the following groups: C 1～10 alkyl, halogen-substituted C 1～10 alkyl, -(C 0～10 alkylene)-OH, -(C 0～10 alkylene)-O-(C 1～10 alkyl), -(C 0～10 alkylene)-SH, -(C 0～10 alkylene)-S-(C 1～10 alkyl), -(C 0～10 alkylene)-NH2, -(C 0～10 alkylene)-NH(C 1～10 alkyl), -(C 0～10 alkylene)-N(C 1～10 alkyl)(C 1～10 alkyl);

[0048] Each L 2 is independently selected from straight-chain or branched linking groups having 1 to 50 carbon atoms in length, wherein one or more methylene groups may optionally be replaced by one or more of the following groups: -C(O)-, -NH-, O, S, -S(O)-, -S(O)2-, -P(O)(OH)-, -P(S)(OH)-, -CH=CH-, -C≡C-, -CH=N-, a carbocyclic group, a heterocyclic group; and may each optionally be substituted by one or more of the following groups: C 1～10 alkyl, halogen-substituted C 1～10 alkyl, -(C 0～10 alkylene)-OH, -(C 0～10 alkylene)-O-(C 1～10 alkyl), -(C 0～10 alkylene)-SH, -(C 0～10 alkylene)-S-(C 1～10 alkyl), -(C 0～10 alkylene)-NH2, -(C 0～10 alkylene)-NH(C 1～10 alkyl), -(C 0～10 alkylene)-N(C 1～10 alkyl)(C 1～10 alkyl).

[0049] More specifically,

[0050] The said L 1 Specifically selected from:

[0051]

[0052]

[0053] where p is 0, 1, 2, 3, 4, 5 or 6;

[0054] The said L 2 Specifically selected from:

[0055]

[0056] where q is 0, 1, 2, 3, 4, 5 or 6.

[0057] More specifically, -L 1 -L 2 - Specifically:

[0058]

[0059] where the aa end is connected to Nu.

[0060] The present invention also provides a pharmaceutical composition comprising any of the above-mentioned oligonucleotide conjugates and a pharmaceutically acceptable carrier thereof.

[0061] The present invention also provides the use of any of the above-mentioned oligonucleotide conjugates and / or the above-mentioned composition in the preparation of a drug for inhibiting the expression of a target gene in cells.

[0062] On the other hand, the present invention also provides a method for inhibiting the expression level of a target gene in cells, the method comprising contacting the cells with an effective amount of the siRNA, pharmaceutical composition and / or siRNA conjugate of the present invention.

[0063] The oligonucleotide conjugate provided by the present invention is conjugated by an oligonucleotide and a drug delivery compound, and comprises an oligonucleotide and a conjugation group conjugated to the oligonucleotide. The conjugation group comprises a linker and a pharmaceutically acceptable targeting group and / or delivery assisting group. Moreover, the oligonucleotide, the linker and the targeting group or the delivery assisting group are covalently or non-covalently connected in sequence. Each targeting group is selected from ligands capable of binding to the transferrin receptor on the cell surface, and each delivery assisting group is selected from groups capable of increasing the biocompatibility of the siRNA conjugate in the target organ or tissue to be delivered.

[0064] In some embodiments, the siRNA drug comprising the present invention is double-stranded. For a double-stranded siRNA drug, the lengths of the sense and antisense strands of the siRNA drug are independently 15-30 nucleotides. In some embodiments, the double-stranded siRNA drug comprises a sense strand and an antisense strand that are at least partially complementary to each other (at least 70% complementary). The antisense strand comprises a region having a sequence that is perfectly complementary (100% complementary) or at least partially complementary (at least 85% complementary) to a sequence in the target mRNA. The sense and antisense strands can be of the same length or different lengths.

[0065] In some embodiments, the length of the sense strand is about 19 nucleotides, while the length of the antisense strand is about 21 nucleotides. In some embodiments, the length of the sense strand is about 21 nucleotides, while the length of the antisense strand is about 23 nucleotides. The region of perfect or partial complementarity between the sense strand and the antisense strand is typically 15-25 nucleotides in length and is located at or near the 5'-end of the antisense strand (e.g., the region is 1, 2, 3, or 4 nucleotides from the 5'-end of the antisense strand and is imperfect or partially complementary).

[0066] For a double-stranded siRNA drug, if there are other sense strand nucleotides, they can be the same as or different from the corresponding sequences in the target mRNA. If there are other antisense strand nucleotides, they can be complementary or non-complementary to the other nucleotides of the corresponding sense strand (if any).

[0067] In some embodiments, the sense and antisense strands of the double-stranded siRNA drug comprising the modified nucleotides of the present invention contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the siRNA drug of the present invention contain different numbers of nucleotides.

[0068] In some embodiments, both the sense and antisense strands contain 1 to 4 phosphorothioate linkages.

[0069] In some embodiments, in the siRNAs of the present invention, each nucleotide is a modified or unmodified nucleotide. In the context of the present disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by substituting the hydroxyl group at the 2'-position of the ribose of the nucleotide with another group, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide does not cause a significant weakening or loss of the function of the siRNA to regulate gene expression. For example, the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov. Today, 2008, 13(19-20):842-55 can be selected.

[0070] In some embodiments, the siRNA drug containing the modified nucleotide inhibits the expression of the target mRNA in cells, tissues or in vivo. In some embodiments, a therapeutically effective amount of the siRNA drug containing the modified nucleotide of the present invention is administered in vivo to inhibit the expression of the target mRNA in vivo.

[0071] In some embodiments, the siRNA drug is used to treat, prevent or control the clinical manifestations related to the expression of the target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more siRNA drugs is administered to a subject in need of such treatment, prevention or control.

[0072] Definition of the terms used in the present invention: Unless otherwise specified, the initial definitions provided for the groups or terms herein apply to such groups or terms throughout the specification; for terms not specifically defined herein, their meanings should be given according to the disclosure content and context by those skilled in the art.

[0073] In the present invention, unless otherwise specified, the capital letters C, G, U, A, T represent the base composition of nucleotides, including modified and unmodified nucleotides; m represents that the nucleotide adjacent to the right of the identifier m is a 2'-methoxy nucleotide; f represents that the nucleotide adjacent to the right of the identifier f is a 2'-fluoro nucleotide; d represents that the nucleotide adjacent to the right of the identifier d is a 2'-deoxy nucleotide; gn represents that the nucleotide adjacent to the right of the identifier gn is a glycerol nucleotide (GNA); the identifier * represents that there is a phosphorothioate linkage between the two nucleotides adjacent to the left and right of the identifier * (or between the nucleotide and the linker-targeting ligand moiety); eVP represents that the nucleotide adjacent to its right is a (E)-vinyl phosphate modified nucleotide; iab represents an inverse abasic residue.

[0074] As used herein, "oligonucleotide" refers to a polymer of linked nucleosides, where each nucleoside can be independently modified or unmodified, and includes an oligonucleotide sequence having about 10 - 50 single-stranded nucleotides or double-stranded nucleobase pairs. In some embodiments, the oligonucleotide has a nucleobase sequence that is at least partially complementary to the core sequence of a target gene expressed intracellularly. In some embodiments, the oligonucleotide can regulate the expression of the corresponding target gene after being delivered to a cell expressing the gene. The expression of the target gene can be regulated in vitro or in vivo. "Oligonucleotide" includes, but is not limited to: single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0075] As used herein, "siRNA" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains an oligonucleotide capable of reducing or inhibiting messenger RNA (mRNA) translation in a sequence-specific manner.

[0076] siRNA can act through the RNA interference mechanism (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) in mammalian cells), or any other mechanism or pathway. Although it is believed that the siRNA drugs used in the present invention act mainly through the RNA interference mechanism, the siRNA drugs are not limited to or restricted to any specific action pathway or mechanism. siRNA drugs include, but are not limited to: single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and dicer enzyme substrates. The siRNA drugs described in the present invention are composed of an oligonucleotide chain having at least partial complementarity to the mRNA serving as the target. In some embodiments, the siRNA drugs described in the present invention are double-stranded and composed of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

[0077] The terms "silence", "reduce", "inhibit", "downregulate", or "knockdown" mean that when a given gene is expressed, the expression level of the gene is reduced or decreased when the cell, tissue, organ, or animal in vivo is treated with the siRNA drug molecules described in the present invention, compared to the cell, tissue, organ, or animal in vivo that has not been so treated.

[0078] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, represented alphabetically using standard nucleotide nomenclature.

[0079] The term "complementary" when used to describe the relationship between a first nucleotide sequence (such as the sense strand of an siRNA drug or target mRNA) and a second nucleotide sequence (such as a single-stranded antisense oligonucleotide or the antisense strand of a double-stranded siRNA drug) refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence and an oligonucleotide or polynucleotide containing the second nucleotide sequence to hybridize (under mammalian physiological conditions or similar in vitro conditions) to form base pairs and form a duplex or double helix structure. Complementary sequences contain Watson-Crick base pairs or non-Watson-Crick base pairs and contain natural or modified nucleotides or nucleotide analogs to an extent sufficient to meet the requirements of the above hybridization. For example, for the purpose of determining identity or complementarity, monomer a and Af are complementary to U (or T) and are equivalent to A.

[0080] The term "sense strand" refers to the strand on an RNA molecule that carries the nucleotide sequence encoding the amino acids of a protein, also known as the coding strand, sense strand, or plus strand, and the other nucleotide sequence complementary to it is the antisense strand.

[0081] The term "antisense strand" is substantially reverse complementary or essentially reverse complementary to a nucleotide sequence of the same length as the antisense strand in the mRNA expressed by the target gene.

[0082] The term "optionally" or "optionally" means that the subsequent described event or circumstance may but need not occur, and this description includes the occasions where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted by an alkyl group" means that the alkyl group may but need not be present, and this description includes the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group.

[0083] The term "substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, are independently replaced by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine what substitutions are possible or impossible through existing technical means and under experimental conditions (by experiment or theory). For example, an amino or hydroxyl group with a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (such as olefinic) bond.

[0084] The minimum and maximum carbon atom contents in a hydrocarbon group are indicated by a prefix. For example, the prefix C a～ C b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, C 1～6 Alkyl refers to a straight-chain or branched-chain alkyl group containing 1 to 6 carbon atoms.

[0085] An alkyl group refers to a straight-chain or branched hydrocarbon group in an alkane molecule, such as methyl -CH3, ethyl -CH2CH3, methylene -CH2-; an alkyl group can also be part of other groups, such as C1 - C6 alkoxy groups and C1 - C6 alkylamino groups. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available attachment point, and the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxy, carboxyl or carboxylate groups.

[0086] “Alkenyl” refers to a straight-chain or branched hydrocarbon group having at least 2 carbon atoms and having at least 1 vinyl unsaturation site (>C=C<). For example, C a-b An alkenyl group refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0087] “Alkynyl” refers to a straight-chain monovalent hydrocarbon group or a branched monovalent hydrocarbon group containing at least one triple bond. The term “alkynyl” is also intended to include those hydrocarbon groups having one triple bond and one double bond. For example, C 2-6 Alkynyl is intended to include ethynyl, propynyl, etc.

[0088] “Alkoxy” means -O-(alkyl) or -O-(cycloalkyl), where the alkyl is defined as above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate groups.

[0089] “Halogen” is fluorine, chlorine, bromine or iodine.

[0090] “Halogen-substituted alkyl” refers to an alkyl group substituted by one or more halogens, where the alkyl is defined as above.

[0091] “Ester group” means -C(O)O(alkyl) or -C(O)O(cycloalkyl), where the alkyl and cycloalkyl are defined as above.

[0092] "Acyl" refers to a compound containing a -C(O)R group, where R is an alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl group.

[0093] "Heteroatom": A heteroatom in an organic compound refers to an atom other than carbon and hydrogen. It generally refers to an atom that replaces carbon in the molecular skeleton (especially the ring system). Nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, iodine, etc. are all common heteroatoms. If an organic compound contains a ring including heteroatoms, it is called a heterocyclic compound.

[0094] Certain atoms (such as N, O, or S atoms) in the compounds and compositions described in the present invention can be in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Thus, as used in the present invention, the structures described in the present invention take into account that certain functional groups, such as OH, SH, or NH, can be protonated or deprotonated. The disclosure of the present invention is intended to cover the above-mentioned compounds and compositions, regardless of their protonation state based on the environmental pH, which is readily understood by those of ordinary skill in the art.

[0095] The terms "salt" and "usable salt" refer to acid and / or base salts formed by the above-mentioned compounds or their stereoisomers with inorganic and / or organic acids and bases, also including zwitterionic salts (inner salts), and also including quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained in the final separation and purification of the compound. They can also be obtained by appropriately (e.g., equimolarly) mixing the above-mentioned compound or its stereoisomer with a certain amount of acid or base. These salts may form a precipitate in solution and be collected by filtration, or recovered after evaporation of the solvent, or prepared by lyophilization after reaction in an aqueous medium.

[0096] The following is a further detailed description of the above content of the present invention by way of specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0097] Figure 1 1H NMR spectrum of compound A3; 1

[0098] Figure 2 LCMS spectrum of compound A5;

[0099] Figure 3 1H NMR spectrum of compound A5; 1

[0100] Figure 4 LCMS spectrum of conjugate C2. Specific Embodiments ​​

[0101] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0102] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0103] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0104] The compounds disclosed in the present invention can be prepared by the following synthetic methods.

[0105] In the following, to help understand the present disclosure, exemplary embodiments will be provided. However, the following embodiments are provided only for easier understanding of the present disclosure and do not limit the present disclosure.

[0106] Example 1. Synthesis of Compound A3

[0107]

[0108] At room temperature, dissolve Compound A1 (1.9 g, 7.88 mmol) and Compound A2 (1.89 g, 8.67 mmol) in a mixed solvent of 34 mL of DMSO solution and 11 mL of water, stir well to dissolve, add SSphos-Pd and CsOH, stir at 90 °C for 4 - 6 hours. After detecting the completion of the reaction by LCMS, add DDTC, stir at 60 °C for 20 minutes, filter the reaction solution, and obtain 2.4 g of Compound A3 after HPLC preparation.

[0109] Example 2. Synthesis of Compound A5

[0110]

[0111] At room temperature, fully dissolve 440 mg of Compound A3 in 10 mL of DMF solvent, slowly add 6.5 mg of Compound A4, then add 1.2 equivalents of HATU, add 2.5 equivalents of DIPEA under ice bath conditions, gradually warm up to room temperature and stir for 2 hours. After detecting the completion of the reaction by LCMS, dilute the reaction solution with water to 80 mL, add 20 mL of ethyl acetate for extraction, repeat 3 times, wash the extraction solution with saturated brine, and obtain 380 mg of Compound A5 after concentration and MPLC preparation.

[0112] Example 3. Preparation of siRNA Conjugate C2

[0113]

[0114] Compound A3 was prepared as a 200 mM DMSO stock solution, and compound C1 was prepared as a 1 mM boric acid buffer stock solution. At room temperature, 1 μmol of the C1 stock solution and 10 μmol of the A3 stock solution were added to an EP tube, and then 20 μmol of the DMSO stock solutions of HATU and DIPEA were added. After shaking at room temperature for 2 hours, the reaction progress was detected by LCMS. After the reaction was complete, 10% v / v saturated sodium chloride solution was added, and 300% v / v absolute ethanol was added and mixed well, followed by freezing at -20 °C for 2 hours. After centrifugation, the precipitate was redissolved in 1 mL of water, and 0.42 μmol (quantified by OD) of conjugate C2 was obtained by HPLC preparation.

Claims

1. A compound of formula I, or a stereoisomer thereof, or a deuterated compound thereof, or a fluorinated compound thereof, or a pharmaceutically acceptable salt thereof: Wherein, n is 1, 2, 3 or 4; X 1 , X 2 , X 3 , X 4 are each independently selected from N or CR X ; Each R X is independently selected from hydrogen, halogen, cyano, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, -OH, -NH2, -O(C 1～6 alkyl), -O(halogen-substituted C 1～6 alkyl), -NH(C 1～6 alkyl), -N(C 1～6 alkyl)(C 1～6 alkyl), a 3- to 6-membered carbocyclic group; R 1 and R 2 are each independently selected from hydrogen, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, 3- to 6-membered carbocyclic group.

2. The compound according to claim 1, characterized in that: Each R X is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl, trifluoromethyl, -OH, -NH2, -O(methyl), -O(ethyl), -NH(methyl), -NH(ethyl), -N(methyl)(methyl), cyclopropyl; R 1 and R 2 are each independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, and cyclopropyl.

3. The compound according to claims 1 to 2, characterized in that: The compound of formula I is specifically:

4. Use of the compound according to any one of claims 1 to 3 as a delivery ligand targeting the transferrin receptor in the preparation of a drug.

5. Use of the compound according to any one of claims 1 to 3 as an intermediate in the preparation of a drug.

6. The use according to claims 4 to 5, characterized in that: The drug is an oligonucleotide drug; preferably, the oligonucleotide drug is an antisense nucleotide, siRNA or saRNA.

7. An oligonucleotide conjugate of formula II, or a pharmaceutically acceptable salt thereof: Wherein, n is 1, 2, 3 or 4; X 1 、X 2 、X 3 、X 4 are each independently selected from N or CR X ; Each R X is independently selected from hydrogen, halogen, cyano, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, -OH, -NH2, -O(C 1～6 alkyl), -O(halogen-substituted C 1～6 alkyl), -NH(C 1～6 alkyl), -N(C 1～6 alkyl)(C 1～6 alkyl), a 3- to 6-membered carbocyclic group; R 1 、R 2 are each independently selected from hydrogen, C 1～6 alkyl, C 2～6 alkenyl, C 2～6 alkynyl, halogen-substituted C 1～6 alkyl, 3- to 6-membered carbocyclic group; m is 1, 2, 3, 4, 5 or 6; L 1 and L 2 are each independently a linking group; Nu is an oligonucleotide moiety.

8. The oligonucleotide conjugate according to claim 7, characterized in that: Each R X is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl, trifluoromethyl, -OH, -NH2, -O(methyl), -O(ethyl), -NH(methyl), -NH(ethyl), -N(methyl)(methyl), cyclopropyl; R 1 and R 2 are each independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, and cyclopropyl.

9. The oligonucleotide conjugate according to claims 7 to 8, wherein: The oligonucleotide conjugate of formula II is specifically: Wherein, m is 1, 2, 3, 4, 5 or 6; L 1 and L 2 are each independently a linking group; Nu is an oligonucleotide moiety.

10. The oligonucleotide conjugate according to claims 7 to 9, characterized in that: The Nu is a single-stranded oligonucleotide moiety or a double-stranded oligonucleotide moiety.

11. The oligonucleotide conjugate according to claim 10, wherein: The double-stranded oligonucleotide moiety comprises a sense strand and an antisense strand, and the sense strand and the antisense strand each comprise 15 to 30 nucleotides, wherein each of the nucleotides is independently a modified or unmodified nucleotide.

12. The oligonucleotide conjugate according to claim 11, characterized in that: The double-stranded oligonucleotide moiety is linked to L through the sense strand 2 or the double-stranded oligonucleotide moiety is linked to L through the antisense strand 2 .

13. The oligonucleotide conjugate according to claim 12, wherein: At least one of the 5'-end or 3'-end of the sense strand of the double-stranded oligonucleotide moiety is linked to L 2 linked 14. The oligonucleotide conjugate according to claim 13, wherein: The sense strand and the antisense strand are at least partially complementary to form a double-stranded region, and the antisense strand is at least partially reverse complementary to the target mRNA, and the target mRNA is the mRNA expressed by the target gene in the target cells in the target tissue or target organ, and the target cells are cells with transferrin receptors on the cell surface.

15. The oligonucleotide conjugate according to claim 14, wherein: At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

16. The oligonucleotide conjugate according to any one of claims 11 to 15, wherein: The double-stranded oligonucleotide in the double-stranded oligonucleotide moiety is siRNA or saRNA.

17. The oligonucleotide conjugate according to claims 7 to 16, characterized in that: Each L 1 is independently selected from linking groups having 1 to 30 carbon atoms, wherein one or more methylene groups may optionally be replaced by one or more of the following groups: -C(O)-, -NH-, O, S, -S(O)-, -S(O)2-, -P(O)(OH)-, -P(S)(OH)-, -CH=CH-, -C≡C-, -CH=N-, a carbocyclic group, a heterocyclic group; and may each optionally be substituted by one or more of the following groups: C 1～10 alkyl, halogen-substituted C 1～10 alkyl, -(C 0～10 alkylene)-OH, -(C 0～10 alkylene)-O-(C 1～10 alkyl), -(C 0～10 alkylene)-SH, -(C 0～10 alkylene)-S-(C 1～10 alkyl), -(C 0～10 alkylene)-NH2, -(C 0～10 alkylene)-NH(C 1～10 alkyl), -(C 0～10 alkylene)-N(C 1～10 alkyl)(C 1～10 alkyl); Each L 2 is independently selected from straight-chain or branched-chain linking groups having 1 to 50 carbon atoms, wherein one or more methylene groups may optionally be replaced by one or more of the following groups: -C(O)-, -NH-, O, S, -S(O)-, -S(O)2-, -P(O)(OH)-, -P(S)(OH)-, -CH=CH-, -C≡C-, -CH=N-, a carbocyclic group, a heterocyclic group; and may each optionally be substituted by one or more of the following groups: C 1～10 alkyl, halogen-substituted C 1～10 alkyl, -(C 0～10 alkylene)-OH, -(C 0～10 alkylene)-O-(C 1～10 alkyl), -(C 0～10 alkylene)-SH, -(C 0～10 alkylene)-S-(C 1～10 alkyl), -(C 0～10 alkylene)-NH2, -(C 0～10 alkylene)-NH(C 1～10 alkyl), -(C 0～10 alkylene)-N(C 1～10 alkyl)(C 1～10 alkyl).

18. The oligonucleotide conjugate according to claim 17, characterized in that: The said L 1 Specifically selected from: Where p is 0, 1, 2, 3, 4, 5 or 6; The said L 2 Specifically selected from: Where q is 0, 1, 2, 3, 4, 5 or 6.

19. The oligonucleotide conjugate according to claim 17, characterized in that: -L 1 -L 2 -Specifically: Wherein, the aa terminus is linked to Nu.

20. A pharmaceutical composition comprising the oligonucleotide conjugate according to any one of claims 7 to 19 and a pharmaceutically acceptable carrier thereof.

21. Use of the oligonucleotide conjugate according to any one of claims 7 to 19 and / or the composition according to claim 20 in the preparation of a drug for inhibiting the expression of a target gene in cells.

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