Modified nucleoside compounds and oligonucleotides comprising the same

Abstract

The present application aims to provide a new type of 5'-modified nucleoside based on selenophosphonate chemistry. Specifically, the present application designs a modified nucleoside compound, which can be efficiently introduced into the 5' end of an oligonucleotide chain by a solid-phase synthesis method. Compared with the prior art, the oligonucleotide synthesis step containing the modified nucleoside compound of the present application is more concise, can resist phosphatase hydrolysis, maintain the binding with Ago2 protein, and produce good gene silencing activity verified by in vitro activity. By utilizing the unique chemical properties of selenium atoms, the internalization behavior and cytoplasmic bioavailability of the modified oligonucleotide in extrahepatic tissue cells are potentially enhanced.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to modified nucleoside compounds and oligonucleotides prepared therefrom or pharmaceutical compositions containing such oligonucleotides. Background Technology

[0002] Oligonucleotide drugs target specific intracellular gene transcripts through complementary base pairing. The main types include antisense oligonucleotides (ASO), small interfering nucleotides (siRNA), microRNAs (miRNA), and aptamers. Among these, siRNA drugs exert their effects through RNA interference (RNAi).

[0003] Studies have found that the efficacy of siRNA drugs is highly dependent on the phosphorylation state of the 5' end of its antisense strand (also known as the guide strand). When a phosphate group is present at the 5' end of the antisense strand, it can stabilize the complex formed by Ago2 and siRNA by forming an electrostatic interaction with positively charged cationic amino acids adjacent to the MID and PIWI domains of the Ago2 protein. However, when the antisense strand loses its 5' phosphate group and exposes a hydroxyl group, the activity of siRNA is greatly reduced. Elkayam E, et al. Nucleic Acids Res. 2017, 45, 6 Since the chemical modification of the 5' end of siRNA after it enters the cell involves a dynamic process: on the one hand, Clp1 kinase can recognize the naked hydroxyl group at the 5' end and phosphorylate it ( Weitzer S. and Martinez J. Nature. 2007, 447, 7141 On the other hand, various endogenous phosphatases can recognize the natural 5' phosphate group and rapidly catalyze the hydrolysis of phosphodiester bonds, exposing the 5' hydroxyl group. Reka AH et al., Nucleic Acids Res. 2017, 45, 7581 Since phosphorylation is an energy-consuming process, most siRNAs in the body are in the low-activity 5' hydroxyl form, which affects drug efficacy.

[0004] Previous studies have found that replacing the natural phosphate group at the 5' end of the antisense strand with a phosphate ester analog can overcome stability issues. Furthermore, phosphate ester analogs with specific structures obtained through screening can maintain or even enhance the binding activity of the siRNA antisense strand to Ago2, thereby significantly improving in vivo stability and drug activity. Prakash TP et al. Nucleic Acids Res. 2015, 43, 6; Parmar RG, et al. J Med Chem. 2018, 61, 3For example, WO 2011 / 139702 A2 discloses a nucleotide containing a 5'-vinylphosphonate (VP) structure, especially the E-configuration VP nucleotide (5'-E-VP), which exhibits high resistance to nucleases while retaining good Ago2 binding ability, thereby maintaining good activity. WO 2018 / 045317 A1 interchanged the positions of the 5' oxygen atom and the 4' carbon atom to obtain a 4' nucleoside analog forming 4'-oxymethylphosphonate (4'-MeMOP), which can improve stability and maintain efficacy to some extent. WO 2021 / 178885 A1 deuterated multiple hydrogen atoms in 5'-E-VP, finding that it can further improve the persistence of in vivo efficacy.

[0005] In improving the technical solution, the design of 5' non-natural phosphonate analogs needs to consider the following points: (a) stability, by modifying the structure of the natural phosphate ester to prevent it from being recognized by phosphatases or hydrolyzed as a substrate; (b) functionality, by preserving as much as possible the tetrahedral structure and charge of the natural phosphate group and its relative position to the first nucleotide to maintain effective binding to the Ago2 protein; (c) based on satisfying the first two points, consider introducing some medicinally chemistry-related atoms or designs that can increase uptake or prolong half-life (such as deuteration). Existing 5' end modification methods sometimes require specific configurations, have complicated synthetic steps, or use expensive synthetic reagents. Some have a significant impact on Ago2 binding activity and poor in vitro activity, while others face insufficient resistance to exonucleases. Therefore, it is necessary to develop novel 5' non-natural phosphonate analogs to make up for these shortcomings.

[0006] Selenium has great potential applications in medicinal chemistry. 4'-Selenonucleotides, obtained by selenizing the oxygen atom at the 4' position of the nucleotide sugar ring, have already been used in biotechnology and pharmaceuticals. Sahu PK, et al. Eur. J. Org. Chem. 2015, 2015, 28 For example, WO 2017 / 052322 A1 synthesized 4'-selenoside analogs for cancer treatment. Phosphoroselenoate is an excellent analog of natural phosphate esters and has been used in nucleic acid labeling and structural analysis. Carrasco N, et al. Nucleosides, Nucleotides and Nucleic Acids. 2001, 20, 9 ), or non-site-specific modifications to the DNA strand backbone to enhance nuclease resistance ( Doddridge ZA, et al. Biochemistry, 2003, 42, 11 ). Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a novel class of 5' phosphonate-modified nucleosides, with the modification site located between the 5' phosphate group and the sugar ring of the nucleotide. This novel modified nucleoside compound can significantly improve stability while still maintaining good biological activity.

[0008] A first aspect of the present invention provides a modified nucleoside compound having the structure shown in formula (I) or formula (II), or being a pharmaceutically acceptable salt or stereoisomer of a compound having the structure shown in formula (I) or (II): The X=PLA phosphonate chain structure contains selenium atom substitution (X=PL-Se), (X=P-Se-A), (Se=PLA), or nitrogen atom substitution (X=PLN). Furthermore, R1 and R2 are each independently selected from one of H, halogen, hydroxyl, C1-C6 alkyl, any substituted C1-C6 alkyl, C1-C6 alkoxy, any substituted C1-C6 alkoxy, sulfonyl, sulfinyl, amide and any substituted amide; R3 is hydrogen, a protecting group (such as benzoyl), or a phosphorus-containing reactive group (such as phosphoramide). R4 and R5 are each independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, amino or any substituted amino, or phosphorus-containing reactive group, -CH2CH2CN and -CH2O(CO)C(CH3)3; M1 is O, S, Se, or -CH2-; M2 is a nucleoside containing phosphorus amide, sugar, or a sugar substitute. B is H, a heterocyclic base or a base substitution group, wherein the heterocyclic base is uracil, cytosine, adenine or guanine, and the amino group on the base may optionally be protected by a protecting group (such as benzoyl), and the base substitution group is phenyl or substituted phenyl. X is selected from O, Se, or S.

[0009] Furthermore, X in equations (I) and (II) can be arbitrarily chosen as: (a) X is Se, and L and A are each independently selected from C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, C2-C6 alkyneyl or any isotope or halogen substituted group above; (b) X is O or S, A is -N(CH3)- or Se, and L is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by an isotope or halogen. (c) X is O or S, L is Se, and A is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by isotopes or halogens.

[0010] According to some preferred embodiments of the present invention, R4 and R5 may be the same or different, and at least one of them is a C1-C6 alkyl group.

[0011] According to some particularly preferred embodiments of the present invention, R4 and R5 are both ethyl, or R4 is ethyl and R5 is H.

[0012] According to some preferred embodiments of the present invention, the phosphorus-containing active reactive group is any one of phosphorus amide, H-phosphate ester, triphosphate ester or phosphorus-containing chiral auxiliaries.

[0013] According to some preferred embodiments of the present invention, in the compound formula (II) provided herein, the sugar or sugar-substituted nucleoside has a sugar-substituted moiety that is morpholino, cyclohexenyl, cyclohexyl, cyclopentyl, pyranyl, or cyclohexanehexaolyl. In some embodiments, the sugar moiety in the sugar or sugar-substituted nucleoside is a furanose. In some embodiments, the sugar or sugar-substituted nucleoside contains a nonlocked nucleobase analog (UNA), a glycerol nucleic acid base analog (GNA), a locked nucleic acid (LNA), or a bridged nucleic acid (BNA).

[0014] According to some particularly preferred embodiments of the present invention, the compound of formula (I) is any one or a combination of at least two of the following compounds having the following structures: .

[0015] Furthermore, the present invention also provides the use of the modified nucleoside compound described in the first aspect in improving the stability of oligonucleotides (e.g., resistance to exonuclease degradation).

[0016] Based on this, the present invention also provides a method for improving the stability of oligonucleotides (e.g., improving resistance to exonuclease degradation), the method comprising replacing at least a portion of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0017] Preferably, 1-3 nucleotides, more preferably 1 nucleotide, are used to replace the 5' end of the oligonucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0018] Furthermore, the present invention also provides the use of the modified nucleoside compound described in the first aspect in enhancing the in vivo function of oligonucleotides (e.g., enhancing the silencing effect of siRNA).

[0019] Based on this, the present invention also provides a method for improving the in vivo function of oligonucleotides (e.g., improving the silencing effect of siRNA), the method comprising replacing at least a portion of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0020] Preferably, 1-3 nucleotides, more preferably 1 nucleotide, are used to replace the 5' end of the oligonucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0021] Furthermore, the present invention also provides the use of the modified nucleoside compound described in the first aspect in simultaneously improving the stability of oligonucleotides (e.g., resistance to exonuclease degradation) and the in vivo function of oligonucleotides (e.g., enhancing the silencing effect of siRNA).

[0022] Based on this, the present invention also provides a method for simultaneously improving the stability of oligonucleotides (e.g., improving resistance to exonuclease degradation) and the in vivo function of oligonucleotides (e.g., improving the silencing effect of siRNA), the method comprising replacing at least a portion of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0023] Preferably, 1-3 nucleotides, more preferably 1 nucleotide, are used to replace the 5' end of the oligonucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0024] A second aspect of the present invention provides an oligonucleotide, wherein the structural units of the oligonucleotide include nucleotides containing the modified nucleoside compound described in the first aspect.

[0025] In some preferred embodiments, for oligonucleotides with a length not exceeding 50 nucleotides (e.g., 15-30 nucleotides), the number of structural units (nucleotides) containing the modified nucleoside compound provided by the present invention does not exceed 3, for example, it can be 1, 2 or 3.

[0026] According to some preferred embodiments of the present invention, the nucleotide containing the modified nucleoside compound is located at the 5' end of the oligonucleotide.

[0027] According to a particularly preferred embodiment of the present invention, the first nucleotide at the 5' end of the oligonucleotide is a nucleotide containing the modified nucleoside compound.

[0028] According to some preferred embodiments of the present invention, the oligonucleotide is an oligonucleotide as shown in formula (III) or a pharmaceutically acceptable salt thereof. in, R4 and R5 are each independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, amino or any substituted amino group, or phosphorus-containing reactive group, -CH2CH2CN and -CH2O(CO)C(CH3)3.

[0029] M2 is a nucleoside containing phosphorus amide, sugar, or a sugar substitute.

[0030] X is O, Se, or S, and optionally: (a) X is Se, and L and A are each independently selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by an isotope or halogen. (b) X is O or S, A is -N(CH3)- or Se, and L is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by an isotope or halogen. (c) X is O or S, L is Se, and A is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl or C2-C6 alkyne, or the above groups are substituted at any position by an isotope or halogen.

[0031] According to some preferred embodiments of the present invention, R4 and R5 may be the same or different, and at least one of them is a C1-C6 alkyl group.

[0032] According to some particularly preferred embodiments of the present invention, R4 and R5 are both ethyl, or R4 is ethyl and R5 is H.

[0033] According to some preferred embodiments of the present invention, in the nucleoside of the sugar or sugar-substitute portion, the sugar-substituted portion is morpholino, cyclohexenyl, cyclohexyl, cyclopentyl, pyranyl, or cyclohexanehexaolyl. In some embodiments, the sugar portion of the nucleoside of the sugar or sugar-substitute portion is furanose. In some embodiments, the nucleoside of the sugar or sugar-substitute portion contains a nonlocked nucleobase analog (UNA), or a glycerol nucleic acid base analog (GNA), or a locked nucleic acid (LNA), or a bridged nucleic acid (BNA).

[0034] According to a preferred embodiment of the present invention, the oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides (siRNA), antisense oligonucleotides (ASO), microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), and aptamers.

[0035] According to some preferred embodiments of the present invention, the oligonucleotide is siRNA.

[0036] Preferably, the siRNA is a double-stranded siRNA or a single-stranded siRNA.

[0037] According to some particularly preferred embodiments of the present invention, the siRNA is a double-stranded siRNA comprising a sense strand and an antisense strand.

[0038] Preferably, the antisense strand in the double-stranded siRNA is an oligonucleotide as shown in formula (III) or a pharmaceutically acceptable salt thereof.

[0039] According to a preferred embodiment of the present invention, each nucleotide in the oligonucleotide is independently a modified or unmodified nucleotide.

[0040] According to some preferred embodiments of the present invention, the oligonucleotide can be used to regulate the expression of a target gene (e.g., it can be used to inhibit the expression of a target gene).

[0041] According to some particularly preferred embodiments of the present invention, the oligonucleotide is any one or a combination of at least two of the following siRNAs: (i) The sense chain has the sequence shown in Table 1 CGBN-0344 (SEQ ID No 2); and the antisense chain has the sequence shown in Table 1 CGBN-0344 (SEQ ID No 5); (ii) The sense chain has the sequence shown in Table 1 CGBN-0346 (SEQ ID No 2); and the antisense chain has the sequence shown in Table 1 CGBN-0346 (SEQ ID No 6).

[0042] A third aspect of the present invention provides a nucleic acid conjugate, characterized in that the nucleic acid conjugate comprises the oligonucleotide or a pharmaceutically acceptable salt thereof described in the second aspect, and a targeting ligand bound thereto. The targeting ligand comprises carbohydrates (such as N-acetylgalactosamine), cholesterol, fatty acid chains (such as C... 16 -C 22Any one or a combination of at least two of the following: alkyl groups, polypeptides, or antibodies.

[0043] A fourth aspect of the present invention provides a pharmaceutical composition comprising the oligonucleotide described in the second aspect or a pharmaceutically acceptable salt thereof; and / or, the pharmaceutical composition comprising the nucleic acid conjugate described in the third aspect.

[0044] The positive and progressive effects of this invention are that it designs a series of nucleoside analogs with different chemical structures, readily available raw materials, and simple steps compared to the 5' phosphorylated compounds of the prior art; and siRNA containing these 5' phosphate compounds retains better target gene inhibitory activity.

[0045] The 5'-phosphonate-modified nucleoside analogs designed in this invention introduce selenium into the compound. On the one hand, the atomic radius, polarizability, and P-Se bond length of the selenium atom make its stereostructure and electronic properties closer to those of a phosphate group in a transition state. On the other hand, the larger radius of the selenium atom increases its steric hindrance, and its electronegativity and redox properties are different from those of oxygen and sulfur in the same group. Therefore, the nucleoside compound with selenium atom substitution has higher resistance to exonuclease degradation than naturally occurring oxygen- or sulfur-containing nucleotides, significantly improving the stability of oligonucleotides. At the same time, the siRNA sequence containing this 5'-phosphonate-modified nucleoside analog has good target gene repression activity. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 The curves show the inhibition rates of the MSTN reporter gene obtained 48 hours after transfection of human 293T cells with siRNA sequences CGBN-0287, CGBN-0288, CGBN-0307, ​​CGBN-0308, CGBN-0344, and CGBN-0346 containing the target compound. Detailed Implementation

[0048] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0049] The first aspect of the present invention provides a modified nucleoside compound having the structure shown in formula (I) or (II), or a pharmaceutically acceptable salt or stereoisomer of a compound having the structure shown in formula (I) or (II), characterized in that a non-natural phosphonate chain (X=PLA) is connected between the phosphorus atom and the sugar ring, and the phosphonate chain is a selenized (X=PL-Se), (X=P-Se-A), (Se=PLA), or nitrogen-substituted (X=PLN) chain.

[0050] in, R1 and R2 are each independently selected from any one of H, halogen, hydroxyl, C1-C6 alkyl, any substituted C1-C6 alkyl, C1-C6 alkoxy, any substituted C1-C6 alkoxy, sulfonyl, sulfinyl, amide and any substituted amide. R3 is hydrogen, a protecting group (such as benzoyl), or a phosphorus-containing reactive group; R4 and R5 are each independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, amino or any substituted amino, or phosphorus-containing reactive group, -CH2CH2CN and -CH2O(CO)C(CH3)3; M1 is O, S, Se, or -CH2-; M2 is a nucleoside containing phosphorus amide, sugar, or a sugar substitute. B is H, a heterocyclic base or a base substitution group, wherein the heterocyclic base includes uracil, cytosine, adenine, guanine, and the amino group on the base may optionally be protected by a protecting group (such as benzoyl), and the base substitution group is phenyl or substituted phenyl. X is O, Se, or S, and optionally: (a) X is Se, and L and A are each independently selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by an isotope or halogen. (b) X is O or S, A is -N(CH3)- or Se, and L is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by an isotope or halogen. (c) X is O or S, L is Se, and A is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl or C2-C6 alkyne, or the above groups are substituted at any position by an isotope or halogen.

[0051] In this application, "protecting group" refers to a group used in a conventional chemical sense to reversibly render a functional group unreactive under certain conditions of the desired reaction. After the desired reaction, the protecting group can be removed to deprotect the protected functional group. All protecting groups should be removable without degrading a significant proportion of the synthesized molecule. "Phosphorus-containing reactive group" refers to a phosphorus-containing group capable of reacting nucleophilically with a hydroxyl or amino group contained in another molecule, particularly in another nucleotide unit or another nucleotide analog. Typically, such a reaction produces an ester-type nucleoside bond linking a nucleotide unit or nucleotide analog unit to another nucleotide unit or nucleotide analog unit.

[0052] Specifically, R4 and R5 may be the same or different, and at least one is a C1-C6 alkyl group. Preferably, both R4 and R5 are ethyl groups, or R4 is ethyl and R5 is H.

[0053] According to some preferred embodiments of the present invention, the phosphorus-containing active reactive group is any one of phosphorus amide, H-phosphate ester, triphosphate ester or phosphorus-containing chiral auxiliaries.

[0054] According to some preferred embodiments of the present invention, in the compound formula (II) provided herein, the sugar or sugar-substituted nucleoside has a sugar-substituted moiety that is morpholino, cyclohexenyl, cyclohexyl, cyclopentyl, pyranyl, or cyclohexanehexaolyl. In some embodiments, the sugar moiety in the sugar or sugar-substituted nucleoside is a furanose. In some embodiments, the sugar or sugar-substituted nucleoside contains a nonlocked nucleobase analog (UNA), a glycerol nucleic acid base analog (GNA), a locked nucleic acid (LNA), or a bridged nucleic acid (BNA).

[0055] According to some particularly preferred embodiments of the present invention, the compound of formula (I) is any one or a combination of at least two of the following compounds having the following structures: .

[0056] Furthermore, the present invention also provides the use of the modified nucleoside compound described in the first aspect in improving the stability of oligonucleotides (e.g., resistance to exonuclease degradation).

[0057] Based on this, the present invention also provides a method for improving the stability of oligonucleotides (e.g., improving resistance to exonuclease degradation), the method comprising replacing at least a portion of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0058] Preferably, 1-3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are replaced with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0059] Furthermore, the present invention also provides the use of the modified nucleoside compound described in the first aspect in enhancing the in vivo function of oligonucleotides (e.g., enhancing the silencing effect of siRNA).

[0060] Based on this, the present invention also provides a method for improving the in vivo function of oligonucleotides (e.g., improving the silencing effect of siRNA), the method comprising replacing at least a portion of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0061] Preferably, 1-3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are replaced with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0062] Furthermore, the present invention also provides the use of the modified nucleoside compound described in the first aspect in simultaneously improving the stability of oligonucleotides (e.g., resistance to exonuclease degradation) and the in vivo function of oligonucleotides (e.g., enhancing the silencing effect of siRNA).

[0063] Based on this, the present invention also provides a method for simultaneously improving the stability of oligonucleotides (e.g., improving resistance to exonuclease degradation) and the in vivo function of oligonucleotides (e.g., improving the silencing effect of siRNA), the method comprising replacing at least a portion of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0064] Preferably, 1-3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are replaced with nucleotides containing the modified nucleoside compound described in the first aspect of the present invention.

[0065] A second aspect of the present invention provides an oligonucleotide, wherein the structural units of the oligonucleotide include nucleotides containing the modified nucleoside compound described in the first aspect. That is, the oligonucleotide provided by the present invention may be formed by replacing some structural units (nucleotides) of a conventional oligonucleotide with nucleotides synthesized from the modified nucleoside compound of the present invention (i.e., a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof) and phosphate.

[0066] The oligonucleotides provided by this invention may contain the modified nucleoside compound provided in the first aspect in some structural units (nucleotides), or all structural units may contain the modified nucleoside compound. When some structural units contain the modified nucleoside compound, the other structural units in the oligonucleotide may be provided by conventional nucleotides or by nucleotides containing other existing modified nucleoside compounds.

[0067] In this invention, the structural unit of an oligonucleotide refers to a nucleotide. As is known in the art, a nucleotide is a compound composed of a base (purine or pyrimidine), a pentose sugar (ribose or deoxyribose), and a phosphate group. The base condenses with the pentose sugar to form a nucleoside, which is then further reacted with phosphate to synthesize a nucleotide. "The structural unit of an oligonucleotide includes a nucleotide containing the modified nucleoside compound described in the first aspect" means that the nucleoside portion of at least one nucleotide in the oligonucleotide sequence is provided by the modified nucleoside compound provided in this invention; that is, in the oligonucleotide comprising several nucleotides, the nucleoside portion of at least one nucleotide is replaced by the modified nucleoside compound provided in this invention.

[0068] In some preferred embodiments, for oligonucleotides with a length not exceeding 50 nucleotides (e.g., 15-30 nucleotides), the number of structural units (nucleotides) containing the modified nucleoside compound provided by the present invention does not exceed 3 (e.g., 1, 2 or 3).

[0069] According to some preferred embodiments of the present invention, the nucleotide containing the modified nucleoside compound is located at the 5' end of the oligonucleotide.

[0070] According to a particularly preferred embodiment of the present invention, the first nucleotide at the 5' end of the oligonucleotide is a nucleotide containing the modified nucleoside compound.

[0071] According to some preferred embodiments of the present invention, the oligonucleotide is an oligonucleotide as shown in formula (III) or a pharmaceutically acceptable salt thereof. in, R4 and R5 are each independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, amino or any substituted amino group, or phosphorus-containing reactive group, -CH2CH2CN and -CH2O(CO)C(CH3)3.

[0072] M2 is a nucleoside containing phosphorus amide, sugar, or a sugar substitute.

[0073] X is O, Se, or S, and optionally: (a) X is Se, and L and A are each independently selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by an isotope or halogen. (b) If X is O or S, and A is -N(CH3)- or Se, then L is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or any position of the above groups is replaced by isotopes or halogens. (c) X is O or S, L is Se, and A is selected from any one of C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted at any position by an isotope or halogen.

[0074] According to some preferred embodiments of the present invention, R4 and R5 may be the same or different, and at least one of them is a C1-C6 alkyl group.

[0075] According to some particularly preferred embodiments of the present invention, R4 and R5 are both ethyl, or R4 is ethyl and R5 is H.

[0076] According to some preferred embodiments of the present invention, in the nucleoside of the sugar or sugar-substitute portion, the sugar-substituted portion is morpholino, cyclohexenyl, cyclohexyl, cyclopentyl, pyranyl, or cyclohexanehexaolyl. In some embodiments, the sugar portion of the nucleoside of the sugar or sugar-substitute portion is furanose. In some embodiments, the nucleoside of the sugar or sugar-substitute portion contains a nonlocked nucleobase analog (UNA), or a glycerol nucleic acid base analog (GNA), or a locked nucleic acid (LNA), or a bridged nucleic acid (BNA).

[0077] It should be noted that the structure shown in formula (III) above is an oligonucleotide whose first 5' end contains the modified nucleoside compound provided by the present invention. That is, the part linked to Oligo is the structure of the first nucleotide of the complete oligonucleotide, while Oligo represents the remaining part of the oligonucleotide starting from the second 5' end.

[0078] The modified nucleoside compound provided by this invention, by replacing part of the nucleoside structure in oligonucleotides, can make oligonucleotides more stable and promote and enhance their function. Any oligonucleotide in the art can be modified using the modified nucleoside compound provided by this invention (i.e., replacing part of the nucleoside structure of the nucleotides in the oligonucleotide with the modified nucleoside compound). According to a preferred embodiment of this invention, the oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides (siRNA), antisense oligonucleotides (ASO), microRNAs (miRNA), small activating RNAs (saRNA), small guide RNAs (sgRNA), transfer RNAs (tRNA), and aptamers.

[0079] In this invention, the oligonucleotide can be a single-stranded oligonucleotide or a double-stranded oligonucleotide. Unless otherwise specified, in this invention, the length of an oligonucleotide refers to the number of nucleotides (also known as the number of bases, nt) for a single-stranded oligonucleotide, and to the number of nucleotide pairs (also known as the number of base pairs, bp) for a double-stranded oligonucleotide.

[0080] According to some preferred embodiments of the present invention, the oligonucleotide is siRNA.

[0081] Preferably, the siRNA is a double-stranded siRNA or a single-stranded siRNA.

[0082] According to some particularly preferred embodiments of the present invention, the siRNA is a double-stranded siRNA comprising a sense strand and an antisense strand.

[0083] Preferably, the antisense strand in the double-stranded siRNA is an oligonucleotide as shown in formula (III) or a pharmaceutically acceptable salt thereof.

[0084] According to a preferred embodiment of the present invention, each nucleotide in the oligonucleotide is independently a modified or unmodified nucleotide.

[0085] According to some preferred embodiments of the present invention, the oligonucleotide can be used to regulate the expression of a target gene (e.g., it can be used to inhibit the expression of a target gene).

[0086] According to some particularly preferred embodiments of the present invention, the oligonucleotide is any one or a combination of at least two of the following siRNAs: (i) The justice chain has a sequence as shown in CGBN-0344SS; and the antisense chain has a sequence as shown in CGBN-0344AS; (ii) The justice chain has a sequence as shown in CGBN-0346SS; and the antisense chain has a sequence as shown in CGBN-0346AS.

[0087] A third aspect of the present invention provides a nucleic acid conjugate, characterized in that the nucleic acid conjugate comprises the oligonucleotide or a pharmaceutically acceptable salt thereof described in the second aspect, and a targeting ligand bound thereto. The targeting ligand comprises carbohydrates (such as N-acetylgalactosamine), cholesterol, fatty acid chains (such as C... 16 -C 22 Any one or a combination of at least two of the following: alkyl groups, polypeptides, or antibodies.

[0088] A fourth aspect of the present invention provides a pharmaceutical composition comprising the oligonucleotide described in the second aspect or a pharmaceutically acceptable salt thereof; and / or, the pharmaceutical composition comprising the nucleic acid conjugate described in the third aspect.

[0089] In the pharmaceutical compositions provided by this invention, the oligonucleotides and / or nucleic acid conjugates provided by this invention serve as the main active ingredients. In some embodiments, the oligonucleotides and / or nucleic acid conjugates provided by this invention may be the sole active ingredient in the pharmaceutical composition; in some embodiments, the pharmaceutical composition may contain other active ingredients in addition to the oligonucleotides and / or nucleic acid conjugates of this invention.

[0090] The following detailed description is provided in conjunction with specific embodiments. This embodiment details the preparation methods of representative compounds (Cpd 1, Cpd 10, Cpd 10-3, Cpd 10-5), the preparation methods of siRNA containing these compounds, and the in vitro pharmacological effects of siRNA containing representative compounds (Cpd 10, Cpd 10-3).

[0091] Example 1: Synthesis of the compound 1. Synthesis of Compound 1 (Cpd 1) Step 1: Synthesis of Compounds 1-2 Compound SM-1-1 (13.5 g, 99.17 mmol) was dissolved in toluene (150.0 mL), and selenium powder (23.49 g, 297.51 mmol) was added. The mixture was stirred at 70 °C for 16 hours. The reaction solution was filtered, the filtrate was evaporated to dryness, and the residue was passed through a silica gel column (petroleum ether / ethyl acetate = 20:1) to give a yellow oily compound 1-2 (15 g, 70.3% yield).

[0092] 1H NMR (400 MHz, CDCl3) δ 4.27 - 3.99 (m, 4H), 1.97 (d, J = 15.6 Hz, 3H), 1.36 - 1.24 (m, 6H). Step 2: Synthesis of compounds 1-3 Compound 2 (5.0 g, 23.25 mmol) was dissolved in dry tetrahydrofuran (50.0 mL) under nitrogen saturation. The mixture was cooled to -70 °C, and n-butyllithium (2.5 M in hexane, 9.76 mL, 24.41 mmol) was slowly added dropwise. The mixture was stirred for 30 min, and a solution of diisopropylaminophosphine chloride (7.44 g, 27.90 mmol) in tetrahydrofuran (50.0 mL) was slowly added. The mixture was stirred at room temperature for 2 h. The reaction mixture was poured into ice water (100 mL) of ammonium chloride and extracted with ethyl acetate (100 mL × 2). The organic phase was washed with saturated aqueous sodium chloride solution (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give compounds 1-3 (7.0 g, 67.6% yield). The crude product was used directly in the next step.

[0093] Step 3: Synthesis of compounds 1-4 Compound 3 (7.0 g, 15.72 mmol) was dissolved in acetonitrile (40.0 mL), and ethanol (2.90 g, 62.86 mmol) and tetrazolium (4.4 g, 62.86 mmol) were added. The mixture was stirred at room temperature for 16 hours under nitrogen protection to obtain compounds 1-4 (5 g). The crude solution was used directly in the next step.

[0094] Step 4: Synthesis of compounds 1-5 The crude solution of compounds 1-4 (5 g) was diluted with tetrahydrofuran (40.0 mL), and the mixture of selenium powder (8.25 g, 104.42 mmol) was added. The mixture was stirred at room temperature for 2 hours, filtered, evaporated to dryness, and the residue was passed through a silica gel column (petroleum ether / ethyl acetate = 20:1) to give compounds 1-5 (4 g, 46.3% yield).

[0095] LCMS m / z = 417.0 [M+H]+ Step 5: Synthesis of compounds 1-6 Compounds 1-5 (4.19 g, 95.4 mmol) were dissolved in dry tetrahydrofuran (20.0 mL), and sodium hydride (323.9 mg, 60% in mineral oil, 8.10 mmol) was added at 0 °C under nitrogen protection. The reaction mixture was stirred for 30 min, and a solution of INT-B (2.5 g, 6.75 mmol) in tetrahydrofuran (20.0 mL) was added dropwise. The mixture was stirred at room temperature for 2 h. The mixture was poured into a saturated ammonium chloride (NH4Cl, 100 mL) solution and extracted with ethyl acetate (100 mL × 2). The organic phase was washed with saturated sodium chloride solution (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was passed through a silica gel column (petroleum ether / ethyl acetate = 5:1) to give compounds 1-6 (1.5 g, 39.2% yield).

[0096] LCMS m / z = 569.2 [M+H]+ Step 6: Synthesis of compounds 1-7 Compounds 1-6 (600 mg, 1.06 mmol) were dissolved in formic acid (HCOOH, 5 mL) and water (5 mL), and reacted at 40 °C for 3 hours. After the reaction was completed, the mixture was evaporated to dryness, and the residue was passed through a silica gel column (dichloromethane / ethyl acetate = 3:2) to give compounds 1-7 (400 mg, 83.5% yield).

[0097] LCMS m / z=455.0 [M+H]+ 1 H NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 7.04 - 6.76 (m, 1H), 6.43 -6.19 (m, 1H), 5.95 (d, J = 1.6 Hz, 1H), 5.79 (d, J = 8.0 Hz, 1H), 4.58 - 4.39(m, 1H), 4.19 - 4.11 (m, 4H), 3.99 (dd, J = 7.6, 5.6 Hz, 1H), 3.82 (dd, J =5.6, 1.6 Hz, 1H), 3.63 (s, 3H),1.34 (td, J = 7.2, 6.8, 0.8 Hz, 6H). Step 7: Synthesis of Compound 1 Compounds 1-7 (250 mg, 0.55 mmol) were dissolved in dry dichloromethane (5 mL), and tetrazolium (323.88 mg, 1.10 mmol) was added. Then, bis(diisopropylamino)(2-cyanoethoxy)phosphine (332.5 mg, 1.10 mmol) in dichloromethane (5 mL) was added dropwise to the mixture. The mixture was stirred at room temperature for 6 hours, and water (50 mL) was added. Extraction was performed with dichloromethane (50 mL), and the organic phase was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by reversed-phase column chromatography (water / acetonitrile = 60%) to give compound 1 (220 mg, 61.0% yield).

[0098] LCMS m / z = 655.2 [M+H]+ 1 H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 7.78 - 7.57 (m, 1H), 6.85- 6.59 (m, 1H), 6.45 - 6.16 (m, 1H), 5.84 - 5.56 (m, 2H), 4.68 - 4.26 (m,2H), 4.21 - 3.95 (m, 5H), 3.89 - 3.68 (m, 2H), 3.69 - 3.52 (m, 2H), 3.47 -3.35 (m, 3H), 2.85 - 2.75 (m, 2H), 1.33 - 0.97 (m, 18H). 31 P NMR (162 MHz, DMSO-d6) δ 149.25 (s), 149.15 (s), 89.41 (d, J = 4.2Hz). 2. Synthesis of Compound 10 (Cpd 10) Step 1: Synthesis of Compound 2A Compound 1A (diethyl iodomethylphosphonate) (8.0 g, 28.77 mmol) was dissolved in acetone (100.0 mL), and potassium selenocyanate (8.3 g, 57.55 mmol) was added. The mixture was stirred at 50 °C for 16 hours. The reaction solution was evaporated to dryness, and water (200 mL) was added. The solution was extracted with ethyl acetate (100 mL × 2), and the organic phase was washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give compound 2A, diethyl selenocyanomethylphosphonate (4 g, 54.3% yield).

[0099] 1 H NMR (400 MHz, CDCl3) δ 4.27 - 4.15 (m, 4H), 3.19 (d, J = 11.6 Hz, 2H), 1.38 (t, J = 7.2, 6.8 Hz, 6H). Step 2: Synthesis of Compound 3A Compound 2A (3.0 g, 11.7 mmol) was dissolved in anhydrous ethanol (50.0 mL), and sodium borohydride (1.8 g, 46.8 mmol) was added in portions under nitrogen protection at 0 °C. The reaction was allowed to proceed for 1 hour to obtain a crude solution of compound 3A, which was directly used in the next step.

[0100] LCMS m / z = 233.0 [M+H]+ Step 3: Synthesis of compound 10-0-2 INT-A (2.0 g, 5.38 mmol) was dissolved in dichloromethane (40 mL), and pyridine (4.2 g, 53.69 mmol) was added. The reaction mixture was cooled to -70 °C, and methanesulfonyl chloride (MsCl, 1.25 mL, 16.11 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 6 hours, and water (100 mL) was added. The mixture was extracted with dichloromethane (50 mL), and the organic phase was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give compound 10-O-2 (2 g, crude product). The crude product was used directly in the next step.

[0101] LCMS m / z = 451.2 [M+H]+ Step 4: Synthesis of compound 10-O-3 A tetrahydrofuran (20 mL) solution of compound 10-O-2 (2 g, crude, 4.44 mmol) was added to an ethanol solution of the prepared compound 3A (diethyl selenide, methylphosphonate) (3 g, crude, 13.32 mmol). The reaction was carried out at 70 °C for 16 hours under nitrogen saturation. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 2). The organic phase was washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and the residue was passed through a C18 reversed-phase column (acetonitrile / water = 1 / 1) to give compound 10-O-3 (1.5 g, 57.7% yield).

[0102] 1 H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), 5.83 - 5.61 (m, 2H), 4.19 - 3.97 (m, 5H), 3.93 - 3.81 (m, 1H), 3.56 (dd, J =5.2, 2.4 Hz, 1H), 3.41 (s, 3H), 3.07 (dd, J = 40.3, 5.4 Hz, 2H), 2.82 - 2.70(m, 1H), 2.52 (dd, J = 14.4, 12.8 Hz, 1H), 1.28 - 1.18 (m, 6H), 0.80 (s, 9H),-0.00 (d, J = 2.4 Hz, 6H). Step 5: Synthesis of compound 10-O-4 Compound 10-O-3 (700 mg, 1.2 mmol) was dissolved in formic acid (HCOOH, 5 mL) and water (5 mL), and reacted at 40 °C for 3 hours. After the reaction was completed, the mixture was evaporated to dryness, and the residue was passed through a C18 reversed-phase column (acetonitrile / water = 1 / 4) to give compound 10-O-4 (400 mg, 70.9% yield).

[0103] LCMS m / z = 473.0[M+H]+ 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 5.83 (d, J = 5.2 Hz, 1H), 5.66 (d, J = 8.4 Hz, 1H), 5.32 (d, J = 5.2 Hz, 1H), 4.10 - 3.96 (m, 6H), 3.90 (t, J = 4.8 Hz, 1H), 3.35 (s, 3H), 3.12 - 2.99 (m,2H), 2.96 - 2.78 (m, 2H), 1.23 (td, J = 7.2, 1.2 Hz, 6H). 31 P NMR (162 MHz, DMSO-d6) δ 25.97 (s). Step 6: Synthesis of Compound 10 Compound 10-0-4 (350 mg, 0.74 mmol) was dissolved in dry dichloromethane (5 mL), and tetrazolium (104.1 mg, 1.49 mmol) was added. Then, a solution of compound A, bis(diisopropylamino)(2-cyanoethoxy)phosphine (447.7 mg, 1.49 mmol), in dichloromethane (5 mL) was added dropwise to the reaction mixture. The mixture was stirred at room temperature for 6 hours, and water (50 mL) was added. The mixture was extracted with dichloromethane (50 mL), and the organic phase was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was passed through a silica gel column (dichloromethane (containing 0.5% triethylamine) / acetonitrile = 1 / 1) to give compound 10 (220 mg, 50.1% yield).

[0104] LCMS m / z = 671.2 [MH]- 1H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 5.84 (dd, J = 5.6, 3.6 Hz, 1H), 5.68 (d, J = 8.0 Hz, 1H), 4.42 - 4.09 (m,3H), 4.08 - 3.97 (m, 4H), 3.86 - 3.55 (m, 4H), 3.36 (d, J = 16.0 Hz, 3H), 3.15 - 3.03 (m, 2H), 2.94 - 2.77 (m, 4H), 1.26 - 1.15 (m, 18H). 31 P NMR (162 MHz, DMSO-d6) δ 149.56 (s), 148.85 (s), 25.84 (s), 25.70 (s). 3. Synthesis of compound 10⁻³ (Cpd 10⁻³) Step 1: Synthesis of Compound 10-3-1 Compound INT-B (1.5 g, 4.05 mmol) was dissolved in 1,2-dichloroethane (30.0 mL), and diethyl (aminomethyl)phosphonate (676.4 mg, 4.05 mmol) and sodium borocyanide acetate (2.6 g, 12.2 mmol) were added. The mixture was stirred at room temperature for 16 h. The reaction solution was then added to dichloromethane (50 mL) and water (50 mL). The organic phase was washed with saturated sodium bicarbonate solution (50 mL × 2). The mixture was dried, filtered, and the crude product was evaporated to dryness. The crude product was then passed through a C18 column (acetonitrile / water = 1 / 1) to give a yellow solid compound 10-3-1 (1.5 g, 71.1% yield).

[0105] LCMS [M+H]+ m / z=522.2[M+H]+ 1H NMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 5.70 (d, J = 5.0 Hz, 1H), 5.55 (d, J = 8.0 Hz, 1H), 4.25 - 4.11 (m, 1H), 4.00- 3.83 (m, 4H), 3.81 (s, 2H), 3.80 - 3.71 (m, 2H), 3.22 - 3.21 (m, 3H), 2.86(d, J = 11.3 Hz, 2H), 2.78 - 2.65 (m, 2H), 1.22 - 1.05 (m, 6H), 0.78 (s, 9H), -0.03 (s, 6H). Step 2: Synthesis of compound 10-3-2 Compound 10⁻³⁻¹ (1.2 g, 2.3 mmol) was dissolved in methanol (20.0 mL), and formaldehyde (559.4 mg, 6.9 mmol, 37 wt% in water) and sodium borocyanide acetate (1.5 g, 6.9 mmol) were added. The mixture was stirred at room temperature for 16 hours. The reaction solution was evaporated to dryness, and ethyl acetate (150 mL) and water (50 mL) were added. The organic phase was washed with saturated sodium bicarbonate solution (50 mL × 2). After drying and filtration, the solution was evaporated to dryness to give a yellow liquid compound 10⁻³⁻² (1.0 g, 81.3% yield). The crude product was used directly in the next step.

[0106] LCMS: m / z = 536.2. [M+H]+. Step 3: Synthesis of compound 10-3-3. Compound 10-3-2 (0.8 g, 1.49 mmol) was dissolved in tetrahydrofuran (2.0 mL), and then added to formic acid (2 mL) and water (2 mL). The reaction was carried out at 70 °C for 3 hours. After the reaction was completed, the mixture was evaporated to dryness, and the residue was passed through a reverse-phase column (C18, (water / acetonitrile = 35%)) to give compound 10-3-3 (500 mg, 79.6% yield).

[0107] LCMS: m / z = 422.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 5.80 (d, J = 4.4 Hz, 1H), 5.64 (d, J = 8.0 Hz, 1H), 5.25 - 5.17 (m, 1H), 4.08- 3.94 (m, 5H), 3.91 - 3.86 (m, 1H), 3.82 - 3.78 (m, 1H), 3.37 (s, 3H), 2.91- 2.66 (m, 4H), 2.38 (s, 3H), 1.26 - 1.19 (m, 6H). Step 4: Synthesis of Compound 10⁻³ Compound 10⁻³⁻ (250 mg, 0.59 mmol) was dissolved in dry dichloromethane (5 mL), and tetrazolium (70.8 mg, 1.18 mmol) was added. Then, compound A, bis(diisopropylamino)(2-cyanoethoxy)phosphine (355.4 mg, 1.18 mmol), was added dropwise to the mixture in dichloromethane (1 mL). The mixture was stirred at room temperature for 6 hours, water (30 mL) was added, and the mixture was extracted with dichloromethane (50 mL). The organic phase was washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was passed through a silica gel column (dichloromethane (containing 0.5% triethylamine) / acetonitrile = 1 / 1) to give a colorless oily compound 10⁻³ (150 mg, 40.9%).

[0108] LCMS: m / z = 622.5[M+H]+. Purity: 99.9% (214 nm). 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.74 (dd, J = 8.1, 2.3 Hz, 1H), 5.83 (dd, J = 5.3, 2.6 Hz, 1H), 5.66 (d, J = 8.0 Hz, 1H), 4.27 (d, J =10.4 Hz, 1H), 4.15 - 3.95 (m, 6H), 3.84 - 3.57 (m, 4H), 3.36 (d, J = 18.4 Hz, 3H), 2.95 - 2.69 (m, 6H), 2.38 (d, J = 7.5 Hz, 3H), 1.29 - 1.10 (m, 18H). 31 P NMR (162 MHz, DMSO-d6) δ 149.46 (s), 149.18 (s), 24.60 (d, J =15.6 Hz). 4. Synthesis of compound 10⁻⁵ (Cpd 10⁻⁵) Step 1: Synthesis of compound 10-5-2 Compound 10-5-1 (30.0 g, 131.46 mmol) was dissolved in ultradry pyridine (300 mL) and 4,4'-bismethoxytriphenylmethyl chloride (53.45 g, 157.75 mmol). The mixture was stirred at room temperature for 16 hours. Thin-layer chromatography (TLC) showed that the reaction was complete. The reaction solution was evaporated to dryness and purified by rapid column chromatography (dichloromethane / methanol = 95 / 5) to give compound 2 (35.0 g, 50.18% yield) as a white solid.

[0109] 1H NMR (400 MHz, DMSO) δ 10.44 (s, 1H), 7.96 (d, J = 7.4 Hz, 1H), 7.35 - 7.10 (m, 9H), 6.90 - 6.80 (m, 4H), 6.40 - 6.30 (m, 1H), 6.05 (d, J =4.5 Hz, 1H), 5.88 (d, J = 7.4 Hz, 1H), 5.23 (d, J = 5.7 Hz, 1H), 4.35 - 4.17(m, 2H), 3.73 (s, 6H), 2.98 - 2.79 (m, 2H). Step 2: Synthesis of compound 10-5-4 Compound 10⁻⁵⁻³ (16.8 g, 188.5 mmol) was dissolved in dry ethylene glycol dimethyl ether (80.0 mL). Under argon protection, trimethylaluminum (40.52 mL, 81.04 mmol) was added at 0 °C. The mixture was stirred at 110 °C for half an hour. Compound 10⁻⁵⁻² (10.0 g, 18.85 mmol) was added at 0 °C, and the mixture was stirred overnight at 125 °C. Thin-layer chromatography (TLC) showed that the reaction was complete. The crude product was directly concentrated and evaporated to dryness. The residue was purified by rapid column chromatography (dichloromethane / methanol = 95:5) to give compound 10⁻⁵⁻⁴ (5.0 g, 36.25% yield) as a white solid.

[0110] 1 H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.91 - 7.82 (m, 1H), 7.72(d, J = 8.1 Hz, 1H), 7.41 - 7.21 (m, 10H), 6.91 (d, J = 8.8 Hz, 4H), 5.81 (d,J = 2.2 Hz, 1H), 5.47 (d, J = 7.7 Hz, 1H), 5.31 - 5.23 (m, 1H), 4.27 - 4.19(m, 1H), 4.19 - 4.13 (m, 1H), 4.06 - 3.99 (m, 3H), 3.74 (s, 6H), 3.29 - 3.23(m, 1H), 2.65 (d, J = 4.7 Hz, 3H). Step 3: Synthesis of compound 10-5-5 Compound 10⁻⁵⁻⁴ (8.0 g, 10.93 mmol) and imidazole (2.98 g, 43.72 mmol) were dissolved in dry N,N-dimethylformamide (80 mL). Under nitrogen protection, tert-butyldimethylchlorosilane (3.29 g, 21.86 mmol) was added at 0 °C, and the mixture was stirred overnight at room temperature. Thin-layer chromatography (TLC) showed that the reaction was complete. The reaction mixture was poured into ice water and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (300 mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give a white solid compound 10⁻⁵⁻⁵ (7.0 g, crude product).

[0111] LCMS m / z = 730.2 [MH]-. Step 4: Synthesis of compound 10-5-6 Compound 10⁻⁵⁻⁵ (7 g, 9.56 mmol) was dissolved in dichloromethane (60 mL), and formic acid (10.0 mL) was added at 0 °C under nitrogen protection. The mixture was stirred for 1 hour. After the reaction was complete, the reaction solution was poured into ice water and extracted with dichloromethane (100 mL × 3). The organic phases were combined, washed with saturated sodium chloride (300 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was passed through a silica gel column (dichloromethane / methanol = 92:8) to give a white solid compound 10⁻⁵⁻⁶ (3.0 g, 73.03% yield).

[0112] LCMS m / z = 430.2 [M+H]+. Step 5: Synthesis of compound 10-5-7 Compound 10-5-6 (1.5 g, 3.49 mmol) was dissolved in ultradry pyridine (15 mL). The reaction solution was cooled to 0 °C, and methanesulfonyl chloride (MsCl, 1.03 mL, 6.98 mmol) was slowly added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. LC-MS showed that the reaction was complete. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound 10-5-7 (1.5 g, crude product). The crude product was used directly in the next step.

[0113] LCMS m / z = 508.2 [M+H]+. Step 6: Synthesis of compound 10-5-8 A tetrahydrofuran (15 mL) solution of compound 10-5-7 (1.5 g, crude) was added to an ethanol solution of the prepared compound 3A (diethyl selenide-methylphosphonate) (1.37 g, crude). The reaction was carried out at 60 °C for 16 hours under nitrogen protection. LC-MS showed that the reaction was complete. The reaction solution was added to water (100 mL), extracted with ethyl acetate (100 mL × 2), the organic phase was washed with saturated brine (200 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 92:8) to give a white solid compound 10-5-8 (1.1 g, 49.00% two-step yield).

[0114] LCMS m / z = 644.2 [M+H]+. Step 7: Synthesis of compound 10-5-9 Compound 10-5-8 (1.1 g, 1.71 mmol) was dissolved in formic acid (10 mL) and water (10 mL) and reacted at 40 °C for 2 h. After the reaction was complete, the mixture was evaporated to dryness, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 94:6) to give compound 10-5-9 (500 mg, 55.28% yield).

[0115] LCMS m / z = 530.2 [M+H]+ Step 8: Synthesis of Compound 10-5 Compound 10-5-9 (500 mg, 0.95 mmol) was dissolved in dry dichloromethane (5 mL), and tetrazolium (132.6 mg, 1.89 mmol) was added. Then, compound 10-5-10 bis(diisopropylamino)(2-cyanoethoxy)phosphine (570.5 mg, 1.89 mmol) was added dropwise to the mixture. The mixture was stirred at room temperature for 3 hours, and ice water (50 mL) was added. Extraction was performed with dichloromethane (50 mL), and the organic phase was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was passed through a silica gel column (acetonitrile / dichloromethane = 0–100%, containing 0.1% triethylamine) to give compound 10-5 (370 mg, 53.66% yield).

[0116] LCMS m / z = 728.1 [MH]- 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 4.0 Hz, 1H), 5.88 - 5.81 (m, 1H), 5.64 (d, J = 8.0 Hz, 1H), 4.38- 4.17 (m, 3H), 4.14 - 3.93 (m, 6H), 3.87 - 3.69 (m, 2H), 3.67 - 3.51 (m,2H), 3.20 - 3.02 (m, 2H), 2.95 - 2.74 (m, 4H), 2.61 (t, J = 5.2 Hz, 3H), 1.31- 1.09 (m, 18H). 31 P NMR (162 MHz, DMSO-d6) δ 149.10 (s), 148.71 (s), 25.83 (d). 5. Synthesis of intermediates INT-A and INT-B Step 1: Synthesis of Compound 2 Compound SM-INT-1 (50.0 g, 193.8 mmol) was dissolved in pyridine (660 mL), the mixture was evaporated to dryness, and then pyridine (660 mL) and 4,4'-bismethoxytriphenylmethyl chloride (DMTrCl, 71.9 g, 232.6 mmol) were added. The mixture was stirred at room temperature for 12 hours. The reaction solution was evaporated to dryness and passed through a silica gel column (petroleum ether / ethyl acetate = 1 / 1) to give compound 2 (69.0 g, 63.9% yield) as a white solid.

[0117] 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.34- 7.32 (m, 2H), 7.28 - 7.23 (m, 5H), 6.91 (d, J= 8.4 Hz, 4H), 5.29 - 5.22 (m, 2H), 4.24- 4.17 (m, 1H), 3.99 - 3.93 (m, 1H), 3.85 - 3.77 (m, 1H), 3.74 (s, 6H), 3.41 (s, 3H), 3.31 - 3.22 (m, 2H). Step 2: Synthesis of Compound 3 Compound 2 (69.0 g, 123.2 mmol) and imidazole (16.7 g, 246.4 mmol) were dissolved in dry N,N-dimethylformamide (500.0 mL). Under nitrogen protection, tert-butyldimethylchlorosilane (TBSCl, 24.0 g, 160.2 mmol) was added at 0 °C, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was poured into ice water and extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with saturated brine (250 mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was passed through a silica gel column (petroleum ether / ethyl acetate = 5:1) to give compound 3 (80.0 g, 96.4% yield) as a white solid.

[0118] LCMS: m / z = 697.3 [M+Na]+. Step 3: Synthesis of compound INT-A Compound 3 (80.0 g, 118.7 mmol) was dissolved in dichloromethane (80.0 mL), and formic acid (20.0 mL) was added at 0 °C under nitrogen protection. The mixture was stirred for 4 h. After the reaction was complete, the reaction solution was poured into ice water and extracted with dichloromethane (200 mL × 3). The organic phases were combined, washed with saturated brine (250 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was passed through a silica gel column (petroleum ether / ethyl acetate = 2:1) to give compound 4 (80.0 g, 51.4% yield) as a white solid. LCMS: m / z = 373.2 [M+H]+. Step 4: Synthesis of compound INT-B Compound INT-A (10.0 g, 26.9 mmol) was dissolved in 280 mL of acetonitrile, and 2-iodobenzoic acid (IBX, 18.8 g, 67.2 mmol) was added. The reaction mixture was heated under reflux for 2 hours. After cooling to 0 °C, the mixture was stirred for 30 minutes. The mixture was filtered, and the solid was rinsed with ethyl acetate (50 mL × 3). The filtrate was evaporated under reduced pressure to dryness to give a white solid, compound 5. The crude product was used directly in the next step.

[0119] LCMS: m / z = 371.0 [M+H]+. Example 2: Preparation of siRNA containing 5' phosphate-modified nucleoside analogs This embodiment uses siRNA targeting the MSTN gene as an example to illustrate how to prepare 5'-terminal modified siRNA using the compounds of this invention. The sense (SS) and antisense (AS) sequences of the parent sequence are CGBN-0287, as shown in Table 1. In this embodiment, the siRNA sequences synthesized other than the parent sequence are CGBN-0288, CGBN-0308, CGBN-0344, and CGBN-0346, respectively. The sense strands of these sequences are the same as the parent sequence. The antisense strand is modified by phosphonate esters of the first nucleotide at the 5' end of the antisense strand of the parent sequence CGBN-0287, such as 5'-E-VP, 4' MeMOP, Cpd10 (deethylated protecting group), and Cpd10-3 (deethylated protecting group). The resulting siRNA double-stranded sequences are shown in Table 1, and the corresponding end-modified structures are shown in Table 2.

[0120] Table 1. siRNA double-stranded sequences The meanings of the abbreviations in this article are as follows: The distributions A, U, G, and C represent naturally occurring adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.

[0121] m indicates that the nucleotide adjacent to it on the left is a nucleotide modified with 2'-OMe (2'-methoxynucleoside). For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OMe, respectively.

[0122] f indicates that the nucleotide adjacent to it on the left is a 2'-F modified nucleotide (2'-fluoronucleoside). For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0123] 's' indicates that the two adjacent nucleotides on its left and right sides and / or the delivery carrier are linked by a thiophosphate.

[0124] EVP indicates that the terminal of its adjacent nucleotide is modified with 5'-(E)-vinylphosphonate.

[0125] MeMOP indicates that its adjacent nucleotide is modified with 4'-oxymethylphosphonate.

[0126] Cpd10 and Cpd10-3 indicate that their adjacent nucleotides are modified with 5' phosphonates.

[0127] Table 2. Compound structure diagrams All siRNA single strands were synthesized using the standard solid-phase phosphoramidite chemistry method, employing commercially available phosphoramidite monomers on a chemical synthesizer from the 3' to the 5' end to produce the corresponding sense and antisense strands. In the final step of antisense strand synthesis, the phosphoramidite monomers of Compound 10, Compound 10-3 of this invention, or other specially modified 5' phosphoramidite monomers disclosed in Table 2 were used instead of conventional monomers for the final coupling reaction to complete the modification of the 5' end of the antisense strand. The specific steps are as follows: (1) Preparation of reagents and monomers A monomeric acetonitrile solution (0.10 M), a 0.30 M acetonitrile solution of 5-ethylthiotetrazole, a pyridine solution of 0.20 M hydroxanthin, a tetrahydrofuran / water / pyridine (70 / 10 / 20, v / v / v) solution of iodine, an oxidizing agent, a 10% acetic anhydride acetonitrile solution (v / v) as capping agent A, a 1-methylimidazolium / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v) as capping agent B, and a 3% trichloroacetic acid solution in dichloromethane (w / v) as a deprotecting agent for the DMTr group were used. A universal carrier with controllable microporous glass beads (Universal Unylinker support 500A, Chemgenes) was selected as the solid-phase carrier and loaded into the designated reagent positions in the HJ12 model DNA / RNA automated synthesizer.

[0128] (2) Crude product synthesis Input the specified oligonucleotide sequence and set the synthesis program. After verifying that everything is correct, begin the synthesis of the cyclic oligonucleotides, with each synthesis scale being 2 μmol. Prepare the oligonucleotides according to the following steps: a. Deprotection The DMTr protecting group was removed using a 3% trichloroacetic acid solution in dichloromethane, followed by washing with acetonitrile.

[0129] b. Coupling The acetonitrile solutions of each nucleotide monomer were coupled using 0.30 M 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0130] c. Oxidation / Sulfidation Oxidation: Oxidation was performed using a 0.05 M solution of tetrahydrofuran / water / pyridine (70 / 10 / 20, v / v / v) as the oxidant, followed by rinsing with acetonitrile.

[0131] Vulcanization: Vulcanization was carried out using a pyridine solution of 0.20 M hydroflavin as the sulfiding agent, followed by rinsing with acetonitrile.

[0132] d. Hydroxyl protection Hydroxyl protection was performed using capping agent A and capping agent B as hydroxyl protecting agents, followed by rinsing with acetonitrile.

[0133] Repeat the above operations in a cyclical manner according to the set sequence to obtain a fully protected product.

[0134] (3) Deprotection a. Preparation of the sense strand and the antisense strand of siRNA without special 5' end modification. The solid support was transferred to a glass vial, concentrated ammonia (25-28%) was added, and the mixture was kept at 45°C for 16 h for ammonolysis. The system was then cooled to room temperature, and the mixture was filtered. The solid support was washed with purified water, and the filtrates were combined and freeze-dried to obtain the crude product of the target sequence.

[0135] b. Preparation of 5' phosphine-modified siRNA antisense strand The solid support was transferred to a centrifuge tube, and a trimethyliodosilane / pyridine / acetonitrile (2 / 13 / 40, v / v / v) solution was added. After reacting at room temperature for 3–9 hours, the solid support was washed with acetonitrile. The solid support was then transferred to a glass vial, and concentrated ammonia (25–28%) was added. After ammonolysis at 45°C for 16 hours, the system was cooled to room temperature, and the mixture was filtered. The solid support was washed with purified water, and the filtrates were combined and freeze-dried to obtain the crude product of the target sequence.

[0136] (4) Purification The crude residue after deprotection was dissolved in purified water, and the sequence was purified using RP-IP-HPLC (WATERS 2489, 3767) (purification column: XBridge BET C18 2.5 μm, mobile phase A: 0.1% triethylamine + 2% hexafluoroisopropanol aqueous solution, mobile phase B: methanol). The combined fractions were lyophilized to obtain the target sequence. The product peak solution was collected and the content was determined by ultra-micro UV spectrophotometry, and the purity and molecular weight were confirmed by LC / MS (see Table 3). The product peak solution was concentrated and lyophilized to obtain the product.

[0137] (5) Annealing The synthesized sense and antisense strands were mixed in distilled water at an equimolar ratio. The resulting solution was allowed to stand at room temperature for 15 minutes. The product was detected by SEC-HPLC to ensure complete annealing (Table 3). The annealed solution was then freeze-dried to obtain the siRNA conjugate.

[0138] Table 3. Molecular weight and purity of siRNA double strands Example 3: In vitro biological activity assessment To verify the biological function of the 5'-phosphonate-modified oligonucleotides provided by this invention, we evaluated the silencing efficiency of the target genes by the siRNAs with different modifications at the 5' end of the antisense strand synthesized in Example 2 through in vitro cell experiments.

[0139] We designed the DualGlo reporter plasmid (Promega, psiCHECK™-2 Vector) for screening siRNAs. This plasmid contains the coding sequence of human MSTN mRNA (ENST00000260950.5) in the 3'-UTR of the reporter luciferase.

[0140] Human 293T cells were grown in DMEM supplemented with a mixture of 10% v / v heat-inactivated fetal bovine serum and 1% v / v penicillin-streptomycin. For transfection, cells were seeded at a density of 4,000 cells / well in 96-well opaque cell culture plates and transfected within 24 hours.

[0141] Cells were co-transfected using MSTN-DualGlo reporter plasmid and siRNA at specified dilutions, mixed according to the manufacturer's recommendations, using Lipofectamine® 3000 (ThermoFisher). In multiple-dose experiments, siRNA concentrations were 100,000 pmol, 10,000 pmol, 1,000 pmol, 100 pmol, 10 pmol, 1 pmol, 0.1 pmol, 0.01 pmol, and 0.001 pmol. Transfected cells were incubated in 5% CO2 at 37°C for 2 days. Using the Dual-Glo® luciferase assay (Promega) and the Dual-Glo® Stop & Glo® assay, the appropriate reagents were added sequentially according to the manufacturer's instructions, and the corresponding fluorescence readings for Firefly luminescence and Renilla luminescence were measured. In screening experiments, Renilla luminescence values ​​were normalized relative to Firefly luminescence values ​​for each sample. Quantification of MSTN mRNA downregulation was calculated using the manufacturer's recommended analytical method. All experiments were performed in triplicate, and the average of the three replicates was calculated. In the two-dose screening, the remaining amount of MSTN mRNA was determined by comparing the experimental group with the control group. Figure 1 As shown in Table 4, in the multi-dose validation experiment, the theoretical maximum knockout efficiency KDmax% was calculated by nonlinear regression analysis of the data using a 4-parameter dose-response inhibition function (GraphPadPrism 8.4.3). The IC50 results were calculated by inserting 50% into the function.

[0142] Table 4. In vitro silencing activity of MSTN reporter gene with siRNAs modified at different 5' ends. Data shows that the nucleotide CGBN-0344 modified with the core compound Cpd 10 of this invention maintained good in vitro silencing activity, with an IC50 value of [missing information]. 50 The activity values ​​were on the same order of magnitude as the positive control compound (E)-VP and the unmodified sequence, and significantly superior to another prior art positive control compound, MeMOP. Although the activity of the slightly modified Cpd 10-3 was significantly lower than that of Cpd 10, it was still slightly better than the MeMOP control, indicating the unique structure-activity relationship of this type of compound. In summary, the Cpd 10 selenophosphonate modification of this invention can efficiently mimic the function of 5'-phosphate, and can be effectively recognized by intracellular processing mechanisms in vitro to mediate efficient gene silencing, achieving the expected results in balancing stability and biological activity.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A modified nucleoside compound having the structure shown in formula (I) or (II), or being a pharmaceutically acceptable salt or stereoisomer of a compound having the structure shown in formula (I) or (II): in, R1 and R2 are each independently selected from one of H, halogen, hydroxyl, C1-C6 alkyl, any substituted C1-C6 alkyl, C1-C6 alkoxy, any substituted C1-C6 alkoxy, sulfonyl, sulfinyl, amide and any substituted amide. R3 is hydrogen, a protecting group, or a phosphorus-containing reactive group; R4 and R5 are each independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, amino or any substituted amino, or phosphorus-containing reactive group, -CH2CH2CN and -CH2O(CO)C(CH3)3; M1 is O, S, Se, or -CH2-; M2 is a nucleoside containing phosphorus amide, sugar, or a sugar substitute. B is H, a heterocyclic base, or a base substitution group, wherein the heterocyclic base includes uracil, cytosine, adenine, and guanine, and the base substitution group is phenyl or a substituted phenyl group; X is O, Se, or S; L is selected from Se, C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 ynynyl, or the above groups are substituted by isotopes or halogens at any position. A is selected from one of the following groups: -N(CH3)-, Se, C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, and C2-C6 alkyneyl, or the above groups are substituted by isotopes or halogens at any position.

2. The modified nucleoside compound according to claim 1, characterized in that, X is Se, and L and A are each independently selected from one of the following groups: C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, and C2-C6 alkyneyl, or the above groups are substituted by isotopes or halogens at any position.

3. The modified nucleoside compound according to claim 1, characterized in that, X is O or S, and A is -N(CH3)- or Se, and L is selected from one of the following groups: C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, C2-C6 alkyneyl, or the above groups are substituted by isotopes or halogens at any position.

4. The modified nucleoside compound according to claim 1, characterized in that, X is O or S, and L is Se. A is selected from one of the following groups: C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, and C2-C6 alkyneyl, or the above groups are substituted by isotopes or halogens at any position.

5. The modified nucleoside compound according to claims 1-4, characterized in that, R4 and R5 may be the same or different, and at least one of them is a C1-C6 alkyl group.

6. The modified nucleoside compound according to claim 5, characterized in that, Both R4 and R5 are ethyl groups.

7. The modified nucleoside compound according to claim 5, characterized in that, R4 is ethyl and R5 is H.

8. The modified nucleoside compound according to claim 1, characterized in that, The phosphorus-containing reactive group is phosphorous amide, H-phosphate ester, triphosphate ester, or a phosphorus-containing chiral auxiliary agent.

9. The modified nucleoside compound according to claim 1, characterized in that, In the nucleoside of the sugar or sugar-substituted moiety described in M2, the sugar-substituted moiety is any one of morpholino, cyclohexenyl, cyclohexyl, cyclopentyl, pyranyl, and cyclohexanehexaolyl; and the sugar moiety is furanose.

10. The modified nucleoside compound according to claim 9, characterized in that, The nucleosides of the sugar or sugar-substitute portion include non-locked nucleoside analogs, glycerol nucleic acid base analogs, locked nucleic acids, or bridged nucleic acids.

11. The modified nucleoside compound according to claim 1, characterized in that, in, The compound of formula (I) is any one or a combination of at least two of the following compounds having the following structures: 。 12. An oligonucleotide, characterized in that, The structural units of the oligonucleotide include nucleotides containing the modified nucleoside compound as described in any one of claims 1-11.

13. The oligonucleotide of claim 12, characterized in that, The nucleotide containing the modified nucleoside compound is located at the 5' end of the oligonucleotide.

14. The oligonucleotide of claim 12, characterized in that, The oligonucleotide is an oligonucleotide as shown in formula (III) or a pharmaceutically acceptable salt thereof: R4 and R5 are each independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, amino or any substituted amino, or phosphorus-containing reactive group, -CH2CH2CN and -CH2O(CO)C(CH3)3; M2 is a nucleoside containing phosphorus amide, sugar, or a sugar substitute. X is O, Se, or S; L is selected from Se, C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 ynynyl, or the above groups are substituted by isotopes or halogens at any position. A is selected from one of the following groups: -N(CH3)-, Se, C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, and C2-C6 alkyneyl, or the above groups are substituted by isotopes or halogens at any position.

15. The oligonucleotide of claim 12, characterized in that, If X is Se, then L and A are each independently selected from one of the following groups: C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, and C2-C6 alkyneyl, or the above groups are substituted by isotopes or halogens at any position.

16. The oligonucleotide of claim 12, characterized in that, X is O or S, and A is -N(CH3)- or Se, and L is selected from one of the following groups: C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl and C2-C6 alkyneyl, or the above groups are substituted by isotopes or halogens at any position.

17. The oligonucleotide of claim 12, characterized in that, X is O or S, and L is Se. A is selected from one of the following groups: C1-C6 alkylene, C1-C4 cycloalkyl, C2-C6 alkenyl, and C2-C6, or the above groups are substituted by isotopes or halogens at any position.

18. The oligonucleotide of claim 12, characterized in that, The oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, small guide RNAs, transfer RNAs, and aptamers.

19. The oligonucleotide of claim 18, characterized in that, The oligonucleotide is siRNA.

20. The oligonucleotide of claim 19, characterized in that, The siRNA is a double-stranded siRNA or a single-stranded siRNA, wherein the double-stranded siRNA comprises a sense strand and an antisense strand.

21. The oligonucleotide of claim 20, characterized in that, The antisense strand of the double-stranded siRNA is an oligonucleotide as shown in formula (III) or a pharmaceutically acceptable salt thereof.

22. The oligonucleotide according to any one of claims 12-21, characterized in that, Each nucleotide in the oligonucleotide is independently either modified or unmodified.

23. The oligonucleotide as claimed in claim 22, characterized in that, The oligonucleotide is any one or a combination of at least two of the following siRNAs: (i) The sense chain has the sequence shown in SEQ ID No. 2; and the antisense chain has the sequence shown in SEQ ID No. 5; (ii) The sense chain has the sequence shown in SEQ ID No 2; and the antisense chain has the sequence shown in SEQ ID No 6.

24. A nucleic acid conjugate comprising an oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 12-23, and a targeting ligand bound thereto.

25. The nucleic acid conjugate as described in claim 24, characterized in that, The targeting ligand comprises any one or a combination of at least two of carbohydrates, cholesterol, fatty acid chains, peptides, or antibodies.

26. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1223; And / or, the pharmaceutical composition comprises the nucleic acid conjugate as described in claim 24 or 25.

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