Nucleoside analogue and application thereof in reducing off-target activity of oligonucleotide medicine

Abstract

The invention relates to the field of biological medicine, in particular to a nucleoside analogue and application thereof to reduction of off-target activity of oligonucleotide drugs. The nucleoside analogue provided by the invention can be used for preparing oligonucleotide drugs, and can be coupled to a seed region of siRNA, so that the specificity of siRNA is efficiently improved, and the silence effect of a target gene can be maintained while the off-target effect of siRNA is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nucleoside analog and an application thereof in reducing the off-target activity of an oligonucleotide drug. Background Art

[0002] RNAi (RNA interference) technology is a very important technological breakthrough in the field of molecular biology. It has been widely used in agriculture, forestry, pharmaceutical industry and other fields. RNA interference is a sequence-specific gene post-transcription silencing process that is triggered by double-stranded RNA (dsRNA) homologous to the target gene sequence and widely exists in organisms. In this process, Dicer, a member of the RNase III family of ribonucleases in cells, cleaves dsRNA into small interfering RNA (siRNA) consisting of 21-25 nucleotides. Subsequently, siRNA acts as a mediator to specifically degrade mRNA of the same sequence, thereby blocking the expression of the corresponding gene. However, researchers have found that in actual applications, there are problems such as off-target effects of siRNA, resulting in unsatisfactory silencing effects.

[0003] Since the natural RNA double strand is unstable, it is necessary to adjust the siRNA design by chemical modification. The chemical modification needs to eliminate or reduce the off-target effect of siRNA while not affecting the ability of gene silencing. However, the anti-off-target effect of siRNA obtained by chemical modification in the current art is still unsatisfactory. Summary of the invention

[0004] In order to overcome the off-target effect of siRNA in the field of small nucleic acid drugs, the present invention provides a nucleoside analog and its application in reducing the off-target activity of oligonucleotide drugs. The nucleoside analog provided by the present invention has a triazole structure or an amide structure, and the oligonucleotide and conjugate prepared therefrom have the effect of preventing off-target.

[0005] The inventors of the present invention cleverly discovered in their research that by using a triazole structure or an amide structure as a linker, a nucleoside monomer compound based on a triazole structure or an amide structure can be modularly, simply and quickly constructed. The compound can be coupled to the seed region of siRNA through solid phase synthesis, thereby efficiently improving the specificity of siRNA, while reducing the off-target effect of siRNA and maintaining the silencing effect on the target gene.

[0006] In the present invention, unless otherwise specified, "nucleoside analogue" refers to the nucleoside analogue provided by the present invention, that is, the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0007] Based on the above findings, the present invention provides the following technical solutions: In a first aspect, the present invention provides a nucleoside analog, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0008] in, L1 is hydrogen, a hydroxyl protecting group or a phosphorus-containing group reactive with nucleophiles; L2 is hydrogen or a hydroxyl protecting group; L3 is -CH2HNC(O)(CH2) n -、-C(O)NH(CH2) n -、-(CH2) n -、-(CH2) n O(CH2) m -or , wherein n and m are each independently any integer from 0 to 5, R4 and R5 are each independently hydrogen, oxygen, alkylene, alkenylene, -R 4' -OR 4'' , -R 4' -CO-R 4'' , -R 4' -N(R 4''' )-R 4'' , where R 4' , R 4'' and R 4''' Each is independently hydrogen, C1-C6 alkylene, C1-C6 alkyl, C1-C6 acyl or C1-C6 alkyleneoxy; L4 is a substituted or unsubstituted base or a salt thereof; R1, R2 and R3 are each independently hydrogen, alkyl, alkenyl or aryl.

[0009] In the present invention, "hydroxyl protecting group" refers to a protecting group that can protect the hydroxyl group in the compound / oligonucleotide / conjugate provided by the present invention from reacting or recovering after the reaction is completed during the reaction process (e.g., in the liquid phase / solid phase nucleotide synthesis reaction process, in the oligonucleotide synthesis reaction process, in the reaction process of preparing a conjugate between an oligonucleotide and a ligand, etc.). Any hydroxyl protecting group commonly used in the art can be applied to the present invention as long as it can achieve the above-mentioned purpose.

[0010] In the present invention, "nucleophilic reagent" can also be referred to as "nucleophilic group", which is an electron pair donor, i.e., a Lewis base. The nucleophilic reagent involved in the present invention can be any nucleophilic reagent in the art, for example, it can be a lone-pair nucleophile, a σ-bond nucleophile, and a π-bond nucleophile, etc. "Phosphorus-containing group reactive with nucleophilic reagent" is a group containing the element P that can react with the above-mentioned nucleophilic reagent. Any active phosphorus group in the art that can be used in oligonucleotides and can react with nucleophilic reagents can be applied to the present invention. In the present invention, "active phosphorus group" can be used to form internucleoside linkages (including, for example, phosphodiester and thiophosphate internucleoside linkages), and its meaning is known in the art and includes groups in the p III or P V Phosphorus atoms in a valence state, examples of active phosphorus groups include but are not limited to phosphoramidites, phosphotriesters, H-phosphates or phosphorus-containing chiral auxiliary agents. Among them, the phosphorus-containing chiral auxiliary agent refers to a phosphorus-containing group with chirality, for example, it can be , , , , , , In some embodiments of the present invention, when L3 is When R4 and R5 are each independently selected from an ether bond-containing group or a carbonyl-containing group.

[0011] In the present invention, the "ether bond-containing group" may include an ether bond, a group having an ether bond at any position in a straight or branched alkylene group, etc., for example, it may be -CH2-O-CH2-, -CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-, -CH2-O-CH2CH2-O-CH2, or -CH2-O-CH2CH2-O-CH2CH2-O-CH2-, etc. Preferably, the number of ether bonds in the ether bond-containing group may be 1-3 (for example, 1, 2 or 3).

[0012] In the present invention, "carbonyl-containing group" may include a carbonyl group, a group having a carbon-oxygen double bond connected to a carbon at any position in a straight or branched alkylene group, etc. Preferably, the number of carbonyl groups in the carbonyl-containing group may be 1-2 (e.g., 1 or 2). For example, it may be -CH2-CO-CH2-, -CH2-CO-CH2CH2-, -CH2CH2-CO-CH2CH2-, or -CH2-CO-CH2CH2-CO-CH2, etc.

[0013] In the present invention, "nitrogen-containing groups" may include groups containing nitrogen-oxygen double bonds, nitrogen-oxygen single bonds, amino groups, amides and the like. Preferably, the number of N atoms in the nitrogen-containing groups may be 1-2 (e.g., 1 or 2). For example, it may be -CH2-N(CH3)-OCH2CH2-, -CH2-N(CH3)-CH2-, -CH2-N(CH3)-CH2CH2-, -CH2-N(CH2CH3)-CH2CH2-, -CH2-C(O)NH-CH2-, -CH2-C(O)NH-CH2CH2- or -CH2CH2-C(O)NH-CH2CH2-2.

[0014] According to a preferred embodiment of the present invention, L1 is an active phosphorus group, and preferably the active phosphorus group is a phosphoramidite, a phosphotriester, an H-phosphate or a phosphorus-containing chiral auxiliary.

[0015] According to a preferred embodiment of the present invention, L1 is a hydroxyl protecting group, preferably the hydroxyl protecting group is selected from any one of 4,4'-dimethoxytrityl, monomethoxytrityl, trityl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl and isopropyldimethylsilyl.

[0016] Preferably, L1 is , " ” indicates the connection location.

[0017] According to a preferred embodiment of the present invention, L2 is a hydroxyl protecting group, preferably the hydroxyl protecting group is selected from any one of 4,4'-dimethoxytrityl, monomethoxytrityl, trityl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl and isopropyldimethylsilyl.

[0018] Preferably, L2 is 4,4'-dimethoxytrityl.

[0019] According to a preferred embodiment of the present invention, wherein L3 is -CH2HNC(O)(CH2)2- or , " ” indicates the connection location.

[0020] According to a preferred embodiment of the present invention, L4 is any one of substituted or unsubstituted purine or pyrimidine or a salt thereof.

[0021] Preferably, L4 is substituted or unsubstituted adenine or a salt thereof, guanine or a salt thereof, cytosine or a salt thereof, thymine or a salt thereof, or uracil or a salt thereof; More preferably, L4 is substituted adenine or a salt thereof, cytosine or a salt thereof, or thymine or a salt thereof.

[0022] According to a preferred embodiment of the present invention, R1, R2 and R3 are all hydrogen.

[0023] According to a preferred embodiment of the present invention, wherein in formula (I) Some of them are (R)-configuration, (S)-configuration or (rac)-configuration (i.e., they can be single chiral compounds or racemic compounds).

[0024] According to a preferred embodiment of the present invention, the compound is a compound having the structure of any one of the following YK-GNA-001, YK-GNA-002, YK-GNA-003, YK-GNA-004, YK-GNA-005 and YK-GNA-006: , , , , , .

[0025] The second aspect of the present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof modified by the compound of formula (I) or a pharmaceutically acceptable salt thereof as described in the first aspect, wherein the oligonucleotide comprises a structural unit formed by the compound or a pharmaceutically acceptable salt thereof as described in the first aspect. That is, the oligonucleotide provided by the present invention may be formed by replacing some structural units in a conventional oligonucleotide with a nucleotide synthesized by the nucleoside analog of the present invention (i.e., the compound of formula (I) or a pharmaceutically acceptable salt thereof) and phosphoric acid.

[0026] In the oligonucleotide provided by the present invention, some of the structural units (nucleotides) may contain the nucleoside analogs of the present invention, or all of the structural units may contain the nucleoside analogs of the present invention. When some of the structural units contain the nucleoside analogs of the present invention, the other structural units in the oligonucleotide may be provided by conventional nucleotides, or by nucleotides containing other existing nucleoside analogs.

[0027] In the present invention, the structural unit of the oligonucleotide refers to a nucleotide. As is well known in the art, a nucleotide is a compound composed of a base (purine base or pyrimidine base), a pentose (ribose or deoxyribose) and phosphoric acid. The base and the pentose condense to form a nucleic acid, and then further synthesize a nucleotide with phosphoric acid. In the aforementioned first aspect, the compound of formula (I) or a pharmaceutically acceptable salt thereof provided by the present invention is a nucleoside analog. "The oligonucleotide includes a structural unit formed by the compound described in the first aspect or a pharmaceutically acceptable salt thereof" means that the nucleoside structure of at least one nucleotide in the oligonucleotide sequence is as shown in the compound of formula (I) or a pharmaceutically acceptable salt thereof provided by the present invention, that is, among the several nucleotides contained in the oligonucleotide, the nucleoside portion of at least one nucleotide is replaced by the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0028] In some preferred embodiments, for an oligonucleotide with a length not exceeding 30 nucleotides (such as 15-30 nucleotides), the number of structural units (nucleotides) containing the nucleoside analogs provided by the present invention does not exceed 3.

[0029] According to some preferred embodiments of the present invention, the oligonucleotide comprises a structural unit having a structure of formula (II):

[0030] in, represents the position where the phosphate bond is formed; L3 is -CH2HNC(O)(CH2) n -、-C(O)NH(CH2) n -、-(CH2) n -、-(CH2) n O(CH2) m -or , wherein n and m are each independently any integer from 0 to 5, R4 and R5 are each independently hydrogen, alkylene, alkenylene, -R 4' -OR 4'' , -R 4' -CO-R 4'' , -R 4' -N(R 4''' )-R 4'' , where R 4' , R 4'' , R 4''' Each is independently hydrogen, oxygen, C1-C6 alkylene, C1-C6 alkyl, C1-C6 acyl or C1-C6 alkyleneoxy; R1, R2 and R3 are each independently hydrogen, alkyl, alkenyl or aryl; L4 , or , " ” indicates the connection location.

[0031] According to a preferred embodiment of the present invention, each structural unit in the oligonucleotide is independently subjected to or not subjected to other modifications. "Other modifications" refers to any modification performed in addition to the modification of the oligonucleotide by the compound of formula (I) or a pharmaceutically acceptable salt thereof provided by the present invention. The present invention has no limitation on the specific method of the other modification, and any method that can be used for oligonucleotide modification in the art can be applied to the present invention. Preferably, the other modification includes modification of the linking modification group and / or structural unit skeleton. "Linking modification group" refers to the connection of an additional modification group to the structural unit of the oligonucleotide, and any group that can modify the oligonucleotide by connecting to the structural unit of the oligonucleotide in the art can be applied to the present invention, for example, 2'-OMe modification, 2'-F modification, 5'-(E)-VP modification, etc. "Structural unit skeleton modification" refers to the modification of the skeleton of the structural unit (e.g., nucleotide) of the oligonucleotide to change its structure, or the modification of the phosphate bond formed between two adjacent nucleotides in the oligonucleotide (e.g., replacing the phosphate bond with a phosphorothioate bond, etc.).

[0032] According to a preferred embodiment of the present invention, the type of the oligonucleotide includes any one of the group consisting of small interfering RNA (siRNA), DNA, microRNA (microRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense oligonucleotide (AON) or aptamer (Aptamer), or a combination of at least two thereof; Preferably, the oligonucleotide is of the type of small interfering RNA.

[0033] The term "small interfering RNA" or "siRNA" is well known in the art. It is a class of double-stranded RNA molecules, also referred to in the art as short interfering RNA or silencing RNA. siRNA generally comprises a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a leading strand), wherein each strand is 17 to 30 nucleotides in length, typically 19 to 25 nucleosides in length, wherein the antisense strand is complementary to the target nucleic acid sequence (such as at least 95% complementary, such as fully complementary) (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand, so that the sense strand and the antisense strand form a fully complementary duplex or a partially complementary duplex region. The siRNA strand may form a flat-ended duplex, or preferably, the 3' ends of the sense strand and the antisense strand may each form a 3' overhang, for example, an overhang with 1, 2 or 3 nucleosides. In some embodiments, both the sense strand and the antisense strand have a 3' overhang of 2 nucleotides. Thus, the duplex region may be, for example, 17 to 25 nucleotides in length, such as 19, 20, 21, 22 or 23 nucleotides in length.

[0034] The term "antisense strand" refers to a strand of siRNA that includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not completely complementary to the target sequence, the mismatch may be in the interior or terminal regions of the molecule. Typically, the most tolerated mismatch is in the terminal region, e.g., within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0035] The term "sense strand" as used herein refers to the strand of a siRNA that includes a region that is substantially complementary to a region of the antisense strand (as that term is defined herein).

[0036] The terms "complementary," "fully complementary," and "substantially complementary" can be used with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA and a target sequence, as one skilled in the art would understand from the context of their use.

[0037] According to a preferred embodiment of the present invention, the small interfering nucleotides include a sense strand and an antisense strand.

[0038] Preferably, the length of the small interfering RNA is 15-30 nucleotides (for example, it can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides). More preferably, the nucleotide structural unit at at least one position in the sense strand and / or antisense strand of the small interfering RNA is replaced with the structural unit shown in formula (II).

[0039] In the present invention, the nucleotide comprising the structural unit represented by formula (II) may be on the sense strand or the antisense strand of the siRNA, or both the sense strand and the antisense strand of the siRNA may contain at least one nucleotide comprising the structural unit represented by formula (II).

[0040] Preferably, the nucleotides comprising the structural unit represented by formula (II) are located in the antisense strand of the siRNA, preferably in the seed region at the 5' end.

[0041] More preferably, the nucleotide structural unit at at least one of the 2nd to 8th positions in the 5' region of the antisense strand of the small interfering RNA is replaced with a structural unit represented by formula (II).

[0042] According to a preferred embodiment of the present invention, the oligonucleotide is a double-stranded RNAi agent that reduces the expression of PCSK9. Preferably, the oligonucleotide comprises any one or a combination of at least two selected from the group consisting of the following oligonucleotide duplexes formed by pairing of a sense strand and an antisense strand: (1) The sense strand has a sequence as shown in SEQ ID NO. 5 or a fragment thereof, or a modified sequence thereof; the antisense strand has a sequence as shown in SEQ ID NO. 6 or a fragment thereof, or a modified sequence thereof; (2) The sense strand has a sequence as shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence thereof; the antisense strand has a sequence as shown in SEQ ID NO. 8 or a fragment thereof, or a modified sequence thereof; (3) The sense strand has the sequence shown in SEQ ID NO. 9 or a fragment thereof, or a modified sequence thereof; the antisense strand has the sequence shown in SEQ ID NO. 10 or a fragment thereof, or a modified sequence thereof; (4) The sense strand has a sequence as shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence thereof; the antisense strand has a sequence as shown in SEQ ID NO. 12 or a fragment thereof, or a modified sequence thereof; (5) The sense strand has a sequence as shown in SEQ ID NO. 13 or a fragment thereof, or a modified sequence thereof; the antisense strand has a sequence as shown in SEQ ID NO. 14 or a fragment thereof, or a modified sequence thereof; (6) The sense strand has a sequence as shown in SEQ ID NO. 15 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 16 or a fragment thereof, or a modified sequence thereof.

[0043] The third aspect of the present invention provides a conjugate comprising a ligand and an oligonucleotide sequence, wherein the oligonucleotide sequence comprises a structural unit provided by the compound described in the first aspect or a pharmaceutically acceptable salt thereof.

[0044] "Conjugate" refers to a compound formed by covalent bonding. The conjugate in the present invention refers to a nucleic acid conjugate, which includes at least one oligonucleotide molecule and one other compound molecule (such as the "ligand" in the present invention). In the conjugate, the role of the oligonucleotide molecule is to exert its biological function (for example, when siRNA is selected, its function is an RNAi agent), and the role of the ligand includes providing targeting (enabling the conjugate to target tissues / cells), protecting the oligonucleotide, etc. (Nucleic acid) conjugates are widely used in biological and clinical medical research (such as detection, diagnosis, treatment, etc. as biomarkers), drug development, bioengineering technology (such as biocatalysis, biosensors, biomaterials, etc.) and other fields.

[0045] According to a preferred embodiment of the present invention, the oligonucleotide is a double-stranded RNAi agent, which comprises any one or a combination of at least two selected from the group consisting of the following oligonucleotide duplexes formed by pairing of a sense strand and an antisense strand: (i) the sense strand has the sequence shown in SEQ ID NO. 31; the antisense strand has the sequence shown in SEQ ID NO. 8; (ii) the sense strand has the sequence shown in SEQ ID NO. 32; the antisense strand has the sequence shown in SEQ ID NO. 10; (iii) the sense strand has the sequence shown in SEQ ID NO. 33; the antisense strand has the sequence shown in SEQ ID NO. 12; (iv) The sense strand has the sequence shown in SEQ ID NO. 34; and the antisense strand has the sequence shown in SEQ ID NO. 14.

[0046] According to a preferred embodiment of the present invention, the ligand is G4, G5, G6 or G7 having the following structure: , , , .

[0047] The fourth aspect of the present invention provides a conjugate comprising a ligand and the oligonucleotide or a pharmaceutically acceptable salt thereof according to the second aspect.

[0048] According to a preferred embodiment of the present invention, the oligonucleotide may be the double-stranded RNAi agent described in the third aspect above, and its specific features and preferred features are as described above and will not be repeated here.

[0049] The fifth aspect of the present invention provides a pharmaceutical composition, which includes the oligonucleotide or a pharmaceutically acceptable salt thereof as described in the second aspect, or the conjugate as described in the third aspect or the fourth aspect.

[0050] It should be understood that in the pharmaceutical composition provided by the present invention, the oligonucleotide provided by the present invention or its pharmaceutically acceptable salt, and the conjugate provided by the present invention exist as a pharmaceutically active component or one of the pharmaceutically active components. On this basis, the pharmaceutical composition provided by the present invention may also include other ingredients required for preparing drugs in the art, and may further include other active ingredients. For example, preferably, the pharmaceutical composition provided by the present invention may also include pharmaceutically acceptable carriers, adjuvants, auxiliary materials, etc., and may also include other active ingredients or active ingredient adjuvants. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which participates in carrying or transporting the nucleoside analogs, oligonucleotides, conjugates or their compositions of the present invention in or to the patient's body so that it can perform its intended function. Typically, such a construct is carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable", i.e., compatible with the other ingredients of the formulation, including the nucleoside analogs, oligonucleotides, conjugates, or combinations thereof of the invention, and not harmful or substantially harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic, compatible substances used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents and absorption delaying agents, etc., which are compatible with the activity of the nucleoside analogs, oligonucleotides, conjugates or their compositions of the present invention and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the composition. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of compounds that can be used in the present invention. Other additional ingredients that can be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described in, for example, Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.Any carrier, adjuvant, and auxiliary material that can be used for pharmaceutical preparation in the art can be applied to the present invention. For example, auxiliary materials can include but are not limited to binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, chelating agents, colorants, flavoring agents, coating agents, buffers, protective agents, preservatives, solubilizers, pH adjusters, etc.

[0051] Oligonucleotides, especially siRNA, comprising nucleotides formed by nucleoside analogs provided by the present invention as structural units have better specificity than siRNA and also greatly reduce off-target effects.

[0052] Based on this, the sixth aspect of the present invention provides the use of the compound described in the first aspect and its pharmaceutically acceptable salt in reducing the off-target effect of oligonucleotides.

[0053] Preferably, the oligonucleotide is siRNA.

[0054] The present invention further provides a method for reducing off-target effects of siRNA, which comprises synthesizing siRNA using the nucleoside analog provided by the present invention (ie, the compound provided in the first aspect or a pharmaceutically acceptable salt thereof).

[0055] The seventh aspect of the present invention provides use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof, or the oligonucleotide described in the second aspect or a pharmaceutically acceptable salt thereof, or the conjugate described in the third aspect in the preparation of an oligonucleotide drug.

[0056] According to a preferred embodiment of the present invention, the oligonucleotide drug is a drug for treating diseases related to PCSK9.

[0057] Preferably, the PCSK9-related disease is selected from any one of hypercholesterolemia, atherosclerosis, dyslipidemia and cardiovascular and cerebrovascular diseases, or a combination of at least two thereof.

[0058] Preferably, the oligonucleotide drug is a drug comprising a double-stranded RNAi agent. The characteristics and preferred characteristics of the double-stranded RNAi agent are as described above and will not be repeated here.

[0059] Through the above technical scheme, the present invention designs a series of novel anti-off-target nucleoside compounds, and the siRNA prepared from the anti-off-target nucleoside compounds of the present invention can achieve high targeting activity and significant anti-off-target effect.

[0060] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The structure is completely different from the existing anti-off-target nucleoside compounds.

[0061] The anti-off-target nucleoside compound designed by the present invention introduces an amide or triazole structure in the connecting arm, which is completely different from the commonly used structure in the prior art and is a brand-new anti-off-target nucleoside compound. The compound can be coupled to the seed region of siRNA through solid phase synthesis, thereby efficiently improving the specificity of siRNA, reducing the off-target effect of siRNA while maintaining the silencing effect of the target gene.

[0062] 2. Compared with the prior art anti-off-target nucleoside compounds, the siRNA molecules modified by the anti-off-target nucleoside compounds of the present invention have higher targeting activity.

[0063] For example, the 6th position of D84-DV27P is modified, and D84-DV27P-GA1-6 (modified with the compound YK-GNA-001 of the present invention) has an IC of PCSK9. 50 It is lower than the parent sequence, only 35.4% of D84-DV27P-GNA-6 (modified with the existing compound GNA) and 33.2% of D84-DV27P-G1-2-6 (modified with the existing compound 1-2).

[0064] The inhibition rate of PCSK9 protein expression in mouse serum by D84-DV27PG5-GA1-6 (modified with the compound YK-GNA-001 of the present invention) on days 7 and 14 was comparable to that of the parent sequence, and was 25.4% and 21.6% higher than that of D84-DV27PG5-G1-2-6 (modified with the existing compound 1-2), respectively.

[0065] D84-DV27P-GA1-6 (modified with the compound YK-GNA-001 of the present invention) reduced LDL-C in mouse serum by 38.1% and 40.4% on the 7th and 14th days, respectively, which was higher than the parent sequence, and the reduction levels were 9.3% and 10.2% higher than D81-DV25G5-GNA-6 (modified with the existing compound GNA), respectively.

[0066] 3. Compared with the prior art anti-off-target nucleoside compounds, the siRNA molecules modified by the anti-off-target nucleoside compounds of the present invention have better anti-off-target effects.

[0067] For example, compared with the parent sequence D84-DV27P without anti-off-target design, D84-DV27P-GA1-6 (modified with the compound YK-GNA-001 of the present invention) can reduce the inhibition rate of off-target gene ITGAV by 77%, and is 76% lower than D84-DV27P-G1-2-6 (modified with the existing compound 1-2). BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the following briefly introduces the drawings required for use in the specific implementation or the prior art description. Obviously, the drawings described below are some implementations of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0069] Figure 1 It is the on-target activity of siRNA molecules D84-DV27P, D84-DV27P-GNA-6, D84-DV27P-GNA-7, D84-DV27P-GA1-6, D84-DV27P-GT6-6, D84-DV27P-GT3-6, D84-DV27P-G1-2-6, D84-DV27P-G1-2-7, D81-DV25, D81-DV25-GNA-6, D81-DV25-GNA-7, D81-DV25-GA2-6, D81-DV25-GT4-6 and D81-DV25-GT5-7 on the PCSK9 gene in HepG2 cells, that is, the IC50 for PCSK9 mRNA.

[0070] Figure 2 This is the inhibition rate of siRNA molecules D84-DV27P, D84-DV27P-GNA-6, D84-DV27P-GA1-6, D84-DV27P-GT6-6, D84-DV27P-GT3-6 and D84-DV27P-G1-2-6 on ITGAV off-target genes.

[0071] Figure 3 The inhibition rate of PCSK9 protein expression in mouse serum by siRNA molecules D84-DV27PG5, D84-DV27PG5-GA1-6, D84-DV27PG5-GT3-6, D84-DV27PG5-G1-2-6, D81-DV25G5, D81-DV25G5-GNA-6 and D81-DV25G5-GT4-6 on the 7th and 14th days after administration.

[0072] Figure 4 On the 7th and 14th days after administration, siRNA molecules D84-DV27PG5, D84-DV27PG5-GA1-6, D84-DV27PG5-GT3-6, D84-DV27PG5-G1-2-6, D81-DV25G5, D81-DV25G5-GNA-6 and D81-DV25G5-GT4-6 reduced the level of LDL-C in mouse serum.

[0073] Figure 5This is a schematic diagram of the structure of the conjugate prepared in Example 3. DETAILED DESCRIPTION

[0074] In order to make the present invention more easily understood, some terms are first defined. In addition, it should be noted that whenever a value or a parameter range is listed, the purpose is to indicate that the intermediate values ​​and ranges of these cited values ​​are also within the scope of the present invention.

[0075] The embodiments of the present invention are intended to further illustrate the invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0076] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0077] All publications and patents mentioned in the present invention are hereby incorporated into the present invention by reference in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in the present invention, then the purposes and terms of the present invention shall prevail.

[0078] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0079] Unless otherwise specified, all technical and scientific terms used in the present invention have the common meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition in the present invention shall prevail.

[0080] Except in the working examples or otherwise indicated, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited in the present invention is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange.

[0081] The words "include", "contain" or "comprises" and the like used in the present invention mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes" used in the present invention may be open, semi-closed or closed. In other words, the term also includes "essentially consisting of" or "consisting of".

[0082] The oligonucleotides in the present invention include single-stranded oligonucleotides (such as antisense oligonucleotides, referred to as ASOs) and double-stranded oligonucleotides (such as oligonucleotide duplexes consisting of two oligonucleotide chains with complementary base pairing, such as small interfering nucleotides, etc.).

[0083] In one embodiment, the oligonucleotide is selected from small interfering nucleotides (siRNA), DNA, micro RNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotides or aptamers, preferably the oligonucleotide is an antisense oligonucleotide or a small interfering nucleotide.

[0084] The oligonucleotides in the present invention include natural oligonucleotides and chemically modified oligonucleotides. Chemical modifications herein include nucleoside modifications (including ribose moiety modifications and nucleobase modifications) and phosphate backbone modifications. Chemical modifications of oligonucleotides do not include situations where there are differences only in the nucleobase sequence. Natural herein refers to situations corresponding to naturally occurring RNA or DNA.

[0085] In the oligonucleotide sequence of the present invention, the abbreviations of each base and modification refer to the conventional definition in the art. In the art, "G", "C", "A", "T" and "U" usually represent the bases of guanine, cytosine, adenine, thymine, and uracil, respectively, but it is also generally known in the art that "G", "C", "A", "T" and "U" each usually also represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively, which is a common way to represent deoxyribonucleic acid sequences and / or ribonucleic acid sequences. Therefore, in the context of the present invention, the meaning of "G", "C", "A", "T", "U" represents includes the above-mentioned various possible situations. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide (as further described below) or an alternative replacement part. Those skilled in the art can appreciate that guanine, cytosine, adenine and uracil can be replaced by other parts without substantially changing the base pairing characteristics of an oligonucleotide (including a nucleotide with such a replacement part). For example, without limitation, a nucleotide comprising inosine as its base can be base paired with a nucleotide comprising adenine, cytosine or uracil. Therefore, a nucleotide containing uracil, guanine or adenine can be replaced by a nucleotide containing, for example, inosine in the nucleotide sequence of the dsRNA characterized by the present invention. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a GU wobble base pairing with the target mRNA. Sequences containing such replacement parts are suitable for the compositions and methods characterized by the present invention.

[0086] Unless otherwise specified, in the oligonucleotide sequence of the present invention, A, U, C, and G each generally represent a nucleotide containing adenine, uracil, cytosine, and guanine as bases, respectively; m represents that the nucleotide adjacent to its left is a 2'-OMe-modified nucleotide (such as Am represents a 2'-OMe-modified adenine nucleotide); f represents that the nucleotide adjacent to its left is a 2'-F-modified nucleotide (such as Af represents a 2'-F-modified adenine nucleotide); s represents that the two adjacent nucleotides on its left and right are connected by a thiophosphate bond (such as Us-A represents that the uracil nucleotide and the adenine nucleotide are connected by a thiophosphate bond); EVP represents that the nucleotide adjacent to its left is a 5'-(E)-VP-modified nucleotide (such as UEVP represents a 5'-(E)-VP-modified uracil nucleotide).

[0087] Example The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the materials, reagents, etc. used, if not otherwise specified, can be obtained through commercial channels. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0088] Example 1: Synthesis of anti-off-target nucleoside phosphoramidite compounds The following abbreviations represent the following reagents: DCM: dichloromethane; PE: petroleum ether; EA: ethyl acetate; THF: tetrahydrofuran; DMF: N, N-dimethylformamide; MeCN: acetonitrile; HBTU: O-benzotriazole-tetramethyluronium hexafluorophosphate; DIPEA: N, N-Diisopropylethylamine; NaHCO3: sodium bicarbonate; NaCl: sodium chloride; Na2SO4: sodium sulfate; Pd / C: palladium on carbon; N2: nitrogen; PPh3: triphenylphosphine; DIAD: diisopropyl azodicarboxylate; HOAc: glacial acetic acid; H2O: water; DMSO: dimethyl sulfoxide; Py: pyridine; DMTrCl: 4,4'-bismethoxytrityl chloride; TEA: triethylamine; MeOH: methanol; Heptane: n-heptane; MTBE: methyl tert-butyl ether; 1,4-dioxane: 1,4-dioxane; NaOH: sodium hydroxide; NH4HCO3: ammonium bicarbonate; TBAI: tetrabutylammonium iodide; SiO2: silicon dioxide; CuSO4: copper sulfate; sodium VC: sodium ascorbate; MgCl2: magnesium chloride; THPTA: tris(3-hydroxypropyltriazolylmethyl)amine; HCl: hydrochloric acid.

[0089] 1. Synthesis of YK-GNA-001 The synthetic route is as follows:

[0090] Step 1: Synthesis of 1-3 Add 1-2 (103.80 g, 0.58 mol, 1.20 eq.) to the reaction bottle, co-rotate with ultra-dry acetonitrile twice, add ultra-dry DCM (630 mL), and stir to dissolve. Control the temperature at 0-10℃, add HBTU (218.61 g, 0.58 mol, 1.20eq.) and DIPEA (93.11 g, 0.72 mol, 1.50 eq.), and stir for about 30 minutes. Add 1-1 (63.04 g, 0.48mol, 1.00 eq.), warm to room temperature, and stir for 15 hours. The reaction solution is diluted with DCM (700 mL), washed with saturated NaHCO3 solution (1.0 L × 3) and saturated NaCl solution (1.0 L × 2) in sequence, dried over anhydrous Na2SO4, and the solvent is removed to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 4 / 1 to 1 / 1) to obtain 80.00 g of an oily substance with a yield of 56.8%. 1 H NMR(400 MHz, CDCl3) δ 7.35 – 7.26 (m, 5 H), 6.61 (s, 1 H), 4.51 (d, J = 1.1 Hz, 2 H), 3.98 (dd, J = 8.4, 6.4 Hz, 1 H), 3.75 – 3.45 (m, 5 H), 3.28 (dt, J =14.0, 5.9 Hz, 1 H), 2.49 (dd, J = 6.2, 5.4 Hz, 2 H), 1.35 (s, 3 H), 1.30 (d,J = 0.7 Hz, 3 H). MS[M+H] + : 294.2.

[0091] Step 2: Synthesis of 1-4 Add 1-3 (35.00 g, 0.12 mol, 1.00 eq.) and methanol (350 mL) to the reaction flask and stir to dissolve. Add Pd / C (8.00 g, 10%), replace with N2 three times, introduce hydrogen into the reaction system, and stir at room temperature for 15 hours. Filter out Pd / C, wash the filter cake with methanol (50 mL × 2), and remove the solvent to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 4 / 1 to 0 / 1) to obtain 18.50 g of brown oil with a yield of 75.8%. 1 H NMR (400 MHz, CDCl3) δ 6.23 (s, 1H), 4.23 (qd, J = 6.3, 3.4 Hz, 1 H), 4.04 (dd, J = 8.5, 6.5 Hz, 1 H), 3.87 (dd, J = 5.9, 5.0 Hz, 2 H), 3.78 – 3.69 (m, 1 H), 3.64 (dd, J = 8.5, 6.2 Hz, 1 H), 3.53 (ddd, J = 14.0, 6.0, 3.5 Hz, 1 H), 3.33 (dt, J = 14.0, 6.0 Hz, 1H), 2.45 (dd, J = 5.9, 5.0 Hz, 2 H), 1.43 (d, J =0.7 Hz, 3 H), 1.33 (d, J =0.8 Hz, 3 H). MS[M+H] + : 204.2.

[0092] Step 3: Synthesis of 1-6 Add PPh3 (19.41 g, 74.05 mmol, 1.00 eq.) and 1-5 (29.00 g, 121.38 mmol, 1.64 eq.) to a reaction flask and co-rotate with ultra-dry acetonitrile (100 mL) twice. Transfer to a 1 L three-necked flask and add ultra-dry THF (600 mL) and 1-4 (15.00 g, 73.80 mmol, 1.00 eq.). Control the temperature at 0-10°C, add DIAD (22.40 g, 115.73 mmol, 1.57 eq. diluted with 100 mL ultra-dry THF), warm to room temperature after the addition, and stir for 15 hours. Concentrate to remove the solvent, add acetonitrile (300 mL), stir for 20 minutes, filter, and concentrate the filtrate to dryness to obtain the residue. The residue was purified by column chromatography (SiO2, PE / EA = 4 / 1~0 / 1) to obtain 18.20 g of the product with a yield of 58.1%. 1 H NMR (400 MHz, DMSO-d 6) δ8.60 (s, 1 H), 8.51 (s, 1 H), 8.14 (d, J = 6.9 Hz, 2 H), 8.07 (t, J = 5.9 Hz, 1 H), 7.62 (t, J = 7.3 Hz, 1 H), 7.53 (t, J = 7.6 Hz, 2 H), 4.61 (s, 2 H), 3.88 (t, J = 6.0 Hz, 1 H), 3.78 (dd, J = 8.3, 6.3 Hz, 1 H), 3.43 – 3.36 (m, 1H), 3.31 (s, 1 H), 3.07 (q, J = 5.6 Hz, 2 H), 2.79 (s, 2 H), 1.23 (s, 3 H), 1.20 (s, 3 H). MS [M+H] + : 425.2.

[0093] Step 4: Synthesis of 1-7 Add 1-6 (12.60 g, 29.7 mmol, 1.00 eq.) and 80% HOAc (315 mL) to the reaction flask. Heat to 50°C and stir for 4 hours. Concentrate the reaction solution to dryness, add acetonitrile (50 mL) and stir for 1 hour. Solid precipitates and filter. Add the filter cake to a mixed solution of ethanol (210 mL), DMF (80 mL) and water (54 mL), heat to 70-75°C, stir and dissolve. Cool to 30°C within 1 hour, continue stirring for about 30 minutes, solid precipitates and filter. Add the filter cake to a mixed solution of ethanol (190 mL), DMF (50 mL) and water (54 mL) again, heat to 70-75°C, stir and dissolve. Cool to 30°C within 1 hour, continue stirring for about 1 hour, solid precipitates and filter to obtain 4.22 g of white solid with a yield of 36.8%. 1 H NMR (400 MHz, DMSO- d6) δ 11.14 (s, 1 H), 8.74 (s, 1 H), 8.37 (s, 1 H), 8.08–8.02(m, 2 H), 7.98 (t, J = 5.7 Hz, 1 H), 7.69–7.60 (m, 1 H), 7.60–7.51 (m, 2 H), 4.73 (d, J = 4.9 Hz, 1 H), 4.55–4.44 (m, 3 H), 3.47–3.39 (m, 1 H), 3.30–3.24 (m, 1 H), 3.24–3.14 (m, 2 H), 2.93 (ddd, J = 13.5, 7.0, 5.5 Hz, 1 H), 2.78(t, J = 6.8 Hz, 2 H). MS [M +H] + : 385.0.

[0094] Step 5: Synthesis of 1-8 Add 1-7 (6.30 g, 16.40 mmol, 1.00 eq.) to the reaction flask and co-rotate with ultra-dried Py (30 mL) twice. Add ultra-dried Py (126 mL) and stir to dissolve. Add DMTrCl (7.20 g, 21.31 mmol, 1.30 eq.) under ice bath conditions, warm to room temperature, and stir to react for 15 hours. The reaction solution was quenched with water (40 mL), extracted with ethyl acetate, TEA (10 mL) was added, and concentrated to dryness to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 4 / 1-0 / 1, DCM / MeOH= 20 / 1) to obtain 5.81 g of white solid, with a yield of 51.5%. 1 H NMR (400 MHz, DMSO- d6) δ 10.08 (s, 1 H), 8.63 (s, 1 H), 8.52 (s, 1 H), 8.15 (d, J = 7.5 Hz, 2 H), 7.92 (t, J = 5.4 Hz, 1 H), 7.59 (t, J = 7.3 Hz, 1 H), 7.51 (t, J = 7.4 Hz, 2 H), 7.41–7.32 (m, 2 H), 7.32–7.11 (m, 7 H), 6.85 (dd, J = 8.9, 1.6 Hz, 4 H), 4.93 (d, J = 5.3 Hz, 1 H), 4.62 (s, 2 H), 3.71 (d, J = 1.3 Hz, 6 H), 3.59 (s, 1 H), 3.26 (s, 1 H), 2.87 (dd, J = 9.3, 5.3 Hz, 2 H), 2.78 (dd, J = 9.2, 5.9 Hz, 3 H). MS [M+H] + : 687.2。

[0095] Step 6: Synthesis of YK-GNA-001 1-8 (4.80 g, 7.00 mmol, 1.00 eq.) was added to the reaction flask, and it was first rotary evaporated with ultra-dry acetonitrile (50 mL) once, and then rotary evaporated with ultra-dry DCM (50 mL) twice. Ultra-dry DCM (50 mL) was added and stirred to dissolve. Tetrazolyl (0.41 g, 5.85 mmol, 0.80 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.84 g, 9.42 mmol, 1.30 eq.) were added under ice bath conditions, and the mixture was warmed to room temperature and stirred for 2 hours. The reaction solution was diluted with DCM (50 mL) under ice bath conditions, washed with saturated NaHCO3 solution (80 mL) and saturated NaCl solution (100 mL × 2), dried over anhydrous Na2SO4, and the solvent was removed to obtain a residue. The residue was purified by column chromatography (SiO2, 0.5% TEA alkalization, n-heptane / EA = 4 / 1-0 / 1, EA / MeCN = 25 / 1, 0.1% TEA alkalization) to obtain 3.02 g of white solid. The solid was added to MTBE / DCM mixed solution (10 / 1, 10 mL), stirred to dissolve, and n-heptane (50 mL) was added dropwise. Stir for about 10 minutes, and solid precipitated and filtered. Repeat this operation once for the filter cake. The filter cake was rotary evaporated with acetonitrile (20 mL) twice to obtain 2.50 g of white solid. Dissolve 2.50 g of white solid in DCM (30 mL) and purify by column chromatography (SiO2, eluent: 30 mL DCM, 30 mL DCM / MeCN = 1 / 1, 30 mL MeCN). Collect the DCM phase and remove the solvent to obtain a residue. The residue was rotary evaporated with acetonitrile (20 mL) twice to obtain 1.50 g of white solid with a yield of 36.8%. 1 H NMR (400 MHz, CDCl3) δ 14.67 (s, 1 H), 8.31 (s, 1 H), 8.24–8.10 (m, 3 H), 7.53–7.32 (m, 5 H), 7.28–7.20 (m, 6 H), 7.16–7.08 (m, 1 H), 6.81–6.70(m, 4 H), 6.42 - 6.14 (d, 1 H), 4.85 (s, 2 H), 3.92 (dd, J = 9.7, 4.4 Hz, 1 H),3.71 (d, J= 1.7 Hz, 6 H), 3.69–3.52 (m, 3 H), 3.52–3.33 (m, 3 H), 3.28–3.07(m, 2 H), 3.01–2.81 (m, 2 H), 2.39 (td, J = 6.1, 2.0 Hz, 2 H), 1.14–0.97 (m,12 H). 31 P NMR (162 MHz, CDCl3) δ149.71, 149.12 ppm. MS[M-H] - : 885.6.

[0096] 2. Synthesis of YK-GNA-002 The synthetic route is as follows:

[0097] Step 1: Synthesis of 2-2 Using 1-4 (27.00 g, 132.80 mmol, 1.00 eq.) and 2-1 (31.45 g, 146.10 mmol, 1.10 eq.) as raw materials, according to the method for synthesizing compound 1-6, 9.04 g of white solid was obtained with a yield of 16.9%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.13 (s, 1 H), 8.12 (t, J = 5.9 Hz, 1 H), 8.00 (d, J =7.2 Hz, 1 H), 7.97 – 7.92 (m, 2 H), 7.62 – 7.53 (m, 1 H), 7.51 – 7.40 (m, 2H), 7.21 (d, J = 7.0 Hz, 1 H), 3.96 (dt, J = 10.4, 6.1 Hz, 3 H), 3.85 (dd, J = 8.3, 6.2 Hz, 1 H), 3.48 (dd, J = 8.3, 6.1 Hz, 1 H), 3.11 (td, J = 5.9, 2.9Hz, 2H), 2.54 (t, J = 6.5 Hz, 2 H), 1.25 (s, 3 H), 1.17 (s, 3 H). MS[M+H] + :401.2.

[0098] Step 2: Synthesis of 2-3 Using 2-2 (9.00 g, 22.50 mmol) as raw material, according to the method for synthesizing compound 1-7, 6.98 g of white solid was obtained with a yield of 86.4%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.17 (s, 1 H), 8.06 (d, J = 7.3Hz, 1 H), 8.02–7.96 (m, 3 H), 7.66–7.58 (m, 1 H), 7.51 (dd, J = 8.4, 7.0 Hz,2 H), 7.24 (d, J = 7.2 Hz, 1 H), 4.73 (s, 1 H), 4.51 (s, 1 H), 4.01 (t, J =6.6 Hz, 2 H), 3.45 (t, J = 5.9 Hz, 1 H), 3.25–3.16 (m, 3 H), 2.95 (ddd, J =13.0, 6.9, 5.5 Hz, 1 H), 2.58 (t, J = 6.6 Hz, 2 H). MS [M +H] + : 361.0.

[0099] Step 3: Synthesis of 2-4 Using 2-3 (7.00 g, 19.40 mmol, 1.00 eq.) and DMTrCl (7.24 g, 21.40 mmol, 1.10 eq.) as raw materials, according to the method for synthesizing compound 1-8, 10.04 g of white solid was obtained with a yield of 77.8%. 1 H NMR (400 MHz, DMSO- d6) δ 11.14 (s, 1 H), 8.01 (d, J = 7.3 Hz, 1 H), 7.99–7.92 (m,2 H), 7.88 (t, J = 5.6 Hz, 1 H), 7.62–7.54 (m, 1 H), 7.51–7.42 (m, 2 H),7.42–7.31 (m, 2 H), 7.27 (d, J = 7.4 Hz, 2 H), 7.25–7.12 (m, 6 H), 6.88–6.80(m, 4 H), 4.93 (d, J = 5.3 Hz, 1 H), 3.94 (t, J = 6.7 Hz, 2 H), 3.69 (s, 6H), 3.66–3.59 (m, 1 H), 3.24 (d, J = 5.4 Hz, 1 H), 2.98–2.83 (m, 2 H), 2.79 (dd, J = 9.2, 5.7 Hz, 1 H), 2.51 (d, J = 6.7 Hz, 2 H). MS[M+Na] + :685.2.

[0100] Step 4: Synthesis of YK-GNA-002 Using 2-4 (5.00 g, 7.54 mmol, 1.00 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.96 g, 9.82 mmol, 1.30 eq.) as raw materials, according to the method for synthesizing YK-GNA-001, 2.03 g of white solid was obtained with a yield of 30.8%. 1H NMR (400 MHz, CDCl3) δ 8.86 (s, 1 H), 7.91–7.83 (m, 3 H), 7.60–7.51 (m, 1 H), 7.49–7.36 (m, 5 H), 7.29–7.21 (m, 6 H), 7.20–7.12 (m, 1H), 6.83–6.73 (m, 4 H), 6.43 - 6.07 (dd, J = 6.4, 3.7 Hz, 1 H), 4.18–4.05 (m,2 H), 3.97 (dt, J = 10.5, 5.0 Hz, 1 H), 3.85 (ddt, J = 10.4, 7.4, 6.2 Hz, 1H), 3.74 (d, J = 2.9 Hz, 6 H), 3.73–3.35 (m, 5 H), 3.26–3.17 (m, 1 H), 3.08 (ddd, J = 51.5, 9.7, 5.4 Hz, 1 H), 2.80–2.55 (m, 3 H), 2.41 (t, J = 6.3 Hz, 1H), 1.18–1.01 (m, 12H). 31 P NMR (400 Hz, CDCl3) δ149.81, 149.23 ppm. MS[M-H] - : 861.6.

[0101] 3. Synthesis of YK-GNA-003 The synthetic route is as follows:

[0102] Step 1: Synthesis of 3-3 Add 3-1 (50.00 g, 378.33 mmol, 1.00 eq.) and DCM (1.3 L) to the reaction flask and stir to dissolve. Add TBAI (4.20 g, 11.37 mmol, 0.03 eq.) and 40% NaOH solution (1.2 L). Stir for half an hour at room temperature, add 3-2 (47.3 g, 397.61 mmol, 1.05 eq). Stir overnight at room temperature. Add water (1.5 L) to dilute the reaction solution, separate the liquids, extract the aqueous phase with DCM (1.5 L), combine the organic phases and wash them with water twice, dry them over anhydrous Na2SO4, and concentrate to remove the solvent to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 20 / 1~5 / 1) to obtain 38.08 g of a colorless oil with a yield of 59.0%. 1 H NMR (400 MHz, DMSO- d 6) δ 4.20 – 4.08 (m, 3 H), 3.98 –3.90 (m, 1 H), 3.64 – 3.55 (m, 1 H), 3.45 – 3.38 (m, 3 H), 1.27 (d, J = 0.7Hz, 3 H), 1.22 (d, J = 0.7 Hz, 3 H). MS[M+H] + :171.2.

[0103] Step 2: Synthesis of 3-5 Using 1-5 (20.00 g, 83.60 mmol, 1.10 eq) and 3-4 (6.50 g, 74.64 mmol, 1.00 eq) as raw materials, according to the method for synthesizing compound 1-6, 4.97 g of white solid was obtained with a yield of 21.7%. MS [M + H] + :309.2.

[0104] Step 3: Synthesis of 3-6 Add a mixed solution of 3-5 (5.00 g, 16.22 mmol, 1.00 eq.) and THF / H2O (V / V, 1 / 1, 40.0 mL) to a reaction flask and stir to dissolve. Add 3-3 (2.90 g, 17.04 mmol, 1.05 eq.). Add anhydrous CuSO4 (0.40 g, 2.51 mmol, 0.15 eq) and sodium VC (1.61 g, 8.13 mmol, 0.5 eq) in turn under ice bath conditions. Warm to room temperature and stir for 3 hours. Add methanol (50 mL) to the reaction solution and concentrate the reaction solution to obtain a residue. Purify the residue by column chromatography (SiO2, PE / EA = 1 / 1 – 1 / 7) to obtain a green solid. The solid was dissolved in methanol and rotary evaporated with acetonitrile once. Solid precipitated and was filtered off with suction. The filtrate was concentrated to obtain 6.02 g of white solid with a yield of 73.3%. 1 H NMR (400 MHz, CDCl3) δ 15.42 (s,1 H), 8.34 (s, 1 H), 8.18 (d, J = 7.0 Hz, 2 H), 7.85 – 7.64 (m, 1 H), 7.46(s, 3 H), 7.26 (d, J = 6.1 Hz, 1 H), 5.23 (s, 2 H), 5.03 (s, 2 H), 4.59 (d, J= 6.0 Hz, 2 H), 4.21 (qd, J = 6.2, 3.6 Hz, 1 H), 4.00 (dd, J = 8.3, 6.5 Hz, 1H), 3.71 – 3.57 (m, 1 H), 3.48 (dd, J = 7.7, 5.5 Hz, 2 H), 1.36 (s, 3 H), 1.30 (s, 3 H). MS[M+H] + :479.2.

[0105] Step 4: Synthesis of 3-7 Using 3-6 (6.00 g, 12.54 mmol, 1.00 eq.) as the raw material, according to the synthesis method of compound 1-7, 2.80 g of white solid was obtained with a yield of 50.9%. MS [M + H] + :439.2.

[0106] Step 5: Synthesis of 3-8 Using 3-7 (2.80 g, 6.39 mmol, 1.00 eq.) and DMTrCl (2.60 g, 7.67 mmol, 1.20eq) as raw materials, according to the method for synthesizing compound 1-8, 3.51 g of white solid was obtained with a yield of 74.0%. 1 H NMR (400 MHz, CDCl3) δ 8.31 (s, 1 H), 8.22 – 8.16 (m, 1 H), 7.70 (s, 1 H), 7.59 – 7.43 (m, 3 H), 7.42 – 7.35 (m, 2 H), 7.31 – 7.20 (m, 7 H), 7.20 – 7.13 (m, 1 H), 7.12 (s, 1 H), 6.78 (d, J = 9.0 Hz, 4 H), 5.24 – 5.10 (m, 1 H), 5.02 – 4.88 (m, 2H), 4.62 – 4.49 (m, 2 H), 3.97 – 3.86 (m, 1 H), 3.75 (s, 6 H), 3.66 – 3.48 (m, 3 H), 3.23 –3.07 (m, 2 H). MS[M-H] - :739.2.

[0107] Step 6: Synthesis of YK-GNA-003 Using 3-8 (3.50 g, 4.72 mmol, 1.00 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.71 g, 5.67 mmol, 1.2 eq) as raw materials, according to the method for synthesizing YK-GNA-001, 1.69 g of white solid was obtained with a yield of 38.3%. 1H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 3.7 Hz, 1 H), 8.17 (d, J = 7.8Hz, 2 H), 7.73 (d, J = 11.7 Hz, 1 H), 7.52 – 7.47 (m, 1 H), 7.45 – 7.34 (m, 5H), 7.28 – 7.18 (m, 7 H), 7.16 – 7.10 (m, 1 H), 6.75 (d, J = 8.7 Hz, 4 H), 5.15 – 5.11 (m, 2 H), 4.99 – 4.94 (m, 2 H), 4.66 – 4.55 (m, 1 H), 4.54 (s, 1H), 4.16 – 4.05 (m, 1 H), 3.80 – 3.44 (m, 12 H), 3.22 – 3.02 (m, 2 H), 2.54(t, J = 6.4 Hz, 1 H), 2.43 – 2.39 (m, 1 H), 1.12 (d, J = 6.8 Hz, 3 H), 1.09 (dd, J = 6.8, 2.0 Hz, 6 H), 0.97 (d, J = 6.8 Hz, 3 H). 31 P NMR (400 Hz, CDCl3) δ150.10, 149.51 ppm. MS[M-H] - :940.6.

[0108] 4. Synthesis of YK-GNA-004 The synthetic route is as follows:

[0109] Step 1: Synthesis of 4-1 Add 2-1 (53.40 g, 248.13 mmol, 1.20 eq) and Ph3P (65.00 g, 247.85 mmol, 1.20 eq) to a reaction flask and co-evaporate with ultra-dry acetonitrile twice. Add ultra-dry DMF (1.5 L) and 3-4 (18.00 g, 206.71 mmol, 1.00 eq) and stir for 10 minutes. Cool to 0-10°C and drop DIAD (62.66 g, 310.04 mmol, 1.50 eq. Dilute with 270 mL ultra-dry THF). After dropping, warm to room temperature and stir for 15 hours. Water (500 mL) and ethyl acetate (500 mL) were added to the reaction solution, stirred for 10 minutes, filtered through diatomaceous earth, the filtrate was separated, the organic phase was washed with water (1 L) and saturated NaCl solution (1 L) in sequence, dried over anhydrous Na2SO4, and concentrated to remove the solvent to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 4 / 1 – 1 / 4) to obtain 2.60 g of a white solid with a yield of 4.4%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.19 (s, 1H), 8.11 (d, J = 7.3 Hz, 1 H), 7.97 (d, J = 7.1 Hz, 2 H), 7.58 (d, J = 7.4Hz, 1 H), 7.48 (d, J = 8.0 Hz, 2 H), 7.29 (d, J = 7.3 Hz, 1 H), 3.98 (t, J =5.7 Hz, 2 H), 3.66 (dd, J = 6.4, 5.0 Hz, 2 H). MS[M+H] + :285.0.

[0110] Step 2: Synthesis of 4-2 Using 4-1 (2.60 g, 9.15 mmol, 1.00 eq.) and 3-3 (1.65 g, 9.69 mmol, 1.05 eq.) as raw materials, according to the method for synthesizing compound 3-6, 4.04 g of white solid was obtained with a yield of 96.2%. 1 H NMR (400 MHz, DMSO- d6) δ 11.22 (s, 1 H), 8.12 (s, 1 H), 8.02 – 7.95 (m, 2 H), 7.64 – 7.60 (m, 2 H), 7.57 – 7.48 (m, 3 H), 4.76 (t, J = 5.6 Hz, 2 H), 4.54 (s, 2 H),4.30 (t, J = 5.7 Hz, 2 H), 4.19 – 4.13 (m, 1 H), 3.99 – 3.92 (m, 1 H), 3.60 –3.54 (m, 1 H), 3.48 – 3.41 (m, 2 H), 1.29 (s, 3 H), 1.23 (s, 3 H). MS[M+H] + :455.5.

[0111] Step 3: Synthesis of 4-3 Using 4-2 (4.00 g, 8.80 mmol) as raw material, according to the method for synthesizing compound 1-7, 2.01 g of white solid was obtained with a yield of 54.8%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.17 (s, 1 H), 8.08 (s, 1 H), 7.98 –7.88 (m, 2 H), 7.65 (d, J = 7.3 Hz, 1 H), 7.62 – 7.53 (m, 1 H), 7.51 – 7.40(m, 2 H), 7.13 (d, J = 7.3 Hz, 1 H), 4.71 (dd, J = 6.5, 4.9 Hz, 2 H), 4.63 (d, J = 5.1 Hz, 1 H), 4.50 – 4.43 (m, 3 H), 4.25 (dd, J = 6.5, 5.0 Hz, 2 H), 3.53 (dt, J = 6.2, 4.9 Hz, 1 H), 3.39 (dd, J = 9.8, 4.6 Hz, 1 H), 3.30 – 3.21 (m, 3 H). MS[M+H] + :415.2.

[0112] Step 4: Synthesis of 4-4 Using 4-3 (2.00 g, 4.83 mmol, 1.00 eq.) and DMTrCl (1.80 g, 5.31 mmol, 1.10 eq.) as raw materials, according to the method for synthesizing compound 1-8, 2.40 g of white solid was obtained with a yield of 69.4%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.17 (s, 1 H), 8.02 (s, 1 H), 7.99 – 7.87 (m, 2 H), 7.69 – 7.53 (m, 2 H), 7.51 – 7.41 (m, 2 H), 7.39 – 7.31 (m, 2 H), 7.29 – 7.06 (m, 8 H), 6.88 – 6.73 (m, 4 H), 4.86 (s, 1 H), 4.69 (t, J = 5.7 Hz, 2 H), 4.46 (s, 2H), 4.24 (t, J = 5.8 Hz, 2 H), 3.81 – 3.70 (m, 1 H), 3.68 (s, 6 H), 3.47 (dd,J = 9.9, 4.5 Hz, 1 H), 3.38 (dd, J = 9.9, 5.9 Hz, 1 H), 2.98 – 2.78 (m, 2 H). MS[M-H] - :715.2.

[0113] Step 5: Synthesis of YK-GNA-004 Using 4-4 ​​(2.40 g, 3.35 mmol, 1.00 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.21 g, 4.01 mmol, 1.20 eq) as raw materials, according to the method for synthesizing YK-GNA-001, 1.52 g of white solid was obtained with a yield of 48.9%. 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 1.7 Hz, 2 H), 7.58 (s, 1 H), 7.47 (s, 2 H), 7.44 – 7.36 (m, 3 H), 7.34 – 7.12 (m, 9 H), 6.77 (d, J = 8.9Hz, 4 H), 4.72 (d, J = 6.1 Hz, 2 H), 4.67 – 4.54 (m, 2 H), 4.41 (s, 2 H), 4.15 – 4.05 (m, 1 H), 3.85 – 3.70 (d, J = 2.9 Hz, 8 H), 3.70 – 3.41 (m, 4 H),3.28 – 3.02 (m, 2 H), 2.59 (t, J = 6.4 Hz, 1 H), 2.41 (d, J = 4.4 Hz, 1 H), 1.17 – 0.92 (m, 12 H). 31 P NMR (400 Hz, CDCl3) δ150.13, 149.67 ppm. MS[M-H] - :915.6.

[0114] 5. Synthesis of YK-GNA-005 The synthetic route is as follows:

[0115] Step 1: Synthesis of 5-2 Using 5-1 (14.60 g, 63.42 mmol, 1.10 eq) and 3-4 (5.00 g, 57.42 mmol, 1.00 eq) as raw materials, according to the method for synthesizing compound 1-5, 14.07 g of light red oil was obtained with a yield of 81.4%. 1 H NMR (400MHz, DMSO- d 6) δ 7.96 – 7.85 (m, 2 H), 7.80 – 7.72 (m, 2 H), 7.61 – 7.53 (m, 2H), 3.88 (dd, J = 6.2, 5.3 Hz, 2 H), 3.61 (t, J = 5.7 Hz, 2 H), 1.81 (d, J =1.1 Hz, 3 H). MS [M + NH4] + :317.2.

[0116] Step 2: Synthesis of 5-3 Using 5-2 (7.00 g, 23.39 mmol, 1.00 eq.) and 3-3 (4.20 g, 24.68 mmol, 1.05 eq.) as raw materials, according to the method for synthesizing compound 3-6, 8.41 g of white solid was obtained with a yield of 76.5%. 1 H NMR (400MHz, CDCl3) δ 7.91 – 7.80 (m, 2 H), 7.67 – 7.59 (m, 1 H), 7.56 (s, 1 H), 7.54– 7.43 (m, 2 H), 6.76 (d, J = 1.3 Hz, 1 H), 4.65 (dd, J = 6.4, 4.9 Hz, 4 H), 4.25 (td, J = 6.2, 5.0 Hz, 3 H), 4.00 (dd, J = 8.3, 6.5 Hz, 1 H), 3.65 (dd, J= 8.3, 6.4 Hz, 1 H), 3.61 – 3.42 (m, 2 H), 1.80 (d, J = 1.2 Hz, 3 H), 1.39 (s, 3 H), 1.32 (s, 3 H). MS[M+H] + :470.0.

[0117] Step 3: Synthesis of 5-4 Add 5-3 (8.40 g, 17.89 mmol, 1.00 eq.) and 1,4-dioxane solution (100 mL) to the reaction flask. Add 1 M NaOH solution (30 mL) dropwise under ice bath conditions. Warm to room temperature and stir for 5.5 hours. Adjust the pH to about 8-9 with 1 M HCl solution under ice bath conditions, and concentrate the reaction solution to dryness to obtain an oil. The oil was purified by column chromatography (SiO2, DCM / MeOH = 20 / 1 –5 / 1) to obtain 3.80 g of white solid, with a yield of 58.1%. MS [M + H] + :366.2. Step 4: Synthesis of 5-5 Using 5-4 (3.80 g, 10.4 mmol) as raw material, according to the method for synthesizing compound 1-7, 1.92 g of white solid was obtained with a yield of 56.2%.

[0118] Step 5: Synthesis of 5-6 Using 5-5 (1.90 g, 5.84 mmol, 1.00 eq.) and DMTrCl (2.30 g, 6.79 mmol, 1.20 eq.) as raw materials, according to the method for synthesizing compound 1-8, 2.71 g of white solid was obtained with a yield of 73.6%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.24 (s, 1 H), 8.00 (s, 1 H), 7.40 – 7.33 (m, 2 H), 7.30 – 7.13 (m, 7 H), 7.06 (d, J = 1.3 Hz, 1 H), 6.88 – 6.77 (m, 4 H), 4.86 (d, J = 5.5Hz, 1 H), 4.64 – 4.52 (m, 2 H), 4.45 (s, 2 H), 4.08 – 3.92 (m, 2 H), 3.82 –3.70 (m, 1 H), 3.69 (s, 6 H), 3.45 (dd, J = 9.9, 4.6 Hz, 1 H), 3.43 – 3.35(m, 1 H), 2.88 (d, J = 5.4 Hz, 2 H), 1.57 (d, J = 1.2 Hz, 3 H). MS [M + Na] + :650.2.

[0119] Step 6: Synthesis of YK-GNA-005 Using 5-6 (2.70 g, 4.30 mmol, 1.00 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.56 g, 5.18 mmol, 1.20 eq) as raw materials, according to the method for synthesizing YK-GNA-001, 2.02 g of white solid was obtained with a yield of 56.2%. 1H NMR (400 MHz, CDCl3) δ 7.58 – 7.35 (m, 3 H), 7.34 – 7.11 (m, 7H), 6.78 (dd, J = 8.7, 4.8 Hz, 4 H), 6.59 (dd, J = 9.4, 1.3 Hz, 1 H), 4.73 –4.46 (m, 4 H), 4.34 – 4.01 (m, 3 H), 3.93 – 3.38 (m, 12 H), 3.30 – 2.99 (m, 2H), 2.58 (t, J = 6.4 Hz, 1 H), 2.43 (td, J = 6.4, 4.5 Hz, 1 H), 1.77 – 1.59(m, 3 H), 1.31 – 0.81 (m, 12 H). 31 P NMR (400 Hz, CDCl3) δ150.14, 149.63 ppm. MS[M-H] - :826.7.

[0120] 6. Synthesis of YK-GNA-006 The synthetic route is as follows:

[0121] Step 1: Synthesis of 6-2 Using 6-1 (8.00 g, 60.8 mmol, 1.00 eq.) and 3-2 (7.20 g, 60.8 mmol, 1.00 eq.) as raw materials, according to the method for synthesizing compound 3-3, 8.30 g of light yellow oil was obtained with a yield of 79.8%. 1 H NMR (400 Hz, DMSO- d 6) δ 4.18 - 4.24 (m, 3 H), 4.01 (t, J = 7.40 Hz, 1 H), 3.64 (t, J =7.25 Hz, 1 H), 3.50 (d, J = 5.12 Hz, 2 H), 3.46 (t, J = 2.30 Hz, 1 H), 1.34 (s, 3 H), 1.29 (s, 3 H).

[0122] Step 2: Synthesis of 6-3 Add THPTA (4.12 g, 9.48 mmol, 1.5 eq.), anhydrous CuSO4 (3.00 g, 18.8 mmol, 3.0 eq.) and water (15 mL) to the reaction flask and stir to dissolve. Add VCNa (5.00 g, 25.2 mmol, 4.0 eq.) to prepare a solution for later use.

[0123] Add 6-2 (1.54 g, 9.08 mmol, 1.40 eq.), 3-5 (2.00 g, 6.48 mmol, 1.00 eq.) and DMSO (48 mL) to the reaction flask and stir to dissolve. Cool to 0-10°C and add the above solution dropwise. After the addition, warm to room temperature and stir for 2 hours. Dilute the reaction solution with water (200 mL), extract with DCM (200 mL × 2), combine the organic phases, wash with water and saturated NaCl solution in turn, dry over anhydrous Na2SO4, and remove the solvent to obtain a residue. The residue was purified by column chromatography (SiO2, MeOH / DCM = 0 / 1 - 1 / 19) to obtain 0.77 g of light blue solid with a yield of 24.8%. 1 H NMR (400 Hz, DMSO- d 6) δ11.13 (s, 1 H), 8.70 (s, 1 H), 8.22 (s, 1 H), 8.07 (s, 1 H), 8.05 (s, 1 H),7.62-7.68 (m, 2 H), 7.55-7.59 (m, 2 H), 4.95 (t, J = 5.70 Hz, 2 H), 4.81 (t,J = 5.44 Hz, 2 H), 4.51 (s, 2 H), 4.14-4.20 (m, 1 H), 3.98 (t, J = 7.33 Hz, 1H), 3.58 (t, J = 7.40 Hz, 1 H), 3.38-3.44 (m, 2 H), 1.32 (s, 3 H), 1.27 (s, 3H). MS [M - H] - :477.3.

[0124] Step 3: Synthesis of 6-4 Using 6-3 (1.23 g, 2.57 mmol) as raw material, according to the method for synthesizing compound 1-7, 1.07 g of light blue solid was obtained with a yield of 95.0%. 1 H NMR (400 Hz, DMSO- d6) δ 11.19 (s, 1 H), 8.72 (s, 1 H), 8.22 (s, 1 H), 8.05-8.09 (m, 2 H), 7.56-7.66 (m, 4 H), 4.96 (s, 2 H), 4.81 (s, 2 H), 4.49-4.55 (m, 3 H), 3.59 (s, 2 H), 3.41-3.44 (m, 2 H), 2.94 (d, J =6.39 Hz, 2 H). MS[M-H] - :437.3.

[0125] Step 4: Synthesis of 6-5 Using 6-4 (0.86 g, 1.96 mmol, 1.00 eq.) and DMTrCl (0.80 g, 2.35 mmol, 1.20 eq.) as raw materials, according to the method for synthesizing compound 1-8, 0.73 g of white solid was obtained with a yield of 50%. 1 H NMR (400 Hz, DMSO- d 6) δ 11.17 (s, 1 H), 8.70 (s, 1 H), 8.21 (s, 1 H), 8.04-8.07 (m, 2 H), 7.66 (t, J = 7.00 Hz, 1 H), 7.56 (t, J = 7.30 Hz, 2 H), 7.40 (d, J = 7.00 Hz, 2 H), 7.22-7.33 (m, 8 H), 6.89 (d, J = 8.72 Hz, 4 H), 5.78 (s, 1 H), 4.91-4.95 (m, 3 H), 4.79 (t, J = 5.50 Hz, 1 H), 4.48 (s, 1 H), 3.74 (s, 6 H),3.49-3.53 (m, 1 H), 3.37-3.44 (m, 3 H), 2.94 (d, J = 5.13 Hz, 2 H). MS[M-H] - :739.5.

[0126] Step 5: Synthesis of YK-GNA-006 Add 6-5 (0.44 g, 0.57 mmol, 1.00 eq.) and ultra-dry DCM (18 mL) to the reaction flask and stir to dissolve. Then add DIPEA (0.37 g, 2.87 mmol, 5.0 eq.) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.34 g, 1.46 mmol, 2.50 eq.). Stir for about 1.5 hours at room temperature. The reaction solution was diluted with DCM (50 mL), washed with saturated NaHCO3 solution (100 mL × 2) and saturated NaCl solution (100 mL) in turn, dried over anhydrous Na2SO4, and the solvent was removed by rotation to obtain a residue. The residue was purified by column (SiO2, PE / EA = 0 / 1, 2.0% TEA alkalization) to obtain 0.26 g of a white solid with a yield of 45.8%. 1 H NMR (400 Hz, DMSO- d 6) δ 12.46 (s, 1 H), 8.24 (s, 3 H), 8.15 (s, 1 H), 8.05 (d, J = 7.93 Hz, 1 H), 7.58 (t, J = 7.00 Hz, 1 H), 7.51(t, J = 7.35 Hz, 2 H), 7.40 (t, J = 6.00 Hz, 2 H), 7.20-7.33 (m, 7 H), 6.88 (t, J = 7.44 Hz, 4 H), 4.96 (t, J = 5.37 Hz, 2 H), 4.85 (s, 2 H), 4.50 (d, J= 3.63 Hz, 1 H), 4.45 (s, 1 H), 4.00-4.07 (m, 1 H), 3.74 (d, J = 2.13 Hz, 6H), 3.47-3.70 (m, 6 H), 2.96-3.12 (m, 2 H), 2.73 (t, J = 6.12 Hz, 1 H), 2.64(t, J = 5.80 Hz, 1 H), 1.13 (t, J = 6.62 Hz, 6 H), 1.08 (d, J = 6.76 Hz, 3H), 1.00 (d, J = 6.77 Hz, 3 H). 31 P NMR (400 Hz, DMSO- d 6) δ148.79, 148.26 ppm. MS[M-H] - :939.6.

[0127] 7. Synthesis of Compound 1-2

[0128] According to the synthesis method in WO2022028462A1, pages 76-77, 537 mg of product was obtained with a total yield of 10.6%.

[0129] Example 2: Synthesis of oligonucleotides without GalNAc conjugation In this example, two siRNA molecules were synthesized, named D84-DV27P and D81-DV25, and then anti-off-target modifications were performed at positions 6 and 7, respectively. The specific synthesized and modified siRNA molecules are shown in Table 1.

[0130] The sequence of D84-DV27P is as follows: Sense strand (SS) (SEQ ID NO.1): 5'-Gms-Ums-Um-Um-Um-Gm-Uf-Am-Gf-Cf-Af-Um-Um-Um-Um-Um-Af-Um -Um-Am-Am-3' Antisense strand (AS) (SEQ ID NO.2): 5'-UmsEVP-Ufs-Am-Af-Uf-Af-Am-Am-Am-Am-Um-Gm-Cm-Uf-Am-Cf-Am-Am -Am-Am-Cms-Cms-Cm-3' The D81-DV25 sequence is as follows: Sense strand (SS) (SEQ ID NO.3): 5'-Cms-Ums-Um-Um-Um-Gm-Uf-Am-Af-Cf-Uf-Um-Gm-Am-Am-Gm-Af-Um -Am-Um-Um-3' Antisense strand (AS) (SEQ ID NO.4): 5'-Ams-Afs-Um-Am-Um-Cf-Um-Um-Cm-Am-Am-Gm-Um-Uf-Am-Cf-Am-Am -Am-Am-Gms-Cms-Am-3' Table 1 siRNA double-stranded molecules without GalNAc conjugation

[0131] Among them, A, U, C, and G each generally represent nucleotides containing adenine, uracil, cytosine, and guanine as bases, respectively; m represents that the nucleotide adjacent to its left is a 2'-OMe-modified nucleotide; f represents that the nucleotide adjacent to its left is a 2'-F-modified nucleotide; s represents that the two adjacent nucleotides on its left and right are connected by a thiophosphate group; EVP represents that the nucleotide adjacent to the left of EVP is a 5'-(E)-VP-modified nucleotide.

[0132] Among them, the structures of GA1(A), GA2(C), GT3(A), GT4(C), GT5(T), GT6(A) and G1-2(A) are as follows: .

[0133] In this article, GA1(A), GA2(C), GT3(A), GT4(C), GT5(T), GT6(A) and G1-2(A) will be repeated in the structure shown above, wherein GA1(A) represents a nucleotide obtained by replacing adenine nucleoside in a conventional adenine nucleotide with YK-GNA-001, GA2(C) represents a nucleotide obtained by replacing cytosine nucleoside in a conventional cytosine nucleotide with YK-GNA-002, and GT3(A) represents a nucleotide obtained by replacing a conventional adenine nucleotide with YK-GNA-003. , GT4(C) represents the nucleotide obtained by replacing the cytosine nucleoside in the conventional cytosine nucleotide by YK-GNA-004, GT5(T) represents the nucleotide obtained by replacing the thymidine nucleoside in the conventional thymidine nucleotide by YK-GNA-005, GT6(A) represents the nucleotide obtained by replacing the adenine nucleoside in the conventional adenine nucleotide by YK-GNA-006, and G1-2(A) represents the nucleotide obtained by replacing the adenine nucleoside in the conventional adenine nucleotide by 1-2.

[0134] 1. Preparation of Antisense Oligonucleotide The oligonucleotide antisense strand was synthesized on a solid support according to the phosphoramidite solid phase synthesis method.

[0135] When synthesizing the antisense strand of sequence number D84-DV27P, conventional monomers (i.e., phosphoramidite monomers without anti-off-target nucleoside) were used for solid phase synthesis; when synthesizing the antisense strands of sequence number D84-DV27P-GNA-6 and D84-DV27P-GNA-7, purchased ( S)-GNA (A base) was used as the 6th or 7th monomer for solid phase synthesis respectively; when synthesizing the antisense chains with sequence numbers D84-DV27P-GT6-6, D84-DV27P-GA1-6 and D84-DV27P-GT3-6, universal CPG was used as the solid phase carrier, and the anti-off-target nucleoside phosphoramidite monomers YK-GNA-006, YK-GNA-001 and YK-GNA-003 synthesized in Example 1 were used as the 6th monomer for solid phase synthesis respectively; when synthesizing the antisense chains with sequence numbers D84-DV27P-G1-2-6 and D84-DV27P-G1-2-7, the anti-off-target nucleoside phosphoramidite monomer compound 1-2 synthesized in Example 1 was used as the 6th or 7th monomer for solid phase synthesis respectively, and the synthesis scale was 200 nmol.

[0136] When synthesizing the antisense strand of sequence number D81-DV25, conventional monomers (i.e., phosphoramidite monomers without anti-off-target nucleoside) were used for solid phase synthesis; when synthesizing the antisense strand of sequence number D81-DV25-GNA-6, purchased ( S )-GNA (C base) was used as the sixth monomer for solid phase synthesis; when synthesizing the antisense strand with sequence number D81-DV25-GNA-7, the purchased ( S )-GNA (T base) was used as the seventh monomer for solid phase synthesis; when synthesizing the antisense chains with sequence numbers D81-DV25-GA2-6 and D81-DV25-GT4-6, universal CPG was used as the solid phase carrier, and the anti-off-target nucleoside phosphoramidite monomers YK-GNA-002 and YK-GNA-004 synthesized in Example 1 were used as the sixth monomers for solid phase synthesis respectively; when synthesizing the antisense chain with sequence number D81-DV25-GT5-7, the anti-off-target nucleoside phosphoramidite monomer YK-GNA-005 synthesized in Example 1 was used as the seventh monomer for solid phase synthesis, and the synthesis scale was 200 nmol.

[0137] 1) Reagent and monomer preparation By using an acetonitrile solution of the monomer (0.15 M), a 0.25 M 5-benzylthiotetrazolyl solution in acetonitrile as an activating agent, a 3% solution of hydrogenated xanthine in pyridine as a thiolation reagent, a 0.05 M solution of iodine in water / pyridine (1 / 9, v / v) as an oxidizing reagent, a 10% acetic anhydride solution in tetrahydrofuran as a capping agent A, tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) as a capping agent B, and 3% dichloroacetic acid in toluene (v / v) as a de-DMT reagent, and loading them into the designated reagent position of a 192 P model DNA / RNA automatic synthesizer.

[0138] 2) Crude product synthesis Enter the designated oligonucleotide sequence, set the synthesis program, and after checking that everything is correct, start the synthesis of cyclic oligonucleotides. Prepare according to the following steps: a. Deprotection 3% dichloroacetic acid toluene solution was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0139] b. Coupling Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolyl as an activating agent, followed by rinsing with acetonitrile.

[0140] c. Oxidation / sulfurization Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (9 / 1) as an oxidant, followed by rinsing with acetonitrile.

[0141] Sulfurization: Use 3% hydrogenated xanthan gum in pyridine as the sulfurizing agent for sulfurization, and then use acetonitrile for washing.

[0142] d. Hydroxyl protection Hydroxyl protection was performed using 10% acetic anhydride tetrahydrofuran solution (CAP A) and tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting reagents, followed by rinsing with acetonitrile.

[0143] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[0144] e. Use 3% dichloroacetic acid toluene solution as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile to wash.

[0145] 3) Deprotection After the synthesis is completed, the solid phase carrier is transferred to a reactor. After the oligonucleotide is cleaved from the solid phase carrier with concentrated ammonia (25-28%) at 60°C for 16 hours, the system is cooled to room temperature and then filtered, rinsed with a mixed solution of purified water and ethanol, the filtrates are combined, and the filtrates are concentrated at low temperature to obtain a crude residue.

[0146] 4) Purification The crude residue after deprotection was dissolved in purified water and purified by HPLC. The product peak solution was collected and the content was measured by an enzyme marker, and the molecular weight was confirmed by ESI MS.

[0147] 2. Preparation of the positive oligonucleotide strand The siRNA sense chain was synthesized according to the method for synthesizing the siRNA antisense chain, using a universal CPG solid phase carrier, and the synthesis scale of each sense chain complementary to the antisense chain was 200 nmol.

[0148] 3. Preparation of oligonucleotide duplexes The siRNA sense strand and the complementary antisense strand were mixed in a ratio of 1:1 according to the UV absorption content, heated to 95°C, and cooled to room temperature after 3 minutes to form a double strand. The obtained double-stranded solution was characterized by HPLC, and the content was determined by an ELISA instrument after the product purity was qualified, and lyophilized to obtain a solid powder for storage.

[0149] The molecular weight and purity of the obtained siRNA double-strands are shown in Table 2.

[0150] Table 2 Molecular weight and purity of non-GalNAc conjugated siRNA double-stranded molecules

[0151] Example 3: Synthesis of GalNAc-conjugated oligonucleotide conjugates The GalNAc-conjugated oligonucleotide conjugate synthesized in this example has the same oligonucleotide sequence as the oligonucleotide prepared in Example 2, except that the 3' end of the sense chain is connected to GalNAc, and the sequence numbers are D84-DV27PG5 and D81-DV25G5, respectively, and D84-DV27PG5 and D81-DV25G5 are sequences modified to prevent off-target at positions 6 and 7, respectively. The specific sequences are shown in Table 3.

[0152] The sequence of D84-DV27PG5 is as follows: Sense strand (SS) (SEQ ID NO.29): 5'-Gms-Ums-Um-Um-Um-Gm-Uf-Am-Gf-Cf-Af-Um-Um-Um-Um-Um-Af-Um -Um-Am-Am-G5-3' Antisense strand (AS) (SEQ ID NO.2): 5'-UmsEVP-Ufs-Am-Af-Uf-Af-Am-Am-Am-Am-Um-Gm-Cm-Uf-Am-Cf-Am-Am -Am-Am-Cms-Cms-Cm-3' The sequence of D81-DV25G5 is as follows: Sense strand (SS) (SEQ ID NO.30): 5'-Cms-Ums-Um-Um-Um-Gm-Uf-Am-Af-Cf-Uf-Um-Gm-Am-Am-Gm-Af-Um -Am-Um-Um-G5-3' Antisense strand (AS) (SEQ ID NO.4): 5'-Ams-Afs-Um-Am-Um-Cf-Um-Um-Cm-Am-Am-Gm-Um-Uf-Am-Cf-Am-Am -Am-Am-Gms-Cms-Am-3' Among them, A, U, C, G each generally represent nucleotides containing adenine, uracil, cytosine and guanine as bases respectively; m represents that the nucleotide adjacent to its left is a 2'-OMe modified nucleotide; f represents that the nucleotide adjacent to its left is a 2'-F modified nucleotide; s represents that the two adjacent nucleotides on the left and right are connected by thiophosphate groups; EVP represents that the nucleotide adjacent to its left is a 5'-(E)-VP modified nucleotide; G5 represents a GalNAc ligand, and its structure is as follows:

[0153] Table 3 G5-conjugated siRNA double-stranded molecules

[0154] 1. Preparation of G5-conjugated oligonucleotide antisense strand The preparation method is the same as Example 2.

[0155] 2. Preparation of G5-conjugated oligonucleotide sense strand A CPG-G5 solid phase carrier was used, and the preparation method was the same as in Example 2. The CPG-G5 solid phase carrier refers to a solid phase carrier formed by connecting the GalNAc ligand G5 to the CPG carrier, and its structure is shown below:

[0156] 3. Preparation of G5-conjugated oligonucleotide duplexes The preparation method is the same as Example 2.

[0157] The oligonucleotide forms a ligand G5 Figure 5 Conjugates as shown.

[0158] The molecular weight and purity of the obtained G5-conjugated siRNA double-strand are shown in Table 4.

[0159] Table 4 Molecular weight and purity of G5-conjugated siRNA double-stranded molecules

[0160] Example 4: Detection of the efficacy and off-target effects of the anti-off-target modified siRNA molecules in HepG2 cells After the siRNA molecules synthesized in Example 2 were transfected into HepG2 cells, the expression of the target gene PCSK9 and the off-target gene ITGAV was detected by qPCR.

[0161] 1. Experimental Materials Test samples: double-stranded siRNA molecules listed in Table 1.

[0162] Cell type: HepG2 cells Drug solvent: sterile enzyme-free water, Gibco Opti-MEM (Gibco-31985070).

[0163] 2. Experimental Methods 2.1 Cell Culture: HepG2 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin, and placed in a cell culture incubator at 37°C with 5% CO2, and subcultured every three days. The cells were digested and subcultured with 0.25% trypsin, centrifuged at 800 r / min for 3 minutes, the supernatant was discarded, and fresh medium was added for subculture.

[0164] 2.2 Cell transfection siRNA solution dilution: The above siRNA molecules were dissolved in sterile enzyme-free water to 20 μM as the stock solution. The stock solution was diluted with Opti-MEM to specific concentration points ranging from 100 nM to 0.128 pM by gradient dilution.

[0165] Preparation of transfection mixture: Lipofectamine RNAiMAX (Invitrogen-13778150) and Opti-MEM were mixed at a ratio of 2:98 and vortexed to mix.

[0166] Transfection complex preparation: According to the ratio of 1:1 (v / v), 60 μL of siRNA solution diluted in Opti-MEM was added to 60 μL of transfection mixture, vortexed and allowed to stand at room temperature for 15 minutes to obtain a transfection complex.

[0167] Add the prepared transfection complex to a 24-well cell culture plate (100 μL per well), and the well containing only the transfection reagent is used as the transfection control group. Add 500 μL of cell suspension (containing 75,000 cells) to each well. Mix well using the cross method and culture in a 37°C, 5% CO2 cell culture incubator for 48 hours.

[0168] 2.3 RNA extraction and reverse transcription 48 hours after transfection, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN-74106) according to the kit instructions, and RNA was finally eluted with 30 μL of sterile enzyme-free water. Subsequently, 8 μL of RNA was taken and reverse transcribed into cDNA using the FastKing RT Kit (TIANGEN-KR116-02) according to the kit instructions for qPCR.

[0169] 2.4 qPCR Dilute the reverse transcription product 5 times with sterile enzyme-free water, and perform qPCR using TB Green Premix Ex Taq (Takara-RR420W) according to the kit instructions. Prepare 10 μL of the reaction system as follows, and perform 3 replicates for each sample:

[0170] qPCR reaction procedure: Heat at 95°C for 30 seconds, then enter the cycle mode, heat at 95°C for 5 seconds, then heat at 60°C for 34 seconds, for a total of 40 cycles.

[0171] 2.5 Data Analysis The target gene mRNA expression level in each sample was calculated by the ΔΔCT relative quantitative method. The calculation formula is as follows: ΔCT = average Ct value of target gene – average Ct value of GAPDH; ΔΔCT = ΔCT (experimental group) – ΔCT (transfection control group); Relative expression of target gene mRNA = 2 -ΔΔCT ; Inhibition rate = (relative expression of mRNA in transfection control group - relative expression of target gene mRNA) / relative expression of mRNA in transfection control group × 100%.

[0172] IC 50 The IC values ​​were calculated by fitting the dose-effect curve using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis using the "log (inhibitor) vs. response-variable slope" function module of the analysis software GraphPad Prism 8 to obtain the IC values ​​of each siRNA molecule. 50 value.

[0173] 3. Experimental Results 3.1 Inhibitory effect of anti-off-target modified siRNA molecules on PCSK9 in HepG2 cells The IC50 (IC50 )See Table 5 for details.

[0174] Table 5 Inhibitory effect of siRNA molecules on PCSK9 in HepG2 cells

[0175] 1) The siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention can significantly inhibit the expression of the PCSK9 gene.

[0176] From Table 5 and Figure 1 It can be seen that after the anti-off-target nucleoside compound of the present invention is modified at the 6th position of D84-DV27P, the IC50IC of D84-DV27P-GA1-6, D84-DV27P-GT6-6 and D84-DV27P-GT3-6 against PCSK9 50 Both were lower than D84-DV27P without anti-off-target design; after modification at the 6th position of D81-DV25, the IC values ​​of D81-DV25-GA2-6 and D81-DV25-GT4-6 for PCSK9 50 IC50 was significantly lower than that of D81-DV25 without anti-off-target modification. For example, the IC 50 Only 16.9% of D81-DV25.

[0177] The anti-off-target nucleoside compound of the present invention is modified at the 7th position of D81-DV25, and the IC of D81-DV25-GT5-7 to PCSK9 is 50 , equivalent to D81-DV25.

[0178] The experimental results show that modifying siRNA molecules with the anti-off-target nucleoside compound of the present invention will not reduce the activity of the siRNA molecules, and may even enhance their activity.

[0179] 2) Compared with the existing anti-off-target technology, the siRNA molecules modified by the anti-off-target nucleoside compounds of the present invention have a better inhibitory effect on the PCSK9 gene.

[0180] The anti-off-target nucleoside compound of the present invention is modified at the 6th position of D84-DV27P, and the IC values ​​of D84-DV27P-GA1-6, D84-DV27P-GT6-6 and D84-DV27P-GT3-6 for PCSK9 are 50 , which are lower than D84-DV27P-GNA-6 and D84-DV27P-G1-2-6 modified by the prior art anti-off-target compound GNA and compound 1-2. For example, the IC 50It is only 35.4% of D84-DV27P-GNA-6 and 33.2% of D84-DV27P-G1-2-6.

[0181] The anti-off-target nucleoside compound of the present invention is used to modify the 6th position of D81-DV25, and D81-DV25-GA2-6 and D81-DV25-GT4-6 are effective for PCSK9IC 50 Compared with D84-DV27P-GNA-6 modified by the prior art anti-off-target compound GNA, IC 50 For example, the IC of D81-DV25-GA2-6 50 It is only 6.5% of D84-DV27P-GNA-6.

[0182] It can be seen from this that the siRNA molecules modified by the anti-off-target nucleoside compounds of the present invention have better on-target activity compared with the existing anti-off-target technologies.

[0183] 3.2 Inhibitory effect of siRNA on off-target gene ITGAV The inhibition rate of each siRNA molecule at a concentration of 10 nM on the off-target gene ITGAV is shown in Table 6.

[0184] Table 6 Inhibitory effect of siRNA molecules on off-target gene ITGAV

[0185] From Table 6 and Figure 2 It can be seen that the parent sequence D84-DV27P without anti-off-target modification has obvious off-target activity, and the inhibition rate of off-target gene ITGAV is as high as 88%. The siRNA sequence modified by the anti-off-target nucleoside compound of the present invention has a significantly reduced inhibition rate of off-target genes. For example, D84-DV27P-GA1-6 has a 77% reduction in the inhibition rate of off-target gene ITGAV compared to the parent sequence D84-DV27P, indicating that the anti-off-target modification can significantly reduce the off-target effect of siRNA.

[0186] The anti-off-target effect of the D84-DV27P-GNA-6 conjugate conjugated with GNA in the prior art and the D84-DV27P-G1-2-6 conjugate conjugated with compound 1-2 is very weak. For example, the inhibition rate of the D84-DV27P-G1-2-6 conjugate on the off-target gene ITGAV is only 1% lower than that of the D84-DV27P conjugate. Therefore, compared with the prior art, the anti-off-target nucleoside compound of the present invention has a better anti-off-target effect. For example, the inhibition rate of the D84-DV27P-GA1-6 conjugate on the off-target gene ITGAV is 76% lower than that of the D84-DV27P-G1-2-6 conjugate.

[0187] Example 5: Effect of anti-off-target modified siRNA molecules on the inhibition of PCSK9 and LDL-C levels in mouse serum In this example, the inhibition rate of the GalNAc-conjugated siRNA molecules in Table 3 on PCSK9 in mouse serum and the effect on LDL-C levels were investigated.

[0188] Test drugs: Anti-off-target modified siRNA molecules listed in Table 3.

[0189] Drug solvent: PBS buffer Experimental Animal Information:

[0190] Feeding and management: Feeding conditions: The experimental animals were received and raised in Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and the animals were free to eat and drink. Standard SPF-grade irradiated sterilized experimental transgenic mouse feed was purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. The production unit provides a feed quality certificate for each batch of feed, and a third-party test report is provided every year; the test standards refer to the national standards GB 14924.3-2010 "Nutritional Composition of Laboratory Animal Feed" and GB14924.2-2001 "General Hygiene Standards for Laboratory Animal Feed". The appearance and bacterial indicators of drinking water are tested once a month, tested within the company, and the test report of the tap water company in the district is provided every year, referring to the national standard GB5749-2015 "Sanitary Standards for Drinking Water".

[0191] 1. Inhibition rate of PCSK9 protein expression in mouse serum by siRNA molecules with different anti-off-target modifications Experimental methods: Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the inner canthus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and centrifuged at about 4 °C and a centrifugal force of about 3000 g for 10 minutes. The PCSK9 protein level in the serum was detected using a PCSK9 kit.

[0192] Experimental results: The inhibition rates of PCSK9 protein in serum 7 days and 14 days after administration are shown in Table 7.

[0193] Table 7 PCSK9 protein expression inhibition rate in mouse serum

[0194] 1) The siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention can effectively inhibit the expression of PCSK9 protein in mouse serum.

[0195] From Table 7 and Figure 3 It can be seen that the siRNA molecules modified by the anti-off-target nucleoside compounds of the present invention have a significant inhibitory effect on PCSK9 protein expression in mouse serum. For example, the inhibition rates of D84-DV27PG5-GA1-6 and D84-DV27PG5-GT3-6 are comparable to the parent sequence D84-DV27PG5, and the inhibition rate of D81-DV25G5-GT4-6 is comparable to the parent sequence D81-DV25G5. This shows that the use of the anti-off-target nucleoside modified siRNA of the present invention will not affect the on-target activity.

[0196] 2) The siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention have a higher inhibitory effect on PCSK9 protein expression in mouse serum than the siRNA molecules modified with the prior art compounds.

[0197] For example, the inhibition rates of D84-DV27PG5-GA1-6 modified with YK-GNA-001 on days 7 and 14 were 25.4% and 21.6% higher than those of D84-DV27PG5-G1-2-6 modified with compound 1-2, respectively.

[0198] 2. Effects of different anti-off-target modified siRNA molecules on LDL-C levels in mouse serum Experimental process: Before administration (D0), 7 days after administration (D7), and 14 days after administration (D14), about 200 μL of blood (without anticoagulation) was collected from the inner canthus of the experimental animals. The whole blood samples were temporarily stored in an ice box before centrifugation, and centrifuged at about 4 °C and a centrifugal force of about 3000 g for 10 minutes to detect the level of serum LDL-C.

[0199] Experimental results: The serum LDL-C reduction levels were observed 7 days and 14 days after administration. The specific results are shown in Table 8.

[0200] Table 8 Reduction level of LDL-C in mouse serum

[0201] 1) The siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention can effectively reduce the LDL-C level in mouse serum.

[0202] From Table 8 and Figure 4 It can be seen that the siRNA molecules modified by the anti-off-target nucleoside compounds of the present invention can significantly reduce the LDL-C level in mouse serum. For example, D84-DV27PG5-GA1-6 reduced the level by 38.1% and 40.4% on the 7th day and 14th day, respectively, which is higher than the parent sequence D84-DV27PG5.

[0203] 2) The siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention have a higher reduction effect on the level of LDL-C in mouse serum than the siRNA molecules modified with the prior art compounds.

[0204] For example, the reduction levels of D81-DV25G5-GT4-6 modified by YK-GNA-004 on days 7 and 14 were 9.3% and 10.2% higher than those of D81-DV25G5-GNA-6 modified by GNA, respectively.

[0205] Although the present invention has been described in detail above using general descriptions, specific implementation methods and experiments, it is not intended to limit the present invention. On this basis, those skilled in the art may make modifications or improvements thereto. These modifications or improvements made without departing from the spirit of the present invention shall all fall within the scope of protection claimed by the present invention.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in, L1 is hydrogen, a hydroxyl protecting group or a phosphorus-containing group reactive with nucleophiles; L2 is hydrogen or a hydroxyl protecting group; L3 is -CH2HNC(O)(CH2) n -、-C(O)NH(CH2) n -、-(CH2) n -、-(CH2) n O(CH2) m -or , wherein n and m are each independently any integer from 0 to 5, R4 and R5 are each independently hydrogen, oxygen, alkylene, alkenylene, -R 4' -OR 4'' , -R 4' -CO-R 4'' or -R 4' -N(R 4''' )-R 4'' , where R 4' , R 4'' and R 4''' Each is independently hydrogen, C1-C6 alkylene, C1-C6 alkyl, C1-C6 acyl or C1-C6 alkyleneoxy; L4 is a substituted or unsubstituted base or a salt thereof; R1, R2 and R3 are each independently hydrogen, alkyl, alkenyl or aryl.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: L1 is an active phosphorus group; And / or, L1 is a hydroxyl protecting group.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 2, wherein: The active phosphorus group is a phosphoramidite, a phosphotriester, an H-phosphate or a phosphorus-containing chiral auxiliary; And / or, the hydroxyl protecting group is selected from any one of 4,4'-dimethoxytrityl, monomethoxytrityl, trityl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl and isopropyldimethylsilyl.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 2, wherein: L1 is , " ” indicates the connection location.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: L2 is a hydroxyl protecting group.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 5, wherein: The hydroxy protecting group is selected from any one of 4,4'-dimethoxytrityl, monomethoxytrityl, trityl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl and isopropyldimethylsilyl.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 5, wherein: L2 is 4, 4'-dimethoxytrityl.

8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: L3 is -CH2HNC(O)(CH2)2- or , " ” indicates the connection location.

9. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: L4 is substituted or unsubstituted purine or pyrimidine, or any salt thereof.

10. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 9, wherein: L4 is substituted or unsubstituted adenine or a salt thereof, guanine or a salt thereof, cytosine or a salt thereof, thymine or a salt thereof, or uracil or a salt thereof.

11. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 10, wherein: L4 is substituted adenine or a salt thereof, cytosine or a salt thereof, or thymine or a salt thereof.

12. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: R1, R2 and R3 are all hydrogen.

13. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: In formula (I) Some are in (R)-, (S)- or (rac)-configuration.

14. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein: The compound is a compound having the structure of any one of the following YK-GNA-001, YK-GNA-002, YK-GNA-003, YK-GNA-004, YK-GNA-005 and YK-GNA-006: 、 、 、 、 、 。 15. An oligonucleotide modified with a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, characterized in that: The oligonucleotide comprises a structural unit formed by the compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

16. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 15, wherein: The oligonucleotide comprises a structural unit having the structure of formula (II): in, represents the position where the phosphate bond is formed; L3 is -CH2HNC(O)(CH2) n -、-C(O)NH(CH2) n -、-(CH2) n -、-(CH2) n O(CH2) m -or , wherein n and m are each independently any integer from 0 to 5, R4 and R5 are each independently hydrogen, alkylene, alkenylene, -R 4' -OR 4'' , -R 4' -CO-R 4'' , -R 4' -N(R 4''' )-R 4'' , where R 4' , R 4'' , R 4''' Each is independently hydrogen, oxygen, C1-C6 alkylene, C1-C6 alkyl, C1-C6 acyl or C1-C6 alkyleneoxy; R1, R2 and R3 are each independently hydrogen, alkyl, alkenyl or aryl; L4 , or , " ” indicates the connection location.

17. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 15, wherein: Each structural unit in the oligonucleotide may or may not be modified independently.

18. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 17, wherein: The other modifications include connecting modifying groups and / or structural unit backbone modifications.

19. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 15, wherein: The type of the oligonucleotide includes any one of the group consisting of small interfering RNA, DNA, microRNA, small activating RNA, small guide RNA, transfer RNA, antisense oligonucleotide or aptamer, or a combination of at least two thereof.

20. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 19, wherein: The type of oligonucleotide is small interfering RNA.

21. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 20, wherein: The small interfering RNA includes a sense strand and an antisense strand.

22. The oligonucleotide according to claim 20 or a pharmaceutically acceptable salt thereof, wherein: The length of the small interfering RNA is 15-30 nucleotides.

23. The oligonucleotide according to claim 20 or a pharmaceutically acceptable salt thereof, wherein: The nucleotide structural unit at at least one position in the sense strand and / or antisense strand of the small interfering RNA is replaced by the structural unit represented by formula (II).

24. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 23, wherein: The nucleotide structural unit at at least one of the 2nd to 8th positions in the 5' region of the antisense strand of the small interfering RNA is replaced with the structural unit represented by formula (II).

25. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 15 to 24, wherein: The oligonucleotide is a double-stranded RNAi agent that reduces the expression of PCSK9.

26. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 25, wherein: The oligonucleotide comprises any one or a combination of at least two selected from the group consisting of the following oligonucleotide duplexes formed by pairing of a sense strand and an antisense strand: (1) The sense strand has a sequence as shown in SEQ ID NO. 5 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 6 or a fragment thereof, or a modified sequence thereof; (2) The sense strand has a sequence as shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 8 or a fragment thereof, or a modified sequence thereof; (3) The sense strand has the sequence shown in SEQ ID NO. 9 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 10 or a fragment thereof, or a modified sequence thereof; (4) The sense strand has a sequence as shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 12 or a fragment thereof, or a modified sequence thereof; (5) The sense strand has a sequence as shown in SEQ ID NO. 13 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 14 or a fragment thereof, or a modified sequence thereof; (6) The sense strand has a sequence as shown in SEQ ID NO. 15 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 16 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

27. A conjugate comprising a ligand and an oligonucleotide, wherein: The oligonucleotide comprises a structural unit provided by the compound of any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

28. A conjugate comprising a ligand and the oligonucleotide according to any one of claims 15 to 26 or a pharmaceutically acceptable salt thereof.

29. The conjugate according to claim 27 or 28, wherein The oligonucleotide is a double-stranded RNAi agent, which comprises any one or a combination of at least two selected from the group consisting of the following oligonucleotide duplexes formed by pairing of a sense strand and an antisense strand: (i) the sense strand has the sequence shown in SEQ ID NO. 31; and the antisense strand has the sequence shown in SEQ ID NO. 8; (ii) the sense strand has the sequence shown in SEQ ID NO. 32; and the antisense strand has the sequence shown in SEQ ID NO. 10; (iii) the sense strand has the sequence shown in SEQ ID NO. 33; and the antisense strand has the sequence shown in SEQ ID NO. 12; (iv) the sense strand has the sequence shown in SEQ ID NO. 34; and the antisense strand has the sequence shown in SEQ ID NO. 14; And / or, the ligand is a compound having the following structure G4, G5, G6 or G7: ， ， ， 。 30. A pharmaceutical composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 15 to 26, or the conjugate according to any one of claims 27 to 29.

31. Use of the compound according to any one of claims 1 to 14 and a pharmaceutically acceptable salt thereof in reducing off-target effects of oligonucleotides.

32. Use of the compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, or the oligonucleotide according to any one of claims 15 to 26 or a pharmaceutically acceptable salt thereof, or the conjugate according to any one of claims 27 to 29 in the preparation of an oligonucleotide drug.

33. The use according to claim 32, wherein: The oligonucleotide drug is a drug for treating PCSK9-related diseases.

34. The use according to claim 33, wherein: The PCSK9-related disease is selected from any one or a combination of at least two of the group consisting of hypercholesterolemia, atherosclerosis, dyslipidemia and cardiovascular and cerebrovascular diseases.

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