A nucleoside analog and its application in reducing the off-target activity of oligonucleotide drugs

Abstract

The present invention relates to the field of biomedicine, and more particularly to a nucleoside analog and its use in reducing the off-target activity of oligonucleotide drugs. The nucleoside analog provided by the present invention can be used in the preparation of oligonucleotide drugs and can be coupled to the seed region of siRNA, thereby effectively improving the specificity of the siRNA, reducing the off-target effects of the siRNA while maintaining the silencing effect of the target gene.

Description

Technical Field

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

[0002] RNAi (RNA interference) technology is a significant breakthrough in molecular biology and is now widely used in a variety of fields, including agriculture, forestry, and medicine. RNAi is a sequence-specific post-transcriptional gene silencing process that occurs widely in organisms and is initiated by double-stranded RNA (dsRNA) homologous to the target gene sequence. During this process, Dicer, a member of the RNase III family of ribonucleases, cleaves dsRNA into small interfering RNAs (siRNAs) consisting of 21-25 nucleotides. The siRNAs then act as mediators to specifically degrade mRNAs with the same sequence, thereby blocking the expression of the corresponding gene. However, researchers have discovered that in practical applications, siRNAs can have off-target effects, resulting in suboptimal silencing results.

[0003] Because natural RNA double strands are unstable, siRNA design needs to be adjusted through chemical modification. This chemical modification needs to eliminate or reduce the off-target effects of siRNA while not affecting the ability of gene silencing. However, the anti-off-target effect of siRNA obtained by chemical modification methods currently in the art is still unsatisfactory. Summary of the Invention

[0004] To overcome the off-target effects of siRNA in the field of small nucleic acid drugs, the present invention provides a nucleoside analog and its use 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 oligonucleotides and conjugates prepared from them have the effect of preventing off-target effects.

[0005] The inventors of this invention cleverly discovered that by using triazole or amide structures as linkers, they can modularly, simply, and rapidly construct nucleoside monomer compounds based on these structures. These compounds can be coupled to the seed region of siRNA via solid-phase synthesis, effectively enhancing the specificity of the siRNA and maintaining target gene silencing while minimizing off-target effects.

[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:

[0008] In a first aspect, the present invention provides a nucleoside analogue, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] in,

[0011] L1 is hydrogen, a hydroxyl protecting group or a phosphorus-containing group reactive with nucleophiles;

[0012] L2 is hydrogen or a hydroxyl protecting group;

[0013] 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;

[0014] L4 is a substituted or unsubstituted base or a salt thereof;

[0015] R1, R2 and R3 are each independently hydrogen, alkyl, alkenyl or aryl.

[0016] In the present invention, a "hydroxyl-protecting group" refers to a protecting group that protects the hydroxyl group in the compounds / oligonucleotides / conjugates provided herein from reaction during a reaction (e.g., during liquid-phase / solid-phase nucleotide synthesis, during oligonucleotide synthesis, during the preparation of a conjugate between an oligonucleotide and a ligand, etc.), preventing the reaction or allowing the group to be restored after the reaction is complete. Any hydroxyl-protecting group commonly used in the art may be used in the present invention, as long as it can achieve the aforementioned purpose.

[0017] In the present invention, "nucleophilic reagent" can also be called "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 an unshared electron pair nucleophilic reagent (lone-pair nucleophiles), σ-bond nucleophiles (σ-bond nucleophiles) and π-bond nucleophiles (π-bond nucleophiles), etc. "Phosphorus-containing group reactive with nucleophilic reagent" is a group containing P element that can react with the above-mentioned nucleophilic reagent. Any active phosphorus group that can be used in oligonucleotides in the art 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, phosphorus-containing chiral auxiliary agents refer to phosphorus-containing groups with chirality, for example, 、 、 、 、 、 、 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.

[0018] 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 linear or branched alkylene group, and the like, for example, -CH2-O-CH2-, -CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-, -CH2-O-CH2CH2-O-CH2, or -CH2-O-CH2CH2-O-CH2CH2-O-CH2-. Preferably, the number of ether bonds in the ether bond-containing group may be 1-3 (for example, 1, 2, or 3).

[0019] In the present invention, a "carbonyl-containing group" may include a carbonyl group, a group having a carbon-oxygen double bond connected to any carbon position in a straight-chain 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.

[0020] 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 nitrogen atoms in the nitrogen-containing group may be 1-2 (e.g., 1 or 2). For example, the group 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.

[0021] 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.

[0022] According to a preferred embodiment of the present invention, wherein 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.

[0023] Preferably, L1 is , " ” indicates the connection location.

[0024] According to a preferred embodiment of the present invention, wherein 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.

[0025] Preferably, L2 is 4,4'-dimethoxytrityl.

[0026] According to a preferred embodiment of the present invention, wherein L3 is -CH2HNC(O)(CH2)2- or , " ” indicates the connection location.

[0027] According to a preferred embodiment of the present invention, L4 is any one of substituted or unsubstituted purine or pyrimidine or a salt thereof.

[0028] 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;

[0029] More preferably, L4 is substituted adenine or a salt thereof, cytosine or a salt thereof, or thymine or a salt thereof.

[0030] According to a preferred embodiment of the present invention, R1, R2 and R3 are all hydrogen.

[0031] 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).

[0032] 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:

[0033] 、 、 、

[0034] 、 、 .

[0035] The second aspect of the present invention provides an oligonucleotide modified with the compound of formula (I) or a pharmaceutically acceptable salt thereof as described in the first aspect, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises structural units formed from the compound of the first aspect or a pharmaceutically acceptable salt thereof. That is, the oligonucleotide provided by the present invention can be formed by replacing some structural units in a conventional oligonucleotide with nucleotides synthesized from a nucleoside analog of the present invention (i.e., a compound of formula (I) or a pharmaceutically acceptable salt thereof) and phosphoric acid.

[0036] In the oligonucleotides provided herein, some or all of the structural units (nucleotides) may contain the nucleoside analogs of the present invention. When some of the structural units contain the nucleoside analogs of the present invention, the remaining structural units in the oligonucleotide may be provided by conventional nucleotides or by nucleotides containing other existing nucleoside analogs.

[0037] In the present invention, the structural unit of an oligonucleotide refers to a nucleotide. As is well known in the art, a nucleotide is a compound composed of a base (purine or pyrimidine), a pentose (ribose or deoxyribose), and phosphate. The base and the pentose condense to form nucleic acids, which are then further condensed with phosphate to form nucleotides. 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. "An oligonucleotide comprising a structural unit formed from 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 multiple nucleotides contained in the oligonucleotide, the nucleoside portion of at least one nucleotide is replaced with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0038] In some preferred embodiments, for an oligonucleotide with a length of no more than 30 nucleotides (eg, 15-30 nucleotides), the number of structural units (nucleotides) containing the nucleoside analogs provided by the present invention is no more than 3.

[0039] According to some preferred embodiments of the present invention, the oligonucleotide comprises a structural unit having a structure of formula (II):

[0040]

[0041] in, represents the position where the phosphate bond is formed;

[0042] 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;

[0043] R1, R2 and R3 are each independently hydrogen, alkyl, alkenyl or aryl;

[0044] L4 is 、 or , " ” indicates the connection location.

[0045] According to a preferred embodiment of the present invention, each structural unit in the oligonucleotide may or may not have undergone other modifications. "Other modifications" refer to any modifications performed on the oligonucleotide in addition to the aforementioned modifications performed with the compound of formula (I) or a pharmaceutically acceptable salt thereof provided by the present invention. The present invention does not limit the specific manner of such other modifications, and any method known in the art for modifying oligonucleotides is applicable to the present invention. Preferably, such other modifications include linker modification groups and / or structural unit backbone modifications. "Linker modification groups" refer to additional modification groups attached to the structural units of the oligonucleotide. Any group known in the art for modifying oligonucleotides by attaching to the structural units of the oligonucleotide is applicable to the present invention, such as 2'-OMe modification, 2'-F modification, 5'-(E)-VP modification, etc. "Structural unit backbone modifications" refer to modifications to the backbone of the structural units (e.g., nucleotides) of the oligonucleotide, resulting in structural changes, or modifications to the phosphate bonds between two adjacent nucleotides in the oligonucleotide (e.g., replacing a phosphate bond with a phosphorothioate bond).

[0046] 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;

[0047] Preferably, the oligonucleotide is of the type of small interfering RNA.

[0048] The term "small interfering RNA" or "siRNA" is well known in the art. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silencing RNA. siRNA typically comprises a sense strand (also known as a passenger strand) and an antisense strand (also known 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 (such as at least 95% complementary, such as fully complementary) to the target nucleic acid sequence (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 strands can form blunt-ended duplexes, or preferably, the 3' ends of the sense strand and the antisense strand can 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.

[0049] The term "antisense strand" refers to a strand of an 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, mismatches can be within the interior or terminal regions of the molecule. Typically, the most tolerated mismatches are within the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' and / or 3' ends.

[0050] The term "sense strand," as used herein, refers to the strand of an siRNA that includes a region that is substantially complementary to a region of the antisense strand (as that term is defined herein).

[0051] 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.

[0052] According to a preferred embodiment of the present invention, the small interfering nucleotides include a sense strand and an antisense strand.

[0053] Preferably, the length of the small interfering RNA is 15-30 nucleotides (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 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 represented by formula (II).

[0054] 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).

[0055] 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.

[0056] More preferably, the nucleotide structural unit at at least one of positions 2 to 8 in the 5' region of the antisense strand of the small interfering RNA is replaced with a structural unit represented by formula (II).

[0057] 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 oligonucleotide duplexes formed by pairing the following sense strand and antisense strand:

[0058] (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;

[0059] (2) The sense strand has the sequence shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence thereof; the antisense strand has the sequence shown in SEQ ID NO. 8 or a fragment thereof, or a modified sequence thereof;

[0060] (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;

[0061] (4) The sense strand has the sequence shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence thereof; the antisense strand has the sequence shown in SEQ ID NO. 12 or a fragment thereof, or a modified sequence thereof;

[0062] (5) The sense strand has the sequence shown in SEQ ID NO. 13 or a fragment thereof, or a modified sequence thereof; the antisense strand has the sequence shown in SEQ ID NO. 14 or a fragment thereof, or a modified sequence thereof;

[0063] (6) The sense strand has a sequence as shown in SEQ ID NO. 15 or a fragment thereof, or a modified sequence thereof; the antisense strand has a sequence as shown in SEQ ID NO. 16 or a fragment thereof, or a modified sequence thereof.

[0064] 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 according to the first aspect or a pharmaceutically acceptable salt thereof.

[0065] A "conjugate" refers to a compound formed by covalent bonding. In the present invention, a conjugate is a nucleic acid conjugate, comprising at least one oligonucleotide molecule and one other compound molecule (e.g., a "ligand" in the present invention). In a conjugate, the oligonucleotide molecule functions to exert its biological function (e.g., in the case of siRNA, it functions as an RNAi agent), while the ligand's functions include providing targeting (enabling the conjugate to target tissues / cells) and protecting the oligonucleotide. (Nucleic acid) conjugates are widely used in biological and clinical medical research (e.g., as biomarkers for detection, diagnosis, and treatment), drug development, and bioengineering technologies (e.g., biocatalysis, biosensors, and biomaterials).

[0066] 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:

[0067] (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;

[0068] (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;

[0069] (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;

[0070] (iv) The sense strand has the sequence shown in SEQ ID NO. 34; the antisense strand has the sequence shown in SEQ ID NO. 14.

[0071] According to a preferred embodiment of the present invention, the ligand is G4, G5, G6 or G7 having the following structure:

[0072] ,

[0073] ,

[0074] ,

[0075] .

[0076] The fourth aspect of the present invention provides a conjugate comprising a ligand and the oligonucleotide according to the second aspect or a pharmaceutically acceptable salt thereof.

[0077] 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.

[0078] The fifth aspect of the present invention provides a pharmaceutical composition comprising 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.

[0079] 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 ingredient or one of the pharmaceutically active ingredients. 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", that is, compatible with the other ingredients of the formulation, including the nucleoside analogs, oligonucleotides, conjugates, or combinations thereof of the present invention, and not injurious or substantially injurious 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, absorption delaying agents, etc., which are compatible with the activity of the nucleoside analogs, oligonucleotides, conjugates, or compositions thereof of the present invention and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the composition. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compounds useful in the present invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.Any carrier, adjuvant, or auxiliary material that can be used for pharmaceutical preparations 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 regulators, and the like.

[0080] Oligonucleotides, especially siRNAs, comprising nucleotides formed by the nucleoside analogs provided by the present invention as structural units have better specificity than siRNAs and also significantly reduce off-target effects.

[0081] 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.

[0082] Preferably, the oligonucleotide is siRNA.

[0083] 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).

[0084] 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 oligonucleotide drugs.

[0085] According to a preferred embodiment of the present invention, the oligonucleotide drug is a drug for treating diseases related to PCSK9.

[0086] 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.

[0087] 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.

[0088] Through the above technical scheme, the present invention designs a series of novel anti-off-target nucleoside compounds. 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.

[0089] Compared with the prior art, the present invention has at least the following beneficial effects:

[0090] 1. The structure is completely different from the existing anti-off-target nucleoside compounds.

[0091] The novel nucleoside compound designed by the present invention incorporates an amide or triazole structure in its linker arm, which is completely different from the commonly used structures in the prior art. This compound can be coupled to the seed region of siRNA via solid-phase synthesis, effectively enhancing the specificity of the siRNA and maintaining target gene silencing while reducing off-target effects.

[0092] 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.

[0093] For example, the 6th position of D84-DV27P is modified, and D84-DV27P-GA1-6 (modified by 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).

[0094] 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.

[0095] D84-DV27P-GA1-6 (modified with the present compound YK-GNA-001) reduced LDL-C levels in mouse serum by 38.1% and 40.4% on days 7 and 14, respectively, which was higher than the parent sequence. The reduction levels were 9.3% and 10.2% higher than those of D81-DV25G5-GNA-6 (modified with the existing compound GNA), respectively.

[0096] 3. Compared with the prior art anti-off-target nucleoside compounds, the siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention have better anti-off-target effects.

[0097] 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 the off-target gene ITGAV by up to 77%, and by 76% compared with D84-DV27P-G1-2-6 (modified with the existing compound 1-2). BRIEF DESCRIPTION OF THE DRAWINGS

[0098] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0099] Figure 1 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 on PCSK9 mRNA.

[0100] Figure 2 The inhibition rates 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.

[0101] 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.

[0102] 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.

[0103] Figure 5Schematic diagram of the structure of the conjugate prepared in Example 3. DETAILED DESCRIPTION

[0104] 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 range of values ​​of a parameter is listed, its purpose is to indicate that the intermediate values ​​and ranges of these quoted values ​​are also within the scope of the present invention.

[0105] The embodiments of the present invention are intended to further illustrate the present 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 are within the scope of the present invention.

[0106] The present invention may be implemented in other specific forms without departing from the essential 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 of any other embodiment or multiple other embodiments to produce additional embodiments. The present invention includes additional embodiments resulting from such combinations.

[0107] All publications and patents mentioned in the present invention are hereby incorporated into the present invention in their entirety by reference. 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.

[0108] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0109] Unless otherwise specified, all technical and scientific terms used in this invention have the common meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition in this invention shall prevail.

[0110] Except in the working examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified in all cases by the term "about". It should also be understood that any numerical range recited herein is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

[0111] As used herein, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the terms also encompass "consisting essentially of" or "consisting of."

[0112] The oligonucleotides of the present invention include single-stranded oligonucleotides (such as antisense oligonucleotides, abbreviated as ASOs) and double-stranded oligonucleotides (such as oligonucleotide duplexes composed of two oligonucleotide chains with complementary base pairing, such as small interfering nucleotides, etc.).

[0113] In one embodiment, the oligonucleotide is selected from small interfering nucleotides (siRNA), DNA, microRNA (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.

[0114] The oligonucleotides of 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 only differences in the nucleobase sequence. Natural herein refers to situations corresponding to naturally occurring RNA or DNA.

[0115] In the oligonucleotide sequence of the present invention, the abbreviations of each base and modification are defined with reference to conventional definitions in the art. In the art, "G", "C", "A", "T" and "U" generally 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 generally 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", and "U" 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 described in further detail below) or an alternative replacement portion. Those skilled in the art will appreciate that guanine, cytosine, adenine and uracil can be replaced by other parts without substantially changing the base pairing properties of an oligonucleotide (including a nucleotide with such a replacement portion). For example, without limitation, nucleotides comprising inosine as their bases can base pair with nucleotides comprising adenine, cytosine or uracil. Thus, nucleotides containing uracil, guanine or adenine can be replaced by nucleotides 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 moieties are suitable for use in the compositions and methods characterized by the present invention.

[0116] 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 a base, respectively; m represents that the nucleotide adjacent to its left is a 2'-OMe-modified nucleotide (e.g., Am represents a 2'-OMe-modified adenine nucleotide); f represents that the nucleotide adjacent to its left is a 2'-F-modified nucleotide (e.g., Af represents a 2'-F-modified adenine nucleotide); s represents that the two adjacent nucleotides on its left and right are connected by a phosphorothioate bond (e.g., Us-A represents that a uracil nucleotide and an adenine nucleotide are connected by a phosphorothioate bond); EVP represents that the nucleotide adjacent to its left is a 5'-(E)-VP-modified nucleotide (e.g., UEVP represents a 5'-(E)-VP-modified uracil nucleotide).

[0117] Example

[0118] The present invention will be further described below with reference to 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. The implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the materials, reagents, etc. used can be obtained through commercial channels unless otherwise specified. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0119] Example 1: Synthesis of off-target nucleoside phosphoramidite compounds

[0120] 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.

[0121] 1. Synthesis of YK-GNA-001

[0122] The synthetic route is as follows:

[0123]

[0124] Step 1: Synthesis of 1-3

[0125] 1-2 (103.80 g, 0.58 mol, 1.20 eq.) was added to a reaction flask and rotary evaporated twice with ultra-dry acetonitrile. Ultra-dry DCM (630 mL) was added and stirred to dissolve. HBTU (218.61 g, 0.58 mol, 1.20 eq.) and DIPEA (93.11 g, 0.72 mol, 1.50 eq.) were added while maintaining the temperature at 0-10°C. The mixture was stirred for approximately 30 minutes. 1-1 (63.04 g, 0.48 mol, 1.00 eq.) was added, the mixture was warmed to room temperature, and stirred for 15 hours. The reaction mixture was diluted with DCM (700 mL) and washed sequentially with saturated NaHCO₃ solution (1.0 L × 3) and saturated NaCl solution (1.0 L × 2). The mixture was dried over anhydrous Na₂SO₄, and the solvent was removed to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 4 / 1 to 1 / 1) to give 80.00 g of an oily product 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.

[0126] Step 2: Synthesis of 1-4

[0127] Compound 1-3 (35.00 g, 0.12 mol, 1.00 eq.) and methanol (350 mL) were added to a reaction flask and stirred to dissolve. Pd / C (8.00 g, 10%) was added, and the atmosphere was replaced with nitrogen three times. Hydrogen was introduced into the reaction system, and the reaction was stirred at room temperature for 15 hours. The Pd / C was removed by filtration, and the filter cake was washed with methanol (50 mL × 2). The solvent was removed 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 a 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.

[0128] Step 3: Synthesis of 1-6

[0129] PPh3 (19.41 g, 74.05 mmol, 1.00 eq.) and 1-5 (29.00 g, 121.38 mmol, 1.64 eq.) were added to a reaction flask and co-evaporated twice with ultra-dry acetonitrile (100 mL). The mixture was transferred to a 1 L three-necked flask, and ultra-dry THF (600 mL) and 1-4 (15.00 g, 73.80 mmol, 1.00 eq.) were added. DIAD (22.40 g, 115.73 mmol, 1.57 eq., diluted in 100 mL of ultra-dry THF) was added dropwise at 0-10°C. The mixture was allowed to warm to room temperature and stirred for 15 hours. The solvent was removed by concentration, and acetonitrile (300 mL) was added. The mixture was stirred for 20 minutes, filtered, and the filtrate was concentrated to dryness to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 4 / 1 to 0 / 1) to obtain 18.20 g of the product in a yield of 58.1%. 1H 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.

[0130] Step 4: Synthesis of 1-7

[0131] Compound 1-6 (12.60 g, 29.7 mmol, 1.00 eq.) and 80% HOAc (315 mL) were added to a reaction flask. The mixture was heated to 50°C and stirred for 4 hours. The reaction mixture was concentrated to dryness, acetonitrile (50 mL) was added, and the mixture was stirred for 1 hour. A solid precipitated and was filtered. The filter cake was added to a mixture of ethanol (210 mL), DMF (80 mL), and water (54 mL). The mixture was heated to 70-75°C and stirred until dissolved. The mixture was cooled to 30°C over 1 hour and stirred for approximately 30 minutes. A solid precipitated and was filtered. The filter cake was added to a mixture of ethanol (190 mL), DMF (50 mL), and water (54 mL). The mixture was heated to 70-75°C and stirred until dissolved. The mixture was cooled to 30°C over 1 hour and stirred for approximately 1 hour. A solid precipitated and was filtered to obtain 4.22 g of a 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.

[0132] Step 5: Synthesis of 1-8

[0133] Compound 1-7 (6.30 g, 16.40 mmol, 1.00 eq.) was added to a reaction flask and co-rotated with ultra-dried pyrolyz ... 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

[0134] Step 6: Synthesis of YK-GNA-001

[0135] Compound 1-8 (4.80 g, 7.00 mmol, 1.00 eq.) was added to a reaction flask and rotary evaporated once with ultra-dry acetonitrile (50 mL), then twice with ultra-dry DCM (50 mL). Ultra-dry DCM (50 mL) was added and stirred to dissolve. Tetrazole (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-cooling conditions. The mixture was warmed to room temperature and stirred for 2 hours. The reaction solution was diluted with DCM (50 mL) under ice-cooling conditions and washed with saturated NaHCO₃ solution (80 mL) and saturated NaCl solution (100 mL × 2). The residue was dried over anhydrous Na₂SO₄, and the solvent was removed to obtain a residue. The residue was purified by column chromatography (SiO2, alkalized with 0.5% TEA, n-heptane / EA = 4 / 1-0 / 1, EA / MeCN = 25 / 1, alkalized with 0.1% TEA) to yield 3.02 g of a white solid. The solid was added to a 10 mL mixture of MTBE / DCM (10 / 1), stirred to dissolve, and n-heptane (50 mL) was added dropwise. Stir for approximately 10 minutes until a solid precipitated, which was filtered. This process was repeated once for the filter cake. The filter cake was rotary evaporated twice with acetonitrile (20 mL) to yield 2.50 g of a white solid. Dissolve 2.50 g of the white solid in DCM (30 mL) and purify it 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 twice with acetonitrile (20 mL) to obtain 1.50 g of a 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.

[0136] 2. Synthesis of YK-GNA-002

[0137] The synthetic route is as follows:

[0138]

[0139] Step 1: Synthesis of 2-2

[0140] 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.

[0141] Step 2: Synthesis of 2-3

[0142] Using 2-2 (9.00 g, 22.50 mmol) as starting 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.

[0143] Step 3: Synthesis of 2-4

[0144] Using 2-3 (7.00 g, 19.40 mmol, 1.00 eq.) and DMTrCl (7.24 g, 21.40 mmol, 1.10 eq.) as starting materials, following the method for synthesizing compound 1-8, 10.04 g of a 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.

[0145] Step 4: Synthesis of YK-GNA-002

[0146] 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 starting materials, following the method for synthesizing YK-GNA-001, 2.03 g of a 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.

[0147] 3. Synthesis of YK-GNA-003

[0148] The synthetic route is as follows:

[0149]

[0150] Step 1: Synthesis of 3-3

[0151] Compound 3-1 (50.00 g, 378.33 mmol, 1.00 eq.) and DCM (1.3 L) were added to a reaction flask and stirred to dissolve. TBAI (4.20 g, 11.37 mmol, 0.03 eq.) and 40% NaOH solution (1.2 L) were added. The mixture was stirred at room temperature for half an hour, and compound 3-2 (47.3 g, 397.61 mmol, 1.05 eq.) was added. The mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (1.5 L), separated, and the aqueous phase was extracted with DCM (1.5 L). The combined organic phases were washed twice with water, dried over anhydrous Na2SO4, and concentrated to remove the solvent to obtain a residue. The residue was purified by column chromatography (SiO2, PE / EA = 20 / 1 to 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.

[0152] Step 2: Synthesis of 3-5

[0153] 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 starting materials, following the method for synthesizing compound 1-6, 4.97 g of a white solid was obtained with a yield of 21.7%. MS [M + H] + :309.2.

[0154] Step 3: Synthesis of 3-6

[0155] A mixture of 3-5 (5.00 g, 16.22 mmol, 1.00 eq.) and THF / H₂O (v / v, 1 / 1, 40.0 mL) was added to a reaction flask and stirred to dissolve. Then, 3-3 (2.90 g, 17.04 mmol, 1.05 eq.) was added. Anhydrous CuSO₄ (0.40 g, 2.51 mmol, 0.15 eq.) and sodium VC (1.61 g, 8.13 mmol, 0.5 eq.) were added sequentially under ice-cooling. The mixture was warmed to room temperature and stirred for 3 hours. Methanol (50 mL) was added to the reaction mixture, and the mixture was concentrated to obtain a residue. The residue was purified 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. 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.

[0156] Step 4: Synthesis of 3-7

[0157] Using 3-6 (6.00 g, 12.54 mmol, 1.00 eq.) as starting material and following the synthesis method of compound 1-7, 2.80 g of a white solid was obtained with a yield of 50.9%. MS [M + H] + :439.2.

[0158] Step 5: Synthesis of 3-8

[0159] Using 3-7 (2.80 g, 6.39 mmol, 1.00 eq.) and DMTrCl (2.60 g, 7.67 mmol, 1.20 eq.) as starting materials, according to the method for synthesizing compound 1-8, 3.51 g of a 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.

[0160] Step 6: Synthesis of YK-GNA-003

[0161] 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.

[0162] 4. Synthesis of YK-GNA-004

[0163] The synthetic route is as follows:

[0164]

[0165] Step 1: Synthesis of 4-1

[0166] 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 add DIAD (62.66 g, 310.04 mmol, 1.50 eq. diluted with 270 mL ultra-dry THF) dropwise. Warm to room temperature and stir for 15 hours. Water (500 mL) and ethyl acetate (500 mL) were added to the reaction mixture, stirred for 10 minutes, and filtered through celite. The filtrate was separated, and the organic phase was washed sequentially with water (1 L) and saturated NaCl solution (1 L), 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.

[0167] Step 2: Synthesis of 4-2

[0168] 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.

[0169] Step 3: Synthesis of 4-3

[0170] Using 4-2 (4.00 g, 8.80 mmol) as the starting material, according to the method for synthesizing compound 1-7, 2.01 g of a 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.

[0171] Step 4: Synthesis of 4-4

[0172] 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.

[0173] Step 5: Synthesis of YK-GNA-004

[0174] 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 starting materials, following the method for synthesizing YK-GNA-001, 1.52 g of a 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.

[0175] 5. Synthesis of YK-GNA-005

[0176] The synthetic route is as follows:

[0177]

[0178] Step 1: Synthesis of 5-2

[0179] 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 (400 MHz, DMSO- d6) 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.

[0180] Step 2: Synthesis of 5-3

[0181] 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.

[0182] Step 3: Synthesis of 5-4

[0183] Compound 5-3 (8.40 g, 17.89 mmol, 1.00 eq.) and a 1,4-dioxane solution (100 mL) were added to a reaction flask. 1 M NaOH solution (30 mL) was added dropwise under ice-cooling conditions. The mixture was warmed to room temperature and stirred for 5.5 hours. The pH was adjusted to approximately 8-9 with 1 M HCl under ice-cooling conditions. The reaction mixture was concentrated to dryness to obtain an oil. The oil was purified by column chromatography (SiO2, DCM / MeOH = 20 / 1–5 / 1) to yield 3.80 g of a white solid, with a yield of 58.1%. MS [M + H]+ :366.2.

[0184] Step 4: Synthesis of 5-5

[0185] Using 5-4 (3.80 g, 10.4 mmol) as starting material, according to the method for synthesizing compound 1-7, 1.92 g of white solid was obtained with a yield of 56.2%.

[0186] Step 5: Synthesis of 5-6

[0187] Using 5-5 (1.90 g, 5.84 mmol, 1.00 eq.) and DMTrCl (2.30 g, 6.79 mmol, 1.20 eq.) as starting materials, following the method for synthesizing compound 1-8, 2.71 g of a 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.

[0188] Step 6: Synthesis of YK-GNA-005

[0189] Using compound 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 starting materials, following the method for synthesizing YK-GNA-001, 2.02 g of a 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.

[0190] 6. Synthesis of YK-GNA-006

[0191] The synthetic route is as follows:

[0192]

[0193] Step 1: Synthesis of 6-2

[0194] 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 a 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).

[0195] Step 2: Synthesis of 6-3

[0196] 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 a reaction flask and stir to dissolve. Add VCNa (5.00 g, 25.2 mmol, 4.0 eq.) to prepare a solution for later use.

[0197] Compound 6-2 (1.54 g, 9.08 mmol, 1.40 eq.), compound 3-5 (2.00 g, 6.48 mmol, 1.00 eq.), and DMSO (48 mL) were added to a reaction flask and stirred to dissolve. The temperature was lowered to 0-10°C, and the above solution was added dropwise. The mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was diluted with water (200 mL) and extracted with DCM (200 mL × 2). The combined organic phases were washed sequentially with water and saturated NaCl solution, dried over anhydrous Na2SO4, and the solvent was removed by vortexing to obtain a residue. The residue was purified by column chromatography (SiO2, MeOH / DCM = 0 / 1 - 1 / 19) to obtain 0.77 g of a 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.

[0198] Step 3: Synthesis of 6-4

[0199] Using 6-3 (1.23 g, 2.57 mmol) as starting 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.

[0200] Step 4: Synthesis of 6-5

[0201] 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 a 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.

[0202] Step 5: Synthesis of YK-GNA-006

[0203] Compound 6-5 (0.44 g, 0.57 mmol, 1.00 eq.) and ultra-dry DCM (18 mL) were added to a reaction flask and stirred to dissolve. Subsequently, DIPEA (0.37 g, 2.87 mmol, 5.0 eq.) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.34 g, 1.46 mmol, 2.50 eq.) were added. The mixture was stirred at room temperature for approximately 1.5 hours. The reaction mixture was diluted with DCM (50 mL), washed sequentially with saturated NaHCO₃ solution (100 mL × 2) and saturated NaCl solution (100 mL), dried over anhydrous Na₂SO₄, and the solvent was removed by vortexing to obtain a residue. The residue was purified by column chromatography (SiO₂, PE / EA = 0 / 1, basified with 2.0% TEA) to afford 0.26 g of a white solid in 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.

[0204] 7. Synthesis of Compound 1-2

[0205]

[0206] According to the synthesis method in WO2022028462A1, pages 76-77, 537 mg of the product was obtained with a total yield of 10.6%.

[0207] Example 2: Synthesis of oligonucleotides without GalNAc conjugation

[0208] In this example, two siRNA molecules were synthesized, named D84-DV27P and D81-DV25, and then modified at positions 6 and 7 to prevent off-target. The specific synthesized and modified siRNA molecules are shown in Table 1.

[0209] The sequence of D84-DV27P is as follows:

[0210] Sense strand (SS) (SEQ ID NO. 1):

[0211] 5'-Gms-Ums-Um-Um-Um-Gm-Uf-Am-Gf-Cf-Af-Um-Um-Um-Um-Um-Af-Um -Um-Am-Am-3'

[0212] Antisense strand (AS) (SEQ ID NO. 2):

[0213] 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'

[0214] The D81-DV25 sequence is as follows:

[0215] Sense strand (SS) (SEQ ID NO. 3):

[0216] 5'-Cms-Ums-Um-Um-Um-Gm-Uf-Am-Af-Cf-Uf-Um-Gm-Am-Am-Gm-Af-Um -Am-Um-Um-3'

[0217] Antisense strand (AS) (SEQ ID NO. 4):

[0218] 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'

[0219] Table 1 siRNA double-stranded molecules without GalNAc conjugation

[0220]

[0221] Among them, A, U, C, and G each generally represent a nucleotide containing adenine, uracil, cytosine, and guanine as a base, 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.

[0222] Among them, the structures of GA1(A), GA2(C), GT3(A), GT4(C), GT5(T), GT6(A) and G1-2(A) are as follows:

[0223] .

[0224] 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 by YK-GNA-001, GA2(C) represents a nucleotide obtained by replacing cytosine nucleoside in a conventional cytosine nucleotide by YK-GNA-002, and GT3(A) represents a nucleotide obtained by replacing a conventional adenine nucleotide by YK-GNA-003. , GT4(C) represents a nucleotide obtained by replacing the cytosine nucleoside in a conventional cytosine nucleotide by YK-GNA-004, GT5(T) represents a nucleotide obtained by replacing the thymidine nucleoside in a conventional thymidine nucleotide by YK-GNA-005, GT6(A) represents a nucleotide obtained by replacing the adenine nucleoside in a conventional adenine nucleotide by YK-GNA-006, and G1-2(A) represents a nucleotide obtained by replacing the adenine nucleoside in a conventional adenine nucleotide by 1-2.

[0225] 1. Preparation of antisense oligonucleotides

[0226] The antisense oligonucleotide chain was synthesized on a solid support using the phosphoramidite solid phase synthesis method.

[0227] When synthesizing the antisense strand of sequence number D84-DV27P, conventional monomers (i.e., nucleoside phosphoramidite monomers without anti-off-targeting) 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.

[0228] When synthesizing the antisense strand of sequence number D81-DV25, conventional monomers (i.e., nucleoside phosphoramidite monomers without anti-off-targeting) 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 strands 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 monomer for solid phase synthesis, respectively; when synthesizing the antisense strand 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. The synthesis scale was 200 nmol.

[0229] 1) Reagent and monomer preparation

[0230] The monomer solution (0.15 M) was prepared by using an acetonitrile solution of the monomer, a 0.25 M solution of 5-benzylthiotetrazole in acetonitrile as an activating agent, a 3% solution of xanthan gum 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.

[0231] 2) Crude product synthesis

[0232] Enter the designated oligonucleotide sequence and set the synthesis program. After checking that everything is correct, start the cyclic oligonucleotide synthesis. Prepare according to the following steps:

[0233] a. Deprotection

[0234] 3% dichloroacetic acid in toluene was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0235] b. Coupling

[0236] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolium as an activating agent, followed by acetonitrile washing.

[0237] c. Oxidation / sulfurization

[0238] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (9 / 1) as an oxidant, followed by rinsing with acetonitrile.

[0239] Sulfurization: Use 3% hydrogenated xanthan gum in pyridine as the sulfurizing agent for sulfurization, and then use acetonitrile for washing.

[0240] d. Hydroxyl protection

[0241] Hydroxyl protection was performed using 10% acetic anhydride in tetrahydrofuran (CAP A) or tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting reagents, followed by rinsing with acetonitrile.

[0242] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[0243] e. Use 3% dichloroacetic acid in toluene as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile to wash.

[0244] 3) Deprotection

[0245] After the synthesis is completed, the solid phase support is transferred to a reactor. After the oligonucleotide is cleaved from the solid phase support with concentrated ammonia (25-28%) at 60°C for 16 hours, the system is cooled to room temperature and then filtered and rinsed with a mixed solution of purified water and ethanol. The filtrates are combined and concentrated at low temperature to obtain a crude residue.

[0246] 4) Purification

[0247] The crude residue after deprotection was dissolved in purified water and subjected to HPLC purification. The product peak solution was collected and the content was measured using an enzyme reader, and the molecular weight was confirmed by ESI MS.

[0248] 2. Preparation of the Sense Strand of Oligonucleotides

[0249] 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.

[0250] 3. Preparation of oligonucleotide duplexes

[0251] Mix the siRNA sense strand and complementary antisense strand in a 1:1 ratio based on UV absorption, heat to 95°C for 3 minutes, and cool to room temperature to form a double strand. The resulting double strand solution is characterized by HPLC for purity, and the content is determined using a microplate reader. The solution is lyophilized to obtain a solid powder for future storage.

[0252] The molecular weight and purity of the obtained siRNA double-strands are shown in Table 2.

[0253] Table 2 Molecular weight and purity of non-GalNAc conjugated siRNA double-stranded molecules

[0254]

[0255] Example 3: Synthesis of GalNAc-conjugated oligonucleotide conjugates

[0256] 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. The sequence numbers are D84-DV27PG5 and D81-DV25G5, respectively. 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.

[0257] The sequence of D84-DV27PG5 is as follows:

[0258] Sense strand (SS) (SEQ ID NO. 29):

[0259] 5'-Gms-Ums-Um-Um-Um-Gm-Uf-Am-Gf-Cf-Af-Um-Um-Um-Um-Um-Af-Um -Um-Am-Am-G5-3'

[0260] Antisense strand (AS) (SEQ ID NO. 2):

[0261] 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'

[0262] The sequence of D81-DV25G5 is as follows:

[0263] Sense strand (SS) (SEQ ID NO. 30):

[0264] 5'-Cms-Ums-Um-Um-Um-Gm-Uf-Am-Af-Cf-Uf-Um-Gm-Am-Am-Gm-Af-Um -Am-Um-Um-G5-3'

[0265] Antisense strand (AS) (SEQ ID NO. 4):

[0266] 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'

[0267] Among them, A, U, C, and G each generally represent a nucleotide containing adenine, uracil, cytosine, and guanine as a base, 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 phosphorothioate group; 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:

[0268]

[0269] Table 3 G5-conjugated siRNA double-stranded molecules

[0270]

[0271] 1. Preparation of G5-conjugated oligonucleotide antisense strand

[0272] The preparation method is the same as Example 2.

[0273] 2. Preparation of G5-conjugated oligonucleotide sense strand

[0274] A CPG-G5 solid phase support was used, and the preparation method was the same as in Example 2. The CPG-G5 solid phase support refers to a solid phase support formed by connecting the GalNAc ligand G5 to the CPG support, and its structure is shown below:

[0275] 3. Preparation of G5-conjugated oligonucleotide duplexes

[0276] The preparation method is the same as Example 2.

[0277] The oligonucleotide forms a complex with the ligand G5. Figure 5 The conjugates shown.

[0278] The molecular weight and purity of the obtained G5-conjugated siRNA double-strands are shown in Table 4.

[0279] Table 4 Molecular weight and purity of G5-conjugated siRNA double-stranded molecules

[0280]

[0281] Example 4: Detection of the efficacy and off-target effects of anti-off-target modified siRNA molecules in HepG2 cells

[0282] 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.

[0283] 1. Experimental Materials

[0284] Test samples: double-stranded siRNA molecules listed in Table 1.

[0285] Cell type: HepG2 cells

[0286] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM (Gibco-31985070).

[0287] 2. Experimental Methods

[0288] 2.1 Cell Culture

[0289] HepG2 cells were subcultured in DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin in a 37°C incubator with 5% CO2, with passages occurring every three days. Cells were digested with 0.25% trypsin, centrifuged at 800 rpm for 3 minutes, the supernatant discarded, and fresh medium added for subculture.

[0290] 2.2 Cell transfection

[0291] siRNA solution dilution:

[0292] Dissolve the above siRNA molecules in sterile enzyme-free water to 20 μM as a stock solution. Then, serially dilute the stock solution with Opti-MEM to specific concentrations ranging from 100 nM to 0.128 pM.

[0293] Preparation of transfection mixture:

[0294] Lipofectamine RNAiMAX (Invitrogen-13778150) and Opti-MEM were mixed at a ratio of 2:98 and vortexed to mix thoroughly.

[0295] Transfection complex preparation:

[0296] At a 1:1 (v / v) ratio, 60 μL of siRNA solution diluted in Opti-MEM was added to 60 μL of transfection mixture, vortexed to mix, and then allowed to stand at room temperature for 15 minutes to obtain a transfection complex.

[0297] Add the prepared transfection complex to a 24-well cell culture plate (100 μL per well). A control well containing only the transfection reagent was used. Add 500 μL of the cell suspension (75,000 cells) to each well. Mix thoroughly using the cross-mixing method and incubate in a 37°C, 5% CO2 incubator for 48 hours.

[0298] 2.3 RNA extraction and reverse transcription

[0299] 48 hours after transfection, the culture medium was removed and the cells were harvested for RNA extraction. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN-74106) according to the kit instructions, and RNA was eluted with 30 μL of sterile, enzyme-free water. Subsequently, 8 μL of RNA was reverse-transcribed into cDNA using the FastKing RT Kit (TIANGEN-KR116-02) according to the kit instructions for qPCR.

[0300] 2.4 qPCR

[0301] Dilute the reverse transcription product 5-fold with sterile enzyme-free water and perform qPCR using TB Green Premix Ex Taq (Takara-RR420W) according to the kit instructions. Prepare a 10 μL reaction system as follows, performing three replicates for each sample:

[0302]

[0303] qPCR reaction procedure:

[0304] Heat at 95°C for 30 seconds, then enter the cycle mode and heat at 95°C for 5 seconds, followed by heating at 60°C for 34 seconds, for a total of 40 cycles.

[0305] 2.5 Data Analysis

[0306] The target gene mRNA expression level in each sample was calculated using the ΔΔCT relative quantification method. The calculation formula is as follows:

[0307] ΔCT = average Ct value of target gene – average Ct value of GAPDH;

[0308] ΔΔCT = ΔCT (experimental group) – ΔCT (transfection control group);

[0309] Relative expression of target gene mRNA = 2 -ΔΔCT ;

[0310] Inhibition rate = (relative mRNA expression level in the transfection control group - relative mRNA expression level of the target gene) / relative mRNA expression level in the transfection control group × 100%.

[0311] IC 50 The IC values ​​were calculated by fitting the dose-effect curve using the logarithmic value of the 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.

[0312] 3. Experimental Results

[0313] 3.1 Inhibitory effect of off-target modified siRNA molecules on PCSK9 in HepG2 cells

[0314] The IC50 (IC 50 ) See Table 5 for details.

[0315] Table 5 Inhibitory effect of siRNA molecules on PCSK9 in HepG2 cells

[0316]

[0317] 1) The siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention can significantly inhibit PCSK9 gene expression.

[0318] From Table 5 and Figure 1It 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 were 50 IC50 was significantly lower than that of D81-DV25 without anti-off-target modification, for example, IC 50 It is only 16.9% of D81-DV25.

[0319] 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 on PCSK9 is 50 , equivalent to D81-DV25.

[0320] The experimental results show that modifying siRNA molecules with the anti-off-target nucleoside compound of the present invention does not reduce the activity of the siRNA molecules, and may even enhance their activity.

[0321] 2) Compared with existing anti-off-target technologies, the siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention have a better inhibitory effect on the PCSK9 gene.

[0322] 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 as follows: 50 , which are lower than those of 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 50 It is only 35.4% of D84-DV27P-GNA-6 and 33.2% of D84-DV27P-G1-2-6.

[0323] The anti-off-target nucleoside compound of the present invention is modified at the 6th position of D81-DV25, and D81-DV25-GA2-6 and D81-DV25-GT4-6 are effective for PCSK9 IC 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-GA2-650 It is only 6.5% of D84-DV27P-GNA-6.

[0324] It can be seen from this that the siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention have better on-target activity compared with existing anti-off-target technologies.

[0325] 3.2 Inhibitory effect of siRNA on the off-target gene ITGAV

[0326] The inhibition rate of each siRNA molecule at a concentration of 10 nM on the off-target gene ITGAV is shown in Table 6.

[0327] Table 6 Inhibitory effect of siRNA molecules on off-target gene ITGAV

[0328]

[0329] From Table 6 and Figure 2 It can be seen that the parent sequence D84-DV27P without the anti-off-target modification has significant off-target activity, with an inhibition rate of up to 88% on the off-target gene ITGAV. The siRNA sequence modified with the anti-off-target nucleoside compound of the present invention significantly reduces the inhibition rate of off-target genes. For example, D84-DV27P-GA1-6 has a 77% lower inhibition rate on the off-target gene ITGAV than the parent sequence D84-DV27P, indicating that the anti-off-target modification can significantly reduce the off-target effect of siRNA.

[0330] The prior art GNA-conjugated D84-DV27P-GNA-6 conjugate and the compound 1-2-conjugated D84-DV27P-G1-2-6 conjugate had very weak off-target protection. For example, the D84-DV27P-G1-2-6 conjugate had only a 1% lower inhibition rate against the off-target gene ITGAV than the D84-DV27P conjugate. Therefore, compared with the prior art, the present invention's anti-off-target nucleoside compound has a superior off-target protection effect. For example, the D84-DV27P-GA1-6 conjugate had a 76% lower inhibition rate against the off-target gene ITGAV than the D84-DV27P-G1-2-6 conjugate.

[0331] Example 5: Effects of Anti-Off-Target Modified siRNA Molecules on PCSK9 Inhibition and LDL-C Levels in Mouse Serum

[0332] In this example, the inhibition rate of PCSK9 in mouse serum and the effect on LDL-C levels of the GalNAc-conjugated siRNA molecules in Table 3 were investigated.

[0333] Test drugs: Anti-off-target modified siRNA molecules listed in Table 3.

[0334] Drug solvent: PBS buffer

[0335] Experimental animal information:

[0336]

[0337] Feeding and management:

[0338] Animal husbandry: Animals were housed at Jiangsu Jicui Yaokang Biotechnology Co., Ltd. upon receipt, with free access to food and water. Standard SPF-grade, irradiated, sterilized experimental transgenic mouse feed was purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. The manufacturer provided a quality certificate for each batch of feed, along with annual third-party testing reports. Testing standards adhered to national standards GB 14924.3-2010, "Nutritional Composition of Compound Feeds for Laboratory Animals," and GB 14924.2-2001, "General Hygiene Standard for Compound Feeds for Laboratory Animals." Drinking water was tested monthly for appearance and bacterial indicators, performed in-house, with annual testing reports provided to the local water company. This adhered to national standard GB 5749-2015, "Hygiene Standard for Drinking Water."

[0339] 1. Inhibition rate of PCSK9 protein expression in mouse serum by siRNA molecules with different anti-off-target modifications

[0340] Experimental methods:

[0341] Approximately 200 μL of blood (without anticoagulation) was collected from the inner canthus of the eyes of the experimental animals before administration (D0), 7 days after administration (D7), and 14 days after administration (D14). Whole blood samples were temporarily stored in an ice box before centrifugation at approximately 3000 g for 10 minutes at approximately 4°C. Serum PCSK9 protein levels were measured using a PCSK9 kit.

[0342] Experimental results:

[0343] The inhibition rates of PCSK9 protein in serum 7 days and 14 days after administration are shown in Table 7.

[0344] Table 7 PCSK9 protein expression inhibition rate in mouse serum

[0345]

[0346] 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.

[0347] From Table 7 and Figure 3As can be seen, siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention exhibited significant inhibitory effects on PCSK9 protein expression in mouse serum. For example, the inhibition rates of D84-DV27PG5-GA1-6 and D84-DV27PG5-GT3-6 were comparable to those of the parent sequence D84-DV27PG5, while the inhibition rate of D81-DV25G5-GT4-6 was comparable to that of the parent sequence D81-DV25G5. This demonstrates that modifying siRNA with the anti-off-target nucleoside compounds of the present invention does not affect on-target activity.

[0348] 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.

[0349] 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.

[0350] 2. Effects of different off-target modified siRNA molecules on LDL-C levels in mouse serum

[0351] Experimental process:

[0352] Approximately 200 μL of blood (without anticoagulation) was collected from the inner canthus of the eye of the experimental animals before administration (D0), 7 days after administration (D7), and 14 days after administration (D14). Whole blood samples were temporarily stored in an ice box before centrifugation at approximately 3000 g for 10 minutes at approximately 4°C. Serum LDL-C levels were then measured.

[0353] Experimental results:

[0354] The serum LDL-C reduction levels were observed 7 days and 14 days after administration. Specific results are shown in Table 8.

[0355] Table 8 Reduction level of LDL-C in mouse serum

[0356]

[0357] 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.

[0358] From Table 8 and Figure 4As can be seen, siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention significantly reduced LDL-C levels in mouse serum. For example, D84-DV27PG5-GA1-6 achieved reductions of 38.1% and 40.4% on days 7 and 14, respectively, exceeding those achieved with the parent sequence D84-DV27PG5.

[0359] 2) The siRNA molecules modified with the anti-off-target nucleoside compounds of the present invention have a higher effect on reducing the level of LDL-C in mouse serum than the siRNA molecules modified with the compounds of the prior art.

[0360] For example, the reduction levels of YK-GNA-004-modified D81-DV25G5-GT4-6 on days 7 and 14 were 9.3% and 10.2% higher than those of GNA-modified D81-DV25G5-GNA-6, respectively.

[0361] Although the present invention has been described in detail above using general explanations, 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. Such modifications or improvements, without departing from the spirit of the present invention, shall fall within the scope of protection claimed by the present invention.

Claims

1. A nucleoside analogue, which is a compound having the structure of any one of the following YK-GNA-003, YK-GNA-004, YK-GNA-005 and YK-GNA-006, or a pharmaceutically acceptable salt thereof: 、 、 、 。 2. The nucleoside analog-modified oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The oligonucleotide comprises a structural unit formed by the nucleoside analogue of claim 1.

3. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 2, 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.

4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 3, wherein The type of oligonucleotide is small interfering RNA.

5. The oligonucleotide or pharmaceutically acceptable salt thereof according to claim 4, wherein The small interfering RNA includes a sense strand and an antisense strand.

6. The oligonucleotide or pharmaceutically acceptable salt thereof according to claim 4, wherein The length of the small interfering RNA is 15-30 nucleotides.

7. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 2 to 6, wherein: The oligonucleotide is a double-stranded RNAi agent that reduces the expression of PCSK9.

8. The oligonucleotide or pharmaceutically acceptable salt thereof according to claim 7, 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 the sequence shown in SEQ ID NO. 5 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 6 or a fragment thereof, or a modified sequence thereof; (2) The sense strand has the sequence shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence 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 the sequence shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 12 or a fragment thereof, or a modified sequence thereof; (5) The sense strand has the sequence shown in SEQ ID NO. 13 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence 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.

9. A conjugate comprising a ligand and an oligonucleotide, wherein: The oligonucleotide comprises a structural unit provided by the nucleoside analog of claim 1.

10. A conjugate comprising a ligand and the oligonucleotide according to any one of claims 2 to 8 or a pharmaceutically acceptable salt thereof.

11. The conjugate according to claim 9 or 10, wherein The oligonucleotide is a double-stranded RNAi agent, comprising 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: ， ， ， 。 12. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 2 to 8 or a pharmaceutically acceptable salt thereof, or the conjugate according to any one of claims 9 to 11.

13. Use of the nucleoside analogue according to claim 1 in reducing off-target effects of oligonucleotides, wherein the use is non-therapeutic and non-diagnostic.

14. Use of the nucleoside analogue or pharmaceutically acceptable salt thereof according to claim 1, the oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 2 to 8, or the conjugate according to any one of claims 9 to 11 in the preparation of an oligonucleotide drug.

15. The use according to claim 14, wherein: The oligonucleotide drug is a drug for treating PCSK9-related diseases.

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