5'-Phosphate-modified nucleosides, their preparation methods, and use

Abstract

This invention provides a novel modified nucleoside compound that is useful as a raw material for the production of siRNA, exhibiting excellent silencing effects and superior resistance to exonuclease degradation. [Solution] The modified nucleoside compound is represented by the following formula (I). TIFF2026127613000036.tif46168 [R1 is H; R2 is -OCH3; R3 is a protecting group or a phosphorus-containing active reactive group, and the phosphorus-containing active reactive group is one of the following: phosphoramidite, H-phosphate ester, phosphate triester, or phosphorus-containing chiral auxiliary; R4 and R5 are each independently C] 1-6 Alkyl groups, etc.; L is -CH2- or -CH2CH2-; A is O; X is O or S; B is a modified or unmodified base or its salt; D is O]

Description

[Technical Field]

[0001] This invention relates to the biopharmaceutical field, and more specifically to 5'-phosphate ester-modified nucleosides, methods for preparing them, and their use. [Background technology]

[0002] Small nucleic acid drugs select targets through complementary base pairing and directly target specific genes, thus possessing high specificity. They extend the drug target to mRNA, which is upstream of pathogenic proteins, and control the expression of target genes from the post-transcriptional level. Compared to antibody drugs and small molecule drugs, small nucleic acid drugs have advantages such as high target diversity, short research and development cycles, and sustained efficacy. There are many types of small nucleic acid drugs, mainly including antisense oligonucleotides (ASOs), small interfering oligonucleotides (siRNAs), microoligonucleotides (miRNAs), and nucleic acid aptamers. Of these, research on ASOs, siRNAs, and aptamers is the most active.

[0003] siRNA drugs exert their effects through an RNA interference mechanism. The 5' end of the siRNA antisense strand, which enters the cytoplasm via the endocytosis pathway, is phosphorylated by the intracellular kinase Clp1, forming a phosphate group at the 5' end of the antisense strand. This phosphate group binds to the Ago2 protein, facilitating the entry of siRNA into the RNA-induced silencing complex (RISC). As the siRNA sense strand is cleaved and removed, the formed active RISC complementarily binds to the target mRNA, which is then cleaved by an endonuclease within the RISC, reducing the expression of the target mRNA. The released active RISC complex continues to search for and bind to the target mRNA, entering a catalytic cycle and continuing to exert its therapeutic effect. Therefore, a phosphate group at the 5' end of the antisense strand in the siRNA double-stranded siRNA is essential, as it allows for specific binding to the side-chain residues of the Ago2 domain, which is crucial for RNA interference activity. However, studies have shown that when siRNA modified with a native phosphate group at its 5' end enters the endocytosis pathway, the terminal phosphate is rapidly degraded by acid phosphatases in lysosomes, which limits their bioavailability in the body (Reka AH, et al. Nucleic Acids Res., 2017, 45, 7581), thereby affecting their efficacy.

[0004] In some cases, the efficacy of siRNA in the body can be improved by substituting the native phosphate group at the 5' end of the siRNA antisense chain with a phosphate ester analog. Since the modified phosphate ester is not a substrate for phosphatases in the body, it resists degradation by exonucleases after modification and enhances the silencing effect of siRNA in the body. However, siRNA obtained by conventional modification methods still lacks sufficient resistance to exonuclease degradation, and problems exist such as the enhancement of the silencing effect on siRNA not being significant or even adversely affecting the silencing effect of siRNA.

[0005] Therefore, it is expected to develop modified nucleoside compounds, and by introducing these into siRNA, the efficacy of siRNA drugs can be improved more effectively and stably.

Summary of the Invention

[0006] To solve the above problems existing in the prior art, the present invention provides a 5'-phosphate ester modified nucleoside, its preparation method and use. Specifically, the present invention designs a modified nucleoside compound, and by means of a solid-phase synthesis method, the modified nucleoside compound of the present invention can be efficiently introduced to the 5' end of an oligonucleotide chain. Compared with the prior art, the oligonucleotide containing the modified nucleoside compound of the present invention has a stronger inhibitory efficiency against the target gene.

[0007] The inventors of the present invention surprisingly found that a nucleoside compound formed by introducing an alkyl group between the phosphorus atom of phosphoric acid and the 5'-hydroxyl group of the sugar ring can effectively improve the nuclease degradation resistance of siRNA and the like, and at the same time enhance the silencing effect of siRNA in vivo. By introducing a nucleotide containing the nucleoside compound modified in this way into an oligonucleotide, a nucleic acid drug with improved efficacy can be obtained.

[0008] Based on this, the first aspect of the present invention provides a modified nucleoside compound, and the modified nucleoside compound has a structure shown in formula (I), or is a pharmaceutically acceptable salt or stereoisomer of a compound having a structure shown in formula (I).

[0009] <​​​​​​​​​​​​​​​​1-6 Selected from alkyl groups, -CH2CH2CN, or -CH2O(CO)C(CH3)3, X is either O or S, L is C 1-6 Alkyl alkyl group, C 2-5 Alkenyl group or C 2-5 It is an alkynyl group, A is either O or S, B is H, a modified or unmodified base, or a salt thereof. D is either O, S, or -CH2-.

[0010] According to some preferred embodiments of the present invention, R4 and R5 are not simultaneously isopropyl groups.

[0011] According to some preferred embodiments of the present invention, here, R1 is H, and R2 is -OCH3. R4 and R5 are each independently selected from -CH2CH3, -CH3, or H. L is CH2.

[0012] According to some preferred embodiments of the present invention, where R1 is H.

[0013] According to some preferred embodiments of the present invention, where R2 is -OCH3.

[0014] According to some preferred embodiments of the present invention, R3 is a phosphorus-containing active reactive group.

[0015] According to some preferred embodiments of the present invention, where R4 is -CH3 or -CH2CH3.

[0016] According to some preferred embodiments of the present invention, where R5 is -CH3 or -CH2CH3.

[0017] According to some preferred embodiments of the present invention, where X is O.

[0018] According to some preferred embodiments of the present invention, where L is -CH2- or -CH2CH2-.

[0019] According to some preferred embodiments of the present invention, where A is O.

[0020] According to some preferred embodiments of the present invention, where B is

[0021] [ka] That is.

[0022] [ka] The symbol "" indicates the connection point. "Bz" refers to the benzoyl group.

[0023] According to some preferred embodiments of the present invention, where D is O.

[0024] According to some preferred embodiments of the present invention, the phosphorus-containing active reactive group is one of a phosphoramidite, an H-phosphate ester, a phosphate triester, or a phosphorus-containing chiral auxiliary agent.

[0025] According to some preferred embodiments of the present invention, where R3 is

[0026] [ka] That is.

[0027] [ka] The symbol " indicates the connection point.

[0028] According to some preferred embodiments of the present invention, R4 and R5 are identical.

[0029] According to some particularly preferred embodiments of the present invention, the compound of formula (I) is one or at least two of the following compounds YK-VP-001, YK-VP-002, YK-VP-003, YK-VP-004, and YK-VP-005 having the following structures.

[0030] [ka] "Bz" refers to a benzoyl group, and "OMe" refers to a methoxy group.

[0031] Furthermore, the present invention further provides the use of the modified nucleoside compound described in the first embodiment to improve the stability of oligonucleotides (e.g., resistance to degradation by exonucleases).

[0032] Based on this, the present invention further provides a method for improving the stability of oligonucleotides (e.g., resistance to degradation by exonucleases), the method comprising substituting at least some constituent units (nucleotides) of the oligonucleotide with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0033] Preferably, 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are substituted with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0034] Furthermore, the present invention further provides the use of the modified nucleoside compound described in the first embodiment to improve the in vivo function of oligonucleotides (e.g., improving the silencing effect of siRNA).

[0035] Based on this, the present invention further provides a method for improving the in vivo function of oligonucleotides (e.g., improving the silencing effect of siRNA), the method comprising substituting at least some constituent units (nucleotides) of an oligonucleotide with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0036] Preferably, 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are substituted with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0037] Furthermore, the present invention further provides the use of the modified nucleoside compound described in the first embodiment for simultaneously improving the stability of oligonucleotides (e.g., resistance to degradation by exonucleases) and the in vivo function of oligonucleotides (e.g., improved silencing effect of siRNAs).

[0038] Based on this, the present invention further provides a method for simultaneously improving the stability of oligonucleotides (e.g., improved resistance to degradation by exonucleases) and the in vivo function of oligonucleotides (e.g., improved silencing effect of siRNA), the method comprising substituting at least some constituent units (nucleotides) of an oligonucleotide with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0039] Preferably, 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are substituted with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0040] A second aspect of the present invention provides an oligonucleotide, wherein the constituent units of the oligonucleotide include nucleotides containing the modified nucleoside compound described in the first aspect. In other words, the oligonucleotide provided in the present invention is formed by substituting some constituent units (nucleotides) of an oligonucleotide produced by a conventional method with nucleotides synthesized from the modified nucleoside compound of the present invention (i.e., the compound of formula (I) or a pharmaceutically acceptable salt thereof) and phosphate.

[0041] In the oligonucleotide provided by the present invention, some of the constituent units (nucleotides) may contain the modified nucleoside compound provided in the first embodiment, or all of the constituent units may contain the modified nucleoside compound. If some of the constituent units contain the modified nucleoside compound, the other constituent units in the oligonucleotide may be provided from nucleotides obtained by conventional methods, or from nucleotides containing other existing modified nucleoside compounds.

[0042] In the present invention, the constituent 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 base or pyrimidine base), a pentose (ribose or deoxyribose), and phosphate. The base and the pentose condense to form a nucleoside, and then phosphate and nucleotide are synthesized. The statement that "the constituent unit of an oligonucleotide includes a nucleotide containing the modified nucleoside compound described in the first embodiment" means that the nucleoside portion of at least one nucleotide in the oligonucleotide sequence is provided by the modified nucleoside compound provided in the present invention, that is, the nucleoside portion of at least one nucleotide among the multiple nucleotides contained in the oligonucleotide is replaced by the modified nucleoside compound provided in the present invention.

[0043] In some preferred embodiments, in the case of an oligonucleotide having a length of 30 nucleotides or less (for example, 15 to 30 nucleotides), the number of constituent units (nucleotides) containing the modified nucleoside compound provided by the present invention is 3 or less (for example, it may be 1, 2, or 3).

[0044] According to some preferred embodiments of the present invention, here, the nucleotide containing the modified nucleoside compound is located at the 5'-end of the oligonucleotide.

[0045] According to a particularly preferred embodiment of the present invention, here, the outermost nucleotide at the 5'-end of the oligonucleotide is the nucleotide containing the modified nucleoside compound. <000024O> According to some preferred embodiments of the present invention, here, the oligonucleotide is the oligonucleotide represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0047] [Chemical formula] In the formula, Oligo is an oligonucleotide, R1 and R2 are each independently H, halogen, C 1-6 alkyl group or C 1-6 alkoxy group, R4 and R5 are each independently H, C 1-6 alkyl group, -CH2CH2CN or -CH2O(CO)C(CH3)3, X is O or S, L is C 1-6 alkyl group, C 2-5 alkenyl group or C 2-5 alkynyl group, A is O or S, D is O, S or -CH2, X1 is O or S, B is

[0048] [ka] It should be noted that the structure shown in formula (II) above is an oligonucleotide in which the final position at the 5' end contains the modified nucleoside compound provided in the present invention. In other words, the part that connects to Oligo is the structure of the final nucleotide in a full-length oligonucleotide, and Oligo represents the remainder of the oligonucleotide starting from the second position at the 5' end.

[0049] The modified nucleoside compounds provided in the present invention can further stabilize oligonucleotides by substituting some of the nucleoside structures in the oligonucleotide, and have the effect of enhancing and improving the function of the oligonucleotide itself. Any oligonucleotide in the art can be modified using the modified nucleoside compounds provided in the present invention (i.e., the modified nucleoside compounds substitute for some of the nucleoside structures of nucleotides in the oligonucleotide). According to a preferred embodiment of the present invention, the oligonucleotide is one or at least two selected from small interfering nucleotides (siRNA), antisense oligonucleotides (ASO), microRNA (miRNA), small activated RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), and aptamers.

[0050] In the present invention, the oligonucleotide may be a single-stranded oligonucleotide or a double-stranded oligonucleotide. Unless otherwise specified, in the present invention, the length of an oligonucleotide refers to the number of nucleotides contained therein in the case of a single-stranded oligonucleotide (which can also be expressed as the number of bases, nt), and refers to the number of nucleotide pairs contained therein in the case of a double-stranded oligonucleotide (which can also be expressed as the number of base pairs, bp).

[0051] According to some preferred embodiments of the present invention, the oligonucleotide is siRNA.

[0052] Preferably, the siRNA is double-stranded siRNA or single-stranded siRNA.

[0053] According to some particularly preferred embodiments of the present invention, the siRNA is a double-stranded siRNA comprising a sense strand and an antisense strand.

[0054] Preferably, the antisense strand in the double-stranded siRNA is an oligonucleotide shown in formula (II) or a pharmaceutically acceptable salt thereof.

[0055] According to a preferred embodiment of the present invention, each nucleotide in the oligonucleotide is independently modified or unmodified.

[0056] According to some preferred embodiments of the present invention, the oligonucleotide can be used to regulate the expression of a target gene (for example, it can be used to suppress the expression of a target gene).

[0057] According to some particularly preferred embodiments of the present invention, the oligonucleotide is one or at least two of the following siRNAs. (i) The sense strand has the sequence shown in SEQ ID NO. 5, and the antisense strand has the sequence shown in SEQ ID NO. 6. (ii) The sense strand has the sequence shown in SEQ ID NO. 7, and the antisense strand has the sequence shown in SEQ ID NO. 8. (iii) The sense strand has the sequence shown in SEQ ID NO. 9, and the antisense strand has the sequence shown in SEQ ID NO. 10. (iv) The sense strand has the sequence shown in SEQ ID NO. 15, and the antisense strand has the sequence shown in SEQ ID NO. 16. (v) The sense strand has the sequence shown in SEQ ID NO. 17, and the antisense strand has the sequence shown in SEQ ID NO. 18. (vi) The sense strand has the sequence shown in SEQ ID NO. 19, and the antisense strand has the sequence shown in SEQ ID NO. 20.

[0058] A third aspect of the present invention provides a nucleic acid complex, characterized in that the nucleic acid complex comprises an oligonucleotide or a pharmaceutically acceptable salt thereof as described in the second aspect, and a target ligand that binds thereto.

[0059] In the present invention, any target ligand usable for nucleic acid complexes in the art can be selected and used. According to some preferred embodiments of the present invention, the target ligand comprises one or at least two of the following: carbohydrates, cholesterol, lipids, polypeptides, or antibodies.

[0060] Preferably, the target ligand includes an N-acetylgalactosamine (GalNAc) moiety.

[0061] In the art, any GalNAc moiety commonly used as a target ligand for nucleic acid complexes can be applied to the present invention. According to some preferred embodiments of the present invention, the N-acetylgalactosamine moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0062] According to some particularly preferred embodiments of the present invention, the target ligand is G5, and its structure is as follows.

[0063] [ka] Here

[0064] [ka] This indicates that it is connected to the 3' end of the siRNA sense strand via a phosphate ester group or a thiophosphate ester group.

[0065] A fourth aspect of the present invention provides a pharmaceutical composition comprising an oligonucleotide or a pharmaceutically acceptable salt thereof as described in the second aspect, and / or the pharmaceutical composition comprises the nucleic acid complex described in the third embodiment.

[0066] In the pharmaceutical composition provided by the present invention, the oligonucleotide and / or nucleic acid complex provided by the present invention serves as the main active ingredient. In some embodiments, the oligonucleotide and / or nucleic acid complex provided by the present invention may be the sole active ingredient in the pharmaceutical composition, and in some embodiments, the pharmaceutical composition may contain other active ingredients in addition to the oligonucleotide and / or nucleic acid complex of the present invention.

[0067] According to some preferred embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials. “pharmaceutically acceptable auxiliary materials” refers to any non-active component material that can be added in the process. For example, the pharmaceutical composition provided in the present invention may include pharmaceutically acceptable carriers, adjuvants, auxiliary materials, etc., or other active components or active component adjuvants. The term “pharmaceutically acceptable carrier” refers to 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 is involved in transporting or transferring the nucleoside analogs, oligonucleotides, complexes, or compositions thereof of the present invention within or into the patient’s body so that they can perform the desired function. Typically, such structures are transported or transferred from one organ or part of the body to another organ or part of the body. The various carriers must be "acceptable," that is, they must be compatible with the other components of the formulation (including the nucleoside analogs, oligonucleotides, complexes, or compositions thereof of the present invention) and harmless or substantially harmless to the patient. Some specific 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; celluloses and their derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth gum, malt, gelatin, talc, cocoa butter, and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyhydric alcohols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffers such as agar, magnesium hydroxide, and aluminum hydroxide; surfactants; alginic acid, non-pyrogenic water, isotonic saline, Ringer's solution, ethanol, phosphate buffer, and other non-toxic and suitable substances used in pharmaceutical formulations.Where used herein, “pharmaceutically acceptable carrier” further includes any and all coating agents, antimicrobial agents, antifungal agents, and absorption retarders, etc., which are compatible with the activity of the nucleoside analogs, oligonucleotides, complexes, or compositions thereof of the present invention and are physiologically acceptable to the patient. Furthermore, auxiliary active compounds may be incorporated into the composition. “pharmaceutically acceptable carrier” further includes pharmaceutically acceptable salts usable with the compounds of the present invention. Other additional components 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 are incorporated herein by reference. Any carrier, adjuvant, or auxiliary material usable in pharmaceuticals within the relevant technical field can be applied to the present invention. For example, auxiliary materials include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancers, adsorbents, chelating agents, colorants, flavoring agents, coating agents, buffers, protective agents, preservatives, solvent co-solvents, and pH adjusters.

[0068] A fifth aspect of the present invention provides the use of an oligonucleotide or a pharmaceutically acceptable salt thereof as described in the second aspect, or a nucleic acid complex as described in the third aspect, or a pharmaceutical composition as described in the fourth aspect, in the preparation of a drug for treating and / or preventing a pathological condition or disease caused by the expression of the proprotein convertase subtilisin / kexin 9 gene (PCSK9 gene) in hepatocytes.

[0069] The PCSK9 gene is located on chromosome 1, and the protein it encodes plays an important role in cholesterol and fatty acid metabolism. Abnormal expression of this gene is associated with metabolic diseases such as autosomal dominant familial hypercholesterolemia.

[0070] According to some preferred embodiments of the present invention, the pathological condition or disease is one or at least two selected from hypercholesterolemia, dyslipidemia, atherosclerosis, and cardiovascular disease.

[0071] Furthermore, the present invention further provides a method for treating a pathological condition or disease caused by the expression of the PCSK9 gene, the method comprising administering to a subject in need an oligonucleotide or pharmaceutically acceptable salt thereof as described in a second aspect of the present invention, or a nucleic acid complex as described in a third aspect, or a pharmaceutical composition as described in a fourth aspect, or administering a drug prepared from an oligonucleotide or pharmaceutically acceptable salt thereof as described in a second aspect of the present invention, or a nucleic acid complex as described in a third aspect, or a pharmaceutical composition as described in a fourth aspect.

[0072] According to some preferred embodiments of the present invention, the subject may be a mammal. For example, it may be a human, a non-human primate, a rodent (e.g., a rabbit, rat, mouse, guinea pig, etc.), a horse, a sheep, a pig, a cat, a dog, etc.

[0073] A sixth aspect of the present invention provides a kit comprising an oligonucleotide or a pharmaceutically acceptable salt thereof as described in the second aspect, or a nucleic acid complex as described in the third aspect, or a pharmaceutical composition as described in the fourth aspect.

[0074] The kit provided in this invention can be used for therapeutic or non-therapeutic purposes, and can also be used for diagnostic or non-diagnostic purposes. [Effects of the Invention]

[0075] The significant advancements of the present invention are as follows: The present invention designs a series of 5'-phosphate ester modified nucleosides, which have a completely different chemical structure compared to conventional 5'-terminal phosphorylated compounds. Oligonucleotides containing the 5'-phosphate ester modified nucleosides of the present invention can significantly improve the repressive activity of target genes by siRNA.

[0076] Compared to the prior art, the present invention has at least the following beneficial effects. 1. The compound structure is completely different from that of existing 5'-terminal phosphate modification techniques. The 5'-phosphate ester-modified nucleoside designed in this invention is completely different from conventional technologies and is an entirely novel 5'-phosphate ester-modified nucleoside compound, as it introduces an alkyl group between the phosphorus atom of the phosphate and the 5'-hydroxyl group of the sugar ring. This compound can be rapidly constructed using commercially available nucleosides as raw materials, by reacting the 5'-hydroxyl group of the nucleoside with a phosphate ester-containing alkyl / alkenylated compound in only one step of nucleophilic substitution / addition reaction. 2. Compared to compounds in the prior art, the 5'-phosphate ester-modified siRNA sequence of the present invention has higher inhibitory activity. For example, compared to D84-DV27-PG5 (modified with the existing compound (E)-VP-Um), D84-DV27-5MMPG5 (modified with the compound YK-VP-002 of the present invention) increased the suppression rate of PCSK9 protein expression in mouse serum by 11.7%, 7.3%, and 9.8% on days 7, 14, and 21, respectively. Compared to D84-DV27-4MMPG5 (modified with the existing compound Phosphoramidite 3), D84-DV27-5MMPG5 (modified with the compound YK-VP-002 of the present invention) increased the suppression rate of PCSK9 protein expression in mouse serum by 11.8%, 16.5%, and 15.3% on days 7, 14, and 21, respectively. Compared to D84-DV27-PG5, D84-DV27-5EMPG5 (modified with the compound YK-VP-001 of the present invention) increased the reduction level of LDL-C in mouse serum by 9.9%, 8.5%, and 8.4% on days 7, 14, and 21, respectively. Compared to D84-DV27-4MMPG5, D84-DV27-5MMPG5 increased the reduction level by 11.2%, 11.6%, and 16.0% on days 7, 14, and 21, respectively. [Brief explanation of the drawing]

[0077] To more clearly describe specific embodiments of the present invention or technical solutions in the prior art, the following briefly introduces drawings that may be used to describe specific embodiments or the prior art. Clearly, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain further drawings based on these without any creative work. [Figure 1] This shows the suppression rates of PCSK9 protein expression in mouse serum by siRNA sequences D84-DV27-G5, D84-DV27-PG5, D84-DV27-5EMPG5, D84-DV27-5MMPG5, D84-DV27-5MEPG5, D84-DV27-4MMPG5, D82-DV29-G5, D82-DV29-5MMPG5, D82-DV29-5EMPG5, and D82-DV29-5MPG5 on days 7, 14, and 21 after administration. [Figure 2] These are the levels of LDL-C reduction in the serum of mice administered with siRNA sequences D84-DV27-G5, D84-DV27-PG5, D84-DV27-5EMPG5, D84-DV27-5MMPG5, D84-DV27-5MEPG5, D84-DV27-4MMPG5, D82-DV29-G5, D82-DV29-5MMPG5, D82-DV29-5EMPG5, and D82-DV29-5MPG5, on days 7, 14, and 21 after administration. [Modes for carrying out the invention]

[0078] To further clarify the object, technical solution, and advantages of the embodiments of the present invention, the technical solution of the embodiments of the present invention will be described clearly and completely below. Clearly, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained based on the embodiments of the present invention described, without requiring creative work by those skilled in the art, are all within the scope of the protection of the present invention.

[0079] The present invention can be implemented in other specific forms without departing from its fundamental attributes. To the extent that it does not create a contradiction, any one and all embodiments of the present invention should be understood to be able to be combined with the technical features of any one or more other embodiments to obtain new embodiments. The present invention includes new embodiments obtained by such combinations.

[0080] All publications and patents referenced herein are incorporated herein by reference in their entirety. In the event of any use or terminology used in any publication or patent incorporated by reference that conflicts with any use or terminology used herein, the use and terminology in this invention shall prevail.

[0081] The chapter headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter described herein.

[0082] Unless otherwise specified, all technical and scientific terms used herein have their common meanings in the art to which the subject matter for which protection is claimed pertains. If there are multiple definitions for a term, the definition provided herein shall prevail.

[0083] Unless explicitly stated in the examples or otherwise indicated, all numerical values ​​relating to quantitative properties such as dosage described in the specification and claims should be understood to be modified in all cases by the term "approximately." Furthermore, any numerical range enumerated in this application should be understood to include all subranges within that range, and any combination of endpoints of that range or subrange.

[0084] In this invention, similar terms such as “contain,” “include,” or “incorporate” mean that the element preceding the term includes the elements and their equivalents listed after the term, and do not exclude any unlisted elements. The terms “contain” or “include (incorporate)” as used herein may be in open form, semi-closed form, or closed form. In other words, the terms also include “substantially consist of…” or “consist of….”

[0085] In this invention, "protecting group" refers to a group used, in the usual chemical definition, to reversibly deactivate a functional group under specific conditions of a desired reaction. After the desired reaction is complete, the protected functional group can be deprotected by removing the protecting group. All protecting groups should be removable without decomposing most of the molecules during synthesis.

[0086] In the present invention, "phosphorus-containing active reactive group" refers to a phosphorus-containing group that can react with hydroxyl groups or amino groups contained in other molecules, particularly other nucleotide units or other nucleotide analogs, via nucleophilic reactions. Typically, such reactions generate ester-type nucleoside bonds in which a nucleotide unit or nucleotide analog unit is linked to another nucleotide unit or nucleotide analog unit. These phosphorus-containing active reactive groups are known groups in the art and contain a phosphorus atom with a valence of P(III) or P(V). The phosphorus-containing active reactive groups include, but are not limited to, phosphoramidites, H-phosphate esters, phosphate triesters, and phosphorus-containing chiral additives, for example.

[0087] [ka] Here, a phosphorus-containing chiral auxiliary refers to a phosphorus-containing group that has chiral properties, for example.

[0088] [ka] It may be based on various possible forms such as those listed above.

[0089] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0090] The term “C 1-6 " means that the main chain and side chains of the group have a total of any integer number of carbon atoms within the range of 1 to 6, for example, when the number of carbon atoms is 1, 2, 3, 4, 5, or 6. Similarly, the term "C" 2-5 "This means that the main chain and side chains of the group have a total of any integer number of carbon atoms within the range of 2 to 5, for example, when the number of carbon atoms is 2, 3, 4, or 5.

[0091] The term "alkyl group" refers to a group with a specified number of carbon atoms (for example, C 1-6 This refers to linear or branched alkyl groups having methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, and n-hexyl groups, but is not limited to these. The term "alkyl group" also includes heteroalkyl groups, that is, a group formed by the substitution of one or more carbon atoms (e.g., 1, 2, 3, or 4) in an alkyl group with heteroatoms (atoms other than carbon atoms and hydrogen atoms, such as oxygen, sulfur, fluorine, nitrogen, phosphorus, etc.).

[0092] The term "alkoxy group" refers to the group RO-, where R is the alkyl group as defined above.

[0093] The term "alkenyl group" refers to a hydrocarbon group having at least one carbon-carbon double bond at one or more locations on the carbon chain of an alkyl group. Alkenyl groups include, but are not limited to, vinyl groups, propenyl groups, and butenyl groups.

[0094] The term "alkynyl group" refers to a hydrocarbon group having at least one carbon-carbon triple bond at one or more locations on the carbon chain of an alkyl group. Alkynyl groups include, but are not limited to, ethynyl, propynyl, and butynyl groups.

[0095] The term "pharmaceutically acceptable salt" refers to a salt obtained by the reaction of a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for patient use) acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, and ammonium salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride salts, sulfate salts, and methanesulfonate salts.

[0096] In this invention, the term "pharmaceutically acceptable excipient" refers to all substances contained in a drug formulation other than the active ingredient.

[0097] The term "treatment" refers to any one of the following: (1) alleviating one or more biological signs of a disease; (2) interfering with one or more sites in the biological cascade response that causes the disease; or (3) slowing the progression of one or more biological signs of a disease.

[0098] The term "prevention" refers to reducing the risk of developing a disease, as well as reducing the severity of the disease after it develops.

[0099] The oligonucleotides in this invention include single-stranded oligonucleotides (e.g., antisense oligonucleotides, abbreviated as ASOs) and double-stranded oligonucleotides (e.g., small interfering nucleotides, abbreviated as siRNAs).

[0100] The oligonucleotides in this invention include native oligonucleotides and chemically modified oligonucleotides. Chemical modification as used herein includes nucleoside modification (including modification of sugar moieties and modification of nucleic acid bases) and modification of internucleoside bonds. Chemical modification of oligonucleotides does not include cases where only the nucleic acid base sequence differs. Natural type as used herein refers to cases corresponding to naturally occurring RNA or DNA.

[0101] Provided that they conform to common sense in the relevant technical field, each preferred embodiment of the present invention can be obtained by arbitrarily combining the above-mentioned preferential conditions.

[0102] All reagents and raw materials used in this invention are commercially available.

[0103] Examples To further clarify the object, technical solution, and advantages of the embodiments of the present invention, the technical solution of the embodiments of the present invention will be described clearly and completely below. Clearly, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained based on the embodiments of the present invention described, without requiring creative work by those skilled in the art, are all within the scope of the protection of the present invention.

[0104] The present invention can be implemented in other specific forms without departing from its fundamental attributes. To the extent that it does not create a contradiction, any one and all embodiments of the present invention should be understood to be able to be combined with the technical features of any one or more other embodiments to obtain new embodiments. The present invention includes new embodiments obtained by such combinations.

[0105] The present invention will be further described below with reference to examples. However, the present invention is not limited to the following examples. The conditions of implementation adopted in the examples can be further adjusted based on different requirements for specific use, and conditions of implementation not specified are common conditions in the industry. All raw materials used in the specific examples of the present invention are commercially available. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features of each embodiment of the present invention can be combined with each other, insofar as they do not conflict with each other.

[0106] Example 1: Compound Synthesis The following abbreviations represent the following reagents: TBDMSCl: tert-butyldimethylchlorosilane, DCM: dichloromethane, MeOH: methanol, NaH: sodium hydride, PE: petroleum ether, EA: ethyl acetate, TEA: triethylamine, Et3SiH: triethylsilane, TCA: trichloroacetic acid, DCI: 4,5-dicyanoimidazole.

[0107] 1. Synthesis of YK-VP-001 The synthesis route is as follows:

[0108] [ka]

[0109] Step 1: Synthesis of YK-VP-001-PM1 YK-VP-001-SM (365.00 g, 0.64 mol) was dissolved in 1460 mL of super-dehydrated dichloromethane and stirred in an ice bath under a nitrogen atmosphere. Imidazole (130.70 g, 1.92 mol) and TBDMSCl (192.90 g, 1.28 mol) were then added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 5 hours. The reaction status was detected by TLC, and it was found that the starting material had disappeared and new, low-polarity spots had appeared, so the reaction was stopped. Water (730 mL) was added to quench the reaction, and the mixture was extracted and washed once with another 730 mL of water. The organic phase was dried over anhydrous sodium sulfate, filtered, and the resulting filtrate was used directly in the next step.

[0110] Step 2: Synthesis of YK-VP-001-PM2 To a solution of YK-VP-001-PM1 (0.64 mol) in dichloromethane (4V, 1460 mL), methanol (1V, 365 mL) and p-toluenesulfonic acid monohydrate (12.20 g) were sequentially added and the mixture was stirred at room temperature. The reaction status was detected by TLC, and the starting material disappeared after 2 hours. 182 mL of TEA (0.5V) was added to quench the reaction, and the mixture was stirred for 1 hour. Then, 730 mL of saturated sodium bicarbonate aqueous solution was added and the mixture was stirred for 15 minutes. Sublimation was then performed, and the aqueous phase was further extracted once with 365 mL of dichloromethane. The combined organic phase was washed once with 730 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain a pale yellow solid. The crude product was slurryed with 1460 mL of n-heptane (4V), filtered, and yielded 183.20 g of a white solid with a yield of 75.5%. MS m / z[M+H] + =373.0. 1 H NMR(400MHz,CDCl3)δ 9.68(s,1H),7.74(d,J=8.1Hz,1H),5.75-5.67(m,2H),4.33(t,J=5.3Hz,1H),4.07-4.01(m,1H),4.00-3.8 9(m,2H),3.80-3.68(m,1H),3.47(s,3H),3.08(dd,J=6.6,3.6Hz,1H),0.89(s,9H),0.08(d,J=5.0Hz,6H).

[0111] Step 3: Synthesis of YK-VP-001-PM3 YK-VP-001-PM2 (20.00 g, 53.70 mmol) was dissolved in 200 mL of super-dehydrated tetrahydrofuran. Under a nitrogen atmosphere, tert-butoxysodium (20.64 g, 214.80 mmol) was added at 5°C and the mixture was stirred for 1 hour, followed by stirring at room temperature for 1 hour. p-toluenesulfonyloxymethylphosphonate diethyl (43.26 g, 134.20 mmol) was added and the mixture was reacted at room temperature for 2 hours. The reaction was detected by TLC, and the reaction was stopped when the starting materials had reacted almost completely and a new spot had formed below the starting materials. The reaction solution was diluted with 500 mL of ethyl acetate, and then quenched with 500 mL of saturated ammonium chloride aqueous solution. Liquid-liquid extraction was then performed, the aqueous phase was further extracted once with 500 mL of ethyl acetate, the combined organic phase was washed once with 500 mL of saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, PE / EA=1 / 3) yielded 13.10 g of a pale yellow oily substance, with a yield of 46.4%. MS m / z [MH] - = 521.2. 1 1H NMR (400MHz, CDCl3)δ 9.77(s,1H),8.01(d,J=8.1Hz,1H),5.91(d,J=2.0Hz,1H),5.78(dd,J=8.1,1. 8Hz,1H),4.24-4.10(m,5H),4.09-4.04(m,1H),3.94(dd,J=10.8,2.2Hz,1H), 3.89-3.74(m,2H),3.67(dd,J=10.9,1.9Hz,1H),3.60(dd,J=4.8,2.1Hz,1H), 3.52(s,3H),1.32(td,J=7.1,4.5Hz,6H),0.87(s,9H),0.07(d,J=5.3Hz,6H). 31 P NMR(162MHz, CDCl3)δ 21.25.

[0112] Step 4: Synthesis of YK-VP-001-PM4 YK-VP-001-PM3 (12.00 g, 23.00 mmol) was dissolved in 240 mL of tetrahydrofuran, and triethylamine hydrofluoric acid (18.51 g, 114.80 mmol) was added. The mixture was stirred at 40°C for 19 hours, and the reaction was stopped when the starting material disappeared and the target molecular weight was found by TLC and LC / MS analysis. The reaction mixture was concentrated by rotary evaporation, and then concentrated once more with acetonitrile to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH=20 / 1) yielded 9.30 g of a pale yellow oily substance with a yield of 99.14%. MS m / z[M+H] + = 409.0. 1 H NMR(400MHz,CDCl3)δ 9.13(s,1H),7.97(d,J=8.1Hz,1H),5.98(d,J=2.5Hz,1H),5.78(dd,J=8.2,1.2Hz,1H),4.28(dd,J=6.8,5.1Hz,1H),4.19-4.11(m ,4H),4.05(dt,J=6.9,2.1Hz,1H),3.99(dd,J=10.8,2.2Hz,1H),3.90-3.74(m,4H),3.58(s,3H),3.46(s,1H),1.35-1.34(m,6H). 31 P NMR(162MHz, CDCl3)δ 21.54.

[0113] Step 5: Synthesis of YK-VP-001 YK-VP-001-PM4 (3.00 g, 7.30 mmol) was concentrated three times by azeotropic rotational evaporation with super-dehydrated dichloromethane. The dried YK-VP-001-PM4 was dissolved in 30 mL of super-dehydrated dichloromethane, cooled to 5°C under a nitrogen atmosphere, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (3.32 g, 11.00 mmol) and tetrazole (0.62 g, 8.80 mmol) were added sequentially. The mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour, and the reaction was stopped after determining the target molecular weight by LC / MS and TLC analysis. The reaction mixture was washed twice with saturated sodium bicarbonate aqueous solution (15 mL x 2), the aqueous phase was further extracted once with 30 mL of dichloromethane, the combined organic phase was washed once with 10% sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated by rotational evaporation to obtain the crude product. Rapid purification yielded 1.80 g of a colorless oily substance, with a yield of 40.5%. MS m / z [MH] - = 607.5. 1 H NMR(400MHz,CDCl3)δ 9.22(s,1H),7.94(dd,J=9.1,5.7Hz,1H),6.00-5.97(m,1H),5.78(dd,J=8.2,3.7Hz,1H),4.49-4.04(m,6H), 3.98-3.73(m,6H),3.72-3.55(m,3H),3.49-3.42(m,3H),2.65(m,2H),1.32-1.26(m,6H),1.19-1.10(m,12H). 31 P NMR(162MHz, CDCl3)δ 151.41,150.47,21.49,21.25.

[0114] 2. Synthesis of YK-VP-002 The synthesis route is as follows:

[0115] [ka]

[0116] Step 1: Synthesis of YK-VP-002-PM1 YK-VP-001-PM2 (34.00 g, 91.20 mmol) was dissolved in 340 mL of super-dehydrated tetrahydrofuran. Under a nitrogen atmosphere, tert-butoxysodium (35.08 g, 364.80 mmol) was added at 5°C and the mixture was stirred for 1 hour, followed by stirring at room temperature for 1 hour. Dimethyl p-toluenesulfonyloxymethylphosphonate (67.14 g, 228.00 mmol) was added and the mixture was reacted at room temperature for 2 hours. The reaction was detected by TLC, and the reaction was stopped when the starting materials had reacted almost completely and a new spot had formed below the starting materials. The reaction solution was diluted with 500 mL of ethyl acetate, and then quenched with 500 mL of saturated ammonium chloride aqueous solution. Liquid-liquid extraction followed, the aqueous phase was further extracted once with 500 mL of ethyl acetate, the combined organic phase was washed once with 500 mL of saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, PE / EA=1 / 8) yielded 12.00 g of a pale yellow oily substance with a yield of 26.58%. MS m / z[M+NH4] + = 512.2. 1 H NMR(400MHz,CDCl3)δ 8.69(s,1H),7.99(dd,J=8.1,3.8Hz,1H),5.91(d,J=2.3Hz,1H),5.77(d,J=8.1Hz,1H),4.26-4.22(m,1H),4.09-4.07(m,1H),3.94(dd,J=11 .1,2.4Hz,1H),3.89-3.73(m,8H),3.68(dd,J=10.7,1.9Hz,1H),3.61-3.60(m,1H),3.54-3.53(m,3H),0.90-0.88(m,9H),0.10-0.06(m,6H). 31 P NMR(162MHz, CDCl3)δ 23.69.

[0117] Step 2: Synthesis of YK-VP-002-PM2 YK-VP-002-PM1 (12.00 g, 24.20 mmol) was dissolved in 240 mL of tetrahydrofuran, and triethylamine hydrofluoric acid (19.56 g, 126.00 mmol) was added. The mixture was stirred at 40°C for 19 hours, and the reaction was stopped when the starting material disappeared and the target molecular weight was found by TLC and LC / MS analysis. The reaction mixture was concentrated by rotary evaporation, and then concentrated once more with acetonitrile to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH=50 / 1) yielded 4.80 g of a pale yellow solid with a yield of 52.0%. MS m / z[M+NH4] + =398.0. 1 H NMR(400MHz,DMSO-d6)δ 11.37(s,1H),7.82(d,J=8.1Hz,1H),5.86(d,J=4.8Hz,1H),5.62(d,J=8.1Hz,1H),5.31(d,J=5.0Hz ,1H),4.10(d,J=4.6Hz,1H),3.97-3.95(m,3H),3.82-3.73(m,2H),3.72-3.64(m,7H),3.35(s,3H). 31 P NMR(162MHz,DMSO-d6)δ 24.49.

[0118] Step 3: Synthesis of YK-VP-002 YK-VP-002-PM2 (1.60 g, 4.20 mmol) was concentrated three times by azeotropic rotational evaporation with super-dehydrated dichloromethane. The dried YK-VP-002-PM2 was dissolved in 16 mL of super-dehydrated dichloromethane, cooled to 5°C under a nitrogen atmosphere, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.90 g, 6.30 mmol) and tetrazole (0.35 g, 5.04 mmol) were added sequentially. The mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour, and the reaction was stopped after determining the target molecular weight by LC / MS and TLC analysis. The reaction mixture was washed twice with saturated sodium bicarbonate aqueous solution (15 mL x 2), the aqueous phase was further extracted once with 30 mL of dichloromethane, the combined organic phase was washed once with 10% sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated by rotational evaporation to obtain the crude product. Rapid purification yielded 1.60 g of a colorless oily substance, with a yield of 65.6%. MS m / z [MH] - = 579.4. 1 H NMR(400MHz,DMSO-d6)δ 11.39(s,1H),7.84(dd,J=8.1,3.2Hz,1H),5.88(t,J=5.2Hz,1H),5.64(d,J=8.1Hz,1H),4.44-4.33(m,1H),4.19-4.11(m,1H),4.04-3 .88(m,3H),3.85-3.73(m,3H),3.72-3.67(m,6H),3.65-3.57(m,3H),3.39(s,2H),3.33(s,1H),2.84-2.76(m,2H),1.19-1.14(m,12H). 31 P NMR(162MHz,DMSO-d6)δ 150.66,149.83,24.45,24.34.

[0119] 3. Synthesis of YK-VP-003 The synthesis route is as follows:

[0120] [ka]

[0121] Step 1: Synthesis of YK-VP-003-PM1 YK-VP-001-PM2 (6.50 g, 17.45 mmol) was dissolved in 105 mL of super-dehydrated tetrahydrofuran. Under a nitrogen atmosphere, NaH (2.09 g, 52.35 mmol) was added in fractions at 5°C and the mixture was stirred for 1 hour, followed by stirring at room temperature for 1 hour. Dimethyl vinylphosphonate (23.75 g, 174.50 mmol) was added under ice water bath conditions, and the mixture was reacted overnight at room temperature. Analysis by TLC and LC / MS confirmed that the starting materials had reacted almost completely and the target molecular weight was found, so the reaction was stopped. The mixture was diluted with 300 mL of ethyl acetate, washed twice with saturated ammonium chloride aqueous solution (300 mL x 2), and the combined aqueous phase was back-extracted once with 300 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, PE / EA=1 / 8) yielded 4.02 g of a pale yellow oily substance, with a yield of 45.32%. MS m / z[M+NH4] + = 526.2. 1 H NMR(400MHz,CDCl3)δ 9.45(s,1H),7.92(d,J=8.1Hz,1H),5.87(d,J=1.9Hz,1H),5.71(d,J=8.1Hz,1H),4.20(dd,J=7.6,4.8Hz,1H),4.15-4.01(m,1H),3.83(dd,J =10.9,2.2Hz,1H),3.80-3.70(m,8H),3.61(dd,J=4.9,2.1Hz,1H),3.57-3.50(m,4H),2.04-2.14(m,2H),0.87(s,9H),0.06(d,J=5.3Hz,6H). 31 P NMR(162MHz, CDCl3)δ 31.05.

[0122] Step 2: Synthesis of YK-VP-003-PM2 YK-VP-003-PM1 (4.00 g, 7.86 mmol) was dissolved in 80 mL of tetrahydrofuran, and triethylamine hydrofluoric acid (6.34 g, 39.30 mmol) was added. The mixture was stirred at 40°C for 19 hours, and the reaction was stopped when the starting material disappeared and the target molecular weight was found by TLC and LC / MS analysis. The reaction mixture was concentrated by rotary evaporation, and then concentrated once more with acetonitrile to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH=30 / 1) yielded 1.80 g of a pale yellow solid with a yield of 58.2%. MS m / z[M+H] + =395.0. 1 H NMR(400MHz,CDCl3)δ 8.79(s,1H),7.93(d,J=8.1Hz,1H),5.95(s,1H),5.73(d,J=8.1Hz,1H),4.31-4.26(m,1H),4.0 4(d,J=7.2Hz,1H),3.90-3.73(m,11H),3.69(d,J=9.7Hz,1H),3.61(s,3H),2.15-2.07(m,2H). 31 P NMR(162MHz, CDCl3)δ 31.36.

[0123] Step 3: Synthesis of YK-VP-003 YK-VP-003-PM2 (1.80 g, 4.56 mmol) was concentrated three times by azeotropic rotational evaporation with super-dehydrated dichloromethane. The dried YK-VP-003-PM2 was dissolved in 18 mL of super-dehydrated dichloromethane, cooled to 5°C under a nitrogen atmosphere, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.06 g, 6.84 mmol) and tetrazole (0.38 g, 5.47 mmol) were added sequentially. The mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour, and the reaction was stopped after determining the target molecular weight by LC / MS and TLC analysis. The reaction mixture was washed twice with saturated sodium bicarbonate aqueous solution (15 mL x 2), the aqueous phase was further extracted once with 30 mL of dichloromethane, the combined organic phase was washed once with 10% sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated by rotational evaporation to obtain the crude product. Rapid purification yielded 1.20 g of a colorless oily substance, with a yield of 44.3%. MS m / z [MH] - = 593.5. 1 H NMR(400MHz,DMSO-d6)δ 11.38(s,1H),7.81(dd,J=8.2,1.6Hz,1H),5.87(dd,J=5.7,4.2Hz,1H),5.71(dd,J=8.1,2.2Hz,1H),4.42-4.33(m,1H),4.17-4.02(m,2H) ),3.82-3.76(m,2H),3.71-3.51(m,12H),3.39(s,2H),3.33(s,1H),2.80(td,J=5.8,1.1Hz,2H),2.20-2.10(m,2H),1.17-1.13(m,12H). 31 P NMR(162MHz,DMSO-d6)δ 150.42,149.68,31.90,31.69.

[0124] 4. Synthesis of YK-VP-004 The synthesis route is as follows:

[0125] [ka]

[0126] Step 1: Synthesis of YK-VP-004-PM1 YK-VP-004-SM (20.00 g, 29.08 mmol) was dissolved in 100 mL of super-dehydrated N,N-dimethylformamide, and then TBDMSCl (6.57 g, 43.62 mmol) and imidazole (4.95 g, 72.70 mmol) were added in fractions. The mixture was stirred at room temperature under a nitrogen atmosphere for 21 hours. Analysis by TLC revealed that the starting material had disappeared and a new, low-polarity spot had appeared, so the reaction was stopped. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL), washed four times with water (100 mL x 4), and the aqueous phase was back-extracted once with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain the crude product, which was 25.29 g of a white solid and was used directly in the next step. MS m / z[M+H] + = 802.4.

[0127] Step 2: Synthesis of YK-VP-004-PM2 YK-VP-004-PM1 (1.60 g, 2.00 mmol) was dissolved in 80 mL of 3% TCA / DCM (50V) solution, then Et3SiH (1.60 mL, 10.00 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Analysis by TLC and LC / MS revealed the disappearance of the starting material and the discovery of the target molecular weight, thus terminating the reaction. Water (80 mL) was added for quenching, followed by extraction, and the mixture was washed twice with dichloromethane (80 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain an oily substance. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0-94 / 6) yielded 0.89 g of a white solid with a yield of 89.6%. MS m / z[M+H] + = 500.1. 1H NMR(400MHz,CDCl3)δ 8.81(s,1H),8.15(s,1H),8.09-8.02(m,2H),7.68-7.59(m,1H),7.55(t, J=7.6Hz,2H),5.98(d,J=6.7Hz,1H),4.63(d,J=6.6Hz,2H),4.25(d,J=1.6 Hz,1H),4.00(dd,J=13.1,1.7Hz,1H),3.77(dd,J=13.1,1.7Hz,1H),3.30 (s,3H),2.98(s,1H),2.90(s,1H),0.99(s,9H),0.19(s,3H),0.17(s,3H).

[0128] Step 3: Synthesis of YK-VP-004-PM3 YK-VP-004-PM2 (7.03 g, 14.10 mmol) was dissolved in 105 mL of super-dehydrated N,N-dimethylformamide. Under ice bath conditions, NaH (1.69 g, 70.50 mmol) was added in fractional amounts, and the mixture was stirred under a nitrogen atmosphere for 10 minutes, followed by stirring at room temperature for 30 minutes. Under ice bath conditions, p-toluenesulfonyloxymethylphosphonate dimethyl (12.43 g, 42.30 mmol) was added, and the mixture was reacted at room temperature for 17 hours. Analysis by TLC and LC / MS revealed that the starting material had disappeared and the target molecular weight had been found, at which point the reaction was stopped. The mixture was diluted with 100 mL of ethyl acetate, washed twice with saturated ammonium chloride aqueous solution (100 mL x 2), and then washed twice again with water (100 mL x 2). The combined aqueous phase was back-extracted once with 100 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated by rotary evaporation to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0-96 / 4) yielded 4.01 g of a white solid, with a yield of 45.7%. MS m / z[M+H] + = 622.2. 1H NMR(400MHz,DMSO-d6)δ 11.12(br,1H),8.77(s,1H),8.62(s,1H),8.08-8.01(m,2H),7.69-7.62(m,1H),7.57-7. 54(m,2H),6.18(d,J=5.6Hz,1H),4.65(t,J=5.2Hz,1H),4.37(t,J=4.3Hz,1H),4.19(q,J =4.0Hz,1H),4.10-4.04(m,2H),3.90(dd,J=11.5,4.2Hz,1H),3.81(dd,J=11.4,4.2Hz,1 H),3.74(d,J=1.7Hz,3H),3.72(d,J=1.7Hz,3H),3.42(s,3H),0.89(s,9H),0.08(s,6H).

[0129] Step 4: Synthesis of YK-VP-004-PM4 YK-VP-004-PM3 (4.01 g, 6.45 mmol) was dissolved in 80 mL of super-dehydrated tetrahydrofuran, and triethylamine hydrofluoric acid (5.25 mL, 32.20 mmol) was added. The mixture was stirred at 40°C for 16 hours under a nitrogen atmosphere. Analysis by LC / MS and TLC revealed that the starting material had disappeared and the target molecular weight had been found, at which point the reaction was stopped. The mixture was concentrated by rotary evaporation to remove the solvent, and further concentrated by a single azeotropic rotary evaporation with acetonitrile to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0-94 / 6) yielded 1.97 g of a white solid with a yield of 60.2%. MS m / z[M+H] + = 508.1. 1 H NMR(400MHz,DMSO-d6)δ 11.22(s,1H),8.77(s,1H),8.76(s,1H),8.09-8.01(m,2H),7.70-7.60( m,1H),7.57-7.53(m,2H),6.17(d,J=6.0Hz,1H),5.30(t,J=5.5Hz,1H), 4.65(t,J=5.4Hz,1H),4.38(dd,J=4.7,3.2Hz,1H),4.19(q,J=3.7Hz,1H ),4.15-4.02(m,2H),3.75-3.69(m,7H),3.66-3.61(m,1H),3.40(s,3H). 31P NMR(162MHz,DMSO-d6)δ 23.22.

[0130] Step 5: Synthesis of YK-VP-004 YK-VP-004-PM4 (1.02 g, 2.00 mmol) was concentrated with super-dehydrated dichloromethane by triple azeotropic rotational evaporation. The dried YK-VP-004-PM4 was dissolved in 20 mL of super-dehydrated dichloromethane, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.08 g, 1.14 mL, 3.60 mmol) and DCI (354 mg, 3.00 mmol) were added sequentially. The mixture was stirred at room temperature under a nitrogen atmosphere for 1.5 hours. Analysis by LC / MS revealed that the starting materials had disappeared and the target molecular weight had been found, at which point the reaction was stopped. The mixture was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (30 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain an oily substance. This oily substance was then purified by medium-pressure column chromatography (SiO2, PE + 1%TEA / EA + 1%TEA = 100 / 0 - 0 / 100) to obtain 630 mg of a white solid, with a yield of 43.6%. MS m / z [MH] - = 706.2. 1 1H NMR (400MHz, DMSO-d6)δ 11.22(s,1H),8.76(s,1H),8.65(d,J=4.3Hz,1H),8.08-8.01(m,2H),7.67-7. 63(m,1H),7.57-7.53(m,2H),6.18(dd,J=5.9,2.6Hz,1H),4.75-4.70(m,1H),4 .45-4.40(m,1H),4.34-4.29(m,1H),4.17-3.89(m,3H),3.84-3.71(m,9H),3. 65-3.51(m,2H),3.41(d,J=2.0Hz,3H),2.81-2.77(m,2H),1.16-1.10(m,12H). 31 P NMR(162MHz,DMSO-d6)δ 147.79,147.63,23.02,22.99.

[0131] 5. Synthesis of YK-VP-005 The synthesis route is as follows:

[0132] [ka]

[0133] Step 1: Synthesis of YK-VP-005-PM1 YK-VP-004-PM2 (0.43 g, 0.85 mmol) was dissolved in 5 mL of super-dehydrated tetrahydrofuran, and tert-butoxysodium (0.33 g, 3.40 mmol) was added under ice bath conditions. The mixture was stirred for 1 hour under a nitrogen atmosphere, and then continued stirring at room temperature for 1 hour. Diethyl p-toluenesulfonyloxymethylphosphonate (1.1 mL, 1.37 g, 4.25 mmol) was added, and the mixture was reacted overnight at room temperature. Analysis by TLC and LC / MS confirmed that the starting material did not further convert, and the reaction was stopped. The mixture was diluted with 10 mL of dichloromethane, and the reaction was quenched with saturated ammonium chloride aqueous solution (10 mL). The mixture was extracted, washed twice with dichloromethane (10 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated by rotary evaporation to obtain an oily substance. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0-95 / 5) yielded 0.19 g of a white solid, with a yield of 34.6%. MS m / z[M+H] + = 650.0. 1 H NMR(400MHz,CDCl3)δ 9.13(s,1H),8.86(s,1H),8.60(s,1H),8.10-8.03(m,2H),7.70-7.61(m,1H),7.57( dd,J=8.4,6.9Hz,2H),6.28(d,J=4.0Hz,1H),4.56(t,J=5.0Hz,1H),4.28-4.20(m,6 H),4.01(dd,J=10.7,3.0Hz,1H),3.91(d,J=8.3Hz,2H),3.84(dd,J=10.8,3.0Hz,1H ),3.53(s,3H),1.39(td,J=7.1,3.3Hz,6H),0.96(s,9H),0.16(s,3H),0.15(s,3H). 31 P NMR(162MHz, CDCl3)δ 20.82.

[0134] Step 2: Synthesis of YK-VP-005-PM2 YK-VP-005-PM1 (0.14 g, 0.21 mmol) was dissolved in 1.8 mL of super-dehydrated tetrahydrofuran, and triethylamine hydrofluoric acid (0.17 g, 173 μL, 1.06 mmol) was added. The mixture was stirred at 40°C for 16 hours under a nitrogen atmosphere, and the reaction was stopped when the starting material disappeared and the target molecular weight was found by TLC and LC / MS analysis. Saturated sodium bicarbonate aqueous solution was slowly added to quench the reaction, and the mixture was extracted with dichloromethane (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0-91 / 9) yielded 0.09 g of a white solid with a yield of 79.2%. MS m / z[M+H] + = 536.0. 1 H NMR(400MHz,CDCl3)δ 9.27(br,1H),8.79(s,1H),8.56(s,1H),8.05-8.00(m,2H),7.61-7.56(m,1H),7.52-7.48(m,2H),6.28(d,J=3.5Hz,1H),4.52(t,J=5 .3Hz,1H),4.24-4.15(m,6H),4.01(dd,J=10.8,2.5Hz,1H),3.92-3.82(m,3H),3.56(s,3H),2.43(s,1H),1.33(td,J=7.1,5.2Hz,6H).

[0135] Step 3: Synthesis of YK-VP-005 YK-VP-005-PM2 (0.69 g, 1.30 mmol) was concentrated with super-dehydrated dichloromethane by three azeotropic rotational evaporations. Dried YK-VP-002-PM2 was dissolved in 14 mL of super-dehydrated dichloromethane, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.69 g, 730 uL, 2.30 mmol) and DCI (0.23 g, 1.95 mmol) were added sequentially. The mixture was stirred at room temperature under a nitrogen atmosphere for 2.5 hours, and the reaction was stopped when the starting material disappeared and the target molecular weight was found by LC / MS and TLC analysis. Saturated sodium bicarbonate aqueous solution was added for quenching, and the mixture was extracted with dichloromethane (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotational evaporation to obtain the crude product. Purification by medium-pressure column chromatography (SiO2, PE + 1%TEA / EA + 1%TEA = 100 / 0 - 0 / 100) yielded 0.70 g of a white solid, with a yield of 73.1%. MS m / z [MH] - = 734.4. 1 H NMR(400MHz,Acetonitrile-d3)δ 9.47(s,1H),8.70(d,J=1.8Hz,1H),8.64(d,J=10.5Hz,1H),8.05(d,J=7.2Hz,2H),7 .68(t,J=7.4Hz,1H),7.58(t,J=7.7Hz,2H),6.24(t,J=5.8Hz,1H),4.77-4.65(m,1H ),4.56-4.46(m,1H),4.44-4.34(m,1H),4.24-4.09(m,4H),3.95-3.87(m,4H),3.84 -3.67(m,3H),3.49(s,1H),3.43(s,1H),2.74(t,J=6.0Hz,2H),1.39-1.19(m,20H). 31 P NMR (162MHz, Acetonitrile-d3) δ 150.86,149.73,20.83,20.69.

[0136] 6. Synthesis of Phosphoramidite 3

[0137] [ka] Following the synthesis method for Phosphoramidite 3 in WO2018045317A1, 438 mg of the product was obtained, with a total yield of 1.9%.

[0138] Example 2: Preparation of siRNA In this example, two types of siRNA parent sequences were synthesized and named D84-DV27-G5 and D82-DV29-G5, respectively. The final positions of D84-DV27-G5 and D82-DV29-G5 were modified with phosphate esters, and the resulting siRNA double-stranded sequences are shown in Table 1.

[0139] [Table 1] TIFF2026127613000020.tif30170

[0140] The meanings of the abbreviations used in this specification are as follows: A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively. The 'm' indicates that the nucleotide adjacent to its left is a 2'-OMe modified nucleotide (2'-methoxynucleoside). For example, Am, Um, Gm, and Cm represent 2'-OMe modified A, U, G, and C, respectively. The 'f' indicates that the nucleotide adjacent to its left is a 2'-F modified nucleotide (2'-fluoronucleoside). For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively. The 's' indicates that the two nucleotides and / or delivery carriers adjacent to it are connected via thiophosphate esters. EVP indicates that the nucleotide adjacent to its left is a 5'-(E)-VP modified nucleotide; see Table 2 for the structure of UmEVP. 5'-O-EtMP indicates that the nucleoside structure of the nucleotide adjacent to its left side is provided by YK-VP-001, and the structure of Ums-5'-O-EtMP is shown in Table 2. 5'-O-MeMP indicates that the nucleoside structure of the nucleotide adjacent to its left side is provided by YK-VP-002; see Table 2 for the structure of Ums-5'-O-MeMP. 5'-O-MeEP indicates that the nucleoside structure of the nucleotide adjacent to its left side is provided by YK-VP-003; see Table 2 for the structure of Ums-5'-O-MeEP. 4'-O-MeMP indicates that the nucleoside structure of the nucleotide adjacent to its left side is provided by Phosphoramidite 3; see Table 2 for the structure of Ums-4'-O-MeMP. 5'-O-MeMP indicates that the nucleoside structure of the nucleotide adjacent to its left side is provided by YK-VP-004; see Table 2 for the structure of Am-5'-O-MeMP. 5'-O-EtMP indicates that the nucleoside structure of the nucleotide adjacent to its left side is provided by YK-VP-005. See Table 2 for the structure of Am-5'-O-EtMP. 5'-O-MP indicates that the nucleoside structure of the nucleotide adjacent to its left side is provided by YK-VP-004, and the structure of Am-5'-O-MP is shown in Table 2. G5 shows a GalNAc delivery carrier with the following structure, where

[0141] [ka] This indicates that it connects to the 3' end of the siRNA sense strand via a phosphate ester group or a thiophosphate ester group, and can be referred to as CN116854754A.

[0142] [ka]

[0143] 1. Preparation of GalNAc-unbound siRNA antisense strands siRNA antisense chains were synthesized on corresponding solid-phase supports based on the phosphoramidite chemistry method. When synthesizing sequence 2 (D84-DV27-PG5), a commercially available (E)-VP-Um monomer was used as the final coupling monomer of the antisense chain. When synthesizing sequences 3-5 (D84-DV27-5EMPG5, D84-DV27-5MMPG5, and D84-DV27-5MEPG5), the 5'-phosphate ester-modified nucleoside phosphoramidite monomers (YK-VP-001, YK-VP-002, and YK-VP-003) synthesized in Example 1 were used as the final coupling monomers of the antisense chains, respectively. When synthesizing sequence 6 (D84-DV27-4MMPG5), the Phosphoramidite 3 monomer from Example 1 was used as the final coupling monomer of the antisense chain.

[0144] When synthesizing sequences 8-10 (D82-DV29-5MMPG5, D82-DV29-5EMPG5, and D82-DV29-5MPG5), the 5'-phosphate ester-modified nucleoside phosphoramidite monomers (YK-VP-004 and YK-VP-005) synthesized in Example 1 were used as the final coupling monomers of the antisense chains, and the synthesis scale for each was 200 nmol. Table 2 shows the antisense chain terminal structures of the synthesized siRNA.

[0145] [Table 2] TIFF2026127613000024.tif220170TIFF2026127613000025.tif60170

[0146] (1) Preparation of reagents and monomers A monomer acetonitrile solution (0.15 M) and a 0.25 M 5-ethylthiotetrazole acetonitrile solution were used as activators, a 3% pyridine solution of xanthogen hydride was used as a vulcanizing agent, a 0.05 M water / pyridine (10 / 90, v / v) solution of iodine was used as an oxidizing agent, a 10% anhydrous acetic acid acetonitrile solution (v / v) was used as capping agent A, a 1-methylimidazole / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v) was used as capping agent B, a 3% dichloroacetic acid toluene solution (v / v) was used as a DMTr removal agent, and a general-purpose carrier of cross-linked polystyrene beads (Primer support 5G Unylinker 350, Cytiva) was selected as the solid phase carrier. Each of these was loaded into the designated reagent positions of the 192 P-type DNA / RNA automated synthesizer.

[0147] (2) Synthesis of crude products After inputting the specified oligonucleotide sequence, setting the synthesis procedure, and confirming that there were no errors, the cyclic synthesis of oligonucleotides was started. The preparation was then carried out according to the following steps. a. Deprotection A 3% toluene dichloroacetate solution was used as a deprotection reagent to deprotect the DMTr protecting group, and then the mixture was washed with acetonitrile. b. Coupling Using 0.25M 5-ethylthiotetrazole as an activator, acetonitrile solutions of each nucleotide monomer were coupled, and then rinsed with acetonitrile. c. Oxidation / vulcanization Oxidation: Oxidation was carried out using a 0.05 M iodine-water / pyridine (90 / 10) solution as the oxidizing agent, followed by rinsing with acetonitrile. Vulcanization: A 3% pyridine solution of xanthogen hydride was used as a vulcanizing agent, followed by rinsing with acetonitrile. d. Protection of hydroxyl groups Capping agents A and B were used as hydroxyl group protecting reagents to perform hydroxyl group protection, and then the samples were rinsed with acetonitrile. The above operations were repeated cyclically according to the set sequence to obtain a fully protected product.

[0148] (3) Deprotection a. Preparation of monomethyl ester-protected 5'-phosphate-modified siRNA antisense chains The solid support was transferred to a reactor, concentrated aqueous ammonia (25-28%) was added, and ammonia decomposition was maintained at 60°C for 12 hours. After that, the reaction system was cooled to room temperature, and the mixture was transferred to a pressure filter tank. Elution was carried out with a mixed solution of purified water and ethanol, the filtrates were combined, and the filtrate was concentrated at low temperature to obtain the crude product of a single methyl ester-protected 5'-phosphate ester-modified siRNA antisense chain. b. Preparation of fully deprotected 5'-phosphate-modified siRNA antisense strands The solid support was transferred to a reactor, TMSI / Py / DCM solution was added, and the reaction was allowed to proceed at room temperature for 1 hour. Then, the reaction was quenched by adding 2-mercaptoethanol TEA / ACN solution. Subsequently, the reaction was carried out according to method a of deprotection to obtain the crude product of a completely deprotected 5'-phosphate ester-modified siRNA antisense chain.

[0149] (4) Purification The crude product residue after deprotection was dissolved in purified water and purified by HPLC. The peak solution of the product was collected, its content was measured using a microplate reader, and its purity and molecular weight were confirmed by LC / MS. The peak solution of the product was concentrated and freeze-dried to obtain the final product.

[0150] 2. Preparation of GalNAc-conjugated siRNA sense strands siRNA sense strands were synthesized according to the synthesis method for siRNA antisense strands. A G5-GalNAc support was used as the solid phase support, and the synthesis volume of each complementary sense strand to the antisense strand was 200 nmol.

[0151] 3. Preparation of double-stranded siRNA siRNA sense strands and complementary antisense strands were mixed in a 1:1 UV absorption ratio, heated to 95°C and held for 3 minutes, then slowly cooled to room temperature to form double strands. The purity of the resulting double-stranded solution was measured by HPLC to meet specifications, and the content was measured using a microplate reader. The solution was then freeze-dried to obtain a solid powder, which was stored for use. The molecular weight and purity of the obtained siRNA double strands are shown in Table 3.

[0152] [Table 3]

[0153] Example 3: Inhibitory effect of modified oligonucleotide sequences on PCSK9 in mouse serum and effect on LDL-C levels This example investigated the inhibitory effect of the siRNA sequences shown in Table 1 on PCSK9 and the effect on LDL-C levels in mouse serum. Experimental materials Test drug: Modified siRNA sequences listed in Table 1 Preparation of the test drug: Drug solvent: PBS buffer Preparation conditions: sterile environment Labeling method: The prepared dosage formulations were labeled by affixing labels, and the outer packaging clearly indicated the test number, name, concentration, quantity, preparation date, preparer, and storage conditions. Storage conditions: Use immediately after preparation, and store any remaining sample at -20°C. Laboratory animal information: Animal species / lineage: B6-hPCSK9-UTR(T053388) mouse Grade: SPF Gender: Male Quantity: 59 animals Age: 6-8 weeks Source: Jiangsu Jiyakang Biological Technology Co., Ltd.

[0154] Rearing and management: Rearing conditions: After receiving the experimental animals, they were reared at Jiangsu Jicui Yaokang Biotechnology Co., Ltd., where the animals were allowed to feed and drink freely. Standard SPF grade radiation-sterilized transgenic mouse feed was used and purchased from Jiangsu Cooperative Medical and Biotechnology Co., Ltd. For each batch of feed, the manufacturer provided a feed quality assurance certificate, and an annual third-party inspection report was provided. The detection standards referred to the national standards GB14924.3-2010 "Nutritional Components of Formulated Feed for Laboratory Animals" and GB14924.2-2001 "General Hygienic Standards for Formulated Feed for Laboratory Animals". For drinking water, the appearance and bacterial indicators were detected once a month in-house, and an annual inspection report from the local water bureau was provided, referring to the national standard GB5749-2006 "Hygienic Standards for Drinking Water".

[0155] 1. Repression rate of PCSK9 protein expression in mouse serum by different modified siRNA sequences Experimental method: The day of administration was designated as D0, and approximately 200 μL of blood was collected from the orbital venous plexus on D-3. The whole blood samples were temporarily stored with a cooling pack before centrifugation, and then centrifuged for 10 minutes at approximately 4°C with a centrifugal force of approximately 3000 g. PCSK9 levels were detected using a PCSK9 kit (purchased from Proteintech), and based on these levels, the mice were divided into groups of 6, ensuring uniformity of PCSK9 levels in each group. The drug was administered by subcutaneous injection on D0 (day 0) at a dose of 2 mg / kg. Blood was collected from the orbital venous plexus on D7 (day 7), D14 (day 14), and D21 (day 21), serum was separated, and PCSK9 protein levels were detected. Experimental results: Table 4 shows the inhibition rates of PCSK9 protein in serum on days 7, 14, and 21 after administration.

[0156] [Table 4]

[0157] 1) All of the 5'-phosphate-modified siRNA sequences of the present invention can efficiently suppress PCSK9 protein expression in mouse serum. As is clear from Table 4 and Figure 1, the 5'-phosphate ester modified siRNA sequences of the present invention have a significant inhibitory effect on PCSK9 protein expression in mouse serum. For example, on days 7, 14, and 21, the inhibition rates of D84-DV27-5EMPG5, D84-DV27-5MMPG5, and D84-DV27-5MEPG5 were all higher than those of D84-DV27-G5. Here, the inhibition rates of YK-VP-002 modified D84-DV27-5MMPG5 on days 7, 14, and 21 were 16.5%, 18.1%, and 19.7%, respectively, higher than those of D84-DV27-G5. For example, on days 7, 14, and 21, the inhibition rates of D82-DV29-5MMPG5 and D82-DV29-5EMPG5 were all higher than those of D82-DV29-G5. Here, the inhibition rates of YK-VP-005 modified D82-DV29-5EMPG5 on days 7, 14, and 21 were 8.6%, 13.6%, and 13.3%, respectively, higher than those of D82-DV29-G5.

[0158] 2) The 5'-phosphate ester-modified siRNA sequence of the present invention significantly improves the suppression rate of PCSK9 protein expression in mouse serum compared to siRNA sequences modified with conventional compounds. Conventional siRNA sequences modified with compounds, including D84-DV27-PG5 modified with (E)-VP-Um and D84-DV27-4MMPG5 modified with Phosphoramidite 3, showed lower inhibition rates on days 7, 14, and 21 compared to the 5'-phosphate ester modified siRNA sequences of the present invention. For example, compared to D84-DV27-PG5, the inhibition rates of D84-DV27-5MMPG5 on days 7, 14, and 21 were increased by 11.7%, 7.3%, and 9.8%, respectively. Compared to D84-DV27-4MMPG5, the inhibition rates of D84-DV27-5MMPG5 on days 7, 14, and 21 were increased by 11.8%, 16.5%, and 15.3%, respectively.

[0159] Structurally, compared to YK-VP-002, Phosphoramidite 3 lacks one CH2 at the 5'-position of the ribose ring, and the rest of its structure is completely identical. However, the correspondingly modified siRNA showed a significant difference in the suppression rate of PCSK9 protein expression in mouse serum. Thus, the 5'-phosphate ester modified siRNA sequence of the present invention yielded an unexpected effect.

[0160] 2. Effects of different modified siRNA sequences on LDL-C levels in mouse serum Experimental process: The day of administration was designated as D0 (Day 0), and approximately 200 μL of blood was collected from the orbital venous plexus on D3 (Day 3). The whole blood samples were temporarily stored with a cooling pack before centrifugation, and then centrifuged for 10 minutes at approximately 4°C with a centrifugal force of approximately 3000 g. LDL-C levels were detected using a biochemical analyzer, and based on these levels, the mice were divided into groups of 6, ensuring that the LDL-C levels were uniform in each group. The drug was administered by subcutaneous injection on D0 (Day 0) at a dose of 2 mg / kg. Blood was collected from the orbital venous plexus on D7 (Day 7), D14 (Day 14), and D21 (Day 21), serum was separated, and serum LDL-C levels were detected.

[0161] Experimental results: Serum LDL-C levels were reduced on days 7, 14, and 21 after administration, and the specific results are shown in Table 5.

[0162] [Table 5]

[0163] 1) All of the 5'-phosphate ester modified siRNA sequences of the present invention can efficiently reduce LDL-C levels in mouse serum. As is clear from Table 5 and Figure 2, the 5'-phosphate ester modified siRNA sequences of the present invention can significantly reduce LDL-C levels in mouse serum. For example, YK-VP-002 modified D84-DV27-5MMPG5 reached reduction levels of 35.8%, 40.5%, and 36.3% on days 7, 14, and 21, respectively, which were 15.0%, 14.2%, and 16.6% higher than the parent sequence D84-DV27-G5, respectively. For example, the YK-VP-005 modified D82-DV29-5EMPG5 showed degradation levels of 30.8%, 33.6%, and 28.2% on days 7, 14, and 21, respectively, which are significantly higher than those of the parent sequence D82-DV29-G5.

[0164] 2) The 5'-phosphate ester-modified siRNA sequence of the present invention significantly improves the level of LDL-C reduction in mouse serum compared to siRNA sequences modified with conventional compounds. siRNA sequences modified with conventional compounds, specifically D84-DV27-PG5 and D84-DV27-4MMPG5, showed degradation levels of 20% to 31% on days 7, 14, and 21, respectively, all lower than the 30% to 41% of the 5'-phosphate ester modified siRNA sequences of the present invention. For example, compared to D84-DV27-PG5, the degradation levels of D84-DV27-5EMPG5 on days 7, 14, and 21 were increased by 9.9%, 8.5%, and 8.4%, respectively. Compared to D84-DV27-4MMPG5, the degradation levels of D84-DV27-5MMPG5 on days 7, 14, and 21 were increased by 11.2%, 11.6%, and 16.0%, respectively.

[0165] As is clear from the experimental results above, the 5'-phosphate-modified siRNA of the present invention is more effective than existing phosphate-modified siRNA in lowering LDL-C levels in mouse serum, yielding unexpected results.

Claims

1. A modified nucleoside compound characterized by having the structure shown in formula (I), or being a pharmaceutically acceptable salt or stereoisomer of a compound having the structure shown in formula (I). 【Chemistry 1】 (In the formula, R １ H is R ２ ha-OCH ３ And, R ３ is a protecting group or a phosphorus-containing active reactive group, and the phosphorus-containing active reactive group is one of the following: phosphoramidite, H-phosphate ester, phosphate triester, or phosphorus-containing chiral auxiliary, and the phosphorus-containing chiral auxiliary is 【Chemistry 2】 Selected from, R ４ and R ５ are each independently H, C １－６ alkyl group, -CH ２ CH ２ CN or -CH ２ O(CO)C(CH ３ ) ３ selected from, X is either O or S, L is -CH ２ - or -CH ２ CH ２ - and A is O, B is a modified or unmodified base or a salt thereof. D is O.

2. R ４ and R ５ It is not possible for it to be an isopropyl group at the same time, or R ４ and R ５ Each is independently -CH ２ CH ３ ien-CH ３ The modified nucleoside compound according to claim 1, characterized in that it is selected from H.

3. R ４ ha-CH ３ or -CH ２ CH ３ And, Or, R ５ ha-CH ３ or -CH ２ CH ３ And, Alternatively, X is O, Alternatively, B is 【Transformation 3】 The modified nucleoside compound according to claim 1, characterized in that it is such.

4. R ３ teeth 【Chemistry 4】 The modified nucleoside compound according to claim 1, characterized in that it is such.

5. R ４ and R ５ The modified nucleoside compound according to claim 3, characterized in that the two are identical.

6. The modified nucleoside compound according to claim 1, characterized in that the compound shown in formula (I) is one of the following compounds YK-VP-001, YK-VP-002, YK-VP-003, YK-VP-004, and YK-VP-005 having the following structures. 【Transformation 5】

7. An oligonucleotide characterized in that the constituent unit of the oligonucleotide includes a nucleotide containing the modified nucleoside compound described in claim 1.

8. The oligonucleotide according to claim 7, characterized in that the nucleotide containing the modified nucleoside compound is located at the 5' end of the oligonucleotide.

9. The oligonucleotide according to claim 7, characterized in that the oligonucleotide is an oligonucleotide shown in formula (II) or a pharmaceutically acceptable salt thereof. 【Transformation 6】 (In the formula, Oligo is an oligonucleotide, R １ H is R ２ ha-OCH ３ And, R ４ and R ５ H and C are independent of each other. １－６ Alkyl alkyl group, -CH ２ CH ２ CN or -CH ２ O(CO)C(CH ３ ) ３ Selected from, X is either O or S, L is -CH ２ - or -CH ２ CH ２ - and A is O, D is O, X １ is O or S, and B is 【Transformation 7】 (That is the case.)

10. The oligonucleotide according to claim 7, characterized in that the oligonucleotide is one selected from small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, small guide RNAs, transfer RNAs, and aptamers.

11. The oligonucleotide according to claim 10, characterized in that the oligonucleotide is siRNA.

12. The oligonucleotide according to claim 11, characterized in that the siRNA is double-stranded siRNA or single-stranded siRNA.

13. The oligonucleotide according to claim 12, characterized in that the siRNA is a double-stranded siRNA and includes a sense strand and an antisense strand.

14. The oligonucleotide according to claim 13, characterized in that the antisense strand in the double-stranded siRNA is an oligonucleotide shown in formula (II) or a pharmaceutically acceptable salt thereof.

15. The oligonucleotide according to claim 7, characterized in that each nucleotide in the oligonucleotide is independently modified or unmodified.

16. A nucleic acid complex comprising an oligonucleotide as described in claim 7 or a pharmaceutically acceptable salt thereof, and a target ligand that binds thereto.

17. The nucleic acid complex according to claim 16, characterized in that the target ligand comprises one of a carbohydrate, a lipid, a polypeptide, or an antibody.

18. The nucleic acid complex according to claim 17, characterized in that the lipids include cholesterol.

19. The nucleic acid complex according to claim 17, characterized in that the target ligand includes an N-acetylgalactosamine moiety.

20. The nucleic acid complex according to claim 19, characterized in that the N-acetylgalactosamine portion is a monovalent N-acetylgalactosamine portion, a divalent N-acetylgalactosamine portion, a trivalent N-acetylgalactosamine portion, or a tetravalent N-acetylgalactosamine portion.

21. A pharmaceutical composition for treating or preventing a pathological condition or disease caused by the expression of the proprotein convertase subtilisin / kexin 9 gene in hepatocytes, comprising an oligonucleotide according to any one of claims 7 to 15 or a pharmaceutically acceptable salt thereof, or a nucleic acid complex according to any one of claims 16 to 20, preferably the pharmaceutical composition further comprising a pharmaceutically acceptable auxiliary material.

22. The pharmaceutical composition for treating or preventing a pathological condition or disease caused by the expression of the proprotein convertase subtilisin / kexin 9 gene in hepatocytes, as described in 21, characterized in that the aforementioned pathological condition or disease is one or at least two combinations selected from dyslipidemia and cardiovascular disease.

23. The aforementioned dyslipidemia includes hypercholesterolemia. Alternatively, the cardiovascular disease includes atherosclerosis, and the pharmaceutical composition for treating or preventing a condition or disease caused by the expression of the proprotein convertase subtilisin / kexin 9 gene in hepatocytes, as described in 22.

24. A kit comprising an oligonucleotide according to any one of claims 7 to 15 or a pharmaceutically acceptable salt thereof, or a nucleic acid complex according to any one of claims 16 to 20.

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