5'-phosphate modified nucleoside as well as preparation method and application thereof

Abstract

The present invention relates to the field of biomedical science, specifically providing a 5'-phosphate ester-modified nucleoside and a method for its preparation and applications. The present invention provides a modified nucleoside compound having a structure represented by formula (I), or a pharmaceutically acceptable salt or stereoisomer of a compound having a structure represented by formula (I). The oligonucleotide of the modified nucleoside compound provided in the present invention has significantly enhanced pharmacological efficacy.

Description

Technology Field

[0001] The present invention relates to the field of biomedical science, and specifically to a 5'-phosphate ester-modified nucleoside and a method for manufacturing the same and its applications. Background Technology

[0002] Small nucleic acid drugs achieve target selection through base complementary binding and possess high specificity by directly targeting specific genes. They also regulate target gene expression at the post-transcriptional level by extending the drug's target to the upstream mRNA of pathogenic proteins. Compared to antibody drugs or small molecule drugs, small nucleic acid drugs have advantages such as abundant targets, short development cycles, and long-term efficacy. There are various types of small nucleic acid drugs, including antisense oligonucleotides (ASOs), small interfering oligonucleotides (siRNAs), micro oligonucleotides (miRNAs), and nucleic acid aptamers; among these, three types of small nucleic acid drugs—ASOs, siRNAs, and aptamers—are being most actively researched.

[0003] siRNA drugs act through the 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 to form a phosphate group. This phosphate group at the 5' end can bind to the Ago2 protein, inducing the siRNA to enter the RNA-induced silencing complex (RISC). Once the siRNA sensor strand is cleaved and removed, the formed active RISC matches and binds to the target mRNA; the target mRNA is then cleaved by endonucleases within the RISC, thereby reducing its expression. The released active RISC continues to seek out and bind to target mRNAs, entering the catalytic cycle to sustain pharmacological effects. Therefore, the presence of a phosphate group at the 5' end of the antisense strand of the siRNA double strand is essential for specific binding to the side chain residues of the Ago2 domain, which is vital for RNA interference activity. However, according to research, siRNA with a modified natural phosphate group at the 5' end is rapidly degraded by lysosomal acid phosphatase after entering through the endocytosis pathway, which limits bioavailability (Reka AH, et al. Nucleic Acids Res., 2017, 45, 7581) and consequently affects efficacy.

[0004] In some cases, replacing the natural phosphate group at the 5' end of the siRNA antisense strand with a phosphate ester analog can improve the in vivo efficacy of siRNA, and since the modified phosphate ester is not a substrate of in vivo phosphatase, it can resist exonuclease degradation after modification, thereby enhancing the in vivo silencing effect of siRNA. However, siRNA obtained by conventional modification methods still has problems such as insufficient resistance to exonuclease degradation, minimal improvement in siRNA silencing effect, or negative effects on siRNA silencing effect.

[0005] Therefore, it is desirable to develop modified nucleoside compounds and introduce them into siRNA to more effectively and stably enhance the efficacy of siRNA drugs. The problem to be solved

[0006] To solve the above problems existing in the prior art, the present invention provides a 5'-phosphate ester-modified nucleoside, a method for manufacturing the same, and applications. Specifically, the present invention designs a modified nucleoside compound, and the modified nucleoside compound of the present invention can be effectively introduced into the 5' end of an oligonucleotide chain through a solid-phase synthesis method. Compared to the prior art, an oligonucleotide containing the modified nucleoside compound of the present invention exhibits stronger inhibitory efficiency against a target gene. means of solving the problem

[0007] The inventors of the present invention unexpectedly discovered that a nucleoside compound formed by introducing an alkyl group between the phosphorus atom of phosphate and the 5'-hydroxyl group of a sugar ring can effectively enhance resistance to nuclease degradation such as siRNA, while simultaneously strengthening the in vivo silencing effect of siRNA. By introducing a nucleotide containing a nucleoside compound modified in this way into an oligonucleotide, a nucleic acid drug with enhanced efficacy can be obtained.

[0008] Based on this, a first aspect of the present invention provides a modified nucleoside compound, said modified nucleoside compound having a structure represented by formula (I), or a pharmaceutically acceptable salt or stereoisomer of a compound having a structure represented by formula (I), and

[0009]

[0010] In the above formula,

[0011] R1 and R2 are independently H, halogen, and C, respectively. 1-6 alkyl or C 1-6Selected from alkoxy;

[0012] R3 is an active reactor containing hydrogen, a protecting group, or phosphorus;

[0013] R4 and R5 each independently contain hydrogen, C 1-6 Selected from alkyl, -CH2CH2CN or -CH2O(CO)C(CH3)3;

[0014] X is O or S and;

[0015] L is C 1-6 Alkyl, C 2-5 Alkenyl or C 2-5 It is alkynyl;

[0016] A is O or S;

[0017] B is H, a modified or unmodified base or a salt thereof;

[0018] D is O, S, or -CH2-.

[0019] According to some preferred embodiments of the present invention, R4 and R5 are not simultaneously isopropyl.

[0020] According to some preferred embodiments of the present invention, here

[0021] R1 is H, R2 is -OCH3 and;

[0022] R4 and R5 are each independently selected from -CH2CH3, -CH3 or H;

[0023] L is CH2.

[0024] According to some preferred embodiments of the present invention, R1 is H.

[0025] According to some preferred embodiments of the present invention, R2 is -OCH3.

[0026] According to some preferred embodiments of the present invention, R3 is a phosphorus-containing active reactor.

[0027] According to some preferred embodiments of the present invention, R4 is -CH3 or -CH2CH3.

[0028] According to some preferred embodiments of the present invention, R5 is -CH3 or -CH2CH3.

[0029] According to some preferred embodiments of the present invention, here X is O.

[0030] According to some preferred embodiments of the present invention, where L is -CH2- or -CH2CH2-.

[0031] According to some preferred embodiments of the present invention, where A is O.

[0032] According to some preferred embodiments of the present invention, where B is or am. " ” indicates the connection location. “Bz” means benzoyl group.

[0033] According to some preferred embodiments of the present invention, where D is O.

[0034] According to some preferred embodiments of the present invention, the phosphorus-containing active reactor is any one of phosphoramidite, H-phosphate ester, phosphate tryster, or phosphorus-containing chiral auxiliary.

[0035] According to some preferred embodiments of the present invention, where R3 is am. " ” indicates the connection location.

[0036] According to some preferred embodiments of the present invention, R4 and R5 are identical.

[0037] According to some particularly preferred embodiments of the present invention, the compound represented by formula (I) above is any one or a combination of at least two of the compounds YK-VP-001, YK-VP-002, YK-VP-003, YK-VP-004, and YK-VP-005 having the following structure.

[0038] “Bz” means benzoyl group, and “OMe” means methoxy.

[0039] In addition, the present invention further provides a use of the modified nucleoside compound according to the first embodiment in the process of improving oligonucleotide stability (e.g., resistance to exonuclease degradation effects).

[0040] Based on this, the present invention further provides a method for improving oligonucleotide stability (e.g., improved resistance to exonuclease degradation effects), said method comprising the step of using a nucleotide containing a modified nucleoside compound according to the first aspect of the present invention instead of at least a partial structural unit (nucleotide) of the oligonucleotide.

[0041] Preferably, instead of 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide, a nucleotide containing a modified nucleoside compound according to the first embodiment of the present invention is used.

[0042] In addition, the present invention further provides a use of a modified nucleoside compound according to the first embodiment in the process of enhancing the in vivo function of oligonucleotides (e.g., enhancing the silencing effect of siRNA).

[0043] Based on this, the present invention further provides a method for improving the in vivo function of an oligonucleotide, wherein the method comprises the step of using a nucleotide containing a modified nucleoside compound according to the first aspect of the present invention instead of at least a partial structural unit (nucleotide) among the oligonucleotides.

[0044] Preferably, instead of 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide, a nucleotide containing a modified nucleoside compound according to the first embodiment of the present invention is used.

[0045] In addition, the present invention further provides a use of a modified nucleoside compound according to the first embodiment in the process of improving oligonucleotide stability (e.g., resistance to exonuclease degradation effects) while simultaneously improving the in vivo function of the oligonucleotide (e.g., improvement of the silencing effect of siRNA).

[0046] Based on this, the present invention further provides a method for improving the stability of an oligonucleotide (e.g., improving resistance to exonuclease degradation effects) while simultaneously improving the in vivo function of the oligonucleotide (e.g., improving the silencing effect of siRNA), and the method comprises the step of using a nucleotide containing a modified nucleoside compound according to the first aspect of the present invention instead of at least a partial structural unit (nucleotide) of the oligonucleotide.

[0047] Preferably, instead of 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide, a nucleotide containing a modified nucleoside compound according to the first embodiment of the present invention is used.

[0048] A second aspect of the present invention provides an oligonucleotide, wherein the structural unit of the oligonucleotide comprises a nucleotide containing a modified nucleoside compound according to the first aspect. That is, the oligonucleotide provided in the present invention can be formed by replacing a partial structural unit (nucleotide) of a general oligonucleotide with a nucleotide synthesized with a modified nucleoside compound of the present invention (i.e., a compound represented by formula (I) or a pharmaceutically acceptable salt thereof) and phosphoric acid.

[0049] In the oligonucleotide provided in the present invention, a partial structural unit (nucleotide) may contain the modified nucleoside compound provided in the first embodiment, and all structural units may contain the modified nucleoside compound. When the modified nucleoside compound is contained in a partial structural unit, other structural units of the oligonucleotide may be provided by a normal nucleotide or by another nucleotide containing the existing modified nucleoside compound.

[0050] In the present invention, the structural unit of an oligonucleotide refers to a nucleotide. As is known in the art, a nucleotide is a compound composed of a base (purine base or pyrimidine base), a pentose (ribose or deoxyribose), and a phosphate. The base condenses with the pentose to form a nucleoside, and then reacts with the phosphate to synthesize a nucleotide. The statement “the structural unit of the oligonucleotide comprises a nucleotide containing a modified nucleoside compound according to 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 various nucleotides included in the oligonucleotide is replaced by the modified nucleoside compound provided in the present invention.

[0051] In some preferred embodiments, for an oligonucleotide composed of 30 or fewer nucleotides (e.g., 15 to 30 nucleotides), the number of structural units (nucleotides) of the modified nucleoside compound provided in the present invention is 3 or fewer (e.g., 1, 2, or 3).

[0052] According to some preferred embodiments of the present invention, the nucleotide containing the modified nucleoside compound is located at the 5' end of the oligonucleotide.

[0053] According to a particularly preferred embodiment of the present invention, the last nucleotide at the 5' end of the oligonucleotide is a nucleotide containing the modified nucleoside compound.

[0054] According to some preferred embodiments of the present invention, the oligonucleotide is an oligonucleotide represented by formula (II) or a pharmaceutically acceptable salt thereof, and

[0055]

[0056] In the above formula,

[0057] Oligo is an oligonucleotide, and

[0058] R1 and R2 are independently H, halogen, and C, respectively. 1-6 alkyl or C 1-6 Selected from alkoxy;

[0059] R4 and R5 each independently H, C 1-6 Selected from alkyl, -CH2CH2CN or -CH2O(CO)C(CH3)3;

[0060] X is O or S and;

[0061] L is C 1-6 Alkyl, C 2-5 Alkenyl or C 2-5 It is alkynyl;

[0062] A is O or S;

[0063] D is O, S, or -CH2;

[0064] X1 is O or S, and B is or It should be noted that the structure represented by the above formula (II) is an oligonucleotide in which the last position of the 5' end is a nucleotide containing the modified nucleoside compound provided in the present invention, that is, the part connected to Oligo is the structure of the nucleotide at the last position of the complete oligonucleotide, and Oligo represents the remaining part of the oligonucleotide starting from the second position of the 5' end.

[0065] By using the modified nucleoside compound provided in the present invention instead of the partial nucleoside structure of the oligonucleotide, the oligonucleotide can be made more stable and the function of the oligonucleotide itself can be promoted and enhanced. Any oligonucleotide in the art can be modified using the modified nucleoside compound provided in the present invention (i.e., using the modified nucleoside compound instead of the nucleoside structure portion of the oligonucleotide). According to a preferred embodiment of the present invention, the oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides (siRNA), antisense oligonucleotides (ASO), microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), and aptamers.

[0066] 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 the oligonucleotide refers to the number of nucleotides (also called base number, nt) contained therein in the case of a single-stranded oligonucleotide, and the number of nucleotide pairs (also called base pair number, bp) contained therein in the case of a double-stranded oligonucleotide.

[0067] According to some preferred embodiments of the present invention, the oligonucleotide is siRNA.

[0068] Preferably, the siRNA is double-stranded siRNA or single-stranded siRNA.

[0069] According to some particularly preferred embodiments of the present invention, the siRNA is a double-stranded siRNA and comprises a sensor strand and an antisense strand.

[0070] Preferably, the antisense strand of the double-stranded siRNA is an oligonucleotide represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0071] According to a preferred embodiment of the present invention, each nucleotide of the oligonucleotide is independently a modified or unmodified nucleotide.

[0072] According to some preferred embodiments of the present invention, the oligonucleotide may be used to regulate the expression of a target gene (e.g., may be used to inhibit the expression of a target gene).

[0073] According to some particularly preferred embodiments of the present invention, the oligonucleotide is any one of the following siRNAs or a combination of at least two.

[0074] (i) The sensor strand has a sequence denoted by SEQ ID NO. 5; the antisense strand has a sequence denoted by SEQ ID NO. 6.

[0075] (ii) The sensor strand has the sequence denoted by SEQ ID NO. 7; the antisense strand has the sequence denoted by SEQ ID NO. 8.

[0076] (iii) The sensor strand has a sequence denoted by SEQ ID NO. 9; the antisense strand has a sequence denoted by SEQ ID NO. 10.

[0077] (iv) The sensor strand has the sequence denoted by SEQ ID NO. 15; the antisense strand has the sequence denoted by SEQ ID NO. 16.

[0078] (v) The sensor strand has the sequence denoted by SEQ ID NO. 17; the antisense strand has the sequence denoted by SEQ ID NO. 18.

[0079] (vi) The sensor strand has the sequence denoted by SEQ ID NO. 19; the antisense strand has the sequence denoted by SEQ ID NO. 20.

[0080] A third aspect of the present invention provides a nucleic acid conjugate, said nucleic acid conjugate comprising an oligonucleotide according to the second aspect or a pharmaceutically acceptable salt thereof, and a targeting ligand bound thereto.

[0081] In the present invention, a targeting ligand that can be used in any nucleic acid conjugate of the art may be selected. According to some preferred embodiments of the present invention, the targeting ligand comprises any one or a combination of at least two of a carbohydrate, cholesterol, lipid, polypeptide, or antibody.

[0082] Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) portion.

[0083] All GalNAc portions generally used as targeting ligands for nucleic acid conjugates in the art are applicable to the present invention. According to some preferred embodiments of the present invention, the N-acetylgalactosamine portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0084] According to some particularly preferred embodiments of the present invention, the targeting ligand is G5, and its structure is as shown below.

[0085]

[0086] In the above formula, This indicates that it is connected to the 3' end of the siRNA sensor strand through the phosphate ester gene or the thiophosphate ester gene.

[0087] A fourth aspect of the present invention provides a pharmaceutical composition, said pharmaceutical composition comprising an oligonucleotide according to a second aspect or a pharmaceutically acceptable salt thereof;

[0088] and / or, the above pharmaceutical composition comprises a nucleic acid conjugate according to a third embodiment.

[0089] In the pharmaceutical composition provided by the present invention, the oligonucleotide and / or nucleic acid conjugate provided by the present invention may be used as a major active ingredient. In some embodiments, the oligonucleotide and / or nucleic acid conjugate provided by the present invention may be used as the only active ingredient in the pharmaceutical composition; and in some embodiments, the pharmaceutical composition may further contain other active ingredients in addition to the oligonucleotide and / or nucleic acid conjugate of the present invention.

[0090] According to some preferred embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable auxiliary material. “Pharmaceutically acceptable auxiliary material” means any material that may be added to the process as an inactive component. For example, the pharmaceutical composition provided in the present invention may include a pharmaceutically acceptable carrier, an adjuvant, an auxiliary material, etc., and may further 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 enables the nucleoside analogs, oligonucleotides, conjugates, or compositions thereof of the present invention to be introduced into or transported into the patient’s body to perform their intended function. Generally, such structures are transported or transported from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable,” which means that it is compatible with other components of the formulation, including nucleoside analogs, oligonucleotides, conjugates, or compositions thereof, of the present invention, and is harmless or substantially harmless to the patient.Some embodiments 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 derivatives thereof such as carboxymethylcellulose sodium, ethylcellulose, and cellulose acetate; gum tragacanth in powder form; malt; gelatin; talc; excipients such as 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; diols such as glycerol, sorbitol, mannitol, and polyethylene glycol; and polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol. It includes esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; sterile distilled water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; and other non-toxic compatible materials used in drug formulations. As used herein, the “pharmaceutically acceptable carrier” further comprises any coating agent, antimicrobial and antifungal agent, or absorption retardant that is compatible with the activity of the nucleoside analogs, oligonucleotides, conjugates, or compositions thereof of the present invention and is physiologically acceptable to the patient. A supplemented active compound may also be mixed into the composition. The “pharmaceutically acceptable carrier” may further comprise a pharmaceutically acceptable salt of a compound that can be used in the present invention. Other additional ingredients that may be included in the pharmaceutical composition used in the practice of the present invention are known in the art, for example, Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., cited by reference herein)As described in , 1985, Easton, PA). Any carrier, adjuvant, or auxiliary material used in the manufacture of drugs in the art may be applied to the present invention, and, for example, auxiliary materials may include, but are not limited to, adhesives, suspending agents, emulsifiers, diluents (or fillers), granulators, adhesives, disintegrants, lubricants, anti-adhesives, wetting agents, gelling agents, absorption retardants, solubility inhibitors, reinforcing agents, adsorbents, chelating agents, coloring agents, fragrances, coating agents, buffers, protective agents, preservatives, solubility aids, pH adjusters, etc.

[0091] A fifth aspect of the present invention provides for the use of an oligonucleotide according to the second aspect or a pharmaceutically acceptable salt thereof, or a nucleic acid conjugate according to the third aspect, or a pharmaceutical composition according to the fourth aspect in the manufacture of a drug, said drug is used to treat and / or prevent a pathological condition or disease caused by the expression of the progenitor protein convertase subtilisin / kexin 9 gene (PCSK9 gene) in hepatocytes.

[0092] The PCSK9 gene is located on chromosome 1, and the protein it codes for plays an important role in cholesterol and fatty acid metabolism. Abnormal expression of the said gene is associated with metabolic diseases such as autosomal dominant hereditary hypercholesterolemia.

[0093] According to some preferred embodiments of the present invention, the pathological condition or disease is selected from any one or a combination of at least two of hypercholesterolemia, dyslipidemia, atherosclerosis, and cardiovascular disease.

[0094] Additionally, the present invention provides a method for treating a pathological condition or disease caused by PCSK9 gene expression, the method comprising the step of administering an oligonucleotide according to a second embodiment or a pharmaceutically acceptable salt thereof, or a nucleic acid conjugate according to a third embodiment, or a pharmaceutical composition according to a fourth embodiment to a subject in need thereof, or administering a drug prepared from an oligonucleotide according to a second embodiment or a pharmaceutically acceptable salt thereof, or a nucleic acid conjugate according to a third embodiment, or a pharmaceutical composition according to a fourth embodiment.

[0095] 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.

[0096] A sixth aspect of the present invention provides a kit, said kit comprising an oligonucleotide according to a second aspect or a pharmaceutically acceptable salt thereof, or a nucleic acid conjugate according to a third aspect, or a pharmaceutical composition according to a fourth aspect.

[0097] The kit provided in the present invention can be used for therapeutic or non-therapeutic purposes as well as for diagnostic or non-diagnostic purposes.

[0098] The active enhancement effect of the present invention is as follows. That is, the present invention has designed a series of 5'-phosphate ester-modified nucleosides, which have a completely different chemical structure compared to 5'-terminal phosphorylated compounds of the prior art. Oligonucleotides containing the 5'-phosphate ester-modified nucleosides of the present invention can significantly enhance the inhibitory activity of siRNA against target genes. Effects of the invention

[0099] Compared to conventional technology, the present invention has at least the following beneficial effects.

[0100] 1. It is completely different from the compound structure of the existing 5' terminal phosphorylation modification technology.

[0101] The 5'-phosphate ester-modified nucleoside designed in the present invention is a completely new 5'-phosphate ester-modified nucleoside compound that is completely different from the prior art and has an alkyl group introduced between the phosphorus atom of the phosphate and the 5'-hydroxyl of the sugar ring. The above compound can be rapidly produced by using a commercially available and easily obtainable nucleoside as a raw material and reacting the nucleoside 5'-hydroxyl with an alkyl / alkenyl compound containing a phosphate ester system through a one-step nucleophilic substitution / addition reaction.

[0102] 2. Compared to compounds of the prior art, the 5'-phosphate ester modified siRNA sequence of the present invention has higher inhibitory activity.

[0103] For example, compared to D84-DV27-PG5 (modified by the existing compound (E)-VP-Um), the inhibition rate of PCSK9 protein expression in mouse serum in D84-DV27-5MMPG5 (modified by the compound YK-VP-002 of the present invention) increased by 11.7%, 7.3%, and 9.8% on day 7, day 14, and day 21, respectively. Compared to D84-DV27-4MMPG5 (modified by the existing compound Phosphoramidite 3), the inhibition rate of PCSK9 protein expression in mouse serum in D84-DV27-5MMPG5 (modified by the compound YK-VP-002 of the present invention) increased by 11.8%, 16.5%, and 15.3% on day 7, day 14, and day 21, respectively.

[0104] Compared to D84-DV27-PG5, the reduction level of LDL-C in mouse serum in D84-DV27-5EMPG5 (modified by the compound YK-VP-001 of the present invention) increased by 9.9%, 8.5%, and 8.4% on day 7, day 14, and day 21, respectively. Compared to D84-DV27-4MMPG5, the reduction level in D84-DV27-5MMPG5 increased by 11.2%, 11.6%, and 16.0% on day 7, day 14, and day 21, respectively. Brief explanation of the drawing

[0105] To more clearly explain specific embodiments of the present invention or technical solutions of the prior art, drawings to be used in describing specific embodiments or prior art are briefly introduced below. It should be noted that the drawings described below are merely some embodiments of the present invention, and a person skilled in the art can obtain other drawings based on these drawings without inventive effort. Figure 1 shows the inhibition rates of 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 PCSK9 protein expression in mouse serum on days 7, 14, and 21 of administration. Figure 2 shows the reduced levels of LDL-C in the serum of mice administered 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 of administration. Specific details for implementing the invention

[0106] To make the purpose, technical solution, and advantages of the embodiments of the present invention clearer, the technical solution of the embodiments of the present invention is described below in a clear and complete manner. It is evident that the described embodiments are only some embodiments of the present invention and not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by a person skilled in the art without inventive effort fall within the scope of protection of the present invention.

[0107] The present invention may be embodied in other specific forms without departing from the basic attributes of the present invention. It should be understood that, unless contradictory, any embodiment of the present invention may be combined with any other embodiment or the technical features of a plurality of other embodiments to obtain additional embodiments. The present invention includes additional embodiments obtained through such combinations.

[0108] All publications and patents mentioned in this invention are incorporated herein by reference in their entirety. If any use or term used in any publication or patent cited by reference conflicts with the use or term used in this invention, the use and term used in this invention shall prevail.

[0109] The chapter and section titles used in this specification are for organizational purposes only and should not be understood as a limitation on the subject matter.

[0110] Unless otherwise specified, all technical and scientific terms used herein have their ordinary meanings in the field to which the subject matter to be protected belongs. If multiple definitions exist for a particular term, the definition in this specification shall be used as the standard.

[0111] Except in working examples or as otherwise specified, all numbers of a quantitative nature, such as dosages stated in the specification and claims, should be understood to be modified by the term “about” in all circumstances. It should also be understood that any numerical range listed in the present invention is intended to include any combination of all sub-ranges within said range and any combination of each endpoint of said range or sub-range.

[0112] The terms “include,” “contain,” or “include,” and similar words used in the present invention, mean that the elements mentioned before such words include the elements listed after such words and their equivalents, without excluding elements not described. The terms “contain” or “include (include)” used in this specification may be open, semi-closed, and closed. In other words, the terms also include the meaning of “composed of ... basically” or “composed of ...

[0113] In the present invention, a “protecting group” refers to a group that allows a reactor to be used in a reversibly inactive state under certain conditions of a desired reaction in the conventional chemical sense. After the desired reaction has passed, the protecting group can be removed to return the reactor from protected to unprotected. All protecting groups must be capable of being removed under conditions where a significant proportion of the positively synthesized molecule is not degraded.

[0114] In the present invention, “phosphorus-containing active reactive group” refers to a phosphorus-containing group that reacts with a hydroxyl or amine group contained in another molecule, particularly another nucleotide unit or another nucleotide analog, through a nucleophilic reaction. Generally, such a reaction produces an esterified nucleoside-nucleoside bond that links a nucleotide unit or nucleotide analog to another nucleotide unit or nucleotide analog. Such phosphorus-containing active reactive group is a phosphorus atom known in the art, including the P(III) or P(V) state, and said phosphorus-containing active reactive group includes, but is not limited to, phosphoramidites, H-phosphate esters, phosphate triesters, and phosphorus-containing chiral auxiliaries, for example is. Here, a phosphorus-containing chiral auxiliary refers to a phosphorus-containing group having chirality, for example It can be a group of various forms such as.

[0115] The term “halogen” refers to fluorine, chlorine, bromine, or iodine.

[0116] term “C 1-6 " means that the main chain and side chains of the group contain a total of any integer number of carbon atoms within the range of 1 to 6, such as 1, 2, 3, 4, 5, or 6 carbon atoms. Similarly, the term “C 2-5 " means that the group's main chain and side chain contain a total of any integer number of carbon atoms within the range of 2 to 5, such as 2, 3, 4, or 5 carbon atoms.

[0117] The term “alkyl” refers to a specified number of carbon atoms (e.g., C 1-6It means a straight-chain or branched-chain alkyl having ). Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, etc. The term “alkyl” further includes heteroalkyl, that is, a group formed in which one or more carbon atoms of the alkyl (e.g., 1, 2, 3, or 4) are replaced by heteroatoms (i.e., atoms other than carbon or hydrogen, such as oxygen, sulfur, fluorine, nitrogen, or phosphorus).

[0118] The term “alkoxy” refers to a group of the RO- form, where R is an alkyl defined above.

[0119] The term “alkenyl” means a hydrocarbon group having at least one carbon-carbon double bond at one or more positions along the carbon chain of an alkyl group, and alkenyls include but are not limited to vinyl, propenyl, butenyl, etc.

[0120] The term “alkynyl” means a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the carbon chain of an alkyl group, and alkynyl includes but is not limited to ethinyl, propynyl, butynyl, etc.

[0121] The term “pharmaceuticalally acceptable salt” means a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base (relatively non-toxic, safe, and suitable for use by patients). If the compound contains a relatively acidic reactive group, a base-added salt may be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base-added salts include, but are not limited to, sodium, potassium, calcium, aluminum, magnesium, bismuth, and ammonium salts. If the compound contains a relatively basic reactive group, an acid-added salt may be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid-added salts include, but are not limited to, hydrochlorides, sulfates, and mesylates.

[0122] In the present invention, the term “pharmaceuticalally acceptable excipient” refers to all substances contained in a drug formulation excluding the active ingredient.

[0123] The term “treatment” means any one of the following situations: (1) alleviating one or more biological symptoms of the disease; (2) disrupting one or more points of the biological chain reaction that causes the disease; or (3) slowing the progression of one or more biological symptoms of the disease.

[0124] The term “prevention” means reducing the risk of disease occurrence and reducing the severity of the disease when it occurs.

[0125] The oligonucleotides of the present invention include single-stranded oligonucleotides (e.g., antisense oligonucleotides, abbreviated as ASO) and double-stranded oligonucleotides (e.g., small interfering nucleotides, abbreviated as siRNA).

[0126] The oligonucleotides of the present invention include natural oligonucleotides and chemically modified oligonucleotides. Herein, chemical modification includes nucleoside modification (including sugar portion modification and nucleobase modification) and modification of inter-nucleoside bonding connections. Chemical modification of oligonucleotides does not include cases where only the nucleobase sequence differs. Here, "natural" refers to cases corresponding to naturally occurring RNA or DNA.

[0127] Based on common sense in the art, each of the above priority conditions can be arbitrarily combined to obtain each preferred embodiment of the present invention.

[0128] All reagents and raw materials used in this invention are commercially available.

[0129] Examples

[0130] To make the purpose, technical solution, and advantages of the embodiments of the present invention clearer, the technical solution of the embodiments of the present invention is described below in a clear and complete manner. It is evident that the described embodiments are only some embodiments of the present invention and not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by a person skilled in the art without inventive effort fall within the scope of protection of the present invention.

[0131] The present invention may be embodied in other specific forms without departing from the basic attributes of the present invention. It should be understood that, unless contradictory, any embodiment of the present invention may be combined with any other embodiment or the technical features of a plurality of other embodiments to obtain additional embodiments. The present invention includes additional embodiments obtained through such combinations.

[0132] The present invention is described in more detail in conjunction with the following examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples may be further adjusted according to different specific requirements, and implementation conditions not specified are general conditions of the company. In specific examples of the present invention, all raw materials used are commercially available. Unless otherwise stated, all temperatures are provided in degrees Celsius. Technical features associated with each embodiment of the present invention may be combined with one another unless they are inconsistent with one another.

[0133] Example 1: Synthesis of a compound

[0134] The following abbreviations each represent the reagents listed below. 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-dicyanomidazole.

[0135] 1. Synthesis of YK-VP-001

[0136] The synthesis path is as follows.

[0137]

[0138] Step 1: Synthesis of YK-VP-001-PM1

[0139] YK-VP-001-SM (365.00 g, 0.64 mol) was dissolved in 1460 mL of ultra-dry dichloromethane and stirred in an ice bath under nitrogen protection. Subsequently, imidazole (130.70 g, 1.92 mol) and TBDMSCl (192.90 g, 1.28 mol) were added in portions, and the mixture was stirred at room temperature for 5 hours under nitrogen protection. Upon detection of the reaction status by TLC, it was found that the raw material had disappeared and a new small polar spot had appeared, so the reaction was stopped. Water (730 mL) was added for quenching, extraction was performed, and then washed once with 730 mL of water. The organic phase was dried with anhydrous sodium sulfate, and the filtrate obtained after filtration was used directly in the next step.

[0140] Step 2: Synthesis of YK-VP-001-PM2

[0141] Methanol (1 V, 365 mL) and p-toluenesulfonic acid monohydrate (12.20 g) were sequentially added to a dichloromethane (4 V, 1460 mL) solution of YK-VP-001-PM1 (0.64 mol) and stirred at room temperature. As a result of detecting the reaction status by TLC, the raw material disappeared after 2 hours. 182 mL of TEA (0.5 V) was added to quench the reaction and stirred for 1 hour, and then 730 mL of saturated sodium bicarbonate aqueous solution was added and stirred continuously for 15 minutes. The liquids were then separated, and the aqueous phase was extracted once with 365 mL of dichloromethane. The combined organic phase was washed once with 730 mL of saturated sodium chloride aqueous solution, dried again with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated to obtain a pale yellow solid. The crude product was slurried in 1460 mL of n-heptane (4 V), and after filtration, 183.20 g of white solid was obtained, with a yield of 75.5%. MS m / z [M + H] + = 373.0. 1 H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 7.74 (d, J= 8.1 Hz, 1H), 5.75 - 5.67 (m, 2H), 4.33 (t, J = 5.3 Hz, 1H), 4.07 - 4.01 (m, 1H), 4.00 - 3.89 (m, 2H), 3.80 - 3.68 (m, 1H), 3.47 (s, 3H), 3.08 (dd, J = 6.6, 3.6 Hz, 1H), 0.89 (s, 9H), 0.08 (d, J = 5.0 Hz, 6H).

[0142] Step 3: Synthesis of YK-VP-001-PM3

[0143] YK-VP-001-PM2 (20.00 g, 53.70 mmol) was dissolved in 200 mL of ultra-dry tetrahydrofuran, and under nitrogen protection, sodium tert-butoxide (20.64 g, 214.80 mmol) was added at 5°C and stirred for 1 hour, followed by stirring at room temperature for 1 hour. Diethyl p-toluenesulfonyloxymethylphosphonate (43.26 g, 134.20 mmol) was added and reacted at room temperature for 2 hours. As a result of monitoring the reaction status by TLC, the raw material was basically completely reacted, and the reaction was stopped when a new spot formed beneath the raw material. The reaction mixture was diluted with 500 mL of ethyl acetate, and then quenched by adding 500 mL of saturated aqueous ammonium chloride solution. The liquids were separated, and the aqueous phase was extracted once with 500 mL of ethyl acetate. The combined organic phase was then washed once with 500 mL of saturated sodium chloride aqueous solution, dried again with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated 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 [M - H] - = 521.2. 1H NMR (400 MHz, CDCl3) δ 9.77 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 5.91 (d, J = 2.0 Hz, 1H), 5.78 (dd, J = 8.1, 1.8 Hz, 1H), 4.24 - 4.10 (m, 5H), 4.09 - 4.04 (m, 1H), 3.94 (dd, J = 10.8, 2.2 Hz, 1H), 3.89 - 3.74 (m, 2H), 3.67 (dd, J = 10.9, 1.9 Hz, 1H), 3.60 (dd, J = 4.8, 2.1 Hz, 1H), 3.52 (s, 3H), 1.32 (td, J = 7.1, 4.5 Hz, 6H), 0.87 (s, 9H), 0.07 (d, J = 5.3 Hz, 6H). 31 P NMR (162 MHz, CDCl3) δ 21.25.

[0144] Step 4: Synthesis of YK-VP-001-PM4

[0145] YK-VP-001-PM3 (12.00 g, 23.00 mmol) was dissolved in 240 mL of tetrahydrofuran, and triethylamine trihydrofluoric acid (18.51 g, 114.80 mmol) was added. The mixture was stirred at 40°C for 19 hours, and analysis by TLC and LC / MS showed that the raw material had disappeared and the target molecular weight was found, at which point the reaction was stopped. The reaction solution was directly rotary evaporated and concentrated, and then concentrated once with the addition of 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 (400 MHz, CDCl3) δ 9.13 (s, 1H), 7.97 (d, J= 8.1 Hz, 1H), 5.98 (d, J = 2.5 Hz, 1H), 5.78 (dd, J = 8.2, 1.2 Hz, 1H), 4.28 (dd, J = 6.8, 5.1 Hz, 1H), 4.19 - 4.11 (m, 4H), 4.05 (dt, J = 6.9, 2.1 Hz, 1H), 3.99 (dd, J = 10.8, 2.2 Hz, 1H), 3.90 - 3.74 (m, 4H), 3.58 (s, 3H), 3.46 (s, 1H), 1.35 - 1.34 (m, 6H). 31 P NMR (162 MHz, CDCl3) δ 21.54.

[0146] Step 5: Synthesis of YK-VP-001

[0147] YK-VP-001-PM4 (3.00 g, 7.30 mmol) was rotary evaporated a total of three times with ultra-dry dichloromethane. The dried YK-VP-001-PM4 was dissolved in 30 mL of ultra-dry dichloromethane and cooled to 5°C under nitrogen protection, after which 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 for 1 hour under nitrogen protection, and the reaction was stopped when the target molecular weight was found as a result of analysis by LC / MS and TLC. The reaction mixture was washed twice with a saturated aqueous sodium bicarbonate solution (15 mL × 2), and the aqueous phase was extracted once with 30 mL of dichloromethane. The combined organic phase was then washed once with a 10% aqueous sodium chloride solution, dried again with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated 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 [M - H] -= 607.5. 1 H NMR (400 MHz, CDCl3) δ 9.22 (s, 1H), 7.94 (dd, J = 9.1, 5.7 Hz, 1H), 6.00 - 5.97 (m, 1H), 5.78 (dd, J = 8.2, 3.7 Hz, 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 (162 MHz, CDCl3) δ 151.41, 150.47, 21.49, 21.25.

[0148] 2. Synthesis of YK-VP-002

[0149] The synthesis path is as follows.

[0150]

[0151] Step 1: Synthesis of YK-VP-002-PM1

[0152] YK-VP-001-PM2 (34.00 g, 91.20 mmol) was dissolved in 340 mL of ultra-dry tetrahydrofuran, and under nitrogen protection, sodium tert-butoxide (35.08 g, 364.80 mmol) was added at 5°C and 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 reacted at room temperature for 2 hours. As a result of monitoring the reaction status by TLC, the raw material was basically completely reacted, and the reaction was stopped when a new spot formed beneath the raw material. The reaction mixture was diluted with 500 mL of ethyl acetate, and then quenched by adding 500 mL of saturated aqueous ammonium chloride solution. The liquids were separated, and the aqueous phase was extracted once with 500 mL of ethyl acetate. The combined organic phase was then washed once with 500 mL of saturated sodium chloride aqueous solution, dried again with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated 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 (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.99 (dd, J = 8.1, 3.8 Hz, 1H), 5.91 (d, J = 2.3 Hz, 1H), 5.77 (d, J = 8.1 Hz, 1H), 4.26 - 4.22 (m, 1H), 4.09 - 4.07 (m, 1H), 3.94 (dd, J = 11.1, 2.4 Hz, 1H), 3.89 - 3.73 (m, 8H), 3.68 (dd, J= 10.7, 1.9 Hz, 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 (162 MHz, CDCl3) δ 23.69.

[0153] Step 2: Synthesis of YK-VP-002-PM2

[0154] YK-VP-002-PM1 (12.00 g, 24.20 mmol) was dissolved in 240 mL of tetrahydrofuran, and triethylamine trihydrofluoric acid (19.56 g, 126.00 mmol) was added. The mixture was stirred at 40°C for 19 hours, and analysis by TLC and LC / MS showed that the raw material had disappeared and the target molecular weight was found, at which point the reaction was stopped. The reaction solution was directly rotary evaporated and concentrated, and then concentrated once with the addition of 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 (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.82 (d, J = 8.1 Hz, 1H), 5.86 (d, J = 4.8 Hz, 1H), 5.62 (d, J = 8.1 Hz, 1H), 5.31 (d, J = 5.0 Hz, 1H), 4.10 (d, J = 4.6 Hz, 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 (162 MHz, DMSO-d6) δ 24.49.

[0155] Step 3: Synthesis of YK-VP-002

[0156] YK-VP-002-PM2 (1.60 g, 4.20 mmol) was rotary evaporated a total of three times with ultra-dry dichloromethane. The dried YK-VP-002-PM2 was dissolved in 16 mL of ultra-dry dichloromethane and cooled to 5°C under nitrogen protection, after which 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 for 1 hour under nitrogen protection, and the reaction was stopped when the target molecular weight was found as a result of analysis by LC / MS and TLC. The reaction mixture was washed twice with a saturated aqueous sodium bicarbonate solution (15 mL × 2), and the aqueous phase was extracted once with 30 mL of dichloromethane. The combined organic phase was then washed once with a 10% aqueous sodium chloride solution, dried again with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated 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 (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.84 (dd, J = 8.1, 3.2 Hz, 1H), 5.88 (t, J = 5.2 Hz, 1H), 5.64 (d, J = 8.1 Hz, 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 (162 MHz, DMSO-d6) δ 150.66, 149.83, 24.45, 24.34.

[0157] 3. Synthesis of YK-VP-003

[0158] The synthesis path is as follows.

[0159]

[0160] Step 1: Synthesis of YK-VP-003-PM1

[0161] YK-VP-001-PM2 (6.50 g, 17.45 mmol) was dissolved in 105 mL of ultra-dry tetrahydrofuran. Under nitrogen protection, NaH (2.09 g, 52.35 mmol) was added in portions at 5°C and stirred for 1 hour, followed by stirring at room temperature for 1 hour. Dimethyl vinyl phosphate (23.75 g, 174.50 mmol) was added under ice bath conditions, and the reaction was carried out overnight at room temperature. Analysis by TLC and LC / MS showed that the raw materials were essentially completely reacted 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 a saturated aqueous ammonium chloride solution (300 mL × 2), and the combined aqueous phase was back-extracted once with 300 mL of ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated 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 (400 MHz, CDCl3) δ 9.45 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 1.9 Hz, 1H), 5.71 (d, J = 8.1 Hz, 1H), 4.20 (dd, J = 7.6, 4.8 Hz, 1H), 4.15 - 4.01 (m, 1H), 3.83 (dd, J= 10.9, 2.2 Hz, 1H), 3.80 - 3.70 (m, 8H), 3.61 (dd, J = 4.9, 2.1 Hz, 1H), 3.57 - 3.50 (m, 4H), 2.04 - 2.14 (m, 2H), 0.87 (s, 9H), 0.06 (d, J = 5.3 Hz, 6H). 31 P NMR (162 MHz, CDCl3) δ 31.05.

[0162] Step 2: Synthesis of YK-VP-003-PM2

[0163] YK-VP-003-PM1 (4.00 g, 7.86 mmol) was dissolved in 80 mL of tetrahydrofuran, and triethylamine trihydrofluoric acid (6.34 g, 39.30 mmol) was added. The mixture was stirred at 40°C for 19 hours, and analysis by TLC and LC / MS showed that the raw material had disappeared and the target molecular weight was found, at which point the reaction was stopped. The reaction solution was directly rotary evaporated and concentrated, and then concentrated once with the addition of 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 (400 MHz, CDCl3) δ 8.79 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H), 5.95 (s, 1H), 5.73 (d, J = 8.1 Hz, 1H), 4.31 - 4.26 (m, 1H), 4.04 (d, J = 7.2 Hz, 1H), 3.90 - 3.73 (m, 11H), 3.69 (d, J = 9.7 Hz, 1H), 3.61 (s, 3H), 2.15 - 2.07 (m, 2H). 31 P NMR (162 MHz, CDCl3) δ 31.36.

[0164] Step 3: Synthesis of YK-VP-003

[0165] YK-VP-003-PM2 (1.80 g, 4.56 mmol) was rotary evaporated a total of three times with ultra-dry dichloromethane. The dried YK-VP-003-PM2 was dissolved in 18 mL of ultra-dry dichloromethane and cooled to 5°C under nitrogen protection, after which 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 for 1 hour under nitrogen protection, and the reaction was stopped when the target molecular weight was found as a result of analysis by LC / MS and TLC. The reaction mixture was washed twice with a saturated aqueous sodium bicarbonate solution (15 mL × 2), the aqueous phase was extracted once with 30 mL of dichloromethane, the combined organic phase was washed once with a 10% aqueous sodium chloride solution, dried again with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated 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 (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 7.81 (dd, J = 8.2, 1.6 Hz, 1H), 5.87 (dd, J = 5.7, 4.2 Hz, 1H), 5.71 (dd, J = 8.1, 2.2 Hz, 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, 1.1 Hz, 2H), 2.20 - 2.10 (m, 2H), 1.17 - 1.13 (m, 12H). 31P NMR (162 MHz, DMSO-d6) δ 150.42, 149.68, 31.90, 31.69.

[0166] 4. Synthesis of YK-VP-004

[0167] The synthesis path is as follows.

[0168]

[0169] Step 1: Synthesis of YK-VP-004-PM1

[0170] YK-VP-004-SM (20.00 g, 29.08 mmol) was dissolved in 100 mL of ultra-dry N,N-dimethylformamide, followed by the addition of TBDMSCl (6.57 g, 43.62 mmol) and imidazole (4.95 g, 72.70 mmol) in portions, and the mixture was stirred at room temperature for 21 hours under nitrogen protection. Analysis by TLC revealed that the raw material had disappeared and a new small polar spot had appeared, so the reaction was stopped. The mixture was quenched with water, extracted with ethyl acetate (100 mL), washed four times with water (100 mL × 4), and the aqueous phase was back-extracted once with ethyl acetate (100 mL). The organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated to obtain 25.29 g of a white solid crude product, which was used directly in the next step. MS m / z [M + H] + = 802.4.

[0171] Step 2: Synthesis of YK-VP-004-PM2

[0172] YK-VP-004-PM1 (1.60 g, 2.00 mmol) was dissolved in 80 mL of 3% TCA / DCM (50 V) solution, followed by the addition of Et3SiH (1.60 mL, 10.00 mmol) and stirring at room temperature for 2 hours. Analysis by LC and LC / MS revealed that the raw material had disappeared and the target molecular weight was detected, so the reaction was stopped. The mixture was quenched with water (80 mL), extracted, and then washed twice with dichloromethane (80 mL × 2). The organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated to obtain the oil phase. This oil phase was purified by medium-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0 - 94 / 6) to yield 0.89 g of a white solid, with a yield of 89.6%. MS m / z [M + H] + = 500.1. 1 H NMR (400 MHz, 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 = 7.6 Hz, 2H), 5.98 (d, J = 6.7 Hz, 1H), 4.63 (d, J = 6.6 Hz, 2H), 4.25 (d, J = 1.6 Hz, 1H), 4.00 (dd, J = 13.1, 1.7 Hz, 1H), 3.77 (dd, J = 13.1, 1.7 Hz, 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).

[0173] Step 3: Synthesis of YK-VP-004-PM3

[0174] YK-VP-004-PM2 (7.03 g, 14.10 mmol) was dissolved in 105 mL of ultra-dry N,N-dimethylformamide, and NaH (1.69 g, 70.50 mmol) was added in portions under ice bath conditions and stirred for 10 minutes under nitrogen protection, followed by stirring for 30 minutes at room temperature. Dimethyl p-toluenesulfonyloxymethylphosphonate (12.43 g, 42.30 mmol) was added under ice bath conditions and reacted for 17 hours at room temperature. As a result of analysis by TLC and LC / MS, the raw material disappeared and the target molecular weight was found, so the reaction was stopped. The mixture was diluted with 100 mL of ethyl acetate, washed twice with a saturated aqueous ammonium chloride solution (100 mL × 2), and then washed twice with water (100 mL × 2). The combined aqueous phase was back-extracted once with 100 mL of ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated to obtain the crude product. Purified by high-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0 - 96 / 4), 4.01 g of white solid was obtained, with a yield of 45.7%. MS m / z [M + H] + = 622.2. 1 H NMR (400 MHz, 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 = 5.6 Hz, 1H), 4.65 (t, J = 5.2 Hz, 1H), 4.37 (t, J = 4.3 Hz, 1H), 4.19 (q, J = 4.0 Hz, 1H), 4.10 - 4.04 (m, 2H), 3.90 (dd, J = 11.5, 4.2 Hz, 1H), 3.81 (dd, J = 11.4, 4.2 Hz, 1H), 3.74 (d,J = 1.7 Hz, 3H), 3.72 (d, J = 1.7 Hz, 3H), 3.42 (s, 3H), 0.89 (s, 9H), 0.08 (s, 6H).

[0175] Step 4: Synthesis of YK-VP-004-PM4

[0176] YK-VP-004-PM3 (4.01 g, 6.45 mmol) was dissolved in 80 mL of ultra-dry tetrahydrofuran, and triethylamine trihydrofluoric acid (5.25 mL, 32.20 mmol) was added. The mixture was stirred for 16 hours at 40°C under nitrogen protection. Analysis by LC / MS and TLC revealed that the raw material had disappeared and the target molecular weight was detected, so the reaction was stopped. The mixture was rotary evaporated and concentrated to remove the solvent, and then rotary evaporated once 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 (400 MHz, 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 = 6.0 Hz, 1H), 5.30 (t, J = 5.5 Hz, 1H), 4.65 (t, J = 5.4 Hz, 1H), 4.38 (dd, J = 4.7, 3.2 Hz, 1H), 4.19 (q, J = 3.7 Hz, 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 (162 MHz, DMSO-d6) δ 23.22.

[0177] Step 5: Synthesis of YK-VP-004

[0178] YK-VP-004-PM4 (1.02 g, 2.00 mmol) was rotary evaporated a total of three times with ultra-dry dichloromethane. The dried YK-VP-004-PM4 was dissolved in 20 mL of ultra-dry dichloromethane, followed by the sequential addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.08 g, 1.14 mL, 3.60 mmol) and DCI (354 mg, 3.00 mmol). The mixture was stirred at room temperature for 1.5 hours under nitrogen protection. As a result of LC / MS analysis, the raw material disappeared and the target molecular weight was detected, so the reaction was stopped. The mixture was quenched with an aqueous solution of saturated sodium bicarbonate and extracted with dichloromethane (30 mL × 3). The organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated to obtain an oily substance. This was purified by high-pressure column chromatography (SiO2, PE + 1% TEA / EA + 1% TEA = 100 / 0 - 0 / 100) to yield 630 mg of a white solid, with a yield of 43.6%. MS m / z [M - H] - = 706.2. 1 H NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 8.76 (s, 1H), 8.65 (d, J = 4.3 Hz, 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, 2.6 Hz, 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= 2.0 Hz, 3H), 2.81 - 2.77 (m, 2H), 1.16 - 1.10 (m, 12H). 31 P NMR (162 MHz, DMSO-d6) δ 147.79, 147.63, 23.02, 22.99.

[0179] 5. Synthesis of YK-VP-005

[0180] The synthesis path is as follows.

[0181]

[0182] Step 1: Synthesis of YK-VP-005-PM1

[0183] YK-VP-004-PM2 (0.43 g, 0.85 mmol) was dissolved in 5 mL of ultra-dry tetrahydrofuran, sodium tert-butoxide (0.33 g, 3.40 mmol) was added under ice bath conditions, and the mixture was stirred for 1 hour under nitrogen conditions, followed by continued stirring for 1 hour at room temperature. Diethyl p-toluenesulfonyloxymethylphosphonate (1.1 mL, 1.37 g, 4.25 mmol) was added, and the reaction was carried out overnight at room temperature. As a result of analysis by TLC and LC / MS, the reaction was stopped as no further conversion of the raw material occurred. The mixture was diluted with 10 mL of dichloromethane and quenched with a saturated aqueous ammonium chloride solution (10 mL). After extraction, the mixture was washed twice with dichloromethane (10 mL × 2). The organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated to obtain the oil phase. 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 (400 MHz, 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, 6.9 Hz, 2H), 6.28 (d, J = 4.0 Hz, 1H), 4.56 (t, J = 5.0 Hz, 1H), 4.28 - 4.20 (m, 6H), 4.01 (dd, J = 10.7, 3.0 Hz, 1H), 3.91 (d, J = 8.3 Hz, 2H), 3.84 (dd, J = 10.8, 3.0 Hz, 1H), 3.53 (s, 3H), 1.39 (td, J = 7.1, 3.3 Hz, 6H), 0.96 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H). 31 P NMR (162 MHz, CDCl3) δ 20.82.

[0184] Step 2: Synthesis of YK-VP-005-PM2

[0185] YK-VP-005-PM1 (0.14 g, 0.21 mmol) was dissolved in 1.8 mL of ultra-dry tetrahydrofuran, and triethylamine trihydrofluoric acid (0.17 g, 173 μL, 1.06 mmol) was added. The mixture was stirred at 40°C for 16 hours under nitrogen protection; analysis by TLC and LC / MS revealed that the raw material had disappeared and the target molecular weight was found, at which point the reaction was stopped. Saturated sodium bicarbonate aqueous solution was slowly added and quenched. After extraction with dichloromethane (10 mL × 3), the organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated 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. 1H NMR (400 MHz, 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 = 3.5 Hz, 1H), 4.52 (t, J = 5.3 Hz, 1H), 4.24 - 4.15 (m, 6H), 4.01 (dd, J = 10.8, 2.5 Hz, 1H), 3.92 - 3.82 (m, 3H), 3.56 (s, 3H), 2.43 (s, 1H), 1.33 (td, J = 7.1, 5.2 Hz, 6H).

[0186] Step 3: Synthesis of YK-VP-005

[0187] YK-VP-005-PM2 (0.69 g, 1.30 mmol) was rotary evaporated a total of three times with ultra-dry dichloromethane. Dried YK-VP-002-PM2 was dissolved in 14 mL of ultra-dry dichloromethane, followed by the sequential addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.69 g, 730 μL, 2.30 mmol) and DCI (0.23 g, 1.95 mmol). The mixture was stirred at room temperature for 2.5 hours under nitrogen protection; analysis by LC / MS and TLC revealed that the raw material had disappeared and the target molecular weight was detected, at which point the reaction was stopped. The mixture was quenched with an aqueous solution of saturated sodium bicarbonate and extracted with dichloromethane (20 mL × 3). The organic phase was dried with anhydrous sodium sulfate, filtered, rotary evaporated, and concentrated to obtain the crude product. 0.70 g of white solid was obtained by purification by medium-pressure column chromatography (SiO2, PE + 1% TEA / EA + 1% TEA = 100 / 0 - 0 / 100), and the yield was 73.1%. MS m / z [M - H] - = 734.4. 1H NMR (400 MHz, Acetonitrile-d3) δ 9.47 (s, 1H), 8.70 (d, J = 1.8 Hz, 1H), 8.64 (d, J = 10.5 Hz, 1H), 8.05 (d, J = 7.2 Hz, 2H), 7.68 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.7 Hz, 2H), 6.24 (t, J = 5.8 Hz, 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 = 6.0 Hz, 2H), 1.39 - 1.19 (m, 20H). 31 P NMR (162 MHz, Acetonitrile-d3) δ 150.86, 149.73, 20.83, 20.69.

[0188] 6. Synthesis of Phosphoramidite 3

[0189]

[0190] 438 mg of product was obtained according to the synthesis method of Phosphoramidite 3 of WO2018045317A1, and the total yield is 1.9%.

[0191] Example 2: Preparation of siRNA

[0192] In this example, two siRNA parent sequences were synthesized and named D84-DV27-G5 and D82-DV29-G5, respectively, and phosphate ester modifications were performed at the end positions of D84-DV27-G5 and D82-DV29-G5, respectively, and the obtained siRNA double-strand sequences are as shown in Table 1.

[0193] Table 1: siRNA double-stranded sequences

[0194]

[0195]

[0196] In this specification, the meaning of each abbreviation is as follows.

[0197] The A, U, G, and C distributions represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0198] m indicates that the nucleotide adjacent to the 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.

[0199] f indicates that the nucleotide adjacent to the left is a 2'-F modified nucleotide (2'-fluoronucleoside). For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0200] s indicates that two adjacent nucleotides and / or carriers are connected by a thiophosphate ester bond.

[0201] EVP indicates that the nucleotide adjacent to the left is a 5'-(E)-VP modified nucleotide, and refer to Table 2 for the structure of UmEVP.

[0202] 5'-O-EtMP indicates that the nucleoside structure of the adjacent nucleotide on the left is provided by YK-VP-001, and refer to Table 2 for the structure of Ums-5'-O-EtMP.

[0203] 5'-O-MeMP indicates that the nucleoside structure of the adjacent nucleotide on the left is provided by YK-VP-002, and refer to Table 2 for the structure of Ums-5'-O-MeMP.

[0204] 5'-O-MeEP indicates that the nucleoside structure of the adjacent nucleotide on the left is provided by YK-VP-003, and refer to Table 2 for the structure of Ums-5'-O-MeEP.

[0205] 4'-O-MeMP indicates that the nucleoside structure of the adjacent nucleotide on the left is provided by Phosphoramidite 3, and refer to Table 2 for the structure of Ums-4'-O-MeMP.

[0206] 5'-O-MeMP indicates that the nucleoside structure of the adjacent nucleotide on the left is provided by YK-VP-004, and refer to Table 2 for the structure of Am-5'-O-MeMP.

[0207] 5'-O-EtMP indicates that the nucleoside structure of the adjacent nucleotide on the left is provided by YK-VP-005, and refer to Table 2 for the structure of Am-5'-O-EtMP.

[0208] 5'-O-MP indicates that the nucleoside structure of the adjacent nucleotide on the left is provided by YK-VP-004, and refer to Table 2 for the structure of Am-5'-O-MP.

[0209] G5 represents a GalNAc transport carrier with the following structure, where indicates that it is connected to the 3' end of the siRNA sensor strand through a phosphate ester group or a thiophosphate ester group, and CN116854754A may be referenced.

[0210] .

[0211] 1. Preparation of siRNA antisense strands without GalNAc conjugation

[0212] siRNA antisense strands were synthesized on a corresponding solid-phase carrier according to the phosphoramidite chemistry method.

[0213] When synthesizing sequence 2 (D84-DV27-PG5), an externally purchased (E)-VP-Um monomer is used as the final conjugation monomer of the antisense strand, and when synthesizing sequences 3-5 (D84-DV27-5EMPG5, D84-DV27-5MPG5 and D84-DV27-5MEPG5), the 5'-phosphate ester-modified nucleoside phosphoramidite monomers (YK-VP-001, YK-VP-002, and YK-VP-4003) synthesized in Example 1 are each used as the final conjugation monomer of the antisense strand; when synthesizing sequence 6 (D84-DV27-4MMPG5), the Phosphoramidite 3 monomer of Example 1 is used as the final conjugation monomer of the antisense strand.

[0214] 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 are used as the final conjugation monomers of the antisense strand, and the synthesis scale is about 200 nmol.

[0215] The terminal structure of the antisense strand of the synthesized siRNA is as shown in Table 2.

[0216] Table 2: Terminal structure of the antisense strand

[0217]

[0218]

[0219]

[0220] (1) Preparation of reagents and monomers

[0221] Select the acetonitrile solution of the monomer (0.15 M), the acetonitrile solution of 5-ethylthiotetrazole used as an activator, the pyridine solution of 3% hydroxyflavin used as a sulfating agent, the water / pyridine solution of iodine (10 / 90, v / v) used as an oxidizing reagent, the acetonitrile solution of 10% acetic anhydride used as capping agent A, the 1-methylimidazole / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v) used as capping agent B, the toluene solution of 3% dichloroacetic acid used as a de-DMTr reagent (v / v), and a standard carrier of cross-linked polystyrene beads used as a solid-phase carrier (Primer support 5G Unylinker 350, manufactured by Cytiva) and place them at the designated reagent locations of the 192 P model DNA / RNA automated synthesizer. Each contained one.

[0222] (2) Synthesis of crude products

[0223] The specified oligonucleotide sequence was entered, the synthesis procedure was set up, and after verifying for errors, the synthesis cycle of the oligonucleotide was started. It was manufactured according to the following steps.

[0224] a. Deprotection

[0225] The DMTr protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotection reagent and washed with acetonitrile.

[0226] b. Joining

[0227] Using 0.25 M 5-ethylthiotetrazole as an activator, the acetonitrile solutions of each nucleotide monomer were conjugated and then rinsed with acetonitrile.

[0228] c. Oxidation / Sulfurization

[0229] Oxidation: Oxidized using a 0.05 M iodine water / pyridine (90 / 10) solution as an oxidizing agent, then rinsed with acetonitrile.

[0230] Sulfation: Sulfation was performed using a 3% hydroxyflavin pyridine solution as a sulfating agent, followed by rinsing with acetonitrile.

[0231] d. Hydroxyl protection

[0232] After protecting the hydroxyl using capping agents A and B as a hydroxyl protection reagent, the hydroxyl was rinsed with acetonitrile.

[0233] The abnormal operation was repeated, and the process was performed repeatedly according to the set sequence to obtain a completely protected product.

[0234] (3) Deprotection

[0235] a. Preparation of monomethyl ester-protected 5'-phosphate ester-modified siRNA antisense strands

[0236] A solid-phase carrier was transferred to a reactor, concentrated ammonia water (25-28%) was added, and an ammonia decomposition reaction was carried out at a temperature of 60°C for 12 hours, after which it was cooled to room temperature and the mixture was transferred to a filter press tank, washed with a mixed solution of purified water and ethanol, the filtrate was combined, and the filtrate was concentrated at a low temperature to obtain a crude product of a 5'-phosphate ester modified siRNA antisense strand protected by a monomethyl ester.

[0237] b. Preparation of completely deprotected 5'-phosphate ester-modified siRNA antisense strands

[0238] The solid-phase support was transferred to a reactor, and a TMSI / Py / DCM solution was added and reacted at room temperature for 1 hour. Then, a 2-mercaptoethanol TEA / ACN solution was added to quench the reaction. Subsequent treatment was performed according to the method of deprotection a to obtain a crude product of a completely deprotected 5'-phosphate ester-modified siRNA antisense strand.

[0239] (4) Refining

[0240] The deprotected crude product residue was dissolved in purified water and purified by HPLC. The product peak solution was collected and its content measured using a microplate reader, and its purity and molecular weight were confirmed by LC / MS. The product peak solution was concentrated and freeze-dried to obtain the product.

[0241] 2. Preparation of GalNAc-conjugated siRNA sensor strands

[0242] SiRNA sensor strands were synthesized according to the synthesis method of siRNA antisense strands. Here, a G5-GalNAc carrier was used as the solid-phase carrier, and the synthesis scale of each sensor strand that complements the antisense strand was approximately 200 nmol.

[0243] 3. Preparation of double-stranded siRNA

[0244] The siRNA sensor strand and the complementary antisense strand were mixed in a 1:1 ratio according to UV absorption, heated to 95°C for 3 minutes, and then slowly cooled to room temperature to form a double strand. The purity of the resulting double strand solution was characterized by HPLC, and after passing the purity test, the content was measured using a microplate reader. The solution was then freeze-dried to obtain and store a solid powder. The molecular weight and purity of the obtained siRNA double strand are shown in Table 3.

[0245] Table 3: siRNA double-stranded molecular weight and purity

[0246]

[0247] Example 3: Effect of Modified Oligonucleotide Sequence on PCSK9 Inhibitory Effect and LDL-C Levels in Mouse Serum

[0248] In this example, the effects of the siRNA sequences in Table 1 on the inhibition rate of PCSK9 and LDL-C levels in mouse serum were observed.

[0249] Experimental materials

[0250] Test drug:

[0251] Modified siRNA sequences listed in Table 1

[0252] Preparation of test drugs:

[0253] Drug solvent: PBS buffer

[0254] Preparation conditions: Sterile environment

[0255] Identification Method: Identify prepared drug formulations by labeling, and clearly indicate the subject number, name, concentration, quantity, date of manufacture, manufacturer, and storage conditions on the outer packaging.

[0256] Storage conditions: Prepare immediately before use, and store remaining samples at -20℃.

[0257] Experimental Animal Information:

[0258] Species / Strain: B6-hPCSK9-UTR (T053388) mouse

[0259] Grade: SPF

[0260] Gender: Male

[0261] Quantity: 59

[0262] Age: 6-8 weeks

[0263] Source: Jiangsu GemPharmatech LLC

[0264] Rearing and Management:

[0265] Rearing Conditions: After acquiring the experimental animals, they were reared at Jiangsu Gem Pharmatech Co., Ltd., and feed and water were provided for free consumption. The feed used was SPF grade radiation-sterilized transgenic laboratory rat feed purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. Each batch of feed was accompanied by a quality certificate from the manufacturer, and an annual third-party inspection report was provided; the inspection standards refer to the national standards GB 14924.3-2010 "Nutritional Components of Compound Feed for Experimental Animals" and GB 14924.2-2001 "General Hygiene Standards for Compound Feed for Experimental Animals." The appearance and bacterial indicators of drinking water were inspected monthly within the company, and an annual inspection report issued by the local waterworks company was provided, referencing the national standard GB 5749-2006 "Hygiene Standards for Domestic Drinking Water."

[0266] 1. Inhibition rates of various modified siRNA sequences on PCSK9 protein expression in mouse serum

[0267] Experimental Method:

[0268] Day 0 (D0) was designated as the drug administration day, and approximately 200 μL of blood was collected from the inner corner of the eye on Day 3 (D-3). The collected blood was stored in an ice box until centrifugation, and centrifuged for 10 minutes at approximately 4°C and a centrifugal force of approximately 3000 g. PCSK9 levels were detected using a PCSK9 kit (Proteintech product), and accordingly, mice were divided into groups of 6 to ensure uniformity of PCSK9 levels within each group. A dose of 2 mg / kg was administered subcutaneously on Day 0 (D0). Blood was collected from the inner corner of the eye on Day 7 (D7), Day 14 (D14), and Day 21 (D21), serum was isolated, and PCSK9 protein expression levels were detected.

[0269] Experimental Results:

[0270] The inhibition rates of PCSK9 protein in serum on days 7, 14, and 21 of administration are shown in Table 4.

[0271] Table 4: PCSK9 protein expression inhibition rate in mouse serum

[0272]

[0273] 1) The 5'-phosphate ester modified siRNA sequence of the present invention can effectively inhibit PCSK9 protein expression in mouse serum.

[0274] As can be seen from Table 4 and Figure 1, the 5'-phosphate ester-modified siRNA sequences of the present invention exhibited 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 D84-DV27-5MMPG5 modified by YK-VP-002 were 16.5%, 18.1%, and 19.7% higher than those of D84-DV27-G5 on days 7, 14, and 21, respectively.

[0275] 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 D82-DV29-5EMPG5 modified by YK-VP-005 were 8.6%, 13.6%, and 13.3% higher than those of D82-DV29-G5 on days 7, 14, and 21, respectively.

[0276] 2) The siRNA sequence modified by the 5'-phosphate ester of the present invention significantly improves the inhibition rate of PCSK9 protein expression in mouse serum compared to the siRNA sequence modified by the compound of the prior art.

[0277] The siRNA sequences modified by existing compounds included D84-DV27-PG5 modified by (E)-VP-Um and D84-DV27-4MMPG5 modified by Phosphoramidite 3, and the inhibition rates on days 7, 14, and 21 were all lower than those of the 5'-phosphate ester modified siRNA sequence of the present invention. For example, compared to D84-DV27-PG5, the inhibition rates of D84-DV27-5MMPG5 increased by 11.7%, 7.3%, and 9.8% on days 7, 14, and 21, respectively. Compared to D84-DV27-4MMPG5, the inhibition rate of D84-DV27-5MMPG5 increased by 11.8%, 16.5%, and 15.3% on day 7, day 14, and day 21, respectively.

[0278] As can be seen from the structure, compared to YK-VP-002, Phosphoramidite 3 has one less CH2 group at the 5'-position of the ribosomal ring, and the rest of the structure is completely identical; however, the modified siRNA showed a significant difference in the inhibition rate of PCSK9 protein expression in mouse serum. Therefore, the 5'-phosphate ester modified siRNA sequence of the present invention achieved an unexpected effect.

[0279] 2. Effects of Various Modified siRNA Sequences on LDL-C Levels in Mouse Serum

[0280] Experimental Procedure:

[0281] Day 0 (D0) was designated as the drug administration day, and approximately 200 μL of blood was collected from the inner corner of the eye on Day 3 (D-3). The collected blood was stored in an ice box until centrifugation, and centrifuged for 10 minutes at approximately 4°C and a centrifugal force of approximately 3000 g. LDL-C levels were detected using a biochemical analyzer, and mice were divided into groups of 6 to ensure uniformity of LDL-C levels within each group. A dose of 2 mg / kg was administered subcutaneously on Day 0 (D0). Blood was collected from the inner corner of the eye on Day 7 (D7), Day 14 (D14), and Day 21 (D21), serum was separated, and LDL-C levels in the serum were detected.

[0282] Experimental Results:

[0283] The serum LDL-C reduction levels on days 7, 14, and 21 of administration are as shown in Table 5.

[0284] Table 5: Reduced LDL-C levels in mouse serum

[0285]

[0286] 1) The 5'-phosphate ester modified siRNA sequence of the present invention can effectively reduce LDL-C levels in mouse serum.

[0287] As can be seen from Table 5 and Figure 2, the 5'-phosphate ester modified siRNA sequence of the present invention significantly reduced LDL-C levels in mouse serum. For example, the reduction levels of D84-DV27-5MMPG5 modified by YK-VP-002 reached 35.8%, 40.5%, and 36.3% on days 7, 14, and 21, respectively, and increased by 15.0%, 14.2%, and 16.6%, respectively, compared to the parent sequence D84-DV27-G5.

[0288] For example, the reduction levels of D82-DV29-5EMPG5 modified by YK-VP-005 reached 30.8%, 33.6%, and 28.2% on days 7, 14, and 21, respectively, and were significantly higher than those of the parent sequence D82-DV29-G5.

[0289] 2) The siRNA sequence modified by the 5'-phosphate ester of the present invention significantly improves the reduction level of LDL-C in mouse serum compared to the siRNA sequence modified by the compound of the prior art.

[0290] The reduction levels of the siRNA sequences D84-DV27-PG5 and D84-DV27-4MMPG5 modified by compounds of the prior art ranged from 20% to 31% on days 7, 14, and 21, all of which were 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 reduction levels of D84-DV27-5MMPG5 increased by 9.9%, 8.5%, and 8.4% on days 7, 14, and 21, respectively. Compared to D84-DV27-4MMPG5, the reduction levels of D84-DV27-5MMPG5 increased by 11.2%, 11.6%, and 16.0% on days 7, 14, and 21, respectively.

[0291] As can be seen from the experimental results above, the 5'-phosphate ester modified siRNA of the present invention had a superior effect in reducing LDL-C levels in mouse serum compared to conventional phosphate ester modified siRNA, and an unexpected effect was obtained.

Claims

Claim 1 In the modified nucleoside compound, the modified nucleoside compound has a structure represented by formula (I), or is a pharmaceutically acceptable salt or stereoisomer of a compound having a structure represented by formula (I); In the above formula, R1 is H, R2 is -OCH3; R3 is a protecting group or a phosphorus-containing active reactive group, the phosphorus-containing active reactive group is any one of phosphoramamidite, H-phosphate ester, phosphate tryster, or a phosphorus-containing chiral auxiliary, and the phosphorus-containing chiral auxiliary is Selected from; R4 and R5 are independently H, C 1-6 A modified nucleoside compound characterized by being selected from alkyl, -CH2CH2CN, or -CH2O(CO)C(CH3)3; X is O or S; L is -CH2- or -CH2CH2-; A is O; B is a modified or unmodified base or a salt thereof; and D is O. Claim 2 A modified nucleoside compound in which R4 and R5 are not simultaneously isopropyl, or R4 and R5 are each independently selected from -CH2CH3, -CH3 or H. Claim 3 In paragraph 1, R4 is -CH3 or -CH2CH3; or, R5 is -CH3 or -CH2CH3; or, X is O; or, B is or Phosphorus, modified nucleoside compound. Claim 4 In paragraph 1, R3 is Phosphorus, modified nucleoside compound. Claim 5 In paragraph 3, R4 and R5 are identical, modified nucleoside compounds. Claim 6 In claim 1, the compound represented by the above formula (I) is a modified nucleoside compound having any one of the following structures: YK-VP-001, YK-VP-002, YK-VP-003, YK-VP-004, and YK-VP-005. Claim 7 An oligonucleotide characterized in that, in the structural unit of the oligonucleotide, a nucleotide containing a modified nucleoside compound according to claim 1 is included. Claim 8 In claim 7, the nucleotide containing the modified nucleoside compound is an oligonucleotide located at the 5' end of the oligonucleotide. Claim 9 In claim 7, the oligonucleotide is an oligonucleotide represented by formula (II) or a pharmaceutically acceptable salt thereof; In the above formula, Oligo is an oligonucleotide, R1 is H, and R2 is -OCH3; R4 and R5 are independently H and C, respectively. 1-6 Selected from alkyl, -CH2CH2CN or -CH2O(CO)C(CH3)3; X is O or S; L is -CH2- or -CH2CH2-; A is O; D is O; X1 is O or S, and B is or Phosphorus, oligonucleotide. Claim 10 In claim 7, the oligonucleotide is an oligonucleotide selected from any one of small interfering nucleotides, antisense oligonucleotides, microRNA, small activating RNA, small guide RNA, transfer RNA, and aptamers. Claim 11 In item 10, the above oligonucleotide is an oligonucleotide that is siRNA. Claim 12 In paragraph 11, the siRNA is an oligonucleotide that is double-stranded siRNA or single-stranded siRNA. Claim 13 In paragraph 12, the siRNA is a double-stranded siRNA and is an oligonucleotide comprising a sense strand and an antisense strand. Claim 14 In paragraph 13, the antisense strand of the double-stranded siRNA is an oligonucleotide represented by formula (II) or a pharmaceutically acceptable salt thereof. Claim 15 In claim 7, the oligonucleotide wherein each nucleotide of the oligonucleotide is independently a modified or unmodified nucleotide. Claim 16 A nucleic acid conjugate, wherein the nucleic acid conjugate comprises an oligonucleotide according to claim 7 or a pharmaceutically acceptable salt thereof, and a targeting ligand bound thereto. Claim 17 In paragraph 16, the targeting ligand is a nucleic acid conjugate comprising any one of a carbohydrate, a lipid, a polypeptide, or an antibody. Claim 18 In paragraph 17, the above lipid is a nucleic acid conjugate containing cholesterol. Claim 19 In paragraph 17, the targeting ligand is a nucleic acid conjugate comprising an N-acetylgalactosamine moiety. Claim 20 A nucleic acid conjugate according to paragraph 19, wherein 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. Claim 21 A pharmaceutical composition used to treat or prevent a pathological condition or disease caused by the expression of the proprotein-converting enzyme subtilisin / quexin 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 conjugate according to any one of claims 16 to 20, and preferably, said pharmaceutical composition further comprising a pharmaceutically acceptable auxiliary substance. Claim 22 A pharmaceutical composition used to treat or prevent a pathological condition or disease resulting from the expression of the proprotein-converting enzyme subtilisin / quexin 9 gene in hepatocytes, wherein the pathological condition or disease is selected from either dyslipidemia and cardiovascular disease or a combination of at least two. Claim 23 A pharmaceutical composition used to treat or prevent a pathological condition or disease caused by the expression of the proprotein-converting enzyme subtilisin / quexin 9 gene in hepatocytes, wherein, in paragraph 22, the dyslipidemia includes hypercholesterolemia; or the cardiovascular disease includes atherosclerosis. Claim 24 A kit characterized in that the kit comprises an oligonucleotide according to any one of claims 7 to 15 or a pharmaceutically acceptable salt thereof, or a nucleic acid conjugate according to any one of claims 16 to 20.

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