5 apos; phosphate modified nucleoside as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicines, and particularly provides 5 '-phosphate modified nucleoside as well as a preparation method and application thereof. The invention provides a modified nucleoside compound, which has a structure as shown in a formula (I), or is a pharmaceutically acceptable salt or stereoisomer of the compound with the structure as shown in the formula (I). The oligonucleotide containing the modified nucleoside compound provided by the invention has a remarkably enhanced drug curative effect. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to 5'-phosphate modified nucleosides and preparation methods and applications thereof. Background Art

[0002] Small nucleic acid drugs achieve target selection and directly target specific genes through base complementary pairing. They have high specificity, expand drug targets to upstream mRNA of pathogenic proteins, and regulate the expression of target genes at the post-transcriptional level. Compared with antibody drugs and small molecule drugs, small nucleic acid drugs have the advantages of rich targets, short R&D cycle, and long-lasting efficacy. There are many types of small nucleic acid drugs, mainly including antisense oligonucleotides (ASO), small interfering oligonucleotides (siRNA), micro oligonucleotides (miRNA) and nucleic acid aptamers, among which the research on three types of small nucleic acid drugs, ASO, siRNA, and aptamer, is the most popular.

[0003] siRNA drugs work through the RNA interference mechanism. The 5' end of the antisense strand of the siRNA that enters the cytoplasm through the endocytosis pathway is phosphorylated by the intracellular kinase Clp1, forming a phosphate group at the 5' end of the antisense strand. The phosphate group at the 5' end can bind to the Ago2 protein, thereby prompting the siRNA to enter the RNA-induced silencing complex (RISC). As the sense strand of the siRNA is cut and removed, the active RISC formed pairs with the target mRNA, and the target mRNA is cut by the nuclease in the RISC, thereby reducing the expression of the target mRNA. The released active RISC species will continue to search for the target mRNA for binding, enter the catalytic cycle, and continue to exert its pharmacological effect. Therefore, the 5' end of the antisense strand in the siRNA double strand must have a phosphate group in order to specifically bind to the side chain residues of the Ago2 domain, which is very important for the activity of RNA interference. However, studies have shown that after siRNA modified with a natural phosphate group at the 5' end enters the endocytic pathway, the lysosomal acid phosphatase will quickly degrade the terminal phosphate, which will limit their bioavailability in the body (Reka AH, et al. Nucleic Acids Res., 2017, 45, 7581), thereby affecting the efficacy.

[0004] In some cases, replacing the natural phosphate group with a phosphate analog at the 5' end of the siRNA antisense strand can improve the efficacy of siRNA in vivo. Since the modified phosphate is not a substrate for phosphatases in the body, the modified phosphate can resist exonuclease degradation and enhance the silencing effect of siRNA in vivo. However, the siRNA obtained by the existing modification method still has problems such as insufficient resistance to exonuclease degradation, no obvious enhancement of the silencing effect of siRNA, or adverse effects on the silencing effect of siRNA.

[0005] Therefore, it is expected to develop modified nucleoside compounds that, after being introduced into siRNA, can more effectively and stably improve the efficacy of siRNA drugs. Summary of the invention

[0006] In order to solve the above problems existing in the prior art, the present invention provides 5'-phosphate modified nucleosides and preparation methods and applications thereof. Specifically, the present invention designs modified nucleoside compounds, and the modified nucleoside compounds of the present invention can be efficiently introduced into the 5' end of the oligonucleotide chain through a solid phase synthesis method. Compared with the prior art, the oligonucleotide containing the modified nucleoside compounds of the present invention has a stronger inhibition efficiency on the target gene.

[0007] The inventors of the present invention unexpectedly discovered that the nucleoside compound formed by introducing an alkyl group between the phosphorus atom of the phosphate and the 5'-hydroxyl group of the sugar ring can effectively improve the resistance of siRNA to nuclease degradation while enhancing the in vivo silencing effect of siRNA. By introducing nucleotides containing nucleoside compounds modified in this way into oligonucleotides, nucleic acid drugs with improved efficacy can be obtained.

[0008] Based on this, the first aspect of the present invention provides a modified nucleoside compound, wherein 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] in, R1 and R2 are each independently selected from H, halogen, C 1-6 Alkyl or C 1-6 Alkoxy; R3 is hydrogen, a protecting group or a phosphorus-containing active reactive group; R4 and R5 are each independently selected from hydrogen, C 1-6 Alkyl, -CH2CH2CN or -CH2O(CO)C(CH3)3; X is O or S; L is C 1-6 Alkyl, C 2-5 Alkenyl or C 2-5 Alkynyl; A is O or S; B is H, a modified or unmodified base or a salt thereof; D is O, S or -CH2-.

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

[0011] According to some preferred embodiments of the present invention, R1 is H, 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, R1 is H.

[0013] According to some preferred embodiments of the present invention, 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, R4 is -CH3 or -CH2CH3.

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

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

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

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

[0020] According to some preferred embodiments of the present invention, wherein B is or . " ” indicates the attachment position. “Bz” refers to benzoyl.

[0021] According to some preferred embodiments of the present invention, D is O.

[0022] According to some preferred embodiments of the present invention, the phosphorus-containing active reaction group is any one of phosphoramidite, H-phosphate, phosphotriester or phosphorus-containing chiral auxiliary.

[0023] According to some preferred embodiments of the present invention, wherein R3 is . " ” indicates the connection location.

[0024] According to some preferred embodiments of the present invention, R4 is the same as R5.

[0025] According to some particularly preferred embodiments of the present invention, the compound of formula (I) is any one of compounds YK-VP-001, YK-VP-002, YK-VP-003, YK-VP-004, YK-VP-005 having the following structures, or a combination of at least two thereof: , , , , "Bz" refers to benzoyl and "OMe" refers to methoxy.

[0026] Furthermore, the present invention also provides use of the modified nucleoside compound described in the first aspect in improving the stability of oligonucleotides (eg, resistance to exonuclease degradation, etc.).

[0027] Based on this, the present invention also provides a method for improving the stability of oligonucleotides (for example, improving resistance to nuclease degradation, etc.), which comprises replacing at least part of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compounds described in the first aspect of the present invention.

[0028] Preferably, 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are replaced with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0029] Furthermore, the present invention also provides use of the modified nucleoside compound described in the first aspect in improving the in vivo function of an oligonucleotide (eg, improving the silencing effect of siRNA, etc.).

[0030] Based on this, the present invention also provides a method for improving the in vivo function of an oligonucleotide (for example, improving the silencing effect of siRNA, etc.), which comprises replacing at least part of the structural units (nucleotides) in the oligonucleotide with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0031] Preferably, 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are replaced with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0032] Furthermore, the present invention also provides the use of the modified nucleoside compound described in the first aspect in simultaneously improving the stability of oligonucleotides (such as resistance to exonuclease degradation, etc.) and the in vivo function of oligonucleotides (such as improving the silencing effect of siRNA, etc.).

[0033] Based on this, the present invention also provides a method for simultaneously improving the stability of oligonucleotides (for example, improving resistance to nuclease degradation, etc.) and the in vivo function of oligonucleotides (for example, improving the silencing effect of siRNA, etc.), the method comprising replacing at least part of the structural units (nucleotides) in the oligonucleotide with nucleotides containing the modified nucleoside compounds described in the first aspect of the present invention.

[0034] Preferably, 1 to 3 (most preferably 1) nucleotides at the 5' end of the oligonucleotide are replaced with a nucleotide containing the modified nucleoside compound described in the first aspect of the present invention.

[0035] The second aspect of the present invention provides an oligonucleotide, wherein the structural unit of the oligonucleotide includes a nucleotide containing the modified nucleoside compound described in the first aspect. That is, the oligonucleotide provided by the present invention can be formed by replacing some structural units (nucleotides) in a conventional oligonucleotide 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 phosphoric acid.

[0036] In the oligonucleotide provided by the present invention, some of the structural units (nucleotides) may contain the modified nucleoside compound provided in the first aspect, or all of the structural units may contain the modified nucleoside compound. When some of the structural units contain the modified nucleoside compound, the other structural units in the oligonucleotide may be provided by conventional nucleotides, or by nucleotides containing other existing modified nucleoside compounds.

[0037] In the present invention, the structural unit of the oligonucleotide refers to a nucleotide. As is well known in the art, a nucleotide is a compound composed of a base (purine base or pyrimidine base), a pentose (ribose or deoxyribose) and phosphoric acid. The base and the pentose are condensed to form a nucleoside, and then further synthesized with phosphoric acid to form a nucleotide. "The structural unit of the oligonucleotide includes a nucleotide containing the modified nucleoside compound described in the first aspect" means that the nucleoside portion of at least one nucleotide in the oligonucleotide sequence is provided as the modified nucleoside compound provided by the present invention, that is, among the several nucleotides contained in the oligonucleotide, the nucleoside portion of at least one nucleotide is replaced by the modified nucleoside compound provided by the present invention.

[0038] In some preferred embodiments, for an oligonucleotide with a length not exceeding 30 nucleotides (such as 15-30 nucleotides), the number of structural units (nucleotides) containing the modified nucleoside compounds provided by the present invention does not exceed 3 (for example, it can be 1, 2 or 3).

[0039] 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.

[0040] 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.

[0041] According to some preferred embodiments of the present invention, the oligonucleotide is an oligonucleotide as shown in formula (II) or a pharmaceutically acceptable salt thereof,

[0042] in, Oligo is an oligonucleotide. R1 and R2 are each independently selected from H, halogen, C 1-6 Alkyl or C 1-6 Alkoxy; R4 and R5 are each independently selected from H, C 1-6 Alkyl, -CH2CH2CN or -CH2O(CO)C(CH3)3; X is O or S; L is C 1-6 Alkyl, C 2-5 Alkenyl or C 2-5 Alkynyl; A is O or S; D is O, S or -CH2; X1 is O or S, B is or It should be noted that the structure shown in the above formula (II) is an oligonucleotide whose last nucleotide at the 5' end is a nucleotide containing the modified nucleoside compound provided by the present invention, that is, the part connected to Oligo is the structure of the last nucleotide in the complete oligonucleotide, and Oligo represents the rest of the oligonucleotide starting from the second nucleotide at the 5' end.

[0043] The modified nucleoside compound provided by the present invention replaces part of the nucleoside structure in the oligonucleotide, which can make the oligonucleotide more stable, and has a promoting effect on the function of the oligonucleotide itself. Any oligonucleotide in the art can be modified by the modified nucleoside compound provided by the present invention (i.e., the modified nucleoside compound is used to replace the nucleoside structure part of part of the nucleotide in the oligonucleotide). According to a preferred embodiment of the present invention, the oligonucleotide is selected from any one of small interfering nucleotides (siRNA), antisense oligonucleotides (ASO), micro RNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA) and aptamers (Aptamer) or a combination of at least two.

[0044] 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 (also known as the number of bases, nt) contained in a single-stranded oligonucleotide, and refers to the number of nucleotide pairs (also known as the number of base pairs, bp) contained in a double-stranded oligonucleotide.

[0045] According to some preferred embodiments of the present invention, the oligonucleotide is siRNA.

[0046] Preferably, the siRNA is double-stranded siRNA or single-stranded siRNA.

[0047] 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.

[0048] Preferably, the antisense strand in the double-stranded siRNA is an oligonucleotide represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0049] According to a preferred embodiment of the present invention, each nucleotide in the oligonucleotide is independently a modified or unmodified nucleotide.

[0050] According to some preferred embodiments of the present invention, the oligonucleotide can be used to regulate the expression of the target gene (for example, can be used to inhibit the expression of the target gene).

[0051] 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 of them: (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.

[0052] The third aspect of the present invention provides a nucleic acid conjugate, characterized in that the nucleic acid conjugate comprises the oligonucleotide or a pharmaceutically acceptable salt thereof according to the second aspect, and a targeting ligand combined therewith.

[0053] In the present invention, any targeting ligand that can be used for nucleic acid conjugates in the art can be selected. According to some preferred embodiments of the present invention, the targeting ligand comprises any one of carbohydrates, cholesterol, lipids, polypeptides or antibodies, or a combination of at least two of them.

[0054] Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0055] The GalNAc moieties commonly 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 moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety.

[0056] According to some particularly preferred embodiments of the present invention, the targeting ligand is G5, and its structure is shown below: ; in Indicates that it is linked to the 3' end of the siRNA sense strand through a phosphate group or a phosphorothioate group.

[0057] The fourth aspect of the present invention provides a pharmaceutical composition, comprising the oligonucleotide or a pharmaceutically acceptable salt thereof according to the second aspect; And / or, the pharmaceutical composition comprises the nucleic acid conjugate described in the third aspect.

[0058] In the pharmaceutical composition provided by the present invention, the oligonucleotide and / or nucleic acid conjugate provided by the present invention is used as the main active ingredient. In some embodiments, the oligonucleotide and / or nucleic acid conjugate provided by the present invention can be used as the only active ingredient in the pharmaceutical composition; in some embodiments, the pharmaceutical composition may contain other active ingredients in addition to the oligonucleotide and / or nucleic acid conjugate of the present invention.

[0059] According to some preferred embodiments of the present invention, wherein, the pharmaceutical composition also comprises a pharmaceutically acceptable adjuvant. "Pharmaceutically acceptable adjuvant" refers to any material of an inactive ingredient that can be added during the process. For example, the pharmaceutical composition provided by the present invention may include pharmaceutically acceptable carriers, adjuvants, adjuvants, etc., and may also include other active ingredients or active ingredient adjuvants. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which participates in carrying or transporting the nucleoside analogs, oligonucleotides, conjugates or their compositions of the present invention in or to the patient's body, so that it can perform its intended function. Typically, such a construct is carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable", i.e. compatible with other ingredients of the preparation, including nucleoside analogs, oligonucleotides, conjugates or their compositions of the present invention, and harmless or substantially harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic compatible substances used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents and absorption delaying agents, etc., which are compatible with the activity of the nucleoside analogs, oligonucleotides, conjugates or their compositions of the present invention and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the composition. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of compounds that can be used in the present invention. Other additional ingredients that can be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described in, for example, Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.Any carrier, adjuvant, and auxiliary material that can be used for pharmaceutical preparation in the art can be applied to the present invention. For example, auxiliary materials can include but are not limited to binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, chelating agents, colorants, flavoring agents, coating agents, buffers, protective agents, preservatives, solubilizers, pH adjusters, etc.

[0060] The fifth aspect of the present invention provides the use of the oligonucleotide or a pharmaceutically acceptable salt thereof according to the second aspect, or the nucleic acid conjugate according to the third aspect, or the pharmaceutical composition according to the fourth aspect in the preparation of a drug, wherein the drug is used to treat and / or prevent a disease caused by a proprotein convertase subtilisin 9 gene ( PCSK9 A pathological condition or disease caused by the expression of a gene.

[0061] PCSK9 The gene is located on chromosome 1. 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.

[0062] According to some preferred embodiments of the present invention, the pathological condition or disease is selected from any one of hypercholesterolemia, dyslipidemia, atherosclerosis and cardiovascular and cerebrovascular diseases, or a combination of at least two thereof.

[0063] Furthermore, the present invention also provides a method for treating PCSK9 A method for treating a pathological condition or disease caused by the expression of a gene, the method comprising administering the oligonucleotide or a pharmaceutically acceptable salt thereof according to the second aspect of the invention, or the nucleic acid conjugate according to the third aspect, or the pharmaceutical composition according to the fourth aspect to a subject in need thereof, or administering a drug prepared from the oligonucleotide or a pharmaceutically acceptable salt thereof according to the second aspect of the invention, or the nucleic acid conjugate according to the third aspect, or the pharmaceutical composition according to the fourth aspect.

[0064] According to some preferred embodiments of the present invention, the subject may be a mammal, such as a human, a non-human primate, a rodent (such as a rabbit, a rat, a mouse, a guinea pig, etc.), a horse, a sheep, a pig, a cat, a dog, etc.

[0065] The sixth aspect of the present invention provides a kit, which includes the oligonucleotide or a pharmaceutically acceptable salt thereof described in the second aspect, or the nucleic acid conjugate described in the third aspect, or the pharmaceutical composition described in the fourth aspect.

[0066] The kit provided by the present invention can be used for therapeutic purposes or non-therapeutic purposes, and can also be used for diagnostic purposes or non-diagnostic purposes.

[0067] The positive progress of the present invention is that the present invention designs a series of 5'-phosphate modified nucleosides, which have completely different chemical structures compared with the prior art 5' end phosphorylated compounds. Oligonucleotides containing the present invention's 5'-phosphate modified nucleosides can significantly improve the inhibitory activity of siRNA on target genes.

[0068] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The structure of the compounds is completely different from that of the existing 5'-end phosphate modification technology.

[0069] The 5'-phosphate modified nucleoside designed by the present invention introduces an alkyl group between the phosphorus atom of the phosphate and the 5'-hydroxyl group of the sugar ring, which is completely different from the prior art and is a brand-new 5'-phosphate modified nucleoside compound. The compound uses commercially available nucleosides as raw materials, uses the 5'-hydroxyl group of the nucleoside to react with the alkyl / alkenyl compound containing the phosphoric acid ester, and only requires a one-step nucleophilic substitution / addition reaction to quickly construct a series of 5'-phosphate modified nucleoside compounds.

[0070] 2. Compared with the prior art compounds, the siRNA sequence modified by the 5'-phosphate of the present invention has higher inhibitory activity.

[0071] For example, compared with 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 inhibition rate of PCSK9 protein expression in mouse serum by 11.7%, 7.3% and 9.8% on the 7th, 14th and 21st days, respectively. Compared with 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 inhibition rate of PCSK9 protein expression in mouse serum by 11.8%, 16.5% and 15.3% on the 7th, 14th and 21st days, respectively.

[0072] Compared with 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 the 7th day, 14th day and 21st day, respectively. Compared with D84-DV27-4MMPG5, D84-DV27-5MMPG5 increased the reduction level by 11.2%, 11.6% and 16.0% on the 7th day, 14th day and 21st day, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work. Figure 1 The inhibition rate 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 the 7th, 14th and 21st days after administration.

[0074] Figure 2 The levels of LDL-C 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 were reduced on days 7, 14 and 21 after administration. DETAILED DESCRIPTION

[0075] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

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

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

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

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

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

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

[0082] In the present invention, "protecting group" refers to a group used in the conventional chemical sense to reversibly render a functional group unreactive under certain conditions of the desired reaction. After the desired reaction, the protecting group can be removed to deprotect the protected functional group. All protecting groups should be removable under conditions that will not degrade a significant proportion of the molecule being synthesized.

[0083] In the present invention, "phosphorus-containing reactive groups" refer to phosphorus-containing groups that can react with hydroxyl or amine groups contained in another molecule, especially in another nucleotide unit or another nucleotide analog, through a nucleophilic reaction. Typically, such a reaction produces an ester-type internucleoside bond connecting a nucleotide unit or a nucleotide analog unit to another nucleotide unit or a nucleotide analog unit. These phosphorus-containing reactive groups are known in the art and contain phosphorus atoms in the P(III) or P(V) valence state, and the phosphorus-containing reactive groups include but are not limited to phosphoramidites, H-phosphates, phosphotriesters, and phosphorus-containing chiral auxiliaries, such as Among them, the phosphorus-containing chiral auxiliary refers to a phosphorus-containing group with chirality, for example, , , , , , , And other possible forms of groups.

[0084] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0085] The term "C 1-6 " refers to a group having a total of any integer value of carbon atoms in the main chain and the branched chain within the range of 1 to 6, such as 1, 2, 3, 4, 5, 6 carbon atoms. Similarly, the term "C 2-5 ” means that the main chain and the branched chains of the group have a total of any integer value of carbon atoms in the range of 2 to 5, such as 2, 3, 4, 5 carbon atoms.

[0086] The term "alkyl" refers to a group having a specified number of carbon atoms (e.g., C 1-6 ) of a straight or branched alkyl group. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, etc. The term "alkyl" also includes heteroalkyl groups, i.e., groups formed by replacing one or more (e.g., 1, 2, 3, or 4) carbon atoms in an alkyl group with heteroatoms (i.e., atoms other than carbon or hydrogen, such as oxygen, sulfur, fluorine, nitrogen, phosphorus, etc.).

[0087] The term "alkoxy" refers to the group RO-, wherein R is alkyl as defined above.

[0088] The term "alkenyl" refers to a hydrocarbon group having at least one carbon-carbon double bond at one or more positions along the carbon chain of an alkyl group, including but not limited to ethenyl, propenyl, butenyl, and the like.

[0089] The term "alkynyl" refers to a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the carbon chain of the alkyl group, including but not limited to ethynyl, propynyl, butynyl, and the like.

[0090] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can 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 addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When the compound contains a relatively basic functional group, an acid addition salt can 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 addition salts include, but are not limited to, hydrochlorides, sulfates, methanesulfonates, and the like.

[0091] The term "pharmaceutically acceptable excipient" in the present invention refers to all substances contained in the pharmaceutical preparation except the active ingredient.

[0092] The term "treat" refers to any of the following: (1) ameliorating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that leads to a disease; or (3) slowing the progression of one or more biological manifestations of a disease.

[0093] The term "prevention" refers to reducing the risk of developing a disease or reducing the severity of a disease if it occurs.

[0094] The oligonucleotides in the present invention include single-stranded oligonucleotides (such as antisense oligonucleotides, referred to as ASOs) and double-stranded oligonucleotides (such as small interfering nucleotides, referred to as siRNAs).

[0095] The oligonucleotides in the present invention include natural oligonucleotides and chemically modified oligonucleotides. Chemical modifications herein include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. Chemical modifications of oligonucleotides do not include situations where there are differences only in the nucleobase sequence. Natural herein refers to situations corresponding to naturally occurring RNA or DNA.

[0096] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0097] The reagents and raw materials used in the present invention are commercially available.

[0098] Example In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0099] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0100] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the raw materials used can be obtained commercially. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0101] Example 1: Synthesis of compounds 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.

[0102] 1. Synthesis of YK-VP-001 The synthetic route is as follows:

[0103] Step 1: Synthesis of YK-VP-001-PM1 Dissolve YK-VP-001-SM (365.00 g, 0.64 mol) in 1460 mL of ultra-dry dichloromethane and stir in an ice-water bath under nitrogen protection. Then add imidazole (130.70 g, 1.92 mol) and TBDMSCl (192.90 g, 1.28 mol) in batches and stir at room temperature under nitrogen protection for 5 hours. TLC detects the reaction. The raw material disappears and new spots with low polarity appear, and the reaction is stopped. Add water (730 mL) to quench, extract, and wash once with 730 mL of water. The organic phase is dried over anhydrous sodium sulfate, and the filtrate obtained after filtration is directly used in the next step.

[0104] Step 2: Synthesis of YK-VP-001-PM2 Methanol (1V, 365 mL) and p-toluenesulfonic acid monohydrate (12.20 g) were added to a solution of YK-VP-001-PM1 (0.64 mol) in dichloromethane (4 V, 1460 mL) in turn and stirred at room temperature. TLC was used to monitor the reaction and the starting material disappeared after 2 hours. 182 mL of TEA (0.5 V) was added to quench the reaction and stirred for 1 hour, followed by the addition of 730 mL of saturated sodium bicarbonate aqueous solution and continued stirring for 15 minutes. The liquids were then separated and the aqueous phase was extracted once with 365 mL of dichloromethane. The combined organic phases were 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 light yellow solid. The crude product was slurried in 1460 mL of n-heptane (4 V) and filtered to obtain 183.20 g of a white solid 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).

[0105] Step 3: Synthesis of YK-VP-001-PM3 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. TLC was used to detect the reaction. The raw material was basically reacted completely, and a new spot was generated below the raw material, and the reaction was stopped. The reaction solution was diluted with 500 mL of ethyl acetate, and then quenched by adding 500 mL of saturated ammonium chloride aqueous solution. Then the liquid was separated, the aqueous phase was extracted once with 500 mL of ethyl acetate, the combined organic phase was washed once with 500 mL of saturated sodium chloride aqueous solution, and then 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 / 3) gave 13.10 g of a light yellow oil with a yield of 46.4%. MS m / z [M - H] - = 521.2. 1 H 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.

[0106] Step 4: Synthesis of YK-VP-001-PM4 Dissolve YK-VP-001-PM3 (12.00 g, 23.00 mmol) in 240 mL of tetrahydrofuran and add triethylamine trihydrofluoride (18.51 g, 114.80 mmol). Stir at 40°C for 19 hours. After TLC and LC / MS analysis, the raw material disappears and the target molecular weight is found, and the reaction is stopped. The reaction solution is directly concentrated by rotary evaporation, and then acetonitrile is added and concentrated once to obtain a crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 20 / 1) gives 9.30 g of a light yellow oil 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 PNMR (162 MHz, CDCl3) δ 21.54.

[0107] Step 5: Synthesis of YK-VP-001 YK-VP-001-PM4 (3.00 g, 7.30 mmol) was co-rotated with ultra-dry dichloromethane for three times. The dried YK-VP-001-PM4 was dissolved in 30 mL of ultra-dry dichloromethane, cooled to 5 °C under nitrogen protection, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (3.32 g, 11.00 mmol) and tetrazole (0.62 g, 8.80 mmol) were added in sequence. Stirring was carried out at room temperature for 1 hour under nitrogen protection. After LC / MS and TLC analysis, the target molecular weight was found and the reaction was stopped. The reaction solution was washed twice with saturated sodium bicarbonate aqueous solution (15 mL × 2), the aqueous phase was extracted once with 30 mL of dichloromethane, the combined organic phase was washed once with 10% sodium chloride aqueous solution, and then dried over anhydrous sodium sulfate, filtered and concentrated by rotary evaporation to obtain the crude product. After rapid purification, 1.80 g of colorless oil was obtained 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, 3.7Hz, 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 PNMR (162 MHz, CDCl3) δ 151.41, 150.47, 21.49, 21.25.

[0108] 2. Synthesis of YK-VP-002 The synthetic route is as follows:

[0109] Step 1: Synthesis of YK-VP-002-PM1 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. TLC was used to detect the reaction. The raw material was basically reacted completely, and a new spot was generated below the raw material, and the reaction was stopped. The reaction solution was diluted with 500 mL of ethyl acetate, and then quenched by adding 500 mL of saturated ammonium chloride aqueous solution. Then the liquid was separated, the aqueous phase was extracted once with 500 mL of ethyl acetate, the combined organic phase was washed once with 500 mL of saturated sodium chloride aqueous solution, and then 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) gave 12.00 g of a light yellow oil 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) δ23.69.

[0110] Step 2: Synthesis of YK-VP-002-PM2 Dissolve YK-VP-002-PM1 (12.00 g, 24.20 mmol) in 240 mL of tetrahydrofuran and add triethylamine trihydrofluoride (19.56 g, 126.00 mmol). Stir at 40°C for 19 hours. After TLC and LC / MS analysis, the starting material disappears and the target molecular weight is found, and the reaction is stopped. The reaction solution is directly concentrated by rotary evaporation, and then acetonitrile is added and concentrated once to obtain a crude product. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 50 / 1) gives 4.80 g of a light yellow solid with a yield of 52.0%. MS m / z [M + NH4] + =398.0. 1 H NMR (400 MHz, DMSO- d 6) 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- d 6) δ 24.49.

[0111] Step 3: Synthesis of YK-VP-002 YK-VP-002-PM2 (1.60 g, 4.20 mmol) was co-rotated with ultra-dry dichloromethane for three times. The dried YK-VP-002-PM2 was dissolved in 16 mL of ultra-dry dichloromethane, cooled to 5 °C under nitrogen protection, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.90 g, 6.30 mmol) and tetrazole (0.35 g, 5.04 mmol) were added in sequence. Stirring was carried out at room temperature for 1 hour under nitrogen protection. After LC / MS and TLC analysis, the target molecular weight was found and the reaction was stopped. The reaction solution was washed twice with saturated sodium bicarbonate aqueous solution (15 mL × 2), the aqueous phase was 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 rotary evaporation to obtain the crude product. After rapid purification, 1.60 g of colorless oil was obtained with a yield of 65.6%. MS m / z [MH] -= 579.4. 1 H NMR (400 MHz, DMSO- d 6) 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 = 8.1Hz, 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 (162 MHz, DMSO- d 6)δ 150.66, 149.83, 24.45, 24.34.

[0112] 3. Synthesis of YK-VP-003 The synthetic route is as follows:

[0113] Step 1: Synthesis of YK-VP-003-PM1 YK-VP-001-PM2 (6.50 g, 17.45 mmol) was dissolved in 105 mL of ultra-dry tetrahydrofuran, and under nitrogen protection, NaH (2.09 g, 52.35 mmol) was added in batches at 5°C and stirred for 1 hour, followed by stirring at room temperature for 1 hour. Vinyl dimethyl phosphate (23.75 g, 174.50 mmol) was added under ice-water bath conditions, and then reacted at room temperature overnight. TLC and LC / MS analysis showed that the raw material was basically reacted and the target molecular weight was found, and the reaction was stopped. 300 mL of ethyl acetate was added for dilution, and the mixture was washed twice with 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 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) gave 4.02 g of a light yellow oil with a yield of 45.32%. MS m / z [M + NH4] + = 526.2.1 H NMR (400 MHz, CDCl3) δ9.45 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 1.9 Hz, 1H), 5.71 (d, J = 8.1Hz, 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.

[0114] Step 2: Synthesis of YK-VP-003-PM2 Dissolve YK-VP-003-PM1 (4.00 g, 7.86 mmol) in 80 mL of tetrahydrofuran and add triethylamine trihydrofluoride (6.34 g, 39.30 mmol). Stir at 40°C for 19 hours. After TLC and LC / MS analysis, the raw material disappears and the target molecular weight is found, and the reaction is stopped. The reaction solution is directly concentrated by rotary evaporation, and then acetonitrile is added and concentrated once to obtain a crude product. Purify by medium pressure column chromatography (SiO2, DCM / MeOH = 30 / 1) to obtain 1.80 g of light 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 PNMR (162 MHz, CDCl3) δ 31.36.

[0115] Step 3: Synthesis of YK-VP-003 YK-VP-003-PM2 (1.80 g, 4.56 mmol) was co-rotated with ultra-dry dichloromethane for three times. The dried YK-VP-003-PM2 was dissolved in 18 mL of ultra-dry dichloromethane, cooled to 5 °C under nitrogen protection, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.06 g, 6.84 mmol) and tetrazole (0.38 g, 5.47 mmol) were added in sequence. Stirring was carried out at room temperature for 1 hour under nitrogen protection. After LC / MS and TLC analysis, the target molecular weight was found and the reaction was stopped. The reaction solution was washed twice with saturated sodium bicarbonate aqueous solution (15 mL × 2), the aqueous phase was 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 rotary evaporation to obtain the crude product. After rapid purification, 1.20 g of colorless oil was obtained with a yield of 44.3%. MS m / z [MH] - = 593.5. 1 H NMR (400 MHz, DMSO- d 6) 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, 1.1Hz, 2H), 2.20 – 2.10 (m, 2H), 1.17 – 1.13 (m, 12H). 31 P NMR (162 MHz, DMSO- d 6)δ 150.42, 149.68, 31.90, 31.69.

[0116] 4. Synthesis of YK-VP-004 The synthetic route is as follows: Step 1: Synthesis of YK-VP-004-PM1 YK-VP-004-SM (20.00 g, 29.08 mmol) was dissolved in 100 mL of ultra-dry N, N-dimethylformamide, followed by adding TBDMSCl (6.57 g, 43.62 mmol) and imidazole (4.95 g, 72.70 mmol) in batches, and stirred at room temperature for 21 hours under nitrogen protection. According to TLC analysis, the raw material disappeared and new spots with small polarity appeared, and the reaction was stopped. Water was added to quench, extracted with ethyl acetate (100 mL), washed with water four times (100 mL × 4), and the aqueous phase was back-extracted with ethyl acetate once (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain a crude product of 25.29 g of white solid, which was used directly in the next step. MSm / z [M + H] + = 802.4.

[0117] 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 (50 V) solution, followed by the addition of Et3SiH (1.60 mL, 10.00 mmol) and stirring at room temperature for 2 hours. According to TLC and LC / MS analysis, the starting material disappeared and the target molecular weight was found, and the reaction was stopped. Water (80 mL) was added for quenching, followed by extraction and washing with dichloromethane twice (80 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain an oily substance, which was purified by medium pressure column chromatography (SiO2, DCM / MeOH = 100 / 0 – 94 / 6) to obtain 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).

[0118] Step 3: Synthesis of YK-VP-004-PM3 YK-VP-004-PM2 (7.03 g, 14.10 mmol) was dissolved in 105 mL of ultra-dry N, N-dimethylformamide. NaH (1.69 g, 70.50 mmol) was added in batches under ice-water bath conditions. Stirring was carried out under nitrogen protection for 10 minutes, and then stirring at room temperature for 30 minutes. Dimethyl p-toluenesulfonyloxymethylphosphonate (12.43 g, 42.30 mmol) was added under ice-water bath conditions, and then reacted at room temperature for 17 hours. According to TLC and LC / MS analysis, the raw material disappeared and the target molecular weight was found, and the reaction was stopped. 100 mL of ethyl acetate was added for dilution, and the mixture was washed twice with 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 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 – 96 / 4) gave 4.01 g of a white solid with a yield of 45.7%. MS m / z [M + H] + = 622.2. 1 H NMR (400 MHz, DMSO- d 6) 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 = 1.7Hz, 3H), 3.42 (s, 3H), 0.89 (s, 9H), 0.08 (s, 6H).

[0119] Step 4: Synthesis of YK-VP-004-PM4 Dissolve YK-VP-004-PM3 (4.01 g, 6.45 mmol) in 80 mL of ultra-dry tetrahydrofuran, add triethylamine trihydrofluoride (5.25 mL, 32.20 mmol), and stir at 40 °C for 16 hours under nitrogen protection. LC / MS and TLC analysis showed that the raw material disappeared and the target molecular weight was found, and the reaction was stopped. The solvent was removed by rotary evaporation, and then the crude product was obtained by co-evaporation with acetonitrile. Purification by medium pressure column chromatography (SiO2, DCM / MeOH = 100 / 0 – 94 / 6) gave 1.97 g of white solid with a yield of 60.2%. MS m / z [M + H] + = 508.1. 1 H NMR (400 MHz, DMSO- d 6) 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). 31 P NMR (162 MHz, DMSO- d 6) δ 23.22.

[0120] Step 5: Synthesis of YK-VP-004 YK-VP-004-PM4 (1.02 g, 2.00 mmol) was co-rotated with ultra-dry dichloromethane for 3 times. The dried YK-VP-004-PM4 was dissolved in 20 mL of ultra-dry dichloromethane, followed by the addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.08 g, 1.14 mL, 3.60 mmol) and DCI (354 mg, 3.00 mmol). Stir at room temperature for 1.5 hours under nitrogen protection. LC / MS analysis showed that the starting material disappeared and the target molecular weight was found, and the reaction was stopped. Add saturated sodium bicarbonate aqueous solution to quench, extract with dichloromethane (30 mL × 3), dry the organic phase with anhydrous sodium sulfate, filter and concentrate by rotary evaporation to obtain an oily substance, which is 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 [M - H] - = 706.2. 1 H NMR (400 MHz, DMSO- d 6) 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- d 6) δ 147.79, 147.63, 23.02, 22.99.

[0121] 5. Synthesis of YK-VP-005 The synthetic route is as follows:

[0122] Step 1: Synthesis of YK-VP-005-PM1 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-water bath conditions, stirred for 1 hour under nitrogen conditions, and then continued to stir at room temperature for 1 hour. Diethyl p-toluenesulfonyloxymethylphosphonate (1.1 mL, 1.37 g, 4.25 mmol) was added and reacted at room temperature overnight. TLC and LC / MS analysis showed that the raw material was not further converted, and the reaction was stopped. 10 mL of dichloromethane was added to dilute, and the reaction was quenched with saturated aqueous ammonium chloride solution (10 mL). Extraction was performed, and the dichloromethane was washed twice (10 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain an oil. Purification by medium-pressure column chromatography (SiO2, DCM / MeOH = 100 / 0 – 95 / 5) gave 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.

[0123] 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 ultra-dry tetrahydrofuran, and triethylamine trihydrofluoride (0.17 g, 173 uL, 1.06 mmol) was added. Stir at 40 °C for 16 hours under nitrogen protection. After TLC and LC / MS analysis, the raw material disappeared and the target molecular weight was found, and the reaction was stopped. Slowly add saturated sodium bicarbonate aqueous solution to quench, extract with dichloromethane (10 mL × 3), dry the organic phase with anhydrous sodium sulfate, filter and concentrate by rotary evaporation to obtain the crude product. Purify by medium pressure column chromatography (SiO2, DCM / MeOH = 100 / 0 – 91 / 9) to obtain 0.09 g of white solid with a yield of 79.2%. MS m / z [M + H] + = 536.0. 1 H 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 = 3.5Hz, 1H), 4.52 (t, J = 5.3 Hz, 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 = 7.1,5.2 Hz, 6H).

[0124] Step 3: Synthesis of YK-VP-005 YK-VP-005-PM2 (0.69 g, 1.30 mmol) was co-rotated with ultra-dry dichloromethane for 3 times. The dried YK-VP-002-PM2 was dissolved in 14 mL of ultra-dry dichloromethane, followed by the addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.69 g, 730 uL, 2.30 mmol) and DCI (0.23 g, 1.95 mmol). Stirring was carried out at room temperature for 2.5 hours under nitrogen protection. After LC / MS and TLC analysis, the raw material disappeared and the target molecular weight was found, and the reaction was stopped. Saturated sodium bicarbonate aqueous solution was added to quench, and the mixture was extracted with dichloromethane (20 mL × 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, PE + 1%TEA / EA + 1%TEA = 100 / 0 – 0 / 100) gave 0.70 g of a white solid with a yield of 73.1%. MS m / z [M - H] - = 734.4. 1 H NMR (400 MHz, Acetonitrile- d 3) 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- d 3) δ 150.86, 149.73, 20.83, 20.69.

[0125] 6. Synthesis of Phosphoramidite 3

[0126] According to the synthesis method of Phosphoramidite 3 in WO2018045317A1, 438 mg of product was obtained with a total yield of 1.9%.

[0127] Example 2: Preparation of siRNA In this example, two siRNA mother sequences were synthesized, named D84-DV27-G5 and D82-DV29-G5, respectively. The last position of D84-DV27-G5 and D82-DV29-G5 were phosphate-modified, and the resulting siRNA double-stranded sequences are shown in Table 1.

[0128] Table 1: siRNA double-stranded sequences

[0129] In this article, the meanings of the abbreviations are as follows: The A, U, G and C distribution represents the natural adenine, uracil, guanine and cytosine ribonucleotides.

[0130] m indicates that the adjacent nucleotide on the left is a 2'-OMe modified nucleotide (2'-methoxy nucleoside). For example, Am, Um, Gm and Cm represent 2'-OMe modified A, U, G and C, respectively.

[0131] f means that the nucleotide adjacent to its left side is a 2'-F modified nucleotide (2'-fluoro nucleoside). For example, Af, Uf, Gf and Cf respectively represent 2'-F modified A, U, G and C.

[0132] s indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0133] EVP represents a nucleotide whose adjacent nucleotide on the left is a 5'-(E)-VP modified nucleotide. The structure of UmEVP is shown in Table 2.

[0134] 5'-O-EtMP represents that the nucleoside structure of the adjacent nucleotide on its left side is provided by YK-VP-001. The structure of Ums-5'-O-EtMP is shown in Table 2.

[0135] 5'-O-MeMP represents that the nucleoside structure of the adjacent nucleotide on its left side is provided by YK-VP-002. The structure of Ums-5'-O-MeMP is shown in Table 2.

[0136] 5'-O-MeEP represents that the nucleoside structure of the adjacent nucleotide on its left side is provided by YK-VP-003. The structure of Ums-5'-O-MeEP is shown in Table 2.

[0137] 4'-O-MeMP represents that the nucleoside structure of the adjacent nucleotide on its left side is provided by Phosphoramidite 3. The structure of Ums-4'-O-MeMP is shown in Table 2.

[0138] 5'-O-MeMP represents that the nucleoside structure of the adjacent nucleotide on its left side is provided by YK-VP-004. The structure of Am-5'-O-MeMP is shown in Table 2.

[0139] 5'-O-EtMP represents that the nucleoside structure of the adjacent nucleotide on its left side is provided by YK-VP-005. The structure of Am-5'-O-EtMP is shown in Table 2.

[0140] 5'-O-MP represents that the nucleoside structure of the nucleotide adjacent to its left side is provided by YK-VP-004. The structure of Am-5'-O-MP is shown in Table 2.

[0141] G5 represents a GalNAc delivery vector of the following structure, wherein It means that the 3' end of the siRNA sense strand is connected via a phosphate group or a thiophosphate group, as shown in CN116854754A: .

[0142] 1. Preparation of siRNA antisense strand without GalNAc conjugation The siRNA antisense strand was synthesized on the corresponding solid phase support according to the phosphoramidite chemistry method.

[0143] When synthesizing sequence 2 (D84-DV27-PG5), the purchased (E)-VP-Um monomer was used as the last coupling monomer of the antisense chain. When synthesizing sequences 3-5 (D84-DV27-5EMPG5, D84-DV27-5MMPG5 and D84-DV27-5MEPG5), the 5'-phosphate modified nucleoside phosphoramidite monomers (YK-VP-001, YK-VP-002 and YK-VP-003) synthesized in Example 1 were used as the last coupling monomer of the antisense chain respectively; when synthesizing sequence 6 (D84-DV27-4MMPG5), the Phosphoramidite 3 monomer in Example 1 was used as the last 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 modified nucleoside phosphoramidite monomers (YK-VP-004 and YK-VP-005) synthesized in Example 1 were used as the last coupling monomer of the antisense chain, respectively, and the synthesis scale was 200 nmol.

[0145] The terminal structures of the antisense strands of the synthesized siRNAs are shown in Table 2.

[0146] Table 2: Antisense strand terminal structure

[0147] (1) Preparation of reagents and monomers Acetonitrile solution (0.15 M) of the monomer, 0.25 M 5-ethylthiotetrazolyl solution in acetonitrile was used as the activating agent, 3% hydrogenated xanthan gum solution in pyridine was used as the sulfurizing agent, 0.05 M iodine solution in water / pyridine (10 / 90, v / v) was used as the oxidizing agent, 10% acetic anhydride solution in acetonitrile (v / v) was used as the capping agent A, 1-methylimidazole / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v) was used as the capping agent B, 3% dichloroacetic acid solution in toluene (v / v) was used as the DMTr removal reagent, and a universal support of cross-linked polystyrene beads (Primer support 5G Unylinker 350, Cytiva manufacturer) was selected as the solid phase support and were respectively loaded into the designated reagent positions in the 192 P model DNA / RNA automatic synthesizer.

[0148] (2) Crude product synthesis Enter the designated oligonucleotide sequence, set the synthesis program, and after checking that everything is correct, start the synthesis of cyclic oligonucleotides. Prepare according to the following steps: a. Deprotection 3% dichloroacetic acid toluene solution was used as a deprotection reagent to remove the DMTr protecting group, followed by washing with acetonitrile.

[0149] b. Coupling Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolyl as an activating agent, followed by rinsing with acetonitrile.

[0150] c. Oxidation / sulfurization Oxidation: Oxidation was performed using 0.05 M iodine in water / pyridine (90 / 10) as the oxidant, followed by rinsing with acetonitrile.

[0151] Sulfurization: Use 3% hydrogenated xanthan gum in pyridine as the sulfurizing agent for sulfurization, and then use acetonitrile for washing.

[0152] d. Hydroxyl protection Capping agent A and capping agent B were used as hydroxyl protecting agents to protect the hydroxyl group, and then acetonitrile was used for washing.

[0153] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[0154] (3) Deprotection a. Preparation of monomethyl ester protected 5'-phosphate modified siRNA antisense strand The solid phase carrier was transferred to a reactor, concentrated ammonia water (25-28%) was added, and after aminolysis was maintained at 60°C for 12 h, the system was cooled to room temperature and the mixture was transferred to a filter press, eluted with a mixed solution of purified water and ethanol, the filtrates were combined, and the filtrates were concentrated at low temperature to obtain a crude product of the monomethyl ester-protected 5'-phosphate-modified siRNA antisense chain.

[0155] b. Preparation of fully deprotected 5'-phosphate-modified siRNA antisense strand The solid phase carrier was transferred to a reactor, and TMSI / Py / DCM solution was added. After reacting at room temperature for 1 hour, a 2-mercaptoethanol TEA / ACN solution was added to quench the reaction. Subsequent treatment was then performed according to the deprotection method a to obtain a completely deprotected 5'-phosphate-modified siRNA antisense strand crude product.

[0156] (4) Purification The crude residue after deprotection was dissolved in purified water, purified by HPLC, the product peak solution was collected and the content was measured by an ELISA instrument, and the purity and molecular weight were confirmed by LC / MS. The product peak solution was concentrated and freeze-dried to obtain the product.

[0157] 2. Preparation of GalNAc-conjugated siRNA sense strand The siRNA sense strand was synthesized according to the method for synthesizing the siRNA antisense strand, wherein the G5-GalNAc carrier was used as a solid phase carrier, and the synthesis scale of each sense strand complementary to the antisense strand was 200 nmol.

[0158] 3. Preparation of double-stranded siRNA The siRNA sense strand and the complementary antisense strand were mixed in a ratio of 1:1 according to the UV absorption content, heated to 95°C and maintained for 3 minutes, and then slowly cooled to room temperature to form a double strand. The obtained double-stranded solution was characterized by HPLC and the content was determined by an ELISA instrument after the product purity was qualified, and lyophilized to obtain a solid powder for storage. The molecular weight and purity of the obtained siRNA double strand are shown in Table 3.

[0159] Table 3: siRNA double-strand molecular weight and purity

[0160] Example 3: Effect of modified oligonucleotide sequences on the inhibition of PCSK9 and LDL-C levels in mouse serum In this example, the siRNA sequences in Table 1 were used to investigate the inhibition rate of PCSK9 in mouse serum and the effects on LDL-C levels.

[0161] Experimental Materials Tested drugs: Modified siRNA sequences listed in Table 1 Preparation of test drug: Drug solvent: PBS buffer Preparation conditions: Sterile environment Labeling method: The prepared drug preparations are labeled with labels, and the outer packaging is marked with the topic number, name, concentration, quantity, preparation date, preparer, and storage conditions; Storage conditions: Prepare immediately before use and store remaining samples at -20℃.

[0162] Experimental Animal Information: Species / Strain: B6-hPCSK9-UTR (T053388) mice Rating: SPF Gender: Male Quantity: 59 Age: 6-8 weeks Source: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Feeding and management: Feeding conditions: The experimental animals were received and raised in Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and the animals were free to eat and drink. Standard SPF-grade irradiated sterilized experimental transgenic mouse feed was purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. The production unit provides a feed quality certificate for each batch of feed, and a third-party test report is provided every year; the test standards refer to the national standards GB 14924.3-2010 "Nutritional Composition of Laboratory Animal Feed" and GB14924.2-2001 "General Hygiene Standards for Laboratory Animal Feed". The appearance and bacterial indicators of drinking water are tested once a month, tested within the company, and the test report of the tap water company in the district is provided every year, referring to the national standard GB5749-2006 "Sanitary Standards for Drinking Water".

[0163] 1. Inhibition rate of PCSK9 protein expression in mouse serum by different modified siRNA sequences Experimental methods: The day of administration was set as D0, and about 200 μL of blood was collected from the inner canthus of the eye on D-3. The whole blood samples were temporarily stored in an ice box before centrifugation, and centrifuged at about 4 °C and a centrifugal force of about 3000 g for 10 min. The PCSK9 level was detected using a PCSK9 kit (purchased from Proteintech), and the mice were divided into groups accordingly, with 6 mice in each group to ensure that the PCSK9 level of each group of mice was uniform. The drug was administered by subcutaneous injection on D0 (day 0), with a dose of 2 mg / kg. Blood was collected from the inner canthus of the eye on D7 (day 7), D14 (day 14), and D21 (day 21), and the serum was separated to detect the PCSK9 protein level.

[0164] Experimental results: The inhibition rates of PCSK9 protein in serum on the 7th, 14th and 21st days of administration are shown in Table 4.

[0165] Table 4: PCSK9 protein expression inhibition rate in mouse serum

[0166] 1) The 5'-phosphate-modified siRNA sequences of the present invention can effectively inhibit the expression of PCSK9 protein in mouse serum.

[0167] From Table 4 and Figure 1 It can be seen that the siRNA sequence modified by the 5'-phosphate of the present invention has a significant inhibitory effect on PCSK9 protein expression in mouse serum. For example, on the 7th day, the 14th day and the 21st day, the inhibition rates of D84-DV27-5EMPG5, D84-DV27-5MMPG5 and D84-DV27-5MEPG5 are all higher than that of D84-DV27-G5. Among them, the inhibition rates of D84-DV27-5MMPG5 modified by YK-VP-002 on the 7th day, the 14th day and the 21st day are 16.5%, 18.1% and 19.7% higher than those of D84-DV27-G5, respectively.

[0168] For example, the inhibition rates of D82-DV29-5MMPG5 and D82-DV29-5EMPG5 were higher than those of D82-DV29-G5 on days 7, 14, and 21. Among them, the inhibition rates of D82-DV29-5EMPG5 modified by YK-VP-005 on days 7, 13.6%, and 13.3% higher than those of D82-DV29-G5, respectively.

[0169] 2) The siRNA sequence modified with the 5'-phosphate of the present invention significantly improves the inhibition rate of PCSK9 protein expression in mouse serum compared with the siRNA sequence modified with the prior art compound.

[0170] The inhibition rates of siRNA sequences modified by existing compounds, including D84-DV27-PG5 modified by (E)-VP-Um and D84-DV27-4MMPG5 modified by Phosphoramidite 3, on the 7th day, 14th day and 21st day were lower than those of the 5'-phosphate modified siRNA sequences of the present invention. For example, compared with D84-DV27-PG5, the inhibition rates of D84-DV27-5MMPG5 on the 7th day, 14th day and 21st day increased by 11.7%, 7.3% and 9.8%, respectively. Compared with D84-DV27-4MMPG5, the inhibition rates of D84-DV27-5MMPG5 on the 7th day, 14th day and 21st day increased by 11.8%, 16.5% and 15.3%, respectively.

[0171] From the structural point of view, compared with YK-VP-002, Phosphoramidite 3 has one less CH2 at the 5'-position of the ribose ring, and the other structures are exactly the same, but the corresponding modified siRNA has a significant difference in the inhibition rate of PCSK9 protein expression in mouse serum. Therefore, the 5'-phosphate modified siRNA sequence of the present invention has achieved unexpected effects.

[0172] 2. Effects of different modified siRNA sequences on LDL-C levels in mouse serum Experimental process: The day of administration was set as D0 (Day 0), and about 200 μL of blood was collected from the inner canthus of the eye on D3 (Day 3). The whole blood sample was temporarily stored in an ice box before centrifugation, and centrifuged at about 4°C and a centrifugal force of about 3000 g for 10 min. The LDL-C level was detected by a biochemical analyzer, and the mice were divided into groups accordingly, with 6 mice in each group to ensure that the LDL-C level of each group of mice was uniform. The drug was administered by subcutaneous injection on D0 (Day 0), and the dosage was 2 mg / kg. Blood was collected from the inner canthus of the eye on D7 (Day 7), D14 (Day 14), and D21 (Day 21), and the serum was separated to detect the level of serum LDL-C.

[0173] Experimental results: The serum LDL-C reduction levels on the 7th, 14th and 21st days after administration are shown in Table 5.

[0174] Table 5: LDL-C reduction levels in mouse serum

[0175] 1) The 5'-phosphate-modified siRNA sequences of the present invention can effectively reduce the LDL-C level in mouse serum.

[0176] From Table 5 and Figure 2It can be seen that the siRNA sequence modified by the 5'-phosphate of the present invention can significantly reduce the LDL-C level in mouse serum. For example, the reduction level of D84-DV27-5MMPG5 modified by YK-VP-002 reached 35.8%, 40.5% and 36.3% on the 7th day, 14th day and 21st day, respectively, which was 15.0%, 14.2% and 16.6% higher than that of the parent sequence D84-DV27-G5, respectively.

[0177] For example, the reduction levels of D82-DV29-5EMPG5 modified by YK-VP-005 reached 30.8%, 33.6% and 28.2% on the 7th, 14th and 21st days, respectively, which were significantly higher than those of the parent sequence D82-DV29-G5.

[0178] 2) Compared with siRNA sequences modified with compounds in the prior art, the siRNA sequences modified with the 5'-phosphate of the present invention significantly improve the level of LDL-C reduction in mouse serum.

[0179] The reduction levels of siRNA sequences modified by prior art compounds, D84-DV27-PG5 and D84-DV27-4MMPG5, on the 7th day, 14th day and 21st day were between 20% and 31%, which were all lower than the 30% to 41% of the 5'-phosphate modified siRNA sequences of the present invention. For example, compared with D84-DV27-PG5, the reduction levels of D84-DV27-5EMPG5 on the 7th day, 14th day and 21st day increased by 9.9%, 8.5% and 8.4%, respectively. Compared with D84-DV27-4MMPG5, the reduction levels of D84-DV27-5MMPG5 on the 7th day, 14th day and 21st day increased by 11.2%, 11.6% and 16.0%, respectively.

[0180] From the above experimental results, it can be seen that the siRNA modified by 5'-phosphate of the present invention has a better effect of reducing the LDL-C level in mouse serum than the siRNA modified by existing phosphate, and has achieved an unexpected effect.

Claims

1. A modified nucleoside compound, characterized in that: 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): in, R1 and R2 are each independently selected from H, halogen, C 1-6 Alkyl or C 1-6 Alkoxy; R3 is hydrogen, a protecting group or a phosphorus-containing active reactive group; R4 and R5 are each independently selected from H, C 1-6 Alkyl, -CH2CH2CN or -CH2O(CO)C(CH3)3; X is O or S; L is C 1-6 Alkyl, C 2-5 Alkenyl or C 2-5 Alkynyl; A is O or S; B is H, a modified or unmodified base or a salt thereof; D is O, S or -CH2-.

2. The modified nucleoside compound according to claim 1, wherein R4 and R5 are not isopropyl at the same time.

3. The modified nucleoside compound according to claim 1, wherein R1 is H, R2 is -OCH3; R4 and R5 are each independently selected from -CH2CH3, -CH3 or H; L is CH2.

4. The modified nucleoside compound according to claim 1, wherein R1 is H; and / or, R2 is -OCH3; and / or, R3 is a phosphorus-containing active reactive group; and / or, R4 is -CH3 or -CH2CH3; and / or, R5 is -CH3 or -CH2CH3; and / or, X is O; and / or, L is -CH2- or -CH2CH2-; and / or, A is O; and / or, B is or ; And / or, D is O.

5. The modified nucleoside compound according to claim 4, wherein The phosphorus-containing active reactive group is any one of phosphoramidite, H-phosphate, phosphotriester or phosphorus-containing chiral auxiliary.

6. The modified nucleoside compound according to claim 4, wherein R3 is .

7. The modified nucleoside compound according to claim 4, wherein R4 is the same as R5.

8. The modified nucleoside compound according to claim 1, wherein The compound of formula (I) is any one of compounds YK-VP-001, YK-VP-002, YK-VP-003, YK-VP-004, YK-VP-005 or a combination of at least two of the following structures: ， ， ， ， 。 9. An oligonucleotide, characterized in that The structural unit of the oligonucleotide includes a nucleotide containing the modified nucleoside compound according to any one of claims 1 to 8.

10. The oligonucleotide according to claim 9, wherein The nucleotide containing the modified nucleoside compound is located at the 5' end of the oligonucleotide.

11. The oligonucleotide according to claim 9, wherein The oligonucleotide is an oligonucleotide as shown in formula (II) or a pharmaceutically acceptable salt thereof, in, Oligo is an oligonucleotide. R1 and R2 are each independently selected from H, halogen, C 1-6 Alkyl or C 1-6 Alkoxy; R4 and R5 are each independently selected from H, C 1-6 Alkyl, -CH2CH2CN or -CH2O(CO)C(CH3)3; X is O or S; L is C 1-6 Alkyl, C 2-5 Alkenyl or C 2-5 Alkynyl; A is O or S; D is O, S or CH2; X1 is O or S, B is or .

12. The oligonucleotide according to claim 9, wherein The oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, small guide RNAs, transfer RNAs and aptamers.

13. The oligonucleotide according to claim 12, wherein The oligonucleotide is siRNA.

14. The oligonucleotide according to claim 13, wherein The siRNA is a double-stranded siRNA or a single-stranded siRNA.

15. The oligonucleotide according to claim 14, wherein The siRNA is a double-stranded siRNA, which comprises a sense strand and an antisense strand.

16. The oligonucleotide according to claim 15, wherein The antisense strand in the double-stranded siRNA is an oligonucleotide represented by formula (II) or a pharmaceutically acceptable salt thereof.

17. The oligonucleotide according to any one of claims 9 to 16, wherein Each nucleotide in the oligonucleotide is independently a modified or unmodified nucleotide.

18. A nucleic acid conjugate, characterized in that The nucleic acid conjugate comprises the oligonucleotide according to any one of claims 9 to 17 or a pharmaceutically acceptable salt thereof, and a targeting ligand bound thereto.

19. The nucleic acid conjugate according to claim 18, wherein The targeting ligand comprises any one of carbohydrates, cholesterol, lipids, polypeptides or antibodies, or a combination of at least two of them.

20. The nucleic acid conjugate according to claim 19, wherein The targeting ligand comprises an N-acetylgalactosamine moiety.

21. The nucleic acid conjugate according to claim 20, wherein The N-acetylgalactosamine moiety is a monovalent N-acetylgalactosamine moiety, a divalent N-acetylgalactosamine moiety, a trivalent N-acetylgalactosamine moiety, or a tetravalent N-acetylgalactosamine moiety.

22. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 9 to 17; And / or, the pharmaceutical composition comprises the nucleic acid conjugate according to any one of claims 18-21.

23. The pharmaceutical composition according to claim 22, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

24. Use of the oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 9 to 17, or the nucleic acid conjugate according to any one of claims 18 to 21, or the pharmaceutical composition according to claim 22 or 23 in the preparation of a medicament for treating and / or preventing a pathological condition or disease caused by the expression of the proprotein convertase subtilisin 9 gene in hepatocytes.

25. The use according to claim 24, wherein The pathological condition or disease is selected from any one of hypercholesterolemia, dyslipidemia, atherosclerosis and cardiovascular and cerebrovascular diseases, or a combination of at least two thereof.

26. A kit, characterized in that The kit comprises the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 9 to 17, or the nucleic acid conjugate according to any one of claims 18 to 21, or the pharmaceutical composition according to claim 22 or 23.

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