2' ribose modified nucleoside monomer and its synthesis method and application

Abstract

The present invention discloses a 2' ribose-modified nucleoside monomer and its synthesis method and application. The present invention provides a compound as shown in formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof. The double-stranded oligonucleotide molecule prepared from the compound of the present application can reduce the melting temperature (Tm) value of the double-stranded oligonucleotide molecule, reduce the off-target effect of the dsRNA sequence or maintain good pharmacodynamic activity by using a modified nucleoside monomer at a specific position of the dsRNA sequence.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid drugs, and in particular to a series of double-stranded oligonucleotide molecules with specific modified groups capable of reducing off-target effects and uses thereof. Background Art

[0002] Small nucleic acid drugs refer to oligonucleotide sequences with a length of less than 30 nt, including small interfering nucleic acids (siRNA), messenger RNA (mRNA), micro RNA (miRNA), antisense nucleic acids (ASO) and nucleic acid aptamers (Aptamer). These drugs bind to the RNA of the target molecule and inhibit its translation or regulation, thereby achieving the therapeutic effect. Small nucleic acid drugs have high targeting and specificity, and can accurately regulate gene expression. Therefore, they have broad application prospects in gene therapy and gene editing.

[0003] To be used as effective therapeutic drugs, natural oligonucleotide molecules still have many problems that must be overcome, such as: (1) poor stability in vivo. The phosphodiester bonds of oligonucleotides are easily rapidly degraded by various ribozymes that are widely present in the blood and cells in the body; (2) low affinity and specificity for binding to target genes, which can easily lead to "off-target" effects, immune stimulation and toxic side effects; (3) low bioavailability. Oligonucleotides are usually polyvalent anionic macromolecules, so it is difficult for them to enter target organs and tissues, and it is not easy for them to penetrate the lipophilic cell membrane and enter the cell.

[0004] Chemical modification of nucleic acids is a key technology and bottleneck in the development of nucleic acid drugs. In response to the above problems, since the 1970s, many successful oligonucleotide structure modification strategies have been developed by modifying the phosphate backbone, sugar group, and base, such as thiophosphate, 2'-OMe, 2'-OMOE, 2'-F modified at the 2' position, Morpholino, PNA, etc., which greatly improve the tolerance of oligonucleotides to nucleases, affinity and specificity for target genes, reduce immune stimulation and toxic side effects, and promote the development of nucleic acid drugs.

[0005] However, currently approved small nucleic acid drugs are mainly concentrated in the field of rare diseases, and further breakthroughs are still expected in major diseases. Off-target toxicity is one of the key issues and challenges in the development of small nucleic acid drugs. Those skilled in the art have been committed to solving the drug-making barriers of small nucleic acid drugs caused by off-target effects. The off-target toxicity of small nucleic acid drugs mainly comes from the toxic reaction caused by the binding of drug molecules to non-target RNA, while the main off-target effect of siRNA comes from the pairing of the antisense chain seed region (positions 1 to 9 at the 5' end) with non-target mRNA, and causes abnormal gene expression (miRNA-like off-target effect) through a mechanism similar to that of microRNA (miRNA). Many nucleic acid drugs with excellent activity in preclinical efficacy studies are difficult to use in actual drug development due to the toxicity caused by their off-target effects. Research and development to maintain good pharmaceutical activity and T of siRNA drugs. m While increasing the value, the need to minimize the off-target effects of this type of drug is an important problem that needs to be solved in this field. It is an important development direction of the new generation of nucleic acid chemical modification and has important theoretical and practical significance. Summary of the invention

[0006] The present invention aims to overcome the poor pharmaceutical activity of small interfering nucleic acid (siRNA) drugs in the prior art, m In order to overcome the technical defects of high ribose value or off-target effect, a 2' ribose modified nucleoside monomer and its synthesis method and application are provided. The novel modified nucleoside monomer provided by the present invention introduces different modification groups at the 2' position of ribose. The double-stranded oligonucleotide (dsRNA) molecule prepared by the modified nucleoside of the present application can reduce the melting temperature (Tm) value of the double-stranded oligonucleotide (dsRNA) molecule, reduce the off-target effect of the dsRNA sequence, or maintain good pharmacodynamic activity by using modified nucleoside monomers at specific positions of the dsRNA sequence.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides a compound having the formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0009]

[0010] in,

[0011] W1 is H, a hydroxyl protecting group or an active phosphorus group;

[0012] W2 is H or a hydroxyl protecting group;

[0013] R1 is optionally replaced by 1, 2 or 3 R a A substituted base or a salt thereof, wherein the base is , , or ;

[0014] R a H, C1-C6 alkyl, -CH2R a-1 、-C(=O)OR a-1 or -C(=O)R a-1 ;

[0015] R a-1 is C1-C6 alkyl or C6-C 10 Aryl;

[0016] R2 is halogen, -OR4 or -NR5R6;

[0017] R4 is H or C1-C6 alkyl;

[0018] R5 and R6 are each independently H, C1-C8 alkyl, -CH2CH2OR7 or -C(=O)R7;

[0019] R7 is H or C1-C8 alkyl;

[0020] R3 is H or forms a carbonyl group with the carbon atom to which it is attached.

[0021] In some embodiments of the present invention, when R5 is H or C1-C8 alkyl, R6 is C1-C8 alkyl, -CH2CH2OR7 or -C(=O)R7.

[0022] In some embodiments of the present invention, R2 is halogen, -OC 1-6 Alkyl, -NHCH2CH2OC1-C8alkyl, -NHC(=O)C1-C8alkyl, -NHC1-C8alkyl or -N(C1-C8alkyl)2.

[0023] In some embodiments of the present invention, R2 is fluoro, methoxy, ethoxy, -NHC(=O)CH3, -NHCH2CH2OCH3, -N(CH2CH3)2 or -NHCH2CH3.

[0024] In some embodiments of the present invention, when R3 is hydrogen, R2 is fluoro, methoxy, ethoxy or -NHC(=O)CH3.

[0025] In some embodiments of the present invention, when R3 and the carbon atom to which it is attached form a carbonyl group, R2 is -N(CH2CH3)2, -NHCH2CH3 or -NHCH2CH2OCH3.

[0026] In some embodiments of the present invention, R1 is substituted with an amino protecting group. , , or .

[0027] In some embodiments of the present invention, R1 is , , or .

[0028] In some embodiments of the present invention, R1 is .

[0029] In some embodiments of the present invention, W1 is a reactive phosphorus group.

[0030] In some embodiments of the present invention, W1 is a phosphoramidite, an H-phosphate, a phosphotriester, or a phosphorus-containing chiral auxiliary.

[0031] In some embodiments of the present invention, W1 is .

[0032] In some embodiments of the present invention, W2 is a hydroxy protecting group.

[0033] In some embodiments of the invention, W2 is benzyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, methanesulfonyl, p-toluenesulfonyl, dimethoxytrityl (DMTr), 9-phenylxanthine-9-yl or 9-(p-methoxyphenyl)xanthine-9-yl.

[0034] In some embodiments of the invention, W2 is dimethoxytrityl ( ).

[0035] In some embodiments of the present invention, the compound represented by formula (I) is a compound represented by formula (Ia), ; wherein, the definitions of R1, R2, R3, W1 and W2 are as described in any embodiment of the present invention.

[0036] In some embodiments of the present invention, the compound represented by formula (I) is a compound represented by formula (Ib), ; wherein, the definitions of R1, R2, R3, W1 and W2 are as described in any embodiment of the present invention.

[0037] In some embodiments of the present invention, the compound represented by formula (I) is any of the following structures:

[0038] , , , , , , , or .

[0039] Another aspect of the present invention provides a method for preparing a compound as shown in formula (I), and the synthetic route is as follows:

[0040]

[0041] It includes the following steps:

[0042] Compound 1-1 is protected from hydroxyl group to obtain 1-2, which is oxidized to obtain 1-3, which is subjected to witting reaction to obtain 1-4, 1-4 is subjected to hydroboration oxidation to obtain 1-5, 1-5 is subjected to different reaction steps to obtain 1-6, which is subjected to glycosylation reaction to obtain 1-7, and 1-7 is subjected to different reaction steps to obtain the compound shown in formula (I),

[0043] in;

[0044] R 11 is a hydroxy substituent, preferably a methyl group or an acetyl group;

[0045] The definitions of R1, R2, R3, W1 and W2 are as described in any embodiment of the present invention.

[0046] Another aspect of the present invention provides a nucleic acid polymer, which is prepared by polymerization reaction of the compound represented by formula (I) as described above.

[0047] In some embodiments of the present invention, the nucleic acid polymer is a double-stranded oligonucleotide molecule, which comprises a sense strand and an antisense strand, wherein the nucleotide sequences of the sense strand and the antisense strand are at least partially reverse complementary; positions 1 to 9 based on the 5'-end of the antisense strand contain at least one modified nucleotide N M , the nucleotide N M for , , , or a tautomer thereof; R1, R2 and R3 are as defined above.

[0048] In some embodiments of the present invention, R1 is , , or .

[0049] In some embodiments of the present invention, the double-stranded oligonucleotide molecule further comprises a ligand;

[0050] Preferably, the ligand is selected from galactose, galactosamine, N-acetylgalactosamine or derivatives thereof.

[0051] In some embodiments of the present invention, the ligand is covalently attached to the 5'-end or 3'-end of the sense strand of the double-stranded oligonucleotide molecule via a linker.

[0052] In some embodiments of the present invention, the 3'-end and / or 5'-end of the sense strand and the antisense strand comprise one or more overhang regions and / or capping groups.

[0053] In some embodiments of the present invention, the nucleic acid polymer is a double-stranded oligonucleotide molecule formed by pairing a sense strand and an antisense strand of any of the following:

[0054] (1) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 5;

[0055] (2) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 6;

[0056] (3) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 7;

[0057] (4) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 8;

[0058] (5) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 9;

[0059] (6) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 10;

[0060] (7) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 11;

[0061] (8) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 12; or

[0062] (9) The sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 13.

[0063] In some embodiments of the present invention, the sense strand is SEQ ID NO: 3: 5'-Cms-Ums-Um-Um-Um-Gm-Uf-Am-Af-Cf-Uf-Um-Gm-Am-Am-Gm-Af-Um-Am-Um-Um-3'; the antisense strand is shown in the following table:

[0064]

[0065] Where: m = 2'-OMe, f = 2'-F, s = 3'-phosphorothioate, ms = 2'-OMe, 3'-phosphorothioate; fs = 2'-F, 3'-phosphorothioate, G num-101 : ; A num-102 s: ; U num-103 : ; C num-104 : ; U num-105 : ; U num-106 : ; U num-107 : ; U num-108 : ; U num-109 : .

[0066] Another aspect of the present invention provides a pharmaceutical composition, which comprises the nucleic acid polymer as described above and a pharmaceutically acceptable excipient.

[0067] Another aspect of the present invention provides use of the nucleic acid polymer as described above or the pharmaceutical composition as described above in the preparation of a nucleic acid diagnostic agent and / or a nucleic acid therapeutic agent.

[0068] Another aspect of the present invention provides the use of the nucleic acid polymer as described above or the pharmaceutical composition as described above in the preparation of a drug for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene (eg, PCSK9 gene).

[0069] In some embodiments of the present invention, the disease is adult primary hypercholesterolemia (heterozygous familial and non-familial) or mixed dyslipidemia.

[0070] Another aspect of the present invention provides a method for inhibiting target gene expression, comprising administering an effective amount of the above-mentioned double-stranded oligonucleotide molecule or the above-mentioned pharmaceutical composition to a subject; wherein the administration comprises administration by subcutaneous or intravenous administration; wherein the subject is a mammal, preferably a human.

[0071] Unless otherwise defined, all terms (including technical and scientific terms) used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined in the present invention.

[0072] In the present invention, "stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including but not limited to cis-trans isomers, enantiomers, and the like.

[0073] In the present invention, "C1-C8" refers to any integer value of carbon atoms in the main chain of the group in the range of 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8 carbon atoms. Similarly, the term "C1-C6" refers to any integer value of carbon atoms in the main chain of the group in the range of 1 to 6, such as 1, 2, 3, 4, 5, 6 carbon atoms.

[0074] In the present invention, "hydroxyl protecting group" refers to a protecting group commonly used to protect the hydroxyl group of the ribose structure in the synthesis of RNA or its derivatives, such as acetyl, phenoxyacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4, 4'-dimethoxytrityl (DMr, 4, 4'-dimethoxytrityl), monomethoxytrityl (MMTr, monomethoxytrityl), 9-phenyl-xanthen-9-yl (9-phenylxanthen-9-yl), 9-p-tolyl-xanthen-9-yl (9-(p-tolyl)-xanthen-9-yl), trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanoethoxymethyl, etc.

[0075] In the present invention, "phosphorus-containing reactive groups" refer to phosphorus-containing groups that can react with a hydroxyl or amine group contained in another molecule, especially in another nucleotide unit or another nucleotide analog, through a nucleophilic attack reaction. Typically, such reactions produce ester-type nucleoside bonds 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 include P III or P V The phosphorus atom in a valence state, the phosphorus-containing reactive groups include but are not limited to phosphoramidites, H-phosphonates, phosphotriesters and phosphorus-containing chiral auxiliary agents, such as .

[0076] In the present invention, "halogen" means fluorine, chlorine, bromine or iodine.

[0077] In the present invention, "alkyl" refers to a straight or branched alkyl group with a specified number of carbon atoms (e.g., C1-C6, C1-C8). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0078] In the present invention, "aryl" refers to a group having a specified number of ring carbon atoms (e.g., C6-C 10 ), an aromatic ring whose ring atoms consist only of carbon atoms, which is a cyclic or monocyclic ring. For example, phenyl or naphthyl.

[0079] In the present invention, "salt" refers to a corresponding salt of the modified nucleoside compound (or nucleotide compound) in the present invention that can be conveniently or desirably prepared, purified and / or processed, for example, a pharmaceutically acceptable salt. Unless otherwise indicated, reference to a specific compound in the present invention also includes its salt form.

[0080] In the present invention, "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.

[0081] In the present invention, "pharmaceutically acceptable excipients" refer to all substances contained in the pharmaceutical preparation except the active ingredient.

[0082] In the present invention, "nucleic acid polymer" may refer to any nucleic acid molecule, including but not limited to DNA, RNA and their hybrids, including but not limited to single-stranded and double-stranded, etc. The number of nucleotides polymerized to form an oligonucleotide is 2, 3 or more, and may be a polymer with less than 20 nucleotides or a polymer with more than 20 nucleotides.

[0083] In the present invention, "pharmaceutical composition" refers to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and a pharmaceutically acceptable excipient, which is intended for administration to a mammal, such as a human, in need thereof.

[0084] In the present invention, "treatment" refers to any of the following situations: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that triggers the disease; (3) slowing the development of one or more biological manifestations of a disease.

[0085] In the present invention, "prevention" refers to reducing the risk of developing a disease.

[0086] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0087] The reagents and raw materials used in the present invention are commercially available.

[0088] The positive improvement effect of the present invention is that the modified nucleosides provided by the present invention have at least one of the following advantages:

[0089] 1) The modified nucleosides provided in the present application introduce a modification group at the 2' position of the sugar ring, which is completely different from the modified nucleosides in the prior art.

[0090] 2) The double-stranded oligonucleotide (dsRNA) molecule prepared by the modified nucleoside in the present application has a relatively lower Tm value compared with the conventional 2'-F or 2'-OMe modified double-stranded oligonucleotide (dsRNA) molecule.

[0091] 3) The double-stranded oligonucleotide (dsRNA) molecule prepared by the modified nucleoside in the present application has better target gene interference activity compared with the conventional 2'-F or 2'-OMe modified double-stranded oligonucleotide (dsRNA) molecule.

[0092] 4) The double-stranded oligonucleotide (dsRNA) molecule prepared by the modified nucleoside in the present application has lower off-target activity compared with the conventional 2'-F or 2'-OMe modified double-stranded oligonucleotide (dsRNA) molecule.

[0093] When the double-stranded oligonucleotide for inhibiting target gene expression provided by the present invention is used to prepare a pharmaceutical composition or a method for inhibiting target gene expression, since dsRNA can effectively inhibit target gene expression and its anti-off-target effect is more excellent, the drug containing the above dsRNA molecule can effectively reduce the drug side effects caused by off-target. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] 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.

[0095] Figure 1 This is a curve for determining the melting temperature Tm values ​​of differently modified double-stranded oligonucleotide (dsRNA) molecules ON1, ON4, ON6, ON7, ON8, ON12, and ON14.

[0096] Figure 2 Circular dichroism spectra of differently modified double-stranded oligonucleotide (dsRNA) molecules ON1, ON4, ON6, ON7, ON8, ON12, and ON14.

[0097] Figure 3 The inhibition rate of double-stranded oligonucleotide (dsRNA) molecules ON1, ON2, ON3, ON4, ON5, ON6, ON7, ON8, ON9, ON10, ON11, ON12, ON13, ON14 and ON15 on the target gene PCSK9 at a concentration of 15 nM.

[0098] Figure 4 The inhibition rate of double-stranded oligonucleotide (dsRNA) molecules ON1, ON4, ON6, ON7, ON8, ON9, ON10 and ON14 on off-target genes PCYOX1-5 at a concentration of 15 nM.

[0099] Figure 5 The inhibition rate of double-stranded oligonucleotide (dsRNA) molecules ON1, ON4, ON6, ON7, ON8, ON9, ON10 and ON14 on the off-target gene hDSE at a concentration of 15 nM. DETAILED DESCRIPTION

[0100] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0101] 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, the percentages in the context are weight percentages, and 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.

[0102] The following abbreviations represent the following reagents:

[0103] IBX: 2-iodoacetylbenzoic acid; Dess-Martin: Dess-Martin periodinane; DEA: diethylamine; TEA: triethylamine; Ac2O: acetic anhydride; HOAc: acetic acid; conc H2SO4: concentrated sulfuric acid; HDMS: hexamethyldisilazane; BSA: N,O-bis(trimethylsilyl)acetamide; TMSOTf: trimethylsilyl trifluoromethanesulfonate; Toluene: toluene; MeOH: methanol; DIEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DMSO: dimethyl sulfoxide; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; THF: tetrahydrofuran; TIPDSCl: 1,3-dichloro-1,1,3,3-tetraisopropyldimethylsilyloxyether; Imid azole: imidazole; DMF: N,N-dimethylformamide; TBAF: tetrabutylammonium fluoride; DMTrCl: 4,4'-dimethoxytriphenylmethane; DIAD: azodiisopropyldicarboxylic acid; pyridine: pyridine; IH-tetrazole: 1H-tetrazole; DCM: dichloromethane; EA: ethyl acetate; DAST: diethylaminosulfur trifluoride; PPh3: triphenylphosphine; ACN: acetonitrile; s-BuLi: sec-butyllithium; t-BuONa: sodium tert-butoxide; DCE: dichloroethane; TEMPO: tetramethylpiperidinium oxide; Ph3P + CH3Br - : Methyltriphenylphosphonium bromide; H2O2: hydrogen peroxide; U: uracil; Phthalimide: phthalimide; Hydraziniumhydroxide solution: hydrazine hydrate; Acetyl chloride: acetyl chloride; Ethylamine: ethylamine.

[0104] Example 1: Synthesis of modified nucleosides

[0105] The modified nucleosides YK-NUM-101~109 designed in this application were synthesized via the following route.

[0106] Synthesis route:

[0107] In this example, the key intermediate 1-5 hydroxyl groups were converted into halogen, amino, methyl ether and acid to obtain modified nucleosides YK-NUM-101-109. This route can quickly screen different modification groups through the functional group conversion of hydroxyl groups.

[0108] ;

[0109] 1. Synthesis of intermediate INT-I

[0110]

[0111] Step 1: Synthesis of INT-I-PM1

[0112] SM, i.e. (2R, 3R, 4S, 5R) -2- (hydroxymethyl) -5-methoxytetrahydrofuran-3, 4-diol (60.00 g, 365.50 mmol) was dissolved in pyridine (300.0 mL), cooled in an ice bath under nitrogen atmosphere, and 1,3-dichloro-1,1,3,3-tetraisopropyldimethylsiloxy ether (115.30 g, 365.53 mmol) was added dropwise, and the reaction was stirred at room temperature overnight. After the reaction was completed, the organic phase was removed by vacuum concentration, dissolved in water, extracted with dichloromethane, the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum concentration to remove the solvent. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate) to obtain INT-I-PM1 (75.00 g, 184.42 mmol, 50.46%).

[0113] Step 2: Synthesis of INT-I-PM2

[0114] Dissolve INT-I-PM1 (35.00 g, 86.06 mmol) and Dess-Martin periodinane (91.30 g, 215.26mmol) in acetonitrile and stir the reaction at 45°C for 19 h under nitrogen atmosphere. TLC shows that the reaction is complete. Cool the reaction solution, filter out the insoluble matter, concentrate the filtrate under vacuum pressure, dissolve it with methyl tert-butyl ether, and filter out the insoluble matter again. Wash with saturated sodium thiosulfate, saturated sodium bicarbonate, saturated brine, and dry over anhydrous sodium sulfate. Filter the mixture and concentrate the filtrate under vacuum pressure. Obtain a colorless liquid target product INT-I-PM2 crude product (29.30g, 100% purity for the next step).

[0115] Step 3: Synthesis of INT-I-PM3

[0116] Methyltriphenylphosphonium bromide (56.90 g, 159.29 mmol) was dissolved in tetrahydrofuran, and the temperature was lowered to -78 °C under nitrogen atmosphere. 1.3 Ms-BuLi (128.1 mL, 166.53 mmol) was added dropwise, and the temperature was kept for 1 hour after the addition was completed. INT-I-PM2 (29.30 g, 72.41 mmol) was added dropwise, and the temperature was kept for 1 hour after the addition was completed. The mixture was heated to room temperature and stirred to react overnight. After the reaction was completed, the organic phase was removed by vacuum concentration, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum concentration to remove the solvent. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate) to obtain INT-I-PM3 (12.67 g, 31.46 mmol, 43.45%).

[0117] Step 4: Synthesis of INT-I

[0118] Add 1 M borane tetrahydrofuran complex (58.3 ml, 58.30 mmol) to the reaction flask, add INT-I-PM3 (9.67 g, 24.01 mmol) tetrahydrofuran solution dropwise at 0 °C, and react at room temperature for 2 h. Add THF:H2O=1:1 (20 mL) dropwise to the above solution in an ice bath, react at room temperature for 3 h under nitrogen, and react at 40 °C overnight. Cool the reaction solution in an ice bath, and add 2M sodium hydroxide solution (48.0 mL, 96.00 mmol) and 30% hydrogen peroxide (36.8 mL, 360.21 mmol) dropwise in sequence, and react at room temperature for 15 h. TLC shows that the reaction is complete, and the reaction solution is concentrated under vacuum, and the concentrated aqueous phase is extracted with EA and dried over anhydrous sodium sulfate. Filter the mixture, and the filtrate is concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product INT-I (5.09 g, 12.10 mmol, 50.38%) as a colorless liquid.

[0119] 2. Synthesis of YK-NUM-101

[0120]

[0121] Step 1: Synthesis of YK-NUM-101-PM1

[0122] INT-I (8.00 g, 19.02 mmol) and sodium tert-butoxide (5.47 g, 56.92 mmol) were dissolved in tetrahydrofuran, reacted at room temperature for 1h under nitrogen protection, cooled in an ice bath, and iodomethane (13.49 g, 95.04 mmol) was added dropwise, and reacted at room temperature overnight. After the reaction was completed, a saturated aqueous solution of ammonium chloride was added, the organic phase was removed by vacuum decompression concentration, a saturated aqueous solution of sodium chloride was added, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum decompression to remove the solvent. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate) to obtain YK-NUM-101-PM1 (5.63 g, 12.95mmol, 68.10%).

[0123] Step 2: Synthesis of YK-NUM-101-PM2

[0124] Dissolve N2-isobutyrylguanine (7.20 g, 32.55 mmol) and ammonium sulfate (143.4 mg, 1.10 mmol) in HDMS (80 mL) and pyridine (25 mL), dissolve at 130°C, keep warm for 1.5 h, remove the solvent by vacuum concentration, vacuumize and replace nitrogen three times, add YK-NUM-101-PM1 (4.72 g, 10.86 mmol) and 1,2-dichloroethane (80 mL), cool in an ice bath under nitrogen protection, add trimethylsilyl trifluoromethanesulfonate (7.24 g, 32.57 mmol) dropwise, and stir at 80°C for 3 h. After the reaction is completed, concentrate, dissolve in dichloromethane, wash with saturated sodium bicarbonate aqueous solution, add diatomaceous earth and filter under reduced pressure, extract the aqueous phase with dichloromethane, combine the organic phases and wash with saturated brine, dry over anhydrous sodium sulfate, filter, and remove the solvent by vacuum concentration of the filtrate. The residue was purified by silica gel chromatography (dichloromethane / methanol) to give YK-NUM-101-PM2 (3.36 g, 5.39 mmol, 49.60%). 28 H 49 N5O7Si2, MS(ES): m / z(M+H + ) 624.3.

[0125] Step 3: Synthesis of YK-NUM-101-PM3

[0126] YK-NUM-101-PM2 (3.35 g, 5.37 mmol), 1 M TBAF in THF solution (21.6 mL, 21.60 mmol) and tetrahydrofuran (30 mL) were mixed and stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated under vacuum and the residue was purified by C18 reverse column chromatography (acetonitrile / water) to obtain YK-NUM-101-PM3 (1.82 g, 4.77 mmol, 88.87%). 16 H 23 N5O6, MS(ES): m / z(M+H + ) 382.2.

[0127] Step 4: Synthesis of YK-NUM-101-PM4

[0128] Dissolve YK-NUM-101-PM3 (1.82 g, 4.77 mmol) in pyridine (20.0 mL), remove pyridine by rotary evaporation, add DMTrCl (2.59 g, 7.64 mmol) and pyridine (20.0 mL), and stir at room temperature overnight. After the reaction is completed, concentrate, wash with saturated sodium bicarbonate aqueous solution, extract with dichloromethane, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to remove the solvent. The residue is purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-101-PM4 (1.11 g, 1.62 mmol, 34.01%). 37 H 41 N5O8, MS(ES):m / z(M+H + ) 684.3.

[0129] Step 5: Synthesis of YK-NUM-101

[0130] YK-NUM-101-PM4 (1.11 g, 1.62 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.15g, 4.86 mmol), N,N-diisopropylethylamine (627.7 mg, 4.86 mmol), and N-methylimidazole (132.9 mg, 1.62 mmol) were dissolved in dichloromethane (11.0 mL) and stirred at room temperature for 3 hours. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, and the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-101 (719.0 mg, 0.81 mmol, 50.10%).46 H 58 N7O9P, MS(ES): m / z(M+H + ) 884.4.

[0131] YK-NUM-101: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.37 (d, J = 8.6, 1H),7.29 - 7.25 (m, 9H), 6.89 (d, J = 7.2 Hz, 4H), 5.85 (d, J = 7.2 Hz, 1H), 4.60(d, J = 7.8 Hz, 1H), 3.90 (t, J = 10.8 Hz, 2H), 3.80 - 3.75(m, 7H), 3.61 (d,J = 8.4 Hz, 2H), 3.40 - 3.17(m, 5H), 2.83 - 2.60 (m, 5H), 2.0 (t, J = 12.4Hz, 1H), 1.12 - 1.06 (m, 18H). 31 P NMR (162 MHz, DMSO-d6) δ 148.40 (s, 1P).

[0132] 3. Synthesis of YK-NUM-102

[0133]

[0134] Step 1: Synthesis of YK-NUM-102-PM1

[0135] N6-benzoyl adenine (5.59 g, 23.37 mmol) and ammonium sulfate (463.2 mg, 3.51 mmol) were dissolved in HDMS (60 mL) and pyridine (15 mL), and the mixture was dissolved at 130°C, kept warm for 1.5 h, and the solvent was removed by vacuum concentration, and the mixture was replaced with nitrogen three times. YK-NUM-101-PM1 (5.08 g, 11.69 mmol) and 1,2-dichloroethane (60 mL) were added, and the mixture was cooled in an ice bath under nitrogen protection, and trimethylsilyl trifluoromethanesulfonate (9.87 g, 44.41 mmol) was added dropwise, and the mixture was stirred at 80°C for 4 h. After the reaction was completed, the mixture was concentrated, dissolved in dichloromethane, quenched and washed with saturated sodium bicarbonate aqueous solution, filtered under reduced pressure with diatomaceous earth, the aqueous phase was extracted with dichloromethane, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / ethyl acetate) to give YK-NUM-102-PM1 (3.70 g, 5.76 mmol, 49.31%). 31 H 47 N5O6Si2, MS(ES): m / z(M+H + ) 642.3.

[0136] Step 2: Synthesis of YK-NUM-102-PM2

[0137] YK-NUM-102-PM1 (2.69 g, 4.19 mmol), 1 M TBAF in THF solution (16.8 mL, 16.80 mmol), and tetrahydrofuran (30 mL) were added to the reaction flask in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated, and the residue was purified by reverse phase chromatography (water / acetonitrile), and the solvent was removed by vacuum concentration. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-102-PM2 (1.56 g, 3.91 mmol, 93.20%). 19 H 21 N5O5, MS(ES): m / z(M+H + ) 400.2.

[0138] Step 3: Synthesis of YK-NUM-102-PM3

[0139] Dissolve YK-NUM-102-PM2 (1.50 g, 3.76 mmol) in pyridine (15 mL), remove pyridine by rotary evaporation, add DMTrCl (2.04 g, 6.02 mmol) and pyridine (10.0 mL), and stir at room temperature overnight. After the reaction is completed, concentrate, wash with saturated sodium bicarbonate aqueous solution, extract with dichloromethane, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to remove the solvent. The residue is purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-102-PM3 (968.3 mg, 1.38 mmol, 36.74%). 40 H 39 N5O7, MS(ES): m / z(M+H + ) 702.3.

[0140] Step 4: Synthesis of YK-NUM-102

[0141] YK-NUM-102-PM3 (968.3 mg, 1.38 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (979.9 mg, 4.14 mmol), N,N-diisopropylethylamine (535.1 mg, 4.14 mmol), and N-methylimidazole (113.3 mg, 1.38 mmol) were dissolved in dichloromethane and stirred at room temperature for 3 hours. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, the organic phases were combined and then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-102 (790.1 mg, 0.88 mmol, 63.48%). 49 H 56 N7O8P, MS(ES): m / z(M+H + ) 902.4.

[0142] YK-NUM-102: 1H NMR (CDCl3, 400 MHz, 298 K) δ 8.35 (s, 1H), 8.19 (s,1H), 7.94 (d, J = 7.2 Hz, 2H), 7.68 - 7.56 (m, 3H), 7.30 - 7.25 (m, 9H), 6.91 (d, J = 7.2 Hz, 4H), 5.90 (d, J = 7.8 Hz, 1H), 4.60 (d, J = 8.6 Hz, 1H), 3.90(t, J = 10.8 Hz, 2H), 3.80 (s, 6H), 3.75 -3.57 (m, 3H), 3.23 - 3.17 (m, 5H),2.83 - 2.60 (m, 4H), 2.01(m, 1H), 1.46 - 1.39 (m, 12H). 31 P NMR (162 MHz, DMSO-d6) δ 148.90 (s, 1P).

[0143] 4. Synthesis of YK-NUM-103

[0144]

[0145] Step 1: Synthesis of YK-NUM-103-PM1

[0146] Dissolve uracil (2.04 g, 18.20 mmol) and ammonium sulfate (96.0 mg, 0.73 mmol) in HDMS (63.6 mL) and pyridine (12.6 mL), dissolve at 130°C, keep warm for 1.5 h, remove the solvent by vacuum concentration, vacuumize and replace nitrogen three times, add YK-NUM-101-PM1 (3.16 g, 7.27 mmol) and acetonitrile (64.0 mL), cool in an ice bath under nitrogen protection, add tin tetrachloride (4.73 g, 18.16 mmol) dropwise, and stir at 0°C for 8 h. After the reaction is completed, concentrate, dissolve in dichloromethane, wash with saturated sodium bicarbonate aqueous solution, add diatomaceous earth and filter under reduced pressure, extract the aqueous phase with dichloromethane, combine the organic phases and wash with saturated brine, dry over anhydrous sodium sulfate, filter, and remove the solvent by vacuum concentration of the filtrate. The residue was purified by silica gel chromatography (dichloromethane / methanol) to give YK-NUM-103-PM1 (1.79 g, 3.48 mmol, 47.84%). 23 H 42 N2O7Si2,MS(ES): m / z(M+H + ) 515.2.

[0147] Step 2: Synthesis of YK-NUM-103-PM2

[0148] YK-NUM-103-PM1 (1.79 g, 3.48 mmol), 1 M TBAF in THF solution (13.9 mL, 13.90 mmol), and tetrahydrofuran (18.0 mL) were added to the reaction flask in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated, and the residue was purified by reverse phase chromatography (water / acetonitrile), and the solvent was removed by vacuum concentration. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-103-PM2 (0.86 g, 3.16 mmol, 90.84%). 11 H 16 N2O6, MS(ES): m / z(M+H + ) 273.1.

[0149] Step 3: Synthesis of YK-NUM-103-PM3

[0150] Dissolve YK-NUM-103-PM2 (836.0 mg, 3.07 mmol) in pyridine (10.0 mL), remove pyridine by rotary evaporation, add DMTrCl (1.66 g, 4.90 mmol) and pyridine (10.0 mL), and stir at room temperature overnight. After the reaction is completed, concentrate, wash with saturated sodium bicarbonate aqueous solution, extract with dichloromethane, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to remove the solvent. The residue is purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-103-PM3 (631.0 mg, 1.10 mmol, 35.76%). 32 H 34 N2O8, MS(ES):m / z(M+H + ) 575.2.

[0151] Step 4: Synthesis of YK-NUM-103

[0152] YK-NUM-103-PM3 (570.0 mg, 0.99 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (704.3 mg, 2.98 mmol), N,N-diisopropylethylamine (384.6 mg, 2.98 mmol), and N-methylimidazole (81.4 mg, 0.99 mmol) were dissolved in 10.0 mL of dichloromethane and stirred at room temperature for 3 hours. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-103 (590.0 mg, 0.76 mmol, 76.76%). 41 H 51 N4O9P, MS(ES): m / z(M+H + ) 775.3.

[0153] YK-NUM-103: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.45 (d, J = 8.6, 1H), 7.28 - 7.26 (m, 9H), 6.89 (d, J = 7.2 Hz, 4H), 5.75 (d, J = 7.8 Hz, 1H), 5.47(d, J = 8.4 Hz, 1H), 4.60 (m, 1H), 3.90 (t, J = 10.8 Hz, 2H), 3.80 (s, 6H), 3.61 – 3.40 (m, 5H), 3.23 (s, 3H), 2.83 – 2.60 (m, 5H), 1.16 – 1.05 (m, 12H). 31 P NMR (162 MHz, DMSO-d6) δ 148.63 (s, 1P).

[0154] 5. Synthesis of YK-NUM-104

[0155]

[0156] Step 1: Synthesis of YK-NUM-104-PM1

[0157] N4-acetylcytosine (5.28 g, 34.48 mmol), ammonium sulfate (0.15 g, 1.15 mmol), pyridine (20 mL), HDMS (100 mL) were added to the reaction flask in sequence, and activated at 130°C for 2 h under nitrogen protection. The above solution was cooled and concentrated under vacuum to obtain a white solid. A solution of YK-NUM-101-PM1 (5.00 g, 11.50 mmol) in acetonitrile was added to the above white solid. Tin tetrachloride (8.99 g, 34.51 mmol) was added dropwise to the above solution under an ice bath. After the addition was completed, the reaction was stirred under an ice bath for 2 h, and then transferred to 37°C for 24 h. TLC showed that the reaction was complete. The reaction solution was placed under an ice bath and saturated sodium bicarbonate was added dropwise to quench the reaction, the pH was adjusted to 7~8, and an equal volume of dichloromethane was added and stirred. The insoluble matter was filtered off, the organic phase was separated, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-104-PM1 (3.90 g, 7.02 mmol, 61.01%) as an off-white solid. 25 H 45 N3O7Si2, MS(ES): m / z(M+H + ) 556.3.

[0158] Step 2: Synthesis of YK-NUM-104-PM2

[0159] YK-NUM-104-PM1 (3.90 g, 7.02 mmol), ultra-dry THF (40 mL), 1 M TBAF in THF solution (28.1 mL, 28.10 mmol) were added to the reaction flask in sequence. The mixture was stirred at room temperature for 4 h under nitrogen protection. LCMS showed that the reaction was complete. The reaction solution was concentrated under vacuum. The residue was purified by C18 reverse column chromatography (acetonitrile / water) to obtain the target product YK-NUM-104-PM2 (1.60 g, 5.11 mmol, 72.78%) as a white solid. 13 H 19 N3O6, MS(ES): m / z(M+H + ) 314.1.

[0160] Step 3: Synthesis of YK-NUM-104-PM3

[0161] YK-NUM-104-PM2 (1.60 g, 5.11 mmol), ultra-dry pyridine (10 mL), and DMTrCl (2.77 g, 8.18 mmol) were added to the reaction flask in sequence. The mixture was stirred at room temperature for 2 h under nitrogen protection. LCMS showed that the reaction was complete. The reaction solution was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-104-PM3 (1.55 g, 2.52 mmol, 49.30%) as a white solid. 34 H 37 N3O8, MS(ES): m / z(M+H + ) 616.3.

[0162] Step 4: Synthesis of YK-NUM-104

[0163] YK-NUM-104-PM3 (1.29 g, 2.10 mmol), ultra-dry dichloromethane (13 mL), N,N-diisopropylethylamine (616.5 mg, 4.77 mmol), N-methylimidazole (130.6 mg, 1.59 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.13 g, 4.77 mmol) were added to the reaction bottle in sequence. The reaction was stirred at room temperature for 1.5 h under nitrogen protection. LCMS showed that the reaction was complete. 10 mL of saturated sodium bicarbonate solution was added to the above reaction solution to quench the reaction. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-104 (756.5 mg, 0.93mmol, 44.25%) as a white solid. 43 H 54 N5O9P, MS(ES): m / z(M+H + )816.4.

[0164] YK-NUM-104: 1H NMR (CDCl3, 400 MHz, 298 K) δ 8.85 (d, J = 8.6, 1H), 7.28 - 7.26 (m, 9H), 6.89 (d, J = 7.2 Hz, 4H), 5.74 (d, J = 7.8 Hz, 1H), 5.53(d, J = 8.4 Hz, 1H), 4.62 - 4.60 (m, 1H), 3.93 (t, J = 10.8 Hz, 2H), 3.80 (s,6H), 3.61 - 3.40 (m, 5H), 3.23 (s, 3H), 2.83 - 2.60 (m, 5H), 1.93 (s, 3H),1.16 - 1.05 (m, 12H). 31 P NMR (162 MHz, DMSO-d6) δ 148.75 (s, 1P).

[0165] 6. Synthesis of YK-NUM-105

[0166]

[0167] Step 1: Synthesis of YK-NUM-105-PM1

[0168] INT-I (10.00 g, 23.77 mmol) was dissolved in dichloromethane (100.0 mL), and the temperature was lowered to -78°C under nitrogen atmosphere. DAST reagent (9.58 g, 59.43 mmol) was slowly added dropwise to the above system. After the addition was completed, the temperature was raised to 0°C and stirred for 4 h. After the reaction was completed, the reaction solution was quenched with saturated sodium bicarbonate solution in an ice bath, filtered, washed with saturated brine (40 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 0-3%) to obtain YK-NUM-105-PM1 (5.90 g, 13.96 mmol, 58.72%).

[0169] Step 2: Synthesis of YK-NUM-105-PM2

[0170] Dissolve uracil (3.91 g, 34.88 mmol) and ammonium sulfate (368.6 mg, 2.79 mmol) in pyridine (24.0 mL) and hexamethyldisilazane (120.0 mL), activate in an oil bath at 130°C for 1.5 h under nitrogen protection, cool down, and concentrate under vacuum to obtain a white solid. Then add YK-NUM-105-PM1 (5.90 g, 13.96 mmol) and acetonitrile (60.0 mL), cool to 0°C in an ice-water bath under nitrogen atmosphere, slowly add anhydrous tin tetrachloride (9.09 g, 34.90 mmol), and stir and react for 22 h. After the reaction was completed, the reaction solution was quenched with saturated sodium bicarbonate in an ice bath, filtered, washed with saturated brine (40 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure in vacuo, and the residue was purified by silica gel chromatography (dichloromethane / ethyl acetate = 0-80%) to obtain YK-NUM-105-PM2 (3.42 g, 6.80 mmol, 48.74%). 22 H 39 FN2O6Si2,MS(ES): m / z(M+H + ) 503.2.

[0171] Step 3: Synthesis of YK-NUM-105-PM3

[0172] Dissolve YK-NUM-105-PM2 (3.42 g, 6.80 mmol) in tetrahydrofuran (35.0 mL), cool to 0°C in an ice-water bath under nitrogen atmosphere, slowly drop 1 M TBAF in THF (27.2 ml, 27.2 mmol) to the above system, and stir and react at room temperature for 22 h after the addition is complete. After the reaction is complete, the reaction mixture is concentrated under vacuum, and the residue is purified by C18 reverse column chromatography (acetonitrile / water) to obtain YK-NUM-105-PM3 (1.36 g, 5.23 mmol, 76.83%). 10 H 13 FN2O5,MS(ES): m / z(M+H + ) 261.1.

[0173] Step 4: Synthesis of YK-NUM-105-PM4

[0174] Dissolve YK-NUM-105-PM3 (1.36 g, 5.23 mmol) and DMTrCl (2.64 g, 7.80 mmol) in pyridine (14.0 mL). After the addition is complete, stir at room temperature under nitrogen atmosphere for 2 h. After the reaction is complete, the reaction mixture is concentrated under vacuum, then washed with EA (80.0 mL) and saturated brine (20 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue is purified by silica gel chromatography (dichloromethane / methanol = 0-3%) to obtain YK-NUM-105-PM4 (1.10 g, 1.96 mmol, 37.48%). 31 H 31 FN2O7, MS(ES): m / z(M+H + ) 563.2.

[0175] Step 5: Synthesis of YK-NUM-105

[0176] YK-NUM-105-PM4 (1.10 g, 1.96 mmol), N,N-diisopropylethylamine (759.9 mg, 5.88mmol), and N-methylimidazole (160.9 mg, 1.96 mmol) were dissolved in dichloromethane (12.0 mL). Under nitrogen atmosphere, the temperature was cooled to 0°C in an ice-water bath. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.39 g, 5.87mmol) was added to the above system. After the addition was completed, the temperature was raised to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was washed with saturated brine (20mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 0-100%) to obtain YK-NUM-105 (763.2 mg, 1.00 mmol, 51.08%). 40 H 48 FN4O8P, MS(ES): m / z(M+H + ) 763.3.

[0177] YK-NUM-005: 1H NMR (CDCl3, 400 MHz, 298 K) δ 7.43 (d, J = 8.6, 1H), 7.27 - 7.24 (m, 9H), 6.86 (d, J = 7.2 Hz, 4H), 5.75 (d, J = 7.8 Hz, 1H), 5.46(d, J = 8.4 Hz, 1H), 4.60 (m, 1H), 3.92 (t, J = 10.8 Hz, 2H), 3.80 (s, 6H), 3.61 - 3.38 (m, 5H), 2.85 - 2.61 (m, 5H), 1.16 - 1.05 (m, 12H). 31 P NMR (162MHz, DMSO-d6) δ 148.55 (s, 1P).

[0178] 7. Synthesis of YK-NUM-106

[0179]

[0180] Step 1: Synthesis of YK-NUM-106-PM1

[0181] INT-I (4.00 g, 9.51 mmol), iodophenyl diacetic acid (6.13 g, 19.03 mmol), sodium bicarbonate (1.44 g, 17.14 mmol), TEMPO (0.27 g, 1.73 mmol), acetonitrile (40.0 mL) and water (40.0 mL) were reacted at room temperature for 3 h under nitrogen protection. After the reaction was completed, the reaction solution was quenched with sodium thiosulfate solution, extracted with EA (40 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent to obtain YK-NUM-106-PM1 (4.62 g, 10.63mmol, calculated as 100.0%).

[0182] Step 2: Synthesis of YK-NUM-106-PM2

[0183] Dissolve YK-NUM-106-PM1 (4.62 g, 10.63 mmol) in acetonitrile (46.0 mL), cool to 0°C in an ice-water bath under nitrogen atmosphere, add HATU (4.85 g, 12.76 mmol), DIEA (4.12 g, 31.88 mmol), and 2-methoxyethylamine (1.76 g, 23.43 mmol) to the above system, and heat to room temperature and stir for 2 hours after the addition. After the reaction is completed, the reaction mixture is concentrated under vacuum, and the residue is purified by silica gel chromatography (petroleum ether / ethyl acetate = 0-70%) to obtain YK-NUM-106-PM2 (5.16 g, 10.49 mmol, 98.7%). 22 H 45 NO7Si2, MS(ES): m / z(M+H + )492.3.

[0184] Step 3: Synthesis of YK-NUM-106-PM3

[0185] Dissolve uracil (3.53 g, 31.49 mmol) and ammonium sulfate (277.2 mg, 2.10 mmol) in pyridine (20.0 mL) and hexamethyldisilazane (100.0 mL), activate in an oil bath at 130°C for 1.5 h under nitrogen protection, cool, and concentrate under vacuum to obtain a white solid. Then add YK-NUM-106-PM2 (5.16 g, 10.49 mmol) and acetonitrile (50.0 mL), cool to 0°C in an ice-water bath under nitrogen atmosphere, and slowly add anhydrous tin tetrachloride (8.20 g, 31.48 mmol) dropwise. After completion, stir and react for 16 h under heat preservation, and then react in an oil bath at 37°C for about 18 h. After the reaction was completed, the reaction solution was quenched with saturated sodium bicarbonate in an ice bath, filtered, washed with saturated brine (40 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and the residue was purified by silica gel chromatography (dichloromethane / ethyl acetate = 0-90%) to obtain YK-NUM-106-PM3 (3.72 g, 6.51 mmol, 62.00%). 25 H 45 N3O8Si2, MS(ES): m / z(M+H + ) 572.3.

[0186] Step 4: Synthesis of YK-NUM-106-PM4

[0187] Dissolve YK-NUM-106-PM3 (1.70 g, 2.97 mmol) in tetrahydrofuran (17.0 mL), cool to 0°C in an ice-water bath under nitrogen atmosphere, slowly drop 1 M TBAF in THF (11.9 ml, 11.90 mmol) into the above system, and stir and react at room temperature for 22 h after the addition is complete. After the reaction is complete, the reaction mixture is concentrated under vacuum, and the residue is purified by C18 reverse column chromatography (acetonitrile / water) to obtain YK-NUM-106-PM4 (952.4 mg, 2.89 mmol, 97.28%). 13 H 19 N3O7, MS(ES): m / z(M+H + ) 330.1.

[0188] Step 5: Synthesis of YK-NUM-106-PM5

[0189] Dissolve YK-NUM-106-PM4 (952.4 mg, 2.89 mmol) and DMTrCl (1.57 g, 4.63 mmol) in pyridine (10.0 mL). After the addition is complete, stir at room temperature under nitrogen atmosphere for 2 h. After the reaction is complete, the reaction mixture is concentrated under vacuum, then washed with EA (60.0 mL) and saturated brine (20 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue is purified by silica gel chromatography (dichloromethane / methanol = 0-3%) to obtain YK-NUM-106-PM5 (921.3 mg, 1.46 mmol, 50.43%). 34 H 37 N3O9, MS(ES): m / z(M+H + ) 632.3.

[0190] Step 6: Synthesis of YK-NUM-106

[0191] YK-NUM-106-PM5 (921.3 mg, 1.46 mmol), N,N-diisopropylethylamine (566.1 mg, 4.38 mmol), and N-methylimidazole (119.9 mg, 1.46 mmol) were dissolved in dichloromethane (10.0 mL). Under nitrogen atmosphere, the temperature was cooled to 0°C in an ice-water bath. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (863.9 mg, 3.65 mmol) was added to the above system. After the addition was completed, the temperature was raised to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 0-100%) to obtain YK-NUM-106 (885.5 mg, 1.06 mmol, 72.98%). 43 H 54 N5O 10 P, MS(ES): m / z(M+H + ) 832.4.

[0192] YK-NUM-106: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.41 (d, J = 8.6, 1H), 7.29 - 7.26 (m, 9H), 6.89 (d, J = 7.2 Hz, 4H), 6.15 (d, J = 7.8 Hz, 1H), 5.46 (d, J = 8.4 Hz, 1H), 4.61 (m, 1H), 4.15 (m, 1H), 3.92 (t, J = 10.8 Hz, 2H), 3.81 (s, 6H), 3.61 - 3.40 (m, 8H), 3.28 (s, 3H), 2.83 - 2.60 (m, 4H), 1.16 -1.05 (m, 12H). 31 P NMR (162 MHz, DMSO-d6) δ 148.83 (s, 1P).

[0193] 8. Synthesis of YK-NUM-107

[0194]

[0195] Step 1: Synthesis of YK-NUM-107-PM1

[0196] YK-NUM-106-PM1 (7.24 g, 16.66 mmol) was dissolved in ultra-dry acetonitrile (72.0 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (11.41 g, 30.00 mmol), N,N-diisopropylethylamine (3.69 g, 28.55 mmol) and diethylamine (1.53 g, 20.92 mmol) were added to the above solution in an ice bath. The mixture was stirred at room temperature for 15 h under nitrogen protection. TLC showed that the reaction was complete. The reaction solution was concentrated under vacuum, and the concentrated oil was added with EA (100.0 mL) to dissolve, washed with saturated sodium bicarbonate solution (50.0 mL), washed with saturated brine (50.0 mL), and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the yellow liquid target product YK-NUM-107-PM1 (3.20 g, 6.53 mmol, 39.20%).

[0197] Step 2: Synthesis of YK-NUM-107-PM2

[0198] Uracil (2.56 g, 22.84 mmol), ammonium sulfate (172.6 mg, 1.31mmol), pyridine (12.8 mL), hexamethyldisilazane (64.0 mL) were added to the reaction bottle in sequence, and activated at 130°C for 1.5 h under nitrogen protection. The above solution was cooled and concentrated under vacuum to obtain a white solid. A solution of YK-NUM-107-PM1 (3.20 g, 6.53 mmol) in acetonitrile (64.0 mL) was added to the above white solid. Anhydrous tin tetrachloride (7.66 g, 29.40 mmol) was added dropwise to the above solution under ice bath. After the addition was completed, the reaction was stirred under ice bath for 2 h, and then transferred to 37°C for 24 h. TLC showed that the reaction was complete. The reaction solution was placed under ice bath and saturated sodium bicarbonate was added dropwise to adjust the pH to 7~8 to quench the reaction, and an equal volume of dichloromethane was added and stirred. The insoluble matter was filtered off, the organic phase was separated, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-107-PM2 (1.43 g, 2.51 mmol, 38.41%) as an off-white solid. 26 H 47 N3O7Si2,MS(ES): m / z(M+H + )570.3.

[0199] Step 3: Synthesis of YK-NUM-107-PM3

[0200] YK-NUM-107-PM2 (1.43 g, 2.51 mmol), ultra-dry THF (1.0 mL), and 1 M TBAF in THF (1.2 mL, 1.20 mmol) were added to the reaction flask in sequence. The reaction was stirred at room temperature for 4 h under nitrogen protection. MS showed that the reaction was complete. The reaction solution was concentrated under vacuum. The residue was purified by C18 reverse column chromatography (acetonitrile / water) to obtain the target product YK-NUM-107-PM3 (668.5 mg, 2.04 mmol, 81.38%) as a white solid. 14 H 21 N3O6, MS (ES): m / z (M+H + ) 328.1.

[0201] Step 4: Synthesis of YK-NUM-107-PM4

[0202] YK-NUM-107-PM3 (668.5 mg, 2.04 mmol), ultra-dry pyridine (3 mL), and DMTrCl (899.6 mg, 2.66 mmol) were added to the reaction bottle in sequence. The mixture was stirred at room temperature for 2 h under nitrogen protection. LCMS showed that the reaction was complete. The reaction solution was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-107-PM4 (802.0 mg, 1.27 mmol, 62.36%) as a white solid. 35 H 39 N3O8, MS (ES): m / z (M+H + ) 630.3.

[0203] Step 5: Synthesis of YK-NUM-107

[0204] YK-NUM-107-PM4 (802.0 mg, 1.27 mmol), ultra-dry dichloromethane (8 mL), N,N-diisopropylethylamine (445.2 mg, 3.44 mmol), N-methylimidazole (94.3 mg, 1.15 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (815.2 mg, 3.44 mmol) were added to the reaction bottle in sequence, and the reaction was stirred at room temperature for 2.5 h under nitrogen protection. LCMS showed that the reaction was complete. 8.0 mL of saturated sodium bicarbonate solution was added to the above reaction solution to quench the reaction. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-107 (663.0 mg, 0.80 mmol, 62.72%) as a white solid. C 44 H 56 N5O9P, MS (ES): m / z (M+H + ) 830.4.

[0205] YK-NUM-107: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.41 (d, J = 8.6, 1H), 7.28 - 7.24 (m, 9H), 6.89 (d, J = 7.2 Hz, 4H), 6.15 (d, J = 7.8 Hz, 1H), 5.46(d, J = 8.4 Hz, 1H), 4.61 - 4.55 (m, 1H), 4.15 - 4.13 (m, 1H), 3.92 (t, J =10.8 Hz, 2H), 3.81 (s, 6H), 3.61 - 3.40 (m, 7H), 2.83 - 2.60 (m, 4H), 1.16 -1.05 (m, 18H). 31 P NMR (162 MHz, DMSO-d6) δ 148.88 (s, 1P).

[0206] 9. Synthesis of YK-NUM-108

[0207]

[0208] Step 1: Synthesis of YK-NUM-108-PM1

[0209] INT-I (5.00 g, 11.89 mmol), triphenylphosphine (6.24 g, 23.79 mmol), phthalimide (2.10 g, 14.27 mmol) were dissolved in tetrahydrofuran (75.0 mL), cooled to 0°C in an ice-water bath, and DIAD (3.61 g, 17.85 mmol) was added to the above system. After the addition was completed, the mixture was heated to room temperature and stirred for 2 hours. After the reaction was completed, the reaction mixture was concentrated under vacuum, extracted with EA (100 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 0-20%) to obtain YK-NUM-108-PM1 (5.87 g, 10.68 mmol, 89.83%). 27 H 43 NO7Si2, MS(ES): m / z(M+H + ) 550.3.

[0210] Step 2: Synthesis of YK-NUM-108-PM2

[0211] Dissolve YK-NUM-108-PM1 (5.87 g, 10.68 mmol) and 85% hydrazine hydrate (10.69 g, 213.60mmol) in ethanol (120.0 mL), stir in an oil bath at 80°C for 1 hour under nitrogen atmosphere. After the reaction is completed, the reaction mixture is concentrated under vacuum, extracted with EA (160 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under vacuum to remove the solvent to obtain YK-NUM-108-PM2 (4.19 g, 9.98 mmol, 93.51%). 19 H 41 NO5Si2, MS(ES):m / z(M+H + ) 420.3.

[0212] Step 3: Synthesis of YK-NUM-108-PM3

[0213] YK-NUM-108-PM2 (4.19 g, 9.98 mmol) and triethylamine (3.03 g, 29.94 mmol) were dissolved in dichloromethane (85.0 mL). Under nitrogen atmosphere, the temperature was cooled to 0°C in an ice-water bath. Acetyl chloride (1.18 g, 15.03 mmol) was added to the above system. After the addition was completed, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was completed, the reaction mixture was concentrated under vacuum, extracted with DCM (150 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 0-80%) to obtain YK-NUM-108-PM3 (4.56 g, 9.88mmol, 98.92%). 21 H 43 NO6Si2, MS(ES): m / z(M+H + ) 462.3.

[0214] Step 4: Synthesis of YK-NUM-108-PM4

[0215] Dissolve uracil (2.77 g, 24.71 mmol) and ammonium sulfate (261.1 mg, 1.98 mmol) in pyridine (20.0 mL) and hexamethyldisilazane (100.0 mL), activate in an oil bath at 130°C for 1.5 h under nitrogen protection, cool down, and concentrate under vacuum to obtain a white solid. Then add YK-NUM-108-PM3 (4.56 g, 9.88 mmol) and acetonitrile (50.0 mL), cool to 0°C in an ice-water bath under nitrogen atmosphere, slowly add anhydrous tin tetrachloride (6.43 g, 24.68 mmol), and stir and react for 21 h. After the reaction was completed, the reaction solution was quenched with saturated sodium bicarbonate in an ice bath, filtered, washed with saturated brine (80 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure in vacuo, and the residue was purified by silica gel chromatography (dichloromethane / ethyl acetate = 0-90%) to obtain YK-NUM-108-PM4 (3.72 g, 6.87 mmol, 69.53%). 24 H 43 N3O7Si2, MS(ES): m / z(M+H + ) 542.3.

[0216] Step 5: Synthesis of YK-NUM-108-PM5

[0217] Dissolve YK-NUM-108-PM4 (3.72 g, 6.87 mmol) in tetrahydrofuran (37.0 mL), cool to 0°C in an ice-water bath under nitrogen atmosphere, slowly drop 1 M TBAF in THF (27.5 ml, 27.50 mmol) into the above system, and stir and react at room temperature for 22 h after the addition is complete. After the reaction is complete, the reaction mixture is concentrated under vacuum, and the residue is purified by C18 reverse column chromatography (acetonitrile / water) to obtain YK-NUM-108-PM5 (1.68 g, 5.61 mmol, 81.76%). 12 H 17 N3O6, MS(ES): m / z(M+H + ) 300.1.

[0218] Step 6: Synthesis of YK-NUM-108-PM6

[0219] Dissolve YK-NUM-108-PM5 (1.68 g, 5.61 mmol) and DMTrCl (3.06 g, 9.03 mmol) in pyridine (20.0 mL). After the addition is complete, stir at room temperature under nitrogen atmosphere for 2 h. After the reaction is complete, the reaction mixture is concentrated under vacuum, then washed with EA (80.0 mL) and saturated brine (15 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue is purified by silica gel chromatography (dichloromethane / methanol = 0-3%) to obtain YK-NUM-108-PM6 (2.45 g, 4.07 mmol, 72.54%). 33 H 35 N3O8, MS(ES): m / z(M+H + ) 602.2.

[0220] Step 7: Synthesis of YK-NUM-108

[0221] YK-NUM-108-PM6 (1.30 g, 2.16 mmol), DIEA (837.5 mg, 6.48 mmol), and N-methylimidazole (177.4 mg, 2.16 mmol) were dissolved in dichloromethane (13.0 mL). Under nitrogen atmosphere, the temperature was cooled to 0°C in an ice-water bath. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.53 g, 6.46 mmol) was added to the above system. After the addition was completed, the temperature was raised to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 0-100%) to obtain YK-NUM-108 (1.10 g, 1.37 mmol, 63.49%). 42 H 52 N5O9P, MS(ES): m / z(M+H + )802.4.

[0222] YK-NUM-108: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.84 (d, J = 6.8, 1H), 7.68 - 7.65 (m, 9H), 7.29 (d, J = 7.2 Hz, 4H), 6.55 (d, J = 7.8 Hz, 1H), 5.87(d, J = 8.4 Hz, 1H), 5.00 (d, J = 8.6 Hz, 1H), 4.55 (dd, J = 10.4 Hz, 1H), 4.40 (t, J = 10.8 Hz, 2H), 4.21 (s, 6H), 4.01 - 3.64 (m, 6H), 3.23 - 3.20 (m,2H), 3.01 (t, J = 12.4 Hz, 2H), 1.46 - 1.39 (m, 15H). 31 P NMR (162 MHz, DMSO-d6) δ 148.78 (s, 1P).

[0223] 10. Synthesis of YK-NUM-109

[0224]

[0225] Step 1: Synthesis of YK-NUM-109-PM1

[0226] YK-NUM-106-PM1 (7.24 g, 16.66 mmol) was dissolved in ultra-dry acetonitrile (72.0 mL) as the raw material. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (11.41 g, 24.71 mmol), N,N-diisopropylethylamine (3.69 g, 28.55 mmol) and 2 M ethylamine (10.46 mL, 20.92 mmol) were added to the above solution in an ice bath. The mixture was stirred at room temperature for 15 h under nitrogen protection. TLC showed that the reaction was complete. The reaction solution was concentrated under vacuum, and the concentrated oil was dissolved in EA, washed with saturated sodium bicarbonate solution, washed with saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-109-PM1 (4.00 g, 8.66 mmol, 50.75%) as a white solid. 21 H 43 NO6Si2, MS(ES): m / z(M+H + ) 462.3.

[0227] Step 2: Synthesis of YK-NUM-109-PM2

[0228] Uracil (3.40 g, 30.33 mmol), ammonium sulfate (229.1 mg, 1.73mmol), pyridine (16.0 mL), hexamethyldisilazane (80.0 mL) were added to the reaction flask in sequence, and activated at 130°C for 2 h under nitrogen protection. The above solution was cooled and concentrated under vacuum to obtain a white solid. A solution of YK-NUM-109-PM1 (4.00 g, 8.66 mmol) in acetonitrile (80.0 mL) was added to the above white solid. Tin tetrachloride (10.16 g, 39.00 mmol) was added dropwise to the above solution under ice bath. After the addition was completed, the reaction was stirred under ice bath for 2 h, and then transferred to 37°C for 24 h. TLC showed that the reaction was complete. The reaction solution was placed under ice bath and saturated sodium bicarbonate was added dropwise to quench the reaction, the pH was adjusted to 7~8, and an equal volume of dichloromethane was added and stirred. The insoluble matter was filtered off, the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-109-PM2 (2.24 g, 4.13 mmol, 47.73%) as an off-white solid. 24 H 43 N3O7Si2, MS(ES): m / z(M+H+ )542.3.

[0229] Step 3: Synthesis of YK-NUM-109-PM3

[0230] YK-NUM-109-PM2 (2.24 g, 4.13 mmol), ultra-dry THF (20.0 mL), and 1 M TBAF in THF (16.5 mL, 16.50 mmol) were added to the reaction flask in sequence. The mixture was stirred at room temperature for 16 h under nitrogen protection. MS showed that the reaction was complete. The reaction solution was concentrated under vacuum. The residue was purified by C18 reverse column chromatography (acetonitrile / water) to obtain the target product YK-NUM-109-PM3 (1.06 g, 3.54 mmol, 85.67%) as a white solid. 12 H 17 N3O6, MS(ES): m / z(M+H + ) 300.1.

[0231] Step 4: Synthesis of YK-NUM-109-PM4

[0232] YK-NUM-109-PM3 (1.06 g, 3.54 mmol), ultra-dry pyridine (3.0 mL), and DMTrCl (1.92 g, 5.67 mmol) were added to the reaction flask in sequence. The mixture was stirred at room temperature for 2 h under nitrogen protection. MS showed that the reaction was complete. The reaction solution was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-109-PM4 (926.4 mg, 1.54 mmol, 43.47%) as a white solid. 33 H 35 N3O8, MS(ES): m / z(M+H + ) 602.2.

[0233] Step 5: Synthesis of YK-NUM-109

[0234] YK-NUM-109-PM4 (926.4 mg, 1.54 mmol), ultra-dry dichloromethane (9.0 mL), N,N-diisopropylethylamine (537.4 mg, 4.16 mmol), N-methylimidazole (113.8 mg, 1.39 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (984.1 mg, 4.16 mmol) were added to the reaction bottle in sequence. The reaction was stirred at room temperature for 2.5 h under nitrogen protection. MS showed that the reaction was complete. 8.0 mL of saturated sodium bicarbonate solution was added to the above reaction solution to quench the reaction. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-109 (754.6 mg, 0.94 mmol, 61.11%) as a white solid. C 42 H 52 N5O9P, MS(ES): m / z(MH + ) 802.4.

[0235] YK-NUM-109: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.84 (d, J = 6.8, 1H), 7.68 - 7.65 (m, 9H), 7.29 (d, J = 7.2 Hz, 4H), 6.55 (d, J = 7.8 Hz, 1H), 5.87(d, J = 8.4 Hz, 1H), 5.00 (d, J = 8.6 Hz, 1H), 4.55 (dd, J = 10.4 Hz, 1H), 4.40 (t, J = 10.8 Hz, 2H), 4.21 (s, 6H), 4.01 - 3.64 (m, 5H), 3.23 - 3.20 (m,2H), 3.01 (t, J = 12.4 Hz, 2H), 1.46 - 1.39 (m, 15H). 31 P NMR (162 MHz, DMSO-d6) δ 148.85 (s, 1P).

[0236] 11. Synthesis of Compound 29

[0237]

[0238] Step 1: Synthesis of compound 29-PM1

[0239] INT-I (8.00 g, 19.01 mmol), DMAP (6.97 g, 57.04mol), and DCM (80.0 mL) were added to the reaction flask in sequence. A solution of acetic anhydride (3.88 g, 38.03 mmol) in pyridine (5.0 mL) was slowly added under nitrogen protection, and the reaction was carried out at room temperature for 3 h. TLC showed that the reaction was complete. After quenching with water under an ice bath, the mixture was concentrated to dryness, and ethyl acetate and water were added for extraction. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product compound 29-PM1 (8.26 g, 17.85 mmol, 93.87%) as a colorless transparent liquid. 21 H 42 O7Si2, MS(ES): m / z(M+H + ) 463.3.

[0240] Step 2: Synthesis of compound 29-PM2

[0241] N4-acetylcytosine (8.20 g, 53.55 mmol), ammonium sulfate (0.47 g, 3.57 mmol), pyridine (20.0 mL), HDMS (100.0 mL), compound 29-PM1 (8.26 g, 17.85 mmol), tin tetrachloride (13.95 g, 53.55 mmol), acetonitrile (50.0 mL) were added according to the synthetic method of YK-NUM-104-PM1 to obtain the target product compound 29-PM2 (5.63 g, 9.64 mmol, 54.02%) as an off-white solid. 26 H 45 N3O8Si2, MS(ES): m / z(M+H + ) 584.3.

[0242] Step 3: Synthesis of compound 29-PM3

[0243] Compound 29-PM2 (5.63 g, 9.64 mmol), ultra-dry THF (50.0 mL), and 1 M TBAF THF solution (28.9 mL, 28.92 mmol) were added according to the synthetic method of YK-NUM-104-PM2 to obtain the target product compound 29-PM3 (2.81 g, 8.23 ​​mmol, 85.37%) as a white solid. 14 H 19 N3O7, MS(ES): m / z(M+H + ) 342.1.

[0244] Step 4: Synthesis of compound 29-PM4

[0245] Compound 29-PM3 (2.81 g, 8.23 ​​mmol), ultra-dry pyridine (10.0 mL), and DMTrCl (3.35 g, 9.88 mmol) were added according to the synthetic method of YK-NUM-104-PM3 to obtain the target product compound 29-PM4 (2.56 g, 3.98 mmol, 48.31%) as a white solid. 35 H 37 N3O9, MS(ES): m / z(M+H + ) 644.3

[0246] Step 5: Synthesis of compound 29

[0247] Compound 29-PM4 (2.56 g, 3.98 mmol), ultra-dry dichloromethane (25.0 mL), N,N-diisopropylethylamine (1.54 g, 11.94 mmol), N-methylimidazole (326.8 mg, 3.98 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (2.83 g, 11.94 mmol) were prepared according to the synthetic method of YK-NUM-104 to obtain the target product compound 29 (1.24 g, 1.47 mmol, 36.95%) as a white solid. 44 H 54 N5O 10 P, MS(ES): m / z(M+H + ) 844.4.

[0248] Compound 29: 1H NMR (CDCl3, 400 MHz, 298 K) δ 8.99 (d, J = 8.2, 1H), 7.35- 7.30 (m, 9H), 6.93 (d, J = 7.0 Hz, 4H), 5.79 (d, J = 7.6 Hz, 1H), 5.57 (d,J = 8.2 Hz, 1H), 4.66 - 4.64 (m, 1H), 4.53 - 4.28 (m, 2H), 3.98 (t, J = 8.8,2H), 3.85 (s, 6H), 3.65 - 3.40 (m, 3H), 3.10 - 2.87 (m, 3H), 2.64 (t, J = 7.6Hz, 2H), 2.08 (s, 3H), 1.90 (s, 3H), 1.10 (m, 12H). 31 P NMR (162 MHz, DMSO-d6)δ 148.96 (s, 1P).

[0249] 12. Synthesis of NA0133

[0250]

[0251] Step 1: Synthesis of NA0133-PM1

[0252] INT-I (8.00 g, 19.01 mmol) and THF (80.0 mL) were added to the reaction flask in sequence. NaH (1.37 g, 57.03 mmol) was slowly added in batches under an ice bath and reacted at room temperature for 16 h. TLC showed that the reaction was complete. The reaction solution was placed under an ice bath and saturated brine was added dropwise to quench the reaction. An equal volume of ethyl acetate was added. After separation, the aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the target product NA0133-PM1 (6.36 g, 9.86mmol, 51.84%) as an oily liquid. 35 H 72 O6Si2, MS(ES): m / z(M+H + ) 645.5.

[0253] Step 2: Synthesis of NA0133-PM2

[0254] Uracil (3.32 g, 29.58 mmol), ammonium sulfate (0.26 g, 1.97 mmol), pyridine (15.0 mL), HDMS (75.0 mL), NA0133-PM1 (6.36 g, 9.86 mmol), tin tetrachloride (7.71 g, 29.58 mmol), acetonitrile (80.0 mL) were added according to the synthetic method of YK-NUM-104-PM1 to obtain the target product NA0133-PM2 (3.83 g, 5.28 mmol, 53.57%) as an off-white solid. 38 H 72 N2O7Si2, MS(ES): m / z(M+H + ) 725.5.

[0255] Step 3: Synthesis of NA0133-PM3

[0256] NA0133-PM2 (3.83 g, 5.28 mmol), ultra-dry THF (40.0 mL), 1 M TBAF in THF solution (16.0 mL, 15.84 mmol) were added according to the synthetic method of YK-NUM-104-PM2 to obtain the target product NA0133-PM3 (1.98 g, 4.10 mmol, 77.67%) as a white solid. 26 H 46 N2O6, MS(ES): m / z(M+H + ) 483.3.

[0257] Step 4: Synthesis of NA0133-PM4

[0258] NA0133-PM3 (1.98 g, 4.10 mmol), ultra-dry pyridine (20.0 mL), and DMTrCl (2.22 g, 6.56 mmol) were added according to the synthetic method of YK-NUM-104-PM3 to obtain the target product NA0133-PM4 (1.49 g, 1.90 mmol, 46.27%) as a white solid. 47 H 64 N2O8, MS(ES): m / z(M+H + ) 785.5.

[0259] Step 5: Synthesis of NA0133

[0260] NA0133-PM4 (1.49 g, 1.90 mmol), ultra-dry dichloromethane (15.0 mL), N,N-diisopropylethylamine (736.7 mg, 5.70 mmol), N-methylimidazole (156.0 mg, 1.90 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.35 g, 5.70 mmol) were prepared according to the synthetic method of YK-NUM-104 to obtain the target product NA0133 (812.6 mg, 0.82 mmol, 43.45%) as a white solid. 56 H 81 N4O9P, MS(ES): m / z(M+H + ) 985.6.

[0261] NA0133: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.46 (d, J = 8.2, 1H), 7.33 -7.31 (m, 9H), 6.94 (d, J = 7.0 Hz, 4H), 5.80 (d, J = 7.6 Hz, 1H), 5.52 (d, J= 8.2 Hz, 1H), 4.65 (d, J = 8.4, 1H), 3.95 (t, J = 8.6, 2H), 3.86 (s, 6H), 3.40 - 3.22 (m, 7H), 2.88 (m, 2H), 2.72 - 2.65 (m, 3H), 1.55 - 1.48 (m, 4H),1.36 - 1.31 (m, 24H), 1.11 (s, 12H), 0.93 (s, 3H). 31 P NMR (162 MHz, DMSO-d6)δ 148.68 (s, 1P).

[0262] 13. Synthesis of Compound 18c

[0263]

[0264] Step 1: Synthesis of compound 18c-PM1

[0265] YK-NUM-108-PM2 (14.00 g, 33.34 mmol), DMAP (12.22 g, 100.06 mol), DCM (140.0 mL), trifluoroacetic anhydride (21.00 g, 100.02 mmol), and pyridine (20.0 mL) were added according to the synthetic method of compound 29-PM1 to obtain the target product compound 18c-PM1 (7.24 g, 14.04 mmol, 42.09%) as a transparent liquid. 21 H 40 F3NO6Si2, MS(ES): m / z(M+H + ) 516.2.

[0266] Step 2: Synthesis of compound 18c-PM2

[0267] Thymine (5.32 g, 42.12 mmol), ammonium sulfate (0.37 g, 2.80 mmol), pyridine (20.0 mL), HDMS (100.0 mL), compound 18c-PM1 (7.24 g, 14.04 mmol), tin tetrachloride (10.98 g, 42.12 mmol), acetonitrile (120.0 mL) were added according to the synthetic method of YK-NUM-104-PM1 to obtain the target product compound 18c-PM2 (3.83 g, 6.28 mmol, 44.74%) as an off-white solid. 25 H 42 F3N3O7Si2, MS(ES): m / z(M+H + ) 610.3.

[0268] Step 3: Synthesis of compound 18c-PM3

[0269] Compound 18c-PM2 (3.83 g, 6.28 mmol), ultra-dry THF (40.0 mL), and 1 M TBAF THF solution (19.0 mL, 18.84 mmol) were added according to the synthetic method of YK-NUM-104-PM2 to obtain the target product compound 18c-PM3 (1.84 g, 5.01 mmol, 79.76%) as a white solid. 13 H 16 F3N3O6, MS(ES): m / z(M+H + ) 368.1.

[0270] Step 4: Synthesis of compound 18c-PM4

[0271] Compound 18c-PM3 (1.84 g, 5.01 mmol), ultra-dry pyridine (18.0 mL), and DMTrCl (2.72 g, 8.02 mmol) were added according to the synthetic method of YK-NUM-104-PM3 to obtain the target product compound 18c-PM4 (1.24 g, 1.85 mmol, 36.96%) as a white solid. 34 H 34 F3N3O8, MS(ES): m / z(M+H + ) 670.2.

[0272] Step 5: Synthesis of compound 18c

[0273] Compound 18c-PM4 (1.24 g, 1.85 mmol), ultra-dry dichloromethane (13.0 mL), N,N-diisopropylethylamine (717.3 mg, 5.55 mmol), N-methylimidazole (151.9 mg, 1.85 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.31 g, 5.55 mmol) were prepared according to the synthetic method of YK-NUM-104 to obtain the target product compound 18c (754.3 mg, 0.87 mmol, 46.83%) as a white solid. 43 H 51 F3N5O9P, MS(ES): m / z(M+H + )870.3.

[0274] Compound 18c: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.30 - 7.21 (m, 10H), 6.91(d, J = 7.6 Hz, 4H), 5.77 (d, J = 7.8 Hz, 1H), 4.62 (d, J = 8.4 Hz, 1H), 3.92(d, J = 8.8 Hz, 2H), 3.83 (s, 6H), 3.77 - 3.38 (m, 3H), 3.11 - 3.02 (m, 3H), 2.83 (t, J = 10.8 Hz, 2H), 2.62 (t, J = 11.8 Hz, 2H), 2.36 (s, 3H), 1.08 (s,12H). 31 P NMR (162 MHz, DMSO-d6) δ 148.58 (s, 1P).

[0275] Example 2: Synthesis of oligonucleotide sequences

[0276] Synthesis method:

[0277] Using nucleoside phosphoramidite monomers as raw materials, the modified nucleosides in Table 1 were embedded into the corresponding oligonucleotide sequences using the standard phosphoramidite method on a nucleic acid automatic synthesizer to obtain oligonucleotides ON1-7 (Table 2).

[0278] Table 1 Structures of modified nucleosides

[0279]

[0280]

[0281] It can be seen from Table 1 that the chemical structures of the compounds YK-NUM-101~109 of the present application are somewhat similar, while others are quite different. Compared with the modified nucleosides disclosed in the prior art, some of the compounds in this series have similar structures, while others are quite different.

[0282] Using different modified nucleosides as raw materials, the sense strands and antisense strands of double-stranded oligonucleotide (dsRNA) molecules with different modifications are synthesized, as shown in Table 2. The synthesized sense strands and antisense strands are annealed and purified to obtain the corresponding small interfering oligonucleotide siRNA.

[0283] The basic sequences of the small interfering RNA in Table 2 are:

[0284] Sense strand: SEQ ID NO: 1: 5'-CUUUUGUAACUUGAAGAUAUU-3'

[0285] Antisense strand: SEQ ID NO: 2: 5'-AAUAUCUUCAAGUUACAAAAGCA-3'

[0286] The above sequences are derived from the small interfering oligonucleotide siRNA targeted to regulate PCSK9 gene expression reported in CN117210468B, wherein sequence ON1 in Table 2 is the sequence numbered P92-si81 D81-DV25 in the patent, and sequence ON12 in Table 2 is the sequence numbered P92-si81 D81-DV25+ in the patent.

[0287] Instruments and reagents: Qingke 192 P model DNA / RNA automatic synthesizer, whose solid phase carrier is a universal carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (cytiva manufacturer).

[0288] Preparation method:

[0289] According to the monomer concentration of 0.15M, the following nucleotide monomer solutions were prepared with acetonitrile: DMT-A-OMe phosphoramidite monomer, DMT-C-OMe phosphoramidite monomer, DMT-G-OMe phosphoramidite monomer, DMT-U-OMe phosphoramidite monomer, DMT-AF phosphoramidite monomer, DMT-CF phosphoramidite monomer, DMT-GF phosphoramidite monomer, DMT-UF phosphoramidite monomer (the structures of the above eight monomers refer to patent CN117210468), compound 29 phosphoramidite monomer, NA0133 Phosphoramidite monomer, compound 18c phosphoramidite monomer, YK-NUM-101 phosphoramidite monomer, YK-NUM-102 phosphoramidite monomer, YK-NUM-103 phosphoramidite monomer, YK-NUM-104 phosphoramidite monomer, YK-NUM-105 phosphoramidite monomer, YK-NUM-106 phosphoramidite monomer, YK-NUM-107 phosphoramidite monomer, YK-NUM-108 phosphoramidite monomer, YK-NUM-109 phosphoramidite monomer, (S)-GNA-U phosphoramidite monomer.

[0290] Prepared by the following steps:

[0291] (1) Deprotection

[0292] 3% dichloroacetic acid toluene solution was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0293] (2) Coupling

[0294] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolyl as an activating agent, followed by washing with acetonitrile.

[0295] (3) Oxidation / sulfurization

[0296] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (90 / 10) as the oxidant, followed by rinsing with acetonitrile.

[0297] Sulfurization: Use 3% hydrogenated xanthan gum in pyridine as the sulfurizing agent for sulfurization, and then use acetonitrile for washing.

[0298] (4) Hydroxyl protection

[0299] Hydroxyl protection was performed using 10% acetic anhydride tetrahydrofuran solution (CAP A) and tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting reagents, followed by rinsing with acetonitrile.

[0300] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[0301] (5) Use 3% dichloroacetic acid toluene solution as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile for washing.

[0302] (6) Aminolysis and purification

[0303] The solid phase carrier is transferred to the reactor, concentrated ammonia water (25-28%) is added, and after maintaining the aminolysis at 60°C for 12 hours, the system is cooled to room temperature and the mixture is transferred to a filter press, eluted with a mixed solution of purified water and ethanol, the filtrate is combined, passed through a chromatography column, concentrated, and freeze-dried to obtain the product.

[0304] (7) Annealing

[0305] The purified sense strand and antisense strand were mixed in a 1:1 ratio, heated to 95°C and maintained for 3 min, then slowly cooled to room temperature to form a double strand.

[0306] The other sequences listed in Table 2 were synthesized according to the above method.

[0307] Table 2 Sequences of double-stranded oligonucleotides (dsRNA) with different modifications and their corresponding mass spectra

[0308]

[0309]

[0310]

[0311] Note: m = 2'-OMe, f = 2'-F, s = 3'-phosphorothioate, ms = 2'-OMe, 3'-phosphorothioate; fs = 2'-F, 3'-phosphorothioate, G num-101 : ; A num-102 s: ; U num-103 : ; C num-104 : ; U num-105 : ; U num-106 : ; U num-107 : ; U num-108 : ; U num-109 : ; Cgna: ;Ugna: ; C 29 : ; U NA0133 : ; T 18c : , bold letters represent modification sites, and subscripts correspond to the modified nucleoside monomer numbers. The phosphoramidite monomers in the table were used for sequence synthesis.

[0312] Example 3: Thermal stability experiment (Tm)

[0313] Experimental methods:

[0314] Annealing Buffer: 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 8 mM KH2PO4, pH 7.4.

[0315] Annealing method: The two oligonucleotide single strands were diluted with annealing buffer to a final concentration of 2 μM, heated in a 95°C water bath for 5 min, slowly cooled to room temperature, and placed in a 4°C refrigerator overnight.

[0316] Melting temperature (Tm) determination method: Add 800 μL of the sample to be tested to the cuvette and cover it with a heat-insulating lid. Select 15°C as the starting temperature, 90°C as the ending temperature, the temperature rise rate is 1°C / min, the A260 reading rate is 2 times / °C, and the instrument gives the Tm value. Repeat the measurement 3 times for each sample, and take the average value as the final result.

[0317] Experimental results:

[0318] The specific experimental results are shown in Table 3 and Figure 1 .

[0319] Table 3 Comparison of thermal stability of double-stranded small interfering oligonucleotide sequences with different modifications

[0320]

[0321]

[0322]

[0323] As shown in Table 3, ON1 was conventionally modified with 2'-F and 2'-OMe groups, and the remaining 14 siRNA double strands were introduced with the modified nucleoside monomers of the present invention and commercially available ethylene glycol nucleoside monomers (gna), etc. The above-mentioned modified monomers were introduced into the antisense strand of siRNA, and the double strands formed after annealing had significantly lower melting temperatures compared with the conventionally modified ON1.

[0324] For example, siRNA double-stranded ON1, ON4, ON7, ON8, and ON10, different functional group modifications were introduced at the same position of the antisense strand, that is, the seventh uridine sugar ring at the 5'-end of the seed region. Among them, ON1 was modified with conventional 2'-Ome, and its melting temperature was 74.4℃; ON4 was modified with 2'-CH2OCH3, and its melting temperature was 65.2℃, which was 9.2℃ lower than that of ON1; ON7 was modified with -C(O)NHCH2CH2OCH3, and its melting temperature was 64.5℃, which was 9.9℃ lower than that of ON1; ON8 was modified with -C(O)N(CH2CH3)2, and its melting temperature was 62.9℃, which was 11.5℃ lower than that of ON1; ON10 was modified with -C(O)NHCH2CH3, and its melting temperature was 63.3℃, ​​which was 9.1℃ lower than that of ON1.

[0325] Example 4: Circular dichroism study

[0326] Experimental methods:

[0327] Annealing buffer: 10 mM Na3PO4, 100 mM NaCl, pH 7.2.

[0328] Annealing method: The two oligonucleotide single strands were diluted with annealing buffer to a final concentration of 2 μM, heated in a 95°C water bath for 5 min, slowly cooled to room temperature, and placed in a 4°C refrigerator overnight.

[0329] The circular dichroism spectrometer was used for measurement, with a scanning range of 200-350nm, a scanning speed of 50nm / min, a scanning interval of 0.5nm, a cuvette optical path of 1mm, and a measurement temperature of 15°C. Each sample was scanned three times in a row and the average value was automatically taken, and the instrument's built-in software was used to smooth the data before drawing the graph. The test results are shown in Figure 1. Figure 2 shown.

[0330] Experimental results:

[0331] As can be seen from the CD diagram, the fully modified siRNA chains, including ON1, ON2, ON3, ON4, ON5, ON6, ON7, ON8, ON9, ON10, ON11, ON12, ON13, ON14, ON15 complementary RNA double chains, all have typical A-form conformational characteristics, with a maximum negative absorption peak (trough) near ~210nm, a maximum positive absorption peak (peak) between 260nm-280nm, and a strong peak signal. The present invention has monomers modified with different functional groups embedded in the antisense oligonucleotide chain without affecting the double-stranded formation ability of the complementary RNA chain (ssRNA), and the embedded modified nucleoside monomers at different positions (5' end, seed region or middle position) of the antisense oligonucleotide chain also do not affect the double-stranded formation ability of the complementary RNA chain (ssRNA). For example, siRNA double strands ON1, ON2, ON3, wherein YK-NUM-101 phosphoramidite monomer is embedded in the middle of ON2 antisense strand, and YK-NUM-102 phosphoramidite monomer is embedded in the 5' end of ON3 antisense strand, but these three siRNA double strands have the same typical A-form conformation characteristics; siRNA double strands ON1, ON4, ON6, ON7, ON8, ON9, ON10, ON12, ON14, ON15 indicate that nucleoside monomers modified with different functional groups are used at the same position of siRNA antisense strand (the seventh position at the 5' end of the antisense strand seed region), and these 10 siRNA double strands also have typical A-form conformation characteristics, with a maximum negative absorption peak (trough) near 210nm, and a maximum positive absorption peak (peak) between 260nm-280nm. The siRNA antisense strand synthesized by the oligonuclear monomer modified by the present invention has good hybridization ability with its complementary RNA strand (ssRNA).

[0332] Example 5: Inhibitory effect of modified sequences on PCSK9 gene

[0333] After the 15 modified siRNA sequences synthesized in Example 2 were transfected into Hep3B cells via lipid nanoparticles (LNP), the inhibitory effect of each sequence on the PCSK9 gene was detected using qPCR technology.

[0334] 1. Experimental Materials

[0335] Test samples: modified small interfering RNA sequences listed in Table 2 (synthesized in Example 2).

[0336] Cell types: Hep3B cells were cultured in EMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010), 1% non-essential amino acid solution (Gibco-11140-050), and 1% GlutaMAX supplement (Gibco-35050-061).

[0337] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0338] 2. Experimental methods

[0339] qRT-PCR was used to detect the inhibitory effect of the test samples on the expression of PCSK9 gene mRNA in HEP3B cell line.

[0340] 2.1 Cell culture

[0341] Take the subcultured Hep3B cell line, and place the logarithmically growing cells in 10% fetal bovine serum RPMI1640 culture medium (supplemented with 100 μL / mL of penicillin and streptomycin), in a cell culture incubator at 37°C with 5% CO2, and change the medium once a day. Digest and subculture with 0.25% trypsin, centrifuge at 1000 r / min for 5 min, discard the supernatant, and add fresh culture medium for subculture.

[0342] 2.2 Cell transfection

[0343] Preparation of transfection mixture: Mix Lipofectamine RNAi MAX and Opti-MEM in a ratio of 1.5:98.5 and vortex to mix.

[0344] Preparation of transfection reagent: Add 60 μL of transfection mixture to 60 ml of siRNA solution diluted in Opti-MEM at a ratio of 1:1 (v / v), vortex and let stand at room temperature for 15 min to obtain lipid nanoparticles (LNP). Take 12.5 μL for encapsulation efficiency detection.

[0345] Take 2 μL siRNA (1 μg / μL) and add 18 μL sterile enzyme-free water according to 1:0.06 (wt / wt), mix well, and then add 22 μL of prepared LNP. Mix well and let stand at room temperature for 15 min. Take 12.5 μL for encapsulation efficiency detection. The final transfection volume per well = (1 μg theoretical sample volume / siRNA purity) / drug loading concentration, and the final siRNA concentration is 0.05 nM.

[0346] Negative control group: Add 22 μL of prepared LNP to 20 μL of sterile enzyme-free water, mix well, and let stand at room temperature for 15 minutes.

[0347] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well) to make the final siRNA concentration in each well 1 nM. Add 500 μL of cell suspension (6×10 cells per ml) 4 After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell culture incubator for 40 h.

[0348] 2.3 PCSK9 mRNA detection:

[0349] RNA extraction: a. Aspirate the culture medium in the 12-well plate, add 0.5 mL 1× PBS to each well to wash the cells, and aspirate the PBS. Add 0.5 mL TRIzol reagent to the wells, blow the cells with the pipette tip to fully lyse, transfer to a 1.5 mL RNase-free EP tube, and let stand at room temperature for 5 min.

[0350] b. Add 0.1 mL of chloroform to each tube, shake vigorously for 15 sec, and let stand at room temperature for 5 min. Centrifuge at 4°C, 12,000×g for 15 min, and take 200 μL of the supernatant into a new EP tube.

[0351] c. Add an equal volume of isopropanol, gently mix the liquid in the tube by inverting it upside down, let it stand at -20℃ for 10 min, centrifuge at 4℃, 12,000×g for 15 min, and discard the supernatant.

[0352] d. Add 0.5 mL of 75% ethanol, gently wash the RNA precipitate, centrifuge at 12,000 × g at 4°C for 5 min, and remove the supernatant. Repeat the rinse once, centrifuge at 12,000 × g at 4°C for 1 min, and remove the residual ethanol with a micropipette tip.

[0353] e. Dry the residual ethanol at room temperature for 2-3 min, then add 40 μL RNase-free ddH2O to dissolve.

[0354] RNA concentration detection: Use nanodrop to detect RNA concentration. Use 2 μL RNase-free ddH2O as blank control and 2 μL RNA sample for detection each time. Record the sample concentration.

[0355] PCSK9 mRNA quantitative detection: In a 15 mL centrifuge tube, add the remaining components except primers and template according to 7.5 × 84 = 630 portions, marked as A.

[0356] Label 84 1.5 mL EP tubes, add 77 μL of A and 420 ng of total RNA to each tube, mix well and set aside (labeled as B).

[0357] The upstream and downstream primers of the internal reference gene GAPDH and the target gene PCSK9 were mixed and set aside (marked as C).

[0358] Add 11 μL of B + 1 μL of C to each well of the PCR plate, cover with sealing film, centrifuge at 3000 rpm for 1 min, and put into the machine.

[0359] *This step is performed on ice to maintain low temperature conditions.

[0360] Place the plate into the qPCR instrument and run the following program:

[0361] Reverse transcription: 55°C, 15 min;

[0362] Pre-denaturation: 95°C, 30 sec;

[0363] Cyclic reaction: 95°C, 10 sec; 60°C, 35 sec; 40 cycles;

[0364] Melting curve: 95℃, 15 sec; 60℃, 60 sec; 95℃, 15 sec.

[0365] The running time is about 2h. Analyze the experimental results and calculate 2 -Δ Δ Ct .

[0366] 2.4 Data Processing

[0367] Expression rate = (PCSK9 mRNA expression in the experimental group / PCSK9 mRNA expression in the blank control group) × 100%;

[0368] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0369] 3. Experimental results

[0370] The inhibition rate in this example is the average of three experiments. The inhibition rate of each sequence on the expression of PCSK9 mRNA in Hep3B cells is shown in Table 4 and Figure 3 .

[0371] Table 4 The inhibition efficiency of PCSK9 gene by different modified small interfering oligonucleotide sequences

[0372]

[0373]

[0374]

[0375] As can be seen from the above table, at a concentration of 15 nM, the fully modified siRNA chains, including ON1, ON2, ON3, ON4, ON5, ON6, ON7, ON8, ON9, ON10, ON11, ON12, ON13, ON14, and ON15, have a significant inhibitory effect on the expression of PCSK9 mRNA in Hep3B cells. Among them, the specific position of the antisense chain in ON4, ON6, ON7, and ON8 (the seventh position from the 5' end of the seed region) adopts the modified nucleoside monomer designed by the present invention, and its inhibition efficiency on the PCSK9 gene exceeds 85%. Compared with ON12, the inhibition efficiency of the PCSK9 gene is significantly improved. Compared with the siRNA chains ON14 and ON15 that introduce the prior art nucleoside monomers (NA0133, 18c) at the specific position of the antisense chain, the inhibition efficiency of the PCSK9 gene is significantly improved.

[0376] 1) Embedding modified nucleoside monomers at different positions (5' end or middle position) of the antisense oligonucleotide chain, for example, siRNA double strands ON1, ON2, ON3, where YK-NUM-101 phosphoramidite monomer is embedded in the middle of the ON2 antisense chain, and YK-NUM-102 phosphoramidite monomer is embedded at the 5' end of the ON3 antisense chain. The inhibition efficiency of ON1 on the PCSK9 gene is 88.6%; the inhibition efficiency of ON2 on the PCSK9 gene is 77.5%, which is 11.1% lower than that of ON1; the inhibition efficiency of ON3 on the PCSK9 gene is 78.9%, which is 9.7% lower than that of ON1. It can be seen that compared with the 2'-OCH3 modification of the nucleoside sugar ring at different positions (5' end or middle position) of the ON1 antisense oligonucleotide chain, the nucleosides at the corresponding positions of ON2 and ON3 are modified with 2'-CH2OCH3. The change of the modification group leads to a decrease of about 10% in the inhibition efficiency of ON2 and ON3 on the PCSK9 gene;

[0377] 2) A modified nucleoside monomer is embedded in the sixth position of the 5' end of the seed region of the antisense oligonucleotide chain, for example, siRNA double strands ON1, ON5, ON11, ON13, wherein the YK-NUM-104 phosphoramidite monomer is embedded in the corresponding position of the ON5 antisense chain, the (S)-GNA-C phosphoramidite monomer is embedded in the position of ON11, and the phosphoramidite monomer of the prior art compound 29 is embedded in the position of ON13. The inhibition efficiency of ON1 on the PCSK9 gene is 88.6%; the inhibition efficiency of ON5 on the PCSK9 gene is 81.5%, which is 9.0% higher than ON11, 3.2% higher than ON13, and 7.0% lower than ON1; the inhibition efficiency of ON11 on the PCSK9 gene is 72.5%, which is 14.1% lower than ON1, and 9.0% lower than ON5; the inhibition efficiency of ON13 on the PCSK9 gene is 78.3%, which is 10.3% lower than ON1, and 3.2% lower than ON5. It can be seen that a nucleoside monomer containing different modification groups is embedded in the sixth position of the 5' end of the seed region of the antisense oligonucleotide chain. Compared with ON1, the nucleoside sugar ring at this position is modified with 2'-F, and the nucleoside at the corresponding position of ON5 is modified with 2'-CH2OCH3; the nucleoside at the corresponding position of ON13 is modified with 2'-CH2OC(O)CH3 of the prior art; ON11 is modified with ethylene glycol nucleoside with a large change in sugar ring structure. This shows that ON5 using the modified YK-NUM-104 phosphoramidite monomer has a significantly improved inhibition efficiency of the PCSK9 gene compared with ON11 and ON13 of the prior art.

[0378] 3) A modified nucleoside monomer is embedded in the seventh position of the 5' end of the seed region of the antisense oligonucleotide chain, for example, in the siRNA double-stranded ON1, ON4, ON6, ON7, ON8, ON9, ON10, ON12, ON14, ON15, a YK-NUM-103 phosphoramidite monomer is embedded in the corresponding position of the ON4 antisense chain, a YK-NUM-105 phosphoramidite monomer is embedded in the corresponding position of the ON6 antisense chain, and a YK-NUM-106 phosphoramidite monomer is embedded in the corresponding position of the ON7 antisense chain , the corresponding position of the ON8 antisense chain is embedded with a YK-NUM-107 phosphoramidite monomer, the corresponding position of the ON9 antisense chain is embedded with a YK-NUM-108 phosphoramidite monomer, the corresponding position of the ON10 antisense chain is embedded with a YK-NUM-109 phosphoramidite monomer, the corresponding position of the ON12 antisense chain is embedded with a (S)-GNA-U phosphoramidite monomer, the corresponding position of the ON14 antisense chain is embedded with a NA0133 phosphoramidite monomer, and the corresponding position of the ON15 antisense chain is embedded with a compound 18c phosphoramidite monomer.The inhibition efficiency of ON1 on PCSK9 gene was 88.6%; the inhibition efficiency of ON4 on PCSK9 gene was 85.7%, which was 2.9% lower than ON1, 2.4% higher than ON12, 33.5% higher than ON14, and 18.8% higher than ON15; the inhibition efficiency of ON6 on PCSK9 gene was 86.4%, which was 2.2% lower than ON1, 3.1% higher than ON12, 34.2% higher than ON14, and 19.5% higher than ON15; the inhibition efficiency of ON7 on PCSK9 gene was 86.2%, which was 2.4% lower than ON1, 2.9% higher than ON12, 34.0% higher than ON14, and 19.3% higher than ON15; the inhibition efficiency of ON8 on PCSK9 gene was 87.1%, which was 1.5% lower than ON1, 3.8% higher than ON12, and 19.5% higher than ON15. ON14 increased by 34.9%, which was 20.2% higher than ON15; ON9 had an inhibitory efficiency of 79.2% on PCSK9 gene, which was 9.4% lower than ON1, 5.1% lower than ON12, 27.0% higher than ON14, and 12.3% higher than ON15; ON10 had an inhibitory efficiency of 81.5% on PCSK9 gene, which was 7.1% lower than ON1, 2.8% lower than ON12, 29.3% higher than ON14, and 14.6% higher than ON15; it can be seen that nucleoside monomers containing different modification groups were embedded in the seventh position of the 5' end of the seed region of the antisense oligonucleotide chain. Compared with ON1, the nucleoside sugar ring at this position was modified with 2'-OCH3, and the corresponding position of ON4 was modified with 2'-CH2OCH3; the nucleoside at the corresponding position of ON6 was modified with 2'-CH2F; the nucleoside at the corresponding position of ON7 was modified with 2'- C(O)NHCH2CH2OCH3 modification; the nucleoside at the corresponding position of ON8 is modified with 2'- C(O)N(CH2CH3)2; the nucleoside at the corresponding position of ON9 is modified with 2'- CH2NHC(O)CH3; the nucleoside at the corresponding position of ON10 is modified with 2'- C(O)NHCH2CH3; ON12 is modified with ethylene glycol nucleoside with a large change in sugar ring structure; the nucleoside at the corresponding position of ON14 is modified with the existing technology 2'-CH2O (CH2). 15 CH3 modification; the nucleoside at the corresponding position of ON15 is modified by the prior art 2'- CH2NHC(O)CF3. Table 4 shows that the specific position of the antisense chain in ON4, ON6, ON7, and ON8 (the seventh position from the 5' end of the seed region) adopts the modified nucleoside monomer designed by the present invention, and its inhibition efficiency on the PCSK9 gene exceeds 85%. Compared with ON12, the inhibition efficiency on the PCSK9 gene is significantly improved. Compared with the siRNA chains ON14 and ON15 that introduce the prior art nucleoside monomers (NA0133, 18c) at the specific position of the antisense chain, the inhibition efficiency on the PCSK9 gene is significantly improved.

[0379] Example 6: Inhibitory effect of modified sequences on off-target genes

[0380] In the actual application of siRNA, there are a lot of cases where the expression of non-target mRNA that is only partially complementary to the guide strand (antisense strand) is inhibited. Research by Alnylam has shown that the hepatotoxicity of n-acetylgalactosamine (GalNAc)-coupled siRNA is mainly attributed to the gene inhibition of the wrong target caused by the off-target effect through a recognition mechanism similar to microRNA (miRNA).

[0381] To solve this problem, Alnylam placed a glycol nucleic acid (GNA) modification at position 7 of the antisense strand of siRNA in its latest fifth-generation template design to disrupt the seed region of the antisense strand, thereby significantly reducing off-target effects and alleviating hepatotoxicity. Such modifications can affect the binding of siRNA to non-designed targets through seed region recognition, thereby inhibiting off-target effects.

[0382] In the modified dsRNA of the present invention, at least one nucleotide modified with a 2'-large steric bulk group is included in the seed region of the antisense strand (i.e., within positions 1-9 at the 5'-end of the antisense strand), wherein the steric bulk of the 2'-large steric bulk group in the modified nucleotide is greater than the steric bulk of the 2'-F and 2'-OMe groups. By performing the above-mentioned group modification in the antisense strand seed region, the binding and recognition of the dsRNA antisense strand with the off-target gene can be weakened due to steric hindrance, thereby reducing the off-target effect.

[0383] 1. Experimental Materials

[0384] Test samples: modified small interfering RNA sequences listed in Table 2 (synthesized in Example 2).

[0385] Cell types: Hep3B cells were cultured in EMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010), 1% non-essential amino acid solution (Gibco-11140-050), and 1% GlutaMAX supplement (Gibco-35050-061).

[0386] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0387] Off-target gene selection: PCYOX1-5, hDSE.

[0388] 2. Experimental methods

[0389] qRT-PCR was used to detect the inhibitory effect of the test samples on the expression of off-target gene mRNA in the HEP3B cell line.

[0390] 2.1 Compound dilution

[0391] For off-target experiments, the test samples were diluted to a concentration of 15 nM and tested in triplicate wells.

[0392] Hep3B cell digestion count:

[0393] Hep3B cells with a density of 80% were taken out, rinsed with Dulbecco's phosphate buffered saline (DPBS), and digested with 0.05% trypsin for 3-10 min. After collecting the cells, they were counted with Countstar Rigel S2 and the number of cells was adjusted to 2×10 5 / mL density.

[0394] 2.2 Preparation of transfection reagent

[0395] Prepare RNAiMAX transfection reagent, and prepare an appropriate volume in a 15mL centrifuge tube at a ratio of RNAiMAX: Opti-MEM = 3:97, vortex for 15 seconds, mix, and incubate at room temperature for 15 minutes. Add 40 μL RNAiMAX Opti-MEM to each well at the corresponding position, and take 40 μL of the diluted compound of the corresponding concentration and add it to the corresponding well of the dilution plate, mix and incubate for 15 minutes.

[0396] 2.3 Cell plating

[0397] Hep3B cells (2×10 4 cells / well) to a 96-well cell plate, and the siRNA compound was mixed with RNAiMAX Opti-MEM transfection reagent and added to the cells in each well. At the same time, a control group without siRNA compound containing RNAiMAX Opti-MEM was set up.

[0398] Anti-off-target experiment: Take the compound and RNAiMAX Opti-MEM mixture from the dilution plate and add it to a 96-well cell culture plate (20 μL / well), then add 100 μL / well cells to the 96-well plate, and the final volume per well is 120 μL. After plating, place the plate in a 5% CO2, 37 ℃ incubator for 24 hours.

[0399] 2.4 RNA extraction and reverse transcription

[0400] 24 h after transfection, the medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using RNeasy® 96 Kit (QIAGE N-74182) according to the kit instructions. Subsequently, cDNA was synthesized using FastKing RT Kit (WithgDNase) (TIANGEN-KR116-02) according to the instructions.

[0401] 2.5 RT-qPCR

[0402] The target cDNA was detected by qPCR, and GAPDH cDNA was detected in parallel as an internal control. 8 μL of the prepared qPCR reaction solution and 2 μL of sample cDNA were added to the 384 wells. The TaqMan qPCR reaction program was: heating at 95 °C for 10 min, then entering the cycle mode, heating at 95 °C for 15 s, then 60 °C for 1 min, for a total of 40 cycles. The SYBR qPCR reaction program was: heating at 50 °C for 2 min, heating at 95 °C for 10 min, then entering the cycle mode, heating at 95 °C for 15 sec, then 60 °C for 1 min, for a total of 40 cycles; the final melting curve was heated at 95 °C for 15 sec, 60 °C for 1 min, and 95 °C for 15 sec.

[0403] 2.6 Data Analysis

[0404] The target gene RNA expression level in each sample was calculated based on the Ct value of each sample using the Δ Δ Ct relative quantitative method. The relative expression of the target gene was calculated using 2 -Δ Δ CT express.

[0405] ΔCT = average Ct value of target gene - average Ct value of reference gene;

[0406] ΔΔ CT = Δ CT (drug-treated group) - Δ CT (RNAiMAX control group);

[0407] Relative expression of target gene mRNA = 2 -Δ Δ CT

[0408] Inhibition rate = (1-relative expression level of sample / average expression level of RNAiMAX control) × 100%

[0409] 3. Experimental results

[0410] The inhibition rate in this example is the average of three experiments. The inhibition rate of each sequence on the expression of off-target genes in Hep3B cells is shown in Table 5. Figure 4 and Figure 5 .

[0411] Table 5 The inhibition efficiency of off-target genes by different modified small interfering oligonucleotide sequences

[0412]

[0413] As can be seen from Table 5, at a concentration of 15 nM, ON1 in the fully modified siRNA chain did not adopt an anti-off-target design (modified nucleosides that reduce the thermal stability of the siRNA double strand were introduced at the seventh position of the 5' end of the antisense oligonucleotide chain seed region), and ON1 had a significant inhibitory effect on the expression of off-target genes PCYOX1-5 and hDSE mRNA in Hep3B cells. For example, the inhibition rate of ON1 on PCYOX1-5 was 46.0%, and the inhibition rate on hDSE was 50.8%. The seventh position of the 5' end of the seed region of ON14 introduced a modified nucleoside in the prior art. After adopting the anti-off-target design, the inhibition rate of ON14 on PCYOX1-5 was 30.6%, which was 15.4% lower than that of ON1; the inhibition rate on hDSE was 35.1%, which was 15.7% lower than that of ON1. After adopting the anti-off-target design, the inhibition rate of off-target genes was significantly reduced, and the anti-off-target efficiency was significantly improved.

[0414] The above-mentioned anti-off-target design is adopted, and the modified nucleoside of the present invention is introduced at the seventh position of the 5' end of the seed region of the antisense oligonucleotide chain. For example, siRNA double-stranded ON4, ON6, ON7, ON8, ON9, ON10, wherein the corresponding position of the ON4 antisense chain is embedded with a YK-NUM-103 phosphoramidite monomer, the corresponding position of the ON6 antisense chain is embedded with a YK-NUM-105 phosphoramidite monomer, the corresponding position of the ON7 antisense chain is embedded with a YK-NUM-106 phosphoramidite monomer, the corresponding position of the ON8 antisense chain is embedded with a YK-NUM-107 phosphoramidite monomer, the corresponding position of the ON9 antisense chain is embedded with a YK-NUM-108 phosphoramidite monomer, and the corresponding position of the ON10 antisense chain is embedded with a YK-NUM-109 phosphoramidite monomer. The inhibition efficiency of ON4 on PCYOX1-5 and hDSE genes were 1.9% and 20.0%, respectively, which were 44.1% and 30.8% lower than ON1, and 28.7% and 15.1% lower than ON14; the inhibition efficiency of ON6 on PCYOX1-5 and hDSE genes were 5.0% and 19.6%, respectively, which were 41.0% and 31.2% lower than ON1, and 25.6% and 15.5% lower than ON14; the inhibition efficiency of ON7 on PCYOX1-5 and hDSE genes were 4.8% and 12.3%, respectively, which were 41.2% and 38.5% lower than ON1, and 25.8% and 22.8% lower than ON14; The inhibition efficiency of N8 on PCYOX1-5 and hDSE genes was 6.1% and 19.3%, respectively, which was 39.9% and 31.5% lower than that of ON1, and 24.5% and 15.8% lower than that of ON14; the inhibition efficiency of ON9 on PCYOX1-5 and hDSE genes was 14.2% and 20.6%, respectively, which was 31.8% and 30.2% lower than that of ON1, and 16.4% and 14.5% lower than that of ON14; the inhibition efficiency of ON10 on PCYOX1-5 and hDSE genes was 18.7% and 25.1%, respectively, which was 27.3% and 25.7% lower than that of ON1, and 11.9% and 10.0% lower than that of ON14. The anti-off-target design of the seventh position at the 5' end of the seed region of the antisense oligonucleotide chain is introduced into the modified nucleoside monomer of the present invention, so that the anti-off-target efficiency of the modified siRNA double-stranded ON4, ON6, ON7, ON8, ON9, and ON10 is significantly improved compared with ON1 which does not adopt the anti-off-target design. Compared with ON14 using the existing anti-off-target technology, ON4, ON6, ON7, and ON8 using the modified nucleosides of the present invention show better anti-off-target efficiency for PCYOX1-5 and hDSE genes.

[0415] In summary, the present invention has monomers with different functional groups modified to embed into antisense oligonucleotide chains without affecting the double-stranded formation ability of the complementary RNA chain (ssRNA), and has a complete double-stranded structure, and the melting temperature of the modified siRNA double-stranded is reduced, that is, the thermodynamic stability is reduced. The siRNA double-stranded using the modified nucleoside monomer of the present invention has a significant inhibitory effect on the expression of the target gene PCSK9 mRNA. Through the anti-off-target design, the modified nucleoside monomer of the present invention is introduced into the seventh position of the 5' end of the seed region of the antisense oligonucleotide chain, which shows a better anti-off-target performance compared with the existing anti-off-target technology.

[0416] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound represented by formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof: ； in, W1 is a hydroxyl protecting group or an active phosphorus group; W2 is a hydroxyl protecting group; R1 is , , or ; R2 is fluorine, methoxy, ethoxy, -NHC(=O)CH3, -NHCH2CH2OCH3, -N(CH2CH3)2 or -NHCH2CH3; R3 is H or forms a carbonyl group with the carbon atom to which it is attached.

2. The compound of formula (I) according to claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that: W1 is an active phosphorus group.

3. The compound of formula (I) according to claim 2, its pharmaceutically acceptable salt or its stereoisomer, characterized in that: The compound represented by formula (I) satisfies one or both of the following conditions: (1) W1 is a phosphoramidite, an H-phosphate, a phosphate triester or a phosphorus-containing chiral auxiliary; and (2) W2 is benzyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, methanesulfonyl, p-toluenesulfonyl, dimethoxytrityl, 9-phenylxanthine-9-yl or 9-(p-methoxyphenyl)xanthine-9-yl.

4. The compound of formula (I) according to claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that: The compound represented by formula (I) satisfies one or more of the following conditions: (1) R1 is ; (2) When R3 is hydrogen, R2 is fluorine, methoxy, ethoxy or -NHC(=O)CH3; (3) When R3 forms a carbonyl group with the carbon atom to which it is connected, R2 is -N(CH2CH3)2, -NHCH2CH3 or -NHCH2CH2OCH3; (4) W1 is ;and (5) W2 is dimethoxytrityl.

5. The compound of formula (I) according to any one of claims 1 to 4, its pharmaceutically acceptable salt or its stereoisomer, characterized in that: The compound represented by formula (I) is a compound represented by formula (Ia), .

6. The compound of formula (I) according to any one of claims 1 to 4, its pharmaceutically acceptable salt or its stereoisomer, characterized in that: The compound represented by formula (I) is a compound represented by formula (Ib), 。 7. The compound of formula (I) according to claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that: The compound represented by formula (I) is any of the following structures: , , , , , , , or .

8. A method for preparing a compound of formula (I) as claimed in any one of claims 1 to 7, wherein the synthetic route is as follows: ； It includes the following steps: Compound 1-1 is protected from hydroxyl group to obtain 1-2, which is oxidized to obtain 1-3, which is subjected to a witting reaction to obtain 1-4, 1-4 is subjected to hydroboration oxidation to obtain 1-5, 1-5 is subjected to different reaction steps to obtain 1-6, which is subjected to a glycosylation reaction to obtain 1-7, and 1-7 is subjected to different reaction steps to obtain a compound as shown in formula (I), in R 11 is methyl or acetyl; R1, R2, R3, W1 and W2 are as defined in any one of claims 1-7.

9. A nucleic acid polymer, characterized in that The compound is prepared by polymerization reaction of a compound represented by formula (I) as claimed in any one of claims 1 to 7.

10. The nucleic acid polymer according to claim 9, characterized in that The nucleic acid polymer is a double-stranded oligonucleotide molecule, which comprises a sense strand and an antisense strand, wherein the nucleotide sequences of the sense strand and the antisense strand are at least partially reverse complementary; positions 1 to 9 of the antisense strand, based on the 5'-end, contain at least one modified nucleotide N M , the nucleotide N M for , , , or a tautomer thereof; R1 is , , or ; R2 and R3 are defined as described in any one of claims 1-7.

11. The nucleic acid polymer according to claim 10, characterized in that The double-stranded oligonucleotide molecule further comprises a ligand.

12. The nucleic acid polymer according to claim 11, characterized in that The ligand is selected from galactose, galactosamine, N-acetylgalactosamine or derivatives thereof.

13. The nucleic acid polymer according to claim 11, characterized in that The ligand is covalently attached to the 5'-end or 3'-end of the sense strand of the double-stranded oligonucleotide molecule via a linker.

14. The nucleic acid polymer according to claim 13, characterized in that The 3'-end and / or 5'-end of the sense strand and the antisense strand contain one or more overhang regions and / or capping groups.

15. The nucleic acid polymer according to claim 9, characterized in that The nucleic acid polymer is a double-stranded oligonucleotide molecule formed by pairing any of the following sense strands and antisense strands: (1) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 5; (2) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 6; (3) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 7; (4) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 8; (5) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 9; (6) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 10; (7) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 11; (8) the sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO: 12; or (9) The sense strand has the sequence shown in SEQ ID NO: 3; and the antisense strand has the sequence shown in SEQ ID NO:

13.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nucleic acid polymer according to any one of claims 9 to 15 and a pharmaceutically acceptable excipient.

17. Use of a nucleic acid polymer as described in any one of claims 9 to 15 or a pharmaceutical composition as described in claim 16 in the preparation of a drug for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene; the specific gene is the PCSK9 gene; the disease is adult primary hypercholesterolemia or mixed dyslipidemia.

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