2apos having increased in vivo stability of oligonucleotides; ribose-modified nucleotide compound as well as synthesis method and application thereof

Abstract

The invention discloses a 2'ribose modified nucleotide compound capable of improving in vivo stability of oligonucleotide, and a synthesis method and application thereof, and particularly discloses a ribose 2 'and 5' site synergistically modified nucleoside compound with a structure as shown in a formula (I), or pharmaceutically acceptable salt or stereoisomer thereof, and a synthesis method and application of the ribose 2 'and 5' site synergistically modified nucleoside compound. The modified nucleoside compound provided by the invention is connected to the 5 '-terminal of siRNA, so that the gene expression inhibition activity of siRNA can be improved, and the druggability of siRNA in vivo is effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nucleic acid drugs, in particular, to a 2' ribose-modified nucleotide compound with increased in vivo stability of oligonucleotides, and a synthesis method and application thereof. BACKGROUND

[0002] Unmodified oligonucleotide molecules have many problems in terms of drugability, for example: (1) poor in vivo stability, the phosphodiester bond of oligonucleotides is easily degraded by various nucleases widely existing in blood and cells in vivo; (2) low affinity and specificity for binding to target genes, prone to "off-target" effects, and produce immune stimulation and toxic side effects; (3) low bioavailability, oligonucleotides are usually multivalent anionic macromolecules, therefore, it is difficult for them to enter target organs and tissues, and to penetrate into cells through lipophilic cell membranes. Therefore, modification of oligonucleotides is necessary for drugability.

[0003] From the perspective of the mechanism of siRNA molecules exerting RNA interference activity in vivo, although the 5' end of the antisense strand in the siRNA double strand must have a phosphate group to specifically bind to the side chain residues of the MID domain of the AGO2 protein, studies have shown that in the case of modification of the 5' end of the antisense strand with a natural phosphate group, after the siRNA enters the endocytosis pathway, the acid phosphatase in the lysosome rapidly degrades the phosphate group, thus limiting the bioavailability of siRNA in vivo (Reka AH, et al. Nucleic Acids Res., 2017, 45, 7581), thereby affecting the efficacy. In some cases, replacing the natural phosphate group at the 5' end of the antisense strand of siRNA with a phosphate analog can improve the in vivo efficacy of siRNA, as the modified phosphate does not belong to the substrate of phosphatase in vivo, so after modification, it can resist exonuclease degradation and enhance the in vivo silencing effect of siRNA. It has been reported that the phosphate analog 5'-(E)-vinyl phosphate (5'-(E)-VP) is a modification group that can be used to increase the stability of single-stranded RNAi (ssRNAi) (Prakash TP et al., Nucleic Acids Res. 2015; 43(6): 2993-3011.). The widely used 5'-(E)-VP modified nucleotides mostly use 2'-O-methyl modification. However, in some cases, the existing modification method still has problems such as insufficient resistance to exonuclease degradation and poor gene silencing effect of siRNA, and even produces cytotoxicity related to off-target effects. Therefore, there is a need for siRNA drugs that have better target gene inhibition activity, higher bioavailability, and lower toxicity. SUMMARY

[0004] The present application provides a new 5'-(E)-VP modified nucleoside compound, using the modified nucleoside compound of the present application in the synthesis of siRNA, which can enhance the activity of oligonucleotide in inhibiting gene expression, or has low toxicity, thus has improved efficacy.

[0005] The present application adopts the following technical solutions:

[0006] The present application provides a compound represented by formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0007] ;

[0008] wherein,

[0009] X is O or S;

[0010] R1 is a base optionally substituted with 1, 2 or 3 R a , or a salt thereof, the base being 、 、 or ;

[0011] R a is H, C1-C6 alkyl, -CH2R a-1 , -C(=O)OR a-1 or -C(=O)R a-1 ;

[0012] R a-1 is C1-C6 alkyl or C6-C 10 aryl;

[0013] R2 is H or halogen;

[0014] R3 is halogen, -OR b or -NR c R d ;

[0015] R b is H or C1-C6 alkyl;

[0016] R c and R d are each independently H, C1-C8 alkyl, -CH2CH2OR e or -C(=O)R e ;

[0017] R e is H or C1-C8 alkyl;

[0018] R4 is H, halogen, C1-C3 alkyl or forms a carbonyl group with the carbon atom to which it is attached;

[0019] R5 is H, a hydroxyl protecting group, or a reactive phosphorus group;

[0020] R6 and R7 are each independently C2-C6 alkyl, -CH2CH2CN, or -CH2O(CO)C(CH3)3.

[0021] In some embodiments, X is O.

[0022] In some embodiments, R1 is substituted a-1 with -C(=O)R , , or ; R a-1 is C1-C6 alkyl or C6-C 10 aryl.

[0023] In some embodiments, R1 is , , or .

[0024] In some embodiments, R1 is .

[0025] In some embodiments, R2 is H or F.

[0026] In some embodiments, R3 is halogen, -OC1-C3 alkyl, -NHC(=O)R e or -NH(CH2)5CH3.

[0027] In some embodiments, R3 is F, methoxy, ethoxy, -NHC(=O)CH3, -NHCH2CH2OCH3, -N(CH2CH3)2, -N(CH3)2, or -NH(CH2)5CH3.

[0028] In some embodiments, R4 is H, F, methyl, or forms a carbonyl with the carbon atom to which it is attached.

[0029] In some embodiments, when R4 is H, R3 is F, methoxy, ethoxy, or -NHC(=O)CH3.

[0030] In some embodiments, when R4 is F, R3 is F.

[0031] In some embodiments, when R4 is methyl, R3 is F or methoxy.

[0032] In some embodiments, when R4forms a carbonyl with the carbon atom to which it is attached, R3is -N(CH2CH3)2, -N(CH3)2, -NH(CH2)5CH3, or -NHCH2CH2OCH3; preferably -NH(CH2)5CH3.

[0033] In some embodiments, is , , , , , , , , or ; preferably , , , , , or .

[0034] In some embodiments, when R2is F, R3is fluoro or methoxy, and R4is H or fluoro.

[0035] In some embodiments, when R2is H, R4forms a carbonyl with the carbon atom to which it is attached, and R3is -NHCH2CH2OCH3, -N(CH2CH3)2, -N(CH3)2, or -NH(CH2)5CH3; preferably -NH(CH2)5CH3.

[0036] In some embodiments, when R2is H, R4is methyl, and R3is methoxy or fluoro.

[0037] In some embodiments, when R2is H, R4is H, and R3is -NHC(=O)CH3.

[0038] In some embodiments, R5is a reactive phosphorus group.

[0039] In some embodiments, R5is a phosphoramidite, H-phosphonate, phosphotriester, or phosphorus-containing chiral auxiliary.

[0040] In some embodiments, R5is .

[0041] In some embodiments, R6and R7are independently C2-C6alkyl.

[0042] In some embodiments, R6and R7are ethyl.

[0043] In some embodiments, the compound of Formula (I) is a compound of Formula (II),

[0044] wherein R1, R2, R3, R4, R5, R6and R7are as defined in any of the aspects of the application.

[0045] In some embodiments, X is O;

[0046] R1is ;

[0047] R2is H or halo;

[0048] R3is halo, -OC1-C3alkyl, -NHC(=O)R e or -NH(CH2)5CH3;

[0049] R4is H, halo, C1-C3alkyl or forms a carbonyl with the carbon atom to which it is attached;

[0050] R5is ;

[0051] R6and R7are ethyl.

[0052] In some embodiments, the compound of formula (II) is any one of the following:

[0053] , , , , , , , , or .

[0054] In another aspect, the present application provides a method for preparing a compound of formula (I) as described above, the synthetic route is as follows:

[0055]

[0056] which comprises the following steps:

[0057] Compound 1-1 is protected by hydroxyl to obtain 1-2, which is oxidized to obtain 1-3, and then subjected to witting reaction to obtain 1-4, and 1-4 is subjected to a series of reactions to obtain 1-5, and 1-5 is subjected to different reaction steps to obtain 1-6, and 1-6 is subjected to glycosylation reaction to obtain 1-7, and 1-7 is subjected to different reaction steps to obtain a compound of formula (I),

[0058] wherein R 11 is a hydroxyl substituent;

[0059] W1and W2are hydroxyl protecting groups;

[0060] X, R1, R2, R3, R4, R5, R6and R7are defined as in any of the aspects of the application.

[0061] In some embodiments, R 11 is methyl or acetyl.

[0062] In some embodiments, W1and W2are trityl, benzoyl, 2,6-dichlorobenzoyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, tetraisopropyl disiloxanyl, methoxymethyl ether, or dimethoxytrityl.

[0063] Another aspect of the application provides a nucleic acid polymer prepared by polymerization of a compound comprising Formula (I) as described above.

[0064] In some embodiments, the nucleic acid polymer is a double-stranded oligonucleotide molecule comprising a sense strand and an antisense strand, the nucleotide sequences of which are at least partially reverse-complementary; containing a modified nucleotide N M at the 5'-terminal position of the antisense strand, the modified nucleotide being ; X1is O or S; R1, R2, R3, R4and X are defined as in any of the aspects of the application.

[0065] In some embodiments, the double-stranded oligonucleotide molecule further contains a ligand; preferably, the ligand is selected from galactose, galactosamine, N-acetylgalactosamine, or a derivative thereof.

[0066] In some embodiments, the ligand is L96, which is a well-known GalNac delivery vehicle in the art, wherein represents the position of attachment to the siRNA via a phosphonate or thiophosphonate group, see, e.g., PCT Publication Nos. WO2009073809 and WO2009082607;

[0067] .

[0068] In some embodiments, the ligand is covalently attached to the 5'-terminal or 3'-terminal end of the sense strand of the double-stranded oligonucleotide molecule via a linker.

[0069] In some embodiments, the 3'-terminal and / or 5'-terminal end of the sense strand and the antisense strand comprise one or more overhang regions and / or capping groups.

[0070] In some embodiments, the nucleic acid polymer is a double-stranded oligonucleotide molecule formed by any of the following sense and antisense strand pairings:

[0071] (1) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 4;

[0072] (2) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 5;

[0073] (3) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 6;

[0074] (4) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 7;

[0075] (5) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 8;

[0076] (6) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 9;

[0077] (7) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 10;

[0078] (8) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 11;

[0079] (9) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 12;

[0080] (10) the sense strand has the sequence of SEQ ID NO: 3; and the antisense strand has the sequence of SEQ ID NO: 13.

[0081] In some embodiments, the sense strand is SEQ ID NO: 3: Cfs-Cms-Uf-Gm-Gf-Am-Cf-Am-Uf-Uf-Cf-Am-Gf-Am-Af-Cm-Af-Am-Gf-Am-Af-L96 (5'- 3'); the antisense strand is shown in the following table:

[0082]

[0083] Another aspect of the present application provides a pharmaceutical composition comprising the nucleic acid polymer as described above and a pharmaceutically acceptable excipient.

[0084] Another aspect of the present application 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.

[0085] Another aspect of the present application provides use of the nucleic acid polymer as described above or the pharmaceutical composition as described above in the preparation of a medicament for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene (e.g. ApoB gene).

[0086] In some embodiments, the disease is adult primary hyperlipidemia.

[0087] Another aspect of the present application also provides a method for inhibiting the expression of a target gene, administering to a subject an effective amount of the double-stranded oligonucleotide molecule as described above or the pharmaceutical composition as described above; wherein the administration comprises subcutaneous or intravenous administration.

[0088] Wherein the subject is a mammal, preferably a human.

[0089] It should be understood that in the above technical solutions, the use provided by the present application can include therapeutic, diagnostic, non-therapeutic and non-diagnostic uses. For example, the therapeutic use can include using the nucleic acid polymer provided by the present application or using the composition of the present application to achieve the effects of treating diseases, improving conditions, regulating physiological activities in the body, etc.; the diagnostic use can include using the nucleic acid polymer provided by the present application or using the composition of the present application to achieve the purpose of disease diagnosis; the non-therapeutic and non-diagnostic purpose can include using the nucleic acid polymer provided by the present application or using the composition of the present application for scientific research, detection and other non-therapeutic and non-diagnostic purposes (such as disease mechanism research, drug mechanism research, new drug development, drug screening, etc.).

[0090] Unless otherwise defined, all terms (including technical and scientific terms) used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined in the present application.

[0091] In the present application, "stereoisomer" refers to isomers resulting from the different spatial arrangement of atoms in a molecule, including but not limited to cis-trans isomers, enantiomers, etc.

[0092] As used herein, the term "C1-C8" refers to any integer value of carbon atoms in the backbone of the group ranging from 1 to 8, e.g., 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 backbone of the group ranging from 1 to 6, e.g., 1, 2, 3, 4, 5, 6 carbon atoms.

[0093] As used herein, "hydroxyl protecting group" refers to a protecting group commonly used in the synthesis of RNA or its derivatives to protect the hydroxyl group of the ribose structure, for example, acetyl, phenoxyacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4, 4'-dimethoxytrityl (DMr, 4,4'-dimethoxytrityl), monomethoxytrityl (MMTr), 9-phenylxanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanoethoxymethyl, and the like.

[0094] As used herein, "active phosphorus group" refers to a phosphorus-containing group capable of reacting with a hydroxyl or amine group contained in another molecule, especially another nucleotide unit or another nucleotide analog, through a nucleophilic attack reaction. Typically, such a reaction results in an ester-type internucleosidic linkage connecting the nucleotide unit or nucleotide analog unit to another nucleotide unit or nucleotide analog unit. These active phosphorus groups are known in the art and include, but are not limited to, phosphoramidite groups, H-phosphonate groups, phosphotriester groups, and phosphorus-containing chiral auxiliary groups, for example III or P V valent phosphorus atoms, including, but not limited to, phosphoramidite groups, H-phosphonate groups, phosphotriester groups, and phosphorus-containing chiral auxiliary groups, for example .

[0095] As used herein, "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0096] As used herein, "alkyl" refers to a straight-chain or branched-chain alkyl group having the specified number of carbon atoms (e.g., C1-C6, C1-C8). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, s-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0097] As used herein, "aryl" refers to an aromatic ring having the specified number of ring carbon atoms (e.g., C6-C 10 ), composed of only carbon atoms, which is either fused or non-fused. For example, phenyl or naphthyl.

[0098] The term "salt" refers to a corresponding salt that can facilitate or desirably modify preparation, purification, and / or handling of the modified nucleoside compounds (or nucleotide compounds) of the present application, e.g., a pharmaceutically acceptable salt. Unless otherwise specified, reference to a particular compound in the present application is also meant to include salt forms of the compound.

[0099] The "pharmaceutically acceptable salt" in the present application refers to a salt of a compound with a pharmaceutically acceptable (relatively non-toxic, safe, suitable for patient use) acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of the pharmaceutically acceptable base in a suitable inert solvent. The 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 a 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 the pharmaceutically acceptable acid in a suitable inert solvent. The pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, sulfate, methanesulfonate, and the like.

[0100] The "pharmaceutically acceptable excipient" in the present application refers to all substances other than the active ingredient that are contained in a pharmaceutical preparation.

[0101] The term "nucleic acid polymer" can refer to any nucleic acid molecule, including but not limited to DNA, RNA, and hybrids thereof, including but not limited to single-stranded and double-stranded, and the like. The number of nucleotides polymerized to form an oligonucleotide is 2, 3, or more, and can be a polymer of 20 or fewer nucleotides, or a polymer of 20 or more nucleotides.

[0102] The "pharmaceutical composition" in the present application means a mixture or solution prepared for administration to a mammal, e.g., a human, in need thereof, which contains a therapeutically effective amount of an active pharmaceutical ingredient and a pharmaceutically acceptable excipient.

[0103] The "treatment" in the present application means any of the following: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in a biological cascade that leads to a disease; (3) slowing the development of one or more biological manifestations of a disease.

[0104] The "prevention" in the present application means reducing the risk of developing a disease.

[0105] The term "nucleic acid polymer" can refer to any nucleic acid molecule, including but not limited to DNA, RNA, and hybrids thereof, including but not limited to single-stranded and double-stranded, and the like. The number of nucleotides polymerized to form an oligonucleotide is 2, 3, or more, and can be a polymer of 20 or fewer nucleotides, or a polymer of 20 or more nucleotides.

[0106] The above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present application without departing from the common general knowledge in the art.

[0107] The modified nucleosides of the present application have at least the following advantages:

[0108] 1) The modified nucleosides of the present application introduce modification groups at the 2' and 5' positions of ribose, which are completely different from the structures of prior art modified nucleosides.

[0109] 2) The double-stranded oligonucleotide (dsRNA) molecules synthesized from the modified nucleosides of the present application have significantly improved target gene interference activity compared with the double-stranded oligonucleotide (dsRNA) molecules with conventional 2'-F or 2'-OMe modifications.

[0110] 3) The double-stranded oligonucleotide (dsRNA) molecules synthesized from the modified nucleosides of the present application have higher target gene interference activity compared with the double-stranded oligonucleotide (dsRNA) molecules synthesized from prior art modified nucleosides. BRIEF DESCRIPTION OF DRAWINGS

[0111] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings required to be used in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only used to exemplarily illustrate some examples of the present application, but not limit the present application.

[0112] Figure 1 The inhibition rates of the modified double-stranded oligonucleotides ON3, ON5, ON6, ON7, ON11 and ON14 on the expression of ApoB protein in the serum of mice on the 7th day, the 14th day and the 21st day after administration.

[0113] Figure 2 The percentage of reduction of LDL-C in the serum of mice by the modified double-stranded oligonucleotides ON3, ON5, ON6, ON7, ON11 and ON14 on the 7th day, the 14th day and the 21st day after administration. DETAILED DESCRIPTION

[0114] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If no specific conditions are indicated in the examples, the operations are carried out according to the conventional conditions or the conditions recommended by the manufacturers, for example, the solvents and the corresponding volume ratios selected when used as the mobile phase, etc. can be carried out according to the conventional operations in the art. If no manufacturer of the reagents or instruments is indicated, they are all products that can be obtained through commercial channels. The technical features involved in each embodiment of the present application can be combined with each other as long as they do not conflict with each other.

[0115] The following abbreviations represent the following reagents, respectively:

[0116] IBX: 2-lodoxybenzoic acid; Dess-Martin: Dess-Martin periodinane; DEA: diethylamine; TEA: triethylamine; Ac20: acetic anhydride; HOAc: acetic acid; conc H2S04: concentrated sulfuric acid; HDMS: hexamethyldisilazane; BSA: N,0-bis(trimethylsilyl)acetamide; TMSOTf: trimethylsilyl triflate; Toluene: toluene; MeOH: methanol; DIEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DMSO: dimethyl sulfoxide; HATU: 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; THF: tetrahydrofuran; TIPDSCl: 1, 3-dichloro-l, 1, 3, 3-tetraisopropyldisiloxane; Imidazole: imidazole; DMF: N,N-dimethylformamide; TBAF: tetrabutylammonium fluoride; DMTrCl: 4,4'-dimethoxytrityl chloride; DIAD: diisopropyl azodicarboxylate; 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: 2,2,6,6-tetramethyl-l-piperidinyloxy, 1-oxyl; Ph3P + CH3Br - : methyltriphenylphosphonium bromide; H202: hydrogen peroxide; U: uracil; Phthalimide: phthalimide; Hydrazinium hydroxide solution: hydrazine hydrate; Acetyl chloride: acetyl chloride; Ethylamine: ethylamine; Dimethylamine hydrochloride: dimethylamine hydrochloride; BH3: 1 M borane tetrahydrofuran complex; SnCl4: tin tetrachloride; TBDPSCl: tert-butyldiphenylsilyl chloride; TsOH: p-toluenesulfonic acid; NaH: sodium hydride; Dimethyl sulfate: dimethyl sulfate; NIS: N-iodosuccinimide; DMM: dimethyl malonate; TfOH: trifluoromethanesulfonic acid.

[0117] Example 1: Synthesis of modified nucleosides

[0118] The modified nucleosides of the present application, YK-NUM-201 to YK-NUM-210, were synthesized by the following routes.

[0119] Synthetic routes:

[0120] This example prepares YK-NUM-201, YK-NUM-202, YK-NUM-204, YK-NUM-207 by oxidation of the hydroxyl group of the alkenyl group of key intermediate INT-I via boron hydride, followed by condensation with different amino compounds; YK-NUM-203 by oxidation of the hydroxyl group of the alkenyl group of INT-I via boron hydride, further amination, followed by reaction with acids, acid anhydrides or acid halides; YK-NUM-205 and YK-NUM-206 by oxidation of the hydroxyl group of the alkenyl group of INT-I via boron hydride, oxidation to aldehyde group, followed by reaction with Grignard reagent to prepare secondary alcohol, further fluorination or methylation; YK-NUM-208, YK-NUM-209, YK-NUM-210 by asymmetric dihalogenation of the alkenyl group of INT-I, followed by hydroxylation, then methylation, fluorination, oxidation and other steps. This route can quickly realize the screening of different modified groups through the functional group transformation of the alkenyl group of INT-I.

[0121]

[0122] 1. Synthesis of intermediate INT-I

[0123]

[0124] Step 1: Synthesis of INT-I-PM1

[0125] SM, (2R, 3R, 4S, 5R)-2-(hydroxymethyl)-5-methoxytetrahydrofuran-3, 4-diol (100.00 g, 609.16 mmol) was dissolved in pyridine (500.0 mL) under nitrogen atmosphere protection, and 1, 3-dichloro-1, 1, 3, 3-tetraisopropyl disiloxane (192.15 g, 609.17 mmol) was added dropwise under ice bath cooling. The reaction was stirred at room temperature overnight. After the reaction was completed, the organic phase was removed by vacuum concentration, dissolved in water and extracted with dichloromethane, and the combined organic phase was 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 (125.12 g, 306.91 mmol, 50.38%).

[0126] Step 2: Synthesis of INT-I-PM2

[0127] INT-I-PM1 (55.00 g, 134.91 mmol), Dess-Martin Oxidizing Reagent (143.06 g, 337.29 mmol) were dissolved in acetonitrile (600 mL) and stirred at 45 °C overnight under nitrogen atmosphere. TLC showed the reaction was complete. The reaction was cooled down and filtered to remove the insoluble material. The filtrate was concentrated under vacuum and redissolved in methyl tert-butyl ether. The insoluble material was removed by filtration. The filtrate was washed with saturated sodium thiosulfate, saturated sodium bicarbonate, and saturated brine. The organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The crude product INT-I-PM2 (46.34 g, 100% purity) was obtained as a colorless liquid.

[0128] Step 3: Synthesis of INT-I

[0129] Methyl triphenylphosphonium bromide (89.72 g, 251.30 mmol) was dissolved in tetrahydrofuran and cooled to -78 °C under nitrogen atmosphere. 1.3 M s-BuLi (202.1 mL, 262.74 mmol) was added dropwise and the mixture was stirred for 1 h. INT-I-PM2 (46.34 g, 114.23 mmol) was added dropwise and the mixture was stirred for 1 h. The reaction was stirred overnight at room temperature. After the reaction was complete, the organic phase was removed by concentration under vacuum. The residue was extracted with ethyl acetate and the organic phase was washed with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate) to give INT-I (20.86 g, 51.67 mmol, 45.23%).

[0130] 2. YK-NUM-201 was synthesized according to the following route

[0131]

[0132]

[0133] Step 1: Synthesis of YK-NUM-201-PM1

[0134] To the reaction flask was added 1 M borane tetrahydrofuran complex (185.8 mL, 185.79 mmol) and a solution of INT-I (30.00 g, 74.31 mmol) in tetrahydrofuran was added dropwise at 0 °C under a nitrogen atmosphere. After the addition was complete, the reaction was allowed to warm to room temperature and stirred for 2 h. To the above solution was added THF:H2O = 1:1 (60.0 mL) dropwise at 0 °C under a nitrogen atmosphere. After the addition was complete, the reaction was allowed to warm to room temperature and stirred for 3 h. The reaction was then heated to 40 °C and stirred overnight. The reaction was cooled in an ice bath and 2 M sodium hydroxide solution (148.6 mL, 297.26 mmol) and 30% hydrogen peroxide (114.2 mL, 1.19 mol) were added dropwise. After the addition was complete, the reaction was allowed to warm to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The reaction was concentrated under reduced pressure in vacuo. The aqueous phase was extracted with EA (400 mL x 2) and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether / ethyl acetate) to give the target product YK-NUM-201-PM1 (16.73 g, 39.67 mmol, 53.38%) as a colorless liquid.

[0135] Step 2: Synthesis of YK-NUM-201-PM2

[0136] YK-NUM-201-PM1 (16.00 g, 37.94 mmol), iodobenzene diacetate (25.66 g, 79.66 mmol), sodium bicarbonate (4.78 g, 56.90 mmol), TEMPO (0.89 g, 5.70 mmol), acetonitrile (70.0 mL), and water (70.0 mL) were stirred at room temperature under a nitrogen atmosphere for 3 h. After the reaction was complete, the reaction was quenched with a sodium thiosulfate solution, extracted with EA (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure in vacuo to remove the solvent to give YK-NUM-201-PM2 (19.68 g, 45.27 mmol, calculated based on 100.0% purity). C 19 H 38 O7Si2, MS(ES): m / z(M-H - )433.2.

[0137] Step 3: Synthesis of YK-NUM-201-PM3

[0138] YK-NUM-201-PM2 (19.68 g, 45.27 mmol) was dissolved in acetonitrile (200.0 mL), under nitrogen atmosphere, ice water bath cooling to 0°C, to the above system was added HATU (20.65 g, 54.32 mmol), DIEA (17.55 g, 135.81 mmol), 2-methoxyethylamine (7.48 g, 99.59 mmol), after the addition was completed, the temperature was raised to room temperature, and the reaction was stirred for 2 h. After the reaction was completed, the reaction mixture was concentrated under vacuum and reduced pressure, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-NUM-201-PM3 (15.73 g, 31.99 mmol, 70.65%). 22 H 45 NO7Si2, MS(ES): m / z(M+H + )492.3.

[0139] Step 4: Synthesis of YK-NUM-201-PM4

[0140] Uracil (4.13 g, 36.85 mmol), ammonium sulfate (389.6 mg, 2.95 mmol) was dissolved in pyridine (30.0 mL) and hexamethyldisilazane (150.0 mL), under nitrogen protection, oil bath 130°C activation for 1.5 h, cooling, vacuum and reduced pressure concentration to obtain white solid. Then YK-NUM-201-PM3 (7.25 g, 14.74 mmol), acetonitrile (80.0 mL) was added, under nitrogen atmosphere, ice water bath cooling to 0°C, slowly droped anhydrous tin tetrachloride (9.60 g, 36.85 mmol), after completion, the temperature was kept and the reaction was stirred for 16 h, and then the oil bath was reacted at 37°C for about 18 h. After the reaction was completed, the reaction solution was quenched with saturated sodium bicarbonate under ice bath, filtered, washed with saturated brine (80 mL x 2), dried over anhydrous sodium sulfate, filtered, vacuum and reduced pressure concentration, the residue was purified by silica gel chromatography (dichloromethane / ethyl acetate) to obtain YK-NUM-201-PM4 (3.08 g, 5.39 mmol, 36.54%). 25 H 45 N3O8Si2, MS(ES): m / z(M-H - )570.3.

[0141] Step 5: Synthesis of YK-NUM-201-PM5

[0142] YK-NUM-201-PM4 (3.08 g, 5.39 mmol) was dissolved in tetrahydrofuran (30.0 mL), under the protection of nitrogen atmosphere, the above system was slowly added with 1 M TBAF in THF (215 mL, 21.54 mmol) dropwise at 0 °C under ice water bath cooling, and then the reaction was stirred at room temperature for 22 h after being added. After the reaction was completed, the reaction mixture was concentrated under vacuum and reduced pressure, and the residue was purified by C18 reverse phase column chromatography (acetonitrile / water) to obtain YK-NUM-201-PM5 (1.62 g, 4.92 mmol, 91.33 %). C 13 H 19 N3O7, MS (ES): m / z (M-H - ) 328.1.

[0143] Step 6: Synthesis of YK-NUM-201-PM6

[0144] YK-NUM-201-PM5 (1.62 g, 4.92 mmol) and DMTrCl (2.33 g, 6.89 mmol) were dissolved in pyridine (18.0 mL), and then the reaction was stirred at room temperature for 2 h after being added under the protection of nitrogen atmosphere. After the reaction was completed, the reaction mixture was concentrated under vacuum and reduced pressure, followed by washing with EA (100.0 mL), saturated brine (40 mL x 2), drying over anhydrous sodium sulfate, filtration, vacuum and reduced pressure concentration, and purification of the residue by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-201-PM6 (1.97 g, 3.12 mmol, 63.40 %). C 34 H 37 N3O9, MS (ES): m / z (M-H - ) 630.3.

[0145] Step 7: Synthesis of YK-NUM-201-PM7

[0146] YK-NUM-201-PM6 (1.97 g, 3.12 mmol), imidazole (2.12 g, 31.14 mmol) were dissolved in DMF (18.0 mL), under the protection of nitrogen atmosphere, the above system was slowly added with TBDPSCl (5.14 g, 18.70 mmol) at 0 °C under ice water bath, after the addition was completed, the temperature was raised to room temperature, and the reaction was stirred for 22 h, and then the reaction was further carried out at 37 °C under oil bath for about 3 h. After the reaction was completed, the reaction mixture was added into EA (300.0 mL), washed with water (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum and reduced pressure to remove the solvent to obtain YK-NUM-201-PM7 (8.92 g, 10.25 mmol, calculated according to 100.0% purity). C 50 H 55 N3O9Si, MS (ES): m / z (M-H - ) 868.4.

[0147] Step 8: Synthesis of YK-NUM-201-PM8

[0148] YK-NUM-201-PM7 (8.92 g, 10.25 mmol) was dissolved in DCM (100.0 mL), under the protection of nitrogen atmosphere, the above system was slowly added with TsOH buffer solution (2.00 g dissolved in 70.0 mL DCM and 30.0 mL MeOH) at 0 °C under ice water bath, and the reaction was stirred for 0.5 h. After the reaction was completed, saturated sodium bicarbonate was slowly added dropwise under ice bath to quench, followed by washing with DCM (300.0 mL), saturated brine (50 mL x 2), drying over anhydrous sodium sulfate, filtering, concentrating under vacuum and reduced pressure, and purifying the residue by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-201-PM8 (1.64 g, 2.89 mmol, 28.18%). C 29 H 37 N3O7Si, MS (ES): m / z (M-H - ) 566.2.

[0149] Step 9: Synthesis of YK-NUM-201-PM9

[0150] YK-NUM-201-PM8 (1.64 g, 2.89 mmol) was dissolved in DCM (16.0 mL), and the mixture was cooled to 0 °C in an ice-water bath under a nitrogen atmosphere. Dess-Martin (1.23 g, 2.90 mmol) was added slowly to the above system, and the mixture was stirred at 0 °C for 1 h and then at room temperature for 3 h. After the reaction was completed, the reaction mixture was added to EA (200.0 mL), washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to give YK-NUM-201-PM9 (2.06 g, 3.64 mmol, calculated based on 100.0% purity). C 29 H 35 N3O7Si, MS (ES): m / z (M-H - ) 564.2.

[0151] Step 10: Synthesis of YK-NUM-201-PM10

[0152] Sodium hydride (437.0 mg, 10.92 mmol) was dissolved in THF (10.0 mL) under a nitrogen atmosphere, and the mixture was cooled to -80 °C. Tetraethyl methylenediphosphonate (2.62 g, 9.09 mmol) was added slowly dropwise to the above system, and the mixture was stirred at -80 °C for 15 min. YK-NUM-201-PM9 (2.06 g, 3.64 mmol) was added, and the mixture was stirred at -80 °C for 10 min and then allowed to warm to room temperature. After the reaction was completed, saturated aqueous ammonium chloride solution was added slowly dropwise to the mixture under ice-bath cooling, and the mixture was washed with EA (200.0 mL), saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane / methanol) to give YK-NUM-201-PM10 (1.94 g, 2.77 mmol, 76.13%). C 34 H 46 N3O9PSi, MS (ES): m / z (M-H - ) 698.3.

[0153] Step 11: Synthesis of YK-NUM-201-PM11

[0154] YK-NUM-201-PM10 (1.94 g, 2.77 mmol) was dissolved in tetrahydrofuran (20.0 mL), under the protection of nitrogen atmosphere, the above system was slowly added with 1 M TBAF in THF (11.1 mL, 11.09 mmol) dropwise at 0 °C under ice water bath cooling, after the addition was completed, it was stirred at room temperature for 22 h. After the reaction was completed, the reaction mixture was concentrated under vacuum and reduced pressure, and the residue was purified by C18 reverse phase column chromatography (acetonitrile / water) to obtain YK-NUM-201-PM11 (602.5 mg, 1.31 mmol, 47.10 %). C 18 H 28 N3O9P, MS(ES): m / z(M-H - )460.2.

[0155] Step 12: Synthesis of YK-NUM-201

[0156] YK-NUM-201-PM11 (602.5 mg, 1.31 mmol), DIEA (506.3 mg, 3.92 mmol), 1- methylimidazole (107.2 mg, 1.31 mmol) were dissolved in dichloromethane (6.0 mL), under the protection of nitrogen atmosphere, 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (927.2 mg, 3.92 mmol) was added to the above system at 0 °C under ice water bath cooling, after the addition was completed, it was stirred at room temperature for 1 h. After the reaction was completed, the reaction liquid was washed with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum and reduced pressure to remove the solvent, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-NUM-201 (415.6 mg, 0.63 mmol, 48.11 %). C 27 H 45 N5O 10 P2, MS(ES): m / z(M-H - )660.3.

[0157] YK-NUM-201: 1H NMR (CDCI3, 400 MHz, 298 K) δ 8.37 (d, J = 8.6, 1H), 7.45 (d, J = 7.2 Hz, 1H), 7.31 - 7.20 (m, 4H), 6.65 (d, J = 7.2 Hz, 5H), 5.72 (d, J = 7.2 Hz, 1H), 5.47 (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, 5H), 3.40 - 3.17 (m, 6H), 2.83 - 2.60 (m, 5H), 2.0 (t, J = 12.4 Hz, 1H), 1.12 - 1.06 (m, 12H); 31 P NMR (162 MHz, DMSO-d6) δ 149.18, 148.81, 16.33, 16.07.

[0158] 3. YK-NUM-202 synthesis route as follows

[0159]

[0160]

[0161] Step 1: Synthesis of YK-NUM-202-PM1

[0162] YK-NUM-201-PM2 (17.48 g, 40.12 mmol) was dissolved in acetonitrile (200 mL) and cooled in an ice bath under nitrogen protection. N, N-diisopropyl ethylamine (15.56 g, 120.40 mmol) was added dropwise, followed by the addition of HATU (18.31 g, 48.15 mmol) after 10 min of reaction at room temperature. Diethylamine (6.46 g, 88.32 mmol) was added and the reaction was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by vacuum concentration. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain the liquid target product YK-NUM-202-PM1 (8.99 g, 18.32 mmol, 45.65%). C 23 H 48 NO6Si2, MS (ES): m / z (M+H + ) 491.3.

[0163] Step 2: Synthesis of YK-NUM-202-PM2

[0164] Starting from uracil (4.92 g, 43.89 mmol) and YK-NUM-202-PM1 (8.62 g, 17.56 mmol), following the synthetic procedure of YK-NUM-201-PM4, the solid target product YK-NUM-202-PM2 (6.51 g, 11.40 mmol, 64.93%) was obtained. 26 H 48 N3O7Si2, MS (ES): m / z (M-H - ) 569.3.

[0165] Step 3: Synthesis of YK-NUM-202-PM3

[0166] Starting from YK-NUM-202-PM2 (6.51 g, 11.40 mmol), following the synthetic procedure of YK-NUM-201-PM5, the solid target product YK-NUM-202-PM3 (1.53 g, 4.67 mmol, 40.99%) was obtained. 14 H 21 N3O6, MS (ES): m / z (M-H - ) 326.1.

[0167] Step 4: Synthesis of YK-NUM-202-PM4

[0168] Starting from YK-NUM-202-PM3 (1.53 g, 4.67 mmol) and DMTrCl (2.06 g, 6.08 mmol), following the synthetic procedure of YK-NUM-201-PM6, the solid target product YK-NUM-202-PM4 (2.51 g, 3.99 mmol, 85.28%) was obtained. 35 H 39 N3O8, MS (ES): m / z (M-H - ) 628.3.

[0169] Step 5: Synthesis of YK-NUM-202-PM5

[0170] Using YK-NUM-202-PM4 (2.51 g, 3.99 mmol) and TBDPSCl (6.57 g, 23.90 mmol) as raw materials, the liquid target product YK-NUM-202-PM5 (8.12 g, 9.35 mmol, calculated at 100% purity) was obtained by following the synthesis method of YK-NUM-201-PM7. 51 H 57 N3O8Si, MS(ES): m / z(MH - 866.4.

[0171] Step 6: Synthesis of YK-NUM-202-PM6

[0172] Using YK-NUM-202-PM5 (8.12 g, 9.35 mmol) as the starting material, the liquid target product YK-NUM-202-PM6 (1.30 g, 2.30 mmol, 24.57%) was obtained by following the synthesis method of YK-NUM-201-PM8. 30 H 39 N3O6Si, MS (ES): m / z (MH) - 564.3.

[0173] Step 7: Synthesis of YK-NUM-202-PM7

[0174] Using YK-NUM-202-PM-PM6 (1.30 g, 2.30 mmol) as the starting material, YK-NUM-202-PM7 (2.02 g, 3.58 mmol, calculated at 100.0% purity) was obtained by following the synthesis method of YK-NUM-201-PM9. 30 H 37 N3O6Si, MS (ES): m / z (MH) - )562.3.

[0175] Step 8: Synthesis of YK-NUM-202-PM8

[0176] Using YK-NUM-202-PM7 (2.02 g, 3.58 mmol) and tetraethyl methylene diphosphate (2.58 g, 8.95 mmol) as raw materials, the solid target product YK-NUM-202-PM8 (1.38 g, 1.98 mmol, 55.19%) was obtained by following the synthesis method of YK-NUM-201-PM10. 35 H 48 N3O8PSi, MS(ES): m / z(MH- ) 696.3.

[0177] Step 9: Synthesis of YK-NUM-202-PM9

[0178] Starting from YK-NUM-202-PM8 (1.38 g, 1.98 mmol), following the procedure for the synthesis of YK-NUM-201-PM11, the solid target product YK-NUM-202-PM9 (676.0 mg, 1.47 mmol, 74.40%) was obtained. 19 H 30 N3O8P, MS (ES): m / z (M-H - ) 458.2.

[0179] Step 10: Synthesis of YK-NUM-202

[0180] Starting from YK-NUM-202-PM9 (407.0 mg, 0.89 mmol) and 2-cyanoethyl-N,N- diisopropyl chlorophosphoramidite (629.0 mg, 2.66 mmol), following the procedure for the synthesis of YK-NUM-201, the solid target product YK-NUM-202 (305.3 mg, 0.46 mmol, 52.24%) was obtained. 28 H 47 N5O9P2, MS (ES): m / z (M-H - )658.3.

[0181] YK-NUM-202: 1 H NMR (CDC13, 400 MHz, 298 K) δ 7.58 (d, J = 8.6, 1H), 7.38 - 7.26 (m, 7H), 6.86 (d, J = 7.2 Hz, 4H), 6.18 (d, J = 7.8 Hz, 1H), 5.48 (d, J = 8.4 Hz, 1H), 4.65 - 4.58 (m, 1H), 4.17 - 4.13 (m, 1H), 3.93 (t, J = 10.8 Hz, 2H), 3.80 (s, 6H), 3.60 - 3.43 (m, 7H), 2.83 - 2.60 (m, 4H), 1.18 - 1.05 (m, 12H); 31P NMR (162 MHz, DMSO-d6) δ 150.08, 149.77, 15.87, 15.57.

[0182] 4. The synthetic route of YK-NUM-203 is as follows

[0183]

[0184]

[0185] Step 1: Synthesis of YK-NUM-203-PM1

[0186] YK-NUM-201-PM1 (13.00 g, 30.90 mmol), triphenylphosphine (16.21 g, 61.80 mmol), phthalimide (5.46 g, 37.11 mmol) were dissolved in tetrahydrofuran (200.0 mL), and the system was cooled to 0 °C in an ice water bath. DIAD (10.62 g, 52.52 mmol) was added to the above system, and the system was stirred at room temperature for 2 h after the addition was completed. The reaction mixture was concentrated under vacuum and reduced pressure, extracted with EA (800 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum and reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-NUM-203-PM1 (14.64 g, 26.63 mmol, 86.17%).

[0187] Step 2: Synthesis of YK-NUM-203-PM2

[0188] YK-NUM-203-PM1 (14.64 g, 26.63 mmol), 85% hydrazine hydrate (31.36 g, 532.48 mmol) were dissolved in ethanol (300.0 mL), and the system was stirred at 80 °C in an oil bath under nitrogen atmosphere for 1 h. The reaction mixture was concentrated under vacuum and reduced pressure after the reaction was completed, extracted with EA (900 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum and reduced pressure to remove the solvent to obtain YK-NUM-203-PM2 (10.94 g, 26.07 mmol, 97.89%).

[0189] Step 3: Synthesis of YK-NUM-203-PM3

[0190] YK-NUM-203-PM2 (10.94 g, 26.07 mmol), triethylamine (7.91 g, 78.17 mmol) were dissolved in dichloromethane (100.0 mL), under the protection of nitrogen atmosphere, the above system was added acetyl chloride (3.07 g, 39.11 mmol) at 0 °C with ice water bath cooling, after the addition was completed, it was raised to room temperature and stirred for 1 h. After the reaction was completed, the reaction mixture was concentrated under vacuum and reduced pressure, extracted with DCM (600 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum and reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to give YK-NUM-203-PM3 (10.49 g, 22.72 mmol, 87.16%). 21 H 43 NO6Si2, MS(ES): m / z(M+H + )462.3.

[0191] Step 4: Synthesis of YK-NUM-203-PM4

[0192] YK-NUM-203-PM4 (6.24 g, 11.52 mmol) as raw material, according to the synthesis method of YK-NUM-201-PM4, YK-NUM-203-PM4 (6.24 g, 11.52 mmol, 50.70%) was obtained.C 24 H 43 N3O7Si2, MS(ES): m / z(M-H - )540.3.

[0193] Step 5: Synthesis of YK-NUM-203-PM5

[0194] YK-NUM-203-PM4 (6.24 g, 11.52 mmol) as raw material, according to the synthesis method of YK-NUM-201-PM5, YK-NUM-203-PM5 (3.25 g, 10.86 mmol, 94.29%) was obtained.C 12 H 17 N3O6, MS(ES): m / z(M-H - )298.1.

[0195] Step 6: Synthesis of YK-NUM-203-PM6

[0196] YK-NUM-203-PM6 was obtained (4.89 g, 8.13 mmol, 74.84%) from YK-NUM-203-PM5 (3.25 g, 10.86 mmol) and DMTrCl (5.89 g, 17.38 mmol) following the procedure for the synthesis of YK-NUM-201-PM6.C 33 H 35 N3O8, MS (ES): m / z (M-H - ) 600.2.

[0197] Step 7: Synthesis of YK-NUM-203-PM7

[0198] YK-NUM-203-PM7 was obtained (9.78 g, 11.64 mmol, calculated as 100.0% purity) from YK-NUM-203-PM6 (4.89 g, 8.13 mmol) and TBDPSCl (13.40 g, 48.75 mmol) following the procedure for the synthesis of YK-NUM-201-PM7.C 49 H 53 N3O8Si, MS (ES): m / z (M-H - ) 838.4.

[0199] Step 8: Synthesis of YK-NUM-203-PM8

[0200] YK-NUM-203-PM8 was obtained (3.47 g, 6.45 mmol, 55.43%) from YK-NUM-203-PM7 (9.78 g, 11.64 mmol) following the procedure for the synthesis of YK-NUM-201-PM8.C 28 H 35 N3O6Si, MS (ES): m / z (M-H - ) 536.2.

[0201] Step 9: Synthesis of YK-NUM-203-PM9

[0202] YK-NUM-203-PM9 was obtained (4.03 g, 7.52 mmol, calculated as 100.0% purity) from YK-NUM-203-PM8 (3.47 g, 6.45 mmol) following the procedure for the synthesis of YK-NUM-201-PM9.C 28 H 33 N3O6Si, MS (ES): m / z (M-H - ) 534.2.

[0203] Step 10: Synthesis of YK-NUM-203-PM10

[0204] YK-NUM-203-PM10 (1.26 g, 1.88 mmol, 25.00%) was obtained by the method for the synthesis of YK-NUM-201-PM10 from tetraethyl methylenediphosphonate (5.42 g, 18.81 mmol) and YK-NUM-203-PM9 (4.03 g, 7.52 mmol). C 33 H 44 N3O8PSi, MS (ES): m / z (M-H - ) 668.3.

[0205] Step 11: Synthesis of YK-NUM-203-PM11

[0206] YK-NUM-203-PM11 (654.1 mg, 1.52 mmol, 80.60%) was obtained by the method for the synthesis of YK-NUM-201-PM11 from YK-NUM-203-PM10 (1.26 g, 1.88 mmol). C 17 H 26 N3O8P, MS (ES): m / z (M-H - ) 430.2.

[0207] Step 12: Synthesis of YK-NUM-203

[0208] YK-NUM-203 (413.7 mg, 0.66 mmol, 43.20%) was obtained by the method for the synthesis of YK-NUM-201 from YK-NUM-203-PM11 (654.1 mg, 1.52 mmol) and 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (1.31 g, 4.55 mmol). C 26 H 43 N5O9P2, MS (ES): m / z (M-H - ) 630.3.

[0209] YK-NUM-203: 1H NMR (CDCI3, 400 MHz, 298 K) δ 7.84 (d, J = 6.8, 1H), 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), 4.01 - 3.64 (m, 5H), 3.23 - 3.20 (m, 2H), 3.01 (t, J = 12.4 Hz, 2H), 1.46 - 1.39 (m, 21H). 31 P NMR (162 MHz, DMSO-d6) δ 151.66, 151.27, 16.17, 15.82.

[0210] 5. The synthetic route of YK-NUM-204 is as follows

[0211]

[0212]

[0213] Step 1: Synthesis of YK-NUM-204-PM1

[0214] YK-NUM-201-PM2 (19.68 g, 45.27 mmol) and dimethylamine hydrochloride (9.23 g, 113.20 mmol) as raw materials, according to the synthetic method of YK-NUM-202-PM3, YK-NUM-204-PM1 (12.43 g, 26.92 mmol, 59.46%) was obtained.C 21 H 43 NO6Si2, MS (ES): m / z (M+H + ) 462.3.

[0215] Step 2: Synthesis of YK-NUM-204-PM2

[0216] YK-NUM-201-PM2 (19.68 g, 45.27 mmol) and dimethylamine hydrochloride (9.23 g, 113.20 mmol) as raw materials, according to the synthetic method of YK-NUM-202-PM3, YK-NUM-204-PM1 (12.43 g, 26.92 mmol, 59.46%) was obtained.C 24 H43 N3O7Si2, MS (ES): m / z (M-H - ) 540.3.

[0217] Step 3: Synthesis of YK-NUM-204-PM3

[0218] YK-NUM-204-PM3 (3.48 g, 11.63 mmol, 94.31%) was obtained from YK-NUM-204-PM2 (6.68 g, 12.33 mmol) following the procedure for the synthesis of YK-NUM-201-PM5.C 12 H 17 N3O6, MS (ES): m / z (M-H - ) 298.1.

[0219] Step 4: Synthesis of YK-NUM-204-PM4

[0220] YK-NUM-204-PM4 (4.84 g, 8.04 mmol, 69.18%) was obtained from YK-NUM-204-PM3 (3.48 g, 11.63 mmol) and DMTrCl (6.30 g, 18.59 mmol) following the procedure for the synthesis of YK-NUM-201-PM6.C 33 H 35 N3O8, MS (ES): m / z (M-H - ) 600.2.

[0221] Step 5: Synthesis of YK-NUM-204-PM5

[0222] YK-NUM-204-PM5 (9.83 g, 11.70 mmol, calculated as 100.0% purity) was obtained from YK-NUM-204-PM4 (4.84 g, 8.04 mmol) and TBDPSCl (13.27 g, 48.28 mmol) following the procedure for the synthesis of YK-NUM-201-PM7.C 49 H 53 N3O8Si, MS (ES): m / z (M-H - ) 838.4.

[0223] Step 6: Synthesis of YK-NUM-204-PM6

[0224] Starting from YK-NUM-204-PM5 (9.83 g, 11.70 mmol), following the synthetic procedure of YK-NUM-201-PM8, YK-NUM-204-PM6 (3.97 g, 7.38 mmol, 63.10%) was obtained.C 28 H 35 N3O6Si, MS (ES): m / z (M-H - ) 536.2.

[0225] Step 7: Synthesis of YK-NUM-204-PM7

[0226] Starting from YK-NUM-204-PM6 (3.97 g, 7.38 mmol), following the synthetic procedure of YK-NUM-201-PM9, YK-NUM-204-PM7 (4.03 g, 7.52 mmol, calculated as 100.0% purity) was obtained.C 28 H 33 N3O6Si, MS (ES): m / z (M-H - ) 534.2.

[0227] Step 8: Synthesis of YK-NUM-204-PM8

[0228] Starting from tetraethyl methylenediphosphonate (5.42 g, 18.8 mmol) and YK-NUM-204-PM7 (crude 4.03 g, 7.52 mmol), following the synthetic procedure of YK-NUM-201-PM10, YK-NUM-204-PM8 (2.26 g, 3.37 mmol, 44.85%) was obtained.C 33 H 44 N3O8PSi, MS (ES): m / z (M-H - ) 668.3.

[0229] Step 9: Synthesis of YK-NUM-204-PM9

[0230] Starting from YK-NUM-204-PM8 (2.26 g, 3.37 mmol), following the synthetic procedure of YK-NUM-201-PM11, YK-NUM-204-PM9 (974.8 mg, 2.26 mmol, 66.97%) was obtained.C 17 H 26 N3O8P, MS (ES): m / z (M-H - ) 430.2.

[0231] Step 10: Synthesis of YK-NUM-204

[0232] YK-NUM-204 was obtained following the synthetic procedure of YK-NUM-201 starting from YK-NUM-204-PM9 (500.0 mg, 1.16 mmol) and 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (823.6 mg, 3.48 mmol) (512.6 mg, 0.81 mmol, 70.02 %). C 26 H 43 N5O9P2, MS (ES): m / z (M-H - ) 630.3.

[0233] YK-NUM-204: 1 H NMR (CDC13, 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), 3.61 - 3.40 (m, 7H), 2.83 - 2.60 (m, 4H), 1.16 - 1.05 (m, 12H). 31 P NMR (162 MHz, DMSO-d6) δ 151.23, 150.81, 16.48, 16.02.

[0234] 6. Synthesis route of YK-NUM-205 is as follows

[0235]

[0236]

[0237] Step 1: Synthesis of YK-NUM-205-PM1

[0238] YK-NUM-201-PM1 (25.00 g, 59.28 mmol), 2-iodoxybenzoic acid (21.58 g, 77.06 mmol) were dissolved in acetonitrile (250.0 mL) and stirred at 70 °C overnight under nitrogen atmosphere. TLC showed the reaction was complete. The reaction was cooled down and filtered to remove the insoluble. The filtrate was concentrated under vacuum. The residue was dissolved in methyl tert-butyl ether and filtered again to remove the insoluble. The filtrate was washed with saturated sodium thiosulfate, saturated sodium bicarbonate and saturated brine successively. The organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The crude product YK-NUM-205-PM1 (28.25 g, 67.31 mmol, calculated as 100% purity) was obtained as a colorless liquid.

[0239] Step 2: Synthesis of YK-NUM-205-PM2

[0240] YK-NUM-205-PM1 (28.25 g, 67.31 mmol) was dissolved in THF (200.0 mL) and cooled to 0 °C under nitrogen atmosphere. 1M MgMeBr in THF (87.6 mL, 87.60 mmol) was added slowly. The reaction was allowed to warm to room temperature and stirred for 3 h. TLC showed the reaction was complete. The reaction was washed with saturated ammonium chloride solution successively. The organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to give the target product YK-NUM-205-PM2 (16.59 g, 38.07 mmol, 56.56%) as a liquid.

[0241] Step 3: Synthesis of YK-NUM-205-PM3

[0242] YK-NUM-205-PM2 (16.59 g, 38.07 mmol) was dissolved in DCM (150.0 mL) and cooled to 0 °C under nitrogen atmosphere. Diethylaminosulfur trifluoride (7.99 g, 49.57 mmol) was added slowly. The reaction was allowed to warm to room temperature and stirred for 3 h. TLC showed the reaction was complete. The reaction was washed with saturated sodium bicarbonate solution successively. The organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to give the target product YK-NUM-205-PM3 (7.12 g, 16.27 mmol, 42.72%) as a liquid.

[0243] Step 4: Synthesis of YK-NUM-205-PM4

[0244] Starting from uracil (4.56 g, 40.68 mmol) and YK-NUM-205-PM3 (7.12 g, 16.27 mmol), following the synthetic procedure of YK-NUM-201-PM4, the liquid target product YK-NUM-205-PM4 (4.47 g, 8.63 mmol, 53.07%) was obtained.C 23 H 42 N2O6FSi2, MS (ES): m / z (M-H - ) 516.3.

[0245] Step 5: Synthesis of YK-NUM-205-PM5

[0246] Starting from YK-NUM-205-PM4 (4.47 g, 8.63 mmol), following the synthetic procedure of YK-NUM-201-PM5, the solid target product YK-NUM-205-PM5 (2.30 g, 8.39 mmol, 97.14%) was obtained.C 11 H 15 N2O5F, MS (ES): m / z (M-H - ) 273.1.

[0247] Step 6: Synthesis of YK-NUM-205-PM6

[0248] Starting from YK-NUM-205-PM5 (2.30 g, 8.39 mmol) and DMTrCl (4.55 g, 13.43 mmol), following the synthetic procedure of YK-NUM-201-PM6, the solid target product YK-NUM-205-PM6 (2.89 g, 5.02 mmol, 59.76%) was obtained.C 32 H 33 FN2O7, MS (ES): m / z (M-H - ) 575.2.

[0249] Step 7: Synthesis of YK-NUM-205-PM7

[0250] Starting from YK-NUM-205-PM6 (2.89 g, 5.02 mmol) and TBDPSCl (11.03 g, 40.13 mmol), following the synthetic procedure of YK-NUM-201-PM7, the liquid target product YK-NUM-205-PM7 (7.51 g, 9.21 mmol, calculated as 100% purity) was obtained.C48 H 51 FN2O7Si, MS(ES): m / z(M-H - )813.3.

[0251] Step 8: Synthesis of YK-NUM-205-PM8

[0252] Starting from YK-NUM-205-PM7 (7.51 g, 9.21 mmol), following the procedure for the synthesis of YK-NUM-201-PM8, the target product YK-NUM-205-PM8 (1.47 g, 2.87 mmol, 31.12%) was obtained.C 27 H 33 FN2O5Si, MS(ES): m / z(M-H - )511.2.

[0253] Step 9: Synthesis of YK-NUM-205-PM9

[0254] Starting from YK-NUM-205-PM8 (1.47 g, 2.87 mmol), following the procedure for the synthesis of YK-NUM-201-PM9, YK-NUM-205-PM9 (2.03 g, 3.98 mmol, calculated as 100.0% purity) was obtained.C 27 H 31 N2O5FSi, MS(ES): m / z(M-H - )509.2.

[0255] Step 10: Synthesis of YK-NUM-205-PM10

[0256] Starting from tetraethyl methylenediphosphonate (2.86 g, 9.92 mmol) and YK-NUM-205-PM9 (2.03 g, 3.98 mmol), following the procedure for the synthesis of YK-NUM-201-PM10, the solid target product YK-NUM-205-PM10 (1.07 g, 1.66 mmol, 41.75%) was obtained.C 32 H 42 FN2O7SiP, MS(ES): m / z(M-H - )643.3.

[0257] Step 11: Synthesis of YK-NUM-205-PM11

[0258] YK-NUM-205-PM10 (1.07 g, 1.66 mmol) as a raw material, according to the synthetic method of YK-NUM-201-PM11, the solid target product YK-NUM-205-PM11 (593.2 mg, 1.46 mmol, 87.96%) was obtained. 16 H 24 FN2O7P, MS(ES): m / z(M-H - ) 405.1.

[0259] Step 12: Synthesis of YK-NUM-205

[0260] YK-NUM-205-PM11 (593.2 mg, 1.46 mmol) and 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (1.04 g, 4.39 mmol) as a raw material, according to the synthetic method of YK-NUM-201, the solid target product YK-NUM-205 (363.6 mg, 0.60 mmol, 41.06%) was obtained. 25 H 41 FN4O8P2, MS(ES): m / z(M-H - ) 605.2.

[0261] YK-NUM-205: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 8.89 (d, J = 8.6,1H), 7.73 (d, J = 7.2 Hz, 1H), 7.58 - 7.26 (m, 3H), 6.89 (d, J = 7.2Hz, 4H), 5.63 (d, J = 7.2 Hz, 1H), 5.31 (d, J = 7.2 Hz, 1H), 3.96(t, J = 10.8 Hz, 2H), 3.82 - 3.63(m, 5H), 3.31 - 3.19(m, 5H), 2.73- 2.58 (m, 5H), 1.82 (t, J = 12.4Hz, 1H), 1.28 - 1.07 (m, 12H). 31 P NMR(162 MHz, DMSO-d6) δ 153.31, 152.97, 16.68, 16.42.

[0262] 7. The synthetic route of YK-NUM-206 is as follows:

[0263]

[0264]

[0265] Step 1: Synthesis of YK-NUM-206-PM1

[0266] To the reaction flask was added YK-NUM-205-PM2 (16.41 g, 37.75 mmol), super dry THF (170.0 mL), followed by the dropwise addition of 60% NaH (9.06 g, 226.49 mmol) in portions under ice bath, and the mixture was stirred at room temperature for 20 min. Then dimethyl sulfate (19.04 g, 150.96 mmol) was added, and the mixture was stirred at room temperature for 6 h. TLC showed that the reaction was complete. The reaction was quenched by the slow addition of saturated NaHC03under ice bath, and extracted with ethyl acetate (400 mL x 2). The organic phase was dried and concentrated under vacuum. The residue was purified by column chromatography (petroleum ether / ethyl acetate) to give the target product YK-NUM-206-PM1 (14.36 g, 32.00 mmol, 84.77%) as a colorless transparent liquid.

[0267] Step 2: Synthesis of YK-NUM-206-PM2

[0268] The target product YK-NUM-206-PM2 (12.43 g, 23.51 mmol, 73.46%) was obtained as a white solid from uracil (7.17 g, 63.97 mmol) and YK-NUM-206-PM1 (14.36 g, 32.00 mmol) according to the synthetic method of YK-NUM-201-PM4. 24 H 44 N2O7Si2, MS (ES): m / z (M-H - ) 527.3.

[0269] Step 3: Synthesis of YK-NUM-206-PM3

[0270] The target product YK-NUM-206-PM3 (6.12 g, 21.38 mmol, 90.94%) was obtained as a white solid from YK-NUM-206-PM2 (12.43 g, 23.51 mmol) according to the synthetic method of YK-NUM-201-PM5. 12 H 18 N2O6, MS (ES): m / z (M-H - ) 285.1.

[0271] Step 4: Synthesis of YK-NUM-206-PM4

[0272] Starting from YK-NUM-206-PM3 (6.12 g, 21.38 mmol) and DMTrCl (10.14 g, 29.93 mmol), following the synthetic procedure of YK-NUM-201-PM6, the white solid target product YK-NUM-206-PM4 (7.02 g, 11.93 mmol, 55.78%) was obtained. C 33 H 36 N2O8, MS (ES): m / z (M-H - ) 587.3.

[0273] Step 5: Synthesis of YK-NUM-206-PM5

[0274] Starting from YK-NUM-206-PM4 (7.02 g, 11.93 mmol) and TBDPSCl (39.33 g, 143.09 mmol), following the synthetic procedure of YK-NUM-201-PM7, the yellow oily liquid target product YK-NUM-206-PM5 (26.34 g, 31.85 mmol, calculated as 100.0% purity) was obtained. C 49 H 54 N2O8Si, MS (ES): m / z (M-H - ) 825.4.

[0275] Step 6: Synthesis of YK-NUM-206-PM6

[0276] Starting from YK-NUM-206-PM5 (26.34 g, 31.85 mmol), following the synthetic procedure of YK-NUM-201-PM8, the white solid target product YK-NUM-206-PM6 (2.33 g, 4.44 mmol, 13.94%) was obtained. C 28 H 36 N2O6Si, MS (ES): m / z (M-H - ) 523.2.

[0277] Step 7: Synthesis of YK-NUM-206-PM7

[0278] To a reaction flask was added YK-NUM-206-PM6 (2.33 g, 4.44 mmol), IBX (1.62 g, 5.79 mmol) and acetonitrile (240.0 mL) sequentially, and the reaction was stirred at 40 °C for 4 h, MS showed the reaction was complete. After cooling to room temperature, filtration, the filtrate was concentrated, then dissolved in ethyl acetate (300.0 mL), washed with saturated NaHCO3solution and saturated Na2S2O3solution sequentially, dried and concentrated to give the target product YK-NUM-206-PM7 (2.52 g, 4.82 mmol, calculated as 100.0% purity) as a yellow oily liquid. 28 H 34 N2O6Si, MS(ES): m / z(M-H - )521.2.

[0279] Step 8: Synthesis of YK-NUM-206-PM8

[0280] Using tetraethyl methylenediphosphonate (3.20 g, 11.10 mmol) and YK-NUM-206-PM7 (2.52 g, 4.82 mmol) as starting materials, the target product YK-NUM-206-PM8 (2.06 g, 3.14 mmol, 65.05%) was obtained as a white solid according to the synthesis method of YK-NUM-201-PM10.C 33 H 45 N2O8PSi, MS(ES): m / z(M-H - )655.3.

[0281] Step 9: Synthesis of YK-NUM-206-PM9

[0282] Using YK-NUM-206-PM8 (2.06 g, 3.14 mmol) as starting material, the target product YK-NUM-206-PM9 (0.94 g, 2.25 mmol, 71.63%) was obtained as a white solid according to the synthesis method of YK-NUM-201-PM11.C 17 H 27 N2O8P, MS(ES): m / z(M-H - )417.2.

[0283] Step 10: Synthesis of YK-NUM-206

[0284] Starting from YK-NUM-206-PM9 (0.94 g, 2.25 mmol) and 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (1.60 g, 6.76 mmol), following the synthetic procedure of YK-NUM-201, the target product YK-NUM-206 was obtained as a white solid (0.62 g, 1.00 mmol, 44.61%). C 26 H 44 N4O9P2, MS (ES): m / z (M-H - ) 617.3.

[0285] YK-NUM-206: 1 H NMR (CDC13, 400 MHz, 298 K) δ 7.59 (d, J = 6.8 Hz, 1H), 6.95 (d, J = 6.6 Hz, 1H), 6.08 (d, J = 6.2 Hz, 1H), 5.93 (d, J = 5.8 Hz, 1H), 5.65 (d, J = 5.6 Hz, 1H), 4.69-4.61 (m, 5H), 4.08-4.06 (m, 3H), 3.59 (s, 3H), 3.38-3.35 (m, 1H), 3.03-3.01 (m, 2H), 2.78-2.75 (m, 3H), 1.38-1.24 (m, 21H); 31 P NMR (162 MHz, DMSO-d6) δ 152.19, 151.86, 15.97, 15.66.

[0286] 8. YK-NUM-207 synthesis route is as follows

[0287]

[0288]

[0289] Step 1: Synthesis of YK-NUM-207-PM1

[0290] Starting from YK-NUM-201-PM2 (28.88 g, 66.29 mmol) and n-hexylamine (11.45 g, 132.88 mmol), following the synthetic procedure of YK-NUM-201-PM3, the target product YK-NUM-207-PM1 was obtained as a liquid (16.05 g, 30.93 mmol, 46.67%). C 25 H 52NO6Si2, MS (ES): m / z (M+H + ) 519.3.

[0291] Step 2: Synthesis of YK-NUM-207-PM2

[0292] The solid target product YK-NUM-207-PM2 (15.30 g, 25.55 mmol, 82.59%) was obtained from uracil (15.60 g, 139.19 mmol) and YK-NUM-207-PM1 (16.05 g, 30.93 mmol) according to the synthetic method of YK-NUM-201-PM4. C 28 H 52 N3O7Si2, MS (ES): m / z (M-H - ) 597.3.

[0293] Step 3: Synthesis of YK-NUM-207-PM3

[0294] The solid target product YK-NUM-207-PM3 (7.54 g, 21.22 mmol, 83.05%) was obtained from YK-NUM-207-PM2 (15.30 g, 25.55 mmol) according to the synthetic method of YK-NUM-201-PM5. C 16 H 25 N3O6, MS (ES): m / z (M-H - ) 354.2.

[0295] Step 4: Synthesis of YK-NUM-207-PM4

[0296] The solid target product YK-NUM-207-PM4 (13.47 g, 20.48 mmol, 96.52%) was obtained from YK-NUM-207-PM3 (7.54 g, 21.22 mmol) and DMTrCl (10.07 g, 29.71 mmol) according to the synthetic method of YK-NUM-201-PM6. C 37 H 43 N3O8, MS (ES): m / z (M-H - ) 656.3.

[0297] Step 5: Synthesis of YK-NUM-207-PM5

[0298] Starting from YK-NUM-207-PM5 (31.44 g, 35.08 mmol) and TBDPSCl (19.59 g, 71.27 mmol), following the synthetic procedure of YK-NUM-201-PM8, the liquid target product YK-NUM-207-PM6 (4.59 g, 7.73 mmol, 22.03%) was obtained.C 53 H 61 N3O6Si, MS (ES): m / z (M-H - ) 592.3.

[0299] Step 7: Synthesis of YK-NUM-207-PM7

[0300] Starting from YK-NUM-207-PM6 (4.59 g, 7.73 mmol), following the synthetic procedure of YK-NUM-206-PM7, the liquid target product YK-NUM-207-PM7 (4.60 g, 7.77 mmol, calculated on 100.0% purity) was obtained.C 32 H 43 N3O6Si, MS (ES): m / z (M-H - ) 590.3.

[0301] Step 7: Synthesis of YK-NUM-207-PM7

[0302] Starting from YK-NUM-207-PM6 (4.59 g, 7.73 mmol), following the synthetic procedure of YK-NUM-206-PM7, the liquid target product YK-NUM-207-PM7 (4.60 g, 7.77 mmol, calculated on 100.0% purity) was obtained.C 32 H 41 N3O6Si, MS (ES): m / z (M-H - ) 590.3.

[0303] Step 8: Synthesis of YK-NUM-207-PM8

[0304] Starting from tetraethyl methylenediphosphonate (5.60 g, 19.43 mmol) and YK-NUM-207-PM7 (4.60 g, 7.77 mmol), following the synthetic procedure of YK-NUM-201-PM10, the solid target product YK-NUM-207-PM8 (2.49 g, 3.43 mmol, 44.13%) was obtained.C 37 H 52N3O8PSi, MS (ES): m / z (M-H - )724.3.

[0305] Step 9: Synthesis of YK-NUM-207-PM9

[0306] Starting from YK-NUM-207-PM8 (1.71 g, 2.37 mmol), following the procedure for the synthesis of YK-NUM-201-PM11, the solid target product YK-NUM-207-PM9 (795.4 mg, 1.63 mmol, 69.26%) was obtained. 21 H 34 N3O8P, MS (ES): m / z (M-H - )486.2.

[0307] Step 10: Synthesis of YK-NUM-207

[0308] Starting from YK-NUM-207-PM9 (795.4 mg, 1.63 mmol) and 2-cyanoethyl-N,N- diisopropyl chlorophosphoramidite (1737.2 mg, 7.34 mmol), following the procedure for the synthesis of YK-NUM-201, the white solid powder YK-NUM-207 (678.3 mg, 0.99 mmol, 60.45%) was obtained. 30 H 51 N5O9P2, MS(ES): m / z(M-H - )686.3.

[0309] YK-NUM-207: 1H NMR (CDCI3, 400 MHz, 298 K): δ 8.23 (d, J = 8.4 Hz, 1H), 7.63-7.59 (m, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.77-6.67 (m, 1H), 6.29 (d, J = 7.6 Hz, 1H), 5.44-5.22 (m, 4H), 5.25 (t, J = 7.4 Hz, 1H), 5.16-5.04 (m, 1H), 4.76-4.68 (m, 2H), 4.23 (t, J = 6.2 Hz, 1H), 4.04-3.96 (m, 2H), 3.68-3.62 (m, 2H), 3.42 (t, J = 6.8 Hz, 2H), 2.14-2.02 (m, 14H), 1.88 (d, J = 4.8 Hz, 12H), 1.73-1.67 (m, 3H); 31 P NMR (162 MHz, DMSO-d6) δ 153.96, 153.71, 16.98, 16.52.

[0310] 9. YK-NUM-208 synthesis route as follows

[0311]

[0312]

[0313] Step 1: Synthesis of YK-NUM-208-PM1

[0314] INT-1 (25.00 g, 61.93 mmol), 1M Tetrabutylammonium fluoride in tetrahydrofuran (248.0 mL, 247.71 mmol) was dissolved in THF (125.0 mL) and allowed to react at room temperature overnight. After the reaction was completed, the solvent was removed by vacuum concentration. The residue was purified by reverse phase preparative column (water / acetonitrile) to obtain a liquid product, which was purified by silica gel chromatography (dichloromethane / methanol) to obtain the solid target product YK-NUM-208-PM1 (9.43 g, 58.87 mmol, 95.07%).

[0315] Step 2: Synthesis of YK-NUM-208-PM2

[0316] Into a reaction flask was placed acetonitrile (250.0 mL), triethylamine trifluoride (9.50 g, 58.91 mmol), N-iodosuccinimide (15.90 g, 70.69 mmol), cooled to 0 °C, under nitrogen atmosphere, YK-NUM-208-PM1 (9.43 g, 58.87 mmol, dissolved in 100.0 mL of acetonitrile) was added slowly dropwise, the reaction was stirred at room temperature for 1 h after 30 min, TLC monitoring reaction was completed, saturated sodium sulfite solution (100.0 mL) was added, extracted with ethyl acetate (300 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum and reduced pressure to remove the solvent, the residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain the target product YK-NUM-208-PM2 (12.62 g, 41.23 mmol, 70.03%).

[0317] Step 3: Synthesis of YK-NUM-208-PM3

[0318] YK-NUM-208-PM2 (12.62 g, 41.23 mmol) was added to a reaction flask, respectively dissolved in dichloromethane (300.0 mL), dimethyl malonate (11.98 g, 90.68 mmol), trifluoromethanesulfonic acid (0.62 g, 4.13 mmol), stirred at room temperature for 6 h, TLC monitoring reaction was completed, saturated aqueous sodium carbonate solution was added to quench, extracted with dichloromethane (200 mL x 3), washed with saturated brine (20 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum and reduced pressure to remove the solvent, the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-208-PM3 (14.18 g, 35.97 mmol, 87.25%).

[0319] Step 4: Synthesis of YK-NUM-208-PM4

[0320] Into separate reaction flasks were added dichloromethane (300.0 mL), water (200.0 mL), tetrabutylammonium hydrogen sulfate (10.99 g, 32.37 mmol), di-potassium hydrogen phosphate (12.53 g, 71.94 mmol), m-chlorobenzoic acid (5.63 g, 35.96 mmol) and YK-NUM-208-PM3 (14.18 g, 35.97 mmol), cooled to 0 °C, slowly added benzoyl chloride (15.17 g, 85%, 107.92 mmol) dropwise under nitrogen atmosphere, warmed to room temperature and stirred for 8 h. The reaction was monitored by TLC. Slowly added saturated sodium sulfite aqueous solution dropwise. The solution changed from colorless to blue and then colorless. Extracted with ethyl acetate (200 mL x 3), washed with saturated brine (20 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to give the target product YK-NUM-208-PM4 (11.06 g, 26.16 mmol, 72.71%).

[0321] Step 5: Synthesis of YK-NUM-208-PM5

[0322] Into separate reaction flasks were added YK-NUM-208-PM4 (11.06 g, 26.16 mmol), saturated ammonia in methanol (750.0 mL), stirred at room temperature for 10 h. The reaction was monitored by TLC. Concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to give the target product YK-NUM-208-PM5 (5.72 g, 20.12 mmol, 76.92%).

[0323] Step 6: Synthesis of YK-NUM-208-PM6

[0324] YK-NUM-208-PM5 (5.72 g, 20.12 mmol), sodium tert-butoxide (8.31 g, 86.47 mmol) were dissolved in THF (50.0 mL), stirred at room temperature for 2 h under nitrogen atmosphere, cooled in ice bath, iodomethane (17.14 g, 120.76 mmol) was added dropwise, and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, saturated ammonium chloride solution was added, and the solvent was removed by vacuum concentration. Saturated sodium chloride solution was added, and dichloromethane (150.0 mL) was added for extraction. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum concentration. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain the target product YK-NUM-208-PM6 (3.96 g, 13.27 mmol, 65.97%) as a liquid.

[0325] Step 7: Synthesis of YK-NUM-208-PM7

[0326] YK-NUM-208-PM6 (3.96 g, 13.27 mmol) was added to 50% aqueous formic acid solution (80.0 mL), and the reaction was allowed to proceed at 80°C for 6 h. After the reaction was completed, the solvent was removed by vacuum concentration. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain the target product YK-NUM-208-PM7 (1.99 g, 9.47 mmol, 71.32%) as a white solid.

[0327] Step 8: Synthesis of YK-NUM-208-PM8

[0328] YK-NUM-208-PM7 (1.99 g, 9.47 mmol) was dissolved in pyridine (50.0 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane (2.99 g, 9.48 mmol) was added dropwise under nitrogen atmosphere. The reaction was allowed to proceed at room temperature overnight. After the reaction was completed, the organic phase was removed by vacuum concentration, dissolved in water, and extracted with dichloromethane (100.0 mL). The combined organic phase was washed with saturated sodium chloride aqueous solution (30.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum concentration. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate) to obtain YK-NUM-208-PM8 (3.42 g, 7.54 mmol, 79.62%).

[0329] Step 9: Synthesis of YK-NUM-208-PM9

[0330] Starting from uracil (1.69 g, 15.08 mmol) and YK-NUM-208-PM8 (3.42 g, 7.54 mmol), following the synthetic procedure of YK-NUM-201-PM4, the target product YK-NUM-208-PM9 (3.32 g, 6.22 mmol, 82.52%) was obtained.C 23 H 42 FN2O7Si2, MS (ES): m / z (M-H - ) 532.3.

[0331] Step 10: Synthesis of YK-NUM-208-PM10

[0332] Starting from YK-NUM-208-PM9 (3.32 g, 6.22 mmol), following the synthetic procedure of YK-NUM-201-PM5, the solid target product YK-NUM-208-PM10 (1.72 g, 5.93 mmol, 95.27%) was obtained.C 11 H 15 FN2O6, MS(ES): m / z (M-H - ) 289.1.

[0333] Step 11: Synthesis of YK-NUM-208-PM11

[0334] Starting from YK-NUM-208-PM10 (1.72 g, 5.93 mmol) and DMTrCl (2.81 g, 8.29 mmol), following the synthetic procedure of YK-NUM-201-PM6, the solid target product YK-NUM-208-PM11 (3.38 g, 5.70 mmol, 96.25%) was obtained.C 32 H 33 FN2O8, MS (ES): m / z (M-H - ) 591.2.

[0335] Step 12: Synthesis of YK-NUM-208-PM12

[0336] Starting from YK-NUM-208-PM11 (3.38 g, 5.70 mmol) and TBDPSCl (12.54 g, 45.62 mmol), following the synthetic procedure of YK-NUM-201-PM7, the liquid target product YK-NUM-208-PM12 (5.30 g, 6.38 mmol, calculated as 100.0% purity) was obtained.C 48 H51 FN2O8Si, MS (ES): m / z (M-H - ) 829.3.

[0337] Step 13: Synthesis of YK-NUM-208-PM13

[0338] Starting from YK-NUM-208-PM12 (5.30 g, 6.38 mmol), following the synthetic procedure of YK-NUM-201-PM8, the liquid target product YK-NUM-208-PM13 (1.46 g, 2.76 mmol, 43.30%) was obtained. 27 H 33 FN2O6Si, MS (ES): m / z (M-H - ) 527.2.

[0339] Step 14: Synthesis of YK-NUM-208-PM14

[0340] Starting from YK-NUM-208-PM13 (1.46 g, 2.76 mmol), following the synthetic procedure of YK-NUM-201-PM9, YK-NUM-208-PM14 (2.12 g, 4.03 mmol, calculated as 100.0% purity) was obtained. 27 H 31 N2O6SiF, MS(ES): m / z(M-H - ) 525.2.

[0341] Step 15: Synthesis of YK-NUM-208-PM15

[0342] Starting from tetraethyl methylenediphosphonate (2.90 g, 10.06 mmol) and YK-NUM-208-PM14 (2.12 g, 4.03 mmol), following the synthetic procedure of YK-NUM-201-PM10, the solid target product YK-NUM-208-PM15 (1.07 g, 1.62 mmol, 40.23%) was obtained. 32 H 42 FN2O8PSi, MS(ES): m / z(M-H - ) 659.2.

[0343] Step 16: Synthesis of YK-NUM-208-PM16

[0344] Starting from YK-NUM-208-PM15 (1.07 g, 1.62 mmol), following the synthetic procedure of YK-NUM-201-PM11, the solid target product YK-NUM-208-PM16 (624.4 mg, 1.48 mmol, 91.29%) was obtained. C 16 H 24 FN2O8P, MS (ES): m / z (M-H - ) 421.1

[0345] Step 17: Synthesis of YK-NUM-208

[0346] Starting from YK-NUM-208-PM16 (624.4 mg, 1.48 mmol) and 2-cyanoethyl-N,N- diisopropyl chlorophosphoramidite (1.05 g, 4.44 mmol), following the synthetic procedure of YK-NUM-201, the solid target product YK-NUM-208 (451.3 mg, 0.72 mmol, 49.03%) was obtained. C 25 H 41 FN4O9P2, MS (ES): m / z (M-H - ) 621.2.

[0347] YK-NUM-208: 1 H NMR (CDC13, 400 MHz, 298 K) δ 9.13 (d, J = 8.6, 1H), 8.26 (d, J = 7.2 Hz, 1H), 7.93 - 7.76 (m, 3H), 7.25 (d, J = 7.2 Hz, 4H), 6.34 (d, J = 7.2 Hz, 1H), 6.11 (d, J = 7.2 Hz, 1H), 5.80 (t, J = 10.8 Hz, 2H), 4.93 - 4.66 (m, 5H), 3.88 - 3.69 (m, 5H), 2.83 - 2.68 (m, 5H), 1.97 (t, J = 12.4 Hz, 1H), 1.33 - 1.17 (m, 12H); 31 P NMR (162 MHz, DMSO-d6) δ 148.83, 148.57, 17.03, 16.89.

[0348] 10. YK-NUM-209 synthesis route as follows

[0349]

[0350]

[0351] Step 1: Synthesis of YK-NUM-209-PM1

[0352] YK-NUM-208-PM5 (15.00 g, 52.76 mmol) was dissolved in DCM (150.0 mL) and cooled to 0 °C under a nitrogen atmosphere. Diethylaminosulfur trifluoride (11.06 g, 68.61 mmol) was added dropwise and the reaction was allowed to warm to room temperature and stirred for 3 h. The reaction was shown to be complete by TLC and was quenched with saturated sodium bicarbonate solution (3 x 100 mL). The organic phase was washed with saturated brine solution and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to give the target product YK-NUM-209-PM1 (7.75 g, 27.07 mmol, 51.31%) as a liquid.

[0353] Step 2: Synthesis of YK-NUM-209-PM2

[0354] YK-NUM-209-PM1 (7.75 g, 27.07 mmol) was used as the starting material and the synthesis was carried out according to the procedure described for YK-NUM-208-PM7 to give the target product YK-NUM-209-PM2 (3.12 g, 15.74 mmol, 58.16%) as a white solid.

[0355] Step 3: Synthesis of YK-NUM-209-PM3

[0356] YK-NUM-209-PM2 (3.12 g, 15.74 mmol) and 1,3-dichloro-1,1,3,3- tetraisopropyl disiloxane (4.97 g, 15.76 mmol) were used as the starting materials and the synthesis was carried out according to the procedure described for YK-NUM-208-PM8 to give YK-NUM-209-PM3 (4.83 g, 10.94 mmol, 69.46%).

[0357] Step 4: Synthesis of YK-NUM-209-PM4

[0358] Uracil (2.45 g, 21.86 mmol) and YK-NUM-209-PM3 (4.83 g, 10.94 mmol) were used as the starting materials and the synthesis was carried out according to the procedure described for YK-NUM-201-PM4 to give the target product YK-NUM-209-PM4 (3.57 g, 6.84 mmol, 62.57%).22 H 39 F2N2O6Si2, MS (ES): m / z (M-H - ) 520.2.

[0359] Step 5: Synthesis of YK-NUM-209-PM5

[0360] Starting from YK-NUM-209-PM4 (3.57 g, 6.84 mmol), following the procedure for the synthesis of YK-NUM-201-PM5, the solid target product YK-NUM-209-PM5 (1.74 g, 6.25 mmol, 91.40%) was obtained.C 10 H 12 F2N2O5, MS (ES): m / z (M-H - ) 277.1.

[0361] Step 6: Synthesis of YK-NUM-209-PM6

[0362] Starting from YK-NUM-209-PM5 (1.74 g, 6.25 mmol) and DMTrCl (2.97 g, 8.77 mmol), following the procedure for the synthesis of YK-NUM-201-PM6, the solid target product YK-NUM-209-PM6 (3.22 g, 5.55 mmol, 88.68%) was obtained.C 31 H 30 F2N2O7, MS (ES): m / z (M-H - ) 579.2.

[0363] Step 7: Synthesis of YK-NUM-209-PM7

[0364] Starting from YK-NUM-209-PM6 (3.22 g, 5.55 mmol) and TBDPSCl (12.20 g, 44.39 mmol), following the procedure for the synthesis of YK-NUM-201-PM7, the liquid target product YK-NUM-209-PM7 (6.13 g, 7.48 mmol, calculated as 100.0% purity) was obtained.C 47 H 48 F2N2O7Si, MS (ES): m / z (M-H - ) 817.3.

[0365] Step 8: Synthesis of YK-NUM-209-PM8

[0366] Starting from YK-NUM-209-PM7 (6.13 g, 7.48 mmol), following the synthetic procedure of YK-NUM-201-PM8, the liquid target product YK-NUM-209-PM8 (1.91 g, 3.70 mmol, 49.39%) was obtained.C 26 H 30 F2N2O5Si, MS (ES): m / z (M-H - ) 515.2.

[0367] Step 9: Synthesis of YK-NUM-209-PM9

[0368] Starting from YK-NUM-209-PM8 (1.91 g, 3.70 mmol), following the synthetic procedure of YK-NUM-201-PM9, YK-NUM-209-PM9 (2.36 g, 4.59 mmol, calculated as 100.0% purity) was obtained.C 26 H 28 N2F2O5Si, MS(ES): m / z(M-H - ) 513.2.

[0369] Step 10: Synthesis of YK-NUM-209-PM10

[0370] Starting from tetraethyl methylenediphosphonate (2.86 g, 9.92 mmol) and YK-NUM-209-PM9 (2.36 g, 4.59 mmol), following the synthetic procedure of YK-NUM-201-PM10, the solid target product YK-NUM-209-PM10 (1.23 g, 1.90 mmol, 41.34%) was obtained.C 31 H 39 F2N2O7PSi, MS(ES): m / z(M-H - ) 647.2.

[0371] Step 11: Synthesis of YK-NUM-209-PM11

[0372] Starting from YK-NUM-209-PM10 (1.23 g, 1.90 mmol), following the synthetic procedure of YK-NUM-201-PM11, the solid target product YK-NUM-209-PM11 (717.3 mg, 1.75 mmol, 92.20%) was obtained.C 15 H 21 F2N2O7P,MS(ES): m / z(M-H - ) 409.1.

[0373] Step 12: Synthesis of YK-NUM-209

[0374] YK-NUM-209 was obtained as a solid following the synthetic procedure of YK-NUM-201 starting from YK-NUM-209-PM11 (717.3 mg, 1.75 mmol) and 2-cyanoethyl-N,N- diisopropyl chlorophosphoramidite (1.24 g, 5.24 mmol). C 24 H 38 F2N4O8P2, MS (ES): m / z (M-H - ) 609.2.

[0375] YK-NUM-209: 1 H NMR (CDC13, 400 MHz, 298 K) δ 8.35 (s, 1H), 7.68-7.56 (m, 3H), 7.30-7.25 (m, 5H), 6.34 (d, J = 7.2 Hz, 1H), 6.11 (d, J = 7.2 Hz, 1H), 5.68 (t, J = 10.8 Hz, 2H), 4.76 - 4.58 (m, 4H), 3.92 - 3.79 (m, 4H), 2.58 - 2.38 (m, 4H), 1.87 (t, J = 12.4 Hz, 1H), 1.23 - 1.07 (m, 12H); 31 P NMR (162 MHz, DMSO-d6) δ 149.53, 149.07, 16.83, 16.56.

[0376] 11. YK-NUM-210 synthesis route as follows

[0377]

[0378]

[0379] Step 1: Synthesis of YK-NUM-210-PM1

[0380] YK-NUM-208-PM5 (20.00 g, 70.35 mmol), 2-iodoxybenzoic acid (25.61 g, 91.46 mmol) were dissolved in acetonitrile, the reaction was stirred at 75 °C overnight under nitrogen atmosphere. TLC showed the reaction was complete, the reaction was cooled, the insoluble was filtered out, the filtrate was concentrated under vacuum, dissolved in methyl tert-butyl ether, the insoluble was filtered out again. The mixture was washed with saturated sodium thiosulfate, saturated sodium bicarbonate, saturated brine in turn, the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, the filtrate was concentrated under vacuum. The crude product YK-NUM-210-PM1 (21.13 g, 74.86 mmol, calculated as 100% purity) was obtained as a colorless liquid.

[0381] Step 2: Synthesis of YK-NUM-210-PM2

[0382] YK-NUM-210-PM1 (21.13 g, 74.86 mmol) was dissolved in DCM (200.0 mL), the reaction was cooled to 0 °C under nitrogen atmosphere, diethylaminosulfur trifluoride (36.20 g, 224.58 mmol) was added dropwise, the reaction was kept for 4 h after the addition was completed, TLC showed the reaction was complete, the mixture was washed with saturated sodium bicarbonate for 5 times, saturated brine, the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to give the target product YK-NUM-210-PM2 (9.64 g, 31.68 mmol, 42.32%) as a liquid.

[0383] Step 3: Synthesis of YK-NUM-210-PM3

[0384] YK-NUM-210-PM2 (9.64 g, 31.68 mmol) was used as raw material, according to the synthesis method of YK-NUM-208-PM7, the target product YK-NUM-210-PM3 (4.21 g, 19.48 mmol, 61.47%) was obtained as a white solid.

[0385] Step 4: Synthesis of YK-NUM-210-PM4

[0386] YK-NUM-210-PM3 (4.21 g, 19.48 mmol) and 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane (6.14 g, 19.47 mmol) were used as raw materials, according to the synthesis method of YK-NUM-208-PM8, YK-NUM-210-PM4 (5.13 g, 11.16 mmol, 57.30%) was obtained.

[0387] Step 5: Synthesis of YK-NUM-210-PM5

[0388] The target product YK-NUM-210-PM5 (4.21 g, 7.80 mmol, 69.89%) was obtained from uracil (2.50 g, 22.30 mmol) and YK-NUM-210-PM4 (5.13 g, 11.16 mmol) following the procedure for the synthesis of YK-NUM-201-PM4. 22 H 38 F3N2O6Si2, MS (ES): m / z (M-H - ) 538.2.

[0389] Step 6: Synthesis of YK-NUM-210-PM6

[0390] The solid target product YK-NUM-210-PM6 (2.03 g, 6.85 mmol, 87.86%) was obtained from YK-NUM-210-PM5 (4.21 g, 7.80 mmol) following the procedure for the synthesis of YK-NUM-201-PM5. 10 H 11 F3N2O5, MS (ES): m / z (M-H - ) 295.1.

[0391] Step 7: Synthesis of YK-NUM-210-PM7

[0392] The solid target product YK-NUM-210-PM7 (3.51 g, 5.86 mmol, 85.56%) was obtained from YK-NUM-210-PM6 (2.03 g, 6.85 mmol) and DMTrCl (3.25 g, 9.59 mmol) following the procedure for the synthesis of YK-NUM-201-PM6. 31 H 29 F3N2O7, MS (ES): m / z (M-H - ) 597.2.

[0393] Step 8: Synthesis of YK-NUM-210-PM8

[0394] Starting from YK-NUM-210-PM7 (3.51 g, 5.86 mmol) and TBDPSCl (12.89 g, 46.90 mmol), following the synthetic procedure of YK-NUM-201-PM7, the liquid target product YK-NUM-210-PM8 (6.92 g, 8.27 mmol, calculated as 100.0% purity) was obtained.C 47 H 47 F3N2O7Si, MS (ES): m / z (M-H - ) 835.3.

[0395] Step 9: Synthesis of YK-NUM-210-PM9

[0396] Starting from YK-NUM-210-PM8 (6.92 g, 8.27 mmol), following the synthetic procedure of YK-NUM-201-PM8, the liquid target product YK-NUM-210-PM9 (2.35 g, 4.40 mmol, 53.17%) was obtained.C 26 H 29 F3N2O5Si, MS (ES): m / z (M-H - ) 533.2.

[0397] Step 10: Synthesis of YK-NUM-210-PM10

[0398] Starting from YK-NUM-210-PM9 (2.35 g, 4.40 mmol), following the synthetic procedure of YK-NUM-201-PM9, YK-NUM-210-PM10 (2.98 g, 5.60 mmol, calculated as 100.0% purity) was obtained.C 26 H 27 N2F3O5Si, MS (ES): m / z (M-H - ) 531.2.

[0399] Step 11: Synthesis of YK-NUM-210-PM11

[0400] Starting from tetraethyl methylenediphosphonate (4.03 g, 13.98 mmol) and YK-NUM-210-PM10 (2.98 g, 5.60 mmol), following the synthetic procedure of YK-NUM-201-PM10, the solid target product YK-NUM-210-PM11 (1.50 g, 2.25 mmol, 40.21%) was obtained.C 31 H 38F3N2O7PSi, MS (ES): m / z (M-H - ) 665.2.

[0401] Step 12: Synthesis of YK-NUM-210-PM12

[0402] Starting from YK-NUM-210-PM11 (1.50 g, 2.25 mmol), following the procedure for the synthesis of YK-NUM-201-PM11, the solid target product YK-NUM-210-PM12 (657.1 mg, 1.53 mmol, 68.19%) was obtained. 15 H 20 F3N2O7P, MS (ES): m / z (M-H - ) 427.1.

[0403] Step 12: Synthesis of YK-NUM-210

[0404] Starting from YK-NUM-210-PM12 (657.1 mg, 1.53 mmol) and 2-cyanoethyl-N,N- diisopropylchlorophosphoramidite (1.09 g, 4.61 mmol), following the procedure for the synthesis of YK-NUM-201, the solid target product YK-NUM-210 (471.8 mg, 0.75 mmol, 48.93%) was obtained. 24 H 37 F3N4O8P2, MS (ES): m / z (M-H - ) 627.2.

[0405] YK-NUM-210: 1 H NMR (CDC13, 400 MHz, 298 K) δ 8.41 (s, 1H), 7.77-7.62 (m, 3H), 7.41-7.25 (m, 5H), 6.38 (d, J = 7.2 Hz, 1H), 5.59 (t, J = 10.8 Hz, 2H), 4.92-4.68 (m, 4H), 3.81-3.69 (m, 4H), 2.77-2.58 (m, 4H), 1.68 (t, J = 12.4 Hz, 1H), 1.31-1.18 (m, 12H); 31 P NMR (162 MHz, DMSO-d6) δ 149.38, 148.89, 16.51, 16.27.

[0406] 12. Synthesis of compound 6

[0407]

[0408] According to the synthesis method of compound 6 in CN 118063533 B (paragraphs

[0146] -

[0155] of the specification), the product was obtained in 526 mg.

[0409] 13. Synthesis of compound 6H

[0410]

[0411] According to the synthesis method of compound 6H in CN 118063535 A (paragraphs

[0171] -

[0184] of the specification), the product was obtained in 603 mg.

[0412] Example 2: Synthesis of oligonucleotide

[0413] Instruments and reagents: Genesee 192 P model DNA / RNA automatic synthesizer, the solid phase carrier used for synthesis of antisense strand is a general carrier of cross-linked polystyrene beads, the model is Primer support 5G Unylinker 350 (cytiva manufacturer), and the solid phase carrier used for synthesis of sense strand is L96-PS carrier, the manufacturer is WuXi AppTec(Tianjin) Co., Ltd.

[0414] For example, the preparation method can include:

[0415] According to the monomer concentration of 0.15 M, the following nucleotide monomer solutions were prepared with acetonitrile: DMT-A-OMe phosphoramidite monomer (formula 1), DMT-C-OMe phosphoramidite monomer (formula 2), DMT-G-OMe phosphoramidite monomer (formula 3), DMT-U-OMe phosphoramidite monomer (formula 4), DMT-A-F phosphoramidite monomer (formula 5), DMT-C-F phosphoramidite monomer (formula 6), DMT-G-F phosphoramidite monomer (formula 7), DMT-U-F phosphoramidite monomer (formula 8), (E)-VP-Um, compound 6, compound 6H, YK-NUM-201, YK-NUM-202, YK-NUM-203, YK-NUM-204, YK-NUM-205, YK-NUM-206, YK-NUM-207, YK-NUM-208, YK-NUM-209 and YK-NUM-210.

[0416]

[0417] The structures of compound 6, compound 6H, YK-NUM-201, YK-NUM-202, YK-NUM-203, YK-NUM-204, YK-NUM-205, YK-NUM-206, YK-NUM-207, YK-NUM-208, YK-NUM-209 and YK-NUM-210 are shown in Example 1.

[0418] The 3' end of the siRNA sense strand is connected with the ligand L96, which is a GalNAc delivery carrier well known in the art (N-[tris(GalNAc-alkyl)-dodecanamido]-4-hydroxyprolinol, see US10465194B2, claim 10), wherein represents the position of connection with the siRNA via a phosphonate group or a phosphorothioate group.

[0419]

[0420] The siRNA antisense strand is not conjugated with GalNAc, and the siRNA antisense strand is synthesized on the corresponding solid support by phosphoramidite chemistry.

[0421] The nucleoside monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction by solid-phase phosphoramidite method. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and hydroxyl protection. When a phosphonate is used between two nucleotides, the four steps of deprotection, coupling, oxidation, and hydroxyl protection are included when connecting the next nucleoside monomer; when a phosphorothioate is used between two nucleotides, the four steps of deprotection, coupling, sulfurization, and hydroxyl protection are included when connecting the next nucleoside monomer.

[0422] (1) Deprotection

[0423] A 3% dichloroacetic acid toluene solution is used as a deprotection reagent to remove the DMT protecting group, and then acetonitrile is used for washing.

[0424] (2) Coupling

[0425] A 0.25M 5-ethylthiotetrazole is used as an activator for the acetonitrile solution of each nucleotide monomer for coupling, and then acetonitrile is used for washing.

[0426] (3) Oxidation / Sulfurization

[0427] Oxidation: A 0.05M iodine solution in pyridine / water (90 / 10) is used as an oxidizing agent for oxidation, and then acetonitrile is used for washing.

[0428] Sulfurization: A 3% hydrogenated xanthate solution in pyridine is used as a sulfurizing agent for sulfurization, and then acetonitrile is used for washing.

[0429] (4) Hydroxyl protection

[0430] Hydroxyl protection was performed using 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / n-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protection reagent, followed by washing with acetonitrile.

[0431] The above operation was repeated, and the above steps were performed in a set nucleotide arrangement sequence to obtain a sense strand product or an antisense strand product with a specific sequence arrangement.

[0432] (5) Deprotection of the last nucleotide DMT protecting group was performed using 3% dichloroacetic acid toluene solution as the deprotection reagent, followed by washing with acetonitrile.

[0433] (6) Amination and purification

[0434] After the reaction, the solid carrier was transferred to a reactor, and a TMSI / Py / DCM solution was added. After reaction at room temperature for 1 h, a 2-mercaptoethanol TEA / ACN solution was added to quench the reaction. Subsequently, concentrated ammonia water (25-28%) was added, and after amination at 60°C for 12 h, the system was reduced to room temperature, and the mixture was transferred to a pressure filter tank. The filter cake was washed with a mixture of purified water and ethanol, and the filtrate was combined and passed through a chromatography column. After concentration, freeze-drying was performed to obtain the product.

[0435] (7) Annealing and freeze-drying

[0436] After the purified sense strand and antisense strand were mixed at a molar ratio of 1:1, heating was performed to 95°C and maintained for 3 min, and then slow cooling to room temperature was performed to form an siRNA duplex. Subsequently, ultrafiltration concentration tubes were used for concentration, and the selection was 3KDa (Cobetter) or 1KDa (Pall). After completion of the concentration, a centrifugal concentrator was used for concentration to a freeze-dried state.

[0437] According to the above method, the freeze-dried products of 14 siRNAs (i.e., siRNAs numbered ON1-ON14) in Table 1 were prepared. Their common base sequence is as follows:

[0438] Sense strand: SEQ ID NO: 1: 5'-CCUGGACAUUCAGAACAAGAA-3'

[0439] Antisense strand: SEQ ID NO: 2: 5'-UUCUUGUUCUGAAUGUCCAGGGU-3'

[0440] The above base sequence is reported in the published patent WO2023241587A1 (the corresponding base sequence of siRNA with double-chain number DR005961 in the table on pages 45-46 of the specification) and the research paper published by Alnylam Pharmaceuticals in J. Med. Chem. 2018, 61, 734-744 (the corresponding base sequence of siRNA 2 in Table 1 on page 737), which can target and regulate the expression of mouse liver ApoB gene.

[0441] Table 1 Double-stranded oligonucleotides (dsRNA) with different modifications and corresponding mass spectrometry verification results

[0442]

[0443] wherein m represents 2'-OMe (2'-methoxy) modification, f represents 2'-F (2'-fluoro) modification, s represents that the nucleotide at this position is connected with the adjacent nucleotide on the right side by 3', 5'-thiophosphoric acid ester bond, the underlined bold letters represent the corresponding nucleotide after the modified nucleoside monomer YK-NUM-201 of the application is synthesized into the oligonucleotide molecule, and the subscript corresponds to the serial number of the modified nucleoside monomer, for example U num-201 represents that the nucleotide at this position is the corresponding nucleotide after the modified nucleoside monomer YK-NUM-201 of the application is synthesized into the oligonucleotide molecule.

[0444] All modified nucleoside monomers are connected with adjacent nucleosides in the antisense strand of the siRNA through thiophosphoric acid ester groups at the 2' position of the ribose ring and (E)-vinyl phosphate groups at the 5' position at the end of siRNA synthesis, that is, the protecting groups R6 and R7 in formula (I) are both removed. The structures of different modified nucleosides in the antisense strand are as follows:

[0445] U num-201 s: ; U num-202 s: ; U num-203 s: ; U num-204 s: ; U num-205 s: ; U num-206 s: ; U num-207 s: ; U num-208 s: ; U num-209 s: ; U num-210s: ; U m s: ; U (E)-VP-Um s: ; U6s: ; U 6H s: , wherein represents a linkage to an adjacent nucleoside.

[0446] It can be seen that, for the modified nucleoside monomers of the dsRNA in Table 1, U m s is a single modification at the ribose 2' position, and the others are a coordinated modification at the ribose 2' and 5' positions. The nucleoside monomers with a coordinated modification at the ribose 2' and 5' positions have the same structure at the 5' position, but different structures at the 2' position.

[0447] Example 3: Primary hepatocyte activity screening in C57BL / 6 wild-type mice

[0448] (1) Purpose of the experiment: test the efficiency of the modified nucleotides of the application in regulating gene expression.

[0449] (2) Free uptake or transfection

[0450] C57BL / 6 wild-type mouse liver primary cells were isolated, counted, plated in 24-well plates at 900 μL / well, 8x10 4 cells / well; and plated in 96-well plates at 100 μL / well, 1x10 4 cells / well. Then, free uptake or transfection was selected.

[0451] Free uptake: the siRNA lyophilisate prepared in Example 2 was dissolved using UltraPure TM DNase- / RNase-free Distilled Water (Invitrogen) to prepare a 100 μM siRNA stock solution, 10 μL of the stock solution was added to 90 μL Opti-MEM and mixed, and then added to the corresponding well, and incubated in a 37°C, 5% CO2 incubator for 24 h. The control group did not add siRNA.

[0452] Transfection: the siRNA lyophilisate prepared in Example 2 was dissolved using UltraPure TMDNase- / RNase-free Distilled Water (Invitrogen) was dissolved to prepare a 100 μΜ siRNA starting solution. 10 μΐ^of the starting solution was added to 40 μΐ^of Opti-MEM to prepare solution Z for standby; 3 μΐ^of RNAiMax transfection reagent (Invitrogen, 13778-150) was added to 47 μΐ^of Opti-MEM to mix and incubate for 5 min, and then mixed with solution Z, and left to stand at room temperature for 10 min. The mixture was added to the corresponding well, and incubated in a 37℃, 5% CO2 incubator for 24 h. The control group did not add siRNA.

[0453] (3) Fluorescent quantitative PCR

[0454] Total RNA was extracted using a high-throughput nucleic acid extractor-magnetic bead method (Fanneng Medical, FG0412; Hangzhou Aosheng, Auto-pure96), and after reverse transcription (PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B)), fluorescent quantitative PCR detection (TaqMan™ Fast Advanced Master Mix (ABI, 4444965)) was performed.

[0455] Table 2 Primer information

[0456]

[0457] (4) Data processing:

[0458] The ΔΔCt relative quantification method was used to calculate the RNA expression level of the target gene in the sample according to the Ct value of each sample. The relative expression of the target gene was expressed by 2 -ΔΔCt

[0459] The calculation formula is as follows:

[0460] ΔCt = average Ct value of target gene - average Ct value of internal reference gene;

[0461] ΔΔCt = ΔCt (experimental group) - ΔCt (blank control group);

[0462] Relative expression of target gene mRNA = 2 -ΔΔCt

[0463] Inhibition rate = (1 - relative expression of target gene mRNA) x 100%

[0464] The target gene is mAPOB, and the control gene is mGAPDH. ​

[0465] (5) mAPOB gene experimental results

[0466] Using the transfection method of step (2), C57BL / 6 wild-type mouse primary hepatocytes were selected, and the cells were plated in a 24-well plate according to the method of step (2). The siRNA lyophilisate prepared in Example 2 and UltraPure TM DNase- / RNase-free Distilled Water (Invitrogen) was used to prepare a siRNA stock solution with a concentration of 40 nM, and 10-fold gradient dilution was performed to prepare working solutions with 5 concentrations (40 nM, 4 nM, 0.4 nM, 0.04 nM, and 0.004 nM). C57BL / 6 wild-type mouse primary hepatocyte IC 50 Activity screening.

[0467] Table 3 IC of siRNA targeting mAPOB gene 50 Activity screening results

[0468]

[0469] It can be seen that the siRNA with coordinated modification of ribose 2' and 5' positions of the present application, ON3, ON5, ON6, ON7, ON8, ON9, and ON10, can efficiently inhibit the expression of ApoB gene in mouse primary hepatocytes.

[0470] As can be seen from Table 3, the IC 50 values of the siRNA sequences with coordinated modification of ribose 2' and 5' positions of the present application, ON3, ON5, ON6, ON7, ON8, ON9, and ON10, are all less than 0.02 nM, among which the IC 50 value of ON5 is the smallest, reaching 0.0093 nM. The IC 50 values of ON1, ON2, and ON4 are all more than 0.10 nM, which are 17 times, 61 times, and 17 times the IC 50 value of ON5, respectively. This indicates that ON3, ON5, ON6, ON7, ON8, ON9, and ON10 can efficiently inhibit the expression of ApoB gene in mouse primary hepatocytes.

[0471] Furthermore, compared with the siRNA sequences with compound modification of the prior art, the siRNA sequences with coordinated modification of ribose 2' and 5' positions of the present application, ON3, ON5, ON6, ON7, ON8, ON9, and ON10, have significantly improved inhibition rate on the expression of ApoB gene in mouse primary hepatocytes.

[0472] Existing siRNA modifications include Um-modified ON11, (E)-VP-Um-modified ON12, U6-modified ON13, and U... 6H Modified ON14 IC 50 The values ​​are 0.4068 nM, 0.1304 nM, 0.3371 nM, and 0.3135 nM, respectively. It can be seen that the ICs of ON11, ON12, ON13, and ON14... 50 The values ​​are significantly higher than those of ON3, ON5, ON6, ON7, ON8, ON9, and ON10, with the IC values ​​of ON11, ON12, ON13, and ON14 being even higher. 50 The values ​​all exceed 0.10 nM, which are for ON5 ICs. 50 The values ​​are 43 times, 14 times, 36 times, and 33 times.

[0473] Example 4: Effects of modified oligonucleotides on the inhibition of ApoB in mouse serum and on LDL-C levels

[0474] This embodiment investigates the inhibitory rate of siRNA on ApoB in mouse serum and its effect on LDL-C levels. Experimental Materials

[0475] Test sample: lyophilized siRNA prepared in Example 2

[0476] Preparation of test drug: The drug solvent was PBS buffer.

[0477] Preparation conditions: sterile environment

[0478] Storage conditions: Prepare fresh before use, and store remaining samples at -20℃.

[0479] The experimental animals were 45 male C57BL / 6 mice, aged 6-8 weeks, SPF grade, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and raised in accordance with ethical principles and common methods of experimental animal husbandry and management.

[0480] 1. Inhibition rate of modified siRNA on ApoB protein expression in mouse serum

[0481] In this embodiment, the ELISA method was used to determine the ApoB protein level.

[0482] The day of administration is recorded as day 0 (D0). About 200 μL of blood is taken from the medial canthus on day 3 (D-3) before the day of administration. The whole blood sample is temporarily stored in an ice box before centrifugation, centrifuged at about 3000 g at about 4°C for 10 min, and the ApoB protein level is determined using an ApoB kit (Mouse ApoB ELISA Kit, Abeam, ab230932), and the mice are grouped according to the ApoB protein level, with 6 mice in each group to ensure that the ApoB protein level of each group of mice is the same. On D0 (day 0), the mice are administered by subcutaneous injection, and the administration dose is 2 mg / kg. Blood is taken from the medial canthus on D7 (day 7), D14 (day 14), and D21 (day 21), respectively, and the serum is separated to detect the ApoB protein level.

[0483] The corresponding ratio is calculated according to the ApoB protein level on the determination day (D7, D14, D21) and the ApoB protein level on day 3 (D-3) before the day of administration, and the corresponding inhibition rate is obtained, as shown in Table 4 below.

[0484] Table 4: Inhibition rate of ApoB protein expression in mouse serum

[0485]

[0486] 1) The siRNAs with coordinated modification of ribose at 2' and 5' positions, ON3, ON5, ON6, and ON7, can efficiently inhibit the expression of ApoB protein in mouse serum.

[0487] From Table 4 and Figure 1 It can be seen that the siRNAs with coordinated modification of ribose at 2' and 5' positions, ON3, ON5, ON6, and ON7, all have a significant inhibitory effect on the expression of ApoB protein in mouse serum. On day 7, day 14, and day 21, the inhibition rates of ON3, ON5, ON6, and ON7 are all higher than those of ON11 and ON14. Among them, the inhibition rates of ON5 modified by YK-NUM-205 on day 7, day 14, and day 21 are 33.8%, 41.7%, and 42.9% higher than those of ON11 (2' single modification), respectively.

[0488] 2) The siRNAs with coordinated modification of ribose at 2' and 5' positions, ON3, ON5, ON6, and ON7, have a significantly improved inhibition rate on the expression of ApoB protein in mouse serum compared with the siRNAs modified by the compounds of the prior art.

[0489] Compared with the siRNAs modified by the compounds of the prior art, for example, U 6HThe modified ON14, the ON3, ON5, ON6 and ON7 of the present application have significantly increased inhibitory effect on the expression of ApoB protein in mouse serum. For example, compared with ON14, the inhibition rate of ON3 at 7th day, 14th day and 21st day is increased by 34.3%, 34.5%, 28.3% respectively, and the inhibition rate of ON5 at 7th day, 14th day and 21st day is increased by 40.9%, 41.9%, 33.6% respectively.

[0490] 2. Effect of siRNAs with different modifications on the LDL-C level in mouse serum

[0491] Experimental method: The day of administration is set as day 0 (D0), and about 200 μL of blood is taken from the medial canthus at 3 days before the administration day (D-3). The whole blood sample is temporarily stored in an ice box before centrifugation, centrifuged at about 3000 g at about 4°C for 10 min, and the LDL-C level is detected using a biochemical instrument (Dongsen full-automatic biochemical analyzer, model NT-1000) according to the manufacturer's instructions, and the mice are grouped according to the LDL-C level, with 6 mice in each group to ensure that the LDL-C level of the mice in each group is relatively uniform. The mice are subcutaneously injected with the drug at D0 (day 0) at a dose of 2 mg / kg. Blood is taken from the medial canthus at D7 (7th day), D14 (14th day) and D21 (21st day), and the serum LDL-C level is detected.

[0492] The percentage reduction of serum LDL-C is calculated according to the serum LDL-C level on the determination day (D7, D14, D21) and the serum LDL-C level at 3 days before the administration day (D-3), and the results are shown in Table 5 below.

[0493] Table 5: Percentage reduction of serum LDL-C

[0494]

[0495] It can be seen that the siRNA sequences with 2' and 5' position coordinated modification of ribose, ON3, ON5, ON6 and ON7 can significantly reduce the serum LDL-C protein level.

[0496] From Table 5 and Figure 2 It can be seen that the siRNA sequences with 2' and 5' position coordinated modification of ribose, ON3, ON5, ON6 and ON7 can significantly reduce the serum LDL-C protein level in mice. For example, the YK-NUM-205 modified ON5 at 7th day, 14th day and 21st day, the percentage reduction is 37.1%, 43.2% and 40.6% respectively, which is increased by 13.4%, 13.6% and 20.5% respectively compared with ON11 (2' position single modification).

[0497] In addition, the siRNAs of the present application, ON3, ON5, ON6 and ON7, have a significant increase in the percentage of serum LDL-C reduction compared to the prior art siRNAs with 2' and 5' position coordinated modification.

[0498] Compared to the prior art modified siRNAs, such as U 6H The siRNAs of the present application, ON3, ON5, ON6 and ON7, have a significant increase in the percentage of serum LDL-C reduction compared to the prior art siRNAs with 2' and 5' position coordinated modification. For example, compared to ON14, ON3 has an increase of 11.7%, 17.2%, 13.6% in the percentage of serum LDL-C reduction at day 7, day 14 and day 21, respectively, and ON5 has an increase of 18.2%, 20.8%, 16.9% in the percentage of serum LDL-C reduction at day 7, day 14 and day 21, respectively.

[0499] In summary, the present application introduces the 2' and 5' position coordinated modified ribose nucleoside at the first position of the 5' end of the antisense strand of the siRNA, which can significantly improve the stability of the siRNA and the activity of silencing the target gene.

[0500] The above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the present application; although the present application has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the present application. The whole scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A compound of Formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: ###0001### wherein, X is O or S; R1 is H or halo; R2 is H or halo; R4 is H, halo, C1-C3 alkyl, or forms a carbonyl with the carbon atom to which it is attached; R5 is H, a hydroxyl protecting group, or an active phosphorus group; R6 and R7 are each independently C2-C6 alkyl, -CH2CH2CN, or -CH2O(CO)C(CH3)3. ； The compound of Formula (I) satisfies one or more of the following conditions: (1) X is O; R1is optionally substituted with 1, 2, or 3 R a a substituted base, or a salt thereof, the base being , , or ; R a H, C1-C6 alkyl, -CH2R a-1 , -C(=O)OR a-1 , or -C(=O)R a-1 ; R a-1 C1-C6alkyl or C6-C 10 aryl; (3) R2 is H or F; R3is halogen, -OR b or -NR c R d ; R b R is H or C1-C6 alkyl; R c and R d each independently is H, C1-C8alkyl, -CH2CH2OR e or -C(=O)R e ; R e R is H or C1-C8alkyl; (5) R4 is H, F, methyl, or forms a carbonyl with the carbon atom to which it is attached; (6) R5 is a phosphoramidite group, a H-phosphonate group, a phosphotriester group, or a phosphorus-containing chiral auxiliary group; and (7) R6 and R7 are each independently C2-C6 alkyl. 2.The compound of formula (I) according to claim 1, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The compound of Formula (I) satisfies one or more of the following conditions: (1) when R2 is F, R3 is fluoro or methoxy, and R4 is H or fluoro; (2) R1is ; (2) when R2 is H, R4 is H, and R3 is -NHC(=O)CH3; (4) R3is halogen, -OCi-C3alkyl, -NHC(=0)R e or -NH(CH2)5CH3; R e is Ci-C3alkyl; (3) when R2 is H, R4 is methyl, and R3 is methoxy or fluoro; (4) when R2 is H, R4 forms a carbonyl with the carbon atom to which it is attached, and R3 is -NHCH2CH2OCH3, -N(CH2CH3)2, -N(CH3)2, or -NH(CH2)5CH3; and 3. The compound of formula (I) according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, characterized in that, (6) R6 and R7 are ethyl. The compound of Formula (I) is a compound of Formula (II), ###0002### The compound of Formula (II) is any one of the following structures: ###0003### The synthesis route of the compound of Formula (I) is shown below: which comprises the following steps: hydroxyl protection of compound 1-1 to obtain 1-2, oxidation to obtain 1-3, Wittig reaction to obtain 1-4, a series of reactions of 1-4 to obtain 1-5, different reaction steps of 1-5 to obtain 1-6, glycosylation reaction of 1-6 to obtain 1-7, and different reaction steps of 1-7 to obtain a compound of Formula (I), (5) R5 is ; W1 and W2 are hydroxyl protecting groups; X, R1, R2, R3, R4, R5, R6, and R7 are as defined in any one of claims 1-5.

4. The compound of formula (I) according to any one of claims 1-3, its pharmaceutically acceptable salt or its stereoisomer, characterized in that, which is obtained by polymerization of a compound of Formula (I) comprising any one of claims 1-5. 。 5. The compound of formula (I) according to claim 4, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The double-stranded oligonucleotide molecule further comprises a ligand covalently attached to the 5'-end or 3'-end of the sense strand of the double-stranded oligonucleotide molecule via a linker. 、 、 、 、 、 、 、 、 or .

6. A process for the preparation of a compound of formula (I) as claimed in any one of claims 1 to 5, characterized in that, The ligand is galactose, galactosamine, N-acetylgalactosamine, or a derivative thereof. ; The pharmaceutical composition comprises the nucleic acid polymer of any one of claims 8-11 and a pharmaceutically acceptable excipient. wherein R 11 is a hydroxyl substituent; 13. Use of the nucleic acid polymer of any one of claims 8-11 or the pharmaceutical composition of claim 12 in the preparation of a nucleic acid diagnostic agent and / or a nucleic acid therapeutic agent. ​ 7. The method for preparing the compound represented by formula (I) according to claim 6, characterized in that, R 11 is methyl or acetyl; W1and W2are trityl, benzoyl, 2,6-dichlorobenzoyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tetraisopropyldisiloxanyl, methoxymethyl ether, or dimethoxytrityl.

8. A nucleic acid polymer, characterized in that, ​ 9. The nucleic acid polymer of claim 8, wherein, The nucleic acid polymer is a double-stranded oligonucleotide molecule comprising a sense strand and an antisense strand, the nucleotide sequences of which are at least partially reverse complementary; wherein the 5'-end of the antisense strand contains a modified nucleotide, which is ; X1 is O or S.

10. The nucleic acid polymer of claim 9, wherein, ​ 11. The nucleic acid polymer of claim 10, wherein, ​ 12. A pharmaceutical composition, characterized by, ​ ​ 14. Use of a nucleic acid polymer according to any one of claims 8-11 or a pharmaceutical composition according to claim 12 for the manufacture of a medicament for the treatment and / or prevention of a pathological condition or disease caused by the expression of a specific gene.

Images