Nucleotide compounds with 2' ribose modifications that increase in vivo stability of oligonucleotides, and methods of synthesis and use thereof

Abstract

The application discloses a nucleotide compound with 2' ribose modification for increasing in-vivo stability of oligonucleotide and a synthesis method and application thereof, and particularly discloses a ribose 2' and 5' position synergistically modified nucleoside compound with a structure of formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof, a synthesis method and application thereof. The modified nucleoside compound is connected to the 5'-end of siRNA, so that the activity of the siRNA for inhibiting gene expression is improved, and the drug property of the siRNA in the body 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 the lipophilic cell membrane into cells. Therefore, the 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 cell toxicity related to off-target effects. Therefore, there is a need for siRNA drugs that have better target gene inhibition activity, higher bioavailability, and low toxicity. SUMMARY

[0004] This application provides a new class of 5'-(E)-VP modified nucleoside compounds. When using the modified nucleoside compounds of this application in the synthesis of siRNA, the activity of oligonucleotides in inhibiting gene expression can be enhanced or the toxicity can be reduced, thus improving the efficacy.

[0005] The technical solution adopted in this application is as follows:

[0006] This application provides, in one aspect, a compound of formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0007] ;

[0008] in,

[0009] X is either O or S;

[0010] R1 can be arbitrarily determined by one, two, or three Rs. a Substituted bases or their salts, wherein the base is , , or ;

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

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

[0013] R2 is H or a halogen;

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

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

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

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

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

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

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

[0021] In some implementations, X is O.

[0022] In some implementations, R1 is the -C(=O)R a-1 Replacement , , or ;R a-1 It is a C1-C6 alkyl or C6-C 10 Aryl.

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

[0024] In some implementations, R1 is .

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

[0026] In some embodiments, R3 is a halogen, -OC1-C3 alkyl, or -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 implementations, R4 is H, F, methyl, or forms a carbonyl group with the carbon atom to which it is attached.

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

[0030] In some implementations, 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 R4 forms a carbonyl group with the carbon atom it is attached to, R3 is -N(CH2CH3)2, -N(CH3)2, -NH(CH2)5CH3 or -NHCH2CH2OCH3; preferably -NH(CH2)5CH3.

[0033] In some implementation schemes, for , , , , , , , , or Preferred , , , , , or .

[0034] In some implementations, when R2 is F, R3 is fluorine or methoxy, and R4 is H or fluorine.

[0035] In some embodiments, when R2 is H, R4 forms a carbonyl group with the carbon atom it is attached to, and R3 is -NHCH2CH2OCH3, -N(CH2CH3)2, -N(CH3)2 or -NH(CH2)5CH3; preferably -NH(CH2)5CH3.

[0036] In some implementations, when R2 is H, R4 is methyl, and R3 is methoxy or fluorine.

[0037] In some implementations, when R2 is H, R4 is H, and R3 is -NHC(=O)CH3.

[0038] In some implementations, R5 is an active phosphorus group.

[0039] In some implementations, R5 is phosphorus amide, H-phosphate ester, triphosphate ester, or a phosphorus-containing chiral adjuvant.

[0040] In some implementations, R5 is .

[0041] In some implementations, R6 and R7 are independently C2-C6 alkyl groups.

[0042] In some implementations, R6 and R7 are ethyl.

[0043] In some embodiments, the compound shown in formula (I) is a compound shown in formula (II).

[0044] The definitions of R1, R2, R3, R4, R5, R6 and R7 are as described in any embodiment of the present invention.

[0045] In some implementations, X is 0;

[0046] R1 is ;

[0047] R2 is H or a halogen;

[0048] R3 is a halogen, -OC1-C3 alkyl group, or -NHC(=O)R. e or -NH(CH2)5CH3;

[0049] R4 is H, halogen, C1-C3 alkyl, or a carbonyl group formed with the carbon atom to which it is attached;

[0050] R5 is ;

[0051] R6 and R7 are ethyl groups.

[0052] In some embodiments, the compound represented by formula (II) has any of the following structures:

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

[0054] Another aspect of the present invention provides a method for preparing the compound as shown in formula (I) as described above, the synthetic route of which is shown below:

[0055]

[0056] It includes the following steps:

[0057] Compound 1-1 was protected with a hydroxyl group to give 1-2, which was then oxidized to give 1-3. A witting reaction was then performed to give 1-4. 1-4 underwent a series of reactions to give 1-5. 1-5 underwent different reaction steps to give 1-6. A glycosylation reaction was then performed to give 1-7. 1-7 underwent different reaction steps to give the compound shown in formula (I).

[0058] Where R 11 It is a hydroxyl substituent;

[0059] W1 and W2 are hydroxyl protecting groups;

[0060] X, R1, R2, R3, R4, R5, R6, and R7 are defined as described in any embodiment of the present invention.

[0061] In some implementation schemes, R 11 It can be methyl or acetyl.

[0062] In some embodiments, W1 and W2 are triphenylmethyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, tetraisopropyldisiloxyl, methoxymethyl ether or dimethoxytriphenylmethyl.

[0063] Another aspect of this application provides a nucleic acid polymer prepared by polymerization of a compound comprising the 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 anticomplementary; and a modified nucleotide N is contained at the 5'-terminus of the antisense strand. M The modified nucleotide is X1 is O or S; R1, R2, R3, R4 and X are defined as described in any embodiment of the present invention.

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

[0066] In some embodiments, the ligand L96 is a GalNac delivery vector well known in the art, wherein The location where the siRNA is attached via a phosphate ester group or a thiophosphate ester group can be seen, for example, in PCT publications WO2009073809 and WO2009082607.

[0067] .

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

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

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

[0071] (1) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 4;

[0072] (2) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 5;

[0073] (3) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 6;

[0074] (4) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 7;

[0075] (5) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 8;

[0076] (6) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 9;

[0077] (7) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 10;

[0078] (8) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 11;

[0079] (9) The sense chain has the sequence shown in SEQ ID NO: 3; and the antisense chain has the sequence shown in SEQ ID NO: 12;

[0080] (10) The positive chain has a sequence as shown in SEQ ID NO: 3; and the negative chain has a sequence as shown in SEQ ID NO: 13.

[0081] In some implementations, the sense chain 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 chains are shown in the table below:

[0082]

[0083] This application also provides a pharmaceutical composition comprising, as described above, a nucleic acid polymer and pharmaceutically acceptable excipients.

[0084] In another aspect, this application provides the use of the nucleic acid polymers or pharmaceutical compositions described above in the preparation of nucleic acid diagnostic agents and / or nucleic acid therapeutic agents.

[0085] In another aspect, this application provides the use of the nucleic acid polymers or pharmaceutical compositions described above in the preparation of medicaments for treating and / or preventing pathological conditions or diseases caused by the expression of a specific gene (e.g., the ApoB gene).

[0086] In some implementations, the disease is primary hyperlipidemia in adults.

[0087] This application also provides a method for inhibiting target gene expression, wherein an effective amount of the above-mentioned double-stranded oligonucleotide molecule or the above-mentioned pharmaceutical composition is administered to a subject; wherein the administration includes administration via subcutaneous or intravenous route;

[0088] The subjects are mammals, preferably humans.

[0089] It should be understood that the uses provided in this application for the above-described technical solutions can include both therapeutic and diagnostic uses, as well as non-therapeutic and non-diagnostic uses. For example, therapeutic uses may include using the nucleic acid polymers or compositions provided in this disclosure to treat diseases, improve symptoms, or regulate physiological activities in the body; diagnostic uses may include using the nucleic acid polymers or compositions provided in this disclosure to achieve the purpose of disease diagnosis; non-therapeutic / non-diagnostic uses may include using the nucleic acid polymers or compositions provided in this disclosure for non-therapeutic and non-diagnostic purposes such as scientific research and testing (e.g., conducting disease mechanism research, drug action mechanism research, new drug development, drug screening, etc.).

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

[0091] In this invention, "stereoisomer" refers to isomers produced by different spatial arrangements of atoms in a molecule, including but not limited to cis-trans isomers and enantiomers.

[0092] As used in this invention, the term "C1-C8" refers to a group having any integer number of carbon atoms in the main chain, ranging from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. Similarly, the term "C1-C6" refers to a group having any integer number of carbon atoms in the main chain, ranging from 1 to 6, such as 1, 2, 3, 4, 5, or 6 carbon atoms.

[0093] In this invention, "hydroxyl protecting group" refers to the protecting group that is commonly used to protect the hydroxyl group of the ribose structure in the synthesis of RNA or its derivatives, such as acetyl, phenoxyacetyl, neopentyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4,4'-dimethoxytriphenylmethyl (DMr, 4,4'-dimethoxytrityl), monomethoxytriphenylmethyl (MMTr, monomethoxytrityl), 9-phenyl-xanthen-9-yl (9-phenylxanthen-9-yl), 9-p-tolyl-xanthen-9-yl (9-(p-tolyl)-xanthen-9-yl), trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanoethoxymethyl, etc.

[0094] In this invention, "active phosphorus group" refers to a phosphorus-containing group capable of reacting with a hydroxyl or amino group contained in another molecule, particularly in another nucleotide unit or another nucleotide analog, via a nucleophilic attack reaction. Typically, such a reaction produces an ester-type nucleoside bond linking a nucleotide unit or nucleotide analog unit to another nucleotide unit or nucleotide analog unit. These active phosphorus groups are known in the art and contain P III or P V The phosphorus atom is in a valence state, and the active phosphorus group includes, but is not limited to, phosphoramidite group, H-phosphonate group, phosphate triester group, and phosphorus-containing chiral auxiliary group, such as... .

[0095] In this invention, "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0096] In this invention, "alkyl" refers to a straight-chain or branched alkyl group having a specified number of carbon atoms (e.g., C1-C6, C1-C8). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0097] In this invention, "aryl" refers to a ring with a specified number of carbon atoms (e.g., C6-C). 10 Aromatic rings, whose ring atoms consist only of carbon atoms, are either fused or monocyclic. Examples include phenyl or naphthyl.

[0098] The term "salt" refers to the corresponding salt that allows for the convenient or desirable preparation, purification, and / or treatment of the modified nucleoside compounds (or nucleotide compounds) of this invention, for example, a pharmaceutically acceptable salt. Unless otherwise stated, references to specific compounds in this invention also include their salt forms.

[0099] In this invention, "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for patient use) acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, and ammonium salts. 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 a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride salts, sulfate salts, and methanesulfonate salts.

[0100] In this invention, "pharmaceuticalally acceptable excipients" refers to all substances contained in a pharmaceutical preparation other than the active ingredient.

[0101] The term "nucleic acid polymer" can refer to any nucleic acid molecule, including but not limited to DNA, RNA, and their hybrids, including but not limited to single-stranded and double-stranded molecules. The number of nucleotides in the oligonucleotides formed by polymerization is 2, 3, or more; it can be a polymer with fewer than 20 nucleotides or a polymer with more than 20 nucleotides.

[0102] In this invention, "pharmaceutical composition" means a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and a pharmaceutically acceptable excipient, intended for use on mammals, such as humans, in need of such treatment.

[0103] In this invention, “treatment” refers to any of the following situations: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that triggers the disease; (3) slowing down the development of one or more biological manifestations of a disease.

[0104] In this invention, "prevention" 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 their hybrids, including but not limited to single-stranded and double-stranded molecules. The number of nucleotides in the oligonucleotides formed by polymerization is 2, 3, or more; it can be a polymer with fewer than 20 nucleotides or a polymer with more than 20 nucleotides.

[0106] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

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

[0108] 1) The modified nucleosides of this application introduce modifying groups at the 2' and 5' positions of the ribose, which is completely different from the structure of modified nucleosides in the prior art.

[0109] 2) The double-stranded oligonucleotide (dsRNA) molecules synthesized from the modified nucleosides in this application have significantly enhanced target gene interference activity compared with conventionally modified double-stranded oligonucleotide (dsRNA) molecules with ribose 2'-F or 2'-OMe.

[0110] 3) The double-stranded oligonucleotide (dsRNA) molecules synthesized by modifying nucleosides in this application have higher target gene interference activity compared with the double-stranded oligonucleotide (dsRNA) molecules synthesized by modifying nucleosides using existing technologies. Attached Figure Description

[0111] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are only used to illustrate some examples of this application, and are not intended to limit this application.

[0112] Figure 1 The modified double-stranded oligonucleotides ON3, ON5, ON6, ON7, ON11, and ON14 were used to inhibit the expression of ApoB protein in mouse serum on days 7, 14, and 21 after drug administration.

[0113] Figure 2 The percentage reduction in serum LDL-C in mice by the modified double-stranded oligonucleotides ON3, ON5, ON6, ON7, ON11, and ON14 on days 7, 14, and 21 after drug administration. Detailed Implementation

[0114] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. For example, the solvent selected and the corresponding volume ratio when used as the mobile phase can be performed according to conventional practices in the art. Reagents or instruments whose manufacturers are not specified are all products that can be obtained through commercial channels. The technical features involved in the various embodiments of this 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:

[0116] IBX: 2-Iodobenzoic acid; Dess-Martin: Dess-Martin oxidant; DEA: Diethylamine; TEA: Triethylamine; Ac2O: Acetic anhydride; HOAc: Acetic acid; conc H2SO4: Concentrated sulfuric acid; HDMS: Hexamethyldisilazane; BSA: N,O-bis(trimethylsilyl)acetamide; TMSOTf: Trimethylsilyl trifluoromethanesulfonate; Toluene: Toluene; MeOH: Methanol; DIEA: N,N-Diisopropylethylamine; DMAP: 4-Dimethylaminopyridine; DMSO: Dimethyl sulfoxide; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-Tetramethylurea hexafluorophosphate; THF: Tetrahydrofuran; TIPDSCl: 1,3-Dichloro-1,1,3, 3-Tetraisopropyldimethylsiloxane; Imidazole; DMF; TBAF; DMTrCl; DIAD; pyridine; IH-tetrazole; DCM; EA; DAST; PPh3; ACN; s-BuLi; t-BuONa; DCE; TEMPO; Ph3P + CH3Br - : Methyltriphenylphosphine bromide; H2O2: 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-butyldiphenylchlorosilane; 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 YK-NUM-201 to YK-NUM-210 of this application were synthesized via the following route.

[0119] Synthesis route:

[0120] In this embodiment, YK-NUM-201, YK-NUM-202, YK-NUM-204, and YK-NUM-207 are prepared by converting the alkenyl group of the key intermediate INT-I to a hydroxyl group via hydroboration oxidation, followed by oxidation to a carboxylic acid, and then condensation with different amino compounds. YK-NUM-203 is prepared by converting the alkenyl group of INT-I to a hydroxyl group via hydroboration oxidation, followed by further amylation and reaction with an acid, anhydride, or acyl halide. YK-NUM-205 and YK-NUM-206 are prepared by converting the alkenyl group of INT-I to a hydroxyl group via hydroboration oxidation, followed by oxidation to an aldehyde group, and then reaction with a Grignard reagent to prepare a secondary alcohol, followed by further fluorination or methylation. YK-NUM-208, YK-NUM-209, and YK-NUM-210 are prepared by asymmetric dihalogenation of the alkenyl group of INT-I, followed by hydroxylation, and then methylation, fluorination, oxidation, and other steps. This route enables rapid screening of different modifying 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). The mixture was cooled in an ice bath under nitrogen atmosphere, and 1,3-dichloro-1,1,3,3-tetraisopropyldimethylsiloxane (192.15 g, 609.17 mmol) was added dropwise. The mixture was stirred overnight at room temperature. After the reaction was complete, the organic phase was removed by vacuum concentration. The product was dissolved in water and extracted with dichloromethane. The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate) to give 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) and Desmartin oxidant (143.06 g, 337.29 mmol) were dissolved in acetonitrile (600 mL). The reaction mixture was stirred overnight at 45 °C under a nitrogen atmosphere. TLC showed complete reaction. The reaction solution was cooled, and insoluble matter was filtered off. The filtrate was concentrated under vacuum, dissolved in methyl tert-butyl ether, and insoluble matter was filtered off again. The solution was washed successively 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 colorless liquid target product, crude INT-I-PM2 (46.34 g, added to the next step at 100% purity), was obtained.

[0128] Step 3: Synthesis of INT-I

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

[0130] 2. The synthesis route of YK-NUM-201 is as follows:

[0131]

[0132]

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

[0134] Add 1 M boranetetrahydrofuran complex (185.8 mL, 185.79 mmol) to the reaction flask. Under nitrogen atmosphere, add INT-I (30.00 g, 74.31 mmol) in tetrahydrofuran solution dropwise at 0 °C. After the addition is complete, react at room temperature for 2 h. Then, add THF:H₂O (60.0 mL) in a 1:1 ratio to the above solution dropwise in an ice bath. After the addition is complete, react at room temperature for 3 h, then raise the temperature to 40 °C and react overnight. Cool the reaction solution in an ice bath, then add 2 M sodium hydroxide solution (148.6 mL, 297.26 mmol) and 30% hydrogen peroxide (114.2 mL, 1.19 mol) dropwise. After the addition is complete, react at room temperature for 15 h. TLC showed that the reaction was complete. Concentrate the reaction solution under vacuum. Extract the concentrated aqueous phase with EA (400 mL × 2) and dry it with anhydrous sodium sulfate. Filter the mixture, and concentrate the filtrate under vacuum. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the colorless liquid target product YK-NUM-201-PM1 (16.73 g, 39.67 mmol, 53.38%).

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

[0136] YK-NUM-201-PM1 (16.00 g, 37.94 mmol), iodophenyldiacetic acid (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 reacted at room temperature for 3 h under nitrogen protection. After the reaction was complete, the reaction solution was quenched with sodium thiosulfate solution, extracted with EA (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent, yielding YK-NUM-201-PM2 (19.68 g, 45.27 mmol, calculated at 100.0% purity). C 19 H 38 O7Si2, MS(ES): m / z(MH - )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, the mixture was cooled to 0°C in an ice-water bath. HATU (20.65 g, 54.32 mmol), DIEA (17.55 g, 135.81 mmol), and 2-methoxyethylamine (7.48 g, 99.59 mmol) were added to the mixture. After the addition was complete, the mixture was brought to room temperature and stirred for 2 h. After the reaction was complete, the reaction mixture was concentrated under vacuum. 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) and ammonium sulfate (389.6 mg, 2.95 mmol) were dissolved in pyridine (30.0 mL) and hexamethyldisilazane (150.0 mL). The mixture was activated in an oil bath at 130 °C for 1.5 h under nitrogen protection, cooled, and concentrated under vacuum to obtain a white solid. Then, YK-NUM-201-PM3 (7.25 g, 14.74 mmol) and acetonitrile (80.0 mL) were added. Under nitrogen protection, the mixture was cooled to 0 °C in an ice-water bath, and anhydrous tin tetrachloride (9.60 g, 36.85 mmol) was slowly added dropwise. After the reaction was completed, the mixture was kept at this temperature and stirred for 16 h, and then reacted in an oil bath at 37 °C for about 18 h. After the reaction was complete, the reaction solution was quenched with saturated sodium bicarbonate in an ice bath, filtered, washed with saturated brine (80 mL × 2), dried over anhydrous sodium sulfate, filtered again, concentrated under vacuum, and 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(MH - )570.3.

[0141] Step 5: Synthesize YK-NUM-201-PM5

[0142] YK-NUM-201-PM4 (3.08 g, 5.39 mmol) was dissolved in tetrahydrofuran (30.0 mL). Under nitrogen atmosphere, the mixture was cooled to 0 °C in an ice-water bath. 1 M TBAF in THF (215 mL, 21.54 mmol) was slowly added dropwise to the system. After the addition was complete, the mixture was brought to room temperature and stirred for 22 h. After the reaction was complete, the reaction mixture was concentrated under vacuum. The residue was purified by C18 reversed-phase column chromatography (acetonitrile / water) to obtain YK-NUM-201-PM5 (1.62 g, 4.92 mmol, 91.33%). 13 H 19 N3O7, MS(ES): m / z(MH - )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). After the addition was complete, the mixture was stirred at room temperature for 2 h under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was concentrated under vacuum, washed with EA (100.0 mL) and saturated brine (40 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and the residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-201-PM6 (1.97 g, 3.12 mmol, 63.40%). 34 H 37 N3O9, MS(ES): m / z(MH - 630.3.

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

[0146] YK-NUM-201-PM6 (1.97 g, 3.12 mmol) and imidazole (2.12 g, 31.14 mmol) were dissolved in DMF (18.0 mL). Under nitrogen atmosphere, the mixture was cooled to 0°C in an ice-water bath. TBDPSCl (5.14 g, 18.70 mmol) was slowly added dropwise to the above system. After the addition was complete, the mixture was brought to room temperature and stirred for 22 h, followed by reaction in an oil bath at 37°C for about 3 h. After the reaction was complete, the reaction mixture was added to EA (300.0 mL), washed with water (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent, yielding YK-NUM-201-PM7 (8.92 g, 10.25 mmol, calculated at 100.0% purity). C 50 H 55 N3O9Si, MS(ES): m / z(MH - )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 nitrogen atmosphere, the mixture was cooled to 0°C in an ice-water bath. TsOH buffer solution (2.00 g dissolved in 70.0 mL DCM and 30.0 mL MeOH) was slowly added dropwise to the system, and the mixture was stirred for 0.5 h. After the reaction was complete, saturated sodium bicarbonate was slowly added dropwise under ice bath conditions to quench the reaction. The mixture was then washed with DCM (300.0 mL) and saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and the residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-201-PM8 (1.64 g, 2.89 mmol, 28.18%). 29 H 37 N3O7Si, MS(ES): m / z(MH - )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). Under nitrogen atmosphere, the mixture was cooled to 0°C in an ice-water bath. Dess-Martin (1.23 g, 2.90 mmol) was slowly added to the system. After the addition was complete, the mixture was kept at this temperature and stirred for 1 h, then heated to room temperature and stirred for 3 h. After the reaction was complete, the reaction mixture was added to EA (200.0 mL), washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent, yielding YK-NUM-201-PM9 (2.06 g, 3.64 mmol, calculated at 100.0% purity). C 29 H 35 N3O7Si, MS(ES):m / z(MH - )564.2.

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

[0152] 60% sodium hydride (437.0 mg, 10.92 mmol) was dissolved in THF (10.0 mL). Under a nitrogen atmosphere, the mixture was cooled to -80°C, and tetraethylmethylene diphosphate (2.62 g, 9.09 mmol) was slowly added dropwise to the system. The mixture was stirred and kept at this temperature for 15 min. YK-NUM-201-PM9 (2.06 g, 3.64 mmol) was added, and the mixture was stirred and kept at this temperature for 10 min. The mixture was then allowed to rise naturally to room temperature. After the reaction was complete, the reaction was quenched by slowly adding saturated ammonium chloride aqueous solution in an ice bath. The mixture was then washed with EA (200.0 mL) and saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and the residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-NUM-201-PM10 (1.94 g, 2.77 mmol, 76.13%). 34 H 46 N3O9PSi, MS(ES): m / z(MH - 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 nitrogen atmosphere, the mixture was cooled to 0 °C in an ice-water bath. 1 M TBAF in THF (11.1 mL, 11.09 mmol) was slowly added dropwise to the system. After the addition was complete, the mixture was brought to room temperature and stirred for 22 h. After the reaction was complete, the reaction mixture was concentrated under vacuum. The residue was purified by C18 reversed-phase column chromatography (acetonitrile / water) to obtain YK-NUM-201-PM11 (602.5 mg, 1.31 mmol, 47.10%). 18 H 28 N3O9P, MS(ES): m / z(MH - 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), and 1-methylimidazole (107.2 mg, 1.31 mmol) were dissolved in dichloromethane (6.0 mL). Under a nitrogen atmosphere, the mixture was cooled to 0°C in an ice-water bath. 2-Cyanoethyl N,N-diisopropylchlorophosphine (927.2 mg, 3.92 mmol) was added to the mixture. After the addition was complete, the mixture was brought to room temperature and stirred for 1 h. After the reaction was complete, the reaction solution was washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain YK-NUM-201 (415.6 mg, 0.63 mmol, 48.11%). 27 H 45 N5O 10 P2, MS(ES): m / z(MH - 660.3.

[0157] YK-NUM-201: 1H NMR (CDCl3, 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 = 12.4Hz,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. The synthesis route for YK-NUM-202 is 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), cooled in an ice bath under nitrogen protection, and N,N-diisopropylethylamine (15.56 g, 120.40 mmol) was added dropwise. After the addition was complete, HATU (18.31 g, 48.15 mmol) was added, and the reaction was carried out at room temperature for 10 min. Diethylamine (6.46 g, 88.32 mmol) was then added, and the reaction was stirred overnight at room temperature. After the reaction was complete, 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%). 23 H 48 NO6Si2, MS(ES): m / z(M+H) + 491.3.

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

[0164] Using uracil (4.92 g, 43.89 mmol) and YK-NUM-202-PM1 (8.62 g, 17.56 mmol) as raw materials, the solid target product YK-NUM-202-PM2 (6.51 g, 11.40 mmol, 64.93%) was obtained by following the synthesis method of YK-NUM-201-PM4. 26 H 48 N3O7Si2, MS(ES): m / z(MH - )569.3.

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

[0166] Using YK-NUM-202-PM2 (6.51 g, 11.40 mmol) as the starting material, the solid target product YK-NUM-202-PM3 (1.53 g, 4.67 mmol, 40.99%) was obtained by following the synthesis method of YK-NUM-201-PM5. 14 H 21 N3O6, MS(ES): m / z(MH - )326.1.

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

[0168] Using YK-NUM-202-PM3 (1.53 g, 4.67 mmol) and DMTrCl (2.06 g, 6.08 mmol) as raw materials, the solid target product YK-NUM-202-PM4 (2.51 g, 3.99 mmol, 85.28%) was obtained by following the synthesis method of YK-NUM-201-PM6. 35 H 39 N3O8, MS(ES): m / z(MH - )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] Using YK-NUM-202-PM8 (1.38 g, 1.98 mmol) as the starting material, the solid target product YK-NUM-202-PM9 (676.0 mg, 1.47 mmol, 74.40%) was obtained by following the synthesis method of YK-NUM-201-PM11. 19 H 30 N3O8P, MS(ES): m / z(MH) - 458.2.

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

[0180] Using YK-NUM-202-PM9 (407.0 mg, 0.89 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramide (629.0 mg, 2.66 mmol) as starting materials, the solid target product YK-NUM-202 (305.3 mg, 0.46 mmol, 52.24%) was obtained according to the synthesis method of YK-NUM-201. 28 H 47 N5O9P2, MS(ES): m / z (MH - )658.3.

[0181] YK-NUM-202: 1 H NMR (CDCl3, 400MHz, 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) δ150.08, 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), and phthalimide (5.46 g, 37.11 mmol) were dissolved in tetrahydrofuran (200.0 mL), cooled to 0°C in an ice-water bath, and DIAD (10.62 g, 52.52 mmol) was added to the system. After the addition was complete, the mixture was brought to room temperature and stirred for 2 h. After the reaction was complete, the reaction mixture was concentrated under vacuum, extracted with EA (800 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) 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) and 85% hydrazine hydrate (31.36 g, 532.48 mmol) were dissolved in ethanol (300.0 mL) and reacted in an oil bath at 80 °C for 1 h under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was concentrated under vacuum, extracted with EA (900 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent, yielding 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) and triethylamine (7.91 g, 78.17 mmol) were dissolved in dichloromethane (100.0 mL). The mixture was cooled to 0°C in an ice-water bath under nitrogen atmosphere. Acetyl chloride (3.07 g, 39.11 mmol) was added to the mixture, and the mixture was then stirred at room temperature for 1 h. After the reaction was complete, the reaction mixture was concentrated under vacuum, extracted with DCM (600 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain 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] Using uracil (6.37 g, 56.83 mmol) and YK-NUM-203-PM3 (10.49 g, 22.72 mmol) as raw materials, YK-NUM-201-PM4 was synthesized according to the same method as YK-NUM-203-PM4 to obtain YK-NUM-203-PM4 (6.24 g, 11.52 mmol, 50.70%). 24 H 43 N3O7Si2, MS(ES): m / z(MH - 540.3.

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

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

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

[0196] Using YK-NUM-203-PM5 (3.25 g, 10.86 mmol) and DMTrCl (5.89 g, 17.38 mmol) as raw materials, YK-NUM-203-PM6 (4.89 g, 8.13 mmol, 74.84%) was synthesized according to the method for YK-NUM-201-PM6. 33 H 35 N3O8, MS(ES): m / z(MH - 600.2.

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

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

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

[0200] Using YK-NUM-203-PM7 (9.78 g, 11.64 mmol) as a raw material, YK-NUM-203-PM8 (3.47 g, 6.45 mmol, 55.43%) was synthesized according to the method for YK-NUM-201-PM8. C 28 H 35 N3O6Si, MS(ES): m / z(MH - )536.2.

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

[0202] Using YK-NUM-203-PM8 (3.47 g, 6.45 mmol) as the starting material, YK-NUM-203-PM9 (4.03 g, 7.52 mmol, calculated at 100.0% purity) was obtained following the synthesis method of YK-NUM-201-PM9. 28 H 33 N3O6Si, MS(ES): m / z(MH - )534.2.

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

[0204] Using tetraethylmethylene diphosphate (5.42 g, 18.81 mmol) and YK-NUM-203-PM9 (4.03 g, 7.52 mmol) as raw materials, YK-NUM-203-PM10 (1.26 g, 1.88 mmol, 25.00%) was synthesized according to the same method as YK-NUM-201-PM10. 33 H 44 N3O8PSi, MS(ES): m / z(MH - )668.3.

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

[0206] Using YK-NUM-203-PM10 (1.26 g, 1.88 mmol) as a starting material, YK-NUM-203-PM11 (654.1 mg, 1.52 mmol, 80.60%) was synthesized according to the method for YK-NUM-201-PM11. 17 H 26 N3O8P, MS(ES): m / z(MH - )430.2.

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

[0208] Using YK-NUM-203-PM11 (654.1 mg, 1.52 mmol) and 2-cyanoethyl N,N-diisopropylphosphorimide chloride (1.31 g, 4.55 mmol) as starting materials, YK-NUM-203 (413.7 mg, 0.66 mmol, 43.20%) was obtained by synthesizing YK-NUM-201 according to the same method. 26 H 43 N5O9P2, MS(ES): m / z(MH - 630.3.

[0209] YK-NUM-203: 1H NMR (CDCl3, 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] Using YK-NUM-201-PM2 (19.68 g, 45.27 mmol) and dimethylamine hydrochloride (9.23 g, 113.20 mmol) as raw materials, YK-NUM-204-PM1 (12.43 g, 26.92 mmol, 59.46%) was obtained by following the synthesis method of YK-NUM-202-PM3. 21 H 43 NO6Si2, MS(ES): m / z(M+H + )462.3.

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

[0216] Using uracil (7.54 g, 67.27 mmol) and YK-NUM-204-PM1 (12.43 g, 26.92 mmol) as raw materials, YK-NUM-204-PM2 (6.68 g, 12.33 mmol, 45.80%) was obtained by synthesizing YK-NUM-201-PM4 according to the same method. 24 H43 N3O7Si2, MS(ES): m / z(MH - 540.3.

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

[0218] Using YK-NUM-204-PM2 (6.68 g, 12.33 mmol) as a raw material, YK-NUM-204-PM3 (3.48 g, 11.63 mmol, 94.31%) was synthesized according to the method for YK-NUM-201-PM5. 12 H 17 N3O6, MS(ES): m / z(MH - )298.1.

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

[0220] Using YK-NUM-204-PM3 (3.48 g, 11.63 mmol) and DMTrCl (6.30 g, 18.59 mmol) as raw materials, YK-NUM-204-PM4 (4.84 g, 8.04 mmol, 69.18%) was obtained by synthesizing YK-NUM-201-PM6 according to the same method. 33 H 35 N3O8, MS(ES): m / z(MH - 600.2.

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

[0222] Using YK-NUM-204-PM4 (4.84 g, 8.04 mmol) and TBDPSCl (13.27 g, 48.28 mmol) as raw materials, YK-NUM-204-PM5 (9.83 g, 11.70 mmol, calculated at 100.0% purity) was obtained by following the synthesis method of YK-NUM-201-PM7. 49 H 53 N3O8Si, MS(ES): m / z(MH - )838.4.

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

[0224] Using YK-NUM-204-PM5 (9.83 g, 11.70 mmol) as a starting material, YK-NUM-204-PM6 (3.97 g, 7.38 mmol, 63.10%) was obtained by following the synthesis method of YK-NUM-201-PM8. 28 H 35 N3O6Si, MS(ES): m / z(MH - )536.2.

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

[0226] Using YK-NUM-204-PM6 (3.97 g, 7.38 mmol) as a starting material, YK-NUM-204-PM7 (4.03 g, 7.52 mmol, calculated at 100.0% purity) was obtained by following the synthesis method of YK-NUM-201-PM9. 28 H 33 N3O6Si, MS(ES):m / z(MH - )534.2.

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

[0228] Using tetraethylmethylene diphosphate (5.42 g, 18.8 mmol) and YK-NUM-204-PM7 (crude product 4.03 g, 7.52 mmol) as raw materials, YK-NUM-204-PM8 (2.26 g, 3.37 mmol, 44.85%) was synthesized according to the same method as YK-NUM-201-PM10. 33 H 44 N3O8PSi, MS(ES): m / z(MH - )668.3.

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

[0230] Using YK-NUM-204-PM8 (2.26 g, 3.37 mmol) as a starting material, YK-NUM-204-PM9 (974.8 mg, 2.26 mmol, 66.97%) was synthesized according to the same method as YK-NUM-201-PM11. 17 H 26 N3O8P, MS(ES): m / z(MH - )430.2.

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

[0232] Using YK-NUM-204-PM9 (500.0 mg, 1.16 mmol) and 2-cyanoethyl N,N-diisopropylphosphorimide chloride (823.6 mg, 3.48 mmol) as starting materials, YK-NUM-204 (512.6 mg, 0.81 mmol, 70.02%) was obtained by synthesizing YK-NUM-201 according to the same method. 26 H 43 N5O9P2, MS(ES): m / z(MH - 630.3.

[0233] YK-NUM-204: 1 H NMR (CDCl3, 400 MHz, 298 K) δ 7.41 (d, J = 8.6, 1H), 7.28 - 7.24 (m, 9H), 6.89 (d, J = 7.2 Hz, 4H), 6.15 (d, J = 7.8 Hz, 1H), 5.46(d, J = 8.4 Hz, 1H), 4.61 - 4.55 (m, 1H), 4.15 - 4.13 (m, 1H), 3.92 (t, J =10.8 Hz, 2H), 3.61 - 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. The 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) and 2-iodobenzoic acid (21.58 g, 77.06 mmol) were dissolved in acetonitrile (250.0 mL) and reacted overnight at 70 °C under nitrogen atmosphere with stirring. TLC showed complete reaction. The reaction solution was cooled, and insoluble matter was filtered off. The filtrate was concentrated under vacuum, dissolved in methyl tert-butyl ether, and insoluble matter was filtered off again. The solution was washed successively 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 colorless liquid target product, crude YK-NUM-205-PM1 (28.25 g, 67.31 mmol, calculated at 100% purity), was obtained.

[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), cooled to 0 °C under nitrogen atmosphere, and 1 M MgMeBr in tetrahydrofuran solution (87.6 mL, 87.60 mmol) was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 h. TLC showed that the reaction was complete. The mixture was washed successively with saturated ammonium chloride solution, extracted with ethyl acetate (300 mL × 2), and 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 obtain the liquid target product YK-NUM-205-PM2 (16.59 g, 38.07 mmol, 56.56%).

[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), cooled to 0 °C under a nitrogen atmosphere, and diethylaminosulfur trifluoride (7.99 g, 49.57 mmol) was slowly added dropwise. After the addition was complete, the reaction was maintained at this temperature for 3 h. TLC showed that the reaction was complete. The mixture was washed three times with saturated sodium bicarbonate and then with saturated brine. 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 obtain the liquid target product YK-NUM-205-PM3 (7.12 g, 16.27 mmol, 42.72%).

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

[0244] Using uracil (4.56 g, 40.68 mmol) and YK-NUM-205-PM3 (7.12 g, 16.27 mmol) as raw materials, the liquid target product YK-NUM-205-PM4 (4.47 g, 8.63 mmol, 53.07%) was obtained according to the synthesis method of YK-NUM-201-PM4. 23 H 42 N₂O₆FSi₂, MS(ES): m / z(MH) - )516.3.

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

[0246] Using YK-NUM-205-PM4 (4.47 g, 8.63 mmol) as the starting material, the solid target product YK-NUM-205-PM5 (2.30 g, 8.39 mmol, 97.14%) was obtained by following the synthesis method of YK-NUM-201-PM5. 11 H 15 N₂O₅F, MS(ES): m / z(MH) - )273.1.

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

[0248] Using YK-NUM-205-PM5 (2.30 g, 8.39 mmol) and DMTrCl (4.55 g, 13.43 mmol) as raw materials, the solid target product YK-NUM-205-PM6 (2.89 g, 5.02 mmol, 59.76%) was obtained by following the synthesis method of YK-NUM-201-PM6. 32 H 33 FN2O7, MS(ES): m / z(MH - )575.2.

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

[0250] Using YK-NUM-205-PM6 (2.89 g, 5.02 mmol) and TBDPSCl (11.03 g, 40.13 mmol) as raw materials, the liquid target product YK-NUM-205-PM7 (7.51 g, 9.21 mmol, calculated at 100% purity) was obtained according to the synthesis method of YK-NUM-201-PM7.48 H 51 FN2O7Si, MS(ES): m / z(MH - )813.3.

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

[0252] Using YK-NUM-205-PM7 (7.51 g, 9.21 mmol) as the starting material, the target product YK-NUM-205-PM8 (1.47 g, 2.87 mmol, 31.12%) was obtained by following the synthesis method of YK-NUM-201-PM8. 27 H 33 FN2O5Si, MS(ES): m / z(MH - )511.2.

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

[0254] Using YK-NUM-205-PM8 (1.47 g, 2.87 mmol) as the starting material, YK-NUM-205-PM9 (2.03 g, 3.98 mmol, calculated at 100.0% purity) was obtained following the synthesis method of YK-NUM-201-PM9. 27 H 31 N₂O₅FSi, MS(ES): m / z(MH) - )509.2.

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

[0256] Using tetraethyl methylene diphosphate (2.86 g, 9.92 mmol) and YK-NUM-205-PM9 (2.03 g, 3.98 mmol) as raw materials, the solid target product YK-NUM-205-PM10 (1.07 g, 1.66 mmol, 41.75%) was obtained by following the synthesis method of YK-NUM-201-PM10. 32 H 42 FN2O7SiP, MS(ES): m / z(MH - )643.3.

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

[0258] Using YK-NUM-205-PM10 (1.07 g, 1.66 mmol) as the starting material, the solid target product YK-NUM-205-PM11 (593.2 mg, 1.46 mmol, 87.96%) was obtained by following the synthesis method of YK-NUM-201-PM11. 16 H 24 FN2O7P, MS(ES): m / z(MH - 405.1.

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

[0260] Using YK-NUM-205-PM11 (593.2 mg, 1.46 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphine (1.04 g, 4.39 mmol) as starting materials, the solid target product YK-NUM-205 (363.6 mg, 0.60 mmol, 41.06%) was obtained according to the synthesis method of YK-NUM-201. 25 H 41 FN4O8P2, MS(ES): m / z(MH - 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 for YK-NUM-206 is as follows:

[0263]

[0264]

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

[0266] YK-NUM-205-PM2 (16.41 g, 37.75 mmol), ultra-dry THF (170.0 mL), and 60% NaH (9.06 g, 226.49 mmol) were added in portions under ice bath conditions, followed by reaction 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 complete reaction. The reaction was quenched by slow addition of saturated NaHCO3 under ice bath conditions, extracted with ethyl acetate (400 mL × 2), dried, and concentrated under vacuum. The residue was purified by column chromatography (petroleum ether / ethyl acetate) to obtain the colorless, transparent liquid target product YK-NUM-206-PM1 (14.36 g, 32.00 mmol, 84.77%).

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

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

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

[0270] Using YK-NUM-206-PM2 (12.43 g, 23.51 mmol) as a starting material, the target product YK-NUM-206-PM3 (6.12 g, 21.38 mmol, 90.94%) was synthesized as a white solid according to the method for YK-NUM-201-PM5. 12 H 18 N₂O₆, MS(ES): m / z(MH) - )285.1.

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

[0272] Using YK-NUM-206-PM3 (6.12 g, 21.38 mmol) and DMTrCl (10.14 g, 29.93 mmol) as raw materials, the target product YK-NUM-206-PM4 (7.02 g, 11.93 mmol, 55.78%) was synthesized as a white solid according to the method for YK-NUM-201-PM6. 33 H 36 N₂O₈, MS(ES): m / z(MH) - )587.3.

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

[0274] Using YK-NUM-206-PM4 (7.02 g, 11.93 mmol) and TBDPSCl (39.33 g, 143.09 mmol) as raw materials, the target product YK-NUM-206-PM5 (26.34 g, 31.85 mmol, calculated at 100.0% purity) was obtained by following the synthesis method of YK-NUM-201-PM7. 49 H 54 N₂O₈Si, MS(ES): m / z(MH) - )825.4.

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

[0276] Using YK-NUM-206-PM5 (26.34 g, 31.85 mmol) as the starting material, the target product YK-NUM-206-PM6 (2.33 g, 4.44 mmol, 13.94%) was synthesized as a white solid according to the method for YK-NUM-201-PM8. 28 H 36 N₂O₆Si, MS(ES): m / z(MH) - )523.2.

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

[0278] YK-NUM-206-PM6 (2.33 g, 4.44 mmol), IBX (1.62 g, 5.79 mmol), and acetonitrile (240.0 mL) were added sequentially to a reaction flask. The mixture was stirred at 40 °C for 4 h, and MS analysis showed that the reaction was complete. The mixture was cooled to room temperature and filtered. The filtrate was concentrated and dissolved in ethyl acetate (300.0 mL). The solution was washed successively with saturated NaHCO3 solution and saturated Na2S2O3 solution, dried, and concentrated to obtain the yellow oily liquid target product YK-NUM-206-PM7 (2.52 g, 4.82 mmol, calculated at 100.0% purity). C 28 H 34 N₂O₆Si, MS(ES): m / z(MH) - )521.2.

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

[0280] Using tetraethylmethylene diphosphate (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 synthesized as a white solid according to the same method as YK-NUM-201-PM10. 33 H 45 N₂O₈PSi, MS(ES): m / z(MH) - 655.3.

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

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

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

[0284] Using YK-NUM-206-PM9 (0.94 g, 2.25 mmol) and 2-cyanoethyl N,N-diisopropylphosphorimide chloride (1.60 g, 6.76 mmol) as starting materials, the target product YK-NUM-206 (0.62 g, 1.00 mmol, 44.61%) was obtained as a white solid following the synthesis method of YK-NUM-201. 26 H 44 N4O9P2, MS(ES): m / z(MH - )617.3.

[0285] YK-NUM-206: 1 H NMR (CDCl3, 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. The synthesis route for YK-NUM-207 is as follows:

[0287]

[0288]

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

[0290] Using YK-NUM-201-PM2 (28.88 g, 66.29 mmol) and n-hexylamine (11.45 g, 132.88 mmol) as raw materials, the liquid target product YK-NUM-207-PM1 (16.05 g, 30.93 mmol, 46.67%) was obtained according to the synthesis method of YK-NUM-201-PM3. 25 H 52NO6Si2, MS (ES): m / z(M+H + )519.3.

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

[0292] Using uracil (15.60 g, 139.19 mmol) and YK-NUM-207-PM1 (16.05 g, 30.93 mmol) as raw materials, the solid target product YK-NUM-207-PM2 (15.30 g, 25.55 mmol, 82.59%) was obtained by following the synthesis method of YK-NUM-201-PM4. 28 H 52 N3O7Si2, MS (ES): m / z (MH) - 597.3.

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

[0294] Using YK-NUM-207-PM2 (15.30 g, 25.55 mmol) as the starting material, the solid target product YK-NUM-207-PM3 (7.54 g, 21.22 mmol, 83.05%) was obtained by following the synthesis method of YK-NUM-201-PM5. 16 H 25 N3O6, MS(ES): m / z (MH - 354.2.

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

[0296] Using YK-NUM-207-PM3 (7.54 g, 21.22 mmol) and DMTrCl (10.07 g, 29.71 mmol) as raw materials, the solid target product YK-NUM-207-PM4 (13.47 g, 20.48 mmol, 96.52%) was obtained by following the synthesis method of YK-NUM-201-PM6. 37 H 43 N3O8, MS (ES): m / z (MH - )656.3.

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

[0298] Using YK-NUM-207-PM4 (5.86 g, 8.91 mmol) and TBDPSCl (19.59 g, 71.27 mmol) as raw materials, the liquid target product YK-NUM-207-PM5 (31.44 g, 35.08 mmol, calculated at 100.0% purity) was obtained by following the synthesis method of YK-NUM-201-PM7. 53 H 61 N3O8Si, MS (ES): m / z (MH - )894.4.

[0299] Step 6: Synthesis of YK-NUM-207-PM6

[0300] Using YK-NUM-207-PM5 (31.44 g, 35.08 mmol) as the starting material, the liquid target product YK-NUM-207-PM6 (4.59 g, 7.73 mmol, 22.03%) was obtained by following the synthesis method of YK-NUM-201-PM8. 32 H 43 N3O6Si, MS (ES): m / z (MH) - )592.3.

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

[0302] Using YK-NUM-207-PM6 (4.59 g, 7.73 mmol) as the starting material, the liquid target product YK-NUM-207-PM7 (4.60 g, 7.77 mmol, calculated at 100.0% purity) was obtained by following the synthesis method of YK-NUM-206-PM7. 32 H 41 N3O6Si, MS (ES): m / z (MH - 590.3.

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

[0304] Using tetraethyl methylene diphosphate (5.60 g, 19.43 mmol) and YK-NUM-207-PM7 (4.60 g, 7.77 mmol) as raw materials, the solid target product YK-NUM-207-PM8 (2.49 g, 3.43 mmol, 44.13%) was obtained by following the synthesis method of YK-NUM-201-PM10. 37 H 52N3O8PSi, MS (ES): m / z (MH - )724.3.

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

[0306] Using YK-NUM-207-PM8 (1.71 g, 2.37 mmol) as the starting material, the solid target product YK-NUM-207-PM9 (795.4 mg, 1.63 mmol, 69.26%) was obtained by following the synthesis method of YK-NUM-201-PM11. 21 H 34 N3O8P, MS (ES): m / z (MH) - 486.2.

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

[0308] Using YK-NUM-207-PM9 (795.4 mg, 1.63 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramide chloroform (1737.2 mg, 7.34 mmol) as raw materials, a white solid powder YK-NUM-207 (678.3 mg, 0.99 mmol, 60.45%) was obtained by following the synthesis method of YK-NUM-201. 30 H 51 N5O9P2, MS(ES): m / z(MH - 686.3.

[0309] YK-NUM-207: 1H NMR (CDCl3, 400 MHz, 298 K): δ8.23 (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. The synthesis route for YK-NUM-208 is as follows:

[0311]

[0312]

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

[0314] INT-I (25.00 g, 61.93 mmol) and 1 M tetrabutylammonium fluoride tetrahydrofuran solution (248.0 mL, 247.71 mmol) were dissolved in THF (125.0 mL) and reacted overnight at room temperature. After the reaction was complete, the solvent was removed by vacuum concentration. The residue was purified by reversed-phase preparative column chromatography (water / acetonitrile) to obtain a liquid product, which was then 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] Acetonitrile (250.0 mL), triethylamine trifluoride (9.50 g, 58.91 mmol), and N-iodosuccinimide (15.90 g, 70.69 mmol) were added to the reaction flask. The mixture was cooled to 0 °C, and under nitrogen atmosphere, YK-NUM-208-PM1 (9.43 g, 58.87 mmol, dissolved in 100.0 mL of acetonitrile) was slowly added dropwise. After reacting for 30 min, the mixture was heated to room temperature and stirred for 1 h. The reaction was monitored by TLC until completion. A saturated sodium sulfite solution (100.0 mL) was added, and the mixture was extracted with ethyl acetate (300 mL × 3). The extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum 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 dissolved in dichloromethane (300.0 mL), dimethyl malonate (11.98 g, 90.68 mmol), and trifluoromethanesulfonic acid (0.62 g, 4.13 mmol) respectively in a reaction flask. The mixture was stirred at room temperature for 6 h. After the reaction was completed by TLC, saturated sodium carbonate aqueous solution was added to quench the reaction. The mixture was extracted with dichloromethane (200 mL × 3), washed with saturated brine (20 mL × 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 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] Dichloromethane (300.0 mL), water (200.0 mL), tetrabutylammonium hydrogen sulfate (10.99 g, 32.37 mmol), dipotassium 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) were added to the reaction flask, respectively. The mixture was cooled to 0 °C, and under a nitrogen atmosphere, benzoyl chloride (15.17 g, 85%, 107.92 mmol) was slowly added dropwise. The mixture was then heated to room temperature and stirred for 8 h. The reaction was monitored by TLC until completion. A saturated sodium sulfite aqueous solution was slowly added dropwise. After the solution changed from colorless to blue and then back to colorless, it was extracted with ethyl acetate (200 mL × 3). The extract was then further purified with saturated saline solution (20 mL × 3). 3) Wash, dry with anhydrous sodium sulfate, filter, concentrate under vacuum to remove solvent, and purify the residue by silica gel chromatography (petroleum ether / ethyl acetate) to obtain 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] YK-NUM-208-PM4 (11.06 g, 26.16 mmol) and saturated ammonia-methanol solution (750.0 mL) were added to the reaction flask, and the mixture was stirred at room temperature for 10 h. The reaction was monitored by TLC until it was complete. The solvent was removed by vacuum concentration, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain 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) and sodium tert-butoxide (8.31 g, 86.47 mmol) were dissolved in THF (50.0 mL). The mixture was stirred at room temperature for 2 h under nitrogen protection, cooled in an ice bath, and iodomethane (17.14 g, 120.76 mmol) was added dropwise. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete, a saturated ammonium chloride solution was added, and the solvent was removed by vacuum concentration. A saturated sodium chloride solution was added, and the mixture was extracted with dichloromethane (150.0 mL). The extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate) to obtain the liquid target product YK-NUM-208-PM6 (3.96 g, 13.27 mmol, 65.97%).

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

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

[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), cooled in an ice bath under nitrogen atmosphere, and 1,3-dichloro-1,1,3,3-tetraisopropyldimethylsiloxane (2.99 g, 9.48 mmol) was added dropwise. The mixture was stirred overnight at room temperature. After the reaction was complete, the organic phase was removed by vacuum concentration, dissolved in water, and extracted with dichloromethane (100.0 mL). The combined organic phases were washed with saturated sodium chloride aqueous solution (30.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate) to give YK-NUM-208-PM8 (3.42 g, 7.54 mmol, 79.62%).

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

[0330] Using uracil (1.69 g, 15.08 mmol) and YK-NUM-208-PM8 (3.42 g, 7.54 mmol) as raw materials, the target product YK-NUM-208-PM9 (3.32 g, 6.22 mmol, 82.52%) was obtained by following the synthesis method of YK-NUM-201-PM4. 23 H 42 FN2O7Si2, MS (ES): m / z (MH) - 532.3.

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

[0332] Using YK-NUM-208-PM9 (3.32 g, 6.22 mmol) as the starting material, the solid target product YK-NUM-208-PM10 (1.72 g, 5.93 mmol, 95.27%) was obtained by following the synthesis method of YK-NUM-201-PM5. 11 H 15 FN2O6, MS(ES): m / z (MH - )289.1.

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

[0334] Using YK-NUM-208-PM10 (1.72 g, 5.93 mmol) and DMTrCl (2.81 g, 8.29 mmol) as raw materials, the solid target product YK-NUM-208-PM11 (3.38 g, 5.70 mmol, 96.25%) was obtained by following the synthesis method of YK-NUM-201-PM6. 32 H 33 FN2O8, MS (ES): m / z (MH - )591.2.

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

[0336] Using YK-NUM-208-PM11 (3.38 g, 5.70 mmol) and TBDPSCl (12.54 g, 45.62 mmol) as raw materials, the liquid target product YK-NUM-208-PM12 (5.30 g, 6.38 mmol, calculated at 100.0% purity) was obtained by following the synthesis method of YK-NUM-201-PM7. 48 H51 FN2O8Si, MS (ES): m / z (MH - )829.3.

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

[0338] Using YK-NUM-208-PM12 (5.30 g, 6.38 mmol) as the starting material, the liquid target product YK-NUM-208-PM13 (1.46 g, 2.76 mmol, 43.30%) was obtained by following the synthesis method of YK-NUM-201-PM8. 27 H 33 FN2O6Si, MS (ES): m / z (MH) - 527.2.

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

[0340] Using YK-NUM-208-PM13 (1.46 g, 2.76 mmol) as the starting material, YK-NUM-208-PM14 (2.12 g, 4.03 mmol, calculated at 100.0% purity) was obtained following the synthesis method of YK-NUM-201-PM9. 27 H 31 N₂O₆SiF, MS(ES): m / z(MH) - )525.2.

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

[0342] Using tetraethyl methylene diphosphate (2.90 g, 10.06 mmol) and YK-NUM-208-PM14 (2.12 g, 4.03 mmol) as raw materials, the solid target product YK-NUM-208-PM15 (1.07 g, 1.62 mmol, 40.23%) was obtained by following the synthesis method of YK-NUM-201-PM10. 32 H 42 FN2O8PSi, MS(ES): m / z(MH - 659.2.

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

[0344] Using YK-NUM-208-PM15 (1.07 g, 1.62 mmol) as the starting material, the solid target product YK-NUM-208-PM16 (624.4 mg, 1.48 mmol, 91.29%) was obtained by following the synthesis method of YK-NUM-201-PM11. 16 H 24 FN2O8P, MS(ES): m / z(MH) - 421.1

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

[0346] Using YK-NUM-208-PM16 (624.4 mg, 1.48 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphine (1.05 g, 4.44 mmol) as starting materials, the solid target product YK-NUM-208 (451.3 mg, 0.72 mmol, 49.03%) was obtained according to the synthesis method of YK-NUM-201. 25 H 41 FN4O9P2, MS(ES): m / z(MH - 621.2.

[0347] YK-NUM-208: 1 H NMR (CDCl3, 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 = 12.4Hz, 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. The synthesis route for YK-NUM-209 is 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), cooled to 0°C under a nitrogen atmosphere, and diethylaminosulfur trifluoride (11.06 g, 68.61 mmol) was slowly added dropwise. After the addition was complete, the reaction was maintained at this temperature for 3 h. TLC showed that the reaction was complete. The mixture was washed three times with saturated sodium bicarbonate and then with saturated brine. 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 obtain the liquid target product YK-NUM-209-PM1 (7.75 g, 27.07 mmol, 51.31%).

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

[0354] Using YK-NUM-209-PM1 (7.75 g, 27.07 mmol) as the starting material, the white solid target product YK-NUM-209-PM2 (3.12 g, 15.74 mmol, 58.16%) was obtained by following the synthesis method of YK-NUM-208-PM7.

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

[0356] YK-NUM-209-PM3 (4.83 g, 10.94 mmol, 69.46%) was obtained by using YK-NUM-209-PM2 (3.12 g, 15.74 mmol) and 1,3-dichloro-1,1,3,3-tetraisopropyldimethoxyether (4.97 g, 15.76 mmol) as raw materials, following the synthesis method of YK-NUM-208-PM8.

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

[0358] Using uracil (2.45 g, 21.86 mmol) and YK-NUM-209-PM3 (4.83 g, 10.94 mmol) as raw materials, the target product YK-NUM-209-PM4 (3.57 g, 6.84 mmol, 62.57%) was obtained by following the synthesis method of YK-NUM-201-PM4.22 H 39 F2N2O6Si2, MS (ES): m / z (MH) - 520.2.

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

[0360] Using YK-NUM-209-PM4 (3.57 g, 6.84 mmol) as the starting material, the solid target product YK-NUM-209-PM5 (1.74 g, 6.25 mmol, 91.40%) was obtained by following the synthesis method of YK-NUM-201-PM5. 10 H 12 F2N2O5, MS(ES): m / z (MH - 277.1.

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

[0362] Using YK-NUM-209-PM5 (1.74 g, 6.25 mmol) and DMTrCl (2.97 g, 8.77 mmol) as raw materials, the solid target product YK-NUM-209-PM6 (3.22 g, 5.55 mmol, 88.68%) was obtained according to the synthesis method of YK-NUM-201-PM6. 31 H 30 F2N2O7, MS (ES): m / z (MH - 579.2.

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

[0364] Using YK-NUM-209-PM6 (3.22 g, 5.55 mmol) and TBDPSCl (12.20 g, 44.39 mmol) as raw materials, the liquid target product YK-NUM-209-PM7 (6.13 g, 7.48 mmol, calculated at 100.0% purity) was obtained according to the synthesis method of YK-NUM-201-PM7. 47 H 48 F2N2O7Si, MS (ES): m / z (MH - 817.3.

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

[0366] Using YK-NUM-209-PM7 (6.13 g, 7.48 mmol) as the starting material, the liquid target product YK-NUM-209-PM8 (1.91 g, 3.70 mmol, 49.39%) was obtained by following the synthesis method of YK-NUM-201-PM8. 26 H 30 F2N2O5Si, MS (ES): m / z (MH) - )515.2.

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

[0368] Using YK-NUM-209-PM8 (1.91 g, 3.70 mmol) as the starting material, YK-NUM-209-PM9 (2.36 g, 4.59 mmol, calculated at 100.0% purity) was obtained following the synthesis method of YK-NUM-201-PM9. 26 H 28 N2F2O5Si, MS(ES): m / z(MH - )513.2.

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

[0370] Using tetraethyl methylene diphosphate (2.86 g, 9.92 mmol) and YK-NUM-209-PM9 (2.36 g, 4.59 mmol) as raw materials, the solid target product YK-NUM-209-PM10 (1.23 g, 1.90 mmol, 41.34%) was obtained according to the synthesis method of YK-NUM-201-PM10. 31 H 39 F2N2O7PSi, MS(ES): m / z(MH - 647.2.

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

[0372] Using YK-NUM-209-PM10 (1.23 g, 1.90 mmol) as the starting material, the solid target product YK-NUM-209-PM11 (717.3 mg, 1.75 mmol, 92.20%) was obtained by following the synthesis method of YK-NUM-201-PM11. 15 H 21 F2N2O7P, MS(ES): m / z(MH) - 409.1.

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

[0374] Using YK-NUM-209-PM11 (717.3 mg, 1.75 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramide (1.24 g, 5.24 mmol) as starting materials, the solid target product YK-NUM-209 (511.3 mg, 0.84 mmol, 47.90%) was obtained by following the synthesis method of YK-NUM-201. 24 H 38 F2N4O8P2, MS(ES): m / z(MH - 609.2.

[0375] YK-NUM-209: 1 H NMR (CDCl3, 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 = 12.4Hz, 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. The synthesis route for YK-NUM-210 is as follows:

[0377]

[0378]

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

[0380] YK-NUM-208-PM5 (20.00 g, 70.35 mmol) and 2-iodobenzoic acid (25.61 g, 91.46 mmol) were dissolved in acetonitrile and reacted overnight at 75 °C under nitrogen atmosphere with stirring. TLC showed complete reaction. The reaction solution was cooled, and insoluble matter was filtered off. The filtrate was concentrated under vacuum, dissolved in methyl tert-butyl ether, and insoluble matter was filtered off again. The solution was washed successively 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 target product YK-NUM-210-PM1 (21.13 g, 74.86 mmol, calculated at 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), cooled to 0 °C under a nitrogen atmosphere, and diethylaminosulfur trifluoride (36.20 g, 224.58 mmol) was slowly added dropwise. After the addition was complete, the reaction was maintained at this temperature for 4 h. TLC showed that the reaction was complete. The mixture was washed five times with saturated sodium bicarbonate and then with saturated brine. 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 obtain the liquid target product YK-NUM-210-PM2 (9.64 g, 31.68 mmol, 42.32%).

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

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

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

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

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

[0388] Using uracil (2.50 g, 22.30 mmol) and YK-NUM-210-PM4 (5.13 g, 11.16 mmol) as raw materials, the target product YK-NUM-210-PM5 (4.21 g, 7.80 mmol, 69.89%) was obtained by following the synthesis method of YK-NUM-201-PM4. 22 H 38 F3N2O6Si2, MS (ES): m / z (MH) - 538.2.

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

[0390] Using YK-NUM-210-PM5 (4.21 g, 7.80 mmol) as the starting material, the solid target product YK-NUM-210-PM6 (2.03 g, 6.85 mmol, 87.86%) was obtained by following the synthesis method of YK-NUM-201-PM5. 10 H 11 F3N2O5, MS(ES): m / z (MH - )295.1.

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

[0392] Using YK-NUM-210-PM6 (2.03 g, 6.85 mmol) and DMTrCl (3.25 g, 9.59 mmol) as raw materials, the solid target product YK-NUM-210-PM7 (3.51 g, 5.86 mmol, 85.56%) was obtained by following the synthesis method of YK-NUM-201-PM6. 31 H 29 F3N2O7, MS (ES): m / z (MH - 597.2.

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

[0394] Using YK-NUM-210-PM7 (3.51 g, 5.86 mmol) and TBDPSCl (12.89 g, 46.90 mmol) as raw materials, the liquid target product YK-NUM-210-PM8 (6.92 g, 8.27 mmol, calculated at 100.0% purity) was obtained according to the synthesis method of YK-NUM-201-PM7. 47 H 47 F3N2O7Si, MS (ES): m / z (MH - 835.3.

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

[0396] Using YK-NUM-210-PM8 (6.92 g, 8.27 mmol) as the starting material, the liquid target product YK-NUM-210-PM9 (2.35 g, 4.40 mmol, 53.17%) was obtained by following the synthesis method of YK-NUM-201-PM8. 26 H 29 F3N2O5Si, MS (ES): m / z (MH) - 533.2.

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

[0398] Using YK-NUM-210-PM9 (2.35 g, 4.40 mmol) as the starting material, YK-NUM-210-PM10 (2.98 g, 5.60 mmol, calculated at 100.0% purity) was obtained following the synthesis method of YK-NUM-201-PM9. 26 H 27 N2F3O5Si, MS (ES): m / z (MH) - )531.2.

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

[0400] Using tetraethyl methylene diphosphate (4.03 g, 13.98 mmol) and YK-NUM-210-PM10 (2.98 g, 5.60 mmol) as raw materials, the solid target product YK-NUM-210-PM11 (1.50 g, 2.25 mmol, 40.21%) was obtained according to the synthesis method of YK-NUM-201-PM10. 31 H 38F3N2O7PSi, MS(ES): m / z(MH - 665.2.

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

[0402] Using YK-NUM-210-PM11 (1.50 g, 2.25 mmol) as the starting material, the solid target product YK-NUM-210-PM12 (657.1 mg, 1.53 mmol, 68.19%) was obtained by following the synthesis method of YK-NUM-201-PM11. 15 H 20 F3N2O7P, MS(ES): m / z(MH - 427.1.

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

[0404] Using YK-NUM-210-PM12 (657.1 mg, 1.53 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphine (1.09 g, 4.61 mmol) as starting materials, the solid target product YK-NUM-210 (471.8 mg, 0.75 mmol, 48.93%) was obtained by following the synthesis method of YK-NUM-201. 24 H 37 F3N4O8P2, MS(ES): m / z(MH - 627.2.

[0405] YK-NUM-210: 1 H NMR (CDCl3, 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 = 12.4Hz, 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] Following the synthetic method of compound 6 in CN 118063533 B (paragraphs

[0146] -

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

[0409] 13. Synthesis of compound 6H

[0410]

[0411] Following the synthetic method of compound 6H in CN 118063535 A (paragraphs

[0171] -

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

[0412] Example 2: Synthesis of Oligonucleotides

[0413] Instruments and reagents: Qingke 192 P model DNA / RNA automated synthesizer. The solid-phase carrier used for antisense strand synthesis was a general-purpose carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (manufacturer: Cytiva). The solid-phase carrier used for sense strand synthesis was L96-PS carrier, manufactured by WuXi AppTec (Tianjin) Co., Ltd.

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

[0415] Based on a monomer concentration of 0.15 M, prepare solutions of the following nucleotide monomers using acetonitrile: DMT-A-OMe phosphorus amide monomer (Formula 1), DMT-C-OMe phosphorus amide monomer (Formula 2), DMT-G-OMe phosphorus amide monomer (Formula 3), DMT-U-OMe phosphorus amide monomer (Formula 4), DMT-AF phosphorus amide monomer (Formula 5), ​​DMT-CF phosphorus amide monomer (Formula 6), DMT-GF phosphorus amide monomer (Formula 7), DMT-UF phosphorus amide 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 compounds 6, 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's positive strand is linked to ligand L96, which is a well-known GalNAc delivery vector (N-[tris(GalNAc-alkyl)-dodecanoyl]-4-hydroxyproline, see US10465194B2, claim 10), wherein This indicates the location where the siRNA is linked via a phosphate ester group or a thiophosphate ester group.

[0419]

[0420] The siRNA antisense strand is not conjugated with GalNAc and is synthesized on a corresponding solid-phase support by phosphoramide chemistry.

[0421] Nucleoside monomers are linked sequentially from 3' to 5' along the nucleotide arrangement using a solid-phase phosphorous amide method. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and hydroxyl protection. Specifically, when two nucleotides are linked using a phosphate ester, the linkage of the subsequent nucleoside monomer involves these four steps; when two nucleotides are linked using a phosphate thioester, the linkage of the subsequent nucleoside monomer involves these four steps.

[0422] (1) Deprotection

[0423] The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0424] (2) Coupling

[0425] The acetonitrile solutions of each nucleotide monomer were coupled using 0.25M 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0426] (3) Oxidation / sulfidation

[0427] Oxidation: Oxidation was performed using a 0.05 M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile.

[0428] Vulcanization: Vulcanization is carried out using a pyridine solution of 3% hydroxanthin as a vulcanizing agent, followed by rinsing with acetonitrile.

[0429] (4) Hydroxyl protection

[0430] Hydroxyl protection was performed using a 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / azirmethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protecting agent, followed by rinsing with acetonitrile.

[0431] Repeat the above steps in a cyclical manner according to the set nucleotide arrangement order to obtain a sense or antisense product with a specific sequence arrangement.

[0432] (5) Use 3% dichloroacetic acid toluene solution as a deprotection agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0433] (6) Ammonolysis and purification

[0434] The reacted solid support was transferred to a reactor, and a TMSI / Py / DCM solution was added. After reacting at room temperature for 1 hour, the reaction was quenched by adding a 2-mercaptoethanol TEA / ACN solution. Subsequently, concentrated ammonia (25-28%) was added, and ammonolysis was maintained at 60°C for 12 hours. The system was then cooled to room temperature, and the mixture was transferred to a filter press. The filter cake was washed with a mixture of purified water and ethanol. The filtrates were combined, passed through a chromatography column, concentrated, and lyophilized to obtain the product.

[0435] (7) Annealing and freeze drying

[0436] The purified sense and antisense strands were mixed in a 1:1 molar ratio, heated to 95°C and held for 3 minutes, then slowly cooled to room temperature to form siRNA double strands. The mixture was then concentrated using an ultrafiltration concentrator, selecting either 3 kDa (Corbott) or 1 kDa (Pall). After concentration, it was further concentrated into a lyophilized state using a centrifuge.

[0437] The lyophilized products of the 14 siRNAs (i.e., siRNAs numbered ON1 to ON14) in Table 1 were prepared according to the method described above. Their common basic sequence is:

[0438] Chain of Justice: SEQ ID NO: 1: 5'-CCUGGACAUUCAGAACAAGAA-3'

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

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

[0441] Table 1. Different modified double-stranded oligonucleotides (dsRNA) and corresponding mass spectrometry verification results

[0442]

[0443] Wherein, m represents 2'-OMe (2'-methoxy) modification, f represents 2'-F (2'-fluorinated) modification, s indicates a nucleotide starting from the 5' end, where the nucleotide at this position is linked to its adjacent nucleotide to the right by a 3',5'-thiophosphate bond, the underlined bold letters represent the corresponding nucleotides synthesized from the modified nucleoside monomers in this application into the oligonucleotide molecule, and the subscripts correspond to the sequence numbers of the modified nucleoside monomers, for example... U num-201 The nucleotide at this position is the corresponding nucleotide synthesized from the modified nucleoside monomer YK-NUM-201 of this application into the oligonucleotide molecule.

[0444] At the end of siRNA synthesis, all modified nucleoside monomers have their ribose rings linked to adjacent nucleosides in the antisense strand of the siRNA via a thiophosphate group at the 2' position, and a (E)-vinyl phosphate group at the 5' position. This means that both the R6 and R7 protecting groups in structure (I) are removed. The different modified nucleoside structures 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: ,in This indicates a connection with an adjacent nucleoside.

[0446] As can be seen, for the modified nucleoside monomers of dsRNA in Table 1, U m s represents a single modification at the 2' position of the ribose, while the others represent co-modifications at the 2' and 5' positions of the ribose. Nucleoside monomers with co-modifications at the 2' and 5' positions of the ribose have the same 5' structure, differing only in the 2' position.

[0447] Example 3: Screening of the viability of primary hepatocytes from C57BL / 6 wild-type mice

[0448] (1) Experimental objective: To test the efficiency of the modified nucleotides in this application in regulating gene expression.

[0449] (2) Free intake or transfection

[0450] C57BL / 6 wild-type mouse primary liver cells were isolated, counted, seeded in 24-well plates, 900 μL / well, 8 × 10⁸ cells / well. 4 Cells / well; 96-well plate deposition, 100 μL / well, 1×10⁻⁶ cells / well; 96-well plate, 100 μL / well, 1×10⁻⁶ cells / well; 4 Cells / wells. Then, choose between free uptake or transfection.

[0451] Free intake: The siRNA lyophilized product prepared in Example 2 was used with UltraPure TM Dissolve 100 μM siRNA in DNase- / RNase-free distilled water (Invitrogen) to prepare a starting solution. Add 10 μL of this starting solution to 90 μL of Opti-MEM, mix well, and then add to the corresponding wells. Incubate at 37°C in a 5% CO2 incubator for 24 h. No siRNA was added to the control group.

[0452] Transfection: The lyophilized siRNA prepared in Example 2 was transfected using UltraPure. TMDissolve 100 μM siRNA in DNase- / RNase-free distilled water (Invitrogen) to prepare a starting solution. Add 10 μL of this starting solution to 40 μL of Opti-MEM and mix well to obtain solution Z. Add 3 μL of RNAiMax transfection reagent (Invitrogen, 13778-150) to 47 μL of Opti-MEM and incubate for 5 min. Mix the incubator with solution Z, incubate at room temperature for 10 min, then add to the corresponding wells and incubate at 37℃ in a 5% CO2 incubator for 24 h. No siRNA was added to the control group.

[0453] (3) Real-time PCR

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

[0455] Table 2 Primer Information

[0456]

[0457] (4) Data processing:

[0458] The relative expression level of the target gene RNA in each sample was calculated using the ΔΔCt relative quantification method based on the Ct value. The relative expression level of the target gene was determined using a 2-ΔCt method. -ΔΔCt express.

[0459] The calculation formula is as follows:

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

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

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

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

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

[0465] (5) mAPOB gene experimental results

[0466] Using the transfection method in step (2), primary C57BL / 6 wild-type mouse hepatocytes were selected and plated in 24-well plates according to the method in step (2). The lyophilized siRNA prepared in Example 2 and UltraPure were used. TM DNase- / RNase-free distilled water (Invitrogen) was used to prepare a 40 nM siRNA stock solution, which was then serially diluted 10-fold to prepare five working solutions (40 nM, 4 nM, 0.4 nM, 0.04 nM, and 0.004 nM). These solutions were then used for IC50 assays in C57BL / 6 wild-type mouse primary hepatocytes. 50 Activity screening.

[0467] Table 3. IC50 of siRNAs targeting the mAPOB gene 50 Activity screening results

[0468]

[0469] It is evident that the siRNAs ON3, ON5, ON6, ON7, ON8, ON9, and ON10, which are co-modified at the 2' and 5' positions of the ribosome in this application, can effectively inhibit the expression of the ApoB gene in primary mouse hepatocytes.

[0470] As shown in Table 3, the IC50 values ​​of the siRNA sequences co-modified at the 2' and 5' positions of the ribose in this application, specifically ON3, ON5, ON6, ON7, ON8, ON9, and ON10, are... 50 The values ​​are all less than 0.02 nM, among which the ON5 IC 50 The minimum value was 0.0093 nM. The ICs for ON1, ON2, and ON4... 50 The values ​​all exceed 0.10 nM, which are for ON5 ICs. 50 The values ​​were 17, 61, and 17 times higher. This indicates that ON3, ON5, ON6, ON7, ON8, ON9, and ON10 can effectively inhibit the expression of the ApoB gene in primary mouse hepatocytes.

[0471] Furthermore, the siRNA sequences ON3, ON5, ON6, ON7, ON8, ON9, and ON10, which are co-modified at the 2' and 5' positions of the ribose in this application, significantly enhance the inhibition rate of ApoB gene expression in mouse primary hepatocytes compared with siRNA sequences modified by prior art compounds.

[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 drug administration was recorded as day 0 (D0). On day 3 prior to drug administration (D-3), approximately 200 μL of blood was collected via the inner canthus of the eye. The whole blood sample was temporarily stored on ice before centrifugation at approximately 4°C and approximately 3000 g for 10 min. ApoB protein levels were measured using an ApoB ELISA Kit (Mouse ApoB ELISA Kit, Abea, ab230932), and mice were grouped accordingly, with 6 mice per group to ensure uniform ApoB levels within each group. The drug was administered subcutaneously on day 0 (D0) at a dose of 2 mg / kg. Blood was collected via the inner canthus of the eye on days 7 (D7), 14 (D14), and 21 (D21), and serum was separated to detect ApoB protein levels.

[0483] The corresponding inhibition rates were calculated based on the ApoB protein levels on the measurement days (D7, D14, D21) and the ApoB protein levels on day 3 before the administration date (D-3), as shown in Table 4 below.

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

[0485]

[0486] 1) The siRNAs with co-modified ribose positions 2' and 5' of this application, ON3, ON5, ON6 and ON7, can effectively inhibit the expression of ApoB protein in mouse serum.

[0487] From Table 4 and Figure 1 It can be seen that the siRNAs with synergistic modifications at the 2' and 5' positions of the ribose in this application, namely ON3, ON5, ON6, and ON7, all exhibited significant inhibitory effects on ApoB protein expression in mouse serum. On days 7, 14, and 21, the inhibition rates of ON3, ON5, ON6, and ON7 were all higher than those of ON11 and ON14. Specifically, the inhibition rate of ON5 modified with YK-NUM-205 on days 7, 14, and 21 was 33.8%, 41.7%, and 42.9% higher than that of ON11 (single modification at the 2' position), respectively.

[0488] 2) The siRNAs ON3, ON5, ON6 and ON7, which are co-modified at the 2' and 5' positions of the ribose in this application, significantly enhance the inhibition rate of ApoB protein expression in mouse serum compared with siRNAs modified by compounds in the prior art.

[0489] Compared to existing technologies that modify siRNA, such as U 6HThe modified ON14, and the ON3, ON5, ON6, and ON7 of this application, significantly increased the inhibitory effect on ApoB protein expression in mouse serum. For example, compared with ON14, the inhibition rates of ON3 on days 7, 14, and 21 were increased by 34.3%, 34.5%, and 28.3%, respectively, and the inhibition rates of ON5 on days 7, 14, and 21 were increased by 40.9%, 41.9%, and 33.6%, respectively.

[0490] 2. Effects of different modified siRNAs on LDL-C levels in mouse serum

[0491] Experimental Methods: Day 0 (D0) was designated as the day of drug administration. Approximately 200 μL of blood was collected via the inner canthus of the eye on day 3 (D-3) prior to drug administration. Whole blood samples were temporarily stored on ice before centrifugation at approximately 4°C and 3000 g for 10 min. LDL-C levels were measured using a biochemical analyzer (Neusoft NT-1000 fully automated biochemical analyzer) according to the manufacturer's instructions, and mice were grouped accordingly (6 mice per group) to ensure relatively homogeneous LDL-C levels within each group. Drug administration was performed subcutaneously on day 0 (D0) at a dose of 2 mg / kg. Blood samples were collected via the inner canthus of the eye on days 7 (D7), 14 (D14), and 21 (D21), and serum was separated to measure serum LDL-C levels.

[0492] The percentage reduction in serum LDL-C was calculated based on the serum LDL-C levels on the measurement days (D7, D14, D21) and the serum LDL-C levels on the 3rd day before the administration day (D-3), and the results are shown in Table 5 below.

[0493] Table 5: Percentage decrease in serum LDL-C

[0494]

[0495] It is evident that the siRNA sequences co-modified at the 2' and 5' positions of the ribose in this application, namely ON3, ON5, ON6, and ON7, can significantly reduce serum LDL-C protein levels.

[0496] From Table 5 and Figure 2 As can be seen, the siRNA sequences with co-modification at the 2' and 5' positions of the ribose in this application, ON3, ON5, ON6, and ON7, can significantly reduce the level of LDL-C protein in mouse serum. For example, the ON5 modified by YK-NUM-205 reduced the protein levels by 37.1%, 43.2%, and 40.6% on days 7, 14, and 21, respectively, which were 13.4%, 13.6%, and 20.5% higher than those modified by ON11 (single modification at the 2' position).

[0497] Furthermore, the siRNAs ON3, ON5, ON6, and ON7 with co-modified ribose positions 2' and 5' of this application significantly increase the percentage reduction in serum LDL-C compared to siRNAs with co-modified ribose positions 2' and 5' of existing technologies.

[0498] Compared to existing technologies that modify siRNA, such as U 6H The modified ON14, and the ON3, ON5, ON6, and ON7 of this application, significantly enhanced the percentage reduction in serum LDL-C. For example, compared with ON14, ON3 increased the percentage reduction rate by 11.7%, 17.2%, and 13.6% on days 7, 14, and 21, respectively, while ON5 increased the percentage reduction rate by 18.2%, 20.8%, and 16.9% on days 7, 14, and 21, respectively.

[0499] In summary, this application introduces a riboside at the first position of the antisense strand 5' of siRNA and a 2' and 5' co-modified riboside at the first position, which can significantly improve the stability of siRNA and the activity of silencing target genes.

[0500] The above embodiments are merely illustrative of the technical solutions of this application and are not intended to limit it. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the scope of this application. The full scope of this application is given by the appended claims and any equivalents.

Claims

1. A compound, its pharmaceutically acceptable salt, or a stereoisomer thereof, characterized in that, The compound has any of the following structures: , , , , , or .

2. A method for preparing the compound as described in claim 1, characterized in that, The synthetic route of the compound is shown below: ； It includes the following steps: hydroxyl protection of compound 1-1 yields 1-2, oxidation yields 1-3, a witting reaction yields 1-4, 1-4 undergoes a series of reactions to yield 1-5, 1-5 undergoes different reaction steps to yield 1-6, glycosylation yields 1-7, and 1-7 undergoes different reaction steps to yield the compound shown in formula (I). Where R 11 It is either methyl or acetyl; W1 and W2 are hydroxyl protecting groups; The compound represented by formula (I) is the compound as described in claim 1.

3. The method for preparing the compound according to claim 2, characterized in that, R 11 It is methyl or acetyl; W1 and W2 are triphenylmethyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, tetraisopropyldisiloxyl, methoxymethyl ether or dimethoxytriphenylmethyl.

4. A nucleic acid polymer, characterized in that, It is obtained by polymerization of a compound as described in claim 1.

5. The nucleic acid polymer according to claim 4, characterized in that, The nucleic acid polymer is a double-stranded oligonucleotide molecule comprising a sense strand and an antisense strand, wherein the nucleotide sequences of the sense strand and the antisense strand are at least partially inversely complementary; wherein the 5'-terminus of the antisense strand contains a modified nucleotide, the modified nucleotide being... X1 is either O or S; X is O; R1 is ; When R2 is H for , , or ; When R2 is F for , or .

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

7. The nucleic acid polymer according to claim 6, characterized in that, The ligand is galactose, galactosamine, N-acetylgalactosamine, or a derivative thereof.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nucleic acid polymer as described in any one of claims 4-7 and pharmaceutically acceptable excipients.

9. The use of a nucleic acid polymer as described in any one of claims 4-7 or a pharmaceutical composition as described in claim 8 in the preparation of nucleic acid diagnostic agents and / or nucleic acid therapeutic agents.

10. The use of a nucleic acid polymer as described in any one of claims 4-7 or a pharmaceutical composition as described in claim 8 in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by the expression of a specific gene, wherein the specific gene is the ApoB gene.

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