Modified nucleoside, nucleoside monomer, oligonucleotide containing modified nucleoside monomer and application thereof

Abstract

The invention discloses a modified nucleoside, a nucleoside monomer, oligonucleotide containing the modified nucleoside monomer and application of the oligonucleotide. The modified nucleoside and the phosphoramidite monomer thereof are obtained by introducing some lipophilic / hydrophilic groups or fragments with pharmacological activity and a targeted receptor and the like to chemically modify nucleoside, and the physicochemical properties and bioavailability of the modified nucleoside are remarkably improved; according to the invention, the modified nucleoside is further doped into oligonucleotide, so that the cell free uptake efficiency of oligonucleotide drugs can be remarkably enhanced, target mRNA can be silenced, and the modified nucleic acid drugs are expected to be efficiently delivered to different organs and tissues for treatment of related indications.

Description

TECHNICAL FIELD

[0001] The present application relates to modified nucleoside monomers, in particular to the modification of the sugar ring and base of nucleoside monomers to obtain modified nucleoside monomers, antisense nucleic acids or siRNAs containing the modified nucleoside monomers and their use in the preparation of nucleic acid drugs, belonging to the field of modified nucleoside monomers and their applications. BACKGROUND

[0002] Oligonucleotide drugs (including antisense nucleic acids, miRNAs, siRNAs, gRNAs, etc.) have brought great hope to the treatment of currently intractable diseases for humans, but the use of nucleic acid drugs as a therapeutic means is also challenging, mainly including immune response triggered by exogenous nucleic acid drugs; immunogenicity and non-immunogenic toxicity of nucleic acid delivery vectors; unpredictable activity due to off-target effects; target gene knockdown activity of nucleic acid drugs accumulated in non-target tissues. In addition, unmodified nucleic acid drugs are not easily systemically administered due to their exogenous nature, unfavorable physicochemical properties, low stability in serum, rapid kidney clearance, reduced target cell uptake, phagocyte uptake and the ability to activate immune responses, which greatly hinders the clinical development of nucleic acid drugs. Therefore, the complete release and enhancement of the therapeutic potential of nucleic acid drugs not only depends on chemical modification and / or aims to protect nucleic acids from degradation and ensure their stability in circulation, enabling them to be localized to the target tissue, but also requires a safe, effective and reliable delivery platform technology to ensure efficient intracellular delivery and tissue distribution. For example, Alnylam introduced a C16 group at the 2' position of the nucleoside ring of siRNA drugs, and through a single intrathecal administration, the drug was widely distributed in neurons, astrocytes and microglia. At the highest dose of 0.9 mg SOD1 siRNA, more than 75% of the target gene can be silenced. Currently, oligonucleotide drugs based on alkyl chain modification have been widely used in the treatment of diseases in different organs and tissues such as the brain, eyes, skin, fat, liver, lungs, kidneys, etc.

[0003] Therefore, the introduction of modified nucleosides into nucleic acid drugs not only enhances their own chemical and biological stability and reduces or avoids the use of delivery lipid materials, but also is expected to directly endow them with the ability to target specific tissues / organs, thereby decoupling the use of nucleic acid drugs from their own or external adverse risks, promoting the more extensive clinical application of nucleic acid drugs. SUMMARY

[0004] One of the purposes of the present application is to modify the structure of the sugar ring backbone and the base part of the nucleoside respectively to obtain modified nucleosides and their phosphoramidite monomer compounds;

[0005] The second purpose of the present application is to provide a method for preparing the modified nucleosides and their phosphoramidite monomer compounds;

[0006] A third object of the present application is to incorporate the modified nucleoside monomers into oligonucleotides to obtain modified oligonucleotides.

[0007] A fourth object of the present application is to prepare the modified oligonucleotides into nucleic acid drugs.

[0008] Based on the above considerations, the present application modifies the sugar ring backbone and the base moiety of the nucleoside monomers respectively, and then prepares antisense nucleic acids and siRNAs modified by these nucleoside monomers, and further studies the cell uptake, drug efficacy, and tissue distribution of these modified antisense nucleic acids and siRNAs, in order to obtain a modification strategy with further research potential, to provide new candidate antisense nucleic acid and siRNA drugs for clinical use, thereby completing the present application.

[0009] An aspect of the present application is to provide a modified nucleoside monomer compound or a pharmaceutically acceptable salt thereof, which has the following general formula I-a or I-b:

[0010]

[0011] wherein Base is selected from cytosine (C), uracil (U), adenine (A), thymine (T), guanine (G), or pseudouracil (ΨU) or derivatives thereof;

[0012] R 1 selected from hydrogen, 4,4'-dimethoxytriphenylmethane (DMTr), or a phosphoramidite analogue;

[0013] R 2 selected from hydrogen, phosphoramidite, or a phosphate analogue;

[0014] R 3 selected from hydroxyl, methoxy, methylethoxy, fluorine, chlorine, bromine, iodine, or

[0015] L 1 or L 2 is simultaneously or separately a covalent bond or a bivalent saturated or unsaturated straight-chain or branched C 1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -CH2C(O)NHNH-, -(CH2) n C(O)NH-, -V 1 CR 2 W 1 - or wherein m is any integer from 1 to 50, and n is any integer from 1 to 50; each -Cy- is independently an optionally substituted bivalent ring; the bivalent ring is selected from phenylene, 8-10 membered bicyclic arylene, 4-7 membered saturated or partially unsaturated carbocyclylene, 4-11 membered saturated or partially unsaturated spiro carbocyclylene, 8-10 membered bicyclic saturated or partially unsaturated carbocyclylene, 4-7 membered saturated or partially unsaturated heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 4-11 membered saturated or partially unsaturated spiro heterocyclylene having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 8-10 membered bicyclic saturated or partially unsaturated heterocyclylene having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 5-6 membered heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 8-10 membered bicyclic heteroarylene having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur; V 1 or W 1 independently -C(R)2-, -OR, -0-, -S-, -Se-, or -NR-; R is independently at each occurrence hydrogen, straight chain or branched C1-C 50 alkyl, aryl, hydroxyl, alkoxy, halogen, methoxy alkoxy, alkylthio, amino, alkylamino, alkynyl, aminoalkyl, or aminoalkoxy;

[0016] R 4 is vitamin E, hexadecyl, or n is any integer from 1 to 20.

[0017] Preferably, the pharmaceutically acceptable salt is a sodium salt, a phosphate salt, a quaternary ammonium salt, a sulfate salt, a hydrochloride salt, a nitrate salt, or an acetate salt.

[0018] In a preferred embodiment of the application, the modified nucleoside monomer compound is selected from any one of the following phosphoramidite monomer compounds shown in II-a or II-b:

[0019]

[0020] B1 is cytosine, adenine, uracil, guanine, uracil or pseudouracil or derivatives thereof, including but not limited to 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2- aminoadenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil and cytosine, 5- propynyluracil, 6-azo uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyaldehyde, other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8- azadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine;

[0021] X is selected from oxygen (O), nitrogen (N), sulfur (S), or selenium (Se);

[0022] R 3 selected from hydroxyl, methoxy, methylethoxy, fluoro, chloro, bromo, iodo, or

[0023] L 1 or L 2 is simultaneously or separately a covalent bond or a bivalent saturated or unsaturated straight-chain or branched C 1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -0-, -C(O)NR-, -NR-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -CH2C(O)NHNH-, -(CH2) n C(O)NH-, -V 1 CR 2 W 1 - or substituted bivalent ring; the bivalent ring is selected from phenylene, 8-10 membered bicyclic arylene, 4-7 membered saturated or partially unsaturated carbocyclylene, 4-11 membered saturated or partially unsaturated spiro carbocyclylene, 8-10 membered bicyclic saturated or partially unsaturated carbocyclylene, 4-7 membered saturated or partially unsaturated heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 4-11 membered saturated or partially unsaturated spiro heterocyclylene having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 8-10 membered bicyclic saturated or partially unsaturated heterocyclylene having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 5-6 membered heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 8-10 membered bicyclic heteroarylene having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; V 1 or W 1 independently of each other selected from -C(R)2-, -OR, -0-, -S-, -Se-, or -NR-; R is independently at each occurrence hydrogen, straight-chained or branched C1-C 24 alkyl, aryl, hydroxyl, alkoxy, halogen, methoxyalkoxy, alkylthio, amino, alkylamino, alkynyl, aminoalkyl, or aminoalkoxy;

[0024] R 4 is vitamin E, hexadecyl, or n is any integer from 1 to 20.

[0025] Another aspect of the present application is to provide a method for preparing the monomer compound represented by the above II-a and II-b, comprising:

[0026] (1) In II-a, when B1 is A and G, respectively using N-benzoyl adenosine and N-isobutyryl guanosine as starting materials, using sodium hydride as base, using a mixed solution of dimethyl sulfoxide and N,N-dimethylformamide as reaction solvent, 2'-O-L 1 modified intermediate is obtained by substitution reaction; the intermediate is further subjected to multi-step reactions to prepare 2'-O-L 1 VE-A / G phosphoramidite monomer.

[0027] (2) In II-a, when B1 is C and ΨU, respectively using cytosine nucleoside and pseudouracil nucleoside as starting materials, using sodium hydride as base, using a mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide as reaction solvent, 2'-O-L 1 modified intermediate is obtained by substitution reaction of 2'-OH; the intermediate is further subjected to multi-step reactions to prepare 2'-O-L 1 VE-C / ΨU phosphoramidite monomer.

[0028] (3) In II-a, when B1is uracil (U), first protect the sugar ring 3', 5'-OH with silyl protection, then react with the halogenated compound of the corresponding substituent in claim 1 in acetonitrile in the presence of (tert-butylimino)tris(pyrazolyl)phosphine (BTTP) to obtain NH-L 2 modified nucleoside intermediate, which can be prepared by further multi-step reactions of the above intermediate 1 - the phosphoramidite monomer of VE-U;

[0029] (4) In II-b, when B1is adenine (A), use 6-chloroadenosine as the starting material, triethylamine as the base, and react with the amino derivative of the corresponding substituent in claim 1 in methanol or ethanol under reflux to obtain 6-NH-L 2 modified intermediate; the intermediate can be prepared by further multi-step reactions of the above intermediate 2 - the phosphoramidite monomer of R 4

[0030] (5) In II-b, when B1is pseudouracil (ΨU), thymine (T) or deoxyuracil, use pseudouridine as the starting material, sodium hydride as the base, and react with the halogenated compound of the corresponding substituent in the above content in the mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide to obtain 5-N-L of ΨU 2 modified important intermediate of ΨU or intermediate 3-N-L of T or deoxy U 2 , which can be prepared by further multi-step reactions of the above intermediate 2 - the phosphoramidite monomer of R 4

[0031] In some specific embodiments, using pseudouridine as an example, first protect the sugar ring -OH with silyl protection, then use N,O-bis(trimethylsilyl)acetamide (BSA) as the base, and react with the alkyne halogenated hydrocarbon under reflux in dichloromethane to obtain the alkyne-modified important intermediate, and the product obtained by reacting the above intermediate with an azide compound can be prepared by further multi-step reactions to obtain 5-N-L 2 - the phosphoramidite monomer of R 4

[0032] (6) In II-b, when B1is cytosine (C), use N-benzoylcytidine as the starting material, sodium hydride as the base, and react with the halogenated compound of the corresponding substituent in the above content in the mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide as the reaction solvent to obtain Ph-N-L 2 modified nucleoside intermediate, which can be prepared by further multi-step reactions of the above intermediate​​​2 -R 4 phosphoramidite monomer of Formula (I)

[0033] (7) In II-b, when B1is guanine (G), the intermediate 2',3',5'-triacetyl-2- bromoguano sine is prepared by refluxing 2',3',5'-triacetylguanosine with n- pentyl nitrite in bromoform, and then the intermediate is reacted with the amino derivative of the corresponding substituent as described above in ethylene glycol monomethyl ether to produce 2-NH-L 2 modified nucleoside intermediate, and the above intermediate is subjected to a plurality of reactions to produce 2-NH-L 2 -R 4 phosphoramidite monomer of Formula (I).

[0034] (8) In II-b, when B1is an alkynyl-modified nucleoside, the azido compound of the corresponding substituent as described above is reacted in dimethyl sulfoxide / water to produce a triazole-linked modified nucleoside intermediate, and the intermediate is subjected to a plurality of reactions to produce L 2 -R 4 phosphoramidite monomer of the modified nucleoside.

[0035] Another aspect of the present application is to provide an oligonucleotide or siRNA containing or doped with the modified nucleoside monomer compound; the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length; wherein the sense strand and the antisense strand form a duplex region, the antisense strand has a complementary region complementary to a target sequence, and the complementary region is at least 15 consecutive nucleotides in length, with a difference of no more than 3 nucleotides; in a more preferred embodiment of the present application, the complementary region is completely complementary to the target sequence.

[0036] In a preferred embodiment of the present application, the antisense strand is 19 to 27 nucleotides in length.

[0037] In a preferred embodiment of the present application, the oligonucleotide comprises at least one modified nucleotide; preferably, the oligonucleotide or siRNA is partially or completely thio-modified in the backbone portion, or the 5'-end of the antisense strand of the siRNA includes, but is not limited to, phosphate, oxymethylphosphonate, vinylphosphonate, or malonylphosphonate modification.

[0038] The siRNA-APP obtained by doping the modified nucleoside monomer into the siRNA double strand shows good uptake effect in HepG2 and ARPE-19 cells, and shows gradient dependence with the siRNA concentration and transfection time. The siRNA doped with the modified nucleoside monomer is further verified in the transfection and target gene sequence knockdown level on U87-MG cells, and the verification result shows that the integration of the modified nucleoside monomer into the siRNA does not affect the biological activity of the siRNA, but is beneficial to enhance the cell uptake effect, and meanwhile, the liposolubility, metabolic stability and targeting of the modified group can promote the siRNA to better achieve the target mRNA knockdown, and finally achieve the desired treatment purpose.

[0039] Another aspect of the present application provides a pharmaceutical composition comprising any one of the above-mentioned oligonucleotide doped with the modified nucleoside monomer compound, or the oligonucleotide doped with the modified nucleoside monomer compound and the ligand conjugate, and a pharmaceutically acceptable carrier, delivery agent or excipient.

[0040] The present application introduces some lipophilic / hydrophilic groups or fragments with pharmacological activity, target receptors, etc. to chemically modify the nucleoside monomer to obtain the modified nucleoside monomer compound, and the physical and chemical properties and bioavailability of the modified nucleoside monomer compound are significantly improved. The modified nucleoside monomer compound is doped into the siRNA to enhance the cell uptake effect and promote the siRNA to better achieve the target mRNA knockdown, thereby providing a nucleic acid drug which is independent of the delivery carrier and has the tissue targeting specificity. The nucleic acid drug has the advantages of good chemical / biological stability, good serum stability, low kidney clearance rate, etc. when administered systemically or locally, and is expected to realize the nucleic acid drug targeting in different tissues and provide a new direction for the development of nucleic acid drugs. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 NO. 3 (Loop_4a_APP_3) in Table 1 is used to knock down the APP mRNA level in U87-MG.

[0042] Figure 2 The cell free uptake efficiency and fluorescence intensity experimental results of NO. 3 (Loop_4a_APP_3) in Table 1 in HepG2 cells, ARPE-19 cells and U87-MG cells are shown; wherein A and B are the cell uptake efficiency and fluorescence intensity (1, 6 and 12h) of NO. 3 in HepG2, respectively; C and D are the cell uptake efficiency and fluorescence intensity (12h) of NO. 3 in ARPE-19, respectively.

[0043] Figure 3Free uptake of NO. 1-15 in Table 1 in U87-MG cells for 24 hours (1 nM, without transfection reagent).

[0044] Figure 4 Free uptake of NO. 1-15 in Table 1 in U87-MG and ACHN cells for 24 hours (100 nM, without transfection reagent). DETAILED DESCRIPTION

[0045] The examples provided herein are intended to be illustrative of the disclosure and should not be construed as limiting thereof. Temperatures are given in degrees Celsius (°C). The structure of final products, intermediates and starting materials was confirmed by standard analytical methods, such as microanalysis and spectroscopic characteristics, for example MS, NMR. Abbreviations used are those conventional in the art.

[0046] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesis the modified nucleosides or analogs thereof of the present patent are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art (METHODS OF ORGANIC SYNTHESIS, Thieme, Vol. 21 (Houben-Weyl 4th Ed. 1952)). Unless otherwise indicated, all reactions were conducted under nitrogen or argon. Proton NMR 1 H) was conducted in deuterated solvents. In certain nucleic acids or analogs thereof disclosed herein, one or more 1 H shifts overlapped with residual solvent signals; these signals were not reported in the experiments provided below. As described in the following embodiments, in certain exemplary embodiments, nucleic acids or analogs thereof were prepared according to the following general procedures. It will be appreciated that while the general methods describe the synthesis of certain nucleosides or analogs thereof of the present patent, the following general methods, as well as other methods known to one of ordinary skill in the art, can be applied to all nucleic acids or analogs thereof, as well as to subgroups and species of each of these nucleic acids or analogs thereof, as described herein.

[0047] Preparation of Example 1 Compound 4a

[0048]

[0049] Reagents and conditions (i) NaH, DMSO, DMF 50 °C, 0 °C - r.t.; (ii) DMTrCl, Py., r.t.; (iii) tetrazole, DCM, N2, 3-((bis(diisopropyl amino)phosphaneyl)oxy)propanenitrile

[0050] Preparation of compound 2a: N-benzoyl adenosine was dissolved in 20 mL of a mixed solvent of N,N-dimethylformamide and dimethyl sulfoxide, and NaH was added under ice bath with stirring for 10 minutes. Ve-C2H5I solution in N,N-dimethylformamide was added dropwise into the reaction bottle. The reaction was continued at room temperature for 36 hours. Extraction was performed with ethyl acetate and saturated NaCl solution, and the organic phase was dried with anhydrous Na2SO4. After removing the solvent under reduced pressure, the crude product was purified by column chromatography (eluent: DCM / EA / MeOH = 5 / 1 / 0.05 ~ 0.2) to obtain compound 2a. 1 H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.77 (d, 2H), 8.07 - 8.03 (m, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.55 (t, J = 7.6 Hz, 2H), 6.25 (d, J = 5.8 Hz, 1H), 5.32 (d, J = 5.0 Hz, 1H), 5.20 (bs, 1H), 4.70 (t, J = 5.3 Hz, 1H), 4.43 (m, 1H), 4.06 (q, J = 3.8 Hz, 1H), 3.95 (m, 1H), 3.73 (m, 5H), 2.45 (t, J = 6.9 Hz, 2H), 1.93 (s, 3H), 1.90 (s, 3H), 1.86 (s, 3H), 1.72 - 1.67 (m, 2H), 1.50 - 1.04 (m, 27H), 0.82 (m, 12H) ppm.

[0051] Preparation of compound 3a: Compound 2a was dissolved in super dry pyridine, and 4,4'-dimethoxytrityl chloride was added with stirring at room temperature. The reaction was continued for 12 hours. The reaction solution was extracted with ethyl acetate and saturated NaCl solution, and the organic phase was dried with anhydrous Na2SO4. After removing the solvent under reduced pressure, the crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 3a. 1H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 11.65 (s, 1H), 8.15 (s, 1H), 7.39 - 7.32 (m, 2H), 7.28 - 7.18 (m, 7H), 6.87 - 6.78 (m, 4H), 6.03 (d, J = 5.4 Hz, 1H), 5.31 (s, 1H), 4.60 (t, J = 5.3 Hz, 1H), 4.37 (m, 1H), 4.13 - 4.06 (m, 1H), 3.96 - 3.88 (m, 1H), 3.82 - 3.75 (m, 1H), 3.72 (s, 8H), 3.29 - 3.33 (m, 1H), 3.22 - 3.18 (m, 1H), 2.81 - 2.71 (m, 1H), 2.49 - 2.41 (m, 2H), 1.95 (s, 3H), 1.93 (s, 3H), 1.90 (s, 3H), 1.69 (t, J = 5.2 Hz, 2H), 1.51 - 1.02 (m, 32H), 0.83 (m, 12H) ppm.

[0052] Preparation of compound 4a: Compound 3a and tetrazole were added to super dry dichloromethane, and after bis(diisopropylamino)(2-cyanoethoxy)phosphine was added under nitrogen protection, the crude product was purified by column chromatography (eluent: DCM / EA / TEA = 15-8 / 1 / 0.01) under nitrogen protection after reaction at room temperature for 6 hours to obtain compound 4a. 1 H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 11.65 (s, 1H), 8.15 (s, 1H), 7.39 - 7.32 (m, 2H), 7.28 - 7.18 (m, 7H), 6.87 - 6.78 (m, 4H), 6.03 (d, J = 5.4 Hz, 1H), 5.31 (s, 1H), 4.60 (t, J = 5.3 Hz, 1H), 4.37 (m, 1H), 4.13 - 4.06 (m, 1H), 3.96 - 3.88 (m, 1H), 3.82 - 3.75 (m, 1H), 3.72 (s, 8H), 3.29 - 3.33 (m, 1H), 3.22 - 3.18 (m, 1H), 2.81 - 2.71 (m, 1H), 2.49 - 2.41 (m, 2H), 1.95 (s, 3H), 1.93 (s, 3H), 1.90 (s, 3H), 1.69 (t, J = 5.2 Hz, 2H), 1.51 - 1.02 (m, 32H), 0.83 (m, 12H) ppm. 31 P NMR (162 MHz, DMSO-d6) δ 149.25, 149.10 ppm.

[0053] Preparation of compound 9 of Example 2

[0054]

[0055] Reagents and conditions: (i) NaH, DMSO, DMF, 0 °C-35 °C; (ii) t-Bu2Si(OTf)2, DMF, 0 °C; (iii) acetic anhydride, Py. 0 °C-r.t.; (ii) HF, Py., DCM, 0 °C-r.t.; (iv) DMTrCI, Py., r.t.; (iii) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0056] Preparation of compound 5: using cytosine as starting material, the reaction conditions are the same as compound 2a, the crude product was purified by column chromatography (eluent: DCM / EA / MeOH = 5 / 1 / 0.05~0.2), respectively to obtain compound 5. 1 H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 7.4 Hz, 1H), 7.21 (d, 2H), 5.90 (d, J = 3.5 Hz, 1H), 5.73 (d, J = 7.4 Hz, 1H), 5.11 (d, J = 5.4 Hz, 1H), 4.97 (d, J = 6.1 Hz, 1H), 4.12 - 4.08 (m, 1H), 3.93 - 3.87 (m, 4H), 3.76 - 3.68 (m, 3H), 3.59 (m, 1H), 2.51 (s, 2H), 2.06 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H), 1.70 (d, J = 7.3 Hz, 2H), 1.50 - 1.03 (m, 27H), 0.83 (m, 12H) ppm.

[0057] Preparation of compound 6: compound 5 was dissolved in 20 mL of super dry N,N- dimethylformamide, di-tert-butylsilyl bis(trifluoromethanesulfonic acid) was added at 0 °C. After 8 hours of reaction, extraction was performed with ethyl acetate and saturated NaCl solution, the organic phase was dried with anhydrous Na2SO4, after removing the solvent, the intermediate was obtained, which was directly used in the next step without further purification. The above crude product was dissolved in 10 mL of super dry N,N-dimethylformamide, acetic anhydride was slowly added under ice bath, then it was restored to room temperature for reaction. After the reaction was completed, extraction was performed with ethyl acetate and saturated NaCl solution, the organic phase was dried with anhydrous Na2SO4, after removing the solvent, the crude product of compound 6 was obtained, which was directly used in the next step without further purification.

[0058] Preparation of compound 7: Compound 6 above was dissolved in super dry dichloromethane, hydrogen fluoride pyridine solution was added under stirring at 0°C. After the reaction was completed, extraction was performed with dichloromethane and saturated NaHC03 aqueous solution, the organic phase was dried with anhydrous Na2S04, after the solvent was removed, the crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 7. 1 H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.50 (d, J = 7.4 Hz, 1H), 7.20 (d, J = 7.4 Hz, 1H), 5.91 (d, J = 2.5 Hz, 1H), 5.22 (t, J = 5.0 Hz, 1H), 5.04 (d, J = 6.4 Hz, 1H), 4.13 - 4.08 (m, 1H), 4.05 - 4.01 (m, 1H), 3.97 - 3.90 (m, 3H), 3.82 - 3.73 (m, 3H), 3.66 - 3.61 (m, 1H), 2.10 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.97 (s, 3H), 1.71 (s, 2H), 1.48 (m, 4H), 1.42 - 0.94 (m, 28H), 0.83 (m, 12H) ppm.

[0059] Preparation of compound 8: Compound 7 was dissolved in 10 mL of super dry pyridine, 4,4'-dimethoxytrityl chloride was added under stirring at room temperature, and the reaction was carried out for 12 hours. After the reaction was completed, extraction was performed with ethyl acetate and saturated NaCl solution, the organic phase was dried with anhydrous Na2S04, after the solvent was removed, the crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 8. 1 H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.50 (d, J = 7.4 Hz, 1H), 7.20 (d, J = 7.4 Hz, 1H), 5.91 (d, J = 2.5 Hz, 1H), 5.22 (t, J = 5.0 Hz, 1H), 5.04 (d, J = 6.4 Hz, 1H), 4.13 - 4.08 (m, 1H), 4.05 - 4.01 (m, 1H), 3.97 - 3.90 (m, 3H), 3.82 - 3.73 (m, 3H), 3.66 - 3.61 (m, 1H), 2.10 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.97 (s, 3H), 1.71 (s, 2H), 1.48 (m, 4H), 1.42 - 0.94 (m, 28H), 0.83 (m, 12H) ppm.

[0060] Preparation of compound 9: Compound 8 and tetrazole were added to super dry dichloromethane, after bis(diisopropylamino)(2-cyanoethoxy)phosphane was added under nitrogen protection, the reaction was carried out at room temperature for 6 hours, part of the solvent was removed, and the crude product was purified by column chromatography under nitrogen protection to obtain compound 9.

[0061] Preparation of compound 14a of example 3

[0062]

[0063] Reagents and conditions: (i) NaH, DMSO, DMF, 0 °C-35 °C; (ii) t-Bu2Si(OTf)2, DMF, 0 °C; (iii) HF, Py., DDCM, 0 °C; (iv) DMTrCI, Py., r.t. (v) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0064] Preparation of compound 10a: The pseudouridine was dissolved in 20 mL of N,N- dimethylformamide and dimethyl sulfoxide mixed solvent, NaH (1.5 eq.) was added under ice bath, and stirred for 10 minutes, then the solution of bromohexadecane (1.8 eq.) in N,N- dimethylformamide was added dropwise into the reaction bottle, and the reaction was continued at room temperature for 36 hours. After the reaction was completed, the reaction mixture was extracted with ethyl acetate and saturated NaCl solution, and the organic phase was dried with anhydrous Na2SO4. After removing the solvent, the crude product was purified by column chromatography (eluent: DCM / EA / MeOH = 5 / 1 / 0.05~0.2) to obtain compound 10a. 1 H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 7.80 (s, 1H), 4.97 (d, J = 5.0 Hz, 1H), 4.82 (dd, J = 6.6, 4.8 Hz, 1H), 4.72 (d, J = 5.8 Hz, 1H), 4.47 (d, J = 4.2 Hz, 1H), 3.95 (q, J = 4.8 Hz, 1H), 3.89 (q, J = 5.6 Hz, 1H), 3.72 - 3.58 (m, 4H), 3.50 - 3.44 (m, 3.6 Hz, 1H), 1.60 - 1.52 (m, 2H), 1.24 (s, 27H), 0.86 (t, J = 6.8 Hz, 3H) ppm.

[0065] Preparation of compound 11a: Compound 10a was dissolved in 20 mL of super dry N,N-dimethylformamide, di-tert-butylsilyl bis(trifluoromethanesulfonic acid) (1.5 eq.) was added at 0 °C, after 6 hours of reaction, imidazole and tert-butyldimethylsilyl chloride were added to the reaction solution. The reaction was continued at room temperature for 10 hours. After the reaction was completed, extraction was performed with ethyl acetate and saturated NaCl solution, the organic phase was dried with anhydrous Na2SO4, and after removing the solvent, compound 11a was obtained, which was used in the next step without further purification.

[0066] Preparation of compound 12a: Compound 11a was dissolved in super dry dichloromethane, hydrogen fluoride pyridine solution was added at 0 °C while stirring, and the reaction was continued for 1.5 hours. After the reaction was completed, extraction was performed with dichloromethane and saturated NaHCO3 aqueous solution, the organic phase was dried with anhydrous Na2SO4, and after removing the solvent under reduced pressure, the crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 12a. 1 H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 7.96 (s, 2H), 7.84 (s, 1H), 4.83 (s, 1H), 4.53 (s, 1H), 4.46 (d, J = 4.3 Hz, 1H), 4.13 (d, J = 4.6 Hz, 1H), 3.89 (m, 1H), 3.74 (m, 1H), 3.65 (m, 3H), 3.48 (m, 1H), 1.55 (s, 2H), 1.24 (m, 30H), 0.85 (m, 12H), 0.04 (s, 6H) ppm.

[0067] Preparation of compound 13a: Compound 12a was dissolved in 10 mL of super dry pyridine, 4,4'-dimethoxytrityl chloride was added at room temperature while stirring, and the reaction was continued for 12 hours. After the reaction was completed, extraction was performed with ethyl acetate and saturated NaCl solution, the organic phase was dried with anhydrous Na2SO4, and after removing the solvent, the crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 13a. 1H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 7.49 (s, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.33 - 7.28 (m, 7H), 6.89 (d, J = 8.3 Hz, 5H), 4.67 (d, J = 6.3 Hz, 1H), 4.59 (d, J = 3.1 Hz, 1H), 4.12 (t, J = 3.9 Hz, 1H), 3.98 (m, 1H), 3.91 (m, 1H), 3.74 (s, 8H), 3.47 (m, 1H), 3.26 (m, 1H), 3.13 (m, 2H), 1.23 (s, 33H), 0.90 - 0.82 (m, 16H), 0.09 (s, 3H), 0.07 (s, 4H) ppm.

[0068] Preparation of compound 14a: Compound 13a and tetrazole were added to super dry dichloromethane, and bis(diisopropylamino)(2-cyanoethoxy)phosphane was added under nitrogen protection, and then reacted at room temperature for 6 hours. After the reaction was completed, part of the solvent was removed, and the crude product was purified by column chromatography under nitrogen protection to obtain compound 14a. 31 P NMR (162 MHz, Chloroform-d) δ 149.72 ppm.

[0069] Preparation of compounds 19a and 19b of Example 4

[0070]

[0071] Reagents and conditions: (i) TEA, CH3(CH2) n NH2, MeOH, reflux; (ii) t-Bu2Si(OTf)2, imdizole, TBDMSCI, DMF, 0 °C-r.t.; (iii) HF·Py, DCM, Py, r.t.; (iv) DMTrCI, Py, r.t.; (v) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0072] Preparation of compounds 15a and 15b: 6-chloroadenine nucleoside was dissolved in methanol, triethylamine was added, and then dodecylamine (preparation 15a) and hexadecylamine (preparation 15b) were added, respectively. After refluxing for 10 hours, the solid was suction filtered, washed with cold methanol and dried to obtain compounds 15a and 15b, respectively. 15a: 1H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 8.20 (s, 1H), 7.87 (s, 1H), 5.89 (d, J = 6.2 Hz, 1H), 5.45 (m, 2H), 5.20 (d, J = 4.6 Hz, 1H), 4.62 (m, 1H), 4.18 - 4.09 (m, 2H), 3.98 (m, 1H), 3.67 (m, 1H), 3.56 (m, 1H), 3.47 (m, 2H), 3.18 (d, J = 5.1 Hz, 2H), 1.58 (m, 2H), 1.23 (m, 22H), 0.85 (t, J = 6.7 Hz, 3H) ppm. 1 H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 8.20 (s, 1H), 7.87 (s, 1H), 5.89 (d, J = 6.2 Hz, 1H), 5.45 (m, 2H), 5.20 (d, J = 4.6 Hz, 1H), 4.62 (m, 1H), 4.18 - 4.09 (m, 2H), 3.98 (m, 1H), 3.67 (m, 1H), 3.56 (m, 1H), 3.47 (m, 2H), 3.18 (d, J = 5.1 Hz, 2H), 1.58 (m, 2H), 1.23 (m, 22H), 0.85 (t, J = 6.7 Hz, 3H) ppm.

[0073] Preparation of compounds 16a and 16b: Compound 15a and 15b were dissolved in 20 mL of super dry N,N-dimethylformamide, di-tert-butylsilyl bis(trifluoromethanesulfonic acid) was added at 0 °C. After 6 min, imidazole and tert-butyldimethylsilyl chloride were added to the reaction solution, and the reaction was allowed to proceed at room temperature for 10 h. After the reaction was completed, extraction was performed with ethyl acetate and saturated NaCl solution, and the organic phase was dried over anhydrous Na2SO4. After removing the solvent under reduced pressure, intermediates 16a and 16b were obtained, which were used in the next step without further purification.

[0074] Preparation of compounds 17a and 17b: Compound 16a and 16b were dissolved in super dry dichloromethane, and the reaction was performed under the same conditions as in the synthesis of compound 12a. The crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compounds 17a and 17b. 17a: 1H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H), 5.91 (d, J = 6.2 Hz, 1H), 5.60 (m, 1H), 5.10 (d, J = 4.9 Hz, 1H), 4.75 (m, 1H), 4.13 (bs, 1H), 4.04 - 3.98 (m, 1H), 3.75 - 3.66 (m, 1H), 3.59 (m, 1H), 3.46 (bs, 2H), 1.58 (t, J = 9.7 Hz, 2H), 1.22 (s, 25H), 0.85 (m, 3H), 0.70 (s, 9H), -0.13 (s, 3H), -0.23 (s, 3H) ppm. 1 H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H), 5.91 (d, J = 6.2 Hz, 1H), 5.60 (m, 1H), 5.10 (d, J = 4.9 Hz, 1H), 4.75 (m, 1H), 4.13 (bs, 1H), 4.04 - 3.98 (m, 1H), 3.75 - 3.66 (m, 1H), 3.59 (m, 1H), 3.46 (bs, 2H), 1.58 (t, J = 9.7 Hz, 2H), 1.22 (s, 25H), 0.85 (m, 3H), 0.70 (s, 9H), -0.13 (s, 3H), -0.23 (s, 3H) ppm.

[0075] Preparation of compounds 18a and 18b: Compound 17a and 17b were dissolved in 10 mL of super dry pyridine, reaction condition was same as synthesis of compound 13a. The crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to give compounds 18a and 18b. 18a: 1HNMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.15 (s, 1H), 7.81 (s, 1H), 7.44 - 7.36 (m, 2H), 7.26 (m, 8H), 6.85 (m, 4H), 5.95 (d, J = 4.9 Hz, 1H), 5.12 (d, J = 5.9 Hz, 1H), 4.85 (t, J = 5.0 Hz, 1H), 4.26 s 1H), 4.10 (q, J = 4.5 Hz, 1H), 3.73 (s, 6H), 3.46 (s, 1H), 3.27 (m, 2H), 1.57 (m, 2H), 1.29 - 1.21 (m, 16H), 0.88 - 0.80 (m, 3H), 0.75 (s, 9H), -0.04 (s, 3H), -0.13 (s, 3H) ppm.18b: 1 H NMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.15 (s, 1H), 7.81 (s, 1H), 7.44 - 7.36 (m, 2H), 7.26 (m, 8H), 6.85 (m, 4H), 5.95 (d, J = 4.9 Hz, 1H), 5.12 (d, J = 5.9 Hz, 1H), 4.85 (t, J = 5.0 Hz, 1H), 4.26 s 1H), 4.10 (q, J = 4.5 Hz, 1H), 3.73 (s, 6H), 3.46 (s, 1H), 3.27 (m, 2H), 1.57 (m, 2H), 1.29 - 1.21 (m, 16H), 0.88 - 0.80 (m, 3H), 0.75 (s, 9H), -0.04 (s, 3H), -0.13 (s, 3H) ppm.

[0076] Preparation of compounds 19a and 19b: using compounds 18a and 18b as raw materials, the reaction conditions are the same as the synthesis of compound 14a, and the crude product is purified by column chromatography under nitrogen protection to obtain compounds 19a and 19b.19a: 1H NMR (400 MHz, Chloroform-d) δ 8.27 (s, 1H), 7.96 (s, 1H), 7.48 (d, J = 7.6 Hz, 2H), 7.39 - 7.34 (m, 4H), 7.30 (s, 1H), 7.22 (t, J = 7.2 Hz, 1H), 6.82 (d, J = 8.5 Hz, 4H), 6.02 (d, J = 6.5 Hz, 1H), 5.65 (s, 1H), 5.11 (dd, J = 6.5, 4.6 Hz, 1H), 4.42 (ddd, J = 12.4, 4.1, 2.1 Hz, 1H), 4.36 (t, J = 3.5 Hz, 1H), 4.02 - 3.95 (m, 1H), 3.95 - 3.86 (m, 1H), 3.80 (s, 6H), 3.70 - 3.54 (m, 5H), 3.34 (dd, J = 10.5, 4.1 Hz, 1H), 2.67 (td, J = 6.5, 4.2 Hz, 2H), 1.70 (p, J = 7.2 Hz, 2H), 1.29 (s, 16H), 1.21 (s, 3H), 1.20 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 0.90 (t, J = 6.7 Hz, 3H), 0.78 (s, 9H), -0.04 (s, 3H), -0.18 (s, 3H) ppm. 31 P NMR (162 MHz, Chloroform-d) δ 148.88 ppm.19b: 1 P NMR (162 MHz, Chloroform-d) δ 150.84, 149.89 ppm.

[0077] Preparation of compound 25 of example 5

[0078]

[0079] Reagents and conditions: (i) NaH, DMSO, DMF, 0 °C-35 °C; (ii) NaOH aq, THF, r.t.; (iii) t-Bu2Si(OTf)2, imidizole, TBDMSC1, DMF, 0 °C; (iv) HF, Py., DCM, 0 °C-r.t.; (v) DMTrCI, Py., r.t.; (vi) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0080] Preparation of compound 20: Using N-benzoylcytidine and bromohexadecane as starting materials, the reaction conditions were the same as those in the synthesis of compound 10a. The crude product was purified by column chromatography (eluent: DCM / EA / MeOH = 5 / 1 / 0.05-0.2) to obtain compound 20, respectively. 1 H NMR (400 MHz, DMSO-d6) δ 8.04 (d, J = 7.6 Hz, 2H), 7.91 (d, J = 8.2 Hz, 1H), 7.57 (t, J = 7.3 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 6.45 (d, J = 8.1 Hz, 1H), 5.83 (d, J = 4.5 Hz, 1H), 5.46 (d, J = 5.3 Hz, 1H), 5.11 (d, J = 5.0 Hz, 2H), 4.07 (m, 3H), 3.98 (q, J = 4.5 Hz, 1H), 3.88 (q, J = 3.7 Hz, 1H), 3.69 - 3.65 (m, 1H), 3.61 - 3.53 (m, 1H), 1.68 (t, J = 7.1 Hz, 2H), 1.41 - 1.12 (m, 29H), 0.85 (t, J = 6.6 Hz, 3H) ppm.

[0081] Preparation of compound 21: Compound 20 was dissolved in 4 mL of tetrahydrofuran, 5 M sodium hydroxide solution 2 mL was added and reacted at room temperature for 2 hours. After the reaction was completed, it was extracted with ethyl acetate and dilute hydrochloric acid aqueous solution, saturated NaCl solution, the organic phase was dried with anhydrous Na2SO4, after removing the solvent, the crude product was purified by column chromatography (eluent: EA / MeOH = 5-1 / 1) to obtain compound 21. 1 H NMR (400 MHz, DMSO-d6) δ 8.04 (d, J = 7.6 Hz, 2H), 7.91 (d, J = 8.2 Hz, 1H), 7.57 (t, J = 7.3 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 6.45 (d, J = 8.1 Hz, 1H), 5.83 (d, J = 4.5 Hz, 1H), 5.46 (d, J = 5.3 Hz, 1H), 5.11 (d, J = 5.0 Hz, 2H), 4.07 (m, 3H), 3.98 (q, J = 4.5 Hz, 1H), 3.88 (q, J = 3.7 Hz, 1H), 3.69 - 3.65 (m, 1H), 3.61 - 3.53 (m, 1H), 1.68 (t, J = 7.1 Hz, 2H), 1.41 - 1.12 (m, 29H), 0.85 (t, J = 6.6 Hz, 3H) ppm.

[0082] Preparation of compound 22: Using compound 21 as the starting material, the reaction conditions were the same as those in the synthesis of compound 11a. Compound 22 was obtained without further purification and was directly used in the next step.

[0083] Preparation of compound 23: using compound 22 as material, the reaction condition was same with the synthesis of compound 12a. The crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 23. 1 H NMR (400 MHz, DMSO-d6) δ 7.78 (d, J = 8.1 Hz, 1H), 7.40 - 7.37 (m, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.27 - 7.24 (m, 5H), 6.91 (d, J = 9.0 Hz, 4H), 5.83 (d, J = 4.2 Hz, 1H), 5.44 (d, J = 8.1 Hz, 1H), 5.13 (d, J = 6.1 Hz, 1H), 4.21 (t, J = 4.6 Hz, 1H), 4.08 (q, J = 5.5 Hz, 1H), 4.03 - 4.02 (m, 1H), 3.84 - 7.71 (m, 10H), 1.49 (m, 2H), 1.23 (s, 26H), 0.87 - 0.85 (m, 12H), 0.07 (s, 3H), 0.05 (s, 3H) ppm.

[0084] Preparation of compound 24: using compound 23 as material, the reaction condition was same with the synthesis of compound 13a. The crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 24. 1 H NMR (400 MHz, DMSO-d6) δ 7.78 (d, J = 8.1 Hz, 1H), 7.40 - 7.37 (m, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.27 - 7.24 (m, 5H), 6.91 (d, J = 9.0 Hz, 4H), 5.83 (d, J = 4.2 Hz, 1H), 5.44 (d, J = 8.1 Hz, 1H), 5.13 (d, J = 6.1 Hz, 1H), 4.21 (t, J = 4.6 Hz, 1H), 4.08 (q, J = 5.5 Hz, 1H), 4.03 - 4.02 (m, 1H), 3.84 - 7.71 (m, 10H), 1.49 (m, 2H), 1.23 (s, 26H), 0.87 - 0.85 (m, 12H), 0.07 (s, 3H), 0.05 (s, 3H) ppm.

[0085] Preparation of compound 25: using compound 24 as material, the reaction condition was same with the synthesis of compound 14a. The crude product was purified by column chromatography to obtain compound 25 under nitrogen protection.

[0086] Preparation of compound 32 of example 6

[0087]

[0088] Reagents and conditions: (i) n-amyl nitrite, CHBr3, 90 °C; (ii) 1-hexadecylamine 2-methoxyethanol, reflux; (iii) ammoniac-methanol, r.t.; (v) t-Bu2Si(OTf)2, imidizole, TBDMSC1, DMF, 0 °C; (vi) HF, Py., DCM, 0 °C-r.t.; (vii) DMTrCI, Py., r.t. (viii) tetrazole, DCM, N2, 3-((bis(diisopropylaminophosphaneyl)oxy)propanenitrile

[0089] Preparation of compound 26: Compound 2', 3', 5'-triacetylguanosine was dissolved in 10 mL of bromoform, after the addition of n-amyl nitrite, it was reacted at 90 °C for 5 hours. After the reaction was completed, the solvent was removed, and the crude product was purified by column chromatography (eluent: DCM / MeOH = 60 / 1) to obtain compound 26. 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 0.7 Hz, 1H), 6.30 (dd, J = 4.2, 0.6 Hz, 1H), 5.58 (dd, J = 6.3, 4.1 Hz, 1H), 5.28 (dd, J = 6.3, 4.1 Hz, 1H), 4.77 (dt, J = 4.1, 3.4 Hz, 1H), 4.33 (qd, J = 12.2, 3.4 Hz, 2H), 2.00 (d, J = 3.1 Hz, 9H) ppm.

[0090] Preparation of compound 27: Compound 26 was dissolved in ethylene glycol monomethyl ether, then hexadecylamine was added, and it was reacted at reflux. After the reaction was completed, the solvent was removed, and the crude product was purified by column chromatography (eluent: DCM / MeOH = 30 / 1) to obtain compound 27. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.32 (d, J = 0.5 Hz, 1H), 7.26 (t, J = 4.3 Hz, 1H), 6.31 (dd, J = 4.1, 0.6 Hz, 1H), 5.60 (dd, J = 6.3, 4.2 Hz, 1H), 5.28 (dd, J = 6.3, 4.1 Hz, 1H), 4.77 (dt, J = 4.2, 3.4 Hz, 1H), 4.33 (qd, J = 12.2, 3.4 Hz, 2H), 3.22 (qd, J = 5.1, 4.3 Hz, 2H), 2.09 - 1.92 (m, 9H), 1.65 - 1.44 (m, 2H), 1.39 - 1.18 (m, 21H), 0.92 - 0.83 (m, 3H) ppm.

[0091] Preparation of compound 28: Compound 27 was dissolved in ammonia-methanol solution and reacted at room temperature. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the crude product was purified by column chromatography (eluent: DCM / MeOH = 30 / 1) to obtain compound 28. 1 H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.32 (d, J = 0.5 Hz, 1H), 7.26 (t, J = 4.3 Hz, 1H), 6.31 (dd, J = 4.1, 0.6 Hz, 1H), 5.60 (dd, J = 6.3, 4.2 Hz, 1H), 5.28 (dd, J = 6.3, 4.1 Hz, 1H), 4.77 (dt, J = 4.2, 3.4 Hz, 1H), 4.33 (qd, J = 12.2, 3.4 Hz, 2H), 3.22 (qd, J = 5.1, 4.3 Hz, 2H), 2.09 - 1.92 (m, 9H), 1.65 - 1.44 (m, 2H), 1.39 - 1.18 (m, 21H), 0.92 - 0.83 (m, 3H) ppm.

[0092] Preparation of compound 29: Compound 28 was used as the raw material, and the reaction conditions were the same as those in the synthesis of compound 11a to obtain compound 29, which was directly used in the next step without further purification.

[0093] Preparation of compound 30: Compound 29 was used as the raw material, and the reaction conditions were the same as those in the synthesis of compound 12a. The crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 30. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.28 (d, J = 0.6 Hz, 1H), 7.26 (t, J = 4.3 Hz, 1H), 6.02 (dd, J = 4.0, 0.6 Hz, 1H), 4.85 (d, J = 3.9 Hz, 1H), 4.71 (t, J = 4.5 Hz, 1H), 4.47 (dd, J = 6.2, 4.0 Hz, 1H), 4.20 - 3.98 (m, 2H), 3.73 - 3.52 (m, 2H), 3.22 (qd, J = 5.2, 4.3 Hz, 2H), 1.60 - 1.42 (m, 2H), 1.41 - 1.20 (m, 21H), 0.95 - 0.80 (m, 12H), 0.09 (s, 3H), 0.06 (s, 3H) ppm.

[0094] Preparation of compound 31 : using compound 30 as raw material, the reaction conditions are the same as the synthesis of compound 13a. The crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 31. 1 H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.28 (d, J = 0.6 Hz, 1H), 7.26 (t, J = 4.3 Hz, 1H), 6.02 (dd, J = 4.0, 0.6 Hz, 1H), 4.85 (d, J = 3.9 Hz, 1H), 4.71 (t, J = 4.5 Hz, 1H), 4.47 (dd, J = 6.2, 4.0 Hz, 1H), 4.20 - 3.98 (m, 2H), 3.73 - 3.52 (m, 2H), 3.22 (qd, J = 5.2, 4.3 Hz, 2H), 1.60 - 1.42 (m, 2H), 1.41 - 1.20 (m, 21H), 0.95 - 0.80 (m, 12H), 0.09 (s, 3H), 0.06 (s, 3H) ppm.

[0095] Preparation of compound 32: using compound 31 and tetrazole as raw materials, the reaction conditions are the same as the synthesis of compound 13a. The crude product was purified by column chromatography under nitrogen protection to obtain compound 32.

[0096] Preparation of compound 38b of example 7

[0097]

[0098] Reagents and conditions: (i) TEA, MeOH, reflux; (ii) EEDQ, EtOH, 60 °C; (iii) t-Bu2Si(OTf)2, imdizole, TBDMSC1, DMF, 0 °C - r.t.; (iv) HF Py, DCM, Py, r.t.; (v) DMTrCI, Py, r.t.; (vi) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0099] Synthesis of compound 33: Compound 33 was synthesized by dissolving 6-chloroadenine nucleoside in methanol, then adding triethylamine (0.3 eq.) and propanediamine (10 eq.). After refluxing for 10 hours, the solid was filtered and washed with methanol, then dried to obtain compound 33. Without further purification, it was directly used in the next step.

[0100] Synthesis of compound 34b: Compound 33 was dissolved in anhydrous ethanol, then 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and VE-CH2CH2COOH were added, and the reaction was carried out at 60 °C. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the crude product was purified by column chromatography (eluent: DCM / EA = 5 / 1) to obtain compound 34b. 1 H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 19.9 Hz, 2H), 8.17 (s, 1H), 7.97 (s, 1H), 5.88 (d, J = 6.1 Hz, 1H), 5.43 (t, J = 8.0 Hz, 2H), 5.19 (s, 1H), 4.61 (s, 1H), 4.15 (s, 1H), 4.00 (d, J = 26.4 Hz, 3H), 3.61 (d, J = 40.1 Hz, 4H), 3.35 (s, 9H), 2.21 - 1.89 (m, 10H), 1.76 (d, J = 21.3 Hz, 5H), 1.60 - 0.95 (m, 29H), 0.83 (d, J = 6.8 Hz, 13H) ppm.

[0101] Synthesis of compound 35b: Compound 35b was synthesized from compound 34b under the same reaction conditions as the synthesis of compound 11a, and was directly used in the next step without further purification.

[0102] Preparation of compound 36b: Compound 36b was prepared from compound 35b under the same reaction conditions as the synthesis of compound 12a. 1H NMR (400 MHz, DMSO-d6) δ 8.39 (m, 2H), 8.15 (s, 1H), 7.95 (s, 1H), 5.91 (d, J = 6.0 Hz, 1H), 5.55 (s, 1H), 5.09 (d, J = 4.9 Hz, 1H), 4.73 (t, J = 5.4 Hz, 1H), 4.13 (q, J = 4.3 Hz, 1H), 4.05 - 3.99 (m, 3H), 3.71 (d, J = 11.1 Hz, 1H), 3.56 (s, 3H), 3.26 (q, J = 6.6 Hz, 2H), 2.09 (s, 3H), 2.06 (s, 3H), 1.99 (s, 3H), 1.83 - 1.76 (m, 2H), 1.72 (q, J = 6.7 Hz, 2H), 1.50 - 1.00 (m, 27H), 0.81 (t, J = 7.2 Hz, 12H), 0.71 (s, 9H), -0.12 (s, 3H), -0.22 (s, 3H) ppm.

[0103] Preparation of compound 37b: using compound 36b as material, the reaction conditions were same as the synthesis of compound 13a. The crude product was purified by column chromatography (eluent: DCM / EA / TEA = 5 / 1 / 0.01) to obtain compound 37b. 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.26 (s, 1H), 8.11 (d, J = 2.5 Hz, 2H), 7.91 (s, 1H), 7.28 - 7.12 (m, 9H), 6.82 (m, 7H), 5.90 (d, J = 4.8 Hz, 1H), 5.37 (d, J = 6.0 Hz, 1H), 4.84 (q, J = 5.5 Hz, 2H), 4.55 (s, 1H), 4.26 (d, J = 5.9 Hz, 1H), 4.03 (dd, J = 6.1, 3.6 Hz, 5H), 3.71 (d, J = 3.3 Hz, 11H), 3.57 (s, 4H), 3.26 (q, J = 7.4, 6.0 Hz, 5H), 2.08 (dd, J = 11.4, 4.0 Hz, 11H), 1.99 (d, J = 2.2 Hz, 6H), 1.76 (d, J = 10.6 Hz, 4H), 1.33 - 1.16 (m, 27H), 0.89 - 0.78 (m, 21H), 0.07 (s, 3H), 0.03 (s, 3H).

[0104] Preparation of compound 38b: using compound 37b and bis(diisopropylamino)(2-cyanoethoxy)phosphine as materials, the reaction conditions were same as the synthesis of compound 14a. The crude product was purified by column chromatography under nitrogen protection to obtain compound 38b.

[0105] Preparation of compound 44a

[0106]

[0107] Reagents and conditions: (i) TEA, MeOH reflux; (ii) EEDQ EtOH, 60 °C; (iii) t-Bu2Si(OTf)2imidizole, TBDMSC1, DMF 0 °C - r.t.; (iv) HF Py, DCM, Py, r.t.; (v) DMTrCI, Py, r.t.; (vi) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0108] Synthesis of compound 39: Compound 39 was synthesized by dissolving 6-chloroadenine nucleoside in ethanol, adding hydrazine hydrate and reacting for 10 hours. After the reaction was completed, the solid was suction filtered, washed with cold methanol and dried to obtain compound 39. No further purification was needed and it was directly used in the next step.

[0109] Synthesis of compound 40a: Compound 40a was synthesized by dissolving compound 39 in anhydrous ethanol, adding EEDQ and hexadecanoic acid and reacting at 60 °C. After the reaction was completed, the solvent was removed by distillation under reduced pressure. After the solid precipitated, it was filtered to obtain compound 40a.

[0110] Synthesis of compound 41a: Compound 41a was synthesized using compound 40a as the starting material and the same reaction conditions as those for the synthesis of compound 11a. Compound 41a was obtained without further purification and was directly used in the next step.

[0111] Synthesis of compound 42a: Compound 42a was synthesized using compound 41a as the starting material and the same reaction conditions as those for the synthesis of compound 12a. The crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 42a. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 8.21 (s, 1H), 7.87 (s, 1H), 7.80 (t, J = 5.8 Hz, 1H), 5.92 (d, J = 6.0 Hz, 1H), 5.55 (s, 1H), 5.08 (d, J = 4.9 Hz, 1H), 4.75 (t, J = 5.5 Hz, 1H), 4.14 (q, J = 4.1 Hz, 1H), 4.03 - 3.93 (m, 2H), 3.72 (dd, J = 12.3, 3.4 Hz, 1H), 3.59 (d, J = 12.4 Hz, 1H), 3.48 (s, 2H), 3.34 (s, 1H), 3.07 (dq, J = 23.9, 6.6 Hz, 2H), 2.05 (t, J = 7.4 Hz, 1H), 1.71 (q, J = 6.8 Hz, 2H), 1.56 - 1.40 (m, 2H), 1.23 (d, J = 6.2 Hz, 21H), 0.85 (td, J = 6.8, 2.5 Hz, 3H), 0.73 (s, 9H), -0.11 (s, 3H), -0.21 (s, 3H) ppm.

[0112] Preparation of compound 43a: using compound 42a and 4,4'-dimethoxytrityl chloride as material, the reaction conditions are same as the synthesis of compound 13a, to obtain compound 43a.

[0113] Preparation of compound 44a: using compound 43a and bis(diisopropylamino)(2-cyanoethoxy) phosphane as material, the reaction conditions are same as the synthesis of compound 14a. The crude product is purified by column chromatography under nitrogen protection to obtain compound 44a.

[0114] Preparation of compound 50b of example 9

[0115]

[0116] Reagents and conditions: (i) TEA, MeOH, reflux; (ii) CuSO4, L-ascorbic acid sodium salt, N2, r.t.; (iii) t-Bu2Si(OTf)2, imdizole, TBDMSCl, DMF, 0°C-r.t.; (iv) HF·Py, DCM, Py, r.t.; (v) DMTrCI, Py, r.t.; (vi) tetrazole, DCM, N2. 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0117] Preparation of compound 45: 6-chloroadenine nucleoside was dissolved in methanol, triethylamine (0.3 eq.) and propargylamine (10 eq.) were added. After refluxing for 10 hours, the solid was filtered and washed with methanol, and then dried to obtain compound 45. 1 H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 1H), 8.28 (d, J = 17.2 Hz, 2H), 5.92 (dd, J = 6.2, 1.9 Hz, 1H), 5.47 (dd, J = 6.4, 2.3 Hz, 1H), 5.35 (td, J = 4.6, 2.2 Hz, 1H), 5.20 (t, J = 3.5 Hz, 1H), 4.62 (q, J = 5.7 Hz, 1H), 4.27 (s, 1H), 4.17 (dt, J = 7.2, 3.6 Hz, 1H), 3.98 (q, J = 3.3 Hz, 1H), 3.69 (dt, J = 11.9, 4.2 Hz, 1H), 3.57 (ddd, J = 11.9, 7.0, 3.6 Hz, 1H) ppm.

[0118] Preparation of compound 46b: compound 45 was dissolved in a mixed solvent of dimethyl sulfoxide and water, copper sulfate pentahydrate and sodium ascorbate were added, and then a dimethyl sulfoxide solution of VE-C2H5N3 was added, and the reaction was carried out at room temperature under nitrogen protection. After the reaction was completed, extraction was carried out with ethyl acetate and saturated NaCl solution, the organic phase was dried with anhydrous Na2SO4, the solvent was removed by reduced pressure distillation, and the crude product was purified by column chromatography (eluent: DCM / EA = 5 / 1) to obtain compound 46b. 1 H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 1H), 8.28 (d, J = 17.2 Hz, 2H), 5.92 (dd, J = 6.2, 1.9 Hz, 1H), 5.47 (dd, J = 6.4, 2.3 Hz, 1H), 5.35 (td, J = 4.6, 2.2 Hz, 1H), 5.20 (t, J = 3.5 Hz, 1H), 4.62 (q, J = 5.7 Hz, 1H), 4.27 (s, 1H), 4.17 (dt, J = 7.2, 3.6 Hz, 1H), 3.98 (q, J = 3.3 Hz, 1H), 3.69 (dt, J = 11.9, 4.2 Hz, 1H), 3.57 (ddd, J = 11.9, 7.0, 3.6 Hz, 1H) ppm.

[0119] Synthesis of compound 47b: using compound 46b as the raw material, the reaction conditions were same as the synthesis of compound 11a. Compound 47b was obtained and used directly for the next step without further purification.

[0120] Preparation of compound 48b: using compound 47b as the raw material, the reaction conditions were same as the synthesis of compound 12a. The crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 48b. 1 H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 2H), 8.25 (s, 1H), 7.99 (s, 1H), 5.93 (d, J = 5.9 Hz, 1H), 5.54 (dd, J = 7.6, 4.2 Hz, 1H), 5.09 (d, J = 4.9 Hz, 1H), 4.81 - 4.70 (m, 3H), 4.67 (t, J = 5.0 Hz, 2H), 4.14 (q, J = 4.6 Hz, 1H), 4.01 (q, J = 3.3 Hz, 1H), 3.87 (t, J = 5.1 Hz, 2H), 3.74 - 4.70 (m, 1H), 3.55 - 4.60 (m, 1H), 2.42 (t, J = 6.9 Hz, 2H), 1.91 (s, 3H), 1.76 (s, 3H), 1.72 (s, 3H), 1.67 (d, J = 7.4 Hz, 2H), 1.53 - 0.98 (m, 31H), 0.82 (dd, J = 9.0, 6.6 Hz, 15H), 0.69 (s, 9H), -0.15 (s, 3H), -0.25 (s, 3H) ppm.

[0121] Preparation of compound 49b: using compound 48b and 4,4'-dimethoxytrityl chloride as the raw material, the reaction conditions were same as the synthesis of compound 13a. The crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 49b. 1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 16.9 Hz, 2H), 8.17 (s, 1H), 8.00 (s, 1H), 7.35 (d, J = 7.7 Hz, 2H), 7.21 (td, J = 12.2, 8.3 Hz, 7H), 6.82 (dd, J = 8.7, 2.5 Hz, 4H), 5.91 (d, J = 5.1 Hz, 1H), 5.39 (d, J = 6.2 Hz, 1H), 4.82 (q, J = 5.5 Hz, 1H), 4.75 (s, 1H), 4.65 (d, J = 6.1 Hz, 2H), 4.47 (t, J = 4.6 Hz, 1H), 4.03 (q, J = 4.6 Hz, 1H), 3.87 (t, J = 5.1 Hz, 2H), 3.70 (s, 6H), 3.12 (dd, J = 10.6, 4.9 Hz, 1H), 2.39 (t, J = 6.9 Hz, 2H), 1.90 (s, 3H), 1.76 (s, 3H), 1.66 (d, J = 3.7 Hz, 5H), 1.52 - 0.97 (m, 28H), 0.85 - 0.78 (m, 21H), 0.07 (s, 3H), 0.04 (s, 3H) ppm.

[0122] Preparation of compound 50b: using compound 49b and bis(diisopropylamino)(2- cyanoethoxy)phosphane as raw materials, the reaction conditions are the same as those of the synthesis of compound 12a, and the crude product is purified by column chromatography under nitrogen protection to obtain compound 50b.

[0123] Preparation of compound 56b of Example 10

[0124]

[0125] Reagents and conditions: (i) t-Bu2Si(OTf)2, imdizole, TBDMSCI, DMF, 0 °C-r.t.; (ii) BSA, DCM, reflux; (iii) CuSO4, L-ascorbic acid sodium salt; (iv) HF Py, DCM, Py, r.t.; (v) DMTrCI, Py, r.t.; (vi) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0126] Preparation of compound 51: using pseudouridine as raw material, the reaction conditions are the same as those of the synthesis of compound 11a. The crude product is purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 51. 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 11.02 (s, 1H). 7.57 (dd, J = 6.4, 1.8 Hz, 1H), 4.66 (dd, J = 6.0, 1.8 Hz, 1H), 4.33 (t, J = 5.9 Hz, 1H), 4.25 (dd, J = 5.7, 4.6 Hz, 1H), 4.03 (dd, J = 2.1, 0.6 Hz, 2H), 3.97 (dt, J = 4.4, 2.1 Hz, 1H), 1.00 (s, 18H), 0.87 (s, 9H), 0.07 (s, 6H).

[0127] Preparation of compound 52: Compound 51 was dissolved in super dry dichloromethane, N, O-bis(trimethylsilyl)acetamide (BSA) and bromo propyl alcohol were added and the reaction was refluxed, the progress of the reaction was monitored by TLC. After the reaction was completed, part of the solvent was removed, and the crude product was purified by column chromatography to obtain compound 52. 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 7.61 (d, J = 1.1 Hz, 1H), 4.59 (s, 1H), 4.56 (d, J = 2.5 Hz, 2H), 4.38 (dd, J = 8.9, 4.6 Hz, 1H), 4.21 (d, J = 4.2 Hz, 1H), 3.97 (dd, J = 9.3, 4.5 Hz, 2H), 3.93 - 3.85 (m, 1H), 3.43 (t, J = 2.5 Hz, 1H), 1.02 (s, 9H), 1.00 (s, 9H), 0.90 (s, 9H), 0.14 (s, 3H), 0.10 (s, 3H) ppm.

[0128] Preparation of compound 53b: Compound 52 was used as raw material, and the reaction conditions were the same as the synthesis of compound 46b. The crude product of compound 53b was obtained without further purification and was directly used in the next step.

[0129] Preparation of compound 54b: Compound 53b was used as raw material, and the reaction conditions were the same as the synthesis of compound 12a. The crude product of compound 54b was obtained without further purification and was directly used in the next step.

[0130] Preparation of compound 55b: Compound 54b and 4,4'-bis-methoxy trityl chloride were used as raw materials, and the reaction conditions were the same as the synthesis of compound 13a. The crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 55b. 1H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H), 8.58 (d, J = 4.7 Hz, 1H), 8.06 (s, 1H), 7.66 (s, 1H), 7.45 (d, J = 7.7 Hz, 2H), 7.36 - 7.25 (m, 7H), 6.91 - 6.86 (m, 4H), 4.75 (d, J = 15.3 Hz, 1H), 4.69 (d, J = 5.4 Hz, 3H), 4.56 (d, J = 2.6 Hz, 1H), 4.39 (m, 1H), 4.10 (d, J = 3.8 Hz, 1H), 3.90 (m, 4H), 3.72 (s, 6H), 3.26 (d, J = 9.2 Hz, 1H), 3.16 (d, J = 10.2 Hz, 1H), 2.44 (d, J = 6.8 Hz, 2H), 1.92 (s, 3H), 1.76 (s, 3H), 1.68 (s, 3H), 1.40 - 1.04 (m, 28H), 0.88 - 0.81 (m, 21H), 0.06 (s, 3H), 0.04 (s, 3H) ppm.

[0131] Preparation of compound 56b: Compound 55b and bis(diisopropylamino)(2- cyanoethoxy)phosphane were used as starting materials, and the reaction conditions were the same as those for the synthesis of compound 14a. The crude product was purified by column chromatography under nitrogen protection to obtain compound 56b.

[0132] Preparation of compound 63a

[0133]

[0134] Reagents and conditions: (i) TIPDSiCI2, Py., r.t.; (ii) PomCI, TBAHS, aq. 0.2~0.4M Na2CO3, DCM; (ii) BTTP, THF, MeCN; (iv) Et3N(HF)3, THF (v) DMTrCI, Py, r.t.; (vi) tetrazole, DCM N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0135] Preparation of compound 58a: Uracil nucleoside was dissolved in 20 mL of super dry pyridine, and 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane was added. After the reaction was completed, the product was extracted with ethyl acetate and 10% hydrochloric acid aqueous solution, and the organic phase was dried over anhydrous Na2SO4. After removing the solvent under reduced pressure, the crude product of compound 58a was obtained without further purification and was directly used in the next step.

[0136] Preparation of compound 59a: Compound 58a was dissolved in two-phase solvent of dichloromethane and aqueous solution of Na2CO3(0.2-0.4 M) (V / V, 1 / 3), then chloromethyl pivalate (2 eq.) and tetrabutylammonium hydrogen sulfate (TBAHS) were added successively, and the reaction was carried out at room temperature for 48 h. After the reaction was completed, the product was extracted with ethyl acetate and 5% aqueous NaHCO3solution, and the organic phase was dried over anhydrous Na2SO4. After the solvent was removed by distillation under reduced pressure, the crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 59a. 1 HNMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 8.5 Hz, 1H), 5.99 - 5.88 (m, 2H), 5.86 - 5.67 (m, 3H), 5.01 - 4.82 (m, 1H), 4.28 - 4.14 (m, 4H), 4.09 - 3.96 (m, 1H), 3.88 - 3.65 (m, 4H), 2.73 - 2.61 (m, 2H), 2.27 - 2.09 (m, 9H), 1.79 - 1.14 (m, 36H), 0.92 - 0.72 (m, 12H) ppm.

[0137] Preparation of compound 60a: Compound 59a was dissolved in a mixed solution of acetonitrile and tetrahydrofuran, and (tert-butylimino)tris(pyrrrolidine)phosphine and VE-C2H5I were added successively, and the reaction was continued at room temperature. After the reaction was completed, the solvent was removed by distillation under reduced pressure, then extracted with ethyl acetate and saturated aqueous NaCl solution, and the organic phase was dried over anhydrous Na2SO4. After the solvent was removed by distillation under reduced pressure, the crude product of compound 60a was obtained without further purification and was directly used in the next step.

[0138] Preparation of compound 61a: Compound 60a was dissolved in super-dry tetrahydrofuran, and triethylamine trihydrofluoride solution was added under stirring at 0°C. After the reaction was completed, the product was extracted with ethyl acetate and saturated aqueous NaHCO3solution, and the organic phase was dried over anhydrous Na2SO4. After the solvent was removed by distillation under reduced pressure, the crude product was purified by column chromatography (eluent: DCM / MeOH = 9 / 1) to obtain compound 61a. 1 HNMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 8.5 Hz, 1H), 5.99 - 5.88 (m, 2H), 5.86 - 5.67 (m, 3H), 5.01 - 4.82 (m, 1H), 4.28 - 4.14 (m, 4H), 4.09 - 3.96 (m, 1H), 3.88 - 3.65 (m, 4H), 2.73 - 2.61 (m, 2H), 2.27 - 2.09 (m, 9H), 1.79 - 1.14 (m, 36H), 0.92 - 0.72 (m, 12H) ppm.

[0139] Preparation of compound 62a: using compound 61a and 4,4'-dimethoxytrityl chloride as the starting material, the reaction conditions are the same as those in the synthesis of compound 13a to obtain compound 62a.

[0140] Preparation of compound 63a: using compound 62a and bis(diisopropylamino)(2-cyanoethoxy)phosphine as the starting material, the reaction conditions are the same as those in the synthesis of compound 14a. The crude product is purified by column chromatography under nitrogen protection to obtain compound 63a.

[0141] Preparation of compound 69a in Example 12

[0142]

[0143] Reagents and conditions: (i) TIPDSiCI2, Py., r.t.; (ii) K2CO3MeCN, r.t.-40 °C; (iii) Et3N(HF)3, THF; (iv) DMTrCI, Py, r.t.; (v) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0144] Preparation of compound 65a: using 2'-methoxyuracil nucleoside as the starting material, the reaction conditions are the same as those in the synthesis of compound 58a. The crude product of compound 65a is obtained without further purification and is directly used in the next step.

[0145] Preparation of compound 66a: compound 65a is dissolved in acetonitrile, potassium carbonate is added, and stirred at room temperature for 30 minutes, then bromohexadecane is added, and the reaction is continued at 40 °C. After the reaction is completed, ethyl acetate and saturated NaCl aqueous solution are used for extraction, and the organic phase is dried with anhydrous Na2SO4. The solvent is removed, and the crude product is purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 66a.

[0146] Preparation of compound 67a: using compound 66a as the starting material, the reaction conditions are the same as those in the synthesis of compound 61a. The crude product is purified by column chromatography (eluent: DCM / MeOH = 9 / 1) to obtain compound 67a.

[0147] Preparation of compound 68a: using compound 67a and 4,4'-dimethoxytrityl chloride as the starting material, the reaction conditions are the same as those in the synthesis of compound 13a to obtain compound 68a. 1H NMR (400 MHz, DMSO-d6) δ 7.54 (d, J = 8.1 Hz, 1H), 7.37 (dd, J = 6.7, 3.0 Hz, 2H), 7.28 - 7.23 (m, 5H), 7.10 (dd, J = 8.8, 4.1 Hz, 4H), 6.85 (t, J = 8.4 Hz, 4H), 6.81 - 6.74 (m, 4H), 5.87 (d, J = 2.9 Hz, 1H), 5.24 (d, J = 8.0 Hz, 1H), 4.03 - 3.91 (m, 2H), 3.71 (d, J = 1.3 Hz, 6H), 3.24 (d, J = 10.8 Hz, 1H), 3.19 (s, 3H), 3.13 (dd, J = 11.0, 4.6 Hz, 1H), 1.47 (t, J = 7.2 Hz, 2H), 1.23 (m, 29H), 0.88 - 0.80 (m, 3H).

[0148] Preparation of compound 69a: using compound 68a and bis(diisopropylamino)(2- cyanoethoxy)phosphane as raw materials, the reaction conditions are the same as those in the synthesis of compound 14a, and the crude product is purified by column chromatography under nitrogen protection to obtain compound 69a.

[0149] Preparation of compounds 75a and 75b

[0150]

[0151] Reagents and conditions (i) t-Bu2Si(OTf)2, imdizole, TBDMSCI, DMF, 0 °C-r.t.; (ii) CuSO4, L-ascorbic acid sodium salt; (iii) HF Py, DCM, Py, r.t.; (iv) DMTrCI, Py, r.t.; (v) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0152] Preparation of compound 71: using 5-alkynyl uracil nucleoside as raw material, the reaction conditions are the same as those in the synthesis of compound 11a, to obtain compound 71.

[0153] Preparation of compound 72b: using compound 71 and VE-C2H5N3 as raw materials, the reaction conditions are the same as those in the synthesis of compound 46b. The crude product of compound 72b is obtained without further purification and is directly used in the next step.

[0154] Preparation of compound 73b: using compound 72b as raw material, the reaction conditions are the same as those in the synthesis of compound 12a, to obtain compound 73b.

[0155] Preparation of compound 74b: using compound 73b and 4,4'-dimethoxybenzhydryl chloride as the raw material, the reaction conditions were the same as those in the synthesis of compound 13a. The crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 74b. 1 H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.47 (s, 1H), 8.39 (s, 1H), 7.42 (d, J = 7.4 Hz, 2H), 7.34 - 7.28 (m, 4H), 7.25 (t, J = 7.7 Hz, 2H), 7.14 (t, J = 7.3 Hz, 1H), 6.88 - 6.82 (m, 4H), 5.92 (d, J = 5.4 Hz, 1H), 5.11 (d, J = 6.2 Hz, 1H), 4.75 (t, J = 5.0 Hz, 2H), 4.32 (t, J = 5.4 Hz, 1H), 4.02 (m, 1H), 3.91 (m, 3H), 3.68 (d, J = 3.7 Hz, 6H), 3.28 (d, J = 4.0 Hz, 2H), 1.93 (s, 3H), 1.85 (s, 3H), 1.81 (s, 3H), 1.69 (q, J = 6.5 Hz, 2H), 1.51 - 0.97 (m, 28H), 0.85 - 0.76 (m, 22H), 0.03 (s, 3H), 0.01 (s, 3H) ppm.

[0156] Preparation of compound 75b: using compound 74b and bis(diisopropylamino)(2-cyanoethoxy) phosphine as the raw material, the reaction conditions were the same as those in the synthesis of compound 14a to obtain compound 75b.

[0157] Experimental Example 1 Evaluation of biological activity of siRNA sequence doped with modified nucleosides

[0158] 1 Experimental method

[0159] 1.1 siRNA sequence doped with modified nucleosides

[0160] The small interfering RNA (siRNA-APP) targeting amyloid precursor protein (APP) reported in the literature was modified to obtain the target sequence: 3'-u·a·uga(X)GuUCAucaucaaa·a·a(SS) and 5'-VPu·U·uuug AugaugaAcUucaua·u·c (AS), where SS and AS represent sense and antisense strands, respectively; capital and lowercase letters represent 2'-fluoro (2'-F) and 2'-O-methyl (2'-OMe) ribose sugar modifications, respectively; underlined capital letters represent glycol nucleic acid (GNA) modifications; · indicates phosphorothioate (PS) modification; (X) represents modified nucleotide doping; VP represents 5'-(E)-vinylphosphonate.

[0161] Table 1. Doping pattern of RNA sequences of modified nucleotide pairs

[0162]

[0163]

[0164] Note: 4a in Table 1 is compound 4a prepared in Example 1; 2-Hd-A is 2'-C16 modified adenosine already reported in the literature (doi.org / 10.1038 / s41587-022-01334-x); 14a is compound 14a prepared in Example 3; 19a is compound 19a prepared in Example 4; 19b is compound 19b prepared in Example 4; 25 is compound 25 prepared in Example 5; 32 is compound 32 prepared in Example 6.

[0165] 1.2 Cell culture and transfection

[0166] HepG2 (human hepatocarcinoma cells), ARPE-19 (human retinal epithelial cells), U87-MG (human malignant glioma cells) and ACHN (human renal adenocarcinoma cells) were chosen for cell level activity evaluation.

[0167] HepG2, U87-MG, ACHN and ARPE-19 cells were thawed in 25 cm 2Add 5 mL of DMEM (10% FBS; ARPE-19 cells use ARPE-DMEM) to a permeable cell culture flask and place it in a cell culture incubator (constant temperature 37℃, constant CO2 concentration 5%) for proliferation. When the cell density in the culture flask reaches approximately 90%, passage the cells. Wash twice with 1×PBS (2 mL / wash), then add 1 mL of trypsin and digest in the cell culture incubator for 2–3 min until the cells are completely digested. Add 3 mL of DMEM (10% FBS) to stop the digestion. Use a 1 mL pipette to remove the cells from the bottom of the flask, then transfer the cell suspension to a 15 mL centrifuge tube and centrifuge for 5 min (1000 rpm). Discard the supernatant and resuspend the cells in fresh DMEM (10% FBS) medium. Take 1 / 3 of the cell resuspended medium and add it to a 75 cm⁻¹ tube containing 15 mL of DMEM (10% FBS). 2 Mix well in a breathable cell culture flask and continue proliferation in an incubator. Count cells using a cell counting chamber and prepare 2×10⁶ cells / flasks. 5 Add 0.5 mL of cell resuspension to each well of a 24-well plate and 0.25 mL of cell resuspension to each well of a 48-well plate. Mix well using the cross-hatching method and incubate in a cell incubator for 24 hours.

[0168] When the cell density in the well plate was approximately 70%, no commercial transfection reagents were used for cell transfection experiments. After the target transfection time point, the well plate was removed, the culture medium was removed, and the cells were washed three times with 1×PBS. Trypsin was then added, and the plate was placed in a cell culture incubator. After complete cell digestion, DMEM (10% FBS) medium was added to terminate the digestion. The cell suspension was transferred to a 1.5 ml centrifuge tube, centrifuged for 5 min (1000 rpm), the supernatant was discarded, and the cells were resuspended in 1×PBS before being analyzed by flow cytometry.

[0169] 1.3 Real-time quantitative PCR

[0170] Total RNA was extracted according to the instructions of the TRIZOL total RNA extraction reagent, and RNA concentration and absorption peak type were detected using a NanoDrop 2000. Then, following the instructions of the Novizan HiScript III 1st Strand cDNASynthesis Kit (+gDNAwiper), 300 ng of total RNA was used for reverse transcription. The cDNA obtained from reverse transcription was diluted in 100 μL of enzyme-free water and then processed according to the Promega kit instructions. Following the instructions for the qPCR Master Mix Real-Time PCR Kit, after preparing the qPCR system, perform detection on an ABI Real-Time PCR instrument. The fluorescence signal is SYBR Green. The housekeeping genes GAPDH / β-actein are selected as internal control genes.

[0171] 2Experimental results

[0172] The experimental results are shown in Table 1. Figure 1 As shown in Table 1, the knockdown effect of compound 4a modified NO.3 siRNA on APP mRNA is better than that of 2-Hd-A modified NO.7 siRNA, which indicates that the biological activity of siRNA will not be affected after the structural modification of nucleoside monomers and then integrating them into siRNA. At the concentration of 10 nM and 50 nM, NO.3 siRNA is better than NO.7 siRNA.

[0173] Compound 4a modified NO.3 siRNA shows good uptake effect in HepG2 Figure 2 (A and B) and ARPE-19 Figure 2 (C and D) cells, and shows gradient dependence with the change of siRNA concentration and transfection time, especially in ARPE-19 cells. Figure 3 The results of U87-MG cell uptake of various modified nucleoside doped siRNAs at the concentration of 1 nM without the assistance of transfection reagent are shown: at the concentration of 1 nM, NO.3, NO.7 and NO.8 sequences are slightly better than the control sequence NO.15, but the uptake efficiency of NO.1-2, NO.4-6 and NO.9-14 modified sequences in U87-MG cells is increased by 2-3 times. Figure 4 The results of free uptake of various modified siRNAs in U87-MG and ACHN cells at the concentration of 100 nM without the action of transfection reagent are shown: compared with sugar ring 2' C16 modified siRNA (NO.7), all alkyl chain modified siRNAs on the base and sugar ring 2' vitamin E modified siRNAs show better free uptake efficiency.

Claims

1. A modified nucleoside monomer compound, characterized in that, Its structural formula is shown in the following general formula Ia or Ib: Wherein, Base is a base, preferably selected from cytosine, uracil, adenine, thymine, guanine, or pseudouracil; or derivatives of any of the bases; preferably, the derivatives include, but are not limited to: 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, and 5-propynyl. Uracil, 6-azouracil, 4-thionuracil; adenine or guanine of 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, or 8-hydroxyaldehyde; 5-halogenated uracil, 5-trifluoromethyluracil, wherein the 5-halogenated uracil is preferably 5-bromouracil; 7-methylguanine, 7-methyladenine; 8-azaguanine, 8-azaadenine; 7-deazoguanine, 7-azaadenine; 3-deazoguanine or 3-deazoadenine; R 1 Selected from hydrogen, 4,4'-dimethoxytriphenylmethane, phosphoramide, or phosphoric acid analogs; R 2 Selected from hydrogen, phosphorous amide, or phosphoric acid analogs; R 3 Selected from hydroxyl, methoxy, methylethoxy, fluorine, chlorine, bromine, iodine or L 1 or L 2 Simultaneously, they may be covalent bonds, divalent saturated or unsaturated straight chains or branched chains C. 1-50 A hydrocarbon chain wherein the 0-10 methylene units of the hydrocarbon chain are independently converted by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -CH2C(O)NHNH-, -(CH2) n C(O)NH-、 -V 1 CR 2 W 1 -or Substitution; where m is any integer from 1 to 50, and n is any integer from 1 to 50; each -Cy- is independently an optionally substituted divalent ring; the divalent ring is selected from phenylene, 8-10 membered bicyclic arylene, 4-7 membered saturated or partially unsaturated carbocyclic, 4-11 membered saturated or partially unsaturated spirocyclic, 8-10 membered bicyclic saturated or partially unsaturated carbocyclic, and 4-7 membered saturated or partially unsaturated carbocyclic with 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Partially unsaturated heterocyclic groups, including 4-11 saturated or partially unsaturated spirocyclic groups having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 8-10 saturated or partially unsaturated heterocyclic groups having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 5-6 aryl heterocyclic groups having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or 8-10 aryl heterocyclic groups having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; V 1 or W 1 Each of the following is independently selected from -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; R is selected from hydrogen, straight-chain or branched C. 1-50 Alkyl, aryl, hydroxyl, alkoxy, halogen, methoxyalkoxy, alkylthio, amino, alkylamino, alkynyl, aminoalkyl or aminoalkoxy; R 4 Vitamin E, hexadecyl or n is any integer from 1 to 20.

2. The modified nucleoside monomer compound according to claim 1, characterized in that, The modified nucleoside monomer compound is selected from any one of the phosphoramidite monomer compounds shown in II-a or II-b below: Wherein, B1 is a base, preferably selected from any one of the bases of cytosine, uracil, adenine, thymine, guanine, or pseudouracil; or a derivative selected from any one of the bases; X is selected from oxygen, nitrogen, sulfur, or selenium; R 3 Selected from hydroxyl, methoxy, methylethoxy, fluorine, chlorine, bromine, iodine or L 1 or L 2 Simultaneously, they may be covalent bonds or divalent saturated or unsaturated straight or branched chains C. 1-50 A hydrocarbon chain; wherein the 0-10 methylene units of the hydrocarbon chain are independently converted by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-, -CH2C(O)NHNH-, -(CH2) n C(O)NH-、 -V 1 CR 2 W 1 -or Replacement, where m is any integer from 1 to 50, and n is any integer from 1 to 50; each -Cy- is independently an optionally substituted divalent ring; the divalent ring is selected from phenylene, 8-10 membered bicyclic arylene, 4-7 membered saturated or partially unsaturated carbocyclic, 4-11 membered saturated or partially unsaturated spirocyclic, 8-10 membered bicyclic saturated or partially unsaturated carbocyclic, and 4-7 membered saturated or partially unsaturated carbocyclic with 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Partially unsaturated heterocyclic groups, including 4-11 saturated or partially unsaturated spirocyclic groups having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 8-10 saturated or partially unsaturated heterocyclic groups having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 5-6 aryl heterocyclic groups having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or 8-10 aryl heterocyclic groups having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; V 1 or W 1 Each of the following is independently selected from -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; R is selected from hydrogen, straight-chain or branched C1-C. 24 Alkyl, aryl, hydroxyl, alkoxy, halogen, methoxyalkoxy, alkylthio, amino, alkylamino, alkynyl, aminoalkyl, or aminoalkoxy; R 4 Vitamin E, hexadecyl or n is any integer from 1 to 20.

3. The pharmaceutically acceptable salt of the modified nucleoside monomer compound according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts are sodium salts, phosphates, quaternary ammonium salts, sulfates, hydrochlorides, nitrates, or acetates.

4. A method for preparing the modified nucleoside monomer compound of claim 2, characterized in that, include: (1) A method for preparing the phosphorous amide monomer compound shown in II-a includes: using a nucleoside as a starting material, in a mixed solution of dimethyl sulfoxide and N,N-dimethylformamide, using sodium hydride as a base, a substitution reaction is carried out to obtain 2'-OL. 1 The modified intermediate was further subjected to hydroxyl protection and phosphoramidation to prepare 2'-OL. 1 -VE-modified nucleosides and their phosphoramidides monomers; wherein the nucleosides are selected from, but not limited to, uracil nucleosides, pseudouracil nucleosides, cytosine nucleosides, thymidine nucleosides, adenine nucleosides, or guanine nucleosides; Preferably, using uracil nucleoside as the starting material, the sugar ring -3',5'-OH is first silica-protected, and then methyl neopentanoate is refluxed in dichloromethane in the presence of (tert-butylimino)tris(pyrrolidine)phosphine to prepare an intermediate. The intermediate is then subjected to hydroxyl protection and phosphoramidation to prepare 2'-OL. 1 -phosphite monomers of VE; (2) A method for preparing the phosphoramidite monomer compound shown in II-b, wherein B1 in II-b is adenine or guanine, the preparation method comprising: using a nucleoside derivative with chlorine or bromine substitution on the corresponding base as a starting material, using triethylamine as a base, and reacting it with the amino derivative with the corresponding substituent in claim 1 under reflux in an alcohol or ethylene glycol monomethyl ether to obtain NH-L 2 Modified nucleoside intermediate; the intermediate is then subjected to hydroxyl protection and phosphoramidation to prepare NH-L 2 -R 4 Modified nucleosides or their phosphoramide monomers; (3) A method for preparing the phosphoramidite monomer compound shown in II-b, wherein B1 in II-b is pseudouracil, uracil, thymine, or cytosine, the preparation method comprising: using pseudouracil nucleoside, uracil nucleoside, thymine nucleoside, or cytosine nucleoside as starting material, using sodium hydride as base, reacting with the halogenated compound with the corresponding substituent in claim 1 in a mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide to obtain NH-L 2 Modified intermediate; the intermediate is then subjected to hydroxyl protection and phosphoramidation to prepare NH-L 2 -R 4 Modification of nucleosides or their phosphoramide monomers; Preferably, using uracil and thymidine as starting materials, the sugar ring -OH is first protected with silica, and then reacted with the halogenated compound with the corresponding substituent in claim 1 in acetonitrile in the presence of (tert-butylimino)tris(pyrrolidine)phosphine to obtain NL with an imine-modified base. 2 Modified intermediate; the intermediate is then subjected to a multi-step reaction to prepare NL. 2 -R 4 Phosphite monomers that modify nucleosides; (4) When a nucleoside containing an alkynyl group is used, it reacts with the corresponding azide compound in dimethyl sulfoxide / water to give NH-L 2 Modified intermediate; the intermediate is then subjected to hydroxyl protection and phosphoramidation to prepare NH-L 2 -R 4 Phosphoramide monomers that modify nucleosides.

5. An oligonucleotide containing or doped with a nucleoside monomer compound modified as described in claim 1 or 2, preferably, the oligonucleotide comprising: miRNA, gRNA, antisense nucleic acid, or siRNA.

6. The oligonucleotide according to claim 5, characterized in that, The antisense nucleic acid or siRNA comprises an antisense strand of 15 to 30 nucleotides in length and / or a sense strand of 15 to 40 nucleotides in length.

7. The oligonucleotide according to claim 6, characterized in that, The sense strand and antisense strand form a double-stranded region, wherein the antisense strand has a complementary region that is complementary to the target sequence, and the length of the complementary region is at least 15 consecutive nucleotides, differing by no more than 3 nucleotides; preferably, the complementary region is completely complementary to the target sequence; the length of the antisense strand is 19 to 27 nucleotides.

8. The oligonucleotide according to claim 5, characterized in that, The oligonucleotide comprises at least one modified nucleoside as described in claim 1 or 2; preferably, the oligonucleotide is also partially or completely thiomodified on the backbone, and / or modified on the sugar ring, or modified on the base, or the 5'-end of the antisense strand of the siRNA includes, but is not limited to, phosphate ester, oxymethylphosphonate, vinylphosphonate or malonylphosphonate modification.

9. Use of the modified nucleoside monomer compound of claim 1 or 2, or the oligonucleotide of any one of claims 5-8, in the preparation of nucleic acid drugs.

10. A pharmaceutical composition, characterized in that, The product comprises any oligonucleotide doped with the modified nucleoside monomer compound of claim 1 or 2, or a conjugate formed by an oligonucleotide doped with the modified nucleoside monomer compound of claim 1 or 2 and a ligand, as well as a pharmaceutically acceptable carrier, delivery agent, or excipient.

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