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

Abstract

Disclosed in the present invention are a modified nucleoside and nucleoside monomer, an oligonucleotide containing the modified nucleoside monomer and the use thereof. According to the present invention, lipophilic / hydrophilic groups, fragments with a pharmacological activity and receptor-targeting properties, etc., are introduced to modify a nucleoside, thereby obtaining a modified nucleoside and a phosphoramidite monomer thereof. The modified nucleoside has significantly improved physicochemical properties and bioavailability. According to the present invention, the modified nucleoside is further incorporated into an oligonucleotide, such that the cellular free uptake efficiency of an oligonucleotide drug can be significantly enhanced, and a target mRNA can be silenced. The present invention holds promise for achieving efficient delivery of a modified nucleic acid drug to various organs and tissues for the treatment of relevant indications.

Description

Modified nucleosides, nucleoside monomers, oligonucleotides containing modified nucleoside monomers and their applications Technical Field

[0001] This invention relates to modified nucleoside monomers, and more particularly to modified nucleoside monomers obtained by modifying the sugar ring and base of nucleoside monomers, antisense nucleic acids or siRNAs containing the modified nucleoside monomers, and their application in the preparation of nucleic acid drugs, belonging to the field of modified nucleoside monomers and their applications. Background Technology

[0002] Oligonucleotide drugs (including antisense nucleic acids, miRNAs, siRNAs, gRNAs, etc.) have brought great hope to the treatment of diseases for which humanity currently has no effective cure. However, using nucleic acid drugs as a therapeutic approach is also challenging, mainly due to the following: immunogenic reactions triggered by exogenous nucleic acid drugs; immunogenic and non-immunogenic toxicity of nucleic acid delivery carriers; unpredictable activity due to off-target effects; and the target gene knockdown activity of nucleic acid drugs accumulating in non-target tissues. Furthermore, unmodified nucleic acid drugs, due to their exogenous nature, unfavorable physicochemical properties, low stability in serum, rapid renal clearance, reduced uptake by target cells, and inability to be taken up by phagocytes and activate immune responses, are not easily administered systemically, which greatly hinders their clinical development. Therefore, the complete release and enhancement of the therapeutic potential of nucleic acid drugs depends not only on chemical modification and / or on protecting nucleic acids from degradation and ensuring their stability in circulation so that they can be localized to target tissues, but also on safe, efficient, and reliable delivery platform technologies to ensure effective intracellular delivery and tissue distribution. For example, Alnylam introduced a C16 group at the 2' position of the nucleotide sugar ring in its siRNA drug, enabling widespread distribution of the drug in neurons, astrocytes, and microglia via a single intrathecal administration. At the highest dose of 0.9 mg, SOD1 siRNA can silence over 75% of target genes. Currently, alkyl chain-modified oligonucleotide drugs are widely used for indications in various organs and tissues, including the brain, eyes, skin, fat, liver, lungs, and kidneys.

[0003] Therefore, introducing modified nucleosides into nucleic acid drugs can not only enhance their chemical and biological stability and reduce or avoid the use of lipid delivery materials, but also potentially endow them with the ability to directly target specific tissues / organs. This would decouple the use of nucleic acid drugs from their own or external adverse risks and promote their wider clinical application. Summary of the Invention

[0004] One objective of this invention is to modify the structure of nucleosides by performing structural modifications on the sugar ring skeleton and the base moiety, respectively, to obtain modified nucleosides and their phosphoridamide monomer compounds.

[0005] A second objective of this invention is to provide a method for preparing the modified nucleoside and its phosphoramide monomer compound;

[0006] The third objective of this invention is to dope the modified nucleoside monomer into oligonucleotides to obtain modified oligonucleotides;

[0007] The fourth objective of this invention is to prepare the modified oligonucleotides into nucleic acid drugs.

[0008] Based on the above considerations, this invention modifies the structure of the glycan backbone and base portion of nucleoside monomers and then prepares antisense nucleic acids and siRNAs modified by these nucleoside monomers. Furthermore, it studies the cellular uptake, efficacy, and tissue distribution of these modified antisense nucleic acids and siRNAs in order to obtain modification strategies with further research potential and provide new candidate antisense nucleic acid and siRNA drugs for clinical use. This completes the invention.

[0009] One aspect of the present invention provides a modified nucleoside monomer compound or a pharmaceutical salt thereof, having the structural formula shown in the following general formula Ia or Ib:

[0010] Base is selected from cytosine (C), uracil (U), adenine (A), thymine (T), guanine (G), or pseudouracil (ΨU) or their derivatives;

[0011] R 1 Selected from hydrogen, 4,4'-dimethoxytriphenylmethane (DMTr) or phosphoramide analogs;

[0012] R 2 Selected from hydrogen, phosphorous amide, or phosphoric acid analogs;

[0013] R 3 Selected from hydroxyl, methoxy, methylethoxy, fluorine, chlorine, bromine, iodine or

[0014] 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 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 independently hydrogen, straight-chain or branched C1-C each time it appears. 50 Alkyl, aryl, hydroxyl, alkoxy, halogen, methoxyalkoxy, alkylthio, amino, alkylamino, alkynyl, aminoalkyl or aminoalkoxy;

[0015] R 4 Vitamin E, hexadecyl or n is any integer from 1 to 20.

[0016] Preferably, the pharmaceutically acceptable salt is a sodium salt, phosphate, quaternary ammonium salt, sulfate, hydrochloride, nitrate, or acetate.

[0017] In a preferred embodiment of the present invention, the modified nucleoside monomer compound is selected from any one of the phosphoramidide monomer compounds shown in II-a or II-b below:

[0018] Wherein, 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-thiothymidine, 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyluracil, 6-azouracil, 4-thiouracil, etc. Uracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyaldehyde, other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromine, 5-trifluoromethyl and other 5-substituted uracil, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-azaadenine, and 3-deadenine and 3-deadenine;

[0019] X is selected from oxygen (O), nitrogen (N), sulfur (S), or selenium (Se);

[0020] R 3 Selected from hydroxyl, methoxy, methylethoxy, fluorine, chlorine, bromine, iodine or

[0021] 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 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 independently hydrogen, straight-chain or branched C1-C each time it appears. 24 Alkyl, aryl, hydroxyl, alkoxy, halogen, methoxyalkoxy, alkylthio, amino, alkylamino, alkynyl, aminoalkyl or aminoalkoxy;

[0022] R 4 Vitamin E, hexadecyl or n is any integer from 1 to 20.

[0023] Another aspect of the present invention is to provide a method for preparing the monomeric compounds shown in II-a and II-b above, comprising:

[0024] (1) In II-a, when B1 is A and G, N-benzoyl adenosine and N-isobutyrylguanosine are used as starting materials, respectively, sodium hydride is used as base, and a mixed solution of dimethyl sulfoxide and N,N-dimethylformamide is used as reaction solvent. 2'-OL is obtained through substitution reaction. 1 The modified intermediate can be further reacted with the intermediate to prepare 2'-OL. 1 -VE-A / G phosphoramide monomer.

[0025] (2) In II-a, when B1 is C and ΨU, cytosine nucleoside and pseudouridine nucleoside are used as starting materials, sodium hydride is used as base, and a mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide is used as reaction solvent. 2'-OL is obtained through the substitution reaction of 2'-OH. 1 Modified intermediate; 2'-OL can be prepared by further reacting the intermediate through multiple steps. 1 -VE-C / ΨU phosphoridamide monomer;

[0026] (3) In II-a, when B1 is uracil (U), the sugar ring 3',5'-OH is first silane protected, 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 (BTTP) to obtain NH-L. 2 The modified nucleoside intermediate, through a series of further reactions, can be used to prepare 2'-OL. 1 -VE-U phosphoramide monomer;

[0027] (4) In II-b, when B1 is adenine (A), 6-chloroadenosine is used as the starting material, triethylamine is used as the base, and the amino derivative with the corresponding substituent in claim 1 is refluxed in methanol or ethanol to give 6-NH-L 2 Modified intermediate; 6-NH-L can be prepared by further reacting the intermediate through multiple steps. 2 -R 4 Phosphite monomers;

[0028] (5) In II-b, when B1 is pseudouracil (ΨU), thymine (T)T, or deoxyuracil, pseudouracil nucleoside is used as the starting material, sodium hydride is used as the base, and it reacts with the corresponding halogenated compound with the substituents mentioned above in a mixed solvent of dimethyl sulfoxide and N,N-dimethylformamide to obtain ΨU 5-NL. 2 The important intermediate for modification, or the intermediate for T or deoxygenated U, 3-NL 2 The above intermediate can be further reacted through multiple steps to prepare 3- or 5-NL. 2 -R 4 Phosphite monomers;

[0029] In some specific implementation cases, taking pseudouridine as an example, the sugar ring -OH is first silicon protected, and then N,O-bis(trimethylsilyl)acetamide (BSA) is used as a base to react with an alkyne haloalkane under reflux in dichloromethane to prepare an important alkyne-modified intermediate. The product obtained by reacting the above intermediate with an azide compound can be further reacted through multiple steps to prepare 5-NL. 2 -R 4 Phosphite monomers;

[0030] (6) In II-b, when B1 is cytosine (C), N-benzoylcytidine is used as the starting material, sodium hydride is used as the base, and the halogenated compound with the corresponding substituent mentioned above is reacted with a mixture of dimethyl sulfoxide and N,N-dimethylformamide as the reaction solvent, via Ph-NL. 2 The modified nucleoside intermediate, through a series of further reactions, can be used to prepare 4-NH-L.2 -R 4 Phosphite monomers;

[0031] (7) In II-b, when B1 is guanine (G), 2′,3′,5′-triacetylguanosine is used as the starting material and reacted with n-amyl nitrite in bromoform under reflux to prepare the intermediate 2′,3′,5′-triacetyl-2-bromoguanosine. Then, it is reacted with the amino derivative with the corresponding substituent in the above-mentioned contents under reflux in ethylene glycol monomethyl ether to prepare 2-NH-L 2 The modified nucleoside intermediate, through a series of further reactions, can be used to prepare 2-NH-L. 2 -R 4 The phosphorus amide monomer.

[0032] (8) In II-b, when B1 is an alkynyl-modified nucleoside, reacting it with the azide compound corresponding to the substituents mentioned above in dimethyl sulfoxide / water yields a triazole-linked modified nucleoside intermediate. This nucleoside intermediate can then be further reacted in multiple steps to prepare L. 2 -R 4 Phosphoramide monomers that modify nucleosides.

[0033] Another aspect of the present invention is to provide oligonucleotides or siRNAs containing or doped with the modified nucleoside monomeric compound described above; the oligonucleotides comprising 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 double-stranded region, the antisense strand having a complementary region complementary to the target sequence, and the complementary region being at least 15 consecutive nucleotides in length, differing by no more than 3 nucleotides; in a more preferred embodiment of the present invention, the complementary region is completely complementary to the target sequence.

[0034] In a preferred embodiment of the present invention, the antisense strand has a length of 19 to 27 nucleotides.

[0035] In a preferred embodiment of the present invention, the oligonucleotide comprises at least one modified nucleotide; preferably, the oligonucleotide or siRNA backbone is partially or completely thiomodified, or the 5'-end of the siRNA antisense strand includes, but is not limited to, modifications such as phosphate ester, oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0036] This invention describes siRNA-APP, obtained by incorporating modified nucleoside monomers into siRNA double strands. This siRNA-APP exhibited good uptake in HepG2 and ARPE-19 cells, showing a gradient dependence with varying siRNA concentration and transfection time. Furthermore, this invention validated the target gene sequence knockdown level of the siRNA-doped with modified nucleoside monomers in U87-MG cells through transfection. The results showed that structural modification of the nucleoside monomers before integration into siRNA did not affect the biological activity of the siRNA; on the contrary, it enhanced its cellular uptake. Simultaneously, the lipophilicity, metabolic stability, and targeting properties of the modified groups facilitated better knockdown of target mRNAs, ultimately achieving the desired therapeutic effect.

[0037] Another aspect of the present invention is to provide a pharmaceutical composition comprising any one or more of the oligonucleotides doped with modified nucleoside monomer compounds, or oligonucleotides doped with modified nucleoside monomer compounds and ligand conjugates, and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0038] This invention chemically modifies nucleoside monomers by introducing lipophilic / hydrophilic groups or pharmacologically active fragments containing target receptors, resulting in modified nucleoside monomer compounds with significantly improved physicochemical properties and bioavailability. The modified nucleoside monomer compounds are then incorporated into siRNA to enhance its cellular uptake, promoting better knockdown of target mRNA. This provides a delivery vector-independent, tissue-specific nucleic acid drug. When administered systemically or locally, this nucleic acid drug exhibits advantages such as good chemical / biological stability, good serum stability, and low renal clearance, potentially enabling targeted delivery of nucleic acid drugs to different tissues and providing a new direction for the development of nucleic acid drugs. Attached Figure Description

[0039] Figure 1 shows the knockdown of APP mRNA levels in U87-MG by NO.3 (Loop_4a_APP_3) in Table 1.

[0040] Figure 2 shows the cellular uptake efficiency and fluorescence intensity of NO.3 (Loop_4a_APP_3) in HepG2, ARPE-19 and U87-MG cells as shown in Table 1. In the figure, A and B are the cellular uptake efficiency and fluorescence intensity of NO.3 in HepG2 cells (1, 6 and 12 h), respectively; C and D are the cellular uptake efficiency and fluorescence intensity of NO.3 in ARPE-19 cells (12 h), respectively.

[0041] Figure 3 shows the free uptake of NO.1–15 in U87-MG cells over 24 hours (1 nM, without transfection reagent).

[0042] Figure 4 shows the free uptake of NO.1–15 in U87-MG and ACHN cells over 24 hours (100 nM, without transfection reagent). Detailed Implementation

[0043] The embodiments provided in this invention are intended to fully disclose the specification and should not be construed as limiting it. Temperatures are given in degrees Celsius (°C). The structures of the final products, intermediates, and starting materials were confirmed by standard analytical methods, such as trace analysis and spectroscopic characterization, such as MS and NMR. Abbreviations used are those conventional in the art.

[0044] All starting materials, structural units, reagents, acids, bases, dehydrating agents, solvents, and catalysts used in the synthesis of the modified nucleosides or analogues involved in this patent are commercially available or can be produced by organic synthetic methods known to those skilled in the art (METHODS OF ORGANIC SYNTHESIS, Thieme, Vol. 21 (Houben-Weyl, 4th ed. 1952)). Unless otherwise stated, all reactions are carried out under nitrogen or argon atmosphere. Proton NMR ( 1 H NMR is performed in a deuterated solvent. In some nucleic acids or analogues disclosed herein, one or more... 1 The H-shift overlaps with residual solvent signals; these signals have not been reported in the experiments provided below. As described in the embodiments below, in some exemplary embodiments, nucleic acids or analogues are prepared according to the following general procedure. It should be understood that while the general method describes the synthesis of certain nucleosides or analogues involved in this patent, the following general method, as well as other methods known to those skilled in the art, can be applied to all nucleic acids or analogues and subclasses and species of each of these nucleic acids or analogues, as described herein.

[0045] Example 1: Preparation of Compound 4a

[0046] Reagents and conditions: (1) NaH, DMSO, DMF, 50℃, 0℃-rt; (ii) DMTrCI, Py., rt; (iii) tetrazole, DCM, N2, 3-((bis(diisopropyl amino)phosphanyl)oxy)propanenitrile

[0047] Preparation of compound 2a: N-benzoyl adenosine was dissolved in 20 mL of a mixed solvent of N,N-dimethylformamide and dimethyl sulfoxide. NaH was added and stirred for 10 minutes in an ice bath. Then, a solution of Ve-C₂H₅I in N,N-dimethylformamide was added dropwise to the reaction flask. The reaction was allowed to proceed for 36 hours at room temperature. Extraction was performed with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na₂SO₄. After removing the solvent by vacuum distillation, 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(400MHz,DMSO-d6)δ11.23(s,1H),8.77(d,2H),8.07–8.03(m,2H),7.65(t,J=7.4Hz,1H),7 .55(t,J=7.6Hz,2H),6.25(d,J=5.8Hz,1H),5.32(d,J=5.0Hz,1H),5.20(bs,1H),4.70(t,J=5.3 Hz,1H),4.43(m,1H),4.06(q,J=3.8Hz,1H),3.95(m,1H),3.73(m,5H),2.45(t,J=6.9Hz,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.

[0048] Preparation of compound 3a: Compound 2a was dissolved in ultradry pyridine, and 4,4'-bismethoxytriphenylmethyl chloride was added under stirring at room temperature. The reaction mixture was reacted for 12 hours. The reaction solution was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4. After removing the solvent by vacuum distillation, the crude product was purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 3a. 1H NMR(400MHz,DMSO-d6)δ12.09(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=5.4Hz,1H),5.31(s,1H),4.60(t,J=5.3Hz,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=5.2Hz,2H),1.51–1.02(m,32H),0.83(m,12H)ppm.

[0049] Preparation of compound 4a: Compound 3a and tetrazolium were added to ultra-dry dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine was added under nitrogen protection. After reacting at room temperature for 6 hours, the crude product was purified by column chromatography under nitrogen protection (eluent: DCM / EA / TEA = 15-8 / 1 / 0.01) to obtain compound 4a. 1 H NMR (400MHz, DMSO-d6) δ11.23(s,1H),8.69–8.60(m,2H),8.08–8.01(m,2H),7.64(s,1H),7.54(t,J=7.6Hz ,2H),7.37(td,J=6.0,5.6,2.3Hz,2H),7.29–7.15(m,8H),6.84–6.80(m,4H),6.25(t,J=6.0Hz,1H),5.05( t,J=5.1Hz,1H),4.79–4.74(m,1H),4.31(dd,J=27.8,4.6Hz,1H),3.73–3.56(m,12H),3.53–3.43(m,2H),2 .66–2.58(m,1H),2.45(s,2H),1.99(s,6H),1.50–1.41(m,4H),1.21–1.11(m,36H),0.84–0.79(m,12H)ppm; 31 P NMR (162MHz, DMSO-d6) δ149.25, 149.10ppm.

[0050] Example 2 Preparation of Compound 9

[0051] Reagents and conditions: (i) NaH, DMSO, DMF, 0℃-35℃; (ii) t-Bu2Si(OTf)2, DMF, 0℃; (iii) acetic anhydride,Py.0℃-rt; (iii)HF,Py.,DCM,0℃-rt; (iv)DMTrCl,Py.,rt; (ii)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0052] Preparation of compound 5: Using cytosine as a raw material, the reaction conditions were the same as those for compound 2a. The crude product was purified by column chromatography (elution buffer: DCM / EA / MeOH = 5 / 1 / 0.05~0.2) to obtain compound 5. 1 H NMR(400MHz,DMSO-d6)δ7.95(d,J=7.4Hz,1H),7.21(d,2H),5.90(d,J=3.5Hz,1H),5 .73(d,J=7.4Hz,1H),5.11(d,J=5.4Hz,1H),4.97(d,J=6.1Hz,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),2.0 4(s,3H),1.97(s,3H),1.70(d,J=7.3Hz,2H),1.50–1.03(m,27H),0.83(m,12H)ppm.

[0053] Preparation of Compound 6: Compound 5 was dissolved in 20 mL of ultradry N,N-dimethylformamide, and di-tert-butylsilylbis(trifluoromethanesulfonic acid) was added at 0 °C. After reacting for 8 hours, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na₂SO₄ to remove the solvent, yielding the intermediate, which could be used directly in the next step without further purification. The crude product was dissolved in 10 mL of ultradry N,N-dimethylformamide, and acetic anhydride was slowly added under ice bath conditions, followed by a return to room temperature. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na₂SO₄ to remove the solvent, yielding the crude compound 6. The crude compound 6 could be used directly in the next step without further purification.

[0054] Preparation of compound 7: Compound 6 was dissolved in ultra-dry dichloromethane, and a solution of pyridine hydrogen fluoride was added with stirring at 0°C. After the reaction was complete, the mixture was extracted with dichloromethane and saturated NaHCO3 aqueous solution. The organic phase was dried over anhydrous Na2SO4 to remove the solvent. The crude product was purified by column chromatography (elution buffer: DCM / EA = 10 / 1) to obtain compound 7. 1 H NMR (400MHz, DMSO-d6) δ10.93(s,1H),8.50(d,J=7.4Hz,1H),7.20(d,J=7.4Hz,1H),5.91(d ,J=2.5Hz,1H),5.22(t,J=5.0Hz,1H),5.04(d,J=6.4Hz,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.

[0055] Preparation of compound 8: Compound 7 was dissolved in 10 mL of ultradry pyridine, and 4,4'-bismethoxytriphenylmethyl chloride was added with stirring at room temperature. The reaction was allowed to proceed for 12 hours. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4 to remove the solvent. The crude product was then purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 8. 1 H NMR (400MHz, DMSO-d6) δ10.94(s,1H),8.33(d,J=8Hz,1H),7.41(d,J=7.8Hz,2H),7.35–7.26(m,7 H),7.03(d,J=8Hz,1H),6.91(d,J=8.3Hz,4H),5.91(s,1H),5.13(d,J=6.9Hz,1H),4.30(m,1H),4 .13–4.08(m,2H),4.00–3.95(m,2H),3.81–3.80(m,2H),3.41–3.43(m,3H),2.11(s,3H),2.09(s, 3H),2.06(s,3H),1.98(s,3H),1.71(t,J=8.4Hz,2H),1.53–0.98(m,28H),0.85–0.80(m,12H)ppm.

[0056] Preparation of compound 9: Compound 8 and tetrazolium were added to ultra-dry dichloromethane. After adding bis(diisopropylamino)(2-cyanoethoxy)phosphine under nitrogen protection, the mixture was reacted at room temperature for 6 hours. After removing part of the solvent, the crude product was purified by column chromatography under nitrogen protection to obtain compound 9.

[0057] Example 3 Preparation of compound 14a

[0058] Reagents and conditions: (i) NaH, DMSO, DMF, 0℃-35℃; (ii) t-Bu2Si(OTf)2, DMF, 0℃; (ii) HF, Py., DCM, 0℃; (iv) DMTrCl, Py., rt (v) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0059] Preparation of compound 10a: Pseudorazine nucleoside was dissolved in 20 mL of a mixed solvent of N,N-dimethylformamide and dimethyl sulfoxide. NaH (1.5 eq.) was added under ice bath conditions and stirred for 10 minutes. Then, a solution of N,N-dimethylformamide containing 1.8 eq. of hexadecane bromide was added dropwise to the reaction flask. The mixture was allowed to return to room temperature and the reaction continued for 36 hours. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na₂SO₄ to remove 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 (400MHz, DMSO-d6) δ11.32(s,1H),7.80(s,1H),4.97(d,J=5.0Hz,1H),4.82(dd,J=6.6,4.8Hz,1H),4.72(d,J=5.8Hz,1H),4.47(d,J=4.2Hz,1H),3 .95(q,J=4.8Hz,1H),3.89(q,J=5.6Hz,1H),3.72–3.58(m,4H),3.50–3.44 (m,3.6Hz,1H),1.60–1.52(m,2H),1.24(s,27H),0.86(t,J=6.8Hz,3H)ppm.

[0060] Preparation of compound 11a: Compound 10a was dissolved in 20 mL of ultradry N,N-dimethylformamide. Di-tert-butylsilylbis(trifluoromethanesulfonic acid) (1.5 eq.) was added at 0 °C. After reacting for 6 hours, imidazole and tert-butyldimethylchlorosilane were added to the reaction solution. The reaction was continued at room temperature for 10 hours. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na₂SO₄ to remove the solvent, yielding compound 11a, which could be used directly in the next step without further purification.

[0061] Preparation of compound 12a: Compound 11a was dissolved in ultra-dry dichloromethane, and a solution of pyridine hydrogen fluoride was added with stirring at 0°C. The reaction was continued for 1.5 hours. After the reaction was completed, the mixture was extracted with dichloromethane and saturated NaHCO3 aqueous solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 12a. 1 H NMR (400MHz, DMSO-d6) δ11.32(s,1H),7.96(s,2H),7.84(s,1H),4.83(s,1H),4.53(s,1H),4.46(d,J=4.3Hz,1H),4.13(d,J =4.6Hz,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.

[0062] Preparation of compound 13a: Compound 12a was dissolved in 10 mL of ultradry pyridine, and 4,4'-bismethoxytriphenylmethyl chloride was added with stirring at room temperature. The reaction was allowed to proceed for 12 hours. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4 to remove the solvent. The crude product was then purified by column chromatography (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 13a. 1H NMR (400MHz, DMSO-d6) δ11.34(s,1H),7.49(s,1H),7.43(d,J=7.8Hz,2H),7.33–7 .28(m,7H),6.89(d,J=8.3Hz,5H),4.67(d,J=6.3Hz,1H),4.59(d,J=3.1Hz,1H),4. 12(t,J=3.9Hz,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.

[0063] Preparation of compound 14a: Compound 13a and tetrazolium were added to ultra-dry dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine was then added under nitrogen protection, and the reaction was carried out at room temperature for 6 hours. After the reaction was complete, some solvent was removed, and the crude product was purified by column chromatography under nitrogen protection to obtain compound 14a. 31 P NMR(162MHz,Chloroform-d)δ149.72ppm.

[0064] Example 4 Preparation of compounds 19a and 19b

[0065] Reagents and conditions:(i)TEA,CH3(CH2) n NH2MeOH,reflux;(ii)t-Bu2Si(OTf)2,imdizole,TBDMSCl,DMF,0℃-rt;(iii)HF·Py,DCM,Py,rt;(iv)DMTrCl,Py,rt;(v)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0066] Preparation of compounds 15a and 15b: 6-Chloroadenine nucleoside was dissolved in methanol, triethylamine was added, followed by dodecylamine (for preparation of 15a) and hexadecamine (for preparation of 15b), respectively. After reflux for 10 hours, the solids were filtered, washed with cold methanol, and dried to obtain compounds 15a and 15b, respectively. 15a: 1H NMR (400MHz, DMSO-d6) δ8.34(s,1H),8.20(s,1H),7.87(s,1H),5.89(d,J=6.2Hz,1H),5.44(m,2H),5.18(d,J=4.6Hz,1H),4.62(m,1H),4.1 6(m,1H),4.10(m,1H),3.98(m,1H),3.69(m,1H),3.56(m,1H),3.18(d,J=5.0Hz,2H),1.59(m,2H),1.24(s,16H),0.90–0.81(m,3H)ppm.15b: 1 H NMR (400MHz, DMSO-d6) δ8.34(s,1H),8.20(s,1H),7.88(s,1H),5.89(d,J=6.2Hz,1H),5.45(m,2H),5.20(d,J=4.6Hz,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=5.1Hz,2H),1.58(m,2H),1.23(m,22H),0.85(t,J=6.7Hz,3H)ppm.

[0067] Preparation of compounds 16a and 16b: Compounds 15a and 15b were dissolved separately in 20 mL of ultradry N,N-dimethylformamide, and di-tert-butylsilylbis(trifluoromethanesulfonic acid) was added at 0 °C. After reacting for 6 hours, imidazole and tert-butyldimethylchlorosilane were added to the reaction solution, and the mixture was allowed to react at room temperature for 10 hours. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na₂SO₄, and the solvent was removed by vacuum distillation to obtain intermediates 16a and 16b, which could be used directly in the next step without further purification.

[0068] Preparation of compounds 17a and 17b: Compounds 16a and 16b were dissolved in ultradry dichloromethane under the same reaction conditions as in the synthesis of compound 12a. The crude products were purified by column chromatography (elution buffer: DCM / EA = 10 / 1) to give compounds 17a and 17b. 17a: 1H NMR (400MHz, DMSO-d6) δ8.36 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H), 5.92 (d, J = 6.3Hz, 1H), 5. 59(m,1H),5.09(d,J=4.9Hz,1H),4.76(m,1H),4.14(td,J=4.9,2.6Hz,1H),4.02(m,1H),3 .72–3.66(m,1H),3.63–3.54(m,1H),3.47(s,2H),1.57(d,J=7.8Hz,2H),1.27(d,J=6.6Hz ,5H),1.23(s,14H),0.89–0.81(m,3H),0.71(s,9H),-0.12(s,3H),-0.22(s,3H)ppm.17b: 1 H NMR (400MHz, DMSO-d6) δ8.36(s,1H),8.19(s,1H),7.89(s,1H),5.91(d,J=6.2 Hz,1H),5.60(m,1H),5.10(d,J=4.9Hz,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=9.7Hz, 2H),1.22(s,25H),0.85(m,3H),0.70(s,9H),-0.13(s,3H),-0.23(s,3H)ppm.

[0069] Preparation of compounds 18a and 18b: Compounds 17a and 17b were dissolved in 10 mL of ultradry pyridine under the same reaction conditions as in the synthesis of compound 13a. The crude product was purified by column chromatography (elution buffer: DCM / EA / TEA = 10 / 1 / 0.01) to give compounds 18a and 18b. 18a: 1HNMR(400MHz,DMSO-d6)δ8.25(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=4.9Hz,1H),5.12(d,J=5.9Hz,1H),4.85(t,J=5.0Hz,1H),4.26(s 1H),4.10(q,J=4.5Hz,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(400MHz,DMSO-d6)δ8.25(s,1H),8.14(s,1H),7.83(s,1H),7.39(d,J=7.7Hz,2H), 7.23(m,8H),6.84(m,4H),5.94(d,J=4.9Hz,1H),5.13(d,J=5.9Hz,1H),4.84(t,J=5.0H z,1H),4.26(s,1H),4.09(d,J=4.6Hz,1H),3.72(s,6H),3.45(s,2H),3.34(s,2H),1.63 –1.51(m,2H),1.22(s,20H),0.84(m,6H),0.75(s,9H),-0.04(s,3H),-0.14(s,3H)ppm.

[0070] Preparation of compounds 19a and 19b: Using compounds 18a and 18b as starting materials, the reaction conditions were the same as those for the synthesis of compound 14a. The crude products were purified by column chromatography under nitrogen protection to obtain compounds 19a and 19b. 19a: 11H 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 31P NMR (162 MHz, Chloroform-d) δ 148.88 ppm. 19b: 1 31P NMR (162 MHz, Chloroform-d) δ 150.84, 149.89 ppm.

[0071] Preparation of Compound 25 in Example 5

[0072] Reagents and conditions: (i) NaH, DMSO, DMF, 0 °C - 35 °C; (ii) NaOH aq, THF, r.t.; (iii) t-Bu2Si(OTf)2, imidizole, TBDMSCl, DMF, 0 °C; (iv) HF, Py., DCM, 0 °C - r.t.; (v) DMTrCl, Py., r.t.; (vi) tetrazole, DCM, N2, 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile

[0073] Preparation of compound 20: Using N-benzoylcytidine and hexadecane bromide as raw materials, the crude product of compound 10a was purified by column chromatography under the same reaction conditions (eluent: DCM / EA / MeOH = 5 / 1 / 0.05~0.2) to obtain compound 20. 1 H NMR (400MHz, DMSO-d6) δ8.04(d,J=7.6Hz,2H),7.91(d,J=8.2Hz,1H),7.57(t,J=7.3Hz,1H), 7.47(t,J=7.5Hz,2H),6.45(d,J=8.1Hz,1H),5.83(d,J=4.5Hz,1H),5.46(d,J=5.3Hz,1H),5. 11(d,J=5.0Hz,2H),4.07(m,3H),3.98(q,J=4.5Hz,1H),3.88(q,J=3.7Hz,1H),3.69–3.65(m, 1H),3.61–3.53(m,1H),1.68(t,J=7.1Hz,2H),1.41–1.12(m,29H),0.85(t,J=6.6Hz,3H)ppm.

[0074] Preparation of compound 21: Compound 20 was dissolved in 4 mL of tetrahydrofuran, and 2 mL of 5M sodium hydroxide solution was added. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was extracted with ethyl acetate, dilute hydrochloric acid aqueous solution, and saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4 to remove the solvent. The crude product was purified by column chromatography (elution buffer: EA / MeOH = 5-1 / 1) to obtain compound 21. 1 H NMR (400MHz, DMSO-d6) δ7.95(d,J=8.1Hz,1H),5.81(d,J=5.0Hz,1H),5.76(d,J= 8.1Hz,1H),5.38(d,J=5.5Hz,1H),5.14–5.06(m,2H),4.03(q,J=5.2Hz,1H),3.97 (q,J=4.8Hz,1H),3.86(q,J=3.4Hz,1H),3.80–3.73(m,2H),3.67–3.62(m,1H),3 .58–3.53(m,1H),1.49(t,J=7.2Hz,2H),1.24(m,27H),0.85(t,J=6.7Hz,3H)ppm.

[0075] Preparation of compound 22: Using compound 21 as a starting material, the reaction conditions were the same as those for the synthesis of compound 11a. Compound 22 was obtained and used directly in the next step without further purification.

[0076] Preparation of compound 23: Using compound 22 as the starting material, the reaction conditions were the same as those for the synthesis of compound 12a. The crude product was purified by column chromatography (elution buffer: DCM / EA = 10 / 1) to obtain compound 23. 1 H NMR(400MHz,DMSO-d6)δ7.78(d,J=8.1Hz,1H),7.40–7.37(m,2H),7.33(t,J=7.6Hz,2H),7 .27–7.24(m,5H),6.91(d,J=9.0Hz,4H),5.83(d,J=4.2Hz,1H),5.44(d,J=8.1Hz,1H),5.13 (d,J=6.1Hz,1H),4.21(t,J=4.6Hz,1H),4.08(q,J=5.5Hz,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.

[0077] Preparation of compound 24: Using compound 23 as the starting material, the crude product of compound 13a was purified by column chromatography under the same reaction conditions (eluent: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 24. 1 H NMR(400MHz, DMSO-d6)δ7.78(d,J=8.1Hz,1H),7.40–7.37(m,2H),7.33(t,J=7.6Hz, 2H),7.27–7.24(m,5H),6.91(d,J=9.0Hz,4H),5.83(d,J=4.2Hz,1H),5.44(d,J=8.1 Hz,1H),5.13(d,J=6.1Hz,1H),4.21(t,J=4.6 Hz,1H),4.08(q,J=5.5Hz,1H),4.03–4.02(m,1H),3.84–7.71(m,10H),1.4 9(m,2H),1.23(s,26H),0.87–0.85(m,12H),0.07(s,3H),0.05(s,3H)ppm.

[0078] Preparation of compound 25: Using compound 24 as the starting material, 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 25.

[0079] Example 6 Preparation of Compound 32

[0080] Reagents and conditions:(i)n-amyl nitrite,CHBr3,90℃; (ii)1-hexadecylamine,2-methoxyethanol,reflux; (ii)ammoniac-methanol,rt; (v)t-Bu2Si(OTf)2,imidizole,TBDMSCl, DMF, 0℃; (vi)HF,Py.,DCM,0℃-rt; (vii)DMTrCl,Py.,rt(vii)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0081] Preparation of compound 26: Compound 2′,3′,5′-triacetylguanine nucleoside was dissolved in 10 mL of bromoform, and then nitroglycerin was added. The mixture 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 (elution buffer: DCM / MeOH = 60 / 1) to obtain compound 26. 1 H NMR(400 MHz, DMSO-d6) δ8.31(d,J=0.7Hz,1H),6.30(dd,J=4.2,0.6Hz,1H),5.58(dd,J=6.3,4.1Hz,1H),5.28(dd ,J=6.3,4.1Hz,1H),4.77(dt,J=4.1,3.4Hz,1H),4.33(qd,J=12.2,3.4Hz,2H),2.00(d,J=3.1Hz,9H)ppm.

[0082] Preparation of compound 27: Compound 26 was dissolved in ethylene glycol monomethyl ether, and then hexadecylamine was added, followed by reflux. After the reaction was completed, the solvent was removed, and the crude product was purified by column chromatography (elution buffer: DCM / MeOH = 30 / 1) to obtain compound 27. 1H NMR (400MHz, DMSO-d6) δ9.43(s,1H),8.32(d,J=0.5Hz,1H),7.26(t,J=4.3Hz,1H),6.31(dd,J=4.1,0.6Hz,1H ),5.60(dd,J=6.3,4.2Hz,1H),5.28(dd,J=6.3,4.1Hz,1H),4.77(dt,J=4.2,3.4Hz,1H),4.33(qd,J=12.2,3.4 Hz,2H),3.22(qd,J=5.1,4.3Hz,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.

[0083] Preparation of compound 28: Compound 27 was dissolved in an ammonia-methanol solution and reacted at room temperature. After the reaction was complete, the solvent was removed by vacuum distillation, and the crude product was purified by column chromatography (eluent: DCM / MeOH = 30 / 1) to obtain compound 28. 1 H NMR (400MHz, DMSO-d6) δ9.43 (s, 1H), 8.29 (d, J = 0.5Hz, 1H), 7.26 (t, J = 4.3Hz, 1H), 5 .94(dd,J=4.0,0.7Hz,1H),5.58(d,J=4.5Hz,1H),5.27(d,J=3.8Hz,1H),4.94(t,J= 4.5Hz,1H),4.54(m,1H),4.22(m,1H),4.05(dt,J=5.0,2.5Hz,1H),3.70–3.55(m,2H ),3.42–3.10(m,2H),1.57–1.45(m,2H),1.34–1.18(m,21H),1.00–0.76(m,3H)ppm.

[0084] Preparation of compound 29: Using compound 28 as a starting material, the reaction conditions were the same as those for the synthesis of compound 11a, to obtain compound 29, which can be used directly in the next step without further purification.

[0085] Preparation of compound 30: Using compound 29 as the starting material, the reaction conditions were the same as those for the synthesis of compound 12a. The crude product was purified by column chromatography (elution buffer: DCM / EA = 10 / 1) to obtain compound 30. 1H NMR (400MHz, DMSO-d6) δ9.43 (s, 1H), 8.28 (d, J = 0.6Hz, 1H), 7.26 (t, J = 4.3Hz, 1H), 6. 02(dd,J=4.0,0.6Hz,1H),4.85(d,J=3.9Hz,1H),4.71(t,J=4.5Hz,1H),4.47(dd,J=6. 2,4.0Hz,1H),4.20–3.98(m,2H),3.73–3.52(m,2H),3.22(qd,J=5.2,4.3Hz,2H),1.6 0–1.42(m,2H),1.41–1.20(m,21H),0.95–0.80(m,12H),0.09(s,3H),0.06(s,3H)ppm.

[0086] Preparation of compound 31: Using compound 30 as the starting material, the reaction conditions were the same as those for the synthesis of compound 13a. The crude product was purified by column chromatography (elution buffer: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 31. 1 H NMR(400MHz, DMSO-d6)δ9.43(s,1H),8.28(d,J=0.6Hz,1H),7.46–7.42(m,2H),7.36–7.31(m, 2H),7.30–7.24(m,2H),7.15–7.10(m,4H),6.99–6.95(m,4H),6.06(dd,J=4.0,0.6Hz,1H),4.7 8–4.75(m,1H),4.58–4.53(m,1H),4.22–4.15(m,2H),3.77(s,7H),3.72–3.67(m,1H),3.22(m, 2H),1.57–1.47(m,2H),1.36–1.22(m,21H),0.92–0.83(m,12H),0.09(s,3H),0.06(s,3H)ppm.

[0087] Preparation of compound 32: Using compound 31 and tetrazolium as raw materials, the reaction conditions were the same as those for the synthesis of compound 13a. The crude product was purified by column chromatography under nitrogen protection to obtain compound 32.

[0088] Example 7 Preparation of compound 38b

[0089] Reagents and conditions: (i) TEA, MeOH, reflux; (ii) EEDQ, EtOH, 60℃; (iii) t-Bu2Si(OTf)2, imdizole, TBDMSCl, DMF, 0℃-rt; (iv) HF·Py ,DCM,Py,rt; (v)DMTrCl,Py,rt; (vi)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0090] Synthesis of compound 33: 6-Chloroadenine nucleoside was dissolved in methanol, and then triethylamine (0.3 eq.) and propylenediamine (10 eq.) were added. After reflux for 10 hours, the solid was filtered, washed with methanol, and dried to obtain compound 33. No further purification was required; it was used directly in the next step.

[0091] Synthesis of compound 34b: Compound 33 was dissolved in anhydrous ethanol, and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and VE-CH2CH2COOH were added. The reaction was carried out at 60°C. After the reaction was completed, the solvent was removed by vacuum distillation, and the crude product was purified by column chromatography (elution buffer: DCM / EA = 5 / 1) to obtain compound 34b. 1 H NMR(400MHz,DMSO-d6)δ8.38(d,J=19.9Hz,2H),8.17(s,1H),7.97(s,1H),5.8 8(d,J=6.1Hz,1H),5.43(t,J=8.0Hz,2H),5.19(s,1H),4.61(s,1H),4.15(s,1 H),4.00(d,J=26.4Hz,3H),3.61(d,J=40.1Hz,4H),3.35(s,9H),2.21–1.89(m ,10H),1.76(d,J=21.3Hz,5H),1.60–0.95(m,29H),0.83(d,J=6.8Hz,13H)ppm.

[0092] Synthesis of compound 35b: Using compound 34b as the starting material, the reaction conditions were the same as those for the synthesis of compound 11a, to obtain compound 35b, which can be used directly in the next step without further purification.

[0093] Preparation of compound 36b: Using compound 35b as a starting material, compound 36b was obtained under the same reaction conditions as that for the synthesis of compound 12a. 1H NMR (400MHz, DMSO-d6) δ8.39(m,2H),8.15(s,1H),7.95(s,1H),5.91(d,J=6.0Hz,1H),5.55(s,1H),5.09 (d,J=4.9Hz,1H),4.73(t,J=5.4Hz,1H),4.13(q,J=4.3Hz,1H),4.05–3.99(m,3H),3.71(d,J=11.1Hz,1H ),3.56(s,3H),3.26(q,J=6.6Hz,2H),2.09(s,3H),2.06(s,3H),1.99(s,3H),1.83–1.76(m,2H),1.72(q ,J=6.7Hz,2H),1.50–1.00(m,27H),0.81(t,J=7.2Hz,12H),0.71(s,9H),-0.12(s,3H),-0.22(s,3H)ppm.

[0094] Preparation of compound 37b: Using compound 36b as the starting material, the reaction conditions were the same as those for the synthesis of compound 13a. The crude product was purified by column chromatography (elution buffer: DCM / EA / TEA = 5 / 1 / 0.01) to obtain compound 37b. 1 H NMR (400MHz, DMSO-d6) δ8.31(s,1H),8.26(s,1H),8.11(d,J=2.5Hz,2H),7.91(s,1H),7.28–7.12(m,9H),6.82(m,7 H),5.90(d,J=4.8Hz,1H),5.37(d,J=6.0Hz,1H),4.84(q,J=5.5Hz,2H),4.55(s,1H),4.26(d,J=5.9Hz,1H),4.03(d d,J=6.1,3.6Hz,5H),3.71(d,J=3.3Hz,11H),3.57(s,4H),3.26(q,J=7.4,6.0Hz,5H),2.08(dd,J=11.4,4.0Hz,11H ),1.99(d,J=2.2Hz,6H),1.76(d,J=10.6Hz,4H),1.33–1.16(m,27H),0.89–0.78(m,21H),0.07(s,3H),0.03(s,3H).

[0095] Preparation of compound 38b: Using compound 37b and bis(diisopropylamino)(2-cyanoethoxy)phosphine as raw materials, 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 38b.

[0096] Example 8 Preparation of compound 44a

[0097] Reagents and conditions: (i) TEA, MeOH, reflux; (ii) EEDQ, EtOH, 60℃; (iii) t-Bu2Si(OTf)2, imdizole, TBDMSCl, DMF, 0℃-rt; (iv) HF·P y,DCM,Py,rt(v)DMTrCl,Py,rt;(vi)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0098] Synthesis of compound 39: 6-Chloroadenine nucleoside was dissolved in ethanol, hydrazine hydrate was added, and the reaction was allowed to proceed for 10 hours. After the reaction was complete, the solid was filtered, washed with cold methanol, and dried to obtain compound 39. No further purification was required; it was used directly in the next step.

[0099] Synthesis of compound 40a: Compound 39 was dissolved in anhydrous ethanol, and EEDQ and hexadecanoic acid were added and reacted at 60°C. After the reaction was completed, the solvent was removed by vacuum distillation, and after solid precipitated, it was filtered to obtain compound 40a.

[0100] Synthesis of compound 41a: Using compound 40a as the starting material, the reaction conditions were the same as those for the synthesis of compound 11a. Compound 41a was obtained and could be used directly in the next step without further purification.

[0101] Preparation of compound 42a: Using compound 41a as the starting material, the crude product of compound 12a was purified by column chromatography (elution buffer: DCM / EA = 10 / 1) under the same reaction conditions to obtain compound 42a. 1H NMR (400MHz, DMSO-d6) δ8.38(s,1H),8.21(s,1H),7.87(s,1H),7.80(t,J=5.8Hz,1H),5.92(d,J=6.0Hz,1H),5.55(s,1H ),5.08(d,J=4.9Hz,1H),4.75(t,J=5.5Hz,1H),4.14(q,J=4.1Hz,1H),4.03–3.93(m,2H),3.72(dd,J=12.3,3.4Hz,1H),3 .59(d,J=12.4Hz,1H),3.48(s,2H),3.34(s,1H),3.07(dq,J=23.9,6.6Hz,2H),2.05(t,J=7.4Hz,1H),1.71(q,J=6.8Hz,2 H),1.56–1.40(m,2H),1.23(d,J=6.2Hz,21H),0.85(td,J=6.8,2.5Hz,3H),0.73(s,9H),-0.11(s,3H),-0.21(s,3H)ppm.

[0102] Preparation of compound 43a: Compound 43a was obtained by using compound 42a and 4,4'-dimethoxytriphenylmethyl chloride as raw materials and reacting under the same conditions as the synthesis of compound 13a.

[0103] Preparation of compound 44a: Compound 43a and bis(diisopropylamino)(2-cyanoethoxy)phosphine 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 44a.

[0104] Example 9 Preparation of compound 50b

[0105] Reagents and conditions: (i) TEA, MeOH, reflux; (ii) CuSO4, L-ascorbic acic sodium salt, N2,rt; (ii)t-Bu2Si(OTf)2,imdizole,TBDMSCl,DMF,0℃-rt; (iv)HF·Py,DCM,Py,rt; (v)DMTrCl,Py,rt; (vi)tetrazole,DCM,N2.3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0106] Preparation of compound 45: 6-Chloroadenine nucleoside was dissolved in methanol, and triethylamine (0.3 eq.) and propargylamine (10 eq.) were added. After reflux for 10 hours, the solid was filtered, washed with methanol, and dried to obtain compound 45. 1 H NMR (400MHz, DMSO-d6) δ8.42(s,1H),8.28(d,J=17.2Hz,2H),5.92(dd,J=6.2,1.9 Hz,1H),5.47(dd,J=6.4,2.3Hz,1H),5.35(td,J=4.6,2.2Hz,1H),5.20(t,J=3.5H z,1H),4.62(q,J=5.7Hz,1H),4.27(s,1H),4.17(dt,J=7.2,3.6Hz,1H),3.98(q,J =3.3Hz,1H),3.69(dt,J=11.9,4.2Hz,1H),3.57(ddd,J=11.9,7.0,3.6Hz,1H)ppm.

[0107] 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, followed by a dimethyl sulfoxide solution of VE-C2H5N3. The reaction was carried out under nitrogen protection at room temperature. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography (eluent: DCM / EA = 5 / 1) to obtain compound 46b. 1 H NMR (400MHz, DMSO-d6) δ8.39 (s, 2H), 8.24 (s, 1H), 8.01 (s, 1H), 5.90 (d, J = 6.1Hz, 1H), 5.49–5.36 (m, 2H), 5. 21(d,J=4.4Hz,1H),4.77(s,2H),4.64(dt,J=25.5,5.4Hz,3H),4.16(q,J=4.1Hz,1H),3.97(q,J=3.4Hz,1H) ,3.89(t,J=5.1Hz,2H),3.67(dt,J=12.1,4.1Hz,1H),3.55(ddd,J=11.6,7.3,3.6Hz,1H),2.43(t,J=7.0Hz, 2H),1.92(s,3H),1.80(s,3H),1.71(d,J=14.6Hz,5H),1.55–0.94(m,26H),0.82(dd,J=9.0,6.4Hz,12H)ppm.

[0108] Synthesis of compound 47b: Using compound 46b as the starting material, the reaction conditions were the same as those for the synthesis of compound 11a. Compound 47b was obtained and could be used directly in the next step without further purification.

[0109] Preparation of compound 48b: Using compound 47b as the starting material, the reaction conditions were the same as those for the synthesis of compound 12a. The crude product was purified by column chromatography (elution buffer: DCM / EA = 10 / 1) to obtain compound 48b. 1 H NMR (400MHz, DMSO-d6) δ8.42(s,2H),8.25(s,1H),7.99(s,1H),5.93(d,J=5.9Hz,1H),5.54(dd,J=7.6,4.2Hz,1H),5 .09(d,J=4.9Hz,1H),4.81–4.70(m,3H),4.67(t,J=5.0Hz,2H),4.14(q,J=4.6Hz,1H),4.01(q,J=3.3Hz,1H),3.87(t, J=5.1Hz,2H),3.74–4.70(m,1H),3.55–4.60(m,1H),2.42(t,J=6.9Hz,2H),1.91(s,3H),1.76(s,3H),1.72(s,3H),1 .67(d,J=7.4Hz,2H),1.53–0.98(m,31H),0.82(dd,J=9.0,6.6Hz,15H),0.69(s,9H),-0.15(s,3H),-0.25(s,3H)ppm.

[0110] Preparation of compound 49b: Compound 48b and 4,4'-dimethoxytriphenylmethyl chloride were used as starting materials, and the reaction conditions were the same as those for the synthesis of compound 13a. The crude product was purified by column chromatography (elution buffer: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 49b. 1H NMR (400MHz, DMSO-d6) δ8.35(d,J=16.9Hz,2H),8.17(s,1H),8.00(s,1H),7.35(d,J=7.7Hz,2H),7.21(td,J=12.2,8.3Hz,7H),6 .82(dd,J=8.7,2.5Hz,4H),5.91(d,J=5.1Hz,1H),5.39(d,J=6.2Hz,1H),4.82(q,J=5.5Hz,1H),4.75(s,1H),4.65(d,J=6.1Hz,2 H),4.47(t,J=4.6Hz,1H),4.03(q,J=4.6Hz,1H),3.87(t,J=5.1Hz,2H),3.70(s,6H),3.12(dd,J=10.6,4.9Hz,1H),2.39(t,J=6. 9Hz,2H),1.90(s,3H),1.76(s,3H),1.66(d,J=3.7Hz,5H),1.52–0.97(m,28H),0.85–0.78(m,21H),0.07(s,3H),0.04(s,3H)ppm.

[0111] Preparation of compound 50b: Using compound 49b and bis(diisopropylamino)(2-cyanoethoxy)phosphine as raw materials, the reaction conditions were the same as those for the synthesis of compound 12a. The crude product was purified by column chromatography under nitrogen protection to obtain compound 50b.

[0112] Example 10 Preparation of compound 56b

[0113] Reagents and conditions: (i) t-Bu2Si(OTf)2, imdizole, TBDMSCl, DMF, 0℃-rt; (ii) BSA, DCM, reflux; (ii) CuSO4, L-ascorbic acid sodium salt; (iv)HF·Py,DCM,Py,rt; (v)DMTrCl,Py,rt; (vi)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0114] Preparation of compound 51: Using pseudouridine as a starting material, the reaction conditions were the same as those for the synthesis of compound 11a. The crude product was purified by column chromatography (elution buffer: DCM / EA = 10 / 1) to obtain compound 51. 1H NMR (400MHz, DMSO-d6) δ11.49(s,1H),11.02(s,1H).7.57(dd,J=6.4,1.8Hz,1H),4.66(dd,J=6.0,1.8Hz,1H),4.33(t,J=5.9Hz,1 H),4.25(dd,J=5.7,4.6Hz,1H),4.03(dd,J=2.1,0.6Hz,2H),3.97(dt,J=4.4,2.1Hz,1H),1.00(s,18H),0.87(s,9H),0.07(s,6H).

[0115] Preparation of compound 52: Compound 51 was dissolved in ultra-dry dichloromethane, and N,O-bis(trimethylsilyl)acetamide (BSA) and bromopropyne were added and refluxed. The reaction progress was monitored by TLC. After the reaction was completed, some solvent was removed, and the crude product was purified by column chromatography to obtain compound 52. 1 H NMR (400MHz, DMSO-d6) δ11.52(s,1H),7.61(d,J=1.1Hz,1H),4.59(s,1H),4.56(d,J=2.5Hz,2H),4.38(dd,J=8.9,4.6Hz,1H),4.21(d,J=4.2Hz, 1H),3.97(dd,J=9.3,4.5Hz,2H),3.93–3.85(m,1H),3.43(t,J=2.5Hz,1 H),1.02(s,9H),1.00(s,9H),0.90(s,9H),0.14(s,3H),0.10(s,3H)ppm.

[0116] Preparation of compound 53b: Using compound 52 as the starting material, the reaction conditions were the same as those for the synthesis of compound 46b. Crude compound 53b was obtained, which, without further purification, was used directly in the next step.

[0117] Preparation of compound 54b: Using compound 53b as a starting material, the reaction conditions were the same as those for the synthesis of compound 12a. Crude compound 54b was obtained, which, without further purification, was used directly in the next step.

[0118] Preparation of compound 55b: Compound 54b and 4,4'-bismethoxytriphenylmethyl chloride were used as starting materials, and the reaction conditions were the same as those for the synthesis of compound 13a. The crude product was purified by column chromatography (elution buffer: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 55b. 1H NMR (400 MHz, DMSO-d6) δ11.45(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=10.2Hz,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.

[0119] Preparation of compound 56b: Compound 55b and bis(diisopropylamino)(2-cyanoethoxy)phosphine 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.

[0120] Example 11 Preparation of compound 63a

[0121] Reagents and conditions: (i)TIPDSiCl2,Py.,rt; (ii)PomCl,TBAHS.aq.0.2~0.4M Na2CO3,DCM; (iii)BTTP,THF,MeCN; (iv)Et3N(HF)3,THF; (v)DMTrCl,Py,rt; (vi)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0122] Preparation of compound 58a: Uracil nucleoside was dissolved in 20 mL of ultradry pyridine, and then 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane was added. After the reaction was complete, the mixture was extracted with ethyl acetate and 10% hydrochloric acid aqueous solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by vacuum distillation to obtain crude compound 58a, which could be used directly in the next step without further purification.

[0123] Preparation of compound 59a: Compound 58a was dissolved in a two-phase solvent (V / V, 1 / 3) of dichloromethane and Na₂CO₃ (0.2–0.4 M) aqueous solution. Then, methyl valerate (2 eq.) and tetrabutylammonium hydrogen sulfate (TBAHS) were added sequentially, and the reaction was carried out at room temperature for 48 hours. After the reaction was completed, the mixture was extracted with ethyl acetate and 5% NaHCO₃ aqueous solution. The organic phase was dried over anhydrous Na₂SO₄, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 59a. 1 H NMR(400 MHz, DMSO-d6)δ7.81(d,J=8.5Hz,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.

[0124] Preparation of compound 60a: Compound 59a was dissolved in a mixed solution of acetonitrile and tetrahydrofuran. (tert-butylimino)tris(pyrrolidine)phosphine and VE-C2H5I were added sequentially, and the reaction was continued at room temperature. After the reaction was complete, the solvent was removed by vacuum distillation, followed by extraction with ethyl acetate and saturated NaCl aqueous solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by vacuum distillation to obtain crude compound 60a, which could be used directly in the next step without further purification.

[0125] Preparation of compound 61a: Compound 60a was dissolved in ultra-dry tetrahydrofuran, and a triethylamine trifluoride solution was added with stirring at 0°C. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated NaHCO3 aqueous solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography (eluent: DCM / MeOH = 9 / 1) to obtain compound 61a. 1 H NMR(400MHz, DMSO-d6)δ7.81(d,J=8.5Hz,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.

[0126] Preparation of compound 62a: Compound 62a was obtained by using compound 61a and 4,4′-dimethoxytriphenylmethyl chloride as raw materials and under the same reaction conditions as the synthesis of compound 13a.

[0127] Preparation of compound 63a: Compound 62a and bis(diisopropylamino)(2-cyanoethoxy)phosphine 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 63a.

[0128] Example 12 Preparation of compound 69a

[0129] Reagents and conditions:(i)TIPDSiCl2,Py.,rt;(ii)K2CO3,MeCN,rt-40℃;(iii)Et3N(HF)3,THF;(iv)DMTrCl,Py,rt;(v)tetrazole,DCM,N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0130] Preparation of compound 65a: Using 2′-methoxyuracil nucleoside as a starting material, the reaction conditions were the same as those for the synthesis of compound 58a. Crude compound 65a was obtained, which, without further purification, was used directly in the next step.

[0131] Preparation of compound 66a: Compound 65a was dissolved in acetonitrile, potassium carbonate was added and stirred at room temperature for 30 minutes, then hexadecane bromo was added, and the reaction was continued at 40°C. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated NaCl aqueous solution, and the organic phase was dried over anhydrous Na2SO4. After removing the solvent, the crude product was purified by column chromatography (eluent: DCM / EA = 10 / 1) to obtain compound 66a.

[0132] Preparation of compound 67a: Using compound 66a as the starting material, the reaction conditions were the same as those for the synthesis of compound 61a. The crude product was purified by column chromatography (elution buffer: DCM / MeOH = 9 / 1) to obtain compound 67a.

[0133] Preparation of compound 68a: Compound 68a was obtained by using compound 67a and 4,4'-dimethoxytriphenylmethyl chloride as raw materials and reacting under the same conditions as the synthesis of compound 13a. 1H NMR(400MHz,DMSO-d6)δ7.54(d,J=8.1Hz,1H),7.37(dd,J=6.7,3.0Hz,2H),7.28–7.23(m,5H) ,7.10(dd,J=8.8,4.1Hz,4H),6.85(t,J=8.4Hz,4H),6.81–6.74(m,4H),5.87(d,J=2.9Hz,1H), 5.24(d,J=8.0Hz,1H),4.03–3.91(m,2H),3.71(d,J=1.3Hz,6H),3.24(d,J=10.8Hz,1H),3.19 (s,3H),3.13(dd,J=11.0,4.6Hz,1H),1.47(t,J=7.2Hz,2H),1.23(m,29H),0.88–0.80(m,3H).

[0134] Preparation of compound 69a: Using compound 68a and bis(diisopropylamino)(2-cyanoethoxy)phosphine as raw materials, 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 69a.

[0135] Example 13 Preparation of compounds 75a and 75b

[0136] Reagents and conditions: (i) t-Bu2Si(OTf)2imdizole, TBDMSCl, DMF0℃-rt; (ii) CuSO4, L-ascorbic acid sodium salt; (iii) HF·Py, DCM, Py, rt; (iv) DMTrCl, Py, rt; (v) tetrazole, DCM, N2,3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

[0137] Preparation of compound 71: Compound 71 was obtained by using 5-alkynyluracil nucleoside as a starting material and under the same reaction conditions as the synthesis of compound 11a.

[0138] Preparation of compound 72b: Using compound 71 and VE-C2H5N3 as starting materials, the reaction conditions were the same as those for the synthesis of compound 46b. Crude compound 72b was obtained, which, without further purification, was used directly in the next step.

[0139] Preparation of compound 73b: Using compound 72b as a starting material, compound 73b was obtained under the same reaction conditions as that for the synthesis of compound 12a.

[0140] Preparation of compound 74b: Compound 73b and 4,4'-dimethoxytriphenylmethyl chloride were used as starting materials, and the reaction conditions were the same as those for the synthesis of compound 13a. The crude product was purified by column chromatography (elution buffer: DCM / EA / TEA = 10 / 1 / 0.01) to obtain compound 74b. 1 H NMR (400MHz, DMSO-d6) δ11.82(s,1H),8.47(s,1H),8.39(s,1H),7.42(d,J=7.4Hz,2H),7.34–7.28(m,4H),7.25(t,J =7.7Hz,2H),7.14(t,J=7.3Hz,1H),6.88–6.82(m,4H),5.92(d,J=5.4Hz,1H),5.11(d,J=6.2Hz,1H),4.75(t,J=5.0Hz ,2H),4.32(t,J=5.4Hz,1H),4.02(m,1H),3.91(m,3H),3.68(d,J=3.7Hz,6H),3.28(d,J=4.0Hz,2H),1.93(s,3H),1. 85(s,3H),1.81(s,3H),1.69(q,J=6.5Hz,2H),1.51–0.97(m,28H),0.85–0.76(m,22H),0.03(s,3H),0.01(s,3H)ppm.

[0141] Preparation of compound 75b: Compound 75b was obtained by using compound 74b and bis(diisopropylamino)(2-cyanoethoxy)phosphine as raw materials and under the same reaction conditions as the synthesis of compound 14a.

[0142] Experiment Example 1: Evaluation of the Bioactivity of Nucleoside-Doped siRNA Sequences

[0143] 1 Experimental Methods

[0144] 1.1 Modified nucleoside-doped siRNA sequences

[0145] Small interfering RNA (siRNA-APP) targeting amyloid precursor protein (APP) as reported in the literature was used as an example. After modification with nucleoside doping, the target sequences were obtained: 3'-u·a·uga(X)GuUCAucaucaaa·a·a(SS) and 5'-VPu·U·uuugAugaugaAcUucaua·u·c(AS). The biological activity of these sequences was then preliminarily evaluated. In this example, SS and AS represent the sense and antisense strands, respectively; uppercase and lowercase letters represent 2'-fluoro(2'-F) and 2'-O-methyl(2'-OMe) ribose modifications, respectively; underlined uppercase letters represent ethylene glycol nucleic acid (GNA) modifications; · indicates phosphate thioester (PS) modifications; (X) indicates modified nucleotide doping; and VP represents 5'-(E)-vinylphosphonate.

[0146] Table 1. Doping methods of RNA sequences modified with nucleoside pairs Note: 4a in Table 1 is compound 4a prepared in Example 1; 2-Hd-A is a 2'-C16 modified adenosine 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; and 32 is compound 32 prepared in Example 6.

[0147] 1.2 Cell Culture and Transfection

[0148] HepG2 (human hepatocellular carcinoma cells), ARPE-19 (human retinal epithelial cells), U87-MG (human malignant glioma cells), and ACHN (human renal adenocarcinoma cells) were selected for cell-level activity evaluation.

[0149] HepG2, U87-MG, ACHN, and ARPE-19 cells were revived at 25 cm⁻¹ 2Add 5 mL of DMEM (10% FBS, ARPE-19 cells use dedicated ARPE-DMEM) medium 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.

[0150] 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.

[0151] 1.3 Real-time quantitative PCR

[0152] 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 cDNA Synthesis Kit (+gDNA wiper), 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.

[0153] 2 Experimental Results

[0154] The experimental results are shown in Figure 1. The NO3 siRNA modified with compound 4a exhibited a better knockdown effect on APP mRNA than the NO7 siRNA modified with 2-Hd-A. This indicates that structural modification of the nucleoside monomer before its integration into the siRNA does not affect the siRNA's biological activity. At concentrations of 10 nM and 50 nM, NO3 siRNA was superior to NO7 siRNA.

[0155] Compound 4a-modified NO.3 siRNA showed good uptake in HepG2 (Fig. 2A and 2B) and ARPE-19 (Fig. 2C and 2D) cells, exhibiting a gradient-dependent effect with varying siRNA concentration and transfection time, particularly in ARPE-19 cells. Figure 3 shows the uptake results of various modified nucleoside-doped siRNAs in U87-MG cells at a concentration of 1 nM without transfection reagent assistance: at a concentration of 1 nM, sequences NO.3, NO.7, and NO.8 were slightly superior to the control sequence NO.15, but modified sequences NO.1-2, NO.4-6, and NO.9-14 showed a 2-3 fold increase in uptake efficiency in U87-MG cells. Figure 4 shows the free uptake of various modified siRNAs in U87-MG and ACHN cells without transfection reagent at a concentration of 100 nM. The results show that compared with siRNA modified with 2′C16 glycan ring (NO.7), siRNAs with alkyl chain modifications on all bases and vitamin E modifications on the 2′ glycan ring all exhibited 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 phosphoramide 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 reacted with (tert-butylimino)tris(pyrrolidine)phosphine in dichloromethane under reflux 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.

Images