Carbocyclic nucleoside, oligonucleotide, preparation method therefor, and medical uses thereof

Abstract

Disclosed in the present invention are a carbocyclic nucleoside, an oligonucleotide, a preparation method therefor, and medical uses thereof. The carbocyclic nucleoside is a compound or salt represented by formula (I). The carbocyclic nucleoside or oligonucleotide of the present invention is used in the preparation of a drug used for gene therapy, genetic vaccination, antisense therapy, interfering RNA, or nucleic acid transfer. The oligonucleotide of the present invention has high binding affinity to single-stranded RNA, and has nuclease resistance and plasma stability.

Description

Carbocyclic nucleoside, oligonucleotide and preparation method and medical use thereof Technical Field

[0001] The present invention relates to a nucleoside and a nucleotide, a preparation method and medical use thereof, and in particular to a carbocyclic nucleoside and an oligonucleotide, a preparation method and medical use thereof. Background Art

[0002] Nucleic acids are one of the fundamental building blocks of life, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleotides are the monomers that make up nucleic acid molecules. They are a class of compounds composed of nitrogenous bases, pentose sugars, and phosphates, and are the phosphate esters of nucleosides. Nucleic acid therapy involves the administration of modified single- or double-stranded nucleotides to the human body as a type of drug or vaccine. Nucleic acid therapy is based on a nucleotide sequence that interacts with messenger RNA (mRNA) through complementary base pairing, inhibiting target protein expression through gene silencing, thereby achieving disease treatment and prevention. Nucleic acid-based therapeutic modalities include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), aptamers, messenger RNA (mRNA), ribozymes, antibody-nucleic acid drug conjugates (ARCs), plasmid DNA, and CRISPR / Cas9. Short chains of nucleotides containing approximately 20 bases are called oligonucleotides, and they primarily include ASOs, siRNAs, aptamers, miRNAs, and saRNAs. ASOs are synthetic single-stranded oligonucleotides, typically consisting of 12 to 30 nucleotides. They bind to target mRNA or pre-mRNA through Watson-Crick base pairing, inducing RNase H nuclease degradation of the RNA strand or sterically interfering with pre-mRNA processing and splicing to regulate gene expression. Fomivirsen was the first commercialized ASO drug, approved by the FDA in 1998. Currently, there are 10 ASO drugs approved for marketing worldwide. siRNAs are double-stranded oligonucleotides, consisting of a sense strand and an antisense strand. They bind to the RNA-induced silencing complex (RISC) in the cytoplasm. Under the action of the argouate 2 protein, the double strand depolymerizes, the sense strand cleaves, and the antisense strand binds to the mRNA in the RISC, causing mRNA degradation, thereby regulating gene expression. In 2018, the FDA approved the first siRNA drug, Patisiran, for marketing, and by 2023, five siRNA drugs had been approved for marketing. Aptamers are single-stranded nucleic acids, typically 25 to 50 bases in length, with a unique three-dimensional conformation. They can target biological components such as proteins on the surface or within diseased tissue cells, blocking them from performing their functions through steric hindrance. They can be used as antibody analogs. Pegaptanib is a nucleic acid aptamer drug approved for marketing by the FDA in 2006, and several such drugs are currently in clinical trials. miRNAs are non-coding RNAs of 19 to 24 nucleotides in length. They bind to mRNA within cells, regulating its transcription and inhibiting protein translation to regulate target gene expression.saRNA is a short double-stranded oligonucleotide with a structure similar to siRNA. It can activate gene transcription and exert its therapeutic effect by recruiting endogenous transcription complexes to target genes, increasing mRNA expression, and upregulating target proteins.

[0003] Synthetic oligonucleotides for gene regulation have been used for half a century. However, unmodified oligonucleotides are unstable and easily degraded by endogenous nucleases in the blood. They are rapidly cleared by the liver and kidneys, have a short half-life, and have low bioavailability, making them difficult to develop into drugs. Chemical modification has been used to improve the resistance of oligonucleotides to enzymatic degradation and increase their affinity for their targets, leading to the first oligonucleotide drug being marketed in 1998. Chemical modifications of nucleic acids primarily include phosphate backbone modifications, base modifications, and ribose modifications. The most typical backbone modification is phosphorothioation, in which a non-bridging oxygen atom of the phosphate group in the nucleotide chain is replaced by a sulfur atom, significantly enhancing resistance to nuclease degradation. Another backbone modification is the replacement of the 3' oxygen atom of the ribose ring with a 3' amine. Thiocarbamates and phosphoramidites have demonstrated high affinity and nuclease resistance. Base modifications typically introduce groups such as carboxyl or amino groups at the 5' position of pyrimidine bases and the 8' position of purine bases. Ribose modifications primarily occur at the 2' position, including 2'-O-methyl, 2'-O-methoxyethyl, and 2'-F modifications, which can lead to varying degrees of improved affinity and nuclease resistance. Linking the 4' and 2' riboses to form bicyclic systems such as locked nucleic acids (LNA) and bridged nucleic acids (BNA) significantly improves affinity. Drug researchers are improving drugability through various modifications, enabling the continuous development and marketing of new nucleic acid drugs. However, there is no ideal molecule, and existing nucleic acid drugs still have room for improvement in terms of affinity, nuclease resistance, and in vivo stability.

[0004] Summary of the Invention

[0005] Objectives of the invention: The objective of the present invention is to provide a carbocyclic nucleoside and an oligonucleotide having high binding affinity for single-stranded nucleic acids (RNA or DNA), nuclease resistance and plasma stability; another objective of the present invention is to provide an oligonucleotide and a method for preparing the same; another objective of the present invention is to provide a pharmaceutical composition; another objective of the present invention is to provide a use of an oligonucleotide in the preparation of a drug for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

[0006] Technical solution: The present invention provides a compound or salt represented by the following formula (I):

[0007] Where,

[0008] R1 and R2 are independently hydrogen, an alkyl group containing 1 to 6 carbon atoms, a phenyl group, 1-3 )alkyl-phenyl, heterocyclic or (C 1-3 ) alkyl-heterocycle, a protecting group for a hydroxyl group, a silicon group containing one or more substituents, a phosphate group containing a protecting group for oligonucleotide synthesis, phosphate ester, phosphorothioate, chiral phosphorothioate, phosphotriester, aminoalkylphosphotriester and alkyl phosphate;

[0009] A is O, S or NR3; R3 is a hydrogen atom, an alkyl group containing 1 to 6 carbon atoms, a haloalkyl group containing 1 to 6 carbon atoms, -C(O)R4, -C(O)OR4 or -C(O)N(R4)2; R4 is H or an alkyl group containing 1 to 6 carbon atoms or a haloalkyl group containing 1 to 6 carbon atoms;

[0010] Y is C=O, CHR5 or CHOR5; R5 is H or an alkyl group containing 1 to 6 carbon atoms or a haloalkyl group containing 1 to 6 carbon atoms;

[0011] Base is the base.

[0012] As a further improvement of the above scheme, the formula (I) is represented by any one of the following formulas (I-1) to (I-4):

[0013] Preferably, Base is selected from any of the following groups:

[0014] Preferably, the alkyl group containing 1 to 6 carbon atoms is a straight chain alkyl group, a branched chain alkyl group, a cycloalkyl group or a (C 1-4 )alkyl-(C 3-7 )cycloalkyl.

[0015] Preferably, the compound of formula (I) has any of the structures shown below:

[0016] In another aspect, the present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof, comprising at least one structure represented by the following formulas (II) to (IV):

[0017] wherein Z is phosphate, phosphorothioate, chiral phosphorothioate, phosphate triester, aminoalkyl phosphate triester, and alkyl phosphate.

[0018] In the above scheme, the oligonucleotide is a linear polymer compound formed by covalently linking adjacent nucleotides via phosphate groups. Alternatively, the oligonucleotide is a spacer polymer formed by linking to DNA or RNA via phosphates, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters, and alkyl phosphates.

[0019] As used herein, oligonucleotides preferably contain about 12 to 50 nucleotides, preferably with a backbone containing a phosphorus atom, such as phosphate, phosphorothioate, chiral phosphorothioate, phosphotriester, aminoalkylphosphotriester, or alkylphosphate.

[0020] In the above oligonucleotide structure, the conformationally locked carbocyclic nucleoside can be linked to DNA through phosphates, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters and alkyl phosphates to form a polymer, or the conformationally locked carbocyclic nucleoside can be linked to RNA through phosphates, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters and alkyl phosphates to form a polymer, or the conformationally locked carbocyclic nucleoside can be linked to DNA and RNA through phosphates, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters and alkyl phosphates to form a spacer polymer.

[0021] In the above scheme, a conformationally locked carbocyclic nucleoside for nucleic acid modification is provided. Oligonucleotides are synthetic nucleotide polymers with single-stranded or double-stranded structures. Oligonucleotide therapies include: antisense oligonucleotides designed through Watson-Crick base pairing to regulate gene expression; small interfering RNA (siRNA) that cleaves target transcripts using RNA-induced silencing complexes (RISC); aptamers that inhibit protein function by interacting with target proteins through their three-dimensional structures. The advantages of oligonucleotide therapies include the simple design of drug molecules based on known genomic information, high target specificity, and less off-target toxicity.

[0022] In order to improve the stability and pharmacokinetic properties of oligonucleotides, chemical modifications (phosphosulfate backbone) and sugar-modified nucleic acids, such as 2'-O-methyl, 2'-O-methoxyethyl, 2'-F, locked nucleic acids (LNA) and bridged nucleic acids (BNA) have been successfully introduced. Oligonucleotide therapeutics are usually small in size, easily filtered by the kidneys, have poor blood pharmacokinetic properties, and low target tissue concentrations. The oligonucleotides developed in this application are expected to have high binding affinity for the target gene, but a small polar surface, which can improve blood pharmacokinetic properties.

[0023] The oligonucleotides of the present invention include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), small activating RNAs (saRNAs), nucleic acid aptamers (Aptamers), etc. The conformationally locked carbocyclic nucleosides may appear at one or more positions of the oligonucleotide.

[0024] In another aspect, the present invention provides a method for preparing the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, comprising the step of synthesizing the oligonucleotide using a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:

[0025] The present invention provides a method for preparing an oligonucleotide or a pharmaceutically acceptable salt thereof, preferably comprising the step of synthesizing the oligonucleotide using at least one of the following compounds or a pharmaceutically acceptable salt thereof:

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the above-mentioned compounds or salts, and a pharmaceutically acceptable excipient.

[0027] In another aspect, the present invention provides a use of the above-mentioned compound and salt in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the above-mentioned oligonucleotides or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable excipient.

[0029] In another aspect, the present invention provides a use of the above oligonucleotide or a pharmaceutically acceptable salt thereof in the preparation of a drug for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: According to the present invention, a conformationally locked carbocyclic nucleoside or nucleotide is provided, and an oligonucleotide comprising the conformationally locked carbocyclic nucleoside or nucleotide has high binding affinity to single-stranded RNA, nuclease resistance and plasma stability, and is expected to be applied to nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a statistical diagram of the results of the anti-nuclease degradation experiment. DETAILED DESCRIPTION

[0032] First, the terms used in this specification are defined.

[0033] In this specification, the term "C 1-4 "Alkyl" refers to a straight or branched chain alkyl group containing 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, tert-butyl, etc. The term "C 3-7 "Cycloalkyl" refers to a saturated or partially saturated monocyclic ring containing 3 to 7 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. The term "(C 1-4 )alkyl-(C3-7 The term "cycloalkyl" refers to a hydrocarbon compound containing 4 to 11 carbon atoms in which a straight or branched chain alkyl group containing 1 to 4 carbon atoms is connected to a saturated or partially saturated monocyclic ring containing 3 to 7 carbon atoms.

[0034] In this specification, the term "C 1-3 "Alkyl" refers to a straight or branched chain alkyl group containing 1 to 3 carbon atoms, including methyl, ethyl, n-propyl, isopropyl, etc. The term "C 1-3 "Alkyl-phenyl" refers to a compound in which a straight-chain or branched alkyl group containing 1 to 3 carbon atoms is attached to a phenyl group.

[0035] In this specification, the term "heterocycle" refers to a saturated or partially saturated monocyclic ring containing 5 to 6 carbon atoms, wherein one or more (1 to 3) carbon atoms are replaced by heteroatoms (such as O, N or S). 1-3 "Alkyl-heterocycle" refers to a compound in which a straight-chain or branched alkyl group containing 1 to 3 carbon atoms is attached to a heterocycle.

[0036] In the present specification, the term "haloalkyl group" refers to an alkyl group in which a hydrogen atom is substituted by a halogen atom (such as F, Cl, Br and I).

[0037] In this specification, the term "salt thereof" refers to a salt of the compound represented by formula I of the present invention, and preferably includes alkali metal salts such as sodium salt, potassium salt, lithium salt, alkaline earth metal salts such as calcium salt, magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt and the like; inorganic salts such as ammonium salt, tert-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucosamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzylphenethylamine salt, piperazine salt, Amine salts such as tetramethylammonium salt, organic salts such as tris(hydroxymethyl)aminomethane salt; inorganic acid salts such as halogenated hydrogen salts such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide, nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate, aryl sulfonates such as benzenesulfonate and p-toluenesulfonate, organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine, lysine, arginine, ornithine, glutamate, and aspartate.

[0038] In this specification, the term "pharmaceutically acceptable salt" means a salt or zwitterion of a compound disclosed herein that is water-soluble, oil-soluble or dispersible and pharmaceutically acceptable. Salts can be prepared by the final separation and purification process of the compound, or by reacting an appropriate compound in the form of a free base with a suitable acid. The acid forming a pharmaceutically acceptable salt can be an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, or an organic acid such as oxalic acid, maleic acid, succinic acid and citric acid. Salts can also be formed by coordination of the compound with alkali metal or alkaline earth ions (e.g., sodium), alkaline earth (e.g., magnesium and calcium), ammonium and NX4+ (wherein X is a C1-C4 alkyl group). For therapeutic use, the active ingredient compound salts of the present invention are generally physiologically acceptable, i.e., they are derived from a physiologically acceptable acid or base. However, salts of physiologically unacceptable acids or bases can also be used to prepare or purify physiologically acceptable compounds. All salts, whether or not derived from a physiologically acceptable acid or base, are within the scope of the present invention.

[0039] In this specification, the term "pharmaceutically acceptable excipient" refers to an additive other than the main drug in a pharmaceutical preparation, which is a carrier that is not accompanied by significant side effects when administered, including but not limited to solvents, cosolvents, preservatives, antioxidants, solubilizers, emulsifiers, and osmotic pressure regulators in liquid preparations, and binders, fillers, disintegrants, and lubricants in solid preparations.

[0040] The conformationally locked carbocyclic nucleosides or nucleotides of the present invention can be present at any position of the nucleic acid drug, and the number can be one or more. The position and number are not particularly limited and can be designed according to the purpose. Nucleic acid drugs are a type of drug consisting of a sequence of multiple nucleotides that specifically act on the target nucleic acid in a complementary base pairing manner to regulate gene expression and achieve disease treatment. Nucleic acid drugs include but are not limited to ASO, siRNA, miRNA, saRNA, Aptamer, Ribozymes, mRNA, ARC, Plasmid DNA, CRISPR / Cas9, etc. ASO is usually a single-stranded oligonucleotide composed of 12 to 30 nucleotides. After entering the cell, it forms a double-stranded structure with the target mRNA and regulates the function of RNA through different mechanisms. siRNA is a linear double-stranded RNA with a length of 21 to 25 nucleotides, including a sense chain and an antisense chain. It binds to the RNA-induced silencing complex (RISC) in the cytoplasm and finally pairs with the target mRNA. The mRNA degradation silences the target gene. miRNA is a non-coding RNA with a length of 19 to 24 nucleotides. Mature miRNA is a single-stranded RNA. After binding to mRNA, it regulates post-transcription and inhibits protein translation to regulate target gene expression. saRNA is a short double-stranded oligonucleotide with a structure similar to siRNA. It exerts a therapeutic effect by recruiting endogenous transcription complexes to target genes, increasing mRNA expression, and upregulating target proteins. Aptamers are single-stranded nucleic acids with a stable secondary structure and a length of 25 to 50 nucleotides. They can specifically bind to target molecules such as small molecules, proteins, bacteria, and viruses of the same length. Experienced professionals can use general nucleic acid synthesis methods, not limited to the methods mentioned in the examples, to synthesize nucleotide analogs containing the conformationally locked carbon ring structure of the present invention, and then synthesize various nucleic acid drugs.

[0041] When a nucleic acid containing the conformationally locked carbon ring of the present invention forms a double strand with a complementary nucleic acid, because the carbon ring-containing ribose analogue portion is locked, it is easy to form a stable double strand with the target nucleic acid chain, hindering the transcription and translation process, affecting the synthesis of pathogenic proteins, or inhibiting the proliferation of infectious viruses, obtaining high binding affinity and resistance to nucleases, and prolonging the residence time in the organism.

[0042] The conformationally locked carbocyclic nucleic acids of the present invention are preferably administered to living organisms in the form of pharmaceutical preparations. These preparations can be formulated into injectable formulations along with preservatives, antioxidants, excipients, and other excipients commonly used in the field of pharmaceutical preparations. Furthermore, these preparations can be formulated into liquids, solids, ointments, and other formulations along with pharmaceutical excipients commonly used in this field for administration via the gastrointestinal tract or the skin.

[0043] The conformationally locked carbocyclic nucleic acid of the present invention can be expected to be used for the treatment or prevention of certain diseases, such as antiviral and antitumor effects, by inhibiting the expression of specific genes.

[0044] The compound of the present invention is a compound or salt represented by the following formula (I):

[0045] Where,

[0046] R1 and R2 are independently hydrogen, an alkyl group containing 1 to 6 carbon atoms, a phenyl group, 1-3 )alkyl-phenyl, heterocyclic or (C 1-3 ) alkyl-heterocycle, a protecting group for a hydroxyl group, a silicon group containing one or more substituents, a phosphate group containing a protecting group for oligonucleotide synthesis, phosphate ester, phosphorothioate, chiral phosphorothioate, phosphotriester, aminoalkylphosphotriester and alkyl phosphate;

[0047] A is O, S or NR3; R3 is a hydrogen atom, an alkyl group containing 1 to 6 carbon atoms, a haloalkyl group containing 1 to 6 carbon atoms, -C(O)R4, -C(O)OR4 or -C(O)N(R4)2; R4 is H or an alkyl group containing 1 to 6 carbon atoms or a haloalkyl group containing 1 to 6 carbon atoms;

[0048] Y is C=O, CHR5 or CHOR5; R5 is H or an alkyl group containing 1 to 6 carbon atoms or a haloalkyl group containing 1 to 6 carbon atoms;

[0049] Base is the base.

[0050] Preferably, the formula (I) is represented by any one of the following formulas (I-1) to (I-4):

[0051] Preferably, the compound of formula (I) has any of the structures shown below:

[0052] In another aspect, the present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof, comprising at least one structure represented by the following formulas (II) to (IV):

[0053] wherein Z is phosphate, phosphorothioate, chiral phosphorothioate, phosphate triester, aminoalkyl phosphate triester, and alkyl phosphate.

[0054] Example

[0055] The synthesis of the conformationally locked carbocyclic nucleosides and analogs thereof of the present invention will be described in more detail below based on examples.

[0056] Example 1: Synthesis of Compound I-1-1

[0057] (1) Synthesis of compound 5

[0058] Under a nitrogen atmosphere, compound 4 (575 g, 2 mol) (synthesis reference: Journal of Medicinal Chemistry, 2014, vol. 57, #5, pp. 2107-2120) was dissolved in 10 L of dichloromethane and cooled to -20-25°C. 5 L of 0.4 M sodium methoxide in methanol was slowly added dropwise, and stirring was continued for 30 minutes after the addition was complete. After completion of the reaction, as determined by thin-layer chromatography, 4 M HCl in 1,4-dioxane was added dropwise to quench the reaction, adjusting the pH to <7. The mixture was filtered and concentrated, and the crude product was purified by column chromatography using a 5:1 to 3:1 ratio of petroleum ether to ethyl acetate as the eluent to yield 228 g of an oil, an 80% yield. [M+1] + =143.1;1H NMR(400MHz,Chloroform-d)δ5.98–5.87(m,1H),5.76(dq,J=5.9,2.1Hz,1H),5.12(dp,J=5.6,1.8Hz,1H ),3.75(s,3H),2.96(ddd,J=9.0,6.7,5.2Hz,1H),2.78(ddq,J=15.9,9.0,2.3Hz,1H),2.66–2.55(m,1H).

[0059] (2) Synthesis of Compound 6

[0060] Under a nitrogen atmosphere, compound 5 (50 g, 0.352 mol) was dissolved in 500 ml of dry tetrahydrofuran and 170 ml of hexamethylphosphoric triamide, cooled to -78°C, and 530 ml of a 2M solution of lithium diisopropylamide in tetrahydrofuran was slowly added dropwise. After the addition was complete, the temperature was raised to -20°C and stirring was continued for 30 to 45 minutes. The temperature was then further lowered to -78°C and a solution of benzyl chloromethyl ether (66 g, 0.422 mol) in dry tetrahydrofuran was slowly added dropwise. After the addition was complete, the temperature was slowly raised to -40°C and the reaction was allowed to proceed for 2 hours. After the reaction was complete, acetic acid was added dropwise to quench the reaction, the temperature was raised to room temperature, saturated ammonium chloride solution was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 5:1 to 4:1 as eluent to obtain 63 g of an oily substance with a yield of 68%. [M+1] +=263.2;1H NMR(400MHz,Chloroform-d)δ7.40–7.29(m,5H),5.99(dt,J=5.3,2.3Hz,1H),5.76(dq,J=6.8,2.2Hz,1H),4.57(d,J=2.3Hz,1H),4 .54(s,2H),3.79(d,J=8.7Hz,1H),3.77(s,3H),3.39(d,J=8.7Hz,1H),3.12(dt,J=17.8,2.4Hz,1H),2.55(dt,J=17.8,2.3Hz,1H).

[0061] (3) Synthesis of Compound 7

[0062] Under a nitrogen atmosphere, compound 6 (63 g, 0.24 mol) was dissolved in 600 ml of dichloromethane, cooled to 0°C, and 85% m-chloroperbenzoic acid (121.8 g, 0.6 mol) was added portionwise. The mixture was stirred at room temperature overnight. After completion of the reaction, as determined by LCMS, saturated sodium sulfite solution was added to quench the reaction. The organic phase was then washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a mixture of petroleum ether:ethyl acetate in a ratio of 5:1 to 4:1 as eluent to afford 45 g of an oily substance in a 67% yield. [M+1] + =288.1.

[0063] (4) Synthesis of Compound 8

[0064] Under a nitrogen atmosphere, compound 7 (35.2 g, 0.126 mol) was dissolved in 250 ml of dry N,N-dimethylformamide, and benzyl bromide (32.3 g, 0.189 mol) was added. The temperature was cooled to -40 to -50°C, and 189 ml of a 1M solution of potassium bis(trimethylsilyl)amide in tetrahydrofuran was slowly added dropwise. After the addition was complete, the reaction was allowed to continue for 1 hour. After completion of the reaction, acetic acid was added dropwise to quench the reaction, and the temperature was raised to room temperature. Saturated ammonium chloride solution was added, and the reaction was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 5:1 to 4:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 40 g of an oil with a yield of 84%. [M+1] + =369.2.

[0065] (5) Synthesis of Compound 9

[0066] Under a nitrogen atmosphere, compound 8 (39 g, 0.106 mol) was dissolved in 1.2 L of dry dichloromethane. The temperature was lowered to -78°C, and 254 ml of a 1 M solution of diisobutylaluminum hydride in n-hexane was slowly added dropwise. After the addition was complete, the reaction was continued at -78°C for 1 hour. LCMS confirmed the reaction was complete, and acetic acid was added dropwise to quench the reaction. The temperature was raised to room temperature, and the organic phase was washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 4:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 28 g of an oil, with a yield of 77.7%. [M+1] + =341.2;1H NMR (400MHz, Chloroform-d) δ7.41–7.29(m,7H),4.80(d,J=11.9Hz,1H),4.60(d,J=11.9Hz,1H),4.47(q,J=12.1Hz,2H),4.20(s,1H),3.87(d ,J=11.2Hz,1H),3.60(d,J=2.7Hz,1H),3.47(d,J=8.3Hz,3H),3.37(d,J=9.1Hz,1H),2.98(s,1H),2.05–1.99(m,1H),1.92(d,J=14.9Hz,1H).

[0067] (6) Synthesis of Compound 10

[0068] Under a nitrogen atmosphere, compound 9 (22 g, 0.065 mol), thymine (32.8 g, 0.26 mol), and potassium carbonate (26.9 g, 0.195 mol) were suspended in 220 ml of dry dimethyl sulfoxide, heated to 140°C, and stirred for 3 hours. After completion of the reaction as determined by LCMS, the mixture was cooled to room temperature, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a dichloromethane:methanol ratio of 50:1 to 20:1 as the eluent to afford 24.4 g of a white solid in an 81% yield. [M+1] +=467.2;1H NMR (400MHz, DMSO-d6) δ11.19(s,1H),7.55–7.16(m,11H),5.09(d,J=6.5Hz,1H),4.81(d,J=11.7Hz,1H),4.71(q,J=9.7Hz,1H),4.59–4.45(m,4H ),4.39(dt,J=9.7,5.8Hz,1H),3.80(d,J=4.9Hz,1H),3.61–3.39(m,4H), 1.93–1.82(m,1H),1.68(d,J=1.1Hz,3H),1.35(dd,J=13.5,10.1Hz,1H).

[0069] (7) Synthesis of Compound 11

[0070] Under a nitrogen atmosphere, compound 10 (17.3 g, 0.037 mol) and triethylamine (15 g, 0.148 mol) were dissolved in 170 ml of dry tetrahydrofuran, cooled to -10°C, and a solution of methanesulfonic anhydride (7.7 g, 0.044 mol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, the reaction was continued at -10°C for 30 min. After completion of the reaction, LCMS analysis confirmed the addition of saturated aqueous sodium bicarbonate solution, followed by extraction twice with ethyl acetate. The combined organic phases were washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 100:1 ratio of dichloromethane to methanol as the eluent to afford 14.5 g of a white foamy solid in a 72.5% yield. [M+1] + =545.2.

[0071] (8) Synthesis of Compound 12

[0072] Under a nitrogen atmosphere, carefully add 60% sodium hydride (3.2 g, 0.081 mol) to 100 ml of dry N,N-dimethylformamide. Cool to 0°C and slowly add a solution of compound 11 (14 g, 0.027 mol) in N,N-dimethylformamide dropwise until the addition is complete. Continue the reaction for 1 hour. LCMS confirms the reaction is complete. Add saturated ammonium chloride solution and extract twice with ethyl acetate. The combined organic phases are washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product is purified by column chromatography using a 40:1 ratio of dichloromethane to methanol as the eluent to afford 8.5 g of an oily liquid, yielding 74%. [M+1] + =449.2.

[0073] (9) Synthesis of Compound I-1-1

[0074] Compound 12 (4 g, 8.9 mmol) was dissolved in 40 ml of anhydrous methanol. 50 μl of acetic acid and 400 mg of 10% palladium on carbon (60% water content) were added. The atmosphere was replaced with hydrogen three times and stirred at room temperature overnight. The reaction was monitored by LCMS. The mixture was filtered and concentrated to yield 2.2 g of a white solid, a 90% yield. [M+1] + =269.2;1H NMR (400MHz, DMSO-d6) δ11.27(s,1H),7.43(d,J=1.3Hz,1H),5.76(s,2H),4.23(dd,J=9.7,5.6Hz,1H),3.91(d,J=14 .6Hz,2H),3.70–3.61(m,2H),3.53–3.43(m,3H),2.05(dd,J=13.6,9.7Hz,1H),1.81–1.78(m,3H),1.78–1.72(m,1H).

[0075] Example 2: Synthesis of Compound I-1-2

[0076] (1) Synthesis of compound 15

[0077] Under a nitrogen atmosphere, compound 12 (8.5 g, 0.019 mol), 4-dimethylaminopyridine (4.6 g, 0.038 mol), and triethylamine (7.7 g, 0.076 mol) were dissolved in 100 ml of dry acetonitrile. 2,4,6-triisopropylbenzenesulfonyl chloride (11.5 g, 0.038 mol) was carefully added and stirred at room temperature for 2 h. The temperature was lowered to 0°C, and 28.5 ml of concentrated aqueous ammonia was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. LCMS confirmed the reaction was complete. Water was added and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 30:1 ratio of dichloromethane to methanol as the eluent to obtain 6 g of an oily liquid with a yield of 70.7%. [M+1] + =448.2.

[0078] (2) Synthesis of Compound 16

[0079] Under a nitrogen atmosphere, compound 15 (4.6 g, 0.010 mol) was dissolved in 35 ml of dry N,N-dimethylformamide, and benzoic anhydride (3.2 g, 0.014 mol) was added. The mixture was stirred at room temperature overnight. LCMS monitored the reaction until completion. Saturated sodium bicarbonate solution was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 3:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 3.8 g of an oily liquid, with a yield of 68.9%. [M+1] + =552.2.

[0080] (3) Synthesis of Compound I-1-2

[0081] Under a nitrogen atmosphere, compound 16 (2 g, 3.6 mmol) was dissolved in 20 ml of anhydrous methanol. 4 ml of formic acid and 960 mg of 10% palladium on carbon (60% water content) were added, and the mixture was stirred at 60°C for 6 h. The reaction was monitored by LCMS. The mixture was filtered, washed, and concentrated to yield 1.1 g of a white solid, with a yield of 85%. [M+1] + =372.2; 1H NMR (400MHz, DMSO-d6) δ13.17(s,1H),8.21(s,2H),7.79(s,1H),7.60(t,J=7.3Hz ,1H),7.50(t,J=7.6Hz,2H),5.13(d,J=4.5Hz,1H),4.56(t,J=5.3Hz,1H),4.31(d d,J=9.7,5.7Hz,1H),4.06(s,1H),3.93(d,J=4.5Hz,1H),3.75–3.61(m,2H),3.52 (dd,J=15.8,5.9Hz,2H),2.19–2.09(m,1H),2.06(s,3H),1.88(d,J=13.1Hz,1H).

[0082] Example 3: Synthesis of Compound I-1-3

[0083] (1) Synthesis of compound 19

[0084] Under a nitrogen atmosphere, compound 9 (24 g, 0.07 mol), cesium fluoride (31.9 g, 0.21 mol), and O-6-benzylguanine (33.8 g, 0.14 mol) were suspended in 240 ml of dry DMF and heated to 90°C for 3 h. The reaction was monitored by LCMS until completion. The temperature was then lowered, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a solvent ratio of petroleum ether:ethyl acetate (1:2 to 1:3) to afford 23.3 g of an oil, with a yield of 56.8%. [M+1] + =582.2;1H NMR(400MHz,Chloroform-d)δ7.64(s,1H),7.57–7.47(m,2H),7.46–7.29(m,13H),5 .58(s,2H),4.94(d,J=11.4Hz,1H),4.88(s,2H),4.70(dd,J=10.8,3.0Hz,2H),4.57( dd,J=8.4,5.6Hz,1H),4.52(s,2H),4.15(d,J=5.6Hz,1H),3.85(d,J=11.3Hz,1H),3 .66(d,J=11.3Hz,1H),3.41(s,2H),2.38(dd,J=13.3,8.7Hz,1H),2.09–1.96(m,2H).

[0085] (2) Synthesis of Compound 20

[0086] Under a nitrogen atmosphere, compound 19 (23.2 g, 0.04 mol) was dissolved in 230 ml of dry pyridine, cooled to 0°C, and trimethylsilyl chloride (26.1 g, 0.24 mol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 2 h. The temperature was then lowered to 0°C, and isobutyryl chloride (4.7 g, 0.044 mol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 2 h. 30 ml of concentrated aqueous ammonia was added dropwise, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Water was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 to 1:2 ratio of petroleum ether to ethyl acetate as the eluent to afford 26.3 g of an oily liquid, with a yield of 97%. [M+1] + =652.3.

[0087] (3) Synthesis of Compound 21

[0088] Under a nitrogen atmosphere, compound 20 (25.5 g, 0.039 mol) was dissolved in 200 ml of dry pyridine and cooled to 0°C. A solution of methanesulfonic anhydride (8.7 g, 0.051 mol) in tetrahydrofuran was slowly added dropwise. Stirring was continued for 2 h after the addition was complete. LCMS monitored the reaction until the product content reached a maximum. Water was added to quench the reaction, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 19.1 g of an oily substance, yielding 56.8%. 4.5 g of the raw material was recovered. [M+1] + =730.3.

[0089] (4) Synthesis of Compound 22

[0090] Under a nitrogen atmosphere, 60% sodium hydride (3.5 g, 87.9 mmol) was carefully added to 210 ml of dry tetrahydrofuran and stirred for 10 min. The temperature was lowered to 0°C, and a solution of compound 21 (21.4 g, 29.3 mmol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. LCMS monitored the reaction completion, quenched with acetic acid, and saturated sodium bicarbonate solution was added. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of petroleum ether to ethyl acetate as the eluent to give 15.8 g of an oily substance with a yield of 85%. [M+1] + =634.3.

[0091] (5) Synthesis of Compound I-1-3

[0092] Under a nitrogen atmosphere, compound 22 (5 g, 7.9 mmol) was dissolved in 50 ml of dichloromethane and cooled to -78°C. 71 ml of a 1 M solution of boron tribromide in dichloromethane was slowly added dropwise. After the addition was complete, the temperature was slowly raised to -40°C and stirred for 2 h. The reaction was monitored by LCMS. Methanol was added dropwise to quench the reaction, and the mixture was allowed to warm to room temperature and concentrated. The crude product was purified by column chromatography using a 10:1 ratio of dichloromethane to methanol as the eluent to afford 2.5 g of a white solid in an 87.2% yield. [M+1] +=364.1;1H NMR (400MHz, DMSO-d6) δ8.14(s,1H),7.36–7.21(m,1H),5.17(d,J=4.2Hz,1H),4.59(t,J=5.2Hz,1H),4.44(dd,J=9.7,5.4Hz,1H),4.03(d,J=4.4Hz,1 H),3.96(s,1H),3.78–3.68(m,2H),2.79(p,J=6.8Hz,1H),2.23(ddd,J=26 .1,14.8,10.2Hz,2H),1.99(dd,J=10.2,6.6Hz,1H),1.12(d,J=6.8Hz,5H).

[0093] Example 4: Synthesis of Compound I-1-4

[0094] (1) Synthesis of compound 25

[0095] Under a nitrogen atmosphere, compound 9 (26 g, 0.076 mol), adenine (20.5 g, 0.15 mol), and potassium carbonate (31.5 g, 0.23) were suspended in 260 ml of dry dimethyl sulfoxide and heated to 120°C for 4 h. Upon completion of the reaction, the temperature was lowered, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 20:1 ratio of dichloromethane to methanol as the eluent to afford 29.2 g of a white solid in an 80.4% yield. [M+1] + =476.2;1H NMR(400MHz,DMSO-d6)δ8.14(s,1H),8.10(s,1H),7.43–7.24(m,10H),7.16(s, 2H),5.19(d,J=6.2Hz,1H),4.84(d,J=11.8Hz,1H),4.81–4.71(m,2H),4.61–4. 53(m,3H),4.50(t,J=4.9Hz,1H),3.90(d,J=4.3Hz,1H),3.65–3.56(m,2H),3.5 4(d,J=4.6Hz,2H), 2.10(dd,J=13.5,8.6Hz,1H), 1.82(dd,J=13.4,9.2Hz,1H).

[0096] (2) Synthesis of Compound 26

[0097] Under a nitrogen atmosphere, compound 25 (22 g, 0.046 mol) was dissolved in 200 ml of dry pyridine, cooled to 0°C, and trimethylsilyl chloride (25 g, 0.231 mol) was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 2 h. The mixture was cooled to 0°C, and benzoyl chloride (13.5 g, 0.097 mol) was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 2 h. The reaction was monitored by LCMS for completion. The mixture was cooled to 0°C, 35 ml of concentrated aqueous ammonia was added, and the mixture was slowly warmed to room temperature and stirred for 1 h. The reaction was monitored by LCMS for completion. Water was added and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using 100% ethyl acetate as the eluent to obtain 20 g of the product, with a yield of 73.7%. [M+1] + =560.3.

[0098] (3) Synthesis of Compound 27

[0099] Under a nitrogen atmosphere, compound 26 (20 g, 0.034 mol) and methanesulfonic anhydride (7.2 g, 0.041 mol) were dissolved in 200 ml of dry tetrahydrofuran, cooled to -10°C, and triethylamine (10.4 g, 0.103 mol) was slowly added dropwise. After the addition was complete, the temperature was raised to 0°C and the reaction was allowed to proceed for 2 h. LCMS confirmed the substantial reaction of the starting material. Saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 17 g of a white solid in a 65% yield. [M+1] + =658.2.

[0100] (4) Synthesis of Compound 28

[0101] Under a nitrogen atmosphere, carefully add 60% sodium hydride (1.87 g, 77.7 mmol) to 170 ml of dry N,N-dimethylformamide, stir for 10 minutes, cool to 0°C, and slowly add a solution of compound 27 (17 g, 25.9 mmol) in N,N-dimethylformamide dropwise. After the addition is complete, continue stirring for 2 hours. LCMS monitors the reaction for completion, add saturated ammonium chloride solution, and extract twice with ethyl acetate. The combined organic phases are washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product is purified by column chromatography using 100% ethyl acetate as the eluent to obtain 12.3 g of a white solid, with a yield of 85%. [M+1] + =562.2.

[0102] (5) Synthesis of Compound I-1-4

[0103] Under a nitrogen atmosphere, compound 28 (5.3 g, 10.7 mmol) was dissolved in 50 ml of dry dichloromethane solution, cooled to -78°C, and 64 ml of a 1 M boron trichloride solution in dichloromethane was slowly added dropwise. After the addition was complete, the temperature was raised to -20°C and stirred overnight. LCMS monitored the reaction for completion, and methanol was added to quench the reaction. The mixture was concentrated under reduced pressure, slurried with ethyl acetate, and filtered to obtain 3.4 g of a white solid, the hydrochloride salt of compound Example 4. [M+1] + =382.2;1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),8.71(s,1H),8.55(s,1H),8.11–8.02(m,2H),7.71–7.63( m,1H),7.56(dd,J=8.3,7.0Hz,2H),7.35(d,J=6.1Hz,5H),7.32–7.23(m,5H),4.79–4.70(m,1H) ,4.66–4.59(m,1H),4.55(d,J=4.7Hz,2H),4.52(d,J=3.1Hz,1H),4.29(s,1H),3.85(d,J=6.7Hz ,1H),3.80(d,J=9.5Hz,1H),3.73(d,J=6.5Hz,1H),3.65(d,J=9.5Hz,1H),2.44(d,J=9.1Hz,2H).

[0104] Example 5: Synthesis of Compound I-1-5

[0105] (1) Synthesis of compound 14

[0106] Under a nitrogen atmosphere, compound I-1-1 (1.6 g, 6.0 mmol) and 4,4′-dimethoxytrityl chloride (4.65 g, 13.8 mmol) were dissolved in 30 ml of dry dichloromethane. The temperature was lowered to 0°C and N,N-diisopropylethylamine (2.3 g, 18 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 2 hours. LCMS monitored the reaction completion. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 40:1 ratio of dichloromethane to methanol as the eluent to afford 2.8 g of a light yellow solid in an 86% yield. [M+1] +=571.2;1H NMR(400MHz, DMSO-d6)δ11.26(s,1H),7.42(d,J=1.4Hz,1H),7.38–7.28(m,4H),7.22( ddt,J=6.9,4.8,2.2Hz,5H),6.92–6.86(m,4H),5.10(d,J=4.6Hz,1H),4.30(dd,J=9.9 ,5.8Hz,1H),3.96(d,J=4.7Hz,1H),3.91(s,1H),3.74(s,6H),3.67(s,2H),3.29(d,J= 9.0Hz, 1H), 3.12 (d, J = 9.0Hz, 1H), 2.30 (dd, J = 13.5, 9.8Hz, 1H), 1.78 (d, J = 1.1Hz, 4H).

[0107] (2) Synthesis of Compound I-1-5

[0108] Under a nitrogen atmosphere, compound 14 (700 mg, 1.2 mmol) and 4,5-dicyanoimidazole (354 mg, 3 mmol) were dissolved in 10 ml of dry dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (904 mg, 3 mmol) was added dropwise at room temperature and stirring was continued for 2 h. LCMS monitored the reaction completion, and dichloromethane was added. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated using high-pressure preparative liquid chromatography on a C18 column with a mobile phase of acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) = 8:2, yielding 560 mg of a white solid with a yield of 60%. [M+1] + =771.3; 31 PNMR:148.30,148.04.

[0109] Example 6: Synthesis of Compound I-1-6

[0110] (1) Synthesis of Compound 18

[0111] Under a nitrogen atmosphere, compound I-1-2 (1.1 g, 3.12 mmol) and 4,4′-dimethoxytrityl chloride (2.3 g, 6.88 mmol) were dissolved in 20 ml of dry dichloromethane. The temperature was lowered to 0°C and N,N-diisopropylethylamine (2.0 g, 15.6 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. After completion of the reaction, the organic phase was washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a solvent ratio of petroleum ether:ethyl acetate:TEA = 3:1:0.1% to obtain 1.4 g of a white solid, with a yield of 70%. [M+1] + =674.3;1H NMR (400MHz, DMSO-d6) δ13.18(s,1H),8.20(s,2H),7.79(s,1H),7.59(t,J=7.3Hz,1H),7.50( t,J=7.5Hz,2H),7.40–7.30(m,5H),7.24(dd,J=8.9,3.2Hz,4H),6.93–6.89(m,4H),5.15(d,J= 4.7Hz,1H),4.38(dd,J=9.7,5.8Hz,1H),4.04(d,J=7.1Hz,1H),4.02–3.98(m,1H),3.75(s,8H ),3.29(s,1H),3.16(d,J=9.0Hz,1H),2.40(t,J=11.7Hz,1H),1.99(s,3H),1.92–1.86(m,1H).

[0112] (2) Synthesis of Compound I-1-6

[0113] Under a nitrogen atmosphere, compound 18 (500 mg, 0.74 mmol), tetrazole (154 mg, 2.2 mmol), and N-methylimidazole (61 mg, 0.74 mmol) were dissolved in 5 ml of dry N,N-dimethylformamide. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (446 mg, 1.48 mmol) was added dropwise at room temperature, and stirring was continued for 2 h. LCMS monitored the reaction completion. The mixture was diluted with dry acetonitrile and separated using a high-pressure preparative liquid chromatography column with a C18 column and 100% acetonitrile as the mobile phase, yielding 320 mg of a white solid in a 49% yield. [M+1] + =874.4; 31 PNMR:148.4,148.2.

[0114] Example 7: Synthesis of Compound I-1-7

[0115] (1) Synthesis of Compound 24

[0116] Under a nitrogen atmosphere, compound I-1-3 (3.1 g, 9.1 mmol) and 4,4'-bismethoxytrityl chloride (6.8 g, 20.2 mmol) were dissolved in 30 ml of dry dichloromethane. The temperature was lowered to 0°C, and N,N-diisopropylethylamine (5.89 g, 45.5 mmol) was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 2 hours. LCMS monitored the reaction completion, followed by concentration. The crude product was purified by column chromatography using a solvent ratio of dichloromethane:methanol:triethylamine (30:1:0.1%) to afford 4.6 g of a light yellow solid, a yield of 76%. [M+1] + =666.3;1H NMR(400MHz,Chloroform-d)δ12.10(s,1H),9.40(s,1H),7.75(s,1H),7.46–7.40(m,2H),7.34–7.25 (m,5H),7.23–7.17(m,2H),6.83(ddd,J=9.0,4.2,2.0Hz,4H),4.66(s,1H),4.48(t,J=7.6Hz,1H),4. 27(s,1H),3.95(d,J=7.1Hz,1H),3.82(s,1H),3.78(s,6H),3.68(d,J=7.0Hz,1H),3.46(d,J=9.6Hz, 1H), 3.27 (d, J = 9.6Hz, 1H), 2.53 (p, J = 6.9Hz, 1H), 2.32 (d, J = 7.6Hz, 2H), 1.19 (dd, J = 9.7, 6.8Hz, 6H).

[0117] (2) Synthesis of Compound I-1-7

[0118] Under a nitrogen atmosphere, compound 24 (1.0 g, 1.5 mmol) and 4,5-dicyanoimidazole (443 mg, 3.75 mmol) were dissolved in 10 ml of dry dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.1 g, 3.75 mmol) was added dropwise at room temperature and stirring continued for 2 h. LCMS monitored the reaction until completion. Dichloromethane and 0.1 ml of triethylamine were added, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated using high-pressure preparative liquid chromatography on a C18 column with a mobile phase of acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) = 7:3, yielding 660 mg of a white solid with a yield of 51%. [M+1] + =866.4; 31 PNMR:147.27,147.17.

[0119] Example 8: Synthesis of Compound I-1-8

[0120] (1) Synthesis of compound 30

[0121] Under a nitrogen atmosphere, compound I-1-4 (1 g, 2.6 mmol) and 4,4'-bismethoxytrityl chloride (1.9 g, 5.72 mmol) were dissolved in dry dichloromethane. The temperature was lowered to 0°C, and triethylamine (1.3 g, 13 mmol) was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 2 hours. LCMS monitored the reaction completion, and the mixture was concentrated under reduced pressure. The crude product was purified by column chromatography using a solvent ratio of dichloromethane:ethyl acetate:triethylamine (1:1:0.1%) to afford 1.3 g of a light yellow solid, a yield of 72.2%. [M+1] + =684.3;1H NMR (400MHz, DMSO-d6) δ11.16(s,1H),8.76(s,1H),8.62(s,1H),8.05(d,J=7.7Hz,2H),7.65(t,J=7.4Hz,1H),7.56(t,J=7.6Hz,2H),7.41( d,J=7.8Hz,2H),7.33(t,J=7.6Hz,2H),7.30–7.21(m,5H),6.96–6.87(m,4H),5.20(d,J=4.6Hz,1H),4.75(dd,J=9.8,5.5Hz,1H),4.36(d,J =4.7Hz,1H),4.11(s,1H),3.81(d,J=6.5Hz,1H),3.75(s,6H),3.40(d,J=9.1Hz,1H),3.21–3.16(m,2H),2.57–2.52(m,1H),2.46-2.39(m,1H).

[0122] (2) Synthesis of Compound I-1-8

[0123] Under a nitrogen atmosphere, compound 30 (800 mg, 1.17 mmol) and 4,5-dicyanoimidazole (345 mg, 2.92 mmol) were dissolved in 10 ml of dry dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (880 mg, 2.92 mmol) was added dropwise at room temperature and stirring continued for 2 h. LCMS monitored the reaction completion, followed by the addition of dichloromethane and 0.1 ml of triethylamine. The organic phase was washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated using high-pressure preparative liquid chromatography on a C18 column with a mobile phase of acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) = 8:2, yielding 550 mg of a white solid with a yield of 53%. [M+1] + =884.4; 31 PNMR:148.40,148.31.

[0124] Example 9: Synthesis of Compound I-1-9

[0125] Under a nitrogen atmosphere, compound I-1-2 (30 mg, 0.081 mmol) was dissolved in 3 ml of 7N ammonia-methanol solution and stirred at room temperature for 4 h. After completion of the reaction, as monitored by LCMS, the mixture was concentrated, slurried with a small amount of methanol, and filtered to yield 18 mg of a white solid, with a yield of 83%. [M+1] + =268.1;1H NMR (400MHz, DMSO-d6) δ7.34 (s, 1H), 6.90 (d, J = 171.7Hz, 2H), 5.04 (d, J = 4.1Hz, 1H), 4.53 (t, J = 5.3Hz, 1H), 4.27 (dd, J = 9.7, 5.6Hz, 1H), 3.97–3.78(m,2H),3.72–3.56(m,2H),3.54–3.40(m,2H),2.02(dd,J=13.6,9.7Hz,1H),1.86(s,3H),1.72(ddd,J=13.9,5.8,2.6Hz,1H).

[0126] Example 10: Synthesis of Compound I-1-10

[0127] Under nitrogen atmosphere, compound I-1-3 (50 mg, 0.14 mmol) was dissolved in 5 ml of 7N ammonia methanol solution and stirred at room temperature for 3 days. After completion of the reaction, as monitored by LCMS, the mixture was concentrated, slurried with a small amount of methanol, and filtered to obtain 35 mg of a white solid, with a yield of 87%. [M+1] +=294.1;1H NMR (400MHz, DMSO-d6) δ10.58(s,1H),7.81(s,1H),6.48(s,2H),5.21–5.02(m,1H),4.56(s,1H),4.33(dd,J=9.6,5 .7Hz,1H),4.02(s,1H),3.90(s,1H),3.79–3.63(m,2H),3.53(dd,J=13.5,8.5Hz,2H),2.16(qd,J=13.7,7.2Hz,2H).

[0128] Example 11: Synthesis of Compound I-2-1

[0129] (1) Synthesis of compound 31

[0130] Under a nitrogen atmosphere, compound 10 (1.6 g, 3.4 mmol) was dissolved in 30 ml of dichloromethane and 30 ml of water. 2,2,6,6-tetramethylpiperidinium oxide (55 mg, 0.35 mmol) and potassium bromide (40 mg, 0.34 mmol) were added. The temperature was lowered to 0°C, and 10% sodium hypochlorite solution (3.5 g, 4.8 mmol) (adjusted to pH 9 with saturated sodium bicarbonate solution) was slowly added dropwise. After the addition was complete, stirring was continued for 1 hour. After completion of the reaction, monitored by LCMS, sodium thiosulfate solution was added to quench the reaction. The mixture was separated, washed with water, dried, and concentrated. The crude product was purified by column chromatography using ethyl acetate:petroleum ether in a ratio of 1:2 to 1:1 as eluent to obtain 950 mg of a solid with a yield of 60%. [M+1] + =465.2;1H NMR (400MHz, DMSO-d6) δ11.22(d,J=2.2Hz,1H),9.57(d,J=2.1Hz,1H),7.85–7.40(m,1H),7.39–7.14 (m,10H),5.37(dd,J=5.8,2.1Hz,1H),4.86(dd,J=11.8,2.0Hz,1H),4.65(d,J=9.8Hz,1H),4.58–4.5 4(m,1H),4.54–4.45(m,2H),4.40(dt,J=5.3,2.4Hz,1H),4.12(dd,J=4.7,2.0Hz,1H),3.88(dd,J=9. 2, 2.1Hz, 1H), 3.61 (dd, J = 9.2, 2.1Hz, 1H), 1.83–1.71 (m, 3H), 1.55–1.42 (m, 1H), 1.37–1.11 (m, 1H).

[0131] (2) Synthesis of Compound I-2-1

[0132] Under nitrogen, compound 31 (400 mg, 0.86 mmol) was dissolved in 5 ml of methanol, and 20 μl of 30% methanolic hydrogen chloride solution was added dropwise. The mixture was stirred at room temperature for 4 h. The mixture was concentrated, and the crude product was purified by column chromatography using ethyl acetate:petroleum ether (1:5) as the eluent to obtain 200 mg of the compound (yield: 49%). [M+1] + =479.2;1H NMR(400MHz,DMSO-d6)δ11.28(s,1H),7.37–7.27(m,10H),7.16(d,J=1.4Hz,1H),4 .97(s,1H),4.69–4.57(m,2H),4.53(d,J=2.5Hz,2H),4.41(s,1H),4.36(dd,J=9.6, 6.0Hz,1H),4.06(s,1H),3.69(d,J=9.8Hz,1H),3.46(d,J=9.8Hz,1H),3.39(s,3H) ,2.38(dd,J=13.4,9.6Hz,1H), 1.75(d,J=1.1Hz,3H), 1.66(dd,J=13.3,6.1Hz,1H).

[0133] Example 12: Synthesis of Compound I-2-2

[0134] Under nitrogen, compound 31 (400 mg, 0.86 mmol) was dissolved in 5 ml of anhydrous ethanol, and 20 μl of 30% ethanolic hydrogen chloride solution was added dropwise. The mixture was stirred at room temperature for 4 h. The mixture was concentrated, and the crude product was purified by column chromatography using ethyl acetate:petroleum ether (1:5) as the eluent to obtain 210 mg of the product (50% yield). [M+1] + =493.2;1H NMR(400MHz,DMSO-d6)δ11.27(s,1H),7.38–7.26(m,10H),7.15(d,J=1.4Hz,1H),5 .08(s,1H),4.68–4.57(m,2H),4.57–4.49(m,2H),4.40–4.33(m,2H),4.03(s,1H), 3.78–3.67(m,2H),3.58–3.48(m,1H),3.46(d,J=9.8Hz,1H),2.41(dd,J=13.4,9.6 Hz, 1H), 1.74 (d, J = 1.1Hz, 3H), 1.66 (dd, J = 13.4, 6.0Hz, 1H), 1.14 (t, J = 7.0Hz, 3H).

[0135] Example 13: Synthesis of Compound I-4-1

[0136] (1) Synthesis of compound 32

[0137] Under a nitrogen atmosphere, compound 8 (10 g, 27.1 mmol) and thymine (13.5 g, 108.4 mmol) were suspended in 100 ml of dry dimethyl sulfoxide. 1,8-diazobisspiro[5.4.0]undec-7-ene (41 g, 271 mmol) was added, and the reaction was stirred at 120°C for 3 h. LCMS analysis showed that the reaction was approximately halfway complete. The temperature was then lowered, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 50:1 ratio of dichloromethane to methanol as the eluent to afford 4.2 g of the product, yielding 31%. 5.4 g of compound 8 was recovered. [M+1] + =495.2;1H NMR(400MHz,DMSO-d6)δ11.21(s,1H),7.48(s,1H),7.40–7.24(m,10H),5.35(d ,J=6.0Hz,1H),4.86(d,J=11.4Hz,1H),4.74(q,J=10.0Hz,1H),4.56(d,J=12.1 Hz,1H),4.51–4.40(m,4H),3.86(d,J=8.5Hz,1H),3.78(d,J=4.2Hz,1H),3.70( d,J=8.5Hz,1H),3.54(s,3H),2.70(dd,J=13.7,9.7Hz,1H),1.80–1.68(m,4H).

[0138] (2) Synthesis of compound 33

[0139] Under a nitrogen atmosphere, compound 32 (4.2 g, 8.5 mmol) and 4-dimethylaminopyridine (4.1 g, 33.6 mmol) were dissolved in 50 ml of dichloromethane and cooled to 0°C. Trifluoromethylsulfonyl chloride (4.3 g, 25.5 mmol) was slowly added dropwise. After the addition was complete, the mixture was naturally warmed to room temperature and stirred for 2 h. After completion of the reaction, monitored by LCMS, water was added, the layers separated, dried over anhydrous sodium sulfate, and concentrated. The crude product was directly carried into the next step without further purification. [M+1] + =477.2.

[0140] (3) Synthesis of compound 34

[0141] Under a nitrogen atmosphere, the crude compound 33 obtained in the previous step was added to 100 ml of acetone and 100 ml of 1 M aqueous sulfuric acid and heated under reflux overnight. Upon completion of the reaction, the acetone was concentrated under reduced pressure. The residue was extracted twice with ethyl acetate. The combined organic phases were washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 2:1 ratio of ethyl acetate to petroleum ether as the eluent to yield 3.4 g, for a two-step yield of 80%. [M+1] + =495.2;1H NMR(400MHz,Chloroform-d)δ8.34(s,1H),7.42–7.23(m,10H),5.18(ddd,J=1 0.6,8.9,5.6Hz,1H),4.69(d,J=11.9Hz,1H),4.58(s,2H),4.56(d,J=11.9Hz,1 H),4.39(dd,J=5.8,3.3Hz,1H),3.98–3.87(m,2H),3.77–3.72(m,1H),3.69(s, 3H), 2.67 (dd, J=13.6, 8.9Hz, 1H), 2.22 (dd, J=13.6, 10.7Hz, 1H), 1.81 (s, 3H).

[0142] (4) Synthesis of Compound 35

[0143] Under a nitrogen atmosphere, compound 34 (3.4 g, 6.9 mmol), potassium carbonate (2.8 g, 20.6 mmol), and 4-methoxybenzyl chloride (1.6 g, 10.4 mmol) were dissolved in 25 ml of dry N,N-dimethylformamide and stirred at 50°C for 2 h. After completion of the reaction, the temperature was lowered and diluted with water, followed by extraction twice with ethyl acetate. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:3 ratio of ethyl acetate to petroleum ether as the eluent to afford 3.9 g of the product, yielding 93%. [M+1] + =615.3.

[0144] (5) Synthesis of Compound 36

[0145] Under a nitrogen atmosphere, compound 35 (500 mg, 0.3 mmol) and potassium hydroxide (138 mg, 0.9 mmol) were dissolved in a mixture of 5 ml of methanol and 1 ml of purified water and heated under reflux overnight. After completion of the reaction, as monitored by LCMS, the temperature was lowered and the reaction mixture was poured into water. The pH was adjusted to 3-4 with hydrochloric acid and extracted twice with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of ethyl acetate to petroleum ether as the eluent to obtain 280 mg of the product, with a yield of 65%. [M+1] + =601.2.

[0146] (6) Synthesis of Compound 37

[0147] Under a nitrogen atmosphere, compound 36 (280 mg, 0.2 mmol), triphenylphosphine (366 mg, 0.6 mmol), and diisopropyl azodicarboxylate (280 mg, 0.6 mmol) were dissolved in 5 ml of dry tetrahydrofuran and stirred at room temperature overnight. After completion of the reaction, as monitored by LCMS, the reaction solution was concentrated and the crude product was purified by column chromatography using ethyl acetate:petroleum ether (1:4) as the eluent to obtain 240 mg of the product, a 90% yield. [M+1] + =583.2.

[0148] (7) Synthesis of Compound 38

[0149] Under a nitrogen atmosphere, compound 37 (240 mg, 0.16 mmol) and cerium ammonium nitrate (670 mg, 0.48 mmol) were dissolved in 3 ml of acetonitrile and 1 ml of purified water, heated to 60°C, and stirred for 6 h. Upon completion of the reaction, the temperature was lowered, and the reaction mixture was added to water. The mixture was extracted twice with ethyl acetate. The combined organic phases were washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 1:1 ratio of ethyl acetate to petroleum ether as the eluent to afford 65 mg of the product, a 33% yield. [M+1] + =463.2.

[0150] (8) Synthesis of Compound I-4-1

[0151] Compound 38 (65 mg, 0.05 mmol) and 65 mg of 10% palladium on carbon (55% aqueous solution) were suspended in 5 ml of methanol and 0.5 ml of glacial acetic acid. The atmosphere was replaced with hydrogen three times and stirred at room temperature for 4 days. LCMS indicated that the reaction was nearly complete. The mixture was filtered, the filter cake washed with 50% aqueous methanol, and concentrated. The crude product was purified by column chromatography using a 20:1 ratio of dichloromethane to methanol as the eluent to afford 8 mg of a white solid (20% yield). [M+1] +=283.1.1H NMR (400MHz, DMSO-d6) δ11.39(s,1H),7.45(s,1H),5.84(d,J=3.4Hz,1H),4.83(s,1H),4.78(t,J=5.5Hz,1H),4.41–4.36(m,1H),4.36–4.33 (m,1H),3.70(dd,J=11.6,5.3Hz,1H),3.62(dd,J=11.6,5.5Hz,1H),2.22(dd,J=14.0,5.5Hz,1H),2.03(dd,J=14.0,9.5Hz,1H),1.81(s,3H).

[0152] Example 14: Synthesis of Compound I-3-1

[0153] (1) Synthesis of compound 39

[0154] Under a nitrogen atmosphere, compound 34 (2.11 g, 4.27 mmol) and 4-dimethylaminopyridine (2.08 g, 17.07 mmol) were dissolved in 24 ml of dry dichloromethane and 6 ml of pyridine. Trifluoromethanesulfonic anhydride (2.41 g, 8.54 mmol) was slowly added dropwise to the mixture at 0°C. Stirring was continued for 3 h after the addition was complete. After completion of the reaction, the organic phase was washed sequentially with 10% citric acid aqueous solution and saturated brine, dried, and concentrated. The crude product was purified by column chromatography using ethyl acetate:petroleum ether in a ratio of 1:3 to 1:1 as eluent to afford 1.62 g of a foamy solid in a 61% yield. [M+1] + =627.2.

[0155] (2) Synthesis of Compound 40

[0156] Under a nitrogen atmosphere, compound 39 (870 mg, 1.39 mmol) and trimethylsilyl azide (480 mg, 4.17 mmol) were dissolved in 8 ml of dry tetrahydrofuran. 4.2 ml of a 1M tetrabutylammonium fluoride solution in tetrahydrofuran was added dropwise, and the mixture was stirred overnight at room temperature. After completion of the reaction, the mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography using ethyl acetate:petroleum ether in a ratio of 1:3 to 1:1 as eluent to yield 630 mg. [M+1] +=520.2.1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),7.46–7.30(m,10H),7.03(s,1H),5.02(q,J=10.1Hz ,1H),4.83(d,J=10.8Hz,1H),4.64(d,J=11.7Hz,1H),4.59(d,J=10.9Hz,1H),4.53(d,J=11.8Hz ,1H),4.20(dd,J=10.7,4.6Hz,1H),4.09(d,J=4.7Hz,1H),3.85(d,J=8.7Hz,1H),3.71(d,J=8.7 Hz, 1H), 3.62 (s, 3H), 3.02 (dd, J=14.0, 10.0Hz, 1H), 2.11 (dd, J=13.9, 9.7Hz, 1H), 1.75 (s, 3H).

[0157] (3) Synthesis of compound 41

[0158] Under a nitrogen atmosphere, compound 40 (1.0 g, 1.92 mmol) and triphenylphosphine (0.6 g, 2.3 mmol) were dissolved in 6 ml of tetrahydrofuran and 2 ml of purified water. The mixture was stirred at room temperature for 2 hours and then at 50°C for another 6 hours. After completion of the reaction, the mixture was concentrated and the crude product was purified by column chromatography using a dichloromethane:methanol ratio of 50:1 to 10:1 as the eluent to obtain 600 mg of the product. [M+1] + =494.2.

[0159] (4) Synthesis of Compound 42

[0160] Under a nitrogen atmosphere, compound 41 (180 mg, 0.35 mmol) and sodium methoxide (95 mg, 1.7 mmol) were dissolved in 10 ml of anhydrous ethanol and stirred at reflux for 10 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature, the pH was adjusted to <7 with hydrochloric acid, and the mixture was concentrated. The crude product was purified by column chromatography using a dichloromethane:methanol ratio of 100:1 to 20:1 as the eluent to obtain 80 mg. [M+1] +=462.2;1H NMR (400MHz, DMSO-d6) δ11.33(s,1H),8.13(s,1H),7.43–7.26(m,10H),7.22(s,1H),4.69–4.48(m,4H),4.21(s,1H),4.18(dd,J= 8.9, 4.5Hz, 1H), 3.97 (s, 1H), 3.64 (d, J = 10.2Hz, 1H), 3.57 (d, J = 10.2Hz, 1H), 2.05–1.99 (m, 1H), 1.96–1.88 (m, 1H), 1.75 (s, 3H).

[0161] (5) Synthesis of Compound I-3-1

[0162] Compound 42 (25 mg, 0.054 mmol) and 25 mg of 10% palladium on carbon (containing 55% water) were suspended in 5 mL of methanol and 1 mL of acetic acid. The atmosphere was replaced with hydrogen three times and stirred overnight at room temperature. The reaction was monitored by LCMS. The mixture was filtered, the filter cake washed with 50% aqueous methanol, and concentrated. The crude product was purified by column chromatography using a 5:1 ratio of dichloromethane to methanol as the eluent to afford 8 mg of a white solid in a 60% yield. [M+1] + =282.1;1H NMR (400MHz, DMSO-d6) δ11.33(s,1H),7.96(s,1H),7.50(d,J=1.4Hz,1H),5.35( d, J=3.8Hz, 1H), 4.51 (t, J=5.5Hz, 1H), 4.13 (dd, J=9.2, 4.3Hz, 1H), 4.07 (dd, J= 3.5,1.8Hz,1H),3.79(d,J=2.1Hz,1H),3.63(dd,J=11.6,5.5Hz,1H),3.55(dd,J =11.6,5.6Hz,1H),1.96(dd,J=14.0,4.4Hz,1H),1.82(s,3H),1.81–1.75(m,1H).

[0163] Example 15 Synthesis and purification of oligonucleic acid analogs

[0164] An oligonucleotide automatic synthesizer was used to perform multi-step solid-phase synthesis of an oligonucleotide analog containing the 10mer obtained in the above-mentioned compound I-1-5 using a solid phase support and a phosphoramidite nucleoside monomer at a 0.2 μmol level, including deprotection, coupling, capping, oxidation, and aminolysis, as shown in Table 1, where X is represented in the sequence.

[0165] The crude oligonucleic acid analog was purified by ion-pair reversed-phase liquid chromatography using a NanoQ-15L 9.5 ml (8 x 190 mm) ion exchange column at a flow rate of 2 ml / min. Mobile phase A consisted of 10 mM sodium hydroxide solution and mobile phase B consisted of 10 mM sodium hydroxide and 2.0 M sodium chloride solution. Gradient elution was performed to obtain oligonucleic acids with the required purity.

[0166] Table 1

[0167] Example 16 Melting Temperature (Tm) Test

[0168] After annealing treatment of the antisense strand 1, antisense strand 2, control antisense strand, and sense strand (5'-AAAAAAAAAA-3') synthesized in Example 15, the hybridization strand formation ability of the antisense strand was tested by measuring Tm.

[0169] 1 mL (0.5 OD / mL) of the sense strand and 1 mL (0.5 OD / mL) of the antisense strand were mixed and stirred evenly, heated at 90°C for 10 minutes, and then cooled to room temperature. The Tm value of the sample was then determined using a UV spectrophotometer (Agilent, Cary 3500 UV-Vis). The sample was placed in a cuvette with a lid and a temperature probe. The temperature was set to increase from 20-25°C to 70-80°C at a rate of 0.5°C or 1.0°C per minute. The UV absorbance at 260 nm was measured at 0.5°C intervals.

[0170] Table 2

[0171] The results in Table 2 show that compared with natural oligonucleotides, the oligonucleotides containing the carbocyclic nucleoside compound X of the present invention have higher Tm values ​​relative to single-stranded oligoRNA, indicating high binding affinity.

[0172] Example 17 Evaluation of nuclease resistance

[0173] The resistance of each oligonucleotide to exonucleases that degrade from the 3' side was examined for the antisense strand 1 and antisense strand 2 synthesized in Example 15. An oligonucleotide (5'-TTTTTTTTTT-3') was used as a control.

[0174] A buffer solution (65 μL) containing 750 pmol of oligonucleotide was kept at 37°C for 5 minutes, and then mixed with a buffer solution (35 μL) containing 5 μg / mL snake venom phosphodiesterase (Crotalus admanteus venom phosphodiesterase (CAVP): Merck KGaA, Darmstadt, Germany). The results were analyzed by HPLC (Thermo UltiMate 3000, Waters XBridge analytical column). TM Oligonucleotide degradation was measured over time using a Shield RP18 3.5 μm (4.6 mm × 150 mm). The buffer used (final concentration) was 50 mM TrisHCl (pH 8.0), 10 mM MgCl2, and was thoroughly degassed before measurement. HPLC quantification conditions are shown below.

[0175] [HPLC quantitative conditions]

[0176] Mobile phase: Liquid A 0.1M triethylammonium acetate buffer, pH 7.0

[0177] Solution B: 0.1M triethylammonium acetate buffer: acetonitrile = 1:1 (v / v), pH 7.0

[0178] Gradient: 15→40% (v / v) Solution B (7 minutes)

[0179] Column used: Waters XBridge TM Shield RP18 3.5μm(4.6mm×150mm)

[0180] Flow rate: 1.0 mL / min

[0181] Column temperature: 50°C

[0182] Detection: UV (268nm)

[0183] The results are shown in Figure 1. In Figure 1, "Remaining oligonucleotide (%)" indicates the remaining ratio of undegraded oligonucleotide (10mer) at the time of measurement relative to the undegraded oligonucleotide (10mer) at time 0.

[0184] The results in Figure 1 show that even after 40 minutes of nuclease treatment, oligonucleotides containing the carbocyclic nucleoside compound X of the present invention at the 3' end still showed over 70% unreacted residues, indicating resistance to degradation. Oligonucleotides containing compound X at the third position on the 3' end also showed over 10% unreacted residues after 5 minutes of nuclease treatment. In contrast, oligonucleotides containing all-natural thymidine were almost completely degraded after 5 minutes of nuclease treatment. Therefore, oligonucleotides containing the carbocyclic nucleoside compound X of the present invention exhibited superior enzyme resistance to oligonucleotides containing all-natural thymidine.

[0185] Industrial applicability

[0186] According to the present invention, a conformationally locked carbocyclic nucleoside and an oligonucleotide are provided. The oligonucleotide comprising the conformationally locked carbocyclic nucleoside has high binding affinity and nuclease resistance, and is expected to be used in nucleic acid drugs.

Claims

1. A compound represented by the following formula (I) or a salt thereof: In the formula, R1 and R2 are independently hydrogen, an alkyl group containing 1 to 6 carbon atoms, a phenyl group, 1-3 )alkyl-phenyl, heterocyclic or (C 1-3 ) alkyl-heterocycle, a protecting group for a hydroxyl group, a silicon group containing one or more substituents, a phosphate group containing a protecting group for oligonucleotide synthesis, phosphate esters, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters and alkyl phosphates; A is O, S or NR3; R3 is a hydrogen atom, an alkyl group containing 1 to 6 carbon atoms, a haloalkyl group containing 1 to 6 carbon atoms, -C(O)R4, -C(O)OR4 or -C(O)N(R4)2; R4 is H or an alkyl group containing 1 to 6 carbon atoms or a haloalkyl group containing 1 to 6 carbon atoms; Y is C=O, CHR5 or CHOR5; R5 is H or an alkyl group containing 1 to 6 carbon atoms or a halogenated alkyl group containing 1 to 6 carbon atoms; Base is the base.

2. The compound or salt thereof according to claim 1, characterized in that The formula (I) is represented by any one of the following formulas (I-1) to (I-4):

3. The compound or salt thereof according to claim 1, characterized in that Base is selected from any of the following groups:

4. The compound or salt thereof according to claim 1, characterized in that The alkyl group containing 1 to 6 carbon atoms is a straight chain alkyl group, a branched chain alkyl group, a cycloalkyl group or (C 1-4 )alkyl-(C 3-7 )cycloalkyl.

5. The compound or salt thereof according to claim 1, characterized in that The compound of formula (I) has any structure shown below:

6. An oligonucleotide or a pharmaceutically acceptable salt thereof, characterized in that: It contains at least one structure represented by the following formula (II) to (IV): Wherein, Z is phosphate, phosphorothioate, chiral phosphorothioate, phosphate triester, aminoalkyl phosphate triester and alkyl phosphate.

7. A method for preparing the oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in claim 6, characterized in that: The method comprises the step of synthesizing an oligonucleotide using the compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

8. A method for preparing the oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 6-7, characterized in that: The method comprises the step of synthesizing an oligonucleotide using at least one compound as claimed in claim 5 or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising a therapeutically effective amount of one or more compounds or salts according to any one of claims 1 to 5, and a pharmaceutically acceptable excipient.

10. Use of the compound and salt according to any one of claims 1 to 5 in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

11. A pharmaceutical composition comprising a therapeutically effective amount of one or more oligonucleotides according to any one of claims 6 to 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

12. Use of the oligonucleotide according to any one of claims 6 to 8 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

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