Carbocyclic nucleoside, oligonucleotide and preparation method and medical application thereof

Abstract

The invention discloses carbocyclic nucleoside, oligonucleotide and a preparation method and medical application of the carbocyclic nucleoside and oligonucleotide. The carbocyclic nucleoside is a compound or salt as shown in a formula (I). The carbocyclic nucleoside or oligonucleotide of the present invention has the use in the preparation of drugs for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer. The oligonucleotide provided by the invention has relatively high binding affinity, nuclease resistance and plasma stability to single-stranded RNA. # imgabs0 #

Description

[0001] This application claims the priority of Chinese patent application CN202310902267.9, filed on July 21, 2023. This application cites the full text of the above Chinese patent application. Technical Field

[0002] The present invention relates to a nucleoside and a nucleotide and a preparation method and medical use thereof, and in particular to a carbocyclic nucleoside, an oligonucleotide and a preparation method and medical use thereof. Background Art

[0003] Nucleic acid is one of the basic substances of life, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleotides are monomers that make up nucleic acid molecules. They are a class of compounds composed of nitrogenous bases, pentoses and phosphates, and are phosphate esters of nucleosides. Nucleic acid therapy is a class of drugs or vaccines composed of modified single-stranded or double-stranded nucleotides applied to the human body. Its basic composition is a nucleotide sequence, which acts on mRNA through base complementary pairing, inhibits the expression of target proteins through gene silencing, and achieves the effect of disease treatment and prevention. Nucleic acid-based treatment models include antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), nucleic acid aptamers (Aptamer), messenger RNA (mRNA), ribozymes (Ribozymes), antibody nucleic acid conjugates (ARC), plasmid DNA (Plasmid DNA), CRISPR / Cas9, etc. Short-chain nucleotides containing about 20 bases are called oligonucleotides, mainly including ASO, siRNA, Aptamer, miRNA, saRNA, etc. ASO is a synthetic single-stranded oligonucleotide, usually composed of 12 to 30 nucleotides. It binds to the target mRNA or pre-mRNA through the Watson-Crick base complementary pairing principle, induces RNase H nuclease to degrade the RNA chain, or affects the processing and splicing of pre-mRNA in a steric manner to regulate gene expression. Fomivirsen is the first commercialized ASO drug, which was approved by the FDA in 1998. Currently, there are 10 ASO drugs approved for marketing worldwide. siRNA is a double-stranded oligonucleotide containing a sense strand and an antisense strand. It binds to the RNA-induced silencing complex (RISC) in the cytoplasm. Under the action of argouate 2 protein, the double strand depolymerizes, the sense strand cleaves and leaves, and the antisense strand in RISC binds to mRNA, 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 have been approved for marketing. Nucleic acid aptamers are single-stranded nucleic acids, usually 25 to 50 bases long, with a unique three-dimensional conformation. They can target biological components such as proteins on the surface or inside diseased tissue cells, and block proteins from performing their corresponding functions through steric hindrance. They can be used as antibody analogs. Pegaptanib is a nucleic acid aptamer drug approved by the FDA in 2006. Currently, there are several such drugs in the clinical research stage. miRNA is a non-coding RNA with a length of 19 to 24 nucleotides. After binding to mRNA in cells, it regulates post-transcription, inhibits protein translation, and regulates target gene expression.saRNA is a short double-stranded oligonucleotide with a structure similar to siRNA. It can activate gene transcription and exert a therapeutic effect by recruiting endogenous transcription complexes to target genes, increasing mRNA expression, and upregulating target proteins.

[0004] As early as half a century ago, synthetic oligonucleotides were used to regulate targeted genes, but unmodified oligonucleotides have poor stability, are easily degraded by endogenous nucleases in the blood, are quickly cleared by the liver and kidneys, have short half-lives, and low bioavailability, making them difficult to make into drugs. Chemical modification can improve the resistance of oligonucleotides to enzymatic hydrolysis and increase their affinity for targets, and the first oligonucleotide drug was launched in 1998. Chemical modification of nucleic acids mainly includes phosphate backbone modification, base modification, and ribose modification. The most typical backbone modification is thiophosphorylation, in which a non-bridging oxygen atom of the phosphate group in the nucleotide chain is replaced by a sulfur atom, and the ability to resist nuclease degradation is greatly increased after modification. The replacement of the 3' oxygen atom on the ribose ring with the 3' amine is another backbone modification. Thiocarbamates and phosphoramidite salts show high affinity and resistance to nucleases. Base modification usually introduces carboxyl, amino and other groups at the 5' position of pyrimidine bases and the 8' position of purine bases. Ribose modification mainly occurs at the 2' position, including 2'-O-methyl, 2'-O-methoxyethyl, 2'-F and other modifications, which can produce different degrees of affinity and nuclease resistance. Linking ribose 4' and 2' together to form double-ring systems such as locked nucleic acid (LNA) and bridged nucleic acid (BNA) can significantly help improve affinity. Drug researchers improve drugability through various modifications, allowing new nucleic acid drugs to be continuously developed and marketed, but there is no ideal molecule. There is room for improvement in the affinity, nuclease resistance, and in vivo stability of existing nucleic acid drugs. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a carbocyclic nucleoside and an oligonucleotide having high binding affinity to single-stranded nucleic acid (RNA or DNA), nuclease resistance and plasma stability; another purpose of the present invention is to provide an oligonucleotide and a method for preparing the same; another purpose of the present invention is to provide a pharmaceutical composition; another purpose 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: A compound or salt of the present invention is represented by the following formula (I):

[0007]

[0008] In the formula,

[0009] 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;

[0010] 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;

[0011] 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;

[0012] Base is the base.

[0013] 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):

[0014]

[0015] Preferably, Base is selected from any of the following groups:

[0016]

[0017] 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 (C 1-4 )alkyl-(C 3-7 )cycloalkyl.

[0018] Preferably, the compound of formula (I) has any structure shown below:

[0019]

[0020] In another aspect, the present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof, which contains at least one structure represented by the following formula (II) to (IV):

[0021]

[0022] Wherein, Z is phosphate, phosphorothioate, chiral phosphorothioate, phosphate triester, aminoalkyl phosphate triester and alkyl phosphate.

[0023] In the above scheme, the oligonucleotide is a linear polymer compound formed by covalently linking adjacent nucleotides through phosphate groups. Alternatively, the oligonucleotide is linked to DNA or RNA through phosphate, phosphorothioate, chiral phosphorothioate, phosphotriester, aminoalkylphosphotriester and alkyl phosphate to form a spacer polymer.

[0024] As described herein, the oligonucleotide preferably contains about 12 to 50 nucleotides, preferably a backbone containing a phosphorus atom, such as phosphate, phosphorothioate, chiral phosphorothioate, phosphotriester, aminoalkylphosphotriester or alkylphosphate.

[0025] In the above oligonucleotide structure, the conformationally locked carbocyclic nucleosides can be connected to DNA through phosphates, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters and alkyl phosphates to form polymers, or the conformationally locked carbocyclic nucleosides can be connected to RNA through phosphates, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters and alkyl phosphates to form polymers, or the conformationally locked carbocyclic nucleosides can be connected to DNA and RNA through phosphates, phosphorothioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters and alkyl phosphates to form spacer polymers.

[0026] In the above scheme, a conformationally locked carbocyclic nucleoside for nucleic acid modification is provided. Oligonucleotides are synthetic nucleotide polymers with a single-stranded or double-stranded structure. Oligonucleotide therapy includes: antisense oligonucleotides designed by 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, etc. The advantages of oligonucleotide therapy include simple design of drug molecules based on known genomic information, high target specificity and less off-target toxicity.

[0027] 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 to the target gene, but a small polar surface, which can improve blood pharmacokinetic properties.

[0028] 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.

[0029] On the other hand, 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,

[0030]

[0031] 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:

[0032]

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the above compounds or salts, and a pharmaceutically acceptable excipient.

[0034] In another aspect, the present invention provides a use of the above-mentioned compound and salt in the preparation of a drug for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

[0035] 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.

[0036] 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.

[0037] 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 a higher 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

[0038] Figure 1 This is a statistical chart of the results of the anti-nuclease degradation experiment. DETAILED DESCRIPTION

[0039] First, the terms used in this specification are defined.

[0040] 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-(C 3-7 The term "cycloalkyl" refers to a hydrocarbon containing 4 to 11 carbon atoms in which a straight or branched alkyl group containing 1 to 4 carbon atoms is connected to a saturated or partially saturated monocyclic ring containing 3 to 7 carbon atoms.

[0041] 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 chain alkyl group containing 1 to 3 carbon atoms is attached to a phenyl group.

[0042] 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 or branched chain alkyl group containing 1 to 3 carbon atoms is attached to a heterocycle.

[0043] In the present specification, the term "haloalkyl group" refers to an alkyl group in which a hydrogen atom is substituted by a halogen atom (eg, F, Cl, Br, and I).

[0044] In the present 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 salts, potassium salts, and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; inorganic salts such as ammonium salts, tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzylphenethylamine salts, piperazine salts, 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, hydroiodide, nitrate, perchlorate, sulfate, phosphate, etc.; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, aryl sulfonates such as benzenesulfonate, p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc.; and amino acid salts such as glycine, lysine, arginine, ornithine, glutamate, aspartate.

[0045] In this specification, the term "pharmaceutically acceptable salt" means a salt or zwitterion of a compound disclosed herein, which is water-soluble or oil-soluble or dispersible and pharmaceutically acceptable. The salt 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 compounds with alkali metals or alkaline earth ions (e.g., sodium), alkaline earths (e.g., magnesium and calcium), ammonium and NX4+ (wherein X is a C1-C4 alkyl). For therapeutic use, the active ingredient compound salts of the present invention are generally physiologically acceptable, i.e., they are derived from physiologically acceptable acids or bases. 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 physiologically acceptable acids or bases, are within the scope of the present invention.

[0046] In this specification, the term "pharmaceutically acceptable excipient" refers to an additive in a pharmaceutical preparation other than the main drug, a carrier that is not accompanied by significant side effects when administered, including but not limited to solvents, cosolvents, preservatives, antioxidants, solubilizers, emulsifiers, osmotic pressure regulators in liquid preparations, and binders, fillers, disintegrants, and lubricants in solid preparations.

[0047] 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 1 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 that specifically acts on target nucleic acids in a manner of complementary base pairing 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 of 21 to 25 nucleotides in length, including a sense strand and an antisense strand. 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 transcription and inhibits protein translation to regulate target gene expression. saRNA is a short double-stranded oligonucleotide with a structure similar to siRNA. It recruits endogenous transcription complexes to target genes, increases mRNA expression, and upregulates target proteins to exert therapeutic effects. 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.

[0048] When the nucleic acid containing the conformationally locked carbon ring of the present invention forms a double-stranded chain with a complementary nucleic acid, because the ribose analog containing the carbon ring is partially locked, it is easy to form a stable double-stranded chain 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.

[0049] The conformationally locked carbocyclic nucleic acid of the present invention is preferably administered to a living body in the form of a pharmaceutical preparation, and can be prepared into an injection preparation together with preservatives, antioxidants, excipients and other auxiliary materials used in the field of pharmaceutical preparation technology. In addition, it can also be formulated into liquid preparations, solid preparations, ointments and other preparations with pharmaceutical excipients used in this technical field, and administered through the gastrointestinal tract or skin.

[0050] The conformationally locked carbocyclic nucleic acid of the present invention can be expected to be used for the treatment or prevention of certain diseases by inhibiting the expression of specific genes, such as antiviral and antitumor effects.

[0051] The compound of the present invention is a compound or salt represented by the following formula (I):

[0052]

[0053] In the formula,

[0054] 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;

[0055] 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;

[0056] 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;

[0057] Base is the base.

[0058] Preferably, the formula (I) is represented by any one of the following formulas (I-1) to (I-4):

[0059]

[0060] Preferably, the compound of formula (I) has any structure shown below:

[0061]

[0062] In another aspect, the present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof, comprising at least one structure represented by the following formula (II) to (IV),

[0063]

[0064] Wherein, Z is phosphate, phosphorothioate, chiral phosphorothioate, phosphate triester, aminoalkyl phosphate triester and alkyl phosphate.

[0065] Example

[0066] 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.

[0067] Example 1: Synthesis of Compound I-1-1

[0068]

[0069] (1) Synthesis of Compound 5

[0070]

[0071] Under nitrogen atmosphere, compound 4 (575 g, 2 mol) (synthesis reference: Journal of Medicinal Chemistry, 2014, vol. 57, #5, p. 2107-2120) was dissolved in 10 L of dichloromethane, cooled to -20 to -25 °C, and 5 L of 0.4 M sodium methoxide methanol solution was slowly added dropwise. After the addition was complete, stirring was continued for 30 min. After the reaction was completed by thin layer chromatography, 4 M HCl 1,4-dioxane solution was added dropwise to quench, and the pH was adjusted to <7. Filter, concentrate, and purify the crude product by column chromatography. The eluent was petroleum ether: ethyl acetate = 5:1 to 3:1, and 228 g of oil was obtained, with a yield of 80%. [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).

[0072] (2) Synthesis of Compound 6

[0073]

[0074] Under nitrogen atmosphere, compound 5 (50g, 0.352mol) was dissolved in 500ml dry tetrahydrofuran and 170ml hexamethylphosphoric acid triamide, cooled to -78°C, and 530ml 2M tetrahydrofuran solution of lithium diisopropylamide was slowly added dropwise. After the addition was complete, the temperature was raised to -20°C and stirring was continued for 30-45min. Then, the temperature was further lowered to -78°C and the dry tetrahydrofuran solution of benzyl chloromethyl ether (66g, 0.422mol) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to -40°C and the reaction was carried out for 2h. After the reaction was completed by gas chromatography, acetic acid was added dropwise to quench, the temperature was raised to room temperature, a saturated ammonium chloride solution was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography with petroleum ether:ethyl acetate = 5:1-4:1 as the eluent to obtain 63 g of an oily substance with a yield of 68%.

[0075] [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).

[0076] (3) Synthesis of Compound 7

[0077]

[0078] Under nitrogen atmosphere, compound 6 (63g, 0.24mol) was dissolved in 600ml dichloromethane, cooled to 0°C, and 85% m-chloroperbenzoic acid (121.8g, 0.6mol) was added in batches, and stirred at room temperature overnight. After the reaction was completed by LCMS detection, saturated sodium sulfite solution was added to quench, and the organic phase was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was petroleum ether: ethyl acetate = 5:1 to 4:1, to obtain 45g of oil, with a yield of 67%. [M+1] + =288.1.

[0079] (4) Synthesis of Compound 8

[0080]

[0081] Under nitrogen atmosphere, compound 7 (35.2 g, 0.126 mol) was dissolved in 250 ml of dry N, N-dimethylformamide, benzyl bromide (32.3 g, 0.189 mol) was added, the temperature was lowered to -40 to -50 °C, 189 ml of 1M tetrahydrofuran solution of potassium bis(trimethylsilyl)amide was slowly added dropwise, and the reaction was continued for 1 hour after the addition was completed. After the reaction was completed by LCMS detection, acetic acid was added dropwise to quench, and saturated ammonium chloride solution was added after the temperature was raised to room temperature, and the mixture was extracted twice with ethyl acetate, the organic phases were combined, washed twice with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was petroleum ether:ethyl acetate.

[0082] =5:1~4:1, 40g of oily substance was obtained, with a yield of 84%. [M+1] + =369.2.

[0083] (5) Synthesis of Compound 9

[0084]

[0085] Under nitrogen atmosphere, compound 8 (39 g, 0.106 mol) was dissolved in 1.2 L dry dichloromethane, cooled to -78 ° C, and 254 ml 1M diisobutylaluminum hydride n-hexane solution was slowly added dropwise. After the addition was completed, the reaction was continued at -78 ° C for 1 h. LCMS detected that the reaction was complete, acetic acid was added dropwise to quench, and the temperature was raised to room temperature. The organic phase was washed with saturated sodium bicarbonate aqueous solution and saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was petroleum ether: ethyl acetate = 4:1, to obtain 28 g of 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).

[0086] (6) Synthesis of Compound 10

[0087]

[0088] Under nitrogen atmosphere, compound 9 (22 g, 0.065 mol), thymine (32.8 g,

[0089] 0.26mol), potassium carbonate (26.9g, 0.195mol) were suspended in 220ml dry dimethyl sulfoxide, heated to 140℃ and stirred for 3h. After the reaction was completed by LCMS, the temperature was lowered to room temperature, 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, and the eluent was dichloromethane: methanol = 50:1-20:1, to obtain 24.4g of white solid, with a yield of 81%. [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).

[0090] (7) Synthesis of Compound 11

[0091]

[0092] Under 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 tetrahydrofuran solution of methylsulfonic anhydride (7.7 g, 0.044 mol) was slowly added dropwise. After the addition was complete, the reaction was continued at -10 ° C for 30 min. After the reaction was completed by LCMS detection, a saturated sodium bicarbonate aqueous solution was added, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was dichloromethane: methanol = 100: 1, to obtain 14.5 g of a white foamy solid with a yield of 72.5%. [M+1] + =545.2.

[0093] (8) Synthesis of Compound 12

[0094]

[0095] Under nitrogen atmosphere, carefully add 60% sodium hydride (3.2g, 0.081mol) to 100ml dry N,N-dimethylformamide, cool to 0°C, slowly drop compound 11 (14g, 0.027mol) in N,N-dimethylformamide solution, and continue to react for 1h. LCMS detection reaction is complete, add saturated ammonium chloride solution, extract twice with ethyl acetate, combine organic phases, wash twice with saturated sodium chloride, dry over anhydrous sodium sulfate, filter, and concentrate. The crude product is purified by column chromatography, the eluent is dichloromethane: methanol = 40:1, and 8.5g of oily liquid is obtained, with a yield of 74%. [M+1] + =449.2.

[0096] (9) Synthesis of Compound I-1-1

[0097]

[0098] 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 carbon (60% water content) were added, hydrogen was replaced three times, and stirred at room temperature overnight. LCMS monitored the completion of the reaction, filtered, and concentrated to obtain 2.2 g of a white solid, with a yield of 90%. [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).

[0099] Example 2: Synthesis of Compound I-1-2

[0100]

[0101] (1) Synthesis of Compound 15

[0102]

[0103] Under nitrogen atmosphere, compound 12 (8.5 g, 0.019 mol), 4-dimethylaminopyridine (4.6 g, 0.038 mol), 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) was carefully added, and stirred at room temperature for 2 h. The temperature was lowered to 0 ° C, 28.5 ml of concentrated ammonia water was added dropwise, and after the addition was complete, it was stirred at room temperature overnight. LCMS detected that the reaction was complete, water was added, and ethyl acetate was used to extract twice. The organic phases were combined, washed with saturated ammonium chloride solution and saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was dichloromethane: methanol = 30:1, and 6 g of oily liquid was obtained, with a yield of 70.7%. [M+1] + =448.2.

[0104] (2) Synthesis of Compound 16

[0105]

[0106] Under 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, and stirred at room temperature overnight. After the reaction was completed by LCMS monitoring, a saturated sodium bicarbonate solution 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, and the eluent was petroleum ether: ethyl acetate = 3:1, to obtain 3.8 g of an oily liquid with a yield of 68.9%.

[0107] [M+1] + =552.2.

[0108] (3) Synthesis of Compound I-1-2

[0109]

[0110] Under 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 carbon (60% water content) were added, and stirred at 60°C for 6 h. After LCMS monitoring, the reaction was completed, filtered, washed, and concentrated to obtain 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).

[0111] Example 3: Synthesis of Compound I-1-3

[0112]

[0113] (1) Synthesis of Compound 19

[0114]

[0115] Under nitrogen atmosphere, compound 9 (24 g, 0.07 mol), cesium fluoride (31.9 g, 0.21 mol), O-6-benzylguanine (33.8 g, 0.14 mol) were suspended in 240 ml of dry DMF, heated to 90 ° C for 3 h, and the reaction was completed after LCMS monitoring. The temperature was lowered, 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, and the eluent was petroleum ether: ethyl acetate = 1:2 to 1:3, to obtain 23.3 g of 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).

[0116] (2) Synthesis of Compound 20

[0117]

[0118] Under 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 mixture was cooled 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 ammonia was added dropwise, and the mixture was stirred at room temperature for 1 h. After the reaction was completed by LCMS monitoring, 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, and the eluent was petroleum ether:ethyl acetate.

[0119] =1:1~1:2, 26.3g of oily liquid was obtained, with a yield of 97%. [M+1] + =652.3.

[0120] (3) Synthesis of Compound 21

[0121]

[0122] Under nitrogen atmosphere, compound 20 (25.5 g, 0.039 mol) was dissolved in 200 ml dry pyridine, cooled to 0 ° C, and a tetrahydrofuran solution of methanesulfonic anhydride (8.7 g, 0.051 mol) was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. LCMS monitored the reaction until the product content was maximum, water was added to quench, ethyl acetate was extracted twice, 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, the eluent was petroleum ether: ethyl acetate = 1:1, and 19.1 g of oil was obtained, with a yield of 56.8%. 4.5 g of raw material was recovered. [M+1] + =730.3.

[0123] (4) Synthesis of Compound 22

[0124]

[0125] Under 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 tetrahydrofuran solution of compound 21 (21.4 g, 29.3 mmol) was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. LCMS monitored the completion of the reaction, quenched with acetic acid, added saturated sodium bicarbonate solution, extracted twice with ethyl acetate, combined the organic phases, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, the eluent was petroleum ether: ethyl acetate = 1:1, and 15.8 g of oil was obtained, with a yield of 85%. [M+1] + =634.3.

[0126] (5) Synthesis of Compound I-1-3

[0127]

[0128] Under nitrogen atmosphere, compound 22 (5 g, 7.9 mmol) was dissolved in 50 ml of dichloromethane, cooled to -78 °C, and 71 ml of 1M dichloromethane solution of boron tribromide was slowly added dropwise. After the addition was complete, the temperature was slowly raised to -40 °C and stirred for 2 h. After the reaction was completed by LCMS monitoring, methanol was added dropwise to quench, the temperature was raised to room temperature, and the mixture was concentrated. The crude product was purified by column chromatography with the eluent being dichloromethane:methanol=10:1, to obtain 2.5 g of a white solid with a yield of 87.2%. [M+1] + =364.1; 1H NMR (400MHz, DMSO-d6) δ8.14 (s, 1H), 7.36-

[0129] 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,1H),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).

[0130] Example 4: Synthesis of Compound I-1-4

[0131]

[0132] (1) Synthesis of Compound 25

[0133]

[0134] Under nitrogen atmosphere, compound 9 (26 g, 0.076 mol), adenine (20.5 g, 0.15 mol), potassium carbonate (31.5 g, 0.23) were suspended in 260 ml of dry dimethyl sulfoxide, and the temperature was raised to 120 ° C for 4 h. After the reaction was completed, the temperature was lowered, water was added, and ethyl acetate was extracted twice. 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, and the eluent was dichloromethane: methanol = 20:1, to obtain 29.2 g of white solid, with a yield of 80.4%.

[0135] [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).

[0136] (2) Synthesis of Compound 26

[0137]

[0138] Under nitrogen atmosphere, compound 25 (22g, 0.046mol) was dissolved in 200ml dry pyridine, cooled to 0℃, and trimethylsilyl chloride (25g, 0.231mol) was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 2h. After the temperature was cooled to 0℃, benzoyl chloride (13.5g, 0.097mol) was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 2h. After the reaction was completed by LCMS monitoring, the mixture was cooled to 0℃, 35ml of concentrated ammonia water was added, the mixture was slowly heated to room temperature, and stirring was continued for 1h. After the reaction was completed by LCMS monitoring, 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 with 100% ethyl acetate as the eluent to obtain 20g with a yield of 73.7%.

[0139] [M+1] + =560.3.

[0140] (3) Synthesis of Compound 27

[0141]

[0142] Under nitrogen atmosphere, compound 26 (20 g, 0.034 mol), methanesulfonic anhydride (7.2 g,

[0143] 0.041mol) was dissolved in 200ml of dry tetrahydrofuran, cooled to -10°C, triethylamine (10.4g, 0.103mol) was slowly added dropwise, and the temperature was raised to 0°C for reaction for 2h after the addition was complete. LCMS monitored that the raw material was basically reacted completely, saturated sodium bicarbonate aqueous solution was added, ethyl acetate was extracted twice, the organic phases were combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, the eluent was petroleum ether: ethyl acetate = 1:1, and 17g of white solid was obtained, with a yield of 65%. [M+1] + =658.2.

[0144] (4) Synthesis of Compound 28

[0145]

[0146] Under nitrogen atmosphere, carefully add 60% sodium hydride (1.87 g, 77.7 mmol) to 170 ml of dry N, N-dimethylformamide, stir for 10 min, cool to 0°C, slowly drop compound 27 (17 g, 25.9 mmol) in N, N-dimethylformamide, and continue stirring for 2 h. After the addition is complete, LCMS monitors the reaction, add saturated ammonium chloride solution, extract twice with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify the crude product by column chromatography with 100% ethyl acetate as the eluent to obtain 12.3 g of white solid with a yield of 85%. [M+1] + =562.2.

[0147] (5) Synthesis of Compound I-1-4

[0148]

[0149] Under 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 1M dichloromethane solution of boron trichloride was slowly added dropwise. After the addition was complete, the temperature was raised to -20°C and stirred overnight. After the reaction was completed by LCMS monitoring, methanol was added to quench, and ethyl acetate was added to slurry after concentration under reduced pressure, and filtered to obtain 3.4 g of white solid, which was the hydrochloride form 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).

[0150] Example 5: Synthesis of Compound I-1-5

[0151]

[0152] (1) Synthesis of Compound 14

[0153]

[0154] Under 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, and N,N-diisopropylethylamine (2.3 g, 18 mmol) was slowly added dropwise at 0°C. After the addition was complete, the mixture was stirred at room temperature for 2 h. LCMS monitored the completion of the reaction, and the organic phase was washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, with the eluent being dichloromethane:methanol=40:1, to obtain 2.8 g of a light yellow solid with a yield of 86%. [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).

[0155] (2) Synthesis of Compound I-1-5

[0156]

[0157] Under 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, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (904 mg, 3 mmol) was added dropwise at room temperature, and stirring was continued for 2 h. After the reaction was completed by LCMS monitoring, dichloromethane was added, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by high-pressure preparative liquid phase, the chromatographic column was C18, and the mobile phase was acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) = 8:2, and 560 mg of white solid was obtained, with a yield of 60%.

[0158] [M+1] + =771.3; 31 PNMR:148.30,148.04.

[0159] Example 6: Synthesis of Compound I-1-6

[0160]

[0161] (1) Synthesis of Compound 18

[0162]

[0163] Under 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, and N,N-diisopropylethylamine (2.0 g, 15.6 mmol) was slowly added dropwise at 0°C. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was completed, the organic phase was washed with saturated sodium bicarbonate solution, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was: petroleum ether: ethyl acetate: TEA = 3:1:0.1%, to obtain 1.4 g of white solid, with a yield of 70%. [M+1] + =674.3; 1HNMR(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-

[0164] 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).

[0165] (2) Synthesis of Compound I-1-6

[0166]

[0167] Under nitrogen atmosphere, compound 18 (500 mg, 0.74 mmol), tetrazole (154 mg,

[0168] 2.2mmol), N-methylimidazole (61mg, 0.74mmol) were dissolved in 5ml dry N,N-dimethylformamide, bis(diisopropylamino)(2-cyanoethoxy)phosphine (446mg, 1.48mmol) was added dropwise at room temperature, and stirring was continued for 2h. After LCMS monitoring, the reaction was completed, and the mixture was diluted with dry acetonitrile and separated by high pressure preparative liquid phase, with a chromatographic column of C18 and a mobile phase of 100% acetonitrile, to obtain 320mg of a white solid with a yield of 49%.

[0169] [M+1] + =874.4; 31PNMR:148.4,148.2.

[0170] Example 7: Synthesis of Compound I-1-7

[0171]

[0172] (1) Synthesis of Compound 24

[0173]

[0174] Under 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, cooled 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 heated to room temperature and stirred for 2 h. The reaction was completed after LCMS monitoring, and the mixture was concentrated. The crude product was purified by column chromatography, and the eluent was dichloromethane: methanol: triethylamine = 30: 1: 0.1%, to obtain 4.6 g of light yellow solid, with 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).

[0175] (2) Synthesis of Compound I-1-7

[0176]

[0177] Under 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, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.1 g, 3.75 mmol) was added dropwise at room temperature, and stirring was continued for 2 h. After the reaction was completed by LCMS monitoring, 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 by high pressure preparative liquid phase, the chromatographic column was C18, and the mobile phase was acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) = 7:3, and 660 mg of white solid was obtained, with a yield of 51%. [M+1] + =866.4; 31 PNMR:147.27,147.17.

[0178] Example 8: Synthesis of Compound I-1-8

[0179]

[0180] (1) Synthesis of Compound 30

[0181]

[0182] Under nitrogen atmosphere, compound I-1-4 (1g, 2.6mmol) and 4,4'-bismethoxytrityl chloride (1.9g, 5.72mmol) were dissolved in dry dichloromethane, cooled to 0°C, and triethylamine (1.3g, 13mmol) was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 2h. After the reaction was completed by LCMS monitoring, the mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography. The eluent was dichloromethane: ethyl acetate: triethylamine.

[0183] =1:1:0.1%, 1.3 g of light yellow solid was obtained, with 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).

[0184] (2) Synthesis of Compound I-1-8

[0185]

[0186] Under 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, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (880 mg, 2.92 mmol) was added dropwise at room temperature, and stirring was continued for 2 h. After the reaction was completed by LCMS monitoring, 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 by high pressure preparative liquid phase, the chromatographic column was C18, and the mobile phase was acetonitrile (containing 0.0035% diisopropylamine): water (containing 0.0035% diisopropylamine) = 8:2, and 550 mg of white solid was obtained, with a yield of 53%. [M+1] + =884.4; 31 PNMR:148.40,148.31.

[0187] Example 9: Synthesis of Compound I-1-9

[0188]

[0189] Compound I-1-2 (30 mg, 0.081 mmol) was dissolved in 3 ml of 7N ammonia methanol solution under nitrogen atmosphere and stirred at room temperature for 4 h. After the reaction was completed, the mixture was concentrated, slurried with a small amount of methanol, and filtered to obtain 18 mg of a white solid with a yield of 83%. [M+1] +=268.1; 1H NMR (400MHz, DMSO-d6)δ

[0190] 7.34(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).

[0191] Example 10: Synthesis of Compound I-1-10

[0192]

[0193] Compound I-1-3 (50 mg, 0.14 mmol) was dissolved in 5 ml of 7N ammonia methanol solution under nitrogen atmosphere and stirred at room temperature for 3 days. After the reaction was completed, 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).

[0194] Example 11: Synthesis of Compound I-2-1

[0195]

[0196] (1) Synthesis of compound 31

[0197]

[0198] Under 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-tetramethylpiperidinoxide (55 mg, 0.35 mmol) and potassium bromide (40 mg, 0.34 mmol) were added, and 10% sodium hypochlorite solution (3.5 g, 4.8 mmol) (adjust pH=9 with saturated sodium bicarbonate solution) was slowly added dropwise at 0°C, and stirring was continued for 1 h after the addition was completed. After the reaction was completed by LCMS monitoring, sodium thiosulfate solution was added to quench, the mixture was separated, washed with water, dried, and concentrated. The crude product was purified by column chromatography, and the eluent was ethyl acetate: petroleum ether = 1:2 to 1:1, to obtain 950 mg of solid, with a yield of 60%.

[0199] [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).

[0200] (2) Synthesis of Compound I-2-1

[0201]

[0202] Under nitrogen atmosphere, compound 31 (400 mg, 0.86 mmol) was dissolved in 5 ml methanol, 20 μl of 30% hydrogen chloride methanol solution was added dropwise, and stirred at room temperature for 4 h. After concentration, the crude product was purified by column chromatography with ethyl acetate: petroleum ether = 1:5 as eluent to obtain 200 mg, 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).

[0203] Example 12: Synthesis of Compound I-2-2

[0204]

[0205] Under nitrogen atmosphere, compound 31 (400 mg, 0.86 mmol) was dissolved in 5 ml of anhydrous ethanol, and 20 μl of 30% hydrogen chloride ethanol solution was added dropwise, and stirred at room temperature for 4 h. Concentrated, the crude product was purified by column chromatography, and the eluent was ethyl acetate: petroleum ether = 1:5, to obtain 210 mg, yield: 50%. [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).

[0206] Example 13: Synthesis of Compound I-4-1

[0207]

[0208] (1) Synthesis of compound 32

[0209]

[0210] Under 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-diazobispiro[5.4.0]undec-7-ene (41 g, 271 mmol) was added, the temperature was raised to 120°C, and the reaction was stirred for 3 h. LCMS monitored that the raw material reacted about halfway, the temperature was lowered, 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, the eluent was dichloromethane: methanol = 50:1, and 4.2 g was obtained, the yield was 31%, and compound 8 was recovered.

[0211] 5.4g. [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).

[0212] (2) Synthesis of Compound 33

[0213]

[0214] Under 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, cooled to 0°C, and trifluoromethylsulfonyl chloride (4.3 g, 25.5 mmol) was slowly added dropwise. After the addition was completed, the temperature was naturally raised to room temperature and stirred for 2 h. After the reaction was completed as monitored by LCMS, water was added, the liquids were separated, dried over anhydrous sodium sulfate, and concentrated. The crude product was directly used for the next step without purification. [M+1] + =477.2.

[0215] (3) Synthesis of Compound 34

[0216]

[0217] Under nitrogen atmosphere, the crude compound 33 obtained in the previous step was added with 100 ml of acetone and 100 ml of 1M sulfuric acid aqueous solution, and heated under reflux to react overnight. After the reaction was completed by LCMS monitoring, the acetone was concentrated under reduced pressure, and the residue was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography with an eluent of ethyl acetate: petroleum ether = 2:1 to obtain 3.4 g, with 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).

[0218] (4) Synthesis of Compound 35

[0219]

[0220] Under nitrogen atmosphere, compound 34 (3.4 g, 6.9 mmol), potassium carbonate (2.8 g, 20.6 mmol), 4-methoxybenzyl chloride (1.6 g, 10.4 mmol) were dissolved in 25 ml of dry N, N-dimethylformamide, heated to 50 ° C and stirred for 2 h. After the reaction was completed, the temperature was lowered and water was added for dilution, 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, the eluent was ethyl acetate: petroleum ether = 1:3, and 3.9 g was obtained, with a yield of 93%. [M+1] + =615.3.

[0221] (5) Synthesis of Compound 36

[0222]

[0223] Under nitrogen atmosphere, compound 35 (500 mg, 0.3 mmol) and potassium hydroxide (138 mg, 0.9 mmol) were dissolved in a mixed solvent of 5 ml methanol and 1 ml purified water and heated under reflux overnight. After the reaction was completed, the temperature was lowered by LCMS monitoring, the reaction solution was poured into water, the pH was adjusted to 3-4 with hydrochloric acid, and ethyl acetate was extracted twice. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, the eluent was ethyl acetate: petroleum ether = 1:1, and 280 mg was obtained, with a yield of 65%. [M+1] + =601.2.

[0224] (6) Synthesis of Compound 37

[0225]

[0226] Under nitrogen atmosphere, compound 36 (280 mg, 0.2 mmol), triphenylphosphine (366 mg, 0.6 mmol), diisopropyl azodicarboxylate (280 mg, 0.6 mmol) were dissolved in 5 ml of dry tetrahydrofuran and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and the crude product was purified by column chromatography with ethyl acetate: petroleum ether = 1:4 as the eluent to obtain 240 mg with a yield of 90%. [M+1] + =583.2.

[0227] (7) Synthesis of Compound 38

[0228]

[0229] Under nitrogen atmosphere, compound 37 (240 mg, 0.16 mmol) and ammonium cerium 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. After the reaction was completed, the temperature was lowered, water was added to the reaction solution, and it was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, and the eluent was ethyl acetate: petroleum ether = 1:1, to obtain 65 mg, with a yield of 33%. [M+1] + =463.2.

[0230] (8) Synthesis of Compound I-4-1

[0231]

[0232] Compound 38 (65 mg, 0.05 mmol) and 65 mg of 10% palladium carbon (55% water content) were suspended in 5 ml of methanol and 0.5 ml of glacial acetic acid, replaced with hydrogen three times, and stirred at room temperature for 4 days. LCMS monitored that the raw material was almost reacted, filtered, the filter cake was washed with 50% methanol aqueous solution, concentrated, and the crude product was purified by column chromatography, the eluent was dichloromethane: methanol = 20:1, and 8 mg of white solid was obtained, with a yield of 20%.

[0233] [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).

[0234] Example 14: Synthesis of Compound I-3-1

[0235]

[0236] (1) Synthesis of compound 39

[0237]

[0238] Under 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, and trifluoromethanesulfonic anhydride (2.41 g, 8.54 mmol) was slowly added dropwise at 0°C. After the addition was completed, stirring was continued for 3 h. After the reaction was completed, the organic phase was washed with 10% citric acid aqueous solution and saturated brine, dried, concentrated, and the crude product was purified by column chromatography. The eluent was ethyl acetate: petroleum ether = 1:3 to 1:1, and 1.62 g of foamy solid was obtained, with a yield of 61%. [M+1] + =627.2.

[0239] (2) Synthesis of Compound 40

[0240]

[0241] Under 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, and 4.2 ml of 1M tetrabutylammonium fluoride solution in tetrahydrofuran was added dropwise, and stirred at room temperature overnight. After the reaction was completed by LCMS monitoring, the mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography, with the eluent being ethyl acetate: petroleum ether = 1:3 to 1:1, to obtain 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).

[0242] (3) Synthesis of Compound 41

[0243]

[0244] Under nitrogen atmosphere, compound 40 (1.0 g, 1.92 mmol) and triphenylphosphine (0.6 g, 2.3 mmol) were dissolved in 6 ml tetrahydrofuran and 2 ml purified water, stirred at room temperature for 2 h and then continued to stir at 50 ° C for 6 h. After the reaction was completed by LCMS monitoring, the mixture was concentrated and the crude product was purified by column chromatography with the eluent being dichloromethane:methanol=50:1-10:1 to obtain 600 mg. [M+1] + =494.2.

[0245] (4) Synthesis of Compound 42

[0246]

[0247] Under 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 refluxed for 10 h. After the reaction was completed by LCMS monitoring, 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 with the eluent being dichloromethane:methanol = 100:1 to 20:1, 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).

[0248] (5) Synthesis of Compound I-3-1

[0249]

[0250] Compound 42 (25 mg, 0.054 mmol) and 25 mg 10% palladium carbon (containing 55% water) were suspended in 5 ml methanol and 1 ml acetic acid, replaced with hydrogen three times, and stirred at room temperature overnight. LCMS monitored the reaction, filtered, the filter cake was washed with 50% methanol aqueous solution, concentrated, and the crude product was purified by column chromatography, the eluent was dichloromethane: methanol = 5:1, and 8 mg of white solid was obtained, with a yield of 60%. [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).

[0251] Example 15 Synthesis and purification of oligonucleic acid analogs

[0252] An oligonucleotide automatic synthesizer was used to perform deprotection, coupling, capping, oxidation, aminolysis and other multi-step solid phase synthesis of an oligonucleotide analog containing 10mer obtained in the above compound I-1-5 using a solid phase carrier and phosphoramidite nucleoside monomers at the order of 0.2 umol, as shown in Table 1, wherein X is represented in the sequence.

[0253] The crude oligonucleic acid analog was purified by ion-pair reversed-phase liquid chromatography. The chromatographic column was a NanoQ-15L9.5ml (8X190mm) ion exchange column. The flow rate was 2ml / min. The mobile phase A was 10mM sodium hydroxide solution. The mobile phase B was 10mM sodium hydroxide and 2.0M sodium chloride solution. Gradient elution was performed to obtain oligonucleic acids with purity meeting the requirements.

[0254] Table 1

[0255]

[0256] Example 16 Melting Temperature (Tm) Test

[0257] After annealing 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 detected by measuring Tm.

[0258] 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 min, and then cooled to room temperature. The Tm value of the sample was then determined using an ultraviolet spectrophotometer (Agilent, Cary3500 UV-Vis). The sample was placed in a cuvette with a lid and a temperature probe, and the temperature was set to rise from 20-25°C to 70-80°C at 0.5°C or 1.0°C per minute, and the ultraviolet absorption at 260nm was measured at intervals of 0.5°C.

[0259] Table 2

[0260] Antisense strand Tm(℃) ΔTm / mod.(℃) Antisense strand 1 46.3 +3.3 Antisense strand 2 45.1 +2.1 Control antisense strand 43 -

[0261] 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, showing high binding affinity.

[0262] Example 17 Evaluation of nuclease resistance

[0263] The resistance of each oligonucleotide to an exonuclease that decomposes from the 3' side was examined for each of the antisense strand 1 and antisense strand 2 synthesized in Example 15. An oligonucleotide (5'-TTTTTTTTTT-3') was used as a control.

[0264] After the buffer solution (65 μL) containing 750 pmol of oligonucleotide was kept at 37°C for 5 minutes, a buffer solution (35 μL) containing 5 μg / mL snake venom phosphodiesterase (Crotalus admanteus venom phosphodiesterase (CAVP): Merck KGaA, Darmstadt, Germany) was mixed. The HPLC (Thermo UltiMate 3000, analytical column Waters XBridge TM Shield RP18 3.5 μm (4.6 mm×150 mm) was used to measure the degradation of oligonucleotides over time. The buffer composition (final concentration) used was 50 mM TrisHCl (pH 8.0), 10 mM MgCl2, and it was thoroughly degassed before measurement. The quantitative conditions of HPLC are as follows.

[0265] [HPLC quantitative conditions]

[0266] Mobile phase: Liquid A 0.1M triethylammonium acetate buffer, pH 7.0

[0267] Solution B: 0.1M triethylammonium acetate buffer: acetonitrile = 1:1 (v / v), pH 7.0

[0268] Gradient: 15→40% (v / v) Solution B (7 minutes)

[0269] Column used: Waters XBridge TM Shield RP18 3.5μm(4.6mm×150mm)

[0270] Flow rate: 1.0mL / min

[0271] Column temperature: 50°C

[0272] Detection: UV (268nm)

[0273] exist Figure 1 The results are displayed in . Figure 1 In the table, "remaining oligonucleotide (%)" indicates the remaining ratio of undegraded oligonucleotide (10mer) at the measurement time relative to the undegraded oligonucleotide (10mer) at time 0.

[0274] Figure 1The results show that the oligonucleotide containing the carbocyclic nucleoside compound X of the present invention at the 3' side end still has more than 70% unreacted even after 40 minutes of nuclease treatment, and is difficult to decompose. The oligonucleotide containing compound X at the third position of the 3' side also has more than 10% unreacted after 5 minutes of nuclease treatment. In contrast, the oligonucleotide containing all-natural thymidine is basically completely decomposed after 5 minutes of nuclease treatment. Therefore, the oligonucleotide containing the carbocyclic nucleoside compound X of the present invention shows higher enzyme resistance than the oligonucleotide containing all-natural thymidine.

[0275] Industrial Applicability

[0276] According to the present invention, a carbocyclic nucleoside and an oligonucleotide having a locked conformation are provided. The oligonucleotide containing the carbocyclic nucleoside having a locked conformation has high binding affinity and nuclease resistance, and is expected to be applied to 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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