Carbocyclic nucleosides, oligonucleotides, their production methods and medical uses

Abstract

The present invention discloses a carbocyclic nucleoside, an oligonucleotide, and a method for producing the same and a pharmaceutical use thereof. The carbocyclic nucleoside is a compound or salt shown in the following formula (I). The carbocyclic nucleoside or oligonucleotide of the present invention is useful in the production of drugs for gene therapy, gene vaccination, antisense therapy, interfering RNA, or nucleic acid transfer. The oligonucleotide of the present invention has high binding affinity for single-stranded RNA, nuclease resistance, and plasma stability. [C76] TIFF2025529338000083.tif42170

Description

[Technical Field]

[0001] The present invention relates to nucleosides and nucleotides, their production methods, and their medical uses, and more particularly to carbocyclic nucleosides and oligonucleotides, their production methods, and their medical uses. [Background technology]

[0002] Nucleic acids are one of the basic components of life and include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleotides are the monomers that make up nucleic acid molecules. They are compounds composed of a nitrogenous base, a pentose, and phosphate, and are the phosphate esters of nucleosides. Nucleic acid therapy is a type of drug or vaccine administered to the human body, consisting of modified single- or double-stranded nucleotides. Nucleic acid therapy is based on a nucleotide sequence and acts on mRNA through complementary base pairing, suppressing target protein expression through gene silencing, thereby achieving disease treatment and prevention. Nucleic acid-based therapeutic models include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), nucleic acid aptamers, messenger RNA (mRNA), ribozymes, antibody-oligonucleotide conjugates (AOCs), plasmid DNA, and CRISPR / Cas9. Oligonucleotides, short chains of nucleotides containing approximately 20 bases, are called oligonucleotides and include ASOs, siRNAs, aptamers, miRNAs, and saRNAs. ASOs are synthetic single-stranded oligonucleotides, typically consisting of 12–30 nucleotides. They bind to target mRNAs or pre-mRNAs through Watson-Crick complementary base pairing and regulate gene expression by inducing RNase H nuclease to degrade the RNA strand or by affecting the pre-mRNA processing and splicing process through steric hindrance. Fomivirsen was the first commercialized ASO drug, approved by the FDA in 1998. Currently, there are 10 ASO drugs approved for sale worldwide. siRNA is a double-stranded oligonucleotide containing one sense strand and one antisense strand that binds to the RNA-induced silencing complex (RISC) in the cytosol. Under the action of argonaute 2 protein, the duplex is depolymerized, the sense strand dissociates, and the antisense strand binds to mRNA in RISC, causing mRNA degradation, thereby regulating gene expression.In 2018, the FDA approved the launch of the first siRNA drug, Patisiran, and is expected to approve five siRNA drugs by 2023. Nucleic acid aptamers are single-stranded nucleic acids, typically 25–50 bases long, with unique three-dimensional conformations. They can target biological components, such as proteins, on the cell surface or intracellularly of diseased tissues, blocking the corresponding protein function through steric hindrance, making them analogous to antibodies. Pegaptanib is a nucleic acid aptamer drug approved for sale by the FDA in 2006, and several such drugs are currently in clinical research. miRNAs are non-coding RNAs, 19–24 nucleotides long, that regulate transcription after binding to mRNA in cells, suppressing protein translation and regulating target gene expression. saRNAs are short double-stranded oligonucleotides with a structure similar to siRNAs that can activate gene transcription, recruiting endogenous transcription complexes to target genes, increasing mRNA expression, and upregulating target proteins for therapeutic effects.

[0003] Half a century ago, synthetic oligonucleotides were available for target gene regulation. However, unmodified oligonucleotides were difficult to formulate into drugs due to their poor stability, susceptibility to degradation by endogenous nucleases in the blood, rapid clearance from the liver and kidneys, short half-lives, and low bioavailability. Chemical modifications increased the enzymatic resistance of oligonucleotides and enhanced their affinity for their targets, leading to the launch of the first oligonucleotide drugs in 1998. Nucleic acid chemical modifications include phosphate backbone modifications, base modifications, and ribose modifications. Thiophosphorylation, the most common backbone modification, replaces one non-bridging oxygen atom in the phosphate chain with a sulfur atom, significantly improving nuclease resistance after modification. Another backbone modification is the replacement of the 3' oxygen atom on the ribose ring with a 3' ammonia. Thiocarbamates and phosphorylimide salts exhibit high affinity and nuclease resistance. Base modifications typically involve the introduction of carboxyl or amino groups at the 5' position of pyrimidine bases and the 8' position of purine bases. Ribose modifications, primarily at the 2' position, include 2'-O-methyl, 2'-O-methoxypropyl, and 2'-F, which can improve affinity and nuclease resistance to varying degrees. Linking the 4' and 2' ends of ribose to form bicyclic systems, such as locked nucleic acid (LNA) and bridged nucleic acid (BNA), significantly improves affinity. Drug researchers can continually develop and market new nucleic acid drugs by improving their drug properties through various modifications. However, there is no ideal molecule, and existing nucleic acid drugs still have room for improvement in various aspects, including affinity, nuclease resistance, and in vivo stability. Summary of the Invention [Problem to be solved by the invention]

[0004] Objects of the invention: The object of the present invention is to provide carbocyclic nucleosides and oligonucleotides having high binding affinity to single-stranded nucleic acids (RNA or DNA), nuclease resistance and plasma stability; another object of the present invention is to provide oligonucleotides and methods for their preparation; still another object of the present invention is to provide pharmaceutical compositions; and still another object of the present invention is to provide uses of oligonucleotides in the manufacture of drugs for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

[0005] Technical solution: The compound or salt of the present invention is represented by the following formula (I): [ka] In the formula: R1 and R2 are each independently a hydrogen atom, an alkyl having 1 to 6 carbon atoms, a phenyl, (C 1-3 ) alkyl-phenyl, heterocycle or (C 1-3 ) alkyl-heterocycles, hydroxy protecting groups, silicon containing one or more substituents, phosphates, phosphate esters, thiophosphate esters, chiral thiophosphate esters, phosphate triesters, aminoalkyl phosphate triesters and alkyl phosphate esters containing protecting groups for oligonucleotide synthesis, A is O, S, or NR3, where R3 is a hydrogen atom, an alkyl containing 1 to 6 carbon atoms, a halogenated alkyl containing 1 to 6 carbon atoms, -C(O)R4, -C(O)OR4, or -C(O)N(R4)2, where R4 is H, an alkyl containing 1 to 6 carbon atoms, or a halogenated alkyl containing 1 to 6 carbon atoms; Y is C=O, CHR5 or CHOR5, where R5 is H or an alkyl containing 1 to 6 carbon atoms or a halogenated alkyl containing 1 to 6 carbon atoms; Base is a base.

[0006] As a further improvement of the above embodiment, the formula (I) is represented by any one of the following formulas (I-1) to (I-4): [ka] Preferably, Base is selected from any of the following groups:

[0007] [ka] Preferably, the alkyl containing 1 to 6 carbon atoms is a straight chain alkyl, a branched alkyl, a cycloalkyl or (C 1-4 ) alkyl-(C 3-7 ) cycloalkyl.

[0008] Preferably, the compound of formula (I) has any of the structures shown in the following formulae: [ka] TIFF2025529338000089.tif160170

[0009] The present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof, which comprises at least one structure shown in the following formulas (II) to (IV): [ka] Further provided are oligonucleotides or pharmaceutically acceptable salts thereof, wherein Z is a phosphate ester, a thiophosphate ester, a chiral thiophosphate ester, a phosphate triester, an aminoalkyl phosphate triester, and an alkyl phosphate ester.

[0010] In the above embodiment, the oligonucleotide is a linear polymer formed by covalently linking adjacent nucleotides via phosphate groups, or a spacer polymer formed by linking phosphate esters, thiophosphate esters, chiral thiophosphate esters, phosphate triesters, aminoalkyl phosphate triesters, and alkyl phosphate esters to DNA or RNA.

[0011] As described herein, an oligonucleotide preferably contains about 12 to 50 nucleotides and preferably contains a phosphorus atom backbone, such as a phosphate, a thiophosphate, a chiral thiophosphate, a phosphate triester, an aminoalkyl phosphate triester, or an alkyl phosphate.

[0012] Within the oligonucleotide structure, the conformationally restricted carbocyclic nucleoside may be a polymer formed by linking phosphate esters, thiophosphate esters, chiral thiophosphate esters, phosphate triesters, aminoalkyl phosphate triesters, alkyl phosphate esters, etc. to DNA, or a polymer formed by linking phosphate esters, thiophosphate esters, chiral thiophosphate esters, phosphate triesters, aminoalkyl phosphate triesters, alkyl phosphate esters, etc. to RNA, or a spacer polymer formed by linking phosphate esters, thiophosphate esters, chiral thiophosphate esters, phosphate triesters, aminoalkyl phosphate triesters, alkyl phosphate esters, etc. to DNA or RNA.

[0013] The above-described embodiments provide conformationally restricted carbocyclic nucleosides for nucleic acid modification. Oligonucleotides are synthetic nucleotide polymers with single- or double-stranded structures. Oligonucleotide therapy includes regulating gene expression with antisense oligonucleotides designed using Watson-Crick base pairing, degrading small interfering RNA (siRNA) from target transcripts using RNA-induced silencing complexes (RISCs), and inhibiting protein function through the interaction of the three-dimensional structure of aptamers with target proteins. Advantages of oligonucleotide therapy include the ease of designing drug molecules based on known genomic information, as well as high target specificity and minimal off-target toxicity.

[0014] To improve the stability and pharmacokinetic properties of oligonucleotides, chemical modifications (phosphosulfate backbone) and sugar modifications, such as 2'-O-methyl, 2'-O-methoxypropyl, 2'-F, locked nucleic acid (LNA), and bridged nucleic acid (BNA), have been successfully introduced. Oligonucleotide therapeutics typically have small volumes, are easily filtered by the kidney, and have poor blood pharmacokinetic properties and low target tissue concentrations. The oligonucleotides developed in this application are expected to have high binding affinity to target genes, but have a small polar surface, which can improve blood pharmacokinetic properties.

[0015] 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. Conformationally rigid carbocyclic nucleosides can appear at one or more positions in the oligonucleotide.

[0016] In another aspect, the present invention provides a method for producing the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, the method comprising the step of synthesizing an oligonucleotide using a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: [ka]

[0017] The method for producing an oligonucleotide or a pharmaceutically acceptable salt thereof according to the present invention preferably comprises the step of synthesizing an oligonucleotide with at least one of the compounds shown below or a pharmaceutically acceptable salt thereof. [ka] TIFF2025529338000093.tif157170

[0018] 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.

[0019] In another aspect, the present invention provides the use of the above compounds and salts in the manufacture of a medicament for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the above-described oligonucleotides or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable excipient.

[0021] In another aspect, the present invention provides use of the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof in the manufacture of a drug for gene therapy, gene vaccination, antisense therapy, interference RNA or nucleic acid transfer. [Effects of the Invention]

[0022] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides conformationally restricted carbocyclic nucleosides or nucleotides, and oligonucleotides containing these conformationally restricted carbocyclic nucleosides or nucleotides have high binding affinity to single-stranded RNA, nuclease resistance, and plasma stability, making them promising candidates for nucleic acid drugs. [Brief explanation of the drawings]

[0023] [Figure 1] Statistical graph of the results of nuclease-resistant degradation experiments. DETAILED DESCRIPTION OF THE INVENTION

[0024] First, the terms used in this specification are defined.

[0025] In this specification, "C 1-4The term "alkyl" means a straight or branched chain alkyl of carbon atoms containing 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, t-butyl, etc. 3-7 The term "cycloalkyl" means a saturated or partially saturated monocyclic ring containing 3 to 7 carbon atoms, and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. 1-4 ) alkyl-(C 3-7 The term "cycloalkyl" means a hydrocarbon compound containing 4 to 11 carbon atoms in which a straight or branched alkyl group containing 1 to 4 carbon atoms is attached to a saturated or partially saturated monocyclic ring containing 3 to 7 carbon atoms.

[0026] In this specification, "C 1-3 The term "alkyl" means a straight or branched alkyl containing 1 to 3 carbon atoms, and alkyl includes methyl, ethyl, n-propyl, isopropyl, and the like. 1-3 The term "alkyl-phenyl" refers to a compound in which a straight or branched chain alkyl containing 1 to 3 carbon atoms is attached to a phenyl.

[0027] As used herein, the term "heterocycle" refers to a saturated or partially saturated monocyclic ring containing 5 to 6 carbon atoms, in which one or more (1 to 3) carbon atoms are replaced by a heteroatom (e.g., O, N, or S). 1-3 The term "alkyl-heterocycle" refers to a compound in which a straight or branched alkyl containing 1 to 3 carbon atoms is attached to a heterocycle.

[0028] As used herein, "halogenated alkyl" refers to an alkyl in which a hydrogen atom is replaced with a halogen atom (eg, F, Cl, Br, and I).

[0029] As used herein, the term "salts thereof" refers to salts of the compounds of formula I of the present invention, and preferably includes metal salts such as alkali metal salts such as sodium salts, potassium salts, and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; inorganic salts such as ammonium salts, tert-octylamine salts, dibenzylamine salts, morpholino 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-benzylbenzene salts, and the like. inorganic acid salts such as hydrofluoride, hydrochloride, hydrobromide, hydrohalogenated salts of hydroiodic acids, nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; organic acid salts such as arylsulfonates of benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0030] As used herein, the term "pharmaceutically acceptable salt" refers to a salt or zwitterion of a compound disclosed herein that is water- or oil-soluble or dispersible and pharmaceutically acceptable. Salts may be prepared by the final isolation and purification process of the compound, or by reacting the free base form of the appropriate compound with a suitable acid. Acids that form pharmaceutically acceptable salts may be inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc., or organic acids such as oxalic acid, maleic acid, succinic acid, citric acid, etc. Salts may also be prepared by reacting a compound with an alkali metal or alkaline earth ion (e.g., sodium), alkaline earth ions (e.g., magnesium and calcium), ammonium, and NX4. +(wherein X is C1-C4 alkyl), etc. For therapeutic use, salts of the active ingredient compounds of the present invention are usually physiologically acceptable, i.e., derived from a physiologically acceptable acid or alkali. However, salts of acids or alkalis that are not physiologically acceptable can also be used in the preparation or purification of physiologically acceptable compounds. All salts, whether derived from a physiologically acceptable acid or alkali, are within the scope of the present invention.

[0031] As used herein, the term "pharmaceutically acceptable excipient" refers to an additive other than the active ingredient in a drug formulation, which is a carrier that does not cause significant side effects when administered, and includes, but is not limited to, solvents, co-solvents, preservatives, antioxidants, solvent enhancers, emulsifiers, osmotic pressure adjusters in liquid formulations, and binders, fillers, disintegrants, and lubricants in solid formulations.

[0032] The conformationally restricted carbocyclic nucleoside or nucleotide of the present invention can be present at any position in a 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 drugs in which a sequence consisting of multiple nucleotides acts specifically on a target nucleic acid through complementary base pairing to regulate gene expression and treat diseases. Nucleic acid drugs include, but are not limited to, ASOs, siRNAs, miRNAs, saRNAs, aptamers, ribozymes, mRNAs, AOCs, plasmid DNA, and CRISPR / Cas9. ASOs are single-stranded oligonucleotides typically consisting of 12 to 30 nucleotides. Upon entering cells, they form a duplex with target mRNA and regulate RNA function through different mechanisms. siRNAs are linear double-stranded RNAs 21 to 25 nucleotides long, containing one sense strand and one antisense strand. They bind to the RNA-induced silencing complex (RISC) in the cytoplasm and ultimately pair with target mRNA, which degrades the silenced target gene. miRNAs are non-coding RNAs 19–24 nucleotides long. Mature miRNAs are single-stranded RNAs that regulate transcription after binding to mRNA and suppress protein translation, thereby regulating target gene expression. saRNAs are short double-stranded oligonucleotides similar in structure to siRNAs. They recruit endogenous transcription complexes to target genes, thereby increasing mRNA expression and upregulating target proteins for therapeutic effects. Aptamers are single-stranded nucleic acids 25–50 nucleotides long with stable secondary structures that can specifically bind to target molecules of the same length, such as small molecules, proteins, bacteria, and viruses. Experienced professionals can use common nucleic acid synthesis methods, not limited to the methods described in the examples, to synthesize nucleotide analogs containing the conformationally fixed carbon ring structure of the present invention and further synthesize various nucleic acid drugs.

[0033] The conformationally fixed carbon ring-containing nucleic acid of the present invention, when forming a duplex with a complementary nucleic acid, easily forms a stable duplex with the target nucleic acid strand because the ribose analog moiety containing the carbon ring is fixed, inhibiting the transcription and translation processes, affecting the synthesis of pathogenic proteins, and suppressing the proliferation of infectious viruses, while achieving high binding affinity and resistance to nucleases and extending the in vivo residence time.

[0034] The nucleic acids containing conformationally restricted carbon rings of the present invention are preferably administered to living organisms in the form of pharmaceutical preparations, and can be prepared into injection preparations together with auxiliary materials used in the pharmaceutical formulation field, such as preservatives, antioxidants, excipients, etc. Alternatively, they can be prepared into preparations such as liquid preparations, solid preparations, and ointments together with pharmaceutical auxiliary materials used in this field, and administered via the gastrointestinal tract or skin.

[0035] The nucleic acids containing the conformationally restricted carbon ring of the present invention are expected to be used in the treatment and prevention of diseases, such as antiviral and antitumor, by inhibiting the expression of specific genes.

[0036] The compound according to the present invention is a compound or salt represented by the following formula (I): [ka] In the formula: R1 and R2 are each independently a hydrogen atom, an alkyl having 1 to 6 carbon atoms, a phenyl, (C 1-3 ) alkyl-phenyl, heterocycle or (C 1-3 ) alkyl-heterocycles, hydroxy protecting groups, silicon containing one or more substituents, phosphates, phosphate esters, thiophosphate esters, chiral thiophosphate esters, phosphate triesters, aminoalkyl phosphate triesters and alkyl phosphate esters containing protecting groups for oligonucleotide synthesis, A is O, S or NR3, where R3 is a hydrogen atom, an alkyl containing 1 to 6 carbon atoms, a halogenated alkyl containing 1 to 6 carbon atoms, -C(O)R4, -C(O)OR4 or -C(O)N(R4)2, where R4 is H, an alkyl containing 1 to 6 carbon atoms or a halogenated alkyl containing 1 to 6 carbon atoms; Y is C=O, CHR5 or CHOR5, where R5 is H or an alkyl containing 1 to 6 carbon atoms or a halogenated alkyl containing 1 to 6 carbon atoms; Base is a base.

[0037] Preferably, the formula (I) is represented by any one of the following formulas (I-1) to (I-4): [ka] Preferably, the compound of formula (I) has any of the structures shown in the following formulae: [ka] TIFF2025529338000097.tif92170

[0038] The present invention provides an oligonucleotide or a pharmaceutically acceptable salt thereof, which comprises at least one structure shown in the following formulas (II) to (IV): [ka] Further provided are oligonucleotides or pharmaceutically acceptable salts thereof, wherein Z is a phosphate ester, a thiophosphate ester, a chiral thiophosphate ester, a phosphate triester, an aminoalkyl phosphate triester, and an alkyl phosphate ester.

[0039] Example The synthesis of the conformationally restricted carbocyclic nucleosides and analogs thereof of the present invention will be described in more detail below with reference to examples.

[0040] Example 1: Synthesis of Compound I-1-1 [ka]

[0041] (1) Synthesis of Compound 5 [ka]

[0042] 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 under a nitrogen atmosphere and cooled to -20 to -25°C. 5 L of 0.4 M sodium chloride in methanol was slowly added dropwise, and stirring was continued for 30 min after the completion of the addition. After completion of the reaction was confirmed by thin-layer chromatography, the reaction mixture was quenched by the dropwise addition of 4 M HCl / 1,4-dioxane solution and the pH was adjusted to <7. After filtration and concentration, the crude product was purified by column chromatography using a petroleum ether:ethyl acetate ratio of 5:1 to 3:1 to obtain 228 g of an oil in 80% yield. [M+1] + =143.1;1H NMR (400 MHz, Chloroform-d) δ 5.98 - 5.87 (m, 1H), 5.76 (dq, J = 5.9, 2.1 Hz, 1H), 5.12 (dp, J = 5.6, 1.8 Hz, 1H), 3.75 (s, 3H), 2.96 (ddd, J = 9.0, 6.7, 5.2 Hz, 1H), 2.78 (ddq, J = 15.9, 9.0, 2.3 Hz, 1H), 2.66 - 2.55 (m, 1H).

[0043] (2) Synthesis of Compound 6 [ka]

[0044] Under a nitrogen gas 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 2 M lithium diisopropylamide in tetrahydrofuran was slowly added dropwise. After the addition was complete, the temperature was raised to -20 ° C and the mixture was stirred for 30 to 45 min. The mixture was then cooled 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 mixture was reacted for 2 h. After the completion of the reaction was confirmed by gas phase chromatography, the mixture was quenched by adding acetic acid dropwise, warmed to room temperature, added saturated ammonium chloride solution, and 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 an eluent of petroleum ether:ethyl acetate = 5:1 to 4:1 to give 63 g of an oily product in 68% yield. [M+1] + =263.2;1H NMR (400 MHz, Chloroform-d) δ 7.40 - 7.29 (m, 5H), 5.99 (dt, J = 5.3, 2.3 Hz, 1H), 5.76 (dq, J = 6.8, 2.2 Hz, 1H), 4.57 (d, J = 2.3 Hz, 1H), 4.54 (s, 2H), 3.79 (d, J = 8.7 Hz, 1H), 3.77 (s, 3H), 3.39 (d, J = 8.7 Hz, 1H), 3.12 (dt, J = 17.8, 2.4 Hz, 1H), 2.55 (dt, J = 17.8, 2.3 Hz, 1H).

[0045] (3) Synthesis of Compound 7 [ka]

[0046] Under a nitrogen atmosphere, compound 6 (63 g, 0.24 mol) was dissolved in 600 ml of dichloromethane and cooled to 0°C. 85% metachloroperbenzoic acid (121.8 g, 0.6 mol) was added in batches and stirred overnight at room temperature. After completion of the reaction was confirmed by LCMS, it was quenched by adding saturated sodium sulfite solution. The organic phase was washed sequentially with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a petroleum ether:ethyl acetate ratio of 5:1 to 4:1 to obtain 45 g of an oily product in 67% yield. [M+1] + =288.1.

[0047] (4) Synthesis of Compound 8 [ka]

[0048] 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 mixture was cooled to -40 to -50 °C. 189 mL of 1 M potassium bis(trimethylsilyl)amide in tetrahydrofuran was slowly added dropwise. The reaction was continued for 1 h after the completion of the addition. After completion of the reaction was confirmed by LCMS, the mixture was quenched by adding acetic acid dropwise. After warming to room temperature, saturated ammonium chloride solution was added and 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 petroleum ether:ethyl acetate ratio of 5:1 to 4:1 to obtain 40 g of an oil in 84% yield. [M+1] + =369.2.

[0049] (5) Synthesis of Compound 9 [ka]

[0050] Under a nitrogen atmosphere, compound 8 (39 g, 0.106 mol) was dissolved in 1.2 L of dry dichloromethane and cooled to -78 °C. 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 h. After completion of the reaction was confirmed by LCMS, the reaction was quenched by adding acetic acid dropwise. The temperature was then raised to room temperature. The organic phase was washed sequentially with saturated aqueous sodium bicarbonate, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 4:1 petroleum ether:ethyl acetate mixture to obtain 28 g of an oily product in 77.7% yield. [M+1] + =341.2;1H NMR (400 MHz, Chloroform-d) δ 7.41 - 7.29 (m, 7H), 4.80 (d, J = 11.9 Hz, 1H), 4.60 (d, J = 11.9 Hz, 1H), 4.47 (q, J = 12.1 Hz, 2H), 4.20 (s, 1H), 3.87 (d, J = 11.2 Hz, 1H), 3.60 (d, J = 2.7 Hz, 1H), 3.47 (d, J = 8.3 Hz, 3H), 3.37 (d, J = 9.1 Hz, 1H), 2.98 (s, 1H), 2.05 - 1.99 (m, 1H), 1.92 (d, J = 14.9 Hz, 1H).

[0051] (6) Synthesis of Compound 10 [ka]

[0052] 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 h. After completion of the reaction was confirmed 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 mixture (50:1 to 20:1) as an eluent to obtain 24.4 g of a white solid in 81% yield. [M+1] + =467.2;1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 7.55 - 7.16 (m, 11H), 5.09 (d, J = 6.5 Hz, 1H), 4.81 (d, J = 11.7 Hz, 1H), 4.71 (q, J = 9.7 Hz, 1H), 4.59 - 4.45 (m, 4H), 4.39 (dt, J = 9.7, 5.8 Hz, 1H), 3.80 (d, J = 4.9 Hz, 1H), 3.61 - 3.39 (m, 4H), 1.93 - 1.82 (m, 1H), 1.68 (d, J = 1.1 Hz, 3H), 1.35 (dd, J = 13.5, 10.1 Hz, 1H).

[0053] (7) Synthesis of Compound 11 [ka]

[0054] 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 and cooled to -10 °C. A solution of methylsulfonic anhydride (7.7 g, 0.044 mol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, the reaction was continued for 30 min at -10 °C. After completion of the reaction was confirmed by LCMS, saturated aqueous sodium bicarbonate was added and 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 dichloromethane:methanol (100:1) mixture as an eluent to obtain 14.5 g of a white foamy solid in 72.5% yield. [M+1] + =545.2.

[0055] (8) Synthesis of Compound 12 [ka]

[0056] Under a nitrogen atmosphere, 60% sodium hydride (3.2 g, 0.081 mol) was carefully added to 100 mL of dry N,N-dimethylformamide and cooled to 0 °C. Compound 11 (14 g, 0.027 mol) in N,N-dimethylformamide was slowly added dropwise. After the addition was complete, the reaction was continued for 1 h. After the completion of the reaction was confirmed by LCMS, saturated ammonium chloride solution was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 40:1 dichloromethane:methanol mixture as the eluent to obtain 8.5 g of an oily liquid in 74% yield. [M+1] + =449.2.

[0057] (9) Synthesis of Compound I-1-1 [ka]

[0058] 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) were added. The mixture was purged with hydrogen three times and stirred overnight at room temperature. After monitoring the completion of the reaction by LCMS, the mixture was filtered and concentrated to give 2.2 g of a white solid in 90% yield. [M+1] + =269.2;1H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H), 7.43 (d, J = 1.3 Hz, 1H), 5.76 (s, 2H), 4.23 (dd, J = 9.7, 5.6 Hz, 1H), 3.91 (d, J = 14.6 Hz, 2H), 3.70 - 3.61 (m, 2H), 3.53 - 3.43 (m, 3H), 2.05 (dd, J = 13.6, 9.7 Hz, 1H), 1.81 - 1.78 (m, 3H), 1.78 - 1.72 (m, 1H).

[0059] Example 2: Synthesis of Compound I-1-2 [ka]

[0060] (1) Synthesis of Compound 15 [ka]

[0061] 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 mixture was cooled to 0 °C and 28.5 mL of concentrated aqueous ammonia was added dropwise. After the addition was complete, the mixture was stirred overnight at room temperature. After completion of the reaction was confirmed by LCMS, water was added and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed sequentially with saturated ammonium chloride solution, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a dichloromethane:methanol = 30:1 mixture as an eluent to obtain 6 g of an oily liquid in 70.7% yield. [M+1] + =448.2.

[0062] (2) Synthesis of Compound 16 [ka]

[0063] 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 overnight at room temperature. After completion of the reaction was monitored by LCMS, 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 using a 3:1 petroleum ether:ethyl acetate mixture to give 3.8 g of an oily liquid in 68.9% yield. [M+1] + =552.2.

[0064] (3) Synthesis of Compound I-1-2 [ka]

[0065] 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) were added, and the mixture was stirred at 60 °C for 6 h. After completion of the reaction was monitored by LCMS, the mixture was filtered, washed, and concentrated to give 1.1 g of a white solid in 85% yield. [M+1] + =372.2;1H NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H), 8.21 (s, 2H), 7.79 (s, 1H), 7.60 (t, J = 7.3 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 5.13 (d, J = 4.5 Hz, 1H), 4.56 (t, J = 5.3 Hz, 1H), 4.31 (dd, J = 9.7, 5.7 Hz, 1H), 4.06 (s, 1H), 3.93 (d, J = 4.5 Hz, 1H), 3.75 - 3.61 (m, 2H), 3.52 (dd, J = 15.8, 5.9 Hz, 2H), 2.19 - 2.09 (m, 1H), 2.06 (s, 3H), 1.88 (d, J = 13.1 Hz, 1H).

[0066] Example 3: Synthesis of Compound I-1-3 [ka]

[0067] (1) Synthesis of Compound 19 [ka]

[0068] 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 for completion, after which the suspension was cooled, 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 petroleum ether:ethyl acetate ratio of 1:2 to 1:3 to obtain 23.3 g of an oil in 56.8% yield. [M+1] + =582.2;1H NMR (400 MHz, Chloroform-d) δ 7.64 (s, 1H), 7.57 - 7.47 (m, 2H), 7.46 - 7.29 (m, 13H), 5.58 (s, 2H), 4.94 (d, J = 11.4 Hz, 1H), 4.88 (s, 2H), 4.70 (dd, J = 10.8, 3.0 Hz, 2H), 4.57 (dd, J = 8.4, 5.6 Hz, 1H), 4.52 (s, 2H), 4.15 (d, J = 5.6 Hz, 1H), 3.85 (d, J = 11.3 Hz, 1H), 3.66 (d, J = 11.3 Hz, 1H), 3.41 (s, 2H), 2.38 (dd, J = 13.3, 8.7 Hz, 1H), 2.09 - 1.96 (m, 2H).

[0069] (2) Synthesis of Compound 20 [ka]

[0070] Under a nitrogen gas atmosphere, compound 19 (23.2 g, 0.04 mol) was dissolved in 230 mL of dry pyridine and cooled to 0 °C. Trimethylchlorosilane (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. After the addition was complete, 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 aqueous ammonia was added dropwise and the mixture was stirred at room temperature for 1 h. After the completion of 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 petroleum ether:ethyl acetate ratio of 1:1 to 1:2 to obtain 26.3 g of an oily liquid in 97% yield. [M+1] + =652.3.

[0071] (3) Synthesis of Compound 21 [ka]

[0072] 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 methylsulfonic anhydride (8.7 g, 0.051 mol) in tetrahydrofuran was slowly added dropwise. After the addition was complete, the mixture was stirred for 2 h. The reaction was monitored by LCMS until the product content reached its maximum. The mixture was then quenched by adding water and 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 1:1 petroleum ether:ethyl acetate mixture to give 19.1 g of an oil in 56.8% yield. 4.5 g of starting material was recovered. [M+1] + =730.3.

[0073] (4) Synthesis of Compound 22 [ka]

[0074] 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. After cooling to 0 °C, 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. After completion of the reaction, the reaction was monitored by LCMS, followed by quenching with acetic acid, adding saturated sodium bicarbonate solution, and extracting 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 mixture of petroleum ether and ethyl acetate to obtain 15.8 g of an oily product in 85% yield. [M+1] + =634.3.

[0075] (5) Synthesis of Compound I-1-3 [ka]

[0076] 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 mixture was slowly warmed to -40 °C and stirred for 2 h. After monitoring the completion of the reaction by LCMS, the mixture was quenched by adding methanol dropwise, warmed to room temperature, and concentrated. The crude product was purified by column chromatography using a dichloromethane:methanol ratio of 10:1 as an eluent to obtain 2.5 g of a white solid in 87.2% yield. [M+1] += 364.1;1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.36 - 7.21 (m, 1H), 5.17 (d, J = 4.2 Hz, 1H), 4.59 (t, J = 5.2 Hz, 1H), 4.44 (dd, J = 9.7, 5.4 Hz, 1H), 4.03 (d, J = 4.4 Hz, 1H), 3.96 (s, 1H), 3.78 - 3.68 (m, 2H), 2.79 (p, J = 6.8 Hz, 1H), 2.23 (ddd, J = 26.1, 14.8, 10.2 Hz, 2H), 1.99 (dd, J = 10.2, 6.6 Hz, 1H), 1.12 (d, J = 6.8 Hz, 5H).

[0077] Example 4: Synthesis of Compound I-1-4 [ka]

[0078] (1) Synthesis of Compound 25 [ka]

[0079] 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 mol) were suspended in 260 mL of dry dimethyl sulfoxide and heated to 120 °C for 4 hours. After completion of the reaction was monitored by LCMS, the mixture was cooled, 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 dichloromethane:methanol mixture to give 29.2 g of a white solid in 80.4% yield. [M+1] +=476.2;1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 8.10 (s, 1H), 7.43 - 7.24 (m, 10H), 7.16 (s, 2H), 5.19 (d, J = 6.2 Hz, 1H), 4.84 (d, J = 11.8 Hz, 1H), 4.81 - 4.71 (m, 2H), 4.61 - 4.53 (m, 3H), 4.50 (t, J = 4.9 Hz, 1H), 3.90 (d, J = 4.3 Hz, 1H), 3.65 - 3.56 (m, 2H), 3.54 (d, J = 4.6 Hz, 2H), 2.10 (dd, J = 13.5, 8.6 Hz, 1H), 1.82 (dd, J = 13.4, 9.2 Hz, 1H).

[0080] (2) Synthesis of Compound 26 [ka]

[0081] Under a nitrogen gas atmosphere, compound 25 (22 g, 0.046 mol) was dissolved in 200 mL of dry pyridine and cooled to 0 °C. Trimethylchlorosilane (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. After cooling to 0 °C, 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. After the completion of the reaction was monitored by LCMS, the mixture was cooled to 0 °C, 35 mL of concentrated aqueous ammonia was added, the mixture was slowly warmed to room temperature, and stirred for 1 h. After the completion of 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 100% ethyl acetate as an eluent to obtain 20 g in 73.7% yield. [M+1] + =560.3.

[0082] (3) Synthesis of Compound 27 [ka]

[0083] Under a nitrogen atmosphere, compound 26 (20 g, 0.034 mol) and methylsulfonic anhydride (7.2 g, 0.041 mol) were dissolved in 200 mL of dry tetrahydrofuran and cooled to -10 °C. Triethylamine (10.4 g, 0.103 mol) was slowly added dropwise. After the addition was complete, the mixture was warmed to 0 °C and reacted for 2 h. After monitoring by LCMS that the reaction was essentially complete, saturated aqueous sodium bicarbonate 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 petroleum ether:ethyl acetate mixture to obtain 17 g of a white solid in 65% yield. [M+1] + =658.2.

[0084] (4) Synthesis of Compound 28 [ka]

[0085] Under a nitrogen atmosphere, 60% sodium hydride (1.87 g, 77.7 mmol) was carefully added to 170 mL of dry N,N-dimethylformamide, stirred for 10 min, cooled to 0 °C, and a solution of compound 27 (17 g, 25.9 mmol) in N,N-dimethylformamide was slowly added dropwise. After the addition was complete, stirring was continued for 2 h. After monitoring the completion of the reaction by LCMS, saturated ammonium chloride 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 100% ethyl acetate as an eluent to obtain 12.3 g of a white solid in 85% yield. [M+1] + =562.2.

[0086] (5) Synthesis of Compound I-1-4 [ka]

[0087] Under a nitrogen gas atmosphere, compound 28 (5.3 g, 10.7 mmol) was dissolved in 50 ml of dry dichloromethane and cooled to -78°C. 64 ml of a 1 M solution of boron trichloride in dichloromethane was slowly added dropwise. After the addition was complete, the mixture was warmed to -20°C and stirred overnight. After monitoring the completion of the reaction by LCMS, the mixture was quenched by adding methanol, concentrated under reduced pressure, and then triturated with ethyl acetate. 3.4 g of a white solid in the form of the hydrochloride salt of compound Example 4 was obtained by filtration. [M+1] + =382.2;1H NMR (400 MHz, DMSO-d6) δ 11.16 (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, 7.0 Hz, 2H), 7.35 (d, J = 6.1 Hz, 5H), 7.32 - 7.23 (m, 5H), 4.79 - 4.70 (m, 1H), 4.66 - 4.59 (m, 1H), 4.55 (d, J = 4.7 Hz, 2H), 4.52 (d, J = 3.1 Hz, 1H), 4.29 (s, 1H), 3.85 (d, J = 6.7 Hz, 1H), 3.80 (d, J = 9.5 Hz, 1H), 3.73 (d, J = 6.5 Hz, 1H), 3.65 (d, J = 9.5 Hz, 1H), 2.44 (d, J = 9.1 Hz, 2H).

[0088] Example 5: Synthesis of Compound I-1-5 [ka]

[0089] (1) Synthesis of Compound 14 [ka]

[0090] Under a nitrogen gas 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 cooled to 0 °C. 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 h. After the completion of the reaction was monitored by LCMS, the organic phase was washed successively with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a dichloromethane:methanol ratio of 40:1 as an eluent to obtain 2.8 g of a pale yellow solid in 86% yield. [M+1] + =571.2;1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 7.42 (d, J = 1.4 Hz, 1H), 7.38 - 7.28 (m, 4H), 7.22 (ddt, J = 6.9, 4.8, 2.2 Hz, 5H), 6.92 - 6.86 (m, 4H), 5.10 (d, J = 4.6 Hz, 1H), 4.30 (dd, J = 9.9, 5.8 Hz, 1H), 3.96 (d, J = 4.7 Hz, 1H), 3.91 (s, 1H), 3.74 (s, 6H), 3.67 (s, 2H), 3.29 (d, J = 9.0 Hz, 1H), 3.12 (d, J = 9.0 Hz, 1H), 2.30 (dd, J = 13.5, 9.8 Hz, 1H), 1.78 (d, J = 1.1 Hz, 4H).

[0091] (2) Synthesis of Compound I-1-5 [ka]

[0092] Under a nitrogen atmosphere, compound 14 (700 mg, 1.2 mmol) and 4,5-dicyanoimidazolyl (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 the mixture was stirred for 2 h. After completion of the reaction was monitored by LCMS, 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-performance liquid chromatography using a C18 chromatography column and a mobile phase mixture of acetonitrile (containing 0.0035% diisopropylamine):water (containing 0.0035% diisopropylamine) = 8:2 to obtain 560 mg of a white solid in 60% yield. [M+1] + =771.3; 31 PNMR: 148.30, 148.04.

[0093] Example 6: Synthesis of Compound I-1-6 [ka]

[0094] (1) Synthesis of Compound 18 [ka]

[0095] 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 and cooled to 0 °C. N,N-diisopropylethylamine (2.0 g, 15.6 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred overnight at room temperature. After the completion of the reaction was monitored by LCMS, the organic phase was washed successively with saturated sodium bicarbonate solution, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using a 3:1:0.1% mixture of petroleum ether, ethyl acetate, and triethanolamine (TEA) to give 1.4 g of a white solid in 70% yield. [M+1] +=674.3;1H NMR (400 MHz, DMSO-d6) δ 13.18 (s, 1H), 8.20 (s, 2H), 7.79 (s, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.50 (t, J = 7.5 Hz, 2H), 7.40 - 7.30 (m, 5H), 7.24 (dd, J = 8.9, 3.2 Hz, 4H), 6.93 - 6.89 (m, 4H), 5.15 (d, J = 4.7 Hz, 1H), 4.38 (dd, J = 9.7, 5.8 Hz, 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 = 9.0 Hz, 1H), 2.40 (t, J = 11.7 Hz, 1H), 1.99 (s, 3H), 1.92 - 1.86 (m, 1H).

[0096] (2) Synthesis of Compound I-1-6 [ka]

[0097] Under a nitrogen atmosphere, compound 18 (500 mg, 0.74 mmol), tetrazolium (154 mg, 2.2 mmol), and N-methylimidazolyl (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 stirred for 2 h. After completion of the reaction was monitored by LCMS, the mixture was diluted with dry acetonitrile and separated by high-performance liquid chromatography (HPLC) using a C18 chromatography column with 100% acetonitrile as the mobile phase. 320 mg of a white solid was obtained in 49% yield. [M+1] + =874.4; 31 PNMR: 148.4, 148.2.

[0098] Example 7: Synthesis of Compound I-1-7 [ka]

[0099] (1) Synthesis of Compound 24 [ka]

[0100] 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, 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 warmed to room temperature and stirred for 2 h. After monitoring the completion of the reaction by LCMS, the mixture was concentrated and the crude product was purified by column chromatography using a dichloromethane:methanol:triethylamine (30:1:0.1%) eluent to obtain 4.6 g of a pale yellow solid in 76% yield. [M+1] + =666.3;1H NMR (400 MHz, Chloroform-d) δ 12.10 (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, 2.0 Hz, 4H), 4.66 (s, 1H), 4.48 (t, J = 7.6 Hz, 1H), 4.27 (s, 1H), 3.95 (d, J = 7.1 Hz, 1H), 3.82 (s, 1H), 3.78 (s, 6H), 3.68 (d, J = 7.0 Hz, 1H), 3.46 (d, J = 9.6 Hz, 1H), 3.27 (d, J = 9.6 Hz, 1H), 2.53 (p, J = 6.9 Hz, 1H), 2.32 (d, J = 7.6 Hz, 2H), 1.19 (dd, J = 9.7, 6.8 Hz, 6H).

[0101] (2) Synthesis of Compound I-1-7 [ka]

[0102] Under a nitrogen atmosphere, compound 24 (1.0 g, 1.5 mmol) and 4,5-dicyanoimidazolyl ether (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 the mixture was stirred for 2 h. After completion of the reaction was monitored by LCMS, dichloromethane and 0.1 mL of triethylamine were added. The organic phase was washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by high-performance liquid chromatography using a C18 chromatography column and a 7:3 mixture of acetonitrile (containing 0.0035% diisopropylamine):water (containing 0.0035% diisopropylamine) as the mobile phase. 660 mg of a white solid was obtained in 51% yield. [M+1] + =866.4; 31 PNMR: 147.27, 147.17.

[0103] Example 8: Synthesis of Compound I-1-8 [ka]

[0104] (1) Synthesis of Compound 30 [ka]

[0105] 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, cooled 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 h. After monitoring the completion of the reaction by LCMS, the mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography using an eluent of dichloromethane:ethyl acetate:triethylamine = 1:1:0.1% to obtain 1.3 g of a pale yellow solid in 72.2% yield. [M+1] + =684.3;1H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.76 (s, 1H), 8.62 (s, 1H), 8.05 (d, J = 7.7 Hz, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.30 - 7.21 (m, 5H), 6.96 - 6.87 (m, 4H), 5.20 (d, J = 4.6 Hz, 1H), 4.75 (dd, J = 9.8, 5.5 Hz, 1H), 4.36 (d, J = 4.7 Hz, 1H), 4.11 (s, 1H), 3.81 (d, J = 6.5 Hz, 1H), 3.75 (s, 6H), 3.40 (d, J = 9.1 Hz, 1H), 3.21 - 3.16 (m, 2H), 2.57 - 2.52 (m, 1H), 2.46-2.39(m, 1H).

[0106] (2) Synthesis of Compound I-1-8 [ka]

[0107] Under a nitrogen atmosphere, compound 30 (800 mg, 1.17 mmol) and 4,5-dicyanoimidazolyl ether (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 the mixture was stirred for 2 h. After completion of the reaction was monitored by LCMS, dichloromethane and 0.1 mL of triethylamine were added. The organic phase was washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by high-performance liquid chromatography using a C18 chromatography column and a mobile phase mixture of acetonitrile (containing 0.0035% diisopropylamine):water (containing 0.0035% diisopropylamine) = 8:2 to obtain 550 mg of a white solid in 53% yield. [M+1] + =884.4; 31 PNMR: 148.40, 148.31.

[0108] Example 9: Synthesis of Compound I-1-9 [ka]

[0109] Compound I-1-2 (30 mg, 0.081 mmol) was dissolved in 3 mL of 7N ammonia-methanol solution under a nitrogen atmosphere and stirred at room temperature for 4 h. After completion of the reaction was monitored by LCMS, the mixture was concentrated, triturated with a small amount of methanol, and filtered to obtain 18 mg of a white solid in 83% yield. [M+1] +=268.1;1H NMR (400 MHz, DMSO-d6) δ 7.34 (s, 1H), 6.90 (d, J = 171.7 Hz, 2H), 5.04 (d, J = 4.1 Hz, 1H), 4.53 (t, J = 5.3 Hz, 1H), 4.27 (dd, J = 9.7, 5.6 Hz, 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, 9.7 Hz, 1H), 1.86 (s, 3H), 1.72 (ddd, J = 13.9, 5.8, 2.6 Hz, 1H).

[0110] Example 10: Synthesis of Compound I-1-10 [ka]

[0111] Compound I-1-3 (50 mg, 0.14 mmol) was dissolved in 5 mL of 7N ammonia-methanol solution under a nitrogen atmosphere and stirred at room temperature for 3 days. After completion of the reaction was monitored by LCMS, the mixture was concentrated, triturated with a small amount of methanol, and filtered to obtain 35 mg of a white solid in 87% yield. [M+1] + =294.1;1H NMR (400 MHz, DMSO-d6) δ 10.58 (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, 5.7 Hz, 1H), 4.02 (s, 1H), 3.90 (s, 1H), 3.79 - 3.63 (m, 2H), 3.53 (dd, J = 13.5, 8.5 Hz, 2H), 2.16 (qd, J = 13.7, 7.2 Hz, 2H).

[0112] Example 11: Synthesis of Compound I-2-1 [ka]

[0113] (1) Synthesis of Compound 31 [ka]

[0114] Under a nitrogen atmosphere, compound 10 (1.6 g, 3.4 mmol) was dissolved in 30 mL of dichloromethane and 30 mL of water, and 2,2,6,6-tetramethylpiperidine oxide (55 mg, 0.35 mmol) and potassium bromide (40 mg, 0.34 mmol) were added. The mixture was cooled 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, the mixture was stirred for 1 h. After completion of the reaction, the reaction was monitored by LCMS, quenched with sodium thiosulfate solution, separated, washed with water, dried, and concentrated. The crude product was purified by column chromatography using ethyl acetate:petroleum ether (1:2 to 1:1) as an eluent to obtain 950 mg of a solid in 60% yield. [M+1] + =465.2;1H NMR (400 MHz, DMSO-d6) δ 11.22 (d, J = 2.2 Hz, 1H), 9.57 (d, J = 2.1 Hz, 1H), 7.85 - 7.40 (m, 1H), 7.39 - 7.14 (m, 10H), 5.37 (dd, J = 5.8, 2.1 Hz, 1H), 4.86 (dd, J = 11.8, 2.0 Hz, 1H), 4.65 (d, J = 9.8 Hz, 1H), 4.58 - 4.54 (m, 1H), 4.54 - 4.45 (m, 2H), 4.40 (dt, J = 5.3, 2.4 Hz, 1H), 4.12 (dd, J = 4.7, 2.0 Hz, 1H), 3.88 (dd, J = 9.2, 2.1 Hz, 1H), 3.61 (dd, J = 9.2, 2.1 Hz, 1H), 1.83 - 1.71 (m, 3H), 1.55 - 1.42 (m, 1H), 1.37 - 1.11 (m, 1H).

[0115] (2) Synthesis of Compound I-2-1 [ka]

[0116] Under a nitrogen atmosphere, compound 31 (400 mg, 0.86 mmol) was dissolved in 5 mL of methanol, and 20 μL of 30% hydrochloric acid in methanol 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 an eluent to obtain 200 mg in a 49% yield. [M+1] + =479.2;1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 7.37 - 7.27 (m, 10H), 7.16 (d, J = 1.4 Hz, 1H), 4.97 (s, 1H), 4.69 - 4.57 (m, 2H), 4.53 (d, J = 2.5 Hz, 2H), 4.41 (s, 1H), 4.36 (dd, J = 9.6, 6.0 Hz, 1H), 4.06 (s, 1H), 3.69 (d, J = 9.8 Hz, 1H), 3.46 (d, J = 9.8 Hz, 1H), 3.39 (s, 3H), 2.38 (dd, J = 13.4, 9.6 Hz, 1H), 1.75 (d, J = 1.1 Hz, 3H), 1.66 (dd, J = 13.3, 6.1 Hz, 1H).

[0117] Example 12: Synthesis of Compound I-2-2 [ka]

[0118] Under a nitrogen atmosphere, compound 31 (400 mg, 0.86 mmol) was dissolved in 5 mL of deionized water, and 20 μL of 30% ethanolic hydrochloric acid solution was added dropwise. The mixture was stirred at room temperature for 4 hours. The mixture was concentrated, and the crude product was purified by column chromatography using ethyl acetate:petroleum ether = 1:5 as an eluent, yielding 210 mg in 50% yield. [M+1] +=493.2;1H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H), 7.38 - 7.26 (m, 10H), 7.15 (d, J = 1.4 Hz, 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 = 9.8 Hz, 1H), 2.41 (dd, J = 13.4, 9.6 Hz, 1H), 1.74 (d, J = 1.1 Hz, 3H), 1.66 (dd, J = 13.4, 6.0 Hz, 1H), 1.14 (t, J = 7.0 Hz, 3H).

[0119] Example 13: Synthesis of Compound I-4-1 [ka]

[0120] (1) Synthesis of Compound 32 [ka]

[0121] 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, and 1,8-diazabicyclo[5.4.0]undec-7-ene (41 g, 271 mmol) was added. The mixture was heated to 120 °C and stirred for 3 hours. After monitoring by LCMS that the starting material was about half-reacted, the mixture was cooled, 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 dichloromethane:methanol mixture to obtain 4.2 g of compound 8 in 31% yield. 5.4 g of compound 8 were recovered. [M+1] +=495.2;1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 7.48 (s, 1H), 7.40 - 7.24 (m, 10H), 5.35 (d, J = 6.0 Hz, 1H), 4.86 (d, J = 11.4 Hz, 1H), 4.74 (q, J = 10.0 Hz, 1H), 4.56 (d, J = 12.1 Hz, 1H), 4.51 - 4.40 (m, 4H), 3.86 (d, J = 8.5 Hz, 1H), 3.78 (d, J = 4.2 Hz, 1H), 3.70 (d, J = 8.5 Hz, 1H), 3.54 (s, 3H), 2.70 (dd, J = 13.7, 9.7 Hz, 1H), 1.80 - 1.68 (m, 4H).

[0122] (2) Synthesis of Compound 33 [ka]

[0123] 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 allowed to warm to room temperature and stirred for 2 h. After monitoring the completion of the reaction by LCMS, water was added, the mixture was separated, dried over anhydrous sodium sulfate, and concentrated. The crude product was directly used in the next reaction without further purification. [M+1] + =477.2.

[0124] (3) Synthesis of Compound 34 [ka]

[0125] Under a nitrogen atmosphere, 100 ml of acetone and 100 ml of 1M aqueous sulfuric acid solution were added to the crude compound 33 obtained in the previous step, and the mixture was heated under reflux overnight. After monitoring the completion of the reaction by LCMS, the acetone was concentrated under reduced pressure, and 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 an eluent of ethyl acetate:petroleum ether = 2:1, yielding 3.4 g in 80% yield over two steps. [M+1] + =495.2;1H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.42 - 7.23 (m, 10H), 5.18 (ddd, J = 10.6, 8.9, 5.6 Hz, 1H), 4.69 (d, J = 11.9 Hz, 1H), 4.58 (s, 2H), 4.56 (d, J = 11.9 Hz, 1H), 4.39 (dd, J = 5.8, 3.3 Hz, 1H), 3.98 - 3.87 (m, 2H), 3.77 - 3.72 (m, 1H), 3.69 (s, 3H), 2.67 (dd, J = 13.6, 8.9 Hz, 1H), 2.22 (dd, J = 13.6, 10.7 Hz, 1H), 1.81 (s, 3H).

[0126] (4) Synthesis of Compound 35 [ka]

[0127] 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, heated to 50 °C, and stirred for 2 h. After completion of the reaction was monitored by LCMS, the mixture was cooled, diluted with water, and 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 1:3 ethyl acetate:petroleum ether mixture to obtain 3.9 g in 93% yield. [M+1] + =615.3.

[0128] (5) Synthesis of Compound 36 [ka]

[0129] Under a 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 of methanol and 1 mL of purified water and heated to reflux overnight. After completion of the reaction was monitored by LCMS, the mixture was cooled, poured into water, and the pH was adjusted to 3-4 with hydrochloric acid. The mixture was 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 ethyl acetate:petroleum ether mixture to obtain 280 mg in 65% yield. [M+1] + =601.2.

[0130] (6) Synthesis of Compound 37 [ka]

[0131] 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 overnight at room temperature. After completion of the reaction was monitored by LCMS, the reaction mixture was concentrated, and the crude product was purified by column chromatography using ethyl acetate:petroleum ether = 1:4 as an eluent to obtain 240 mg in 90% yield. [M+1] + =583.2.

[0132] (7) Synthesis of Compound 38 [ka]

[0133] 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. After completion of the reaction was monitored by LCMS, the mixture was cooled, water 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 ethyl acetate:petroleum ether mixture to obtain 65 mg in a 33% yield. [M+1] + =463.2.

[0134] (8) Synthesis of Compound I-4-1 [ka]

[0135] Compound 38 (65 mg, 0.05 mmol) and 65 mg of 10% palladium on carbon (55% water) were suspended in 5 mL of methanol and 0.5 mL of glacial acetic acid, purged with hydrogen three times, and stirred at room temperature for 4 days. After monitoring by LCMS that the reaction of the raw material was nearly complete, the mixture was filtered, the filter cake was washed with 50% aqueous methanol, concentrated, and the crude product was purified by column chromatography using dichloromethane:methanol = 20:1 as eluent to obtain 8 mg of a white solid in 20% yield. [M+1] + =283.1.1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.45 (s, 1H), 5.84 (d, J = 3.4 Hz, 1H), 4.83 (s, 1H), 4.78 (t, J = 5.5 Hz, 1H), 4.41 - 4.36 (m, 1H), 4.36 - 4.33 (m, 1H), 3.70 (dd, J = 11.6, 5.3 Hz, 1H), 3.62 (dd, J = 11.6, 5.5 Hz, 1H), 2.22 (dd, J = 14.0, 5.5 Hz, 1H), 2.03 (dd, J = 14.0, 9.5 Hz, 1H), 1.81 (s, 3H).

[0136] Example 14: Synthesis of Compound I-3-1 [ka]

[0137] (1) Synthesis of Compound 39 [ka]

[0138] 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, cooled to 0 °C, and trifluoromethanesulfonic anhydride (2.41 g, 8.54 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 3 h. After completion of the reaction was monitored by LCMS, the organic phase was washed sequentially with 10% aqueous citric acid and saturated brine, dried, and concentrated. The crude product was purified by column chromatography using ethyl acetate:petroleum ether (1:3 to 1:1) as an eluent to obtain 1.62 g of a foamy solid in 61% yield. [M+1] + =627.2.

[0139] (2) Synthesis of Compound 40 [ka]

[0140] 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, and 4.2 mL of a 1 M tetrabutylammonium fluoride solution in tetrahydrofuran was added dropwise. The mixture was stirred overnight at room temperature. After monitoring the completion of the reaction by LCMS, the mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography using an eluent of ethyl acetate:petroleum ether = 1:3 to 1:1 to obtain 630 mg. [M+1] +=520.2.1H NMR (400 MHz, Chloroform-d) δ 8.66 (s, 1H), 7.46 - 7.30 (m, 10H), 7.03 (s, 1H), 5.02 (q, J = 10.1 Hz, 1H), 4.83 (d, J = 10.8 Hz, 1H), 4.64 (d, J = 11.7 Hz, 1H), 4.59 (d, J = 10.9 Hz, 1H), 4.53 (d, J = 11.8 Hz, 1H), 4.20 (dd, J = 10.7, 4.6 Hz, 1H), 4.09 (d, J = 4.7 Hz, 1H), 3.85 (d, J = 8.7 Hz, 1H), 3.71 (d, J = 8.7 Hz, 1H), 3.62 (s, 3H), 3.02 (dd, J = 14.0, 10.0 Hz, 1H), 2.11 (dd, J = 13.9, 9.7 Hz, 1H), 1.75 (s, 3H).

[0141] (3) Synthesis of Compound 41 [ka]

[0142] 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, stirred at room temperature for 2 hours, and then stirred at 50 °C for 6 hours. After monitoring the completion of the reaction by LCMS, the mixture was concentrated and the crude product was purified by column chromatography using a dichloromethane:methanol ratio of 50:1 to 10:1 to obtain 600 mg. [M+1] + =494.2.

[0143] (4) Synthesis of Compound 42 [ka]

[0144] Under a nitrogen atmosphere, compound 41 (180 mg, 0.35 mmol) and sodium methanolate (95 mg, 1.7 mmol) were dissolved in 10 mL of deionized water and stirred at reflux for 10 h. After completion of 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 mixture (100:1 to 20:1) to give 80 mg of [M+1]. + =462.2;1H NMR (400 MHz, DMSO-d6) δ 11.33 (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, 4.5 Hz, 1H), 3.97 (s, 1H), 3.64 (d, J = 10.2 Hz, 1H), 3.57 (d, J = 10.2 Hz, 1H), 2.05 - 1.99 (m, 1H), 1.96 - 1.88 (m, 1H), 1.75 (s, 3H).

[0145] (5) Synthesis of Compound I-3-1 [ka]

[0146] Compound 42 (25 mg, 0.054 mmol) and 25 mg of 10% palladium on carbon (55% water) were suspended in 5 mL of methanol and 1 mL of acetic acid, purged with hydrogen three times, and stirred overnight at room temperature. After monitoring the completion of the reaction by LCMS, the mixture was filtered, the filter cake was washed with 50% aqueous methanol, concentrated, and the crude product was purified by column chromatography using a dichloromethane:methanol = 5:1 eluent to give 8 mg of a white solid in 60% yield. [M+1] +=282.1;1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.96 (s, 1H), 7.50 (d, J = 1.4 Hz, 1H), 5.35 (d, J = 3.8 Hz, 1H), 4.51 (t, J = 5.5 Hz, 1H), 4.13(dd,J=9.2,4.3Hz,1H),4.07 (dd, J = 3.5, 1.8 Hz, 1H), 3.79 (d, J = 2.1 Hz, 1H), 3.63 (dd, J = 11.6, 5.5 Hz, 1H), 3.55 (dd, J = 11.6, 5.6 Hz, 1H), 1.96 (dd, J = 14.0, 4.4 Hz, 1H), 1.82 (s, 3H), 1.81 - 1.75 (m, 1H).

[0147] Example 15 Synthesis and purification of oligonucleic acid analogues Using an automated oligonucleotide synthesizer, the 10-mer oligonucleotide analogue obtained in the above compound I-1-5 was synthesized in solid phase using a solid support and phosphoramidite nucleoside monomers at 0.2 μmol / L through multiple steps such as deprotection, coupling, capping, oxidation, and aminolysis, and the sequence is indicated by X, as shown in Table 1.

[0148] The resulting crude oligonucleotide 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. The gradient elution was performed using a mobile phase A of 10 mM sodium hydroxide solution and a mobile phase B of 10 mM sodium hydroxide and 2.0 M sodium chloride solution to obtain oligonucleotides of a purity that met the required standards.

[0149] [Table 1]

[0150] Example 16 Melting Temperature (Tm) Test The antisense strand 1, antisense strand 2, control antisense strand, and sense strand (5'-AAAAAAAAAA-3') synthesized in Example 15 above were annealed, and the Tm was measured to detect the hybridization ability of the antisense strands.

[0151] 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 to homogeneity, heated at 90°C for 10 min, and then cooled to room temperature. The Tm values ​​of the samples were then measured using a UV spectrophotometer (Agilent, Cary 3500 UV-Vis). The samples were placed in a cuvette with a lid and a temperature probe, and the temperature was raised from 20-25°C to 70-80°C at 0.5°C or 1.0°C per minute. UV absorbance at 260 nm was measured at 0.5°C intervals.

[0152] [Table 2]

[0153] According to the results in Table 2, compared to natural oligonucleic acids, all of the oligonucleotides containing the carbocyclic nucleoside compound X of the present invention had high Tm values ​​and high binding affinity to single-stranded oligoRNAs.

[0154] Example 17 Evaluation of nuclease resistance The resistance of each of the antisense strands 1 and 2 oligonucleotides synthesized in Example 15 to exonuclease degradation from the 3' end was investigated. The oligonucleotide (5'-TTTTTTTTTT-3') was used as a control.

[0155] A buffer solution (65 μL) containing 750 pmol of oligonucleotide was incubated at 37°C for 5 minutes and then mixed with a buffer solution (35 μL) containing 5 μg / mL of snake venom phosphodiesterase (Crotalus admanteus venom phosphodiesterase (CAVP): Merck KGaA, Darmstadt, Germany). The oligonucleotides were analyzed by HPLC (Thermo UltiMate 3000, analytical column: Waters XBridge). TM The degradation of oligonucleotides was measured over time using a Shield RP18 3.5 μm (4.6 mm × 150 mm) column. The buffer composition (final concentration) used was 50 mM TrisHCl (pH 8.0) and 10 mM MgCl, which was thoroughly degassed before measurement. The HPLC quantification conditions are shown below.

[0156] [HPLC quantitative conditions] Mobile phase: Solution A 0.1M triethylammonium acetate buffer, pH 7.0 Solution B: 0.1 M triethylammonium acetate buffer: acetonitrile = 1:1 (v / v), pH 7.0 Gradient: 15 to 40% (v / v) solution B (7 min) Column used: Waters XBridge TM Shield RP18 3.5μm(4.6mm×150mm) Flow rate: 1.0mL / min Temperature: 50℃ Detection: UV (268 nm) 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.

[0157] According to the results shown in Figure 1, oligonucleotides containing the carbocyclic nucleoside compound X of the present invention at the 3'-terminus remained unreacted for 40 minutes after treatment with nuclease, demonstrating their resistance to degradation. Oligonucleotides containing compound X at the third position of the 3'-terminus remained unreacted for 5 ... In contrast, oligonucleotides containing all-natural thymidines were almost completely degraded after 5 minutes of treatment with nuclease. Therefore, oligonucleotides containing the carbocyclic nucleoside compound X of the present invention exhibited higher enzyme resistance than oligonucleotides containing all-natural thymidines.

[0158] Industrial Applicability The present invention provides nucleosides and oligonucleotides having a conformationally restricted carbocyclic ring. Oligonucleotides of carbocyclic nucleosides containing this conformationally restricted carbocyclic ring have high binding affinity and nuclease resistance, and are expected to be used in nucleic acid drugs.

Claims

1. A compound represented by the following formula (I) or a salt thereof: 【Chemical 71】 In the formula: R １ and R ２ are each independently a hydrogen atom, an alkyl having 1 to 6 carbon atoms, a phenyl, (C １－３ ) alkyl-phenyl, heterocycle or (C １－３ ) alkyl-heterocycles, hydroxy protecting groups, silicon containing one or more substituents, phosphates, phosphate esters, thiophosphate esters, chiral thiophosphate esters, phosphate triesters, aminoalkyl phosphate triesters and alkyl phosphate esters containing protecting groups for oligonucleotide synthesis; A is O, S or NR ３ and R ３ is a hydrogen atom, an alkyl having 1 to 6 carbon atoms, a halogenated alkyl having 1 to 6 carbon atoms, —C(O)R ４ , -C(O)OR ４ or -C(O)N(R ４ ) ２ and R ４ is H or an alkyl containing 1 to 6 carbon atoms or an alkyl halide containing 1 to 6 carbon atoms; Y is C=O, CHR ５ or CHOR ５ and R ５ is H or an alkyl containing 1 to 6 carbon atoms or an alkyl halide containing 1 to 6 carbon atoms; Base is a compound or a salt thereof that is a base.

2. The compound or salt thereof according to claim 1, wherein the formula (I) is represented by any one of the following formulas (I-1) to (I-4): 【Chemical Formula 72】

3. 2. The compound or salt thereof according to claim 1, wherein Base is selected from the following groups: 【Chemical 73】

4. The alkyl containing 1 to 6 carbon atoms can be a straight chain alkyl, a branched alkyl, a cycloalkyl or (C １－４ ) alkyl-(C ３－７ 2. The compound or salt thereof according to claim 1, wherein:

5. 2. The compound of claim 1, wherein the compound of formula (I) has any of the structures shown in the following formulae: 【Chemical 74】 【change】

6. An oligonucleotide or a pharmaceutically acceptable salt thereof, comprising at least one structure shown in formula (II) to (IV): 【Chemistry 75】 An oligonucleotide or a pharmaceutically acceptable salt thereof, wherein Z is a phosphate ester, a thiophosphate ester, a chiral thiophosphate ester, a phosphate triester, an aminoalkyl phosphate triester, or an alkyl phosphate ester.

7. 7. A method for producing the oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 6, comprising a step of synthesizing an oligonucleotide using a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

8. A method for producing the oligonucleotide or a pharmaceutically acceptable salt thereof according to claims 6 to 7, comprising a step of synthesizing an oligonucleotide using at least one compound or a pharmaceutically acceptable salt thereof according to claim 5.

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 or salt according to any one of claims 1 to 5 in the manufacture of a drug for gene therapy, gene vaccination, antisense therapy, interference 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 pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable excipient.

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

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