JP6767011B2 - Nucleoside derivative with physiological activity such as anti-DNA virus activity - Google Patents

Description

The present invention relates to a nucleoside derivative having physiological activity such as anti-DNA virus activity, and more specifically, a 2'-deoxypurine nucleoside derivative having antiviral activity against at least hepatitis B virus, and an active ingredient of the derivative. Regarding anti-DNA virus agents.

Infection with hepatitis B virus (HBV) causes acute or fulminant hepatitis, which can be fatal. In addition, hepatitis may develop chronically and progress to cirrhosis and hepatocellular carcinoma. The number of infected people is estimated to be about 400 million worldwide, and the prevalence is extremely high mainly in Southeast Asia, and the development of effective treatment methods is sought worldwide.

HBV is an incomplete double-stranded DNA virus and is known to perform reverse transcription that synthesizes DNA from RNA in its life cycle. On the other hand, in humans as hosts, reverse transcription is not performed, and by inhibiting this stage, it is possible to prevent only HBV replication. Nucleoside derivative preparations have been developed as therapeutic agents for HBV infection from such a viewpoint (Patent Documents 1 and 2).

Japanese Unexamined Patent Publication No. 2004-244422 Japanese Unexamined Patent Publication No. 2008-273960

Most of the current nucleoside derivative preparations are also toxic to host cells, that is, human cells to be taken, and side effects due to medium- to long-term administration have become a problem. In addition, resistant strains to nucleoside derivatives may develop during the period of administration. Therefore, at present, no effective treatment method for DNA virus infections such as HBV has been established.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a nucleoside derivative having at least antiviral activity against HBV and low toxicity to host cells. Another object of the present invention is to provide a nucleoside derivative that exhibits antiviral activity against HBV that is resistant to existing nucleoside derivatives (entecavir and the like).

As a result of intensive research to solve the above problems, the present inventors have set the 2nd and 6th positions of purine bases and the 4th position of ribose sugars as specific functional groups in 2'-deoxypurine nucleosides. It has been found that the substituted nucleoside derivative exhibits excellent antiviral activity against HBV. In particular, in the 2'-deoxypurine nucleoside, the 6-position of the purine base may have a relatively bulky functional group (amino group substituted with an alkyl group having 1 or more carbon atoms, or a substituent which may have a substituent). A nucleoside derivative substituted with 2 or more alkoxy groups or a mercapto group substituted with an alkyl group having 1 or more carbon atoms) shows antiviral activity against HBV, but generally has low cytotoxicity. Revealed. Further, they have found that the 2'-deoxypurine nucleoside can exert antiviral activity against entecavir-resistant HBV, and have completed the present invention.

That is, the present invention provides at least a nucleoside derivative having antiviral activity against hepatitis B virus and an antiviral agent containing the derivative as an active ingredient, as follows.
<1> A nucleoside derivative represented by the following general formula (1).

[In the above formula, R ¹ represents a hydroxy group, an amino group which may have a substituent, an alkoxy group which may have a substituent, or a mercapto group which may have a substituent. .. R ² represents a hydrogen atom, a halogen atom, or an amino group. R ³ represents an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group. However, in the nucleoside derivative represented by the general formula (1), when R ¹ is an amino group, R ² is a halogen atom or a hydrogen atom, and R ³ is a cyano group, R ¹ is When it is a hydroxy group, R ² is an amino group and R ³ is an azide group, R ¹ is a hydroxy group, R ² is an amino group and R ³ is an azide group, and Except when R ¹ is an amino group, R ² is a hydrogen atom, and R ³ is an azide group. ]
<2> In the above formula, R ¹ is an amino group substituted with an alkyl group having 1 or more carbon atoms, an alkoxy group having 2 or more carbon atoms which may have a substituent, or an alkyl having 1 or more carbon atoms. The nucleoside derivative according to <1>, which is a mercapto group substituted with a group.
<3> R ¹ is one functional group selected from the group consisting of a methylamino group, a dimethylamino group, an ethylamino group, a cyclopropylamino group, an ethoxy group, an allyloxy group, a benzyloxy group and a methyl mercapto group. and R ² is a hydrogen atom or an amino group, a nucleoside derivative according to <2>.
<4> An anti-DNA virus agent containing the nucleoside derivative according to any one of <1> to <3> as an active ingredient.
<5> The antiviral agent according to <4>, which is an anti-hepatitis B virus agent.

According to the present invention, it is possible to provide a nucleoside derivative having antiviral activity against at least HBV and having low toxicity to host cells. It is also possible to provide a nucleoside derivative capable of exerting antiviral activity against HBV which is resistant to existing nucleoside derivatives (entecavir and the like).

(Nucleoside derivative)
As shown in Examples described later, it was revealed that the nucleoside derivative represented by the following formula has antiviral activity against hepatitis B virus. Therefore, the present invention provides a nucleoside derivative exhibiting anti-DNA virus activity, and more specifically, providing a nucleoside derivative represented by the following general formula (1), which has antiviral activity against hepatitis B virus at least. Is.

[In the above formula, R ¹ represents a hydroxy group, an amino group which may have a substituent, an alkoxy group which may have a substituent, or a mercapto group which may have a substituent. .. R ² represents a hydrogen atom, a halogen atom, or an amino group. R ³ represents an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group. However, in the nucleoside derivative represented by the general formula (1), when R ¹ is an amino group, R ² is a halogen atom or a hydrogen atom, and R ³ is a cyano group, R ¹ is When it is a hydroxy group, R ² is an amino group and R ³ is an azide group, R ¹ is a hydroxy group, R ² is an amino group and R ³ is an azide group, and Except when R ¹ is an amino group, R ² is a hydrogen atom, and R ³ is an azide group. ].

The nucleoside derivative of the present invention has at least antiviral activity against hepatitis B virus (HBV). In the present invention, "HBV" means a virus having the ability to develop hepatitis B. As HBV, genotypes of A (A2 / Ae, A1 / Aa), B (Ba, B1 / Bj), C (Cs, Ce), D to H and J are known, and the nucleoside of the present invention is known. The derivative may have antiviral activity against at least one genotype of HBV. Among the above genotypes, HBV / Ce is known to be a genotype that exhibits resistance to the existing nucleoside derivative preparation, entecavir. Therefore, the nucleoside derivative of the present invention is preferably a nucleoside derivative having antiviral activity against HBV / Ce.

In the present invention, "anti-virus activity" means an activity of eliminating or suppressing the growth of a virus-infected cell (host cell) such as HBV, and for example, suppressing virus replication in the host cell. Activity is mentioned. Further, when the target of such suppression or the like is a virus having DNA as a genome (DNA virus), it is referred to as "anti-DNA virus activity". Further, such activity can be evaluated by, as shown in the examples below, EC ₅₀ values calculated copy number of the virus, etc. in the host cells as the indicator. The nucleoside derivative of the present invention preferably has an EC ₅₀ value of antiviral activity of 0.1 μM or less, more preferably 0.05 μM or less, further preferably 0.01 μM or less, and 0.005 μM. More preferably, it is less than or equal to (for example, 0.004 μM or less, 0.003 μM or less, 0.002 μM or less, 0.001 μM or less).

Further, the nucleoside derivative of the present invention preferably has low cytotoxicity. In the present invention, "cytotoxicity" means an activity of killing a cell, inhibiting its function, or suppressing its proliferation. Such activity can be evaluated by the CC50 value calculated using the number of surviving cells as an index, as shown in Examples described later. The nucleoside derivative of the present invention preferably has a CC50 value of 50 μM or more, more preferably 100 μM or more, and even more preferably 200 μM or more.

From the viewpoint that the nucleoside derivative of the present invention can exhibit at least antiviral activity against HBV and reduce the cytotoxicity of the derivative, it is preferable to select each substituent as shown below.

As the substituent in the "amino group which may have a substituent", an alkyl group having 1 or more carbon atoms is preferable, and a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms is more preferable. A linear, branched or cyclic alkyl group having 1 to 4 carbon atoms is more preferable, and a methyl group, an ethyl group or a cyclopropyl group is more preferable. More specifically, the "amino group which may have a substituent" is preferably a methylamino group, a dimethylamino group, an ethylamino group or a cyclopropylamino group.

As the alkoxy group in the "alkoxy group which may have a substituent", an alkoxy group having 1 or more carbon atoms is preferable, and an alkoxy group having 1 to 5 carbon atoms (methoxy, ethoxy, propoxy, butoxy, pentyloxy) is preferable. More preferred. As the substituent in the "alkoxy group which may have a substituent", a linear, branched or cyclic alkenyl group having 2 to 6 carbon atoms (ethenyl group, propa-1-enyl group, pig- 1-enyl group, but-2-enyl group, penta-2-enyl group, isopropenyl group, 3-methylbuta-1-enyl group, cyclohex-2-enyl group, cyclohexa-2,5-dienyl group, dicyclo Pentaenyl group, dicyclopentadienyl group, etc.) and aryl groups having 6 to 10 carbon atoms (phenyl group, naphthyl group) are preferable. More specifically, the "alkoxy group which may have a substituent" is preferably an ethoxy group, an allyloxy group or a benzyloxy group.

As the substituent in the "mercapto group which may have a substituent", an alkyl group having 1 or more carbon atoms is preferable, and a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms is more preferable. More specifically, the "mercapto group which may have a substituent" is preferably a methyl mercapto group.

The "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and a fluorine atom, a chlorine atom, or an iodine atom is preferable.

The alkyl group in the "alkyl group which may have a substituent" is not particularly limited, but a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms is preferable, and a methyl group or an ethyl group is preferable. More preferred. The substituent in the "alkyl group which may have a substituent" is not particularly limited, and examples thereof include a halogen atom, a hydroxy group, an alkoxy group, a cyano group, and an amino group. A halogen atom is preferable, and fluorine is preferable. Atoms are more preferred. More specifically, the "alkyl group which may have a substituent" is preferably a monofluoromethyl group.

The alkenyl group in the "alkenyl group which may have a substituent" is not particularly limited, but a linear, branched or cyclic alkenyl group having 2 or more carbon atoms is preferable, and the alkenyl group has 2 to 6 carbon atoms. A linear, branched or cyclic alkenyl group is more preferred, and an ethenyl group is even more preferred. The substituent in the "alkenyl group which may have a substituent" is not particularly limited, and examples thereof include a halogen atom, a hydroxy group, an alkoxy group, a cyano group, and an amino group.

Examples of nucleoside derivatives with suitable functional groups, R ¹ is an amino group, carbon atoms which may have a substituent two or more alkoxy groups substituted by the number 1 or more alkyl group having a carbon or Examples thereof include a nucleoside derivative represented by the general formula (1), which is a mercapto group substituted with an alkyl group having 1 or more carbon atoms (note that "a substituent may have a substituent having 2 or more carbon atoms". The "alkoxy group" means that the number of carbon atoms in the alkoxy group which may have a substituent is 2 or more).

Further, as an example of a nucleoside derivative having a suitable combination of functional groups, R ¹ is a methylamino group, a dimethylamino group, an ethylamino group, a cyclopropylamino group, an ethoxy group, an allyloxy group, a benzyloxy group and a methyl mercapto. A functional group selected from the group consisting of groups, R ² is a hydrogen atom or an amino group, and R ³ is a group consisting of a methyl group, a monofluoromethyl group, an ethenyl group, a cyano group and an azide group. is one functional group selected from and R ⁴ is a hydrogen atom, a nucleoside derivative represented by the general formula (1).

As an example of a nucleoside derivative having a more suitable combination of functional groups, R ¹ is a methyl amino group, R ² is an amino group, and R ³ is an ethenyl group or a cyano group. ), Examples of the nucleoside derivative.

The nucleoside derivatives of the present invention also include pharmacologically acceptable salts, hydrates or solvates. Such a pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on the structure of the nucleoside derivative and the like. For example, an acid addition salt (hydrochloride, sulfate, hydrogen bromide, etc.) Nitrate, hydrosulfate, phosphate, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluenesulfone Acids, cerebral sulfonates, sulfamates, mandelates, propionates, glycolates, stearate, malate, ascorbate, pamonate, phenylacetate, glutamate, benzoate , Salicylate, sulfanylate, 2-acetoxybenzoate, ethanedisulfonate, oxalate, isethionate, formate, trifluoroacetate, ethylsuccinate, lactobionate, gluconate, glucoheptonic acid Salt, 2-hydroxyethanesulfonate, benzenesulfonate, lauryl sulfate, asparagate, adipate, hydroiodide, nicotinate, oxalate, picrate, thiocyanate, undecane Acids, etc.), base addition salts (sodium salt, potassium salt, zinc salt, calcium salt, bismuth salt, barium salt, magnesium salt, aluminum salt, copper salt, cobalt salt, nickel salt, cadmium salt, ammonium salt, ethylenediamine salt) , N-dibenzylethylenediamine salt). The hydrate or solvate is not particularly limited, and examples thereof include those obtained by adding 0.1 to 3 molecules of water or a solvent to one molecule of a nucleoside derivative or a salt thereof.

The nucleoside derivatives of the present invention include all isomers and isomer mixtures such as tautomers, geometric isomers, asymmetric carbon-based optical isomers, stereoisomers and the like. Furthermore, the nucleoside derivative of the present invention is still desired after undergoing metabolism such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydration, alkylation, dealkylation, and conjugation in vivo. The present invention also includes compounds exhibiting the activity of the present invention, and the present invention also includes compounds (so-called prodrug forms) that undergo metabolism such as oxidation, reduction, and hydrolysis in vivo to produce the nucleoside derivative of the present invention. .. Furthermore, the nucleoside derivative of the present invention can be formulated by a known pharmaceutical method as described later.

Further, in the synthesis of the nucleoside derivative of the present invention, for example, ribose sugar (D-ribofuranose protected by substituting a hydroxy group with an acetyl group, a benzyl group, etc.) and a purine base (adenine) are silylated. It is reacted via the body and further reduced via a phenoxythiocarbonyl derivative to deoxylate the 2-position of the ribose sugar, and if necessary, a substituent is added to the desired position of the ribose sugar and / or purine base by a known method. It can be done by introducing it. Such a method for synthesizing the nucleoside derivative of the present invention is shown in detail in Examples described later. Therefore, those skilled in the art can refer to the description of Examples and refer to the reaction raw materials, reaction reagents, and reaction conditions (for example). , Solvent, reaction temperature, catalyst, reaction time) and the like, and by appropriately modifying or modifying these methods as necessary, it is possible to synthesize the nucleoside derivative of the present invention. In addition, the nucleoside derivative synthesized in this manner can be appropriately subjected to methods (reverse phase chromatography, ion exchange chromatography, adsorption chromatography, recrystallization method) used for isolation and purification of general nucleosides and nucleotides. It can be separated and purified by using it alone or in combination.

(Antiviral agents, methods for preventing and treating viral infections)
As shown in Examples described later, the nucleoside derivative of the present invention has at least antiviral activity against hepatitis B virus. Therefore, it is possible to provide an anti-DNA virus agent containing the nucleoside derivative of the present invention as an active ingredient.

The infectious disease targeted by the anti-DNA virus agent of the present invention and the preventive method and the therapeutic method described later is not particularly limited, and examples thereof include HBV infection, and more specifically, hepatitis B (chronic hepatitis, Acute hepatitis, fulminant hepatitis), cirrhosis, liver fibrosis, hepatocellular carcinoma.

The anti-DNA virus agent of the present invention can be formulated by a known pharmaceutical method. For example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublinguals, chews, buccal agents, pastes, syrups, suspensions, Orally or parenterally as an elixir, emulsion, coating, ointment, ointment, poultice, transdermal preparation, lotion, inhalant, aerosol, injection, suppository, etc. Can be done.

In these formulations, pharmacologically acceptable carriers or vehicles, specifically sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents. , Fragrances, Excipients, Vehicles, Preservatives, Binders, Diluents, Isotonic Agents, Painless Agents, Bulking Agents, Disintegrants, Buffers, Coatings, Lubricants, Colorants, Sweeteners It can be appropriately combined with a thickener, a flavoring agent, a solubilizing agent, or other additives. More specifically, as carriers, solid carriers such as lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride, glycerin, peanut oil, polyvinyl Liquid carriers such as pyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol and water can also be mentioned.

Moreover, the antiviral agent of the present invention may be used in combination with other known antiviral agents. Examples of such known antiviral agents include known nucleoside analog preparations such as entecavir, 3TC (lamivudine), and adefovir, and interferon (IFN) when the target disease is HBV infection. In addition to antiviral therapy using such drugs, immunotherapy (corticosteroid hormone withdrawal therapy, oral propagelnium preparation, etc.), liver protection therapy (intravenous injection of glycyrrhizin preparation, oral administration of bile acid preparation, etc.) The anti-DNA virus agent of the present invention can also be used for combination therapy with.

The preferred administration form of the anti-DNA virus agent of the present invention is not particularly limited, and is oral administration or parenteral administration, more specifically, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, Examples include intra-airway administration, rectal administration and intramuscular administration, and administration by infusion.

The anti-DNA virus agent of the present invention can be used mainly for humans, but can also be used for non-human animals such as laboratory animals.

When the anti-DNA virus agent of the present invention is administered, the dose thereof is appropriately selected according to the age, body weight, symptom, health condition, serious condition, tolerability of the drug, administration form, etc. of the subject. The daily dose of the anti-DNA virus agent of the present invention is usually 0.00001 to 1000 mg / kg body weight, preferably 0.0001 to 100 mg / kg body weight as the amount of the nucleoside derivative as an active ingredient, and once. Alternatively, it is administered to the subject in multiple doses. The product of the anti-DNA virus agent of the present invention or a description thereof may be labeled to be used for treating or preventing a viral infection. Here, "marked on a product or instruction manual" means that a label is attached to the main body, container, packaging, etc. of the product, or an instruction manual, package insert, promotional material, or other printed matter that discloses product information. It means that the display is attached to. In addition, in the indication that it is used for treating a viral infection, it is also possible to inhibit the reverse transcriptase reaction of the virus and suppress the replication of the virus by administering the nucleoside derivative of the present invention. It can be included as information on the mechanism of action of antiviral agents.

As described above, the present invention can prevent or treat an infectious disease by administering the anti-DNA virus agent of the present invention to a subject. Therefore, the present invention also provides a method for preventing or treating a DNA virus infection, which comprises administering the nucleoside derivative of the present invention.

The subject to which the nucleoside derivative of the present invention is administered is not particularly limited, and examples thereof include patients with a virus infection such as HBV, virus carriers before the onset of the infection, and those before the infection.

In order to obtain a nucleoside derivative having anti-DNA virus activity, a nucleoside derivative represented by the following general formula (1) having a functional group in the combination shown in Table 1 below was synthesized by the method shown below. The numbers in the table indicate the numbers of the compounds shown below.

Furthermore, the compounds were thus synthesized is a it is a compound having the desired structure was confirmed by measuring the ^{1 H} nuclear magnetic resonance (NMR) spectra. The results are also shown below.

Synthesis Example 1: Synthesis of 2-amino-2'-deoxy-4'-fluoromethyladenosine In order to synthesize 2-amino-2'-deoxy-4'-fluoromethyladenosine (Compound 5), first, the following compound 2 (2'-O-Acetyl-2-amino-3', 5'-di-O-benzyl-4'-fluoromethyladenosine) was synthesized as follows.

That is, compound 1 (see Tetrahedron, Vol. 53, No. 39, pp. 13315-13322 (1997)) (5.78 g, 12.9 mmol), 2,6-diaminopurine (3.87 g, 25.8 mmol). , 1,2-Dichloroethane (104 mL) was added to bis (trimethylsilyl) acetamide (37.8 mL, 0.155 mol), and the mixture was heated under reflux for 1 hour. The reaction mixture was cooled to 0 ° C., trimethylsilyl trifluoromethanesulfonate (4.66 mL, 25.8 mmol) was added, and the mixture was heated under reflux for 8 hours. The reaction mixture was cooled to 0 ° C., a saturated aqueous sodium hydrogen carbonate solution was added and stirred, and the resulting insoluble matter was removed by filtration through Celite, and the filtrate organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to give compound 2 (5.15 g, 9.60 mmol, 74.4%).
^{1 1} H-NMR (CD ₃ CN): δ7.65 (1H, s, H-8), 7.24-7.08 (10H, m, aromatic), 6.02 (1H, d, H-1' ), 5.98 (1H, t, H-2'), 5.70 (2H, br.s, NH ₂ ), 4.95 (2H, br.s, NH ₂ ), 4.82 (1H, d, H-3'), 4.71 (1H, dd, 4'-CH ^a F), 4.61 (1H, dd, 4'-CH ^b F), 4.62 (1H, d, Ph- CH ^a ), 4.59 (2H, d, Ph-CH ^b ), 4.56 (1H, d, Ph-CH ^c ), 4.53 (1H, d, Ph-CH ^d ), 3.70 ( 2H, m, H-5'), 2.01 (3H, s, Ac).

Next, using the compound 2 obtained as described above, compound 3 (2-Amino-3', 5'-di-O-benzyl-4'-fluoromethyladenenocine) was synthesized as follows.

That is, compound 2 (5.15 g, 9.60 mmol) was dissolved in methanol (100 mL), a 1 M aqueous sodium hydroxide solution (20 mL, 20 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was neutralized with acetic acid, concentrated, and the residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1-10: 1) to give compound 3 (4.70 g, 9.50 mmol, 99.0%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.84 (1H, s, H-8), 7.38-7.28 (10H, m, aromatic), 6.72 (2H, br.s, NH) ₂ ), 5.86 (1H, d, H-1'), 5.81 (2H, br.s, NH ₂ ), 5.74 (1H, d, 2'-OH), 5.01 (1H) , Dd, H-2'), 4.89 (1H, d, Ph-CH ^a ), 4.68-4.53 (5H, m, Ph-CH, 4'-CH ₂ F), 4.28 (1H, d, H-3 '), 3.68 (1H, dd, H-5' a), 3.64 (1H, dd, H-5 'b).

Next, using the compound 3 obtained as described above, compound 4 (2-Amino-3', 5'-di-O-benzyl-2'-deoxy-4'-fluoromethyladenocine) was added as follows. Synthesized.

That is, compound 3 (4.70 g, 9.50 mmol) and 4-dimethylaminopyridine (1.74 g, 14.2 mmol) were dissolved in acetonitrile (38.0 mL) and phenyl chlorothionostate (1.54 mL) at 0 ° C. 11.4 mmol) was added, and the mixture was stirred for 2 hours. After adding methanol (2 mL) and stirring, the reaction solution was diluted with ethyl acetate and washed (saturated saline solution → 0.1 M hydrochloric acid → saturated saline solution → saturated aqueous sodium hydrogen carbonate solution). The organic layer was dried over magnesium sulfate, concentrated, and the residue was azeotropically boiled with toluene to obtain a crude thiocarbonate ester.

Crude thiocarbonate ester, tributyltin hydride (10.2 mL, 37.9 mmol) is dissolved in toluene (95.0 mL), heated to 85 ° C., azobis (isobutyronitrile) (20 mg) is added, and 2 hours. Stirred. After concentrating the reaction solution, the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain Compound 4 (3.23 g, 6.75 mmol, 71.1%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.87 (1H, s, H-8), 7.37-7.26 (10H, m, aromatic), 6.71 (2H, br.s, NH) ₂ ), 6.23 (1H, d, H-1'), 5.80 (2H, br.s, NH ₂ ), 4.70-4.50 (7H, m, 4'-CH ₂ F, _{Ph-CH 2 & H-3} '), 3.64 (2H, dd, H-5' a), 3.60 (2H, dd, H-5 'b), 2.91 (1H, m, H- ^{2 'a), 2.59 (1H} , m, H-2' b).

Next, using the compound 4 obtained as described above, compound 5 (2-Amino-2'-deoxy-4'-fluoromethyladenocine) was synthesized as follows.

That is, naphthalene (9.08 g, 70.8 mmol) was dissolved in dehydrated tetrahydrofuran (76.1 mL), metallic lithium (369 mg, 53.2 mmol) was added, and the mixture was stirred at room temperature for 3 hours. After cooling the solution to −78 ° C., a dehydrated tetrahydrofuran solution (35.6 mL) of compound 4 (2.12 g, 4.33 mmol) was added, and the mixture was stirred at −45 ° C. for 2 hours. After adding methanol (5 mL), the reaction mixture was diluted with ethyl acetate and extracted with deionized water. The aqueous layers were combined and concentrated to a small amount, then purified by ODS column chromatography (deionized water to 5% methanol), vacuum dried to give compound 5 (0.90 g, 3.0 mmol, 68%). ..
^{1 1} H-NMR (DMSO-d ₆ ): δ7.91 (1H, s, H-8), 6.70 (2H, br.s, NH ₂ ), 6.24 (1H, dd, H-1' ), 5.72 (2H, br.s, NH ₂ ), 5.36-5.35 (2H, m, 3'-OH &5'-OH), 4.60 (1H, dd, 4'-CH ^a F), 4.50 (1H, dd , 4'-CH b F), 4.51 (1H, m, H-3 '), 3.54 (1H, br.s, H-5' a), 3.53 (1H, br.s, H- 5 'b), 2.81 (1H, m, H-2' a), 2.22 (1H, m, H-2 'b).

Synthesis Example 2: Synthesis of 4'-fluoromethyl-2'-deoxyguanosine Using the compound 5 obtained in Synthesis Example 1, 6, 4'-fluoromethyl-2'-deoxyguanosine (Compound) as described below. 6) was synthesized.

That is, compound 5 (500 mg, 1.68 mmol) was dissolved in 50 mM Tris-hydrochloric acid buffer (pH 7.5) (45.0 mL), calf spleen-derived adenosine deaminase (50 uL, 6.5 units) was added, and the temperature was 40 ° C. The mixture was stirred for 2 hours. The mixture was allowed to stand at 5 ° C. overnight, and the precipitated compound 5 was collected by filtration and dried (409 mg). Further, the filtrate was concentrated to a small amount and purified (44 mg) by ODS column chromatography (ODS 50 cc, 0 to 5% MeOH). Combined with those previously obtained, compound 6 was obtained (453 mg, 1.51 mmol, 89.9%).
^{1 1} H-NMR (DMSO-d ₆ ): δ10.58 (1H, br.s, NH), 7.91 (1H, s, H-8), 6.42 (2H, br.s, NH ₂ ) , 6.19 (1H, dd, H-1'), 5.37 (1H, 3'-OH), 5.09 (1H, t, 5'-OH), 4.59 (1H, dd, 4) '-CH ^a F), 4.49 (1H, dd, 4'-CH ^b F), 4.49 (1H, m, H-3'), 3.50 (2H, m, H-5') , 2.71 (1H, m, H -2 'a), 2.25 (1H, m, H-2' b).

Synthesis Example 3: Synthesis of 2-amino-2'-deoxy-4'-fluoromethyl-N ⁶ -methyladenosine 2-amino-2'-deoxy-4'-fluoromethyl-N ⁶ -methyladenosine (Compound 8) First, the following compound 7 (3', 5'-Di-O-Acetyl-4'-fluoromethyl-2'-deoxyguanosine) was synthesized as follows.

That is, compound 6 (409 mg, 1.37 mmol) obtained in Synthesis Example 2 was suspended in acetonitrile (13.7 mL), acetic anhydride (285 uL, 3.0 mmol), triethylamine (629 uL, 4.51 mmol), 4 -Dimethylaminopyridine 10 mg (82 umol) was added, and the mixture was stirred at room temperature for 4 hours. Methanol (0.5 mL) was added to the reaction mixture, and the mixture was stirred and concentrated. Deionized water was added to the residue, and the precipitated compound 7 was collected by filtration and dried (397 mg). The filtrate was further concentrated to a small amount and then purified (80 mg) by ODS column chromatography (ODS 50 cc, 0 to 20% to 30% MeOH). Combined with those previously obtained, compound 7 was obtained (477 mg, 1.24 mmol, 90.5%).
^{1 1} H-NMR (DMSO-d ₆ ): δ10.60 (1H, s, NH), 7.89 (1H, s, H-8), 6.42 (2H, br.s, NH ₂ ), 6 .19 (1H, t, H-1'), 5.59 (1H, dd, H-3'), 4.59 (1H, dd, 4'-CH ^a F), 4.50 (1H, dd) ^{, 4'-CH b F),} 4.26 (1H, dd, H-5 'a), 4.16 (1H, dd, H-5' b), 3.02 (1H, m, H-2 ^{'a), 2.48 (1H,} m, H-2' b), 2.07 (3H, s, Ac), 2.00 (3H, s, Ac).

Next, using the compound 7 obtained as described above, compound 8 (2-Amino-2'-deoxy-4'-fluoromethyl-N ^6- methyladenocine) was synthesized as follows.

That is, compound 7 (0.26 g, 0.68 mmol) was suspended in dichloromethane (6.8 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (618 mg, 2.04 mmol) and triethylamine (569 uL, 4.08 mmol). ), 4-Dimethylaminopyridine (70 mg, 0.57 mmol) was added, and the mixture was stirred at room temperature for 25 hours. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to give a sulfonyl ester (0.38 g, 0.58 mmol, 85%).

The obtained sulfonyl ester (0.38 g, 0.58 mmol) was dissolved in a 40% methylamine methanol solution (20 mL), and the mixture was stirred at room temperature for 17 hours. The reaction mixture was concentrated, the residue was purified by silica gel column chromatography (chloroform: methanol = 10: 1), and the residue was dissolved in deionized water. After lyophilization, compound 8 was obtained (94 mg, 0.30 mmol, 52%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.87 (1H, s, H-8), 7.20 (1H, s, NH), 6.25 (1H, dd, H-1'), 5 .76 (2H, br.s, NH ₂ ), 5.37 (1H, t, 5'-OH), 5.34 (1H, d, 3'-OH), 4.60 (1H, dd, 4) '-CH ^a F), 4.50 (1H, dd, 4'-CH ^b F), 4.50 (1H, m, H-3'), 3.53 (1H, br.s, H-5) ^{'a), 3.52 (1H,} br.s, H-5' b), 2.88 (3H, br.s, Me), 2.80 (1H, m, H-2 'a), 2 .22 (1H, m, H- 2 'b).

Synthesis Example 4: Synthesis of 2-amino-N ⁶ -cyclopropyl-2'-deoxy-4'-fluoromethyladenosine 2-amino-N ⁶ -cyclopropyl-2'-deoxy-4'-fluoromethyladenosine (compound) 9) was synthesized as follows.

That is, compound 7 (150 mg, 0.391 mmol) obtained in Synthesis Example 3 was suspended in dichloromethane (3.9 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (357 mg, 1.18 mmol) and triethylamine. (329 uL, 2.36 mmol) and 4-dimethylaminopyridine (40 mg, 0.32 mmol) were added, and the mixture was stirred at room temperature for 17 hours. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to give a sulfonyl ester (240 mg, 0.369 mmol, 94.4%).

The obtained sulfonyl ester (240 mg, 0.369 mmol) was dissolved in cyclopropylamine (1.5 mL) and methanol (2.2 mL), and the mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated, the residue was purified by silica gel column chromatography (chloroform: methanol = 20: 1), and the residue was dissolved in deionized water. After lyophilization, compound 9 was obtained (115 mg, 0.340 mmol, 92.1%).
^{1 1} H-NMR (DMSO-d ₆ ): 7.89 (1H, s, H-8), 7.33 (1H, s, NH), 6.25 (1H, dd, H-1'), 5 .77 (2H, br.s, NH ₂ ), 5.36-5.35 (2H, m, 5'-OH &3'-OH), 4.59 (1H, dd, 4'-CH ^a F), ^{4.50 (1H, dd, 4'-} CH b F), 4.50 (1H, m, H-3 '), 3.53 (1H, br.s, H-5' a), 3.52 (1H, br.s, H-5 'b), 3.02 (1H, br.s, cyclopropyl), 2.79 (1H, m, H-2' a), 2.22 (1H, m, ^{H-2 'b), 0.62} (4H, m, cyclopropyl).

Synthesis Example 5: Synthesis of 4'-fluoromethyl-2'-deoxy-O ⁶ -methylguanosine 4'-fluoromethyl-2'-deoxy-O ⁶ -methylguanosine (Compound 10) is synthesized as follows. did.

That is, compound 7 (100 mg, 0.261 mmol) obtained in Synthesis Example 3 was suspended in dichloromethane (2.6 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (238 mg, 0.786 mmol) and triethylamine. (219 uL, 1.57 mmol) and 4-dimethylaminopyridine (27 mg, 0.22 mmol) were added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give a sulfonyl ester (168 mg, 0.258 mmol, 98.9%).

The resulting sulfonyl ester (168 mg, 0.258 mmol) was dissolved in dehydrated dioxane (5.6 mL), molecular sieves 4A (282 mg), 1,4-diazabicyclo [2.2.2] octane (58 mg, 0.52 mmol). ) Was added, and the mixture was stirred at room temperature for 30 minutes. 1,8-Diazabicyclo [5.4.0] undec-7-ene (96uL, 0.64 mmol) and dehydrated methanol (105 uL, 2.59 mmol) were added, and the mixture was stirred at room temperature for 24 hours. After removing the insoluble matter by filtration, the filtrate was concentrated. The residue was dissolved in methanol (10.0 mL), 28% aqueous ammonia (5.0 mL) was added, and the mixture was stirred at room temperature for 24 hours. The residue was azeotroped with dioxane, and the residue was purified by silica gel column chromatography (chloroform: methanol = 20: 1). After lyophilization, compound 10 was obtained (59 mg, 0.19 mmol, 74%).
^{1 1} H-NMR (DMSO-d ₆ ): 8.05 (1H, s, H-8), 6.37 (1H, s, NH), 6.29 (1H, dd, H-1'), 5 .36 (1H, d, 3'-OH), 5.12 (1H, t, 5'-OH), 4.59 (1H, dd, 4'-CH ^a F), 4.51 (1H, m) , H-3 '), 4.49 (1H, dd, 4'-CH b F), 3.96 (3H, s, OMe), 3.52 (1H, br.s, H-5' a) , 3.51 (1H, br.s, H -5 'b), 2.78 (1H, m, H-2' a), 2.26 (1H, m, H-2 'b).

Synthesis Example 6: Synthesis of 2'-deoxy-2-fluoro-4'-fluoromethyladenosine In order to synthesize 2'-deoxy-2-fluoro-4'-fluoromethyladenosine (Compound 12), first, the following compound 11 (3', 5'-Di-O-acetyl-2-amino-2'-deoxy-4'-fluoromethyladenocine) was synthesized as follows.

That is, compound 5 (143 mg, 0.479 mmol) obtained in Synthesis Example 1 was suspended in dehydrated acetonitrile (2.4 mL), acetic anhydride (136 uL, 1.44 mmol), triethylamine (401 uL, 2.88 mmol), and the like. A small amount of 4-dimethylaminopyridine was added, and the mixture was stirred at room temperature for 25 hours. The reaction mixture was diluted with ethyl acetate, washed with water, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1-25: 1) to give compound 11 (0.15 g, 0.39 mmol, 81%).
^{1 1} H-NMR (CDCl ₃ ): 7.61 (1H, s, H-8), 6.27 (1H, t, H-1'), 5.81 (1H, dd, H-3'), _{5.35 (2H, br.s, NH 2} ), 4.82 (2H, br.s, NH 2), 4.68 (1H, dd, H-5 'a), 4.64 (1H, dd ^{, 4'-CH a F),} 4.55 (1H, dd, 4'-CH b F), 4.32 (1H, dd, H-5 'b), 3.27 (1H, m, H- ^{2 'a), 2.55 (1H} , m, H-2' b), 2.15 (3H, s, Ac), 2.07 (3H, s, Ac).

Next, using the compound 11 obtained as described above, compound 12 (2'-Deoxy-2-fluoro-4'-fluoromethyladenosine) was synthesized as follows.

That is, compound 11 (0.15 g, 0.39 mmol) was dissolved in dehydrated pyridine (2 mL) and then cooled to −30 ° C. 65% hydrogen fluoride-pyridine (2 mL), then dehydrated pyridine (2 mL) and tert-butyl nitrite (0.23 mL, 1.9 mmol) were added and stirred at the same temperature for 1 hour. After adding 65% hydrogen fluoride-pyridine (4 mL) and tert-butyl nitrite (0.46 mL, 3.9 mmol), the mixture was further stirred for 2 hours, and then deionized water (20 mL) was added. After extraction with chloroform twice, the organic layer was washed (saturated aqueous sodium hydrogen carbonate), dried (anhydrous magnesium sulfate), and concentrated. The residue was dissolved in dioxane (30 mL) and 28% aqueous ammonia (10 mL) and stirred at room temperature for 15 minutes. After concentrating the reaction solution, the residue was dissolved in methanol (30 mL) and 28% aqueous ammonia (10 mL), and the mixture was stirred at room temperature for 2 hours. The residue was purified by ODS column chromatography (2-4-6-8% methanol) and the resulting residue was crystallized from deionized water to give compound 12 (34 mg, 0.11 mmol, 28%).
^{1 1} H-NMR (DMSO-d ₆ ): 8.31 (1H, s, H-8), 7.81 (2H, br. S, NH ₂ ), 6.30 (1H, dd, H-1' ), 5.43 (1H, d, 3'-OH), 5.10 (1H, t, 5'-OH), 4.62 (1H, dd, CH ^a F), 4.56 (1H, m) , H-3 '), 4.56 (1H, dd, CH b F), 3.54 (2H, m, H-5'), 2.87 (1H, m, H-2 'a), 2 .32 (1H, m, H- 2 'b).

Synthesis Example 7: Synthesis of 2-chloro-2'-deoxy-4'-fluoromethyladenosine 2-Chloro-2'-deoxy-4'-fluoromethyladenosine (Compound 13) was synthesized as follows.

That is, acetyl chloride (260 uL, 3.64 mmol) was dissolved in dehydrated dichloromethane (15.6 mL), cooled to −5 ° C., benzyltriethylammonium nitrite (774 mg, 3.25 mmol), and then in Synthesis Example 6. A dehydrated dichloromethane solution (6.5 mL) of the obtained compound 11 (0.25 g, 0.65 mmol) was added, and the mixture was stirred at the same temperature for 3 hours. Saturated aqueous sodium hydrogen carbonate was added to the reaction solution, and the mixture was stirred, and then the organic layer was dried (anhydrous magnesium sulfate) and concentrated. The residue was dissolved in methanol (40 mL) and dioxane (40 mL), 28% aqueous ammonia (30 mL) was added, and the mixture was stirred at room temperature for 21 hours. The reaction mixture was concentrated, and the residue was purified by ODS column chromatography (deionized water to 5 to 10 to 20% methanol). After drying, the residue was suspended in diethyl ether, collected by filtration, and washed. The obtained solid was vacuum dried to give compound 13 (51 mg, 0.16 mmol, 25%).
^{1 1} H-NMR (DMSO-d ₆ ): 8.34 (1H, s, H-8), 7.78 (2H, br. S, NH ₂ ), 6.33 (1H, dd, H-1' ), 5.42 (1H, d, 3'-OH), 5.10 (1H, t, 5'-OH), 4.62 (1H, dd, CH ^a F), 4.57-4.49 (2H, m, H-3 '& CH b F), 3.55 (1H, s, H-5' a), 3.54 (1H, s, H-5 'b), 2.85 (1H, ^{m, H-2 'a)} , 2.34 (1H, m, H-2' b).

Synthesis Example 8: Synthesis of 2-amino-2'-deoxy-4'-methyladenosine In order to synthesize 2-amino-2'-deoxy-4'-methyladenosine (compound 18), first, the following compound 15 (2) '-O-Acetyl-2-amino-3', 5'-di-O-benzyl-4'-methyladenosine) was synthesized as follows.

That is, compound 14 (see Bioscience. Biotech. Biochem. 57, 1433 (1993)) (5.01 g, 11.7 mmol), 2,6-diaminopurine (3.51 g, 23.4 mmol), N, O-bis. Dehydrated 1,2-dichloroethane (94.4 mL) was added to (trimethylsilyl) acetamide (34.4 mL, 0.140 mol), and the mixture was stirred at 85 ° C. for 1 hour. After cooling the solution to 0 ° C., trimethylsilyl trifluoromethanesulfonate (4.23 mL, 23.4 mmol) was added, and the mixture was stirred at 85 ° C. for 23 hours. The reaction mixture was cooled to 0 ° C., saturated aqueous sodium hydrogen carbonate was added, and the mixture was stirred, and insoluble solids were removed by filtration through Celite. The filtrate The organic layer was dried (anhydrous magnesium sulfate) and concentrated, and the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain Compound 15 (5.03 g, 9.70 mmol, 82.9). %).
^{1 1} H-NMR (CDCl ₃ ): δ7.76 (1H, s, H-8), 7.35-7.29 (10H, m, aromatic), 6.10 (1H, d, H-1') , 5.83 (1H, dd, H-2'), 5.41 (2H, br.s, NH ₂ ), 4.62 (2H, br. S, NH ₂ ), 4.60 (1H, d) , PhCH ^a ), 4.53 (1H, d, H-3'), 4.52 (1H, d, PhCH ^b ), 4.50 (1H, d, PhCH ^c ), 4.47 (1H, d) ^{, PhCH d), 3.55 (1H} , d, H-5 'a), 3.37 (1H, d, H-5' b), 2.08 (3H, s, Ac), 1.34 ( 3H, s, 4'-Me).

Next, using the compound 15 obtained as described above, compound 16 (2-Amino-3', 5'-di-O-benzyl-4'-methylaldehyde) was synthesized as follows.

That is, compound 15 (5.03 g, 9.70 mmol) was dissolved in methanol (48.5 mL), a 2N aqueous sodium hydroxide solution (9.70 mL, 19.4 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was neutralized with acetic acid, concentrated, and the residue was dissolved in ethyl acetate. The solution was washed (saturated saline), dried (anhydrous magnesium sulfate), concentrated, and the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain Compound 16 (4.35 g, 9). .13 mmol, 94.1%).
^{1 1} H-NMR (CDCl ₃ ): δ7.68 (1H, s, H-8), 7.35-7.24 (10H, m, aromatic), 5.91 (1H, d, H-1') , 5.83 (2H, br.s, NH ₂ ), 4.96 (2H, br.s, NH ₂ ), 4.79 (1H, d, PhCH ^a ), 4.74 (1H, t, H) -2'), 4.66 (1H, d, PhCH ^b ), 4.52 (1H, d, PhCH ^c ), 4.47 (1H, d, PhCH ^d ), 4.23 (1H, d, H) -3 '), 3.50 (1H, d, H-5' a), 3.37 (1H, d, H-5 'b), 2.51 (1H, br.s, 2'-OH) , 1.36 (3H, s, 4'-Me).

Next, using the compound 16 obtained as described above, compound 17 (2-Amino-3', 5'-di-O-benzyl-2'-deoxy-4'-methylaldehyde) was added as follows. Synthesized.

That is, compound 16 (4.35 g, 9.13 mmol) and 4-dimethylaminopyridine (1.61 g, 13.2 mmol) were dissolved in dehydrated acetonitrile (76.0 mL) and phenyl chlorothionostate (1.48 mL, 11.2 mmol). 0 mmol) was added, and the mixture was stirred for 1 hour. After adding methanol (5 mL) and stirring, the reaction solution was diluted with ethyl acetate and washed (saturated brine-> 0.1 M brine-> saturated brine-> saturated brine). The organic layer was dried (magnesium sulfate) and concentrated, and the residue was azeotropically heated with toluene to obtain a crude thiocarbonate ester.

The crude thiocarbonate ester, tributyltin hydride (9.82 mL, 36.5 mmol) and azobis (isobutyronitrile) (10 mg) were dissolved in toluene (30.4 mL) and then stirred at 85 ° C. for 1 hour. After concentrating the reaction solution, the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain Compound 17 (2.71 g, 5.71 mmol, 62.5%).
^{1 1} H-NMR (CDCl ₃ ): δ7.84 (1H, s, H-8), 7.35-7.28 (10H, m, aromatic), 6.26 (1H, t, H-1') , 5.25 (2H, br.s, NH ₂ ), 4.61 (1H, d, PhCH ^a ), 4.59 (2H, br.s, NH ₂ ), 4.56 (1H, d, PhCH) ^{b), 4.51 (2H, d} , PhCH c & PhCH d), 4.39 (1H, t, H-3 '), 3.57 (1H, d, H-5' a), 3.45 ( 1H, d, H-5 ' b), 2.71 (1H, m, H-2' a), 2.57 (1H, m, H-2 'b), 1.32 (3H, s, 4 '-Me).

Next, using the compound 17 obtained as described above, compound 18 (2-Amino-2'-deoxy-4'-methyladenocine) was synthesized as follows.

That is, naphthalene (11.25 g, 87.8 mmol) was dissolved in dehydrated tetrahydrofuran (94.3 mL), metallic lithium (458 mg, 66.0 mmol) was added, and the mixture was stirred at room temperature for 3 hours. After cooling the solution to −78 ° C., a dehydrated tetrahydrofuran solution (11.0 mL) of compound 17 (2.61 g, 5.50 mmol) was added, and the mixture was stirred at −45 ° C. for 3 hours. Methanol (1 mL) was added, the reaction solution was neutralized with 1N hydrochloric acid, hexane was added, and the mixture was stirred. The organic layer was extracted with deionized water, and the aqueous layers were combined and concentrated to a small amount. Purification was performed by ODS column chromatography (deionized water to 5% methanol), and the fraction containing the desired product was concentrated. The precipitated solid was collected by filtration, washed with a little deionized water, and vacuum dried to obtain Compound 18 (640 mg, 2.28 mmol, 41.5%). After concentrating the filtrate, the residue was dissolved in methanol, silica gel was added, and the mixture was concentrated. Purification by silica gel column chromatography (chloroform: methanol = 10: 1) gave compound 18 (210 mg, 0.749 mmol, 13.6%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.90 (1H, s, H-8), 6.70 (2H, br.s, NH ₂ ), 6.13 (1H, t, H-1' ), 5.69 (2H, br.s, NH ₂ ), 5.36 (1H, dd, 5'-OH), 5.12 (1H, d, 3'-OH), 4.31 (1H, 1H, q, H-3 '), 3.48 (1H, m, H-5' a), 3.34 (1H, dd, H-5 'b), 2.71 (1H, m, H-2' ^{a), 2.21 (1H, m} , H-2 'b), 1.09 (3H, s, 4'-Me).

Synthesis Example 9: Synthesis of 2'-deoxy-4'-methylguanosine 2'-deoxy-4'-methylguanosine (Compound 19) was synthesized as follows.

That is, compound 18 (0.59 g, 2.1 mmol) was dissolved in Tris-hydrochloric acid buffer (50 mM, pH 7.5, 61.8 mL), calf spleen-derived adenosine deaminase (14 uL, 16 units) was added, and the temperature was 40 ° C. The mixture was stirred for 20 hours. The precipitated solid was collected by filtration, washed with a small amount of deionized water and vacuum dried to give compound 19 (452 mg, 1.61 mmol, 77%).
^{1 1} H-NMR (DMSO-d ₆ ): δ10.59 (1H, br.s, NH), 7.93 (1H, s, H-8), 6.43 (2H, br.s, NH ₂ ) , 6.08 (1H, t, H-1'), 5.16 (1H, d, 3'-OH), 5.00 (1H, t, 5'-OH), 4.31 (1H, q) , H-3 '), 3.44 (1H, dd, H-5' a), 3.33 (1H, dd, H-5 'b), 2.61 (1H, m, H-2' a ), 2.25 (1H, m, H-2 'b), 1.09 (3H, s, 4'-Me).

Synthesis Example 10: 2-Amino-2'-deoxy-4'-methyl -N ⁶ - Synthesis of methyl adenosine 2-amino-2'-deoxy-4'-methyl -N ⁶ - Synthesis of methyl adenosine (Compound 21) To this end, first, the following compound 20 (3', 5'-Di-O-Acetyl-2'-deoxy-4'-methylguanocine) was synthesized as follows.

That is, compound 19 (452 mg, 1.61 mmol) obtained in Synthesis Example 9 was suspended in dehydrated acetonitrile (16.1 mL), acetic anhydride (381 uL, 4.03 mmol), triethylamine (843 uL, 6.05 mmol), and the like. 4-Dimethylaminopyridine (10 mg, 82 umol) was added, and the mixture was stirred at room temperature for 24 hours. The precipitated solid was collected by filtration, washed with a small amount of acetonitrile and vacuum dried to give compound 20 (262 mg, 0.712 mmol, 44.2%). The filtrate was concentrated and dried in vacuum, and the residue was purified by silica gel column chromatography (chloroform: methanol = 10: 1) to obtain compound 20 (211 mg, 0.578 mmol, 35.9%).
^{1 1} H-NMR (DMSO-d ₆ ): δ10.65 (1H, br.s, NH), 7.91 (1H, s, H-8), 6.48 (2H, br.s, NH ₂ ) , 6.13 (1H, t, H -1 '), 5.42 (1H, dd, H-3'), 4.18 (1H, dd, H-5 'a), 4.05 (1H, ^{dd, H-5 'b)} , 3.03 (1H, m, H-2' a), 2.50 (1H, m, H-2 'b), 2.11 (3H, s, Ac), 2.02 (3H, s, Ac), 1.18 (3H, s, 4'-Me).

Next, using the compound 20 obtained as described above, compound 21 (2-Amino-2'-deoxy-4'-methyl-N ^6- methyladenocine) was synthesized as follows.

That is, compound 20 (473 mg, 1.29 mmol) was suspended in dehydrated dichloromethane (12.9 mL), triethylamine (1.08 mL, 7.75 mmol), 4-dimethylaminopyridine (131 mg, 1.07 mmol), 2, chloride 2. 4,6-Triisopropylbenzenesulfonyl (1.17 g, 3.86 mmol) was added, and the mixture was stirred at room temperature for 17 hours. After adding methanol (1 mL), the reaction solution was washed (saturated aqueous sodium hydrogen carbonate) and dried. After concentrating the solution, the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1). The obtained residue was dissolved in a 40% methylamine methanol solution (20.0 mL) and stirred at room temperature for 16 hours. After concentrating the reaction solution, the residue was purified by silica gel column chromatography (chloroform: methanol = 20: 1). The residue was dissolved in a small amount of deionized water and then lyophilized to give compound 21 (262 mg, 0.890 mmol, 74.2%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.90 (1H, s, H-8), 7.24 (1H, br.s, NH), 6.14 (1H, dd, H-1') , 5.79 (2H, br.s, NH ₂ ), 5.34 (1H, t, 5'-OH), 5.14 (1H, d, 3'-OH), 4.31 (1H, q) , H-3 '), 3.47 (1H, dd, H-5' a), 3.33 (1H, dd, H-5 'b), 2.87 (3H, br.s, N 6 - Me), 2.70 (1H, m , H-2 'a), 2.21 (1H, m, H-2' b), 1.09 (3H, s, 4'-Me).

Synthesis Example 11: Synthesis of 2-amino-2'-deoxy-4'-vinyl adenosine In order to synthesize 2-amino-2'-deoxy-4'-vinyl adenosine (compound 24), first, the following compound 23 (2) -Benzamido-N ^6- benzoyl-3', 5'-di-O-tert-butyldimethylyl-2'-deoxy-4'-vinyladenocine) was synthesized as follows.

That is, compound 22 (see Nucleosides, Nucleotides & Nucleic Acids, Vol. 23, No. 4, pp. 671-690, 2004) (0.42 g, 0.57 mmol) was mixed with dry dimethyl sulfoxide (2.5 mL) and dry toluene (1). Dissolve in (3 mL), add 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (328 mg, 1.71 mmol), dry pyridine (104 uL), and trifluoroacetic acid (52 uL), and stir at room temperature for 2 hours. did. The reaction mixture was diluted with ethyl acetate, washed (saturated brine), dried (anhydrous magnesium sulfate), and concentrated. The residue was azeotroped with tetrahydrofuran 3 times and then vacuum dried to give a crude aldehyde.

Methyltriphenylphosphonium bromide (1.02 g, 2.86 mmol) was suspended in dry tetrahydrofuran (7.1 mL) and cooled to −78 ° C. An n-butyllithium hexane solution (1.60 M, 1.79 mL, 2.86 mmol) was added, and the mixture was stirred at 0 ° C. for 1 hour, the crude aldehyde-dried tetrahydrofuran solution (5.9 mL) was added, and the mixture was stirred at room temperature for 1 hour. .. A saturated aqueous solution of ammonium chloride was added and stirred, and the product was extracted with ethyl acetate. The organic layer was dried (anhydrous magnesium sulfate) and concentrated, and the residue was dissolved in a small amount of dichloromethane, silica gel was added and concentrated. Purification by silica gel column chromatography (hexane: ethyl acetate = 1: 1) gave compound 23 (293 mg, 0.402 mmol, 71%).
^{1 1} H-NMR (CDCl ₃ ): δ9.29 (1H, br.s, NH), 9.24 (1H, br.s, NH), 8.44 (1H, s, H-8), 8. 04-7.49 (10H, m, aromatic), 6.49 (1H, t, H-1'), 5.98 (1H, dd, vinyl), 5.57 (1H, dd, vinyl), 5 .34 (1H, dd, vinyl) , 4.90 (1H, t, H-3 '), 3.72 (1H, d, H-5' a), 3.68 (1H, dd, H-5 ' ^b ), 2.87 (3H, br.s, N ^6- Me), 2.49 (2H, m, H-2'), 0.94 (9H, s, tert-Bu-Si), 0 .91 (9H, s, tert-Bu-Si), 0.04, 0.03 (12H, s, Me-Si).

Next, using the compound 23 obtained as described above, compound 24 (2-Amino-2'-deoxy-4'-vinyladenocine) was synthesized as follows.

That is, compound 23 (293 mg, 0.402 mmol) was dissolved in dry tetrahydrofuran (1.3 mL), tetrabutylammonium fluoride tetrahydrofuran solution (1.0 M, 2.01 mL, 2.01 mmol) was added, and the mixture was added at room temperature for 1 hour. Stirred. After concentrating the reaction solution, the residue was purified by silica gel column chromatography (chloroform: methanol = 20: 1) to obtain a crude diol (293 mg, 0.402 mmol, 71%).
The crude diol was dissolved in methanol (10 mL) and a 40% aqueous methylamine solution (10 mL), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (chloroform: methanol = 10: 1 to 5: 1). The residue was recrystallized from deionized water to give compound 24 (83 mg, 0.28 mmol, 70%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.94 (1H, s, H-8), 6.71 (1H, br.s, NH ₂ ), 6.16 (1H, t, H-1' ), 5.95 (1H, dd, vinyl), 5.69 (2H, br.s, NH ₂ ), 5.49 (1H, t, 5'-OH), 5.35 (1H, dd, vinyl) ), 5.19-5.16 (2H, m, 3'-OH & vinyl), 5.18 (1H, dd, vinyl), 4.58 (1H, q, H-3'), 3.50 (1H) ^{, dd, H-5 'a} ), 3.41 (1H, dd, H-5' b), 2.54 (1H, m, H-2 'a), 2.17 (1H, m, H- ^2'b ).

Synthesis Example 12: Synthesis of 2'-deoxy-4'-vinylguanosine 2'-deoxy-4'-vinylguanosine (Compound 25) was synthesized as follows.

That is, compound 24 (30 mg, 0.10 mmol) obtained in Synthesis Example 11 was dissolved in Tris-hydrochloric acid buffer (50 mM, pH 7.5, 2.8 mL), and calf spleen-derived adenosine deaminase (3 uL, 3. 5 units) was added, and the mixture was stirred at 40 ° C. for 5 hours. After allowing to stand at 5 ° C. overnight, the precipitated solid was collected by filtration and washed with a small amount of deionized water. The resulting solid was vacuum dried to give compound 25 (25 mg, 0.085 mmol, 83%).
^{1 1} H-NMR (DMSO-d ₆ ): δ10.60 (1H, br.s, NH), 7.98 (1H, s, H-8), 6.44 (1H, br.s, NH ₂ ) , 6.10 (1H, dd, H-1'), 5.95 (1H, dd, vinyl), 5.34 (1H, dd, vinyl), 5.24 (1H, br.s, OH), 5.19 (1H, dd, vinyl), 5.11 (1H, br.s, OH &), 4.57 (1H, t, H-3'), 3.50 (1H, d, H-5') ^{a), 3.40 (1H, d} , H-5 'b), 2.44 (1H, m, H-2' a), 2.19 (1H, m, H-2 'b).

Synthesis Example 13: Synthesis of 2-amino-2'-deoxy-N ⁶ -methyl-4'-vinyl adenosine 2-amino-2'-deoxy-N ⁶ -methyl-4'-vinyl adenosine (Compound 27), It was synthesized as follows.

That is, the compound 25 (102 mg, 0.348 mmol) obtained in Synthesis Example 12 was suspended in dehydrated acetonitrile (3.5 mL), acetic anhydride (72 uL, 0.76 mmol), triethylamine (159 uL, 1.14 mmol), and the like. 4-Dimethylaminopyridine (2 mg, 16 umol) was added, and the mixture was stirred at room temperature for 18 hours. The precipitated solid was collected by filtration, washed with a small amount of acetonitrile and vacuum dried to give compound 26 (93 mg, 0.25 mmol, 72%).

Next, as shown in the above reaction formula, compound 26 (93 mg, 0.25 mmol) was suspended in dehydrated dichloromethane (2.5 mL), and triethylamine (209 uL, 1.50 mmol) and 4-dimethylaminopyridine (26 mg, 0.21 mmol) were suspended. ), 2,4,6-Triisopropylbenzenesulfonyl chloride (227 mg, 0.750 mmol) was added, and the mixture was stirred at room temperature for 18 hours. Methanol (1 mL) was added to the reaction solution, diluted with ethyl acetate, and washed (saturated aqueous sodium hydrogen carbonate). The organic layer was dried (anhydrous magnesium sulfate), concentrated, and the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1). The obtained residue was dissolved in a 40% methylamine methanol solution (10.0 mL) and stirred at room temperature for 15 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (chloroform: methanol = 20: 1 to 10: 1). The residue was dissolved in a small amount of deionized water and then lyophilized to give compound 27 (47 mg, 0.15 mmol, 60.0%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.94 (1H, s, H-8), 7.26 (1H, br.s, NH), 6.18 (1H, t, H-1') , 5.96 (1H, dd, vinyl), 5.82 (2H, br.s, NH ₂ ), 5.48 (1H, t, 5'-OH), 5.35 (1H, dd, vinyl) , 5.22 (1H, d, 3'-OH), 5.18 (1H, dd, vinyl), 4.58 (1H, q, H-3'), 3.50 (1H, dd, H- ^{5 'a), 3.42 (1H} , dd, H-5' b), 2.87 (3H, br.s, N 6 -Me), 2.53 (1H, m, H-2 'a) , 2.18 (1H, m, H -2 'b), 1.09 (3H, s, 4'-Me).

Synthesis Example 14: Synthesis of 2'-deoxy-2-fluoro-4'-vinyl adenosine In order to synthesize 2'-deoxy-2-fluoro-4'-vinyl adenosine (Compound 29), first, the following compound 28 (3) ', 5'-Di-O-acetyl-2-amino-2'-deoxy-4'-vinyladenosine) was synthesized as follows.

That is, compound 24 (100 mg, 0.342 mmol) obtained in Synthesis Example 11 was suspended in dehydrated acetonitrile (3.4 mL), acetic anhydride (71 uL, 0.75 mmol), triethylamine (157 uL, 1.13 mmol), and the like. 4-Dimethylaminopyridine (2 mg, 16 umol) was added, and the mixture was stirred at room temperature for 18 hours. The precipitated solid was collected by filtration, washed with a small amount of acetonitrile and vacuum dried to give compound 28 (115 mg, 0.306 mmol, 89.5%).
^{1 1} H-NMR (CDCl ₃ ): δ7.73 (1H, s, H-8), 6.28 (1H, t, H-1'), 5.85 (1H, dd, vinyl), 5.72 (1H, t, H-3'), 5.61 (1H, dd, vinyl), 5.37 (1H, dd, vinyl), 5.32 (2H, br.s, NH ₂ ), 4.77 _{(2H, br.s, NH 2)} , 4.47 (1H, d, H-5 'a), 4.25 (1H, d, H-5' b), 3.07 (1H, m, H ^{-2 'a), 2.48 (1H} , m, H-2' b), 2.11 (3H, s, Ac), 2.08 (3H, s, Ac).

Next, using the compound 28 obtained as described above, compound 29 (2'-Deoxy-2-fluoro-4'-vinyladenosine) was synthesized as follows.

That is, 70% hydrogen fluoride pyridine (1.6 mL) was cooled to −10 ° C., and dehydrated pyridine (0.7 mL) was added. Compound 28 (115 mg, 0.306 mmol) was dissolved therein, tert-butyl nitrite (181 uL, 1.53 mmol) was added, and the mixture was stirred for 3 hours. After adding deionized water (10 mL) to the reaction solution, the mixture was extracted with ethyl acetate, and the organic layer was washed 3 times (saturated sodium bicarbonate solution). The organic layer was dried (anhydrous magnesium sulfate) and concentrated, and the residue was dissolved in dioxane (9 mL) and 28% aqueous ammonia (3 mL), and the mixture was stirred at room temperature for 30 minutes. After concentrating the reaction solution, the residue was dissolved in methanol (15 mL) and 28% aqueous ammonia (5 mL), and the mixture was stirred at room temperature for 2 hours. The residue was dissolved in chloroform-methanol, silica gel was added, and the mixture was concentrated. Purification was performed by silica gel column chromatography (chloroform: methanol = 10: 1), and the obtained residue was crystallized from deionized water to obtain compound 29 (54 mg, 0.18 mmol, 59%).
^{1 1} H-NMR (DMSO-d ₆ ): δ8.36 (1H, s, H-8), 7.83 (2H, br.s, NH ₂ ), 6.22 (1H, dd, H-1' ), 5.96 (1H, dd, vinyl), 5.38 (1H, dd, vinyl), 5.29 (1H, d, 3'-OH), 5.21 (1H, dd, vinyl), 5 .11 (1H, t, 5'- OH), 4.64 (1H, q, H-3 '), 3.50 (1H, dd, H-5' a), 3.45 (1H, dd, ^{H-5 'b), 2.58} (1H, m, H-2' a), 2.25 (1H, m, H-2 'b).

Synthesis Example 15: Synthesis of 2-amino-2'-deoxy-4'-ethyladenosine 2-Amino-2'-deoxy-4'-ethyladenosine (Compound 30) was synthesized as follows.

That is, compound 24 (50 mg, 0.17 mmol) obtained in Synthesis Example 11 was dissolved in methanol (3.4 mL), 5% palladium carbon (50 mg) was added, and the mixture was stirred under a hydrogen atmosphere for 15 hours. After removing the catalyst by filtration through Celite, the filtrate was concentrated. The residue was purified by silica gel column chromatography (chloroform: methanol = 10: 1-5: 1). The resulting residue was crystallized from deionized water to give compound 30 (43 mg, 0.15 mmol, 88%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.93 (1H, s, H-8), 6.81 (2H, br.s, NH ₂ ), 6.13 (1H, dd, H-1' ), 5.79 (2H, br.s, NH ₂ ), 5.27 (1H, br.s, 5'-OH), 5.10 (1H, d, 3'-OH), 4.36 ( 1H, q, H-3 ' ), 3.48 (1H, d, H-5' a), 3.41 (1H, d, H-5 'b), 2.75 (1H, m, H- ^{2 'a), 2.18 (1H} , m, H-2' b), 1.63 (1H, m, CH 3 -CH a), 1.55 (1H, m, CH 3 -CH b), 0.87 (3H, t, CH ₃ ).

Synthesis Example 16: Synthesis of 2-amino-2'-deoxy-4'-ethylguanosine 2-Amino-2'-deoxy-4'-ethylguanosine (Compound 31) was synthesized as follows.

That is, the compound 30 (25 mg, 85 umol) obtained in Synthesis Example 15 was dissolved in Tris-hydrochloric acid buffer (50 mM, pH 7.5, 6.4 mL), and calf spleen-derived adenosine deaminase (3 uL, 3.5 units) was dissolved. Was added, and the mixture was stirred at 40 ° C. for 3 hours. After allowing to stand at 5 ° C. overnight, the precipitated solid was collected by filtration and washed with a small amount of deionized water. The obtained solid was vacuum dried to give compound 31 (18 mg, 61 umol, 72%).
^{1 1} H-NMR (DMSO-d ₆ ): δ10.59 (1H, br.s, NH), 7.93 (1H, s, H-8), 6.44 (2H, br.s, NH ₂ ) , 6.08 (1H, dd, H-1'), 5.12 (1H, d, 3'-OH), 4.91 (1H, t, 5'-OH), 4.35 (1H, q) , H-3 '), 3.42 (2H, m, H-5'), 2.65 (1H, m, H-2 'a), 2.21 (1H, m, H-2' b) , 1.60 (1H, m, CH _3- CH ^a ), 1.55 (1H, m, CH _3- CH ^b ), 0.86 (3H, t, CH ₃ ).

Synthesis Example 17: Synthesis of 2-amino-4'-cyano-2'-deoxy-N ⁶ -methyladenosine 2-Amino-4'-cyano-2'-deoxy-N ⁶ -methyladenosine (Compound 35) was synthesized. To this end, first, the following compound 33 (3', 5'-Di-O-tert-butyldimethylyl-4'-cyano-2'-deoxyguanosine) was synthesized as follows.

That is, compound 32 (see Nucleosides, Nucleosides & Nucleic Acids Vol. 23, N0.4, pp. 671-690, 2004) (355 mg, 1.21 mmol), imidazole (824 mg, 12.1 mmol) in N, N-dimethylformamide (see). It was dissolved in 2.4 mL), tert-butylchlorodimethylsilane (912 mg, 6.05 mmol) was added, and the mixture was heated and stirred at 60 ° C. for 7 hours. Methanol (5 mL) was added to the reaction solution, the mixture was stirred, and the precipitated solid was collected by filtration. The obtained solid was washed with a small amount of methanol and dried under vacuum to obtain Compound 33 (473 mg, 0.908 mmol, yield 75.0%).
^{1 1} H-NMR (DMSO-d ₆ ): δ10.69 (1H, br.s, NH), 7.99 (1H, s, H-8), 6.55 (2H, br.s, NH ₂ ) , 6.30 (1H, dd, H-1'), 4.68 (1H, dd, H-3'), 3.86 (1H, d, H-5'a), 3.79 (1H, 1H, d, H-5'b), 2.99 (1H, m, H-2'a), 2.41 (1H, m, H-2 'b), 0.92 (9H, s, tert-Bu ), 0.87 (9H, s, tert-Bu), 0.17 (3H, s, Me), 0.16 (3H, s, Me), 0.06 (3H, s, Me), 0. 05 (3H, s, Me).

Next, using compound 33 obtained as described above, compound 34 (2-Amino-3', 5'-di-O-tert-butyldimethylyl-4'-cyano-2'-deoxy-N ^6- Methyladenocine) was synthesized as follows.

That is, compound 33 (300 mg, 0.576 mmol) was suspended in dichloromethane (2.9 mL), triethylamine (482 uL, 3.46 mmol), 4-dimethylaminopyridine (58 mg, 0.47 mmol), 2,4,6 chloride. -Triisopropylbenzenesulfonyl (523 mg, 1.73 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was washed with saturated aqueous sodium hydrogen carbonate solution, and the organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 5: 1). The obtained residue was dissolved in a 40% methylamine methanol solution (20 mL) and stirred at room temperature for 3 hours. After concentrating the reaction solution, the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain Compound 34 (253 mg, 0.474 mmol, 96.1%).
^{1 1} H-NMR (CDCl ₃ ): δ7.55 (1H, s, H-8), 6.32 (1H, t, H-1'), 5.59 (1Hbr.s, 6-NH), 4 .92 (1H, t, H- 3 '), 4.66 (2H, br.s, 2-NH 2), 4.01 (1H, d, H-5' a), 3.86 (1H, ^{d, H-5 'b)} , 3.11-3.07 (4H, m, N 6 -Me &H-2' a), 2.48 (1H, m, H-2 'b), 0.96 ( 9H, s, tert-Bu), 0.89 (9H, s, tert-Bu), 0.19 (3H, s, Me), 0.17 (3H, s, Me), 0.08 (3H, 3H, s, Me), 0.04 (3H, s, Me).

Next, using the compound 34 obtained as described above, compound 35 (2-Amino-4'-ciano-2'-deoxy-N ^6- methyladenocine) was synthesized as follows.

That is, compound 34 (253 mg, 0.474 mmol) was dissolved in tetrahydrofuran (2.4 mL), a 1 M tetrabutylammonium fluoride tetrahydrofuran solution (1.42 mL, 1.42 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (chloroform: methanol = 20: 1 to 10: 1). The residue was dissolved in a small amount of deionized water, pre-frozen and lyophilized to give compound 35 (253 mg, 0.474 mmol, 96.1%).
^{1 1} H-NMR (DMSO-d ₆ ): δ7.91 (1H, s, H-8), 7.29 (1H, br.s, 6-NH), 6.34 (1H, t, H-1) '), 6.26 (1H, d, 3'-OH), 5.93 (2H, br.s, 2-NH ₂ ), 5.85 (1H, t, 5'-OH), 4.63 (1H, q, H-3 '), 3.77 (1H, dd, H-5' a), 3.65 (1H, dd, H-5 'b), 2.87 (4H, m, N ^{6 -Me & H-2 'a} ), 2.37 (1H, m, H-2' b).

Synthesis Example 18: Synthesis of 4'-azido-2'-deoxy-O ⁶ -methylguanosine In order to synthesize 4'-azido-2'-deoxy-O ⁶ -methylguanosine (Compound 39), first, the following compound 36 (4'-Azido-N ² , O ^3'- dinitrogenzoil-5'-(3-chlorobenzoyl) -2'-deoxyguanosine) was synthesized as follows.

That is, first, compound 36 was subjected to J. Med. Chem. It was synthesized according to the method described in 1992, 35, 1440-451. Then, the obtained compound 36 (1.02 g, 1.63 mmol) was dissolved in a solvent (dichloromethane: water = 50 mL: 30 mL), dipotassium hydrogen phosphate (1.70 g, 9.76 mmol), tetrabutylammonium sulfate. Hydrogen salt (1.66 g, 4.89 mmol) and m-chlorobenzoic acid (762 mg, 4.87 mmol) were added and stirred. After cooling to 0 ° C., m-chloroperbenzoic acid (72%, 2.50 g, 10.1 mmol) was added, and stirring was continued overnight at room temperature. After the reaction, a 10% aqueous sodium disulfide solution was added and extracted with dichloromethane. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: ethyl acetate = 3: 1-1: 3) to give compound 37 (638 mg, 0.974 mmol, 60%).
_{^{1 H NMR (CDCl 3, 500MHz}} ) δ12.08 (brs, 1H), 9.62 (s, 1H), 8.17-8.16 (m, 2H), 8.04-8.02 (m, 2H), 7.76 (s, 1H), 7.73-7.70 (m, 2H), 7.64-7.57 (m, 4H), 7.48-7.43 (m, 3H) , 7.17 (t, J = 8.0Hz, 1H), 7.01 (dd, J = 9.2,7.5Hz, 1H), 6.37 (dd, J = 8.0, 2.3Hz) , 1H), 5.12 (d, J = 12.0Hz, 1H), 4.66 (d, J = 12.0Hz, 1H), 3.26 (ddd, J = 13.2,7.5, 2.3Hz, 1H), 2.90-2.84 (m, 1H).

Next, using the compound 37 obtained as described above, the following compound 38 (4'-Azido-N ² , O ^3'- dinitrogenzoyl-5'-(3-chromobenzyl) -2'-deoxy-O ⁶ -(2,4,6-triisopropylbenzensulfonyl) guanosine) was synthesized.

That is, triethylamine (781uL, 5.60 mmol), 2,4,6-triisopropylbenzenesulfonyl chloride (848 mg, 2.80 mmol), 4-dimethyl in a solution of compound 37 (612 mg, 0.934 mmol) in dichloromethane (15 mL). Aminopyridine (23 mg, 0.188 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane and washed with saturated aqueous ammonium chloride solution and saturated brine. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: ethyl acetate = 97: 3 to 95: 5) to give compound 38 (421 mg, 0.456 mmol, 49%).
¹ H NMR (CDCl ₃ , 500 MHz) δ8.59 (bs, 1H), 8.12-8.10 (m, 2H), 8.03 (s, 1H), 7.89 (t, J = 1. 7Hz, 1H), 7.79-7.74 (m, 3H), 7.63-7.41 (m, 7H), 7.23 (s, 2H), 7.19 (t, J = 8. 0Hz, 1H), 6.85 (t, J = 8.0Hz, 1H), 6.52 (dd, J = 8.0, 3.4Hz, 1H), 5.05 (d, J = 12.0) , 1H), 4.88 (d, J = 12.0, 1H), 4.28 (sep, J = 6.9Hz, 2H), 3.54-3.48 (m, 1H), 2.96 -2.87 (m, 2H), 1.29 (d, J = 6.9, 6H), 1.28 (d, J = 6.9, 6H), 1.26 (d, J = 6. 9,6H).

Next, using the compound 38 obtained as described above, compound 39 (4'-Azido-2'-deoxy-O ^6- methylguanocine) was synthesized as follows.

That is, in a solution of compound 38 (200 mg, 0.217 mmol) in dioxane (5 mL), 1,4-diazabicyclo [2.2.2] octane (51.5 mg, 0.459 mmol), molecular sieves (3A) (250 mg). Was added, and the mixture was stirred at room temperature for 30 minutes. Then, methanol (99 uL, 2.30 mmol) and diazabicycloundecene (85 uL, 0.569 mmol) were added, and the mixture was stirred at room temperature for 14 hours. After the reaction, the insoluble material was filtered off and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a 5M ammonia-methanol solution (10 mL), and the mixture was heated and stirred overnight at 55 ° C. in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane: methanol = 97: 3 to 9: 1) to obtain Compound 39 (47.0 mg, 0.146 mmol, 67%). ..
^{1 1} H NMR (DMSO-d 6,500 MHz) δ8.08 (s, 1H), 6.54 (brs, 2H), 6.38 (dd, J = 7.5, 5.2 Hz, 1H), 5.78 (D, J = 4.6,1H), 5.48 (dd, J = 6.3,5.8Hz, 1H), 4.66 (dt, J = 6.3,5.2Hz, 1H), 3.96 (s, 3H), 3.67 (dd, J = 12.0, 6.3Hz, 1H), 3.57 (dd, J = 12.0, 5.8Hz, 1H) 2.75- 2.70 (m, 1H), 2.47-2.41 (m, 1H).

Synthesis Example 19: 4'-azido-2'-deoxy -O ⁶ - Synthesis of ethyl guanosine 4'-azido-2'-deoxy -O ⁶ - ethyl guanosine (compound 40) was synthesized as follows.

That is, compound 38 (200 mg, 0.217 mmol) obtained in Synthesis Example 18, 1,4-diazabicyclo [2.2.2] octane (48.7 mg, 0.434 mmol), molecular sieves (3A) (250 mg). ) Was added to the eggplant flask (5 mL), and the mixture was stirred at room temperature for 30 minutes. Then, ethanol (127 uL, 2.17 mmol) and diazabicycloundecene (81 uL, 0.543 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction, the insoluble material was filtered off and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a 9M ammonia-methanol solution (10 mL) and heated and stirred overnight at 55 ° C. in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane: methanol = 98: 2-94: 6) to obtain Compound 40 (47.1 mg, 0.140 mmol, 65%). ..
_{^{1 H NMR (DMSO-d 6}} , 500MHz) δ8.06 (s, 1H), 6.48 (brs, 2H), 6.38 (dd, J = 7.5,5.2Hz, 1H), 5. 77 (d, J = 5.7Hz, 1H), 5.48 (t, J = 5.7Hz, 1H), 4.66 (dd, J = 10.9, 5.7Hz, 1H), 4.45 (Q, J = 6.9Hz, 2H), 3.67 (dd, J = 12.0, 5.7Hz, 1H), 3.57 (dd, J = 12.0, 5.7Hz, 1H), 2.74-2.68 (m, 1H), 2.47-2.40 (m, 1H), 1.35 (t, J = 6.9Hz, 3H).

Synthesis Example 20: Synthesis of O ⁶ -allyl-4'-azido-2'-deoxyguanosine O ⁶ -allyl-4'-azido-2'-deoxyguanosine (Compound 41) was synthesized as follows.

That is, 1,4-diazabicyclo [2.2.2] octane (49.2 mg, 0.438 mmol), in a dioxane (5 mL) solution of compound 38 (202 mg, 0.219 mmol) obtained in Synthesis Example 18. Molecular Sieves (3A) (250 mg) was added and stirred at room temperature for 30 minutes. Then, allyl alcohol (149 uL, 2.19 mmol) and diazabicycloundecene (82 uL, 0.548 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After the reaction, the insoluble material was filtered off and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a 7M ammonia-methanol solution (15 mL) and heated and stirred at 60 ° C. overnight in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (hexane: ethyl acetate = 1: 9 to 8:92) to obtain Compound 41 (41 mg, 0.118 mmol, 54%).
^{1 1} H NMR (Methanol-d 4,500 MHz) δ8.05 (s, 1H), 6.48 (dd, J = 7.5, 5.2 Hz, 1H), 6.18-6.08 (m, 1H) , 5.48-5.42 (m, 1H), 5.29-5.24 (m, 1H), 5.01-4.98 (m, 2H), 4.81 (t, J = 6. 9Hz, 1H), 3.83 (d, J = 12.0Hz, 1H), 3.71 (d, J = 12.0Hz, 1H), 2.91-2.84 (m, 1H), 2. 56-2.51 (m, 1H).

Synthesis Example 21: Synthesis of 4'-azido-O ⁶ -benzyl-2'-deoxyguanosine 4'-Azide-O ⁶ -benzyl-2'-deoxyguanosine (Compound 42) was synthesized as follows.

That is, 1,4-diazabicyclo [2.2.2] octane (76.0 mg, 0.682 mmol), in a dioxane (5 mL) solution of compound 38 (314 mg, 0.341 mmol) obtained in Synthesis Example 18. Molecular Sieves (3A) (250 mg) was added and stirred at room temperature for 30 minutes. Then, benzyl alcohol (239 mg, 3.41 mmol) and diazabicycloundecene (127 uL, 0.852 mmol) were added, and the mixture was stirred at room temperature for 14 hours. After the reaction, the insoluble material was filtered off and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a 7M ammonia-methanol solution (15 mL) and heated and stirred overnight at 65 ° C. in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by amino silica gel chromatography (dichloromethane: methanol = 98: 2-95: 5) to obtain compound 42 (47 mg, 0.118 mmol, 35%).
^{1 1} H NMR (Methanol-d ₄ , 500 MHz) δ; 8.03 (s, 1H), 7.50-7.49 (m, 2H), 7.37-7.28 (m, 3H), 6. 47 (dd, J = 6.9, 5.2Hz, 1H), 5.53 (s, 2H), 4.81 (t, J = 6.9Hz, 1H), 3.82 (d, J = 12) .0Hz, 1H), 3.70 (d, J = 12.0Hz, 1H), 2.87 (ddd, J = 12.0, 6.9, 5.2Hz, 1H), 2.56-2. 50 (m, 1H).

Synthesis Example 22: Synthesis of 2-amino-4'-azido-2'-deoxy-N ⁶ -methyladenosine 2-amino-4'-azido-2'-deoxy-N ⁶ -methyladenosine (Compound 43), It was synthesized as follows.

That is, the compound 38 (231 mg, 0.251 mmol) obtained in Synthesis Example 18 was dissolved in a 30% methylamine-ethanol solution (10 mL), and the mixture was heated and stirred overnight at 55 ° C. in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane: methanol = 98: 2-9: 1) to obtain Compound 43 (21 mg, 0.0653 mmol, 26%).
^{1 1} H NMR (Methanol-d 4,500 MHz) δ7.89 (s, 1H), 6.44 (t, J = 6.3 Hz, 1H), 4.75 (dd, J = 6.3, 5.7 Hz, 1H), 3.82 (d, J = 12.0Hz, 1H), 3.66 (d, J = 12.0Hz, 1H), 3.03 (brs, 3H), 2.92-2.87 ( m, 1H), 2.53-2.48 (m, 1H).

Synthesis Example 23: 2-amino-4'-azido-2'-deoxy -N ⁶ - Synthesis of ethyl adenosine 2-amino-4'-azido-2'-deoxy -N ⁶ - ethyl adenosine (Compound 44), It was synthesized as follows.

That is, the compound 38 (201 mg, 0.218 mmol) obtained in Synthesis Example 18 was dissolved in a 2M ethylamine-methanol solution (15 mL), and the mixture was heated and stirred at 70 ° C. overnight in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane: methanol = 98: 2-93: 7) to obtain Compound 44 (35 mg, 0.104 mmol, 48%).
1H NMR (Methanol-d4,500MHz) δ7.89 (s, 1H), 6.44 (t, J = 6.3Hz, 1H), 4.78 (dd, J = 6.3,5.7Hz, 1H) ), 3.82 (d, J = 12.0Hz, 1H), 3.66 (d, J = 12.0Hz, 1H), 3.53 (m, 2H), 2.89 (ddd, J = 13) .2,6.3,6.3Hz, 1H), 2.54-2.48 (m, 1H), 1.25 (t, J = 7.2Hz, 3H).

Synthesis Example 24: Synthesis of 2-amino-4'-azido-2'-deoxy-N ⁶ -cyclopropyladenosine 2-amino-4'-azido-2'-deoxy-N ⁶ -cyclopropyladenosine (Compound 45) Was synthesized as follows.

That is, compound 38 (200 mg, 0.217 mmol) obtained in Synthesis Example 18, 1,4-diazabicyclo [2.2.2] octane (48.7 mg, 0.434 mmol), molecular sieves (3A) (250 mg). ) Was added to the eggplant flask (5 mL), and the mixture was stirred at room temperature for 30 minutes. Then, cyclopropylamine (151 L, 2.17 mmol) and diazabicycloundecene (81 uL, 0.543 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction, the insoluble material was filtered off and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in 10 mL of a 9 M ammonia-methanol solution, and the mixture was heated and stirred overnight at 55 ° C. in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane: methanol = 99: 1 to 95: 5) to obtain Compound 45 (45.1 mg, 0.130 mmol, 60%). ..
^{1 1} H NMR (Methanol-d 4,500 MHz) δ7.90 (s, 1H), 6.44 (t, J = 6.3Hz, 1H), 4.75 (t, J = 6.3Hz, 1H), 3 .81 (d, J = 12.0Hz, 1H), 3.66 (d, J = 12.0Hz, 1H), 2.91-2.84 (m, 1H), 2.90 (brs, 1H) , 2.54-2.47 (m, 1H), 0.85-0.80 (m, 2H), 0.62-0.57 (m, 2H).

Synthesis Example 25: Synthesis of 4'-azido-2'-deoxy-2-fluoro-O ⁶ -methyl inosine 4'-azido-2'-deoxy-2-fluoro-O ⁶ -methyl inosine (Compound 46), It was synthesized as follows.

That is, while stirring a pyridine (1 mL) solution of compound 38 (88.0 mg, 0.273 mmol) obtained in Synthesis Example 18 at 0 ° C., hydrogen fluoride-pyridine (1.8 mL, 64.3 mmol), nitrite. Tert-Butyl nitrate (82 uL, 0.692 mmol) was added, followed by stirring at room temperature for 1 hour. Then, the reaction solution was added to a 50% aqueous potassium carbonate solution, quenched, and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over potassium carbonate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: methanol = 100: 0-96: 4) to give compound 46 (42.7 mg, 0.131 mmol, 48%).
_{^{1 H NMR (DMSO-d 6}} , 500MHz) δ8.55 (d, J = 0.6Hz, 1H), 6.49 (ddd, J = 7.6,4.0,0.6Hz, 1H), 5 .80 (dd, J = 5.7, 0.6Hz, 1H), 5.38 (ddd, J = 6.3, 5.7, 0.6Hz, 1H), 4.74 (ddd, J = 13) .2,7.5,0.6Hz, 1H), 4.12 (s, 3H), 3.69 (ddd, J = 12.0, 5.7, 0.6Hz, 1H), 3.60 ( ddd, J = 12.0, 5.7, 0.6Hz, 1H), 2.84-2.78 (m, 1H), 2.56-2.51 (m, 1H).

Synthesis Example 26: Synthesis of 4'-azido-2'-deoxyinosine 4'-azido-2'-deoxyinosine (Compound 47) was synthesized as follows.

That is, compound 38 (117 mg, 0.400 mmol) obtained in Synthesis Example 18 was dissolved in 10 mM Tris-hydrochloric acid buffer (pH 7.5) (30.0 mL), and calf spleen-derived adenosine deaminase (30 uL, 35 units) was dissolved. Was added, and the mixture was stirred at 37 ° C. for 2 hours. The reaction mixture was concentrated and purified by silica gel chromatography (dichloromethane: methanol = 9: 1 to 85:15) to give compound 47 (94.0 mg, 0.321 mmol, 80%).
_{^{1 H NMR (DMSO-d 6}} , 500MHz) δ12.45 (s, 1H), 8.31 (s, 1H), 8.09 (s, 1H), 6.48 (dd, J = 7.5, 4.7Hz, 1H), 5.83 (brs, 1H), 5.48 (brs, 1H), 4.69 (t, J = 7.2Hz, 1H), 3.68 (d, J = 12. 0Hz, 1H), 3.58 (d, J = 12.0Hz, 1H), 2.77 (ddd, J = 13.4, 7.2, 4.7Hz, 1H), 2.51-2.49 (M, 1H).

Synthesis Example 27: Synthesis of 2-amino-4'-azido-2'-deoxyadenosine In order to synthesize 2-amino-4'-azido-2'-deoxyadenosine (Compound 53), first, the following compound 48 (2) -Amino-2', 5'-dideoxy-5'-iododenocine) was synthesized as follows.

Iodine (14.3 g, 56.3 mmol) was added to a pyridine (100 mL) solution of 2-Amino-2'-deoxydenocine (10.0 g, 37.6 mmol) and triphenylphosphine (14.8 g, 56.3 mmol). Stirring was performed at room temperature. After 4 hours, a 10% aqueous sodium thiosulfate solution was added to the reaction solution for distillation under reduced pressure, and then 100 mL of dichloromethane and 100 mL of water were added to the residue and the mixture was stirred on an ice bath. Then, the precipitate was collected by filtration and vacuum dried at 40 ° C. to obtain Compound 48 (11.7 g, 31.1 mmol, 83%).
^{1 1} H NMR (DMSO-d ₆ , 500 MHz) δ7.94 (s, 1H), 6.72 (brs, 2H), 6.20 (dd, J = 8.0, 6.3 Hz, 1H), 5. 82 (brs, 2H), 5.50 (d, J = 4.0Hz, 1H), 4.37-4.33 (m, 1H), 3.95-3.90 (m, 1H), 3. 53 (dd, J = 10.3, 6.9Hz, 1H), 3.41 (dd, J = 10.3, 6.6Hz, 1H), 2.88-2.80 (m, 1H), 2 .24-2.18 (m, 1H).

Next, using the compound 48 obtained as described above, compound 49 (2-Amino-9- (2,5-Dideoxy-β-D-glycero-pent-4-enoufranosyl) adenine) was added to the following. It was synthesized according to the street.

That is, diazabicycloundecene (13.9 mL, 93.3 mmol) was added to a solution of compound 48 (11.7 g, 31.1 mmol) in acetonitrile (100 mL), and the mixture was heated and stirred at 80 ° C. for 2 hours. After distilling off the solvent under reduced pressure, the residue was purified by silica gel chromatography (dichloromethane: methanol = 90: 10 to 85:15). Then, the mixture was stirred and washed with 20 mL of dichloromethane on an ice bath and vacuum dried at 40 ° C. to obtain Compound 49 (2.09 g, 8.42 mmol, 27%).
^{1 1} H NMR (DMSO-d ₆ , 500 MHz) δ7.90 (s, 1H), 6.74 (brs, 2H), 6.40 (t, J = 6.3Hz, 1H), 5.85 (brs, bs, 2H), 5.57 (d, J = 5.2Hz, 1H), 4.93-4.89 (m, 1H), 4.22 (s, 1H), 4.12 (s, 1H), 2 .92-2.85 (m, 1H), 2.35-2.28 (m, 1H).

Next, using the compound 49 obtained as described above, compound 50 (2-Amino-4'-azido-2', 5'-dideoxy-5'-iodoadenocine) was synthesized as follows. ..

That is, dimethylformamide of compound 49 (2.09 g, 8.41 mmol) in a solution of sodium azide (2.74 g, 42.1 mmol) and iodine monochloride (3.42 g, 21.0 mmol) in dimethylformamide (8 mL). The (16 mL) solution was added slowly and stirred at room temperature for 1 hour. Then, saturated layered water and a 10% aqueous sodium thiosulfate solution were added on an ice bath, extracted with dichloromethane, washed with saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: methanol = 95: 5 to 90:10) to give compound 50 (2.23 g, 5.35 mmol, 64%).
_{^{1 H NMR (DMSO-d 6}} , 500MHz) δ7.97 (s, 1H), 6.75 (brs, 2H), 6.38 (t, J = 6.9Hz, 1H), 6.20 (d, J = 5.2Hz, 1H), 5.87 (brs, 2H), 4.79-4.74 (m, 1H), 3.76 (d, J = 10.9Hz, 1H), 3.61 ( d, J = 10.9Hz, 1H), 3.04-2.97 (m, 1H), 2.48-2.41 (m, 1H).

Next, using the compound 50 obtained as described above, compound 51 (2-Amino-4'-azido-3'-O-benzoyl-2', 5'-dideoxy-5'-iododenocine) was added. It was synthesized as follows.

That is, benzoic anhydride (794 mg, 3.51 mmol) and N, N-dimethylaminopyridine (58.6 mg, 0.479 mmol) were added to a pyridine (10 mL) solution of compound 50 (1.00 g, 2.40 mmol). Was stirred at room temperature for 1 hour. Further, N, N-dimethylaminopyridine (58.6 mg, 0.479 mmol) was added, the mixture was stirred for 1 hour, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with dichloromethane. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: methanol = 99: 1-95: 5) to give compound 51 (722.6 mg, 1.39 mmol, 58%).
_{^{1 H NMR (DMSO-d 6}} , 500MHz) δ8.10-8.07 (m, 2H), 8.02 (s, 1H), 7.76-7.72 (m, 1H), 7.63- 7.59 (m, 2H), 6.81 (brs, 2H), 6.58 (t, J = 6.9Hz, 1H), 6.04 (dd, J = 6.3, 4.0Hz, 1H) ), 5.91 (brs, 2H), 3.99 (d, J = 11.5Hz, 1H), 3.78 (d, J = 11.5Hz, 1H), 3.48-3.41 (m) , 1H), 2.86-2.79 (m, 1H).

Next, using compound 51 obtained as described above, compound 52 (N ^4- acetyl-2-acetyllamino-4'-azido-3'-O-benzoyl-2', 5'-dideoxy-5' -Iodoadenosine) was synthesized as follows.

That is, acetic anhydride (0.17 mL, 1.76 mmol) and N, N-dimethylaminopyridine (58.6 mg, 0.479 mmol) were added to a pyridine (5 mL) solution of compound 51 (230.0 mg, 0.441 mmol). In addition, the mixture was heated and stirred at 40 ° C. overnight. After adding a saturated aqueous sodium hydrogen carbonate solution, extraction was performed with dichloromethane. After washing with saturated brine, the mixture was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: methanol = 97: 3 to 93: 7) to give compound 52 (134 mg, 0.221 mmol, 50%).
^{1 1} H NMR (DMSO-d ₆ , 500 MHz) δ10.66 (brs, 1H), 10.52 (brs, 1H), 8.52 (s, 1H), 8.12-8.08 (m, 2H) , 7.77-7.72 (m, 1H), 7.64-7.59 (m, 2H), 6.76 (t, J = 6.9Hz, 1H), 6.21 (dd, J = 6.3, 4.6Hz, 1H), 4.17 (d, J = 11.5Hz, 1H), 3.85 (d, J = 11.5Hz, 1H), 3.67-3.60 (m) , 1H), 2.89-2.82 (m, 1H), 2.31 (s, 3H), 2.22 (s, 3H).

Next, using the compound 52 obtained as described above, compound 53 (2-Amino-4'-azido-2'-deoxyadenosine) was synthesized as follows.

That is, compound 52 (133.0 mg, 0.220 mmol) was dissolved in a solvent (dichloromethane: water = 7 mL: 4 mL), dipotassium hydrogen phosphate (153.3 mg, 0.880 mmol), and tetrabutylammonium hydrogen sulfate (164). .3 mg, 0.484 mmol) and m-chlorobenzoic acid (75.8 mg, 0.484 mmol) were added and stirred. After cooling to 0 ° C., m-chloroperbenzoic acid (72%, 158.2 mg, 0.660 mmol) was added, and the mixture was stirred at room temperature. After 3 hours, the same amount of each reagent was added and stirring was continued overnight. After the reaction, a 10% aqueous sodium disulfide solution was added and extracted with dichloromethane. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: methanol = 100: 0-92: 8). The resulting product was dissolved in a 30% methylamine-ethanol solution (10 mL) and heated and stirred at 50 ° C. overnight in a sealed tube. Then, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane: methanol = 91: 9 to 85:15) to obtain compound 53 (17.6 mg, 0.0572 mmol, 2 steps 26). %).
^{1 1} H NMR (DMSO-d ₆ , 500 MHz) δ7.92 (s, 1H), 6.77 (brs, 2H), 6.34 (dd, J = 7.5,5.7 Hz, 1H), 5. 83 (brs, 2H), 5.78 (brs, 1H), 5.66 (brs, 1H), 4.64 (t, J = 6.3Hz, 1H), 3.67 (dd, J = 12. 0,4.0Hz, 1H), 3.54 (dd, J = 12.0, 4.6Hz, 1H), 2.76-2.69 (m, 1H), 2.44-2.37 (m) , 1H).

Synthesis Example 30: Synthesis of common intermediates such as compound 80 When synthesizing compounds 80, 82, 84 and 97 described later, intermediates (compound 78) common to these compounds were synthesized by the reaction steps shown below. ..

First, from compound 73 (see Nucleoside & Nucleotide, 1985, 4, 641-649), compound 74 (9- (3-O-tert-butyldimethylsilyl-2-O-deoxy-β-D-ribofuranosyl) -1-hydro. -2-isobutyrylamino-6H-purine-6-one) was synthesized. That is, compound 73 (8.33 g, 13 mmol) was dissolved in N, N-dimethylformamide (65 mL), followed by imidazole (2.83 g, 42 mmol) and tert-butyldimethylsilyl chloride (5.89 g, 39 mmol). They were added sequentially and stirred at room temperature for 20 hours. After completion of the reaction, quenching was performed with saturated aqueous sodium hydrogen carbonate, and then extraction was performed with ethyl acetate. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified 3'hydroxyl protectant (13 mmol). A crudely purified 3'hydroxyl protectant (13 mmol) was dissolved in chloroform (87 mL), and then p-toluenesulfonic acid monohydrate (7.42 g, 39 mmol) dissolved in methanol (37 mL) was added at −15 ° C. And stirred at the same temperature for 1.5 hours. After completion of the reaction, saturated aqueous sodium hydrogen carbonate was added until the reaction solution turned orange to colorless, and extraction was performed with chloroform. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue is purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1 → ethyl acetate → methanol / chloroform = 1/20). This was carried out to obtain compound 74 (5.44 g, 12 mmol, 2 steps 93%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ12.08 (1H, brs), 8.36 (1H, brs), 7.73 (1H, s), 6.19 (1H, dd, J = 9. 0,5.5Hz), 5.21 (1H, d, J = 10.5Hz), 4.61 (1H, d, J = 5.5Hz), 4.12 (1H, d, J = 1.0Hz) ), 3.94 (1H, m), 3.74 (1H, m), 2.79 (1H, m), 2.65 (1H, m), 2.23 (1H, m), 1.28 (3H, d, J = 3.0Hz), 1.27 (3H, d, J = 3.0Hz), 0.93 (9H, s), 0.118 (3H, s), 0.115 (3H) , S).

Next, from the compound 74 thus obtained, compound 75 (9- (3-O-tert-butyldimethylsilyl-2-O-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl)- 1-hydro-2-isobutyrylamino-6H-purine-6-one) was synthesized. That is, compound 74 (103 mg, 0.23 mmol) was dissolved in toluene (0.23 mL) and dimethyl sulfoxide (0.23 mL), followed by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (132 mg). , 0.69 mmol), pyridine (36.9 μL, 0.46 mmol) and trifluoroacetic acid (18 μL, 0.23 mmol) were sequentially added, and the mixture was stirred at room temperature for 14 hours. After completion of the reaction, extraction with ethyl acetate was performed. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crude aldehyde. After dissolving the crude aldehyde in dioxane (4.6 mL), a 37% aqueous formaldehyde solution (100 μL, 1.0 mmol) and a 1N aqueous sodium hydroxide solution (275 μL, 0.28 mmol) are sequentially added, and the temperature is 20 minutes at room temperature. Stirred. Next, 1N aqueous sodium hydroxide solution (275 μL, 0.28 mmol) was added, and the mixture was stirred at room temperature for 35 minutes, sodium borohydride (26.0 mg, 0.69 mmol) was added at 0 ° C., and 45 minutes at room temperature. Stirred. After completion of the reaction, quenching was carried out with 1N hydrochloric acid, and then extraction with ethyl acetate was carried out. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (methanol / chloroform = 1/20) to obtain Compound 75 (38.9 mg, 0. 081 mmol, 2 steps 35%).
_{^{1 H-NMR (CDCl 3,}} 500MHz); δ12.08 (1H, brs), 9.78 (1H, brs), 7.92 (1H, s), 6.27 (1H, t, J = 7. 0Hz), 4.83 (1H, dd, J = 7.0, 4.0Hz), 4.76 (1H, brs), 3.95 (1H, d, J = 12.0Hz), 3.89 ( 1H, d, J = 11.0Hz), 3.68-3.61 (3H, m), 2.85-2.77 (2H, m), 2.41 (1H, m), 1.25 ( 3H, d, J = 7.0Hz), 1.22 (3H, d, J = 7.0Hz), 0.90 (9H, s), 0.13 (3H, s), 0.12 (3H, s).

Next, from the compound 75 thus obtained, compound 76 (9- (3,5-di-O-tert-butyldimethylsilyl-2-O-deoxy-4-C-hydroxymethyl-β-D) -Riboflanosyl) -1-hydro-2-isobutyrylamino-6H-purine-6-one) was synthesized. That is, compound 75 (726 mg, 1.5 mmol) was azeotropically boiled with pyridine, then dissolved in N, N-dimethylformamide (15 mL), and triethylamine (421 μL, 3.0 mmol) and 4,4′- at 0 ° C. Dimethoxytrityl chloride (767 mg, 2.3 mmol) was sequentially added, and the mixture was stirred at room temperature for 20 minutes and at 0 ° C. for 1 hour. After completion of the reaction, quenching was performed with methanol, and then extraction was performed with ethyl acetate. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 2/1 → ethyl acetate), and a crude 6'hydroxyl hydroxyl group was purified. A protected product (958 mg, 1.2 mmol) was obtained. After dissolving the 6'hydroxyl protector (958 mg, 1.2 mmol) in N, N-dimethylformamide (6 mL), imidazole (291 mg, 4.27 mmol) and tert-butyldimethylsilyl chloride (552 mg, 3.7 mmol) Was sequentially added, and the mixture was stirred at room temperature for 25 minutes. After completion of the reaction, quenching was performed with saturated aqueous sodium hydrogen carbonate, and then extraction was performed with ethyl acetate. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified 5'hydroxyl protectant. After dissolving the crude 5'hydroxyl protector in chloroform (8 mL), p-toluenesulfonic acid monohydrate (928 mg, 4.9 mmol) dissolved in methanol (4.5 mL) was added at -15 ° C. The mixture was added dropwise, and the mixture was stirred at the same temperature for 1.5 hours. After completion of the reaction, saturated aqueous sodium hydrogen carbonate was added until the reaction solution turned orange to colorless, and extraction was performed with chloroform. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 2/1) to obtain compound 76 (461 mg, 0). .77 mmol, 3 steps 51%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ12.03 (1H, brs), 8.68 (1H, brs), 7.94 (1H, s), 6.24 (1H, t, J = 6. 0Hz), 4.79 (1H, dd, J = 6.5,5.0Hz), 3.83 (1H, dd, J = 12.0, 4.5Hz), 3.75 (1H, d, J) = 10.5Hz), 3.69-3.65 (2H, m), 2.68-2.58 (3H, m), 2.50-2.45 (1H, m), 1.27 (3H) , D, J = 7.0Hz), 1.24 (3H, d, J = 7.0Hz), 0.92 (9H, s), 0.91 (9H, s), 0.14 (3H, s) ), 0.13 (3H, s), 0.08 (3H, s), 0.07 (3H, s).

Next, from the compound 76 thus obtained, compound 77 (9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D-) Ribofuranosyl) -1-hydro-2-isobutyrylamino-6H-purine-6-one) was synthesized. That is, compound 76 (70.4 mg, 0.12 mmol) was dissolved in toluene (0.12 mL) and dimethyl sulfoxide (0.12 mL), followed by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. (67.9 mg, 0.35 mmol), pyridine (19.0 μL, 0.24 mmol) and trifluoroacetic acid (9.0 μL, 0.12 mmol) were sequentially added, and the mixture was stirred at room temperature for 14 hours. After completion of the reaction, extraction with ethyl acetate was performed. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crude aldehyde. After dissolving the crude aldehyde in pyridine (1.2 mL), hydroxylamine hydrochloride (12.3 mg, 0.18 mmol) was added, and the mixture was stirred at room temperature for 35 minutes. After completion of the reaction, extraction with ethyl acetate was performed. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crude oxime. After dissolving the crude oxime in dichloromethane (1.2 mL), triethylamine (33 μL, 0.24 mmol) and methanesulfonyl chloride (14 μL, 0.18 mmol) were sequentially added at 0 ° C., and the mixture was stirred at the same temperature for 1 hour. .. After completion of the reaction, quenching was performed with saturated aqueous sodium hydrogen carbonate, and then extraction was performed with ethyl acetate. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure, and then the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 2/1) to obtain Compound 77 (61). .1 mg, 0.10 mmol, 3 steps 88%).
_{^{1 H-NMR (CDCl 3,}} 500MHz); δ12.00 (1H, brs), 7.97 (1H, brs), 7.81 (1H, s), 6.36 (1H, t, J = 6. 5Hz), 4.82 (1H, t, J = 6.0Hz), 3.91 (2H, s), 2.66-2.55 (3H, m), 1.31 (6H, d, J = 7.0Hz), 0.97 (9H, s), 0.90 (9H, s), 0.19 (3H, s), 0.17 (3H, s), 0.11 (3H, s), 0.07 (3H, s).

Next, from the compound 77 thus obtained, a common intermediate such as compound 80 (compound 78, 9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-) O-deoxy-β-D-ribofuranosyl) -2-isobutyrylamino-6- (2,4,6-triisopropylbenzenesulfonyloxy) purine) was synthesized. That is, after dissolving compound 77 (46.1 mg, 0.078 mmol) in dichloromethane (1 mL), triethylamine (22 μL, 0.16 mmol), 2,4,6-triisopropylbenzenesulfonyl chloride (47.3 mg, 0) .16 mmol) and 4-dimethylaminopyridine (0.95 mg, 7.8 μmol) were sequentially added, and the mixture was stirred at room temperature for 1.5 hours. After completion of the reaction, quenching was performed with saturated aqueous sodium hydrogen carbonate, and then extraction was performed with ethyl acetate. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/4) to obtain compound 78 (58.5 mg). , 0.068 mmol, 88%).
^{1 1} H-NMR (CDCl ₃ , 500 MHz); δ7.99 (1H, s), 7.95 (1H, brs), 7.23 (2H, s), 6.38 (1H, t, J = 6. 5Hz), 5.18 (1H, t, J = 5.5Hz), 4.25 (2H, m), 4.12 (1H, d, J = 11.5Hz), 3.95 (1H, d, J = 11.0Hz), 3.20 (1H, m), 2.94 (1H, m), 2.69 (1H, brs), 2.51 (1H, m), 1.30-1.26 (18H, m), 1.23 (3H, d, J = 2.0Hz), 1.21 (3H, d, J = 1.5Hz), 0.96 (9H, s), 0.88 (9H) , S), 0.22 (3H, s), 0.14 (3H, s), 0.070 (3H, s), 0.065 (3H, s).

Synthesis Example 31: Synthesis of 2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-dimethylaminopurine 2-amino-9- (4-C-cyano-) In the synthesis of 2-O-deoxy-β-D-ribofuranosyl) -6-dimethylaminopurine (Compound 80), first, the compound 78 obtained in Synthesis Example 30 was used, and the compound 79 (2-amino-9-) was used. (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-dimethylaminopurine) was synthesized as follows.

That is, compound 78 (33.0 mg, 0.039 mmol) was dissolved in tetrahydrofuran (2 mL), a 50% aqueous dimethylamine solution (1 mL) was added, and the mixture was stirred at 50 ° C. for 1 hour. After completion of the reaction, extraction with ethyl acetate was performed. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/2) to obtain compound 79 (18.4 mg). , 0.030 mmol, 77%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ7.69 (1H, s), 7.63 (1H, brs), 6.36 (1H, t, J = 6.5 Hz), 5.03 (1H, 1H, t, J = 5.5Hz), 4.04 (1H, d, J = 11.0Hz), 3.93 (1H, d, J = 11.0Hz), 3.47 (6H, brs), 3. 11 (2H, m), 2.48 (1H, m), 1.26 (6H, d, J = 7.0Hz), 0.97 (9H, s), 0.89 (9H, s), 0 .21 (3H, s), 0.16 (3H, s), 0.083 (3H, s), 0.066 (3H, s).

Next, from the compound 79 thus obtained, compound 80 (2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6) was prepared as follows. -Dimethylaminopurine) was synthesized.

That is, compound 79 (18.4 mg, 0.030 mmol) was dissolved in 2-propanol (0.5 mL), followed by ammonium iodide (4.3 mg, 0.0298 mmol) and hydrazine monohydrate (0.5 mL). ) Was added sequentially, and the mixture was stirred at 70 ° C. for 14.5 hours. After completion of the reaction, extraction with ethyl acetate was performed. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1) to remove the 2-position amino group (0). .030 mmol) was obtained. After dissolving the 2-position amino group deprotected body (0.030 mmol) in tetrahydrofuran (1 mL), tetrabutylammonium fluoride (1.0 M tetrahydrofuran solution, 0.1 mL) was added, and the mixture was stirred at room temperature for 30 minutes. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / chloroform = 1/20) to obtain a crudely purified compound 80. The crudely purified compound 80 was purified by silica gel column chromatography (NH silica, ethyl acetate → methanol / chloroform = 1/10) to obtain compound 80 (7.8 mg, 0.024 mmol, 2 steps 82%).
^{1 1} H-NMR (CD ₃ OD, 500 MHz); δ7.82 (1H, s), 6.43 (1H, t, J = 7.0 Hz), 4.78 (1H, dd, J = 6.0, 4.0Hz), 3.96 (1H, d, J = 12.0Hz), 3.88 (1H, d, J = 12.5Hz), 3.40 (6H, brs), 2.95 (1H, 1H, m), 2.48 (1H, m).

Synthesis Example 32: Synthesis of 2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-cyclopropylaminopurine 2-amino-9- (4-C-cyano) In the synthesis of -2-O-deoxy-β-D-ribofuranosyl) -6-cyclopropylaminopurine (Compound 82), first, the compound 78 obtained in Synthesis Example 30 was used, and the compound 81 (2-amino-) was used. 9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-cyclopropylaminopurine) is as follows. Synthesized.

That is, compound 78 (9.5 mg, 0.011 mmol) was dissolved in tetrahydrofuran (2 mL), cyclopropylamine (0.2 mL) was added, and the mixture was stirred at 50 ° C. for 2 hours. After completion of the reaction, extraction with ethyl acetate was performed. The organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified 6-position cyclopropylamino compound. After dissolving the crudely purified 6-position cyclopropylamino compound (0.011 mmol) in 2-propanol (0.5 mL), ammonium iodide (1.6 mg, 0.011 mmol) and hydrazine monohydrate (0. 5 mL) was added sequentially, and the mixture was stirred at 70 ° C. for 2.5 hours. After completion of the reaction, extraction with ethyl acetate was performed. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1) to obtain compound 81 (5.4 mg). , 0.0097 mmol, 2 steps 87%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ7.56 (1H, s), 6.32 (1H, t, J = 6.5 Hz), 5.73 (1H, brs), 4.92 (1H, 1H, t, J = 6.0Hz), 4.71 (2H, brs), 4.01 (1H, d, J = 11.0Hz), 3.86 (1H, d, J = 11.0Hz), 3. 08 (1H, m), 2.97 (1H, brs), 2.48 (1H, m), 0.96 (9H, s), 0.89 (9H, s), 0.86 (2H, m) ), 0.61 (2H, m), 0.19 (3H, s), 0.17 (3H, s), 0.085 (3H, s), 0.043 (3H, s).

From the compound 81 thus obtained, compound 82 (2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-cyclopropylaminopurine) was added to the following. It was synthesized as follows.

That is, compound 81 (12.9 mg, 0.023 mmol) was dissolved in tetrahydrofuran (1 mL), tetrabutylammonium fluoride (1.0 M tetrahydrofuran solution, 0.1 mL) was added, and the mixture was stirred at room temperature for 10 minutes. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / chloroform = 1/20) to obtain a crudely purified compound 82. The crudely purified compound 82 was purified by silica gel column chromatography (NH silica, ethyl acetate → methanol / chloroform = 1/10) to obtain compound 82 (8.3 mg, 0.0230 mmol, quant.).
^{1 1} H-NMR (CD ₃ OD, 500 MHz); δ7.86 (1H, s), 6.42 (1H, t, J = 6.5 Hz), 4.80 (1H, m), 3.96 (1H) , D, J = 12.0Hz), 3.88 (1H, d, J = 12.5Hz), 2.95 (1H, m), 2.90 (1H, brs), 2.49 (1H, m) ), 0.83 (1H, d, J = 5.0Hz), 0.60 (1H, s).

Synthesis Example 33: Synthesis of 2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-ethylaminopurine 2-amino-9- (4-C-cyano-) In the synthesis of 2-O-deoxy-β-D-ribofuranosyl) -6-ethylaminopurine (Compound 84), first, the compound 78 obtained in Synthesis Example 30 was used, and the compound 83 (2-amino-9-) was used. (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-ethylaminopurine) was synthesized as follows.

That is, compound 78 (29.9 mg, 0.035 mmol) was dissolved in tetrahydrofuran (2 mL), 70% aqueous ethylamine solution (0.5 mL) was added, and the mixture was stirred at 50 ° C. for 1 hour. After completion of the reaction, extraction with ethyl acetate was performed. The organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified 6-position ethylamino compound. After dissolving the crudely purified 6-position ethylamino compound (0.035 mmol) in 2-propanol (0.5 mL), ammonium iodide (5.1 mg, 0.035 mmol) and hydrazine monohydrate (0.5 mL) ) Was added sequentially, and the mixture was stirred at 70 ° C. for 17 hours. After completion of the reaction, extraction with ethyl acetate was performed. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1) to obtain compound 83 (15.0 mg). , 0.027 mmol, 2 steps 79%).
_{^{1 H-NMR (CDCl 3,}} 500MHz); δ7.56 (1H, s), 6.33 (1H, t, J = 6.5Hz), 5.57 (1H, brs), 4.92 (1H, t, J = 6.0Hz), 4.67 (2H, brs), 4.01 (1H, d, J = 11.0Hz), 3.86 (1H, d, J = 11.0Hz), 3. 59 (2H, brs), 3.08 (1H, m), 2.47 (1H, m), 1.26 (3H, t, J = 7.5Hz), 0.96 (9H, s), 0 .89 (9H, s), 0.19 (3H, s), 0.17 (3H, s), 0.078 (3H, s), 0.047 (3H, s).

Next, from the compound 83 thus obtained, compound 84 (2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6) was prepared as follows. -Ethylaminopurine) was synthesized.

That is, compound 83 (15.0 mg, 0.027 mmol) was dissolved in tetrahydrofuran (1 mL), tetrabutylammonium fluoride (1.0 M tetrahydrofuran solution, 0.1 mL) was added, and the mixture was stirred at room temperature for 10 minutes. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / chloroform = 1/20) to obtain a crudely purified compound 84. The crudely purified compound 84 was purified by silica gel column chromatography (NH silica, ethyl acetate → methanol / chloroform = 1/10) to obtain compound 84 (7.6 mg, 0.024 mmol, 87%).
^{1 1} H-NMR (CD ₃ OD, 500 MHz); δ7.85 (1H, s), 6.42 (1H, t, J = 7.0 Hz), 4.80 (1H, dd, J = 6.5) 4.5Hz), 3.97 (1H, d, J = 12.0Hz), 3.89 (1H, d, J = 12.0Hz), 3.54 (2H, brs), 2.96 (1H, 1H, m), 2.49 (1H, m), 1.25 (3H, t, J = 7.0Hz).

Synthesis Example 35: Synthesis of 2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-thiomethoxyaminopurine 2-amino-9- (4-C-cyano) -2-O-deoxy-β-D-ribofuranosyl) -6-thiomethoxyaminopurine (Compound 97) was synthesized as follows.

First, from compound 78 synthesized in Synthesis Example 30, compound 96 (2-amino-9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β) -D-ribofuranosyl) -6-thiomethoxypurine) was synthesized. That is, compound 78 (72.8 mg, 0.085 mmol) was dissolved in tetrahydrofuran (2 mL), sodium thiomethoxydo (29.8 mg, 0.43 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, extraction with ethyl acetate was performed. The organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified 6-position thiomethoxyated product. After dissolving the crudely purified 6-position thiomethoxyylated product (<0.085 mmol) in 2-propanol (1 mL), ammonium iodide (12.3 mg, 0.085 mmol) and hydrazine monohydrate (1 mL) were sequentially added. In addition, it was stirred at room temperature for 4 hours. After completion of the reaction, extraction with ethyl acetate was performed. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/3) to obtain compound 96 (21.3 mg). , 2 steps 46%).
_{^{1 H-NMR (CDCl 3,}} 500MHz); δ7.74 (1H, s), 6.37 (1H, t, J = 6.5Hz), 4.91 (3H, m), 3.98 (1H, d, J = 11.0Hz), 3.87 (1H, d, J = 11.0Hz), 3.03 (1H, m), 2.62 (3H, s), 2.51 (1H, m) , 0.97 (9H, s), 0.88 (9H, s), 0.20 (3H, s), 0.17 (3H, s), 0.09 (3H, s), 0.04 ( 3H, s).

Next, from the compound 96 thus obtained, compound 97 (2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -6-thiomethoxyaminopurine) Was synthesized. That is, compound 96 (32.2 mg, 0.059 mmol) was dissolved in methanol (1 mL), acidic ammonium fluoride (33.4 mg, 0.59 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, acidic ammonium fluoride (33.4 mg, 0.59 mmol) was added, and the mixture was stirred at room temperature for 7 hours. Further, ammonium fluoride (66.8 mg, 1.2 mmol) was added, and the mixture was stirred for 15 hours, then ammonium fluoride (66.8 mg, 1.2 mmol) was added, and the mixture was stirred at room temperature for 96 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / chloroform = 1/10) to obtain Compound 97 (16.6 mg, 88%).
^{1 1} H-NMR (CD ₃ OD, 500 MHz); δ7.99 (1H, s), 6.42 (1H, t, J = 6.5 Hz), 4.83 (1H, t, J = 6.0 Hz) , 3.92 (1H, d, J = 12.5Hz), 3.84 (1H, d, J = 12.0Hz), 2.95 (1H, m), 2.55 (3H, s), 2 .51 (1H, m).

In the nucleoside derivatives obtained synthetically as described above, with respect to the compounds 5, 18, 30, 24, 12, 29, 25, 31, 19, 10, 8, 27, 35, 21 and 9 shown in Table 1. , Antiviral activity and cytotoxicity were evaluated by the methods shown below.

Test Example 1: Evaluation of anti-HBV activity HepG2 2.2 prepared to continuously produce HBV by introducing the HBV gene into a human liver cancer-derived cell line (HepG2 cell) as a test cell. .15 cells were used. HepG2 2.2.15 cells were maintained in continuous culture in DMEM containing 10% fetal bovine serum. Moreover, since the cell has an HBV gene produced as an episome, the DNA of the episome HBV was quantified, and the anti-HBV activity was evaluated by the degree of decrease in the amount in the presence of the nucleoside derivative. The results obtained are shown in Tables 2-4.

More specifically, HepG2 2.2.15 cells were seeded into each well of a 12-well cell culture dish to a concentration of 1.5 × 10 ⁵ cells / 2 mL. When the cells reached 80% confluence, each nucleoside derivative was added at various concentrations. The culture medium to which each nucleoside derivative was added was changed every 4 days, and the cells were cultured in the presence of the derivative for 12 days. Then, from each HepG2 2.2.15 cell, whole cell DNA was extracted using QIAamp DNA Blood Mini Kit (manufactured by QIAGEN) and dissolved in 200 μL of 1 × TE buffer. Then, using the DNA extracted in this way as a template, HBV DNA was quantified by real-time PCR. That is, 2 μL of the DNA extracted from HepG2 2.2.15 cells was amplified using 2 × SYBR PCR master mix (manufactured by Applied Biosystems). The following primer set for detecting the HBV polymerase region was used for the amplification (PCR) reaction:
5'-GCGAGGACTGGGACCCTGTGGACGAAC-3'(SEQ ID NO: 1), and 5'-GTCCACCACGAGTTACGACTGC-3'(SEQ ID NO: 2).
The PCR reaction was carried out at 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute.

The data obtained by such a PCR reaction was analyzed with StepOneTM Software Version 2.0 (manufactured by Applied Biosystems) to obtain a CT value. Next, the CT value was determined by the number of copies of HBV (HBV) in the presence of each nucleoside derivative by a calibration curve prepared by diluting a known concentration of HBV plasmid every 10 times (20 to 2 × ¹⁰⁸ copies). It was converted to the amount of DNA of. Then, compared to that in control cultured in the absence of nucleoside derivatives, calculated EC ₅₀ values from the rate of decrease was evaluated anti-HBV activity of each nucleoside derivatives. The results obtained are shown in Tables 2-4.

Test Example 2: Cytotoxicity test Regarding the above nucleoside derivative, a cytotoxicity test was also performed on HepG2 cells and HepG2 2.2.15 cells. With medium was added to each nucleoside derivatives of various concentrations, to a concentration of 2 × ¹⁰ 3 cells / 100μL respect HepG2 cells, and the concentration of 1.5 × ¹⁰ 4 / 100μL respect HepG2 2.2.15 cells Each was sown so as to be. After culturing these cells for 5 days in the presence of various concentrations of each nucleoside derivative in this way, the number of surviving cells in each well was measured using the Cell Counting Kit-8. Then, based on the obtained number of viable cells, CC ₅₀ was calculated for each nucleoside derivative. The results obtained are shown in Tables 2-4.

Further, in the nucleoside derivatives obtained by synthesizing as described above, the compounds 13, 53, 47, 6, 39, 46, 80, 82, 84, 43, 44, 45, 42, 40, shown in Table 1 For 41 and 97, antiviral activity and cytotoxicity were evaluated by the methods shown below.

Test Example 3: Evaluation of anti-HBV activity HepG2 2.2.1.7 cells having the above HepG2 2.2.15 cells as a parent strain were used as test cells. HepG2 2.2.15 cells were maintained in DMEM containing 10% fetal bovine serum, G418 (500 μg / ml) and antibiotics (penicillin and kanamycin). In addition, HepG2 2.2.1.7 cells, like HepG2 2.2.15 cells, are HBV continuous-producing cells that carry not only the DNA integrated into the genome but also the HBV gene produced as episomes. Therefore, the number of DNA copies of the virus released into the culture supernatant was quantified by co-culturing with each nucleoside derivative, and the degree of decrease was used as an index for evaluating the anti-HBV activity.

More specifically, HepG2 2.2.1.5.7 cells having a cell viability of 90% or more were seeded in a collagen-coated 96-well cell culture dish at a concentration of 2 × 10 ⁴ cells / ml, and on the same day of cell seeding. , Each nucleoside derivative was added at various concentrations. After culturing for 3 days under the standard culture conditions of 37 ° C. and 5% CO _2, the medium was replaced with a fresh medium containing each nucleoside derivative, and HBV DNA was recovered from the culture supernatant on the 3rd day after the replacement. Then, from the collected medium, quantitative PCR was performed in the same manner as described above, the virus copy number was determined from the calibration curve, and the EC ₅₀ for each nucleoside derivative was calculated. The results obtained are shown in Tables 5-7.

Test Example 4: Cytotoxicity test A cytotoxicity test on HepG2 cells was also performed on the above nucleoside derivative. HepG2 cells were seeded to a concentration of 1 × 10 ⁴ cells / ml with a medium supplemented with each concentration of each nucleoside derivative after serial dilution. In this way, after culturing these cells for 7 days under standard culture conditions of 37 ° C. and 5% CO _{2 in} the presence of various concentrations of each nucleoside derivative, the number of surviving cells in each well was quantified by MTT assay. .. Then, based on the obtained number of viable cells, CC ₅₀ was calculated for each nucleoside derivative. The results obtained are shown in Tables 5-7.

Regarding compounds 24, 8, 35 and 21, HBV strains showing resistance to the existing nucleoside derivative preparation entecavir were targeted, and the antiviral activity was evaluated by the method shown below.

Test Example 5: Evaluation of anti-HBV / Ce activity Huh transfected with an ETV resistant strain (genotype: HBV / Ce, referred to as "HBV ETVr" in the table below) with respect to compounds 24, 8, 35 and 21. -7 cells were each added. Then, 72 hours later, DNA was extracted from each cell according to a conventional method, analyzed by Southern blotting using a probe for the HBV gene, the number of DNA copies of the virus was quantified, and the EC ₅₀ value was calculated in the same manner as above. did. The results obtained are shown in Tables 2 and 4.

In the following Table 2-7, items that "* (asterisk)" is assigned with items related to antiviral activity, indicates that The EC ₅₀ values are 0.1μM than at items related cytotoxicity Items marked with "* (asterisk)" indicate that the CC ₅₀ value is less than 50 μM, and items marked with “-” indicate that the test has not been performed.

As is clear from the results shown in Tables 2 to 7, it was revealed that all the nucleoside derivatives synthesized and tested as described above have excellent antiviral activity against HBV, and further, in these nucleoside derivatives. , An amino group in which the 6-position of the purine base is substituted with a relatively bulky functional group (an alkyl group having 1 or more carbon atoms, an alkoxy group having 2 or more carbon atoms which may have a substituent, or 1 carbon number). It was also clarified that the compounds substituted with the above alkyl group-substituted mercapto group) showed antiviral activity against HBV, but generally had low cytotoxicity (Tables 4 and 6). And 7). Furthermore, it has been clarified that these nucleoside derivatives and the like also have antiviral activity against HBV which is resistant to the existing nucleoside derivative entecavir.

As described above, according to the present invention, it is possible to provide a nucleoside derivative having at least excellent antiviral activity against HBV and low toxicity to host cells. The nucleoside derivative of the present invention can also exhibit antiviral activity against HBV which is resistant to existing nucleoside derivatives (entecavir and the like). Therefore, the present invention is extremely useful in the prevention or treatment of DNA virus infections.

SEQ ID NOs: 1 and 2
<223> Sequence of artificially synthesized primers

Claims (4)

A nucleoside derivative represented by the following general formula (1).
[In the above formula, R ¹ represents a hydroxy group, an amino group which may have a substituent, an alkoxy group which may have a substituent, or a mercapto group which may have a substituent. .. R ² represents a hydrogen atom, a halogen atom, or an amino group. R ³ represents an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group. However, in the nucleoside derivative represented by the general formula (1), when R ¹ is an amino group, R ² is a halogen atom or a hydrogen atom, and R ³ is a cyano group, R ¹ is a hydroxy group, R ² is hydrogen atom, and when R ³ is cyano group, R ¹ is hydroxy group, R ² is an amino group, and when R ³ is azido radical, R ^{When 1} is an amino group, R ² is a hydrogen atom and R ³ is an azide group , and R ¹ is a hydroxy group, R ² is a hydrogen atom and R ³ is an azido group. Except for cases . ] In the above formula, R ¹ is substituted with an amino group substituted with an alkyl group having 1 or more carbon atoms, an alkoxy group having 2 or more carbon atoms which may have a substituent, or an alkyl group having 1 or more carbon atoms. The nucleoside derivative according to claim 1, which is a mercapto group. R ¹ is one functional group selected from the group consisting of a methylamino group, a dimethylamino group, an ethylamino group, a cyclopropylamino group, an ethoxy group, an allyloxy group, a benzyloxy group and a methyl mercapto group, and R ² 2. The functional group according to claim 2, wherein is a hydrogen atom or an amino group, and R ³ is a functional group selected from the group consisting of a methyl group, a monofluoromethyl group, an ethenyl group, a cyano group and an azide group. Nucleoside derivative of. An anti -hepatitis B virus agent containing the nucleoside derivative according to any one of claims 1 to 3 as an active ingredient.