WO2018110643A1 - Nucleoside derivative exhibiting antiviral activity - Google Patents

Abstract

[Problem] To provide a nucleoside derivative having antiviral activity against at least HBV and also having low toxicity for host cells. [Solution] A nucleoside derivative represented by general formula (1). In formula (1), R is an optionally substituted alkyl group, an optionally substituted alkenyl group, a cyano group, or an azide group.

Description

Nucleoside derivatives exhibiting antiviral activity

The present invention relates to a nucleoside derivative exhibiting antiviral activity, and more specifically, a 2′-deoxypurine nucleoside derivative having antiviral activity against at least hepatitis B virus, and an antiviral agent comprising the derivative as an active ingredient About.

When hepatitis B virus (HBV) is infected, hepatitis may occur acutely or fulminantly, sometimes leading to death. 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 incidence is extremely high mainly in Southeast Asia, and the development of effective treatment methods is demanded worldwide.

HBV is an incomplete double-stranded DNA virus, and is known to perform reverse transcription to synthesize DNA from RNA in its life cycle. On the other hand, since reverse transcription is not performed in the host human, it is possible to inhibit only HBV replication by inhibiting this step. Nucleoside derivative preparations have been developed as therapeutic agents for HBV infection from such a viewpoint (see, for example, Patent Documents 1 and 2 and Non-Patent Document 1).

JP 2004-244422 A JP 2008-273960 A

Many of the present nucleoside derivative preparations are toxic to host cells, ie, human cells to be taken, and side effects due to medium to long-term use are problematic. In addition, resistant strains to nucleoside derivatives may occur during the period of administration. Therefore, the present condition is that the effective treatment method with respect to viral infections, such as HBV, is not established.

The present invention has been made in view of such circumstances, and an object thereof is to provide a nucleoside derivative having antiviral activity against at least HBV and low toxicity to host cells.

As a result of intensive studies to solve the above problems, the present inventors have found that a nucleoside derivative represented by the following structural formula has antiviral activity against at least HBV, and has particularly low toxicity to host cells. I found out.

 

In formula (1), R is an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group.

That is, in the 2′-deoxyadenosine, the present inventors can obtain an excellent antiviral activity against HBV by setting the 2-position of the purine base as an amino group and the 6-position as a hydrogen atom. At the same time, it has been found that the toxicity to the host cell can be particularly reduced.

That is, the present invention relates to a nucleoside derivative having at least antiviral activity against hepatitis B virus, and an antiviral agent comprising the derivative as an active ingredient, and more specifically provides the following.

<1> A nucleoside derivative represented by the following general formula (1).

In formula (1), R is an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group.

<2> The nucleoside derivative according to <1>, wherein R is a monofluoromethyl group, an ethenyl group, a cyano group, or an azide group.

<3> An antiviral agent comprising the nucleoside derivative according to <1> or <2> as an active ingredient.

<4> The antiviral agent according to <3>, 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 low toxicity to host cells.

(Nucleoside derivatives)
As shown in Examples described later, the nucleoside derivative represented by the following formula was found to have antiviral activity against hepatitis B virus and particularly low toxicity to host cells. Therefore, the present invention relates to a nucleoside derivative exhibiting antiviral activity, and more specifically, to provide a nucleoside derivative represented by the following general formula (1) having at least antiviral activity against hepatitis B virus. is there.

In formula (1), R is an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group.

The nucleoside derivative of the present invention has antiviral activity against at least hepatitis B virus (HBV). In the present invention, “HBV” means a virus having the ability to develop hepatitis B. As HBV, the genotypes of A (A2 / Ae, A1 / Aa), B (Ba, B1 / Bj), C (Cs, Ce), DH and J are known, and the nucleoside of the present invention is used. The derivative only needs to have antiviral activity against at least one genotype of HBV. Among the above genotypes, HBV / Ce is known to be a genotype exhibiting resistance to entecavir, which is an existing nucleoside derivative preparation. Therefore, the nucleoside derivative of the present invention is preferably a nucleoside derivative having antiviral activity against HBV / Ce.

[Correction based on Rule 91 20.12.2017]
In the present invention, the term “antiviral activity” refers to the activity of extinguishing the virus or suppressing its growth in a cell (host cell) infected with a virus such as HBV, for example, suppressing virus replication in the host cell. Activity. In addition, when the target of such suppression 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 an EC ₅₀ value calculated using the virus copy number in the host cell as an index, as shown in the Examples below. 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, still more preferably 0.01 μM or less, and 0.005 μM. More preferably (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 growth. Such activity can be evaluated by the CC ₅₀ value calculated using the number of viable cells as an index, as shown in the Examples described later. The nucleoside derivative of the present invention preferably has a CC ₅₀ value of 10 μM or more, more preferably 50 μM or more, still more preferably 100 μM or more, and particularly 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 the cytotoxicity of the derivative can be reduced, each substituent is preferably selected as shown below.

As described above, R is an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group.

The alkyl group in the “optionally substituted alkyl group” 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 optionally having substituent (s)” is not particularly limited, and examples thereof include a halogen atom, a hydroxy group, an alkoxy group, a cyano group, and an amino group. Atoms are more preferred. More specifically, the “optionally substituted alkyl group” is preferably a monofluoromethyl group.

The alkenyl group in the “optionally substituted alkenyl group” is not particularly limited, but is preferably a linear, branched or cyclic alkenyl group having 2 or more carbon atoms, and having 2 to 6 carbon atoms. A linear, branched, or cyclic alkenyl group is more preferable, and an ethenyl group is more preferable. There is no restriction | limiting in particular as a substituent in "The alkenyl group which may have a substituent", For example, a halogen atom, a hydroxy group, an alkoxy group, a cyano group, an amino group is mentioned.

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. For example, an acid addition salt (hydrochloride, sulfate, hydrobromide, Nitrate, hydrogen sulfate, phosphate, acetate, lactate, succinate, citrate, maleate, hydroxy maleate, tartrate, fumarate, methanesulfonate, p-toluenesulfone Acid salt, camphor sulfonate, sulfamate, mandelate, propionate, glycolate, stearate, malate, ascorbate, pamonate, phenylacetate, glutamate, benzoate , Salicylate, sulfanilate, 2-acetoxybenzoate, ethanedisulfonate, oxalate, isethionate, formate, trifluoroacetate, ethyl Succinate, lactobionate, gluconate, glucoheptonate, 2-hydroxyethanesulfonate, benzenesulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate Oxalate, picrate, thiocyanate, undecanoate, 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). Further, 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, optical isomers based on asymmetric carbon, and stereoisomers. Furthermore, the nucleoside derivative of the present invention is still desirable after undergoing metabolism such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydration oxidation, alkylation, dealkylation, and conjugation in vivo. In addition, the present invention also includes a compound (so-called prodrug form) that produces the nucleoside derivative of the present invention upon metabolism in vivo such as oxidation, reduction, or hydrolysis. . Furthermore, the nucleoside derivative of the present invention can be formulated by a known pharmaceutical method as described later.

Further, the synthesis of the nucleoside derivative of the present invention can be achieved by, for example, combining a ribose sugar (D-ribofuranose protected by substitution of a hydroxy group with an acetyl group, a benzyl group, etc.) and a purine base (adenine, etc.) Reaction via a silyl compound, followed by reduction via a phenoxythiocarbonyl derivative to deoxylate the 2-position of the ribose sugar, and if necessary, a substituent at the desired position of the ribose sugar and / or purine base by known methods Can be done by introducing.
Since the method for synthesizing such a nucleoside derivative of the present invention is shown in detail in the examples described later, those skilled in the art will refer to the description of the examples, reaction raw materials, reaction reagents, reaction conditions (for example, The nucleoside derivative of the present invention can be synthesized by appropriately modifying or modifying these methods as necessary, while appropriately selecting the solvent, reaction temperature, catalyst, reaction time, etc. In addition, the nucleoside derivatives synthesized in this manner can be obtained by appropriately using methods (reverse phase chromatography, ion exchange chromatography, adsorption chromatography, recrystallization methods) used for isolation and purification of general nucleosides and nucleotides. It can be separated and purified by using alone or in combination.

(Antiviral agents, methods for preventing and treating viral infections)
As shown in the Examples described later, the nucleoside derivative of the present invention has at least antiviral activity against hepatitis B virus. Therefore, an antiviral agent comprising the nucleoside derivative of the present invention as an active ingredient can be provided.

The infectious disease targeted by the antiviral agent of the present invention and the preventive and therapeutic methods described below is not particularly limited, and examples thereof include HBV infection. More specifically, hepatitis B (chronic hepatitis, acute Hepatitis, fulminant hepatitis), cirrhosis associated with it, liver fibrosis, and hepatocellular carcinoma.

The antiviral agent of the present invention can be formulated by a known pharmaceutical method. For example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, Use orally or parenterally as elixirs, emulsions, coatings, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. Can do.

In these preparations, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, solvent, base, emulsifier, suspension, surfactant, stabilizer, flavoring agent. , Fragrances, excipients, vehicles, preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrating agents, buffering agents, coating agents, lubricants, coloring agents, sweeteners, It can be appropriately combined with a thickener, a flavoring agent, a solubilizing agent, or other additives. More specifically, as a carrier, 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 Examples include liquid carriers such as pyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, and water.

In addition, 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, propagenium preparation, etc.), liver protection therapy (intravenous injection of glycyrrhizin preparation, oral bile acid preparation, etc.) The antiviral agent of the present invention can also be used in combination therapy.

The preferred dosage form of the antiviral agent of the present invention is not particularly limited, and oral administration or parenteral administration, more specifically, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, airway Examples thereof include internal administration, rectal administration and intramuscular administration, and administration by infusion.

The antiviral agent of the present invention can be used mainly for human subjects, but can also be used for non-human animals such as laboratory animals.

When administering the antiviral agent of the present invention, the dosage is appropriately selected according to the age, weight, symptom, health condition, serious condition, tolerability of the drug, dosage form and the like of the subject. The dose of the antiviral agent of the present invention per day 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 which is an active ingredient, or once or It is administered to the subject in multiple doses. The product of the antiviral agent of the present invention or the instructions thereof may have a label indicating that it is used for treating or preventing a viral infection. Here, “labeled product or description” means that the product body, container, packaging, etc. is labeled, or a description, attached document, 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 to treat viral infections, the administration of the nucleoside derivative of the present invention can inhibit the reverse transcriptase reaction of the virus and suppress the replication of the virus. It can be included as information regarding the mechanism of action of the antiviral agent.

Thus, the present invention can prevent or treat infectious diseases by administering the antiviral agent of the present invention to a subject. Therefore, the present invention also provides a method for preventing or treating a viral infection characterized by 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 viral infections such as HBV, virus holders before onset of infection, and those before infection.

In order to obtain a nucleoside derivative having antiviral activity, a nucleoside derivative represented by the following general formula (1) having a functional group in a combination shown in Table 1 below was synthesized by the method shown below. In addition, the number in a table | surface shows the number of the compound shown below.

 

In addition, compounds C1 and C2 shown in Table 2 below were synthesized as comparative compounds. As reference compounds, compounds R1 to R4 were synthesized in Table 3 below.

In addition, it confirmed that the compound synthesize | combined in this way was a compound which has a desired structure by measuring a < ¹ > H nuclear magnetic resonance (NMR) spectrum. The results are also shown below.

Synthesis Example 1: 9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -2-isobutyrylamino-6- ( Synthesis of 2,4,6-triisopropylbenzenesulfonyloxy) purine (Compound 6) Compound 6 was synthesized by the reaction steps shown below.

First, from compound 1 (see Nucleoside & Nucleotide, 1985, 4, 641-649), compound 2 (9- (3-O-tert-butyldimethylsilyl-2-O-deoxy-β-D-ribofuranosyl) -2-iso Butyrylamino-1H-purin-6-one) was synthesized. That is, after dissolving Compound 1 (8.33 g, 13 mmol) in N, N-dimethylformamide (65 mL), imidazole (2.83 g, 42 mmol) and tert-butyldimethylsilyl chloride (5.89 g, 39 mmol) were added. Sequentially added and stirred at room temperature for 20 hours. After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate and extracted with ethyl acetate. Subsequently, the organic layer was dried with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified 3 'hydroxyl group protector (13 mmol). The crude 3′-hydroxyl-protected product (13 mmol) was dissolved in chloroform (87 mL), and 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 became colorless from orange, and extraction with chloroform was performed. After drying the organic layer with magnesium sulfate and evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1 → ethyl acetate → methanol / chloroform = 1/20). And compound 2 was obtained (5.44 g, 12 mmol, 2 steps 93%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 12.08 (1H, brs), 8.36 (1H, brs), 7.73 (1H, s), 6.19 (1H, dd, J = 9. 0, 5.5 Hz), 5.21 (1 H, d, J = 10.5 Hz), 4.61 (1 H, d, J = 5.5 Hz), 4.12 (1 H, d, J = 1.0 Hz) ), 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 = 3.0 Hz), 1.27 (3H, d, J = 3.0 Hz), 0.93 (9H, s), 0.118 (3H, s), 0.115 (3H , S).

Next, from compound 2 thus obtained, compound 3 (9- (3-O-tert-butyldimethylsilyl-2-O-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl)- 2-isobutyrylamino-1H-purin-6-one) was synthesized. Specifically, Compound 2 (103 mg, 0.23 mmol) was dissolved in toluene (0.23 mL) and dimethyl sulfoxide (0.23 mL), and then 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 with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crude aldehyde. After dissolving the crude aldehyde in 1,4-dioxane (4.6 mL), a 37% formaldehyde aqueous solution (100 μL, 1.0 mmol) and a 1N aqueous sodium hydroxide solution (275 μL, 0.28 mmol) were sequentially added. And stirred at room temperature for 20 minutes. Next, 1N aqueous sodium hydroxide solution (275 μL, 0.28 mmol) was added, and the mixture was stirred at room temperature for 35 minutes, and then sodium borohydride (26.0 mg, 0.69 mmol) was added at 0 ° C. Stir for minutes. After completion of the reaction, the reaction mixture was quenched with 1N hydrochloric acid and extracted 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 (methanol / chloroform = 1/20) to obtain Compound 3 (38.9 mg, 0.00). 081 mmol, 35% for 2 steps).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 12.08 (1H, brs), 9.78 (1H, brs), 7.92 (1H, s), 6.27 (1H, t, J = 7. 0 Hz), 4.83 (1H, dd, J = 7.0, 4.0 Hz), 4.76 (1H, brs), 3.95 (1H, d, J = 12.0 Hz), 3.89 ( 1H, d, J = 11.0 Hz), 3.68-3.61 (3H, m), 2.85-2.77 (2H, m), 2.41 (1H, m), 1.25 ( 3H, d, J = 7.0 Hz), 1.22 (3H, d, J = 7.0 Hz), 0.90 (9H, s), 0.13 (3H, s), 0.12 (3H, s).

Next, from compound 3 thus obtained, compound 4 (9- (3,5-di-O-tert-butyldimethylsilyl-2-O-deoxy-4-C-hydroxymethyl-β-D -Ribofuranosyl) -2-isobutyrylamino-1H-purin-6-one) was synthesized. That is, Compound 3 (726 mg, 1.5 mmol) was azeotroped with pyridine and then dissolved in N, N-dimethylformamide (15 mL), and triethylamine (421 μL, 3.0 mmol) and 4,4′- were dissolved 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, the reaction was quenched with methanol and extracted with ethyl acetate. After drying the organic layer with magnesium sulfate and evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 2/1 → ethyl acetate) to obtain a crude 6 ′ hydroxyl group. The protector (958 mg, 1.2 mmol) was obtained. A 6′-hydroxyl-protected product (958 mg, 1.2 mmol) was dissolved in N, N-dimethylformamide (6 mL), and then imidazole (291 mg, 4.27 mmol) and tert-butyldimethylsilyl chloride (552 mg, 3.7 mmol). Were sequentially added and stirred at room temperature for 25 minutes. After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate and extracted with ethyl acetate. Subsequently, the organic layer was dried with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crude 5′-hydroxyl-protected body. The crude 5′-hydroxyl-protected product was dissolved in chloroform (8 mL), and p-toluenesulfonic acid monohydrate (928 mg, 4.9 mmol) dissolved in methanol (4.5 mL) was added at −15 ° C. The solution was added dropwise 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 became colorless from orange, and extraction with chloroform 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 = 2/1) to obtain Compound 4 (461 mg, 0 .77 mmol, 3 steps 51%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 12.03 (1H, brs), 8.68 (1H, brs), 7.94 (1H, s), 6.24 (1H, t, J = 6. 0 Hz), 4.79 (1H, dd, J = 6.5, 5.0 Hz), 3.83 (1H, dd, J = 12.0, 4.5 Hz), 3.75 (1H, d, J = 10.5 Hz), 3.69-3.65 (2H, m), 2.68-2.58 (3H, m), 2.50-2.45 (1H, m), 1.27 (3H , D, J = 7.0 Hz), 1.24 (3H, d, J = 7.0 Hz), 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 compound 4 thus obtained, compound 5 (9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D- Ribofuranosyl) -2-isobutyrylamino-1H-purin-6-one) was synthesized. That is, after dissolving Compound 4 (70.4 mg, 0.12 mmol) in toluene (0.12 mL) and dimethyl sulfoxide (0.12 mL), 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 with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crude aldehyde. The crude aldehyde was dissolved 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 with 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 stirred at the same temperature for 1 hour. . After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate and extracted with ethyl acetate. Subsequently, the organic layer was dried with 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 5 (61 .1 mg, 0.10 mmol, 3 steps 88%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 12.00 (1H, brs), 7.97 (1H, brs), 7.81 (1H, s), 6.36 (1H, t, J = 6. 5 Hz), 4.82 (1H, t, J = 6.0 Hz), 3.91 (2H, s), 2.66-2.55 (3H, m), 1.31 (6H, d, J = 7.0 Hz), 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 compound 5 thus obtained, compound 6 (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, compound 5 (46.1 mg, 0.078 mmol) was dissolved in dichloromethane (1 mL), then triethylamine (22 μL, 0.16 mmol), 2,4,6-triisopropylbenzenesulfonyl chloride (47.3 mg, 0 .16 mmol), 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, the reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate and extracted 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 6 (58.5 mg). , 0.068 mmol, 88%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.9 (1H, s), 7.95 (1H, brs), 7.23 (2H, s), 6.38 (1H, t, J = 6. 5 Hz), 5.18 (1 H, t, J = 5.5 Hz), 4.25 (2 H, m), 4.12 (1 H, d, J = 11.5 Hz), 3.95 (1 H, d, J = 11.0 Hz), 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 = 2.0 Hz), 1.21 (3H, d, J = 1.5 Hz), 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 2: 9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) -2-isobutyrylaminopurine (Compound 7 ) Was synthesized in the reaction steps shown below.

Compound 6 (626 mg, 0.730 mmol) was dissolved in tetrahydrofuran (3 mL), hydrazine monohydrate (0.2 mL, 4.12 mmol) was added, and the mixture was stirred at room temperature for 1 hr. After completion of the reaction, extraction with ethyl acetate was performed. The organic layer was dried with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crude 6-position hydrazide.
The crude 6-position hydrazide (<0.730 mmol) was dissolved in tetrahydrofuran (4 mL), silver (I) oxide (508 mg, 2.19 mmol) was added, and the mixture was stirred for 15 hours under light shielding and heating under reflux. did. After completion of the reaction, extraction with ethyl acetate was performed. After drying the organic layer with magnesium sulfate and evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1) to obtain compound 7 (180 mg, 0.313 mmol, 2 steps 43%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.91 (1H, s), 8.18 (1H, brs), 8.07 (1H, s), 6.43 (1H, t, J = 6. 5 Hz), 5.16 (1 H, t, J = 6.0 Hz), 4.12 (1 H, d, J = 12 Hz), 3.96 (1 H, d, J = 11 Hz), 3.23 ( 1H, m), 2.76 (1H, brs), 2.55 (1H, m), 1.23 (6H, d, J = 6.5 Hz), 0.97 (9H, s), 0.89 (9H, s), 0.23 (3H, s), 0.16 (3H, s), 0.081 (3H, s), 0.067 (3H, s).

Synthesis Example 3: Synthesis of 2-amino-9- (3,5-di-O-tert-butyldimethylsilyl-4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) purine (Compound 8) Compound 8 was synthesized by the reaction steps shown below.

Compound 7 (180 mg, 0.313 mmol) was dissolved in isopropyl alcohol (2.5 mL), then ammonium iodide (45.3 mg, 0.313 mmol) and hydrazine monohydrate (2.5 mL) were sequentially added, Stir at room temperature for 6.5 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) to obtain Compound 8 (120 mg, 0.238 mmol, 76%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.70 (1H, s), 7.88 (1H, s), 6.40 (1H, dd, J = 5.5, 1.5 Hz), 5. 04 (2H, brs), 4.95 (1H, t, J = 6.0 Hz), 3.99 (1H, d, J = 11 Hz), 3.88 (1H, d, J = 11 Hz), 3. 05 (1H, m), 2.56 (1H, m), 0.97 (9H, s), 0.87 (9H, s), 0.20 (3H, s), 0.18 (3H, s ), 0.083 (3H, s), 0.023 (3H, s).

Synthesis Example 4: Synthesis of 2-amino-9- (4-C-cyano-2-O-deoxy-β-D-ribofuranosyl) purine (Compound P1) Compound P1 was synthesized by the reaction steps shown below.

Compound 8 (120 mg, 0.238 mmol) was dissolved in chloroform (2 mL) and methanol (4 mL), then ammonium acid fluoride (815 mg, 14.3 mmol) was added, and the mixture was stirred at room temperature for 137.5 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 = 1/4) to obtain crude compound P1. The crude compound P1 was purified by silica gel column chromatography (methanol / chloroform = 1/6) to obtain compound P1 (57.5 mg, 0.208 mmol, 88%).

¹ H-NMR (CD ₃ OD, 500 MHz); δ 8.56 (1H, s), 8.21 (1H, s), 6.51 (1H, t, J = 6.5 Hz), 4.90 (1H M), 3.94 (1H, d, J = 12 Hz, 3.87 (1H, d, J = 12 Hz), 3.02 (1H, m), 2.56 (1H, m).

Synthesis Example 5: 2-amino-9- (2-O-acetyl-3,5-di-O-benzyl-4-C-fluoromethyl-β-D-ribofuranosyl) -6-chloropurine (Compound 10) Synthetic compound 9 was synthesized by the reaction steps shown below.

Compound 9 (see Tetrahedron, Vol. 53, No. 39, pp. 13315-13322 (1997)) (117 mg, 0.263 mmol) and 2-amino-6-chloropurine (66.8 mg, 0.394 mmol) in acetonitrile After being dissolved in (1.3 mL), N, O-bis (trimethylsilyl) acetamide (206 μL, 0.842 mmol) was added, and the mixture was stirred with heating under reflux for 1 hour. Subsequently, after returning the reaction solution to room temperature, trimethylsilyl trifluoromethanesulfonate (64 μL, 0.342 mmol) was added, and the mixture was stirred for 5 hours under heating to reflux. After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate and extracted 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 = 1/2 → 1/1) to obtain compound 10 (122 mg, 0.220 mmol, 84%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.9 (1H, s), 7.38-7.29 (10 H, m), 6.16 (1 H, d, J = 5.5 Hz), 5 .91 (1H, dd, J = 5.5, 5.5 Hz), 5.01 (2H, brs), 4.71-4.54 (7H, m), 3.72 (1H, dd, J = 10, 2.0 Hz), 3.66 (1H, dd, J = 10, 2.0 Hz), 2.06 (3H, s).

Synthesis Example 6: 2-Amino-9- (3,5-di-O-benzyl-4-C-fluoromethyl-2-O-phenylthioformyl-β-D-ribofuranosyl) -6-chloropurine (Compound 12 ) Was synthesized in the reaction steps shown below.

Compound 10 (122 mg, 0.220 mmol) was dissolved in methanol (2 mL), aqueous ammonia (0.5 mL) was added, and the mixture was stirred at room temperature for 13 hr. After completion of the reaction, extraction with ethyl acetate was performed. The organic layer was dried with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified compound 11.
Crude compound 11 (<0.220 mmol) was azeotropically dehydrated with dioxane. Next, after dissolving in acetonitrile (3 mL), 4-dimethylaminopyridine (80.6 mg, 0.660 mmol) and phenylchlorothionoformate (45.6 μM, 0.330 mmol) were sequentially added, and at room temperature for 2 hours. Stir. After completion of the reaction, extraction with ethyl acetate was performed. After the organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/2) to obtain compound 12 (110.6 mg, 0. 170 mmol, 2 steps 77%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.00 (1H, s), 7.41-7.33 (13H, m), 6.97 (2H, d, J = 8.0 Hz), 6. 41 (1H, dd, J = 6.3, 5.5 Hz), 6.37 (1H, d, J = 6.5 Hz), 5.00 (2H, brs), 4.88 (1H, d, J = 5.0 Hz), 4.76-4.59 (6 H, m), 3.77 (1 H, dd, J = 10, 2.0 Hz), 3.74 (1 H, dd, J = 10, 2. 0 Hz).

Synthesis Example 7: 2-amino-9- (3,5-di-O-benzyl-2-O-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -6-chloropurine (Compound 13) Synthetic compound 13 was synthesized in the reaction steps shown below.

Compound 12 (110.6 mg, 0.170 mmol) was dissolved in toluene (6 mL), then tributyltin hydride (229 μL, 0.850 mmol) and azobisisobutyronitrile (7.0 mg, 0.0425 mmol) were added. , And stirred at 80 ° C. for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/2 → 1/1) to obtain compound 13 (59.3 mg, 0.119 mmol, 70%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.9 (1H, s), 7.39-7.25 (10 H, m), 6.34 (1 H, dd, J = 6.5, 6.5 Hz) ), 5.02 (2H, brs), 4.67 (2H, d, J = 47.5 Hz), 4.65 (1H, d, J = 12 Hz), 4.55-4.51 (4H, m), 3.63 (1H, s), 3.62 (1H, s), 2.78 (1H, m), 2.63 (1H, m).

Synthesis Example 8 Synthesis of 2-amino-9- (3,5-di-O-benzyl-2-O-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) purine (Compound 14) These were synthesized by the reaction steps shown below.

After adding methanol (3 mL) to compound 13 (59.3 mg, 0.119 mmol) and 10% palladium carbon (29.7 mg, 50 wt%), triethylamine (166 μL, 1.19 mmol) was added, and at room temperature under hydrogen atmosphere For 19 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain Compound 14 (37.6 mg, 0.0811 mmol, 68%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.65 (1H, s), 7.94 (1H, s), 7.39-7.26 (10H, m), 6.39 (1H, dd, J = 6.5, 6.5 Hz), 4.97 (2H, brs), 4.69 (2H, d, J = 47 Hz), 4.66 (1H, d, J = 12 Hz), 4.57− 4.50 (4H, m), 3.65 (1 H, s), 3.64 (1 H, s), 2.81 (1 H, m), 2.62 (1 H, m).

Synthesis Example 9 Synthesis of 2-amino-9- (2-O-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) purine (Compound P2) Compound P2 was synthesized by the following reaction steps. .

Compound 14 (18.0 mg, 0.0388 mmol) was azeotropically dehydrated with toluene, then dissolved in dichloromethane (1 mL), and boron trichloride (1.0 M in CH ₂ Cl ₂ , 0.29 mg, 0.291 mmol) was added. The mixture was further stirred at −78 ° C. for 21 hours. After completion of the reaction, the reaction was quenched with 1M triethylamine-carbon dioxide buffer (4.4 mL) and then azeotroped with methanol. Thereafter, the residue was purified by silica gel column chromatography (methanol / chloroform = 1/5) and preparative thin layer chromatography (methanol / chloroform = 1/4) to obtain compound P2 (6.7 mg, 0.0237 mmol, 61 %).

¹ H-NMR (CD ₃ OD, 500 MHz); δ 8.56 (1H, s), 8.26 (1H, s), 6.42 (1H, dd, J = 6.5, 6.5 Hz), 4.73 (1H, dd, J = 6.5, 4.0 Hz), 4.63 (2H, dd, J = 47.5, 2.5 Hz), 3.76 (2H, s), 2.97 (1H, m), 2.48 (1H, m).

Synthesis Example 10: 9- (5-O-acetyl-3-O-tert-butyldimethylsilyl-2-O-deoxy-β-D-ribofuranosyl) -2-isobutyrylamino-1H-purin-6-one Synthesis of (Compound 15) Compound 15 was synthesized by the reaction steps shown below.

Compound 2 (15.0 mg, 0.0332 mmol) was dissolved in dichloromethane (1 mL), then triethylamine (13.9 μL, 0.0996 mmol), 4-dimethylaminopyridine (0.41 mg, 3.32 μmol), acetic anhydride (4.71 μL, 0.0498 mmol) was sequentially added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, quenching with saturated aqueous sodium hydrogen carbonate and extraction with ethyl acetate were 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 = 2/1 → ethyl acetate) to obtain compound 15 (11.4 mg , 0.0231 mmol, 70%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.57 (1H, brs), 8.02 (1H, s), 7.74 (1H, s), 6.23 (1H, dd, J = 7. 5, 6.0 Hz), 4.60 (1 H, dd, J = 11.5, 5.5 Hz), 4.48 (1 H, m), 4.37 (1 H, dd, J = 11.5, 6) .0Hz), 2.76 (1H, m), 2.67 (1H, m), 2.33 (1H, m), 2.11 (3H, s), 1.30 (3H, d, J = 4.0 Hz), 1.28 (3H, d, J = 4.0 Hz) 0.93 (9 H, s), 0.123 (3 H, s), 0.120 (3 H, s).

Synthesis Example 11 9- (5-O-acetyl-3-O-tert-butyldimethylsilyl-2-O-deoxy-β-D-ribofuranosyl) -2-isobutyrylamino-6- (2,4 Synthesis of 6-triisopropylbenzenesulfonyloxy) purine (Compound 16) Compound 16 was synthesized by the reaction steps shown below.

Compound 15 (5.55 g, 11.2 mmol) was dissolved in dichloromethane (56 mL), and then triethylamine (3.1 mL, 22.4 mmol), 4-dimethylaminopyridine (137 mg, 1.12 mmol), 2, 4 , 6-Triisopropylbenzenesulfonyl chloride (6.78 g, 22.4 mmol) was sequentially added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, quenching with saturated aqueous sodium hydrogen carbonate and extraction with chloroform were 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 = 1/4) to obtain compound 16 (7.26 g, 9. 55 mmol, 85%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.04 (1H, s), 7.85 (1H, brs), 7.23 (2H, s), 6.33 (1H, t, J = 6. 5 Hz), 4.76 (1 H, dd, J = 10.5, 4.5 Hz), 4.39 (1 H, dd, J = 12, 4.5 Hz), 4.30-4.23 (3 H, m ), 4.13 (1H, dd, J = 9.5, 4.5 Hz), 2.93 (2H, m), 2.86 (1H, brs), 2.44 (1H, m), 2. 05 (3H, s), 1.30-1.26 (18H, m), 1.23 (3H, d, J = 3.0 Hz), 1.21 (3H, d, J = 3.5 Hz) 0 .91 (9H, s), 0.13 (3H, s), 0.11 (3H, s).

Synthesis Example 12 Synthesis of 9- (3-O-tert-butyldimethylsilyl-2-O-deoxy-β-D-ribofuranosyl) -2-isobutyrylaminopurine (Compound 18) Synthesized in the reaction step.

Compound 16 (6.92 g, 9.10 mmol) was dissolved in 1,4-dioxane (65 mL), triethylamine (7.6 mL, 54.6 mmol), formic acid (2.1 mL, 54.6 mmol), 1, 3-bis (diphenylphosphino) propane (413 mg, 1.00 mmol) and palladium (II) acetate (204 mg, 0.91 mmol) were sequentially added, and the mixture was stirred at 90 ° C. for 3.5 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate, short) to obtain roughly purified Compound 17.
Crude compound 17 (<9.10 mmol) was dissolved in methanol (46 mL), aqueous ammonia (15 mL) was added, and the mixture was stirred at room temperature for 12.5 hr. 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 = 2/1 → ethyl acetate) to obtain compound 18 (1.82 g, 4.18 mmol, 46% for 2 steps).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.96 (1H, s), 8.30 (1H, brs), 8.09 (1H, s), 6.31 (1H, dd, J = 8. 0, 6.0 Hz), 4.88 (1H, ddd, J = 5.5, 2.5, 2.5 Hz), 4.44 (1H, m), 4.09 (1H, dd, J = 4.5, 2.0 Hz), 3.95 (1H, m), 3.82 (1 H, m), 3.02 (1 H, m), 2.79 (1 H, m), 2.30 ( 1H, m), 1.30 (3H, d, J = 2.0 Hz), 1.28 (3H, d, J = 2.0 Hz), 0.927 (9H, s), 0.138 (3H, s), 0.121 (3H, s).

Synthesis Example 13 Synthesis of 9- (3-O-tert-butyldimethylsilyl-2-O-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl) -2-isobutyrylaminopurine (Compound 20) Compound 20 was synthesized by the reaction steps shown below.

Compound 18 (26.8 mg, 0.0618 mmol) was dissolved in toluene (0.5 mL) and dimethyl sulfoxide (0.5 mL), and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (35 0.5 mg, 0.185 mmol), pyridine (10 μL, 0.124 mmol), and trifluoroacetic acid (4.7 μL, 0.0618 mmol) were sequentially added, and the mixture was stirred at room temperature for 14 hours. Then, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (118 mg, 0.618 mmol), pyridine (30 μL, 0.371 mmol), trifluoroacetic acid (14 μL, 0.186 mmol) were sequentially added, Stir at room temperature for 2 hours. After completion of the reaction, extraction with ethyl acetate was performed. The organic layer was dried with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified compound 19.
Crude compound 19 (<0.0618 mmol) was dissolved in 1,4-dioxane (1 mL), then 37% aqueous formaldehyde solution (27 μL, 0.272 mmol), 1 N aqueous sodium hydroxide solution (155 μL, 0 mL). .155 mmol) was sequentially added, and the mixture was stirred at room temperature for 1 hour. Subsequently, sodium borohydride (7.0 mg, 0.185 mmol) was added at 0 ° C., and the mixture was stirred at the same temperature for 30 minutes. After completion of the reaction, quenching with methanol and extraction with ethyl acetate were 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 → methanol / chloroform = 1/10) to obtain compound 20 (18.3 mg, 0. 1). 0393 mmol, 64% for 2 steps).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.94 (1H, s), 8.75 (1H, brs), 8.09 (1H, s), 6.38 (1H, dd, J = 6. 5, 6.5 Hz), 5.30 (1 H, dd, J = 6.5, 5.0 Hz), 4.28 (1 H, brs), 3.90-3.63 (5 H, m), 3. 09 (1H, m), 2.94 (1H, m), 2.51 (1H, m), 1.29 (3H, d, J = 6.0 Hz), 1.28 (3H, d, J = 7.0 Hz), 0.945 (9H, s), 0.215 (3H, s), 0.152 (3H, s).

Synthesis Example 13 9- (2-O-deoxy-3,5-di-O-tert-butyldimethylsilyl-4-C-hydroxymethyl-β-D-ribofuranosyl) -2-isobutyrylaminopurine (compound 23) Synthesis compound 23 was synthesized by the following reaction steps.

Compound 20 (103.4 mg, 0.222 mmol) was dissolved in N, N-dimethylformamide (2.3 mL), and then triethylamine (62 μL, 0.444 mmol), 4,4′-dimethoxytrityl chloride (113 mg, 0.333 mmol) was sequentially added, and the mixture was stirred at 0 ° C. for 1.5 hours. After completion of the reaction, quenching with methanol and extraction with ethyl acetate were performed. The organic layer was dried with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified compound 21.
Crude compound 21 (<0.222 mmol) was dissolved in N, N-dimethylformamide (2.2 mL), and then imidazole (52.9 mg, 0.777 mmol), tert-butyldimethylsilyl chloride (100 mg, 0.8 mL). 666 mmol) was sequentially added, and the mixture was stirred at room temperature for 16.5 hours. After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate and extracted with ethyl acetate. The organic layer was dried with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified compound 22.
Crude compound 22 (<0.222 mmol) was dissolved in chloroform (1.5 mL), and then p-toluenesulfonic acid monohydrate (127 mg, dissolved in methanol (0.6 mL) at −15 ° C. 0.666 mmol) was added dropwise and stirred at the same temperature for 2 hours. After completion of the reaction, the reaction mixture was quenched with 1N aqueous sodium hydroxide solution and extracted 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) to obtain compound 23 (19.8 mg, 0 0.0341 mmol, 3 steps 15.4%).

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.98 (1H, s), 8.83 (1H, brs), 8.27 (1H, s), 6.48 (1H, dd, J = 7. 0, 6.0 Hz), 4.97 (1 H, dd, J = 6.5, 5.5 Hz), 3.87 (1 H, m), 3.82 (2 H, s), 3.73 (1 H , M), 2.93 (1H, m), 2.78 (1H, m), 2.59 (1H, m), 1.29 (3H, d, J = 2.0 Hz), 1.28 ( 3H, d, J = 2.0 Hz), 0.939 (9H, s), 0.883 (9H, s), 0.165 (3H, s), 0.147 (3H, s), 0.043 (6H, s).

Synthesis Example 14 9- (2-O-deoxy-3,5-di-O-tert-butyldimethylsilyl-4-C-vinyl-β-D-ribofuranosyl) -2-isobutyrylaminopurine (Compound 25 ) Was synthesized in the reaction steps shown below.

Compound 23 (42.7 mg, 0.0737 mmol) was dissolved in toluene (0.5 mL) and dimethyl sulfoxide (0.5 mL), and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (141 .3 mg, 0.737 mmol), pyridine (36 μL, 0.442 mmol), and trifluoroacetic acid (17 μL, 0.221 mmol) were sequentially 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 with magnesium sulfate and the solvent was distilled off under reduced pressure to obtain a crudely purified compound 24.
Methyltriphenylphosphonium bromide (79.0 mg, 0.221 mmol) was dissolved in tetrahydrofuran (2 mL), and then n-butyllithium (1.6 M in hexane, 0.13 mL, 0.214 mmol) was added at −78 ° C. In addition, the mixture was stirred at 0 ° C. for 30 minutes. Subsequently, the crudely purified compound 24 (<0.0737 mmol) dissolved in tetrahydrofuran (4 mL) was dissolved in tetrahydrofuran (4 mL), followed by stirring at room temperature for 11 hours. Subsequently, a separately prepared 9.9 equivalent Wittig reagent was added at 0 ° C. and stirred at room temperature for 3 hours. After completion of the reaction, quenching with a saturated aqueous ammonium chloride solution and extraction with ethyl acetate were 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 25 (14.4 mg, 0. 1). 0250 mmol, 34% for 2 steps) was obtained as a mixture with compound 25 ′ (25: 25 ′ = 1: 0.3). The mixing ratio of compound 25 and compound 25 ′ was determined from the ¹ H-NMR spectrum.
LRMS of compound 25 (ESI ^<+> ); m / z 598.4114 [M + Na] ^< +>.

Synthesis Example 15 Synthesis of 2-amino-9- (2-O-deoxy-3,5-di-O-tert-butyldimethylsilyl-4-C-vinyl-β-D-ribofuranosyl) purine (Compound 26) Compound 26 was synthesized by the reaction step shown below.

A mixture of compound 25 and compound 25 ′ (18.1 mg, 0.0314 mmol) was dissolved in isopropyl alcohol (1 mL), then ammonium iodide (4.6 mg, 0.0314 mmol), hydrazine monohydrate (1 mL). Were sequentially added and stirred at room temperature for 1.5 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 give compound 26 (10.1 mg, 0. 1). 0200 mmol, 64%) was obtained as a mixture with compound 26 ′ (26: 26 ′ = 1: 0.3). The mixing ratio of compound 26 and compound 26 ′ was determined from ¹ H-NMR spectrum.
LRMS of compound 26 (ESI ^<+> ); m / z 528.3378 [M + Na] ^< +>.

Synthesis Example 16 Synthesis of 2-amino-9- (2-O-deoxy-4-C-vinyl-β-D-ribofuranosyl) purine (Compound P3) Compound P3 was synthesized by the reaction steps shown below.

A mixture of compound 26 and compound 26 ′ (10.1 mg, 0.0200 mmol) was dissolved in chloroform (1 mL) and methanol (1 mL), and then acid ammonium fluoride (68.4 mg, 1.2 mmol) was added. Stirred under for 84 hours. Subsequently, acidic ammonium fluoride (68.4 mg, 1.2 mmol) was added, and the mixture was stirred at room temperature for 48 hours. Then, acidic ammonium fluoride (205 mg, 3.6 mmol) was added, and the mixture was stirred at room temperature for 216 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 = 1/4) to give compound P3 (4.4 mg, 0.0159 mmol, 79%) as compound P3. As a mixture (P3: P3 ′ = 1: 0.3). The mixing ratio of compound P3 and compound P3 ′ was determined from ¹ H-NMR spectrum.

¹ H-NMR (CD ₃ OD, 500 MHz) of Compound P3; δ 8.56 (1H, s), 8.36 (1H, s), 6.37 (1H, dd, J = 7.0, 4.5 Hz) ), 6.00 (1H, dd, J = 17.5, 11.0 Hz), 5.53 (1H, dd, J = 17.5, 2.0 Hz), 5.32 (1H, dd, J = 11.0, 2.0 Hz), 4.80 (1 H, t, J = 6.5 Hz), 3.70 (1 H, d, J = 12.0 Hz), 3.60 (1 H, d, J = 12) .0Hz), 2.74 (1H, m), 2.41 (1H, m);
¹ H-NMR (CD ₃ OD, 500 MHz) of Compound P3 ′; δ 8.56 (1H, s), 8.30 (1H, s), 6.33 (1H, t, J = 7.0 Hz), 4 .61 (1H, dd, J = 6.0, 4.0 Hz), 3.71 (2H, d, J = 12.0 Hz), 3.63 (1H, d, J = 12.0 Hz), 2. 93 (1H, m), 2.42 (1H, m), 1.77 (1H, m), 1.68 (1H, m), 0.992 (3H, t, J = 7.5 Hz).

Synthesis Example 17: 9- (3-O-tert-butyldimethylsilyl-2,5-di-O-deoxy-5-O-iodo-β-D-ribofuranosyl) -2-isobutyrylaminopurine (Compound 27) ) Was synthesized in the reaction steps shown below.

Compound 18 (323 mg, 0.742 mmol) was dissolved in pyridine (3.7 mL), triphenylphosphine (584 mg, 2.23 mmol) and iodine (565 mg, 2.23 mmol) were sequentially added, and the mixture was stirred at room temperature for 3 hours. did. Thereafter, triphenylphosphine (389 mg, 1.48 mmol) and iodine (377 mg, 1.48 mmol) were added, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction, quenching with a saturated aqueous sodium thiosulfate solution and extraction with ethyl acetate were performed. The organic layer was dried over magnesium sulfate and the solvent was distilled off under reduced pressure. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 2/1) to give a compound. 27 (352 mg, 0.645 mmol, 87%) was obtained.

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.93 (1H, s), 8.35 (1H, s), 8.18 (1H, s), 6.38 (1H, t, J = 7. 0 Hz), 4.73 (1 H, m), 4.06 (1 H, m), 3.66 (1 H, dd, J = 10.5, 6.5 Hz), 3.47 (1 H, dd, 11, 6.0 Hz), 3.21 (1 H, m), 2.86 (1 H, brs), 2.38 (1 H, m), 1.30 (3 H, d, J = 5.0 Hz), 1 .29 (3H, d, J = 4.5 Hz), 0.93 (9H, s), 0.18 (3H, s), 0.15 (3H, s).

Synthesis Example 18: 9- (3-O-tert-butyldimethylsilyl-2,5-di-O-deoxy-β-D-erythro-pent-4-enofuranosyl) -2-isobutyrylaminopurine (Compound 28 ) Was synthesized by the reaction steps shown below.

Compound 27 (352 mg, 0.645 mmol) was dissolved in acetonitrile (4 mL), diazabicycloundecene (481 μL, 3.23 mmol) was added, and the mixture was stirred at 80 ° C. for 1.5 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 2/1) to obtain Compound 28 (240 mg, 0.574 mmol, 89%). .

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.95 (1H, s), 8.19 (1H, s), 8.06 (1H, s), 6.57 (1H, t, J = 6. 0 Hz), 5.12 (1H, t, J = 5.5 Hz), 4.51 (1H, m), 4.26 (1H, m), 3.05 (1H, brs), 2.95 ( 1H, m), 2.55 (1H, m), 1.29 (3H, d, J = 3.0 Hz), 1.28 (3H, d, J = 3.0 Hz), 0.94 ( 9H, s), 0.18 (3H, s), 0.17 (3H, s).

Synthesis Example 19 9- (4-C-azido-3-O-tert-butyldimethylsilyl-2,5-di-O-deoxy-5-O-iodo-β-D-ribofuranosyl) -2-isobuty Synthesis of Rylaminopurine (Compound 29) Compound 29 was synthesized in the reaction steps shown below.

Sodium azide (187 mg, 2.87 mmol) and iodine monochloride (1.0 M in hexane, 1.44 mL, 1.44 mmol) were dissolved in N, N-dimethylformamide (1 mL) and then N, N-dimethyl. Compound 28 (240 mg, 0.574 mmol) dissolved in formamide (3 mL) was added, and the mixture was stirred at room temperature for 3.5 hours. After completion of the reaction, quenching with a saturated aqueous sodium thiosulfate solution and extraction with ethyl acetate were performed. After drying the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1) to give a compound. 29 (206 mg, 0.351 mmol, 61%) was obtained.

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.94 (1H, s), 8.69 (1H, s), 8.08 (1H, s), 6.38 (1H, dd, J = 7. 0, 6.0 Hz), 5.39 (1 H, t, J = 6.0 Hz), 4.00 (1 H, d, J = 11.5 Hz), 3.73 (1 H, d, J = 11.0 Hz) ), 3.34 (1H, m), 2.74 (1H, brs), 2.58 (1H, m), 1.30 (3H, d, J = 7.0 Hz), 1.28 (3H, d, J = 7.5 Hz), 0.97 (9H, s), 0.25 (3H, s), 0.18 (3H, s).

Synthesis Example 20 Synthesis of 2-amino-9- (4-C-azido-3-O-tert-butyldimethylsilyl-β-D-ribofuranosyl) purine (Compound 31) Compound 31 was subjected to the reaction steps shown below. And synthesized.

Compound 29 (152 mg, 0.259 mmol) was dissolved in N, N-dimethylformamide (5 mL), and then sodium benzoate (373 mg, 2.59 mmol) and 15-crown-5 (514 μL, 2.59 mmol) were added. In addition, the mixture was stirred at 90 ° C. for 24 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 crude compound 30.
Crude compound 30 (<0.113 mmol) was dissolved in isopropyl alcohol (1.5 mL), and then ammonium iodide (16.4 mg, 0.113 mmol) and hydrazine monohydrate (1.5 mL) were added. Sequentially added and stirred at room temperature for 15 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 (methanol / chloroform = 1/10) to obtain compound 31 (38.4 mg, 0.0945 mmol, 2 Step 37%) was obtained.

¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.74 (1H, s), 7.83 (1H, s), 7.41 (1H, s), 6.41 (1H, dd, J = 9. 0, 5.5 Hz), 5.14 (2H, brs), 4.78 (1H, dd, J = 5.0, 1.5 Hz), 3.87 (1H, d, J = 12.5 Hz) ), 3.53 (1H, d, J = 12.5 Hz), 3.21 (1H, m), 2.38 (1H, m), 0.97 (9H, s), 0.21 (3H) , S), 0.18 (3H, s).

Synthesis Example 21 Synthesis of 2-amino-9- (4-C-azido-2-deoxy-β-D-ribofuranosyl) purine (Compound P4) Compound P4 was synthesized by the reaction steps shown below.

Compound 31 (38.4 mg, 0.0945 mmol) was dissolved in chloroform (2 mL) and methanol (2 mL), then ammonium acid fluoride (323 mg, 5.67 mmol) was added, and the mixture was stirred at room temperature for 41 hours. Subsequently, acidic ammonium fluoride (323 mg, 5.67 mmol) was added, and the mixture was stirred at room temperature for 72 hours. Then, it stirred at 50 degreeC for 42 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 = 1/6 → 1/4) to obtain compound P4 (22.6 mg, 0.0773 mmol, 82%). Got.

¹ H-NMR (CD ₃ OD, 500 MHz); δ 8.56 (1H, s), 8.26 (1H, s), 6.52 (1H, dd, J = 7.0, 4.0 Hz), 4 .88 (1H, m), 3.83 (1H, d, J = 12.0 Hz), 3.75 (1H, d, J = 12.0 Hz), 2.90 (1H, m), 2. 59 (1H, m).

Synthesis Example 22 Synthesis of Compounds C1 and C2 Based on the description of Non-Patent Document 1 described above, compounds C1 and C2 represented by the following formula were synthesized.

Synthesis Example 23 Synthesis of 2-amino-2′-deoxy-4′-fluoromethyladenosine (Compound R1) To synthesize 2-amino-2′-deoxy-4′-fluoromethyladenosine (Compound R1), first, The following compound 32 (2′-O-Acetyl-2-amino-3 ′, 5′-di-O-benzyl-4′-fluoromethyladenosine) was synthesized as follows.

That is, 1 in Compound 9 (5.78 g, 12.9 mmol), 2,6-diaminopurine (3.87 g, 25.8 mmol), N, O-bis (trimethylsilyl) acetamide (37.8 mL, 0.155 mol). , 2-dichloroethane (104 mL) was added, and the mixture was heated to reflux for 1 hour. The reaction solution was cooled to 0 ° C., trimethylsilyl trifluoromethanesulfonate (4.66 mL, 25.8 mmol) was added, and the mixture was heated to reflux for 8 hours. The reaction solution was cooled to 0 ° C., saturated aqueous sodium hydrogen carbonate was added and stirred, the resulting insoluble material was removed by Celite filtration, 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 obtain Compound 32 (5.15 g, 9.60 mmol, 74.4%).

¹ H-NMR (CD ₃ CN): δ 7.65 (1H, s), 7.24-7.08 (10H, m), 6.02 (1H, d), 5.98 (1H, t), 5.70 (2H, br.s), 4.95 (2H, br.s), 4.82 (1H, d), 4.71 (1H, dd), 4.61 (1H, dd), 4 .62 (1H, d), 4.59 (2H, d), 4.56 (1H, d), 4.53 (1H, d), 3.70 (2H, m), 2.01 (3H, s).

Next, using Compound 32 obtained as described above, Compound 33 (2-Amino-3 ', 5'-di-O-benzyl-4'-fluoromethyladenosine) was synthesized as follows.

That is, compound 32 (5.15 g, 9.60 mmol) was dissolved in methanol (100 mL), 1M 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 and 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 to 10: 1) to obtain Compound 33 (4.70 g, 9.50 mmol, 99.0%).

¹ H-NMR (DMSO-d ₆ ): δ 7.84 (1H, s), 7.38-7.28 (10H, m), 6.72 (2H, br. S), 5.86 (1H, d), 5.81 (2H, br.s), 5.74 (1H, d), 5.01 (1H, dd), 4.89 (1H, d), 4.68-4.53 (5H) M), 4.28 (1H, d), 3.68 (1H, dd), 3.64 (1H, dd).

Next, using Compound 33 obtained as described above, Compound 34 (2-Amino-3 ′, 5′-di-O-benzoyl-2′-deoxy-4′-fluoromethyladenosine) was obtained as described below. Synthesized.

That is, Compound 33 (4.70 g, 9.50 mmol) and 4-dimethylaminopyridine (1.74 g, 14.2 mmol) were dissolved in acetonitrile (38.0 mL), and phenyl chlorothionoformate (1.54 mL) was added at 0 ° C. 11.4 mmol) was added and stirred for 2 hours. After adding methanol (2 mL) and stirring, the reaction mixture was diluted with ethyl acetate and washed (saturated brine → 0.1 M hydrochloric acid → saturated brine → saturated aqueous sodium bicarbonate solution). The organic layer was dried over magnesium sulfate and concentrated, and the residue was azeotroped with toluene to obtain a crude thiocarbonate.
Crude thiocarbonate, tributyltin hydride (10.2 mL, 37.9 mmol) was dissolved in toluene (95.0 mL), heated to 85 ° C., azobisisobutyronitrile (20 mg) was added, and the mixture was stirred for 2 hours. did. After the reaction solution was concentrated, the residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain Compound 34 (3.23 g, 6.75 mmol, 71.1%).

¹ H-NMR (DMSO-d ₆ ): δ 7.87 (1H, s), 7.37-7.26 (10H, m), 6.71 (2H, br. S), 6.23 (1H, d), 5.80 (2H, br.s), 4.70-4.50 (7H, m), 3.64 (2H, dd), 3.60 (2H, dd), 2.91 (1H) , M), 2.59 (1H, m).

Next, using Compound 34 obtained as described above, Compound R1 (2-Amino-2'-deoxy-4'-fluoromethyladenosine) 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. The solution was cooled to −78 ° C., a dehydrated tetrahydrofuran solution (35.6 mL) of compound 34 (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 was diluted with ethyl acetate and extracted with deionized water. The aqueous layers were combined and concentrated to a small volume, then purified by ODS column chromatography (deionized water to 5% methanol), and after vacuum drying, compound R1 was obtained (0.90 g, 3.0 mmol, 68%). .

¹ H-NMR (DMSO-d ₆ ): δ 7.91 (1H, s), 6.70 (2H, br. S), 6.24 (1H, dd), 5.72 (2H, br. S) , 5.36-5.35 (2H, m), 4.60 (1H, dd), 4.50 (1H, dd), 4.51 (1H, m), 3.54 (1H, br.s) ), 3.53 (1H, br.s), 2.81 (1H, m), 2.22 (1H, m).

Synthesis Example 24 Synthesis of 6,4′-fluoromethyl-2′-deoxyguanosine (Compound R2) Using compound R1 obtained in Synthesis Example 23, 6,4′-fluoromethyl-2′- Deoxyguanosine (Compound R2) was synthesized.

Specifically, Compound R1 (500 mg, 1.68 mmol) was dissolved in 50 mM Tris-HCl buffer (pH 7.5) (45.0 mL), calf spleen-derived adenosine deaminase (50 uL, 6.5 units) was added, and 40 ° C. Stir for 2 hours. The mixture was allowed to stand at 5 ° C. overnight, and the precipitated compound R2 was collected by filtration and dried (409 mg). The filtrate was further concentrated to a small amount and purified by ODS column chromatography (ODS 50 cc, 0 to 5% MeOH) (44 mg). Combined with the previously obtained compound R2 was obtained (453 mg, 1.51 mmol, 89.9%).

¹ H-NMR (DMSO-d ₆ ): δ 10.58 (1H, br.s), 7.91 (1H, s), 6.42 (2H, br. S), 6.19 (1H, dd) , 5.37 (1H, m), 5.09 (1H, t), 4.59 (1H, dd), 4.49 (1H, dd), 4.49 (1H, m), 3.50 ( 2H, m), 2.71 (1H, m), 2.25 (1H, m).

Synthesis Example 25 Synthesis of 2-amino-2′-deoxy-4′-vinyladenosine (Compound R3) To synthesize 2-amino-2′-deoxy-4′-vinyladenosine (Compound R3), first, Compound 36 (2-Benzamido-N ⁶ -benzoyl-3 ′, 5′-di-O-tert-butyldimethylsilyl-2′-deoxy-4′-vinyladenosine) was synthesized as follows.

That is, Compound 35 (see Nucleosides, Nucleotides & Nucleic Acids, Vol. 23, No. 4, pp. 671-690, 2004) (0.42 g, 0.57 mmol) was dried dimethyl sulfoxide (2.5 mL), dry toluene (1 3 mL), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (328 mg, 1.71 mmol), dry pyridine (104 μL), trifluoroacetic acid (52 μL) were added, and the mixture was stirred 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 three times and then vacuum dried to obtain a crude aldehyde.

Methyltriphenylphosphonium bromide (1.02 g, 2.86 mmol) was suspended in dry tetrahydrofuran (7.1 mL) and cooled to -78 ° C. 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 dry tetrahydrofuran solution (5.9 mL) was added, and the mixture was stirred at room temperature for 1 hour. . Saturated aqueous ammonium chloride solution was added and stirred, and the product was extracted with ethyl acetate. The organic layer was dried (anhydrous magnesium sulfate) and concentrated. The residue was dissolved in a small amount of dichloromethane, and concentrated by adding silica gel. Purification by silica gel column chromatography (hexane: ethyl acetate = 1: 1) gave compound 36 (293 mg, 0.402 mmol, 71%).

¹ H-NMR (CDCl ₃ ): δ 9.29 (1H, br.s), 9.24 (1H, br.s), 8.44 (1H, s), 8.04-7.49 (10H, m), 6.49 (1H, t), 5.98 (1H, dd), 5.57 (1H, dd), 5.34 (1H, dd), 4.90 (1H, t), 3. 72 (1H, d), 3.68 (1H, dd), 2.87 (3H, br.s), 2.49 (2H, m), 0.94 (9H, s), 0.91 (9H , S), 0.04, 0.03 (12H, s).

Next, using Compound 36 obtained as described above, Compound R3 (2-Amino-2'-deoxy-4'-vinyladenosine) was synthesized as follows.

That is, compound 36 (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 stirred at room temperature for 1 hour. Stir. After the reaction solution was concentrated, 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 40% aqueous methylamine solution (10 mL) and stirred at room temperature for 48 hours. The reaction solution 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 R3 (83 mg, 0.28 mmol, 70%).

¹ H-NMR (DMSO-d ₆ ): δ 7.94 (1H, s), 6.71 (1H, br. S), 6.16 (1H, t), 5.95 (1H, dd), 5 .69 (2H, br.s), 5.49 (1H, t), 5.35 (1H, dd), 5.19-5.16 (2H, m), 5.18 (1H, dd), 4.58 (1H, q), 3.50 (1H, dd), 3.41 (1H, dd), 2.54 (1H, m), 2.17 (1H, m).

Synthesis Example 26 Synthesis of 2′-deoxy-4′-vinyl guanosine (Compound R4) 2′-Deoxy-4′-vinyl guanosine (Compound R4) was synthesized as follows.

That is, Compound R3 (30 mg, 0.10 mmol) obtained in Synthesis Example 25 was dissolved in Tris-HCl buffer (50 mM, pH 7.5, 2.8 mL), and calf spleen-derived adenosine deaminase (3 μL, 3. 5 units) and stirred at 40 ° C. for 5 hours. After standing 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 H-NMR (DMSO-d ₆ ): δ 10.60 (1H, br.s), 7.98 (1H, s), 6.44 (1H, br.s), 6.10 (1H, dd) , 5.95 (1H, dd), 5.34 (1H, dd), 5.24 (1H, br.s), 5.19 (1H, dd), 5.11 (1H, br.s), 4.57 (1H, t), 3.50 (1H, d), 3.40 (1H, d), 2.44 (1H, m), 2.19 (1H, m).

In the nucleoside derivatives obtained by synthesis as described above, antiviral activity and cytotoxicity were evaluated by the following methods. The compound P3 was evaluated in the form of a mixture with the compound P3 ′.

Test Example 1: Evaluation of anti-HBV activity (2-week evaluation system)
As test cells, HepG2 2.2.15 cells prepared to continuously produce HBV by introducing the HBV gene into a human liver cancer-derived cell line (HepG2 cells) were used. In addition, HepG2 2.2.15 cells were maintained by continuous culture in DMEM containing 10% fetal bovine serum. Moreover, since the said cell has the HBV gene produced as an episome, the DNA of this episomal 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 obtained results are shown in Tables 4 to 6.

More specifically, HepG2 2.2.15 cells were seeded in 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 solution to which each nucleoside derivative was added was changed every 4 days and cultured for 12 days in the presence of the derivative. Thereafter, total cell DNA was extracted from each HepG2 2.2.15 cell using QIAamp DNA Blood Mini Kit (manufactured by QIAGEN) and dissolved in 200 μL of 1 × TE buffer. Subsequently, HBV DNA was quantified by real-time PCR using the DNA thus extracted as a template. That is, 2 μL of DNA extracted from HepG2 2.2.15 cells was amplified using 2 × SYBR PCR master mix (manufactured by Applied Biosystems). The amplification (PCR) reaction used the following primer set that detects the HBV polymerase region:
5′-GCGAGGACTGGGGGACCCTGTGACGAAC-3 ′ (SEQ ID NO: 1) and 5′-GTCCACCACGAGTTCTAGACTCTGC-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.

Data obtained by such a PCR reaction was analyzed with StepOne ™ Software Version 2.0 (Applied Biosystems) to obtain a CT value. Subsequently, the CT value was calculated from the HBV copy number (HBV) in the presence of each nucleoside derivative by using a calibration curve prepared by diluting the HBV plasmid at a known concentration every 10 times (20 to 2 × 10 ⁸ copies). The amount of DNA). Then, the EC ₅₀ value was calculated from the degree of decrease compared with that in the control cultured in the absence of the nucleoside derivative, and the anti-HBV activity of each nucleoside derivative was evaluated. The obtained results are shown in Tables 4 to 6.

Test Example 2: Evaluation of anti-HBV activity (one week evaluation system)
As test cells, HepG2 2.2.5.7 cells were used, which were the above HepG2 2.2.15 cells as a parent strain. HepG2 2.2.15 cells were maintained by continuous culture in DMEM containing 10% fetal bovine serum, G418 (500 μg / ml) and antibiotics (penicillin and kanamycin). Moreover, the HepG2 2.2.5.7 cell is an HBV continuous production cell that retains not only DNA integrated into the genome but also an HBV gene produced as an episome, like the HepG2 2.2.15 cell. Therefore, the nucleoside derivatives were co-cultured, the number of viral DNA copies released into the culture supernatant was quantified, and the degree of decrease was used as an index for evaluating anti-HBV activity.

More specifically, a collagen-coated 96-well cell culture dish was seeded with HepG2 2.2.15.7 cells having a cell viability of 90% or more at a concentration of 2 × 10 ⁴ cells / ml, and the same day of cell seeding. Each nucleoside derivative was added at various concentrations. After culturing for 3 days under standard culture conditions of 37 ° C. and 5% CO ₂ , the culture medium was further 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, quantitative PCR was performed from the collected medium in the same manner as described above, and the virus copy number was determined from the calibration curve, and the EC ₅₀ for each nucleoside derivative was calculated. The obtained results are also shown in Tables 4 to 6.

In the table, 1W represents a one-week evaluation type result, and 2W represents a two-week evaluation type result. The difference between the two is not only the culture period, but also whether the DNA to be evaluated is total DNA including intracellular (2-week evaluation system) or only extracellular DNA (1-week evaluation system).

Test Example 3: Cytotoxicity test With respect to the nucleoside derivative, a cytotoxicity test against HepG2 cells was also conducted. HepG2 cells were seeded to a concentration of 1 × 10 ⁴ cells / ml together with a medium supplemented with each concentration of each nucleoside derivative after serial dilution. Thus, after culturing these cells for 7 days at 37 ° C. and 5% CO _{2 in} the presence of various concentrations of each nucleoside derivative, the number of viable 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 obtained results are also shown in Tables 4 to 6.

Tables 4 to 6 also show values obtained by dividing the CC ₅₀ value by the EC ₅₀ value as the selectivity index. The greater the selectivity index, the greater the toxicity / activity ratio, and the better the drug is.

As is clear from the results shown in Tables 4 to 6, it was revealed that the compounds P1 to P4 are less cytotoxic than the compounds C1 and C2 and R1 to R4. It was also revealed that the compounds P1 to P4 have excellent antiviral activity against HBV.

As described above, according to the present invention, it is possible to provide a nucleoside derivative that has at least excellent antiviral activity against HBV and has low toxicity to host cells. Therefore, the present invention is extremely useful in the prevention or treatment of viral infections.

SEQ ID NO: 1 and 2
<223> Artificially synthesized primer sequences

Claims (4)

1. A nucleoside derivative represented by the following general formula (1).
   

   

   In formula (1), R is an alkyl group which may have a substituent, an alkenyl group which may have a substituent, a cyano group, or an azide group.
2. The nucleoside derivative according to claim 1, wherein R is a monofluoromethyl group, an ethenyl group, a cyano group, or an azide group.
3. An antiviral agent comprising the nucleoside derivative according to claim 1 or 2 as an active ingredient.
4. The antiviral agent according to claim 3, which is an anti-hepatitis B virus agent.