JP7125714B2 - 2'-Deoxy-7-deazapurine nucleoside derivatives with antiviral activity - Google Patents

Description

The present invention relates to 2'-deoxy-7-deazapurine nucleoside derivatives having antiviral activity, more particularly 2'-deoxy-7-deazapurine nucleoside derivatives having antiviral activity against at least hepatitis B virus. The present invention relates to a purine nucleoside derivative and an antiviral agent containing the derivative as an active ingredient.

Hepatitis B virus (HBV) infection can cause acute or fulminant hepatitis and sometimes death. In some cases, chronic hepatitis develops and progresses to cirrhosis and hepatocellular carcinoma. It is estimated that about 400 million people are infected with the disease worldwide, and the prevalence rate is extremely high mainly in Southeast Asia.

HBV is a defective 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 does not occur in the human host, inhibition of this step makes it possible to block only HBV replication. Nucleoside derivative preparations have been developed as therapeutic agents for HBV infection from such a point of view (Patent Documents 1 and 2).

Japanese Patent Application Laid-Open No. 2004-244422 Japanese Patent Application Laid-Open No. 2008-273960

Many of the current nucleoside derivative preparations are toxic to host cells, ie, human cells to which they are administered, and side effects caused by medium- to long-term administration have become a problem. Therefore, at present, no effective treatment method for viral 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 that has antiviral activity at least against HBV and low toxicity to host cells.

The present inventors have made intensive studies to solve the above problems, and found that, in 2′-deoxy-7-deazapurine nucleosides, the 2nd, 6th and 7th positions of the purine base and the 4th position of the ribose sugar was substituted with a specific functional group, while exhibiting excellent antiviral activity against HBV, they generally have low cytotoxicity, leading to the completion of the present invention.

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

[In the above formula, R ¹ represents a hydroxy group or an optionally substituted amino group. ^R2 represents a hydrogen atom, a halogen atom or an amino group. R3 represents an optionally substituted alkyl group ^, cyano group, azide group or hydrogen atom. R ⁴ represents a hydrogen atom, a halogen atom, an optionally substituted alkynyl group, or an optionally substituted heterocyclic group. ]
<2> An antiviral agent comprising the nucleoside derivative according to <1> as an active ingredient.
<3> The antiviral agent according to <2>, which is an anti-hepatitis B virus agent.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a nucleoside derivative that has antiviral activity against at least HBV and low toxicity to host cells.

(Nucleoside derivative)
As shown in Examples below, the nucleoside derivative represented by the following formula was found to have antiviral activity against hepatitis B virus. Accordingly, the present invention relates to nucleoside derivatives exhibiting antiviral activity, and more specifically, to provide nucleoside derivatives represented by the following general formula (1) having antiviral activity against at least hepatitis B virus. be.

[In the above formula, R ¹ represents a hydroxy group or an optionally substituted amino group. ^R2 represents a hydrogen atom, a halogen atom or an amino group. R3 represents an optionally substituted alkyl group ^, a cyano group, or a hydrogen atom. R ⁴ represents a hydrogen atom, a halogen atom, an optionally substituted alkynyl group, or an optionally substituted heterocyclic group. ].

The nucleoside derivatives of the invention have antiviral activity at least against hepatitis B virus (HBV). In the present invention, "HBV" means a virus capable of causing hepatitis B. As HBV, genotypes A (A2/Ae, A1/Aa), B (Ba, B1/Bj), C (Cs, Ce), DH and J are known. Derivatives may have antiviral activity against at least one genotype of HBV.

In the present invention, the term "antiviral activity" means an activity that eliminates or suppresses the growth of a virus such as HBV in a cell (host cell) infected with the virus, such as suppressing viral replication in the host cell. activity to In addition, when the target of such suppression or the like is a virus having DNA as its genome (DNA virus), it is referred to as "anti-DNA virus activity". Furthermore, such activity can be evaluated by an _EC50 value calculated using the number of virus copies in host cells as an index, as shown in the Examples below. The nucleoside derivative of the present invention preferably has an antiviral activity EC50 value of less than 1 μM, more preferably _0.5 μM or less, even more preferably 0.1 μM or less, and 0.05 μM or less ( For example, it is more preferably 0.04 μM or less, 0.03 μM or less, 0.02 μM or less, 0.01 μM or less).

Also, the nucleoside derivative of the present invention preferably has low cytotoxicity. In the present invention, "cytotoxicity" means an activity that kills cells, inhibits their function, or suppresses their proliferation. Such activity can be evaluated by a _CC50 value calculated using the number of viable cells or the like as an index, as shown in Examples below. The nucleoside derivative of the present invention preferably has a CC ₅₀ value of 10 μM or higher, _more preferably 25 μM or higher, even more preferably ₅₀ μM or higher, and 100 μM or higher. is more preferred.

From the viewpoint that the nucleoside derivative of the present invention can exhibit at least antiviral activity against HBV while reducing the cytotoxicity of the derivative, each substituent is preferably selected as shown below.

The substituent in the "optionally substituted amino group" is preferably an alkyl group having 1 or more carbon atoms, more preferably a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, A linear, branched or cyclic alkyl group having 1 to 4 carbon atoms is more preferred, and a methyl group is more preferred. The "amino group optionally having substituent(s)" is more specifically preferably an amino group or a methylamino group.

The alkyl group in the "alkyl group optionally having substituent(s)" 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 preferred. more preferred. The substituent in the "alkyl group optionally having substituent(s)" is not particularly limited and includes, for example, a halogen atom, a hydroxy group, an alkoxy group, a cyano group and an amino group, preferably a halogen atom, fluorine Atoms are more preferred. More specifically, the "optionally substituted alkyl group" is preferably a monofluoromethyl group.

A "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom, a chlorine atom or an iodine atom, more preferably a chlorine atom or a fluorine atom.

The alkynyl group in the "alkynyl group optionally having substituents" is not particularly limited, but linear, branched or cyclic alkynyl groups having 2 or more carbon atoms are preferable, and A linear, branched or cyclic alkynyl group is more preferred, and an ethynyl group is even more preferred. The substituents in the "alkynyl group optionally having substituent(s)" are not particularly limited, and examples thereof include halogen atoms, hydroxy groups, alkoxy groups, cyano groups, and amino groups.

The heterocyclic group in the "optionally substituted heterocyclic group" is not particularly limited, and examples thereof include aromatic or aliphatic heterocyclic rings containing a nitrogen atom, an oxygen atom and a sulfur atom. Among these, nitrogen-containing heterocyclic groups are preferred, such as pyrrolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, imidazolyl, imidazolinyl, pyrazolyl, (1,3,5 -) triazinyl group, pyrimidinyl group, pyridazinyl group, pyrazinyl group, indolyl group, quinolyl group, isoquinolyl group, purinyl group, benzimidazolyl group, benzoxazolyl group, benzthiazolyl group, tetrazolyl group, tetrazinyl group, triazolyl group, carbazolyl group , an acridinyl group, a quinoxalyl group, and a quinazolyl group, and among these, a pyrazolyl group is preferred. The substituents in the "optionally substituted heterocyclic group" are not particularly limited, and examples thereof include halogen atoms, hydroxy groups, alkoxy groups, cyano groups, and amino groups.

Examples of ^{nucleoside derivatives} having suitable functional groups include those of the above general ^{formula (} Nucleoside derivatives represented by 1) are included.

The nucleoside derivatives of the present invention also include pharmacologically acceptable salts, hydrates or solvates. Such pharmacologically acceptable salts are not particularly limited and may be appropriately selected depending on the structure of the nucleoside derivative. Examples include acid addition salts (hydrochloride, sulfate, hydrobromide, Nitrate, hydrogen sulfate, phosphate, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluenesulfone acid, camphor sulfonate, sulfamate, mandelate, propionate, glycolate, stearate, malate, ascorbate, pamonate, phenylacetate, glutamate, benzoate , salicylate, sulfanilate, 2-acetoxybenzoate, ethanedisulfonate, oxalate, isethionate, formate, trifluoroacetate, ethylsuccinate, lactobionate, gluconate, glucoheptonic acid salt, 2-hydroxyethanesulfonate, benzenesulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate, oxalate, picrate, thiocyanate, undecane acid salts, etc.), base addition salts (sodium salts, potassium salts, zinc salts, calcium salts, bismuth salts, barium salts, magnesium salts, aluminum salts, copper salts, cobalt salts, nickel salts, cadmium salts, ammonium salts, ethylenediamine salts , 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 1 molecule of the nucleoside derivative or 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 carbons, and stereoisomers. Furthermore, the nucleoside derivative of the present invention undergoes metabolism such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydration-oxidation, alkylation, dealkylation, conjugation, etc. in vivo, and is still desired. The present invention also includes compounds that undergo metabolism such as oxidation, reduction and hydrolysis in vivo to produce the nucleoside derivatives of the present invention (so-called prodrug forms). . Furthermore, the nucleoside derivative of the present invention can be formulated by known pharmaceutical methods, as described below.

Further, the synthesis of the nucleoside derivative of the present invention is carried out, for example, by combining a ribose sugar (D-ribofuranose in which the hydroxy group is substituted with an acetyl group, a benzyl group, etc.) and a purine base (7-deazaadenine). , reacted via a silyl derivative, further reduced via a phenoxythiocarbonyl derivative to deoxylate the 2-position of the ribose sugar, and if necessary, substitute at the target position of the ribose sugar and / or purine base by a known method. It can be done by introducing a group. The method for synthesizing such a nucleoside derivative of the present invention is described in detail in the examples below. , solvent, reaction temperature, catalyst, reaction time), etc., and by appropriately modifying or altering these methods as necessary, it is possible to synthesize the nucleoside derivative of the present invention. In addition, the nucleoside derivative synthesized in this way can be appropriately processed by methods (reverse phase chromatography, ion exchange chromatography, adsorption chromatography, recrystallization method) used for isolation and purification of general nucleosides and nucleotides. They can be separated and purified by using them alone or in combination.

(Antiviral agents, methods for preventing and treating viral infections)
As shown in the examples below, the nucleoside derivatives of the present invention have antiviral activity at least against hepatitis B virus. Therefore, it is possible to provide an antiviral agent containing the nucleoside derivative of the present invention as an active ingredient.

The infectious diseases targeted by the antiviral agent of the present invention and the prophylactic and therapeutic methods described below are not particularly limited. hepatitis, fulminant hepatitis), liver cirrhosis, liver fibrosis, and hepatocellular carcinoma.

The antiviral agent of the present invention can be formulated by known pharmaceutical methods. For example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coating agents, pellets, lozenges, sublingual agents, chewing agents, buccal agents, pastes, syrups, suspensions, Use orally or parenterally as elixirs, emulsions, ointments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. can be done.

In these formulations, pharmacologically acceptable carriers or media, specifically sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents , flavoring agents, excipients, vehicles, preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, sweetening agents, It can be appropriately combined with a thickening agent, a flavoring agent, a solubilizing agent, or other additives. More specifically, solid carriers such as lactose, kaolin, sucrose, crystalline cellulose, cornstarch, 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, water and the like are also included.

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 (adrenal corticosteroid hormone withdrawal therapy, oral propagernium preparation, etc.), liver support therapy (intravenous administration of glycyrrhizin preparation, oral administration of bile acid preparation, etc.) The antiviral agent of the present invention can also be used for combination therapy with.

Preferred administration forms of the antiviral agent of the present invention are not particularly limited, and are oral administration or parenteral administration, more specifically, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, and respiratory tract administration. Intra-, rectal and intramuscular administration, and administration by infusion.

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

When administering the antiviral agent of the present invention, the dosage is appropriately selected according to the subject's age, body weight, symptoms, health condition, serious condition, drug tolerance, dosage form, and the like. The daily dose of the antiviral agent of the present invention is usually 0.00001 to 1000 mg/kg body weight, preferably 0.0001 to 100 mg/kg body weight, in terms of the amount of the nucleoside derivative as an active ingredient. Multiple doses are administered to the subject.

The product of the antiviral agent of the present invention or its instructions may be labeled as being used for treating or preventing viral infections. Here, "labeled on the product or instruction manual" means that the label is attached to the body, container, packaging, etc. of the product, or instruction manuals, attached documents, advertising materials, and other printed materials that disclose product information It means that the display is attached to etc. In addition, in the indication that the nucleoside derivative of the present invention is used for treating viral infections, 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 on the mechanism of action of the antiviral agent.

Thus, according to the present invention, infectious diseases can be prevented or treated by administering the antiviral agent of the present invention to a subject. Accordingly, the present invention also provides a method for preventing or treating viral infections, which comprises administering the nucleoside derivative of the present invention.

Subjects to which the nucleoside derivative of the present invention is administered are not particularly limited, and include, for example, patients with viral infections such as HBV, viral carriers before the onset of infectious diseases, and those before being infected.

In order to obtain a nucleoside derivative having antiviral activity, nucleoside derivatives represented by the following general formula (1) having functional groups in the combinations shown in Table 1 below were synthesized by the method shown below. The numbers in the table indicate the numbers of the compounds shown below.

Furthermore, it was confirmed by measuring ¹ H nuclear magnetic resonance (NMR) spectrum that the compound thus synthesized had the desired structure. These results are also shown below.

Synthesis Example 1: Synthesis of 4-amino-7-(2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl-pyrrolo[2,3-d]pyrimidine 4-amino-7-(2-deoxy- 4-C-fluoromethyl-β-D-ribofuranosyl-pyrrolo[2,3-d]pyrimidine (compound 8) was synthesized by the reaction steps shown below.

First, Biosci. Biotechnol. Biochem. 1999, 63, 736-742 (ref. 1 above), from 1,2:5,6-di-O-isopropylidene-α-D-allofuranose (Compound 1) to 1, 2-O-isopropylidene-3-O-p-methoxybenzyl-α-D-allofuranose (compound 2) was synthesized.

Next, compound 3 (3,5-di-O-benzyl-4-C-fluoromethyl-1,2-O-isopropylidene-α-D-ribofuranose) is obtained from compound 2 thus obtained. Synthesized. That is, after dissolving compound 2 (10.5 mg, 0.026 mmol) in toluene (1 mL), N,N-diethylaminosulfur trifluoride (6.9 μL, 0.052 mmol) was added and stirred at 60° C. for 2 hours. did. Subsequently, N,N-diethylaminosulfur trifluoride (6.9 μL, 0.052 mmol) was added, and the mixture was stirred at 60° C. for 4 hours. After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium bicarbonate solution and then 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=1/5) to obtain compound 3 (6.1 mg , 0.015 mmol, 58%).
¹ H-NMR (CDCl ₃ , 500 MHz); , J=48.5, 10.0 Hz), 4.74-4.60 (3H, m), 4.57-4.48 (3H, m), 4.26 (1H, dd, J=5. 0, 1.5Hz), 3.61 (1H, dd, J = 10.5, 2.5Hz), 3.55 (1H, dd, J = 10.5, 2.0Hz), 1.63 (3H , s), 1.35(3H, s).

Next, from compound 3 thus obtained, compound 4 (1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-fluoromethyl-D-ribofuranose) is obtained. Synthesized. That is, after dissolving compound 3 (269 mg, 0.67 mmol) in acetic acid (4.2 mL), water (1.3 mL) and trifluoroacetic acid (0.42 mL) were sequentially added and stirred for 5.5 hours. After completion of the reaction, the solvent was distilled off to obtain a crude deacetonide (0.67 mmol). The crude deacetonide (0.67 mmol) was azeotroped three times with toluene. After the obtained residue was dissolved in pyridine (3.5 mL), acetic anhydride (0.19 mL, 2.0 mmol) and 4-dimethylaminopyridine (8.15 mg, 67 μmol) were added and stirred for 1 hour. After completion of the reaction, 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 4 (293 mg, 0 .66 mmol, 98%).
¹ H-NMR (CDCl ₃ , 500 MHz); 4.70-4.64 (1H, dd), 4.61-4.48 (5H, m), 4.43-4.34 (1H, m), 3.70-3.62 (1H, dd , J=10.0, 2.0 Hz), 3.51-3.49 (1H, dd, J=9.5, 2.0 Hz), 2.10 (3H, s), 2.04-1. 90 (3H, s).

Next, from compound 4 thus obtained, compound 5 (7-(2-O-acetyl-3,5-di-O-benzyl-4-C-fluoromethyl-β-D-ribofuranosyl)- 4-chloro-5-iodopyrrolo[2,3-d]pyrimidine) was synthesized. That is, acetonitrile (0.2 mL) was added to 6-chloro-7-iodo-7-deazapurine (15.1 mg, 0.054 mmol), followed by N,O-bis(trimethylsilyl)acetamide (15.9 μL, 0.05 μL). 065 mmol) was added and stirred at room temperature for 20 minutes. Subsequently, compound 4 (15.4 mg, 0.035 mmol) dissolved in acetonitrile (0.3 mL) and trimethylsilyl trifluoromethanesulfonate (12.5 μL, 0.069 mmol) were sequentially added and stirred at room temperature for 10 minutes. After that, the mixture was stirred under heating under reflux for 21 hours. After completion of the reaction, the reaction mixture was quenched with saturated aqueous sodium bicarbonate solution and then 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=1/6) to obtain compound 5 (9.8 mg , 0.015 mmol, 43%).
¹ H-NMR (CDCl ₃ , 500 MHz); J = 6.5 Hz), 5.69 (1H, t, J = 5.5 Hz), 4.71-4.48 (8H, m), 3.76 (1H, dd, J = 10.0, 2 .0 Hz), 3.70 (1 H, dd, J=10.0, 2.5 Hz), 2.02 (3 H, s).

Next, from compound 5 thus obtained, compound 6 (4-amino-7-(3,5-di-O-benzyl-4-C-fluoromethyl-2-O-phenylthionoformyl- β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine) was synthesized. That is, after dissolving compound 5 (9.8 mg, 0.015 mmol) in 1,4-dioxane (0.5 mL), aqueous ammonia (1 mL) was added and the mixture was stirred at 120° C. for 20 hours. After completion of the reaction, extraction was performed 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 6-aminated compound (9.5 mg, 0.015 mmol). After dissolving the crude 6-aminated product (9.5 mg, 0.015 mmol) in acetonitrile (1 mL), 4-dimethylaminopyridine (3.0 mg, 0.025 mmol) and phenyl chlorothionoformate (2. 3 μL, 0.016 mmol) was sequentially added and stirred at 0° C. for 1 hour. Subsequently, phenyl chlorothionoformate (1.0 μL, 7.1 μmol) was added and stirred at 0° C. for 1 hour, then phenyl chlorothionoformate (1.0 μL, 7.1 μmol) was added and stirred at room temperature for 1 hour. . Furthermore, 4-dimethylaminopyridine (2.2 mg, 0.018 mmol) and phenyl chlorothionoformate (2.0 μL, 0.014 mmol) were sequentially added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was quenched with water and then 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=1/1) to obtain compound 6 (8.2 mg , 0.011 mmol, 2 steps 75%).
¹ H-NMR (CDCl ₃ , 500 MHz); J = 6.5 Hz), 6.21 (1H, dd, J = 7.0, 5.5 Hz), 5.74 (2H, brs), 4.86-4.46 (8H, m), 3. 78 (2H, m).

Compound 7 (4-amino-7-(3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl )-pyrrolo[2,3-d]pyrimidine) were synthesized. That is, after dissolving compound 6 (133 mg, 0.18 mmol) in toluene (6 mL), tributyltin hydride (0.24 mL, 0.90 mmol) and azobisisobutyronitrile (7.4 mg, 0.045 mmol) were sequentially added and stirred at 80° C. for 35 minutes. Subsequently, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain compound 7 (42.3 mg, 0.092 mmol, 51%).
¹ H-NMR (CDCl ₃ , 500 MHz); 74 (1H, t, J = 6.5Hz), 6.31 (1H, d, J = 4.0Hz), 5.12 (2H, brs), 4.77-4.49 (8H, m), 3.72 (1H, dd, J = 9.5, 1.5Hz), 3.65 (1H, dd, J = 10.0, 2.0Hz), 2.69-2.64 (1H, m) , 2.61-2.56 (1H, m).

Compound 8 (4-amino-7-(2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-pyrrolo[2,3-d] is then obtained from compound 7 thus obtained. pyrimidines) were synthesized. That is, after dissolving compound 7 (25.6 mg, 0.055 mmol) in dichloromethane (2 mL), boron trichloride (1.0 M dichloromethane solution, 0.28 mL, 0.28 mmol) was added at -78°C, The mixture was stirred at the same temperature for 3 hours. After completion of the reaction, quenching with saturated aqueous sodium bicarbonate and evaporation of the solvent under reduced pressure were performed. After azeotroping with methanol, the residue was purified by silica gel column chromatography (methanol/chloroform=1/6) to obtain compound 8 (13.4 mg, 0.048 mmol, 77%).
¹ H-NMR (CD ₃ OD, 500 MHz); δ 8.06 (1 H, s), 7.31 (1 H, d, J = 3.5 Hz), 6.60 (1 H, d, J = 3.5 Hz) , 6.55 (1H, dd, J = 8.5, 6.0 Hz), 4.68 (1H, dd, J = 33.5, 10.0 Hz), 4.65 (1H, dd, J = 6 .0, 3.0 Hz), 4.58 (1H, dd, J = 34.0, 10.0 Hz), 3.74 (2H, m), 2.89-2.83 (1H, m), 2 .39-2.34 (1H, m).

Synthesis Example 2: 4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine and 7-(4-C-cyano-2-deoxy- Synthesis of β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3-d]pyrimidine 4-Amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)pyrrolo[2,3 -d]pyrimidine (Compound 18) and 7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3-d]pyrimidine (Compound 19) are described below. Synthesized by the indicated reaction steps.

First, from compound 9 (see Synthesis 1988(9):670-674), compound 10 (7-(3-O-tert-butyldimethylsilyl-2-deoxy-β-D-ribofuranosyl)-4-chloropyrrolo[2 , 3-d]pyrimidines) were synthesized. That is, compound 9 (3.65 g, 6.4 mmol) was dissolved in N,N-dimethylformamide (64 mL), imidazole (1.30 g, 19 mmol) was added, and then tert-butyldimethylsilyl was added under ice cooling. Chloride (1.44 g, 9.6 mmol) was added and stirred at room temperature for 19.5 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (150 mL), and a methanol solution (60 mL) of tosylic acid monohydrate (2.43 g, 13 mmol) was added at -15°C over 10 minutes. and stirred for 5 minutes. A saturated aqueous solution of sodium bicarbonate was added to stop the reaction, and the mixture was partitioned, extracted with chloroform, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=9:1-4:1) to obtain Compound 10 (2.32 g, 2 steps 95%).
¹ H-NMR (CDCl ₃ ): δ 8.63 (1H, s), 7.35 (1H, d, J = 3.7 Hz), 6.62 (1H, d, J = 3.7 Hz), 6. 29 (1H, dd, J = 5.5Hz, 9.3Hz), 5.38 (1H, dd, J = 2.0Hz, 9.2Hz), 4.70 (1H, d, J = 5.3Hz) , 4.13 (1H, d, J = 1.4 Hz), 3.97-3.93 (1H, m), 3.80-3.74 (1H, m), 3.04-2.99 ( 1 H, m), 2.24-2.20 (1 H, m), 0.93 (9 H, s), 0.12 (6 H, s).

Next, from compound 10 thus obtained, compound 11 (7-(3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl)-4- chloropyrrolo[2,3-d]pyrimidines) were synthesized. That is, compound 10 (2.25 g, 5.9 mmol) was dissolved in dimethyl sulfoxide (18 mL) and toluene (12 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ( 4.49 g, 23 mmol), pyridine (630 μL, 7.8 mmol) and trifluoroacetic acid (292 μL, 3.9 mmol) were added and stirred for 5 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in 1,4-dioxane (18 mL), and 37% formaldehyde aqueous solution (3.3 mL, 41 mmol) and 1 M sodium hydroxide aqueous solution (6 .2 mL, 6.2 mmol) was added and stirred for 22 hours. After quenching the reaction by adding acetic acid, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in ethanol (30 mL), sodium borohydride (226 mg, 6.0 mmol) was added under ice-cooling, and the mixture was stirred for 30 minutes. After quenching the reaction by adding acetic acid, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=9:1-1:1) to obtain compound 11 (1.02 g, 3 process 42%).
¹ H-NMR (CDCl ₃ ): δ 8.63 (1H, s), 7.34 (1H, d, 3.7 Hz), 6.61 (1H, d, J = 3.7 Hz), 6.40 ( 1H, dd, J = 6.1 Hz, 8.5 Hz), 5.12 (1 H, d, J = 9.3 Hz), 4.93 (1 H, dd, J = 2.1 Hz, 6.4 Hz), 3 .85-3.80 (2H, m), 3.71-3.66 (2H, m), 3.20-3.14 (1H, m), 2.67 (brs, 1H), 2.37 -2.33 (1H,m), 0.96 (9H,s), 0.18 (3H,s), 0.17 (3H,s).

Next, from compound 11 thus obtained, compound 12 (7-(3-O-tert-butyldimethylsilyl-2-deoxy-4-C-dimethoxytrityloxymethyl-β-D-ribofuranosyl)- 4-chloropyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 11 (1.02 g, 2.5 mmol) was dissolved in dichloromethane (15 mL), triethylamine (515 μL, 3.7 mmol) was added, and 4,4′-dimethoxytrityl chloride (918 mg, 2 .7 mmol) in dichloromethane (10 mL) was added dropwise over 20 minutes and then stirred for 4 hours. After the reaction was stopped by adding methanol, chloroform and water were added for partition, and the organic layer was washed with saturated aqueous sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified with a medium-pressure fractionator (n-hexane/ethyl acetate = 4:1-2:1), and then again subjected to silica gel chromatography (n-hexane/ Ethyl acetate=5:1) to obtain compound 12 (1.44 g, 82%).
¹ H-NMR (CDCl ₃ ): δ 8.67 (1H, s), 7.47-7.19 (10H, m), 6.83-6.80 (4H, m), 6.64 (1H, d, J = 3.6 Hz), 6.30 (1H, dd, J = 6.1 Hz, 8.3 Hz), 4.71 (1H, dd, J = 2.6 Hz, 6.1 Hz), 4.66 (1H, dd, J = 3.0Hz, 10.7Hz), 4.20 (1H, dd, J = 3.0Hz, 12.3Hz), 3.79 (3H, s), 3.78 (3H, s), 3.64 (1H, dd, J = 10.7Hz, 12.3Hz), 3.53 (1H, d, J = 10.7Hz), 3.05-3.03 (2H, m), 2.24-2.29 (1H,m), 0.76 (9H,s), -0.03 (3H,s), -0.12 (3H,s).

Compound 13 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4-C-hydroxymethyl -β-D-ribofuranosyl)-4-chloropyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, compound 12 (1.41 g, 2.0 mmol) and imidazole (408 mg, 6.0 mmol) were dissolved in N,N-dimethylformamide (20 mL), and tert-butyldiphenylsilyl chloride (768 μL, 3.0 mmol) was added and stirred at room temperature for 24 hours, imidazole (136 mg, 2.0 mmol) and tert-butyldiphenylsilyl chloride (256 μL, 1.0 mmol) were added and the mixture was further stirred for 3 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (40 mL), and a methanol solution (20 mL) of tosylic acid monohydrate (761 mg, 4.0 mmol) was added at -15°C for 15 minutes. and stirred for 10 minutes. A saturated aqueous sodium bicarbonate solution was added to stop the reaction, and the mixture was partitioned. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=7:1-1:1) to obtain Compound 13 (1.25 g, 2 steps 96%).
¹ H-NMR (CDCl ₃ ): δ 8.55 (1H, s), 7.65-7.28 (11H, m), 6.70 (1H, t, J = 6.6 Hz), 6.47 ( 1H, d, J = 3.7Hz), 4.87 (1H, dd, J = 4.2Hz, 6.5Hz), 3.90-3.74 (4H, m) 2.75-2.70 ( 1H, m), 2.48-2.43 (1H, m), 2.34 (1H, dd, J = 5.1 Hz, 8.7 Hz), 1.08 (9H, s), 0.93 ( 9H, s), 0.13 (3H, s), 0.13 (3H, s).

Compound 14 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy- β-D-ribofuranosyl)-4-chloropyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 13 (326 mg, 0.50 mmol) was dissolved in dimethylsulfoxide (1.5 mL) and toluene (1.0 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (288 mg, 1 .5 mmol), pyridine (40 μL, 0.50 mmol), and trifluoroacetic acid (19 μL, 0.25 mmol) were added and stirred for 23 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in pyridine (2.5 mL), hydroxylamine hydrochloride (52 mg, 0.75 mmol) was added, and the mixture was stirred for 1.5 hours. The reaction mixture was concentrated under reduced pressure and partitioned with ethyl acetate and water. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in dichloromethane (2.5 mL), triethylamine (139 μL, 1.0 mmol) and mesyl chloride (58 μL, 0.75 mmol) were added, and the mixture was stirred for 1 hour. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=9:1) to obtain compound 14 (266 mg, 3 steps 82%).
¹ H-NMR (CDCl ₃ ): δ 8.50 (1H, s), 7.62-7.29 (11H, m), 6.63 (1H, t, J = 6.5 Hz), 6.55 ( 1H, d, J = 3.7 Hz), 4.97 (1H, t, J = 5.9 Hz), 4.04 (1H, d, J = 11.2 Hz), 3.87 (1H, d, J = 11.2Hz), 3.00-2.95 (1H, m), 2.56-2.51 (1H, m), 1.05 (9H, s), 0.96 (9H, s), 0.18 (3H,s), 0.16 (3H,s).

Next, compound 15 (pyridinium salt) was synthesized from compound 14 thus obtained. That is, compound 14 (191 mg, 0.30 mmol) was dissolved in pyridine (4.5 mL) and water (1.5 mL) and stirred at 50°C for 65 hours. The reaction solution was concentrated under reduced pressure to obtain compound 15 as a crude product (0.30 mmol).

Compound 16 (4-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-deoxy-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, crudely purified compound 15 (0.15 mmol) was dissolved in 1,4-dioxane (1.5 mL), aqueous ammonia (3 mL) was added, and the mixture was stirred for 48 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=5:1-1:2) to obtain compound 16 (34 mg, 36%).
¹ H-NMR (CDCl ₃ ): δ 8.16 (1H, s), 7.64-7.32 (10H, m), 7.01 (1H, d, J = 3.7 Hz), 6.61 ( 1H, t, J = 6.5Hz), 6.30 (1H, d, J = 3.7Hz), 5.41 (2H, brs), 4.96 (1H, t, J = 5.8Hz), 4.03 (1H, d, J = 11.1 Hz), 3.86 (1H, d, J = 11.1 Hz), 2.99-2.94 (1H, m), 2.51-2.46 (1 H, m), 1.06 (9 H, s), 0.95 (9 H, s), 0.17 (3 H, s), 0.15 (3 H, s).

Compound 18 (4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine) is then obtained from compound 16 thus obtained. was synthesized.

Compound 16 (42 mg, 0.067 mmol) was dissolved in tetrahydrofuran (1 mL), 1M tetrabutylammonium fluoride solution in tetrahydrofuran (150 μL, 0.15 mmol) was added, and the mixture was stirred at room temperature for 45 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=5-7%) to obtain compound 18 (16 mg, 86%).
¹ H-NMR (DMSO-d ₆ ): δ 8.07 (1H, s), 7.34 (1H, d, J = 3.7 Hz), 7.08 (2H, brs), 6.64 (1H, t, J = 7.0 Hz), 6.61 (1H, d, J = 3.7 Hz), 6.27 (1H, d, J = 5.1 Hz), 5.82 (1H, t, J = 6 .1Hz), 4.60 (1H, dd, J = 5.1Hz, 6.1Hz), 3.75 (1H, dd, J = 5.9Hz, 11.8Hz), 3.62 (1H, dd, J=6.3 Hz, 11.8 Hz), 2.80-2.75 (1 H, m), 2.38-2.34 (1 H, m).

Further, from compound 15 obtained as described above, compound 17 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy- β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, crudely purified compound 15 (0.15 mmol) was dissolved in 1,4-dioxane (1.5 mL), an aqueous methylamine solution (3 mL) was added, and the mixture was stirred for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=2:1-1:2), and then re-chromatographed on silica gel (n-hexane/ethyl acetate=3: 1-1:2) to obtain compound 17 (50 mg, 0.078 mmol, 52%).
¹ H-NMR (CDCl ₃ ): δ 8.23 (1H, s), 7.64-7.28 (10H, m), 6.95 (1H, d, J = 3.7 Hz), 6.63 ( 1H, t, J = 6.6Hz), 6.29 (1H, d, J = 3.7Hz), 5.10 (1H, brs), 4.95 (1H, t, J = 5.7Hz), 4.04 (1H, d, J = 11.1 Hz), 3.86 (1H, d, J = 11.1 Hz), 3.16 (3H, d, J = 5.0 Hz), 2.99-2 .94 (1 H, m), 2.49-2.44 (1 H, m), 1.07 (9 H, s), 0.95 (9 H, s), 0.17 (3 H, s), 0. 15 (3H, s).

Compound 19 (7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3-d]pyrimidine) is then obtained from compound 17 thus obtained. ) was synthesized. That is, compound 17 (50 mg, 0.078 mmol) was dissolved in tetrahydrofuran (1 mL), 1M tetrabutylammonium fluoride solution in tetrahydrofuran (171 μL, 0.17 mmol) was added, and the mixture was stirred at room temperature for 50 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=3-7%) to obtain compound 19 (19 mg, 85%).
¹ H-NMR (DMSO-d ₆ ): δ 8.16 (1H, s), 7.56 (1H, d, J = 4.6 Hz), 7.34 (1H, d, J = 3.7 Hz), 6.65 (1H, t, J = 7.0Hz), 6.60 (1H, d, J = 3.6Hz), 6.28 (1H, d, J = 5.1Hz), 5.82 (1H , t, J = 6.1 Hz), 4.61 (1H, dd, J = 4.8 Hz, 11.0 Hz), 3.76 (1H, dd, J = 5.9 Hz, 11.7 Hz), 3. 62 (1H, dd, J = 6.3Hz, 11.7Hz), 2.95 (3H, d, J = 4.6Hz), 2.81-2.75 (1H, m), 2.39-2 .34(1H,m).

Synthesis Example 3: Synthesis of 7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-2,4-diaminopyrrolo[2,3-d]pyrimidine 7-(4-C-cyano-2 -deoxy-β-D-ribofuranosyl)-2,4-diaminopyrrolo[2,3-d]pyrimidine (Compound 30) was synthesized by the reaction steps shown below.

First, compound 22 (4-chloro-7-(2- deoxy-3,5-di-Op-toluoyl-β-D-ribofuranosyl)-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 21 (3.41 g, 14 mmol) was dissolved in acetonitrile (70 mL), sodium hydride (1.08 g, 27 mmol) was added, and compound 20 (5.52 g, 14 mmol) was added under ice cooling. Acetonitrile solution (70 mL) was added dropwise over 15 minutes and stirred for 1 hour under ice-cooling, then sodium hydride (540 mg, 14 mmol) was added and stirred at room temperature for 1.5 hours. 270 mg, 7.1 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After stopping the reaction by adding an aqueous solution of ammonium chloride under ice-cooling, the reaction solution was concentrated to half under reduced pressure and partitioned with ethyl acetate and water. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=9:1-2:1) to obtain compound 22 (4.76 g, 58% ).
¹ H-NMR (CDCl ₃ ): δ 8.18 (1H, brs), 8.00-7.22 (9H, m), 6.75 (1H, d, J = 6.2 Hz, 8.0 Hz), 6.51 (1H, d, J = 3.8Hz), 5.81-5.79 (1H, m), 4.74 (1H, d, J = 4.2Hz, 11.9Hz), 4.63 (1H, d, J = 4.4Hz, 11.9Hz), 4.57 (1H, dd, J = 4.2Hz, 7.1Hz), 3.05-3.00 (1H, m), 2. 81-2.77 (1H, m), 2.44 (3H, s), 2.42 (3H, s), 1.35 (9H, s).

Compound 23 (4-chloro-7-(2-deoxy-5-O-dimethoxytrityl-β-D-ribofuranosyl)-2-pivaloylaminopyrrolo [ 2,3-d]pyrimidines) were synthesized. That is, compound 22 (182 mg, 0.30 mmol) was dissolved in dichloromethane (1.5 mL) and methanol (1.5 mL), sodium methoxide (162 mg, 3.0 mmol) was added at -10°C, and ice The mixture was stirred for 4 hours under cooling. After 1M hydrochloric acid was added to stop the reaction, the reaction mixture was concentrated under reduced pressure, partitioned with chloroform and water, the aqueous layer was extracted with chloroform, and the chloroform layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was azeotroped with pyridine, dissolved in pyridine (3 mL), and dimethoxytrityl chloride (122 mg, 0.36 mmol) was added under ice-cooling. Stirred for an hour. Additional dimethoxytrityl chloride (31 mg, 0.090 mmol) was added and stirred for an additional 16 hours. After the reaction was stopped by adding methanol, the reaction solution was concentrated under reduced pressure, partitioned with ethyl acetate and water, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1-2:1) to obtain compound 23 (92 mg, 46% in 2 steps). ).
¹ H-NMR (CDCl ₃ ): δ 7.99 (1H, brs), 7.43-7.17 (9H, m), 7.11 (1H, d, J = 3.7 Hz), 6.84 ( 1H, d, J = 6.3 Hz, 7.3 Hz), 6.80-6.77 (4 H, m), 6.41 (1 H, d, J = 3.7 Hz), 4.66 (1 H, d , J = 3.0 Hz, 5.9 Hz), 4.15 (1 H, d, J = 3.4 Hz, 7.5 Hz), 4.08 (3 H, s), 3.77 (3 H, s), 3 .77 (3H, s), 3.42 (1H, d, J = 3.4Hz), 3.36-3.34 (1H, m), 2.59-2.57 (1H, m), 2 .54-2.52 (1 H, m), 1.30 (9 H, s).

Compound 24 (7-(3-O-tert-butyldimethylsilyl-2-deoxy-β-D-ribofuranosyl)-4-chloro-2-pivaloyl)-4-chloro-2-pivaloyl is then obtained from compound 23 thus obtained. aminopyrrolo[2,3-d]pyrimidines) were synthesized. That is, compound 23 (282 mg, 0.42 mmol) was dissolved in N,N-dimethylformamide (4 mL), imidazole (86 mg, 1.3 mmol) was added, and tert-butyldimethylsilyl chloride ( 95 mg, 0.63 mmol) was added and stirred at room temperature for 2 hours, imidazole (86 mg, 1.3 mmol) and tert-butyldimethylsilyl chloride (95 mg, 0.63 mmol) were added and stirred for 17 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (8 mL), and a methanol solution (4 mL) of tosylic acid monohydrate (160 mg, 0.84 mmol) was added at -15°C for 10 minutes. and stirred for 1 hour. A saturated aqueous sodium bicarbonate solution was added to stop the reaction, and then chloroform was added for partition. The organic layer was washed with a saturated aqueous sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=6:1-3:1) to obtain Compound 24 (189 mg, 93% in two steps). ).
¹ H-NMR (CDCl ₃ ): δ 8.11 (1H, brs), 7.20 (1H, d, 3.7 Hz), 6.52 (1H, d, J = 3.7 Hz), 6.24 ( 1H, t, J = 6.7Hz), 4.88-4.85 (1H, m), 4.22-4.20 (1H, m), 4.03 (1H, d, J = 3.5Hz ), 3.92 (1H, dd, J = 2.1Hz, 12.5Hz), 3.80 (1H, dd, J = 4.6Hz, 12.5Hz), 2.97-2.92 (1H, m), 2.30-2.25 (1H, m), 1.34 (9H, s), 0.92 (9H, s), 0.13 (3H, s), 0.12 (3H, s ).

Then, from compound 24 thus obtained, compound 25 (7-(3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl)-4- Chloro-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 24 (418 mg, 0.87 mmol) was dissolved in dimethylsulfoxide (5 mL) and toluene (3.5 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (995 mg, 5.2 mmol) was ), pyridine (141 μL, 1.7 mmol), and trifluoroacetic acid (97 μL, 1.3 mmol) were added and stirred for 2 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in 1,4-dioxane (3 mL), and 37% formaldehyde aqueous solution (495 μL, 6.1 mmol) and 1 M sodium hydroxide aqueous solution (910 μL) were added under ice-cooling. , 0.91 mmol) was added and stirred for 2 hours. An ethanol solution (3 mL) of sodium borohydride (34 mg, 0.89 mmol) was added under ice-cooling, and the mixture was stirred for 10 minutes. After quenching the reaction by adding acetic acid, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=5:1-2:1) to obtain compound 25 (220 mg, 2 steps 49 %).
¹ H-NMR (CDCl ₃ ): δ 8.14 (1H, brs), 7.19 (1H, d, 3.7 Hz), 6.51 (1H, d, J = 3.7 Hz), 6.31 ( 1H, dd, J = 5.2Hz, 7.5Hz), 3.87-3.64 (5H, m), 3.09-3.06 (1H, m), 2.68 (1H, brs), 2.50-2.46 (1H,m), 1.34 (9H,s), 0.94 (9H,s), 0.21 (3H,s), 0.15 (3H,s).

Compound 26 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4-C-hydroxymethyl -β-D-ribofuranosyl)-4-chloro-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, compound 25 (71 mg, 0.14 mmol) was dissolved in N,N-dimethylformamide (1.5 mL), triethylamine (39 μL, 0.28 mmol) was added, and 4,4'-dimethoxytrityl was added under ice-cooling. Chloride (56 mg, 0.17 mmol) was added and stirred for 2 hours, 4,4′-dimethoxytrityl chloride (9 mg, 0.03 mmol) was added and stirred for another 2 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was partitioned with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in N,N-dimethylformamide (1.5 mL), imidazole (29 mg, 0.42 mmol) was added, and tert-butyldiphenylsilyl was added under ice cooling. After adding chloride (55 μL, 0.21 mmol) and stirring at room temperature for 7 hours, imidazole (29 mg, 0.42 mmol) and tert-butyldiphenylsilyl chloride (55 μL, 0.21 mmol) were added and stirring for 2 hours. , and further imidazole (29 mg, 0.42 mmol) and tert-butyldiphenylsilyl chloride (55 μL, 0.21 mmol) were added and stirred for 14.5 hours. A saturated aqueous solution of sodium bicarbonate was added under ice-cooling to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (3 mL), and a solution of tosylic acid monohydrate (53 mg, 0.28 mmol) in methanol (1.5 mL) was added at -15°C. It was added dropwise over a period of 5 minutes and stirred for 5 minutes. A saturated aqueous sodium bicarbonate solution was added to stop the reaction, and the mixture was partitioned, partitioned with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=9:1) to obtain Compound 26 (76 mg, 3 steps 72%).
¹ H-NMR (CDCl ₃ ): δ 8.00 (1H, brs), 7.61-7.20 (11H, m), 6.61 (1H, dd, J=4.9Hz, 7.4Hz), 6.36 (1H, d, J = 3.7Hz), 5.09 (1H, dd, J = 6.3Hz, 7.4Hz), 3.92-3.77 (4H, m), 2.70 -2.64 (1H, m), 2.58-2.51 (1H, m), 1.26 (9H, s), 1.05 (9H, s), 0.92 (9H, s), 0.13 (3H,s), 0.12 (3H,s).

Compound 27 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy- β-D-ribofuranosyl)-4-chloro-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 26 (113 mg, 0.50 mmol) was dissolved in dimethylsulfoxide (1 mL) and toluene (0.5 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (173 mg, 0.90 mmol) was ), pyridine (24 μL, 0.30 mmol) and trifluoroacetic acid (11 μL, 0.15 mmol) were added and stirred for 2 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in pyridine (1 mL), hydroxylamine hydrochloride (16 mg, 0.23 mmol) was added, and the mixture was stirred for 1 hour. The reaction mixture was concentrated under reduced pressure and partitioned with ethyl acetate and water. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in dichloromethane (1 mL), triethylamine (42 μL, 0.30 mmol) and mesyl chloride (17 μL, 0.23 mmol) were added, and the mixture was stirred for 1 hour. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=9:1) to obtain compound 27 (86 mg, 77% in 3 steps).
¹ H-NMR (CDCl ₃ ): δ 7.97 (1H, brs), 7.60-7.14 (11H, m), 6.46 (1H, d, 3.7Hz), 6.42 (1H, dd, J = 4.0 Hz, 8.2 Hz), 5.37 (1H, t, J = 7.4 Hz), 4.07 (1H, d, J = 11.5 Hz), 4.02 (1H, d , J = 11.5 Hz), 2.83-2.80 (1H, m), 2.63-2.59 (1H, m), 1.20 (9H, s), 1.04 (9H, s ), 0.93 (9H, s), 0.14 (3H, s), 0.09 (3H, s).

Compound 28 (4-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-deoxy-β-D-ribofuranosyl)-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 27 (80 mg, 0.30 mmol) was dissolved in pyridine (2 mL) and water (1 mL), and stirred at 50°C for 62.5 hours. The reaction solution was concentrated under reduced pressure, the residue was dissolved in 1,4-dioxane (1 mL), an aqueous methylamine solution (1 mL) was added, and the mixture was stirred for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1-2:1) to obtain Compound 28 (53 mg, 68% in two steps).
¹ H-NMR (CDCl ₃ ): δ 7.80 (1H, brs), 7.61-7.26 (10H, m), 6.88 (1H, d, J = 3.7 Hz), 6.56 ( 1H, dd, J = 5.1Hz, 7.6Hz), 6.23 (1H, t, J = 3.7Hz), 5.24 (2H, brs), 5.11 (1H, t, J = 6 .7Hz), 3.98 (1H, d, J = 11.3Hz), 3.93 (1H, d, J = 11.3Hz), 2.72-2.68 (1H, m), 2.56 -2.52 (1H, m), 1.26 (9H, s), 1.06 (9H, s), 0.94 (9H, s), 0.15 (3H, s), 0.13 ( 3H, s).

Compound 29 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy- β-D-ribofuranosyl)-2,4-diaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 28 (44 mg, 0.060 mmol) and ammonium iodide (8.7 mg, 0.060 mmol) were dissolved in chloroform (1 mL) and hydrazine monohydrate (2 mL), and the mixture was heated at 60°C for 19.5 hours. Stirred. After adding water to stop the reaction, the mixture was extracted with chloroform three times, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=1:1-1:3) to obtain compound 29 (19 mg, 50%). .
¹ H-NMR (CDCl ₃ ): δ 7.66-7.34 (10H, m), 6.68 (1H, d, J = 3.7 Hz), 6.50 (1H, t, J = 6.8 Hz ), 6.14 (1H, d, J = 3.7Hz), 4.98 (2H, brs), 4.87 (1H, dd, J = 5.2Hz, 5.7Hz), 4.45 (2H , brs), 3.99 (1H, d, J = 11.0 Hz), 3.86 (1H, d, J = 11.0 Hz), 2.90-2.85 (1H, m), 2.42 -2.37 (1H,m), 1.08 (9H,s), 0.96 (9H,s), 0.18 (3H,s), 0.16 (3H,s).

Compound 30 (7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-2,4-diaminopyrrolo[2,3-d] is then obtained from compound 29 thus obtained. pyrimidines) were synthesized. That is, compound 29 (29 mg, 0.045 mmol) was dissolved in tetrahydrofuran (1 mL), a 1 M tetrahydrofuran solution of tetrabutylammonium fluoride (99 μL, 0.099 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=3-7%) to obtain compound 30 (11.5 mg, 88%).
¹ H-NMR (DMSO-d ₆ ): 6.89 (1H, d, J = 3.7 Hz), 6.58 (2H, brs), 6.51 (1H, t, J = 7.1 Hz), 6.40 (1H, d, J = 3.7Hz), 6.25 (1H, d, J = 3.8Hz), 5.84 (1H, t, J = 5.8Hz), 5.64 (1H , d, J = 5.8 Hz), 4.53 (1H, d, J = 3.8 Hz), 3.70 (1H, dd, J = 5.5 Hz, 11.7 Hz), 3.60 (1H, dd, J=6.2 Hz, 11.7 Hz), 2.64-2.60 (1 H, m), 2.28-2.23 (1 H, m).

Synthesis Example 4: Synthesis of 2-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-4-one 2-amino-7 -(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-4-one (compound 34) was synthesized by the reaction steps shown below.

First, from compound 27 obtained in Synthesis Example 3, compound 31 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy- β-D-ribofuranosyl)-4-methoxy-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 27 (100 mg, 0.13 mmol) was dissolved in pyridine (1.5 mL) and water (1 mL), and stirred at 60°C for 48 hours. The reaction solution was concentrated under reduced pressure, the residue was suspended in methanol (1.9 mL), 28% sodium methoxide methanol solution (0.1 mL) was added, and the mixture was stirred for 8 hours. A saturated ammonium chloride aqueous solution was added to stop the reaction, and the mixture was concentrated under reduced pressure. After extraction with ethyl acetate, the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=9:1-8:1) to obtain compound 31 (29 mg, 2 steps 33 %).
¹ H-NMR (CDCl ₃ , 500 MHz); 54 (1H, dd, J = 4.8Hz, 7.7Hz), 6.39 (1H, d, J = 3.6Hz), 5.20 (1H, t, J = 6.9Hz), 4.10 (3H, s), 4.01 (1H, d, J = 11.3 Hz), 3.96 (1H, d, J = 11.3 Hz), 2.77-2.72 (1H, m), 2 .59-2.53 (1H, m), 1.27 (9H, s), 1.05 (9H, s), 0.94 (9H, s), 0.15 (3H, s), 0. 12 (3H, s).

Compound 32 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy- β-D-ribofuranosyl)-2-pivaloylamino-3H-pyrrolo[2,3-d]pyrimidin-4-one) was synthesized. That is, compound 31 (28 mg, 0.038 mmol) was dissolved in pyridine (0.5 mL), pyridine hydrochloride (13 mg, 0.11 mmol) was added, stirred at 90°C for 2 hours, and pyridine hydrochloride (30 mg, 0.26 mmol) and pyridine (0.5 mL) were added, and the mixture was further stirred for 15 hours. The reaction mixture was concentrated under reduced pressure, partitioned with ethyl acetate and water, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=3:1-1:2) to obtain compound 32 (16 mg, 58%). .
¹ H-NMR (CDCl ₃ , 500 MHz); J = 3.6 Hz), 6.60-6.57 (2H, m), 4.80 (1 H, t, J = 5.9 Hz), 3.89-3.86 (2H, m), 2. 49-2.45 (2H, m), 1.35 (9H, s), 1.08 (9H, s), 0.96 (9H, s), 0.17 (3H, s), 0.15 (3H, s).

Compound 33 (2-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-deoxy-β-D-ribofuranosyl)-3H-pyrrolo[2,3-d]pyrimidin-4-one) was synthesized. That is, compound 32 (16 mg, 0.022 mmol) and ammonium iodide (3 mg, 0.022 mmol) were dissolved in 2-propanol (1 mL), hydrazine monohydrate (500 µL) was added, and the mixture was stirred at room temperature for 70 minutes. did. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=1:1-1:2) to obtain compound 33 (14 mg, quant).
¹ H-NMR (CDCl ₃ , 500 MHz); 48 (1H, t, J = 6.7Hz), 6.39 (1H, d, J = 3.7Hz), 5.15 (2H, brs), 4.82 (1H, t, J = 5.6Hz ), 3.97 (1H, d, J = 11.1 Hz), 3.86 (1H, d, J = 11.1 Hz), 2.76-2.71 (1H, m), 2.42-2 .37 (1 H, m), 1.09 (9 H, s), 0.96 (9 H, s), 0.18 (9 H, s), 0.16 (3 H, s).

Compound 34 (2-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine- 4(3H)-one) were synthesized. Specifically, compound 33 (14 mg, 0.022 mmol) was dissolved in tetrahydrofuran (1 mL), a tetrabutylammonium fluoride solution in tetrahydrofuran (48 μL, 0.048 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (methanol/chloroform=5-7%), and crystallized from acetonitrile and water to obtain compound 34 (3.9 mg, 61 %).
¹ H-NMR (DMSO-d ₆ , 500 MHz); ), 6.35 (2H, brs), 6.29 (1H, d, J = 3.7 Hz), 6.25 (1H, d, J = 4.7 Hz), 5.73 (1H, t, J = 6.0Hz), 4.51 (1H, dd, J = 4.7Hz, 5.7Hz), 3.69 (1H, dd, J = 6.0Hz, 11.7Hz), 3.60 (1H, dd, J=6.0 Hz, 11.7 Hz), 2.64-2.58 (1 H, m), 2.31-2.26 (1 H, m).

Synthesis Example 5: Synthesis of 2-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3-d]pyrimidine 2-amino-7-( 4-C-cyano-2-deoxy-β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3-d]pyrimidine (compound 37) was synthesized by the reaction steps shown below.

First, from compound 27 obtained in Synthesis Example 3, compound 35 (7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy- β-D-ribofuranosyl)-4-methylamino-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, compound 27 (46 mg, 0.062 mmol) was dissolved in 1,4-dioxane (1 mL), an aqueous methylamine solution (1 mL) was added, and the mixture was stirred for 3.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=4:1-3:1) to obtain compound 35 (45 mg, 98%).
¹ H-NMR (CDCl ₃ , 500 MHz); 55 (1H, dd, J = 4.9Hz, 7.7Hz), 5.17-5.14 (2H, m), 4.00 (1H, d, J = 11.3Hz), 3.94 (1H , d, J = 11.3 Hz), 3.17 (3H, d, J = 4.7 Hz), 2.73-2.69 (1H, m), 2.57-2.51 (1H, m) , 1.26 (9H,s), 1.06 (9H,s), 0.94 (9H,s), 0.14 (3H,s), 0.12 (3H,s).

Compound 36 (2-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-deoxy-β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 35 (41 mg, 0.055 mmol) and ammonium iodide (8 mg, 0.055 mmol) were dissolved in 2-propanol (0.5 mL), hydrazine monohydrate (0.5 mL) was added, and the temperature was adjusted to 50°C. After stirring for 1 hour at , the mixture was heated to 70° C. and further stirred for 4.5 hours. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=2:1-1:1) to obtain compound 36 (20 mg, 56%). .
¹ H-NMR (CDCl ₃ , 500 MHz); .7Hz), 6.16 (1H, d, J = 3.7Hz), 5.00 (1H, brs), 4.86 (1H, t, J = 5.3Hz), 4.44 (2H, brs ) 3.99 (1H, d, J = 11.0 Hz), 3.86 (1H, d, J = 11.0 Hz), 3.07 (3H, d, J = 4.9 Hz), 2.88- 2.85 (1H, m), 2.41-2.37 (1H, m), 1.08 (9H, s), 0.96 (9H, s), 0.18 (3H, s), 0 .15 (3H, s).

Compound 37 (2-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-4-methylaminopyrrolo[2,3 -d]pyrimidine) were synthesized. Specifically, compound 36 (20 mg, 0.030 mmol) was dissolved in tetrahydrofuran (1 mL), a tetrabutylammonium fluoride solution in tetrahydrofuran (67 μL, 0.067 mmol) was added, and the mixture was stirred at room temperature for 20 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/chloroform = 5%) and then purified again by silica gel column chromatography (methanol/chloroform = 5%) to give compound 37. Obtained (8.9 mg, 97%).
¹ H-NMR (CD ₃ OD, 500 MHz); J = 3.7Hz), 4.69 (1H, dd, J = 4.4Hz, 6.4Hz), 3.89 (1H, d, J = 12.0Hz), 3.83 (1H, d, J = 12.0 Hz), 2.98 (3H, brs), 2.82-2.77 (1H, m), 2.43-2.38 (1H, m).

Synthesis Example 6: Synthesis 2 of 2-amino-7-(2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-1,7-dihydropyrrolo[2,3-d]pyrimidin-4-one -amino-7-(2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-1,7-dihydropyrrolo[2,3-d]pyrimidin-4-one (Compound 47) is Synthesized in the reaction process.

First, from compound 22 obtained in Synthesis Example 3, compound 38 (7-(2-deoxy-β-D-ribofuranosyl)-4-methoxy-2-pivaloylaminopyrrolo[2,3-d]pyrimidine ) was synthesized. That is, compound 22 (7.87 g, 13 mmol) was dissolved in dichloromethane (63 mL) and methanol (32 mL), 5 M sodium methoxide methanol solution (31 mL, 160 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 10 minutes. did. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain compound 38 (3.69 g, 78%).
¹ H-NMR (CDCl ₃ , 500 MHz); 6.37 (1H, dd, J=8.1Hz, 6.0Hz), 5.08 (1H, brs), 4.79 (1H, s), 4.12 (3H, s), 3.91 ( 1H, d, J = 12.2 Hz), 3.79 (1H, d, J = 11.1 Hz), 2.90-2.84 (1H, m), 2.35-2.31 (1H, m ) 1.34 (9H, s).

Compound 39 (7-(5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl)-4-methoxy-2-pivaloyl)-4-methoxy-2-pivaloyl is then obtained from compound 38 thus obtained. aminopyrrolo[2,3-d]pyrimidines) were synthesized. Specifically, compound 38 (100 mg, 0.27 mmol) was dissolved in pyridine (2.7 mL), tert-butyldiphenylsilyl chloride (210 μL, 0.82 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 24 hours. . After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=2:1) to obtain compound 39 (150 mg, 91%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.99 (1H, brs), 7.64-7.30 (10H, m), 7.12 (1H, s), 6.80 (1H, s) , 6.40 (1H, d, J = 2.9Hz), 4.74 (1H, s), 4.09 (3H, s), 4.07 (1H, s), 3.86 (2H, dd , J=4.7 Hz, 4.3 Hz), 2.56-2.51 (2H, m), 1.33 (9 H, s), 1.08 (9 H, s).

Compound 40 (7-(3-O-benzyl-5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl)-4-methoxy) is then obtained from compound 39 thus obtained. -2-pivaloylaminopyrrolo[2,3-d]pyrimidine) were synthesized. That is, compound 39 (491 mg, 0.82 mmol) was dissolved in 1,4-dioxane (8 mL), molecular sieves 5 Å (491 mg), 2,4,6-tris(benzyloxy)-1,3,5-triazine. (163 mg, 0.41 mmol) was added and stirred at room temperature for 30 minutes, then trifluoromethanesulfonic acid (72 μL, 0.82 mmol) was added and stirred for 24 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=2:1) to obtain compound 40 (361 mg, 64%).
¹ H-NMR (CDCl ₃ , 500 MHz); 67 (1H, m), 6.39 (1H, d, J = 3.7Hz), 4.58 (2H, d, J = 4.3Hz), 4.45-4.43 (1H, m), 4.19-4.17 (1H, m) 4.12 (3H, s), 3.83-3.78 (2H, m), 2.56-2.44 (1H, m), 1.34 (9H, s), 1.07 (9H, s).

Compound 41 (7-(3-O-benzyl-2-deoxy-β-D-ribofuranosyl)-4-methoxy-2-pivaloylaminopyrrolo[2 , 3-d]pyrimidines) were synthesized. That is, compound 40 (1.3 g, 1.9 mmol) was dissolved in tetrahydrofuran (19 mL), 1M tetrabutylammonium fluoride solution in tetrahydrofuran (2.1 mL, 2.1 mmol) was added, and the mixture was stirred at room temperature for 3.5 hours. Stirred. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=2:1-1:1) to obtain compound 41 (714 mg, 84%). .
¹ H-NMR (CDCl ₃ , 500 MHz); 43 (1H, d, J = 3.7Hz), 5.88 (1H, brs), 6.39 (1H, d, J = 3.7Hz), 4.58 (2H, d, J = 4.3Hz ), 4.45-4.43 (1H, m), 4.19-4.17 (1H, m) 4.12 (3H, s), 3.98 (1H, dd, J = 13.4 Hz, 1.6Hz), 3.76 (1H, dd, J = 10.7Hz, 10.8), 2.99-2.94 (1H, m), 2.43-2.40 (1H, s), 1.34 (9H, s).

Compound 42 (7-(3-O-benzyl-2-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl)-4-methoxy-pivalo) is then obtained from compound 41 thus obtained. ylaminopyrrolo[2,3-d]pyrimidines) were synthesized. That is, compound 41 (714 mg, 1.6 mmol) was dissolved in dimethyl sulfoxide (4.5 mL) and toluene (3 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ( 1.8 g, 9.4 mmol), pyridine (253 μL, 3.1 mmol) and trifluoroacetic acid (120 μL, 1.6 mmol) were added and stirred at room temperature for 2.5 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in 1,4-dioxane (16 mL), and 37% formaldehyde aqueous solution (585 μL, 3.1 mmol) and 1 M sodium hydroxide aqueous solution (1 .7 mL, 1.7 mmol) was added and stirred for 1 hour. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in ethanol (7.7 mL), sodium borohydride (119 mg, 3.1 mmol) was dissolved in ethanol (8.0 mL), and the mixture was cooled with ice. and stirred at room temperature for 10 minutes. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=2:1-1:1-1:2) to obtain Compound 42 (525 mg , 3 steps 68%).
¹ H-NMR (CDCl ₃ , 500 MHz); 43 (1H, d, J = 3.6Hz), 6.23 (1H, dd, J = 7.9Hz, 6.4Hz), 5.39 (1H, brs), 4.87 (2H, dd, J = 6.4 Hz, 2.7 Hz), 4.72 (1H, d, J = 11.6 Hz), 4.57 (1H, d, J = 11.6 Hz), 4.13 (3H, s), 3 .81-3.74 (2H, m), 3.10-3.05 (1H, m), 2.62 (1H, brs), 2.55-2.51 (1H, m), 1.35 (9H, s).

Then, from compound 42 thus obtained, compound 43 (7-(3,5-di-O-benzyl-2-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl)-4- Methoxy-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 42 (30 mg, 0.061 mmol) was dissolved in 1,4-dioxane (0.6 mL) and molecular sieves 5 Å (30 mg), 2,4,6-tris(benzyloxy)-1,3,5 -Triazine (12.3 mg, 0.031 mmol) was added and stirred at room temperature for 30 minutes, then trifluoromethanesulfonic acid (5.4 μL, 0.061 mmol) was added and stirred for 24 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=5:1) to obtain compound 43 (7.4 mg, 21%).
¹ H-NMR (CDCl ₃ , 500 MHz); 41 (1H, d, J = 3.6Hz), 6.17 (1H, dd, J = 8.6Hz, 6.0Hz), 5.66 (1H, d, J = 7.4Hz), 4.66 -4.52 (5H, m) 4.14 (3H, s), 3.99 (1H, d, J = 11.8 Hz), 3.84 (1H, d, J = 11.9 Hz), 3. 76 (1H, d, J = 9.8Hz), 3.68 (1H, d, J = 9.8Hz), 3.05-2.99 (1H, m), 2.48-2.44 (1H , m), 1.35 (9H, s).

Compound 44 (7-(3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-4- Methoxy-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, after dissolving compound 43 (10.3 mg, 0.016 mmol) in dichloromethane (0.15 mL), Deoxo Fluor (registered trademark, 8.8 mg, 0.048 mmol) was added and stirred at -78°C for 1 hour. After that, the temperature was raised to 0° C. and stirred for 1 hour, and then the temperature was raised to room temperature and stirred for 30 minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=5:1) to obtain compound 44 (5.1 mg, 49%).
¹ H-NMR (CDCl ₃ , 500 MHz); 1H, t, J = 6.9 Hz), 6.46 (1H, d, J = 3.7 Hz), 4.77-4.53 (7H, m), 4.09 (3H, s), 3. 81 (1H, d, J = 2.4Hz), 3.73 (1H, d, J = 1.6Hz), 2.77-2.71 (1H, m), 2.64-2.59 (1H , m), 1.36 (9H, s).

Compound 45 (7-(3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-2- pivaloylamino-3H-pyrrolo[2,3-d]pyrimidin-4-one) was synthesized. That is, compound 44 (11.7 mg, 0.020 mmol) was dissolved in pyridine (0.4 mL), pyridine hydrochloride (7.0 mg, 0.060 mmol) was added, and the mixture was stirred at 80°C for 24 hours. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=1:1) to obtain compound 45 (10.7 mg, 94%).
¹ H-NMR (CDCl ₃ , 500 MHz); J = 3.7Hz), 6.67 (1H, d, J = 3.6Hz), 6.53 (1H, dd, J = 7.3Hz, 7.1Hz), 4.74-4.49 (6H , m), 4.39 (1H, dd, J=5.2 Hz, 2.6 Hz), 3.80 (1 H, d, J=2.7 Hz), 3.69 (1 H, d, J=1. 5Hz), 2.54-2.48 (2H, m), 1.31 (9H, s).

Compound 46 (2-amino-7-(3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl )-3H-pyrrolo[2,3-d]pyrimidin-4-one) was synthesized. That is, compound 45 (10.7 mg, 0.019 mmol) was dissolved in 1 M sodium hydroxide aqueous solution (0.2 mL) and methanol (0.2 mL), and stirred at 80° C. for 80 minutes. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with chloroform. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain compound 46 (6.2 mg, 70%).
¹ H-NMR (CDCl ₃ , 500 MHz); 51 (1H, dd, J = 7.1Hz, 7.1Hz), 6.48 (1H, d, J = 3.7Hz), 5.26 (2H, s), 4.73-4.50 (6H , m), 4.40 (1H, dd, J=4.5 Hz, 4.2 Hz), 3.81 (1 H, d, J=2.3 Hz), 3.79 (1 H, d, J=2. 2 Hz), 2.54-2.50 (2H, m).

Compound 47 (2-amino-7-(2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-3H-pyrrolo[2,3- d]pyrimidin-4-one) was synthesized. That is, after dissolving compound 46 (6.2 mg, 0.013 mmol) in dichloromethane (0.3 mL), boron trichloride (1.0 M dichloromethane solution, 130 μL, 0.13 mmol) was added at −78° C., Stir at 0° C. for 10 minutes. After stopping the reaction by adding methanol, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/chloroform = 1:3) and then desalted using SP207 resin to obtain Compound 47. (3.3 mg, 88%).
¹ H-NMR (CD ₃ OD, 500 MHz); J = 3.6 Hz), 4.58-4.57 (1H, m), 4.56 (1H, dd, J = 43.9 Hz, 9.8 Hz), 4.47 (1H, dd, J = 43 .5Hz, 9.8Hz), 3.75-3.73 (2H, m), 2.60-2.57 (1H, m), 2.35-2.32 (1H, m).

Synthesis Example 7: Synthesis of 2,4-diamino-7-(2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine 2,4-diamino-7- (2-Deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (compound 51) was synthesized by the following reaction steps.

First, from compound 45 obtained in Synthesis Example 6, compound 48 (7-(3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-4- (2,4,6-Triisopropylbenzenesulfonyloxy)-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) was synthesized. That is, after dissolving compound 45 (29.0 mg, 0.061 mmol) in dichloromethane (0.6 mL), triethylamine (17 μL, 0.12 mmol), 2,4,6-triisopropylbenzenesulfonyl chloride (27.5 mg) , 0.091 mmol) and 4-dimethylaminopyridine (7.4 mg, 0.061 mmol) were sequentially added, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with chloroform. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=5:1) to obtain compound 48 (44.9 mg, 75%).
¹ H-NMR (CDCl ₃ , 500 MHz); 6.65 (1H, t, J = 7.0Hz), 6.60 (1H, d, J = 3.6Hz), 6.28 (1H, d, J = 5.1Hz), 5.82 (1H , t, J = 6.1 Hz), 4.61 (1H, dd, J = 4.8 Hz, 11.0 Hz), 3.76 (1H, dd, J = 5.9 Hz, 11.7 Hz), 3. 62 (1H, dd, J = 6.3Hz, 11.7Hz), 2.95 (3H, d, J = 4.6Hz), 2.81-2.75 (1H, m), 2.39-2 .34(1H,m).

From compound 48 thus obtained, compound 49 (4-amino-7-(3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl )-2-pivaloylaminopyrrolo[2,3-d]pyrimidine) were synthesized. That is, compound 48 (56.9 mg, 0.069 mmol) was dissolved in tetrahydrofuran (3 mL), aqueous ammonia (3 mL) was added, and the mixture was stirred at 90° C. for 24 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=1:2) to obtain compound 49 (22.9 mg, 59%).
¹ H-NMR (CDCl ₃ , 500 MHz); 41 (1H, d, J = 3.8Hz), 4.72-4.55 (7H, m), 4.24-4.11 (2H, m), 3.80-3.71 (2H, m ), 1.28-1.23 (27H, m).

Compound 50 (2,4-diamino-7-(3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D -ribofuranosyl)-pyrrolo[2,3-d]pyrimidine) were synthesized. That is, compound 49 (15.7 mg, 0.028 mmol) was dissolved in 1 M sodium hydroxide aqueous solution (1 mL) and methanol (1 mL), and stirred at 90° C. for 2 hours. A saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with chloroform. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain compound 50 (4.5 mg, 70%).
¹ H-NMR (CDCl ₃ , 500 MHz); .1Hz), 6.25 (1H, d, J = 3.8Hz), 5.70 (2H, brs), 4.94 (2H, brs), 4.73-4.51 (6H, m), 4.40 (1H, t, J = 4.4Hz), 3.80 (1H, dd, J = 10.2Hz, 2.5Hz), 3.69 (1H, dd, J = 10.1Hz, 1. 7 Hz), 2.55-2.52 (2H, m).

Compound 51 (2,4-diamino-7-(2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl)-pyrrolo[2,3- d]pyrimidine) was synthesized. That is, after dissolving compound 50 (4.5 mg, 9.4 μmol) in dichloromethane (0.1 mL), boron trichloride (1.0 M dichloromethane solution, 94 μL, 0.09 mmol) was added at −78° C., Stirred at 0° C. for 30 minutes. After the reaction was stopped by adding methanol, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/chloroform=1:3) and then desalted using SP207 resin to obtain compound 51. (2.3 mg, 82%).
¹ H-NMR (CD ₃ OD, 500 MHz); J = 3.8Hz), 4.61-4.60 (1H, m), 4.58 (1H, dd, J = 51.0Hz, 9.7Hz), 4.49 (1H, dd, J = 51 .7Hz, 9.7Hz), 3.78 (2H, dd, J = 12.2Hz, 2.5Hz), 2.66-2.60 (1H, m), 2.38-2.33 (1H, m).

Synthesis Example 8: Synthesis of 4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine 4-amino-7-(4- C-cyano-2-deoxy-β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine (compound 60) was synthesized by the reaction steps shown below.

First, from compound 52 (Eur. Pat. Appl., 710667) to compound 53 (4-benzylamino-7-(2-deoxy-β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 52 (2.27 g, 6.0 mmol) was dissolved in pyridine (15 mL), chlorotrimethylsilane (7.66 mL, 60 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 3 hours. Chloride (714 μL, 6.2 mmol) was added and stirred for 2 hours. After adding water under ice-cooling and stirring for 20 minutes, aqueous ammonia (12 mL) was added and the mixture was further stirred at room temperature for 1 hour. After concentrating the reaction solution under reduced pressure, it was crystallized from methanol and water to obtain Compound 53 (1.83 g, 63%).
¹ H-NMR (DMSO-d ₆ ): δ 8.69 (1H, s), 8.10-7.54 (6H, m), 6.66 (1H, t, J = 7.0 Hz), 5. 33 (1H, d, J = 4.1Hz), 5.00 (1H, t, J = 5.5Hz), 4.38 (1H, s), 3.86 (1H, d, J = 2.6Hz) ), 3.60-3.52 (2H, m), 2.56-2.54 (1 H, m), 2.28-2.26 (1 H, m).

Compound 54 (4-benzylamino-7-(2-deoxy-5-O-dimethoxytrityl-β-D-ribofuranosyl)-5-iodopyrrolo[2,3 -d]pyrimidine) were synthesized. That is, after azeotroping compound 53 (1.79 g, 3.7 mmol) with pyridine, it was dissolved in pyridine (20 mL), and a solution of 4,4'-dimethoxytrityl chloride (1.52 g, 4.5 mmol) in pyridine ( 20 mL) was added dropwise over 30 minutes under ice-cooling, and the mixture was stirred at room temperature for 17 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the reaction mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane/ethyl acetate=1:1-1:4) to obtain compound 54 (2.88 g, 99%). .
¹ H-NMR (CDCl ₃ ): δ 9.34 (1H, brs), 8.75 (1H, s), 8.09-6.83 (19H, m), 6.76 (1H, t, J = 6.5Hz), 4.67 (1H, dd, J = 2.5Hz, 5.8Hz), 4.13-4.10 (1H, m), 3.79 (6H, s), 3.46- 3.38 (2H,m), 2.66-2.62 (1H,m), 2.55-2.51 (1H,m).

Compound 55 (4-benzoylamino-7-(3-O-tert-butyldimethylsilyl-2-deoxy-β-D-ribofuranosyl)-5-iodopyrrolo [ 2,3-d]pyrimidines) were synthesized. That is, compound 54 (2.79 g, 3.6 mmol) was dissolved in N,N-dimethylformamide (35 mL), imidazole (969 mg, 14 mmol) was added, and tert-butyldimethylsilyl chloride ( 1.07 g, 7.1 mmol) was added and stirred at room temperature for 21 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (40 mL), and a solution of tosylic acid monohydrate (1.36 g, 7.1 mmol) in methanol (80 mL) was added at -15°C. It was added dropwise over minutes and stirred for 1.5 hours. A saturated aqueous solution of sodium bicarbonate was added to stop the reaction, and the mixture was partitioned, extracted with chloroform, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1-1:1) to obtain compound 55 (1.99 g, 2 steps 94%).
¹ H-NMR (CDCl ₃ ): δ 9.39 (1H, brs), 8.75 (1H, s), 8.09-7.53 (5H, m), 7.40 (1H, s), 6 .27 (1H, dd, J=5.6 Hz, 9.3 Hz), 5.34 (1 H, d, J=10.0 Hz), 4.68 (1 H, d, J=5.2 Hz), 4. 12 (1H, dd, J = 3.7Hz, 7.3Hz), 3.95 (1H, d, J = 12.6Hz), 3.78-3.73 (1H, m), 3.03-2 .97 (1 H, m), 2.23-2.19 (1 H, m), 0.93 (9 H, s), 0.12 (6 H, s).

Compound 56 (4-benzoylamino-7-(3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-β-D- Ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 55 (115 mg, 0.19 mmol) was dissolved in dimethylsulfoxide (1.2 mL) and toluene (0.7 mL), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added under ice cooling. A salt (222 mg, 1.2 mmol), pyridine (31.5 μL, 0.39 mmol) and trifluoroacetic acid (21.5 μL, 0.29 mmol) were added and stirred at room temperature for 2 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in 1,4-dioxane (2 mL), and 37% formaldehyde aqueous solution (108 μL, 1.3 mmol) and 1 M sodium hydroxide aqueous solution (200 μL) were added under ice-cooling. , 0.20 mmol) was added and stirred for 7.5 hours. Sodium borohydride (22 mg, 0.57 mmol) was added under ice-cooling, and the mixture was stirred for 1 hour. After quenching the reaction by adding acetic acid, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=2:1-1:1) to obtain compound 56 (85 mg, 2 steps 72 %).
¹ H-NMR (CDCl ₃ ): δ 9.39 (1H, brs), 8.75 (1H, s), 8.09-7.53 (5H, m), 7.39 (1H, s), 6 .36 (1H, dd, J = 6.2 Hz, 8.4 Hz), 5.15 (1 H, d, J = 9.3 Hz), 4.91 (1 H, dd, J = 1.6 Hz, 6.2 Hz ), 3.84-3.80 (2H, m), 3.71-3.64 (2H, m), 3.19-3.16 (1H, m), 2.61 (d, 1H, J = 4.8 Hz), 2.35-2.31 (1 H, m), 0.95 (9 H, s), 0.18 (3 H, s), 0.16 (3 H, s).

Compound 57 (4-benzoylamino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4 -C-hydroxymethyl-β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 56 (85 mg, 0.14 mmol) was dissolved in N,N-dimethylformamide (1.4 mL), triethylamine (38 µL, 0.27 mmol) was added, and 4,4'-dimethoxytrityl was added under ice-cooling. Chloride (55 mg, 0.16 mmol) was added and stirred at room temperature for 2 hours, triethylamine (19 μL, 0.14 mmol) and 4,4′-dimethoxytrityl chloride (14 mg, 0.040 mmol) were added and further stirred for 2 hours. did. After ice was added to stop the reaction, ethyl acetate and saturated aqueous sodium bicarbonate were added for partition, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in N,N-dimethylformamide (1.4 mL), imidazole (37 mg, 0.54 mmol), tert-butyldiphenylsilyl chloride ( 71 μL, 0.27 mmol) was added and stirred at room temperature for 17 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (3 mL), and a solution of tosylic acid monohydrate (52 mg, 0.27 mmol) in methanol (1.5 mL) was added at -15°C. It was added dropwise over minutes and stirred for 1 hour. A saturated aqueous sodium bicarbonate solution was added to stop the reaction, followed by extraction with chloroform. The organic layer was washed with a saturated aqueous sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1-1:1) to obtain compound 57 (65 mg, 3 steps 55% ).
¹ H-NMR (CDCl ₃ ): δ 9.35 (1H, brs), 8.73 (1H, s), 8.09-7.35 (16H, m), 6.74 (1H, t, J = 6.6Hz), 4.81 (1H, d, J = 3.5Hz, 6.0Hz), 3.87-3.72 (4H, m) 2.71-2.65 (1H, m), 2 .49-2.45 (1H,m), 2.29 (1H,brs), 1.11 (9H,s), 0.93 (9H,s), 0.13 (3H,s), 0. 11 (3H, s).

Compound 58 (4-benzylamino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano -2-deoxy-β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 57 (63 mg, 0.073 mmol) was dissolved in dimethylsulfoxide (0.45 mL) and toluene (0.25 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (84 mg, 0 .44 mmol), pyridine (11.8 μL, 0.15 mmol), and trifluoroacetic acid (5.4 μL, 0.073 mmol) were added and stirred for 1.5 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in pyridine (1 mL), hydroxylamine hydrochloride (7.6 mg, 0.11 mmol) was added, and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure and partitioned with ethyl acetate and water. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in dichloromethane (1 mL), triethylamine (20.3 μL, 0.15 mmol) and mesyl chloride (8.5 μL, 0.11 mmol) were added, and the mixture was cooled with ice. and stirred for 2.5 hours. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=5:1-2:1) to obtain compound 58 (47 mg, 3 steps 74 %).
¹ H-NMR (CDCl ₃ ): 9.31 (1H, brs), 8.67 (1H, s), 8.08-7.35 (116H, m), 6.67 (1H, t, J = 6.6 Hz), 4.89 (1H, t, J = 5.5 Hz), 4.06 (1H, d, J = 11.2 Hz), 3.88 (1H, d, J = 11.2 Hz), 2.93-2.87 (1H, m), 2.55-2.50 (1H, m), 1.09 (9H, s), 0.96 (9H, s), 0.16 (3H, s), 0.15 (3H, s).

Compound 59 (4-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-deoxy-β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, compound 58 (44 mg, 0.051 mmol) was dissolved in methanol (3 mL), aqueous ammonia (1 mL) was added, and the mixture was stirred for 48 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1-1:1) to obtain compound 59 (36 mg, 94%).
¹ H-NMR (CDCl ₃ ): δ 8.15 (1H, s), 7.64-7.34 (10H, m), 7.11 (1H, s), 6.59 (1H, t, J = 6.5Hz), 5.70 (2H, brs), 4.89 (1H, t, J = 5.8Hz), 4.02 (1H, d, J = 11.2Hz), 3.85 (1H, d, J = 11.2 Hz), 2.87-2.81 (1H, m), 2.50-2.45 (1H, m), 1.08 (9H, s), 0.94 (9H, s), 0.15 (3H, s), 0.14 (3H, s).

Compound 60 (4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-iodopyrrolo[2,3-d ] pyrimidines) were synthesized. Compound 59 (36 mg, 0.048 mmol) was dissolved in tetrahydrofuran (1 mL), 1M tetrabutylammonium fluoride solution in tetrahydrofuran (103 μL, 0.10 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=3-5%) to obtain compound 60 (19 mg, 97%).
¹ H-NMR (DMSO-d ₆ ): δ 8.13 (1H, s), 7.67 (1H, s), 6.71 (2H, brs), 6.63 (1H, t, J=6. 9Hz), 6.28 (1H, d, J = 5.0Hz), 5.76 (1H, t, J = 6.1Hz), 4.59 (1H, dd, J = 5.0Hz, 10.9Hz ), 3.75 (1H, dd, J = 5.9Hz, 11.8Hz), 3.62 (1H, dd, J = 6.1Hz, 11.8Hz), 2.77-2.72 (1H, m), 2.38-2.33 (1H, m).

Synthesis Example 9: Synthesis of 4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine 4-amino-7-(4 —C-cyano-2-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine (compound 69) was synthesized by the reaction steps shown below.

First, from compound 61 (see Synthesis, 2006, 12, 2005), compound 62 (4y-benzylamino-7-(2-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine ) was synthesized. That is, compound 61 (383 mg, 1.4 mmol) was dissolved in pyridine (4 mL), chlorotrimethylsilane (1.81 mL, 14 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 2.5 hours. Chloride (174 μL, 1.5 mmol) was added and stirred for 4.5 hours. After adding water under ice-cooling and stirring for 15 minutes, aqueous ammonia (3 mL) was added and the mixture was further stirred at room temperature for 1.5 hours. After concentrating the reaction solution under reduced pressure, it was crystallized from methanol and water to obtain compound 62 (436 mg, 82%).
¹ H-NMR (DMSO-d6): δ 11.24 (1H, brs), 8.67 (1H, s), 8.05-7.54 (6H, m), 6.73 (1H, t, J = 6.5 Hz), 5.33 (1H, d, J = 4.1 Hz), 4.98 (1H, t, J = 5.5 Hz), 4.36 (1H, d, J = 5.0 Hz) , 3.85-3.83 (1 H, m), 3.58-3.50 (2 H, m), 2.53-2.24 (2 H, m).

Compound 63 (4-benzylamino-7-(2-deoxy-5-O-dimethoxytrityl-β-D-ribofuranosyl)-5-fluoropyrrolo[2, 3-d]pyrimidine) were synthesized. That is, after azeotroping compound 62 (421 mg, 1.1 mmol) with pyridine, it was dissolved in pyridine (26 mL), and a pyridine solution (6 mL) of 4,4'-dimethoxytrityl chloride (460 mg, 1.4 mmol) was added with ice. The mixture was added dropwise over 15 minutes under cooling, and stirred at room temperature for 24 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the reaction mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane/ethyl acetate=1:1-1:4) to obtain compound 63 (762 mg, quant.).
¹ H-NMR (CDCl ₃ ): δ 8.62-6.82 (21H, m), 4.60 (1H, d, J = 5.1 Hz), 4.12 (1H, dd, J = 4.2 Hz , 8.1 Hz), 3.79 (6H, s), 3.43-3.35 (2H, m), 2.57-2.44 (2H, m).

Compound 64 (4-benzoylamino-7-(3-O-tert-butyldimethylsilyl-2-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo) is then obtained from compound 63 thus obtained. [2,3-d]pyrimidines) were synthesized. That is, compound 63 (744 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (10 mL), imidazole (300 mg, 4.4 mmol) was added, and tert-butyldimethylsilyl chloride ( 332 mg, 2.2 mmol) was added, and the mixture was stirred at room temperature for 5.5 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (12 mL), and a methanol solution (24 mL) of tosylic acid monohydrate (837 g, 4.4 mmol) was added at -15°C for 5 minutes. and stirred for 30 minutes. A saturated aqueous solution of sodium bicarbonate was added to stop the reaction, and the mixture was partitioned, extracted with chloroform, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1-1:1) to obtain compound 64 (504 mg, 94% in two steps). ).
¹ H-NMR (CDCl ₃ ): δ, 8.63 (1H, s), 8.61 (1H, s), 7.99-7.06 (6H, m), 6.23 (1H, dd, J = 5.7Hz, 9.0Hz), 5.21 (1H, d, J = 9.4Hz), 4.67 (1H, d, J = 5.3Hz), 4.11 (1H, d, J = 1.5 Hz), 3.94 (1H, d, J = 12.6 Hz), 3.76-3.71 (1H, m), 2.99-2.94 (1H, m), 2.25 -2.21 (1 H, m), 0.95 (9 H, s), 0.12 (6 H, s).

Compound 65 (4-benzoylamino-7-(3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-β-D- ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, compound 64 (470 mg, 0.97 mmol) was dissolved in dimethylsulfoxide (6 mL) and toluene (4 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (1. 11 g, 5.8 mmol), pyridine (157 μL, 1.9 mmol) and trifluoroacetic acid (108 μL, 1.5 mmol) were added and stirred at room temperature for 4 hours. After adding ice to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in 1,4-dioxane (3 mL), and under ice-cooling, 37% formaldehyde aqueous solution (552 μL, 6.8 mmol), 1 M sodium hydroxide aqueous solution (1 .94 mL, 1.9 mmol) was added and stirred for 6.5 hours. Sodium borohydride (110 mg, 2.9 mmol) was added under ice-cooling, and the mixture was stirred for 1 hour. After quenching the reaction by adding acetic acid, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=2:1-1:1) to obtain compound 65 (310 mg, 2 steps 62 %).
¹ H-NMR (CDCl ₃ ): δ 8.74 (1H, brs), 8.59 (1H, brs), 7.99-7.52 (5H, m), 7.04 (1H, d, J = 2.4 Hz), 6.35 (1H, t, J=7.1 Hz), 4.99 (1H, brs), 4.89 (1H, dd, J=2.8 Hz, 6.5 Hz), 3. 84-3.79 (2H, m), 3.72-3.66 (2H, m), 3.13-3.07 (1H, m), 2.71 (1H, brs), 2.39- 2.35 (1 H, m), 0.95 (9 H, s), 0.17 (3 H, s), 0.16 (3 H, s).

Compound 66 (4-benzoylamino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4 -C-hydroxymethyl-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 65 (305 mg, 0.59 mmol) was dissolved in N,N-dimethylformamide (3 mL), triethylamine (164 μL, 1.2 mmol) was added, and 4,4'-dimethoxytrityl chloride ( 240 mg, 0.71 mmol) in N,N-dimethylformamide (3 mL) was added dropwise over 1 hour, stirred at room temperature for 4 hours, triethylamine (82 μL, 0.59 mmol), 4,4′-dimethoxytrityl chloride. (60 mg, 0.18 mmol) was added and stirred for an additional 16 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, ethyl acetate and saturated aqueous sodium bicarbonate were added for partition, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in N,N-dimethylformamide (6 mL), imidazole (161 mg, 2.4 mmol), tert-butyldiphenylsilyl chloride (307 μL, 1.2 mmol) was added and stirred at room temperature for 12 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in chloroform (12 mL), and a methanol solution (6 mL) of tosylic acid monohydrate (224 mg, 1.2 mmol) was added at -15°C for 5 minutes. and stirred for 1 hour. A saturated aqueous sodium bicarbonate solution was added to stop the reaction, followed by extraction with chloroform. The organic layer was washed with a saturated aqueous sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1-1:1) to obtain compound 66 (311 mg, 3 steps 70% ).
¹ H-NMR (CDCl ₃ ): δ 8.87 (1H, brs), 8.56 (1H, s), 7.97-7.35 (15H, m), 7.08 (1H, d, J = 6.4Hz), 6.80 (1H, t, J = 6.7Hz), 4.80 (1H, d, J = 4.0Hz, 6.3Hz), 3.89-3.72 (4H, m ) 2.60-2.54 (1H, m), 2.45-2.40 (2H, m), 1.10 (9H, s), 0.93 (9H, s), 0.13 (3H , s), 0.11 (3H, s).

Compound 67 (4-benzylamino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano -2-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 66 (275 mg, 0.36 mmol) was dissolved in dimethylsulfoxide (1 mL) and toluene (2 mL), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (420 mg, 2.2 mmol), Pyridine (58 μL, 0.72 mmol) and trifluoroacetic acid (27 μL, 0.36 mmol) were added and stirred for 1.5 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in pyridine (4 mL), hydroxylamine hydrochloride (38 mg, 0.54 mmol) was added, and the mixture was stirred for 45 minutes. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in dichloromethane (4 mL), triethylamine (100 μL, 0.72 mmol) and mesyl chloride (42 μL, 0.54 mmol) were added, and the mixture was cooled to 2.5% under ice-cooling. Stirred for an hour. After adding water to stop the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane/ethyl acetate=5:1-3:1) to obtain compound 67 (213 mg, 3 steps 79 %).
¹ H-NMR (CDCl ₃ ): 8.57 (1H, s), 8.51 (1H, s), 7.98-7.35 (15H, m), 6.98 (1H, d, J = 2.1 Hz), 6.74 (1H, t, J = 6.5 Hz), 4.87 (1H, t, J = 5.7 Hz), 4.00 (1H, d, J = 11.2 Hz), 3.87 (1H, d, J=11.2Hz), 2.81-2.76 (1H, m), 2.54-2.49 (1H, m), 1.08 (9H, s), 0.96 (9H, s), 0.17 (3H, s), 0.15 (3H, s).

Compound 68 (4-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, compound 67 (75 mg, 0.10 mmol) was dissolved in methanol (6 mL), aqueous ammonia (3 mL) was added, and the mixture was stirred for 40 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane/ethyl acetate=3:1) to obtain compound 68 (47 mg, 73%).
¹ H-NMR (CDCl ₃ ): δ 8.18 (1H, s), 7.65-7.34 (10H, m), 6.70 (1H, s), 6.63 (1H, t, J = 6.4Hz), 5.44 (2H, brs), 4.89 (1H, t, J = 5.8Hz), 3.98 (1H, d, J = 11.2Hz), 3.84 (1H, d, J = 11.2 Hz), 2.83-2.78 (1H, m), 2.50-2.45 (1H, m), 1.07 (9H, s), 0.95 (9H, s), 0.16 (3H, s), 0.15 (3H, s).

Compound 69 (4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3- d]pyrimidine) was synthesized. Compound 68 (45 mg, 0.070 mmol) was dissolved in tetrahydrofuran (1 mL), 1M tetrabutylammonium fluoride solution in tetrahydrofuran (153 μL, 0.15 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=3-5%) to obtain compound 69 (17 mg, 83%).
¹ H-NMR (DMSO-d6): δ 8.10 (1H, s), 7.37 (1H, d, J = 1.7 Hz), 7.08 (2H, brs), 6.69 (1H, dd , J = 6.2 Hz, 6.6 Hz), 6.30 (1H, d, J = 5.1 Hz), 5.76 (1H, t, J = 6.1 Hz), 4.56 (1H, dd, J = 5.0Hz, 10.9Hz), 3.73 (1H, dd, J = 6.0Hz, 11.8Hz), 3.61 (1H, dd, J = 6.2Hz, 11.8Hz), 2 .70-2.65 (1H,m), 2.38-2.33 (1H,m).

Synthesis Example 10: Synthesis of 4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-chloropyrrolo[2,3-d]pyrimidine 4-amino-7-(4- C-cyano-2-deoxy-β-D-ribofuranosyl)-5-chloropyrrolo[2,3-d]pyrimidine (compound 71) was synthesized by the reaction steps shown below.

First, from compound 16 obtained in Synthesis Example 2, compound 70 (4-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2 -deoxy-β-D-ribofuranosyl)-5-chloropyrrolo[2,3-d]pyrimidine) was synthesized. Specifically, compound 16 (24 mg, 0.038 mmol) was dissolved in N,N-dimethylformamide (0.5 mL), and N-chlorosuccinimide (5.1 mg, 0.038 mmol) was dissolved in N,N-dimethylformamide (0 .5 mL) was added dropwise under ice-cooling, and after stirring for 1 hour, the mixture was stirred at room temperature for 2 hours. After the reaction was stopped by adding methanol, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane:ethyl acetate=3:1-1:1) to obtain compound 70 (7 .4 mg, 29%).
¹ H-NMR (CDCl ₃ ): δ 8.16 (1H, s), 7.64-7.34 (10H, m), 6.95 (1H, s), 6.60 (1H, dt, J = 6.5Hz), 5.68 (1H, brs), 4.89 (1H, t, J = 5.9Hz), 4.00 (1H, d, J = 11.2Hz), 3.85 (1H, d, J=11.2 Hz), 2.85-2.81 (1 H, m), 2.52-2.48 (1 H, m).

Compound 71 (4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-chloropyrrolo[2,3-d ] pyrimidines) were synthesized. Compound 70 (7.4 mg, 0.011 mmol) was dissolved in tetrahydrofuran (0.5 mL), 1M tetrabutylammonium fluoride solution in tetrahydrofuran (25 μL, 0.025 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=3%) to obtain compound 71 (3.5 mg, quant.).
¹ H-NMR (DMSO-d6): δ 8.11 (1H, s), 7.40 (1H, s), 6.68 (1H, d, J = 6.7 Hz), 4.73 (1H, dd , J = 5.4 Hz, 6.5 Hz), 3.88 (1H, d, J = 12.0 Hz), 3.83 (1H, d, J = 12.0 Hz), 2.79-2.74 ( 1H, m), 2.53-2.48 (1H, m).

Synthesis Example 11: Synthesis of 4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-ethynylpyrrolo[2,3-d]pyrimidine 4-amino-7-(4 —C-cyano-2-deoxy-β-D-ribofuranosyl)-5-ethynylpyrrolo[2,3-d]pyrimidine (compound 72) was synthesized by the reaction steps shown below.

Compound 59 (15 mg, 0.020 mmol) obtained in Synthesis Example 8, Pd(PPh ₃ ) ₂ Cl ₂ (0.70 mg, 0.0010 mmol) and copper iodide (0.40 mg, 0.0020 mmol) were combined with N, It was dissolved in N-dimethylformamide (1 mL), trimethylsilylacetylene (28 μL, 0.20 mmol) and triethylamine (8 μL, 0.057 mmol) were added, and the mixture was stirred at room temperature for 5 hours. After adding saturated aqueous sodium bicarbonate to stop the reaction, the reaction mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in tetrahydrofuran (1 mL), 1M tetrabutylammonium fluoride solution in tetrahydrofuran (66 μL, 0.066 mmol) was added, and the mixture was stirred for 1 hour. The reaction solution is concentrated under reduced pressure, the residue is purified by silica gel chromatography (methanol/chloroform = 0-4%), compound 72 (4-amino-7-(4-C-cyano-2-deoxy-β -D-ribofuranosyl)-5-ethynylpyrrolo[2,3-d]pyrimidine) (5.9 mg, 2 steps 99%).
¹ H-NMR (DMSO-d6): δ 8.13 (1H, s), 7.62 (1H, s), 6.64 (1H, t, J = 6.7 Hz), 4.76 (1H, dd , J = 5.4 Hz, 6.4 Hz), 3.90 (1H, d, J = 12.0 Hz), 3.84 (1H, d, J = 12.0 Hz), 2.83-2.78 ( 1 H, m), 2.55-2.50 (1 H, m).

Synthesis Example 12: 4-Amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-(1H-pyrazol-3-yl)-pyrrolo[2,3-d]pyrimidine Synthetic 4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-(1H-pyrazol-3-yl)-pyrrolo[2,3-d]pyrimidine (Compound 74) was synthesized in the reaction steps shown below.

First, from compound 59 obtained in Synthesis Example 8, compound 73 (4-amino-7-(3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2 -deoxy-β-D-ribofuranosyl)-5-(1H-pyrazol-3-yl)-pyrrolo[2,3-d]pyrimidine) was synthesized. That is, compound 59 (30 mg, 0.040 mmol), 1H-pyrazol-3-ylboronic acid (7 mg, 0.060 mmol), Pd(PPh ₃ ) ₄ (0.70 mg, 0.0010 mmol) was combined with 1,4-dioxane ( 0.4 mL), 2M aqueous sodium carbonate solution (0.3 mL, 0.60 mmol) was added, and the mixture was stirred at 100° C. for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=0-5%) to obtain compound 73 (9.3 mg, 34%).
¹ H-NMR (CDCl ₃ ): δ 8.12 (1H, s), 7.69-7.32 (12H, m), 6.67 (1H, t, J = 6.8 Hz), 6.27 ( 1H, d, J = 2.4 Hz), 4.96-4.93 (1H, m), 4.06 (1H, d, J = 11.1 Hz), 3.89 (1H, d, J = 11 .1Hz), 3.00-2.94 (1H, m), 2.51-2.46 (1H, m), 1.07 (9H, s), 0.96 (9H, s), 0. 17 (3H, s), 0.16 (3H, s).

Compound 74 (4-amino-7-(4-C-cyano-2-deoxy-β-D-ribofuranosyl)-5-(1H-pyrazole-3- yl)-pyrrolo[2,3-d]pyrimidine) were synthesized. That is, compound 73 (8.9 mg, 0.013 mmol) was dissolved in methanol (1 mL), ammonium hydrogen fluoride (9.5 mg, 0.26 mmol) was added, and the mixture was heated under reflux for 30 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol/chloroform=0-6%) to obtain compound 74 (2.5 mg, 57%).
¹ H-NMR (DMSO-db): δ 8.06 (1H, s), 7.71 (1H, s), 7.66 (1H, d, J = 2.4 Hz), 6.71 (1H, t , J = 6.8 Hz), 6.65 (1H, d, J = 2.4 Hz), 4.78 (1H, dd, J = 5.1 Hz, 6.5 Hz), 3.93 (1H, d, J=12.0 Hz), 3.87 (1H, d, J=12.0 Hz), 2.89-2.84 (1H, m), 2.55-2.50 (1H, m).

Synthesis Example 13: Synthesis of 4-amino-7-(4-C-azido-2-O-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyridine 4-amino-7- (4-C-azido-2-O-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine (compound 85) was synthesized by the reaction steps shown below.

First, after dissolving 2.44 g (4.66 mmol) of compound 75 (see Synthesis, 2006, 12, 2005) in methanol (20 mL), aqueous ammonia (20 mL) was added and stirred at 85°C for 22 hours (shield tube). 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 crude compound 76 (1.06 g, <3.95 mmol). Obtained.

Next, after dissolving the crude compound 76 (1.06 g, <3.95 mmol) in pyridine (11 mL), chlorotrimethylsilane (5.0 mL, 39.5 mmol) was added at 0° C., and 1 Stirred for hours. Subsequently, benzoyl chloride (0.48 mL, 4.15 mmol) was added and stirred for 3 hours, then benzoyl chloride (0.14 mL, 1.19 mmol) was added and stirred for 1 hour. After completion of the reaction, water was added at 0° C. and stirred for 20 minutes, then aqueous ammonia (8 mL) was added and stirred 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 (methanol/chloroform=1/4) to obtain crude compound 77 (<3.95 mmol).

Next, crude compound 77 (<3.95 mmol) that had been subjected to azeotropic dehydration with pyridine was dissolved in pyridine (20 mL), and then 4,4′-dimethoxytrityl chloride (1.61 g, 4 .74 mmol) was added and stirred at room temperature for 1.5 hours. After completion of the reaction, the reaction was quenched with methanol at 0°C. Thereafter, the solvent was distilled off under reduced pressure to obtain crude compound 78 (<3.95 mmol).

Next, crudely purified compound 78 (<3.95 mmol) was dissolved in N,N-dimethylformamide (20 mL) and then imidazole (0.81 g, 11.9 mmol) and tert-butyldimethylchlorosilane TBSCl ( 1.20 g, 7.90 mmol) were sequentially added, and the mixture was stirred at room temperature for 13.5 hours. After completion of the reaction, quenching was performed at 0°C with saturated aqueous sodium bicarbonate. Subsequent extraction with ethyl acetate and evaporation of the solvent under reduced pressure afforded crude compound 79 (<3.95 mmol).

Next, crude compound 79 (<3.95 mmol) was dissolved in chloroform (11 mL), and then p-toluenesulfonic acid monohydrate (2.30 g, 11.9 mmol) was dissolved in methanol (22 mL). ) was added dropwise at -15°C. After stirring at the same temperature for 2 hours, the mixture was neutralized with a 1N sodium hydroxide aqueous solution. Extraction with ethyl acetate followed. 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/hexane = 1/2 → 1/1) to give crude compound 80 ( 943 mg, <1.94 mmol).

Crude compound 80 (429 mg, <0.882 mol) was then dissolved in pyridine (4.4 mL) followed by triphenylphosphine (1.16 g, 4.41 mmol) and iodine (1.12 g, 4.41 mmol). ) were sequentially added and stirred at room temperature for 3.5 hours. After completion of the reaction, quenching was performed at 0° C. with a saturated aqueous solution of sodium thiosulfate. Extraction with ethyl acetate followed. 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/hexane=1/3) to give compound 81 (4-benzylamino-7- (3-O-tert-butyldimethylsilyl-2,5-di-O-deoxy-iodo-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine, 261mg, 0.438mmol, 24 % for 6 steps) was obtained.
¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.72 (1H, s), 8.65 (1H, brs), 7.99 (2H, m), 7.61 (1H, m), 7.54 (2H, m), 7.23 (1 H, s), 6.76 (1 H, t, J=6.5 Hz), 4.45 (1 H, m), 3.86 (1 H, m), 3. 41 (2H, m), 2.62 (1H, m), 2.38 (1H, m), 0.93 (9H, s), 0.16 (3H, s), 0.15 (3H, s ).

Next, compound 81 (213 mg, 0.357 mmol) was dissolved in pyridine (1.8 mL), potassium tert-butoxide (120 mg, 1.07 mmol) was added, and the mixture was stirred at room temperature 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/hexane = 1/2) to give compound 82 (4-benzylamino-7-(3-O -tert-butyldimethylsilyl-2,5-di-O-deoxy-β-D-erythro-pent-enofuranosyl)-5-fluoropyrrolo[2,3-d]pyrimidine, 155mg, 0.330mmol, 93%) got
¹ H-NMR (CDCl ₃ , 500 MHz); t, J = 8.0 Hz), 7.54 (2H, t, J = 7.5 Hz), 7.01 (1H, d, J = 2.0 Hz), 6.97 (1H, t, J = 7 .0Hz), 4.94 (1H, t, J = 5.0Hz), 4.47 (1H, s), 4.21 (1H, d, J = 1.5Hz), 2.56 (1H, m ), 2.51 (1 H, m), 0.94 (9 H, s), 0.16 (6 H, s).

Next, after dissolving compound 82 (86.0 mg, 0.184 mmol) in N,N-dimethylformamide (3 mL), sodium azide (59.7 mg, 0.918 mmol) and iodine chloride (1.0 M in Dichloromethane, 0.46 mL, 0.46 mmol) was added and stirred at room temperature for 1.5 hours. After completion of the reaction, quenching was performed at 0° C. with a saturated sodium thiosulfate aqueous solution. Extraction with ethyl acetate followed. 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/hexane=1/3) to give compound 83 (7-(4-C-azide -3-O-tert-butyldimethylsilyl-2,5-di-O-deoxy-5-O-iodo-β-D-ribofuranosyl)-4-benzylamino-5-fluoropyrrolo[2,3-d] pyrimidine, 50.1 mg, 0.0786 mmol, 43%).
¹ H-NMR (CDCl ₃ , 500 MHz); t, J = 7.5Hz), 7.54 (2H, t, J = 7.5Hz), 7.21 (1H, s), 7.13 (1H, m), 4.35 (1H, d, J = 4.0 Hz), 3.61 (1H, d, J = 11.0 Hz), 3.51 (1H, d, J = 11.0 Hz), 2.79 (1H, m), 2.48 ( 1H, m), 0.98 (9H, s), 0.25 (3H, s), 0.20 (3H, s).

Next, after dissolving compound 83 (50.1 mg, 0.0786 mmol) in N,N-dimethylformamide (1.5 mL), sodium benzoate (113 mg, 0.786 mmol) and 15-crown-5 (0 .16 mL, 0.786 mmol) was added and stirred at 100° C. for 24 hours. After completion of the reaction, 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/hexane = 1/2→1/1→2/1) for crude purification. of compound 84 (10.5 mg, <0.0202 mmol).

Crudely purified compound 84 (10.5 mg, <0.0202 mmol) was dissolved in methanol (1 mL), aqueous ammonia (1 mL) was added, and the mixture was stirred at room temperature for 80 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 = 1/20) to give compound 85 (4-amino-7-(4-C-azido- 2-O-deoxy-β-D-ribofuranosyl)-5-fluoropyrrolo[2,3-d]pyridine, 2.6 mg, 8.41 μmol, 11% for 2 steps).
¹ H-NMR (CD ₃ OD, 500 MHz); , t, J = 7.0 Hz), 3.77 (1H, d, J = 12 Hz), 3.69 (1H, d, J = 12 Hz), 2.66 (1H, m), 2.52 (1H , m).

In addition, the antiviral activity and cytotoxicity of the nucleoside derivatives synthesized as described above were evaluated by the methods described below.

Test Example 1: Evaluation of anti-HBV activity 1
HepG2 2.2.15.7 cells were used as test cells. HepG2 2.2.15.7 cells are 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). and HepG2 2.2.15.7 cells were maintained in continuous culture in DMEM containing 10% fetal bovine serum, G418 (500 μg/ml) and antibiotics (penicillin and kanamycin).

HepG2 2.2.15.7 cells are HBV persistent producing cells that carry HBV genes that are produced episomally as well as DNA integrated into the genome. Therefore, by co-cultivating with each nucleoside derivative, the DNA copy number of the virus released in the culture supernatant and the DNA copy number of the virus present in the HepG2 2.2.15.7 cells were quantified, and the degree of reduction was evaluated by the antimicrobial method. It was used as an index for evaluating HBV activity.

More specifically, HepG2 2.2.15.7 cells with 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 as the 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 medium was replaced with a fresh medium containing each nucleoside derivative. assay), HBV DNA was recovered. In addition, a fresh medium containing each nucleoside derivative was added to the cell plate after collecting the supernatant, and the culture was continued for 7 days and 14 days, respectively, 2W assay (assay on day 14 from the start of culture) and Intracellular HBV DNA was harvested from HepG2 2.2.15.7 cells for the 3W assay (assay on day 21 of culture initiation). Quantitative PCR was performed using these DNAs as templates, and the virus copy number was obtained from a calibration curve to calculate the 50% effect (EC ₅₀ ) for each nucleoside derivative. The results obtained are shown in Tables 2 and 3.

DNA extraction from HepG2 2.2.15.7 cell supernatant and cells was performed using QIAamp MiniElute virus Spin Kit (manufactured by QIAGEN), and 5 μL of the extracted DNA was used for qPCR. Specific TaqMan probe primers from PrimerDesign that detect the HBV core protein region were used in the PCR reaction. The PCR reaction was carried out at 95°C for 15 minutes, followed by 50 cycles of 95°C for 10 seconds and 60°C for 60 seconds. The resulting CT was converted to HBV copy number using a standard curve generated from reactions of 10- _fold dilutions (20 to 2×10 ⁸ copies) of known concentrations of HBV DNA fragments.

Test Example 2: Cytotoxicity test 1
A cytotoxicity test against HepG2 cells was also performed for the above nucleoside derivatives. HepG2 cells were maintained by continuous culture in DMEM containing 10% fetal bovine serum and antibiotics (penicillin and kanamycin).

For the cytotoxicity test, HepG2 cells were inoculated at a concentration of 1×10 ⁴ cells/ml together with a medium supplemented with serially diluted nucleoside derivatives at various concentrations. After culturing these cells for 7 days at standard culture conditions of 37°C and 5% _CO2 in the presence of various concentrations of each nucleoside derivative in this manner, the number of viable cells in each well was quantified by MTT assay. . Then, _CC50 was calculated for each nucleoside derivative based on the number of viable cells obtained. The results obtained are shown in Tables 2 and 3.

Test Example 3: Cytotoxicity test 2
PXB cells were used as test cells. PXB cells were fresh human hepatocytes derived from human hepatocyte chimeric mice and maintained in dHCGM medium for PXB cells. Both the cells and the medium are manufactured by Phoenix Bio Co., Ltd.

PXB cells were seeded at 3×10 ⁵ cells/ml on a collagen-coated 96-well plate, and cultured for 7 days under standard culture conditions of serially diluted compounds at each concentration and 37° C., 5% CO ₂ . Viable cell numbers in the wells were quantified with the MTT assay. Then, based on the obtained values, the degree of cytotoxicity was determined, and the _CC50 value was calculated as the cytotoxicity of each compound. The results obtained are shown in Tables 2 and 3.

In Tables 2 and 3 below, items marked with "* (asterisk)" in items related to antiviral activity indicate that the _EC50 value is greater than 1 μM, and items related to cytotoxicity "* Items marked with (asterisk)” indicate CC ₅₀ values less than 10 μM, items marked with “-” indicate not tested.

As shown in Tables 2 and 3, all of the nucleoside derivatives synthesized and tested as described above were found to have excellent antiviral activity against HBV. It was also confirmed from the results of the 2W assay that the effect was sustained (although not shown in the table, the results of performing the 3W assay on compound 69 showed an _EC50 of 0.060, a high antiviral activity). is maintained). Furthermore, as shown in Tables 2 and 3, the cytotoxicity of these nucleoside derivatives was also found to be generally low.

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

Claims (3)

An anti-hepatitis B virus agent containing a nucleoside derivative represented by the following general formula (1) as an active ingredient .

[In the above formula, R ¹ represents a hydroxy group or an optionally substituted amino group. ^R2 represents a hydrogen atom, a halogen atom or an amino group. R3 represents an optionally substituted alkyl group ^, cyano group or azide group. R ⁴ represents a hydrogen atom, a halogen atom, an optionally substituted alkynyl group, or an optionally substituted heterocyclic group. ] The nucleoside derivative is
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a monofluoromethyl group, and R ⁴ is a hydrogen atom, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a cyano group, and R ⁴ is an iodine atom, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a cyano group, and R ⁴ is a fluorine atom, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a cyano group, and R ⁴ is an ethynyl group, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is an amino group and R ³ is a cyano group, and R ⁴ is a hydrogen atom, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a cyano group, and R ⁴ is a chlorine atom, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is an azide group and R ⁴ is a fluorine atom, a nucleoside derivative, or
In the general formula (1), R ¹ is a hydroxy group and R ² is an amino group and R ³ is a cyano group, and R ⁴ The anti-hepatitis B virus agent according to claim 1, which is a nucleoside derivative, wherein is a hydrogen atom. The nucleoside derivative is
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a monofluoromethyl group, and R ⁴ is a hydrogen atom, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a cyano group, and R ⁴ is an iodine atom, a nucleoside derivative,
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a cyano group, and R ⁴ is a fluorine atom, a nucleoside derivative, or
In the general formula (1), R ¹ is an amino group and R ² is a hydrogen atom, and R ³ is a cyano group, and R ⁴ The anti-hepatitis B virus agent according to claim 1, which is a nucleoside derivative, wherein is an ethynyl group.