WO2018110591A1 - 2'-deoxy-7-deazapurine nucleoside derivative having antiviral activity - Google Patents

Abstract

The present invention makes it clear that a nucleoside derivative which shows excellent antiviral activity against HBV and which has low cytotoxicity can be obtained by substituting specific functional groups at each of positions 2, 6, and 7 of the purine base and position 4 of the ribose sugar, in 2'-deoxy-7-deazaadenosine.

Description

2'-Deoxy-7-deazapurine nucleoside derivatives having antiviral activity

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

When hepatitis B virus (HBV) is infected, acute or fulminant hepatitis occurs, sometimes resulting in death. Hepatitis may also develop chronically and may progress to cirrhosis and hepatocellular carcinoma. The number of infected people is estimated to be about 400 million in the whole world, the morbidity rate is very high mainly in Southeast Asia, and the development of its effective treatment method is required worldwide.

HBV is an incomplete double-stranded DNA virus and is known to perform reverse transcription to synthesize DNA from RNA in its life cycle. On the other hand, since reverse transcription is not performed in the host human, it is possible to inhibit only HBV replication by inhibiting this step. And a nucleoside derivative preparation is developed as a therapeutic drug of HBV infection from such a viewpoint (patent documents 1 and 2).

JP 2004-244422 A JP 2008-273960 A

In the current nucleoside derivative preparations, many of them are also toxic to host cells, that is, human cells to be taken, and the side effects due to medium- and long-term taking are problematic. 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 thereof is to provide a nucleoside derivative which has at least antiviral activity against HBV and low toxicity to host cells.

As a result of intensive studies to solve the above problems, the present inventors have found that in the 2'-deoxy-7-deazapurine nucleoside, the 2-, 6- and 7-positions of the purine base and the 4-position of the ribose sugar Thus, the inventors have found that, while the nucleoside derivatives each substituted to a specific functional group exert an excellent antiviral activity against HBV, they generally have low cytotoxicity, and have completed the present invention.

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

[In the above formula, R ¹ represents a hydroxy group or an amino group which may have a substituent. R ² represents a hydrogen atom, a halogen atom or an amino group. R ³ represents an alkyl group which may have a substituent, a cyano group, an azide group or a hydrogen atom. R ⁴ represents a hydrogen atom, a halogen atom, an alkynyl group which may have a substituent, or a heterocyclic group which may have a substituent. ]
The antiviral agent which uses the nucleoside derivative as described in <2><1> as an active ingredient.
<3> The antiviral agent according to <2>, which is an anti-hepatitis B virus agent.

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

(Nucleoside derivative)
As shown in the following examples, it was revealed that the nucleoside derivatives represented by the following formula have antiviral activity against hepatitis B virus. Accordingly, the present invention relates to a nucleoside derivative exhibiting antiviral activity, and more particularly to provide a nucleoside derivative represented by the following general formula (1) having antiviral activity against at least hepatitis B virus. is there.

[In the above formula, R ¹ represents a hydroxy group or an amino group which may have a substituent. R ² represents a hydrogen atom, a halogen atom or an amino group. R ³ represents an alkyl group which may have a substituent, a cyano group or a hydrogen atom. R ⁴ represents a hydrogen atom, a halogen atom, an alkynyl group which may have a substituent, or a heterocyclic group which may have a substituent. ].

The nucleoside derivative of the present invention has antiviral activity at least against hepatitis B virus (HBV). In the present invention, "HBV" means a virus having the ability to develop hepatitis B. As HBV, genotypes of A (A2 / Ae, A1 / Aa), B (Ba, B1 / Bj), C (Cs, Ce), DH and J are known, but the nucleosides of the present invention The derivative may be one having antiviral activity against HBV of at least one genotype.

In the present invention, "antiviral activity" means an activity to eliminate the virus or suppress its growth in a cell (host cell) infected with a virus such as HBV, for example, to suppress viral replication in the host cell Activity is included. In addition, when the subject such as suppression is a virus having a DNA as a genome (DNA virus), it is referred to as "anti-DNA virus activity". Furthermore, such an activity can be evaluated by an EC ₅₀ value calculated using, as an index, the copy number of the virus in the host cell, as shown in the examples described later. The nucleoside derivative of the present invention preferably has an EC ₅₀ value of antiviral activity of less than 1 μM, more preferably 0.5 μM or less, still more preferably 0.1 μM or less, and 0.05 μM or less For example, it is more preferable that the concentration is 0.04 μM or less, 0.03 μM or less, 0.02 μM or less, and 0.01 μM or less.

Moreover, it is preferable that the nucleoside derivative of this invention has low cytotoxicity. In the present invention, "cytotoxicity" means an activity to kill cells, inhibit their functions or inhibit their proliferation. Such activity can be evaluated by a CC ₅₀ value calculated using the number of viable cells of the cell as an indicator, as described in the examples below. The nucleoside derivative of the present invention preferably has a CC ₅₀ value of 10 μM or more, more preferably a CC ₅₀ value of 25 μM or more, still more preferably a CC ₅₀ value of 50 μM or more, and 100 μM or more Is more preferred.

From the viewpoint that the cytotoxicity of the derivative can be reduced while at least exerting antiviral activity on HBV in the nucleoside derivative of the present invention, 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 preferable, and a methyl group is more preferable. More specifically, the "amino group which may have a substituent (s)" is preferably an amino group or a methylamino group.

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

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

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

The heterocyclic group in the “optionally substituted heterocyclic group” is not particularly limited and includes, for example, an aromatic or aliphatic heterocyclic ring containing a nitrogen atom, an oxygen atom, and a sulfur atom. Among them, a nitrogen-containing heterocyclic group is preferable. For example, pyrrolyl group, thiazolyl group, isothiazolyl group, oxazolyl group, isoxazolyl group, pyridyl group, imidazolyl group, imidazolinyl group, pyrazolyl group, (1,3,5 -) Triazinyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, quinolyl, isoquinolyl, isoquinolyl, purinyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, tetrazolyl, tetrazinyl, triazolyl, carbazolyl And acridinyl groups, quinoxalyl groups and quinazolyl groups, and among these, pyrazolyl groups are preferable. There is no restriction | limiting in particular as a substituent in "the heterocyclic group which may have a substituent", For example, a halogen atom, a hydroxyl group, an alkoxy group, a cyano group, an amino group is mentioned.

As an example of a nucleoside derivative having a suitable functional group, a compound represented by the above general formula (R ¹ is an amino group, R ² is a hydrogen, R ³ is a cyano group, and R ⁴ is a fluorine or ethynyl group) The nucleoside derivative represented by 1) is mentioned.

The nucleoside derivatives of the present invention also include pharmacologically acceptable salts, hydrates or solvates. Such a pharmacologically acceptable salt is not particularly limited and can be appropriately selected according to the structure of the nucleoside derivative etc. For example, acid addition salt (hydrochloride, sulfate, hydrobromide, Nitrate, hydrogen sulfate, phosphate, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluene sulfone Acid, camphor sulfonate, sulfamate, mandelic acid, propionate, glycolate, stearate, malate, ascorbate, pamoate, phenylacetate, glutamate, benzoate Salicylate, Sulfanilate, 2-Acetoxybenzoate, Ethanedisulfonate, Oxalate, Isethionate, Formate, Trifluoroacetate, Ethyl Co Cuprate, lactobionate, gluconate, glucoheptonate, 2-hydroxyethane sulfonate, benzene sulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate , Oxalate, picrate, thiocyanate, undecanoate etc., base addition salt (sodium salt, potassium salt, zinc salt, calcium salt, bismuth salt, barium salt, magnesium salt, aluminum salt, copper salt, Cobalt salts, nickel salts, cadmium salts, ammonium salts, ethylenediamine salts, N-dibenzylethylenediamine salts) can be mentioned. Further, the hydrate or the solvate is not particularly limited, and, for example, one obtained by adding 0.1 to 3 molecules of water or a solvent to one molecule of a nucleoside derivative or a salt thereof can be mentioned.

Nucleoside derivatives of the present invention include all isomers and isomer mixtures such as tautomers, geometric isomers, optical isomers based on asymmetric carbon, stereoisomers and the like. Furthermore, the nucleoside derivative of the present invention is further desired in vivo by metabolism such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydration, alkylation, dealkylation, conjugation and the like. The present invention also encompasses compounds that undergo metabolism, such as oxidation, reduction, hydrolysis, etc., in vivo to produce the nucleoside derivative of the present invention (so-called prodrug form). . Furthermore, the nucleoside derivative of the present invention can be formulated by known pharmaceutical methods as described later.

Furthermore, synthesis of the nucleoside derivative of the present invention includes, for example, a ribose sugar (D-ribofuranose protected by substituting a hydroxy group with an acetyl group, a benzyl group or the like) and a purine base (7-deazaadenine) And silyl form, and further reduction via a phenoxythio carbonyl derivative to desoxylate the 2-position of the ribose sugar, and, if necessary, substitution at a target position of the ribose sugar and / or purine base by known methods It can be done by introducing a group. The synthesis method of such a nucleoside derivative of the present invention is shown in detail in the examples described later, so those skilled in the art will refer to the description of the examples, referring to reaction raw materials, reaction reagents, reaction conditions (for example, It is possible to synthesize the nucleoside derivative of the present invention by appropriately modifying or modifying these methods as necessary while appropriately selecting the solvent, reaction temperature, catalyst, reaction time) and the like. Moreover, the nucleoside derivative thus synthesized can be selected from methods generally used for isolation and purification of nucleosides and nucleotides (reverse phase chromatography, ion exchange chromatography, adsorption chromatography, recrystallization method) as appropriate. It can be separated and purified by using alone or in combination.

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

There is no particular limitation on the infection targeted by the antiviral agent of the present invention and the preventive method and therapeutic method described later, and examples thereof include HBV infection, and more specifically, hepatitis B (chronic hepatitis, acute Hepatitis, fulminant hepatitis), liver cirrhosis, hepatic fibrosis, and hepatocellular carcinoma.

The antiviral agent of the present invention can be formulated by known pharmaceutical methods. For example, capsules, tablets, pills, solutions, powders, granules, fine granules, film coatings, pellets, troches, sublingual agents, lozenges, buccal agents, pastes, syrups, suspensions, Use orally or parenterally as elixirs, emulsions, coatings, ointments, salves, patches, patches, transdermal preparations, lotions, aspirants, aerosols, injections, suppositories, etc. Can.

In these formulations, a pharmacologically acceptable carrier or vehicle, specifically, sterile water or saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent Fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, coloring agent, sweetener, It can be appropriately combined with a thickener, a flavoring agent, a solubilizer, or other additives. More specifically, solid carriers such as lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride, glycerin, peanut oil, polyvinyl alcohol Liquid carriers such as pyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, water and the like can also be mentioned.

In addition, the antiviral agent of the present invention may be used in combination with other known antiviral agents. As such known antiviral agents, when the target disease is an HBV infection, for example, known nucleoside analogue preparations such as entecavir, 3TC (lamivudine), adefovir, interferon (IFN) and the like can be mentioned. In addition to antiviral therapy using such drugs, immunotherapy (corticosteroid withdrawal therapy, oral administration of propagernium preparation, etc.), liver protection therapy (intravenous injection of glycyrrhizin preparation, oral administration of bile acid preparation, etc.) The antiviral agent of the present invention can also be used in combination therapy with

The preferred administration form of the antiviral agent of the present invention is not particularly limited, and orally or parenterally, more specifically, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, airway Internal administration, rectal administration and intramuscular administration, administration by infusion is included.

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

When the antiviral agent of the present invention is administered, the dosage is appropriately selected according to the age, body weight, symptoms, health condition, serious condition, tolerance to drugs, administration form of the subject, and the like. The dose of the antiviral agent of the present invention per day is usually 0.00001 to 1000 mg / kg body weight, preferably 0.0001 to 100 mg / kg body weight as the amount of nucleoside derivative which is the active ingredient, either once or It is administered to a subject divided into multiple doses.

The antiviral agent product of the present invention or instructions therefor may be labeled with an indication that it is used to treat or prevent a viral infection. Here, "indicating the indication on the product or instruction" means that the indication is attached to the main body of the product, the container, the package or the like, or the instruction, the package insert, the advertisement, the other printed matter disclosing the information of the product. It means that the display was attached to etc. In addition, in the indication that it is used to treat a viral infection, 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 antiviral agents.

Thus, the present invention can prevent or treat an infectious disease by administering the antiviral agent of the present invention to a subject. Accordingly, the present invention also provides a method for preventing or treating a viral infection, which comprises administering the nucleoside derivative of the present invention.

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

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

Furthermore, it was confirmed by measuring the ¹ H nuclear magnetic resonance (NMR) spectrum that the compound thus synthesized is a compound having the desired structure. The 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 in the following reaction steps.

First, Biosci. Biotechnol. Biochem. According to the method described in 1999, 63, 736-742 (Ref. 1 above), 1,2: 5,6-di-O-isopropylidene-α-D-allofuranose (compound 1), 2-O-isopropylidene-3-Op-methoxybenzyl-α-D-allofuranose (compound 2) was synthesized.

Next, from compound 2 thus obtained, compound 3 (3,5-di-O-benzyl-4-C-fluoromethyl-1,2-O-isopyridene-α-D-ribofuranose) 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) is 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 was quenched with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. After drying of the organic layer with magnesium sulfate and evaporation of 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); δ 7.34-7.25 ( ¹⁰ H, m), 5.76 (1 H, d, J = 3.5 Hz), 4.91-4. 81 (1 H, dd) , J = 48.5, 10.0 Hz), 4.74-4.60 (3H, m), 4.57-4. 48 (3H, m), 4.26 (1 H, dd, J = 5. 0, 1.5 Hz), 3.61 (1 H, dd, J = 10.5, 2.5 Hz), 3.55 (1 H, dd, J = 10.5, 2.0 Hz), 1.63 (3 H) , S), 1.35 (3H, s).

Next, compound 4 (1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-fluoromethyl-D-ribofuranose) was obtained from compound 3 thus obtained. Synthesized. That is, Compound 3 (269 mg, 0.67 mmol) was dissolved in acetic acid (4.2 mL), water (1.3 mL) and trifluoroacetic acid (0.42 mL) were sequentially added, and the mixture was stirred for 5.5 hours. After completion of the reaction, the solvent was distilled off to obtain a crudely purified deacetonide (0.67 mmol). The crude deacetonide (0.67 mmol) was azeotroped with toluene three times. 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 the mixture was stirred for 1 hour. After completion of the reaction, extraction with ethyl acetate was performed. After drying of the organic layer with magnesium sulfate and evaporation of 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); δ 7.36-7.24 (10 H, m), 6.42-6.17 (1 H, m), 5.35-5. 22 (1 H, m), 4.74-4.64 (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 (1 H, dd, J = 9.5, 2.0 Hz), 2.10 (3 H, s), 2.04-1. 90 (3H, s).

Then, 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, after adding acetonitrile (0.2 mL) to 6-chloro-7-iodo-7-deazapurine (15.1 mg, 0.054 mmol), N, O-bis (trimethylsilyl) acetamide (15.9 μL, 0. 065 mmol) was added and stirred at room temperature for 20 minutes. Subsequently, Compound 4 (15.4 mg, 0.035 mmol) and trimethylsilyl trifluoromethanesulfonate (12.5 μL, 0.069 mmol) dissolved in acetonitrile (0.3 mL) were sequentially added, and stirred at room temperature for 10 minutes. After that, it was stirred for 21 hours while heating under reflux. After completion of the reaction, the reaction was quenched with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. After drying of the organic layer with magnesium sulfate and evaporation of 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); δ 8.58 (1 H, s), 7.7 1 (1 H, s), 7.44-7. 32 (10 H, m), 6.59 (1 H, d, 7) J = 6.5 Hz), 5.69 (1 H, t, J = 5.5 Hz), 4.71-4. 48 (8 H, m), 3. 76 (1 H, dd, J = 10.0, 2 .0 Hz), 3.70 (1 H, dd, J = 10.0, 2.5 Hz), 2.02 (3 H, s).

Next, compound 6 (4-amino-7- (3,5-di-O-benzyl-4-C-fluoromethyl-2-O-phenylthionoformyl-) was obtained from compound 5 thus obtained. β-D-ribofuranosyl) -5-iodopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 5 (9.8 mg, 0.015 mmol) was dissolved 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 with ethyl acetate was performed. Subsequently, the organic layer was dried with magnesium sulfate and the solvent was evaporated under reduced pressure to obtain a crudely purified 6-aminated product (9.5 mg, 0.015 mmol). The crude 6-aminated product (9.5 mg, 0.015 mmol) was dissolved in acetonitrile (1 mL) and then 4-dimethylaminopyridine (3.0 mg, 0.025 mmol) and phenyl chlorothionoformate (2. 3 μL, 0.016 mmol) were sequentially added, and the mixture was stirred at 0 ° C. for 1 hour. Subsequently, after adding phenylchlorothionoformate (1.0 μL, 7.1 μmol) and stirring at 0 ° C. for 1 hour, phenyl chlorothionoformate (1.0 μL, 7.1 μmol) was added and stirred at room temperature for 1 hour . Further, 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 reaction was quenched with water and extracted with ethyl acetate. After drying of the organic layer with magnesium sulfate and evaporation of the solvent under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1/1) to give compound 6 (8.2 mg , 0.011 mmol, 2 steps 75%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.24 (1 H, s), 7.40-7. 26 (13 H, m), 6. 92 (2 H, m), 6.69 (1 H, d, d) J = 6.5 Hz), 6.21 (1 H, dd, J = 7.0, 5.5 Hz), 5.74 (2 H, brs), 4.86-4. 46 (8 H, m), 78 (2H, m).

Next, compound 7 (4-amino-7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) was obtained from compound 6 thus obtained. ) -Pyrrolo [2,3-d] pyrimidine) was synthesized. That is, after dissolving compound 6 (133 mg, 0.18 mmol) in toluene (6 mL), tributyl tin hydride (0.24 mL, 0.90 mmol) and azobisisobutyronitrile (7.4 mg, 0.045 mmol) Were sequentially added and stirred at 80.degree. C. for 35 minutes. Subsequently, after the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate) to obtain Compound 7 (42.3 mg, 0.092 mmol, 51%).
⁶ H-NMR (CDCl ₃ , 500 MHz); δ 8.29 (1 H, s), 7. 38-7. 28 (10 H, m), 7. 19 (1 H, d, J = 3.5 Hz), 6. 74 (1 H, t, J = 6.5 Hz), 6.31 (1 H, d, J = 4.0 Hz), 5.12 (2 H, brs), 4.77-4. 49 (8 H, m), 3.72 (1 H, dd, J = 9.5, 1.5 Hz), 3. 65 (1 H, dd, J = 10.0, 2.0 Hz), 2.69-2.64 (1 H, m) , 2.61-2.56 (1 H, m).

Then, from compound 7 thus obtained, compound 8 (4-amino-7- (2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -pyrrolo [2,3-d] is obtained. Pyrimidine) was synthesized. That is, Compound 7 (25.6 mg, 0.055 mmol) is dissolved in dichloromethane (2 mL), and then boron trichloride (1.0 M solution in dichloromethane, 0.28 mL, 0.28 mmol) is added at -78.degree. It stirred under 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 (1 H, dd, J = 8.5, 6.0 Hz), 4.68 (1 H, dd, J = 33.5, 10.0 Hz), 4.65 (1 H, dd, J = 6 .0, 3.0 Hz), 4.58 (1 H, dd, J = 34.0, 10.0 Hz), 3.74 (2 H, m), 2.89-2.83 (1 H, m), 2 .39-2.34 (1 H, 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 prepared according to It synthesize | combined in the reaction process shown.

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] pyrimidine) was synthesized. That is, compound 9 (3.65 g, 6.4 mmol) is dissolved in N, N-dimethylformamide (64 mL), imidazole (1.30 g, 19 mmol) is added, and then tert-butyldimethylsilyl under ice cooling. Chloride (1.44 g, 9.6 mmol) was added and stirred at room temperature for 19.5 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (150 mL), and methanol solution (60 mL) of tosic acid monohydrate (2.43 g, 13 mmol) is added over 10 minutes at -15 ° C The solution was added dropwise and stirred for 5 minutes. Saturated aqueous sodium bicarbonate solution was added to terminate the reaction, and the reaction solution was partitioned, 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 chromatography (n-hexane / ethyl acetate = 9: 1-4: 1) to give compound 10 (2.32 g, 2 steps) 95%).
¹ H-NMR (CDCl ₃ ): δ 8.63 (1 H, s), 7. 35 ( ¹ H, d, J = 3.7 Hz), 6.62 (1 H, d, J = 3.7 Hz), 6. 29 (1 H, dd, J = 5.5 Hz, 9.3 Hz), 5. 38 (1 H, dd, J = 2.0 Hz, 9.2 Hz), 4. 70 (1 H, d, J = 5.3 Hz) , 4.13 (1 H, d, J = 1.4 Hz), 3.97-3. 93 (1 H, m), 3.80-3.74 (1 H, 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- 4 Chloropyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 10 (2.25 g, 5.9 mmol) is dissolved in dimethyl sulfoxide (18 mL) and toluene (12 mL), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (with ice-cooling) 4.49 g (23 mmol), pyridine (630 μL, 7.8 mmol) and trifluoroacetic acid (292 μL, 3.9 mmol) were added and the mixture was stirred for 5 hours. The reaction was quenched with water and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in 1,4-dioxane (18 mL), 37% aqueous formaldehyde solution (3.3 mL, 41 mmol) under ice-cooling, 1 M aqueous solution of sodium hydroxide (6 .2 mL, 6.2 mmol) was added and stirred for 22 hours. Acetic acid was added to quench the reaction, extraction was performed 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. Acetic acid was added to quench the reaction, extraction was performed 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 to 1: 1) to obtain compound 11 (1.02 g, 3 Process 42%).
¹ H-NMR (CDCl ₃ ): δ 8.63 (1 H, s), 7.34 (1 H, d, 3.7 Hz), 6.61 (1 H, d, J = 3.7 Hz), 6.40 ( 1 H, 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 (1 H, m), 0.96 (9 H, s), 0.18 (3 H, s), 0.17 (3 H, s).

Next, compound 12 (7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-dimethoxytrityloxymethyl-β-D-ribofuranosyl)-) was obtained from compound 11 thus obtained. 4-Chloropyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 11 (1.02 g, 2.5 mmol) is dissolved in dichloromethane (15 mL), triethylamine (515 μL, 3.7 mmol) is added, and 4,4′-dimethoxytrityl chloride (918 mg, 2) is added under ice cooling. After dropwise addition of a solution of 7 mmol) in dichloromethane (10 mL) over 20 minutes, the mixture was stirred for 4 hours, and 4,4′-dimethoxytrityl chloride (83 mg, 0.25 mmol) was further added and stirred for another 2 hours. Methanol was added to stop the reaction, chloroform and water were added for partitioning, and the organic layer was washed with saturated aqueous sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, and the residue is purified by a medium-pressure separator (n-hexane / ethyl acetate = 4: 1-2: 1), and then re-chromatographed on silica gel (n-hexane / The residue was purified with ethyl acetate = 5: 1) to give compound 12 (1.44 g, 82%).
¹ H-NMR (CDCl ₃ ): δ 8.67 (1 H, s), 7.47-7. 19 (10 H, m), 6.83-6. 80 (4 H, m), 6.64 (1 H, 1 H, d, J = 3.6 Hz), 6.30 (1 H, dd, J = 6.1 Hz, 8.3 Hz), 4.71 (1 H, dd, J = 2.6 Hz, 6.1 Hz), 4.66 (1H, dd, J = 3.0 Hz, 10.7 Hz), 4.20 (1 H, dd, J = 3.0 Hz, 12.3 Hz), 3.79 (3 H, s), 3.78 (3 H, s), 3.64 (1 H, dd, J = 10.7 Hz, 12.3 Hz), 3.53 (1 H, d, J = 10.7 Hz), 3.05-3.03 (2 H, m), 2.24-2.29 (1 H, m), 0.76 (9 H, s), -0.03 (3 H, s), -0.12 (3 H, s).

Then, from compound 12 thus obtained, 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. That is, compound 12 (1.41 g, 2.0 mmol) and imidazole (408 mg, 6.0 mmol) are dissolved in N, N-dimethylformamide (20 mL), and tert-butyldiphenylsilyl chloride (768 μL, under ice cooling). The mixture was added with 3.0 mmol), stirred at room temperature for 24 hours, added with imidazole (136 mg, 2.0 mmol) and tert-butyldiphenylsilyl chloride (256 μL, 1.0 mmol) and further stirred for 3 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (40 mL), and a methanol solution (20 mL) of tosic acid monohydrate (761 mg, 4.0 mmol) at -15 ° C is added over 15 minutes The solution was added dropwise and stirred for 10 minutes. Saturated aqueous sodium bicarbonate solution was added to terminate 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 give compound 13 (1.25 g, 2 steps) 96%).
¹ H-NMR (CDCl ₃ ): δ 8.55 (1 H, s), 7.65-7. 28 (11 H, m), 6.70 (1 H, t, J = 6.6 Hz), 6.47 ( 1H, d, J = 3.7 Hz), 4.87 (1 H, dd, J = 4.2 Hz, 6.5 Hz), 3.90-3.74 (4 H, m) 2.75-2.70 ( 1 H, m), 2.48-2. 43 (1 H, m), 2. 34 (1 H, dd, J = 5.1 Hz, 8.7 Hz), 1.08 (9 H, s), 0.93 (0.92) 9 H, s), 0.13 (3 H, s), 0.13 (3 H, s).

Next, from compound 13 thus obtained, compound 14 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy-) is obtained. β-D-ribofuranosyl) -4-chloropyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 13 (326 mg, 0.50 mmol) is dissolved in dimethyl sulfoxide (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. The reaction was quenched with water and 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 solution was concentrated under reduced pressure, partitioned between 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. The reaction was quenched with water and 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 give compound 14 (266 mg, 82% of three steps).
¹ H-NMR (CDCl ₃ ): δ 8.50 (1 H, s), 7.62-7. 29 (11 H, m), 6.63 (1 H, t, J = 6.5 Hz), 6.55 ( 1H, d, J = 3.7 Hz), 4.97 (1 H, t, J = 5.9 Hz), 4.04 (1 H, d, J = 11.2 Hz), 3.87 (1 H, d, J = 11.2 Hz), 3.00 to 2.95 (1 H, m), 2.56 to 2.51 (1 H, m), 1.05 (9 H, s), 0.96 (9 H, s), 0.18 (3 H, s), 0.16 (3 H, 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 give Compound 15 as a crudely purified product (0.30 mmol).

Next, compound 16 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano) was obtained from compound 15 thus obtained. 2-Deoxy-β-D-ribofuranosyl) pyrrolo [2,3-d] pyrimidine) was synthesized. That is, crude 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 to 1: 2) to give compound 16 (34 mg, 36%).
¹ H-NMR (CDCl ₃ ): δ 8.16 (1 H, s), 7.64-7. 32 (10 H, m), 7.01 (1 H, d, J = 3.7 Hz), 6.61 1H, t, J = 6.5 Hz), 6.30 (1 H, d, J = 3.7 Hz), 5.41 (2 H, brs), 4.96 (1 H, t, J = 5.8 Hz), 4.03 (1 H, d, J = 11.1 Hz), 3.86 (1 H, d, J = 11.1 Hz), 2.99-2.94 (1 H, 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).

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

Compound 16 (42 mg, 0.067 mmol) was dissolved in tetrahydrofuran (1 mL), a 1 M solution of tetrabutylammonium fluoride 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 give compound 18 (16 mg, 86%).
¹ H-NMR (DMSO-d ₆ ): δ 8.07 (1 H, s), 7.34 (1 H, d, J = 3.7 Hz), 7.08 (2 H, brs), 6.64 (1 H, 1 H, t, J = 7.0 Hz), 6.61 (1 H, d, J = 3.7 Hz), 6. 27 (1 H, d, J = 5.1 Hz), 5.82 (1 H, t, J = 6) .1 Hz), 4.60 (1 H, dd, J = 5.1 Hz, 6.1 Hz), 3.75 (1 H, dd, J = 5.9 Hz, 11.8 Hz), 3.62 (1 H, dd, J = 6.3 Hz, 11.8 Hz), 2.80-2.75 (1 H, m), 2.38-2.34 (1 H, m).

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

Then, from compound 17 thus obtained, compound 19 (7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -4-methylaminopyrrolo [2,3-d] pyrimidine is obtained. Was synthesized. That is, Compound 17 (50 mg, 0.078 mmol) was dissolved in tetrahydrofuran (1 mL), a 1 M solution of tetrabutylammonium fluoride 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 give compound 19 (19 mg, 85%).
¹ H-NMR (DMSO-d ₆ ): δ 8.16 (1 H, s), 7.56 (1 H, d, J = 4.6 Hz), 7.34 (1 H, d, J = 3.7 Hz), 6.65 (1H, t, J = 7.0 Hz), 6.60 (1 H, d, J = 3.6 Hz), 6.28 (1 H, d, J = 5.1 Hz), 5.82 (1 H) , T, J = 6.1 Hz), 4.61 (1 H, dd, J = 4.8 Hz, 11.0 Hz), 3.76 (1 H, dd, J = 5.9 Hz, 11.7 Hz), 3. 62 (1 H, dd, J = 6.3 Hz, 11.7 Hz), 2.95 (3 H, d, J = 4.6 Hz), 2.81-2.75 (1 H, m), 2.39-2 .34 (1 H, 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 in the following reaction steps.

First, compound 22 (4-chloro-7- (2-) was prepared from compound 20 (see Synthetic Commun. 1997, 27, 3505-3511) and compound 21 (see J. Med. Chem. 2012, 55, 7786-7795). Deoxy-3,5-di-O-p-toluoyl-β-D-ribofuranosyl) -2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 21 (3.41 g, 14 mmol) is dissolved in acetonitrile (70 mL), sodium hydride (1.08 g, 27 mmol) is added, and then Compound 20 (5.52 g, 14 mmol) is added under ice cooling. Acetonitrile solution (70 mL) was added dropwise over 15 minutes and stirred under ice-cooling for 1 hour, then sodium hydride (540 mg, 14 mmol) was added and stirred at room temperature for 1.5 hours, and sodium hydride ( Further, 270 mg (7.1 mmol) was added and stirred at room temperature for 1 hour. The reaction mixture was concentrated by 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 to 2: 1) to give compound 22 (4.76 g, 58%) ).
¹ H-NMR (CDCl ₃ ): δ 8.18 (1 H, brs), 8.00-7. 22 (9 H, m), 6.75 (1 H, d, J = 6.2 Hz, 8.0 Hz), 6.51 (1 H, d, J = 3.8 Hz), 5.81-5. 79 (1 H, m), 4.74 (1 H, d, J = 4.2 Hz, 11.9 Hz), 4.63 (1H, d, J = 4.4 Hz, 11.9 Hz), 4.57 (1 H, dd, J = 4.2 Hz, 7.1 Hz), 3.05-3.00 (1 H, m), 81-2.77 (1 H, m), 2.44 (3 H, s), 2.42 (3 H, s), 1. 35 (9 H, s).

Next, from the compound 22 thus obtained, compound 23 (4-chloro-7- (2-deoxy-5-O-dimethoxytrityl-β-D-ribofuranosyl) -2-pivaloylaminopyrrolo [ 2,3-d] pyrimidine) was synthesized. That is, Compound 22 (182 mg, 0.30 mmol) is dissolved in dichloromethane (1.5 mL) and methanol (1.5 mL), sodium methoxide (162 mg, 3.0 mmol) is added at -10 ° C, and then ice is added. It stirred under cooling for 4 hours. After the reaction was stopped by adding 1 M hydrochloric acid, the reaction solution 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 is concentrated under reduced pressure, and the residue is azeotroped with pyridine, then dissolved in pyridine (3 mL), dimethoxytrityl chloride (122 mg, 0.36 mmol) is added under ice cooling, and the solution is cooled to room temperature 7 Stir for hours. Dimethoxytrityl chloride (31 mg, 0.090 mmol) was added and stirred for a further 16 hours. After the reaction was quenched by adding methanol, the reaction mixture was concentrated under reduced pressure, 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 = 3: 1-2: 1) to give compound 23 (92 mg, two steps 46%) ).
¹ H-NMR (CDCl ₃ ): δ 7.99 (1 H, brs), 7.43-7. 17 (9 H, m), 7.1 ¹ ( ¹ H, d, J = 3.7 Hz), 6.84 1 H, 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 (3 H, s), 3.42 (1 H, d, J = 3.4 Hz), 3.36-3.34 (1 H, m), 2.59-2.57 (1 H, m), 2 54-2.52 (1 H, m), 1.30 (9 H, s).

Next, from compound 23 thus obtained, compound 24 (7- (3-O-tert-butyldimethylsilyl-2-deoxy-β-D-ribofuranosyl) -4-chloro-2-pivaloyl is obtained. Aminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 23 (282 mg, 0.42 mmol) is dissolved in N, N-dimethylformamide (4 mL), imidazole (86 mg, 1.3 mmol) is added, and then tert-butyldimethylsilyl chloride (I After 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 the mixture was stirred for 17 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (8 mL), and a methanol solution (4 mL) of tosic acid monohydrate (160 mg, 0.84 mmol) is added over 10 minutes at -15.degree. The solution was added dropwise and stirred for 1 hour. Saturated aqueous sodium bicarbonate solution was added to stop the reaction, chloroform was 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 by silica gel chromatography (n-hexane / ethyl acetate = 6: 1 to 3: 1) to give compound 24 (189 mg, two steps 93%) ).
¹ H-NMR (CDCl ₃ ): δ 8.11 (1 H, brs), 7.20 (1 H, d, 3.7 Hz), 6.52 (1 H, d, J = 3.7 Hz), 6.24 ( 1 H, t, J = 6.7 Hz), 4.88-4.85 (1 H, m), 4.22-4. 20 (1 H, m), 4.03 (1 H, d, J = 3.5 Hz) ), 3.92 (1 H, dd, J = 2.1 Hz, 12.5 Hz), 3.80 (1 H, dd, J = 4.6 Hz, 12.5 Hz), 2.97-2.92 (1 H, 1 H, dd) m), 2.30-2.25 (1 H, m), 1. 34 (9 H, s), 0.92 (9 H, s), 0.13 (3 H, s), 0.12 (3 H, s) ).

Next, from compound 24 thus obtained, compound 25 (7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl) -4- 4 Chloro-2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 24 (418 mg, 0.87 mmol) is dissolved in dimethyl sulfoxide (5 mL) and toluene (3.5 mL), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (995 mg, 5.2 mmol) ), Pyridine (141 μL, 1.7 mmol) and trifluoroacetic acid (97 μL, 1.3 mmol) were added and stirred for 2 hours. The reaction was quenched with water and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in 1,4-dioxane (3 mL), 37% aqueous formaldehyde solution (495 μL, 6.1 mmol) under ice-cooling, 1 M aqueous solution of sodium hydroxide (910 μL) , 0.91 mmol) was added and stirred for 2 hours. Under ice-cooling, a solution of sodium borohydride (34 mg, 0.89 mmol) in ethanol (3 mL) was added and stirred for 10 minutes. Acetic acid was added to quench the reaction, extraction was performed 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 (1 H, brs), 7.19 (1 H, d, 3.7 Hz), 6.51 (1 H, d, J = 3.7 Hz), 6.31 (6 1H, dd, J = 5.2 Hz, 7.5 Hz), 3.87-3.64 (5 H, m), 3.09-3. 06 (1 H, m), 2.68 (1 H, brs), 2.52-2.46 (1 H, m), 1. 34 (9 H, s), 0.94 (9 H, s), 0.21 (3 H, s), 0.15 (3 H, s).

Next, compound 26 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4-C-hydroxymethyl) was obtained from compound 25 thus obtained. -Β-D-ribofuranosyl) -4-chloro-2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 25 (71 mg, 0.14 mmol) is dissolved in N, N-dimethylformamide (1.5 mL), triethylamine (39 μL, 0.28 mmol) is added, and 4,4′-dimethoxytrityl is cooled under ice cooling. Chloride (56 mg, 0.17 mmol) was added and the mixture was stirred for 2 hours, and 4,4′-dimethoxytrityl chloride (9 mg, 0.03 mmol) was further added and stirred for another 2 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution, partitioned with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in N, N-dimethylformamide (1.5 mL), imidazole (29 mg, 0.42 mmol) is added, and tert-butyldiphenylsilyl is added under ice cooling. Add chloride (55 μL, 0.21 mmol), stir at room temperature for 7 hours, add imidazole (29 mg, 0.42 mmol), tert-butyldiphenylsilyl chloride (55 μL, 0.21 mmol) and stir for 2 hours Further, imidazole (29 mg, 0.42 mmol) and tert-butyldiphenylsilyl chloride (55 μL, 0.21 mmol) were further added, and the mixture was stirred for 14.5 hours. The reaction was quenched with saturated aqueous sodium bicarbonate under ice cooling, extracted with ethyl acetate, the organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (3 mL), and a methanol solution (1.5 mL) of tosylate monohydrate (53 mg, 0.28 mmol) at -15 ° C. It dripped over 5 minutes and stirred for 5 minutes. Saturated aqueous sodium bicarbonate solution was added to terminate the reaction, and the mixture was partitioned. Ethyl acetate 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 = 9: 1) to give compound 26 (76 mg, 3 steps 72%).
¹ H-NMR (CDCl ₃ ): δ 8.00 (1 H, brs), 7.61-7.20 (11 H, m), 6.61 (1 H, dd, J = 4.9 Hz, 7.4 Hz), 6.36 (1 H, d, J = 3.7 Hz), 5.09 (1 H, dd, J = 6.3 Hz, 7.4 Hz), 3.92-3.77 (4 H, m), 2.70 -2.64 (1H, m), 2.58-2.51 (1 H, m), 1.26 (9 H, s), 1.05 (9 H, s), 0.92 (9 H, s), 0.13 (3 H, s), 0.12 (3 H, s).

Next, compound 27 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy-) was obtained from compound 26 thus obtained. β-D-ribofuranosyl) -4-chloro-2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 26 (113 mg, 0.50 mmol) is dissolved in dimethyl sulfoxide (1 mL) and toluene (0.5 mL), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (173 mg, 0.90 mmol) ), Pyridine (24 μL, 0.30 mmol) and trifluoroacetic acid (11 μL, 0.15 mmol) were added and stirred for 2 hours. The reaction was quenched with water and 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 solution was concentrated under reduced pressure, partitioned between 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. The reaction was quenched with water and 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 give compound 27 (86 mg, 3 steps 77%).
¹ H-NMR (CDCl ₃ ): δ 7.97 (1 H, brs), 7.60-7.14 (11 H, m), 6.46 (1 H, d, 3.7 Hz), 6.42 (1 H, 1 H, dd, J = 4.0 Hz, 8.2 Hz), 5.37 (1 H, t, J = 7.4 Hz), 4.07 (1 H, d, J = 11.5 Hz), 4.02 (1 H, d , J = 11.5 Hz), 2.83-2.80 (1 H, m), 2.63-2.59 (1 H, m), 1.20 (9 H, s), 1.04 (9 H, s) ), 0.93 (9 H, s), 0.14 (3 H, s), 0.09 (3 H, s).

Next, compound 28 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-) was obtained from compound 27 thus obtained. 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), 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 give compound 28 (53 mg, 68% over two steps).
¹ H-NMR (CDCl ₃ ): δ 7.80 (1 H, brs), 7.61-7.26 (10 H, m), 6.88 (1 H, d, J = 3.7 Hz), 6.56 (6 1 H, dd, J = 5.1 Hz, 7.6 Hz), 6.23 (1 H, t, J = 3.7 Hz), 5. 24 (2 H, brs), 5.11 (1 H, t, J = 6) .7 Hz), 3.98 (1 H, d, J = 11.3 Hz), 3.93 (1 H, d, J = 11.3 Hz), 2.72-2.68 (1 H, m), 2.56. −2.52 (1 H, m), 1.26 (9 H, s), 1.06 (9 H, s), 0.94 (9 H, s), 0.15 (3 H, s), 0.13 ( 3H, s).

Then, from compound 28 thus obtained, 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) are dissolved in chloroform (1 mL) and hydrazine monohydrate (2 mL), and the solution is heated at 60 ° C. for 19.5 hours It stirred. The reaction was quenched with water and extracted three times with chloroform, 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 give compound 29 (19 mg, 50%) .
¹ H-NMR (CDCl ₃ ): δ 7.66-7.34 ( ¹⁰ H, m), 6.68 (1 H, d, J = 3.7 Hz), 6.50 (1 H, t, J = 6.8 Hz ), 6.14 (1 H, d, J = 3.7 Hz), 4.98 (2 H, brs), 4.87 (1 H, dd, J = 5.2 Hz, 5.7 Hz), 4.45 (2 H) , Brs), 3.99 (1 H, d, J = 11.0 Hz), 3.86 (1 H, d, J = 11.0 Hz), 2.90-2.85 (1 H, m), 2.42 -2.37 (1 H, m), 1.08 (9 H, s), 0.96 (9 H, s), 0.18 (3 H, s), 0.16 (3 H, s).

Next, from the compound 29 thus obtained, the compound 30 (7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -2,4-diaminopyrrolo [2,3-d] is obtained. Pyrimidine) was synthesized. That is, Compound 29 (29 mg, 0.045 mmol) was dissolved in tetrahydrofuran (1 mL), a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (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 give compound 30 (11.5 mg, 88%).
¹ H-NMR (DMSO-d ₆ ): 6.89 (1 H, d, J = 3.7 Hz), 6.58 (2 H, brs), 6.51 (1 H, t, J = 7.1 Hz), 6.40 (1 H, d, J = 3.7 Hz), 6. 25 (1 H, d, J = 3.8 Hz), 5. 84 (1 H, t, J = 5.8 Hz), 5.64 (1 H , D, J = 5.8 Hz), 4.53 (1 H, d, J = 3.8 Hz), 3.70 (1 H, dd, J = 5.5 Hz, 11.7 Hz), 3.60 (1 H, 1 H, 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 in the following reaction step.

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-) was obtained. 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. The reaction was quenched with saturated aqueous ammonium chloride solution and concentrated under reduced pressure. It 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 8: 1) to give compound 31 (29 mg, 2 steps 33) %).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.86 (1 H, brs), 7.60-7.22 (10 H, m), 6.93 (1 H, d, J = 3.6 Hz), 6. 54 (1H, dd, J = 4.8 Hz, 7.7 Hz), 6.39 (1 H, d, J = 3.6 Hz), 5.20 (1 H, t, J = 6.9 Hz), 4.10 (3 H, s), 4.01 (1 H, d, J = 11.3 Hz), 3.96 (1 H, d, J = 11.3 Hz), 2.77-2.72 (1 H, m), 2 .59-2.53 (1 H, m), 1.27 (9 H, s), 1.05 (9 H, s), 0.94 (9 H, s), 0.15 (3 H, s), 0.. 12 (3H, s).

Next, compound 32 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy-) was obtained from compound 31 thus obtained. β-D-ribofuranosyl) -2-pivaloylamino-3H-pyrrolo [2,3-d] pyrimidin-4-one) was synthesized. That is, compound 31 (28 mg, 0.038 mmol) is dissolved in pyridine (0.5 mL), pyridine hydrochloride (13 mg, 0.11 mmol) is added, and the mixture is stirred at 90 ° C. for 2 hours to obtain pyridine hydrochloride (30 mg, 30 mg, 0.26 mmol) and pyridine (0.5 mL) were added and further stirred for 15 hours. The reaction solution was concentrated under reduced pressure, 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 column chromatography (n-hexane / ethyl acetate = 3: 1-1: 2) to give compound 32 (16 mg, 58%) .
¹ H-NMR (CDCl ₃ , 500 MHz); δ 11.82 (1 H, brs), 8.06 (1 H, brs), 7.64-7.26 (10 H, m), 6.73 (1 H, d, J = 3.6 Hz), 6.60-6.57 (2 H, m), 4. 80 (1 H, t, J = 5.9 Hz), 3.89-3. 86 (2 H, m), 49-2.45 (2H, m), 1.35 (9 H, s), 1.08 (9 H, s), 0.96 (9 H, s), 0.17 (3 H, s), 0.15 (3H, s).

Next, from compound 32 thus obtained, 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) are dissolved in 2-propanol (1 mL), hydrazine monohydrate (500 μL) is added, and the mixture is stirred at room temperature for 70 minutes. did. The reaction solution 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 to 1: 2) to give compound 33 (14 mg, quant).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 11.71 (1 H, brs), 7.67-7. 35 (10 H, m), 6.63 (1 H, d, J = 3.7 Hz), 6. 48 (1 H, t, J = 6.7 Hz), 6.39 (1 H, d, J = 3.7 Hz), 5. 15 (2 H, brs), 4.82 (1 H, t, J = 5.6 Hz ), 3.97 (1 H, d, J = 11.1 Hz), 3.86 (1 H, d, J = 11.1 Hz), 2.76-2.71 (1 H, 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).

Next, compound 34 (2-amino-7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) pyrrolo [2,3-d] pyrimidine-was obtained from compound 33 thus obtained. 4 (3H) -on) was synthesized. That is, Compound 33 (14 mg, 0.022 mmol) was dissolved in tetrahydrofuran (1 mL), a solution of tetrabutylammonium fluoride 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, and the residue was purified by silica gel column chromatography (methanol / chloroform = 5 to 7%), and crystallization was performed from acetonitrile and water to obtain compound 34 (3.9 mg, 61) %).
¹ H-NMR (DMSO-d ₆ , 500 MHz); δ 10.42 (1 H, brs), 6.93 (1 H, d, J = 3.7 Hz), 6.46 (1 H, t, J = 7.1 Hz ), 6.35 (2H, brs), 6.29 (1H, d, J = 3.7 Hz), 6.25 (1 H, d, J = 4.7 Hz), 5. 73 (1 H, t, J = 6.0 Hz), 4.51 (1 H, dd, J = 4.7 Hz, 5.7 Hz), 3.69 (1 H, dd, J = 6.0 Hz, 11.7 Hz), 3.60 (1 H, 1 H, 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 in the following reaction steps.

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-) was obtained. β-D-ribofuranosyl) -4-methylamino-2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 27 (46 mg, 0.062 mmol) was dissolved in 1,4-dioxane (1 mL), 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 give compound 35 (45 mg, 98%).
⁶ H-NMR (CDCl ₃ , 500 MHz); δ 7.91 (1 H, brs), 7.60-7. 25 (11 H, m), 6.83 (1 H, d, J = 3.7 Hz), 6. 55 (1 H, dd, J = 4.9 Hz, 7.7 Hz), 5.17-5.14 (2 H, m), 4.00 (1 H, d, J = 11.3 Hz), 3.94 (1 H) , D, J = 11.3 Hz), 3.17 (3 H, d, J = 4.7 Hz), 2.73-2.69 (1 H, m), 2.57-2.51 (1 H, m) , 1.26 (9H, s), 1.06 (9H, s), 0.94 (9H, s), 0.14 (3H, s), 0.12 (3H, s).

Next, compound 36 (2-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano) was obtained from compound 35 thus obtained. 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) are dissolved in 2-propanol (0.5 mL), hydrazine monohydrate (0.5 mL) is added, and the temperature is 50 ° C. After stirring for 1 hour, the temperature was raised to 70.degree. C. and stirring was further continued for 4.5 hours. The reaction solution 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 to 1: 1) to obtain Compound 36 (20 mg, 56%) .
¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.66-7.34 ( ¹⁰ H, m), 6.63 (1 H, d, J = 3.7 Hz), 6.52 (1 H, t, J = 6) .7 Hz), 6.16 (1 H, d, J = 3.7 Hz), 5.00 (1 H, brs), 4.86 (1 H, t, J = 5.3 Hz), 4.44 (2 H, brs) ) 3.99 (1 H, d, J = 11.0 Hz), 3.86 (1 H, d, J = 11.0 Hz), 3.07 (3 H, d, J = 4.9 Hz), 2.88- 2.85 (1 H, m), 2.41-2. 37 (1 H, m), 1.08 (9 H, s), 0.96 (9 H, s), 0.18 (3 H, s), 0 .15 (3H, s).

Next, compound 37 (2-amino-7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -4-methylaminopyrrolo [2,3] was obtained from compound 36 thus obtained. -D] pyrimidine was synthesized. That is, Compound 36 (20 mg, 0.030 mmol) was dissolved in tetrahydrofuran (1 mL), a tetrahydrofuran solution of tetrabutylammonium fluoride (67 μL, 0.067 mmol) was added, and the mixture was stirred at room temperature for 20 minutes. The reaction solution is concentrated under reduced pressure, and the residue is 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); δ 6.84 (1 H, d, J = 3.7 Hz), 6.47 (1 H, t, J = 7.0 Hz), 6.37 (1 H, d, J J = 3.7 Hz), 4.69 (1 H, dd, J = 4.4 Hz, 6.4 Hz), 3.89 (1 H, d, J = 1 2.0 Hz), 3.83 (1 H, d, J = 12.0 Hz), 2.98 (3 H, brs), 2.82-2.77 (1 H, m), 2.43-2.38 (1 H, m).

Synthesis Example 6: Synthesis of 2-amino-7- (2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -1,7-dihydropyrrolo [2,3-d] pyrimidin-4-one 2 -Amino-7- (2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -1,7-dihydropyrrolo [2,3-d] pyrimidin-4-one (compound 47), It synthesize | combined at 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 obtained. Was synthesized. That is, compound 22 (7.87 g, 13 mmol) is dissolved in dichloromethane (63 mL) and methanol (32 mL), 5 M sodium methoxide methanol solution (31 mL, 160 mmol) is added under ice-cooling, and stirred at room temperature for 10 minutes did. The reaction was quenched by the addition of a saturated aqueous ammonium chloride solution, and 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 give compound 38 (3.69 g, 78%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.06 (1 H, brs), 6.99 (1 H, d, J = 3.4 Hz), 6.44 (1 H, d, J = 3.4), 6.37 (1 H, dd, J = 8.1 Hz, 6.0 Hz), 5.08 (1 H, brs), 4.79 (1 H, s), 4.12 (3 H, s), 3. 91 ( 1H, d, J = 12.2 Hz), 3.79 (1 H, d, J = 11.1 Hz), 2.90-2.84 (1 H, m), 2.35-2.31 (1 H, m) ) 1.34 (9 H, s).

Then, from compound 38 thus obtained, compound 39 (7- (5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl) -4-methoxy-2-pivaloyl is obtained. Aminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, 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. . The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and 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 give compound 39 (150 mg, 91%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.99 (1 H, brs), 7.64-7.30 (10 H, m), 7.12 (1 H, s), 6. 80 (1 H, s) , 6.40 (1 H, d, J = 2.9 Hz), 4. 74 (1 H, s), 4.09 (3 H, s), 4.07 (1 H, s), 3.86 (2 H, dd) , J = 4.7 Hz, 4.3 Hz), 2.56-2.51 (2 H, m), 1.33 (9 H, s), 1.08 (9 H, s).

Next, from the compound 39 thus obtained, the compound 40 (7- (3-O-benzyl-5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl) -4-methoxy is obtained. 2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 39 (491 mg, 0.82 mmol) is dissolved in 1,4-dioxane (8 mL), and molecular sieve 5 Å (491 mg), 2,4,6-tris (benzyloxy) -1,3,5-triazine After adding (163 mg, 0.41 mmol) and stirring at room temperature for 30 minutes, trifluoromethanesulfonic acid (72 μL, 0.82 mmol) was added and stirred for 24 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and 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) to obtain Compound 40 (361 mg, 64%).
⁶ H-NMR (CDCl ₃ , 500 MHz); δ 7.94 (1 H, brs), 7.61-7.20 (10 H, m), 7.08 (1 H, d, J = 3.7 Hz), 67 (1 H, m), 6.39 (1 H, d, J = 3.7 Hz), 4.58 (2 H, d, J = 4.3 Hz), 4.45-4. 43 (1 H, m), 4.19-4.17 (1 H, m) 4.12 (3 H, s), 3.83-3. 78 (2 H, m), 2.56-2.44 (1 H, m), 1.34 (9 H, s), 1.07 (9 H, s).

Next, from compound 40 thus obtained, compound 41 (7- (3-O-benzyl-2-deoxy-β-D-ribofuranosyl) -4-methoxy-2-pivaloylaminopyrrolo [2 , 3-d] pyrimidine) was synthesized. That is, compound 40 (1.3 g, 1.9 mmol) is dissolved in tetrahydrofuran (19 mL), a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (2.1 mL, 2.1 mmol) is added, and the reaction is continued for 3.5 hours at room temperature. It stirred. The reaction was quenched with saturated aqueous ammonium chloride solution and 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 to 1: 1) to obtain Compound 41 (714 mg, 84%) .
⁶ H-NMR (CDCl ₃ , 500 MHz); δ 8.03 (1 H, brs), 7.38-7. 31 (10 H, m), 6.93 (1 H, d, J = 3.6 Hz), 6. 43 (1 H, d, J = 3.7 Hz), 5.88 (1 H, brs), 6.39 (1 H, d, J = 3.7 Hz), 4.58 (2 H, d, J = 4.3 Hz ), 4.45-4.43 (1H, m), 4.19-4.17 (1H, m) 4.12 (3H, s), 3.98 (1 H, dd, J = 13.4 Hz, 1.6 Hz), 3.76 (1 H, dd, J = 10.7 Hz, 10.8), 2.99-2.94 (1 H, m), 2.43-2.40 (1 H, s), 1.34 (9 H, s).

Then, from compound 41 thus obtained, compound 42 (7- (3-O-benzyl-2-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl) -4-methoxy-pivalo Ylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 41 (714 mg, 1.6 mmol) is 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 the mixture was stirred at room temperature for 2.5 hours. The reaction was quenched by the addition of water and extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in 1,4-dioxane (16 mL), 37% aqueous formaldehyde solution (585 μL, 3.1 mmol) under ice-cooling, 1 M aqueous sodium hydroxide solution (1 .7 mL, 1.7 mmol) was added and stirred for 1 hour. The reaction was quenched with saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in ethanol (7.7 mL), sodium borohydride (119 mg, 3.1 mmol) is dissolved in ethanol (8.0 mL) and cooled with ice. The solution was added dropwise and stirred at room temperature for 10 minutes. The reaction was quenched with saturated aqueous ammonium chloride solution and 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 to 1: 1 to 1: 2) to give compound 42 (525 mg) , 3 steps 68%).
⁶ H-NMR (CDCl ₃ , 500 MHz); δ 8.02 (1 H, brs), 7.40-7.33 (10 H, m), 6.94 (1 H, d, J = 3.6 Hz), 6. 43 (1H, d, J = 3.6 Hz), 6.23 (1 H, dd, J = 7.9 Hz, 6.4 Hz), 5.39 (1 H, brs), 4.87 (2 H, dd, J = 6.4 Hz, 2.7 Hz), 4.72 (1 H, d, J = 11.6 Hz), 4.57 (1 H, d, J = 11.6 Hz), 4.13 (3 H, s), 3 .81-3.74 (2H, m), 3.10-3.05 (1 H, m), 2.62 (1 H, brs), 2.55-2.51 (1 H, m), 1.35 (9H, s).

Next, from compound 42 thus obtained, compound 43 (7- (3,5-di-O-benzyl-2-deoxy-4-C-hydroxymethyl-β-D-ribofuranosyl) -4-) was obtained. Methoxy-2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 42 (30 mg, 0.061 mmol) is dissolved in 1,4-dioxane (0.6 mL), and molecular sieve 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. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and 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 = 5: 1) to give compound 43 (7.4 mg, 21%).
⁶ H-NMR (CDCl ₃ , 500 MHz); δ 8.04 (1 H, s), 7.34-7. 27 (10 H, m), 6.92 (1 H, d, J = 3.6 Hz), 6. 41 (1H, d, J = 3.6 Hz), 6.17 (1 H, dd, J = 8.6 Hz, 6.0 Hz), 5.66 (1 H, d, J = 7.4 Hz), 4.66 -4.52 (5 H, m) 4.14 (3 H, s), 3.99 (1 H, d, J = 11.8 Hz), 3.84 (1 H, d, J = 11.9 Hz), 3. 76 (1 H, d, J = 9.8 Hz), 3.68 (1 H, d, J = 9.8 Hz), 3.05-2.99 (1 H, m), 2.48-2.44 (1 H , M), 1.35 (9 H, s).

Next, Compound 44 (7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -4-) was obtained from Compound 43 thus obtained. 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) is added, and the mixture is stirred at -78 ° C for 1 hour Then, the temperature was raised to 0 ° C. and stirred for 1 hour, and 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 give compound 44 (5.1 mg, 49%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.93 (1 H, s), 7.47-7. 28 (10 H, m), 7.13 (1 H, J = 3.7 Hz), 6.64 1H, t, J = 6.9 Hz), 6.46 (1 H, d, J = 3.7 Hz), 4.77-4. 53 (7 H, m), 4.09 (3 H, s), 3. 81 (1 H, d, J = 2.4 Hz), 3.73 (1 H, d, J = 1.6 Hz), 2.77-2.71 (1 H, m), 2.64-2.59 (1 H , M), 1.36 (9 H, s).

Next, Compound 45 (7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -2-) was obtained from Compound 44 thus obtained. 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), then pyridine hydrochloride (7.0 mg, 0.060 mmol) was added, and the mixture was stirred at 80 ° C. for 24 hours. The reaction was quenched with saturated aqueous ammonium chloride solution and 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 give compound 45 (10.7 mg, 94%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 11.71 (1 H, s), 7.97 (1 H, s), 7. 38-7. 29 (10 H, m), 6.99 (1 H, d, J = 3.7 Hz), 6.67 (1 H, d, J = 3.6 Hz), 6.53 (1 H, dd, J = 7.3 Hz, 7.1 Hz), 4.74-4. 49 (6 H) , M), 4.39 (1 H, dd, J = 5.2 Hz, 2.6 Hz), 3.80 (1 H, d, J = 2.7 Hz), 3.69 (1 H, d, J = 1. 5 Hz), 2.54-2.48 (2 H, m), 1.31 (9 H, s).

Then, from compound 45 thus obtained, 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 aqueous sodium hydroxide solution (0.2 mL) and methanol (0.2 mL), and stirred at 80 ° C. for 80 minutes. The reaction was quenched with saturated aqueous ammonium chloride solution, and 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 give compound 46 (6.2 mg, 70%).
6. ¹ H-NMR (CDCl ₃ , 500 MHz); δ 11.84 (1 H, s), 7. 36-7. 27 (10 H, m), 6. 84 (1 H, d, J = 3.7 Hz), 51 (1 H, dd, J = 7.1 Hz, 7.1 Hz), 6.48 (1 H, d, J = 3.7 Hz), 5.26 (2 H, s), 4.73-4. 50 (6 H , M), 4.40 (1 H, 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 (2 H, m).

Next, compound 47 (2-amino-7- (2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -3 H-pyrrolo [2,3-] was obtained from compound 46 thus obtained. d] Pyrimidin-4-one was synthesized. That is, Compound 46 (6.2 mg, 0.013 mmol) is dissolved in dichloromethane (0.3 mL), and then boron trichloride (1.0 M solution in dichloromethane, 130 μL, 0.13 mmol) is added at -78 ° C, Stir at 0 ° C. for 10 minutes. After the reaction is stopped by adding methanol, the reaction solution is concentrated under reduced pressure, and the residue is 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); δ 6.88 (1 H, d, J = 3.8 Hz), 6.47 (1 H, t, J = 6.9 Hz), 6.41 (1 H, d, J J = 3.6 Hz), 4.58-4.57 (1 H, m), 4.56 (1 H, dd, J = 43.9 Hz, 9.8 Hz), 4.47 (1 H, dd, J = 43) .5 Hz, 9.8 Hz), 3.75 to 3.73 (2 H, m), 2.60 to 2.57 (1 H, m), 2.35 to 2. 32 (1 H, 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 in 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-) was used. (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), 4-dimethylaminopyridine (7.4 mg, 0.061 mmol) were sequentially added, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, and 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 give compound 48 (44.9 mg, 75%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.16 (1 H, s), 7.56 (1 H, d, J = 4.6 Hz), 7.34 (1 H, d, J = 3.7 Hz), 6.65 (1H, t, J = 7.0 Hz), 6.60 (1 H, d, J = 3.6 Hz), 6.28 (1 H, d, J = 5.1 Hz), 5.82 (1 H) , T, J = 6.1 Hz), 4.61 (1 H, dd, J = 4.8 Hz, 11.0 Hz), 3.76 (1 H, dd, J = 5.9 Hz, 11.7 Hz), 3. 62 (1 H, dd, J = 6.3 Hz, 11.7 Hz), 2.95 (3 H, d, J = 4.6 Hz), 2.81-2.75 (1 H, m), 2.39-2 .34 (1 H, m).

Next, 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) was 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); δ 7.71 (1 H, s), 7.34-7.20 (11 H, m), 6.58 (1 H, t, J = 6.8 Hz), 6. 41 (1 H, d, J = 3.8 Hz), 4.72-4. 55 (7 H, m), 4.24-4. 11 (2 H, m), 3.80-3.71 (2 H, m) ), 1.28 to 1.23 (27H, m).

Next, from the compound 49 thus obtained, the compound 50 (2,4-diamino-7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl-β-D) -Ribofuranosyl) -pyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 49 (15.7 mg, 0.028 mmol) was dissolved in 1 M aqueous sodium hydroxide solution (1 mL) and methanol (1 mL), and stirred at 90 ° C. for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution, and 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 give compound 50 (4.5 mg, 70%).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 7.36-7.27 ( ¹⁰ H, m), 6.92 (1 H, d, J = 3.8 Hz), 6.55 (1 H, t, J = 7 .1 Hz), 6.25 (1 H, d, J = 3.8 Hz), 5. 70 (2 H, brs), 4. 94 (2 H, brs), 4.73 to 4.51 (6 H, m), 4.40 (1 H, t, J = 4.4 Hz), 3.80 (1 H, dd, J = 10.2 Hz, 2.5 Hz), 3.69 (1 H, dd, J = 10.1 Hz, 1. 7 Hz), 2.55-2.52 (2 H, m).

Next, Compound 51 (2,4-diamino-7- (2-deoxy-4-C-fluoromethyl-β-D-ribofuranosyl) -pyrrolo [2,3-] was obtained from compound 50 thus obtained. d) pyrimidine was synthesized. That is, Compound 50 (4.5 mg, 9.4 μmol) is dissolved in dichloromethane (0.1 mL), and then boron trichloride (1.0 M solution in dichloromethane, 94 μL, 0.09 mmol) is added at -78 ° C, Stir at 0 ° C. for 30 minutes. After the reaction is stopped by adding methanol, the reaction solution is concentrated under reduced pressure, and the residue is 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); δ 6.93 (1 H, d, J = 3.8 Hz), 6.52 (1 H, t, J = 6.8 Hz), 6.43 (1 H, d, J J = 3.8 Hz), 4.61-4.60 (1 H, m), 4.58 (1 H, dd, J = 51.0 Hz, 9.7 Hz), 4.49 (1 H, dd, J = 51) .7 Hz, 9.7 Hz), 3.78 (2 H, dd, J = 12.2 Hz, 2.5 Hz), 2.66-2.60 (1 H, m), 2.38-23.3 (1 H, 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 in the following reaction steps.

First, compound 53 (4-benzylamino-7- (2-deoxy-β-D-ribofuranosyl) -5-iodopyrrolo [2,3-d] pyrimidine) was prepared from compound 52 (Eur. Pat. Appl., 710667). Was synthesized. That is, compound 52 (2.27 g, 6.0 mmol) is dissolved in pyridine (15 mL), chlorotrimethylsilane (7.66 mL, 60 mmol) is added under ice-cooling, and the mixture is 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. The reaction mixture was concentrated under reduced pressure and crystallized from methanol and water to give compound 53 (1.83 g, 63%).
¹ H-NMR (DMSO-d ₆ ): δ 8.69 (1 H, s), 8.10-7.54 (6 H, m), 6.66 (1 H, t, J = 7.0 Hz); 33 (1 H, d, J = 4.1 Hz), 5.00 (1 H, t, J = 5.5 Hz), 4.38 (1 H, s), 3.86 (1 H, d, J = 2.6 Hz ), 3.65-3.52 (2H, m), 2.56-2.54 (1 H, m), 2.28-2.26 (1 H, m).

Next, from the compound 53 thus obtained, compound 54 (4-benzylamino-7- (2-deoxy-5-O-dimethoxytrityl-β-D-ribofuranosyl) -5-iodopyrrolo [2, 3 -D] pyrimidine was synthesized. That is, Compound 53 (1.79 g, 3.7 mmol) was azeotroped with pyridine and then dissolved in pyridine (20 mL) to give 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 stirred at room temperature for 17 hours. After the reaction was quenched by adding saturated aqueous sodium bicarbonate solution, the reaction solution 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 give compound 54 (2.88 g, 99%) .
¹ H-NMR (CDCl ₃ ): δ 9.34 (1 H, brs), 8.75 (1 H, s), 8.09-6.83 (19 H, m), 6.76 (1 H, t, J = 6.5 Hz), 4.67 (1 H, dd, J = 2.5 Hz, 5.8 Hz), 4.13-4.10 (1 H, m), 3.79 (6 H, s), 3.46- 3.38 (2H, m), 2.66-2.62 (1 H, m), 2.55 to 2.51 (1 H, m).

Next, from compound 54 thus obtained, compound 55 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy-β-D-ribofuranosyl) -5-iodopyrrolo [ 2,3-d] pyrimidine) was synthesized. That is, Compound 54 (2.79 g, 3.6 mmol) is dissolved in N, N-dimethylformamide (35 mL), imidazole (969 mg, 14 mmol) is added, and then tert-butyldimethylsilyl chloride (I 1.07 g (7.1 mmol) was added and stirred at room temperature for 21 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (40 mL) and methanol solution (80 mL) of tosic acid monohydrate (1.36 g, 7.1 mmol) at -15 ° C. It was added dropwise over a minute and stirred for 1.5 hours. Saturated aqueous sodium bicarbonate solution was added to terminate the reaction, and the reaction solution was partitioned, 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 chromatography (n-hexane / ethyl acetate = 3: 1-1: 1) to give compound 55 (1.99 g, 2 steps) 94%).
¹ H-NMR (CDCl ₃ ): δ 9.39 (1 H, brs), 8.75 (1 H, s), 8.09-7.53 (5 H, m), 7.40 (1 H, s), 6 4.27 (1 H, 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 = 3.7 Hz, 7.3 Hz), 3.95 (1 H, d, J = 12.6 Hz), 3.78-3.73 (1 H, 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).

Next, compound 56 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-β-D-) was obtained from compound 55 thus obtained. Ribofuranosyl) -5-iodopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 55 (115 mg, 0.19 mmol) is dissolved in dimethyl sulfoxide (1.2 mL) and toluene (0.7 mL), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is cooled with ice cooling. The 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 the mixture was stirred at room temperature for 2 hours. The reaction was quenched with water and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in 1,4-dioxane (2 mL), 37% aqueous formaldehyde solution (108 μL, 1.3 mmol) under ice-cooling, 1 M aqueous solution of sodium hydroxide (200 μL) (0.20 mmol) was added and stirred for 7.5 hours. Under ice-cooling, sodium borohydride (22 mg, 0.57 mmol) was added and it stirred for 1 hour. Acetic acid was added to quench the reaction, extraction was performed 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 1: 1) to obtain Compound 56 (85 mg, two steps 72) %).
¹ H-NMR (CDCl ₃ ): δ 9.39 (1 H, brs), 8.75 (1 H, s), 8.09-7.53 (5 H, m), 7. 39 (1 H, s), 6 .36 (1 H, 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).

Next, compound 57 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4) was obtained from compound 56 thus obtained. -C-hydroxymethyl-β-D-ribofuranosyl) -5-iodopyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 56 (85 mg, 0.14 mmol) is dissolved in N, N-dimethylformamide (1.4 mL), triethylamine (38 μL, 0.27 mmol) is added, and 4,4′-dimethoxytrityl is cooled under ice cooling. Add chloride (55 mg, 0.16 mmol) and stir at room temperature for 2 hours, add triethylamine (19 μL, 0.14 mmol) and 4,4'-dimethoxytrityl chloride (14 mg, 0.040 mmol) and stir for another 2 hours did. Ice was added to stop the reaction, ethyl acetate and saturated aqueous sodium bicarbonate were added for partitioning, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in N, N-dimethylformamide (1.4 mL), and imidazole (37 mg, 0.54 mmol), tert-butyldiphenylsilyl chloride (I 71 μL, 0.27 mmol) was added and stirred at room temperature for 17 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (3 mL) and methanol solution (1.5 mL) of tosylate monohydrate (52 mg, 0.27 mmol) at -15 ° C. It was added dropwise over a minute and stirred for 1 hour. The reaction was quenched with saturated aqueous sodium bicarbonate solution, extracted with chloroform, 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 by silica gel chromatography (n-hexane / ethyl acetate = 3: 1-1: 1) to give compound 57 (65 mg, 3 steps 55%) ).
¹ H-NMR (CDCl ₃ ): δ 9.35 (1 H, brs), 8.73 (1 H, s), 8.09-7. 35 (16 H, m), 6.74 (1 H, t, J = 6.6 Hz), 4.81 (1 H, d, J = 3.5 Hz, 6.0 Hz), 3.87-3. 72 (4 H, m) 2.7 1-2. 65 (1 H, 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).

Next, Compound 58 (4-benzylamino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano) was obtained from compound 57 thus obtained. 2-Deoxy-β-D-ribofuranosyl) -5-iodopyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 57 (63 mg, 0.073 mmol) is dissolved in dimethyl sulfoxide (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. The reaction was quenched with water and 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 solution was concentrated under reduced pressure, partitioned between ethyl acetate and water, the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in dichloromethane (1 mL), triethylamine (20.3 μL, 0.15 mmol) and mesyl chloride (8.5 μL, 0.11 mmol) are added and the mixture is ice-cooled. The mixture was stirred for 2.5 hours. The reaction was quenched with water and 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 (1 H, brs), 8.67 (1 H, s), 8.08-7. 35 (116 H, m), 6.67 (1 H, t, J = 6.6 Hz), 4.89 (1 H, t, J = 5.5 Hz), 4.06 (1 H, d, J = 11.2 Hz), 3.88 (1 H, d, J = 11.2 Hz), 2.93-2.87 (1 H, m), 2.55-2. 50 (1 H, m), 1.09 (9 H, s), 0.96 (9 H, s), 0.16 (3 H, 3) s), 0.15 (3 H, s).

Next, from the compound 58 thus obtained, 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. That is, 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 give compound 59 (36 mg, 94%).
¹ H-NMR (CDCl ₃ ): δ 8.15 (1 H, s), 7.64-7. 34 (10 H, m), 7.1 ¹ ( ¹ H, s), 6.59 (1 H, t, J = 6.5 Hz), 5.70 (2H, brs), 4.89 (1 H, t, J = 5.8 Hz), 4.02 (1 H, d, J = 11.2 Hz), 3.85 (1 H, 1 H, d, J = 11.2 Hz), 2.87-2.81 (1 H, m), 2.50-2.45 (1 H, m), 1.08 (9 H, s), 0.94 (9 H, 9 H, m) s), 0.15 (3H, s), 0.14 (3H, s).

Next, compound 60 (4-amino-7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -5-iodopyrrolo [2,3-d] was obtained from compound 59 thus obtained. Pyrimidine) was synthesized. Compound 59 (36 mg, 0.048 mmol) was dissolved in tetrahydrofuran (1 mL), a 1 M solution of tetrabutylammonium fluoride 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 give compound 60 (19 mg, 97%).
¹ H-NMR (DMSO-d ₆ ): δ 8.13 (1 H, s), 7.67 (1 H, s), 6.71 (2 H, brs), 6.63 (1 H, t, J = 6. 9 Hz), 6.28 (1 H, d, J = 5.0 Hz), 5. 76 (1 H, t, J = 6.1 Hz), 4.59 (1 H, dd, J = 5.0 Hz, 10.9 Hz ), 3.75 (1 H, dd, J = 5.9 Hz, 11.8 Hz), 3.62 (1 H, dd, J = 6.1 Hz, 11.8 Hz), 2.77-2.72 (1 H, 1 H, m), 2.38-2.33 (1 H, 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 in the following reaction step.

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 is obtained. Was synthesized. That is, compound 61 (383 mg, 1.4 mmol) is dissolved in pyridine (4 mL), chlorotrimethylsilane (1.81 mL, 14 mmol) is added under ice-cooling, and the mixture is stirred at room temperature for 2.5 hours, Chloride (174 μL, 1.5 mmol) was added and stirred for 4.5 hours. After water was added under ice-cooling and the mixture was stirred for 15 minutes, aqueous ammonia (3 mL) was added and the mixture was further stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure and crystallized from methanol and water to give Compound 62 (436 mg, 82%).
¹ H-NMR (DMSO-d6): δ 11.24 (1 H, brs), 8.67 (1 H, s), 8.05 to 7.54 (6 H, m), 6.73 (1 H, t, J = 6.5 Hz), 5.33 (1 H, d, J = 4.1 Hz), 4.98 (1 H, t, J = 5.5 Hz), 4. 36 (1 H, 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).

Next, from the compound 62 thus obtained, compound 63 (4-benzylamino-7- (2-deoxy-5-O-dimethoxytrityl-β-D-ribofuranosyl) -5-fluoropyrrolo [2, 3-d] pyrimidine was synthesized. That is, Compound 62 (421 mg, 1.1 mmol) was azeotroped with pyridine and then dissolved in pyridine (26 mL), and a solution of 4,4'-dimethoxytrityl chloride (460 mg, 1.4 mmol) in pyridine (6 mL) was ice-cooled. The solution was added dropwise over 15 minutes under cooling, and stirred at room temperature for 24 hours. After the reaction was quenched by adding saturated aqueous sodium bicarbonate solution, the reaction solution 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 give compound 63 (762 mg, quant.).
¹ H-NMR (CDCl ₃ ): δ 8.62 to 6.82 (21 H, m), 4.60 (1 H, d, J = 5.1 Hz), 4.12 (1 H, dd, J = 4.2 Hz , 8.1 Hz), 3.79 (6 H, s), 3.43-3. 35 (2 H, m), 2.57-2.44 (2 H, m).

Next, from compound 63 thus obtained, compound 64 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy-β-D-ribofuranosyl) -5-fluoropyrrolo is obtained. [2,3-d] pyrimidine) was synthesized. That is, Compound 63 (744 mg, 1.1 mmol) is dissolved in N, N-dimethylformamide (10 mL), imidazole (300 mg, 4.4 mmol) is added, and then tert-butyldimethylsilyl chloride (ice-cold) 332 mg (2.2 mmol) was added and stirred at room temperature for 5.5 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (12 mL), and a methanol solution (24 mL) of tosylate monohydrate (837 g, 4.4 mmol) is added over 5 minutes at -15 ° C. It was added dropwise and stirred for 30 minutes. Saturated aqueous sodium bicarbonate solution was added to terminate the reaction, and the reaction solution was partitioned, 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 chromatography (n-hexane / ethyl acetate = 3: 1-1: 1) to give compound 64 (504 mg, two steps 94%) ).
¹ H-NMR (CDCl ₃ ): δ, 8.63 (1 H, s), 8.61 (1 H, s), 7.99-7. 06 (6 H, m), 6.23 (1 H, dd, J = 5.7 Hz, 9.0 Hz), 5.21 (1 H, d, J = 9.4 Hz), 4.67 (1 H, d, J = 5.3 Hz), 4.11 (1 H, d, J = 1.5 Hz), 3.94 (1 H, d, J = 12.6 Hz), 3.76 to 3.71 (1 H, m), 2.99 to 2.94 (1 H, m), 2.25 -2.21 (1 H, m), 0.95 (9 H, s), 0.12 (6 H, s).

Next, compound 65 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-β-D-) was obtained from compound 64 thus obtained. Ribofuranosyl) -5-fluoropyrrolo [2,3-d] pyrimidine) was synthesized. That is, compound 64 (470 mg, 0.97 mmol) is dissolved in dimethyl sulfoxide (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. The reaction was quenched with ice and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in 1,4-dioxane (3 mL), 37% aqueous formaldehyde solution (552 μL, 6.8 mmol) under ice-cooling, 1 M aqueous sodium hydroxide solution (1 .94 mL, 1.9 mmol) was added and stirred for 6.5 hours. Under ice cooling, sodium borohydride (110 mg, 2.9 mmol) was added and the mixture was stirred for 1 hour. Acetic acid was added to quench the reaction, extraction was performed 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 1: 1) to obtain compound 65 (310 mg, two steps 62) %).
¹ H-NMR (CDCl ₃ ): δ 8.74 (1 H, brs), 8.59 (1 H, brs), 7.99-7.52 (5 H, m), 7.04 (1 H, d, J = 2.4 Hz), 6.35 (1 H, t, J = 7.1 Hz), 4.99 (1 H, brs), 4.89 (1 H, 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 (1 H, brs), 2.39- 2.35 (1 H, m), 0.95 (9 H, s), 0.17 (3 H, s), 0.16 (3 H, s).

Next, compound 66 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4) was obtained from compound 65 thus obtained. -C-hydroxymethyl-β-D-ribofuranosyl) -5-fluoropyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 65 (305 mg, 0.59 mmol) is dissolved in N, N-dimethylformamide (3 mL), triethylamine (164 μL, 1.2 mmol) is added, and 4,4′-dimethoxytrityl chloride (ice-cooled) A solution of 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 An additional (60 mg, 0.18 mmol) was added and stirred for additional 16 hours. Saturated aqueous sodium bicarbonate solution was added to stop the reaction, ethyl acetate and saturated aqueous sodium bicarbonate solution were added for partitioning, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in N, N-dimethylformamide (6 mL), imidazole (161 mg, 2.4 mmol) under ice-cooling, tert-butyldiphenylsilyl chloride (307 μL, 1.2 mmol) was added and stirred at room temperature for 12 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in chloroform (12 mL), and a methanol solution (6 mL) of tosic acid monohydrate (224 mg, 1.2 mmol) at -15 ° C is added over 5 minutes The mixture was dropped and stirred for 1 hour. The reaction was quenched with saturated aqueous sodium bicarbonate solution, extracted with chloroform, 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 by silica gel chromatography (n-hexane / ethyl acetate = 3: 1-1: 1) to give compound 66 (311 mg, three steps 70%) ).
¹ H-NMR (CDCl ₃ ): δ 8.87 (1 H, brs), 8.56 (1 H, s), 7.97-7. 35 (15 H, m), 7.08 (1 H, d, J = 6.4 Hz), 6.80 (1 H, t, J = 6.7 Hz), 4. 80 (1 H, d, J = 4.0 Hz, 6.3 Hz), 3.89-3. 72 (4 H, m) 2.60-2.54 (1 H, m), 2.45-2.40 (2 H, m), 1.10 (9 H, s), 0.93 (9 H, s), 0.13 (3 H) , S), 0.11 (3H, s).

Next, from the compound 66 thus obtained, 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) is dissolved in dimethyl sulfoxide (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. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate and 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. The reaction was quenched with water and extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under reduced pressure, the residue is dissolved in dichloromethane (4 mL), triethylamine (100 μL, 0.72 mmol) and mesyl chloride (42 μL, 0.54 mmol) are added and the mixture is ice-cooled 2.5. Stir for hours. The reaction was quenched with water and 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 to 3: 1) to obtain Compound 67 (213 mg, 3 steps 79) %).
¹ H-NMR (CDCl ₃ ): 8.57 ( ¹ H, s), 8.5 ¹ ( ¹ H, s), 7.98-7. 35 (15 H, m), 6.98 (1 H, d, J = 2.1 Hz), 6.74 (1 H, t, J = 6.5 Hz), 4.87 (1 H, t, J = 5.7 Hz), 4.00 (1 H, d, J = 11.2 Hz), 3.87 (1 H, d, J = 11.2 Hz), 2.81-2. 76 (1 H, m), 2.54-2.49 (1 H, m), 1.08 (9 H, s), 0.96 (9 H, s), 0.17 (3 H, s), 0.15 (3 H, s).

Next, compound 68 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-) was obtained from compound 67 thus obtained. 2-Deoxy-β-D-ribofuranosyl) -5-fluoropyrrolo [2,3-d] pyrimidine) was synthesized. That is, 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 give compound 68 (47 mg, 73%).
¹ H-NMR (CDCl ₃ ): δ 8.18 (1 H, s), 7.65-7.34 (10 H, m), 6. 70 (1 H, s), 6.63 (1 H, t, J = 6.4 Hz), 5.44 (2 H, brs), 4.89 (1 H, t, J = 5.8 Hz), 3.98 (1 H, d, J = 11.2 Hz), 3.84 (1 H, 1 H, d, J = 11.2 Hz), 2.83-2.78 (1 H, m), 2.50-2.45 (1 H, m), 1.07 (9 H, s), 0.95 (9 H, m) s), 0.16 (3 H, s), 0.15 (3 H, s).

Next, compound 69 (4-amino-7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -5-fluoropyrrolo [2,3-] was obtained from compound 68 thus obtained. d) pyrimidine was synthesized. Compound 68 (45 mg, 0.070 mmol) was dissolved in tetrahydrofuran (1 mL), a 1 M solution of tetrabutylammonium fluoride 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 give compound 69 (17 mg, 83%).
¹ H-NMR (DMSO-d6): δ 8.10 (1 H, s), 7.37 (1 H, d, J = 1.7 Hz), 7.08 (2 H, brs), 6.69 (1 H, dd) , J = 6.2 Hz, 6.6 Hz), 6.30 (1 H, d, J = 5.1 Hz), 5.76 (1 H, t, J = 6.1 Hz), 4.56 (1 H, dd, J = 5.0 Hz, 10.9 Hz), 3.73 (1 H, dd, J = 6.0 Hz, 11.8 Hz), 3.61 (1 H, dd, J = 6.2 Hz, 11.8 Hz), 2 70-2.65 (1 H, m), 2.38-2.33 (1 H, 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 in the following reaction steps.

First, compound 70 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2) was obtained from compound 16 obtained in Synthesis Example 2. -Deoxy-β-D-ribofuranosyl) -5-chloropyrrolo [2,3-d] pyrimidine) was synthesized. That is, Compound 16 (24 mg, 0.038 mmol) is dissolved in N, N-dimethylformamide (0.5 mL), and a solution of N-chlorosuccinimide (5.1 mg, 0.038 mmol) in N, N-dimethylformamide (0%) .5 mL) was added dropwise under ice-cooling and stirred for 1 hour, and then stirred at room temperature for 2 hours. Methanol was added to quench the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane: ethyl acetate = 3: 1-1: 1) to give compound 70 (7 .4 mg, 29%).
¹ H-NMR (CDCl ₃ ): δ 8.16 (1 H, s), 7.64-7. 34 (10 H, m), 6.95 (1 H, s), 6.60 (1 H, dt, J = 6.5 Hz), 5.68 (1 H, brs), 4.89 (1 H, t, J = 5.9 Hz), 4.00 (1 H, d, J = 11.2 Hz), 3.85 (1 H, d, J = 11.2 Hz), 2.85-2.81 (1 H, m), 2.52-2.48 (1 H, m).

Next, from compound 70 thus obtained, compound 71 (4-amino-7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -5-chloropyrrolo [2,3-d] is obtained. Pyrimidine) was synthesized. Compound 70 (7.4 mg, 0.011 mmol) was dissolved in tetrahydrofuran (0.5 mL), a 1 M solution of tetrabutylammonium fluoride 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 give compound 71 (3.5 mg, quant.).
¹ H-NMR (DMSO-d6): δ 8.11 (1 H, s), 7.40 (1 H, s), 6.68 (1 H, d, J = 6.7 Hz), 4.73 (1 H, dd) , J = 5.4 Hz, 6.5 Hz), 3.88 (1 H, d, J = 12.0 Hz), 3.83 (1 H, d, J = 12.0 Hz), 2.79-2.74 (2. 1 H, m), 2.53-2.48 (1 H, 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 in the following reaction steps.

Compound 59 (15 mg, 0.020 mmol) obtained in Synthesis Example 8, Pd (PPh ₃ ) ₂ Cl ₂ (0.70 mg, 0.0010 mmol), copper iodide (0.40 mg, 0.0020 mmol), 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. The reaction mixture was quenched with saturated aqueous sodium bicarbonate and 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), a 1 M solution of tetrabutylammonium fluoride 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, and the residue is purified by silica gel chromatography (methanol / chloroform = 0-4%) to give compound 72 (4-amino-7- (4-C-cyano-2-deoxy-β). -D-ribofuranosyl) -5-ethynylpyrrolo [2,3-d] pyrimidine) was obtained (5.9 mg, two steps 99%).
¹ H-NMR (DMSO-d6): δ 8.13 (1 H, s), 7.62 (1 H, s), 6.64 (1 H, t, J = 6.7 Hz), 4.76 (1 H, dd) , J = 5.4 Hz, 6.4 Hz), 3.90 (1 H, d, J = 12.0 Hz), 3.84 (1 H, d, J = 12.0 Hz), 2.83-2. 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 Synthesis 4-Amino-7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -5- (1H-pyrazol-3-yl) -pyrrolo [2,3-d] pyrimidine (Compound 74) Were synthesized in the reaction steps shown below.

First, compound 73 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2) was obtained from compound 59 obtained in Synthesis Example 8. -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) in 1,4-dioxane The mixture was dissolved in 0.4 mL), 2 M 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 give compound 73 (9.3 mg, 34%).
¹ H-NMR (CDCl ₃ ): δ 8.12 (1 H, s), 7.69-7. 32 (12 H, m), 6.67 (1 H, t, J = 6.8 Hz), 6.27 1H, d, J = 2.4 Hz), 4.96-4. 93 (1 H, m), 4.06 (1 H, d, J = 11.1 Hz), 3.89 (1 H, d, J = 11) 1), 3.00-2.94 (1 H, m), 2.51-2. 46 (1 H, m), 1.07 (9 H, s), 0.96 (9 H, s), 0.. 17 (3 H, s), 0.16 (3 H, s).

Next, compound 74 (4-amino-7- (4-C-cyano-2-deoxy-β-D-ribofuranosyl) -5- (1H-pyrazole-3-) was obtained from compound 73 thus obtained. Yl) -pyrrolo [2,3-d] pyrimidine) was 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 to 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 give compound 74 (2.5 mg, 57%).
¹ H-NMR (DMSO-db): δ 8.06 (1 H, s), 7.71 (1 H, s), 7. 66 ( ¹ H, d, J = 2.4 Hz), 6.71 (1 H, t) , J = 6.8 Hz), 6.65 (1 H, d, J = 2.4 Hz), 4.78 (1 H, dd, J = 5.1 Hz, 6.5 Hz), 3.93 (1 H, d, J) J = 12.0 Hz), 3.87 (1 H, d, J = 12.0 Hz), 2.89-2.84 (1 H, m), 2.55-2.50 (1 H, 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 in the following reaction step.

First, 2.44 g (4.66 mmol) of compound 75 (see Synthesis, 2006, 12, 2005) was dissolved in methanol (20 mL), aqueous ammonia (20 mL) was added, and the mixture was stirred at 85 ° C. for 22 hours (shield Use a tube). After completion of the reaction, the solvent is distilled off under reduced pressure, and the residue is purified by silica gel column chromatography (methanol / chloroform = 1/4) to give crudely purified compound 76 (1.06 g, <3.95 mmol). Obtained.

Next, crude compound 76 (1.06 g, <3.95 mmol) is dissolved in pyridine (11 mL) and chlorotrimethylsilane (5.0 mL, 39.5 mmol) is added at 0 ° C., 1 at room temperature Stir for hours. Subsequently, benzoyl chloride (0.48 mL, 4.15 mmol) was added and stirred for 3 hours, and then benzoyl chloride (0.14 mL, 1.19 mmol) was further 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 the mixture was stirred for 1 hour. After completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / chloroform = 1/4) to obtain roughly purified compound 77 (<3.95 mmol).

Next, crude compound 77 (<3.95 mmol) which was subjected to azeotropic dehydration with pyridine was dissolved in pyridine (20 mL), and then 4,4'-dimethoxytrityl chloride (1.61 g, 4) at 0 ° C. .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. After that, the solvent was distilled off under reduced pressure to give crude Compound 78 (<3.95 mmol).

The crude compound 78 (<3.95 mmol) is then dissolved in N, N-dimethylformamide (20 mL) and then imidazole (0.81 g, 11.9 mmol) and tert-butyldimethylchlorosilane TBSCl (0 ° C.) 1.20 g, 7.90 mmol) were sequentially added, and stirred at room temperature for 13.5 hours. After completion of the reaction, quenching with saturated aqueous sodium bicarbonate was performed at 0 ° C. Subsequently, extraction with ethyl acetate and evaporation of the solvent under reduced pressure gave 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, neutralization was performed with 1N aqueous sodium hydroxide solution. Subsequently, extraction with ethyl acetate was performed. After drying of the organic layer with magnesium sulfate and evaporation of the solvent under reduced pressure, the residue is purified by silica gel column chromatography (ethyl acetate / hexane = 1/2 → 1/1) to give crudely purified compound 80 ( 943 mg, <1.94 mmol) were obtained.

The crude compound 80 (429 mg, <0.882 mol) is then dissolved in pyridine (4.4 mL) and triphenylphosphine (1.16 g, 4.41 mmol) and iodine (1.12 g, 4.41 mmol) ) Was 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. Subsequently, extraction with ethyl acetate was performed. After drying of the organic layer with magnesium sulfate and evaporation of the solvent under reduced pressure, the residue is 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, 261 mg, 0.438 mmol, 24 % For 6 steps).
¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.72 (1 H, s), 8. 65 ( ¹ H, brs), 7.99 (2 H, m), 7.61 (1 H, 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), 41 (2H, m), 2.62 (1 H, m), 2.38 (1 H, m), 0.93 (9 H, s), 0.16 (3 H, s), 0.15 (3 H, 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 is distilled off under reduced pressure, and the residue is 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-pento-enofuranosyl) -5-fluoropyrrolo [2,3-d] pyrimidine, 155 mg, 0.330 mmol, 93%) I got
¹ H-NMR (CDCl ₃ , 500 MHz); 8.69 (1 H, s), 8. 50 (1 H, brs), 7.98 (2 H, d, J = 7.0 Hz), 7.62 (1 H, 1 H, t, J = 8.0 Hz), 7.54 (2 H, t, J = 7.5 Hz), 7.01 (1 H, d, J = 2.0 Hz), 6.97 (1 H, t, J = 7) .0 Hz), 4.94 (1 H, t, J = 5.0 Hz), 4.47 (1 H, s), 4.21 (1 H, d, J = 1.5 Hz), 2.56 (1 H, m) ), 2.51 (1 H, m), 0.94 (9 H, s), 0.16 (6 H, s).

Next, Compound 82 (86.0 mg, 0.184 mmol) is dissolved 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 with saturated aqueous sodium thiosulfate solution was performed at 0 ° C. Subsequently, extraction with ethyl acetate was performed. After drying of the organic layer with magnesium sulfate and evaporation of the solvent under reduced pressure, the residue is 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] The pyrimidine, 50.1 mg, 0.0786 mmol, 43%) was obtained.
¹ H-NMR (CDCl ₃ , 500 MHz); δ 8.69 (1 H, s), 8. 65 ( ¹ H, brs), 7.99 (2 H, d, J = 6.5 Hz), 7.63 (1 H, s t, J = 7.5 Hz), 7.54 (2 H, t, J = 7.5 Hz), 7.21 (1 H, s), 7. 13 (1 H, m), 4. 35 (1 H, d, J = 4.0 Hz), 3.61 (1 H, d, J = 1 1.0 Hz), 3.51 (1 H, d, J = 1 1.0 Hz), 2.79 (1 H, m), 2.48 (2. 1 H, m), 0.98 (9 H, s), 0.25 (3 H, s), 0.20 (3 H, s).

Compound 83 (50.1 mg, 0.0786 mmol) is then dissolved in N, N-dimethylformamide (1.5 mL) and sodium benzoate (113 mg, 0.786 mmol) and 15-crown-5 (0 .16 mL, 0.786 mmol) was added and stirred at 100.degree. C. for 24 hours. After completion of the reaction, extraction with ethyl acetate was performed. After drying of the organic layer with magnesium sulfate and evaporation of the solvent under reduced pressure, the residue is purified by silica gel column chromatography (ethyl acetate / hexane = 1/2 → 1/1 → 2/1) and crudely purified Of Compound 84 (10.5 mg, <0.0202 mmol).

After crude compound 84 (10.5 mg, <0.0202 mmol) was dissolved in methanol (1 mL), aqueous ammonia (1 mL) was added and stirred at room temperature for 80 hours. After completion of the reaction, the solvent is distilled off under reduced pressure, and the residue is 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) was obtained.
¹ H-NMR (CD ₃ OD, 500 MHz); δ 8.09 (1 H, s), 7. 20 ( ¹ H, d, J = 1.0 Hz), 6.73 (1 H, m), 4.69 (1 H , T, J = 7.0 Hz), 3.77 (1 H, d, J = 12 Hz), 3.69 (1 H, d, J = 12 Hz), 2.66 (1 H, m), 2.52 (1 H) , M).

Moreover, antiviral activity and cytotoxicity were evaluated by the method shown below regarding the nucleoside derivative obtained by synthesize | combining as mentioned above.

Test Example 1: Evaluation of anti-HBV activity 1
HepG2 2.2.15.7 cells were used as test cells. In addition, HepG2 2.2.15 cells were prepared so as to produce HBV continuously by introducing the HBV gene into human liver cancer cell line (HepG2 cells). 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 producer cells that carry not only DNA integrated into their genome but also the HBV gene produced as an episome. Therefore, co-culturing 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 HepG2 2.2.15.7 cells are quantified, and the degree of reduction is It was used as an index for evaluation of HBV activity.

More specifically, HepG2 2.2. 15.7 cells with a cell viability of 90% or more are seeded at a concentration of 2 x 10 ⁴ cells / ml on collagen-coated 96-well cell culture dishes, and the day on which cells are seeded 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 is further replaced with fresh medium containing each nucleoside derivative, and 1 W assay (7 days from the start of culture) HBV DNA was recovered for the assay. In addition, fresh medium containing each nucleoside derivative is further added to the cell plate after recovery of the supernatant, and culture is continued for 7 more days and 14 days, and 2 W assay (assay on day 14 from the start of culture) and Intracellular HBV DNA was recovered from HepG2 2.2.15.7 cells for 3W assay (assay at day 21 after culture start). Then, using these DNAs as templates, quantitative PCR was performed to determine the virus copy number from the calibration curve, and the 50% effect (EC ₅₀ ) of each nucleoside derivative was calculated. The obtained results are shown in Tables 2 and 3.

In addition, 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. For the PCR reaction, specific TaqMan probe primers of PrimerDesign Inc. for detecting the HBV core protein region were used. After 15 minutes at 95 ° C., the PCR reaction was carried out 50 cycles of 10 seconds at 95 ° C. and 60 seconds at 60 ° C. The resulting _{C T,} using (from 20 2 × 10 ⁸ copies) diluted HBV DNA fragment of known concentration for each 10-fold resulting from reaction calibration curve was converted to HBV copy number.

Test Example 2: Cytotoxicity test 1
The above-mentioned nucleoside derivatives were also subjected to cytotoxicity tests on HepG2 cells. HepG2 cells were maintained by continuous culture in DMEM containing 10% fetal bovine serum and antibiotics (penicillin and kanamycin).

In performing the cytotoxicity test, HepG2 cells were seeded at a concentration of 1 × 10 ⁴ cells / ml together with the medium to which each concentration of each nucleoside derivative after serial dilution was added. Thus, after culturing these cells for 7 days in the presence of various concentrations of each nucleoside derivative at 37 ° C., 5% CO ₂ standard culture conditions, the number of viable cells in each well was quantified by MTT assay . Then, CC ₅₀ was calculated for each nucleoside derivative based on the obtained number of viable cells. The obtained results are shown in Tables 2 and 3.

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

PXB cells are seeded at 3 × 10 ⁵ cells / ml in collagen-coated 96-well plates and cultured for 7 days under standard culture conditions of 37 ° C., 5% CO ₂ with each concentration of compound after serial dilution, The number of surviving cells in the wells was quantified by MTT assay. Then, based on the obtained values, the degree of cell damage was determined, and the CC ₅₀ value was calculated as the cytotoxicity of each compound. The obtained results are shown in Tables 2 and 3.

In Tables 2 and 3 below, items with “* (asterisk)” in the item regarding antiviral activity show that the EC ₅₀ value is more than 1 μM, and “* in the item regarding cytotoxicity Items marked with (asterisk) indicate that the CC ₅₀ value is less than 10 μM, and items marked with “-” indicate that the test has not been conducted.

As shown in Tables 2 and 3, all the nucleoside derivatives synthesized and tested as described above were found to have excellent antiviral activity against HBV. The results of 2W assay, the effect was also confirmed to be sustained (Although not shown in Table, Compound 69 results of 3W assayed for, EC ₅₀ is 0.060 higher antiviral activity Have confirmed that is maintained). Furthermore, as shown in Tables 2 and 3, the cytotoxicity of these nucleoside derivatives was also found to be generally low.

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

Claims (3)

1. The nucleoside derivative represented by following General formula (1).
   

   

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