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WO2018110591A1 - Dérivé de 2'-désoxy-7-déazapurine nucléoside ayant une activité antivirale - Google Patents

Dérivé de 2'-désoxy-7-déazapurine nucléoside ayant une activité antivirale Download PDF

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WO2018110591A1
WO2018110591A1 PCT/JP2017/044684 JP2017044684W WO2018110591A1 WO 2018110591 A1 WO2018110591 A1 WO 2018110591A1 JP 2017044684 W JP2017044684 W JP 2017044684W WO 2018110591 A1 WO2018110591 A1 WO 2018110591A1
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compound
mmol
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PCT/JP2017/044684
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Japanese (ja)
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裕明 満屋
山田　浩平
晃太 苫谷
裕太郎 大野
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ヤマサ醤油株式会社
国立研究開発法人国立国際医療研究センター
国立大学法人熊本大学
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Priority to JP2018556715A priority Critical patent/JP7125714B2/ja
Publication of WO2018110591A1 publication Critical patent/WO2018110591A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/14—Pyrrolo-pyrimidine radicals

Definitions

  • 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.
  • hepatitis B virus HBV
  • 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).
  • nucleoside derivative preparations 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.
  • 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.
  • 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.
  • 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.
  • the antiviral agent according to ⁇ 2> which is an anti-hepatitis B virus agent.
  • 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.
  • 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).
  • HBV hepatitis B virus
  • "HBV” means a virus having the ability to develop hepatitis B.
  • 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.
  • 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.
  • 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”.
  • anti-DNA virus 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 ⁇ M, more preferably 0.5 ⁇ M or less, still more preferably 0.1 ⁇ M or less, and 0.05 ⁇ M or less
  • concentration is 0.04 ⁇ M or less, 0.03 ⁇ M or less, 0.02 ⁇ M or less, and 0.01 ⁇ M or less.
  • the nucleoside derivative of this invention has low cytotoxicity.
  • 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 ⁇ M or more, more preferably a CC 50 value of 25 ⁇ M or more, still more preferably a CC 50 value of 50 ⁇ M or more, and 100 ⁇ M or more Is more preferred.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • R 1 is an amino group
  • R 2 is a hydrogen
  • R 3 is a cyano group
  • R 4 is a fluorine or ethynyl group
  • the nucleoside derivatives of the present invention also include pharmacologically acceptable salts, hydrates or solvates.
  • a pharmacologically acceptable salt is not particularly limited and can be appropriately selected according to the structure of the nucleoside derivative etc.
  • acid addition salt hydroochloride, 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, Triflu
  • 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.
  • 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.
  • a ribose sugar D-ribofuranose protected by substituting a hydroxy group with an acetyl group, a benzyl group or the like
  • purine base 7.deazaadenine
  • nucleoside derivative of the present invention 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.
  • 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.
  • 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.
  • HBV infection 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.
  • 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.
  • 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.
  • 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.
  • the antiviral agent of the present invention may be used in combination with other known antiviral agents.
  • 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.
  • 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.
  • 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.
  • "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.
  • 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.
  • 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.
  • 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.
  • surface shows the number of the compound shown below.
  • 1,2 5,6-di-O-isopropylidene- ⁇ -D-allofuranose (compound 1), 2-O-isopropylidene-3-Op-methoxybenzyl- ⁇ -D-allofuranose (compound 2) was synthesized.
  • compound 3 (3,5-di-O-benzyl-4-C-fluoromethyl-1,2-O-isopyridene- ⁇ -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 ⁇ L, 0.052 mmol) is added and stirred at 60 ° C. for 2 hours did. Subsequently, N, N-diethylaminosulfur trifluoride (6.9 ⁇ L, 0.052 mmol) was added and the mixture was stirred at 60 ° C. for 4 hours.
  • 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.
  • compound 5 (7- (2-O-acetyl-3,5-di-O-benzyl-4-C-fluoromethyl- ⁇ -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 ⁇ L, 0. 065 mmol) was added and stirred at room temperature for 20 minutes.
  • compound 6 (4-amino-7- (3,5-di-O-benzyl-4-C-fluoromethyl-2-O-phenylthionoformyl-) was obtained from compound 5 thus obtained.
  • ⁇ -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.
  • compound 7 (4-amino-7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl- ⁇ -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.
  • compound 8 (4-amino-7- (2-deoxy-4-C-fluoromethyl- ⁇ -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.
  • compound 10 (7- (3-O-tert-butyldimethylsilyl-2-deoxy- ⁇ -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.
  • 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 ⁇ L, 7.8 mmol) and trifluoroacetic acid (292 ⁇ L, 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.
  • compound 12 (7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-dimethoxytrityloxymethyl- ⁇ -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 ⁇ L, 3.7 mmol) is added, and 4,4′-dimethoxytrityl chloride (918 mg, 2) is added under ice cooling.
  • compound 13 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4-C-hydroxymethyl) - ⁇ -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 ⁇ L, 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 ⁇ L, 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.
  • compound 14 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy-) is obtained.
  • 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 ⁇ L, 0.50 mmol) and trifluoroacetic acid (19 ⁇ L, 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.
  • 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).
  • compound 16 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano) was obtained from compound 15 thus obtained.
  • compound 18 (4-amino-7- (4-C-cyano-2-deoxy- ⁇ -D-ribofuranosyl) pyrrolo [2,3-d] pyrimidine) was obtained from compound 16 thus obtained.
  • 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.
  • ⁇ -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.
  • compound 19 (7- (4-C-cyano-2-deoxy- ⁇ -D-ribofuranosyl) -4-methylaminopyrrolo [2,3-d] pyrimidine is obtained.
  • Compound 17 50 mg, 0.078 mmol
  • tetrahydrofuran 1 mL
  • tetrabutylammonium fluoride 171 ⁇ L, 0.17 mmol
  • Synthesis example 3 Synthesis of 7- (4-C-cyano-2-deoxy- ⁇ -D-ribofuranosyl) -2,4-diaminopyrrolo [2,3-d] pyrimidine 7- (4-C-cyano-2 -Deoxy- ⁇ -D-ribofuranosyl) -2,4-diaminopyrrolo [2,3-d] pyrimidine (compound 30) was synthesized in the following reaction steps.
  • 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- ⁇ -D-ribofuranosyl) -2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized.
  • compound 23 (4-chloro-7- (2-deoxy-5-O-dimethoxytrityl- ⁇ -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.
  • 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.
  • compound 25 (7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl- ⁇ -D-ribofuranosyl) -4- 4 Chloro-2-pivaloylaminopyrrolo [2,3-d] pyrimidine) was synthesized.
  • 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 ⁇ L, 1.7 mmol) and trifluoroacetic acid (97 ⁇ L, 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.
  • compound 26 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4-C-hydroxymethyl) was obtained from compound 25 thus obtained.
  • - ⁇ -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 ⁇ L, 0.28 mmol) is added, and 4,4′-dimethoxytrityl is cooled under ice cooling.
  • compound 27 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy-) was obtained from compound 26 thus obtained.
  • 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 ⁇ L, 0.30 mmol) and trifluoroacetic acid (11 ⁇ L, 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.
  • compound 28 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-) was obtained from compound 27 thus obtained.
  • 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.
  • compound 29 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy-) ⁇ -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 compound 30 (7- (4-C-cyano-2-deoxy- ⁇ -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 ⁇ L, 0.099 mmol) was added, and the mixture was stirred at room temperature for 30 minutes.
  • 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.
  • compound 32 (7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-2-deoxy-) was obtained from compound 31 thus obtained.
  • ⁇ -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.
  • compound 33 (2-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-Deoxy- ⁇ -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 ⁇ L) is added, and the mixture is stirred at room temperature for 70 minutes. did.
  • compound 34 (2-amino-7- (4-C-cyano-2-deoxy- ⁇ -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 ⁇ L, 0.048 mmol) was added, and the mixture was stirred at room temperature for 30 minutes.
  • compound 36 (2-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano) was obtained from compound 35 thus obtained.
  • compound 37 (2-amino-7- (4-C-cyano-2-deoxy- ⁇ -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 ⁇ L, 0.067 mmol) was added, and the mixture was stirred at room temperature for 20 minutes.
  • compound 39 (7- (5-O-tert-butyldiphenylsilyl-2-deoxy- ⁇ -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 ⁇ L, 0.82 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 24 hours. .
  • compound 39 (491 mg, 0.82 mmol) is dissolved in 1,4-dioxane (8 mL), and molecular sieve 5 ⁇ (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 ⁇ L, 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.
  • compound 41 (7- (3-O-benzyl-2-deoxy- ⁇ -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.
  • compound 42 (30 mg, 0.061 mmol) is dissolved in 1,4-dioxane (0.6 mL), and molecular sieve 5 ⁇ (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 ⁇ L, 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.
  • Compound 45 (7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl- ⁇ -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.
  • compound 46 (2-amino-7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl- ⁇ -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.
  • compound 47 (2-amino-7- (2-deoxy-4-C-fluoromethyl- ⁇ -D-ribofuranosyl) -3 H-pyrrolo [2,3-] was obtained from compound 46 thus obtained.
  • 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 ⁇ L, 0.13 mmol) is added at -78 ° C, Stir at 0 ° C. for 10 minutes.
  • compound 49 (4-amino-7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl- ⁇ -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.
  • the compound 50 (2,4-diamino-7- (3,5-di-O-benzyl-2-deoxy-4-C-fluoromethyl- ⁇ -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.
  • Compound 51 (2,4-diamino-7- (2-deoxy-4-C-fluoromethyl- ⁇ -D-ribofuranosyl) -pyrrolo [2,3-] was obtained from compound 50 thus obtained.
  • pyrimidine was synthesized. That is, Compound 50 (4.5 mg, 9.4 ⁇ mol) is dissolved in dichloromethane (0.1 mL), and then boron trichloride (1.0 M solution in dichloromethane, 94 ⁇ L, 0.09 mmol) is added at -78 ° C, Stir at 0 ° C. for 30 minutes.
  • compound 53 (4-benzylamino-7- (2-deoxy- ⁇ -D-ribofuranosyl) -5-iodopyrrolo [2,3-d] pyrimidine) was prepared from compound 52 (Eur. Pat. Appl., 710667).
  • 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 ⁇ L, 6.2 mmol) was added and stirred for 2 hours.
  • compound 54 (4-benzylamino-7- (2-deoxy-5-O-dimethoxytrityl- ⁇ -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.
  • compound 55 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy- ⁇ -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.
  • 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.
  • compound 56 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl- ⁇ -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 ⁇ L, 0.39 mmol) and trifluoroacetic acid (21.5 ⁇ L, 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.
  • 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- ⁇ -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 ⁇ L, 0.27 mmol) is added, and 4,4′-dimethoxytrityl is cooled under ice cooling.
  • compound 59 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano- 2-Deoxy- ⁇ -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.
  • compound 60 (4-amino-7- (4-C-cyano-2-deoxy- ⁇ -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 ⁇ L, 0.10 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours.
  • Synthesis example 9 Synthesis of 4-amino-7- (4-C-cyano-2-deoxy- ⁇ -D-ribofuranosyl) -5-fluoropyrrolo [2,3-d] pyrimidine 4-amino-7- (4 -C-Cyano-2-deoxy- ⁇ -D-ribofuranosyl) -5-fluoropyrrolo [2,3-d] pyrimidine (compound 69) was synthesized in the following reaction step.
  • compound 63 (4-benzylamino-7- (2-deoxy-5-O-dimethoxytrityl- ⁇ -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.
  • compound 64 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy- ⁇ -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.
  • 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.
  • compound 65 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl- ⁇ -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.
  • compound 66 (4-benzoylamino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-2-deoxy-4) was obtained from compound 65 thus obtained.
  • 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.
  • compound 67 (4-benzylamino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano) 2-Deoxy- ⁇ -D-ribofuranosyl) -5-fluoropyrrolo [2,3-d] pyrimidine) was synthesized.
  • 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 ⁇ L, 0.72 mmol) and trifluoroacetic acid (27 ⁇ L, 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.
  • 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 420 mg, 2.2 mmol
  • Pyridine 58 ⁇ L, 0.72 mmol
  • trifluoroacetic acid 27 ⁇ L, 0.36 mmol
  • compound 68 (4-amino-7- (3-O-tert-butyldimethylsilyl-5-O-tert-butyldiphenylsilyl-4-C-cyano-) was obtained from compound 67 thus obtained.
  • compound 69 (4-amino-7- (4-C-cyano-2-deoxy- ⁇ -D-ribofuranosyl) -5-fluoropyrrolo [2,3-] was obtained from compound 68 thus obtained.
  • 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 ⁇ L, 0.15 mmol) was added, and the mixture was stirred at room temperature for 2 hours.
  • 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- ⁇ -D-ribofuranosyl) -5-chloropyrrolo [2,3-d] pyrimidine) was synthesized.
  • compound 71 (4-amino-7- (4-C-cyano-2-deoxy- ⁇ -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 ⁇ L, 0.025 mmol) was added, and the mixture was stirred at room temperature for 2 hours.
  • 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- ⁇ -D-ribofuranosyl) -5- (1H-pyrazol-3-yl) -pyrrolo [2,3-d] pyrimidine) was synthesized.
  • compound 74 (4-amino-7- (4-C-cyano-2-deoxy- ⁇ -D-ribofuranosyl) -5- (1H-pyrazole-3-) was obtained from compound 73 thus obtained.
  • 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).
  • 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.
  • antiviral activity and cytotoxicity were evaluated by the method shown below regarding the nucleoside derivative obtained by synthesize
  • 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 ⁇ g / 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.
  • 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.
  • 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 ⁇ L of the extracted DNA was used for qPCR.
  • 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).
  • 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.
  • the number of viable cells in each well was quantified by MTT assay .
  • 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.
  • 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.

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Abstract

La présente invention montre clairement qu'un dérivé de nucléoside qui présente une excellente activité antivirale contre le VHB et qui a une faible cytotoxicité peut être obtenu par substitution de groupes fonctionnels spécifiques à chacune des positions 2, 6, et 7 de la base purine et de la position 4 du sucre ribose, en 2'-désoxy-7-déazaadénosine.
PCT/JP2017/044684 2016-12-13 2017-12-13 Dérivé de 2'-désoxy-7-déazapurine nucléoside ayant une activité antivirale WO2018110591A1 (fr)

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Citations (10)

* Cited by examiner, † Cited by third party
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