Anti-hepatitis B virus agents
阅读说明：本技术 抗乙型肝炎病毒剂 (Anti-hepatitis B virus agents ) 是由 滨田智仁 岸川洋介 稻生佳菜子 池田正德 武田绿 于 2019-09-27 设计创作，主要内容包括：本发明所要解决的技术问题在于,提供一种含有核酸类似物作为有效成分的抗乙型肝炎病毒剂和乙型肝炎病毒相关疾病的预防或治疗剂。该技术问题可以通过2’-脱氧-2’-氟-β-2-氟-D-腺苷或其前药、或它们在药学上可接受的盐、或者它们的溶剂化物而得到解决。(The present invention has been made to solve the above problems, and an object of the present invention is to provide an anti-hepatitis B virus agent and a prophylactic or therapeutic agent for hepatitis B virus-related diseases, each of which contains a nucleic acid analog as an active ingredient. The technical problem can be solved by 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof.)
1. An anti-hepatitis B virus agent comprising 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate of either as an active ingredient.
2. A prophylactic or therapeutic agent for a hepatitis B virus-related disease, which comprises 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, a pharmaceutically acceptable salt thereof, or a solvate of either as an active ingredient.
3. The prophylactic or therapeutic agent for hepatitis B virus according to claim 2, wherein the hepatitis B virus-related disease is 1 or more selected from the group consisting of hepatitis B, hepatitis B cirrhosis and hepatitis B liver cancer.
Technical Field
The present invention relates to an anti-hepatitis B virus agent and a prophylactic or therapeutic agent for hepatitis B virus-related diseases, which contain a nucleic acid analog as an active ingredient.
Background
As an active ingredient of an anti-hepatoma virus agent and a preventive or therapeutic agent for diseases associated with hepatoma virus, for example, a nucleic acid analog represented by the following formula is known (patent document 1):
[ CHEM 1 ]
(in the formula, R represents fluorine or hydrogen).
As hepatitis viruses, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), and the like are known. In which the life cycle of HBV is shown in FIG. 1. HBV is an incompletely double-stranded DNA virus belonging to the genus orthopathophilus of the family hepadnaviridae. HBV, if invaded into cells, forms a complete double-stranded DNA in the nucleus, forming cccDNA. 3.5, 2.4, 2.1 and 0.7kb mRNA was transcribed using DNA as a template, and translated into polymerase, HBcAg (core), HBsAg and protein X. The 3.5kb pregenomic RNA (pgRNA) is assembled with the core, polymerase. After reverse transcription of pgRNA into DNA, the viral particles are released extracellularly. HBV is not a retrovirus, but since polymerase has reverse transcription activity, reverse transcriptase inhibitors against HIV-1 are used in the treatment against it.
Documents of the prior art
Patent document
Patent document 1 International publication No. 2017/155082
Disclosure of Invention
Technical problem to be solved by the invention
An object of the present invention is to provide an anti-hepatitis B virus agent and a prophylactic or therapeutic agent for hepatitis B virus-related diseases, each of which contains a nucleic acid analog as an active ingredient.
Means for solving the problems
The present invention includes the following aspects.
Item 1.
An anti-hepatitis B virus agent comprising 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate of either as an active ingredient.
Item 2.
A prophylactic or therapeutic agent for a hepatitis B virus-related disease, which comprises 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, a pharmaceutically acceptable salt thereof, or a solvate of either as an active ingredient.
Item 3.
The prophylactic or therapeutic agent according to item 2, wherein the hepatitis B virus-related disease is 1 or more selected from the group consisting of hepatitis B, hepatitis B cirrhosis and hepatitis B liver cancer.
The present invention further includes the following aspects.
2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, which is useful as a prophylactic or therapeutic agent for a disease associated with hepatitis b virus.
2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate of either thereof, for use in the manufacture of a medicament for the prophylaxis or treatment of a disease associated with hepatitis b virus.
Effects of the invention
According to the present invention, there are provided an anti-hepatitis B virus agent and a prophylactic or therapeutic agent for hepatitis B virus-related diseases, each containing a nucleic acid analog as an active ingredient.
Drawings
Fig. 1 is a diagram showing a life cycle of HBV.
FIG. 2 is a graph comparing the relative amounts of extracellular HBs antigen in the case of using the compound of example 1 and a conventional anti-hepatitis B virus agent.
Detailed Description
The foregoing summary of the invention is not intended to describe each disclosed embodiment or every implementation of the present invention.
The following description of the invention more particularly exemplifies illustrative embodiments.
Guidance is provided throughout this specification by way of illustration, and the illustration can be used in a wide variety of combinations.
The illustrated groups may serve as non-exclusive and representative groups for each case.
All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.
Term(s) for
Unless otherwise defined, the terms "a" and "an" in the present specification shall be interpreted as meaning generally used in the technical field of the present invention in the context of the present specification.
In this specification, the phrase "including" is intended to include the phrases "consisting essentially of … …" and "consisting of … …" for use.
The steps, treatments, or operations described in this specification may be performed at room temperature unless otherwise specified.
In the present specification, the room temperature may be a temperature in the range of 10 to 40 ℃.
In this specification, the symbol "Cn-m"(where n and m are each a number) indicates that the number of carbon atoms is n or more and m or less, as is generally understood by those skilled in the art.
In the present specification, examples of the halogen atom include fluorine, chlorine, bromine and iodine.
Examples of the alkyl group in the present specification include straight-chain or branched C1-20Alkyl [ for example: methyl, ethyl, propyl (n-propyl, isopropyl), butyl (n-butyl, sec-butyl, isobutyl, tert-butyl), pentyl, hexyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl and octadecyl]。
In the present specification, examples of the cycloalkyl group include C3-10Cycloalkyl [ for example: cyclopentyl and cyclohexyl]。
In the present specification, examples of the aryl group include C6-10Aryl [ for example: phenyl and naphthyl]。
In the present specification, examples of the aralkyl group include C6-10Aryl-straight or branched chain C1-10Alkyl [ for example: benzyl and phenethyl]。
Examples of the alkoxy group in the present specification include straight-chain or branched C1-10Alkoxy [ for example: methoxy, ethoxy and propoxy groups]。
Examples of the alkoxyalkoxy group in the present specification include straight-chain or branched C1-20Alkoxy-straight or branched C1-4Alkoxy [ for example: methoxymethoxy, methoxyethoxy, ethoxyethoxy, hexadecyloxypropoxy and octadecyloxyethoxy]。
Examples of the acyloxy group in the present specification include straight-chain or branched-chain C1-10Alkylcarbonyloxy [ for example: methylcarbonyloxy, ethylcarbonyloxy and propylcarbonyloxy]。
Examples of the substituent which the aforementioned alkyl group, cycloalkyl group, aryl group, aralkyl group, alkoxy group, alkoxyalkoxy group, acyloxy group and steroid group may have each include halogen and an organic group, and
suitable examples thereof include fluorine, chlorine, bromine, alkoxy, alkylcarbonyloxy, alkylcarbonylthio, alkyloxycarbonyl and alkyldithio, and
more preferable examples thereof include halogen, straight chain or branched C1-20Alkyloxy, straight or branched C1-10Alkylcarbonyloxy, straight or branched C1-10Alkylcarbonylthio, straight or branched C1-10Alkyloxycarbonyl, and straight or branched C1-10An alkyl disulfide group.
Anti-hepatitis B virus agents
The "anti-hepatitis B virus agent" in the present invention means an agent for delaying or inhibiting the proliferation of hepatitis B virus.
The hepatitis B virus may be a virus strain having resistance to existing anti-hepatitis B virus agents (e.g., entecavir, tenofovir, adefovir dipivoxil).
The "virus strain having tolerance" against a hepatitis b virus agent refers to a virus strain in which the effect of proliferation delay or proliferation inhibition exhibited by a normal virus strain is not exhibited by the anti-hepatitis b virus agent, or a virus strain in which the effect is exhibited to a lesser extent than that exhibited by a normal virus strain.
An anti-hepatitis B virus agent according to one embodiment of the present invention is an anti-hepatitis B virus agent containing 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine [ alias (2R,3R,4S,5R) -5- (6-amino-2-fluoro-9H-purin-9-yl) -4-fluoro-2- (hydroxymethyl) tetrahydrofuran-3-ol ] or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.
<2 '-deoxy-2' -fluoro-beta-2-fluoro-D-adenosine >
The method for producing 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine is not particularly limited, and may be, for example, a method comprising the steps of:
step A: reacting a compound of the following formula (I):
[ CHEM 2 ]
(in the formula, Q1And Q2Identical or different, is a protecting group for hydroxyl, X is bromine or iodine. )
Reacting the compound with 2-fluoroadenine; and
and B: the protecting group for the hydroxyl group of the product obtained by the aforementioned reaction is deprotected.
[ step A ]
In the formula (I), provided that Q1And Q2Each is a functional group capable of protecting a hydroxyl group, and is not particularly limited, and examples thereof include ether-type protecting groups (e.g., t-butyl, benzyl, trityl), acetal-type protecting groups (e.g., tetrahydropyranyl), acyl-type protecting groups (e.g., acetyl, benzoyl) and silyl ether-type protecting groups (e.g., t-butyldimethylsilyl, etc.).
The amount of each of the compound represented by the formula (I) and 2-fluoroadenine to be used is not particularly limited as long as the reaction proceeds, and the molar ratio of the compound represented by the formula (I) to 2-fluoroadenine may be, for example, in the range of 1:1 to 1: 5.
The reaction of step A is generally carried out in the presence of a base. The base is preferably a non-nucleophilic base, and examples thereof include metal alkoxides (e.g., sodium t-butoxide and potassium t-butoxide).
The reaction of step A is generally carried out in a solvent.
Examples of the solvent include
Halogen-based solvents (e.g., methylene chloride, chloroform, dichloroethane),
Alcohol solvents (e.g., ethanol, propanol, butanol, pentanol),
A nitrile-based solvent (e.g., acetonitrile), and
a mixed solvent thereof.
The reaction temperature in step A is not particularly limited as long as the reaction can proceed, and may be, for example, a temperature in the range of 15 to 80 ℃.
The reaction time in step A may be a time sufficient to obtain the target compound, and may be carried out until the reaction is completed.
[ step B ]
The method and conditions for deprotecting the protecting group of the hydroxyl group of the product obtained by the reaction in step A can be appropriately selected depending on the kind of the protecting group, and, for example, if the protecting group is benzoyl, deprotection can be carried out by a reaction with a metal alkoxide (e.g., sodium methoxide).
The reaction temperature in step B is not particularly limited as long as the reaction can proceed, and may be, for example, a temperature in the range of 15 to 80 ℃.
The reaction time in step B may be a time sufficient to obtain the target compound, and may be carried out until the reaction is completed.
The product obtained by the reaction in steps A and B can be purified by filtration, column chromatography, or the like, if necessary.
The method for producing the compound represented by the formula (I) is not particularly limited, and may be, for example, a method including the steps of:
reacting the following formula (II):
[ CHEM 3 ]
(in the formula, Q1And Q2Each have the same meanings as described above, and Q3Being a protecting group for hydroxy groups)
The compounds shown are reacted with hydrogen bromide or hydrogen iodide.
The amount of the compound represented by the formula (II) and the hydrogen bromide or hydrogen iodide used is not particularly limited as long as the reaction proceeds, and the molar ratio of the compound represented by the formula (II) to the hydrogen bromide or hydrogen iodide is, for example, in the range of 1:1 to 1: 5.
The reaction of the compound represented by the formula (II) with hydrogen bromide or hydrogen iodide is usually carried out in a solvent.
Examples of the solvent include halogen-based solvents (e.g., methylene chloride, chloroform, and dichloroethane), carboxylic acid-based solvents (e.g., acetic acid), and mixed solvents thereof.
The reaction temperature is not particularly limited as long as the reaction can proceed, and may be, for example, a temperature in the range of 15 to 30 ℃.
< prodrug >
The prodrug of 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine is not particularly limited as long as it can be converted into its active metabolite or 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine in vivo, and any prodrug used as a prodrug of a nucleic acid analog can be used.
Representative examples of such prodrugs include esters and ester amides.
Examples of the aforementioned esters include phosphate esters. Suitable examples thereof include:
a phosphoric monoester represented by the following formula:
[ CHEM 4 ]
(in the formula, R1And R2The same or different, is a hydrogen atom, an alkyl group which may have 1 or more substituents, a cycloalkyl group which may have 1 or more substituents, an aryl group which may have 1 or more substituents, or an aralkyl group which may have 1 or more substituents; or bonded to each other to form a ring together with the phosphorus atom and oxygen atom constituting the phosphate ester moiety in the formula), and
a phosphodiester or triester represented by the following formula:
[ CHEM 5 ]
(in the formula, wherein,
R3and R at each occurrence4The same or different, is a hydrogen atom, an alkyl group which may have 1 or more substituents, a cycloalkyl group which may have 1 or more substituents, an aryl group which may have 1 or more substituents, or an aralkyl group which may have 1 or more substituents,
R5is an alkyl group which may have 1 or more substituents, an alkoxy group which may have 1 or more substituents, or an alkoxy group which may have 1 or more substituentsAn alkoxy group, an acyloxy group which may have 1 or more substituents, or a steroid-like alcohol group which may have 1 or more substituents (a group containing cyclopentaphenanthrene or hydrogenated cyclopentaphenanthrene), and
n is 1 or 2. ).
R1Preferred is an alkyl group which may have 1 or more substituents, and more preferred is an alkyl group which may have a substituent selected from the group consisting of an alkoxy group, an alkylcarbonyloxy group, an alkylcarbonylthio group and an alkyldithio group.
R2The alkyl group may have 1 or more substituents, the aryl group may have 1 or more substituents, or the aralkyl group may have 1 or more substituents is preferable, and the hydrogen atom, the alkyl group, the aryl group, or the aralkyl group is more preferable.
R1And R2The aforementioned rings bonded to each other to form together with the phosphorus atom and the oxygen atom constituting the phosphate ester moiety may be a single ring or a fused ring.
The number of the constituent atoms of the ring is, for example, an integer in the range of 6 to 10.
Specific examples of the aforementioned ring include the following formulae:
[ CHEM 6 ]
The rings shown.
The aforementioned ring may have 1 or more substituents. Examples of such substituents include halogen, alkyl, cycloalkyl, aryl and aralkyl.
R3And R4The alkyl group may have 1 or more substituents, the aryl group may have 1 or more substituents, or the aralkyl group may have 1 or more substituents is preferable, and the hydrogen atom, the alkyl group, the aryl group, or the aralkyl group is more preferable.
R5Preferably an alkyl, alkoxy, alkoxyalkoxy, acyloxy, or steroid-like group.
Specific examples of the aforementioned ester amides include, for example, phosphoric ester amides. Suitable examples thereof include compounds represented by the following formulae:
[ CHEM 7 ]
(in the formula, wherein,
R8is-NR8aR8bOR-OR8cAnd is and
R6、R7、R8a、R8band R8cThe same or different, is a hydrogen atom, an alkyl group which may have 1 or more substituents, a cycloalkyl group which may have 1 or more substituents, an aryl group which may have 1 or more substituents, or an aralkyl group which may have 1 or more substituents. ).
R6And R8aThe alkyl group may have 1 or more substituents is preferable, and the alkyl group may have a substituent selected from the group consisting of halogen and an alkyloxycarbonyl group is more preferable.
R7And R8bThe alkyl group may preferably have 1 or more substituents, and more preferably a hydrogen atom or an alkyl group.
R8cPreferably, the alkyl group may have 1 or more substituents, the aryl group may have 1 or more substituents, or the aralkyl group may have 1 or more substituents, and more preferably, the hydrogen atom, the alkyl group, the aryl group, or the aralkyl group.
Further suitable examples of such prodrugs include compounds of the following formula.
[ CHEM 8 ]
(in the formula, wherein,
R1a、R6aand R8dAre each an alkyl group, or a substituted alkyl group,
R1bis a halogen or an alkyl group,
R2and R7The same meaning as that of the above-mentioned,
ar is an aryl group, and Ar is an aryl group,
nu is the following formula:
[ CHEM 9 ]
The radicals shown are, for example,
m1 is an integer in the range of 1 to 18, and
m2 is an integer in the range of 1 to 10)
The prodrug can be produced by a known method (for example, the method described in Chemical Reviews 2014,114, 9154-9218) according to its Chemical structure and based on the technical common knowledge.
< salt >
Examples of pharmaceutically acceptable salts of 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof include
(1) With inorganic acids [ for example: salts of hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, nitric acid, pyrosulfuric acid, metaphosphoric acid, and the like ]; and
(2) with organic acids [ e.g.: salts of citric, benzoic, acetic, propionic, fumaric, maleic and sulfonic acids (e.g., methanesulfonic, p-toluenesulfonic and naphthalenesulfonic acids); and
(3) alkali metal salts [ for example: sodium and potassium salts ].
< solvate >
Examples of solvates of 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or prodrugs thereof, or pharmaceutically acceptable salts thereof include hydrates and organic solvates (e.g., methanolate, ethanolate, dimethylsulfoxide, etc.).
< other active ingredients >
The anti-hepatitis B virus agent may further contain other active ingredients.
Examples of such "other active ingredients" include other nucleic acid analogs (2 '-deoxy-2' -fluoro-nucleosides, etc.) and other anti-hepatitis B virus agents.
The anti-hepatitis B virus agent may contain 2 or more agents.
2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof may be formulated with the "other active ingredient" each as a separate formulation.
2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate of either, may be administered to a subject simultaneously, sequentially, or alternately with the "other active ingredient".
< content of active ingredient >
The lower limit of the content of the active ingredient may be set to, for example, 0.001 mass%, preferably 0.01 mass%, and more preferably 0.05 mass% with respect to the total mass of the anti-hepatitis b virus agent, from the viewpoint of activity. The upper limit of the content of the active ingredient is not particularly limited, and may be set to, for example, 99.99 mass%, preferably 90 mass%, and more preferably 80 mass% with respect to the total mass of the anti-hepatitis b virus agent. The content of the active ingredient may be, for example, 0.001 to 99.99 mass%, preferably 0.01 to 90 mass%, and more preferably 0.05 to 80 mass% within a range in which the lower limit and the upper limit are arbitrarily selected.
< additive >
The anti-hepatitis B virus agent may contain a pharmaceutically acceptable additive.
Examples of the form of the anti-hepatitis B virus agent include solid preparations (e.g., granules, powders, tablets, capsules, dry syrups), semi-solid preparations (e.g., creams, ointments, gels) and liquid preparations (e.g., solutions, suspensions).
The solid preparation can be produced, for example, by mixing the active ingredient and additives (e.g., excipient, binder, disintegrant, lubricant, colorant), and granulating, compressing, and/or coating as desired.
Examples of such excipients include lactose, lactose hydrate, sucrose, mannitol, sorbitol, crystalline cellulose, starch (e.g., corn starch), hydrated silicon dioxide, and combinations thereof.
Examples of the binding agent include agar, gum arabic, hyaluronic acid, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and combinations thereof.
Examples of the disintegrant include alginic acid, carboxymethyl cellulose (carboxymethylcellulose), croscarmellose sodium, low-substituted hydroxypropyl cellulose, polyvinylpyrrolidone (povidone), crospovidone, and combinations thereof.
Examples of the lubricant include stearic acid, magnesium stearate, calcium stearate, talc, and combinations thereof.
Examples of the colorant include iron sesquioxide, titanium oxide, and combinations thereof. The semisolid preparation can be produced, for example, by mixing the active ingredient, the semisolid carrier and, depending on the desired further additives.
The liquid preparation can be prepared, for example, by mixing an active ingredient, a liquid carrier [ for example: an aqueous carrier (e.g., purified water), an oily carrier ], and other additives as desired (e.g., an emulsifier, a dispersant, a suspending agent, a buffer, an antioxidant, a surfactant, an osmotic pressure regulator, a chelating agent, and an antibacterial agent), and is sterilized as necessary.
< form of administration >
The anti-hepatitis B virus agent can be administered orally or parenterally (for example, intravenously, intramuscularly, subcutaneously).
The method of administration of the anti-hepatitis B virus agent may be topical administration.
The subject to which the anti-hepatitis B virus agent is administered may be any of humans, non-human mammals (e.g., monkeys, sheep, dogs, mice, rats), and non-mammals.
The number of administration of the anti-hepatitis b virus agent may be appropriately selected depending on the age, body weight, condition, etc. of the subject to be administered, and may be, for example, 1, 2, or 3 times per day; 1 time 2 days, 1 time 3 days, 1 time 4 days, 1 time 5 days, 1 time 6 days, or 1 week.
The dose of the anti-hepatitis B virus agent administered 1 time may be in the range of 0.1mg to 1000mg depending on the administration subject, the number of times of administration, and the like.
Suitable examples of the anti-hepatitis B virus agent are orally administered preparations, and specific examples thereof include:
a tablet comprising an active ingredient, crystalline cellulose, hydroxypropylmethylcellulose, povidone, magnesium stearate, and titanium oxide; and
a capsule comprising the active ingredient, povidone and magnesium stearate in a hard gelatin capsule.
Prophylactic or therapeutic agent for hepatitis B virus-related disease
"hepatitis B virus-related disease" refers to a disease caused by infection with hepatitis B virus. The hepatitis B virus-related disease may be, for example, 1 or more selected from hepatitis B (e.g., acute hepatitis B, chronic hepatitis B), hepatitis B cirrhosis, and hepatitis B liver cancer.
The prophylactic or therapeutic agent for a hepatitis b virus-related disease according to one embodiment of the present invention contains 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate of either as an active ingredient.
The other active ingredients and additives contained in the prophylactic or therapeutic agent, and the dosage form and administration form (for example, administration route, administration subject, administration frequency and administration amount) of the prophylactic or therapeutic agent can be selected from those described for the anti-hepatitis B virus agent.
Method for delaying or inhibiting proliferation of hepatitis B virus, or preventing or treating hepatitis B virus-related diseases Method (2)
A method for delaying or inhibiting the proliferation of a hepatitis b virus or a method for preventing or treating a hepatitis b virus-related disease according to one embodiment of the present invention includes a step of administering 2 '-deoxy-2' -fluoro- β -2-fluoro-D-adenosine or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject in need thereof.
The form of administration (e.g., route of administration, subject to be administered, number of administrations, and dose) can be selected from those described for the anti-hepatitis B virus agent.
Examples
Hereinafter, one embodiment of the present invention will be described in further detail by way of examples, but the present invention is not limited thereto.
Example 1 (Synthesis example)
(1)
2g of 2-deoxy-2-fluoro-1, 3, 5-tri-O-benzoyl-alpha-D-arabinofuranose was dissolved in 20mL of methylene chloride, and 4mL of a hydrobromic acid-acetic acid solution (5.1mol/L) was added thereto and the mixture was stirred. After the reaction was completed, a saturated aqueous sodium bicarbonate solution was added to neutralize the unreacted HBr.
Thereafter, the solution obtained by extraction with dichloromethane was subjected to liquid separation and concentrated to obtain 1.7g of a crude product of 1-bromo-2-deoxy-2-fluoro-3, 5-di-O-benzoyl- α -D-arabinofuranose. It was used in the following steps.
(2) Reaction with 2-fluoroadenine
1g of 2-fluoroadenine was dispersed in a cosolvent of t-amyl alcohol acetonitrile.
Potassium tert-butoxide was added thereto, and the mixture was heated to 50 ℃ and dissolved by stirring.
1.7g of crude 1-bromo-2-deoxy-2-fluoro-3, 5-di-O-benzoyl- α -D-arabinofuranose dissolved in acetonitrile was added dropwise thereto to effect a reaction.
After completion of the reaction, the solid was diluted with dichloroethane and filtered, and then the filtrate was concentrated and purified by column chromatography.
To obtain 200mg of a reaction product (2 '-deoxy-2' -fluoro-3 ',5' -di-O-benzoyl-beta-2-fluoro-D-adenosine).
(3) Deprotection of the amino acid
100mg of 2 '-deoxy-2' -fluoro-3 ',5' -di-O-benzoyl-. beta. -2-fluoro-D-adenosine was dissolved in methanol, and 10mg of sodium methoxide was added thereto and the mixture was stirred.
After completion of the reaction, the reaction product was purified by preparative TLC to obtain 60mg of 2 '-deoxy-2' -fluoro-. beta. -2-fluoro-D-adenosine (the following formula).
[ CHEM 10 ]
1H NMR (400MHz, acetone-D6) δ 8.14-8.19(m,1H),7.08(br,2H),6.36(dd, J ═ 15.3,4.3Hz,1H),5.14-5.29(m,2H),4.61-4.68(m,1H),4.29(br,1H)3.71-3.99(m,3H)
Comparative example 1 (synthetic example)
In the same manner as in example 1 except for using 2, 6-dichloropurine instead of 2-fluoroadenine in step (2) of example 1, 2, 6-dichloro-9- ((2R,3S,4R,5R) -3-fluoro-4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purine (formula below) was obtained.
[ CHEM 11 ]
1H NMR(400MHz,DMSO-D6)δ8.53(s,1H),6.41(dd,J＝12.3,4.6Hz,1H),5.97(s,1H),5.25(dt,J＝52.8,4.6Hz,1H),5.09(t,J＝5.5Hz,1H),4.38-4.43(m,1H),3.81-3.91(m,1H),3.58-3.68(m,2H)
Test example 1 (anti-HBV activity)
In the evaluation of anti-HBV activity, the amount of HBV DNA in cells on day 7 after the addition of a drug was evaluated by the quantitative PCR method using hepg2.2.15 cell line, which is an HBV-producing cell obtained by introducing a gene 2 times as long as the HBV genome into a human liver cancer cell line HepG2. In the PCR of HBV DNA, a forward primer (HBV-S190F; 5'-GCT CGT GTT ACA GGC GGG-3': SEQ ID No:1) and a reverse primer (HBV-S703R; 5'-GAA CCA CTG AAC AAA TGG CAC TAG TA-3': SEQ ID No:2) were used. PCR was carried out for 35 cycles using 1 reaction at 95 ℃ for 10 seconds to 62 ℃ for 10 seconds to 72 ℃ for 30 seconds.
Specifically, HepG2.2.15 cells were plated to 1X 105The inoculation was performed in a single cell/well format. After 24 hours, the compounds of example 1 and comparative example 1 were diluted to 0, 0.49, 0.97, 1.95, 3.90, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000nM and added to the cells. After the cells were cultured for 7 days, the cytoplasmic fraction was collected and extracted with phenol/chloroform to purify the DNA. The amount of HBV DNA in the cells was measured by quantitative PCR using 20ng of the purified DNA.
Test example 2(HBs antigen amount)
The HBs antigen content was measured by CLIA method (chemiluminescence immunoassay) using HepG2.2.15 cell line, which is an HBV-producing cell obtained by introducing a gene 2 times as long as the HBV genome into a human hepatoma cell line HepG2, and the culture supernatant on day 4 after the addition of the drug.
Test example 3 (cytotoxicity test)
HepG2 NTCP-myc cells to reach 2X104Individual cells/well. After 24 hours, the agents were diluted to 0, 0.49, 0.97, 1.95, 3.90, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000nM and added to the cells. HepG2 NTCP-myc cells were cultured for 7 days. After the culture, 10. mu.l of Premix WST-1Cell Proliferation Assay System (TaKaRa) was added, and after culturing at 37 ℃ for 2 hours, absorbance was measured at 450nm using a microplate analyzer.
Results of test examples 1 to 3
1. anti-HBV activity
EC50(50% effective concentration) was calculated from a graph showing the relationship between the concentration of the compound and the anti-HBV activity.
[ TABLE 1 ]
EC50(nM)
Example 1
1.58
Comparative example 1
＞10000
As can be seen from table 1, the compound of example 1 has significantly higher anti-HBV activity than the compound of comparative example 1.
HBs antigen amount
The measurement results of the HBs antigen amount are shown in FIG. 2. As can be seen from fig. 2, the compound of example 1 significantly reduced the amount of HBs antigen compared to the existing anti-hepatitis b virus agents (entecavir and tenofovir disoproxil fumarate).
3. Cytotoxicity
CC50(50% cytotoxic concentration) was calculated from a graph showing the relationship between the concentration of the compound and cytotoxicity.
[ TABLE 2 ]
CC50(μM)
Example 1
337.19
As can be seen from table 2, the compound of example 1 has low cytotoxicity.
SI value (Selectivity index)
The SI value is an index of the possibility of use of a drug by comparing the Cytotoxic Concentration (CC)50) Divided by the Effective Concentration (EC)50) And then calculated. I.e. SI ═ CC50/EC50。
[ TABLE 3 ]
SI(CC50/EC50)
Example 1
213411
As is clear from table 3, the compound of example 1 has a large SI value (high possibility of use as a drug).
Sequence listing
<110> Dajin Industrial Co., Ltd (DAIKIN INDUSTRIES, LTD.)
National UNIVERSITY of legal people deer island UNIVERSITY (KAGOSHIMA UNIVERSITY)
<120> anti-hepatitis B Virus agent
<130> FP210329JP（P19-189WO）
<150> JP 2018-189216
<151> 2018-10-04
<150> JP 2019-078616
<151> 2019-04-17
<160> 2
<170> PatentIn version 3.5
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<213> Artificial sequence
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<210> 2
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<212> DNA
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