JP2002322192A - 2'-o,4'-crosslinked nucleoside triphosphate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate possible to amplify a gene simply and accurately in a gene amplification using RNA polymerase. SOLUTION: The compound represented by general formula (1): (wherein A is a 1-4C alkylene group, B is purin-9-yl group, 2-oxo-1,2-dihydropyrimidin-1-yl group or substituted purin-9-yl group or substituted 2-oxo-1,2-dihydropyrimidin-1- yl group, and X is oxygen atom or sulfur atom) and its salt are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a 2'-O, 4'-C- which enables simple and accurate amplification when performing gene amplification.
The present invention relates to a crosslinked nucleoside triphosphate.

[0002]

2. Description of the Related Art Polymerase chain reaction (PCR)
It is known as an excellent gene amplification method using a polymerase. However, it is known that an error in complementary strand formation occurs with a certain probability between natural genes. Therefore, PC
In the R method, after an error in complementary strand formation occurs, the erroneous gene is amplified, and there is a limit on the amplification rate in performing accurate amplification.

[0003] On the other hand, when a gene is amplified using RNA polymerase, complementary RNA is continuously synthesized using DNA as a template, and the complementary RNA is not formed as a template, as in PCR, in that the amplified RNA is not used as a template. Errors do not matter. Therefore, the gene amplification method using RNA polymerase is used for gene expression monitoring, mutation analysis, gene polymorphism analysis, etc. in combination with a recently developed DNA chip (Fumio Kimizuka, Kato Kato Ikuyuki Susumu, Protein Nucleic Acid Enzyme, 1998, Vol 43, 2004-2011). However, when RNA polymerase is used, there is a disadvantage that RNA produced by amplification is less stable than DNA and is difficult to handle.

[0004] Compounds that inhibit influenza polymerase are expected to be effective against influenza, but no compounds having sufficient activity have been known so far.

[0005]

SUMMARY OF THE INVENTION The present inventors have conducted intensive studies on a technique for enabling simple and accurate gene amplification, and as a result, have found that 2'-O, 4 'having an intramolecular ether bond has been obtained.
-C-crosslinked nucleoside triphosphate has been found to be an excellent substrate that enables simple and accurate amplification in gene amplification methods using RNA polymerase, and has an activity to inhibit influenza polymerase. Heading, the present invention has been completed.

[0006]

Means for Solving the Problems 2'-O, 4'- of the present invention
The C-bridged nucleoside triphosphate has the general formula (1)

[0007]

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In the formula, A represents an alkylene group having 1 to 4 carbon atoms, and B represents a purine-9-yl group, a 2-oxo-1,2-dihydropyrimidin-1-yl group or the following α: A substituted purin-9-yl group or a substituted 2-oxo-1,2-dihydropyrimidine- having a substituent selected from the group
X represents a 1-yl group, and X represents an oxygen atom or a sulfur atom. Or a salt thereof, wherein (1) A is a methylene group, (2) B is 6-aminopurin-9-yl (ie, adenyl), 2,6
-Diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-fluoropurine-
9-yl, 2-amino-6-bromopurin-9-yl,
2-amino-6-hydroxypurin-9-yl (ie, guaninyl), 6-amino-2-methoxypurine-
9-yl, 6-amino-2-chloropurin-9-yl,
6-amino-2-fluoropurin-9-yl, 2,6-
Dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (ie, cytosinyl) 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo -4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-
4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (ie, uracinyl), 2-oxo-4-hydroxy-5-methyl Pyrimidin-1-yl (ie, thyminyl) or 4-amino-5-methyl-
A 2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl) group, and (3) X is an oxygen atom. In the general formula (1), A is arbitrarily selected from the above (1), B is arbitrarily selected from (2), X is arbitrarily selected from (3), and these are arbitrarily combined. The compounds and salts thereof obtained by the above method are also suitable, and more preferably, compounds or salts thereof selected from the following compound group. (Compound group) 2'-O, 4'-C-ethyleneguanosine 5 '
-O-1-thiotriphosphoric acid, 2'-O, 4'-C-ethylene adenosine 5'-O-1-thiotriphosphoric acid, 2'-O, 4'-
C-ethylene uridine 5′-O-1-thiotriphosphate,
2'-O, 4'-C-ethylene 5-methyluridine 5'-O
-1-thiotriphosphoric acid, 2'-O, 4'-C-ethylenecytidine 5'-O-1-thiotriphosphoric acid, 2'-O, 4'-C-ethylene-5-methylcytidine 5'-O-1 -Thiotriphosphate, 2'-O, 4'-C-ethylene guanosine 5'-triphosphate, 2'-O, 4'-C-ethylene adenosine 5'-
Triphosphoric acid, 2'-O, 4'-C-ethyleneuridine 5 '
-Triphosphoric acid, 2'-O, 4'-C-ethylene 5-methyluridine 5'-triphosphoric acid, 2'-O, 4'-C-ethylenecytidine 5'-triphosphoric acid, and 2'-O, 4'-C-ethylene-5-methylcytidine 5'-triphosphoric acid. Still more preferably, it is a compound selected from the following compound group or a salt thereof. (Compound group) 2'-O, 4'-C-ethyleneguanosine 5 '
-Triphosphoric acid, 2'-O, 4'-C-ethylene adenosine
5'-triphosphoric acid, 2'-O, 4'-C-ethylene uridine 5'-triphosphoric acid, 2'-O, 4'-C-ethylene 5-methyluridine 5'-triphosphoric acid, 2'-O, 4'-C-ethylene cytidine 5'-triphosphate, and 2'-O, 4'-
C-ethylene-5-methylcytidine 5'-triphosphoric acid. (Α group) hydroxyl group, alkoxy group having 1 to 4 carbon atoms, mercapto group, alkylthio group having 1 to 4 carbon atoms, amino group, amino group substituted with alkyl group having 1 to 4 carbon atoms, carbon number 1 to 4 alkyl groups and halogen atoms.

In the above general formula (1), examples of the “alkylene group having 1 to 4 carbon atoms” of A include methylene, ethylene, trimethylene and tetramethylene groups. It is.

In the above general formula (1), the “alkoxy group having 1 to 4 carbon atoms” in the α group includes, for example, methoxy,
Examples include ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy or tert-butoxy, preferably a methoxy or ethoxy group.

In the general formula (1), the “alkylthio group having 1 to 4 carbon atoms” in the α group includes, for example, methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, tert- Examples thereof include butylthio, preferably a methylthio or ethylthio group.

In the general formula (1), the “amino group substituted by an alkyl group having 1 to 4 carbon atoms” in the α group includes:
For example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, s-butylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di ( Examples thereof include s-butyl) amino and di (tert-butyl) amino, preferably a methylamino, ethylamino, dimethylamino, diethylamino or diisopropylamino group.

In the general formula (1), the “alkyl group having 1 to 4 carbon atoms” in the α group includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s
-Butyl and tert-butyl, and preferably a methyl or ethyl group.

In the general formula (1), examples of the “halogen atom” in the α group include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom or a chlorine atom. is there.

In the above general formula (1), the “purine-9-” of B
Suitable groups throughout the "yl group" and "substituted purin-9-yl groups" are 6-aminopurin-9-yl (ie, adenyl), 2,6-diaminopurin-9-yl, 2-amino- 6-chloropurin-9-yl, 2-amino-6-
Fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurine-
9-yl (ie, guaninyl), 6-amino-2-
Methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurine-9
-Yl, 2,6-dimethoxypurin-9-yl, 2,6
-Dichloropurin-9-yl or 6-mercaptopurin-9-yl group, more preferably 6-benzoylaminopurin-9-yl, adenyl, 2-isobutyrylamino-6-hydroxypurine-9. -An yl or guaninyl group.

In the above general formula (1), “2-oxo-
1,2-dihydropyrimidin-1-yl group "and" substituted 2
-Oxo-l, 2-dihydropyrimidin-l-yl group ", suitable groups are 2-oxo-4-amino-l, 2-dihydropyrimidin-l-yl (ie, cytosinyl), 2-oxo -4-amino-5-fluoro-1,2-
Dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2 -Oxo-4-mercapto-1,2-
Dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (ie, uracinyl), 2-oxo-4-hydroxy-5-
A methylpyrimidin-1-yl (ie, thyminyl) or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl) group, more preferably, 2-oxo-4-benzoylamino-1,2-dihydropyrimidin-1-yl, cytosinyl, thyminyl, uracinyl, 2-oxo-
4-benzoylamino-5-methyl-1,2-dihydropyrimidin-1-yl or a 5-methylcytosinyl group.

The "salt" refers to the salt of the compound of the present invention, which is not particularly limited as long as it does not hinder gene amplification. Such salts are preferably sodium salts,
Metal salts such as alkali metal salts such as potassium salts and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts and cobalt salts; ammonium salts Inorganic salts, such as t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine Organic salts such as salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) aminomethane salt Amines such as Salts; inorganic acid salts such as hydrohalides such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide, nitrates, perchlorates, sulfates and phosphates; Lower alkane sulfonates such as methane sulfonate, trifluoromethane sulfonate and ethane sulfonate, aryl sulfonates such as benzene sulfonate and p-toluene sulfonate, acetate and malate , Fumarate, succinate, citrate, tartrate, oxalate, maleate, etc .; and glycine, lysine, arginine, ornithine, ornithine, glutamate, aspartate And more preferably an alkali metal salt and an amine salt, and still more preferably a sodium salt and a triethylamine salt.

Tables 1 and 2 show specific compounds of the above formula (1) included in the compounds of the present invention. However, the compound of the present invention is not limited to these.

[0019]

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[0020]

[Table 1]

[0021]

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[0022]

[Table 2] In Table 1 or Table 2, preferred compounds are (1-5)
(1-7), (1-25), (1-27), (1-4)
5), (1-47), (1-65), (1-67),
(1-85), (1-87), (1-105), (1-
107), (1-125), (1-127), (1-1)
45), (1-147), (2-1), (2-2),
(2-3), (2-4), (2-9), (2-10),
(2-11), (2-12), (2-17), (2-1)
8), (2-19), (2-20), (2-25),
(2-26), (2-27), (2-28), (2-3)
3), (2-34), (2-35), (2-36),
(2-41), (2-42), (2-43), (2-4)
4), (2-49), (2-50), (2-51),
(2-52), (2-57), (2-58), (2-5)
9) and (2-60), and more preferably, 2′-O,
4'-C-ethylene guanosine 5'-O-1-thiotriphosphate (1-45), 2'-O, 4'-C-ethylene adenosine 5'-O-1-thiotriphosphate (1-47), 2 '-
O, 4'-C-ethyleneuridine 5'-O-1-thiotriphosphate (2-33), 2'-O, 4'-C-ethylene5-methyluridine 5'-O-1-thiotriphosphate (2 -3
4) 2'-O, 4'-C-ethylenecytidine 5'-O-1
-Thiotriphosphoric acid (2-35), 2'-O, 4'-C-ethylene-5-methylcytidine 5'-O-1-thiotriphosphoric acid (2-36), 2'-O, 4'-C- Ethylene guanosine
5'-triphosphoric acid (1-5), 2'-O, 4'-C-ethylene adenosine 5'-triphosphoric acid (1-7), 2'-O,
4'-C-ethyleneuridine 5'-triphosphoric acid (2-
1) 2'-O, 4'-C-ethylene 5-methyluridine
5′-triphosphoric acid (2-2), 2′-O, 4′-C-ethylenecytidine 5′-triphosphoric acid (2-3), and 2′-
O, 4'-C-ethylene-5-methylcytidine 5'-triphosphoric acid (2-4), still more preferably 2'-O,
4'-C-ethylene guanosine 5'-triphosphate (1-
5) 2'-O, 4'-C-ethyleneadenosine 5'-triphosphate (1-7), 2'-O, 4'-C-ethyleneuridine 5'-triphosphate (2-1), 2 '-O, 4'-C-ethylene 5-methyluridine 5'-triphosphoric acid (2-
2) 2'-O, 4'-C-ethylene cytidine 5'-triphosphoric acid (2-3) and 2'-O, 4'-C-ethylene-
5-methylcytidine 5′-triphosphoric acid (2-4).

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The compound (1) of the present invention can be produced by the following Method A.

[0024]

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In the method A, X represents an oxygen atom or a sulfur atom, Y ¹ and Y ² represent the same or different protecting groups,
Has the same meaning as described above, and B ¹ is the aforementioned B or
B represents a group in which a hydroxyl group or an amino group on B is protected with a protecting group as necessary, B has the same meaning as described above, R ⁷ represents a group forming a leaving group together with an oxygen atom, and R ⁸ represents And an aliphatic acyl group having 1 to 4 carbon atoms.

The “protecting group” in the definition of Y ¹ and Y ² is not particularly limited as long as it can stably protect a hydroxyl group during nucleic acid synthesis. A protecting group that is stable under acidic conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis and photolysis, such as formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, Valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 3,13- dimethyl-tetradecanoyl, heptadecanoyl, 15-methyl hexadecanoyl, octadecanoyl, 1-methyl-hept decanoyl,
Alkylcarbonyl groups such as nonadecanoyl, eicosanoyl and henicosanoyl, carboxylated alkylcarbonyl groups such as succinoyl, glutaroyl, adipoyl, chloroacetyl, dichloroacetyl, trichloroacetyl, halogeno lower alkylcarbonyl groups such as trifluoroacetyl, methoxyacetyl; Lower alkoxy lower alkylcarbonyl group such as (E) -2
An "aliphatic acyl group" such as an unsaturated alkylcarbonyl group such as -methyl-2-butenoyl; an arylcarbonyl group such as benzoyl, α-naphthoyl and β-naphthoyl; Halogenated arylcarbonyl groups, lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl, 4-toluoyl, lower alkoxylated arylcarbonyl groups such as 4-anisoyl, 2-carboxybenzoyl Carboxylated arylcarbonyl groups such as, 3-carboxybenzoyl and 4-carboxybenzoyl; nitrated arylcarbonyl groups such as 4-nitrobenzoyl and 2-nitrobenzoyl; 2- (methoxycarbonyl) benzoyl Lower alkoxycarbonylated arylcarbonyl , An acylated protecting group such as an "aromatic acyl group" such as an arylated arylcarbonyl group such as 4-phenylbenzoyl; methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl,
1-methylpentyl, 3,3-dimethylbutyl, 2,2
-Dimethylbutyl, 1,1-dimethylbutyl, 1,2-
"Lower alkyl" such as dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl; ethenyl, 1-propenyl, 2-propenyl,
1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-
Propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl-1-butenyl, 3-methyl-2-butenyl, 1-
Ethyl-2-butenyl, 3-butenyl, 1-methyl-3
-Butenyl, 2-methyl-3-butenyl, 1-ethyl-
3-butenyl, 1-pentenyl, 2-pentenyl, 1-
Methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2
-Methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1
-Hexenyl, 2-hexenyl, 3-hexenyl, 4-
“Lower alkenyl groups” such as hexenyl, 5-hexenyl; arylcarbonyl groups such as benzoyl, α-naphthoyl, β-naphthoyl, 2-bromobenzoyl,
Halogenoarylcarbonyl groups such as 4-chlorobenzoyl, lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl, 4-toluoyl,
Lower alkoxylated arylcarbonyl group such as 4-anisoyl, 2-carboxybenzoyl, 3-carboxybenzoyl, carboxylated arylcarbonyl group such as 4-carboxybenzoyl, 4-nitrobenzoyl,
A nitrated arylcarbonyl group such as 2-nitrobenzoyl; a lower alkoxycarbonylated arylcarbonyl group such as 2- (methoxycarbonyl) benzoyl;
"Aromatic acyl group" such as arylated arylcarbonyl group such as 4-phenylbenzoyl; tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydro "Tetrahydropyranyl or tetrahydrothiopyranyl group" such as thiopyran-2-yl and 4-methoxytetrahydrothiopyran-4-yl; tetrahydrofuran
"Tetrahydrofuranyl or tetrahydrothiofuranyl group" such as -2-yl, tetrahydrothiofuran-2-yl; trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl And tri-lower alkylsilyl groups such as triisopropylsilyl, tri-lower alkylsilyl groups substituted with one or two aryl groups such as diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, and phenyldiisopropylsilyl. "Silyl group";
"Lower alkoxymethyl groups" such as methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, t-butoxymethyl; "such as 2-methoxyethoxymethyl" Lower alkoxylated lower alkoxymethyl group "; 2,2,2-trichloroethoxymethyl, bis (2
"Halogeno lower alkoxymethyl" such as -chloroethoxy) methyl; "lower alkoxylated ethyl groups" such as 1-ethoxyethyl, 1- (isopropoxy) ethyl; "such as 2,2,2-trichloroethyl Ethyl halide group "; benzyl, α-naphthylmethyl, β-
"Methyl group substituted with 1 to 3 aryl groups" such as naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl; 4-methylbenzyl, 2,4,6 -Trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, "Lower alkyl, lower alkoxy, halogen, methyl group substituted with one to three aryl groups substituted with an aryl ring with a cyano group" such as 4-bromobenzyl and 4-cyanobenzyl; methoxycarbonyl, ethoxycarbonyl Lower amines such as, t-butoxycarbonyl and isobutoxycarbonyl "Coxycarbonyl group";"aryl group substituted by halogen atom, lower alkoxy group or nitro group" such as 4-chlorophenyl, 2-chlorophenyl, 4-methoxyphenyl, 4-nitrophenyl, and 2,4-dinitrophenyl "A lower alkoxycarbonyl group substituted by a halogen or a tri-lower alkylsilyl group" such as 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl; "alkenyloxycarbonyl" such as vinyloxycarbonyl and aryloxycarbonyl Group "; benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-
One or two "aralkyloxycarbonyl groups in which the aryl ring may be substituted with a lower alkoxy or nitro group" such as dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and 4-nitrobenzyloxycarbonyl. .

Examples of the protecting group for a hydroxyl group in the definition of B ¹ include the protecting groups for Y ¹ and Y ² described above.
The protecting group for the amino group is not particularly limited as long as it can stably protect the amino group during nucleic acid synthesis.
Specifically, a protecting group that is stable under acidic or neutral conditions and that can be cleaved by a chemical method such as hydrogenolysis, hydrolysis, electrolysis and photolysis, such as formyl, acetyl, propionyl, Butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl,
Dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,1
3-dimethyltetradecanoyl, heptadecanoyl, 1
5-methylhexadecanoyl, octadecanoyl, 1-
Alkylcarbonyl groups such as methylheptadecanoyl, nonadecanoyl, eicosanoyl and henicosanoyl, succinoyl, glutaroyl, carboxylated alkylcarbonyl groups such as adipoyl, chloroacetyl,
Halogeno lower alkylcarbonyl groups such as dichloroacetyl, trichloroacetyl, trifluoroacetyl,
“Aliphatic acyl group” such as lower alkoxy lower alkylcarbonyl group such as methoxyacetyl, unsaturated alkylcarbonyl group such as (E) -2-methyl-2-butenoyl; benzoyl, α-naphthoyl and β-naphthoyl Arylcarbonyl group, 2-bromobenzoyl,
Halogenoarylcarbonyl groups such as 4-chlorobenzoyl, lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl, 4-toluoyl,
Lower alkoxylated arylcarbonyl group such as 4-anisoyl, 2-carboxybenzoyl, 3-carboxybenzoyl, carboxylated arylcarbonyl group such as 4-carboxybenzoyl, 4-nitrobenzoyl,
A nitrated arylcarbonyl group such as 2-nitrobenzoyl; a lower alkoxycarbonylated arylcarbonyl group such as 2- (methoxycarbonyl) benzoyl;
An "aromatic acyl group" such as an arylated arylcarbonyl group such as 4-phenylbenzoyl; a "lower alkoxycarbonyl group" such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl; 2,2,2 -Trichloroethoxycarbonyl,
A "lower alkoxycarbonyl group substituted with a halogen or a tri-lower alkylsilyl group" such as 2-trimethylsilylethoxycarbonyl; vinyloxycarbonyl,
“Alkenyloxycarbonyl group” such as aryloxycarbonyl; 1 such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl And two or more aralkyloxycarbonyl groups in which the aryl ring may be substituted with a lower alkoxy or nitro group, and preferably an "aliphatic acyl group" or an "aromatic acyl group". And more preferably,
It is a benzoyl group.

As the “group forming a leaving group” of R ⁷ ,
Examples thereof include lower alkylsulfonyl groups such as methanesulfonyl and ethanesulfonyl; halogen-substituted lower alkylsulfonyl groups such as trifluoromethanesulfonyl; and arylsulfonyl groups such as p-toluenesulfonyl. It is a sulfonyl group or a p-toluenesulfonyl group.

The "C2-C4 aliphatic acyl group" for R ⁸ includes, for example, acetylpropionyl, butyryl and the like, and is preferably an acetyl group.

Hereinafter, each step of the method A will be described in detail. (Step A-1) In this step, the compound (3) produced by a method B to D described below in an inert solvent in the presence of a base catalyst
And reacting a leaving group-introducing reagent to produce a compound (4).

Examples of the solvent used include aliphatic hydrocarbons such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform,
Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran; Ethers such as dioxane, dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; acetonitrile and isobutyronitrile Nitriles; formamide, N, N
Amides such as -dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, hexamethylphosphorotriamide; sulfoxides such as sulfolane; and pyridines. , Preferably pyridine.

As the base catalyst used, preferably,
Bases such as triethylamine, pyridine, dimethylaminopyridine.

Examples of the leaving group-introducing reagent to be used include alkylsulfonyl halides such as methanesulfonyl chloride and ethanesulfonyl bromide; and arylsulfonyl halides such as p-toluenesulfonyl chloride. Are methanesulfonyl chloride and p-toluenesulfonyl chloride.

The reaction temperature varies depending on the starting compound, solvent, leaving group-introducing reagent and base catalyst used.
It is 0 to 50 ° C, preferably 10 to 40 ° C.

The reaction time varies depending on the starting compound used, the solvent, the leaving group-introducing reagent, the base catalyst and the reaction temperature, but is usually from 10 minutes to 24 hours, preferably from 1 to 10 hours. .

After completion of the reaction, the target compound (4) of this reaction is
For example, neutralize the reaction solution, concentrate the reaction mixture, add an immiscible organic solvent such as water and ethyl acetate, wash with water,
The organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step A-2) This step is carried out in a solvent in the presence of an acid catalyst.
-1 The step of reacting the compound (4) produced in the step 1 with an acid anhydride to produce the compound (5).

Examples of the solvent used include ethers such as diethyl ether, dioxane and tetrahydrofuran; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethyl Examples include amides such as acetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, and hexamethyl phosphorotriamide; organic acids such as acetic acid, and acetic acid is preferred.

Examples of the acid catalyst to be used include inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and preferred is sulfuric acid (particularly concentrated sulfuric acid).

Examples of the acid anhydride to be used include anhydrides of lower aliphatic carboxylic acids such as acetic anhydride and propionic anhydride, and acetic anhydride is preferred.

The reaction temperature varies depending on the starting compound, solvent, acid catalyst and acid anhydride used, but is usually from 0 ° C to 50 ° C, preferably from 10 to 40 ° C.

The reaction time varies depending on the starting compounds used, the solvent, the acid catalyst, the acid anhydride and the reaction temperature, but is usually from 10 minutes to 12 hours, preferably from 30 minutes to 3 hours.

After completion of the reaction, the target compound (5) of this reaction is
For example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. can get.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step A-3) In this step, compound (5) produced in step A-2 in an inert solvent in the presence of an acid catalyst is prepared according to literature (H.
Vorbrggen, K. Krolikiewicz and B, Bennua, Chem. B
er., 114, 1234-1255 (1981)), and reacting a trimethylsilylated compound corresponding to a purine or pyrimidine which may have a desired substituent, to obtain a compound (6).
This is the step of manufacturing.

Examples of the solvent used include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, chlorobenzene and dichlorobenzene. Nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N
-Methyl-2-pyrrolidone, N-methylpyrrolidinone,
Amides such as hexamethyl phosphorotriamide; carbon sulfide and the like, but preferably 1,2-dichloroethane.

Examples of the acid catalyst used include, for example, AlCl
₃ , SnCl ₄ , TiCl ₄ , ZnCl ₂ , BF ₃ , Lewis acid catalysts such as trimethylsilyl trifluoromethanesulfonate, and the like, preferably trimethylsilyl trifluoromethanesulfonate.

The reaction temperature varies depending on the starting compound, solvent and acid catalyst used, but is usually from 0 ° C. to 100 ° C., preferably from 50 ° C. to 80 ° C.

The reaction time varies depending on the starting compound used, the solvent, the acid catalyst and the reaction temperature, but is usually 1 hour to 24 hours, preferably 1 hour to 8 hours.

After completion of the reaction, the target compound (6) of this reaction is
For example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. can get.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step A-4) This step is a step of cyclizing the compound (6) produced in the step A-3 in an inert solvent in the presence of a base catalyst to produce a compound (7).

Examples of the solvent used include water; pyridines; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-methyl-2-amide.
Amides such as pyrrolidone, N-methylpyrrolidinone, and hexamethylphosphorotriamide; or a mixed solvent thereof, preferably a mixed solvent of water and pyridine.

Examples of the base catalyst used include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; sodium methoxide and sodium ethoxide. Such alkali metal alkoxides; ammonia water and the like can be mentioned, and preferred are alkali metal hydroxides (particularly sodium hydroxide).

The reaction temperature varies depending on the starting compound, solvent and acid catalyst used, but is usually from 0 ° C. to 50 ° C., preferably from 10 ° C. to 30 ° C.

The reaction time varies depending on the starting compound used, the solvent, the acid catalyst, and the reaction temperature.
Time, preferably 1 minute to 30 minutes.

After completion of the reaction, the target compound (7) of this reaction is
For example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. can get.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography or the like. (Step A-5) This step is a step of reacting the compound (7) obtained in Step A-4 with a deprotection reagent in an inert solvent to produce a compound (8).

The method of deprotection varies depending on the type of protecting group, but is not particularly limited as long as it does not cause other side reactions. For example, "Protective Groups in Organic Syn
thesis ”(by Theodora W. Greene, Peter GMWuts,
1999, published by A Wiley-Interscience Publication).

In particular, when the protecting group is (1) an “aliphatic acyl group or an aromatic acyl group”, (2) a “methyl group substituted with 1 to 3 aryl groups” or a “lower alkyl, lower alkoxy, A methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a halogen or cyano group ",
(3) In the case of a "silyl group", it can be carried out by the following method. (1) In the case of an aliphatic acyl group and an aromatic acyl group, the reaction is usually carried out by reacting a base in an inert solvent.

The solvent to be used is not particularly limited as long as it is easily mixed with water, does not inhibit the reaction, and dissolves the starting material to a certain extent or more.
Amides such as dimethylformamide, dimethylacetamide; methylene chloride, chloroform, 1,2-
Halogenated hydrocarbons such as dichloroethane or carbon tetrachloride; ethers such as tetrahydrofuran, diethyl ether and dioxane are preferred, and ethers are preferred, and tetrahydrofuran is more preferred.

Examples of the base to be used include alkali metal hydroxides such as lithium hydroxide, potassium hydroxide and sodium hydroxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; sodium methoxide and sodium ethoxide Alkali metal alkoxides; ammonia water, and ammonia solutions such as ammonia / methanol solutions.

The reaction temperature is 0 ° C. to 60 ° C., preferably 20 ° C. to 40 ° C.

The reaction time is from 10 minutes to 24 hours,
Preferably, it is 1 to 3 hours.

After completion of the reaction, the target compound (8) of this reaction is
For example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. can get.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization or silica gel column chromatography. (2) a protective group in which the protecting group is substituted by "1 to 3 aryl groups substituted by 1 to 3 aryl groups" or "1 to 3 aryl groups in which the aryl ring is substituted by lower alkyl, lower alkoxy, halogen or cyano group"; In the case of “a methyl group”, the reaction is performed using a reducing agent in an inert solvent.

Examples of the solvent used include alcohols such as methanol, ethanol and isopropanol; ethers such as diethyl ether, tetrahydrofuran and dioxane; toluene, benzene and xylene. Such as aromatic hydrocarbons; aliphatic hydrocarbons such as hexane and cyclohexane; esters such as ethyl acetate and propyl acetate; organic acids such as acetic acid; or mixed solvents of these organic solvents and water are preferred. It is.

The reducing agent to be used is not particularly limited as long as it is usually used in a catalytic reduction reaction.
Preferably, palladium carbon, Raney-nickel, platinum oxide, platinum black, rhodium-aluminum oxide, triphenylphosphine-rhodium chloride, and palladium-barium sulfate are used.

The pressure is not particularly limited, but is usually 1 to 1
Performed at 0 atm.

The reaction temperature is from 0 ° C. to 60 ° C., preferably from 20 ° C. to 40 ° C.

The reaction time is from 10 minutes to 24 hours,
Preferably, it is 1 to 3 hours.

After completion of the reaction, the target compound (8) of this reaction is
For example, the reducing agent is removed from the reaction mixture, an immiscible organic solvent such as water and ethyl acetate is added, and after washing with water, the organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off. Obtained by leaving.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

In the case of "a methyl group substituted with three aryl groups", that is, a trityl group, the reaction can be carried out using an acid.

In this case, the solvent to be used includes aromatic hydrocarbons such as benzene, toluene and xylene;
Methylene chloride, chloroform, carbon tetrachloride, 1,2
-Halogenated hydrocarbons such as dichloroethane, chlorobenzene, dichlorobenzene; methanol, ethanol-
Alcohols such as toluene, isopropanol and tert-butanol; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-
Amides such as 2-pyrrolidone, N-methylpyrrolidinone, and hexamethyl phosphorotriamide; organic acids such as acetic acid; and organic acids (particularly acetic acid) or alcohols (particularly tert. -Butanol).

The acid used is preferably acetic acid or trifluoroacetic acid.

The reaction temperature is from 0 ° C. to 60 ° C., preferably from 20 ° C. to 40 ° C.

The reaction time is from 10 minutes to 24 hours,
Preferably, it is 1 to 3 hours.

After completion of the reaction, the target compound (8) of this reaction is
For example, neutralizing the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate, etc., and distilling off the solvent. Is obtained. The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (3) When the protecting group is a “silyl group”, it is usually treated with a compound that generates a fluorine anion, such as tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, and potassium fluoride. Or acetic acid, methanesulfonic acid,
It can be removed by treatment with an organic acid such as paratoluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid or an inorganic acid such as hydrochloric acid.

In the case of removal by fluorine anion,
The reaction may be accelerated by adding organic acids such as formic acid, acetic acid and propionic acid.

The solvent to be used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent. Preferably, diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxy Ethers such as ethane and diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; water; organic acids such as acetic acid; and mixed solvents thereof.

The reaction temperature is from 0 ° C. to 100 ° C., preferably from 20 ° C. to 70 ° C.

The reaction time is 5 minutes to 48 hours, preferably 1 to 24 hours.

After completion of the reaction, the target compound (8) of this reaction was
For example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. can get. If necessary, the obtained compound can be further purified by a conventional method, for example, recrystallization or silica gel column chromatography. (Step A-6) This step is a step of producing a compound (9) by reacting the compound (8) obtained in the step A-5 with a deprotection reagent in an inert solvent.

The method of deprotection varies depending on the type of the protecting group, but is not particularly limited as long as it does not cause other side reactions.
thesis ”(Theodora W. Greene, 1981, A Wiley-
Interscience Publication).

In particular, when the protecting group is an aliphatic acyl group or an aromatic acyl group, it can be carried out by the following method.

That is, when the protecting group is an aliphatic acyl group or an aromatic acyl group, the reaction is usually carried out by reacting a base in an inert solvent.

The solvent used is not particularly limited as long as it is easily mixed with water, does not inhibit the reaction, and dissolves the starting material to a certain extent or more.
Alcohols such as methanol and ethanol; amides such as dimethylformamide and dimethylacetamide; halogenated hydrocarbons such as methylene chloride, chloroform, 1,2-dichloroethane or carbon tetrachloride;
Examples include ethers such as tetrahydrofuran, diethyl ether and dioxane, preferably alcohols, and more preferably methanol.

Examples of the base used include alkali metal hydroxides such as lithium hydroxide, potassium hydroxide and sodium hydroxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; sodium methoxide and sodium ethoxide And an alkali metal alkoxide such as ammonia; and ammonia is preferred.

The reaction temperature is from 0 ° C. to 50 ° C., preferably from 10 ° C. to 40 ° C.

The reaction time is from 10 minutes to 24 hours,
Preferably, it is 10 to 15 hours. After completion of the reaction, for example, the reaction mixture is concentrated, an immiscible organic solvent such as water and ethyl acetate is added, and after washing with water, the organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate and the like, and the solvent is distilled off. Obtained by leaving.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step A-7) This step is a step of producing a compound (10) by introducing a phosphate group or a thiophosphate group into the 5′-hydroxyl group of the compound (9) obtained in step A-6 in a solvent. It is.

Examples of the solvent used include ethers such as diethyl ether, dioxane and tetrahydrofuran; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethyl Amides such as acetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, and hexamethyl phosphorotriamide; phosphoric acid triesters such as trimethyl phosphate; It is.

The phosphorylating agent to be used is not particularly limited as long as it is usually used for nucleic acid synthesis. Preferably, chlorides, anhydrides and amides, which are active species of phosphoric acid, and corresponding salts are used. A thiophosphoric acid derivative, more preferably,
Phosphoryl chloride or thiophosphoryl chloride.

The reaction temperature varies depending on the starting compound, solvent and phosphorylating agent used, but is usually from −40 ° C. to 3 ° C.
It is 0 ° C, preferably -5 to 5 ° C.

The reaction time varies depending on the starting compound, solvent and phosphorylating agent used, but is usually from 30 minutes to 12 minutes.
Time, preferably 3 to 6 hours.

After completion of the reaction, the target compound of this reaction is neutralized with, for example, an aqueous solution of sodium hydrogen carbonate,
An aqueous solution is obtained by adding an organic solvent such as chloroform and washing away water-insoluble substances.

The obtained compound can be further purified, if necessary, by a conventional method, for example, reverse phase liquid chromatography, ion exchange chromatography and the like. (A-8) This step is a step of reacting the compound (10) obtained in the step A-5 with pyrophosphoric acid in a solvent to produce the compound (1) of the present invention.

Examples of the solvent to be used include ethers such as diethyl ether, dioxane and tetrahydrofuran; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethyl Amides such as acetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, and hexamethylphosphorotriamide can be mentioned, and N, N-dimethylformamide is preferred.

As the phosphorylating agent to be used, pyrophosphoric acid and various salts thereof are preferable, and pyrophosphoric acid-tributylamine salt is preferable.

The condensing agent to be used is not particularly limited as long as it is used for phosphorylation of nucleic acid. Examples of such a dehydrating condensing agent include (a) 1,3-dicyclohexylcarbodiimide, ,
3-diisopropylcarbodiimide, 1-ethyl-3-
Carbodiimides such as (3-dimethylaminopropyl) carbodiimide; combinations of the carbodiimides with the following bases; the carbodiimides and N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-di A combination of N-hydroxys such as carboximide; (b) N, N'-disuccinimidyl carbonate, di-2-pyridyl carbonate, S, S'-bis (1-
Carbonates such as phenyl-1H-tetrazol-5-yl) dithiocarbonate; (c) N, N'-disuccinimidyl oxalate;
N, N'-diphthalimide oxalate, N, N'-bis (5-norbornene-2,3-dicarboximidyl) oxalate, 1,1'-bis (benzotriazolyl) oxalate, 1,1 Oxalates such as' -bis (6-chlorobenzotriazolyl) oxalate and 1,1'-bis (6-trifluoromethylbenzotriazolyl) oxalate; (d) N-ethyl-5-phenylisoxa Zolium-
N-lower alkyl-5-arylisoxazolium-3'-sulfonates such as 3'-sulfonate; (e) diheteroaryl diselenides such as di-2-pyridyldiselenide; (f ) 1,1'-oxalyldiimidazole, 1,1 '
Imidazoles such as -carbonyldiimidazole, N, N'-carbonyldiimidazole; (g) 3-ethyl-2-chloro-benzothiazolium
3-h-lower alkyl-2-halogen-benzothiazolium fluoroborates such as fluoroborate; (h) 3-lower alkyl-benzothiazole-2-cellones such as 3-methyl-benzothiazole-2-cellone (I) phosphates such as phenyldichlorophosphate and polyphosphate esters; (j) halogenosulfonyl isocyanates such as chlorosulfonyl isocyanate; and (k) halogenosilanes such as trimethylsilyl chloride and triethylsilyl chloride. Preferably, they are imidazoles, more preferably 1,1'-carbonyldiimidazole.

The reaction temperature varies depending on the starting compound, solvent and phosphorylating agent used, but it is usually from −40 ° C. to 3 ° C.
It is 0 ° C, preferably -5 to 5 ° C.

The reaction time varies depending on the starting compound, solvent and phosphorylating agent used, but is usually from 4 hours to 40 hours.
Hours, preferably 20 hours.

After completion of the reaction, the target compound of this reaction can be purified by a conventional method, for example, ion exchange chromatography.

The above-mentioned intermediate (3) has the following structure
To D method.

[0104]

Embedded image

In the methods B to D, A, X, Y ¹ and Y ² have the same meanings as described above, R ⁹ represents a group forming a leaving group together with an oxygen atom, and R ¹¹ and R ¹² represent Are the same and represent a hydrogen atom or together represent an oxygen atom, and Z represents a single bond, a methylene or ethylene group.

Examples of the group forming a leaving group together with the oxygen atom of R ⁹ include the same groups as those described above for R ⁷ , preferably a trifluoromethanesulfonyl group.

R^TenIs R¹¹And R¹²Together with oxygen
When referring to an atom, methyl, ethyl, propyl, iso
Propyl, butyl, isobutyl, sec-butyl, tert-butyl
An alkyl group having 1 to 4 carbon atoms such as tyl;
Suitably, it is a methyl group, ¹¹And R¹²Are the same
In the case of a hydrogen atom, an aralkyl such as a benzyl group
Groups; alkoxyalkyl groups such as methoxymethyl groups;
Benzyloxymethyl, such as a benzyloxymethyl group
Or an aralkyloxy group such as a benzyloxymethyl group
Alcohol such as methoxyethoxymethyl group
Xyalkoxyalkyl group; trimethylsilyl, t-butyl
Dimethylsilyl, diphenylmethylsilyl, diphenyl
Rubutylsilyl, diphenylisopropylsilyl,
Include silyl groups such as nildiisopropylsilyl
Can be.

Compound (11), which is a starting compound used in Method B or Method C, can be produced by the following method.

That is, commercially available 1,2,5,6-diisopropylidene D-glucose was used as a starting material, and a known method (RDYou
ssefyeh, JPHVerheyden, JGMoffatt. J.Org.Che
m., 44, 1301-1309 (1979)) to produce a compound in which the "X" portion of compound (11) corresponds to a hydrogen atom, and then according to a known method (Japanese Patent Application Laid-Open No. 10-304889). Can be manufactured. Further, a commercially available 1,2,5,6-diisopropylidene D-glucose is used as a starting material, and a known method (Mersmaeke
r, Alain De, Leberton, Jacques, Jouanno, Chantal,
Fritsh, Valerie, Wolf, Romain M., Wedenborn, Sebas
tian, Syn. Lett., 11, 1287-1290) (1997)).
-Diisopropylidene D-allofuranose was synthesized and used in a known manner (Wood, William W., Watson, Gra
ham M., J. Chem. Soc. Chem. Commun., 21, 1599-1600.
(1986))

[0110]

Embedded image

The aldehyde group is then synthesized by a conventional method (eg, Hudlicky "Reductionsin Organic Chemi
stry ", a method described in Ellis Horwood (1984), etc.) and a known method (Japanese Patent Laid-Open No. 10-304889).
Can be obtained according to

The steps B to D will be described in detail below. (Method B) (Step B-1) In this step, a compound (11) produced by the above-mentioned method is reacted with a leaving group-introducing reagent in an inert solvent in the presence of a base catalyst to give a compound ( This is the step of manufacturing 12).

Examples of the solvent used include amides such as dimethylformamide and dimethylacetamide; halogenated hydrocarbons such as methylene chloride, chloroform, 1,2-dichloroethane and carbon tetrachloride;
Tetrahydrofuran, diethyl ether, ethers such as dioxane can be mentioned, preferably,
Methylene chloride.

The base catalyst used is preferably
Bases such as triethylamine, pyridine, dimethylaminopyridine.

The leaving group-introducing reagent used is preferably trifluoromethanesulfonic acid chloride or trifluoromethanesulfonic anhydride.

The reaction temperature varies depending on the starting compound, solvent and acid catalyst used, but is usually from -100 ° C to -5 ° C.
0 ° C, and preferably -100 ° C to -70 ° C.

The reaction time varies depending on the used starting compound, solvent, acid catalyst and reaction temperature, but is usually 30 minutes to 12 hours, preferably 30 minutes to 3 hours.

After completion of the reaction, the desired compound (12) of the present reaction is obtained.
For example, after concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. Obtained by:

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step B-2) This step is a step of producing a compound (13) by reacting the compound (12) produced in the step B-1 with a cyanating reagent in an inert solvent.

Examples of the solvent used include amides such as dimethylformamide and dimethylacetamide; halogenated hydrocarbons such as methylene chloride, chloroform, 1,2-dichloroethane and carbon tetrachloride;
Ethers such as tetrahydrofuran, diethyl ether and dioxane; acetonitrile; dimethylsulfoxide and the like can be mentioned, and amides (dimethylformamide) are preferred.

Examples of the cyanating reagent used include KCN, NaCN, and trimethylsilane cyanide. NaCN is preferred.

The reaction temperature varies depending on the starting compound, solvent and cyanating reagent used, but is usually from 0 ° C. to 10 ° C.
0 ° C. and 30 ° C. to 70 ° C.

The reaction time varies depending on the used starting compound, solvent, cyanating reagent and reaction temperature.
It is from 0 minutes to 12 hours, preferably from 1 to 3 hours.

After completion of the reaction, the desired compound (13) of the present reaction is obtained.
For example, after concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. Obtained by:

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step B-3) This step is a step of producing a compound (14) by reacting the compound (13) produced in the step B-2 with a reducing agent in an inert solvent.

Examples of the solvent used include halogenated hydrocarbons such as methylene chloride, chloroform, 1,2-dichloroethane or carbon tetrachloride; hexane,
Aliphatic hydrocarbons such as heptane, ligroin and petroleum ether; aromatic hydrocarbons such as benzene, toluene and xylene; diethyl ether, diisopropyl ether
Ethers such as ter, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; and the like. Hydrogens (especially methylene chloride).

Examples of the reducing agent to be used include diisobutylaluminum hydrogen, triethoxyaluminum hydrogen and the like, and preferred is diisobutylaluminum hydride.

The reaction temperature is different from -100 ° C to -50 ° C, preferably from -90 ° C to -70, depending on the starting compound, solvent and reducing agent used.

The reaction time varies depending on the used starting compound, solvent, reducing agent and reaction temperature, but is usually 30 minutes to 12 hours, and preferably 1 to 5 hours.

After completion of the reaction, the desired compound (14)
For example, after concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. Obtained by:

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step B-4) In this step, compound (14) produced in step B-3 is reacted with a reducing agent in an inert solvent to give compound (3a) which is one of the starting compounds for method A This is the step of manufacturing.

Examples of the solvent used include methanol, ethanol, n-propanol and isopropanol.
, N-butanol, isobutanol, t-butanol
Alcohols such as toluene, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol and methyl cellosolve; acetic acid and the like, and preferably alcohols (particularly ethanol).

Examples of the reducing agent used include alkali metal borohydrides such as sodium borohydride and lithium borohydride; aluminum hydride compounds such as lithium aluminum hydride and lithium triethoxide aluminum. Borane and the like, but preferably sodium borohydride.

The reaction temperature varies depending on the starting compound, solvent and reducing agent used, but is usually from 0 ° C to 50 ° C, preferably from 10 ° C to 40 ° C.

The reaction time varies depending on the starting compound used, the solvent, the reducing agent and the reaction temperature, but is usually from 10 minutes to 12 hours, preferably from 30 minutes to 5 hours.

After completion of the reaction, the desired compound (3a) of this reaction
For example, after decomposing the reducing agent, concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate, etc. Obtained by distilling off the solvent.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Method C) (Step C-1) This step is a step of producing a compound (15) by reacting the compound (11) produced by the above-mentioned method with an oxidizing agent in an inert solvent.

Examples of the solvent used include aliphatic hydrocarbons such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform,
Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran; Ethers such as dioxane, dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; and preferred are halogenated hydrocarbons (particularly, , Methylene chloride).

As the oxidizing agent used, Swer (Swer)
n) Oxidizing reagent, Dess-Martin oxidizing reagent,
A chromium trioxide complex such as pyridine hydrochloride / chromium trioxide complex (pyridinium chlorochromate, pyridinium dichromate) can be mentioned, but a preferable reagent is a reagent for swan oxidation (that is, dimethyl sulfoxide-oxalyl). Chloride).

Although the reaction temperature varies depending on the starting compound, solvent and oxidizing agent used, it is usually -100 ° C to -5 ° C.
0 ° C., preferably -100 to −70 ° C.

The reaction time varies depending on the used starting compound, solvent, oxidizing agent and reaction temperature, but is usually 30 minutes to 12 hours, preferably 1 to 5 hours.

After the completion of the reaction, the desired compound (15)
For example, decompose the oxidizing agent, concentrate the reaction mixture, add an immiscible organic solvent such as water and ethyl acetate, wash with water, separate the organic layer containing the target compound, dry with anhydrous magnesium sulfate, etc. Obtained by distilling off the solvent.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step C-2) In this step, the compound (15) produced in Step C-1 is reacted with a carbon-enhancing reagent in an inert solvent,
This is a step of producing a compound (16).

Examples of the solvent to be used include aliphatic hydrocarbons such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform,
Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran; Ethers such as dioxane, dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; and preferred are halogenated hydrocarbons (particularly , Methylene chloride).

The reagents used were Wittig (W
ittig) reagent, Horner-Emmons reagent, Peterson reaction reagent, TiCl ₄ -CH ₂ Cl ₂ -Zn
Examples of the reagent include a system reagent, a Tebbe reagent, and the like, and preferred are a Wittig reagent, a Horner-Emmons reagent, and a Thebe reagent.

The reaction temperature depends on the starting compound, solvent and carbon-enriching reagent used, but it is usually from -20 ° C to 2 ° C.
It is 0 ° C, preferably 0 ° C.

The reaction time varies depending on the starting compound used, the solvent, the carbon-enhancing reagent and the reaction temperature, but is usually 30 minutes to 12 hours, preferably 1 to 5 hours.

After completion of the reaction, the desired compound (16)
For example, after concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. Obtained by:

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step C-3) This step is a step of selectively introducing a hydroxyl group into the terminal carbon of the olefin of compound (16) produced in step C-2 in an inert solvent to produce compound (3a). It is.

Examples of the solvent to be used include aliphatic hydrocarbons such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform,
Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran; Ethers such as dioxane, dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone, and preferably ethers (particularly tetrahydrofuran) is there.

Examples of the reaction reagent used include borane, disiamylborane, sexylborane, 9-BBN (9-borabicyclo [3.3.1] nonane) and the like, preferably 9-BBN.

The reaction temperature varies depending on the starting compounds, solvents and reagents used, but is from 0 ° C to 50 ° C, preferably from 10 ° C to 40 ° C.

The reaction time varies depending on the starting compounds used, the solvent, the reagent and the reaction temperature, but is usually from 6 to 48.
Hours, preferably 12 to 24 hours.

After completion of the reaction, the target compound (3a)
For example, after concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. Obtained by:

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Method D) (Step D-1) In this step, a compound (15) produced in step C-1 is reacted with an increasing carbon reagent in an inert solvent,
This is a step of producing a compound (17).

Examples of the solvent used include aliphatic hydrocarbons such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform,
Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran; Ethers such as dioxane, dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone, preferably ethers (particularly tetrahydrofuran) And more preferably halogenated hydrocarbons (particularly methylene chloride).

Examples of the carbon-enriching reagent used include Wittig reagent, Horner-Emmon.
s) Reagents and the like can be mentioned.

The reaction temperature varies depending on the starting compounds, solvents and reagents used, but is usually from -20 ° C to 40 ° C, preferably from 0 to 20 ° C.

The reaction time varies depending on the starting compounds used, the solvent, the reagent and the reaction temperature.
Time, preferably 1 to 5 hours.

After completion of the reaction, the desired compound (17) of the present reaction is obtained.
For example, after concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent. Obtained by:

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization or silica gel column chromatography. (Step D-2) This step is a step of producing a compound (18) by reacting the compound (17) produced in the step D-1 with a reducing agent in an inert solvent.

This step can be carried out according to (2) of step A-5. However, when R ¹⁰ is a benzyl group which may have a substituent and R ¹¹ and R ¹² are hydrogen atoms, compound (3b) can be directly produced by this step. . (Step D-3) In this step, the compound (18) produced in Step D-2 is reacted with a reducing agent in an inert solvent to give A
This is a step of producing a compound (3b) which is one of the starting compounds of the method. (A) When R ¹¹ and R ¹² are Together an Oxygen Atom The solvents used include, for example, methanol, ethanol, n-propanol, isopropanol, and n-butanol. Alcohols such as toluene, isobutanol, t-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol and methyl cellosolve; acetic acid and the like, preferably alcohol. (Especially ethanol).

The reducing agent used includes, for example, an alkali metal borohydride such as lithium borohydride;
Aluminum hydride compounds such as lithium aluminum hydride and lithium triethoxide aluminum; borane and the like can be mentioned, and preferably borane or lithium aluminum hydride.

The reaction temperature varies depending on the starting compound, solvent and reducing agent used, but is usually from 0 ° C to 50 ° C, preferably from 10 ° C to 40 ° C.

The reaction time varies depending on the starting compound used, the solvent, the reducing agent and the reaction temperature, but is usually 10 minutes to 12 hours, preferably 30 minutes to 5 hours.

After completion of the reaction, the desired compound (3b) of the reaction is obtained.
For example, after decomposing the reducing agent, concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate, etc. Obtained by distilling off the solvent.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (B) When R ¹¹ and R ¹² are hydrogen and R ¹⁰ is other than a benzyl group When R ¹⁰ is a silyl group, it can be carried out according to the method (3) of Step A-5. .

R ¹⁰ is an aralkyl group such as a benzyl group;
In the case of an alkoxyalkyl group such as a methoxymethyl group; an aralkyloxymethyl group such as a benzyloxymethyl group or a benzyloxymethyl group such as a benzyloxymethyl group; and an alkoxyalkoxyalkyl group such as a methoxyethoxymethyl group. , using an acid catalyst, the acid catalyst used in this case p- toluenesulfonic acid, trifluoroacetic acid, an organic acid such as dichloroacetic acid, BF _3, can be mentioned Lewis acids such as AlCl _3.

Examples of the solvent used include aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform, carbon tetrachloride, 1,2-
Halogenated hydrocarbons such as dichloroethane, chlorobenzene and dichlorobenzene; nitriles such as acetonitrile and isobutyronitrile; formamide, N,
Amides such as N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, hexamethylphosphorotriamide; and carbon sulfide.

The reaction temperature varies depending on the starting compound, solvent and acid catalyst used, but is usually from 0 ° C. to 50 ° C., preferably from 10 ° C. to 40 ° C.

The reaction time varies depending on the used starting compound, solvent, acid catalyst and reaction temperature, but is usually 10 minutes to 12 hours, preferably 30 minutes to 5 hours.

After completion of the reaction, the desired compound (3b) of this reaction
For example, neutralize the reaction mixture, add an immiscible organic solvent such as water and ethyl acetate, wash with water, separate the organic layer containing the target compound, dry over anhydrous magnesium sulfate, and distill off the solvent. It is obtained by doing.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

The gene amplification method using RNA polymerase is as follows. (Test method) Gene amplification method using RNA polymerase Double-stranded DNA having a promoter sequence corresponding to the RNA polymerase to be used is dissolved in an RNA polymerase reaction buffer. At least one compound represented by the general formula (1) and, if necessary, a natural nucleoside dotriphosphate are added (however, the compound represented by the general formula (1) and the natural nucleoside triphosphate are added). Are four in total.) Then, RNA polymerase is added and the mixture is heated. After incubation for the required time (30 minutes to 4 hours),
Loading 2.5 μl part of sample (2.5 μl)
Solution (formamide, 0.01% bromophenol blue, 0.01% xylene cyanol) and analyze by 10% polyacrylamide gel electrophoresis containing 7M urea.
After the electrophoresis, the gel is wrapped in a wrap, placed on a TLC containing a fluorescent agent, and the gene generated by UV irradiation is analyzed. Also,
The reaction product was recovered by ethanol precipitation and ion-exchange HPLC.
Alternatively, the production of the target substance can be quantitatively analyzed by capillary electrophoresis.

When examining gene expression, (1) mRNA is extracted from cells or tissues, and cDNA is prepared by a reverse transcription reaction using a reverse transcriptase. (2) PCR was performed on the cDNA using a primer containing an RNA polymerase promoter (S
aiki et al. Science (1988) 239, 487-491)
Double-stranded DNA. (3) Using this DNA as a template, the gene can be amplified and gene expression analyzed by the above-described test method.

When performing mutation analysis or gene polymorphism analysis, DNA is extracted from cells or tissues, and then the steps (2) and (3) described above are performed. Can be analyzed.

Examples, reference examples and test examples are given below.
The present invention will be described in more detail.

[0178]

EXAMPLES (Example 1) 2'-O, 4'-C-Ethyleneuridine-5'-ammonium triphosphate
Unsalted (Compound No. 2-1) The compound obtained in Reference Example 32 (about 15 micromol) was dissolved in anhydrous DMF (2 ml), 1,1'-carbonyldiimidazole (16 mg, 0.1 mmol) was added, The mixture was stirred at 0 ° C under a nitrogen stream for 12 hours. Next, 1M tributylamine pyrophosphate (100 mg) was added, and the mixture was stirred at 0 ° C. under a nitrogen stream for 60 hours. The reaction solution is concentrated under reduced pressure and subjected to ion exchange high performance liquid chromatography (HPLC: LC- manufactured by Shimadzu Corporation).
VP; Column: Tosoh TSKgel DEAE-2SW; 20% CH ₃ CN-H ₂ O,
5 → 40% 1M HCOONH ₄ / 20min: linear gradient; 40
(1 ° C .; 1 ml / min), and fractions eluted at 15.8-17.4 minutes were collected. This compound is ion-exchange high performance liquid chromatography (column: TSKgel DEAE-2SW; 20% CH ₃ CN,
5 → 40% 1M HCOONH ₄ / 20min, linear gradient; 40
(° C; 1 ml / min), eluted at 17.84 minutes. The eluate was desalted using a gel filtration column (sephadex G-25, eluate:
Water), and after lyophilization, the target compound (4.2 µmol) was obtained (the yield was calculated from UV absorbance). UV: max = 265nm, min = 232nm ¹ H-NMR (400MHz, D ₂ O): 1.48 (1H, m), 2.09 (1H, dt, 9.
5 and 13Hz), 3.93 (2H, m), 4.06 (1H, d, 3.0Hz), 4.08
(2H, m), 4.21 (1H, d, 3.0Hz), 5.83 (1H, d, 8Hz), 5.9
7 (1H, s), 8.04 (1H, d, 8Hz). (Reference Example) (Reference Example 1) 3,5-di-O-benzyl-4-trifluoromethanesulfoni
Roxymethyl-1,2-O-isopropylidene-α-D-ery
Ro pentofuranosyl nitrogen stream, 3,5-di -O- benzyl-4-hydroxymethyl -1, 2-O-isopropylidene--.alpha.-D-erythro pentofuranosyl (2000 mg, 5.0 mmol) in anhydrous dichloromethane 50ml Dissolve and cool to -78 ° C. There, anhydrous pyridine (0.60 ml, 7.5 mmol) and trifluoromethanesulfonic anhydride (1010 m
g, 6.0 mmol) and stirred for 40 minutes.

After completion of the reaction, a saturated aqueous solution of sodium hydrogencarbonate (about 100 ml) was added to the reaction solution, and the mixture was separated. The organic layer was washed with a saturated aqueous solution of sodium hydrogencarbonate (about 100 ml) and a saturated saline solution (about 100 ml). , And dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain a white powder (2520 mg, 4.73 mmol, 95%), which was used for the next reaction as it was.

¹ H-NMR (400 MHz, CDCl ₃ ): 1.34 (3H, s),
1.63 (3H, s), 3.48 (1H, d, 10Hz), 3.53 (1H, d, 10Hz),
4.21 (1H, d, 5.0Hz), 4.5 (4H, m), 4.74 (1H, d, 12Hz),
4.80 (1H, d, 12Hz), 5.01 (1H, d, 12Hz), 5.73 (1H, d, 12Hz)
4.6Hz), 7.3 (10H, m) (Reference Example 2) 3,5-di-O-benzyl-4-cyanomethyl-1,2-O-isopro
Pyridene-α-D-erythropentofuranose 3,5-di-O-benzyl-4-trifluoromethanesulfonyloxymethyl-1,2-O-isopropylidene-α-D-erythropentofuranose obtained in Reference Example 1 (2520m
g, 4.73 mmol) in dimethyl sulfoxide (5
0 ml) and dissolved at 90 ° C. After returning to room temperature,
Sodium cyanide (463 mg, 9.46 mmol)
Was added and stirred at 50 ° C. for 3 hours.

After completion of the reaction, water (about 100 m
l) and ethyl acetate (about 100 ml) were added, and the mixture was separated.
The organic layer was washed with saturated saline (about 100 ml) and dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (hexane: ethyl acetate = 4: 1) to obtain a colorless oil (1590 mg, 3.89 mmol, 82%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.34 (3H, s),
1.62 (3H, s), 2.88 (1H, d, 17Hz), 3.15 (1H, d, 17Hz),
3.50 (1H, d, 10Hz), 3.58 (1H, d, 10Hz), 4.08 (1H, d, 10Hz)
5.1Hz), 4.52 (1H, d, 12Hz), 4.56 (1H, d, 12Hz), 4.57
(1H, m), 4.58 (1H, d, 12Hz), 4.76 (1H, d, 12Hz), 5.7
3 (1H, d, 3.7Hz), 7.3 (10H, m). (Reference Example 3) 3,5-di-O-benzyl-4-formylmethyl-1,2-O-isop
3,5-Di-O-benzyl-4 obtained in Reference Example 2 under a nitrogen stream of lopylidene-α-D-erythropentofuranose.
-Cyanomethyl-1,2-O-isopropylidene-α-D-erythropentofuranose (610 mg, 1.49 mmol)
Was dissolved in dichloromethane (10 ml) and cooled to -78 ° C. There, 1.5M diisobutylaluminum hydride / toluene solution (2 ml, 3.0 mmol)
l) was slowly added dropwise, followed by stirring at -78 ° C for 1 hour.
Thereafter, the temperature was returned to room temperature, methanol (5 ml) was added to the reaction solution, and a saturated aqueous ammonium chloride solution (about 20 ml) was added.
ml) and stirred for 30 minutes.

After the completion of the reaction, the reaction mixture was added to ethyl acetate (about 3
0 ml), and the layers are separated. The organic layer is separated from a saturated aqueous solution of sodium hydrogencarbonate (about 30 ml) and then a saturated aqueous solution of sodium chloride (about 3 ml).
0 ml) and dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, this was used for the next reaction as it was. (Reference Example 4) 3,5-di-O-benzyl-4-hydroxyethyl-1,2-O-iso
Propylidene-α-D-erythropentofuranose 3,5-di-O-benzyl-4-formylmethyl-1,2-O-isopropylidene-α-D-erythropentofuranose obtained in Reference Example 3 (154 mg, 0.377 mmol) was dissolved in 5 ml of ethanol, and NaBH ₄ (7.6 mg,
0.2 mmol) and stirred at room temperature for 1 hour.

After completion of the reaction, the reaction solution was added with ethyl acetate (about 1
0 ml) and water (about 10 ml) were added, the layers were separated, and the organic layer was washed with saturated saline (about 10 ml) and dried over anhydrous magnesium sulfate.

After distilling off the solvent under reduced pressure, the obtained residue was
Purification by silica gel chromatography (hexane: ethyl acetate = 2: 1) gave a colorless oil (117m).
g, 0.284 mmol, 75%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.33 (3H, s),
1.66 (3H, s), 1.78 (1H, ddd, 4.0,8.5, 15Hz), 2.51 (1
H, ddd, 3.4, 6.4, 15Hz), 3.31 (1H, d, 10Hz), 3.54 (1
H, d, 10Hz), 3.80 (2H, m), 4.13 (1H, d, 5.3Hz), 4.43
(1H, d, 12Hz), 4.52 (1H, d, 12Hz), 4.55 (1H, d, 12H
z), 4.65 (1H, dd, 4.0, 5.3Hz), 4.77 (1H, d, 12Hz), 5.
77 (1H, d, 4.0 Hz), 7.3 (10H, m). FABMS (mNBA): 415 (M + H) ⁺ , [α] _D + 57.4 ° (0.91, methanol). (Reference Example 5) 3, 5-di-O-benzyl-4-formyl-1,2-O-isopropyl
Oxalyl chloride (6.02 ml, 6 ml) was added to anhydrous dichloromethane (200 ml) cooled to -78 ° C under a nitrogen stream of den-α-D-erythropentofuranose.
9.0 mmol), and dimethyl sulfoxide (7. 0 mmol) dissolved in anhydrous dichloromethane (100 ml) was added thereto.
87 ml, 110 mmol) was added dropwise. After stirring for 20 minutes, 3,5-di-O-benzyl-1,2-O-isopropylidene dissolved in anhydrous dichloromethane (100 ml) was added to the reaction solution.
-α-D-erythropentofuranose (9210 mg, 2
3.02 mmol) was added dropwise, and the mixture was further stirred for 30 minutes. Furthermore, triethylamine (28 ml, 200 ml
mmol) and slowly warmed to room temperature. Water (about 300 ml) was added to the reaction solution, liquid separation was performed, and the organic layer was separated with water (about 30 ml).
0 ml) and saturated saline (about 300 ml), and dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel chromatography (hexane:
Ethyl acetate = 5: 1), colorless oil (8310 mg,
20.88 mmol, 91%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.35 (3H, s),
1.60 (3H, s), 3.61 (1H, d, 11Hz), 3.68 (1H, d, 11Hz),
4.37 (1H, d, 4.4Hz), 4.46 (1H, d, 12Hz), 4.52 (1H, d, 4.4Hz)
12Hz), 4.59 (1H, d, 12Hz), 4.59 (1H, dd, 3.4, 4.4H
z), 4.71 (1H, d, 12Hz), 5.84 (1H, d, 3.4Hz), 7.3 (10
H, m), 9.91 (1H, s). FABMS (mNBA): 397 (MH) ⁺ , 421 (M +
(Na) ⁺ , [α] _D + 27.4 ° (0.51, methanol). (Reference Example 6) 3,5-di-O-benzyl-4-vinyl-1,2-O-isopropylide
3,5-di-O-benzyl-4 obtained in Reference Example 5 under a nitrogen stream of 1 -α-D-erythropentofuranose.
-Formyl-1,2-O-isopropylidene-α-D-erythropentofuranose (8310 mg, 20.88 mmol)
Was dissolved in anhydrous tetrahydrofuran (300 ml), and 0
Cooled to ° C. Thereto, a 0.5M-Thebe reagent / toluene solution (44 ml, 22 mmol) was added dropwise, and the mixture was added at 0 ° C for 1 hour.
Stirred for hours.

After completion of the reaction, diethyl ether (300 m
l), and then a 0.1N aqueous sodium hydroxide solution (2
0m) was added slowly. The resulting precipitate was filtered using celite, and the filtered product was filtered in diethyl ether (about 100 m).
The organic layer was washed with 1) and separated, and the organic layer was dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was roughly purified by alumina (basic) chromatography (dichloromethane), and the obtained crude product was further purified by silica gel chromatography (hexane:
Ethyl acetate = 8: 1, then 5: 1) to give a colorless oil (5600 mg, 14.14 mmol, 68%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.28 (3H, s),
1.52 (3H, s), 3.31 (1H, d, 11Hz), 3.34 (1H, d, 11Hz),
4.25 (1H, d, 4.9Hz), 4.40 (1H, d, 12Hz), 4.52 (1H, d, 4.9Hz)
12Hz), 4.57 (1H, dd, 3.9, 4.9Hz), 4.59 (1H, d, 12H
z), 4.76 (1H, d, 12Hz), 5.25 (1H, dd, 1.8, 11Hz), 5.
52 (1H, dd, 1.8, 18Hz), 5.76 (1H, d, 3.9Hz), 6.20 (1
H, dd, 11, 18Hz), 7.3 (10H, m). FABMS (mNBA): 419 (M
+ Na) ^+. (Example 7) 3,5--O- benzyl-4-hydroxyethyl -1, 2-O-iso
3,5-Di-O-benzyl- obtained in Reference Example 6 under a nitrogen stream of propylidene-α-D-erythropentofuranose.
4-vinyl-1,2-O-isopropylidene-α-D-erythropentofuranose (5500 mg, 13.89 mmol
l) was dissolved in anhydrous tetrahydrofuran (200 ml), and a 0.5 M 9-BBN (9-borabicyclo [3.3.1] nonane) / tetrahydrofuran solution (8) was added thereto.
0 ml, 40 mmol) was added dropwise and stirred at room temperature overnight.

Water was added until no bubbles appeared in the reaction solution, and then a 3N aqueous sodium hydroxide solution (30 ml) was added. Further, a 30% aqueous hydrogen peroxide solution (30 ml) was slowly added so that the temperature of the reaction solution became 30 to 50 ° C., and the mixture was stirred for 30 minutes.

After completion of the reaction, a saturated saline solution (about 200 ml) and ethyl acetate (about 200 ml) were added to the reaction mixture, and the mixture was separated. The organic layer was separated from a neutral phosphate buffer (about 200 ml).
ml) and then with saturated saline (about 200 ml)
It was dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (hexane: ethyl acetate = 2: 1, then
1: 1), colorless oil (5370 mg, 12.97 m)
mol, 93%).

[0192] ^{1 H-NMR (400MHz, CDCl} 3): 1.33 (3H, s),
1.66 (3H, s), 1.78 (1H, ddd, 4.0,8.5, 15Hz), 2.51 (1
H, ddd, 3.4, 6.4, 15Hz), 3.31 (1H, d, 10Hz), 3.54 (1
H, d, 10Hz), 3.80 (2H, m), 4.13 (1H, d, 5.3Hz), 4.43
(1H, d, 12Hz), 4.52 (1H, d, 12Hz), 4.55 (1H, d, 12H
z), 4.65 (1H, dd, 4.0, 5.3Hz), 4.77 (1H, d, 12Hz), 5.
77 (1H, d, 4.0 Hz), 7.3 (10H, m). FABMS (mNBA): 415
(M + H) ⁺ , [α] _D + 57.4 ° (0.91, methanol). (Reference Example 8) 3,5-di-O-benzyl-4- (p-toluenesulfonyloxy
Ethyl) -1,2-O-isopropylidene-α-D-erythropene
The 3,5- obtained in Reference Example 4 was azeotropically distilled with toluene under a nitrogen stream of tofuranose.
Di-O-benzyl-4-hydroxyethyl-1,2-O-isopropylidene-α-D-erythropentofuranose (1035
mg, 2.5 mmol) in anhydrous dichloromethane (35 mg).
ml) and cooled to 0 ° C. There, triethylamine (1.8 ml, 13 mmol), dimethylaminopyridine (30 mg, 0.25 mmol), and p-toluenesulfonyl chloride (858 mg, 4.5 mmol) were added, and the mixture was stirred at room temperature overnight.

After completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate (about 100 ml) was added to the reaction solution, and the mixture was separated. The organic layer was separated with a saturated aqueous solution of sodium hydrogen carbonate (about 100 ml).
The extract was washed with saturated saline (about 100 ml) and dried over anhydrous magnesium sulfate.

After distilling off the solvent under reduced pressure, the obtained residue was
Purification by silica gel chromatography (hexane: ethyl acetate = 3: 1) gave a colorless oil (1340m).
g, 2.6 mmol, 94%).

¹ H-NMR (400 MHz, DCl ₃ ): 1.33 (3H, s),
1.49 (3H, s), 1.99 (1H, dt, 7.6 and15 Hz), 2.47 (3H, s)
s), 2.60 (1H, ddd, 5.7, 7.6, 15Hz), 3.28 (1H, d, 10H
z), 3.45 (1H, d, 10Hz), 4.11 (1H, d, 5.3Hz), 4.32 (2H,
m), 4.42 (1H, d, 12Hz), 4.50 (1H, d, 12Hz), 4.54 (1
H, d, 12Hz), 4.62 (1H, dd, 4.0, 5.2Hz), 4.76 (1H, d,
12Hz), 5.74 (1H, d, 4.0 Hz), 7.3 (12H, m), 7.78 (2H,
d, 8.3 Hz). FAB-MAS (mNBA): 569 (M + H) ⁺ (Reference Example 9) 1,2-di-O-acetyl-3,5-di-O-benzyl-4- (p- Torue
N-sulfonyloxyethyl) -α-D-erythropentofura
North 3,5-di-O-benzyl-4- (p-toluenesulfonyloxyethyl) -1,2-O-isopropylidene- obtained in Reference Example 8
α-D-erythropentofuranose (1340 mg, 2.
36 mmol) was dissolved in 15 ml of acetic acid, and 1.
88 ml (20 mmol) and 0.01 ml of concentrated sulfuric acid were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was poured into ice-cold water (60 ml) and stirred for another 30 minutes. Saturated saline (about 100m
l), ethyl acetate (about 100 ml) was added, and the organic layer was neutralized with a neutral phosphate buffer, a saturated aqueous solution of sodium hydrogen carbonate,
The extract was washed with saturated saline and dried over anhydrous magnesium sulfate. After evaporating the solvent, the obtained residue was purified by silica gel chromatography (hexane: ethyl acetate =
2: 1), 1290 mg of a colorless oil (2.11 mm
ol, 89%, α: β = 1: 5).

¹ H-NMR (400 MHz, CDCl ₃ ): (β form) 1.86
(3H, s), 2.05 (3H, s), 2.08 (1H, m), 2.18 (1H, m), 2.
42 (3H, s), 3.30 (1H, d, 10Hz), 3.33 (1H, d, 10Hz),
4.23 (1H, d, 5.1Hz), 4.24 (2H, m), 4.42 (2H, s), 4.45
(1H, d, 12Hz), 4.55 (1H, d, 12Hz), 5.28 (1H, d, 5.1H
z), 6.01 (1H, s), 7.3 (12H, m), 7.73 (2H, d, 8.3Hz).
FAB-MAS (mNBA): 613 (M + H) ⁺ (Reference Example 10) 2'-O-acetyl-3 ', 5'-di-O-benzyl-4'-p-toluene
Sulfonyloxy-ethyl-5-methyluridine nitrogen stream, 1 obtained in Reference Example 9 at room temperature, 2-di -O- acetyl-3,5-di -O- benzyl-4-(p-toluenesulfonyloxy ethyl ) -α-D-erythropentofuranose (650
mg, 1.06 mmol) was dissolved in anhydrous 1,2-dichloroethane (15 ml).
Vorbrgen, K .; Krolikiewicz
and B, Bennua, Chem. Ber. , 1
14,1234-1255 (1981)) (500 mg, about 2 mm).
ol) was added. Further, there, trimethylsilyl trifluoromethanesulfonate (0.36 ml, 2 mmol
l) was added dropwise, and the mixture was stirred at 50 ° C for 1 hour.

After completion of the reaction, a saturated aqueous solution of sodium hydrogencarbonate (about 50 ml) was added to the reaction solution, and the mixture was filtered using celite. ),
After washing with a saturated saline solution (about 50 ml), it was dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (hexane: ethyl acetate = 1.2: 1) to obtain an amorphous colorless substance (432 mg, 0.64 mmol, 60%). .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.52 (3H, d,
0.9Hz), 1.94 (1H, dt, 7.5 and 15Hz), 2.06 (3H, s),
2.23 (1H, dt, 6.0 and 15Hz), 2.42 (3H, s), 3.38 (1H,
d, 10Hz), 3.67 (1H, d, 10Hz), 4.17 (2H, m), 4.36 (1H,
d, 6.0Hz), 4.41 (1H, d, 12Hz), 4.44 (1H, d, 12Hz),
4.48 (1H, d, 12Hz), 4.58 (1H, d, 12Hz), 5.39 (1H, dd,
5.1 and 6.0Hz), 6.04 (1H, d, 5.1Hz), 7.3 (12H, m),
7.73 (2H, dt, 1.8 and8.3Hz), 8.18 (1H, s). FAB-MAS (m
NBA): 679 (M + H) ⁺ (Reference Example 11) 2'-O-acetyl-3 ', 5'-di-O-benzyl-4'-p-toluene
Sulfonyloxy ethyl -4-N-benzo Irushi cytidine 1,2-di -O- acetyl-3,5-di -O- benzyl-4-(p-toluenesulfonyloxy-ethyl) obtained in Reference Example 9-.alpha.- D
-Erythropentofuranose (383 mg, 0.626
mmol) in anhydrous 1,2-dichloroethane (4 ml)
Was dissolved. There, the aforementioned document (H. Vorbrgg)
en, K .; Krolikiewicz and B, B
ennua, Chem. Ber. , 114,1234-
1255 (1981)), trimethylsilylated benzoylcytosine (300 mg, ca.
ol), cooled to 0 ° C., and further added trimethylsilyl trifluoromethanesulfonate (0.18 ml, 0.1 mL).
995 mmol), followed by stirring at 50 ° C. for 1 hour. The reaction solution was returned to room temperature, and a saturated aqueous solution of sodium hydrogen carbonate (about 10 ml) was added.

After completion of the reaction, methylene chloride (about 20 ml) was added to the reaction mixture, and the mixture was stirred, and the precipitated white insoluble matter was filtered using celite. The organic layer was separated from the obtained filtrate, and the organic layer was washed with saturated saline (about 20 ml) and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain a colorless amorphous substance (397 mg, 83
%).

¹ H-NMR (400 MHz, CDCl ₃ ): 8.70 (1H, br),
8.18 (1H, d, 7.4Hz), 7.87 (2H, d, 7.5Hz), 7.72 (2H, d,
8.3Hz), 7.61-7.57 (1H, m), 7.51-7.48 (2H, m), 7.43-
7.21 (13H, m), 6.02 (1H, d, 2.9Hz), 5.40 (1H, dd, 5.8,
2.9Hz), 4.57 (1H, d, 11Hz), 4.39 (1H, d, 11Hz), 4.3
2-4.28 (3H, m), 4.19-4.16 (2H, m), 3.69 (1H, d, 11Hz),
3.31 (1H, d, 11Hz), 2.40 (3H, s), 2.30-2.23 (1H, m),
2.06 (3H, s), 1.95-1.89 (1H, m) FAB-MAS (mNBA): 768 (M + H) ⁺ (Reference Example 12) 2'-O-acetyl-3 ', 5'-di-O -Benzyl-4'-p-toluene
Sulfonyloxy ethyl -6-N-benzoyl adenosine Under a nitrogen stream, at room temperature, 1 obtained in Reference Example 9, 2-di -O- acetyl-3,5-di -O- benzyl-4-(p-toluene Sulfonyloxyethyl) -α-D-erythropentofuranose (60
0 mg, 0.98 mmol) were dissolved in anhydrous 1,2-dichloroethane (15 ml), and the solution was added to the above-mentioned literature (H. Vorbrgen, K. Krolikiwiwi).
cz and B, Bennua, Chem. Be
r. , 114,1234-1255 (1981)) was added (500 mg, ca. 2 mmol). Trimethylsilyl trifluoromethanesulfonate (0.36 ml, 2 mmol) was added dropwise to the obtained reaction solution, followed by stirring at 50 ° C. for 4 hours.

After completion of the reaction, a saturated aqueous solution of sodium hydrogencarbonate (about 50 ml) was added to the reaction solution, dichloromethane (about 50 ml) was further added, and the mixture was separated. The organic layer was separated with a saturated aqueous solution of sodium hydrogencarbonate (about 50 ml) and then. After washing with saturated saline (about 50 ml), the extract was dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (dichloromethane: methanol = 50: 1) to obtain an amorphous colorless substance (405 mg, 0.51 mmol, 52%).

¹ H-NMR (400 MHz, CDCl ₃ ): 2.0 (1 H, m),
2.06 (3H, s), 2.32 (1H, dt, 6.0 and15Hz), 2.40 (3H,
s), 3.36 (1H, d, 10Hz), 3.58 (1H, d, 10Hz), 4.22 (2H,
m), 4.39 (1H, d, 12Hz), 4.45 (1H, d, 12Hz), 4.47 (1H, d, 12Hz)
d, 12Hz), 4.59 (1H, d, 12Hz), 4.62 (1H, d, 5.6Hz),
5.94 (1H, dd, 4.5 and 5.6Hz), 6.21 (1H, d, 4.5Hz), 7.
2-7.3 (12H, m), 7.54 (2H, m), 7.62 (1H, dt, 1.2 and
6.2Hz), 7.72 (2H, d, 8.3Hz), 8.02 (2H, m), 8.21 (1H,
s), 8.75 (1H, s), 8.97 (1H, brs). FAB-MAS (mNBA): 792
(M + H) ⁺ (Reference Example 13) 2'-O-acetyl-3 ', 5'-di -O- benzyl-4'-p-toluene
Sulfonyloxyethyl-uridine 1,2-di-O-acetyl-3,5-di-O-benzyl-4- (p-toluenesulfonyloxyethyl) -α obtained in Reference Example 9 at room temperature under a nitrogen stream. -D-erythropentofuranose (200
mg, 0.327 mmol) was dissolved in anhydrous 1,2-dichloroethane (8 ml).
Vorbrgen, K .; Krolikiewicz
and B, Bennua, Chem. Ber. , 1
14,1234-1255 (1981)), trimethylsilylated uracil (200 mg, ca.
8 mmol) was added. Further, there, trimethylsilyl trifluoromethanesulfonate (0.145 ml,
0.8 mmol) was added dropwise, and the mixture was stirred at 70 ° C. for 1 hour.

After completion of the reaction, a saturated aqueous solution of sodium hydrogencarbonate (about 10 ml) was added to the reaction solution, and the mixture was filtered using celite. Dichloromethane (about 10 ml) was added to the filtrate, and the organic layer was separated. After washing with water, it was dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (dichloromethane: methanol = 10).
0: 2), an oily colorless substance (199 mg, 0.299 mm)
ol, 92%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.94 (1H, dt, 7.
4 and 15Hz), 2.07 (3H, s), 2.23 (1H, dt, 5.9 and 15Hz),
2.43 (3H, s), 3.36 (1H, d, 10Hz), 3.65 (1H, d, 10Hz), 4.1
7 (2H, dd, 6 and 7Hz), 4.31 (1H, d, 5.9Hz), 4.38 (1H, d, 1
1Hz), 4.39 (1H, d, 11Hz), 4.40 (1H, d, 11Hz), 4.58 (1H, d, 11Hz)
11Hz), 5.29 (1H, dd, 2.4 and 8.2Hz), 5.33 (1H, dd, 4.5an
d 6Hz), 6.00 (1H, d, 4.5Hz), 7.2-7.4 (12H, m), 7.61 (1H,
d, 8.2Hz), 7.74 (1H, d, 8.3Hz), 8.14 (1H, brs). FAB-MAS (mNBA): 665 (M + H) ⁺ (Reference Example 14) 2'-O-acetyl-3 ', 5'-di-O-benzyl-4'-p-toluene
Sulfonyloxyethyl-4-N-benzoyl-5-methylsi
Cytidine obtained in Reference Example 9 1,2-di -O- acetyl-3,5-di -O- benzyl-4-(p-toluenesulfonyloxy-ethyl)-.alpha.-D
-Erythropentofuranose (400mg, 0.653
mmol) in anhydrous 1,2-dichloroethane (6 ml)
Was dissolved. There, the aforementioned document (H. Vorbrgg)
en, K .; Krolikiewicz and B, B
ennua, Chem. Ber. , 114,1234-
1255 (1981)), trimethylsilylated benzoyl 5-methylcytosine (about 400 mg,
About 1.2 mmol), cooled to 0 ° C., and trimethylsilyl trifluoromethanesulfonate (180
μl, 1.0 mmol) and then stirred at 50 ° C. for 1 hour. The reaction solution was returned to room temperature, and a saturated aqueous solution of sodium hydrogen carbonate (about 5 ml) was added.

After the completion of the reaction, methylene chloride (about 10 ml) was added to the reaction mixture, and the mixture was stirred, and the precipitated white insoluble matter was filtered using celite. An organic layer was separated from the obtained filtrate, and the organic layer was washed with saturated saline and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain a colorless amorphous substance (320 mg, 0.409 mmol, 6
3%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.68 (3H, s), 1.
95 (1H, dt, 7.3 and 15Hz), 2.07 (3H, s), 2.25 (1H, dt, 6 a
nd 15Hz), 2.43 (3H, s), 3.40 (1H, d, 10Hz), 3.71 (1H, d, 1
0Hz), 4.18 (2H, m), 4.37 (1H, d, 5.8Hz), 4.42 (1H, d, 12H
z), 4.46 (1H, d, 12Hz), 4.51 (1H, d, 12Hz), 4.61 (1H, d, 1Hz)
2Hz), 5.42 (1H, dd, 4.9 and 5.8Hz), 6.07 (1H, d, 4.9Hz),
7.2-7.6 (17H, m), 7.74 (2H, d, 8.3Hz), 8.28 (2H, d, 7.0H
z). FAB-MAS (mNBA): 782 (M + H) ⁺ (Reference Example 15) 2'-O-acetyl-3 ', 5'-di-O-benzyl-4'-p-toluene
Sulfonyloxyethyl-2-N-isobutyrylguanosine Under a nitrogen stream, at room temperature, the 1,2-di-O-acetyl-3,5-di-O-benzyl-4- (p) obtained in Reference Example 9 was obtained. -Toluenesulfonyloxyethyl) -α-D-erythropentofuranose (400
mg, 0.65 mmol) was dissolved in anhydrous 1,2-dichloroethane (10 ml), and the solution was added to the above-mentioned literature (H. Vor.
brgen, K .; Krolikiewicz and
B, Bennua, Chem. Ber. , 114,1
234-1255 (1981)), trimethylsilylated isobutyrylguanosine (about 650 mg,
About 1.5 mmol) was added. To the obtained reaction solution, trimethylsilyl trifluoromethanesulfonate (0.2 ml,
(1.2 mmol) was added dropwise, followed by stirring at 50 ° C. for 4 hours.

After completion of the reaction, a saturated aqueous solution of sodium hydrogencarbonate (about 5 ml) was added to the reaction solution, and the mixture was separated. The organic layer was washed with a saturated aqueous solution of sodium hydrogencarbonate and then with a saturated saline solution, and dried over anhydrous magnesium sulfate. . After distilling off the solvent,
This was used for the next reaction as it was. (Reference Example 16) 3 ', 5'-di-O-benzyl-2'-O, 4'-C-ethylene-4-N-beta
2'-O-acetyl-3 ', 5'-di-O-benzyl-4'-p-toluenesulfonyloxyethyl-4-N- obtained in nzoylcytidine reference example 11
Benzoylcytidine (6.80 g, 8.86 mmol)
Was dissolved in pyridine (136 ml) and cooled to 0 ° C.
A 2N aqueous sodium hydroxide solution (68 ml) was added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, 20% aqueous acetic acid was added dropwise to the reaction solution to neutralize the reaction solution, followed by extraction with chloroform and washing with saturated saline. After distilling off the solvent under reduced pressure,
The residue was purified using silica gel chromatography (dichloromethane: methanol = 100: 3) to obtain the desired compound (3.33 g, 6.02 mmol, 68%).

¹ H-NMR (400 MHz, CDCl ₃ ): 8.64 (2H, br
s), 7.89 (2H, d, 7.6Hz), 7.64-7.60 (1H, m), 7.54-7.5
1 (2H, m), 7.48-7.37 (3H, m), 7.36-7.26 (8H, m), 6.18
(1H, s), 4.70 (1H, d, 11Hz), 4.60 (1H, d, 11Hz), 4.55
(1H, d, 11Hz), 4.46 (1H, d, 2.9Hz), 4.42 (1H, d, 11H
z), 4.10-4.02 (2H, m), 3.89 (1H, d, 2.9Hz), 3.75 (1H,
d, 11Hz), 3.62 (1H, d, 11Hz), 2.34-2.26 (1H, m), 1.3
9-1.36 (1H, m). FAB-MAS (mNBA): 554 (M + H) ⁺ (Reference Example 17) 2′-O, 4′-C-ethylene-4-N-benzoylcytidine Reference Example 16 The resulting 3 ', 5'-di-O-benzyl-2'-O, 4'-
C-ethylene-4-N-benzoylcytidine (2.06 g,
3.72 mmol) in anhydrous dichloromethane (317 m
l) and cooled to -78 ° C when trichloroborane (1.0 Min dichloromethane) (31.7 m
l) was added dropwise. After stirring at −78 ° C. for 1 hour, −2
The temperature was slowly raised to 0 ° C, and the reaction vessel was placed in an ice-salt bath and stirred between -20 ° C and -10 ° C for 2 hours. After methanol (12 ml) was slowly added dropwise, and the mixture was stirred for 10 minutes, a saturated aqueous solution of sodium hydrogen carbonate was added little by little to adjust the pH to 7 to 8, and the temperature was returned to room temperature. This mixed solution was concentrated under reduced pressure, and the obtained residue was purified using silica gel chromatography (dichloromethane: methanol = 100: 5), and the target product as a white solid (1.21 g, 3.
24 mmol, 87%).

¹ H-NMR (500 MHz, DMSO-d ₆ ): 11.23 (1H, b
r.), 8.70 (1H, d, 7.2Hz), 8.00 (2H, d, 7.5Hz), 7.3-6 (4H,
m), 5.97 (1H, s), 5.35 (1H, dd, 5 and 10Hz), 4.10 (1H, d
d, 5 and 10Hz), 4.03 (1H, d, 3.2Hz), 3.95-3.85 (2H, m)
3.83 (1H, d, 3.2Hz), 3.65-3.51 (2H, m), 2.06-1.98 (1H,
m), 1.26 (1). FAB-MAS (mNBA): 374 (M + H) ⁺ (Reference Example 18) 2′-O, 4′-C-ethylene-cytidine 2′- obtained in Reference Example 17 O, 4'-C-ethylene-4-N-benzoylcytidine (0.1 g, 0.268 mmol) was dissolved in a saturated ammonia-methanol solution (12 ml),
Left overnight. The solvent was distilled off, and the target substance (0.
(054 g, 75%).

¹ H-NMR (500 MHz, DMSO-d ₆ ): 8.18 (1H, d,
7.4Hz), 7.10 (2H, br.), 5.84 (1H, s), 5.69 (1H, d, 7.
6Hz), 5.27-5.24 (2H, m), 3.86 (1H, d, 3.2Hz), 3.90-
3.78 (2H, m), 3.76 (1H, d, 3.2Hz), 3.56 (1H, dd, 5.5
and 12Hz), 3.49 (1H, dd, 5.5 and 12Hz), 2.01-1.93 (1
H, dt, 7.5 and 12Hz), 1.22 (1H, dd, 3.6 and 13Hz). FAB-MAS (mNBA): 270 (M + H) ⁺ (Reference Example 19) 3 ', 5'-di-O-benzyl -2'-O, 4'-C-ethylene 5-methyl
Obtained in Reference Example 10 (418 mg, 0.62 mmol
l) was dissolved in a mixed solution (5 ml) of pyridine: methanol: water = 65: 30: 5. Thereto, 2N sodium hydroxide / same mixed solution (5 ml) was added at 0 ° C., and the mixture was stirred at room temperature for 15 minutes.

After the completion of the reaction, the reaction solution was neutralized with 1N hydrochloric acid.
Ethyl acetate (about 30 ml) was added, the layers were separated, and the organic layer was washed with a saturated aqueous solution of sodium hydrogen carbonate (about 30 ml) and a saturated saline solution (about 30 ml), and then dried over anhydrous magnesium sulfate.

After distilling off the solvent under reduced pressure, the obtained residue was
Purification by silica gel chromatography (hexane: ethyl acetate = 1: 1) gave an amorphous colorless substance (228 mg, 0.49 mmol, 79%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.35 (1H, d, 1
3Hz), 1.41 (3H, s), 2.28 (1H, dt, 9.4 and 13Hz), 3.60
(1H, d, 11Hz), 3.76 (1H, d, 11Hz), 3.94 (1H, d, 3.0H
z), 4.10 (1H, d, 7.0Hz), 4.14 (1H, d, 7.0Hz), 4.31 (1H
H, d, 3.0Hz), 4.51 (1H, d, 12Hz), 4.54 (1H, d, 12Hz),
4.58 (1H, d, 12Hz), 4.75 (1H, d, 12Hz), 6.06 (1H,
s), 7.3 (10H, m), 7.91 (1H, s), 8.42 (1H, brs). FAB-MAS (mNBA): 465 (M + H) ⁺ (Reference Example 20) 2'-O, 4 ' -C-ethylene 5-methyluridine 3 ′, 5′-di-O-benzyl-2′-O, 4′- obtained in Reference Example 19
C-ethylene 5-methyluridine (195 mg, 0.1%).
42 mmol) was dissolved in methanol (10 ml), and 160 mg of 20% Pd (OH) ₂ on carbon was added to the obtained reaction solution.
The mixture was stirred at normal pressure for 5 hours under a hydrogen stream.

After completion of the reaction, the catalyst was filtered off, the solvent of the filtrate was distilled off under reduced pressure, and the obtained residue was purified by silica gel chromatography (dichloromethane: methanol =
10: 1), white powder (76 mg, 0.268 mmol)
1, 64%).

¹ H-NMR (400 MHz, CD ₃ OD): 1.33 (1H, dd,
3.8 and 13Hz), 1.86 (3H, d, 0.9Hz), 1.94 (1H, ddd,
7.5, 11.7 and 13Hz), 3.68 (1H, d, 12Hz), 3.75 (1H,
d, 12Hz), 3.9-4.0 (2H, m), 4.05 (1H, d, 3.2Hz), 4.09
(1H, d, 3.2Hz), 6.00 (1H, s), 8.28 (1H, d, 1.1Hz). FAB-MAS (mNBA): 285 (M + H) ⁺ (Reference Example 21) 3 ', 5'-Di-O-benzyl-2'-O,4'-C-ethylene-6-N-be
Emissions benzoyl adenosine Reference Example 12 2'-O-acetyl -3 obtained in ', 5'-di -O- benzyl-4'-p-toluenesulfonyloxy-ethyl -6-N-
Benzoyl adenosine (238 mg, 0.30 mmol
l) was dissolved in a mixed solution (5 ml) of pyridine: methanol: water = 65: 30: 5. Thereto, 2N sodium hydroxide / same mixed solution (5 ml) was added at 0 ° C, and the mixture was stirred at room temperature for 15 minutes.

After the completion of the reaction, the reaction solution was neutralized with 1N hydrochloric acid.
The mixture was extracted with ethyl acetate (about 30 ml), saturated aqueous sodium hydrogen carbonate solution (about 30 ml), and saturated saline (about 30 m).
After washing in 1), the mixture was dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (dichloromethane: methanol = 50: 1) to obtain an amorphous colorless substance (133 m
g, 0.23 mmol, 78%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.44 (1H, d, 1
3Hz), 2.31 (1H, dd, 13 and 19Hz), 3.56 (1H, d, 11Hz),
3.70 (1H, d, 11Hz), 4.10 (2H, m), 4.24 (1H, s), 4.45
(1H, d, 12Hz), 4.53-4.67 (4H, m), 6.52 (1H, s), 7.3
(10H, m), 7.53 (2H, m), 7.62 (1H, m), 8.03 (2H, d, 7.
6Hz), 8.66 (1H, s), 8.78 (1H, s), 9.00 (1H, brs). FAB-MAS (mNBA): 578 (M + H) ⁺ (Reference Example 22) 2'-O, 4 ' -C- ethylene -6-N-benzoyl adenosine) under a nitrogen stream, obtained in reference example 21 3 ', 5'-di -O- benzyl -2'-O, 4'-C- ethylene -6-N -Benzoyladenosine (116 mg, 0.20 mmol) was dissolved in 5 ml of anhydrous dichloromethane and cooled to -78 ° C. There 1M
-Boron trichloride / dichloromethane solution (1.5 ml,
1.5 mmol) was slowly added dropwise, and the mixture was stirred at -78 ° C for 3 hours. Further, a 1M-boron trichloride / dichloromethane solution (1.5 ml, 1.5 mmol) was added, and the mixture was stirred for 2 hours. Then, the temperature was slowly raised to room temperature, and again -78.
After rapidly cooling to ℃, methanol (5 ml) was added, and
The temperature was slowly raised to room temperature.

After completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel chromatography (dichloromethane: methanol = 9: 1) to obtain a white powder (49 mg, 0.17 mmol, 84%). Was.

¹ H-NMR (400 MHz, CD ₃ OD): 1.45 (1H, dd,
4.3 and 13Hz), 2.12 (1H, m), 3.72 (1H, d, 12Hz), 3.7
9 (1H, d, 12Hz), 4.04 (1H, dd, 7.3 and 12Hz), 4.15 (1
H, dt, 4.3 and 9.4Hz), 4.36 (1H, d, 3.2Hz), 4.43 (1
H, d, 3.2Hz), 6.57 (1H, s), 7.57 (2H, m), 7.66 (1H,
m), 8.09 (2H, d, 8.0Hz), 8.72 (1H, s), 8.85 (1H, s). FAB-MAS (mNBA): 398 (M + H) ⁺ (Reference Example 23) 2'-O 2,4'-C-ethyleneadenosine 2'-O, 4'-C-ethylene-6-N-benzoyladenosine obtained in Reference Example 22 (14 mg, 0.035 mmol)
Was dissolved in a saturated ammonia / methanol solution (1 ml) and left overnight.

After the completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel chromatography (dichloromethane: methanol = 10: 1) to obtain a white powder (10 mg, 0.034 mmol, 98%). Was.

¹ H-NMR (400 MHz, CD ₃ OD): 1.32 (1H, dd,
4 and 13Hz), 2.04 (1H, dt, 7.4 and 12Hz), 3.53 (1H,
(dd, 5 and 12Hz), 3.61 (1H, dd, 5.2 and 12Hz), 3.90
(1H, dd, 7.4 and 12Hz), 3.97 (1H, dt, 4 and 12Hz),
4.15 (1H, d, 3.1Hz), 4.21 (1H, d, 3.1Hz), 5.27 (1H, d, 3.1Hz)
t, 5.2Hz), 5.39 (1H, d, 3.1Hz), 6.33 (1H, s), 7.29 (2
H, s), 7.66 (1H, m), 8.14 (1H, s), 8.42 (1H, s) .FAB-M
AS (mNBA): 294 (M + H) ⁺ UV (λmax): 260 (pH7), 260 (pH1), 258 (pH13) (Reference Example 24) 3 ′, 5′-di-O-benzyl-2 ′ -O, 4'-C-ethyleneuridine 2'-O-acetyl-3 ', 5'-di-O-benzyl-4'-p-toluenesulfonyloxyethyl- uridine obtained in Reference Example 13 (194 mg, 0.292 mmol) was dissolved in pyridine (3 ml). Thereto, 1N sodium hydroxide (2 ml) was added at 0 ° C, and the mixture was stirred at room temperature for 30 minutes.

After the completion of the reaction, the reaction solution was neutralized with 1N hydrochloric acid.
Ethyl acetate (10 ml) was added, the layers were separated, and the organic layer was washed with a saturated aqueous solution of sodium bicarbonate and brine, and dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (dichloromethane: methanol = 100:
3) oily colorless substance (105 mg, 0.233 mmol,
80%).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.36 (1H, m),
2.29 (1H, m), 3.63 (1H, d, 11Hz), 3.74 (1H, d, 11Hz), 3.8
7 (1H, d, 2.9Hz), 4.03 (2H, m), 4.29 (1H, d, 2.9Hz), 4.49
(1H, d, 12Hz), 4.50 (1H, d, 11Hz), 4.53 (1H, d, 11Hz), 4.7
3 (1H, d, 12Hz), 5.20 (1H, dd, 2and 8Hz), 6.04 (1H, s),
7.2-7.4 (10H, m), 8.13 (1H, d, 8.2Hz), 8.57 (1H, br.). FAB-MAS (mNBA): 451 (M + H) ⁺ (Reference Example 25) 2'-O 3,4'-C-ethyleneuridine 3 ', 5'-di-O-benzyl-2'-O, 4'- obtained in Reference Example 24
C-ethylene uridine (100 mg, 0.222 mmol
l) was dissolved in methanol (4 ml), and 90 mg of 20% Pd (OH) ₂ on carbon was added to the obtained reaction solution.
The mixture was stirred at normal pressure for 5 hours.

After completion of the reaction, the catalyst was filtered off, the solvent of the filtrate was distilled off under reduced pressure, and the obtained residue was purified by silica gel chromatography (dichloromethane: methanol =
10: 1), colorless oil (45 mg, 0.167 mm)
ol, 75%).

¹ H-NMR (400 MHz, CD ₃ OD): 1.35 (1H, dd, 4
and 13Hz), 2.13 (1H, ddd, 7,11 and13Hz), 3.66 (1H, d, 1
2Hz), 3.73 (1H, d, 12Hz), 3.91-4.08 (2H, m), 4.01 (1H, d,
3.2Hz), 4.12 (1H, d, 3.2Hz), 5.66 (1H, d, 8.2Hz), 6.00 (1
H, s), 8.37 (1H, d, 8.2 Hz). FAB-MAS (mNBA): 271 (M + H) ⁺ (Reference Example 26) 3 ', 5'-di-O-benzyl-2'-O , 4'-C-Ethylene-4-N-B
Nzoiru 5- methylcytidine Reference Example 14 2'-O-acetyl -3 obtained in ', 5'-di -O- benzyl-4'-p-toluenesulfonyloxy-ethyl -4-N-
Benzoyl-5-methylcytidine (310 mg, 0.39
6 mmol) was dissolved in pyridine (5 ml), and after cooling to 0 ° C., a 1N aqueous sodium hydroxide solution (5 ml) was added, followed by stirring at room temperature for 20 minutes. After the reaction, add 20%
Aqueous acetic acid was added dropwise to neutralize the reaction solution, followed by extraction with dichloromethane and washing with saturated saline. After evaporating the solvent under reduced pressure, the residue was purified by silica gel chromatography (dichloromethane: methanol = 100:
2), the target compound (190 mg, 0.334 mmol,
84%).

[0226] ^{1 H-NMR (400MHz, CDCl} 3): 1.37 (1H, m),
1.58 (3H, s), 2.30 (1H, dt, 10 and 13Hz), 3.64 (1H, d, 11H
z), 3.79 (1H, d, 11Hz), 3.95 (1H, d, 3.0Hz), 4.04 (2H, dd,
2.3 and 10Hz), 4.37 (1H, d, 3.0Hz), 4.50 (1H, d, 12Hz),
4.56 (1H, d, 11Hz), 4.61 (1H, d, 11Hz), 4.76 (1H, d, 12Hz),
6.11 (1H, s), 7.2-7.5 (13H, m), 8.09 (1H, s), 8.29 (2H,
m). FAB-MAS (mNBA): 568 (M + H) ⁺ (Reference Example 27) 2'-O, 4'-C-ethylene-4-N-benzoyl-5-methylsi
Obtained in lysophosphatidic Reference Example 26 3 ', 5'-di -O- benzyl -2'-O, 4'
C-ethylene-4-N-benzoyl-5-methylcytidine (1
20 mg, 0.211 mmol) in anhydrous dichloromethane (5 ml) and cooled to -78 ° C when trichloroborane (1.0 M in dichloromethane) (1.6).
ml) was added dropwise. After stirring at −78 ° C. for 4 hours, methanol (1 ml) was slowly added dropwise, and after stirring for 10 minutes, a saturated aqueous solution of sodium hydrogen carbonate was added little by little to adjust the pH to 7 to 8, and then returned to room temperature. The mixed solution is concentrated under reduced pressure, and the obtained residue is purified using silica gel chromatography (dichloromethane: methanol =
100: 6), the desired product as a white solid (29 mg, 0.07
(5 mmol, 36%).

¹ H-NMR (400 MHz, d-DMSO): 1.24 (1 H, m),
2.01 (3H, s), 2.0 (1H, m), 3.54 (1H, dd, 5.4 and 12Hz),
3.64 (1H, dd, 5.4 and 12Hz), 3.88 (3H, m), 4.10 (1H, m),
5.36 (1H, d, 5.4Hz), 5.49 (1H, t, 5.0Hz), 5.95 (1H, s), 7.
4-7.6 (3H, m), 8.21 (2H, m), 8.49 (1H, s), 13.17 (1H, b
r.). FAB-MAS (mNBA): 388 (M + H) ⁺ (Reference Example 28) 2′-O, 4′-C-ethylene-5-methylcytidine 2′-O obtained in Reference Example 27 , 4'-C-ethylene-4-N-benzoyl-5-methylcytidine (11.6 mg, 0.030
mmol) in a saturated ammonia-methanol solution (2 m
1) and left overnight. The solvent was distilled off to obtain the target product as a white solid (8.5 mg, 0.03 mmol).

¹ H-NMR (400 MHz, d-DMSO): 1.20 (1 H, m),
1.82 (3H, s), 1.97 (1H, m), 3.49 (1H, dd, 5 and 12Hz), 3.
58 (1H, dd, 5 and 12Hz), 3.85 (2H, m), 5.23 (1H, d, 5Hz),
5.32 (1H, t, 5Hz), 5.84 (1H, s), 6.7 (1H, brs), 7.2 (1H, br
s), 8.08 (1H, s ) .FAB-MAS (mNBA): 284 (M + H) + UV (λmax): 279 (pH7), 289 (pH1), 279 (pH13) ( Reference Example 29) 3 ' , 5'-Di-O-benzyl-2'-O, 4'-C-ethylene-2-N-i
Sobutyrylguanosine 2′-O-acetyl-3 ′, 5′-di-O-benzyl-4′-p-toluenesulfonyloxyethyl-2-N- obtained in Reference Example 15
Isobutyrylguanosine (200 mg) was dissolved in pyridine (2 ml). Thereto, 1N sodium hydroxide (2 ml) was added, and the mixture was stirred at room temperature for 15 minutes.

After the completion of the reaction, the reaction solution was neutralized with 1N hydrochloric acid.
Extract with ethyl acetate, saturated aqueous sodium bicarbonate,
After washing with saturated saline, it was dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel chromatography (dichloromethane:
Methanol = 50: 1), amorphous colorless substance (2
0 mg, 0.036 mmol, 6% (2 steps)).

¹ H-NMR (400 MHz, CDCl ₃ ): 1.27 (3H, s),
1.29 (3H, s), 1.43 (1H, dd, 3 and 13Hz), 2.28 (1H, m), 2.
59 (1H, qui, 6.9Hz), 3.54 (1H, d, 11Hz), 3.68 (1H, d, 11H
z), 4.03 (2H, m), 4.15 (1H, d, 3.0Hz), 4.31 (1H, d, 3.0H
z), 4.45 (1H, d, 12), 4.56 (1H, d, 12Hz), 4.61 (1H, d, 12H
z), 4.63 (1H, d, 12Hz), 6.18 (1H, s), 7.2-7.4 (10H, m),
8.19 (1H, s), 11.93 (1H, brs). FAB-MAS (mNBA): 560 (M + H) ⁺ (Reference Example 30) 2'-O, 4'-C-ethylene-2-N-iso Butyrylguanosine 3 ', 5'-di-O-benzyl-2'-O, 4'- obtained in Reference Example 29
C-ethylene-2-N-isobutyrylguanosine (10m
g, 0.018 mmol) in methanol (2 ml), and the resulting reaction solution was added with 160 mg of 20% Pd (OH) ₂ on carbon.
Was added, and the mixture was stirred under a hydrogen stream at normal pressure for 5 hours.

After completion of the reaction, the catalyst was filtered off, the solvent of the filtrate was distilled off under reduced pressure, and the obtained residue was purified by silica gel chromatography (dichloromethane: methanol =
10: 2), colorless oil (5 mg, 0.013 mmol)
1, 72%).

¹ H-NMR (400 MHz, CD ₃ OD): 1.21 (3H, s), 1.
22 (3H, s), 1.41 (1H, dd, 4 and 13Hz), 2.18 (1H, m), 2.6
9 (1H, qui, 6.9Hz), 3.69 (1H, d, 12Hz), 3.76 (1H, d, 12Hz),
4.0 (2H, m), 4.26 (1H, d, 3.2Hz), 4.30 (1H, d, 3.2Hz), 6.
30 (1H, s), 8.40 (1H, s). FAB-MAS (mNBA): 380 (M + H) ⁺ (Reference Example 31) 2′-O, 4′-C-ethylene guanosine obtained in Reference Example 30. 2′-O, 4′-C-ethylene-2-N-isobutyrylguanosine (0.5 mg) was dissolved in a saturated ammonia / methanol solution (0.5 ml),
Allowed to react for hours.

After the completion of the reaction, the solvent was distilled off under reduced pressure to obtain a white powder (0.4 mg).

FAB-MAS (mNBA): 310 (M + H) ⁺ UV (λmax): 2
55 (pH7), 256 (pH1), 258-266 (pH13) (Reference Example 32) 2'-O, 4'-C-ethyleneuridine-5'-triethyl monophosphate
Min salt Under a nitrogen stream, 2'-O at 0 ℃, 4'-C- ethylene uridine (15
mg, 0.055 mmol) in trimethyl phosphate (2 ml)
And phosphoryl chloride (37 μl, 0.4 mmol) was added thereto, and the mixture was allowed to stand at 0 ° C. for 12 hours. After a saturated aqueous solution of sodium hydrogen carbonate (2 ml) was added to neutralize the reaction solution, the reaction solution was washed with dichloromethane (5 ml). The aqueous layer is subjected to reversed-phase high-performance liquid chromatography (HPLC: LC-V manufactured by Shimadzu Corporation).
P; Column: GL Science Inertsil Prep-ODS (20 × 250m
m); 0.1 M triethylamine acetate aqueous solution (TEAA), pH7; 1
0 → 40% CH ₃ CN / 30min, linear gradient; 50 ℃, 10ml / m
in; 254 nm), and fractions eluted at 2.8-11.9 minutes were collected. This compound was analyzed by HPLC (column: Wakosil DNA (4.6
× 150mm), 0.1M TEAA, pH7, 10 → 50% CH ₃ CN / 20min, li
When analyzed by near gradient, 60 ° C, 1ml / min), it was eluted at 4.26 minutes (λmax = 263nm). Lyophilize the eluate,
The target compound (19.2 μmol) was obtained (the yield was calculated from UV absorbance). ¹ H-NMR (400MHz, D ₂ O): 1.45 (1H, m), 2.06 (1H, m), 3.8
7 (1H, dd, 4.8 and 12Hz), 3.92 (2H, m), 3.98 (1H, dd,
4.8 and 12Hz), 4.01 (1H, d, 3.0Hz), 4.19 (1H, d, 3.0H
z), 5.79 (1H, d, 8.1Hz), 5.96 (1H, s), 8.07 (1H, d,
(8.1 Hz). FAB-MAS (mNBA): 351 (M + H) ⁺ (Test example) (Test example 1) Gene amplification by T7 RNA polymerase T7 promoter sequence 5′-taatacgactcactatag -3 ′ (SEQ ID NO: 1 in Sequence Listing) Is dissolved in 50 μl of T7 RNA polymerase reaction buffer. After adding the compound of Example 1, adenosine-5'-triphosphate, guanosine-5'-triphosphate, and cytidine-5'-triphosphate to a final concentration of 5 mM, T7 RNA polymerase (1000 U ) And add 3
Heat at 7 ° C. After one hour, a portion of the sample (2.5 μ
l) 2.5 μl of loading solution (formamide,
0.01% bromophenol blue, 0.01% xylene cyanol) and analyze by 10% polyacrylamide gel electrophoresis containing 7M urea. After electrophoresis, wrap the gel in wrap, place on TLC containing fluorescent agent, and generate RNA generated by UV irradiation
To analyze.

The compounds of the present invention are useful as substrates for RNA polymerase. (Test Example 2) (Measurement of nuclease enzyme resistance) An exonuclease or an endonuclease is mixed with an oligonucleotide buffer solution kept at 37 ° C for 15 minutes. The mixture is kept at 37 ° C., and after a certain period of time, a part of the mixture is taken, ethylenediaminetetraacetic acid (EDTA) is added, and the reaction is stopped by heating at 100 ° C. for 2 minutes. The remaining amount of the oligonucleotide in the mixture is quantified by reversed-phase high-performance liquid column chromatography, and the change over time in the amount of the oligonucleotide in the presence of nuclease is measured.

Oligonucleotides amplified using the compounds of the present invention exhibit significant nuclease resistance. Therefore, the gene amplification method using the compound of the present invention is superior to the conventional method in that the amplified product is more stable than the natural type and easy to handle. (Test Example 3) (Measurement of influenza polymerase inhibitory activity) Purification method of influenza virus The virus to be propagated was inoculated into fertilized eggs (10-day eggs) using a syringe, cultured at 37 ° C for 40 hours, and then cultured at 4 ° C for 60 hours. Cultured for hours. After that, the upper shell of the egg was cracked to collect allantoic fluid,
It was concentrated by ultracentrifugation, purified by sucrose gradient, dissolved in NTE buffer, and used as purified influenza virus. The amount of virus used was controlled by the TopCounter under the following assay conditions.
A dilution factor of -3000 was used. Influenza polymerase assay A Millipore multiscreen plate is used. After pretreatment of the plate, 25 ml of the reaction solution (50 mM Tris-HCl [p
H 7.8], 5 mM MgCl ₂ , 120 mM KAc, 0.25% TN101 and 35
mg tRNA), 10 ml of virus diluent, 10 ml of NTPs mix
(5 mM CTP, 5 mM GTP, 10 mM ATP, 7.4 kBq ³³ P-UTP,
30 ng ALMV RNA4 and 1 U RNase inhibitor) were added to each well of the plate. Then, the compound of Example 1 was added to 5
In addition, 5 ml of 4% DMSO was prepared as a control, and a well containing no ALMV RNA4 was prepared as a background. Incubate the prepared plate at 30 ° C for 2 hours, then 100 ml 12% TCA / 0.12 M sodium pyrophospha
te was added to form a precipitate at 4 ° C. After a lapse of 30 minutes or more, the precipitate was washed three times with 150 ml of 5% TCA / 0.05 M sodium pyrophosphate, and the wells were further washed with 100 ml of 80% EtOH. After air-drying the plate, Microscint-20 was added, and measurement was performed using a TopCounter. The background value was subtracted from the obtained value, and the control value was set to 0%, and the inhibition rate of the sample to be evaluated was calculated.

The influenza polymerase inhibitory activity was
The concentration of the test compound that inhibited the elongation of the RNA chain in the absence of the test compound by 50% was defined as the IC _50, and this value was used to carry out the test.

The results are shown below.

[0239]

[Table 3] Test compound IC ₅₀ (μg / ml) Example 1 0.43 As is clear from the above, the compounds of the present invention have remarkable influenza polymerase inhibitory activity and are useful as therapeutic agents for influenza.

[0240]

Industrial Applicability The 2'-O, 4'-C-bridged nucleoside triphosphate of the present invention is a substrate or a therapeutic agent for influenza which enables simple and accurate amplification in a gene amplification method using RNA polymerase. Useful as

[0241]

[Sequence List] SEQUENCE LISTING <110> Sankyo Company, Limited <120> 2'-O, 4'-C-Bridged Nucleoside Triphosphate <130> 2001081SL <150> JP2000-242233 <151> 2000-08-10 <160> 1 <170> PatentIn version 3.0 <210> 1 <211> 18 <212> DNA <213> Bacteriophage T7 <400> 1 taatacgact cactatag 18

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 ZNA A61K 48/00 ZNA C12N 15/09 C12N 15/00 A (72) Inventor Masakatsu Kaneko Tokyo 1-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Takeshi Imanishi 2-2-1-18 Chiyogaoka, Nara-shi, Nara F-term (reference) 4B024 AA20 CA01 HA14 HA19 4C057 LL22 LL45 4C084 AA13 NA14 ZC202 4C086 AA03 EA17 EA18 NA14 ZC20

Claims (6)

[Claims] 1. A compound of the general formula (1) [In the formula, A represents an alkylene group having 1 to 4 carbon atoms, and B represents a purine-9-yl group, a 2-oxo-1,2-dihydropyrimidin-1-yl group or the following α group. Represents a substituted purin-9-yl group or a substituted 2-oxo-1,2-dihydropyrimidin-1-yl group having a substituent, and X represents an oxygen atom or a sulfur atom. Or a salt thereof. (Α group) hydroxyl group, alkoxy group having 1 to 4 carbon atoms, mercapto group, alkylthio group having 1 to 4 carbon atoms, amino group, amino group substituted with alkyl group having 1 to 4 carbon atoms, carbon number 1 to 4 alkyl groups and a halogen atom. 2. The compound according to claim 1, wherein A is a methylene group, and a salt thereof. 3. B is 6-aminopurin-9-yl (ie, adenyl), 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino- 6-fluoropurin-9-yl, 2-amino-6
-Bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl (ie guaninyl), 6-amino-2-methoxypurin-9-yl, 6-amino-2
-Chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurine-9- Yl, 2-oxo-4-amino-1,2-
Dihydropyrimidin-1-yl (ie, cytosinyl), 2-oxo-4-amino-5-fluoro-1,2-
Dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2 -Oxo-4-mercapto-1,2-
Dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (ie, uracinyl), 2-oxo-4-hydroxy-5-
3. A methylpyrimidin-1-yl (ie, thyminyl) or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl) group. A compound or a salt thereof according to any one of the above. 4. The compound or a salt thereof according to claim 1, wherein X is an oxygen atom. 5. A compound selected from the following group or a salt thereof:
2'-O, 4'-C-ethylene guanosine 5'-O-1-thiotriphosphoric acid, 2'-O, 4'-C-ethylene adenosine
5′-O-1-thiotriphosphate, 2′-O, 4′-C-ethyleneuridine 5′-O-1-thiotriphosphate, 2′-O,
4'-C-ethylene 5-methyluridine 5'-O-1-thiotriphosphate, 2'-O, 4'-C-ethylene cytidine 5 '
-O-1-thiotriphosphoric acid, 2'-O, 4'-C-ethylene-5
-Methylcytidine 5'-O-1-thiotriphosphate 2'-O,
4'-C-ethylene guanosine 5'-triphosphate, 2'-
O, 4'-C-ethylene adenosine 5'-triphosphate,
2'-O, 4'-C-ethylene uridine 5'-triphosphoric acid, 2'-O, 4'-C-ethylene 5-methyluridine
5'-triphosphoric acid, 2'-O, 4'-C-ethylene cytidine 5'-triphosphoric acid and 2'-O, 4'-C-ethylene-
5-Methylcytidine 5'-triphosphate. 6. A compound selected from the following group and a salt thereof:
2'-O, 4'-C-ethylene guanosine 5'-triphosphate, 2'-O, 4'-C-ethylene adenosine 5'-triphosphate, 2'-O, 4'-C-ethylene uridine 5 ' -Triphosphoric acid, 2'-O, 4'-C-ethylene 5-methyluridine 5'-triphosphoric acid, 2'-O, 4'-C-ethylenecytidine 5'-triphosphoric acid, and 2'-O, 4'-C-ethylene-5-methylcytidine 5'-triphosphoric acid.