DESCRIPTION
METHOD FOR PRODUCING CYTIDINE DERIVATIVES
Technical Field
The present invention relates to a method for producing
cytidine derivatives. The cytidine derivatives are useful
not only as pharmaceuticals, such as antitumor agents and
antiviral agents, and agricultural chemicals, but also as a
raw material of antisense DNAs that are being developed now.
Background Art
Examples of the known method for producing cytidine
derivatives are as follows.
(1) The method in which 5'-O-(dimethoxytrityl)uridine
derivatives are condensed with 1,2,4-triazole to give
1-[5'-0-(dimethoxytrityl)-ß-D-ribofuranosyl]-4-(1,2,4-tri
azol-l-yl)pyrimidin-2-(1H)-one derivatives, which are then
reacted with an amine in dioxane solvent to yield
5'-O-(dimethoxytrityl) cytidine derivatives (Journal of
Organic Chemistry, 47, 3623 (1982)).
(2) The method in which 2'-deoxy-2'-methyluridine
derivatives are reacted with 4-(N,N-dimethylamino) pyridine
(thereinafter referred to as DMAP) to give
4-[4-(N,N-dimethylamino)pyridinium] derivatives, which are
then treated with 28% NH4OH to yield
2'-deoxy-2'-methylcytidine derivatives (Arch. Pharm., 329,
66 (1996)).
As for the above methods, the method (1) using
1,2,4-triazole is unsuitable for mass production, since the
reaction for the synthesis of
4-(l,2,4-triazol-l-yl)pyrimidin-2-(1H)-one derivatives is
extremely slow and time-consuming, and the extraction of the
product is required, thereby taking a lot of time and effort;
and the method (2) via 4-[4-(dimethylamino)pyridinium]
derivatives is also unsuitable for mass production, since the
reaction is extremely slow and time-consuming, and, if less
than 2.0 equivalents of DMAP is used based on the reaction
substrate, then the unchanged substrates remain and the
manipulation to separate them from the product become necessary,
resulting in an increase in the number of processes, as
described in the comparative examples later.
Disclosure of Invention
Thus, any efficient methods for producing cytidine
derivatives capable of supplying them in large quantities have
been absent previously. The present invention provides a
method for producing cytidine derivatives efficiently.
The inventors have intensively investigated in order to
solve the above problems, and then found that uridine
derivatives can be reacted with a tertiary amine and a
dehydrating reactant in the presence of a deacidifying agent,
followed by ammonia, or a primary or secondary amine to yield
cytidine derivatives in a short period of time by means of
an easy manipulation. That is, the reaction time, which was
long in the conventional methods, was largely reduced.
Furthermore, it was found that the tertiary amine to be used
could be reduced to nearly the same mole based on the uridine
derivative, and then the present invention was completed.
Accordingly, the method for producing cytidine
derivatives of the present invention is the method,
characterized in that a uridine derivative represented by
formula (1):

wherein, X represents a hydrogen atom, a halogen atom,
an alkyl group having 1 to 4 carbon atoms, an alkyl group having
1 to 4 carbon atoms substituted with a halogen atom(s), or
an alkenyl group having 2 to 4 carbon atoms, and Rl and R2
each independently represent either a hydrogen atom or a
hydroxyl-protecting group, and R3 represents a hydrogen atom,
a halogen atom, a hydroxyl group, an alkyl group having 1 to
4 carbon atoms, a cyano group, an alkenyl group, an alkynyl
group, an alkoxy group having 1 to 4 carbon atoms, a hydroxyl
group substituted with a hydroxyl-protecting group,
is reacted with a tertiary amine and dehydrating reactant,
followed by ammonia, or a primary or secondary amine
represented by formula (2):
(2)
wherein, R4 and R5 each independently represent a hydrogen
atom, an alkyl group having 1 to 4 carbon atoms, a cycloalkyl
group having 5 to 8 carbon atoms, an alkyl group having 1 to
4 carbon atoms substituted with a halogen atom (s) , or analkenyl
group having 2 to 4 carbon atoms, or R4 and R5 may be linked
together to form a ring,
for producing a cytidine derivative represented by formula
(3) :

wherein, X, R1, R2, R3, R4 and R5 are as defined above.
Preferably, in the above formulas, examples of the
hydroxyl-protecting groups, i.e. R1 and R2, include an
aliphatic acyl group having 1 to 4 carbon atoms, an aromatic
acyl group, an aromatic acyl group substituted with an alkyl
group(s) having 1 to 4 carbon atoms, an aromatic acyl group
substituted with a halogen atom(s) , an aromatic acyl group
substituted with an alkoxy group (s) having 1 to 4 carbon atoms,
or a trialkylsilyl group, R3 may be a hydrogen atom, an alkoxy
group having 1 to 4 carbon atoms, an aliphatic alkyloxy group
having 1 to 4 carbon atoms substituted with an alkoxy group (s)
having 1 to 4 carbon atoms, an aliphatic acyloxy group having
1 to 4 carbon atoms, an aromatic acyloxy group, an aromatic
acyloxy group substituted with an alkyl group(s) having 1 to
4 carbon atoms, an aromatic acyloxy group substituted with
a halogen atom(s) , or an aromatic acyloxy group substituted
with an alkoxy group(s) having 1 to 4 carbon atoms.
The preferred combination of X and R3 in the above formulas
(1) and (3) includes the combination in which X represents
a hydrogen atom or a methyl group, and R3 is a hydrogen atom,
a methoxy group, or a methoxyethyl group.
Examples of the above tertiary amine include an alicyclic
amine represented by formula (4) :
wherein, n and m each independently represent an integer
of 1 to 4, Y represents hydrogen atom, carbon atom, nitrogen
atom, oxygen atom, or sulfur atom, Z represents a hydrogen
atom, an alkyl group having 1 to 4 carbon atoms, an alkyl group
having 1 to 4 carbon atoms substituted with a halogen atom(s) ,
an alkenyl group having 2 to 4 carbon atoms, or Z may be linked
to A to form a ring, A represents an alkyl group having 1 to
4 carbon atoms, an alkyl group having 1 to 4 carbon atoms
substituted with a halogen atom(s) , an alkenyl group having
2 to 4 carbon atoms, or A may be linked to Z to form a ring;
and an aliphatic amine represented by formula (6):
wherein, R6, R7 and R8 each independently represent an
alkyl group having 1 to 4 carbon atoms, a cycloalkyl group
having 5 to 8 carbon atoms, an alkyl group having 1 to 4 carbon
atoms substituted with a halogen atom(s) , or an alkenyl group
having 2 to 4 carbon atoms.
The specific examples of the tertiary amine include
N-methylpiperidine, N-methylmorpholine,
1,4-diazabicyclo[2.2.2]octane, N, N'-dimethylpiperazine,
and trimethylamine.
On the other hand, the method for producing cytidine
derivatives of the present invention can possess the reaction
route capable of obtaining a cytidine derivative represented
by formula (5):
wherein, X, R1, R2, R3, n, m, A, Y and Z are as defined
above,
as a reaction intermediate, in reacting the uridine derivative
with the tertiary amine and the dehydrating reactant.
Examples of the dehydrating reactants in the above
reaction include acid halides or acid anhydrides, and in this
case, the above reaction is preferably conducted in the
presence of a deacidifying agent. The specific examples of
the above dehydrating reactant include p-toluenesulfonyl
chloride.
The molar ratio of the above tertiary amine to the uridine
derivative represented by formula (1) may be, for example,
1.2 or less.
A cytidine derivative represented by formula (5):
wherein, X, Rl, R2, R3, n, m, A, Y and Z are as defined
above, or salt thereof is a novel compound, and included in
the present invention. The compounds of the above formula
(5) include one, where X represents a hydrogen atom or a methyl
group, Rl and R2 are a hydrogen atom or a hydroxyl-protecting
group, R3 is a hydrogen atom, a methoxy group, a methoxyethyloxy
group, n and m are 2, A is a methyl group, and Y is a methylene
group or an oxygen atom.
Another aspect of the method for producing cytidine
derivatives according to the present invention is the method
for producing a cytidine derivative represented by formula
(3) :
wherein, X, R1, R2, R3, R4 and R5 are as defined above,
characterized in that a cytidine derivative represented by
the above formula (5) or a salt thereof is reacted with ammonia,
or the above primary or secondary amine.
The use of the method suitable for mass production
according to the present invention made it possible to produce
cytidine derivatives more efficiently as compared with the
conventional methods.
Best Mode for Carrying Out the Invention
Now, the present invention will be described in detail.
X in a uridine derivative represented by formula (1)
(thereinafter referred to as the uridine derivative (1)) is,
but not limited to, a hydrogen atom; a halogen atom, such as
a fluorine atom, a chlorine atom, a bromine atom and an iodine
atom; an alkyl group, i.e. a methyl group, an ethyl group,
a propyl group, a 2-propyl group, and a tertiary butyl group;
an alkyl group having 1 to 4 carbon atoms substituted with
a halogen atom (s) , i.e. a chloromethyl group, a dichloromethyl
group, and a trifluoromethyl group; an alkenyl group having
2 to 4 carbon atoms, i.e. a bromovinyl group.
R1 and R2 in the uridine derivative (1) independently
represent a hydrogen atom or a hydroxyl-protecting group.
Hydroxyl-protecting groups include, for example, alkyl ethers ,
aralkyls, acyls, carbonates, sulfonates, silyls.
Alkyl ethers, such as a methoxymethyl group, a
2-methoxyethyl group, a 1-ethoxyethyl group, a
2-methoxyethoxymethyl group, and a tetrahydropyranyl group
can be used.
Aralkyls, such as a benzyl group, a 4-methoxybenzyl group,
a 2-nitrobenzyl group, a trityl group, a 4-methoxytrityl group
and a 4,4'-dimethoxytrityl group can be used.
Both of an aliphatic acyl group and an aromatic acyl group
can be used as the acyls. Such acyl groups may have one or
more substituents, such as halogen, an alkyloxy group, a nitro
group, an acyl group, and an alkyl group, if necessary.
More preferably, the above acyl groups include an
aliphatic acyl group, such as an acetyl group and a propionyl
group, and an aromatic acyl group, such as a benzoyl group,
a toluoyl group, a nitrobenzoyl group, a 4-chlorobenzoyl group,
a 3-chlorobenzoyl group, a 2-chlorobenzoyl group and a
4-methoxybenzoyl group.
Carbonates, such as a methoxycarbonyl group, an
ethoxycarbonyl group, a tertiary butyloxycarbonyl group, a
benzyloxycarbonyl group, and a phenyloxycarbonyl group can
be used.
Sulfonates, such as a p-toluenesulfonyl group, a
methanesulf onyl group, a2,4,6-trimethylphenylsulfonyl group,
a and a 2,4,6-triisopropylphenylsulfonyl group can be used.
Silyls, such as a trimethylsilyl group, a triethylsilyl
group, a dimethyl-tert-butylsilyl group, a
diphenylmethylsilyl group, a triphenylsilyl group, and a
1, l,3,3-tetraisopropyldisiloxan-l,3-diyl group can be used.
R3 in the uridine derivative (1) includes, for example,
a hydrogen atom; a halogen atom, such as a fluorine atom, a
chlorine atom, a bromine atom and an iodine atom; a hydroxyl
group; an alkyl group having 1 to 4 carbon atoms, i.e. a methyl
group, an ethyl group, a propyl group, a 2-propyl group, and
a tertiary butyl group; a cyano group; an alkenyl group, such
as a bromovinyl group; an alkynyl group, such as ethynyl group;
an alkoxy group having 1 to 4 carbon atoms, i.e. a methoxy
group, an ethyloxy group, a n -propyl oxy group, and a n-butyloxy
group; or a hydroxyl group substituted with the above
hydroxyl-protecting group. More preference is given to a
hydrogen atom, a methoxy group, and a methoxyethyloxy group.
Examples of tertiary amines represented by formulas (4)
and (6) include a trimethylamine, a triethylamine, a
tripropylamine, a tri(2-propyl)amine, a tributylamine, a
tri(2-butyl)amine, a tri(tert-butyl)amine, a
di(2-propyl)ethylamine, a N-methylpyrrolidine, a
N-methylpiperidine, a N-methylpyrrole, a
N,N'-dimethylpiperazine, a l,4-diazabicyclo[2.2.2]octane, a
N-methylmorpholine, a N-methylthiomorpholine. The tertiary
amines are not limited to these concrete groups, so far as
the nucleophilicity of nitrogen atom is sufficient for the
reaction to proceed.
Particular preference is given to trimethylamine,
N-methylpyrrolidine, 1,4-diazabicyclo[2.2.2]octane,
N-methylpiperidine, N-methylmorpholine, and
N,N'-dimethylpiperazine.
R4 and R5 in ammonia, or a primary or secondary amine
represented by formula (2) independently represent a hydrogen
atom; an alkyl group having 1 to 4 carbon atoms, i.e. a methyl
group, an ethyl group, a propyl group, a 2-propyl group, and
a tert-butyl group; a cycloalkyl group having 5 to 8 carbon
atoms, i.e. a cyclopentyl group, a cyclohexyl group, a
cycloheptyl group, and a cyclooctyl group; an alkyl group
having 1 to 4 carbon atoms substitutes with a halogen atom(s) ,
i.e. a chloromethyl group and a dichloromethyl group; an
alkenyl group having 2 to 4 carbon atoms, such as a bromovinyl
group, etc. Additionally, R4 and R5 may be linked together
to form a ring, and in this case, R4(R5)N group may be, but
not limited to, a pyrrolidine group or a piperidine group.
Particular preference is given to ammonia and a piperidine.
Dehydrating reactants include, but not limited to, acid
halides, acid anhydrides, esterifying-amidating agent,
acidic catalysts and fluorinating agents.
Acid halides, such as a p-toluenesulfonyl chloride, a
methanesulfonyl chloride, a 2,4,6-trimethylphenylsulfonyl
chloride, a 2,4,6-triisopropylphenylsulfonyl chloride, a
phosphorus oxychloride, a thionyl chloride, a 4-chlorophenyl
dichlorophosphate, an oxalyl chloride, and a malonyl
dichloride can be used. Acid anhydrides, such as
trifluoromethanesulfonic anhydride, acetic anhydride, and
trifluoroacetic anhydride can be used.
Esterifying-amidating agents, for example, carbodiimides
such as 1,3-dicyclohexylcarbodiimide; phosphoniums, such as
bromo(tris-pyrrolidine)phosphonium hexafluorophosphate;
pyridiniums, such as 2-chloro-N-methylpyridinium iodide; and
azodicarboxylates, such as diethyl azodicarboxylate can be
used. Acidic catalysts, such as boron trifluoride diethyl
ether, tin tetrachloride, and aluminium trichloride can be
used. Fluorinating agents, such as diethylaminosulfur
trifluoride, cyanuric fluoride, and
l,3-dimethyl-2,2-difluoroimidazoline can be used.
Preference is given to acidhalide, and particular preference
is given to p-toluenesulfonyl chloride.
Reaction solvents, such as ethers, such as diethyl ether,
diisopropyl ether, tetrahydrofuran, anddioxane; an aliphatic
hydrocarbon-type solvent, such as n-pentane, n-hexane and
cyclohexane; an aromatic hydrocarbon-type solvent, such as
benzene, toluene, xylene, and a halogenated benzene; a
halogenated hydrocarbon-type solvent, such as
dichloromethane, chloroform, and dichloroethane; or
acetonitrile can be used in this reaction, but the reaction
solvent was not limited to them. Particular preference is
given to acetonitrile and chloroform.
The reaction of the uridine derivative (1) according to
the present invention with a tertiary amine and a dehydrating
reactant is carried out by adding the dehydrating reactant
or a solution thereof to the solution of the uridine derivative
(1) and the tertiary amine.
When the acidity within the reaction system affect the
reaction, in addition to the tertiary amine, a deacidifying
agent can be added. In addition to the above tertiary amine,
examples of the deacidifying agent include, but not limited
to, organic bases, such as pyridine, lutidine,
N,N-dimethylaniline, or inorganic bases, such as potassium
carbonate, sodium carbonate, and sodium hydrogencarbonate;
and ion-exchange resin. Particular preference is given to
triethylamine.
The molar ratio of the tertiary amine to the uridine
derivative (1) is in the range between 0.5 and 3.0, preferably
in the range between 1.0 and 2.0, and more preferably 1.2.
The molar ratio of the dehydrating reactant to the uridine
derivative (1) used in the reaction of the uridine derivative
(1) with the tertiary amine and the dehydrating reactant is
in the range between 0.5 and 5.0, preferably 1.0 to 3.0, and
more preferably 2.0.
The molar ratio of the deacidifying agent, if used, to
the uridine derivative (1) in the reaction of the uridine
derivative (1) with a tertiary amine and dehydrating reactant
is in the range between 0.5 and 5.0, preferably 1.0 to 3.0,
and more preferably 2.0.
The temperature at the time when the dehydrating reactant
or its solution is added to the solution of the uridine
derivative (1) and tertiary amine can be in the range between
-10°C and 50°C, preferably in the range between -5°C and 10°C.
The reaction of the uridine derivative (1) with the
tertiary amine and the dehydrating reactant takes 0.5 to 27
hours, but the reaction time is preferably between 0.5 and
3 hours.
The synthesis of the cytidine derivative (3) according
to the present invention is carried out by reacting the uridine
derivative (1) with a tertiary amine and dehydrating reactant,
followed by ammonia, or a primary or a secondary amine. The
molar ratio of ammonia, or the primary or the secondary amine
(2) to the uridine derivative (1) is in the range between 0.5
and 60, preferably 1.0 to 40, and more preferably 2.0 to 37.
As for the condition in the reaction with ammonia, or
a primary or secondary amine after reacting the uridine
derivative (1) with a tertiary amine and a dehydrating reactant,
the temperature can be in the range between -10 t and 50 ºC,
preferably in the range between -5 t and 10 t, and it takes
0.5 to 10 hours to carry out this reaction, but the reaction
time is preferably between 0.5 and 6 hours . In this reaction,
the activation of the 4-position of the uridine derivative
by the dehydrating reactant firstly proceeds, and then the
activated uridine derivative (1) is reacted with the tertiary
amine to form the cytidine derivative represented by formula
(5) or the ammonium salt represented by formula (7):

wherein, R1, R2, R3, R6, R7 , R8 and X are as defined above,
which are then reacted with ammonia, or a primary or secondary
amine to undergo amination at the 4-position.
The cytidine derivative (3) can be also produced by
treating with ammonia, or a primary or secondary amine after
the isolation of the cytidine derivative (5) or (7).
The present invention, therefore, made it possible to
produce the cytidine derivative (3) more efficiently.
Examples
Now, the present invention will be specifically described
by the following examples, but is not limited to them.
Example 1
To 10.0 g of 3',5'-O-bis(4-chlorobenzoyl)
-2' -deoxyuridine was added 50 mL of acetonitrile, 4.0 g of
triethylamine, and 2.4 g (1.2 times moles based on the uridine
derivative) of 1-methylpiperidine, and the resulting mixture
was stirred in an ice bath. To this, the solution of 7.5 g
of p-toluenesulfonyl chloride in 25 ml of acetonitrile was
added dropwise over 1 hour at -1.5 t or below —1.5 ºC. After
the completion of addition, the mixture was stirred for 1 hour.
Then, 50 mL of 28% ammonia water was added dropwise over 20
minutes at 4.5 t or below 4.5 t. After the completion of
addition, the mixture was stirred for 2.5 hours. The
precipitated crystals were filtered and washed with
acetonitrile to give 7.93g of 3',5'-O-bis(4-chlorobenzoyl)
-2'-deoxycytidine. Yield 79.5%.
1H-NMR (400 MHz, DMS0-d6) 8 2.47-2.55 (m, 2H) , 3.35 (s,
1H), 4.50-4.63 (m, 3H), 5.59-5.61 (m, 1H), 5.70 (d, J =7.6
Hz, 1H) , 6.29 (t, J = 7.0 Hz, 1H) , 7.23 (d, J = 7.8 Hz, 1H) ,
7.59-7.66 (m, 5H), 7.96-8.04 (m, 4H).
IR (KBr) cm-1 1719, 1655, 1491, 1271, 1095.
Comparative Example 1
The method given in the conventional method (2) using
DMAP was examined. The results are as follows. To 0.283 g
of 3',5'-O-bis(4-chlorobenzoyl)-2'-deoxyuridine was added
8.5 mL of acetonitrile, 0.221 g of p-toluenesulfonyl chloride,
and 0.141 g (2.1 moles base on the uridine derivative) of DMAP,
followed by adding dropwise 0.117 g of triethylamine, and the
resulting mixture was stirred at room temperature for 22 hours.
To this, 5.7 mL of 28% ammonia water was added and the mixture
was stirred at room temperature for 2 hours, and then ice-cooled.
Crystals were filtered and washed with 60% acetonitrile in
water to give 0.17 g of
3',5'-O-bis(4-chlorobenzoyl)-2'-deoxycytidine. Yield 60% .
In the conventional method, thus, the reaction mixture
must be stirred at room temperature for 22 hours before the
addition of 28% ammonia water, although only one hours is
required in the method according to the present invention,
which made it possible to reduce the overall reaction time
largely.
Comparative Example 2
Experiments with varying the equivalent of DMAP are
described below.
To 0.2 g of
3',5'-0-bis(4-chlorobenzoyl)-2'-deoxyuridine was added 3. 0
mL of acetonitrile, 0.151 g of p-toluenesulfonyl chloride,
and the equivalent shown in Table 1 of DMAP, followed by adding
dropwise 80.2 mg of tri ethyl amine, and the resulting mixture
was stirred at room temperature for 30 hours. The reaction
solution was analyzed by High Performance Liquid
Chromatography to determine the residual rate of the reaction
substrate, 3',5'-O-bis(4-chlorobenzoyl)-2'-deoxyuridine.
The results are shown in Table 1.
As described above, if 1.2 equivalents or less of DMAP
is used, the unchanged raw material remains, and the separation
of it from the desired product become necessary, resulting
in an increase in the number of processes. This seems to be
due to the deactivation of DMAP by hydrogen chloride generated
in the reaction system. On the other hand, since tertiary
amines shown in the present invention are not deactivated by
hydrogen chloride, no unchanged reaction substrates remains
even when 1.2 equivalents of the tertiary amine is used, and
the desired product can be obtained in a short time.
Example 2
To 1.0 g of
3',5'-O-bis(4-chlorobenzoyl)-2'-deoxyuridine was added 5 mL
of acetonitrile, 0.40 g of triethylamine, 0.24 g (1.2 times
moles based on the uridine derivative) of 1-methylpiperidine
and 0.76 g of p-toluenesulfonyl chloride, and the reaction
mixture was stirred in an ice bath for 2 hours. To this, 7.5
mL of a solution of ammonia in 2-propanol was added, and the
mixture was stirred in an ice bath for 4 hours, and at room
temperature for 1 hour. The crystals were filtered and washed
with acetonitrile to give 0.54 g of
3',5'-O-bis(4-chlorobenzoyl)-2'-deoxycytidine. Yield 54%.
Example 3
To 2.0 g of
3', 5'-O-bis (4-chlorobenzoyl) -2' -deoxyuridine was added 60 mL
of acetonitrile, 0.81 g of triethylamine, 0.54. g (1.2 times
moles based on the uridine derivative) of
1,4-diazabicyclo [2.2.2] octane and 1.5 g of p-toluenesulfonyl
chloride, and the resulting mixture was stirred at room
temperature for 3 hours. Ammonia gas was bubbled into the
reaction mixture at room temperature for 1 hour, and the mixture
was stirred in an ice bath for 30 minutes. The crystals were
filtered and washed with acetonitrile to give 1.52 g of
3',5'-O-bis(4-chlorobenzoyl)-2'-deoxycytidine. Yield
75.2%.
Example 4
To 3.0 g of
3', 5' -O-bis (4-chlorobenzoyl) -2' -deoxyuridine was added 30mL
of chloroform, 0 . 72 g of triethylamine, 0 . 80 g (1.2 times moles
of uridine derivative) of l,4-diazabicyclo[2 .2 .2] octane and
1.36 gof p-toluenesulf onyl chloride, and the resulting mixture
was stirred at room temperature for 30 minutes. Ammonia gas
was bubbled into the reaction mixture at room temperature for
1 hour, and the mixture was stirred in an ice bath for 1 hour.
After sludging by the addition of 100 mL of water and 100 mL
of methanol, the crystals were filtered and washed with the
mixed solution of water and methanol (1:1) to give 2.42 g of
3',5'-O-bis(4-chlorobenzoyl)-2'-deoxycytidine. Yield
80.9%.
Reference Example 1
The suspension of 5.0 g of thymine in 41.8 mL of HMDS
was heated to reflux for 3 hours. After cooling, the excess
HMDS was evaporated off. The residue was dissolved by the
addition of 60 mL of chloroform. To this was added 11.4 g
of 3,5-O-bis(4-chlorobenzoyl)-2-deoxyribofuranos-1-yl
chloride (purity 85%) , followed by 60 mL of chloroform, the
mixture was heated at 50 °C for 4 hours with stirring. After
cooling, the solution of 4.46 g of sodium hydrogencarbonate
in 70 ml of water and 70 mL of methanol were added, and the
mixture was stirred at room temperature for 1 hour. After
the removal of the aqueous layer, the solution of 300 mg of
sodium carbonate in 30 mL of water was added, and the mixture
was stirred at room temperature for 10 minutes. After the
separation of the layers, the solvent of the organic layer
was evaporated off, and the resulting residue was purified
by silica gel chromatography (chloroform:methanol = 30:1) to
give 10.5g of 3' ,5'-O-bis (4-chlorobenzoyl) thymidine. Yield
90%.
1H-NMR (400 MHz, CDCl3) 8 1. 65 (d, J= 0. 7 Hz, 3H) , 2 .55-2 . 66
(m, 2H) , 4.48-4.68 (m, 3H) , 5.61-5.65 (m, 1H) , 6.28-6.32 (m,
1H) , 7.50 (d, J = 0.7 Hz, 1H) , 7.57-7.64 (m, 4H) , 7.99-8.04
(m, 4H), 11.4 (s, 1H).
IR (KBr) cm-1 3193, 3067, 1720, 1680, 1593, 1488, 1276,
1013, 761.
Example 5
To the suspension of 3.00 g of
3' , 5' -0-bis (4-chlorobenzoyl) thymidine (a uridine derivative,
where X is methyl group) in 15 mL of acetonitrile was added
0.85 mL (1.2 times moles based on the uridine derivative) of
1-methylpiperidine and 1.70 mL of triethylamine, and the
resulting mixture was cooled. The solution of 2.31 g of
p-toluenesulfonyl chloride in 15 ml of acetonitrile was added
dropwise with keeping the temperature at 0 ºC or below 0 ºC,
and the mixture was stirred for 3 hours with keeping the
temperature at 0 ºC or below 0 ºC. With keeping the temperature
at 0 ºC or below 0 ºC, 15 ml of 28% ammonia water was added,
and the mixture was stirred for 6 hours . After the filtration
of the solvent, 10 mL of methanol was added to the resulting
residue, and themixture was stirred for 2 hours with ice cooling.
After the filtration of the solvent, the resulting solid was
dried to give 1.59 g of
3',5'-O-bis(4-chlorobenzoyl)-5-methyl-2'-deoxycytidine as
a light brown solid. Yield 52%.
Reference Example 2
To the suspension of 0.71 g of
3' , 5'-O-bis(4-chlorobenzoyl)-5-methyl-2'-deoxycytidine in
4 mL of methanol was added 0.7 mL of the solution of sodium
hydroxide in methanol (prepared by dissolving 100 mg of sodium
hydroxide in 4 mL of methanol) , and the mixture was heated
at 45 ºC for 5 hours with stirring. After cooling, the mixture
was neutralized with a solution of hydrogen chloride in
methanol, and methanol was evaporated off. After chloroform
and water were added to the residue, the layers were separated,
and the aqueous layer was washed with chloroform. The aqueous
layer was concentrated and acidified with 6N aqueous hydrogen
chloride solution, and after the addition of 5 mL of acetone,
allowed to stand overnight at -140 ºC. The precipitated solid
was filtered and dried to give 0.37 g of
5-methyl-2'-deoxycytidine hydrochloride as a light brown
solid. Yield 97%.
Reference Example 3
To the solution of 1.01 gof 2'-O-methyluridine and 0.67
g of imidazole in 20 mL of DMF was added 1.3 g of
chloro (t-butyl) dimethyl si lane, and the resulting mixture was
stirred at room temperature for 7 hours. After the completion
of the reaction, the reaction mixture was concentrated, and
the residue was purified by silica gel chromatography (ethyl
acetate:n-hexane =3:7) to give 1.44 g of
3',5'-O-bis(t-butyldimethylsilyl)-2'-O-methyluridine as a
colorless powder. Yield 76%.
1H-NMR (400 MHz, DMSO-d6) 8 0.085 (s, 3H) , 0.10 (s, 3H) ,
0.12 (s, 3H) , 0.13 (s, 3H) , 0.91 (s, 9H) , 0.94 (s, 9H) , 3.55
(s, 3H) ,3.60 (dd, J = 1.7 & 4.9 Hz, 1H) , 3.77 (dd, J = 2.0
& 12.0 Hz, 1H) , 4.18 (dd, J = 2.0 & 12.0 Hz, 1H) , 4.02-4.06
(m, 1H) ,4.24 (dd, J = 4 .9 & 7.1 Hz, 1H) , 5.68 (dd, J = 2.0
& 8.3 Hz, 1H) , 5.94 (d, J = 2.0 Hz, 1H) , 8.07 (d, J = 8.3 Hz,
1H), 8.77 (br s, 1H).
IR (KBr) cm-1 3463, 2953, 2930, 2858, 1691, 1630, 1542,
1463, 1192, 1124, 1035, 1012, 839, 683.
Example 6
To the solution of 750 mg of
3', 5 ' -O-bis (t-butyldimethylsilyl) -2 ' -O-methyl uridine in 10
mL of acetonitrile was added 0.23 mL (1.2 times moles based
on the uridine derivative) of 1-methylpiperidine and 0.45 mL
of triethylamine, and the mixture was cooled. The solution
of 614 mg of p-toluenesulfonyl chloride in 5 ml of acetonitrile
was added dropwise with ice cooling, and the mixture was stirred
for 1 hour. After the disappearance of the starting material,
an 1 mL aliquot of the reaction solution was concentrated,
and the residue was triturated with ether to remove the
precipitated crystals by filtration. The filtrate was
concentrated, and the residue was then washed with n-hexane
and dried to give
[[3,5-0-bis(t-butyldimethylsilyl)-2-0-methylribofuranos-l
-yl]-2-oxo-l,2-dihydro-4-pyrimidinyl]-1-methylpiperidiniu
m chloride (a reaction intermediate):
To the remaining reaction solution was added 3.5 mL of
28% ammonia water, and the mixture was stirred for 2 hours.
The reaction solution was concentrated, diluted with ethyl
acetate and washed with water and saturated aqueous sodium
chloride solution. The organic layer was dried over magnesium
sulfate and concentrated, and the residue was purified by
silica gel chromatography (methanol :chloroform=l:25) to give
544 mg of
3', 5' -O-bis (t-butyldimethylsilyl) -2' -O-methylcytidine as a
colorless powder. Yield 73%.
1H-NMR (400 MHz, DMSO-d6) 5 0.054 (s, 3H) , 0.076 (s, 3H) ,
0.11 (s, 3H) , 0.13 (s, 3H) , 0.89 (s, 9H) , 0.94 (s, 9H) , 3.61
(d, J = 4.9 Hz, 1H) , 3.64 (s, 3H) , 3.78 (d, J = 12.0 Hz, 1H) ,
4.04 (d, J = 9.0 Hz, 1H) , 4.09 (d, J = 12.0 Hz, 1H) , 4.18 (dd,
J = 4 . 9 & 8 . 8 Hz, 1H) , 5 . 61 (d, J = 7 .3 Hz, 1H) , 5 . 93 (s, 1H) ,
8.17 (d, J = 7.3 Hz, 1H).
IR (KBr) cm-1 3355, 3200, 2955, 2930, 2859, 1647, 1491,
1255, 1129, 1073, 838, 781.
WE CLAIM:
1. A method for producing cytidine derivative represented by
the formula
wherein X, represents a hydrogen atom, a halogen atom, an alkyl
group having 1 to 4 carbon atoms, an alkyl group having 1 to 4
carbon atoms substituted with a halogen atom(s), or an alkenyl
group having 2 to 4 carbon atoams and R1 and R2 each
independently represent either a hydrogen atom or a hydroxyl-
protecting group, and R3 represents a hydrogen atom, a halogen
atom, a hydroxy1 group, an alkyl group having 1 to 4 carbon
atoms, a cyano group, an alkanyl group, an alkynyl group, an
alkoxy group having 1 to 4 carbon atoms, a hydroxy1 group
substituted with a hydroxyl-protacting group by reacting a
uridina derivative represented by formula (i)

with tertiary amine and dehydrating reactent, followed by
ammonis, or a primary or a secondary amine represented by formula
(2);
HNR4R5
(2)
wherein R4 and R5 each independently represent a hydrogen atom,
an alkyl group having 1 to 4 carbon atoms a cycloalkyl group
having 5 to 8 carbon atoms, an alkyl group having 1 to 4 carbon
atoms substituted with halogen atom(s), or an alkenyl group
having 2 to 4 carbon atoms, or R4 and R5 linked together may form
a ring.
2. The method for producing cytidine derivatives as claimed
in claim 1, wherein R1 and R2 each independently are an aliphatic
acyl group having 1 to 4 carbon atoms, an aromatic acyl group, an
aromatic acyl group substituted with an alkyl group(s) having 1-4
carbon atoms, an aromatic acyl group substituted with a halogen
atom(s), an aromatic acyl group substituted with an alkoxy
group(s) having 1 to 4 carbon atoms, or a trialkylsilyl group, R3
is a hydrogen atom, an alkoxy group having 1 to 4 carbon atoms,
an aliphatic alkyloxy group having 1 to 4 carbon atoms
substituted with an alkoxy group(s) having 1 to 4 carbon atoms,
an aliphatic acyloxy group having 1 to 4 carbon atoms an
aromatic acyloxy group, an aromatic........................
acyloxy group substituted with an alkyl group(s) having 1 to
4 carbon atoms, an aromatic acyloxy group substituted with
a halogen atom(s) , or an aromatic acyloxy group substituted
with an alkoxy group(s) having has 1 to 4 carbon atoms.
3. The method for producing cytidine derivatives
as claimed in Claim 2, where X represents a hydrogen atom or
a methyl group, R3 is a hydrogen atom, a methoxy group, or
a methoxy ethyloxy group.
4. The method for producing cytidine derivatives
as claimed in claim 1 to 3, where said tertiary amine is an
alicyclic amine represented by formula (4) :
wherein, n and m each independently represent an integer
of 1 to 4, Y represents hydrogen atom, carbon atom, nitrogen
atom, oxygen atom, sulfur atom, Z represents hydrogen atom,
an alkyl group having 1 to 4 carbon atoms, an alkyl group having
1 to 4 carbon atoms substituted with a halogen atom(s), an
alkenyl group haying 2 to 4, carbon atoms, or Z attached to
A may form a ring, A represents an alkyl group having 1 to
4 carbon atoms, an alkyl group having 1 to 4 carbon atoms
substituted with a halogen atom(s) , an alkenyl group having
2 to 4 carbon atoms, or A attached to Z may form a ring.
5. The method for producing cytidine derivatives
according to Claim 4, characterized in that a reaction
intermediate, in reacting uridine derivatives with a tertiary
amine and a dehydrating reactant, such as herein described
is a cytidine derivative represented by formula (5) :
wherein, X, Rl, R2, R3, n, m, A, Y and Z are as defined
above.
6. The method for producing cytidine derivatives
according to Claim 1 to 3, where said tertiary amine is an
aliphatic amine represented by formula (6):
wherein, R6, R7 and R8 each independently represent an
alkyl group having 1 to 4 carbon atoms, a cycloalkyl group
having 5 to 8 carbon atoms, an alkyl group having 1 to 4 carbon
atoms substitutad with a halogen atom(s), or an alsenyl group
having 2 to 4 carbon atoms.
7. The method for producing cytidine derivatives as claimed
in claims 1 to 6 wherein said tartiary having is N-
metyhylpiperidine, N-methylmorpholine, 1,4-diazabicyclo [2.2.2]
octane N.N'-dimethylpiperazine, or trimethylamine.
8. The method for producing cytidine derivatives as claimed
in claim 1 to 7 wherein said dehydrating reactant is acid halidas
or acid anhydridas, and said reaction is carried out in the
presence of a deacidifying agant.
9. The method for producing cytidine derivatives as claimed
in claims 1 to 8 wherein said dehydrating reactant is p-toluene
sulfonyl chloride.
10. The method for producing cytidine derivatives as claimed
in claiam 1 to 9 wherein tha solar ratio of said tartiary amine
to said uridina darivative represented by foroula (1) is in the
range of l.0 to 2.0.
11. A cytidina derivative represented by formula (5):
wherein, X, Rl, R2, R3, n, m, A, Y and Z are as defined
above, or salts thereof.
12. The cytidine derivative or salts thereof as claimed

in Claim 11, where X represents a hydrogen atom or a methyl
group, Rl and R2 are a hydrogen atom or a hydroxyl-protecting
group, R3 is a hydrogen atom, a methoxy group, or a
methoxyethyloxy group, n and m are 2, A is a methyl group,
and Y is a methylene group or an oxygen atom.
13. A method for producing a cytidine derivative
represented by formula (3):
wherein, X, Rl, R2, R3, R4 and R5 are as defined above,
characterized in that the cytidine derivative or salts thereof
according to Claim 11 and 12 is reacted with ammonia or a primary
or secondary amine.

A method for producing cytidine derivative represented by the
formula (3)
wherein X, represents a hydrogen atom, a halogen atom, an alkyl
group having 1 to 4 carbon atoms, an alkyl group having 1 to 4
cartoon atoms substituted with a halogen atom(s), or an alkenyl
group having 2 to 4 carbon atoms, and R1 and R2 each
independently represent either a hydrogen atom or a hydroxyl-
protecting group and R3 represents a hydrogen atom, a halogen
atom, a hydroxyl group, an alkyl group having 1 to 4 carbon
atoms, a cyano group, an alkenyl group, alkynyl group, an
alkoxy group having 1 to 4 carbon atoms, a hydroxyI group
substituted with a hydroxyl-protecting group by reacting a
uridina derivative represented by formula (1)
with a tertiary amine and dehydrating reactant, followed by
ammonia, or a primary or a secondry amine represented by formula
(2):t
HNR4R5
(2)
wherein R4 and RS each independently represent a hydrogen atom,
an alkyl group having 1 to 4 carbon atoms, a cycloalkyl group,
having 9 to 8 carbon atoms, an alkyl group having 1 to 4 carbon
atoms substituted with a halogaen atom(s), or an alkenyl group
having 2 to 4 carbon atoms, or R4 and R5 linked together may form
a ring.