Title of Invention

A NOVEL PYRAZINE NUCLEOTIDE OR PIRAZINE NUCLEOSIDE ANALOG

Abstract wherein each of R1, R2, R3, R4, R5, R6, R7, R8, R9 and A has the same meaning as given in the specification. The present invention relates to a method for exhibiting a virus growth-inhibiting effect and/or a virucidal effect, in which pyrazine nucleotide analog [2] and pyrazine nucleoside analog [3z] are subjected to biotransformation, decomposed and then phosphorylated, so that they become a pyrazine nucleotide analog [lb] exhibiting the aforementioned effect. This method is useful as a method for treating virus infections. Moreover, the pyrazine carboxamide analog or a salt thereof of the present invention is useful as an agent for preventing or treating virus infections.
Full Text DESCRIPTION
TECHNICAL FIELD
The present invention relates to a virus
growth inhibition and/or virucidal method characterized
in that it uses a pyrazine nucleotide or pyrazine
nucleoside analog generated by kinase or a salt
thereof, a novel pyrazine nucleotide or pyrazine
nucleoside analog or a salt thereof, and a method for
treating virus infection using them.
BACKGROUND ART
Infectious virus diseases (e.g., influenza
infection, herpesvirus infection, acquired
immunodeficiency syndrome (AIDS), viral hepatitis,
viral hemorrhagic fever, etc.) have been recognized to
be medically important problems, and a wide range of
treatments such as prevention of the diseases with
vaccines or therapeutic methods of using drugs have
been studied. A large number of nucleic acids having
purine bases and pyrimidine bases and derivatives
thereof have been developed to date as agents used for
drug treatment of virus infections. These agents have
an action mechanism such that they are
triphosphorylated in cells and inhibit virus
polymerase. Examples of such agents may include
azidothymidine and acyclovir [Proceedings of the
National Academy of Science of the United States of
America (Proc. Natl. Acad. Sci. USA), Vol. 83, pp. 8333
to 8337 (1986); the same publication, Vol. 74, pp. 5716
to 5720 (1977)].
Moreover, it has been reported that the
active form of a compound whose antiviral effect is
exhibited when its portion corresponding to bases of
nucleic acid is converted into an unnatural chemical
structure is a monophosphorylated form obtained by
converting the compound in a cell, and that it inhibits
inosine monophosphate dehydrogenase (IMPDH) in the
cell, thereby exhibiting effects. Examples of such a
compound may include ribavirin and EICAR [Proceedings
of the National Academy of Science of the United States
of America (Proc. Natl. Acad. Sci. USA), Vol. 70, pp.
1174 to 1178 (1973); The Journal of Biological
Chemistry (J. Biol. Chem.), Vol. 268, pp. 24591 to
24598 (1993)].
Moreover, as an example of nucleoside and
nucleotide analogs having a pyrazine ring as a base,
the following general formula has been known:
wherein R16 represents a hydrogen atom, a methyl group
or decyl group. However, this compound exhibits no
antiviral activity (no anti-Visna virus activity)
[Nucleosides & Nucleotides, Vol. 15, Nos. 11 and 12,
pp.. 1849 to 1861 (1996) ] .
On the other hand, nucleoside and nucleotide
analogs having a pyrazine ring that is substituted with
a carbamoyl group have not been known.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to
provide a highly safe antiviral agent with low
toxicity, which has an unnatural chemical structure in
a portion corresponding to bases of nucleic acid, and a
novel virus growth inhibition and/or virucidal method
using the antiviral agent.
The present inventors have found that a
pyrazine nucleotide or pyrazine nucleoside analog
represented by the following general formula [1] or a
salt thereof:
wherein R1 represents a hydrogen atom, or a substituent
of a pyrazine ring; R2 represents a hydrogen atom, an
acyl group, or a carbamoylalkyl or carboxyalkyl group
that may be substituted; each of R3, R4, Rs and R6
identically or differently represents a hydrogen atom,
or a hydroxyl group that may be substituted or
protected; A represents an oxygen atom or a methylene
group; Y represents an oxygen atom or an imino group;
and n represents an integer of 0 to 3, especially, a
triphosphorylated pyrazine nucleotide analog or a salt
thereof exhibits a highly safe, excellent virus growth
inhibiting and/or virucidal effect with low toxicity
which inhibits virus polymerase, especially RNA
polymerase directly or in the form of a substance
converted therefrom in vivo.
Moreover, the present inventors have found a
novel method of hydrolyzing or decomposing in vivo or
in a cell a pyrazine nucleotide analog represented by
the following general formula [2] or a salt thereof:
wherein each of R1, R2, R3, R4, R5, R6, A and Y has the
same meaning as given above; and each of R7 and R8 in
phosphoric acid or phosphonic acid independently
represents a protected or unprotected, substituted or
unsubstituted hydroxyl group to be decomposed under
physiological conditions, and then inducing the product
thus obtained into the pyrazine nucleotide or pyrazine
nucleoside analog represented by general formula [1] or
a salt thereof by the effect of kinase such as
nucleotide kinase, so as to make it exhibit a virus
growth-inhibiting effect and/or a virucidal effect.
Furthermore, the present inventors have found
that a pyrazine nucleotide analog represented by the
following general formula [lz] or a salt thereof:
wherein R1 represents a hydrogen atom, or a substituent
of a pyrazine ring; R2 represents a hydrogen atom, an
acyl group, or a carbamoylalkyl or carboxyalkyl group
that may be substituted; each of R3, R4, R5 and R6
identically or differently represents a hydrogen atom,
or a hydroxyl group that may be substituted or
protected; R represents a hydroxyl group that may be
protected or substituted with a group decomposed under
physiological conditions; A represents an oxygen atom
or a methylene group; and n represents an integer of 1
to 3, is a compound, which is converted under
physiological conditions and inhibits virus RNA
polymerase in the same manner as the general formula
[1], thus exhibiting a virus growth-inhibiting effect
and/or a virucidal effect.
Any of the aforementioned compounds is
converted in vivo or in a cell into a pyrazine
triphosphate nucleotide analog represented by the
following general formula [ly]:
wherein R1 represents a hydrogen atom, or a substituent
of a pyrazine ring; and each of Z10, Z11, Z12 and Z13
identically or differently represents a hydrogen atom
or hydroxyl group, and it exhibits an RNA polymerase
inhibitory effect. In addition, a compound represented
by general formula [1x] indicated below is an RNA
polymerase inhibitor precursor that is converted into
the compound represented by general formula [1y] in
vivo or in a cell:
wherein R1 represents a hydrogen atom, or a substituent
of a pyrazine ring; R2 represents a hydrogen atom, an .
acyl group, or a carbamoylalkyl or carboxyalkyl group
that may be substituted; each of R3, R4, R5 and R6
identically or differently represents a hydrogen atom,
or a hydroxyl group that may be substituted or
protected; A represents an oxygen atom or a methylene
group; Y represents an oxygen atom or an imino group;
and m represents an integer of 0 to 2.
The RNA polymerase inhibitor precursor of the
present invention inhibits virus-derived RNA polymerase
with selectivity much more higher than host-derived RNA
polymerase. The present RNA polymerase inhibitor
precursor can inhibit virus-derived RNA polymerase with
selectivity preferably 200 times or more, more
preferably 1,000 times or more, and further more
preferably 2,000 times or more, higher than that for
host-derived RNA polymerase. Moreover, the RNA
polymerase inhibitor precursor of the present invention
hardly inhibits inosine monophosphate dehydrogenase,
and after it is converted in vivo into a
triphosphorylated form, it inhibits virus polymerase.
Accordingly, the present RNA polymerase inhibitor
precursor is characterized in that it has an extremely
strong virus polymerase inhibitory effect after it is
converted in vivo, and it also has high selectivity,
while cytotoxicity caused by inhibition of inosine
monophosphate dehydrogenase is extremely reduced.
Using this high selectivity, a highly safe agent can be
obtained.
The RNA polymerase inhibitor precursor of the
present invention has extremely high selectivity to RNA
polymerase and inosine monophosphate dehydrogenase. In
a pyrazine nucleoside or pyrazine mononucleotide analog
structure represented by the following general formula
[lw] :
wherein R1 represents a hydrogen atom, or a substituent
of a pyrazine ring; R2 represents a hydrogen atom, an
acyl group, or a carbamoylalkyl or carboxyalkyl group
that may be substituted; each of R3, R4, R5 and R6
identically or differently represents a hydrogen atom,
or a hydroxyl group that may be substituted or
protected; Y represents an oxygen atom or an imino
group; and p represents 0 or 1, the ratio of the
inhibitory effect on virus-derived RNA polymerase of
the precursor after it is converted in vivo that on the
host cell-derived inosine monophosphate dehydrogenase
of the precursor is preferably 900 : 1 or more, more
preferably 5,000 : 1 or more , and further more
preferably 10,000 : 1 or more.
The present inventors have confirmed that
when each of R1, R3 and R5 is a hydrogen atom and each of
R4Z, R6Z and Rz is a hydroxyl group, for example, in a
pyrazine nucleotide derivative represented by the
following general formula [3z]:
wherein each of R1, R2, R3, R5 and Y has the same meaning
as given above; Rz represents a protected or
unprotected, substituted or unsubstituted hydroxyl
group to be decomposed under physiological conditions;
each of R4Z and R6Z identically or differently represents
a hydrogen atom or a hydroxyl group that may be
substituted or protected, or R4Z and R6Z together
represent a group represented as -O-alkylene-O- that
may be substituted, and when such a compound is
administered to an animal, there is generated 4-[(2R,
3R, 4S, 5R)-3,4-dihydroxy-5~(hydroxymethyl)tetrahydro-
2-furanyl]-3-oxo-3,4-dihydro-2-pyrazinecarboxamide
(substitution nomenclature: 3,4-dihydro-3-oxo-4-p-D-
ribofuranosyl-2-pyrazinecarboxamide) in the blood
plasma of the animal.
What is more, the present inventors have
confirmed that when the pyrazine nucleoside analog
represented by general formula [3z] or a salt thereof
such as 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-(hydroxy-
methyl) tetrahydro-2-furanyl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide is administered to an animal, there
is generated {(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-
oxo-1(2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl}
methyl triphosphate in the organ of the animal.
Thus, the present inventors have found that a
method of administering the compound represented by
general formula [3z] or a salt thereof to mammals, or
administering the compound represented by general
formula [2] or a salt thereof to mammals, so as to
induce the pyrazine nucleotide or pyrazine nucleoside
analog represented by general formula [1] or a salt
thereof in vivo, exhibits a novel virus growth-
inhibiting effect and/or a novel virucidal effect,
thereby completing the present invention.
The method of the present invention is useful
as a method for treating patients infected with virus,
which comprises a step of administering to a patient
infected with virus the aforementioned pyrazine
nucleotide or pyrazine nucleoside analog or a salt
thereof such as the compound represented by general
formula [3z] or a salt thereof. More preferably, the
method of the present invention further comprises a
step of converting the above compound or a salt thereof
into the pyrazine triphosphate nucleotide analog
represented by general formula [1y].
Moreover, it is preferably that, in the body
of a patient infected with virus, the general formula
[3z] is converted into the pyrazine triphosphate
nucleotide analog represented by general formula [1y]
through a pyrazine nucleotide analog represented by the
following general formula [Iv]:
wherein R1 represents a hydrogen atom, or a substituent
of a pyrazine ring; and each of Z10, Z11, Z12 and Z13
identically or differently represents a hydrogen atom
or hydroxyl group. The pyrazine nucleotide analog
represented by general formula [1v] is characterized in
that it does not substantially inhibit inosine
monophosphate dehydrogenase derived from host cells.
Moreover, the pyrazine triphosphate nucleotide analog
represented by general formula [1y] is characterized in
that it inhibits virus-derived RNA polymerase more
selectively than host-derived RNA polymerase.
Furthermore, as a result of intensive studies
of the present inventors regarding anhydrides of 4-
[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-dihydro-
2-pyrazinecarboxamide that is a representative compound
of the present invention, they have found 4-[(2R, 3R,
4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-2-
furanyl]-3-oxo-3,4-dihydro-2-pyrazinecarboxamide
monohydrate, which has excellent stability during the
process of pharmaceutical preparation. This hydrate is
a crystal that is stable during the process of
pharmaceutical preparation by common methods, and
during the process of pharmaceutical preparation, and
it has less dustability and does not adhere to
instruments when compared with anhydrides.
Accordingly, it enables good mixing and granulation.
Wet granulation is generally used as
granulation during the process of pharmaceutical
preparation. During such wet granulation, water and an
aqueous solution containing a binder are generally
used. However, it is known that when an anhydride is
used, a part of the anhydride is converted into a
hydrate depending on conditions. Moreover, an
amorphous substance generated in this process causes a
problem from the viewpoint of pharmaceutical
preparation or stability. Accordingly, when a hydrate
exists as a crystal polymorph, strict conditions are
required for preparation of a pharmaceutical from an
anhydride. However, a monohydrate is a stable crystal
during the common pharmaceutical preparation process,
and therefore, it is an excellent compound that does
not cause the aforementioned problem.
In addition, such a monohydrate does not need
an organic solvent in the final preparation process,
but it can be crystallized from water. Accordingly,
the risk that organic solvents are remained in the
finally obtained crystal is low. Moreover, since the
monohydrate does not need an organic solvent, it does
not need explosion-proof equipment. Thus, it can be
said that this compound has great advantages on the
production process.
The compound of the present invention will be
described in detail below.
BEST MODE FOR CARRYING OUT THE INVENTION
In the specification, unless otherwise
mentioned, a halogen atom means a fluorine atom, a
chlorine atom, and an iodine atom; an alkyl group means
a lower alkyl group, for example, a C1-5 alkyl group such
as methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, tert-butyl, and pentyl; an alkoxy
group means a lower alkoxy group, for example, a C1-5
alkoxy group such as methoxy, ethoxy, n-propoxy,
isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-
butoxy, and pentyloxy; an alkoxycarbonyl group means a
lower alkoxycarbonyl group, for example, a C1-5
alkoxycarbonyl group such as methoxycarbonyl,
ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl,
n-butoxycarbonyl, isobutoxycarbonyl, sec-
butoxycarbonyl, tert-butoxycarbonyl, 4-
hydroxybutoxycarbonyl, and pentyloxycarbonyl; an
alkylamino group means, for example, a mono- or di-C1-5
alkylamino group such as methylamino, ethylamino,
propylamine, dimethylamino, diethylamino, and
methylethylamino; a halogenoalkyl group means, for
example, a halogeno-C1-5 alkyl group such as
fluoromethyl, chloromethyl, bromomethyl,
dichloromethyl, trifluoromethyl, trichloromethyl,
chloroethyl, dichloroethyl, trichloroethyl, and
chloropropyl; a carbamoylalkyl group means, for
example, a C1-5 carbamoylalkyl group, such as
carbamoylmethyl, carbamoylethyl, carbamoyl-n-propyl,
carbamoylisopropyl, carbamoyl-n-butyl,
carbamoylisobutyl, and carbamoylpentyl; a carboxyalkyl
group means, for example, a C1-5 carboxyalkyl group such
as carboxymethyl, carboxyethyl, carboxy-n-propyl,
carboxyisopropyl, carboxy-n-butyl, carboxyisobutyl, and
carboxypentyl; an alkenyl group means, for example, a
C2-5 alkenyl group such as vinyl and allyl; a cycloalkyl
group means, for example, a C3-6 cycloalkyl group such as
cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; a
cycloalkyloxy group means, for example, a C3-6
cycloalkyloxy group such as cyclopropyloxy,
cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy; an
aryl group means, for example, a group such as phenyl
and naphthyl; a heterocyclic group means, for example,
a 4- to 6-membered or condensed heterocyclic group
containing at least one heteroatom selected from an
oxygen atom, a nitrogen atom, and a sulfur atom such as
azetidinyl, thienyl, furyl, pyrrolyl, imidazolyl,
pyrazolyl, thiazolyl, isothiazolyl, oxazolyl,
isoxazolyl, furazanyl, pyrrolidinyl, pyrrolynyl,
imidazolydinyl, imidazolynyl, pyrazolidinyl,
pyrazolinyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-
triazolyl, 1,2,4-triazolyl, thiatriazolyl, pyridyl,
pyrazinyl, pyrimidinyl, pyridazinyl, pyranyl,
morpholinyl, 1,2,4-triazinyl, benzothienyl,
naphthothienyl, benzofuryl, isobenzofuryl, chromenyl,
indolizinyl, isoindolyl, indolyl, indazolyl, purinyl,
quinolyl, isoquinolyl, phthalazinyl, naphthylidinyl,
quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl,
isochromanyl, chromanyl, indolinyl, isoindolinyl,
benzoxazolyl, triazolopyridyl, tetrazolopyridazinyl,
tetrazolopyrimidinyl, thiazolopyridazinyl,
thiadiazolopyridazinyl, triazolopyridazinyl,
benzimidazolyl, benzothiazolyl, 1,2,3,4-
tetrahydroquinolyl, imidazo[1,2-b][1,2,4]triazinyl, and
quinuclidinyl; an alkylene group means, for example, a
straight or branched C1-5 alkylene group such as
methylene, ethylene, and propylene; an alkylthio group
means, for example, a straight or branched C1-5 alkylthio
group such as methylthio, ethylthio, n-propylthio,
isopropylthio, n-butylthio, isobutylthio, sec-
butylthio, tert-butylthio, and pentylthio; an aryloxy
group means, for example, a group represented by aryl-
0- such as phenoxy and naphthoxy; an arylthio group
means, for example, a group represented by aryl-S- such
as phenylthio and naphthylthio; an arylamino group
means, for example, phenylamino and naphthylamino; a
cycloalkylamino group means, for example, a C3-6
cycloalkylamino group such as cyclopropylamino,
cyclobutylamino, cyclopentylamino, and cyclohexylamino;
an acyl group means, for example, C2-6 alkanoyl group
such as formyl, acetyl, or propionyl, an aroyl group
such as benzoyl or naphthoyl, and a heterocyclic
carbonyl group such as nicotinoyl, thenoyl,
pyrrolidinocarbonyl, or furoyl; an acyloxy group means,
for example, a C2-6 alkanoyloxy group such as acetyloxy
or propionyloxy, an aroyloxy group such as benzoyloxy
or naphthoyloxy, and a heterocyclic carbonyloxy group
such as nicotinoyloxy, thenoyloxy,
pyrrolidinocarbonyloxy or furoyloxy; an arylsulfonyloxy
group means, for example, a group such as
phenylsulfonyloxy and p-toluenesulfonyloxy; an
alkylsulfonyloxy group means, for example, a straight
or branched C1-5 alkylsulfonyloxy group such as
methylsulfonyloxy, ethylsulfonyloxy, n-
propylsulfonyloxy, isopropylsulfonyloxy, n-
butylsulfonyloxy, isobutylsulfonyloxy, sec-
butylsulfonyloxy, tert-butylsulfonyloxy, and
pentylsulfonyloxy, respectively.
In a general formula described in the
specification, substituents of a pyrazine ring of R1
include a group selected from a halogen atom; an alkyl
group that may be substituted by hydroxyl, alkoxy,
alkylthio, aryl, amino, or alkylamino group; an alkyl
or alkenyl group that may be substituted by a halogen
atom; a cycloalkyl group; a hydroxyl group; an alkoxy
group; a cycloalkyloxy group; an alkoxycarbonyl group;
a mercapto group; an alkylthio group that may be
substituted by an aryl group; an aryl group; an aryloxy
group; an arylthio group; an arylamino group; a cyano
group; a nitro group; an amino group that may be
substituted by an acyl group; an alkylamino group; a
cycloalkylamino group; an acyl group; a carboxyl group;
a carbamoyl group; a thiocarbamoyl group; an
alkylcarbamoyl group; and a heterocyclic group, and one
or more such groups may be substituted.
Protecting groups and substituents of a
hydroxyl group of Rz include, for example, an acyl group
that may be substituted, a lower alkoxycarbonyl group,
and an acyloxyalkyl group, more specifically, an acyl
group that may be substituted, such as acetyl,
propionyl, valeryl, benzoyl, pivaloyl, 2-aminoacetyl,
2-aminopropionyl, 2-aminovaleryl, and 2-aminocaprolyl;
a lower alkoxycarbonyl group such as methoxycarbonyl,
ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl,
n-butoxycarbonyl, isobutoxycarbonyl, sec-
butoxycarbonyl, tert-butoxycarbonyl, and 4-
hydroxybutoxycarbonyl; and an acyloxyalkyl group such
as acetyloxymethyl, propionyloxymethyl,
isopropionyloxymethyl, butyryloxymethyl,
isobutyryloxymethyl, valeryloxymethyl,
isovaleryloxymethyl, pivaloyloxymethyl, and 1-
pivaloyloxyethyl.
R7 and R8, each independently a protecting
group or substituent of a hydroxyl group to be
decomposed under the physiological condition in the
phosphate or phosphonate, and the R group decomposable
under such condition are, for example, a protecting
group or substituent of the phosphate or phosphonate
described in Progress in Medicinal Chemistry, Vol. 34,
pp. 111-147 (1997), Elsevier Science B.V., and Current
Medicinal Chemistry, Vol. 7, pp. 995-1039 (2000). The
specific examples include an aryl group such as phenyl,
chlorophenyl, nitrophenyl, cyanophenyl, naphthyl; a
cyclosaligenyl group such as cyclosaligenyl, 5-
methylcyclosaligenyl; an amidate group such as
methoxyalaninyl and phenoxyalaninyl; a haloethyl group
such as trichloroethyl; an acyloxyalkyl group such as
acetyloxymethyl, propionyloxymethyl,
isopropionyloxymethyl, butyryloxymethyl,
isobutyryloxymethyl, valeryloxymethyl,
isovaleryloxymethyl, pivaloyloxymethyl, and 1-
pivaloyloxyethyl; an acyloxybenzyl group such as
acetyloxybenzyl, propionyloxybenzyl,
isopropionyloxybenzyl, butyryloxybenzy1,
isobutyryloxybenzyl, valeryloxybenzyl,
isovaleryloxybenzyl, pivaloyloxybenzyl; an s-lower
acylthioalkyl group such as acetylthioethyl,
propionylthioethyl, isopropionylthioethyl,
butyrylthioethyl, isobutyrylthioethyl,
valerylthioethyl, isovalerylthioethyl,
pivaloylthioethyl, and pivaloylthiobutyl; an s-higher
acylthioalkyl group such as lauroylthioethyl; an s-
aroylthioalkyl group such as benzoylthioethyl and
naphthoylthioethyl; and dithiodiethyl group.
The expression "decomposed under the
physiological condition" means to be decomposed by an
enzyme such as esterase, phosphodiesterase,
phosphonamidase, hydrolase, aminohydrolase,
transaminase, or reductase, as well as a physiological
oxidation, hydrolysis, and/or reduction reaction.
Examples of a kinase include a nucleotide
kinase, nucleoside kinase, nucleoside
phosphotransferase, and 5'-nucleotidase.
A precursor means a substance that produces a
pharmacologically active substance itself by
conversion/decomposition in vivo.
Examples of a protecting group of a carboxyl
group include all groups that can be used as a usual
protecting group of a carboxyl group, for example, a
lower alkyl group such as methyl, ethyl, n-propyl,
isopropyl, 1,1-dimethylpropyl, n-butyl, and tert-butyl;
an aryl group such as phenyl and naphthyl; an aryl-
lower alkyl group such as benzyl, diphenylmethyl,
trityl, p-nitrobenzyl, p-methoxybenzyl, and bis(p-
methoxyphenyl)methyl; an acyl-lower alkyl group such as
acetylmethyl, benzoylmethyl, p-nitrobenzoylmethyl, p-
bromobenzoylmethyl, and p-methanesulfonylbenzoylmethyl;
an oxygen-containing heterocyclic group such as 2-
tetrahydropyranyl and 2-tetrahydrofuranyl; a halogeno-
lower alkyl group such as 2,2,2-trichloroethyl; a lower
alkylsilylalkyl group such as 2-(trimethylsilyl)ethyl;
an acyloxyalkyl group such as acetoxymethyl,
propionyloxymethyl, and pivaloyloxymethyl; a nitrogen-
containing heterocyclic-lower alkyl group such as
phthalimidemethyl and succinimidemethyl; a cycloalkyl
group such as cyclohexyl; a lower alkoxy-lower alkyl
group such as methoxymethyl, methoxyethoxymethyl, and
2-(trimethylsilyl)ethoxymethyl; an aryl-lower alkoxy-
lower alkyl group such as benzyloxymethyl; a lower
alkylthio-lower alkyl group such as methylthiomethyl
and 2-methylthioethyl; an arylthio-lower alkyl group
such as phenylthiomethyl; a lower alkenyl group such as
1,1-dimethyl-2-propenyl, 3-methyl-3-butynyl, and aryl;
and a substituted silyl group such as trimethylsilyl,
triethylsilyl, triisopropylsilyl,
diethylisopropylsilyl, tert-butyldimethylsilyl, tert-
butyldiphenylsilyl, diphenylmethylsilyl, and tert-
butylmethoxyphenylsilyl.
Examples of a protecting group of an amino
group and an imino group include all groups that can be
used as a usual amino protecting group, for example, an
acyl group such as trichloroethoxycarbonyl,
tribromoethoxycarbonyl, benzyloxycarbonyl, p-
nitrobenzyloxycarbonyl, o-bromobenzyloxycarbonyl,
(mono-, di-, tri-) chloroacetyl, trifluoroacetyl,
phenylacetyl, formyl, acetyl, benzoyl, tert-
amyloxycarbonyl, tert-butoxycarbonyl, p-
methoxybenzyloxycarbonyl, 3,4-
dimethoxybenzyloxycarbonyl, 4-
(phenylazo)benzyloxycarbonyl, 2-furfuryloxycarbonyl,
diphenylmethoxycarbonyl, 1,1-dimethylpropoxycarbonyl,
isopropoxycarbonyl, phthaloyl, succinyl, alanyl,
leucyl, 1-adamantyloxycarbonyl, and 8-
quinolyloxycarbonyl; an aryl-lower alkyl group such as
benzyl, diphenylmethyl, and trityl; an arylthio group
such as 2-nitrophenylthio and 2,4-dinitrophenylthio; an
alkane- or arene-sulfonyl group such as methanesulfonyl
and p-toluenesulfonyl; a di-lower alkylamino-lower
alkylidene group such as N,N-dimethylaminomethylene; an
aryl-lower alkylidene group such as benzylidene, 2-
hydroxybenzylidene, 2-hydroxy-5-chlorobenzylidene, and
2-hydroxy-l-naphthylmethylene; a nitrogen-containing
heterocyclic alkylidene group such as 3-hydroxy-4-
pyridylmethylene; a cycloalkylidene group such as
cyclohexylidene, 2-ethoxycarbonylcyclohexylidene, 2-
ethoxycarbonylcyclopentylidene, 2-
acetylcyclohexylidene, and 3,3-dimethyl-5~
oxycyclohexylidene; a diaryl- or diaryl-lower
alkylphosphoryl group such as diphenylphosphoryl and
dibenzylphosphoryl; an oxygen-containing heterocyclic
alkyl group such as 5-methyl-2-oxo-2H-l,3-dioxol-4-yl-
methyl; and a lower alkyl substituted silyl group such
as trimethylsilyl.
Examples of a protecting group of a hydroxyl
group include all groups that can be used as a usual
hydroxyl protecting group, for example, an acyl group
such as benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-
bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, methoxycarbonyl,
ethoxycarbonyl, tert-butoxycarbonyl, 1,1-
dimethylpropoxycarbonyl, isopropoxycarbonyl,
isobutyloxycarbonyl, diphenylmethoxycarbonyl, 2,2,2-
trichloroethoxycarbonyl, 2,2,2-tribromoethoxycarbonyl,
2-(trimethylsilyl)ethoxycarbonyl, 2-
(phenylsulfonyl)ethoxycarbonyl, 2-
(triphenylphosphonio)ethoxycarbonyl, 2-
furfuryloxycarbonyl, 1-adamantyloxycarbonyl,
vinyloxycarbonyl, allyloxycarbonyl, S-
benzylthiocarbonyl, 4-ethoxy-l-naphthyloxycarbonyl, 8-
quinolyloxycarbonyl, acetyl, formyl, chloroacetyl,
dichloroacetyl, trichloroacetyl, trifluoroacetyl,
methoxyacetyl, phenoxyacetyl, pivaloyl, and benzoyl; a
lower alkyl group such as methyl, tert-butyl, 2,2,2-
trichloroethyl, and 2-trimethylsilylethyl; a lower
alkenyl group such as allyl; an aryl-lower alkyl group
such as benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl,
diphenylmethyl, and trityl; an oxygen- and sulfur-
containing heterocyclic group such as tetrahydrofuryl,
tetrahydropyranyl, and tetrahydrothiopyranyl; a lower
alkoxy- and lower alkylthio-lower alkyl group such as
methoxymethyl, methylthiomethyl, benzyloxymethyl, 2-
methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl, 2-
(trimethylsilyl)ethoxymethyl, and 1-ethoxyethyl; an
alkyl- and aryl-sulfonyl group-such as raethanesulfonyl
and p-toluenesulfonyl; and a substituted silyl group
such as trimethylsilyl, triethylsilyl,
triisopropylsilyl, diethylisopropylsilyl, tert-
butyldimethylsilyl, tert-butyldiphenylsilyl,
diphenylmethylsilyl, and tert-butylmethoxyphenylsilyl,
and in the case of a dihydroxyl group, further include
a lower alkylidene group such as methylene,
benzylidene, and isopropylidene, a lower alkoxy-lower
alkylidene group such as methoxymethylene, and a lower
alkyl substituted silyl group such as 1,1,3,3-
tetraisopropyldisiloxanylidene.
A carbamoylalkyl group and a carboxyalkyl
group of R2 may be substituted by one or more
substituents selected from a halogen atom; an alkyl
group that may be substituted by a hydroxyl, alkoxy,
alkylthio, aryl, amino, or alkylamino group; a
halogenoalkyl group; an alkenyl group; a cycloalkyl
group; hydroxyl group; alkoxy group; a cycloalkyloxy
group; an alkoxycarbonyl group; a mercapto group; an
alkylthio group that may be substituted by an aryl
group; an aryl group; an aryloxy group; an arylthio
group; an arylamino group; cyano group; nitro group; an
amino group that may be substituted by an acyl group;
an alkylamino group; a cycloalkylamino group; an acyl
group; a carboxyl group; a carbamoyl group; a
thiocarbamoyl group; an alkylcarbamoyl group, and a
heterocyclic group.
A hydroxyl group of R3, R4, R5, and R6 may be
substituted by one or more substituents selected from a
carboxyl group that may be protected, an alkyl group,
an alkoxycarbonyl group, an aryl group, a cycloalkyl
group, an alkenyl group, a halogenoalkyl group, and a
heterocyclic group.
Examples of salts of general formula [1] and
general formula [2] include salts of a usually known
basic group such as an amino group, or an acidic group
such as a hydroxyl, phosphoryl, phosphonyl, or carboxyl
group. Salts of basic groups include, for example, a
salt with a mineral acid such as hydrochloric acid,
hydrobromic acid, and sulfuric acid; a salt with an
organic carboxylic acid such as tartaric acid, formic
acid, and citric acid; and a salt with a sulfonic acid
such as methanesulfonic acid, benzenesulfonic acid, p-
toluenesulfonic acid, mesitylenesulfonic acid, and
naphthalenesulfonic acid. Salts of acidic groups
include, for example, a salt with an alkali metal such
as sodium and potassium; a salt with an alkali earth
metal salt such as calcium and magnesium; an ammonium
salt; as well as a salt with a nitrogen-containing
organic base such as trimethylamine, triethylamine,
tributylamine, pyridine, N,N-dimethylaniline, N-
methylpiperidine, N-methylmorpholine, diethylamine,
dicyclohexylamine, procaine, dibenzylamine, N-benzyl-p-
phenethylamine, 1-ephenamine, and N,N'-
dibenzylethylenediamine.
Moreover, when the compounds represented by
general formulas [11, [Iv], [lwl, [1x1, [1y], [1Z], [2]
and [3z], and salts thereof have isomers (e.g., optical
isomers, geometric isomers, tautomers, etc.), the
present invention includes these isomers, solvates,
hydrates, and various forms of crystals.
Examples of virus to which the virus growth
inhibition and/or virucidal method of the present
invention is applied may include influenza virus, RS
virus, AIDS virus, papilloma virus, adenovirus,
hepatitis A virus, hepatitis B virus, hepatitis C
virus, hepatitis E virus, poliovirus, echovirus,
Coxsackie virus, enterovirus, rhinovirus, rotavirus,
Newcastle disease virus, mumps virus, vesicular
stomatitis virus, rabies virus, Lassa fever virus,
measles virus, Filovirus, Japanese encephalitis virus,
yellow fever virus, dengue fever virus or West Nile
virus. Preferably, such examples may include influenza
virus, RS virus, hepatitis A virus, hepatitis C virus,
hepatitis E virus, poliovirus, echovirus, Coxsackie
virus, enterovirus, rhinovirus, rotavirus, Newcastle
disease virus, mumps virus, vesicular stomatitis virus,
rabies virus, Lassa fever virus, measles virus,
Filovirus, Japanese encephalitis virus, yellow fever
virus, dengue fever virus or West Nile virus.
Particularly preferably, such examples may include
influenza virus and hepatitis C virus.
Preferred compounds of the present invention
may include compounds having the below mentioned
substituents.
In each of the general formulas described in
the present invention, examples of a preferred
substituent of R1 may include a hydrogen atom, a halogen
atom, a lower alkyl group and a hydroxyl group, more
preferably a hydrogen atom, a fluorine atom and a
chlorine atom, and further more preferably a hydrogen
atom.
Examples of a preferred substituent of R2 may
include a hydrogen atom, an acetyl group, a benzoyl
group, a pivaloyl group, a carbamoylmethyl group and a
carboxymethyl group, more preferably a hydrogen atom,
an acetyl group and a carboxymethyl group, and further
more preferably a hydrogen atom.
Examples of a preferred substituent of each
of R3, R4, R5 and R5 may include a hydrogen atom and a
hydroxyl group that may be substituted with a lower
alkoxycarbonyl, acetyl, benzoyl or pivaloyloxymethyl
group, more preferably a hydrogen atom and a hydroxyl
group that may be substituted with an acetyl or benzoyl
group, and further more preferably a hydrogen atom and
a hydroxyl group.
Examples of a preferred substituent of each
of R4Z and R6Z may include the same substituents
described as for R4 and R6, and a methylene group in
which both R4Z and R5Z may be substituted.
Examples of a preferred substituent of Rz may
include a hydroxyl group that may be substituted with
an acyl that may be substituted, lower alkoxycarbonyl,
or acyloxyalkyl group, more preferably a hydroxyl group
that may be substituted with an isovaleryl, acetyl or
propionyl group that may be substituted with an amino
group that may be protected, benzoyl group, pivaloyl
group, ethoxycarbonyl group, isopropyloxycarbonyl
group, or pivaloyloxymethyl group, and further more
preferably a hydroxyl group that may be substituted
with an isovaleryl, acetyl or benzoyl group that may be
substituted with an amino group.
Examples of a preferred substituent of each
of R1 and R8 may include a hydroxyl group that may be
substituted with a cyclosaligenyl, pivaloyloxymethyl,
1-pivaloyloxyethyl or S-pivaloyl-2-thioethyl group.
An example of a preferred substituent of Y
may be an oxygen atom. An example of a preferred
substituent of A may be an oxygen atom.
In particular, a preferred example of the
compound represented by general formula [3z] is a
compound represented by general formula [3z']:
wherein Ra represents a hydrogen or halogen atom; and
each of Rb and Rc identically or differently represents
a hydrogen atom or hydroxyl protecting group, or Rb and
Rc together represent an alkylene group that may be
substituted. A compound wherein, in the general
formula [3z'], each of Ra, Rb and Rc represents a
hydrogen atom is more preferable.
The compounds indicated below are
representative compounds of the present invention.
Codes in the formulas have the following meanings.
Ac: acetyl, Bz: benzoyl, Me: methyl, and Et:
ethyl
In addition, sugar chains in the following
formulas of the representative compounds are described
in commonly used expressions. For example, the
configuration of a compound represented by the
following formula:

means each of the compounds represented by the
following formulas:
Next, a method for producing the compound of
the present invention will be explained.
The pyrazine nucleotide analog represented by-
general formula [2] or a salt thereof can be produced
by known methods, methods equivalent thereto, or a
combined use of these methods. Examples of
publications which describe the production methods may
include Antiviral Research, Vol. 24, pp. 69 to 77
(1994); Antiviral Chemistry, Vol. 9, pp. 389 to 402
(1998); Journal of Chemical Society Perkin Transaction
(J. Chem. Soc. PERKIN TRANS.) Vol. 1, pp. 1239 to 1245
(1993); and US Patent No. 5770725. Moreover, the
compound of the present invention can also be produced
in accordance with the routes of production methods 1
to 5 as shown below.
[Production method 1]
wherein each of R1, R2, R7 and R8 has the meanings as
described above; and each of Z1, Z2, Z3 and Z4
identically or differently represents a hydrogen atom
or protected hydroxyl group, however, when Z1, Z2, Z3 and
Z4 have hydroxyl groups binding to two or more different
carbon atoms, oxygen atoms of each hydroxyl group and
carbon atoms to which each hydroxyl group binds form a
ring together with protecting groups, so that they may
be protected.
(a) The compound represented by general formula [2a]
or a salt thereof can be obtained by (1) reacting the
compound represented by general formula [3a] or a salt
thereof with a phosphorylation agent in the presence or
absence of additives, or (2) reacting the same above
compound or a salt thereof with a phosphitylation agent
in the presence or absence of additives, followed by
reacting with an oxidizing agent, according to the
method described in e.g., the 4th Jikken Kagaku Koza,
Vol. 22, pp. 313 to 438 (1992).
In the method according to (1) above, a
solvent used in this reaction is not particularly
limited as long as it does not affect the reaction.
Examples of such a solvent may include: aromatic
hydrocarbons such as benzene, toluene or xylene; ethers
such as dioxane, tetrahydrofuran, anisole, diethylene
glycol diethyl ether or dimethyl cellosolve; nitriles
such as acetonitrile; amides such as N,N-
dimethylformamide or N,N-dimethylacetamide; sulfoxides
such as dimethyl sulfoxide; phosphoric esters such as
trimethyl phosphate; and pyridine. One or more types
of these solvents may be used in combination.
Any phosphorylation agent can be used in this
reaction as long as it is generally used in
phosphorylation of hydroxyl groups. Examples of such a
reagent may include phosphoric diesters such as
dibenzyl phosphate; phosphoric dithioesters such as
S, S'-diphenyl phosphorodithioate monocyclohexylammonium;
and phosphoric chlorides such as phosphoryl chloride or
methylchlorophenylphosphoryl P?N-L-alaninate. Such a
phosphorylation agent may be used in an amount
equimolar or greater, and more preferably at a molar
ratio of 1.0 : 1.0 to 5.0 : 1.0, with respect to the
compound represented by general formula [3a] or a salt
thereof.
Examples of an additive used in this reaction
may include azo compounds such as diethyl
azodicarboxylate or diisopropyl azodicarboxylate,
phosphines such as triphenyl phosphine, allene sulfonyl
chlorides such as 2,4,6-triisopropyl benzenesulfonyl
chloride, and bases such as pyridine or tert-butyl
magnesium chloride. These may be used in combination.
Such an additive may be used in an amount equimolar or
greater, and more preferably at a molar ratio of 1.0 :
1.0 to 5.0 : 1.0, with respect to the compound
represented by general formula [3a] or a salt thereof.
This reaction may be carried out generally at
-50°C to 170°C, preferably at 0°C to 100°C and for 1
minute to 72 hours, preferably for 5 minutes to 24
hours.
In the method according to (2) above, a
solvent used in this reaction is not particularly
limited as long as it does not affect the reaction.
Examples of such a solvent may include: aromatic
hydrocarbons such as benzene, toluene or xylene; ethers
such as dioxane, tetrahydrofuran, anisole, diethylene
glycol diethyl ether or dimethyl cellosolve; nitriles
such as acetonitrile; amides such as N,N-
dimethylformamide or N,N-dimethylacetamide; sulfoxides
such as dimethyl sulfoxide; and pyridine. One or more
types of these solvents may be used in combination.
Any phosphitylation agent can be used in this
reaction as long as it is generally used in
phosphitylation of hydroxyl groups. Examples of such a
reagent may include phosphoramidites such as diallyl
diisopropyi phosphoramidite or bis(S-pivaloyl-2-
thioethyl)N,N-diisopropyl phosphoramidite, and
phosphorous chlorides such as diallyl
phosphorochloridite. Such a phosphitylation agent may
be used in an amount equimolar or greater, and more
preferably at a molar ratio of 1.0 : 1.0 to 3.0 : 1.0,
with respect to the compound represented by general
formula [3a] or a salt thereof.
Examples of an additive used in this reaction
may include nitrogen-containing heterocyclic rings such
as 1H-tetrazole, 4-dimethylaminopyridine, pyridine or
collidine, and these may be used in combination. Such
an additive may be used in an amount equimolar or
greater, and more preferably at a molar ratio of 1.0 :
1.0 to 5.0 : 1.0, with respect to the compound
represented by general formula [3a] or a salt thereof.
Examples of an oxidizing agent used in this
reaction may include peroxides such as
metachloroperbenzoic acid or tert-butylhydroperoxide,
and halogenated compounds such as iodine. Such an
oxidizing agent may be used in an amount eguimolar or
greater, and more preferably at a molar ratio of 1.0 :
1.0 to 5.0 : 1.0, with respect to the compound
represented by general formula [3a] or a salt thereof.
This reaction may be carried out generally at
-78°C to 100°C, preferably at -50°C to 50°C and for 1
minute to 24 hours, preferably for 5 minutes to 6
hours.

wherein each of R1, R2, Z1, Z2, Z3 and Z4 has the same
meaning as given above; each of R10 and R11 identically
or differently represents a protecting group of
phosphoric acid decomposed under physiological
conditions; and X represents a halogen atom.
The compound represented by general formula
[2b] or a salt thereof can be obtained by reacting the
compound represented by general formula [3b] or a salt
thereof with the compound represented by general
formula [6a] according to the method described in e.g.,
Journal of Medicinal Chemistry, Vol. 37, pp. 3902 to
3909 (1994).
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride or chloroform; ethers such as dioxane,
tetrahydrofuran, anisole, diethylene glycol diethyl
ether or dimethyl cellosolve; nitriles such as
acetonitrile; amides such as N,N-dimethylacetamide;
alcohols such as methanol, ethanol or propanol;
sulfoxides such as dimethyl sulfoxide; and water. One
or more types of these solvents may be used in
combination.
The compound represented by general formula
[6a] is used in an amount equimolar or greater, and
more preferably at a molar ratio of 1.0 : 1.0 to 3.0 :
1.0, with respect of the compound represented by
general formula [3b] or a salt thereof.
This reaction may be carried out generally at
0°C to 170°C, preferably at 20°C to 120°C and for 5
minutes to 24 hours, preferably for 1 to 10 hours.
[Production method 3]
wherein each of R1, R2, R3, R4, R5, R6, R7, R8, Z1, Z2, Z3
and Z4 has the same meaning as given above.
The compound represented by general formula
[2c] or a salt thereof can be obtained by subjecting
the compound represented by general formula [2a] or a
salt thereof to a deprotection reaction.
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; ethers such as dioxane, tetrahydrofuran,
anisole, diethylene glycol diethyl ether or dimethyl
cellosolve; nitriles such as acetonitrile; amides such
as N,N-dimethylacetamide; alcohols such as methanol,
ethanol or propanol; sulfoxides such as dimethyl
sulfoxide; and water. One or more types of these
solvents may be used in combination.
Any deprotecting agent that is generally used
in deprotection of hydroxyl groups may be used in this
reaction, and preferred examples of such a reagent may
include: bases such as sodium methoxide, ammonia gas,
ammonia water, butylamine or hydrazine; acids such as
formic acid, acetic acid aqueous solution,
trifluoroacetic acid aqueous solution, hydrochloric
acid, bromotrimethyl silane, Dowex 50WX4-200 ion
exchange resin, or Amberlite IR-120 ion exchange resin;
palladium catalysts such as tetrakis triphenyl
phosphine palladium (0); and phosphines such as
triphenyl phosphine. These may be used in combination,
or may be produced in the reaction system. Such a
deprotecting agent may be used at a molar ratio of
0.01 : 1 or more with respect to the compound
represented by general formula [2a] or a salt thereof,
and the deprotecting agent may also be used as a
solvent.
This deprotection reaction may be carried out
generally at -50°C to 170°C, preferably at -20°C to 100°C
and for 1 minute to 100 hours, preferably for 5 minutes
to 50 hours.
[Production method 4]
wherein each of R1, R7, R8, Z1, Z2, Z3 and Z4 has the same
meaning as given above; and R2a represents an acyl
group.
The compound represented by general formula
[2d] or a salt thereof can be obtained by subjecting
the compound represented by general formula [2a'] or a
salt thereof to an acylation reaction in the presence
of a deacidification agent, in the presence or absence
of an additive, according to the method described in
e.g., the 4th Jikken Kagaku Koza, Vol. 22, pp. 137 to
151 and 166 to 169 (1992); Journal of Medicinal
Chemistry, Vol. 44, pp. 777 to 786 (2001); or JP-A-10-
195075.
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
ethers such as dioxane, tetrahydrofuran, anisole,
diethylene glycol diethyl ether or dimethyl cellosolve;
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as
dichloromethane, chloroform or dichloroethane; amides
such as N,N-dimethylformamide or N,N-dimethylacetamide;
and water. These solvents may also be used in
combination.
Examples of an acylating agent used in this
reaction may include: carboxylic acids such as acetic
acid; protected amino acids such as N- (tert-
butoxycarbonyl)-L-valine; acid halides such as pivalic
acid chloride; acid anhydrides such as acetic
anhydride; imidazoles such as 1, 1'-carbonyldiimidazole;
carboxylates such as methyl acetate; and amide acetals
such as N,N-dimethylacetamide dimethylacetal. These
agents may be produced in the reaction system. Such an
acylating agent may be used in an amount equimolar or
greater, and preferably at a molar ratio of 1.0 : 1.0
to 2.0 : 1.0, with respect to the compound represented
by general formula [2a']•
Examples of a deacidification agent used in
this reaction may include pyridine, triethylamine,
sodium hydride, potassium tert-butoxide, potassium
carbonate and sodium bicarbonate. Such a
deacidification agent may be used in an amount
equimolar or greater, and preferably at a molar ratio
of 1.0 : 1.0 to 2.0 : 1.0, with respect to the compound
represented by general formula [2a'].
Examples of an additive used in this reaction
may include 1,3-dicyclohexylcarbodiimide, diethyl
azodicarboxylate and triphenyl phosphine. Such an
additive may be used in an amount equimolar or greater,
and preferably at a molar ratio of 1.0 : 1.0 to 2.0 :
1.0, with respect to the compound represented by
general formula [2a'].
This reaction may be carried out generally at
0°C to 100°C, preferably at 20°C to 60°C and for 5
minutes to 24 hours, preferably for 30 minutes to 10
hours.
wherein each of R1, R2, R3, R4, R5, R6, R7 and R8 has the
same meaning as given above.
The compound represented by general formula
[2e] or a salt thereof can be obtained by reacting the
compound represented by general formula [7b] or a salt
thereof with a reactive agent in the presence or
absence of an additive according to the method
described in e.g., the 4th Jikken Kagaku Koza, Vol. 22,
pp. 371 to 424 (1992); and Journal of Medicinal
Chemistry (J. Med. Chem), Vol. 38, pp. 1372 to 1379
(1995).
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; ethers such as dioxane, tetrahydrofuran,
anisole, diethylene glycol diethyl ether or dimethyl
cellosolve; nitriles such as acetonitrile; amides such
as N,N-dimethylformamide or N,N-dimethylacetamide;
ureas such as N,N'-dimethylpropylene urea; sulfoxides
such as -dimethylsulfoxide; and pyridine. One or more
types of these solvents may also be used in
combination.
Any reactive agent commonly used in
substitution reaction of phosphate groups may be used
in this reaction. Examples of such a reactive agent
used in this reaction may include: halogenated alkyl
compounds such as pivaloyloxymethyl chloride or 1-
(pivaloyloxy) ethyl chloride; alcohols and phenols such
as 4-bromophenol, 4-chlorophenol, S-(2-hydroxyethyl)
thiopivaloate or S-(4-hydroxybutyl) thioisobutylate;
and amines such as alanine methyl ester. Such a
reactive agent may be used in an amount equimolar or
greater, and preferably at a molar ratio of 1.0 : 1.0
to 5.0 : 1.0, with respect to the compound represented
by general formula [7b] or a salt thereof.
Examples of an additive used in this reaction
may include: halogenated compounds such as phosphorus
pentachloride or sodium iodide; nitrogen-containing
heterocyclic compounds such as 1-methylimidazole or
1, l'-carbonyldiimidazole; azo compounds such as diethyl
azodicarboxylate or diisopropyl azodicarboxylate;
phosphines such as triphenyl phosphine; allene sulfonyl
chlorides such as 2,4,6-triisopropyl benzenesulfonyl
chloride; and bases such as triethylamine, pyridine or
tert-butyl magnesium chloride. These may be used in
combination. Such an additive may be used at a molar
ratio of 0.01 : 1 to 10 : 1, and preferably at a molar
ratio of 0.05 : 1 to 5.0 : 1, with respect to the
compound represented by general formula [7b] or a salt
thereof.
This reaction may be carried out generally at
-50°C to 170°C, preferably at 0°C to 100°C and for 1
minute to 72 hours, preferably for 5 minutes to 24
hours.
[Production method 6]
wherein each of R1, R2, R7, R8, Z1, Z2, Z3, Z4 and Y has
the same meaning as given above.
The compound represented by general formula
[2i] or a salt thereof can be obtained by performing
the reaction according to the production method 1,
using the compound represented by general formula [3i]
or a salt thereof.
[Production method 7]
wherein each of R1, R2, Z1, Z2, Z3, Z4, R10, R11, X and Y
has the same meaning as given above.
The compound represented by general formula
[2j] or a salt thereof can be obtained by performing
the reaction according to the production method 2,
using the compound represented by general formula [3j]
or a salt thereof.
[Production method 8]

wherein each of R1, R2, R3, R4, Rs, R6, R1, R8, Z1, Z2, Z3,
Z4 and Y has the same meaning as given above.
The compound represented by general formula
[2k] or a salt thereof can be obtained by performing
the reaction according to the production method 3,
using the compound represented by general formula [2i]
or a salt thereof.
[Production method 9]
wherein each of R1, R2a, R7, R8, Z1, Z2, Z3, Z4 and Y has
the same meaning as given above.
The compound represented by general formula
[2m] or a salt thereof can be obtained by performing
the reaction according to the production method 4,
using the compound represented by general formula [21]
or a salt thereof.
Next, method for producing compounds
represented by general formulas [3a], [3e] and [3f], or
salts thereof will be explained.
[Production method A]
wherein each of R1, R2, Z1, Z2, Z3 and Z4 has the meanings
as given above; R12 represents a lower alkyl or aryl
group; R13 represents a halogen atom, acyloxy group,
alkylsulfonyloxy group or arylsulfonyloxy group; each
of Z5, Z6, Z7 and Z8 identically or differently
represents a hydrogen atom or protected hydroxyl group;
Z9 represents a hydrogen atom, or a protecting group of
a hydroxyl group; and each of Z10, Z11, Z12 and Z13
identically or differently represents a hydrogen atom
or hydroxyl group.
(a) The compound represented by general
formula [3f] or a salt thereof can be obtained by (1)
inducing the compound represented by general formula
[4b] or a salt thereof into the compound represented by
general formula [4a] or a salt thereof in the presence
or absence of an additive according to a commonly used
silylation method, and then (2) reacting the obtained
compound represented by general formula [4a] or a salt
thereof with the compound represented by general
formula [6b] or a salt thereof in the presence or
absence of Lewis acid.
A solvent used in these reactions is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; ethers such as dioxane, tetrahydrofuran,
anisole, diethylene glycol diethyl ether or dimethyl
cellosolve; nitriles such as acetonitrile; amides such
as N,N-dimethylformamide or N,N-dimethylacetamide;
sulfoxides such as dimethyl sulfoxide; and halogenated
hydrocarbons such as methylene chloride, chloroform or
dichloroethane. One or more types of these solvents
may be used in combination.
Any silylation agent may be used in the
reaction of (1) above, as long as it is commonly used
in conversion of a carbonyl group into silyl enol
ether. Examples of such a silylation agent may include
1,1,1,3,3,3-hexamethyldisilazane, N,0-
bis(trimethylsilyl) acetamide, and trimethylsilyl
chloride. Such a silylation agent may be used in an
amount equimolar or greater, and preferably at a molar
ratio of 1.0 : 1.0 to 10.0 : 1.0, with respect to the
compound represented by general formula [4b] or a salt
thereof.
Ammonium sulfate is an example of the
additive used in this reaction as necessary. Such an
additive may be used at a molar ratio of 0.01 : 1.0 to
10.0 : 1.0, and preferably at a molar ratio of 0.05 :
1.0 to 5.0 : 1.0, with respect to the compound
represented by general formula [4b] or a salt thereof.
This reaction may be carried out generally at
0°C to 200°C, preferably at 0°C to 150°C and for 5
minutes to 24 hours, preferably for 5 minutes to 12
hours.
The compound represented by general formula
[6b] or a salt thereof used in the reaction of (2)
above may be used at a molar ratio of 0.5 : 1 to 10 :
1, and preferably at a molar ratio of 0.5 : 1 to 5 : 1,
with respect to the compound represented by general
formula [4a] or a salt thereof.
Examples of Lewis acid used in this reaction
as necessary may include trimethylsilyl
trifluoromethane sulfonate, tin (IV) chloride,
titanium(IV) chloride and zinc chloride. Such Lewis
acid may be used in an amount of 0.5 mole or greater,
and preferably at a molar ratio of 0.5 : 1 to 10 : 1,
with respect to the compound represented by general
formula [4a] or a salt thereof.
This reaction may be carried out generally at
0°C to 100°C, preferably at 0°C to 50°C and for 1 minute
to 72 hours, preferably for 5 minutes to 24 hours.
(b) The compound represented by general
formula [3e] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [3f] or a salt thereof.
(c) The compound represented by general
formula [3a] or a salt thereof can be obtained by
protecting the compound represented by general formula
[3e] or a salt thereof using a reagent in the presence
or absence of an acid catalyst or base.
A solvent used in this reaction is not
particularly limited, as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; ethers such as dioxane, tetrahydrofuran,
anisole, diethylene glycol diethyl ether or dimethyl
cellosolve; nitriles such as acetonitrile; amides such
as N,N-dimethylacetamide; alcohols such as methanol,
ethanol or propanol; sulfoxides such as dimethyl
sulfoxide; ketones such as acetone; and water. One or
more types of these solvents may be used in
combination.
Any reagent commonly used in protection of
hydroxyl groups may be used in this reaction.
Preferred examples may include 2,2-dimethoxypropane,
acetyl chloride and benzoyl chloride. These reagents
may also be produced in the reaction system. Such a
reagent may be used in an amount equimolar or greater,
and preferably at a molar ratio of 1.0 : 1.0 to 10 :
1.0, with respect to the compound represented by
general formula [3e] or a salt thereof.
Examples of an acid catalyst or base used in
this reaction may include pyridinium
paratoluenesulfonate, paratoluenesulfonic acid, and
triethylamine. Such an acid catalyst or base may be
used at a molar ratio of 0.01 : 1 to 10 : 1, and
preferably at a molar ratio of 0.05 : 1 to 10 : 1, with
respect to the compound represented by general formula
[3e] or a salt thereof.
This reaction may be carried out generally at
-50°C to 170°C, preferably at 0°C to 150°C and for 1
minute to 24 hours, preferably for 5 minutes to 10
hours.
The compound represented by general formula
[4b] or a salt thereof can be acquired by purchasing
commercially available products, or can be produced by
known methods, methods equivalent thereto, or the
combined use of them. The production methods are
described in publications such as Journal of American
Chemical Society (J. Am. Chem. Soc), Vol. 71, p. 78
(1949); the same publication, Vol. 78, pp. 242 to 244
(1956); Journal of Heterocyclic Chemistry (J.
Heterocycl. Chem.), Vol. 15, No. 4, pp. 665 to 670
(1978); Journal of Chemical Society (J. Chem. Soc), p,
1379 (1955); US Patent No. 5597823; or International
Patent Publication WO00/10569.
Next, a method for producing a compound
represented by general formula [3b] or a salt thereof
will be explained.
[Production method B]
wherein R9 represents an alkyl group; and each of R1, R2,
R12, R13, Z1, Z2, Z3, Z4, Z5, Z6, Z% Z8, Z9, Z10, Z11, Z12, Z13
and X has the same meaning as given above.
(a) The compound represented by general
formula [3h] or a salt thereof can be obtained by
performing the reaction according to the production
method A(a), using the compound represented by general
formula [4d] or a salt thereof.
(b) The compound represented by general
formula [3g] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [3h] or a salt thereof.
(c) The compound represented by general
formula [3d] or a salt thereof can be obtained by
performing the reaction according to the production
method A(c), using the compound represented by general
formula [3g] or a salt thereof.
(d) The compound represented by general
formula [3c] or a salt thereof can be obtained,
according to the method described in e.g., the 4th
Jikken Kagaku Koza, Vol. 19, pp. 416 to 482 (1992), (1)
by reacting the compound represented by general formula
[3d] or a salt thereof with a halogenating agent in the
presence or absence of an additive, or (2) by reacting
the same compound or a salt thereof with a sulfonating
agent in the presence of an deacidification agent and
then reacting with a halogenating agent.
In the method according to (1) above, a
solvent used in this reaction is not particularly
limited as long as it does not affect the reaction.
Examples of such a solvent may include: aromatic
hydrocarbons such as benzene, toluene or xylene;
halogenated hydrocarbons such as methylene chloride,
chloroform; ethers such as dioxane, tetrahydrofuran,
anisole, diethylene glycol diethyl ether or dimethyl
cellosolve; nitriles such as acetonitrile; amides such
as N,N-dimethylformamide or N,N-dimethylacetamide;
alcohols such as methanol, ethanol or propanol;
sulfoxides such as dimethyl sulfoxide; and water. One
or more types of these solvents may be used in
combination.
A halogenating agent used in this reaction is
not particularly limited as long as it is a reagent
commonly used in halogenation reaction of hydroxyl
groups. Preferred examples of such a halogenating
agent may include iodine, bromine, chlorine, hydriodic
acid, hydrobromic acid, sodium bromide, potassium
iodide, sulfuryl chloride, N-bromosuccinimide, N-
chlorosuccinimide, carbon tetrabromide, or phosphorus
compounds such as triphenyl iodine phosphonate. Such a
halogenating agent may be used at a molar ratio of 1 :
1 to 50 : 1, and preferably at a molar ratio of 1 : 1
to 20 : 1, with respect to the compound represented by
general formula [3d] or a salt thereof.
An additive used in this reaction as
necessary is not particularly limited as long as it is
a reagent commonly used in halogenation reaction of
hydroxyl groups. Preferred examples of such an
additive may include: phosphines such as
triphenylphosphine; azo compounds such as diethyl
azodicarboxylate; and silanes such as trimethyl silyl
chloride or hexamethyldisiloxane. One or more types of
these additives may be used in combination. Such an
additive may be used at a molar ratio of 0.01 : 1 to
10 : 1, and preferably at a molar ratio of 0.1 : 1 to
10 : 1, with respect to the compound represented by
general formula [3d] or a salt thereof.
This reaction may be carried out generally at
-80°C to 170°C, preferably at -80°C to 100°C and for 1
minute to 72 hours, preferably for 5 minutes to 48
hours.
In the method according to (2) above, a
solvent used in the sulfonation reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride or chloroform; ethers such as dioxane,
tetrahydrofuran, anisole, diethylene glycol diethyl
ether or dimethyl cellosolve; nitriles such as
acetonitrile; amides such as N,N-dimethylformamide or
N,N-dimethylacetamide; alcohols such as methanol,
ethanol or propanol; sulfoxides such as dimethyl
sulfoxide; and water. One or more types of these
solvents may be used in combination.
A sulfonating agent used in this reaction is
not particularly limited as long as it is a reagent
commonly used in sulfonation reaction of hydroxyl
groups. Preferred examples of such a sulfonating agent
may include halogenated sulfonyls such as
methanesulfonyl chloride or p-toluenesulfonyl chloride;
sulfonic acid anhydrides such as trifluoromethane-
sulfonic acid anhydride; and sulfonic acids such as
trifluoromethanesulfonic acid. Such a sulfonating
agent may be used at a molar ratio of 1 : 1 to 20 : 1,
and preferably at a molar ratio of 1.0 : 1.0 to 5.0 :
1.0, with respect to the compound represented by
general formula [3d] or a salt thereof.
A deacidification agent used in this reaction
as necessary is not particularly limited as long as it
is a reagent commonly used in sulfonation reaction of
hydroxyl groups. Preferred examples of such a
deacidification agent may include bases such as
pyridine, 2,6-lutidine, triethylamine or sodium
methoxide, and one or more types of these deacidifica-
tion agents may be used in combination. Such a
deacidification agent may be used at a molar ratio of
0.01 : 1 to 20 : 1, and preferably at a molar ratio of
0.1 : 1 to 10 : 1, with respect to the compound
represented by general formula [3d] or a salt thereof.
This reaction may be carried out generally at
-80°C to 100°C, preferably at -20°C to 50°C and for 1
minute to 24 hours, preferably for 5 minutes to 12
hours.
A solvent used in halogenation reaction is
not particularly limited as long as it does not affect
the reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride or chloroform; ethers such as dioxane,
tetrahydrofuran, anisole, diethylene glycol diethyl
ether or dimethyl cellosolve; nitriles such as
acetonitrile; amides such as N,N-dimethylformamide or
N,N-dimethylacetamide; ketones such as acetone;
alcohols such as methanol, ethanol or propanol;
sulfoxides such as dimethyl sulfoxide; and water. One
or more types of these solvents may be used in
combination.
A halogenating agent used in this reaction is
not particularly limited as long as it is a reagent
commonly used in halogenation reaction of sulfonate
groups. Preferred examples of such a halogenating
agent may include sodium bromide, sodium iodide,
potassium iodide and magnesium iodide. Such a
halogenating agent may be used at a molar ratio of 1 :
1 to 50 : 1, and preferably at a molar ratio of 1 : 1
to 20 : 1, with respect to the compound represented by
general formula [3d] or a salt thereof.
This reaction may be carried out generally at
-80°C to 170°C, preferably at -80°C to 100°C and for 1
minute to 72 hours, preferably for 5 minutes to 12
hours.
(e) The compound represented by general
formula [3b] or a salt thereof can be obtained by
subjecting the compound represented by general formula
[3c] or a salt thereof to ammonolysis reaction of
carboxylate in the presence or absence of a catalyst.
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride or chloroform; ethers such as dioxane,
tetrahydrofuran, anisole, diethylene glycol diethyl
ether or dimethyl cellosolve; nitriles such as
acetonitrile; amides such as N,N-dimethylformamide or
N,N-dimethylacetamide; alcohols such as methanol,
ethanol or propanol; sulfoxides such as dimethyl
sulfoxide; and water. One or more types of these
solvents may be used in combination. This reaction may
be carried out, using a reagent and conditions that are
commonly used in ammonolysis reaction of aromatic
carboxylate. Ammonia gas, liquid ammonia or ammonia
water may be preferably used. Such a reagent may be
used in an amount equimolar or greater with respect to
the compound represented by general formula [3c] or a
salt thereof. In addition, the reagent may also be
used as a solvent. Examples of a catalyst used in this
reaction as necessary may include: acid ammonium salts
such as ammonium chloride; bases such as sodium
methoxide or butyllithium; and alkali metal amide such
as sodium amide. Such a catalyst may be used at a
molar ratio of 0.01 : 1 to 100 : 1, and preferably at a
molar ratio of 0.01 : 1 to 20 : 1, with respect to the
compound represented by general formula [3c] or a salt
thereof.
This reaction may be carried out generally at
-100°C to 250°C, preferably at -78°C to 100°C and for 1
minute to 72 hours, preferably for 30 minutes to 50
hours.
The compound represented by general formula
[4d] or a salt thereof can be acquired by purchasing
commercially available products, or can be produced by
known methods, methods equivalent thereto, or the
combined use of them. The publication as described
above for the production method of the compound
represented by general formula [4b] is an example of
publications describing the production methods of the
compound represented by general formula [4d] or a salt
thereof.
Next, a method for producing a compound
represented by general formula [7b] or a salt thereof
will be explained.
wherein each of R1, R2, R3, R4, R5, R6, R9, Z1, Z2, Z3 and
Z4 has the same meaning as given above.
(a) The compound represented by general
formula [8d] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [8a] or a salt thereof.
(b) The compound represented by general
formula [7b] or a salt thereof can be obtained: (1) by
deprotecting the compound represented by general
formula [8d] or a salt thereof according to the
production method 3, and then amidating the obtained
product according to the production method B(e); or (2)
by amidating the above compound or a salt thereof
according to the production method B(e), and then
deprotecting the obtained product according to the
production method 3.
(c) The compound represented by general
formula [8b] or a salt thereof can be obtained by
performing the reaction according to the production
method 4, using the compound represented by general
formula [8c] or a salt thereof.
(d) The compound represented by general
formula [8f] or a salt thereof can be obtained by
deprotecting the compound represented by general
formula [8b] according to the production method 3.
Next, a method for producing compounds
represented by general formulas [8a] and [8b'] or salts
thereof will be explained.
wherein each of R1, R2, R9, R12, R13, z1, Z2, Z3 and Z4 has
the same meaning as given above.
(a) The compound represented by general
formula [8a] or a salt thereof can be obtained by-
reacting the compound represented by general formula
[9a] or a salt thereof with the compound represented by
general formula [10a] or a salt thereof according to
the production method B(a).
(b) The compound represented by general
formula [8b'] or a salt thereof can be obtained by
reacting the compound represented by general formula
[9c] or a salt thereof with the compound represented by
general formula [10a] or a salt thereof according to
the production method B(a).
Next, a method for producing a compound
represented by general formula [10a] or a salt thereof
will be explained.
[Production method E]

wherein each of R9, R13, Z1, Z2, Z3, Z4, Z10, Z11, Z12 and Z13
has the same meaning as given above.
(a) The compound represented by general
formula [10c] or a salt thereof can be obtained by
performing the reaction according to the production
method A(c), using the compound represented by general
formula [10b] or a salt thereof.
(b) The compound represented by general
formula [10a] or a salt thereof can be obtained by
subjecting the compound represented by general formula
[10c] or a salt thereof to a substitution reaction
according to the method described in e.g., Journal of
Organic Chemistry (J. Org. Chem.) Vol. 55, pp. 3853 to
3857 (1990) .
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride or chloroform; ethers such as dioxane,
tetrahydrofuran, anisole, diethylene glycol diethyl
ether or dimethyl cellosolve; nitriles such as
acetonitrile; amides such as N,N-dimethylacetamide;
alcohols such as methanol, ethanol or propanol;
sulfoxides such as dimethyl sulfoxide; and water. One
or more types of these solvents may be used in
combination.
A reactive agent used in this reaction may be
one commonly used in a substitution reaction on lactol
under acidic conditions. Examples of such a reactive
agent may include: organic acids and acid anhydrides
such as acetic acid or acetic acid anhydride; inorganic
acids such as hydrogen chloride gas, hydrochloric acid,
hydrobromic acid, sulfuric acid or hydrofluoric acid;
halogen compounds such as chlorine or bromine; Lewis
acids such as titanium tetrabromide or chlorotrimethyl
silane; and thio compounds such as thiophenol or
methylthiotrimethyl silane. Such a reactive agent may
be used at a molar ratio of 1 : 1 to 20 : 1, and
preferably at a molar ratio of 1 : 1 to 10 : 1, with
respect to the compound represented by general formula
[10c] or a salt thereof and may be employed as a
solvent.
This reaction may be carried out generally at
0°C to 200°C, preferably at 0°C to 100°C and for 5
minutes to 48 hours, preferably for 30 minutes to 24
hours.
The compound represented by general formula
[10b] can be obtained according to the method described
in Journal of Chemical Society Chemical Communication
(J. Chem. Soc, Chem. Commun.) pp. 40 to 41 (1989).
Next, a method for chemically synthesizing
the pyrazine nucleotide analog represented by general
formula [1] in which A is an oxygen atom will be
explained.
[Production method F]
wherein each of R1, R2, R3, R4, R5, R6, Z1, Z2, Z3 and Z4
has the same meaning as given above; R14 represents a
monophosphate group or monophosphoric chloride that may
be protected; and R15 represents a diphosphate acid or
triphosphate group that may be protected.
(a) The compound represented by general
formula [5c] or a salt thereof can be obtained by
performing the reaction according to the production
method 1, using the compound represented by general
formula [3v] or a salt thereof.
(b) The compound represented by general
formula [5b] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [5c] or a salt thereof.
(c) The compound represented by general
formula [5a] or a salt thereof can be obtained by
reacting the compound represented by general formula
[5b] or a salt thereof with a phosphorylation agent in
the presence or absence of a condensation agent
according to the methods described in e.g., Chemical
Review (Chem. Rev.), Vol. 100, pp. 2047 to 2059 (2000);
Journal of Organic Chemistry (J. Org. Chem.), Vol. 55,
pp. 1834 to 1841 (1990); or Acta Biochimica Biophysica
Academia Scientiarum Hungaricae (Acta Biochim. et
Biophys. Acad. Sci. Hung.), Vol. 16, pp. 131 to 133
(1981) .
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; ethers such as dioxane, tetrahydrofuran,
anisole, diethylene glycol diethyl ether or dimethyl
cellosolve; nitriles such as acetonitrile; amides such
as N,N-dimethylformamide or N,N-dimethylacetamide;
sulfoxides such as dimethylsulfoxide; phosphoric esters
such as trimethyl phosphate; and pyridine. One or more
types of these solvents may be used in combination.
A phosphorylation agent used in this reaction
may be one commonly used in phosphorylation of
monophosphate groups. Examples of such a
phosphorylation agent may include phosphates such as
tri-n-butyl ammonium phosphate or tri-n-butyl ammonium
pyrophosphate. These agents may also be produced in .
the reaction system. Such a phosphorylation agent may
be used in an amount equimolar or greater, and
preferably at a molar ratio of 1 : 1 to 10 : 1, with
respect to the compound represented by general formula
[5b] or a salt thereof. Examples of a condensation
agent may include imidazoles such as 1,1'-
carbonyldiimidazole or N-methylimidazole, and amines
such as morpholine or diisopropylamine. These may also
be used in combination. Such a condensation agent may
be used in an amount equimolar or greater, and
preferably at a molar ratio of 1 : 1 to 5 : 1, with
respect to the compound represented by general formula
[5b] or a salt thereof.
This reaction may be carried out generally at
-50°C to 100°C, preferably at 0°C to 50°C and for 1
minute to 72 hours, preferably for 5 minutes to 24
hours.
(d) The compound represented by general
formula [5a] or a salt thereof can be obtained by
performing the reaction according to the production
method F(c), using the compound represented by general
formula [5c] or a salt thereof.
(e) The compound represented by general
formula [5a] or a salt thereof can be obtained by
reacting the compound represented by general formula
[3v] or a salt thereof according to the production
method 1, so as to induce it into the compound
represented by general formula [5c] or a salt thereof,
and then reacting the obtained compound or a salt
thereof in the same system according to the production
method F(d).
[Production method G]
wherein each of R1, R2, R12, R13, Rz, Z1, Z2, Z3, Z4, Z5, Z6,
Z7, Z8, Z9, Z10, Z11, Z12, Z13 and Y has the same meaning as
given above.
(a) The compounds represented by general
formulas [31], [3m] and [3i] or salts thereof can be
obtained by performing the reaction according to the
production method A, using the compound represented by
general formula [4f] or a salt thereof.
(b) The compound represented by general
formula [3n] or a salt thereof can be obtained by
performing the reaction according to the production
method 4, using the compound represented by general
formula [3i] or a salt thereof; or it can be obtained
by subjecting the compound represented by general
formula [3i] or a salt thereof to an alkylation
reaction in the presence or absence of acid or base
according to the method described in e.g., Shin Jikken
Kagaku Koza, Vol. 14, pp. 567 to 587 (edited by The
Chemical Society of Japan, 1977).
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
ethers such as dioxane, tetrahydrofuran, anisole,
diethylene glycol diethyl ether or dimethyl cellosolve;
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as
dichloromethane, chloroform or dichloroethane; amides
such as N,N-dimethylformamide or N,N-dimethylacetamide;
sulfoxides such as dimethylsulfoxide; and water. These
solvents may be used in combination.
Examples of an alkylating agent used in this
reaction may include: halogenated alkyls such as benzyl
bromide; esters such as diethyl sulfate; diazo
compounds such as diphenyldiazomethane; olefins such as
2-methylpropene; and amide acetals such as N,N-
dimethylacetamide dimethylacetal. Such an. alkylating
agent may be used in an amount equimolar or greater,
and preferably at a molar ratio of 1.0 : 1.0 to 2.0 :
1.0, with respect to the compound represented by
general formula [3i].
Examples of acid used in this reaction may
include p-toluenesulfonic acid and sulfuric acid.
Examples of base used in this reaction may include
triethylamine, sodium methoxide, sodium hydride,
potassium tert-butoxide, potassium carbonate and
metallic sodium. Such acid or base may be used in an
amount equimolar or greater, and preferably at a molar
ratio of 1.0 : 1.0 to 2.0 : 1.0, with respect to the
compound represented by general formula [3i].
This reaction may be carried out generally at
0°C to 100°C, preferably at 20°C to 60°C and for 5
minutes to 24 hours, preferably for 30 minutes to 10
hours.
(c) The compound represented by general
formula [3o] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [3n] or a salt thereof.
(d) The compound represented by general
formula [3j'] or a salt thereof can be obtained by
performing the reaction according to the production
method B(d), using the compound represented by general
formula [3m] or a salt thereof.
[Production method H]
wherein each of R1, R2, Z5, Z6, Z7, Z8, Z10, Z11, Z12, Z13
and Y has the same meaning as given above; R14
represents a protecting group of an amino group; and R18
represents an amino acid residue that may be protected.
(a) The compound represented by general
formula [3p] or a salt thereof can be obtained by
reacting the compound represented by general formula
[3n'] or a salt thereof with a deprotecting agent in
the presence or absence of a catalyst according to
common methods such as one described in Protective
Groups in Organic Synthesis, Third Edition, Theodora W.
Greene, pp. 494 to 653 (1999).
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
water; alcohols such as methanol, ethanol or propanol;
thioalcohols such as ethanethiol or thiophenol;
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride, chloroform or 1,2-dichloroethane; ethers such
as dioxane, tetrahydrofuran, anisole, diethylene glycol
diethyl ether or dimethyl cellosolve; thioethers such
as dimethyl sulfide; ketones such as acetone or methyl
ethyl ketone; nitriles such as acetonitrile; amides
such as N,N-dimethylformamide or N,N-dimethylacetamide;
sulfoxides such as dimethylsulfoxide; inorganic acids
such as sulfuric acid or hydrochloric acid; carboxylic
acids such as acetic acid or trifluoroacetic acid;
sulfonic acids such as trifluoromethanesulfonic acid;
nitroalkanes such as nitromethane; and organic bases
such as pyridine or triethylamine. One or more types
of these solvents may be used in combination.
A deprotecting agent used in this reaction is
not particularly limited, and those commonly used in
deprotection of protected amino groups may be used
herein. Preferred examples of such a deprotecting
agent may include: hydrogen gas; ammonium formate;
zinc; sodium; acid chlorides such as vinylchloroformate
or acetyl chloride; organic silanes such as
triethylsilane or trimethylsilyliodide; tributyltin
hydride; alkali metal alkoxide such as potassium tert-
butoxide; alkali metal thioalkoxide such as sodium
thiomethoxide; 2,3-dichloro-5, 6-dicyano-l,4-
benzoquinone; sodium borohydride; alkali metal salts
such as potassium fluoride or sodium iodide; Lewis
acids such as boron tribromide, aluminum chloride,
ruthenium chloride or zinc chloride; inorganic acids
such as hydrochloric acid, hydrobromic acid or sulfuric
acid; organic acids such as trifluoroacetic acid,
methanesulfonic acid or paratoluenesulfonic acid;
inorganic bases such as potassium carbonate, sodium
bicarbonate or sodium hydroxide; organic bases such as
piperidine; amines such as ammonia or hydrazine;
organic lithium such as methyllithium; cerium
diammonium nitrate; and peroxides such as hydrogen
peroxide, ozone or permanganic acid. Such a
deprotecting agent may be used at a molar ratio of
0.01 : 1 to 1000 : 1, and preferably 0.1 : 1 to 100 :
1, with respect to the compound represented by general
formula [3n'] or a salt thereof.
A catalyst used in this reaction as necessary
is not particularly limited, as long as it is commonly
used in deprotection of protected amino groups.
Preferred examples of such a catalyst may include:
palladium catalysts such as palladium-carbon; rhodium;
Raney nickel; and platinum oxide (IV). A catalyst such
as palladium-carbon or Raney nickel may be used at a
weight ratio of 0.01 : 1 to 10 : 1, and more preferably
of 0.01 : 1 to 5 : 1, with respect to the compound
represented by general formula [3n'] or a salt thereof.
Catalysts other than palladium-carbon and Raney nickel
may be used at a molar ratio of 0.01 : 1 to 10 : 1, and
preferably of 0.01 : 1 to 5 : 1, with respect to the
compound represented by general formula [3n'] or a salt
thereof.
This reaction may be carried out generally at
-80°C to 200°C, preferably at 0°C to 160°C and for 1
minute to 48 hours, preferably for 5 minutes to 12
hours.
(b) The compound represented by general
formula [3o'] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [3p] or a salt thereof.
(c) The compound represented by general
formula [3q] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [3n'] or a salt thereof.
(d) The compound represented by general
formula [3o'] or a salt thereof can be obtained by
performing the reaction according to the production
method H(a), using the compound represented by general
formula [3q] or a salt thereof.
(e) The compound represented by general
formula [3o'] or a salt thereof can be obtained by
performing the reaction according to the production
method 3 or the production method H(a), using the
compound represented by general formula [3n'] or a salt
thereof.
[Production method I]
wherein each of R1, R2a, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12,
Z13 and Y has the same meaning as given above.
(a) The compound represented by general
formula [31'] or a salt thereof can be obtained by
performing the reaction according to the production
method 4, using the compound represented by general
formula [3r] or a salt thereof.
(b) The compound represented by general
formula [3m'] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [31'] or a salt thereof.
[Production method J]
wherein each of R1, R2 and R12 has the same meaning as
given above.
(a) The compound represented by general
formula [4h] or a salt thereof can be obtained by
reacting the compound represented by general formula
[4g] or a salt thereof with alcohol in the presence or
absence of an acid catalyst or base according to the
method described in e.g., Shin Jikken Kagaku Koza, Vol.
14, pp. 1599 to 1602 (1978).
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride, chloroform or dichloroethane; ethers such as
dioxane, tetrahydrofuran, anisole, diethylene glycol
diethyl ether or dimethyl cellosolve; amides such as
N,N-dimethylformamide or N,N-dimethylacetamide; and
sulfoxides such as dimethylsulfoxide. One or more
types of these solvents may be used in combination.
Examples of alcohol used in this reaction may
include methanol, ethanol and phenol. Such alcohol may
be used in an amount equimolar or greater with respect
to the compound represented by general formula [4g] or
a salt thereof. Moreover, alcohol may also be used as
a solvent.
A reagent commonly used in imidation of
nitrile may be used as an acid catalyst used in this
reaction. Hydrogen chloride is an example of such an
acid catalyst. Such an acid catalyst may be used at a
molar ratio of 0.1 : 1 or more with respect to the
compound represented by general formula [4g] or a salt
thereof.
Examples of a base used in this reaction may
include metal alkoxides such as sodium methoxide,
sodium ethoxide or sodium phenoxide. These bases may
also be produced in the reaction system. Such a base
may be used in this reaction at a molar ratio of 0.01 :
1 or more, and preferably at a molar ratio of 1.0 : 1.0
to 5.0 : 1.0, with respect to the compound represented
by general formula [4g] or a salt thereof.
This reaction may be carried out generally at
-78°C to 170°C, preferably at -40°C to 120°C and for
minute to 72 hours, preferably for 5 minutes to 24
hours.
(b) The compound represented by general
formula [4f] or a salt thereof can be obtained by
reacting the compound represented by general formula
[4h] or a salt thereof with a reagent according to the
method described in e.g., Shin Jikken Kagaku Koza, Vol.
14, pp. 1614 to 1617 (1978).
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride, chloroform or dichloroethane; ethers such as
dioxane, tetrahydrofuran, anisole, diethylene glycol
diethyl ether or dimethyl cellosolve; amides such as
N,N-dimethylformamide or N,N-dimethylacetamide; and
sulfoxides such as dimethylsulfoxide. One or more
types of these solvents may be used in combination.
A reagent commonly used in amidination of
imidates may be used in this reaction. Examples of
such a reagent may include: ammonia gas, ammonia
alcohol solution, ammonia water, or acid ammonium salts
such as ammonium chloride; and amino acids that may be
protected, such as glycine ethyl ester, or salts
thereof. Such a reagent may be used in this reaction
in an amount equimolar or greater with respect to the
compound represented by general formula [4h] or a salt
thereof, and it may also be used as a solvent.
This reaction may be carried out generally at
-78°C to 170°C, preferably at 0°C to 120°C and for 1
minute to 72 hours, preferably for 5 minutes to 24
hours.
wherein each of R1, R2 and R9 has the same meaning as
given above.
The compound represented by general formula
[4b] or a salt thereof can be obtained by subjecting
the compound represented by general formula [4i] or a
salt thereof to a condensation reaction with
carboxylate and amines such as ammonia or a primary
amine in the presence or absence of a catalyst.
A solvent used in this reaction is not
particularly limited as long as it does not affect the
reaction. Examples of such a solvent may include:
aromatic hydrocarbons such as benzene, toluene or
xylene; halogenated hydrocarbons such as methylene
chloride or chloroform; ethers such as dioxane,
tetrahydrofuran, anisole, diethylene glycol diethyl
ether or dimethyl cellosolve; nitriles such as
acetonitrile; amides such as N,N-dimethylformamide or
N,N-dimethylacetamide; alcohols such as methanol,
ethanol or propanol; sulfoxides such as
dimethylsulfoxide; and water. One or more types of
these solvents may be used in combination. This
reaction may be carried out, using a reagent and
conditions that are commonly used in a condensation
reaction with aromatic carboxylate and amines.
Examples of amines preferably used herein may include
ammonia such as ammonia gas, liquid ammonia or ammonia
water, and primary amines such as L-aspartic acid
diethyl ester. Such amine may be used in an amount
equimolar or greater with respect to the compound
represented by general formula [4i] or a salt thereof.
These reagents may also be used as solvents. Examples
of a catalyst used in this reaction as necessary may
include: acid ammonium salts such as ammonium chloride;
bases such as triethylamine, sodium methoxide or
butyllithium; and alkali metal amides such as sodium
amide. Such a catalyst may be used at a molar ratio of
0.01 : 1 to 100 : 1, and preferably at a molar ratio of
0.01 : 1 to 20 : 1, with respect to the compound
represented by general formula [4i] or a salt thereof.
This reaction may be carried out generally at
-100°C to 250°C, preferably at -78°C to 100°C and for 1
minute to 72 hours, preferably for 30 minutes to 50
hours.

wherein each of R1, R2a and Y has the same meaning as
given above.
The compound represented by general formula
[4f"] or a salt thereof can be obtained by performing
the reaction according to the production method 4,
using the compound represented by general formula [4j]
or a salt thereof.
[Production method M]
wherein each of R1, Rz, R3, R4, R5, R6, R9, Rz, Z1, Z2, Z3
and Z4 has the same meaning as given above.
(a) The compound represented by general
formula [3t] or a salt thereof can be obtained by
performing the reaction according to the production
method G(b), using the compound represented by general
formula [3d] or a salt thereof.
(b) The compound represented by general
formula [3s] or a salt thereof can be obtained by
performing the reaction according to the production
method 3, using the compound represented by general
formula [3t] or a salt thereof.
(c) The compound represented by general
formula [3a'] or a salt thereof can be obtained by
performing the reaction according to the production
method K, using the compound represented by general
formula [3s] or a salt thereof.
wherein each of R1, R2, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13
and Y has the same meaning as given above.
The compound represented by general formula
[3u] or a salt thereof can be obtained by performing
the reaction according to the production method 3,
using the compound represented by general formula [31]
or a salt thereof.
When the compound obtained by the above
production method has isomers (e.g., optical isomers,
geometric isomers, tautomers, etc.), these isomers may
also be used. In addition, solvates, hydrates, and
various forms of crystals may also be used. Moreover,
after completion of the reaction, the reaction product
of interest may directly be used in the following
reaction without isolating it.
Where the compound obtained by the above
production method has an amino, hydroxyl or carboxyl
group, it is also possible that these groups are
previously protected by common protecting groups, and
that after completion of the reaction, these protecting
groups are removed by common methods.
The compound represented by general formula
[1] or a salt thereof can be isolated, purified or
recrystallized according to common methods such as
extraction, crystallization and/or column
chromatography.
When the compound of the present invention is
used as a pharmaceutical, it can be prepared as a
pharmaceutical composition by an ordinary method using
a pharmaceutical carrier used in common pharmaceutical
preparation. Various types of carriers that are
commonly used for ordinary pharmaceuticals, such as an
excipient, binder, disintegrator, lubricant, coloring
agent, corrective agent, flavoring agent or surfactant,
may be used herein.
The administration form of the compound of
the present invention is not particularly limited, but
it can be appropriately selected depending on
therapeutic purposes. More specifically, examples of
such an administration form may include: parenteral
agents such as an injection, suppository or external
preparation (ointment, fomentation, etc.); aerosols;
and oral agents such as a tablet, powder, fine granule,
granule, capsule, liquid, pill, suspension, syrup or
emulsion.
Various types of agents described above can
be prepared as pharmaceuticals by common methods.
When the present compound is prepared into
the form of a solid pharmaceutical for oral
administration, such as a tablet, powder, fine granule
or granule, examples of a carrier used herein may
include: excipients (lactose, sucrose, sodium chloride,
glucose, starch, calcium carbonate, kaolin, crystalline
cellulose, calcium diphosphate anhydride, alginic acid,
etc.); binders (simple syrup, glucose solution, starch
solution, gelatin solution, polyvinyl alcohol,
polyvinyl ether, polyvinylpyrrolidone, carboxymethyl
cellulose, shellac, methylcellulose, ethylcellulose,
sodium alginate, gum Arabic,
hydroxypropylmethylcellulose, hydroxypropylcellulose,
their water and/or ethanol solution, etc.);
disintegrators (starch, alginic acid, crosslinked
polyvinylpyrrolidone, crosslinked
carboxymethylcellulose sodium, carboxymethylcellulose
calcium, sodium glycolate starch, etc.); release-
controlling agents (higher fatty acid, higher fatty
alcohol, cacao butter, hydrogenated oil, water-soluble
polymer, polymer soluble in gastric juice, polymer
soluble in intestinal juice, etc.); absorbefacients
(surfactants such as quaternary ammonium salt, sodium
lauryl sulfate or sorbitan monooleate); absorbents
(starch, lactose, kaolin, bentonite, silicic acid
anhydride, hydrated silicon dioxide, magnesium
aluminometasilicate, colloidal silicic acid, etc.); and
lubricants (purified talc, stearate, silicic acids,
polyethylene glycol, etc.)
Tablets may be converted, as necessary, into
those coated with common coatings, such as a sugar-
coated tablet, gelatin-coated tablet, tablet coated
with a coating that is soluble in gastric juice, tablet
coated with a coating that is soluble in intestinal
juice, or tablet coated with a water-soluble film.
Capsules can be prepared by mixing the
compound with the aforementioned various types of
carriers and then filling the obtained mixture into a
hard gelatin capsule, a soft capsule, etc.
Liquid pharmaceutical can be a water or oil
suspension, solution, syrup or elixir, and these can be
prepared by common methods using ordinary additives.
When the compound of the present invention is
prepared into the form of an injection, examples of a
carrier used herein may include: diluents (water, ethyl
alcohol, Macrogol, propylene glycol, etc.); pH
controllers or buffers (citric acid, acetic acid,
phosphoric acid, lactic acid and their salts, sulfuric
acid, sodium hydroxide, etc.); and stabilizers (sodium
pyrosulfite, ethylenediaminetetraacetic acid,
thioglycolic acid, thiolactic acid, etc.). In this
case, common salts, glucose, mannitol or glycerine may
be contained in the pharmaceutical composition in an
amount sufficient to prepare an isotonic solution. In
addition, a common solubilizer, soothing agent or local
anesthetic may also be added thereto.
The administration method, the dosage, and
the number of doses can be appropriately selected
depending on a patient's age, body weight and symptom.
When the patient is an adult, the compound of the
present invention may be administered orally or
parenterally (e.g., injection, infusion, administration
to the rectum, etc.) at a dosage of 0.1 to 1000 mg/kg,
once per day or divided into several times.
The virus growth inhibition and/or virucidal
method of the present invention is characterized in
that it comprises the following steps.
wherein each of R1, R2, R3, R4, R5, R6, R7, R8, Rz, A and Y
has the same meaning as given above.
Step A: the pyrazine nucleotide analog represented by
general formula [2] or a salt thereof is converted in
vivo into the compound represented by general formula
[2a] or a salt thereof.
Step B: the pyrazine nucleoside analog represented by
general formula [3z] or a salt thereof is converted in
vivo into the compound represented by general formula
[2a] or a salt thereof.
Step C: (1) the pyrazine nucleoside analog represented
by general formula [3z] or a salt thereof is converted
in vivo by enzyme such as nucleosidase into the
compound represented by general formula [4f], and then
(2) the obtained compound is converted in vivo by
enzyme such as phosphoribosyltransferase into the
compound represented by general formula [2a] or a salt
thereof.
With regard to steps B, C(l) and C(2), the
reverse conversion may also occur in vivo.
The compound represented by general formula
[2a] or a salt thereof generated as a result of the
above steps is further converted in vivo by enzyme such
as nucleotide kinase [Advances in Antiviral Drug
Design, Vol. 2, pp. 167 to 172 (1996)] into the
compound represented by general formula [lb] (a
pyrazine nucleotide triphosphate) or a salt thereof.
This compound or a salt thereof exhibits a virus growth
inhibition and/or virucidal effect by inhibiting virus
polymerase. Moreover, the reverse conversion may also
occur in vivo.
Furthermore, the compound wherein, in the
above steps, Y is an imino group is converted in vivo
into a compound as an oxygen atom, thereby exhibiting
pharmacological effects.
EXAMPLES
The present invention will be described in
the following Test Examples. However, the present
invention is not limited thereto. In the following
test examples, compound A represents the compound 4-
[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-dihydro-
2-pyrazinecarboxamide that is obtained in Example 29;
compound B represents the compound 6-chloro-4-[(2R, 3R,
4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-2-
furanyl]-3-oxo-3,4-dihydro-2-pyrazinecarboxamide that
is obtained in Reference Example 7; and compounds shown
in tables showing test results indicate products
obtained in Reference Examples and Examples.
Test Example 1
[Detection of phosphate compound in cell]
5 ml of an E'-MEM culture medium (containing
1% bovine serum albumin and 3% vitamin solution)
containing a compound, 6-f luoro-3-hydroxy-2-[2-14C]
pyrazinecarboxamide, which had been 14C-labeled at
position 2 of the pyrazine ring, was added to MDCK
cells that had been monolayer-cultured on a 55 cm2
culture plate. After completion of culture under
conditions of 35°C and 5% C02 for 20 hours, 6-fluoro-3-
hydroxy-2-[2-14C] pyrazinecarboxamide and a converted
compound were extracted from cell fractions, using a
66.6% acetonitrile solution. The extract was
lyophilized and concentrated, and then analysis was
carried out under the HPLC analysis conditions
indicated below.
As a result, a monophosphate (the compound in
Reference Example 15; recovery time: 23.3 minutes) and
a triphosphate (the compound in Reference Example 16;
recovery time: 34.0 minutes) were detected.
HPLC analysis conditions
Instruments
Pump: HITACHI L-6200
Detector: HITACHI L-4000
Radioactivity detector: Packard FLO-ONE500
Analysis conditions
Separation column: Develosil ODS-MG-5 (4.6 x
250 mm)
Mobile phase A: 0.2 M TEAA, pH 6.6
Mobile phase B: 10% acetonitrile, 0.2 M TEAA,
pH 6. 6
Mixing ratio: 0 to 10 minutes; B 5%
10 to 35 minutes; B 5% to 75% (linear)
35 to 50 minutes; B 75%
Test Example 2
[Detection of deprotected compound in cell]
The compound of Example 2 was added to MDCK
cells suspended in a Hank's balanced salt solution, and
the mixture was incubated at 37°C for 1 hour.
Thereafter, the obtained product was layered on silicon
oil (KF-99), followed by centrifugation at 4°C. Cell
fractions from the precipitate were suspended and
frozen-thawed in a mobile phase indicated below, and
the obtained solution was analyzed under the HPLC
analysis conditions indicated below.
As a result, a monophosphate (recovery time:
14.3 minutes) was detected as a deprotected compound.
HPLC analysis conditions
Instruments
Pump: HITACHI L-6000
Detector: HITACHI L-7500
Analysis conditions
Separation column: Develosil ODS-MG-5 (4.6 x
250 mm)
Mobile phase: 0.02 M phosphate buffer
solution, pH 3.0
Test Example 3-1
[Polymerase inhibition test (influenza virus)]
Influenza virus particles were treated with a
dissolving solution (100 mM Tris-HCl (pH 8), 100 mM
KC1, 5 mM MgCl2, 1.5 mM DTT, 5% glycerol, 1.5% Triton
N101, 1% LPC), and these particles were used as
polymerase crude enzymes. Each of the test compounds
with different concentrations was added to a reaction
buffer (100 mM Tris-HCl (pH 8.0), 100 mM KC1, 5 mM
MgCl2, 1 mM DTT, 0.25% Triton N101, 0.25 mM ApG, 0.1 mM
ATP, 0.05 mM CTP, 0.05 mM UTP, 0.0005 mM GTP, 32P-GTP,
crude enzyme), followed by incubation at 30°C for 60
minutes. Thereafter, 10% trichloroacetic acid (TCA)
was added thereto, and the mixture was retained on ice
for 60 minutes. Thereafter, it was dropped on a GF/C
filter, and the filter was then washed with 5% TCA.
The filter was dried, and scintillation cocktail was
added thereto. The radioactivity was determined using
a liquid scintillation counter. The measurement value
was given as a 50% inhibition concentration, providing
that a group in which no test compounds were added was
defined as 100%. The results are shown in Table 1.
[Table 1]
Compound 50% inhibition concentration (µM)
Reference Example 16 0.14
Reference Example 17 0.33
Reference Example 22 0.07 6
Reference Example 23 0.25
Reference Example 25 28
Test Example 3-2
[Polymerase inhibition test (hepatitis C virus (HCV))]
The NS5B region of hepatitis C virus was
produced in Escherichia coli, and it was used as HCV
polymerase for the test. The sequence of the 3'-region
of HCV was prepared by the in vitro transcription
method, and it was used as an RNA template for the
test.
Each of the test compounds with different
concentrations was added to a reaction buffer (20 mM
Tris-HCl (pH 8.0), 0.05 mM MnCl2, 1 mM DTT, 20 units
RNase inhibitor, [a-32P]GTP, 0.05 mM each of ATP, CTP
and UTP, and 2 µg/mL RNA template), followed by
incubation at 30°C for 2 hours. Thereafter, 10% TCA was
added to terminate the reaction. Thereafter, the
reaction solution was dropped on a DE81 filter, and the
filter was then washed with 5% TCA. The filter was
dried, and scintillation cocktail was added thereto.
The radioactivity was determined using a liquid
scintillation counter. The measurement value was given
as a 50% inhibition concentration, providing that a
group in which no test compounds were added was defined
as 100%. The results are shown in Table 1-2.
[Table 1-2]
Compound 50% inhibition concentration (µM)
Reference Example 16 0.75
Reference Example 17 2.7
Reference Example 22 0.088
Reference Example 23 2.2
Reference Example 25 1.6
Test Example 3-3
[Human RNA polymerase inhibition test]
A Hela cell nucleus extract (Promega) was
used as human RNA polymerase for the test. The pCMP
script was cleaved with restriction enzymes and then
purified, and it was used as a DNA template for the
test.
Each of the test compounds with different
concentrations was added to a reaction buffer (Hela
cell nucleus extract, 3 mM MgCl2, 0.4 mM each of ATP,
UTP and CTP, 0.016 mM GTP, 16 µg/mL DNA template, 0.4
mCi/mL [a-32P]GTP), followed by incubation at 30°C for 1
hour. After completion of the reaction, the reaction
solution was dropped on a DE81 filter, and the filter
was then washed with 5% Na2HP04 solution 3 times for 30
minutes, and then with distilled water once for 1
minute. The filter was dried, and scintillation
cocktail was added thereto. The radioactivity was
determined using a liquid scintillation counter. The
measurement value was given as a 50% inhibition
concentration, providing that a group in which no test
compounds were added was defined as 100%. The results
are shown in Table 1-3.
[Table 1-3]
Compound 50% inhibition concentration (u.M)
Reference Example 16 >200
Reference Example 17 >200
Reference Example 22 >200
Reference Example 23 >200
Reference Example 25 >200
Test Example 4
[Anti-influenza effect]
MDCK cells that had fully grown in a 6-well
culture plate were infected with influenza virus
A/PR/8/34 at 70 PFU/well. After 60 minutes, the
infection solution was removed, and an E'-MEM culture
medium containing 0.6% agar noble, 1% bovine serum
albumin and 3 µg/mL acetyltrypsin that contained a 100
µg/mL test compound was added thereto. The mixture was
fully solidified and then turned upside down. It was
cultured for 3 days under conditions of 35°C, a humidity
of 100% and 5% C02. After completion of the culture,
surviving cells were stained with 1% neutral red, and
then fixed with 10% formalin. Thereafter, the agar
medium was removed with running water, and then the
number of plaques was counted. The plaque inhibition
rate was expressed by the percentage obtained by-
comparing with a control to which no test compounds
were added. The results are shown in Table 2.
[Table 2]
Compound Inhibition rate (%)
Reference Example 7 42
Reference Example 12 78
Example 2 100
Example 6 100
Example 11 24
Example 22 33
Example 2 9 10 0
Example 31 4 4
Example 32 100
Test Example 5
[Anti-BVDV effect]
MDBK cells that had fully grown in a 6-well
culture plate were infected with bovine diarrhea virus
(BVDV) NADL at 70 PFU/well. After 60 minutes, the
infection solution was removed, and a test culture
solution (E'-MEM) containing 5% horse serum and 1% agar
(SeaPlaque Agar) that contained a 100 µg/mL test
compound was added thereto. The mixture was fully
solidified and then cultured for 3 days under
conditions of 37°C, a humidity of 100% and 5% C02.
After completion of the culture, the test plate was
fixed with a 3% formaldehyde solution, and the agar
medium was removed with running water, followed by
staining with a 1% crystal violet solution, so as to
count the number of plaques. The plaque inhibition
rate was expressed by the percentage obtained by
comparing with a control to which no test compounds
were added. The results are shown in Table 3.
[Table 3]
Compound Inhibition rate (%)
Example 2 100
Example 4 68
Example 6 57
Example 2 9 100
Example 32 100
Test Example 6
[Test to confirm presence of phosphate form in the
liver of mouse administered with compound A]
Compound A was administered into the caudal
vein of a mouse at a dose of 300 mg/kg. 30 minutes
after the administration, 1.6 g of the liver was
excised, and it was then ground under ice cooling,
while adding thereto 22.5 mL of 70 % methanol that had
been cooled to -20°C, so that compound A and a
phosphate(s) form were extracted. 10 mL of the
supernatant of the extract centrifuged at 4°C for 10
minutes was purified under the condition indicated
below, using a solid extraction cartridge (Varian BOND
ELUT SAX HF 2 g/12 mL; eluted with 0.01 M to 1.0 M KC1)
pretreated with 12 mL of methanol and 12 mL each of 1.0
M and 0.005 M KC1.
The phosphate form contained in 5 mL of No. 3
0.05 M KC1 eluate and a synthetic monophosphate
compound (a compound obtained in Reference Example 30)
were identical in terms of HPLC retention time and UV
spectrum in HPLC analysis (HPLC-1) described in HPLC
conditions. In addition, the phosphate form contained
in 5 mL of No. 1 0.5 M KC1 eluate and a synthetic
diphosphate compound (a compound obtained in Reference
Example 31) were identical in terms of HPLC retention
time in HPLC analysis (HPLC-2) described in HPLC
conditions. Moreover, the phosphate form contained in
5 mL of No. 2 0.5 M KC1 eluate and a synthetic
triphosphate compound (a compound obtained in Reference
Example 22) were identical in terms of HPLC retention
time in HPLC analysis (HPLC-3) described in HPLC
conditions.
0.5 mL of 0.5 M Tris-HCl (pH 8.0) and 0.5 mL
of 0.1 M MgCl2 were added to 4 mL of the above No. 3
0.05 M KC1 eluate, and then 0.5 mL of alkali
phosphatase prepared from the intestine of a calf
(EC3.1.3.1., 20 U/mL) was further added thereto,
followed by incubation at 37°C for 1 hour. Likewise,
0.5 rnL of 0.5 M Tris-HCl (pH 8.0) and 0.5 mL of 0.1 M
MgCl2 were added to 4 mL of the above No. 1 0.5 M KC1
eluate, and then 0.5 mL of alkali phosphatase prepared
from the intestine of a calf (EC3.1.3.1., 20 U/mL) was
further added thereto, followed by incubation at 37°C
for 1 hour. Likewise, 0.2 mL of 0.5 M Tris-HCl (pH
8.0) and 0.2 mL of 0.1 M MgCl2 were added to 1.6 mL of
the above No. 2 0.5 M KC1 eluate, and then 0.1 mL of
alkali phosphatase prepared from the intestine of a
calf (EC3.1.3.1., 20 U/mL) was further added thereto,
followed by incubation at 37°C for 1 hour.
By the HPLC analysis (HPLC-4) described in
HPLC conditions, it was confirmed that the
monophosphate disappeared from the No. 3 0.05 M KCl
eluate, and that a newly generated compound and
compound A were identical in terms of HPLC retention
time and UV spectrum. Likewise, by the HPLC analysis
(HPLC-4) described in HPLC conditions, it was confirmed
that the diphosphate form disappeared from the No. 1
0.5 M KCl eluate, and that a newly generated compound
and compound A were identical in terms of HPLC
retention time and UV spectrum. Likewise, by the HPLC
analysis (HPLC-4) described in HPLC conditions, it was
confirmed that the triphosphate disappeared from the
No. 2 0.5 M KCl eluate, and that a newly generated
compound and compound A were identical in terms of HPLC
retention time and UV spectrum.
Based on these results, it was confirmed that
compound A was converted in vivo into a monophosphate,
then into a diphosphate form, and then into a
triphosphate.
Purification conditions using solid extraction
cartridge
Washing: washing was carried out using the
following 3 solvents in the following order.
Water (cooled with ice) 12 mL
60% methanol (cooled with ice) 12 mL
Water (cooled with ice) 12 mL
Elution: elution was successively carried out
under the following concentration conditions. (Elution
was carried out by unit of 5 mL. "x 2" and "x 3" mean
that elution of 5 mL was performed two and three times,
respectively with each concentration. Fractions were
defined as No. 1 eluate, No. 2 eluate, and No. 3
eluate, successively.)
0.01 M KC1 (5 mL x 2)
0.05 M KC1 (5 mL x 3)
0.1 M KC1 (5 mL x 3)
0.5 M KC1 (5 mL x 2)
1.0 M KC1 (5 mL x 2)
HPLC analysis conditions
HPLC-1
Column: 4.6 x 250 mm, Nomura Chemical Co.,
Ltd., Develosil ODS-MG-5
Mobile phase: 0.02 M phosphate buffer
solution (pH 7.0)
Detection: UV 200 to 400 nm
HPLC-2
Column: 4.6 x 250 mm, Whatman Partisil 10-SAX
Mobile phase: 0.2 M phosphate buffer solution
(pH 3.5)
Detection: UV 200 to 400 nm
HPLC-3
Column: 4.6 x 250 mm, Whatman Partisil 10-SAX
Mobile phase: 0.6 M phosphate buffer solution
(pH 3.5)
Detection: UV 200 to 400 nm
HPLC-4
Column: 4.6 x 250 mm, Nomura Chemical Co.,
Ltd., Develosil ODS-MG-5
Mobile phase: 2% acetonitrile 0.02 M
phosphate buffer solution (pH 5.0)
Detection: UV 200 to 400 nm
Measuring instruments used
Diode Array Detector Agilent 1100 Series
Quaternary Pump Agilent 1100 Series
Autosampler Agilent 1100 Series
ChemStation Agilent 1100 Series
Test Example 7
[Determination of concentration of compound A in plasma
of mouse orally administered with test compound]
The test compound was orally administered
once to two mice (ICR) in a group. Blood was collected
30 minutes after the administration. 400 µL of
acetonitrile was added to 200 µL of the centrifuged
plasma. The mixture was centrifuged, and the
precipitated protein was removed. The obtained
supernatant was concentrated under reduced pressure,
and then the concentration of compound A in the plasma
was determined under the following HPLC conditions.
The results are shown in Table 4.
HPLC conditions
Column: Develosil ODS-MG-5, 4.6 x 250 mm
(Nomura Chemical Co., Ltd.)
Guard column: Develosil ODS-MG-5, 4.6 x 10 mm
(Nomura Chemical Co., Ltd.)
Detection: UV 350 nm
Mobile phase: 2% acetonitrile 0-02 M
phosphate buffer solution (pH 5.0)
Measuring instruments
Detector: Shimadzu SPD-6A
Pump: HITACHI L-6000
Test Example 8
[Determination of concentration of compound B in plasma
of mouse orally administered with test compound]
The compound in Reference Example 6 was
orally administered at 200 mg/kg once to two ICR mice
in a group. Blood was collected 30 minutes and 60
minutes after the administration. The same operation
as in Test Example 7 was carried out, and the
concentration of compound B in the plasma was
determined by HPLC. The results are shown in Table 5.
HPLC conditions
Column: Develosil ODS-MG-5, 4.6 x 250 mm
(Nomura Chemical Co., Ltd.)
Guard column: Develosil ODS-MG-5, 4.6 x 10 mm
(Nomura Chemical Co., Ltd.)
Detection wavelength: UV 350 nm
Mobile phase: 5% acetonitrile 0.04 M
phosphate buffer solution (pH 6.0)
[Table 5]
Concentration of compound B in
Test compound Plasma (µg/mL)
30 minutes later 60 minutes later
Reference Example 6 1.6 4.6
Text example 9
[Detection of phosphate compound in cell by addition of
3-hydroxy-2-pyrazinecarboxamide]
5 ml of an E'-MEM culture medium (containing
1% bovine serum albumin and 3% vitamin solution)
containing 3-hydroxy-2-pyrazinecarboxamide (final
concentration: 5000 µM) was added to MDCK cells that
had been monolayer-cultured on a 55 cm2 culture plate,
and culture was carried out under conditions of 37°C and
5% C02 for 24 hours. 1 mL of 70% methanol that had been
cooled to -20°C was added to cell fractions under ice
cooling, and 3-hydroxy-2-pyrazinecarboxamide, compound
A, and a phosphate(s) were extracted therefrom. 0.7 mL
of the supernatant of the extract centrifuged at 4°C for
10 minutes was purified under the condition indicated
below, using a solid extraction cartridge (Varian BOND
ELUT SAX HF 100 mg/1 mL; eluted with 0.01 M to 1.0 M
KC1) pretreated with 1 mL of methanol and 1 mL each of
1.0 M and 0.005 M KC1.
The phosphate form contained in 1 mL of 0.05
M KC1 eluate and a synthetic monophosphate compound (a
compound obtained in Reference Example 30) were
identical in terms of HPLC retention time and UV
spectrum in HPLC analysis (HPLC-5). In addition, the
phosphate form contained in 1 mL of 0.25 M KC1 eluate
and a synthetic diphosphate compound (a compound
obtained in Reference Example 31) were identical in
terms of HPLC retention time in HPLC analysis (HPLC-6).
Moreover, the phosphate form contained in 1 mL of 0.5 M
KC1 eluate and a synthetic triphosphate compound (a
compound obtained in Reference Example 22) were
identical in terms of HPLC retention time in HPLC
analysis (HPLC-6).
To 0.8 mL each of the above KCl eluates, 0.1
mL of 0.5 M Tris-HCl (pH 8.0) and 0.1 mL of 0.1 M MgCl2
were added. Then 0.8 mL of the mixture was taken, to
which 0.08 mL of alkali phosphatase prepared from the
intestine of a calf (EC3.1.3.1., 20 U/mL) was added,
followed by incubation at 37°C for 1 hour.
By the HPLC analysis (HPLC-7), it was
confirmed that the monophosphate disappeared from the
0.05 M KCl eluate, and that a newly generated compound
and compound A were identical in terms of HPLC
retention time. Moreover, by the HPLC analysis (HPLC-
8), it was confirmed that a newly generated compound
and compound A were identical in terms of UV spectrum.
Likewise, by the HPLC analysis (HPLC-7), it was
confirmed that the diphosphate form disappeared from
the 0.25 M KC1 eluate, and that a newly generated
compound and compound A were identical in terms of HPLC
retention time. Furthermore, by the HPLC analysis
(HPLC-8), it was confirmed that a newly generated
compound and compound A were identical in terms of UV
spectrum. Likewise, by the HPLC analysis (HPLC-7), it
was confirmed that the triphosphate disappeared from
the 0.5 M KC1 eluate, and that a newly generated
compound and compound A were identical in terms of HPLC
retention time.
Based on these results, it was confirmed that
3-hydroxy-2-pyrazinecarboxamide, was converted in a
cell into compound A, a monophosphate (a compound
obtained in Reference Example 30), then into a
diphosphate form (a compound obtained in Reference
Example 31), and then into a triphosphate (a compound
obtained in Reference Example 22).
Purification conditions using solid extraction
cartridge
Washing: Washing was carried out using the
following 3 solvents in the following order.
Water (cooled with ice) 1 mL
60% methanol (cooled with ice) 1 mL
Water (cooled with ice) 1 mL
Elution: Elution was successively carried out
under the following concentration conditions. (Elution
was carried out by unit of 1 mL.)
0.01 M KC1 (1 mL)
0.05 M KC1 (1 mL)
0.1 M KC1 (1 mL)
0.25 M KC1 (1 mL)
0.5 M KC1 (1 mL)
1.0 M KC1 (1 mL)
HPLC analysis conditions
HPLC-5
Column: 4.6 x 250 mm, Nomura Chemical Co.,
Ltd., Develosil ODS-MG-5
Mobile phase: 0.02 M phosphate buffer
solution (pH 7.0)
Detection: UV 200 to 400 nm
Measuring instruments used
Diode Array Detector Agilent 1100 Series
Quaternary Pump Agilent 1100 Series
Autosampler Agilent 1100 Series
ChemStation Agilent 1100 Series
HPLC-6
Column: 4.6 x 250 mm, Whatman Partisil 10-SAX
Mobile phase: 0.75 M phosphate buffer
solution (pH 3.5)
Detection: UV 350 nm
Measuring instruments used
Shimadzu SPD-6A UV Spectrophotometry
Detector
HITACHI L-6000 Pump
HPLC-7
Column: 4.6 x 2 50 mm, Nomura Chemical Co.,
Ltd., Develosil ODS-MG-5
Mobile phase: 5% acetonitrile 0.02 M
phosphate buffer solution (pH 5.0)
Detection: UV 350 ran.
Measuring instruments used
Shimadzu SPD-6A UV Spectrophotometry
Detector
HITACHI L-6000 Pump
HPLC-8
Column: 4.6 x 250 mm, Nomura Chemical Co.,
Ltd., Develosil ODS-MG-5
Mobile phase: 2% acetonitrile 0.02 M
phosphate buffer solution (pH 5.0)
Detection: UV 200 to 400 nm
Measuring instruments used
Diode Array Detector Agilent 1100 Series
Quaternary Pump Agilent 1100 Series
Autosampler Agilent 1100 Series
ChemStation Agilent 1100 Series
Text example 10
[Detection of triphosphate compound in cell by addition
of compound A]
10 ml of an E'-MEM culture medium (containing
5% fetal bovine serum) containing compound A was added
to MDBK cells that had been monolayer-cultured on a 55
cm2 culture plate. After completion of culture under
conditions of 37°C and 5% C02 for 24 hours, the culture
was' removed using a cell scraper, and the culture
medium was removed by centrifugation. Thereafter, 5%
trichloroacetic acid was added to the obtained cell
fractions, so that a converted compound was extracted.
An equal amount of 20% trioctylamine-containing pentane
was added to the extract, and the obtained water layer
was then concentrated using a centrifugal concentrator.
In the above concentrate, a component whose HPLC
retention time matches that of a synthetic compound of
triphosphates (a compound obtained in Reference Example
22) was detected.
HPLC conditions
Instruments
Pump: HITACHI L-6000
Detector: HITACHI L-4000
Analysis conditions
Separation column: Develosil ODS-MG-5 (4.6 x
250 mm)
Mobile phase: 7% acetonitrile, 5 mM
tetrabutylammonium bromide, 0.1 M phosphate buffer
solution (pH 7.0)
Measurement wavelength: 350 nm
Text example 11
[Detection of monophosphate and diphosphate compounds
in cell by addition of compound A]
5 ml of an E'-MEM culture medium (containing
1% bovine serum albumin and 3% vitamin solution)
containing compound A (final concentration: 5000 µM)
was added to MDCK cells that had been monolayer-
cultured on a 55 cm2 culture plate, and culture was
carried out under conditions of 37°C and 5% C02 for 24
hours. 1 mL of 7 0% methanol that had been cooled to -
20°C was added to cell fractions under ice cooling, and
compound A, and a phosphate(s) were extracted
therefrom. 0.7 mL of the supernatant of the extract
centrifuged at 4°C for 10 minutes was purified under the
condition indicated below, using a solid extraction
cartridge (Varian BOND ELUT SAX HF 100 mg/1 mL; eluted
with 0.01 M to 1.0 M KC1) pretreated with 1 mL of
methanol and 1 mL each of 1.0 M and 0.005 M KC1.
The phosphate form contained in 1 mL of 0.05
M KC1 No. 1 eluant and a synthetic monophosphate
compound (a compound obtained in Reference Example 30)
were identical in terms of HPLC retention time and UV
spectrum in HPLC analysis (HPLC-9). In addition, the
phosphate form contained in 1 mL of 0.5 M KC1 eluate
and a synthetic diphosphate compound (a compound
obtained in Reference Example 31) were identical in
terms of HPLC retention time in HPLC analysis (HPLC-
10) .
To 0.8 mL of the above 0.05 M KC1 No. 1
eluant, 0.1 mL of 0.5 M Tris-HCl (pH 8.0) and 0.1 mL of
0.1 M MgCl2 were added. Then 0.5 mL of the mixture was
taken, to which 0.06 mL of alkali phosphatase prepared
from the intestine of a calf (EC3.1.3.1., 20 U/mL) was
added, followed by incubation at 37°C for 1 hour. By
the HPLC analysis (HPLC-11), it was confirmed that the
monophosphate disappeared from the 0.05 M KC1 eluate,
and that a newly generated compound and compound A were
identical in terms of HPLC retention time and UV
spectrum.
Based on these results, it was confirmed that
compound A was converted in a cell into a monophosphate
(a compound obtained in Reference Example 30), and a
diphosphate form (a compound obtained in Reference
Example 31).
Purification conditions using solid extraction
cartridge
Washing: Washing was carried out using the
following 3 solvents in the following order.
Water (cooled with ice) 1 mL
60% methanol (cooled with ice) 1 mL
Water (cooled with ice) 1 mL
Elution: elution was successively carried out
under the following concentration conditions. (Elution
was carried out by unit of 1 mL. "x 2" means that
elution of 1 mL was performed twice with each
concentration twice. Fractions were defined as No. 1
eluate and No. 2 eluate, successively.)
0.01 M KC1 (1 mL)
0.05 M KC1 (1 mL x 2)
0.1 M KC1 (1 mL)
0.5 M KC1 (1 mL)
1.0 M KC1 (1 mL)
HPLC analysis conditions
HPLC-9
Column: 4.6 x 250 mm, Nomura Chemical Co.,
Ltd., Develosil ODS-MG-5
Mobile phase: 0.02 M phosphate buffer
solution (pH 7.0)
Detection: UV 200 to 400 nm
Measuring instruments used
Diode Array Detector Agilent 1100 Series
Quaternary Pump Agilent 1100 Series
Autosampler Agilent 1100 Series
ChemStation Agilent 1100 Series
HPLC-10
Column: 4.6 x 250 mm, Whatman Partisil 10-SAX
Mobile phase: 0.2 M phosphate buffer solution
(pH 3.5)
Detection: UV 350 nm
Measuring instruments used
Shimadzu SPD-6A UV Spectrophotometric
Detector
HITACHI L-6000 Pump
HPLC-11
Column: 4.6 x 250 mm, Nomura Chemical Co.,
Ltd., Develosil ODS-MG-5
Mobile phase: 2% acetonitrile 0.02 M
phosphate buffer solution (pH 5.0)
Detection: UV 200 to 400 nm
Measuring instruments used
Diode Array Detector Agilent 1100 Series
Quaternary Pump Agilent 1100 Series
Autosampler Agilent 1100 Series
ChemStation Agilent 1100 Series
Test Example 12
[Inosine monophosphate dehydrogenase (IMPDH) inhibition
test]
MDCK cells monolayer-cultured on a culture
plate were suspended in 0.05 M Tris-HCl (pH 8.0), and
the suspension was homogenated with a Downs homogenizer
to obtain a cell homogenate. The cell homogenate was
centrifuged at 16000 x g, and the thus obtained
supernatant was used as an IMPDH enzyme solution.
As reaction compositions, 0.1 M Tris-HCl (pH
8.0), 0.1 M KC1, 30 mM EDTA, 5 mM NAD, 5 mg/mL bovine
serum albumin, and 0.04 mM [8-14C] -inosine 5'-
monophosphate were used. After completion of reaction
at 37°C for 1 hour, 2 volumes of acetonitrile were added
to terminate the reaction, and the reaction product was
concentrated. The obtained concentrate was analyzed
under the HPLC conditions indicated below. The ratio
between the reaction substrate ( [14C]-inosine 5'-
monophosphate) and the reaction product ([14C]-
xanthosine 5'-monophosphate) was obtained, and the
reaction rate was calculated. Ribavirin monophosphate
was used as a control compound. The results are shown
in Table 6.
HPLC analysis conditions
Separation column: Develosil ODS-MG-5 (4.6 x
250 mm)
Mobile phase: 20% acetonitrile, 5 mM
butylammonium bromide, 0.02 M phosphate buffer solution
(pH 7.0)
Radiation detector: Packard FLO-ONE500
[Table 6]
Test compound 50% inhibition concentration (jjM)
Reference Example 30 980
Example 37 74 0
Control compound 1.9
Next, the compound of the present invention
will be explained in Reference Examples and examples.
However, the present invention is not limited thereto.
Mixing ratios in eluants are all expressed by
volume ratios. Silica gel BW-127ZH (Fuji Silysia
Chemical Ltd.) was used as a medium for column
chromatography; YMC GEL ODS-AM 120-S50 (YMC Co., Ltd.)
was used as a carrier for reverse phase silica gel
column chromatography; and DEAE cellulose (Wako Pure
Chemical Industries, Ltd.) was used as a carrier for
ion exchange column chromatography. The symbols used
in Reference Examples and Examples mean the following:
DMSO-d6: Deuterated dimethyl sulfoxide, Ms:
Methanesulfonyl group, Ph: Phenyl group, and Et: Ethyl
group
1.52 g of methyl 3-hydroxy-2-
pyrazinecarboxylate was suspended in 12.2 mL of
1, 1,1, 3, 3, 3-hexarnethyldisilazane, and the suspension
was heated under reflux for 1 hour. After standing to
cool, the solvent was removed under reduced pressure,
and the obtained residue was dissolved in 30 mL of
dichloroethane under nitrogen atmosphere. 4.98 g of p-
D-ribofuranose-l-acetate-2,3,5-tribenzoate and 1.7 3 mL
of tin(IV) chloride were successively added thereto,
and the mixture was further stirred at room temperature
for 14 hours. The reaction mixture was diluted with 30
mL of chloroform and 30 mL of a saturated sodium
bicarbonate aqueous solution, and the precipitate was
removed by filtration, so that the organic layer was
obtained. The obtained organic layer was successively
washed with water and then with a saturated saline
solution. Thereafter, the layer was dried with
anhydrous magnesium sulfate, and the solvent was
removed under reduced pressure. The obtained residue
was purified by silica gel column chromatography
[eluant; n-hexane : ethyl acetate =1 : 1], so as to
obtain 3.4 g of a white solid, methyl 4-{(2R, 3R, 4R,
5R)-3,4-bis(benzoyloxy)-5-
[(benzoyloxy)methyl]tetrahydro-2-furanyl}-3-oxo-3,4-
dihydro-2-pyrazinecarboxylate.
IR(KBr)cm-1: 1734, 1660
1H-NMR(CDCl3)d: 3.96(3H,s), 4 . 71 (1H, dd, J=4 . 0, 12 . 4Hz) ,
4.8-4.9(2H,m), 5.8-5.9(2H,m), 6.45(1H,d,J=4.0Hz),
7.34(lH,d,J=4.2Hz), 7.3-7.6(9H,m), 7.70(1H,d,J=4.2Hz),
7.9-8.0(4H,m), 8.0-8.1(2H,m)
Reference Example 2
36.3 g of methyl 4-{(2R, 3R, 4R, 5R)-3,4-
bis(benzoyloxy)-5-[(benzoyloxy)methyl]tetrahydro-2-
furanyl}-3-oxo-3,4-dihydro-2-pyrazinecarboxylate was
suspended in 400 mL of methanol, and 11.7 g of a 28%
sodium methoxide methanol solution was added thereto
under ice cooling. The mixture was stirred at the same
temperature for 1 hour. The reaction solution was
adjusted to pH 4 using 2M hydrochloric acid, and the
solvent was then removed under reduced pressure. The
obtained residue was purified by reverse phase silica
gel column chromatography [eluant; acetonitrile : water
= 1 : 4], so as to obtain 12.6 g of a light yellow
solid, methyl 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-dihydro-
2-pyrazinecarboxylate.
IR(KBr)cm-1: 1740
1H-NMR(DMSO-d6)d: 3. 6-3.65 (lH,m) , 3 . 75-3 . 8 (lH,m) ,
3.83(3H,s), 3.9-4.0(3H,m), 5.13(1H,d,J=5.2Hz),
5.29(lH,t,J=5.2Hz), 5.64(1H,d,J=2.4Hz),
5.91(lH,d,J=2.4Hz), 7.48(1H,d,J=4.4Hz),
8.31(lH,d,J=4.4Hz).
Reference Example 3
0.5 g of methyl 4-[(2R, 3R, 4S, 5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxylate was suspended in 5 mL
of acetone, and 1 mL of trimethyl orthoformate and 33
mg of paratoluenesulfonic acid monohydrate were
successively added thereto. The mixture was heated
under reflux for 1 hour, and the solvent was then
removed under reduced pressure. The obtained residue
was purified by column chromatography [eluant; ethyl
acetate], so as to obtain 0.49 g of a white solid,
methyl 4-[(3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-
dimethyltetrahydrofuro[3,4-d] [1,3]dioxol-4-yl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxylate.
IR(KBr)cm"1: 1728
1H-NMR(CDCl3)d: 1.35(3H,s), 1.60(3H,s),
2.55(lH,t,J=4.6Hz), 3 . 8-3.9(lH,m) , 3.97(3H,s), 3.95-
4.0(lH,m), 4.4-4.5(lH,m), 4.97(1H,dd,J=3.2,6.3Hz),
5.01(lH,dd,J=2.4,6.3Hz), 5.80(1H,d,J=2.4Hz),
7.49(lH,d,J=4.3Hz), 7.69(1H,d,J=4.3Hz).
Reference Example 4

6.78 g of methyl 4-f(3aR,4R,6R,6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate was dissolved in 68 mL of methanol,
and ammonia gas was then introduced therein under ice
cooling for saturation. After reaction at the same
temperature for 1.5 hours, the deposited solid was
collected by filtration, so as to obtain 2.34 g of a
light yellow solid, 4-[(3aR,4R,6R,6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][l,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide. The filtrate was concentrated, so
as to further obtain 2.54 g of the above compound.
IR(KBr)cm_1: 1701, 1654
1H-NMR(DMSO-d6)d: 1.29(3H,s), 1.51(3H,s), 3. 5-3. 6 (lH,m) ,
3.6-3.7(lH,m), 4.3-4.4(lH,m), 4.7-4.8(lH,m), 4.8-
4.9(1H,m), 5.22(1H,t,J=4.7Hz), 5.98(1H,s),
7.55(1H,d,J=4.0Hz), 7.76(1H,brs), 8.04(1H,d,J=4.0Hz),
8.36(1H,brs).
Reference Example 5
15 mL of sulfuryl chloride was dropped into
80 mL of an N,N-dimethylformamide suspension containing
20 g of 3-hydroxy-2-pyrazinecarboxamide at a
temperature between 80°C and 90°C. The mixture was
stirred at a temperature between 95°C and 100ºC for 1
hour, and it was then poured into a mixed solution of
200 mL of ice water and 200 mL of ethyl acetate. The
organic layer was separated. The aqueous layer was
extracted with 100 mL of ethyl acetate 5 times, and it
was then combined with the organic layer. The mixture
was washed with a saturated saline solution.
Thereafter, the mixture was treated with activated
carbon, and the solvent was removed under reduced
pressure. The obtained residue was suspended in 50 mL
of water, and 3.2 g of sodium bicarbonate was added
thereto and dissolved. Thereafter, concentrated
hydrochloric acid was added thereto to adjust the
solution to pH 2. The deposit was collected by
filtration, so as to obtain 4.8 g of a white solid, 6-
chloro-3-hydroxy-2-pyrazinecarboxamide.
IR(KBr)cm"1: 1660
1H-NMR(DMSO-d6)d: 8.51 (2H, brs) , 8.73 (1H,s),
13.60(1H,brs)
Reference Example 6

7.5 mL of a 1,1,1,3,3,3-hexamethyldisilazane
suspension containing 1.5 g of 6-chloro-3-hydroxy-2-
pyrazinecarboxamide was heated under reflux for 30
minutes. After cooling, it was concentrated under
reduced pressure. 5 mL of toluene was added thereto,
and the solvent was removed under reduced pressure.
Thereafter, 5 mL of toluene was added thereto, and the
solvent was removed under reduced pressure again. 15
mL of acetonitrile was added to the obtained residue
and dissolved, and under ice cooling, p-D-ribofuranose-
1,2,3,5-tetraacetate and tin(IV) chloride were
successively added thereto, followed by stirring at
room temperature for 5 hours. The reaction mixture was
diluted with 30 mL of ethyl acetate and 20 mL of water,
and it was then adjusted to pH 7 by addition of a
saturated sodium bicarbonate aqueous solution.
Thereafter, the precipitate was removed by filtration,
and the organic layer was separated. The aqueous layer
was extracted with 10 mL of ethyl acetate 3 times, and
it was then combined with the organic layer. The
obtained mixture was washed with a saturated saline
solution and then dried with anhydrous magnesium
sulfate, and the solvent was removed under reduced
pressure. Diethyl ether was added to the obtained
residue, and the mixture was collected by filtration,
so as to obtain 2.8 g of a light yellow solid, (2R, 3R,
4R, 5R)-4-(acetyloxy)-2-[(acetyloxy)methyl]-5-[3-
(aminocarbonyl)-5-chloro-2-oxo-l(2H)-pyrazinyl]
tetrahydro-3-furanyl acetate.
IR(KBr)cm-1: 1756, 1733, 1701, 1648
^-NMRlCDClaJS: 2.09(3H,s), 2.18(3H,s), 2.23(3H,s),
4.45(2H,s), 4.5-4.6(1H,m), 5.2-5.3(1H,m), 5.45-
5.5(1H,m), 6.14(1H,d,J=2.0Hz), 6.22(1H,brs),
8.06(1H,s), 8.84(1H,s).
Reference Example 7
1.8 g of (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[(acetyloxy)methyl]-5-[3-(aminocarbonyl)-5-chloro-2-
oxo-1(2H)-pyrazinyl] tetrahydro-3-furanyl acetate was
suspended in 27 mL of methanol, and 2.4 g of a 28%
sodium methoxide methanol solution was added thereto
under ice cooling, followed by stirring at the same
temperature for 30 minutes. 0.95 mL of acetic acid was
added thereto, and the solvent was removed under
reduced pressure. The obtained residue was purified by
silica gel column chromatography [eluant; chloroform :
methanol =3 : 1], so as to obtain 0.73 g of a light
yellow solid, 6-chloro-4-[(2R,3R,4S,5R)-3,4-dihydroxy-
5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-
dihydro-2-pyrazinecarboxamide.
IR(KBr)cm-1: 1693
1H-NMR(DMSO-d6)d: 3 . 35 (3H, brs) , 3 . 65 (1H, d, J=12 . 0Hz) ,
3.8-3.9(1H,m), 3.9-4.0(3H,m), 5.81(1H,s), 7.92(1H,brs),
8.44 (1H,brs), 8.70(1H,s).
Reference Example 8
0.3 g of 6-chloro-4-[(2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxamide was dissolved in a
mixed solvent of 0.6 mL of acetone and 1.5 mL of N,N-
dimethylformamide, and then, 3 mL of 2,2-
dimethoxypropane and 0.12 g of p-toluenesulfonic acid
pyridinium salts were successively added thereto,
followed by stirring at 50°C for 5 hours. After
cooling, a mixed solvent of 5 mL of ethyl acetate and 5
mL of water was added thereto, and the organic layer
was separated. The layer was washed with water and
then with a saturated saline solution, and it was then
dried with anhydrous magnesium sulfate, and thereafter,
the solvent was removed under reduced pressure. The
obtained residue was purified by silica gel column
chromatography [eluant; chloroform : methanol = 10 :
1], so as to obtain 0.18 g of a light yellow solid, 4-
[(3aR, 4R, 6R, 6aR)-6-(hydroxymethyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-6-
chloro-3-oxo-3,4-dihydro-2-pyrazinecarboxamide.
IR(KBr)cm_1: 1700
1H-NMR(DMSO-d6)d: 1.29(3H,s), 1.50(3H,s), 3.5-3. 6 (1H,m) ,
3.7-3.8(1H,m), 4.3-4.4(1H,m), 4.74(1H,dd,J=2.9,6.1Hz),
4.8 8(1H,dd,J=2.0,6.1Hz), 5.37(1H,t,J=4.6Hz),
5.95(1H,d,J=1.7Hz), 7.93(1H,brs), 8.32(1H,s),
8.41(1H,brs).
Reference Example 9
1.0 g of methyl 4-[(3aR,4R,6R,6aR)-6-
{hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate was dissolved in 5.0 mL of
pyridine, and 0.36 mL of methanesulfonyl chloride was
then added thereto at 10°C, followed by stirring at room
temperature for 0.5 hours. The reaction mixture was
poured into a mixed solution of 20 mL of ethyl acetate
and 20 mL of water. The organic layer was separated,
and the aqueous layer was extracted with 20 mL of ethyl
acetate 3 times. It was then combined with the organic
layer. The mixture was washed with a saturated saline
solution and then dried with anhydrous magnesium
sulfate. Thereafter, the solvent was removed under
reduced pressure, so as to obtain 1.2 g of a colorless
oil product, methyl 4-[(3aR, 4R, 6R, 6aR)-2,2-dimethyl-
6-[[(methylsulfonyl)oxy]methyl]tetrahydrofuro[3,4-
d][1,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate.
IR(KBr)cm"1: 1734, 1669
1H-NMR(CDCl3)d: 1.35(3H,s), 1.58(3H,s), 3.02(3H,s),
3.98(3H,s), 4.51(2H,s), 4.8-5.2(3H,m), 5.73(1H,brs),
7.43 (2H,brs)
Reference Example 10
1.2 g of methyl 4-[(3aR, 4R, 6R, 6aR)-2,2-
dimethyl-6-
[[(methylsulfonyl)oxyjmethyl]tetrahydrofuro[3,4-
d][l,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate was dissolved in 12 mL of acetone.
2.3 g of sodium iodide was added thereto, and the
mixture was heated under reflux for 2 hours. The
reaction mixture was cooled to room temperature, and it
was then poured into a mixed solution of 20 mL of ethyl
acetate and 20 mL of water. The organic layer was
separated, and the aqueous layer was extracted with 20
mL of ethyl acetate. It was then combined with the
organic layer. The mixture was successively washed
with a sodium thiosulfate aqueous solution and then
with a saturated saline solution, and it was then dried
with anhydrous magnesium sulfate. Thereafter, the
solvent was removed under reduced pressure. The
obtained residue was purified by silica gel column
chromatography [eluant; toluene : ethyl acetate = 2 :
1], so as to obtain 1.0 g of a yellow oil product,
methyl 4-[(3aR, 4R, 6S, 6aR)-6-(iodomethyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-3-oxo-
3, 4-dihydro-2-pyrazinecarboxylate.
IR(KBr)cm"1: 1734,1670,1654
1H-NMR(CDCl3)d: 1.36{3H,s), 1.59(3H,s), 3.3-3.7 (2H,m) ,
3.98(3H,s), 4.3-4.5(1H,m), 4.9-5.1(2H,m),
5.76(1H,d,J=1.7Hz), 7.5-7.6(2H,m)
0.55 g of methyl 4-[(3aR, 4R, 6S, 6aR)-6-
(iodomethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][l,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate was dissolved in 5 mL of methanol,
and ammonia gas was introduced therein under ice
cooling for saturation. After stirring at the same
temperature for 1 hour, the solvent was removed under
reduced pressure. Diisopropyl ether was added to the
obtained residue, and the precipitate was collected by
filtration, so as to obtain 0.45 g of a yellow solid,
4-[(3aR,4R,6S,6aR)-6-(iodomethyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxamide.
IR(KBr) cnf1: 1684, 1654
1H-NMR(DMSO-d6)d: 1.30(3H,s), 1.51(3H,s), 3 . 3-3 . 5 (2H, m) ,
4.3-4.4(1H,m), 4.79(1H,dd,J=3.6,6.4Hz),
5.13(1H,dd,J=1.2,6.0Hz), 6.00(1H,d,J=l.6Hz),
7.53(1H,d,J=4.4Hz), 7.76(1H,brs), 7.90(1H,d,J=4.4Hz),
8.20(1H,brs)
5.3 g of 6-fluoro-3-hydroxy-2~
pyrazinecarboxamide was suspended in 53 mL of
acetonitrile under nitrogen current, and 8.4 mL of N,0-
bis(trimethylsilyl) acetamide was added thereto under
ice cooling, followed by stirring at room temperature
for 1.5 hours. 53 mL of an acetonitrile solution
containing 9.4 g of (2R, 3R, 4R)-4,5-bis(acetyloxy)-2-
(hydroxymethyl)tetrahydro-3-furanyl acetate that had
been separately prepared by the method described in
Carbohydrate Research (Carbohydr. Res.), Vol. 203, No.
9, pp. 324 to 329 (1990), and 7.2 mL of tin(IV)
chloride were successively added to the reaction
mixture under ice cooling, and the thus obtained
mixture was stirred at room temperature for 20 minutes.
The reaction mixture was poured into a mixed solution
of 100 mL of ethyl acetate and 300 mL of a saturated
sodium bicarbonate aqueous solution. The organic
layers were separated, and the aqueous layer was then
extracted with 700 mL of ethyl acetate. Such organic
layers were combined, and the organic layers were then
dried with anhydrous magnesium sulfate. Thereafter,
the solvent was removed under reduced pressure. The
obtained residue was dissolved in 200 mL of methanol,
and 100 mL of an 80% acetic acid aqueous solution was
added thereto, followed by stirring at room temperature
for 2 hours. The solvent was removed under reduced
pressure, and the obtained residue was purified by
silica gel column chromatography [eluant; chloroform:
methanol =40 : 1]. Thereafter, chloroform and
diisopropyl ether were added thereto, and the mixture
was collected by filtration, so as to obtain 9.3 g of a
light yellow solid, (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]-5-
(hydroxymethyl)tetrahydro-3-furanyl acetate.
IR(KBr)cm"1: 1752,1686
1H-NMR(DMSO-d6)d: 2.04(3H,s), 2.10(3H,s),
3.64 (1H,ddd, J=2. 5, 5.0,13Hz) ,
3.86(1H,ddd,J=2.5,5.0,13Hz), 4.29(1H,d,J=6.0Hz),
5.35(1H,t,J=6.0Hz), 5.4 9(1H,dd,J=3.0,5.0Hz),
5.65(1H,t,J=5.0Hz), 6.11(1H,d,J=3.0Hz), 7.96(1H,brs),
8.42(1H,d,J=5.0Hz), 8.49(1H,brs)
Reference Example 13
1.5 g of (2R,3R,4R,5R)-4-(acetyloxy)-2-[3-
(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinylj-5-
(hydroxymethyl)tetrahydro-3-furanyl acetate and 0.84 g
of 1H-tetrazole were dissolved in 30 mL of acetonitrile
under nitrogen current. Thereafter, 20 mL of an
acetonitrile solution containing 1.4 mL of diallyl
diisopropyl phosphoramidite was added thereto under ice
cooling, and the mixture was stirred for 20 minutes.
10 mL of an acetonitrile solution containing 1.4 g of
m-chloroperbenzoic acid was added to the reaction
mixture, followed by stirring for 10 minutes. 60 mL of
ethyl acetate was added to the reaction mixture, and
the obtained mixture was then poured into 60 mL of
water. The organic layers were separated, and the
aqueous layer was extracted with 90 mL of ethyl
acetate. The organic layers were combined, and 30 mL
of water was added thereto. The mixture was adjusted
to pH 8 by addition of a saturated sodium bicarbonate
aqueous solution, and then the aqueous layer was
separated. The organic layer was washed with a
saturated saline solution and then dried with anhydrous
magnesium sulfate, and thereafter, the solvent was
removed under reduced pressure. The obtained residue
was purified by silica gel column chromatography
[eluant; chloroform : methanol =40 : 1], so as to
obtain 1.3 g of a yellow solid, (2R,3R,4R,5R)-4-
(acetyloxy)-2-[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-
pyrazinyl]-5-([[bis(allyloxy)phosphoryl]oxy]methyl)-
tetrahydro-3-furanyl acetate.
IR(KBr)cm_1: 1753, 1694,
1H-NMR(CDCl3)d: 2.11(3H,s), 2.15(3H,s), 4 . 32-4 . 35 (1H, m) ,
4.4 7-4.52(2H,m), 4.58-4.64(4H,m),
5.27(2H,dt,J=1.0,10.5Hz), 5.37-5.44(4H,m), 5.90-
6.00(2H,m), 6.28{1H,d,J=4.0Hz), 6.32(1H,brs),
7.99(1H,d,J=6.0Hz), 9.02(1H,brs)

0.23 g of (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]-5-
([[bis(allyloxy)phosphoryl]oxy]methyl)tetrahydro-3-
furanyl acetate was dissolved in 4.0 mL of methanol,
and 0.17 g of a 28% sodium methoxide methanol solution
was added thereto under ice cooling, followed by
stirring for 5 minutes. 0.15 mL of acetic acid was
added thereto, and the solvent was removed under
reduced pressure. 1.0 g of (2R, 3R, 4R, 5R)-4-
(acetyloxy)-2-[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-
pyrazinyl]-5-
([[bis(allyloxy)phosphoryl]oxy]methyl)tetrahydro-3-
furanyl acetate was subjected to the same above
reaction. The reaction mixtures were combined, and the
obtained mixture was purified by silica gel column
chromatography [eluant; chloroform : methanol =40 :
1], so as to obtain 0.35 g of a yellow solid, [(2R, 3S,
4R, 5R)-5-[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-
pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl] methyl
diallyl phosphate.
IR{KBr)cm_1: 168 4
1H-NMR (DMSO-d6, D20)d: 3 . 1-4 . 7 ( 9H,m) , 5 .1-5 . 5 (4H, m) , 5.7-
6.2(3H,m), 7.94(1H,d,J=6.0Hz)
Reference Example 15
0.82 g of [(2R, 3S, 4R, 5R)-5-[3-
(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl] methyl diallyl phosphate
was dissolved in a mixed solution of 8.2 mL of methanol
and 8.2 mL of tetrahydrofuran under nitrogen current.
0.11 g of tetrakis triphenyl phosphine palladium (0)
and 0.28 g of triphenyl phosphine were successively
added thereto, and the mixture was stirred at room
temperature for 30 minutes. 1.9 mL of a
tetrahydrofuran solution containing 0.68 mL of formic
acid and 8.2 mL of a tetrahydrofuran solution
containing 1.1 mL of n-butylamine were successively
added to the reaction mixture under water cooling, and
the obtained mixture was stirred at a temperature
between 30°C and 35°C for 1 hour, and at a temperature
between 40°C and 45°C for 2 hours. The reaction mixture
was diluted with 10 mL of water, and the organic
solvent was then removed under reduced pressure. The
obtained aqueous solution was washed with 20 mL of
chloroform, and the washing was extracted with 30 mL of
water. All the aqueous layers were combined, and the
solvent was removed under reduced pressure. The
obtained residue was purified by reverse phase silica
gel column chromatography [eluant; water], so as to
obtain 0.29 g of n-butyl ammonium salts of a yellow
solid, [ (2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-5-fluoro-
2-oxo-l(2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-
furanyl] methyl dihydrogen phosphate.
IR(KBr)cm-1: 1685
1H-NMR(DMSO-d6)d: 0 . 75-0 . 90 (3H, m) , 1. 25-1. 40 (2H,m) ,
1.45-1.70(2H,m), 2.70-2.80(2H,m), 3.3-4.7(8H,m),
5.33(1H,d,J=10Hz), 5.42(1H,d,J=17Hz), 5.90(2H,brs),
7.95(1H,brs), 8.34(1H,d,J=5.0Hz), 8.63(1H,brs)
Reference Example 16

0.21 g of n-butyl ammonium salts of [(2R, 3S,
4R, 5R)-5-[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H) -
pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl] methyl
dihydrogen phosphate was suspended in a mixed solution
of 4.2 mL of acetonitrile and 8.4 mL of N,N-
dimethylformamide, and thereafter, 0.15 g of 1,1'-
carbonyldiimidazole was added thereto, followed by
stirring at room temperature for 2 hours. Thereafter,
19 µL of methanol was added to the reaction mixture,
followed by stirring for 30 minutes. Thereafter, 2.0
mL of an N,N-dimethylformamide solution containing 0.8 6
g of tri-n-butyl ammonium pyrophosphate was added to
the reaction mixture, and the obtained mixture was
further stirred for 14 hours. The solvent was removed
under reduced pressure, and the obtained residue was
successively purified by ion exchange column
chromatography [eluant; 0.10 mol/L triethylammonium
bicarbonate solution] and reverse column chromatography
[eluant; water]. 0.90 mL of methanol was added to the
obtained solid, and 4.5 mL of an acetone solution
containing 0.17 g of sodium perchlorate was added
thereto. The precipitate was centrifuged and then
washed with acetone, so as to obtain 60 mg of sodium
salts of a light yellow solid, [ [2R, 3S, 4R, 5R]-5-[3-
(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl] methyl triphosphate.
IR(KBr)cm-1: 3422,1686,1252,1108
1H-NMR(D20)d: 4 . 3-4 . 5 (5H, m) , 6.09(1H,s),
8.41(1H,d,J=5.1Hz)
0.12 g of 6-chloro-4-[(2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxamide was suspended in 1.2
mL of trimethyl phosphate, and 38 µL of phosphorus
oxychloride was added thereto under ice cooling,
followed by stirring at the same temperature for 1
hour. The reaction mixture was poured into 3.0 mL of a
dimethylformamide solution containing 0.30 mL of n-
tributylamine and 0.72 g of n-tributyl ammonium
pyrophosphate under ice cooling, and the obtained
solution was stirred at the same temperature for 5
minutes. 10 mL of a 0.1 mol/L triethyl ammonium
bicarbonate solution and 10 mL of water were
successively added to the reaction mixture, and the
obtained mixture was purified by ion exchange column
chromatography [eluant; 0.07 mol/L triethyl ammonium
bicarbonate solution] and reverse phase silica gel
column chromatography [eluant; water] , so as to obtain
41 mg of a solid consisting of triethyl ammonium salts
of {(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-5-chloro-2-
oxo-1(2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl}
methyl triphosphate. 41 mg of the obtained triethyl
ammonium salts of {(2R, 3S, 4R, 5R)-5-[3-
(aminocarbonyl)-5-chloro-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl} methyl triphosphate was
dissolved in 0.43 mL of methanol, and thereafter, 2.2
mL of an acetone solution containing 78 mg of sodium
perchlorate was added thereto. The obtained solid was
centrifuged and then washed with 2.2 mL of acetone, so
as to obtain 26 mg of sodium salts of a white solid,
{(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-5-chloro-2-oxo-
1(2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-
furanyl}methyl triphosphate.
IR(KBr)cm-1: 1700, 1654
1H-NMR(D20)d: 4 . 25-4 . 5 (5H, m) , 6.08(1H,s), 8.44(1H,s)
Reference Example 18
43 mg of diethyl 2-[(2R, 3S, 4R, 5R)-3,4-
dihydroxy-5-methoxytetrahydro-2-furanyl] ethyl
phosphonate that had been prepared by the method
described in Journal of Chemical Society Chemical
Communication (J. Chem. Soc, Chem. Commun.), pp. 40 to
41 (1989) and 82 µL of triethylamine were dissolved in
1 mL of dichloromethane. Thereafter, 0.21 mL of
benzoyl chloride and 10 mg of 4-dimethylaminopyridine
were successively added thereto, and the mixture was
stirred at room temperature for 1 hour. 0.80 g of
diethyl 2-[(2R, 3S, 4R, 5R)-3,4-dihydroxy-5-
methoxytetrahydro-2-furanyl] ethyl phosphonate was
treated in the same above manner. Thereafter, 10 mL of
water was added to the obtained reaction mixture, the
organic layers were separated, and the aqueous layer
was extracted twice with 20 mL of chloroform. The
organic layers were combined and then successively
washed with water and with a saturated saline solution.
Thereafter, it was dried with anhydrous magnesium
sulfate, and the solvent was removed under reduced
pressure. The obtained residue was purified by silica
gel column chromatography [eluant; chloroform], so as
to obtain 1.38 g of a colorless oil product, (2R, 3R,
4R, 5R)-4-(benzoyloxy)-5-[2-(diethoxyphosphoryl)ethyl]-
2-methoxytetrahydro-3-furanyl benzoate.
IR (neat) cm1-1: 1729
1H-NMR(CDCl3)d: 1. 3-1. 35 (6H,m) , 1. 8-2 .2 (4H,m) ,
3.46(3H,s), 4.0-4.2(4H,m), 4.3-4.45(1H,m), 5.10(1H,s),
5.52(1H,t,J=5.1Hz), 5.59(1H,d,J=5.1Hz),
7.33(2H,t,J=7.8Hz), 7.41(2H,t,J=7.8Hz) , 7.5-7.6(2H,m),
7.90(2H,d,J=7.3Hz), 7.99(2H,d,J=7.3Hz)
Reference Example 19
1.34 g of (2R, 3R, 4R, 5R)-4- (benzoyloxy)-5-
[2-(diethoxyphosphoryl)ethyl]-2-methoxytetrahydro-3-
furanyl benzoate and 1.30 mL of acetic anhydride were
dissolved in 20 mL of acetic acid, and thereafter, 0.13
mL of concentrated sulfuric acid was added thereto
under ice cooling. After raising the temperature, the
mixture was left at room temperature for 16 hours. The
obtained reaction mixture was poured into a mixed
solution of 50 mL of ethyl acetate, 50 mL of ice and
100 mL of a saturated sodium bicarbonate aqueous
solution. The organic layers were separated, and the
aqueous layer was extracted twice with 50 mL of ethyl
acetate. The organic layers were combined and then
washed with a saturated saline solution. Thereafter,
they were dried with anhydrous magnesium sulfate, and
the solvent was removed under reduced pressure. The
obtained residue was purified by silica gel column
chromatography [eluant; toluene : ethyl acetate = 1 :
1], so as to obtain 1.19 g of a colorless oil product,
(2S, 3R, 4R, 5R)-2-(acetyloxy)-4-(benzoyloxy)-5-[2-
(diethoxyphosphoryl)ethyl] tetrahydro-3-furanyl
benzoate.
IR (neat) cm-1: 1729
1H-NMR(CDCl3)d: 1. 3-1. 35 (6H,m) , 1. 8-2 .2 (4H,m) ,
2.11,2.16(3H,2s), 4.05-4.2(4H,m), 4.45-4.5(1H,m), 5.45-
5.7(2H,m), 6.37,6.62(1H,2d,J=1.0,3.9Hz), 7.3-7.5(4H,m),
7.5-7.6(2H,m), 7.85-7.9(2H,m), 7.95-8.05(2H,m)
50 mg of methyl 3-hydroxy-2-
pyrazinecarboxylate was suspended in 1.6 mL of
1,1,1,3,3,3-hexamethyldisilazane, and the solution was
heated under reflux for 1 hour under nitrogen
atmosphere. After standing to cool, the solvent was
removed under reduced pressure, and an acetonitrile
solution containing 0.17 g of (2S, 3R, 4R, 5R)-2-
(acetyloxy)-4-(benzoyloxy)-5-[2-
(diethoxyphosphoryl)ethyl]tetrahydro-3-furanyl benzoate
was added thereto. Thereafter, the solvent was removed
under reduced pressure. The obtained residue was
suspended in 2.00 mL of acetonitrile under nitrogen
atmosphere, and thereafter, 67 µL of tin (IV) chloride
was added thereto under ice cooling, followed by
leaving at room temperature for 24 hours. 300 mg of
methyl 3-hydroxy-2-pyrazinecarboxylate was treated in
the same above manner, and the obtained reaction
mixture was poured into a mixed solution of 50 mL of
ethyl acetate, 50 mL of ice and 100 mL of a saturated
sodium bicarbonate aqueous solution. The precipitate
was removed by filtration, the organic layers were
separated, and the aqueous layer was extracted with 50
mL of ethyl acetate. The organic layers were combined,
and the obtained organic layers were washed with a
saturated saline solution and then dried with anhydrous
magnesium sulfate. The solvent was then removed under
reduced pressure. The obtained residue was purified by
silica gel column chromatography [eluant; ethyl
acetate : methanol = 100 : 1], so as to obtain 0.76 g
of a colorless oil product, methyl 4-{(2R, 3R, 4R, 5R) -
3, 4-bis(benzoyloxy)-5-[2-
(diethoxyphosphoryl)ethyl]tetrahydro-2-furanyl}-3-oxo-
3,4-dihydro-2-pyrazinecarboxylate.
IR(neat) cm"1: 1734, 1670
1H-NMR(CDCl3)d: 1. 3-1. 35 ( 6H,m) , 1. 85-2 . 3 (4H,m) ,
3.97(3H,s), 4.05-4.2(4H,m), 4.45-4.55(1H,m),
5.65(1H,t,J=6.5Hz), 5.7 4(1H,dd,J=3.6,5.9Hz),
6.24(1H,d,J=3.6Hz), 7.3-7.4(4H,m), 7.5-7.6(4H,m), 7.85-
7.95(4H,m).
Reference Example 21
6-f luoro-3-hydroxy-2- [2-14C]
pyrazinecarboxamide (radiochemical purity: 99.0%) was
produced from diethyl [2-14C] malonate as a starting
material by known methods, methods equivalent thereto,
or a combined use thereof. Such a production method is
described in International Patent Publication
WO00/10569, for example.
140 mg of sodium salts of a white solid,
{(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl} methyl
triphosphate was obtained from 136 mg of 4-[(2R, 3R,
4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-2-
furanyl]-3-oxo-3,4-dihydro-2-pyrazinecarboxamide in the
same manner as in Reference Example 17.
1H-NMR(D20)d: 4 . 30-4 . 39 (4H,m) , 4 . 45-4 . 48 (1H,m) ,
6.14(1H,s), 7.86(1H,d,J=3.6Hz), 8.34(1H,d,J=3.6Hz)

0.06 g of sodium salts of a white solid,
{(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-5-methyl-2-oxo-
1(2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl}
methyl triphosphate was obtained from 0.1 g of 4-[(2R,
3R, 4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
2-furanyl]-6-methyl-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide in the same manner as in Reference
Example 17.
IR(KBr)cm-1: 1684,1654
1H-NMR(D20)d: 2.44(3H,s), 4 . 31-4 . 47 ( 5H, m) , 6.12(1H,s),
8.20(1H,s)
Reference Example 24
0.02 g of sodium salts of a yellow solid,
{(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-5-phenyl-
1(2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl}
methyl triphosphate was obtained from 0.06 g of 4-[(2R,
3R, 4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
2-furanyl]-3-oxo-6-phenyl-3,4-dihydro-2-
pyrazinecarboxamide in the same manner as in Reference
Example 17.
IR(KBr)cm"1: 1684,1654
1H-NMR(D20)d: 4.07-4.41(5H,m) , 6.22(1H,s), 7.47-
7.60(3H,m), 7.99(2H,d,J=7.8Hz), 8.58(1H,s)
146 mg of sodium salts of a white solid,
{(2S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl]-4-hydroxytetrahydro-2-furanyl} methyl
triphosphate was obtained from 128 mg of 4-[(2R, 3R,
5S)-3-hydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-
oxo-3,4-dihydro-2-pyrazinecarboxamide in the same
manner as in Reference Example 17.
1H-NMR (D2O)d: 2 . 04-2 . 18 (2H,m) , 4 . 23-4 . 29 (1H,m) , 4.50-
4.58(2H,m), 4.78-4.88(1H,m), 6.03(1H,s),
7.86(1H,d,J=3.8Hz), 8.41(1H,d,J=3.8Hz)
Reference Example 2 6

0.9 g of methyl 4-[(2R, 3R, AS, 5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxylate was suspended in 9 mL
of N,N-dimethylacetamide. 2.3 mL of benzaldehyde
dimethylacetal and 160 mg of pyridinium-p-
toluenesulfonate were added thereto, and the mixture
was stirred at 65°C for 7 hours. Subsequently, the
reaction solution was poured into a mixed solution of
10 mL of ethyl acetate and 5 mL of water. The
deposited solid was collected by filtration, so as to
obtain 0.24 g of a white solid, methyl 4-[(3aR, 4R, 6R,
6aR)-6-(hydroxymethyl)-2-phenyltetrahydrofuro[3,4-
d][l,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate. Thereafter, the filtrate was
separated, and the obtained organic layer was
successively washed with 5 mL of water and 5 mL of a
saturated sodium chloride aqueous solution, and then
dried with anhydrous magnesium sulfate. The solvent
was removed under reduced pressure, and the residue was
washed with ethyl acetate, so as to obtain 0.40 g of a
white solid, methyl 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2-phenyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate.
IR(KBr)cm-1: 3440, 1731
1H-NMR(DMSO-d6)d: 3 . 61-3 . 71 (2H,m) , 3.83(3H,s), 4.50-
4.53(1H,m), 4.86(1H,dd,J=2.2, 6.6Hz), 5.01(1H,dd,J=2.0,
6.3Hz), 5.23(1H,t,J=4.9Hz), 5.95(1H,s),
6.07(1H,d,J=2.0Hz), 7.4 5-7.47(3H,m),
7.49(1H,d,J=4.4Hz), 7.54-7.57(2H,m), 8.09(1H,d,J=4.4Hz)
0.30 g of methyl 3-hydroxy-2-
pyrazinecarboxylate was dissolved in 1.5 mL of dimethyl
sulfoxide, and thereafter, 0.70 mL of triethylamine and
0.54 g of L-aspartic acid diethyl ester hydrochloride
were successively added thereto, followed by stirring
at room temperature for 8 hours. After chloroform and
water were added to the reaction solution, the mixture
was adjusted to pH 2 with 2 mol/L hydrochloric acid,
and the organic layer was separated. The obtained
organic layer was washed with water and then dried with
anhydrous magnesium sulfate, and the solvent was
removed under reduced pressure. Toluene and n-hexane
were added to the obtained residue, and the precipitate
was collected by filtration, so as to obtain 0.18 g of
diethyl (2S)-2-{[(3-hydroxy-2-pyrazinyl)carbonyl]amino}
butanedioate.
1H-NMR(CDCl3)d: 1. 27 (3H, t, J=7 . 2Hz) , 1.30(3H,t,J=7Hz),
2.94(1H,dd,J=4.4,17.2Hz), 3.15(1H,dd,J=4.8,17.2Hz),
4.14-4.23(2H,m), 4.24-4.32(2H,m), 4.99-5.02(1H,m),
8.15(1H,d,J=2.6Hz), 8.4 0(1H,d,J=l.5Hz),
8.78(1H,d,J=5.9Hz), 12.4(1H,brs)

2.0 g of 3-oxo-3,4-dihydro-2-
pyrazinecarbonitrile was dissolved in 20 mL of
methanol, and hydrogen chloride gas was then introduced
therein under ice cooling for saturation. After
stirring at the same temperature for 6 hours, ethyl
acetate was added thereto, and the precipitate was
collected by filtration, so as to obtain 2.3 g of a
yellow solid, methyl 3-oxo-3,4-dihydro-2-pyrazine
carboximidate hydrochloride.
1H-NMR(DMSO-d6)d: 4.27 (3H,s), 7.88(1H,d,J=3.4Hz),
7.91 (1H,brs), 8.07(1H,brs), 8.15(1H,d,J=3.4Hz),
8.71(1H,brs)

0.40 g of methyl 3-oxo-3,4-dihydro-2-pyrazine
carboximidate hydrochloride was dissolved in 4 mL of a
25% ammonia water, followed by stirring at room
temperature for 2 hours. Methanol was added thereto,
and the deposit was collected by filtration, so as to
obtain 0.21 g of a light yellow solid, 3-oxo-3,4-
dihydro-2-pyrazine carboximidamide.
1H-NMR(DMSO-d6,D20)d: 7.60(1H,s), 8.19(1H,s)
Reference Example 30
0.2 g of 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-dihydro-
2-pyrazinecarboxamide was suspended in 4 mL of
acetonitrile, and thereafter, 0.2 mL of diphosphoryl
chloride was added thereto under ice cooling, followed
by stirring at the same temperature for 20 minutes.
The reaction solution was adjusted to pH 7 with a 1
mol/L triethyl ammonium bicarbonate solution, and it
was then concentrated under reduced pressure. The
obtained residue was purified by reverse phase silica
gel column chromatography [eluant; water], so as to
obtain 0.29 of triethyl ammonium salts of a solid,
{(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl} methyl
phosphate.
1H-NMR(D20)d: 1. 28 ( 9H, t, J=7 . 3Hz) , 3 . 20 ( 6H, q, J=7 . 3Hz) ,
4.15-4.20(1H,m), 4.28-4.40(4H,m), 6.11(1H,d,J=2.0Hz),
7.8 0(1H,d,J=4.2Hz), 8.34(1H,d,J=4.2Hz)
7 ml of an acetone solution containing 0.35 g
of sodium perchlorate was added to 1.4 mL of a methanol
suspension containing 0.28 g of the above triethyl
ammonium salts of monophosphoric acid at room
temperature, and the mixture was stirred at the same
temperature for 1 hour. The deposit was collected by
filtration, and it was then washed with acetone, so as
to obtain 0.19 g of sodium salts of a white solid,
{ (2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl} methyl
phosphate.
IR(KBr)cm_1: 1662
1H-NMR(D2O)d: 4.15-4.19(1H,m) , 4 . 29-4 . 38 (4H,m) ,
6.12(1H,s), 7.80(1H,d,J=3.8Hz), 8.35(1H,d,J=3.8Hz)

0.11 g of 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-
5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-
dihydro-2-pyrazinecarboxamide was suspended in 2.0 mL
of trimethyl phosphate, and thereafter, 0.11 mL of
phosphorus oxychloride was added thereto under ice
cooling, followed by stirring at the same temperature
for 2 hours. 6.0 mL of a dimethylformamide solution
containing 1.2 mL of tributylamine and 1.12 g of
tributyl ammonium phosphate was added to the reaction
mixture, and the obtained mixture was stirred at the
same temperature for 1 hour. A 0.1 mol/L triethyl
ammonium bicarbonate solution was added to the reaction
mixture, and the obtained mixture was left at room
temperature for 12 hours. The solvent was removed
under reduced pressure. The obtained residue was
purified by ion exchange column chromatography [eluant;
0.07 mol/L triethyl ammonium bicarbonate solution], to
collect both fractions containing triethyl ammonium
salts of {(2R, 3S, 4R, 5R)-5-[3-( aminocarbonyl)-2-oxo-
1(2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl}
methyl diphosphate, and fractions containing triethyl
ammonium salts of {(2R, 3S, 4R, 5R)-5-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl} methyl triphosphate, and
as a result, 143 mg of a solid and 113 mg of another
solid were obtained. 110 mg out of 143 mg of the
obtained triethyl ammonium salts of {(2R, 3S, 4R, 5R)-
5-[3-(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl} methyl diphosphate was
dissolved in 3.0 mL of methanol, and thereafter, 7.5 mL
of an acetone solution containing 0.28 g of sodium
perchlorate was added thereto. The solid was
centrifuged and then washed with acetone, to obtain 64
mg of sodium salts of a white solid, {(2R, 3S, 4R, 5R)-
5-[3-(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl} methyl diphosphate.
IR(KBr)cm-1: 3418, 1682, 1236, 983, 905
1H-NMR(D20)d: 4.2-4 . 5 (5H,m) , 6.12(1H,s),
7.83(1H,d,J=3.7Hz), 8.35(1H,d,J=3.7Hz)
Example 1
65 mg of 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3, 4-
d][1,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide, 44 mg of IH-tetrazole, and 10 mg
of molecular sieves 4A were suspended in 2.0 mL of
acetonitrile. 3.0 mL of an acetonitrile solution
containing 0.16 g of bis(S-pivaloyl-2-thioethyl)-N,N-
diisopropyl phosphoramidite, which had been separately
prepared by the method described in Journal of
Medicinal Chemistry (J.Med.Chem.) Vol. 38, No. 20, pp.
3941 to 3950 (1995), was added thereto in portions
under ice cooling, and the obtained mixture was stirred
at the same temperature for 4 0 minutes. 1.0 mL of an
acetonitrile solution containing 78 mg of m-
chloroperbenzoic acid was added to the reaction
mixture, and the obtained mixture was further stirred
at the same temperature for 10 minutes. 10 mL of ethyl
acetate and 10 mL of water were added to the reaction
mixture. The organic layers were separated, and the
aqueous layer was extracted with 20 mL of ethyl
acetate. All the organic layers were combined, and the
combined layer was washed with a saturated sodium
bicarbonate aqueous solution and then dried with
anhydrous magnesium sulfate. The solvent was then
removed under reduced pressure. The obtained residue
was purified by silica gel column chromatography
[eluant; chloroform : methanol =50 : 1], to obtain 68
mg of a yellow oil product, 2,2-dimethyl-thiopropionic
acid S-(2-{[(3aR, 4R, 6R, 6aR)-6-(3-carbamoyl-2-oxo-2H-
pyrazine-1-yl)-2,2-dimethyltetrahydrofuro[3,4-
d] [1,3]dioxol-4-ylmethoxy]-[2-(2,2-dimethyl-
propionylsulfanyl)-ethoxy]-phosphoryloxy}-ethyl)-ester.
IR (neat) cm"1: 1684
1H-NMR(CDCl3)d: 1.22(9H,s), l,23(9H,s), 1.40(3H,s),
1.65(3H,s), 3.08(2H,t,J=,6.9Hz), 3.10(2H,t,J=6.8Hz),
4.04-4.12(4H,m), 4.29-4.36(1H,m), 4.39-4.45(1H,m),
4.59-4.64(1H,m), 4.86-4.88(2H,m), 6.02(1H,brs),
6.05(1Hrd,J=1.5Hz), 7.8 4(1H,d,J=4.3Hz),
7.87(1H,d,J=4.3Hz), 9.17(1H,brs)
Example 2

60 mg of 2,2-dimethyl-thiopropionic acid S-
(2-{[(3aR, 4R, 6R, 6aR)-6-(3-carbamoyl-2-oxo-2H-
pyrazine-l-yl)'-2, 2-dimethyl-tetrahydrofuro [3, 4-
d][1,3]dioxol-4-ylmethoxy]-[2-(2,2-dimethyl-
propionylsulfanyl)-ethoxy]-phosphoryloxy}-ethyl)-ester
was dissolved in a mixed solvent of 2.4 mL of water and
2.4 mL of methanol, and thereafter, 1.2 g of a Dowex
50WX4-200 ion exchange resin (H+form) was added thereto
in portions, and the mixture was stirred at room
temperature for 3 hours. The reaction mixture was
filtrated, the removed resin was washed with methanol,
and the filtrate and the washings were combined. The
organic solvent was then removed under reduced
pressure. The deposit was collected by filtration, to
obtain 36 mg of a white solid, 2,2-dimethyl-
thiopropionic acid S-(2-[[(2R, 3S, 4R, 5R)-5-(3-
carbamoyl-2-oxo-2H-pyrazine-l-yl)-3,4-dihydroxy-
tetrahydro-furan-2-ylmethoxy]-[2-(2,2-dimethyl-
propionylsulfanyl)-ethoxy]-phosphoryloxy]-ethyl) ester.
IR(KBr)cm1: 1677, 1660
1H-NMR(CDCl3)d: 1.22(9H,s), l,23(9H,s),
3.11(4H,t,J=7.0Hz), 3.67(1H,brs), 4.05-4.15(4H,m),
4.25-4.38(3H,m), 4.44-4.52(2H,m), 4.67(1H,brs),
6.04(1H,d,J=3.7Hz), 6.10(1H,brs), 7.90(1H,d,J=4.1Hz),
8.08(1H,d,J=4.1Hz), 8.95(1H,brs)
12 mg of a yellow oil product, 2,2-dimethyl-
thiopropionic acid S-(2-{[(3aR, 4R, 6R, 6aR)-6-(3-
carbamoyl-5-chloro-2-oxo-2H-pyrazine-l-yl)-2,2-
dimethyl-tetrahydrofuro f3,4-d] [1,3]dioxol-4-ylmethoxy]-
[2-(2,2-dimethyl-propionylsulfanyl)-ethoxy]-
phosphoryloxy}-ethyl) ester was obtained from 40 mg of
4-[(3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-6-
chloro-3-oxo-3,4-dihydro-2-pyrazinecarboxamide in the
same manner as in Example 1.
IR (neat) cm"1: 1687
1H-NMR(CDCl3)d: 1.21(9H,s), l,22(9H,s), 1.40(3H,s),
1.64(3H,s), 3.07(2H,dt,J=7.0,1.5Hz),
3.14(2H,t, J=6.8Hz), 4.06-4.16(4H,m), 4.34-4.39(1H,m),
4.42-4.47(1H,m), 4.63-4.65(1H,m),
4.85(1H,dd,J=6.4,2.2Hz), 4.89(1H,dd,J=6.2,2.8Hz),
6.04(1H,d,J=2.2Hz), 6.17(1H,brs), 7.98(1H,s),
9.07(1H,brs)

7 mg of a yellow oil product, 2,2-dimethyl-
thiopropionic acid S-(2-{[(2R, 3S, 4R, 5R)-5-(3-
carbamoyl-5-chloro-2-oxo-2H-pyrazine-l-yl)-3,4-
dihydroxy-tetrahydro-furan-2-ylmethoxy]- [2- (2, 2-
dimethyl-propionylsulfanyl)-ethoxy]-phosphoryloxy}-
ethyl) ester was obtained from 12 mg of 2,2-dimethyl-
thiopropionic acid S-(2-{[(3aR, 4R, 6R, 6aR)-6-(3-
carbamoyl-5-chloro-2-oxo-2H-pyrazine-l-yl)-2,2-
dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-ylmethoxy]-
[2-(2,2-dimethyl-propionylsulfanyl)-ethoxy]-
phosphoryloxy}-ethyl) ester in the same manner as in
Example 2.
IR(neat)cm"1: 1686, 1654
1H-NMR(CDCl3)d: 1.21(9H,s), l,23(9H,s), 3.00 (1H,brs) ,
3.10-3.16(4H,m), 3.83(1H,brs), 4.05-4.20(4H,m), 4.25-
4.55(5H,m), 6.02(1H,d,J=2.4Hz), 6.40(1H,brs),
8.15(1H,s), 8.81(1H,brs)
0.05 g of 4-[(3aR, 4R, 6S, 6aR)-6-
(iodomethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][l,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide was suspended in 2 mL of toluene.
70 mg of silver salts of bis(2,2-dimethyl-
propionyloxymethyl) phosphate, which had been
separately prepared by the method described in Journal-
of Medicinal Chemistry (J. Med. Chem.) Vol. 37, pp.
3902 to 3909 (1994), was added thereto, and the mixture
was stirred at 60°C for 2 hours. 0.15 g of 4-[(3aR, 4R,
6S, 6aR)-6-(iodomethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][1.3]dioxol-4-yl]-3-OXO-3,4-dihydro-2-
pyrazinecarboxamide was treated in the same above
manner. The obtained reaction mixtures were combined,
and the solvent was removed under reduced pressure.
The obtained residue was purified by silica gel column
chromatography [eluant; chloroform : methanol = 10 :
1], to obtain 0.12 g of a yellow solid, 2,2-dimethyl-
propionic acid [(3aR, 4R, 6R, 6aR)-6-(3-carbamoyl-2-
oxo-2H-pyrazine-l-yl)-2,2-dimethyl-tetrahydrofuro[3,4-
d][1,3]dioxol-4-ylmethoxy]-(2,2-dimethyl-
propionyloxymethoxy)-phosphoryloxymethyl ester.
IR(KBr)cm-1: 1750, 1684, 1654
1H-NMR(DMSO-d5)d: 1.14(18H,s), 1.29(3H,s), 1.51(3H,s),
3.5-3.7(2H,m), 4.34(1H,dd,J=4.0,7.2Hz),
4.75(1H,dd,J=2.8,6.0Hz), 4.86(1H,dd,J=2.0,6.0Hz),
5.23(1H,t,J=4.8Hz), 5.31(2H,s), 5.34(2H,s),
5.97(1H,d,J=2.0Hz), 7.56 (1H,d,J=4.4Hz), 7.83(1H,brs),
8.06(1H,d,J=4.4Hz) , 8.43{1H,brs)
Example 6
0.1 g of 2,2-dimethyl-propionic acid[(3aR,
4R, 6R, 6aR)-6-(3-carbamoyl-2-oxo-2H-pyrazine-l-yl)-
2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-
ylmethoxy]-{2,2-dimethyl-propionyloxymethoxy)-
phosphoryloxymethyl ester was dissolved in a mixed
solution of 1.5 mL of methanol and 1.5 mL of water, and
thereafter, 5 mL of a Dowex 50WX4-200 ion exchange
resin (H+form) was added thereto, followed by stirring
at room temperature for 1.5 hours. Thereafter, the
resin was removed by filtration and then washed with a
mixed solution of 2.5 mL of acetonitrile and 2.5 mL of
water. The obtained washings and the filtrate were
combined, and the organic solvent was removed under
reduced pressure. The obtained residue was
lyophilized, to obtain 0.07 g of a light yellow solid,
2,2-dimethyl-propionic acid[(2R, 3S, 4R, 5R)-5-(3-
carbamoyl-2-oxo-2H-pyrazine-l-yl)-3,4-dihydroxy-
tetrahydro-furan-2-ylmethoxy]-(2,2-dimethyl-
propionyloxymethoxy)-phosphoryloxymethyl ester.
IR(KBr)cm_1: 1751, 1670
1H-NMR(DMSO-d6)d: 1.16(18H,s), 3 . 64(1H, dd,J=2.0,12.4Hz),
3.81(1H,dd,J=2.0,12.4Hz), 3.9-4.0(3H,m), 5.46(2H,s),
5.49(2H,s), 5.92(1H,d,J=2.4Hz), 7.54(1H,d,J=4.4Hz),
7.75 (1H,brs), 8.29(1H,d,J=4.4Hz), 8.35(1H,brs)
Example 7
0.50 g of 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][l,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide was suspended in 50 mL of pyridine.
Thereafter, 43 mL of a tetrahydrofuran solution
containing 2.2g of methyl chloro phenylphosphoryl P—»N-
L-alaninate, which had been separately prepared by the
method described in Antiviral Research, Vol. 43, pp. 37
to 53 (1999), and 1.3 mL of N-methylimidazole were
successively added to the suspension, and the mixture
was stirred at the same temperature for 3 hours. The
solvent was removed from the reaction mixture under
reduced pressure. The obtained residue was purified by
silica gel column chromatography [eluant; chloroform :
methanol =40 : 1], to obtain 0.43 g of a light yellow
solid, (2S)-[[(3aR, 4R, 6R, 6aR)-6-(3-carbamoyl-2-oxo-
2H-pyrazine-l-yl)-2,2-dimethyl-tetrahydrofuro[3,4-
d][1,3]dioxol-4-ylmethoxy]-phenoxy-phosphorylamino]-
propionic acid methyl ester.
IR(KBr)cm_1: 1749,1684
1H-NMR(DMSO-d6)d: 1.22, 1.23(3H,d,J=7.1Hz), 1.28,
1.30(3H,s), 1.50, 1.51(3H,s), 3.58,3.60(3H,s), 3.80-
3.88(1H,m), 4.15-4.34(2H,m), 4.42-4.45, 4.50-
4.54(1H,m), 4.74, 4.81(1H,dd,J=2.0,6.3Hz), 4.67,
4.93(1H,dd,J=3.0,6.1Hz), 5.94-5.97(1H,m), 6.09-
6.15(1H,m), 7.08-7.20(3H,m), 7.32-7.39(2H,m), 7.46,
7.51(1H,d,J=4.0Hz) , 7.75-7.80(1H, brs), 7.84-7.87(1H,m),
8.26-8.32(1H,m)
Example 8
49 mg of a white solid, (2S)-{[(2R, 3S, 4R,
5R)-5-(3-carbamoyl-2-oxo-2H-pyrazine-l-yl)-3,4-
dihydroxy-tetrahydro-furan-2-ylmethoxy]-phenoxy-
phosphorylaraino}-propionic acid methyl ester was
obtained from 190 mg of (2S)-{[(3aR, 4R, 6R, 6aR)-6-(3-
carbamoyl-2-oxo-2H-pyrazine-l-yl)-2,2-dimethyl-
tetrahydrofuro[3,4-d][1,3]dioxol-4-ylmethoxy]-phenoxy-
phosphorylamino}-propionic acid methyl ester in the
same manner as in Example 2.
IR(KBr)cm_1: 1735, 1676, 1661
1H-NMR(DMSO-d6)d: 1.21, 1,23(3H,d,J=7.2Hz),
3.57,3.58(3H,s), 3.81-3.98(2H,m), 4.02-4.04(1H,m),
4.12-4.41(3H,m), 5.60(2H,brs), 5.91-5.94(1H,m), 6.10-
6.20(1H,m), 7.15-7.24(3H,m), 7.34-7.45(3H,m),
7.73(1H,brs), 7.81,7.86(1H,d,J=4.2Hz), 8.29(1H,brs)
Example 9

0.20 g of methyl 4-[(2R, 3R, 4R, 5R)-3,4-
bis(benzoyloxy)-5-[2-
(diethoxyphosphoryl)ethyl]tetrahydro-2-furanyl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxylate was dissolved in 2.0
mL of acetonitrile, and thereafter, 0.33 mL of
bromotrimethyl silane was added thereto under ice
cooling, followed by stirring at 0°C for 30 minutes, and
then at room temperature for 1 hour. The solvent was
removed from the reaction mixture under reduced
pressure. 2.0 mL of methanol was added to the obtained
solid and dissolved, and thereafter, ammonia gas was
blown therein under ice cooling for saturation,
followed by stirring at room temperature for 3 hours.
The solvent was removed from the reaction mixture under
reduced pressure, 10 mL of water was added, and the
mixture was washed with chloroform. After insoluble
products were filtrated, the solvent was removed under
reduced pressure. The obtained residue was purified by
reverse phase silica gel column chromatography [eluant;
water], to obtain 28 mg of a light yellow solid, 2-
[(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl] ethyl
phosphoric acid.
IR(KBr)cm"1: 167 6
1H-NMR(D2O)d: 1. 7-1. 95 (2H, m) , 2 . 0-2 . 2 (2H,m) , 3.95-
4.0(1H,m), 4.2-4.25(1H,m), 4.33(1H,d,J=4.6Hz),
6.06(1H,s), 7.79(1H,d,J=4.0Hz), 8.03(1H,d,J=4.0Hz)
Example 10

1.2 g of a yellow oil product, [(2R, 3R, 4R,
5R)-5-[3-(aminocarbonyl)-5-methyl-2-oxo-l(2H)-
pyrazinyl]-3,4-bis(benzoyloxy)tetrahydro-2-furanyl]
methyl benzoate was obtained from 0.43 g of 3-hydroxy-
6-methyl-2-pyrazinecarboxamide in the same manner as in
Reference Example 6.
IR(KBr)cm-1: 1727, 1686, 1654
1H-NMR(CDCl3)d: 2.15(3H,s), 4.67(1H,dd,J=3.4, 12.7Hz),
4.85-4.88(1H,m), 4.99(1H,dd,J=2.4,12.7Hz), 5.88-
5.95(2H,m), 6.03(1H, d, J=3.2Hz) , 6.47(1H,d,J=3.9Hz),
7.36-7.65{10H,m), 7.93-8.00(4H,m), 8.10-8.13(2H,m),
9.12(1H,d,J=3.9Hz)

0.4 g of a light yellow solid, 4-[(2R, 3R,
4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-2-
furanyl]-6-methyl-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide was obtained from 1.05 g of [(2R,
3R, 4R, 5R)-5-[3-(aminocarbonyl)-5-methyl-2-oxo-l(2H)-
pyrazinyl]-3,4-bis(benzoyloxy)tetrahydro-2-furanyl]
methyl benzoate in the same manner as in Reference
Example 7.
IR(KBr)cm: 1698, 1654
1H-NMR(DMSO-d6)d: 2.24(3H,s), 3 . 62-3 . 67 (1H,m) , 3.80-
3.85(1H,m), 3.94-4.01(3H,m), 5.10(1H,d,J=5.1Hz),
5.32(1H,t,J=4.8Hz), 5.62(1H,d,J=3.2Hz),
5.92(1H,d,J=1.7Hz), 7.76(1H,brs), 8.16(1H,s),
8.48(1H,brs)
0.5 g of a yellow oil product, [(2R, 3R, 4R,
5R)-5-[3-(aminocarbonyl)-2-oxo-5-phenyl-l(2H)-
pyrazinyl]-3,4-bis(benzoyloxy)tetrahydro-2-furanyl]
methyl benzoate was obtained from 0.35 g of 3-hydroxy-
6-phenyl-2-pyrazinecarboxamide in the same manner as in
Reference Example 6.
IR(KBr)cm~1: 1720, 1717, 1684, 1654
1H-NMR(CDCl3)d: 4.77(1H,dd,J=3.6, 12.4Hz), 4.90-
4.97(2H,m), 5.93-5.99(2H,m), 6.03(1H,d,J=3.7Hz),
6.58(1H,d,J=4.2Hz), 7.32-7.68(14H,m), 7.95-8.05{6H,m),
8.11(1H,s), 8.93{1H,d,J=3.4Hz)

0.12 g of a yellow solid, 4-[(2R, 3R, 4S,
5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-2-
furanyl]-3-oxo-6-phenyl-3,4-dihydro-2-
pyrazinecarboxamide was obtained from 0.48 g of [(2R,
3R, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-5-phenyl-l(2H)-
pyrazinyl]-3,4-bis(benzoyloxy)tetrahydro-2-furanyl]
methyl benzoate in the same manner as in Reference
Example 7.
IR(KBr)cm_1: 1685, 1670, 1654
1H-NMR(DMSO-d6)d: 3 . 71-3.75(1H,m), 3.93-3.97(1H,m) ,
4.02-4.15(3H,m), 5.10(1H,d,J=6.6Hz),
5.67(1H,t, J=4.0Hz) , 5.73(1H,d,J=4.6Hz) , 5.96(1H,s),
7.32-7.46(3H,m), 7.82(1H,brs), 7.88(2H, d, J=7.8Hz) ,
8.43(1H,brs), 9.07(1H,s)

0.24 g of 3-hydroxy-2-pyrazinecarboxamide was
suspended in 5 mL of 1,1,1-3,3,3-hexamethyldisilazane,
and the suspension was under reflux for 2 hours. After
cooling, the solvent was removed under reduced
pressure. 5 mL of xylene was added, and the solvent
was removed under reduced pressure. To the obtained
residue, 12.5 mL of acetonitrile, 0.50 g of [(2S,4R)-
4,5-bis(acetyloxy)tetrahydro-2-furanyl] methyl
benzoate, and 0.29 mL of tin(IV) chloride were
successively added, and the mixture was then stirred at
room temperature for 30 minutes. Water was added to
the reaction solution, and the organic solvent was
removed under reduced pressure. Thereafter, a
saturated sodium bicarbonate aqueous solution and ethyl
acetate were added to the residue, and insoluble
products were removed by filtration. The organic layer
was separated, and the aqueous layer was extracted with
ethyl acetate. The obtained organic layer was dried
with anhydrous magnesium sulfate, and the solvent was
removed under reduced pressure. The obtained residue
was purified by silica gel chromatography [eluant;
chloroform : methanol =16 : 1], to obtain 0.61 g of a
light yellow solid, { (2S, 4R, 5R)-4-(acetyloxy)-5- [3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]tetrahydro-2-
furanyl} methyl benzoate.
IR(KBr)cm"1: 1743, 1706, 1667
1H-NMR(CDCl3)d: 2.13-2.23(5H,m), 4.67(1H,dd,J=4.0,
12.8Hz), 4.78-4.85(2H,m), 5.48(1H,d,J=4.0Hz),
6.05(1H,s), 6.61(1H,brs), 7.47-7.51(2H,m),
7.58(1H,d,J=4.0Hz), 7.62-7.66(1H,m),
7.96(1H,d,J=4.0Hz), 8.02-8.04(2H,m), 9.06(1H,brs)
Example 15
1.21 g of { (2S, 4R, 5R)~4-(acetyloxy)-5-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]tetrahydro-2-
furanyl} methyl benzoate was dissolved in 36 mL of
methanol, and thereafter, 1.28 g of a 28% sodium
methoxide methanol solution was added thereto under ice
cooling, followed by stirring at the same temperature
for 1 hour. The reaction solution was adjusted to pH 5
by adding 1 mol/L hydrochloric acid, and the solvent
was then removed under reduced pressure. The obtained
residue was purified by silica gel chromatography
[eluant; n-hexane : ethyl acetate =4 : 1], and a mixed
solvent of 2-propanol and ethyl acetate was added
thereto. The precipitate was collected by filtration,
to obtain 0.47 g of a white solid, 4-[(2R, 3R, 5S)-3-
hydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxamide.
IR(KBr)cm"1: 1682, 1654
1H-NMR(DMSO-d6)d: 1. 72 (1H, dd, J=5 . 2, 13. 2Hz) , 1.86-
1.93(1H,m), 3.61-3.66(1H,m), 3. 88-3.93(1H,m),
4.25(1H,t,J=4.0Hz), 4.43-4.48(1H,m),
5.28(1H,t,J=5.0Hz), 5.77(1H,d,J=4.0Hz), 5.82(1H,s),
7.56(1H,d,J=4.4Hz), 7.76(1H,brs), 8.37(1H,d,J=4.4Hz),
8.42(1H,brs)
0.5 g of 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3, 4-
d] [1, 3]dioxol-4-yl]-3-OXO-3,4-dihydro-2-
pyrazinecarboxamide was dissolved in 5 mL of N,N-
dimethylformamide, and thereafter, 0.29 g of 1,1'-
carbonyldiimidazole was added thereto, followed by
stirring at room temperature for 1 hour. Thereafter,
0.21 mL of 1,4-butanediol was added thereto, and the
mixture was stirred at room temperature for 1 hour, and
then at 60°C for 1 hour. 0.29 mL of 1,4-butanediol was
further added thereto, and the mixture was stirred at
60°C for 3 hours. The reaction solution was
concentrated under reduced pressure, and then, 30 mL of
ethyl acetate, 30 mL of water, and 5 mL of a saturated
saline solution were added to the concentrate. The
organic layers were separated, and the aqueous layer
was then extracted with 20 mL of ethyl acetate. The
organic layers were combined and then dried with
anhydrous magnesium sulfate, and the solvent was
removed under reduced pressure. The obtained residue
was purified by silica gel chromatography [eluant;
ethyl acetate : methanol = 10 : 1], to obtain 0.31 g of
a light yellow oil product, {(3aR, 4R, 6R, 6aR)-6-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-2, 2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl} methyl
4-hydroxybutyl carbonate.
IR(KBr)cm-1: 1750, 1684, 1654
1H-NMR(CDCl3)d: 1.41(3H,s), 1. 47-1. 54 (2H, m) , 1.62-
1.68(5H,m), 3.60-3.66(2H,m), 4.04-4.16(2H,m),
4.32(1H,dd,J=3.2, 12.4Hz), 4.48(1H,dd,J=2.2, 12.2Hz),
4.73-4.76(1H,m), 4.79(1H,dd,J=2.4, 6.0Hz),
4.92 (1H,dd, J=1.8, 6.2Hz), 5.98(1H,d,J=2.0Hz) ,
6.31(1H,d,J=23.6Hz) , 7.77(1H,s), 7.75(1H,s),
8.78(1H,br), 9.25(1H,brs)
Example 17
1.2 g of {(3aR, 4R, 6R, 6aR)-6-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-2,2-dimethyl-
tetrahydrofuro[3,4-d][1,3]dioxol-4-yl} methyl 4-
hydroxybutyl carbonate was dissolved in a mixed solvent
of 8 mL of methanol and 60 mL of water, and thereafter,
19 g of a Dowex 50WX4-200 ion exchange resin (H+form)
was added thereto, followed by stirring at room
temperature for 3 hours. Thereafter, the resin was
removed by filtration, and it was washed with methanol
and then with water. The obtained filtrate and the
washings were combined, and the solvent was removed
under reduced pressure. The residue was dissolved in
water, and the solution was then lyophilized. The
obtained solid was washed with acetonitrile and then
with chloroform, to obtain 0.16 g of a colorless solid,
{(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl} methyl 4-
hydroxybutyl carbonate.
IR(KBr)cm_1: 1745, 1664
1H-NMR(DMSO-d6)d: 1. 45-1. 51 (2H,m) , 1. 64-1. 67 (2H,m) ,
3.40-3.42(2H,m), 3.90(1H,d,J=4.8Hz), 4.08-4.15(4H,m),
4.36-4.47(3H,m) , 5.38(1H,d,J=5.2Hz), 5.74(1H,brs),
5.92(1H,s), 7.50(1H,d,J=3.6Hz), 7.75(1H,brs),
7.81(1H,d,J=3.6Hz), 8.30(1H,brs)
Example 18

200 mg of (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[(acetyloxy)methyl]-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl] tetrahydro-3-furanyl acetate was dissolved
in 2 mL of pyridine, and thereafter, 0.1 mL of
hydrazine monohydrate was added thereto, followed by
stirring at room temperature for 1 hour. Thereafter,
acetone was added thereto, and the solvent was then
removed at reduced pressure. The obtained residue was
purified by silica gel chromatography [eluant; ethyl
acetate : methanol =10 : 1], to obtain 42 mg of a
white solid, {{2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-
oxo-1 (2H)-pyrazinyl]-3,4-dihydroxytetrahydro-2-furanyl}
methyl acetate.
1H-NMR(DMSO-d6)d: 3.20(3H,s), 3 . 90 (1H,dd, J=6. 8, 11. 5) ,
4.0 6-4.0 9(1H,m), 4.12-4.17(1H,m),
4.31(1H,dd,J=5.6,12.7), 4.35(1H,dd,J=3.4,12.7),
5.33(1H,d, J=6.6) , 5.73,(1H,d,J=5.1), 5 . 90(1H,d,J=2 . 0) ,
7.57(1H,d,J=4.4), 7.75(1H,brs), 7.85(1H,d,J=4.4),
8.31{1H,brs)
Example 19
5 g of 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3, 4-dihydro-
2-pyrazinecarboxamide was suspended in 25 mL of acetic
anhydride and 12.5 mL of pyridine, and the suspension
was stirred at 50°C for 1 hour and then at 70°C for 1
hour. After cooling the suspension to room
temperature, insoluble products were removed by
filtration. The residue was concentrated under reduced
pressure, and the concentrate was then purified by
silica gel column chromatography [eluant; ethyl
acetate], to obtain 1.26 g of a light yellow solid,
(2R, 3R, 4R, 5R)-2-[3-[(acetylamino)carbonyl]-2-oxo-
1(2H)-pyrazinyl]-4-(acetyloxy)-5-[(acetyloxy)methyl]
tetrahydro-3-furanyl acetate.
IR(KBr)cm_1: 1750, 1709
1H-NMR(DMSO-d6)d: 2.06(6H,s), 2.09(3H,s), 2.17(3H,s),
4.26-4.42(3H,m), 5.35(1H,t,J=6.1Hz),
5.50(1H,dd,J=3.7,6.1Hz), 6. 06(1H,d,J=3.4Hz),
7.50(1H,d,J=4.4Hz) , 7.83(1H,d,J=4.4Hz), 11.44(1H,s)
Example 20
0.2 g of 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-
d] [1,3]dioxol-4-yl]-3-OXO-3,4-dihydro-2-
pyrazinecarboxamide was suspended in 1 mL of acetic
anhydride and 4 mL of pyridine, and the suspension was
stirred at room temperature for 1 hour. The reaction
solution was concentrated under reduced pressure, 5 mL
of water was added to the residue, and the mixture was
extracted with 5 mL of ethyl acetate 10 times. The
organic layers were combined, 1 mL of water was added
thereto, and the obtained solution was adjusted to pH 3
with 2 mol/L hydrochloric acid. The organic layer was
separated, washed with a saturated sodium chloride
aqueous solution, and then dried with anhydrous
magnesium sulfate. Then, the solvent was removed under
reduced pressure. The obtained residue was purified by
silica gel column chromatography [eluant; chloroform :
methanol = 10 : 1], to obtain 0.15 g of a light yellow
oil. product, { (3aR, 4R, 6R, 6aR) -6- [3- (aminocarbonyl) -
2-oxo-l(2H)-pyrazinyl]-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl} methyl acetate.
IR(KBr)cm-1: 1743, 1685
1H-NMR(CDCl3)d: 1.41(3H,s), 1.65(3H,s), 1.94(3H,s),
4.32(1H,dd,J=4.4,12.5Hz) , 4.40(1H, dd,J=2.9,12.5Hz) ,
4.68-4.71(1H,m), 4.76(1H,dd,J=2.9,6.1Hz),
4.87(1H,dd,J=2.1,6.2Hz) , 5.95(1H,d,J=2.0Hz),
6.17(1H,brs), 7.70(1H,d,J=4.2Hz), 7.80(1H,d,J=4.2Hz),
9.13(1H,brs)
Example 21
0.5 g of methyl 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2-phenyltetrahydrofuro [3,4-
d] [1,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxylate was suspended in 15 mL of a
methanol solution containing approximately 5 mol/L dry
ammonia, and the suspension was stirred at room
temperature for 3 hours. The deposit was collected by
filtration and then washed with methanol, to obtain
0.34 g of a colorless solid, 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2-phenyltetrahydrofuro[3, 4-
d][1,3]dioxol-4-yl]-3-OXO-3,4-dihydro-2-
pyrazinecarboxamide.
IR(KBr) cm"1: 1684
1H-NMR (DMSO-d6)d: 3 . 61-3 . 75 (2H,m) , 4 . 52-4.54(1H,m) ,
4.87(1H,dd,J=2.4,6.3Hz), 5.00(1H,dd,J=2.2,6.6Hz),
5.25(1H,t,J=4.9Hz), 5.96(1H,s), 6.10(1H,d,J=2.0Hz),
7.45-7.48(3H,m), 7.54-7.57(3H,m), 7.76(1H,brs),
8.06(1H,d,J=4.4Hz), 8.34(1H,brs)
Example 22

1.39 g of 3-hydroxy-2-pyrazinecarboxamide was
suspended in 10 mL of 1,1,1,3,3,3-hexamethyldisilazane,
and the suspension was under reflux for 1 hour. After
cooling, the solvent was removed under reduced
pressure. Then, 5 mL of toluene was added, and the
solvent was removed under reduced pressure. 15 mL of
acetonitrile, 4.14 g of |3-D-ribofuranose 1,2,3,5-
tetraacetate, and 1.99 mL of trimethylsilyl
trifluoromethane sulfonate were successively added to
the obtained residue, and the obtained mixture was
stirred at room temperature for 2 hours. Thereafter,
0.95 g of p-D-ribofuranose 1,2,3,5-tetraacetate was
added thereto, and the mixture was further stirred at
room temperature for 1 hour. The solvent was removed
under reduced pressure, and chloroform and water were
added. The organic layer was separated, and it was
washed with a saturated sodium bicarbonate aqueous
solution and then dried with anhydrous magnesium
sulfate. The solvent was removed under reduced
pressure. Ethyl acetate was added to the residue, and
the precipitate was collected by filtration, to obtain
1.73 g of (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[(acetyloxy)methyl]-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl] tetrahydro-3-furanyl acetate.
1H-NMR(CDCl3)d: 2.10(3H,s), 2.16(3H,s), 2.17(3H,s),
4.40-4.52(3H,m), 5.29(1H,t,J=6.2Hz),
5.4 8(1H,dd,J=3.2,5.1Hz) , 6.05(1H,brs),
6.16(1H,d,J=3.2Hz), 7.79(1H,d,J=4.2Hz),
7.81(1H,d,J=4.2Hz), 8.98(1H,brs),
0.40 g of (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[(acetyloxy)methyl]-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl] tetrahydro-3-furanyl acetate was suspended
in tetrahydrofuran, and thereafter, 0.06 g of sodium
hydride (60% mineral oil suspension) and 0.14 mL of
pivaloyl chloride were successively added to the
suspension under ice cooling. The mixture was stirred
at room temperature for 2 hour. Thereafter, 0.0 6 g of
sodium hydride (60% mineral oil suspension) and 0.14 mL
of pivaloyl chloride were further added thereto, and
the mixture was stirred at room temperature for 1 hour.
Further, 0.06 g of sodium hydride (60% mineral oil
suspension) and 0.14 mL of pivaloyl chloride were
further added thereto, and the mixture was stirred at
room temperature for 1 hour. Ethyl acetate and water
were added to the reaction solution. The organic layer
was separated, washed with water, and dried with
anhydrous magnesium sulfate. The solvent was removed
under reduced pressure, and the obtained residue was
purified by silica gel chromatography [eluant; n-
hexane : ethyl acetate =3 : 1], to obtain 0.14 g of a
yellow solid, (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[ (acetyloxy)methyl]-5-[3-{[(2,2-
dimethylpropanoyl)amino]carbonyl}-2-oxo-l(2H)-
pyrazinyl] tetrahydro-3-furanyl acetate.
1H-NMR(CDCl3) 5: 1.30(9H,s), 2.13(3H,s), 2.16(6H,s),
4.39-4.49(2H,m), 4.52-4.55(1H,m), 5.29(1H,t,J=5.9Hz),
5.4 8(1H,dd,J=3.7,5.6Hz), 6.22(1H,d,J=3.4Hz),
7.84(1H,d,J=4.2Hz), 7.86(1H,d,J=4.2Hz), 11.82(1H,brs)
Example 24
0.16 g of diethyl (2S)-2-{[(3-hydroxy-2-
pyrazinyl)carbonyl]amino} butanedioate was suspended in
2 mL of 1,1,1,3,3,3-hexamethyldisilazane, and the
suspension was under reflux for 1 hour. After cooling,
the solvent was removed under reduced pressure. 1 mL
of toluene was added, and the solvent was removed under
reduced pressure. 2 mL of acetonitrile, 0.24 g of P~D-
ribofuranose 1,2,3,5-tetraacetate, and 0.11 mL of
trimethylsilyl trifluoromethane sulfonate were
successively added to the obtained residue, and the
obtained mixture was stirred at room temperature for 1
hour. Chloroform and water were added thereto, and the
organic layer was separated. The obtained organic
layer was washed with a saturated sodium bicarbonate
aqueous solution, and then dried with anhydrous
magnesium sulfate. The solvent was removed under
reduced pressure. The obtained residue was purified by
silica gel chromatography [eluant; chloroform :
methanol =40 : 1], to obtain 0.25 g of a light yellow
solid, diethyl (2S)-2-{[(4-{(2R, 3R, 4R, 5R)-3,4-
bis(acetyloxy)-5-[(acetyloxy)methyl]tetrahydro-2-
furanyl}-3~oxo-3,4-dihydro-2-pyrazinyl)carbonyl]amino}
butanedioate.
1H-NMR(CDCl3)d: 1. 25 (3H, t, J=7 . 2Hz) , 1.28(3H,t,J=7.2Hz),
2.10(3H,s), 2.15(3H,s), 2.17(3H,s),
2.98(1H,dd,J=4.8,17.2Hz), 3.11(1H,dd,J=4.8Hz,17.2Hz),
4.13-4.18(2H,m), 4.21-4.28(2H,m), 4.40-4.47(2H,m),
4.49-4.51(1H,m), 5.09-5.12(1H,m), 5.26-5.28(1H,m),
5.43-5.44(1H,m), 6.27(1H,d,J=3.3Hz),
7.77(1H,d,J=4.0Hz), 7.7 9(1H,d,J=4.0Hz),
10.13(1H,d,J=8.1Hz)

0.21 g of diethyl (2S)-2-{[(4-{(2R, 3R, 4R,
5R)-3,4-bis(acetyloxy)-5-[(acetyloxy)methyl]tetrahydro-
2-furanyl}-3-oxo-3,4-dihydro-2-
pyrazinyl)carbonyl]amino} butanedioate was dissolved in
ethanol, and thereafter, 75 mg of sodium ethoxide was
added thereto under ice cooling, followed by stirring
at room temperature for 1 hour. Thereafter, the
stirring was terminated, and the reaction mixture was
left overnight at room temperature. 0.13 mL of acetic
acid and 1 mL of water were successively added thereto,
and the solvent was removed under reduced pressure.
The obtained residue was purified by silica gel
chromatography [eluant; chloroform : methanol =40 :
1]. Ethyl acetate and n-hexane were added to the
obtained residue, and the precipitate was collected by
filtration, to obtain 39 mg of a yellow solid, diethyl
(2S}-2-{[{4-{(2R, 3R, 4S, 5R)-5-[(acetyloxy)methyl]-
3, 4-dihydroxytetrahydro-2-furanyl}-3-oxo-3,4-dihydro-2-
pyrazinyl)carbonyl]amino} butanedioate.
IR(KBr)cm-1: 1739, 1670
1H-NMR(DMSO-d6)d: 1.18 ( 6H, t, J=6. 8Hz) , 2.10£3H,s),
2.89(2H,brs), 3.90(1H,brs), 4.07-4.15(6H,m), 4.30-
4.38(2H,m), 4.87-4.92(1H,m), 5.30-5.34(1H,m), 5.77-
5.79(1H,m), 5.91(1H,s), 7.70(1H,d,J=3.4Hz),
7.96(1H,d,J=3.4Hz), 9.83(1H,d,J=7.6Hz)
0.31 g of 4-[(3aR, 4R, 6R, 6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-
d] [l,3]dioxol-4-yl]-3-oxo-3,4-dihydro-2-
pyrazinecarboxamide was dissolved in 2 mL of N,N-
dimethylformamide, and thereafter, 0.33 g of N-(t-
butoxycarbonyl)-L-valine, 12 mg of 4-
dimethylaminopyridine, and 0.41 g of 1,3-
dicyclohexylcarbodiimide were successively added
thereto, followed by stirring at room temperature for 2
hours. 0.5 mL of acetic acid was added to the mixture,
and it was stirred at room temperature for 30 minutes.
Thereafter, ethyl acetate and water were added thereto,
and insoluble products were removed by filtration. The
organic layer was separated, washed with 1 mol/L
hydrochloric acid, and dried with anhydrous magnesium
sulfate. The solvent was then removed under reduced
pressure. The obtained residue was purified by silica
gel chromatography [eluant; ethyl acetate], to obtain
0.40 g of a yellow solid, {(3aR, 4R, 6R, 6aR)-6-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl} methyl
(2S)-2-[(tert-butoxycarbonyl)amino]-3-methyl butanoate
1H-NMR(DMSO-d5)d: 0 . 83-0 . 86 ( 6H, m) , 1.30(3H,s),
1.38(9H,s), 1.52(3H,s), 1.86-1.95(1H,m),
3.79(1H,t,J=7.2Hz), 4.27-4.34(2H,m), 4.46(1H,brs),
4.78-4.80(1H,m), 5.03(1H,d,J=6.1Hz), 6.00(1H,s),
7.24(1H,d,J=7.2Hz), 7.54(1H,d,J=4.3Hz), 7.77(1H,brs),
7.86(1H,d,J=4.3Hz), 8.28(1H,brs)
Example 27
0.26 g of {(3aR, 4R, 6R, 6aR)-6-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl} methyl
(2S)-2-[(tert-butoxycarbonyl)amino]-3-methyl butanoate
was dissolved in a mixed solvent of 3 mL of methanol
and 3 mL of water, and thereafter, 2.6 g of a Dowex
50WX4-200 ion exchange resin (H+form) was added
thereto, followed by stirring at room temperature for 3
hours. Thereafter, the resin was removed by
filtration, and it was then washed with methanol and
then with water. The obtained filtrate and the
washings were combined, and the solvent was removed
under reduced pressure. 2 mL of toluene was added, and
the solvent was removed under reduced pressure, to
obtain 70 mg of a light yellow solid, { (2R, 3S, 4R,
5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl}methyl (2S)-2-[(tert-
butoxycarbonyl)amino]-3-methyl butanoate.
1H-NMR(DMSO-d6)d: 0 . 87-0 . 91 ( 6H,m) , 1.38 (9H,s), 1.97-
2.05(1H,m), 3.53(2H,br), 3.85-3.93(2H,m), 4.07(1H,brs),
4.19(1H,brs), 4.31-4.35(1H,m), 4.39-4.42(1H,m),
5.92(1H,s), 7.32(1H,d,J=7.6Hz), 7.60(1H,d,J=4.1Hz),
7.76(1H,brs), 7.87(1H,d,=4.1Hz), 8.32(1H,brs)
Example 2 8
14.1 g of 3-hydroxy-2-pyrazinecarboxamide was
suspended in 150 mL of toluene, and the suspension was
stirred under reflux for 1 hour, followed by azeotropic
dehydration. After cooling it to room temperature, the
solvent was removed under reduced pressure. 42.3 mL of
1,1,1,3,3,3-hexamethyldisilazane was added to the
residue, and the obtained mixture was heated under
reflux for 2 hours. The reaction mixture was
concentrated under reduced pressure, 45 mL of toluene
was added thereto, and the solvent was removed under
reduced pressure. Using 45 mL of toluene, the same
above operation was repeated twice. The residue was
dissolved in 40 mL of acetonitrile, and it was then
added to 100 mL of an acetonitrile solution containing
48.4 g of p-D-ribofuranose 1-acetate 2,3,5-tribenzoate.
25 g of tin(IV) chloride was dropped into the mixture
at room temperature. The obtained solution was stirred
at 50°C for 4.5 hours. After cooling it to room
temperature, it was added to a mixed solution
consisting of 100 g of sodium bicarbonate, 500 mL of
water and 280 mL of methylene chloride. Insoluble
products were filtrated, and the organic layer was
separated. The obtained organic layer was washed with
50 mL of water and then with 50 mL of a saturated
sodium chloride aqueous solution, and the solvent was
removed under reduced pressure. 280 mL of ethyl
acetate and 30 mL of water were added to the obtained
residue, and the mixture was heated to 50°C, and it was
then cooled to 5°C. The deposit was collected by
filtration, to obtain 40.9 g of a grayish-white solid,
[(2R, 3R, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-l(2H)-
pyrazinyl]-3,4-bis(benzoyloxy)tetrahydro-2-furanyl]
methyl benzoate.
IR(KBr)cm-1: 1729, 1683
1H-NMR(DMSO-d6)d: 4 . 69-4 . 78 (2H,m) , 4 . 88-4 . 92 (1H,m) ,
5.97-6.05(2H,m), 6. 34 (1H, d, J=2 . 4Hz) , 7.40-7.54(7H,m),
7.62-7.71(3H,m), 7.80(1H,brs), 7.84-7.86(2H,m), 7.95-
8.03(5H,m), 8.22(1H,m)
17.5 mL of water containing 2.2 g of sodium
hydroxide was added to 330 mL of a methanol suspension
containing 35 g of [(2R, 3R, 4R, 5R)-5-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
bis(benzoyloxy)tetrahydro-2-furanyl] methyl benzoate at
room temperature. The mixture was stirred at the same
temperature for 2 hours, and then cooled to 5°C. The
obtained deposit was collected by filtration, to obtain
12.5 g of a light yellow solid, 4-[(2R, 3R, 4S, 5R)-
3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-
oxo-3,4-dihydro-2-pyrazinecarboxamide (water content:
0.4%).
IR(KBr)cm_1: 3406, 1654
1H-NMR(DMSO-d5) d: 3 . 65 (1H, ddd, J=2 . 6, 5 .1, 12 . 5Hz) ,
3.81(1H,ddd,J=2.2,4.8,12.1Hz), 3.96-4.00(2H,m), 4.01-
4.03(1H,m), 5.10(1H,d,J=5.9Hz), 5.28(1H,t,J=4.9Hz),
5.64(1H,d,J=4.8Hz), 5.93(1H,d,J=2.6Hz),
7.54(1H,d,J=4.0Hz), 7.74(1H,s), 8.29(1H,d,J=4.0Hz),
8.36(1H,S)
Example 29(2)
11 g of the 4-[(2R, 3R, 4S, 5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-
3,4-dihydro-2-pyrazinecarboxamide obtained in Example
29 was dissolved in 300 mL of water (45°C), and then 1.1
g of activated carbon was added thereto, followed by
stirring for 10 minutes. The activated carbon was
filtrated and washed with 20 mL of water twice. The
filtrate and the washings were combined, and 1.1 g of
activated carbon was added thereto, followed by
stirring for 10 minutes. The activated carbon was
filtrated, washed with 20 mL of water twice, and then
concentrated under reduced pressure. 90 mL of water
was added to the concentrate, and thereafter the
mixture was filtrated, to obtain 9.35 g of a colorless
crystal, 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-dihydro-
2-pyrazinecarboxamide monohydrate (water content:
6.8%) .
IR(KBr)cm1: 3578, 3399, 3091, 2929, 1674
1H-NMR(DMSO-d6) d: 3 . 65 (1H, ddd, J=2 . 6, 5 .1, 12 . 5Hz) ,
3.81(1H,ddd,J=2.2,4.8,12.1Hz), 3.96-4.00(2H,m), 4.01-
4.03(1H,m), 5.10(1H,d,J=5.9Hz), 5 . 28(1H, t,J=4.9Hz) ,
5.64(1H,d,J=4.8Hz), 5.93(1H,d,J=2.6Hz),
7.54(1H,d,J=4.0Hz), 7.74(1H,s), 8.29(1H,d,J=4.0Hz),
8.36(1H,s)
0.47 g of a light yellow solid, [(2R, 3R, 4R,
5R)-5-[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-
pyrazinyl]-3, 4-bis(benzoyloxy)tetrahydro-2-furanyl]
methyl benzoate was obtained from 0.23 g of 6-fluoro-3-
hydroxy-2-pyrazinecarboxamide in the same manner as in
Reference Example 6.
IR(KBr)cm1: 1726, 1690
1H-NMR(DMSO-d6)d: 4 . 6-5. 0 (3H,m) , 5 . 9-6.1 (2H,m) ,
6.33(1H,s), 7.3-8.2(17H,m), 8.53(1H,brs)
Example 31

7.37 g of a light yellow solid, (2R, 3R, 4R,
5R)-4-(acetyloxy)-2-[(acetyloxy)methyl]-5-[3-
(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]
tetrahydro-3-furanyl acetate was obtained from 4.0 g of
6-fluoro-3-hydroxy-2-pyrazinecarboxamide in the same
manner as in Reference Example 6.
IR(KBr)cm-1: 1748, 1715, 1662
1H-NMR(CDCl3)d: 2.04(3H,s), 2.08(3H,s), 2.18(3H,s) 4.47-
4.58(3H,m), 5.20-5.34(1H,m), 5.51(1H,dd,J=2.3,5.0Hz),
6.16(1H,d,J=2.2Hz), 6.41(1H,brs), 7.95(1H,d,J=5.6Hz),
8.94(1H,brs)
Example 32

0.15 g of t(2R, 3R, 4R, 5R)-5-[3-
(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]-3,4-
bis(benzoyloxy)tetrahydro-2-furanyl] methyl benzoate
was dissolved in 2.0 mL of methanol, and thereafter,
0.14 g of a 28% sodium methoxide methanol solution was
added thereto under ice cooling. The obtained mixture
was stirred at the same temperature for 20 minutes and
then at room temperature for 30 minutes. 0.75 mL of 1
mol/L hydrochloric acid was added to the reaction
mixture, and the solvent was removed under reduced
pressure. The obtained residue was purified by column
chromatography [eluant; chloroform : methanol =5 : 1],
and thereafter, isopropanol and diethyl ether were
added thereto. The mixture was then filtrated, to
obtain 40 mg of 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-6-fluoro-3-oxo-
3, 4-dihydro-2-pyrazinecarboxamide.
0.20 g of 3-oxo-3,4-dihydro-2-
pyrazinecarboximidamide and 10 mg of ammonium sulfate
were suspended in 2.0 mL of 1,1,1,3,3,3-
hexamethyldisilazane, and the suspension was heated
under reflux for 10 minutes under nitrogen current.
9.0 mg of ammonium sulfate was added thereto, and the
mixture was further heated under reflux for 2 hours.
The reaction mixture was cooled, and the solvent was
then removed under reduced pressure. The obtained
residue was dissolved in 4.0 mL of acetonitrile, and
thereafter, 0.46 g of p-D-ribofuranose-1,2,3,5-
tetraacetate and 0.34 mL of tin(IV) chloride were
successively added thereto, followed by stirring at
room temperature for 3 hours. 10 |iL of trifluoroacetic
acid and 1.0 mL of water were added to the reaction
mixture, and the solvent was then removed under reduced
pressure. Using 0.05 g of 3-oxo-3,4-dihydro-2-
pyrazinecarboximidamide, the same above reaction was
repeated to obtain a reaction mixture. The obtained
reaction mixtures were combined and then purified by
reverse phase silica gel column chromatography [eluant;
acetonitrile : water =1 : 4], to obtain 0.34 g of a
light yellow solid, (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[(acetyloxy)methyl]-5-[3-[amino(imino)methyl]-2-oxo-
1(2H)-pyrazinyl] tetrahydro-3-furanyl acetate.
IR(KBr)cm-1: 3392, 1750, 1685
1H-NMR(CDCl3)d: 2.11(3H,s), 2.16(6H,s), 4 . 4-4 . 7 (3H,m) ,
5.31 (1H,t, J=5.4Hz) , 5.5-5.6(1H,m) , 6.22(1H,d,J=3.0Hz),
7.8-8.0(1H,m), 8.1-8.3(1H,m), 8.67(1H,brs),
10.45(2H,brs)
Example 34

0.10 g of (2R, 3R, 4R, 5R)-4-(acetyloxy)-2-
[(acetyloxy)methyl]-5-[3-[amino(imino)methyl]-2-oxo-
1(2H)-pyrazinyl] tetrahydro-3-furanyl acetate was added
to 5.0 mL of 25% ammonia water under ice cooling, and
the mixture was stirred at the same temperature for 2
hours. 4.9 mL of acetic acid was added to the reaction
mixture, and the solvent was removed under reduced
pressure. Using 20 mg of (2R, 3R, 4R, 5R)-4-
(acetyloxy)-2-[(acetyloxy)methyl]-5-[3-
[amino(imino)methyl]-2-oxo-l(2H)-pyrazinyl] tetrahydro-
3-furanyl acetate, the same above reaction was carried
out. The obtained reaction mixtures were combined and
then purified by reverse phase silica gel column
chromatography [eluant; water]. 5.0 mL of 1 mol/L
hydrochloric acid was added to the obtained solid, and
the solvent was removed under reduced pressure.
Moreover, 5.0 mL of 1 mol/L hydrochloric acid was
further added thereto, and the solvent was removed
under reduced pressure. Ethanol was added to the
obtained residue, and the solid was collected by
filtration, to obtain 30 mg of hydrochloride of a light
yellow solid, 4-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydro-2-furanyl]-3-oxo-3,4-dihydro-
2-pyrazinecarboximidamide.
IR(KBr)cm1: 3374, 3281, 1690
1H-NMR(DMSO-d6) d: 3 . 7-3 . 9 (2H, m) , 3 . 9-4 . 2 (3H,m) , 5.1-
5.3(1H,m), 5.3-5.6(1H,m), 5.6-5.8(1H,m), 5.90(1H,s),
7.86(1H,d,J=4.0Hz), 8.76(1H,d,J=4.0Hz), 9.44(4H,brs)
Example 35

37 mg of {(2R, 3S, 4R, 5R)-5-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl} methyl benzoate was
obtained from 200 mg of {(2R, 3R, 4R, 5R)-5-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
bis(benzoyloxy)tetrahydro-2-furanyl} methyl benzoate in
the same manner as in Example 18.
1H-NMR(DMSO-d6)d: 4.06 (1H, dd, J= 6.6,12.7), 4.12-
4.15(1H,m), 4.28-4.32(1H,m), 4.59(1H,dd,J=5.1,12.2),
4.67(1H,dd,J=2.7,12.5), 5.40(1H,d,J=6.3),
5.76(1H,d,J=4.9), 5.91(1H,d,J=2.0), 7.37(1H,d,J=4.4),
7.57(2H,t,J=7.8), 7.71(1H,dd,J=7.1,7.6), 7.75(1H,brs),
7.85(1H,d,J=4.2), 8.01(2H,dd,J=l.0,7.4), 8.30(1H,brs)
Example 36
100 mg of { (2R, 3S, 4R, 5R)-5-[3-
(aminocarbonyl)-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl}methyl (2S)-2-[(tert-
butoxycarbonyl)amino]-3-methyl butanoate was dissolved
in a mixed solvent of 1 mL of trifluoroacetic acid and
0.1 mL of water, and the mixture was stirred at room
temperature for 1 hour. Trifluoroacetic acid was
removed under reduced pressure, and water was added.
The mixture was adjusted to pH 8 with a saturated
sodium bicarbonate aqueous solution, and it was then
purified by reverse phase silica gel chromatography
[eluant; 10% acetonitrile aqueous solution], followed
by azeotropy with ethanol, to obtain 59 mg of a white
solid, {(2R, 3S, 4R, 5R)-5-[3-(aminocarbonyl)-2-oxo-
1(2H)-pyrazinyl]-3, 4-dihydroxytetrahydro-2-furanyl}
methyl (2S)-2-amino-3-methyl butanoate.
IR(KBr)cm1: 1724, 1670, 1653
1H-NMR(DMSO-d6) d: 0.84 (3H, d, J=6. 6Hz) ,
0.89(3H,d,J=6.6Hz), 1.82-1.90(1H,m),
3.22(1H,d,J=5.2Hz), 3.88-3.94(1H,m), 4.09(1H,s), 4.17-
4.20(1H,m), 4.36-4.38(2H,m), 5.91(1H,s),
7.58(1H,d,J=4.0Hz), 7 . 90(1H,d,J=4.0)
58 mg of 4-[(2R, 3R, 4S, 5R)]-3,4-dihydroxy-
5-(hydroxymethyl)tetrahydro-2-furanyl]-6-fluoro-3-oxo-
3,4-dihydro-2-pyrazinecarboxamide was suspended in 1 mL
of trimethyl phosphate, and thereafter, 37 µL of
pyridine and 49 µL of phosphorus oxychloride were
successively added thereto under ice cooling, followed
by stirring at the same temperature for 1 hour. 3 mL
of a 1 mol/L triethyl ammonium bicarbonate aqueous
solution was added thereto, and the mixture was stirred
for 15 minutes. The solvent was removed under reduced
pressure, and the residue was purified by ion exchange
column chromatography [eluant; 0.1 mol/L triethyl
ammonium bicarbonate aqueous solution]. Subsequently,
it was purified by reverse phase silica gel column
chromatography [eluant; water], to obtain 39 mg of
triethyl ammonium salts of a solid, [(2R, 3S, 4R, 5R) -
5-[3-(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]-
3,4-dihydroxytetrahydro-2-furanyl] methyl phosphate.
3 mL of an acetone solution containing 0.14 g
of sodium perchlorate was added to 0.5 mL of a methanol
suspension containing 36 mg of the above obtained
triethyl ammonium salts of monophosphoric acid at room
temperature, and the mixture was stirred for 30
minutes. The deposit was collected by filtration and
washed with acetone, to obtain 27 mg of sodium salts of
a light yellow solid, [(2R, 3S, 4R, 5R)-5-[3-
(aminocarbonyl)-5-fluoro-2-oxo-l(2H)-pyrazinyl]-3,4-
dihydroxytetrahydro-2-furanyl] methyl phosphate.
1H-NMR(D20)d: 4 . 13-4 .18 (1H,m) , 4 . 29-4 . 40 (4H,m) ,
6.10(1H,s), 8.46(1H,d,J=4.9Hz)
INDUSTRIAL APPLICABILITY
From the above test examples, it was found
that a pyrazine nucleotide triphosphate analog has an
activity to specifically inhibit virus polymerase in
the virus polymerase inhibition test, that the pyrazine
carboxamide nucleotide modified with a substituent
given in the present invention moves into a cell and
has an antiviral activity therein, although it has been
generally known that nucleotide cannot move into a cell
through a cell membrane, and that pyrazine nucleoside
is converted into a pyrazine nucleotide triphosphate
analog in the body of an animal administered with the
pyrazine nucleoside. Accordingly, the virus growth
inhibition and/or virucidal method of the present
invention, which is characterized by the use of a
pyrazine nucleotide or pyrazine nucleoside analog
generated by the effect of kinase such as nucleotide
kinase or a salt thereof, is useful as a method for
treating virus infection. Moreover, the novel pyrazine
nucleotide or pyrazine nucleoside analog or a salt
thereof of the present invention is useful as an agent
for preventing and/or treating virus infection.
razine nucleotide analog represented by the following general formula
[1z] or a salt thereof:

wherein R1 represents a hydrogen atom, or a substituent of a pyrazine
ring; R2 represents a hydrogen atom, a)n acyl group, or a C1-5 carbamoylalkyl or
C1-5 carboxyalkyl group that may be substituted with at least one group selected
from the group consisting of a halogen from;
an C1-5 alkyl group that may be substituted by a hydroxyl, C1-5 alkoxy, C1-5
alkylthio, aryl, amino or mono-or di-C1-5 alkylamino group; a halogeno-C1-5 alkyl
group;
an C2-5 alkenyl group;
a C3-6 cycloalkyl group;
a hydroxyl group;
an C1-5 alkoxy group;
a C3-6 cycloalkyloxy group;
an C1-5 alkoxycarbonyl group;
a mercapto group;
an C1-5 alkylthio goup that may be substjtuted by an aryl group;
an aryl group;
an aryloxy group;
an arylthio group;
an arylamino group;
a cyano group;
a nitro group;
an amino group that may be substituted by an acyl group;
an mono-or di -C1-5 alkylamino group;
a C3-6 cycloalkylamino group;
an acyl group;
a carboxyl group;
a carbamoyl group;
a thiocarbamoyl group;
an C1-5 alkylcarbamoyl group;
and a heterocyclic group; each of R3, R4, R5 and R6, which may be the same or
different, represents a hydrogen atolm, or a hydroxyl group that may be
substituted with at least one group selected from the group consisting of
a carboxyl group that may be protected;
an C1-5 alkyl group;
an C1-5 alkoxycarbonyl group;
an aryl group;
a C3-6 cycloalkyl group;
an C2-5 alkenyl group;
a halogeno-C1-5 alkyl group;
and a heterocyclic group;
or that may be protected; R represents a hydroxyl group that may be protected or
substituted with a group decomposed under physiological conditions; A
represents an oxygen atom or a methylene group; and n represents an integer of
1 to 3;
the substituent of a pyrazine of R1 represents one or more groups selected from
a halogen atom;
an C1-5 alkyl group that may be substituted by hydroxyl, C1-5 alkoxy, C1-5 alkylthio,
aryl, amino or mono- or di-C1-5 alkylamino group;
an C1-5 alkyl or C2-5 alkenyl group that may be substituted by a halogen atom;
a C3-6 cycloalkyl group;
a hydroxyl group;
an C1-5 alkoxy group;
a C3-6 cycloalkyloxy group;
2. A pyrazine nucleothide analog represented by the following general
formula [1z] or a salt thereof:
wherein R1 represents a hydrogen atom, or a substituent of a pyrazine
ring; R2 represents a hydrogen atom, an acyl group, or a C1-5 carbamoylalkyl or
C1-5 carboxyalkyl group that may be substituted with the group as defined in
claim 1; each of R3, R4, R5 and R6, which may be the same or different,
represents a hydrogen atom, or a hydroxyl group that may be substituted with the
group as defined in claim 1 or that may be protected; R represents a hydroxyl
group that may be protected or substituted with a group decomposed under
physiological conditions; A represents an oxygen atom or a methylene group;
and n represents an integer of 1 to 3; provided that a case where R is a hydroxyl
goup, A is an oxygen atom, and R1 is a hydrogen atom or halogen atom is
excluded; the substituent of a pyrazine of R1 represents the group as defined in
claim 1;
the R group decomposable under the physiological condition represents the
group as defined in claim 1.
3. A pyrazine nucleotide analog represented by the following general formula
[2] or a salt thereof:
wherein R1 represents a hydrogen atom, or a substituent of a pyrazine
ring; R2 represents a hydrogen atom, an acyl group, or a C1-5 carbamoylalkyl or
C1-5 carboxyalkyl group that may be substituted with the group as defined in
claim 1; each of R3 , R4, R5 and R6, which may be the same or different,
represents a hydrogen atom, or a hydroxyl group that may be substituted with the
group as defined in claim 1 or that may be protected; each of R7 and R8 in
phosphoric acid or phosphonic acid independently represents a protected or
unprotected, substituted or unsubstituted hydroxyl group to be decomposed
under physiological conditions; A represents an oxygen atom or a methylene
group; and Y represents an oxygen atom or an imino group; the substituent of a
pyrazine of R1 represents the group as defined in claim 1;
R7 and R8, each independently a protecting group or substituent of a hydroxyl
group to be decomposed under the physiological condition in the phosphate or
phosphonate are an aryl group; a cyclosaligenyl group; an amidate group; a
haloethyl group; an acyloxyalkyl group; an acyloxybenzyl group; an s-lower
acylthioalkyl group; an s-higher acylthioalkyl group; an s-aroylthioalkyl group; and
dithiodiethyl group.
4. The pyrazine nucleotide analog or a salt thereof as claimed in claim 3,
wherein Y is an oxygen atom.
5. A pyrazine nucleoside analog represented by the following general
formula [3z] or a salt thereof:
or unprotected, substituted or unsubstituted hydroxy! group to be decomposed
under physiological conditions; and Y represents an oxygen atom or an imino
group; the substituent of a pyrazine of R1 represents the group as defined in
claim 1; protecting groups and substituents of a hydroxyl group of Rz represents
an acyl group that may be substituted, a C1-5alkoxycarbonyl group, and an
acyloxy-C1-5alkyl group.
6. The pyrazine nucleoside analog or a salt thereof as claimed in claim 5,
wherein, in the pyrazine nucleotide analog represented by general formula
[3z] or a salt thereof, Y is an imino group.
7. The pyrazine nucleoside analog represented by general formula [3z] or a
salt thereof as claimed in claim 5, represented by general formula [3z']:
wherein Ra represents a hydrogen or halogen atom; and each of Rb and Rc ,
which may be the same or different, represents a hydrogen atom or a hydroxyl
protecting group, or Rb and Rc together represent an C1-5alkylene group that may
be substituted.
8. An RNA polymerase inhibitor precursor having a pyrazine nucleotide
analog structure represented by general formula [1x] :
wherein R1 represents a hydrogen atom, or a substituent of a pyrazine ring; R2
represents a hydrogen atom, an acyl group, or a C1-5carbamoylalkyl or C1-
5carboxyalkyl group that may be substituted with the group as defined in claim 1;
each or R3, R4, R5 and R6, which may be the same or different,, represents a
hydrogen atom, or a hydroxyl group that may be substituted with the group as
defined in claim 1 or that may be protected; A represents an oxygen atom or a
methylene group; Y represents an oxygen atom or an imino group; and m
represents an integer of 0 to 2, said RNA polymerase inhibitor precursor being
converted in vivo into a pyrazine triphosphate nucleotide analog represented by
general formula [1y]:
wherein R1 represents a hydrogen atom, or a substituent of a pyrazine ring; each
of Z10, Z11, Z12 and Z13 which may be the same or different, represents a
hydrogen atom or a hydroxyl group, and thereby exhibiting an RNA polymerase
inhibitory effect; the substituent of a pyrazine of R1 represents the group as
defined in claim 1.
9.The RNA polymerase inhibitor precursor as claimed in claim 8 wherein, in
the pyrazine nucleotide analog structure represented by general formula
[1x], Y is an oxygen atom.
10.The RNA polymerase inhibitor precursor as claimed in any one of claims 8
and 9 wherein, in the pyrazine nucleotide analog structure represented by
general formula [1x], the substituent of a pyrazine ring is one or more
groups selected from the group consisting of: a halogen atom; an C1-5alkyl
group that may be substituted with a hydroxyl, C1-5alkoxy, C1-5alkylthio,
aryl, amino or mono - or di-C1-5alkylamino group; an C1-5alkyl or C2-
5alkenyl group that may be substituted with a halogen atom; a C3-
6cycloalkyl group; a hydroxyl group; an C1-5alkoxy group; a C3-
6cycloalkyloxy group; an C1-5 alkoxycarbonyl group; a mercapto group; an
C1-5 alkylthio group that may be substituted with an aryl group; an aryl
group; an aryloxy group; an arylthio group; an arylamino group; a cyano
group; a nitro group; an amino group that may be substituted with an acyl
group; an mono - or di - C1-5 alkylamino group; a C3-6 cycloalkylamino
group; an acyl group; a carboxyl group; a carbamoyl group; a
thiocarbamoyl group; an C1-5alkylcarbamoyl group; and a heterocyclic
group.
11.The RNA polymerase inhibitor precursor as claimed in any one of claims 8
to 10, wherein each of R1, R3, and R5 represents a hydrogen atom; and
each of R4 and R6 represents a hydroxyl group, in the pyrazine nucleotide
analog structure represented by general formula [1x] that is converted in
vivo into the pyrazine triphosphate nucleotide analog represented by
general formula [1y] wherein each of R1, Z10 and Z12 represents a
hydrogen atom; and each of Z11 and Z13 represents a hydroxyl group.
12. The RNA polymerase inhibitor precursor as claimed in any one of claims 8
to 11, wherein the pyrazine triphosphate nucleotide analog represented by
general formula [1y] is generated by a kinase.
13. The RNA polymerase inhibitor precursor as claimed in any one of claims 8
to 12, wherein the pyrazine triphosphate nucleotide analog represented by
general formula [1y] is generated by a nucleotide kinase.
14.The RNA polymerase inhibitor precursor as claimed in any one of claims 8
to 13, wherein it inhibits virus-derived RNA polymerase with selectivity 200
times or more higher than that for host-derived RNA polymerase.
15. The RNA polymerase inhibitor precursor as claimed in any one of claims 8
to 13, which is a pyrazine nucleoside or pyrazine mononucleotide analog
structure represented by general formula [1w]:
wherein R1 represents a hydrogen atom, or a substituent of a pyrazine ring; R2
represents a hydrogen atom, an acyl group, or a C1-5carbamoylalkyl or C1-
5carboxyalkyl group that may be substituted with the group as defined in claim 1;
each of R3, R4, R5 and R6, which may be the same or different, represents a
hydrogen atom, or a hydroxyl group that may be substituted with the group as
defined in claim 1 or that may be protected; Y represents an oxygen atom or an
imino group; and p represents 0 or 1, wherein the ratio of an inhibitory effect of
the RNA polymerase inhibitor precursor on the virus-derived RNA polymerase to
an inhibitory effect of the RNA polymerase inhibitor precursor on host cell-derived
inosine monophosphate dehydrogenase is 900 : 1 or more; the substituent of a
pyrazine of R1 represents the group as defined in claim 1.
16. The RNA polymerase inhibitor precursor as claimed in claim 15 wherein,
in the pyrazine nucleoside or pyrazine mononucleotide analog
represented by general formula [1w], Y is an oxygen atom.
17.The RNA polymerase inhibitor precursor as claimed in any one of claims
15 and 16 wherein, in the pyrazine nucleoside or pyrazine mononucleotide
analog structure represented by general formula [1w], the substituent of a
pyrazine ring is one or more groups selected from the group consisting of:
a halogen atom; an C1-5 alkyl group that may be substituted with a
hydroxyl, C1-5 alkoxy, C1-5alkylthio, aryl, amino or mono- or di-C1-5
alkylamino group; an C1-5alkyI or C2-5 alkenyl group that may be
substituted with a halogen atom; a C3-6 cycloalkyl group; a hydroxyl group;
an C1-5 alkoxy group; a C3-6 cycloalkyloxy group; an C1-5 alkoxycarbonyl
group; a mercapto group; an C1-5alkylthio group that may be substituted
with an aryl group; an aryl group; an aryloxy group; an arylthio group; an
arylamino group; a cyano group; a nitro group; an amino group that may
be substituted with an acyl group; an mono- or di-C1-5 alkylamino group; a
C3-6 cycloalkylamino group; an acyl group; a carboxyl group; a carbamoyl
group; a thiocarbamoyl group; an C1-5alkylcarbamoyl group; and a
heterocyclic group.
18. The RNA polymerase inhibitor precursor as claimed in any one of
claims 15 to 17, which is the pyrazine nucleoside or pyrazine
mononucleotide analog structure represented by general formula [1w],
wherein each of R1, R3 and R5 represents a hydrogen atom; and each
of R4 and R6 represents a hydroxyl group.
wherein R1 represents a hydrogen atom, or a substituent of a pyrazine ring; and
each of Z10, Z11, Z12, and Z13, which may be the same or different, represents a
hydrogen atom or hydroxyl group; the substituent of a pyrazine of R1 represents
the group as defined in claim 1,
23. The RNA polymerase inhibitor having a pyrazine triphosphate nucleotide
analog structure as claimed in claim 22, wherein its inhibitory effect on
virus-derived RNA polymerase is 200 times or more selective than the
inhibitory effect on host-derived RNA polymerase.
24. The RNA polymerase inhibitor having a pyrazine triphosphate
nucleotide analog structure as claimed in claim 23, wherein the virus-
derived RNA polymerase is derived from influenza virus, RS virus,
hepatitis A virus, hepatitis C virus, hepatitis E virus, poliovirus,
echovirus, Coxsackie virus, enterovirus, rhinovirus, rotavirus,
Newcastle disease virus, mumps virus, vesicular stomatitis virus,
rabies virus, Lassa fever virus, measles virus, Filovirus, Japanese
encephalitis virus, yellow fever virus, dengue fever virus or West Nile
virus.
25. The RNA polymerase inhibitor having a pyrazine triphosphate nucleotide
analog structure as claimed in claim 24, wherein the virus-derived RNA
polymerase is influenza virus-derived RNA polymerase or hepatitis C virus-
derived RNA polymerase.

wherein each of R1, R2, R3, R4, R5, R6, R7, R8, R9 and A
has the same meaning as given in the specification.
The present invention relates to a method for
exhibiting a virus growth-inhibiting effect and/or a
virucidal effect, in which pyrazine nucleotide analog
[2] and pyrazine nucleoside analog [3z] are subjected
to biotransformation, decomposed and then
phosphorylated, so that they become a pyrazine
nucleotide analog [lb] exhibiting the aforementioned
effect. This method is useful as a method for treating
virus infections. Moreover, the pyrazine carboxamide
analog or a salt thereof of the present invention is
useful as an agent for preventing or treating virus
infections.

Documents:

173-kolnp-2004-abstract.pdf

173-kolnp-2004-claims.pdf

173-kolnp-2004-correspondence.pdf

173-kolnp-2004-description (complete).pdf

173-kolnp-2004-examination report.pdf

173-kolnp-2004-form 1.pdf

173-kolnp-2004-form 18.pdf

173-kolnp-2004-form 2.pdf

173-kolnp-2004-form 26.pdf

173-kolnp-2004-form 3.pdf

173-kolnp-2004-form 5.pdf

173-KOLNP-2004-FORM-27.pdf

173-kolnp-2004-reply to examination report.pdf

173-kolnp-2004-specification.pdf

173-kolnp-2004-translated copy of priority document.pdf


Patent Number 235048
Indian Patent Application Number 173/KOLNP/2004
PG Journal Number 26/2009
Publication Date 26-Jun-2009
Grant Date 24-Jun-2009
Date of Filing 09-Feb-2004
Name of Patentee TOYAMA CHEMICAL CO. LTD.
Applicant Address 2-5, 3-CHOME, NISHISHINJUKU, SHINJUKU-KU, TOKYO
Inventors:
# Inventor's Name Inventor's Address
1 TAKAHASHI KAZUMI 21-15, KAKINOKIZAWA, TATEYAMA-MACHI, NAKANIIKAWA-GUN, TOYAMA 930-0237
2 TSUTSUI YASUHIRO 1-6-27, SHIMOOKUI, TOYAMA-SHI, TOYAMA 930-0817
3 UEHARA SAYURI 748-1, HORIKAWAKOIZUMICHO, TOYAMA-SHI, TOYAMA 939-8081
4 MURAKAMI MAKOTO 1-6-27, SHIMOOKUI, TOYAMA-SHI, TOYAMA 930-0817
5 FURUTA YOUSUKE 255-18, HONGOMACHI, TOYAMA-SHI, TOYAMA 939-8045
6 EGAWA HIROYUKI 612, KANAYAMASHINHIGASHI, TOYAMA-SHI, TOYAMA 930-2205
PCT International Classification Number A61K 31/706
PCT International Application Number PCT/JP2002/08250
PCT International Filing date 2002-08-13
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2001-245896 2001-08-14 Japan