U.S. patent application number 14/409318 was filed with the patent office on 2016-03-10 for intermediate for production of nucleoside analog and method for producing the same.
This patent application is currently assigned to Daiichi Sankyo Company, Limited. The applicant listed for this patent is Daiichi Sankyo Company, Limited. Invention is credited to Makoto Koizumi, Koji Morita, Miho Sato.
Application Number | 20160068561 14/409318 |
Document ID | / |
Family ID | 49768725 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160068561 |
Kind Code |
A9 |
Koizumi; Makoto ; et
al. |
March 10, 2016 |
Intermediate for Production of Nucleoside Analog and Method for
Producing the Same
Abstract
A compound represented by the general formula (III) which serves
as an intermediate of an oligonucleotide analog having stable and
excellent antisense or antigene activity or having excellent
activity as a detection reagent (probe) for a specific gene or as a
primer for the initiation of amplification of a specific gene can
be produced at high yields regardless of the type of nucleobase by
a method comprising reacting a compound represented by the general
formula (II) or a salt thereof with a trimethylsilylated compound
obtained from a compound represented by the general formula (IVb),
wherein X, Y, Z, A, R, and B are as defined in claim 1.
Inventors: |
Koizumi; Makoto; (Tokyo,
JP) ; Morita; Koji; (Tokyo, JP) ; Sato;
Miho; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Daiichi Sankyo Company, Limited |
Tokyo |
|
JP |
|
|
Assignee: |
Daiichi Sankyo Company,
Limited
Tokyo
JP
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150152132 A1 |
June 4, 2015 |
|
|
Family ID: |
49768725 |
Appl. No.: |
14/409318 |
Filed: |
June 17, 2013 |
PCT Filed: |
June 17, 2013 |
PCT NO: |
PCT/JP2013/066578 PCKC 00 |
371 Date: |
December 18, 2014 |
Current U.S.
Class: |
536/27.11;
536/27.23 |
Current CPC
Class: |
C07H 23/00 20130101;
Y02P 20/55 20151101; C07H 19/16 20130101; C07F 7/10 20130101 |
International
Class: |
C07H 23/00 20060101
C07H023/00; C07H 19/16 20060101 C07H019/16; C07F 7/10 20060101
C07F007/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2012 |
JP |
2012-137049 |
Claims
1. A method of producing a compound of formula (III): ##STR00016##
or a salt thereof, comprising reacting a compound of formula (IVb):
##STR00017## with a trimethylsilylating agent to produce a
trimethylsilylated compound; and reacting the trimethylsilylated
compound with a compound of formula (II): ##STR00018## or a salt
thereof, wherein A is an alkylene group having 1 to 4 carbon atoms;
B is a purin-9-yl group or a purin-9-yl group having one or more
substituents selected from group .alpha.; X, Y, and Z are each
independently a protective group for the hydroxy group; R is a
silyl protective group; R.sup.4, R.sup.5, and R.sup.6 are each
independently a hydrogen atom, a hydroxy group, a protected hydroxy
group, an alkoxy group having 1 to 4 carbon atoms, a mercapto
group, a protected mercapto group, an alkylthio group having 1 to 4
carbon atoms, an amino group, a protected amino group, an amino
group substituted by one or more alkyl groups having 1 to 4 carbon
atoms, an alkyl group having 1 to 4 carbon atoms, or a halogen
atom; and group .alpha. is a hydroxy group, a protected hydroxy
group, an alkoxy group having 1 to 4 carbon atoms, a mercapto
group, a protected mercapto group, an alkylthio group having 1 to 4
carbon atoms, an amino group, a protected amino group, an amino
group substituted by one or more alkyl groups having 1 to 4 carbon
atoms, an alkyl group having 1 to 4 carbon atoms, and a halogen
atom.
2. The method of claim 1, wherein X is a methyl group substituted
by 1 to 3 aryl groups, or a methyl group substituted by 1 to 3 aryl
groups wherein the aryl ring is substituted by a lower alkyl, lower
alkoxy, halogen, or cyano group.
3. The method of claim 1, wherein X is a benzyl group, a
p-methoxybenzyl group, a dimethoxytrityl group, or a
monomethoxytrityl group.
4. The method of claim 1, wherein Y is a methyl group substituted
by 1 to 3 aryl groups, or a methyl group substituted by 1 to 3 aryl
groups wherein the aryl ring is substituted by a lower alkyl, lower
alkoxy, halogen, or cyano group.
5. The method of claim 1, wherein Y is a benzyl group, a
.beta.-naphthylmethyl group, or a p-methoxybenzyl group.
6. The method of claim 1, wherein Z is an aliphatic acyl group
having 2 to 4 carbon atoms.
7. The method of claim 1, wherein Z is an acetyl group.
8. The method of claim 1, wherein A is a methylene group or an
ethylene group.
9. The method of claim 1, wherein A is a methylene group.
10. The method of claim 1, wherein B is a 6-aminopurin-9-yl group,
a 6-aminopurin-9-yl group with the amino group protected, a
2,6-diaminopurin-9-yl group, a 2-amino-6-chloropurin-9-yl group, a
2-amino-6-chloropurin-9-yl group with the amino group protected, a
2-amino-6-fluoropurin-9-yl group, a 2-amino-6-fluoropurin-9-yl
group with the amino group protected, a 2-amino-6-bromopurin-9-yl
group, a 2-amino-6-bromopurin-9-yl group with the amino group
protected, a 2-amino-6-hydroxypurin-9-yl group, a
2-amino-6-hydroxypurin-9-yl group with the amino group protected, a
2-amino-6-hydroxypurin-9-yl group with the amino group and hydroxy
group protected, a 6-amino-2-methoxypurin-9-yl group, a
6-amino-2-chloropurin-9-yl group, a 6-amino-2-fluoropurin-9-yl
group, a 2,6-dimethoxypurin-9-yl group, a 2,6-dichloropurin-9-yl
group, or a 6-mercaptopurin-9-yl group.
11. The method of claim 1, wherein B is a 6-benzoylaminopurin-9-yl
group, an adeninyl group, a 2-isobutyrylamino-6-hydroxypurin-9-yl
group, or a guaninyl group.
12. The method of claim 1, wherein R is a tri-lower alkylsilyl
group, a monoaryl di-lower alkylsilyl group, or a diaryl mono-lower
alkylsilyl group.
13. The method of claim 1, wherein R is a monoaryl di-lower
alkylsilyl group or a diaryl mono-lower alkylsilyl group.
14. The method of claim 1, wherein R is a t-butyldiphenylsilyl
group.
15. A compound of formula (III): ##STR00019## or a salt thereof,
wherein X, Y, and Z are each independently a protective group for
the hydroxy group; A is an alkylene group having 1 to 4 carbon
atoms; R is a silyl protective group; B is a purin-9-yl group or a
purin-9-yl group having one or more substituents selected from
group .alpha.; and group .alpha. is a hydroxy group, a protected
hydroxy group, an alkoxy group having 1 to 4 carbon atoms, a
mercapto group, a protected mercapto group, an alkylthio group
having 1 to 4 carbon atoms, an amino group, a protected amino
group, an amino group substituted by (an) alkyl group(s) having 1
to 4 carbon atoms, an alkyl group having 1 to 4 carbon atoms, and a
halogen atom.
16. A method of producing a compound of formula (I): ##STR00020##
or a salt thereof, comprising reacting a compound of formula (IVb):
##STR00021## with a trimethylsilylating agent to produce a
trimethylsilylated compound; reacting the trimethylsilylated
compound with a compound of formula (II): ##STR00022## or a salt
thereof, to produce a compound of formula (III): ##STR00023## or a
salt thereof, wherein A is an alkylene group having 1 to 4 carbon
atoms; B is a purin-9-yl group or a purin-9-yl group having one or
more substituents selected from group .alpha.; X, Y, and Z are each
independently a protective group for the hydroxy group; R is a
silyl protective group, R.sup.4, R.sup.5, and R.sup.6 are each
independently a hydrogen atom, a hydroxy group, a protected hydroxy
group, an alkoxy group having 1 to 4 carbon atoms, a mercapto
group, a protected mercapto group, an alkylthio group having 1 to 4
carbon atoms, an amino group, a protected amino group, an amino
group substituted by one or more alkyl groups having 1 to 4 carbon
atoms, an alkyl group having 1 to 4 carbon atoms, or a halogen
atom; and group .alpha. is a hydroxy group, a protected hydroxy
group, an alkoxy group having 1 to 4 carbon atoms, a mercapto
group, a protected mercapto group, an alkylthio group having 1 to 4
carbon atoms, an amino group, a protected amino group, an amino
group substituted by one or more alkyl groups having 1 to 4 carbon
atoms, an alkyl group having 1 to 4 carbon atoms, and a halogen
atom.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel intermediate for
production of a nucleoside analog which serves as an intermediate
for production of an oligonucleotide analog having stable and
excellent antisense or antigene activity or having excellent
activity as a detection reagent (probe) for a specific gene or as a
primer for initiation of amplification of a specific gene, and a
method for producing the same.
BACKGROUND ART
[0002] Patent Literature 1 describes an oligonucleotide analog
having stable and excellent antisense or antigene activity or
having excellent activity as a detection reagent (probe) for a
specific gene or as a primer for initiation of amplification of a
specific gene, and the following nucleoside analog:
##STR00001##
which serves as an intermediate for production of the
oligonucleotide analog. The patent literature also describes steps
A-3 and A-4 as a method for producing the nucleoside analog.
##STR00002##
[0003] Patent Literature 1, however, makes no mention about a silyl
protective group, though it specifically describes only an example
wherein R.sup.7 is a p-toluenesulfonyloxy group. Particularly, the
production method described in Patent Literature 1
disadvantageously has a significantly reduced reaction yield, when
the nucleobase is a 2-isobutyrylamino-6-hydroxypurin-9-yl group
(yield of 6% in two steps of Reference Example 15 and Example 24).
In addition, it is less than industrially satisfactory, even when
the nucleobase is any of other groups, for example, when the
nucleobase is a 6-benzoylaminopurin-9-yl group, the yield of step
A-3 is as low as 52%.
[0004] Patent Literature 2 describes a nucleoside analog with the
2'- and 4'-positions bridged by a NHCOCH.sub.2 group, and discloses
the step of synthesizing compound 40 from compound 39 in Example
5(6).
##STR00003##
CITATION LIST
Patent Literature
[0005] Patent Literature 1: Japanese Patent Laid-Open No.
2000-297097 [0006] Patent Literature 2: International Publication
No. WO2011/052436 A1
SUMMARY OF INVENTION
Technical Problem
[0007] Thus, the present inventors have conducted diligent studies
to solve the problems mentioned above and consequently completed
the present invention by finding that use of a production
intermediate having a specific protective group can improve the
reaction yield regardless of the type of nucleobase.
Solution to Problem
[0008] The production method of the present invention is a
production method for producing a compound represented by the
general formula (III) or a salt thereof, comprising reacting a
trimethylsilylated compound, which was obtained by reaction of a
compound represented by the following general formula:
##STR00004##
wherein R.sup.4, R.sup.5, and R.sup.6 are the same or different and
each represents a hydrogen atom, a hydroxy group, a protected
hydroxy group, an alkoxy group having 1 to 4 carbon atoms, a
mercapto group, a protected mercapto group, an alkylthio group
having 1 to 4 carbon atoms, an amino group, a protected amino
group, an amino group substituted by (an) alkyl group(s) having 1
to 4 carbon atoms, an alkyl group having 1 to 4 carbon atoms, or a
halogen atom, with a trimethylsilylating agent, with a compound
represented by the following general formula:
##STR00005##
wherein X, Y, and Z are the same or different and each represents a
protective group for the hydroxy group; A represents an alkylene
group having 1 to 4 carbon atoms; and R represents a silyl
protective group, or a salt thereof: to obtain the compound
represented by the following general formula or a salt thereof:
##STR00006##
wherein X, Y, Z, A, and R are as defined above; and B represents a
purin-9-yl group or a substituted purin-9-yl group having (a)
substituent(s) selected from group .alpha. below.
[0009] The group .alpha. is a group consisting of a hydroxy group,
a protected hydroxy group, an alkoxy group having 1 to 4 carbon
atoms, a mercapto group, a protected mercapto group, an alkylthio
group having 1 to 4 carbon atoms, an amino group, a protected amino
group, an amino group substituted by (an) alkyl group(s) having 1
to 4 carbon atoms, an alkyl group having 1 to 4 carbon atoms, and a
halogen atom.
[0010] The compound of the present invention is a compound
represented by the above general formula (III) or a salt
thereof.
[0011] Also, the production method of the present invention is a
production method for producing a compound represented by the
general formula (I) or a salt thereof, comprising the step of
producing a compound represented by the general formula (III) or a
salt thereof, the step comprising reacting a trimethylsilylated
compound, which was obtained by reaction of a compound represented
by the following general formula:
##STR00007##
wherein R.sup.4, R.sup.5, and R.sup.6 are as defined above, with a
trimethylsilylating agent, with a compound represented by the
following general formula:
##STR00008##
wherein X, Y, Z, A, and R are as defined above, or a salt thereof:
to obtain the compound represented by the following general formula
or a salt thereof:
##STR00009##
wherein X, Y, Z, A, R, and B are as defined above:
##STR00010##
wherein X, Y, A, and B are as defined above.
[0012] In the present invention, examples of the "alkylene group
having 1 to 4 carbon atoms" represented by A can include methylene,
ethylene, trimethylene, and tetramethylene groups. The alkylene
group is preferably a methylene or ethylene group, more preferably
a methylene group.
[0013] In the present invention, the "protective group for the
hydroxy group" represented by X, Y, or Z and the protective group
in the "protected hydroxy group" included in the group .alpha.
refer to protective groups that are cleavable by a chemical method
such as hydrogenolysis, hydrolysis, electrolysis, or photolysis or
by a biological method such as hydrolysis in the human body.
Examples of such protective groups can include: "aliphatic acyl
groups" such as alkylcarbonyl groups (e.g., formyl, acetyl,
propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl,
isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl,
8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl,
undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl,
hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl,
13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl,
octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl, and
heneicosanoyl), carboxylated alkylcarbonyl groups (e.g., succinoyl,
glutaroyl, and adipoyl), halogeno-lower alkylcarbonyl groups (e.g.,
chloroacetyl, dichloroacetyl, trichloroacetyl, and
trifluoroacetyl), lower alkoxy lower alkylcarbonyl groups (e.g.,
methoxyacetyl), and unsaturated alkylcarbonyl groups (e.g.,
(E)-2-methyl-2-butenoyl); "aromatic acyl groups" such as
arylcarbonyl groups (e.g., benzoyl, .alpha.-naphthoyl, and
.beta.-naphthoyl), halogenoarylcarbonyl groups (e.g.,
2-bromobenzoyl and 4-chlorobenzoyl), lower alkylated arylcarbonyl
groups (e.g., 2,4,6-trimethylbenzoyl and 4-toluoyl), lower
alkoxylated arylcarbonyl groups (e.g., 4-anisoyl), carboxylated
arylcarbonyl groups (e.g., 2-carboxybenzoyl, 3-carboxybenzoyl, and
4-carboxybenzoyl), nitrated arylcarbonyl groups (e.g.,
4-nitrobenzoyl and 2-nitrobenzoyl), lower alkoxycarbonylated
arylcarbonyl groups (e.g., 2-(methoxycarbonyl)benzoyl), and
arylated arylcarbonyl groups (e.g., 4-phenylbenzoyl);
"tetrahydropyranyl or tetrahydrothiopyranyl groups" such as
tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl,
4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran-2-yl, and
4-methoxytetrahydrothiopyran-4-yl; "tetrahydrofuranyl or
tetrahydrothiofuranyl groups" such as tetrahydrofuran-2-yl and
tetrahydrothiofuran-2-yl; "lower alkoxymethyl groups" such as
methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl,
propoxymethyl, isopropoxymethyl, butoxymethyl, and t-butoxymethyl;
"lower alkoxylated lower alkoxymethyl groups" such as
2-methoxyethoxymethyl; "halogeno-lower alkoxymethyl" such as
2,2,2-trichloroethoxymethyl and bis(2-chloroethoxy)methyl; "lower
alkoxylated ethyl groups" such as 1-ethoxyethyl and
1-(isopropoxy)ethyl; "halogenated ethyl groups" such as
2,2,2-trichloroethyl; a "methyl group substituted by 1 to 3 aryl
groups" such as benzyl, .alpha.-naphthylmethyl,
.beta.-naphthylmethyl, diphenylmethyl, triphenylmethyl,
.alpha.-naphthyldiphenylmethyl, and 9-anthrylmethyl; a "methyl
group substituted by 1 to 3 aryl groups with the aryl ring
substituted by lower alkyl, lower alkoxy, halogen, or a cyano
group" such as 4-methylbenzyl, 2,4,6-trimethylbenzyl,
3,4,5-trimethylbenzyl, 4-methoxybenzyl,
4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl,
2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, and
4-cyanobenzyl; "lower alkoxycarbonyl groups" such as
methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, and
isobutoxycarbonyl; "halogen-substituted lower alkoxycarbonyl
groups" such as 2,2,2-trichloroethoxycarbonyl; "alkenyloxycarbonyl
groups" such as vinyloxycarbonyl and aryloxycarbonyl; and
"aralkyloxycarbonyl groups with the aryl ring optionally
substituted by 1 or 2 lower alkoxy or nitro groups" such as
benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and
4-nitrobenzyloxycarbonyl. The "protective group for the hydroxy
group" represented by X or Y is preferably a "methyl group
substituted by 1 to 3 aryl groups" or a "methyl group substituted
by 1 to 3 aryl groups with the aryl ring substituted by lower
alkyl, lower alkoxy, halogen, or a cyano group", more preferably a
benzyl, .beta.-naphthylmethyl, p-methoxybenzyl, dimethoxytrityl, or
monomethoxytrityl group. The "protective group for the hydroxy
group" represented by Z is preferably an aliphatic acyl group
having 2 to 4 carbon atoms (e.g., an acetyl, propionyl, or butyryl
group), more preferably an acetyl group. The "protected hydroxy
group" included in the group .alpha. is preferably an "ethyl group
substituted by a nitrated aryl group" or an "arylated aminocarbonyl
group", more preferably a 1-(4-nitrophenyl)ethyl group or a
diphenylaminocarbonyl group.
[0014] In the present invention, the "silyl protective group"
represented by R can be a "silyl protective group" such as a
tri-lower alkylsilyl group (e.g., trimethylsilyl, triethylsilyl,
isopropyldimethylsilyl, t-butyldimethylsilyl,
methyldiisopropylsilyl, methyl-di-t-butylsilyl, or
triisopropylsilyl); or a monoaryl di-lower alkylsilyl or diaryl
mono-lower alkylsilyl group (e.g., diphenylmethylsilyl,
diphenylbutylsilyl, diphenylisopropylsilyl, t-butyldiphenylsilyl,
or phenyldiisopropylsilyl). The silyl protective group is
preferably a monoaryl di-lower alkylsilyl or diaryl mono-lower
alkylsilyl group, more preferably a t-butyldiphenylsilyl group.
[0015] In the present invention, examples of the "alkoxy group
having 1 to 4 carbon atoms" included in the group .alpha. can
include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,
isobutoxy, s-butoxy, and t-butoxy groups. The alkoxy group is
preferably a methoxy or ethoxy group.
[0016] In the present invention, examples of the protective group
in the "protected mercapto group" included in the group .alpha. can
include those listed above as the protective group for the hydroxy
group as well as "disulfide-forming groups" such as alkylthio
groups (e.g., methylthio, ethylthio, and t-butylthio) and arylthio
groups (e.g., benzylthio). The protective group is preferably an
"aliphatic acyl group" or an "aromatic acyl group", more preferably
a benzoyl group.
[0017] In the present invention, examples of the "alkylthio group
having 1 to 4 carbon atoms" included in the group .alpha. can
include methylthio, ethylthio, propylthio, isopropylthio,
butylthio, isobutylthio, s-butylthio, and t-butylthio groups. The
alkylthio group is preferably a methylthio or ethylthio group.
[0018] In the present invention, examples of the protective group
in the "protected amino group" included in the group .alpha. can
include: "aliphatic acyl groups" such as alkylcarbonyl groups
(e.g., formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl,
pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl,
3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl,
3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl,
tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl,
14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl,
15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl,
nonadecanoyl, eicosanoyl, and heneicosanoyl), carboxylated
alkylcarbonyl groups (e.g., succinoyl, glutaroyl, and adipoyl),
halogeno-lower alkylcarbonyl groups (e.g., chloroacetyl,
dichloroacetyl, trichloroacetyl, and trifluoroacetyl), lower alkoxy
lower alkylcarbonyl groups (e.g., methoxyacetyl), aryloxy lower
alkylcarbonyl groups (e.g., a phenoxyacetyl group), aryloxy lower
alkylcarbonyl groups (e.g., a 4-(t-butyl)phenoxyacetyl group), and
unsaturated alkylcarbonyl groups (e.g., (E)-2-methyl-2-butenoyl);
"aromatic acyl groups" such as arylcarbonyl groups (e.g., benzoyl,
.alpha.-naphthoyl, and .beta.-naphthoyl), halogenoarylcarbonyl
groups (e.g., 2-bromobenzoyl and 4-chlorobenzoyl), lower alkylated
arylcarbonyl groups (e.g., 2,4,6-trimethylbenzoyl and 4-toluoyl),
lower alkoxylated arylcarbonyl groups (e.g., 4-anisoyl),
carboxylated arylcarbonyl groups (e.g., 2-carboxybenzoyl,
3-carboxybenzoyl, and 4-carboxybenzoyl), nitrated arylcarbonyl
groups (e.g., 4-nitrobenzoyl and 2-nitrobenzoyl), lower
alkoxycarbonylated arylcarbonyl groups (e.g.,
2-(methoxycarbonyl)benzoyl), and arylated arylcarbonyl groups
(e.g., 4-phenylbenzoyl); "lower alkoxycarbonyl groups" such as
methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, and
isobutoxycarbonyl; "lower alkoxycarbonyl groups substituted by
halogen or a tri-lower alkylsilyl group" such as
2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl;
"alkenyloxycarbonyl groups" such as vinyloxycarbonyl and
aryloxycarbonyl; and "aralkyloxycarbonyl groups with the aryl ring
optionally substituted by 1 or 2 lower alkoxy or nitro groups" such
as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and
4-nitrobenzyloxycarbonyl. The protective group is preferably an
"aliphatic acyl group" or an "aromatic acyl group", more preferably
an isobutyryl or benzoyl group, most preferably an isobutyryl
group.
[0019] In the present invention, examples of the "amino group
substituted by (an) alkyl group(s) having 1 to 4 carbon atoms"
included in the group .alpha. can include methylamino, ethylamino,
propylamino, isopropylamino, butylamino, isobutylamino,
s-butylamino, t-butylamino, dimethylamino, diethylamino,
dipropylamino, diisopropylamino, dibutylamino, diisobutylamino,
di(s-butyl)amino, and di(t-butyl) amino groups. The amino group
substituted by (an) alkyl group(s) is preferably a methylamino,
ethylamino, dimethylamino, diethylamino, or diisopropylamino
group.
[0020] In the present invention, examples of the "alkyl group
having 1 to 4 carbon atoms" included in the group .alpha. can
include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl,
and t-butyl. The alkyl group is preferably a methyl or ethyl
group.
[0021] In the present invention, the "halogen atom" included in the
group .alpha. refers to a fluorine atom, a chlorine atom, a bromine
atom, or an iodine atom and is preferably a fluorine atom or a
chlorine atom.
[0022] In the present invention, the "purin-9-yl group" or the
"substituted purin-9-yl group" represented by B is preferably a
6-aminopurin-9-yl (i.e., adeninyl) group, a 6-aminopurin-9-yl group
with the amino group protected, a 2,6-diaminopurin-9-yl group, a
2-amino-6-chloropurin-9-yl group, a 2-amino-6-chloropurin-9-yl
group with the amino group protected, a 2-amino-6-fluoropurin-9-yl
group, a 2-amino-6-fluoropurin-9-yl group with the amino group
protected, a 2-amino-6-bromopurin-9-yl group, a
2-amino-6-bromopurin-9-yl group with the amino group protected, a
2-amino-6-hydroxypurin-9-yl (i.e., guaninyl) group, a
2-amino-6-hydroxypurin-9-yl group with the amino group protected, a
2-amino-6-hydroxypurin-9-yl group with the amino group and hydroxy
group protected, a 6-amino-2-methoxypurin-9-yl group, a
6-amino-2-chloropurin-9-yl group, a 6-amino-2-fluoropurin-9-yl
group, a 2,6-dimethoxypurin-9-yl group, a 2,6-dichloropurin-9-yl
group, or a 6-mercaptopurin-9-yl group, more preferably a
6-benzoylaminopurin-9-yl group, an adeninyl group, a
2-isobutyrylamino-6-hydroxypurin-9-yl group, or a guaninyl
group.
[0023] In the present invention, the "salt thereof" refers to a
salt of compound (II) or (III), because these compounds can form
salts. Such salts can be preferably metal salts such as alkali
metal salts (e.g., sodium salt, potassium salt, and lithium salt),
alkaline earth metal salts (e.g., calcium salt and magnesium salt),
aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and
cobalt salt; amine salts such as inorganic salts (e.g., ammonium
salt) and organic salts (e.g., t-octylamine salt, dibenzylamine
salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester
salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt,
diethylamine salt, triethylamine salt, dicyclohexylamine salt,
N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine
salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine
salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane
salt); inorganic acid salts such as hydrohalides (e.g.,
hydrofluoride, hydrochloride, hydrobromide, and hydroiodide),
nitrate, perchlorate, sulfate, and phosphate; organic acid salts
such as lower alkanesulfonates (e.g., methanesulfonate,
trifluoromethanesulfonate, and ethanesulfonate), arylsulfonates
(e.g., benzenesulfonate and p-toluenesulfonate), acetate, malate,
fumarate, succinate, citrate, tartrate, oxalate, and maleate; and
amino acid salts such as glycine salt, lysine salt, arginine salt,
ornithine salt, glutamate, and aspartate.
[0024] Preferred embodiments of the production method of the
present invention are as follows:
[0025] (1) The production method, wherein X is a methyl group
substituted by 1 to 3 aryl groups, or a methyl group substituted by
1 to 3 aryl groups with the aryl ring substituted by lower alkyl,
lower alkoxy, halogen, or a cyano group.
[0026] (2) The production method, wherein X is a benzyl group, a
p-methoxybenzyl group, a dimethoxytrityl group, or a
monomethoxytrityl group.
[0027] (3) The production method, wherein Y is a methyl group
substituted by 1 to 3 aryl groups, or a methyl group substituted by
1 to 3 aryl groups with the aryl ring substituted by lower alkyl,
lower alkoxy, halogen, or a cyano group.
[0028] (4) The production method, wherein Y is a benzyl group, a
.beta.-naphthylmethyl group, or a p-methoxybenzyl group.
[0029] (5) The production method, wherein Z is an aliphatic acyl
group having 2 to 4 carbon atoms.
[0030] (6) The production method, wherein Z is an acetyl group.
[0031] (7) The production method, wherein A is a methylene group or
an ethylene group.
[0032] (8) The production method, wherein A is a methylene
group.
[0033] (9) The production method, wherein B is a 6-aminopurin-9-yl
(i.e., adeninyl) group, a 6-aminopurin-9-yl group with the amino
group protected, a 2,6-diaminopurin-9-yl group, a
2-amino-6-chloropurin-9-yl group, a 2-amino-6-chloropurin-9-yl
group with the amino group protected, a 2-amino-6-fluoropurin-9-yl
group, a 2-amino-6-fluoropurin-9-yl group with the amino group
protected, a 2-amino-6-bromopurin-9-yl group, a
2-amino-6-bromopurin-9-yl group with the amino group protected, a
2-amino-6-hydroxypurin-9-yl (i.e., guaninyl) group, a
2-amino-6-hydroxypurin-9-yl group with the amino group protected, a
2-amino-6-hydroxypurin-9-yl group with the amino group and hydroxy
group protected, a 6-amino-2-methoxypurin-9-yl group, a
6-amino-2-chloropurin-9-yl group, a 6-amino-2-fluoropurin-9-yl
group, a 2,6-dimethoxypurin-9-yl group, a 2,6-dichloropurin-9-yl
group, or a 6-mercaptopurin-9-yl group.
[0034] (10) The production method, wherein B is a
6-benzoylaminopurin-9-yl group, an adeninyl group, a
2-isobutyrylamino-6-hydroxypurin-9-yl group, or a guaninyl
group.
[0035] (11) The production method, wherein R is a tri-lower
alkylsilyl group, a monoaryl di-lower alkylsilyl group, or a diaryl
mono-lower alkylsilyl group.
[0036] (12) The production method, wherein R is a monoaryl di-lower
alkylsilyl group or a diaryl mono-lower alkylsilyl group.
[0037] (13) The production method, wherein R is a
t-butyldiphenylsilyl group.
[0038] The larger number between or among (1) and (2), (3) and (4),
(5) and (6), (7) and (8), (9) and (10), or (11) to (13) mentioned
above represents a more preferred embodiment of the production
method. The production method of the present invention is also
preferably a production method, wherein X is arbitrarily selected
from (1) and (2); Y is arbitrarily selected from (3) and (4); Z is
arbitrarily selected from (5) and (6); A is arbitrarily selected
from (7) and (8); B is arbitrarily selected from (9) and (10); or R
is arbitrarily selected from (11) to (13); or these forms are
arbitrarily combined.
[0039] Preferred embodiments of the compound (III) of the present
invention are as follows:
[0040] (1) The compound or a salt thereof, wherein X is a methyl
group substituted by 1 to 3 aryl groups, or a methyl group
substituted by 1 to 3 aryl groups with the aryl ring substituted by
lower alkyl, lower alkoxy, halogen, or a cyano group.
[0041] (2) The compound or a salt thereof, wherein X is a benzyl
group, a p-methoxybenzyl group, a dimethoxytrityl group, or a
monomethoxytrityl group.
[0042] (3) The compound or a salt thereof, wherein Y is a methyl
group substituted by 1 to 3 aryl groups, or a methyl group
substituted by 1 to 3 aryl groups with the aryl ring substituted by
lower alkyl, lower alkoxy, halogen, or a cyano group.
[0043] (4) The compound or a salt thereof, wherein Y is a benzyl
group, a .beta.-naphthylmethyl group, or a p-methoxybenzyl
group.
[0044] (5) The compound or a salt thereof, wherein Z is an
aliphatic acyl group having 2 to 4 carbon atoms.
[0045] (6) The compound or a salt thereof, wherein Z is an acetyl
group.
[0046] (7) The compound or a salt thereof, wherein A is a methylene
group or an ethylene group.
[0047] (8) The compound or a salt thereof, wherein A is a methylene
group.
[0048] (9) The compound or a salt thereof, wherein B is a
6-aminopurin-9-yl (i.e., adeninyl) group, a 6-aminopurin-9-yl group
with the amino group protected, a 2,6-diaminopurin-9-yl group, a
2-amino-6-chloropurin-9-yl group, a 2-amino-6-chloropurin-9-yl
group with the amino group protected, a 2-amino-6-fluoropurin-9-yl
group, a 2-amino-6-fluoropurin-9-yl group with the amino group
protected, a 2-amino-6-bromopurin-9-yl group, a
2-amino-6-bromopurin-9-yl group with the amino group protected, a
2-amino-6-hydroxypurin-9-yl (i.e., guaninyl) group, a
2-amino-6-hydroxypurin-9-yl group with the amino group protected, a
2-amino-6-hydroxypurin-9-yl group with the amino group and hydroxy
group protected, a 6-amino-2-methoxypurin-9-yl group, a
6-amino-2-chloropurin-9-yl group, a 6-amino-2-fluoropurin-9-yl
group, a 2,6-dimethoxypurin-9-yl group, a 2,6-dichloropurin-9-yl
group, or a 6-mercaptopurin-9-yl group.
[0049] (10) The compound or a salt thereof, wherein B is a
6-benzoylaminopurin-9-yl group, an adeninyl group, a
2-isobutyrylamino-6-hydroxypurin-9-yl group, or a guaninyl
group.
[0050] (11) The compound or a salt thereof, wherein R is a
tri-lower alkylsilyl group, a monoaryl di-lower alkylsilyl group,
or a diaryl mono-lower alkylsilyl group.
[0051] (12) The compound or a salt thereof, wherein R is a monoaryl
di-lower alkylsilyl group or a diaryl mono-lower alkylsilyl
group.
[0052] (13) The compound or a salt thereof, wherein R is a
t-butyldiphenylsilyl group.
[0053] The larger number between or among (1) and (2), (3) and (4),
(5) and (6), (7) and (8), (9) and (10), or (11) to (13) mentioned
above represents a more preferred embodiment of the compound. The
production intermediate of the present invention is also preferably
a compound or a salt thereof, wherein X is arbitrarily selected
from (1) and (2); Y is arbitrarily selected from (3) and (4); Z is
arbitrarily selected from (5) and (6); A is arbitrarily selected
from (7) and (8); B is arbitrarily selected from (9) and (10); or R
is arbitrarily selected from (11) to (13); or these forms are
arbitrarily combined, particularly preferably
2'-O-acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-6-N-ben-
zoyladenosine,
2'-O-acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-2-N-iso-
butyrylguanosine, or a salt thereof, most preferably
2'-O-acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-2-N-iso-
butyrylguanosine or a salt thereof.
[0054] The compound (IVb) is purine, or substituted purine
optionally having a substituent selected from the group consisting
of (a) hydroxy group(s), (a) protected hydroxy group(s), (an)
alkoxy group(s) having 1 to 4 carbon atoms, (a) mercapto group(s),
(a) protected mercapto group(s), (an) alkylthio group(s) having 1
to 4 carbon atoms, (an) amino group(s), (a) protected amino
group(s), (an) amino group(s) substituted by (an) alkyl group(s)
having 1 to 4 carbon atoms, (an) alkyl group(s) having 1 to 4
carbon atoms, and (a) halogen atom(s) and is preferably a compound
or a salt thereof selected from the following group:
(Compound Group)
[0055] Adenine with the amino group protected, guanine with the
amino group protected, and salts thereof, preferably adenine with
the amino group protected with an "aliphatic acyl group" or an
"aromatic acyl group", guanine with the amino group protected with
an "aliphatic acyl group" or an "aromatic acyl group", and salts
thereof, more preferably N6-benzoyladenine, N6-acetyladenine,
N6-phenoxyacetyladenine, N6-(t-butyl)phenoxyacetyladenine,
N2-isobutyrylguanine, N2-acetylguanine, N2-phenoxyacetylguanine,
N2-(t-butyl)phenoxyacetylguanine, and salts thereof, even more
preferably N6-benzoyladenine, N2-isobutyrylguanine, and salts
thereof.
[0056] The trimethylsilylated compound according to the present
invention refers to a compound that is produced through the
reaction of compound (IVb) with a trimethylsilylating agent
resulting in the bonding of a trimethylsilyl group in place of a
hydrogen atom bonded to a nitrogen atom of an amino group, a
hydrogen atom bonded to a nitrogen atom in the heterocyclic ring
and/or a hydrogen atom bonded to an oxygen atom of a hydroxy group
in the compound (IVb). The trimethylsilylated compound is
relatively unstable and is therefore used in the subsequent
reaction without being isolated. The trimethylsilylated compound is
preferably a compound in which a trimethylsilyl group is bonded in
place of a hydrogen atom bonded to a nitrogen atom of an amino
group, a hydrogen atom bonded to a nitrogen atom in the
heterocyclic ring and/or a hydrogen atom bonded to an oxygen atom
of a hydroxy group, in an adenine in which the amino group is
protected with an "aliphatic acyl group" or an "aromatic acyl
group" or a guanine in which the amino group is protected with an
"aliphatic acyl group" or an "aromatic acyl group", or a salt
thereof, more preferably a compound in which a trimethylsilyl group
is bonded in place of a hydrogen atom bonded to a nitrogen atom of
an amino group, a hydrogen atom bonded to a nitrogen atom in the
heterocyclic ring and/or a hydrogen atom bonded to an oxygen atom
of a hydroxy group, in an N6-benzoyladenine, N6-acetyladenine,
N6-phenoxyacetyladenine, N6-(t-butyl)phenoxyacetyladenine,
N2-isobutyrylguanine, N2-acetylguanine, N2-phenoxyacetylguanine, or
N2-(t-butyl)phenoxyacetylguanine, or a salt thereof, particularly
preferably a compound in which a trimethylsilyl group is bonded in
place of a hydrogen atom bonded to a nitrogen atom of an amino
group, a hydrogen atom bonded to a nitrogen atom in the
heterocyclic ring and/or a hydrogen atom bonded to an oxygen atom
of a hydroxy group, in an N6-benzoyladenine or
N2-isobutyrylguanine, or a salt thereof.
[0057] The trimethylsilylated compound is, for example,
N6-benzoyl-N6,N9-bis(trimethylsilyl)adenine or
O6,N2,N9-tris(trimethylsilyl)-N2-isobutyrylguanine.
[0058] The compound (III), the compound (IVb), or the
trimethylsilylated compound of the compound (IVb) according to the
present invention may have tautomerism. Any tautomeric structure of
the following substructure in these compounds is included in the
scope of the present invention:
##STR00011##
wherein R.sup.7 represents a hydrogen, a protective group for the
hydroxy group, a protective group for the amino group, or a
trimethylsilyl group.
Advantageous Effects of Invention
[0059] According to the present invention, an oligonucleotide
analog having stable and excellent antisense or antigene activity
or having excellent activity as a detection reagent (probe) for a
specific gene or as a primer for initiation of amplification of a
specific gene, and intermediate compound (III) for the production
of nucleoside analog compound (I) which serves as an intermediate
for the production thereof can be produced at high yields
regardless of the type of nucleobase.
DESCRIPTION OF EMBODIMENTS
[0060] The production method of the present invention is a method
for producing compound (III), comprising reacting compound (IVb)
with a trimethylsilylating agent, and then reacting the obtained
trimethylsilylated compound with compound (II).
##STR00012##
wherein R.sup.4, R.sup.5, R.sup.6, X, Y, Z, A, R, and B are as
defined above.
(1) Step of Producing Compound (II)
[0061] Compound (II), which is a starting material in this step,
can be produced through the reaction of compound (3) of method A
described in Japanese Patent Laid-Open No. 2000-297097 with a silyl
protecting agent in the presence of a base catalyst in an inert
solvent.
[0062] The silyl protecting agent used is a tri-lower alkylsilyl
chloride such as trimethylsilyl chloride, triethylsilyl chloride,
isopropyldimethylsilyl chloride, t-butyldimethylsilyl chloride,
methyldiisopropylsilyl chloride, methyl-di-t-butylsilyl chloride,
or triisopropylsilyl chloride; or a monoaryl di-lower alkylsilyl or
diaryl mono-lower alkylsilyl chloride such as diphenylmethylsilyl
chloride, diphenylbutylsilyl chloride, diphenylisopropylsilyl
chloride, t-butyldiphenylsilyl chloride, or phenyldiisopropylsilyl
chloride.
[0063] Examples of the solvent used can include: aromatic
hydrocarbons such as benzene, toluene, and xylene; halogenated
hydrocarbons such as methylene chloride, chloroform, carbon
tetrachloride, 1,2-dichloroethane, chlorobenzene, and
dichlorobenzene; nitriles such as acetonitrile and
isobutyronitrile; amides such as formamide, N,N-dimethylformamide,
N,N-dimethylacetamide, N-methyl-2-pyrrolidone,
N-methylpyrrolidinone, and hexamethylphosphortriamide; and carbon
sulfide. The solvent is preferably N,N-dimethylformamide.
[0064] Examples of the base catalyst used include organic bases
such as triethylamine, pyridine, N-methylmorpholine, DBU, and
imidazole. The base catalyst is preferably imidazole.
[0065] The reaction temperature differs depending on the starting
compound, solvent, and base catalyst used and is usually 0.degree.
C. to 100.degree. C., preferably 0.degree. C. to 50.degree. C.
[0066] The reaction time differs depending on the starting
compound, solvent, and base catalyst used, and the reaction
temperature and is usually 0.5 hours to 24 hours, preferably 1 hour
to 8 hours.
[0067] After the completion of the reaction, the compound (II) of
interest of this reaction is obtained, for example, by:
concentrating the reaction mixture; adding water and an immiscible
organic solvent such as ethyl acetate to the residue; after washing
with water, separating the organic layer containing the compound of
interest; drying the organic layer over anhydrous magnesium sulfate
or the like; and then distilling off the solvent.
[0068] The obtained compound can be further purified, if necessary,
by a routine method, for example, recrystallization or silica gel
column chromatography.
(2) Step of Producing Trimethylsilylated Compound
[0069] The trimethylsilylated compound used in this step can be
produced through the reaction of compound (IVb) with a
trimethylsilylating agent.
[0070] The trimethylsilylating agent used can be trimethylsilyl
chloride, N,O-bis(trimethylsilyl)acetamide (BSA),
N,O-bis(trimethylsilyl)trifluoroacetamide,
N-methyl-N-(trimethylsilyl)trifluoroacetamide,
1,1,1,3,3,3-hexamethyldisilazane (HMDS), or trimethylsilyl
trifluoromethanesulfonate and is preferably trimethylsilyl chloride
or N,O-bis(trimethylsilyl)acetamide (BSA).
[0071] The trimethylsilylating agent is usually used at 1 to 100
equivalents, preferably 2 to 50 equivalents, particularly
preferably 5 to 30 equivalents, with respect to one reaction site
of 1 to 4 hydrogen atoms bonded to a nitrogen atom of the N--H
group or an oxygen atom of a hydroxy group in the compound
(IVb).
[0072] Examples of the solvent used can include: aromatic
hydrocarbons such as benzene, toluene, and xylene; halogenated
hydrocarbons such as methylene chloride, chloroform, carbon
tetrachloride, 1,2-dichloroethane, chlorobenzene, and
dichlorobenzene; nitriles such as acetonitrile and
isobutyronitrile; amides such as formamide, N,N-dimethylformamide,
N,N-dimethylacetamide, N-methyl-2-pyrrolidone,
N-methylpyrrolidinone, and hexamethylphosphortriamide;
1,1,1,3,3,3-hexamethyldisilazane (HMDS); and carbon sulfide. The
solvent is preferably toluene, acetonitrile, or HMDS.
[0073] In this step, for example, an organic base such as
triethylamine, pyridine, N-methylmorpholine, DBU, or imidazole may
be used as a base catalyst according to the need. The base catalyst
is preferably triethylamine.
[0074] The reaction temperature differs depending on the starting
compound, solvent, and base catalyst used and is usually 0.degree.
C. to 180.degree. C., preferably 20.degree. C. to 120.degree.
C.
[0075] After the completion of the reaction, the compound of
interest of this reaction is used in the subsequent step without
being isolated, for example, after concentration of the reaction
mixture and drying of the residue under reduced pressure.
(3) Step of Producing Compound (III)
[0076] This step involves reacting the said trimethylsilylated
compound with the said compound (II) in the presence of an acid
catalyst in an inert solvent to produce compound (III).
[0077] Examples of the solvent used can include: aromatic
hydrocarbons such as benzene, toluene, and xylene; halogenated
hydrocarbons such as methylene chloride, chloroform, carbon
tetrachloride, 1,2-dichloroethane, chlorobenzene, and
dichlorobenzene; nitriles such as acetonitrile and
isobutyronitrile; amides such as formamide, N,N-dimethylformamide,
N,N-dimethylacetamide, N-methyl-2-pyrrolidone,
N-methylpyrrolidinone, and hexamethylphosphortriamide; and carbon
sulfide. The solvent is preferably an aromatic hydrocarbon or a
nitrile, more preferably toluene or acetonitrile.
[0078] Examples of the acid catalyst used include Lewis acid
catalysts such as aluminum chloride, tin tetrachloride, titanium
tetrachloride, trifluoroboron, and trimethylsilyl
trifluoromethanesulfonate. The acid catalyst is preferably
trimethylsilyl trifluoromethanesulfonate.
[0079] The reaction temperature differs depending on the starting
compound, solvent, and acid catalyst used and is usually 0.degree.
C. to 150.degree. C., preferably 70.degree. C. to 120.degree.
C.
[0080] The reaction time differs depending on the starting
compound, solvent, and acid catalyst used, and the reaction
temperature and is usually 0.5 hours to 24 hours, preferably 1 hour
to 8 hours.
[0081] After the completion of the reaction, the compound (III) of
interest of this reaction is obtained, for example, by:
concentrating the reaction mixture; adding water and an immiscible
organic solvent such as ethyl acetate to the residue; after washing
with water, separating the organic layer containing the compound of
interest; drying the organic layer over anhydrous magnesium sulfate
or the like; and then distilling off the solvent.
[0082] The obtained compound can be further purified, if necessary,
by a routine method, for example, recrystallization or silica gel
column chromatography.
[0083] In this step of producing compound (III), the
trimethylsilylated compound can also be formed in a reaction system
containing compound (IVb) and a trimethylsilylating agent and then
reacted with compound (II) in one pot without being isolated.
[0084] The trimethylsilylating agent used in the one-pot reaction
is, for example, N,O-bis(trimethylsilyl)acetamide (BSA) or
1,1,1,3,3,3-hexamethyldisilazane (HMDS) and is preferably
N,O-bis(trimethylsilyl)acetamide.
EXAMPLES
[0085] Hereinafter, the present invention will be described more
specifically with reference to Examples and Reference Examples.
Example 1
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-6-N-benz-
oyladenosine
##STR00013##
[0086] (1)
3,5-di-O-Benzyl-4-(2-t-butyldiphenylsilyloxyethyl)-1,2-O-isopro-
pylidene-.alpha.-D-erythropentofuranose
[0087]
3,5-di-O-Benzyl-4-(2-hydroxyethyl)-1,2-O-isopropylidene-.alpha.-D-e-
rythropentofuranose (18.3 g, 44.2 mmol) was dissolved in dry
dimethylformamide (DMF, 55 mL). Under a nitrogen stream, imidazole
(15.7 g, 218 mmol) was added to this solution, and the mixture was
cooled to 0.degree. C. t-Butyldiphenylchlorosilane (23.0 mL, 88.4
mmol) was added thereto, and the mixture was stirred for 15
minutes. After the completion of the reaction, the reaction
solution was diluted with ether (ca. 3 mL). A saturated aqueous
solution of sodium bicarbonate (ca. 50 mL) was added thereto, and
the mixture was concentrated under reduced pressure. Ether and DMF
were distilled off. Water (ca. 10 mL) was added to the residue,
followed by extraction with ethyl acetate (30 mL.times.3). The
organic layer was washed with saturated saline, dried over
anhydrous magnesium sulfate, and then concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (elution solvent: hexane/ethyl acetate=5/1) to
obtain the title compound (28.8 g).
(2)
3,5-di-O-Benzyl-4-(2-t-butyldiphenylsilyloxyethyl)-1,2-di-O-acetyl-.al-
pha.-D-erythropentofuranose
[0088] The compound (containing an amount corresponding to 28.8 g,
44.2 mmol) obtained in step (1) was dissolved in acetic acid (150
mL), and this solution was cooled to 0.degree. C. Acetic anhydride
(82.2 mL, 871 mmol) and concentrated sulfuric acid (cat. 10 .mu.L)
were added thereto, and the mixture was then stirred for 30
minutes. After the completion of the reaction, the reaction
solution was put in ice water (50 mL), and the mixture was stirred
for 1 hour. Saturated saline (ca. 50 mL) was added thereto,
followed by extraction with ethyl acetate (50 mL.times.3). The
organic layer was washed with a saturated aqueous solution of
sodium bicarbonate and saturated saline in this order, dried over
anhydrous magnesium sulfate, and then concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (elution solvent: hexane/ethyl acetate=10/1) to
obtain the title compound (mixture of .alpha. and .beta. forms,
26.0 g, yield: 84% (2 steps)). The NMR data of the a form obtained
by the further purification of a portion of the obtained compound
will be shown below.
[0089] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.02 (9H,
s), 1.84 (3H, s), 1.93 (3H, s), 1.95-2.18 (2H, m), 3.40 (1H, d),
3.50 (1H, d), 3.80-3.93 (2H, m), 4.35-4.60 (5H, m), 5.29 (1H, d),
6.03 (1H, s), 7.20-7.40 (16H, m), 7.60-7.70 (4H, m).
(3)
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-6-N--
benzoyladenosine
[0090] HMDS (20 mL) and trimethylsilyl chloride (4 mL) were added
to N6-benzoyladenine (268 mg, 1.12 mmol), and the mixture was
refluxed overnight, concentrated under reduced pressure, and dried
to obtain trimethylsilylated N6-benzoyladenine.
[0091] The compound (521 mg, 0.748 mmol) obtained in step (2) was
dissolved in dry toluene (10 mL). Under a nitrogen stream, the
above-mentioned trimethylsilylated N6-benzoyladenine and
trimethylsilyl trifluoromethanesulfonate (TMSOTf, 166 .mu.L, 0.901
mmol) were added to this solution, and the mixture was stirred.
After 5 minutes, the consumption of the starting material was
confirmed by thin-layer chromatography (TLC), followed by heating
to reflux for 2 hours. After the completion of the reaction, a
saturated aqueous solution of sodium bicarbonate (ca. 2 mL) was
added to the reaction solution, and the mixture was filtered
through celite. The filtrate was subjected to extraction with
dichloromethane (ca. 10 mL). The organic layer was washed with a
saturated sodium bicarbonate solution and saturated saline in this
order, dried over anhydrous magnesium sulfate, and then
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (elution solvent:
dichloromethane/methanol=100/1) to obtain the title compound (482
mg, yield: 74%).
[0092] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.02 (9H,
s), 1.88-1.97 (1H, m), 2.02 (3H, s), 2.15-2.25 (1H, s), 3.44 (1H,
d), 3.75-3.90 (3H, m), 4.37-4.61 (5H, m), 5.86 (1H, t), 6.26 (1H,
d), 7.20-7.65 (23H, m), 8.03 (2H, d), 8.31 (1H, s), 8.77 (1H, s),
9.04 (1H, s).
Example 2
3',5'-di-O-Benzyl-2'-O-4'-C-ethylene-6-N-benzoyladenosine
(1)
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-hydroxyethyl)-6-N-benzoyladenosine
[0093] The compound (475 mg, 0.542 mmol) obtained in Example 1(3)
was dissolved in dry tetrahydrofuran (THF, 5 mL). A solution of
tetrabutylammonium fluoride in THF (1 mol/L, 705 .mu.L, 0.705 mmol)
was added to this solution, and the mixture was stirred at room
temperature for 12 hours. After the completion of the reaction, the
reaction solution was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (elution
solvent: dichloromethane/methanol=50/1) to obtain the title
compound (269 mg, yield: 78%).
[0094] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.88-1.95
(1H, m), 2.08 (3H, s), 2.20-2.30 (2H, m), 3.47 (1H, d), 3.74 (1H,
d), 3.76-3.85 (2H, m), 4.42-4.68 (5H, m), 5.97 (1H, t), 6.35 (1H,
d), 7.24-7.65 (13H, m), 8.03 (2H, d), 8.26 (1H, s), 8.76 (1H, s),
9.06 (1H, s).
(2) 3',5'-di-O-Benzyl-2'-O-4'-C-ethylene-6-N-benzoyladenosine
[0095] Under a nitrogen stream, the compound (102 mg, 0.106 mmol)
obtained in step (1) was dissolved in dry dichloromethane (2 mL),
pyridine (100 .mu.L) and methanesulfonyl chloride (36 .mu.L, 0.465
mmol) were added to this solution, and the mixture was stirred at
room temperature for 12 hours. Then, water (ca. 1 mL) was added
thereto, followed by extraction with dichloromethane (5 mL). The
organic layer was washed with saturated saline, dried over
anhydrous magnesium sulfate, and then concentrated under reduced
pressure. The residue was dissolved in a mixed solvent of pyridine
(3 mL) and methanol (2 mL). This solution was cooled to 0.degree.
C. A 5 mol/L aqueous sodium hydroxide solution (5 mL) was added
thereto, and the mixture was stirred for 10 minutes. After the
completion of the reaction, the reaction solution was subjected to
extraction with dichloromethane (5 mL.times.3). The organic layer
was washed with a phosphate buffer (pH 6.86, 0.025 M) and saturated
saline in this order, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The obtained residue was
purified by silica gel column chromatography (elution solvent:
dichloromethane/methanol=50:1) to obtain the title compound (84.3
mg, yield: 91%). This compound was completely consistent with the
compound described in Example 10 of Japanese Patent Laid-Open No.
2000-297097 in analysis by .sup.1H-NMR.
Example 3
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-2-N-isob-
utyrylguanosine
##STR00014##
[0097] HMDS (500 mL) and trimethylsilyl chloride (125 mL) were
added to N2-isobutyrylguanine (7.14 g, 32.3 mmol), and the mixture
was refluxed overnight, concentrated under reduced pressure, and
dried to obtain trimethylsilylated N2-isobutyrylguanine.
[0098] The compound (15.0 g, 21.5 mmol) obtained in Example 1(2)
was dissolved in dry toluene (200 mL). Under a nitrogen stream, the
above-mentioned trimethylsilylated N2-isobutyrylguanine and TMSOTf
(4.80 mL, 26.1 mmol) were added to this solution, and the mixture
was stirred. After 5 minutes, the consumption of the starting
material was confirmed by TLC, followed by heating to reflux for 2
hours. After the completion of the reaction, a saturated aqueous
solution of sodium bicarbonate (ca. 50 mL) was added to the
reaction solution, and the mixture was filtered through celite. The
filtrate was subjected to extraction with dichloromethane (ca. 300
mL). The organic layer was washed with a saturated sodium
bicarbonate solution and saturated saline in this order, dried over
anhydrous magnesium sulfate, and then concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (elution solvent: dichloromethane/methanol=100/1.5)
to obtain the title compound (16.0 g, yield: 86%; which was a
mixture containing an isomer glycosylated at the 7-position of
guanine).
Example 4
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-2-N-isob-
utyrylguanosine
[0099] The compound (6.18 g, 8.87 mmol) obtained in Example 1(2)
was dissolved in dry toluene (120 mL). Under a nitrogen stream,
N2-isobutyrylguanine (2.83 g, 13.3 mmol) and
N,O-bis(trimethylsilyl)acetamide (BSA, 9.60 mL, 38.8 mmol) were
added to this solution, and the mixture was heated to reflux for 1
hour. Subsequently, TMSOTf (3.40 mL, 18.5 mmol) was added to the
reaction solution, and the mixture was further heated to reflux for
45 minutes. After the completion of the reaction, the title
compound (6.44 g, yield: 85%; which was a mixture containing an
isomer glycosylated at the 7-position of guanine) was obtained in
the same way as in Example 3.
Example 5
3',5'-di-O-Benzyl-2'-O-4'-C-ethylene-2-N-isobutyrylguanosine
(1)
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-hydroxyethyl)-2-N-isobutyrylguanos-
ine
[0100] The compound (16.0 g, 18.6 mmol) obtained in Example 3 was
dissolved in THF (160 mL). A solution of tetrabutylammonium
fluoride in THF (1 mol/L, 25 mL, 25.0 mmol) was added to this
solution, and the mixture was stirred at room temperature for 12
hours. After the completion of the reaction, the reaction solution
was concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (elution solvent:
dichloromethane/methanol=20/1) to obtain the title compound (9.97
g, yield: 75%; which was a mixture containing an isomer
glycosylated at the 7-position of guanine).
(2)
3',5'-di-O-Benzyl-2'-O-4'-C-ethylene-2-N-isobutyrylguanosine
[0101] The compound (3.00 g, 4.84 mmol) obtained in step (1) was
dissolved in dry dichloromethane (25 mL). Under a nitrogen stream,
pyridine (4 mL) and methanesulfonyl chloride (750 .mu.L, 9.69 mmol)
were added to this solution, and the mixture was stirred at room
temperature for 12 hours. After the completion of the reaction,
water (ca. 5 mL) was added thereto, followed by extraction with
dichloromethane (ca. 30 mL). The organic layer was washed with
saturated saline, dried over anhydrous magnesium sulfate, and then
concentrated under reduced pressure. The residue was dissolved in a
mixed solvent of pyridine (25 mL) and methanol (10 mL), and the
solution was cooled to 0.degree. C. An aqueous sodium hydroxide
solution (5 mol/L, 25 mL) was added to this solution, and the
mixture was stirred for 30 minutes. Then, the reaction solution was
subjected to extraction with a dichloromethane (ca. 50 mL.times.3)
solution. The organic layer was washed with saturated saline, dried
over anhydrous magnesium sulfate, and concentrated under reduced
pressure. Then, the residue was completely separated and purified
from an isomer glycosylated at the 7-position of guanine by silica
gel column chromatography (elution solvent:
dichloromethane/methanol=100/1.5) to obtain the title compound
(1.81 g, yield: 43% (4 steps)). This compound was completely
consistent with the compound described in Example 24 of Japanese
Patent Laid-Open No. 2000-297097 in analysis by .sup.1H-NMR.
Reference Example 1
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-t-butyldiphenylsilyloxyethyl)-5-methyl-
uridine
##STR00015##
[0103] The compound (206 mg, 0.336 mmol) obtained in Example 1(2)
was dissolved in dry acetonitrile (4 mL). Under a nitrogen stream,
thymine (62.8 mg, 0.498 mmol) and N,O-bis(trimethylsilyl)acetamide
(BSA, 0.37 mL, 1.5 mmol) were added to this solution, and the
mixture was heated to reflux for 1 hour. Subsequently, TMSOTf
(0.125 mL, 0.678 mmol) was added to the reaction solution, and the
mixture was further heated to reflux for 40 minutes. After the
completion of the reaction, a saturated aqueous solution of sodium
bicarbonate was added to the reaction solution, followed by
extraction with dichloromethane. The organic layer was washed with
saturated saline, dried over anhydrous magnesium sulfate, and then
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (elution solvent: hexane/ethyl
acetate=1/2) to obtain the title compound (190 mg, yield: 74%).
[0104] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.04 (9H,
s), 1.47 (3H, s), 1.74-1.85 (1H, m), 2.02 (3H, s), 2.03-2.08 (1H,
m), 3.39 (1H, d, J=10.3 Hz), 3.69-3.83 (2H, m), 3.86 (1H, d, J=11
Hz), 4.32-4.56 (5H, m), 5.33 (1H, t), 6.06 (1H, d, J=5.1 Hz),
7.19-7.61 (20H, m), 7.93 (1H, s).
Reference Example 2
3',5'-di-O-Benzyl-2'-O-4'-C-ethylene-5-methyluridine
(1)
2'-O-Acetyl-3',5'-di-O-benzyl-4'-(2-hydroxyethyl)-5-methyluridine
[0105] The compound (185 mg, 0.242 mmol) obtained in Reference
Example 1 was dissolved in THF (2 mL). A solution of
tetrabutylammonium fluoride in THF (1 mol/L, 0.34 mL, 0.34 mmol)
was added to this solution, and the mixture was stirred overnight
at room temperature. After the completion of the reaction, the
reaction solution was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (elution
solvent: hexane/ethyl acetate=1/4) to obtain the title compound
(121 mg, yield: 95%).
[0106] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.49 (3H,
s), 1.73-1.80 (1H, m), 2.06 (3H, s), 2.11-2.17 (1H, m), 3.24-3.28
(1H, m), 3.41 (1H, d, J=10.3 Hz), 3.72-3.75 (2H, m), 3.77 (1H, d,
J=10.3 Hz), 4.34-4.62 (5H, m), 5.39 (1H, t), 6.16 (1H, d, J=5.1
Hz), 7.20-7.41 (11H, m).
(2) 3',5'-di-O-Benzyl-2'-O-4'-C-ethylene-5-methyluridine
[0107] The compound (55.4 mg, 0.106 mmol) obtained in step (1) was
dissolved in dry dichloromethane (1 mL). Under a nitrogen stream,
pyridine (0.1 mL) and methanesulfonyl chloride (16.5 .mu.L, 0.213
mmol) were added to this solution, and the mixture was stirred at
room temperature for 3 hours. After the completion of the reaction,
water was added thereto, followed by extraction with
dichloromethane. The organic layer was washed with saturated
saline, dried over anhydrous magnesium sulfate, and then
concentrated under reduced pressure. The residue was dissolved in a
mixed solvent of pyridine (0.5 mL) and methanol (0.5 mL), and the
solution was cooled to 0.degree. C. An aqueous sodium hydroxide
solution (5 mol/L, 1 mL) was added to this solution, and the
mixture was stirred for 5 minutes. Then, the reaction solution was
subjected to extraction with dichloromethane. The organic layer was
washed with saturated saline, dried over anhydrous magnesium
sulfate, and concentrated under reduced pressure. Then, the residue
was purified by silica gel column chromatography (elution solvent:
dichloromethane/methanol=100/1.5.fwdarw.100/2) to obtain the title
compound (40.1 mg, yield: 82%). This compound was completely
consistent with the compound described in Example 6 of Japanese
Patent Laid-Open No. 2000-297097 in analysis by .sup.1H-NMR.
INDUSTRIAL APPLICABILITY
[0108] According to the present invention, an oligonucleotide
analog having stable and excellent antisense or antigene activity
or having excellent activity as a detection reagent (probe) for a
specific gene or as a primer for initiation of amplification of a
specific gene, and intermediate compound (III) for the production
of nucleoside analog compound (Ia) or (I) which serves as an
intermediate for the production thereof can be produced at high
yields regardless of the type of nucleobase.
* * * * *