Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
CROSSLINKED NUCLEIC ACID GUNA, METHOD FOR PRODUCING SAME,
AND INTERMEDIATE COMPOUND
TECHNICAL FIELD
[0001]
The present invention relates to monomer or oligomer
of bridged nucleic acid GuNA, a method for preparing the
same, as well as an intermediate compound for preparing
the same.
BACKGROUND ART
[0002]
Examples of methods for treating diseases using
nucleic acid drugs include antisense therapies, antigen
therapies, aptamer-based therapies, siRNA-based therapies,
and the like. Among these therapies, the antisense
therapies are approaches for treating or preventing
diseases, involving inhibiting a translation process of
pathogenic RNAs by externally introducing
oligonucleotides (antisense strands) that are
complementary to disease-associated mRNAs to form double
strands. The siRNA-based therapies are similar to the
antisense therapies, involving inhibiting translation
from mRNAs to proteins by administering double-stranded
RNAs into a living body. Meanwhile, the antigen
therapies suppress transcription from DNAs to RNAs by
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externally introducing triplex-forming oligonucleotides
corresponding to DNA sites that are to be transcribed
into pathogenic RNAs. Also, aptamers exert their
function by binding specifically to biological components
such as disease-associated proteins and thereby adjusting
the protein activity.
[0003]
Although various artificial nucleic acids have been
developed as materials for such the nucleic acid drugs,
no ideal molecule has been found yet. Examples of the
materials developed for nucleic acid drugs to date
include phosphorothioate (S¨P03) oligonucleotide (S-
oligo), 2',4'-bridged nucleic acid (BNA)/21,47-locked
nucleic acid (LNA) (see Patent Documents 1 to 4 and Non-
Patent Documents 1 to 6), and the like. S-oligo is
commercially available in the United States as an
antisense drug for cytomegalovirus. S-oligo has a high
nuclease resistance, but is problematic and needs
improvement in that its binding affinity to the target
nucleic acid strands is low. 2',4'-BNA/LNA developed to
date has a high binding affinity to the target nucleic
acid strands, and provides the most promising molecules
as the materials for the future nucleic acid drugs.
However, there is still room for improvement in that the
nuclease resistance is not sufficient and the stability
in a living body is poor.
[0004]
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As one of artificial nucleic acid, 2'-amino LNA has
been reported (see Non-Patent Document 7. It has a
nuclease resistance, and has a high binding affinity to a
target nucleic acid strand, and thus is the most expected
molecule to be a material for nucleic acid drugs like the
above-mentioned LNA. However, for the nucleotide with
appropriate substituents that can be a starting material
for synthesizing oligonucleotide, only nucleotides
containing thyminyl or 5-methyl-cytosinyl have been known
(see Non-patent document 8).
Also, as another artificial nucleic acid, Aza-ENA
has been reported (see Non-Patent Document 9). The Aza-
ENA has a structure in which the number of the carbon
atom that is composed of the bridging is one more than
the 2'-amino LNA, and only the nucleic acid in which the
base moiety thereof is thyminyl has been known.
[0005]
As further artificial nucleic acid, a guanidine
bridged artificial nucleic acid (GuNA) is included.
Hitherto, for the guanidine bridged artificial nucleic
acid (GuNA), GuNA that contains thyminyl as a base moiety
of nucleic acid has been known (see Non-Patent Document
5). However, GuNA that contains the base moiety of
nucleic acid other than thyminyl has never been known.
Also, since the preparation of GuNA requires multi steps,
it has not been easy to prepare GuNA effectively. Also,
as artificial nucleic acid being analogous to GuNA, GENA
has been reported (see Non-Patent Document 10). The GENA
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has a structure in which the number of the carbon atom
that is composed of the bridging is one more than GuNA,
and only the nucleic acid in which the base moiety
thereof is thyminyl has been known.
[0006]
Though the preparation method of GuNA that has been
known so far is described in Non-Patent document 11, the
method needs multi steps, and the production yields was
not sufficient.
The preparation route is shown below.
[chem.1]
H2SO4
HO BnBrBo TBDPSCI 8,0 Ac20 Bn0..."µ
NaH Het ,.0
---)." H0/4:3-4 ,, ----41.- TBDPS04 AcOH51_,,,
DAIF Bod -' A' DMF end "-\ Bnd '0Ac
commercially available
64% quant
0
'-ellIFI
BSA TICI
Bo()
TMSOTI 8n0 PH Al¨ 404 MeNH2 aq BuO e NH pm's.
____ > I ) IBOPS0/....N/'.1-
/
CH,CN TBDPSO % THF TBDPSCA P . -VO
CH,CI,
and µ0,4c Bud OH
q
quant uant
2steps74%
0 0 0
Irl(APY. 1112 \-4
ireloa0Hao >--q
Bn0 NH 2) NEIN DA4F BrIO
'14NH Na BH, NICI2 Ein0 e NH
____ $ _________________ > b" 40 __ A. ,bN-0
TtiF
TBDPSCC-- N-\C' TBDPS07 Me01.4 TBDPSO/ -
Bod OH and ..tsi, Bnd NH,
97% 2 steps 66% 76%
[chem.2]
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(tr4)
Bno1)0. PY C82C12
\F4:
\ P
BocHN'jk NHBac
712
Bo0 _,<NK EDC=HCI DIPEA Bee K/4111 TEIAF HO/ 2)612,1C11202
TBOPSO/b" yelp t\/N
N80c
IBDPS0/*Y
NBoc
,
13n0
Br10 11N-4
BnCr NH/
NHEloc NHBoe
86% 50301
Pr, N,Pr
BYO H Hi C 14.N) HkPr 13MTrO e
ci Pd(OH) HO H CMTrO2,C b3 N440 DMTrCI 13,7--tso
rPr
(1314461
Me0H Pp CH,CN
HO
NBoc >-=NBoc )-=NBoc I >-0µ/Boc
BocHN a ocHN BocHN (P02" BocHN
77% 2 steps 56% GuNA-T (Boc)
86%
CITATION LIST
PATENT DOCUMENT
[0007]
Patent Document 1: WO 98/39352 pamphlet
Patent Document 2: WO 2005/021570 pamphlet
Patent Document 3: WO 2003/068795 pamphlet
Patent Document 4: WO 2011/052436 pamphlet
Patent Document 5: WO 2014/046212 pamphlet
NON-PATENT DOCUMENT
[0008]
Non-Patent Document 1: C. Wahlestedt et al., Proc.
Natl. Acad. Sci. USA, 2000, 97, 10, 5633-5638
Non-Patent Document 2: Y. Hari et al., Bioorg. Med.
Chem., 2006, 14, 1029-1038
Non-Patent Document 3: K. Miyashita et al., Chem.
Commun., 2007, 3765-3767
Non-Patent Document 4: S.M.A. Rahman et al., J. Am.
Chem. Soc., 2008, 130, 14, 4886-4896
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Non-Patent Document 5: M. Kuwahara et al., Nucleic
Acids Res., 2008, 36, 13, 4257-4265
Non-Patent Document 6: S. Obika et al., Bioorg. Med.
Chem., 2001, 9, 1001-1011
Non-Patent Document 7: S.K. Singh et al., J. Org.
Chem. 1998, 63, 6078-6079
Non-Patent Document 8: Christoph Rosenbohm et al.,
Org. Biomol. Chem., 2003, 1, 655-663
Non-Patent Document 9: Oommen P. Vargheseet al., J.
AN. CHEM. SOC. 2006, 128, 15173-15187
Non-Patent Document 10: Jharna Barman et al., RSC
Advances 2015, 16, 12257-12260
Non-Patent Document 11: Ajaya R. Shrestha et al.,
Chem. Commun. 2014, 50, 575-577
SUMMARY OF INVENTION
(PROBLEMS TO BE SOLVED BY INVENTION)
[0009]
The present invention relates to a method for
preparing a guanidine bridged artificial nucleic acid
(hereinafter, abbreviated as "GuNA"), and intermediate
compound for preparing the same, and monomer or oligomer
of GuNA.
(MEANS TO SOLVE PROBLEMS)
[0010]
In order to solve the above-mentioned problems to be
solved, the inventors have intensively studied to find
out a novel method for preparing GuNA, which has lower
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steps as the number of steps compared to the conventional
method and has the improved yield of GuNA, and novel
intermediate compound for preparing GuNA, thereby
completed the present invention as follows.
[0011]
That is, the present invention includes the
following Items [1] to [89], and should not be limited
thereto.
Item [1]
A method for preparing a compound represented by
general formula I:
[chem.3]
R4
R1'0 _R3
0
A
õNH
Rs
0
R6-
NN,N
0
R2 H
[wherein
121 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom or a C2_6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a
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6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
a cycle A represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
or salts thereof,
said method comprising a step of reacting a compound
represented by formula II:
[chem.4]
R1
0 R3
/
1.---'
A'
%:=7)
[wherein,
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each of R1, R2, R3, R4, R5, R6 and m is the same as
defined in the above-mentioned general formula I, a cycle
A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle Al.]
with a reducing agent to cleave an oxazolidine ring fused
to a cycle A'.
Item [2]
The method according to Item [1] wherein each of R1
and R2 represents a Bn group, each of R3, R4, Rs, and R6
represents a hydrogen atom, cycle A represents a thyminyl
group or an uracinyl group, and cycle A' represents the
following structural formula II-1 or 11-2:
[chem.5]
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/
0
N
(11-1) (11-2)
, and the reducing agent is Ph3P.
Item [3]
A method for preparing a compound represented by
general formula VII:
[chem.6]
R3
R7
0 R4
0
R5
[R6'"
0
R8
\N __________________________ R9
R10 - N
Ril
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represent independently
of each other a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
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atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom or a
protecting group for hydroxy group, R8 represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, a
thiophosphate group which may be optionally substituted
with one or more substituents, Rg, Rn, and Rn represent
independently of each other a hydrogen atom, a C1_6 alkyl
group which may be optionally substituted with one or
more substituents, or a protecting group for amino group,
and m is an integer of 1 to 3.]
, said method comprising the step according to Item [1]
or [2].
Item [4]
A compound represented by formula I:
[chem.7]
R4
R,
R3
rnA
0 NH
'N\N\
RI5 vi0
R2 H
4D ni
0
[wherein,
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R1 and R2 represent independently of each other a
benzyl (Bn) group, a 2-naphthylmethyl group, a p-
methoxybenzyl group, a 3,4-dimethoxybenzyl group, a 2,6-
dimethoxybenzyl group, or a p-phenylbenzyl group, R3 and
R4 represent independently of each other a hydrogen atom,
or a Ci_6 alkyl group which may be optionally substituted
with one or more substituents, each R5 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, each R6 represents independently a hydrogen
atom, or a C1-6 alkyl group which may be optionally
substituted with one or more substituents, m is an
integer of 1 to 3, and a cycle A represents a five to
seven membered unsaturated heterocyclic group which may
be optionally substituted with one or more substituents
selected from the group consisting of a C1-6 alkyl group
which may be optionally substituted with one or more
substituents, a C2-6 alkenyl group which may be optionally
substituted with one or more substituents, a C2_6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A.]
or salts thereof.
Item [5]
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The compound according to Item [4] or salts thereof
wherein each of R1 and R2 represents a En group, each of
R3, R4, R5, and R6 represents a hydrogen atom, and the
cycle A represents a thyminyl group or an uracinyl group.
Item [6]
A compound represented by general formula II:
[chem.8]
R1
R4 0 R3
Rs R6
rn
N3
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom or a C1_6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
a cycle A represents a five to seven membered
unsaturated heterocyclic group which may be optionally
substituted with one or more substituents selected from
the group consisting of a C1_6 alkyl group which may be
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optionally substituted with one or more substituents, a
C2_6 alkenyl group which may be optionally substituted
with one or more substituents, a C2-6 alkynyl group which
may be optionally substituted with one or more
substituents, an amino group which may be optionally
substituted with one or more suhstituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A'.]
or salts thereof.
Item [7]
The compound according to Item [6] or salts thereof
wherein R1 and R2 represent a Bn group, each of R3, R4, R51
and R6 represents a hydrogen atom, and cycle A'
represents the following structural formula II-1 or 11-2:
[chem.9]
N A,
V
0 0
N
N
(11-)) (11-2)
Item [8]
A method for preparing a compound represented by
general formula II:
[chem.10]
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Ri,__ R.4
0 R3
RE, R6
j
N3 In ,C)
N\-----.rsr'-'----
R2- ----- 0
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1-6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
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oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A'.]
, said method comprising a step of reacting a compound
represented by general formula III:
[chem.11]
R4
RI ----,0
R3
'. R5 R6
HO
in 7.0
.7
R2 --- 0
[wherein, each of R1, R2, R3, R4, R5, R6, m and cycle A' is
the same as defined in the above-mentioned general
formula II.]
with an azide agent.
Item [9]
The method according to Item [8] wherein each of R1
and R2 represents a Bn group, each of R3, R4, R5, and R6
is a hydrogen atom, cycle A' represents the following
structural formula II-1 or 11-2:
[chem.12]
A'
\ / o
N N
lif--
(11-1) (11-2)
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, and the azide agent is DPPA.
Item [10]
A method for preparing a compound represented by
general formula VII:
[chem.13]
R3
R4
R5
Rd/
N.,õ.õ,
0
N
Rio ¨N
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represents independently
of each other a hydrogen atom or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom or a
protecting group for hydroxy group, R8 represents a
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hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, R9, Rio, and RH represent
independently of each other a hydrogen atom, a C1_6 alkyl
group which may be optionally substituted with one or
more substituents, or a protecting group for amino group,
and m is an integer of 1 to 3.11
or salts thereof, the method comprising the steps
according to Item [8] or [9].
Item [11]
A compound represented by general formula III:
[chem.14]
R4
R1----0 .---,_ R3
Ho, m E, Rf5
\ /
1R
-----'
0
7
n A'
/
0
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
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6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituent selected from the group
consisting of a C1-6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A'.]
or salts thereof.
Item [12]
The compound according to Item [11] or salts thereof
wherein each of R1 and R2 represents a Bn group, each of
R3, R4, R5, and R6 represents a hydrogen atom, and cycle
A represents the following structural formula II-1 or
11-2:
[chem.15]
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A, A,
/
0
(11-0 (11-2)
Item [13]
A method for preparing a compound represented by
general formula IV:
[chem.16]
Ri. R4
0
R3
Rs R4i.
0
X
R2 ¨
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each Rs represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a C1-
6 alkyl group which may be optionally substituted with
one or more substituents, X represents a leaving group, m
is an integer of 1 to 3, and cycle A' represents a five
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21
to seven membered unsaturated heterocyclic group which
may be optionally substituted with one or more
substituents selected from the group consisting of a C1-6
alkyl group which may be optionally substituted with one
or more substituents, a C2-6 alkenyl group which may be
optionally substituted with one or more substituents, a
C2_6 alkynyl group which may be optionally substituted
with one or more substituents, an amino group which may
be optionally substituted with one or more substituents,
a hydroxy group which may be optionally substituted with
one or more substituents, an oxo group, a thioxo group,
and a halogen atom, and the unsaturated heterocyclic
group may be further fused with another cycle to form a
cycle A'.]
, the method comprising a step of reacting a compound
represented by general formula V:
[chem.17]
R4
--0 ¨R3
\\\
R5 Re A
0 y N H
0
R2 OH
[wherein,
each of RI, R2, 123, 124, R5, R6, X and m is the same as
defined in the above-mentioned general formula IV, and
cycle A represents a five to seven membered unsaturated
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heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a Ci_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2_6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
with an activating agent for hydroxy group to activate an
unprotected hydroxy group which is substituted on a
tetrahydrofuran ring in the general formula V.
Item [14]
The method according to Item [13] wherein each of R1
and R2 represents a Bn group, each of 123, R4, R5 and R6
represents a hydrogen atom, X represents a mesyloxy group
(Ms-0-), cycle A represents a thyminyl group or an
uracinyl group, cycle A' represents the following
structural formula II-1 or 11-2:
[chem.18]
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23
/ 0
t-711-1
(n-n (11-2)
, and the activating agent for hydroxy group represents a
trifluoromethanesulfonyl chloride.
Item [15]
A method for preparing a compound represented by
general formula VII:
[chem.19]
R3
0 R4
R5 iYN
s,
R,
-
Rir-"-14
R11
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represent independently
of each other a hydrogen atom, or a C1-6 alkyl group which
may be optionally substituted with one or more
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24
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom, or a
protecting group for hydroxy group, R8 represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, and R9, Rn and Rn
represent independently of each other a hydrogen atom, a
C1-6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3.1
, the method comprising the step according to Item [13]
or [14].
Item [16]
A compound represented by general formula IV:
[chem.20]
RI ... R4
t.,
..,-
in 0
X --- R5 R6
R, ¨0 '0
[wherein,
24
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R1 and R2 represents independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1_6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, X represents a leaving group, m
is an integer of 1 to 3, and cycle A' represents a five
to seven membered unsaturated heterocyclic group which
may be optionally substituted with one or more
substituents selected from the group consisting of a C1-6
alkyl group which may be optionally substituted with one
or more substituents, a C2-6 alkenyl group which may be
optionally substituted with one or more substituents, a
C2_6 alkynyl group which may be optionally substituted
with one or more substituents, an amino group which may
be optionally substituted with one or more substituents,
a hydroxy group which may be optionally substituted with
one or more substituents, an oxo group, a thioxo group,
and a halogen atom, and the unsaturated heterocyclic
group may be further fused with another cycle to form a
cycle A'.]
or salts thereof.
Item [17]
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26
The compound according to Item [16] or salts thereof
wherein each of RI and R2 represents a Bn group, and each
of R3, Rg, R5, and R6 represents a hydrogen atom, X
represents a mesyloxy group (Ms-0-), and cycle A'
represents the following structural formula II-1 or 11-2:
[chem.21]
----N -------
7\---::
0 -----N///1---
v
\ N
(11-1) (11-2)
Item [18]
A compound represented by general formula V:
[chem.22]
R4
_
R5 RE;
0 H
X ----
-------- - .. m
0
/
/
OH
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and Rg represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
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27
hydrogen atom, or a C1-6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, X represents a leaving group, m
is an integer of 1 to 3, and cycle A represents a five to
seven membered unsaturated heterocyclic group which may
be optionally substituted with one or more substituents
selected from the group consisting of a C1-6 alkyl group
which may be optionally substituted with one or more
substituents, a C2-6 alkenyl group which may be optionally
substituted with one or more substituents, a C2-6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A.]
or salts thereof.
Item [19]
The compound according to Item [18] or salts thereof
wherein each of R1 and R2 represents a Bn group, each of
R3, R4, R5 and R6 represents a hydrogen atom, X represents
a mesyloxy group (Ms-0-), and cycle A represents a
thyminyl group or an uracinyl group.
Item [20]
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28
A method for preparing a compound represented by
general formula II:
[chem.23]
R, R4
'----'0
7 R6
,4
N3 70
R2----0 07--2
[wherein,
R1 and R2 represents independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
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29
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A'.]
, the method comprising a step of reacting a compound
represented by formula IV:
[chem.24]
R" R4----0 ¨
N3
.0
,.....-- 7".
m
X
4R6 R6
,
R,---0 0'--
[wherein,
X represents a leaving group, and each of RI, R2, R3,
RI, R5, R6, m and cycle A' is the same as defined in the
above-mentioned general formula II.]
with an azide agent.
Item [21]
The method according to the Item [20] wherein each
of R1 and R2 represent a Bn group, each of R3, R4, R5, and
R6 represents a hydrogen atom, and X represents a
mesyloxy group (Ms-0-), cycle A7 represents the following
structural formula II-1 or 11-2:
[chem.25]
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S¨N
0 0
(11-1) (11-2)
, and the azide agent represents NBu4NN3 or sodium azide.
Item [22]
A method for preparing a compound represented by
general formula VII:
[chem.26]
R3
/
0
-0NyB
R5 \
R6
R8
Rio ¨N
Ril
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represents independently
of each a hydrogen atom, or a C1_6 alkyl group which may
be optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a C]_
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31
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, R7 represents a hydrogen atom, or a
protecting group for hydroxy group, R8 represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, R9, Rn and RI1 represent
independently of each other a hydrogen atom, a C1_6 alkyl
group which may be optionally substituted with one or
more substituents, or a protecting group for amino group,
and m is an integer of 1 to 3.11
, the method comprising the step according to Item [20]
or [21].
Item [23]
A method for preparing a compound represented by
general formula II:
[chem.27]
R1 A
R ---- 0 R3
ov
)ni
[wherein,
31
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32
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1_6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2_6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A'.]
or salts thereof, the method comprising a step of
reacting a compound represented by general formula VI:
[chem.28]
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33
R4
-0 R,
,
R5 REI ( A
0
N3 3 m
0
OH
[wherein,
each of R1, R2, R3/ R4, R5, R6 and m is the same as
defined in the above-mentioned general formula II, and
cycle A represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
with an activating agent for hydroxy group to activate an
unprotected hydroxy group which is substituted on a
tetrahydrofuran ring in the general formula VI.
Item [24]
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34
The method according to Item [23] wherein each of R1
and R2 represents a Bn group, each of R3, R4, R5 and RG
represents a hydrogen atom, cycle A represents a thyminyl
group or an uracinyl group, cycle A' represents the
following structural formula II-1 or the 11-2:
[chem.29]
------N///7/ ---= N/ --------
1
0
'-1111 N
'-'1117 N
(H-1) (1.1-2)
, and the activating agent for hydroxy group represents a
trifluoromethanesulfonyl chloride.
Item [25]
A method for preparing a compound represented by
general formula VII:
[chem.30]
R3
R7 /R4.---,.. _0 _
"-1 0
B
R5, /
[
-In /"..,,,, 1.
/
0 N
_--
N/_...__N ___________________ R9
RIO ___________
\
Rit
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[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represents independently
of each other a hydrogen atom or a C1-6 alkyl group which
may be optionally substituted with one or more
substituents, each R6 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1_6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom or a
protecting group for hydroxy group, R8 represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, and R9, R10 and R11
represent independently of each other a hydrogen atom, a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3.]
, the method comprising the step according to Item [23]
or [24].
Item [26]
A compound represented by general formula VII:
[chem.31]
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36
R3
0
0
R,
b _m õNN
0
=
RB-
RV) N
Ril
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represents independently
of each other a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1.6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom, or a
protecting group for hydroxy group, R8 represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, and R9, Rn and R11
represent independently of each other a hydrogen atom, a
36
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37
C1_6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3 (with the proviso
that the following cases are excluded:
the case where m is 1, B represents a thyminyl group,
each of R3, R4, R5 and R6 represents a hydrogen atom, R7
represents a DMTr group, R8 represents a hydrogen atom or
a -P(O(CH2)2CN)(N(iPr)2) group, R9 represents a Boc group,
R10 represents a Boc group, and R11 represents a hydrogen
atom, and
the case where m is 2, B represents a thyminyl group,
each of R3, R4, R5 and R6 represents a hydrogen atom, R7
represents a DMTr group, Ra represents a hydrogen atom or
a -P(O(C112)2CN)(N(iPr)2) group, Rg represents a Ceoc group,
R10 represents a Ceoc group, and Rn represents a hydrogen
atom.)]
or salts thereof.
Item [27]
The compound according to Item [26] or salts thereof
wherein B represents an adeninyl group which may
optionally have one or more protecting groups, a guaninyl
group which may optionally have one or more protecting
groups, a cytosinyl group which may optionally have one
or more protecting groups, a 5-methylcytosinyl group
which may optionally have one or more protecting groups,
or an uracinyl group which may optionally have one or
more protecting groups, each of R3, R4, R5 and R6
represents a hydrogen atom, R7 represents a DMTr group,
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38
R8 represents a hydrogen atom or a -P(O(C1-12)2CN)(N(iPr)2)
group, R9 represents a Teoc group or a Boc group, R10
represents a Teoc group or a Boc group, and Ril
represents a hydrogen atom.
Item [28]
The compound according to Item [26] or salts thereof
wherein B represents a thyminyl group which may
optionally have one or more protecting groups, each of R3,
R4, Rs and R6 represents a hydrogen atom, R7 represents a
DMTr group, R8 represents a hydrogen atom or a -
P(O(CP2)2CN)(N(iPr)2) group, Rg represents a Teoc group,
R10 represents a Teoc group, and Rn represents a hydrogen
atom.
Item [29]
A method for preparing a compound represented by
general formula VIII:
[chem.32]
R3
R12
-0 R4
0
- R5 -V Nre'
R
-m
R13--
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
38
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39
or more substituents, R3 and R4 represents independently
of each other a hydrogen atom, or a C1-6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, and R12 and Rn represents
independently of each other a hydrogen atom or a
protecting group for hydroxy group, and m is an integer
of 1 to 3.]
, the method comprising a step of substituting a cycle A
in a compound represented by a general formula IX:
[chem.33]
R ,R3
0 // R4
0 NH
'
N
R- 5 ,
0
RB/
_ m
0
[wherein,
each of R3, R4, R5, R5, and m is the same as defined
in the above-mentioned general formula VIII, each of R14
and R15 represents independently of each other a hydrogen
atom, or a protecting group for hydroxy group, and cycle
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A represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2_6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2_6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
with a B.
Item [30]
The method according to Item [29] wherein B
represents an adeninyl group which may optionally have
one or more protecting groups, a guaninyl group which may
optionally have one or more protecting groups, a
cytosinyl group which may optionally have one or more
protecting groups, a 5-methylcytosinyl group which may
optionally have one or more protecting groups, a thyminyl
group which may optionally have one or more protecting
groups, or an uracinyl group which may optionally have
one or more protecting groups, each of Rn, Rn, R14, and
R15 represents a Bn group, and cycle A represents a
thyminyl group or an uracinyl group.
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41
Item [31]
The method according to Item [29] wherein B
represents an adeninyl group which may optionally have
one or more protecting groups, a guaninyl group which may
optionally have one or more protecting groups, a
cytosinyl group which may optionally have one or more
protecting groups, a 5-methylcytosinyl group which may
optionally have one or more protecting groups, a thyminyl
group which may optionally have one or more protecting
groups, or an uracinyl which may optionally have one or
more protecting groups, R12 represents a DMTr group, Rn
represents a TMS group, Rn represents a DMTr group, R15
represents a hydrogen atom, and cycle A represents a
thyminyl group or an uracinyl group.
Item [32]
The method according to Item [29] wherein B
represents an adeninyl group which may optionally have
one or more protecting groups, a guaninyl group which may
optionally have one or more protecting groups, a
cytosinyl group which may optionally have one or more
protecting groups, a 5-methylcytosinyl group which may
optionally have one or more protecting groups, a thyminyl
group which may optionally have one or more protecting
groups, or an uracinyl group which may optionally have
one or more protecting groups, Rn represents a TMS group,
Rfl represents a TMS group, Rn represents a hydrogen atom,
R15 represents a hydrogen atom, and the cycle A
represents a thyminyl group or an uracinyl group.
41
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42
Item [33]
A method for preparing a compound represented by
general formula VII:
[chem.34]
R3
- R4
0
r5/1/Ni\
ni NNN
0
R8
R10 N
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represent independently
of each other a hydrogen atom, or a C1..6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1.5 alkyl group which may be optionally
substituted with one or more substituents, each RG
represents independently a hydrogen atom, or a Cl_6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom or a
protecting group for hydroxy group, R8 represents a
hydrogen atom, a phosphate group which may be optionally
42
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43
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, and R9, R10 and Rn
represent independently of each other a hydrogen atom, a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3.]
, the method comprising the step according to any one of
Item [29] to [32].
Item [34]
A compound represented by general formula VIII:
[chem.35]
R3
R12 > R
0 4
m 0
R5
NN,/7
=
0
N
R13-
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represent independently
of each other a hydrogen atom, or a C1-6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
43
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44
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1_6 alkyl
group which may be optionally substituted with one or
more substituents, R12 represents a Bn group, a DMTr
group, or a TMS group, and Rn represents a Bn group, a
hydrogen atom, or a TMS group, and m is an integer of 1
to 3.]
or salts thereof.
Item [35]
The compound according to Item [34] or salts thereof
wherein B represents an adeninyl group which may
optionally have one or more protecting groups, a guaninyl
group which may optionally have one or more protecting
groups, a cytosinyl group which may optionally have one
or more protecting groups, a 5-methylcytosinyl group
which may optionally have one or more protecting groups,
a thyminyl group which may optionally have one or more
protecting groups, or an uracinyl group which may
optionally have one or more protecting groups, each of R3,
R4, R5, and R6 represents a hydrogen atom, and each of R12
and Rn represents a Bn group.
Item [36]
The compound according to Item [34] or salts thereof
wherein B represents an adeninyl group which may
optionally have one or more protecting groups, a guaninyl
group which may optionally have one or more protecting
groups, a cytosinyl group which may optionally have one
or more protecting groups, a 5-methylcytosinyl group
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which may optionally have one or more protecting groups,
a thyminyl group which may optionally have one or more
protecting groups, or an uracinyl group which may
optionally have one or more protecting groups, and each
of 123, R4, Rs and R6 represents a hydrogen atom, R12
represents a DMTr group, and Rn represents a hydrogen
atom.
Item [37]
A method for preparing an oligonucleotide containing
one or more nucleosides represented by general formula X:
[chem.36]
R3
0 RA
0
N
[R5
4
R6/
111 y--
or
Jsr
0
I
H +
N¨R
N¨R16 16
18
R ¨N
\Ru Ru
[wherein,
R3 and R4 represent independently of each other a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a C,
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
CA 02999199 2018-03-16
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46
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, B represents a base moiety of nucleic acid
wherein said base moiety may be optionally substituted
with one or more substituents, R16, Rfl and R18 represent
independently of each other a hydrogen atom, a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, or a protecting group for amino group,
and m is an integer of 1 to 3.]
or salts thereof,
(with the proviso that when m =1 or 2, the
oligonucleotide containing as a bridged nucleic acid only
the nucleoside wherein B represents a thyminyl group,
each of 12.3, R4, R5, R5, R15, R17 and R18 represents a
hydrogen atom, is excluded),
said method comprising a preparation of the same by a
phosphoramidite method from a compound represented by
general formula XI:
[chem.37]
46
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47
R3
R,
0
B
7
[ R5\
I
e
/
R
6 ille/N./
N
D ----d
%===N ______________________ R21
R23----- N
\
R22
[wherein,
each of B, R3, RI, R5, R6 and m is the same as
defined in the above-mentioned general formula X, R7
represents a hydrogen atom, or a protecting group for
hydroxy group, R20 represents a phosphate group which may
be optionally substituted with one or more substituents
or a thiophosphate group which may be optionally
substituted with one or more substituents, and Rn, R22
and Rn represents independently of each other a hydrogen
atom, a C1_6 alkyl group which may be optionally
substituted with one or more substituents, or a
protecting group for amino group, and m is an integer of
1 to 3.]
Item [38]
The method according to Item [37] wherein B
represents an adeninyl group which may optionally have
one or more protecting groups, a guaninyl group which may
optionally have one or more protecting groups, a
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48
cytosinyl group which may optionally have one or more
protecting groups, a 5-methylcytosinyl group which may
optionally have one or more protecting groups, or an
uracinyl group which may optionally have one or more
protecting groups, R7 represents a protecting group for
hydroxy group, each of Rn, R17 and Rn represents a
hydrogen atom, R20 represents a -P(O(C112)2CN)(N(iPr)2)
group, and each of 19.21, R22 and Rn represent a protecting
group for amino group.
Item [39]
An oligonucleotide containing one or more
nucleosides represented by general formula X:
[chem.38]
al(111(1A
R3
0 R4
0
4
R6.
_ m Y- or
0
/
N¨R1s
R18, ________________________________________ N
\R t.?
[wherein,
R3 and R4 represent independently of each other a
hydrogen atom, or a C1-6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
48
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49
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, B represents a base moiety of nucleic acid
wherein said base moiety may be optionally substituted
with one or more substituents, R16, Rn and Rie represent
independently of each other a hydrogen atom, a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, or a protecting group for amino group,
and m is an integer of 1 to 3.]
(with the proviso that when m =1 or 2, the
oligonucleotide containing as a bridged nucleic acid only
the nucleoside wherein B represents a thyminyl group,
each of R3, R4, R5, R6, R16, Rn and R18 represents a
hydrogen atom is excluded).
Item [40]
The oligonucleotide according to Item [39] or salts
thereof wherein B represents an adeninyl group, a
guaninyl group, a cytosinyl group, a 5-methylcytosinyl
group, a thyminyl group, or an uracinyl group, and each
of R16, Rn and Ris represents a hydrogen atom].
Item [41]
A compound represented by general formula XII:
[chem.39]
49
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R4
0 R3
1R6
\ A'
Di
,/ NN
0
[wherein,
R1 and R2 represents independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1_6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, D1 represents N3, a hydroxy
group, or -X (wherein -X represents a leaving group,
preferably a leaving group for hydroxy group, and
exemplifies a methanesulfonyloxy (mesyloxy; Ms-O-) group,
a trifluoromethanesulfonyloxy group, a p-
toluenesulfonyloxy group and the like, which should not
be limited thereto), m is an integer of 1 to 3, and cycle
A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents, and the unsaturated
heterocycle may be fused with another cycle to form the
cycle A' (wherein, the substituents for "five to seven
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51
membered unsaturated heterocycle" represent preferably a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, a C2-6 alkenyl group which may
be optionally substituted with one or more substituents,
a C2-6 alkynyl group which may be optionally substituted
with one or more substituents, an amino group which may
be optionally substituted with one or more substituents,
a hydroxy group which may be optionally substituted with
one or more substituents, an oxo group, a thioxo group,
or a halogen atom.)],
or salts thereof.
Item [42]
The compound according to Item [41] or salts thereof
wherein R3, R4, Rs and R6 represent independently of each
other a hydrogen atom, or a C1_6 alkyl group.
Item [43]
The compound according to Item [41] or [42] or salts
thereof wherein each of R3, R4, R5 and R6 represents a
hydrogen atom.
Item [44]
The compound according to any one of Items [41] to
[43] or salts thereof wherein R1 and R2 represent
independently of each other a En group, a 4,41-
dimethoxytrityl (DMTr) group, a t-butyldimethylsilyl
group, a t-butyldiphenylsilyl group, a
trifluoromethansulfonyl group, a trimethylsilyl group, or
a methanesulfonyl group, or the like.
Item [45]
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52
The compound according to any one of Items [41] to
[44] or salts thereof wherein cycle A' represents a 6
membered unsaturated heterocyclic group which may be
optionally substituted with one or more substituents,
preferably a heterocyclic group having the following
structural formula II-1 or 11-2:
[chem.40]
A,
A, 0
N
(11-1) (11-2)
Item [46]
The compound according to any one of Items [41] to
[45] wherein D1 represents N3.
Item [47]
The compound according to any one of Items [41] to
[45] wherein D1 represents a hydroxy group.
Item [48]
The compound according to any one of Items [41] to
[45] wherein D1 represents -X (a leaving group), and the
leaving group represents preferably a leaving group for
hydroxy group, and exemplifies a methanesulfonyloxy
(mesyloxy; Ms-O-) group, a trifluoromethanesulfonyloxy
group, or a p-toluenesulfonyloxy group and the like,
which should not be limited thereto.
Item [49]
52
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53
A method for preparing a compound represented by
general formula I:
[chem.41]
R4
R
0 R3
0
R64,,
0
R6
0
R2-""
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C16 alkyl group which may be
optionally substituted with one or more substituents,
each RG represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1.6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2_6 alkynyl group which may be
53
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54
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocycle may be fused with another cycle
to form the cycle A.]
, said method comprising a step of reacting the compound
according to Item [46] with a reducing agent, and the
reducing agent used in the present preparation method
includes phosphines, preferably Ph3P, and the cycle A
includes preferably a thyminyl group or an uracinyl group.
Item [51]
A method preparing the compound according to Item
[46] or salts thereof, said method comprising reacting
the compound according to Item [47] with an azide agent
to prepare the same, and the azide agent used in the
present preparation method includes hydrazoic acid,
sodium azide, lithium azide, tetrabutylammonium azide,
trimethylsilyl azide, diphenylphosphoryl azide (DPPA),
nicotinyl azide, zinc azide (Zn(N3)2), and preferably
DPPA.
Item [52]
A method for preparing the compound according to
Item [46] or salts thereof, said method comprising
reacting the compound according to Item [48] or salts
thereof with an azide agent to prepare the same, and the
azide agent used in the present preparation method
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includes nBu4NN3 or sodium azide, which should not be
limited thereto.
Item [53]
A method for preparing the compound according to
Item [46] or salts thereof, said method comprising
reacting the compound represented by general formula VI:
[chem.42]
R4
0 R3
R5 RG
=0 A
m Ni,
1,43 ,
0
R2 OH
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each Rs represents independently a
hydrogen atom, or a C1-6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, X represents a leaving group, m
is an integer of 1 to 3, and cycle A represents a five to
seven membered unsaturated heterocyclic group which may
be optionally substituted with one or more substituents
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56
selected from the group consisting of a C1_6 alkyl group
which may be optionally substituted with one or more
substituents, a C2_6 alkenyl group which may be optionally
substituted with one or more substituents, a C2-6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A.]
with an activating agent for hydroxy group to prepare the
compound according to Item [46] or salts thereof, and the
activating agent for hydroxy group used in the present
preparation method includes, for example,
trifluoromethanesulfonyl chloride, methanesulfonyl
chloride, and trifluoromethanesulfonic anhydride, which
should not be limited thereto.
Item [54]
A method for preparing the compound according to
Item [48] or salts thereof, said method comprising
reacting a compound represented by general formula v:
[chem.43]
56
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57
R4
0 ¨R3
R5 RG ( A \\)
NVN
,/
OH
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, X represents a leaving group, m
is an integer of 1 to 3, and cycle A represents a five to
seven membered unsaturated heterocyclic group which may
be optionally substituted with one or more substituents
selected from the group consisting of a C1_6 alkyl group
which may be optionally substituted with one or more
substituents, a C2_6 alkenyl group which may be optionally
substituted with one or more substituents, a C2-6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
57
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58
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocycle may be
further fused with another cycle to form a cycle A.]
with an activating agent for hydroxy group to prepare the
compound according to Item [48], and the activating agent
for hydroxy group used in the present preparation method
includes, for example, methanesulfonyl chloride, which
should not be limited thereto.
Item [55]
A method for preparing a compound represented by
general formula VII:
[chem.44]
R3
R7
r 0 R4
0
[RR:41
0
R9
Rin-N
RI/
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represent independently
of each other a hydrogen atom, or a C1_6 alkyl group which
58
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59
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a Ci_E, alkyl
group which may be optionally substituted with one or
more substituents, R1 represents a hydrogen atom or a
protecting group for hydroxy group, Rg represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, and R9, Rlp, and R11
represent independently of each other a hydrogen atom, a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3.]
or salts thereof,
said method comprising the preparation method
according to any one of Items [49] to [54].
Item [56]
A method for preparing guanidine bridged artificial
nucleic acid, said method comprising the preparation
method according to any one of Items [49] to [55],
wherein the guanidine bridged artificial nucleic acid
represents an oligonucleotide containing one or more
nucleosides represented by general formula X:
[chem.45]
59
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JVN_AJNA
R3
0
0
,/
{R5
4
R6
Y= or
0
L17 cS I
16
_____________________________ ¨R16
Ria¨N
[wherein,
R3 and R4 represent independently of each other a
hydrogen atom, or a Ci_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1-6 alkyl group which
may be optionally substituted with one or more
substituents, B represents a base moiety of nucleic acid
wherein said base moiety may be optionally substituted
with one or more substituents, R16, R17 and R18 represent
independently of each other a hydrogen atom, a Ci_6 alkyl
group which may be optionally substituted with one or
more substituents, or a protecting group for amino group,
and m is an integer of 1 to 3.1
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61
, and the nucleic acid is a nucleic acid which may be
prepared by a phosphoramidite method from the compound
represented by general formula XI:
[chem.46]
R3
R7
0
0
R6 \A/ Nr7
R6
0
- R2.1
R22
[wherein,
each of B, R3, 124, R5, R6 and m is the same as
defined in the above-mentioned general formula X, R7
represents a hydrogen atom, or a protecting group for
hydroxy group, Rn represents a phosphate group which may
be optionally substituted with one or more substituents
or a thiophosphate group which may be optionally
substituted with one or more substituents, R21, R22 and Rn
represent independently of each other a hydrogen atom, a
C1_6 alkyl group which may be optionally substituted with
one or more substituents or a protecting group for amino
group.].
Item [57]
A compound represented by general formula XIII:
61
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62
[chem.47]
R3
R4
0
0
R5/). D B or cycle A
H
RA /
va
111
0
R2
[wherein,
R1 and R2 represent independently of each other a
hydrogen atom, or a protecting group for hydroxy group,
R3 and R4 represent independently of each other a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1-6 alkyl group which
may be optionally substituted with one or more
substituents, m is an integer of 1 to 3, and D represents
a cycle A (wherein the cycle A represents a five to seven
membered unsaturated heterocyclic group which may be
optionally substituted with one or more substituents
selected from the group consisting of a C1.6 alkyl group
which may be optionally substituted with one or more
substituents, a C2-6 alkenyl group which may be optionally
substituted with one or more substituents, a C2_6 alkynyl
62
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63
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A.)
or
a B (said B represents a base moiety of nucleic acid
wherein said base moiety may be optionally substituted
with one or more substituents).]
or salts thereof.
Item [58]
The compound according to Item [57] or salts thereof
wherein each of R3, R4, R5 and R6 represents a hydrogen
atom.
Item [59]
The compound according to Item [57] or [58] or salts
thereof wherein D represents a cycle A.
Item [60]
The compound according to Item [59] or salts thereof
wherein cycle A represents a thyminyl group or an
uracinyl group.
Item [61]
The compound according to Item [59] or [60] or salts
thereof wherein each of R1 and R2 represents a protecting
group for hydroxy group.
Item [62]
63
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64
The compound according to Item [61] or salts thereof
wherein R1 and R2 represent independently of each other a
En group, a 2-naphthylmethyl group, a p-methoxybenzyl
group, a 3,4-dimethoxybenzyl group, a 2,6-dimethoxybenzyl
group, or a p-phenylbenzyl group.
Item [63]
The compound according to any one of Items [59] to
[62] selected from the group consisting of
1-{(1R, 3R, 4R, 75)-7-(benzyloxy)-1-
[(benzyloxy)methy1]-2-oxa-5-azabicyclo[2.2.1]hepta-3-yll-
5-methylpyrimidine-2,4(1H,3H)-dione, and
1-{(1R, 3R, 4R, 75)-7-(benzyloxy)-1-
[(benzyloxy)methy1]-2-oxa-5-azabicyclo[2.2.1]hepta-3-y1}-
pyrimidine-2,4(1H,3H)-dione,
or salts thereof.
Item [64]
The compound according to Item [57] or [58] wherein
D represents B (said B represents a base moiety of
nucleic acid wherein said base moiety may be optionally
substituted with one or more substituents).
Item [65]
The compound according to Item [64] or salts thereof
wherein B represents an adeninyl group which may be
optionally substituted with one or more substituents, a
guaninyl group which may be optionally substituted with
one or more substituents, a cytosinyl group which may be
optionally substituted with one or more substituents, a
5-methylcytosinyl group which may be optionally
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substituted with one or more substituents, a thyminyl
group which may be optionally substituted with one or
more substituents, or an uracinyl group which may be
optionally substituted with one or more substituents.
Item [66]
The compound according to Item [64] or [65] wherein
R1 represents a protecting group for hydroxy group.
Item [67]
The compound according to Item [66] or salts thereof
wherein R1 represents a Bn group, a DMTr group, or a TMS
group.
Item [68]
The compound according to any one of Item [64] to
[67] wherein R2 represents a protecting group for hydroxy
group.
Item [69]
The compound according to Item [68] or salts thereof
wherein R2 represents a En group.
Item [70]
The compound according to Item [68] or salts thereof
wherein R2 represents a TMS group.
Item [71]
The compound according to any one of Items [64] to
[67] wherein R2 represents a hydrogen atom.
Item [72]
The compound according to any one of Items [64] to
[71] selected from
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66
1-1(1R, 3R, 4R, 7S)-7-(benzyloxy)-1-
[(benzyloxy)methy1]-2-oxa-5-azabicyclo[2.2.1]hepta-3-yll-
5-methylpyrimidine-2,4(1H,3H)-dione.
Item [73]
A method for preparing the compound according to
Item [64] or salts thereof, said method comprising a
replacement of a cycle A in a compound represented by
general formula IX:
[chem.48]
R3
R14
0 R4
rTh
0 NH
5/
0
.n1
0
115`-'
[wherein,
R3 and R4 represents independently of each other a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, R1,1 and R15 represents independently of each
other a hydrogen atom, or a protecting group for hydroxy
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67
group, m is an integer of 1 to 3, and cycle A represents
a five to seven membered unsaturated heterocyclic group
which may be optionally substituted with one or more
substituents selected from the group consisting of a C1_6
alkyl group which may be optionally substituted with one
or more substituents, a C2_6 alkenyl group which may be
optionally substituted with one or more substituents, a
C2_6 alkynyl group which may be optionally substituted
with one or more substituents, an amino group which may
be optionally substituted with one or more substituents,
a hydroxy group which may be optionally substituted with
one or more substituents, an oxo group, a thioxo group,
and a halogen atom, and the unsaturated heterocyclic
group may be further fused with another cycle to form a
cycle A.],
in the present preparation method, the B represents
preferably an adeninyl group which may optionally have
one or more protecting groups, a guaninyl group which may
optionally have one or more protecting groups, a
cytosinyl group which may optionally have one or more
protecting groups, a S-methylcytosinyl group which may
optionally have one or more protecting groups, a thyminyl
group which may optionally have one or more protecting
groups, or an uracinyl group which may optionally have
one or more protecting groups; the cycle A represents
preferably a thyminyl group or an uracinyl group; all of
the R14, Rn and R16 represent a Bn group, alternatively
the R14 represents a DMTr group, the Rn represents a TMS
67
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68
group, and the R16 represents a hydrogen atom, which
should not be limited thereto. In the present
preparation method, the replacement method of the cycle A
includes, for example, a method using a Lewis acid,
preferably, a method using a Lewis acid and a silylating
agent, which should not be limited thereto.
Item [74]
A method for preparing a compound represented by
general formula VII:
[chem.49]
R3
R7
0 R4
0
,-- B
--- ,,
R5
R6/
[
rn N-NN
/
0 N
R8 \
Rio ---N7
\
R11
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represent independently
of each other a hydrogen atom, or a C1-6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
68
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69
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom or a
protecting group for hydroxy group, R6 represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, and R9, Rn and R11
represent independently of each other a hydrogen atom, a
C1-6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3.]
,
said method comprising the preparation method according
to Item [73].
Item [75]
A method for preparing a guanidine bridged
artificial nucleic acid, said method comprising the
preparation method according to Item [62] or [63],
wherein the guanidine bridged artificial nucleic acid
represents an oligonucleotide containing one or more
nucleosides represented by general formula X:
[chem.50]
69
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j-V-V-Vtrt_
R3
0 ----- 4
_0
R5
nyNN or
0 V,CLIAP
H +
__________________________________________________ N ¨ R16
RIB _________________________________________ N=
N
R
Rq
[wherein,
R3 and R4 represent independently of each other a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, B represents a base moiety of nucleic acid
wherein said base moiety may be optionally substituted
with one or more substituents, and R16, R17 and R18
represent independently of each other a hydrogen atom, a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3.]
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71
, and the nucleic acid is a nucleic acid which may be
prepared by a phosphoramidite method from a compound
represented by general formula XI:
[chem. 51]
R3
R7 -
----- 0 R4
0
R5*
[Re
111
0 N.
R21
R.23 N
R22
[wherein,
each of B, 123, 124, 125, R6 and m is as the same
defined in the above-mentioned general formula X, R7
represents a hydrogen atom or a protecting group for
hydroxy group, and Rn represents a phosphate group which
may be optionally substituted with one or more
substituents, or a thiophosphate group which may be
optionally substituted with one or more substituents, Rn,
1222, and R23 represent independently of each other a
hydrogen atom, a C1_6 alkyl group which may be optionally
substituted with one or more substituents, or a
protecting group for amino group].
Item [76]
A compound according to general formula VII:
71
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72
[chem.52]
R3
0 ///_R4
R6/
[R5\xr
rn
"Ns N
RI(
N ¨R9
Rio----N
Rll
[wherein,
B represents a base moiety of nucleic acid wherein
said base moiety may be optionally substituted with one
or more substituents, R3 and R4 represent independently
of each other a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, each R5 represents independently a hydrogen
atom, or a C1_6 alkyl group which may be optionally
substituted with one or more substituents, each R6
represents independently a hydrogen atom, or a C1_6 alkyl
group which may be optionally substituted with one or
more substituents, R7 represents a hydrogen atom or a
protecting group for hydroxy group, R8 represents a
hydrogen atom, a phosphate group which may be optionally
substituted with one or more substituents, or a
thiophosphate group which may be optionally substituted
with one or more substituents, and R9, R13, and Rll
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represent independently of each other a hydrogen atom, a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group, and m is an integer of 1 to 3
(with the proviso that the following cases are excluded:
the case where m = 1, B represents a thyminyl group,
each of R3, R4, R5 and RG represents a hydrogen atom, R7
represents a DMTr group, R8 represents a hydrogen atom or
a -P(O(CH2)2CN)(N(iPr)2) group, ,R9 represents a Boc group,
R10 represents a Boc group, and R11 represents a hydrogen
atom, and
the case where m = 2, B represents a thyminyl group,
each of R3, R4, R5 and R6 represents a hydrogen atom, R7
represents a DMTr group, R8 represents a hydrogen atom or
a -P(0(0-12)2CN)(N(iPr)2) group, R9 represents a Ceoc group,
R.,0 represents a Ceoc group, and Rn represents a hydrogen
atom.)]
or salts thereof.
Item [77]
The compound according to Item [76] or salts thereof,
wherein B represents an adeninyl group, a guaninyl group,
a cytosinyl group, a 5-methylcytosinyl group, or an
uracinyl group, each of which may optionally have one or
more protecting groups.
Item [78]
The compound according to Item [76] or [77] or salts
thereof, wherein each of R3, R4, R5 and R6 represent
independently a hydrogen atom.
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Item [79]
The compound according to any one of Items [76] to
[78] or salts thereof, wherein R7 represents a protecting
group for hydroxy group, and the protecting group for
hydroxy group as the R7 represents preferably a methyl
group which is substituted with one to three aryl groups
wherein the aryl group is substituted with an alkyl group
having 1 to 6 carbon atoms, an alkoxy group having 1 to 6
carbon atoms, a halogen atom, and/or a cyano group, more
preferably a DMTr group, which should not be limited
thereto.
Item [80]
The compound according to any one of Item [76] to
[79] or salts thereof, wherein R8 represents a hydrogen
atom.
Item [81]
The compound according to any one of Items [76] to
[79] or salts thereof, wherein R8 represents a phosphate
group which may be optionally substituted with one or
more substituents, and the phosphate group includes,
preferably a -P(O(C1-12)2CN)(N(iPr)2) group, which should
not be limited thereto.
Item [82]
The compound according to any one of Items [76] to
[81] or salts thereof, wherein each of Rg and 1210
represents independently a protecting group for amino
group, and the protecting group for amino group includes,
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preferably, a Teoc group or a Boc group, which should not
be limited thereto.
Item [83]
The compound according to any one of Items [76] to
[82] or salts thereof, wherein R11 represents a hydrogen
atom.
Item [84]
The compound according to Item [76] or salts thereof,
wherein B represents a thyminyl group which may
optionally have one or more protecting groups.
Item [85]
The compound according to Item [84], wherein each of
R9 and R10 represents independently a Teoc group.
Item [86]
An oligonucleotide containing one or more
nucleosides represented by general formula X:
[chem.53]
R3
0 R4
R6)>CN,
¨ 411 \Nõ, y= or
-Trs'
0
SS,
,S
H+
____________________________ N ¨ R16
____________________________________________ N/
R1
R-
[wherein,
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R3 and R4 represent independently of each other a
hydrogen atom, or a C1-6 alkyl group which may be
optionally substituted with one or more substituents,
each Rs represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, B represents a base moiety of nucleic acid
wherein said base moiety may be optionally substituted
with one or more substituents, R16, R17, and R18 represents
independently of each other a hydrogen atom, a C1_6 alkyl
group which may be optionally substituted with one or
more substituents, or a protecting group for amino group,
and m is an integer of 1 to 3.11
(with the proviso that the oligonucleotide containing as
a bridged nucleic acid only nucleoside wherein B
represents a thyminyl group, and each of R3, 124, Rs, RIG,
R17 and R18 represent a hydrogen atom is excluded.),
or salts thereof.
Item [87]
The oligonucleotide according to Item [86] or salts
thereof, wherein B represents an adeninyl group, a
guaninyl group, a cytosinyl group, a 5-methylcytosinyl
group, a thyminyl group, or an uracinyl group.
Item [88]
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The oligonucleotide according to Item [86] or [87],
wherein each of R16, R17, and RIB represent independently a
hydrogen atom.
Item [89]
The method for preparing the oligonucleotide
according to any one of Items [86] to [89] or salts
thereof, said method comprising the preparation of the
same by a phosphoramidite method from a compound
represented by general formula XI:
[chem.541
R3
0
Rn-0-
Rn---N
R22
[wherein,
each of B, R3, 124, R5, R6 and m is the same as
defined in the above-mentioned general formula X, R7
represents a hydrogen atom, or a protecting group for
hydroxy group, R20 represents a phosphate group which may
be optionally substituted with one or more substituents,
or a thiophosphate group which may be optionally
substituted with one or more substituents, and R21, R22
and Rn represent independently of each other a hydrogen
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78
atom, a C1_6 alkyl group which may be optionally
substituted with one or more substituents, or a
protecting group for amino group.).
(EFFECT OF INVENTION)
[0012]
The preparation method of the present invention can
reduce largely the number of steps compared to the
conventional method, and also improved the yield of GuNA
largely. The replacement method of nucleic acid base of
the present invention can prepare GuNA that contains
various nucleic acid bases other than a thymine group
effectively and also a preferred 3-form selectively. The
preparation method of the present invention can prepare
GuNA oligomer which contains GuNA monomer having various
kinds of nucleic acid bases, for example, GuNA monomer
having a base moiety of adeninyl, guaninyl, cytosinyl, 5-
methylcytosinyl or uracinyl, or GuNA oligomer containing
these GuNA monomers. The intermediate compound of the
present invention is useful for preparing GuNA of various
kinds of nucleic acid base.
[0013]
The intermediate compound of the present invention
includes 2'-amino LNA monomer. The replacement of
nucleic acid base of the present invention can prepare
2'-amino LNA having not only adeninyl or thyminyl, but
also the other various kinds of nucleic acid base, for
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79
example, 21-amino LNA having a base moiety of guaninyl,
cytosinyl, 5-methylcytosinyl or uracinyl.
Using the monomer of the 2'-amino LNA having various
kinds of nucleic acid base that is prepared by the
present invention, an oligomer of 2'-amino LNA can be
prepared. The oligomer of 2'-amino LNA is resistant to
nuclease, and also binds strongly to a nucleic acid
having a complementary sequence, and accordingly, it can
be used as pharmaceuticals containing nucleic acid for
treatment, alleviation, prevention, relapse prevention
and diagnosis of various diseases, and can be used widely
as reagents containing nucleic acid for various
inspections or tests.
[0014]
The GuNA that is prepared by the present invention
is remarkably resistant to nuclease, and also binds
strongly to nucleic acid having a complementary sequence,
and further, migrates effectively into cells, and
accordingly, it can be used as pharmaceuticals containing
nucleic acid for treatment, alleviation, prevention,
relapse prevention and diagnosis for various diseases,
and can be used widely as reagents containing nucleic
acid for various inspections or tests.
MODE FOR CARRYING OUT THE INVENTION
[0015]
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Hereinafter, the present invention is further
explained in details. All of the publications cited
herein are incorporated herein by reference.
(Definition)
First, the definitions of the terms as used herein
are described.
[0016]
The term of "C1_6 alkyl that may be optionally
substituted with one or more substituents" as used herein
encompasses any straight chain alkyl group having 1 to 6
carbon atoms (C1-6), preferably 1 to 4 carbon atoms (C1_4),
any branched chain alkyl group having 3 to 6 carbon atoms
in which the identical or different branched chains are
contained, any cyclic alky group having 3 to 6 carbon
atoms, and any combinations thereof which have 4 to 6
carbon atoms. Specific examples of the straight chain
alkyl group having 1 to 6 carbon atoms include methyl,
ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl.
Specific examples of the branched chain alkyl group
having 3 to 6 carbon atoms in which the identical or
different branched chains are contained include iso
(which is abbreviated as "i")- propyl, isobutyl, tert
(which is abbreviated as "t")- butyl, sec (which is
abbreviated as "s")- butyl, neopentyl, isopentyl, and the
others, and also examples of the cyclic alkyl group
having 3 to 6 carbon atoms include preferably a 3 to 6
membered monocyclic cycloalkyl group, and specific
examples thereof include cyclopropyl, cyclobutyl,
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cyclopentyl, cyclohexyl, and the others, which are not
limited thereto. Examples of the substituents include
one or more (or preferably one to three) identical or
different groups selected from the group consisting of a
C1_6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group,
a C1_6 alkoxy group, an amino group, a hydroxy group, an
oxo group, a thioxo group, and a halogen atom.
[0017]
The term of "C2_6 alkenyl that may be optionally
substituted with one or more substituents" as used herein
encompasses any straight chain alkenyl group having 2 to
6 carbon atoms (C2_6), preferably having 2 to 4 carbon
atoms (C2_4), any branched chain alkenyl group having 3 to
6 carbon atoms in which the identical or different
branched chains are contained, any cyclic alkenyl group
having 3 to 6 carbon atoms, and any combinations thereof
that have 4 to 6 carbon atoms. Specific examples of the
straight chain alkenyl group having 2 to 6 carbon atoms
include ethenyl, 1-propenyl, 2-propenyl, 1-buteny1, 2-
butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,
1-hexenyl, and the others. Specific examples the
branched chain alkenyl group having 3 to 6 carbon atoms
in which the identical or different branched chains are
contained include isopropenyl, 1-methyl-l-propenyl, 1-
methy1-2-propenyl, 2-methyl-l-propenyl, 2-methy1-2-
propenyl, 1-methyl-2-butenyl and the others, and also
examples of the cyclic alkenyl group having 3 to 6 carbon
atoms include a three to six membered monocyclic
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82
cycloalkenyl group, and specific examples thereof include
cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl,
and the others. Examples of the substituents include one
or more (or preferably one to three) identical or
different groups selected from the group consisting of a
c1_6 alkyl group, a C2_6 alkenyl group, a C2-6 alkynyl group,
a C1_6 alkoxy group, an amino group, a hydroxy group, an
oxo group, a thioxo group, and a halogen atom.
[0018]
The term of "a C2-6 alkynyl that may be optionally
substituted with one or more substituents" as used herein
encompasses any straight chain alkynyl group having 2 to
6 carbon atom (C2-6), preferably any straight chain
alkynyl group having 2 to 4 carbon atoms (C2-4), any
branched chain alkynyl group having 3 to 6 carbon atoms
which have the identical or different branched chains,
any cyclic alkynyl group having 3 to 6 carbon atoms, and
any combinations thereof which have 4 to 6 carbon atoms.
Specific examples of the straight chain alkynyl group
having 2 to 6 carbon atoms include ethynyl, 1-propynyl,
2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,
3-pentynyl, 4-pentynyl, 1-hexynyl and the others.
Specific examples of the branched chain alkynyl group
having 3 to 6 carbon atoms in which the identical or
different branched chains are contained include
isopropynyl, 1-methyl-l-propynyl, 1-methyl-2-propynyl, 2-
methyl-1-propynyl, 2-methyl-2-propynyl, 1-methy1-2-
butynyl, and the others, and also examples of the cyclic
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83
alkynyl group having 3 to 6 carbon atoms include
preferably a three to six membered monocyclic
cycloalkynyl group, and specific examples thereof include
cyclobutynyl, cyclopentynyl, cyclohexynyl and the others,
which are not limited thereto. Examples of the
substituents include one or more (or preferably one to
three) identical or different group selected from the
group consisting of a C1-6 alkyl group, a C2-6 alkenyl
group, a C2-6 alkynyl group, a C1_6 alkoxy group, an amino
group, a hydroxy group, an oxo group, a thioxo group, and
halogen atom.
[0019]
The term of "C1_6 alkoxy group" as used herein
represents a monovalent group in which the C1_6 alkyl
binds to an oxygen atom, and represents a C1_6 alkyl-0
group. Specific examples thereof include methoxy, ethoxy,
n-propoxy, i-propoxy, n-butoxy, t-butoxy, s-butoxy, 3-
methylbutoxy, and the others, which are not limited
thereto.
[0020]
Examples of the term of "halogen (atom)" as used
herein include fluorine atom (fluoro), chlorine atom
(chloro), bromine atom (bromo), and iodine atom (iodo).
[0021]
The term of "aryl (group)" as used herein represents
a functional group or a substituent group which is
derivatized from aromatic hydrocarbon group and
encompasses a group composed of a plurality of cycles,
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84
and specifically, represents a monovalent group having 6
to 14 carbon atoms in which one hydrogen atom is excluded
from aromatic hydrocarbon atom, and include, for example,
phenyl, indenyl, naphthyl, phenanthrenyl, anthracenyl and
the others. Also the aryl ring may be substituted with
one or more groups selected from a halogen atom, a C1_6
alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a
hydroxy group, a C1_6 alkoxy group, an aryloxy group, an
amino group, a nitro group, a trifluoromethyl group, or a
phenyl group. Examples of the aryl group which may be
optionally substituted include 2-methylphenyl, 3-
methylphenyl, 4-methylphenyl, 2,6-dimethylphenyl, 2,4-
dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-
dichlorophenyl, 2,5-dichlorophenyl, 2,6-dichlorophenyl,
2-bromophenyl, 4-methoxyphenyl, 4-chloro-2-nitrophenyl,
4-nitrophenyl, 2-nitrophenyl, 2,4-dinitrophenyl, biphenyl
and the others. Preferred examples of the aryl group
include a phenyl group which is substituted with halogen
atom, C1.6 alkoxy group or nitro group, or an
unsubstituted phenyl group.
[0022]
The term of "heteroaryl (group)" as used herein
represents a monovalent group in which one hydrogen atom
is excluded from optional heteroaromatic compound
containing heteroatoms (such as nitrogen atom, oxygen
atom, and/or sulfur atom) in a ring structure and having
3 to 12 carbon atoms, and include, for example, pyridyl,
pyrimidinyl, pyridazinyl, pyrrolyl, isoquinolyl, quinolyl,
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indolyl, imidazolyl, triazolyl, furyl, thienyl and the
others. Also, the heteroaryl ring may be optionally
substituted with one or more groups selected from a
halogen atom, a C1_6 alkyl group, a C2-6 alkenyl group, a
C2_6 alkynyl group, a hydroxy group, a C1.6 alkoxy group,
an aryloxy group, an amino group, a nitro group, a
trifluoromethyl group, or a phenyl group and the others.
[0023]
Herein when the compound of the present invention,
an intermediate compound, or a starting material and the
others has a functional group (such as, a hydroxy group,
an amino group, a carboxyl group and the others), the
functional group may be protected with a protecting group
that is usually used in an organic synthetic chemistry
according to the method described in Theodora W. Greene,
Peter G. M. Wuts, "Protective Groups in Organic
Synthesis" 4th. ed., John Wiley & Sons, Inc. 1999, and
after the reaction, the protecting group may be then
removed to obtain the desirable compound. Examples of
the protecting group include the protecting group that is
usually used in an organic synthetic chemistry as
described in the same document, and is also usually used
in an organic synthetic chemistry, and examples of each
of the protecting group depending on the functional group
are described below.
[0024]
The term of "protecting group" described in terms of
"a protecting group for hydroxy group", "a protecting
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group for amino group", "a protecting group for phosphate
group", or "a protecting group for mercapto group" as
used herein is not particularly limited to any group as
long as it can protect amino group, hydroxy group,
phosphate group or mercapto group stably during a nucleic
acid synthesis. Specific examples thereof include a
protecting group that is stable under acidic or neutral
condition and also can be cleaved by a chemical method
such as a hydrogenolysis, a hydrolysis, an electrolysis,
and a photolysis. Examples of the protecting group
includes an alkyl group having 1 to 6 carbon atoms; an
alkenyl group having 2 to 6 carbon atoms; an alkynyl
group having 2 to 6 carbon atoms; an acyl group; a
tetrahydropyranyl group or a tetrahydrothiopyranyl group;
a tetrahydrofuranyl group or a tetrahydrothiofuranyl
group; a silyl group; a methyl group substituted with
alkoxy group having 1 to 6 carbon atoms; a methyl group
substituted with alkoxy group having 1 to 6 carbon atoms,
said alkoxy group being substituted with alkoxy group
having 1 to 6 carbon atoms; a methyl group substituted
with alkoxy group having 1 to 6 carbon atoms said alkoxy
group being substituted with halogen atom; an ethyl group
substituted with alkoxy group having 1 to 6 carbon atoms;
an ethyl group substituted with halogen atom; a methyl
group substituted with one to three aryl groups; a methyl
group substituted with one to three aryl groups, said
aryl groups being substituted with alkyl group having 1
to 6 carbon atoms, alkenyl group having 2 to 6 carbon
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87
atoms, alkynyl group having 2 to 6 carbon atoms, alkoxy
group having 1 to 6 carbon atoms, halogen atom, and/or
cyano group; a carbonyl group substituted with alkoxy
group having 1 to 6 carbon atoms; an aryl group
substituted with halogen atom, alkoxy group having 1 to 6
carbon atoms and/or nitro group; a carbonyl group
substituted with alkoxy group having 1 to 6 carbon atoms,
said alkoxy group being substituted with halogen atom
and/or silyl group substituted with alkyl group having 1
to 6 carbon atoms; an alkenyloxycarbonyl group; an
aralkyloxycarbony group which may be optionally
substituted with alkoxy group having 1 to 6 carbon atoms
and/or aryl group substituted with nitro group; and the
others.
[0025]
The term of "protecting group for hydroxy group"
represents a protecting group that is usually used in an
organic synthetic chemistry (in particular, nucleic acid
synthesis), and includes, for example, an aliphatic acyl
group; an aromatic acyl group; an aminocarbonyl group
which may be substituted; an alkoxycarboyl group which
may be substituted; an aliphatic sulfonyl group; an
aromatic sulfonyl group; a methyl group substituted with
one to three aryl groups; a methyl group substituted with
one to three aryl groups, said aryl group being
substituted with alkyl group having 1 to 6 carbon atoms,
alkoxy group having 1 to 6 carbon atoms, halogen atom
and/or cyano group; or a silyl group. Specific examples
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thereof include benzyl (En), 4,4'-dimethoxytrityl (DMTr),
4-methoxytrityl, triphenylmethyl, 2-naphthylmethyl,
diphenylaminocarbonyl (DPC), cyanoethoxycarbonyl,
tetrahydropyranyl, trimethylsilyl, triethylsilyl,
triisopropylsilyl, t-butyldimethylsilyl, t-
butyldiphenylsilyl, triphenylsilyl, 4-methoxybenzyl (p-
methoxybenzyl), 3,4-dimethoxybenzyl, 2,6-dimethoxybenzyl,
p-phenylbenzyl, methanesulfonyl, trifluoromethansulfonyl,
methoxymethyl, benzoyl (Br), and acetyl and the others,
which should not be limited thereto. Preferably, benzyl
(En), 4,4'-dimethoxytrityl (DMTr), t-butyldimethylsilyl,
t-butyldiphenylsilyl, trimethylsilyl (TMS),
diphenylaminocarbonyl (DPC), methanesulfonyl, and
trifluoromethansulfonyl are included.
[0026]
The term of "hydroxy group which may be optionally
substituted with one or more substituents" as used herein
encompasses a hydroxy group which may optionally have a
protecting group, and examples of the substituents
includes the above-mentioned protecting group for hydroxy
group, C16 alkyl group, aryl group which may be
optionally substituted, and heteroaryl group which may be
optionally substituted. Preferred examples of protecting
group for hydroxy group include En, DPC, DMTr, 4-
methoxytrityl, triphenylmethyl, 2-naphthylmethyl,
cyanoethoxycarbonyl, tetrahydropyranyl, trimethylsilyl,
triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, trimethylsilyl, 4-methoxybenzyl,
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3,4-dimethoxybenzyl, benzoyl (Bz), and acetyl, and
preferred examples of C1-6 alkyl include methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl,
pentyl, and isopentyl, and preferred examples of aryl
include phenyl, biphenyl, naphthyl, and anthracenyl, and
preferred examples of heteroaryl include pyridyl,
pyrimidinyl, pyridazinyl, pyrrolyl, isoquinolyl, quinolyl,
indolyl, imidazolyl, benzimidazolyl, triazolyl, furyl,
thienyl and the others. Also, examples of the
substituents of the aryl group which may be optionally
substituted or the heteroaryl group which may be
optionally substituted include a halogen atom, a C1-6
alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group,
hydroxy group, a C1_6 alkoxy group, an aryloxy group, an
amino group, a nitro group, a trifluoromethyl group, a
phenyl group and the others, which may be further
optionally substituted with one or more of these groups.
[0027]
The term of "protecting group for amino group"
represents a protecting group that is usually used in an
organic synthetic chemistry (in particular, nucleic acid
synthesis), and include, for example, an aliphatic acyl
group; an aromatic acyl group; an alkoxycarboyl group
which may be optionally substituted; a methyl group
substituted with one to three aryl groups; and a methyl
group substituted with aryl groups, said aryl group being
substituted with halogen atom and/or cyano group.
Specific examples thereof include acetyl (Ac),
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phenoxyacetyl (Pac), propionyl group, isobutyryl, benzoyl
(Bz), methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl
(Boo), trimethylsilylethoxycarbonyl (Teoc),
cyanoethoxycarbonyl (Ceoc), benzyloxycarbonyl (Cbz),
allyloxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),
2,2,2-trichloroethoxycarbonyl, t-amyloxycarbonyl, 4-
methoxybenzyl, triphenylmethyl, 2-nitrobenzenesulfonyl,
2,4-dinitrobenzenesulfonyl or 2-
(trimethylsilyl)ethoxymethyl and the others, which are
not limited thereto. Preferably, isobutyryl, benzoyl
(Bz), t-butoxycarbonyl (Boo), and
trimethylsilylethoxycarbonyl (Teoc) are included.
[0028]
The term of "amino group which may be optionally
substituted with one or more substituents" as used herein
encompasses an amino group which may optionally have a
protecting group, and the examples of substituents
include the above-mentioned protecting group for amino
group, a C1-6 alkyl group, an aryl group which may be
optionally substituted, and a heteroaryl group which may
be optionally substituted. Preferred examples of the
protecting group for amino group include Bn, DPC, CMTr,
4-methoxytrityl, triphenylmethyl, 2-naphthylmethyl,
cyanoethoxycarbonyl, tetrahydropyranyl, trimethylsilyl,
triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, trimethylsilyl, 4-methoxybenzyl,
3,4-dimethoxybenzyl, benzoyl (Bz), and acetyl, and
preferred examples of C1_6 alkyl group include methyl,
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ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-
butyl, pentyl, and isopentyl, and preferred examples of
aryl group include phenyl, biphenyl, naphthyl, and
anthracenyl, and preferred examples of heteroaryl group
include pyridyl, pyrimidinyl, pyridazinyl, pyrrolyl,
isoquinolyl, quinolyl, indolyl, imidazolyl,
benzimidazolyl, triazolyl, furyl, thienyl and the others.
Also, examples of the substituents of the aryl group
which may be optionally substituted, or the heteroaryl
group which may be optionally substituted include a
halogen atom, a C1_6 alkyl group, a C2-6 alkenyl group, a
C2_6 alkynyl group, a hydroxy group, a ci..6 alkoxy group,
an aryloxy group, an amino group, a nitro group, a
trifluoromethyl group, and a phenyl group, and the others,
which may be further optionally substituted with one or
more of these groups.
[00293
The term of "protecting group for a phosphate group"
represents a protecting group that is usually used in an
organic synthetic chemistry (in particularly, nucleic
acid synthesis), and examples thereof include an alkyl
group having 1 to 6 carbon atoms and/or an alkyl group
having 1 to 6 carbon atoms substituted with cyano group;
an aralkyl group; an aralkyl group substituted with aryl
group, said aryl group being substituted with nitro group
and/or halogen atom; an aryl group substituted with alkyl
group having 1 to 6 carbon atoms, halogen atom, or nitro
group; 2-cyanoethyl group; 2,2,2-trichloroethyl group;
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benzyl group; 2-chlorophenyl group; and 4-chlorophenyl
group, and the others, which are not limited thereto.
[0030]
The term of "protecting group for mercapto group"
represents a protecting group that is usually used in an
organic synthetic chemistry (in particularly, nucleic
acid synthesis), and includes, for example, an aliphatic
acyl group, an aromatic acyl group, a benzoyl group (Bz),
and the others, which are not limited thereto.
[0031]
The term of "leaving group" as used herein
represents a partial substrate having electron pairs that
is created when the substrate is cleaved by a cleavage of
heterolysis during reaction(s), and encompasses a halogen
atom (such as fluorine atom, chlorine atom, bromine atom,
iodine atom), and "a leaving group for hydroxy".
Examples of the leaving group for hydroxyl include a
sulfonyloxy group (such as paratolunesulfonyloxy,
mesyloxy, and trifluoromethanesulfonyloxy), an acyloxy
group (preferably, saturated or unsaturated acyloxy group
having 1 to 8 carbon atoms, for example, aryl group
represented by R'-C(-0)-0- wherein the RL represents an
aryl group which may be optionally substituted with alkyl
group (the total number of carbon atom is preferably 6 to
8, for example, phenyl, or p-tolyl), an aryloxy group
which may be optionally substituted with alkyl group (the
total number of carbon atom is preferably 6 to 8, for
example, phenoxy, or p-tolyloxy), an aralkyl group (the
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total number of carbon atom is preferably 7 to 9, for
example, benzyl), an arylalkenyl group (the total number
of carbon atom is preferably 8 or 9, for example,
cinnamyl), an aralkyloxy group (the total number of
carbon atom is 7 to 15, for example, benzyloxy, or 9-
fluorenylmethyloxy), an alkoxy group (a straight chain or
branched chain alkoxy group, for example, methoxy, ethoxy,
t-butoxy), and specific examples of the leaving group
include iodo, bromo, chloro, fluoro, mesyloxy,
methanesulfonyloxy, trifluoromethanesulfonyloxy,
ethanesulfonyloxy, 2,2,2-trifluoroethanesulfonyloxy,
propanesulfonyloxy, iso-propanesulfonyloxy,
butanesulfonyloxy, nonafluorobutanesulfonyloxy,
heptafluoropropan-l-sulfonyloxy, pentanesulfonyloxy,
pentafluoroethanesulfonyloxy, pentanesulfonyloxy,
cyclopentanesulfonyloxy, hexanesulfonyloxy,
cyclohexanesulfonyloxy, o-toluenesulfonyloxy, m-
toluenesulfonyloxy, p-toluenesulfonyloxy,
benzenesulfonyloxy, o-bromobenzenesulfonyloxy, m-
bromobenzenesulfonyloxy, p-bromobenzenesulfonyloxy, o-
nitrobenzenesulfonyloxy, m-nitrobenzenesulfonyloxy, and
p-nitrobenzenesulfonyloxy, and the others, which are not
limited thereto. Preferred examples of the leaving group
include methanesulfonyloxy (mesyloxy; Ms-0-),
trifluoromethanesulfonyloxy, and p-toluenesulfonyloxy.
[0032]
The term of "phosphate group which may be optionally
substituted" as used herein represents a phosphate, a
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phosphite, or a hydrophosphite, each of which may have
optionally substituents (which encompasses protecting
group). The term encompasses a phosphate group
represented by a formula of -P(R1)R2, wherein RP1 and RP2
represent independently of each other a hydroxy group, a
hydroxy group that is protected with a protecting group
for nucleic acid synthesis, a mercapto group, a mercapto
that is protected with a protecting group for nucleic
acid synthesis, an amino group, an alkoxy group having 1
to 5 carbon atoms, an alkylthio group having 1 to 6
carbon atoms, a cyanoalkoxy group having 1 to 6 carbon
atoms, or an amino group that is substituted with alkyl
group having 1 to 6 carbon atoms. Here the groups of the
above-mentioned formula wherein RP' represents ORPla and
RP2 represents NRP2a is referred to as "phosphoramidite
group", which is a preferred example. The lela represents
an alkyl group having 1 to 5 carbon atoms or a cyanoalkyl
group having 1 to 6 carbon atoms, and the RP2a represents
an alkyl group having 1 to 6 carbon atoms. Specific
examples of "phosphoramidite group" include a group
represented by a formula of -P(O(CH2)2CN)(N(iPr)2), or a
group represented by a formula of -P(0CH3)(N(iPr)2) and
the others, which is not limited thereto. Herein "iPr"
represents an isopropyl group.
[0033]
The term of "thiophosphate group which may be
optionally substituted" as used herein encompasses a
thiophosphate group which may have optionally
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substituents (which encompasses protecting group). The
term may encompass a phosphate group represented by
formula -P(=S)(RP3)RP4, and in the formula, RP3 and RP4
represent independently of each other a hydroxy group, a
hydroxy group that is protected with a protecting group
for nucleic acid synthesis, a mercapto group, a mercapto
group that is protected with a protecting group for
nucleic acid, an amino group, alkoxy group having one to
five carbon atoms, an alkylthio group having 1 to 6
carbon atoms, a cyanoalkoxy group having 1 to 6 carbon
atoms, or an amino group that is substituted with alkyl
group having 1 to 6 carbon atoms.
[0034]
Examples of the term of "acyl group" as used herein
include an aliphatic acyl group and aromatic acyl group.
Specific examples of the aliphatic acyl group include an
alkylcarbonyl group, such as formyl, acetyl, propionyl,
butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl,
isovaleryl, octanoyl, nonanoyl, decanoyl, 3-
methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-
dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl,
tetradecanoyl, pentdecanoyl, hexadecanoyl, 1-
methylpentdecanoyl, 14-methylpentadecanoyl, 13,13-
dimethyltetradecanoyl, heptadecanoyl, 15-
methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl,
nonadecanoyl, eicosanoyl, and henaicosanoyl; an
aryloxyalkycarbonyl group such as phenoxyacetyl (pac); a
carboxylated alkylcarbonyl group such as succinoyl,
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glutaroyl, and adipoyl; a carbonyl group that is
substituted with alkyl group having 1 to 6 carbon atoms,
said alkyl group being substituted with halogen atom,
such as chloroacetyl group, dichloroacetyl,
trichloroacetyl, trifluoroacetyl; an alkoxyalkylcarbonyl
group having 1 to 6 carbon atoms such as methoxyacetyl;
and an unsaturated alkylcarbonyl group such as (E)-2-
methy1-2-butenoyl. Also, examples of the aromatic acyl
group include an arylcarbonyl group such as benzoyl, a-
naphthoyl, and p-naphthoyl; a halogenoarylcarbonyl group
such as 2-bromobenzoyl and 4-chlorobenzoyl; an
arylcarbonyl group that is substituted with alkyl group
having 1 to 6 carbon atoms, such as 2,4,6-
trimethylbenzoyl, and 4-toluoyl; an arylcarbonyl group
that is substituted with alkoxy group having 1 to 6
carbon atoms, such as 4-anisoyl; a carboxylated
arylcarbonyl group such as 2-carboxybenzoyl, 3-
carboxybezoyl, and 4-carboxybenzoyl; a nitrolated
arylcarbonyl group such as 4-nitrobenzoly1 and 2-
nitrobenzoyl; a carbonylated arylcarbonyl group that is
substituted with alkoxy group having 1 to 6 carbon atoms,
such as 2-(methoxycarbonyl)benzoyl; and an arylated
arylcarbonyl group such as 4-phenylbenzoyl, which are not
limited thereto.
[0035]
The term of "aralkyl group" as used herein
represents an alkyl group of 1 to 6 carbon atoms,
preferably 1 to 4 carbon atoms, more preferably 1 to 3
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carbon atoms, said alkyl group being substituted with
aromatic hydrocarbon group (such as, 6 to 14 membered
monocyclic, bicyclic, or tricyclic aromatic hydrocarbon
group). Specific examples thereof include benzyl,
phenethyl, 1-naphthylmethyl, 2-naphthylmethyl, and the
others, but which are not limited thereto.
[0036]
Examples of tetrahydropyranyl group or
tetrahydrothiopyranyl group include more specifically,
tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-
methoxytetrahydropyran-4-yl, tetrahydrothiopyran-4-yl, 4-
methoxytetrahydrothiopyran-4-yl, and the others, but
which are not limited thereto.
[0037]
Examples of tetrahydrofuranyl group or
tetrahydrothiofuranyl group include more specifically,
tetrahydrofuran-2-yl, tetrahydrothiofuran-2-yl, and the
others, but which are not limited thereto.
[0038]
Examples of term of "silyl group" as used herein
include a silyl group that is substituted with alkyl
group having 1 to 6 carbon atoms, such as trimethylsilyl,
triethylsilyl, isopropyldimethylsilyl, t-
butyldimethylsilyl, methyldiisopropylsilyl, methyl di-t-
butylsilyl, triisopropylsilyl; a silyl group that is
substituted with one or two aryl group and also three
alkyl groups having 1 to 6 carbon atoms, such as t-
butyldiphenylsilyl, diphenylmethylsilyl,
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butyldiphenylbutylsilyl, diphenylisopropylsilyl, and
phenyldiisopropylsilyl; and triphenylsilyl, but which are
not limited thereto.
[0039]
Examples of the term of "methyl group that is
substituted with alkoxy group having 1 to 6 carbon atoms"
as used herein include methoxymethyl, 1,1-simethyl-l-
methoxymethyl, ethoxymethyl, propoxymethyl,
isopropoxymethyl, butoxymethyl, t-butoxymethyl and the
others, but which are not limited thereto.
[0040]
Examples of the term of "methyl group that is
substituted with alkoxy group having 1 to 6 carbon atoms,
said alkoxy group being substituted with alkoxy group
having 1 to 6 carbon atoms" as used herein include 2-
methoxyethoxymethyl, but which is not limited thereto.
[0041]
Examples of the term of "methyl group that is
substituted with alkoxy group having 1 to 6 carbon atoms,
said alkoxy group being substituted with halogen atom" as
used herein include 2,2,2-trichloroethoxymethyl, and
bis(2-chloroethoxy)methyl and the others, but which are
not limited thereto.
[0042]
Examples of the term of "ethyl group that is
substituted with alkoxy group having 1 to 6 carbon atoms"
as used herein include 1-ethoxyethyl, 1-(isopropoxy)ethyl
and the others, but which are not limited thereto.
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[0043]
Examples of the term of "ethyl group that is
substituted with halogen atom" as used herein include
2,2,2-trichloroethyl and the others, but which is not
limited thereto.
[0044]
Examples of the term of "methyl group that is
substituted with one to three aryl groups" as used herein
include benzyl, a-naphthylmethyl, 13-naphthylmethyl,
diphenylmethyl, triphenylmethyl, 4,4'-dimethoxytrityl, 4-
methoxytrityl, trityl, a-naphthyldiphenylmethyl, 9-
anthrylmethyl and the others, but which are not limited
thereto.
[0045]
Examples of the term of "methyl group that is
substituted with one to three aryl groups, said aryl
groups being substituted with alkyl group having 1 to 6
carbon atoms, alkoxy group having 1 to 6 carbon atoms,
halogen atoms and/or cyano groups" as used herein include
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, 4-cyanobenzyl and the
others, but which are not limited thereto.
[0046]
Examples of the term of "carbonyl group that is
substituted with alkoxy group having 1 to 6 carbon atoms"
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as used herein include methoxycarbonyl, ethoxycarbonyl,
t-butoxycarbonyl, isobutoxycarbonyl, Teoc, Ceoc, Fmoc and
the others, which are not limited thereto.
[0047]
Examples of the term of "aryl group that is
substituted with halogen atoms, alkoxy group having 1 to
6 carbon atoms, and/or nitro group" as used herein
include 4-chlorophenyl, 2-flnorophenyl, 4-methoxyphenyl,
4-nitrophenyl, 2,4-dinitrophenyl and the others, which
are not limited thereto.
[0048]
Examples of the term of "carbonyl group that is
substituted with alkoxy group having 1 to 6 carbon atoms,
said alkoxy group being substituted with silyl group and
said silyl group being substituted with halogen atoms
and/or three alkyl group having 1 to 6 carbon atoms" as
used herein include 2,2,2-trichloroethoxycarbonyl, 2-
trimethylsilylethoxycarbonyl and the others, which are
not limited thereto.
[0049]
Examples of the term of "alkenyloxycarbonyl group"
as used herein include vinyloxycarbonyl, allyloxycarbonyl
and the others, which are not limited thereto.
[0050]
Examples of the term of "aralkyloxycarbonyl group
that is substituted with aryl group, said aryl group
being substituted with alkoxy group having 1 to 6 carbon
atoms and/or nitro groups" as used herein include
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benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-
dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-
nitrobenzyloxycarbonyl and the others, which are not
limited thereto.
[0051]
The term of "unsaturated heterocycle" as used herein
encompasses an unsaturated aliphatic heterocyclic group
or an aromatic heterocyclic group. The unsaturated
aliphatic heterocyclic group represents an unsaturated
aliphatic heterocyclic group in which at least one double
bonds are contained in the ring, and one to three
identical or different heteroatoms independently selected
from oxygen atom, sulfur atom and nitrogen atom are
contained, preferably, an unsaturated aliphatic
heterocyclic group in which at least one double bonds are
contained in the ring, at least one nitrogen atoms is
contained, and further at least one (preferably one to
two (when two or more thereof are present, they may be
the identical or different from each other), particularly
preferably one) heteroatoms independently selected from
the group consisting of oxygen atom, sulfur atom and
nitrogen atom. The unsaturated aliphatic heterocyclic
group is, for example, a four to eight membered cycle,
preferably a five to seven membered cycle, more
preferably a five or six membered cycle, and particularly
preferably a six membered cycle. Specific examples
thereof include dihydropyrimidinyl, pyrrolidinyl,
imidazolidinyl, pyrazolidinyl and the others, which are
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not limited thereto. Also, the aromatic heterocyclic
group represents an aromatic heterocyclic group wherein
the heterocyclic moiety contains one to three identical
or different heteroatoms independently selected from the
group consisting of an oxygen atom, a sulfur atom and a
nitrogen atom, preferably an aromatic heterocyclic group
wherein the heterocyclic moiety contains at least one
nitrogen atoms, and further at least one (preferably one
to two (when two or more thereof are present, they are
identical or different from each other), particularly
preferably one) heteroatoms selected from the group
consisting of an oxygen atom, a sulfur atom and a
nitrogen atom. The aromatic heterocyclic group is, for
example, a four to eight membered cycle, preferably a
five to seven membered cycle, more preferably a five or
six membered cycle, and particularly preferably a six
membered cycle. Specific examples thereof include
pyrrolyl, furanyl, thienyl, pyridinyl, pyrimidinyl,
pyridazinyl, and the others, which are not limited
thereto. Particularly preferably, a pyrimidinyl is
included. The unsaturated heterocycle may be optionally
substituted with substituent(s), and examples of the
substituent include a C1_6 alkyl group which may be
optionally substituted, a C2-6 alkenyl group which may be
optionally substituted, a 02-6 alkynyl group which may be
optionally substituted, an amino group which may be
optionally substituted, a hydroxy group which may be
optionally substituted, an oxo group, a thio group, and a
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halogen atom, and the others, which are not limited
thereto.
[0052]
The unsaturated heterocycle may be further fused
with another cycle. Examples of the another cycle which
may be fused include a saturated or unsaturated
nonaromatic carbon cycle, an aromatic carbon cycle, a
saturated or unsaturated nonaromatic heterocycle, and an
aromatic heterocycle, preferably, an aromatic cycle or an
aromatic heterocycle. The saturated carbon cycle
represents an alkane forming a cycle, for example, the
above-mentioned cycloalkane having 3 to 6 carbon atoms.
The unsaturated nonaromatic carbon cycle represents an
alkene forming a cycle, for example, the above-mentioned
cyclic alkene having 3 to 6 carbon atoms. The aromatic
carbon cycle (which is referred to as "aryl") encompasses,
for example, a six to fourteen membered monocyclic,
bicyclic, or tricyclic hydrocarbon group, and specific
examples thereof include benzene, naphthalene,
phenanthrene, anthracene and the other, which are not
limited thereto. The saturated or unsaturated
nonaromatic heterocycle represents a saturated five to
fourteen monocyclic or bicyclic nonaromatic heterocycle
containing one to three heteroatoms independently
selected from the group consisting of an oxygen atom, a
sulfur atom, and a nitrogen atom, in which at least one
double bond may be contained in the ring. Specific
examples thereof include azetidine, pyrrolidine,
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pyrazolidine, piperidine, piperazine, morpholine,
thiomorpholine, homopiperidine, oxazolidine,
isoxazolidine, thiazolidine, isothiazolidine,
imidazolidine, imidazoline, tetrahydrofuran, dihydrofuran,
tetrahydrothien, dihydrothien, dihydropyran,
tetrahydropyran, indoline, isoindoline, chroman,
isochroman, dihydrobenzofuran, dihydrofuropyridine,
dihydrobenzothien, and the others, which are not limited
thereto.
[0053]
Also, the five to seven membered unsaturated
heterocycle in "five to seven membered unsaturated
heterocycle which may be optionally substituted" as
"cycle A" may be a base moiety of nucleic acid in the
term of "a base moiety of nucleic acid that may
optionally have substituents" as used herein as long as
it contains two or more nitrogen atoms and also one or
more oxygen atoms are substituted. They may be a cyclic
moiety derived from natural nucleic acid base or
unnatural nucleic acid base, for example, purine ring
derived from adenine or guanine, or derivatives thereof,
or mono or di-one ring derived from uracil, cytosine or
thymine, or derivatives thereof, preferably thyminyl or
uracinyl, which are not limited thereto. The substituent
is the same as defined in the above-mentioned unsaturated
heterocycle. Preferred substituents for cyclic A include
a C1_6 alkyl group which may be optionally substituted
with one or more substituents, a C2_6 alkenyl group which
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may be optionally substituted with one or more
substituents, a C2_6 alkynyl group which may be optionally
substituted with one or more substituents, an amino group
which may be optionally substituted with one or more
substituents, a hydroxy group which may be optionally
substituted with one or more substituents, an oxo group,
a thioxo group, and a halogen atom, which are not limited
thereto.
[0054]
The "cycle A'" being an unsaturated heterocycle as
used herein may be fused with tetrahydrofuran ring or
oxazolidine ring to form a polycyclic structure. The
cycle A' is not limited to any cycle, as long as it is an
unsaturated heterocycle containing two or more nitrogen
atoms, however, is preferably "five to seven membered
unsaturated heterocycle which may be optionally
substituted". The unsaturated heterocycle may be further
fused with another cycle. Examples of preferred
substituents for cycle A include a C1-6 alkyl group which
may be optionally substituted with one or more
substituents, a C2-6 alkenyl group which may be optionally
substituted with one or more substituents, a C2_6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, which are not limited thereto.
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[0055]
In the preparation method as described herein, when
the cycle A is changed into cycle A' by a reaction with
an activating agent for hydroxy group, or the fused
oxazolidine ring for the cycle A' is cleaved by a
reaction with a reducing agent to change into a cycle A,
the structurally non-limited moiety that is connected to
two nitrogen atoms are identical for the cases of cycle A
and cycle A'. The cycle A is not limited to any cycle as
long as it is an unsaturated heterocycle containing two
or more nitrogen atom, and is preferably a five to seven
membered unsaturated heterocycle which may be optionally
substituted, more preferably a pyrimidine group which may
be optionally substituted, and most preferably a thyminyl
group or an uracinyl group. The unsaturated heterocycle
may be further fused with another ring.
[0056]
The "a base moiety of nucleic acid" in the term of
"a base moiety of nucleic acid which may optionally have
substituents" includes, for example, a base moiety of
natural nucleic acid, and a base moiety of unnatural
nucleic acid, which contains an aromatic heterocycle, and
encompasses a monocyclic ring, a bicyclic ring, and a
tricyclic ring. Here it should be clearly understood
that base moieties of various nucleic acid that has been
now considered "unnatural" to a skilled person in the art,
could be found in nature hereafter. Thus "nucleic acid
base moiety" includes not only any heterocycle contained
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107
in publicly known purine and pyrimidine, but also their
heterocyclic analogues and tautomers. Specific examples
of the base moiety of nucleic acid include adeninyl,
guaninyl, thyminyl, cytosinyl, uracinyl, purinyl,
xanthinyl, diaminopurinyl, 8-oxo-N6-methyladeninyl, 7-
deazaxanthinyl, 7-deazaguaninyl, N4,N4-ethanocytosinyl,
N6,N6-ethano-2,6-diaminopurinyl, 5-methylcytosinyl, 5-
(C3-C6)-alkynylcytosinyl, 5-fluorocytosinyl, 5-bromoan
uracinyl, pseudoisocytosinyl, 2-hydroxy-5-methy1-4-
triazolopyridinyl, isocytosinyl, isoguaninyl, inosinyl,
N6-arylpurinyl, N6-acylpurinyl, N6-benzylpurinyl, N6-
halopurinyl, N6-vinylpurinyl, N6-acetylenic purinyl, N6-
acylpurinyl, N6-hydroxyalkylpurinyl, N6-thioalkylpurinyl,
N2-alkylpurinyl, N6-alkylpyrimidinyl, N4--acylpyrimidinyl,
N4-benzylpurinyl, N4-halopyrimidinyl, N4-vinylpyrimidinyl,
N4-acetylenic pyrimidinyl, N4-acetylpyrimidinyl, N4-
hydroxyalkylpyrimidinyl, N6-thioalkylpyrimidinyl, 6-
azapyrimidinyl, 6-azacytosinyl, 2-and/or 4-
mercaptopyrimidinyl, uracinyl, e-alkylpyrimidinyl, C5-
benzylpyrimidinyl, C5-halopyrimidinyl, C5-
vinylpyrimidinyl, C5-acetylenicpyrimidinyl, C5-
acylpyrimidinyl, C5-hydroxyalkylpurinyl, C5-
amidopyrimidinyl, C5-cyanopyrimidinyl, C5-
nitropyrimidinyl, C5-aminopyrimidinyl, N2-alkylpurinyl,
N2-alkyl-6-thiopurinyl, 5-cytidinyl, 5-azauracinyl,
trazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl,
pyrazolopyrimidinyl, and the others, which are not
limited thereto. Preferred examples of the base moiety
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108
of nucleic acid include adeninyl, guaninyl, 2,6-
diaminopurinyl, thyminyl, 2-thiothyminyl, cytosinyl, 5-
methylcytosinyl, uracinyl, 5-fluorocytosinyl, xanthinyl,
6-aminopurinyl, 2-aminopurinyl, 6-chloro-2-aminopurinyl,
and 6-chloropurinyl, and particular preferred examples of
the base moiety of nucleic acid include adeninyl,
guaninyl, cytosinyl, 5-methylcytosinyl, thyminyl, and
uracinyl. These base moieties of nucleic acid may
further have one or more substituents, and examples of
the substituents include hydroxy group, C1_6 alkoxy group,
mercapto group, C1_6 alkylthio group, amino group, amino
group substituted with C1_6 alkyl group, C1-6 alkyl group,
C1_6 alkynyl group, oxo group, thioxo group, and halogen
atom. If necessary or if desirable, the functional
oxygen atom, sulfur atom and nitrogen atom on the base
moiety may be protected and/or deprotected. The
appropriate protecting group is well known to a skilled
person in the art, and encompasses, for example, the
above-mentioned protecting group for hydroxy group and
protecting group for amino group, and includes
diphenylaminocarbonyl group, silyl group (such as
trimethylsilyl group, dimethylhexylsilyl group, t-
butyldimethylsily1 group, and t-butyldiphenylsilyl group),
trityl group, alkyl group, acyl group (such as acetyl
group, propionyl group, isobutyryl group, benzoyl group
(Bz), phenoxyacetyl group (Pac)), alkoxycarboyl group
(such as t-butoxycarbonyl group (Boc), benzyloxycarbonyl
group (Cbz), diphenylaminocarbonyl group (DPC),
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cyanoethoxycarbonyl group (Ceoc)), sulfonyl group (such
as methanesulfonyl group, and p-toluenesulfonyl group),
and the others, which are not limited thereto.
[0057]
The term of "azide agent" as used herein represents
a reagent for adding an azide group, and for example,
includes, preferably hydrazoic acid, sodium azide,
lithium azide, tetrabutylammonium azide (Bu4NN3),
trimethylsilyl azide, diphenylphosphoryl azide (DPPA),
nicotinyl azide, zinc azide (Zn(N3)2), and more
preferably tetrabutylammonium azide, sodium azide, and
diphenylphosphoryl azide (DPPA).
[0058]
The term of "activating agent for hydroxy group" as
used herein represents a reagent for increasing a leaving
ability by activating a non-protected hydroxy group, and
includes, for example, methanesulfonylating agent,
trifluoromethansulfonylating agent, ethanesulfonylating
agent, 2,2,2-trifluoroethanesulfonylating agent,
propanesulfonylating agent, isopropanesulfonylating agent,
butanesulfonylating agent, nonafluorobutanesulfonylating
agent, heptafluoropropane-l-sulfonylating agent,
pentanesulfonylating agent,
pentafluoroethanesulfonylating agent,
cyclopentanesulfonylating agent, hexanesulfonylating
agent, cyclohexanesulfonylating agent, o-
toluenesulfonylating agent, m-toluenesulfonylating agent,
p-toluenesulfonylating agent, benzenesulfonylating agent,
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o-bromobenzenesulfonylating agent, m-
bromobenzenesulfonylating agent, p-
bromobenzenesulfonylating agent, o-
nitrobenzenesulfonylating agent, m-
nitrobenzenesulfonylating agent, and p-
nitrobenzenesulfonylating agent, and specific examples
thereof include methanesulfonyl chloride, methanesulfonic
anhydride, trifluoromethanesulfonyl chloride,
trifluoromethanesulfonic anhydride,
ethanesulfonylchloride, 2,2,2-
trifluoroethanesulfonylchloride, 2,2,2-
trifluoroethanesulfonylfluoride, propanesulfonylchloride,
propanesulfonylfluoride, isopropylsulfonylchloride,
isopropylsulfonylfluoride, butanesulfonylchloride,
butanesulfonylfluoride, nonafluorobutanesulfonylchloride,
nonafluorobutanesulfonylfluoride, nonafluorobutane
sulfonic acid anhydride, heptafluoropropane-1-
sulfonylchloride, heptafluoropropane-l-sulfonylfluoride,
pentanesulfonylchloride, pentanesulfonylfluoride,
pentafluoroethanesulfonylchloride,
pentafluoroethanesulfonylfluoride,
cyclopentanesulfonylchloride,
cyclopentanesulfonylfluoride, hexanesulfonylchloride,
hexanesulfonylfluoride, cyclohexanesulfonylchloride,
cyclohexanesulfonylfluoride, o-toluenesulfonylchloride,
o-toluenesulfonylfluoride, m-toluenesulfonylchloride, m-
toluenesulfonylfluoride, p-toluenesulfonylchloride, p-
toluenesulfonylfluoride, benzenesulfonylchloride,
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benzenesulfonylfluoride, o-bromobenzenesulfonylchloride,
o-bromobenzenesulfonylfluoride, m-
bromobenzenesulfonylchloride, m-
bromobenzenesulfonylfluoride, p-
bromobenzenesulfonylchloride, p-
bromobenzenesulfonylfluoride, o-
nitrobenzenesulfonylchloride, o-
nitrobenzenesulfonylfluoride, m-
nitrobenzenesulfonylchloride, m-
nitrobenzenesulfonylfluoride, p-
nitrobenzenesulfonylchloride, p-
nitrobenzenesulfonylfluoride, and the others, which are
not limited thereto. For example, preferably,
trifluoromethanesulfonyl chloride,
trifluoromethanesulfonic anhydride, methanesulfonyl
chloride, methanesulfonic anhydride, p-toluenesulfonyl
chloride, nonafluoro-l-butanesulfonylfluoride,
heptafluoropropane-1-sulfonylfluoride, 2,2,2-
trifluoroethanesulfonylchloride, and
pentafluoroethanesulfonylchloride are preferably included,
and trifluoromethanesulfonyl chloride,
trifluoromethanesulfonic anhydride, methanesulfonyl
chloride, methanesulfonic anhydride, and p-
toluenesulfonylchloride are more preferably included.
[0059]
For example, the term of "reducing agent" as used
herein includes preferably phosphines such as
trialkylphosphine and triarylphosphine, metal hydrides,
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112
and transition metal catalysts in the presence of
hydrogen gas, and more preferably triphenylphosphine,
tributylphosphine, lithium aluminum hydride, sodium
borohydride, nickel borohydride, palladium on carbon in
the presence of hydrogen gas, and palladium hydroxide in
the presence of hydrogen gas.
[0060]
The term of "a phosphoramidite method" represents a
solid-phase synthesis method of nucleic acid using
nucleotide monomer, which comprises, for example, a step
of coupling the 5' hydroxy group or 3' hydroxy group of a
nucleotide, or the amino group of the base moiety of
nucleic acid, further, for RNA, a phosphoramidite monomer
in which the 2' hydroxy group is protected, with
tetrazole type compounds or imidazole type compounds etc.,
followed by oxidative reaction or oxidative sulfuration
reaction to form a phosphodiester bond. The
"phosphoramidite method" as used herein is used
preferably phosphoramidite monomer, which is at least one
kind of bridged nucleic acid, and the bridged nucleic
acid is more preferably GuNA.
[0061]
The "replacement of a cyclic A with a B" represents
a phenomenon in which the cycle A that binds to a sugar
moiety of nucleic acid is dissociated from the sugar
moiety, and is then replaced with a B, which is a base
moiety of nucleic acid which may be optionally
substituted with one or more substituents,
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(transglycosylation). Here the cycle A that binds to the
sugar moiety of nucleic acid is dissociated by treating
with Lewis acid, and a B binds to the sugar moiety of the
nucleic acid in which the cycle A is dissociated.
Examples of the Lewis acid used for dissociation include
TMSOTf, and TBSOTf, which are not limited thereto.
Silylating agent can be used to facilitate the
transglycosylation. Examples of the silylating agent
include BSA, and hexamethyldisilazane, which are not
limited thereto. The transglycosylation is preferably
conducted in one step.
[0062]
In the transglycosylation, the B may be provided as
any reactant as long as the B is in the form that can be
replaced, and the reactant is preferably "nucleic acid
base". Said "nucleic acid base" includes not only any
heterocycle contained in publicly known purine and
pyrimidine, but also their heterocyclic analogues and
tautomers. Specific examples of the base moiety of
nucleic acid include adenine, guanine, thymine, cytosine,
uracine, purine, xanthine, diaminopurine, 8-oxo-N6-
methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-
ethanocytosine, N6,N6-ethano-2,6-diaminopurine, 5-
methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-
fluorocytosine, 5-bromoan uracine, pseudoisocytosine, 2-
hydroxy-5-methyl-4-triazolopyridine, isocytosine,
isoguanine, inosine, N6-arylpurine, N6-acylpurine, N6-
benzylpurine, N6-halopnrine, N6-vinylpurine, N6-
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acetylenic purine, N6-acylpurine, N6-hydroxyalkylpurine,
N6-thioalkylpurine, N2-alkylpurine, N6-alkylpyrimidine,
N4--acylpyrimidine, N4-benzylpurine, N4-halopyrimidine,
N4-vinylpyrimidine, N4-acetylenic pyrimidinel, N4-
acetylpyrimidine, N4-hydroxyalkylpyrimidine, N6-
thioalkylpyrimidine, 6-azapyrimidine, 6-azacytosine, 2-
and/or 4-mercaptopyrimidine, uracine, Cs-alkylpyrimidine,
Cs-benzylpyrimidine, Cs-halopyrimidine, Cs-
vinylpyrimidine, Cs-acetylenicpyrimidine, Cs-
acylpyrimidine, Cs-hydroxyalkylpurine, Cs-amidopyrimidine,
Cs-cyanopyrimidinyl, Cs-nitropyrimidine, Cs-
aminopyrimidine, N2-alkylpurine, N2-alkyl-6-thiopurine,
5-cytidine, 5-azauracine, trazolopyridine,
imidazolopyridine, pyrrolopyrimidine, pyrazolopyrimidine,
and the others, which are not limited thereto. Preferred
examples of the nucleic acid base include adenine,
guanine, 2,6-diaminopurine, thymine, 2-thiothymine,
cytosine, 5-methylcytosine, uracine, 5-fluorocytosine,
xanthine, 6-aminopurine, 2-aminopurine, 6-chloro-2-
aminopurine, and 6-chloropurine, and particular preferred
examples of the base moiety of nucleic acid include
adenine, guanine, cytosine, 5-methylcytosine, thymine,
and uracine. These nucleic acid bases may further have
one or more substituents, and examples of the
substituents include hydroxy group, C1_6 alkoxy group,
mercapto group, C1_6 alkylthio group, amino group, amino
group substituted with C1.6 alkyl group, C1_6 alkyl group,
C1_6 alkynyl group, oxo group, thioxo group, and halogen
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115
atom. If necessary or if desirable, the functional
oxygen atom, sulfur atom and nitrogen atom on the base
moiety may be protected and/or deprotected. The
appropriate protecting group is well known to a skilled
person in the art, and encompasses, for example, the
above-mentioned protecting group for hydroxy group and
protecting group for amino group, and includes
diphenylaminocarbonyl group, silyl group (such as
trimethylsilyl group, dimethylhexylsilyl group, t-
butyldimethylsily1 group, and t-butyldiphenylsilyl group),
trityl group, alkyl group, acyl group (such as acetyl
group, propionyl group, isobutyryl group, benzoyl group
(Bz), phenoxyacetyl group (Pac)), alkoxycarboyl group
(such as t-butoxycarbonyl group (Boc), benzyloxycarbonyl
group (Cbz), diphenylaminocarbonyl group (DPC),
cyanoethoxycarbonyl group (Ceoc)), sulfonyl group (such
as methanesulfonyl group, and p-toluenesulfonyl group),
and the others, which are not limited thereto. These
nucleic acid bases are commercially available or can be
prepared using commercially available starting materials
according to organic synthesis methods.
[0063]
The term of "13 form" as used herein represents the a
compound having a stereochemistry in which the direction
of substituting the substituted base moiety of nucleic
acid at 1' position of a (deoxy) ribose of nucleic acid
and the direction of substituting the substituted 5'-
position side chain at 4' position of a (deoxy) ribose of
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116
nucleic acid are the same direction of the substitution.
For a bridged artificial nucleic acid 2',4'-LNA, the p
form represents the compound having the stereo
configuration in which the direction of substituting the
substituted base moiety of nucleic acid at 1 position of
a (deoxy) ribose of nucleic acid and the direction of
substituting the substituted 5'-position side chain that
is not used for bridging substituted at 4' position of
(deoxy) ribose of nucleic acid are the same direction of
the substitution.
The term of "a form" represents the a compound
having a stereochemistry in which the direction of
substituting the substituted base moiety of nucleic acid
at 1' position of a ribose of nucleic acid and the
direction of substituting the substituted 5'-position
side chain at 4' position of a ribose of nucleic acid are
the different direction of the substitution.
[0064]
The term of up selective" as used herein represents
that a p form can be obtained. It means that the 13 form
is obtained in its contents of preferably 80 % or more,
more preferably 90 % or more, most preferably 99% or more,
as opposed to the total contents of the a form and the p
form as a product obtained by the transglycosylation
[0065]
The term of "artificial nucleic acid" as used herein
encompasses artificial nucleoside, artificial nucleotide
(herein, one nucleoside or nucleotide is sometimes
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117
referred to as a monomer) or artificial oligonucleotide.
These artificial nucleic acids are not natural nucleic
acids, but nucleic acids that can be produced only
artificially. Examples of these artificial nucleic acids
include those in which the nucleic acid base moiety
contains an unnatural base, those having a sugar in which
the sugar moiety is modified, and/or those containing an
unnatural phosphate group as a phosphate moiety, and the
artificial nucleic acids as used herein represents those
in which the sugar moiety contains an unnatural sugar, in
particular, those containing a (deoxy) ribose in which
the carbon atoms at 2' position and 4' position are
bridged.
[0066]
The term of "artificial oligonucleotide" as used
herein represents a substance in which two or more of the
identical or different "artificial nucleotides" are
bonded with each other via a phosphodiester bond or a
thiophosphodiester bond and the like, and includes a
substance in which preferably 2 up to 100, more
preferably 5 up to 50, most preferably 10 up to 30
artificial nucleotides are bonded, or a substance in
which the nucleotides together with these complementary
strands forms a double strand. Herein, an
oligonucleotide in which two or more nucleotides are
bonded with each other is sometimes referred to as
oligomer.
[0067]
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118
Embodiments of the preparation methods and the
compounds of the present invention (including
intermediate compounds) are described.
[0068]
Preparation method of compound represented by general
formula I
According to one aspect of the present application,
the present invention provides a method for preparing a
compound represented by general formula I:
[chem.55]
R4
Ri
0
-R3
A
0NH
R54,
0
R-
0
[wherein
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each Rs represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
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a cycle A represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a Cl_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
or salts thereof,
said method comprising a step of reacting a compound
represented by formula II:
[chem.56]
R1, R4
R3
1R-
)
ON
Ny"-rn
-
R2-0
[wherein,
each of R1, R2, R3, 124, Rs, R6 and m is the same as
defined in the above-mentioned general formula I, a cycle
A' represents a five to seven membered unsaturated
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120
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2_6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A'.]
with a reducing agent to cleave an oxazolidine ring fused
to a cycle A'.
[0069]
According to one embodiment of the present
application, the reducing agent is preferably phosphines,
more preferably triphenylphosphine (Ph3P) .
[0070]
According to one embodiment, a compound represented
by formula I:
(chem.57)
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121
R4
o
Ri
R3
0
R5 11
0 (I)
Pt,
N
0
R2'
[wherein,
R1 and R2 represent independently of each other a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1_6
alkyl group which may be optionally substituted with one
or more substituents, each Rs represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2_6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
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122
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.
Here, when m .1 or 2, and each of R3, R4, R5 and R6
represents a hydrogen atom, or
when m =1, and each of R3 and R5 represents a methyl
group, each of R4 and R6 represents a hydrogen atom,
cycle A is preferably not a thyminyl group.]
or salts thereof is included as a novel intermediate
compound.
[0071]
According to one embodiment of the present
application, the compound represented by the above-
mentioned general formula I, wherein R1 and R2 represent
independently of each other a benzyl (Bn) group, a 2-
naphthylmethyl group, a 4-methoxybenzyl group, a 3,4-
dimethoxybenzyl group, a 2,6-dimethoxybenzyl group, or a
p-phenylbenzyl group, R3, R4, R5, and R6 represent
independently of each other a hydrogen atom, or a C1_6
alkyl group which may be optionally substituted with one
or more substituents, and cycle A represents a five to
seven membered unsaturated heterocyclic group which may
be optionally substituted with one or more substituents
selected from the group consisting of a C1_6 alkyl group
which may be optionally substituted with one or more
substituents, a C2_6 alkenyl group which may be optionally
substituted with one or more substituents, a C2_6 alkynyl
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123
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A,
or salts thereof is included.
[0072]
According to one embodiment of the present
application, the compound represented by general formula
I or salts thereof is preferably a compound represented
by general formula I-a:
[chem.58]
R4
R3,....--------
0 rµi A
, H
A (I-a)
0
-
6- N
R2"-- H
(wherein, R1, R2, R3, R4, Rs, R6, m and cycle A are the
same as defined in the above-mentioned formula I.
Here, when m =1 or 2, and each of R3, R4, R5 and R6
represents a hydrogen atom, or
when m =1, and each of R3 and R5 represents a methyl
group, each of R4 and R6 represents a hydrogen atom,
cycle A is preferably not a thyminyl group.
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124
[0073]
According to one embodiment of the present
application, in the general formula I (formula I-a), when
each of R1 and R2 represents a protecting group for
hydroxy group, the protecting group for hydroxy group
includes, for example, a methyl group substituted with
one to three aryl group(s), and preferably a benzyl (Bn)
group, a 2-naphthylmethyl group, a p-methoxybenzyl group,
a 3,4-dimethoxybenzyl group, a 2,6-dimethoxybenzyl group,
and a p-phenylbenzyl group, and more preferably a benzyl
group.
[0074]
According to one embodiment of the present
application, in the general formula I (formula I-a), each
of R3, R4, R5, and R6 represents independently a hydrogen
atom.
[0075]
According to one embodiment of the present
application, in the general formula I (formula I-a), when
each of R3, R4, Rs, and R6 represents a Cl_G alkyl group
which may be optionally substituted with one or more
substituents, the C1_6 alkyl group is preferably methyl
group, ethyl group, propyl group, isopropyl group, butyl
group, isobutyl group, pentyl group, or hexyl group, more
preferably methyl group. Examples of the substituents
include one or more (or preferably one to three)
identical or different groups selected from the group
consisting of a C1_6 alkyl group, a C26 alkenyl group, a
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125
C2-6 alkynyl group, a C1-6 alkoxy group, an amino group, a
hydroxy group, an oxo group, a thioxo group, and a
halogen atom).
[0076]
According to one embodiment of the present
application, in the general formula I (formula I-a), the
cycle A is preferably a 6 membered unsaturated
heterocyclic group, and examples of the substituents of
the cycle A include one or more (preferably 2 or 3)
groups selected from the group consisting of a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkenyl group which may be
optionally substituted with one or more substituents, a
C2-6 alkynyl group which may be optionally substituted
with one or more substituents, a halogen atom, and an oxo
group. The cycle A is more preferably a thyminyl group
or an uracinyl group. The substituents are preferably a
methyl group, a halogen atom (such as fluoro atom, chloro
atom, or bromo atom).
[0077]
According to one embodiment of the present
application, the compound represented by general formula
I or salts thereof is particular preferably the compound
represented by the above-mentioned general formula 1
(preferably formula I-a) wherein each of R1 and R2
represents a Bn group, each of R3, R4, Rs, and R6
represents a hydrogen atom, cycle A represents a thyminyl
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126
group or an uracinyl group, and the cycle A' represents
the following structure UU-1 or 11-2:
[chem.59]
-------
-7.---
0 -.-11// -------
11 t-N N
(11-1) (11-2)
, or salts thereof.
As used herein, a wave line:
[chem.60]
al_nrus
in the structure II-1 or 11-2 represents a binding point
to the remaining moiety of the fused tetrahydrofuran ring
or oxazolidine ring.
[0078]
According to one embodiment of the present
application, preferred compound represented by general
formula II or salts thereof represents the compound
represented by general formula II wherein RI, 122, R3, R4,
R5 and R6 are the same as defined in the gnarl formula I
(formula I-a), and the cycle Al represents an unsaturated
heterocyclic group in which the oxazolinone ring fused to
the cycle A' is cleaved by the reduction step in the
preparation method to form the cycle A represented by
general formula I and also two or more nitrogen atoms are
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127
contained, and one or more oxygen atom is substituted, or
salts thereof.
[0079]
Preparation method of compound represented by general
formula II
According to one aspect, the present invention
provides a method for preparing a compound represented by
general formula II:
[chem.61]
R,4
R3
N3 Rs R6
rn
(11)
0
[wherein, each of Pl, R2, R3, R4, R5, R6 and m are the same
as defined in the general formula I, and the cycle A'
represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2_6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
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128
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
or salts thereof,
said method comprising a step of reacting a compound
represented by formula III:
[chem.62]
R4
R 0
HO
R3
Rs R6
0
)
R2
[wherein,
each of RI, R2, R3, P4, 125, R6, m and cycle A' are the
same as defined in general formula II.]
with an azide agent.
[0080]
According to one embodiment of the present
application, the azide agent represents preferably
hydrazoic acid, sodium azide, lithium azide,
tetrabutylammonium azide, trimethylsilyl azide,
diphenylphosphoryl azide (DPPA), nicotinyl azide, and
zinc azide (Zn(N3)2), more preferably diphenylphosphoryl
azide (DPPA).
[0081]
According to one aspect, a compound represented by
general formula II:
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129
[chem.63]
R4
0
R3
R5 R6
,0
14--õLv
N3 fli
(H)
[wherein,
each of R1 and R2 represents independently a
protecting group for hydroxy group, m is an integer of 1
to 3, R3 and R4 represents independently of each other a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, and cycle A' represents a five to seven
membered unsaturated heterocyclic group which may be
optionally substituted with one or more substituents
selected from the group consisting of a C1_6 alkyl group
which may be optionally substituted with one or more
substituents, a C2_6 alkenyl group which may be optionally
substituted with one or more substituents, a C2-6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
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130
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A'.]
or salts thereof is includes as a novel intermediate
compound.
[0082]
According to one embodiment of the present
application, the compound represented by general formula
II or salts thereof is preferably a compound represented
by general formula II-a:
[chem.64]
R4
R1 ----- 0
N3
1.--v in 0
N,
\\, Ar
Oki)
,-
=-".'\
R,¨u 0
or salts thereof (wherein, each of RI, R2, 123, R4, Rs, 126,
m and cycle A' is the same as defined in the general
formula II).
[0083]
According to one embodiment of the present
application, in the general formula II (formula II-a),
when each of R1 and R2 represents a protecting group for
hydroxy group, the protecting group for hydroxy group
includes, for example, a methyl group substituted with
one to three aryl groups, preferably a benzyl group, a 2-
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131
naphtylmethyl group, a 4-methoxybenzyl group, more
preferably a benzyl group.
[0084]
According to one embodiment of the present
application, in the general formula II (general formula
II-a), each of R3, R4, Rs, and R6 represents independently
a hydrogen atom.
[0085]
According to one embodiment of the present
application, in the general formula II (formula II-a),
when each of R3, R4, R5 and R6 represents independently a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, the C1-C6 alkyl group
represents preferably methyl group, ethyl group, propyl
group, isopropyl group, butyl group, isobutyl group,
pentyl group, or hexyl group, more preferably methyl
group. Examples of the substituents include one or more
(or preferably one to three) identical or different
groups selected from the group consisting of a Cl_s alkyl
group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C1-6
alkoxy group, an amino group, a hydroxy group, an oxo
group, a thioxo group, and a halogen atom.
[0086]
According to one embodiment of the present
application, in the general formula II (formula II-a),
the cycle At represents preferably a six membered
unsaturated heterocyclic group, and further, the cycle At
represents more preferably the above-mentioned structure
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II-1 or 11-2. The substituents of the cycle A'
represents preferably one or more substituents
(preferably two or three) groups selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2_6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, a
halogen atom and an oxo group, and the substituent
represents more preferably methyl group, or halogen atom
(such as fluoro atom, chloro atom, bromo atom).
[0087]
According to one embodiment of the present
application, the compound represented by general formula
II or salts thereof represents particularly preferably a
compound represented by the above-mentioed general
formula II (preferably the compound represented by
general formula II-a or salts thereof, more preferably
the compound represented by the above-mentioned general
formula II or general formula II-a wherein the cycle A'
represents the above-mentioned structure 11-1 or 11-2)
wherein each of R1 and R2 represents a Bn group, each of
R3, Rg, R5 and R6 represents a hydrogen atom, and the
cycle A' represents the following structure II-1 or 11-2,
or salts thereof.
[0088]
According to one aspect, a compound represented by
general formula III:
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[diem. 65]
R4
R 0
R3
IR5 R6
0
HO m hr-DAP
(III)
R2
[wherein,
each of R1 and R2 represents independently a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
the cycle A' represents a a five to seven membered
unsaturated heterocyclic group which may be optionally
substituted with one or more substituents selected from
the group consisting of a C1_6 alkyl group which may be
optionally substituted with one or more substituents, a
C2_6 alkenyl group which may be optionally substituted
with one or more substituents, a C2-6 alkynyl group which
may be optionally substituted with one or more
substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
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group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A']
or salts thereof is included as intermediate compound.
[0088]
According to one embodiment of the present
application, the compound represented by general formula
III or salts thereof represents preferably a compound
represented by general formula III-a:
[chem.66]
R R4
R3
P
Witil.... 0
HO- ,m A'
Nae¨D (II1-a)
,-
-$'
R2¨ 0
or salts thereof (wherein, each of RI, R2, 123, R4, R5, R6,
m and cycle A' is the same as defined in general formula
III.).
[0090]
According to one embodiment of the present
application, preferred compound represented by general
formula III (or general formula III-a) represents the
compound represented by the above-mentioned general
formula III (or the above-mentioned general formula III-
a) wherein each of RI, 122, R3, R4, 125, R6 and cycle A' is
the same as defied in the above-mentioned formula II (or
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general formula II-a), for example, take the above-
mentioned structure II-1 or 11-2, or salts thereof.
[0091]
Preparation method of a compound represented by general
formula IV
According to one aspect, the present invention
provides a method for preparing a compound represented by
general formula IV:
[chem.67]
R1 4
R ----- 0 R3
R5 R6
X 77.LnA'N.
(IV)
R, 0
[wherein,
each of R1 and R2 represents a protecting group for
hydroxy group, R3 and R4 represent independently of each
other a hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, X represents a leaving group, m is an
integer of 1 to 3, and the cycle A' represents a five to
seven membered unsaturated heterocyclic group which may
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be optionally substituted with one or more substituents
selected from the group consisting of a C1_6 alkyl group
which may be optionally substituted with one or more
substituents, a C2_6 alkenyl group which may be optionally
substituted with one or more substituents, a C2_6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A'.]
or salts thereof,
said method comprising a step of reacting a compound
represented by general formula V:
[chem.68]
R4
¨0 R3
R5 Re
j A -s)H
(V)
X<
0
R2 ¨0 OH
[wherein,
each of RI, 122, 123, RI, R5, R6, m and X is the same as
defined in the general formula IV, and the cycle A'
represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
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with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2_6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2_6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
with an activating agent for hydroxy group to activate an
unprotected hydroxy group which is substituted on
tetrahydrofuran ring in the general formula V.
[0092]
According to one embodiment of the present
application, the activating agent for hydroxy group
represents preferably trifluoromethansulfonylating agent
or methanesulfonylating agent, more preferably
trifluoromethanesulfonyl chloride, methanesulfonyl
chloride, or trifluoromethanesulfonic anhydride,
particularly preferably trifluoromethanesulfonyl chloride.
[0093]
According to one aspect, a compound represented by
general formula IV:
[chem.69]
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R4
0 R3
Rs R6
N`--Din s
R2¨ 0
[wherein,
each of R1 and R2 represents independently a
protecting group for hydroxy group, R3 and R4 represents
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each Rs represents independently a
hydrogen atom, or a CI_G alkyl group which may be
optionally substituted with one or more substituents,
each RG represents independently a hydrogen atom, or a
6 alkyl group which may be optionally substituted with
one or more substituents, X represents a leaving group, m
is an integer of 1 to 3, and cycle A' represents a five
to seven membered unsaturated heterocyclic group which
may be optionally substituted with one or more
substituents selected from the group consisting of a C1-6
alkyl group which may be optionally substituted with one
or more substituents, a C2-6 alkenyl group which may be
optionally substituted with one or more substituents, a
C2-6 alkynyl group which may be optionally substituted
with one or more substituents, an amino group which may
be optionally substituted with one or more substituents,
a hydroxy group which may be optionally substituted with
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one or more substituents, an oxo group, a thioxo group,
and a halogen atom, and the unsaturated heterocyclic
group may be further fused with another cycle to form a
cycle A']
or salts thereof is included as intermediate compound.
[0094]
According to one embodiment of the present
application, the compound represented by general formula
IV or salts thereof is preferably a compound represented
general formula IV-a:
[chem.70]
R4
0 R3
(N7-0
0
X
0
s,
R, OH
(wherein, each of R1, R2/ R3/ R4, Rs, R6, X, m and cycle A'
is the same as defined in the general formula IV).
[0094]
According to one embodiment of the present
application, the compound represented by general formula
IV or salts thereof is preferably a compound represented
by the above-mentioned general formula IV (preferably
general formula IV-a) wherein more preferably, each of R1
and R2 is a Bn group, each of R3, R4, R5, and R6 is a
hydrogen atom, and cycle A' is the cycle A represented
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140
by the above-mentioned structure II-1 or 11-2, further
preferably, cycle A' may optionally have one or
substituents, and said substituents is preferably methyl
group, or a halogen atom (fluoro atom, chloro atom, or
bromo atom and the like).
[0096]
According to one embodiment of the present
application, in the general formula IV (general formula
IV-a), a leaving group as X is preferably mesyloxy group
(Ms-0-).
[0097]
According to one aspect, a compound represented by
general formula V:
[chem.71]
R4
R
0 R3
R5 R6 A
0 (
X
0
R2 ¨ OH
[wherein,
each of R1 and R2 represents independently a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
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optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, X represents a leaving group, m
is an integer of 1 to 3, and cycle A represents a five to
seven membered unsaturated heterocyclic group which may
be optionally substituted with one or more substituents
selected from the group consisting of a C1_6 alkyl group
which may be optionally substituted with one or more
substituents, a C2-6 alkenyl group which may be optionally
substituted with one or more substituents, a C2-6 alkynyl
group which may be optionally substituted with one or
more substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A.]
is included as a novel intermediate compound.
[0098]
According to one embodiment of the present
application, the compound represented by general formula
V or salts thereof represents preferably a compound
represented by general formula V-a:
[chem.72]
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142
R4
R5 Rs
rA
..
...,hõ,, 0
X -
0
OH
or salts thereof (wherein, each of RI, R2, R3, R4, Rs, R6,
X, m and cycle A is the same as defined in general
formula V.).
[0099]
According to one embodiment of the present
application, when each of R1 and R2 represents
independently a protecting group for hydroxy group in a
general formula V (a general formula V-a), examples of R1
and R2 include a methyl group substituted with 1 to 3
aryl group(s), and for example, benzyl, 2-naphthylmethyl,
or 4-methoxybenzyl is preferred, and benzyl is more
preferred.
[0100]
According to one embodiment of the present
application, in a general formula V (a general formula V-
a), preferably each of R3, R4, R5 and R6 represents
independently a hydrogen atom.
[0101]
According to one embodiment of the present
application, in a general formula V (a general formula V-
a), each of R3, R4, R5 and R6 represents a C1_6 alkyl group
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143
which may be optionally substituted with one or more
substituents, examples of the C1-6 alkyl group include
preferably methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl, and hexyl, and more preferably methyl.
Example of the substituents includes one or more
(preferably 1 to 3 group(s)) the identical or different
groups selected from the group consisting of C1_6 alkyl
group, C2-6 alkenyl group, C2-6 alkynyl group, Cl_6 alkoxy
group, amino group, hydroxy group, oxo group, thioxo
group, and hydrogen atom.
[0102]
According to one embodiment of the present
application, in a general formula V (a general formula V-
a), a leaving group as X group is preferably a mesyloxy
group (Ms-0-).
[0103]
According to one embodiment of the present
application, in a general formula V (a general formula V-
a), the cycle A is preferably a 6 membered unsaturated
heterocycle, and the substituents of the cycle A includes,
for example, one or more (preferably 2 or 3) substituents
selected from the group consisting of C1_6 alkyl group,
C2_6 alkenyl group, C2-6 alkynyl group, halogen atom, and
oxo group. The cycle A is more preferably a thymine
group or an uracil group. The substituents include
preferably methyl group, halogen atom (such as fluoro
atom, chloro atom, or bromo atom).
[0104]
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According to one embodiment of the present
application, the compound represented by general formula
V or salts thereof is particularly preferably a compound
represented by the above-mentioned general formula V
(preferably a general formula V-a) wherein each of R1 and
R2 represents a Bn group, each of R3, R4, R5 and R6
represents a hydrogen atom, and the cycle A represents a
thymine group or an uracil group, or salts thereof.
[0105]
Alternative method for preparing a compound represented
by general formula II
[0106]
According to one embodiment of the present
application, a method for preparing a compound
represented by general formula II:
RR4
1
RE, R6
,...,70
\
N3-< 111
( 11)
/
R2 ¨ 0
[wherin,
each of R1 and R2 represents independently a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1-6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
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optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2_6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2_6 alkynyl group which may be
optionally substituted with one or more substituents, an
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
or salts thereof is provided, and the method comprising a
step of reacting a compound represented by general
formula IV:
[chem.74]
R4
,
-m R6 (IV)
111
õõ Nr
R,--0
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146
[wherein,
X represents a leaving group, and each of R1, R2/ R3/
R4, Rs, R6, m and cycle A' is the same defined in the
above-mentioned general formula II]
with an azide agent.
[0107]
According to one embodiment of the present
application, the azide agent for the hydroxy group is
preferably nBu4NN3 or sodium azide.
[0108]
According to one embodiment of the present
application, preferred compound represented by general
formula II or salts thereof, and preferred compound
represented by general formula IV or salts thereof
respectively is as described elsewhere herein.
[0109]
According to one embodiment of the present
application, for a method for preparing the above-
mentioned compound represented by general formula II or
salts thereof, preferably, each of R1 and R2 represents a
Bn group, each of R2, R4, Rs and RG represents a hydrogen
atom, X represents a mesyloxy group (Ms-0-), the cycle A'
represents the above-mentioned structure formula II-1 or
11-2, and the azide agent represents nBu4NN3 or sodium
azide.
[0110]
Further alternative method for preparing a compound
represented by general formula II
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According to one aspect, a method for preparing a
compound represented by general formula II:
[chem.75]
R4
R3
RE, R6
N3
(II)
R2-
[wherein,
each of R1 and R2 represents independently a
protecting group for hydroxy group, R3 and R4 represent
independently of each other a hydrogen atom, or a C1_6
alkyl group which may be optionally substituted with one
or more substituents, each R5 represents independently a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R6 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, m is an integer of 1 to 3, and
cycle A' represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1_6 alkyl group which may be optionally
substituted with one or more substituents, a C2-6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
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148
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A'.]
or salts thereof is provided, said method comprising a
step of reacting a compound represented by general
formula VI:
[chem.76]
R4
0 R3
R5 Rg
C) )rMH (NI)
In 'Tht7
0
R2 ¨0 OH
[wherein,
each of R1, R2, R3, R4, R5, R6 and m is the same
defined in the general formula II, and cycle A represents
a five to seven membered unsaturated heterocyclic group
which may be optionally substituted with one or more
substituents selected from the group consisting of a ci_6
alkyl group which may be optionally substituted with one
or more substituents, a C2_6 alkenyl group which may be
optionally substituted with one or more substituents, a
C2_6 alkynyl group which may be optionally substituted
with one or more substituents, an amino group which may
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149
be optionally substituted with one or more substituents,
a hydroxy group which may be optionally substituted with
one or more substituents, an oxo group, a thioxo group,
and a halogen atom, and the unsaturated heterocyclic
group may be further fused with another cycle to form a
cycle A.]
with an activating agent for hydroxy group to activate an
unprotected hydroxy group that is substituted on
tetrahydrofuran ring in a general formula VI.
[0111]
According to one embodiment of the present
application, the activating agent for hydroxy group is
preferably a trifluoromethansulfonylating agent or a
methanesulfonylating agent, more preferably
trifluoromethanesulfonyl chloride, methanesulfonyl
chloride, or trifluoromethanesulfonic anhydride, and
particularly preferably trifluoromethanesulfonyl chloride.
[0112]
According to one embodiment of the present
application, preferred compound represented by general
formula II or salts thereof are as described elsewhere
herein.
[0113]
According to one embodiment of the present
application, for a method for preparing a method for
preparing the above-mentioned compound represented by
general formula II or salts thereof, preferably, each of
R1 and R2 represents a Bn group, each of R3, R4, R5 and R6
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150
represents a hydrogen atom, cycle AT represents a thymine
group or an uracil group, cycle A' represents the above-
mentioed general formula II-1 or 11-2, and the activating
agent for hydroxy group represents
trifluoromethanesulfonyl chloride.
[0114]
According to one embodiment of the present
application, preferred compound represented by general
formula VI or salts thereof represents preferably a
compound represented by general formula VI-a:
[chem.77]
R4
R1
R
R5 Re rnH
--- 3
(VI-a)
m
N3-- .
0
=
[0115]
According to one embodiment of the present
application, preferred compound represented by general
formula VI (general formula VI-a) or salts thereof
represents a compound represented by general formula VI
wherein each of R1 and R2 represents a Bn group, each of
R3, R4, Rs and R6 represents a hydrogen atom, and cycle A
represents a thymine group or an uracil group.]
or salts thereof.
[0116]
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According to one embodiment of the present
application, preferred compound represented by general
formula II or salts thereof is described elsewhere herein.
[0117]
Method for preparing a compound represented by general
formula VIII
According to one aspect, a method for preparing a
compound represented by general formula VIII:
[chem.78]
R3
o
R 12
- R4
0
[
R5 13
(VIII)
R(
= rn
N
0
,13
[wherein,
B represents a base moiety of nucleic acid that may
be optionally substituted with one or more substituents,
R3 and R4 represent independently of each other a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
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152
substituents, R12 and R12 represent independently of each
other a hydrogen atom or a protecting group for hydroxy
group, and m is an integer of 1 to 3.]
or salts thereof is provided, said method comprising a
step of replacing a cycle A in a compound represented by
general formula IX:
[chem.79]
R3
R1 4
0 4
CMA
0 NH
R5
(IX)
0
Rtr
_ m
pQ
-15
[wherein,
each of R3, RI, R5/ R6 and m is the same as defined
in general formula VIII, and R14 and R15 represent
independently of each other a hydrogen atom or a
protecting group for hydroxy group, and cycle A
represents a five to seven membered unsaturated
heterocyclic group which may be optionally substituted
with one or more substituents selected from the group
consisting of a C1-6 alkyl group which may be optionally
substituted with one or more substituents, a C2_6 alkenyl
group which may be optionally substituted with one or
more substituents, a C2-6 alkynyl group which may be
optionally substituted with one or more substituents, an
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153
amino group which may be optionally substituted with one
or more substituents, a hydroxy group which may be
optionally substituted with one or more substituents, an
oxo group, a thioxo group, and a halogen atom, and the
unsaturated heterocyclic group may be further fused with
another cycle to form a cycle A.]
wth B.
[0118]
According to one embodiment of the present
application, Lewis acid for use in the replacement
reaction of cycle A (transglycosylation) is preferably
TMSOTf. Also, in order to facilitate the replacement
reaction, a silylating agent may be used, and the
silylating agent is preferably BSA.
[0119]
According to one aspect, a compound represented by
general formula VIII:
[chem.80]
R3
R12
"-----0 //________. R4
.---
õ7- B
_ _ . (VIII)
R5 /
..-,)"-.,
_ e,. . ¨111 ''-õ,..,,,, ,
m N..
//
0 N
m .---- H
r..13
[wherein,
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154
B represents a base moiety of nucleic acid that may
be optionally substituted with one or more substituents,
R3 and R4 represent independently of each other a
hydrogen atom, or a CI_G alkyl group which may be
optionally substituted with one or more substituents,
each R5 represents independently a hydrogen atom, or a CI_
6 alkyl group which may be optionally substituted with
one or more substituents, each R6 represents
independently a hydrogen atom, or a C1_6 alkyl group which
may be optionally substituted with one or more
substituents, and R12 and R13 represent independently of
each other a hydrogen atom or a protecting group for
hydroxy group, and m is an integer of 1 to 3.
Here, when m =1 or 2, and each of R3, R4, R5 and R6
represents a hydrogen atom, or
when m =1, and each of R3 and R4 represents a methyl
group, and each of R4 and R5 represent a hydrogen atom,
the cycle A is not preferably a thyminyl group.]
or salts thereof is included as a novel intermediate
compound.
[0120]
According to one embodiment of the present
application, a compound represented by general formula
VIII or salts thereof is preferably a compound
represented by general formula VIII-a:
[chem.81]
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155
/R3
/ ¨R4
R- Vt11-a)
RB,>N1-fl N
N
0
,13
or salts thereof.
Here, when m =1 or 2, and each of R3, R4, R5 and R6
represents a hydrogen atom, or
when m =1, and each of R3 and R4 represents a methyl
group, and each of R4 and R5 represent a hydrogen atom,
the cycle A is not preferably a thyminyl group.
[0121]
According to one embodiment of the present
application, a compound represented by a general formula
VIII (a general formula VIII-a) wherein B represents a
base moiety of nucleic acid that may be optionally
substituted with one or more substituents, R3, R4, R5, and
R6 represent independently of each other a hydrogen atom
or a C1_6 alkyl group which may be optionally substituted
with one or more substituents, R12 represents a En group,
a DMTr group, or a TMS group, and R13 represents a Bn
group, a hydrogen atom, or a TMS group, or salts thereof
is included.
[0122]
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According to one embodiment of the present
application, in general formula VIII (general formula
VIII-a), each of R3, Rg, R5 and R6 represents
independently a hydrogen atom.
[0123]
According to one embodiment of the present
application, in the general formula VIII (formula VIII-a),
when each of R3, Rg, R5, and R6 represents a C1-6 alkyl
group which may be optionally substituted with one or
more substituents, the C1-6 alkyl group represents
preferably methyl group, ethyl group, propyl group,
isopropyl group, butyl group, isobutyl group, pentyl
group, or hexyl group, more preferably methyl group.
Examples of the substituents include one or more (or
preferably one to three) identical or different groups
selected from the group consisting of C1_6 alkyl group,
C2_6 alkenyl group, C2-5 alkynyl group, C1_6 alkoxy group,
amino group, hydroxy group, oxo group, thioxo group, and
halogen atom.
[0124]
According to one embodiment of the present
application, in the general formula VIII (formula VIII-a),
B represents a base moiety of nucleic acid, preferably a
base moiety of natural nucleic acid or a base moiety of
unnatural nucleic acid, more preferably a purine ring
derived from adenine or guanine or derivatives thereof,
or a mono- or di-one ring of pyrimidine derived from
uracil, cytosine or thymine or derivatives thereof,
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particularly preferably adeninyl, guaninyl, cytosinyl, 5-
methylcytosinyl, thyminyl or uracinyl. The base moiety
of the nucleic acid may optionally have one or more
substituents, and the preferred substituents represent
preferably hydroxy group, C1_6 alkoxy group, mercapto
group, C1-6 alkylthio group, amino group, amino group
substituted with C1_6 alkyl group, C1_6 alkyl group, C2-6
alkenyl group, C2-6 alkynyl group, oxo group, thioxo group,
or halogen atom, more preferably one or more (preferably
two or three) substituents selected from the group
consisting of C1-6 alkyl group, and oxo group. The base
moiety of the nucleic acid may optionally have one or
more substituents, and examples of the preferred
substituents represents preferably hydroxy group, C1-6
alkoxy group, mercapto group, Ci_6 alkylthio group, amino
group, amino group substituted with C1-6 alkyl group, C1-6
alkyl group, C2_6 alkenyl group, C2-6 alkynyl group, oxo
group, thioxo group, and halogen atom, more preferably
one or more (preferably two or three) substituents
selected from the group consisting of C1_6 alkyl group and
oxo group. The hydroxy group, amino group or thiol group
of the base moiety of nucleic acid may be optionally
protected, and the protecting group represents preferably
a protecting group for hydroxy group or a protecting
group for amino group, each of which is usually used in
nucleic acid synthesis, more preferably acetyl group,
phenoxyacetyl group, isobutyryl group, benzoyl group,
cyanoethoxycarbonyl group, or diphenylaminocarbonyl group,
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particularly preferably isobutyryl group, benzoyl group,
or diphenylaminocarbonyl group.
[0125]
According to one embodiment of the present
application, the compound represented by general formula
VIII or salts thereof represents particularly preferably
the compound represented by the above-mentioned general
formula VIII (preferably a general formula VIII-a)
wherein B represents an adeninyl group which may be
optionally substituted with one or more substituents, a
guaninyl group which may be optionally substituted with
one or more substituents, a cytosinyl group which may be
optionally substituted with one or more substituents, or
a 5-methylcytosinyl group which may be optionally
substituted with one or more substituents, or a thyminyl
group which may be optionally substituted with one or
more substituents, or an uracinyl group which may be
optionally substituted with one or more substituents,
each of R3, R4, Rs, and R6 represents a hydrogen atom, and
each of R12 and Rn represents a En group, or salts
thereof.
[0126]
According to one embodiment of the present
application, the compound represented by general formula
VIII or salts thereof represents particularly preferably
the compound represented by the above-mentioned general
formula VIII (preferably, a general formula VIII-a)
wherein B represents an adeninyl group which may be
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optionally substituted with one or more substituents, a
guaninyl group which may be optionally substituted with
one or more substituents, a cytosinyl group which may be
optionally substituted with one or more substituents, a
5-methylcytosinyl group which may be optionally
substituted with one or more substituents, a thyminyl
group which may be optionally substituted with one or
more substituents, or an uracinyl group which may be
optionally substituted with one or more substituents,
each of R3, R4, R5, and R6 represents a hydrogen atom, R12
represents a DMTr group, and Rn represents a hydrogen
atom, or salts thereof.
[0127]
According to one aspect, a compound represented by
general formula IX:
[chem.82]
R3
R 14
0
NH
/ (Ix)
- R5
Rcir,
- rn
/N
0
R15'
[wherein,
each of R3, R4, R5, and R6 is the same as defined in
the general formula VIII, R14 and R15 represent
independently of each other a hydrogen atom, or a
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protecting group for hydroxy group, m is an integer of 1
to 3, and cycle A represents a five to seven membered
unsaturated heterocyclic group which may be optionally
substituted with one or more substituents selected from
the group consisting of a C1-6 alkyl group which may be
optionally substituted with one or more substituents, a
C2_6 alkenyl group which may be optionally substituted
with one or more substituents, a C2-6 alkynyl group which
may be optionally substituted with one or more
substituents, an amino group which may be optionally
substituted with one or more substituents, a hydroxy
group which may be optionally substituted with one or
more substituents, an oxo group, a thioxo group, and a
halogen atom, and the unsaturated heterocyclic group may
be further fused with another cycle to form a cycle A.
Here, when m =1 or 2, and each of R3, R4, R5 and R6
represents a hydrogen atom, or
when m =1, and each of R3 and R5 represents a methyl
group, each of R4 and R6 represents a hydrogen atom,
cycle A is preferably not a thyminyl group.]
or salts thereof is included as a novel intermediate
compound.
[0128]
According to one embodiment of the present
application, the compound represented by general formula
IX or salts thereof represents preferably a compound
represented by general formula IX-a:
[chem.83]
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161
R3
R14 D
"4
17- A
0
H
r
(IX-a )
0
m
D
or salts thereof (wherein, each of R3, 124, Rs, 126, R14, R15,
m and cycle A is the same as defined in the general
formula IX. Here when m =1 or 2, and each of R3, Rg, R5
and R6 represents a hydrogen atom, or when m =-1, and each
of R3 and R5 represents a methyl group, each of Rg and R6
represents a hydrogen atom, cycle A is preferably not a
thyminyl group.)
[0129]
According to one embodiment of the present
application, in the general formula IX (a general formula
IX-a), each of R3, R4, Rs, and R6 is the same as defined
in the general formula VIII (a general formula VIII-a).
[0130]
According to one embodiment of the present
application, in the general formula IX (a general formula
IX-a), when R15 represents a protecting group for hydroxy
group, the protecting group for hydroxy group is
preferably the same as defined in the general formula I.
[0131]
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According to one embodiment of the present
application, in the general formula IX (a general formula
IX-a), the cycle A represents preferably a pyrimidine
base moiety of nucleic acid which may be optionally
substituted with one or more (preferably two or three)
substituents selected from the group consisting of C1-6
alkyl group an oxo group.
[0132]
According to one embodiment of the present
application, the compound represented by general formula
IX or salts thereof represents particularly preferably a
compound represented by general formula IX (a general
formula IX-a) wherein B represents an adeninyl group
which may be optionally substituted with one or more
substituents, a guaninyl group which may be optionally
substituted with one or more substituents, a cytosinyl
group which may be optionally substituted with one or
more substituents, a 5-methylcytosinyl group which may be
optionally substituted with one or more substituents, a
thyminyl group which may be optionally substituted with
one or more substituents, or an uracinyl group which may
be optionally substituted with one or more substituents,
each of R14 and R15 represents a Bn group, and the cycle A
represents a thyminyl group or an uracinyl group, or
salts thereof.
[0133]
According to one embodiment of the present
application, the compound represented by general formula
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IX or salts thereof represents particularly preferably a
compound represented by general formula IX (a general
formula IX-a) wherein B represents an adeninyl group
which may be optionally substituted with one or more
substituents, a guaninyl group which may be optionally
substituted with one or more substituents, a cytocinyl
group which may be optionally substituted with one or
more substituents, a 5-methylcytosinyl group, a thyminyl
group which may be optionally substituted with one or
more substituents, or an uracinyl group which may be
optionally substituted with one or more substituents, R14
represents a DMTr group, R15 represents a hydrogen atom,
and the cycle A represents a thyminyl group or a uracinyl
group, or salts thereof.
[0134]
According to one embodiment of the present
application, the compound represented by general formula
IX or salts thereof represents particularly preferably a
compound represented by general formula IX (a general
formula IX-a) wherein B represents an adeninyl group
which may be optionally substituted with one or more
substituents, a guaninyl group which may be optionally
substituted with one or more substituents, a cytocinyl
group which may be optionally substituted with one or
more substituents, a 5-methylcytosinyl group, a thyminyl
group which may be optionally substituted with one or
more substituents, or an uracinyl group which may be
optionally substituted with one or more substituents, R14
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164
represents a hydrogen atom, R15 represents a hydrogen
atom, and the cycle A represents a thyminyl group or an
uracinyl group, or salts thereof.
[0135]
Method for preparing a compound represented by
general formula VII
According to one aspect, a compound represented by
general formula VII:
[chem.84]
R3
R7
0 R4
0
B
N./
[R5// (VII)
R6 NN
M
0
¨ R9
Rio- N
Rit
[wherein,
B represents a base moiety of nucleic acid that may
be optionally substituted with one or more substituents,
R3, R4, R5, and RG represent independently of each other a
hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents, R7
represents a hydrogen atom or a protecting group for
hydroxy group, R8 represents a hydrogen atom, a phosphate
group which may be optionally substituted with one or
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165
more substituents, or a thiophosphate group which may be
optionally substituted with one or more substituents, R9,
R10 and Rn represent independently of each other a
hydrogen atom, a C1-6 alkyl group which may be optionally
substituted with one or more substituents, or a
protecting group for amino group, and m is an integer of
1 to 3. (with the proviso that the following cases are
excluded:
the case where m is I, B represents a thyminyl group,
each of R3, R4, R5 and R6 represents a hydrogen atom, R7
represents a DMTr group, R8 represents a hydrogen atom or
a -P(O(CH2)2CN) (N(iPr)2) group, R9 represents a Boc group,
R10 represents a Boc group, and R11 represents a hydrogen
atom, and
the case where m is 2, B represents a thyminyl group,
each of R3, R4, R5 and RG represents a hydrogen atom, R7
represents a DMTr group, R8 represents a hydrogen atom or
a -P(O(CH2)2CN) (N(iPr)2) group, P.9 represents a Ceoc group,
Rn represents a Ceoc group, and Ril represents a hydrogen
atom.)]
is included as a novel compound.
[0136]
According to one embodiment of the present
application, the compound represented by general formula
VII or salts thereof represents preferably a compound
represented by general formula VII-a:
[chem.85]
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166
R3
0
0
B (VII-a)
R5
[
R6 N
m=::' -
-
6 N
RE('
\----i, _______________ N ___ R9
/
R10 ----- N
\
Ril
or salts thereof (wherein, each of B, R3, R4, R5, R6, R7,
R8, R9, Rn, RH, and m is the same as defined in general
formula VII.).
[0137]
In the proviso of the general formula VII, the
exclusion of the case where B represents a thyminyl group,
each of R3, R4, R6 and R6 represents a hydrogen atom, R7
represents a DMTr group, Rg represents a hydrogen atom or
-P(O(CH2)2CN)(N(iPr)2), Rg represents a Boc group, Rn
represents a Boc group, and Ril represents a hydrogen
atom is intended to exclude the publicly known compounds
described in WO 2014/046212.
[0138]
According to one embodiment of the present
application, in the general formula VII (a general
formula VII-a), more preferably, each of R3, R4, R5, and
R6 represents independently a hydrogen atom.
[0139]
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According to one embodiment of the present
application, in the general formula VII (a general
formula VII-a), when each of R3, R4, R5 and R6 represents
a C1.6 alkyl group which may be optionally substituted
with one or more substituents, the C1_6 alkyl group
represents preferably methyl group, ethyl group, propyl
group, isopropyl group, butyl group, isobutyl group,
pentyl group, or hexyl group, more preferably methyl
group. Examples of the substituents include one or more
(or preferably one to three) identical or different
groups selected from the group consisting of C1_6 alkyl
group, C2-6 alkenyl group, C2-6 alkynyl group, C1-.6 alkoxy
group, amino group, hydroxy group, oxo group, thioxo
group, and halogen atom.
[0140]
According to one embodiment of the present
application, in the general formula VII (a general
formula VII-a), R7 represents a protecting group for
hydroxy group, more preferably trityl type of protecting
group, particularly preferably a DMTr.
[0141]
According to one embodiment of the present
application, in the general formula VII (a general
formula VII-a), when the compound or salts thereof
represents phosphoramidite, R8 represents preferably a
formula: -P(O(CH2)2CN)(N(iPr)2).
[0142]
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168
According to one embodiment of the present
application, in the general formula VII (a general
formula VII-a), preferably, each of Rg and R10 represents
a protecting group for amino group, RI1 represents a
hydrogen atom, and more preferably, the protecting group
for amino group represents a Teoc group or a Boc group.
[0143]
According to one embodiment of the present
application, in the general formula VII (a general
formula VII-a), B represents a base moiety of nucleic
acid that may be optionally substituted with one or more
substituents, and the preferred substituents represents
preferably a hydroxy group, Ci_6 alkoxy group, mercapto
group, C1-6 alkylthio group, amino group, amino group
substituted with C1-6 alkyl group, C1-6 alkyl group, C2-6
alkenyl group, C2-6 alkynyl group, oxo group, thioxo group,
halogen atom, more preferably one or more (preferably two
or three) substituents selected from the group consisting
of C1_6 alkyl group and oxo group. The hydroxy group,
amino group or thiol group of the base moiety of nucleic
acid may be optionally protected, and examples of the
protecting group include preferably a protecting group
for hydroxy group and a protecting group for amino group,
each of which is usually used in a nucleic acid synthesis,
more preferably acetyl group, phenoxyacetyl group,
isobutyryl group, benzoyl group, cyanoethoxycarbonyl
group, and diphenylaminocarbonyl group, more preferably
cyanoethoxycarbonyl group and diphenylaminocarbonyl group,
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169
and particularly preferably isobutyryl group, benzoyl
group, and diphenylaminocarbonyl group. More preferred
embodiment of B includes adeninyl, guaninyl, cytosinyl,
5-methylcytosinyl, thyminyl or uracinyl, each of which
may optionally have a protecting group.
[0144]
According to one embodiment of the present
application, the compound represented by general formula
VII or salts thereof represents preferably a compound
represented by general formula VII (a general formula
VII-a) wherein B represents an adeninyl which may
optionally have one or more protecting groups, a guaninyl
which may optionally have one or more protecting groups,
a cytosinyl which may optionally have one or more
protecting groups, a 5-methylcytosinyl which may
optionally have one or more protecting groups, or an
uracil which may optionally have one or more protecting
groups, each of R3, R4, Rs, and R6 represents a hydrogen
atom, R7 represents a DMTr group, R8 represents a
hydrogen atom or -P(O(C1-12)2CN)(N(iPr)2), Rg represents a
Teoc group or a Boc group, R10 represents a Teoc group or
a Hoc group, and Rn represents a hydrogen atom.
[0145]
According to one embodiment of the present
application, the general formula VII represents
preferably a compound represented by general formula
represented by general formula VII (general formula VII-
a) wherein B represents an adeninyl which may optionally
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170
have one or more protecting groups, a guaninyl which may
optionally have one or more protecting groups, a
cytosinyl which may optionally have one or more
protecting groups, a 5-methylcytosinyl which may
optionally have one or more protecting groups, or an
uracil which may optionally have one or more protecting
groups, each of rt3, R4, R5 and R6 represents a hydrogen
atom, R7 represents DMTr group, R8 represents a hydrogen
atom or -P(O(C112)2CN)(N(iPr)2), R9 represents Teoc group,
R10 represents Teoc group, and RH represents a hydrogen
atom, or salts thereof.
[0146]
According to one embodiment of the present
application, the method for preparing the compound
represented by general formula VII or salts thereof may
comprise at least one step of the followings among the
steps described herein.
According to one embodiment of the present
application, the method for preparing the compound
represented by general formula VII or salts thereof
comprises the step of reacting the compound represented
by general formula II with a reducing agent to prepare
the compound represented by general formula I or salts
thereof.
[0147]
According to one embodiment of the present
application, the method for preparing the compound
represented by general formula VII or salts thereof
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171
comprises a step of reacting the compound represented by
general formula III with an azide agent to prepare the
compound represented by general formula II or salts
thereof.
[0148]
According to one embodiment of the present
application, the method for preparing the compound
represented by general formula VII or salts thereof
comprises a step of reacting the compound represented by
general formula V with an activating agent for hydroxy
group to activate an unprotected hydroxy group which is
substituted on tetrahydrofuran ring in the general
formula V to prepare the compound represented by general
formula IV or salts thereof.
[0149]
According to one embodiment of the present
application, the method for preparing the compound
represented by general formula VII or salts thereof
comprises a step of reacting the compound represented by
general formula IV with an azide agent to prepare the
compound represented by general formula II or salts
thereof.
[0150]
According to one embodiment of the present
application, the method for preparing the compound
represented by general formula VII or salts thereof
comprises a step of reacting the compound represented by
general formula VI with an activating agent for hydroxy
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172
group to activate an unprotected hydroxy group which is
substituted on tetrahydrofuran ring in the general
formula VI to prepare the compound represented by general
formula II or salts thereof.
[0151]
According to one embodiment of the present
application, the method for preparing the compound
represented by general formula VII or salts thereof
comprises a step of replacing the cycle A in the compound
represented by general formula IX with B to prepare the
compound represented by general formula VIII or salts
thereof.
[0152]
Method for preparing artificial oligonucleotide
represented by general formula X
According to one aspect, a method for preparing an
oligonucleotide containing one or more nucleosides
represented by general formula X:
[chem.86]
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173
R3
o
4
ON B
(X)
[R5
4i
Ri3 Ns.
111 or
0 dIPLP
H +
____________________________ N¨R16 ______________ N¨R16
R18N R1B¨N
R17 R17
[wherein,
R3, R4, R5, and R6 represent independently of each
other a hydrogen atom, or a C1_6 alkyl group which may be
optionally substituted with one or more substituents, B
represents a base moiety of nucleic acid that may be
optionally substituted with one or more substituents,
R17 and R18 represent independently of each other a
hydrogen atom, a C1-6 alkyl group which may be optionally
substituted with one or more substituents, or a
protecting group for amino group, and m is an integer of
1 to 3.]
(with the proviso that when m =1 or 2, the
oligonucleotide containing as a bridged nucleic acid only
the nucleoside wherein B represents a thyminyl group,
each of R3, RI, R5, R5, RIG, R17 and R18 represents a
hydrogen atom, is excluded),
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174
is provided, said method comprising a preparation of the
same by a phosphoramidite method from a compound
represented by general formula XI:
[chem.87]
/ 3
IR?
/ Ra
0
(XI)
R5
R5
111
0
RV-
N ¨R21
R23¨ IN
R22
[wherein,
each of B, R3, R4, R5, R6, and m is the same as
defined in general formula X, R7 represents a hydrogen
atom or a protecting group for hydroxy group, R20
represents a phosphate group which may be optionally
substituted with one or more substituents or a
thiophosphate group which may be optionally substituted
with one or more substituents, and
1222 and R23
represent independently of each other a hydrogen atom, a
C1_6 alkyl group which may be optionally substituted with
one or more substituents, or a protecting group for amino
group.].
[0153]
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175
According to one embodiment of the present
application, the phosphoramidite method is preferably
conducted according to the method described in WO
2014/046212 Al.
[0154]
According to one embodiment of the present
application, for the preparation method of the
oligonucleotide represented by the above-mentioned
general formula X, in general formula X or general
formula XI, preferably, B represents an adeninyl which
may optionally have one or more protecting groups, a
guaninyl which may optionally have one or more protecting
groups, a cytosinyl which may optionally have one or more
protecting groups, a 5-methylcytosinyl which may
optionally have one or more protecting groups or an
uracinyl which may optionally have one or more protecting
groups, R7 represents a protecting group for hydroxy
group, each of R16, R17 and R18 represents a hydrogen atom,
R20 represents -P(O(C1-12)20N)(N(iPr)2), and each of R21, R22
and Rn represents independently a protecting group for
amino group.].
[0155]
According to one aspect, the present invention
provides a novel GuNA oligonucleotide or salts thereof,
and according to one embodiment of the present
application, the present invention provides an
oligonucleotide containing one or more nucleosides
represented by general formula X:
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176
[chem.88]
R3
0 /1 Rd
=
(X)
RN/
Ra/
Y= or
0IN
I
-N¨Ri6 H4,
__________________________________________________ N --R16
Rm __________________
R17.
[wherein,
R3, R4, R5, and R6 represent independently of each
other a hydrogen atom, or a C1-6 alkyl group which may be
optionally substituted with one or more substituents, B
represents a base moiety of nucleic acid that may be
optionally substituted with one or more substituents, R16,
R17 and Rn represent independently of each other a
hydrogen atom, a C1_6 alkyl group which may be optionally
substituted with one or more substituents, or a
protecting group for amino group, and m is an integer of
1 to 3.]
(with the proviso that the oligonucleotide containing as
a bridged nucleic acid only the nucleoside wherein when m
or 2, B represents a thyminyl group, each of R3, R4, R5,
R5, R16, R17 and R18 represents a hydrogen atom, is
excluded),
or salts thereof.
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177
[0156]
According to one embodiment of the present
application, the compound represented by general formula
X or salts thereof represents preferably an
oligonucleotide containing one or more nucleosides
represented by general formula X-a:
[chem.89]
R3
0
NB
CK-0
R6 ,s=
R-
b
In$ y=
or
..)%r
H+
__________________________________________________ N¨R.16
____________________________ N ¨ R
Ri6 16
RIB ¨ IN
- IN
R
R"
or salts thereof.
[0157]
According to one embodiment of the present
application, in the general formula X (a general formula
X-a), each of R3, Rg, R5 and R6 represents a hydrogen atom.
[0158]
According to one embodiment of the present
application, in the general formula X (a general formula
X-a), when each of R3, Rg, R5 and R6 represents a C1-6
alkyl group which may be optionally substituted with one
or more substituents, the C1-6 alkyl group represents
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178
preferably methyl group, ethyl group, propyl group,
isopropyl group, butyl group, isobutyl group, pentyl
group, or hexyl group, more preferably methyl group.
Examples of the substituents include one or more (or
preferably one to three) groups selected from the group
consisting of C1-6 alkyl group, C2-6 alkenyl group, C2_6
alkynyl group, Ci_6 alkoxy group, amino group, hydroxy
group, oxo group, thioxo group, and halogen atom.
[0159]
According to one embodiment of the present
application, in the general formula X (a general formula
X-a), each of R16, R17 and Rn represents independently a
hydrogen atom.
[0160]
According to one embodiment of the present
application, in the general formula X (a general formula
X-a), when each of R16, R17 and Rn represents a C1_6 alkyl
group which may be branched chain or may be formed to a
cycle, and said c1_6 alkyl group may be optionally
substituted with one or more substituents, the C1-6 alkyl
group represents preferably methyl group, ethyl group,
propyl group, isopropyl group, butyl group, isobutyl
group, pentyl group, or hexyl group, more preferably
methyl group. Examples of the substituents include one
or more (or preferably one to three) identical or
different groups selected from the group consisting of
C1_6 alkyl group, C2-6 alkenyl group, C2_6 alkynyl group,
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C1-6 alkoxy group, amino group, hydroxy group, an oxo
group, a thioxo group, and a halogen atom.
[0161]
According to one embodiment of the present
application, oligonucleotide wherein in the general
formula X (the general formula X-a), B represents
adeninyl group, guaninyl group, cytosinyl group, 5-
methylcytosinyl group, thyminyl group or uracinyl group,
and each of R16, R17 and R16 represent a hydrogen atom,
or salts thereof is preferred.
[0162]
According to one embodiment of the present
application, the oligonucleotide or salts thereof is made
by binding two or more the nucleotides continuously or
discontinuously, and preferably contains 2 to 100
nucleotides, more preferably 5 to 50 nucleotides, and
particularly preferably those in which 10 to 30
artificial nucleotides are bonded or are combined
together with these complementary strands forms a double
strand. When the nucleotides are bonded discontinuously,
the nucleotide(s) which is/are positioned between the
nucleotides is/are not particularly limited, and the
nucleotide(s) is/are preferably natural nucleotide(s)
without bridged structure.
[0163]
The term of "salt" in the expression of "salts of
compound" used herein alkali metal salts such as sodium
salt, potassium salt, lithium salt; alkaline earth metal
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salts such as calcium salt, and magnesium salts; metal
salts such as aluminium salt, iron salt, zinc salt,
copper salt, nickel salt, and cobalt salt; inorganic
salts such as ammonium salt; amine salts, for example,
organic salts such as t-octylamine salt, dibenzylamine
salt, morpholine salt, glucosamine salt, phenylglysine
alkylester salt, etylenediamine salt, N-methylglucamine
salt, guanidine salt, diethylamine salt, triethylamine
salt, dicyclohexylamine salt, N,N'-dibenzyletylenediamine
salt, chloroprocaine salt, procaine salt, diethanolamine
salt, N-benzyl phenethylamine salt, piperazine salt,
tetramethylammonium salt, and
tris(hydroxymethyl)aminomethane salt; inorganic acid
salts, for example, hydrohalic acids (such as
hydrofluoric acid, hydrochloric acid, hydrobromic acid,
and hydroiodic acid), nitrate salt, perchloric acid salt,
sulfate salt, and phosphate salt; salts of alkanesulfonic
acid having one to six carbon atoms, such as
methanesulfonate salt, trifluoro methanesulfonate salt,
ethanesulfonate salt; arylsulfonic acid salts such as
benzene sulfonate salt, and p-toluene sulfonate salt;
organic acid salts such as acetate salt, malate salt,
fumarate salt, succinate salt, citrate salt, tartrate
salt, oxalate salt, and maleate salt; and amino acid
salts such as glycine salt, lysine salt, arginine salt,
ornithine salt, glutamic acid salt, and aspartic acid
salt, which should not be limited thereto. The salts are
encompassed by pharmaceutically acceptable salts.
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[0164]
Each compound represented by general formulae I to
XI respectively described herein encompasses enantiomer
form, diastereomer form, or mixtures thereof. For
example, a stereo configuration of sugar moiety in the
structure of each compound encompasses a form and p form,
however, p form is preferred. When the above-mentioned
compounds represented by formula I to XI is obtained in
the form of diastereomer or enantiomer, they can be
separated by well-known methods in organic synthesis (for
example, sugar synthesis), such as chromatography method
or fractional crystallization method. For example, the
compound 16a represented by the following formula:
[chem.90]
IDNMO
IfTrdr4r
\tr_NH
002N,
p ¨0
NC(H2C)26
R"¨NH
16a:R1"7-Teoc
has [3 form as a stereo configuration of sugar moiety.
[0165]
Each compound represented by general formulae I to
XI described herein compasses the compounds labeled with
isotopes (such as 3H, 13C, 14C, 15N, 18F, 32p,
"S or 1251)
and deuterated transformers.
[0166]
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The oligonucleotide or analogues thereof described
herein may be mixed with auxiliary agents that are
usually used in the formulation of pharmaceuticals, such
as excipients, binders, preservatives, antioxidants,
disintegrants, lubricants, and flavors, to make
parenterally-administered formulation or liposome
formulations. Also, the oligonucleotide or analogues
thereof is mixed with pharmaceutical carriers that are
usually used in the pharmaceutical arts to formulate into
topical formulations such as solutions, creams and
ointments.
[0167]
(Preparation method)
Hereinafter, the preparation method of bridged
nucleic acid GuNA described herein is described.
Optically active substances having absolute configuration
in each compound represented by general formulae I to XI
may be prepared by using as starting material optically
active substance(s) or separating the isomers that
created in the intermediate stages for synthesis. Also,
the below-mentioned replacement reaction of nucleic acid
base in which nucleic acid base is replaced may proceed a
13-selective transglycosylation effectively, and the
obtained compound represented by general formula VIII may
be obtained as a desired p former selectively.
[0168]
The compound of the present invention or
pharmaceutically acceptable salts thereof may be prepared
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183
according to the following method, but which should not
limited thereto.
For starting materials, unless specific processes
therefor are stated, they may be used as commercially
available ones, or can be prepared according to the
publicly known method or a similar method thereto.
[0169]
The abbreviated symbols as used herein mean the
following meanings respectively.
THF: tetrahydrofuran
TBAF: tetra-n-butylammonium fluoride
Ph3P: triphenylphosphine
DMF: N,N-dimethylformamide
MsCl: methanesulfonyl chloride
DMAP: N,N-dimethy1-4-aminopyridine
iPr2NEt, DIPEA: N,N-diisopropylethylamine
Tf20: trifluoromethanesulfonic anhydride
NaN3: sodium azide
Bu4NN3: tetrabutylammonium azide
TfC1: trifluoromethanesulfonyl chloride
Ph3PO: triphenylphosphineoxide
BSA: N,0-bis(trimethylsilyl)acetamide
TMSOTf: trifluoro methanesulfonic acid
trimethylsilyl
TBSOTf: t-butyldimethylsilyl trifluoro
methanesulfonic acid
Bn: benzyl
TMS: trimethylsilyl
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TBDPS: tert-butyldiphenylsilyl
Ac: acetyl
Teoc: trimethylsilylethoxycarbonyl
Boc: tert-butoxycarbonyl
CH2C12: dichloroethane
MeCN: acetonitrile
DPPA: diphenylphosphoryl azide
DIAD: diisopropyl azodicarboxylate
DMTr: 4,4'-dimethoxytrityl
BHT: dibutylhydroxytoluene
[0170]
First, the summary of a preparation method for
bridged nucleic acid GuNA of the present invention is
described.
In the main production route ("A method"), a sugar
compound 1 is used as a starting material, and a
protecting group for hydroxy group is introduced as R1
variable to prepare a compound 2. Next, multi-step (six
steps) reaction routes are carried out using the compound
2 as a starting material to prepare a precursor of a
_
compound represented by general formula III (for example,
compound 7). The compound is deprotected to obtain a
compound represented by general formula III (for example,
compound 8) as a novel intermediate. Successively,
Mitsunobu reaction is carried out using an appropriate
azidating reagent to obtain a compound represented by
general formula II (for example, compound 9 (a derivative
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on a thymine group) and compound 36 (a derivative on a
uracil group) as a novel intermediate.
[0171]
Further, the resulting compound is reacted with
appropriate reducing agent to obtain a compound
represented by general formula I (for exmaple, compound
12).
[0172]
Next, the compound represented by general formula I
is deprotected, and is then introduced by novel another
protecting group, to obtain a compound represented by
general formula IX (for example, compound 14).
Successively, the resulting compound is reacted with a
guanidination reagent to obtain a compound represented by
general formula VII (for example, compound 15) as a novel
intermediate, which can be further reacted with a
phosphoramidite reagent to obtain a GuNA phosphoramidite
compound represented by general formula VII (or general
formula XI) (for example, compound 16) as a novel
compound. The compounds may be used as a monomer
starting material for a preparation of artificial
oligonucleotide of bridged nucleic acid GuNA.
[0173]
Also, in the above-mentioned main production route
("A method"), any alternation may be carried out by
adopting another production route. Examples of the
alternation method include a mesylation reaction route
("B method"), and an azidation reaction route ("C
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method"), and a nucleic acid base replacement reaction
route ("D method").
[0174]
A summary of the production route for the
preparation method of the present invention is described.
[chem.91]
..0 P...1
R1--0 R4 R3 one step Ri... R3
..--30. FR6 X: Ms elc.
R2-0 OH R2-0
Mesylation route Compound
[B method] Novel intermediate compound
General formula IV
General formula V one step
four steps
five steps RI --0 R3
__________________________ I. R5R6 0 CH one step
N3 . m I-
Azidation route
IC method] 132-o OH
Y
Major preparation route Novel intermediate compound
General formula VI
[A method] r
one step or
HO-'0 H3 one step R.4 R1...0 R4 R3 RI ..0 R4 two steps Ri -- 0
H4
six steps R3 R3
---41' *42,..
, 0 _ 0 ¨ . R 0
R 40_ )..50
HO l 0 HO n 0
.
Nr---;) N3 n
14-C)
R2-0 0 HO n
)K R2-0 lar( -L
R2-0 0.. R2-0 0.-'''
1 2
Novel intermediate compound Novel intermediate compound
General formula III General formula II
[chem.92]
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R4 R3
R Ri 1-0 R3 R14---0 __,R4
R
1-0 ' R3 0 0
1/7)
"R5R{I one step , H inH
0 'Thy two steps
.m 0 _______________ Y
N3
--).' R5 iff, -R5
RG 4
. 0
()
36 I N
R2-0 0) R2- H R.I.5" ri
Novel intermediate compound Novel intermediate compound Novel
compound
General formula II General formula I General formula IX
stul.A.,
R3 R3
R7,0 R4
R2-.....0
R4 R4
0 0 0
one step 8 one step B one step B
--11.- [ R54 - R5.4 --lb. =R
54,
R6 ,n -,N Rti M N.,
6-'0 N 0 N
R 0 V
>=N¨R9 R2,3"
N----R2-1 "2.----
i.
R10¨N R23 ¨ N\
s'IR11 Novel oligomer compound
Novel compound R22 General formula X
Novel compound General formula XI
General formula VII Y-- ,s 1,-- or 5 1
<)----N ?-----N
/
RiS¨N 1:218¨N
\ \
Ril R17
[chem . 931
R3
RI4 s, 0 R4
0 NH
.,-
R5 Novel compound one step
Novel oligomer compound
0
General formula VII General formula X
R6
- in ...N.
,O N
Rls H
Novel intermediate compound one step
General formula IX
Base exchange Novel compound
reaction route General formula VII
[D method]
one step 1 A
R3
one step
0
--- N B
one step
Intermediate compound
R /
A in, ,,,,
H
[0175]
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[A method]
A preparation of precursors of novel intermediate
compound represented by general formula III
First, a preparation method for precursors of a
compound represented by general formula III (for example,
compound 7) from a compound 1 as a starting material is
carried out via seven steps according to a method known
in the reference (for example, described in J. Org. Chem.
2011, 76, 9891-9899).
The representative preparation method is described
below.
[chem. 94]
Bn0 Bn0 Acid
catalyst
NaH BnBr
TBDPSCI
1,0 AC20
H(7)*())-,0
____________________ HO/:1)5..0 imidazole
_________________________________________ TBDPSO
Bn d. .
Bn0 DM F 6 AcOH
Bn
1
2 3
commercially available
H or Me 0
Bn0 thymine or uracil
BSA TMSOTf Bn0 / NH 40910aq. MeNI-12
TBDPSO4
0),OAc
MeON TBDPSO/ O
Brio up.c
Bnd Ac
4 5 (thymine):
30 (uracyl)
H or Me
TfCl BriO H or Me
Bn0 NH DMAP
TBDPSO
TBDPSO Bn n,'"\N
d ¨
8n0 bH
7 (thymine);
6 (thymine);
32 (uracyl)
31 (uracyl)
[0176]
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189
Compound 1 is commercially available or can be
prepared using commercially available compounds according
to a generally known method in an organic synthetic
chemistry (in particular, nucleic acid synthesis). For
exmaple, the compound 1 that is described in the above-
mentioned reference can be reacted with appropriate
reagents according to an organic synthesis to obtain the
other compound 1 represented by a general formula.
Each of the preparation steps may be carried out
under a reaction condition according to the methods
described in J. Org. Chem. 2011, 76, 9891-9899, but the
reaction condition of each of the reaction step (for
example, reagent to be used, and reaction period) may be
varied appropriately depending on kinds of the compound 1
as a starting material. Also, according to the method
described in J. Org. Chem. 2011, 76, 9891-9899, the
intermediate compound 4 is reacted with the other nucleic
acid base (for example, uracil group (U)) as a nucleic
acid base as below-mentioned in place of a thymine group
(T) in a reaction with a thymine group (T) to prepare
some analogues of various kinds of nucleic acid base
other than thymine group.
[0177]
A preparation for novel intermediate compound represented
by general formula II
[chem. 95]
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190
R1_0 R4 R3 Deprotection 1-0 R3 R4 R3
-R5R6 .0 -R3 group -R5R6 0 Azidation -R5R6
0
Ra - m
-)17) HO -
12,2)
R2¨(3 0 R2-0' 0 R2-0 0
Novel intermediate compound
Precursors of compound General formula ill Novel intermediate compound
of general formula III General formula II
Introduction of
R1- R4
leaving group X 0 _R3 TAzidaton
(ex. Mesylation)
m
X: Ms-0- etc.
R2-0/ 0
Intermediate compound
General formula IV
(in a general formula of precursors of the compound
represented by general formula III, Ra represents a
protecting group or hydroxy group.)
Next, the precursors of the above-prepared compound
represented by general formula III is deprotected in
appropriate solvent to obtain intermediate compounds
represented by novel general formula III (for example,
compound 8 (for a thymine group) and compound 34 (for an
uracil group)). The deprotection reaction may be carried
out in appropriate reaction condition (for example,
reagents to be used) depending on the kinds of the
protecting group. For example, when Ra variable as a
protecting group for hydroxy group is a silyl type of
protecting group (for example, TBDPS), the deprotection
can be carried out by treating the resulting compound
with a hydrolysis reaction under acidic condition (for
example, acetic acid-THF-water) or a fluoride ion -
donating reagent (for example, TBSF). The solvents may
be any ones unless they affect the reaction adversely,
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and the reaction may be carried out in an appropriate
solvents (for example, ethers such as THF, halogenated
hydrocarbon atoms such as dichloromethane, water, or
mixed solvents thereof). The reaction may proceed
preferably at 0 C to 100 C, particularly preferably at
0 C to 50 C as a reaction temperature.
[0178]
Successively, the resulting compound is reacted with
an appropriate azidating reagent under Mitsunobu reaction
condition to obtain novel intermediate compounds
represented by general formula II (for example, compound
9(for thymine group) and compound 36 (for uracil group)).
_
The azidation reaction may be carried out by reacting the
compound represented by general formula III under
Mitsunobu reaction condition (for example, which is
carried out by using azidating reagent (for example,
diphenylphosphoryl azide (DPP)) in the presence of
azodicarboxylic acid diester (for example, diethyl
azodicarboxylate (DEAD), or diisopropyl azodicarboxylate
(DIaD)) or triphenylphosphine (Ph3P)) .
The solvents may be any ones unless they affect the
reaction adversely, and the reaction may be carried out
in appropriate solvent (for example, ethers such as THF,
halogenated hydrocarbons such as dichloromethane,
hydrocarbons such as toluene, water, or mixed solvents
thereof). The reaction may proceed preferably at -78 C
to 100 C, particularly preferably at 0 C to 50 C as a
reaction temperature.
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[0179]
The progress of the reaction for obtaining azide
compound represented by general formula II from the
compound represented by general formula III may be easily
affected by the degree of purification of the reactant(s)
and etc., and the reaction is not easy to handle.
Without preparing the compound represented by general
formula II directly, once the compound represented by
general formula III is converted into an intermediate
compound represented by general formula IV in which the
hydroxy group is converted into a leaving group) (for
example, compound 10 (for a thymine group), compound 35
(for an uracil group)), and thereafter the leaving group
is removed and an appropriate azide agent is then
introduced to lead easily the compound represented by
general formula II.
The leaving groups X are not particularly limited
unless they are groups that have been generally known in
organic synthesis, and include, for example,
methanesulfonyloxy group (mesyloxy; Ms-0-),
trifluoromethanesulfonyloxy group, and p-
toluenesulfonyloxy group, and mesyloxy group is preferred.
Examples of the reagents for introducing a leaving group
include trifluoromethanesulfonyl chloride,
methanesulfonyl chloride, p-toluenesulfonylchloride, and
trifluoromethanesulfonic anhydride, and methanesulfonyl
chloride is preferred.
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Examples of the azide agent include NEu4NN3 and
sodium azide.
[0180]
The introduction of the leaving group may be carried
out under basic condition (for example,
dimethylaminopyridine (DMAP)) using the reaction
condition (for example, using a sulfonylating agent (for
example, MsC1)) that is well known in organic synthetic
chemistry (for example, sugar synthesis).
The solvents may be any ones unless they affect the
reaction adversely, and the reaction may be carried out
in appropriate solvents (for example, ethers such as THF,
halogenated hydrocarbons such as dichloromethane, water,
or mixed solvents thereof). The reaction may proceed
preferably at -25 C to room temperature, particularly
preferably at 0 C to room temperature as a reaction
temperature.
On the other hand, the reactions associated with a
deprotection and an azidation reaction may be carried out
using appropriate azidating agent (for example,
tetrabutylammonium azide) depending on the kinds of
leaving group under the reaction condition that has been
well known in organic synthetic chemistry.
The solvents may be any ones unless they affect the
reaction adversely, and the reaction may be conducted in
appropriate solvent (for example, ethers such as dioxane,
halogenated hydrocarbons such as dichloromethane, water,
or mixed solvents thereof). The reaction may proceed
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194
preferably at room temperature to a ref lux temperature of
the used solvents, for example, at 100 C or more.
[0181]
That is, the method for preparing the compound
represented by general formula II comprises the following
steps:
a) deprotecting the precursors of the compound
represented by general formula III (that is, deprotecting
of the protecting group of the hydroxy group at 5'
position) to obtain the compound represented by general
foimula III; and
b) subjecting the obtained compound represented by
general formula III to Mitsunobu reaction using an
appropriate azidating reagent to obtaining the compound
represented by general formula II.
As an alternative preparation method, the following
steps b') and c') comprises in place of the above-
mentioned step b);
b') reacting the obtained compound represented by
general formula III with an appropriate reagent to
introduce the leaving group (X) (on the hydroxy group at
5' position), thereby a compound represented by general
formula IV is obtained; and
c') reacting the obtained compound represented by
general formula IV with an appropriate azidating reagent
to obtain a compound represented by general formula II.
[0182]
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195
Alternative method for preparing the novel compound
represented by general formula II
An alternative method for preparing a compound
represented by general formula II is described.
[chem.96]
Riõ.0 R4 R3 RI R4 R3
1R5R6 A -R5R61
_________________________________________ 10-
X one step
-
X . \ A'
Mesylation route
; Ms-0-
[B method] R2-0 OH X R2-0
Novel intermediate compound Intermediate compound
four steps General formula V General formula IV
one step
01nsi L
R1-0 R4 R3 R10 R4 R3 1 0 R4 R
R5R6 0 six steps -R-R
6 0 one step oN
HO cg) N3 3 __ N 07r-75::-)
R2 -0 R2 ¨0
2 Novel intermediate compound
Novel intermediate compound General formula II
General formula III
As aforementioned, first, a hydroxy group is
subjected to a mesylation reaction by using the compound
2 that is prepared from the compound 1, successively, the
reactions may be carried out via multi-step (four steps)
reaction routes according to the method described in the
above-mentioned J. Org. Chem. 2011, 76, 9891-9899 (that
is, a preparation of the compound 6 from the compound 3)
to prepare the novel intermediate compound represented by
general formula IV (specifically, compound 25).
The mesylation reaction may be carried out under
basic condition (for example, in the presence of
triethylamine) using a reaction condition (for example,
using a mesylating agent such as methanesulfonyl chloride,
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and methanesulfonic anhydride) that has been generally
known in organic synthetic chemistry (for example,
nucleic acid synthesis).
The solvent may be any ones unless they affect the
reaction adversely, and the reaction may be conducted in
appropriate solvents (for exmaple, ethers such as THF,
halogenated hydrocarbons such as dichloromethane, water,
or mixed solvents thereof). The reaction may proceed
preferably at -25 C to 100 C, and particularly preferably
at 0 C to room temperature.
[0183]
That is, the method for preparing a compound
represented by general formula IV comprises the following
steps:
The compound represented by general formula V as a
novel intermediate compound is reacted with an activating
agent for hydroxy group (for exmaple,
trifluoromethansulfonylating agent) to activate an
unprotected hydroxy group that is substituted in a
tetrahydrofuran ring in a general formula V, thereby the
compound represented by general formula IV is obtained.
[0184]
As aforementioned, the compound represented by
general formula IV (for example, compound 10 (for a
thymine group) or compound 35 (for an uracil group)) is
reacted with an azide agent (for example, nBuINN3 or
sodium azide) to obtain the compound represented by
general formula II.
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[0185]
Alternative method for preparing a novel intermediate
compound represented by general formula II
Further alternative method for preparing the
compound represented by general formula II is described.
[chem.97]
R4
1 =-= 0
-R5RÃ 0
Azidation route Th{
N3 ni 0
[C method]
R2-0 OH
one step
Compound
five steps General formula VI
V
õ0 R4 R3 R 0 R4 R, 1 R4R
3
R5R 0 - -1R5R6 0 RfR61T
I 0
= - )0.
HO N3
120
R20 0.-)K ¨ 0 0 R2 ¨0 0
2 Novel intermediate compound
Compound General formula II
General formula III
As aforementioned, the reaction may be carried out
via multi-steps (five steps) reaction routes according to
the method described in the reference (Henrik M.
Pfundheller, H. M., Bryld, T., Olsen, C. E., Wengel,
J. Helvetica Chimica Acta 2000, 83, 128-151) using as a
starting material the compound 2 that is prepared from
the compound 1 to obtain the azide compound represented
by general formula VI (specifically, compound 21) as an
intermediate compound.
Next, the resulting compound is reacted with an
activating agent for hydroxy group under basic condition
(for example, in the presence of DMAP) using a the same
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198
reaction condition as those used in the reaction for
preparing the compound 7 from the compound 6 (or the
compound 10 from the compound 25) to obtain the novel
intermediate compound represented by general formula II
(specifically, compound 9).
Examples of the activating agent for hydroxy group
as used herein include trifluoromethanesulfonyl chloride,
methanesulfonyl chloride, and trifluoromethanesulfonic
anhydride, and trifluoromethanesulfonyl chloride is
preferred.
The solvent may be any ones unless they affect the
reaction adversely, and the reaction may be carried out
in appropriate solvents (for example, ethers such as THF,
halogenated hydrocarbons such as dichloromethane), water,
or mixed solvents thereof).
The reaction may proceed preferably at 0 C to 100 C,
and particularly preferably at 0 C to room temperature.
[0186]
That is, the method for preparing the compound
represented by general formula II as a novel intermediate
compound from the compound represented by general formula
VI comprises the following steps:
The compound represented by general formula VI is
reacted with an activating agent for hydroxy group to
activate an unprotected hydroxy group that is substituted
to a tetrahydrofuran ring in the general formula VI to
obtain the compound represented by general formula II.
[0187]
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199
Preparation of novel intermediate compound represented by
general formula I
[chem. 98]
R R4 R4 R1. R4
R3 -- 0 R3 R1-0
R5R6 0 Reduction -R5f% o
R3
0
[R5 .
N3 - R2-0 H2N
R2-0
ones tep
o 0
/ [R6 n
0 N
Ri H H
Novel intermediate compound
¨ Novel intermediate
General formula II Intermediate compound 2 compound
General formula t
Further, the obtained compound represented by
general formula II may be subjected to a reduction
reaction to obtain novel intermediate compound
represented by general formula I wherein the azide group
is reduced (for example, compound 12 (for a thymine
group), compound 37 (for an uracil group)) (herein which
referred to as "2'-amino-LNA").
The reduction reaction of the azide group may be
carried out in the presence of a reducing agent (for
example, phosphines such as triphenylphosphine).
The solvents may be any ones unless they affect the
reaction adversely, and the reaction may be carried out
in appropriate solvents (for example, ethers such as THF,
halogenated hydrocarbons such as dichloromethane, water,
or mixed solvents thereof). The reaction may proceed
preferably at 0 C to high temperature, and particularly
preferably at room temperature to 100 C or less.
[0188]
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That is, the method for preparing novel intermediate
compound represented by general formula I comprises the
following steps:
reacting the above obtained novel intermediate
compound represented by general formula II with a
reducing agent to prepare the compound represented by
general formula I or salts thereof.
[0189]
Here, the present reaction is novel reaction step
comprising introducing 2'-position amino group by
expanding an azide methyl group from 4'-position carbon
atom and thereby bridging with 2'-position carbon atom.
[0190]
Preparation of precursors of novel monomer compound
represented by general formula VII
[chem. 99]
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R4
RI R3
k- A R3
'\AH )
- Deprotection A
-ON ti.j _NH
"R5 - R1 ,R2 groups Rs
one step "6 n
R2'0
R N
2
Novel intermediate compound Intermediate compound 3
General formula I R2: Hydrogen atom
R14 0
R3
Introduction of another R4
1( A
protecting for hydroxy 0
0 NH
+R7 group [R5
--)fe
0
[Ro n R6 "IN
M
one step
RON
I 0 N
R15'
Intermediate compound 4 a Intermediate compound
(Precursors) 78 General formula IX
ty
R7: Another protecting group
for hydroxy V
R2: Hydrogen atom
Novel compound
General formula VII
[0191]
The above obtained compound represented by general
formula I is deprotected at 5 ' -position and 3 ' -position
respectively to deprotected R1 variable and R2 variable
to obtain an intermediate compound 3 ( for example,
compound of the example 13) . Successively, a new
protecting group for hydroxy group having different
deprotection condition than the deprotected protecting
group is introduced to only 5 ' -position hydroxy group
(R7) to obtain an intermediate compound 4 ( for example,
compound 14) as a precursor.
Here the deprotection reaction may be carried out
under any reaction conditions that have been generally
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known in organic synthetic chemistry (for example,
nucleic acid synthesis) depending on the kinds of the
protecting group. For example, when the benzyl type
protecting group (for exmaple, Bn group) is used, the
deprotection reaction may be carried out by hydrogenation
reaction with hydrogen in the presence of organic
metallic catalyst (for example, palladium-carbon)
The solvents may be any ones unless they affect the
reaction adversely and the reaction may be carried out in
appropriate solvents (for example, ethers such as THF,
acetates such as ethyl acetate, halogenated hydrocarbons
such as dichloromethane, acids such as acetic acid,
alcohols such as methanol, water, or mixed solvents
thereof) or by using bases itself as solvent. The
reaction may proceed preferably at room temperature to
high temperature, and particularly preferably at room
temperature to 50 C or less.
[0192]
Also, examples of the newly introduced protecting
group for hydroxy group include trityl type of protecting
group (for exmaple, DMTR group). The introduction
reaction of the protecting group may be carried out under
the reaction condition that has been well known in
organic synthetic chemistry (for example, nucleic acid
synthesis) depending on the kinds of the protecting group.
For when the protecting group for hydroxy group is a
trityl type protecting group (for example, DMTr group),
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he reaction may be carried out under basic condition (for
example, in the presence of pyridine).
The solvents may be any ones unless they affect the
reaction adversely, and the reaction may be carried out
in appropriate solvents (for example, ethers such as THF,
halogenated hydrocarbons such as dichloromethyl, water,
or mixed solvents thereof) or by using bases itself as a
solvent. The reaction may proceed preferably at 0 C to
50 C, and particularly preferably at room temperature.
[0193]
That is, the method for preparing the intermediate
compound 4 comprises the following steps:
a) subjecting the above obtained compound represented
by general formula I (for example, R1 variable and R3
variable at 5'-position and 3'-position) to a
deprotection reaction (which provides an intermediate
compound 3);
b) introducing a protecting group for hydroxy group
into the reaction product (at 5'-position).
[0194]
As below-mentioned herein, the obtained intermediate
compound 4 is carried out by guanidination, followed by
phosphoramiditation to obtain a novel compound
represented by general formula VII.
[0195]
Exchange reaction of nucleic acid base
[chem. 100]
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R3
Ri4 R4 R3
R12 0 / R4
0 NI NH
Base exchange 0
0 reaction route
. Novel Compound
/
m General formula
VII
0 N 6 m NN
.õ0 N
Novel Intermediate compound R13
General formula IX
Compound
General formula VIII
According to a nucleic acid exchange reaction
(transglycosylation reaction) using as a starting
material, the above obtained compound represented by
general formula I or the intermediate compound 4 (which
collectively referred to as compound represented by
general formula IX) (for exmaple, compound 12 and
compound 14), a pyrimidine base moiety of nucleic acid
base such as thymine group (T) and uracil group (U) may
be replaced with another nucleic acid base moiety (for
exmaple, adenine (A), guanine (G), uracil (U), thymine
(T), cytosine (C), or 5-methylcytosine (meC) to obtain as
a novel intermediate compound the compound represented by
general formula VIII or salts thereof (for example,
compound 26, compound 28 and compound 29).
The base exchange reaction may be carried out, for
exmaple, by reacting the compound represented by general
formula IX in the presence of Lewis acid, and may be
facilitated by reacting with a silylating agent.
Examples of the silylating agent include BSA, and
hexamethyldisilazane, and examples of Lewis acid includes
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TMSOTf, TBSOTf, tin chloride, and the others, which are
not limited thereto.
The silylating agent may be used in about 1 to about
20 molar equivalents, and the Lewis acid may be used in a
catalytic amount (about 0.05 molar equivalents) to about
2 molar equivalents as opposed to 1 mole of the reaction
substrates.
The solvents may be any ones unless they affect the
reaction adversely, and the reaction may be carried out
in appropriate solvents (for exmaple, ethers such as THF;
halogenated hydrocarbons such as dichloromethane,
dichloroethane, and chloroform; hydrocarbons such as
benzene and toluene; acetonitrile; water; or mixed
solvents thereof). The reaction may proceed preferably
at 0 C to high temperature, and particularly preferably
at room temperature to about 60 C.
[0196]
Preparation of monomer compound represented by general
formula VII
[chem. 101]
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R3
R14 =-=-. 0 R4
0 NH
R Y R3 R3
0 R7
R4 R7- R4
R6qi-
m
N 0 0
R16 Phosphoramiditation
Guanidination reagent 1R6 reagent
or Compound ________ 7, 4 1R5 4
General formula IX R6
' m ER,
R3 ON N
/ R4 Rsa
\N¨FR11 RBb N-
0
IR"
R5 Ril
Novel compound Novel compound
R6 General formula VII General formula VII
m
N Raa: Hydrogen atom
R13--H R6b: Phosphate
group which may be
optionally substituted
Novel intermediate compound
General formula VIII
[0197]
The above obtained compound represented by general
formula IX or compound represented by general formula
VIII is reacted with guanidination reagent to obtain the
guanidinated compound represented by general formula VII
(wherein R8a represents a hydrogen atom) (for exmaple,
compound 15), which corresponds to the compound wherein a
bridged amino group in the compound represented by
general formula VIII is guanidined.
Here, the guanidination reaction may be carried out
under reaction conditions that have generally known in
organic synthesis (in particular, nucleic acid synthesis)
(for example, using a reagent). Examples of the
guanidination reagent include a compound represented by
following formula:
[chem. 102]
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207
FRPm RPm
N'
[wherein, RPr represents a protecting group for amino
group (for example, Teoc, and Bac)]
, and the reaction may be carried out in appropriate
activating reagent (for example, silver trif late (Ag0Tf)).
[0198]
Further, the above obtained compound represented by
general formula VII may be reacted with
phosphoramiditation reagent to obtain a compound
represented by general formula VII or salts thereof
(which herein referred to as "GuNA phosphoramidite") (for
example, compound 16).
Here the phosphoramiditation reaction may be
conducted under reaction conditions that have been
generally known in organic synthesis (in particular,
nucleic acid synthesis) (for exmaple, in terms of
reagent(s)). Examples of the phosphoramiditation reagent
include agents by the following formula:
[chem.103]
(PI)2N CI ('Pr)2N, _N(Pr)2
= -P
and
0
NC-------
, which should not be limited thereto. The
phosphoramiditation reaction may be conducted in the
presence of appropriate bases (such as DIPEA) or
appropriate acid (such as diisopropyl ammonium
tetrafluoride, and 4,5-dicyanoimidazole).
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[0199]
That is, the method for preparing the compound
represented by general formula VII or salts thereof may
comprise the following steps:
a) a step of guanidination of the compound represented
by general formula IX or salts thereof or the compound
represented by general foimula VIII or salts thereof to
prepare the compound represented by general formula VII
(wherein, Rga represents a hydrogen atom);
b) a step of phosphoramiditation of the reaction
products to prepare the compound represented by general
formula VII (wherein, Rgb represents a phosphate group
which may be optionally substituted).
[0200]
Also, the method for preparing the compound
represented by general formula VII or salts thereof may
comprise at least one of the following steps among the
steps described herein:
a step of reacting the compound represented by
general formula II with a reducing agent to prepare the
compound represented by general formula I or salts
thereof;
a step of reacting the compound represented by
general formula III with an azide agent to prepare the
compound represented by general formula II or salts
thereof;
a step of reacting the compound represented by
general formula V with an activating agent for hydroxy
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209
group to prepare the compound represented by general
formula IV or salts thereof;
a step of reacting the compound represented by
general formula IV with an azide agent to prepare the
compound represented by general formula II or salts
thereof;
a step of reacting the compound represented by
general formula VI with an activating agent for hydroxy
group to prepare the compound represented by general
formula II or salts thereof;
a step of replacing cycle A in the compound
represented by general formula IX to prepare the
compounds represented by general formula VIII or salts
thereof.
[0201]
Preparation of GuNA oligonucleotide represented by
general formula X
[chem.104]
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210
R3
----- 0 R4
B
Rs
4
_Rs m
,rvvvs.r
)3 N
R3
N¨R,
R4
R10¨N.
0
Novel compound RH
General formula VII Rs
Amidaitation 4
Of R3
m Nss,
If necess ,
ary
0 R4 deprolection 0 Y Novel oligomer compound
General formula X
0
Y-
4
R6 m Ns, or I
N
R20 N¨ R21 N¨R16 _________ 114L--R16
R23--N
\Ru R17
R22
Novel compound
General formula XI
The compound represented by general formula VII or
salts thereof can be used as a monomer starting material
for preparing GuNA oligonucleotide. The GuNA
oligonucleotide can be synthesized by oligomerization of
the compound represented by general formula VII or
general formula XI or salts thereof, if necessary,
followed by deprotecting an amino protecting group.
For example, the oligomerization is not limited to
any method as long as it is the method that has been
generally known in the synthetic chemistry (in particular,
nucleic acid synthesis), and it can be used by an
phosphoramidite method. The phosphoramidite method can
be conducted, for example, according to the method
described in WO 2014/046212 Al.
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Using this oligomerization, oligonucleotide
containing one or more nucleotides represented by general
formula X can be prepared.
[0202]
That is, the method for preparing oligonucleotide
represented by general formula X comprises the following
step.
a) a step of oligomerization of the compound
represented by general formula VII or general formula XI
or salts thereof, if necessary, followed by deprotecting
an amino protecting group.
[0203]
Also, the method for preparing the compound
represented by general formula VII or general formula XI
or salts thereof may comprise at least one of the
following steps among the steps described herein.
a step of reacting the compound represented by
general formula II with a reducing agent to prepare the
compound represented by general formula I or salts
thereof;
a step of reacting the compound represented by
general formula III with an azide agent to prepare the
compound represented by general formula II or salts
thereof;
a step of reacting the compound represented by
general formula V with an activating agent for hydroxy
group to prepare the compound represented by general
formula IV or salts thereof;
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a step of reacting the compound represented by
general formula IV with an azide agent to prepare the
compound represented by general formula II or salts
thereof;
a step of reacting the compound represented by
general formula VI with an activating agent for hydroxy
group to prepare the compound represented by general
formula II or salts thereof;
a step of replacing the cycle A in the compound
represented by general formula IX to prepare the compound
represented by general formula VIII or salts thereof;
a step of guanidination of the compound represented
by general formula IX or salts thereof or the compound
represented by general formula VIII or salt thereof to
prepare the compound represented by general formula VII
(wherein Rn represents a hydrogen atom) or salts
thereof;
a step of phosphoramiditation of the compound
represented by general formula VII (wherein R8a
represents a hydrogen atom) or salts thereof to prepare
the compound represented by general formula VII (wherein
Rn represents a phosphate group which may be optionally
substituted) to prepare the compound represented by
general formula (wherein Rn represents a phosphate group
which may be optionally substituted) or salts thereof;
a step of deprotecting at least one of amino
protecting group as R9, Rn or Ril in the compound
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213
represented by general formula VII to prepare the
compound represented by general formula XI.
Examples
[0204]
Hereinafter, the present invention is further
specifically described by Examples, however, the present
invention is not limited thereto. The identification of
compound was carried out by Elemental analysis, Mass
spectrum, High performance liquid chromatography mass
spectrometer; LCMS, IR spectrum, NMR spectrum, High-
performance liquid chromatography (HPLC) and the like.
In NMR spectrum, for proton nuclear magnetic resonance
('H-NMR), an apparatus with 400 MHz of the resonance
frequency was used, and for phosphorus nuclear magnetic
resonance (31P-NMR), an apparatus with 161.8 MHz of the
resonance frequency was used. As symbols used in NMR
spectrum, "s" is "singlet", "d" is "doublet", "dd" is
"double of doublet", "t" is "triplet", "td" is "double of
triplet", "q" is "quartet", "quin" is "quintet", "sept"
is "septet", "m" is "multiplet", "br" is "broad", "brs"
is "broad singlet", "brd" is "broad doublet", "brt" is
"broad triplet", and "J" is "coupling constant".
[0205]
One example of preparation method of compound 12 is
described below, however, which is not limited thereto.
Specifically, compound 12 was prepared using a
commercially available compound 1 as a starting material
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214
according to the method described in J. Org. Chem. 2011,
76, 9891-9899.
[chem.105]
HOBn0 TBDPSCI Bn0 H2SO4
13
NaH, BnBr im TBDPSO -0 idazole Ac20
DMF
,
' HO/ Bn DMF AcOHd Bnd ... )( Bnd
'Ov\'''
1 2 3
)
Bn0 thymine
BSA TMSOTf Bn0 e NH aq. MeNH2
TBDPSO OAc ___ .. _____ ,.. z,b7--4õ ____ >
MeCN 0 THE
- TBDPSO
Brld' bAc
Bnd '0A0
4 5
0
/
Bn0 TtC1 Bn0
=I.--4NH DMAP
/ TSDPSO
4_01-i ____
0 CH2Cl2
TBDPSO Bnd N
: =
Bnd -OH
6 7
[chem.106]
Bn0 Bn0 DPPA Bn0
TBAF Ph3P. MAD
TBDPSO IZ-11 _________ ' HO/1r N=Z-----0
THE THE Bnd N Lind N Bnd 0 N
7 8 9
&
msci
Bn0 ^8u4NN,
DMAP. CH2C/2dioxane
Ms0
sr,d. 0 N
- _
Bn0 Bn0
PPh, rj).,0
9 _________ ' H2t4/trC--f ___________
N
THE, H20 , ,,,f0 ' (i...,_t =71,..NH
Bnd - Bnr _.,1 0
o.,... -NH
11
_ _
12
[0206]
Example 1
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((2R,35,3a5,9aR)-3-(benzyloxy)-2-[(benzyloxy)methyl]-2-
(hydroxymethyl)-7-methy1-2,3,3a,9a-tetrahydro-6H-
furo[2',3':4,5] [1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 8)
Compound 7 (prepared from 3,5-di-O-benzy1-4-C-
hydroxymethyl-1,2-0-isopropylidene-a-D-ribofurahose 2
according to the method described in J. Org. Chem. 2011,
76, 9891) (2.00 g, 2.903 mmol) and THF (14.5 mL) were
mixed, and thereto was added TBAF (3.77 mL, 3.774 mmol)
dropwise under cooling at 0 C. The mixtures were stirred
for 130 minutes at room temperature, and the solvents
were evaporated. The resulting residues were purified by
silica gel chromatography (chloroform/methanol, 100/0 to
85/15) to obtain compound 8 (1260 mg, yield 96%).
MS (APCI): m/z = 451 (M+H)+
1H-NMR (CDC13) 6: 7.41-7.24 (8H, m), 7.21-7.11 (3H, m),
6.25 (1H, d, J 6.2 Hz), 5.32
(1H, dd, J = 6.2, 2.6 Hz),
4.79 (1H, d, J = 11.8 Hz), 4.61 (1H, d, J = 11.8 Hz),
4.39 (1H, d, J = 12.3 Hz), 4.34 (1H, d, J = 2.6 Hz), 4.34
(1H, d, J = 12.3 Hz), 3.86 (1H, dd, J = 12.2, 5.7 Hz),
3.69 (1H, dd, J = 12.2, 7.7 Hz), 3.33 (IH, d, J . 10.3
Hz), 3.30 (1H, d, J = 10.3 Hz), 2.20 (1H, dd, J - 7.7,
5.7 Hz), 1.99-1.95 (3H, m)
[0207]
Example 2
(2R,35,3a5,9aR)-2-(azidomethyl)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-7-methyl-2,3,3a,9a-tetrahydro-6H-
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216
furo[21,31:4,5][1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 9)
Compound 8 (300 mg, 0.6659 mmol) and THF (4.4 mL)
were mixed. Thereto were added PPh3 (524 mg, 1.998 mmol)
and diisopropyl azodicarboxylate (0.413 mL, 2.098 mmol)
under cooling at 0 C, and successively followed by adding
diphenylphosphoryl azide (0.431 mL, 1.998 mmol) dropwise.
The mixtures were stirred at room temperature for 10
minutes, and stirred under heating at 50 C as external
temperature for 30 minutes. Thereto was added DMF (1.2
mL), and the mixtures were stirred under heating at 50 C
as external temperature for 17 hours. Thereto was added
saturated brine at 0 C, and the mixtures were extracted
with ethyl acetate. To the resulting organic layers was
added anhydrous sodium sulfate, and the mixtures were
dried and filtered, and the solvents were then evaporated.
The resulting residues were purified by silica gel
chromatography (ethyl acetate/methanol, 100/0 to 95/5) to
obtain compound 9 (285 mg, yield 90%, containing Ph3P0 as
impurities).
MS (APCI): m/z = 476 (M+H)+
1H-NMR (CDC1.1) 6: 7.41-7.26 (8H, m), 7.21-7.11 (3H, m),
6.19 (1H, d, J = 6.2 Hz), 5.29 (1H, dd, J = 6.2, 2.3 Hz),
4.77 (1H, d, J = 11.8 Hz), 4.61 (1H, d, J = 11.8 Hz),
4.40 (1H, d, J = 12.0 Hz), 4.35 (1H, d, J = 12.0 Hz),
4.29 (1H, d, J = 2.3 Hz), 3.63 (1H, d, J = 12.8 Hz), 3.49
(1H, d, J = 12.8 Hz), 3.33 (1H, d, J = 10.2 Hz), 3.25 (1H,
d, J = 10.2 Hz), 2.01-1.95 (3H, m)
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217
[0208]
Example 3
Also, compound 9 can be prepared also from compound
8 according to the following Examples 3 and 4.
_
{(2S,35,3a5,9aR)-3-(Benzyloxy)-2-[(benzyloxy)methyl]-7-
methy1-6-oxa-2,3,3a,9a-tetrahydro-6H-
furo[21,3':4,5] [1,3]oxazolo[3,2-a]pyrimidin-2-yl}methyl
methanesulfonate (compound 10)
To the suspension of compound 8 (7.00 g, 15.54 mmol)
in methylene chloride (155 mL) were added MsC1 (2.05 mL,
26.42 mmol) and DMAP (5695 mg, 46.61 mmol) under ice-
cooling, and the mixtures were stirred at room
temperature for 1.5 hours. Diisopropyl ether (500 mL)
was added thereto to obtain white solids, and the solids
were collected by filtration, and washed with diisopropyl
ether. The resulting solids were recrystallized from
diisopropyl ether (500 mL) and chloroform (100 mL), and
the obtained crystals were washed with water (IL) and
dried to obtain compound 10 (8.12 g, 15.4 mmol, yield
98.9%).
MS (APCI): m/z = 529 (M+H)+
1H-NMR (CDC13) 6: 7.39-7.27 (81-1, m), 7.21-7.14 (3H, m),
6.22 (1H, d, J = 6.1 Hz), 5.28 (1H, dd, J = 6.1, 2.6 Hz),
4.76 (IH, d, J = 11.8 Hz), 4.60 (1H, d, J - 11.8 Hz),
4.44-4.31 (5H, m), 3.40 (1H, d, J - 10.2 Hz), 3.29 (1H, d,
J - 10.2 Hz), 2.92 (3H, s), 2.00-1.95 (3H, m)
[0209]
Example 4
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(2R,35,3a5,9aR)-2-(Azidomethyl)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-7-methyl-2,3,3a,9a-tetrahydro-6H-
furo[2',3':4,5] [1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 9)
To a solution of compound 10 (7.00 g, 13.24 mmol) in
1,4-dioxane (132 mL) was added tetrabutylammonium azide
(11.3 g, 39.73 mmol), and the mixtures were stirred at
120 C as external temperature for 7 hours. The mixtures
were cooled under ice-cooling, and thereto was added
saturated aqueous sodium bicarbonate solution. The
mixtures were extracted with ethyl acetate twice, and the
organic layers were washed with saturated brine, and
dried over anhydrous sodium sulfate, and the insoluble
materials were removed by filtration. The solvents were
evaporated from the filtrates, and the resulting residues
were purified by silica gel chromatography (ethyl
acetate/methanol, 90/10 to 85/15) to obtain compound 9
(5.80 g, 12 mmol, yield 92%).
MS (APCI): m/z = 476 (M+H)+
1H-NMR data was identical to those of compound 9
obtained in Examples 2.
[0210]
Example 5
1-{(1R,3R,4P,75)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-2-
oxa-5-azabicyclo[2.2.1]hepta-3-y11-5-methylpyrimidine-
2,4(1H,3H)-dione (compound 12)
Compound 9 (1446 mg, 3.041 mmol), THF (20 mL) and
water (4 mL) were mixed, and thereto was added Ph3P (1196
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219
mg, 4.562 mmol), and the mixtures were stirred under
heating at 70 C as external temperature for 17 hours.
The solvents were evaporated, and the resulting residues
were purified by silica gel chromatography (ethyl
acetate/methanol, 95/5 to 85/15) to obtain compound 12
(1267 mg, yield 92%).
MS (APCI): m/z = 450 (M+H)
1H-NMR (CDC13) 6: 8.08 (1H, brs), 7.61-7.57 (1H, m),
7.39-7.23 (10H, m), 5.52 (1H, s), 4.65-4.51 (4H, m), 3.91
(1H, s), 3.87 (IH, d, J = 10.8 Hz), 3.80 (1H, d, J = 10.8
Hz), 3.64 (IH, s), 3.15 (1H, d, J = 9.7 Hz), 2.90 (1H, d,
J = 9.7 Hz), 1.68-1.53 (3H, m) =
[0211]
One example of preparation method of compounds 16a
and 15b is described below, however, which is not limited
thereto. Specifically, using the above compound 12 as a
starting material, compounds 16a and 15b were prepared.
[chem.107]
En() HO DIMTrO
--- 0 H2. Pd(OH)21C
0 01 N*),ir 0 mma 0 r\e
1T--NH
AcOH NH
pyridine ky
0
BnYThilH Hc(N OH HO NH
12 13 14
(Pr)2N 01
R R 'P'
Fi
AgOTtEt3N MATTOb_Nrjµ'r o NC ---"'"--"O DMTrO
, DIPEA
b" NH
/
THFi 0 C1-t2012 (P1924,
p-O N
N-Rr'r
NH
zAV",Thoc
txl,r")=Eloc 158 151) 168
[0212]
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Example 6
1-[(1R,3R,4R,7S)-7-Hydroxy-1-(hydroxymethyl)-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y1]-5-methylpyrimidine-
2,4(1H,3H)-dione acetate salt (compound 13)
Compound 12 (1.00 g, 2.225 mmol) and acetic acid (6
mL) were mixed, and thereto was added palladium hydroxide
(200 mg, 1.425 mmol). The mixtures were stirred under
heating at 50 C as external temperature under hydrogen
atmosphere for 6 hours. The reaction solutions were
purged with nitrogen, and filtered with a membrane filter
using methanol, and the filtrates were concentrated. The
residues were azeotroped with toluene, and dried in
vacuum to obtain compound 13 (774 mg, containing 9.8wt%
of toluene, yield 100 %).
MS (APCI): m/z = 270 (M+H)+
1H-NMR (DMSO-d0 6: 11.31 (1H, brs), 7.75-7.70 (1H, m),
5.33 (1H, s), 3.83 (1H, s), 3.78-3.56 (2H, m), 3.26 (1H,
s), 2.90 (1H, d, J = 10.2 Hz), 2.58 (1H, d, J = 10.2 Hz),
1.91 (3H, s), 1.79-1.75 (3H, m)
[0213]
Example 7
1-[(1R,3R,4R,7S)-1-{[Bis(4-
methoxyphenyl)(phenyl)methoxylmethyll-7-hydroxy-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y1]-5-methylpyrimidine-
2,4(1H,3H)-dione (compound 14)
Under ice-cooling, to the solution of compound 13
(4.00 g, 12.1 mmol) in pyridine (37 mL, 462 mmol) was
added 4,4'-dimethoxytrityl chloride (12.4 g, 36.4 mmol),
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and the mixtures were warmed to room temperature, and
stirred for 1.5 hours. The reaction solutions were added
dropwise to aqueous acetic acid solution (water 250
mL/acetic acid 69.5 ml), and thereto was added ethyl
acetate (300 mL), and the mixtures were stirred for 30
minutes. The mixtures were extracted with ethyl acetate,
and the organic layers were then washed with saturated
brine, saturated aqueous sodium bicarbonate solution, and
saturated brine successively, and dried over anhydrous
sodium sulfate, and the insoluble materials were removed
by filtration. The solvents were evaporated from the
filtrates, and the resulting residues were purified by
silica gel chromatography (chloroform/methanol, 100/0 to
90/10) to obtain compound 14 (5.98 g, yield 86%).
MS (APCI): m/z = 572 (M+H)
1H-NMR (DMSO-d6) 5: 11.33 (1H, brs), 7.63-7.59 (1H, m),
7.47-7.21 (9H, m), 6.94-6.86 (4H, m), 5.37 (1H, d, J =
5.1 Hz), 5.34 (1H, s), 4.05 (1H, d, J = 5.1 Hz), 3.74 (6H,
s), 3.39-3.22 (3H, m), 2.84 (1H, d, J = 9.8 Hz), 2.63 (1H,
d, J = 9.8 Hz), 1.53-1.46 (3H, m)
[0214]
Example 8
Bis[2-(trimethylsilyl)ethy1]{[(1R,3R,4R,7S)-1-1[bis(4-
methoxyphenyl)(phenyl)methoxy]methy11-7-hydroxy-3-(5-
methy1-2,4-dioxa-3,4-dihyropyrimidinel(21-I)-y1)-2-oxa-5-
azabicyclo[2.2.1]hepta-5-yl]methylidene}biscarbamate
(compound 15a)
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A solution of compound 14 (1266 mg, 2.215 mmol), THF
(7.7 mL), and 2-trimethylsilylethyl N-[methylsulfanyl-(2-
trimethylsilylethoxycarbonylamino)methylene]carbamate
(1258 mg, 3.322 mmol) in THF (7 mL) was stirred, and
thereto were added Et3N (1.23 mL, 8.859 mmol) and Ag0Tf
(854 mg, 3.322 mmol) under ice-cooling successively. The
mixtures were warmed to room temperature, and stirred at
room temperature for 4 hours. Under ice-cooling, thereto
was added saturated brine, and the mixtures were
extracted with ethyl acetate. The organic layers were
dried over anhydrous sodium sulfate, and the insoluble
materials were removed by filtration. The solvents were
evaporated from the filtrates, and the resulting residues
were purified by silica gel chromatography (hexane/ethyl
acetate, 55/45 to 45/55) to obtain compound 15a (1563 mg,
containing 1.9wt% of ethyl acetate, yield 78%).
MS (APCI): m/z - 902 (M+H)+
1H-NMR (CDC13) 6: 10.36 (1H, brs), 8.26 (1H, s), 7.59 (1H,
s), 7.49-7.42 (2H, m), 7.39-7.20 (7H, m), 6.89-6.80 (4H,
m), 5.62 (1H, s), 4.71 (1H, s), 4.36-4.07 (5H, m), 3.84-
3.70 (7H, m), 3.62 (1H, d, J = 11.8 Hz), 3.57-3.46 (2H,
m), 1.67 (31-1, s), 1.11-0.97 (4H, m), 0.04 (18H, s)
[0215]
Example 9
Bis[2-(trimethylsilyl)ethyl] {[(1R,3R,4R,7S)-1-([bis(4-
methoxyphenyl)(phenyl)methoxylmethyll-7-{[(2-
cyanoethoxy)(dipropan-2-ylamino)phosphanyl]oxyl-3-(5-
methy1-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-y1)-2-oxa-5-
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azabicyclo[2.2.11hepta-5-yl]methylidene}biscarbamate
(compound 16a)
Compound 15a (60.5 mg, 0.0671 mmol) and
dichloromethane (1.5 mL) were mixed, and under ice-
cooling, thereto were added iPr2NEt (0.0232 mL, 0.134
mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramide
(0.0224 mL, 0.101 mmol) dropwise successively, and the
mixtures were stirred at room temperature for four hours.
The mixtures were cooled again under ice-cooling, and
thereto were additionally added iPr2NEt (0.0232 mL, 0.134
mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramide
(0.0224 mL, 0.101 mmol), and the mixtures were stirred at
room temperature for 2 hours. Under ice-cooling, thereto
was added saturated aqueous sodium bicarbonate solution,
and the mixtures were extracted with chloroform. The
organic layers were washed with water and saturated brine
successively, and dried over anhydrous sodium sulfate,
and the insoluble materials were removed by filtration.
The solvents were evaporated from the filtrates, and the
resulting residues were purified by silica gel
chromatography (hexane/ethyl acetate, 65/35 to 50/50) to
obtain compound 16a (40.1 mg, yield 54%-)
MS (APCI): m/z = 1102 (M+H)'
31P-NMR (CDC13) 6: 149.44, 149.31, 148.87, 148.69
[0216]
Example 10
Di-tert-butyl {[(1R,3R,4R,75)-1-f[bis(4-
me thoxyphenyl ) (phenyl ) methoxy] methyl} - 7 -hydroxy- 3 - ( 5 -
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224
methy1-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-y1)-2-oxa-5-
azabicyclo[2.2.1]hepta-5-yl]methylidene)biscarbamate
(compound 15b)
To a suspension of compound 14 (1.20 g, 2.10 mmol)
in THF (21 mL) were added Et3N (1.2 mL, 8.4 mmol), tert-
butyl N-[(tert-butoxycarbonylamino)-methylsulfanyl-
methylene]carbamate (910 mg, 3.1 mmol) and Ag0Tf (810 mg,
3.1 mmol) successively at room temperature, and the
mixtures were stirred at room temperature for 2 hours.
To the reaction solution was added saturated brine, and
the mixtures were extracted with ethyl acetate. The
ethyl acetate layers were washed with saturated brine,
and then dried over anhydrous sodium sulfate, and the
insoluble materials were removed by filtration. The
solvents were evaporated from the filtrates, and the
resulting residues were purified by silica gel
chromatography (hexane/ethyl acetate, 50/50 to 30/70) to
obtain compound 15b (1.44g, 1.77 mmol, yield 84%).
MS (APCI): m/z = 814 (M+H)+
1H-NMR (CDC13) 6: 10.11 (1H, brs), 8.09 (1H, brs), 7.65-
7.57 (1H, m), 7.52-7.41 (2H, m), 7.40-7.19 (714, m), 6.89-
6.78 (4H, m), 5.57 (1H, s), 4.67 (114, s), 4.32 (1H, s),
4.15 (1H, brs), 3.85-3.72 (7H, m), 3.64-3.40 (3H, m),
1.72-1.65 (314, m), 1.48 (1814, s)
[0217]
Another preparation method (hereinafter, sometimes
referred to as azidation route) of compound 12 is
described.
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[chem.108]
HO Bn0 1120 Bn0 Bn0
0
NaftBnBr 0 pyridine 0 NaN3 0
HO/ .,0 . Ho/ ,. ..,0 ... ,,
- Tf0/ b '-' . =e0
N,
DM!, CH 7C12 A,- DMF
(34 13--V B4 veV (kid '0 . Bne '0-\--
1 2 17 18
0 \..40
Acv0 Bn0 thymine
H2S0,, 0 0A
__________ ' fs4 ' BSA TMS011 Bn0 / NH
________________________________ = ,,,, 0 N- aq MeNH2 800 '
/ NH
AcOH MeCN 0 THF
N3 N6
Brd bft
8116 bft 194 bri
19 N 21
_
MI Bn0 Bn0 Bn0
DMAP PPh3 , 1\r
CH2C12
3 _.,.,..10 THF, H20
_________ N t7N , H2N/'..t,i/---
' el NH
sr
Bn6 0 N 8110. - 0
BnOE1
11
_
- _
[0218]
Example 11 (Azidation route)
The preparation of compound 20 from compound 2 (3,5-
di-O-benzy1-4-C-hydroxymethyl-1,2-0-isopropylidene-a-D-
ribofuranose) was carried out according to the known
literature (Henrik M. Pfundheller, H. M., Bryld, T.,
Olsen, C. E., Wengel, J. Helvetica Chimica Acta 2000, 83,
128-151).
[0219]
3-0-benzy1-4-[(benzyloxy)methy1]-1,2-0-(1-
methylethylidene)-5-0-(trifluoromethylsulfony1)-VL-
lyxofuranose (compound 17)
Compound 2 (70.10 g, 174 mmol) was dissolved in
methylene chloride (700 mL), and thereto was added
pyridine (41.51 g, 524 mmol), and the mixtures were
cooled to -55 C as internal temperature. Thereto was
added Tf20 (78.93 g, 280 mmol) dropwise at -55 to -48 C
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as internal temperature over 30 minutes, and the mixtures
were stirred at the same temperature for 1 hour. The
cooling bath was replaced with an ice-cooling bath, and
the mixtures were stirred for additional 1 hour, and
thereto was added 8% aqueous sodium hydrogen carbonate
solution, and the organic layers were separated, and
washed with 8% aqueous sodium hydrogen carbonate solution
and saturated brine successively. Each of aqueous layers
was back-extracted with ethyl acetate, and the combined
organic layers were dried over anhydrous sodium sulfate,
and the insoluble materials were removed by filtration.
The solvents were evaporated under reduced pressure to
obtain compound 17 (92.96 g).
1H-NMR (CDC13): the data was identical to that
described in the literature.
[0220]
5-Azido-3-0-benzy1-4-[(benzyloxy)methy1]-5-deoxy-1,2-0-
(1-methylethylidene)-13-L-lyxofuranose (compound 18)
Under nitrogen stream, compound 17 (92.96 g, 174
mmol) was dissolved in DMF (465 mL). Thereto was added
NaN3 (45.42 g, 698 mmol) at room temperature, and the
mixtures were stirred at 60 C as external temperature for
4 hours and 30 minutes. After the mixtures were allowed
to cool, the mixtures were partitioned between ethyl
acetate and water. The organic layers were separated,
and washed with 8% aqueous sodium hydrogen carbonate
solution and saturated brine successively. Each of the
aqueous layers was back-extracted with ethyl acetate, and
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227
the combined organic layers were dried over anhydrous
sodium sulfate, and the insoluble materials were removed
by filtration. The solvents were evaporated under
reduced pressure to obtain crude product (75.70 g). The
crude product was purified by column chromatography (n-
hexane/ethyl acetate, 90/10 to 80/20) to obtain compound
18 (45.70 g, yield 60 %).
1H-NMR (CDC13): the data was identical to that
described in the literature.
[0221]
1,2-Di-O-acety1-5-azido-3-0-benzyl-4-[(benzyloxy)methyl]-
5-deoxy-L-lyxofuranose (compound 19)
Under nitrogen stream, compound 18 (45.70 g) was
dissolved in acetic anhydride (46 mL) and acetic acid (92
mL). Thereto was added concentrated sulfuric acid (280
uL) under ice-cooling, and the mixtures were stirred at
room temperature for 2 hours. The mixtures were diluted
with ethyl acetate (420 mL), and thereto was added
saturated aqueous sodium carbonate solution dropwise
under ice-cooling, and the organic layers were separated,
and washed with 8% aqueous sodium hydrogen carbonate
solution and saturated brine successively. Each of the
aqueous layers was back-extracted with ethyl acetate, and
the combined organic layers were dried over anhydrous
sodium sulfate, and the insoluble materials were removed
by filtration. The solvents were evaporated under
reduced pressure, and then azeotroped with toluene to
obtain compound 19 (50.72 g). The obtained compound was
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228
used without purification in next step as quantitative
yield.
114-NMR (CDC13): the data was identical to that
described in the literature.
[0222]
1-{2-0-Acetyl-5-azido-3-0-benzy1-4-[(benzyloxy)methyl]-5-
deoxy-a-L-lyxofuranosyll-5-methylpyrimidine-2,4(1H,3H)-
dione (compound 20)
Under nitrogen stream, compound 19 (50.72 g) was
dissolved in acetonitrile (445 mL), and thereto was added
thymine (26.56 g, 210 mmol). Thereto was added BSA
(171.86 g, 841 mmol) dropwise over 20 minutes at room
temperature, and the mixtures were stirred at 90 C as
external temperature for 1 hour. After the mixtures were
allowed to cool, thereto was added TMSOTf (46.96 g, 210
mmol) dropwise over 15 minutes under ice-cooling, and the
mixtures were stirred at 90 C as external temperature for
1 hour. After the mixtures were allowed to cool, thereto
was added 8% aqueous sodium hydrogen carbonate solution
under ice-cooling, and the participated solids were
filtered off, and acetonitrile was evaporated under
reduced pressure. Thereto was added ethyl acetate, and
the organic layers were separated and washed with
saturated brine. Each of the aqueous layers was back-
extracted with ethyl acetate, and the combined organic
layers were dried over anhydrous sodium sulfate, and the
insoluble materials were removed by filtration. The
solvents were evaporated under reduced pressure to obtain
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compound 20 (66.18 g). The obtained compound was used
without purification in next step as quantitative yield.
MS (ESI): m/z 536 (M+H)+
1H-NMR (CDC13): the data was identical to that
described in the literature.
[0223]
1-{5-Azido-3-0-benzy1-4-[(benzy1oxy)methyl]-5-deoxy-a-L-
lyxofuranosy1}-5-methylpyrimidine-2,4(1H,3H)-dione
(compound 21)
Compound 20 (66.18 g) was dissolved in THF (70 mL),
and azeotroped, and was then dissolved in THF (200 mL),
and thereto was added 4096 aqueous methyl amine solution
(132 mL) at room temperature under nitrogen stream.
After the mixtures were stirred at room temperature for 1
hour and 30 minutes, the solvents were evaporated under
reduced pressure. The concentrated residues were
partitioned among ethyl acetate, water, and saturated
brine, and the organic layers were separated and washed
with saturated brine. Each of the aqueous layers was
back-extracted with ethyl acetate, and the combined
organic layers were dried over anhydrous sodium sulfate,
and insoluble materials were removed by filtration. The
solvents were evaporated under reduced pressure to obtain
compound 21 (56.26 g). The obtained compound was used
without purification in next step as quantitative yield.
MS (ESI): m/z 494 (M+H)+
1H-NMR (CDC13) 6: 7.40-7.26 (12H, m), 5.88 (1H, d, J =
5.1 Hz), 4.74 (1H, d, J = 11.3 Hz), 4.64 (1H, d, J = 11.3
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230
Hz), 4.55 (2H, s), 4.40 (1H, t, J = 5.4 Hz), 4.26 (1H, d,
J = 5.7 Hz), 3.72 (1H, s), 3.69 (1H, d, J = 4.1 Hz), 3.56
(1H, d, J = 9.8 Hz), 3.43 (1H, d, J = 12.8 Hz), 2.81 (1H,
d, J = 5.1 Hz), 1.63 (3H, brd)
[0224]
(2R,3S,3aS,9aR)-2-(Azidomethyl)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-7-methy1-2,3,3a,9a-tetrahydro-6H-
furo[2',31:4,5] [1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 9)
Under nitrogen stream, compound 21 (56.26 g) was
dissolved in methylene chloride (935 mL), and thereto was
added DMAP (51.38 g, 420 mmol). The mixtures were
stirred for 30 minutes under ice-cooling, and thereto was
then added TfC1 (51.44 g, 210 mmol) dropwise over 25
minutes. The mixtures were stirred at room temperature
for 1 hour, and thereto was then added water under ice-
cooling. The organic layers were separated, and washed
with water, 0.1M hydrochloric acid, 4% aqueous sodium
hydrogen carbonate solution, and saturated brine
successively. Each of the aqueous layers was back-
extracted with ethyl acetate and the combined organic
layers were dried over anhydrous sodium sulfate, and the
insoluble materials were removed by filtration. The
solvents were evaporated under reduced pressure to obtain
compound 9 (52.10 g). The obtained compound was used
without purification in next step as quantitative yield.
MS (ESI): m/z 476 (M+H)'
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231
11-1-NMR (CDC13): the data was identical to that of
compound 9 of Examples 2 (DMAP was remained).
[0225]
1-((lR,3R,4R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-2-
oxa-5-azabicyclo[2.2.1]hepta-3-y11-5-methylpyrimidine-
2,4(1H,3H)-dione (compound 12)
Under nitrogen stream, compound 9 was dissolved in
THF (810 mL), and thereto was added water (160 mL).
Thereto was added Ph3P (29.79 g, 113.5 mmol) at room
temperature, and the mixtures were stirred at 70 C as
external temperature for 17 hours. After the mixtures
were allowed to cool, the solvents were evaporated under
reduced pressure, and azeotroped with toluene (200 mL x
2) to obtain crude product (167.79 g). The crude
product was purified by column chromatography (ethyl
acetate/methanol = 100/0 to 90/10 to 6/1 to 4/1) to
obtain crude compound 12 (42.78 g) containing Ph3P0 and
ethyl acetate. Under nitrogen stream, the crude compound
12 (42.78 g) was suspended in ethyl acetate (200 mL) in a
1L egg-plant shaped flask. The mixtures were stirred at
50 C as external temperature for 1 hour, and thereto was
then added diethyl ether (200 mL), and the mixtures were
stirred at 50 C as external temperature for 30 minutes.
After the mixtures were allowed to cool overnight, the
solids were collected by filtration, and washed with
ethyl acetate/diethyl ether = 1/1 (100 mL x 2), and dried
under reduced pressure to obtain compound 12 (30.11 g,
yield 70%: on the basis of compound 18).
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232
MS (ESI): m/z 450 (M+H)f
The data of 1H-NMR was identical to that of compound
12 obtained in Example 5 (CDC13).
[0226]
One of preparation method (hereinafter, sometimes
referred to as mesylation route) of compound 10 is
described.
[chem.109]
Ho Bc,0 wm Bno Ac2o BiO
O 8nBr;1 Et3N H2,504 7) õ0
_________________________________ Ms0/ A
0
_____________________________________________________ kid OAc
HO HOEinci.
CH2Cl2
DMF cOH
BnCi 134 134 tw.
2 22 23
0
thy:nine MeNH, TfCI 8n0
BSA
imS0Tf 6,0 NH ,n Me0H Bno <1 NH DMAP
___________________________________________________________ MsDAZ. 11.
MeCN Ms0/"V-4 CH202
MS0 Brld N
BnCf '0Ac Bnd 'OH
[0227]
Example 12 (Mesylation route)
The preparation of compound 24 from compound 2 was
carried out according to the known literature (Henrik M.
Pfundheller, H. M., Bryld, T., Olsen, C. E., Wengel, J.
Helvetica Chimica Acta 2000, 83, 128-151).
[0228]
3-0-Benzy1-4-[(benzyloxy)methy1]-1,2-0-(1-
methylethylidene)-5-0-(methylsulfony1)-13-L-lyxofuranose
(compound 22)
Pyridine method:
Under nitrogen atmosphere, compound 2 (1.00 g, 2.50
mmol) and pyridine (10 mL) were mixed, and thereto was
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233
added MsC1 (0.23 mL, 2.97 mmol) dropwise at 0 C. After
adding dropwise, the mixtures were stirred at the same
temperature for 1 hour, and then warmed to room
temperature, and stirred for additional 4 hours. To the
reaction solutions was added 10% hydrochloric acid, and
the mixtures were extracted with ethyl acetate. The
organic layers were dried over anhydrous sodium sulfate,
and filtered, and then concentrated. The crude product
was subjected to column chromatography (n-hexane/ethyl
acetate = 100/0 to 2/1) to obtain compound 22 (743 mg,
yield 62%).
1H-NMR (CDC13): the data was identical to that
described in the literature.
[0229]
Triethylamine method
Under nitrogen atmosphere, compound 2 (7.50 g, 18.7
mmol), methylene chloride (150 mL), and Et3N (7.83 mL,
56.2 mmol) were mixed, and thereto was added MsC1 (1.74
mL, 22.5 mmol) dropwise at 0 C. After adding dropwise,
the mixtures were warmed to room temperature, and the
mixtures were stirred for 2 hours. To the reaction
solutions was added water, and the mixtures were
extracted with methylene chloride. The organic layers
were dried over anhydrous sodium sulfate, filtered and
concentrated. The crude product was subjected to column
chromatography (n-hexane/ethyl acetate = 100/0 to 50/50)
to obtain compound 22 (8.20 g, yield 92%).
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234
1H-NMR (CDC13): the data was identical to that
described in the literature.
[0230]
1,2-Di-O-acety1-3-0-benzyl-4-[(benzyloxy)methyl]-5-0-
methylsulfony1)-L-lyxofuranose (compound 23)
Under nitrogen atmosphere, compound 22 (8.20 g, 17.1
mmol), acetic acid (140 mL), acetic anhydride (14 mL),
and concentrated sulfuric acid (0.14 mL) were mixed, and
the mixtures were stirred overnight. To the reaction
solutions was added ice water, and the mixtures were
extracted with ethyl acetate. The organic layers were
washed with saturated aqueous hydrogen carbonate solution,
dried over anhydrous sodium sulfate, filtered and
concentrated. The crude product was subjected to column
chromatography (n-hexane/ethyl acetate = 100/0 to 2/1) to
obtain compound 23 (5.45 g, yield 61%).
1H-NMR (CDC13): the data was identical to that
described in the literature.
[0231]
1-{2-0-Acety1-3-0-benzy1-4-[(benzyloxy)methyl]-5-0-
(methylsulfony1)-a-L-lyxofuranosy1}-5-methy1pyrimidine-
2,4(1H,3H)-dione (compound 24)
Under nitrogen atmosphere, compound 23 (5.15 g, 9.86
mmol) and acetonitrile (25 mL) were mixed, and thereto
was added thymine (1.67 g, 13.2 mmol) at 0 C, and thereto
was added BSA (5.20 mL, 21.3 mmol) dropwise slowly.
After adding dropwise, the mixtures were stirred at 50 C
for 3 hours. After the complete dissolution was
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235
confirmed, the mixtures were cooled to 0 C, and thereto
was added TMSOTf (1.90 mL, 10.5 mmol) slowly. The
reaction solutions were stirred at 50 C overnight.
Because the starting materials were not disappeared, the
reaction mixtures were cooled to 0 C, and thereto was
added TMSOTf (1.90 mL, 10.5 mmol) slowly. After adding
dropwise, the mixtures were stirred at 50 C for 4 hours.
To the reaction solutions was added saturated aqueous
sodium hydrogen carbonate solution, and the mixtures were
extracted with ethyl acetate. The organic layers were
saturated aqueous sodium hydrogen carbonate solution,
dried over anhydrous sodium sulfate, filtered and
concentrated. The crude product was subjected to column
chromatography (n-hexane/ethyl acetate = 100/0 to 50/50)
to obtain compound 24 (5.00 g, yield 86%).
11-1-NMR (CDC13): the data was identical to that
described in the literature.
[0232]
1-f3-0-Benzy1-4-[(benzyloxy)methyl]-5-0-(methylsulfony1)-
a-L-lyxofuranosy1}-5-methylpyrimidine-2,4(1H,3H)-dione
(compound 25)
Under nitrogen atmosphere, compound 24 (5.00 g, 8.49
mmol) and 7N ammonia methanol solution (60 mL) were mixed,
and the mixtures were stirred for 4 to 7 hours. The
reaction solutions were concentrated, and the crude
product was subjected to column chromatography (n-
hexane/ethyl acetate 100/0 to 30/70) to obtain compound
25 (4.12 g, yield 89%).
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MS (ESI): m/z 547 (M+H)+
1H-NMR (CDC13) 6: 9.08 (1H, brs), 7.39-7.26 (11H, m),
5.94 (1H, d, J = 5.0 Hz), 4.80 (1H, d, J = 11.4 Hz), 4.62
(1H, d, J = 11.4 Hz), 4.56-4.43 (4H, m), 4.35 (1H, d, J =
5.8 Hz), 4.29 (1H, d, J = 11.0 Hz), 3.78 (1H, d, J = 10.1
Hz), 3.66-3.61 (2H, m), 2.94 (3H, s), 1.59 (3H, d, J =
0.8 Hz)
[0233]
{(25,3S,3aS,9aR)-3-(Benzyloxy)-2-[(benzyloxy)methyl]-7-
methy1-6-oxa-2,3,3a,9a-tetrahydro-6H-
furo[2',31:4,5] [1,3]oxazolo[3,2-a]pyrimidin-2-yl}methyl
methanesulfonate (compound 10)
Under nitrogen atmosphere, compound 25 (4.10 g, 7.50
mmol), methylene chloride (80 mL) and DMAP (3.67 g, 30.0
mmol) were mixed, and thereto was added TfC1 (2.4 mL,
22.5 mmol) dropwise at 0 C. After adding dropwise, the
mixtures were warmed to room temperature, and stirred for
2 to 4 hours. To the reaction solutions was added water,
and the mixtures were extracted with methylene chloride.
The organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated. The crude product
was subjected column chromatography (chloroform/methanol
= 90/10) twice repeatedly to obtain compound 10 (3.20 g,
yield 8196).
MS (ESI): m/z 529 (M+H)
The data of 1H-NMR was identical to that of compound
obtained in the Example 3.
[0234]
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A preparation method of various nucleic acid base
compounds from thymine compound (compound 12) using a
nucleic acid base replacement method is described.
[chem.110]
NHBz OCONPh2
.N
Bn0 N en
BnO*0_)_. Nrj\r ________________________
)r_NH
i " NHCOPr
$..õ1
Bn0 NH
Bn0 rCIH Bn0 NH
26A 12 26G
N
BnO) Ø rNHBz
=ir-N 4_7"N
"
A ki
Bn0 NH Bn0 NH 0
26m6C 26C
[0235]
Example 13
Preparation of adenine (bz) (compound 26A) from thymine
compound (compound 12)
N-(9-{(1R,3R,4R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-
2-oxa-5-azabicyclo[2.2.1]hepta-3-y1)1-9H-purine-6-
yl)benzamide (compound 26A)
To the suspension of compound 12 (100 mg, 0.2225
mmol) and N-(9H-purin-6-yl)benzamide (159 mg, 0.6674
mmol) in 1,2-dichloroethane (2 mL) was added BSA (0.49 mL,
2.002 mmol), and the mixtures were stirred at 60 C as
external temperature for 15 minutes. After the mixtures
were allowed to cool at room temperature for 15 minutes,
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thereto was added TMSOTf (13 pL, 0.067 mmol), and the
mixtures were stirred at 60 C as external temperature
again for 15 minutes. To the reaction solutions were
added chloroform (10 mL), saturated brine (2 mL), and
saturated aqueous sodium bicarbonate solution (2 mL)
under ice-cooling, and the mixtures were stirred at room
temperature for 10 minutes. The organic layers were
passed through a phase separator. After the solvents
were evaporated, the resulting residues were purified by
silica gel chromatography (chloroform/methanol, 96/4 to
90/10) to obtain compound 26A (119 mg, containing 1.0wt%
of ethyl acetate, yield 95%). Here compound 26A was .13
form", and "a form" as an isomer could not be obtained.
MS (APCI): m/z = 563 (M+H)*
1H-NMR (CDC13) 6: 8.91 (1H, s), 8.77 (1H, s), 8.34 (1H,
s), 8.06-8.01 (2H, m), 7.65-7.59 (1H, m), 7.57-7.51 (2H,
m), 7.40-7.23 (8H, m), 7.22-7.17 (2H, m) , 6.02 (1H, s),
4.67 (1H, d, J = 12.3 Hz), 4.62 (1H, d, J = 12.3 Hz),
4.53 (1H, d, J = 11.8 Hz), 4.50 (1H, d, J = 11.8 Hz),
4.16 (1H, s), 3.89 (1H, s), 3.84 (1H, d, J = 10.8 Hz),
3.79 (1H, d, J = 10.8 Hz), 3.22 (11-1, d, J = 10.3 Hz),
3.02 (1H, d, J = 10.3 Hz), 2.21 (1H, brs)
[0236]
Example 14
Preparation of guanine (ib, dpc) (compound 26G) from
thymine compound (compound 12)
9-{(1R,3R,4R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methy1]-2-
oxa-5-azabicyclo[2.2.1]hepta-3-y1)-2-[(2-
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methylpropanoyl)amino]-9H-purin-6-y1 diphenylcarbamate
(compound 26G)
Similarly to Example 13 using compound 12 (1000 mg,
2.225 mmol) and [2-(2-methylpropanoylamino)-9H-purin-6-
yl] N,N-diphenylcarbamate (2779 mg, 6.674 mmol) (the
preparation method follows the method described in J. Org.
Chem. 1996, 61, 9207), compound 26G (1353 mg, yield 82%)
was prepared. Here compound 26G was "p form", and "a
form" as an isomer could not be obtained.
MS (APCI): m/z = 740 (M+H)+
1H-NMR (CDC13) 6: 8.26 (1H, s), 7.90 (1H, s), 7.52-7.13
(20H, m), 5.92 (1H, s), 4.65 (1H, d, J = 12.3 Hz), 4.61
(1H, d, J = 12.3 Hz), 4.50 (2H, s), 4.10 (1H, s), 3.89
(1H, s), 3.81 (1H, d, J = 10.8 Hz), 3.76 (1H, d, J = 10.8
Hz), 3.19 (1H, d, J = 10.3 Hz), 3.04 (1H, brd), 2.98 (1H,
d, J = 10.3 Hz), 1.26 (6H, d, J = 6.7 Hz)
[0237]
Example 15
Preparation of methylcytosine (bz) (compound 26meC) from
thymine compound (compound 12)
N-(1-{(1R,3R,4R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methyll-
2-oxa-5-azabicyclo[2.2.1]hepta-3-y1}-5-methy1-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (compound 26mC)
To a solution of compound 12 in acetonitrile (6.7
mL) were added N-(5-methy1-2-oxo-1H-pyrimidin-4-
yl)benzamide (the preparation method follows the method
described in European Journal of Organic Chemistry, 2006,
3152; 431 mg, 2.002 mmol) and BSA (1.47 mL, 6.00 mmol),
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and the mixtures were stirred at 60 C as external
temperature for 15 minutes. After the mixtures were
allowed to cool to room temperature, thereto was added
TMSOTf (39 pL, 0.200 mmol) dropwise, and the mixtures
were stirred at 60 C as external temperature again for
150 minutes. The mixtures were cooled under ice-cooling,
and thereto were added saturated aqueous sodium
bicarbonate solution and chloroform successively, and the
mixtures were separated by a separatory funnel. The
aqueous layers were extracted with chloroform twice, and
the organic layers were washed with saturated brine, and
dried over sodium sulfate. The insoluble materials were
removed by filtration, and the solvents were removed by
filtration, and the solvents were evaporated, and the
resulting residues were purified by silica gel
chromatography (chloroform/methanol, 100/0 to 95/5) to
obtain compound 26"C (71 mg, yield 19%).
Here compound 26"C was "p form", and "a form" as an
isomer could not be obtained.
MS (APCI): m/z = 553 (M+H)+
1H-NMR (CDC13) 6: 13.38 (1H, brs), 8.36-8.27 (214, m),
7.81-7.75 (1H, m), 7.58-7.21 (1314, m), 5.57 (114, s),
4.68-4.50 (4H, m), 3.92 (1H, s), 3.89 (1H, d, J = 11.3
Hz), 3.81(1H, d, J = 11.3 Hz), 3.71 (11-1, s), 3.16 (114, d,
J = 9.8 Hz), 2.91 (1H, d, J = 9.8 Hz), 1.80-1.75 (3H, m)
[0238]
Example 16
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N-(1-1(1R,3R,4R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-
2-oxa-5-azabicyclo[2.2.1]hepta-3-y11-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (compound 260)
Similarly to Example 13 from compound 12 (20.0 mg,
0.0445 mmol) and N-(2-oxo-1H-pyrimidin-4-yl)benzamide
(28.7 mg, 0.134 mmol), compound 260 (10.4 mg, yield 43%)
was prepared. Here compound 260 was "p form", and "a
form" as an isomer could not be obtained.
MS (APCI): m/z = 539 (M+H)+
1H-NMR (CDC13) 15: 8.64 (1H, brs), 8.28 (1H, d, J = 7.2
Hz), 7.90 (2H, d, J = 7.7 Hz), 7.62 (1H, m), 7.53 (2H, m),
7.45-7.20 (11H, m), 5.66 (1H, s), 4.66 (1H, d, J = 11.8
Hz), 4.62 (1H, d, J = 11.8 Hz), 4.55 (1H, d, J = 11.8 Hz),
4.47 (1H, d, J = 11.8 Hz), 3.87 (2H, s), 3.84 (1H, d, J =
10.8 Hz), 3.79 (1H, d, J = 10.8 Hz), 3.16 (1H, d, J = 9.8
Hz), 2.95 (1H, d, J = 9.8 Hz)
[0239]
The preparation method of various nucleic acid base
compounds from thymine compound (compound 14) using a
nucleic acid base replacement method is described.
[chem.111]
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NHBz OCONPh2
R5.0
r-c0 N
R5D*N
R5O*0
N
1 N---CNHCO'Pr
RY0--tqH
R3=P'r11-1 123'0r'KIH
, __ 28A: R TMS, R 5 DMTr 14: R = H. R DMIr , 28G:
R TMS, RDMTr 5 =
> 29k S3 = H, R 5 = DMTr __________________ v 29G: R H, H, R = DMTr
N
R5'0 NHSz R5. NHBzJJ 0
N 0
N
Ive-AH
____ 2841*C: 53 - TMS. R 28C: R 3' = TMS, R = DMTr
> 29meC: R - H, R I - DMTr 29C: R3. = R = Divirr
[0240]
Example 17
Preparation of adenine (bz) (compound 28A) from thymine
compound (compound 14)
N-(9-{(1R,3R,4R,75)-1-{[Bis(4-
methoxyphenyl)(phenyl)methoxy]methyll-7-
[(trimethylsilyl)oxy]-2-oxa-5-azabicyclo[2.2.1]hepta-3-
y1}-9H-purin-6-yl)benzamide (compound 28A)
Similarly to Example 13 using compound 14 (1.50 g,
2.60 mmol) and BSA (6.40 mL, 26.0 mmol), compound 28A
(1.92 g, yield 97%) was obtained. Here compound 28A was
.13 form", and "a form" as an isomer could not be obtained.
MS (APCI): m/z = 757 (M+H)+
1H-NMR (CDC13) 6: 9.07(1H, s), 8.82 (1H, s), 8.42 (IH, s),
8.09-8.01 (2H, m), 7.66-7.19 (12H, m), 6.90-6.80 (4H, m),
6.05 (1H, s), 4.40 (1H, s), 3.89 (1H, s), 3.80 (6H, s),
3.46 (1H, d, J = 10.8 Hz), 3.32 (1H, d, J = 10.8 Hz),
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3.11 (1H, d, J = 10.3 Hz), 2.99 (1H, d, J - 10.3 Hz), -
0.02 (9H, s)
[0241]
Example 18
Desilylation of adenine (bz): (compound 29A)
N-(9-{(1R,3R,4R,7S)-1-{[Bis(4-
methoxyphenyl ) (phenyl) me thoxy] methyl} -7 -hydroxy-2 - oxa -5 -
azabicyclo [2.2.11hepta-3-y1} -9H-purin-6-y1) benzamide
(compound 29A)
Compound 28A (110 mg, 0.1453 mmol) and THF (2 mL)
were mixed, and thereto was added TBAF (0.19 mL, 0.1889
mmol) dropwise under ice-cooling, and the mixtures were
stirred at the same temperature for 50 minutes. To the
reaction solutions were chloroform (10 mL) and saturated
brine (2 mL), and the mixtures were stirred and extracted.
The organic layers were washed with saturated brine (2
mL) and then dried over sodium sulfate. The insoluble
materials were removed by filtration, and the solvents
were evaporated, and the resulting residues were then
purified by silica gel chromatography
(chloroform/methanol, 100/0 to 91/9) to obtain compound
29A (96.5 mg, containing 1.4wt% of ethyl acetate, yield
97%).
MS (APCI): m/z = 685 (M+H)
1H-NMR (CDC13) 6: 9.16 (1H, s), 8.78 (1H, s), 8.33 (IH,
s), 8.08-7.99 (2H, m), 7.65-7.21 (12H, m), 6.89-6.81 (4H,
m), 6.05 (1H, s), 4.31 (1H, s), 3.90 (1H, s), 3.80 (6H,
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s), 3.59 (1H, d, J = 10.8 Hz), 3.52 (1H, d, J = 10.8 Hz),
3.16 (1H, d, J = 10.8 Hz), 3.09 (1H, d, J = 10.8 Hz)
[0242]
Example 19
Preparation of guanine (lb, dpc) (compound 28G) from
thymine compound (compound 14)
9-{(1R,3R,4R,75)-1-{[Bis(4-
methoxyphenyl)(phenyl)methoxy]methyl}-7-
[(trimethylsilyl)oxy]-2-oxa-5-azabicyclo[2.2.1]hepta-3-
y1}-2-[(2-methylpropanoyl)amino]-9H-purin-6-yl
diphenylcarbamate (compound 28G)
To a suspension of compound 14 (200 mg, 0.3499 mmol)
and [2-(2-methylpropanoylamino)-9H-purin-6-yl] N,N-
diphenylcarbamate (437 mg, 1.050 mmol) in 1,2-
dichloroethane (4 mL) was added BSA (0.856 mL, 3.499
mmol) at room temperature, and the mixtures were stirred
at 60 C as external temperature for 15 minutes. Thereto
was added TMSOTf (20 pL, 0.1050 mmol) dropwise under ice-
cooling, and the mixtures were stirred at 60 C as
external temperature for 15 minutes. Thereto were added
saturated aqueous sodium bicarbonate solution, saturated
aqueous ammonium chloride solution, saturated sodium
bicarbonate solution, saturated brine, and chloroform
under ice-cooling, and the mixtures were stirred for 10
minutes. The insoluble materials were removed by
filtration through Celite, and the filtrates were passed
through a phase separator. After the solvents were
evaporated, the resulting residues were purified by
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silica gel chromatography (chloroform/methanol, 99/1 to
96/4) to obtain compound 28G (295 mg, containing 4.1wt%
of ethyl acetate, and 1.9wt% of acetamide derived from
BSA, yield 90%). Here compound 28G was "p form", and "a
form" as an isomer could not be obtained.
MS (APCI): m/z = 934 (M+H)4
1H-NMR (CDC13) 5: 8.37 (1H, s), 7.95 (1H, s), 7.52-7.20
(19H, m), 6.88-6.82 (m, 4H), 5.99 (1H, s), 4.29 (1H, s),
3.84 (1H, s), 3.79 (6H, s), 3.46 (1H, d, J = 10.8 Hz),
3.29 (1H, d, J = 10.8 Hz), 3.14-3.00 (1H, m),
3.08 (1H,
d, J = 10.2 Hz), 2.97 (1H, d, J = 10.2 Hz), 1.28 (3H, d,
J = 6.7 Hz), 1.27 (3H, d, J = 6.7 Hz)
[0243]
Example 20
Desilylation of guanine (ib, dpc): (compound 29G)
9-[(1R,3R,4R,7S)-1-{[Bis(4-
methoxyphenyl)(phenyl)methoxy]methy11-7-hydroxy-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y1]-2-[(2-
methylpropanoyl)amino]-9H-purin-6-y1 diphenylcarbamate
(compound 29G)
Similarly to Example 18 from compound 28G (210 mg,
0.2182 mmol), compound 29G (163 mg, yield 87%) was
prepared.
MS (APCI): m/z = 862 (M+H)
1H-NMR (CDC13) 6: 8.20 (1H, s), 8.08 (1H, s), 7.49-7.18
(19H, m), 6.85-6.80 (m, 4H), 5.93 (1H, s), 4.48 (1H, s),
4.01 (1H, s), 3.77 (6H, s), 3.49 (1H, d, J = 10.8 Hz),
3.45 (1H, d, J = 10.8 Hz), 3.19 (1H, d, J = 10.8 Hz),
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3.13 (1H, d, J = 10.8 Hz), 2.80 (1H, brs), 1.24 (3H, d, J
= 6.7 Hz), 1.23 (3H, d, J = 6.7 Hz)
[0244]
Example 21
Preparation of methylcytosine (bz) (compound 28MeC) from
thymine compound (compound 14)
N-(1-{(1R,3R,4R,7S)-1-{[Bis(4-
me thoxyphenyl ) (phenyl) me thoxy] methyl } - 7 -
[ (trimethylsilyl ) oxy] -2- oxa -5- a zabicyclo [2.2.1] hepta -3-
y1}-5-methy1-2-oxo-1,2-dihydropyrimidin-4-y1)benzamide
(compound 28meC)
To a suspension of compound 14 (200 mg, 0.3499 mmol)
and N-(5-methy1-2-oxo-1H-pyrimidin-4-yl)benzamide (241 mg,
1.050 mmol) in 1,2-dichloroethane (3.5 mL) was added BSA
(0.77 mL, 3.149 mmol), and the mixtures were stirred at
40 C as external temperature for 15 minutes. Thereto was
added TMSOTf (10 pL, 0.05248 mmol) under ice-cooling, and
the mixtures were stirred at 40 C as external temperature
again for 15 minutes. To the reaction solutions were
added ethyl acetate (10 mL) and saturated aqueous sodium
bicarbonate solution (2 mL) under ice-cooling, and the
mixtures were stirred and extracted. The organic layers
were washed with saturated brine (2 mL) and dried over
sodium sulfate. The insoluble materials were removed by
filtration, and the solvents were evaporated from the
filtrates, and the resulting residues were then purified
by silica gel chromatography (chloroform/methanol, 100/0
to 95/5) to obtain compound 2811eC (176 mg, yield 67%).
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247
Here compound 28meC was .13 form", and "a form" as an
isomer could not be obtained.
MS (APCI): m/z - 747 (M+H)
1H-NMR (CDC13) 5: 13.44 (1H, brs), 8.36-8.29 (2H, m),
8.05 (1H, s), 7.58-7.21 (12H, m), 6.90-6.80 (4H, m), 5.58
(1H, s), 4.27 (1H, s), 3.81 (6H, s), 3.59 (1H, s), 3.53
(1H, d, J = 10.8 Hz), 3.23 (1H, d, J = 10.8 Hz), 2.97
(1H, d, J = 9.8 Hz), 2.78 (1H, d, J = 9.8 Hz), 1.87 (3H,
s), 0.06 (9H, s)
[0245]
Example 22
Desilylation of methylcytosine (bz): (compound 29meC)
N-(1-{(1R,3R,4R,7S)-1-{[Bis(4-
methoxypheny1)(phenyl)methoxylmethyl}-7-hydroxy-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y1}-5-methyl-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (compound 29meC)
Similarly to Example 18 from compound 28meC (1124 mg,
1.5049 mmol), compound 29meC (1001 mg, yield 999) was
prepared.
MS (APCI): m/z = 675 (M+H)+
1H-NMR (CDC13) 5: 13.40 (1H, brs), 8.36-8.24 (2H, m),
7.91-7.86 (1H, m), 7.56-7.20 (12H, m), 6.92-6.79 (4H, m),
5.56 (1H, s), 4.22 (1H, s), 3.85-3.77 (6H, m), 3.68 (1H,
s), 3.56 (1H, d, J . 10.8 Hz), 3.46 (1H, d, J = 10.8 Hz),
2.97 (1H, d, J = 10.3 Hz), 2.93 (1H, d, J = 10.3 Hz),
1.87-1.84 (3H, m)
[0246]
Example 23
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Preparation of cytosine (bz) (compound 28C) from thymine
compound (compound 14)
N-(1-{(1R,3R,4R,75)-1-{[Bis(4-
methoxyphenyl)(phenyl)methoxy]methyll-7-
[(trimethylsilyloxy1-2-oxa-5-azabicyclo[2.2.1]hepta-3-
y1}-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (compound
28C)
Similarly to Example 21 from compound 14 (200 mg,
0.350 mmol) and N-(2-oxo-1H-pyrimidin-4-yl)benzamide (226
mg, 1.05 mmol), compound 28C (101 mg, yield 39 96.) was
prepared. Here compound 28C was Hp form", and "a form"
as an isomer could not be obtained.
MS (APCI): m/z= 733 (M+H)4
1H-NMR (CDC13) 6: 8.61 (1H, d, J = 7.7 Hz), 7.94-7.89 (2H,
m), 7.66-7.25 (13H, m), 6.92-6.86 (4H, m), 5.70 (1H, s),
4.22 (1H, s), 3.85 (6H, s), 3.70 (1H, s), 3.53 (1H, d, J
= 10.8 Hz), 3.32 (1H, d, J = 10.8 Hz), 3.00 (1H, d, J =
9.8 Hz), 2.82 (1H, d, J = 9.8 Hz), 0.02 (9H, s)
[0247]
Example 24
Desilylation of cytosine (bz): (compound 29C)
N-(1-{(1R,3R,4R,7S)-1-{[Bis(4-
methoxyphenyl)(phenyl)methoxy]methy1}-7-hydroxy-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y11-2-oxo-1,2-dihydropyrimidin-
4-yl)benzamide (compound 29C)
Similarly to Example 18 from compound 28C (101 mg,
0.133 mmol), compound 29C (86.0 mg, yield 98%) was
prepared.
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MS (APCI): m/z= 661 (M+H)+
1H-NMR (CDC13) 5: 8.48 (1H, d, J - 7.7 Hz), 7.82-7.76 (2H,
m), 7.60-7.19 (13H, m), 6.93-6.82 (4H, m), 5.63 (1H, s),
4.24 (1H, s), 3.87 (1H, s), 3.81 (6H, s), 3.53 (1H, d, J
= 10.8 Hz), 3.44 (1H, d, J =10.8 Hz), 3.01 (1H, d, J =
10.3 Hz), 2.88 (1H, d, J = 10.3 Hz)
[0248]
[chem.112]
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250
s-'
(pr),N, CI DMTrO
RP:N -"L'N-RP"' DMTrO P'
DMTrO, H
b..., õ..-õ, 6 O8
Ag0Tf, Et3N NC- -,---
B NC
, --\....0tB
rt _______ THF, 0 `C to rt - .- .
Hd.'--t1 Or
iiH N - RP') ('Pr)2N N(Pr)2 (Pr)2t( N-IRPt
R -NN Rv -NH
29 15 16
29 15 16
Compound
\
DMTrO
- DMTrO).0)...8 DMTrO
,,1
0
NC--\_/,
Ring B \
HCi NH FiCr'N
Rf"-NH N-RPru (pr)2uN, z..... a
RP' NRPra
= Teoc : R'' = Soc RV = TeocR
:
'' h1
=Bo-o
RPT
i
NHBz
A ,I, N
29A 15Aa 15Ab 16Aa 16Ab
N I .)
,..".., N--
OCONPh2
G N_]r\--
29G 15Ga 15Gb 16Ga 16Gb
N
õ,.,, J,
" NHCO'Pr
NHBz 29MeC 1544eCa 15MeCb 16meCa 16meCb
mec v, 4
0
---/M¨NHEiz 290 15Ca 15Cb -16Ca 16Cb
C
o
[0249]
Example 25
Guanidination (teoc) of adenine (bz): (compound 15Aa)
Bis[2-(trimethylsilyl)ethyl] {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methy11-7-hydroxy-3-(6-
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[(phenylcarbonyl)amino]-9H-purin-9-y11-2-oxa-5-
azabicyclo[2.2.1]hepta-5-yl]methylidenelbiscarbamate
(compound 15Aa)
Similarly to Example 8 from compound 29A (91.5 mg,
0.134 mmol), compound 15Aa (111 mg, containing 1.1wt% of
ethyl acetate, yield 82%) was prepared.
MS(APCI): m/z = 1015 (M+H)+
H-NMR (CDC13) 5: 10.66 (1H, brs), 9.01 (1H, s), 8.77 (1H,
s), 8.25 (1H, s), 8.08-7.98 (2H, m), 7.67-7.40 (5H, m),
7.39-7.17 (7H, m), 6.90-6.78 (4H, m), 6.28 (1H, s), 5.13
(1H, brs), 4.63-4.54 (1H, m), 4.32-4.15 (4H, m), 3.96 (1H,
d, J= 11.3 Hz), 3.87-3.74 (7H, m), 3.62-3.50 (2H, m),
3.31-3.09 (1H, m), 1.16-0.98 (4H, m), 0.04 (18H, s)
[0250]
Example 26
Phosphorylation (teoc) of adenine (bz) (compound 16Aa)
Bis[2-(trimethylsilyl)ethyl] {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methy1}-7-1[(2-
cyanoethoxy)(dipropan-2-ylamino)phosphanylloxy}-3-16-
[(phenylcarbonyl)amino]-9H-purin-9-y1}-2-oxa-5-
azabicyclo[2.2.1]hepta-5-yl]methylidenelbiscarbamate
(compound 16Aa)
Similarly to Example 9 from compound 15Aa (104.8 mg,
0.1032 mmol), compound 16Aa (66.8 mg, yield 53%) was
prepared.
MS (APCI): m/z = 1215 (M+H)
31P-NMR (CDC13) 5: 149.67, 149.39, 149.34, 148.01
[0251]
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Example 27
Guanidination (teoc) of guanine (ib, dpc): (compound
15Ga)
Bis[2-(trimethylsilyl)ethyl] 1[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methy1}-3-{6-
[(diphenylcarbamoyl)oxy]-2-[(2-methylpropanoyl)amino]-9H-
purin-9-y1}-7-hydroxy-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yl]methylidene}biscarbamate (compound 15Ga)
Similarly to Example 8 from compound 29G (94.4 mg,
0.110 mmol), compound 15Ga (114 mg, yield 84%) was
prepared.
MS (APCI): m/z = 1192 (M+H)
1H-NMR (CDC13) 5: 10.87 (1H, brs), 8.12 (1H, s), 8.03 (1H,
s), 7.52-7.33 (11H, m), 7.32-7.13 (8H, m), 6.81-6.75 (4H,
m), 6.24 (IH, brs), 5.18 (1H, s), 4.99 (1H, brs), 4.26-
4.03 (6H, m), 3.93-3.81 (1H, m), 3.76 (3H, s), 3.76 (3H,
s), 3.45 (1H, d, J = 10.8Hz), 3.38 (1H, d, J = 10.8Hz),
2.65-2.52 (1H, m), 1.22 (3H, d, J = 7.2Hz), 1.17 (3H, d,
J = 6.7Hz), 1.13-0.87 (4H, m), 0.03 (9H, brs), -0.01 (9H,
brs)
[0252]
Example 28
Phosphorylation (teoc) of guanine (ib, dpc): (compound
16Ga)
Bis[2-(trimethylsilyl)ethyl] {[(1R,3R,4R,75)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxylmethy11-7-{[(2-
cyanoethoxy)(dipropan-2-ylamino)phosphanyl]oxy}-3-{6-
[(diphenylcarbamoyl)oxy]-2-[(2-methylpropanoyl)amino]-9H-
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purin-9-y1}-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yl]methylidenelbiscarbamate (compound 16Ga)
To a solution of compound 15Ga (50.5 mg, 0.0423
mmol) in acetonitrile (1 mL) were added diisopropyl
ammonium tetrazolide (10.9 mg, 0.0635 mmol), 2-cyanoethyl
N,N,N',N'-tetraisopropylphosphorodiamidite (19.1 mg,
0.0635 mmol) and 2-cyanoethyl N,N,N',N'-
tetraisopropylphosphorodiamidite (19.1 mg, 0.0635 mmol)
at room temperature, and the mixtures were stirred at
room temperature for 1 hour. Thereto were added further
diisopropyl ammonium tetrazolide (21.8 mg, 0.127 mmol)
and 2-cyanoethyl N,N,N',N'-
tetraisopropylphosphorodiamidite (38.3 mg, 0.127 mmol),
and the mixtures were stirred at room temperature for 3
hours. Thereto was added further 2-cyanoethyl N,N,N1,N1-
tetraisopropylphosphorodiamidite (6.4 mg, 0.0212 mmol),
and the mixtures were stirred at room temperature for 20
minutes. Thereto were added chloroform (5 mL) and
saturated brine (2 mL) under ice-cooling, and the
mixtures were stirred and passed through a phase
separator. After the solvents were evaporated, the
mixtures were purified by silica gel chromatography
(hexane/ethyl acetate, 80/20 to 60/40) to obtain compound
16Ga (61.4 mg, yield 83%).
MS (APCI): m/z= 1392 (M1-1-1)+
P-NMR (CDC13) 5: 149.16, 148.68
[0253]
Example 29
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Guanidination (teoc) of methylcytosine (bz): (compound
is Ca)
Bis[2-(trimethylsilyl)ethyl] {[(1R,3R,4R,7S)-1-Ohis(4-
methoxyphenyl)(phenyl)methoxy]methyll-7-hydroxy-3-{5-
methy1-2-oxo-4-[(phenylcarbonyl)amino]pyrimidin-1(2H)-
y11-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yl]methylidene}biscarbamate (compound 15" Ca)
2-Trimethylsilylethyl N-M1R,4R,6R,7S)-6-(4-benzamide-5-
methy1-2-oxo-pyrimidin-l-y1)-4-((bis(4-methoxypheny1)-
phenylmethoxy)methyl)-7-hydroxy-5-oxa-2-
azabicyclo(2.2.1)hepta-2-y1)-(2-
trimethylsilylethoxycarbonylamino)methylene)carbamate
(compound 15meCa)
Similarly to Exmaple 8 from compound 29meC (890 mg,
1.319 mmol), compound 15meCa (870 mg, yield 66%) was
prepared.
MS (APCI): m/z= 1005 (M+H)+
1H-NMR (CDC13) 5: 13.41 (1H, brs), 10.37 (1H, brs), 8.37-
8.25 (2H, m), 7.75 (1H, s), 7.58-7.21 (12H, m), 6.93-6.81
(4H, m), 5.67 (1H, s), 4.76 (1H, s), 4.36-4.12 (5H, m),
3.86-3.73 (7H, m), 3.63 (1H, d, J = 11.7 Hz), 3.56 (1H, d,
J = 11.3 Hz), 3.52 (1H, d, J = 11.3 Hz), 1.87 (3H, s),
1.11-1.00 (4H, m), 0.04 (18H, s)
[0254]
Example 30
Phosphorylation (teoc) of methylcytosine (bz): (compound
16M0 Ca)
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255
Bis[2-(trimethylsilyl)ethyl] {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methy11-7-{[(2-
cyanoethoxy)(dipropan-2-ylamino)phosphanyl]oxyl-3-{5-
methyl-2-oxo-4-[(phenylcarbonyl)amino]pyrimidin-1(2H)-
y1}-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yl]methylidene}biscarbamate (compound 16" Ca)
Similarly to Example 9 from compound 15'eCa (50 mg,
0.0497 mmol), compound 16meCa (31 mg, yield 52%) was
prepared.
MS (APCI): m/z= 1205 (M+H)+
P-NMR (CDC13) 5: 149.66, 149.44, 149.03. 148.87
[0255]
Example 31
Guanidination (boc) of adenine (bz): (compound 15Ab)
Di-tert-butyl {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methy1}-7-hydroxy-3-(6-
[(phenylcarbonyl)amino]-9H-purin-9-y11-2-oxa-5-
azabicyclo[2.2.1]hepta-5-yl]methylidenelbiscarbamate
(compound 15Ab)
Similarly to Example 10 from compound 29A (1.74 g,
2.54 mmol), compound 15Ab (1.735 g, containing 2.0wt% of
hexane, yield 74%) was prepared.
MS(APCI): m/z= 927 (M+H)-'
1H-NMR (CDC13) 5: 10.46 (1H, brs), 9.08 (1H, s), 8.76 (1H,
s), 8.26 (1H, s), 8.06-7.99 (2H, m), 7.66-7.18 (12H, m),
6.88-6.80 (41-i, m), 6.22 (1H, s), 5.21 (1H, s), 4.50 (1H,
s), 3.95-3.81 (2H, m), 3.79 (6H, s), 3.58-3.49 (21-1, m),
3.47-3.37 (1H, m), 1.65 (9H, s), 1.59-1.40 (91-1, m)
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256
[0256]
Example 32
Phosphorylation (boc) of adenine (bz): (compound 16)
Di-tert-butyl {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxylmethy11-7-1[(2-
cyanoethoxy)(dipropan-2-ylamino)phosphanyl]oxy1-3-{6-
[(phenylcarbonyl)amino]-9H-purin-9-y1)-2-oxa-5-
azabicyclo[2.2.1]hepta-5-yl]methylidene)biscarbamate
(compound 16Ab)
Similarly to Example 9 from compound 15Ab (100 mg,
0.1079 mmol), compound 16Ab (105 mg, yield 86%) was
prepared.
MS (APCI): m/z=1127 (M+H)+
'P-NMR (CDC13) 6: 149.39, 149.23
[0257]
Example 33
Guanidination (boc) of guanine (ib, dpc): (compound 15Gb)
Di-tert-butyl {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methyl)-3-{6-
[(diphenylcarbamoyl)oxy]-2-[(2-methylpropanoyl)amino]-9H-
purin-9-y11-7-hydroxy-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yl]methylidene)biscarbamate (compound 15Gb)
Similarly to Example 10 from compound 290 (77.8 mg,
0.0903 mol), compound 15Gb (83.2 mg, yield 82%) was
prepared.
MS (APCI): m/z=1104 (M+H)
'H-NMR (CDC13) 6: 10.56 (1H, s), 8.13 (IH, s), 8.01 (1H,
s), 7.49-7.33 (11H, m), 7.32-7.14 (8H, m), 6.77-6.80 (4H,
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257
m), 6.17 (11-I, brs), 5.10 (IH, brs), 4.14-4.00 (2H, m),
3.89-3.81 (1H, m), 3.76 (3H, s), 3.76 (3H, s), 3.50-3.36
(2H, m), 2.73-2.58 (1H, m), 1.55-1.32 (18H, m), 1.22 (3H,
d, J =7.2Hz), 1.17 (3H, d, J =7.2Hz)
[0258]
Example 34
Phosphorylation (boc) of guanine (ib, dpc): (compound
16Gb)
Di-tert-butyl {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxylmethyl}-7-{[(2-
cyanoethoxy)(dipropan-2-ylamino)phosphanyl]oxy}-3-{6-
[(diphenylcarbamoyl)oxy]-2-[(2-methylpropanoyl)amino]-9H-
purin-9-y11-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yllmethylidenelbiscarbamate (compound 16Gb)
Similarly to Example 28 from compound 15Gb (41.8 mg,
0.0379 mmol), compound 16Gb (43.8 mg, yield 84%) was
prepared.
MS (APCI): m/z=1304 (M+H)+
31P-NMR (CDC13) 6: 148.92, 148.80, 148.36
[0259]
Example 35
Guanidination (boc) of methylcytosine (bz): (compound
15"Cb)
Di-tert-butyl {[(1R,3R,4R,75)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methy11-7-hydroxy-3-{5-
methyl-2-oxo-4-[(phenylcarbonyl)amino]pyrimidin-1(2H)-
y1}-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yllmethylidenelbiscarbamate (compound 15MeCb)
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258
Similarly to Example 10 from compound 29meC (1762 mg,
2.612 mmol), compound 15meCb (1467 mg, yield 61%) was
prepared.
MS (APCI): m/z = 917 (M+H)
1H-NMR (CDC13) 6: 13.39 (1H, brs), 10.11 (1H, brs), 8.38-
8.26 (2H, m), 7.77 (1H, s), 7.57-7.21 (12H, m), 6.91-6.80
(4H, m), 5.62 (1H, s), 4.71 (1H, s), 4.34 (1H, s), 3.85-
3.75 (7H, m), 3.58 (1H, d, J - 11.3 Hz), 3.54 (1H, d, J =
11.3 Hz), 3.49 (1H, d, J = 11.3 Hz), 1.88 (3H, s), 1.53-
1.45 (18H, m)
[0260]
Example 36
Phosphorylation (boc) of methylcytosine (bz): (compound
16meCb)
Di-tert-butyl {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxylmethyl}-7-{[(2-
cyanoethoxy)(dipropan-2-y1amino)phosphany1loxyl-3-(5-
methy1-2-oxo-4-[(phenylcarbonyl)amino]pyrimidin-1(2H)-
y1)-2-oxa-5-azabicyclo[2.2.1]hepta-5-
yl]methylidyne}biscarbamate (compound 16meCb)
Similarly to Example 9 from compound 15meCb (142 mg,
0.1549 mmol), compound 16meCb (85.0 mg, yield 49%) was
prepared.
MS (APCI): m/z= 1117 (M+H)+
31P-NMR (CDC13) 6: 149.48, 149.08, 148.56
[0261]
Example 37
Guanidination (boc) of cytosine (bz): (compound 15Cb)
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259
Di-tert-butyl {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxylmethy11-7-hydroxy-3-{2-oxo-
4-[(phenylcarbonyl)amino]pyrimidin-1(2H)-y1)-2-oxa-5-
azabicyclo[2.2.1]hepta-5-yl]methylidenelbiscarbamate
(compound 15Cb)
Similarly to Example 10 from compound 29C (86 mg,
0.130 mmol), compound 15Cb (91 mg, yield 77%) was
prepared.
MS (APCI): m/z= 903 (M+H)+
1H-NMR (CDC13) 5: 10.06 (1H, brs), 8.66 (1H, brs), 8.33
(1H, d, J = 7.7 Hz), 7.92-7.85 (2H, m), 7.65-7.23 (13H,
m), 6.92-6.85 (4H, m), 5.71 (1H, s), 4.86 (1H, s), 4.30
(1H, s), 4.26-4.07 (1H, m), 3.87-3.74 (7H, m), 3.62-3.53
(2H, m), 3.45 (1H, d, J = 10.8 Hz), 1.49 (18H, s)
[0262]
Example 38
Phosphorylation (boc) of cytosine (bz): (compound 16Cb)
Di-tert-butyl {[(1R,3R,4R,7S)-1-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methy1}-7-{[(2-
cyanoethoxy)(dipropan-2-ylamino)phosphanyl]oxyl-3-{2-oxo-
4-[(phenylcarbonyl)aminolpyrimidin-1(2H)-y11-2-oxa-5-
azabicyclo[2.2.11hepta-5-yl]methylidyne}biscarbamate
(compound 16Cb)
Similarly to Exmaple 9 from compound 15Cb (91 mg,
0.100 mmol), compound 16Cb (61 mg, yield 55%) was
prepared.
MS (APCI): m/z= 1103 (M+H)+
31P-NMR (CDC13) 6: 149.58, 149.25, 148.86
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260
[0263]
Preparation method of uracil product (compound 26A) and
guanine product (compound 26G) (hereinafter, sometimes
referred to uracil route) is described.
[chem.113]
Uracil route
HO Bn0TBDPSCI Bn0 H2SO4
NaH, BnBr HO/ 0 imidazole Ac20
- .
- o ' TBDPSO/x. -
DMF
BnCii O DMF AcOH-k- BnC.'- "\ Bn0
1 2 3
0
Bn0 uracil
BSA TMSOM Bn0 (4NH ao, MeNH2
, ___ b,OAc
TBDPSO ... ______ TBDPSO
MeCN 0 THF
Bne bAc
Bnd bAc
4 30
0
TfCI Bn0
Bn0 NH DMAP j.,.'1Øz.
.
, t,N--io T8DPSO N -----
CH2C12 .
TBDPSO 13nd ) N 0
BnCi: bH
M. n
[chem.114]
8n0 8,10 MsCI 8n0
TBAF) DMAP
. n),--- /-C1 TI-IF -
F ._/-----,,, 0 CH2a2 ... Ms0
, 0
Bn0' - N Bnd' - ftd cd-=-N
32 33 34
,
Bn0 Eln0 8n0
-43u4NN3
' IC- THF, H2O' I-12N i. 0
/
dioxane .
en
.i. 0
8n0 NH
35 - 36 - 3T
NHBz OCONPI.2
AiGim'C N.z.õ.1õ. .1V Nx.k
BSA TMSOTf Bn0 c% i ` N Bn0
________________ ' \r.0j...N / _,..-.1
N
1.2-dichlomethane N,, tN 'NHCO'Pr
I
'=v_,' i
Bnd 'NFI Bre.' NH
26A 26G
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261
[0264]
Example 39
1-{2-0-Acety1-3-0-benzy1-4-[(benzyloxy)methyl]-5-0-[tert-
butyl(diphenyl)sily1]-a-L-lyxofuranosyllpyrimidine-
2,4(1H,3H)-dione (compound 30)
Acetonitrile (100 mL) and uracil (5.668 g, 50.57
mmol) were mixed with compound i (25.58 g, 37.46 mmol)
(which was prepared via 2 steps according to the method
described in the literature (J. Org. Chem. 2011, 76,
9891-9899) from 3,5-di-O-benzy1-4-C-hydroxymethyl-1,2-0-
isopropylidene-a-D-ribofuranose (compound 2) (15.0 g,
37.45 mmol)), and thereto was added BSA (21.02 mL, 85.97
mmol) dropwise over 10 minutes. After the reaction
system was heated at 50 C for 13 minutes until the
reaction system became homogeneous, the mixtures were
cooled to 0 C. Thereto was added TMSOTf (7.7 mL, 40.08
mmol) dropwise at 0 C. After the mixtures were stirred
at room temperature for 5 minutes, the mixtures were
stirred at 50 C under heating for 2 hours. After
saturated aqueous sodium bicarbonate solution (200 mL)
was added thereto at 0 C, the reaction solutions were
concentrated. After the residues were extracted with
ethyl acetate, the extracts were washed with saturated
brine, dried over anhydrous sodium sulfate, and the
insoluble materials were removed by filtration, and the
solvents were evaporated. The crude product was
subjected to column chromatography (n-hexane/ethyl
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262
acetate = 65/35 to 50/50) to obtain compound 30 (23.84 g,
yield 87%, via 3 steps from 2).
MS (APCI): m/z= 735 (M+H)+
1H-NMR (CDC13) 6: 8.00-7.95 (1H, m), 7.72 (1H, d, J = 8.2
Hz), 7.64-7.56 (4H, m) 7.46-7.27 (14H, m), 7.22-7.16 (2H,
m), 6.07 (1H, d, J = 5.7 Hz), 5.35-5.27 (2H, m), 4.58-
4.47 (4H, m), 4.35 (1H, d, J = 5.7 Hz), 3.92 (1H, d, J =
10.8 Hz), 3.82-3.63 (3H, m), 1.94 (3H, s), 1.04 (9H, s)
[0265]
Example 40
1-{3-0-Benzy1-4-[(benzyloxy)methyl]-5-0-[tert-
butyl(diphenyl)sily1]-a-L-lyxofuranosyllpyrimidine-
2,4(1H,3H)-dione (compound 31)
((2R,3R,4S,55)-4-Benzyloxy-5-(benzyloxymethyl)-5-
((tert-butyl(diphenyl)silyl)oxymethyl)-2-(2,4-
dioxopyrimidin-l-yl)tetrahydrofuran-3-y1)acetate
(compound 30, 11 g, 14.97 mmol) and tetrahydrofuran (55
mL) were mixed, and thereto was added methylamine (34 mL,
306.5 mmol) dropwise with a dropping funnel at room
temperature, and the mixtures were stirred at room
temperature for 1 hour. After ethyl acetate was added
thereto, the mixtures were extracted with ethyl acetate.
The mixtures were washed with saturated brine, dried over
anhydrous sodium sulfate, and the insoluble materials
were then removed by filtration, and the solvents were
evaporated. The crude product was subjected to column
chromatography (n-hexane/ethyl acetate = 50/50 to 40/60)
to obtain compound 31 (9.23 g, yield 89%).
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263
MS (APCI): m/z= 693 (M+H)
1H-NMR (CDC13) 6: 8.08 (1H, brs), 7.68-7.59 (5H, m) 7.48-
7.26 (14H, m), 7.24-7.19 (2H, m), 5.95 (1H, d, J = 4.1
Hz), 5.40-5.33 (11-1, m), 4.73 (1H, d, J =11.1 Hz), 4.63
(1H, d, J = 11.1 Hz), 4.49-4.40 (2H, m), 4.37-4.26 (2H,
m), 3.82-3.69 (3H, m), 3.59 (1H, d, J = 10.1 Hz), 3.54
(1H, d, J = 10.1 Hz), 1.06 (9H, s)
[0266]
Example 41
(2S,3S,3aS,9aR)-3-(Benzyloxy)-2-[(benzyloxy)methy1]-2-
({[tert-butyl(diphenyl)silyl]oxy}methyl)-2,3,3a,9a-
tetrahydro-6H-furo[2',31:4,5][1,3]oxazolo[3,2-
a]pyrimidine-6-one (compound 32)
Compound 31 (9.23 g, 13.3 mmol), methylene chloride
(66.6 mL) and DMAP (5.70 g, 46.6 mmol) were mixed, and
the mixtures were cooled to 0 C. Thereto was added TfC1
(5.61 g, 33.3 mmol) dropwise at 0 C. The mixtures were
stirred at room temperature for 1 hour. Thereto was
added water at 0 C, and the mixtures were extracted with
chloroform. The mixtures were washed with saturated
brine, and dried over anhydrous sodium sulfate, and the
insoluble materials were removed by filtration, and the
solvents were evaporated. The crude product was
subjected to column chromatography (chloroform/methanol =
100/0 to 90/10) to obtain compound 32 (8.58 g, yield 95%).
MS (APCI): m/z= 675 (M+H)+
1H-NMR (CDC13) 6: 7.67-7.58 (4H, m), 7.48-7.19 (15H, m),
7.13-7.07 (2H, m), 6.21 (1H, d, J = 6.1 Hz), 6.09 (1H, d,
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J = 7.2 Hz), 5.51 (1H, dd, J = 6.1, 4.1 Hz), 4.80 (1H, d,
J = 11.3 Hz), 4.60 (1H, d, J = 11.3 Hz), 4.41-4.28 (3H,
m), 3.82 (1H, d, J = 10.8 Hz), 3.68 (1H, d, J = 10.8 Hz),
3.36-3.27 (2H, m), 1.03 (9H, s)
[0267]
Example 42
(2R,3S,3a5,9aR)-3-(Benzyloxy)-2-[(benzyloxy)methy1]-2-
(hydroxymethyl)-2,3,3a,9a-tetrahydro-6H-furo[2',3':4,5] [1,
3]oxazolo[3,2-a]pyrimidine-6-one (compound 33)
Compound 32 (8.58 g, 12.7 mmol) and THF (63.6 mL)
were mixed, and thereto was added 1M TBAF (16.5 mL, 16.5
mmol) dropwise with a dropping funnel under cooling at
0'C. The mixtures were stirred at room temperature for
2.5 hours. After the solvents were evaporated, the crude
product was subjected to column chromatography
(chloroform/methanol = 100/0 to 85/15) to obtain compound
33 (5.54 g, yield quant.).
MS (APCI): m/z= 437 (M+H)-
1H-NMR (CDC13) 6: 7.41-7.26 (91-1, m), 7.20-7.13 (2H, m),
6.27 (1H, d, J = 6.2 Hz), 6.06 (1H, d, J = 7.7 Hz), 5.35
(114, dd, J = 6.2, 2.6 Hz), 4.78 (1H, d, J = 11.8 Hz),
4.60 (1H, d, J = 11.8 Hz), 4.43-4.31 (314, m), 3.88-3.80
(114, m), 3.72-3.63 (1H, m), 3.40-3.26 (214, m), 2.35-2.21
(1H, m)
[0268]
Example 43
{(2S,3S,3aS,9aR)-3-(Benzyloxy)-2-[(benzyloxy)methyl]-6-
oxo-2,3,3a,9a-tetrahydro-6H-
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furo[21,3':4,5] [1,3]oxazolo[3,2-a]pyrimidin-2-yllmethyl
methanesulfonate (compound 34)
Compound 33 (5.54 g, 12.7 mmol) and methylene
chloride (127 mL) were mixed, and thereto were added MsC1
(1.68 mL, 21.6 mmol) and DMAP (4.65 g, 38.1 mmol) at 0 C.
The mixtures were stirred at room temperature for 1 hour.
After water was added thereto at 0 C, the mixtures were
extracted with chloroform. The extracts were washed with
saturated brine, and dried over anhydrous sodium sulfate,
and the insoluble materials were removed by filtration,
and the solvents were evaporated. The crude product was
subjected to column chromatography (ethyl
acetate/methanol = 100:0 to 90/10) to obtain compound 34
(5.47 g, yield 8490.
MS (APCI): m/z= 515 (M+H)'
1H-NMR (CDC13) 5: 7.41-7.25 (9H, m), 7.21-7.15 (2H, m),
6.23 (1H, d, J = 6.2 Hz), 6.08 (1H, d, J = 7.7 Hz), 5.30
(1H, dd, J = 6.2, 2.6 Hz), 4.76 (1H, d, J = 11.6 Hz),
4.60 (1H, d, J = 11.6 Hz), 4.46-4.29 (51-1, m), 3.39 (1H, d,
J = 10.3 Hz), 3.31 (1H, d, J = 10.3 Hz), 2.92 (3H, s)
[0269]
Example 44
(2R,3S,3a5,9aR)-2-(Azidomethyl)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-2,3,3a,9a-tetrahydro-6H-
furo[2',31:4,5] [1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 35)
Compound 34 (3.00 g, 5.830 mmol) and 1,4-dioxane
(58.3 mL) were mixed, and thereto was added
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tetrabutylammonium azide (4.976 g, 17.49 mmol). The
mixtures were stirred at 120 C under heating for 4.5
hours. After the mixtures were cooled to room
temperature, thereto was added saturated aqueous sodium
bicarbonate solution at 0 C, and the mixtures were
extracted with ethyl acetate. The extracts were washed
with saturated brine, and dried over anhydrous sodium
sulfate, and the insoluble materials were removed by
filtration, and the solvents were evaporated. The crude
product was subjected to column chromatography (ethyl
acetate/methanol = 100/0 to 90/10) to obtain compound 35
(2.264 g, yield 84%).
MS (APCI): m/z= 462 (M+H)+
1H-NMR (CDC13) 5: 7.41-7.24 (9H, m), 7.20-7.13 (2H, m),
6.21 (1H, d, J = 6.1 Hz), 6.07 (1H, d, J = 7.7 Hz), 5.32
(1H, dd, J = 6.1, 2.1 Hz), 4.76 (1H, d, J = 11.8 Hz),
4.60 (1H, d, J = 11.8 Hz), 4.40 (1H, d, J = 12.1 Hz),
4.34 (1H, d, J = 12.1 Hz), 4.29 (1H, d, J = 2.1 Hz), 3.62
(1H, d, J = 12.8 Hz), 3.47 (1H, d, J = 12.8 Hz), 3.32 (1H,
d, J = 10.2 Hz), 3.27 (1H, d, J = 10.2 Hz)
[0270]
Example 45
1-1(1R,3R,4R,75)-7-(Benzyloxy)-1-[(benzyloxy)methy1]-2-
oxa-5-azabicyclo[2.2.1]hepta-3-y1}-pyrimidine-2,4(1H,3H)-
dione (compound 37)
Compound 35 (2.2 g, 4.8 mmol), THF (32 mL) and H20
(6.4 mL, 360 mmol) were mixed, and thereto was added Ph3P
(1.90 g, 7.20 mmol), and the mixtures were stirred for
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21.5 hours at 70 C under heating. After the solvents
were evaporated, the crude product was subjected to
column chromatography (ethyl acetate/methanol = 100/0 to
17/2) to obtain compound 37 (2.067 g, yield quant.).
MS (APCI): m/z= 436 (M+H)+
1H-NMR (CDC13) 6: 8.30 (1H, brs), 7.80 (1H, d, J = 8.2
Hz), 7.40-7.21 (10H, m), 5.53 (IH, s), 5.46 (1H, d, J =
8.2 Hz), 4.65-4.49 (4H, m), 3.87 (1H, s), 3.83 (IH, d, J
= 10.9 Hz), 3.78 (1H, d, J = 10.9 Hz), 3.64 (IH, s), 3.14
(1H, d, J = 10.0 Hz), 2.89 (1H, d, J = 10.0 Hz)
[0271]
Example 46
N-(9-{(1R,3R,4R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-
2-oxa-5-azabicyclo[2.2.1]hepta-3-y1)1-9H-purin-6-
yl)benzamide (compound 26A)
To a solution of compound 37 (100 mg, 0.2296 mmol),
N-(9H-purin-6-yl)benzamide (164.8 mg, 0.6889 mmol) in
1,2-dichloromethane (2.296 mL) was added BSA (0.5053 mL,
2.067 mmol), and the reaction solutions were stirred at
60 C for 15 minutes. The mixtures were allowed to cool
to room temperature, and stirred, and thereto was added
TMSOTf (0.01331 mL, 0.06889 mmol) and the mixtures were
stirred at 50 C under heating for 15 minutes. After the
mixtures were diluted with chloroform, thereto was added
saturated aqueous sodium bicarbonate solution at 0 C, and
the mixtures were extracted with chloroform. The
mixtures were washed with saturated brine, and dried over
anhydrous sodium sulfate, and the insoluble products were
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removed by filtration, and the solvents were evaporated.
The crude product was subjected to column chromatography
(chloroform/methanol = 100/0 to 95/5) and reverse phase
preparative chromatography (10 mM aqueous ammonium
carbonate solution/acetonitrile = 90/10 to 60/40) to
obtain compound 26A (129.2 mg, yield 8496). Here compound
26A was "p form", and "a form" as an isomer could not be
obtained.
MS (APCI): m/z= 563 (M+H)
The date of 1H-NMR was identical to that of compound
26A of Example 13.
[0272]
Example 47
Preparation of guanine product from uracil product:
(compound 26G)
9-{(1R,3R,4R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methy1]-2-
oxa-5-azabicyclo[2.2.1]hepta-3-y11-2-[(2-
methylpropanoyl)amino]-9H-purin-6-y1 diphenylcarbamate
(compound 26G)
Similarly to Example 13 from compound 37 (300 mg,
0.689 mmol) and (2-(2-methylpropanoylamino)-9-purin-1-y1)
N,N-diphenylcarbamate (861 mg, 2.07 mmol) (the
preparation method follows the method described in J. Org.
Chem. 1996, 61, 9207), compound 26G (412 mg, yield 81%-).
Here compound 26G was "p form", and "a form" as an isomer
could not be obtained.
MS (APCI): m/z= 740 (M+H)+
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The data of 1H-NMR was identical to that of compound
26G of Example 14.
[0273]
Next, preparation method of pyrimidine derivatives
(compound 38a and compound 38b) is described.
[chem.115]
Bn0 (C0C1)2 Bn0 Bn0 DPPA
DMSO EN .
, la MeMsBr __ ,vb
HO -,0 Ph3P, DiAD .
HO
ft 1
CH2Cl2
e .0,A.' THF '0"V toluene & '-(X Bna w
tmo
2 0 M
VI
Bn0 Ac20 Bn0 thymine
TMSOTf Bn0 ( \./ NH ,q MeN1-12
µ.0K AcOH
a µ+'0Ac MeCN r'61)(C)il- THF
Bn0' Bn0 1 ibac
Bn0
42 43 44
TM Bn0
CNN e \NH
Bn0 DMAP PPh, Bno
bri , N-, ,,:t_)\
N3 ,,,õ 0
) i ''.0H Ci-ItA 1 0
Bn01 N THF, H20 ' .,,,....01Tio
setwateclby
thimMPLC
Bn6 Bfle--,NH,
45 46 38
\_d9 _11
Bn0 e (NH Bno (-- \NH
N--
jOi 0 14-i
bl
El.16-Nr4i B.,(Y1_1
38a 38b
[0274]
Example 48
(5x1)-3-0-Benzy1-4-[(benzyloxy)methyl]-6-deoxy-1,2-0-(1-
methylethylidene)-p-L-lyxo-hexofuranose (compound 41)
Compound 40 (prepared from compound 2 according to
the method described in J. Med. Chem. 2000, 43, 4516-
4525) (2.00 g, 5.02 mmol) and THF (25.1 mL) were mixed,
and the mixtures were cooled to -78 C. Thereto was added
methyl magnesium bromide (0.91 M, 8.3 mL, 7.53 mmol)
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270
dropwise at the same temperature, and the mixtures were
stirred at -78 C for 1 hour. After saturated aqueous
ammonium chloride solution was added thereto, the
mixtures were extracted with ethyl acetate. The mixtures
were washed with saturated brine, and dried over
anhydrous sodium sulfate, and the solvents were
evaporated. The crude product was purified by silica gel
chromatography (n-hexane/ethyl acetate, 80/20 to 75/25)
to obtain compound 41 (1.62 g) as a mixture.
[0275]
(5xi) ¨5 ¨Azido-3-0-benzy1-4- [ (benzyloxy)methyl] -5, 6-
dideoxy-1,2-0-(1-methylethylidene)-p-L-lyxo-hexofuranose
(compound 42)
Compound 41 (1.44 g) and toluene (17.4 mL) were
mixed, and thereto were added Ph3P (2.73 g, 10.4 mmol)
and DIAD (2.15 mL, 10.9 mmol) under cooling at 0 C, and
thereto was added DPPA (2.24 mL, 10.4 mmol) dropwise.
The mixtures were stirred at room temperature for 1 hour,
and stirred at 50 C under heating for 2 hours, and the
solvents were evaporated. The crude product was purified
by silica gel chromatography (n-hexane/ethyl acetate,
85/15 to 80/20) to obtain compound 42 (1.53 g) as a
mixture.
[0276]
(5xi)-1,2-Di-0-acety1-5-azido-3-0-benzyl-4-
[(benzyloxy)methy1]-5,6-dideoxy-L-lyxo-hexofuranose
(compound 43)
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271
Compound 42 (1.47 g), acetic anhydride (4 mL) and
acetic acid (2.31 mL) were mixed, and thereto was added
concentrated sulfuric acid (17.9 pL) at 0 C, and the
mixtures were stirred at room temperature for 2 hours.
Thereto was added saturated aqueous sodium carbonate
solution (8.8 mL) dropwise at 0 C to make the mixtures pH
= 6, and thereto was added saturated aqueous sodium
hydrogen carbonate solution, and the mixtures were
extracted with ethyl acetate. The organic layers were
washed with saturated aqueous sodium hydrogen carbonate
solution, water, and saturated brine successively, and
the mixtures were dried over anhydrous sodium sulfate.
The solvents were evaporated, and then azeotroped with
toluene three times to obtain compound 43 (1.60 g). The
obtained compound was used without purification in next
step.
[0277]
1-{(5xi)-2-0-Acetyl-5-azido-3-0-benzy1-4-
[(benzyloxy)methyl]-5,6-dideoxy-a-L-lyxo-hexofuranosy1}-
5-methylpyrimidine-2,4(1H,3H)-dione (compound 44)
Compound 43 (1.60 g), acetonitrile (11.0 mL) and
thymine (563 mg, 4.467 mmol) were mixed, and thereto was
added BSA (1.86 mL, 7.60 mmol) dropwise. The reaction
system was heated at 50 C for 10 minutes until the
reaction system became homogeneous, the mixtures were
cooled to 0 C. Thereto was added TMSOTf (0.684 mL, 3.54
mmol) dropwise at the same temperature. The mixtures
were stirred at room temperature for 5 minutes, and then
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272
stirred at 50 C under heating for 2 hours. Thereto was
added saturated aqueous sodium hydrogen carbonate
solution dropwise at 0 C, and the reaction solutions were
concentrated. The residues were extracted with ethyl
acetate, and the organic layers were washed with
saturated brine, and dried over anhydrous sodium sulfate,
and the solvents were evaporated. The crude product was
purified by silica gel chromatography (n-hexane: ethyl
acetate, 67/33 to 50/50) to obtain compound 44 (628 mg,
yield 3596-: on the basis of compound 41) as a mixture.
[0278]
1-{(5xi)-5-Azido-3-0-benzy1-4-[(benzyloxy)methyl]-5,6-
dideoxy-a-L-lyxo-hexofuranosyll-5-methylpyrimidine-
2,4(1H,3H)-dione (compound 45)
Compound 44 (620 mg, 1.13 mmol) and THF (4.5 mL)
were mixed, and thereto was added 40% aqueous methyl
amine solution (2.56 mL) dropwise at room temperature,
and the mixtures were stirred at room temperature for 40
minutes. The reaction solutions were extracted with
ethyl acetate. The organic layers were washed with
saturated brine, and dried over anhydrous sodium sulfate,
and the solvents were evaporated. The crude product was
purified by silica gel chromatography (n-hexane/ethyl
acetate, 60/40 to 40/60) to obtain compound 45 (556 mg,
yield 97%) as a mixture.
[0279]
(2R,3S,3aS,9aR)-2-(1-Azidoethyl)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-7-methy1-2,3,3a,9a-tetrahydro-6H-
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273
furo[21,31:4,5] [1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 46)
Compound 45 (541 mg, 1.07 mmol), methylene chloride
(5.3 mL), and DMAP (456 mg, 3.73 mmol) were mixed, and
the mixtures were cooled to 0 C. After TfC1 (0.282 mL,
2.67 mmol) was added dropwise thereto at the same
temperature, the mixtures were stirred at room
temperature for 90 minutes. After water was added to the
reaction solutions at 0 C, the mixtures were extracted
with chloroform. The organic layers were washed with
saturated brine, and dried over anhydrous sodium sulfate,
and the solvents were evaporated. The crude product was
purified by silica gel chromatography
(chloroform/methanol, 100/0 to 89/11) to obtain compound
46 (458 mg, yield 88%) as a mixture.
[0280]
1-{(1R,3R,4R,6S,7S)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-
6-methy1-2-oxa-5-azabicyclo[2.2.1]hepta-3-y1}-5-
methylpyrimidine-2,4(1H,3H)-dione (compound 38a)
1-{(1R,3R,4R,6R,7S)-7-(Benzyloxy)-1-[(benzyloxy)methy1]-
6-methyl-2-oxa-5-azabicyclo[2.2.1]hepta-3-y11-5-
methylpyrimidine-2,4(1H,3H)-dione (compound 38b)
Compound 46 (450 mg, 0.919 mmol), THF (6.1 mL) and
water (1.22 mL) were mixed, and thereto was added Ph3P
(362 mg, 1.38 mmol), and the mixtures were stirred at
70 C under heating for 16 hours. After the solvents were
evaporated, the crude product was purified by silica gel
chromatography (ethyl acetate/methanol, 100/0 to 85/15)
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to obtain compound 38 (406 mg, yield 95%) as a mixture.
Compound 38 (287 mg) was purified by chiral HPLC (DAICEL,
CHIRALPAK IC, o 30 x 250 mm, methanol/THF/diethylamine,
65/35/0/1, flow rate 20 mL/min.) to obtain crude 38a (138
mg) and crude 38b (139 mg), which contain BHT as a
stabilizer of THF. Each of the products was purified by
silica gel chromatography (ethyl acetate/methanol, 100/0
to 85/15) to obtain compound 38a (132 mg) and compound
38b (123 mg).
Compound 38a:
MS (APCI): m/z = 464 (M+H)+
1H-NMR (CDC13) 6: 8.21 (1H, brs), 7.55-7.50 (1H, m),
7.39-7.22 (10H, m), 5.48 (1H, s), 4.66-4.56 (3H, m), 4.49
(1H, d, J = 11.8 Hz), 3.91-3.83 (3H, m), 3.70 (1H, s),
3.25 (1H, q, J = 6.7 Hz), 1.62-1.57 (3H, m), 1.20 (3H, d,
J = 6.7 Hz)
Compound 38b:
MS (APCI): m/z= 464 (M+H)
1H-NMR (CDC13) 6: 8.18 (1H, brs), 7.67-7.62 (1H, m),
7.39-7.22 (10H, m), 5.47 (1H, s), 4.66-4.50 (4H, m), 4.02
(11-1, s), 3.77 (1H, d, J = 11.0 Hz), 3.72 (1H, d, J = 11.0
Hz), 3.55 (1H, s), 3.49 (1H, q, J = 6.7 Hz), 1.58-1.54
(3H, m), 1.14 (3H, d, J = 6.7 Hz)
[0281]
Preparation of adenine product from thymine product:
(compound 39Aa)
[chem.116]
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275
NHBz
_N N
0 HN NHBz
Bn0 NH BSA, Th4S0T1 8n0
0 N
0 N
0 1,2-dicholoroethane 6S ,--
6SI"
Brie"
38a 39Aa
[0282]
Example 49
N-(9-f(1R,3R,4R,6S,7S)-7-(Benzyloxy)-1-
[(benzyloxy)methy1)-6-methy1-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y1))-9H-purin-6-y1)benzamide
(compound 39Aa)
Similarly to Example 13 from compound 38a (50 mg,
0.108 mmol) and (N-(9H-purin-6-yl)benzamide (77.4 mg,
0.324 mmol), compound 39Aa (50.9 mg, yield 82%) was
prepared. Here compound 39Aa was "p form", and "a form"
as an isomer could not be obtained.
MS (APCI): m/z= 577 (M--H)
1H-NMR (CDC13) 6: 9.03 (111, s), 8.78 (111, s), 8.28 (1H,
s), 8.08-8.00 (2H, m), 7.65-7.50 (3H, m), 7.40-7.16 (10H,
m), 5.99 (1H, s), 4.70-4.62 (2H, m), 4.53-4.44 (2H, m),
4.14 (1H, s), 3.96 (1H, s), 3.91-3.82 (2H, m), 3.37 (1H,
q, J - 6.7 Hz), 1.25 (31-i, d, J = 6.7 Hz)
[0283]
Preparation of adenine product from thymine product:
(compound 39Ab)
[chem.117]
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276
NHBz
J431-C
, 0
HN J
N ¨ NHBz
BnoNic/ NH
N-X-IN
.. .i. :
0 BSA, TMSOTf Bn0
1,2-clichromethane ),
6 k 0 <i i NN
sr N---
..q.... i...= 1
Bn0 -NH
BnCr.11H
38b 39Ab
[0284]
Example 50
N-(9-{(1R,3R,4R,6R,7S)-7-(Benzyloxy)-1-
[(benzyloxy)methy1]-6-methyl-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y1)}-9H-purin-6-yl)benzamide
(compound 39Ab)
Similarly to Example 13 from compound 38b (50 mg,
0.108 mmol) and (N-(9H-purin-6-yl)benzamide (77.4 mg,
0.324 mmol), compound 39Ab (48.1 mg, yield 77%) was
prepared. Here compound 39Ab was "p form", and "a form"
as an isomer could not be obtained.
MS (APCI): m/z= 577 (M+H)+
1H-NMR (CDC13) 6: 9.03 (1H, s), 8.77 (1H, s), 8.41 (11-1,
s), 8.08-8.00 (211, m), 7.66-7.49 (3H, m), 7.41-7.14 (10H,
m), 5.99 (1H, s), 4.70 (1H, d, J = 12.3 Hz), 4.60 (1H, d,
J- = 12.3 Hz), 4.56-4.46 (2H, m), 4.29 (1H, s), 3.78-3.68
(3H, m), 3.54 (1H, q, J = 6.7 Hz), 1.20 (3H, d, J = 6.7
Hz)
[0285]
Preparation of guanine product from thymine product:
(compound 39Ga)
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277
[chem.118]
PI' N_Ph
0
Ph
N-Ph
0
N -4y
NH BSA, TMSOTf 8n0 / N
N
6'S(511 µCs 1,2-dicholoroethane
BriCf",N:H
811H
39Ga
38a
[0286]
Example 51
9-{(1R,3R,4R,63,7S)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-
6-methy1-2-oxa-5-azabicyclo[2.2.1]hepta-3-y1}-2-[(2-
methylpropanoyl)amino]-9H-purin-6-y1 diphenylcarbamate
(compound 39Ga)
Similarly to Example 13 from compound 38a (33 mg,
0.0712 mmol) and (2-(2-methylpropanoylamino)-9-purin-l-
y1) N,N-diphenylcarbamate (130 mg, 0.641 mmol) (the
preparation method follows the method described in J. Org.
Chem. 1996, 61, 9207), compound 39Ga (37.6 mg, yield 7096)
was prepared. Here compound 39Ga was form", and "a
form" as an isomer could not be obtained.
MS (APCI): m/z= 754 (M+H)+
1H-N11R (CDC13) a: 8.21 (1H, s), 7.88 (1H, s), 7.49-7.41
(2H, m), 7.40-7.15 (19H, m), 5.87 (1H, s), 4.64 (2H, s),
4.49 (1H, d, J = 11.8 Hz), 4.45 (1H, d, J . 11.8 Hz),
4.07 (1H, s), 3.95 (11-1, s), 3.87-3.80 (2H, m), 3.33 (1H,
q, J = 6.7 Hz), 3.07 (1H, brs), 1.26 (6H, d, J = 6.7 Hz),
1.22 (3H, d, J = 6.7 Hz)
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278
[0287]
Preparation of guanine product from thymine product:
(compound 39Gb)
[chem.119]
Ph
11-Ph
04-0 Ph
,N-Ph
HN _kr
0 0
Irk
Bn0 /111-1 BSA, TMSOTf Bn0 'N
0 N 0 NAm
1.2-d icholoroethane
=¨=.2
B
Br16NH n0 NH
3813 Mb
[0288]
Example 52
9-01R,3R,4R,6R,75)-7-(Benzyloxy)-1-[(benzyloxy)methyl]-
6-methy1-2-oxa-5-azabicyclo[2.2.11hepta-3-y11-2-[(2-
methylpropanoyl)amino]-9H-purin-6-y1 diphenylcarbamate
(compound 39Gb)
Similarly to Example 13 from compound 38b (20 mg,
0.0432 mmol) and (2-(2-methylpropanoylamino)-9-purin-1-y1
N,N-diphenylcarbamate (79.0 mg, 0.388 mmol) (the
preparation method follows the method described in J. Org.
Chem. 1996, 61, 9207), compound 39Gb (27.9 mg, yield 869).
Here compound 39Gb was up form", and "a form" as an
isomer could not be obtained.
MS (APCI): m/z= 754 (M+H)+
1H-NMR (CDC13) 6: 8.33 (1H, s), 7.88 (1H, s), 7.48-7.41
(2H, m), 7.41-7.14 (19H, m), 5.88 (1H, s), 4.69 (1H, d, J
- 12.3 Hz), 4.58 (1H, d, J = 12.3 Hz), 4.52-4.45 (2H, m),
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4.24 (1H, s), 3.74-3.65 (3H, m), 3.50 (1H, q, J = 6.7 Hz),
3.04 (1H, brs), 1.26 (3H, d, J = 7.2 Hz), 1.25 (3H, d, J
= 7.2 Hz), 1.16 (3H, d, J = 6.7 Hz)
[0289]
Preparation of adenine product from 3',5'-hydroxy thymine
product: (compound 47)
[cnem.120]
NHBz
HO
0 TMSO eaCks-N
o
Hu NH AcIOH TNISCr'RH
13 47
[0290]
Example 51
N-(9-{(1R,3R,4R,7S)-7-[(Trimethylsilyl)oxy]-1-
{[(trimethylsily1)oxy]methyll-2-oxa-5-
azabicyclo[2.2.1]hepta-3-y1)}-9H-purin-6-yl)benzamide
(compound 47)
Similarly to Example 13 from compound 13 (50 mg,
0.132 mmol) and BSA (0.36 mL, 1.45 mmol), compound 47
(56.6 mg, containing 2.9wt% of ethyl acetate, yield 79%)
was prepared. Here compound 47 was "p form", and "a
form" as an isomer could not be obtained.
MS (APCI): m/z 527 (M+H)+
1H-NMR (CDC13) 6: 9.07 (1H, 8), 8.80 (1H, s), 8.36 (1H,
s), 8.08-8.02 (2H, m), 7.65-7.59 (1H, m), 7.57-7.51 (2H,
m), 6.04 (1H, s), 4.29 (1H, s), 3.90 (1H, d, J = 11.8 Hz),
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3.84 (1H, d, J = 11.8 Hz), 3.76 (1H, s), 3.16 (1H, d, J =
9.7 Hz), 3.90 (1H, d, J = 9.7 Hz), 0.19 (9H, s), 0.09 (9H,
s)
[0291]
Preparation of GuNA nucleotide monomer having different
ring size of bridged chain by transglycosylation
By modifying the number of carbon in a carbon chain
that is composed of a bridged chain to ribose backbone,
GuNA monomer having different ring size can be prepared.
One example of the preparation thereof is described below,
in which transglycosylation can be carried out using
compound 61 obtained by the below-mentioned scheme to
prepare GuNA monomer having different ring size
containing various nucleic acid base(s).
[chem.121]
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281
Bn0 Et0
sp,'--s-00 Et Bn0 Bn0 Bn0
OHCe,. 0 Et0' v0 2
=n0 NICBIH, ,, 0
EtO2C...-.1% '" )<, t,, ay.., EtO2C--/ ". "i \ __.õ LAH
NaH
Elnd ..'" )< __ 7.- laai ..."'" -2\ ¨41^ HO
Me0H 84 ,., THE
THE
40 48 49 50
BOO Bn0 Bn0 Thymine
WC! NaN, Ac20
El3N
15-Crown-5 cat. H2SO4 BSA
/....../,,:b.,,0
-----Ae. _______________ A ."---// '..0µ A... f.___,"_'"" G .e0Ac
TMSOTI
Et0Ac ' '_,;' .''ecX DMF N3 ."'C" AcOH N3 MeCN
Bn0 BnCT Bnd '0Ac
51 52 53
y0
ri
8n0 (1,....r0 MsCI PPli,
Bn0 ...' an
b.,,,N,e4NH MeNti, Pyridine H20
0 N NH Bn0.µk.0_z_. ...._ -----lir
--g- then ,...../." N"---- DME
N, 8 THF/Et0Ac "-J.:bit'
N, DBU N,
0 H2N
Bnd ..1:1Ac :i ."1. .F.
Bn0 OH Bn0-
54
SS 56 57
BOO ri..f.0 Tt20 0
an ,.., DMA? Bn0 ..rly
Boc20 "I'*()_Z..N.----- Na0Haq
---)..... --1... , 0 riNH Pyricline,õ
.....OzN.,,,r,NH
BocHN 4 ....1., 0 THF
BocHN"--/
8 8
011204 and 0 N CH2Cl2 BocHN
Bnd OHµ
Bn0 OTt
58
59 60
rjy0
Bn0
TEA
--)e. b.,,t1 NH
CHyCly
ano`µ\-6iFi
61
[0292]
Hereinafter, each step of the above-mentioned
preparation method is described.
[chem.122]
Et0.......,..---..,
r CO2Et
Et0
Bn0 ,0 Bn0
0
OHCii\-- . Cktilo
s'
NaH
EtO2C ni"-..." s'?"10
________________________________ VI-
,1
Bny THF Bn0
-i'.
40 48
[0293]
Example 52
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282
Ethyl (5E)-3-0-benzy1-4-[(benzyloxy)methy1]-5,6-dideoxy-
1,2-0-(1-methy1ethylidene)-3-L-1yxo-hepta-5-
enofuranuronate (compound 48)
To a solution of ethyl diethylphosphonoacetate (6.83
mL, 34.43 mmol) in THF (163 mL) was added sodium hydride
(1.38 g, 34.43 mmol, 60% contents) under ice-cooling, and
the mixtures were stirred at room temperature for 30
minutes. The reaction solutions were cooled under ice-
cooling, and thereto was added a solution of compound 40
(10.96 g, 26.48 mmol) in THF (54 mL) dropwise. The ice-
water bath was removed, and the reaction mixtures were
stirred at room temperature for 1 hour. To the reaction
solutions were added ethyl acetate (300 mL) and cold
water (500 mL), and the mixtures were stirred for a while,
and the organic layers were then separated by a
separatory funnel. The aqueous layers were extracted
with ethyl acetate (100 mL) using a separatory funnel,
and the combined organic layers were washed with water
and saturated brine, and then dried over anhydrous sodium
sulfate. The solvents were evaporated under reduced
pressure, and the resulting residues were purified by
silica gel chromatography (hexane/ethyl acetate, 95/5 to
60/40) to obtain compound 48 (12.18 g, yield 97%).
MS (APCI): m/z 486 (M+NH4)+
1H-NMR (CDC13) 6: 7.38-7.19(11H, m), 6.24 (1H, d, J =
15.9 Hz), 5.76 (1H, d, J = 4.1 Hz), 4.77 (1H, d, J = 12.3
Hz), 4.60 (1H, d, J = 12.3 Hz), 4.57 (1H, dd, J = 4.6,
4.1 Hz), 4.50 (1H, d, J = 11.8 Hz), 4.41 (1H, d, J = 11.8
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Hz), 4.27 (1H, t, J = 4.6 Hz), 4.23-4.13 (2H, m), 3.38-
3.30 (2H, m), 1.47 (3H, s), 1.30-1.24 (6H, m)
[0294]
[chem.123]
Bn0 Bn0
o NiCl2
0
EtO2C =110 NaBH4 EtO2C¨.../1" "I
A:f Me0H (
any But'
48 49
Example 53
Ethyl 3-0-benzy1-4-[(benzyloxy)methyl]-5,6-dideoxy-1,2-0-
(1-methylethylidene)-13-L-lyxo-heptofuranuronate (compound
49)
To a solution of compound 48 (12.06 g, 25.50 mmol)
in methanol (241 mL) was added nickel(II) chloride
hexahydrate (1212 mg, 5.100 mmol). The reaction
solutions were cooled in ice-cooling, and thereto was
added sodium borohydride (1930 mg, 51.00 mmol) over 40
minutes. The ice-water bath was removed, and the
mixtures were stirred at room temperature for 2 hours.
The reaction solutions were filtered through Celite,
washed with ethyl acetate (300 mL) and water (300 mL),
and the organic layers of the filtrates were separated by
a separatory funnel. The aqueous layers were extracted
with ethyl acetate (150 mL portions were carried out
twice) by a separatory funnel, and the combined organic
layers were washed with water (300 mL) and saturated
brine (300 mL), and then dried over anhydrous sodium
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284
sulfate. The solvents were evaporated under reduced
pressure to obtain compound 49 (11.90 g, yield 98%).
MS (APCI): m/z 488 (M+NH.04
1H-NMR (CDC13) 5: 7.38-7.21(10H m), 5.75 (1H, d, J = 4.1
Hz), 4.76 (1H, d, J = 12.3 Hz), 4.61 (1H, dd, J = 5.1,
4.1 Hz), 4.55 (1H, d, J = 12.3 Hz), 4.49 (1H, d, J =
12.3 Hz), 4.40 (1H, d, J = 12.3 Hz), 4.14-4.06 (3H, m),
3.40 (1H, d, J = 10.3 Hz), 3.26 (1H, d, J = 10.3 Hz),
2.66-2.50 (2H, m), 2.37-2.26 (1H, m), 1.92-1.81 (1H, m),
1.62 (3H, s), 1.32 (3H, s), 1.23 (3H, t, J = 7.2 Hz)
[0295]
[chem.124]
Bn0 Bn0
LAH
0
EtO2C ).00 HO *4.:)
.110
)1`
THF '''40):
Bnu Bny
49 50
[0296]
Example 54
3-0-Benzy1-4-[(benzyloxy)methy1]-5,6-dideoxy-1,2-0-(1-
methylethylidene)-p-L-1yxo-heptofuranose (compound 50)
TO a suspension of lithium aluminum hydride (1904 mg,
50.17 mmol) in THF (155 mL) was added a solution of
compound 49 (11.90 g, 25.08 mmol) in THF (83 mL) dropwise
over 15 minutes under ice-cooling, and the mixtures were
stirred at room temperature for 1 hour. The reaction
solutions were cooled under ice-cooling, and thereto was
added saturated aqueous sodium sulfate solution (10 mL)
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285
dropwise, and followed by adding water (3 mL)
successively, and the mixtures were stirred at room
temperature for a whole day and night. The reaction
solutions were filtered through Celite, washed with ethyl
acetate (100 mL portions were carried out three times),
and the filtrates were dried over anhydrous sodium
sulfate. The solvents were evaporated under reduced
pressure to obtain compound 50 (10.98 g, 25.04 mmol,
yield 100%).
MS (APCI): m/z 446 (M+NH4)+
1H-NMR (CDC13) 6: 7.37-7.21(10H, m), 5.76 (1H, d, J = 4.1
Hz), 4.76 (1H, d, J = 12.3 Hz), 4.62 (1H, dd, J = 5.7,
4.1 Hz), 4.56 (1H, d, J = 12.3 Hz), 4.51 (1H, d, J
12.3 Hz), 4.41 (1H, d, J = 12.3 Hz), 4.16 (1H, d, J = 5.7
Hz), 3.76-3.58 (2H, m), 3.50 (1H, d, J = 10.3 Hz), 3.32
(1H, d, J = 10.3 Hz), 2.28-2.15 (1H, m), 2.06-2.01 (1H,
m), 1.79-1.67 (2H, m), 1.65-1.55 (4H, m), 1.33 (3H, s)
[0297]
[chem.125]
Bn0 Bn0
MsCI
0
.1 Et3N =7
=.10
HO
4.- 0
Bnv Bn0
50 51
[0298]
Example 55
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286
3-0-Benzy1-4-[(benzyloxy)methy1]-5,6-dideoxy-1,2-0-(1-
methylethylidene)-7-0-(methylsulfony1)-p-L-lyxo-
heptofuranose (compound 51)
To a solution of compound 50 (10.45 g, 23.83 mmol)
in ethyl acetate (209 mL) was added triethylamine (4.64
mL, 33.36 mmol) under ice-cooling, and followed by adding
methanesulfonyl chloride (2.22 mL, 28.6 mmol) dropwise
successively. The mixtures were stirred at the same
temperature for 1 hour. To the reaction solutions was
added methanol (0.5 mL) and the mixtures were stirred for
30 minutes. The reaction solutions were poured into
water (200 mL), and the organic layers were separated by
a separatory funnel. After the organic layers were
washed with saturated brine (100 mL), the mixtures were
dried over anhydrous sodium sulfate. The solvents were
evaporated under reduced pressure and the resulting
residues were purified by silica gel chromatography
(hexane/ethyl acetate, 90/10 to 30/70) to obtain compound
51 (11.72 g, containing 3.3wt% of ethyl acetate, yield
97%).
MS (APCI): m/z 524 (M+NH4)+
1H-NMR (CDC13) 6: 7.37-7.22(10H, m), 5.74 (1H, d, J = 4.1
Hz), 4.76 (1H, d, J = 12.3 Hz), 4.61 (1H, dd, J = 5.7,
4.1 Hz), 4.55 (1H, d, J = 12.3 Hz), 4.51 (1H, d, J =
12.3 Hz), 4.41 (1H, d, J = 12.3 Hz), 4.30-4.12 (2H, m),
4.11 (1H, d, J = 5.7 Hz), 3.41 (1H, d, J = 10.3 Hz), 3.28
(1H, d, J = 10.3 Hz), 2.95 (3H, s), 2.25-2.15 (1H, m),
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287
2.07-1.94 (1H, m), 1.83-1.65 (2H, m), 1.59 (3H, s), 1.33
(3H, s)
[0299]
[chem.126]
Bn0 Bn0
NaN 3 0
/---"Jt) =,t0 15-Crown-5
Ms0
8n6: ** DMF ___ Alt N3
Bnd
51 52
[0300]
Example 56
7-Azido-3-0-benzy1-4-[(benzyloxy)methy1]-5,6,7-trideoxy-
1,2-0-(1-methylethylidene)-13-L-lyxo-heptofuranose
(compound 52)
To a solution of compound 51 (11.72 g, 22.38 mmol)
and 15-crown-5 (4.43 mL, 22.38 mmol) in DMF (117 mL) was
injected sodium azide (4365 mg, 67.15 mmol), and the
mixtures were stirred at 50 C for 16 hours. To the
reaction solutions was added water, and the mixtures were
extracted with ethyl acetate by a separatory funnel. The
combined organic layers were washed with water and
saturated brine, and then dried over anhydrous sodium
sulfate. The solvents were evaporated under reduced
pressure and the resulting residues were purified by
silica gel chromatography (hexane/ethyl acetate, 95/5 to
70/30) to obtain compound 52 (9.94 g, yield 98%).
MS (APCI): m/z 471 (M+NH4)+
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288
1H-NMR (CDC13) 6: 7.36-7.22(10H, m), 5.74 (1H, d, J = 4.1
Hz), 4.76 (1H, d, J = 12.3 Hz), 4.60 (1H, dd, J = 5.1,
4.1 Hz), 4.55 (1H, d, J = 12.3 Hz), 4.51 (1H, d, J =
12.3 Hz), 4.41 (1H, d, J = 12.3 Hz), 4.11 (1H, d, J = 5.1
Hz), 3.42 (1H, d, J = 10.3 Hz), 3.34-3.19 (3H, m), 2.22-
2.11 (1H, m), 1.92-1.80 (1H, m), 1.69-1.51 (2H, m), 1.60
(3H, s), 1.33 (3H, s)
[0301]
[chem.127]
Bn0 Bn0
Ac20
0 1"--/
=,10 cat }-12SO4 N .110Ac
N3 3
.1140)K. AcON 10Ac
Bnu Bnd
52 53
[0302]
Example 57
1,2-Di-O-acetyl-7-azido-3-0-benzy1-4-[(benzyloxy)methyl]-
5,6,7-trideoxyL-lyxo-heptofuranose (compound 53)
To a solution of compound 52 (9.94 g, 21.9 mmol) in
acetic acid (19.9 mL) were added acetic anhydride (9.94
mL, 105 mmol) and concentrated sulfuric acid (0.0497 mL,
0.932 mmol), and the mixtures were stirred at room
temperature for 4 hours. To the reaction solutions was
added ethyl acetate (200 mL). Thereto was added 10 6
aqueous sodium carbonate solution (200 mL) dropwise over
40 minutes while stirring vigorously, and the mixtures
were stirred vigorously insitu for 1 hour. The organic
layers were separated by a separatory funnel and the
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289
aqueous layers were extracted with ethyl acetate (50 mL)
by a separatory funnel. The combined organic layers were
washed with 5% aqueous sodium bicarbonate solution (200
mL) and saturated brine (100 mL), and dried over
anhydrous sodium sulfate. The solvents were evaporated
under reduced pressure, and the residues were azeotroped
with toluene (150 mL portions were carried out twice) and
then dried under reduced pressure to obtain compound 53
(11.40 g, containing 4.9wt% of toluene, yield 100%).
MS (APCI): m/z 515 (M+NH4)+
1H-NMR (CDC13) .5: 7.38-7.13(10H, m), 6.10 (1H, s), 5.33
(1H, d, J = 5.1 Hz), 4.60 (1H, d, J = 11.8 Hz), 4.55-4.42
(3H, m), 4.29 (1H, d, J = 5.1 Hz), 3.40 (1H, d, J = 9.8
Hz), 3.35 (1H, d, J = 9.8 Hz), 3.31-3.18 (2H, m), 2.11
(3H, s), 1.89 (3H, s), 1.89-1.56 (4H, m)
[0303]
[diem. 128]
Bn0 Thymirm reke
Bn0
0
/..10Ac BSA
TMSOTf /..___//h, 0 NII,NH
N3
'9/0Ac MeCN N3 V 0
Brit) Bnd 11100x
53 54
[0304]
Example 58
1-{2-0-Acety1-7-azido-3-0-benzy1-4-[(benzyloxy)methyl]-
5,6,7-trideoxy-a-L-lyxo-heptofuranosy11-5-
methylpyrimidine-2,4(1H,3H)-dione (compound 54)
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290
To a solution of compound 53 (11.40 g, 21.78 mmol)
in acetonitrile (57 mL) was inserted thymine (3022 mg,
23.96 mmol) at room temperature, followed by adding BSA
(16 mL, 47.93 mmol) dropwise slowly successively. The
mixtures were stirred at 60 C as external temperature for
1 hour. Thereto was added TMSOTf (4.337 mL, 23.96 mmol)
dropwise slowly at the same temperature, and the mixtures
were stirred at 80 C as external temperature for 3 hours.
The reaction solutions were cooled under ice-cooling, and
thereto was added 5% aqueous sodium carbonate solution
(50 mL) over 30 minutes, and the reactions were quenched.
Thereto were added ethyl acetate (100 mL) and water (100
mL), and the mixtures were stirred for a while, and the
organic layers were separated by a separatory funnel.
The aqueous layers were extracted with ethyl acetate (50
mL) by a separatory funnel, and the combined organic
layers were washed with 5% aqueous sodium hydrogen
carbonate solution (100 mL) and saturated brine (100 mL),
and dried over anhydrous sodium sulfate. The solvents
were evaporated under reduced pressure, and the resulting
residues were purified by silica gel chromatography
(hexane/ethyl acetate, 95/5 to 50/50) to obtain compound
54 (10.62 g, yield 83%-).
MS (ESI): m/z 564 (M-1-1-)+
1H-NMR (CDC13) 6: 8.14(11i, s), 7.44-7.42 (1H, m), 7.39-
7.27 (10H, m), 6.19 (1H, d, J = 5.7 Hz), 5.39 (1H, t, J =
5.7 Hz), 4.64 (1H, d, J = 11.3 Hz), 4.55-4.42 (3H, m),
4.37 (1H, d, J = 5.7 Hz), 3.66 (1H, d, J = 10.3 Hz), 3.37
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291
(1H, d, J = 10.3 Hz), 3.34-3.17 (2H, m), 2.10 (3H, s),
1.95-1.70 (2H, m), 1.63-1.51 (5H, m)
[0305]
[diem. 129]
0
Bn0 r-j-f Bn0 eLe
MeNH2
x_si/tlyNINH
/"."=====/
N3
THF/Et0Ac N3 = = ti
Bnd l' OAc s
BritY
54 55
[0306]
Example 59
1-(7-Azido-3-0-benzy1-4-[(benzyloxy)methyl]-5,6,7-
trideoxy-a-L-lyxo-heptofuranosyll-5-methylpyrimidine-
2,4(1H,3H)-dione (compound 55)
To a solution of compound 54 (10.62 g, 18.10 mmol)
in THF (32 mL) and ethyl acetate (11 mL) was added methyl
amine (10.62 mL, 120 mmol, 40% aqueous solution) under
ice-cooling, and the mixtures were stirred for 2 hours.
To the reaction solutions was added 1N-hydrochloric acid
(105 mL) to make pH = 8. The mixtures were extracted
with ethyl acetate (200 mL, and 50 mL) by a separatory
funnel. The combined organic layers were washed with
saturated brine (100 mL), and then dried over anhydrous
sodium sulfate. The solvents were evaporated under
reduced pressure, and the resulting residues were
azeotroped with toluene twice, and dried under reduced
pressure to obtain compound 55 (10.74 g, yield 100%).
MS (ESI): m/z 522 (M+H)+
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292
1H-NMR (CDC13) 6: 8.23(1H, brs), 7.41-7.23 (9H, m), 7.19-
7.12 (2H, m), 5.89 (11-I, d, J = 6.2 Hz), 4.71 (1H, d, J =
11.3 Hz), 4.65 (1H, d, J = 11.3 Hz), 4.56 (1H, d, J =
11.3 Hz), 4.52 (1H, d, J = 11.3 Hz), 4.38-4.30 (1H, m),
4.17 (1H, d, J = 6.2 Hz), 3.63 (1H, d, J = 9.8 Hz), 3.41
(1H, d, J = 9.8 Hz), 3.37-3.28 (1H, m), 3.27-3.18 (1H, m),
2.94 (1H, d, J = 8.2 Hz), 1.98-1.71 (2H, m), 1.64-1.50
(SH, m)
[0307]
[chem.130]
rr0Bn0 MsCI Bn0
0
.eoNNH Pyddine N
= m
N3t=,.rf/ g then DBU
Bnd )*N
Bn0 OH
55 6
[0308]
Example 60
(2R,3S,3aS,9aR)-2-(3-Azidopropy1)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-7-methy1-2,3,3a,9a-tetrahydro-6H-
furo[21,3':4,5] [1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 56)
To a solution of compound 55 (10.74 g, 18.14 mol) in
pyridine (32 mL) was added methanesulfonyl chloride (1.83
mL, 23.59 mmol) dropwise under ice-cooling, and the
mixtures were stirred at room temperature for 3.5 hours.
The reaction solutions were cooled under ice-cooling, and
thereto was added 1,8-diazabicyclo[5.4.0]-7-undecene
(8.21 mL, 54.43 mmol) dropwise, and the mixtures were
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293
stirred at room temperature for 4 hours. The reaction
solutions were diluted with ethyl acetate (400 mL), and
were poured into 10% aqueous citric acid solution (500
mL), and the mixtures were stirred for a while. The
organic layers were separated by a separatory funnel, and
the aqueous layers were extracted with ethyl acetate (100
mL) by a separatory funnel. The combined organic layers
were washed with water (500 mL) and saturated brine (300
mL). To the organic layers (brown color) was added
activated carbon (1.00 g), and after the mixtures were
stirred for 20 minutes, the mixtures were filtered
through Celite. The filtrates were concentrated under
reduced pressure, and then azeotroped with toluene. To
the resulting concentrated residues was added ethyl
acetate (40 mL) to prepare a suspension, and thereto was
added hexane (300 mL), and the precipitates were
collected by filtration. The precipitates were air-dried
under nitrogen atmosphere and then dried under reduced
pressure to obtain compound 56 (8.61 g, yield 94%).
MS (ESI): m/z 504 (M+H)*
1H-NMR (CDC13) 6: 7.41 - 7.27 (8H, m), 7.18 (1H , d, J =
1.5 Hz), 7.16 - 7.12 (2H , m), 6.09 (1H , d, J = 6.2
Hz, ), 5.21 (IH, dd, J = 6.2, 1.0 Hz), 4.72 (1H , d, J
11.8 Hz), 4.57 (1H , d, J = 11.8 Hz), 4.37 (1H , d, J =
11.8 Hz), 4.29 (1H , s), 4.26 (1H , d, J = 11.8 Hz), 3.24
(2H , t, J = 6.2 Hz), 3.18 (1H , d, J = 9.8 Hz), 3.13
(1H , d, J = 9.8 Hz), 1.97 (3H , d, J = 1.0 Hz), 1.88 -
1.45 (4H , m)
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[0309]
[chem.131]
Bn0 Bn0
PPh3
0
H20
N3 0 DME H2N
Bnd )*N Brd
56 57
[0310]
Example 61
(2R,3S,3aS,9aR)-2-(3-Aminopropy1)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-7-methyl-2,3,3a,9a-tetrahydro-6H-
furo[2',3':4,5] [1,3]oxazolo[3,2-a]pyrimidine-6-one
(compound 57)
To a solution of compound 56 (8.61 g, 17.1 mmol) in
1,2-dimethoxyethane (198 mL) was added water (17.2 mL),
and thereto was added triphenylphosphine (4930 mg, 18.8
mmol) portion wise at 60 C (external temperature) while
observing a forming phenomenon. The mixtures were
stirred at the same temperature for 20 minutes, and then
stirred at 80 C as external temperature for 2 hours. The
reaction solutions were concentrated under reduced
pressure and the resulting residues were purified by
silica gel chromatography (chloroform/methanol, 100/0 to
70/30) to obtain compound 57 (8.25 g, containing 6.1wt%
of ethyl acetate, yield 100%).
MS (ESI): m/z 478 (Mi-H)
1H-NMR (CDC13) 6: 7.39 - 7.23 (8H , m), 7.18 (1H d, J =
1.0 Hz), 7.15 - 7.10 (2H , m), 6.11 (1H , d, J = 6.2
Hz, ), 5.23 (1H dd, J=6.2, 1.5 Hz), 4.72 (1H, d, J =
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11.8 Hz, ), 4.56 (1H , d, J=11.8 Hz), 4.35 (1H, d, J=11.8
Hz), 4.29 (1H, d, J = 1.5 Hz), 4.26 (1H , d, J = 11.8 Hz),
3.20 (1H , d, J = 10.3 Hz), 3.18 (1H, d, J = 10.3 Hz, ),
2.65 (2H, t, J = 7.2 Hz), 1.95 (3 H , d, J = 1.0 Hz, ),
1.80 - 1.29 (6H , m)
[0311]
[chem.132]
BO Bn0
0
H2N
Boc20
N
BocHN
,-14*-- 0 CH2Cl2
Brd 0 N Bn0
57 58
[0312]
Example 62
Tert-butyl (3-{(2R,3S,3aS,9aR)-3-(benzyloxy)-2-
[(benzyloxy)methy1]-7-methyl-6-oxo-2,3,3a,9a-tetrahydro-
6H-furo[2',3':4,5] [1,3]oxazolo[3,2-a]pyrimidin-2-
yl}propyl)carbamate (compound 58)
To a solution of compound 57 (7.54 g, 14.8 mmol) in
dichloromethane (98 mL) was added di-tert-butyl
bicarbonate (3560 mg, 16.3 mmol), and the mixtures were
stirred at room temperature for 3 hours. The reaction
solutions were concentrated under reduced pressure and
the resulting residues were purified by silica gel
chromatography (ethyl acetate/methanol, 100/0 to 97/3) to
obtain compound 58 (7.67 g, containing 1.1wt% of ethyl
acetate, yield 89%).
MS (ESI): m/z 578 (M+H)
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1H-NME (CDC13) 6: 7.40 - 7.27 (8H, m), 7.17 (1H, d, J =
1.5 Hz), 7.15 - 7.09 (2H , m), 6.09 (1H, d, J = 6.2 Hz),
5.20 (1H, dd, J = 6.4, 1.5 Hz), 4.71 (1H, d, J = 11.8 Hz),
4.56 (11-i, d, J = 11.8 Hz), 4.48 (1H, brs), 4.35 (1H, d, J
= 12.3 Hz), 4.28 (IH, d, J = 1.5 Hz), 4.24 (1H, d, J =
12.3 Hz), 3.17 (1H, d, J - 9.8 Hz), 3.14 (1H, d, J = 9.8
Hz), 3.11 - 3.03 (2H, m), 1.97 (3H, d, J = 1.0 Hz), 1.78
- 1.62 (2H, m), 1.55 - 1.48 (1H, m), 1.43 (9H, s), 1.42 -
1.33 (1H, m)
[0313]
[chem.133]
Bn0
Bn0 roy
N Na0Haq 1,:k- 0 N NH
__________________________________ lliw
BocHN/"--/ ,t ''Ir
BocHN
...:f 0
N THF
Bnd 'OH
58 59
[0314]
Example 63
1-{3-0-Benzy1-4-[(benzyloxy)methy1]-7-[(tert-
butoxycarbonyl)amino]-5,6,7-trideoxy-a-L-xy10-
heptofuranosy11-5-methylpyrimidine-2,4(111,3H)-dione
(compound 59)
To a solution of compound 58 (7.67 g, 13.1 mmol) in
THF (153 mL) was added 1M aqueous sodium hydroxide
solution (32.8 mL, 32.8 mmol) dropwise under ice-cooling,
and the ice bath was removed, and the mixtures were
stirred at room temperature for 6 hours. To the reaction
solutions were added ethyl acetate (200 mL) and 5%
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aqueous citric acid solution (120 mL), and the mixtures
were stirred for a while, and the organic layers were
then separated by a separatory funnel. The aqueous
layers were extracted with ethyl acetate (100 mL) by a
separatory funnel, and the combined organic layers were
washed with water (200 mL) and saturated brine (100 mL),
and dried over anhydrous sodium sulfate. The solvents
were evaporated under reduced pressure and the resulting
residues were purified by silica gel chromatography
(hexane/ethyl acetate, 90/10 to 0/100) to obtain compound
59 (7.82 g, containing 2.4wt% of ethyl acetate, yield
100%).
MS (ESI): 496 [M-Boc+H]
1H-NMR (CDC13) 5: 8.46 (1H, brs), 7.52 (1H, d, J = 1.0
Hz), 7.40 - 7.25 (10H, m), 6.07 (IH, d, J = 4.6 Hz), 4.77
(1H, d, J = 11.8 Hz), 4.61 - 4.46 (4H, m), 4.42 (1H, brs),
4.17-3.86 (2H, m), 3.73 (1H, d, J = 10.3 Hz), 3.47 (1H, d,
J = 10.3 Hz), 3.17-2.98 (2H, m), 1.77-1.45 (7H, m), 1.43
(9H, s)
[0315]
Ichem.134]
Bn0
rY9 Tf20
DMAP Bn0 eLe.0
PyridMe
BocHN CHP2 BocHN 8
Bnd OH Bn0 OTf
59 60
[03161
Example 64
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1-13-0-Eenzy1-4-[(benzyloxy)methyl]-7-[(tert-
butoxycarbonyl)amino]-5,6,7-trideoxy-2-0-
[(trifluoromethyl)sulfony1)-a-L-xylo-heptofuranosyl)-5-
methylpyrimidine-2,4(1H,3H)-dione (compound 60)
To a solution of compound 59 (7.82 g, 12.8 mmol) in
dichloromethane (63 mL) were added pyridine (15.6 mL, 193
mmol) and DMAP (3130 mg, 25.6 mmol), and thereto was
added trifluoromethanesulfonic anhydride (5.39 mL, 32.0
mmol) dropwise over 30 minutes under ice-cooling, and the
mixtures were stirred at the same temperature for 1 hour.
Thereto was added additionally trifluoromethanesulfonic
anhydride (2.16 mL, 12.8 mmol), and the mixtures were
stirred at the same temperature for 3 hours. To the
reaction solutions was added saturated aqueous sodium
bicarbonate solution (200 mL) under ice-cooling, and the
mixtures were stirred for a while. The organic layers
were separated by a separatory funnel, and the aqueous
layers were extracted with dichloromethane (100 mL) by a
separatory funnel. The combined organic layers were
washed with saturated brine, and dried over anhydrous
sodium sulfate. The solvents were evaporated under
reduced pressure and azeotroped with toluene three times.
The resulting residues were purified by silica gel
chromatography (hexane/ethyl acetate, 90/10 to 50/50) to
obtain compound 60 (8.24 g, containing 4.2wt% of ethyl
acetate, yield 88%).
MS (ESI): 628 [M-Boc+H]
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1H-NMR (CDC13) 6: 8.36 (1H, brs), 7.41 - 7.27 (9 H, m),
7.25 - 7.21 (2H, m), 6.31 (1H, d, J = 5.1 Hz), 5.44 -
5.38 (1H, m, ), 4.75 (IH, d, J = 11.8 Hz, ), 4.59 - 4.40
(5H, m), 3.52 (IH, d, J = 9.8 Hz), 3.40 (1H, d, J = 9.8
Hz) , 3.22 - 3.01 (2H, m), 1.79 - 1.51 (7H, m, ), 1.44
(9H, s)
[0317]
[chem.135]
eyBn0 Bn0
TFA
cH,c12
BocHN
Bnd OTf BnO``s--N-H
60 61
[0318]
Example 65
1-{(1R,6R,8R,9S)-9-(Benzyloxy)-6-[(benzyloxy)methyl]-7-
oxa-2-azabicyclo[4.2.11non-8-y1}-5-methylpyrimidine-
2,4(1H,3H)-dione (compound 61)
To a solution of compound 60 (8.24 g, 10.9 mmol) in
dichloromethane (124 mL) was added trifluoroacetic acid
(41.2 mL, 535 mmol) under ice-cooling, and the ice bath
was removed, and the mixtures were stirred at room
temperature for 2 hours. The reaction solutions were
concentrated under reduced pressure, and then azeotroped
with toluene. The resulting residues were dissolved in
dichloromethane (200 mL), and thereto was added saturated
aqueous sodium bicarbonate solution (100 mL) under ice-
cooling while stirring vigorously, and the mixtures were
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300
stirred at room temperature for 13 hours. The organic
layers of the reaction solutions were separated by a
separatory funnel, and the aqueous layers were extracted
with chloroform (50 mL portions were carried out twice)
by a separatory funnel. The combined organic layers were
dried over anhydrous sodium sulfate. The solvents were
evaporated and the resulting residues were purified by
silica gel chromatography (chloroform/methanol, 100/0 to
95/5) to obtain compound 61 (4.96 g, yield 96%).
MS (ESI): 478 [M+H]
1H-NMR (CDC13) .5: 8.24 (1H, brs), 7.97 (1H, d, J = 1.5
Hz), 7.40 - 7.26 (8H, m), 7.25 - 7.20 (2H, m), 5.86 (1H,
s), 4.76 (1H, d, J = 11.8 Hz), 4.56 (1H d , J= 11.8 Hz),
4.53 - 4.46 (2H, m), 4.40 (IH, d, J = 7.2 Hz), 3.72 (1H,
d, J=10.8 Hz), 3.58 (1H, d, J=7.2 Hz), 3.54 (1H, d,
J=10.8 Hz), 3.34 - 3.21 (1H, m), 3.14 - 3.03 (IH, m),
1.90 - 1.55 (4H, m), 1.41 (3H, d, J=1.5 Hz)
[0319]
[chem.136]
N NHBz
rjY Fit;_t N
7
BSAN
Bn0 .0N _Ix)7BZ
..s/b NHY N
TMSOTf N-----j .
,
K...i 0 Toluene .
BnC NH BnCi'X NJ
H
62 63
[0320]
Example 66
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N-(9-1(1R,5R,7R,8S)-8-(Benzyloxy)-5-[(benzyloxy)methy1]-
6-oxa-2-azabicyclo[3.2.1]oct-7-y11-9H-purin-6-
yl)benzamide (compound 63)
Compound 62 (1-{(1R,SR,7R,8S)-8-(benzyloxy)-5-
[(benzyloxy)methyli-6-oxa-2-azabicyclo[3.2.1]oct-7-y1}-5-
methylpyrimidine-2,4(1H,3H)-dione (prepared from 3,5-di-
0-benzy1-4-C-hydroxymethy1-1,2-0-isopropylidene-a-D-
ribofuranose 2 according to the method described in J. Am.
Chem. Soc. 2006, 128, 15173) (49.8 mg, 0.107 mmol) and N-
(9H-purin-6-yl)benzamide (77.1 mg, 0.322 mmol) were
placed in a 10 mL egg-plant shaped flask, and thereto
were added toluene (1 mL) and BSA (0.235 mL, 0.961 mmol).
The mixtures were stirred at 60 C as external temperature
for 15 minutes, and then warmed to 100 C as external
temperature, and thereto was added TMSOTf (0.005 mL,
0.026 mmol). The mixtures were stirred at the same
temperature for 30 minutes, and warmed to room
temperature, and thereto were added 396 aqueous sodium
bicarbonate solution (10 mL) and chloroform (10 mL), and
the mixtures were stirred. The organic layers were
separated, and the aqueous layers were extracted with
chloroform (10 mL). The organic layers were mixed, and
washed with water (10 mL). The aqueous layers were
extracted with chloroform (5 mL), and the extracts were
mixed with the organic layers. The mixed organic layers
were filtered through a cotton plug to remove solids.
The solvents were evaporated from the filtrates, and the
residues were subjected to reverse phase HPLC preparative
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302
chromatography (10mM aqueous ammonium carbonate
solution/acetonitrile = 60/40 to 30/70) and thin layer
chromatography (the development using chloroform/methanol
= 12/1 was carried out twice) to obtain compound 63 (4.9
mg, yield 8%).
MS (ESI): 577 [M+Hr
1H-NMR (CDC13) 5: 9.08 (1H, brs), 8.77 (1H, s), 8.72 (1H,
s), 8.05 (2H, d, J = 7.7 Hz), 7.65-7.59 (1H, m), 7.57-
7.51 (2H, m), 7.39-7.21 (10H, m), 6.45 (IH, s), 4.64 (1H,
d, J 12.3 Hz), 4.58 (1H, d, J = 12.3 Hz), 4.55 (1H, d,
J = 11.8 Hz), 4.47 (1H, d, J = 11.8 Hz), 4.23 (1H, d, J =
3.6 Hz), 3.71 (1H, d, J = 3.6 Hz), 3.66 (IH, d, J - 10.8
Hz), 3.52 (1H, d, J = 10.8 Hz), 3.26-3.16 (1H, m),
3.07(1H, dd, J = 13.9, 6.7 Hz), 2.07-1.97 (IH, m),
1.86(1H, brs), 1.37(111, dd, J = 12.8, 4.6 Hz)
[0321]
[chem.137]
N OCONPh2
N
H N OCONPh2
N "NHCOOr
Nx
Bn0
j\\
BSA
TMSOTf Bn0 N
II NHCO Pr
z 0 Toluene
BnO` `--1'114 BnO\ NH
62 64
[0322]
Example 67
9-{(1R,5R,7R,8S)-8-(Benzyloxy)-5-[(benzyloxy)methy1]-6-
oxa-2-azabicyclo[3.2.1]oct-7-y11-2-[(2-
methylpropanoyl)amino]-9H-purin-6-y1 diphenylcarbamate
(compound 64)
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303
Similarly to Example 66 from compound 62 (49.8 mg,
0.107 mmol) and [2-(2-methylpropanoylamino)-9H-purin-6-
yl] N,N-diphenylcarbamate (133.9 mg, 0.3216 mmol) (the
preparation method follows the method described in J. Org.
Chem. 1996, 61, 9207), compound 64 (24.4 mg, yield 30%)
was prepared.
MS (ESI): 754[M+H] +
1H-NMR (CDC13) 6: 8.65 (1H, s), 8.04 (1H, s), 7.49-7.42
(2H, m), 7.40-7.21 (18H, m), 6.37 (1H, s), 4.64 (1H, d, J
= 12.3 Hz), 4.56 (1H, d, J = 12.3 Hz), 4.53 (1H, d, J
11.8 Hz), 4.44 (1H, d, J = 11.8 Hz), 4.24 (1H, d, J = 3.6
Hz), 3.66 (1H, d, J = 3.6 Hz), 3.63 (1H, d, J = 10.8 Hz),
3.50 (1H, d, J = 10.8 Hz), 3.21-3.12 (1H, m), 3.04(1H, dd,
J = 13.9, 6.7 Hz), 2.90 (1H, brs), 2.07 (1H, brs), 2.06-
1.93(1H, m), 1.35(1H, dd, J = 12.8, 4.6 Hz), 1.27(3H, d,
J = 1.5 Hz), 1.25(3H, d, J = 1.5 Hz)
[0323]
[chem.138]
N NHBz
H(N'-141Y WHBz
an0 rke
Bn0 0 .e14-N
BSA
0 N NH TMSOTf 1--
)0,0
2-dichloroethane y
Bne\--RH
Br?-6H
61 65
[0324]
Example 68
N-(9-{(1R,6R,8R,9S)-9-(Benzyloxy)-6-[(benzyloxy)methy1]-
7-oxa-2-azabicyclo[4.2.11non-8-y11-9H-purin-6-
yl)benzamide (compound 65)
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Compound 61 (49.7 mg, 0.104 mmol) and N-(9H-purin-6-
yl)benzamide (74.7 mg, 0.312 mmol) were placed in an egg-
plant shaped flask, and thereto were added 1,2-
dichloroethane (1 mL) and BSA (0.230 mL, 0.941 mmol).
After the mixtures were stirred at 60 C as external
temperature for 15 minutes, thereto was added TMSOTf
(0.0050 mL, 0.026 mmol), and the mixtures were stirred at
60 C as external temperature for 3.5 hours. The mixtures
were cooled to room temperature, and thereto were added
3% aqueous sodium bicarbonate solution (10 mL) and
chloroform (15 mL), and the mixtures were stirred. The
organic layers were separated, and the aqueous layers
were extracted with CHC13 (10 mL). The organic layers
were mixed, and washed with tap water (10 mL). The
aqueous layers were extracted with CHC13 (5 mL), and the
extracts were mixed with the organic layers. The aqueous
layers were extracted with chloroform (5 mL). The
organic layers were mixed again, concentrated under
reduced pressure at 50 C, and then subjected to reverse
phase HPLC preparative chromatography (10 mM aqueous
ammonium carbonate solution/acetonitrile = 50/50 to
20/80) to obtain compound 65 (19.2 mg, yield 31%).
MS (ESI): 591[M+H]
1H-NMR (CDC13) 6: 9.00 (1H, brs), 8.80 (1H, s), 8.70 (1H,
s), 8.04 (2H, d, J = 7.2 Hz), 7.64-7.59 (1H, m), 7.56-
7.51 (2H, m), 7.37-7.24 (10H, m), 6.28 (1H, s), 4.65 (1H,
d, J = 6.7 Hz), 4.60 (1H, d, J = 11.3 Hz), 4.59 (IH, d, J
= 12.3 Hz), 4.52 (1H, d, J = 12.3 Hz), 4.47 (1H, d, J =
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11.3 Hz), 3.86 (1H, d, J = 6.7 Hz), 3.65 (1H, d, J = 10.8
Hz), 3.51-3.47 (2H, m), 3.36-3.28(1H, m), 3.17-3.08(1H,
m), 1.98-1.89 (2H, m), 1.83-1.67 (2H, m)
[0325]
[chem.139]
N OCONPh2
OCONPh2
Bn0
dy0 NHCO'Pr
Bn0
BSA
v. 0 NTNH TMSOTf 'vkCYNeDrµN
DP- " NHCOIPr
0
BnO`` NH 1, 2-dichloroethane
BnOLNH
61 66
[0326]
Example 69
9-{(1R,6R,8R,95)-9-(Benzyloxy)-6-[(benzyloxy)methyl]-7-
oxa-2-azabicyclo[4.2.1]non-8-y1}-2-[(2-
methylpropanoyl)amino]-9H-purin-6-y1 diphenylcarbamate
(compound 66)
Similarly to Example 66 from compound 61 (50.1 mg,
0.105 mmol) and [2-(2-methylpropanolylamino)-9H-purin-6-
yl] N,N-diphenylcarbamate (131.1 mg, 0.3148 mmol) (the
preparation method follows the method described in J. Org.
Chem. 1996, 61, 9207), compound 66 (42,4 mg, yield 53%-)
was prepared.
MS (ESI): 768 [M+H]*
1H-NMR (CDC13) 5: 8.60 (1H, s), 8.01 (1H, s), 7.47-7.42
(2H, m), 7.40-7.21 (18H, m), 6.18 (1H, s), 4.68 (1H, d, J
= 6.7 Hz), 4.62 (1H, d, J = 11.3 Hz), 4.60 (1H, d, J =
12.3 Hz), 4.51 (IH, d, J = 12.3 Hz), 4.47 (1H, d, J =
11.3 Hz), 3.84 (1H, d, J - 6.7 Hz), 3.63 (1H, d, J - 10.8
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Hz), 3.49 (1H, d, J = 10.8 Hz), 3.36-3.25 (1H, m), 3.13-
3.04(1H, m), 2.92(1H, brs), 1.97 (1H, s), 1.94-1.84 (1H,
m), 1.80-1.65 (3H, m), 1.26 (6H, d, J = 7.2 Hz)
[0327]
Example 70
Preparation and Purification of Oligonucleotide analogues
Using compound 16Ab prepared by Example 32, compound
16meCa prepared by Example 30, compound 16Ga prepared by
Example 28, and various commercially available
phosphoramidites, oligonucleotide analogues having
various nucleic acid base(s) were prepared by DNA/RNA
oligonucleotide automatic synthesizer nS-8 or nS-811 (the
both are manufactured by GeneDesign, Inc.) with 5'
terminal DMTr ON or OFF mode using 0.2 pmol scale of CPG
or polystyrene support. When GuNA monomer was subjected
to a coupling reaction, the coupling time of a solution
of the GuNA monomer in acetonitrile (0.1 M) was 8 minutes,
and the other steps were carried out under usual
oligonucleotide synthesis condition of nS-8 or nS-811.
5-Ethylthio-1H-tetrazole (0.25 M) was used as an
activating agent.
[0328]
The purification and confirmation of purity of the
synthesized oligonucleotide analogues were carried out by
reverse phase HPLC chromatography under the following
conditions.
Mobile phase
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307
A solution: 0.1M triethylammonium acetate buffer, pH
7.0
B solution: 0.1M triethylammonium acetate (water:
acetonitrile = 1:1) solution
Gradient: A:B = 95:5 ¨470:30 (23 min.) or A:B = 95:5 -4
50:50 (22.5 min.)
Used column:
Analysis: Waters XBridgeTM OST 018 2.5 pm (4.6 x 50
mm)
Preparation: Waters XBridgeTM OST C18 2.5 pm (10 x
50 mm)
Flow rate:
Analysis: 1 mL/min.
Preparation: 4 mL/min.
Column temperature: 50 C
Detection: UV (254 nm)
The molecular weight of the synthesized
oligonucleotide analogues was determined by a time-of-
flight mass spectrometry (MALDI ¨TOF ¨MS).
[0329]
Preparation of 5'-d(TTTTATTTTT)-3' (SEQ ID No. 1) (A:
guanidine bridged nucleic acid, compound 48)
Boc-protected oligonucleotide was prepared using
compound 16Ab prepared by Example 32 on 0.2 pmol of
polystyrene support (LV-PS dT 0.2 pmol, manufactured by
Glen Research), and the column after the synthesis was
charged by a solution of 20% trifluoroacetic acid in
methylene chloride and allowed to stand at room
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308
temperature for 1 hour. Methylene chloride and
acetonitrile were passed through the column successively
to wash the support, and the support was dried under
nitrogen stream, and further dried under reduced pressure.
Next, the column was filled with a solution of
ammonia in methanol (7N), and the column was allowed to
stand at room temperature for 1 hour, and the support was
then cleaved, and the support was washed with 28% aqueous
ammonia. The cleavage solution and the washing solution
were combined in a snap vial, and heated at 65 C as
external temperature for 6 hours. The mixtures were
concentrated under reduced pressure to about 80% volume,
and purified by reverse phase HPLC chromatography
(gradient: A:B = 95:5 70:30 (23 min.)).
Yield 27%.
MALDI-TOF-MS: m/z Theoretical value (M-H-) 3057.06,
Measured value (M-H) 3053.00
[0330)
Preparation method 2 of 5f-d(TTTTATTTTT)-3'(SEQ ID No. 1)
(A: guanidine bridged nucleic acid, compound 48)
Boc-protected oligonucleotide was prepared using
compound 16Ab prepared by Example 32 on CPG support (CPG
500A dT 0.2 pmol, manufactured by Prong LLC), and the
column after the synthesis was charged by a Boc-
deprotecting solution (a mixture of trimethylsilyl
triflate 940 pL, 2,6-dimethylpyridine 1,260 pL, and
methylene chloride 10 mL), and allowed to stand at room
temperature for 3 hours. The Boc-deprotecting solution
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was removed, and 10v/v% of 2,6-dimethylpyridine/methylene
chloride solution and methylene chloride were passed
through the column successively to wash the support.
Next, 28% aqueous ammonia was added thereto, and the
column was allowed to stand for 12 hours, and the
cleavage solution was transferred into a snap vial, and
heated at 65 C as external temperature for 5 hours. The
solution was concentrated under reduced pressure to half
volume, and purified by reverse phase simple column (Sep-
Pak (trademark) Plus C18 Cartridges, manufactured by
Waters Inc.) and subjected to reverse phase HPLC
purification (gradient: A:B = 95:5 -4 70:30 (23 min.)).
Yield 12%.
MALDI-TOF-MS: m/z Theoretical value (M-H-) 3057.06,
Measured value (M-H-) 3058.82
[0331]
Preparation method of 5'-d(TTTTmCTTTTT)-3'(SEQ ID No. 2)
(mC: guanidine bridged nucleic acid, compound 49)
Teoc-protected oligonucleotide was prepared using
compound 16meCa prepared by Example 30 on 0.2 pmol of
polystyrene support (LV-PS dT 0.2 pmol, manufactured by
Glen Research), and the support was transfered from the
column to a snap vial. A solution of tetrabutylammonium
fluoride in tetrahydrofuran (0.2 M) was added thereto,
and the mixtures were heated at 65 C as external
temperature for 8 hours, and then 28% aqueous ammonia
was added thereto, and the mixtures were heated at 65 C
as external temperature for 8 hours. The mixtures were
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concentrated under reduced pressure to about half volume,
and the residues were purified by a gel filtration
column ( llustraTM NAP"- 10 Column SephadexTM G-25 DNA
Grade, manufactured by GE Healthcare Inc.) and
successively reverse-phase simple column (Sep-Pak
(trademark) Plus C18 Cartridges, manufactured by Waters
Inc.), and further subjected to reverse phase HPC
purification (gradient: A:B = 95:5 --*50:50 (22.5 min.)).
Yield 19%.
MALDI-TOF-MS: m/z Theoretical value (M-W) 3047.06,
Measured value (M-H") 3046.06
[0332]
Preparation method of 5'-d(TTTTGTTTTT)-3' (SEQ ID No. 3)
(G: guanidine bridged nucleic acid, compound 50)
Teoc-protected oligonucleotide was prepared using
compound 16Ga prepared by Example 28 on CPG support (CPG
500A dT 0.2 pmol, manufactured by Prong LLC) according
to the above-mentioned preparation method 2 of compound
48, and the workup treatments from the deprotection
procedures to the purification procedures were carried
out similarly to compound 48.
Yield 23%.
MALDI-TOF-MS: m/z Theoretical value (M-H-) 3073.06,
Measured value (M-H-) 3071.24
[0333]
Since the result of a molecular weight measurement
by a time-of-flight mass spectrometry (MALDI-TOF¨MS)
showed good match with the theoretical value, it was
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confirmed that each of the desirable oligonucleotides was
obtained.
[0334]
Compounds 51 to 58 were prepared similarly. The
results are shown in Table 1.
Table 1: Oligonucleotide containing guanidine bridged
nucleic acid
[Table 1]
Oligonucleotide Time-of-flight mass
(the underlined part indicates spectrometry
guanidine bridged nucleic acid) calculated Measured
value value
(M-H-) (M-H-)
5'-d(GCGTTTTTTGCT)-3' 3701.45 3702.52
(compound 1.)(SEQ ID No.4)
5'-d(GCGTTATTTGCT)-3' 3710.48 3711.41
(compound 52)(SEQ ID No.5)
5' -d(GCGTTmCTTTGCT) -3' 3700.47 3702.23
(compound 53)(SEQ ID No.6)
5'-d(GCGTTGTTTGCT)-3' 3726.47 3728.41
(compound .-4)(SEQ ID No.7)
5'-d(TTTTTTTTTT)-3' 3186.18 3184.70
(compound 55)(SEQ ID No.8)
5'-d(AAAAAAAAAA)-3' 3276.33 3276.52
(compound 56)(SEQ ID No.9)
5' -d(TTTmCmCmCTTTT) -3' 3183.23 3186.41
(compound 57)(SEQ ID No.10)
5'-d(TTTGGGTTTT)-3' 3261.22 3263.47
(compound 58)(SEQ ID No.11)
As a comparison and contrast, oligonucleotides in
which the base sequences are identical to those of
compounds 51 to 58, and guanidine bridged nucleic acid is
not contained, and only naturally occurring nucleotides
are contained) (compounds 59 to 66 respectively) were
prepared according to the usual synthesis condition of
oligonucleotide, and then purified. Also, as
complementary strands that can form a double strand with
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the above-mentioned oligonucleotide, oligonucleotides in
which the base sequences are complementary sequences
against compounds 51 to 58, and guanidine bridged nucleic
acid is not contained, and only naturally occurring
nucleotides are contained (compounds 67 to 74
respectively), and oligonucleotides being RNA in which
the base sequences are complementary sequences against
compounds 51 to 54, and guanidine bridged nucleic acid is
not contained, and only naturally occurring nucleotides
are contained (compounds 75 to 78 respectively) were
prepared according to the usual synthesis condition of
oligonucleotide, and then purified.
[0335]
Example 71
Measurement of melting temperature (Tm)
Each of various oligonucleotides prepared by Example
70 (compounds 48 to 66) and the complementary
oligonucleotides (compounds 67 to 74, or compounds 75 to
78) were annealed to form a double strand, and then Tm
value that is a temperature at which 50% of the double
strand is deviated, was measured, and the double strand-
forming capacity of oligonucleotide was examined.
Specifically, regarding a sample solution (130 pL)
which contains sodium chloride 100 mM, sodium phosphate
buffer (pH 6.99) 10 mM, oligonucleotide 4 pM and the
complementary strands 4 pM, the compounds indicated in
Table 2 were warmed from room temperature to 95 C at a
rate of 5 C per minute and then cooled to 5 C at a rate
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of 0.5 C per minute, while the compounds indicated in
Table 3 were heated using the boiling water bath and
allowed to stand to cool to room temperature. Nitrogen
stream was passed into a cell chamber of
spectrophotometer (Shimadzu, UV-1650PC or UV-1800) so as
to prevent a condensation phenomenon, and the sample
solution was cooled to 5 C gradually, and kept at 5 C for
1 minute, and the measurement was then started. The
temperature was warmed moderately to 90 C at a rate of
0.5 C per minute, and the ultraviolet absorption at 260
nm was measured at every timing of 0.5 C rise. Here in
order to prevent a concentration change due to a
temperature rise, the cell with a lid was used. The
measurement results are shown in Table 2 as a Tm value
and the difference in Tm value per a modified unit. It
shows that the higher Tm value was, the higher the
capacity of forming double strand was.
[0336]
Table 2: Effect on Tm value by introduction of one
residue of guanidine bridged nucleic acid
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[Table 2]
Oligonucleotide Tm
(the underlined part indicates guanidine (ATm/modified
bridged nucleic acid) unit) ( C)
DNA* RNA*
5'-d(GCGTTTTTTGCT)-3' (compound 59) 53 47
(SEQ ID No.4)
5'-d(GCGTTTTTTGCT)-3' (compound 51) 56(+3) 53(4-6)
5'-d(GCGTTATTTGCT)-3' (compound 60) 50 46
(SEQ ID No.5)
5'-d(GCGTTATTTGCT)-3' (compound 52) 52(+2) 50(+4)
5'-d(GCGTTCTTTGCT)-3' (compound 61) 53 53
(SEQ ID No.6)
5' -d(GCGTTmCTTTGCT) -3' (compound 53) 60(+7) 60(+7)
5'-d(GCGTTGTTTGCT)-3' (compound 62) 55 52
(SEQ ID No.7)
5'-d(GCGTTGTTTGCT)-3' (compound 54) 60(+5) 57(+5)
*: base sequences of complementary strand
oligonucleotides:
' Complementary strands of compounds 51 and 59:
DNA/ 5'-d(AGCAAAAAACGC)-3' (compound 67) (SEQ ID
No.12),
RNA/ 5'-r(AGCAAAAAACGC)-3' (compound 75) (SEQ ID
No.13),
- Complementary strand of compounds 52 and 60:
DNA/ 5'-d(AGCAAATAACGC)-3' (compound 68) (SEQ ID
No.14),
RNA/ 5'-r(AGCAAATAACGC)-3' (compound 76) (SEQ ID
No.15),
= Complementary strand of compounds 53 and 61:
DNA/ 5'-d(AGCAAAGAACGC)-3' (compound 69) (SEQ ID
No.16),
RNA/ 5'-r(AGCAAAGAACGC)-3' (compound 77) (SEQ ID
No.17),
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Complementary strand of compounds 54 and 62:
DNA/ 5'-d(AGCAAACAACGC)-3' (compound 70) (SEQ ID
No.18),
RNA/ 5'-r(AGCAA7sCAACGC)-3' (compound 78) (SEQ ID
No.19).
[0337]
Table 3: Effect on Tm value by introduction of three
residues of guanidine bridged nucleic acid
[Table 3]
Oligonucleotide Complementary strand Tm
(the underlined part (DNA) (ATm/modif
indicates guanidine bridged led unit)
nucleic acid) ( C)
5'-d(TTTTTTTTTT)-3' 5'-d(AAAAAAAAAA)-3' 23
(compound 63) (SEQ ID No.8) (compound 71)
(SEQ ID No.20)
5'-d(TTTTTTTTTT)-3' 5'-d(AAAAAAAAAA)-3' 50(+9.0)
(compound 55) (compound 71)
5'-d(AAAAAAAAAA)-3' 5'-d(TTTTTTTTTT)-3' 23
(compound 64) (SEQ ID No.9) (compound 72)
(SEQ ID No.21)
5'-d(AAAAAAAAAA)-3' 5'-d(TTTTTTTTTT)-3' 37(+4.7)
(compound 56) (compound 72)
5'-d(TTTCCCTTTT)-3' 5'-d(AAAAGGGAAA)-3' 31
(compound 65) (SEQ ID No.10) (compound 73)
(SEQ ID No.22)
5' -d(TTTmCmCmCTTTT) -3' 5'-d(AAAAGGGAAA)-3' 52(+7.0)
(compound 57) (compound 73)
5'-d(TTTGGGTTTT)-3' 5'-d(AAAACCCAAA)-3' 36
(compound 66)(SEQ ID No.11) (compound74)
(SEQ ID No.23)
5'-d(TTTGGGTTTT)-3' 5'-d(AAAACCCAA1)-3' 56(+6.7)
(compound 58) (compound74)
[0338]
As evident from Tables 2 and 3, oligonucleotides
containing guanidine bridged artificial nucleic acid
showed excellent capacity of forming double strand for
all bases in the cases of not only RNA but also DNA.
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Also, it was found that for any base, the higher the
introduction ratio of guanidine bridged artificial
nucleic acid into oligonucleotide was, the higher the
rise of Tm value was. Accordingly, oligonucleotide of
the present invention in which guanidine bridged
artificial nucleic acid is introduced is considered an
oligonucleotide suitable for an antisense method.
Industrial Applicability
[0339]
According to the novel preparation method of bridged
nucleic acid GuNA of the present invention, the number of
steps for preparing bridged nucleic acid GuNA can be
shorten, and the yields of bridged nucleic acid GuNA can
be improved. Also, using the compound as a novel
intermediate of the present invention, bridged artificial
nucleic acid GuNA containing various kinds of nucleic
acid base can be prepared. The preparation method of the
present invention and the intermediate compound of the
present invention are useful for efficient preparation of
guanidine bridged nucleic acid GuNA, and the guanidine
bridged artificial nucleic acid GuNA oligomer can be used
for various pharmaceuticals, diagnosis, and experiment
and research.
[Sequence Listing Free Text]
[0340]
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SEQ ID Nos. 1 to 12, 14, 16, 18, and 20 to 23 show
DNA oligonucleotides.
SEQ ID Nos. 13, 15, 17, and 19 show RNA
oligonucleot ides.
317
