U.S. patent application number 09/941845 was filed with the patent office on 2002-02-21 for 4'-c-ethynyl pyrimidine nucleoside compounds.
Invention is credited to Kodama, Eiichi, Kohgo, Satoru, Machida, Haruhiko, Mitsuya, Hiroaki, Ohrui, Hiroshi, Shigeta, Shiro.
Application Number | 20020022722 09/941845 |
Document ID | / |
Family ID | 27471622 |
Filed Date | 2002-02-21 |
United States Patent
Application |
20020022722 |
Kind Code |
A1 |
Ohrui, Hiroshi ; et
al. |
February 21, 2002 |
4'-C-ethynyl pyrimidine nucleoside compounds
Abstract
The invention provides 4'-C-ethynyl pyrimidine nucleosides
(other than 4'-C-ethynylthymidine) represented by formula [I]: 1
wherein B represents a base selected from the group consisting of
pyrimidine and derivatives thereof; X represents a hydrogen atom or
a hydroxyl group; and R represents a hydrogen atom or a phosphate
residue; and a pharmaceutical composition containing any one of the
compounds and a pharmaceutically acceptable carrier. Preferably,
the composition is used as an anti-HIV agent or a drug for treating
AIDS.
Inventors: |
Ohrui, Hiroshi; (Sendai-shi,
JP) ; Shigeta, Shiro; (Fukushima-shi, JP) ;
Kodama, Eiichi; (Kyoto-shi, JP) ; Machida,
Haruhiko; (Choshi-shi, JP) ; Kohgo, Satoru;
(Sendai-shi, JP) ; Mitsuya, Hiroaki;
(Kumamoto-shi, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
27471622 |
Appl. No.: |
09/941845 |
Filed: |
August 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09941845 |
Aug 30, 2001 |
|
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09570041 |
May 12, 2000 |
|
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6291670 |
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Current U.S.
Class: |
536/26.8 ;
536/28.1 |
Current CPC
Class: |
C07H 19/06 20130101;
C07H 19/16 20130101; A61P 31/18 20180101 |
Class at
Publication: |
536/26.8 ;
536/28.1 |
International
Class: |
C07H 019/10; C07H
019/06 |
Foreign Application Data
Date |
Code |
Application Number |
May 12, 1999 |
JP |
11-131539 |
Jun 22, 1999 |
JP |
11-174920 |
Nov 9, 1999 |
JP |
11-318246 |
Mar 23, 2000 |
JP |
12-81117 |
Claims
1. A 4'-C-ethynyl pyrimidine nucleoside (other than
4'-C-ethynylthymidine) represented by the following formula [1]:
36wherein B represents a base selected from the group consisting of
pyrimidine and derivatives thereof; X represents a hydrogen atom or
a hydroxyl group; and R represents a hydrogen atom or a phosphate
residue.
2. A compound according to claim 1, wherein X is a hydrogen
atom.
3. A compound according to claim 1, wherein X is a hydroxyl
group.
4. A compound according to claim 1, wherein B is cytosine or a
derivative thereof.
5. A compound according to claim 1, wherein B is cytosine or a
derivative thereof, and X is a hydrogen atom.
6. A compound according to claim 1, wherein B is cytosine or a
derivative thereof, and X is a hydroxyl group.
7. A compound according to claim 1, wherein the compound is
4'-C-ethynyl-2'-deoxycytidine.
8. A compound according to claim 1, wherein the compound is
4'-C-ethynyl-2'-deoxy-5-fluorocytidine.
9. A compound according to claim 1, wherein the compound is
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)cytosine.
10. A pharmaceutical composition containing the 4'-C-ethynyl
pyrimidine nucleoside as recited in any one of claims 1 through 9
and a pharmaceutically acceptable carrier.
11. A pharmaceutical composition according to claim 10, which is an
anti-HIV agent.
12. A pharmaceutical composition according to claim 10, which is a
drug for the treatment of AIDS.
13. Use, as a therapeutic drug, of 4'-C-ethynyl pyrimidine
nucleoside as recited in any one of claims 1 through 9.
14. Use according to claim 13, wherein the therapeutic drug is an
anti-HIV agent.
15. Use according to claim 13, wherein the therapeutic drug is a
drug for the treatment of AIDS.
16. A method for the treatment of AIDS comprising administering, to
a mammal including a human, 4'-C-ethynyl pyrimidine nucleoside as
recited in any one of claims 1 through 9.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to 4'-C-ethynyl nucleosides
and the use thereof for producing pharmaceuticals, and more
particularly to the use thereof in treating acquired
immunodeficiency syndrome (AIDS).
[0003] 2. Background Art
[0004] The clinical setting for AIDS has been dramatically changed
by a multi-drug therapy called highly active antiretroviral
therapy, or HAART. In this therapy, nucleoside reverse
transcriptase inhibitors (NRTIs) such as zidovudine (AZT),
didanosine (ddI), zalcitabine (ddC), stavudine (d4T), and
lamivudine (3TC) and protease inhibitors (PIs) are employed in
combination. Application of this therapy has drastically decreased
the number of deaths due to AIDS in many countries (Textbook of
AIDS Medicine, p751 (Williams & Wilkins, Baltimore, 1999)).
[0005] In spite of the decrease in AIDS-related deaths due to
HAART, there has emerged a multi-drug resistant HIV-1 (human
immunodeficiency virus-1) mutant exhibiting cross-resistance to
various drugs. For example, in the early 1990s patients infected
with an HIV exhibiting resistance to both AZT and 3TC were very
rare, whereas the percentage of AIDS patients infected with such an
HIV was as high as 42% in 1995-1996 (AIDS, 11, 1184(1997)).
[0006] It has been reported that such multi-drug resistant viruses
cause 30-60% of drug failure cases in which the viremia level drops
once below the detection limit and then revives to exhibit lasting
viremia (AIDS, 12, 1631(1998)). Thus, the present status of AIDS
treatment is serious.
[0007] Conventionally, in terms of a compound which exhibits potent
antiviral activities against multi-drug resistant viruses, there
have been known only a few protease inhibitors; e.g., JE-2147,
which have potent antiviral activity against a multi-PI resistant
HIV-1 (Proc. Natl. Acad. Sci. USA, 96,8675(1999)). However, no
nucleoside derivative having such potent activities has been
reported yet.
[0008] Ohrui, one of the inventors of the present invention, has
synthesized 1-(4-C-ethynyl-.beta.-D-ribo-pentofuranosyl)thymine,
4'-C-ethynyluridine, and 4'-C-ethynylcytidine and assayed
biological activities such as antiviral and antitumor activities
thereof. However, no such biological activities have been observed
for these compounds (Biosci. Biotechnol. Biochem., 63(4), 736-742,
1999).
[0009] Furthermore, Matsuda et al. have synthesized
4'-C-ethynylthymidine and assayed the anti-HIV activity thereof.
The anti-HIV activity of the compound is weaker than that of AZT.
However, the assay described by Matsuda et al. (Bioorg. Med. Chem.
Lett., 9(1999), 385-388) is drawn to an ordinary assay for
determining anti-HIV activity on the basis of MT-4 cells versus an
HIV-1 III.sub.b strain, and does not use a multi-drug resistant
virus strain.
SUMMARY OF THE INVENTION
[0010] In order to find a compound having more potent antiviral
activity than AZT, the present inventors have synthesized a variety
of 4'-C-ethynyl nucleosides and evaluated the antiviral activity
thereof, and have found that: 1) a 4'-ethynyl nucleoside derviative
having a specific structure exhibits potent anti-HIV activity equal
to or greater than that of AZT; 2) the compound has potent
antiviral activity against a multi-drug resistant virus strain
exhibiting resistance to various anti-HIV drugs such as AZT, ddI,
ddC, d4T, and 3TC; and 3) the compound exhibits no significant
cytotoxicity. The present invention has been accomplished on the
basis of these findings.
[0011] Accordingly, the present invention provides 4'-C-ethynyl
nucleosides (other than 4'-C-ethynylthymidine) represented by
formula [I]: 2
[0012] wherein B represents a base selected from the group
consisting of pyrimidine, purine, and derivatives thereof; X
represents a hydrogen atom or a hydroxyl group; and R represents a
hydrogen atom or a phosphate residue.
[0013] The present invention also provides a pharmaceutical
composition containing any one of the compounds and a
pharmaceutically acceptable carrier.
[0014] Preferably, the composition is employed as an antiviral drug
or a drug for treating AIDS.
[0015] The present invention also provides use, as pharmaceuticals,
of compounds represented by formula [1].
[0016] The present invention also provides a method for treatment
of AIDS, comprising administering a compound of formula [1] to a
vertebrate, including human.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
(1) Compounds
[0017] The compounds of the present invention are represented by
formula [I]. Examples of bases in formula [I] represented by B
include pyrimidines; purines, including azapurines and
deazapurines; and derivatives thereof.
[0018] Examples of substituents in the bases includes a halogen
atom, an alkyl group, a haloalkyl group, an alkenyl group, a
haloalkenyl group, an alkynyl group, an amino group, an alkylamino
group, a hydroxyl group, a hydroxyamino group, an aminoxy group, an
alkoxy group, a mercapto group, an alkylmercapto group, an aryl
group, an aryloxy group, and a cyano groups The number and
substitution site of these substituents are not particularly
limited.
[0019] Examples of halogen atoms serving as substituents include
chlorine, fluorine, iodine, and bromine. Examples of alkyl groups
include C1-C7 alkyl group such as methyl, ethyl, and propyl.
Examples of haloalkyl groups include C1-C7 haloalkyl groups such as
fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl, and
bromoethyl. Examples of alkenyl groups include C2-C7 alkenyl groups
such as vinyl and allyl. Examples of haloalkenyl groups include
C2-C7 haloalkenyl groups such as bromovinyl and chlorovinyl.
Examples of alkynyl groups include C2-C7 alkynyl groups such as
ethynyl and propynyl. Examples of alkylamino groups include C1-C7
alkylamino groups such as methylamino and ethylamino.
[0020] Examples of alkoxy groups include C1-C7 alkoxy groups such
as methoxy and ethoxy. Examples of alkylmercapto groups include
C1-C7 alkylmercapto groups such as methylmercapto and
ethylmercapto. Examples of aryl groups include a phenyl group;
alkylphenyl groups having a C1-C5 alkyl such as methylphenyl and
ethylphenyl; alkoxyphenyl groups having a C1-C5 alkoxy such as
methoxyphenyl and ethoxyphenyl; alkylaminophenyl groups having a
C1-C5 alkyl such as dimethylaminophenyl and diethylaminophenyl; and
halogenophenyl groups such as chlorophenyl and bromophenyl.
[0021] Examples of pyrimidine bases and derivatives thereof include
cytosine, uracil, 5-fluorocytosine, 5-fluorouracil,
5-chlorocytosine, 5-chlorouracil, 5-bromocytosine, 5-bromouracil,
5-iodocytosine, 5-iodouracil, 5-methylcytosine, 5-ethylcytosine,
5-methyluracil (thymine), 5-ethyluracil, 5-fluoromethylcytosine,
5-fluorouracil, 5-trifluorocytosine, 5-trifluorouracil,
5-vinyluracil, 5-bromovinyluracil, 5-chlorovinyluracil,
5-ethynylcytosine, 5-ethynyluracil, 5-propynyluracil,
pyrimidin-2-one, 4-hydroxyaminopyrimidin-2-one,
4-aminoxypyrimidin-2-one, 4-methoxypyrimidin-2-one,
4-acetoxypyrimidin-2-one, 4-fluoropyrimidin-2-one, and
5-fluoropyrimidin-2-one.
[0022] Examples of purine bases and derivatives thereof include
purine, 6-aminopurine (adenine), 6-hydroxypurine, 6-fluoropurine,
6-chloropurine, 6-methylaminopurine, 6-dimethylaminopurine,
6-trifluoromethylaminopurine, 6-benzoylaminopurine,
6-acethylaminopurine, 6-hydroxyaminopurine, 6-aminoxypurine,
6-methoxypurine, 6-acetoxypurine, 6-benzoyloxypurine,
6-methylpurine, 6-ethylpurine, 6-trifluoromethylpurine,
6-phenylpurine, 6-mercaputopurine, 6-methylmercaputopurine,
6-aminopurine-1-oxide, 6-hydroxypurine-1-oxide,
2-amino-6-hydroxypurine (guanine), 2,6-diaminopurine,
2-amino-6-chloropurine, 2-amino-6-iodepurine, 2-aminopurine,
2-amino-6-mercaptopurine, 2-amino-6-methylmercaptopurine,
2-amino-6-hydroxyaminopurine, 2-amino-6-methoxypurine,
2-amino-6-benzoyloxypurine, 2-amino-6-acetoxypurine,
2-amino-6-methylpurine, 2-amino-6-cyclopropylaminomethylpurine,
2-amino-6-phenylpurine, 2-amino-8-bromopurine, 6-cyanopurine,
6-amino-2-chloropurine (2-chloroadenine), 6-amino-2-fluoropurine
(2-fluoroadenine), 6-amino-3-deazapurine, 6-amino-8-azapurine,
2-amino-6-hydroxy-8-azapurine, 6-amino-7-deazapurine,
6-amino-1-deazapurine, and 6-amino-2-azapurine.
[0023] When B is a pyrimidine base and X is a hydrogen atom,
examples of compounds represented by formula [I] include the
following compounds:
[0024] 4'-C-ethynyl-2'-deoxycytidine,
[0025] 4'-C-ethynyl-2'-deoxy-5-halogenocytidine,
[0026] 4'-C-ethynyl-2'-deoxy-5-alkylcytidine,
[0027] 4'-C-ethynyl-2'-deoxy-5-haloalkylcytidine,
[0028] 4'-C-ethynyl-2'-deoxy-5-alkenylcytidine,
[0029] 4'-C-ethynyl-2'-deoxy-5-haloalkenylcytidine,
[0030] 4'-C-ethynyl-2'-deoxy-5-alkynylcytidine,
[0031] 4'-C-ethynyl-2'-deoxy-5-halogenouridine,
[0032] 4'-C-ethynyl-2'-deoxy-5-alkyluridine (other than
4'-C-ethynylthymidine),
[0033] 4'-C-ethynyl-2'-deoxy-5-haloalkyluridine,
[0034] 4'-C-ethynyl-2'-deoxy-5-alkenyluridine,
[0035] 4'-C-ethynyl-2'-deoxy-5-haloalkenyluridine, and
[0036] 4'-C-ethynyl-2'-deoxy-5-alkynyluridine, and 5'-phoshate
esters thereof.
[0037] When B is a pyrimidine base and X is a hydroxyl group,
examples of compounds represented by formula [I] include the
following compounds:
[0038] 1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)cytosine,
[0039]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-halogenocytosine,
[0040]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-alkylcytosine,
[0041]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-haloalkylcytosine-
,
[0042]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-alkenylcytosine,
[0043]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-haloalkenylcytosi-
ne,
[0044]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-alkynylcytosine,
[0045]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-halogenouracil,
[0046]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-alkyluracil,
[0047]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-haloalkyluracil,
[0048]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-alkenyluracil,
[0049]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-haloalkenyluracil-
, and
[0050]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-alkynyluracil,
and 5'-phoshate esters thereof.
[0051] When B is a purine base and X is a hydrogen atom, examples
of compounds represented by formula [I] include the following
compounds:
[0052] 4'-C-ethynyl-2'-deoxyadenosine,
[0053] 4'-C-ethynyl-2'-deoxyguanosine,
[0054] 4'-C-ethynyl-2'-deoxyinosine,
[0055] 9-(4-C-ethynyl-2-deoxy-.beta.-D-ribo-furanosyl)purine,
and
[0056]
9-(4-C-ethynyl-2-deoxy-.beta.-D-ribo-furanosyl)-2,6-diaminopurine,
and 5'-phoshate esters thereof.
[0057] When B is a purine base and X is a hydroxyl group, examples
of compounds represented by formula [I] include the following
compounds:
[0058] 9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)adenine,
[0059] 9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)guanine,
[0060]
9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)hypoxanthine,
[0061] 9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)purine,
and
[0062]
9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-2,6-diaminopurine,
and 5'-phoshate esters thereof.
[0063] Examples of preferred compounds of the present invention
includes the following compounds:
[0064] (i) 4'-C-ethynyl pyrimidine nucleosides including
[0065] (1) a compound represented by formula [I] wherein X is a
hydrogen atom,
[0066] (2) a compound represented by formula [I] wherein X is a
hydroxyl group,
[0067] (3) a compound represented by formula [I] wherein B is
cytosine,
[0068] (4) a compound represented by formula [I] wherein B is
cytosine and X is a hydrogen atom,
[0069] (5) a compound represented by formula [I] wherein B is
cytosine and X is a hydroxyl group,
[0070] (6) 4'-C-ethynyl-2'-deoxycytidine,
[0071] (7) 4'-C-ethynyl-2'-deoxy-5-fluorocytidine, and
[0072] (8) 1-(4-C-ethynyl-.beta.-D-arabinofuranosyl)cytosine,
and
[0073] (ii) 4'-C-ethynyl purine nucleosides including
[0074] (1) a compound represented by formula [I] wherein X is a
hydrogen atom,
[0075] (2) a compound represented by formula [I] wherein X is a
hydroxyl group,
[0076] (3) a compound represented by formula [I] wherein B is
selected from the group consisting of adenine, guanine,
hypoxanthine, and diaminopurine,
[0077] (4) a compound represented by formula [I] wherein B is
selected from the group consisting of adenine, guanine,
hypoxanthine, and diaminopurine and X is a hydrogen atom,
[0078] (5) a compound represented by formula [I] wherein B is
selected from the group consisting of adenine, guanine,
hypoxanthine, and diaminopurine and X is a hydroxyl group,
[0079] (6) 4'-C-ethynyl-2'-deoxyadenosine,
[0080] (7) 4'-C-ethynyl-2'-deoxyguanosine,
[0081] (8) 4'-C-ethynyl-2'-deoxyinosine,
[0082] (9)
9-(4-C-ethynyl-2-deoxy-.beta.-D-ribo-pentofuranosyl)-2,6-diamin-
opurine, and
[0083] (10)
9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)adenine.
[0084] The compounds of the present invention may be salts,
hydrates, or solvates. When R is a hydrogen atom, examples of salts
include acid-adducts such as hydrochlorides and sulfates. When R is
a phosphate residue, examples of salts include alkali metal salts
such as sodium salts, potassium salts, and lithium salts; alkaline
earth metal salts such as calcium salts; and ammonium salts. These
salts are pharmaceutically acceptable.
[0085] Examples of hydrates or solvates include adducts comprising
one molecule of the compound of the present invention or a salt
thereof and 0.1-3.0 molecules of water or a solvent. In addition,
the compounds of the present invention encompass a variety of
isomers thereof such as tautomers.
(2) Method of Production
[0086] One of the compounds of the present invention in which X is
a hydrogen atom; i.e., a 2'-deoxy derivative, can be produced by
the following steps.
[0087] First Step;
[0088] In the first step, a hydroxymethyl group at the 4-position
of the compound represented by [II] is oxidized to thereby form an
aldehyde, which is further converted into an alkyne to thereby
yield a compound represented by formula [III]: 3
[0089] wherein each of R1 and R2 represents a protective group; R3
represents a hydrogen atom or a protective group; and Bn represents
a benzyl group.
[0090] The starting material of the reaction is a known compound
represented by formula [II] (Biosci. Biotech. Biochem., 57,
1433-1438(1993)).
[0091] Each of R1 and R2 may be a protective group which is
typically employed for protecting a hydroxyl group. Examples of
types of a protective moiety containing R1 or R2 include an ether
type, an acyl type, a silyl type, and an acetal type. Specific
examples protective groups include a silyl group, an acetyl group,
a benzyl group, and an isopropylidenyl group.
[0092] When the hydroxymethyl group at the 4-position of the
compound represented by [II] is converted into an aldehyde group by
use of an oxidizing agent, examples of oxidizing agents include a
chromium-containing oxidizing agent such as chromic
anhydride-pyridine-acetic anhydride composite reagent, pyridinium
chlorochromate, or pyridinium dichromate; a high-valency iodine
oxidizing agent such as Dess-Martin reagent; and a
dimethylsulfoxide-based oxidizing agent such as a combination of
dimethylsulfoxide and any one of acetic anhydride, oxalyl chloride,
or dicyclohexyl carbodiimide.
[0093] Reaction conditions vary depending on an employed oxidizing
agent. For example, when oxidation is carried out by use of oxalyl
chloride and dimethyl sulfoxide, oxaly chloride in an amount of
0.5-5 mol and dimethyl sulfoxide in an amount of 1.5-6 mol are
added to 1 mol of a compound represented by formula [II] in an
organic solvent such as dichloromethane optionally under an inert
gas such as argon or nitrogen. The mixture is then allowed to react
for approximately 15 minutes to two hours at -100.degree. C. to
0.degree. C. Subsequently, a base such as triethylamine is added in
an amount of 2-10 mol to the mixture, and the resultant mixture is
further allowed to react at room temperature for approximately 15
minutes to two hours.
[0094] The thus-formed aldehyde can be converted into a
corresponding alkyne through carbon-increasing (i.e., C-C bond
formation) reaction of the aldehyde; treating the resultant
compound with a strong base to thereby form a metal alkynyl
compound; and introducing a protective group to the metal alkynyl
compound.
[0095] Carbon-increasing reaction may be carried out in an organic
solvent such as dichloromethane or dichloroethane, optionally under
an inert gas such as argon or nitrogen. Specifically, 1 mol of the
above-produced aldehyde is reacted with 1-5 mol of carbon
tetrabromide and 2-10 mol of triphenylphosphine at 0-50.degree. C.
for approximately 15 minutes to three hours.
[0096] Treatment with a strong base may be carried out in an
organic solvent such as tetrahydrofuran, 1,4-dioxane, or
dimethoxyethane, optionally under an inert gas such as argon or
nitrogen. Specifically, 1 mol of a compound obtained through
carbon-increasing reaction is reacted with 2-4 mol of a lithium
compound such as n-butyllithium or t-butyllithium at -100.degree.
C. to -20.degree. C. for approximately 5-60 minutes.
[0097] Furthermore, when a silyl protective group represented by R3
is introduced into an alkynyl group in the thus-obtained compound,
the aforementioned treatment is followed by addition of a
silylating agent such as chlorotriethylsilane. A protective group
can be introduced to a hydroxyl group by use of a customary method.
For example, an acetyl group may be introduced through reaction
with an acetylating agent such as acetic anhydride.
[0098] The thus-obtained compound represented by formula [III] may
be isolated and purified through a manner which is employed for
isolating and purifying typical protected saccharides. For example,
the crude compound is partitioned by use of an ethyl
acetate-saturated sodium bicarbonate solution, and the isolated
compound is purified by use of a silica gel column.
[0099] Second step;
[0100] The second step includes condensation of a compound
represented by formula [III] and a base represented by B;
deoxygenation at the 2'-position; removing a protective group of a
saccharide portion; and optionally phosphorylating the hydroxyl
group at the 5'-position, to thereby produce a compound represented
by formula [I]: 4
[0101] wherein B represents a base selected from the group
consisting of pyrimidine; purine, including azapurine or
deazapurine; and a derivative thereof (other than thymine); R
represents a hydrogen atom or a phosphate residue; each of R1 and
R2 represents a protective group; R3 represents a hydrogen atom or
a protective group; and Bn represents a benzyl group.
[0102] Condensation of a compound represented by formula [III] and
a base represented by B can be carried out by reacting the compound
with the base in the presence of a Lewis acid.
[0103] The base represented by B may be silylated, and silylation
may be carried out through a known method. For example, a base is
silylated by use of hexamethylsilazane and trimethylchlorosilane
under reflux.
[0104] Examples of Lewis acids include trimethylsilyl
trifluoromethanesulfonate, tin tetrachloride, zinc chloride, zinc
iodide, and anhydrous aluminum chloride.
[0105] Condensation reaction may be carried out in an organic
solvent such as dichloromethane, 1,2-dichloroethane, acetonitrile,
or toluene, optionally under an inert gas such as argon or
nitrogen. Specifically, 1 mol of a compound represented by formula
[III] is reacted with 1-10 mol of a base represented by B and
0.1-10 mol of Lewis acid at -20.degree. C. to 150.degree. C. for
approximately 30 minutes to three hours.
[0106] Deoxygenation at the 2'-position may be carried out by
converting the derivative having a hydroxyl group to the derivative
having a group such as halogeno, phenoxythiocarbonyl,
thiocarbonylimidazolyl, or methyldithiocarbonyl and reducing the
converted derivative using a radical reducing agent in the presence
of a radical initiator.
[0107] For example, when deoxygenation is carried out through
phenoxythiocarbonate, conversion of a hydroxyl group to a
phenoxythiocarbonyl group may be carried out in an organic solvent,
such as tetrahydrofuran, acetonitrile, or dichloromethane, in the
presence of a base such as dimethylaminopyridine or pyridine,
optionally under an inert gas such as argon or nitrogen.
Specifically, 1 mol of the aforementioned condensation product in
which only the protective group for the hydroxyl group at the
2'-position had been eliminated is reacted under stirring with 1-10
mol, preferably 1.1-2 mol, of a phenyl chlorothionoformate
derivative at 0-50.degree. C. for approximately 0.5-5 hours.
Alternatively, when deoxygenation is carried out via a bromo
compound, the bromination may be carried out in an organic solvent,
such as tetrahydrofuran, acetonitrile, or dichloromethane, by use
of a brominating agent such as acetyl bromide at 0-150.degree. C.
for approximately 0.5-5 hours, optionally under an inert gas such
as argon or nitrogen. The brominating agent is used in an amount of
1-50 mol, preferably 5-20 mol, per mol of the aforementioned
condensate from which a protective group at the 2'-position had
been removed.
[0108] Subsequently, reduction may be carried out in an organic
solvent such as toluene or benzene in the presence of a radical
initiator such as azobisisobutyronitrile, optionally under an inert
gas such as argon or nitrogen. Specifically, 1 mol of the
aforementioned phenoxythiocarbonate or bromide is reacted under
stirring with 1-10 mol, preferably 2-5 mol, of a radical reducing
agent such as tributyltin hydride at 50-150.degree. C. for
approximately 1-5 hours.
[0109] One of the compounds of the present invention in which X is
a hydroxyl group; i.e., an arabino derivative, can be produced by
the following steps.
[0110] First step;
[0111] The first step includes condensation of a compound
represented by formula [III] and a base represented by B;
stereochemically inverting the hydroxyl group at the 2'-position to
be an arabino form; removing a protective group of a saccharide
portion; and optionally phosphorylating the hydroxyl group at the
5'-position, to thereby produce a compound represented by formula
[I]: 5
[0112] wherein B represents a base selected from the group
consisting of pyrimidine, purine including azapurine or
deazapurine, and a derivative thereof; R represents a hydrogen atom
or a phosphate residue; each of R1 and R2 represents a protective
group; R3 represents a hydrogen atom or a protective group; and Bn
represents a benzyl group.
[0113] Condensation of a compound represented by formula [III] and
a base represented by B can be carried out by reacting the compound
with the base in the presence of a Lewis acid.
[0114] The base represented by B may be silylated, and silylation
may be carried out through a known method. For example, a base is
silylated by use of hexamethylsilazane and trimethylchlorosilane
under reflux.
[0115] Examples of Lewis acids include trimethylsilyl
trifluoromethanesulfonate, tin tetrachloride, zinc chloride, zinc
iodide, and anhydrous aluminum chloride.
[0116] Condensation reaction may be carried out in an organic
solvent such as dichloromethane, 1,2-dichloroethane, acetonitrile,
or toluene, optionally under an inert gas such as argon or
nitrogen. Specifically, 1 mol of a compound represented by formula
[III] is reacted with 1-10 mol of a base represented by B and
0.1-10 mol of Lewis acid at -20.degree. C. to 150.degree. C. for
approximately 30 minutes to three hours.
[0117] Stereo-inversion of the hydroxyl group at the 2'-position
can be carried out by converting a compound containing the hydroxyl
into a corresponding 2,2'-anhydrocyclonucleoside and hydrolyzing
the nucleoside. Anhydrocyclization may be carried out through
treatment with a sulfonating agent such as methanesulfonyl
chloride, or through treatment with a fluorinating agent such as
diethylaminosulfur trifluoride.
[0118] For example, when diethylaminosulfur trifluoride is
employed, anhydrocyclization may be carried out in an organic
solvent such as dichloromethane or toluene, optionally under an
inert gas such as argon or nitrogen. Specifically, 1 mol of the
aforementioned condensation product in which the protective group
for the hydroxyl group at the 2'-position was removed is reacted
with 1.1-5 mol, preferably 1.5-2 mol, of diethylaminosulfur
trifluoride at 0.degree. C. to room temperature for approximately
five minutes to 2 hours. Alternatively, when methanesulfonyl
chloride is employed, anhydrocyclization may be carried out in an
organic solvent such as pyridine, optionally under an inert gas
such as nitrogen. Specifically, 1 mol of the aforementioned
condensation product in which the protective group for the hydroxyl
group at the 2'-position had been eliminated is reacted with 1.1-5
mol, preferably 1.5-2 mol, of methanesulfonyl chloride at
0-50.degree. C. for approximately five minutes to 10 hours.
[0119] Subsequently, hydrolysis may be carried out in the presence
of an appropriate base or acid catalyst. For example, when a base
catalyst is employed, hydrolysis may be carried out in a solvent
mixture comprising water and an alcoholic solvent such as ethanol
in the presence of a base such as sodium hydroxide or potassium
hydroxide at room temperature to 100.degree. C. for approximately
30 minutes to 5 hours.
[0120] In the case in which a base represented by B in the target
compound; i.e., 4'-ethynylnucleoside, is a base having an amino
group, the target compound may also be produced from a
hydroxyl-containing base compound through a known method. Four
example, if the 4-position of a pyrimidine base is sought to be
aminated, the hydroxyl group at the 4-position of a pyrimidine base
may be converted into a group such as chloro, silyloxy, alkyloxy,
sulfonyloxy, or triazolyl, and then the converted group is reacted
with ammonia. For example, amination through a triazole derivative
may be carried out with stirring in an organic solvent such as
dichloromethane, acetonitrile, dimethylformamide, or pyridine in
the presence of a base such as triethylamine (triethylamine may be
omitted if pyridine is used as a solvent) and a phosphorylating
agent such as 4-chlorophenylphosphorodichloridate, optionally under
an inert gas such as argon or nitrogen. Specifically, 1 mol of the
aforementioned condensation product is reacted with 1-20 mol,
preferably 2-10 mol, of 1,2,4-triazole at 0.degree. C. to room
temperature for approximately 12-72 hours, followed by addition of
aqueous ammonia in an appropriate amount and further reaction at 0C
to room temperature for approximately 1-12 hours.
[0121] In addition, an amino group in a base may be removed through
a conventional method making use of any of a variety of deaminases,
such as adenosine deaminase or cytidine deaminase.
[0122] Finally, a protective group of the thus-produced nucleoside
is removed, to thereby obtain the compounds (R=H) of the present
invention.
[0123] A protective group may be removed through a method
appropriately selected from a routine procedure such as hydrolysis
under acidic conditions, hydrolysis under basic conditions,
treatment with tetrabutylammonium fluoride, or catalytic reduction,
in accordance with the protective group employed.
[0124] When R in a target compound is a phosphate residue such as
monophosphate or diphosphate, a compound in which R is a hydrogen
atom is reacted with a phosphorylating agent; e.g., phosphorus
oxychloride or tetrachloropyrophosphoric acid, which selectively
phosphorylates the 5'-position of a nucleoside, to thereby produce
a target compound in a free or salt form.
[0125] The compounds of the present invention may be isolated and
purified through conventional methods, in appropriate combination,
which are employed for isolating and purifying nucleosides and
nucleotides; e.g., recrystallization, ion-exchange column
chromatography, and adsorption column chromatography. The
thus-obtained compounds may further be converted to a salt thereof
in accordance with needs.
(3) Use
[0126] As shown in the below-described Test Examples, the compounds
of the present invention exhibit excellent antiviral activity
against herpesvirus or retrovirus. Thus, the compositions of the
present invention containing one of the compounds of the present
invention as an active ingredient can be used as therapeutic drugs.
Specifically, the compositions of the present invention are useful
for the treatment of infectious diseases caused by herpesvirus or
retrovirus, in particular, AIDS, which is caused by HIV
infection.
[0127] Examples of target viruses include viruses belonging to
Herpesviridae such as herpes simplex virus type 1, herpes simplex
virus type 2, or varicella-zoster virus, and Retroviridae such as
human immunodeficiency virus.
[0128] The dose of the compounds of the present invention depends
on and is determined in consideration of conditions such as the
age, body weight, and type of disease of the patient; the severity
of a disease of the patient; the drug tolerance; and the
administration route. However, the dose per day and per body weight
is selected typically within 0.00001-1,000 mg/kg, preferably
0.0001-100 mg/kg. The compounds are administered in a single or
divided manner.
[0129] Any administration route may be employed, and the compounds
may be administered orally, parenterally, enterally, or
topically.
[0130] When a pharmaceutical is prepared from the compounds of the
present invention, the compounds are typically mixed with
customarily employed additives, such as a carrier and an excipient.
Examples of solid carriers include lactose, kaolin, sucrose,
crystalline cellulose, corn starch, talc, agar, pectin, stearic
acid, magnesium stearate, lecitin, and sodium chloride. Examples of
liquid carriers include glycerin, peanut oil, polyvinylpyrrolidone,
olive oil, ethanol, benzyl alcohol, propylene glycol, and
water.
[0131] The dosage form is arbitrarily selected. When the carrier is
solid, examples of dosage forms include tablets, powder, granules,
capsules, suppositories, and troches, whereas when it is liquid,
examples include syrup, emulsion, soft-gelatin-encapsulated, cream,
gel, paste, spray, and injection.
[0132] As shown in the below-described results of Test Examples,
the compounds of the present invention exhibit excellent anti-HIV
activity, particularly against multi-drug resistant HIV strains
having resistance to various of anti-HIV drugs such as AZT, DDI,
DDC, D4T, and 3TC. The compounds have no significant cytotoxicity.
Thus, the compounds of the present invention are expected to be
developed for producing pharmaceuticals, particularly drugs for
treating AIDS.
EXAMPLE
[0133] The present invention will next be described in detail by
way of examples including Synthesis Examples, Test Examples, and
Drug Preparation Examples, which should not be construed as
limiting the invention thereto.
[0134] Synthesis Example 1
[0135] (1) Synthesis of
4-C-formyl-3,5-di-O-benzyl-1,2-O-isopropylidene-.a-
lpha.-D-ribo-pentofuranose (Compound 2) 6
[0136] Oxalyl chloride (3.38 ml, 38.7 mmol) was dissolved in
dichloromethane (80.0 ml), and dimethylsulfoxide (5.50 ml, 77.5
mmol) was added dropwise to the solution at -78.degree. C. in an
argon atmosphere, followed by stirring for 15 minutes at the same
temperature. A solution (100 ml) of
4-C-hydroxymethyl-3,5-di-O-benzyl-1,2-O-isopropylidene-.alpha-
.-D-ribo-pentofuranose (Compound 1) (10.3 g, 25.7 mmol) in
dichloromethane was added dropwise to the solution at -78.degree.
C., and the mixture was stirred for 30 minutes. After triethylamine
(10.9 ml, 77.6 mmol) was added thereto, the reaction mixture was
allowed to warm to room temperature, followed by stirring for 30
minutes. After water was added to the mixture with stirring, the
organic layer was dried over anhydrous magnesium sulfate and was
concentrated through distillation under reduced pressure. The
residue was purified by means of silica gel column chromatography
(silica gel 1500 ml, eluent; n-hexane:ethyl acetate=2:1), to
thereby yield a colorless viscous compound (Compound 2; 9.68 g,
24.3 mmol, 94.1%).
[0137] .sup.1H-NMR(CDCl.sub.3) .delta.9.92 (1H, s, formyl),
7.33-7.24 (10H, m, aromatic), 5.84 (1H, d, H-1 J.sub.1,2=3.30),
4.71, 4.59 (each 1H, d, benzyl, J.sub.gem=12.00), 4.60 (1H, br.t,
H-2), 4.52, 4.46 (each 1H, d, benzyl, J.sub.gem=12.00), 4.37 (1H,
d, H-3, J.sub.2,3=4.50), 3.68, 3.61 (each 1H, d, H-5,
J.sub.gem=10.95), 1.60, 1.35 (each 3H, s, acetonide)
[0138] EIMS m/z: 398(M.sup.+).
[0139] HRMS m/z(M.sup.+): Calcd. for C.sub.23H.sub.26O.sub.6:
398.1729, Found: 398.1732
[0140] [.alpha.].sub.D+24.5.degree. (c=1.03, CHCl.sub.3)
[0141] (2) Synthesis of
4-C-(2,2-dibromoethenyl)-3,5-di-O-benzyl-1,2-O-iso-
propylidene-.alpha.-D-ribo-pentofuranose (Compound 3) 7
[0142] Compound 2 (9.50 g, 23.8 mmol) was dissolved in
dichloromethane (200 ml), and carbon tetrabromide (15.8 g, 47.6
mmol) and triphenylphosphine (25.0 g, 95.3 mmol) were added to the
solution under ice-cooling, followed by stirring at room
temperature for one hour. Triethylamine (20.0 ml, 142 mmol) was
added to the mixture, followed by stirring for 10 minutes. The
reaction mixture was poured into n-hexane (1000 ml) and the
produced precipitates were separated through filtration. The
filtrate was concentrated through distillation under reduced
pressure, and the residue was purified by means of silica gel
column chromatography (silica gel 1500 ml, eluent; n-hexane:ethyl
acetate=3:1), to thereby yield a colorless viscous compound
(Compound 3; 12.6 g, 22.7 mmol, 95.4%).
[0143] .sup.1H-NMR(CDCl.sub.3) .delta.7.34-7.24 (10H, m, aromatic),
7.16 (1H, s, Br.sub.2C.dbd.CH--), 5.76 (1H, d, H-1 J.sub.1,2=3.90),
4.72, 4.60 (each 1H, d, benzyl, J.sub.gem=12.00), 4.53 (1H, br.t,
H-2), 4.60, 4.42 (each 1H, d, benzyl, J.sub.gem=12.00), 4.21 (1H,
d, H-3, J.sub.2,3=4.80), 3.83, 3.39 (each 1H, d, H-5,
J.sub.gem=11.40), 1.59, 1.30 (each 3H, s, acetonide)
[0144] EIMS m/z: 473, 475 (M-Br).
[0145] [.alpha.].sub.D+6.20.degree. (c=1.00, CHCl.sub.3)
[0146] (3) Synthesis of
4-C-ethynyl-3,5-di-O-benzyl-1,2-O-isopropylidene-.-
alpha.-D-ribo-pentofuranose (Compound 4) 8
[0147] Compound 3 (12.4 g, 22.4 mmol) was dissolved in dry
tetrahydrofuran (160 ml), and a 1.6 M n-butyl lithium (30.7 ml,
49.1 mmol) in n-hexane was added to the solution at -78.degree. C.
in an argon atmosphere, followed by stirring for 30 minutes at the
same temperature. After water was added to the mixture with
stirring, the organic layer was dried over anhydrous magnesium
sulfate and concentrated through distillation under reduced
pressure. The residue was purified by means of silica gel column
chromatography (silica gel 1500 ml, eluent; n-hexane:ethyl
acetate=3:1), to thereby yield a colorless viscous compound
(Compound 4; 7.95 g, 20.2 mmol, 90.3%).
[0148] .sup.1H-NMR(CDCl.sub.3) .delta.7.39-7.22 (10H, m, aromatic),
5.70 (1H, d, H-1 J.sub.1,2=3.60), 4.78, 4.69 (each 1H, d, benzyl,
J.sub.gem=12.60), 4.55 (1H, br.t, H-2), 4.53, 4.44 (each 1H, d,
benzyl, J.sub.gem=12.30), 4.16 (1H, d, H-3, J.sub.2,3=4.50), 3.71,
3.56 (each 1H, d, H-5, J.sub.gem=11.40), 1.73, 1.33 (each 3H, s,
acetonide)
[0149] EIMS m/z: 394(M.sup.+).
[0150] HRMS m/z(M.sup.+): Calcd. for C.sub.24H.sub.26O.sub.5:
394.1780, Found: 394.1777
[0151] [.alpha.].sub.D+22.60 (c=1.00, CHCl.sub.3)
[0152] (4) Synthesis of
4-C-triethylsilylethynyl-3,5-di-O-benzyl-1,2-O-iso-
propylidene-.alpha.-D-ribo-pentofuranose (Compound 5) 9
[0153] Compound 4 (5.00 g, 12.7 mmol) was dissolved in dry
tetrahydrofuran (100 ml), and a 1.6 M n-butyl lithium (9.50 ml,
15.2 mmol) in n-hexane was added to the solution at -78.degree. C.
in an argon atmosphere, followed by stirring for five minutes at
the same temperature. Under the same conditions,
chlorotriethylsilane (2.55 ml, 15.2 mmol) was added thereto,
followed by stirring for 30 minutes. After water was added to the
mixture with stirring, the organic layer was dried over anhydrous
magnesium sulfate and concentrated through distillation under
reduced pressure. The residue was purified by means of silica gel
column chromatography (silica gel 1000 ml, eluent; n-hexane:ethyl
acetate=3:1), to thereby yield a colorless oily compound (Compound
5; 6.32 g, 12.4 mmol, 97.6%).
[0154] .sup.1H-NMR(CDCl.sub.3) .delta.7.41-7.22 (10H, m, aromatic),
5.71 (1H, d, H-1, J.sub.1,2=3.85), 4.77, 4.65 (each 1H, d, benzyl,
J.sub.gem=12.09), 4.63 (1H, br.t, H-2), 4.57, 4.48 (each 1H, d,
benzyl, J.sub.gem=12.09), 4.23 (1H, d, H-3, J.sub.2,3=4.67), 1.73,
1.33 (each 3H, s, acetonide), 0.98 (9H, t, Si--CH.sub.2--CH.sub.3,
J=7.83), 0.60 (6H, Si--CH.sub.2--CH.sub.3, J=7.97)
[0155] EIMS m/z: 508(M.sup.+).
[0156] HRMS m/z(M.sup.+): Calcd. for C.sub.30H.sub.40O.sub.5Si:
508, 2645, Found: 508,2642
[0157] [.alpha.].sub.D-27.27.degree. (c=1.045, CHCl.sub.3)
[0158] (5) Synthesis of
4-C-triethylsilylethynyl-1,2-di-O-acetyl-3,5-di-O--
benzyl-D-ribo-pentofuranose (Compound 6) 10
[0159] Compound 5 (5.55 g, 10.9 mmol) was dissolved in acetic acid
(70.0 ml), and trifluoroacetic acid (10.0 ml) and water (30.0 ml)
were added to the solution, followed by stirring overnight at room
temperature. After disappearance of Compound 5 had been confirmed
by means of silica gel thin-layer chromatography, the reaction
mixture was concentrated through distillation under reduced
pressure. The residue was further concentrated by co-boiling with
toluene three times, and then dissolved in pyridine (50.0 ml).
Acetic anhydride (10.3 ml, 0.11 mol) was added thereto, followed by
stirring overnight at room temperature. The reaction mixture was
concentrated through distillation under reduced pressure, and the
residue was dissolved in ethyl acetate. The organic layer was
washed with water, dried over anhydrous magnesium sulfate, and
concentrated through distillation under reduced pressure. The
residue was purified by means of silica gel column chromatography
(silica gel 1000 ml, eluent; n-hexane:ethyl acetate=5:1), to
thereby yield a colorless viscous compound (Compound 6; 4.80 g,
8.68 mmol, 79.6%) as an anomer mixture (.alpha.:.beta.=1:6.6).
[0160] .sup.1H-NMR for .alpha. anomer (CDCl.sub.3) .delta.7.38-7.28
(10H, m, aromatic), 6.39 (1H, d, H-1, J.sub.1,2=4.67), 5.13 (1H,
dd, H-2, J.sub.1,2=4.67, J.sub.2,3=6.87), 4.80, 4.55 (each 1H,
benzyl, d, J.sub.gem=12.09), 4.61, 4.52 (each 1H, d, benzyl,
J.sub.gem=12.09), 4.30 (1H, d, H-3, J.sub.2,3=6.87), 3.62 (2H, d,
H-5, J=0.55), 2.12, 2.07 (each 3H, s. acetyl), 0.94 (9H, t,
Si--CH.sub.2--CH.sub.3, J=7.97), 0.55 (6H, Si--CH.sub.2--CH.sub.3,
J=7.97)
[0161] [.alpha.].sub.D-21.8.degree. (c=1.00, CHCl.sub.3)
[0162] .sup.1H-NMR for .beta. anomer (CDCl.sub.3) .delta.7.35-7.24
(10H, m, aromatic), 6.20 (1H, d, H-1, J.sub.1,2=0.82), 5.33 (1H,
dd, H-2, J.sub.1,2=0.82, J.sub.2,3=4.67), 4.66, 4.61 (each 1H,
benzyl, d, J.sub.gem=11.81), 4.56, 4.47 (each 1H, benzyl, d,
J.sub.gem=11.81), 4.48 (1H, d, H-3, J.sub.2,3=4.67), 3.69, 3.62
(each 1H, d, H-5, J.sub.gem=10.99), 2.09, 1.84 (each 3H, s.
acetyl), 0.96 (9H, t, Si--CH.sub.2--CH.sub.3, J=7.97), 0.58 (6H,
Si--CH.sub.2--CH.sub.3, J=7.97)
[0163] [.alpha.].sub.D-58.0.degree. (c=1.00, CHCl.sub.3)
[0164] EIMS m/z: 552(M.sup.+).
[0165] HRMS m/z(M.sup.+): Calcd. for C.sub.31H.sub.40O.sub.7Si:
552.2543, Found: 552.2551
[0166] (6) Synthesis of 4'-C-triethylsilylethynyl-2'-O-acetyl-3',
5'-di-O-benzyluridine (Compound 7) 11
[0167] Compound 6 (3.00 g, 5.43 mmol) was dissolved in
1,2-dichloroethane (100 ml), and uracil (1.52 g, 13.6 mmol) and
N,O-bis(trimethylsilyl)aceta- mide (9.40 ml, 38.0 mmol) were added
to the solution, followed by refluxing for one hour. After the
reaction mixture was allowed to cool to room temperature,
trimethylsilyl trifluoromethanesulfonate (1.97 ml, 10.9 mmol) was
added thereto, followed by stirring overnight at 50.degree. C. A
saturated aqueous solution of sodium hydrogencarbonate was added to
the mixture, and after stirring, precipitate was filtered. The
organic layer was dried over anhydrous magnesium sulfate and
concentrated through distillation under reduced pressure. The
residue was purified by means of silica gel column chromatography
(silica gel 300 ml, eluent; n-hexane:ethyl acetate=1:1), to thereby
yield a colorless viscous compound (Compound 7; 2.50 g, 4.13 mmol,
76.1%).
[0168] .sup.1H-NMR(CDCl.sub.3) .delta.8.63 (1H, br.s, 3-NH), 7.59
(1H, d, 6-H, J.sub.5',6=8.24), 7.41-7.24 (10H, m, aromatic), 6.31
(1H, d, H-1', J.sub.1',2'=4.95), 5.34 (1H, d, H-5, J.sub.5,6=8.24),
5.21 (1H, dd, H-2', J.sub.1',2'=4.95, J.sub.2',3'=6.04), 4.71, 4.58
(each 1H, d, benzyl, J.sub.gem=11.81), 4.48 (2H, s, benzyl), 4.34
(1H, d, H-3', J.sub.2',3'=6.04), 3.86, 3.67 (each 1H, d, H-5',
J.sub.gem=10.50), 2.05 (3H, s, acetyl), 0.97 (9H, t,
Si--CH.sub.2--CH.sub.3, J=7.95), 0.60 (6H, Si--CH.sub.2--CH.sub.3,
J=7.95).
[0169] FABMS m/z: 605(MH.sup.+).
[0170] HRMS m/z(MH.sup.+): Calcd. for
C.sub.33H.sub.41N.sub.2O.sub.7Si: 605.2683, Found: 605.2683.
[0171] [.alpha.].sub.D-21.97.degree. (c=1.015, CHCl.sub.3).
[0172] (7) Synthesis of
4'-C-triethylsilylethynyl-3',5'-di-O-benzyluridine (Compound 8)
12
[0173] Compound 7 (2.00 g, 3.3 mmol) was dissolved in methanol
(90.0 ml), and triethylamine (10.0 ml) was added to the solution,
followed by stirring for 48 hours at room temperature. The reaction
mixture was concentrated through distillation under reduced
pressure, and the residue was purified by means of silica gel
column chromatography (silica gel 200 ml, eluent; n-hexane:ethyl
acetate=1:1), to thereby yield a white powdery compound (Compound
8; 1.72 g, 3.06 mmol, 92.4%).
[0174] .sup.1H-NMR(CDCl.sub.3) .delta.8.43 (1H, br.s, 3-NH), 7.55
(1H, d, H-6, J.sub.5',6=8.24), 7.41-7.25 (10H, m, aromatic), 6.10
(1H, d, H-1', J.sub.1',2'5.22), 5.37 (1H, dd, H-5,
J.sub.5',6'=8.24), 4.96, 4.66 (each 1H, d, benzyl,
J.sub.gem=11.54), 4.56, 4.50 (each 1H, d, benzyl, J.sub.gem=11.00),
4.21 (1H, m, H-2'), 4.17 (1H, d, H-3', J.sub.2',3'=5.77), 3.87,
3.74 (each 1H, d, H-5', J.sub.gem=10.44), 3.02 (1H, br.d, 2'-OH),
0.97 (9H, t, Si--CH.sub.2--CH.sub.3, J=7.69), 0.60 (6H,
Si--CH.sub.2--CH.sub.3, J=7.69).
[0175] FABMS m/z: 563(MH.sup.+).
[0176] HRMS m/z(MH.sup.+): Calcd. for
C.sub.31H.sub.39N.sub.2O.sub.6Si: 563.2577, Found: 563.2586.
[0177] [.alpha.].sub.D-21.56.degree. (c=1.025, CHCl.sub.3)
[0178] m.p. 119-120.degree. C.
[0179] (8) Synthesis of 4'-C-triethylsilylethynyluridine (Compound
9) 13
[0180] Compound 8 (1.50 g, 2.67 mmol) was dissolved in
dichloromethane (75.0 ml), and a 1.0 M boron trichloride (26.7 ml,
26.7 mmol) in dichloromethane was added to the solution at
-78.degree. C. in an argon atmosphere, followed by stirring for
three hours at the same temperature. A mixture of pyridine (10.0
ml) and methanol (20.0 ml) was added thereto at -78.degree. C.,
followed by stirring for ten minutes. The reaction mixture was
concentrated through distillation under reduced pressure, and the
residue was partitioned with ethyl acetate and water. The organic
layer was dried over anhydrous magnesium sulfate and concentrated
through distillation under reduced pressure. The residue was
purified by means of silica gel column chromatography (silica gel
200 ml, eluent; chloroform:methanol=9:1), to thereby yield a white
powdery compound (Compound 9; 0.95 g, 2.48 mmol, 92.9%).
[0181] .sup.1H-NMR(CDCl.sub.3) .delta.11.36 (1H, d, 3-NH), 7.81
(1H, d, H-6, J.sub.5 , 6=8.24), 5.92 (1H, d, H-1',
J.sub.1',2'=6.32), 5.68 (1H, dd, J.sub.5,6=8.24), 5.55 (1H, t,
5'-OH), 5.33 (1H, d, 2'-OH), 5.16 (1H, d, 3'-OH), 4.13 (1H, dd,
H-2', J.sub.1',2'=6.32, J.sub.2',3'=5.77) 4.07 (1H, t, H-3',
J.sub.2', 3'=5.77), 3.58 (1H, d, H-5'), 0.96 (9H, t,
Si--CH.sub.2--CH.sub.3, J=7.97), 0.57 (6H, Si--CH.sub.2--CH.sub.3,
J=7.97).
[0182] FABMS m/z: 383(MH.sup.+).
[0183] HRMS m/z(MH.sup.+): Calcd. for
C.sub.17H.sub.27N.sub.2O.sub.6Si: 383, 1638, Found: 383.1645.
[0184] [.alpha.].sub.D-4.50.degree. (c=1.00, CH.sub.3OH)
[0185] m.p. 183-186.degree. C.
[0186] (9) Synthesis of
4'-C-triethylsilylethynyl-3',5'-di-O-acetyl-2'-deo- xyuridine
(Compound 11) 14
[0187] Compound 9 (0.80 g, 2.09 mmol) was suspended in acetonitrile
(20.0 ml), and a solution (20.0 ml) of acetyl bromide (1.55 ml,
21.0 mmol) in acetonitrile was added dropwise to the suspension at
85.degree. C. over 30 minutes, followed by refluxing for one hour.
After the reaction mixture was concentrated through distillation
under reduced pressure, the residue was dissolved in ethyl acetate
and the solution was washed with a saturated aqueous solution of
sodium hydrogencarbonate and a saturated aqueous solution of sodium
chloride. The organic layer was dried over anhydrous magnesium
sulfate and concentrated through distillation under reduced
pressure, to thereby yield 4'-C-triethylsilylethynyl-3',5'-di-O-a-
cetyl-2'-bromo-2'-deoxyuridine (Compound 10). After the crude
product (Compound 10) was concentrated by co-boiling with toluene
three times, the product was dissolved in dry toluene (50.0 ml).
Hydrogenated tri(n-butyl)tin (1.08 ml, 4.19 mmol) and
2,2'-azobis(isobutyronitrile) (0.01 g) were added to the solution
at 85.degree. C., and the mixture was heated under stirring for one
hour in an argon atmosphere. After the reaction mixture was
concentrated through distillation under reduced pressure, the
residue was purified by means of silica gel column chromatography
(silica gel 300 ml, eluent; toluene:ethyl acetate), to thereby
yield a colorless viscous compound (Compound 11; 0.40 g,
42.6%).
[0188] .sup.1H-NMR(CDCl.sub.3) .delta.7.49 (1H, d, H-6,
J.sub.5,6=8.24), 6.34 (1H, t, H-1', J.sub.1',2'=6.46), 5.77 (1H,
dd, H-5, J.sub.5,6=8.24), 5.37 (1H, dd, H-3', J.sub.2',3'=4.95,
7.42), 4.42, 4.37 (each 1H, d, H-5', J.sub.gem=11.81), 2.62, 2.32
(each 1H, m, H-2'), 2.13 (6H, s, acetyl), 1.00 (9H, t,
Si--CH.sub.2--CH.sub.3, J=7.82), 0.63 (6H, Si--CH.sub.2--CH.sub.3,
J=7.82).
[0189] FABMS m/z: 451(MH.sup.+).
[0190] HRMS m/z(MH.sup.+): Calcd. for
C.sub.21H.sub.31N.sub.2O.sub.7Si: 451.1900, Found: 451.1934.
[0191] [.alpha.].sub.D-11.7.degree. (c=1.04, CHCl.sub.3)
[0192] (10) Synthesis of 4'-C-ethynyl-2'-deoxycytidine (Compound
13) 15
[0193] Compound 11 (0.30 g, 0.67 mmol) was dissolved in pyridine
(15.0 ml), and p-chlorophenylphosphrodichloridate (0.33 ml, 2.00
mmol) was added to the solution under ice-cooling, followed by
stirring for two minutes. 1,2,4-Triazole (0.46 g, 6.66 mmol) was
added to the mixture, followed by stirring for seven days at room
temperature. After disappearance of raw material had been confirmed
by means of silica gel thin-layer chromatography, the reaction
mixture was concentrated through distillation under reduced
pressure, and the residue was partitioned with ethyl acetate and
water. The organic layer was dried over anhydrous magnesium sulfate
and concentrated through distillation under reduced pressure. The
residue was purified by means of silica gel column chromatography
(silica gel 50 ml, eluent; n-hexane:ethyl acetate=1:3), to thereby
yield colorless viscous Compound 12: 4-(1,2,4-triazolo)-4'-C-ethy-
nyl-2'-deoxyuridine. Compound 12 was dissolved in dioxane (30.0
ml), and 25% aqueous ammonia (10.0 ml) was added to the solution,
followed by stirring overnight at room temperature. After
disappearance of Compound 12 had been confirmed by means of silica
gel thin-layer chromatography, the reaction mixture was
concentrated through distillation under reduced pressure. The
residue was dissolved in methanol (45.0 ml), and an aqueous 1 N
solution of sodium hydroxide (5.00 ml, 5.00 mmol) was added
thereto, followed by stirring for two hours at room temperature.
Acetic acid (0.29 ml, 5.00 mmol) was added to the mixture, and the
reaction mixture was concentrated through distillation under
reduced pressure. The residue was purified by means of
reversed-phase medium-pressure column chromatography (Wakosil 40C18
50 g, eluent; a 5% aqueous solution of acetonitrile). The fractions
containing Compound 13 were brought to dryness under reduced
pressure, and the residue was crystallized from methanol-ether, to
thereby yield a white crystalline compound (Compound 13; 0.12 g,
0.48 mmol, 71.6%).
[0194] .sup.1 H-NMR(DMSO-d.sub.6) .delta.7.78 (1H, d, H-6,
J.sub.5,6=7.50), 7.17 (2H, br.d, NH.sub.2), 6.14 (1H, dd, H-1',
J.sub.1',2'=4.76, 7.20), 5.72 (1H, d, H-5, J.sub.5,6=7.50), 5.49
(1H, d, 3'-OH), 5.42 (1H, t, 5'-OH), 4.30 (1H, t, H-3',
J.sub.2',3'32 7.20), 3.64, 3.58 (each 1H, m, H-5'), 3.48 (1H, s,
ethynyl), 2, 25, 2.07 (each 1H, m, H-2')
[0195] [.alpha.].sub.D+75.0.degree. (c=1.00, CH.sub.3OH)
[0196] FABMS m/z: 252(MH.sup.+).
[0197] HRMS m/z(MH.sup.+): Calcd. for
C.sub.11H.sub.14N.sub.3O.sub.4: 252.0984, Found: 252.0979.
[0198] UV .lambda. max (CH.sub.3OH) nm (.epsilon.): 271 (9227)
[0199] m.p. 220.degree. C. (Dec)
Synthesis Example 2
[0200] 5-Fluorouracil, 5-ethyluracil, 5-bromovinyluracil, and
5-ethynyluracil were employed instead of uracil used in Synthesis
Example 1 (6), and the reactions were carried out in the same
manner as described above (if necessary, amination reaction by use
of triazole described in (10) was omitted), to thereby synthesize
the following compounds:
[0201] 4'-C-ethynyl-2'-deoxy-5-fluorouridine;
[0202] 4'-C-ethynyl-2'-deoxy-5-ethyluridine;
[0203] 4'-C-ethynyl-2'-deoxy-5-bromovinyluridine;
[0204] 4'-C-ethynyl-2'-deoxy-5-ethynyluridine;
[0205] 4'-C-ethynyl-2'-deoxy-5-ethylcytidine;
[0206] 4'-C-ethynyl-2'-deoxy-5-bromovinylcytidine; and
[0207] 4'-C-ethynyl-2'-deoxy-5-ethynylcytidine.
Synthesis Example 3
[0208] (1) Synthesis of
4-C-ethynyl-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribo- -pentofuranose
(Compound 14) 16
[0209] Compound 4 (6.00 g, 15.2 mmol) was dissolved in acetic acid
(70.0 ml), and trifluoroacetic acid (10.0 ml) and water (30.0 ml)
were added to the solution, followed by stirring overnight at room
temperature. After disappearance of Compound 4 had been confirmed
by means of silica gel thin-layer chromatography, the reaction
mixture was concentrated through distillation under reduced
pressure. The residue was concentrated by co-boiling with toluene
three times. The treated residue was dissolved in pyridine (50.0
ml). Acetic anhydride (14.3 ml, 0.15 mol) was added thereto,
followed by stirring overnight at room temperature. The reaction
mixture was concentrated through distillation under reduced
pressure, and the residue was dissolved in ethyl acetate. The
organic layer was washed with water, dried over anhydrous magnesium
sulfate, and concentrated through distillation under reduced
pressure. The residue was purified by means of silica gel column
chromatography (silica gel 1000 ml, eluent; n-hexane:ethyl
acetate=2:1), to thereby yield a colorless viscous compound
(Compound 14; 5.40 g, 12.3 mmol, 80.9%) as an anomer mixture
(.alpha.:.beta.=1:3.0).
[0210] .sup.1H-NMR for .alpha. anomer (CDCl.sub.3) .delta.7.39-7.25
(10H, m, aromatic), 6.42 (1H, d, H-1, J.sub.1,2=4.67), 5.13 (1H,
dd, H-2, J.sub.1,2=4.67, J.sub.2,3=6.87), 4.81, 4.60 (each 1H,
benzyl, d, J.sub.gem=12.09), 4.59, 4.51 (each 1H, d, benzyl,
J.sub.gem=12.09), 4.30 (1H, d, H-3, J.sub.2,3=6.87), 3.63 (2H, d,
H-5, J=0.55), 2.73 (1H, s, ethynyl), 2.10, 2.02 (each 3H, s.
acetyl).
[0211] .sup.1H-NMR for .beta. anomer (CDCl.sub.3) .delta.7.35-7.20
(10H, m, aromatic), 6.21 (1H, d, H-1, J.sub.1,2=0.82), 5.40 (1H,
dd, H-2, J.sub.1,2=0.82, J.sub.2,3=4.67), 4.66, 4.60 (each 1H,
benzyl, d, J.sub.gem=11.81), 4.50, 4.47 (each 1H, benzyl, d,
J.sub.gem=11.81), 4.42 (1H, d, H-3, J.sub.2,3=4.67), 3.70, 3.66
(each 1H, d, H-5, J.sub.gem=10.99), 2.80 (1H, s, ethynyl), 2.08,
1.81 (each 3H, s. acetyl).
[0212] EIMS m/z: 438(M.sup.+).
[0213] HRMS m/z(M.sup.+): Calcd. for C.sub.25H.sub.26O.sub.7:
438.1679, Found: 438.1681
[0214] (2) Synthesis of
4'-C-ethynyl-2'-O-acetyl-3',5'-di-O-benzyluridine (Compound 15)
17
[0215] Compound 14 (2.50 g, 5.70 mmol) was dissolved in
1,2-dichloroethane (80.0 ml), and uracil (1.60 g, 14.27 mmol) and
N,O-bis(trimethylsilyl)ace- tamide (9.86 ml, 39.74 mmol) were added
to the solution, followed by refluxing for one hour. After the
reaction mixture was allowed to cool to room temperature,
trimethylsilyl trifluoromethanesulfonate (2.06 ml, 11.40 mmol) was
added thereto, followed by stirring overnight at 50.degree. C. A
saturated aqueous solution of sodium hydrogencarbonate was added to
the mixture, and after stirring, precipitate was filtered. The
organic layer was dried over anhydrous magnesium sulfate and
concentrated through distillation under reduced pressure. The
residue was purified by means of silica gel column chromatography
(silica gel 300 ml, eluent; n-hexane:ethyl acetate=2:3), to thereby
yield a colorless viscous compound (Compound 15; 2.44 g, 4.97 mmol,
87.2%).
[0216] .sup.1H-NMR(CDCl.sub.3) .delta.8.52 (1H, br. s, 3-NH), 7.55
(1H, d, 6-H, J.sub.5,6=8.24), 7.40-7.22 (10H, m, aromatic), 6.25
(1H, d, H-1', J.sub.1',2'=4.40), 5.33 (1H, d, H-5, J.sub.5,6=8.24),
5.22 (1H, dd, H-2', J.sub.1',2'=4.40, J.sub.2',3'=5,77), 4.63 (2H,
s, benzyl), 4.45, 4.40 (each 1H, d, benzyl, J.sub.gem=10.99), 4.34
(1H, d, H-3', J.sub.2',3'=5.77), 3.84, 3.62 (each 1H, d, H-5',
J.sub.gem=10.58), 2.69 (1H, s, ethynyl), 2.11 (3H, s, acetyl).
[0217] FABMS m/z: 491(MH.sup.+).
[0218] HRMS m/z(MH.sup.+): Calcd. for
C.sub.27H.sub.27N.sub.2O.sub.7: 491.1818, Found: 491.1821.
[0219] [.alpha.].sub.D29.0.degree. (c=1.00, CHCl.sub.3).
[0220] (3) Synthesis of
1-(4-C-ethynyl-2-O-acetyl-3,5-di-O-benzyl-.beta.-D-
-arabino-pentofuranosyl)uracil (Compound 16) 18
[0221] Compound 15 (2.30 g, 4.69 mmol) was dissolved in methanol
(90.0 ml), and a 1 N aqueous solution of sodium hydroxide (10.0 ml)
was added to the solution, followed by stirring for two hours at
room temperature. The reaction mixture was neutralized with acetic
acid and then brought to dryness under reduced pressure. The
residue was dissolved in ethyl acetate. The organic layer was
washed with water, dried over anhydrous magnesium sulfate, and
brought to dryness under reduced pressure. The residue was
concentrated by co-boiling with a small amount of pyridine three
times. The product was dissolved in pyridine (50.0 ml), and
methanesulfonyl chloride (0.73 ml, 9.41 mmol) was added to the
solution under cooling, followed by stirring for three hours. A
small amount of water was added to the reaction mixture, and the
mixture was brought to dryness under reduced pressure. The residue
was dissolved in ethyl acetate, followed by washing with water. The
organic layer was dried over anhydrous magnesium sulfate and then
brought to dryness under reduced pressure. The residue was
dissolved in tetrahydrofuran (30.0 ml), and a 1 N aqueous solution
of sodium hydroxide (50.0 ml) was added to the solution, followed
by refluxing for one hour. After the reaction mixture was
neutralized with acetic acid, the target compound was taken up
through extraction with ethyl acetate. The organic layers were
combined and dried over anhydrous magnesium sulfate. The organic
layer was brought to dryness under reduced pressure, and the
residue was purified by means of silica gel column chromatography
(silica gel 250 ml, eluent; n-hexane:ethyl acetate=1:2), to thereby
yield a white powder compound (Compound 16; 1.54 g, 3.43 mmol,
73.1%).
[0222] .sup.1H-NMR(CDCl.sub.3) .delta.9.82 (1H, br.s, 3-NH), 7.73
(1H, d, 6-H, J.sub.5,6=8.06), 7.41-7.19 (10H, m, aromatic), 6.24
(1H, d, H-1', J.sub.1',2'=5,86), 5.25 (1H, d, H-5, J.sub.5,6=8.06),
4.88, 4.76 (each 1H, d, benzyl, J.sub.gem=12.21), 4.78 (1H, H-2'),
4.52 (1H, 2'-OH), 4.46, 4.39 (each 1H, d, benzyl, J.sub.gem=11.11),
4.19 (1H, d, H-3, J.sub.2',3'=6.59), 3.834, 3.64 (each 1H, d, H-5',
J.sub.gem=10.62), 2.67 (1H, s, ethynyl).
[0223] FABMS m/z: 449(MH.sup.+).
[0224] HRMS m/z(MH.sup.+): Calcd. for
C.sub.25H.sub.25N.sub.2O.sub.6: 449.1712, Found: 449.1713.
[0225] [.alpha.].sub.D40.70 (c=1.00, CHCl.sub.3).
[0226] m.p. 105-106.degree. C.
[0227] (4) Synthesis of
1-(4-C-ethynyl-2,3,5-tri-O-acetyl-.beta.-D-arabino-
-pentofuranosyl)uracil (Compound 17) 19
[0228] Compound 16 (1.40 g, 3.12 mmol) was dissolved in
dichloromethane (40.0 ml), and 1.0 M boron tribromide (15.6 ml,
15.6 mmol) in dichloromethane was added to the solution at
-78.degree. C. in an argon atmosphere, followed by stirring for
three hours at the same temperature. A mixture of pyridine (5.00
ml) and methanol (10.0 ml) was added thereto at -78.degree. C., and
after stirring for ten minutes, the reaction mixture was
concentrated through distillation under reduced pressure. After the
residue was concentrated by co-boiling with a small amount of
methanol three times and by another co-boiling with a small amount
of pyridine three times, the residue was dissolved in pyridine
(50.0 ml), and acetic anhydride (4.42 ml, 46.7 mmol) was added to
the solution, followed by stirring overnight at room temperature.
The reaction mixture was brought to dryness under reduced pressure,
and the residue was concentrated by co-boiling with a small amount
of toluene three times and then partitioned with ethyl acetate and
water. The organic layer was dried over anhydrous magnesium sulfate
and concentrated through distillation under reduced pressure. The
residue was purified by means of silica gel column chromatography
(silica gel 150 ml, eluent; chloroform:methanol=20:1), to thereby
yield a white powdery compound (Compound 17; 1.15 g, 2.92 mmol,
93.6%).
[0229] .sup.1H-NMR(CDCl.sub.3) .delta.8.99 (1H, br. s, 3-NH), 7.42
(1H, d, 6-H, J.sub.5,6=8.24), 6.45 (1H, d, H-1', J.sub.1',2'=4.95),
5.76 (1H, dd, H-5, J.sub.5,6=8.24), 5.55 (1H, dd, H-2',
J.sub.1',2'=4.95, J.sub.2',3'=3.57) 5.34 (1H, d, H-3',
J.sub.2',3'=3.57), 4.51, 4.42 (each 1H, d, H-5', J.sub.gem=11.81),
2.73 (1H, s, ethynyl).
[0230] FABMS m/z: 395(MH.sup.+).
[0231] HRMS m/z(MH.sup.+): Calcd. for
C.sub.17H.sub.19N.sub.2O.sub.9: 395.1090, Found: 395.1092.
[0232] [.alpha.].sub.D 18.2.degree. (c=1.00, CHCl.sub.3).
[0233] m.p. 160-162.degree. C.
[0234] (5) Synthesis of
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)cyt- osine (Compound
19) 20
[0235] Compound 17 (1.00 g, 2.54 mmol) was dossolved in pyridine
(50.0 ml), and p-chlorophenylphosphrodichloridate (1.05 ml, 6.38
mmol) was added to the solution under ice-cooling, followed by
stirring for five minutes. 1,2,4-Triazole (1.75 g, 25.3 mmol) was
added to the mixture, followed by stirring for seven days at room
temperature. After disappearance of raw material had been confirmed
by means of silica gel thin-layer chromatography, the reaction
mixture was concentrated through distillation under reduced
pressure, and the residue was partitioned with ethyl acetate and
water. The organic layer was dried over anhydrous magnesium sulfate
and concentrated through distillation under reduced pressure. The
residue was purified by means of silica gel column chromatography
(silica gel 50 ml, eluent; n-hexane:ethyl acetate=1:3), to thereby
yield a colorless viscous compound (Compound 18:
1-(4-C-ethynyl-2,3,5-tri-O-acetyl-.beta.-D-arabino-pentofuranosyl)-4-(1,2-
,4-triazolo)uracil. Compound 18 was dissolved in dioxane (60.0 ml),
and a 25% aqueous solution of ammonia (20.0 ml) was added to the
solution, followed by stirring overnight at room temperature. After
disappearance of Compound 18 had been confirmed by means of silica
gel thin-layer chromatography, the reaction mixture was
concentrated through distillation under reduced pressure. The
residue was purified by means of reversed-phase medium-pressure
column chromatography (Wakosil 40C18 50 g, eluent; a 3% aqueous
solution of acetonitrile). The fractions containing Compound 19
were brought to dryness under reduced pressure, and the residue was
dissolved in methanol-ether and crystallized from the same medium,
to thereby yield a white crystalline compound (Compound 19; 0.51 g,
1.91 mmol, 75.2%).
[0236] .sup.1H-NMR(DMSO-d.sub.6) .delta.7.52 (1H, d, H-6,
J.sub.5,6=7.42), 7.10 (2H, br. d, NH.sub.2), 6.17 (1H, dd, H-1',
J.sub.1',2'=6.04), 5.66 (1H, d, H-5, J.sub.5,6=7.42), 5.62, 5.49
(each 1H, d, 2'-OH, 3'-OH), 5.42 (1H, t, 5'-OH), 4.16 (1H, q, H-2',
J.sub.1',2'=J.sub.2',3'=6.04), 3.97 (1H, t, H-3',
J.sub.2',3'=6.04), 3.58 (2H, m, H-5'), 3.48 (1H, s, ethynyl).
[0237] [.alpha.].sub.D+95.7.degree. (c=1.00, CH.sub.3OH)
[0238] FABMS m/z: 268(MH.sup.+).
[0239] HRMS m/z(MH.sup.+): Calcd. for
C.sub.11H.sub.14N.sub.3O.sub.5: 268.0933, Found: 268.0965.
[0240] UV .lambda..sub.max (CH.sub.3OH) nm (.epsilon.): 271
(9350)
[0241] m.p. .about.200.degree. C. (Dec)
Synthesis Example 4
[0242] 5-Fluorouracil, 5-ethyluracil, 5-bromovinyluracil, and
5-ethynyluracil were employed instead of uracil used in Synthesis
Example 3 (2), and the reactions were carried out in the same
manner as described above (if necessary, amination reaction by use
of triazole described in (5) was omitted), to thereby synthesize
the following compounds:
[0243]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-fluorouracil;
[0244]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-ethyluracil;
[0245]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-bromovinyluracil;
[0246]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-ethynyluracil;
[0247]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-fluorocytosine;
[0248]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-ethylcytosine;
[0249]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-bromovinylcytosin-
e; and
[0250]
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-5-ethynylcytosine.
Synthesis Example 5
[0251] (1) Synthesis of
2'-O-acetyl-3',5'-di-O-benzyl-4'-C-triethylsilylet- hynyladenosine
(Compound 20) 21
[0252] To a solution of Compound 6 (1.1 g, 2 mmol) in
1,2-dichloroethane (16.5 ml), adenine (0.405 g, 3 mmol) and
N,O-bis(trimethylsilyl)acetamide (2.7 ml, 11 mmol) were added,
followed by refluxing for 1.5 hours. After the mixture was allowed
to cool to room temperature, trimethylsilyl
trifluoromethanesulfonate (0.77 ml, 4 mmol) was added dropwise to
the mixture under stirring at 0.degree. C. in an argon atmosphere.
The mixture was stirred for 15 minutes at room temperature,
refluxed for 24 hours, and allowed to cool to room temperature. A
saturated aqueous solution of sodium hydrogencarbonate was added
thereto at 0.degree. C., followed by stirring for 15 minutes at
room temperature. Insoluble materials were removed through
filtration by use of Celite, and then the organic layer was
separated from the filtrate. After an aqueous layer was extracted
with chloroform, the organic layer was washed once with a saturated
aqueous solution of sodium hydrogencarbonate, dried over anhydrous
sodium sulfate, and concentrated through distillation under reduced
pressure, so as to evaporate the solvent. The residue was applied
to a silica gel column (15 g, eluent; ethyl
acetate:n-hexane:ethanol=20:2- 0:1), to thereby yield Compound 20
in an amount of 0.69 g (55%).
[0253] .sup.1H-NMR(CDCl.sub.3) .delta.8.32 (1H, s, purine-H), 8.01
(1H, s, purine-H), 7.27-7.37 (10H, m, 2.times.Ph), 6.37 (1H, d,
J=5.1 Hz, H-1'), 5.60 (1H, t, J=5.6 Hz, H-2'), 5.59 (2H, br s,
NH.sub.2), 4.75 (1H, d, J=11.0 Hz, CHH'Ph), 4.69 (1H, d, J=5.6 Hz,
H-3'), 4.60 (1H, d, J=11.0 Hz, CHH'Ph), 4.58 (1H, d, J=11.2 Hz,
CHH'Ph), 4.51 (1H, d, J=11.0 Hz, CHH'Ph), 3.84 (1H, d, J=11.1 Hz,
H-5'), 3.69 (1H, d, J=11.1 Hz, H-5') 2.03 (3H, s, Ac), 0.98 (9H, t,
J=8.7 Hz, 3.times.CH.sub.3CH.sub.2), 0.61 (6H, q, J=8.7 Hz,
3.times.CH.sub.3CH.sub.2).
[0254] (2) Synthesis of
3',5'-di-O-benzyl-4'-C-triethylsilylethynyladenosi- ne (Compound
21) 22
[0255] To a solution of Compound 20 (0.354 g, 0.565 mmol) in
methanol (14 ml), triethylamine (3.3 ml) was added, and the mixture
was stirred for one day at room temperature under air-tight
condition. The mixture was concentrated under reduced pressure. The
residue was applied to a silica gel column (10 g, eluent; ethyl
acetate:n-hexane:ethanol 20:10:1), to thereby yield Compound 21 in
an amount of 0.283 g (86%).
[0256] .sup.1H-NMR(CDCl.sub.3) .delta.8.30 (1H, s, purine-H), 8.00
(1H, s, purine-H), 7.30-7.42 (10H, m, 2.times.Ph), 6.17 (1H, d,
J=5.6 Hz, H-1'), 5.55 (2H, br s, NH.sub.2), 4.97 (1H, d, J=11.1 Hz,
CHH'Ph), 4.75-4.80 (1H, m, H-2'), 4.72 (1H, d, J=11.1 Hz, CHH'Ph),
4.59 (1H, d, J=11.6 Hz, CHH'Ph), 4.54 (1H, d, J=11.6 Hz, CHH'Ph),
4.50 (1H, d, J=5.6 Hz, H-3'), 3.84 (1H, d, J=11.1 Hz, H-5'), 3.74
(1H, d, J=11.1 Hz, H-5'), 3.50 (1H, d, J=8.3 Hz, OH), 0.98 (9H, t,
J=7.9 Hz, 3.times.CH.sub.3CH.sub.2), 0.62 (6H, q, J=7.9 Hz,
3.times.CH.sub.3CH.sub.2).
[0257] (3) Synthesis of
3',5'-di-O-benzyl-2'-deoxy-4'-C-triethylsilylethyn- yladenosine
(Compound 22) 23
[0258] To a solution of Compound 21 (0.18 g, 0.308 mmol) and DMAP
(0.113 g, 0.924 mmol) in acetonitrile (10.6 ml),
4-fluorophenylchlorothionoforma- te (0.065 ml, 0.462 mmol) was
added dropwise under stirring at room temperature in an argon
atmosphere and stirred for an hour at room temperature, followed by
condensation under reduced pressure. Water was added to the
residue, and the mixture was extracted with ethyl acetate. The
organic layer was washed with water and was washed with a saturated
aqueous solution of sodium chloride, dried over anhydrous sodium
sulfate, and the solvent was distilled off under reduced pressure.
The residue was applied to a silica gel column (eluent; ethyl
acetate:n-hexane:ethanol=20- :20:1), to thereby yield crude
thiocarbonate.
[0259] The thiocarbonate was dissolved in toluene (9 ml), and
hydrogenated tributyltin (0.41 ml, 1.85 mmol) and
2,2'-azobis(isobutyronitrile) (0.013 g, 0.077 mmol) were added to
the solution. The reaction mixture was stirred at 85.degree. C. for
an hour in an argon atmosphere and allowed to cool to room
temperature. The solvent was evaporated under reduced pressure. The
residue was applied to a silica gel column (20 g, eluent; ethyl
acetate:n-hexane:ethanol=20:10:1), to thereby yield Compound 22 in
an amount of 0.10 g (57%).
[0260] .sup.1H-NMR(CDCl.sub.3) .delta.8.32 (1H, s, purine-H), 8.11
(1H, s, purine-H), 7.26-7.37 (10H, m, 2.times.Ph), 6.51 (1H, t,
J=6.0 Hz, H-1'), 5.54 (2H, br s, NH.sub.2), 4.72 (1H, d, J=12.0 Hz,
CHH'Ph), 4.61 (2H, d, J=10.5 Hz, CH.sub.2Ph), 4.60 (1H, t, J=6.6
Hz, H-3'), 4.55 (1H, d, J=12.0 Hz, CHH'Ph), 3.88 (1H, d, J=10.7 Hz,
H-5'), 3.76 (1H, d, J=10.7 Hz, H-5'), 2.71-2.76 (2H, m, H-2'), 0.99
(9H, t, J=7.8 Hz, 3.times.CH.sub.3CH.sub.2), 0.62 (6H, q, J=7.5 Hz,
3.times.CH.sub.3CH.sub.- 2).
[0261] (4) Synthesis of 2'-deoxy-4'-C-ethynyladenosine (Compound
23) and 9-(2-deoxy-4-C-ethynyl-.beta.-D-ribofuranosyl)purine
(Compound 24) 24
[0262] To a solution of Compound 22 (0.23 g, 0.404 mmol) in
tetrahydrofuran (9.4 ml), a 1.0 M solution of tetrabutylammonium
fluoride (0.44 ml, 0.44 mmol) was added under stirring at room
temperature, and after stirring for 30 minutes at the same
temperature, the solvent was evaporated under reduced pressure. The
residue was applied to a silica gel column and eluted with ethyl
acetate, to thereby yield 0.186 g of a crude compound with no
triethylsilyl group.
[0263] A solution of the above-described compound with no
triethylsilyl group in tetrahydrofuran (1.8 ml) and anhydrous
ethanol (0.18 ml) were fed to a flask. Ammonia gas was condensed at
-78.degree. C. to 18 ml and fed to the flask. Metallic sodium
(0.047 g, 2.02 mmol) were added quickly in an argon atmosphere,
followed by stirring for 15 minutes at the same temperature. In
addition, metallic sodium (0.023 g) was added to the mixture, and
after stirring for 10 minutes, ammonium chloride was added. After
the mixture was stirred for 1.5 hours at room temperature, ethanol
was added thereto. Insoluble materials were separeted through
Celite, and washed with ethanol two times. The resultant filtrate
and the washing liquid were concentrated under reduced pressure.
The residue was applied to a silica gel column (10 g, eluent; ethyl
acetate:methanol=20:1), to thereby yield a mixture of Compound 23
and Compound 24 in an amount of 0.079 g. Subsequently, the mixture
was applied to a reversed-phase ODS silica gel column and eluted
with a 5% aqueous solution of ethanol, to thereby yield Compound 24
in an amount of 0.028 g (27%), and further eluted with a 7.5%
aqueous solution of ethanol, to thereby yield Compound 23 in an
amount of 0.021 g (19%).
[0264] (Compound 23)
[0265] .sup.1H-NMR(DMSO-d.sub.6) .delta.8.33 (1H, s, purine-H),
8.15 (1H, s, purine-H), 7.30 (2H, br s, NH.sub.2), 6.36 (1H, t,
J=6.4 Hz, H-1'), 5.54 (1H, d, J=5.4 Hz, OH), 5.53 (1H, t, J=5.4 Hz,
OH), 4.58 (1H, q, J=5.9 Hz, H-3'), 3.66 (1H, dd, J=12.2, 5.4 Hz,
H-5'), 3.56 (1H, dd, J=11.7, 7.3 Hz, H-5'), 3.50 (1H, s,
ethynyl-H), 2.76 (1H, dt, J=13.2, 6.4 Hz, H-2'), 2.41 (1H, dt,
J=13.2, 6.8 Hz, H-2').
[0266] (Compound 24)
[0267] .sup.1H-NMR(DMSO-d.sub.6) .delta.9.18 (1H, s, purine-H),
8.96 (1H, s, purine-H), 8.79 (1H, s, purine-H), 6.50 (1H, t, J=7.3,
4.9 Hz, H-1'), 5.60 (1H, d, J=5.9 Hz, OH), 5.29 (1H, t, J=5.4 Hz,
OH), 4.67 (1H, q, J=5.9 Hz, H-3'), 3.67 (1H, dd, J=11.7, 5.9 Hz,
H-5'), 3.58 (1H, dd, J=11.7, 6.8 Hz, H-5'), 3.53 (1H, s,
ethynyl-H), 2.85 (1H, ddd, J=13.2, 6.8, 4.9 Hz, H-2'), 2.48-2.56
(1H, m, H-2').
Synthesis Example 6
[0268] (1) Synthesis of
9-(2-O-acetyl-3,5-di-O-benzyl-4-C-triethylsilyleth-
ynyl-.beta.-D-ribofuranosyl)-2,6-diaminopurine
[0269] (Compound 25) 25
[0270] To a solution of Compound 6 (1.1 g, 2 mmol) in
1,2-dichloroethane (16.5 ml), diaminopurine (0.45 g, 3 mmol) and
N,O-bis(trimethylsilyl)acet- amide (4.4 ml, 18 mmol) were added,
followed by refluxing for three hours. After the mixture was cooled
to room temperature, trimethylsilyl trifluoromethanesulfonate (0.77
ml, 4 mmol) was added dropwise to the mixture at 0.degree. C. in an
argon atmosphere. The mixture was stirred for 15 minutes at room
temperature, refluxed for 24 hours, and cooled to room temperature.
A saturated aqueous solution of sodium hydrogencarbonate was added
thereto at 0.degree. C., followed by stirring for 15 minutes at
room temperature. Insoluble materials were separated through
filtration by use of Celite, and then the organic layer was
separated from the filtrate. After an aqueous layer was extracted
with chloroform once, the organic layer was washed with a saturated
aqueous solution of sodium chloride, dried over anhydrous sodium
sulfate, and the solvent was evaporated under reduced pressure. The
residue was applied to a silica gel column (20 g, eluent; ethyl
acetate:n-hexane:ethanol=20:10:1- ), to thereby yield Compound 25
in an amount of 0.85 g (66%).
[0271] .sup.1H-NMR (CDCl.sub.3) .delta.7.68 (1H, s, H-8), 7.26-7.37
(10H, m, 2.times.Ph), 6.17 (1H, d, J=6.5 Hz, H-1'), 5.78 (1H, dd,
J=6.5, 6.0 Hz, H-2'), 5.34 (2H, br s, NH.sub.2), 4.76 (1H, d,
J=11.4 Hz, CHH'Ph), 4.69 (1H, d, J=6.0 Hz, H-3'), 4.61 (1H, d,
J=11.4 Hz, CHH'Ph), 4.60 (1H, d, J=11.9 Hz, CHH'Ph), 4.55 (2H, br
s, NH.sub.2), 4.52 (1H, d, J=11.9 Hz, CHH'Ph), 3.83 (1H, d, J=10.7
Hz, H-5'), 3.70 (1H, d, J=10.7 Hz, H-5'), 2.04 (3H, s, Ac), 0.99
(9H, t, J=8.3 Hz, 3.times.CH.sub.3CH.sub.2), 0.61 (6H, q, J=8.3 Hz,
3.times.CH.sub.3CH.sub.2).
[0272] (2) Synthesis of
2,6-diamino-9-(3,5-di-O-benzyl-4-C-triethylsilylet-
hynyl-.beta.-D-ribofuranosyl)purine (Compound 26) 26
[0273] Compound 25 (0.85 g, 1.32 mmol) was treated in the same
manner as in the synthesis of Compound 21, and the resultant
residue was applied to a silica gel column (15 g, eluent; ethyl
acetate:n-hexane:ethanol=30:10:1- ), to thereby yield Compound 26
in an amount of 0.74 g (93%).
[0274] .sup.1H-NMR(CDCl.sub.3) .delta.7.70 (1H, s, H-8), 7.29-7.42
(10H, m, 2.times.Ph), 6.00 (1H, d, J=4.9 Hz, H-1'), 5.35 (2H, br s,
NH.sub.2), 4.93 (1H, d, J=11.5 Hz, CHH'Ph), 4.74 (1H, d, J=11.5 Hz,
CHH'Ph), 4.73 (1H, t, J=5.8 Hz, H-2'), 4.60 (1H, d, J=12.0 Hz,
CHH'Ph), 4.55 (2H, br s, NH.sub.2), 4.54 (1H, d, J=12.0 Hz,
CHH'Ph), 4.49 (1H, d, J=5.9 Hz, H-3'), 3.81 (1H, d, J=10.7 Hz,
H-5'), 3.72 (1H, d, J=10.7 Hz, H-5'), 3.62 (1H, br s, OH), 0.99
(9H, t, J=7.8 Hz, 3.times.CH.sub.3CH.sub.2), 0.62 (6H, q, J=7.8 Hz,
3.times.CH.sub.3CH.sub.2).
[0275] (3) Synthesis of
2,6-diamino-9-(3,5-di-O-benzyl-2-deoxy-4-C-triethy-
lsilylethynyl-.beta.-D-ribofuranosyl)purine (Compound 27) 27
[0276] Compound 26 (0.103 g, 0.171 mmol) was treated in the same
manner as in the synthesis of Compound 22, and the resultant
residue was applied to a silica gel column (10 g, eluent; ethyl
acetate:n-hexane:ethanol=30:10:1- ), to thereby yield Compound 27
in an amount of 0.055 g (55%).
[0277] .sup.1H-NMR(CDCl.sub.3) .delta.7.79 (1H, s, H-8), 7.26-7.37
(10H, m, 2.times.Ph), 6.34 (1H, dd, J=6.6, 5.5 Hz, H-1'), 5.36 (2H,
br s, NH.sub.2), 4.72 (1H, d, J=11.7 Hz, CHH'Ph), 4.56-4.63 (5H, m,
CH.sub.2Ph, H-3'), 4.57 (1H, d, J=11.7 Hz, CHH'Ph), 3.85 (1H, d,
J=10.1 Hz, H-5'), 3.75 (1H, d, J=10.6 Hz, H-5'), 2.62-2.73 (2H, m,
H-2'), 0.99 (9H, t, J=7.9 Hz, 3.times.CH'CH.sub.2), 0.62 (6H, q,
J=7.9 Hz, 3.times.CH.sub.3CH.sub.2).
[0278] (4) Synthesis of
2,6-diamino-9-(2-deoxy-4-C-ethynyl-.beta.-D-ribofu- ranosyl)purine
(Compound 28) 28
[0279] To a solution of Compound 27 (0.263 g, 0.45 mmol) in
tetrahydrofuran (10.3 ml), a 1.0 M solution of tetrabutylammonium
fluoride (0.5 ml, 0.5 mmol) was added at room temperature, and the
mixture was stirred for 30 minutes at the same temperature. The
solvent was evaporated under reduced pressure. The residue was
applied to a short silica gel column (eluent; ethyl
acetate:ethanol=30:1), to thereby yield 0.214 g of a crude compound
with no triethylsilyl group.
[0280] The above-described compound with no triethylsilyl group in
tetrahydrofuran (2 ml) and anhydrous ethanol (0.1 ml) were fed to a
flask. Ammonia gas was condensed at -78.degree. C. to 20 ml and fed
to the flask. Metallic sodium (0.062 g, 2.7 mmol) was added quickly
in an argon atmosphere, followed by stirring for 30 minutes at the
same temperature. After ammonium chloride was added thereto, the
mixture was stirred for two hours at room temperature, and ethanol
was added to the mixture. Insoluble materials were separated
through filtration by use of Celite and washed with ethanol two
times. The resultant filtrate and the washing liquid were
concentrated under reduced pressure. The residue was applied to a
silica gel column (13 g, eluent; ethyl acetate:methanol=10:1), to
thereby yield Compound 28 in an amount of 0.099 g (76%).
[0281] .sup.1H-NMR(DMSO-d.sub.6) .delta.7.89 (1H, s, H-8), 6.71
(2H, br s, NH.sub.2), 6.20 (1H, t, J=6.3 Hz, H-1'), 5.74 (2H, br s,
NH.sub.2), 5.59 (1H, t, J=5.9 Hz, OH), 5.47 (1H, d, J=4.9 Hz, OH),
4.50 (1H, q, J=5.9 Hz, H-3'), 3.65 (1H, dd, J=11.7, 5.4 Hz, H-5'),
3.56 (1H, dd, J=11.7, 7.3 Hz, H-5'), 3.46 (1H, s, ethynyl-H), 2.64
(1H, dt, J=12.7, 6.4 Hz, H-2'), 2.32 (1H, dt, J=13.2, 6.4 Hz,
H-2').
Synthesis Example 7
[0282] Synthesis of 2'-deoxy-4'-C-ethynylinosine (Compound 29)
29
[0283] To a Tris-HCl buffer solution (6 ml, pH 7.5) of Compound 23
(0.022 g, 0.08 mmol), adenosine deaminase (0.044 ml, 20 unit) was
added, and the mixture was stirred for 2.5 hours at 40.degree. C.,
followed by cooling to room temperature. The reaction mixture was
applied to a reverse-phase ODS silica gel column (50 g), desalted
by water (500 ml) flow, and through use of a 2.5% aqueous ethanol,
Compound 29 was eluted. Subsequently, the Compound 29 was
pulverized with isopropanol, to thereby yield 0.016 g of Compound
29 (72%).
[0284] .sup.1H-NMR (DMSO-d.sub.6) .delta.12.28 (1H, brs, NH), 8.29
(1H, s, purine-H), 8.06 (1H, s, purine-H), 6.32 (1H, dd, J=6.8, 4.9
Hz, H-1'), 5.57 (1H, d, J=5.4 Hz, OH), 5.32 (1H, t, J=5.9 Hz, OH),
4.56 (1H, dt, J=6.4, 5.4 Hz, H-3'), 3.65 (1H, dd, J=12.2, 5.9 Hz,
H-5'), 3.57 (1H, dd, J=11.7, 6.4 Hz, H-5'), 3.50 (1H, s,
ethynyl-H), 2.66 (1H, dt, J=12.2, 5.9 Hz, H-2'), 2.46 (1H, dt,
J=13.2, 6.9 Hz, H-2').
Synthesis Example 8
[0285] Synthesis of 2'-deoxy-4'-C-ethynylguanosine (Compound 30)
30
[0286] To a Tris-HCl buffer solution (7.8 ml, pH 7.5) of Compound
28 (0.03 g, 0.103 mmol), adenosine deaminase (0.057 ml, 20 unit)
was added, and the mixture was stirred for 2 hours at 40.degree.
C., followed by cooling to room temperature. The reaction mixture
was applied to a reverse-phase ODS silica gel column (50 g),
desalted by water (500 ml) flow, and through use of aqueous 2.5%
ethanol, Compound 30 was eluted. Recrystallization from water
yielded Compound 30 in an amount of 0.015 g (50%).
[0287] .sup.1H-NMR(DMSO-d.sub.6) .delta.10.61 (1H, br s, NH), 7.90
(1H, s, H-8), 6.48 (2H, br s, NH.sub.2), 6.13 (1H, dd, J=7.3, 5.9
Hz, H-1'), 5.51 (1H, d, J=4.9 Hz, OH), 5.30 (1H, t, J=5.9 Hz, OH),
4.47 (1H, dt, J=6.4, 5.4 Hz, H-3'), 3.62 (1H, dd, J=12.2, 6.4 Hz,
H-5'), 3.54 (1H, dd, J=12.2, 6.4 Hz, H-5'), 3.47 (1H, s,
ethynyl-H), 2.56 (1H, dt, J=12.2, 6.4 Hz, H-2'), 2.36 (1H, dt,
J=12.7, 6.8 Hz, H-2').
Synthesis Example 9
[0288] Adenine, guanine, and 2,6-diaminopurine were employed
instead of uracil used in Synthesis Example 3 (2), and reaction is
carried out in a manner similar to that described above (amination
by use of triazole described in (5) omitted), to thereby synthesize
the following compounds:
[0289] 9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)adenine;
[0290] 9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)guanine;
and
[0291]
9-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)-2,6-diaminopurine.
Synthesis Example 10
[0292] (1) Synthesis of
2'-O-acetyl-3',5'-di-O-benzyl-4'-C-triethylsilylet-
hynyl-5-fluorouridine (Compound 31) 31
[0293] Compound 6 (2.00 g, 3.62 mmol) was dissolved in
1,2-dichloroethane (60.0 ml), and 5-fluorouracil (0.71 g, 5.46
mmol) and N,O-bis(trimethylsilyl)acetamide (5.37 ml, 21.7 mmol)
were added to the solution, followed by refluxing for one hour.
After the reaction mixture was allowed to cool to room temperature,
trimethylsilyl trifluoromethanesulfonate (0.85 ml, 4.70 mmol) was
added thereto, followed by stirring overnight at 50.degree. C. A
saturated aqueous solution of sodium hydrogencarbonate was added to
the mixture, and after stirring, the organic layer was dried over
anhydrous magnesium sulfate. The residue was purified by means of
silica gel column chromatography (silica gel 300 ml, eluent;
n-hexane:ethyl acetate=3:1), to thereby yield a colorless viscous
compound (Compound 31; 0.80 g, 1.28 mmol, 35.4%).
[0294] .sup.1H-NMR(CDCl.sub.3) .delta.7.86 (1H, d, H-6,
J.sub.6,F=6.35), 7.37-7.29 (10H, m, aromatic), 6.32 (1H, dd, H-1',
J=5.62, 1.47), 5.17 (1H, t, H-2', J.sub.2',3'=5.62), 4.73, 4.55
(each 1H, d, benzyl, J.sub.gem=11.72), 4.55, 4.50 (each 1H, d,
benzyl, J.sub.gem=11.72), 4.32 (1H, d, H-3',J.sub.2',3'=5.86),
3.87, 3.63 (each 1H, d, H-5 J.sub.gem=10.50), 2.04 (3H, s, acetyl),
0.96 (9H, t, Si--CH.sub.2--CH.sub.3, J=8.06), 0.59 (6H,
Si--CH.sub.2--CH.sub.3, J=7.81).
[0295] FABMS m/z:623 (MH.sup.+).
[0296] HRMS m/z(MH.sup.+): Calcd. for
C.sub.33H.sub.40FN.sub.2O.sub.7Si: 623.2589, Found: 623.2589.
[0297] [.alpha.].sub.D-23.3.degree. (c=0.18, CHCl.sub.3). ps (2)
Synthesis of
3',5'-di-O-benzyl-4'-C-triethylsilylethynyl-5-fluoroouridine
(Compound 32): 32
[0298] Compound 31 (0.77 g, 1.24 mmol) was dissolved in methanol
(45.0 ml), and triethylamine (5.00 ml) was added to the solution,
followed by stirring for 48 hours at 30.degree. C. The reaction
mixture was concentrated through distillation under reduced
pressure, and the residue was purified by means of silica gel
column chromatography (silica gel 100 ml, eluent; n-hexane:ethyl
acetate=2:1), to thereby yield a white powdery compound (Compound
32; 0.68 g, 1.17 mmol, 94.4%).
[0299] .sup.1H H-NMR(CDCl.sub.3).delta.8.42 (1H, br. s, 3-NH), 7.80
(1H, d, J.sub.6,F=6.10), 7.38-7.29 (10H, m, aromatic), 6.10 (1H,
dd, H-1', J=5.98, 1.47), 5.00, 4.63 (each 1H, d, benzyl,
J.sub.gem=11.23), 4.58, 4.54 (each 1H, d, benzyl, J.sub.gem=10.99),
4.20 (1H, m, H-2), 4.13 (1H, d, H-3', J.sub.2',3'=5.86), 3.88, 3.70
(each 1H, d ,H-5', J.sub.gem=10.25), 2.99 (1H, d, 2'-OH, J=9.77),
0.96 (9H, t, Si--CH.sub.2--CH.sub.3, J=8.06), 0.58 (6H,
Si--CH.sub.2--CH.sub.3, J=7.82).
[0300] FABMS m/z: 581 (MH.sup.+).
[0301] HRMS m/z(MH.sup.+): Calcd. for
C.sub.31H.sub.38FN.sub.2O.sub.6Si:58- 1.2483, Found:581.2484.
[0302] [.alpha.].sub.D-16.3.degree. (c=1.05, CHCl.sub.3)
[0303] m.p. 138-139.degree. C.
[0304] (3) Synthesis of 4'-C-triethylsilylethynyl-5-fluorouridine
(Compound 33): 33
[0305] Compound 32 (1.00 g, 1.72 mmol) was dissolved in
dichloromethane (50.0 ml), and a solution (26.7 ml, 26.7 mmol) of
1.0 M boron trichloride in dichloromethane was added thereto at
-78.degree. C. in an argon atmosphere, followed by stirring for
three hours at the same temperature. A mixture of pyridine (10.0
ml) and methanol (20.0 ml) was added at -78.degree. C., followed by
stirring for 30 minutes. The reaction mixture was concentrated
through distillation under reduced pressure, and the residue was
partitioned with ethyl acetate and water. The organic layer was
dried over anhydrous magnesium sulfate and concentrated through
distillation under reduced pressure. The residue was purified by
means of silica gel column chromatography (silica gel 150 ml,
eluent; chloroform:methanol=10:1), to thereby yield a white powdery
compound (Compound 33; 0.64 g, 1.60 mmol, 93.0%).
[0306] .sup.1H-NMR(DMSO-d.sub.6) .delta.11.93 (1H, d, 3-NH,
J=5.13), 8.13 (1H, d, H-6, J.sub.6,F=7.08), 5.89 (1H, dd, H-1',
J=6.35,1.95), 5.71 (1H, t, 5'-OH, J=5.37), 5.37, 5.23 (each 1H, d,
2'-OH, 3'-OH, J=6.35), 4.12 (1H, q, H-2, J=6.35), 4.05 (1H, t, H-3,
J=5.61), 3.61-3.57 (2H, m, H-5), 3.35 (1H, s, ethynyl), 0.95 (9H,
t, Si--CH.sub.2--CH.sub.3, J=7.81), 0.55 (6H,
Si--CH.sub.2--CH.sub.3, J=7.81).
[0307] FABMS m/z: 401 (MH.sup.+).
[0308] HRMS m/z(MH.sup.+): Calcd. for
C.sub.17H.sub.26FN.sub.2O.sub.6Si: 401.1544, Found:401.1550.
[0309] [.alpha.].sub.D-2.30.degree. (c=1.00,CH.sub.3OH)
[0310] m.p. 180-183.degree. C.
[0311] (4) Synthesis of
3',5'-di-O-acetyl-2'-deoxy-4'-C-triethylsilylethyn-
yl-5-fluorouridine (Compound 34): 34
[0312] Compound 33 (0.54 g, 1.35 mmol) was suspended in
acetonitrile (30.0 ml), and a solution (20.0 ml) of acetyl bromide
(1.00 ml, 13.5 mmol) in acetonitrile was added dropwise to the
suspension at 85.degree. C. over one hour, followed by refluxing
for a further three hours. After the reaction mixture was
concentrated through distillation under reduced pressure, the
residue was dissolved in ethyl acetate and the solution was washed
sequentially with a saturated aqueous sodium hydrogencarbonate
solution and a saturated aqueous sodium chloride solution. The
organic layer was dried over anhydrous magnesium sulfate and
concentrated through distillation under reduced pressure, to
thereby yield
3',5'-di-O-acetyl-2'-bromo-2'-deoxy-4'-C-triethylsilylethynyl-5-fluorouri-
dine in crude form (Compound 34). After the crude product (Compound
34) was concentrated by co-boiling with toluene three times, the
product was dissolved in dry toluene (20.0 ml). Hydrogenated
tri(n-butyl)tin (0.75 ml, 2.91 mmol) and
2,2'-azobis(isobutyronitrile) (0.01 g) were added to the solution
at 85.degree. C., and the mixture was heated under stirring for 30
minutes in an argon atmosphere. After the reaction mixture was
concentrated through distillation under reduced pressure, the
residue was purified by means of silica gel column chromatography
(silica gel 200 ml, eluent; n-hexane ethyl acetate), to thereby
yield a white powdery compound (Compound 35; 0.41 g, 0.88 mmol,
65.2%).
[0313] .sup.1H-NMR(CDCl.sub.3).delta.9.23 (1H, br.s, 3-NH), 7.70
(1H, d, H-6, J.sub.6,F=6.10), 6.35 (1H, t, H-1, J.sub.1',2'=7.08),
5.36 (1H, t, H-3', J.sub.2',3'=7.57), 4.43, 4.39 (each 1H, d, H-5',
J.sub.gem=12.21), 2.65, 2.33 (each 1H, m, H-2'), 2.17, 2.13 (each
3H, s, acetyl), 1.00 (9H, t, Si--CH.sub.2--CH.sub.3, J=7.82), 0.63
(6H, Si--CH.sub.2--CH.sub.3, J=7.82).
[0314] FABMS m/z: 469 (MH.sup.+).
[0315] HRMS m/z(MH.sup.+): Calcd. for
C.sub.21H.sub.30FN.sub.2O.sub.7Si:46- 9.1806, Found: 469.1810.
[0316] [.alpha.].sub.D-12.9.degree. (c=1.00,CHCl.sub.3)
[0317] m.p. 111-112.degree. C.
[0318] (5) Synthesis of 4'-C-ethynyl-2'-deoxy-5-fluorocytidine
(Compound 37) 35
[0319] Compound 35 (0.35 g, 0.75 mmol) was dissolved in pyridine
(5.00 ml), and p-chlorophenylphosphrodichloridate (0.62 ml, 3.77
mmol) was added to the resultant solution under ice-cooling,
followed by stirring for five minutes. 1,2,4-Triazole (0.78 g, 11.3
mmol) was added to the mixture, followed by stirring for 24 hours
at 30.degree. C. The reaction mixture was concentrated through
distillation under reduced pressure, and the residue was
partitioned with ethyl acetate and water. The organic layer was
dried over anhydrous magnesium sulfate, and concentrated through
distillation under reduced pressure. The residue was purified by
means of silica gel column chromatography (silica gel 50 ml,
eluent; ethyl acetate), to thereby yield a colorless viscous
compound
(4'-C-triethylsilylethynyl-2'-deoxy-5-fluoro-4-(1,2,4-triazolo)uridine;
Compound 36). Compound 36 was dissolved in dioxane (15.0 ml), and
25% aqueous ammonia (5.00 ml) was added to the resultant solution,
followed by stirring overnight at room temperature. After
disappearance of Compound 36 had been confirmed by means of silica
gel thin-layer chromatography (chloroform:methanol=10:1), the
reaction mixture was concentrated through distillation under
reduced pressure. The residue was dissolved in methanol (45.0 ml),
and an aqueous 1 N sodium hydroxide solution (5.00 ml, 5.00 mmol)
was added thereto, followed by stirring for 24 hours at room
temperature. Acetic acid (0.29 ml, 5.00 mmol) was added to the
mixture, and the reaction mixture was concentrated through
distillation under reduced pressure. The residue was purified by
means of silica gel column chromatography (silica gel 50 ml;
chloroform:ethanol=4:1). The fraction containing Compound 37 was
brought to dryness under reduced pressure, and the residue was
crystallized from methanol-ether, to thereby yield a white
crystalline compound (Compound 37; 0.12 g, 0.45 mmol, 60.0%).
[0320] .sup.1H-NMR(DMSO-d.sub.6) .delta.8.06 (1H, d, H-6,
J.sub.6,F=7.08) , 7.79, 7.54 (each 1H, br.s, NH.sub.2), 6.05 (1H,
m, H-1'), 5.57, 5.50 (each 1H, br, 3'-OH, 5'-OH), 4.31 (1H, br.q,
H-3), 3.66, 3.60 (each 1H, d, H-5, J.sub.gem=11.72), 3.51 (1H, s,
ethynyl), 2.25, 2.12 (each 1H, m, H-2')
[0321] [.alpha.].sub.D +77.9.degree. (c=1.00, CH.sub.3OH)
[0322] FABMS m/z: 270 (MH.sup.+).
[0323] HRMS m/z(MH.sup.+): Calcd. for
C.sub.11H.sub.13FN.sub.3O.sub.4: 270.0890, Found: 270.0888.
[0324] m.p. -225.degree. C. (Dec)
Drug Preparation Example 1
Tablets
[0325]
1 Compound of the present invention 30.0 mg Cellulose micropowder
25.0 mg Lactose 39.5 mg Starch 40.0 mg Talc 5.0 mg Magnesium
stearate 0.5 mg
[0326] Tablets are prepared from the above composition through a
customary method.
Drug Preparation Example 2
Encapsulated Drug
[0327]
2 Compound of the present invention 30.0 mg Lactose 40.0 mg Starch
15.0 mg Talc 5.0 mg
[0328] Encapsulated drugs are prepared from the above composition
through a customary method.
Drug Preparation Example 3
Injections
[0329]
3 Compound of the present invention 30.0 mg Glucose 100.0 mg
[0330] Injections are prepared by dissolving the above composition
in purified water for preparing injections.
[0331] Test Examples will next be described. Employed in tests were
the following seven compounds of the present invention and two
known compounds:
[0332] Compound 13: 4'-C-ethynyl-2'-deoxycytidine;
[0333] Compound 19:
1-(4-C-ethynyl-.beta.-D-arabino-pentofuranosyl)cytosin- e;
[0334] Compound 23:
9-(2-deoxy-4-C-ethynyl-.beta.-D-ribo-pentofuranosyl)ad- enine
(4'-C-ethynyl-2'-deoxyadenosine);
[0335] Compound 28:
9-(2-deoxy-4-C-ethynyl-.beta.-D-ribo-pentofuranosyl)-2-
,6-diaminopurine;
[0336] Compound 29:
9-(2-deoxy-4-C-ethynyl-.beta.-D-ribo-pentofuranosyl)hy- poxanthine
(4'-C-ethynyl-2'-deoxyinosine);
[0337] Compound 30:
9-(2-deoxy4-C-ethynyl-.beta.-D-ribo-pentofuranosyl)gua- nine
(4'-C-ethynyl-2'-deoxyguanosine);
[0338] Compound 37: 4'-C-ethynyl-2'-deoxy-5-fluorocytidine);
and
[0339] Known compounds: 4'-C-ethynylthymidine and AZT.
Test Examples
Test Methods
[0340] (1) Anti-HSV-1 Activity
[0341] 1. Human embryonic lung cells are subcultured by splitting
at 1:2 to 1:4 in an Eagle's MEM supplemented with 10% bovine serum
(Mitsubishi Chemical Corporation) at intervals of 4-5 days.
[0342] 2. The suspension of cells obtained from parent cells by
splitting at 1:2 is added to a 96-well-microplate at 200
.mu.l/well, and the cells are cultured in a CO.sub.2-incubator for
four days at 37.degree. C.
[0343] 3. After culture medium is removed, a test agent (100 .mu.l)
in serial fivefold dilution with a Hanks' MEM is added to the
wells.
[0344] 4. An Eagle's MEM (100 .mu.l) supplemented with 5% bovine
serum containing 100-320 TCID.sub.50 of herpes simplex virus
type-1, VR-3 strain is added to the wells to thereby seed the
virus, and the infected cells are cultured at 37.degree. C. in a
CO.sub.2-incubator.
[0345] 5. After the cells are cultured for 2-3 days, the degree of
CPE of each well is observed under a microscope for evaluation on a
scale of 0 to 4. When the cells in test agent-free controls are
completely degenerated through infection with the virus, the CPE
score is 4.
[0346] 6. The antiviral activity is expressed as ED.sub.50 at which
HSV-induced CPE were expressed at least 50%.
[0347] (2) Anti-human Immunodeficiency Virus (HIV) Activity
[0348] 1) MTT Method Using MT-4 Cells
[0349] 1. A test agent (100 .mu.l) is diluted on a 96-well
microplate. MT-4 cells infected with HIV-1 (III.sub.b strain; 100
TCID.sub.50) and non-infected MT-4 cells are added to the
microplate such that the number of cells in each well becomes
10,000. The cells are cultured at 37.degree. C. for five days.
[0350] 2. MTT (20 .mu.l, 7.5 mg/ml) is added to each well, and the
cells are further cultured for 2-3 hours.
[0351] 3. The cultured medium (120 .mu.l) is sampled, and MTT
terminating solution (isopropanol containing 4% Triton X-100 and
0.04N HCl) is added to the sample. The mixture is stirred to form
formazane, which is dissolved. The absorbance at 540 nm of the
solution is measured. Since the absorbance is proportional to the
number of viable cells, the test agent concentration at which a
half value of the absorbance is measured in a test using infected
MT-4 cells represents EC.sub.50, whereas the test agent
concentration at which a half value of the absorbance is measured
in a test using non-infected MT-4 cells represents CC.sub.50.
[0352] 2) MAGI Assay Using HeLa CD4/LTR-beta-Gal Cells
[0353] 1. HeLa CD4/LTR-beta-Gal cells are added to 96 wells such
that the number of cells in each well is 10,000. After 12-24 hours,
the culture medium is removed, and a diluted test agent (100 .mu.l)
is added.
[0354] 2. A variety of HIV strains (wild strain: WT, drug-resistant
strain: MDR, M184V, NL4-3, 104pre, and C; each equivalent to 50
TCID.sub.50) are added, and the cells are further cultured for 48
hours.
[0355] 3. The cells are fixed for five minutes using PBS containing
1% formaldehyde and 0.2% glutaraldehyde.
[0356] 4. After the fixed cells are washed with PBS three times,
the cells are stained with 0.4 mg/ml X-Gal for one hour, and the
number of blue-stained cells of each well is counted under a
transmission stereoscopic microscope. The test agent concentration
at which blue-stained cells decrease to 50% and 90% in number
represents EC.sub.50 and EC.sub.90, respectively.
[0357] 5. In a manner similar to that employed in the MTT method,
cytotoxicity is measured by use of HeLa CD4/LTR-beta-Gal cells.
[0358] The test results are shown in Tables 1 to 7.
Results
[0359] (1) Anti-HSV-1 Activity
4 TABLE 1 Drug HSV-1(ED.sub.50, .mu.g/ml) Compound 13 33
[0360] (2) Anti-human Immunodeficiency Virus (HIV) Activity and
Cytotoxicity
[0361] Each value shown in Tables 2 to 7 represents an average of
two to five assayed values.
[0362] 1. MTT Method Using MT-4 Cells
5 TABLE 2 MT-4 cells HIV-1 Cytotoxicity Drugs (EC.sub.50, .mu.g/ml)
(CC.sub.50, .mu.g/ml) Compound 13 0.0012 0.56 Compound 19 0.0115
0.53 4'-C-ethynyl 0.22 >100 thymidine AZT 0.0016 >0.27
[0363]
6 TABLE 3 MT-4 cells HIV-1 Cytotoxicity Drugs (EC.sub.50, .mu.g/ml)
(CC.sub.50, .mu.g/ml) Compound 23 0.0027 4.4 Compound 28 0.0001
0.26 Compound 29 0.037 38 Compound 30 0.00044 0.41 AZT 0.0011
9.08
[0364]
7 TABLE 4 MT-4 cells HIV-1 Cytotoxicity Drugs (EC.sub.50, .mu.M)
(CC.sub.50, .mu.M) Compound 37 0.033 >500 AZT 0.055
[0365] 2. MAGI Assay Using HeLa CD4/LTR-beta-Gal Cells
8 TABLE 5 HeLa CD4/LTR-beta-Gal cells HIV MDR Cytotoxicity Drugs WT
(EC.sub.50, .mu.g/ml) M184V (CC.sub.50, .mu.g/ml) Compound 13
0.00031 0.00030 0.00054 >100 Compound 19 0.0019 0.021 0.19
>100 4'-C-ethynyl 0.097 0.033 0.049 >100 thymidine AZT 0.0059
4.1 0.0083 >26.7
[0366]
9 TABLE 6 HeLa CD4/LTR-beta-Gal cells HIV MDR Cytotoxicity Drugs WT
(EC.sub.50, .mu.g/ml) M184V (CC.sub.50, .mu.g/ml) Compound 23
0.0012 0.0017 0.013 >100 Compound 28 0.00028 0.00029 0.0017 2.7
Compound 29 0.22 0.14 4.6 >100 Compound 30 0.002 0.0014 0.0023
15.2 AZT 0.0027 5.34 0.0013 >26.7
[0367]
10 TABLE 7 HeLa CD4/LTR-beta-Gal cells HIV 104 pre EC.sub.50, .mu.M
Drugs NL-43 (EC.sub.90, .mu.M) C Compound 37 0.021 0.022 0.122
(0.25 0.19 3.44) AZT 0.109 0.059 3.269 (4.96 9.66 >10)
* * * * *