Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
`` 1 1319932
2'-ALKYLIDENEP~RIMIDINE NUCLEOSIDE DERIVATIVES,
PRO~ESS FOR PRODUCTION THEREOF, AND USES THEREOF
Technical Field
The present invention relates to novel compounds,
2'-alkylidenepyrimidine nucleoside derivatives, a process
for the production thereof, and antiviral agents
comprising the compounds as active ingredients.
Backqround Art
In recent years, the development of preventives and
remedies for various viral infections has attracted
particular attention with the advance of researches into
15 pathogenic viruses.
Antiviral agents for use in chemotherapy heretofore
proposed for clinical purposes are idoxuridine,
cytarabine, vidarabine, acyclovir and the like. (See, for
example, Yutaka Mizushima and Terumasa Miyamoto, The
20 Edition of 1986, "Konnichi no Chiryo-yaku (Present-Day
Remedies), Kaisetsu to Binran (Explanation and Manual)",
Nanko-do, pp. 47-50 (March 10, 1986).)
Mo~t of the above-mentioned drugs, however, have
been accompanied by problems including limited clinical
~5 applicability due, for instance, to antiviral activity
spectra, low absorption, poor solubility, easy
decomposition, the advent of drug-fast virus strains, and
various side effects. Accordingly, there has been an
urgent demand for the development of novel antiviral
30 agents-
A primary object of the present invention is toprovide novel compounds having remarkable antiviral
activities.
3S Disclosure of Invention
As a result of extensive research efforts for the
development of novel compounds useful as antiviral
2 13~9932
agents, we have found that 2'-alkylidenepyrimidine
nucleoside derivatives represented by the following
formula [I] have excellent antiviral activities. On the
basis of this finding, we have arrived at the present
5 invention.
More particularly, the present invention relates to
2'-alkylidenepyrimidine nucleoside derivatives
represented by formula [I];
R
N
11
15O N
~4 V ~ [I]
HO CH-R
25 wherein Rl is an amino or hydroxy group, R2 is a hydrogen
or halogen atom or a lower alkyl group, R~ i~ a hydrogen
atom or a lower alkyl group, and R4 is a hydrogen atom or
a phosphate residue, and salts thereof.
This invention also relates to a process for
30 producing 2'-alkylidenepyrimidine nucleoside derivatives
represented by the above formula [I], which process
comprises Steps (1), (2) and (3~ set forth below
~hereinafter referred to as l'the first process"). The
first process illustrates inclusively the second, third
35 and fourth processes described hereinlater.
~ 3 131~932
lR6 R6
O , ~ oJ`~3~R2
10 ZV ~ ZV ~
~ /
Z ZO \\CH-R3
[II~
[III]
Step (2l
ZO
Rl R6
~ O ~
~ v~ v ~J
\~
30HO CH-R3 HO CH-R3
[I] [IV]
wherein Rl, R2, R3 and R4 are as defined previously, R5
is alkoxy, hydroxy, amino or acylamino (-NHR6, R6 being
-` ~ 13i~2
an acyl group), and Z is a hydroxy protecting group in
the sugar moiety.
In the process shown above, Step (1) involves the
alkylidenation of the 2'-position in the sugar moiety of
5 a compound of formula [II] with Wittig's reagent, Step
(2) involves the removal of the hydroxy protecting groups
in the sugar moiety of a compound of formula tIII] thus
obtained, and Step (3) involves the hydrolysis or
amination of the 4-position in the base moiety of a
10 compound of formula [IV] resulting from Step (2) when R5
is an alkoxy group, or the removal of the acyl protecting
group when R5 is an acylamino group, and the subsequent
optional phosphorylation at the 5'-position in the sugar
moiety irrespective of whether R5 is an alkoxy, hydroxy,
15 amino or acylamino group to obtain a compound of formula
[I]-
The present invention further relates to a processfor producing 2'-alkylidenepyrimidine nucleoside
derivatives represented by formula [I] shown earlier,
20 which process comprises the following Steps (1), (2) and
(3) (hereinafter referred to as "the second process").
`` 5 13~ 9~32
R6~ R5
O ~ 0
10 Z ~V~ ZO ~V~
Zo CH-R3
lII'~
lIII']
Step ~2) 1
Rl IR6'
; ~ Step ~3) N ; ~ R2
V ~ ~V~
HO CH-R3 HO CH-R3
[IV']
wherein Rl, R2, R3, R4 and Z are as defined previously,
and R5 is an alkoxy group.
6 13~ 9'~2
In this process, Steps (1) and (2) are the same
reaction steps as those in the aforementioned first
process, and Step (3) involves the hydrolysis or
amination of the 4-position in the base moiety and the
5 subsequent optional phosphorylation at the 5'-position in
the sugar moiety to obtain a compound of formula [I].
The present invention still further relates to a
process for producing 2'-alkylidenepyrimidine nucleoside
derivatives of formula [I] shown above, which process
10 comprises the followins Steps (1) and (2) (hereinafter
referred to as "the third process").
7 131~32
5 ~ R~ ~
10 ZO~ ZO ~
Z ZO CH R
[II"] lIII"]
Step (2)
R
N
o N
V~
H H-R3
lI]
wherein R1, R2, R3, R4 and Z are as defined hereinbefore.
35In the above process, Step (1) is the sa~e reaction
step as that in the first process, and Step (2) involves
the removal of the hydroxy protecting groups in the sugar
8 i3~ 9~32
moiety and the subsequent optional phosphorylation at the
S'-position in the sugar moiety to obtain a compound of
formula [I].
Furthermore, the present invention relates to a
5 process for producing 2'-alkylidenepyrimidine nucleoside
derivatives represented by formula [I"'] shown below,
which process comprises the following Steps (1), (2) and
(3) (hereinafter referred to as "the fourth process").
1319932
. g
INHR6 NHR6
O ~ o~
10 Z ~V~ ZO ~V~
ZO CH-R3
[II"'] [III"'~
Step (2)
NH2
1 O i ~ R~
R~O V ~ ~
HO CH-R3 H-R3
~: 30 lI 1 [IV"'}
:~
: wherein R2, R3, R4 and Z are as defined hereinbefore, and
R6 is an acyl group.
In the above process, Steps (1) and (2) are the same
reaction steps as those in the first process, and Step
(3) involves the removal of the acyl group represented by
R6 and the subse~uent optional phosphorylation at the 5'-
lo 13~ ~932
position in the sugar moiety to obtain a compound offormula [I"'].
The present invention, in another aspect thereof,
relates to an antiviral agent comprising an effective
5 amount of 2'-alkylidenepyrimidine nucleoside derivatives
of the above shown formula [I] or salts thereof and a
pharmaceutically acceptable carrier or adjuvant.
The present invention, in still another aspect
thereof, relates to a method for the treatment of viral
10 infections in a subject which comprises administering a
therapeutically effective amount of the antiviral agent
described above to said subject.
Detailed Description of the Invention
~ereinafter, the present invention will be set forth
in detail.
ComPound of the present invention
The 2'-alkylidenepyrimidine nucleoside derivatives
according to the present invention are represented by
20 formula [I] shown hereinbefore.
Rl, R2, R3 and R4 in the formula are as defined
previously. Specific examples of the lower alkyl groups
represented by R2 and R3 are those having 1 to 3 carbon
atoms, more specific examples being meth~l, ethyl, propyl
25 and isopropyl. Examples of the halogen atom represented
by R2 are chlorine, fluorine, bromine, and iodine.
Typical examples of the compounds of the present
invention include nucleosides such as 2'-methylidene-2'-
deoxyuridine, 2'-methylidenethymidine, 2'-
30 ethylidenethymidine, 2'-methylidene-2'-deoxycytidine, 2'-
methylidene-2'-deoxy-5-fluorouridine, 2'-methylidene-2'-
deoxy-5-chlorouridine, 2'-methylidene-2'-deoxy-5-
bromouridine, and 2'-methylidene-2'-deoxy-5-iodouridine,
and 5'-phosphates thereof.
Among these nucleosides of the present invention, a
group of compounds of formula lI] wherein R2 is a
hydrogen or halogen atom or a methyl group and R3 is a
`` 11 1319~32
hydrogen atom have a high antiviral activity against
herpes simplex virus ~HSV).
The compounds of the present invention also include
salts thereof. Bxamples of such salts are any
5 pharmaceutically acceptable salts, such as acid addition
salts including hydrochloride and sulfate in the case
where R4 in the aforementioned formula [I] is a hydrogen
atom, and alkali metal salts including sodium, potassium
and lithium salts, alkaline earth metal salts including
10 calcium salt, or ammonium salt in the case where R4 is a
phosphate residue.
Production of the comPounds of the Present invention
The compounds of the present invention are novel
15 compounds, and can be produced by the second to fourth
processes set forth hereinbefore when Rl in formula [I]
is an amino group while by the second and third processes
when Rl is a hydroxy group.
The reaction steps in the respective processes will
20 be described below in detail.
Prearation of the startina compounds:
The pyrimidine nucleoside derivatives used in the
process of the present invention as starting compounds
are represented by formula [II'], lII"] or [II"'] shown
25 earlier.
Rl, R2, R5 , R6 and Z in the formulae are as defined
previously. Specific examples of the alkoxy group
represented by R5 are lower alkoxy groups having 1 to 3
cabon atoms, more specific examples being methoxy, ethoxy
30 and propoxy. Examples of the acyl group represented by
R6 are aliphatic acyl groups such as acetyl,
chloroacetyl, dichloroacetyl, trichloroacetyl,
trifluoroacetyl, methoxyacetyl, propionyl, n-butyryl,
isobutyryl, (E)-2-methyl-2-butenoyl, pentanoyl and
35 pivaloyl, and aromatic acyl groups such as benzoyl, o-
(dibromomethyl)benzoyl, p-phenylbenzoyl, 2,4,6-
trimethylbenzoyl, p-toluoyl, p-anisoyl, p-halobenzoyl, p-
1 3 ~
~2
nitrobenzoyl, and p-methoxybenzoyl. Further, the
protecting group represented by Z may be any of those
customarily used as hydroxy protecting groupsr for
example, acyl groups such as acetyl, propionyl, butyryl,
benzoyl, and naphthoyl; acetal- or ketal-type protecting
groups such as ethylidene, propylidene, isopropylidene,
benzylidene t cyclohexylidene, cyclopentylidene,
methoxymethylidene, ethoxymethylidene, and
dimethoxymethylidene; aralkyl groups such as benzyl, p-
methoxybenzyl t 3 t 4-dimethoxybenzyl, diphenylmethyl,
triphenylmethyl, a- or ~-naphthylmethyl, and a-
naphthyldiphenylmethyl; and silyl groups such as
tr i m e t h y l s i l y l , t - b u t y l d i m e t hy l s i l y l ,
methyldiisopropylsilyl, triisopropylsilyl, and
tetraisopropyldisiloxyl (TIPDS).
1. Preparation of starting compound [II']
These starting compounds can be synthesized by
applying a known method.
The compounds of formula ~ can be prepared, for
example, by the following reaction procéss.
~ 13
i31`9932
OH R6
oJ ~ oJ~ R2
~ ~ *
HO V ~ V ~
HO OH
~A) (B)
R6l
N ~ R2
O N
* ~ Z~VO~
/
zo .0
lII']
wherein R2, R5 and Z are as defined earlier.
More particularly, the hydroxy groups in the sugar
moiety of a uridine derivative of formula (A) are
: protected, the 4-position in the base moiety is
halogenated with a halogenating agent, and then an alkoxy
group is introduced at this position by the reaction with
an alkoxide to obtain a compound of formula (B).
Subsequently, the 3'- and 5'-positions in the sugar
131~32
14
moiety of the 4-alkoxy compound of formula ~B) thus
obtained are protected, and the hydroxy group at the 2'-
position in the sugar moiety is subjected to oxidation,
whereby a compound of formula lII'] can be obtained.
The hydroxy protecting group in the halogenation
reaction is not particularly limited insofar as it does
not hinder the halogenation reaction. Conventional
hydroxy protecting groups such as acyl groups, acetal- or
ketal-type groups, aralkyl groups and silyl groups may be
applied, especially preferred protecting groups being
those which are not removed due to the presence of an
acid such as acyl groups.
The protection with an acyl group, for instance, may
be carried out by a conventional method in which the
compound of formula (A) is reacted with 3- to 10-fold
mols of an acylating agent (e.g., an acid anhydride or
acid chloride of acetic acid, propionic acid, butyric
acid, benzoic acid, or substituted benzoic acid) in a
solvent (e.g., a basic solvent such as pyridine,
picoline, diethylaniline, tributylamine and triethylamine
or a mixture thereof with acetonitrile,
dimethylformamide, dimethylacetamide, formamide,
chloroform, methane dichloride, dioxane, tetrahydrofuran
or dimethylaminopyridine) at a temperature of from 0 to
50C.
The halogenation reaction can be carried out by
causing a halogenating agent to react with the compound
in an inert solvent (e.g., chloroform and methylene
chloride). For the halogenating agent, thionyl chloride,
thionyl bromide, phosphorus oxychloride and the like, if
desired, in the form of a solution in an organic solvent
such as dimethyl sulfoxide, can be used.
The amount of the halogenating agent to be added is
approximately 1- to 5-fold mols per mol of the compound
of formula (A), and the reaction may be carried out under
heat at reflux.
13`1 9932
The introduction of the alkoxy group can be
accomplished by reacting under heat the 4-halogeno-
compound of formula (A) with protecting groups with about
l- to 5-fold mols of an alkoxide (e.g., sodium methoxide,
potassium methoxide, sodium ethoxide, potassium ethoxide,
and sodium propoxide) in a solvent (e.g., methanol,
ethanol, and propanol).`
The protecting groups at the 3'- and 5'-positions
may be those used in the halogenation reaction described
above, silyl groups being preferred and TIPDS group being
especially preferred.
With reference to the protection, for example, by
silylation, the amount of a silylating agent to be used
can be suitably determined within the range of from l- to
3-fold mols per mol of the compound of formula (B), and
the same reaction conditions as in the aforementioned
acylation reaction can be emplo~ed.
As a method for the oxidation of the hydroxy group
at the 2'-position, chromic acid oxidation (Method A)
using a chromic acid-pyridine-acetic anhydride complex
and the like, or activated dimethyl sulfoxide oxidation
(Method B) using activated dimethyl sulfoxide obtained
from oxalyl chloride-dimethyl sulfoxide and the like can
be applied.
The oxidation reaction can be carried out in the
presence of an oxidizing agent in an amount of 1- to lO-
fold mols per mol of the compound for 1 to 10 hours at -
10C to room temperature in the case of Method A or at -
lO to -80C in the case of Method B.
2. Preparation of starting compound [II"]
The compounds of formula ~II"] can be prepared, for
example, by the following reaction process.
16 ~ 3 2
OJ` N Oi I~RI
HO V V ~J
HO OH
(C) [II"]
wherein Rl, R2 and Z are as defined above.
More particularly, the hydroxy groups at the 3'- and
5'-positions in the sugar moiety of a nucleoside of
formula (C) are protected, and thereafter the hydroxy
group at the 2'-position in the sugar moiety is subjected
to oxidation, whereby a compound of formula lII"] can be
obtained.
The protection reaction o~ the hydroxy groups at the
3'- and 5'-positions and the oxidation reaction of the
hydroxy group at the 2'-position can be carried out in
accordance with those performed in the preparation of the
compound of formula [II'] described above.
3. Preparation of starting compound [II"']
The starting compounds [II"'] can be prepared, for
example, by introducing protecting groups on the amino
group at the 4-position in the base moiety and the
hydroxy groups at the 3'- and 5'-positions in the sugar
moiety of a cytidine derivative and then subjecting the
hydroxy group at the 2'-position in the sugar moiety to
oxidation reaction. This reaction process can be
illustrated by the following reaction formulae:
17 131 ~932
INH2 l HR6
N ~R~ N ~ R~
V ~ V
\ \ __/
r
HO OH
~D)
N~R6
N ~R2
Jl
O N
* ~ ZOVO~
'
ZO O
lII"'~
wherein R2, R6 and Z are as defined previously.
The introduction of protecting groups represented by
R6 and Z into the cytidine derivative may be carried out
in accordance with a method ordinarily employed for the
particular protecting groups selected.
For example, an acyl group represented by R6 can be
introduced by causing reaction in the presence of 1- to
5-fold mols of an acylating agent ~an acid anhydride or
18
13~9332
acid chloride of the acid corresponding to R6) per mol of
the cytidine derivative in a solvent (e.g., a basic
solvent such as pyridine, picoline, diethylaniline,
tributylamine and triethylamine or a mixture thereof with
acetonitrile, dimethylformamide, dimethylacetamide,
formamide, chloroform, methane dichloride, dioxane,
tetrahydrofuran or dimethylaminopyridine) at a
temperature of from 0 to 50C for 1 to 30 hours.
~lso, a hydroxy protecting group represented by Z,
for example, a silyl group, can be introduced by causing
reaction in the presence of 1- to 3-fold mols of a
silylating agent per mol of the cytidine derivative under
the same conditions as in the acylation.
Subse~uently, a compound (D) with protecting groups
thus prepared is subjected to oxidation reaction to
obtain the target starting compound.
The oxidation of the hydroxy group at the 2'-
position of the compound (D) can be performed by the
aforementioned chromic acid oxidation (Method A) using a
chromic acid-pyridine-acetic anhydride complex and the
like, or activated dimethyl sulfoxide oxidation (Method
B) using activated dimethyl sulfoxide obtained from
oxalyl chloride-dimethyl sulfoxide and the like under the
conditions described hereinbefore.
The starting compounds [II'], [II"] and [II"'] thus
prepared can be isolated and purified by a suitable
combination of isolation and purification methods
commonly applied to nucleosides (e.g., various
chromatographic procedures such as ion exchange and
adsorption, and the rècrystallization method). For
example, after the solvent has been distilled off, the
compound is subjected to column chromatography and
crystallized from n-hexane or any other suitable organic
solvent.
Second process
19 i31~9~
Step (1) in the second process is the reaction step
of alkylidenating the 2'-position in the sugar moiety of
the compound of formula [II'] with Wittig's reagent.
The Wittig's reagent used in the alkylidenation
reaction is alkylidenephosphorane represented by the
formula
(C6H5)3p = CH-R3
wherein R3 is as defined earlier.
Typically, triphenylphosphine methylene r
triphenylphosphine ethylene and triphenylphosphine
propylene are employed.
The Wittig's reagent used in the reaction is
lS preferably prepared immediately before use from a
triphenylphosphonium compound represented by the formula
[(C6H5)3P+-CH2-R3]X- wherein R3 is as defined earlier and
X~ is a halogen ion such as Br~ or I- (e.g.,
methyltriphenylphosphonium bromide,
methyltriphenylphosphoniUm iodide, and
ethyltriphenylphosphonium bromide) and a strong alkali
(e.g., potass;um hydride, sodium hydride, n-butyl
lithium, sodium methoxide, potassium-t-butoxide, and
sodium amide) by a conventional method.
The amount of Wittig's reagent to be used can be
suitably determined within the range of from 1- to 3-fold
mols per mol of the compound of formula [II'].
The alkylidenation reaction using the above
described Wittig's reagent can be carried out by reacting
the compound of formula [II'] with Wittig's reagent at
3û to 30C for 0.5 to 20 hours in a solvent (e.g.,
tetrahydrofuran, dioxane, ether, benzene, and dimethyl
sulfoxide singly or in a mixture of two or more members).
The compound of formula [III'] thus prepared can be
isolated and purified by means of ordinary silica gel
column chromatography.
~ 20 131993~
Step (2) in the second process is the reaction step
of removing the hydroxy protecting groups in the sugar
moiety of the compound of formula lIII'].
The removal of the protecting groups may be carried
5 out by suitably selecting an ordinary treatment such as
acidic hydrolysis, alkaline hydrolysis, ammonium fluoride
treatment or catalytic reduction depending upon the
protecting groups used. For example r silyl groups, when
used as the hydroxy protecting groups, can be removed by
10 ammonium fluoride treatment or acidic or alkaline
hydrolysis.
The compound of formula [IV'] thus synthesized can
be isolated by ordinary silica gel column chromatography.
In Step (3) in the second process, the compound of
15 formula [IV'] is subjected to amination reaction in order
to obtain the compound of formula [I] in which Rl is an
amino group, or hydrolysis reaction in order to obtain
the compound of the same formula in which Rl is a hydroxy
group.
The amination reaction may be carried out by a
conventional method, for example, by reacting methanolic
ammonia with the compound of formula [IV'] in a sealed
tube. The reaction temperature i5 50 to 150C.
The hydrolysis reaction may also be carried out by a
25 conventional method, especially preferably by acidic
hydrolysis.
In order to produce the compound of formula [I] in
which R4 is a phosphate residue, the compound of formula
[IV'] is reacted, after completion of the amination
30 reaction or the hydrolysis reaction described above, with
a phosphorylating agent commonly used for the selective
phosphorylation at the 5'-position of nucleosides, such
as phosphorus oxychloride or tetrachloropyrophosphoric
acid, by a known method, whereby the desired compound can
35 be obtained in the free acid or salt form.
Third process:
.
~ 21 1~9~32
Step (1) in the third proce~s i8 the reaction step
of alkylidenating the 2'-position in the sugar moiety of
the compound of formula [II"] with Wittig's reagent.
The alkylidenation reaction and the isolation and
5 purification of the compound of formula [III"] can be
accomplished in accordance with Step ll) in the second
process.
Step (2) in the third process is the reaction step
of removing the hydroxy protecting groups in the sugar
10 moiety of the compound of formula [III"] and optionally
phosphorylating the 5'-position in the sugar moiety
thereof.
The removal of the protecting groups and the
phosphorylation can be carried out in accordance with
15 Steps (2) and (3) in the second process.
Fourth Process;
Step (1) in the fourth process is the reaction step
of alkylidenating the 2'-position in the sugar moiety of
the compound of formula [II"'] with Wittig's reagent.
The alkylidenation reaction and the isolation and
purification of the compound of formula [III"'] can be
accomplished in accordance with Step (1) in the second
process.
Step (2) in the fourth process is the reaction step
25 of removing the hydroxy protecting groups in the sugar
moiety of the compound of formula [III"']. This removal
of the protecting groups and the isolation and
purification of the compound of formula [IV"'~ can be
carried out in accordance with Step (2) in the second
30 process.
Step (3) in the fourth process is the reaction step
of removing the acyl group represented by R6 and then
optionally phosphorylating the 5'-position in the sugar
moiety.
The acyl group can be removed by suitably selecting
a removal method customarily used for the particular acyl
group employed, for example, by alkaline hydrolysis using
13~33~
a methanol-ammonia (1:1) mixture, concentrated ammonia
and the like.
The phosphorylation can be carried out in accordance
with that in Step (3) in the second process.
The compound of formula [I] or lI"'] thus synthesized
can be isolated and purified by a suitable combination of
methods ordinarily employed for the isolation and
purification of nucleosides or nucleotides. For example,
in the case of a nucleoside (R4 being a hydrogen atom),
10 the compound may be crystallized from an appropriate
solvent such as ethanol after the reaction solvent has
been distilled off. If desired, the compound can be
obtained in salt form. In the case of a nucleotide (R4
being a phosphate residue), on the other hand, the
15 compound is purified by ion exchange column
chromatography using an ion exchange resin or by
adsorption column chromatography using activated carbon
and the like, and freeze-dried or crystallized, whereby
the compound in free acid form or, if desired, in salt
20 form can be obtained.
In the event that the phosphonium salt of Wittig's
intermediate which is a reaction intermediate (the
structure of this intermediate is not clear, but may be
presumed to be as shown below in view of the reaction
25 process according to the first process:
23
1~19~3`~
o~
~ V\~ I
10 _ ~ I
ZO CHR3P(~6H5)3 J
wherein R2, R3, R5, Z and X~ are as defined earlier)
15 remains in the reaction solution in the alkylidenation
reaction in the process for producing the compounds of
the present invention, i.e., any of the first to fourth
processes, the intermediate can optionally be reacted
with a strong alkali (e.g., potassium hydride, sodium
20 hydride, n-butyl lithium, sodium methoxide, potassium-t-
butoxide, and sodium amide) in a solvent (e.g.,
tetrahydrofuran, dioxane, ethyl ether, benzene, and
dimethyl sulfoxide singly or in a mixture of two or more
members) to obtain the compound of formula [III], lIII'],
[III"] or [III"']. Thus, the production yield of the end
product can be -increased by carrying out the strong
alkali treatment after Step (1) in the respective
processes.
~he 2'-alkylidenepyrimidine nucleoside derivatives
of the present invention can be synthesized by the first
process, and more specifically the second to fourth
processes, as has been set forth in detail. From those
processes, the most suitable one may be selected
depending upon the desired compounds and the starting
compounds used.
In order to produce, for example, the compound
wherein Rl is an amino group, any of the second to fourth
24 i`3~993'~
processes can be employed, but the fourth process wherein
a specific cytidine derivative is used as the starting
compound, has the following advantages over the second
process wherein a uridine derivative is used as such.
(1) The starting compound supplied for the
alkylidenation reaction in the fourth process can be
prepared in a shorter reaction step than in the second
process.
(2) The fourth process substantially comprises two
10 steps, i.e., the alkylidenation and the removal of
protecting groups, and the amination carried out in the
second process can be omitted.
(3) The production yield of the end products can be
improved by the curtailment or omission of the reaction
15 step as mentioned in paragraphs (1) and (2). More
specifically, the overall yield of 2'-alkylidenecytidine
derivatives produced from the starting compound 2'-
ketonucleoside is 53~ in the fourth process and 25% in
the second process, indicating that two-fold or more
20 production yield can be attained according to the fourth
process.
Utility of the compounds of the present invention
The compounds of the present invention or salts
thereof exhibit antiviral activities against DNA viruses
such as herpes simplex virus (HSV) and cytomegalovirus
(CMV~ of the herpesvirus family, and the drugs of the
present invention comprising these compounds as active
ingredients are clinically used for the treatment o~
viral infections.
While the dose level of the compounds of the present
invention incorporated in the drugs of the present
invention as active ingredients may vary depending, for
example, upon the severity of patients' diseases and
their tolerance for the drugs, and should be determined
ultimately by doctors, 0.1 to 10 9, preferably 0.2 to 5
9, per day of the compounds are ordinarily admini~tered
a~
1 319932
to an adult in one portion or several portions. The
drugs can be administered in any mode suited for the
route of administration.
The drugs of the present invention can be prepared
5 for administration by any convéntional method suitable
for the purpose. Thus, the drugs include pharmaceutical
compositions containing 2'-alkylidenepyrimidine
nucleoside derivatives of formula [I~ suitable as
medicines for humans.
Such composition~ are provided for administration by
a known method through any pharmaceutically acceptable
carriers or adjuvants required.
In the case, for example, of a pharmaceutical
composition for oral use, the composition is provided in
the form suitable for absorption through the alimentary
tract and may be formulated as solid preparations such as
tablets, capsules, powders, sugar-coated tablets, and
granules t or liquid preparations such as syrups,
suspensions and elixirs. The solid preparation can be
20 formulated by selecting and adding from a pharmaceutical
viewpoint an adjuvant, for example, a binder such as
syrup, gum arabic, gelatin, sorbitol, tragacanth, or
polyvinyl pyrrolidone; a vehicle such as lactose, sugar,
cornstarch, calcium phosphate, sorbitol, or glycine; a
lubricant such as magnesium stearate, talc, polyethylene
glycol, or silica; a disintegrator such as potato starch;
a wetting agent; a stabilizer; and a taste modifier. For
the liquid preparation, a suspending agent such as
sorbitol, syrup, methyl cellulose, glucose/sugar syrup,
30 gelatin, hydroxyethyl cellulose, carboxymethyl cellulose,
aluminum stearate gel, or a hydrogenated edible oil; an
emulsifier; and an antiseptic such as methyl p-
hydroxybenzoate, propyl p-hydroxybenzoate, or sorbic acid
can be used, if desired, as an adjuvant.
In order to obtain a pharmaceutical preparation for
injection use, a pH adjusting agent, a buf~er, a
stabilizer, a preservative, a solubilizer or the like is
a6
13~9332
added, if desired, to the compounds of the pre~ent
invention which form active ingredients of the drugs of
the invention to formulate a preparation for
subcutaneous, intramuscular or intravenous injection by a
conventional method.
Hereinafter, the methods for testing the anti-HSV
and anti-CMV activities of the compounds of formula [I]
incorporated in the drugs o the present invention as
active ingredients and the results obtained will be set
forth.
Test Method (HSV):
A. Human embryonic lung cells are subcultivated in
Eagle's MEM supplemented with 10~ semi-fetal calf serum.
B. The cells thus subcultivated are used as a parent
culture. A cell suspension obtained from the parent
culture by dividing it at 1:2 split is seeded into 96
microwells in an amount of 150 yl/well and incubated in a
CO2-incubator at 37C for 4 to 5 days.
C. The culture fluid i5 discarded, and 100- to 320-
so% tissue culture infective doses (100-320 TCID50) of
~SV type 1 (HSV-11 strain VR-3 or HSV type 2 (HSV-2)
strain MS is inoculated. After incubation at 37C for 1
hour, the virus fluid is discarded, 150 ~1 of Eagle's MEM
supplemented with 2.5~ semi-fetal calf serum and
containing an appropriate amount of each test compound is
added, and cultivation is carried out at 37C. Each of
the test compounds is usually diluted in the range o
from 10~ to 1 ~g/ml by serial 0.5 log10 decrements.
D. After cultivation for 2 to 3 days, the degree of
the cytopathic effect (CPE) caused by the viral infection
in each of the wells is observed microscopically. At the
time when the control cultures, in which the test
compound is absent, have shown complete CPE by the viral
infection, the degree of CPE in each well is rated from 0
to 4.
E. The minimum concentration at which the CPE is
inhibited at least by 50% (indicated by the CPE score not
27 13~9~
exceeding 2) is defined as the minimum inhibitory
concentration ~MIC) of the test compound.
Test method (CMV):
A. Human embryonic lung cells are subcultivated in
Eagle's MEM supplemented with 10% semi-fetal calf serum.
B. The cells thus subcultivated are used as a parent
culture. A cell suspension obtained from the parent
culture by dividing it at 1:2 split is seeded into 24
semi-microwells in an amount of 400 ~l~well and incubated
in a CO2-incubator at 37C for 4 to 5 days.
C. The culture fluid is discarded, and about 50
plaque forming units of CMV strain AD 169 is inoculated.
After incubation at 37C for 1 hour, 400 ~1 of Eagle's
MEM supplemented with 2.5% semi-fetal calf serum and
containing an appropriate amount of each test compound is
added, and cultivation is carried out at 37C. Each Oc
the test compounds is usually diluted in the range of
from 100 to 1 yg/ml by serial 0.5 log10 decrements.
D. After cultivation for 4 to 6 days, infected cells
are stained with a 0.5% Crystal Violet solution, and the
number of plaques formed is counted under the microscope.
E. The minimum concentration at which the plaque
- formation is inhibited at least by 50%t based on the
number of plaques formed in the control cultures in which
the test compound is absent, is defined as the minimum
inhibitory concentration (MIC) of the test compound.
28
Test results:
Test Compound MIC (~g/ml)
Rl R2 R3 R4 HSV-lHSV-2 CMV
NE~2 ~ H ~1 1 1 O .1
0~1C~3 H H 1 1 5. 6
O~ Cl H H 10 10
OH Br H H 3.2 3.2
OE~ I H ~ 1 1
O~ CH3 CH3 H 10 32
EXAMP~E
The present invention will now be illustrated with
reference to the following examples, it being understood
that these examples are not intended to limit the scope
of the invention.
Example 1
Preparation of 2'-methylidene-2'-deoxycytidine (com~ound
of formula [I] wherein Rl is NH2, R2 is H, R~ is H, and
R4 is ~)~ydrochloride
1) Synthesis of 4-O-ethyluridine (compound of formula
(B) wherein R2 iS H, and R5 is OC2H5)
To a solution of 3.35 g o 2',3',5'-tri-O-
acetyluridine in 50 ml of chloroform were added 8.1 ml of
thionyl chloride and 0.5 ml of dimethylformamide, and the
mixture refluxed for 6.5 hours and evaporated to dryness
under reduced pressure. The residue was dissolved in 20
ml of ethanol and 30 ml of lN sodium ethoxide added. The
mixture was refluxed for 2 hours and neutralized with lN
hydrochloric acid. The salt precipitated was filtered
off and the remaining solution concentrated to dryness.
The concentrate was adsorbed onto a silica gel column
29 13~9932
(4x31 cm), a fraction containing the desired compound
eluted with a 16~ ethanol-chloroform mixture, and the
solvent distilled off to obtain the desired compound in
crude crystal form. Recrystallization from ethanol gave
2.08 9 (yield 84.2%) of the desired compound.
Melting point: 136 - 137.5C
Elemental analysis:
(as CllHl6N2O6 3 H20)
Calcd. C: 46.97%t H: 6.09~,
N: 9.96~ r 0: 36.98%
Found C: 46.91%, ~: 6.02%,
N: 9.98%, O: 37.09%
2) Synthesis of 1-(3,5-O-TIPDS-~-D-erythropentofuran-2-
ulosyl)-4-ethoxy-2-pyrimidinone (compound of formula
[II] wherein R2 is H, R5 is OC2H5, and Z(3')-Z(5')
are TIPDS)
To an ice-cooled solution of 7.04 9 of 4-O-
e~hyluridine in 80 ml of pyridine was added 9.57 9 of
1,1,3,3-dichlorotetraisopropyldisiloxane, and the mixture
stirred at room temperature for 4.5 hours to cause
reaction. To the reaction mixture was added ice water,
and the solvent distilled off. The residue was
partitioned between chloroform and water, the chloroform
layer dried and then the solvent distilled off. The
residue was adsorbed onto a silica gel column (lOx130
cm). Fractions eluted with a 40~ ethyl acetate-hexane
mixture were collected and concentrated to obtain 12.3 g
of a 3',5`-TIPDS compound.
Subsequently, a solution of 2.7 ml of oxalyl
chloride in 40 ml of methylene chloride was cooled to -
70C. To the cooled solution was added dropwise over a
period of 20 minutes under argon 4.8 ml of dimethyl
suloxide dissolved in 20 ml of methylene chloride, and
the mixture stirred for 30 minutes. To the reaction
mixture was added dropwise the above obtained 3',5'-TIPDS
compound (12.3 9) dissolved in 50 ml of methylene
chloride, the resulting mixture stirred at -70C for 2
~ 30 13199~
hours, 20 ml of triethylamine added, and the mixture
further stirred for 1 hour. This reaction solution was
left standing until its temperature reached room
temperature, water added, and the resultin~ solution
partitioned. The methylene chloride layer was removed
and the solvent distilled of~. The residue was dissolved
in ethyl acetate and partitioned from water. The ethyl
acetate layer was concentrated to dryness and adsorbed
onto a silica gel column (5 x 28 cm). Fractions eluted
with a 20% ethyl acetate-n-hexane mixture and containing
the desired compound were collected and the solvent
distilled off. The fractions were then crystalli~ed from
n-hexane to obtain 10.2 g (yield 72.1~) of the desired
compound.
Melting point: 157.5 - 159C
Elemental analysis:
(as C2oH4oN2o7si2)
Calcd. C: 53.87%, H: 7.86%,
N: 5.46%
Found C; 53.73%, H; 7.87%,
N; 5.57%
3) Synthesis of 2'-methylidene-4-O-ethyl-2'-
deoxyuridine (compound of formula [IVl wherein R2 is
H, R3 is H, and R5 is OC2H5)
232 mg of potassium hydride was added to 2.4 ml of
dimethyl sulfoxide under argon and the mixture stirred at
room temperature for ~0 minutes. The resulting potassium
hydride-dimethyl sulfoxide mixture was added dropwise to
a solution of 2.2 9 of methyltriphenylphosphonium bromide
in 8 ml of dimethyl sulfoxide in an ice bath under argon
and the mixture stirred for 10 minutes.
To this mixture was added dropwise under argon a
solution of 1.02 g of the above obtained crystalline 1-
~3,5-O-TIPDS-~-D-erythropentofuran-2-ulosyl)-4-ethoxy-2-
pyrimidinone in 10 ml of dimethyl sulfoxide, and themixture stirred in an ice bath for 2 hours. To the
reaction mixture were added 10 ml of lN aqueous ammonium
31 ~3~3~
chloride solution and then 50 ml of ethyl acet~te and 40
ml of water whereby the mixture was partitioned. The
organic layer was concentrated under reduced pressure,
adsorbed onto a silica gel column (2.4x30 cm), and eluted
with an n-hexane-ethyl acetate mixture to obtain a 2'-
methylidene compound.
To a solution of 320 mg of the thus obtained
compound in 10 ml o tetrahydrofuran was added 1 ml of
tri-n-butylammonium fluoride, and the mixture stirred at
room temperature for 10 minutes. The reaction mixture
was neutralized with acetic acid, adsorbed onto a silica
gel column (2.4x12 cm), and eluted with a chloroform-
ethanol mixture. Fractions containing the desired
compound were collected to obtain 155 mg (yield 30%) of a
deprotected 2'-methylidene-4-O-ethyl compound.
Melting point: 157.5 - 159C
Elemental analysis:
(as Cl2Hl6N25)
Calcd. C: 53.72%, H: 6.01%,
N: 10.44%
Found C: 53.80~, H: 5.99%,
N: 10.37%
4) Synthesis of 2'-methylidene-2'-deoxycytidine
(compound of formula [I] wherein Rl is NH2, R2 is H,
R3 is H, and R4 is H)hydrochloride
150 mg of the 2'-methylidene-4-O-ethyl compound was
dissolved in 10 ml of an ammonia saturated methanol
solution in an ice bath and the resulting solution poured
into a tube which was then sealed and heated at 100C for
2 days. The reaction solution was allowed to cool, 2 ml
of 2N hydrochloric acid added, and the mixture
concentrated. Crystallization from an ethanol-water
mixture gave 125 mg (yield 81.7%) of the title compound.
Melting point: >300C (carbonized at 148 - 155C)
Elemental analysis:
(as CloHl3N3O4 HCl)
Calcd. C: 43.57%, H: 5.19%,
~ 3 ~3~`~9~
N: 15.~4~
Found C: 43.~7~, H: 5.23~,
N: 15.22
Example 2
Preparation of 2'-methylidenethymidine (compound of
formula ~I] wherein R1 is OH, R2 is CH~, R3 is H, and R4
is H)
1) Synthesis of 3',5'-0-TIPDS-2'-ketothymidine
(compound of formula [II] wherein R2 is CH3, and
Z(3')-Z(5') are TIPDS)
To a solution of 4.13 g of 5-methyluridine in 50 ml
of pyridine was added in an ice bath 5.57 9 of 1,1,3,3-
dichlorotetraisopropyldisiloxane, and the mixture stirred
at room temperature for 6 hours. To the resulting
mixture was added a small amount of water, and the
mixture stirred for 30 minutes and concentrated to
dryness under reduced pressure. The residue was
partitioned between chloroform and water, and the organic
layer concentrated and adsorbed onto a silica gel column
(5x21 cm). From fractions eluted with a 2~ ethanol-
chloroform mixture was obtained a 3',5'-protected
compound.
Separately, a solution of 1.7 ml of oxalyl chlorid~`
in 40 ml of methylene chloride was cooled to -70C. To
the cooled solution was added dropwise under argon a
solution mixture of 3 ml of dimethyl sulfoxide and 20 ml
of methylene chloride, and the resulting mixture stirred
for 30 minutes. To the reaction mixture was added
dropwise 8.04 9 of the above obtained 3',5'-protected
compound dissolved in 50 ml of methylene chloride, and
the resulting mixture stirred further at -70C for 2
hours. To this mixture was added dropwise 6.6 ml of
triethylamine, and the solution stirred for 1.5 hours,
left standing until its temperature reached room
temperature and partitioned between chloroform and water.
The organic layer was concentrated to dryness under
33
1 3 ~ 2
reduced pressure and developed through a silica gel
column (4x28 cm). Fractions eluted with a 40~ ethyl
acetate-n-hexane mixture were concentrated and
crystallized from n-hexane to obtain 6.68 g (yield 83.2%)
of a 2'-keto compound.
Melting point: 168 - 170C
Elemental analysis:
(as C22H38N2O7si2)
Calcd. C: 52.98%, H: 7.68
N: 5.62%
Found C: 52.93%, H: 7.71%,
N: 5.61%
2) Synthesis of 2'-methylidenethymidine (compound of
formula [I] wherein Rl is OH, R2 is CH3, R3 is H,
and R4 is H)
455 mg of potassium hydride was added to 5 ml of
dimethyl sulfoxide under argon and the mixture stirred at
room temperature for 50 minutes. The resultant solution
containing potassium hydride was added dropwise to a
separately prepared solution of 4.28 g of
methyltriphenylphosphonium bromide in 10 ml of dimethyl
sulfoxide in an ice bath, and the mixture stirred further
for 20 minutes. To the resultant solution was added
dropwise a solution of 1.5 g of the above obtained 3',5'-
O-TIP~S-2'-ketothymidine in a solvent mixture of 5 ml of
tetrahydrofuran and 5 ml of dimethyl sulfoxide, and the
mixture stirred at room temperature for 10 hours.
The reaction solution was then neutralized with lN
ammonium chloride and partitioned between 120 ml of ethyl
acetate and 120 ml of water. The organic layer was
concentrated to dryness and the residue developed through
a silica gel column (2.4x24 cm). Fractions eluted with a
20% ethyl acetate-n-hexane mixture were collected to
obtain a 2'-methylidene compound. This compound was
dissolved in 10 ml of tetrahydrofuran, 1 ml of 1 M tetra-
n-butylammonium fluoride-tetrahydrofuran solution added,
34 i31~2
and the mixture stirred at room temperature for 10
minutes to deprotect the compound.
Subsequently, the reaction mixture was neutralized
with acetic acid, concentrated to dryness under reduced
pressure, and developed through a silica gel column
(2.4x14 cm). Fractions eluted with a 7% ethanol-
chloroform mixture were collected and concentrated to
obtain 257 mg (yield 85%) of a crystalline powder of 2'-
methylidenethymidine.
Melting point: 161 - 162C
Elemental analysis:
(as CllHl4N2o5)
Calcd. C: 49.58%, H: 5.83%,
N: 11.57%
Found C: 49.46~, H: 5.91%,
N: 11.48%
Example 3
Preparation of 2'-ethylidenethymidine (compound of
formula [I] wherein Rl is OH, R2 is CH , R3 is CH , and R4
3 3
is H)
455 mg of potassium hydride was added to 5 ml of
dimethyl sulfoxide under argon and the mixture stirred at
room temperature for 50 minutes.
The resultant solution containing potassium hydride
was added dropwise to a separately prepared ~olution of
4.44 g of ethyltriphenylphosphonium bromide in 10 ml of
dimethyl sulfoxide in an ice bath and the mixture stirred
further for 20 minutes.
To the resulting solution was added dropwise a
solution of 1.5 g of the 3',5'-O-TIPDS-2'-ketothymidine
obtained in Example 2 in a solvent mixture of 5 ml of
tetrahydrofuran and 5 ml of dimethyl sulfoxide, and the
mixture stirred at room temperature for 12 hours.
The reaction solution was then neutralized with lN
ammonium chloride and partitioned between 140 ml of ethyl
acetate and 140 ml of water. The organic layer was
1319~32
concentrated to dryness and the residue developed through
a silica gel column (2x18 cm). Fractions eluted with a
10% ethyl acetate-n-hexane mixture were combined to
obtain a 2'-ethylidene compound. This compound was
dissolved in 10 ml of tetrahydrofuran, 1 ml of 1 M tetra-
n-butylammonium fluoride-tetrahydrofuran solution added,
and the mixture stirred at room temperature for 30
minutes to deprotect the compound.
Subsequently, the reaction mixture was neutralized
with acetic acid, concentrated to dryness under reduced
pressure, and developed through a silica gel column ~2xlO
cm). Fractions eluted with a 7~ ethanol-chloroform
mixture were collected and concentrated to obtain 190 mg
of an amorphous powder of 2'-ethylidenethymidine.
Elemental analysis:
(as Cl2~l6N20s)
Calcd. C: 53.72~, H: 6.01%,
N: 10.44~
Found C: 53.68%, H: 6.15%,
N: 10.39%
Example 4
Preparation of 2'-methylidene-2'-deoxy-5-fluorouridine
(compound of formula [I] wherein Rl is O~, R2 is F, R3 is
H, and R4 is H)
1) Synthesis of 1-(3,5-O-TIPDS-~-D-erythropentofuran-2-
ulosyl)-5-fluorouracil (compound of formula [II]
wherein R2 is F, and Z(3')-Z(5') are TIPDS)
To a solution of 2.42 g of 5-fluorouridine in 30 ml
of pyridine was added in an ice bath 3.3 g of 1,1,3,3-
dichlorotetraisopropyldisiloxane, and the mixture stirred
for 2 hours. The reaction solution was then left
standing until its temperature reached room temperature
and stirred for 1.5 hours. To the resulting solution was
added a small amount of water, and the mixture stirred,
concentrated to dryness under reduced pressure, and
developed through a silica gel column (2.4x23 cm).
36
1319~32
Fractions eluted with a 25% ethyl acetate-n-hexane
mixture were collected to obtain a 3',5'-O-TIPDS
compound.
To a solution of 3.91 g of the 3',5'-O-TIPDS
compound in 10 ml of methylene chloride was added 4
equivalents of a chromic acid complex ~a mixture of 3 g
of chromium trioxide (CrO3), 5 ml of pyridine and 3 ml of
acetic anhydride with 80 ml of methylene chloride), and
the mixture stirred at room temperature or 1 hour and
then at -4C for 14 hours. To the resulting mixture was
added another 4 equivalents of a chromic acid complex,
and the mixture stirred at room temperature for 1 hour.
The reaction solution was added dropwise to 600 ml of
ethyl acetate and the mixture filtered through silica gel
(6x15 cm). The filtrate was concentrated to dryness
under reduced pressure and the residue developed through
a silica gel column (2.4x21 cm). Fractions eluted with a
20% ethyl acetate-n-hexane mixture were combined to
obtain 2.8 g (yield 71.6%) of a 2'-keto compound.
Melting point: 183 - 186C
Elemental analysis:
(as C2lH35N2O7Fsi2)
Calcd. C: 50.15%, H: 7.01%,
N: 5.57%
Found C: 50.01%, H: 7.22%,
N: 5.49%
2) Synthesis of 2'-methylidene-2'-deoxy-5-fluorouridine
(compound of formula lI] wherein R1 is OH, R2 is F,
R3 is H, and R4 is H)
1.1 g of potassium hydride was added to 12 ml of
dimethyl sulfoxide under argon and the mixture stirred
for 1 hour.
The resultant solution containing potassium hydride
was added dropwise to a separately prepared solution of
11 g of methyltriphenylphosphonium bromide in 25 ml of
dimethyl sulfoxide in an ice bath and the mixture stirred
further for 10 minutes.
37 1 3 ~ 9 3'2
To the resulting solution was added dropwise a
solution of 1.4 g of the above obtained 2'-keto compound
in 25 ml of dimethyl sulfoxide, and the mixture stirred
at room temperature for 10 hours.
The reaction solution was then neutralized with lN
ammonium chloride and partitioned between 200 ml of ethyl
acetate and 200 ml of water. The organic layer was
concentrated to dryness and the residue developed through
a silica gel column (2.4x22 cm). Fractions eluted with a
20% ethyl acetate-n-hexane mixture were collected to
obtain a 2'-methylidene compound. This compound was
dissolved in 5 ml of tetrahydrofuran, 4 ml of 1 M tetra-
n-butylammonium fluoride-tetrahydrofuran solution added,
and the mixture stirred at room temperature for 30
minutes to deprotect the compound.
Subsequently, the reaction mixture was neutralized
with acetic acid, concentrated to dryness under reduced
pressure, and developed through a silica gel column
(2.4x17 cm). Fractions eluted with a 7% ethanol-
chloroform mixture were combined and concentrated to
obtain 0.37 g (yield 54%) of 2'-methylidene-2'-deoxy-5-
fluorouridine.
Melting point: 154 - 156C
Elemental analysis:
(ag Cl0HllN25F)
Calcd. C: 46.55%, H: 4.30%,
N: 10.86%
Found C: 46.49%, H: 4.41%,
N: 10.78%
Example 5
Pre aration of 2'-meth lidene-2'-deoxv-5-iodouridine
P Y
(compound of formula [I] wherein Rl is OH, R2 is I, R3 is
H, and R4 is H)
1) Synthesis of 1-(3,5-O-TIPDS-~-D-erythropentofuran-2-
ulosyl)-5-iodouracil (compound of formula [II]
wherein R2 is I, and Z(3')-Z(5') are TIPDS)
38
1 319~3~
To a solution of 10.0 9 of 5-iodouridine in 100 ml
of pyridine was added in an ice bath 8.94 g of 1,1,3,3-
dichlorotetraisopropyldisiloxane, and the mixture stirred
for 1.5 hours. The reaction solution was then left
standing until its temperature reached room temperature
and stirred further for 3 hours. To the resulting
solution was added a small amount of water, and the
mixture stirred, concentrated to dryness under reduced
pressure, and developed through a silica gel column (3x30
cm). Fractions eluted with a 25% ethyl acetate-n-hexane
mixture were collected to obtain a 3',5'-O-TIPDS
compound.
To a solution of 13.65 g of the 3',5'-O-TIPD~
compound in 30 ml of methylene chloride was added 4
equivalents of a chromic acid complex (a mixture of 9 g
of chromium trioxide (CrO3), 15 ml of pyridine and 9 ml
of acetic anhydride with 230 ml of methylene chloride),
and the mixture stirred at room temperature for 2 hours.
To the resulting mixture was further added 2 equivalents
of a chromic acid complex, and the mixture stirred at
room temperature for another 2 hours. The reaction
solution was then added dropwise to 1.5 liters of ethyl
acetate and the mixture filtered through silica gel
(10x20 cm). The filtrate was concentrated to dryness
under reduced pressure and the residue developed through
a silica gel column (3.0x32 cm). Fractions eluted with a
20~ ethyl acetate-n-hexane mixture were collected to
obtain 4.4 g of a 2'-keto compound.
2) Synthesis of 2'-methylidene-2'-deoxy-5-iodouridine
(compound of formula [I] wherein Rl is OH, R2 is I,
R3 is H, and R4 is H)
To a solution of 22.0 g of methyltriphenyl-
phosphonium bromide in 100 ml of tetrahydrofuran was
added dropwise under argon 37.5 ml of n-butyllithium, and
the mixture stirred for 1 hour.
To this solution was added dropwise at -10C a
solution of 4.0 g of the above obtained 2'-keto compound
1319932
in 20 ml of tetrahydrofuran, and the mixture stirred at
this temperature for 30 minutes and then at room
temperature for 1.5 hours.
Subsequently, the reaction solution was neutralized
with lN ammonium chloride and partitioned between ~oo ml
of ethyl acetate and 200 ml of water. The organic layer
was concentrated to dryness and the residue developed
through a silica gel column (3x23 cm). Fractions eluted
with a 20~ ethyl acetate-n-hexane mixture were collected
to obtain a 2'-methylidene compound.
To a solution of 300 mg of the thus obtained 2'-
methylidene compound in 5 ml of tetrahydrofuran was added
1.1 ml of 1 M tetra-n-butylammonium fluoride-
tetrahydrofuran solution, and the mixture stirred at room
temperature for 30 minutes to deprotect the compound.
The reaction mixture was thereafter neutralized ~-ith
acetic acid, concentrated to dryness under reduced
pressure, and developed through a silica gel column
~2.4x17 cm). Fractions eluted with a 7% ethanol-
chloroform mixture were collected and concentrated toobtain 118 mg of 2'-methylidene-2'-deoxy-5-iodouridine.
Melting point; 169 - 172C
Elemental analysis:
(as CloHllN2O5I)
Calcd. C: 32.82%, H: 3.03%r
N: 7.65%
Found C: 32.76~, H: 3.15~,
N: 7.60%
~xample 6
Preparation of 2'-methylidene-2'-deoxy-5-bromouridine
(compound of formula [I] wherein Rl is OH, R2 is Br, R3
is H, and R4 is H)
To a solution of 3.32 g of 5-bromouridine in 30 ml
of pyridine was added in an ice bath 3.3 g of 1,1,3,3-
dichlorotetraisopropyldisiloxane, and the mixture stirred
for 2 hours. The reaction solution was left standing
13~9~
until its temperature reached room temperature and
stirred for an additional 1~ hours. To the resulting
solution was added a small amount of water, and the
mixture stirred, concentrated to dryness under reduced
pressure, and developed through a silica gel column
(2.4x25 cm). Fractions eluted with a 25% ethyl acetate-
n-hexane mixture were collected to obtain a 3',5'-0-TIPDS
compound.
To a solution of 4.30 g of the 3',5' -O-TIPDS
compound in 10 ml of methylene chloride was added 4
equivalents of a chromic acid complex (a mixture of 3 g
of chromium trioxide (CrO3), 5 ml of pyridine and 3 ml of
acetic anhydride with 80 ml of methylene chloride), and
the mixture stirred at room temperature for 2 hours. The
reaction solution was then added dropwise to 300 ml of
ethyl acetate and the mixture filtered through silica gel
(6xlO cm). The filtrate was concentrated to dryness
under reduced pressure and the residue developed through
a silica gel column (2.4x32 cm). Fractions eluted with a
20% ethyl acetate-n-hexane mixture were collected to
obtain 2.4 g of a 2'-keto compound.
To a solution of 3.3 g o~ methyltriphenylphosphonium
bromide in 20 ml of tetrahydrofuran was added dropwise
under argon in an ice bath 6.6 ml of n-butyllithium, and
the mixture stirred further for 50 minutes~
To the resulting solution was added dropwise at -
10C a solution of 650 mg o~ the above obtained 2'-keto
compound in 10 ml of tetrahydrofuran, and the mixture
stirred at this temperature for 1 hour and then at room
temperature for 4 hours.
Subsequently, the reaction solution was neutralized
with lN ammonium chloride and partitioned between 100 ml
of ethyl acetate and 100 ml of water. The organic layer
was concentrated to dryness and the residue developed
through a silica gel column (2.4x18 cm). Fractions
eluted with a 20% ethyl acetate-n-hexane mixture were
combined to obtain a 2'-methylidene compound. This
41 1 3 1~ ~ 3 2
compound wa~ dissolved in 5 ml of tetrahydrofuran, 1.4 ml
of 1 M tetra-n-butylammonium fluoride-tetrahydrofuran
solution added, and the mixture stirred at room
temperature for 30 minutes to deprotect the compound.
The reaction mixture was then neutralized with
acetic acid, concentrated to dryness under reduced
pressure, and developed through a silica gel column
(2.4x12 cm). Fractions eluted with a 7~ ethanol-
chloroform mixture were collected and concentrated to
obtain 2'-methylidene-2'-deoxy-5-bromouridine as an
amorphous powder.
Elemental analysis:
(as Cl0HllN2o5Br)
Calcd. C: 37.65%, H: 3.48%,
N: 8.78~
Found C: 37.49%, H: 3.55%,
N: 8.79%
Example 7
Preparation of 2'-methylidene-2'-deoxy-uridine (compound
of formula [I~ wherein Rl is OH, R2 is H, R3 is H, and R4
is H)
To a solution of 3.91 9 of uridine in 50 ml of
pyridine was added in an ice bath 5.57 g of 1,1,3,3-
dichlorotetraisopropyldisiloxane, and the mixture stirredat room temperature for 6 hours. To the resulting
solution was added a small amount of water, and the
mixture stirred for 30 minutes and concentrated under
reduced pressure. The residue was partitioned between
chloroform and water, and the organic layer concentrated
and adsorbed onto a silica gel column (5x21 cm). From
fractions eluted with a 2% ethanol-chloroform mixture was
obtained a 3',5'-protected compound.
Separately, a solution of 1.7 ml of oxalyl chloride
in 40 ml of methylene chloride was cooled to -70C. To
the cooled solution was added dropwise under argon a
solution mixture of 3 ml of dimethyl sulfoxide and 20 ml
42
13~9~3~
o methylene chlorider and the resulting mixture stirred
for 30 minutes. To the reaction solution was added
dropwise a solution of 7.8 g of the above obtained 3',5'-
protected compound in 50 ml of methylene chloride, and
the mixture stirred further at -70C for 2 hours. To the
resulting solution was added dropwise 6.6 ml of
triethylamine, and the mixture stirred for 1.5 hours.
The reaction solution was then left standing until its
temperature reached room temperature and partitioned
between chloroform and water. The organic layer was
concentrated to dryness under reduced pressure and
developed through a silica gel column (4x28 cm).
~ractions eluted with a 40~ ethyl acetate-n-hexane
mixture were concentrated and crystallized from n-hexane
to obtain 6.53 9 of a 2'-keto compound.
Separately, to a solution of 22.0 9 of
methyltriphenylphosphonium bromide in 100 ml of
tetrahydrofuran was added dropwise under argon 37.5 ml of
n-butyllithium, and the mixture stirred for 1 hour. To
the resulting solution was added dropwise at -10C a
solution of 3.2 g of the above obtained 2'-keto compound
in 20 ml of tetrahydrofuran, and the mixture stirred at
this temperature for 30 minutes and then at room
temperature for 1.5 hours.
The reaction solution was thereafter neutralized
with lN ammonium chloride and partitioned between 200 ml
of ethyl acetate and 200 ml of water. The organic layer
was concentrated to dryness and the residue developed
through a silica gel column (3x24 cm). Fractions eluted
with a 20% ethyl acetate-n-hexane mixture were collected
to obtain a 2'-methylidene compound.
240 mg of the thus obtained 2'-methylidene compound
was dissolved in 5 ml of tetrahydrofuran, 1 ml of 1 M
tetra-n-butylammonium fluoride-tetrahydrofuran solution
added, and the mixture stirred at room temperature for 30
minutes to deprotect the compound.
9 3 ~
The reaction mixture was then neutralized with
acetic acid, concentrated to dryness under reduced
pressure, and developed through a silica gel column
(2.4x14 cm). Fractions eluted with a 7% ethanol-
chloroform mixture were collected and concentrated to
obtain 77 mg of a crystalline powder of 2'-methylidene-
2'-deoxy-uridine.
Melting point: 163 - 165C
Elemental analysis:
(as CloHl2N2O5)
Calcd. C: 50.00%, H: 5.04%r
N: 11.66%
Found C: 49.88%, H: 5.13%,
N: 11.59%
ExamPle 8
Preparation of 2'-methylidene-2'-deoxy-5-chlorouridine
(compound of formula lI] wherein Rl is OH, R2 is Cl, R3
is H, and R4 is H)
Protection, oxidation, methylidenation and
deprotection reactions were carried out similarly as in
Example 7 except that the uridine was replaced by 5-
chlorouridine to obtain 2'-methylidene-2'-deoxy-5-
chlorouridine.
In this example, the 40% ethyl acetate-n-hexane
mixture was also replaced by a 30% ethyl acetate-n-hexane
mixture.
Melting point: 149 - 152C
Elemental analysis:
(as Cl0HllN2o5cl)
Calcd. C: 43.68%, H: 4.03%,
N: 10.19%
Found C: 43.77%, H: 3.98%,
N: 10.20%
Example 9
Preparation of 2'-methYlidenethYmidine-5'-phosphoric acid
~319932
To an ice-cooled solution of 2.54 g of 2'-
methylidenethymidine in 60 ml of trimethylphosphoric acid
was added dropwise 1.53 g of phosphorus oxychloride, and
the mixture stirred for 1 hour. The reaction solution
was poured into 100 ml of ice-cooled water containing 8 g
of sodium hydrogencarbonate, and the mixture stirred for
1 hour. Then 100 ml of ether was added, and the
resulting solution partitioned. The aqueous layer was
concentratedr adsorbed onto an anion exchange resin,
Dowex 1 (Formic Acid Type), and eluted with 1 M formic
acid solution. Fractions containing the desired compound
were combined, concentrated, and freeze-dried to obtain
2'-methylidenethymidine-5'-phosphoric acid.
ExamPle 10
Preparation of 2'-deoxy-2'-methylidenecytidine (compound
of formula [I] wherein Rl is NH~, R2 is ~, R3 is H, and
R4 i~ H~hydrochloride
1) Synthesis of 3',5'-O-(tetraisopropyldisiloxane-1,3-
diyl)-4-N-benzoylcytidine (compound of formula [D]
wherein R2 is H, R6 is COC6H5, and Zt3')-Z(5') are
TIPDS)
To a solution of 5 9 ~20.6 mmol~ of 4-N-
benzoylcytidine in 50 ml of pyridine was added 7.1 ml
(22.6 mmol) of 1,1,3,3-dichlorotetraisopropyldisiloxane,
and the mixture stirred at 0C for 3 hours and
subsequently at room temperature for 3 hours to cause
reaction. After completion of the reaction, ice water
was added to the reaction solution and the solvent
distilled off under reduced pressure. The residue was
dissolved in ethyl acetate and partitioned three times
from water. The organic layer was dried over sodium
sulfate anhydride and the solvent distilled off under
reduced pressure. The residue was adsorbed onto a silica
gel column (5xlO cm) and eluted with a 3396 ethyl acetate-
hexane solvent mixture to obtain fractions containing the
desired compound. These fractions were concentrated
45 1 3 1 9 ~3 2
under reduced pressure to obtain 7.~ g (yield 86%) of an
amorphous powder of 3',5'-O-(tetraisopropyldisiloxane-
1,3-diyl)-4-N-benzoylcytidine.
Elemental analysis:
(as C28H43N307Si H2O)
Calcd. C: 55.32~, H: 7.46%,
N: 6.91~
Found C: 55.54%, ~: 7.41%,
N: 6.99%
2) Synthesis of 3'5'-O-(tetraisopropyldisiloxane-1,3-
diyl)-2'-keto-4-N-benzoylcytidine (compound of
formula [II] wherein R2 is H, R5 is NHCOC6H5, and
Z(3')-Z(5') are TIPDS)
5 g (40 mmol) of chromium trioxide (CrO3), 8.3 ml
(80 mmol) of pyridine and 5 ml (40 mmol) of acetic
anhydride were dissolved together in 110 ml of methylene
chloride to prepare a chromic acid complex solution. In
this solution was dissolved 5.9 9 (10 mmol) of 3',5'-O-
(tetraisopropyldisiloxane-1,3-diyl)-4-N-benzoylcytidine,
and the solution stirred at room temperature for 1 hour
to cause reaction. After completion of the reaction, 500
ml of ethyl acetate was added dropwise to the reaction
solution and the mixture passed through a silica gel
column (6x1.5 cm) to obtain filtrates. The filtrates
collected were evaporated to dryness under reduced
pressure. The residue was adsorbed onto a silica gel
column (3.0x21 cm), eluted with a 25% ethyl acetate-
hexane solvent mixture, and crystallized from ethyl
acetate-hexane to obtain 4.6 g (yield 78%) of 3',5'-O-
(tetraisopropyldisiloxane-1,3-diyl)-2'-keto-4-N-
benzoylcytidine.
Melting point: 135 - 137C
Elemental analysis:
(as C28H4lN3O7si 2 )
Calcd. C: 57.21%, H~ 7.03%,
N: 7.15%
Found C: 57.08%, H: 7.12%,
~ 46 1319932
N: 7.01~
3) Synthesis of 3',5'-O-(tetraisopropyldisiloxane-1;3-
diyl)-2'-deoxy-2'-methylidene-4-N-benzoylcytidine
(compound of formula [III~ wherein R2 is H, R3 is H,
R5 is NHCOC6H5, and Z(3')-Z(5') are TIPDS)
To a suspension of 10.7 g (30 mmol) of
methyltriphenylphosphonium bromide in 60 ml of
tetrahydrofuran cooled to -20C was added dropwise 15.8
ml (25 mmol) of an n-butyllithium solution, and the
mixture stirred for 1 hour to cause reaction. To the
resulting solution was added dropwise a solution of 2.9 g
(5 mmol) of 3',5'-O-(tetraisopropyldisiloxane~ -diyl)-
2'-keto-4-N-benzoylcytidine in 20 ml of tetrahydrofuran,
and the mixture caused to react at -20C for 1 hour. The
reaction solution was left standing until its temperature
reached room temperature and stirred for a further 2
hours to cause reaction. After completion of the
reaction, 50 ml of lN aqueous solution of ammonium
bromide was added to the reaction solution. Ethyl
acetate was added, and the mixture partitioned. The
organic layer was washed twice with water and dried over
sodium sulfate anhydride, and the solvent distilled off
under reduced pressure. The residue was adsorbed onto a
silica gel column (2.4x20 cm) and eluted with a 25~ ethyl
acetate-hexane solvent mixture to obtain 0.9 g of an
amorphous powder of 3',5'-O-(tetraisopropyldisiloxane-
1,3-diyl)-2'-deoxy-2'-methylidene-4-N-benzoylcytidine.
Furthermore, fractions eluted with a 6.25% ethanol-
dichloromethane mixture in the above-mentioned silica gel
column were collected and concentrated to dryness. The
residue was dissolved in 35 ml of tetrahydrofuran, 6.1 9
of a 60~ powdery reagent of sodium hydride added under
argon, and the mixture stirred at room temperature for 3
hours. In the same manner as was described above, an
aqueous solution of ammonium bromide was added to the
reaction solution, and the mixture partitioned between
ethyl acetate and water and further purified through a
47 1319~2
silica gel column to obtain 1.2 9 (2.1 g in total, yield
72% ) of an amorphous powder of 3 ', 5 ' -O-
(tetraisopropyldisiloxane-l, 3-diyl) -2' -deoxy-2 ' -
methylidene-4-N-benzoylcytidine.
Elemental analysis:
(as C2gH43N3O6si 2 )
Calcd. C: 59.46%, H: 7.40~,
N: 7.17%
Found C: 59.39%t H: 7.52%
N: 7.10%
4) Synthesis of 2 ' -deoxy-2 ' -methylidene-4-N-
benzoylcytidine (compound of formula lIV] wherein R2
is H, R3 is H, and R5 is NHCOC6H5)
To a solution of 343 mg (1 mmol) of 3',5'-O-
15 ( tetraisopropyldisiloxane-l, 3-diyl ) -2 '-deoxy-2 ' -
methylidene-4-N-benzoylcytidine in 10 ml of
tetrahydrofuran was added 2.2 ml of lN tributylammonium
fluoride, and the mixture stirred at 0C for 30 minutes
to cause reaction. The reaction solution was neutralized
20 with acetic acid and the solvent distilled off under
reduced pressure. The residue was developed through a
silica gel column (1.6x30 cm, 896 ethanol-chloroform being
used as a solvent for elution) and crystallized from
ethyl ether-ethanol to obtain 302 mg (yield 88%) of 2'-
25 deoxy-2~-methylidene-4-N-benzoylcytidine~
Melting point: >300C
Elemental analysis:
(as Cl7Hl7N3O5)
Calcd. C: 59.47%, El: 4.99%,
N: 12.24%
Found C: 59.28%, H: 5.05%,
N: 12.11%
5) Synthesis of 2'-deoxy-2'-methylidenecytidine
(compound of formula [I] wherein Rl is HN2, R2 is ~1,
R3 is H, and R4 is H)hydrochloride
A solution of 139 mg (0.5 mmol) of 2'-deoxy-2'-
methylidene-4-N-benzoylcytidine in 10 ml of methanolic
48 131~932
ammonia was stirred at room temperature for 6 hours to
cause reaction and the solvent distilled off under
reduced pressure. The residue was developed through a
silica gel column (1.6xlO cm, 20~ ethanol-chloroform
being used as a solvent for elution), fractions
containing the desired compound collected, 2 ml of lN
hydrochloric acid added, and the solvent distilled off
under reduced pressure. The residue was crystallized
from acetone-methanol to obtain 115 mg (yield 83%) of 2'-
deoxy-2'-methylidenecytidine.
Exam~le 11
Tablet
2'-Methylidenethymidine 10 g
Cornstarch 65 9
Carboxycellulose 20 g
Polyvinyl pyrrolidone 3 9
Calcium stearate 2 q
Total 100 9
Tablets each weighing 100 mg and containing 10 mg of
2'-methylidenethymidine are prepared by a conventional
method.
Example 12
Powder ~ Capsule
2'-Methylidene-2'-deoxycytidine
hydrochloride 20 g
Crystalline cellulose 80 a
Total 100 g
The two substances in powder form are blended
together to formulate a powder.
Further, 100 mg of the powder is charged into a No.5
hard capsule to prepare capsules.
INDUSTRIAL APPLICABILITY
As has been set forth hereinabove, the novel
compounds of the present invention, 2'-
~ ~9
131~3~
alkylidenepyrimidine nucleoside derivatives or salts
thereof, exhibit remarkable antiviral activity,
especially against herpes simplex virus (HSV) and
cytomegalovirus (CMV), and therefore drugs comprising
these compounds as active ingredients are useful as
antiviral agents.
