Note: Descriptions are shown in the official language in which they were submitted.
X-7108A -1- 13 31~ ~ 8
IMPROVEMENTS IN OR RELATING TO 2',2'-DIFLUORONUCLEOSIDES
This invention provides a novel process for
preparing 2',2'-difluoronucleosides and intermediates
thereto.
United States Patent No. 4,526,988 teaches
that 2'-deoxy-2',2'-difluoronucleosides are useful anti-
viral agents. European Patent Application 184,365
teaches the use of the same compounds as oncolytic
agents. The synthetic process disclosed in the pub-
lications produces intermediates containing up to two
centers of chirality. One such intermediate having a
chiral center is a protected lactone consisting of
- erythro and threo enantiomers of the formulae
~ 2o and ~ =
(erythro) (threo)
wherein P is a protecting group. The publications teach
the erythro enantiomer is preferred since it provides a
carbohydrate which has the stereochemistry of naturally
occurring ribose. A carbohydrate which has the stereo- -
-
- . . - .
X--7108A --2- 13 31~ 0 ~ :
chemistry of naturally occurring ribose is preferred
since it provides final product nucleosides which
exhibit superior biological activity. ~ -
United States Patent No. 4,526,988 teaches the
preparation of the above described erythro enantiomer by
first forming an alkyl 2,2-difluoro-3-hydroxy-3-(2,2-
dialkyldioxolan-4-yl)propionate, consisting of 3-R- and
3-S- hydroxy enantiomers, of the formulae
H0
~ \ / COz(C1-C4 alkyl) ~ ~ / t \ / OZ(C1-C4 alkyl)
R4 t - F~ F and R4 ~ O
Rs Rs
::
.. . .
(3-R-) (3-S-)
.
20 wherein R4 and R5 are independently C1-C3 alkyl, in a .
ratio of about 3 parts 3-R- enantiomer to about 1 part : ..... '~
3-S- enantiomer. The publication discloses that the
3-R- hydroxy enantiomer has the proper stereochemistry .:;~
to provide the desired erythro enantiomer and that the
3-R- and 3-S- enantiomers can be separated by expensive,
laborious column chromatography procedures.
The patent teaches that once the 3-R- hydroxy
enantiomer is isolated it is next hydrolyzed under very
mild conditions to form an unprotected lactone; namely,
.... - ,
' ;' '`:,
- , :' . :.. .
1 33~ ~ ~8
X-7108A -3-
2-deoxy-2,2-difluoro-D-erythro-pentofuranos-1-ulose,
which has the formula
. ~ .
H
~ =
H ~ -
The publication teaches that mild conditions useful for
forming the above compound include the use of hydrolysis
reagents such as mildly acidic ion exchange resins or
relatively strong acids, such as aqueous acetic acid or
chloroacetic acid, which have a pKa between about 2.8 to
about 5Ø Both types of hydrolysis reagents can cause
problems in the hydrolysis reaction. For example, the
use of ion exchange resin reguires such large quantities
of water that, especially in larger scale reactions, the
lactone often reverts back to its open chain precursor
because of its sensitivity to water. The relatively
strong acids, on the other hand, are less preferred
hydrolysis reagents for converting the 3-R- hydroxy
enantiomer to the unprotected lactone since they produce
~; large amounts of undesirable reaction products, includ-
~; 25 ing unreacted starting material.
Finally, once the unprotected lactone has been
formed it is converted to the protected erythro lactone
described above by adding an hydroxy protecting group
to the lactone's hydroxy groups.
X-7108A -4 1331~8
A second chiral center is produced at the
anomeric carbon atom when the keto portion of the ~ -
lactone is converted to an alcohol. More specifically,
the two anomers for the desired erythro configuration ' - -~
are identified as a and ~ anomers of the fomulae
(c~) ' (O
The unprotected hydroxy group at the l-position is
ultimately replaced by a heterocyclic base, such as
15 cytosine, to provide protected precursors of the ~;
biologically active 2'-deoxy-2',2'-difluoronucleosides.
The ~ anomer precursor is preferred since it provides
2'-deoxy-2',2'-difluoronucleosides which possess superior
biological activity. -~
United States Patent No. 4,526,988 specifi- -~
cally illustrates the use of t-butyldimethylsilyl as a
protecting group. When this protecting group is used in
the synthesis of 2'-deoxy-2',2'-difluoronucleosides the
product is composed of about a 4:1 /~ anomeric ratio.
This product must be purified by expensive, laborious
column chromatography procedures to isolate the desired -
anomer.
The present invention provides a convenient ~-~
process for obtaining 2'-deoxy-2',2'-difluoronucleosides -
. ,, ~
~` , ': i,:
'". ' ~' 1'
~ ~ ` A
1331~08 -
X-7108A -5-
having the desired erythro and ~ stereochemistry which
eliminates the need for extensive column chromatography
purification, as previously required. In addition, the
present invention provides a process for obtaining
2'-deoxy-2',2'~difluoronucleosides having the desired
- erythro and ~ stereochemistry in higher yields than
previously possible.
According to the present invention, in one aspect, there is provided
provided a process for preparing an enantiomeric mixture .
of erythro and threo lactones of the formula
. ~ .. ..
/Qi ,.. ~ -.~-.
~0~ / \ " '
, ~/-=0 . ~"~
H r
R~ I ~:
wherein R is B or ~ , which comprises
~: hydrolyzing a mixture of 3-R- and 3-S- enantiomers of
:~ an alkyl 2,2-difluoro-3-hydroxy-3-(2,2-dialkyldioxolan-
4-yl)propionate, or a protected derivative thereof, of -~
the formula
OR
CO2(C1-C4 alkyl)
~/ -- F~ \F
R4 t : : -
Rs
.
1331~
X-7108A -6-
wherein R is as defined above and R4 and R5 are inde-
pendently C1-C3 alkyl, by using a strong acid as a
hydrolytic reagent, followed by azeotxopic distillation
of water. The present process prepares a crystalline :~ -
lactone which is stable, therefore minimizing reversion
back to the open chain precursor, as well as minimizing
the formation of undesirable reaction products. : :
The present invention, in another aspect, also provi(les a process ~ -
for selectively isolating, in greater than about 95.0%
purity, 2-deoxy-2,2-difluoro-D-erythro-pentofuranos-
l-ulose-3,5-dibenzoate, which has the formula
~;~
from an enantiomeric mixture of erythro and threo
lactones of the formula
~ / ~ ~ 0
''.', ','. ;~ ',..'',.,.' .~
',,.~ ' . '::
. - ,, ~
' '.,'.'.:;," '' ~.''
1331~
X-7108A -7-
, ,.
comprising dissolving the enantiomeric mixture in
methylene chloride, cooling the solution to a tempera- : : `
ture in the range of about -5C to about 10C, and col- - ~ -
lecting the precipitated erythro enantiomer. Thus the
S desired erythro enantiomer can be obtained without hav-
ing to use expensive, laborious, column chromatography
procedures.
Yet another embodiment of the present inven-
tion is a process for producing a 2'-deoxy-2',2'-
difluoronucleoside of the formula
~Q~ : , .
RO 1~ ,
O
wherein R is H or C ~ and
B is a base of the formula
I~lHRa
20 ~ t g~
11H~
H~ /N~ -~HR2 ~ ~`
~4~- , R ~~ /, or 0~
-.
r -
X-7108A -8-
~ 3 3 ~
X-is N or C-R4; ~: -
R1 is hydroxy or amino;
R2 is bromo, chloro or iodo;
Ol : -
R3 is hydrogen, C1-C4 alkyl or -C-R5; i -
R4 is hydrogen, C~-C4 alkyl, amino, bromo,
fluoro, chloro or iodo; and : - -
R5 is hydrogen or C,-C4 alkyl;
in about a l:l a/~ anomeric ratio comprising reacting a -
protected carbohydrate of the formula
wherein L is a leaving group, with an appropriate base :
B-H to form the species wherein R is . :~
and optionally removing the benzoyl protectinq group by . .
reaction with a strong or moderately strong base to form : .:
the species wherein R is H. The 1 1 a/~ anomeric ratio
obtained by the present process compares favourably to
the 4:1 a/~ ratio produced by prior art process~s. This
embodiment of the invention is also disclosed, and is claimed,in Canadian Patent : - ~
Application No. 575,329 of Chou et al, filed August æ, 1988, of which the :- . ~ .
present application is a divisional.
The present invention also provides a process
for selectively isolating ~-2'-deoxy-2',2'-difluoro- ~ .
cytidine hydrochloride or hydrobromide which is at least ~:~
about 80.0% pure from about a 1:1 a/~ anomeric mixture
of 2'-deoxy-2',2'-difluorocytidine hydrochloride or
hydrobromide comprising dissolving the 1:1 a/~ mixture
13~1~0~
X-7~08A -9-
in hot water, adding acetone, cooling the solution toa temperature in the range of about -10C to about
50C, and collecting the precipitated ~-2'-deoxy-2',2'-
difluorocytidine hydrochloride or hydrobromide salt.
~-2'-Deoxy-2',2'-difluorocytidine hydrochloride or
hydrobromide thus prepared may be further purified by
repeating the process set forth above, on the salt
collected above, to provide approximately 99.0% pure
~-2'-deoxy-2',2'-difluorocytidine hydrochloride or
~0 ~ydrobromide.
Finally, the present invention provides a
process for selectively isolating ~-2'-deoxy-2',2'-
difluorocytidine which is about 99.0% pure from about
a 1:1 a/~ anomeric mixture of 2'-deoxy-2',2'-difluoro-
cytidine, or an organic or inorganic acid addition saltthereof, comprising dissolving the a/~ mixture in hot
water, increasing the pH of the aqueous solution to -;
about 7.0 to about 9.0, cooling the solution to a tem-
perature in the range of from about -10C to about 30C,
20 and collecting the precipitated ~-2'-deoxy-2',2'- -
difluorocytidine free base. ~-2'-Deoxy-2',2'-difluoro-
cytidine free base thus prepared may be converted to a
pharmaceutically acceptable organic cr inorganic acid
addition salt by a process comprising dissolving the -
25 free base collected above in hot water, adding a pharma- - ~-
ceutically acceptable organic or inorganic acid to the
solution, cooling the solution to a temperature in the -
range of from about -10C to about 40C, and collecting
the precipitated, approximately 99.0% pure, ~-2'-deoxy-
2',2'-difluorocytidine acid addition salt.
Both processes described above provide improve-
ments over the prior art since the ~ anomer can be
obtained without having to use expensive, laborious,
column chromatography procedures.
- (~ ~ ::
` ~
1331~08
X-7108A -10- .
' ,'
United States Patent No. 4,526,988 discloses
alkyl 2,2-difluoro-3-hydroxy-3-(2,2-dialkyldioxolan-4-
yl)propionates of the formula
H0
CO2(C1 C- ~Ikyl)
R4
Rs
10 wherein R4 and Rs are independently Cl-C3 alkyl. The ~
compounds consist of 3-R- and 3-S- hydroxy enantiomers - : :
in a ratio of about 3 parts 3-R- enantiomer to 1 part ~ :
3-S- enantiomer. The present invention provides a proc-
ess for converting the above compounds, or protected . - :;
derivatives thereof, of the formula
R-o
~ C02(Cl~ ;31kyl)
R4
Rs
O .' ~' :
wherein R is H or -C_\ ~ , to a lactone of the formula
:' . ~'
HO~
. .: .
.,~,~';' ;:'''
~' ~
X-7108A -11- 1331~8 - ~
The protected derivative starting material
noted above can be prepared by reacting the unprotected
alkyl 2,2-difluoro-3-hydroxy-3-(2,2-dialkyldioxolan-4-
yl)propionate with benzoyl bromide, chloride, cyanide,
S or azide. The reaction is conveniently carried out at
temperatures in the range of from about -10C to about
50C in an inert solvent to which an acid scavenger,
such as a tertiary amine, has been added. The reaction -~
may also be carried out in a basic solvent such as
pyridine, qui~oline, isoquinoline, or lutidine, or in a
tertiary amine solvent such as triethylamine, tributyl-
amine, methylpiperidine, or the like. Additionally, a -
catalyst such as 4-dimethylaminopyridine or
4-pyrrolidinopyridine may be used in the reaction, if -
lS desired.
The 3-hydroxy compounds, or their benzoyl pro-
tected derivatives, are converted to the lactone in the
following manner. First, the isoalkylidene protecting ~ ~-
group is selectively removed to form an alkyl 2,2-
difluoro-3,4,5-trihydroxypentanoate or alkyl 2,2-
difluoro-3-(benzoyloxy)-4,5-dihydroxypentanoate compound
of the formula ;~
OR
-- ~H/~
;
1 3 3 1 ~
X-7108A -12- ~ ~
The selective removal of the isoalkylidene ~ -
protecting group i8 achieved by using a strong acid as a
hydrolytic reagent. The term "strong acids", as defined
herein, are acids which have a pKa at room temperature
(22C) of about -10.0 to about 2Ø Examples of strong -
acids include inorganic acids such as 1 to 8 normal
hydrochloric acid, 1 to 8 normal sulfuric acid, and the
like, and organic acids such as p-toluenesulfonic acid, - ;
trifluoroacetic acid, and the like. Preferred strong -
acids are those acids which have a pKa of about -7.0 to
about 0Ø Parkicularly preferred strong acids are 6N
sulfuric acid, trifluoroacetic acid and ~-toluene-
sulfonic acid. The strong acid is generally employed in
catalytic quantities, although greater than catalytic ~ -
quantities can be employed if desired. Typically the
acid is employed in an amount sufficient to provide
about 0.05 to about 0.5 molar eguivalents of acid
relative to the alkyl 2,2-difluoro-3-hydroxy-3-(2,2-
dialkyldio~olan-4-yl)propionate, or protected
derivative, starting material.
The propionate starting material and the
strong acid are dissolved in a suitable solvent and the
water content of the solution is adjusted to provide
from about 1 to about 5 molar equivalents of water ~ -
relative to the propionate starting material. Suitable
solvents include polar solvents such as the alcohols,
for example methanol, ethanol, isopropanol, and the
like; acetonitrile; and related polar solvents. The
water content of the solution can be adjusted to provide
between about 1 to about 5 equivalents of water in
.. . : ~ . . . ; . `, ' . ; `
l3~la~s .....
X-7108A -13-
~ : .
several ways; by adding additional water to the water
already present in the organic or inorganic strong acid,
by choosing an inorganic acid which has the proper
normality to provide the desired quantity of water, or
5 by choosing a solvent, such as 95% ethanol, which ~ -~
contains a small amount of water. In general, about 1
to 2 molar equivalents of water relative to the pro- -
pionate starting material are preferred since the lower ~-~
water content is easily removed when cyclizing to the --
lactone.
After the propionate starting material, the ~ u~
strong acid, the solvent and water have been mixed, the
solution is heated in order to begin selective removal
of the isoalkylidene protecting group. The solution is ;-
lS preferably heated to the reflux temperature of the
reaction mixture. The isoalkylidene protecting group is ,' :.'':~`~-'.,'.'''',''?
substantially removed after about 2 hours to about 8
hours when the reaction is conducted at the preferred
temperature. ;--
Once the isoalkylidene protecting group has
been substantially removed the resulting pentanoate is
cyclized to the desired lactone. The pentanoate is ` ~ -~
cyclized by distilling a water/alcohol, a water/~
acetonitrile, or a water/acetonitrile/aromatic solvent `
azeotropic mixture in order to remove water from the
reaction solution. When an alcohol is used as the
solvent the water/alcohol distillation preferably should
continue until substantially all of the water and
alcohol have been removed. However, when acetonitrile
- 30 is used as the solvent fresh acetonitrile and/or
~ . ~ - -:.
. . ~ .: , . .
' ,.''; '' `' ~`', `''~
r~ ~- ~
X-7108A -14- 1 3 3 1 ~ ~ 8
aromatic solvent is added in order to ensure that
sufficient solvent is present in order to drive out the
water and any non-solvent volatile components, yet still
maintain a homogeneous liquid solution. Preferably, an
aromatic solvent, such as toluene, is used in place of
fresh acetonitrile when removing water from an aceto-
nitrile solvent solution since less solvent is then
required to azeotropically dry the solution. Once the
water has been substantially removed from the reaction
mixture the pentanoate cyclizes to the lactone in high
yield. This cyclization reaction can be monitored by
high performance liquid chromatography assay techniques
in order to determine when the reaction is substantially
complete. The lactone produced consists of erythro and
15 threo enantiomers in approximately the same enantiomeric - -
proportions as present in the propionate starting
material. ~ - --
The present invention also provides a process
for selectively isolating the erythro enantiomer of a
protected derivative of the above lactone from an
enantiomeric mixture of protected compounds.
Before isolating the erythro enantiomer the ;
unprotected hydroxy groups of the above lactone (C-3 and
C-5 if a 3-hydroxy propionate starting material was used
to prepare the lactone, only C-5 if the benzoyl pro-
tected starting material was used) are protected with - ;
a benzoyl protecting group. The protected lactone is -~
prepared by reacting the unprotected lactone with
benzoyl chloride, bromide, cyanide, or azide using con~
ditions disclosed in ~nited States Patent No. 4,526,988.
, ~
X-7108A -15- 13 31~ ~ 8
Once the protected lactone is prepared the erythro ~ -
enantiomer can be isolated by dissolving the enantio-
meric mixture in methylene chloride. While the erythro
enantiomer can be isolated from methylene chloride alone, ~ -
the use of an isopropanol or hexane counter-solvent will
increase the amount of erythro enantiomer which can be ~ ~ -
recovered. Accordingly, isopropanol/methylene chloride
and hexane/methylene chloride solvent mixtures are pre-
ferred for isolating the erythro enantiomer. -
When an isopropanol or hexane counter-solvent
is used, the isopropanol or hexane may be added to the
solution of enantiomeric mixture dissolved in methylene -~
chloride all at once, or slowly over a period of time ~ -
~ ranging from 5 minutes to 4 hours. The specific time
15 for slowly adding the counter-solvent will, of course, -~
be influenced by the amount of counter-solvent added.
If isopropanol is used as counter-solvent, the amount of
isopropanol added may vary from that needed to obtain an
isopropanol/methylene chloride solvent mixture of from `~
about 5 parts by volume of isopropanol to about 1 part
by volume methylene chloride to about 20 parts by volume - ~ --of isopropanol to about 1 part by volume of methylene
chloride. If hexane is used as counter-solvent, it
may be added in any amount up to that which will produce i
a hexane/methylene chloride solvent mixture of from
about 5 parts by volume of hexane to about 1 part by
volume of methylene chloride. A hexane/methylene
chloride solvent mixture of about 3:2, v:v, hexane:-
methylene chloride is most preferred.
': '
X-7108A -16- 13 31~ a ~
After the protected lactone has substantially
dissolved in the methylene chloride, and any desired
counter-solvent has been added, the solution is seeded
with a crystal of authentic 2-deoxy-2,2-difluoro-D-
erythro-pentofuranos-1-ulose-3,5-dibenzoate, and cooled
to a temperature in the range of about -5C to about
10C, more preferably to about 0C. The cold solution
is stirred, while maintaining the desired temperature,
for about 30 minutes to about S hours and the desired
erythro enantiomer is isolated, typically by filtration,
using standard isolation technigues.
Occasionally, and with greater frequency when
the counter-solvent is added all at once, the erythro
-enantiomer will begin to crystallize immediately upon - -~
15 counter-solvent addition. When this happens, the iso- -
lated product often is less than 95.0% purity 2-deoxy- -~
2,2-difluoro-D-erythro-pentofuranos-l-ulose-3,5-
dibenzoate. The purity of this less than 95.0% material ;~
can be improved by slurrying the impure material in an
aromatic solvent such as toluene. The slurry is heated
to about 40C to 50C, dissolving substantially all of
the desired erythro enantiomer and very little of the
undesired impurities. The non-dissolved impurities are
then removed using any standard isolation technique,
such as filtration, to provide a solution. The aromatic
solvent is removed to provide a residue, which is
dissolved in methylene chloride. The erythro enantiomer
is then recovered in greater than 95.0% purity following
the procedures described above for isolating the erythro
enantiomer from an erythro/threo enantiomeric mixture.
1 3 3 1 ~ 0 8
X-7108A -17-
The isolated erythro enantiomer of the pro-
tected lactone is next converted to a compound of the
formula
S ~ /0\ .~
, , ,
wherein L is a leaving group via procedures disclosed in
United States Patent No. 4,526,988. Appropriate leaving
groups include the sulfonates such as methanesulfonate,
toluenesulfona~e, ethanesulfonate, isopropanesulfonate, :~
15 4-methoxybenzenesulfonate, 4-nitrobenzenesulfonate, -
2-chlorobenzenesulfonate and the like; halogens such as
chloro, bromo and the like; and other related leaving
groups. A preferred leaving group for the process of -~
this invention is methanesulfonate. - ~-
. .. ~. -, .
~ ;'.- ` .: ' . ~'
~,,, . ., ~ ~
. ' . ., ',:
--: :' ' . .: :..,:
X-7108A -18- 13 31~ ~ 8
The above compound, having a leaving group as
noted above, is reacted, per the process of the present
invention, with a base of the formula
S
HT/ \f~ H~
~\X/ R3H~
~ R~
wherein
X is N or C-R4; . ~
is hydroxy or amino; ~ :
R2 is bromo, chloro or iodo;
R3 is hydrogen, C1-C4 alkyl or -C-R5;
R4 is hydrogen, Cl-C4 alkyl, amino, bromo, - .
fluoro, chloro or iodo; and ~ ~ -
R5 is hydrogen or Cl-C4 alkyl,
to produce a 2'-deoxy-2',2'-difluoronucleoside in about
a 1:1 a/~ anomeric ratio.
X-7108A -19- 1 3 3 1 a ~ 8 ~
~: .
The bases set forth above are commonly known
to organic chemists, and no discussion of their synthe-
sis is necessary. However, the primary amino groups,
present on some of the bases, should be protected before
the base is coupled with the carbohydrate. The usual
amino-protecting groups, such as trimethylsilyl, ~ -
isopropyldimethylsilyl, methyldiisopropylsilyl, triiso- -
propylsilyl, t-butyldimethylsilyl, t-butoxycarbonyl,
benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4-nitro-
benzylo~ycarbonyl, formyl, acetyl, and the like, may be
used according to procedures described in standard text-
books, such as Protective GrouPs in Orqanic ChemistrY,
McOmie, Ed., Plenum Press, N.Y. (1973); and Theodora W. ~ ~
Greene,Protective Groups in Organic Synthesis,John Wiley ~ ~ -
& Sons, N.Y. (1981).
It is often advisable to convert keto oxygen
atoms on the bases to the enol form in order to increase ; ,
the base's aromaticity and thereby allow more ready
attack of the base by the carbohydrate. Oxygen atoms ; ~ -~
are preferably enolized with the silyl protecting groups
noted above.
The coupling reaction between base and carbo- ;-
hydrate may be carried out according to any of the pro- -~
cedures described in United States Patent No. 4,526,988.
A preferred coupling procedure uses a reaction initiator
such as trimethylsilyltriflate, and a solvent such as
1,2-dichloroethane, at a temperature in the range of
about 20C to about 100C. The coupling reaction, sub- ~ -
stantially complete within about 2 hours to about 20 - ~;
30 hours when conducted at a temperature in the range of ~ - ~
- ~ :
~- ~ o
X-7108A -20- 1 3 3 1 a ~ 8
about 20C to about 100C, provides a protected nucleo-
side in about a 1~ anomeric ratio.
The same anomeric ratio of unprotected nucleo-
side is obtained by removal of protecting groups. Most
- 5 silyl amino-protecting groups are easily cleaved using
a protic solvent such as water or an alcohol. The
benzoyl hydroxy-protecting group, and any acyl amino-
protecting groups, are removed by hydrolysis with a
strong or moderately strong base at a temperature from
about 0C to about 100C. Strong or moderately strong
bases suitable for use in this reaction are bases which
have a pKa (at 25C) of about 8.5 to about 20Ø Such
bases include alkali metal hydroxides such as sodium or
potassium hydroxide; alkali metal alkoxides such as
15 sodium methoxide or potassium t-butoxide; amines such as ~ ~
diethylamine, hydroxylamine, ammonia and the like; and ~ -
other common bases such as hydrazine and the like.
Preferably, the reaction employs ammonia to remove
protecting groups at a temperature of about 10C. At
20 least one mole equivalent of base is needed for each -
protecting group removed. It is preferable to use an
excess of base in this reaction. However, the amount of
excess base used to remove the protecting groups is not
crucial.
Removal of the hydroxy-protecting groups and
amino-protecting groups is conveniently carried out in
alcoholic solvents, especially aqueous alkanols such as -~
methanol. However, the reaction may also be carried out
in any convenient solvent, such as polyols including
ethylene glycol, ethers such as tetrahydrofuran, ketones
such as acetone and methyl ethyl ketone, or
dimethylsulfoxide.
.
` .
X-7108A -21- 1 3 3 1 ~ ~ 8
The preferred process for producing 2'-deoxy- -~
2',2'-difluoronucleosides employs the base cytosine,
which has the formula
y
~'il .,~
~,. .
:~
to provide 2'-deoxy-2',2'-difluorocytidine in about a ~ ~
1:1 a/~ anomeric mixture. - ~ :
As noted above, the present invention finally ~ :
provides processes for selectively isolating ~-2'-deoxy- ;~
15 2',2'-difluorocytidine, or an organic or inorganic acid ~ ~
addition salt thereof, in approximately 99.0% purity, ---
from about a 1:1 a/~ 2'-deoxy-2',2'-difluorocytidine ; ~
anomeric mixture. ~ -
One process for selectively isolating the ~ ~
20 anomer, utilizes a hydrochloride or hydrobromide salt of -
the 1:1 a/~ anomeric mixture as starting material. The ~
hydrochloride or hydrobromide salt of the a/~ mixture is --
isolated by combining the 1:1 a/~ mixture with isopropanol, - -
and heating when necessary, to dissolve the anomeric ~ -
25 mixture in the solvent. The amount of isopropanol used, - -~ ; while not critical, should be sufficient to effect ~ ~ -
co~plete dissolution of the anomeric mixture once -
hydrochloric or hydrobromic acid addition is complete, - ~
but yet be as minimal as possible to avoid excessive - -~ -
product loss during crystallization and isolation. The
preferred amount of isopropanol used will be from about
~ ,- "': '~ ' ,.
X-7108A -22- 1331~8
2 ml of solvent per gram of anomeric mixture to about
12 ml of solvent per gram of anomeric mixture.
Once the anomeric mixture is substantially
dissolved in the solvent, hydrochloric or hydrobromic
acid is added to form either the hydrochloride or hydro-
bromide salt of the a and ~ anomers. Any undissolved
anomeric mixture will dissolve after the acid is added
to the isopropanol solution. Reagent grade concentrated
liquid hydrochloric acid and forty-eight percent
aqueous hydrobromic acid are preferred forms of hydro-
chloric and hydrobromic acids for use in preparing the
hydrochloride or hydrobromide a and ~ salts. The amount
of acid added is not critical so long as at least a
slight molar excess of acid is used relative to the
anomeric mixture. Preferably two mole eguivalents of
hydrochloric or hydrobromic acid are used for each mole
eguivalent of the anomeric mixture.
After the acid is added the a and ~ hydrochlo- ~-
ride or hydrobromide salts will begin to crystallize.
If a smaller quantity of 1:1 a/~ anomeric mixture, for
instance less than 5.0 grams, is used in preparing the
hydrochloride or hydrobromide salt, the ~ anomer will
selectively crystallize relative to the a anomer. Thus,
small quantities of a 1:1 a/~ anomeric mixture can be
25 purified to provide 2'-deoxy-2',2'-difluorocytidine -
hydrochloride or hydrobromide which has at least about -
-~ a 1:4 a/~ anomeric ratio simply by combining the 1:1 ~
anomeric mixture with isopropanol, adding hydrochloric - ~ -
or hydrobromic acid to the mixture, cooling the solution
to a temperature in the range of about -10C to about
50C, and collecting the precipitated solid.
: :
' :- " ': ;' -
1 3 3 1 ~
X-7108A -23- -
However, when larger quantities of the 1:1 a/~
anomeric mixture are used to prepare the hydrochloride
or hydrobromide salt, the a and ~ salts precipitate in
approximately the same 1:1 ratio as present in the a/~ -
mixture. To obtain the a and ~ salts in high yield the
solution should be cooled to a temperature in the range
of from about -10C to about 50C. The approximately -~
1:1 a/~ 2'-deoxy-2',2'-difluorocytidine hydrochloride or --
hydrobromide thus precipitated is isolated, typically by -
filtration, from the solution using standard isolation
techniques and may be purified to provide approximately
99.0% ~ anomer as set forth below. -
The 1:1 a/~ anomeric salt mixture is first dis- ~; ;
solved in hot water. The temperature of the hot water
lS is not critical, but it is preferred that the water tem-
perature be from about 50C to about reflux (100C). A -
preferred hot water temperature is about 80C. The con- ; ^
centration of anomeric salt mixture in the water is not
critical as long as sufficient water is employed to -
20 ensure total dissolution. It is preferred that the ~
amount of water employed be as minimal as possible to ~;
avoid excessive product loss during crystallization and
isolation. Appropriate concentrations of the anomeric
salt mixture in water vary from about 50 mg of mixture
per ml of water to about 400 mg of mixture per ml of
water. The preferred concentration used in the isolation -~ -
, iof the ~ anomer is about 200 mg of anomeric salt mixture
per ml of water. ~ ;
Once the anomeric salt mixture is dissolved in
the water, acetone is added to the hot solution to form a
solvent mixture. The composition of the solvent mixture
may vary from about 7 parts by volume of acetone to 1
part by volume of water to about 30 parts by volume of
X-7108A -24- 1331~
acetone to 1 part by volume of water. A composition
of about 12:1, v:v, acetone:water is preferred. After
acetone addition, the ~ anomer will begin to crystallize.
In order to obtain the ~ anomer in high yield the
solution should be cooled to a temperature in the range
of about -10C to about 50C, preferably from about 0C
to about 15C. The cooled solution is stirred, while
maintaininq the desired temperature, for about 30
minutes to about 24 hours and 2'-deoxy-2',2'-difluoro-
cytidine hydrochloride or hydrobromide which has atleast about a 1:4 a/~ anomeric ratio is isolated, typi-
cally by filtration, from the solution using standard -
isolation techniques. -
The 1:4 anomeric mixture thus isolated can be
further purified, if desired, by repeating the procedure
used to prepare the 1:4 a/~ anomeric mixture, described
above. Thus, ~-2'-deo~y-2',2'-difluorocytidene hydro-
chloride or hydrobromide may be obtained in approximately
49.0% purity by dissolving ~-2'-deoxy-2',2'-difluorocyti-
20 dine hydrochloride or hydrobromide which is at least -~
80.0X pure in hot water, adding acetone, cooling the
solution to a temperature in the range of about -10C to
about 50C, and collecting the precipitated solid.
A second process for selectively isolating an
25 acid addition salt of ~-2'-deoxy-2',2'-difluorocytidine -~
from about a 1:1 a/~ anomeric mixture of 2'-deoxy-2',2'~
difluorocytidine utilizes the solubility difference
between the free base forms of the a and ~ anomers, in a
slightly basic aqueous solution, to selectively isolate
30 ~-2'-deoxy-2',2'-difluorocytidine. once the free base ~ ; form of the ~ anomer is isolated, it is easily converted
to an organic or inorganic acid addition salt.
::: . ; . ~ .
~ . ~
',
':~' '',': ~
X-7108A -25- 13 31~ 0 8
Isolation of the free base form of the
anomer is a preferred process for selectively isolating ~
acid addition salts of ~-2'-deoxy-2',2'-difluorocytidine ~ -
since the ~ anomer can be recovered in higher yields -
than provided by previously known processes. Addition-
ally, isolation of the free base form of the ~ anomer
can be used to improve product purity since the free
base form of the ~ anomer selectively crystallizes ~ -
relative to both the free base form of the a anomer, as
well as any additional impurities (such as ammonium
triflate and inorganic salts such as magnesium sulfate - -
and the like) present in the 1:1 a/~ anomeric mixture. -
The free base form of the ~ anomer is isolated : ;
by dissolving the 1:1 a/~ anomeric mixture, or an `~ -
15 organic or inorganic acid addition salt thereof, in hot ;
~about 45C to about 90C) water. To aid dissolution of
the non-salt form of the anomeric mixture, the pH of the ~ ~
water may be adjusted to about 2.5 to about 5.0 using ; ;~- -
common organic or inorganic acids such as hydrochloric
acid or the like. If an acid addition salt of the
anomeric mixture is used a common organic or inorganic --
base such as sodium hydroxide or the like may be used to ~ -
adjust the pH of the water to about 2.5 to about 5.0 in
order to aid dissolution. The amount of water used,
25 while not critical, should be suficient to effect ~
complete dissolution of the anomeric mixture or its salt, -;
but yet be as minimal as possible to avoid excessive ~ ; --;
product loss during crystallization and isolation.
Organic or inorganic acid addition salts of
the 1:1 a/~ anomeric mixture included within the scope
of this process include salts formed from organic acids
such as tartaric acid, citric acid, acetic acid and the -
like, as well as salts formed from inorganic acids such
X-7108A -26- 1 3 3 1 ~ a 8
as hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid and the like. The hydrochloride and
hydrobromide acid addition salts are especially pre-
ferred in the process of the present invention. Such
salts of the 1~ anomeric mixture can be prepared
by processes well known to those skilled in the art, for
example, by the process for preparing a hydrochloride
or hydrobromide salt of the 1:1 a/~ anomeric mixture as
discussed above. -
Once the anomeric mixture, or its salt, is
substantially dissolved in the water, the pH of the hot
solution is increased to about 7.0 to about 9.0 using a
common organic or inorganic base such as sodium hydroxide
or the like. Pref erably, the pH of the aqueous solution ---
will be increased to about 8.0 to about 8.5. After the
pH has been increased to the desired value, the solution
is allowed to cool. To obtain the free base form of the
~ anomer in high yield the solution should be cooled to -;
a temperature in the range of from about -10C to about
30C. The solution, optionally seeded with authentic
crystals of ~-2'-deoxy-2',2'-difluorocytidine if desired,
is then stirred for about 30 minutes to about 24 hours.
The ~-2'-deoxy-2',2'-difluorocytidine thus crystallized
is isolated from the solution using standard isolation ~ -
techniques, typically by filtration, in about 99.0% purity.
~-2'-Deoxy-2',2'-difluorocytidine thus prepared
can be converted to a pharmaceutically acceptable organic -
or inorganic acid addition salt by dissolving the ~
anomer in hot (about 45C to about 90C) water. The
amount of water used, while not critical, should be suf-
ficient to effect complete dissolution of the ~ anomer, ;-
but yet be as minimal as possible to avoid excessive
X-7108A -27- 13 31~ ~ 8 :
product loss during crystallization and isolation. The
pH of the water may be adjusted to about 2.5 to about
5.0 using a pharmaceutically acceptable acid to aid in ~ ~-
dissolving the ~ anomer, if desired.
Once the ~ anomer is substantially dissolved,
a pharmaceutically acceptable organic or inorganic acid
is added to form an acid addition salt. Pharmaceutically
acceptable organic or inorganic acids contemplated -
within the scope of this invention include organic acids
such as tartaric acid, citric acid, acetic acid, benzoic
acid and the like, and inorganic acids such as hydro-
chloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid and the like. Hydrochloric acid and hydrobromic
acid are preferred acids for use in the present process.
The amount of acid added is not critical so long as at
least a slight molar excess of acid is used relative to
the free base form of the ~ anomer.
After the acid is added the acid addition salt
of ~-2'-deoxy-2',2'-difluorocytidine will begin to crys-
20 tallize. To obtain the salt of the ~ anomer in high yield -
the solution should be cooled to a temperature in the
range of from about -10C to about 40C, preferably from
about 0C to about 15C. The solution, optionally seeded
with authentic crystals of the 2'-deoxy-2',2'-difluoro-
cytidine salt if desired, is then stirred for about 30
minutes to about 24 hours. Finally, the product is
isolated, typically by filtration, from the solution
using standard isolation techniques to provide a pharma-
ceutically acceptable acid addition salt of 2'-deoxy- -
2',2'-difluorocytidine in about 99.0% purity.
The following Examples illustrate specific
aspects of the present invention. The Examples are not
intended to limit the scope of the invention in any
respect and should not be so construed.
~-'..,,.'.:
:::
X-7108A -28- 1331~
Example 1
Preparation of an enantiomeric mixture of ~ -
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos- - -~
5 1-ulose-3-benzoate ~- - ;
To a 2 liter flask fitted with a reflux con~
denser were added 104 g (0.27 mole) of 96.0% pure ethyl ~ -
2,2-difluoro-3-(benzoyloxy)-3-(2,2-dimethyldioxolan-
4-yl)propionate which consisted of 3 parts of the 3-R-
benzoyloxy enantiomer to 1 part of the 3-S- benzoyloxy
enantiomer. Acetonitrile (1000 ml), deionized water
(25 ml, 1.35 mole), and trifluoroacetic acid (6.4 g,
0.05 mole) were added. The resulting colution was
heated to its reflux temperature (about 78C), and
stirred at that temperature for 4 hours. After 4 hours -~
the condenser was modified in order to allow the boiling --~
liguid to distill rather than reflux. As the volatile -c-~
acetonitrile, water, and trifluoroacetic acid distilled,
~ 20 fresh dry acetonitrile was added in order to maintain --
`~ the solution volume at about 1000 ml. After a total of
3000 ml of liguid had distilled the solution was cooled
to room temperature (22C).
The identity of the major components in the
; 25 solution were characterized by a high perfo D ance liguid
~ chromatographic (HPLC) comparison with authentic
t,:~ I ~ ,,reference standards. The assay sample was prepareq by
placing 125 ~1 of the reaction solution in a 25 ml
flask, adding 2 ml of isopropanol, and then diluting the
30 resulting solution to 25 ml with hexane. The column was ~ -
,.-,, :,..',,i.,."-
X-7108A -29- 1331~
eluted with an elution solvent comprised of 6% by volume
isopropanol and 94% by volume hexane. The column
employed was a 25 cm 'Zorbax CN'*. The detector had a wave-
length of 230 nm, the column flow rate was 2.0 ml/min,
the injection volume was 10 ~1. The ~PLC assay estab-
lished that the reaction solution contained a product
assaying 87.2% by weight of an enantiomeric mixture of - -
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos-
1-ulose-3-benzoate. The HPLC assay also indicated the
major impurities present were 2.7% by weight unreacted
propionate and 5.5% by weight ethyl 2,2-difluoro-3-
(benzoyloxy)-4,5-dihydroxypentanoate.
Examrle 2
- Preparation of an enantiomeric mixture of
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos-
1-ulose-3-benzoate
To a 110 liter glass lined reactor were added
64 1 of acetonitrile, 6.00 kg (16.2 mole) of a 3 part
3-R- to 1 part 3-S- enantiomeric mixture of ethyl
2,2-difluoro-3-(benzoyloxy)-3-(2,2-dimethyldioxolan-4-
yl)propionate, 0.57 kg (4.4 mole) of trifluoroacetic
acid, and lS00 ml (83.3 mole) of purified water. The
resulting solution was heated to its reflux temperature
(about 78C), and stirred at that temperature for 5~
hours. Next, 16 l of a solution of acetonitrile, water,
and trifluoroacetic acid were distilled and replaced by ~-
30 16 1 of toluene. The resulting solution was heated to ;~
*Trademark
. , .. . . . - ~ .
: ' i; - ' . ' , .. ., , ~ " , ~ , , , " , .. .
X-7108A -30- ~3 31~
about 96.5C and an additional 16 l of volatiles were
distilled. Fresh toluene (16 l) was added and the ~ -
solution was heated to 96.5C again. The distillation, - i ~ -
addition of fresh toluene, and heat to 96.5C process
S was repeated until 107 1 of volatile constituents had -
distilled. The remaining solution was cooled to room
temperature (22C) while maintaining a slight nitrogen
bleed into the reactor to ensure that no moist air could
enter the reactor during cooling. The resulting
solution, characterized by the HPLC assay described in
Example 1, contained a product assaying 81.8% by weight~ - `
of an enantiomeric mixture of D-erythro- and D-threo-2-
deoxy-2,2-difluoropentofuranos-1-ulose-3-benzoate. The -~
HPLC assay indicated the major impurities present were
lS 2.2% by weight unreacted propionate and 7.7% by weight
ethyl 2,2-difluoro-3-(benzoyloxy)-4,5-dihydroxypentanoate.
Exam~le 3
:, , ~, :,. . .
A. Preparation of an enantiomeric mixture of
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos-
l-ulose-3,5-dibenzoate `~
~, " ,,,~...
A solution of D-erythro- and D-threo-2-deoxy-
2,2-difluoropentofuranos-1-ulose-3-benzoate dissolved in
toluene was prepared according to the procedure of
! Example 2. This solution, assayed according to the HPLC
assay described in Example 1, contained a product assay-
ing 73.0% by weight D-erythro- and D-threo-2-deoxy-2,2-
. . .~ .
30 difluoropentofuranos-1-ulose-3-benzoate. The solution :~ ~
,, . ~. . ~ ~ .
~`. ~ '' , .
,~, ...
" , ~ .
..
. . .
~ `:
X-7108~ -31- 1331~
was heated to 50C under reduced pressure to remove the
toluene and provide 20.0 g (53.6 mmole) of the D-erythro-
and D-threo- enantiomeric compound as an oil. The oil
was transferred to a 500 ml flask and 100 ml of ethyl
acetate and 11.6 g (146.8 mmole) of pyridine were added.
Benzoyl chloride (10.3 g, 73.5 mmole) was dissolved in
I00 ml of ethyl acetate and the resulting solution was
added dropwise over 2 hours to the contents of the
500 ml flask. The reaction mixture was heated to about
60C and stirred at that temperature for 3~ hours, then
cooled to room temperature (22C) and stirred overnight.
The resulting mixture was washed successively with
200 ml of water, 200 ml of lN hydrochloric acid, 200 ml
of water, 200 ml of a saturated sodium bicarbonate
solution, and 200 ml of a saturated sodium chloride
solution, and then dried over anhydrous magnesium
sulfate. The dried solution was concentrated under
reduced pressure to provide 26.0 g of an oil. A repre-
sentative sample of the oil was dissolved in aceto-
nitrile and assayed using the HPLC procedure describedin Example 1 to establish that the oil was composed of
52.0% by weight 2-deoxy-2,2-difluoro-D-erythro-pento- -
furanos-l-ulose-3,5-dibenzoate and 16.7% by weight of
the corresponding D-threo enantiomer.
B. Isolation of 2-deoxy-2,2-difluoro-D-erythro-
!~ pentofuranos-1-ulose-3,5-dibenzoate
The remaining oil prepared above was dissolved
in 20 ml of methylene chloride. Hexane (30 ml) was
:
~'i'``~'``.`,."`''``,`'`-..`~'`''''`,f,''`"'-`''''' ' ,',
X-7108A -32- 13 31~ ~ 8
added with stirring. The solution was seeded with
authentic 2-deoxy-2,2-difluoro-D-erythro-pentofuranos-
1-ulose-3,5-dibenzoate and an additional 25 ml of a 3:2
(v:v) hexane/methylene chloride solution were added. ~-
The resulting solution was cooled to about 0C for 15
minutes. The precipitated solid was collected by vacuum
filtration and washed with 25 ml of a cold (0C) 3:2
(v:v) hexane/methylene chloride solution. The resulting , -
crystals were dried in a vacuum oven at 40C for 3 hours
0 to provide 9.0 g of the desired enantiomer, which was
identified by N.M.R. analysis on a 300 mHz instrument in ~-
CDCl3: ~ = 4.70 (singlet, 2H); 4.99 (singlet, 1~); 5.76 ~ -
(singlet, lH); 7.4-8.2 (broad multiplet, lOH). A -
representative sample of the dried crystals was dis-
solved in acetonitrile and assayed using the HPLC assay
technigue described in Example 1. The HPLC assay indi- -
cated that the product was 98.0% pure 2-deoxy-2,2- ~ -~
difluoro-D-erythro-pentofuranos-l-ulose-3,5-dibenzoate.-;~
ExamPle 4 `~
A. Preparation of an enantiomeric mixture of
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos- - -
l-ulose-3,5-dibenzoate
A solution of 169.0 g (0.472 mole) of D-erythro-
and D-threo-2-deoxy-2,2-difluoropentofuranos-1-ulose-3-
benzoate (prepared according to the procedure of Example ,,
2) dissolved in 845 ml of ethyl acetate was added to a ~;
2-liter flask. Pyridine (111.5 g, 1.410 mole) was added
. ~p,,.- . :~
.. ., ,-,:
- , :. - ::
,'-,~., ' :,-..::
:- . ~ .: ,
~: ~,,, ,, ~
. :. ...
..., : ,. - ...
~ e~ ; ,
i~ ~
X-7108A _33_ 1331~8
,.: .
to the flask and the solution was cooled to about 5C. '-
Benzoyl chloride (132.2 g, 0.940 mole) wa6 dissolved in
300 ml of ethyl acetate and the resulting solution was
added dropwise over 30 minutes to the 2-liter flask. '
The reaction mixture was allowed to warm to room tempera-
ture (22~C) and stirred at that temperature overnight. -
The next morning the mixture was cooled to about 5C and
the pyridine hydrochloride salts which had formed during
the reaction were removed by filtration. The solution , - ~ '
10 was then concentrated under reduced pressure to provide ~,
249.0 g of an oil. A representative sample of the oil
was dissolved in acetonitrile and assayed using the HPLC ,~ '-
procedure described in Example 1 to establish that the - ,
oil was composed of 52.2% by weight 2-deoxy-2,-2-difluoro-
lS D-erythro-pentofuranos-l-ulo6e-3,5-dibenzoate and 14.9%
by weight of the corresponding D-threo en,antiomer.
B. Isolation of 2-deoxy-2,2-difluoro-D-erythro- ~ , '
pentofuranos-l-ulose-3,5-dibenzoate " ''
,
The remaining oil prepared above was dissolved ~ ,
in 174 ml of methylene chloride. Hexane (249 ml) was
added over,30 minutes while the solution was stirred. , "~
The resulting solution was stirred at room temperature ~'
(22C) for 30 minutes and then cooled to about 5C and `-
stirred at that temperature for an additional 30
minutes. The precipitated solid was collected by vacuum '`'~
' ' filtration and washed with 264 ml'of a cold (0C) 3.2 '~
(v:v) hexane/methylene chloride solution. The~resulting ' ' '
30 crystals were dried in a vacuum oven at room temperature ~
''''''- ~
:-: :
,. .
X-7108A -34- 1 3 3 1 a 08
(22C) to provide 85.8 g of the desired enantiomer. m e
HPLC assay described in Example 1 established that the
recovered crystals were 99.6% pure 2-deoxy-2,2-difluoro-
D-erythro-pentofuranos-1-ulose-3,5-dibenzoate. m.p.
116-118C.
ExamDle 5 -~-
A. Preparation of an enantiomeric mixture of -~ ;
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos-
1-ulose -~
To a 500 ml flask were added 50.0 g (0.2 mole) ;~ -
of a 3 part 3-R- to 1 part 3-S- enantiomeric mixture of
ethyl 2,2-difluoro-3-hydroxy-3-(2,2-dimethyldioxolan~
4-yl)propionate, 250 ml of ethanol, and 6.0 ml of 6N i`-
sulfuric acid. The resulting solution was heated to its ;~
reflux temperature (about 76C), and stirred at that
temperature for 3~ hours. After 3~ hours 100 ml o$ an
ethanol/water mixture were distilled and replaced by
100 ml of fresh ethanol. The solution was cooled to -`- t ~-
room temperature (22C) and 4.5 g of anhydrous sodium
carbonate were added. Ten minutes later 20 g of 3A ~ ~-
molecular sieves were added. The resulting mixture was
refrigerated overnight. The next morning the sodium
carbonate and the molecular sieves were removed by
filtration to provide a solution which, when charac- ~ ~ ~ n~
terized by the HPLC assay described in Example 1, was
found to contain 231 mg of D-erythro- and D-threo-2-
30 deoxy-2,2-difluoropentofuranos-1-ulose per ml of solu- -;~ --
;~:', ' ~' '',' '`,-.
,. . ....- " - , ;:
..,:. .. .:. ~ ,:
:, . : .-,
- : . " :- ~
' !
X-7108A -35- 1331~G8
tion. Karl Fischer analysis of the solution indicated
the water content was 0.9% by weight water.
B. Preparation of an enantiomeric mixture of
D-erythro and D-threo-2-deoxy-2,2-difluoropentofuranos-
1-ulose-3,5-dibenzoate
The ethanol solution above was heated under
reduced pressure to remove the ethanol. The resulting -~ -
oil was dissolved in 100 ml of ethyl acetate. The solu-
tion was heated under reduced pressure to remove the
ethyl acetate. The resulting gum was dissolved in
100 ml of methylene chloride. Karl Fischer analysis of
the methylene chloride solution indicated the water -
15 content waæ about 0.09% by weight. The methylene chlo- `
ride solution was diluted with an additiQnal 225 ml
of methylene chloride, followed by the addition of ~`
2,6-lutidine ~48.3 g, 0.45 mole) and 4-dimethylamino-
pyridine (3.0 g, 0.02 mole). The resulting solution was
20 chilled in an ice bath to about 8C and benzoyl chloride -~
(63.4 g, 0.45 mole) was added dropwise over the next 18
minutes at a rate which kept the reaction solution's tem-
perature below about 15C. After the benzoyl chloride
was added the solution was allowed to warm to room tem-
25 perature (22C) and 2,6-lutidine hydrochloride precipi- ~ ~-
tated. The reaction mixture was washed successively
with 250 ml of water, 250 ml of a 5% by weight sodium
bicarbonate solution, 250 ml of 2N hydrochloric acid,
and 250 ml of a saturated brine solution. The methylene
chloride solution was then dried over anhydrous mag-
X-7108A -36- 1~31~8 -
nesium sulfate and assayed using the HPLC assay de-
scribed in Example 1. The HPLC assay indicated that
the methylene chloride solution contained 28. 3 g of
D-eryt~ro- and D-threo-2-deoxy-2,2-difluoropentofuranos-
1-ulose-3,5-dibenzoate.
C. Isolation of 2-deoxy-2,2-difluoro-D-
erythro-pentofuranos-l-ulose-3,5-dibenzoate - ~ ~
.: ' -~ ~ .' ~ . .
The methylene chloride solution prepared above - -
was concentrated to a thick syrup by distillation. The
syrup was redissolved in 30 ml of fresh methylene
chloride. Isopropanol (300 ml) was added and the
D-erythro product began to çrystallize. Within ten -
minutes the product had precipitated to such an extent
that a viscous slurry had formed. Additional methylene
chloride (10 ml) and isopropanol (100 ml) were added in -
order to reduce the slurry's ~iscosity and the result-
ing mixture was refrigerated at 5C overnight. The ~ -
20 precipitated solids were collected by vacuum filtration -
and washed successively with cold (0C) isopropanol and ~
cold (0C) hexane. The resulting crystals were dried in -- -
a vacuum oven at 22C to provide 17.4 g of the D-erythro -- ~-
product, which was identified by N.M.R. analysis on a ~--
300 mHz instrument in CDCl3: ~ = 4.70 (singlet, 2H);
4.99 (singlet, lH); 5.76 (singlet, lH); 7.4-8.2 (broad
multiplet, lOH). The product, which melted at
119-119.5C, was believed to be greater than 95.0% 5
purity 2-deoxy-2,2-difluoro-D-erythro-pentofuranos-l-
ulose-3,5-dibenzoate.
.', ,. ' ,: - -::
. ' :'. . '-~ '':~ . .
'' . '',; ' " ~ ' ."~'','~ '
X-7108A -37- 13 31~ ~ ~
Example 6
A. Preparation of an enantiomeric mixture of
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos- -
5 1-ulose-3,5-dibenzoate -
To a 250 ml flask were added 73 ml of a 2:1 -
(v:v) methanol/water solution which contained 2.33 g
(13.89 mmole) of an enantiomeric mixture of D-erythro-
and D-threo-2-deoxy-2,2-difluoropentofuranos-1-ulose
(62.4% erythro ,enantiomer) prepared according to the
procedure of Example 5. The solution was heated under
reduced pressure to provide an oil. The oil was dis-
solved in 100 ml of ethyl acetate and the resulting
solution was dried over anhydrous magnesium sulfate.
After the magnesium sulfate was removed by filtration
the solution was again concentrated under reduced
pressure to provide a thick oil. This oil was dissolved
in 18 ml of methylene chloride, followed by the addition
of 0.17 g (1.38 mmol) of 4-dimethylaminopyridine. The
solution was cooled to about 0C and 3.41 g (31.85 mmol)
of 2,6-lutidine and 4.50 g (31.98 mmol) of benzoyl
chloride were added. The solution was allowed to warm
to room temperature (22C) and then stirred for about 64
hours. After stirring the solution volume was increased
to about 50 ml with methylene chloride. The resulting
solution was washed successively with 25 ml of a 5% by
weight hydrochloric acid solution, 25 ml of a 5% by
weight sodium bicarbonate solution, and 25 ml of water.
The methylene chloride solution was dried over anhydrous
, : ~
. .. .
. . ,
X-7108A -38- 13 31~ ~ 8
magnesium sulfate and concentrated under reduced pres-
sure to provide an oil. This oil, while not assayed,
was believed to be the desired enantiomeric mixture of -
D-erythro- and D-threo-2-deoxy-2,2-difluoropentofuranos-
1-ulose-3,5-dibenzoate.
:
B. Isolation of 2-deoxy-2,2-difluoro-D-erythro-
pentofuranos-l-ulose-3,5-dibenzoate ~ ~
., ~ .
The above oil was dissolved in methylene -
chloride (3.5 ml). Isopropanol (35 ml) was added and
the solution cooled to about 0C in an ice bath, then
seeded with a crystal of the authentic compound. After -stirring at about 0C for 3 hours the mixture was ; - -
filtered. The filter cake was washed with cold isopro-
panol and room temperature (22C) hexane and dried in a
vacuum oven at 22C to provide 0.87 g of 97.0% pure
2-deoxy-2,2-difluoro-D-erythro-pentofuranos-l-ulose- ~
3,5-dibenzoate as established by the analytical tech- ~ -
20 nique described in Example 1. mp = 117-118C. The ;
product was also identified by N.M.R. analysis on a
300 mHz instrument in CDCl3: ~ = 4.70 (singlet, 2H);
4.99 (singlet, lH); 5.76 (singlet, lH); 7.4-8.2 (broad -
multiplet, lOH). -
25 ~ -
'~ '''` '' ~'"':
,~, ~,, . ', .:
X-7108A -39- 1331~8
ExamDle 7
Preparation of a 1~ anomeric mixture
of 2'-deoxy-2',2'-difluorocytidine
S ' .' .
To a 500 ml, 3-neck round bottom flask con- - -
taining 250 ml of 1,2-dichloroethane were added 15.00 g
(32.88 mmole) of 2-deoxy-2,2-difluoro-D-erythro-pento-
furanos-3,5-dibenzoate-1-methanesulfonate, 15.65 g (52.60
mmole) of bis-trimethylsilyl-N-acetylcytosine, and
9,50 g (42.74 mmole) of trifluoromethanesulfonyloxy-
trimethylsilane~ The solution was heated to reflux
(84C) for about 8 hours. The reaction solution was - -
cooled to room temperature (22C) and 100 ml of a 5% by ~ ~
15 weight hydrochloric acid solution added. After stirring -
for about 5 minutes the layers were separated and the ~-
water layer washed with 25 ml of methylene chloride.
The organic layers were combined and washed successively
with 100 ml of a 5% by weight sodium bicarbonate solu-
tion and 100 ml of a saturated brine solution. The
resulting organic layer was dried over anhydrous magne- `~
sium sulfate and concentrated under reduced pressure to
provide a foam. ~ -
Methanol (150 ml) was added to dissolve the ~ ~
25 foam. The solution was cooled to about 0C. Ammonia - -
gas was bubbled through the solution for about one
. , .
minute. The volatile constituents were removed under -
reduced pressure to provide a gum. The gum was dis-
solved in 100 ml of ethyl acetate and 100 ml of water.
The layers were separated and the organic layer was
,:: . , . .. ,:
, . 1~ ~ :
X-7108A -40- 1331~08 : ~
washed with 25 ml of water. Both aqueous layers were ~ -
combined, washed with 100 ml of diethyl ether, and the -~ -
aqueous solution was concentrated under reduced pressure
to provide a gum. About 10 ml of methanol were added to
5 dissolve the gum. The resulting solution was concen- ~ --
trated to dryness under reduced pressure to provide -
4.14 g of 2'-deoxy-2',2'-difluorocytidine. -
The product was characterized by an HPLC
comparison with an authentic reference standard. The ;
10 assay sample was prepared by placing 3 mg of product ;
into a 5 ml volumetric flask and then diluting to volume
with O.lN hydrochloric acid. The column was eluted with --
an elution solvent comprised of 5% by volume methanol
and 95% by volume 0.04M sodium acetate solution. The
column employed was a 25 cm YMC type A-303. The
detector had a wavelength of 275 nm, the column flow
rate was 1.0 ml/min, the injection volume wa's 20 ~l, and ~-
the column temperature was ambient (22C). The HPLC
assay disclosed the product as approximately a 1:1 a/~ -;
20 anomeric mixture of 2'-deoxy-2',2'-difluorocytidine. -
ExamPle 8
, " .
Preparation of 90.5% pure ~-2'-deoxy-2',2'-
25 difluorocytidine hydrochloride ~-
' . ' :
An approximately 1:1 a/~ anomeric mixture ; - -
of 2'-deoxy-2',2'-difluorocytidine (1.86 g), prepared ~
according to the procedure of Example 7, was dissolved ~ -
in 6 ml of hot isopropanol (80C). Concentrated hydro-
:
!
X-7108A -41- 13 31~ ~ 8
chloric acid (15 drops) was added to the hot solution.
The solution was seeded with authentic ~-2'-deoxy-2',2'-
difluorocytidine hydrochloride, allowed to cool to room
temperature, and refrigerated over the weekend. The
S mixture was filtered and the filter cake was washed with
isopropanol and vacuum dried at 22C to provide 0.38 g
of a product which assayed as 90.5% pure ~-2'-deoxy-
2',2'-difluorocytidine hydrochloride by the analytical
procedure described in Example 7. The product was also
identified by N.M.R. analysis on a 300 MHz instrument
in CDCl3: ~ = 3.81 (triplet, 2H); 3.93 (doublet, lH);
4.23 (triplet, lH); 4.80 (doublet, 2H); 6.08 (doublet,
lH); 6.32 (doublet, lH); 8.21 (doublet, lH); 9.0 (singlet,
lH); 10.17 (singlet, lH).
Additional crystals formed in the filtrate
upon sitting overnight at room temperature. These ~ -
crystals were recovered and dried as described above to
provide an additional 0.17 g of 82.0% pure ~-2'-deoxy- ~ -
2',2'-difluorocytidine hydrochloride as established by
the above HPLC procedure.
Example 9
Preparation of 99.4% pure ~-2'-deoxy-2',2'-
25 difluorocytidine hydrochloride ; ~ i
: . -. :-. . .~
To a 50 ml flask fitted with a reflux con-
denser were added 100.0 mg of 2'-deoxy-2',2'-difluoro-
cytidine hydrochloride (88.7% pure ~ anomer prepared
30 according to the procedure of Example 8) and 0.5 ml ;
water. The mixture was heated to reflux (100C) and all
`"~'"''.: '''''~.'
: '~ ~''':,'','.'
X-7108A -42- 13 31~ ~ 8
solids dissolved. While the solution was refluxing
10 ml of acetone were added. The solution was cooled
to room temperature and refrigerated overnight. The ~,
mixture was filtered and the resulting crystals were
S dried in a vacuum oven at 22C to provide 69.0 mg of
99.4% pure ~-2'-deoxy-2',2'-difluorocytidine hydro-
chloride as established by the analytical technique
described in Example 7. The N.M.R. spectrum of this , - , -
material was identical to that described in Example 8. ~ ,
' ' '.
ExamDle 10 ~ ,;
- .
Preparation of 79% pure ~-2'-deoxy-2',2'-
difluorocytidine hydrobromide
An, approximately l~ ano,meric mixture
of 2'-deoxy-2',2'-difluorocytidine (300 mg), prepared ~, ~ , ,-
according to the procedure of Example 7, was substan-
tially dissolved in 3 ml of hot isopropanol ~60C). ~
20 Aqueous hydrobromic acid (0.3 ml of a 48% by weight ~ , ,
solution of hydrobromic acid dissolved in water) was
added to the hot solution and all the remaining solids -
dissolved. An additional l ml of isopropanol was added , ,
and the solution was refrigerated overnight. The mix- ,
ture was filtered and the filter cake was washed with
isopropanol and vacuum dried at 22C to provide 110 mg
of a product which assayed as 79.0% pure ~-2'-deoxy-
2',2'-difluorocytidine hydrobromide by the analytical ,~
procedure described in Example 7. (The only change - -
from the analytical procedure of Example 7 was that
' ~,
X-7108A -43- 1331~8
O.lN hydrobromic acid was used to dilute the present
sample to volume rather than O.lN hydrochloric acid.)
The product had the following elemental analysis.
Analysis calc. for CgHl2N304F2Br:
Theory: C, 31.41; H, 3.52; N, 12.21; F, 11.04;
Br, 23.22;
Found: C, 29.54; H, 3.64; N, 11.02; F, 10.90;
Br, 23.16. ~ ~
' '~ "'
ExamDle 11
:- :
Preparation of a 1:1 a/~ anomeric mixture of
2~-deoxy-2',2'-difluorocytidine hydrochloride
A. Preparation of a 1:1 a/~ anomeric mixture -
of 2'-deoxy-2',2'-difluorocytidine -
To a 500 ml, 3-neck round bottom flask contain-
ing 200 ml of 1,2-dichloroethane were added 10.0 g (65.7
mmole) of N-acetylcytosine, 12.0 g (74.5 mmole) of
1,1,1,3,3,3-hexamethyldisilazane (HMDS) and 0.47 g (4.38
mmole) of chlorotrimethylsilane (CTMS). The slurry was
hèated to reflux (84C) and within 15 minutes a solution
25 was obtained. The solution was refluxed for about -~
one hour and then the solvent was removed to provide a
thick residue.
,.-; :,~
X-7108A -44- 13 31~
The thick residue was dissolved in 200 ml of
1,2-dichloroethane. To the solution were added 19.55 g
(88.0 mmole) of trifluoromethanesulfonyloxytrimethylsilane,
3.6 g (22.2 mmole) of HMDS and 2.4 g (22.1 mmole) of CTMS. --
The solution was stirred at room temperature (22C) for
30 minutes and 58 ml of a solution of 2-deoxy-2,2-difluoro-
D-erythro-pentofuranos-3,5-dibenzoate-1-methanesulfonate
dissolved in 1,2-dichloroethane (0.345 grams of methane-
sulfonate per ml of solvent; total methanesulfonate -
20.0 g) were added. The solution was heated to reflux
(84C) for about 18 hours then cooled to room temperature -
(22C) and 10 ml of methanol and 145 ml of water were
added. After stirring for about 5 minutes the layers
were separated and the organic layer was again combined
with 145 ml of water. The layers were separated once
more, and the two water layers were combined and washed
with 25 ml of 1,2-dichloroethane. The organic layer
from above was combined with the dichloroethane wash and
the combination washed successively with 145 ml of a 5%
by weight sodium bicarbonate solution and 145 ml of
water. The resulting organic layer was dried over -
anhydrous magnesium sulfate and concentrated under --
reduced pressure to provide a foam. ; `
Methanol (220 ml) was added to dissolve the
foam. The solution was cooled to about 5C and ammonia
gas (6.0 g) was bubbled through the solution. The
volatile constituents were removed under reduced pressure
to provide an oily residue. The residue was dissolved in
145 ml of ethyl acetate and 145 ml of water. The layers
were separated and the organic layer washed twice with
X-7108A -45- 1331~8 ~:
50 ml of water. The aqueous layers were combined, and
the resulting solution was concentrated to dryness under
reduced pressure to provide 8.4 g of an approximately -
1:1 a/~ anomeric mixture of 2'-deoxy-2',2'-difluorocyti-
dine, as established by the HPLC technique described in
Example 7. -~
' ~
B. Preparation of a 1:1 a/~ anomeric mixture
of 2'-deoxy-2',2'-difluorocytidine hydrochloride -~
,
The gum prepared above was dissolved in hot
(60C) isopropanol. Reagent grade concentrated hydro- ~ ~-
chloric acid (5.1 ml) was added to the hot solution.
The resulting solution was cooled to room temperature
(22C) and refrigerated overnight. The resulting preci-
pitate was collected by vacuum filtration, washed suc-
cessively with cold (5C) isopropanol and room tempera-
ture hexane, and dried in a vacuum oven at 40C to -~
provide 5.15 g of a compound which assayed as approxi-
mately a 1:1 a/~ anomeric mixture of 2'-deoxy-2',2'~
difluorocytidine hydrochloride by the HPLC technique set - ;~
forth in Example 7. ; ~ `
Exam~le 12
Preparation of 97.7% pure ~-2'-deoxy-2',2'- - --
difluorocytidi~e from a 1:1 a/~ 2'-deoxy-2',2'-difluoro-
cytidine hydrochloride anomeric mixture ~,' ,r,~ ", ,,'"
Five grams of the approximately 1:1 a/~ 2'-
deoxy-2',2'-difluorocytidine hydrochloride anomeric
mixture obtained in Example 11 were placed in a 100 ml
'~ '; ':~ ~ `' ''`
- ` ~ ~
:
)
l33laQs
X-7108A -46-
round bottom flask containing 50 ml of hot (50C) water.
Sodium hydroxide (2N) was added until the pH of the water
was about 3.0, at which time all solids were dissolved.
The pH of the aqueous solution was increased to about
8.2 using SN sodium hydroxide. The basic solution was
allowed to cool to room temperature (22C) and then
refrigerated overnight. The precipitated solid was col-
lected by vacuum filtration, washed with 5 ml of cold,
pH 8.5, water, and dried in a vacuum oven at 40C to
provide 1.65 g of a product which assayed as 97.7% pure
~-2'-deoxy-2',2'-difluorocytidine by the HPLC technigue
described in Example 7.
.
Exam~le 13
Preparation of 98.8% pure ~-2'-deoxy-2',2'- -~
difluorocytidine from a 1:1 a/~ 2'-deoxy-2',2'-difluoro-
cytidine anomeric mixture
To 17 ml of hot (50C) water were added 8.4 g
of a 1:1 a/~ anomeric mixture of 2'-deoxy-2',2'-difluoro-
cytidine prepared according to the procedure set forth
in Example 11. The pH of the resulting aqueous solution
was increased to about 8.2 with 2N sodium hydroxide.
The basic solution was cooled to room temperature (22C)
and then refrigerated overnight. The precipitated solid
!~' ' was collected by vacuum filtration, washed with 5 ml of
cold, pH 8.5, water, and dried in a vacuum oven at 40C
to provide 1.4 g of a product which assayed as 98.8%
pure ~-2'-deoxy-2',2'-difluorocytidine by the HPLC assay
technique set forth in Example 7.
,., :
'
:
X-7108A _47- 13 ~
ExamDle 14
~''~. :.
Preparation of 100% pure ~-2'-deoxy-2',2'- ;
difluorocytidine hydrochloride from 99.7% pure ~-2'-
deoxy-2',2'-difluorocytidine
, . . - . ,,:
To a S00 ml four neck round bottom flask con-
taining 100 ml of hot (55C) water were slowly added ~ , ;-
20.0 g of 99.7% pure ~-2'-deoxy-2',2'-difluorocytidine
prepared according to the procedure of Example 12. Con-
centrated reagent grade hydrochloric acid was simultane-
ously added to the flask at a rate such that the pH of
the agueous solution was maintained at about 3.0 during
the difluorocytidine addition. After the difluorocyti- `~
dine addition was complete, an additional 13 ml of
concentrated hydrochloric acid were added. The solution
was cooled ~o about 0C in an ice bath and stirred at
that temperature for about 3 hours. The precipitated
solid was collected by vacuum filtration, washed succes- ~ `
sively with 5 ml of pH 1.0 water and 10 ml of acetone,
and dried in a vacuum oven at 45C to provide 21.3 g of -~
a product which assayed as 100% pure ~-2'-deoxy-2',2'-
difluorocytidine hydrochloride by the HPLC assay tech-
nique described in Example 7. The product was further
characterized by N.M.R. analysis on a 300 mHz instrument
and had the same spectrum as the product described in ~-
Example 8.
. .,}-,. ~.~ .".:
- :, .
. : . .: ~.: .
, , . . . .,: ,: .
~:- :~:-: :.: .: .-.
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