CA1331008C - 2',2'-difluoronucleosides - Google Patents

Abstract

ABSTRACT
The present invention provides a process for preparing lactone intermediates to 2',2'-difluoronucleo-sides whereby reversion back to the lactone's open chain precursor is minimized and the desired erythro enantiomer can be selectively isolated from an enantiomeric mixture of erythro and threo lactones in crystalline form. Also provided is a process for producing 2'-deoxy-2',2'-difluoronucleosides in about a 1:1 .alpha./.beta. anomeric ratio, and processes for selectively isolating .beta.-2'-deoxy-2',2'-difluorocytidine, or an organic or inorganic acid addi-tion salt thereof, from the 1:1 .alpha./.beta. mixture.

Description

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 ~ \ / 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 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 -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~

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~

, ,.
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 -~ 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 CO2(C1 C- ~Ikyl) 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) 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 .....

~ : .
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 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 ~

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 ;~

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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,,.- . :~
.. ., ,-,:
- , :. - ::
,'-,~., ' :,-..::
:- . ~ .: , ~: ~,,, ,, ~

. :. ...
..., : ,. - ...

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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.

.', ,. ' ,: - -::

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'' . '',; ' " ~ ' ."~'','~ ' 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 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.

. .,}-,. ~.~ .".:
- :, .
. : . .: ~.: .
, , . . . .,: ,: .
~:- :~:-: :.: .: .-.
~::; - .
~ . ': . '' .":

: .:
- ,. .. ~:

Claims (2)

1. A process for selectively isolating, in greater than about 95.0% purity, 2-deoxy-2,2-difluoro-D-erythro-pentofuranos-1-ulose-3,5-dibenzoate, which has the formula , from an enantiomeric mixture of erythro and threo lactones of the formula , comprising dissolving the enantiomeric mixture in methylene chloride, cooling the solution to a tempera-ture in the range of about -5°C to about 10°C, and collecting the precipitated erythro enantiomer.

2. A process according to Claim 1 for selectively iso-tional step of adding hexane or isopropanol to the solu-tion of the enantiomeric mixture dissolved in methylene chloride to provide a hexane/methylene chloride or isopropanol/methylene chloride solvent mixture.