TITLE
PROCESS FOR PREPARING 1-(2'-DEOXY-2',2'-DIFLUORO-D-
RIBOFURANOSYL)-4-AMINOPYRIMIDIN-2-ONE) HYDROCHLORIDE
1-(2'-Deoxy-2',2'-difluoro-p-ribofuranosyl)-4-
aminopyrimidin-2-one hydrochloride (also known as 2'-deoxy-
2',2'-difluorocytidine hydrochloride or gemcitabine
hydrochloride - see Formula I) is one of a series of 2'-
deoxy-2',2'-difluoronucleosides known in the art. Such
compounds are disclosed and taught to have antiviral
activity in U.S. Patents No. 4,526,988 and 4,808,614.
European Patent Application Publication 184,365 teaches
that these same difluoronucleoside agents have oncolytic
activity. Gemcitabine hydrochloride is undergoing clinical
evaluation to determine its usefulness as a treatment for a
variety of cancers, such as pancreatic cancer.

The synthesis of gemcitabine is a multi-step
process - see U.S. Patents No. 4,526,988, 4,808,614, and
5,223,608 and European Patent Application Publications
577,303, 577,304, and 587,364. Most of these synthetic
routes go through the penultimate intermediate P-1-(2'-
deoxy-2' ,2 '-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one which is deprotected and salified to
give the final desired product as the hydrochloride salt.
For example, in U.S. Patent No. 5,223,608, at column 10,
line 41 et seg., it is taught that the benzoyl protecting
groups "are removed by hydrolysis with a strong or
moderately strong base". It is also taught that "[a]t
least one mole equivalent of base is needed for each
protecting group." Examples 7 and 11 of this patent teach
the use of anhydrous ammonia in methanol for accomplishing
this deprotection which results in the formation of
ammonium chloride which is insoluble in the organic
solvents used and must be removed by treatment with water.
The preparation of gemcitabine hydrochloride
salt is also disclosed in the literature. U.S. Patent No.
5,223,608, at column 11, line 22 et seg., and Example 8
where hot isopropanol and concentrated hydrochloric acid
are used to generate the crystalline gemcitabine
hydrochloride.
This invention provides a more economical and
higher yielding process for preparing gemcitabine
hydrochloride which avoids the use of excess base and loss
of product due to the presence of water.
This invention provides a process for preparing
gemcitabine hydrochloride which comprises:
a) deblocking ß-1-(2'-deoxy-2',2'-difluoro-
3',5'-di-O-benzoyl-D-ribofuranosyl)-4-aminopyrimidin-2-one
with a catalytic amount of an alkylamine in the presence of
methanol or ethanol in an environment essentially free of
water;
b) treating the resulting solution with
hydrochloric acid and an antisolvent; and
c) recovering the resulting solid gemcitabine
hydrochloride.
During the deprotection of ß-1- (2'-deoxy-2' ,2'-
difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one, the protic solvent employed is the
actual nucleophile which attacks the benzoyl group. This
attack is catalyzed by base and stoichiometric amounts of
base are not required.
When used in this application, the term
"alkylamine" means an organic amine having one, two or
three alkyl groups and which is capable of adjusting the pH
of the reaction mixture to at least 9.5. Examples of such
reagents include, methylamine, ethylamine, propylamine,
isopropylamine, butylamine, dimethylamine, diethylamine,
dipropylamine, diisopropylamine, dibutylamine,
ethanolamine, trimethylamine,* triethylamine,
tripropylamine, N-methyl-N-ethylamine, N-methyl-N-
propylamine, N-methyl-N-butylamine, N-ethyl-N-propylamine,
and the like. As discussed below, this invention requires
that the alkylamine hydrochloride must also be soluble in
the reaction mixture. Further, for effective reaction
conditions, the alkylamine should be sufficiently non-
volatile to allow for effective reaction at elevated
temperatures. For all of these reasons, diethylamine is
the preferred alkylamine in this reaction, although other
alkylamines are operable.
The term "catalytic amount" refers to an amount
of alkylamine which will promote the nucleophilic attack on
the benzoyl protecting groups by the protic solvent.
Typically 0.1-0.5 molar equivalents (relative to the (3-1-
(2'-deoxy-2',2'-difluoro-3',5'-di-O-benzoyl-D-
ribofuranosyl)-4-aminopyrimidin-2-one employed) are used,
most preferably from 0.2-0.4 equivalents - however, as will
be appreciated by those skilled in the art, the optimal
amount of alkylamine employed will depend upon the chemical
and physical properties of the alkylamine so long as the pH
of the reaction mixture is maintained at no less than 9.5.
The phrase "in an environment essentially free
of water" indicates that the reaction is carried out
without any added water. It is therefore preferred that
the reagents and solvents used be essentially free of
water. The reaction mixture can be protected from
atmospheric moisture but this precaution is not necessary.
This limitation, which is not critical to the deblocking
reaction, is preferred because the final desired
gemcitabine hydrochloride is soluble in water and therefore
the presence of water will reduce isolated yield of the
desired product from the crystallization process.
As noted above, the deblocking of the ß-1-(2'-
deoxy-2',2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one is one where the protic nucleophile is
catalyzed by base and therefore it is the protic reagent
that is critical to the reaction. While any protic solvent
can theoretically be employed for the solvolysis to occur,
it is preferred that an alcohol, preferably ethanol and
most preferably methanol, be employed. This choice is
preferred for several reasons - first, the substrate and
alkylamine hydrochloride are soluble in methanol, as is the
resulting product. Second, the resulting by-product is
methyl benzoate which is also soluble in methanol and in
the precipitating solvent isopropanol or acetone. The use
of methanol also allows for its use both as a reagent and
as a solvent. Finally, as noted above, the reagent should
be essentially free of water.
As those familiar with this chemistry will
appreciate, in theory substrates other than ß-1-(2'-deoxy-
2',2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one can be employed - however, the
protecting groups must afford a substrate and by-product
which are soluble as described above. Moreover, the two
protective groups do not have to be the same. Thus,
substrates wherein the 3'- and/or 5'-hydroxy groups are
protected with other blocking groups, such as substituted
benzoyl (e.g., 4-methylbenzoyl) will be operable, if a
judicious choice of alkylamine and protic solvent are
employed - however, they offer no advantages and are, in
fact, more expensive to use.
The solvolysis is best carried out at
temperatures from 0°-80°C; elevated temperatures are
preferred. The reflux temperature of the reaction mixture
(50-60°C when methanol and diethylamine are employed) is
most preferred; under these conditions, the solvolysis is
generally complete in 1-8 hours.
It is preferred that the amount of methanol or
ethanol used be approximately 15-25 volumes relative to the
ß-1- (2'-deoxy-2', 2'-dif luoro-3', 5' -di-O-benzoyl-D-
ribofuranosyl)-4-aminopyrimidin-2-one starting material
(mL/g). The optimal ratio is 20 volumes of methanol. It
is also preferred that the amount of antisolvent be
approximately equal to the amount of methanol or ethanol
used, although other ratios are operable. If the P-1-(2'-
deoxy-2',2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one is generated in situ from 2-deoxy-2,2-
difluoro-3,5-dibenzoyl-D-ribofuranosyl-1-methanesulfonate
and cytosine (e.g., as described in EPO Patent Application
Publication No. 577,303), the yield of ß-1-(2'-deoxy-
2',2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one is usually approximately 70% - if this
reaction scheme is incorporated into the present process,
the amount of methanol or ethanol used is approximately 10-
20 volumes relative to the mesylate starting material -
see, e.g., Example 3 infra.
The formation and isolation of gemcitabine
hydrochloride is usually carried out by cooling the
solvolysis reaction mixture to ambient temperature and
adding an anti-solvent such as acetone, acetonitrile,
tetrahydrofuran, propanol, butanol, isobutanol, sec-
butanol, or preferably isopropanol. Gemcitabine base, the
alkylamine, the alkylamine hydrochloride, and methyl
benzoate are all soluble in this mixture; however, upon
introduction of hydrogen chloride, the desired gemcitabine
hydrochloride will crystallize out while the by-products
and unreacted reagents will remain in solution. The
hydrogen chloride is generally added as gaseous hydrogen
chloride or concentrated hydrochloric acid to a pH of 1.5
to 2.0; excess acid, particularly hydrochloric acid, is
unnecessary and may adversely affect yield. Although
concentrated hydrochloric acid contains water, this minimal
amount of water will not materially affect the yield.
The resulting gemcitabine hydrochloride is then
isolated from the reaction mixture by conventional means,
e.g., filtration, centrifugation, decantation, etc.
The starting materials and intermediates for
the preparation of the compounds of the present
invention are commercially available or can be readily
prepared by other methods known in the literature.
References to specific literature procedures are cited
in the examples and listed following the example section
hereinbelow.
The following examples further illustrate the
preparation of the compounds of this invention. The
examples are provided for purposes of illustration only
and are not to be construed as limiting the scope of the
instant invention in any way.
The terms and abbreviations used in the
instant examples have their normal meaning unless
otherwise designed, for example, "HPLC" refers to high
performance liquid chromatography; "°C" refers to
degrees Celsius; "mmol" refers to millimole; "g" refers
to gram; "L" refers to liter; "mL" means milliliter; "M"
refers to molar or molarity; "eq." means molar
equivalents.
Example 1
1-(2'-deoxy-2',2'-difluoro-D-ribofuranosyl)-4-
aminopyrimidin-2-one hydrochloride
(5-1- (2' -deoxy-2', 2' -dif luoro-3' , 5' -di-O-benzoyl-
D-ribofuranosyl)-4-aminopyrimidin-2-one (0.24 g, 0.51 mmol,
1 eq.) was slurried in methanol (7 mL) then diethylamine
(0.01 mL, 0.102 mmol, 0.2 eq.) was added. The mixture was
heated to 55°C for 2 hours and 20 minutes.
To isolate the product, the mixture was filtered
and the filter was rinsed with isopropanol (3.5 mL). The
filtrate and rinse were combined and adjusted to a pH of
1.5 to 2.0 by adding concentrated hydrochloric acid (0.3
mL). A precipitate formed within 1 to 2 minutes. The
mixture was then stirred at room temperature for 2 hours
and filtered. The solid was washed with isopropyl alcohol
(5 mL) then with acetone (5 mL). HPLC analysis showed the
potency of the above product to be greater than 99 percent.
The identity of the major components of the
solution was characterized by a HPLC comparison with
authentic reference standards. The HPLC assay sample was
prepared by placing 0.6-0.8 g of the reaction solution or
10 mg to 15 mg of 1-(2'-deoxy-2',2'-difluoro-D-
ribofuranosyl)-4-aminopyrimidin-2-one in a 50 mL flask,
then diluting to volume with water. The column was eluted
with eluant: A = 0.05M acetate at pH of 5.0; B =
acetonitrile; flow rate at 1.5 mL/minute. Eluant profile
is 97 percent A, 3 percent B hold for 5 minutes, gradient
to 30 percent A, 70 percent B over 10 minutes, hold at 30
percent A, 70 percent B for 5 minutes, return to 97 percent
A, 3 percent B over 2 minutes and hold for 13 minutes. The
column employed was a 25 cm Zorbax RxC8 column. The
detector had a wavelength of 275 nm, the column flow rate
was 1.5 mL/minute and the injection volume was 10 µ L.
The HPLC assay established retention times as
follows: a) cytosine and other impurities, 2.4 to 2.6
minutes; b) a-1-(2'-deoxy-2',2'-difluoro-D-ribofuranosyl)-
4-aminopyrimidin-2-one, 5 to 6 minutes; c) ß-1-(2'-deoxy-
2',2'-difluoro-D-ribofuranosyl)-4-aminopyrimidin-2-one, 6
to 7 minutes; d) benzoic acid, 10 to 11 minutes; e) 1-(2'-
deoxy-2',2'-difluoro-3' or 5'-mono-O-benzoyl-D-
ribofuranosyl)-4-aminopyrimidin-2-one, 14 to 15 minutes; f)
methyl benzoate; 16.5 to 17.5 minutes; and g) ß-1-(2'-
deoxy-2' ,2 '-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one, 19 to 20 minutes.
Example 2
1-(2'-deoxy-2',2'-difluoro-D-ribofuranosyl)-4-
aminopyrimidin-2-one hydrochloride
ß-1- (2' -deoxy-2' , 2' -difluoro-3' , 5' -di-O-benzoyl-
D-ribofuranosyl) -4-aminopyrimidin-2-one (0.24 g, 0.51 mmol,
1 eq.) was slurried in methanol (7 mL) and propylamine
(0.0084 mL, 0.102 mmol, 0.2 eq.) was added. The mixture
was heated to 55°C for 3 hours.
To isolate the product, the mixture was filtered
the filter was rinsed with isopropanol (3.5 mL) and the
combined filtrate and rinse was adjusted to a pH of 1.5 to
2.0 by adding concentrated hydrochloric acid (0.3 mL). A
precipitate formed in 1 to 2 minutes. The mixture was then
stirred at 0°C to 5°C for 90 minutes and filtered. The
solid was washed with isopropyl alcohol (5 mL) then with
acetone (5 mL). An HPLC analysis, carried out as described
in Example 1, showed the potency of the isolated solid to
be 98.9 percent.
Example 3
1-(2'-deoxy-2',2'-difluoro-D-ribofuranosyl)-4-
aminopyrimidin-2-one hydrochloride
P-1-(2'-deoxy-2',2'-difluoro-3',5'-di-O-benzoyl-
D-ribofuranosyl) -4 -aminopyrimidin-2 -one (obtained from
reaction of 3.83 6 g of 2-deoxy-2,2-difluoro-3,5-dibenzoyl-
D-ribofuranosyl-1-methanesulfonate (8.4 mmol) and 20.0 g
(180 mmol) of cytosine as described in EPO Patent
Application Publication No. 577,3 03) was slurried in
methanol (54 mL). The mixture was heated to 50°C with
stirring and adjusted to a pH of 10 by adding diethylamine
(0.3 mL, 2.9 mmol, 0.34 eq.). The stirring continued and
the mixture was heated to 55°C to 60°C for 14 hours.
To isolate the product, decolorizing charcoal
(0.17 g) and isopropyl alcohol (40 mL) were added and the
mixture was stirred at 20°C to 25°C for 1 hour. The
mixture was cooled to 0°C to 5°C, stirred for 30 minutes,
then filtered through a pad of filter aid. The filter cake
was washed with isopropyl alcohol (14 mL). The combined
filtrate was adjusted to a pH of 1.5 to 1.8 by adding
concentrated hydrochloric acid (0.95 mL). A precipitate
formed and the mixture was stirred at 0°C to 5°C for 2
hours then filtered. The solid was washed with isopropyl
alcohol (2 x 15 mL) then with acetone (1 x 15 mL). An HPLC
analysis, carried out as described in Example 1, showed the
potency of the isolated solid to be 99.4 percent. The
overall yield of the product was 57.6 weight percent (based
on mesylate).
Example 4
1-(2'-deoxy-2',2'-difluoro-D-ribofuranosyl)-4-
aminopyrimidin-2-one hydrochloride
(3-1- (2 ' -deoxy-2 ' , 2 ' -dif luoro-3', 5' -di-O-benzoyl-
D-ribofuranosyl)-4-aminopyrimidin-2-one (2.58 g, 5.48 mmol)
was slurried in methanol (56 mL). The mixture was heated
to 40°C to 45°C with stirring and the pH adjusted to 10 by
adding diethylamine (0.1 mL, 0.97 mmol, 0.2 eq.). The
stirring continued and the mixture was heated to 50°C to
60°C for 6.5 hours. The mixture was then stirred at 20-
25°C overnight (15 hours).
To isolate the product, decolorizing charcoal
(0.2 g) and isopropyl alcohol (42 mL) were added and the
mixture was stirred at room temperature for 1 hour. The
mixture was filtered through a pad of filter aid and the
filter cake was washed with isopropyl alcohol (14 mL).
Methanol was added to the combined filtrate to adjust the
total volume of solution to 112 mL. The solution was then
divided into two equal volumes. One solution was adjusted
to a pH of 1.5 and the other adjusted to a pH of 2.5 by
adding concentrated hydrochloric acid. Both solutions were
cooled to 0°C to 5°C, stirred for 2 hours, then filtered.
The solid filter cake from each solution was washed with
isopropyl alcohol (5 mL) then with acetone (5 mL). After
drying, a product yield of 89.6 weight percent from the 1.5
pH solution and 82.8 weight percent from the 2.5 pH
solution was obtained. An HPLC analysis, carried out as
described in Example 1, showed the product potency of the
material obtained at pH of 1.5 to be 99.1%; the potency of
the material obtained at pH 2.5 was 99.6%.
Example 5
1-(2'-deoxy-2',2'-difluoro-D-ribofuranosyl)-4-
aminopyrimidin-2-one hydrochloride
ß-1-(2'-deoxy-2',2'-difluoro-3',5'-di-O-benzoyl-
D-ribofuranosyl)-4-aminopyrimidin-2-one (0.24 g, 0.51 mmol)
was slurried in methanol (7 mL) containing 0.03 mL
diethylamine (0.26 mmol, 0.5 equiv.). The mixture was
heated to 50°C to 60°C with stirring for 6 hours.
To isolate the product, the mixture was cooled
to room temperature and isopropyl alcohol (7 mL) was added.
The pH of the mixture was adjusted to pH 1.5 by adding
concentrated hydrochloric acid (0.30 mL). After stirring
for 2-3 minutes, a precipitate formed. The stirring
continued for 1 hour at 0°C to 5°C; the mixture was then
filtered. The yield of the product (0.15 g) formed was 98
weight percent.
WE claim:
1. A process for preparing gemcitabine
hydrochloride which comprises:
a) deblocking ß-1-(2'-deoxy-2',2'-
difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one with a catalytic amount of an
alkylamine in the presence of methanol or ethanol in
an environment essentially free of water;
b) treating the resulting solution with
hydrochloric acid and an antisolvent; and
c) recovering the resulting solid
gemcitabine hydrochloride.
2. The process of Claim 1 wherein the
alkylamine is diethylamine.
3. The process of Claim 1 wherein the solvent
of step (a) is methanol.
4. The process of Claim 3 wherein the
antisolvent of step (b) is selected from acetone,
acetonitrile, tetrahydrofuran, propanol, butanol,
isobutanol, sec-butanol, and isopropanol.
5. The process of Claim 4 wherein the
antisolvent is isopropanol.
6. The process of Claim 5 wherein the ratio of
methanol to isopropanol is 1:1.
7. The process of Claim 3 wherein 15-25 mL of
methanol is used per gram of ß-1-(2'-deoxy-2',2'-difluoro-
3',5'-di-O-benzoyl-D-ribofuranosyl)-4-aminopyrimidin-2-one.
8. The process of Claim 7 wherein 20 mL of
methanol is used per gram of ß-1-(2'-deoxy-2', 2'-difluoro-
3',5'-di-O-benzoyl-D-ribofuranosyl)-4-aminopyrimidin-2-one.
9. The process of Claim 1 wherein the ß-1-(2'-
deoxy-2',2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4-
aminopyrimidin-2-one is generated in situ from 2-deoxy-2,2-
difluoro-3,5-dibenzoyl-D-ribofuranosyl-1-methanesulfonate
and cytosine.
10. The process of Claim 9 wherein 10-20 mL of
methanol is used per gram of 2-deoxy-2,2-difluoro-3,5-
dibenzoyl-D-ribofuranosyl-1-methanesulfonate.

This invention provides a process for preparing
gemcitabine hydrochloride which comprises deblocking β-1-
(2'-deoxy-2',2'-difluoro-3',5'-di-O-benzoyl-D-
ribofuranosyl)-4-aminopyrimidin-2-one with a catalytic
amount of an alkylamine in the presence of methanol or
ethanol in an environment essentially free of water;
treating the resulting solution with hydrochloric acid and
an antisolvent; and recovering the resulting solid
gemcitabine hydrochloride.