Optically active and racemic hydrated diacetylesters of .alpha.-glycero-phosphoryl-choline

Monohydrate 1,2-di-O-acetyl-glycero-phosphoryl-choline in optically active and racemic form, processes for their preparation and pharmaceutical compositions containing them as the active principle for treating cerebral involutions of the aged and for treating dislipidemia and hyperlipoproteinemia.

The present invention relates to optically active and racemic mixtures of 
monohydrate 1,2-di-O-acetyl-glycero-3-phosphoryl-choline, their methods of 
preparation and pharmaceutical compositions containing them for treating 
the cerebral involution syndromes of the aged, dyslipidemia, and 
hyperlipoproteinemia. This compound in the anhydrous form is a highly 
hygroscopic paste. This creates considerable problems when it is used to 
prepare pharmaceutical formulations. The Applicant has now found that the 
hydrated form of both the individual optically active isomer and of the 
raceme of 1,2-di-O-acetyl-glycero-3-phosphoryl-choline is a flowable, 
stable and non hygroscopic crystalline powder with a well defined melting 
point between 99.degree. and 101.degree. C., which products can be 
therefore used more easily than the corresponding anhydrous products, to 
obtain more stable pharmaceutical formulations. Specifically, the 
compounds of the present invention are the optically active isomers or the 
raceme of 1,2-di-O-acetyl-glycero-3-phosphoryl-choline monohydrate, whose 
structural formula can be represented in the following manner: 
##STR1## 
wherein the asterisk indicates the presence of an asymmetric carbon. 
The present invention also provides pharmaceutical compositions comprising 
a therapeutically effective amount of the compound of formula (I) suitably 
combined with excipients, vehicles and/or solvents conventionally used for 
preparing pharmaceutical formulations. The pharmaceutical compositions 
according to the present invention are particular advantageous for 
treating cerebral involutions of the aged and for treating dislipidemia 
and hyperlipoproteinemia. The pharmaceutical compositions of the present 
invention are, in particular, suitable for oral and parenteral 
administration. Object of the present invention are also processes for 
preparing the compound of formula (I). For example 
1,2-di-O-acetyl-glycero-phosphoryl-choline may be obtained according to 
the following process which comprises treating the corresponding anhydrous 
form with water until it contains from 5 to 10%, preferably 7%, by weight 
of water, followed by crystallization from an organic solvent of medium 
polarity, preferably alcohols, such as isopropylalcohol, or a mixture of 
an alcohol and a ketone, as for example absolute ethanol and acetone. The 
treatment with water in said process can be obtained (i) by treating the 
anhydrous 1,2-di-O-acetyl-glycero-phosphoryl-choline with the above 
mentioned polar solvent or (ii) by directly treating the anhydrous 
1,2-di-O-acetyl-glycero-phosphoryl-choline with one of the polar solvents 
mentioned above, containing a small quantity of water and then 
crystallizing the hydrated form from the same solvent. For preparing the 
optically active form L, the above mentioned process encompasses the use 
of L-1,2-di-O-acetyl-glycero-phosphoryl-choline, which is obtained in turn 
by acetylating L-.alpha.-glycero-phosphoryl-choline. The product may be 
obtained by the process described in EP217765, by using either deoiled 
soya or egg lecithins as the starting material, and by deacylating with 
metal alkoxides, and complexing the resulting mixture of 
L-.alpha.-glycero-phosphoryl-ethanolamine and 
L-.alpha.-glycero-phosphoryl-choline with zinc halide, decomposing the 
complexes by means of an organic base, purifying and separating 
L-.alpha.-glycero-phosphoryl-choline from 
L-.alpha.-glycero-phosphoryl-ethanolamine by means of an ion exchange 
resin. This process presents considerable disadvantages, including that it 
requires significant quantities of solvents, a long time, and it gives a 
very impure L-.alpha.-glycero-phosphoryl-choline in a low yield, because 
it is difficult to remove L-.alpha.-glycero-phosphoryl-ethanolamine by 
eluting the raw product on a chromatographic column. The Applicant has 
therefore also found new processes selective for preparing the L-form, not 
requiring the use as the starting material of 
L-.alpha.-glycero-phosphoryl-choline, derived from natural products. The 
monohydrate L-1,2-di-O-acetyl-glycero-phosphoryl-choline may be obtained 
according to a process comprising the following steps: 
a) 2-chloro-2-oxa-1,3,2-dioxaphospholane of formula (II) 
##STR2## 
is condensed with L-1,2-di-O-acetylglycerol of formula (III) 
##STR3## 
in an apolar solvent, preferably an ether, more preferably ethyl ether, 
preferably at room temperature in the presence of triethylamine, thereby 
obtaining the intermediate of formula (IV) 
##STR4## 
b) the intermediate of formula (IV) is reacted with an excess of 
trimethylamine under pressure, preferably at 1.5 atmospheres and at 
50.degree. C., thereby obtaining anhydrous raw 
L-1,2-di-O-acetylglycero-phosphoryl-choline; 
c') the raw product obtained in (b) is then dissolved in an alcoholic 
solvent, preferably methanol, filtered on carbon and concentrated to a 
small volume under vacuum, afterwards it is eluted on a cationic exchange 
resin, preferably an Amberlite IR 120 (H.sup.+) type resin, by using as 
the eluent a hydroalcoholic solution, preferably aqueous methanol; the 
eluted product obtained is treated according to one of the following 
operating conditions: (i) it is concentrated under vacuum until containing 
a water amount ranging from 5 to 10%, preferably 7% by weight, and then 
crystallized from a mixture of a ketone and an alcohol, preferably acetone 
and absolute ethanol in a volumetric ratio 10:1; or (ii) it is completely 
evaporated until obtaining a residue, which is directly crystallized from 
an alcoholic solvent, preferably methanol, containing 3% by weight of 
water; or 
c") the raw product obtained in step (b) is separated from the reaction 
mixture by elution on a chromatographic column, by using as the eluent the 
following solvents or mixture of solvents in sequence: chloroform, 
choloroform-methanol in the following volumetric ratio: 100:2, 100:7, 4:1, 
1:1, the product thus purified is treated with carbon in a polar solvent, 
preferably methanol, in order to remove any color, and the solvent is 
completely removed, thereby obtaining pure anhydrous 
L-.alpha.-diacetylglycerophosphorylcholine, which is converted into the 
monohydrate form according to one of the following alternative operating 
conditions: (i') by treating the anhydrous form with from 5 to 10%, 
preferably 7% by weight of water, and crystallizing the hydrated form from 
a polar solvent, preferably methanol, or a mixture of polar solvents; or 
(ii') treating directly the purified anhydrous form with a polar solvent, 
preferably methanol, containing a small amount, preferably 3%, of water, 
and crystallizing the monohydrate form from the same solvent. The 
crystallization of the hydrated form described in step (c') or (c") 
preferably occurs at temperatures lower than 0.degree. C., preferably 
-20.degree. C. A further process for preparing monohydrated 
L-1,2-di-O-acetyl-glycero-phosphoryl-choline comprises the following 
steps: 
a) reacting the choline phosphate salt of formula (V): 
##STR5## 
wherein A is a cation selected from: Li.sup.+, Na.sup.+, K.sup.+, or 
N.sup.(+) (CH.sub.3).sub.4 with the optically active form D of the 
compound of formula (VI) 
##STR6## 
wherein X is selected from Cl, Br, I, and O-tosyl, Y is --COCH.sub.3, or 
the Y groups together form a 
##STR7## 
groups. in polar solvents, preferably alcoholic solvents such as methanol, 
absolute ethanol, apolar solvents selected from ethereal solvents such as 
diglyme, dioxane, tetrahydrofuran, and other apolar solvents such as 
methylcellosolve acetate, or a mixture of both polar and apolar solvents 
optionally in the presence of alkylating adjuvants and solubilizers 
specific for the cations such as crown ethers; 
thereby obtaining the intermediate of formula (VII): 
##STR8## 
b) in the case where the Y groups form together a 
##STR9## 
group hydrolizing the acetalic group of the compound (VII) in a mineral 
acid such as a hydrochloric acid aqueous solution at pH 2,3, or organic 
acid such as glacial acetic acid thereby obtaining 
L-.alpha.-glycero-phosphoryl-choline; 
c) reacting L-.alpha.-glycero-phosphoryl-choline from step (b) with a 
mixture of acetic anhydride and pyridine respectively in molar ratio 3:2 
in the presence of glacial acetic acid as the solvent, thereby obtaining 
anhydrous L-1,2-di-O-acetyl-glycero-phosphoryl-choline; 
d) eluting the anhydrous L-1,2-di-O-acetyl-glycero-phosphoryl-choline, 
coming from step (a) (in case the substituents Y in formula (VII) are 
--COCH.sub.3), or coming from step (c) on a cationic exchange resin, 
preferably an Amberlite type resin IR 120(H.sup.+) using as the eluent an 
aqueous alcoholic solvent, preferably methanol, then concentrating the 
eluted product until it contains an amount of water ranging from 2 to 10% 
by weight, more preferably from 3 to 7%. 
In particular, in the process according to the present invention, when the 
reactant of formula (V) has A=Na, the reactant of formula (VI) has X=Br 
and the substituents Y form together the group 
##STR10## 
the reaction described in step (a) is carried out in methanol at the 
reflux temperature, and the molar ratio of the two reactants is 
respectively of 2:1; 
step (b) is carried out in the presence of hydrochloric aqueous solution 
having a pH 2.3. 
When in the above mentioned process the reactant of formula (V) having 
A=Li.sup.+, K.sup.+, N.sup.(+) (CH.sub.3).sub.4, and the reactant of 
formula (VI) in which the substituents Y forming 
##STR11## 
and X=O-tosyl, are used, step (a) is carried out in absolute ethanol, in 
the presence of crown ethers specific for the cation at temperatures 
ranging from 50.degree. to 70.degree. C.; 
step (b) is carried out in the presence of hydrochloric acid 1N, and the 
reaction temperature is .ltoreq.40.degree. C. 
When in the above mentioned process the reactant of formula (VI) is used 
having Y=--COCH.sub.3 and X=Br or --O-tosyl, step (a) is carried out in 
the presence of a mixture of THF and isopropanol. 
The intermediate of formula (VI) having X=Br, and the substituents Y 
forming together 
##STR12## 
is a product commercially available with the name D-3-bromo-solketal. The 
compound of formula (VI) having X=O-tosyl and the substituents Y forming 
together 
##STR13## 
is obtained by reacting the 1,2 isopropylidene glycerol of formula (VIII): 
##STR14## 
with para-toluen-sulphonyl-chloride in the presence of a hydrochloric acid 
acceptor, preferably pyridine, at 0.degree. C. 
The choline phosphate salt of formula (V) is obtained by reacting the 
choline phosphate acid with the corresponding hydroxide. The following 
examples are reported for illustrative, but not limitative purposes.

EXAMPLE 1 
180 ml of acetone and 20 ml of ethanol are added to 51 g of (+) 
1,2-di-O-acetyl-glycero-3-phosphoryl-choline which has previously been 
made to absorb water in a quantity of 7% by weight. The mixture is stirred 
under reflux until a complete solution has formed. The solution is left to 
crystallize at ambient temperature, and the precipitate obtained is 
filtered off and dried until it reaches constant weight. Forty-two grams 
of a white solid are obtained having the following characterics: 
______________________________________ 
Elemental analysis: 
C H N P 
______________________________________ 
calculated (%): 
40.10 7.29 3.90 8.62 
found (%): 39.9 7.2 4.0 8.7 
M.P. 99-100.degree. C. 
H.sub.2 O K.F. 5.8% 
[.alpha.].sup.20 s.s 
+7.7 (c = 10, water) 
______________________________________ 
EXAMPLE 2 
95 ml of isopropyl alcohol are added to 10 g of (+) 
1,2-di-O-acetyl-glycero-3-phosphoryl-choline which has previously been 
made to absorb water in a quantity of 7% by weight. The mixture is stirred 
under reflux until a complete solution has formed, which is then allowed 
to crystallize at ambient temperature. 
After filtration and drying, 8.2 g of a white solid are obtained having the 
following characteristics: 
______________________________________ 
M.P. 100-101.degree. C. 
H.sub.2 O K.F. 7.2% 
[.alpha.].sup.20 s.s 
+7.6 (c = 10, water) 
______________________________________ 
EXAMPLE 3 
A solution consisting of 300 ml of anhydrous ether containing 46.885 g of 
L-1,2-diacetyl-glycerol (265 mmoles) and 26.7 g of triethylamine is added 
slowly under an anhydrous nitrogen stream to 950 ml of ether containing 
265 mmoles of 2-chloro-2-oxa-1,3,2-dioxophospholan at 22.degree. C. After 
two hours at the same temperature the triethylammonium chloride which 
forms is filtered off under nitrogen and washed with ether. The filtrate 
solution is evaporated and the residue (92% yield with 90% minimum purity) 
is left under vacuum (0.1 mm Hg) for 1 hour. The residue is cooled to 
0.degree. C. and 59.5 g of trimethylamine are added. These are reacted at 
about 50.degree. C. under a pressure of 1.5 atmospheres for 3 hours. The 
minimum quantity of chloroform is added to the product, and the solution 
obtained is transferred to a 4 kg column of silicic acid in chloroform. 
The column is eluted with chloroform (15 liters), then with the following 
quantities of chloroform-methanol mixtures in the stated proportions: 15 
liters of 100:2, 15 liters of 100:7, 15 liters of 7:1, 15 liters of 4:1 
and 30 liters of 1:1. The latter mixture is passed until all the desired 
product has been eluted. The solvent is evaporated under reduced pressure 
and the residue is taken up in 500 ml of methanol and treated with Darco 
G-60 activated carbon. After filtration and evaporation, the colourless 
product is dissolved in 100 ml of methanol containing 3% of water, and 
allowed to crystallize at -20.degree. C. 
41.14 g (57% yield) of L-1,2-diacetyl-glycero-phosphoryl-choline 
monohydrate are obtained. 
EXAMPLE 4 
A solution consisting of 300 ml of anhydrous ether containing 46.885 g of 
L-1,2-diacetyl-glycerol (265 mmoles) and 26.7 g of triethylamine is added 
slowly under an anhydrous nitrogen stream to 950 ml of ether containing 
265 mmoles of 2-chloro-2-oxa-1,3,2-dioxophospholan at 22.degree. C. After 
two hours at the same temperature the triethylammonium chloride which 
forms is filtered off under nitrogen and washed with ether. The filtrate 
solution is evaporated and the residue (92% yield with 90% minimum purity) 
is left under vacuum (0.1 mm Hg) for 1 hour. The residue is cooled to 
0.degree. C. and 59.5 g of trimethylamine are added. These are reacted at 
about 50.degree. C. under a pressure of 1.5 atmospheres for 3 hours. The 
mixture is concentrated by the aid of vacuum and the residue obtained is 
dissolved in methanol. The solution obtained is filtered on carbon, 
concentrated, and eluted on Amberlite resin IR 120(H.sup.+) by using as 
the eluent aqueous methanol. The fraction containing the product obtained 
is concentrated until its content of water is comprised between 5 and 10% 
by weight, then crystallized from a mixture of acetone:ethanol in 
volumetric ratio 10:1. 
EXAMPLE 5 
20.6 g (100 mmoles) of choline phosphate sodium salt are dissolved in 100 
ml of methanol; 9.75 g (50 mmoles) of D-3-bromo-solketal were added to the 
solution. The mixture was heated to reflux temperature for 3 hours under 
stirring. Finally the mixture was cooled and the solvent was removed with 
the aid of vacuum. The residue thus obtained was dissolved in 15 ml of 
water and the pH was brought to 2.3 by means of hydrochloric acid. The 
mixture was heated up to two hours at 50.degree. C. until complete 
hydrolysis of the protecting group consisting of acetone. The solution was 
eluted on a basic resin in the form of a formiate salt. The column was 
eluted with a gradient of concentration of formic acid and the fraction 
containing L-.alpha.-glycero-phosphoryl-choline was recovered. The solvent 
was evaporated with the aid of vacuum until obtaining a solid residue. At 
the end the residue was dissolved in acetone, filtered and the acetone was 
evaporated again; obtained were 9 g of 
L-.alpha.-glycero-phosphoryl-choline (yield 70%) with a minimum title: 
98%. The product thus obtained was treated with a mixture of acetic 
anhydride:pyridine in molar ratio 3:2 respectively using glacial acetic 
acid as the solvent. The reaction mixture was then heated with water and 
eluted on Amberlite resin IR 120(H.sup.+) using as the eluent water or 
aqueous alcohol. The product eluted was concentrated under vacuum at 
T.ltoreq.50%. The product obtained was then treated with water until 
acetic acid was completely removed. A product was obtained containing 
water from 5 to 10% by weight, preferably 7%. The product obtained was 
finally crystallized from a mixture acetone:ethanol in volumetric ratio 
10:1. 
EXAMPLE 6 
20.6 g (100 mmoles) of sodium choline phosphate salt were suspended in 200 
ml of a mixture of THF and isopropanol in volumetric ratio 1:1, 11.95 g of 
D-bromo 1,2-di-O-acetyl glycerol were added and 10 g of 15-crown-5. The 
reaction mixture was heated to 50.degree. C. under stirring for 6 hours, 
following the reaction course by TLC. At the end, the solvent was 
evaporated and the residue obtained was dissolved in methanol and eluted 
on Amberlite resin IR 120 (H.sup.+). The eluate was evaporated until 
containing water in an amount of 7% and then crystallized from 
acetone:ethanol 10:1. 
EXAMPLE 7 
22.2 g (100 mmoles) of potassium choline phosphate salt were suspended in 
200 mg of THF 10 g of 18-crown-6 and the mixture obtained was stirred for 
15', thereafter 12 g (50 mmoles) of D-bromo 1,2-diacetyl-glycerol were 
added. The mixture was heated to 50.degree. C. for 6 hours while 
maintaining a vigorous stirring and following the reaction course by TLC. 
At the end the solvent was evaporated and the residue was dissolved in a 
mixture water:methanol 1:1. The solution obtained was eluted on an 
Amberlite resin IR 120(H.sup.+) by using as the eluent methanol:water 1:1. 
The elution fraction containing the desired product was collected. The 
product obtained was neutralized at pH 7 and the solvent was evaporated 
with the aid of vacuum. The residue obtained was dissolved in absolute 
ethanol and filtered on carbon and the solvent was evaporated by the aid 
of vacuum. The residue obtained was crystallized from methanol having a 
content of water of 3.5% by weight. 
EXAMPLE 8 
Stage a-(+)-1,2-isopropylidene glycerol tosylate (III) 
A solution of 6.8 g of (+)-1,2-isopropylidene-glycerol in 10 ml of pyridine 
cooled to 0.degree. C. was added drop by drop to a solution of 12.95 g of 
p-toluene suphonyl chloride in 15 ml of the same solvent. The mixture was 
then left for 18 hours at 0.degree. C. and constantly agitated, and then 
poured into water to dissolve the salt that had formed (pyridine 
chloride). The raw reaction product was extracted by using chloroform. The 
products extracted were washed in a diluted solution of hydrochloric acid, 
then with sodium bicarbonate, and lastly in water. The solvent was dried 
on anhydrous sodium sulphate, and then evaporated. 
Stage b-Preparation of the intermediate (IV) in which A is tetramethyl 
ammonia 
18.295 g of choline phosphate acid (IV) were dissolved in 100 ml of 
methanol containing 18.630 g of tetramethyl ammonium hydrate. The solvent 
was removed with the aid of vacuum at 40.degree. C. 
Stage c-L-.alpha.-1,2-isopropylidene-glyceryl-phosphoryl-choline (VII) 
34.853 g of tetramethyl ammonium salt of phosphoryl-choline were dissolved 
in 300 ml of absolute alcohol. 25.43 g of (+)-1,2-isopropylidene glycerol 
tosylate were then added. The mixture was then heated to reflux 
temperature for about 18 hours. The salt was filtered and the solvent 
removed with the aid of vacuum. 
Stage d-L-.alpha.-glyceryl-phosphoryl-choline (I) 
The product obtained in the previous stage was dissolved in 100 ml of 95% 
ethanol and hydrolysed at 40.degree. C. with 5 ml of hydrochloric acid 1N 
for one hour. The solvent was evaporated with the aid of vacuum. The 
residue was dissolved in 100 ml of distilled water, and the solution was 
then passed through an Amberlite column (IR-120-H-shape). The column was 
washed with 200 ml of water, the combined eluates were evaporated with the 
aid of a vacuum at 35.degree. to 40.degree. C. in the presence of 
phosphoric anhydride. 16.81 g of 99% pure 
L-.alpha.-glyceryl-phosphoryl-choline were obtained with a yield of 75%. 
Stage (e) L-.alpha.-diacetylglycerophosphorylcholine monohydrate 
The product thus obtained was treated with a mixture of acetic 
anhydride:pyridine in molar ratio 3:2 respectively using glacial acetic 
acid as the solvent. The reaction mixture is then heated with water and 
eluted on Amberlite resin IR 120(H.sup.+) using as the eluent water or 
aqueous alcohol. The product eluted was concentrated under vacuum at 
T.ltoreq.50%. The product obtained was then treated with water until 
acetic acid was completely removed. A product was obtained containing 
water from 5 to 10%, preferably 7%, by weight. The product obtained was 
finally crystallized from a mixture of acetone:ethanol in volumetric ratio 
10:1. 
EXAMPLE 9 
Following the same operating conditions described in example 6 monohydrate 
1,2-di-O-acetyl-glycero-phosphoryl-choline was obtained by reacting D 
1,2-diacetyl-3-glycerol with tetramethylammonium-choline phosphate salt.