DERIVES ACYLES DE LA CYTIDINE-DIPHOSPHATE-CHOLINE, PROCEDE DE PREPARATION ET APPLICATION THERAPEUTIQUE

ACYLATED DERIVATIVES OF CYTIDINE-DIPHOSPHATE-CHOLINE, PROCESS FOR THEIR PREPARATION AND THEIR THERAPEUTIC USE

Abrégé anglais

ABSTRACT OF THE DISCLOSURE
Derivatives of cytidine-diphosphate-choline of the
following formula:
<IMG>
in which R represents an acyl radical chosen from the group
comprised of saturated and unsaturated, linear or branched,
monocarboxylic fatty acids having from 3-7 carbon atoms, and R1
represents H or an acyl radical identical to R, show a thera-
peutic activity analogous to that of cytidine-dlphosphate-choline
and are suitable for oral administration in that they lead to a
gradual release of the active principle in the gastrointestinal
tract. The compounds that respond to these requirements are
mono-, di- and tri-acyl derivatives of CDP-choline that can be
synthetized by acylation of CDP-choline with carboxylic acids
using N,N'-carbonyldiimidazole.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A derivative of cytidine diphosphate choline having
the formula:
<IMG>
in which R represents an acyl radical selected from the group of
saturated or unsaturated, linear or branched monocarboxylic fatty
acids having from 3 to 7 carbon atoms, and R1 represents H, or an
acyl radical indentical to R, or R1 is acyl radical and R is H.
2. A derivative of the formula
<IMG>
in which R1 is an acyl radical of a monocarboxylic fatty acid
having 3 to 7 carbon atoms.
3. A derivative of the formula
12

<IMG>
where R is an acyl radical of a monocarboxylic fatty acid having
3 to 7 carbon atoms.
4. A process for the preparation of the O-monoacylated
derivatives of cytidine diphosphate choline of the formula
<IMG>
where R is an acyl radical of a monocarboxylic fatty acid having
3 to 7 carbon atoms, which comprises reacting cytidine diphos-
phate choline with the acylimidazole (imidazoilde) of the desired
carboxylic acid in an aqueous mixture of a poorly solvating
compound.
5. A process according to claim 4, in which the car-
boxylic acid is a saturated or unsaturated, linear or branched
monocarboxylic fatty acid containing 3 to 7 carbon atoms.
6. A process according to claim 4, in which said
poorly solvating compound is selected among tetrahydrofuran,
acetone and acetonitrile.
13

7. A process according to claim 6, in which the reac-
tion medium is a mixture 1:4 v/v of water and the poorly solvat-
ing compound.
8. A process according to claim 4, in which the molar
ratio between imidazolide and cytidine diphosphate choline is
2:1.
9. A process for preparation of N4-monocylated
derivatives of the formula
<IMG>
in which R1 is an acyl radical of a monocarboxylic fatty acid
having 3 to 7 carbon atoms, in which the tetrabutylammonium salt
of cytidine diphosphate choline, in a homogeneous solution of an
aprotic dipolar solvent and In the presence of an acylation cata-
lyst, is reacted with the imidozoilde of the carboxylic acid to
be introduced at N4.
10. A process according to claim 9, in which said sol-
vent is selected among formamide, pyridine and dimethylformamide.
11. A process according to claim 9, in which said acy-
lation catalyst is 4-N,N-dimethylaminopyridine.
12. A process according to claim 9, in which the reac-
tion is carried out with an excess of imidazolide.
13. A process according to claim 9, in which the reac-
14

tion is carried out at 50°C and for 36 hours.
14. A process for the preparation of N4-acyl-2',3'-di-
0-acyl derivatives of cytidine diphosphate choline of formula I
<IMG>
in which the reaction is carried out according to claim 10, with
an excess of imidazolide, for a time of 96 hours and at a temper-
ature of 50°C.
15. A process for the preparation of a derivative of
cytidine diphosphate choline having the formula
<IMG>
in which R represents an acyl radical selected from the group of
saturated or unsaturated, linear or branched, monocarboxylic
fatty acids having from 3 to 7 carbon atoms, and R1 represents
H, or an acyl radical identical to R, or R1 is acyl radical and R
is H, which comprises (a) reacting cytidine diphosphate choline
with the acylimidazole (imidazolide) of the desired carboxylic
acid in an aqueous mixture of a poorly solvating compound, (b)
reacting in the tetrabutyl ammonia salt of cytidine diphosphate
choline, in a homogeneous solution of an aprotic dipolar solvent
and in the presence of an acylation catalyst with the imidazolide
of the carboxylic acid to be introduced at N4, or (c) reacting in

the tetrabutyl ammonia salt of cytidine diphosphate choline in a
homogeneous solution of an aprotic dipolar solvent and in the
presence of an acylation catalyst with the imidazolide of the
carboxylic acid to be introduced at N4 with an excess of imida-
zolide, for a time of 96 hours and at a temperature of 50°C.
16. Pharmaceutical composition characterized by con-
taining as the active ingredient, a derivative of
<IMG>
together with conventional excipients and carriers.
17. Pharmacological composition according to claim 16,
in the form suitable for oral administration.
18. Pharmaceutical composition according to claim 16 or
17, characterized by containing 200 to 1500 mg of the active
principle, as a unitary dosage.
16

Description

1S7
The present invention relates to acylated ~erivati.ves o~ cyti.dine-
di.phospha~:e-choL:i.n~, proc~ss for ~hei.r prepa~ati.on and ~hei.r
therapeuti.c use.
This inven-tion provides de~ivati.~es of cytidine-di.phosphate-
choline havi.n~ the general ~ormula:
lHRl
~ ~ N ~ OH ~ ~ CH3 (I)
~ ~ H2_o_p_o_l_OC~2CH2N \ 3
; Po R
in which R represents an acyl radical chosen from the group comprised of
monocarboxylic saturated and unsaturated, linear or branched, fatty acids having from
3 to 7 carbon atoms, and Rl represents H or an acyl radical identical to R.
Cytidine-diphosphate-choline, re ~erred to from now on as the abbreviation CDP-
choline, represents the active form of choline and is a key intermediate in the
biosynthesis of complex lipids. The importance of the cytidinic nucleotides in the
formation of diester bonds in phospholipid molecules has been broadly documented in
fact.
As shown in the literature, the lecithins and sphingomyelins are formed by means of a
reaction catalysed by microsomal enzymes, in which the CDP-choline donates the
phosphorylcholinic fragment to the D-d, "~3 -diglyceride or to the N-acylsphingosine
respectively.
Furthermore one should remember that CDP-choline acts as a P-choline donor to 1-alkyl, l'-enyl-2-acyl-sn-glycerol, forming the plasmalogens.
CDP-choline is biosynthetized from P-choline and CTP, by means of the enzyme
choline phosphate-cytidine transferase. This enzymic activity has been found in the
particle free fraction of the cytoplasm and in the microsomal fraction. It is
appropriate to underline, with reference to this, that the formation of CDP-choline
~, ~,};.'

3L2~8~
- 2 --
represents the slowest step and therefore the limitative one in the whole metabolic
pathway. The cellular concentrations of this metabolite therefore have a critical
importance in the regulation of the biosynthesis of phospholipids.
As a consequence of its biochemical roles, CDP-choline finds a use and pharmalogical
indication prevailingly in a series of alterations in the central nervous system. In this
organ, in fact, the structural and functional integrity of phospholipid membranes is
particularly critical. The form of adrninistration used up to now has been parenteral.
The pharmacological activity of the molecule has been demonstrated in various
diseases such as the sequences of cerebral apoplexy, different types o F cerebral
ischaemia, Parkinson's disease, cranial fractures and their sequences.
This invention concerns the use of the acylated analogues of CDP-choline, as
previously given in formula 1, in therapy.
They are obtained by acylation at positions N4 and/or 2', 3' of the molecule with the
already definecl carboxylic acids, in particular and preferably monocarboxylic acids,
naturally present in mammals, of different chain lenghts, linear or branched, saturated
or unsaturated.
Esterification concerns some or all of the reactive positions on the molecule
mentioned previously.
The acylated derivatives of CDP-choline are sufficiently stable in acid or alkaline
environments so as not to be chemically broken down by the variationa in pH
associated with the journey through the gastro-intestinal tract.
The presence of strongly hydrophobic chains significantly modifies the molecularmorphology, impeding, both at the gastric level and at the intestinal level, hydrolysis
of the drug at the pyrophosphoric bond. This i5 possible only after removal of the
acylic groups in sequence by the action of the gastric and intestinal acyl esterases
and/or amidases and peptidases.
Drug absorption is conditioned by the hydrolysis of the acyl groups and there-Fore
acylation of CDP-choline conditions the absorption of the cholinic and cytosiniccomponents of the drug.
The acyl residues that are released can be considered as metabolites normally present
in the organism with absolutely negligible toxicity levels.

r~9~17
-- 3 --
The N4, 2', 3'-triacyl derivatives of CDP-choline administersd orslly act as retard
forms of CDP-choline. The consequence of thi~ process of 810w release of CDP-choline
in the organism is a more uniform and gradual distribution of the active principle.
As already indicated, according to this invention the CDP-choline derivatives have, in
oral form, indications analogous to those for CDP-choline.
More simply9 according to the invention, oral use of the CDP-choline derivatives is
foreseen in table form, each containing from 20û to 1500 mg of the active principle, to
be administered 2-3 times a day in the following illnesses:
- arteriosclerosis especially cerebral.
- short and long term treatment of cerebrovascular accidents.
- short and long term treatment of the consequences of a stroka.
- treatment of Parkinson's and Parkinson-like syndrornes, in particular in the arteriosclerotic form.
- anti-depression treatment.
- treatment of cerebral traumatic coma.
- prevention and therapy of hyaline membrane disease (IRD5).
- therapy in acute and chronic hepatitis (viral hepatitis etc.).
- therapy and prevention of fatty liver in alcoholics.
- coadjuvant therapy in liver cirrhosis.
This inventions also concerns the preparation of the derivatives of CDP-choline of
formula I. More specifically, the processes for the preparation of the derivatives (I)
form another object of this invention, bound to the type of functionalisation oresterification desired. Therefore:
c~ e
~L ~ 1) In~l-monoacylation of CDP~choline at the &mi~e group of the carbon
~the ~i~e nucleus, the process, according to this invention, is characteri~ed
by the fact that the tetrabutylammonium salt of CDP-choline is reacted with an
excess of imidazolide of the desired carboxylic acid in an aprotic dipolar solvent (DMF,
formamide or pyridine, of which DMF represents the best one), at a temperature of
5ûC for 36 hours and in the presence of an acylation catalyst such as 4-(N,N-
dimethylamino) pyridine.
The imidazolide of the carboxylic acid is in turn prepared from the carboxylic acid by

f37
-- 4 -
reaction with N,N'-carbonyldiimidazole in a solution of anhydrous DMF.
2) In the realisation of N -acyl-2'-,3'-di-0-acyl-CDP-choline one can use
the same method described in section 1 above, obviously using an excess of imidazolide
and, as a necessity, prolonging the time of the reaction to 96 hours7 at a reaction
temperature of 50C.
3) Acylation with an aqueous mixture of poorly solvating cornpounds
likeTHF, acetone, acetonitrile, the acylation takes place exclusively at the hydroxyl
groups of ribose.
The best yields are obtained using mixtures H2O/THF or H2O/acetonitrile (1: 4 v/v) as
solvents. The acylation reaction never runs to completion and the best yields are
obtained using a molar ratio of 2:1 between imidazolide (as prepared separately in
anhydrous THF) and CDP-choline.
Under these conditions monoacylation of CDP-choline takes exclusively place,
indistinclty at one of the free hydroxyl groups at 2' or 3' of the ribose, giving 2'(3')-0-
acyl-CDP-choline (Formula ll).
Il O O
/~ N CH20-P-O-l-OCH2cH2cH2N( 3 3
O O
H, RCO
( I I )
The chromatographic purification of the reaction mixtur0 on DOWEX AG 1:8 does not
allow for resolution of the two isomers of CDP-choline acylated at 2' and 3' of the
ribose respectively.
Therefor all the spectroscopic characterizations were carried out on the mixture of
the two isomers that are stable in aqueous solution at pH 6.5 or lyophilized.
The identiFication of all the monoacyl and triacyl derivatives of CDP-choline is

- 5 -
essentially based on the H-NMR and lJV spectroscopic data.
The H-NMR data concisely reported in Table 1 refer to the derivatives of valeric acid
and are consistent with the structural assignations for each derivative (see Table 1).

j9t9B'7
-- 6 ---
_ _ .__ _ - __
~ ~:;" ~ ~
V ~ ... ~ __ . I, Q~ .
3 r
g ~ _ r _ __ ~ r h v
tJ I ~ ~ . ~ u7
a~ 3 ~ ~: ~ >
. ~, 3 > O Q~
_ ., ~ ,tF~ C --I
r ~ ~ ~1 ~ ~n Q ~ 11
r~ _ ,~ ~ I rJ ~
;; ~ ra a 0 ~ o ~j t o L Q ,a) W
t 6~ s I u _ ~r -- r~ tD O n)
I ~ I ~ ~C ~ 0l ~
~ a I ~ I ~ Q ~ r~
.. . æ N _ ~ 0 0
_ ~ _ _ ~ I c ~C a
_ ~ ~ r s ~ c C~ ~ J
.^ ~ .~r, _ S~ ~ _ .
c 7--------
tO / ~ _ t~ ~ ~ D
rn J ~

~2.,599~37
- 7 --
The nuclear magnetic resonance specturm in the case of 2'(3')-0-valeryl CDP-choline
shows that only one acyl group has been introduced and is, localised at the level of the
2' or 3' hydroxyl groups of ribose. One can in fact see a shift to lower fields on the part
of the complex signal to 4,0-4,5, attributable to the H's on the hydroxylic group
carrying carbons, whereas the proton's signals on the carbons 5 and 6 of the cytosinic
nucleus are found at the same values of chemical shiFt that they have in CDP-choline
thus excluding the possibility of an acylation oF the aminic group at the carbon 4 of the
aromatic system.
In the spectrum of N -valeryl CDP-choline, however, the presence of a single unit of
valeric acid per molecule is observed, specifically localised at the nitrogen in position
4 of the cytosinic system. In Fact while the characteristic shiFt to lower fields of
protons 5 and 6 of the chromophore is revealed, the ribose protons show resonance in
the same fields as CDP-choline.
Finally, in the N -valeryl-2', 3'-di-0-valeryl-CDP-choline spectrum, the integration of
the protons of the acylating group shows the presence of three unit of valeric acid per
molecule of CDP-choline. The total acylation of the molecule leads to a shift to low
fields both of the aromatic protons and of the hydrogens of the ribose.
The examples that follow illustrate, without in any way limiting, the processes
according to this invention.
EXAMPLE 1
Synthesis of 3'(2')-0-valeryl-CDP-choline
The chemical synthesis of CDP-choline acylated with valeric acid at the hydroxyl 2' or
3' of ribose, is carried out by condensation of CDP-choline with the imidazolide of
valeric acid.
Ths latter, which is obtained by a reaction between valeric acid and N,N'carbonyldii-
mida~ole in a rigorously anhydrous environment, permits the acylation to be
selectively carried out, in mild conditions and in an aqueous-organic environment, on
one of the two ribose hydroxyl groups.
In a standard procedure, the preparation of the valeric imidazolide is carried out by
reacting 1,3 9 (8 mmoles) of carbonyldiimidazole with 500 mg (4.9 mmoles3 of valeric

g~7
-- 8 --
acid, in 5 mls of tetrahydrofurane, anhydrified on molecular sieves and kept under
anhydrous nitrogen. The reaction, carried out in nitrogen in very anhydrous conditions,
is completed in about 10 minutes.
Like results are obtained if, while keeping the volume of solvent unaltered, double or
triple quantities of the reactants are used or if the THF is substituted with acetonitrile
or anhydrous acetone.
The acylation of CDP-choline is carried out in an aqueous-organic environment bymixing under stirring at room temperature 20 ml of a solution of 250 mM CDP-choline
with 5 ml of the organic solution of imidazolide at different concentrations (1.6, 3.2,
4.8 M). In every case the reaction rnixture initially appears like a stable white coloured
emulsion; it becomes clear as the acylation of CDP-choline takes place. In about 4
hours the reaction reaches maximum yield. Working at 70DC the course of the reaction
is much quicker (2h) but the yield of acylated product i8 not significantly modified. The
3'(2')-û-valeryl-CDP-choline can be isolated by evaporation of the solvent in a vacuum
with a stream of nitrogen. The remaining acqueous solution is brought to pH 8.5 with
0.5N KOH and is then adsorbed onto a DOWEX lX8 (forrniate) column.
The elution is carried out first with H2O and then with a linear gradient of formic acid
from û.0û to 0.02 M.
The 3'(2')-0-valeryl-CDP-choline has a very similar UV spectrum to that of CDP-
choline, showing a maximum of 280 nm (=12.8 x 10 ) in water.
The information that one can deduce from the analysis of the NMR spectrum of C
fully confirms the structural identity of the molecule, excluding once again that the
aminic group on the aromatic system has been involved in the acylation reaction.In fact the cytosinic carbon signals, as in CDP-choline, have chemical shifts of160.49 d (C2) 168.98 ~ (C4), 99.29 ~ (C5), and 144.28 ~ (C6) respectively. The
ribose carbons however give rise to a good ten signals, five for each of the 2 isomers
respectively acylated in positions 2' or 3', while the cholinic carbons are found at the
same chemical shifts as in CDP-choline. On the other hand, at high fields one finds the
aliphatic carbons of the valeric acid.
Structural analogues of CDP-choline, acylated at 2' or 3' with fatty acis from C3 to
C7, saturated or unsaturated, linear or branched, can be obtained according to the

~L2~ 19fl97
- 9
same reaction scheme given in example 1, substituting the valeric acid with the
desired acid.
EXAMPLE 2
Synthesis_f N -valery!-CDP-choline
0.2 mmoles of tetrabutylammonium hydroxide (1 ml 0.2 M) are added to 100 mg of
CDP-choline, dissolved in 5 ml of H2O, and then one proceeds to the Iyophilisation of
the sample. The tetrabutylammonium salt (TEBA) of CDP-choline is then dissolved in 4
ml of anhydrous DMF containing 20 mg of 4-dimethylaminopyridine.
2 mmoles of the imidazolide of valeric acid, ohtained by reacting 204 mg of aciddissolved in 1 ml of anhydrous DMF with 324 mg of N,N'-carbonyl-diimidazole, areadded to the resulting solution.
The reaction, stirred for 36 hours at 50C, is interrupted by removing the solvent in a
vacuum and the oleaginous residue obtained is triturated three tirnes with ethylacetate.
The reaction mixture is purified by chromatography on silica, by elution with H2O in
CH3OH in a linear gradient from û to 20% v/v- Th N -valeryl-CDP-choline is obtained
ith a yield of 50-60% and N -valeryl-2',3'-di-0-valeryl-CDP-choline, N -acylatedwith fatty acids from C3 to C7, saturated or unsaturated, linear or branched, can be
obtained according to the method given in this example, substituting the valeric acid
with the acid desired.
EXAMPLE 3
Synthesis of N -valeryl-2',3'-di-0-valerYI-CDP-choline
One proceeds as described in the former example up to the preparation of the solution
of the TE13A salt of CDP-choline in DMF, containing 4-(N,N-dimethylamine) pyridine.
At time nought and a~ter 48 hours, 2 mmoles of the imidazolide of valeric acid,
obtained by reacting 204 mg of the acid dissolved in 1 ml of anhydrous DMF with 324
mg of N,N'-carbonyl-diimidazole, are added to this solution. The reaction mixture is
stirred for 96 hours at 50C, and the reaction is interrupted by removing the solvent
in a vacuum and triturating the oleaginous residue obtained three times with ethyl
acetate.
The triacylate (105 mg) is isolated pure by chromatography on a silica column by

~2~S9~ 37
- 10 -
elution with H20 in CH30H in a linear gradient from nought to 20% v/v.
Structural analogues of CDP-choline, triacylated with fatty acids from C3 to C7,saturated or unsaturated, linear or branched, can be obtained according to the same
method given in example 3, substituting the valeric acid with the acid desired.
The pharmacokinetic characteristics of N -valeryl-2',3'-di-0-valeryl-CDP-choline have
been defined in rats by oral administration of the product with a double label of H at
5' on the cytosinic nucleus and C on the cholinic methyls. The required data refer to
the distribution of radioactivity in the animal and also to structural identity of the
radioactive molecular species present in some of the more significant biologicalsamples (liver, brain, faeces, gastric and intestinal contents).
A precise analysis of the role played by the functionalisation in the absorption process
and metabolism of the triacylated drug has imposed a comparison of the obtained data
with those of a control experiment, in which rats were given orally equimolecular
doses of (5- H; methyl- C) CDP-choline. The caracterization of the labelled
metabolites present in the liver, brain, faeces, gastric and intestinal contents of the
animals treated orally with pharmacological doses (20 /umoles/kg) of (5- H; methyl-
C)-CDP-choline or of N -valeryl-2-2',3'-di-0-valeryl-(5- H; methyl- C)-CDP-choli-
ne leads to their preventive extraction from the tissues.
The scheme of extraction consents the recovery of three different classes of labelled
metabolites:
1) Hydrosoluble metabolites, subsequently analysed by HPLC.
2) Metabolites with high molecular weights, proteins and nucleic acids,
recovered by thermal denaturation in a hydroalcoholic environment.
3) Lipids, subsequently purified by chromatography on silica.
The experimental results show that N -valeryl-2'a3'-di-0-valeryl CDP-choline is an
interesting oral form of slow release of CDP-choline.
The global analysis of the metabolic picture, as compared to that found in the
experiment with CDP-choline, substantially shows a better and more gradual
distribution of the radioacitivity in the organism. The metabolic pictures of the organs
examined (liver and brain) were substantially equal and acylated cytosinic metabolites
were not found. This indicates that the valeric acid chains are quantitatively removed
~ '

11~7
- 11 -
before the cytosinic component is used wich does not appear to be signi ficantlycompromised in comparison with the control experiment.
The bioavailabili~y in terms of recovery of the structural components of the drug in 24
hours is very similar for CDP-choline and its triacylated analogue, the faecal and
urinary excretions being quite comparable.
The radioactive levels and the structural identity of the labelled species present at
gastric and intestinal level, clearly show the notable consistency of the protective
effect exercised by the valeric acid residues as regards the pyrophosphatases,
intestinal especially, and the indirect control operated on the speed of absorption.
In fact, while after 30 minutes in the control experiment at intestinal level not more
than 5% of the radioactivity present in the dose and only 0.2% of the double labelled
CDP-choline administered is found, on the other hand in the case o-f trivaleryl-CDP-
choline 29% of the variably acylated double labelled molecule is still found after an
hour and a total radioactivity equal to 30% of that present in the administered dose is
detected.
To confirm this datum, in ~he plasmatic component and in the organs, the maximumuptake is definitely moved to longer periods of time (5 hours) with trivaleryl-CDP-
choline.