IT8322283A1 - Derivatives of cytidine diphosphate choline, process for their preparation and their therapeutic use. - Google Patents

Description

DESCRIPTION

of the industrial invention entitled:

"Derivatives of cytidine-disphosphate-choline, process for their preparation and their therapeutic use".

SUMMARY

Iderivatidelia cytidine diphosphate-choline diformula:

(1)

wherein R represents an acyl radical selected from the group comprising 2 to 20 atomic carbon dioxide, saturated and unsaturated, linear or branched, mono and dicarboxylic acids optionally substituted with other functional groups in addition to the carboxylic one; R1 represents H or an acyl radical identical to R and R2 represents hydrogen or, when R and R1 are hydrogen, the residue of a? eventually substituted alkalonamine present activity? therapeutic analogue and that of cytidine-diphosphate-choline and are suitable for oral administration.

Their synthesis occurs by monoacylation with N-acyl-imidazole, by reaction with anhydride chloride of carboxylic acid in the case of di- and triacylated derivatives and by condensation between cytidine-monophosphate and phosphoalkanolamine.

The present invention relates to new derivatives of cytidine diphosphatecholine having the general formula:

(I)

wherein R represents an acyl radical selected from the group comprising fatty acids having from 2 to 20 carbon atoms, saturated and unsaturated, linear or branched, mono or dicarboxylic, optionally substituted with other functional groups in addition to the carboxylic one; R1 represents H or an acyl radical identical to R and R2 represents hydrogen or, when R and R1 are hydrogen, the residue of an optionally substituted alkanalamine.

Cytidine-diphosphate-choline, hereinafter referred to as usual with the abbreviated name of CDP-choline, represents the active form of choline and? a key intermediate in the biosynthesis of complex lipids: the importance of cytidine nucleotides in the formation of the diester bonds of the phospholipid molecule? in fact widely documented.

As demonstrated in the literature, lecithins and sphingomyelines are formed by a reaction, catalyzed by microsomal enzymes, in which CDP-choline donates the phosphorous or Unico fragment to D-? -? diglyceride or N-acylsphingosine respectively.

Furthermore, it should be remembered that CDP-choline acts as a donor of P-choline to 1 alkyl, 1 'enyl-2acyl-sn-qlycerol, forming plasmalogens. CDP-choline is in turn biosynthesized from P-choline and CTP, by the enzyme coiin-phosphate-cytidintransferase. This activity? enzymatic? was found in the non-particulate fraction of the cytoplasm and in the microsomal fraction. In this regard, it should be emphasized that the formation of the CDP-choline represents the most important stage. slow and therefore limiting of the entire metabolic pathway; cellular concentrations of this metabolite are therefore of critical importance in regulating the biosynthesis of phospholipids.

Alongside the "de novo" biosynthesis of lecithins, a collateral pathway should also be mentioned, known as the Bremer pathway, of which? the operation has been demonstrated? in the liver and brain.

In this way the phosphatidyl-ethanolamine is trimethylated on the nitrogenous function by the S-adenosylmethionine, which acts as a donor of the methyl group, forming lecithins.

As a consequence of its biochemical roles, CDP-choline finds use and pharmacological indication mainly in a series of alterations of the central nervous system; in this body, in fact, the integrity? structural and functional of the phospholipid membranes? particularly critical. The route of administration used so far? parenteral.

The activity? drug of the molecule? has been demonstrated in various clinical pictures, including the sequelae of cerebral apoplexy, different types of cerebrovasculopathies, Parkinson's disease, cranial traumatology and its sequelae.

The use of oral CDP-choline raises the problem of integrity? structure of the molecule during its passage through the gastrointestinal tract, and that of its absorption. In fact, the hydrolytic action of intestinal esterases should not be neglected, so that this enzymatic cleavage and absorption can be considered as two competitive processes, in the sense that a rapidly absorbable product will result? evidently less exposed to enzymatic cleavage in the intestinal tract.

It has also been found that the CDP-choline derivatives of formula (I) perform this purpose through a modulation of the degree of polarity. of CDP-choline which is obtained by introducing apolar groups on the molecule or by eliminating the net charge of the cholinic nitrogen.

Pi? specifically, this modulation is carried out through the partial or total acylation of the amino function present on the cytasine with an alkanolamine group, so that? the net charge of the quaternized nitrogen is removed from the molecule.

Taking acylated derivatives into consideration first, they are obtained by acylation on the 2 ', 3' and / or positions of the molecule with the carboxylic acids gi? defined, in particular and d? preferably monocarboxylic acids, naturally present in mammals, of different chain length, linear or branched, saturated or unsaturated.

Esterification affects some or all of the aforementioned reactive positions of the molecule. Such a type of structural modification substantially changes the chemical-physical nature of the molecule, giving it a greater hydrophobic character, the entity of which? ? in relation to the degree of acylation and the nature of the esterifying groups / or groups.

The derivatives of the invention, especially those in which major? the hydrophobic component introduced, show characteristics of amphipathic molecules, being present at the same time strongly polar structural elements, such as the hydrocarbon chain of / or esterifying acyl residues. In consideration of their structural characteristics, the acylated derivatives of CDP-choline according to the invention have a more vast spectrum of solubility, which makes it easy to convey it in both aqueous and predominantly organic media.

In particular, especially for those derivatives in which they are pi? long hydrocarbon chains, the solubilization process? associated with the formation of aggregates of the miscellar type, property? this common to all surfactants.

In turn, the derivatives in which the choline portion of CDP-choline? substituted with an alkanolamine group, which can also be defined as demethyl derivatives of CDP-choline, while representing a modest structural modification are from the chemico-physical point of view, considerably different from CDP-choline, since? the positive charge of the quaternary ammonium function is removed.

The molecule? therefore considerably less polar than CDP-choline, although it is closely related to it from a structural and biochemical point of view.

How already? indicated, the derivatives of CDP-choline according to the present invention have, in oral form, indications similar to the known ones adopted for CDP-choline. However, it is understood that the indication of the oral route does not intend to exclude the other routes of administration and in particular the parenteral route. Pi? simply for the derivatives of CDP-choline according to the invention? intended for use, orally in tablets containing 500 and 1500 mg. of active ingredient, to be administered 2-3 times a day, in the following pathologies:

- Sclerotic vasculopathies with prevalent manifestation in the cerebral district.

- Short and long-term treatment of cerebrovascular accidents.

- Short and long-term therapy of the sequelae of stroke. - Parkinsonian syndrome therapy, in particular in the atherosclerotic form.

- Antidepressant treatment.

-Treatment of traumatic cerebral coma.

-Prevention and therapy of hyaline membrane disease (IRDS).

- Therapy of acute and chronic hepatitis (viral hepatitis, etc ..)

- Therapy and prevention of fatty liver in alcoholics.

- Adjuvant therapy in liver cirrhosis.

Without wishing to impose undue limitations on the scope of the present invention, it seems plausible to attribute the activity? of the derivatives in question of CDP-choline to the factors summarized below:

a) The acylated derivatives of CDP-choline are sufficiently stable in acidic or alkaline environments not to be chemically degraded by the pH changes associated with crossing the gastrointestinal tract.

b) The functionalization of positions 2 ', 3' and / or pu? represent not only an element of resistance to the degradative action of the enzymes of the digestive tract, but also the condition for a better assimilation of the molecule through the intestinal lumen.

c) The amphipathic nature of these acylated derivatives, in fact, authorizes us to foresee that they are able to permeate with pi? ease? structures such as biological membranes, in which the crossing of the lipid layer can? be favored by the presence of a lipophilic component on the molecule.

d) Probably the action of lipase pi? unless specific on mono and acyl CDP-choline or its fragments, deriving from the biological use of the molecule, it produces the release of the acyl residues and / or residues.

In particular, it should be emphasized that the acyl radicals introduced on CDP-choline are molecules naturally present in the body, devoid of any toxicity. intrinsic, easily metabolizable and specifically recoverable in lipid biosynthesis.

As regards the triacylate derivatives, due to the involvement of the amino pyrimidine group in the acylation process, a different behavior is found with respect to the corresponding derivatives of and mono acylates. This is not only due to the different degree of polarity? average of the molecule, but also why? the amino group represents an important structural element in enzyme-substrate recognition. It should also be considered that the presence of an amide bond, in addition to the ester bonds, involves not only the action of lipase in the drug release process, but also other activities. enzymatic able to cleave the amide bond (amidase and peptidase).

e) As regards the derivatives of formula (I) in which the cholinic part? substituted with an alkanolamine radical, in particular N, N-dimethylethanolamine and N-methyl-ethanolamine, and in particular their metabolic utilization, there are two possible schemes.

A first metabolic pathway involves an S-adenosylmethionine-dependent methylation, with the formation of CDP-choline. Can you? also hypothesize that the two analogues are recognized by the CDP-ethanolamine enzyme: diacylglycerol ethanolamine phosphotransferase, thus bringing about? to the synthesis of mono- or N-methylated phosphatidyl-ethanolamine, which through the Bremer pathway? converted into lecithin.

The present invention also relates to the preparation of the CDP-choline derivatives of formula (I). Pi? specifically, the present invention relates to processes for the preparation of derivatives (I) linked to the desired type of functionalization or esterification. Consequentially:

1) -In the monoacylation of CDP-choline at the level of one of the two ribose oxidyls, the process according to the present invention is realized by the fact that CDP-choline is brought to react with N-acylimidazole in which the acyl group? the desired one on CDP-choline, in a solvent mixture comprising water and an organic solvent, with a prevalence in volume of the aqueous phase, at room temperature.

The acylating agent used? an N-acylimidazole, obtained by reaction in anhydrous environment between a fatty acid and N, N carbonyldiimidazole. The organic solvent (acetone, acetanitrile, etc.), in which the synthesis of N-acylimidazole takes place,? the same as then, added to the water,? used in the synthesis of the desired derivative of CDP-choline. N-acylimidazoI? an extremely active acylating agent, capable of giving rise to a reaction with weak nucleophiles such as the 2 'and 3' hydroxyls of ribose, even in the presence of high quantities? of water, working at room temperature.

It is obvious that the reaction conditions in which this acylation occurs are ideal for a molecule such as CDP-choline, which? chemically labile in pi? points and presents, due to its ionic character, problems of solubility? in exclusively organic environments. In fact, aqueous-organic mixtures such as water: acetone (5: 1 v / v) or acetonitrile: water (1: 4 v / v) can be used as reaction solvents in this phase, etc. generally lasts from 2 to 5 hours at room temperature under stirring and the product? recovered under vacuum by evaporation of the solvents. The reaction yields, operating under these conditions, are 50-60%. The characteristics of the aqueous-organic system control the selectivity? acylation, which is directed exclusively on the 2 'or 3' positions of the ribose, operating with strong excesses of water in the presence of weakly solvating aprotic solvents such as acetonitrile, tetrahydrofuran or acetone, while it is also directed on the position by operating in poorly aqueous environments, in which highly solvating aprotic solvents such as dimethylformamide or pyridine prevail.

2) In the realization of the derivatives of and triacylates of CDP-choline of formula (I), the process according to the present invention provides for the reaction of CDP-choline with a reactive derivative of the desired carboxylic acid, preferably the chloride or anhydride , the reaction being carried out in the presence or absence of a catalyst, such as pyridine and sodium acetate, depending on whether the triacyl or diacylated derivative is desired.

This procedure is particularly suitable for the introduction of low molecular weight acid residues.

3) In the preparation of CDP-choline derivatives of formula (I) in which the cholinic portion? substituted with an alkanolamine group, in particular for the synthesis of CDP-N, N-dimethyl-ethanolamine and of CDP-N-methylethanolamine, the process according to the present invention is characterized by the condensation in the presence of a condensing agent such as dicyclohexylcarbodiimide, the phosphoalkanolamine and cytidine monophosphate (CMP).

The following examples illustrate, without limitation, the processes according to the present invention.

EXAMPLE 1

Synthesis of 3 '(2') - O - valeryl - CDP - choline

The chemical synthesis of CDP-choline acylated with valeric acid on the 2 'or 3' hydroxyl of ribose is carried out by condensation of CDP-choline with the imidazolide of valeric acid. The latter compound, which is obtained by reaction between valeric acid and N, N 'carbonyldiimidazole in a strictly anhydrous environment, allows, under mild conditions in an aqueous-organic environment, to selectively carry out the acylation of one of the two hydroxyls of ribose. In a standard procedure, the preparation of the imidazalide of valerianic acid is carried out by reacting in tetrahydro furan (THF) (5 ml), dried on molecular sieves and maintained under anhydrous nitrogen, 1.3 g (8 mmoles) of carbonyldiimidazole with 500 mg (4.9 mmol) of valeric acid. The reaction, carried out under nitrogen under strictly anhydrous conditions,? complete in about 10 minutes; similar results are obtained if, while maintaining the volume of the solvent unchanged, quantities are used. double or triple of reactive or if the THF? replaced with anhydrous acetonitrile or acetone.

Acylation of CDP-choline? carried out in an aqueous-organic environment, mixing under stirring at room temperature 20 ml of a 250 mM solution of CDP-choline with 5 ml of the organic solution of imidazolide at different concentrations (1.6; 3.2; 4.8 M). In all cases, the reaction mixture initially appears as a stable emulsion, white in color, which becomes clearer as the acylation of CDP-choline proceeds. In about 4 hours the reaction reaches the maximum yield. Operating at 70? Is there the course of the reaction? much more? fast (2h), but the yields of acylated product do not change significantly. The 3 '(2') - 0-vaIerii-CDP-coIin, due to the simultaneous presence on its molecular structure of the net charge, associated with cholinic nitrogen, and of the aliphatic component of acyl, bound to ribose, has all the characteristics of an amphipathic species, straining with ease? to foam in solution, especially under vacuum. For this reason the removal of the organic solvent from the reaction mixture is carried out in a nitrogen stream. The residual aqueous solution, brought to pH 8.5 with KOH 0.5 N, is then adsorbed on a Dowex 1x8 column, in formate form (18 cm h, 30 mm internal diameter) to carry out the separation of the species present. The elution is carried out first with 200 ml of H2O and then with a linear gradient of formic acid from 0.00 to 0.02 M (500 ml HO 500 ml HCOOH 0.02 M). The column has a flow of 1.5 ml / min and 5 ml fractions are collected which are analyzed for absorption at 280 nm.

The chromatographic purification of the reaction mixture on Dowex 1x8 does not allow the resolution of the two isomers of CDP-choline, respectively acylated in 2 'or 3' on ribose. Therefore all the spectroscopic characteristics reported refer to the mixture of the two isomers. These are stable for months in the dry state and in aqueous solution at pH 6.5. The 3 '(2') - 0-valerii-CDP-choline has a very similar U.V spectrum to that of CDP-choline, showing a maximum at 280 nm with a molar of 12.8 x 10 <3>.

Overall, the nuclear magnetic resonance spectrum of the proton shows that on the CDP-choline molecule? only one acyl was introduced and that this? specifically localized at the level of V or 3 'hydroxyls of ribose. Indeed the integration of the various signals? consistent with the presence on the CDP-choline molecule of a single acyl chain, whose specific localization on one of the two free hydroxyls of ribose? implicitly demonstrated by the shift to fields pi? low (? 5.3 - 5.4} of part of the complex signal at 4.0-4.5 is ascribable to the hydrogens on the carbons bearing hydroxyl functions. A further confirmation of the localization of the acyl function is given by the fact that the signals of the protons on carbons 5 and 6 of the cytosine nucleus are found at the same Chemical shift values as in CDF-choline, thus excluding that the amino function on carbon 4 of the pyrimidine nucleus is involved in the acylation process.

The information obtained from the analysis of the nuclear-magnetic resonance spectrum of <13> C fully confirm the identity? structure of the molecule, excluding once again that the amino function on the aromatic system was involved in the acylation reaction. In fact, the signals of cytosine carbons, as in CDP-choline, have respectively a chemical shift of 160.49

(C4), 99.29? (C5) and 144.28? (C6) The ribose carbons instead give rise to 10 signals, 5 for each of the two isomers acylated in 2 'or 3' respectively, while the cholinic carbons are found in the same Chemical shifts that in the CDP-choline. On the other hand, the aliphatic carbons of valerianic acid are found in high fields.

Structural analogues of CDP-choline, acylated in 2 'or 3' with fatty acids from C2 to saturated or unsaturated, linear or branched, containing other functional groups in addition to the carboxylic one, can be obtained according to the same reaction scheme reported in Example 1, replacing valeric acid with the desired acid.

It is also possible to involve the amino function on carbon 4 in the cytidine nucleus in the acylation process, using in the reaction traacyl imidazole and CDP-choline solvents strongly solvating aprotic, such as dimethylsol-fossido or dimethylformamide.

EXAMPLE 2

Synthesis of 2 ', 3' - of O-acetyl-CDP-choline

The chemical synthesis of the diacetylated CDP-choline on the 2 'and 3' hydroxyls of ribose is carried out according to the principles of acid acetylation, using acetic anhydride or acetyl chloride as acylating reactive and acetic acid as solvent.

In a standard procedure 300 mg of CDP-choline are suspended in 2 ml of glacial acetic acid, anhydrified by distillation on P2O5.Acylating reagent is added to the suspension, 1 ml in the case of acetyl chloride and 1.5 ml if anhydride is used acetic. The heterogeneous suspension, kept under stirring at room temperature in anhydrous conditions, tends to clear up. The reaction is stopped after twelve hours and the solution is brought to dryness under reduced pressure, repeatedly coevaporating the residue with ethanol. The 1 glass residue is dissolved in H2O and, after correcting the pH to 8.5 with KOH 0.2 N,? purified on a Dowex lx 8 column as described in Example 1. The U.V. of the 2 ', 3'-di O-acetyl-CDP-choline? similar to that of the mono acylated derivative (example 1) and shows the same molar.

As a whole, the NMR spectrum demonstrates the selective introduction of two acetyl residues in position 2 'and 3' on the ribose, while the amino function remains unacetylated in position 4 on the cytosine nucleus.

Structural analogues acylated with a pi? long chain can be prepared using the chlorides of the corresponding acids instead of acetyl chloride.

The triacylate analogues in which the amino function on carbon 4 of the cytosine nucleus is also involved, can be obtained by adding pyridine in quantities to the reaction mixture. catalytic.

Analogues in which a dicarboxylic acid engages both the 2 'and 3' hydroxyls in the ester bond are prepared, according to the scheme of Example 2, using the appropriate acid chloride.

The scheme of example 2, as well as? the generalizations referred to in points (ac) can be applied to functionalize the cytidine monophasfate (CMP) which can? subsequently be condensed with P-choline, operating with cyclohexylcarbodiimide as condensing agent, as described in example 3, or according to industrial synthesis schemes. In this way, undesired hydrolytic phenomena associated with lability can be circumvented. of the pyrophosphoric bridge, which are found in certain types of direct acylations of CDP-choline.

EXAMPLE 3

Synthesis of CDP-N.N-dimethyl-ethanolamine

In a standard procedure 11? Moles of CMP and 11? Moles of N, N-dimethyl-P-ethanolamine are dissolved in 2 ml of a water-pyridine mixture (1: 7), to which 200 mg of DCC are initially added; two further additions of 100 mg each of the condensing agent come

Claims (16)

1) Derivatives of cytidine-diphosphate-choline, having formula (I) wherein R represents an acyl radical selected from the group comprising fatty acids having from 2 to 20 carbon atomides, saturated or unsaturated, linear or branched, mono or dicarboxylic, optionally substituted with other functional groups in addition to the carboxylic one; R1 represents H or an acyl radical identical to R and R2 represents hydrogen or, when R and R1 are hydrogen, the residue of an optionally substituted alkanoiamine. 2) Process for the synthesis of monoacylated derivatives of formula (I) according to claim 1, characterized in that cytidine-diphosphate-choline reacts with N-acyl-imidazole, containing the desired acyl group, in a solvent mixture consisting of from water and an aprotic organic solvent, with an excess by volume of water compared to the organic solvent. 2) Process according to claim 2, characterized in that a slightly solvating apratic organic solvent is used to obtain monoacylated derivatives on one of the two ribose hydroxyls. 4) Process according to claim 3, characterized in that said organic solvent? chosen from acetone, acetonitrile and tetrahydrofuran. 5) Process according to claim 3, characterized in that the ratio between water and organic solvent? between 4: 1 and 3: 1 v / v. 6) Process according to claim 2, characterized in that a strongly solvating organic solvent is used to obtain the monoacylated derivatives on the nitrogen atom in position 4 of the cytidine nucleus. 7) Process according to claim 6, characterized in that said organic solvent? selected from dimethylformamide, dimethyl sulfoxide and pyridine. 8) Process according to claim 7, characterized in that the reaction is carried out at room temperature and for a time of 4-5 hours. 9) Process for the synthesis of diacylated and triacylated derivatives of formula (I) according to claim 1, characterized in that cytidine-diphosphate-choline is reacted at room temperature with a reactive derivative of the desired carboxylic acid. 10) Process according to claim 9 characterized by the fact that said reactive derivative? chosen between chloride and anhydride. 11) Process according to claim 9, characterized in that for obtaining the triacylate derivatives the reaction? conducted in the presence of an acylation catalyst. 12) Process according to claim 11, characterized in that said catalyst? chosen between pyridine and sodium acetate. 13) Process for the synthesis of the derivatives of formula (I) in which the choline portion of the cytidine-diphosphate-choline? replaced with an alkanolamine radical, characterized in that cytidine monophosphate is condensed, in the presence of a condensing agent, with a phosphoalkanolamine. 14. Process according to claim 13, characterized in that said condensing agent? dicyclohexylcarbodiimide. 15) Pharmaceutical composition characterized in that it contains, as active ingredient, a derivative of cytidine-diphosphate-choline according to claim 1 together with the usual excipients and vehicles. 16) Pharmaceutical compositions according to claim 15, characterized in that they are in a form suitable for oral administration.