KR920001689B1 - Process for preparing acylated derivatives of cytidinediphosphate-choline - Google Patents

Abstract

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Description

Method for preparing acylated derivatives of cytidine-diphosphate-choline

The present invention relates to a process for the preparation of novel derivatives of cytidine-diphosphate-choline having the following general structural formula.

In the above formula, R is an acyl group selected from the group consisting of monocarboxylic saturated and unsaturated linear or branched chain fatty acids having 3 to 7 carbon atoms, and R ¹ is hydrogen or the same acyl group as R.

Citidine-diphosphate-choline (hereinafter referred to simply as “CDP-choline”) represents the active form of choline and is an important intermediate in the biosynthesis of complex lipeds. The importance of cytidine nucleotides in the formation of diester bonds in phospholipid molecules has been demonstrated extensively in practice.

As shown in the literature, lecithins and sphingomyelins are produced by reactions promoted by microsomal enzymes, in which CDP-choline is produced by D-α, β. Donate a phosphorylcholine fragment to diglyceride or N-acylsphingosine respectively. Furthermore, it should be remembered that CDP-choline acts as a p-choline donor for 1-alkyl, 1'-enyl-2-acyl-sn-glycerol to form plasmalogen.

CDP-choline is biosynthesized from p-choline and cytidin-triphate (CTP) by the choline phosphate-cytidine transferase. This enzymatic activity has been found in the particleless and microcoal fractions of cytoplasm. With this in mind, it is appropriate to underline that the production of CDP-choline represents the slowest stage, and thus is a limited one of the entire metabolism. Therefore, the cell concentration of this metabolite is very important in the regulation of phospholipid biosynthesis.

As a result of its biochemical role, CDP-choline finds its use and pharmacological adaptability to a series of stool in the central nervous system. In fact, the structural and functional integrity of the phospholipid membrane is of particular importance for this organ. The mode of administration that has been used so far has been parenteral. The pharmacological activity of the molecule has been shown in various diseases such as stroke, different forms of cerebral anemia, Parkinson's syndrome, cranial fractures, and their secondary manifestations.

The present invention relates to the use of acylated derivatives of CDP-choline, represented by the general formula (I) shown above, useful for the treatment of these diseases.

These compounds are of the straight or branched chains of different lengths of the carboxylic acids defined above at the N ⁴ and / or 2 ′, 3 ′ positions of the molecule, particularly preferably the branches [chains] naturally present in the mammal. Obtained by acylating with saturated or unsaturated monocarboxylic acids. Some or all of the reactive sites of the aforementioned molecules are involved in the esterification reaction.

The acylated derivatives of CDP-choline of the present invention are sufficiently stable in the presence of acids or alkalis and therefore do not chemically degrade due to pH changes during the process leading to the intestine.

The presence of strong hydrogenated branches [chains] significantly alters the metabolism of the molecule and prevents hydrolysis of the compound at fatigue phosphoric acid bonds, both at gastric and serous concentrations. This is possible only after the acyl groups have been removed in turn by the action of acyl esterases in gastric and intestinal fluids and / or amidases and peptidases.

Since the absorbency of the compound is controlled by the hydrolysis of the acyl group, the acylation of CDP-choline determines the absorbance of the choline and cytosine components of the compound. The released acyl residues can be thought of as metabolites normally present in vivo with extremely negligible toxicity levels.

N ⁴ , 2 ′, 3′-triacyl derivatives of orally administered CDP-choline act as delayed forms of CDP-choline. The gentle release of CDP-choline in vivo results in a more uniform and smooth distribution of the active ingredient. As already pointed out, the CDP-choline derivatives in oral dosage form according to the invention have similar applicability to CDP-choline. More simply, according to the present invention, oral use of CDP-choline derivatives is expected to be in tablet form, each tablet containing 200 to 1500 mg of the active ingredient and administered 2-3 times a day for the diseases listed below. Can be.

-Atherosclerosis, especially cerebral atherosclerosis,

-Short and long term treatment of cerebrovascular failure,

-Short and long term treatment of seizure secondary,

-In particular, the treatment of Parkinson's syndrome and Parkinson's-like syndrome in the form of atherosclerosis,

-Treatment of hypotension,

-Treatment of traumatic coma in the brain,

Prevention and treatment of hyper-substance diseases (IRDS),

-Acute and chronic hepatitis (such as viral hepatitis),

-Treatment and prevention of fatty liver in alcoholism,

Adjuvant treatment of cirrhosis.

The invention also relates to a process for the preparation of CDP-choline derivatives of general formula (I). More specifically, the process for the preparation of the compound of formula (I) as desired in the present invention relates to the desired functionalization or esterification reaction. therefore,

1) In the first method, the method of the present invention in the N-acylation of CDP-choline in the amine group of the pyrimidine nucleus quaternary carbon has a cationic ion of tetrabutylammonium salt of CDP-choline. Desired excess carboxyl at 50 ° C. for 36 hours in the presence of an acylation catalyst such as 4- (N, N-dimethylamino) pyridine in a bipolar solvent (DMF, formamide or pyridine, of which DMF is most preferred). It is characterized by reacting with acid imidazolide. The carboxylic acid imidazide is obtained by reacting a carboxylic acid with N, N'-carbonyldiimidazole in anhydrous DMF solution.

2) In the second method, in producing n ⁴ -acyl-2 ′, 3-di-O-acyl-CDP-choline, an excess of imidazolide must be used and the reaction time at a reaction temperature of 50 ° C. By increasing the time to 96 hours can be used the same method as in 1) above.

3) In the third method, in acylation using an aqueous mixture of low solubility compounds such as THF, acetone and acetonitrile, acylation occurs only at the hydroxyl group in ribose. Best yields are obtained when using a mixture of H ₂ O / acetonitrile (1: 4 v / v) as the solvent. This acylation reaction is not disturbed and the best yield is obtained when the molar ratio of imidazolide (prepared in anhydrous THF) and CDP-choline molar ratio is 2: 1.

Under these conditions, monoacylation of CDP-choline is unclearly the only occurrence at either of the free hydroxyl groups in the 2 'or 3' position of ribose, resulting in 2 '(3')-O-acyl-CDP- Generate choline (see General Structural Formula (II) below).

The results of the chromatographic purification of the reaction mixture at DOWEX AG 1: 8 do not result in the cleavage of the two isomers of acylated CDP-choline at 2 'and 3' of ribose. Therefore, all spectrophotometric analyzes were performed on mixtures of the two isomers which were stable in pH 6.5 or frozen aqueous solutions. Identification of monoacyl and triacyl derivatives of CDP-choline is based primarily on ¹ H-NMR and UV spectrophotometric data.

The ¹ H-NMR data briefly shown in Table 1 below relates to valeric acid derivatives and is consistent with the structural designations for each derivative (see Table 1).

TABLE 1

^* 1) Spectra were obtained in ² H ₂ O using a Bruker 500 MHz spectrometer.

2) Chemical shifts are represented by δ.

3) Compound 1: CDP-choline

Compound 2: 2 '(3')-O-valeryl-CDP-choline

Compound 3: N ⁴ -Valeryl-CDP-choline

Compound 4: N ⁴ -Valeryl-2 ', 3'-di-O-valeryl-CDP-choline

se (sestet); q (quintet); t (triplet); d (duplet); s (singlet); c (comples signal).

In the case of 2 '(3')-O-valeryl-CDP-choline, nuclear magnetic resonance has only one acyl group introduced and is limited to the position of the 2 'or 3' hydroxyl group of ribose. Is showing. In fact, some displacements can be seen to decrease the fields in the complex singal region to 4.0-4.5 due to the H of the hydroxyl group which is bonded to the carbon atom, while the same value affects the chemical displacement. The proton singal on the fifth and sixth carbons of the cytosic acid nuclei present can be seen to exclude the acylation functionality of the amine groups on the aromatic fourth carbon since those nuclei are in CDP-choline. . However, in the spectrum of n ⁴ -valeryl-CDP-choline, the presence of a single unit of valeric acid per molecule is observed, in particular it is limited to nitrogen at the fourth position of the cytosine system. Indeed, there is a characteristic displacement that lowers the fields of the fifth and sixth positions of the chromophore, but both ribose shows resonance as CDP-choline in the same field.

Finally, in the n ⁴ -valeryl-2 ', 3'-di-O-valeryl-CDP-choline spectrum, the combination of both acylation groups shows that the presence of three units of valeric acid per CDP-choline molecule appear. The total acylation of the molecule leads to displacements that lower the hydrogen fields of both aromatic protons and ribose.

Hereinafter, the present invention will be described in more detail by way of examples. However, the method of the present invention is not limited thereto.

Example 1

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

Chemical synthesis of acylated CDP-choline with valeric acid in the 2 'or 3' hydroxyl of ribose is accomplished by conducting a condensation reaction of CDP-choline with imidazolide of valeric acid.

Imidazolids, prepared by the reaction of valeric acid with N, N'-carbonyldiimidazole under extreme anhydrous conditions, allow the acylation reaction to be carried out selectively in a water-soluble organic medium under mild conditions.

In the standard method, the preparation of valeric acid imidazolide is carried out by dissolving 1.3 g (8 mmol) of carbonyldiimidazole in 5 ml of tetrahydrofuran (THF) stored on anhydrous nitrogen and dehydrated on molecular sieves, 500 mg (4.9 mmol) of valeric acid. Reaction). In an anhydrous bath the reaction in the presence of nitrogen is terminated in about 10 minutes.

Similar results are obtained if the amount of reactant is doubled or tripled without changing the volume of solvent or if acetonitrile or anhydrous acetone is used instead of THF.

Acylation of CDP-choline proceeds in anhydrous organic medium conditions by mixing 20 ml of 250 mM CDP-choline solution and 5 ml of imidazole organic solution at different concentrations (1.6,3.2,4.8 M) under stirring at room temperature. In each case, the reaction mixture initially appears as a stable white, colored emulsion, but it clears as acylation of CDP-choline occurs. In about 4 hours the reaction reaches maximum yield. By proceeding at 70 ° C., the reaction proceeds more rapidly (2 hours), but the yield of acylation product does not change significantly. 3 '(2')-O-valeryl-CDP-choline can be isolated by vacuum evaporation of the solvent with a stream of nitrogen. The remaining aqueous solution is adjusted to pH 8.5 with 0.5N KOH and then adsorbed onto a DOWEX 1 × 8 (formeate) column. The eluate is initially treated with a linear gradient of H ₂ O followed by 0.00 to 0.02 molar formic acid.

3 '(2')-O-Valeryl-CDP-choline has a UV spectrum similar to that of CDP-choline and exhibits a maximum of 280 nm (= 12.8 x 10 ³ ) in water. ^The information deduced from the analytical value of the ¹³ C NMR spectrum confirms the structural identity of the molecule, except that the aromatic amine group is involved in the acylation reaction. In fact, as in CDP-choline, the cytosine carbon symbol has chemical shifts of 160.49δ (C ₂ ), 168.98δ (C ₄ ), 99.29δ (C ₅ ) and 144.28δ (C ₆ ), respectively. However, while ribose carbon has five symbols for each of the two isomers, each of which is acylated at ten 2 'or 3' positions, it is found that choline carbon has the same chemical shift as in CDP-choline. On the other hand, aliphatic carbons of valeric acid are found in high fields.

Structural derivatives of CDP-choline acylated at the 2 'or 3' position by C ₃ -C ₇ saturated or unsaturated, straight or branched fatty acids, by using the desired acid instead of valeric acid, Example 1 It may be prepared according to the same reaction as described in.

Example 2

Synthesis of N ⁴ -valeryl-CDP-choline

0.2 mmol of tetrabutylammonium hydroxide (1 ml, 0.2 M) is added to 100 mg of CDP-choline dissolved in 5 ml of H ₂ O, followed by freezing treatment of the sample. Next, tetrabutylammonium salt (TEBA) of CDP-choline is dissolved in 4 ml of anhydrous DMF containing 20 mg of 4-dimethylaminopyridine. To this solution, 2 mmol of valeric acid imidazolide obtained by reacting 204 mg of valeric acid dissolved in 1 ml of anhydrous DMF with 324 mg of N, N'-carbonyldiimidazole are added. The reaction was terminated by vacuum removal of the solvent from the reaction mass stirred at 50 ° C. for 36 hours, and the resulting oily residue was treated three times with ethyl acetate.

The reaction mixture is purified by chromatography on silica and eluting with a linear gradient of 0-20% v / v using H ₂ O in CH ₃ OH. N ⁴ -Valeryl-CDP-choline is produced in a yield of 50-60%, with the same C ₃ -C ₇ as N ⁴ -Valeryl-2 ′, 3′-di-O-valeryl-CDP-choline With straight or branched chain fatty acids, saturated or unsaturated, N ⁴ -acylated derivatives can be prepared according to the method described in this example by using the desired acid instead of valeric acid.

Example 3

Synthesis of N ⁴ -Valeryl-2 ', 3'-di-O-valeryl-CDP-choline

The method up to the preparation of a solution of the TEBA salt of CDP-choline in DMF containing 4- (N, N-dimethylamine) pyridine can be carried out according to the method described in the previous examples.

After 0 to 48 hours, 2 mmol of imidazolide of valeric acid obtained by reacting 204 mg of valeric acid in which this solution was dissolved in 1 ml of anhydrous DMF with 324 ml of N, N'-carbonyldiimidazole are added. The reaction mass was stirred for 16 h at 50 ° C., then the solvent was removed in vacuo to terminate the reaction and the oily residue was treated three times with ethyl acetate.

Triacyl salt (105 mg) was purified by chromatography on a silica column, eluting with a linear gradient of 0-20% v / v using H ₂ O in CH ₃ OH.

Structural derivatives of CDP-choline triacylated with saturated or unsaturated, straight or branched chain fatty acids of C ₃ -C ₇ can be prepared by repeating the method described in Example 3 by replacing valeric acid with the desired acid. have.

The pharmacological properties of N ⁴ -valeryl-2 ', 3'-di-O-valeryl-CDP-choline are shown by the double labeling of ¹⁴ C of 3H and methyl choline at position 5' above the cytosine nucleotide. The product was identified in rats by administering the product. The data required are in accordance with the origin of radioactivity in animals and also in accordance with the structural identity of the radiomolecular species present in some more important biological samples (liver, brain, stool, stomach and intestinal contents).

Precise analysis of the action by functionalization in the absorption and metabolic processes of the triacylated product requires a comparison of the acquired data with the control experimental data, in which mice (5- ³ H; methyl- ¹⁴ C) -CDP Choline is administered orally in equimolar amounts. Characterization of labeled metabolites present in liver, brain, stool, stomach and intestinal contents of animals orally administered pharmacological doses (20 μmol / kg) of (5- ³ H; methyl- ¹⁴ C) -CDP-choline Leads to their prophylactic extraction from the tissue.

The extraction process is consistent with the recovery of the different classes of labeled metabolites of the following three 1) to 3).

1) Water soluble metabolite, followed by HPLC.

2) Recovered by thermal denaturation in polymer protein, nucleic acid, and aqueous alcohol solution.

3) Lipids, followed by chromatography on silica column.

Experimental results show that N ⁴ -valeryl-2 ', 3'-di-O-valeryl-CDP-choline is an interesting oral dose that slowly releases CDP-choline.

The overall analysis of metabolic properties, compared with those found in experiments with CDP-choline, shows substantially better and more gradual distribution in vivo. The metabolic properties of the tissues (liver and brain) used in the experiments were substantially equivalent, and no acylated cytosine acid metabolites were found. This fact indicates that valeric acid (chain) is removed quantitatively before the cytosine acid component is used. Cytosuccinic acid components do not appear to be significantly problematic when compared to control experiments.

Bioavailability during the recovery period of the structural component of the compound within 24 hours is similar to the bioavailability of CDP-choline, and its triacylated derivatives, feces and urine excretion can be compared significantly.

The radiation levels and structural identity of the labeled species present in the stomach and intestine, the protective effect carried out by pyrophosphatase, especially in the intestine, by indirect counterparts acting on the rate of absorption with valeric acid residues Shows a significant persistence. In fact, less than 5% of the radioactivity present in the dose and only 2% of the double labeled CDP-choline present in the dose was found after 30 minutes in the intestinal control experiment, while on the one hand the trivaleryl-CDP-choline In this case, 29% of the variably acylated label molecules are found even after 1 hour and radioactivity corresponding to 30% of those present at that dose is detected.

In plasma components and biological tissues, in order to confirm this data, the maximum absorption capacity is certainly longer (5 hours) with Trivaleryl-CDP-choline.

Claims (10)

Acylimidazoles of saturated or unsaturated, straight or branched chain monocarboxylic fatty acids having 3 to 7 carbon atoms with cytidine-diphosphate-choline (CDP-choline) as a reaction medium using an aqueous mixture of low-soluble compounds. A method for producing an O-monoacylated derivative of CDP-choline represented by the following general formula (I), which is reacted with Zolid).

Wherein R is an acyl group selected from saturated or unsaturated straight or branched monocarboxylic fatty acids having 3 to 7 carbon atoms and R ¹ is hydrogen. The process according to claim 1, wherein the low solubility reaction compound in the reaction medium is selected from the group consisting of tetrahydrofuran, acetone and acetonitrile. The process according to claim 1 or 2, wherein the reaction medium is a 1: 4 v / v mixture of water and low solubility compounds. The method according to claim 1, wherein the molar ratio of imidazolide to CDP-choline is 2: 1. Imidazolides of saturated or unsaturated monocarboxylic fatty acids having 3-7 carbon atoms to introduce the tetrabutylammonium salt of CDP-choline to the N ⁴ position in the presence of a homogeneous solution of an amphoteric dipolar solvent and an acylation catalyst. A method for producing an N ⁴ -monoacylated derivative of CDP-choline represented by the following general formula (I), which is reacted.

Wherein R ¹ is selected from O 3-7 carbon atoms, a saturated or unsaturated straight or branched chain monocarboxylic fatty acid group and R is hydrogen. The method of claim 5 wherein the solvent is selected from the group consisting of formamide, pyridine and dimethyl formamide. 6. The process of claim 5 wherein the acylation catalyst is 4-N, N-dimethylaminopyridi. The method of claim 5, wherein the reaction is carried out in the presence of excess imidazolide. The method of claim 5, wherein the reaction proceeds at 50 ° C. for 36 hours. The reaction is carried out for 96 hours at 50 ° C. by the method according to claim 5 in the presence of an imidazolide of an saturated or unsaturated straight or branched monocarboxylic fatty acid having 3 to 7 carbon atoms in excess. (I) A method for producing an N ⁴ -acyl-2 ′, 3′-di-O-acyl derivative of CDP-choline.

Wherein R and R1 are acyl groups selected from saturated or unsaturated straight or branched chain carboxylic acids having 3 to 7 carbon atoms.