FI92208B - Process for the preparation of pharmaceutically valuable cytidine 5'-phosphate alkanols and glycerols - Google Patents

Description

92208

The process for the preparation of pharmaceutically valuable cytidine-5'-phosphate alkanols and glycerols - For the preparation of pharmaceutically active cytidine-5'-diphosphatalkanoler and glycerol

The invention relates to a process for the preparation of pharmaceutically valuable cytidine 5'-diphosphate alkanols and glycerols of the formula (I) nh2 * - o- f-O-O-CH2O

HO OH

wherein R is a saturated or unsaturated straight-chain or branched aliphatic group having 6 to 30 carbon atoms or a group ch2-a ch2-a ch2-, optionally substituted by halogen, hydroxy, alkoxy or cyano.

I I I

CH-B II, CH-III, CH-A IV,

I I I

ch2- ch2-b ch2-b wherein A is a saturated or unsaturated, straight-chain or branched alkoxy group having 6 to 30 carbon atoms optionally substituted by halogen, hydroxy, alkoxy or cyano and Β is hydrogen, halogen or a group -OCH3 or -O- (CH2) nCF3, where n is a number from 1 to 3.

Cytidine diphosphate dialkylglycerols are already known to be prepared according to the method of M. Katts et al., Can J. Biochem. 49 (1971), 275, in an analogous manner. These compounds occur in nature. For example, they are substrates for phosphatidylinositol synthetase (E.C.2.6.1.67) and react intracellularly in the presence of inositol to phosphatidylinositol.

(R.H. Raosa, Biochem. Biophys. Acta 348 (1978) 306). These 2,92020 compounds have no cytostatic effect. Also known are cytidine arabinoside (Ara-C) adducts, for example: Ara-C1-palmitoylglycerol (Chung II Hong et al. J. Med. Chem., 31, 1988, 1973), and Ara-C-acylalkyl glycerols (Chung II Hong et al., Ebenda, 29, 1986, 20 39).

These act cytostatically, making this effect attributable to the presence of Ara-C, which is itself a potent cytostat. Ara-C acyl or alkyl glycerol adducts can be considered as depot forms of Ara-C.

New cytidine 5'-diphosphate alkanes and glycerols have now been found to have excellent cytostatic activity.

In formula I, R is a saturated and unsaturated, straight-chain or branched aliphatic hydrocarbon group having 6 to 30 carbon atoms. R is most preferably a saturated or unsaturated, straight-chain or branched hydrocarbon group having 10 to 20 carbon atoms. Examples of such groups are dodecyl, tetra, penta, hexane, hepta, octa, nanadecyl, eicosyl groups or their branched analogues or tetra, penta, hexane, hepta, octa, nonadecylene or eicosylene groups .

R may further denote groups according to general formulas II, III or IV of the formula page.

In formulas II, III or IV A, a straight-chain or branched or branched, saturated or unsaturated, alkoxy group having 6 to 30 carbon atoms, for example 10 to 20 carbon atoms. Examples of such alkoxy groups are tetra, penta, hexa, hepta, octa, nonadecyl, eicosyloxy groups or branched analogs thereof, or tetra, penta, hexa, hepta, octa, nona, dodecadecyloxyoxy, eicosyleneoxy groups .

These groups may optionally also be substituted, such halogens being, for example, chlorine, bromine, iodine and fluorine, or hydroxy, alkoxy or cyano.

II

3 92208 B represents hydrogen or halogen, for example chlorine, bromine, iodine, fluorine, preferably chlorine or -OCHa or -O- (CH2) nCF3, where n is a number from 1 to 3, preferably 1.

Particularly preferred individual compounds are cytidine-5'-diphosphate-hexadecanol cytidine-5'-diphosphate-dodecanol cytidine-5'-diphosphate-2-chloro-2-deoxy-1-O-hexadecyl-glycerol cytidine-5'- diphosphate-1-O-hexadecyl-2-O- (2,2,2-trifluoroethyl) glycerol cytidine-5'-diphosphate-3-chloro-3-deoxy-1-O-hexadecyl glycerol cytidine-5 '-di phosphate-10-hexadecyl-2-deoxyglycerol cytidine-5'-diphosphate-1-O-octadecyl-2-O-methylglycerol

The novel cytidine-5'-diphosphate alkanols and glycerols can be prepared by reacting alkylphosphoric acids or alkylglycerophosphoric acids of general formula V, wherein R is as defined above, with a condensed cytidine-5'-phosphate or an activated derivative of this compound in the presence of anhydrous conditions.

· * As the activated derivative of cytidine-5'-phosphate, for example, cytidine-5'-morpholidophosphate can be used. It is expedient to use an excess of the second reaction component, in which case the molar ratio of cytidine-5 * -phosphate to alkylphosphoric acid or alkylglycerophosphoric acid can be 1: 0.5 to 1: 2. As the condensing agent, for example, dicyclohexylcarbodiimide can be used. As the organic base, for example, 4-dimethylaminohyridine or pyridine is used, in which case, when pyridine is used, the reaction can take place without the addition of an organic solvent.

4 92208

Optionally, an inert organic solvent such as chloroform can be used as the solvent. The reaction takes place at temperatures of about 20 to 60 ° C, preferably 35 to 40 ° C.

After completion of the reaction, the product is isolated from the reaction mixture, for example by extraction. The compounds thus obtained can optionally be purified, for example, by chromatography.

The new cytidine-5'-diphosphate alkanols and glycerols are characterized by a high cytostatic effect.

To determine the cytostatic effect, the antiproliferative effect on Ehrlich's ascites tumor cells was studied. The compounds according to the invention proved to have an excellent cytostatic effect. They can therefore be used alone or in admixture with other active ingredients as cytostatics or viral preparations as conventional galenic preparations.

The compounds of general formula I of the formula page are suitable for human use and can be administered in the customary manner, for example orally or parenterally. Preferably, they are administered orally at a daily dose of about 0.05 to 20 mg / kg body weight, most preferably 0.05 to 5 mg / kg body weight. The attending physician may also prescribe lower or higher doses in this range depending on the general condition and age of the patient, the particular compound of formula I of the formula page, the nature of the disease, and the mode of formulation.

The compounds of formula I of the formula page may be administered alone or in combination with other pharmaceutically active substances, the content of the compounds of general formula I of the formula page being between 0.1 and 99%. In general, the pharmaceutically active compounds are in admixture with suitable inert excipients and / or carriers or diluents which are

II

, For example, pharmaceutically acceptable solvents, gelatin, acacia, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, petrolatum or the like. The pharmaceutical preparations may be in solid form, for example tablets, granules, suppositories, capsules and the like, in semi-solid form, for example ointments, or in liquid form, for example suspensions or emulsions. Optionally, they are sterilized and contain excipients such as preservatives, stabilizers and emulsifiers, osmotic pressure modifying salts, etc. In particular, the pharmaceutical preparations may contain the compounds according to the invention in combination with other therapeutically valuable substances. The compounds according to the invention can be formulated with them, for example together with the above-mentioned auxiliaries and / or carriers or diluents, as combination preparations.

Example 1: • ·

Cytidine-5'-di phosphate-hexadecanol

Hexadecyl phosphate (150 mg, 0.47 mmol) is taken up four times in about 5 ml of pyridine (Camilla dried) and the solution is evaporated to dryness in vacuo.

*

To cytidine-51-morpholidophosphate (4-morpholine-N, N'-dicyclohexylcarboxamidine salt) (404.2 mg, 0.58 mmol) is added 4 ml of toluene four times and the mixture is evaporated to dryness in vacuo on a rotary evaporator. The reaction components are then combined, re-evaporated on a rotary evaporator. in an evaporator with 5 ml of pyridine and taken up in about 10 ml of pyridine. The solution is stirred for 5 days at 37 ° C to prevent air from entering. The reaction mixture is then evaporated to dryness in vacuo. Toluene (3 x 5 ml) is added to the residue, which in each case is distilled off in vacuo. The product obtained is taken up in a mixture of 6 92208 in 15 ml of a chloroform-methanol (1: 1, v / v) mixture and 7 ml of 0.01 N HCl. The aqueous phase is separated and extracted three times with 5 ml of chloroform-methanol. The combined organic phases are washed neutral with a small amount of water and evaporated to dryness. The residue is taken up in a mixture of 45 ml of chloroform, 20 ml of methanol, 9 ml of water and 0.3 ml of 1N methanolic ammonia, the organic phase is separated off and extracted with a further 5 ml of water. Evaporation of the combined aqueous phases gives compound 1 as the diammonium salt, which is further purified by reprecipitation from chloroform-acetone.

Yield 144 mg, thin layer chromatography (Merck Fertigplatte Kieselgel 60 F254); Rt 0.31 (chloroform-methanol-glacial acetic acid-water, 25: 15: 2: 4, v / v / v / v).

UV absorption: λ max x 272.6 nm (water), λ max 282 (0.01 N HCl). Example 2:

Cytidine-5'-disfofaatti-l-O-hexadecyl-2-0- (2,2,2-trifluoro-etyyliJglyseroli

A mixture of 1-O-hexadecyl-2-O- (2,2,2-trifluoroethyl) glycerophosphoric acid (0.12 g, 0.25 mmol) and cytidine-5'-morpholido phosphate (4-morpholine-Ν , Ν * -dicyclohexylcarboxamidine salt) (0.30 g, 0.44 mmol), rotary evaporate four times under high vacuum to dryness with 5 ml of pyridine each time (dried over CaH2). The residue is then taken up again in 6 ml of pyridine and the solution is stirred for 9 days at 37 [deg.] C. to prevent the ingress of moisture. The reaction mixture is then evaporated to dryness in vacuo, about 4 ml of toluene are added several times to the residue and re-evaporated to dryness. The substance obtained is taken up in a mixture of a mixture of chloroform and methanol (1: 1, v / v, 30 ml) and 0.01 N. hydrochloric acid (15 ml). The mixture is adjusted to pH 2-3 by dropwise addition of 1 K hydrochloric acid. The chloroform-methanol phase is separated, washed with water until neutral and evaporated after filtration. The residue in a mixture of chloroform (13 ml), methanol-water (2: 1, v / v, 10 ml) and 1N methanolic ammonia is extracted twice with said methanol-water-methanolic NH3 mixture (in each case about 8 ml). The combined aqueous phases are evaporated. The residue is crystallized with a small amount of methanol and then washed several times with dry acetone. After drying, 30 mg of the diammonium salt of compound 1 are obtained chromatographically as a uniform substance.

Thin layer chromatography (Fertigplaett Kieselgel 60, Merck): Rf 0.39 (n-propanol 25%, aq. NH 3 / water, 8: 2: 1, v / v / v; Rf 0.30 (chloroform / methanol / glacial acetic acid)). water, 25: 15: 2: 4, v / v / v / v).

UV absorption: λ max 275.5 nm (water); λ max 282 nm (0.01 N HCl) Fast Atom bombardment mass spectrometry: m / z 783 (M) m / z 558 (C 16 H 33 OCH 2 -CH ( OCH2Clr3) CH2-O-P (O) -Op (O) -OH),

OH OH

m / z 540 (C 16 H 33 OCH 2 -CH (OCH 2 CF 3) CH 2 -O-P (O) -O 1 - (O) -).

OH O

m / z 477 (C 16 H 33 OCH 2 -CH (OCH 2 CF 3> CH 2 -O 2 (O) -O-),

OH

m / z 384 (M-2H (C 16 H 33 OCH 2 -CH (OCH 2 CF 3) CH 2 -O-))

Example 3: 8 92208

Cytidine 5'-diphosphate-3-chloro-3-deoxy-1-O-hexadecyl glycerol

To a solution of 0.664 g (1.6 mmol) of 3-chloro-3-deoxy-10-hexadecylglycerol-2-phosphoric acid and 0.77 g (6.3 mmol) of 4-dimethylaminopyridine in 10 ml of dry chloroform is added 0.882 g (1.3 mmol) of cytidine 5'-morpholidophosphate (4-morpholine-N, N'-dicyclohexylcarboxamidine salt). The mixture is stirred in a sealed vessel at 35 ° C for 5 days to prevent moisture from entering. The reaction mixture is then taken up in a mixture of chloroform, methanol and water (pH 2), the organic phase is washed with water and evaporated on a rotary evaporator in vacuo. The residue is taken up in a mixture of chloroform, pyridine and formic acid (50: 30: 7, v / v / v) and separated by flash chromatography on 30 g of Kieselgel 60 (Merck, 0.040-0.063 mm). Elute first with 270 ml of the same solvent and then with a mixture of chloroform and methanol (9: 1, 340 ml; 8: 2, 300 ml; in each case v / v).

Fractions 31 to 55 (fraction size 20 ml) are evaporated to dryness in vacuo. The residue obtained is taken up in a mixture of 28 ml of a mixture of chloroform and methanol (1: 1, v / v) and 9 ml of 0.01 N hydrochloric acid, and the mixture is adjusted to pH 2 with 1 N hydrochloric acid. The phase is separated and the organic phase is treated once more with 5 ml of a mixture of chloroform and methanol (1: 1, v / v). The combined organic phases are rotary evaporated by adding methanolic ammonia. The resulting diammonium salt of compound 1 is crystallized by treatment with methanol-diethyl ether, washed with diethyl ether and dried. Yield: 0.340 g. Thin layer chromatography (Merck Fertigplatte Kieselgel 60 F 254); Rf 0.42 (chloroform-methanol-glacial acetic acid-water 50: 30: 4: 8, v / v / v / v); Rf 0.42 (n-propanol-25% aq. Ammonia-water 8: 2: 1, v / v / v). UV absorption: δ {281.5 nm (0.01 N HCl).

, 92208

Example 4:

Cytidine 5'-diphosphate-2-chloro-2-deoxy-1-O-hexadecyl glycerol

A mixture of 664 mg of 2-chloro-2-deoxy-1-O-hexadecyl-1-glycero-3-phosphoric acid, 882 mg of 5'-cytidine morpholidophosphate (4-morpholine-N, N'-dicyclohexylcarboxamidine salt) and 770 mg mg of 4-dimethylaminopyridine, is stirred under anhydrous conditions in 30 ml of dry chloroform (non-alcoholic) for 5 days at 35 ° C, and after the reaction is complete, chloroform, methanol and 0.02% hydrochloric acid (20 ml each) are added to the reaction mixture. The aqueous phase is adjusted to pH 3 with 25% hydrochloric acid. The lower phase is separated, washed with water, dried and evaporated to dryness in vacuo. The residue is dissolved in 3 to 5 ml of a 50: 30: 7, v / v / v, mixture of chloroform / pyridine / formic acid and chromatographed on 25 g of Kieselgel 60 (Merck, 0.04 - 0.063 mm). Elute first with 200 ml of a chloroform / methanol / formic acid mixture and then with chloroform / methanol (in each case with v / v mixtures) 9: 2 (1.1 l), 8: 2 (0.5 l), 7.5: 2 , 5 (0.7 L), 6.5: 3.5 (0.3 L), and 6: 4 (2 L). Chloroform / methanol fractions 70 to 96 (fraction size 50 ml) give 200 mg of compound 1. The substance is then treated with chloroform / methanol / 2.5 N hydrochloric acid, 2: 2: 2, v / v / v, and water. phase is adjusted to pH 2.5. After separation of the lower phase, the aqueous phase is treated once more with chloroform / methanol, 2: 1, v / v. The combined lower phases, to which methanolic ammonia has been added, are rotary evaporated to give the ammonium salt of the title compound.

DC (Fertigplatte Kieselgel 60 F 250, Merck), Rf 0.33 (CHCl 3 / CH 3 OH, CH 3 -COOH, H 2 O, 29: 18: 2; v / v / v / v), UV absorption: / (m »x 275.5 (neutral, Κ2θ), λmax 203.6 (0.02 N HCl).

Example A

10 92208

Concentration-dependent growth inhibition of Ehrlich's ascites tumor cells

Concentration (micromoles) 3 10 30 100

Inhibitory effect (%)

Compound 12 19 46 31 of Example 1

Compound of Example 2 9.5 30 86 100

Compound 12 of Example 3

Compound 18 of Example 4

II

11 92208

Formula page nh2 8 8 JÖ R- 0-P-0-P-0-CH_ ^ r i * | X |

HO OH

CH2- A

“- β II

CHG

CH, - A

I 2

CH - III

CH2- B

CHp -

™ - A IV

CH2- B

0

B

R r- o - P - OH v

I-OH

Claims (2)

A process for the preparation of pharmaceutically valuable cytidine-5'-diphosphate alkanols and glycerols of the general formula (I) nh 2 S Ϊ J 2) R-QpOPO - CH 2 R 0 "Q" 1 HO OH where R is a saturated or unsaturated straight or branched, optionally with halogen, hydroxy, alkoxy or cyano substituted aliphatic 6-30 carbon atomic group or a group of ch2-a ch2-a ch2-III CH-B II, CH-III, CH-A IV, III ch2-ch2-b ch2-b where A is a saturated or unsaturated, straight or branched, optionally with halogen, hydroxy, alkoxy or cyano substituted 6-30 carbon atomic alkox group and B is hydrogen, halogen or a group -OCH3 or 0- (CH2) nCF3, where n is a tai 1-3, characterized in that alkylphosphonic acids or alkylglycerophosphoric acids of the general formula V 0 II R - O - P - OH (V) OH II 92208 where R is the same as above, reacted with cytidine 5'-phosphate or an activated derivative of this compound in the presence of a condensing agent or n organic base in anhydrous conditions. Process according to claim 1, characterized in that the alkanols and glycerols prepared are selected from the group: cytidine-5'-di phosphate-dodecanol cytidine-5'-di phosphate hexadecanol cytidine-5'-di phosphate-2-chloro-2 -desoxyhexadecyclyl glycerol cytidine-5'-diphosphate-1-O-hexadecyl-2-0- (2,2,2-trifluoroethyl) -glycerol cytidine-5'-diphosphate-3-chloro-3-deoxy-1 O-hexadecylglycerol cytidine-5'-di phosphate-1-O-hexadecyl-2-O-methylglycerol cytidine-5'-di-phosphate-1-O-hexadecyl-2-deoxyglycerol.