DD299727A7 - ALKYL AND ALKENYL PHOSPHO-L-SERINES, METHOD FOR THEIR PREPARATION AND USE - Google Patents

Abstract

The invention relates to novel alkyl and alkenyl phospho-L-serines, processes for their preparation and use. The compounds of general formula I, ROO

Description

ROPO-CH ₂ -CH-COOH «I

OH NH ₂

in the

R = a saturated hydrocarbon radical having 14 to 18C atoms, which is substituted by OR ¹ , SR ¹ , NR ¹ R ² , wherein R ¹ and R ² = CH ₃ or an unsaturated hydrocarbon radical having 18 to 20C atoms, and of their salts. The compounds I have some structural relationship with naturally occurring phospholipids, e.g. As the phosphatidylserines, which play a very important role in biological processes. However, they differ from the latter in that they contain no glycerol component and hydrolyzable fatty acid residues in the molecule. They can be regarded as phosphatidylserine analogues, which have an increased biostability compared to the naturally occurring phosphatidylserines, and 3tellen interesting experimental objects in processes of interaction of phospholipids with the cell or the cell components.

Field of application of the invention are the chemical as well as the pharmaceutical industry.

Characteristic of the known state of the art

It is known that Glycerophosphoserine can be produced by condensation of Glycerophosphorsäurealkylestern with serine derivatives whose functional groups are partially substituted by protective groups to avoid Nebehreaktionen, wherein after the reaction, a deprotection of the protecting groups takes place. For example, diacyl, dialkyl and also acylalkylglycerophosphoserines have been prepared in this manner (AJ Slotboom et al., Chem. Phys. Lipids, S [1970] 301-379; H. Elb, Chem. Phys. Lipids, 26 (1980) 405-429; A. Hermetter et al., Chem. Phys. Lipids, 30 (1982) 35-45). Diacylglycerophospho-L-serines have also been prepared by reaction of diacylglycerophosphocholines with unprotected L-serine in the presence of phospholipase D (Comfurius, P., et al., Biochim., Biophys, Acta, 488 [1977], 36-42), but with yields are very low and preferably a conversion to the corresponding Diacylglycerophosphorsäuren. The alkylphosphoserines of the general formula I are novel compounds for which no methods of preparation have hitherto been described. In addition, from DE-OS 3639084 u.a. Alkylphospho-L-serines described, however, which are used as emulsifiers and humectants.

Object of the invention

It is the object of the invention to develop an economical process according to which the alkylphospho-L-serines of the formula I can be synthesized, and to find and to provide pharmaceutically usable compounds.

Explanation of the essence of the invention

According to the invention, the alkyl and alkenylphosphoserines of general formula I and their salts are prepared by

a) phosphoric acid esters of the general formula II,

R-O-P-i

, 0- (CH ₂ I _n -X

o '>

in the R the o.g. Has meaning and

X is H, NH ₂ , NH-CH ₃ , N (CH ₃ I ₂ , N (CH ₃ J ₃ , OH, Cl, Br and η = 1 to 6,

reacted with L-serine in the presence of phospholipase D or

b) by adding a phosphoric acid ester of the general formula III,

R-O-P-A III

in the R the o.g. Meaning,

A = OH, Cl, Br, J and B is OH, Cl, Br, J, alkoxy or aryloxy,

or the salts of these compounds with a serine derivative of the general formula IV,

HO-CH ₂ -CH-COOY IV

NH-Z

in the

Y is benzyl, t-butyl, phthalimidomethyl, isopropyl, benzhydryl or another in a conventional manner, e.g. B. by catalytic Hydrogenolysis, hydrazinolysis, treatment with HCl, Natriumthiophenolat or easily cleavable by hydrolysis Protective group and ' Z = N-benzoyloxycarbonyl, N-t-butoxycarbonyl (t-BOC), N-phthaloyl or another by conventional methods, such as. B.

catalytic hydrogenolysis, hydrazinolysis, treatment with HCl or formic acid removable protecting group,

optionally in the presence of a Kondonsationmittels.

The resulting reaction product is optionally treated with water for hydrolysis. Subsequently, the protecting groups of the resulting substituted alkylphosphoserine derivatives of general formula V,

R-O-P

.'- CH ₂ -CH-COOY V

OH NH-Z

in the R, Y and Z the o.g. Meaning have cleaved and optionally formed the corresponding salts. Essential to the invention are in addition to the phosphoric acid esters of type III (A = B = OH) and the corresponding phosphoric acid dichlorides (III, A = B-CI) as starting materials which are z. B. according to known method, by reaction of alkyl alcohols with Phosphoroxychloiid receives. Condensation of alkylphosphodichloriclene with a protected serine of type IV gives phosphoric acid chlorides of the compounds of type V, which are converted into the alkylphosphoserines I or their salts by hydrolysis and removal of the protective groups Y and Z. A further variant of the method according to the invention which is essential to the invention consists in using compounds V by condensation of the alkylphosphoric acid halides of all; reacting a formula III (A = halogen, preferably chlorine, B = alkoxy, aryloxy) with the doubly protected serines IV and converting the resulting alkyl or aryl esters of the condensation products IV into the alkylphosphoserines !.

The enzymatic method mentioned under a) for preparing the compounds of the formula I is carried out in general by reacting a phosphoric acid ester of the general formula II with excess L-serine in an aqueous acetate or Tris buffer solution or suspension at pH 4.8 to 8.5 in the presence of 0.01 to 0.1 M calcium chloride with addition of diethyl ether or a mixture of diethyl ether with other organic solvents, eg. B. chloroform, and phospholipase D at temperatures between 5 and 60 ° C, preferably 4O ⁰ C, is reacted. During the reaction time, which is between 0.5 and 48 hours, the reaction mixture is stirred vigorously. Thereafter, to deactivate the enzyme, a 0.1 M EDTA solution is added and the resulting alkylphospho-L-serine purified by chromatography. It is remarkable and by no means foreseeable that the naturally occurring compounds of type II, which have no glycerol component in the molecule, ie do not represent actual phospholipids, are substrates for the naturally occurring phos. represent olipase D and can be converted by this into the also not occurring in nature type I compounds. The synthesis variant for the new compounds of type I, which proceeds via the intermediate compounds of type V, mentioned under b), is conveniently carried out by reacting the dried pyridinium salts of the compounds IH (A = B = OH) with the double-protected serine IV (Y = Benzhydryl, Z = Nt-butoxycarbonyl) in a molar ratio of 1: 1 to 1: 1.8 in the presence of an organic base, e.g. As pyridine, wherein further inert organic solvents may be present, in the presence of a condensing agent, for example 2,4,6-Triisopropylbenzolsulfochlorid, at temperatures between 5 and 50 ° C, preferably at room temperature, is reacted. The resulting substituted alkylphosphoserines V are usually isolated from the reaction mixture and, if necessary, purified by chromatography on silica gel. Their conversion into the compounds I takes place by introducing into the solution of the compounds V in an inert organic solvent, preferably chloroform, at -5 to 5 ⁰ C dried HCl gas. After the deprotection has occurred, excess HCl is removed by introducing nitrogen.

Subsequently, the resulting compounds I are converted by treatment with ammonia-containing solutions in the diammonium salts and these are purified by chromatography if necessary.

The compounds according to the invention are new and biologically active. They have a strong cytostatic and inhibit z. B. the cell proliferation of Ehrlich ascites tumor cells already in very low concentrations. 17-Methoxyoctadecylphospho-L-serine proved to be particularly effective; cis-9-n-octadecenylphospho-L-serine, arachylphospho-L-serine, 2-methoxy-tetradecylphospho-L-serine: 2-methylmercapto-tetradecylphospho-L-serine, 2-dimethylthio-tetradecylphospho-L-serine.

The compounds of the general formula I and alkylphospho-L-serines of the general formula VI,

ROPO-CH ₂ -CH-COOH OH NH ₂

Vl

wherein R = an alkyl radical of 10 to 16C atoms are suitable for use in the preparation of cytostatic pharmaceutical agents.

In addition to the abovementioned compounds of the formula I, particularly effective compounds of the formula VI: decylphospho-L are

seri "Oodecylphospho-L-serine, Tetradecylphospho-L-serine, Hexadecylphospho-L-serine.

The compounds of the formula I and VI act strongly cytostatic and inhibit z. For example, the cell proliferation of Ehrlich ascites

already in very low concentrations. Table 1 shows the concentration-dependent growth inhibition of

Ehrlich ascites tumor cells by compounds of general formulas I and VI listed:

Table 1 3 10 Concentration of 100mm. 30 substance 37.9 60.1 95.5 ...- L-serine 22.4 43.9 75.4 98.0 1. hexadecylphospho 19.3 40.0 78.0 100 2. decylphospho · 26.3 38.6 79.4 99.4 3. Tetradecylphospho- 16.0 29.3 82.6 98.7 4. 17-methoxy-octadecylphospho 16.0 28.7 78.4 93.0 5. cis-9-n-octadecenylphospho 18.2 41.5 84.2 97.0 6. Arachylphospho 14.7 28.7 82.3 100 7. 2-methoxy-tetradecylphospho- 16.7 24.3 80.3 97.0 8. P. methylmercapto-tetradecylphospho- 77.5 9. 2-dimethylaminotetradecylphosphono

The dodecylphospho-L-serine exhibited considerable cytostatic activity on HL-60 leukemia cells as well as KB tumor cells, the IC _M values (concentration causing 50% inhibition of cell growth) were 15 and 39 x 10 ^-8 M., respectively The invention will be explained in more detail by the following examples.

example 1 Hexadecylphospho-L-serine (VI, R = C ₁ (H ₃ ))

0.8 g of L-serine (7,6mmol) are in 1,66ml 0.1 M acetate buffer (pH5,6) of 0.09M to CaCl ₂ is dissolved at 45 ⁰ C.

32 mg (0,075mmol) hexadecylphosphocholine To this solution (II, R = C ₁₆ H _33, X = N (CH _{3) 3),} 1.6 ml of diethyl ether / Chloroform (9: 1, v / v, ethanolfroi) and 200 mg of a phospholipase D enzyme preparation prepared from white cabbage, a Activity of 0.9 U / ml (1 unit (U] sets at 27 ° 1 μπιοΙ substrate per min) in the reaction mixture

Stirred for 3 hours at 45 ° C. After cooling to room temperature, 3.31 ml of 0.1 M EDTA solution are added and passed through

Introducing nitrogen to remove the organic solvents. The remaining aqueous phase becomes 4.3 times Volume of chloroform / methanol (5: 8, v / v) stirred for 30 minutes and aspirated from the insoluble (L-series). The filtrate is with

1 volume of water and 3.7 volumes of chloroform, the mixture was shaken for 10 minutes, the organic phase separated, concentrated in vacuo and the resulting residue on carboxymethylcellulose (CM 52 Whatman, Na ⁺ form) separated by column chromatography. Dio elution is carried out successively with 75 ml of chloroform, 300 ml each of chloroform / methanol, 9: 1.8: 2.7: 3, then 1750 ml of chloroform / methanol 1: 1 (v / v in each case). The fraction size is 50ml. 16 mg of pure hexadecylphospho-L-serine (Na salt) were obtained from fractions 18-40.

TLC (Kieselgel 60, Merck precast plate, CHCl ₃ / CH ₃ OH / acetone / acetic acid / H ₂ 0.50: 10: 20: 10: 5, v / v / v / v / v): R f = 0.1 Example 2 Hexadecylphospho-L-serine (VI, R = C ₁₆ H ₃₁ ) A solution of 0,219g (0,679mmol) hexadecyl (III, A = B = OH, R = C ₁₈ H ₃₃₎ in 1.2 mL of pyridine in vacuo

concentrated and dried the pyridinium salt thus obtained over phosphorus pentoxide in vacuo. It is then dissolved in 11 ml of anhydrous pyridine.

0.806 g (2.17 mmol) of protected serine (IV, Y = CH (CjH ₅ J ₂ , Z-CO-O (CH ₃ I ₃ ), dried over phosphorus pentoxide in vacuo, are dissolved in 9 ml of anhydrous pyridine are mixed and added to the mixture 1.32g (4.35mmol)

Triisopropylbenzenesulfochloride added. The reaction mixture is treated under anhydrous conditions at room temperature

Stirred for 24 hours. After adding a few drops of water, stirring is continued for a further 30 minutes. Thereafter, the mixture is concentrated in vacuo and the residue is dried over phosphorus pentoxide in vacuo. The residue obtained is mixed with 20 ml

Extracted diethyl ether and washed 3 times with 4 ml of diethyl ether. The ether solutions are combined and in vacuo

concentrated. The residue is dissolved in chloroform and purified by column chromatography on 36 g of silica gel 60 (particle size 0.04-0.063 mm, 230-400 mesh, Merck). The chloroform used for the column chromatography is saturated with 25% aqueous ammonia. Eluiertwird with 300m! Chloroform, 250ml chloroform / methanol, 98: 2, v / v, 200ml chloroform /

Methanol, 95: 5, v / v, 500ml chloroform / methanol, 90:10, v / v, 400ml chloroform / methanol, 80:20, v / v. The fraction size

is Σ .ml. The fractions 41-47 'contain 152 mg hexadecylphospho-Nf-butoxycarbonyl-L-serinbenziihydryl ester V (R = C ₁₆ U ₃₃ , Y = CH (C ₆ H ₅ I ₂ , Z = CO-OC (CH ₃ I ₃ ), ( Ammonium salt), yield 32%.

TLC (Kieselgel 60, Merck precast plates, chloroform / methanol / 25% aqueous NH ₃ , 65: 35: 5, v / v / v): R f = 0.7.

61 mg (0.09 mmol) of the described compound V are dried in vacuo over phosphorus pentoxide and then dissolved in 12 ml of anhydrous chloroform. Dry nitrogen is passed through this solution for 10 minutes followed by HCIu for 20 minutes under anhydrous conditions at ⁰ ° C. Thereafter, the reaction vessel is tightly closed and the reaction mixture is stirred for 1 hour at ^{0 0} C.

Nitrogen is then passed through the reaction mixture for 1 hour at room temperature to remove the HCl gas. Thereafter, the reaction mixture is concentrated in vacuo. The residue is treated with a mixture of 10 ml of chloroform / methanol, 2: 1, v / v, 1 ml of water and 0.02 ml of 25% aqueous ammonia and separated after phase separation, the resulting organic phase. The aqueous phase is extracted 3x with chloroform / methanol, 2: 1, v / v, the organic solutions are combined and concentrated in vacuo. To remove the remaining water is distilled several times with 1 ml of benzene / ethanol, 2: 3, v / v. The product obtained is dried over phosphorus trioxide in vacuo and then washed with acetone. Yield: 30.8 mg (83%).

The obtained Hexadecylphospho-L-serine (diammonium salt) I (R = Ci ₆ H ₃₃ ) is thin-layer chromatographic unitary. TLC (Kieselgel 60, Merck precast plates, chloroform / methanol / acetone / acetic acid / water, 10: 2: 4: 2: 1, v / v / v / v / v): Rf = 0.1. The visualization is carried out with molybdate reactant as well as with ninhydrin spray.

To remove traces of NH ₄ Cl, the substance (30.8 mg) is chromatographed on 2 g of silica gel 60 (particle size 0.04-0.063 mm, 230-400 mesh, Merck). The column is equilibrated with chloroform / methanol / aqueous 25% ammonia, 80: 20: 0.5, v / v / v. In the same mixture, the substance is dissolved and added to the column. Elution is with 20 ml of chloroform / methanol / 25% aqueous ammonia, 80: 20: 0.5, v / v / v, 20 ml of chloroform / methanol / 25% aqueous ammonia, 75: 25: 0.5, v / v / v, 20ml chloroform / methanol / 25% aqueous ammonia, 70: 30: 0.5, v / v / v, 20ml chloroform / methanol / 25% aqueous ammonia, 65: 35: 0.5, v / v / v / v, 260ml chloroform / methanol / 25% aqueous ammonia, 60: 40: 0.5, v / v / v. The compound is obtained from fraction 7-13 (fraction size 20 ml) by concentration. TLC (Kieselgel 60, Merck precast plates, chloroform / methanol / acetone / acetic acid / water, 10: 2: 4: 2: 1, v / v / v / v / v): Rf = 0.1. The following compounds are prepared analogously to Example 2:

Example 3

n-decylphospho-L-serln (VI, R = C ₁₀ H ₂ i)

From n-decyl phosphate and protected serine (IV, Y = CH (C ₆ H ₆ I ₂ , Z = CO-OC (CH ₃ ) ₃ ), TLC (Kieselgel 60, Merck precast plates, Chloroform / methanol / acetone / acetic acid / water, 10: 2: 4: 2: 1; v / v / v / v / v): Example 4

n-dodecylphospho-L-serine (VI, R = C ₁₂ H ₂₅ )

From n-dodecyl phosphate and protected serine (IV, Y = CH (CeH ₆ J ₂ , Z = CO-OC (CH ₃ I ₃ ), TLC (Kieselgel 60, Merck precast plates, Chloroform / methanol / acetone / acetic acid / water, 10: 2: 4: 2: 1; v / v / v / v / v): Example 5

n-tetradecylphospho-L-serln (VI, R = C _M H ₂₉ )

From n-tetradecyl phosphate and protected serine (IV, Y-CH (C ₆ H ₆ J ₂ , Z = CO-OC (CH ₃ J ₃ ), TLC (Kieselgel 60, Morck precast plates, Chloroform / methanol / acetone / acetic acid / water, 10: 2: 4: 2: 1; v / v / v / v / v):

Example β17-Methoxy-n-octadecylphospho-L-Seiln (I, R = C "H ₃ , OCH _} )

From 17-methoxy-n-octadecyl phosphate and protected serine (IV, Y = CH (CgH ₆ )), Z = CO-OC (CHa) ₃ ), TLC (Kieselgel 60, Merck Finished plates, chloroform / methanol / acetone / acetic acid / water, 10: 2: 4: 2: 1; v / v / v / v / v): Example 7

Cls-9-n-octadecenylphospho-L-serine (I, R = CuH ₃₈ ) From cis-gn-octadecenyl phosphate and protected serine (IV, Y = CH (C ₆ H ₆ J ₂₎ Z = CO-OC (CH ₃ J ₃ ), TLC (Kieselgel 60, Merck precast plates, chloroform / methanol / acetone / acetic acid / water 10: 2: 4: 2: 1, v / v / v / v / v): Rf = 0.1.

Example 8 Arachylphospho-L-serine (I, R = C ₂₀ H ₃₃ ) Arachyl phosphate and protected serine (IV, Y = CH (C ₈ H ₆ ) ₂ , Z = CO-OC (CH ₃ I ₃ ), TLC (Kieselgel 60, Merck precast plates, Chloroform / methanol / acetone / acetic acid / water 10: 2: 4: 2: 1; v / v / v / v / v): Example 9

2-methoxy-n-tetradecylphospho-L-serine (1, R = CmH ₂₈ OCH ₃ ) From 2-methoxy-n-tetradecylphosphate and protected serine (IV, Y = CH (C ₆ H ₅ I ₂ , Z = CO-) OC (CH ₃ J ₃ ), TLC (silica gel 60, Merck precast plates, chloroform / methanol / acetone / acetic acid / water 10: 2: 4: 2: 1, v / v / v / v / v): R f = 0 ,1.

Example 10

2-Methylmercapto-n-tetradecylphoepho-L-enel (I, R "Ci ₄ HnSCHj) From 2-methylmercapto-n-tetradecyl phosphate and protected serine (IV, Y = CH (C _e H ₆ ) ₂ , Z-CO-OC (CHj) ₃ ), TLC (Kieselgel 60, Merck-Grundlgplatten, chloroform / methanol / acetone / Eeslgsäure / water 10: 2: 4: 2: 1, v / v / v / v / v):

Rf = O ₁ L

Example 11

2-Dimethylamino-tetradecylphospho-L-serine (I, R = C ₁₄ H ₂ ) N (CH ₁ )]) From 2-dimethylamino-tetradecyl phosphate and washed serine (IV ₁ Y ° CH (C ₈ Hs) ₂ , Z = CO-OC (CH ₃ ) J), OC (Kieselgel 60, Merck precoated plates, chloroform / methanol / acetone / ethyl acetate / water 10: 2: 4: 2: 1, v / v / v / v / v):

Documents considered:

DE-OS 3639084: (following verb, are novelty damaging described above, as well as their preparation method [Chem.Wegl:

ROPO-CH ₂ -CH-COOH OH NH ₂

R = alkyl Ce-38 [or on the C ₂ atom OH subst.] EP 122152: enzyme. Analogy method for nity-damaging prescribed verb, not protectable)

Claims (9)

1. A process for the preparation of alkyl- and alkenylphospho-L-serines of general formula I, ROPO-CH ₂ -CH-COOH OH NH ₂ NH ₂ in the R is a saturated hydrocarbon radical having 14 to 18C atoms, which is substituted by OR ¹ , SR ¹ , NR ¹ R ² , wherein R ¹ and R ² = CH ₃ , or an unsaturated hydrocarbon radical having 18 to 20C atoms means and their salts, characterized in that one a) a phosphoric acid ester of the general formula II, ROPO- (CH ₂ ) _n -X CT in the R the o.g. Has meaning and X = H, NH ₂ , NH-CH ₃ , N (CH ₃ I ₂ , N (CH ₃ J ₃ , OH, Cl, Br and η = 1 to 6), reacted with L-serine in the presence of phospholipase D or b) a phosphoric acid ester of the general formula III, R-O-rf-A III in the R the o.g. Has meaning, A = OH, Cl, Br, J and B = OH, Cl, Br, J, alkoxy or aryloxy or the salts of these compounds with a serine derivative of the general formula IV, HO-CH ₂ -CH-COOY IV NH-Z in the Y = benzyl, t-butyl, phthalimidomethyl, isopropyl, benzhydryl or another in a conventional manner, e.g. By catalytic hydrogenolysis, hydrazinolysis, treatment with HCl, Sodium thiophenolate or readily cleavable by hydrolysis protecting group and, Z = N-benzoyloxycarbonyl, N-t-butoxycarbonyl (t-BOC), N-phthaloyl or another by conventional methods such. B. catalytic hydrogenolysis, hydrazinolysis, treatment with HCl or formic acid removable protecting group, if appropriate in the presence of a condensing agent, the reaction product is optionally treated with water, the protective groups are split off after completion of the reaction and the product is optionally converted into a salt. 2. Alkyl and Alkenylphospho-L-serines of the general formula I. 3. ly-methoxy-octadecylphospho-L-serine 4. C-Q-n-octadecenylphospho-L-serine 5. Arachylphospho-L-serine 6. 2-Methoxy-tetradecylphospho-L-serine 7. 2-methylmorcapto-tetradecylphospho-L-serine 8. 2-Dimethylamino-tetradecylphospho-L-serine 9. Use of the compounds of the formula I or of a compound of the general formula VI, ROPO-CH ₂ -CH-COOH Vl OH NH ₂ where R is an alkyl radical having 10 to 16C atoms, for the preparation of cytostatic pharmaceutical agents. Field of application of the invention The invention relates to alkyl and alkenylphospho-L-serines of the general formula I,