WO1994005676A1 - Novel phospho and phosphono-(n-acyl)-serines and their production - Google Patents

Abstract

The invention relates to novel phospho and phosphono-(N-acyl)-serines of the general formula (I), including all enantiomeric and diastereomeric forms and all pharmaceutically applicable salts of these compounds. The compounds (I) are produced by acylising the amino group of the corresponding phospho or phosphono serines or by causing a phosphate or phosphanate to react with a carboxyl-protected N-acyl-serine and subsequently separating the protective group. The compounds (I) are cytostatically and virustatically effective.

Description

New phospho- or phosphono- (N-acyl) -serines and their production

1. Description

The invention relates to new phospho- or phosphono- (N-acyl) serines of the general form. I including all enantiomeric and diastereomeric forms as well as all pharmaceutically acceptable salts of these compounds. The invention further relates to a process for the preparation of the compounds I and their pharmaceutical use. The field of application of the invention is therefore the chemical and pharmaceutical industries

It is known that alkyllysophosphatidylserines are physiologically highly effective, naturally occurring bioregulators (A. Bruni. And G. Toffano, Pharmacol. Res. Comm. 14, 469-484 (1982)). It is also known that synthetic alkyllysophosphatidylserines and also alkylphosphoserines (EP 229 128) can have an influence on cell proliferation. So far nothing is known about the synthesis and the physiological activity of N-acyl derivatives of the above-mentioned compounds.

The aim of the invention was to provide new phospho- or phosphono- (N-acyl) -serines which are able to inhibit the cell proliferation of tumor cells and which are therefore suitable for pharmaceutical use.

The objective is solved according to claims 1, 8 and 11, the subclaims are preferred variants

The invention relates to new phospho- or phosphono- (N-acyl) -serines of the general formula I

OH NH-CO-X

wherein is a straight-chain or branched, saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano-substituted hydrocarbon radical having 1-22 C atoms and in which

CH-B CH-B

CH ₂ - ^CH 2 "°"

is what

A saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano-substituted (C ₅ -C ₃₀ ) alkoxy or alkenoxy, or a group of the general formula

0- (CH ₂ ) _n -CF ₃ ,

where n stands for 0-3, means

B has one of the meanings given for A or is hydrogen or

R represents an alkyl or alkoxy radical having 6-30 carbon atoms, which is straight-chain or branched, saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano.

The compounds of the general formula I in which R denotes a glycero analog have two chiral centers in the molecule and can thus form 4 diastereomers. The connections ^ in which R is alkyl, have a chiral center in the serine head group and are therefore known to exist in two enantiomeric forms. The invention also relates to these possible enantiomeric and diastereomeric forms. The compounds of the general formula I can be prepared from O-alkylglycerophosphoserine analogs and alkylphosphoserines or the corresponding phosphono compounds by acylation of the amino group. A gentle process is the conversion with so-called activated esters of the general formula III, which are customary in peptide chemistry. The reaction is carried out in an inert solvent, preferably chloroform or dimethylformamide, with the addition of an equivalent base, preferably triethylamine, at 5-60 ° C., preferably at room temperature.

The compounds of the general formula V used as starting compounds can likewise be obtained in a manner known per se by acylation with the compounds of the general formula III, the OH group not having to be protected on account of the selectivity of the acylating agent. The compounds of the general formula V can also be prepared by silylating the OH group of a carboxyl-protected serine with hexamethyldisilazane, then acylating on nitrogen with an acid chloride or anhydride and splitting off the silyl protective group with methanol. The carboxyl protecting group of the compounds of the general formulas V and VI is a group which can be split off in a suitable manner, preferably a group which can be hydrated off, preferably benzyl.

The resulting compound of the general formula V can be condensed to a compound of the general formula VI at room temperature without prior isolation, but after adequate drying with a compound of the general formula IV in the presence of 2,4,6-triisopropylbenzenesulfochloride in pyridine.

The carboxyl protecting group of the compounds VI is removed in a gentle manner, in the presence of a group which can be split off by hydrogenolysis, primarily with hydrogen over the palladium / activated carbon catalyst, and this is obtained Compounds of the general formula I according to the invention which can then be isolated in a known manner and optionally further purified, for example by chromatography.

The compounds of general formula I and their salts are biologically active and have a pronounced antitumor activity, which was investigated in vitro on Ehrlich ascites tumor cells. In order to determine the selectivity of the antiprolerative effect, the growth inhibition of bone marrow cells was also examined in vitro by the compounds according to the invention. The compounds according to the invention show a surprising selectivity in their anti-proliferation activity. The ID _5Q values for the growth inhibition of bone marrow cells in vitro are many times higher than those for Ehrlich ascites cells, which is advantageous for the use of the compounds according to the invention as anti-cancer agents.

Table I lists the inhibition of cell growth of Ehrlich-Aszi¬ tes tumor cells and bone marrow cells by selected compounds of general formula I:

Table 1

ID ₅₀ values of compounds of the general formula I on Ehrlich ascites tumor cells and bone marrow cells (mouse)

1 hexadecylphospho- (N-acetyl) -L-serine

2 2-chloro-2-deoxy-l-0-hexadecyl-glycero-3-phospho- (N-acetyl) -L-serine

3 1-O-Hexadecy1-2-0- (2, 2,2-trifluoroethyl) glycero-3-phospho- (N-acetyl) -L-serine Since the compounds according to the invention have, in addition to an excellent antitumor activity in vitro, also a surprisingly good selectivity, ie they leave the growth of normal cells in the effective concentration range almost unaffected, advantageous use in human medicine for the treatment and prophylaxis of tumor diseases is possible.

The compounds of general formula I and their salts can preferably be used as cytostatic and virostatic in a 3-5% mixture with pharmaceutically customary carriers and additives.

The invention will be explained in more detail below by means of exemplary embodiments.

EXAMPLES

example 1

Hexadecylphospho- (N-acetyl) -L-serine (Formula I, R = C ₁₆ H ₃₃ 0, X = CH ₃ )

320 mg (0.72 mmol) of hexadecylphospho-L-serine and 167 mg (1.44 mmol) of N-acetoxy-5-norbornene-2,3-dicarboximide are added with 1 ml of triethylamine in 200 ml of chloroform for 48 hours at room temperature stirred and the reaction followed by thin layer chromatography. The resulting amide function can be detected as a bruise by treating the thin layer chromatogram with chlorine gas (5 min at room temperature) and then briefly immersing it in an acetic acid solution of 4,4'-tetramethyldiamino-diphenylmethane in water. The reaction mixture is evaporated at 30 ° C. on a rotary evaporator and taken up in methanol. A pH of about 8 is set with ammoniacal methanol and the solution is evaporated on a rotary evaporator at 30 ° C. The residue obtained is suspended in anhydrous acetone, washed well with acetone and suction filtered. The 300 mg of crude product obtained are purified by column chromatography (CM 52 Whatman, Na ⁺ form). The elution takes place with Chloroform / methanol with 200 ml each 9: 1, 8: 2, 7: 3. The size of the fraction is 50 ml. 205 mg (57% of theory) of chromatographically pure ammonium salt of hexadecylphospho- (N-acetyl) -L-serine are obtained from fractions 10-15. TLC (silica gel 60, Merck prefabricated plates CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.45

Example 2

Hexadecylphospho- (N-palmitoyl) -L-serine (Formula I, R = C ₁₆ H ₃₃ 0, ^{X = C} 15 ^H 31>

(N-Palmitoyl) -L-serine benzyl ester

200 mg of L-serine benzyl ester hydrochloride (0.86 mmol) and 396 mg (0.95 mmol) of N-palmitoyloxy-5-norbornene-2,3-dicarboximide are stirred for 48 hours in dimethylformamide at room temperature with the addition of 2 equivalents of triethylamine. The solution is concentrated in vacuo on a rotary evaporator and taken up in ice-cold acetone. The crude product thus obtained is recrystallized from n-hexane and 202 mg (54% of theory) of chromatographically uniform product are obtained.

Hexadecylphospho- (N-palmitoyl) -L-serine benzyl ester

A solution of 40 mg (0.13 mmol) of hexadecyl phosphate (IV, R = C ₁₆ H ₃₃ 0) in 1.5 ml of pyridine is concentrated in vacuo and the pyridinium salt thus obtained is dried in vacuo over phosphorus pentoxide. Then it is dissolved in 10 ml of anhydrous pyridine. 121 mg (0.27 mmol) (N-palmitoyl) -L-serine benzyl ester, dried over phosphorus pentoxide in vacuo, are dissolved in 5 ml of anhydrous pyridine and this solution is added to the first solution. Add 160 mg (054 mmol)

Triisopropylbenzenesulfochlorid and the mixture is stirred for 24 hours under anhydrous conditions at room temperature. After adding a few drops of water, the mixture is stirred for a further 60 min. The solution is then evaporated to dryness at 30 ° C. on a rotary evaporator and several times with dry toluene rotated again. The residue is extracted with dried ether and washed with ether until no more phosphate-containing compounds appear on the thin layer chromatogram of the washing solution. The ether solutions are combined and concentrated in vacuo. The residue is suspended with acetone and the crystals obtained are filtered off with suction. The crude product obtained in this way is recrystallized from methanol and 51 mg of pure product (52% of theory) are obtained.

Hexadecylphospho- (N-palmitoyl) -L-serine

50 mg hexadecylphospho- (N-palmitoyl) -L-serine benzyl ester (0.07 mmol) are dissolved in 50 ml glacial acetic acid. 0.5 g of palladium-activated carbon catalyst is added to the solution. Hydrogen gas is introduced with stirring for 1 h. After the hydrogenation, nitrogen is passed in for 5 min, then the catalyst is filtered off through a hard paper filter and washed several times with chloroform / methanol 2: 1 (v / v). The washing solution solutions are combined and concentrated in vacuo. The residue is taken up in acetone and 39 mg of hexadecylphospho- (N-palmitoyl) -L-serine are obtained (83% of theory). TLC (silica gel 60, Merck prefabricated plates CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.59

Example 3

Hexadecylphosphono- (N-acetyl) -L-serine

(N-acetyl) -L-serine benzyl ester

500 mg of L-serine benzyl ester hydrochloride (2ramol) are boiled under reflux in 20 ml of hexamethyldisilazane. The solution is concentrated in vacuo on a rotary evaporator. The residue is taken up in 20 ml of pyridine and, after addition of 5 ml of acetic anhydride, stirred at 0 ° C. for 1 hour. After adding 5 ml of methanol, the mixture is stirred for a further hour and then the solution is concentrated on a rotary evaporator. The residue is re-routed twice with toluene and the crude product from dried ether recrystallized. This gives 405 mg (N-acetyl) -L-serine benzyl ester (83% of theory) of a chromatographically uniform product.

Hexadecylphosphono- (N-acetyl) -L-serine benzyl ester

The synthesis is carried out analogously to hexadecylphospho- (N-palmitoyl) -L-serine benzyl ester in Example 2 from hexadecylphosphonate and (N-acetyl) -L-serine benzyl ester. 53% of theory of a chromatographically uniform product is obtained.

Hexadecylphosphono- (N-acetyl) -L-serine

The synthesis is carried out analogously to hexadecylphospho- (N-palmitoyl) -L-serine in Example 2 by hydrogenation of hexadecylphosphono- (N-acetyl) -L-serine benzyl ester. 61% of the theory of a chromatographically uniform product is obtained. TLC (silica gel 60, Merck finished plates) CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.45

Example 4

Dodecylphospho- (N-acetyl) -L-serine

According to Example 1 from dodecylphospho-L-serine and N-acetoxy-5-norbornene-2,3-dicarboximide DC (silica gel 60, Merck finished plates) CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8 : 4, (v / v / v / v): Rf = 0.42

Example 5

2-chloro-2-deoxy-l-0-hexadecyl-glycero-3-phospho- (N-acetyl) -L-serine

According to Example 1 from 2-chloro-2-deoxy-l-0-hexadecyl-glycero-3-phospho-L-serine and N-acetoxy-5-norbornene-2,3-dicarboximide TLC (silica gel 60, Merck finished plates) CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.45

Example 6

l-θ-hexadecyl-2-θ- (2,2,2-trifluoroethyl) glycero-3-phospho- (N-acetyl) -L-serine

Corresponding to example 1 from l-0-hexadecyl-2-0- (2,2,2-trifluoroethyl) -glycero-3-phospho-L-serine and N-acetoxy-5-norbornene-2,3-dicarboximide

TLC (silica gel 60, Merck finished plates) CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.39

Example 7

Dodecylphospho- (N-palmitoyl) -L-serine

According to Example 2 from dodecyl phosphate and (N-palmitoyl) -L-serine benzyl ester

TLC (Kieselgel 60, Merck prefabricated panels)

CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.6

Example 8

2-chloro-2-deoxy-l-0-hexadecyl-glycero-3-phospho- (N-palmitoyl) - L-serine

According to Example 2 from 2-chloro-2-deoxy-l-0-hexadecyl-glycero-3-phosphate and (N-palmitoyl) -L-serine benzyl ester. TLC (silica gel 60, Merck finished plates) CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.55 Example 9

l-θ-hexadecyl-2-θ- (2,2,2-trifluoroethyl) glycero-3-phospho- (N-palmitoyl) -L-serine

According to example 2 from l-0-hexadecyl-2-0- (2,2,2-trifluoroethyl) glycero-3-phosphate and (N-palmitoyl) -L-serine benzyl ester DC (silica gel 60, Merck finished plates) CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.6

Example 10

Dodecylphosphono- (N-palmitoyl) -L-serine

According to example 2 from dodecylphosphonate and (N-palmitoyl) -

L-serine benzyl ester

TLC (Kieselgel 60, Merck prefabricated panels)

CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.6

Example 11

Hexadecylphosphono- (N-palmitoyl) -L-serine

Corresponding to example 2 from hexadecylphosphonate and (N-palmitoyl) -L-serine benzyl ester DC (silica gel 60, Merck finished plates) CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.6

Example 12

Hexadecylphosphono- (N-capryl) -L-serine

According to Example 2 from hexadecylphosphonate and (N-capryl) -

L-serine benzyl ester

TLC (Kieselgel 60, Merck prefabricated panels)

CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.58 Example 13

Dodecylphosphono- (N-capryl) -L-serine

According to Example 2 from dodecylphosphonate and (N-capryl) -L-serine benzyl ester

TLC (Kieselgel 60, Merck prefabricated panels)

CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.55

Example 14

Dodecylphosphono- (N-acetyl) -L-serine

According to Example 3 from dodecylphosphonate and (N-acetyl) -L-serine benzyl ester

TLC (Kieselgel 60, Merck prefabricated panels)

CHC1 ₃ / CH ₃ 0H / H ₂ 0 / acetic acid, 50: 30: 8: 4, (v / v / v / v): Rf = 0.42

Claims

2. Patent claims 1. New phospho- or phosphono- (N-acyl) -serines of the general formula I including all enantiomeric and diastereomeric forms and all pharmaceutically acceptable salts of these compounds,

wherein X denotes a straight-chain or branched, saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano hydrocarbon radical having 1-22 C atoms and in the

CH-B CH-B CH - CH ₂ -0- is what saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano-substituted (C ₅ -C ₃₀ ) alkoxy or alkenoxy, or a group of the general formula 0- (CH ₂ ) _n -CF ₃ , where n stands for 0-3, means B has one of the meanings given for A or is hydrogen or b) R is an alkyl or alkoxy radical having 6-30 C atoms which is straight-chain or branched, saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen , Hydroxy, alkoxy or cyano is substituted. 2. Hexadecylphospho- (N-acetyl) -L-serine 3. Hexadecylphospho- (N-palmitoyl) -L-serine 4. 2-chloro-2-deoxy-l-0-hexadecyl-glycero-3-phospho- (N-acetyl) -L-serine 5. 2-Chloro-2-deoxy-l-0-hexadecyl-glycero-3-phospho- (N-palmitoyl) -L-serine 6. l-O-Hexadecyl-2-O- (2,2,2-trifluoroethyl) glycero-3-phospho- (N-acetyl) -L-serine 7. l-O-Hexadecyl-2-O- (2,2,2-trifluoroethyl) glycero-3-phospho- (N-palmitoyl) -L-serine 8. A process for the preparation of the compounds of general formula I, characterized in that a compound of general formula II,

OH NH. in which R has the meaning given for the compounds I, with an activated ester of the general formula III, Y-O-CO-X III where X has the meaning given for the compounds I and

can be in the presence of a base to compounds of general Formula I implements or a compound of the general formula IV R-P-OH IV OH in which R has the meaning given for the compounds I, or the salt thereof with a compound of the general formula V,

NH-CO-X where X has the meaning given for the compounds I and Z is preferably benzyl or another suitable protective group which can be removed in the presence of a Condensing agent to a compound of general formula VI

implements, then the. Protective group is split off and the product is optionally converted into a salt. 9. The method according to claim 8, characterized in that 2,4,6-triisopropylbenzenesulfochloride is used as the condensing agent in the reaction of the compounds of the general formula IV with compounds of the general formula V. 10. The method according to claim 8, characterized in that the splitting off of the protective group Z from compounds of the general formula VI is carried out by hydrogenation on a palladium / activated carbon catalyst. 11. cytostatic and virostatic which contains one or more compounds of the general formula I as active ingredient. 12. cytostatic and antiviral according to claim 11, characterized in that it contains hexadecylphospho- (N-acetyl) -L-serine as the active ingredient. 13. cytostatic and antiviral according to claim 11, characterized in that it contains 2-chloro-2-deoxy-l-O-hexadecyl-glycero-3-phospho- (N-acetyl) -L-serine as the active ingredient. 14. cytostatic and antiviral according to claim 11, characterized in that it is 1-O-Hexadecy1-2-0- (2,2,2-trifluoroethyl) glycero-3-phospho- (N-acetyl) -L-serine as active ingredient ent¬ contains. 15. cytostatic and antiviral according to claim 11, characterized in that it contains dodecylphosphono- (N-acetyl) -L-serine as the active ingredient. 16. cytostatic and antiviral according to claim 11, characterized in that it contains hexadecylphosphono- (N-acetyl) -L-serine as the active ingredient. 17. cytostatic and antiviral according to claim 11, characterized in that it contains dodecylphosphono- (N-capryl) -L-serine as the active ingredient. 17. cytostatic and antiviral according to claim 11, characterized in that it contains hexadecylphosphono- (N-capryl) -L-serine as the active ingredient. 18. cytostatic and antiviral according to claim 11, characterized in that it contains dodecylphosphono- (N-palmitoyl) -L-serine as the active ingredient. 19. cytostatic and virostatic according to claim 11, characterized in that it contains hexadecylphosphono- (N-palmitoyl) -L-serine as the active ingredient.