NO169171B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE GLYCERO-3 (2) -PHOSPHO-L SERIN DERIVATIVES - Google Patents

Description

The invention relates to an analogue method for the production of new cytostatically active glycero-3(2)-phospho-L-serine derivatives.

From A. Slotboom et al., Chem. Phys. Lipids 5 (1970), 321-325

there are known production methods for various phosphatidylserines which are esterified in the 1- and 2-position with fatty acids and which, in conjunction with phosphatidylethanolamines, are to constitute anti-coagulants for blood coagulation.

In E. Eibl, Chemistry and Physics of Lipids, 26 (1980), 420-422, phosphatidylserines are described which in the 1- and/or 2-position of glycerol are esterified with fatty acids, or in the 1-

and 2-position are esterified with long-chain alcohols and must constitute the active components in blood coagulation.

In DD 222,595 phosphocholine derivatives are described, in EP-A-0 100 499 phospholipid derivatives which show anti-tumor activity. Serine derivatives are not mentioned. From Honma et al., Cancer Research 41 (1981), page 3212 ff, it is known that O-alkyl-lysophosphatidylcholines and -ethanolamines prevent the growth of M1 and H-60 cells, in contrast to this, O-alkyl-lysophosphatidylserines show no such effects.

It has now surprisingly been found that certain new glycero-3-(2)-phosphoserine derivatives exhibit an excellent cytostatic effect.

The object of the invention is consequently an analogue method for the production of new, therapeutically effective, glycero-3(2)-phospho-L-serine derivatives of general formula

in which

A stands for unsubstituted or substituted (C5-C3Q)-alkoxy, unsubstituted or substituted (C5-C30)-alkenoxy, whereby a double bond in the alkenoxy acid residue does not originate from the C atom that is bound to the oxygen atom, halogen or a group of the general the formula

in which

n stands for 0, 1, 2 or 3, or one of the two residues B and C, which is equal to or different from A, one has the meaning indicated for A or stands for hydrogen, and the other residue stands for a phosphatidyl L-serine group of the formula

on the condition that only one of the residues A, B or C stands for (<C>5-C30)-Alkoxy or (C5-<C>30)-Alkenoxy, or their pharmaceutically acceptable salts, which are characterized in that a ) reacts a glycero-3(2)-phosphoric acid derivative of the general formula

in which

A is as defined in formula (I), one of the residues

D and E have a meaning given in formula (I) for A, or stand for hydrogen, and the other residue stands for a group of the general formula

in which

X and Y are either the same and stand for hydroxy or halogen, or Y stands for lower alkoxy or aryloxy, with only one of the residues A, D or E standing for (C5-C30)-alkoxy or (C5-C30)-alkenoxy, or salts thereof with a protected L-serine derivative of the general formula

in which

Zi stands for a carboxyl and

Z2 represents an amino protecting group, preferably in the presence of a condensing agent, then the protecting groups and Z2 are removed in any order or simultaneously and the glycerophosphoric acid ester or halides formed, if any, are saponified, or

by that one

b) brings a glycero-3(2)-phosphoric acid ester of the general formula

in which

A is as defined in formula (I), one of the residues

L and M have the meaning given in formula (I) for A, or stand for hydrogen, and the other residue is a group of the general formula

in which

R^ stands for optionally with hydroxy or halogen-substituted (C1-C6)-alkyl,

R 2 , R 3 and R 4 , which are the same or different, independently represent hydrogen or methyl, and

n stands for an integer from 1 to 6, with only one of the residues Å, L or M standing for (C5-C30)-alkoxy or (C5-C30)<->alkenoxy, for reaction with L-serine in the presence of phospholipase D, and the formed compounds of the general formula I or salts thereof are isolated, and if desired, a compound of the general formula (I) obtained by method variant a) or b) is transferred, or a pharmaceutically unacceptable salt thereof, to a pharmaceutically acceptable sal±.

Glycerophospho-L-serine derivatives of the general formula I have in each case a center of chirality in the serine part of the molecule and, depending on the significance of the residues A, B and C, can exhibit a second center of chirality in the glycerol part of the molecule. Object of the invention and encompassed by the general formula I are consequently also all possible chiral and diastereomeric forms of the compounds produced according to the invention.

The term "alkoxy" used in the present description relates to straight-chain, single or multi-branched alkylated groups which preferably have 14-20 carbon atoms, and most preferably 16-18 carbon atoms. Examples of preferred carboxylic acid residues are tetra-, penta-, hexa-, hepta-, octa-, nonadecyloxy, eicosyloxy or their branched analogues. The term "alkenyloxy" stands for straight-chain, single or multi-branched, single or polyunsaturated alkenyl ether groups, which preferably have 14-20, and most preferably 16-18 carbon atoms, whereby in these alkenyl ether groups enol ether groups in which an olefinic double bond originates from the carbon atom that is bound to the oxygen atom, is expressly excluded. The above-mentioned alkoxy and also alkenoxy acid stones Å, B and C can be substituted one or more times, preferably singly substituted, as substituents preferably halogen, hydroxy, alkoxy or cyano are mentioned. The term "halogen" refers to the four halogen atoms chlorine, bromine, iodine and fluorine, whereby chlorine and fluorine are particularly preferred.

In a preferred class of compounds of the general formula I, C means the phosphatidyl-L-serine group of formula III, one of the residues A and B represents alkoxy or alkenoxy and the other residue represents halogen.

A further preferred class relates to compounds of the general formula I in which C stands for the phosphatidyl-L-serine group of formula III, one of the residues A and B stands for alkoxy or alkenoxy and the other for a terminal fluorinated alkoxy acid residue of formula II. Also preferred are compounds of the general formula I in which A stands for alkoxy or alkenoxy, B stands for hydrogen or alkoxy and C stands for the phosphatidyl-L-serine group of formula III. Finally, preferred is also the class of compounds of the general formula I in which B stands for the phosphatidyl-L-serine group of formula III, one of the residues A and C stands for alkoxy or alkenoxy and the other stands for halogen.

Particularly preferred single compounds of formula I are: 1-0-hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine 1-0-hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho -L-serine l-chloro-l-deoxy-3-0-hexadecyl-glycero-2-phospho-L-serine l-chloro-l-deoxy-2-0-hexadecyl-glycero-3-phospho-L-serine l-0-hexadecyl-2-deoxy-glycero-3-phospho-L-serine 1-0-(2,2,2-trifluoroethyl )-2-0-hexadecyl-glycero-3-phospho-L-serine l- 0-hexadecyl-2-0-(2,2,2-trifluoroethyl)-glycero-3-phospho-L-serine l-chloro-1-deoxy-2-0-octadecyl-glycero-3-phospho-L- serine 1-O-octadecyl-2-(2,2,2-trifluoroethyl)-glycero-3-phospho-L-serine 1-O-octadecy1-2-chloro-2-deoxy-glycero-3-phospho-L- serine. The glycero-3(2)-phospho-L-serine derivatives of the general formula I and pharmaceutically acceptable salts thereof are prepared according to the invention preferably by a) reacting a glycero-3(2)-phosphoric acid derivative of the gene real formula

in which

A is as defined in formula I, one of the residues

D and E have the meaning given in formula I for A, or stand for hydrogen, and the other residue stands for a group of the general formula

in which

X and Y are either equal and stand for hydroxy or halogen, or Y stands for lower alkoxy or aryloxy, provided that only one of the residues A, D or E stands for

(C5-C30)-Alkoxy or (<C>5-<C>30)-Alkenoxy, or salts thereof with a protected L-serine derivative of the general formula

in which

Zi stands for a carboxyl and

Zg represents an amino protecting group, preferably in the presence of a condensing agent, then, in any order or simultaneously, the protecting groups Z^ and Z2 are cleaved off, and the glycerophosphoric acid ester or halides formed are saponified, or

b) a glycero-3(2)-phosphoric acid ester of the general formula

in which

A is as defined in formula I, one of the residues

L and M have the meaning given in formula I for A, or stand for hydrogen, and the other residue stands for a group of the general formula

in which

Ri stands for (C1-C6)-alkyl optionally substituted by hydroxy or halogen,

R 2 , R 3 and R 4 , which are the same or different, independently represent hydrogen or methyl, and

n stands for an integer 1-6, under the condition that only one of the residues A, L or M stands for (C5-C30 )-Alkoxy or (C5-C30 )-Alkenoxy, is reacted with L-serine in the presence of phospholipase D, and the formed compound of the general formula I or its salt is isolated, and a compound of the general formula I or a pharmaceutically unacceptable salt thereof obtained according to method variant a) or b) is transferred to a pharmaceutically acceptable salt.

According to method variant a), the compounds of the general formula I can therefore be prepared from glycero-3(2)-phosphoric acid derivatives of formula IV and correspondingly protected L-serines of formula VI by methods known per se. E.g. is the production of phosphatidylserines with saturated or unsaturated acyl residues by condensation of phosphatidic acids with serines protected on the amino and carboxyl groups, or by reaction of diacylglycerol iodohydrins with protected 0-phosphoserines, whereby the protective groups on the protected phosphatidylserines obtained as intermediates can either be removed simultaneously or one after the other by suitable methods, known. Instead of phosphatidic acids, phosphatidic chlorides have also been used for reaction. Similarly, alkyl- and alkylene-substituted glycerophosphoserines can also be synthesized (A. J. Slotboom and P. P. Bonsen, Chem. Phys. Lipids 5 (1970), 301-398; M. Kates, in: E. D. Korn (publisher) "Methods in Membrane Biology", vol 8, Plenum Press, New York, 1977, page 219; H. Eibl, Chem. Phys. Lipids 26 (1980) 405-429; A. Hermetter, F. Paltauf and H. Hauser, Chem. Phys. Lipids 30 ( 1982) 35-45).

For the preparation of the new glycero-3(2)-phospho-L-serine derivatives of the general formula I, correspondingly substituted glycero-3(2)-phosphoric acids of formula IV are preferably used, in which a residue D or E stands for a group of formula V, wherein X and Y represent hydroxy.

Instead of the free acids, however, the corresponding phosphoric acid dihalides (X and Y in V: halogen) can also be used, preferably the phosphoric acid dichlorides, which are obtained by reacting the corresponding substituted glycerols with phosphoroxy halides. In the reaction of these phosphoric acid dihalides of formula IV (X and Y in V: halogen) with a protected serine of type VI, glycero-phospho-L-serine chlorides of compounds of the general formula I arise as intermediate products at the serine group, which before or after isolation and before, during or after removal of the protecting groups can be converted into compounds of the general formula I or salts thereof.

A further variant of the method according to the invention consists in bringing the protected serines of formula VI into reaction with glycerophosphoric acid ester halides of formula IV, in which a residue D or E is a group of formula V in which X represents halogen, preferably chlorine, and Y represents for lower alkoxy or aryloxy. The alkyl or aryl esters of compounds of the general formula I obtained as intermediates, protected by the serine group, can be converted by usual chemical methods such as hydrolysis and removal of the protective groups to the acids of the general formula I or salts thereof.

Glycerophosphoric acid alkyl(-aryl) ester halides of formula IV are readily available by reaction of correspondingly substituted glycerols with phosphoric acid alkyl (respectively aryl) ester dihalides.

Suitable as protected L-serine derivatives for reaction according to the invention are compounds of formula VI in which Z^ stands for a carboxyl protecting group common in peptide chemistry, usually by catalytic hydrogenolysis, hydrazinolysis, treatment with HCl, sodium thiophenolate or by hydrolysis easily removable carboxyl protecting group, such as benzyl . Such amino protecting groups are e.g. acyl groups, such as formyl, acetyl, trifluoroacetyl; alkoxycarbonyl groups such as methoxycarbonyl, tert-butoxycarbonyl, <p>,<p>,<p->trichloroethoxycarbonyl, p-iodoethoxycarbonyl; aralkyl such as benzyloxycarbonyl, p-methoxybenzyloxycarbonyl; aryloxycarbonyl such as phenoxycarbonyl; silyl groups such as trimethylsilyl; and other groups such as trityl, tetrahydropyranyl, vinyloxycarbonyl, O-nitrophenylsulfenyl, diphenylphosphinyl, p-toluenesulfonyl, benzyl etc. In and of themselves, the protecting groups Z^ and Z2 can be chosen in any way, as a rule, however, it is advantageous for the protection of the L-serine to choose such combinations of protecting groups that can be cleaved off in one reaction step, for example as a protected serine derivative of formula VI uses N-tert.butyloxy-L-serine benzhydryl ester and the like.

The reaction of compounds of formula IV with compounds of formula VI to the derivative of the compounds of the general formula I formed as an intermediate according to process step _a), still protected by the serine group, is generally carried out in such a way that one reacts a glycero-3 (2)-phosphoric acid derivative of formula IV, preferably a well-dried salt of a glycero-3(2)-phosphoric acid of formula IV, for example the pyridinium salt, with the protected serine of formula VI in a molar ratio of 1:1 to 1:5, in presence of a strong base, such as pyridine, triethylamine, Eining base, etc., and optionally a further inert, apolar organic solvent such as chloroform, acetic ester, diethyl ether, diisopropyl ether, benzene, chlorobenzene, tetrahydrofuran, etc. The reaction is preferably carried out in the presence of a suitable condensation agent, e.g. 2,4,6-triisopropylbenzene sulfochloride, at room temperature or at temperatures just above or below room temperature. The removal of the protecting groups from the protected intermediates obtained by this reaction can, depending on the selected protecting groups, take place by methods known to the person skilled in the art. For example, intermediates in the form of benzylhydryl esters protected with alkoxycarbonyl can be transferred to compounds of formula I by treatment with HC1, preferably by introducing EC1 into solutions of the intermediates in organic solvents, during cleavage of both protecting groups, any trityl protecting groups present can be removed by hydrogenolysis, others by hydrolysis, hydrazinolysis etc.

According to process step b), the compounds of the general formula I can be recovered from glycero-3(2)-phosphoric acid esters of the general formula VII by enzymatically catalyzed reaction with the aid of phospholipase D by methods known per se. Certain phospholipids have already been produced using phospholipase D (H. Eibl et al., Methods in Enzymology, 72, 1981, 632-639), whereby partially non-naturally occurring glycerophosphoric acid alkyl esters and analogues thereof are used as substrates in the reaction. In the enzymatic synthesis of phosphatidylserines. from natural phosphatidylcholines (P. Comfurius et al., Biochim. Biophys. Acta 488, 1977, 36-42) so far only modest yields of phosphatidylserines and mainly hydrolysis with formation of phosphatidyl acids have been obtained. Others, as natural substrates, have so far not been used for the production of phosphatidylserines by transesterification with the aid of phospholipase D.

Essentially, the preparation of the new glycero-3(2)-phospho-L-serine derivatives of the general formula I takes place by allowing a compound of the general formula VII to react in aqueous solution or suspension with the addition of organic solvents as dissolution mediators, e.g. ether and/or chloroform and a buffer, e.g. sodium acetate or tris buffer, at a pH of 4.8 to 8 in the presence of a calcium salt (molarity preferably 0.01 to 0.1 mol/liter) with L-serine in the presence of phospholipase D at temperatures between 10 and 50°C. A particular advantage of the method lies in the fact that unprotected serine can be used for turnover. After the reaction has taken place, this can e.g. followed by means of thin-layer chromatography, the enzyme is inactivated, advantageously by the addition of 0.1 M ethylenediaminetetraacetic acid solution, and then the glycero-3(2)-phospho-L-serine derivative of the general formula I formed is isolated and purified in the usual way, f .ex. by means of chromatographic methods, such as thin-layer, column or high-pressure liquid chromatography.

As mentioned at the outset, the general formula I includes all possible chiral and diastereomeric forms of the compounds produced according to the invention. Both with process variant a) and also with process variant b), depending on the steric relationships in the starting materials of formula IV,

respectively formula VII, either chiral or diastereomeric end products are obtained. If, in one of the two process variants mentioned, chiral starting materials or materials that do not have a chirality center are used, chiral forms of compounds of the general formula I are obtained. If, on the other hand, racemic starting materials of formula IV or formula VII are used, the reaction according to the invention with a protected L-serine derivative of formula VI, respectively with L-serines themselves, diastereomer mixtures of compounds of general formula I.

The compounds of the general formula I obtained according to method variant a) or b), or pharmaceutically unacceptable salts thereof, can be transferred to pharmaceutically acceptable salts in the usual way with inorganic or organic bases. The salt formation can, for example, be carried out by dissolving the aforementioned compounds of formula I in a suitable organic solvent, e.g. a lower aliphatic alcohol, add an equivalent amount of the desired base, ensure good thorough mixing and, after completion of salt formation, distill off the solvent in vacuo. Pharmaceutically acceptable salts are e.g. metal salts, especially alkali metal and alkaline earth metal salts, such as sodium, potassium, magnesium or calcium salts. Other pharmaceutically acceptable salts are, for example, also ammonium salts, which are derived from ammonia or organic amines, e.g. mono-, di- or tri-lower (alkyl, cycloalkyl or hydroxyalkyl) amines, lower alkylene diamines or heterocyclic bases, e.g. methylamine, diethylamine, triethylamine, dicyclohexylamine, triethanolamine, ethylenediamine, pyridine, piperidine, piperacin, morpholine, etc. Pharmaceutically unacceptable salts of the compound of the general formula I can be transferred by usual renewed salt formation to pharmaceutically acceptable salts, whereby the pharmaceutically unusable cation is replaced by a pharmaceutically usable cation. Alternatively, one can also neutralize a pharmaceutically unusable salt and then react the thereby obtained free acid with a base which gives a pharmaceutically acceptable salt.

The glycero-3(2)-phosphoric acid derivative of the general formula IV used as starting material for method variant a) is either known or can be prepared according to methods known per se (e.g. H. Brachwitz et al., Chem. Phys. Lipids 31, 1982, 33-52, A. Hermetter et al., Chem. Phys. Lipids, 30, 1982, 35-45), especially according to the details in the embodiment examples. The glycero-3(2)-phosphoric acid ester of the general formula VII used as starting material for method variant b) is also either known or can be produced by known methods (GDR patent no. 222 594 and no. 222 595).

A phospholipase D which is suitable for the enzymatic reaction can be obtained, based on already known methods, in a simple way from head cabbage, by homogenising it, filtering the homogenise and centrifuging the aqueous phase for 45 min. at 25,000 g. The clear supernatant is mixed with 2 volumes of acetone. It is decanted from the formed precipitate using a diving frit and the residue is centrifuged for 20 min. at 13,000 g. The acetone-moist enzyme-containing preparation is dried in vacuum over phosphorus pentoxide.

The compounds of the general formula I and salts thereof are biologically very active and in particular have a pronounced antitumor effect. These valuable pharmacological properties can be demonstrated in vitro and in vivo using standard methods, for example by the inhibition of the proliferation of Ehrlich-Ascites tumor cells in vitro under the influence of glycero-3(2)-phosphorus-L-serine derivatives of the general formula I is determined. In this experiment (table I), the compound according to the invention causes, for example

1-0-hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine

(connection no. 1)

1-0-hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine

(connection no. 2)

1 - chloro-l-deoxy-3-0-hexadecyl-glycero-2-phospho-L-serine

(connection no. 3)

1-0-(2,2,2-trifluoroethyl)-2-0-hexadecyl-glycero-3-phospho-L-serine (compound no. 4),

already at a very low concentration a significant inhibition of the cell growth of Ehrlich-Ascites tumor cells.

The in vitro activity of the compounds produced according to the invention is qualitatively comparable to the cytostatically active 1-0-octadecyl-2-0-methyl-glycero-3-phosphocholine, which has already found clinical use in cancer therapy (P. G. Munder et al. in: " Augmenting Agents in Cancer Therapy", pages 441-458, Raven Press, New York, 1981; W.E. Berdel et al. Cancer, 50, 1982, 2011-2015). Compared to the comparison substance, the compounds produced according to the invention have the advantage that a significant antitumor activity occurs already at significantly lower concentrations, so that in order to achieve the same cytostatic effect, significantly smaller dosage units must be administered. Due to these properties, the compounds produced according to the present invention can be expected to be advantageously used in human medicine for the treatment and prophylaxis of tumor diseases. Overall, the pronounced antitumor effect of the glycero-3(2)-phospho-L derivatives produced according to the invention is surprising, as it has hitherto been assumed

(cf. D. R. Hoffman et al., Research Commun in Chem. Pathology and Pharmacology, 44, 1984, 239-306) that the anti-tumor activity of alkylphospholipid analogues is limited to compounds containing a phosphocholine group.

The compounds of the general formula I can be used as healing agents, e.g. in the form of pharmaceutical preparations containing the compounds in admixture with a pharmaceutical, organic or inorganic inert auxiliary and/or carrier material suitable for enteral or parenteral administration, such as e.g. pharmaceutically acceptable solvents include gelatin, gum arabic, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, petroleum jelly and the like. on speech. The pharmaceutical preparations can be in solid form, for example as tablets, dragées, suppositories, capsules etc., or in liquid form, for example as solutions, suspensions or emulsions. If necessary, they are sterilized and contain auxiliary substances, such as preservatives, stabilizers and emulsifiers, salts for changing the osmotic pressure, etc. In particular, pharmaceutical preparations can contain the compounds produced according to the invention in combination with other therapeutically valuable substances. With these, the compounds produced according to the invention can be formulated with the above-mentioned auxiliary and/or carrier substances for combination preparations.

The following examples illustrate the invention in more detail: Example 1: 1- 0- Hexadecyl- 2- chloro- 2- desoxv- glvsero- 3- phospho- L- serine

1-O-hexadecyl-2-chloro-2-deoxy-glycero-3-phosphoric acid:

335 mg (1 mmol) of 1-0-hexadecyl-2-chloro-2-deoxy-glycerol is dissolved in 10 ml of dry tetrahydrofuran, 0.6 ml of pyridine is added, the resulting solution is added dropwise to a solution of 0.35 ml ( 3.755 mmol) of phosphorus oxychloride in 3.5 ml of anhydrous tetrahydrofuran at 0°C with stirring and stirred for a further 3 hours at 0°C. 16 ml of a 10% sodium bicarbonate suspension is then added, and the mixture is stirred for 15 min. , adjusted with dilute hydrochloric acid to a pH of 7 and extracted several times with ether/chloroform. The extracts are evaporated in vacuum, thereby obtaining 390 mg (92% of theoretical) crude product, which is sufficiently pure for further processing.

Rf: 0.15 ("Kieselgel 60", Alufolie Merck; CHCl3/CH30H/25# NH3 = 50:25:6, v/v/v). 1 -O-hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester: 225 mg (0.54 mmol) 1-O-hexadecyl-2-chloro-2- Desoxy-glycero-3-phosphoric acid, 0.3 g (0.81 mmol) of N-tert.butoxycarbonyl-L-serine benzhydryl ester and 0.657 g (2.35 mmol) of 2,4,6-triisopropylbenzene sulfochloride are stirred in 13 ml of anhydrous pyridine in 36 hours at room temperature. A few drops of water are added, evaporated in vacuo, further distilled several times with toluene, the residue taken up in ether, filtered and evaporated. The thus obtained crude product is absorbed on silica gel (35 g, "KG 60", Merck 40-63 µm) and eluted in two portions with 50 ml of chloroform and chloroform/methanol (9:1). Fractions are extracted after every 15 ml. Fractions 6-12 are collected and evaporated. Thereby 161 mg of pure product is obtained (38$ of theoretical).

Rf: 0.72 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/25# NE3 = 65:35:5, v/v/v).

1-O-hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine:

161 mg (0.21 mmol) of 1-0-hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.butoxycarbonyl-L-serine benzhydryl ester are dissolved in 30 ml of dry chloroform and flowed through with stirring at O^ for 15 min. of dry HCl gas. Dry nitrogen is then introduced for 1 hour. The solution is then washed with dilute aqueous ammonia and water and evaporated. The thus obtained crude product is purified over silica gel (10 g "KG 60", Merck 40-63 jjm; eluent: CHCl3/CH3OH/, 2:1 v/v with increasing methanol gradient). After collection of the fractions uniform by thin-layer chromatography and evaporation of the solvent, 38 mg of pure product are obtained (36$ of theoretical).

Rf: 0.13 ("Kieselgel 60", Alufolie Merck; CECI3/CH3OE/H2O = 50:25:4, v/v/v).

<C>22H45C1PN07(502> 02) Calc. C52.63 H9.04 N2.79

Found C51.76 H8.82 N2.60

Analogous to the procedure "described in example 1, the compounds in examples 2-9 are prepared: Example 2: 1-O-hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine

1-O-hexadecyl-2-fluoro-2-deoxy-glycero-3-phosphoric acid:

from 334 mg (1.05 mmol) l-O-hexadecyl-2-fluoro-2-deoxy-glycerol

Yield: 320 mg ($76 of theoretical)

Rf: 0.15 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/25£ NH3 = 50:25:6, v/v/v). 1-0-hexadecyl-2-fluoro-2-deoxy-glycero-3-f<p>sfo-N-tert-but-oxycarbonyl-L-serine benzhydryl ester: from 184 mg (0.46 mmol) of 1-0-hexadecyl -2-fluoro-2-deoxy-glycero-3-phosphoric acid

Yield: 190 mg of pure product ($54 of theoretical)

Rf: 0.75 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/25# NE3 = 65:35:5, v/v/v).

1-O-hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine: from 190 mg (0.25 mmol) 1-O-hexadecyl-2-fluoro-2-deoxy-glycero-3 -phospho-N-tert.butoxycarbonyl-L-serine benzhydryl ester Yield: 88 mg of pure product (72$ of theoretical)

Rf: 0.12 ("Kieselgel 60", Alufolie Merck; CHC13/CH30H/H20 50:25:4, v/v/v).

Example 3: l-chloro-l-desox. γ- 3- 0- hexadecyl- glycero- 2- phospho- L- serine

1-chloro-1-deoxy-3-0-hexadecyl-glycero-2-phosphoric acid:

from 350 mg (1.05 mmol) l-chloro-l-deoxy-3-0-hexadecyl-glycerol

Yield: 322 mg of pure product ($74 of theoretical)

Rf: 0.18 ("Kieselgel 60", Alufolie Merck; CHCl3/CE30H/25$é NH3 = 50:25:6, v/v/v). 1 - chloro-1-deoxy-3-0-hexadecyl-glycero-2-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester: from 322 mg (0.77 mmol) of 1-chloro-1-deoxy-3-0 -hexadecyl-glycero-2-phosphoric acid

Yield: 318 mg of pure product ($53 of theoretical)

Rf: 0.80 ("Kieselgel 60", Alufolie Merck; CHCl3/CH30H/25# NE3 = 65:35:5, v/v/v).

l-chloro-l-deoxy-3-0-hexadecyl-glycero-2-phospho-L-serine:

from 308 mg (0.40 mmol) l-chloro-l-deoxy-3-0-hexadecyl-glycero-2-phospho-N-tert.butoxycarbonyl-L-serine benzhydryl ester

Yield: 87 mg pure product (4356 of theoretical)

Rf: 0.13 ("Kieselgel 60", Alufolie Merck; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 4: l-chloro-l-deoxy-3-0-octadecyl-glycero-2-phospho-L-serine

l-chloro-l-deoxy-3-0-octadecyl-glycero-2-phosphoric acid:

from 417 mg (1.15 mmol) l-chloro-l-deoxy-3-0-octadecyl-glycerol

Yield: 450 mg pure product (89% of theoretical)

Ef: 0.18 ("Kieselgel 60", Alufolie Merck; CHCl3/CH30H/25# NH3 = 50:25:6, v/v/v). 1 - chlor -1 - de soxy - 3 - 0 -octadecyl -glycero-2-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester: from 320 mg (0.72 mmol) of l-chloro-l-desoxy- 3-O-Octadecyl-glycero-2-phosphoric acid

Yield: 258 mg of pure product ($45 of theoretical)

Ef: 0.80 ("Kieselgel 60", Alufolie Merck; CHCl3/CH30H/25# NH"3 = 65:35:5, v/v/v).

1-chloro-1-deoxy-3-0-octadecyl-glycero-2-phospho-L-serine:

from 258 mg (0.32 mmol) l-chloro-l-deoxy-3-0-octadecyl-glycero-2-phospho-N-tert.butoxycarbonyl-L-serine benzhydryl ester Yield: 61 mg pure product (36$ of theoretical)

Ef: 0.13 ("Kieselgel 60", Alufolie Merck; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 5: 1-O-octadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine

1-O-octadecyl-2-fluoro-2-deoxy-glycero-3-phosphoric acid:

from 346 mg (1 mmol) l-O-octadecyl-2-fluoro-2-deoxy-glycerol Yield: 324 mg pure product (7b% of theoretical)

Ef: 0.15 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/255é NH3 = 50:25:6, v/v/v).

1-O-octadecyl-2-fluoro-2-deoxy-glycero-3-phospho-N-tert.butyl-oxycarbonyl-L-serine benzhydryl ester: from 307 mg (0.72 mmol) 1-O-octadecyl-2-fluoro -2-deoxy-glycero-3-phosphoric acid

Yield: 281 mg of pure product ($50 of theoretical)

Rf: 0.75 ("Kieselgel 60", Alufolie Merck; CECl3/CH3OH/255é NH3 = 65:35:5, v/v/v).

1-O-octadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine:

from 281 mg (0.36 mmol) 1-0-octadecyl-2-fluoro-2-deoxy-glycero-3-phospho-N-tert.butoxycarbonyl-L-serine benzhydryl ester Yield: 54 mg pure product (29% of theoretical)

Ef: 0.13 ("Kieselgel 60", Alufolie Merck; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 6: 1-0-octadecyl-2-chloro-2-desoxy-glycero-3-phospho-L-serine

1-0-octadecyl-2-chloro-2-deoxy-glycero-3-phosphoric acid:

from 544 mg (1.5 mmol) l-O-octadecyl-2-chloro-2-deoxy-glycerol Yield: 597 mg pure product (90% of theoretical)

Ef: 0.16 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/25# NE3 = 50:25:6, v/v/v).

1- O-octadecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester: from 443 mg (1 mmol) 1-O-octadecyl-2-chloro-2- deoxy-glycero-3-phosphoric acid

Yield: 382 mg of pure product ($48 of theoretical)

Rf: 0.80 ("Kieselgel 60", Alufolie Merck; CHCl3/CH30H/25# NE3 = 65:35:5, v/v/v).

1-O-octadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine:

from 382 mg (0.48 mmol) 1-O-octadecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.butoxycarbonyl-L-serine benzhydryl ester

Yield 102 mg pure product (4056 of theoretical)

Ef: 0.13 ("Kieselgel 60", Alufolie Merck; CHC13/CE30H/H20 50:25:4, v/v/v).

Example 7: l-0-tetradecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine l-0-tetradecyl-2-chloro-2-deoxy-glycero-3-phosphoric acid: from 368 mg ( 1.2 mmol) 1-O-tetradecyl-2-chloro-2-deoxy-glycerol

Yield: 445 mg pure product (9656 of theoretical)

Rf: 0.14 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/2556 NH3 = 50:25:6, v/v/v). 1 - O-tetradecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester: from 425 mg (1.1 mmol) 1-0-tetradecyl-2-chloro-2 -deoxy-glycero-3-phosphoric acid

Yield: 423 mg pure product (5256 of theoretical)

Ef: 0.72 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/2556 NH3 = 65:35:5, v/v/v).

1-0-tetradecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine:

from 192 mg (0.26 mmol) 1-0-tetradecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.butoxycarbonyl-L-serine benzhydryl ester Yield: 39.5 mg pure product (3256 of theoretical )

Ef: 0.13 ("Kieselgel 60", Alufolie Merck; CHC13/CE30H/H20 = 50:25:4, v/v/v).

Example 8: 1-0-(1-methyl-heptadecyl)-2-chloro-2-deoxy-glycero-3-phospho-L-serine 1-0-(1-methyl-heptadecyl)-2-chloro-2- Desoxy-glycero-3-phosphoric acid: from 363 mg (1 mmol) 1-O-(1-methyl-heptadecyl)-2-chloro-2-desoxy-glycerol

Yield: 372 mg pure product (8456 of theoretical)

Rf: 0.15 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/2556 NH3 = 50:25:6, v/v/v).

1-0-(1-methyl-heptadecyl)-2-chloro-2-deoxy-glycero-3-phospho-L-serine : from 370 mng (0.59 mmol) 1-0-(1-methyl-heptadecyl) -2-chloro-2-deoxy-glycero-3-phospho-N-tert.butoxycarbonyl-L-serinebenz-hydryl ester

Yield: 122 mg pure product (3956 of theoretical)

Rf: 0.14 ("Kieselgel 60", Alufolie Merck; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 9: 1-0-pentyl-2-chloro-2-desoxy-glycero-3-phospho-L-serine

1-O-pentyl-2-chloro-2-deoxy-glycero-3-phosphoric acid:

from 271 mg (1.5 mmol) of 1-0-pentyl-2-chloro-2-deoxy-glycerol Yield: 250 mg of pure product (6456 of theoretical)

Rf: 0.10 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/2556 NH3 = 50:25:6, v/v/v). 1-0-pentyl-2-chloro-2-deoxy-glycero-3-phospho-N. tert.butoxy-carbonyl-L-ser inbenzhydryl ester : from 208 mg (0.8 mmol) 1-0-pentyl-2-chloro-2-deoxy-glycero-3-phosphoric acid

Yield: 407 mg pure product (8356 of theoretical)

Rf: 0.70 ("Kieselgel 60", Alufolie Merck; CHCl3/CH3OH/255é NH3 = 65:35:5, v/v/v).

1-0-penty1-2-chloro-2-deoxy-glycero-3-phospho-L-serine:

from 406 mg (0.66 mmol) l-O-pentyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.butoxy-L-serine benzhydryl ester Yield: 64 mg pure product (28% of theoretical)

Rf: 0.11 ("Kieselgel 60", Alufolie Merck; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 10:

1- O- hexadecyl- 2- deoxy- glycero- 3- phospho- L- serine

A mixture of 1 g L-serine, 1.9 ml 0.1 M acetate buffer (pH 5.6) with 0.1 M CaCl2, 40 mg l-O-hexadecyl-2-deoxy-glycero-phosphoric acid ethyl ester, 2 ml ether /chloroform (9:1, v/v) and 100 mg of one from approx. 500 g of head cabbage extracted phospholipase-D preparation is stirred intensively for 40 hours at 40°C. After cooling to room temperature, 4.35 ml of 0.1 M ethylenediaminetetraacetic acid is added. The organic solvent is removed by introducing nitrogen. The mixture is stirred with 4.3 parts by volume of chloroform/methanol (5:8, v/v) for 30 min. and the precipitated, unconverted, serine is removed by suction. The filtrate is stirred with 1 volume part of water and 3.7 volume parts of chloroform for 10 min., the organic phase is separated and evaporated. The obtained residue is separated by column chromatography on 20 g of carboxymethylcellulose ("Servacel CM 52"), the elution is carried out step by step with 75 ml of chloroform (fraction 1), then each time 500 ml of chloroform/methanol (9:1, 8:2, 7:3, 1:1, v/v), fractions 2-5. The final product is obtained from fraction 5 in pure form.

Yield: 15 mg pure product (33% of theoretical).

Rf: 0.13 ("Kieselgel 60", finished plate; CHC13/CH30H/E20 = 50:25:4, v/v/v).

Analogous to this method, the compounds in examples 11-20 are prepared.

Example 11: 1-O-hexadecyl-2-O-(2.2,2-trifluoroethyl)-glycero-3-phospho-L-serine

from 40 mg of 1-0-hexadecyl-2-0-(2,2,2-trifluoroethyl)-glycero-3-phospho-choline

Yield: 12 mg pure product (305° of theoretical)

Rf: 0.13 ("Kieselgel 60", finished plate; CHC13/CH30H/E20 = 50:25:4, v/v/v).

Example 12: 1-O-hexadecyl-2-O-(2.2.2-trifluoroethyl)-glycero-3-phospho-L-serine

from 40 mg of 1-0-hexadecyl-2-0-(2,2,2-trifluoroethyl)-glycero-3-phosphoric acid bromomethyl ester

Yield: 13.5 mg of pure product ($35 of theoretical)

Rf: 0.13 ("Kieselgel 60", ready plate; CHC13/CH30H/H20 50:25:4, v/v/v).

Example 13:

1- chloro- l- deoxy- 2- O- hexadecyl- glycero- 3- phospho- L- serine

from 40 mg l-chloro-l-deoxy-2-0-hexadecyl-glycero-3-phospho-choline

Yield: 15 mg pure product (375° of theoretical)

Rf: 0.14 ("Kieselgel 60", finished plate; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 14: 1-O-(2.2.2-trifluoroethyl)-2-O-hexadecyl-glycero-3-phospho-L-serine

from 40 mg 1-0-(2,2,2-trifluoroethyl)-2-0-hexadecyl-glycero-3-phosphocholine ■

Yield: 18 mg pure product (4456 of theoretical)

Rf: 0.13 ("Kieselgel 60", finished plate; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 15:

l- 0- eicosanyl- 2- chloro- 2- deoxy- glycero- 3- phospho- L- serine

from 40 mg of 1-0-eicosanyl-2-chloro-2-deoxy-glycero-3-phosphoric acid-n-butyl ester

Yield: 14.5 mg pure product (3456 of theoretical)

Rf: 0.15 ("Kieselgel 60", finished plate; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 16:

l- 0- triacontyl- 2- chloro- 2- deoxy- glycero- 3- phospho- L- serine

from 60 mg 1-0-triacontyl-2-chloro-2-deoxy-glycero-3-phosphoric acid ethyl ester

Yield: 14 mg pure product (3356 of theoretical)

Rf: 0.20 ("Kieselgel 60", finished plate; CEC13/CE30H/E20 = 50:25:4, v/v/v).

Example 17: 1-O-octadecyl-2-O-(2,2.2-trifluoroethyl)-glycero-3-phospho-L-serine

from 40 mg of 1-0-octadecyl-2-0-(2,2,2-trifluoroethyl)-glycero-3-phosphoric acid-2-bromoethyl ester

Yield: 13.5 g pure product (3256 of theoretical)

Rf: 0.14 ("Kieselgel 60", finished plate; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 18: 1-O-eicosanyI-2-O-(2.2.2-trifluoroethyl)-glycero-3-phospho-L-serine

from 40 mg of 1-0-eicosanyl-2-0-(2,2,2-trifluoroethyl)-glycero-3-phosphocholine

Yield 16 mg pure product (4056 of theoretical)

Rf: 0.14 ("Kieselgel 60", finished plate; CHC13/CH30E/H20 = 50:25:4, v/v/v).

Example 19: l-chloro-l-deoxy-3-0-(cis-9-octadecenyl)-glycero-2-phospho-L-serine

from 40 mg of 1-chloro-1-deoxy-3-0-(cis-9-octadecenyl)-glycero-2-phosphoric acid ethyl ester

Yield: 15.5 mg pure product (3456 of theoretical)

Rf: 0.13 ("Kieselgel 60", finished plate; CHC13/CH30H/H20 = 50:25:4, v/v/v).

Example 20: 1-O-(2-methoxy-octadecyl)2-chloro-2-deoxy-glycero-3-phospho-L-serine

from 50 mg 1-O-(2-methoxy-octadecyl)-2-chloro-2-deoxy-glycero-3-phosphoric acid ethyl ester

Yield: 16 mg pure product (2856 of theoretical)

Rf: 0.15 ("Kieselgel 60", finished plate; CHC13/CH30E/H20 = 50:25:4, v/v/v).

Claims (1)

Patent claim Analogous process for the preparation of therapeutically active glycero-3(2)-phospho-L-serine derivatives with the general formula in which A stands for unsubstituted or substituted (C5-C30)- alkoxy, unsubstituted or substituted (C5-C3Q)-alkenoxy, whereby a double bond in the alkenoxy acid residue does not proceed from the C atom which is bonded to the oxygen atom, halogen or a group of the general formula in which n stands for 0, 1, 2 or 3, or one of the two residues B and C, which are equal A or different from this, one for A has the meaning indicated or represents hydrogen", and the other residue represents a phosphatidyl-L-serine group of the formula where only one of the residues A, B or C represents (C5-C30 )-alkoxy or (C5-C3Q)-alkenoxy, or pharmaceutically acceptable salts thereof, characterized in that a) reacts a glycero-3(2)-phosphoric acid derivative of the general formula in which A is as defined in formula (I), one of the residues D and E has a meaning given in formula (I) for A, or represents hydrogen, and the other residue represents a group of the general formula in which X and Y are either the same and stand for hydroxy or halogen, or Y stands for lower alkoxy or aryloxy, with only one of the residues A, D or E standing for (C5-C3Q)-alkoxy or (C5-C30)-alkenoxy, or salts thereof with a protected L-serine derivative of the general formula in which Zi stands for a carboxyl and Z2 stands for an amino protecting group, preferably in the presence of a condensing agent, then the protecting groups Z^ and Z2 are cleaved off in any order or simultaneously and the glycerophosphoric acid ester or halides possibly formed are saponified, or by a b) bringing a glycero-3( 2)-phosphoric acid ester of the general formula in which Å is as defined in formula (I), one of the residues L and M has the meaning given in formula (I) for A, or stands for hydrogen, and the other residue is a group of the general formula in which R 1 represents (C 1 -C 6 )-alkyl optionally substituted by hydroxy or halogen, R 2 , R 3 and R 4 , which are the same or different, independently represent hydrogen or methyl, and n represents an integer from 1 to 6, wherein only one of the residues A, L or M stands for (C5-C30)-Alkoxy or (C5-C30)-Alkenoxy, for reaction with L-serine in the presence of phospholipase D, and the formed compounds of the general formula I or its salts are isolated, and if desired, a compound of the general formula (I) obtained by method variant a) or b) or a pharmaceutically unacceptable salt thereof is transferred to a pharmaceutically acceptable salt.