DD238979A1 - PROCESS FOR THE PREPARATION OF O-ALKYL GLYCEROPHOSPHOSERIN ANALOG - Google Patents

Abstract

The invention has for its object to develop a suitable, technically applicable method for the preparation of new 0-Alkylglycerophosphoserin analogs. According to the invention, these compounds are obtained by condensing 0-alkylglycerophosphoric acid analogues or their salts, the dihalogen derivatives or the ester halide derivatives of these compounds with a protected serine and the resulting protected O-alkylglycerophosphoserin analogues or their halides or alkyl (aryl ) converts ester into the 0-Alkylglycerophosphoserin analogs or their salts. Field of application of the invention is the chemical or pharamazeutische industry.

Description

OH Wd ₂

I. II

in which R is a saturated or unsaturated alkyl radical having 5 to 30 carbon atoms, which may also be substituted, X is a halogen, preferably chlorine, and their salts, characterized in that O-alkylglycerophosphoric acid analogues of the general formula III and IV, in which R and X have the meanings given above

CHp-O-R CHp-O-R

I ι 0

CH-X ₀ CK-0-ϊ-Λ

Y / i »3

CH ₂ -OPA CHp-X

III IV

and A = B = OH or halogen, preferably chlorine, and B = alkoxy or aryloxy, with a serine of general formula V in which Y is benzyl,

HO-CH ₂ -CI-I-COOX KH-Z

t-butyl, phthalimidomethyl, isopropyl, benzhydryl or other in the usual manner, e.g. B. by catalytic hydrogenolysis, hydrazinolysis, treatment with HCL, sodium thiophenolate or readily cleavable by hydrolysis protecting group, Z is N-benzoxycarbonyl, N-t-butoxycarbonyl, N-phthaloyl or another, by conventional methods, such. catalytic hydrogenolysis, hydrazinolysis, treatment with HCI or formic acid removable protecting group, condenses according to known methods, preferably in the presence of a condensing agent and the protecting groups Y and Z of the resulting compounds, optionally after hydrolysis, in a conventional manner and then optionally cleaved into the salt over.

Field of application of the invention

The invention relates to a process for the preparation of O-alkylglycerophosphoserin analogues of the general formulas I and II,

CH ₀ -OR CH ₀ -OR

I ² . I ² O

CH-X CH-O-i-O-CHp-QH-COOH

"I, I, HH <CHp-O-P-O-CHp-CH-COOH CHp-X

P oH

II

in which R is a saturated or unsaturated alkyl radical having 5-30 C atoms, which may also be substituted, X is a halogen, preferably chlorine, and their salts.

The compounds of this type are analogs of naturally occurring phosphatidylserines. The latter have significant importance as cell components in the biological process and find because of their physiological activity increasingly, for. B. as components of liposomes, use.

Field of application of the invention is the chemical or pharmaceutical industry.

Characteristic of the known technical solutions

It is known that phosphatidylserines with saturated or unsaturated acyl radicals can be prepared by condensation of phosphatidic acids with serines protected by the amino group and carboxyl group or by reaction of diacylglyceroliodohydrins with protected O-phosphoserines, the protecting groups of the protected phosphatidyl series formed as intermediates either simultaneously or sequentially be cleaved with suitable methods. Instead of the phosphatidic acids, phosphatidic acid dichlorides were also used for the reaction. Analogously, alkyl- and alkenyl-substituted glycerophosphoserines can also be synthesized (AJ Slotboom and PP Bonsen, Chem. Phys. Lipids, 5 [1970] 301-398; M. Kates, in: ED Korn [ed.] Methodes in Membrane Biology, Vol. 8. Plenum Press, New York 1977, pp. 119 et seq .; H. Eibl, Chem. Phys. Lipids, 26 [1980] 405-429; A. Hermetter, F. Paltauf and H. Hauser, Chem. Phys. Lipids, 30 [1982] 35-45). Methods for the preparation of the halogen-containing O-alkylglycerophosphoserin analogues of the general formulas I and II are not yet known.

Object of the invention

The object of the invention is the development of a suitable method for the preparation of O-AIkylglycerophosphoserin analogues of the general formulas I and II and their salts.

Explanation of the essence of the invention

According to the invention, the O-alkylglycerophosphoserin analogues of the general formulas I and II are prepared by reacting the O-alkylglycerophosphoric acid analogues of the general formulas III and IV in which A = B = OH and R and X.

GH ₀ -OR CH ₀ -OR

I ² I ^ Q

CH-X GH-O-P-A

IQ I B

CH ₀ -OPA CH ₀ -X

B ^d

III IV

have the meanings already mentioned, or the salts of these compounds, with a serine of the general formula V,

HO-CH ₂ -CH-COOY

NH-Z

in the Y, benzyl, t-butyl, phthalimidomethyl, isopropyl, benzhydryl or another in a conventional manner, e.g. B. by catalytic hydrogenolysis, hydrazinolysis, treatment with HCL, sodium thiophenolate or by hydrolysis easily removable protecting group, Z is an N-benzyloxycarbonyl, 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, according to known methods, preferably in the presence of a condensing agent, condensed and the protective groups Y and Z of the resulting O-Alkylglycerophosphoserin analogs of the general formulas Vl and VII, in R, X, Y, Z have the meanings already mentioned, or their salts, according to methods known per se, which are dependent on the type of protective group used, eg according to the methods already listed above, splits off.

CH ₀ -OR CH ₀ -OR

\ ^d I ² O

CH-X-CH-CH ₀ -CH OPO-COOY

I 0 j OH ^d '

CH ₀ -OPO-CH-COOY _'v

^Urapid NH-Z ^d -

VI VII

Instead of the O-Alkylglycerophosphorsäuren analogs of the type IM and IV (A = B = OH) can be used according to the invention, the corresponding phosphoric dihalides (formula III, IV, A = B = halogen, preferably chlorine), which can be, for. B. according to known methods, by reacting O-Alkyldesoxyhalogenglycerol with phosphorus oxyhalide obtained. For example, condensation of the O-alkyldeoxyhalogenoglycerophosphoric acid dichlorides with a protected type V serine produces the phosphoric acid chlorides of compounds Vl and VII which, optionally after isolation, are converted to the O-alkylglycerophosphoserin analogues I and II or their salts by hydrolysis and deprotection become.

A further variant of the process according to the invention consists in obtaining the protected serines Vl and VII by condensation of the O-alkylglycerophosphoric acid ester halide analogues of the general formula III and IV (A = halogen, preferably chlorine, B = alkoxy, aryloxy), obtainable e.g. by reaction of the corresponding O-Alkyldesoxyhalogenglycerole with phosphoric alkyl (or aryDester halides, with protected serines of type V brings the reaction and the resulting alkyl or aryl esters of the compounds of type VI and VII in the O-alkyl glycerophosphoserin analogues I and II or their salts converts.

The process according to the invention enables both the synthesis of the racemates and the synthesis of the optical stereoisomers.

In general, the preparation of the intermediate compounds of types VI and VII is carried out by mixing the well-dried pyridinium salts of the O-alkyl glycerophosphoric acid analogues III and IV (A = B = OH) with the protected serine V (Y = benzhydryl, Z = N - t - butoxycarbonyl) in a molar ratio of 1: 1 to 1: 5, in the presence of an organic base, for example pyridine, wherein additionally a further organic solvent, for. As chloroform, may be present in the presence of a condensing agent, for. B. 2,4,6-Triisopropylbenzensulfochlorid, advantageously reacted at room temperature. After workup and chromatography of the reaction mixture, the compounds obtained Vl and VII are converted into the compounds I and II by z. B. in the solutions of compounds Vl and VII in organic solvents, for. B. chloroform, at ^{0 0} C dry HCL initiates. After removal of the protective groups HCI is removed by introducing dry nitrogen, the resulting hydrochlorides of the compounds I and II converted by treatment with NH ₃ -containing solutions in the stable ammonium salts and these purified by chromatography.

The compounds prepared are biologically highly active.

You have pronounced antitumor activity z. Against Ehrlich ascites tumor cells.

Thus, their in vitro efficacy is comparable to the cytostatically effective iO-alkyl ^ -O-methylglycero-S-phosphocholine (A, see Table), which has already found clinical application in cancer therapy (PG Münder, M. Modolell, W Bausert, HFOettgen and O.Westphal in: "Augmenting Agents in Cancer Therapy," pp. 441-458, Raven Press, New York 1981; WEBerdel, H. Schlehe, U. Fink, B. Emmerich, PAMaubach, HP Emslander, S.Daum and J. Rastetter, Cancer, 50 [1982] 2011-

In contrast to this compound, an effect already occurs at lower concentrations (see Table 1). This demonstrates that the antitumor effect of alkyl phospholipid analogues is by no means - as widely believed -

(D.R. Hoffman, J.D. Stanley, R. Berchtold and F.Snyder, Research, Commun. In Chem. Pathology and Pharmacology, 44 [1984] 293-306) for compounds having a phosphocholine moiety.

Table 1

Inhibition of proliferation (%) of Ehrlich ascitic tumor cells in vitro by O-alkyl glycerophosphoserine analogs

Compound concentration (μΜ)

100 20 10 5 2 1

(R = C ₁₆ H ₃₃ , 95 80 55 30 10 16

(R = C ₁₆ H ₃₃ , 95 82 51 18 13 10

(R = C ₁₆ H ₃₃ , 93 81 68 38 17 15

A ⁺ 100 62 23 4 0 0

A = 1-O-octadecyl-2-O-methylglycero-3-phosphocholine

The invention will be illustrated by the following examples.

Exemplary embodiments Example 1

(I, R = C ₁₆ H ₃₃ , X = Cl)

a) 2-chloro-2-deoxy-1-O-hexadecylglycero-3-phosphoric acid (III, R = Ci ₆ H ₃₃ , X = Cl) (synthesis based on H. Brachwitz et al., Chem. Phys. Lipids , 31 [1982] 33-52) A solution of 0.335 g (1 mmol) of 2-chloro-2-deoxy-1-O-hexadecylglycerol in 10 ml of dried tetrahydrofuran is added after addition of 0.6 ml of dried pyridine to a solution of 0, 35 ml (3.755 mmol of phosphorus oxychloride in 3.5 ml of tetrahydrofuran are added dropwise with stirring and cooling with ice, the reaction mixture is stirred for a further 3 hours while cooling and then 10% NaHCO ₃ solution is added The solution is extracted repeatedly with ether and chloroform, and after concentration of the combined dried extracts 0.4 g (crude product) of 2-chloro-2-deoxy-1-O-hexadecylglycero-3-phosphoric acid are obtained. TLC (Merk-Alufolie, Kieselgel 60 ): Rf 0.15 (CHCl ₃ ZCH ₃ OH / 25% aq NH ₃ , 50: 25: 6, v / v / v).

b) 2-Chloro-2-deoxy-1-O-hexadecylglycero-3-phospho-Nt-butoxycarbonyl-L-serine benzhydryl ester (VI, R = C ₁₆ H ₃₃ , X = CH (C ₆ Hg) ₂ , Z = t BOC)

(Analog A. Hermetter et al., Chem. Phys. Lipids, 30 [1982] 35-45)

A mixture of 0.225 g (0.54 mmol) of 2-chloro-2-deoxy-1-O-hexadecylglycero-3-phosphoric acid, 0.3 g (0.81 mmol) of Nt-BOC-L-serine benzhydryl ester (V, Y = CH (C ₆ H ₅ J ₂ , Z = t-BOC), 0.657 g (2.35 mmol) of 2,4,6-triisopropylbenzenesulfonyl chloride and 13 ml of dried pyridine are stirred for 36 hours at room temperature After addition of a few drops of water the reaction mixture becomes distilled off in vacuo, distilled several times with toluene, the residue taken up in ether, filtered off from the undissolved and the filtrate concentrated, the resulting crude product is washed with 50 ml CHCl ₃ , 50 ml CHCl ₃ / CH ₃ OH (9: 1, v / v). on 35g silica gel 60 (Merck, 0.040-0.063 mm) chromatographies.

From fractions 6-12 (15 ml each) 0.161 g of the clean compound are obtained. DC (Merck aluminum foil, silica gel 60): Rf 0.72 (CHCl 3 / CH 3 OH / 25% aq NH _3., 65: 35: 5, v / v / v).

c) Cleavage of the protecting groups

Into a solution of 0.161 g (0.21 mmol) of 2-chloro-2-deoxy-1-O-hexadecylglycero-3-phospho-Nt-BOC-L-serine benzhydryl ester in dried chloroform, dry HCL gas is introduced under ice-cooling for 15 minutes and the mixture stirred for 1 hour. Thereafter, HCl is removed by introducing (1 hour) nitrogen. The solution is washed successively with dilute aqueous NH ₃ solution and water and concentrated. The resulting crude phosphatidylserine analog (0.15 g) is purified by column chromatography on 10 g of silica gel 60 (eluant CHCl3 / CH3OH, 2: 1, v / v, with increasing methanol content). From fractions 12-28 (15 ml each), 0.038 g of 2-chloro-2-deoxy-1-O-hexadecylglycero-3-phospho-L-serine are obtained. TLC (Merck precast plates, layer thickness 0.25 mm): Rf 0.13 (CHCl ₃ / CH ₃ OH / H ₂ 0.50: 25: 4, v / v / v). C ₂₂ H ₄₅ CIPNO ₇ (502.02) Ber.C 52.63 H 9.04 N 2.79

Gef.C51.76 H 8.82 N 2.60

Example 2

2-deoxy-2-fluoro-1-0-hexadecylglycero-3-phospho-L-serine (I, R = C ₁₆ H _33, X = F) The reaction of 2-deoxy-2-fluoro-1-O- hexadecylglycerol and phosphorus oxychloride analogously to Example 1 a, the 2-deoxy-2-fluoro-1-0-hexadecylglycero-3-phosphoric acid (III, R = Ci ₆ H ₃₃₁ X = F) is synthesized and this in the form of pyridinium analogously to Example 1 b with the serine V (Y = CH (C ₆ H ₅ I ₂ , Z = t-BOC) to 2-deoxy-2-fluoro-1-O-hexadecylglycero-3-phospho-Nt-BOC-L-serine benzhydryl ester (Vl , R = C ₁₆ H ₃₃ , X = F, Y = CH (C ₆ H ₅ J ₂ , Z = t-BOC) condensed TLC (Merck aluminum foil, silica gel 60): Rf 0.73 (CHCl ₃ ZCH ₃ NH / 25% aqueous NH ₃ , 65: 35: 5, v / v / v). Cleavage of the protective groups analogously to Example 1c results in the formation of the O-alkylglycerophosphoserine analogue I (R =Ci ₆ H ₃₃ , X = F) TLC (Merck precast plate, Kieselgel 60): Rf 0.13 (CHCl ₃ ZCH ₃ OHZh ₂ O, 50: 25: 4, vZvZv).

Example 3

i-Chloro-i-deoxy-SO-hexadecylglycero ^ -phospho-L-serine (II, R = C ₁₆ H ₃₃ , X = CL) By reacting i-chloro-i-deoxy-SO-hexadecylglycerol and phosphorus oxychloride analogously to Example 1 a is the 1-ChIoM-deoxy-SO-hexadecylglycero-S-phosphoric acid (IV, R = C ₁₆ H ₃₃ , X = Cl) synthesized and these in the form of the pyridinium salt alnalog Example 1 b with the serine V (Y = CH (C ₆ H ₅ I ₂ , Z = t-BOC) to the 1-chloro-deoxy-SO-hexadecylglycero ^ -phospho-Nt-BOC-L-serine ester (VII, R = C ₁₆ H ₃₃ , X = Cl, Y = CH (C ₆ Hg) ₂ , Z = t-BOC), TLC (Merck aluminum foil, Kieselgel 60): RF 0.70 (CHCl ₃ ZCH ₃ OH 25% aq NH ₃ , 65: 35: 5, The removal of the protective groups analogously to Example 1c results in the formation of the O-alkylglycerophosphoserine analogue (R =C ₁₆ H ₃ XX = Cl) TLC (Merck precast plate, silica gel 601 → 0.13 (CHCl ₃ ZCH ₃ OHZh ₂ O, 50: 25: 4, vZvZv).

Claims (1)

Invention claim: 1. Process for the preparation of O-alkylglycerophosphoserine analogues of the general formulas I and II, GH ₀ -OR CHp-OR CH-X rs CH-OPO-CH ₀ -CH-GOOH j "0. ι QH CH ₀ -OPO-CH ₀ -CH-COOH CHp-X ^Wd 2