DD289892A7 - PROCESS FOR THE PREPARATION OF ALKYL PHOSPHONESERINES - Google Patents

Abstract

The invention relates to a process for preparing alkylphosphonoserines of the general formula I. The compounds are obtained by reacting alkylphosphonic acids with N-substituted serine esters and subsequent removal of the protective groups of the alkylphosphonoserine esters and by phospholipase D-catalyzed transesterification of alkylphosphonic acid esters with serine. alkylphosphonic; serine ester; Phospholipase D; transesterification}

Description

O RPO-CH ₉ -CH-COOH

OH NH ₂

in the pure aliphatic hydrocarbon radical having 8 to 22 carbon atoms, and of their salts. These compounds represent potential Cytcstatica and Virostatica. Fields of application of the invention are the chemical and plant industries.

Characteristic of the known state of the art

It is known that phosphatidylserines can be synthesized by condensation of phosphatidic acids with doubly protected serine and subsequent cleavage of the protective groups of the dinucleotide-protected phosphatidylserine derivatives formed in the serine part (AJ Slotboom et al., Chem. Phys. Lipids, 5 1970] 301-379). This Mothode has also been applied to other serine-containing phospholipid (H. Brachwitz et al., DDR patent DD 238979 A1 [1986]). It is also known that phosphatidylserine analogs of phosphatidic acid analogues and serine can be prepared by phospholipase D-catalyzed transesterification [P.Comfurius et al., Biochem. Biophys. Acta, 488 [1977] 36-42; Brachwitz et al., DDR patents DD 238978 A1, DD 239208 A1, DD 239209 A1, DD 239405 A1 [all 1986]). The compounds of the invention of type 1 have not been described in the literature so far. These are novel compounds for which no manufacturing methods exist so far.

Object of the invention

It is the object of the invention to develop a process for the preparation of the alkylphosphonoserines of general formula I.

Explanation of the essence of the invention

According to the invention, the alkylphosphonoserines of the general formula I are prepared by reacting alkylphosphonic acids of the general formula II

Q OH Il •

R-P Il

OH

in which R has the meaning already mentioned, with an N-substituted serin ester of the general formula III,

I o-χ

NH-Y

in which X and Y are protective groups whose meanings have already been mentioned, to the Alkylphosphonoserin Derivaien the general formula IV

Q O

It ^

RPO-CH ₃₁ -CH-C II ^N

CIH NH-Y OX

converts and from these the protective groups X and Y cleaved again by methods known per se.

^IV

The Alkylphosplionsäuren used as Ausgangsvorbindungon II can be ζ. Example, by first synthesizing the Alkylphosphonsäuredimethylester from a corresponding alkyl bromide R-Br and trimethyl phosphite, these converted by reaction with a mixture of trimethylsilyl chloride and NaJ In acetonitrile in the alkylphosphonic acid bistrimethylsilylester and hydrolyzed the latter by water at room temperature. The reaction according to the invention of the alkylphosphonic acids II with a serine derivative of the general formula III is generally carried out in such a way that the compounds Ii In the salt of a tertiary base, for. As pyridine, and this is preferably in the presence of a condensing agent, for. B. 2,4,6-Triisopropylbenzolsulfochlorid, under anhydrous conditions, moist in the presence of a solvent, for. As pyridine, brings with the serine derivative to the reaction. It is expedient here to use the reaction components in a molar ratio of 1: 1 to 1: 2 for the reaction. The reaction, which generally takes place already at room temperature, is suitably followed by thin-layer chromatography. After the reaction has ended, water is added to the reaction mixture to remove the remaining acid chloride, stirred for 30 minutes, concentrated to dryness, and the residue is extracted exhaustively with diethyl ether. The Alkylphosphonsäureserinester IV obtained by concentration of the ether extracts are purified by column chromatography on silica gel. The conversion of the compounds IV in the final stage I is carried out according to the invention in such a way that in the solutions of the compounds IV in an inert solvent, for. B. chloroform, under anhydrous conditions with cooling, preferably at temperatures between -5 ° C to + D ⁰ C, dehydrated hydrogen chloride gas, this allowed to act for about 0.5 to 3 hours and finally removed by introducing dried nitrogen from the solutions. The residue obtained after concentration of the solution is treated with a mixture of chloroform / methanol / aqueous ammonia to give the diammonium salts of the compounds I, which are isolated in the usual way. This method allows the synthesis of the L- or D-serine ester I as well as the racemates.

Another variant of the invention consists in the enzymatic synthesis of Alkylphostihonoserine the general formula Here are alkylphosphonic the general formula V,

O Ü-Z

μ y

R-P V

in which R and Z have the meanings already mentioned, transesterified with L-serine in the presence of phospholipase D and the resulting alkylphosphonoserines I isolated by customary methods. In detail, this reaction is carried out by reacting a solution or suspension of Alkylphosphonsäureesters V, z. As the alkylphosphonocholine, in a very highly concentrated solution of L-serine · η an aqueous buffer system, for. D. vigorously shaking acetate or Tris buffer at pH values between 4.8 and 8.5 in the presence of calcium ions (0.01 to 0.1 M) in the presence of phospholipase D at temperatures between 10 ^° C. and 50 ° C. diethyl ether or a mixture of the latter with another organic solvent, preferably choloroform, is added to the reaction mixture, in which case the transesterification takes place in a period of from 0.5 to 24 hours to deactivate the phospholipase D, a 0.1 M EDTA solution added to the reaction mixture and then the resulting final product I isolated in the usual manner.

The phosphonates prepared according to the invention have a certain structural relationship with the physiologically active natural phosphatidylserines, but differ from these in the absence of the glycerol components with the enzymatically readily cleavable fatty acid residues and in the presence of a CP bond instead of a COP bond and thus in an increase in biostability.

The novel compounds prepared according to the invention have a potent antiproliferative effect on Ehrlich ascites tumor cells. Table 1 shows the growth inhibition of these tumor cells under the influence of different concentrations.

Table 1: Growth inhibition (%) of Ehrlich ascites tumor cells under the influence of different concentrations

Compound% inhibition at a concentration of

1x10 " ⁴ 3X10" ⁵ ! ΧΙΟ " ⁵ 3x10" ⁶ M

1. n-hexadecyl 89 45 13 a phosphono-L-serine Second n-Decylphosphono- 99 81.7 38.2 14.7 L-serine Third n-octadecyl 100 78.3 56.7 40.1 phosphono-L-serine 4th Arachylphosphono- 97.2 81.5 35.7 21.5 L-serine

This invention will be illustrated by the following examples.

embodiments example 1

n-hexadecylphosphono-L-serine (I, R = C ₁₁ H ₃₁ )

n-hexadecylphosphonic acid (II, R = C ₁₈ H ₃₃ ):

A mixture of n-hexadecylbromide (4.89g, 16mmol) and freshly distilled trimyl phosphite (2.48g, 20mmol) is stirred for 24 hours at 116 ° C. Subsequently, the excess trimethyl phosphite is distilled off in vacuo and the residue stored for 24 hours over phosphorus pentoxide in vacuo. The ent indene n-Hexadecylphosphonsäuredimethylester

is isolated from the residue by extraction twice with 10mt methanol. The two methanol extracts are combined and concentrated in vacuo. The oily, colorless residue thus obtained, which still contains n-hexadecyl bromide in addition to the n-hexadecylphosphonic acid dimethyl ester, is further reacted in 4.7 ml of anhydrous acetonitrile with trimethylsilyl chloride (1.02 g, 9.4 mmol) and sodium iodide (1.4 g, 9.4 mmol ). The reaction is carried out with stirring at room temperature and lasts 15 minutes,

The sodium chloride formed in the reaction is filtered off. The filtrate is concentrated in vacuo, and the resulting residue is treated with 4 ml of water, whereby n-hexadecylphosphonic acid is formed by hydrolysis. In order to remove the ₃ Me simultaneously formed SiOSiMe _3, several times with ethanol / water (1: 1, V / V) is redistilled in vacuo. After treating the residue with 7 ml of dried acetone, 346 mg of n-hexadecylphosphonic acid II (R = C ₁₆ H ₃₃ ) are kept.

TLC gel column 60, Merck precast plates, chloroform / methanol / water, 65: 25: 4, v / v / v): R f = 0.15 Elemental analysis calculated for C ₁₆ H ₃₃ PO ₃ (306.42): C 62 , 71, H 11,51; found C 62.81, H 11.89 (%).

n-Hexadecylphosphono-Nt-butoxycarbonyl-L-serine benzhydryl ester dV, R = C ₁₆ H ₃₃₁ H = CH (C ₆ H ₆ I ₂ , Y = t-BOC):

A solution of 92mg (0.3 mmol) of n-hexadecylphosphonic (II, R = C ₁₆ H ₃₃ ) in 1 ml of pyridine is concentrated in vacuo and the pyridinium salt thus obtained is dried in vacuo over phosphorus pentoxide in vacuo. Then it is dissolved in 5ml anhydrous pyridine. 356 mg (0.96 mmol) of protected serine (MI ₁ X = CH (C _e H ₆ ) 2.Y = CO-OC (CH ₃ I ₃ ), dried over phosphorus pentoxide in vacuo, are dissolved in 4 ml of anhydrous pyridifene The reaction mixture is stirred under anhydrous conditions at room temperature for 24 hours, after which the mixture is concentrated in vacuo, distilled several times with toluene and the residue is dried over phosphorus pentoxide in vacuo The latter is then extracted with 15 ml of diethyl ether and washed three times with 4 ml of diethyl ether The ether solutions are combined and concentrated under reduced pressure 118 mg of n-hexadecylphosphonoserine derivative IV are obtained from the residue thus obtained by recrystallization from acetonitrile Column chromatography on 8 g of silica gel 60 (particle size 0.04-0.063 mm, 230 to 400 mesh, Merck) used chloroform and the solvent mixtures for elution contain 0.5% aqueous 25% ammonia. Elution is with 50ml chloroform, 50ml chloroform / methanol, 98: 2, v / v, 200ml chloroform / methanol, 95: 5, v / v, 200ml chloroform / methanol, 90:10, v / v. The fraction size is 15ml. Fractions 15-20 contain 72mg of pure hexadecylphosphono-Nt-butoxycarbonyl-L-serine benzhydryl ester.

TLC (Kieselgel 60, Merck precast plates, chloroform / methanol, 80:20, v / v): Rf = 0.2.

Elemental analysis calculated for C ₃₇ H ₆₁ N ₂ PO ₇ (NH ₄ -SaIz, 676.87): C, 65.65; H, 9.08, N, 4.14; found C 65.44, H 9,15 N 3,73 (%).

n-Hexadecylphosphono-L-serine:

69 mg (0.1 mmol) of the described compound IV are dried in vacuo over phosphorus pentoxide and then dissolved in 13 ml of anhydrous chloroform. Dry nitrogen is passed through this solution for 10 minutes, followed by hydrogen chloride gas at ⁰ ° C. for 20 minutes under anhydrous conditions. Thereafter, the reaction vessel is tightly closed and stirred at 0 ° C for 1 hour. To remove the hydrogen chloride, nitrogen is then passed through the reaction mixture for 1 hour at room temperature. Thereafter, the reaction mixture is concentrated in vacuo and 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, shaken and separated after phase separation, the resulting organic phase , The aqueous phase is extracted three times with 5 ml of chloroform / methanol / water, 2: 1 -0.18, v / v / v, the organic solutions are combined and concentrated in vacuo. To remove residual water is distilled several times with 1 ml of benzene / ethanol, 2: 3, V / V.

The resulting product, hexadecylphosphono-L-serine I, we I dried over phosphorus pentoxide in vacuo and then washed with acetone.

Yield: 43mg (95%).

To remove traces of ammonium chloride, the substance is chromatographed on 2.2 g Kieselgcl 60 (particle size 0.04-0.063 mm, 230 to 400 mesh, Merck). The column is equilibrated with chloroform / methanol / aqueous 25% ammonia, 80: 20: 0.5, v / v / v. Elution is with 20ml chloroform / methanol / aqueous 25% ammonia, 80: 20: 0.5, v / v / v, 20ml chloroform / methanol / aqueous 25% ammonia, 75: 25: 0.5, v / v / V, 20ml chloroform / methanol / aqueous 25% ammonia, 70: 30: 0.5, v / v / v, 20ml chloroform / methanol / aqueous 25% ammonia, 65: 35: 0.5, v / v / V, 200ml chloroform / methanol / aqueous 25% ammonia, 60: 40: 0.5, v / v / v. The compound is obtained from fractions 7-19 by concentration. (Fraction size 20ml).

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.

Visualization is with molybdate reagent and with ninhydrin reagent.

Elemental analysis calculated for Ci ₉ H ₄₆ N ₂ PO ₆ (NH ₄ -SaIz, monohydrate, 428.55): C, 53.24, H, 10.58, N, 6.54; found C 53.80, H 10.55, N 5.73 (%).

FAB mass spectrometry (anion spectrum): m / z414, (M - 2H + Na) ⁺ ; m / z 392, (M - H) ⁺ ; m / z 305 (M - CH ₂ CH [NH ₂ ] COOH) ⁺ ; Scale spectrum: m / z416, (M + Na) ⁺ ; m / z438, (M -t-2Na) ⁺ .

Example 2

Enzymatic Synthesis of n-Hexadecylphosphono-L-serine (I, R = C ₁₆ H ₃₃ )

0.8g L-serine (7,6mmol) are dissolved in 1.52 ml of 0.1 M acetate buffer (pH 5.6) containing 0.09 M of CaCl ₂ is dissolved at 45 ⁰ C. To this solution are added 31 mg (0.075 mmol) of n-hexadecylphosphonocholine, 1.6 ml of diethyl ether / chloroform (9: 1, v / v, ethanol free) and 209 mg of a phospholipase D enzyme preparation prepared from white cabbage, having an activity of 0, 9 U / ml (1 unit [U] sets at 27 ⁰ C 1 μΜοΙ substrate per minute) in the reaction mixture. The mixture is stirred at 45 ^° C. for 2.2 hours. After cooling to room temperature, 3.31 ml of 0.1 M EDTA solution are added and the organic solvents are removed by passing in nitrogen. The residue left In the aqueous phase is stirred with 4.3 times the volume of chloroform / methanol (5: 8, V / V) for 30 minutes and sucked from the insoluble (L-serine). The filtrate is treated with 1 volume of water and 3.7 volumes of chloroform, the mixture shaken for 10 minutes, the organic phase separated, concentrated in vacuo and the

residue obtained on carboxymethyl cellulose (CM52 Whatman, Na * form) separated by column chromatography. The elution is carried out successively with 75 ml of chloroform, 300 ml of chloroform / methanol, 9: 1.8: 2.7: 3, then 1750 ml of chloroform / methanol, 1: 1 (each v / v). The fraction size is 60 ml. From fractions 20-39, 6 mg of pure n-hexadecylphosphono-L-serine (I, R "C ₁₈ H ₃₃ ) are obtained

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 3

n-Decylphosphono-L-serln

According to Example 1 from n-decylphosphonic acid and protected serine (formula III, X = CH (C ₆ H ₆ ) ₂ , Y = CO-OC - (CH ₃ J ₃ ), DC

(Silica gel 60, Merck precast plates, chloroform / methanol / acetone / acetic acid / water 10: 2: 4: 2: 1, v / v / v / v / v): Rf = 0.1 Yield: 28.7%

Example 4

n-Octadocylphosphono-L-serln

According to Example 1 from n-octadecyiphosphonic acid and protected serine (formula III, X = CH (C _e H ₆ ) ₂ , Y = 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): Rf = 0.1

Yield: 31.8%

Example 5 Arachylphosphono-L-serine According to Example 1 from arachylphosphonic and protected serine (formula III, X = CH (C ₆ H) ₂ , Y = CO-OC (CH ₃ I ₃ , DC

(Silica gel 60, Meck-Fertic® plates, chloroform / methanol / acetone / acetic acid / Wassor 10: 2: 4: 2: 1 v / v / v / v / v): Rf = 0.1 Yield: 26.0%

Claims (5)

1. Process for the preparation of alkylphosphonoserines of the general formula I, O
RPO-CH.-CH-COOH, OH NH ₂ in which the radical R is an aliphatic hydrocarbon radical having 8 to 22 carbon atoms, and salts thereof, characterized in that
a) an alkylphosphonic acid of the general formula II, O.OH
" / in which the radical R has the meaning already mentioned, with an N-substituted serin ester of the general formula III, HO-CH ₀ -CH-C ... NH-Y ° " ^X in the X benzyl, t-butyl, phthalimidomethyl, isopropyl, benzhydryl or another in a conventional manner, for. B. by catalytic hydrogenolysis, hydroquinolysis, treatment with HCl, Natriumthiophenolat or by hydrolysis readily cleavable protecting group, Y N-benzoxycarbonyl, N-t-butoxycarbonyl, N-phthaloyl or another, by conventional methods, such as. As 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 X and Y of the resulting compounds of the general formula IV O O RPO-CH ₀ -CH-C IV ι ² I OH NH-Y OX splits off in a conventional manner and the resulting alkylphosphoserines optionally subsequently converted into their salts or
b) that an alkylphosphonic ester of the general formula V, 0 0-Z
RP V in which R has the meanings already mentioned and Z is an alkyl radical having 1 to 6 carbon atoms, which may also be substituted by OH, Cl, Br, or the grouping "(CH ₂ ) _n N - B where A, B and C are methyl groups of the hydrogen atoms and η = 2-6, brings with serine in the presence of phospholipase D for the reaction and isolated the resulting compounds of formula I or their salts by conventional methods. 2. n-hexadecylphosphono-L-serine 3. n-decylphosphono-L-serine 4. n-Octadecylphosphono-L-serine 5. Arachylphosphono-L-serine. Field of application of the invention The invention relates to a process for the preparation of novel alkylphosphonoserines of the general formula I