Phosphathizyl hydroxide compound and process for preparing same

The invention relates to novel phosphatidyl-quaternary ammonium compounds which are structurally related to phosphatidylcholine, and to the use of these novel compounds for solubilizing or emulsifying water-insoluble compounds._Z in particular cholesterol, cholesterol esters and triglycerides.

Phospholipids and phosphatidylcholine in particular are amipathic compounds in that they contain hydrophobic and hydrophilic groups or regions within one and the same molecule. Compounds with this amipathic property tend to undergo self-association in aqueous systems with the formation of micelles, which have a hydrophobic interior and a hydrophilic exterior. which have limited or no solubility in water and they can separate such insoluble compounds in the hydrophobic region of the micelle.

The outer, polar, hydrophilic region of the micelle gives the micelle complex or group water solubility. It is a well known fact that such insoluble biological compounds as cholesterol, cholesterol esters and derivatives, triglycerides and other compounds can be solubilized in phospholipid micelles. However, the solubilizing ability of a surfactant is highly dependent on the hydrophobic / hydrophilic balance in the molecule of the substance.

Thus, for example, natural phosphatidylcholine (ie lecithin) is an excellent emulsifier for a variety of insoluble biological compounds such as cholesterol, cholesterol esters and triglycerides and lecithin is widely used for many industrial purposes, for example in the food industry. Lecithin is a natural surfactant and like such, its solubilizing properties derive from its amipathic character, ie. the molecule contains a region

7711362-9 with hydrophobic character (the heterogeneous fatty acid chain) and a region with hydrophilic character (the polar head group ethyl-N-trimethylammonium group). In addition, lecithin is a zwitterion in the pH range 2-12, because it has a positively charged group (the quaternary ammonium group) and a negatively charged group (the phosphate group). This zwitterionic character stabilizes the ionic structure of lecithin against any pH variation, which would lead to a tendency to flocculate other natural detergents, i.e. other phospholipids or bile acid salts.

The naturally occurring phospholipids have limited solubilizing properties. Thus, it is known that the largest amount of cholesterol that phosphatidylcholine can solubilize corresponds to a molar ratio of about 1: 1, while little or no cholesterol ester can be solubilized by phosphatidylcholine. Thus, the new phospholipid compounds, which have modified solubilizing properties (especially those which solubilize a greater amount of both biological and industrial compounds than is possible with the natural compounds or which have other solubilizing properties) would be the most desirable and useful.

The present invention relates to novel synthetic phosphatidyl - quaternary ammonium compounds which have solubilizing, surfactant or other action which is better than or different from that of the heterogeneous, naturally occurring phosphatidylcholine, and to the use of these compounds for solubilizing and emulsifying other compounds. , in particular cholesterol, cholesterol derivatives and triglyceride compounds.

It has been found in the work underlying the present invention that in particular the solubilizing and surfactant properties of the new phosphatidyl compounds can be achieved by varying the distance between the positively and negatively charged groups, i.e. by increasing or

7711362-9 reduce the distance between the groups, for example by increasing the length of the groups of divalent radicals, for example the length of the methylene radical between the charged groups and / or by delocating the positive charge on and around the quaternary nitrogen atom, for example by exchanging one or more of the three methylene groups against other groups, for example other alkyl groups. Thus, by utilizing the zwitterionic character of natural phosphatidylcholine and charging the structure to form new compounds, modified and in some cases unexpected surfactant properties are achieved, in particular by the change and modification of the polar main group (the quaternary anronium group) and the region of the various phosphatidylcholines.

The novel compounds of the present invention are useful and interesting substitutes for lecithin in solubilizing water-insoluble compounds. In particular, the new compounds are useful as stabilizers and emulsifiers, especially in foods. The compounds can be used for emulsifying other compounds.

The novel synthetic phosphatidyl compounds of the invention are represented by the general formula:

<img file="SE442014B_D0001.tif" />

- 0 - CH₀

- 0 - CH,

<img file="SE442014B_D0002.tif" />

7711362-9 wherein a, b and c are integers 0-3 and d is an integer

1-5, however, a, b and c can not simultaneously be 0 and d can not be 2.

In the compounds of the present invention, the long chain hydrocarbon groups are composed of natural or synthetic ^C14-^C 2Q fatty acid or alcohol radicals or combinations of mixtures thereof. Useful fat radicals include both saturated and ethylenically unsaturated hydrocarbon radicals, such as those derived from fatty acids or alcohols, such as myristate, palmitate, oleate, linoleate and stearate radicals, and heterogeneous mixtures found in natural products, such as egg yolk, soybean egg yolk and similar.

By selecting suitable fat radicals and the length of these radicals, this hydrophobic character of the synthetic compound can be changed and modified to a desired, final level, for example by such selection of the said radicals that they have the same or different chain length or degree of saturation or substitution. i.e. Among the novel compounds of the present invention, the following may be mentioned, although the invention is not limited thereto: dioleate-phosphatidyl- (isopropyl-N-triethyl) - (quaternary ammonium hydroxide); dipalmitate phosphatidyl- (ethyl-N-dimethyl, ethyl) - (quaternary ammonium hydroxide); distearyl-phosphatidyl (ethyl-N-dimethylethyl) - (quaternary ammonium hydroxide); oleate palmitate phosphatidyl- (ethyl-N-dimethylethyl) - (quaternary ammonium hydroxide); dimyristate phosphatidyl- (butyl-N-dipropylmethyl) - (quaternary ammonium hydroxide); dipalmitate phosphatidyl (propyl-N-trimethyl) - (quaternary ammonium hydroxide); egg phosphatidyl- (propyl-N-trimethyl) - (quaternary ammonium hydroxide); soybean phosphatidyl- (propyl-N-trimethyl) - (quaternary ammonium hydroxide); and mixtures thereof.

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The new compounds have been designated above with derivative nomenclature, but for example the above-mentioned compound dimyristate-phosphatidyl- (butyl-N-dipropylmethyl) - (quaternary ammonium hydroxide) may also be referred to as dimyristoylphosphatidyl- (tetramethylene-N-dipropylmethyl) - (quaternary ammonium).

The new compounds can be prepared in different ways. In a preferred manner, the synthetic phosphatidyl-alkyl-N is prepared⁺the hydroxide by reaction and coupling of the polar head group with the phosphatidic acid, for example using triisopropylbenzenesulfonyl chloride in pyridine (see R. Anjea and JS Chandra. Biochem. Biophys. Acta 248, 455 (1971) and B. Sears, WC Hutton and TE Thompson , Biochem, Bicphys, Res, Caim. 60, 1141 (1974)). The ebsfatidic acid can be obtained from natural or synthetic phosphatidylcholine by digestion with the enzyme phospholipase D (see RMC Dawson, Biochem. J. 102, 76 (1967)). The modified polar head group compound is then synthesized by the general reaction method, which is represented and exemplified by the use of the quaternary ammonium alcohol as follows:

R_

IN

R₁ N Cl - (CH.) - OH R.

ii 2 η 1

<img file="SE442014B_D0003.tif" />

(CH₂)_n - ^{0H 0]}-^Θ

<img file="SE442014B_D0004.tif" />

<img file="SE442014B_D0005.tif" />

<img file="SE442014B_D0006.tif" />

^(CH2⁾n^{0H kc &amp;}

The salt form, for example the acetate form of the salt, is obtained by subjecting the halide salt to ion exchange, for example the chloride form in an ion exchange column equilibrated with the acetate ions. Thus, the method of the present invention comprises the following: synthesis of synthetic phosphatidylcholine or isolation of natural phosphatidylcholine, subsequent enzymatic cleavage of the phosphatidylcholine to phosphatidic acid, synthesis of a modified hydroxy- (quaternary alkylammonium halide), conversion of the latter into the corresponding acetene solvent, namely pyridine, used for the coupling), and covalent coupling with quaternary alkylammonium acetate at the phosphatidic acid, thereby obtaining the synthetic phospholipid modified in the polar head group. The acetate or weak acid form may also be used with acetonitrile as solvent or the iodide form may be used when the coupling solvent is a mixture of pyridine and acetonitrile in a ratio of about 1: 1.

In the above procedure for the preparation of synthetic phosphatidyl compounds, an N is reacted or coupled⁺salt, preferably a salt of a weak acid or a halogen salt of N⁺the compound with the phosphatidic acid in a common non-aqueous solvent, typically an organic polar solvent such as pyridine or acetonitrile, for example a nitrogen-containing solvent, and phosphatidylalkyl (quaternary hydroxide compound) is recovered and subjected to chromatographic purification.

The invention is described in more detail in the form of examples and illustrations by means of the preparation of certain preferred compounds, but it is of course not limited to the details given therein.

)

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DESCRIPTION OF DETAILS

Synthesis of dipalmitoyl-phosphatidyl-ammonium hydroxide compounds.

Glycerol phosphorylcholine was recovered from crude egg yolk phosphatidylcholine using the method developed by JS Chandra (Chen. Phys. Lipids 2,104 (1970)). Dipalmitoylphosphatidylcholine was synthesized according to the method developed by Cubero Robles, E. and van de Berg, D. (Biochem.

Biophys. Acta 187 5-0 (1909)) Dipalmitoylphosphatidic acid was prepared by enzymatic cleavage of dipalmitoylphosphatidylcholine by cabbage phospholipase D according to Dawson, RBC, Biochem. J. 102 76 (1967) · The correct hydroxyalkylammonium acetate was covalently bound to dipalmitoylphosphate 15 fatty acid using 2,4,6-triisopropylbenzenesulfonyl chloride as a coupling agent in the manner described by Sears and co-workers, Biochem. Biophys. Res. Comm. 60 1141 (1974). The phosphatidylcholine analog was then purified by chromatography on silica. The detailed description of the synthesis of the hydroxyalkylammonium compounds and the corresponding phosphatidylcholine compounds is found below.

A. Bipalmitoylphosphatidyl- (ethyl-K-dimethyl, ethyl) -ammonium hydroxide.

0.925 g (10.9 mmol) of dimethylethanolamine was charged to a 50 ml round bottom flask which was cooled to -10 ° C, then 1.159 g (7.43 mmol) of ethyl iodide were added with stirring. The temperature of the reaction mixture was allowed to rise to 25 ° C and to a value corresponding to room temperature and the mixture was kept in the dark for 72 hours. Then the mixture was dissolved in 2Z ml of 211-DH and the solution was poured into a column (2x40 cm) of cation exchange resin (Bio Rad 50W-X3). The column was washed with 500 ml of RK-NH 4 CH and (2-hydroxyethyl) -1T-dimethyl, the ethylammonium cation was released from the column by the addition of 300 ml of 0.5&gt; M-ITH The solution of the (2-hydroxyethyl) IT-dimethyl-ethyl-ammonium bicarbonate was evaporated to dryness and the evaporation residue was taken up in distilled water. the solution was poured into a column (2x40 cm) of cation exchange resin (Bio Rad AG1-KS) in the acetar form. The column was eluted with distilled water and the (2-hydroxyethyl) N-dimethylethylammonium acetate was evaporated to dryness. Toner layer chromatography on an isopropyl alcohol / water / 14K-luvOH system (7: 2: 1) gave only a single spot on iodine staining. Colorimetric analysis for quaternary ammonium salts gave a total yield of 70 (~, ~ mmol). 3 .5 pmol of the (2-hydroxyethyl) -1T-dimethyl-ethylammonium acetate in methanol were mixed with 275 .mu.mol of dipalmitoylphosphatidic acid and the mixture was then evaporated to dryness. The mixture was dried in high vacuum

7711362-9 ever PpO ^ all night. 760 .mu.l of 2,4,6-triisopropylbenzenesulfonyl chloride in 5 ml of dry pyridine were added to the dry mixture. The flask with the reaction mixture was plugged and heated with stirring for one hour at 65 ° C, then stirring was continued for 4 hours at room temperature. At the end of the reaction, the pyridine was evaporated from the reaction mixture. The residue was taken up in 20 ml of chloroform-methanol (2: 1) and then 5 ml of distilled water were added. The lower phase thus obtained was evaporated to dryness and the residue was taken up in chloroform. The chloroform solution was poured into a silica column (2x30 cm 3) and the phosphatidylcholine was eluted with an increasing amount of methanol in chloroform. The phosphatidylcholine gave only a single spot on thin layer chromatography. The yield based on colorimetric phosphorus analysis was 17% (46.6 μmol). Elemental analysis gave the following results:

Theoretical: G = 64.31%; Ξ = 10.98%; IT = 1.83%; P = 4.05%

Experimental: C = 64.12%; H = 11.14%; N = 1.66%; P = 3.93% B. Dipalmitoylphosphatidyl- (butyl-IT-trimethyl) -ammonium hydroxide.

1.0 g (11.2 mmol) of 4-aminobutanol was charged to a 50 ml flask and cooled to -10 ° C. 1.6 g (11.3 mmol) of methyl iodide were added with stirring. The temperature of the reaction mixture was allowed to rise to a value corresponding to that of the room temperature, and the mixture was kept in the dark for 72 hours, after which it was purified in the manner described for the (2-hydroxylethyl) -1T-dimethylethylammonium acetate. The final yield of (4-hydroxybutyl) -trimethylammonium acetate was 17% (1.8 mmol). 750 Imol (4-hydroxybutyl) -trimethylammonium acetate and 500 μmol of dipalmitoylphosphatidic acid were mixed in methanol and the mixture was evaporated to dryness. The evaporation residue was dried in high vacuum overnight, 1250 gmol of 2,4,6-triisopropylbenzenesulfonyl chloride in 10 ml of pyridine was added, the reaction mixture was heated for 1 hour at 65 ° C, stirred for a further 4 hours at room temperature and then purified as above. The final yield of dipalmitoylphosphatidyl- (butyl-1T-trimethyl) -ammonium hydroxide was 9.7 (48 μmol) based on phosphorus analysis. Only a single spot was observed on thin layer chromatography.

Elemental analysis

Theoretical: C = 64.69%; H = 11.0%; IT = 1.79%; P = 3.98%

Experimental: G = 64.93%; H = 10.72%; IT = 1.70%; P = 4.12%

G. Dipalmitoyl-phosphatidyl- (propyl-IT-trimethyl) -ammonium hydroxide.

g (26.6 mmol) of 3-aminopropanol was charged to a 50 ml round bottom flask and cooled to -10 ° C. With stirring, 3.78 g (26.6 mmol) of methyl iodide were added and the flask was plugged. The mixture temperature was allowed to rise

7711362-9 to a value corresponding to that of the room temperature. The flask was kept in the dark for 48 hours. The (3-hydroxypropyl) -trimethylammonium salt was purified and converted to the acetate salt as above. Only a single spot was observed on thin layer chromatography. The yield was according to kolo5 rimetric analysis 21% (5.5 μmol). 567 mmol (5-hydroxypropyl) -trimethylammonium acetate and 245 μmol dipalmitoylphosphatidic acid were mixed in methanol and the mixture was evaporated to dryness. The residue was dried under high vacuum over P_OFor 12 hours, then 612 μmol of 2,4,6-triisopropylbenzenesulfonyl chloride in 5 ml of pyridine were added. The reaction mixture was heated at 65 ° C^Q0 for 1 hour and then stirred for 4 hours at room temperature. Dipalmitoylphosphatidyl (propyl-N-trimethylammonium hydroxide was purified as above. Only a single spot was observed on thin layer chromatography.

Elemental analysis

Theoretical: C = 64.48.5; H = 10.92.5; IT = 1,? 0, '5; P = 3.83.5

Experimental: C = 64.31.5; H = 10.96 / 5; IT = 1.833; P = 4.053

D. Dipalmitoylphosphatidyl- (ethyl-IT-dimethyl, propyl) -monium hydroxide. 0.975 g (10.9 mmol) of dimethylethanolanine were charged to a 50 ml round bottom flask, cooled to -10 ° C and 5.61 g (33 mmol) of propyl iodide were added with stirring. The temperature of the mixture was allowed to rise to a value corresponding to that of the room temperature and the mixture was then kept in the dark for 72 hours. (2-hydroxy) -1T-dimethyl, the propyl ammonium acetate was purified as above. 50 μmol (2-hydroxy) -IT-dimethyl, propylanmonium acetate and 500 pmol dipalmitoylphosphatidic acid were mixed in methanol and the mixture was evaporated to dryness. The residue was dried under high vacuum over Ρ₉0 ^ all night. 1250 μmol of 2,4,6-triisopropylbenzenesulfonyl chloride in 15 ml of pyridine was added to the residue. The mixture was heated for one hour at 65 ° C and then stirred for 4 hours at room temperature. The purification of the dipalmitoyl-phosphatidyl- (ethyl-IT-dimethylpropyl) -ammonium hydroxide was carried out as above. In thin layer chromatography, only a single spot was obtained.

Elemental analysis

Theoretical: C = 64.69.5; H = 11,003; IT = 1.79 / 5; P = 3.9S, j

Experimental: C = 65.163; H = 11.743; IT = 1.763; F = 3.903 The synthesis of a selected number of preferred phosphatidylcholine compounds in which the hydrophilic region of the molecule has been chemically modified has been described. As a result, the hydrophobic-hydrophilic balance in the molecule changes. One criterion for this change is the relative ease of movement of these new compounds on silica-thin layer chromato40 grams. The agility of the compound is directly dependent on the structure of the molecule.

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Due to the change of the hydrophilic region in the phosphatidylcholine molecule, all the compounds described now have different migration rates. Three of the compounds (A, B, D) have greater mobility than phosphatidyl, whereas one (C) has less mobility than phosphatidylcholine. Consequently, the hydrophobic-hydrophilic balance in each of the phosphatidylcholine molecules has changed.

The synthesis of a selected number of preferred phosphatidylcholine compounds in which the hydrophilic region of the molecule has been chemically modified has been described above. As a result, the hydrophobic-hydrophilic balance in the molecule has changed. In addition, the charge density of the positively charged phosphonium atom is substantially different from that of the quaternary ammonium atom commonly found in phosphatidylcholine.

These new compounds can be used as solubilizers in the industrial and biological production of foods.

7711362-9 ,1

To demonstrate that the synthetic phosphatidylammonium hydroxide compounds of the present invention have unexpected and / or surprising properties compared to lecithin, certain tests have been performed according to the following examples. In these tests, quantitative evaluations and comparisons have been made between the ability of the synthetic compounds and lecithin to solubilize cholesterol.

Criteria for the intended solubilization are the ability of the phospholipid compounds to form micelle complexes with cholesterol that are so small that they pass through a 0.22 μm membrane filter.

Since lecithin is a naturally occurring compound that occurs in close association with cholesterol in natural mammalian membranes, one would expect lecithin to have the ability to solubilize cholesterol to a greater degree than synthetic phosphatidyl-ammonium hydroxide compounds.

Another physical property that is important for the use of phospholipid compounds as a solubilizing agent for cholesterol and other non-aqueous compounds is the ability of the compounds to remove cholesterol from lecithin model membranes saturated with cholesterol.

The predicted result for the natural product lecithin would be that lecithin not only removes more cholesterol but also achieves this at a faster rate than related synthetic compounds. However, the invention shows that with respect to both of these physical properties, the synthetic phospholipid compounds of the present invention are markedly superior to lecithin.

The test results obtained are highly unexpected and could not have been predicted on the basis of prior knowledge of the synthetic modifications carried out according to the invention in the polar head group of the synthetic compounds.

The synthetic phospholipid compounds of the present invention are decidedly superior to lecithin in its most important biological and industrial function, namely in its ability to solubilize aqueous compounds, and in particular cholesterol.

The experiments carried out show that the compounds according to the invention have an improved ability to remove cholesterol from lecithin micelles saturated with cholesterol. This is largely unexpected, for it has been assumed that lecithin was chosen during its natural development because its chemical structure was ideally suited for solubilizing such compounds as cholesterol. It is also quite unexpected that such small changes in the charge density and charge separation in the polar main group in lecithin would lead to such a sharp improvement in the physical properties of the compound, as shown by a comparison between the compound of the invention

<img file="SE442014B_D0007.tif" />

7711362-9

Example 1: Comparison of the ability to solubilize cholesterol. The ability of the synthetic compounds and lecithin to solubilize cholesterol is determined by determining how much radioactive cholesterol could react with each phospholipid to form micelles that were small enough to pass through a 0.22 μτη. filter. This filter was chosen because it is commonly used for sterilization of biological solutions. 5.50 μmol of each phospholipid and Ή-cholesterol in excess (6.12 pmol) was evaporated to dryness. The frogs were dried over F_O0 ^ in high vacuum all night. A sample containing only cholesterol was also prepared in the same manner. 4 ml of distilled water was added to each of the samples and the mixture was centrifuged at 40 ° C^CG for 5 minutes. Each of the samples was then subjected to the action of sound waves for exactly 5 minutes at 40 ° C. Immediately thereafter, 1 ml of the sample was passed through a 0.22 μm membrane filter. 0.5 ml of the upper liquid layer was introduced into 10 ml of a dioxane-based liquid-scintillation fluid (dioxane).

The values ​​given in Table I below show the ability of the tested compounds to solubilize excess amounts of cholesterol, so that phospholipid-cholesterol micelle complexes can pass through a C ', 22 μm filter. Cholesterol alone could not pass through the filter under the test conditions. Dimyristoyl lecithin was the least effective phospholipid for solubilizing cholesterol, whereas dimyristoyl-butyl27- (trimethyl) ammonium hydroxide was the most effective. The synthetic

<td colspan="3">The phospholipid compounds prepared according to the present invention were 5 to 30 times as effective as lecithin in solubilizing</td>

<td rowspan="2">of cholesterol. Association</td><td colspan="2">label I</td>

<td>Filtrate ,, pulse rate / min^a</td><td>Relative efficiency³</td>

<td>Dimyristoyllecitin</td><td></td><td> 1,0</td>

<td>Dimyristoyl-phosphatidyl-propyl 1-IT- (trimethyl) ammonium hydroxide</td><td> 371</td><td> 5,3</td>

<td>Dimyristoyl phosphatidylethyl -IT- (dimethyl, ethyl)^aammonium hydroxide</td><td> 495</td><td> 7,0</td>

Dimyristoyl-phosphatidyl ty 1-IT-C dimethyl, propyl) -

<td>ammonium hydroxide</td><td> 924</td><td> 13,2</td>

<td>Dimyristoyl phosphatidylbutyl-1- (trimethyl) ammonium hydroxide</td><td> 2109</td><td> 30,1</td>

Control sample (no phospholipid) 0

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Footnotes

a. ¾-cholesterol-poly number in the solution, son passes through the 0.22 μη filter.

b. The ratio of 3 H-cholesterol complexed with lecithin to the other phospholipids.

Example 2. Comparison of the ability to remove cholesterol from lecithin cholesterol micelles. The ability of the synthetic compounds of the present invention and lecithin to remove cholesterol from lecithin micelles saturated with cholesterol was tested by determining the amount of 3 H-cholesterol, which could be removed per unit of phospholipid tested.

Η-Cholesterol was removed from preformed membranes as described by Rothman and Davidowicz (Biochemistry 14 2309 (1975)) · Lecithin (10 μmol) and saturated amounts of hcgvakuum. 0.5 ml of distilled water was added to the dried lipids and the mixture was centrifuged for 2 minutes at 40 ° 0. The dispersions thus obtained were referred to as dispersions not subjected to the action of sound waves. 20 jimol of each of the phospholipids and a trace amount of cholesteryl oleate were evaporated to dryness and dried over Ε_ο0 ^ i hcgvakuum. 4 ml of distilled water was added to each of the phospholipid samples. The mixtures were centrifuged at 40 ° C^cC for 5 minutes and then subjected to the influence of sound waves for 15 minutes at 40^c0. These dispersions were referred to as dispersions subject to the action of sound waves. 1.2 ml of the dispersions subjected to the action of sound waves were centrifuged at 10,000 G for 5 minutes to sediment any undispersed lipid. 0.9 ml of the above-mentioned effects of sound waves subjected to the vesicles were incubated with 0.5 ml of the dispersions which were not subjected to the effects of sound waves for various lengths of time. At an appropriate time, the mixture was centrifuged at 10,000 G for 5 minutes to separate the dispersions subjected to sound waves from those not subjected to sound waves. 0.5 ml of the upper liquid layer was then counted in the liquid counting tube. The 3 H: 3 C ratio represents the amount of cholesterol removed per phospholipid unit.

Inheritance data such as the -LiXc ratio were plotted in a graph as a function of time in hours. As in Example 1, dinyristoylphosphatidyl-butyl-N- (trimethyl) -ammonium hydroxide was found to be the most effective compound in terms of both the rate at which the removal occurred and the degree of removal. Also, the other synthetic compounds tested were superior to dimyristoyllecithin in removing cholesterol.