CH636595A5 - DERIVATIVES FOR THE PREPARATION OF ACID N- (phosphonoacetyl) -L-ASPARTIC NEW AND DERIVATIVES OBTAINED. - Google Patents

Description

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1. Compound, characterized by its belonging to the following group: disodium salt of N- (phosphonoacetyl) -L-aspartic acid and the corresponding P-ethyl and dibenzyl esters; the di-benzyl ester of N- (phosphonoacetyl) -L-aspartic acid and the corresponding N.N'-dibenzylethylenediamine and cyclohexylamine salts; the tetraethyl ester of N- (phosphonoacetyl) -L-aspartic acid and the corresponding dimethyl-P, P-diethyl ester; the calcium, piperazine and cyclohexylamine salts of N- (phosphonoacetyl) -L-aspartic acid.

2. Compound according to claim 1, which is the disodium salt of N- (phosphonoacetyl) -L-aspartic acid.

3. The compound according to claim 1, which is the disodium salt of the N-(phosphonoacetyl) -L-aspartic acid P-ethyl ester.

4. The compound according to claim 1, which is the disodium salt of the dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid.

5. A compound according to claim 1, which is the dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid.

6. A compound according to claim 1, which is the salt of N, N'-dibenzylethylenediamine of the dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid.

7. The compound according to claim 1, which is the cyclohexylamine salt of the dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid.

8. A compound according to claim 1, which is the tetraethyl ester of N- (phosphonoacetyl) -L-aspartic acid.

9. A compound according to claim 1, which is the dimethyl-P, P-diethyl ester of N- (phosphonoacetyl) -L-aspartic acid.

10. Compound according to claim 1, which is the calcium salt of N- (phosphonoacetyl) -L-aspartic acid.

11. A compound according to claim 1, which is the piperazine salt of N- (phosphonoacetyl) -L-aspartic acid.

12. Compound according to claim 1, which is the cyclohexylamine salt of N- (phosphonoacetyl) -L-aspartic acid.

13. Process for preparing the dibenzyl ester of PALA, characterized in that the L-aspartic acid is reacted with benzyl alcohol and p-toluenesulfonic acid to obtain the dibenzyl ester of p- toluenesulfonate of L-aspartic acid, then reacting the dibenzyl ester of p-toluenesulfonate of L-aspartic acid with triethylamine, adding phosphonoacetic acid chloride to produce the dibenzyl ester of PALA and finally separating said ester from chloride of unreacted phosphonoacetic acid.

14. The method of claim 13, characterized in that one separates the dibenzyl ester of PALA chloride phosphonoacetic acid which has not reacted by washing with water, to remove the chloride from phosphonoacetic acid.

15. The method of claim 13, characterized by the use of perchlorethylene as an esterification medium for the reaction of L-aspartic acid with benzyl alcohol and p-toluene sulfonic acid.

16. Process for the preparation of the tetrasodium salt of PALA, characterized in that the dibenzyl ester of PALA is prepared according to the process of claim 13, that the dibenzyl ester of PALA is hydrolyzed by an aqueous solution of sodium hydroxide to obtain a mixture containing the tetrasodium salt of PALA.

17. Method for preparing the tetrasodium salt of PALA, characterized in that a mixture containing the tetrasodium salt of PALA is prepared according to the method of claim 16, said mixture is subjected to an ion exchange process for to obtain free PALA acid, the free acid is titrated with sodium hydroxide to a pH of 9.2 to form the tetrasodium salt of PALA and the salt is recovered.

18. Process for the preparation of the disodium salt of PALA, characterized in that a mixture is prepared containing the tetrasodium salt of PALA according to the process of claim 16, the said mixture is dissolved in glacial acetic acid, the solution obtained with ethanol to precipitate the disodium salt of PALA and that said disodium salt of PALA is recovered.

19. Process for the preparation of the disodium salt of PALA, characterized in that the dibenzyl ester of PALA is prepared according to the process of claim 13, said ester is reacted with N, N'-dibenzylethylenediamine to produce the salt of N, N'-dibenzylethylenediamine of the dibenzyl ester of PALA and that said salt is subjected to hydrolysis to produce a mixture containing the tetrasodium salt of PALA, said mixture is dissolved in glacial acetic acid, the solution obtained is diluted with ethanol to precipitate the disodium salt of PALA and the said disodium salt of PALA is recovered.

20. Process for the preparation of the disodium salt of PALA, characterized in that the L-aspartic acid is reacted with benzyl alcohol and p-toluenesulfonic acid to obtain the dibenzyl ester of p-toluenesulfonate L-aspartic acid, said ester is reacted with triethylamine, phosphonoacetic acid chloride is added to produce the dibenzyl ester of PALA, cyclohexylamine is added to produce the cyclohexylamine salt of said dibenzyl ester of PALA, then reacting said cyclohexylamine salt with sodium hydroxide to produce the tetrasodium salt of PALA, reacting the tetrasodium salt of PALA with acetic acid to produce the disodium salt of PALA, and precipitate the disodium salt obtained, twice in water, the second precipitation including the dropwise addition of an aqueous solution of said disodium salt to a vortex of vigorously stirred ethanol, which causes the elimination of impurities retained in the form of acetic acid and sodium acetate.

21. Process for the preparation of the cyclohexylamine salt of the dibenzyl ester of PALA, characterized in that the L-aspartic acid is reacted with benzyl alcohol and p-toluene / sulfonic acid to obtain l The dibenzyl ester of p-toluene sulfonate of L-aspartic acid, phosphonoacetic acid chloride is added to produce the dibenzyl ester of PALA and the said ester is reacted with cyclohexylamine.

The free tetraacid, N- (phosphonoacetyl) -L-aspartic acid (designated in this description by the abbreviation PALA) is a known product. The present invention relates to new compounds of N- (phosphonoacetyl) -L-aspartic acid (PALA), especially the disodium salt of PALA and to processes for the preparation of N- (phosphonoacetyl) -L acid compounds -aspartic. A particular aspect of this invention relates to the large-scale preparation of the known tetrasodium salt and the new disodium salt of N- (phosphonoacetyl) -L-aspartic acid. The known tetraacid, N- (phosphonoacetyl) -L-aspartic acid (PALA), was prepared for the first time by Stark et al., "J. Biol. Chem. ”, 246, 6599 (1971). The tetrasodium salt of PALA is known as an anti-tumor agent, according to the literature and particularly according to "Cancer Research", 36, 2720 (1976), mentioned here for reference. For example, the survival time of mice carrying P388 type intraperitoneal leukemia was extended up to 64% (when treated with the tetrasodium salt of PALA in a range of 188 to 750 mg / kg, i.p.). Lewis sarcoma of the lung in mice is very sensitive to the action of the tetrasodium salt of PALA during i.p. doses of 240 to 490 mg / kg. Mice carrying B16 melanins survived 77 to 86% longer than the controls when they had been treated with tetrasodium salt of PALA (490 mg / kg, i.p.).

While the synthesis of tetraacid PALA is direct, the preparation of tetrasodium salt, especially in quantities of the order of 1 kg, has proven to be a major problem. The methods of this invention are particularly well suited to the production of such amounts.

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The new PALA compounds are the disodium salt of N- (phosphonoacetyl) -L-aspartic acid and the corresponding P-ethyl and dibenzyl esters; the dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid and the corresponding N, N'-dibenzyl-ethylenediamine and cyclohexylamine salts; the tetraethyl ester of N- (phosphonoacetyl) -L-aspartic acid and the corresponding dimethyl- (P, P-diethyl) ester; the calcium salt of N- (phosphonoacetyl) -L-aspartic acid and the salts of piperazine and cyclohexylamine of N- (phosphonoacetyl) -L-aspartic acid.

The new products of this invention have an anti-tumor activity in vivo or are intermediates in the production of PALA compounds which are active against tumors and which, unlike the known tetrasodium salt of PALA, are not hygroscopic or are mobile and non-adherent powders. .

Some of these PALA compounds exist in anhydrous form and some exist in solvated form (including hydrated forms). Both forms are suitable for the applications of this invention. The disodium salt of PALA, produced by the methods of this invention, is a hydrate whose hydration water content differs from one preparation to another between approximately 0.2 to 2 mol of water. The product may contain certain solvents such as ethanol (between 0.1 and 0.5 mol approximately), acetic acid (between 0.03 and 0.4 mol approximately) and sodium acetate (approximately 0.2 mol). Acetic acid and ethanol can be removed by lyophilization. Two or three lyophilizations allow the total elimination of the solvents. The disodium salt of PALA obtained by these methods can also contain significant amounts of trisodium salt, that up to approximately 30 to 40% of trisodium salt. All solvates of the disodium salt of PALA, as well as the mixtures of disodium salt and trisodium salt of PALA, should be understood as included in the term disodium salt of PALA (equivalent term) used here, since the forms of this product are interchangeable in their use as an anti-tumor agent, the formulations of which are covered by this invention. In its use as an antitumor agent, the disodium salt of PALA exhibits an activity and a toxicity equivalent to that of the known tetrasodium salt of PALA. The disodium salt of PALA can be used for its antitumor activity, as described in this presentation, in the form of pharmaceutical compositions and of a suitable pharmaceutical carrier. The compositions may also contain antimicrobial agents or other anti-tumor agents. The compositions can be made in any pharmaceutical form suitable for the route of administration considered. Examples of such compositions include solid forms for oral administration such as tablets, capsules, pills, powders and granules, liquid forms for topical or oral administration such as solutions, suspensions, syrups and elixirs and preparations for parenteral administration such as sterile solutions, suspensions or emulsions. For use as an anti-tumor agent, the compositions are administered in doses such that the growth of the tumor is stopped. A treatment suggested in the use as an antitumor agent in mammals consists of the single parenteral administration, daily, of 50 to 500 mg of disodium salt of PALA per kilo (intravenous injection of a 2% aqueous solution). The new calcium salt of PALA is pharmaceutically equivalent, for the purposes of this invention, to the disodium salt of PALA and can therefore be used instead of or in combination with the disodium salt of PALA in the combinations mentioned above. above. The calcium salt of PALA has interesting solubility properties, it dissolves in water, which makes it possible to design a direct formulation; however, it dissolves relatively slowly, allowing the removal of inorganic impurities by washing with water.

The new disodium salt of PALA is a mobile, non-adherent powder which can be directly handled, analyzed and weighed for the preparation of the desired compositions. In contrast to the known tetrasodium salt of PALA, it is relatively non-hygroscopic. PALA tetrasodium salt absorbs atmospheric moisture 1.5 times faster than PALA disodium salt and is difficult

to manipulate and analyze. While the tetrasodium salt of PALA is water soluble, the disodium salt has a water solubility greater than 950 mg / ml. A 2% (w / v) solution in water of PALA disodium salt has a characteristic pH of about 4; similar solutions of PALA trisodium and tetrasodium salt have pHs of around 6 and 9 respectively.

The disodium salt of PALA can also be characterized by its nuclear magnetic resonance spectrum (NMR) at 60 MHz which typically shows a doublet corresponding to the methylene group (—CH2) which is in position a with respect to the —CH group, while the tetrasodium salt of PALA has a characteristic NMR spectrum which shows a three-line byte corresponding to the methylene group mentioned.

The PALA tetraethyl and dimethyl-P, P-diethyl esters are obtained directly in anhydrous form and their elemental analysis shows excellent agreement between the values obtained and the values calculated. For example, the maximum difference between the values obtained and calculated for hydrogen for these tetraesters is 0.03%, compared to a maximum difference of 1.27% for the tetrasodium salt of PALA.

Cyclohexylamine and piperazine form solid salts of PALA. In addition, the salt of cyclohexylamine is completely non-hygroscopic.

According to an implementation of the invention, a process for the preparation of PALA compounds comprises the step which consists in reacting L-aspartic acid with benzyl alcohol and p-toluenesulfonic acid to obtain the ester dibenzyl p-toluenesulfonate of L-aspartic acid. The p-toluenesulfonate ester of L-aspartic acid is then reacted with triethylamine, phosphonoacetic acid chloride is added to produce the PALA dibenzyl ester and the PALA dibenzyl ester is separated from the chloride unreacted phosphonoacetic acid. The PALA dibenzylester is appropriately isolated. Since it is insoluble in water, the dibenzyl ester of PALA and the unreacted phosphonoacetic acid chloride are preferably separated by washing the mixture with water so as to remove the phosphonoacetic acid chloride.

According to another implementation, the dibenzyl ester is subjected to hydrolysis with sodium hydroxide to obtain a mixture containing the tetrasodium salt of PALA, described below by the term mixing.

Another implementation comprises the step which consists in subjecting the mixture to an ion exchange process to obtain N-phosphonoacetyl-L-aspartic acid in free form, then the free acid is titrated until a pH of 9.2 and the tetrasodium salt of PALA thus purified is recovered.

Another implementation comprises the step which consists in dissolving said mixture in glacial acid, then the solution obtained is diluted with ethanol to precipitate the disodium salt of PALA and thus recover said disodium salt of PALA.

Another implementation includes the step of reacting the PALA dibenzyl ester with N, N'-dibenzylethylenediamine to produce the N, N'-dibenzylethylenediamine salt of PALA dibenzyl ester, and then subjecting said salt to hydrolysis to produce a mixture containing the tetrasodium salt of PALA, then dissolving the mixture in glacial acetic acid, then diluting the solution obtained with ethanol to precipitate the disodium salt of PALA and thereby recovering said disodium salt from PALA.

Another implementation includes the use of perchlorethylene as an esterification medium in the reaction of L-aspartic acid with benzyl alcohol and p-toluenesulfonic acid monohydrate. In the preparation of dibenzylaspartate, perchlorethylene is the preferred solvent. It forms an excellent azeotope with water, its boiling point is quite high, so that a large-scale esterification is quickly obtained (within 3 h) and, at the same time, the stability of the product n 'is not

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affected by temperature, at least during the short thermal contact time.

Another implementation includes the step of reacting the cyclohexylamine salt of the dibenzyl ester of PALA with sodium hydroxide to produce the tetrasodium salt of N-phosphonoacetyl-L-aspartic acid, then said tetrasodium salt is reacted with acetic acid to produce the disodium salt of PALA, and the disodium salt obtained is reprecipitated twice in water, the second precipitation comprising the dropwise addition of an aqueous solution of disodium salt with vigorously stirred ethanol, thus allowing the elimination of impurities which consist of acetic acid and sodium acetate.

The production of disodium salt of PALA according to this invention offers the following advantages: 1) a shorter time is required for the synthesis of the product, this being due to: a) total elimination of the ion exchange columns, and b) a substantial reduction in the volume of water to be evaporated; 2) the expected cost for a quantity of 5 kg of product is at least 30% lower than the cost of 5 kg of tetrasodium salt of PALA; 3) consistent low hydrogen analyzes associated with tetrasodium sodium salt are no longer a problem; 4) synthesis is directly applicable on a large scale; 5) the product obtained is less hygroscopic than the tetrasodium salt of PALA and, unlike the tetrasodium salt, it is a fluid and non-adherent powder which can be directly handled and weighed for the preparation of compositions, and 6) the product is very soluble in water.

Another implementation includes the step of reacting the dibenzyl-p-toluenesulfonate ester of L-aspartic acid with triethylamine, then phosphonoacetic acid chloride is added to produce the dibenzyl ester of PALA and reacting said ester with cyclohexylamine to produce the cyclohexylamine salt of said dibenzyl ester.

The following examples illustrate the present invention.

Example 1:

L-aspartic acid (I) dibenzyl-p-toluenesulfonate ester

A mixture of L-aspartic acid (399 g; 3.00 mol), benzyl alcohol (1.95 kg; 18.0 mol), p-toluenesulfonic acid monohydrate (582 g; 3.06 mol) and anhydrous benzene (1.21) is stirred and heated under reflux for 16 h. The water formed during the reaction (145 ml) is removed by a Dean-Stark apparatus. The resulting solution is cooled to room temperature, then diluted with benzene (1.2 L) and ether (3.61). The solid obtained is collected on a filter, washed with ether (7.01) and dried; yield 1195 g (82%).

The crude product is recrystallized from methanol (1720 ml) to give 959 g (80% recovery) of purified product (I); M.p. 158-159.5; M.p. 158-160 ° given in the literature.

Other similar syntheses were carried out to obtain a total quantity of 4.27 kg of product ready for further processing.

Phosphonoacetic acid (II)

A solution of triethylphosphonoacetate (900 g; 4.01 mol) in 6M hydrochloric acid (6.11) is stirred and heated under reflux for 6.5 h. The solution is then concentrated in vacuo, then the last traces of water are eliminated by coevaporation with benzene (2 x 300 ml). The solid residue is recrystallized twice from 1.01 glacial acetic acid to give 342 g (60.8%) of acid (II); Mp 140-141; P.F. from the literature: 143 °.

Other similar syntheses were carried out to obtain a total amount of 1020 g of product ready for further processing.

Phosphonoacetic acid chloride (III)

A mixture of phosphonoacetic acid (II) (355 g; 2.54 mol) and thionyl chloride (1770 ml) is stirred and heated for 4.5 h between 50 and 55 ". The resulting solution is concentrated in vacuo

(<35; tube pressure 1 mmHg) to give 395.7 g (98.3%) of product. The oily yellow material is used in the following reactions without further characterization.

N- (phosphonoacetyl) -L-aspartic acid (IV) dibenzyl ester To a cold suspension (15) of L-aspartic acid dibenzyl-p-toluenesulfonate (I) (812 g; 1.67 mol ) in anhydrous dioxane (5.21), triethylamine (486 g; 4.80 mol) is added dropwise over 30 min. The resulting solution is stirred for 30 min to 15, then phosphonoacetic acid (III) chloride (395.7 g; 2,500 mol) dissolved in anhydrous dioxane (600 ml) is added dropwise for 1 h . The temperature is kept below 15 'during the addition. The cooling bath is removed and the reaction mixture is then stirred for 1 h. The insoluble products are then removed by filtration and washed with dioxane (2.0 l). The filtrate is concentrated in vacuo and the oily residue is dissolved in benzene (10.1 l). The organic solution is washed with water (6 x 4.01), dried over magnesium sulfate, then evaporated in vacuo to give 568 g (78.1%) of a yellow solid product ready to undergo other transformations.

N-phosphonoacetyl-L-aspartic acid-4.5H20 tetrasodium salt (PALA) (VII).

To a cold solution (10 °) of sodium hydroxide (312 g; 7.80 mol) in 101 of water, dibenzyl ester of N-phosphonoacetyl-L-aspartic acid is added at once ( IV) (568 g; 1.30 mol) while stirring. The mixture is stirred at 10-15 ° for 6 h, then the insoluble parts are removed by filtration. The filtrate is concentrated under vacuum to a volume of 3.0 1 which is then extracted with methylene chloride (1 x 1.3 1) and ether (1 x 1.3 1). The aqueous solution is poured into 12.01 ethanol, which causes the precipitation of a semi-solid. After decantation, the product is dissolved in water (680 ml) and equal portions of solution are poured into two ion exchange columns, filled with cationic resin AG 50W-X8 (hydrogen form) of 9.6 x 25 cm . Each column is eluted with 2.01 of water (20 portions of 100 ml). The fractions 7-14 of each column, which contain the desired product as determined by TLC, are combined and evaporated, under vacuum (bath temperature <30 °). The oily residue is dissolved in acetone (2.0 l), activated charcoal (50 g) is added and the mixture is stirred for 18 h at room temperature. The insoluble parts are removed by filtration and the filtrate is evaporated to dryness under vacuum. The semi-solid residue (tetraacid; 227.1 g) is dissolved in 2.01 of water. The solution is cooled, stirring, to 10 °, then titrated with an aqueous solution of IN sodium hydroxide to a pH of 9.2 (3123 ml). The base solution is evaporated under reduced pressure (1-2 mmHg; <30 °) and the oily residue is triturated with acetone (5.8 l). The solid material is partly dried under vacuum, then triturated with acetone (2.0 l) and ether (2.01). The resulting powder is dried under reduced pressure over phosphorus pentoxide for 9 d at room temperature to give 272.2 g of the desired product.

Analysis for C6H6N08P-4Na-4,5H20:

Calculated: C 16.99 H 3.56 N3.30 P7.30 Na 21.68%

Found: C 16.76 H 2.29 N 3.46 P7.31 Na 21.61%

Spectral data:

Nuclear magnetic resonance (D20)

82.29 (m, 4, - CH2 a of P H-CH2 a of - CH); 4.13 (m, 1, -CH)

Optical rotation

Observed Literature

[a] y + 9.44 (c, 3.743 in water) [a] ^ f + 10.30 (c, 3.798 in water)

Chromatography:

Thin layer chromatography (TLC)

(Cellulose, Quanta / Gram Q2F glass plates)

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Movable phases R / values

1. Ethanol / ammonium hydroxide / water (6/1 / 3) 0.13

2. n-Butanol / acetic acid / water (5/2/3) 0.30

(streaks)

3. Lithium chloride (0.6M) / ethanol / ammonium hydrate (5/5/1) 0.61

4. Ethanol / water (2/3) 0.81

Applied quantity: 112 | xg.

Detection: Phospray (commercial reagent for visualization of compounds containing phosphorus).

Results: The product migrates in a single spot in each test of different mobile phases. The TLC of the free acid released from the tetrasodium salt by hydrochloric acid gives a negative test for aspartic acid when sprayed with ninhydrin.

Example 2:

Disodium salt of N-phosphonoacetyl-L-aspartic-1,1 H20.

Tetrasodium salt of N-phosphonoacetyl-L-aspartic acid 4,5H20 (VII) (10.0 g; 0.236 mol) is dissolved in boiling glacial acetic acid (125 ml) (90 °) . Celite is added to the boiling cloudy solution (5 g), then the insoluble parts are removed by filtration. The clear, dark yellow filtrate is cooled and then diluted in ethanol (300 ml) and the resulting mixture is stirred at room temperature for 30 min. The solid which precipitated is collected on a filter, washed by resuspension in ethanol (3 x 300 ml) and ether (1 x 300 ml), then dried to give 5.7 g (80.8% ) disodium salt in the form of a white powder. A similar 5.0 g of product is similarly prepared. The combined products (10.7 g) which are contaminated with acetic acid and ethanol (determined by NMR) are dissolved in water (250 ml). The solution is clarified by filtration and then lyophilized. The lyophilization process is repeated two more times, then the product is dried to constant weight under vacuum at 40 ° over phosphorus pentoxide; yield of the analytically pure product: 9.0 g (84.1% recovery).

Analysis for C6H8N08P-2Na-l, lH20:

Calculated: C 22.60 H 3.22 N 4.39 P 9.71 Na 14.42%

Found: C 22.68 H 3.21 N 4.36 P 9.62 Na 14.37%

Spectral data:

Nuclear magnetic resonance (D20)

52.75 (d, 2, J = 20 Hz, -CH2 at a of P); 2.80 (d, 2, -CH2 in a of -CH) 4.53 (t, 1, -CH)

Optical rotation

Observed [ol] d + 15.95 (c, 2,000 in water)

Chromatography:

Thin layer chromatography (Cellulose, Quanta / Gram Q2F glass plates)

Movable phases Rf values

1. Lithium chloride (0.6M) / ethanol / ammonium hydroxide (5/5/1) 0.47

2. Ethanol / water (2/3) 0.75

3. n-Butanol / acetic acid / water (5/2/3) 0.19

Detection: Phospray (commercial reagent for visualizing compounds containing phosphorus).

Results: The body migrates in a single spot in each of the different mobile phase systems.

Example 3:

Disodium salt of N-phosphonoacetyl-L-aspartic acid monohydrate'0.3 acetic acid'0.1 ethanol.

To 2.01 boiling glacial acetic acid (85 °), tetrasodium salt of N-phosphonoacetyl-L-aspartic acid tetrahydrate (VII) is added at once (214 g; 0.516 mol). After having stirred the mixture for 30 min at 85-90, celite (50 g) is added, then the insoluble parts are removed by filtration. The dark yellow thinned filtrate is cooled to room temperature and diluted with ethanol (4.5 l). The resulting mixture is stirred for 30 min, then the precipitated solid is collected on a filter. The product is washed by resuspension in ethanol (2 x 3.5 1) and ether (1 x 1.2 1) then dried under vacuum at 55 over phosphorus pentoxide to give 111.8 g ( 63.8%) of analytically pure disodium salt.

Analysis for C6H8N08P-2Na \ 0.3C2H402-0, lC2H6:

Calculated: C 24.04 H 3.50 N4.12 P9.12 Na 13.53% Found: C 24.19 H 3.56 N4.14 P 8.96 Na 13.48%

Spectral data:

Nuclear magnetic resonance

30.92 (t, 0.3, - CH3 ethanol); 1.83 (s, 0.9, - CH3 of acetic acid); 2.55 (d, 2, J = 20 Hz, -CH2 at a of P) 2.58 (d, 2, -CH2 at a of -CH); 3.33 (q, 0.2, -CH2 of ethanol) 4.32 (t, 1, -CH).

Optical rotation

Observed [a] 2ß + 15.31 (c, 2.103 in water)

Chromatography:

Thin layer chromatography (Cellulose, Quanta / Gram Q2F glass plates)

Movable phases Values of Rj

1. Lithium chloride (0.6M) / ethanol / ammonium hydroxide (5/5/1) 0.54

2. Ethanol / water (2/3) 0.69

3. Ethanol / ammonium hydroxide / water (6/1/3) 0.18

(lying down)

4. n-Butanol / acetic acid / water (5/2/3) 0.20

(streaks)

Detection: Phospray (commercial reagent for visualization of compounds containing phosphorus).

Results: The body migrates in a single spot in each of the systems.

Example 4:

Disodium salt of N-phosphonoacetyl-L-aspartic acid monohydrate-0.3 acetic acid'0.1 ethanol

N-phosphonoacetyl-L-aspartic acid 4.5H20 (VII) tetrasodium salt (716.8 g; 1.690 mol) is added in a single portion to boiling glacial acetic acid (95 °) (7 , 01). The mixture is stirred at 90-95 ° for 45 min, then celite (250 g) is added. After stirring the mixture for 20 min at 90-95 ", the insoluble parts are collected on a filter and washed with acetic acid (0.51). The dark orange clear filtrate is cooled to room temperature and diluted with ethanol (16.1 l), the resulting mixture is stirred for one hour, then the precipitate is collected on a filter, the product is suspended in ethanol (7.5 l) and the suspension is stirred vigorously for 3 hours The solid is collected on a filter, then washed as above with ethanol (2 x 7.5 1) and ether (1 x 7.51). The product is dried under vacuum at 50-55 on phosphorus pentoxide to give 426.5 g (74.3%) of analytically pure disodium salt.

Analysis for C6H8N08P-2NaH200,3C2H4020, IC2H60:

Calculated: C 24.04 H 3.50 N4.12 P9.12 Na 13.53%

Found: C 24.35 H 3.46 N4.19 P8.78 Na 13.44%

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Spectral data:

Nuclear magnetic resonance (D20)

80.98 (t, 0.3, -CH3 ethanol); 1.88 (s, 0.9, -CH3 acetic acid); 2.61 (d, 2, J = 20 Hz, - CH2 in a of P); 2.63 (d, 2, - CH2 has a -CH); 3.44 (q, 0.2, -CH2 ethanol); 4.36 (t, 1, -CH).

Optical rotation

Observed Md + (it is 1.998 in water)

Chromatography:

Thin layer chromatography (Cellulose, Quanta / Gram Q2F glass plates)

Movable phases

Rf values

I.

Lithium chloride (0.6M) / ethanol / hydroxide

ammonium (5/5/1)

0.60

2.

Ethanol / water (2/3)

0.78

3.

Ethanol / ammonium hydroxide / water (6/1/3)

0.19

4.

n-Butanol / acetic acid / water (5/2/3)

0.22

(lying down)

Applied quantity: 80 (ig.

Detection: Phospray (commercial reagent used for the visualization of compounds containing phosphorus).

Results: The product migrates in a single spot in the systems considered.

Example 5:

N, N'-dibenzylethylenediamine salt of the dibenzyl ester of N-phosphonoacetyl-L-aspartic acid (XII).

To a cold solution (5 °) of dibenzyl ester of N-phosphonoacetyl-L-aspartic acid (IV) (2449 g; 5.625 mol) in methylene chloride (9.5 i), is added dropwise stirring a solution of N, N'-dibenzylethylenediamine (1488 g; 6.191 mol) in methylene chloride (1.651) for 3.0 h. The temperature is kept below 15 ° during the addition. After removing the cooling bath, the reaction solution is stirred at room temperature for 16 h, then concentrated in vacuo until an oil is obtained. The residue is dissolved in acetone (5.01) and the solution is allowed to stand overnight at room temperature (18 h). A finely divided solid, which precipitated overnight, is removed by filtration, then the filtrate is evaporated under reduced pressure. The raw material is dissolved in ethyl acetate (12.01). The organic solution is washed with water (3 x 3.5 1), dried over magnesium sulphate, stirred with Norit A (125 g) for 45 min, then evaporated with rotation under vacuum. The glassy residue is triturated until a powder is obtained by vigorous mixing with an ether / petroleum ether mixture (m.p. 30-60 °) (5.01 / 7.01). The solid product is collected on a filter and then dried to give 1609 g of a light brown salt; Mp> 300 °.

A further similar reaction was carried out to give a total of 2308.5 g of (XII) ready for further processing.

N-phosphonoacetyl-L-aspartic acid (V) tetrasodium salt.

To a cold solution (14 °) of sodium hydroxide (240 g; 6.00 mol) in water (7.8 1), the salt of N, N'-dibenzylethylenediamine is added in several portions. dibenzyl ester of N-phosphonoacetyl-L-aspartic acid (XII) (675.7 g) which has been finely sprayed, for 5 min. The reaction mixture is stirred for 6 h at 10-15 ', celite (250 g) is added, then the insoluble parts are removed by filtration. The filtrate is extracted with methylene chloride (2 x 1.5 1) and ether (1 x 1.5 1) and then concentrated in vacuo (<40; 3-5 mmHg). The aqueous solution (volume: 3.81) is clarified by filtration and diluted with ethanol (13.51), which causes the precipitation of an oil. After standing for 18 h at room temperature, the aqueous ethanol solution is removed, leaving 600 ml of crude product (V) in the form of an orange oil. Additional hydrolysis was carried out in a similar manner to give a total of 1350 ml of oil ready for further processing.

Disodium salt of N- (phosphonoacetyl) -L-aspartic acid-0.2H2 0-0.2 sodium acetate-0.4 acetic acid-0.15 ethanol

N- (phosphonoacetyl) -L-aspartic acid (V) tetrasodium salt (1350 ml of oil) is dissolved in glacial acetic acid (6.5 l) at room temperature. The orange solution is stirred for 30 min, clarified by filtration, then diluted with ethanol (18.0 l). The resulting mixture is stirred for 1 h, then the solvent is removed by filter candles. The solid is suspended in ethanol (10.5 l) and the mixture is stirred vigorously for 1 h. The ethanol is removed as above, then the product is washed two more times with ethanol (10.51, then 6.01). The solid is collected on three filters, under a nitrogen atmosphere, washed with ether (2 x 1.0 1 per filter), then partially dried by evaporation under rotation at reduced pressure (30-45 °; setting the tube 3-5 mmHg). The solid in pieces is finely pulverized under nitrogen, then dried under vacuum over phosphorus pentoxide (33.5 h at room temperature, then 12.5 h at 50 °) to give 945.1 g of the desired product analytically pure.

Analysis for C6H8N08P-2Na-0.2H20 • 0.2C2H302Na-0.4C2H402-0.15C2H60:

Calculated: C 25.74 H 3.31 N4.00 P 8.85 Na 14.45%

Found: C 25.55 H 3.35 N 4.06 P 9.19 Na 14.30%

Spectral data:

Nuclear magnetic resonance (D20)

50.89 (t, 0.45, -CH3 ethanol); 1.78 (s, 1.8, -CH3 acetate-1-acetic acid); 2.54 (d, 2, - CH2 has a of - CH); 2.54 (d, 2, J = 20.3 Hz -CH2 at a of P); 3.36 (q, 0.30, -CH2 ethanol); 4.28 (, 1, -CH).

Optical rotation

Observed MB + 16.39 (c, 1.885 in water)

Chromatography:

Thin layer chromatography (Cellulose, Quanta / Gram Q2F glass plates)

Movable phases Rf values

1. Lithium chloride (0.6M) / ethanol / ammonium hydroxide (5/5/1) 0.64

2. Ethanol / water (2/3) 0.78

3. Ethanol / ammonium hydroxide / water (6/1/3) 0.24

(lying down)

4. n-Butanol / acetic acid / water (5/2/3) 0.28

(streaks)

Detection: Phospray (commercial reagent for the visualization of compounds containing phosphorus).

Results: The compound migrates in a single spot in the systems considered.

Example 6:

N- (phosphonoacetyl) -L-aspartic acid (IV) dibenzyl ester

To a cold suspension (10 °) of dibenzyl ester of p-toluenesulfonate of L-aspartic acid (I) (5785 g; 11.91 mol) in anhydrous dioxane (30.01), one adds in a single portion of triethylamine (3238 g; 32.00 mol). The resulting solution is stirred for 1 h, then phosphonoacetic acid (III) chloride (2236 g; 14.11 mol) in anhydrous dioxane (5.51 is added dropwise for 3 h. The temperature is maintained below 30 ° during the addition, the cooling bath is removed and the reaction mixture is stirred for 1 hour.

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wheat are removed by filtration and washed with dioxane (12.0 1). The filtrate is concentrated in vacuo, then the oily residue is dissolved in methylene chloride (64.0 l). The organic solution is washed with water (6 x 19.01), dried over sodium sulfate, then evaporated under reduced pressure to a volume of 5.01.

N- (phosphonoacetyl) -L-aspartic acid (V) tetrasodium salt

To a cold solution (15) of sodium hydroxide (1323 g; 33.08 mol) in water (43.0 1), 2.41 of the above prepared solution of ester is added all at once. N- (phosphonoacetyl) -L-aspartic acid (IV) dibenzyl (2.41 = 2400 g; 5.512 mol). The reaction mixture is stirred for 8 h at 10-15 ', then celite (825 g) is added and the insoluble parts are removed by filtration (600 g of celite ball). The filtrate is extracted with methylene chloride (2 x 9.01) and ether (1 x 9.01). The aqueous solution is combined with that coming from an identical preparation and the whole is concentrated under vacuum (865J; 3-5 mmHg). The solution (volume: 25.01) is clarified by filtration (400 g of celite ball) and diluted with ethanol (88.01), which causes the precipitation of an oil. After standing for 7.5 h at room temperature, the aqueous ethanol solution is removed, leaving 4.41 of crude product (V) in the form of an orange oil. An additional quantity of product is similarly prepared from the hydrolysis of 6695 g of (IV) to obtain a total of 9.98 1 of oil ready for further processing.

Disodium salt of N- (phosphonoacetyl) -L-aspartic acid

N- (phosphonoacetyl) -L-aspartic acid (V) tetrasodium salt (4.41 of oil) is dissolved in glacial acetic acid (14.01) at room temperature. The orange solution is stirred for 1 h, clarified by filtration, then diluted with ethanol (44.01). The resulting mixture is stirred for 1.5 h, then the solvent is removed by the use of filter candles. The solid is suspended in ethanol (30.01) and the mixture is stirred vigorously for 2 h. The ethanol is removed as above, then the product is washed by resuspending in ethanol (2 x 30.01) and ether (1 x 14.01). The solid is collected on two filters, under a nitrogen atmosphere, then partially dried by evaporation with rotation under vacuum (30-45 °; tube pressure 3-5 mmHg). An additional 2.93 l of oil (V) is similarly processed. The entire product in pieces is finely ground, under nitrogen, then dried under vacuum over phosphorus pentoxide (40 h at room temperature and 17 h at 45-50 °) to give 4562.5 g of a light yellow powder. The nuclear magnetic resonance spectrum and elemental analysis reveal the presence of sodium acetate (0.1 mol), acetic acid (0.5 mol) and ethanol (0.15 mol). A quantity of 2000 g of the product is poured in several portions into 11.5 1 of glacial acetic acid with vigorous stirring, for 20 min. The mixture is stirred at room temperature for 1 h, then the solution is clarified by filtration. The filtrate is diluted with ethanol (26.01) and the resulting mixture is stirred for 2 h. The solvent is removed (filter candles), then the product is washed twice by suspension in ethanol (7.01 then 15.01), collected on a filter and partially dried by evaporation with rotation under reduced pressure. The white solid (1989 g) contains acetic acid (1.25 mol) and ethanol (0.24 mol) as determined by nuclear magnetic resonance analysis. 1974 g is dissolved in water (4.01) and the aqueous solution is diluted with ethanol (16.01). The resulting mixture is stirred for 30 min, then the oily precipitate is left to settle. The aqueous ethanol solution is removed and the oil is washed once with ethanol (3.5 L). This product, which contains acetic acid (0.03 mol) and ethanol (0.8 mol), as determined by nuclear magnetic resonance, is dissolved in water (32.01). The aqueous solution is clarified by filtration and then lyophilized to give 1512.4 g of a flaky yellow solid.

Analysis for C6H8N08P-2Na-H20-0.03 C2H402-0.35 C2HsO

Calculated: C 24.23 H 3.68 N 4.18 P 9.24 Na 13.72% Found: C 24.39 H 3.56 N 4.15 P 9.03 Na 13.44%

Spectral data:

Nuclear magnetic resonance (D20)

81.19 (t, - CH 3 ethanol); 2.11 (s, - CH3 of acetic acid); 2.83 (d, 2, J = 20 Hz, -CH2 at a of P); 2.87 (d, 2, -CH2 to a of -CH); 3.67 (q, -CH2 ethanol); 4.61 (t, 1, -CH)

Optical rotation

Observed Md + 14.68 (c, 1.873 in water)

Chromatography:

Thin layer chromatography (Cellulose, Quanta / Gram Q2F glass plates)

Movable phases Values of Rj-

1. Lithium chloride (0.6M) / ethanol / ammonium hydroxide (5/5/1) 0.66

2. Ethanol / water (2/3) 0.83

3. Ethanol / ammonium hydroxide / water (6/1/3) 0.31

(lying down)

4. n-Butanol / acetic acid / water (5/2/3) 0.28

Detection: Phospray (commercial reagent used for the visualization of compounds containing phosphorus).

Result: The product migrates in a single spot in the systems considered.

The large-scale development of processes for the preparation of pure salt has overcome potentially serious handling problems. The cyclohexylamine salt of dibenzyl-PALA is prepared by the addition of about 0.9 to 1.0 Eq of cyclohexylamine to a solution of dibenzyl-PALA in acetone. The product is insoluble in acetone while a large percentage of the impurities remains in solution. The purity of the product is improved to an acceptable level by recrystallization from absolute methanol.

Difficulties are encountered in the use of dioxane to prepare dibenzyl-PALA. Triethylamine hydrochloride is an insoluble byproduct of the reaction and large volumes of solvents are required to assure proper stirring. In addition, the reaction is exothermic and the use of dioxane limits the possible range of cooling to about 12 °, the temperature at which dioxane freezes.

The solvent used in this reaction in place of dioxane is methylene chloride. This solvent offers the following advantages: a) it is non-flammable; b) it achieves a lower cooling temperature; c) the volume of solvent is reduced by half; d) the elimination of triethylamine hydrochloride by filtration is eliminated because this product is soluble in the solution of the reaction mixture, and e) evaporation of the solvent before analysis is no longer necessary.

Other process improvements include the fact that the cyclohexylammonium salt is hydrolyzed directly to the tetrasodium salt of PALA. This eliminates the additional manipulation of releasing dibenzyl-PALA from the amino salt before hydrolysis.

A final point is the fact that the volume of water required for hydrolysis has been reduced by 63% compared to that of the initial syntheses. This, of course, allows larger productions from the same equipment. On the test bench, this process made it possible to obtain disodium salt of PALA of about 2 kg using containers of 501 maximum. Incorporating all of the modifications described, preparation was carried out in 50 and 100 gallon pfaudlers (189-3781) with success.

On a large scale, about 15 kg of the desired product can be produced per test using equipment of this size.

The process, as developed, is limited only by the size of the equipment.

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By the methods mentioned herein, the purity of the disodium salt of PALA has been improved to a level satisfactory for parenteral administration in an environment suitable for the treatment of human cancer, particularly for purposes of large scale investigations.

This was accomplished by: 1) complete removal of acetic acid and sodium acetate by swirling precipitation, and 2) isolation of dibenzyl-PALA in the form of its cyclohexyl ammonium salt.

In addition, the process has been optimized to facilitate application on a larger scale and handling problems have been resolved.

Example 7:

Phosphonoacetic acid chloride (III)

To a mixture of phosphonoacetic acid (II) (2000 g; 14.28 mol), N, N-dimethylformamide (208.8 g; 2.856 mol) and dioxane (7.15 1), is added dropwise , stirring, thionyl chloride (3568 g; 29.99 mol) for 1.5 h. The temperature is kept below 30 ° during the addition. The resulting solution is heated at 45 "for 2.5 h, then cooled to 5 °. Water (283 ml; 15.7 mol) dissolved in dioxane (2.5 l) is then added dropwise, over a period of 2 hours The temperature is kept below 10 during the addition This solution of acid chloride (III) is stirred at 5-10 for 40 min, then used in the following reaction without further characterization. A second chlorination is carried out in parallel, under the same conditions and using the same amount of reagents.

N- (phosphonoacetyl) -L-aspartic acid (IV) dibenzyl ester

A suspension of L-aspartic acid (I) p-toluenesulfonate dibenzyl ester (4625 g; 9.525 mol) in dioxane (20.0 l) is cooled with stirring to 15 °, then added triethylamine (4820 g; 47.63 mol) in a thin stream, for 1 h. The resulting solution is stirred for 20 min, then the phosphonoacetic acid (III) chloride solution, prepared above from 14.28 mol of the corresponding acid, is added dropwise over a period of 5 h. The temperature is kept below 20 ° during the addition. An additional portion of triethylamine (1162 g; 11.48 mol) is added and the reaction mixture is stirred for 1 h. After standing for 8 h at room temperature, the mixture is diluted with acetone (5.5 l), stirred for 15 min, then the insoluble parts are collected on a filter and washed with dioxane (10.01) . A second reaction is carried out in parallel, under the same conditions and using the same quantity of reagents. The filtrates of the two reactions are combined and evaporated by rotation under vacuum. The residue (viscous orange oil) is dissolved in methylene chloride (110.01), then the organic solution is gently washed with water (6 x 30.01). After drying the solution over sodium sulphate (11.3 kg) and magnesium sulphate (2.3 kg), the insoluble parts are removed by filtration (celite ball) and the filtrate is evaporated in vacuo to weight constant; dibenzyl-PALA (IV) yield: 7970 g (96.1%). This viscous yellow oil is ready for further processing.

Cyclohexylamine salt of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester

Cyclohexylamine (1815 g; 18.30 mol) is added dropwise to a cold solution (7 °) of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (7970 g; 18.30 mol ) in acetone (24.01) with stirring, this for 1.25 h. The temperature is kept below 15 "during the addition. The cooling bath is removed and the resulting mixture is stirred for 1 h. The mixture is left at room temperature for 6 h, then the solid which has precipitated is collected on a filter, washed with acetone (15.01) and dried; yield 4932 g; PF 176.5-177.5 °. This product is recrystallized from boiling ethanol (35.0 1), then dried to give 1663 g of purified salt; mp 178-181 °.

The mother liquor is concentrated under vacuum to a volume of 20.0 1. The solution is diluted with acetone (16.0 1) and cooled (—10) to give an additional 967 g of product; P.F. 178-180. A third crop of product (429 g) is obtained by evaporating the above methanol / acetone filtrate almost to dryness and by suspending the residue in acetone (5.01); total weight of the amino salt ready to be processed: 3059 g (62.0% recovery).

N- (phosphonoacetyl) -L-aspartic acid (V) tetrasodium salt io A cold solution (5) of sodium hydroxide (1291 g; 32.28 mol) in water (20.5 1) , cyclohexylamine salt of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (3059 g; 5.378 mol) is added with stirring in portions over 30 min. The reaction mixture is stirred at 5-15 for 3.5 h, then is extracted with methylene chloride (2 x 8.51) and ether (1 x 8.51). The aqueous solution is clarified by filtration, concentrated in vacuo (<35 '; 3-5 mmHg) to a volume of 14.6 1 and then diluted with ethanol (51.4 1). The resulting mixture is stirred for 1 h, then allowed to stand at room temperature for 12 h. The aqueous ethanol solution is removed, giving the crude product (V) in the form of a light yellow oil, ready for further processing.

Disodium salt of N- (phosphonoacetyl) -L-aspartic acid

25 Glacial acetic acid (8.0 L) is added to the above precipitated oil (tetrasodium salt of crude N- (phosphonoacetyl) -L-aspartic acid (V), prepared from 3059 g amino salt). The mixture is stirred at room temperature for 30 min, then an insoluble gelatinous part is removed by filtration. The clarified, light yellow filtrate is diluted with ethanol (24.01). The resulting mixture is stirred for 1.75 h then the precipitate is collected on a filter. The solid is suspended in ethanol (14.5 l) and the mixture is stirred vigorously for 1 h. The product is collected on four filters, then partially dried under vacuum (30-45 °; vacuum: 3-5 mmHg) by rotary evaporation. The product in pieces (2870 g) is dissolved in water (5.251), the solution is clarified by filtration, then the filtrate (about 6.91) is diluted with ethanol (21.01). The resulting mixture is stirred for 30 min, then the oil which has precipitated is allowed to settle (1 h). The aqueous ethanol solution 40 is removed and the oil is washed with ethanol (1 x 4.31). This product is dissolved in water (8.15 1) and the solution (9.81) is divided into three portions (two of 4.0 1 and one of 1.8 1). Each portion is poured for 13 h into a vortex of vigorously stirred ethanol (10 x aqueous volume: 2 x 40.01; 1 x 18.01). After vigorously stirring the mixtures for 2 h, the water / ethanol solutions are removed by siphoning and the solid from the three precipitations is collected. The product is stirred for 30 min in ethanol (10.01), collected on a filter, then dried at constant weight under vacuum at room temperature on phosphorus pentoxide 50. The dry product (1748.0 g) is passed through a 150 stainless steel sieve and carefully mixed to give the disodium salt of PALA in the form of a white powder.

Analysis for C6H7 6NO8P'2.4Na-2H2O 0.5C2H <sO: 55 Calculated: C 22.91 H 4.01 N 3.82 P 8.44 Na 15.04% Found: C 23.16 H 3.76 N 3.79 P 8.57 Na 15.18%

Analysis of sodium indicates a composition of:

60% di-Na PALA 40% tri-Na PALA Based on the empirical formula:

H2O = 9.8%

Et0H = 6.3%

Spectral data:

65 Nuclear magnetic resonance (D20)

SI, 17 (t, 1.5, -CH3 of ethanol); 2.74 (d, 2, -CH2 to a of -CH); 2.77 (d, 2, J = 20 Hz, -CH2 at a of P); 3.63 (q, 1, -CH2 ethanol); 4.48 (t, 1, -CH)

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Optical rotation

Observed [a] 2è5 + 14,73 (c, 2,098 in water)

Chromatography:

Thin layer chromatography (Cellulose, Quanta / Gram Q2F glass plates)

Movable phases Rf values

1. Lithium chloride (0.6M) / ethanol / ammonium hydroxide (5/5/1) 0.52

2. Ethanol / water (2/3) 0.72

3. Ethanol / ammonium hydroxide / water (6/1/3) 0.16

(lying down)

4. n-Butanol / acetic acid / water (5/2/3) 0.22

(streaks)

Detection: a) Ninhydrin;

b) Phospray.

Results: The body migrates in a single spot, revealed by the Phospray, in each system considered. No stain due to aspartic acid was observed during the revelation to ninhydrin.

Example 8:

P, P-diethyl ester of phosphonoacetic acid

Triethyl phosphonoacetate (89.7 g; 0.400 mol) is added all at once to a solution of potassium hydroxide (0.408 mol, 26.1 g of 87.6% pure product) in ethanol (450 ml ) and water (150 ml). The solution is stirred for 22 h at room temperature, then evaporated with rotation under vacuum (bath temperature <25 °; 3-5 mmHg). The residue is suspended in ether (400 ml). The crystalline product, potassium salt, is collected on a filter and then dissolved in water (300 ml).

The solution is cooled, with stirring, to 5 °, then concentrated hydrochloric acid (33 ml) is added dropwise for 15 min. The temperature is kept below 10 ° during the addition. The solution is stirred at 5-10 ° for 30 min, then concentrated in vacuo (bath temperature <25; 3-5 mmHg). The residue is suspended in acetone (350 ml). The insoluble potassium chloride is removed by filtration and the filtrate is evaporated under reduced pressure. Ether (500 ml) is added to the waste oil. The resulting mixture is cooled to 10 ° and the insoluble parts are removed by filtration. The filtrate is discolored by passage over active charcoal, dried over magnesium sulfate and then concentrated in vacuo to obtain 69.3 g of a pale yellow oily product (88.2%). The product is ready for further processing.

P, P-diethyl ester chloride of phosphonoacetic acid

Oxalyl chloride (63.5 g; 0.500 mol) is dissolved in anhydrous benzene (100 ml), then poured dropwise with stirring into a cold solution (10) of P, P-diethyl ester of phosphonoacetic acid (19.6 g: 0.100 mol) in anhydrous benzene (350 ml) for 45 min. The temperature is maintained between 5 and 10 during the addition. The cooling bath is removed and the solution is stirred for 2 h. The volatiles are removed in vacuo, then the residue is coevaporated with benzene (2 x 50 ml) to give the acid chloride as a yellow liquid. This product is used in the following reaction without further characterization.

P, P-diethyl ester of N-phosphono-acetyl-L-aspartic acid dibenzyl ester

To a cold suspension (15) of d-benzenzyl ester of p-toluene sulfonate of L-aspartic acid (53.4 g; 0.110 mol) in dioxane (325 ml), is added dropwise with stirring. triethylamine (21.3 g; 0.210 mol) for 10 min. The resulting solution is stirred at 10-15 for 15 min, then a solution of acid chloride, prepared from 0.100 mol of acid in dioxane (100 ml) is poured dropwise for 50 min. The temperature is kept below 15 during the addition. The cooling bath is removed and the reaction mixture is stirred for 2 h. The insoluble parts are collected on a filter, then washed with dioxane (50 ml) and ether (100 ml). The filtrate is evaporated by rotation under vacuum and the residue is taken up in benzene (500 ml). The organic solution is washed with water (4 x 150 ml), bleached by passage over active charcoal (10 g), dried over magnesium sulfate, then concentrated in vacuo until an oil is obtained ; yield 51.6 g (> 100%). A 22.8 g portion of the crude product is dissolved in ethanol (80 ml). The solution is added all at once to a suspension of cationic exchange resin AG50W-X8 (form H +) (550 ml) in ethanol (200 ml) and the mixture is stirred at room temperature for 9 h. The resin is collected on a filter and washed with ethanol (250 ml), then the filtrate is evaporated in vacuo.

The oily residue is dissolved in benzene (400 ml) and the solution is dried over magnesium sulfate and evaporated by rotation under reduced pressure. The product is dried at constant weight under vacuum to give 19.7 g (86.5% recovery) of a homogeneous product, by control on thin layer chromatography (silica gel; acetone / petroleum ether (PE 30 -60) (2/3) or ethyl acetate This viscous yellow oil is ready for further processing.

Disodium salt of N- (phosphonoacetyl) -L-aspartic acid P-ethyl ester, monohydrate

To a cold solution (10) of P, P-diethyl ester of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (19.7 g; 0.0400 mol) in absolute ethanol ( 100 ml), stirred, sodium hydroxide (5.00 g; 0.125 mol) is added dropwise

dissolved in absolute ethanol (60 ml), this for 20 min. The temperature is kept below 15 during the addition. The solution is stirred at 10-15 for 45 min, then is heated with reflux for 30 min. An additional 3.2 g (0.080 mol) of sodium hydroxide dissolved in ethanol (200 ml) is added and the mixture is heated at reflux for 2.5 h. The solid obtained is collected on a filter, washed by suspension in ethanol (70 ml) and ether (150 ml), then is dried. This light brown powder (16.4 g) is dissolved in boiling glacial acetic acid (75) (30 ml). The solution is cooled to room temperature, diluted with ethanol (50 ml), then clarified by filtration. Ethanol (300 ml) is added to the filtrate and the mixture obtained is cooled. The solid which has precipitated is collected on a filter, washed successively by suspension in ethanol (350 ml), acetone (250 ml) and ether (250 ml) and then dried; yield: 9.2 g (66.6%). An 8.3 g portion of this product, which is contaminated with acetic acid (this having been determined by NMR), is suspended in ethanol (200 ml). The suspension is stirred and heated to reflux for 15 min, then cooled to room temperature. This heating / cooling cycle method is repeated two more times and the solid is collected on a filter. The washing process described above is carried out three times in total. The white solid is dried at constant weight under vacuum at 40 over phosphorus pentoxide to give 7.2 g (86.7% recovery) of the analytically pure product, the disodium salt of the P-ethyl ester of PALA.H20 . The body migrates in a single spot on cellulose (Quanta glass plates / Gram Q2F) developed with mobile phases ethanol / water (2/3) (Rf = 0.80), ethanol / ammonium hydroxide / water ( 6/1/3) (Rf = 0.42) or n-butanol / acetic acid / water (5/2/3) (Rf = 0.40); detection by Phospray.

Analysis for C8H, 2N08P-2Na-H20:

Calculated: C 27.84 H 4.09 N 4.06 P 8.97 Na 13.32%

Found: C 27.71 H 4.13 N 4.07 P 8.86 Na 13.45% Spectral data:

Infrared (Nujol)

Main bands: 3300, 2950,2920, 2860, 1700, 1640, 1600, 1455, 1405, 1375, 1215, 1045, 935, 760cm-i

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Nuclear magnetic resonance (DzO)

51.28 (t, 3, - CH3 of the ethyl group); 2.82 (d, 2, - CH2 a -CH); 2.85 (d, 2, J = 20.0 Hz, -CH2 at a of P), 4.00 (5 lines m, 2, —CH2 of the ethyl group); 4.57 (t, 1, - CH).

Example 9:

Disodium salt of dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid, tetrahydrate

A solution of dibenzyl ester of N- (phosphono-acetyl) -L-aspartic acid (26.7 g; 0.610 mol) and triethylamine (25.2 ml; 0.180 mol) in acetone (180 ml) is brewed at room temperature for 1 hour. The volatile parts are removed in vacuo and the semi-solid residue is dissolved in acetone (300 ml). Sodium iodide (27.0 g; 0.180 mol) dissolved in acetone (150 ml) is then added and the reaction mixture is heated at reflux for 3 h.

The solvent is removed by evaporation with rotation under reduced pressure, then the residue is resuspended in water (300 ml). The solid product is collected on a filter, washed with me-thanol (200 ml) and acetone (100 ml), then it is dried to constant weight to give 7.7 g (26%) of analytically pure product ; P.F.> 300 \ The body is either streaked or remains on the baseline in a TLC analysis. Part of the product was therefore transformed into free acid using hydrochloric acid. This product migrates in a single stain on the cellulose (Quanta glass plates / Gram Q2F) developed by methanol / water (9/1), ethanol / acetone / water (5/4/1) or ethanol / ether (1 / 1).

Analysis for C20H20NO8P-2Na-4H2O:

Calculated: C 43.57 H 5.12 N 2.54 P 5.62 Na 8.34%

Found: C 43.95 H 4.69 N2.50 P 5.57 Na8.31%

Spectral data:

Infrared (Nujol)

Main bands: 3600, 3360, 2960, 2920, 2860, 1740,1720, 1635, 1540, 1450, 1375, 1340, 1280, 1200, 1130,1045, 965,

725 cm ~ '.

Example 10:

L-aspartic acid dibenzyl ester hydrochloride

Hydrochloric acid is bubbled through a suspension of L-aspartic acid (133 g; 1.00 mol) in absolute ethanol (1.95 l) while stirring at room temperature for 2 h. The solution obtained is heated to reflux for 5 h, cooled to room temperature and then evaporated under vacuum. The residue is dissolved in benzene (500 ml). The solution is heated to reflux and the water present is removed by a Dean-Stark apparatus.

The solution is concentrated under reduced pressure until an oil is obtained which is left to crystallize slowly. The solid is suspended in ether (1.3 l), collected on a filter, washed with ether (800 ml) and then dried; yield of hydrochloride of dibenzyl ester of L-aspartic acid: 216 g (95.7%); M.p. 99-103 °. The product is recrystallized from 1.25 l of acetone / ether mixture (4/1) to give 164.6 g; (76.2% recovery) of a product ready for further processing; Mp 106.5-107.5 \

N- (phosphonoacetyl) -L-aspartic acid tetraethyl ester

To a cold suspension (10) of hydrochloride of the dibenzyl ester of L-aspartic acid (19.2 g; 0.0850 mol) in dioxane (350 ml), triethylamine (18 , 2 g; 0.180 mol) for 20 min. The mixture is stirred at 10 ° for 15 min, then a solution of chloride of the P, P-diethyl ester of phosphonoacetic acid, prepared from 0.090 mol of acid, in dioxane (90 ml) is added drop by drop for 1 hour. The temperature is maintained between 8 and 10 ° during the addition. The cooling bath is removed and the reaction mixture is stirred for 3.5 h. The insoluble product is collected on a filter, then washed with dioxane (100 ml) and ether (200 ml). The filtrate is evaporated by rotation under vacuum and the residue is dissolved in ethyl acetate (300 ml).

The organic solution is washed with water (3 x 100 ml), dried over magnesium sulphate, then concentrated in vacuo until an oil is obtained; yield of tetraethyl ester of N- (phosphonoacetyl) -L-aspartic acid: 31.1 g (99.6%). A 25.9 g portion of the product is dissolved in ether (500 ml) and the solution is extracted with water (5 x 150 ml). The aqueous extracts are combined and concentrated under reduced pressure until an oil is obtained. This pale yellow product is dried under vacuum over phosphorus pentoxide to give 20.1 g (77.6% recovery) of analytically pure product. The body migrates in a single spot on silica gel (Eastman Chromagram Sheet 13181) developed with acetone, chloroform or ethyl acetate; detection by iodine vapor.

Analysis for C14H26N08P:

Calculated: C 45.77 H 7.13 N3.81 P8.43%

Found: C 45.88 H 7.11 N3.81 P 8.50%

Spectral data:

Infrared (Nujol)

Main bands: 3260, 2980,2920,2900, 1730,1670, 1545, 1525, 1440, 1390, 1365, 1335, 1230, 1200,1090, 1040, 1015, 955, 850 cm-1.

Nuclear magnetic resonance (CDC13)

31.23 (m, 12, -CH3 of the ethyl groups); 2.83 (d of d, 2, -CH2 to a of -CH); 2.85 (d, 2, J = 20.0 Hz, -CH2 at a of P); 4.08 (m, 8, '—CH2 of ethyl groups); A, 11 (wide m, 1, - CH); 7.37 (large d, 1, -NH)

Optical rotation:

Observed Mo "—6.05 (c, 3849 in water)

Example II:

L-aspartic acid dimethyl ester hydrochloride

To absolute methanol (1.901; 1.50 kg; 46.8 mol) stirred at -5 °, thionyl chloride (357 g; 3.00 mol) is added dropwise over 2 h. The temperature is maintained between 0 and -5 ° during the addition. The solution is stirred between 0 and -5 ° for 1.5 h, then L-aspartic acid (133 g; 1.00 mol) is added in portions, this for 35 min. The solution is stirred at -5 'for 2.5 h and at room temperature for 16 h. The volatile parts are removed under vacuum, then the oily residue is evaporated at reduced pressure with benzene (4 x 175 ml). The solid obtained is collected on a filter, washed with ether (400 ml), and dried. The crude product (190.4 g) is recrystallized from 3.51 of boiling acetone to give 135 g (68.3%) of product suitable for further processing; Mp 117-119 ".

Dimethyl ester of P, P-diethyl ester of N- (phosphonoacetyl) -L-aspartic acid

To a cold suspension (10 °) of L-aspartic acid dimethyl ester hydrochloride (18.8 g; 0.0950 mol) in dioxane (350 ml) is added dropwise, with stirring, triethylamine ( 20.2 g; 0.200 mol) for 15 min. The mixture is stirred at 10 'for 15 min, then P, P-diethylester phosphonoacetic acid chloride, prepared from 0.100 mol of acid, dissolved in dioxane (100 ml), is added dropwise for 1 hour. The temperature is maintained at 5-10 ° during the addition.

The cooling bath is removed and the reaction mixture is stirred for 1.25 h. The insoluble parts are collected on a filter, washed with dioxane (100 ml) and ether (200 ml) and the filtrate is concentrated in vacuo. The oily residue is dissolved in benzene (100 ml) and ether (400 ml), then the organic solution is washed with water (4 x 150 ml). Aqueous solutions are collected

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The combined extracts are dried over magnesium sulphate, then evaporated in vacuo to give 33.2 g (103%) of crude product in the form of a yellow oil. An 18.0 g portion of the product is dissolved in water (50 ml) and the solution is passed through a column containing cationic exchange resin AG50W-X8 (hydrogen form). The column (3.8 x 15 cm) is eluted with 500 ml of water. The fractions containing the desired product, this being determined by TLC, are combined and evaporated by rotation under vacuum. The residue is dissolved in chloroform (120 ml) and the solution is dried over magnesium sulfate. The solvent is removed under reduced pressure, then the pale yellow oil is dried under vacuum over phosphorus pentoxide to give 14.4 g (80% recovery) of dimethyl ester of P, P-diethylester of N acid analytically pure phosphonoacetyl-L-aspartic.

The compound migrates in a single spot on silica gel (Eastman Chromagram Sheet 13181) developed with acetone, chloroform or ethyl acetate; detection by iodine vapor.

Analysis for C12H22N08P:

Calculated: C 42.48 H 6.54 N4.12 P9.13%

Found: C 42.50 H 6.51 N 4.09 P 9.06%

Spectral data:

Infrared (pure)

Main bands: 3260, 2980, 2950,1735, 1665, 1530, 1435, 1365, 1220, 1160, 1040, 1015, 955 cm- '.

Nuclear magnetic resonance (CDC13)

81.35 (t, 6, - CH3 of the ethyl groups); 2.88 (d, 2, - CH2 has a CH); 2.90 (d, 2, J = 21.5 Hz, -CH2 at a of P); 3.65 (s, 3, - CH3); 3.70 (s, 3, - CH3); 4.08 (5 lines m, 4, - CH2 of the ethyl groups); 4.80 (wide m, 1, - CH); 7.47 (large d, 1, - NH)

Optical rotation:

Observed [a] fj — 6.89 (c, 3.183 in water)

Example 12:

N- (phosphonoacetyl) -L ~ aspartic acid piperazine salt, tetrahydrate-0.5 C2H5 OH

A cold solution (10) of N- (phosphonoacetyl) -L-aspartic acid (10.2 g; 0.0400 mol) in water (60 ml) is added dropwise with stirring. piperazine (15.2 g; 0.176 mol) dissolved in water (100 ml) for 35 min. The temperature is kept below 15 during the addition. The solution is stirred at 10-15 for 1 h, then evaporated by rotation under vacuum (<35; 2-5 mmHg). The semi-solid residue is triturated with ethanol (2 x 200 ml), then dissolved in water (60 ml). Activated charcoal (1 g) is added and the mixture is stirred at room temperature for 25 min. The insoluble parts are removed by filtration, then the filtrate is poured dropwise into a vortex of vigorously stirred ethanol (2.0 l), this for 40 min. The solid obtained is collected on a filter, washed with acetone (200 ml) and dried to give 5.8 g (25.6%) of piperazine salt of N- (phosphonoacetyl) -L-aspartic acid. , tetrahydrate-0.5C2H5OH; M.p. 92-95; 111-115 (sealed capillary). The compound migrates in a large spot on the cellulose (Quanta glass plates / Gram Q2F) developed with the ethanol / ammonium hydroxide / water phases (6/1/3) (Rf = 0.34), n- butanol / acetic acid water (5/2/3) (Rf = 0.25) or ethanol / water (2/3) (Rf = 0.79); detection by Phospray.

Analysis for C6HION08P-2,5C4HION2'4H20-0,5C2H50H

Calculated: C 36.10 H 8.20 N 14.86 P 5.48%

Found: C 35.74 H 7.20 N 14.62 P 5.48%

Spectral data:

Infrared (Nujol)

Main bands: 3340, 3270, 2950, 2920, 2850, 2720, 1645, 1625, 1580, 1460, 1380, 1295, 1050, 950 cm- '.

Nuclear magnetic resonance (D20)

80.88 (t, 1.5, -CH3 EtOH); 2.40 (3-line m, 4, -CH2 a of P + —CH2 aspartate); 3.12 (s, 20, —CH2 of the piperazine cycle); 3.33 (q, 1, -CH2 EtOH); 4.10 (large t, 1, -CH of aspartate).

Example 13:

N- (phosphonoacetyl) -L-aspartic acid

To a cold solution (10;) of sodium hydroxide (289 g; 7.23 mol) in 9.3.5 1 of water, dibenzyl ester of N- acid is added at once. phosphonoacetyl) -L-aspartic (523 g; 1.20 mol). The mixture is stirred at 10-15 ° for 7 h, then the insoluble parts are removed by filtration. The filtrate is concentrated in vacuo to a volume of 3.01 and extracted with methylene chloride (1 x 1.361) and ether (1 x 1.361). The aqueous solution is poured into 12.0 l of ethanol, which causes the precipitation of a semi-solid product. After decantation, the product is dissolved in water (680 ml), and the solution is passed through a column (8 x 46 cm) containing a cation exchange resin AG50W-X8 (H + form). The column is eluted with 2.7 l of water (18 fractions of 150 ml each). Fractions 8-16, which contain the product as determined by TLC, are combined and evaporated in vacuo (bath temperature <30). The oily residue is dissolved in acetone (2.0 l) and activated charcoal (50 g) is added, then the mixture is stirred at room temperature for 18 h. The insoluble parts are removed by filtration and the filtrate is evaporated by rotation under reduced pressure. The residue is dried under vacuum at room temperature over phosphorus pentoxide for 18 h to give 220 g (71.8%) of the tetraacid. This semi-solid product is suitable for further processing.

Calcium salt of N- (phosphonoacetyl) -L-aspartic acid-2.5H20

To a cold solution (10) of N- (phosphonoacetyl) -L-aspartic acid (20.4 g; 0.0800 mol) in water (300 ml), carbonate of calcium (17.6 g; 0.176 mol) for 30 min. The reaction mixture is stirred at room temperature for 21 h, then the insoluble part (7.2 g) is collected on a filter and washed with water (50 ml). Activated charcoal (5 g) is added to the aqueous filtrate and the mixture is stirred at room temperature for 35 min. The insoluble parts are removed by filtration and the filtrate is diluted with acetone (600 ml). The solid obtained is collected on a filter, washed with acetone (200 ml) and with ether (200 ml) and is then dried. This product (24.7 g) is suspended in water (125 ml) and the mixture is stirred vigorously for 10 min. The insoluble parts are removed by filtration and the filtrate is added to acetone (350 ml). The solid which precipitates is collected on a filter, washed with acetone (200 ml), then dried to give 10.8 g (37.8%) of calcium salt of N- (phosphonoacetyl) -L acid. -aspartic-2.5H20, analytically pure; M.P.> 280.

Analysis for C6H-, N08P'l, 5Ca-2,5H20:

Calculated: C 20.17 H 3.39 N 3.92 P 8.67 Ca 16.86%

Found: C 20.28 H 3.09 N 3.88 P 8.63 Ca 16.85%

Spectral data:

Infrared (Nujol)

Main bands: 3530, 3380, 2950, 2920, 2850, 1590, 1455, 1375, 1140, 1110, 1070.945 cm-1.

Nuclear magnetic resonance (D20)

82.62 (m with 3 lines, 4, —CH2 has of P-1 — CH2 has of of -CH); 4.35 (t, 1, -CH)

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Optical rotation:

Observed [a] a + 8.38 (c, 3.113 in water)

Chromatography:. '5

Thin layer chromatography (TLC)

(Cellulose, Quanta / Gram Q2F glass plates)

Movable phases Values of Rj- Values of Rj-

free acid calcium salt 10

1. n-Butanol / acetic acid / water

(5/2/3) 0.18 0.29

2. Ethanol / ammonium hydroxide / water (6/1/3) 0.00 0.13

3. Ethanol / water (2/3) 0.84 0.75 15

Detection: Phospray (commercial reagent for the visualization of compounds containing phosphorus).

Results: the free acid was obtained from the calcium salt acidified with hydrochloric acid. A baseline spot was observed for the free acid in each system considered and for the calcium salt in system 3.

Example 14:

N- (phosphonoacetyl) -L-aspartic acid cyclohexylamine salt dihydrate

To a cold solution (15) of N- (phosphonoacetyl) -L-aspartic acid (5.1 g; 0.020 mol) in water (50 ml), cyclohexylamine ( 8.7 g; 0.088 mol) dissolved in water (15 ml) for 15 min. The temperature is kept below 20 during the addition. The solution is stirred at room temperature for 30 min, then is diluted with acetone (850 ml). The solid obtained is collected on a filter, washed with acetone (200 ml) and air dried. The crude product is dissolved in water (100 ml) and air dried. The crude product is dissolved in water (100 ml), activated charcoal (5 g) is added and the mixture is stirred at room temperature for 1.5 h. The insoluble parts are removed by filtration and the filtrate is poured into vigorously stirred acetone (1.3 l). The precipitate is collected on a filter, washed with acetone (400 ml), then dried to give 8.0 g (65.7%) of cyclohexylamine salt of N- (phosphonoacetyl) -L-aspartic acid. , dihydrate; M.p. 167.5-170.5 °. The compound migrates in a single spot on cellulose (Quanta glass plates / Gram Q2F) developed with ethanol / ammonium hydroxide / water phases (6/1/3) (Rf = 0.38), n-butanol / acetic acid / water (5/2/3) (Rf = 0.33) and ethanol / water (2/3) (Rf = 0.82); detection: Phospray.

Analysis for C6H10NO8P-3,2C6Hi1NH2-2H2O:

Calculated: C 49.74 H 9.21 N9.67 P 5.09%

Found: C 50.12 H 8.37 N 9.44 P 5.07%

Spectral data:

Infrared (Nujol)

Main bands: 3210, 3050, 2950, 2920, 2860, 2670, 2620, 1640, 1575, 1545, 1460, 1450, 1400, 1380, 1300, 1145, 1115, 1040 cm "1. Nuclear magnetic resonance (D20)

51.50 (m, 32, - CH2 of the cyclohexane cycle); 2.57 (m with 3 lines, 4, - CH2 has P + - CH2 aspartate); 2.97 (broad s, 3.2, —CH of the cyclohexane ring); 4.22 (d of d, 1, —CH of aspartate)

Example 15:

Cyclohexylamine salt of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester

138 g (0.317 mol) of N- (phosphono-acetyl) -L-aspartic acid dibenzyl ester are dissolved in acetone (500 ml). The solution is stirred and cooled to 10 °, then cyclohexylamine (69.2 g; 0.700 mol) is added dropwise over 30 min. The resulting suspension is stirred at room temperature for 18 h. The solid which has precipitated is collected on a filter, washed with acetone (500 ml) and ether (400 ml), then dried to give

47.2 g of cyclohexylamine salt of the dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid. A 24.8 g portion of the product is recrystallized from boiling methanol (500 ml) to give

18.3 g (73.8% recovery) of the purified cyclohexylamine salt; Mp 186-188 °.

R