FI65261B - FOERFARANDE FOER FRAMSTAELLNING AV FARMAKOLOGISKT ANVAENDBART DINATRIUMSALT AV N- (FOSFONOACETYL) -L-ASPARAGINSYRA - Google Patents

Description

P5Sr * l ΓβΙ «« advertisement, r 0 * λ lJ '* UTLÄGGN I NGSSKRI FT 65261 • jSöjS C (45) Fai.v .-. · :, 11.:.; CnnL t ly 10 04 1934 0¾¾ Patent meddelat y (S1) Kv.lk.3 / tnt.C! .3 C 07 F 9/38 SUOMI-FINLAND (21) P * tenttlhalc «rrm * - Ptttntansdknlng 703467 (22) H» keml * ptlvl - An * eknlng * dag l4.ll.78 * '(23) AlkuplivI— Giltlghetsdag 14.11.78 (41) Tullut fulklMktl - BIMt offaotllg 15.05.79

Patent and registration holders .... .... „...

,. (44) Date of issue and date of issue. -

Patent · och registerstyrelaen Anaökan utlagd och utl.akrlftan published. , Buffalo, New York 14213, USA (72) Robert John Schultz, Amherst, New York, Fred William Starks, Kenmore,

New York, USA (74) Oy Jalo Ant-Wuorinen Ab (54) Method for the preparation of a pharmacologically useful disodium salt of N- (phosphonoacetyl) -L-aspartic acid - Förfarande för framställning av pharmacologisktvartbart dinatriumsalt av N- (fos-fono) This invention relates to a new process for the preparation of a novel disodium salt of N- (phosphonoacetyl) -L-aspartic acid (disodium PALA) having # pharmacological properties, having the formula

O O

^ nhcch2p-oh CH0-CH ONa / 2 \ H02c xCO2Na

Free tetrahydric acid, N- (phosphonoacetyl) -L-aspartic acid (hereinafter also referred to as PALA) is a known compound.

Known tetrahydric compound. N- (phosphonoacetyl) -L-Asparagic acid (PALA) was first prepared by Stark et al., J. Biol. Chem. 246, 6599 (1971). The tetrasodium salt of PALA is a known antitumor agent, as described in the literature, especially in Cancer Research, 36. # 2720 (1976). For example, mice with intra-peritoneal P388 leukemia had a lifespan increase of up to 64% when treated with PALA tetrasodium salt at doses of 188-750 mg / kg i.p. Lewis lung sarcoma was highly sensitive to PALA — tetrasodium salt in mice i.p. at doses of 240-490 mg / kg. Mice with B16 melanoma lived 77-86% longer compared to controls when treated with PALA tetrasodium salt 2,6261 (490 mg / kg i.p.).

Although the synthesis of tetrahydro-PALA is simple, the preparation of the triumium salt, especially in kg, has proved problematic. The new process according to the invention is particularly well suited for the preparation of such amounts.

The novel disodium PALA compound prepared according to the invention has antitumor activity in vivo.

The novel disodium PALA compound may exist in anhydrous form or in solvated (including hydrated) form. Both forms are suitable for the purposes of the invention. The process of the invention provides the disodium PALA compound as a hydrate having a hydrogenation-water content that varies in various preparations, typically being about 0.2 to 2 moles of water. The compound may also contain ethanol (e.g., about 0.1 to 0.5 moles), acetic acid (e.g., about 0.03 to 0.4 moles), and sodium acetate (e.g., about 0.2 moles) as a solvate. Acetic acid and ethanol can be removed by freeze-drying. Lyophilization two or three times gives a solvent-free substance. The disodium PALA compound prepared by these methods may also contain significant amounts of trisodium PALA, up to as much as 30-40% trisodium PALA. All such solvates of disodium PALA and mixtures of disodium PALA and trisodium PALA are included herein as "disodium PALA" because these compounds are interchangeable and used as active antitumor agents in the compositions of the invention. When used as an antitumor agent, the new dinatrirone PALA is essentially equivalent in activity and toxicity to the known tetrasodium PALA. Due to its antitumor activity, PALA disodium can be used according to the invention in the form of pharmaceutical compositions together with an acceptable pharmaceutical carrier. The compositions may also contain other antimicrobial agents and other antitumor agents. The compositions can be prepared anywhere in the art. in a pharmaceutical form suitable for the mode of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for topical or oral administration such as solutions, suspensions, syrups and elixirs, and preparations for parenteral administration such as sterile suspensions or emulsions. When the compositions are used as antitumor agents, they are administered at a dose that inhibits tumor growth. A suitable dose range for use as antitumor agents (especially against the solid tumors mentioned above) in mammals is 50-500 mg disodium PALA per kg for a single daily parenteral treatment (e.g. intravenous infusion as a 2% aqueous solution).

The new DISODIUM PIECE is preferably a mobile, free-flowing fine solid that is easy to handle, analyze and weigh when forming compositions. Compared to the known tetra-sodium PALA, it is relatively non-hygroscopic. Tetrasodium PALA absorbs atmospheric moisture 1.5 times faster than disodium PALA, and is thus difficult to handle and analyze. Where tetrasodium PALA is water soluble, the solubility of disodium PALA in water is preferably greater than 950 mg / ml. The pH of a 2% aqueous solution (w / v) of disodium PALA is typically about 4; a comparable trisodium PALA and tetrasodium PALA solution 11a has pH values of about 6 and about 9, respectively. Disodium PALA is also characterized by a 60 Mc nuclear magnetic resonance (NMR) spectrum, typically corresponding to the methylene group (-Cl 2). a doublet in the α-position relative to the -CH group, while tetrasodium PALA is characterized by an NMR spectrum with a three-line multiplet corresponding to the above-mentioned methylene group.

The process according to the invention comprises dissolving tetrasodium PALA or a product mixture containing it in glacial acetic acid, diluting the resulting solution with ethanol to precipitate disodium PALA and recovering disodium PALA.

Tetrasodium-PALA, in turn, can be prepared by reacting L-aspartic acid with benzyl alcohol and p-toluenesulfonic acid to prepare p-toluenesulfonate of L-aspartic acid dibenzyl ester PAL, followed by addition of L-aspartic acid dibenzyl ester p-toluenesulfonate to give to prepare the dibenzyl ester, and separating the PALA dibenzyl ester from the unreacted phosphonoacetyl chloride. The PALA dibenzyl ester is separated by any suitable means. Because it is insoluble in water, PALA dibenzyl ester and unreacted phosphonoacetyl chloride are preferably separated by washing the reaction mixture with water to remove phosphonoacetyl chloride. The dibenzyl ester is subjected to hydrolysis with aqueous sodium hydroxide to prepare a product mixture containing tetrasodium PALA.

The product mixture can be subjected to an ion exchange reaction to prepare N- (phosphonoacetyl) -L-aspartic acid in the free acid form, titrate the resulting free acid to pH 9.2, and separate the purified tetrasodium salt.

The PALA dibenzyl ester can also be reacted with Ν, Ν'-di-benzylethylenediamine to produce the Ν, Ν'-dibenzylethylenediamine salt of the PALA dibenzyl ester, after which said salt is subjected to hydrolysis according to the invention to further react the PALA tetrasodium salt to prepare the disodium salt.

In another embodiment, perchlorethylene is used as the esterification medium in the reaction of L-aspartic acid with benzyl alcohol and p-toluenesulfonic acid monohydrate. In the preparation of dibenzyl aspartate, perchlorethylene is the most preferred solvent. It forms an excellent azeotrope with water; it boils high enough for large-scale esterification to be carried out quickly (in about three hours) and at the same time the temperature does not affect the stability of the product, at least not during this short thermal contact.

According to another embodiment - in the preparation of PALA compounds in which a cyclohexylamine salt of a PALA dibenzyl ester is reacted with sodium hydroxide to prepare a tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid and said tetrasodium salt is reacted with acetic acid - from water, wherein during the second precipitation an aqueous solution of said disodium salt is added dropwise to the center of strongly stirred ethanol, thus removing impurities in the form of acetic acid and sodium acetate.

The cyclohexylamine salt of dibenzyl-PALA, in turn, can be prepared by reacting the p-toluenesulfonate of the dibenzyl ester of L-aspartic acid with triethylamine, adding phosphonoacetyl chloride to prepare the PALA-dibenzyl ester, and reacting said dibenzyl ester with said dibenzyl ester.

The preparation of disodium PALA according to the invention has the following advantages: 5 65261 (1) The time required to synthesize the substance is shorter due to (a) the use of ion exchange columns can be completely eliminated and (b) the volume of water to be evaporated can be significantly reduced; (2) the estimated cost of producing 5 kg of material is at least 39% less than 5 kg of tetrasodium PIA; (3) the persistently low hydrogen analyzes of tetrasodium PALA are no longer a problem; (4) the synthesis is easily applicable on a larger scale; (5) the resulting product is less hygroscopic than tetrasodium PIA, and in contrast to tetrasodium PIA, it is a mobile, free-flowing fine solid that is easy to handle and weigh for formulation purposes, and (6) the substance is very soluble in water.

The following examples illustrate the invention. Temperature is given in degrees Celsius.

Example 1: L-aspartic acid dibenzyl ester p-toluenesulfonate (I) A stirred mixture of L-aspartic acid (399 g, 3.00 moles) benzyl alcohol (1.95 kg, 18.0 moles), p-toluenesulfonic acid monohydrate (582 g, 3.06 mol) and dry benzene (1.2 liters) were heated at reflux for 16 hours. The water formed in the reaction (145 mL) was removed using a Dean-Stark trap. The resulting solution was cooled to room temperature, then diluted with benzene (1.2 liters) and ether (3.6 liters). The resulting solid was collected on a filter, washed with ether (7.0 liters) and dried; yield 1195 g (82%). The crude product was crystallized from methanol (1720 ml) to give 959 g (80% yield) of purified product (I); mp 158 to 159.5 °; literature sul.p. 158-160 °.

Additional reactions were performed to give a total of 4.27 kg of product suitable for the following transformation reactions. Phosphonoacetic acid (II) A stirred solution of triethylphosphonoacetate (900 g; 4.01 mol) in 6M hydrochloric acid (6.1 liters) was heated at reflux for 6.5 hours. The solution was concentrated in vacuo, then the last volumes of water were removed by co-evaporation with Bentene (2 x 300 ml). The solid residue was crystallized twice from 1.0 liter of glacial acetic acid to give 342 g (60.8%) of acid (II); mp 140-141 °; literature sul.p. 143 °. Additional reactions were performed to give a total of 1020 g of product suitable for the following transformation reactions.

6 65261

Phosphonoacetyl chloride (III) A stirred mixture of phosphonoacetic acid (II) (355 g; 2.54 moles) and thionyl chloride (1770 ml) was heated at 50-55 ° for 4.5 hours. The resulting solution was concentrated in vacuo (below 35 °; aspirator pressure then 1 mm Hg) to give 395.7 g (98.3%) of product. an oily yellow substance was used in the next reaction without further characterization.

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

To a cold (15 °), stirred suspension of L-Asparagic acid dibenzyl ester p-toluenesulfonate (I) (812 g; 1.67 moles) in dry dioxane (5.2 liters) was added dropwise triethylamine (486 g; 4.80 moles) within 30 minutes. The resulting solution was stirred at 15 ° for 30 minutes, then phosphonoacetyl chloride (III) (395.7 g; 2,500 mol) dissolved in dry dioxane (600 ml) was added dropwise over 1 hour. The temperature was kept below 15 ° during the addition. The cooling bath was removed and the reaction mixture was stirred for one hour. The insolubles were filtered off and washed with dioxane (2.0 liters). The filtrate was concentrated in vacuo, after which the oily residue was dissolved in benzene (10.1 liters). The organic solution was washed with water (6 x 4.0 liters), dried over magnesium sulphate and then evaporated in vacuo to give 568 g (78.1%) of product as a yellow crude solid which was useful for the following transformation reactions.

N- (phosphonoacetyl) -L-aspartic acid tetrasodium salt · 4.5 H-O (PALA) (VII) - To a cooled (10 °) stirred solution of sodium hydroxide (312 g; 7.80 moles) 10.0 per liter of water was added N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (IV) (568 g; 1.30 moles) in one portion. The mixture was stirred at 10-15 ° for 6 hours, after which the insoluble components were filtered off. The filtrate was concentrated in vacuo to a volume of 3.0 liters and then extracted with methylene chloride (1 x 1.3 liters) and ether (1 x 1.3 liters). The resulting aqueous solution was added to 12.0 liters of ethanol, whereupon a semi-solid precipitated. After decantation, the material was dissolved in water (680 mL) and equal volumes of the solution were added to two AG50W-X8 (hydrogen form) cation exchange resin columns (9.6 cm x 25 cm). Each column was eluted with 2.0 liters of water (20 fractions, 100 ml each). Fractions 7-14 from each column containing the desired 6 5 2 61 7 product as determined by thin layer chromatography were combined and evaporated in vacuo (bath temperature below 30 °). the oily residue was dissolved in acetone (2.0 liters), carbon (50 g) was added and the mixture was stirred at room temperature for 18 hours. The insoluble components were filtered off and then the filtrate was evaporated in vacuo. The semi-solid residue (tetrahydric acid; 227.1 g) was dissolved in 2.0 liters of water. The stirred solution was cooled to 10 ° and then titrated to pH 9.2 with aqueous sodium hydroxide (3123 mL). The basic solution was concentrated under reduced pressure (1-2 mm Hg; below 30 °) and the oily residue was triturated with acetone (5.8 liters). The solid was partially dried in vacuo and then triturated with acetone (2.0 liters) and ether (2.0 liters). The obtained powder was dried under reduced pressure with phosphorus pentoxide for 9 days at room temperature to give 272.2 g of the desired product.

Calculated for C6H6NOgP C H N P Na • 4Na · 4.5 H 2 O 16.99 3.56 3.30 7.30 21.68

Found 16.76 2.29 3.46 7.31 21.61

Spectral data:

Nuclear Magnetic Resonance (D 2 O) δ 2.29 (m, 4, -CH 2 in the α-position to P + -CH 2 in the α-position to -CH); 4.13 (m, 1, -CH).

Optical rotation:

Observed Literature + 9.44 (c, 3.743 in water) (a) ^ + 10.30 (c, 3.798 in water) Chromatography:

Thin layer chromatography (cellulose, Quanta / Gram Q2F glass plates) Solvent system Rf value 1. ethanol-ammonium hydroxide-water (6: 1: 3) 0.13 2. n-butanol-acetic acid-water (5: 2: 3) 0 .30 (tail) 3. lithium chloride (0.6 M) -ethanol-ammonium hydroxide (5: 5: 1) 0.61 4. ethanol-water (2: 3) 0.81 Number of spots: 112 μg.

Determination:

Fospray (a commercial reagent used to detect phosphorus-containing compounds).

8 65261

Score:

The compound moves as a single spot in each solvent system. The thin layer chromatogram of the free acid liberated from the tetrasodium salt with hydrochloric acid was negative for aspartic acid injected with ninhydrin.

Example 2: Disodium salt of N- (phosphonoacetyl) -L-aspartic acid · 1.1 H90 The tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid • 4.5 H2O (VII) (10.1 g; 0.236 mol) was dissolved in hot ( 90 °) to glacial acetic acid (125 ml). Celite (5 g) was added to the hot cloudy solution, after which the insoluble components were filtered off. The clear cooled dark yellow filtrate was diluted with ethanol (300 mL) and the resulting mixture was stirred at room temperature for 30 minutes. The precipitated solid was collected on a filter, washed by resuspension in ethanol (3 x 300 mL) and ether (1 x 300 mL) and dried to give 5.7 g (80.8%) of the disodium salt as a white powder. An additional 5.0 g of product was prepared in a similar manner. The combined material (10.7 g) containing impurities of acetic acid and ethanol (as determined by NMR) was dissolved in water (250 ml). The aqueous solution was clarified by filtration and then lyophilized. The lyophilization procedure was repeated twice more, after which the product was dried to constant weight in vacuo at 40 ° with phosphorus pentoxide; the yield of analytically pure product was 9.0 g (24.1% yield).

Analysis:

Calculated CgHgNOgP · 2 Na · 1.1 H2O C H N P Na 22.60 3.22 4.39 9.71 14.42

Found 22.68 3.21 4.36 9.62 14.37

Spectral data:

Nuclear Magnetic Resonance (D 2 O) δ 2.75 (d, 2, J = 20 Hz, -CH 2 at α to P); 2.80 (d, 2, -CH 2 in the α-position to -CH) and 4.53 (t, 1, -CH).

Optical rotation:

Observed (a) p5 + 15.95 (c, 2,000 in water)

chromatography:

Thin layer chromatography (cellulose, Quanta / Gram Q2F glass plates) 9 65261

Solvent system Rf value 1. lithium chloride (0.6 M) -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 Determination:

Fospray (a commercial reagent used to detect phosphorus-containing compounds).

Score:

The compound moves as a single spot in each solvent system. Example 3: Disodium salt of N- (phosphonoacetyl) -L-aspartic acid, monohydrate · 0.3 acetic acid ♦ 0.1 ethanol _____ 2.0 liters of hot (85 °) glacial acetic acid were added in one portion to N- (phosphonoacetyl) -L-aspartic acid tetrasodium salt, tetrahydrate (VII) (214 g; 0.516 moles). The mixture was stirred at 85-90 for 30 minutes, then Celite (50 g) was added, after which the insoluble components were filtered off. The clear dark yellow filtrate was cooled to room temperature and diluted with ethanol (4.5 liters). The resulting mixture was stirred for 30 minutes, after which the precipitated solid was collected on a filter. The material was washed by resuspension in ethanol (2 x 3.5 liters) and ether (1 x 1.2 liters) and then dried in vacuo at 55 ° with phosphorus pentoxide to give 111.8 g (63.8%) of analytically pure disodium salt.

Analysis:

Calculated CgHgNOgP · 2 Na · H2O · 0.3 C2H4C> 2 · 0.1 C- ^ HgO

C H N P Na 24.04 3.50 4.12 9.12 13.53

Found 24.19 3.56 4.14 8.96 13.48

Spectral data:

Nuclear Magnetic Resonance (D 2 O) δ 0.92 (t, 0.3, -CH 2 of ethanol); 1.83 (s, 0.9, acetic acid -CH 2); 2.55 (d, 2, J = 20 Hz, -CH 2 in the α-position to P); 2.58 (d, 2, -CH 2 in the α-position to -CH); 3.33 (q, 0.2, ethanol-CH 2); 4.32 (t, 1, -CH).

Optical rotation:

Found (a) p + 15.31 (c, 2.103 in water).

6 5 2 61 10

chromatography:

Thin layer chromatography (cellulose, Quanta / Gram Q2F glass plates) solvent system Rf value 1. lithium chloride (0.6 M) -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 (long) 4. n-Butanol-acetic acid-water (5: 2: 3) 0.20 (tail) Determination:

Fospray (a commercial reagent used to detect phosphorus-containing compounds).

Score:

The compound moves as a single spot in each solvent system. Example 4: Disodium salt of N- (phosphonoacetyl) -L-aspartic acid, monohydrate »0.3 acetic acid · 0.1 ethanol_ Tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid * 4.5 H2O (VII) (716.8 g 1.690 moles) was added in one portion to hot (95 °) glacial acetic acid (7.0 L). The mixture was stirred at 90-95 ° for 45 minutes, then Celite (250 g) was added. The hot mixture (90-95 °) was stirred for 20 minutes, the insolubles were collected on a filter and washed with acetic acid (0.5 L). The clear dark orange filtrate was cooled to room temperature and diluted with ethanol (16.1 L). The resulting mixture was stirred for 1 hour and then the precipitated solid was collected on a filter. The material was suspended in ethanol (7.5 L) and the suspension was stirred vigorously for 3 hours. The solid was collected on a filter and washed as above with ethanol (2 x 7.5 L) and ether (1 x 7.5 L). The material was dried in vacuo at 50-55 ° with phosphorus pentoxide to give 426.5 g (74.3%) of analytically pure disodium salt. Analysis:

Calculated CgNgNOgP · 2 Na * HjO · 0.3 C2H4C> 2 · 0.1 C2H60 C H N P Na 24.04 3.50 4.12 9.12 13.53

Found 24.35 3.46 4.19 8.78 13.44 11 65261

Spectral data:

Nuclear Magnetic Resonance (D2O) δ 0.98 (t, 0.3, ethanol -CH3); 1.88 (s, 0.9, acetic acid -CH 2); 2.61 (d, 2, J = 20 Hz, -CH 2 in the α-position to P); 2.63 (d, 2, -CH 2 in the α-position to -CH); 3.44 (q, 0.2, ethanol-CH 2); 4.36 (t, 1, -CH).

Optical rotation:

Found + 14.86 (c, 1.998 in water)

chromatography:

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

Solvent system Rf value 1. lithium chloride (0.6 M) -ethanol-ammonium hydroxide (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 (long) Number of spots: 80 μg.

Determination:

Fospray (a commercial reagent used to detect phosphorus - containing compounds.

Score:

The compound moves as a single spot in each solvent system. Example 5: N, N'-Dibenzylethylenediamine salt of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (XII)

To a cold (5 °) stirred solution of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (IV) (2449 g; 5.625 moles) in methylene chloride (9.5 L) was added dropwise Ν, Ν'-di-benzylethylenediamine. (1488 g; 6.191 mol) dissolved in methylene chloride (1.65 L) for 3.0 hours. The temperature was kept below 15 ° during the addition. After removing the cooling bath, the reaction solution was triturated at room temperature for 16 hours and then concentrated in vacuo to an oil. The residue was dissolved in acetone (5.0 L) and the solution was stored overnight at room temperature. The fine white solid precipitated from the solution was filtered off, then the filtrate was evaporated under reduced pressure. The crude material was dissolved in ethyl acetate (12.0 L). The organic solution was washed with water (3 x 3.5 L), dried over magnesium sulfate, stirred with 12 65261

With Norit A (125 g) for 45 minutes and evaporated on a rotary evaporator in vacuo. The residue (glass) was triturated to a powder by vigorous stirring with ether-petroleum ether (b.p. 30-60 °) (5.0 L; 7.0 L). The solid product was collected on a filter to give 1609 g of a light brown salt; mp over 300 °. Yet another reaction was performed in the same manner to give a total of 2308.5 g of compound (XII) suitable for use in the next conversion reaction.

Tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid (V) To a cooled (14 °) stirred solution of sodium hydroxide (240 g; 6.00 mol) in water (7.8 L) was added portionwise carefully ground N- (phosphonoacetyl). ) L, Ν'-dibenzylethylenediamine salt (XII) (675.7 g) of -L-aspartic acid dibenzyl ester for 5 minutes. The reaction mixture was stirred at 10-15 ° for 6 hours, Celite (250 g) was added, after which the insoluble components were filtered off. The filtrate was extracted with methylene chloride (2 x 1.5 L) and ether (1 x 1.5 L) and then concentrated in vacuo (<40 °; 3.5 mm Hg). The aqueous solution (3.8 L volume) was clarified by filtration and diluted with ethanol (13.5 L) to separate the oil. After standing for 18 hours at room temperature, the aqueous ethanol solution was removed to give 600 ml of crude compound (V) as an orange oil. Further hydrolysis reactions were performed in a similar manner to give a total of 1350 ml of oil suitable for the subsequent transformation reactions.

Disodium salt of N- (phosphonoacetyl) -L-aspartic acid * 0.2 1-0-0.2 sodium acetate · 0.4 acetic acid · 0.15 ethanol_ tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid (V) (1350 ml oil) was dissolved in glacial acetic acid (6.5 L) at room temperature. The orange solution was stirred for 30 minutes and clarified by filtration and then diluted with ethanol (18.0 L). The resulting mixture was stirred for 1 hour, then the solvent was removed using filter candles. The solid was suspended in ethanol (10.5 L) and the mixture was stirred vigorously for 1 hour. The ethanol was removed as above, then the material was washed twice more with ethanol (10.5 L; then 6.0 L). The solid was collected on three filters under a nitrogen atmosphere and washed with ether (2 x 1.0 L / funnel) and then partially dried by evaporation on a rotary evaporator under reduced pressure (30-45 °; 3-5 mm Hg). The agglomerated solid was carefully triturated under a nitrogen atmosphere, then dried in vacuo over phosphorus pentoxide (33.5 hours at room temperature and 12.5 hours at 50 °) to give 945.1 g of analytically pure product.

Analysis:

Calculated CgHgNOgP ♦ 2 Na · 0.2 H2O · 0.2 C2H3O2Na · 0.4 C2H4C> 2 * 0.15 C2H60 C H N P Na 25.74 3.31 4.00 8.85 14.45

Found 25.55 3.35 4.06 9.19 14.30

Spectral data:

Nuclear Magnetic Resonance (D 2 O) δ 0.89 (t, 0.45, -CH 2 of ethanol); 1.78 (s, 1.8, acetate + acetic acid -CH 2); 2.54 (d, 2, -CH 2 in the α-position to -CH); 2.54 (d, 2, J = 20.3 Hz, CH2 at α to P); 3.36 (q, 0.30, ethanol-CH 2)} 4.28 (t, 1 -CH).

Optical rotation:

Found 22 + 16.39 (c, 1.885 in water).

chromatography:

Thin layer chromatography (cellulose, Quanta / Gram Q2F glass plates) solvent system Rf value 1. lithium chloride (0.6 M) -ethanol-ammonium hydroxide (5: 5: 1) 0.64 2. ethanol-water (2: 3) 0.78 3. ethanol-ammonium hydroxide-water (6: 1: 3) (long) 0.24 4. n-butanol-acetic acid-water (5: 2: 3) (tail) 0.28 Determination:

Fospray (a commercial reagent used to detect phosphorus-containing compounds).

Score:

The compound moves as a single spot in each solvent system. Example 6: Dibenzyl ester of N- (phosphonoacetyl) -L-aspartic acid (IV) In a cooled (10 °) stirred suspension of p-toluenesulfonate (I) of L-aspartic acid dibenzyl ester (I) (5785 g; 11.91 moles) in dry dioxane (30.0 L) triethylamine (3238 g; 32.00 mol) was added in one portion. The resulting solution was stirred for 1 hour and then phosphonoacetyl chloride (III) (2236 g; 14.11 moles) dissolved in dry dioxane (5.5 L) was added dropwise over 3 hours. The temperature was kept below 30 ° during the addition. The cooling bath was removed and the reaction mixture was stirred for one hour. The insoluble components were filtered off and washed with dioxane (12.0 L). The filtrate was concentrated in vacuo, after which the oily residue was dissolved in methylene chloride (64.0 L). The organic solution was washed with water (6 x 19.0 L), dried over sodium sulfate and evaporated under reduced pressure to a volume of 5.0 L.

Tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid (V) To a cooled (15 °) stirred solution of sodium hydroxide (1323 g; 33.08 moles) in water (43.0 L) was added in one portion 2.4 liters of the above preparation. A solution of methylbenzyl chloride of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (IV) (2.4 L = 2400 g; 5.512 moles). The reaction mixture was stirred at 10-15 ° for 8 hours, after which Celite (825 g) was added and the insolubles were filtered off (600 g, Celite pad). The filtrate was extracted with methylene chloride (2 x 9.0 L) and ether (1 x 9.0 L). The aqueous solution was combined with an aqueous solution from an identical reaction and concentrated in vacuo (<35 °; 3-5 mm Hg). The solution (25.0 L volume) was clarified by filtration (400 g Celite pad) and diluted with ethanol (88.0 L) to precipitate an oil. After standing for 7.5 hours at room temperature, the aqueous ethanol solution was removed to leave 4.4 liters of crude compound (V) as an orange oil. An additional 6695 g of compound (IV) was hydrolyzed in a similar manner to give a total of 9.98 liters of oil suitable for further reactions.

Disodium salt of N- (phosphonoacetyl) -L-aspartic acid The tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid (V) (4.4 L of oil) was dissolved in glacial acetic acid (14.0 L) at room temperature. The orange solution was stirred for 1 hour, clarified by filtration and then diluted with ethanol (44.0 L). The resulting mixture was stirred for 1.5 hours, after which the solvent was removed using filter candles. The solid was suspended in ethanol (30.0 L) and the mixture was stirred vigorously for 2 hours. The ethanol was removed as above, after which the material was washed by resuspension in ethanol (2 x 30.0 L) and ether (1 x 14.0 L). The solid was collected on two filters under a nitrogen atmosphere and then dried on a partially rotary evaporator under vacuum (30-45 °; aspirator pressure 3-5 mm Hg). An additional 2.93 liters of oil (65261 (V)) was reacted in a similar manner. The combined lump-like material was carefully ground under a nitrogen atmosphere and dried in vacuo over phosphorus pentoxide (40 hours at room temperature and 17 hours at 45-50 °) to give 4562.5 g of a pale yellow powder. The nuclear magnetic resonance spectrum and elemental analysis of this material showed that it contained sodium acetate (0.1 mol), acetic acid (0.5 mol) and ethanol (0.15 mol). 2000 g of the product were added portionwise to 11.5 liters of vigorously stirred glacial acetic acid over 20 minutes. The mixture was stirred at room temperature for 1 hour, then the solution was clarified by filtration. The filtrate was diluted with ethanol (26.0 L) and the resulting mixture was stirred for 2 hours. The solvent was removed (filter candles), then the solid was washed twice by resuspension in ethanol (7.0 L, then 15.0 L), collected on a filter, and dried on a rotary evaporator under reduced pressure. Nuclear magnetic resonance showed that the white solid (1989 g) contained acetic acid (1.25 moles) and ethanol (0.25 moles). 1974 g of this material was dissolved in water (4.0 L) and the aqueous solution was diluted with ethanol (16.0 L). The resulting mixture was stirred for 30 minutes and then the precipitated oil was allowed to settle. The aqueous ethanol solution was removed and the oil was washed once with ethanol (3.5 L). This material containing acetic acid (0.03 moles) and ethanol (0.8 moles) as determined by nuclear magnetic resonance was dissolved in water (32.1 L). The aqueous solution was clarified by filtration and then lyophilized to give 1512.4 g of a fluffy yellow solid.

Analysis:

Calculated CgHgNOgP · 2 Na · H2O - 0.03 C2 ^ / p2 · 0.35 C2H60 C H N P Na 24.23 3.68 4.18 9.24 13.72

Found 24.39 3.56 4.15 9.03 13.44

Spectral data:

Nuclear Magnetic Resonance (D 2 O) δ 1.19 (t, -CH 2 of ethanol); 2.11 (s, acetic acid -CH 2); 2.83 (d, 2, J = 20 Hz, -CH 2 in the α-position to P); 2.87 (d, 2, -CH 2 in the α-position to -CH); 3.67 (q, ethanol-CH 2); 4.61 (t, 1, -CH). Optical rotation:

Found 2 2 (a) D + 14 »68 (c, 1,873 in water).

16 65261

Chromatography:

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

Solvent system Rf value 1. lithium chloride (0.6 M) -ethanol-ammonium hydroxide (5: 5: 1) 0.65 2. ethanol-water (2: 3) 0.33 3. ethanol-ammonium hydroxide-water (6: 1: 3) (long) 0.31 4. n-butanol-acetic acid-water (5: 2: 3) 0.28 Determination:

Fospray (a commercial reagent used to detect phosphorus-containing compounds).

Score:

The compound moves as a single spot in each solvent system. As a result of the development work aimed at large-scale production of pure salt, some potentially difficult processing problems have been solved. The cyclohexylamine salt of dibenzyl PALA is prepared by adding about 0.9 to about 1.0 equivalents of cyclohexylamine to an acetone solution of dibenzyl PALA. The product is insoluble in acetone while much of the impurity remains in solution. The purity of the product can be improved to acceptable by recrystallization from absolute methanol.

Some difficulties exist with the use of dioxane in the preparation of dibenzyl PALA. Triethylamine hydrochloride is an insoluble reaction by-product and large amounts of solvent are required to maintain adequate mixing. In addition, the reaction is exothermic and the use of dioxane limits cooling to about 12 °, which is the freezing point of dioxane.

The solvent used in this reaction instead of dioxane was methylene chloride. This solvent has the following advantages: a) it is non-combustible; (b) it allows the use of lower cooling temperatures; c) the volume of solvent is halved; d) removal of triethylamine hydrochloride by filtration is eliminated because it dissolves in the reaction mixture; and e) evaporation of the solvent before further processing is no longer necessary.

Other improvements include the direct hydrolysis of the cyclohexylammonium salt to the tetrasodium PALA. This eliminates the extra step of liberating dibenzyl PALA from the amine salt prior to hydrolysis.

6 5 2 61 17

As a final aspect, the volume of water required for hydrolysis has been reduced by 63% compared to the amount used in the original synthesis. This, of course, allows larger quantities to be produced using the same equipment. On a laboratory scale, this method has been used to prepare disodium PALA in 2 kg batches, using bottles of a maximum of 50 liters. Using all of the aforementioned enhancements, series of 50 and 100 gallon Pfaudlers have been successfully completed. On a full scale, the substance of the invention can always be produced in amounts of about 15 kg per series of work using equipment of this size.

The method, which is currently under development, is limited only by the size of the equipment.

According to current methods, the purity of the desired disodium PALA can be raised to a level acceptable for parenteral administration in a suitable carrier for the treatment of human cancer, especially for large scale research. This has been achieved by (1) complete elimination of acetic acid and sodium acetate using vortex precipitation; and (2) isolating dibenzyl-PALA as the cyclohexylammonium salt. In addition, the method has been optimized to facilitate the transition to a larger scale and the problems associated with process steps have been solved.

Example 7:

Phosphonoacetyl chloride (III) To a stirred mixture of phosphonoacetic acid (II) (2000 g * 14.28 moles), N, N-dimethylformamide (208.8 g; 2.856 moles) and dioxane (7.15 L) was added dropwise thionyl chloride (3568 g; 29.99 moles) over 1.5 hours. The temperature was kept below 30 ° during the addition. The resulting solution was heated at 45 ° for 2.5 hours and then cooled to 5 °. Water (283 mL; 15.7 moles) dissolved in dioxane (2.5 L) was then added dropwise over 2 hours. The temperature was kept below 10 ° during the addition. This solution of acid chloride (III) was stirred at 5-10 ° for 40 minutes and then used in the next reaction without further characterization. A second chlorination was performed simultaneously under the same conditions using identical amounts of reagent.

N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (IV) To a stirred suspension of N-aspartic acid dibenzyl ester p-toluenesulfonate (I) (4625 g; 9.525 moles) in dioxane (20.0 L) was cooled to 15 ° C. Tri-ethyl acetate (4820 g; 47.63 moles) was then added in a thin stream over 1 hour. The resulting solution was stirred for 20 minutes, then the above solution of phosphonoacetyl chloride (III) prepared from 14.28 moles of the corresponding acid was added dropwise over 5 hours. The temperature was kept below 20 ° during the addition. Additional triethylamine (1162 g; 11.48 moles) was then added and the reaction mixture was stirred for one hour. After standing for 8 hours at room temperature, the mixture was diluted with acetone (5.5 L), stirred for 15 minutes, after which the insoluble components were collected on a filter and washed with dioxane (10.0 L). The second reaction was performed simultaneously under the same conditions, using identical amounts of substances. The filtrates from both series were combined and evaporated on a rotary evaporator in vacuo. The residue (orange viscous oil) was dissolved in methylene chloride (110.0 L), then the organic solution was gently washed with water (6 x 30.0 L). After the solution was dried over sodium sulfate (11.3 kg) and magnesium sulfate (2.3 kg), the insoluble constituents were filtered off (Celite pad), the filtrate was evaporated in vacuo to constant weight; the yield of dibenzyl PALA (IV) was 7970 g (96.1%). This yellow viscous oil was suitable for the following transformation reactions.

N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester cyclohexylamine salt__

Cyclohexylamine (1815 g; 18.30 moles) was added dropwise to a cold (7 °) stirred solution of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester (IV) (7970 g; 18.30 moles) in acetone (24.0 L). ) For 1.25 hours. The temperature was kept below 15 ° during the addition. The cooling bath was removed and the resulting mixture was stirred for one hour. The mixture was stored at room temperature for 6 hours, then the precipitated solid was collected on a filter, washed with acetone (15.0 L) and dried; yield 4932 g; mp 176.5 to 177.5 ° C. This material was recrystallized from boiling methanol (35.0 L) and dried to give 1663 g of purified salt, m.p. 178-181 °; the mother liquor was concentrated in vacuo to a volume of 20.0 liters. The solution was diluted with acetone (6.0 L) and cooled (-10 °) to give an additional 967 g of product, m.p. 177-18o ° C. A third crop (429 g) was obtained by evaporating the above methanol-acetone filtrate to near dryness and suspending the residue in acetone (5.0 L); the total amount of purified amine salt suitable for the following transformation reactions was 3059 g (62.0% recovery).

19 65261 Tetrasodium salt of N- (phosphonoacetyl) -L-aspartic acid (V)

To a cold (5 °), stirred solution of sodium hydroxide (1291 g; 32.28 moles) in water (20.5 L) was added portionwise over 30 minutes the cyclohexylamine salt of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester ( 3059 g; 5.378 mol). The reaction mixture was stirred at 5-15 ° for 3.5 hours, then extracted with methylene chloride (2 x 8.5 L) and ether (1 x 8.5 L). The aqueous solution was clarified by filtration, concentrated in vacuo (<35 °; 3-5 mm Hg) to a volume of 14.6 liters and then diluted with ethanol (51.4 L). The resulting mixture was stirred for one hour and kept at room temperature for 12 hours. The aqueous ethanol solution was removed to give crude compound (V) as a pale yellow oil suitable for the following transformation reactions.

Disodium salt of N- (phosphonoacetyl) -L-aspartic acid Glacial acetic acid (8.9 L) was added to the oil precipitated above (crude N- (phosphonoacetyl) -L-aspartic acid tetrasodium salt (V) prepared from 3059 g of the amine salt). The mixture was stirred at room temperature for 30 minutes, after which the gelatinous insoluble matter was filtered off. The clear pale yellow filtrate was diluted with ethanol (24.0 L). The resulting mixture was stirred for 1.75 hours, after which the precipitated material was collected on a filter cake. The solid was suspended in ethanol (14.5 L) and the mixture was stirred vigorously for one hour. The product was collected on four filters and then dried on a partially rotary evaporator under vacuum (30-45 °; aspirator pressure then 3-5 mm Hg). The lump-like material (2870 g) was dissolved in water (5.25 L), then the solution was clarified by filtration, then the filtrate (approximately 6.9 L volume) was diluted with ethanol (21.0 L). The resulting mixture was then stirred for 30 minutes, after which the precipitated oil was allowed to settle (1 hour). The aqueous ethanol solution was removed and the oil was washed once with ethanol (4.3 L). This material was dissolved in water (8.15 L) and the solution (9.8 L) was divided into 3 portions (2 of which were 4.0 L; one 1.8 L). Each portion was added within 13 hours to the center of vigorously stirred ethanol (10 x water volume: 2 x 40.0 L; 1 x 18.0 L). The mixtures were stirred for 2 hours, after which the aqueous-ethanol solutions were siphoned off and the solid from the three precipitates was combined. This material was stirred for 30 minutes in ethanol (10, 1 L), collected on a filter and dried to constant weight in vacuo at room temperature with phosphorus pentoxide. The dry product (1748.0 g) was passed through a 150 μ stainless steel sieve and mixed gently.

2Q

65261 to give disodium PALA as a white powder. Analysis:

Calculated C6H? gNOgP * 2.4 Na * 2 H 2 O · 0.5 C 2 H 2 O.

C H N P Na 22.91 4.01 3.82 8.44 15.04

Found 23.16 3.76 3.79 8.57 15.18

Sodium analysis shows that the composition contains 60% di-Na-PALA 40% tri-Na-PALA Calculated from the empirical formula:% H 2 O = 9.8%% EtOH = 6.3%

Spectral data:

Nuclear Magnetic Resonance (D 2 O) δ 1.17 (t, 1.5, ethanol -CH-j) and 2.74 (d, 2, -CH 2 α-mass to -CH); 2.77 (d, 2, J = 20 Hz, -CH 2 at ct to P) i 3.63 (q, 1, -CH 2 of ethanol); 4.48 (t, 1, --CH). Optical rotation:

Found (α) + 2 + 14.73 (c, 2.098 in water).

chromatography:

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

Solvent system Rf value 1. lithium chloride (0.6 M) -ethanol-airunonium hydroxide (5: 5: 1) 0.52 2. ethanol-water (2: 3) 0.72 3. ethanol-ammonium hydroxide-water (6: 1: 3) 0.16 (long) 4. n-butanol-acetic acid-water (5: 2: 3) 0.22 (tail) Determination: (a) ninhydrin (b) fospray

Score:

The compound moves as a single phospray-positive spot in each solvent system. No aspartic acid was detected after spraying with ninhydrin.

21 65261

Example 8: Cyclohexylamine salt of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester 138 g (0.317 mol) of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester were dissolved in acetone (500 ml). The stirred solution was cooled to 10 °, then cyclohexylamine (69.2 g; 0.700 mol) was added dropwise over 30 minutes. The resulting suspension was stirred at room temperature for 18 hours. The precipitated solid was collected on a filter and washed with acetone (500 ml) and ether (400 ml) and dried to give 47.2 g of N- (phosphonoacetyl) -L-aspartic acid dibenzyl ester cyclohexylamine salt. 24.8 g of this material was recrystallized from boiling methanol (500 ml) to give 18.3 g (73.8% yield) of the purified cyclohexylamine salt, m.p. 186-188 °.

Claims (3)

22 6 5 2 61 A process for preparing N- (phosphonoacetyl) -L-aspartic acid disodium salt (disodium PALA) having pharmacological properties, characterized in that the N- (phosphonoacetyl) -L-aspartic acid tetrasodium salt (tetrasodium PALA) is dissolved in glacial acetic acid. , the disodium PIAS formed is precipitated by adding ethanol and recovered. Process according to Claim 1, characterized in that the disodium PALA obtained is precipitated twice from water, during which time an aqueous solution of disodium PALA is added dropwise to the middle of a highly stirred ethanol to remove impurities in the form of glacial acetic acid and sodium acetate. Process according to Claim 1, characterized in that the tetrasodium PALA used as starting material is prepared by reacting L-aspartic acid with benzyl alcohol and p-toluenesulfonic acid, preferably in the presence of perchlorethylene, to react To prepare the PALA dibenzyl ester, which is separated, preferably by washing with water, from the unreacted phosphonoacetyl chloride and hydrolyzed as such or in the form of the N, N'-dibenzylethylenediamine salt or cyclohexylamine salt by treatment with sodium hydroxide to 9.2 for the preparation of pure tetrasodium-PALA. li