NO161261B - ANALOGY PROCEDURE FOR THE PREPARATION OF 4-CARBAMOYLOXYOCSAZAPHOSPHORPHORIN DERIVATIVES WITH CYTOSTATIC EFFECT. - Google Patents

Description

The present invention relates to an analogous process for the production of 4-carbamoyloxyoxazaphosphorine derivatives with cytostatic action.

From DE-OS 2 231 311 and 2 552 135 it is known that by introducing a hydroperoxy group -0H in the 4-position in the molecules of the known cytostatics 2-[bis-(2-chloroethyl)-amino]-2-oxo -tetrahydro-2H-1,3,2-oxazophosphorine (cyclophosphamide), 3-(2-chloroethylamino)-2-[bis-(2<1->chloroethyl)-amino]-2-oxo-tetrahydro-2H-1 ,3,2-oxazaphosphorine (trophosphamide), 3-(2-chloroethylamino)-2-(2<1->chloroethylamino)-2-oxo-tetrahydro-2H-1,3,2-oxazaphosphorine (ifosfamide), 3- (2-chloroethylamino)-2-(2<1->methanesulfonylethylamino)-2-oxo-tetrahydro-2H-1,3,2-oxazaphosphorine (sufosfamide) and other cyclophosphamides with a similar structural formula, can obtain compounds with valuable cytostatic properties, that these compounds, however, have such little stability that a processing into pharmaceutical preparations as is necessary in human medicine is not possible. The object of the present invention is therefore to provide cyclophosphamides with a further derived hydroxy group in the 4-position which are particularly distinguished by high cytostatic action and improved stability.

In the analog method of the invention, compounds with the general formula are prepared

where R]_, R 2 and R 3 are the same or different and mean hydrogen or 2-chloroethyl and A is the group -0N(R 4 )- and R 4 is a C 1-6 alkyl group, X is oxygen and Z benzylamino, phenylamino or C 1-4 -alkylamino, or R 4 is hydrogen, X is oxygen and Z is benzyloxy or C 1-4 ~ alkoxy, or where A is the group -

N(OH)- and Z is the group -NRgR7, X is oxygen or sulfur and R6 is hydrogen or C1_4-alkyl, and R7 is hydrogen, benzoyl, cycloalkyl with 3-8 carbon atoms, straight or branched alkyl with 1-18 C- atoms, whereby the alkyl residue can also be substituted with -OH, -halogen, carboxy, carb-C1_2~ alkoxy, benzyloxycarbonyl or -PO(CH3)2. or where R7 means phenyl-C1_4~alkyl, which is optionally substituted with a carboxy group, or where R7 is phenyl, the phenyl residue optionally being substituted with one or two C1_4~alkyl, nitro, halogen or trifluoromethyl, and where R6 and R7 together with the nitrogen atom forms a piperidine ring or a morpholine ring, as well as pharmaceutically acceptable salts thereof.

Because of the particularly good properties and easy availability, there are 4-carbamoyloxy-oxazaphosphorins of the general formula I where Z is the group NR5R7, X is oxygen, R5 and R6 which are the same or different are hydrogen, methyl or ethyl and R7 is hydrogen or straight-chain or branched alkyl with 1-18 carbon atoms, phenyl or benzyl, preferred.

Another group of preferred compounds of formula I are those in which X is oxygen, R 1 , R 2 and R 3 , which are the same or different, are hydrogen or a 2-chloroethyl residue, R 4 , R 5 and R 6 are hydrogen, and R 7 is hydrogen, benzyl, phenyl which is unsubstituted or substituted with one or two carboxy groups, optionally with a carboxy group substituted C1-4-alkyl or phenyl-C1-4-alkyl, which is optionally substituted with 1 or 2 carboxy groups in the phenyl and/or alkyl part of the remainder.

The process according to the invention for the production of the new 4-carbamoyloxy-oxazaphosphorines of the general formula I is characterized by reacting a hydroxy-or 4-methoxy- or 4-ethoxy-oxazaphosphorine of the general formula II

wherein R 1 , R 2 and R 3 have the same meaning as in formula I and Y is hydrogen, methyl or ethyl, with a hydroxycarbamate derivative of the general formula III in which Z, R 4 and X have the same meaning as in formula I in the presence of an inert solvent, whereby compounds of formula I are obtained where A is the group >N(OH), when in formula III R4 stands for hydrogen and Z for -NR5R7. Suitable solvents include water, lower alkyl halides such as methylene chloride in particular, lower alkyl ketones such as acetone in particular, diethyl ether, dimethylformamide (DMF), hexmethylphosphoric acid triamide (HMPT). or similar solvents or mixtures of several such solvents. The reaction is carried out at temperatures in the range -35°C to +50°C, i.e. possibly during cooling, at room temperature or during heating. The reaction can take place in the presence of an acidic catalyst such as an inorganic acid, trichloroacetic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or a Lewis acid such as AlCl3, ZnCl2 or TiCl4.

A further method for the preparation of compounds of formula I, where Z is -NR6R7 with R6 = H and R5 is hydrogen, is characterized by reacting an oxime of the general formula IV^ where RI - R3 has the same meaning as in formula I , in an inert solvent at a temperature in the range -70°C to +50°C, with a compound of the general formula V

where X and R7 have the same meaning as in formula I, whereby compounds of formula I arise where A is the group >N(OH) and Z the group -NHR7.

Optionally, the obtained compounds are converted into the corresponding salts.

The reaction can be followed by thin-layer chromatography. Isolation of thin-layer chromatographically uniform substances is successful by usual processing methods for such products, especially by crystallization or chromatographic purification. The structure is determined by melting point determination, thin-layer chromatography, elemental analysis and/or IR and <1>H-NMR spectral analysis.

The compounds used as starting materials according to the method are largely known and can be used in crystalline form or as raw products and can be synthesized as indicated below in a manner known per se.

4-Hydroxy-oxazaphosphorins are obtained by reduction of the 4-hydroxyperoxy derivatives (eg A. Takamizawa et al., "J. Med. Chem. <11>, 18, 376 (1975)). 4-Methoxy- respectively The 4-ethoxy-oxazaphosphorines arise under acid catalysis from the 4-hydroxy derivatives in methanol or ethanol or in inert solvents containing methanol or

ethanol. The hydroxyurea derivatives are produced by reacting correspondingly substituted isocyanates or carbamide acid chlorides with hydroxylamine or N-monosubstituted

hydroxylamines. In a similar way, the N-hydroxycarbamic acid esters (hydroxycarbamates) can be obtained by reacting the corresponding chlorocarbonic acid esters with hydroxylamine or N-monosubstituted hydroxylamines. For the synthesis of hydroxyurea or N-hydroxycarbamic acid esters with an additional hydroxy, carboxy or sulfonic acid group, a synthesis route with a protecting group is recommended.

The oximes of formula IV can be obtained via various syntheses. Thus, they can be obtained by reacting corresponding 4-hydroxy-oxazaphosphorins with hydroxylamine or with hydroxylamine salts, the pH value should, if possible, be kept between 2 and 7.

From the 4-carbamoyloxy-oxazaphosphorines according to the invention, the racemic cis- and trans-isomers can be prepared (cis = 2R,4R/2S,4S; trans = 2R,4S/2S,4R). The assignment takes place in accordance with the IUPAC nomenclature rules and with the literature on corresponding oxazaphosphorine derivatives. The cis- and respectively the transform can also be produced specifically, depending on the choice of reaction conditions.

Pharmacologically, the isomers show no significant differences.

The compounds according to the invention are distinguished by remarkable chemotherapeutically valuable properties. Compared to the previously known reference substance cyclophosphamide, they have an approximately equally strong cancerotoxic chemotherapeutic effect on experimental transplant tumors in rats. They are directly alkylating in aqueous solution and have a high cytotoxicity in vitro, while cyclophosphamide needs an enzymatic activation and is practically not cytotoxic in vitro. The acute toxicity of the compounds according to the invention is approx. 4 times less than the reference substance cyclophosphamide, the therapeutic application area is thereby considerably expanded. Also with regard to organotoxic side effects such as leukocyte depression and immunosuppression, the compounds according to the invention show clear advantages over the reference substance cyclophosphamide.

The compounds according to the invention are suitable for the treatment of malignant tumors and similar malignant diseases such as leukemia in humans. Quantities in the range 0.01-100 mg/kg are thereby used. The pharmaceutical preparations used here are prepared according to usual methods using usual additives and carriers.

The following examples shall illustrate the invention in more detail without limiting it.

Example 1

2-( bis-(2-chloroethyl)- amino)- 2-oxo-tetrahydro- 2H- 1. 3. 2-oxazaphosphorin- 4- yl- oxvurea 15 g (54 mmol) 4-hydroxycyclophosphamide (that is 2-(bis-(2-chloroethyl)-amino-4-hydroxy-tetrahydro-2H-1,3,2-oxazaphosphorin-2-oxide) and 4.4 g (58 mmol) of hydroxyurea are dissolved in 70 ml of DMF which is acidified with trichloroacetic acid to pH 3-4 and left for 20 hours at 0° C. in a refrigerator. The crystal slurry was then diluted with 70 ml of acetic acid and sucked off after 2 hours, washed, dried and recrystallized from methanol. Yield: 11.3 g ( 62% of the theoretical) of the cis form, melting point 139-143°C (under cleavage).

Example 2

2-( bis- f 2- chloroethyl)- amino)- 2- oxo- tetrahydro- 2H- 1. 3. 2-oxazaphosphorin- 4- yl- oxy- urea. cis form

1.1 g (4.0 mmol) of 4-hydroxycyclophosphamide is dissolved in methanol to which a trace of trichloroacetic acid has been added, and left overnight at -25°C, after which the methanol was gently drawn off, the residue taken up in a little methylene chloride, dried and concentrated to 1.2 g of 4-methoxycyclophosphamide (that is, 2-(bis-(2-chloroethyl)-amino)-4-methoxy-tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide. Then 1.2 g of 4-methoxycyclophosphamide and 3+4 mg of hydroxyurea were dissolved in 3 ml of DMF and stored for 20 hours at -25° C. in a refrigerator. The crystal slurry was diluted with 3 ml of acetic acid, suctioned off, washed, dried and recrystallized from methanol .

Yield: 11.3 g (62% of theory) of the cis form, melting point 139-143°C (under cleavage).

Example 2

2-( bis-(2-chloroethyl)- amino)- 2- oxo- tetrahydro- 2H- 1. 3. 2-oxazaphospharin- 4- yl- oxy-urea. cis form

1.1 g (4.0 mmol) of 4-hydroxycyclophosphamide is dissolved in methanol to which a trace of trichloroacetic acid has been added, and left overnight at -25°C, after which the methanol was gently drawn off, the residue taken up in a little methylene chloride, dried and concentrated to 1.2 g of 4-methoxycyclophosphamide (that is, 2-(bis-(2-chloroethyl)-amino)-4-methoxy-tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide. Then 1.2 g of 4-methoxycyclophosphamide and 304 mg of hydroxyurea were dissolved in 3 ml of DMF and stored for 20 hours at -25° C. in a refrigerator.

Yield: 670 mg (50% of the theoretical) of the same product

as in example 1.

Example 3

2- ( bis- f 2- chloroethyl)- amino)- 2- oxo- tetrahydro- 2H- 1. 3. 2-oxazaphosphorin- 4- yl- oxy- urea. transform

16 g (58 mmol) of 4-hydroxycyclophosphamide and 5.2 g (68 mmol) of hydroxyurea were dissolved in 160 ml of water, acidified with trichloroacetic acid to pH 3-4 and left for 20 hours at 0°C in a refrigerator. The crystal slurry was then sucked off, washed with water, dried over P2O5 in high vacuum and recrystallized from methanol/chloroform. Yield: 12.7 g (65% of theoretical) of the transform of the product according to example 1 with a melting point of 148°C (under cleavage). Example 4 3-(2-chloroethyl)-2-(bis-(21-chloroethyl)-aminoi)-2-oxo-tetrahydro-2H-1.3.2-oxazaphosphorin-4-vl-oxy-urea 20 g (50 mmol) 4-Hydroxytrophosphamide (that is, 3-(2-chloroethyl)-2-bis-(2-chloroethyl)-amino)-4-hydroxy-tetrahydro-2H-1,3,2-oxazaphosphorin-2- oxide and 5.3 (70 mmol) of hydroxyurea f were dissolved in 100 ml of DMF and cooled to -15°C. The whole was then acidified with trichloroacetic acid to pH 3-4 and stirred for 5 hours at -15°C.

After standing overnight at 0°C, the reaction solution was diluted with twice the amount of water. The whole was then shaken out 4 times with 300 ml of acetic acid/methanol 1 ratio 10:1, after which the combined ester phases were washed

2 times with water, dried over sodium sulfate and concentrated in vacuo to 22 g of oil. After absorption in acetic acid/methanol

crystallized 4.2 g with melting point 106-110°C. The mother liquor was separated by column chromatography on silica gel with chloroform/methanol in a ratio of 10:1 and recrystallized together with the first crystallisate from acetic ester/methanol.

Yield: 7.0 g (35% of theoretical), melting point 115-116°C

(during cleavage).

Example 5

3- benzyl-1-\ 2- bis- f 2- chloroethyl)- amino)- 2- oxo- tetrahydro- 2H-1. 3. 2- oxazaphosphorin- 4- yl- oxYl- urea

To 900 mg (3.25 mmol) of 4-hydroxycyclophosphamide in 1 ml of methylene chloride is added 540 mg (3.25 mmol) of 3-benzyl-1-hydroxyurea in 40 ml of acetone and a catalytic amount of trichloroacetic acid. The mixture is stored overnight at -25°C, the crystals are then sucked off, washed with acetone and ether and recrystallized from acetic acid.

Yield: 500 mg (40% of the theoretical), melting point 122-123"C (during cleavage).

Example 6

3-( o- bromophenyl)- l-[ 2-( bis-(2- chloroethyl)- amino)- 2- oxo-tetrahydro- 2H- l. 3. 2- oxazaphosphorin- 4- yl-oxyl- urea

560 g (2 mmol) of 4-hydroxycyclophosphamide in 10 ml of acetone are added to 460 mg of 3-o-bromophenyl-1-hydroxyurea and a catalytic amount of trichloroacetic acid and the mixture is placed at -25°C.

After 2 days, the crystals are sucked off and recrystallized from acetone.

Yield: 320 mg (32% of the theoretical), melting point 110-111'C (during cleavage).

Example_el_7

N- r2- bis-(2- chloroethyl)- amino)- 2- oxo- tetrahydro- 2H- 1. 3. 2-oxazaphosphorin- 4- yl-oxyl- 4- morpholinocarboxamide

1.2 g (4.3 mmol) of 4-hydroxycyclophosphamide in 15 ml of acetone are added to 630 mg (4.3 mmol) of N-hydroxy-morpholino-carboxamide and a trace of trichloroacetic acid and then stored at -25°C. After 4 days, the crystals are sucked off and recrystallized from acetone.

Yield: 780 mg (45% of the theoretical), melting point 123-124°C (under cleavage).

Example 49

Ethyl- 2-( bis-( 2-chloroethyl)- amino- 2- oxo- tetrahydro-- 2H- 1 . 3. 2-oxazaphosphorin- 4- yl- oxv-carbamate

550 mg (2 mmol) of 4-hydroxycyclophosphamide and 210 mg (2 mmol) of ethyl hydroxycarbamate (hydroxyurethane) were dissolved in 5 ml of dry alcohol-free methylene chloride, a catalytic amount of trichloroacetic acid was added, molecular sieve 4 A was added and left at -25°C for 3 days . The solution was then separated from the molecular sieve, washed once in a separatory funnel with dilute sodium bicarbonate solution, the methylene chloride phase dried over sodium sulfate concentrated in vacuo and diluted with ether. After standing for 20 hours at -25°C, the crystals were sucked off, washed and dried.

Yield: 290 mg (40% of the theoretical), melting point 96°C.

Example 50

Benzyl- 2-( bis-( 2- chloroethyl)- amino- 2- oxo- tetrahydro- 2H- 1. 3. 2- oxazaphorin- 4- yl- oxv-carbamate

750 mg (2.7 mmol) of 4-hydroxycyclophosphamide and 450 mg (2.7 mmol) of benzyl hydroxycarbamate were dissolved in 6 ml of alcohol-free methylene chloride to which some trichloroacetic acid had been added, kept at -25°C in a refrigerator, filtered after 3 days, after which the mother liquor was diluted with 5 ml of chloroform, washed with water, with dilute sodium bicarbonate solution and once more with water, dried over sodium sulfate and concentrated in vacuo. The oil-like residue was recrystallized from ethyl acetate/a little methanol.

Yield: 680 mg (59% of the theoretical), melting point 112-114°C.

Example 51

3- r2-( bis-(2- chloroethyl)- amino)- 2- oxo- tetrahydro- 2H- 1. 3. 2-oxazaphosphorin- 4- yl- oxy1- ureido- acetic acid- cyclohexylamine- salt

a) 3-hydroxy-ureido-acetic acid

56.5 g (0.28 mol) glycine benzyl ester hydrochloride was

suspended in 300 ml of toluene and with stirring on a bath

temperature of 14 0°C, a dry phosgene gas was introduced for 2 hours. The reaction mixture was then concentrated in vacuo and the residue distilled in high vacuum.

Yield: 51 g (95% of theory) benzyl isocyanato-acetate with boiling point 0.05: 100-102"C.

To 28.7 g (0.15 mol) of benzyl isocyanato-acetate in 50 ml of dioxane, while stirring and periodically cooling, 6.6 g (0.2 mol) of hydroxylamine in 200 ml of dioxane are added dropwise. After stirring for 1 hour then at room temperature, the whole was concentrated in vacuo and the residue recrystallized from acetic acid.

Yield: 28.1 g (83.6% of the theoretical) benzyl-3-hydroxy-ureido-acetate with melting point 113-120°C.

22.4 g (0.1 mol) of benzyl-3-hydroxy-ureido-acetate in 300 ml of methanol was added to 5 g of palladium-on-charcoal and hydrated in a shaking container. After approx. After 20 minutes, the corresponding hydrogen uptake had ended. The catalyst was sucked off, the filtrate concentrated in vacuo and the solid residue recrystallized from dioxane.

Yield: 9.8 g (73% of theoretical) 3-hydroxy-ureido-acetic acid, melting point 135°C. b) 2.4 g (18 mmol) of 3-hydroxy-ureido-acetic acid in 10 ml of water and 25 ml of acetone were added to 6.1 g (2 mmol) of 4-hydroxy-cyclophosphamide and left overnight at -25°C . Then 25 ml of acetone and 1.8 g or 18 mmol of cyclohexylamine in 10 ml of acetone were added, the whole was sucked off after a 2-hour standstill and recrystallized from acetone with a little methanol.

Yield: 3.1 g (44% of the theoretical), melting point 107-108°C.

Example 52

3-[ N. N-( bis- f 2- chloroethyl)- diamino- phosphinyl- oxy]- propion-aldehyde- oxime (aldophosphamide- oxime

4.0 g (13.7 mmol) of 4-hydroperoxycyclophosphamide is suspended in 50 ml of water under ice cooling and 500 mg of sodium thiophosphate is added. 5 moles of water. While stirring at 5-10°C, the pH value was checked with a pH meter and maintained between pH 4.5 and 5.5 with 2N sulfuric acid, and it was dipped into a concentrated solution of sodium thiosulphate until the pH value no longer increased . It was stirred for half an hour at approx. 10°C, after which it was added dropwise to an aqueous solution of 950 mg of hydroxylamine hydrochloride. The pH value was maintained at 5 with 2N caustic soda, the whole was put overnight in a refrigerator at 5°C, it was extracted 4 times with 50 ml of acetic acid each time, and the organic extracts were, after drying over sodium sulfate, concentrated in vacuo at 30°C. The residue was taken up in methylene chloride, and the crystals were sucked off after 1 day. Yield: 3.4 g (85% of the theoretical), melting point 79-81°C.

Example 53

3- p- bromophenyl- l-\ 2 -( bis- 2- chloroethyl)- amino)- 2- oxo- tetrahydro-2H- 1. 3. 2- oxazaphosphorin- 4- yl- oxy1- urea

5.8 g (20 mmol) of aldophosphamide oxime in 60 ml of acetone was added to 4 g (20 mmol) of p-bromophenyl isocyanate in 40 ml of acetone and then stirred for 5 hours under cooling. After 2 hours, the aspirated crystallisate was dried under vacuum in a rotary evaporator at 40°C and recrystallized in methanol.

Yield: 8.1 g (82.8% of theory), m.p

118-120'C.

Eczema gel_54

m- trifluoromethylphenyl- 1- f 2-( bis-( 2- chloroethyl)- amino)- 2-oxotetrahydro- 2H- 1. 3. 2- oxazaphosphorin- 4- yl- oxy1- urea

7.3 g (25 mmol) of aldophosphamidoxime in 80 ml of acetone was added to 4.7 g (25 mmol) of m-trifluoromethylphenyl isocyanate in 40 ml

acetone. It was stirred for 3 hours at CC, the whole was placed overnight in a refrigerator at -25°C, after which 150 ml of petroleum ether was added, and the whole was sucked off after further standing in a refrigerator overnight at -25°C. The crystallisate was dried at 30°C and recrystallized from isopropanol.

Yield: 9.2 g (76.8% of theory), m.p

91-93"C.

Example_el_55_

3- cyclohexyl- 2-\ 2 -( bis-(2- chloroethyl)- amino)- 2- oxo- tetrahydro- 2H- 1. 3. 2- oxazaphosphorin- 4- yl- oxy]-urea

5 g (18 mmol) of aldophosphamidoxime and 2.2 g of cyclohexyl isocyanate were dissolved in 10 ml of acetone each, combined at 0°C, and after 2 hours the crystals were sucked off and washed with acetone/ether. Yield: 4.2 g (56% of theory), m.p 113°C. Example 56 3-ethyl-1-r2-(bis-(2-chloroethyl)-amino)-2-oxo-tetrahydro-2H-1. 3. 2-oxazaphosphorin-4-yl-oxv-urea 5 g (18 mmol) of aldephosphamidoxime and 1.2 g of ethyl isocyanate were each dissolved in 15 ml of acetone and combined at approx. 0°C. After 5 hours, the crystallisate was sucked off and washed with acetone/ether.

Yield: 3.5 g (54% of theoretical), m.p

lOl-C.

Example_<57>

3-( fluoren- 2- vl)- 1- T2-( bis-( 2- chloroethyl)- amino)- 2- oxo-tetrahydro- 2H- l. 3. 2- oxazaphosphorin- 4- yl- oxyT- urea

2.9 g (10 mmol) of aldophosphamidoxime in 30 ml of acetone was added to 2.1 g (10 mmol) of fluorenyl-2-isocyanate in 20 ml of acetone at 0°C. The next day, the precipitated product was sucked off, dried under vacuum at 60°C and recrystallized from isopropanol/methanol.

Yield: 2.5 g (40.1% of theory), m.p

114°C.

<Ex>emp_el_58

3- benzoyl-1- f 2- fbis-(2- chloroethyl)- amino- 2- oxo- tetrahydro- 2H-1. 3. 2-oxazaphosphorin-4-yl-oxy1-urea

5.8 g (20 mmol) of aldophosphamidoxime in 60 ml of acetone was added to 2.9 g (20 mmol) of benzoisocyanate in 40 ml of acetone. It was stirred for 5 hours in an ice bath under a nitrogen atmosphere, after which the solid was sucked off after 2 hours, dried in a rotary evaporator at 30°C and recrystallized from methanol. Yield: 2.4 g (27.3% of theory), m.p

124-125°C.

Example_el_59

3- p- nitrophenyl- 1- r 2- fbis- f 2- chloroethyl)- amino)- 2- oxo- tetrahydro- 2H- l. 3. 2- oxazaphosphorin- 4- yl- oxy1- urea

To 5.8 g (20 mmol) aldephosphamidoxime in 60 ml acetone was added a solution of 3.3 g (20 mmol) p-nitrophenyl isocyanate in 40 ml acetone, after which after 2 hours the solid product was sucked off, dried in a rotary evaporator at 40°C and recrystallized from dimethylformamide/ethanol.

Yield: 6.7 g (73.5% of theory), m.p

117-118°C.

The cytotoxic activity of compounds produced according to the invention was determined in vitro.

To determine the cytotoxic activity in vitro, according to Schmåhl and Druckrey, "Naturwissen-schaften", 43., 199 (1956), newly obtained cells of the Yoshida-Ascites sarcoma were incubated for 2 hours at 37°C with increasing concentrations of the test substances in Ringer resolution. After washing out the substances, the transplantability of the tumor cells on non-treated experimental animals was examined. By determining the concentration, EC50, at which half of the animals' further development of the tumor cells transplanted after the incubation is prevented, a quantitative determination of the cytotoxic activity in vitro can be achieved.

This incubation experiment has proven particularly beneficial for assessing the direct cytotoxic potency of alkylating compounds and thus for distinguishing between so-called transport and active forms. Cyclophosphamide, which needs enzymatic activation to be effective, is not yet cytotoxic at a concentration of 2600 µg/ml, i.e. the solubility limit. In contrast, the compounds described here are cytotoxically active in vitro at concentrations from 1 to 1000 pg/ml, reference is made to the following table.

Cytotoxic activity in vitro (37'C, 2 hours)

Claims (1)

Analogous process for the preparation of 4-carbamoyloxyox-azaphosphorine derivatives with cytostatic action and with the general formula in which , R 2 and R 3 are the same or different and mean hydrogen or 2-chloroethyl and A is the group -ON(R 4 )- and R 4 is a CX . —D ,,-alkyl group, X is oxygen and > Z benzylamino, phenylamino or C1_4-alkylamino, or R4 is hydrogen, X is oxygen and Z is benzyloxy or C1_4~ alkoxy, or in which A is the group -N(OH)- and Z is the group -NR5R7, X is oxygen or sulfur and Rg is hydrogen or C1_4~alkyl, and R7 is hydrogen, benzoyl, cycloalkyl with 3-8 carbon atoms, straight or branched alkyl with 1-18 C atoms, whereby the alkyl residue can also be substituted with -OH, -halogen, carboxy, carb-C1_2~ alkoxy, benzyloxycarbonyl or -PO(CH3)2, or in which R7 means phenyl-C1_4~alkyl, which is optionally substituted with a carboxy group, or in which R7 is phenyl, whereby the phenyl residue is unsubstituted or may be substituted once or twice with C1_4-alkyl, nitro, halogen or trifluoromethyl, and in which R5 and R7 together with the nitrogen atom form a piperidine ring or a morpholine ring, as well as pharmaceutically acceptable salts thereof, characterized in that a) reacts a 4-hydroxy- or 4-methoxy- or 4-ethoxy-oxazaphosphorine with the general formula (II) in which Rx, R2 and R3 have the same meaning as in formula (I) and Y is hydrogen, methyl or ethyl, with a hydroxycarbamate derivative of the general formula (III) in which Z, R4 and X have the same meaning as in formula (I) in the presence of an inert solvent, thereby obtaining compounds of formula (I) where A is the group >N(OH), when in formula (III) R4 is for hydrogen and Z for -NR6R7, or b) reacts an oxime with the general formula (IV) wherein R x - R 3 have the same meaning as in formula (I), in an inert solvent at a temperature in the range of -70°C to +50°C, with a compound of the general formula (V) wherein X and R7 have the same meaning as in formula (I), whereby compounds of formula (I) arise where A is the group >N(OH) and Z the group -NHR7, and optionally transfer the obtained compounds to the corresponding salts.