IE51784B1 - 4-carbamoyloxy-oxazaphosphorins,method of producing them and pharmaceutical preparations containing them - Google Patents

Abstract

The present invention relates to novel 4-carbamoyloxyoxazaphosphorines of the general formula I <IMAGE> to processes for their preparation, and to pharmaceutical preparations containing these active compounds. [FR2489825A1]

Description

This invention relates to oxazaphosphorins, methods of producing them and pharmaceutical preparations containing them.

It is known from German Offenlegungsschriften 2 231 311 5 and 2 552 135 that the introduction of a hydroperoxy group -OOH into the 4-position of the known cytostatics 2-^is(2-chloroethyl)-amino7-2-oxo-tetrahydro-2H-1,3,2-oxazaphosphorin (Cyclophosphamide), 3-(2-chloroethylaminO)-2/Bis-(2'-chloroethyl)-amino7_2-oxo-tetrahydro-2H-1,3,210 oxazaphosphorin (trofosfamid), 3-(2-chloroethylamino)-2(2'-chloroethylamino)-2-oxo-tetrahydro-2H-1,3,2-oxazaphosphorin (ifosfamid), 3-(2-chlorethylamino)-2-(2'-methanesulfonylamino)-2-oxo-tetrahydro-2H-1,3,2-oxazaphosphorin (sufosfamid) and other similar cyclophosphamides yieldsinto compounds having valuable cytostatic properties. Thes.e compounds, however, have such a low stability that it is not possible to convert them into pharmaceutical preparations as is necessary for their use in human therapy. It is therefor an object of the present invention to provide new cyclophosphamide compounds substituted in the 4-position by a further converted hydroxy group which are characterized in particular by a high cytostatic activity and an improved stability.

The new compounds of the present invention are 4-carb25 amoyloxy-oxazaphosphorins of the formula 7—4 R I '4 /Rg In which Z is the group -N or the group -OR X is oxygen \d ' or sulphur; R^, Rg and Rg, 7 which may be the same or different, represent hydrogen, methyl, ethyl, 2-chloroethyl or 2-methanesulphonyloxyethyl; the groups R4> which may be the same or different, represent hydrogen, methyl or ethyl; Rg and Rg, which may be the same or different, represent hydrogen, C1_4-alkyl, hydroxy-C1_4~alkyl or phenyl; R? is hydrogen, the carbamoyl group, -0Rg (in which Rg is hydrogen, C^^-alkyl, phenyl or benzyl), straight or branched chain C^_^g-alkyl (optionally substituted by 1 to 3 substituents which may be the same or different and are selected from the group consisting of hydroxy, halogen, -COOH, -COORg, -CONHg, -phenyl, benzyloxycarbonyl, -N(Rg)2> -N(Rg)g, ORg, -SRg, -SO-Rg, “SOg-Rg, SOgH or -PO(CHg)g, in which Rg represents methyl or ethyl), phenyl-C1_4-alkyl (optionally substituted by 1 or 2 carboxy groups in the phenyl and/or alkyl part), allyl, Cg_g-cycloalkyl, tetrahydrofuranyl, tetrahydropyranyl, phenyl (optionally substituted by one or two C^_4~alkyl, C^_g-alkoxy, nitro, halogen, trifluoromethyi, -SOgNHg, carboxy, benzyloxycarbonyl and/or carb-C-^-alkoxy), benzhydryl, naphthyl, fluorenyl, fluorenopyl, pyridyl, thienyl, benzoyl or C1-4Rg and Rg or Rg and R? together with the atoms to which they are connected form a saturated heterocyclic ring optionally containing an oxygen atom, a C^_4~alkyl substituted nitrogen atom or an -S-, -SO- or -SOg- group; or Rg and R? together with the adjacent nitrogen form an aziridin ring optionally substituted by a cyano or carbamoyl group, and the pharmaceutically useful salts thereof, but excluding compounds in which RK represents hydrogen and Z is the group -N 6.

XR7 Due to their particularly favourable properties and ease of manufacture, the„preferred compounds of formula I are those /Rg in which Z is -N ; X is oxygen; RK is methyl or ethyl; R_ xR75 6 is hydrogen, methyl or ethyl; and R? is hydrogen, straight or branched chain C1_lg-alkyl, phenyl or benzyl. -351784 Particularly preferred compounds are those of the above preferred compounds in which all groups in formula I represent hydrogen atoms.

Another group of preferred compounds are those of formula I in which X is oxygen, R^, Rg and Rg which may be the same or different, represent hydrogen or the 2-chloroethyl group, R4, Rg and Rg represent hydrogen and R? is hydrogen, benzyl, phenyl (optionally substituted by one or two carboxy groups), C-^-alkyl (optionally substituted by one carboxy group) or phenyl-C4_4~alkyl (optionally substituted by one or two carboxy groups in the phenyl and/or alkyl part of the group).

Oxazaphosphorins are obtained, according to the invention, by reacting a compound of formula II II in which R^, Rg, Rg and R4 are as defined in formula I and Y is hydrogen, methyl or ethyl, with a compound of formula III Rc X 15 II HO - N - C - Z III in which Z, Rg and X are as defined in formula I (except that, inDaddition, Rc may represent hydrogen when Z is the group -N ), in the presence of an inert solvent, optionally with heating or cooling and/or in the presence of an acid catalyst.

Water, lower alkyl halides such as methylene chloride, lower alkyl ketones such as acetone, diethyl ether, dimethylformamide (DMF), hexamethylphosphoric acid triamide (HMPT) or similar solvents or mixtures of such solvents are suitable inert solvents for use in the above process. The reaction -451784 may be carried out at temperatures in the range from -35°C. to +50°C., that is to say possibly with cooling, at room temperature or with heating. The reaction can be carried out in the presence of an acid catalyst such as an inorganic acid, trichloroacetic acid, trifluoromethanesulphonic acid or a Lewis acid such as AlCIg, ZnCl2 or TiCl4· Another embodiment of the process to produce oxazaphosphorins is characterized in that an oxime of formula XV R. R, 13 z\ zNH 0 XV CH ι OH 1θ wherein R^ to R4 are as defined in formula I, is subjected to reaction with a compound of formula V X = C = N - R_ wherein X and R? are as defined in formula I, in an inert solvent at a temperature ranging from -70°C. to +50°C.

The course of the reaction in both embodiments can be followed by thin layer chromatography. The isolation of thin layer chromatography uniform substances is achieved by conventional preparation processes for such products, particularly by crystallisation or chromatographic purification.

Confirmation of structure may be effected by melting point, thin layer chromatography, elementary analysis, infra red and/or ^H-NMR spectral analysis. -551784 Tho compounds of formulae II and III used as raw materials in the method according to the invention are largely known and may be used in crystalline form or as a crude product. They can be synthesized in known manner, for example as follows: 4-Hydroxy-oxazaphosphorins are obtained by reduction of the 4-hydroperoxy-derivatives (see for example A. TAKAMIZAWA et al., J.MED.CHEM. 18, 376 (1975)). The 4-methoxy- or 4-ethoxy-oxazaphosphorins may be formed under acid catalysis 1o from the 4-hydroxy derivatives in methanol or ethanol or in inert solvents which contain methanol or ethanol. The hydroxy urea derivatives are produced by the conversion of suitably substituted isocyanates or carbamic acid chlorides with hydroxylamine or N-monosubstituted hydroxylamines.

Racemic cis- and trans-isomers can be produced from the 4-carbamoyloxy-oxazaphosphorins according to the invention. The cis configuration is 2R, 4R/2S, 4S whilst the trans configuration is 2R, 4S/2S, 4R which correlation is in accordance with the IUPAC nomenclature rules and with the 2o literature concerning corresponding oxazaphosphorin derivatives. The cis- or trans-form can be produced deliberately by selection of the reaction conditions. Pharmacologically, the isomers do not display any significant differences.

The compounds according to the invention possess particularly valuable chemotherapeutic properties. In comparison with the previously known cyclic phosphamides such as cyclophosphamide, trofosfamide, ifosfamide and sufosfamide, they display substantially equal carcinotoxic chemotherapeutic effectiveness on experimental transplant tumours in rats. They have 3o a direct alkylating effect in aqueous solution and have a high cytotoxicity in vitro, unlike the cyclic phosphamides where, for example, cyclophosphamide requires anereymatic activation and has practically no cytotoxic effect in vitro. The acute toxicity of the compounds according to the invention is considerably lower than that of the known cyclic phosphamides, for example it is about 4 times less than that of the reference substance cyclophosphamide, and thus the therapeutic ratio of the compounds of the inven5 tion is considerably improved. The compounds according to the invention also have clear advantages over the prior art cyclic phosphamides with regard to organotoxic side effects such as leukocyte depression and immunosuppression.

The 4-carbamoyloxy-oxazaphosphorins according to the present 1o invention are useful in the treatment of malignant tumors and similar malignant diseases in humans such as leukemia. They are administered in daily dosages in the range of o,o1 to 1oo mg, per kg. of body weight. The pharmaceutical preparations used in such therapy are those usual for cyclo15 phosphamide and the other known cytostatic oxazaphosphorins. They may be produced in a usual manner using usual additives, diluents and/or carrier materials.

The compounds of the present invention may be administered to animals, including humans, in the same manner and against 2o the same disease conditions as those known in connection with the above mentioned cyclic phosphamides. However in view of the lower toxicity of the compounds of the invention they can be used at higher dosage rates and therefore possess a significantly expanded effective therapeutic dosage.range. Normally, the compounds of the invention are administered in conventional formulations produced by mixing the compounds with physiologically acceptable vehicles and/or diluents and by conventional routes for example orally or by injection. The production of such 3o formulations and the methods by which they are administered are well known to those skilled in the art. - 7 51784 The compounds according to the present application are useful in the preparation of pharmaceutical products or drugs containing as active agent one or several of such compounds, possibly together with other pharmacologically or pharmaceutically active agents. The production of the pharmaceutical preparation is effected in manners known per se using known and usual pharmaceutical auxiliary agents or usual carrier materials or diluents.

Useful as carrier and auxiliary materials are for instance 1o compounds which are described in ULLMANNS ENCYKLOPAEDIE DER TECHNISCHEN CHEMIE (1953) vol. 4, pgs. 1 to 39; JOURNAL OF PHARMACEUTICAL SCIENCES, (1963), VOl. 52, pgs. 918 and following? Η. v. CZETSCH-LINDENWALD, Hilfsstoffe fur Pharmazie und angrenzende Gebiete, Pharm. Ind. (1961), vol. 2, pgs. 72 and following; Dr. Η. P. FIEDLER, Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete, Cantor KG. Aulendorf (Wiirttemberg) 1971.

Examples are gelatin, natural sugars such as cane sugar or lactose, lecithin, pectin, starch (such as corn starch), 2o alginic acid, tylose, talcum, lycopodium, silicic acid (for instance colloidal silicic acid), cellulose, cellulose derivatives (for instance cellulose ethers, the hydroxy group whereof being partly etherized with lower aliphatic saturated alcohols and/or lower aliphatic saturated oxy25 alcohols, such as methyloxypropylcellulose), stearates, magnesium and calcium salts of fatty acids having from 12 to 22 C-atoms, in particular of saturated fatty acids (such as stearates), emulsifiers» oils and fats, in particular plant fats (such as peanut oil, castor oil, olive 3o oil, sesaneoil, cottonseed oil, corn oil, wheatgrain oil, sunflower oil, codfish liver oil, mono-, di- and triglycerides of saturated fatty acids ε·}2Η24°2 C'18®36°2 and mixtures thereof), pharmaceutically compatible monoor polyols and polyglycols such as polyethyleneglycol and derivatives thereof, esters of aliphatic saturated or - 8 51784 unsaturated fatty acids (having from 2 to 22) in particular from 1o to 18 carbon atoms) with aliphatic monoalcohols (having from 1 to 2o carbon atoms) or of polyols such as glycols, glycerol, diothyleneglycol, pentaerythritol, sorbitol and mannitol which optionally are etherized; benzylbenzoate, dioxolanes, glycerol formales, tetrahydrofurfuryl alcohol, polyglycol ether with C^C^-alcohols, dimethylacetamide, lactamides, lactates, ethylcarbonates, silicones (in particular dimethylpolysiloxanes having a 1o medium range viscosity) and magnesiumcarbonate.

For preparing solutions there are used for instance water or physiologically compatible organic solvents such as ethanol, 1.2-propyleneglycol-, polyglycols and derivatives thereof, dimethylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerol and paraffins.

In the preparation of the pharmaceutical products there may be used usual and known solubilizers or emulsifiers, for instances polyvinylpyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, lecithin, acacia, 2o tragacanth, polyoxyethyl sorbitan monooleate, polyoxyethylized fats, polyoxyethylized oleotriglycerides, linolized oleotriglycerides, polyethyleneoxide-condensation products of fatty alcohols, alkylphenols or fatty acids . or 1-methyl-3-(2-hydroxyethyl)-imidazolidon-(2). As above used, polyoxyethylized means that the respective compound contains polyoxyethylene chains with a polymerisation degree generally between 2 to 4o and in particular 1o to 2o.

Such polyoxyethylized products may for instance be produced 3o by subjecting the corresponding hydroxy compound (for instance a mono- or diglyceride or unsaturated compound such as containing the unsaturated oleyl group) with ethylene oxide (for instance with 4o moles of ethylene oxide per mole of glyceride). - 9 5178 4 Examples of oleotriglycerides are olive oil, peanut oil, castor oil, sesame oil, cottonseed oil, corn oil (see for instance Dr, Η, P. FIEDLER Lexikon der Hilfsstoffe fiir Pharmazie, Kosmetik und, angrenzende Gebiete (1971), pgs. 191 to 195).

Furthermore, the pharmaceutical preparations according to the present invention may contain preservatives, stabilysing agents, buffering agents, such as CaHPO^, colloidal aluminum hydroxide, flavoring agents, antioxidants and 1o complex forming agents (such as ethylenediaminotetraacetic acid). Optionally, the pH is rendered, to about 3 to 7 by means of physiologically compatible acids or buffers in order to stabilize the active agent.

An almost neutral or weakly acid pH (up to pH 5) is generally preferred. Useful antioxidants are for instance sodium metabisulfite, ascorbic acid, gallic acid, gallic acid alkylesters, butylhydroxyanisol, nordihydroguajaric acid, tocopheroles as well as combinations of tocopherols and synergistically active agents (agents binding heavy 2o metal cations hy complex formation, such as lecithin, ascorbic acid, phosphoric acid). The addition of synergistically active agents considerably increases the antioxidizing activity of tocopherols.

Useful preservatives are for instance sorbic acid, p-hydroxy25 benzoic acid esters (such as lower alkyl esters), benzoic acid, sodium benzoate, trichloroisobutyl alcohol, phenol, cresol, benzethonium chloride and formaldehyde derivatives.

The conversion of the compounds according to the present invention into pharmaceutical products occurs with the 3o application of usual galenic principals and usual methods.

For instance, the active agent or agents and auxiliary and/or carrier materials are thoroughly mixed (for instance by means of usual mixers), applying generally temperatures between 2o and 8o°c., preferably between 2o and 5o°C., in particular at room temperature. For further details see: 1o 51784 SUCKER, FUCHS, SPEISER, Pharmaaeutische Technologie, Stuttgart (1978).

The compounds according to the present invention and, respectively, the pharmaceutical preparations containing the same are administered on the skin or on mucous membranes or for instance orally, enterally, pulmonally, rectally, by way of the nose, vagina or tongue, intravenously, intraarterially, intracardially, intramuscularly, intraperitoneally, intracutanously, subcutanously, intrapleurally, 1o intrathecally and generally intracaviterally.

In view of its very favourable results it is preferred to combine the active compounds according to the present invention with other active compounds usual in drugs, in particular with uroprotectlng agents (for instance and preferably sodium-2-mercapto-ethane sulfonate or the disodium salt of 2.2'-dithiodi-(ethane sulfonic acid), but also with other systemically or regionally detoxifying agents.

The compounds according to the present invention show a 2o good cytostatic and curative activity upon intravenous, intraperitoneal or oral application to rats and mice suffering from various experimental tumors.

For instance, depending upon the dose, a curative activity is achieved with the compounds according to the present invention after administering them intravenously, intraperitoneally or orally in varying dosages one day after intraperitonial implantation of cells of Yoshida-ascitessarkoma AH 13. For instance, tumor ascites is collected under steril conditions(verified by bacteriological control) 3o from rats having a Yoshida-ascites-sarkoma 5. to 7 days old.

The average cell count of the ascites was 2 times 1o5 cells 8 per pi. The cell count is brought to lo cells/ml by the addition of tyrode solution. o.4 ml thereof (= 4 x 1o7 cells) are implanted intraperitoneally to the test animals.

In the untreated control animals the rate at which these tumors start to grow is 95 %, the mean mortality time is 8 days (e = ί o,5 d). The compounds to be tested are administered, also intraperitoneally, 3 hours after implan5 tation of the tumors. 6 animals are used in each test group.

The criterion of curative activity is the cure defined as freedom from relapse and metastasis, for 9o days after treatment. After determination of the per cent rate of cure the dose which produces cure in 5o % of the animals is cal1o culated from the dose activity line by means of probit analysis according to R. FISCHER. This DC 50 is the dose at which 5o % of the tumor infected animals are cured. This dose for the compounds of the present invention in the above test method upon intraperitoneal administration, is for instance between o.o5 and 3o mg/kg in rats. For instance, with the dose DC 5o the mean survival time against leukemia L121o in mice can be prolonged for 1oo %.

For determining the cytotoxic activity in vitro, freshly collected cells of Yoshida ascites sarkoma are incubated 2o for 2 hours at 37°C. with increased concentrations of the compounds to be tested in Ringer solution, as described by SCHmAIIL AND DRUCKREY, Naturwissenschaften, vol. 43 (1956) p. 199. After washing out of the test compound, the transplantability of the tumor cells to untreated test animals is determined. A quantitative value of the cytotoxic activity in vitro can be obtained by determining the concentration EC 5o at which the survival of the transplanted tumor cells in half of the animals is suppressed. With the compounds of the present invention, the above test shows 3o for instance in vitro cytotoxic activity in concentrations ranging from 1 to 1oo jug/ral.

For testing the activity of the compounds of the present invention against leukemia L5222 in rats, the blood of the donar rats of the strain BD IX was withdrawn by cardiopuncture in pentobarbital narcosis (intraperitoneal appli5 cation), The leukocyte number in the blood varied between 5o,ooo and 15o,ooo cells per pi. The blood is diluted with sterile sodium chloride solution to a leukocyte number of o £ x 1o cell/jul. 1 ml (= 5 x 1o cells) are implanted intraperitoneally to test animals of the same strain. 1o The rate at which this tumor starts to grow is 1oo %, the time until death of the test animals varies between 8 and 1o days. 6 animals are used for each test group. The test compounds were administered 5 days after implantation of the leukemia, The criterion of curative effectiveness is the freedom from relapse for 9o days after treatment. After determination of the per cent rate of cure, the DC 5o dose can be determined from the dose activity line by means of a probit analysis according to R. FISCHER.

This is the dose producing cure in 5o % of the test animals. 2o This DC 5o for leukemia L5222 in rats with the compounds according to the present invention upon intraperitoneal administration is for instance in the range of o«5 to 2o mg/kg of rats.

Furthermore, the compounds according to the present inven25 tion have been administered one or several times (4 times) during subsequent days after intraperitoneal implantation of 1o® cells of leukemia L121o of mice at varying dosages and a cytostatic activity was achieved.

The cytostatic activity is the prolongation of the mean 3o survival time of tumor animals, i.e. the dose dependant per cent prolongation of the survival time over an uninfested control group. This test method is described for instance by N.BROCK, PharmakolQgische Grundlagen der KrebsChemotherapie in A. GEORGII, Verhandlungen der Deutschen Krebsgesellschaft, vol. 1 (1978) pgs. 15 to 42.

“ This curative and cytostatic activity is comparable with that of the known cytostatics cyclophosphamide and ifosfamide.

The compounds according to the present invention further5 more show a good therapeutic breadth. Furthermore, they have definite advantages over the known cytostatic cyclophosphamide in view of the lower organotoxic side effects such as decrease of the number of leucocytes and immunosuppression. Furthermore, the urotoxicity of the 1o compounds according to the present invention is considerably lower. It can be further avoided by prophylactic administration of a uroprotector, for instance and preferably of sodium-2-mercapto-ethane sulfonate.

The lowest, already curative or cytostatic dose in the 15 above animal tests is for instance o.o1 mg/kg upon oral administration, o.o1 mg/kg upon intraperitoneal administration, o.o1 mg/kg upon intravenous administration.

The general dose range for the curative and cytotatic 2o activity (animal tests as above) is for instance: o.o1-1oo mg/kg orally, in particular o.1-1o.o mg/kg, 0.0Ι-Ι00 mg/kg intraperitoneally, in particular o„1-1o.o mg/kg, o.o1-1oo mg/kg intravenously, in particular 0.Ι-Ί0.0 25 mg/kg.

The compounds according to the present invention are useful for the treatment of malign diseases in humans and animals.

The pharmaceutical preparations in general contain between mg and 1 g, preferably between 1o and 3oo mg of the active 3o compound or compounds according to the present invention. - 14 51784 The administration may be effected for instance by means of tablets, capsules, pills, dragees, suppositories, ointments, gels, creams or in liquid form. Liquid forms of uppl t»»Hrwi srn Tor Instance oily solution» or solutions in alcohol or water as well as suspensions and emulsions.

The preferred form of administration are tablets, containing between 1o and 2oo mg of the active compound or solutions containing between o.1 and 5 % of the active compound, 1o The single dose at which the compounds according to the present invention are administered may be for instance: a) between 1 and 1oo mg/kg, preferably between 1o and 6o mg/kg for pharmaceutical preprations to be administered orally; b) between 1 and 1oo mg/kg, preferably between 1o and 6o mg/kg for pharmaceutical preparations for parenteral administration (for instance for intravenous or intramuscular administration); c, between 1 and 1oo mg/kg, preferably between 1o and 6o 2o mg/kg for pharmaceutical preparations for rectal or vaginal application; d) between 1 and 1oo mg/kg, preferably between 1o and 6o mg/kg for pharmaceutical preprations for local application upon the skin or upon mucous membranes (for in25 stance in the form of solutions, lotions, emulsions, ointments or the like).

(The above single dosages refer to the free base the active compound.) For instance there may be administered 1 to 1o tablets 3o 1 to 3 times per day, each tablet containing 1o to 3oo mg of the active compound. Or there may be administered one or several ampoules containing 1 to 1o ml with 1o to 5o mg of the active compound 1 or 2 times per day for intravenous injection. The minimal daily dose for oral administration may be for instance 2oo mg; the maximum daily dose for oral administration should not be higher than 5ooo mg.

The compounds may also be administered by continuous intravenous drip over 12 or more hours in particular cases.

For the treatment of dogs and cats the oral single dose in general is between about 1o and 6o mg/kg of body v/eight. 1o The parenteral dose is approximately between 1o and 6o mg/kg of body weight.

For the treatment of horses and cattle the oral single dose in general is between about 1o and 6o mg/kg. The parenteral single dose is between about 1o and 6o mg/kg of body weight.

The acute toxicity of the compounds of the present application in mice (expressed by LD 5o mg/kg; test method according to Miller and Tainter, Proc. Soc. Exper. Biol, a. Med., vol. 57 (1944) p. 261) is for instance upon oral 2o administration between 1oo and 1ooo mg/kg or, respectively above looo mg/kg.

The pharmaceutical preparations according to the present invention may be used in human therapy, in veterinary therapy or in agriculture alone or admixed with other pharmacologically active agents.

The following examples will further illustrate the preparations of the new compounds of the present invention without however limiting the same thereto. - 16 51784 EXAMPLE 1 g (54 mmol) of 4-hydroxycyclophosphamide (that is to say 2-/&is-{2-chloroethyl)-amino7-4-hydroxytetrahydro-2H-1,3,2oxazaphosphorin-2-oxide) and 4.4 g (58 mmol) of hydroxy5 urea were dissolved in 7o ml of DMF, acidified with trichloroacetic acid (pH 3-4) and left for 2o hours at 0°C. in a refrigerator. The resultant crystal sludge was diluted with 7o ml of ethyl acetate and after 2 hours was drawn off by suction, washed, dried and recrystallized from methanol.

Yields 11.3 g (62% of the theoretical) in the cis form, m.p. 139-143°C. (decomposition).

EXAMPLE 2 1,1 g (4 mmol) of 4-hydroxycyclophosphamide were dissolved in methanol, treated with a trace of trichloroacetic acid, left to stand over night at -25°C., then methanol was gently drawn off, the residue dissolved in a little methylenechloride, dried and concentrated to 1.2 g of 4-methoxycyclophosphamide (that is to say 2-^&is-(2-chloroethyl)amino7-4-methoxy-tetrahydro-2H-1,3,2-oxazaphosphorin-220 oxide). The 1.2 g of 4-methoxycyclophosphamide and 3o4 mg of hydroxyurea were dissolved in 3 ml of DMF and kept in a refrigerator at -25°C. for 2o hours. The crystal sludge was diluted with 3 ml of ethyl acetate, drawn off by suction, washed, dried and recrystallized from methanol.

Yield; 67o mg (5o% of the theoretical) of the same product as in Example 1.

S1784 EXAMPLE 3 g (58 mmol) of 4-hydroxycyclophosphamide and 5.2 g (68 mmol) of hydroxyurea were dissolved in 16o ml of water, acidified with trichloracetic acid (pH 3-4) and left to stand for 2o hours at 0°C. in a refrigerator. The crystal sludge was then drawn off by suction, washed with water, dried over P2°5 un^er high vacuum and recrystallized from methanol/chloroform.

Yield: 12.7 g (65% of the theoretical) of the trans form 10 of the product in Example 1, m.p. 148°C. (decomposition) EXAMPLE 4 2o g (5o mmol) of 4-hydroxytrofosfamide (that is to say 3-(2-chloroethyl)-2-/Lis-(2'-chloroethyl)-aminc7~4-hydrox.ytetrahydro-2H-1,3,2-oxazaphosphorin-2-oxide) and 5.3 g (7o mmol) of hydroxyurea were dissolved in 1oo ml of DMP and cooled to -15°C. Then acidification was effected with trichloroacetic acid (pH 3-4) and agitation effected for 5 hours at -15°C. After standing overnight at O°C., the reaction solution was diluted with twice the amount of water. Then extraction by shaking was effected 4 times each with 3oo ml of acetic ester /methanol (1o:1), the combined acetic ester phase was washed twice with water, dried over sodium sulphate and doncentrated to 22 g of oil under vacuum. After absorption in ethyl acetate/methanol, 4.2 g (Schmp. 1o6-11o°C.) crystallized out. The mother liquor vias fractionated by column chromatography on silica 1851784 gel with chloroform/methanol (1o:1, and recrystallized together with the 1st crystallisate from ethyl acetate/ methanol.

Yields 7.0 g (35% of the theoretical), m.p. 115-116°C. (decomposition).

EXAMPLE 5 54o mg (3.25 mmol) of 3-benzy1-1-hydroxyurea in 4o ml of acetone and a catalytic amount of trichloroacetic acid were added to 9oo mg (3.25 mmol, of 4-hydroxycyclophosphamide in 1 ml of methylenechloride. The mixture was stored overnight at -25°C«, the crystals were then drawn off by suction, washed with acetone and ether and recrystallized from ethyl acetate.

Yields 5oo mg (4o% Of the theoretical), m.p. 122-123°C. (decomposition).

EXAMPLE 6 56o mg (2mmol) of 4-hydroxycyclophosphamide in 1o ml of acetone were treated vzith 46o mg of 3-o-bromophenyl-1~ hydroxyurea and a catalytic amount of trichloroacetic acid and let to stand at -25°C, After 2 days, the crystals were drawn off by suction and recrystallized from acetone.

Yields 320 mg (32% of the theoretical), m.p. 11o-111°C. (decomposition,.

EXAMPLE 7 1.2 g (4.3 mmol)of 4-hydroxycyclophosphamide in 15 ml of acetone were treated with 63o mg (4.3 mmol) of N-hydroxymorpholino-carboxamide and a trace of trichloroacetic acid and stored at -25°C. After 4 days the crystals were drawn off by suction and recrystallized from acetone.

Yield; 78o mg (45% of the theoretical), m.p. 123-124°C. (decomposition). - 2o 51784 μ Ό G c •rt d O O. to Φ fafi fi •rt •rt X μ 0 rt 1 Φ CM I £ 1 C fi •rt Φ ti > 0 •rt X3 EC a to Φ o JS JC μ cu d bO N C d •rt X > o d t J3 CM A co co Φ A > i—( rt Λ μ d CM > ι •rt o rt Φ Ό XJ Eh JC A d Φ tl d μ rt o a μ ε 1 t< Eh 0 X «Η O tl rt Ό d Eh tl £i Φ I G x* Φ ho bO fi ho •rt c Ό •rt C £ 0 O a rt (0 rt φ O tl *rt tl O Φ ϋ JC μ E O «rt rt O Μ Ό <0 Φ bfl Φ rt fi > d •rt •rt a. μ μ φ rt d rt d E> & μ •rt CO rt Φ Φ rt o Eh Ό d ο- ι J2 d Φ 00 A Φ 3 S» rt rt co rt a d w rt Eh > *4 d X Di rt 0 «μ EE •rt rt S3 < E ό •rt Eh rt w co X O I O EC Ό C d CC co 1 1 CC—z o \ / Pl ω rt d •H (4 0) μ ω c •fH μ tl d μ co μ c •μ Φ o a D«rt d bo > c •rt «Η μ x e—t Φ tl S 0 cc CO cc CM X rt X φ rt Q. · ε o d z X w N o 0 CD O ϋ 0 rt O o 0 05 0) o 0 o in 1 o N O 0 rt rt rt rt rt ϋ O 0* CD I tn CD o rt ϋ o co co SJ O I CO CM EC EC u u i I X EC Z Z o u CO CO S3 S3 CM O o 1 1 co co m EC z—\ o o CM CM S3 S3 iEC S3 U O ϋ *»✓ 1 1 EC 1 EC 1 S3 X Z Z Z z 1 o 1 o 1 o o ϋ ϋ u t Q 1 1 EC 1 S3 I S3 1 S3 Z Z Z Z 111(1 CM CM CM CM CM S3 a X EC EC ο ο α ϋ o CM Q4 CM CM CM S3 EC S3 EC EC o o O Q u I I I I I rt rt u o u 1 OJ 1 OJ 1 OJ ta ta ta CJ o u CM CM CM ta ta ta υ CJ u rt o ϋ o t I S3 EC ϋ O EC EC Ο ϋ I ( QO 0) O rt CM J EC I CM S3 Q CM EC O I CM EC O CM S I rt a c ϋ □ X o T I II OJ X w cr X Q Q 1 o CM CM X X X O a o 1 X 1 X X z z z 1 o 1 o o a a o 1 X x ' X Z z z I S3 S3 S3 I S3 I EC I S3 I I I CM CM CM CM cq CM X ta w Q ϋ o CM CM CM X ta K u o o rt ϋ ϋ o CM CM X X w ϋ ϋ o OJ CM N X X w ϋ o u I rt rt rH ϋ ϋ Q •m* m cd rt rt rt -2151784 w • »ΰ β k β •rl r· 0 Φ ·« & Λ in β -Ρ ο Ν Η ϋ Λ 0 φ τί § β •Η Φ Λ γΗ Λ •Ρ Η ω 2 0 •rl 6 •Ρ β X. (0 fr β >1 β k k *Η 0 ϋ •Ρ W 0 Φ •Η k ►β τί 0 ♦Ρ Φ Η Λ Λ k ϋ •Ρ φ •rl £ Λ £ η +» •Η ωΓ0 £ Φ α ο Φ A fr β _ O Φ υ υ Η r-1 •Η •Η 0 « β 0 Λ 0 Η S • Λ τί Φ Φ k •Ρ fr ο ο φ •Ρ k φ k e 0 □ 0 Η Ο •Η •Ρ w •Η k Φ •Ρ ϋ Φ τί φ β •Η Π k Φ •Ρ Φ Ό were G k Φ Λ υ ω >1 Μ Φ fr ω Η β & •Η β XJ ω •Η Φ +1 Λ •rl ΰ +1 β Η fr ο fr β ft β •Η •Η +J &ν « ω β β •rl •Η »0 +1 Φ γΗ β k ω r-| Φ Η Φ Λ > •Ρ QJ k χί Ή β &< κ &< EXAMPLE 71 Ethyl-2-/^is-(2-chloroethyl)-amlng7~2-oxo-totrahydro-2H1,3,2-oxazaphosphorln-4-yl-oxy-carhamate 55o mg (2 mmol) of 4-hydroxycyclophosphamide and 21o mg 5 (2 mmol) of ethylliydroxycnr hamate (hydroxyurethanc) arc dissolved in 5 ml of dry methylenechloride free of alcohol.

A catalytic amount of trichloroacetic acid and molecular sieve 4 A are added thereto. The reaction mixture is allowed to stand at -25°C. for 3 days. Thereafter, the molecular lo sieve is separated and washed once with a diluted solution of NaHCO^. The methylenechloride phase is dried over sodium sulfate and part of the solvent is evaporated in a vacuum and thereafter diluted with ether. After standing for 2o hours at -25°C., the separated crystals are filtered off, washed and dried.

Yield; 29o mg (4o% of the theoretical) m.p. 96° C.

EXAMPLE 72 Benzyl-2-Zhis-(2-chloroethyl)-amlno7-2-oxo-tetrahydro-2H2o 1,3,2-oxazaphosphorln-4-yl-oxy-carhamate 75o mg (2.7 mmol) of 4-hydroxycyclophosphamide and 45o mg (2.7 mmol) of benzyl-hydroxycarbamate are dissolved in 6 ml of methylene chloride free of alcohol, h small amount of trichloroacetic acid is added thereto and the solution is allowed to stand at -25°C. in a refrigerator for 3 days.

The resulting solution is filtered off, the mother liquor is diluted with 5 ml of chloroform, then Is diluted with water and thereafter is washed with a dilute solution of NaHCOj and with water. The washed solution is dried over 3o sodium sulfate and is evaporated in a vacuum. The oily residue thereafter is reerystallized from acetic acid ethyl ester containing a small amount of methanol.

Yield: 680 mg (59% of the theoretical) m.p. 112-114°C.

EXAMPLE 73 a) 3-hydroxy-ureido-acetlc acid 56.5 g (0,28 mole) of glycine benzylester-hydrochloride are suspended in 3oo ml of toluene. Dry gaseous phosgene are introduced for 2 hours with stirring while the reaction mixture is heated on an oil bath heated to 14o°C, The reaction mixture thereafter is evaporated in a vacuum and the residue of crude benzyl-isocyanatoacetate is destilled in a high vacuum.

Yield: 51 g (95% of the theoretical).

B.p,. 1oo-1o2°C.

OoOj A solution of 6.6 g (o.2 mole) of hydroxylamine in 2oo ml 15 of dioxane is added to a solution Of 28.7 g (0.15 mole) of benzyl-isocyanato-acetate in 5o ml of dioxane dropwise with stirring and temporary cooling. Stirring is continued for another hour at room temperature. A reaction mixture thereafter is evaporated in a vacuum. The resul20 ting residue of crude benzyl-3-hydroxy-ureido-acetate is recrystallized from ethyl acetate.

Yields 28.1 g (83.6% of the theoretical) m.p.: 113“12o°C. g of palladium on activated charcoal are added to a 25 solution of 22.4 g (0.1 mole) of benzyl-3-hydroxyureido-acetate in 3oo ml of methanol. Hydrogen is intro28 duced with shaking. After about 2o minutes the hydrogen uptake is finished. The catalyst is filtered off with suction and the filtrate is evaporated in a vacuum. The solid residue of crude 3-hydroxy-ureido-acetic acid is recrystallized from dioxane.

Yield: 9.8 g (73% of the theoretical, m.p. 135°C. b) 6.1 g (2mmol) of 4-hydroxycyclophosphamide are added to a solution of 2.4 g (18 mmol) of 3-hydroxy-ureido-acetic 1o acid in 1o ml of water and 25 ml of acetone. The reaction mixture is allowed to stand over night at -25°C. Thereafter, 25 ml of acetone and a solution of 1.8 g (18 mmol, of cyclohexylamine in 1o ml of acetone are added thereto. After standing for 2 hours, the precip15 itate is filtered off with suction and is recrystallized from acetone containing a small amount of methanol.

Yield: 3.1 g (44% of the theoretical) m.p. 1o7-1o8°C.

EXAMPLE 74 2o 3-/Kl,N-(bis-(2-chloroethyl)-diamino)-phosphinyl-oxy7proplonaldohydo-oxlmo (nldophosphamide-oxime) 4.0 g (13.7 mmol) of 4-hydroperoxycyclophosphamide are suspended in 5o ml of water with ice-cooling. 5oo mg Na2S2O3 x 5 H20 are added thereto. During the stirring at to 1o°c. the pH is controlled with a pH-measuring device and is kept between pH 4.5 and 5.5 by the addition of 2nH_S0,. A concentrated solution of sodium thiosulfate is added dropwise thereto until there is no longer observed a continues increase of the pH of the reaction 2951784 mixture. Stirring is continued for half an hour about 1o°C and an aqueous solution of 95o mg of hydroxylamine-hydrochloride is added dropwise keeping the pH at 5 by the addition of 2n-NaOH, The resulting reaction mixture is allowed to stand overnight in a cooling box at 5°C.

Thereafter, the reaction mixture is extracted four times with 5o ml ethyl acetate each and the organic extractes are dried over sodium sulphate and evaporated in a vacuum at 3o°C. The residue is dissolved in methylene chloride and the separated crystals are filtered off after one day.

Yield: 3.4 g (85% of the theoretical) m.p.: 79-81°C.

EXAMPLE 75 g (2o mmole) of p-bromophenylisocyanate in 4o ml of 15 acetone are added to 5.8 g (20 mmole) of aldophosphamideoxime in 6o ml of acetone. The reaction mixture is stirred with cooling for 5 hours. After standing for 2 hours, the separated crystals are filtered off and dried in a vacuum in a rotation evaporator at 4o°C. and are recrystallized in methanol.

Yield: 8.1 g (82.8% of the theoretical) m.p.: 118-12o°C.

EXAMPLE 7G 4.7 g (25 mmole) of m-trifluoromethyl phenylisocyanute dissolved in 4o ml of acetone are added to 7.3 g (25 mmole) of aldophosphamido-oxime in 8o ml of acetone. The reaction mixture is stirred for 3 hours at 0°C. and is allowed to stand overnight in a cooling box at -25°C. Thereafter, 15o ml of petrolether are added thereto and the mixture is allowed to stand for another night in the cooling box at -25°C. The resulting crystals are filtered off, dried at 3o°C, and are recrystallized from isopropanol.

Yield: 9.2 g (76.8% of the theoretical) m.p.: 91-93°C.

EXAMPLE 77 g (18 mmole) of aldophosphamide-oxime and 2.2 g cyclo15 hexylisocyanate are separately dissolved in 1o ini of acetone and the solutions are admixed at O°C, After standing for 2 hours, the resulting crystals are filtered off with suction and are recrystallized from acetone/ether.

Yield: 4.2 g (56% of the theoretical) m.p.: 113°C.

EXAMPLE 78 g (18 mmole) of aldophosphamide-oxime and 1,2 g ethylisocyanate are separately dissolved in 15 ml of acetone each. The solutions are admixed at about 0°C, After standing for 5 hours the separated crystals are filtered off with suction and washed with acetone/ether. yield: 3.5 g {54% of the theoretical) m.p.: 1o1°C, EXAMPLE 79 2.1 g (lommole) of fluorenyl-2-isOcyanate dissolved in 2o ml of acetone are added to 2.9 g (1o mmole) of aldophosphamide-oxime in 3o ml of acetone at 0°C. The separated crystals were filtered off on the next day, are dried in a vacuum at 6o°C. and are recrystallized from isopropanol/methanol.

Yield: 2.5 g (50.1% of the theoretical, m.p.: 114°C.

EXAMPLE 80 2.9 g (2o mmole) of benzoylisocyanate dissolved in 4o ml of acetone are added to 5,8 g (2o mmole) of aldophosphamideoxime in 6o ml of acetone. A reaction mixture is stirred for 5 hours under a nitrogen atmosphere with cooling in an ice bath. The separated solid material is filtered off with suction, is dried in a rotating evaporator at 3o°C. and is recrystallized from methanol.

Yield: 2.4 g (27.3% of the theoretical) m.p.: 124-125°C. - 32 51784 EXAMPLE 81 3.3 g (2o mmole) of p~nitrophenyllsocyanate dissolved ii 4o ml of acetone are added to 5.8 g (2o mmole) of aldophosphamide-oxime in 6o ml of acetone. After standing for 2 hours the separated solid material is filtered off witli suction, is dried in a rotating evaporator at 4o°C. and is recrystallized from DMF/ethanol.

Yield: 6.7 g (73.5% of the theoretical) m.p.: 117-118°C.

EXAMPLE 82 Tablet coated with a coating resistant against stomach •juices loo g of the compound of Example 5 together with 7.0 g of Aerosil*(i.e. finely devided amorphous silicic acid) are passed through a sieve and are thoroughly mixed. To this mixture there are added 76.0 g of Avicel*PH 1o5 (i.e. micro crystalline cellulose product of FMC), 1o g of corn starch and 7,o g of stearic acid. This mixture is mixed until reaching a homogenous distribution of all components.

The mixture is pressed in usual manner to kernels each weighing 2oo mg containing 1oo mg of the active compound.

* Aerosil and Avicel are Trade Marks.

The kernels are coated with a usual stomach juice resistant coating, for instance from suitable cellulose derivatives or from a fully synthetic coating from an organic solution or aqueous dispersion which may contain usual plasticizers, dyestuffs, sweeteners or defoamers. - 33 51784 EXAMPLE 83 Gelatine capsule coated with a stomach resistant coating 25o g of the compound of Example 5 together with 7.5 g of Aerosil are passed through a sieve and thoroughly mixed 4o g of lactose and 2.5 g of magnesium stearate are added to this mixture which is mixed until reaching a homogenous distribution of its components.

This product is filled into gelatine capsules each capsule containing a single dose of 3oo mg of the active compound. 1o The capsules are closed and are coated as described in Example 82.

Claims (5)

1. Or Claim 2, wherein all groups in formula I are hydrogen atoms. 2q 4. 4-Carbamoyloxy-oxazaphosphorins as claimed in Claim 1, wherein X is oxygen; R 1 , R 2 and Rg, which may be the same or different, represent hydrogen or the 2chloroethyl group; R 4 , R g and R g represent hydrogen; and R ? is hydrogen, benzyl, phenyl (optionally substituted by one 2 5 or two carboxy groups), C^_ 4 ~alkyl (optionally substituted by one carboxy group) or phenyl-C^_ 4 -alkyl (optionally substituted by one or two carboxy groups in the phenyl and/ or alkyl part of the group. 5. 4-Carbamoyloxy-oxazaphosphorins as herein before 30 described in example 71 or 72. 6. A method of producing oxazaphosphorins, characterized -3651784 In which Rj^, R 2 , Rg and R 4 are as defined in Claims 1 to 5 and Y is hydrogen, methyl or ethyl, is subjected to reaction with a compound of formula III HO R K X i’-iIII in which Z, R c and X are as defined in Claims 1 to 5 (except o R 5 may represent hydrogen when Z is the iddition, that, group -iT 6 ), in the presence of an inert solvent; optionally with heatiZg or cooling and/or in the presence of an acid catalyst. 7. A method of producing oxazaphosphorins, characterized in that an oxime of formula IV wherein R^ to R^ are as defined in Claims 1 to 5, is subjected to reaction with a compound of formula V x = c = n-r 7 V wherein X and R? are as defined in Claims 1 to 5, in an inert solvent at a temperature ranging from -70°C. to +50°C. 8. Pharmaceutical preparations, comprising as an active ingredient a compound as claimed in any one of Claims 1 to 5 in association with a physiologically acceptable carrier and/or diluent. -3751784 9. A method as claimed in Claim 6 or 7, substantially as hereinbefore described with reference to any of Examples 1 to 73 and 75 to 81.

1. 4-Carbamoyloxy-oxazaphosphorins of the general formula I X II c or the group -OR?; X is oxygen mav Ho Ήίο cawo nr different, represent hydrogen, methyl, ethyl, 2-chloroethyl or 2-methanesulphonyloxyethyl; the groups R^, which may be the same or different, represent hydrogen, methyl 10 or ethyl; R g and R g , which may be the same or different, represent hydrogen, C^^-alkyl, hydroxy-C^^-alkyl or phenyl; R ? is hydrogen, a carbamoyl group, -ORg (in which Rg is hydrogen, C^-alkyl, phenyl or benzyl), straight or branched chain C^g-alkyl (optionally substituted by 1 to 15 3 substituents which may be the same or different and are selected from hydroxy, halogen, -COOH, -COORg, -CONH 2 , -phenyl, benzyloxycarbonyl, -N(R q ) 2 , -N(Rg) 3 , -ORg, -SRg, -SO-Rg, -SO 2 ~R g , -SOgH or-PO(CII 3 ) 2 , in which Rg represents methyl or ethyl), phenyl-C^_^-all:yl 20 (optionally substituted by 1 or 2 carboxy groups in the phenyl and/or alkyl part), allyl, C 3 _ g -cycloalkyl, tetrahydrofuranyl, tetrahydropyranyl, phenyl (optionally substituted by one or two C^^-alkyl, C^_ 2 -alkoxy, nitro, halogen, trifluormethyl, -SO 2 NH 2 , carboxy, benzyloxy 25 carbonyl and/or carb-C^^-alkoxy), benzhydryl, naphthyl, fluorenyl, fluorenonyl, pyridyl, thienyl, benzoyl or C 1 _^-35517 84 alkanoyl; or Rg and Rg or R g and R ? together with the atoms to which they are connected form a saturated heterocyclic ring optionally containing an oxygen atom, a C^_ 4 -alkyl substituted nitrogen atom or an -S-, -SO- or -SOg- group; or 5 R g and R ? together with the adjacent nitrogen form an aziridin ring optionally substituted by a cyano or carbamoyl group, and the pharmaceutically useful salts thereof, but excluding compounds in which R K represents hydrogen Z R~ ΰ and Z is the group -N' b . !0

2. 4-Carbamoyloxy-oxazaphosphorins as claimed in Z R Claim 1, wherein Z is -N °; X is oxygen; is R 7 methyl or ethyl; R g is hydrogen, methyl or ethyl; and R ? is hydrogen, straight or branch chained C^_^g-alkyl, phenyl 15 or benzyl.

3.

4. -Carbamoyioxy-oxazaphosphorins as claimed in Claim

5. 10. Oxazaphosphorins when prepared by a method as claimed in any of Claims 6, 7 and 9.