IE49274B1 - Therapeutic compositions with cytostatic action - Google Patents

Abstract

A therapeutic composition with cytostatic action comprising an isocyanurate having the formula I <IMAGE> in which R, R', and R'' which may be the same or different represent hydrogen or alkyl having from 1 to 4 carbon atoms, preferably hydrogen or methyl in an inert pharmaceutically acceptable diluent, carrier or vehicle. The compositions may also contain other alkylating substances such as derivatives of nitrogen mustard or fluoruracil.

Description

The present invention relates to therapeutic compositions containing specific isocyanurates having three epoxy groups.

It is known that a number of alkylating substances have a cytostatic or cytotoxic effect. The best known compounds are derived from the so-called nitrogen mustards. It is also known to use compounds containing at least two epoxide groups in the molecule as cancer-ostatics. Such compounds are, for example, 4,4'-bis-(Z,3 - epoxypropyl) - di -piperidinyl - and 1,2-15, 16-diepoxy - 4, 7, 10, 13 - tetraoxohexadecane. However these diepoxide compounds bring about no substantial improvement in the cytostatic treatment and are rarely used. They are still used occasionally for the treatment of tumours. The limited solubility of the above mentioned compounds also prevents their wider use.

Various protocols for screening cytostats against animal tumours have been published by Geran, Greenberg, MacDonald, Schumacher and Abbott in Cancer Chemotherapy Reports (Sept. 1972) pages 1 to 87. These procedures will be referred to hereinafter by reference to the page(s) of this report.

An object of the present invention is to find easily-soluble, particularly water-soluble, compounds with cytostatic or cytotoxic action, which are effective against a great number of malignomas and forms of leukemia. They should have a T/C ratio (see page 47) - 1a 49274 of at least 150% in at least one of the following leukemias.

L1210 and P388, melanoma B16 and LL-carcinoma produced in animal experiments according to the specifications of the Cancer Chemotherapy Reports, pages 7, 9, 11 and 15.

The present invention therefore provides a therapeutic composition with a cytostatic action comprising an isocyanurate having the formula I II u R || R CHz-C-CH2 V in which R, R' and R", which may be the same or different, represent 10 hydrogen or an alkyl group having from 1 to 4 carbon atoms, in admixture with an inert, sterile aqueous pharmaceutically acceptable carrier, diluent or vehicle.

The preferred compounds used according to the invention are those in which R, R' and R which may be the same or different, represent hydrogen or a methyl group.

The therapeutic compositions according to the invention - 2 49274 preferably contain from 0.05% to 5% by weight of the isocyanurate of the general formula I and the remainder to 100% of an inert aqueous pharmaceutically acceptable vehicle, diluent or carrier.

These glycidyl compounds containing an isocyanurate ring are, in principle, known substances. They have already been synthetized in more or less pure form and are used for the production of cross-linked plastics. While the preferred compounds, in which R, R1 and R are hydrogen, are characterized by surprisingly good solubility, for a compound of this type, in water, and hydrophilic water-miscible solvents, it has never been proposed to use them in hydrous or aqueous hydrophilic solvents, particularly for pharmaceutical purposes or in drugs.

If the three substituents R, R' and R are identical, two substances are obtained which are diastereomeric. The same is true if there are no alkyl radicals present, and the substituents are hydrogen. The compounds where R, R' and R" are hydrogen are called a - triglycidyl isocyanurate or β - triglycidyl isocyanurate (see Angew. Chemie., (1968), pages 851/2). Their production is described in U.S. Patent No. 3,300,490 and in U.S. Patent No. 3,337,509.

These two compounds are readily obtainable by reacting cyanuric acid with excess epichlorohydrin, and dehydrochlorination with formation of the oxirane ring. The pure products (aandg) can be obtained from the crude product by fractional crystallization from, for example, methanol, methylene chloride, ethylene glycol monomethyl ether or ethylene glycol monoethyl ether. The so-called α-form has, in the pure form, a melting point of 106°C, while the β-form has a melting point of 158°C. Though a separation is not generally necessary for - 3 49274 technical purposes, the therapeutic effectiveness of the two isomers was investigated separately in the present case. Due to the different solubility in water or in mixture with the abovementioned solvents, a clear separation is readily possible. Pure products show an epoxide oxygen content which is between 163! and 16.2% by weight. Naturally the enantioners can also be obtained in more or less pure form from the diastomeric a- and β-triglyceridyl isocyanurate, and the effectiveness can be further increased in some cases.

For use as cancerostatics, the active substances should be administered by means of suitable vehicles. In the present case, the use of water, if necessary together with a compatible glycol ether, such as ethylene glycol monomethyl ether, butylene glycol methyl-ether or propylene glycol methylether, was found to be of advantage, particularly if the active substance is administered parenterally. For oral administration, conventional pharmaceutically acceptable auxiliary substances and vehicles can be used, provided they show a corresponding compatability with the glycidyl compounds. Ordinarily, the glycidyl compounds are employed in amounts of from 0.05% to 5% by weight in the therapeutical compositions.

In animal experiments, the use of freshly prepared aqueous solutions administered intraperitoneally proved advisable. The maximum daily dose of a- or β-triglycidyl isocyanurate in mice can be 100 mg/kg of mouse. Pronounced toxic effects appear only at higher doses. The optimum dose was found to be in many cases to - 4 49274 be 30 mg to 60 mg/kg of mouse per day during an application period of 1 to 9 days.

The above-mentioned compounds are effective against various forms of leukemia and malignant neoplasmas, like lung cancer, cancer of the colon, melanomas, ependymoblastomas and sarcomas. A clear superiority over cyclophosphamide and fluoruracil was found in many cases.

Naturally it is also possible in addition to using a - triglycidyl isocyanurate and ¢- triglycidyl isocyanurates as cancerostatics to employ other compounds of formula I, in which R, R1 and/or R represents a methyl group or groups. As long as the solubility of the compound is sufficient, the groups R, R' and R may represent alkyl groups which contain more carbon atoms.

Combination therapy with other alkylating substances, SUch as derivatives of nitrogen mustard or fluoruracil is naturally also possible.

The invention will now be further described with reference to the following examples, EXAMPLES 20 The following examples were carried out according to the test specifications of the Natural Cancer Institute, Bethesda, Maryland 20014, U. S. A. published in Cancer Chemotherapy Reports part 3, Sept. 72, Vol. 3, No. 2. The active substance used was either a - triglycidyl isocyanurate (mp: 106’C) or 6 - triglycidyl -5 isocyanurate (mp:158°C), both with 16.1% epoxide-oxygen content - 5 49274 (for preparation see U.S. Patent No. 3,337,509). The preparation of tri - (2-methylglycidyl) isocyanurate is described in German Offenlegungsschrift No. 1,954,531, but on repeating the procedure there was only obtained a product which did not show the expected infrared spectrum. Therefore, the desired compound was prepared by reaction of the potassium salt of cyanuric acid and methallylchloride followed by the epoxidation of the triallyl compound intermediate. a) A mixture of 86 g of the potassium salt of cyanuric acid and 95g methallyl chloride in 391 g acetic acid nitrile was heated in an autoclave under nitrogen pressure at 150°C for a period of 3 hours. After cooling, the mixture was filtered and all volatile material was distilled off in vacuo at 0.1 mm pressure. The crude material was dissolved in cyclohexane and, after evaporation, re15 crystallized from the same solvent. The purified material had a melting point of from 84 to 85° C and an iodine number of 264 (calculated: 261.3). b) 20g of the material obtained as described above was dissolved in 300g of CH2C12 and treated with 15.85g m-chloroper20 benzoic acid. The mixture was allowed to stand for 70 hours in a refrigerator at 4°C. Thereafter, the precipitate of 3 - chlorobenzoic acid was filtered off. The organic solution of the epoxide obtained was washed with an aqueous solution of sodium carbonate (10% by weight), and then washed several times with water and dried with anhydrous sodium sulphate. When no more peracid - 6 49274 was present, the CH^CIg was distilled off in vacuo. The residue was recrystallized using diethyl ether. Yield: 12.9g(55.7% of the theory) of white crystals having a m.p. of 69 to 74°C.

Epoxide content: 13.7% by weight (calculated: 14.1%).

The infrared spectrum gives typically strong isocyanurate absorptions at 1700 and 1455cm k ^H-NMR spectroscopy in CDC1g (reference TMS) shows the protons of the CHg groups at 1.4 ppm.

The two protons on the C-atom 1 of the 2-methylglycidyl groups give an AB system at 4.1 ppm, the two protons on C-atom 3 an AB system with a smaller coupling constant at 2.6 ppm. The structure 13 is further confirmed by C-NMR and mass spectroscopy. All aqueous 1% w/v injection solutions were prepared fresh, just prior to administration. Triglycidyl isocyanurate is also referred to by its initials TGI.

EXAMPLE 1 In mice a P388 (leukemia) tumour was emplanted i.p. with 10® cells/mouse according to protocol 1.200 (page 9). The untreated animals have a mean survival time (m.s.t.) of 10.5 days.

After nine days of i.p. treatment with 100 mg/kg of α - TG1 per day, the mean life expectancy increased to 285%, compared to the control group(T/C, extension rate). Half of the treated mice lived longer than 40 days and must be considered as cured.

If only 50 mg/kg of a- TG1 per day were administered i.p. for 9 days, the corresponding values were 200% T/C and 17% cured.

The corresponding value with e - TG1 for 100 mg/kg of mouse per day were Z28% T/C, and for 50 mg/kg per day 179% T/C. Healing - 7 49274 was observed in 17% of the test animals.

Comparison test A leukemia form of P388, which was resistant to cyclophosphamide (NSC 26271), was emplanted in groups of mice and treated with a- TG1 or cyclophosphamide. Each group consisted of 10 mice.

With daily doses of 40 mg/kg of a - TG1 for 1 to 9 days, all mice lived for 60 days (T/C 478%). On the other hand, those treated with 180 mg/kg of mouse of cyclophosphamide did not live for even 30 days (T/C 150%).

EXAMPLE 2 Example 1 was repeated with a dose of 25 mg/kg per day for 9 days and the following observations were made: a - TG1 showed a T/C of 196% and 6- TG1 a T/C of 174%.

EXAMPLE 3 Leukemia L 1210 was produced in mice according to protocol 1.100 (page 7) by i.p. administration of 0.1 ml diluted stimulating c solution corresponding to 10 cells. In the control group the mean survival time was 8 days (m.s.t.).

A) One group (6 mice) received from the first to the 20 gth day 50 mg/kg of mouse per day of o - TG1 i.p. The mean survival time rose to 23.8 days corresponding to a T/C of 297%. 3 mice lived for 30 days, that is, a healing rate of about 50% was achieved.

Another group received from the first to the 9th day either 50 or 100 mg/kg of 6 - TG1 per day, i.p. The mean survival - 8 49 27 4 time was 16.3 or 25.2 days respectively corresponding to a T/C of 203% or 315% respectively.

B) The influence of the treatment plan on the therapeutic effect of a- TGI in tumour L 1210 after 30 days can be seen from the following Table 1.

TABLE 1 Dose per day in mg/kg Surviving / entire group Leukemic control group mice (not emplanted with leukemia) 50 mg for 5 x i.p. 5/10 7/8 (1st to 5th day) 40 mg for 9 x i.p. 8/10 8/8 (1st to 9th day) EXAMPLE 4 1/10 homogenate melanoma B 16 were administered i.p. accord- ing to protocol 1.300 (page 11) at a rate of 0. 5 ml per mouse. The control group had a mean survival time of 15.8 (m.s.t.). Treated groups received from the first to the 9th day various amounts of a- TG1 i.p. The following Table 2 shows the mean survival time and T/C in dependence on the daily dose of active substance. TABLE 2 Effect of a- TG1 as a function of the dose mg a- TGI/kg mouse/day m.s.t. (days) T/C 50 37.0 234% 25 36.0 227% 12.5 29.8 188% - 9 - EXAMPLE 5 Ο According to protocol 1.400 (page 13) cells of a 1mm piece of lung cancer (Lewis) were implanted s.c. in mice. 40 mg/kg of a- TGI and 90 mg/kg of 8- TGI respectively were administered daily i.p. to each mouse from the first to the 11th day.

The inhibition of metastases was 92% for a- TG1 and 87½ for 8- TGI compared to the control group after 23 days.

EXAMPLE 6 q In a test group of mice, 1 mm of a tissue of epen10 dymoblastoma was implanted intracerebrally. The mean survival time in the control group was 19.3 days.

In a treatment with 40 mg/kg a- TG1 daily for 9 days a T/C of 165½ was achieved.

EXAMPLE 7 Sarcoma 180 was produced by i.p. administration of 10θ cells/mouse. The untreated control group had a mean survival time of 20.2 days.

For the treatment, 30 mg/kg of mouse of a - TGI were administered i.p. on a daily basis from 1 to 9 days. A T/C of 183½ was observed.

EXAMPLE 8 q A) Groups of 10 mice were implanted with about 1 mm of colon carcinoma 38, s.c. The mean tumour weight in the untreated control group after 20 days was 512 mg/mouse.

One group received on the 2nd and 9th day 50 mg/kg - 10 4 9 274 daily of a- TG1, and another group additional received the same amount on the 16th day. The mean tumour weight was 153 mg and 183 mg respectively per mouse.

B) A carcinoma was produced by i.p. implantation 0 (1 mm colon carcinoma 26) in groups of 10 mice each. The treatment consisted in the i.p. administration of a- TG1. The following Table 3 shows the results as a function of the amount administered on the 1st, 5th and 9th day. TABLE 3 mg/kg of mouse Result 25 mg i.p. 25 days all mice survived 60 days 6 out of 10 mice survived 50 mg i.p. 60 days 10 mice still alive 100 mg i.p. 49 days all mice still alive 60 days 4 mice still alive A positive comparison by treatment with methyl CCNU (NSC-95441) 10 mg/kg of mouse i.p. showed that only 2 out of 10 mice were still alive on the 60th day (mean survival time 55 days).

EXAMPLE 9 In mice a P 388 (leukemia) tumour was implanted i.p. with 106 cells/mouse according to protocol 1.200 (page 9). The untreated animals had a mean survival time (m.s.t.) of 10 days.

After nine days of i.p. treatment with 100 mg/kg of tri-(2 - methylglycidyl) isocyanurate per day, the mean life expectancy increased to 170%, compared to the control group (T/C, extension rate).

If only 50 mg/kg of tri -(2- methylglycidyl) isocyanurate per day were administered i.p. for 9 days, the corresponding values were 150% T/C.

Claims (6)

1. A therapeutic composition with a cytostatic action containing an isocyanurate having the formula I R CH— 0= c II c \ N-CH a — C — CH 2 i v CHj- C -CH S \ / 5 in which, R, R‘ and R which may be the same or different represent hydrogen or an alkyl group having from 1 to 4 carbon atoms, in admixture with an inert, sterile aqueous pharmaceutically acceptable diluent, carrier or vehicle.

2. A therapeutic composition as claimed in claim 1 in which 10 R, R‘ and R which may be the same or different represent hydrogen or methyl.

3. A composition as claimed in claim 2 in which R, R' and R are hydrogen.

4. A therapeutic composition as claimed in claim 3 in which 15 the isocyanurate is a- triglycidyl isocyanurate, B- trigylcidyl isocyanurate or a mixture thereof, or one of the optically active - 12 49274 forms of the triglycidyl isocyanurates.

5. A therapeutic composition as claimed in any of claims 1 to 4 which contains from 0.05 to 5% by weight of the isocyanurate of formula I. 5

6. A therapeutic composition as claimed in claim 1 substantially as herein described with reference to any of the Examples.