NO763991L - METHOD OF PREPARING CANCEROSTATIC - Google Patents

Abstract

Process for the preparation of carcinostatic agent.

Description

The present invention relates to a method for the production of a cancerostatic and immunostimulating agent.

A number of cancerostatically active compounds are known which, by interfering with the metabolism of the rapidly dividing cancer cells, damage the tumor so severely that growth is reduced or even a regression is achieved. A complete restoration

However, treatment with drugs alone is difficult to achieve as, at the necessary high dosages, healthy body tissue is also attacked and destroyed. On the other hand, it has been shown that most cytostatics at the required high dosage damage the body's own immune system so that it is no longer able to destroy the remaining cancer cells, or to prevent further growth. Furthermore, the reduction of the immune response increases the susceptibility of the weakened body to bacterial and viral infections.

It is therefore a goal to find a cancerostatically active therapeutic that does not affect, or stimulates the body's own immune system. It has now been shown that 4-imino-1,3-diazabicyclo-[3•1.0]-hexan-2-one which can be designated by the three tautomeric structural formulas I-III, respectively. their physiologically acceptable salts have the desired cancerostatic and immunostimulating properties.

However, it is not yet certain whether the compound itself or a metabolite formed in the body constitutes the actual active principle.

It has now been shown that it is possible to isomerize the 1-carboxamido-2-cyano-aziridine known from DL patent no. 110,492 in anhydrous, polar, organic solvents, preferably in lower alcohols with 1-4 carbon atoms, with the addition of catalytic amounts of alkali to the above compound, which crystallizes well, is stable in dry form, but which dissolves well in physiological sodium chloride solution and aqueous alkali hydroxide or other bases.

The structure of the new compounds has not been unequivocally determined, but the physico-chemical investigations (analysis, mass spectrum, infrared spectrum, nuclear resonance spectrum) which have been carried out on the compounds indicate one of the structures I-III. The good water solubility as well as the splitting point for the material of over 250°C indicate ionic interaction and thus also the tautomeric structures J-lii.

The preparation of the new compound is illustrated by the following example.

Example

4-Imino-1,3-diazabicyclo-[3.1.O]-hexan-2-one

1.1 g (10 mmol) of 1-carboxamido-2-cyano-aziridine is dissolved in

5 ml anhydrous methanol. Below, the exclusion of moisture is instilled

56 mg (1 mmol) of potassium hydroxide in 5 ml of anhydrous methanol at 50°C.

Within a few minutes, 0.66 g (6 mmol) of 4-imino-1,3-diazabicyclo-[3.1.O]-hexan-2-one separate, corresponding to a yield of 60% of the theoretical. Eft. cross-linked from anhydrous methanol, the compound has a boiling point of over 250°C.

The infrared spectrum of the compound in potassium bromide shows two broad bands between 3300 to 2500 cm<-1> and 1800 to 1400 cm<-1>, sharper bands for example at 1295 cm 1, 1232 cm. 1 , 1178 cm 1, 1013 cm"<1>, 922 cm"<1>, 855 cm"<1>, 820 cm"1, 682 cm"<1>, 542 cm"<1.>

The mass spectrum shows a molecular peak at 111 as well as peaks at

110, 83, 68 and 41.

The NMR spectrum (deuterodimethylformamide) shows the following bands:

Microelement analysis gives the following values:

C H N

42.62 4.03 37.52 found

43.24 4.53 37.82 calculated (C^bN )

The pharmacological properties of the product were determined as follows:

1• Cancerostatic activity

Sprague-Dawley rats weighing 80 to 120 g and 6-8 weeks of age were inoculated subcutaneously in the neck with tumor cells. 0.1 ml of an aqueous suspension of approx. 10 tumor cells of Walker sarcoma 256 (solid, rat)..

4-imino-1,3-diazabicyclo-[3.1.oj-hexan-2-one was dissolved in

physiological saline solution (the amount desired in each case in 5 ml solution/kg experimental animal). These solutions were administered to the animals intravenously or orally by gavage 6 hours after inoculation of the animals. The control animals received 5 ml/kg physiological saline solution. In each experiment, 20 experimental and 20 control animals were used. The animals were euthanized with ether after 10 days, the tumors were removed and the weight of the tumors from the control group was compared with those from the experimental group.

The following table shows that the tumor growth by intravenous

and oral administration of the compound was significantly inhibited.

2. Immune stimulation

8 Sprague-Dawley rats with an average weight of 200 - 250 g were administered the active substance, dissolved in 5 ml of physiological saline solution, intravenously in a tail vein. As a control, 8 rats that were only treated with 5 ml of physiological saline solution per kg body weight. In the following days there remained from one 1-2 drops of blood were taken from the tail vein, and the corpuscular blood component was determined. Of the 8 values from the individual animals, the mean value was determined statistically. It was found that the number of leukocytes increased strongly, and that the percentage of lymphocytes also increased. Only after 20 - 25 days was the value for the control animals again reached. The number of erythrocytes, on the other hand, did not change significantly over the entire experimental period. The trial results from the 3rd day, which approximately corresponded to the maximum increase, are set out in the following Table 2.

In addition, the number of antibody-forming spleen cells was determined according to Jerne (Jerne et al, Science 140, 1963, p. 405).

20 white male mice weighing 20 - 25 g were each immunized with

1 ml preserved sheep erythrocytes (1 ml contains 5 x 100 erythrocytes) . On the same day, 4-imino-1,3-diazabicyclo-[3 vi.0]-hexan-2-one, resp. comparison active substances, administered intravenously dissolved in 5 ml physiological saline solution/kg mouse. As a control, 20 animals were likewise immunized with 1 ml sheep erythrocytes and treated with 5 ml/kg physiological saline solution. 3 days after the administration, the spleens were removed sterilely from the animals, and the number of antibody-forming spleen cells was determined according to the method of Jerne. The average of the single values was

•calculated. Individual results are summarized in table 3-

From the previous experiments, it can be deduced that for the intended pharmacological effect of immune stimulation, a dose of from approx. 1 to 50 mg/kg body weight required, which can either be administered at once or in several single doses. As the effect slowly wears off after approx. 2 weeks, possibly a renewed treatment may be necessary.

An LD^ of 660 mg/kg in rats and 750 mg/kg in mice was found as acute toxicity with single intravenous administration. LD5q vé<3 oral administration amounts to more than 4>0 g/kg in mice.

For the preparation of the pharmaceutical agent, 4-imino-1,3"diazabicyclo-[3.1.0]-hexan-2-one, or its pharmacologically acceptable salts, are mixed in a manner known per se with suitable pharmaceutical carrier substances and, for example formed into tablets or dragées, or suspended or dissolved with the addition of corresponding excipients in water or oil, e.g. olive oil, and filled in capsules. As the active ingredient is acid-labile, the preparation is provided with a first coating soluble in an alkaline small intestinal environment, or mixed with a corresponding carrier, eg a higher fatty acid or carboxymethylcellulose Solid carriers are eg starch, lactose, mannitol, methylcellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids (such as stearic acid), gelatin, agar-agar, calcium phosphate , magnesium stearate, animal or vegetable fat and solid, high-molecular polymers (such as polyethylene glycols), preparations suitable for oral application may possibly contain flavorings and sweeteners.

Preferably, however, the active ingredient is injected. The injection medium is preferably water, which contains the usual additives for injection solutions such as stabilizer, solubilizing agent or weakly alkaline buffer. Such additions are e.g. phosphate or

in

ca r bonatpuf f er , e~thanol, complexing agents (such as a hylendiamint a ra-acetic acid and its non-toxic salts), high molecular weight polymers (such as liquid polyethylene oxide) for viscosity regulation.

Claims (1)

Procedure for production. a v 4~imino-l., 3-diazabicyclo - [ 3 • 1 .0 ]-hexan-2-one, characterized in that l-carboxamido-2-cyano-aziridine is isomerized in polar, anhydrous solvents with the addition of catalytic amounts of alkali.