FI80259B - Process for the preparation of therapeutically active isothiuronium salts - Google Patents

Description

80259

The present invention relates to a process for the preparation of therapeutically useful isothiuronium salts. Said isothiuronium salts are novel compounds. These compounds have useful antitumor, antiparasitic and immunostimulatory activity and can be used in human and animal therapy.

General formula I

H2N-C-NH-C = N-R2 · HX (I)

Compounds according to Uh Ui, in which R1 represents methyl, ethyl or benzyl and R2 represents hydrogen, are known (FR patent publication 788,429 and DOS 2,426,683). However, in these publications these compounds are mentioned only as intermediates and the publications do not mention anything about their biological activity or the use of these compounds in therapy.

Simons and Davis (Int. J. Rad. Biol., Vol. 10, No. 4, 343-352) have studied the biological activity of the hydrobromide salt of S-ethyl guanylthiourea known from the above-mentioned patents. However, they came to the conclusion that it is not significant.

U.S. Pat. No. 3,960,949 further discloses the use of a compound of formula I as a starting material for the preparation of 1,2-biguanides wherein R 1 is methyl and R 2 is ethyl. However, even in this publication no mention is made of the therapeutic properties of this compound.

According to the present invention there are thus provided novel therapeutically useful novel compounds of general formula I.

H2N-C-NH-C = N-R2 · HX (I) 11 li NH5-R1 2 80259 (wherein R1 is C1-8 alkyl, C2-6 alkenyl / C2-6 "alkynyl or benzyl, wherein said alkyl groups being optionally substituted by one or more hydroxy, mercapto and / or halogen, R 2 represents hydrogen, C 1-6 alkyl, C 2-6 alkenyl or C 3-8 cycloalkyl and X represents a halogen ion, provided that if R 2 represents hydrogen, R 1 is other than methyl, ethyl or benzyl and if R 2 is ethyl, R 1 is other than methyl).

The term "C 1-6 alkyl" denotes straight-chain or branched saturated aliphatic hydrocarbon groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, etc.). The term "C 2-6 alkenyl" means straight-chain or branched olefinic unsaturated groups (e.g. vinyl, allyl, methallyl, etc.). The term "C 2-6 alkynyl" denotes straight-chain or branched aliphatic groups having at least one triple bond (e.g. propargyl, etc.). The term "C 3-10 cycloalkyl" denotes cyclic saturated aliphatic hydrocarbon groups (e.g., cyclobutyl, cyclopentyl, cyclohexyl, etc.). The term "halogen" means fluorine, chlorine, bromine and iodine atoms.

Examples of said substituted alkyl groups include 2-chloroethyl, 3-bromopropyl, 2-hydroxyethyl, 3-chloro-2-hydroxypropyl, and the like.

A preferred group of compounds of general formula I are those derivatives wherein R 1 is allyl.

Another preferred group of compounds of general formula I are those derivatives in which R 1 is 2-haloethyl.

R 2 preferably represents hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, allyl or cyclohexyl.

3 80259 X preferably denotes in particular chloride, bromide or iodide.

Particularly preferred representatives of the compounds of general formula I are the pharmaceutically acceptable salts of the following derivatives: N-aminoiminomethyl-S-propyl-isothiuronium salts, N-aminoiminomethyl-S-allyl-isothiuronium salts, N-aminoiminomethyl-S- (2-chloroethyl) ) -isothiuronium salts, N-aminoiminomethyl-S- (1-chloro-2-hydroxypropyl-isothiuronium salts, N-aminoiminomethyl-S- (3-chloropropyl) -isothiuronium salts and N-aminoiminomethyl-S- ( 2-hydroxyethyl) -isotiuronium salts.

Further preferred compounds of general formula I are N-aminoiminomethyl-N'-alkyl-S-allyl-isothiuronium salts, in particular the corresponding N'-methyl, N'-ethyl, N'-n-propyl and N'-isopropyl compounds.

According to the present invention there is provided a process for the preparation of compounds of general formula I, according to which process

of general formula II

HoN-C-NH-C-NH-R2 (II)

Z II II NH S

(wherein R 2 is as defined above) or a suitable salt thereof is reacted with an alkylating agent capable of attaching an R 1 group, preferably with an alkylating agent of the general formula R 1 -Y (wherein R 1 is as defined above and Y is leaving group, preferably halogen) and, if necessary, converting the product thus obtained into a salt by reacting it with an acid of general formula HX (in which X represents a halogen ion).

4 80259

In said reaction, isothiuronium salts are formed. The reaction is generally followed by the formation of N-alkylated by-products.

According to a preferred embodiment of the process, the starting materials of the general formula II are not used in the free base form, but in the form of a suitable salt, in particular a carbonate. In this way, the formation of N-alkylated derivatives is prevented, which is probably due to the fact that the carbon dioxide evolved strongly lowers the pH of the basic medium.

Corresponding alkyl halides or dialkyl sulfates can be preferably used as the alkylating agent, but other alkylating agents can be used as well.

The reactants may be used in equimolar amounts or the alkylating agent may be added in excess. As the reaction medium, inert organic solvents can be used, preferably an alcohol, an aliphatic ketone, an aliphatic nitrile or an ether.

The reaction can be carried out at atmospheric pressure and at a temperature of 20 to 80 ° C.

According to a preferred embodiment of the process, the starting materials of the general formula II and the alkylating agent are reacted in a molar ratio of 1: 1 to 1: 1.8 in anhydrous ethanol, acetonitrile, dimethylformamide, acetone or ether at a temperature of 20 to 30 ° C.

The starting materials of the general formula II are known or can be prepared according to methods known per se. Thus, N-aminoiminomethylthiourea can be readily prepared by reacting dicyandiamide with hydrogen sulfide (Houben-Weyl VIII., 214). N-Aminoiminomethyl-N'-alkylthiourea derivatives of general formula II can be prepared by reacting a guanidine base with the corresponding alkyl isothiocyanate (U.S. Patent 4,009,163).

5 80259

It is also conceivable to deviate from the method described above for the preparation of the compounds of the general formula I. Thus, the general formula IV

H2N-C-NH = C = NH (IV)

Il I.

NH S-R1

compounds (which form a more limited subgroup of compounds of general formula I, R 2 is hydrogen) can be prepared by reacting compounds of formula III

H2N-C-NH-C = N (III)

NH

to react with a thioalcohol of general formula R1-SH (in formula R1 is as defined above). The reactants may be used in equimolar amounts or an excess of thioalcohol of general formula Ri-SH may be used. Alcohol, especially methanol, can be used as the reaction medium. The reaction can be carried out under pressure, if necessary. It is best to work at a pressure of 1 ... 5 bar and a temperature of 20 ... 100 # C. The starting materials of the formula III can be prepared by methods known per se.

The product thus obtained can be converted into a pharmaceutically acceptable salt formed with hydrohalic acid. For salt formation, it is preferable to use hydrochloric acid or hydrobromic acid.

The compounds of general formula I have antitumor (i.e. antitumor) and antiparasitic immunostimulatory properties. The intensity and direction of activity and toxicity depend on the substituents of the molecule.

It is known that gutimine, which is structurally close to the compounds of general formula I, has been tested as an antihypoxic agent [C.A. 66, 452,929 (1972);

Pathol. Phys. Exp. Ther. 10, (6)] and in pharmacological and clinical tests as an agent that causes microcirculatory disorders (Gematol. Transfusiol. (1984), 29 (1), 49-52).

It has been found that the compounds of general formula I are able to restore the body's reduced natural immune system to normal values or to increase it above normal levels.

Thus, the animal and human body has an immune system (e.g., a tumor-eliminating system). When the activity of said system is reduced or ceases to function, various diseases and tumors appear. The compounds of the general formula I activate the natural immune systems and can therefore be used in all cases in which it is desirable to increase the reduced activity of the immune system and to further increase its normal activity.

The compounds of general formula I have other useful biological effects, e.g. they improve microvessel circulatory disorders, bind harmful radicals, improve the function of oxygen deficient cells and prolong their lifespan.

The biological activity of the compounds of general formula I is demonstrated by the following tests. It should be noted, however, that not all effects of highly active compounds have been reported.

On effects testing Immunological tests:

The lymphocytes. Lymphocytes were isolated from the Ficoll-Uromiro (Böyum 1968) gradient from the venous blood of healthy donors and patients with malignant lymphoid tumors and metastatic solid tumors, respectively. Decreased ADCC-7 80259 and NK activity were taken into account when selecting patients from the latter group. Phagocytes were removed by treatment with iron powder and magnet.

ADCC. ADCC was determined according to the method of Perlamann & Perlamann (1970) with minor variations (Läng et al., 1981). Chicken erythrocytes labeled with 105 51 Cr and coated with anti-chicken erythrocyte rabbit serum were incubated with lymphocytes for 4 hours at 37 ° C. When the reaction was stopped, the activity of the mother liquor of the centrifuged cells was determined with a gamma counter. The rate of chromium release was expressed by the cytotoxicity index (CI%). This value was calculated as defined in one of our previous publications (Läng et al., 1980) - (see Table I).

NK: NK activity was determined by the method of Jondal and Pross (1975) using minor changes (Läng et al., 1981). K-562 tumor cells labeled with 51 Cr were incubated with effector cells in TC 199 liquid nutrient medium containing 10% decomplemented calf serum. When the reaction was complete, the radioactivity (chromium release) of the mother liquor of the centrifuged cells was calculated with a gamma counter. Intended cell death (cytotoxicity index%) was expressed as the difference between experimental release and spontaneous release.

Statistical calculations were performed using a monosample test. Several compounds of general formula I have excellent immunostimulatory activity (e.g. compounds prepared according to Examples 10, 11 and 15). Said compounds are able to increase normal and strongly reduced NK and K cell activity, even above normal levels. Said effect may be so strong that cytotoxic activity reduced to one tenth of the original value can be restored to the original level (Table II).

At a dose range corresponding to the expected human plasma concentration, the compounds of general formula I increase the NK and K cell activity of human lymphocytes and improve the NCMC and ADCC response at a dose-dependent rate, respectively. According to preliminary results from other immunological tests, the compounds increase blast transformation and migration.

When performing a substitution on nitrogen atoms, the molar relative immunostimulatory effect is generally reduced relative to toxicity.

Since the cytotoxic activity of tumor patients decreases in proportion to the disease state and further decreases due to conventional cytostatic therapies, it is very important that the compounds of general formula 1 have a strong immunostimulatory effect and are not immunodepressants. This can be very important for recovery.

Leukocyte migration:

Spontaneous (random) migration of leukocytes was measured with agarose microdroplets according to the method of McCoy et al (1977) by performing a few minor variations (Kalmär and Gergely, 1982).

2 ml of agarose droplets (0.2%) were placed on migration plates (Greiner, Federal Republic of Germany). TC 199 liquid nutrient medium was added and the plates were sealed.

After 24 hours, the migration area was measured by stereomicroscopy. The migration range was expressed as mm ^.

Leukocyte migration:

Direct (random) migration of polymorphonuclear cells from healthy donors was increased with the compounds of Examples 10, 11 and 13. The optimal dose was 1.0 ug / ml. At the higher dose (10 μg / ml), the effect was less pronounced (Table III).

Il 9 80259

Mitogen-induced blastos transformation:

Lymphocytes were cultured in TC 199 liquid medium supplemented with 10% decomplemented calf serum, antibiotics and 25 mH HEPES (Serva).

200 μl of cell suspension containing 2 · ιο5 lymphocytes was placed on a microplate (manufactured by Sterilin, UK); five parallel comparisons were performed.

2 and 10 μg / ml Phytohaemagglutininera (Leucoag-glutinine; manufactured by Pharmacia, Sweden) and 25 μg / ml Conca-navalini A (Carbiochem), respectively, were added to the cultures; the above values are given relative to the final concentration. Cultures were cultured at 37 ° C for 72 hours. Eight hours before the end of the culture period, 0.5 μCi of 3 H-thymidine (Chemapol. Czechoslovakia) was added to the samples. The incubation was stopped by freezing the cultures. After thawing, the samples were filtered in an automatic harvester (Dynatech). The radioactivity of the samples was measured with a scintillation counter. Activity was expressed as cpm (Tables IV and V).

Statistical analysis

Significance was determined using the monosample-2T2 test. The results obtained with the product of Example 10 are shown in Tables I, II and III.

Preliminary rapid test to determine antitumor and immunostimulatory effect This method was performed in our laboratories to test a large number of samples rapidly. Male CFLP mice (mean weight 30 g) were inoculated i.p. with four mil-j Oona ascites lymphoma cells.

The test used those animals that had developed a tumor that was easily visible on the fourth day - i.e. ascites. At least 10 animals were used in each group and mice were treated orally with one-tenth of the LD50 value.

10 80259 24 hours after oral treatment, the results were evaluated with Giemsa-stained ascites lubricant based on cytological analysis. The same number of animals was used in the control group as in the treated group.

Tumor cell damage, organism activation, and potential toxic effects were assessed.

To evaluate the antitumor effect, the percentage of dead and intact tumor cells was estimated. The results were expressed according to the following scale:

Tumor cell death rate effect 0 ... 30 no effect 30 .. .70% medium strong effect 70 .. .100% strong effect (inhibition)

The growth of the body's immune system was assessed by the number and activity of neutrophilic granulocytes, lymphocytes and macrophages present in ascites tumor cells and involved in the immune system. Immune system activity was manifested at the rate at which the above-mentioned cells in the ascites form a plasma bridge bond with tumor cells. According to this evaluation, if there is no difference in the number or activity of said cells, no increase can be observed compared to the control. A moderate effect was observed if the increase in cell number and activity relative to the control was 20%. The effect was strong if the increase in cell number and activity of the immune system was more than 20% higher than that of the control (Table VII).

Toxicity was determined by assessing morphological changes in neutrophilic granulocytes and lymphocytes, plasma vasculature, nuclear fragmentation, polysegment formation, and toxic granulation. Toxicity was expressed according to the following scale: 11 80259

Observed results toxicity no above mentioned changes in own cells no toxicity 20% changes medium toxicity more than 20% changes high toxicity

Antitumor Activity Testing 1. In vitro: Cultures of K-562 human erythroleukemia cells and P-388 mouse lymphoma cells were treated with 1 to 100 μg / ml of test substance: 2. In vivo: a) NK-ly ascites lymphoma 1 million N6meth -Kellner-ascites lymphoma cells were transplanted ip 20 male CFLP mice (average weight 30 g). [A new mouse ascites tumor was used as a screening tool. Neoplasma 8, 337 (1961)]. Half of the animals served as controls and the rest were treated with one-tenth of the LD5Q for various compounds of general formula I p.o. and i.p. one and the following five days, respectively. (If one-tenth of the LD5Q was effective, a lower dose was used). In the treatment group, a cytomorphological assay of the ascites lubricant was performed after 24 hours, and the ratio of damaged to dead cells was evaluated. Survival was determined in animals treated for 5 days.

b) L1210 tumor 106 L1210 tumor cells were transplanted i.p. 20 female BDFi mice (weight 20 g). Treatment was started 24 hours after tumor transplantation and continued for 8 days. Each group consisted of 5 animals; dose: 5, 50 and 500 12 80259 mg / kg p.o. daily. The control group also had 5 animals. Survival rates were monitored.

c) Lewis long-term tumor

6 animals were used in each group. Dose: 5 and 50 mg / kg. The control group also had 6 animals. Female Cs7B1 mice (weight 20 g) were used in the test.

The tumor was transferred s.c. the muscle of the right leg. After 10 days, the leg was amputated, oral treatment was initiated and continued for 9 days. Animals were sacrificed and metastases were counted under a stereomicroscope.

d) Different species of dogs with spontaneous breast cancer (ductus cc.) were treated for 4 weeks with oral doses of 1 to 10 mg / kg of test substances. Test surgery was performed and tissue samples were collected for histological analysis.

The compounds of Examples 10, 12, 13, 14, 22, 23, 29, 30, 31, 33 have excellent antitumor activity. Thus, the compound of Example 10 inhibits human K-562 erythroleukemia cells in tissue cultures at a concentration of 4 μg / ml. On the other hand, this compound inhibits the proliferation of P-388 cells only at a concentration of 40 μg / ml, and when the concentration is increased to 400 μg / ml, the activity does not become stronger. Compounds with high activity have a dose-dependent effect at a concentration of 0.5 to 5 ug / ml, while compounds with low activity are not dose-dependent. According to in vivo tests, the members of this group of compounds are either not active at all against the L-tumor or only to a very small extent.

The compounds significantly reduce the number of Lewis long-term tumor lung metastases. Compounds 10, 12, 13, 14, 22, 23, 39 are effective at a dose lower than one tenth of the LD50 value. The compound of Example 13 reduces the number of lung metastases by 50% even at a dose of 5 mg / kg and this result is very significant due to the fact that LD50 = 2380 mg / kg.

It has been found that the effect can be enhanced to a greater extent by repeating the treatment than by increasing the dose.

i3 80259

The compounds of Examples 10, 12, 13, 17, 22, 39 proved to be active against Nk-ly ascites tumor at a concentration less than one tenth of the LD50 value. The latter compounds increase survival and longevity in a dose-dependent manner. These compounds have an immunostimulatory effect at low doses, while at higher doses a direct antitumor effect is similar. Thus, the compounds of Example 10 have an immunostimulatory effect at doses of 25 mg / kg. The compound of Example 10 is cytotoxic over the dose range of 250-500 mg / kg. The S-allyl-, S-1-chloro-2-hydroxypropyl- or S-1-bromo-2-hydroxy-propyl-substituted compounds of the general formula I are highly active against spontaneous canine breast tumors. After 3 to 4 weeks of treatment, a sample of living tissue was found to infiltrate cellular infiltration of lymphocytes and neutrophilic granulocytes, macrophages, and occasionally eosinophilic granulocytes in tumors (ductus cc.). Degenerative changes and strong degradation were observed in the tumor cells.

NK cells form plasma bridges with tumor cells, and this results in the degradation of the plasma cell cell of the tumor cells and a large thinning of the nuclear DNA structure. The nuclear-to-plasma ratio changes, the nuclei swell strongly, and the plasma basophilicity, or alkalinity, decreases.

In about a quarter of cases, leukocyte infiltration into the tumor is negligible; on the other hand, tumor cells are swollen and vitreous degenerated. A very large amount of vitreous stained material precipitates in the intercellular structure.

When the compound of Example 10 was administered orally at a daily dose of 30 to 120 mg (0.5 to 2 mg / kg) for 6 to 12 11 80259 months, a significant improvement or recovery in human bone metastases from breast, ovarian and pancreatic cancer was observed, respectively.

Testing of antitumor activity in combinations 1) Combinations with known cytostatic agents:

Various cytostatic or cell growth inhibitory agents having a biological alkylating effect were co-administered with compounds of general formula I to CFLP mice bearing an NK-ly ascites tumor. Cytostatic agents were administered orally, intraperitoneally, or intravenously at a dose corresponding to one-fifth or one-tenth of the LD50 value, or the dose most effective in the art. The compounds of general formula I were administered orally in a dose corresponding to one tenth or one twentieth of the LD50 value, in a single dose or several times 48 hours after treatment with a cytostatic agent. Thus, combinations of cyclophosphamide, carnomustine, degranol, dibromodulsitol and dibromomannitol on the one hand and the compounds of Examples 10, 12, 13, 14, 15, 22, 23, 39 on the other hand were administered. Compared with the control group treated with known cytostatic agents alone, the antitumor effect was enhanced and immunodepression was reduced.

2) Combination with known vitamins:

The isothuronium salts of the general formula I can be effectively combined with medium doses of vitamins A and D3 and high doses of vitamin C. Good results were obtained both in pharmacological tests and in human clinical cases.

3) Combination with known hormones: In the treatment of hormone-dependent tumors, the combination proved to be useful both in pharmacological tests and in human clinical cases.

15 80259 4) Use in surgery:

The compounds of general formula I were successfully used in pharmacological tests in the case of solid tumors for surgical pre- and post-treatment (NK-ly solid, Yosida). It can be stated from pharmacological tests that the compounds of general formula I are probably also suitable for use in human surgery for pre- and post-treatment.

Antiparasitic testing:

The compounds of general formula I were administered orally to dogs infected with ticks (Ixodes ricinus) for three days. Thus, N-aminoiminomethyl-S-allyl-iso-thiurenium hydrochloride was administered orally at 1-2 mg / kg and the number of mite deaths was recorded. 6-8 hours after administration of the active ingredient of general formula I, all ticks had died in dogs.

Binding of free radicals:

The compounds of Examples 9, 10, 15 and 24 significantly reduce the toxicity (LD50) of hydrogen peroxide, organic peroxides and biological alkylating (free radical generating) agents. Based on the preferred toxicity values of the compounds of the general formula I, they are more suitable for free radical scavenging and provide better radiation protection than the known isothiurenium derivatives obtained from the corresponding thiourea.

Cardioprotective effect in isoprenaline necrosis: 5 mg / kg isoproterenol was administered i.p. for male CFY rats (weight 250-300 g). There were 10 animals in each group. Some animals served as controls and the rest were treated with 1 to 5 mg / kg i.p. or 10 to 50 mg / kg p.o. of Compound No. 13. After two weeks, the hearts were examined histologically. The ratio of necrosis and healthy areas of myocardium to i6 80259 was determined. It was found that all treatments with N-aminoiminomethyl-S-allyl-iso-thiurenium hydrochloride of general formula I produced a significant reduction in necrosis, i.e. necrosis.

In the damaged area, the test compound is expected to exert its effect by binding hydrogen peroxide produced by neutrophilic granulocytes. In this model, other free radical scavengers and antioxidant molecules have a similar effect.

Effects on blood circulation and respiration:

The compound of Example 10 i.v. At a dose of 10 mg / kg, the haemodynamic parameters and respiratory function of cats with open and closed breasts were not significantly affected. The following parameters were tested: ventricular and peripheral pressure, heart rate (function) ECG, volume / minute, contractility, tidal volume, tidal rate, and resistance. At intravenous doses above 10 mg / kg, left ventricular pressure and heart rate decreased. At a dose of 50 mg / kg, the effects were significant and long-lasting. At a dose of 100 mg / kg i.v., strong hemodynamic changes were observed, but cats did not die.

Effects of N- (aminoiminomethyl) -S-allyl-isothiuronium salts (halides) (Compound A).

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Table VI

22 80259

24-hour acute toxicity of compounds of general formula I.

Eg R2 r! ΙΌ50 mg / kg i.p.

n: o p.o.

9 H propyl 28001260 20 H butyl 27001310 19 H i-propyl 22601210 21 H i-amyl 22601241 12 H 2-chloroethyl 11201140 22 H 2-bromoethyl 10501280 13 H 1-chloro-2-OH-propyl 12401160 14 H 3- chloropropyl 12101180 23 H 3-bromopropyl 9801640 15 H 2-hydroxyethyl 32801460 16801210 24 H 1-oxypropyl 28601180 10 H allyl 23801320 320130 38 H methallyl 16401210 17 methyl allyl 600154 7516.3 29 ethyl allyl 780162 8417 ethyl allyl 780162 8417 allyl allyl 380141 3614

Table VII

23 80259

Test compound antitumor immunostimulatory e.g. no effect lantern res. Impact 9 Medium Medium 0 20 Medium Medium 0 19 Medium Medium 0 21 Medium Medium 0 12 Strong Weak 0 22 Strong Weak 0 13 Strong Weak 0 14 Strong Weak 0 23 Strong Weak 0 15 Medium Strong 0 24 Medium Medium 0 10 Strong Strong 0 38 medium medium 0 17 strong weak 0 29 medium medium 0 18 strong weak 0 39 strong weak 0 24 80259

The pharmaceutical compositions may be prepared by methods known per se in the pharmaceutical industry. The active ingredients of the general formula I can be finished in the form of capsules mixed with customary excipients commonly used (lubricants, etc.); capsules can be prepared using automatic encapsulation machines. The pharmaceutical preparations can also be prepared in the form of powder ampoules, parenteral compositions for injections and infusions; these compositions may also be prepared by methods known per se. Distilled water or physiological saline can be used as a solvent.

The active ingredients of the general formula I can also be finished in the form of an ointment, in particular as lyophilic ointments. The highly fragrant active ingredients of the general formula I can preferably be finished by microencapsulation, e.g. by forming complexes incorporated in the cyclodextrin.

The pharmaceutical compositions may also contain, in addition to the compound of general formula I, one or more known biologically active substances as active ingredient.

The pharmaceutical compositions can be used in both human and animal therapy. When used as an immunostimulant, the oral or rectal dose may preferably be about 0.5 to 25 mg / kg. Higher doses are preferred for topical treatment.

Industrial suitability:

The invention relates primarily to novel compounds having antitumor and immunostimulatory activity.

Other details of the present invention are set forth in the following examples without limiting the scope of the protection to the examples.

80259 Starting materials Example 1 N-aminoiminomethylthiourea carbonate 84.0 g (1.0 mol) of dicyanamide dime and 300 ml of water are placed in an autoclave. 34.0 g (1 mole) of gaseous hydrogen sulphide are introduced into the closed autoclave as follows: At a temperature of 20 ° C, enough hydrogen sulphide gas is introduced so that the pressure in the autoclave is 1,5 bar. The gas conduction is stopped and the autoclave is slowly heated to 80 ° C. Meanwhile, gaseous hydrogen sulfide is passed at such a rate that the autoclave should have a constant pressure of 1.5 bar. After the entire amount of hydrogen sulphide gas has been introduced into the autoclave, the autoclave is kept at 80 ° C until the pressure drops to 0.3 ... 0.4 bar. The reaction mixture is cooled to 20 ° C, purged with nitrogen and filtered. The clear filtrate is cooled to 0 ° C and carbon dioxide gas is introduced. The precipitated white crystalline N- (aminoiminomethyl) thiourea carbonate is filtered off, washed with water and dried at 60-80 ° C. Thus 100 g of the desired compound are obtained, yield 67%, m.p. 127 ... 129aC.

The IR spectrum of the product thus obtained is compared with the iR spectrum of a standard sample of the compound.

Example 2 N-aminoiminomethyl N'-methylthiourea carbonate

To a solution of 5.9 g (0.1 mol) of guanidine base and 30 ml of acetone is added a solution of 7.3 g (0.1 mol) of methyl isothiocyanate in 10 ml of acetone. The isothiocyanate solution is added to the guanidine solution at such a rate that the temperature of the mixture does not rise above 40 ° C. The reaction mixture is allowed to stand at 40 ° C for 4 hours, after which the solvent is removed in vacuo. The remaining pale yellow oil is suspended in water to give an opaque solution. Carbon dioxide gas is introduced into the solution thus obtained and the desired compound precipitates in the form of white crystals. Yield: 12.0 g (73.5%). M.p .: 132 ... 134 ° C.

Examples 3-8 26 80259

In a manner similar to Example 2, the following compounds can be prepared: 3. N-aminoiminomethyl-N'-ethylthiourea carbonate, 4. N-aminoiminomethyl-N'-n-propylthiourea carbonate, 5. N-aminoiminomethyl-N'-isopropylthiourea carbonate , 6. N-aminoiminomethyl-N'-n-butylthiourea carbonate, 7. N-aminoiminomethyl-N'-allylthiourea carbonate, 8. N-aminoiminomethyl-N'-cyclohexylthiourea carbonate.

The following examples explain the preparation of the final products of general formula I.

Example 9 N-Aminoiminomethyl-S-n-propylisothiuronium bromide 14.9 g (0.05 mol) of N-aminoiminomethylthiourea carbonate are suspended in 50 ml of anhydrous ethanol. 14.8 g (0.12 mol) of n-propyl bromide are added to the suspension. The reaction mixture is slowly heated to boiling with stirring while evolving carbon dioxide. The reaction mixture is heated to reflux for 45 minutes and cooled to room temperature.

The mixture is filtered to remove any precipitated material. The clear filtrate is evaporated in vacuo and the oily residue is crystallized from ethyl acetate. The white crystals are filtered off and dried at 40-50 ° C. 19.5 g of the desired compound are thus obtained, yield 81%, m.p .: 122-124 ° C.

Example 10 N-Aminoiminomethyl-S-allyl-isothiuronium bromide 14.9 g (0.05 mol) of N-aminoiminomethylthiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained is added 14.5 g (0.12 mol) of allyl bromide. The reaction mixture is heated to boiling while stirring vigorously. The reaction mixture is heated to reflux for 30 minutes and cooled to room temperature. The mixture is filtered to remove any precipitated material. The filtrate is evaporated in vacuo and the oily residue is crystallized from ethyl acetate. The precipitated crystals are filtered off and dried at 40-50 ° C. 19.6 g of the desired compound are thus obtained, yield 82%, m.p .: 123-125 ° C.

Example 11 N-Aminoiminomethyl-S-allyl-isothiuronium chloride 14.9 g (0.05 mol) of N-aminoiminomethylthiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained, 9.18 g (0.12 mol) of allyl chloride are added and the reaction mixture is slowly heated to boiling while stirring vigorously. The reaction mixture is heated to reflux for one hour and cooled to room temperature. The mixture is filtered to remove any precipitated material. The filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The precipitated crystals are filtered off and dried at 40-50 ° C. 15.7 g of the desired compound are thus obtained, yield 81%, m.p .: 125-127 ° C.

29

Example 12 N-Aminoiminomethyl-S- (2-chloroethyl) isothiuronium bromide 14.9 g (0.05 mol) of N-aminoiminomethylthiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained, 17.2 g (0.12 mol) of 1-bromo-2-chloroethane are added and the reaction mixture is heated to boiling while stirring vigorously. The reaction mixture is heated to reflux and cooled to room temperature. The mixture is filtered to remove any precipitated material. The clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The precipitated white crystals are filtered off and dried at 40-50 ° C. 17.2 g of the desired compound are thus obtained, yield 66%, m.p .: 142-143 ° C.

28 80259

Example 13 N-Aminoiminomethyl-S- (1-chloro-2-hydroxypropyl) iso-fluoronium bromide 14.9 g (0.05 mol) of N-aminoiminomethylthiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained is added 20.8 g (0.12 mol) of 1-bromo-2-hydroxy-3-chloropropane. The reaction mixture is slowly heated to boiling while stirring vigorously. The reaction mixture is heated to reflux for 90 minutes and cooled to room temperature. The mixture is filtered to remove any precipitated material. The clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The precipitated slightly cream-colored crystals are filtered off and dried at 40-50 ° C. 17.8 g of the desired compound are thus obtained, yield 61%, m.p .: 118-119 ° C.

Example 14 N-Aminoiminomethyl-S- (3-chloropropyl) isothiuronium bromide 14.9 g (0.05 mol) of N-aminoiminomethyllourea carbonate are added to 80 ml of acetonitrile. To the suspension thus obtained, 18.9 g (0.12 mol) of 1-bromo-3-chloropropane are added and the reaction mixture is slowly heated to boiling while stirring vigorously. The reaction mixture is heated to reflux for 90 minutes, the solid is removed by filtration of the hot reaction mixture and the filtrate is allowed to cool to room temperature. The precipitated white crystals are filtered off and dried at 40-50 ° C. 17.3 g of the desired compound are thus obtained, yield 65%, m.p .: 129-131 ° C.

Example 15 N-Aminoiminomethyl S- (2-hydroxyethyl) isothiuronium chloride 14.9 g (0.05 mol) of N-aminoiminomethylthiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained is added 9.7 g (0.12 mol) of ethylene chlorohydrin and the reaction mixture is slowly heated to boiling while stirring vigorously. The reaction mixture is heated to reflux for 90 minutes and cooled to room temperature. The mixture is filtered to remove any precipitate and the clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The crystals are filtered off and dried at 40-50 ° C. 13.5 g of the desired compound are thus obtained, yield 68%, m.p .: 134-136 ° C.

Example 16

If desired, the isothuronium salts prepared according to Examples 9-15 can be recrystallized as follows. The method is described using N-aminoiminomethyl-S-allyl-isothiuronium chloride.

10 g of N-aminoiminomethyl-S-allyl-isothiuronium chloride are added to 60 ml of a 5: 1 mixture of ethanol and methanol. A small amount of insoluble crystals are removed by filtration of the hot mixture. If necessary, the filtrate is clarified with activated carbon. The clear filtrate is allowed to cool to room temperature, whereupon white crystals precipitate. The mixture is cooled to 0-5 ° C, allowed to stand at this temperature for one hour and filtered. The crystals are washed with ice-cold ethanol and dried at 40-50 ° C. 8.1 g of the desired compound are thus obtained, yield 81%.

Example 17 N-Aminoiminomethyl-N'-methyl-S-allyl-isothiuronium chloride 16.3 g (0.05 mol) of N-aminoiminomethyl-N'-methylthiourea carbonate prepared according to Example 2 are added to 50 ml of anhydrous ethanol. To the suspension thus obtained, 9.18 g (0.12 mol) of allyl chloride are added and the reaction mixture is heated to boiling while stirring vigorously. The reaction mixture is heated to reflux for one hour and cooled to room temperature. The mixture is filtered to remove any precipitated material. The clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The white crystals are filtered off and dried

Already 80259 at 40-50 ° C. 13.6 g of the desired compound are obtained, yield 61%, m.p .: 137-139 ° C.

Example 18 N-Aminoiminomethyl-N'-isopropyl-S-allyl-isothiuronium chloride 19.1 g (0.05 mol) of N-aminoiminomethyl-N'-isopropyl-thiourea carbonate (prepared according to Example 5) are added in 50 ml: anhydrous ethanol. To the suspension thus obtained, 9.18 g (0.12 mol) of allyl chloride are added and the reaction mixture is heated to boiling while stirring vigorously. The reaction mixture is heated to reflux for one hour and cooled to room temperature. The mixture is filtered to remove any precipitated material. The clear filtrate is evaporated in vacuo and the oily residue is crystallized from ethyl acetate. The precipitated white crystals are filtered off and dried at 40-50 ° C. 13.3 g of the desired compound are thus obtained, yield 56%, m.p .: 134-135 ° C.

Examples 19 to 26

In a manner analogous to Example 11, the following isothiuronium compounds of general formula I are prepared: 19. N-aminoiminomethyl-5-isopropyl-isothiuronium bromide, m.p .: 120-122 ° C; 20. N-aminoiminomethyl-S-n-butylisothiuronium bromide, m.p .: 116-118 ° C; 21. N-aminoiminomethyl-S-isoamyl-isothiuronium bromide, m.p .: 111.. .H3eC; 22. N-aminoiminomethyl-S- (2-bromoethyl) isothiuronium bromide, m.p .: 129-131 ° C; 23. N-aminoiminomethyl-S- (3-bromopropyl) isothiuronium bromide, m.p .: 127-129 ° C; 24. N-aminoiminomethyl-S- (3-hydroxypropyl) isothiuronium bromide, m.p .: 132-135 ° C; 25. N-aminoiminomethyl-S- (3-mercaptopropyl) isothiuronium bromide, m.p .: 141-142 ° C; 3i 80259 26. N-aminoiminomethyl-S-propargyl-isothiuronium bromide, m.p .: 131-133 ° C.

Examples 27 to 34

In a manner similar to Example 18, the following compounds are prepared: 27. N-aminoiminomethyl-N'-methyl-S- (2-hydroxyethyl) iso-thiuronium bromide, m.p .: 147-148 ° C; 28. N-aminoiminomethyl-N'-ethyl-S- (2-chloroethyl) iso-thiuronium bromide, m.p .: 143-144 ° C; 29. N-aminoiminomethyl-N'-ethyl-S-allyl-isothiuronium chloride, m.p .: 138-139 ° C; 30. N-aminoiminomethyl-N'-isopropyl-S- (3-chloropropyl) -isothiuronium bromide, m.p .: 140-142 ° C; 31. N-aminoiminomethyl-N'-isopropyl-S-methallyl-iso-thiuronium chloride, m.p .: 138-139 ° C; 32. N-aminoiminomethyl-N'-cyclohexyl-S-n-propyl-iso-thiuronium bromide, m.p .: 133-135 ° C; 33. N-aminoiminomethyl-N'-cyclohexyl-S-methallyl-iso-thiuronium chloride, m.p .: 130-132 ° C; 34. N-aminoiminomethyl-N'-cyclohexyl-S-benzyl-iso-thiuronium chloride, m.p .: 147-149 ° C.

The melting points and IR spectra of the compounds of general formula I are shown in Table VIII below.

Table VIII

32 80259 R2 R1 X Sp./"C characteristic IR spectral peaks H n-propyl Br 122-124 3300, 3120, 2925, 2850, 1685, 1570, 730 H allyl Br or Cl 123-125 3290, 3260, 3160, 3105, 1640, 1605, 1550, 1375, 1090, 980, 910, 705, 650 H 2-chloroethyl Br 142-143 3300, 3110, 2925, 1640, 920, 720 H 1-chloro-2-hydr-Br 118- 119 3300, 3110, 3000, oxypropyl 2850, 1685, 1610, 720 H 3-chloropropyl Br 129-131 3300, 3275, 3165, 3105, 2900, 2825, 1680, 695 H 2-hydroxyethyl Cl 134-136 3310, 3280, 3110, 3000, 2990, 1675, 1610 H isopropyl Br 120-122 3300, 3120, 1680, 1380, 1365 H n-butyl Br 116-118 3315, 3300, 3210, 3180, 2850, 1690, 1450 H isoamyl Br 111- 113 3305, 3300, 3205, 3175, 2960, 2845, 1695, 1470

II

Table VIII continued 33 80259 R2 R1 X Sp./eC characteristic IR spectral peaks H isopropyl Br 120-122 3300, 3120, 1680, 1380, 1365 H n-butyl Br 116-118 3315, 3300, 3210, 3180, 2850, 1690, 1450 H isoamyl Br 111-113 3305, 3300, 3205, 3175, 2960, 2845, 1695, 1470 H 2-bromoethyl Br 129-131 3300, 3205, 2910, 1670, 1090, 715 H 3-bromopropyl Br 127- 129 3305, 3215, 2920, 1665 H 3-hydroxypropro Br 132-135 3590, 3300, 3220, pill 2925, 1680 H 3-mercaptopro Br 141-142 3320, 3305, 3210, fill 3180, 2920, 2570, 1690 H propargyl Br 131-133 3310, 3300, 3200, 2180, 1680 methyl allyl Cl 137-139 3305, 3290, 2960, 2690, 1640 methyl 2-hydroxyethyl Br 147-148 3583, 3300, 3285, 1680, 1375, 1360 ethyl 2-chloroethyl Br 143-144 3305, 3285, 2965, 1660, 765 ethyl allyl Cl 138-139 3310, 3300, 3210, 3180, 1650, 990, 905

Table VIII continued 34 8 0 2 5 9 R2 R1 X Sp./eC characteristic IR spectral peaks isopropallyl Cl 134-135 3305, 3295, 3200, peak 3170, 1655, 985, 895 isopropomethyl Cl 138-139 3305 , 3295, 3200, ethyl 3175, 1650, 890 isoprop-3-chloropropyl Br 140-142 3320, 3300, 3195, ethyl 3160, 1690, 775 cyclohexn-propyl Br 133-135 3320, 3300, 3210, wil 3175, 2925 cyclohexamethyl Cl 130-132 3315, 3300, 3215, wedge 3180, 885 cyclohexbenzyl Cl 147-149 3300, 3275, 3120, wart 3000, 1675, 1590, 750

K

35 80259

Example 35

Manufacture of tablets and granules

Tablets and granules are made on a compression machine equipped with an automatic feeder. The active ingredient is granulated above with suitable excipients by the dry or wet method. If wet granules are prepared, the granules are preferably dried under vacuum. The granules used to make tablets and granules are pressed through a suitable metal-free screen. In particular, a granule simplex composition according to the Pharmacopoeia or another suitable composition can be used to prepare the granules. Tablets and granules are prepared containing 30 mg of active ingredient to which 70 mg of excipient (e.g. granulatum simplex) has been added.

Example 36 Preparation of capsules

I prefer to use automatic filling machines. The active ingredient is granulated with suitable excipients (e.g. granulatum simplex) or other inert excipient. The granules are brought to a suitable particle size, screened and filled into capsules. The active ingredient content of the capsules is 30 mg.

Example 37

Manufacture of powder ampoules

No separate excipients are used in the manufacture of powder ampoules. The active ingredient content of the powder ampoules is 10 and 20 mg. Ampoules for solution contain 1 or 2 ml of distilled water or physiological saline. It is preferred to dilute at least 100 ml of solution for infusion and use the contents of the ampoules immediately after reconstitution.

36 80259

Example 38 N- (Aminoiminomethyl) -5-methallyl-isothiuronium bromide 14.9 g of N-aminoiminomethyl-thiourea carbonate are suspended in 50 ml of abs. ethanol. 16.1 g of methallyl bromide are added and the reaction mixture is heated to reflux for 30 minutes. After filtration, the solvent is distilled off in vacuo. 21 g of a honey-like product are obtained, which crystallizes on standing.

Example 39 N- (Aminoiminomethyl) -N'-allyl-S-allyl-isothiuronium bromide 17.6 g of N- (aminoiminomethyl) -N'-allyl-thiourea carbonate are suspended in 50 ml of anhydrous ethanol. 14.5 g of allyl bromide are added and the reaction mixture is heated under reflux with vigorous stirring for 1 hour, after which the mixture is filtered and the solvent is distilled off in vacuo. 23 g of oily viscous product are obtained.

Example 40 N-Aminoiminomethyl-S-allyl-isothiuronium chloride

A mixture of 4.2 g of dicyandiamide, 20 ml of ethanol saturated with hydrochloric acid and 3.7 g of allyl mercaptan is slowly heated to boiling with vigorous stirring. The reaction mixture is heated to reflux for 3 hours and cooled to 0 ° C. The precipitated crystals are filtered off and washed with ethanol. This gives 7 g of the desired compound. M.p .: 125-127 ° C.

Il

Claims (4)

37 8 0 2 5 9 A process for the preparation of therapeutically useful compounds of the general formula H 2 N-C-NH-C = N-R 2 · HX (I) NH S-R 1, wherein R 1 is C 1-8 alkyl, C 2-6 alkenyl / C 2-6 alkynyl or benzyl, wherein said alkyl groups are optionally substituted with one or more hydroxy, mercapto and / or halogen, R 2 represents hydrogen, C 1-6 alkyl, C 2-6 alkenyl, or C 3-6 cycloalkyl, and X represents a halogen ion, provided that when R 2 represents v , R 1 is other than methyl, ethyl or benzyl, and if R 2 represents ethyl, R 1 is other than methyl, characterized in that a) HoN-C-NH-C-NH-R 2 (II) Z II II NH of general formula II A compound of formula S wherein R 2 is as defined above, or a suitable salt thereof, is reacted with an alkylating agent capable of attaching an R 1 group, preferably with an alkylating agent of general formula R 1 -Y, wherein R 1 is as defined above and Y is a leaving group, preferably halogen, or b) reacting a compound of formula III H2N-C-NH-C = N (III) z II NH with a thioalcohol of general formula R1 -SH, wherein R1 is as defined above. , and, if necessary, converting the product thus obtained into a salt by reacting it with an acid of the general formula HX, in which formula X represents a halogen ion. Process according to Claim 1, characterized in that the starting material of the general formula II is used in the form of a salt, preferably a carbonate. Process according to Claim 1 or 2, characterized in that an alkyl halide is used as the alkylating agent. Process according to one of Claims 1 to 3, characterized in that the reaction is carried out in the presence of an organic solvent, preferably an alcohol, an aliphatic ketone, a nitrile and / or an ether. 39 80259