Technical field of the invention

This invention relates to isothiuronium salts, a process for the preparation thereof and pharmaceutical compositions comprising the same. The majority of the said isothiuronium salts are new compounds. The said compounds possess useful antitumor and antistimulant effect and can be used in human and veterianary therapy.

Background art

Compounds of the general Formula I

H-N-C-NH-C = N-R ² • HX /I/ II I _λ

NH S-R ^** -

wherein R 1 is methyl, ethyl or benzyl and R2 stands for hydrogen are known /French patent No. 788,429 and

DOS No. 2,426,683/. In these publications however the said compounds were only mentioned as intermediates and the publications were completely silent in disclosing any biological activity thereof or any use of these compounds in therapy.

Statement of the invention

According to an aspect of the present invention there are provided new compounds and salts of the general Formula I

A 3498-77 Y

. HX /I/

/wherein

R is C _1-6 alkyl, C^ ^ alkenyl, C- ^ alkynyl,

C-, _g cycloalkyl, aralkyl or aryl, the said groups being optionally substituted by one or more hydroxy, mercapto and/or halogen;

R ² stands for hydrogen, C, £ alkyl, C_ e alkenyl,

C * alkynyl or C^ cycloalkyl;

X represents an organic or inorganic anion with the proviso that if R 2 stands for hydrogen, R1 is other than methyl, ethyl or benzyl/.

The term "C, _g alkyl" relates to straight or branched chain saturated aliphatic hydrocarbon groups

/e.g. methyl, ethyl, ri-propyl, isopropyl, ri-butyl, iso- butyl etc./. The term "C _c alkenyl" covers straight or branched chain olephinic unsaturated groups /e.g. vinyl, allyl, ethallyl etc./. The term "C ₆ alkynyl" relates to straight or branched chain aliphatic groups comprising at least one triple bond /e.g. propargyl etc./. The term " _{3 c} cycloalkyl" relates to cyclic saturated aliphatic hycrocarbon groups /e.g. cyclobutyl, cyclopentyl, cyclo- hexyl etc./. The term ^" ^aryl" group encompasses mono- and polycyclic aromatic hydrocarbon groups /e.g. phenyl, naphthyl etc./. The term "aralkyl" relates to alkyl groups substituted by at least one aryl group /e.g. benzyl, β-phenyl-ethyl etc./. The term "halogen" encompasses the fluorine, chlorine, bromine and iodine atoms.

The said alkyl, alkenyl, alkynyl, cycloalkyl, aryl and aralkyl groups may optionally bear one or more substituent/s/ selected from the group consisting of

hydroxy, mercapto and halogen. Thus as examples of the said substituted groups the following groups may be mentioned ^' : 2-chloro-ethyl, 3-bromo-propyl, 2-hydroxy-

-ethyl, 3-chloro-2-hydroxy-propyl etc. A preferred group of the compounds of the general

Formula I are those derivatives in which R is allyl.

A further preferred group of the compounds of the general Formula I are those derivatives in which R is

2-halogeno-ethyl. - R stands preferably for hydrogen, methyl, ethyl, ri-propyl, isopropyl, n-butyl, allyl or cyclohexyl.

X stands preferably for a halide anion, particularly for chloride, bromide or iodide.

Particularly preferred representatives of the compounds of the general Formula I are pharmaceutically acceptable salts of the following derivatives:

N-aminoiminomethyl-S-methyl-isothiuronium salts; N-aminoiminomethyl-S-propyl-isothiuronium salts; N-aminoiminomethyl-S-allyl-isothiuronium salts; N-aminoiminomethyl-S-/2-chloroethyl/-isothiuronium salts; N-aminoiminomethy1-S-/1-chloro-2-hydroxy-propyl/-iso¬ thiuronium salts;

N-arainoiminomethyl-S-/3-chloro-propyl/-isothiuronium salts; N-aminoiminomethyl-S-/2-hydroxyethyl/-isothiuronium salts; N-aminoiminomethyl-S-glycidyl-isothiuronium salts;

Further preferred compounds of the general Formula I are the N-aminoiminomethyl-N'-alkyl-S-allyl-isothiuronium salts,, particularly the corresponding N'-methyl, N'-ethyl, N'-n-propyl and N'-isopropyl compounds. According to a further aspect of the present invention there is provided a process for the preparation of compounds of the general Formula I and salts thereof which comprises a/ reacting a compound of the general Formula II

- -

H-N-C-NH-C-NH-R' /II/ II II NH S

/wherein R is as stated above/ or a suitable salt thereof with an alkylating agent capable of introducing a R group, preferably with an alkylating agent of the general Formula R -Y 10 /wherein R is as stated above and Y stands for a leaving group, preferably halogen/; or b/ reacting the compound ^" of the Formula III

NH

with a compound of the general Formula R -SH /wherein R is as stated above/ 20 and if desired converting the product thus obtained into a salt by reacting with an acid of the general Formula HX /wherein X is an anion of an organic or inorganic acid/.

In the said reactions isothiuronium salts are formed. The reaction is generally accompanied by the formation of 25 N-alkylated by-products.

Disclosure of invention

According to a preferred form of realization of 30 process a/ the starting materials of the general Formula II are not used in the free base form but in the form of a suitable salt - particularly carbonate - thereof. Thus the formation of the N-alkylated derivatives is quenched and this is probably due to the fact that the carbon 35 _. dioxide evolved decreases the pH of the strongly basical

medium .

As alkylating agent preferably the corresponding alkyl halidesor dialkyl sulfates can be used, but other alkylating agents may be applied as well. The reactants may be used in equimolar amount or the alkylating agent may be added in an excess. As reaction medium inert organic solvents, preferably an alcohol, aliphatic ketone, aliphatic nitrile or ether may be used. The reaction may be accomplished under athmospheric pressure and at a temperature of 20-80 °C.

According to a preferred form or realization of method a/ the starting material of the general Formula II and the alkylating agent are reacted in a molar ratio of 1:1-1:1.8, in anhydrous ethanol, acetonitrile, dimethyl formamide, acetone or ether, at a temperature between 20 °C and 30 °C.

According to method b/ the compounds of the general Formula IV

H-N-C-NH-C = NH /IV/

² II I _λ

NH S-R

/which form a narrower sub-group of the compounds of the general Formula I; R is hydrogen/ may be prepared by reacting the compound of the Formula III with a thio- alcohol of the general Formula R -SH. The reactants may be used in an equimolar amount or the thioalcohol of the general Formula R -SH may be applied in an excess. As reaction medium preferably an alcohol - particularly ethanol - may be used. The reaction may be accomplished under pressure, if necessary. It is preferred to work under a pressure of 1-5 bar and at a temperature of 20-100 °C.

The starting materials of the general Formula II are known or can be prepared by methods known per se. Thus

N-aminoiminomethyl-thiourea can be readily prepared by reacting dicyane diamide with hydrogen sulfide /Houben- -Weyl VIII., 214/. The N-aminoiminomethyl-N'-alkyl- thiourea derivatives of the general Formula II can be prepared by reacting guanidine base with the corresponding alkyl isothiocyanate /US patent No. 4,009,163/.

The starting material of the Formula III can be prepared by known methods.

The product thus obtained can be converted into a pharmaceutically acceptable salt formed with an organic or inorganic acid. For salt-formation preferably formic acid, lactic acid, citricacid, maleic acid or sulfuric acid, hydrochloric acid, phosphoric acid or hyd- robromic acid may be used. The compounds of the general Formula I possess antitu or and antiparasital immunostimulant properties. The strenght and direction of the activity and the toxicity depend on the substituents of the molecule. It is known that gutimine which is structurally related to the compounds of the general Formula I was • tested as antihypoxial agent /_~C.A. 66 452,929 /1972/; Pathol. Phys. Exp. Ther. 10_, /6/_7 in pharmacological and clinical tests as substance causing microcirculation disorders /Gematol. Transfusiol. /1984/, 29 /l/ , 49-52_7. It has been found that the compounds of the general Formula I are capable of restoring the decreased natural immune system of organism to the normal value or of increasing the same above the normal level.

Thus the organism of animals and humans possess an immune system /e.g. tumor eliminating system/. Once the activity of the said system is reduced or stops functionating, various diseases and tumors appear and manifest themselves. The compounds of the general Formula I activate the natural immune systems and are thus useful in all cases when the increase of the reduced activity of

the immune system and the further raising of the normal activity thereof is desired.

The compounds of the general Formula I exhibit further useful biological effects, e.g. the curing of microcirculation disorders, binding of radicals detrimental to health, improvement of the function and lengthening of the lifetime of cells being in oxygen- -deficient stage.

The biological activity of the compounds of the general Formula I is demonstrated by the following tests. It is stated however that not all effects of the highly active compounds are shown.

Testing of specific effects

Immunological tests

Lymphocytes. Lymphocytes were separated on Ficoll-Uromiro /Bδyum 1968/ gradient from the venal blood of healthy donors and patients suffering from maligne lymphoma and metastatizing solid tumours, respectively. When selecting the patients belonging to the latter group, the decreased ADCC and NK activity was taken into considera¬ tion. The fagocytes were removed by treatment with iron powder and a magnet.

ADCC. ADCC was determined according to the method of Perlmann and Perlmann /1970/ with minor modifications /Lang et al, 1981/. Chicken erythrocytes labelled with 10 Cr and coated with anti-chicken erythrocyte rabbit-serum were incubated with the lymphocytes for 4 hours at 37 C. The reaction having been stopped the activity of the supernatant of the centrifuged cells was determined in a gamma-counter. The rate of chrome-release was expressed as cytotoxicity index /CI %/. This value was calculated as described in one of our earlier publications

/Lang et al, 1980/ - /see Table I/.

NK: NK activity was determined by the method of Jondal and Pross /1975/ by using minor modifications /Lang et al,

51 1981/. K-562 tumor cells labelled by Cr were incubated with effector cells in a TC 199 liquid nutrient medium comprising 10 % of decomplemented calf serum. The reaction having been stopped the radioactivity of the supernatant of the centrifuged cells /chrome release/ was counted in a gamma-counter. The killing of the aimed cells /cyto¬ toxicity index %/ was expressed as the difference of the test release and spontaneous release.

Statistical calculations were carried out by means of a mono-sample test. Several compounds of the general Formula I exhibit outstanding immunostimulant effect /e.g. compounds prepared according to Examples 11, 12 and 16/. The said compounds are capable of significant increase of normal and strongly decreased NK and K cell activity, even ^* above the normal level. The said effect may be so strong that cytotoxical activity decreased to one- -tenth of the original value may be restored to the oroginal level /Table /II/.

In the dose range corresponding to the expected human plasma concentration the compounds of the general Formula I increase the NK and K cell activity of human lymphocytes and enhance NCMC and ADCC reaction, respectively at a dose-dependant rate. According to preliminary results of further immunological tests the compounds increase bastos transformation and migration. On carrying out substitution on the nitrogen atoms the molar proportional immunostimulant effect related to the toxicity is generally decreased.

^* Since the cytotoxical activity of patients suffering from tumor is decreased proportionally with the stage of the disease and oreove it is further reduced by usual

cytostatical treatments, it is of outstanding importance that the compounds of the general Formula I possess strong immunostimulant effect and are no immunodepressors. This may be highly significant from the point of view of reconvalescence. ^' - -

Migration of leucocytes

Spontaneous /random/ migration of leukocytes was measured on agarose microdrops according to the method of McCoy et al /1977/ by carrying out some minor modifications /Kal ar and Gergely, 1982/.

2 ml agarose drops /0.2 %/ were placed on migration plates /Greiner, German Federal Republic/. TC 199 liquid nutrient medium was added and the plates were air-tightly closed. After 24 hours the migration area were measured under a stereomicroscope. The migration area were

2 expressed in mm .

Migration of leucocytes

The direct /random/ migration of polymorphonuclear cells of healthy donors was increased by compounds according to Examples 11, 12 and 14. The optimal dose amounted to 1.0 -ug/ml. At a higher dose /10, ,ug/ml/ the effect was less expressed /Table III/.

Mitogen-induced blastos transformation

Lymphocytes were cultivated in a TC 199 liquid nutrient medium completed by 10 % decomplemented calf serum, antibiotics and 25 mH HEPES /Serva/. 200 ,ul of a cell* suspension comprising 2.10^ lymphocytes were placed on a icroplate /manufacturer: Sterilin Great- -Britain/; five parallels were carried out each.

2 and 10 ,ug/ml, respectively of Phytohaemagglutinine

/Leucoagglutinine; manufacturer Pharmacie Sweden/ and

25 Ug/ml of Concanavalin A /Carbiochem/ were added to the cultures; the above values relate to final concentration. The cultures were cultivated at 37 C for

72 hours. Eight hours before the termination of the end

3 of the cultivating period 0.5 ,uCi H thymidine /Chemapol.

Tschechoslowakia/ was added to the samples. Incorpora¬ tion was stopped by freezing the cultures. After melting the samples were filtered on an automatic harvester

/Dynatech/. The radioactivity of the samples was measured in a scintillator counter. The activity was expressed as c.p.m. /Tables IV and V/.

Statistical analysis

Significance was determined by means of a mono- -sample 2T2 test. The results obtained with the product of Example 11 are disclosed in Tables I, II and III.

Preliminary rapid test for the determination of

-a—n^titumor a"—n—d—i—m—m—u"—no—s^—t—i—mu—l^a-n^â €”t —e—f—fe— ^» -c^t— »

This method was elaborated in our laboratories for the rapid testing of a large number of samples. CFLP male mice /average body-weight 30 g/ were i.p. inoculated with four million ascites lymphoma cells.

In the test those animals were used on which on the fourth day an easily visible tumor - i.e. ascites - was developped. At least ten animals were used in each group and the mice were orally treated with one-tenth of the LD5-0-. value.

^• 24 hours after oral treatment the results were evaluated by means of Giemsa stained ascites smear on the basis of cytological analysis. In the control group

- li ¬

the same number of animals was used as in the treated group.

The damages of the tumor cells, the activation of the organism and the eventual toxical effects were evaluated.

On evaluating anti-tumor effect the percental ratio of killed and intact tumor cells was evaluated. The results were expressed according to the following scale:

Killing rate of tumor cells Effect

0-30 % no effect

30-70 % medium effect

70-100 % strong effect

/inhibition/

The increase of the immune system _. of the organism was evaluated on the basis of the number and activity of neutrophilic granulocytes, lymphocytes and macrophages appearing among the ascites tumor cells and participating at the immune system. The activity of the immune system is also manifested by the rate in which the above cells in the ascites form plasma-bridge bond with the tumor cells. According to this evaluation if there is no difference in the number and activity of the said cells, no increase can be declared over the control. Medium effect is observed if the increase of cell number or activity over the control amounts to 20 %. The effect is strong if the increase of the cell number and activity of the immune system is more than 20 % higher than the control /Table VII/ Toxicity was determined by evaluating the morphological changes of neutrophylic granulocytes and limphocytes, the vasquolization of plasma, the fragmentation of the nucleus, the formation of polysegments and toxical granulation. The toxicity was expressed by the following scale:

- L2 -

Results observed Toxicity

No above changes in own cells no toxicity 20 % change medium toxicity changes above 20 % high toxicity

Testing of antitu or effect

1. In vitro

K-562 human erythroÏŠeukamia cells and P-388 mouse lymphoma cell cultures were treated with 1-100 ,ug/ml of the test material.

2. In vivo

a/ NK-ly-ascites lymphoma

One million of Nemeth-Kellner ascites lymphoma cells were i.p. transplanted into twenty CFLP male mice /average bodyweight 30 g/; _~A new mouse ascites tumor to be used as a screening tool. Neoplasma 8, 337 /1961/ _7. Half of the animals served as control and the other half was treated with one-tenth of the LD,_ ₀ value of various compounds of the general Formula I p.o. and i.p. on one and five consecutive days, respectively. /If one-tenth of the LD _J - _Q value was effective, a lower dose was used/. In the group subjected to one treatment the cytomorphological determination of the ascites smear was carried out after 24 hours and the ratio of damaged and killed cells, respectively was evaluated. In the groups treated for 5 days the survival rate was determined.

^{b/ L} 1210 tumor

10 ^L _] _2io ^tu ro°-f cells were transplanted i.p. into twenty BDF- _j^ female mice /body weight 20 g/. Treatment was started 24 hours after tumor transplantation and was continued for eight days. Each group consisted of 5 animals; dose: 5, 50 and 500 mg/kg p.o. daily. The control group also consisted of 5 animals. The rate of survival was observed.

c/ Lewis long tumor

10

In each group 6 animals were used. Dose: 5 and 50 mg/kg. -The control group also consisted of 6 animals. In the test C- ₇ B1 female mice /weight 20 g/ were used. Tumor was s.c. transplanted into the muscle of 15 the right leg. After 10 days the leg was amputated, the oral treatment was started and continued for 9 days. The animals were sacrificed and the metastases were counted under stereo icroscope.

20 d/ Dogs belonging to various species and having spontaneous mammal tumor /ductus cc./ were treated for 4 weeks with 1-10 mg/kg oral doses of the test materials. Test excision was performed and tissue samples were collected for hystological analysis.

25 Compounds according to Example 11, 13, 14, 15,

27, 28, 34, 35, 36, 38 exhibit an outstanding antitumor effect. Thus the compound according to Example 11 inhibits the K-562 human erythroleukaemia cells in tissue culture in a concentration of 4 ,ug/ml. On the other hand this

30. compound inhibits the proliferation of P-388 cells only in a concentration of 40 ,ug/ml and when increasing the concentration to 400 ,ug/ml the activity does not become stronger. Compounds having a strong activity exhibit a dose-dependant effect in a concentration range of 5 0.5-5 Ug/ml, while compound having a low activity are

not dose-dependant. According to in vivo tests the members of this compound group are active against L ₁₂ 10 ^e i ^tner not at all or but to a very small extent.

The compounds significantly decrease the number of lung metastasis in Lewis long tumor. Compounds 11, 13, 14, 15, 22, 27, 28, 45 are effective in a dose lower than one-tenth of the LD _CQ value. Compound according to Example 14 decreases by 50 % the number of lung metastasis already in a dose of 5 mg/kg and this result is particularly significant in view of the fact that LD _5Q = 2380 mg/kg.

It has been found that the effect can be strenghtened to a larger extent by repeating the treatment than by increasing the dose. Compounds according to Examples 11, 13, 14, 20,

22, 27, 45 proved to be active against Nk-ly-ascites tumor in a concentration below one-tenth of the LD _[ - ₀ value. The latter compounds increase survival rate and lifetime in a dose-dependant manner. These compounds exhibit immuno- stimulant effect in small doses, while in higher doses the direct antitu or effect manifests itself as well. Thus compounds of Example 11 and 22 exhibit an immunostimulant effect in doses of 25 mg/kg and 20 mg/kg, respectively. Compounds of Example 11 and 22 are cytotoxical in a dose range of 250-500 mg/kg and 40-160 mg/kg, respectively. S-allyl-> S-L-chloro-2-hydroxy-propyl- or S-l-bromo-2- -hydroxy-propyl-substituted compounds of the general Formula I are highly active against spontaneous mammal tumors on dogs. In biopsy taken after treatment carried out for 3-4 weeks, cell infiltration of invasive character was observed in tumors /ductus cc./ consisting of lymphocytes and neutrophilic granulocytes, macrophages and occasionally of eoziniphilic granulocytes. On the tumor cells degenerative changes and strong decomposition was observed.

NK cells form plasma bridge bonds with tumor cells and this causes vacoular degeneration of the plasma of tumor cells and large thinning in the DNS stand of the nucleus. The nucleus-plasma relation is changed, the nucleus are strongly swelled and the basophility of the plasma is decreased.

In about one-fourth of the cases the leukocyte infiltration in the tumor is negligible; on the other hand the tumor cells are swelled..and hyalinially degenerated... In the intercellular stand a very large amount of hyaline stained material is deposited.

On administering the compound of Example 11 in an oral daily dose of 30-120 rag /0.5-2 mg/kg/ for 6-12 months a significant improvement or reconvalescence respectively, was observed in human bone metastasis of mammal tumors, ovary tumors and pancreas tumors.

Testing of antitumor effect in combinations 1/ Combinations with known cytostatical agents Various cytostatical agents having biological alkyla ing effect were administered together with compounds of the general Formula I to CFLP mice having NK-ly ascites tumor. The cytostatical agents were administered orally, intra- peritonally or intravenously in a dose corresponding to one-fifth or one-tenth of the LD,_ ₀ value or in the most effective dosage disclosed in prior art. Compounds of the general Formula I were administered orally in a dose of one-tenth or one-twentieth of the L value once or several times, 48 hours after treatment with the cyto- statical agent. Thus the combinations of cyclophosphamide, carnomustine, degranol, dibromo dulcitol and dibromo mannitol on the one hand and compounds of Examples 11, 13 on the other 14, 15, 16, 27, 28, 45 were administered. Compared to the control group treated only with the known cytostatical agent, the antitumor effect was strengthened and immuno-

depression decreased.

2/ Combination with known vitamins The isothiuronium salts of the general Formula I can be effectively combined with medium doses of vitamins A and D~ and with high doses of vitamin C. Good results were obtained both in pharmacological tests and human clinical cases.

3/ Combination with known hormones

In the treatment of hormone—dependant tumors the combination proved to be useful both in pharmacological tests and human clinical cases.

4/ Use in surgery

The compounds of the general Formula I were success¬ fully used in pharmacological tests in the case of solid tumors for surgical pre- and post-treatment /NK-ly solid, Yosida/. It can be stated on the basis of pharmacological tests that the compounds of the general Formula I are probab¬ ly also suitable for use in human surgery for pre- and post- -treatment.

Testing of anti-parasital effect Compounds of the general Formula I were added orally to dogs infected by ticks /Ixodes ricinus/ for three days. Thus N-aminoiminomethyl-S-allyl-isothiuronium hydrochloride was administered in an oral dose of 1-2 mg/kg and the kill¬ ing rate of ticks was registered. Six-eight hours after administration of the active ingredient of the general Formula I all ticks were killed in the do Όgs- .

Binding of free radicals

Compounds of Examples 9, 10, 11, 16, 23 and 29 significantly decrease the toxicity /LOr- _Q / of hydrogen

peroxide, organic peroxides and biological alkylating /free radical forming/ agents. On the basis of their favourable toxicity data the compounds of the general Formula I are more suitable for the binding of free radicals and protec¬ tion against radiation than the known isothiuronium derivatives obtained from the corresponding thiourea. Thus the therapeutical effect of compound of Example 23 is better than that of beta-aminoethyl-isothiuronium hydrochloride.

Cardioprotective effect in isoprenaline necrosis

5 mg/kg of isoproterenol are i.p. administered to male CFY rats /weight 250-300 g/. In each group 10 animals were used. One part of the animals served as control and the other were treated with a 1-5 mg/kg i.p. or 10-50 mg/kg p,o. dose of compound No. 14. After two weeks the hearts were subjected to hystological evaluation. The ratio of necrotized and intact area of heart muscle were determined. It has been found that all treatments with N-aminoiminomethyl-S-allyl-isothiuronium hydrochloride of the general Formula I resulted in a significant decrease of necrosis.

On the damaged area the test compound presumably exhibits the effect thereof through the binding of hydrogen peroxide produced by neutrophilic granulocytes. On this model other free radical binding and antioxidant molecules exert a similar effect.

Effect on circulation and respiration

The compound according to Example 11 does not considerably effect the he odinamical parameters and respiration function of cats with opened and closed chest, when administered in an i.v. dose of 10 mg/kg. The following parameters were tested: ventricular and peripherial pressure, heart frequency /action/ ECG, volume/minute, contractility, respiratory volume, respiration rate and resistance. In a intravenal dose above 10 mg/kg the left venticular pressure and frequency decreased. In a dose of 50 mg/kg these effects were significant and long-lasting. In an i.v. dose of 100 mg/kg strong hemodynamical changes were observed but the cats did not perish. Effects of N-/aminoiminomethyl/- -S-allyl-isothiuronium salts /halogenides/. /Compound A/

Table I

Effect on ADDC. Cytotoxicity index /%/.

Ratio of effector cells; aimed cells = 5:1. Incubation time 4 hours.

Average: S.E.

x significant /p<0.05/

Table II

Effect on NK activity.

Ratio of effector cells: aimed cells = 50:1. Incubation time 4 hours.

Cytotpxicity index /%/. Average values: * S.E.

x significant /p <.0.05/

Table III

In vitro effect on direct /random/ migration of human polymorphonuclear leucocytes. Migration area /mm /. Average values of seven healthy controls: *

non significant significant, /p-^0.01/ significant /p 0.05/.

Table IV

In vitro effect on blastos transformation of human lymphocytes stimulated by PHA and Con A. Healthy controls of normal reactivity; n = 6. Average c.p.m. * 'S.E.M.

Table V

In vitro effect on blastos transformation of human lymphocytes stimulated by PHA and Con A. Patients of decreased reactivity /tumor and SLE/; n = 6. ^' Average c.p.m. * S.E.M.

Mytogenic agent Control Dose /~μg/ml_7

0.1 1.0 10.0

1. Compound A PHA 2 _/ ug/ml 3034 * 1293 2877 * 984 4344 i 1153 4716 * 1287 PHA 10 /U /ml 5267 * 1392 5802 * 1504 6323 * 1161 6968 * 1669 Con A 25 ,ug/ml 804 * 307 1097 i 354 935 * 363 1023 * 361

Tabelle VI

24 hours' acute toxicity of compounds of the general

Formula I

Example R ² R ¹ LD ₅₀ mg/kg No. p.o. i.p.

9 H methyl 2320 + 220

23 H ethyl 3080 +

290

10 H propyl 2800 + 260

25 H butyl 2700 + 310

24 H i-propyl 2260 + 210

26 H i-amyl 2260 + 241

13 H 2-chloroethyl 1120 + 140

27 H 2-bromo-ethyl 1050 + 280

14 H l-chloro-2-OH- 1240 + 160 propyl

15 H 3-chloro-propyl 1210 + 180

28 H 3-bromo-propyl 980 + 640

16 H 2-hydroxy-ethyl 3280 + 460 1680 * 210

29 H l.-oxypropyl 2860 + 180

11 H allyl 2380 + 320 320 t 30

44 H methallyl 1640 + 210

18 H benzyl 2420 + 260

17 H propargyl 90 + 7,6

20 methyl allyl 600 + 54 75 * 6.3

34 ethyl allyl 780 + 62 84 i 7.6

22 cyclohexyl allyl 450 + 51 65 * 5.1

21 isopropyl allyl 280 + 26 32 i 4

45 allyl allyl 380 + 41 36 i 4

22 cyclohexyl allyl 400 + 38 40 i 6

- Table VII

Test compound Antitumor Immunostimulant Toxical

Example No. effect effect effect

9 medium medium medium

23 medium strong 0

10 medium medium 0

25 medium medium 0

24 medium medium 0

26 medium medium 0

13 strong weak 0

27 strong weak 0

14 strong weak 0

15 strong weak 0

28 st-rong weak 0

16 medium strong 0

29 medium medium 0

11 strong strong 0

44 medium medium 0

18 increase 0 0

17 weak weak medium

20 strong weak 0

34 medium medium 0

22 strong weak 0

21 strong weak 0

45 strong weak 0

22 weak weak 0

According to a still further feature of the present invention there are provided pharmaceutical compositions comprising as active ingredient at least one compound of the general Formula I /wherein

R is C, ₆ alkyl, C _g alkenyl, C- g alkynyl, C ₃ _ ₆ cyclo¬ alkyl, aralkyl or aryl, the said groups being optionally substituted by one or more hydroxy, mercapto and/or halogen; 2 R stands for hydrogen, C, _β alkyl, C alkenyl,

C ₂ _g- alkynyl or C _g cycloalkyl;

X represents an organic or inorganic anion/.

The pharmaceutical compositions of the present invention can be prepared by methods of pharmaceutical industry known per se. The active ingredients of the general Formula I can be preferably finished in the form of capsules in admixture with conventional generally used auxiliary agents /sliding agents etc./; the capsules may be manufactured by using automatical encapsulating machines. The pharmaceutical compositions can also prepared in the form of powder ampouls, parenteral compositions for injection and infusion purposes; these compositions may also prepared by methods known per se. As solvent e.g. distilled water or physiological saline solution can be used.

The active ingredients of the general Formula I can also be finished in the form of an ointment, particularly as lypophilic ointments. Active ingredients of the general Formula I having a stronger odour may be preferably finished by means of microencapsulation, e.g. by means of the formation of cyclodextrine inclusion complexes.

The pharmaceutical compositions of the present invention may also contain in addition to che compound of the general Formula I one or more of known biologically

2 1

active substance/s/ as active ingredient.

The pharmaceutical compositions of the present invention can be used both in human and veterinary therapy. When used as immunostimulant the oral or rectal dose may be preferably about 0.5-25 mg/kg. For local treatment it is preferred to apply higher doses.

Industrial applicability

The invention relates to predominantly new compounds having antitumor and immunostimulent effect.

Modes of carrying out the invention

Further details of the present invention are to be found in the Examples without limiting the scope of protection to the said Examples.

Starting materials

Example 1

N-aminoiminomethyl-thiourea carbonate -

84.0 g /1.0 mole/ of dicyan diamide and 300 ml of water are filled into an autoclave. Into the closed auto- clave 34.0 g /l mole/ of gaseous hydrogen sulfide are introduced in the following manner: at a temperature of 20 C so much hydrogen sulfide gas is introduced that the pressure in the autoclave amounts to 1.5 bar. The introduc¬ tion of gas is stopped and the autoclave is slowly heated to 80 C. In the mean time the gaseous hydrogen sulfide is introduced at such a rate that the pressure in the autoclave should be constantly 1.5 bar. After the complete amount of hydrogen sulfide gas having been introduced the autoclave is kept at 80 °C until the pressure decreases to 0.3-0.4 bar. The reaction mixture is cooled to 20 °C, flushed with nitrogen and filtered. The clear filtrate is cooled to 0 °C, and carbon dioxide gas is let in. The precipitated white crystalline N-/aminoiminomethyl/-thiourea carbonate is filtered off, washed with cold water and dried at 60-80 °C, Thus 100 g of the desired compound are obtained, yield 67 %, mp.: 127-129 °C.

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

Example 2

N-aminoiminomethyl-N'-methyl-thiourea carbonate To a solution of 5.9 g /0.1 mole/ of guanidine base and 30 ml of acetone a solution of 7.3 g /0.1 mole/ of methyl isothiocyanate in 10 ml of acetone is added. The isothiocyanate solution is added at such a rate to the guanidine solution that the temperature of the mixture should not exceed 40 °C. The reaction mixture is allowed to stand at 40 C for 4 hours, whereupon the solvent is removed in vacuo. The residual light yellow oil is suspended in water - to yield an opalescent 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 %/ . Mp . : 132-134 °C .

Examples 3-8

In an analogous manner to Example 2 the following compounds are prepared:

3. N-aminoiminomethyl-N'-ethyl-thiourea-carbonate

4. N-aminoiminomethyl-N'-n-propyl-thiourea-carbonate . N-aminoiminomethyl-N'-isopropyl-thiourea-carbonate 6. N-aminoiminomethyl-N'-n-butyl-thiourea-carbonate 7. N-aminoiminomethyl-N'-allyl-thiourea-carbonate

8. N-aminoiminomethyl-N'-cyclohexyl-thiourea-carbonate

In the following Examples the preparation of the end-products of the general Formula I is disclosed.

Example 9

N-aminoiminomethyl-S-methyl-isothiuronium iodide 14.9 g /0.05 mole/ of N-aminσiminomethyl-thiourea carbonate are suspended in 50 ml of anhydrous ethanol. To the suspension thus obtained 17.0 g /0.12 mole/ of methyl iodide are added. The reaction mixture is slowly heated to boiling, whereby carbon dioxide is evolved. The reaction mixture is heated to boiling for 30 minutes and cooled to room temperature. The mixture is filtered in order to remove the eventual crystalline precipitate. The clear filtrate is eveporated in vacuo and the residual oil is recrystalliz- ed from ethyl acetate. The yellow crystals are filtered off and dried at 40-50 °C. Thus 22.1 g of the desired compound are obtained, yield 85 %, mp.: 124-125 °C.

Example 10

N-aminoiminomethyl-S-n-propyl-isothuronium bromide 14.9 g /0.05 mole/ of N-aminoiminomethyl-thiourea carb are suspended in 50 ml of anhydrous ethanol. To the suspension 14.8 g /0.12 mole/ of _n-propyl bromide are added. The re- - action mixture is slowly heated to boiling under stirring,

whereby carbon dioxide is evol.ved. The reaction mixture is heated to boiling for 45 minutes, and -cooled to room temperature. The mixture is filtered in order to remove the eventually precipitated substance. The clear filtrate is evaporated in vacuo and the oily residue is crystallized from ethyl acetate. The white crystals are filtered and dried aatt 4400--5500 °CC.. TThhuuss 1199..55 gg ooff tthhee <desired compound are obtain- ed, yield 81 %. Mp.: 122-124 °C.

Example 11

N-aminoiminomethy1-S-allyl-isothiuronium bromide 14.9 g /0.05 mole/ of N-aminoiminomethyl-thiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 14.5 g /0.12 mole/ of allyl bromide are added. The reaction mixture is slowly heated to boiling under vigorous stirring. The reaction mixture is refluxed for 30 minutes and cooled to room temperature. The mixture is filtered in order to remove the eventually precipitated substance. The filtrate is evaporated in vacuo and the oily residue is crystallized from ethyl acetate. The pre¬ cipitated crystals are filtered and dried at 40-50 C. Thus 19.6 g of the desired compound are obtained, yield 82 %. Mp. : 123-125 °C.

Example 12

N-aminoiminomethyl-S-allyl-isothiuronium chloride 14.9 g /0.05 mole/ of N-aminoiminomethyl-thiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 9.18 g /0.12 mole/ of allyl chloride are added and the reaction mixture is slowly heat¬ ed to boiling under vigorous stirring. The reaction mixture is refluxed for an hour and cooled to room temperature. The mixture is filtered in order to remove the eventually pre- - cipitated substance. The filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The

precipitated crystals are filtered and dried at 40-50 C. Thus 15.7 g of the desired compound are obtained, yield 81 %, mp.: 125-127 °C.

Example 13

N-aminoiminometh l-S-/2-chloroethyl/-isothiuronium bromide

14.9 g /0.05 ^" mole/ of N-aminoiminoraethyl-thiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 17.2 g /0.12 mole/ of 1-bromo- -2-chloro-ethane are added and the reaction mixture is heated to boiling under intensive stirring. The reaction mixture is refluxed for 90 minutes and cooled to room temperature. The mixture is filtered in order to remove the eventually precipitated substance. The clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The precipitated white crystals are filtered and dried at 40-50 °C. Thus 17.2 g of the desired compound are obtained, yield 66 mp 142-143 ^U C.

ExamDle 14

N-aminoiminomethyl-S-/l-chloro-2-hydroxy-p opyl/- -isothiuronium bromide

14.9 g /0.05 mole/ of N-aminoiminomethyl-thiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 20.8 g /0.12 mole/ of l-bromo-2- -hydroxy-3-chloro-propane are added. The reaction mixture is slowly heated to boiling under vigorous stirring. The re¬ action mixture is refluxed for 90 minutes and cooled to room temperature. The mixture is filtered in order to remove the eventually precipitated substance. The clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The precipitated slightly creme-coloured crystals are filtered and dried at 40-50 C. Thus 17.8 g of the desired compound are obtained, yield

61 %. Mp.: 118-119 °C.

Example 15

N-aminoiminomethyl-S-/3-chloropropyl/-isothiuronium bromide

14.9 g /0.05 mole/ of N-aminoiminomethyl-thiourea carbonate are added to 80 ml of acetonitrile. To the suspension thus obtained 18.9 g /0.12 mole/ of l-bromo-3- -chloro-propane are added and the reaction mixture is slow- ly heated to boiling under vigorous stirring. The reaction mixture is refluxed for 90 minutes, the solid substance is removed by filtering the hot reaction mixture and the filtrate is allowed to cool to room temperature. The precipitated white crystals are filtered and dried at 40-50 °C. Thus 17.3 g of the desired compound are obtained, yield 65 %. Mp.: 129-131 °C.

Example 16 N-aminoiminomethyl-S-/2-hydroxyethyl/-isothiuronium chloride

14.9 g /0.05 mole/ of N-aminoiminomethyl-thiourea carbonate are added to 50 ml of anhydrous ethanol. To the suspension 9.7 g /0.12 mole/ of ethylene chlorohydrine are added and the reaction mixture is slowly heated to boil- ing under vigorous stirring. The reaction mixture is refluxed for 90 minutes and cooled to room temperature. The mixture is filtered in order to remove the eventually precipitated substance and the clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The crystals are filtered and dried at 40-50 °C. Thus 13.5 g of the desired compound are obtained, yield 68 %. Mp.: 134-136 °C.

Example 17 N-aminoiminomethyl-S-glycidyl-isothiuronium bromide

14.9 ^' S /0.05 mole/ of N-aminoiminomethyl-thiourea carbonate are added to 80 ml of acetonitrile. To the suspen¬ sion thus obtained 16.5 g /0.12 mole/ of epibromo hydrine are added and the reaction mixture is slowly heated to boil- 5 ing under vigorous stirring. The reaction mixture is reflux¬ ed for 2 hours and filtered until hot. The clear filtrate is allowed to cool to room temperature. The precipitated white crystals are filtered and dried at 40-50 °C. Thus 13.5 g of the desired compound are obtained, yield 53 %. 10 Mp.: 138-139 °C.

Example 18

N-aminoiminomethy1-S- enzyl-thiuronium chloride 14.9 g /0.05 mole/ of N-aminoiminomethyl-thiourea 15 carbonate are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 15.2 g /0.12 mole/ of benzyl _t chloride are added and the reaction mixture is heated to boiling under vigorous stirring. The reaction mixture is refluxed for an hour and cooled to room temperature. The ^■ 20 mixture is filtered in order to remove the eventually pre¬ cipitated substance. The clear filtrate is evaporated in vacuo and the oily residue is crystallized from ethyl acetate. The precipitated white crystals are filtered and dried at 40-50 °C. Thus 19.1 g of the desired compound are obtained, 25 yield 78 %. Mp.: 159-161 °C.

Example 19

The isothiuronium salts prepared according to Examples 9-13 can be recrystallized by the following method, 30 if desired. The process is shown by 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. The small amount of undissolved crystals is 35 removed by filtering the hot mixture. The filtrate is

clarified with activated charcoal, if necessary. The clear filtrate is allowed to cool to room temperature, whereby white crystals precipitate. The mixture is cooled to 0-5 °C, allowed to stand at this temperature for an hour and filter- ed. The crystals are washed with icecold ethanol and dried at 40-50 C. Thus 8.1 g of the desired compound are obtain¬ ed, yield 81 %.

Example 20 N-aminoiminomethyl-N'-methyl-S-allyl-isothiuronium chloride

16.3 g /0.05 mole/ of N-aminoiminomethyl-N'-methyl- -thiourea carbonate prepared according to Example 2 are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 9.18 g /0.12 mole/ of allyl chloride are added and the reaction mixture is heated to boiling under vigorous stirring. The reaction mixture is refluxed for an hour and cooled to room temperature. The mixture is filtered in order to remove the eventually precipitated substance. The clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The white crystals are filtered and dried at 40-50 °C. Thus 13.6 g of the desired compound are obtained, yield 61 %.Mp.:

137-139 C.

Example 21

N-aminoiminomethyl-N'-isopropyl-S-allyl-isothiuronium chloride

19.1 g /0.05 mole/ of N-aminoiminomethyl- '-isopropyl- -thiourea carbonate prepared according to Example 5, are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 9.18 g /0.12 mole/ of allyl chloride are added and the reaction mixture is heated to boiling under vigorous stirring. The reaction mixture is refluxed for an hour and cooled to room temperature. The mixture is filtered

in order to remove the eventually precipitated substance. The clear filtrate is evaporated in vacuo and the oily residue is crystallized from ethyl acetate. The precipita¬ ted white crystals are filtered and dried at 40-50 °C. Thus 13.3 g of the desired compound are obtained, yield 56 %. Mp.: 134-135 °C

Example 22

N-aminoiminomethyl-N'-cyclohexyl-S-allyl-iso- thiuronium chloride

23.1 g /0.05 mole/ of N-aminoiminomethyl-N'-cyclo- hexyl-thiourea carbonate prepared according to Example 8 are added to 50 ml of anhydrous ethanol. To the suspension thus obtained 9.18 g /0.12 mole/ of allyl chloride are added and the reaction mixture is heated to boiling under vigorous stirring. The reaction mixture is refluxed for an hour and cooled to room temperature. The mixture is filtered in order to remove the eventually precipitated substance and the clear filtrate is evaporated in vacuo. The oily residue is crystallized from ethyl acetate. The precipitat¬ ed white crystals are filtered and dried at 40-50 C. Thus 20.85 g of the desired compound are obtained, yield 85 %. Mp.: 129-130 °C.

Examples 23-31

In an analogous manner to Example 12 the following isothiuronium compounds of the general Formula I are prepared:

23. N-aminoiminomethyl-S-ethyl-isothiuronium bromide, mp.: 123-124 °C;

24. N-aminoiminomethyl-S-isopropyl-isothiuronium bromide, mp.: 120-122 °C;

25. N-aminoiminoraethyl-S-n-butyl-isothiuronium bromide, mp.: 116-118 °C; 26. N-aminoiminomethyl-S-isoamyl-isothiuronium bromide,

mp . : 111-113 °C ;

27. N-aminoiminomethyl-S-/2-bromo-ethyl/-isothiuronium bromide, mp.: 129-131 °C;

28. N-aminoiminomethyl-S-/3-bromo-propyl/-isothiuronium bromide, mp.: 127-129 °C;

29. N-aminoiminomethyl-S-/3-hydroxy-propyl/-isothiuronium bromide, mp.: 132-135 C;

30. N-aminoiminomethyl-S-/3-mercapto-propyl/-isothiuronium bromide, mp.: 141-142 °C; 31. N-aminoiminomethyl-S-propargyl-isothuronium bromide* ^• mp.: 131-133 °C.

Examples 32-40

In an analogous manner to Example 20 the following compounds are prepared:

32. N-aminoiminomethyl-N'-methyl-S-/2-hydroxyethyl/-iso- thiuronium bromide, mp.: 147-148 °C;

3 . N-aminoiminomethyl- '-ethyl-S-/2-chloroethyl/-iso- thiuronium bromide, mp.: 143-144 C; 34. N-aminoiminomethyl-N'-ethyl-S-allyl-isothiuronium chloride p.: 138-139 °C;

35. N-aminoiminomethyl-N'-isopropyl-S-/3-chloropropyl/-iso- thiuronium bromide, mp.: 140-142 °C;

36. N-aminoiminomethyl-N'-isopropyl-S-methallyl-isothiuronium chloride, mp.: 138-139 °C;

37• N-aminoiminomethyl-N'-cyclohexyl-S-n-propyl-isothiuronium bromide, mp.: 133-135 °C; 38. N-aminoiminomethyl-N'-cyclohexyl-S-methallyl-isothiuronium chloride, mp.: 130-132 °C; 39. N-aminoiminomethyl-N'-cyclohexyl-S-glycidyl-isothiuronium bromide, mp.: 143-145 °C; 40. N-aminoiminomethyl-N'-cyclohexyl-S-benzyl-isothiuronium chloride, mp.: 147-149 °C.

The melting point and IR spectrum of the compounds of the general Formula I are summarized in the following Table VIII.

Table VIII

R- Op/ C/ Characterized peaks of IR spectrum

H methyl J 124-125 3300, 3120, 1685, 1570

H n-propyl Br 122-124 3300, 3120, 2925, 2 1685, 1570, 730 H allyl Br or CI 123-125 3290, 3260, 3160, 3105, 1640, 1605, 1550, 1375, 1090, 980, 910, 705, 650

H 2-chloroethyl Br 142-143 3300, 3110, 2925, 1 920, 720,

H l-chloro-2-hydroxy- Br 118-119 3300, 3110, 3000, -propyl 2850, 1685, 1610, 720

H 3-chloro-propyl Br 129-131 3300, 3275, 3165, 31 2900, 2825, 1680, 69

H 2-hydroxy-ethyl CI 134-136 3310, 3280, 3110, 3000, 2990, 1675, 1610,

H glycidyl Br 138-139 3300, 3275, 3110, 3000, 1690, 1600

865,

H benzyl CI 159-161 3290, 3280, 3115, 3005, 1680, 1590, 1485, 755

H ethyl Br 123-124 3300, 3120, 1685, 1570,

H isopropyl Br 120-122 3300, 3120, 1680, 1380, 1365

H n-butyl Br 116-118 3315, 3300, 3210, 3180, 2850, 1690, 14

Op/ C/ Characterized peaks of IR spectrum

H isoamyl Br 111-113 3305 , 3300, 3205,

3175 2960, 2845,

1695 1470

H 2-bromo-ethyl Br 129-131 3300 3205, 2910,

1670 , 1090, 715 H 3-bromo-propyl Br 127-129 3305i 3215, 2920,

1665 H 3-hydroxy-propyl Br 132-135 3590 , 3300, 3220,

2925 1680 H 3-mercapto-propyl Br 141-142 3320 3305, 3210,

3180 , 2920, 2570,

1690

H propargyl Br 131-133 3310 , 3300, 3200,

2180 1680 methyl allyl CI 137-139 3305 3290, 2960,

2690 1640 methyl 2-hydroxy-ethyl Br 147-148 3585 3300, 3285,

1680 1375, 1360 ethyl 2-chloro-ethyl Br 143-144 3305 3285, 2965,

1660 765 ethyl allyl CI 138-139 3310 3300, 3210,

3180 1650, 990,

905 isopropyl allyl CI 134-135 3305 3295, 3200,

3170 1655, 985, isopropyl methallyl CI 138-139 3305 3295, 3200,

3175 1650, 890 isopropyl 3-chloro-propyl Br 140-142 3320 , 3300, 3195,

3160 1690, 775 cyclohexyl allyl CI 129-130 3315 , 3305, 3200,

3170 . 2830, 1695,

985, 900 cyclohexyl n-propyl Br 133-135 3320 , 3300, 3210,

3175 , 2925

R' Op /°C/ Characterized peaks of IR spectrum

5 cyclo-hexyl methallyl CI 130-132 3315, 3300, 3215, 3180, 885 cyclnhexyl glycidyl Br 143-145 3325, 3315, 3210,

3190, 1690, 870 cyclohexyl benzyl CI 147-149 3300, 3275, 3120,

3000, 1675, 1590, 750.

Example 41

Preparation of tablets and dragees Tablets and dragees are prepared in pressing machine equipped with an automatic feeder. The active ingredient is previously granulated with suitable auxiliary agents by means of the dry or wet procedure. If wet granules are prepared, the granules are preferably dried in vacuo. The granules used for the preparation of tablets or dragee core are pressed through a suitable metal-free sieve. For the preparation of granules preferably a granule simplex composition according to the Phar acopiea or other suitable composition may be used. Tablets and dragee cores compris¬ ing 30 mg of the active ingredient are prepared and 70 mg of vehiculum /e.g. granulatum simplex/ are added.

Example 42 Preparation of capsules

Preferably automatic filling machines are used. The active ingredient is granulated with suitable auxiliary - agents /e.g. granulatum simplex/ or any other inert additive. The granules are brought to suitable particle size, sieved and filled into capsules. The active ingredient content of

the capsules amounts to 30 mg.

Examples 43

Preparation of powder ampouls No separate auxiliary agents are used in the preparation of the powder ampouls. The active ingredient content of the powder ampouls amounts to 10 and 20 mg, respectively. The solvent ampouls contain 1 or 2 ml of distilled water or a physiological saline solution, respectively. It is preferred to dilute with at least

100 ml of an infusion and to use the contents of the ampouls directly after dissolving.

Example 44 , N-/aminoimino ethy1/-S-metally1-isothiuronium-

-bromide

14.9 g N-/aminoiminomethyl/-thiourea carbonate are suspended in 50 ml of abs. ethanol. 16.1 g of metally1- -bromide are added and the reaction mixture is refluxed for 30 minutes. After filtration the solvent is distilled off in vacuo. 21 g of a honey like product are obtained which cristallizes on standing.

Example 45 N-/aminoiminomethyl/-N'-allyl-S-allyl-isothiuronium bromide

17.6 g N-/aminoiminomethyl/- '-allyl-thiourea carbonate are suspended in 50 ml of anhydrous ethanol. 14.5 g of allyl-bromide are added and the reaction mixture is refluxed while stirring intensively for 1 hour where¬ upon the mixture is filtered and the solvent is distilled off in vacuo. 23 g of an oily viscous product are obtained.

Example 46

N-aminoiminomethyl-S-allyl-isothiuronium chloride A mixture of 4.2 g of dicyane diamide, 20 ml of ethano saturated with hydrochloric acid and 3.7 g of allyl mercaptane is slowly heated to boiling under vigorous stirr¬ ing. The reaction mixture is refluxed for 3 hours and . cooled to 0 °C. The precipitated crystals are filtered off and washed with ethanol. Thus 7 g of the desired compound are obtained. Mp.: 125-127 °C.