PHARMACOLOGICAL STUDIES OF SOME ORGANIC THIOCYANATES

YASUO YOKOI

Department of Pharmacology, Faculty of Medicine, University of Tokyo and the National Hygienic Laboratory, Tokyo

Received for publication October 19, 1953

Strikingly rapid toxic symptoms of organic thiocyanates were repeatedly ob served in various kinds of animals by Traubmann (1930)(1), von Oettingen et al. (1936)(2), Cameron et al. (1939)(3), Ohashi and Kimizuka (1947)(4), though not confirmed by Moriki (1936)(5). Such a violent action as above, however, can not be expected in regard to inorganic thiocyanates, such as KSCN, clinically available as a sedative or a blood pressure depressant. The SCN radical has a close similarity in its chemical property to halogens, which may be named "pseudohalogen". Then, Traubmann naturally presupposed an ethyl chloride C2H5Cl-like narcotic action as a pharmacological property of alkyl thiocyanates, i.e. methyl thiocyanate CH3SCN, ethyl thiocyanate C2H5SCN, or propyl thiocyanate C3H7SCN. But it was found that the animals poisoned either by these alkyl thiocyanates or by aromatic thiocyanates (phenyl thiocyanate C6H5SCN and aniline thiocyanate NH2C6H4SCN) caused respiratory excitement and convulsions. Thus he came to a postulation for the toxic particularity of organic thiocyanates. Von Oettingen and others noticed the following phenomena : hyperemia, hemor rhage and edema in internal organs, when animals succumbed to the acute poison ing by lower alkyl thiocyanates, respiratory excitement and a rise in blood pres sure when methyl or ethyl thiocyanate was administered subcutaneously ; appear ance of a AgNO3-consuming substance in vitro when these thiocyanates were placed for 48 hours with pieces of liver. These observations gave the impression that hydrocyanic acid might be liberated from methyl and ethyl thiocyanates in living animals. In order to scrutinize more carefully the above-mentioned assumption, the present study was designed with six compounds of organic thiocyanates* as listed in Table 1.

*The author owes the preparation of these compounds to Dr . T. Itai in the National Hygienic Laboratory, Tokyo TABLE 1. Organic thiocyanates adopted for experiments

METHODS AND RESULTS

Distilled water was used to solve inorganic cyanides and ethyl thiocyano acetate, and olive oil or 50 per cent ethylenglycol monoethyl ether to the other compounds. Their respective solubilities are shown in Table 1. Lethal doses were expressed in gram per kilogram and also in millimol per kilogram. From the figures of the molecular lethal dose of various compounds in question, their lethal effect was calculated, as expressed by the ratio of the molecular lethal dose of each substance divided by that of HCN.

I. Systemic symptoms and taxi ci ti es

Toxic symptoms : When ethyl thiocyanoacetate or potassium cyanide was ad ministered through three routes to hundreds of mice and some rabbits and pige ons, no particular difference was noticed between the toxic symptoms developed respectively by them: marked respiratory stimulation, heavy convulsions and then respiratory failure along with general paresis. With other organic thiocyanates, especially when administered intraperitoneally, the symptoms yielded were nearly same as mentioned above, although the convulsive manifestations were less mar ked in most cases when administered subcutaneously. In regard to the halogenated organic compounds, in which SCN radical of the mother thiocyanates was substituted with a halogen, the toxic symptoms became different losing a cyanide-like symptom : chloro-, bromo or iodobenzene appeared to have some toxic resemblances in their symptoms to carbolic acid, and ethyl iodide yielded such narcotic effect as alchol or ether, and thymol chloride caused a toxic symptom like that of thymol itself. Ethyl monochloroacetate was excep tional, because it resembled to cyanide in toxic symptom, when administered in much larger dose than that of ethyl thiocyanoacetate. Lethal dose : The molecular lethal doses of organic thiocyanates, indeed, varied with species of animal or method of administration, but they tended to approach to one in their ratios to that of hydrocyanic acid, especially when they were ad ministered intraperitoneally as shown in Table 2. On the contrary, the halogena ted corresponding compounds were much larger in their ratios to cyanide, although they had many physical properties common to their mother compounds and were prepared for testing in a solvent common to the mother ones.

IL Respiratory movement and blood pressure

Organic thiocyanates and sodium cyanide were administered into ear vein of rabbit, about 2 Kg. of body weight, anesthetized with urethane. Kymographic tracings of respiratory movement were recorded by a Marey's tambour connected to tracheal canula, and blood pressure by an ordinary mercury manometer at common carotid artery.

TABLE 3. Effects of cyanide and organic thiocyanates on respiratory movement of rabbit Ethyl thiocyanoacetate for testing was dissolved in distilled water, and other organic thiocyanates were all in 50 per cent ethyl o.nglycol monoethyl ether. The effect of organic thiocyanates was proved quite similar to that of sodium cyanide (Fig. 1). As to the respiratory movement, the only finding with smaller dose was of excitatory one and the movement ceased transiently for several minutes when used in more dose, resulting in its standstill in a final large dose (Table 3 and Fig. 1, A).

FIG. 1 A. Effect of intravenous phenyl thiocyanate on respiration of urethanized rabbits.

There was no marked dfference between the toxicity of sodium cyanide and that of organic thiocyanate ; the doses causing the transient cessation of the movement were determined by intravenous administration as shown in Table 4: 0.02 millimol per Kg. of body weight for ethyl thiocyanoacetate, phenyl, benzyl, and thymol thiocyanates as well as sodium cyanide respectively , and 0.04 millimol for ethyl thiocyanate. Regarding the blood pressure, the effects appeared more complicated than the above and yet there was no remarkable difference between sodium cyanide and organic thiocyanates. With smaller dose, in the above-mentioned stage o respira tory excitement, an initial rise in blood pressure was mostly followed by a tran sient fall even below the starting level and then a second rise in the pressure . When used in larger dose, blood pressure fell rapidly and respiration stopped long before the heart did (Fig. 1, B).

TABLE 4. Intravenous millimol doses causing transient cessation of respiration*

FIG. 1 B. Effect of intravenous phenyl thiocyanate on blood pressure of urethanized rabbits

It was thus revealed that organic thiocyanates and sodium cyanide acted alike qualitatively upon respiration and blood pressure and also quantitatively upon the former. As for halogen compounds, ethyl monochloroacetate was adopted for testing in place of ethyl thiocyanoacetate, chlorobenzene for phenyl thiocyanate and thymol chloride for thymol thio;yanate. These compounds caused no remarkable change in respiratory movement but only a transient fall in blood pressure, when administered to rabbits intravenously even in the dose with which thiocyanates would cause noticeable effects.

III. Gas metabolism

fin order to analize the air expired from rabbit under the influence of organic thiocyanates or potassium cyanide, in a rabbit, comparatively deeply anesthetized with urethane, one end of a T-shaped glass-tube was inserted into the trachea. This T-canula was provided with an inflow valve at one of the other ends to conduct fresh air into lungs and an outflow valve at another end letting expired air out. The expired air was caught in a Douglass' bag with every 10 minutes frac

TABLE 5. VoI ime of oxygen consumption and expired air of rabbits intoxicated by cyanide and organic thiocyanates tion ; its volume was measured by a gas-meter (Table 5) and the percentages of its components were obtained by a modified Haldane's gas-analyser(6) and then calculation showed the volumes of oxygen consumed (Table 5) and of carbon dio xide produced. When organic thiocyanates and potassium cyanide were given in the doses listed in Table 5, a rapid and marked decrease of oxygen consumed and a decre ase of carbon dioxide produced were both proved in all samples, and the expired air did not change pararell to them; in fact, it increased mostly very much, resul ting in a consequent hyperpnea and in some cases convulsions too. All changes in volume as observed above were recovered as the poisoning subsided. The organic thiocyanates' doses causing actually such an impressive phenomenon were nearly same as in case of cyanide : 0.07 millimol per kilogram with KCN, ethyl thiocyanoacetate and benzyl thiocyanate, respectively, 0.07 to 0.11 millimol with phenyl thiocyanate and 0.19 millimol with ethyl thiocyanate. IV. Antidotes

Sodium thiosulfate and nitrite, well-known antidotes against cyanide poison ing, were tried respectively as a treatment to the poisoning of organic thiocyanates.

TABLE 6 A. Effect of sodium thiosulfate pretreatment on de-ith-rate of mice h poisoning of organic thioz yanates and other chemicals (1) Sodium thiosulfate The experimental method for the organic thiocyanates was designed in accor dance with the R. Hunt's antidotal experiment(7) in which sodium thiosulfate was used against "cyanide" poisoning: first, 10 per cent sodium thiosulfate solution, 0.5 cc. per animal, was administered subcutaneously on the ventral part of a mouse, and then, about 10 minutes later, a certain dose of one of the organic thiocyanates for testing was given subcutaneously on its dorsal part. The death-rate caused by all these compounds but thymol thiocyanate fell down under the effect of thio sulfate, as shown in Table 6, A. As for thymol thiocyanate, the time of death was prolonged under thiosulfate. Thus sodium thiosulfate was proved effective in counteracting the poisoning of the compounds of the above series including the last one. When SCN group of organic thiocyanates was substituted by halogen, the death-rate of these halogen-containing compounds became, contrarily, higher under the influence of thiosulfate. (2) Sodium nitrite Sollmann and Hanzlik's experimental method(8) regarding to the antidote sodium nitrite against '-cyanide" poisoning, was adopted in cases of the organic compounds : I per cent sodium nitrite solution, 2 cc. per Kg. of body weight, was administered into a pigeon's vein, and just before or after, one of the organic thiocyanates for testing was intramuscularly given. The antidote against cyanide was proved effective to counteract these poisons as shown in Table 6, B.

TABLE 6 B. Effect of intravenous sodium nitrite treatment on intramuscular toxicity of organic thiocyanates in pigeons

One of the organic thiocyanates to be tested, 0.1 or 0.5 Gm., and blood of rab bit, about 50 Gm., were placed together in vitro and in order to avoid bubbling 0.2 to 0.4 Gm. of cityl alcohol was added to the mixture, which was stirred by a motor-driven agitator for one hour at 38'C and then distilled at 50 to 60 C in carbon dioxide gas: the receiver filled with distilled water was cooled with ice from its outside. Chemical reactions for hydrocyanic acid out of the sample-fluid were obtained as shown in Table 7: Prussian blue reaction was positive in case of all samples except p-thiocyano.dimethyl-aniline, and Schonbein's and AgNO3 reaction were both positive with all samples. As for the control experiment the chemical reactions for hydrocyanic acid were negative either for sodium thiocyan ate added with blood or for organic thiocyanates without blood.

TABLE 7. Decomposition of organic and inorganic thiocyanates in vitro, (stirred at 33CC for one hour), and identification of CN, by chemical reactions

To determine how much the organic compounds were to be decomposed, or ganic thiocyanate, 0.02 to 0.03 Gm., and blood of rabbit, about 50 Gm., were pla ced in vitro for one hour in such conditions as mentioned above : the gas was liberated from the mixture by carbon dioxide gas and transferred into a silver nitrate solution. The quantity of silver nitrate consumed was determined by means of a back titration with ammonium thiocyanate and the production of hydrocyanic acid was estimated from these readings. Assuming that these or ganic thiocyanates liberate one molecule of hydrocyanic acid per molecule of the compound in question, the decomposition will run up to about 80 per cent in case of ethyl thiocyanoacetate, 30 per cent or so in phenyl and benzyl thiocyanate, and 10 per cent or so in thymol thiocyanate, as shown in Table 8. The percen tages of those compounds decomposed without blood were actually measured as about one tenth as compared with the samples to which blood was added.

TABLE S. Percentage of decomposed organic and inorganic thiocysnates in vitro, stirred at 3S 'C for one hour

It was found that fine black particles were slightly produced in silver nitrate solution during the above procedure determining the decomposition of organic thiocyanates to which blood was added, and in case of ethyl thiocyanoacetate, a lead acetate paper turned into brown, indicating that these organic thiocyanates can possibly produce not only hydrocyanic acid but also hydrogen sulfide or the like. Furthermore, the above experiments disclosed that sodium sulfide was to be easily decomposed up to 95 per cent or more and on the other hand sodium cyanide was rather too stable to be decomposed more than 20 to 30 per cent at most.

VI. Additional Sulfides : Hydrogen sulfide and cyanide resembled each other in general toxic symptoms(9), and sodium sulfide had the similar effect on respiratory movement and blood pressure to cyanide(10). The toxicity of sodium sulfide, however, was to be regarded in a different light, for the ratio of its molecular lethal dose to that of cyanide was 22: 1 when administered subcutaneously, and was 13: 1 when given intraper itoneally (Table 2), and the treatment with sodium thiosulfate, the antidote against cyanide, was proved harmful to the poisoning of sodium sulfide , although the treatment was evidently effective against that of organic thioc yanates (Table 6, A). Now it should be concluded that the toxicity of these organic thiocyanates is substantially due to the action of hydrocyanic acid even when HCN and H2S are both liberated from the organic compounds . Inorganic thiocyanate : Toxic symptoms of sodium thiocyanate began to appear usually later as compared with cyanides or organic thiocyanates, and they con sisted of spastic paresis, stiff and clumsy in motions , hypersensitive to external stimuli, e.g. sound. This compound was really less toxic ; 55 times larger in mo lecular lethal dose than hydrocyanic acid when administered subcutaneously, and 64 times larger when given intraperitoneally (Table 2). The treatment with so dium thiosulfate was proved rather harmful to the poisoning of this compound (Table 6, A). Thus it was disclosed that inorganic thiocyanate such as NaSCN was entirely different from organic thiocyanates or cyanides. To the author's interest, Zeze(10) stated that the effect of NaSCN on respiratory movement and blood pressure was different from that of NaCN.

SUMMARY

Comparative studies on six representative organic thiocyanates with hydrocy anic acid were carried out from the pharmacological or toxicological point of view. 1. The toxic symptoms of the organic thiocyanates in mice, rabbits and pige ons bore a good likeness to those of cyanide. The 50 per cent molecular lethal doses of the organic compounds came mostly near to cyanide, especially when administered intraperitoneally. 2. The feature in kymographic tracings of respiratory movement and blood pressure obtained under the intravenous administration of the organic thiocyana tes was, for most of them, almost alike to that of cyanide ; a transient cessation of respiratory movement with 0.02 millimol per Kg. of body-weight. 3. When the organic thiocyanates were Administered subcutaneously to rabbits, the oxygen consumption decreased markedly in most cases, while on the contrary the volume of air expired from lungs much increased. Seemingly paradoxical

phenomenon of this sort, characteristic to the cyanide poisoning, was produced by most of them in the nearly same molecular dose with potassium cyanide. 4. Sodium thiosulfate and nitrite, representative antidotes against cyanide poisoning, relieved successfully mice and pigeons from death in spite of the lethal doses of organic thiocyanates. Unlike inorganic thiocyanates or organic halogen compounds, organic thiocy anates had a close resemblance to cyanides in their pharmacological or toxicolo gical properties. 5. It was identified in vitro by Prussian blue reaction and other two chemi cal reactions that hydrocyanic acid could be liberated more or less from all samples of the organic thiocyanates except one, so long as they were mixed with blood. The results obtained thus in vitro as well as in vivo were indicative of a pos sible liberation of hydrocyanic acid from organic thiocyanates, and therefore the author came to the conclusion that the toxicity of these organic compounds should be originated by the hydrocyanic acid liberated from the compounds. Acknowledgements Sincere appreciations are due to Prof. Y. Kebayashi for his kind guidance.

REFERENCES

1) TRAUBMANN. G.: Arch. exper. Path. u. Pharmckol. 150, 257 (1930) 2) VON OETT1NGEN, W. F., HUPER, W. C. AND DEICHMANN-GRUEBLER, W.: J. Industr. Hyg. 18, 311 (1936) 3) CAMERON, G. R., DONINGEN, C. R. AND MCKENNEY HUGHES, A. W.: J. Path. & Bact. 49 304 (1939) , 4) OHASHI, S. AND KIMIZUKA, M.: Unpublished (1947) 5) MORIKI, H.: Folia pharmacol. japon. 22, 157 (1936) 6) NUMAJIRI, K,: J. Science of Lcbour 23, No. 4, (19 ) 7) HUNT, R.: Arch. internat. pharmccodyn. 12, 449 (1904) 8) SOLLMAN, AND HANZLIK, F. J.: Fundamentals of Experimental Pharmacology 198 (1939) 9) POHL, J.: Heffiers Hanabuch 3, 433 (1927) 10) ZEZE, M.: Folia pharmacol. japon. 33, 201 (1941)

ADDENDUM The preliminary report was presented at the 2nd Regional Meeting of the Kanto Branch of the Japanese Pharmacological Society on June 6, 1950. I am much obliged that the reports concerning the conversion of inorganic thiocyanate to cyanide were just in time for printing this English report through the kindness of the following authors :

GOLDSTEIN, F. AND RIEDERS, F.: Am. J. Physiol. 167, 47 (1951) BOXER, G. E. AND RICKARD, J. C.: Arch. Biockem. & Biophysics 39, 7 (1952) PINES, K. L. AND CRYMBLE, M. M.: Proc. Soc. Exper. Biol. & Med. 81, 160 (1952) GOLDSTEIN, F. AND RIEDERS, F.: Am. J. Physiol. 173, 287 (1953)