COMPLEX COMPOUNDS OF TRYPAFLAVINE AND THEIR ANTI-BRUCELLOSIS ACTIVITY

L. P. Kulev, A. G. Pechenkin

Among acridine derivatives, trypaflavine (10-chloromethyl-3,6-diaminoacridine) stands out as a preparation with a broad bactericidal spectrum. It is considerably more vigorous than sulema; it acts on diphtheria bacilli, streptococci, staphylococci, meningococci, and other microbes [1]. The toxicity of trypaflavine is relatively low.

In 1926 Izar established that trypaflavine, outside the organism, kills cultures of brucellae at a dilution of 1:500,000–1:1,000,000. However, in the treatment of brucellosis animals this preparation in most cases proved to be insufficiently effective.

Among therapeutically active preparations obtained on the basis of trypaflavine, compounds of complex character are of particular interest. Thus, the preparation “septacrol” [2] was proposed long ago; it is a complex compound of nitrate of 2,7-dimethyl-3,6-diamino-10-methylacridinium with silver nitrate.

This preparation is more active than trypaflavine, but is poorly soluble in water. Later septacrol was proposed [3] in a new modification in the form of a solution of the complex salt of 3,6-diamino-10-methylacridinium lactate and silver lactate. In the Soviet literature this preparation is described under the name “flavargin” [4]. Flavargin has been used in clinics and has been recognized as a highly active bactericidal and chemotherapeutic agent [5].

In 1946 A. M. Grigorovskii and T. A. Veselitskaya [6] isolated the complex salt of flavargin in dry form and named it “dry flavargin,” which has been successfully used intravenously for septic diseases, endocarditis, arthritis, cystitis, etc. However, in brucellosis, flavargin and preparations analogous to it apparently have not been tested.

The high antimicrobial activity of complex compounds of this type is due to the presence in them of two strong bactericidal substances—trypaflavine and a silver salt.

It is probable that, upon cleavage of the complex in the organism, the components entering into its composition are liberated, and each of them manifests its own action.

Taking into account the high physiological activity of complex compounds of the above-indicated type, we set ourselves the task of broadening the scope of investigation in this series. For this purpose we obtained and tested several complex compounds of trypaflavine of the general formula

In choosing the second component (\(\mathrm{MeX}\)) of the complex, its capacity for complex formation with heterocyclic amines and its anti-brucellosis activity were taken into account.

Our considerations were based on the fact that free trypaflavine, through its active amino group, probably interacts with substances of the macroorganism, as a result of which it is inactivated before it has time to display its strong antimicrobial properties. In the bound state, i.e., with the amino group protected in one way or another, it loses its antimicrobial activity and regains it in the organism only after decomposition of the complex compound. The most favorable therapeutic effect in this case may be achieved at an optimal rate of hydrolysis or cleavage of the preparation.

To protect the amino group of trypaflavine, we used the complex-formation reaction, which makes it possible to obtain a large number of diverse compounds with different hydrolytic stability. In most cases the complex compounds were obtained in the form of crystalline precipitates by interaction of concentrated aqueous or alcoholic solutions of trypaflavine with the corresponding double or simple metal salts.

The starting product—10-chloromethyl-3,6-diaminoacridine—was obtained by Bend’s method [7], by methylation of the medicinal preparation proflavine, followed by drying of the product at \(110\)—\(115^\circ\) for 24 hours and recrystallization from alcohol. The composition of the complex compounds was determined by chemical and physicochemical methods (elemental analysis, electrical conductivity, and colorimetry), which gave well-concordant results.

The electrical conductivity of the systems was determined according to Kohlrausch’s scheme in a \(TC\)-15M thermostat; the optical density of the systems was determined with an “FEK-2” electrophotocolorimeter.

All microbiological and pharmacological tests of the synthesized preparations were carried out at the Novosibirsk Scientific-Research Veterinary Experimental Station (NIVOS) by K. F. Lamikhov and S. S. Nazarov.

The list of synthesized preparations, the results of their analysis, and their activity against various strains of brucellae outside the organism are given in Table 1.

In experiments outside the organism, the greatest activity against brucellae of the goat and bovine types was shown by complex compounds of trypaflavine with sodium iodide (No. 12), with potassium iodide (No. 11), with mercuric iodide potassium (No. 15), and with acid mercuric iodide potassium (No. 50A).

From the results of microbiological tests outside the organism it is evident that not a single one of the preparations we obtained showed any appreciable increase in activity in comparison with trypaflavine itself, whose anti-brucellosis action in our experiments was \(1:500{,}000\).

On the contrary, in the overwhelming majority of cases a sharp decrease in activity was obtained, which is in accordance with the considerations we have expressed regarding the inactivation of trypaflavine when one or both amino groups are protected.

It was noted above that the most favorable therapeutic effect, in our opinion, will be achieved at an optimal rate of hydrolysis (cleavage) of the preparation inside the organism. Therefore, along with microbiological tests of the synthesized substances, we determined the comparative hydrolytic stability of some of them by the electrical-conductivity method.

Table 1

The rate of hydrolysis is characterized by the increase in the electrical conductivity of the solutions over time at constant temperature (Fig. 1).

Fig. 1

1. Dependence of anti-brucellosis activity on the rate of hydrolysis of preparations (percent increase in electrical conductivity).

Curve I—prep. No. 12—bacteriostatic effect at a dilution of 1:500,000; II—prep. No. 11—1:400,000; III—prep. No. 15—1:400,000; IV—prep. No. 23—1:200,000; V—prep. No. 50A—1:200,000; VI—prep. No. 50—1:200,000; VII—prep. No. 24—1:100,000; VIII—prep. No. 57—1:200,000.

It is evident from Fig. 1 that among complex compounds of the same type (11 and 12; 23 and 24; 15 and 57; 50 and 50A), the preparations having a higher rate of hydrolysis also possess greater antibacterial activity.

Some of the compounds we obtained were tested on guinea pigs infected with brucellosis, and the activity of these compounds manifested itself differently, depending on their hydrolytic stability. Thus, the relatively readily hydrolyzed preparations Nos. 11, 12, and 50A proved effective in the acute form of brucellosis and only slightly active in the chronic form. Conversely, preparations that are difficult to cleave, for example Nos. 24, 57, etc., gave a positive result in the treatment of chronic brucellosis. It should be noted that the toxicity of the preparations tested is 5–10 times lower than the toxicity of trypaflavine itself.

Experimental Part

I. Preparation No. 23. To 4.96 g of hyposulfite dissolved in 20 ml of water, 1.43 g of freshly precipitated silver chloride is gradually added. The resulting clear solution of silver hyposulfite is gradually added, with vigorous stirring, to a solution of 5 g of trypaflavine in 100 ml of water. The complex immediately precipitates in the form of orange crystals with m.p. 215°. The yield is quantitative. The product is poorly soluble in water and alcohol.

II. Preparation No. 24. To a solution of 2 g of potassium rhodanide in 5 ml of water, 1.65 g of silver rhodanide is added in small portions. The resulting solution is poured dropwise, with vigorous stirring, into a solution of 5 g of trypaflavine in 100 ml of water. The complex compound immediately precipitates in the form of orange crystals. After recrystallization from hot water, the product has m.p. 135°. Yield 4.7 g (90%).

III. Preparation No. 22. 3.32 g of ignited potassium iodide is dissolved in 10 ml of water. To the resulting solution, 2.37 g of silver iodide is added in small portions. The final portions of silver iodide dissolve upon heating. The solution thus obtained is added dropwise, with vigorous stirring, to a solution of 5 g of trypaflavine in 200 ml of water. In this process the complex precipitates in the form of orange-colored crystals; thoroughly washed with hot water and dried at 100–105°, it has m.p. 233–235°. Yield 7.4 g.

IV. Preparation No. 59. A solution of 3.66 g of cadmium iodide in 50 ml of water is added, with vigorous stirring, to a solution of 2.5 g of trypaflavine in 100 ml of water. Orange crystals (from water), m.p. 148–150°. Yield 4.97 g (82%).

V. Preparation 13. Obtained analogously to IV from a solution of 5.9 g of bismuth iodide in 300 ml of alcohol and a solution of 2.5 g of trypaflavine in 50 ml of alcohol. Quantitative yield. Insoluble in water and alcohol. Decomp. t. 285–290°.

VI. Preparation No. 31. Obtained analogously to IV from a solution of 5.03 g of mercuric iodide in 100 ml of 20% tartaric acid and a solution of 2.5 g of trypaflavine in 50 ml of water. Yield 7.0 g (94%). Red crystals, moderately soluble in water. M.p. 195°.

VII. Preparation No. 15. Obtained analogously to III from a solution of 7.86 g of mercuriodide potassium in 10 ml of water and a solution of 2.5 g of trypaflavine in 100 ml of water. Quantitative yield. Yellow crystals, poorly soluble in water. M.p. 210°.

VIII. Preparation No. 57. Obtained analogously to II from a solution of 6.16 g of mercurorhodanide potassium in 20 ml of water and a solution of 2.5 g of trypaflavine in 100 ml of water. Yield of the product, recrystallized from 40% aqueous alcohol, 5.55 g (85%). Red powder. M.p. 175°.

IX. Preparation No. 50. To a solution of 5 g of trypaflavine in 50 ml of water, with stirring, a solution of 5 g of acid potassium pyrosulfurous mercurate in 200 ml of water is added. On standing, a precipitate of the complex compound gradually forms. Upon recrystallization from a small amount of water, red crystals with m.p. 160° are obtained. Yield 3.29 g (36%).

X. Preparation No. 50 A. To a solution of 2 g of preparation No. 50 in 50 ml of water, a solution of 1 g of potassium iodide in 20 ml of water is added. Red-brown crystals (from water) with m.p. 245–247°. Yield 1.98 g (87%).

XI. Preparation No. 11. Obtained analogously to IV from a solution of 5 g of potassium iodide in 20 ml of water and a solution of 2.4 g of trypaflavine in 50 ml of water. The complex precipitates in the form of orange-red crystals, which, after repeated recrystallization from water, have decomp. t. 240–242°. Yield 1.9 g.

XII. Preparation 12. Obtained analogously to XI from a solution of 5 g of sodium iodide in 20 ml of water and a solution of 2.4 g of trypaflavine in 50 ml of water. Decomp. t. 243–245°. Yield 2.1 g.

Conclusions

1. Some complex compounds of trypaflavine have been obtained, their composition has been determined, and microbiological tests have been carried out on various strains of brucellae of the goat and bovine types. It was found that the majority of the complexes obtained have a composition of 1:1.

1. The relative hydrolytic stability of the compounds was determined by the electrical-conductivity method. It was shown that the anti-brucellosis activity of these compounds depends on their composition and hydrolytic stability. The less stable compounds show activity in the acute form of experimental brucellosis, while the more stable ones do so in the chronic form.

2. Considerations are advanced concerning the inactivation of trypaflavine within the organism and ways of eliminating it by protecting the amino group. It has been established that, in a number of cases, complex formation is accompanied by a considerable reduction in the toxicity of trypaflavine and of the second component of the complex associated with it.

REFERENCES

1. Mankovskii M. D., Medicinal Agents, Medgiz, 1955.
2. S. Browning, J. Cohen. — Brit. Med. J. II, 695, 1921.
3. G. P. No. 619067, No. 644646. Frdl., 22, 534—536, 1939.
4. Proceedings of the Pharmacopoeial Committee of the Scientific Medical Council, issues 1 and 2, 90, 1940.
5. Mankovskii M. D., Farmatsiya, 4, 41, 1941; Anbel’ S. I., Klinicheskaya meditsina, XX, 1—2, 89, 1942.
6. Griporovskii A. M., Veselitskaya T. A., Farmatsiya, 6, 1946.
7. L. Benda. — Berichte, 1913, 45, 1787.