Complex-Forming Ability and Antitubercular Activity of Azo Derivatives of p-Aminosalicylic Acid

L. P. Kulev, L. I. Ariston

Previously we [1] obtained a series of azo derivatives of isomeric aminosalicylic acids. It was of undoubted interest to determine the relationship between the complex-forming ability of these compounds and their antitubercular activity. The complex-formation reaction of the compounds obtained with salts of heavy metals was studied by the colorimetric method on an FEK-2 photocolorimeter. Microbiological studies were carried out at the Tomsk Scientific Research Institute of Vaccines and Sera by L. A. Egorina under the direction of Corresponding Member of the USSR Academy of Medical Sciences S. P. Karpov. The studies were conducted by the surface-film method on the “Akademiya” strain.

The complex-forming ability of the substances, as follows from the data presented in the table, depends primarily on the structural features of the compounds, on the nature and mutual arrangement of the substituents, and on the nature of the metal. The relative rate of the complex-formation reaction of azo derivatives is the higher, the more functional groups participating in the formation of inner-complex salts the compound contains.

Thus, in the series of azo derivatives of p-aminosalicylic acid with a naphthalene ring, the compound No. 5, obtained by coupling with 8-hydroxyquinoline, proved to be the most reactive toward copper and iron. Close to it in activity with these same metals is the azo compound containing a β-naphthol residue (1, Table 3). Conversely, the azo dye analogous to it in structure, with α-naphthol as the azo component (3), is somewhat inferior in this respect to both the first and the second azo compound.

Azo dyes in which, instead of hydroxyl, an ethoxy group stands in the para position to the azo group react with metal salts at almost the same rate (4).

The introduction of electronegative substituents into the molecule of the azo component (orthohydroxyquinoline or β-naphthol) markedly slows the rate of the complex-formation reaction (1 and 7; 5 and 6, table), although not with all metals.

These substituents exert the opposite effect when introduced into the benzene rings of the azo components. Thus, for example, azo compounds (9, 10, 11), which in addition to the hydroxyl group contain a nitro group or a halogen, react faster than (8). The introduction of alkyl radicals has an insignificant effect in phenolic derivatives of p-aminosalicylic acid (8 and 14, 15).

Among the azo derivatives of resorcinol that we studied, high activity was shown by (13), which has a hexyl radical in the benzene ring of the azo component, whereas the azo derivative without this radical (12) reacted with copper and iron salts much more slowly than all the other phenolic derivatives tested by us.

It is noteworthy that the azo derivatives of aminosalicylic acids in which the hydroxyl group is located adjacent to the azo group proved to be the most reactive toward nickel. This regularity is observed for almost all the compounds we studied, regardless of the nature of the azo and diazo components. According to Mackenzie’s data [2], this phenomenon is explained by the tendency of nickel to form inner-complex compounds.

An exception to this rule, however, is the azo compound with alpha-naphthol as the azo component (3), which, in the rate of complex formation with nickel, far surpasses most of the compounds we studied. The fact that the analogous azo derivative of p-aminosalicylic acid (4), containing an ethoxy group instead of a hydroxyl, reacts with nickel sulfate much more slowly indicates that it is precisely the hydroxyl group in the naphthalene ring that participates in complex formation with this metal.

This circumstance is further confirmed by the fact that with other metals (copper, iron) both compounds react at almost the same rate (Tables 3, 3, 4).

It is possible that the effect of the pH of the medium is manifested here, creating more favorable conditions for hydrolysis of the nickel salt, as a result of which the reaction proceeds with the formation of an Ar–OMe compound, as was established by V. I. Kuznetsov [3] in the study of inner-complex and cyclic salts. This probably also explains the analogous behavior, under the same conditions, of the resorcinol derivative of p-aminosalicylic acid (12). For the corresponding phenolic derivative (8), reduced activity toward nickel is observed, since this azo compound has a stronger acidic character in comparison with the compounds indicated above. Thus, the nature of the metal plays a very substantial role in the formation of inner-complex compounds of p-aminosalicylic acid and has a very noticeable effect on the rate of the complex-formation reaction. The similarity noted above in the behavior of azo compounds (3 and 4) with iron and copper salts, and the sharp difference in the behavior of the same compounds with nickel sulfate, point to the selective action of these metals with respect to the complex-forming groups of the azo derivative. In the formation of complex compounds with iron and copper salts, the adjacent hydroxyl and carboxyl groups predominantly take part, whereas with nickel salts it must be assumed that the hydroxyl groups located in the benzene or naphthalene rings of the azo component react mainly.

The mechanism of action of medicinal substances, especially in the living organism, is very complex and depends on many factors whose role has not yet been fully studied. Therefore it can hardly be stated with certainty which of them plays the primary role in this mechanism. However, in evaluating the physiological activity of a medicinal preparation, its chemical nature, including its complex-forming ability, must undoubtedly be taken into account.

The comparative complex-forming ability of the compounds that we studied and the stability of the complexes make it possible to draw certain conclusions concerning the relationship between these properties and the antibacterial activity of the substances. In the overwhelming majority of cases, the antibacterial activity of azo derivatives of aminosalicylic acids in experiments “outside the organism” is directly dependent on the rate of complex formation and on the stability of the complexes formed, although this dependence cannot be expressed quantitatively. As an illustration, we present the following data on the comparative complex-forming ability and antitubercular activity of azo derivatives of p-aminosalicylic acid with the azo components: 8-oxyquinoline (1), hexyl-

resorcinol (2), alpha-naphthol (3), alpha-ethoxynaphthol (4), thymol (5), resorcinol (6), presented in Table 1.

Table 1

The most active against tubercle bacteria proved to be the azo derivative of hydroxyquinoline, and the least active—the azo derivative of resorcinol; the corresponding values characterizing their complex-forming ability also coincide for these compounds.

The existence of a definite relationship between antitubercular activity and complex-forming ability can also be judged from the results of our experiments with physiologically well-studied medicinal preparations, “PASK,” “Tibon,” and others.

This relationship for these substances is presented in Table 2.

Table 2

It is interesting to note that, among the isomeric aminosalicylic acids, p-aminosalicylic acid (1) has the greatest complex-forming ability; as is known, it far surpasses 3- and 5-aminosalicylic acids (2 and 3) in antibacterial respect.

Also noteworthy is the circumstance that, in terms of complex-forming ability, thiosemicarbazone of salicylaldehyde (4) occupies first place among the substances indicated in the graph. According to Donovick [4], this compound exhibits high antibacterial activity outside the organism, but has almost no effect in the living organism. It is possible that such a sharp difference in the antibacterial activity of thiosemicarbazone of salicylaldehyde depending on the experimental conditions is explained by its tendency toward rapid formation of complex compounds with metals present in the macroorganism itself. According to our data, this compound has the greatest complex-forming ability with respect to heavy metals and forms more stable complexes with them than “Tibon” and “PASK,” i.e., upon combining with metals in the living organism it may immediately lose its activity. The above-mentioned difference in the antitubercular activity of thiosemicarbazone was also observed for other substances by many authors, who, however, did not take into account the complex-forming ability.

Table 5

compounds and the properties of the metal-containing complexes formed by them. Speaking of the role of complex-formation reactions in the mechanism of antitubercular action, we do not limit this role to the action of substances only on the metals present in the pathogen itself. It seems quite probable to us that the chemical activity of antitubercular agents is closely connected with their stimulating effect on the protective forces of the organism, which may also be due to the formation of unstable complexes with metals that are constituents of living tissues.

We do not exclude the possibility that this is precisely what explains the interesting observations of F. V. Shebanov, E. F. Sidelnikova, R. I. Rozina, and N. P. Skakun [5, 6, 7] on the stimulating action of “PASK” on the liver of patients with tuberculosis.

The results of the colorimetric and microbiological studies we carried out are presented in Table 3.

Experimental Part

For a comparative assessment of the rate of complex formation of azo dyes with heavy metals, we used the following procedure: into four flasks were introduced 20 ml each of a 0.001 M solution of the substance under investigation in a 0.125% solution of caustic soda; 1 ml of a 0.01 M solution of a copper salt (or of other salts—nickel, iron) was added. The flasks were connected to a reflux condenser and placed in a boiling water bath. After 10–12 minutes, after a constant temperature of the reaction mixture had been established, the first flask was removed from the bath, rapidly cooled with cold water, and the contents of the flask were filtered. The filtrate was acidified with 1 ml of 0.5 N sulfuric acid. The precipitated azo dye was filtered off, and the metal remaining in solution, which had not reacted with the compound under investigation, was determined colorimetrically by the dimethylglyoxime method for nickel and by sodium diethyldithiocarbamate for copper and iron.

From calibration curves previously constructed for these metals, the percentage content of metal bound by the azo dye was determined. After 60, 120, and 180 minutes from the start of the first analysis, analogous determinations were carried out for the subsequent samples. The determination data are given in summary Table 3.

Conclusions

1. Experiments were carried out to determine, by a colorimetric method, the comparative complex-forming ability of azo derivatives of aminosalicylic acid. It was found that the properties of metal-containing complex compounds depend on the structural features of the azo derivatives, the nature of the metal, and the pH of the medium. The rate of the complex-formation reaction of the azo derivatives studied depends primarily on the presence in the molecule of active complex-forming groups and on their mutual arrangement.

2. It was established that between the complex-forming ability of the substances and their antibacterial activity in “in vitro” experiments there exists a direct relationship, which, however, cannot be expressed in strictly quantitative terms. The most active antibacterial among the azo derivatives of para-aminosalicylic acid proved to be the azo derivative with orthooxyquinoline as the azo component, and the least active—the azo dye obtained from the same acid and resorcinol. The complex-forming ability of these azo compounds was greatest for the former and least for the latter.

1. It was found that the highest rate of the complex-formation reaction corresponds also to the known active antitubercular drugs themselves: p-aminosalicylic acid (“PASK”) and thiosemicarbazone of p-aminobenzaldehyde (“Tibone”).

2. On the basis of experimental data on determining the complex-forming ability of the substances and the stability of the complexes formed by them, considerations are put forward concerning the mechanism of action of antitubercular drugs that exhibit different antibacterial activity in the living organism and outside it.

REFERENCES

1. Kuznetsov L. P., Aristov L. I. Azo derivatives of aminosalicylic acids and their inner-complex salts. Izvestiya Tomskogo politekhnicheskogo in-ta, vol. 102; the article is placed in this collection.
2. N. Mackenzie et al. Khimiya i khimicheskaya tekhnologiya, 4, 209, 1953.
3. Kuznetsov V. I. ZhOKh, 20, 176, 1950.
4. R. Donowick et al. J. Bact., 59, 667, 1950.
5. Shebanov F. V. Problemy tuberkuleza, 5, 22, 1950.
6. Smelyannikova E. F. and Rozina R. I. Problemy tuberkuleza, 3, 43, 1952.
7. Saksun N. P. Byulleten’ eksperimental’noi biologii i meditsiny, 10, 45, 1952.

CORRECTIONS AND ERRATA

Izv. TPI, vol. 102.