[In vitro antimycobacterial activities of pyrazinamide analogs: results of screening tests]

Attempting to find new drugs, which are more effective than pyrazinamide against Mycobacterium tuberculosis and also active against M. avium complex (MAC), we synthesized various pyrazinamide analogs and pyrazine derivatives and assayed their antimycobacterial activities in vitro against M. tuberculosis, M. avium and M. intracellulare. As is well known, pyrazinamide is more active in acidic medium than in neutral medium, but the growth of mycobacteria in acidic media is poor and inconsistent. Our preliminary experiments revealed that the relative antimycobacterial activities of test-drugs compared with pyrazinamide were essentially the same in pH5.5 medium as in pH6.0 medium. Therefore, Middlebrook 7H9 broth adjusted to pH6.0 was used throughout the present studies. Among 39 compounds synthesized, four drugs were insoluble in any of the solvent suitable for culture experiments and could not be tested, and remaining 35 compounds were screened. The growth of mycobacteria was followed by measuring the optical density at 530 nm (OD), and the OD of the culture in the presence of 200 micrograms/ml of the test-drug (OD-TD) was compared with that in the presence of pyrazinamide (OD-PZA). Each test-drug was ranked as A, B, C, D or E according to the ratio (OD-TD/OD-PZA) x 100%, if the ratio was equal to or less than 10%, 11-20%, 21-40%, 41-60% or 61-80%, respectively. Any drugs showing the ratio above 80% were excluded from further examinations. For M. tuberculosis, 11 drugs were ranked as A and 4 more as B. For M. avium, 2 drugs were ranked as A and 2 more as B. For M. intracellulare, 5 drugs were ranked as A and 2 more as B. Among highly ranked ones, 4 compounds, namely, pyrazinoic acid noctyl ester, pyrazinoic acid pivaloyloxymethyl ester, pyrazine thiocarboxamide and N-hydroxymethyl pyrazine thiocarboxamide were ranked as A against M. tuberculosis and M. intracellulare, and ranked as A, B or C against M. avium, and considered as hopeful candidates of new antimycobacterial drugs. Their bacteriostatic and bacteriocidal activities against M. tuberculosis as well as M. avium and M. intracellulare have been studied in details and reported in a separate paper. In vivo activities against murine experimental tuberculosis of these 4 drugs is now under investigation. Further, two drugs, N-hydroxy pyrazinamide and N-hydroxy pyrazinamide-4-oxide were ranked as A against M. tuberculosis and ranked A or B against M. intracellulare, and their more precise in vitro antimycobacterial activities are now under examination.