Yamamoto S, Toida I, Watanabe N, Ura T
Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Tokyo.
Kekkaku. 1996 Mar;71(3):253-8.
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.
为了寻找比吡嗪酰胺更有效地抗结核分枝杆菌且对鸟分枝杆菌复合群（MAC）也有活性的新药，我们合成了各种吡嗪酰胺类似物和吡嗪衍生物，并在体外测定了它们对结核分枝杆菌、鸟分枝杆菌和胞内分枝杆菌的抗分枝杆菌活性。众所周知，吡嗪酰胺在酸性培养基中比在中性培养基中更具活性，但分枝杆菌在酸性培养基中的生长较差且不稳定。我们的初步实验表明，与吡嗪酰胺相比，受试药物在pH5.5培养基中的相对抗分枝杆菌活性与在pH6.0培养基中基本相同。因此，本研究全程使用调至pH6.0的Middlebrook 7H9肉汤培养基。在合成的39种化合物中，有4种药物不溶于任何适合培养实验的溶剂，无法进行测试，其余35种化合物进行了筛选。通过测量530nm处的光密度（OD）来跟踪分枝杆菌的生长，并将受试药物浓度为200微克/毫升时培养物的OD（OD-TD）与吡嗪酰胺存在时的OD（OD-PZA）进行比较。根据（OD-TD/OD-PZA）×100%的比值，若该比值分别等于或小于10%、11-20%、21-40%、41-60%或61-80%，则每种受试药物分别被评为A、B、C、D或E级。任何比值高于80%的药物都被排除在进一步检查之外。对于结核分枝杆菌，11种药物被评为A级，另有4种被评为B级。对于鸟分枝杆菌，2种药物被评为A级，另有2种被评为B级。对于胞内分枝杆菌，5种药物被评为A级，另有2种被评为B级。在排名靠前的药物中，有4种化合物，即吡嗪酸正辛酯、吡嗪酸新戊酰氧甲酯、吡嗪硫代甲酰胺和N-羟甲基吡嗪硫代甲酰胺，对结核分枝杆菌和胞内分枝杆菌被评为A级，对鸟分枝杆菌被评为A、B或C级，被认为是有希望的新型抗分枝杆菌药物候选物。它们对结核分枝杆菌以及鸟分枝杆菌和胞内分枝杆菌的抑菌和杀菌活性已进行了详细研究，并在另一篇论文中报道。这4种药物对小鼠实验性结核病的体内活性目前正在研究中。此外，两种药物，N-羟基吡嗪酰胺和N-羟基吡嗪酰胺-4-氧化物，对结核分枝杆菌被评为A级，对胞内分枝杆菌被评为A或B级，它们更精确的体外抗分枝杆菌活性目前正在研究中。
