The most potent compounds from our previous studies were subjected to the following set of tests: determination of (i) the MICs for different single-drug-resistant strains of M. tuberculosis, (ii) the MBCs, (iii) the activities against nonreplicating bacteria, (iv) the MTDs, (v) the levels of oral bioavailability, and (vi) the in vivo efficacies.
Table shows the MICs obtained for single-drug-resistant strains of M. tuberculosis, including those resistant to INH, rifampin, thiacetazone, ethambutol (EMB), ciprofloxacin (CIP), kanamycin (KAM), ethionamide, and p-aminosalicylic acid. In general, all the compounds showed good MICs for resistant strains; MICs ranged between 0.1 and 6.25 μg/ml, with the exception of that of 3,7-dimethylquinoxaline-2-carboxylate 1,4-di-N-oxide (compound 2), a drug which showed poor activity against EMB-resistant and KAM-resistant strains, with MICs of 25 μg/ml. The susceptibilities of resistant strains can be considered comparable to those of H37Rv, as indicated by the ratios of MICs for resistant and nonresistant M. tuberculosis strains (calculations not shown). This finding suggests that there is minimal if any cross-resistance with the current anti-TB drugs, thereby supporting the notion of a novel mechanism of action. These results are promising for the development of new effective compounds against the growing number of drug-resistant strains. The quinoxaline-2-carboxylate derivative with a benzyl group in the carboxylate (compound 10) was the most active compound, with MICs of 0.4 μg/ml or less for the resistant strains.
MICs for strains of single-drug-resistant M. tuberculosis
Compound 7 is likely activated via bioreduction in bacteria, similar to the reduction observed for other N
). Since PA-824, a nitroimidazole in clinical trials for treating TB, is bioreduced into an active intermediate (4
), we tested the activity of compound 7 against an isogenic set of M. bovis
strains with defined resistance to PA-824. PA-824 is bioreduced into an active form by a pathway involving the deazaflavin F420
cofactor-dependent glucose dehydrogenase (Fdg1) and other cellular factors, including molybdopterin, a cofactor for many oxidoreductase enzymes. Thus, the loss of function of Fdg1 or the loss of the ability to synthesize the F420
cofactor leads to resistance to PA-824 (Table ). Compound 7 was active against all PA-824-resistant M. bovis
strains tested, thus showing the lack of cross-resistance and supporting the evidence for a different pathway of drug activation. Niclosamide (an anthelmintic agent that shows poor absorption from the host intestine) was included as another control compound that is structurally different from PA-824 and activated by a different pathway (1
In vitro activities against susceptible and genetically defined PA-824-resistant mycobacteria
Compound 7 was also tested under aerobic and anaerobic (LORA) conditions for activity against M. tuberculosis strain H37Rv containing the luciferase reporter. Compound 7 was active under aerobic growth conditions (MIC = 0.24 μg/ml) and under anaerobic conditions (LORA MIC = 0.42 μg/ml). The corresponding values for PA-824 and niclosamide were as follows: MICs under aerobic growth conditions, 0.10 and 0.28 μg/ml, respectively, and LORA MICs, 1.4 and 0.32 μg/ml, respectively. Activity against nonreplicating bacteria is another unique property of quinoxaline 1,4-di-N-oxides. Although the idea is speculative, activity targeting nonreplicating bacteria may translate into faster sterilization of infected tissues, and we are in the process of testing this possibility in animal models. The long continuation phase for the treatment of TB is believed to be due in part to the presence of nonreplicating organisms that persist even in the presence of antitubercular drugs. PA-824 is an experimental nitroimidazole that is in phase I clinical trials. PA-824 is bioreduced by M. tuberculosis into an active component. Niclosamide is another bioreduced drug that is active in the LORA. Compound 7 was slightly more active than PA-824 in the LORA on a molar basis (1.5 μM versus 4.85 μM).
The MBCs of select compounds for H37
Rv, along with the MICs, were determined using the MABA (10
). The MIC of compound 7 tested as 0.6 μg/ml in this assay, and the MBC was 2.5 μg/ml (MBC/MIC ratio = 4.2). The MIC of compound 6 was 0.2 μg/ml, with an MBC of 3.13 μg/ml (MBC/MIC ratio = 15.7), and the MIC of compound 10 was 0.39 μg/ml, with an MBC of >6.25 μg/ml (MBC/MIC ratio > 16.0). A compound is generally considered to be bactericidal if the ratio of the MBC to the MIC is ≤4 (30
), so the ratios for these compounds against H37
Rv indicated bacteriostatic rather than bactericidal activity by this definition. However, bactericidal activity in vivo is determined by peak levels in blood and tissues and exposure times following oral administration. The in vivo peak levels of compound 7 in serum reached 5 to 8 μg/ml 15 to 30 min following the administration of 100- to 200-mg/kg doses, as determined by bioassays, and bactericidal activity was observed in vivo (see below for more on in vivo efficacy).
Selected compounds 6 to 12 were evaluated in in vivo assays, including tests for efficacy in the mouse model of TB infection. The MTD of each compound was determined by using an escalating single dose of the drug given to mice by oral gavage. No acute adverse effects, reactions, or toxicity were observed at oral doses of up to 1,000 mg/kg (the highest dose tested). Subsequent in vivo evaluations of the efficacies of compounds 6 to 12 were done with doses of 300 mg/kg administered via oral gavage to infected GKO C57BL/6 mice (18
). Six of the seven compounds were found to be inactive in that they did not effectively reduce the bacterial numbers in the lungs and spleens with respect to those in the untreated controls (Table ). While we have not definitively determined the reason for the lack of in vivo efficacy of compounds 6 and 8 to 12, it appears that the lack of activity of compound 10 may be due to extensive serum binding, since the MIC is reduced 200-fold in the presence of serum. Likewise, the lack of efficacy of compound 6 appears to be due to a short in vivo half-life, based on the results of preliminary studies. The lack of activity was not due to the potency of M. tuberculosis
strain Erdman, as all of the compounds were active against this strain (Table ).
Evaluation of in vivo efficacies in the GKO mouse modela
However, the ethyl 7-chloroquinoxaline derivative (compound 7) afforded significant reductions of 3.2 and 2.62 log10
CFU in lungs and spleens, respectively. Generally, compounds are considered to be active if they yield at least a 0.75-log10
reduction in bacterial counts compared to day 24 counts. The protection shown by compound 7 is similar to that afforded by clinically available compounds and other compounds classified as very active (17
). INH reduced the numbers of CFU by 3.10 and 4.17 log units in the same experiment. Since the majority of compounds showing good in vitro activity do not show in vivo efficacy in mice (23
), the in vivo activity of ethyl 7-chloro-3-methylquinoxaline-2-carboxylate 1,4-di-N
-oxide is promising. Bactericidal activity of compound 7 was observed, since the final lung CFU count following 9 days of treatment was below the starting lung CFU count on day 15, the day therapy was initiated (6.81 log CFU untreated lung, compared to 4.55 log CFU following 9 days of treatment) (Table ). Similar evidence for bactericidal activity was obtained from the spleen data, with an initial count of 5.37 log CFU untreated spleen compared to 3.95 log CFU following 9 days of treatment (Table ).
In a second experiment (Table ), the dose-response relationship for compound 7 in vivo was determined using the GKO mouse model and doses of 25, 100, and 300 mg/kg (Table ). Compound 7 was weakly active in the lung and spleen at a dose of 25 mg/kg (CFU readings for tissues from treated mice on day 24 were lower than those for tissues from the untreated controls on day 24, but the activity appeared to be bacteriostatic). At 100 and 300 mg/kg (P, <0.001 by a one-way analysis of variance followed by a multiple-comparison analysis of variance using a one-way Tukey test in the SigmaStat software program), compound 7 appeared to be bactericidal, since the numbers of CFU recovered from both the lungs and spleens were well below the CFU levels observed on day 15 (Table ), except for the spleens from the 100-mg/kg-dose group, in which the mean number of CFU was reduced by only 0.28 logs, which was not significant. Activity at a dose of 300 mg/kg was striking in that it lowered the numbers of CFU by 5.58 and 5.51 logs, respectively, in the lungs and spleens. Although no clear toxicity was apparent in the single-dose MTD test and in the first in vivo experiment, some toxicity was observed in the second dose-response experiment; three mice in the 300-mg/kg group died during treatment, which was truncated to 7 days instead of the usual 9 days. No CFU were recovered from the lung and spleen of one of the surviving mice dosed at 300 mg/kg when the entire organs were plated for CFU enumeration. Although it appears that multiple dosing at 300 mg/kg may result in some toxicity in infected animals, data collected to date on this series (quinoxaline-2-carboxylate 1,4-di-N-oxide derivatives) indicate that both in vitro (cytotoxicity) and in vivo toxicity can be separated from the antitubercular activity, as determined by plotting MICs versus 50% inhibitory concentrations for over 100 analogs, wherein no correlation was observed. Although the MTD is high (>1,000 mg/kg for normal mice), we have observed that some compounds can show toxicity in infected GKO mice at lower levels, due likely to the added stress of disease. Thus, 300 mg/kg may be on the borderline for tolerability in GKO infected mice and may therefore show toxicity in some experiments. Experiments with normal C57BL/6 mice and longer treatment times are being scheduled.
Dose-response data for compound 7 (TAACF 151985) in the GKO mouse model
In conclusion, an extended evaluation of the in vitro and in vivo antimycobacterial activities of quinoxaline 1,4-di-N-oxide derivatives was performed. Most of these compounds displayed good inhibitory activity against resistant strains, and only compound 2 was associated with apparent cross-resistance in EMB-resistant and KAM-resistant strains, although we have not yet confirmed these data. Compound 7 was the only analog active in vivo. This activity was clearly due to adequate oral bioavailability and levels in the blood (measured at 3, 6, and 9 μg/ml for the 25-, 100-, and 300-mg/kg doses, respectively) and to a terminal elimination half-life of 4 to 8 h (unpublished data). Finally, we did detect bactericidal activity when we tested the sera of animals dosed orally with 200 mg of compound 7/kg and bled at 30 min. Sera diluted twofold killed 2.51 logs, and when diluted fourfold, they still killed 1.15 logs after 7 days of incubation. Thus, compound 7 has all of the necessary characteristics of a validated lead that can serve as the starting point for more advancement in medicinal chemical and preclinical development.