## 4 From BTZ043 to PBTZ169  
At the time of its discovery, BTZ043 was the most efficient inhibitor of in vitro mycobacterial growth ever described. While MICs for first line drugs isoniazid or ethambutol for _M. tuberculosis_ were 0.02-0.2 \(\upmu\)g/ml or 1-5 \(\upmu\)g/ml, respectively, the value for BTZ043 was just 1 ng/ml. The compound proved to be active also in intracellular _M. tuberculosis_ infection in Raw 264.7 macrophages and in a mouse model of chronic tuberculosis and it did not show any signs of toxicity or mutagenicity in concentrations well above the amounts used for the experiments. Nevertheless, the extremely low MIC did not translate to comparably high efficiencies in animal studies [9]. In the effort to improve pharmacological properties of benzothiazinone series, structure-activity relationship (SAR) analysis was performed, with main focus on position 2 of BTZ scaffold [20]. It was previously shown that the rest of the positions harbor substituents critical for the antimyobacterial activity, e.g., replacement of 8-nitro-group with any other group, including hydroxylamino-, amino- and nitryl-, led to at least a 500-fold increase in the MIC against _M. tuberculosis_[9]. The new series contained a piperazine at the position 2 of the BTZ structure, which allowed extensive modifications of the N-4 position of this substituent. Evaluation of the 60 prepared piperazine-benzothizinones (PBTZ) for in vitro activity on _M. tuberculosis_ pointed to a strong correlation between the MICs and lipophilicity. The alkyl-PBTZ derivatives were most active and some of them reached the MICs between 0.19-0.75 ng/ml for _M. tuberculosis_. Five of these derivatives were tested in the chronic TB mouse model, which allowed for selection of PBTZ169 as the most promising candidate [20]. Comparison of BTZ043 with PBTZ169 confirmed superior properties of the latter in all tested parameters. It was three to seven times more active in vitro against _M. tuberculosis_, _M. bovis_ BCG, _M. marinum_, _M. smegmatis_, _Corynebacterium diphtherie_ and _C. glutamicum_ as established by resazurin microtiter assay (REMA). The molecule was also highly active against a panel of 9 MDR- and XDR-clinical isolates of _M. tuberculosis_[20]. Cross-resistance between BTZ043 and PBTZ169 was confirmed for BTZ-resistant strains of _M. tuberculosis_, _M. bovis_ BCG and _M. smegmatis_, indicating the common mechanism of action. Interestingly, PBTZ169 was less susceptible to inactivation by nitro-reduction by the nitroreductase NfnB from _M. smegmatis_ compared to BTZ043, as confirmed by the liquid chromatography-mass spectrometry analyses. This property could be critical for in vivo activity, due to inevitable actions of host nitroreductases or microbial nitroreductases in the digestive tract of the patients, which would potentially reduce the amount of the available active drug. Enzymatic studies confirmed that PBTZ169 is more efficient inhibitor of its DprE1 target than BTZ043; the enzyme was completely inactivated after 5 min of incubation with 5 \(\upmu\)m PBTZ169, while four times higher concentration was needed to achieve the same result with BTZ043. The crystal structure of  
Figure 2: Mechanism of action of macozinone. _Upper line:_ Reactions catalyzed by DprE1 and DprE2. _Bottom line:_ Activation of macozinone and its covalent attachment to DprE1 from _M. tuberculosis_ through Cys387 of the enzyme’s active site. The scheme is based on mechanistic studies performed initially with benzothiazinone BTZ043 [17; 18; 19] and subsequently confirmed for piperazine-benzothizinone PBTZ169 (macozinone) [20]. _M. tuberculosis_ DprE1 with covalently bound PBTZ169 showed similarities with the BTZ043-bound structure of the _M. smegmatis_ enzyme. However, higher flexibility of the methyl-cyclohexyl group on piperazine, could account for better affinity of this drug for its target and explain its higher capacity to inactivate the enzyme [20]. Efficacies of PBTZ169 and BTZ043 were also evaluated against _M. marinum_ in a zebrafish embryo model.