## 4 Conclusions  
The anticancer drug triapazamine (TPZ (**1**)) has previously been reported to be an efficient antifungal and antibacterial agent [4]. In the present work, TPZ (**1**) and a series of TPZ analogues, produced by means of a relatively simple, fast and low-cost synthetic procedure, were examined as antibacterial agents towards representative Gram-negative and -positive strains. Amid the most potent compounds (MICs of \(<\)10 \(\upmu\)g/mL), acetyl- and methoxycarbonyl-substituted TPZs were the strongest agents, exhibiting approximately 4-30 fold greater activities compared to those of TPZ (**1**), along with the conventional antibiotics chloramphenicol and nitrofurantoin used as reference drugs.  
QSAR studies showed that the activities of the tested TPZs were highly impacted by their structural features. The bulky/steric, hydrophobic and polar substituents of the compounds in general led to their lower inhibitory potencies. For TPZ drug (**1**) along with a virtually uniform set of molecules (**2-6**), their activities increased with an increase in their electrophilic/electron-accepting potencies, which implies that the activity of the compounds may be partially related to their reductive activation.  
TPZ (**1**) is known to be more active under low oxygen tension ([4; 51], and refs. therein). Hence, in the current work, the studies performed for TPZs under aerobic conditions seemingly underestimate their antibacterial effects. It should be noted that antibacterials require a drug safety profile which differs from that of the compounds used as antitumor agents. One should point out that the tolerated dose of TPZ (**1**) has previously been found to be 390 mg m\({}^{-2}\) ([4], and refs. therein) corresponding to approximately 85 \(\upmu\)M (or 15 \(\upmu\)g/mL), which is twice as high as the dose estimated in the current work against Gram-negative bacteria strains and close to that as defined with respect to the Gram-positive strain. In addition, the combined antibacterial studies performed in the current work gave clear evidence that TPZ compounds coupled with conventional antibiotics can generate additive and synergistic effects, allowing for the use of a lower dosage of the compounds, which could be advantageous as this may decrease the risk of their toxic effects.  
Considering TPZs as a source of potential antibacterial, our further research will focus on the synthesis of a broader series of novel di-\(N\)-oxides based on the structures of the most potent analogues revealed in our work by the introduction of substituents into the benzene ring of the benzotriazine scaffold, along with the estimation of the inhibitory potencies of the compounds used as single agents and in combination with conventional antibiotics. It must be noted, however, that it remains to be proven that TPZs could be safe antibacterial agents. Thus, the assessment of their toxicity and selectivity, as well as the investigation of the mechanistic aspects of their action will also be one of the most important subjects of our forthcoming work.  
Conceptualization, E.P.-S. and Z.A.; quantum mechanical computation and regression analysis, Z.A. and K.K.; synthesis and spectral characterization of TPZs, J.S. and Z.S.; antibacterial study, E.P.-S. and Z.L.; original draft preparation, Z.A. All authors have read and agreed to the published version of the manuscript.  
This research was partially supported by the European Social Fund (Measure No. 09.33-LMT-K-712, grant no. DOTSUT-34 (09.3.3-LMT-K712-01-0058/LSS-600000-58).  
The authors thank [PERSON] (Department of Biochemistry and Molecular Biology, Life Sciences Center, Vilnius University) for the technical assistance in microbiological experiments and helpful discussion. The authors also thank [PERSON] (Lecture at Vilnius University) for proofreading the manuscript and highly appreciate the constructive comments and suggestions of the reviewers.  
The authors declare no conflict of interest.