**2. Discovery of Benzothiazinones as Highly Potent Killers of** *Mycobacterium Tuberculosis*

The very first scientific report on benzothiazinones (BTZs) with extremely high antimycobacterial activity was published in 2009 [9]. These compounds emerged as a result of several years of efforts focused on investigation of a family of ditiocarbamate (DTC) derivatives with antimicrobial properties, which was carried out at the Research Center for Antibiotics in Moscow and the Hans Knöll Institute in Jena [10]. DTCs gained their fame primarily as additives in industry and pesticides, which have been in use since 1930. However, some of them were tested as pharmacological agents for treatment of alcoholism, human immunodeficiency virus (HIV) or cancer [11]. It occurred that selected DTC derivatives had minimal inhibitory concentrations (MICs) between 3–30 μg/ml for *M. tuberculosis*, but were active also on several species of fast-growing mycobacteria and multiresistant staphylococci [10]. Thorough structure-activity studies focused on improvement of the activity and selectivity of these compounds for mycobacteria was performed. Selected compounds derived from the basic active ortho nitro-dialkyldithiocarbamate structure showed enhanced activity towards fast growing mycobacteria, with *M. vaccae* being the most sensitive reaching MICs 0.4–1.56 μg/ml [12]. In addition, 3 out of 7 tested molecules were efficient and well tolerated also in the mouse foot pad model of leprosy [12]. Nevertheless, it was clear that reactivity of DTCs poses a significant problem in further development of these compounds despite their activity and rather unexpected low level of toxicity as exemplified by studies in mice [12]. Consequently, a thorough investigation of the metabolic conversion of the most active DTCs containing spiroamine moiety, was undertaken. The drug was incubated with the non-pathogenic strain *M. smegmatis* and all isolated metabolites were tested for antimycobacterial activity. One of these metabolites was particularly active and its structure established by nuclear magnetic resonance (NMR) revealed the principal BTZ scaffold. A set of novel BTZ derivatives was synthesized and tested against both fast-growing and pathogenic mycobacteria in collaboration with Prof. Stewart Cole at Pasteur Institute in Paris [13]. Surprisingly, these compounds were highly active and selective against mycobacteria in vitro, with MIC values 0.195–1.56 μg/ml for *M. tuberculosis*, including the resistant strains. The lowest MIC value, 0.023 pg/ml, was obtained for *M. fortuitum* and 2,2-[2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-nitro- 6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one,

later named BTZ038 [9]. The pilot in vivo studies with this compound revealed its potency in the murine model of TB, which was comparable to isoniazid used in the same dose of 25 mg/kg [13]. These encouraging results warranted further exploration of BTZs as perspective compounds for development of a new TB drug.
