Research Article | Volume: 11, Issue: 2, February, 2021

New benzothiazinone linked 1, 2, 4-triazoles: Design, synthesis, characterization, and evaluation of antitubercular activity

Kannampally Ajmal Gurubasavaraj V. Pujar Sethu Arun Kumar Subhankar P. Mandal Manisha Singh   

Open Access   

Published:  Feb 05, 2021

DOI: 10.7324/JAPS.2021.110204
Abstract

Introduction: In a quest for developing active molecules to treat tuberculosis, we reported the design, synthesis, and antitubercular activity evaluation of benzo[1, 4]thiazin-3(4H)-one encompassed 1, 2, 4-triazoles targeting the DprE1 (Decaprenylphosphoryl-beta-D-ribose oxidase) enzyme involved in cell wall component biosynthesis.

Methodology: The antitubercular potential of the title compounds was screened against the standard strain of Mycobacterium tuberculosis H37Rv by Microplate Alamar Blue Assay (MABA) and in vitro cytotoxicity was screened against in human embryonic kidney 293 (HEK293T) cells. The prediction of ligand interactions to the target DprE1 enzyme’s binding site was realized through a molecular docking study.

Results: Synthesized molecules have shown good antitubercular activity with reference to the standard antitubercular drugs. The 5c, 5e, and 6c are the most active antitubercular compounds having a minimum inhibitory concentration of 12.5 μg/ml. The synthesized molecules’ cytotoxic data reveal that tested compounds exhibited low in vitro cytotoxicity (higher IC50 values) in human embryonic kidney 293 (HEK293T) cells. Moreover, the prediction of ligand interactions to the target DprE1 enzyme’s binding site was realized through a molecular docking study. The compounds 6a–d have shown good docking scores, and their interactions with amino acids with active site pockets of the DprE1 were in line with the reference ligand.

Conclusion: Thus, the adopted docking protocol is in good correlation to the in vitro antitubercular activity. Hence, benzo[1, 4]thiazin-3(4H)-one encompassed 1, 2, 4-triazole scaffolds may provide the basis for developing new DprE1 inhibitors.


Keyword:     1 2 4-triazole benzo[14]thiazin-3(4H)-one MABA Mycobacterium tuberculosis MTT assay DprE1 inhibitors.


Citation:

Ajmal K, Pujar GV, Kumar SA, Mandal SP, Singh M. New benzothiazinone linked 1, 2, 4-triazoles: Design, synthesis, characterization, and evaluation of antitubercular activity. J Appl Pharm Sci, 2021; 11(02):029–038.

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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INTRODUCTION

Tuberculosis (TB) is continuing to affect humankind with broad population statistics. It reached approximately 1.9 million deaths and is increasing in number every year (Rojano et al., 2019). The recent emergence of the disease with multidrug-resistant tuberculosis, total drug resistance, and extensively drug-resistant TB strains (Prasad et al., 2017) has increased the mortality rate by several folds, which in turn prompts the necessity for the discovery of drug which can reduce the load of the resistant strains of TB (Akkerman et al., 2019). Recently, several antitubercular drugs were in the drug discovery pipeline, and few were in the developmental stage (Libardo et al., 2018). However, due to enormous cases of failure in the early stage of drug development, there is a constant need for novel drug candidates with possibly novel scaffold possessing efficacy to combat all kinds of resistant TB cases.

Mycobacterium tuberculosis (Mtb) owns an elegant cell wall due to the presence of arabinogalactan and mycolic acid along with the peptidoglycan layer, which is reversibly connected to the outer layer of protein and polysaccharide (Abrahams and Besra, 2018). The well-known first-line anti-TB drug isoniazid (INH) inhibits a vital enzyme involved in mycolic acid biosynthesis, and ethambutol targets the enzymes involved in arabinan biosynthesis. Likewise, few among the best active antibiotics in contrast to TB act by suppressing the cell wall synthesis (Wolucka et al., 1994). Validation of the new target DprE1, a key enzyme in producing an intermediate decaprenyl-phospho-arabinose (DPA) in cell wall components’ biosynthesis, has emerged as a promising target in developing antitubercular agents (Brecik et al., 2015). DprE1 is a vital Mtb flavoenzyme encoded by the dprE1 gene (rv3790) named decaprenophosphoryl-beta-D-ribose-2-oxidase (Makarov et al., 2009). DprE1 works in harmony with the DprE2 (Decaprenylphosphoryl-2-keto-beta-D-erythro-pentose reductase) enzyme that helps in the epimerization of decaprenyl-phosphoribose (DPR) to DPA. DprE1 utilizes FAD (Flavin Adenine Dinucleotide) to oxidate DPR to a ketonic derivative and later undergoes reduction to DPA, an exclusive precursor in the synthesis of lipopolysaccharides (lipoarabinomannan and arabinogalactan) by NADH (reduced Nicotinamide Adenine Dinucleotide) dependent DprE2. Both DprE1 and DprE2 are indispensable for cell growing and Mtb’s existence (Brecik et al., 2015). Benzothiazinones (BTZ) (Batt et al., 2012), dinitrobenzamides (Li et al., 2018), nitroimidazoles (Kim et al., 2009), and nitroquinoxalines (Magnet et al., 2010) with chemically diverse molecules were found to be active as covalent DprE1 inhibitors. They displayed significant antitubercular activity in both in vitro and in vivo efficacy studies. Few studies have shown that the replacement of the nitro group on BTZ by the azide group or pyrrole ring also offered substantial antitubercular activity, while their mode of inhibition changed from a covalent to non-covalent fashion (Tiwari et al., 2016; Zhang et al., 2014). As a result of cell-based screening, TCA1 (a benzothiazole derivative) demonstrated potency against non-replicating and replicating Mtb was identified with an effective in vivo activity alone or combined with the first-line drugs (Wang et al., 2013). Keeping TCA1 as a lead, a structure-based drug discovery approach had witnessed the report of several non-covalent DprE1 inhibitors in the last few years (Makarov et al., 2015; Manina et al., 2010; Naik et al., 2014; Shirude et al., 2013; Tiwari et al., 2016; Wang et al., 2013).

Triazole (1, 2, 3-triazole/1, 2, 4-triazole) moiety is a critical five-membered heterocycle, an integral moiety in various clinically approved drugs (Maddila et al., 2013). It has been explored to synthesize new molecules with varied infection-related activities (Keri et al., 2015). Several studies have revealed the promising effect of triazole as antitubercular agents (Bangalore et al., 2019; Karabanovich et al., 2019; Reddyrajula et al., 2020; Shaikh et al., 2019; Zampieri et al., 2019). Furthermore, the conjunction of 1, 2, 4-triazole with other azoles (heterocyclic systems) has resulted in potential antitubercular activity against DprE1 target (Karabanovich et al., 2019; Shaikh et al., 2016, 2019), including our earlier work on exploring the antitubercular potential of triazoles (Krishna et al., 2014; Neenu et al., 2020). The strategy for the design of title compounds (Fig. 1) was based on the antitubercular potentials of two heterocyclic ring systems, benzo[1, 4]thiazin-3(4H)-one and 1, 2, 4-triazole. The introduction of aryl/cycloalkyl groups (R) at the fourth position of triazole and the nitro benzoyl group at thiol of triazoles increases the lipophilicity of the prefinal and final compounds (Arnott et al., 2012; Shaikh et al., 2016). Furthermore, the introduction of nitro benzoyl functionality realizes antitubercular potentials. Based on these findings, an effort is conceived to explore the in vitro efficacy of benzothiazinone conjugate of 1,2,4-triazoles against Mtb.


MATERIALS AND METHODS

Solvents and chemicals were procured from Sigma-Aldrich, TCI chemicals and were utilized as received to prepare intermediates and final compounds. The synthesized compounds’ melting point was determined using the one-end open capillary tube method using a digital melting point apparatus. The reaction’s progress was determined on thin-layer chromatography (TLC) using aluminum plates (Merck) with silica gel 60 F254. Infrared (IR) spectra were recorded using Shimadzu FT-IR (Fourier Transform-Infrared spectroscopy). 13C and 1HNMR spectra were recorded using a Bruker AMX-300 NMR spectrometer at 400 MHz. Chemical shifts (δ) were reported in parts per million (ppm) using tetramethylsilane as the reference. Mass spectra were recorded on liquid chromatography–mass spectrometry (LCMS) from Agilent technologies in Electrospray ionization mode. Elemental analysis was conducted for the synthesized compounds, and the values obtained were within 0.3% of the calculated values.

Synthesis of 2H-benzo[b][1, 4]thiazin-3(H)-one (1)

Equimolar quantities (0.04 mol) of 2-amino thiophenol and chloroacetic acid were taken in a 150 ml round bottom flask (RBF) and glacial acetic acid (40 ml) and sodium acetate (equimolar) were added. The reaction content was heated at a boiling temperature for 24 hours. The reaction’s progress was checked by TLC using hexane and ethyl acetate as mobile phase (1:2). Then, the final content was poured into ice cold water. The solids were separated by filtration, and then dried and recrystallized using 70% ethanol.

Figure 1. Design strategy for the proposed compounds.

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Yield: (5.2 g, 78%). mp: 179°C–181°C (Literature value:176°C–178°C); IR (KBr) cm−1: 3,024 (Ar C-H), 3,359 (N-H), 1,702 (C=O); 1H NMR dimethyl sulfoxide (DMSO-d6, δ ppm): 3.42 (s, 2H, CH2), 6.80–7.31 (m, 4H, Ar H:), 8.61 (s, 1H, NH); 13C NMR (DMSO-d6, δ ppm): 40.6 (CH2), 123.5, 124.6, 125.8, 130.0, 136.0, 143.1 (C-N), 166.88 (C=O); LCMS: 165.02 [M+].

Synthesis of ethyl 2-(2, 3-dihydro-3-oxobenzo[b][1, 4]thiazin-4-yl)acetate (2)

An equimolar quantity of 2H-benzo[b][1,4]thiazin-3(4H)one (0.04 mol) and ethyl bromoacetate (0.04 mol) were transferred to dry acetone (15 ml) in the presence of anhydrous potassium carbonate (0.04 mol). The reaction content was heated to a refluxing temperature for 22 hours. The progress of the reaction was observed by TLC using mobile phase hexane and ethyl acetate in the ratio of 1:2 and once the reaction was completed, it was filtered off the potassium carbonate. The filtrate acetone was removed using a rotary vacuum evaporator to get the desired product (2).

Yield: (4.52 g, 67%). mp: (ND); IR (KBr) cm−1: 3,026 (Ar C-H), 1,654 and 1,702 (C=O); 1H NMR (DMSO-d6, δ ppm): 1.46 (t, 2H, CH3), 3.52 (s, 2H, CH2), 4.23 (m, 2H, CH2), 4.43 (s, 2H, CH2), 6.92–7.30 (m, 4H, ArH),13C NMR (DMSO-d6, δ ppm):15.3 (CH3), 40.6 (CH2), 51.0 (CH2), 61.4 (CH2), 123.5, 124.1, 125.1, 130.0, 133.0, 138.1 (C-N), 166.23 (C=O); LCMS m/z: 251.03 [M+].

Synthesis of 2-(2, 3-dihydro-3-oxobenzo[b][1, 4]thiazin-4yl)acetohydrazide (3)

Ethyl 2-(2,3-dihydro-3-oxobenzo[b][1, 4] thiazin-4yl)acetate (0.01 mol) was taken in an RBF and dissolved in ethanol (10 ml). To this, 99% hydrazine hydrate (0.01 mol) was added dropwise and the reaction mixture was stirring overnight at room temperature. The progress of the reaction was observed by TLC using chloroform and methanol as a mobile phase (4:1). The solid separated was filtered, dried, and recrystallized using ethanol.

Yield: (6.73 g, 71%). mp: 186°C–189°C; IR (KBr) ν cm−1: 3,365 and 3,630 (N-H), 3,021 (Ar C-H), 1,654 and 1,765 (C=O); 1H NMR (DMSO-d6, δ ppm): 3.52 (s, 2H, CH2), 4.43 (s, 2H, CH2), 7.1–7.3 (m, 4H, ArH), 9.63 (bs, 2H, NH), 10.30 (s, 1H, NH), 13C NMR (DMSO-d6, δ ppm): 41.1 (CH2), 52.0 (CH2), 123.5, 124.1, 125.1, 131.1, 133.3, 138.5 (C-N), 167.27 (C=O); LCMS m/z: 237.06 [M+].

Synthesis of 4-((5-mercapto-4-aryl/cyclohexyl-4H-1, 2, 4-triazol-3-yl)methyl)-2H-benzo[b][1,4]thiazin-3(4H)-ones (5a–5f)

An equimolar quantity (0.004 mol) of acetohydrazide derivative (3) and different aryl/cyclohexyl isothiocyanates were dissolved in solvent ethanol, and the reaction content was refluxed for 3–8 hours. The white solids were obtained on cooling the reaction content, then filtered and dried to obtain carbothioamide derivatives (4a–4f). The product formed was directly used for the subsequent reaction step. Compound 4a (0.004 mol) was taken in an RBF and dissolved in 2 M NaOH solution (12 ml) and refluxed to a boiling temperature for 8 hours. TLC monitored the completion of the reaction. The mixture was then cooled and filtered. Upon acidification of filtrate using 2 M HCl, the product precipitated out. The obtained precipitate was filtered, washed with water, dried, and recrystallized from absolute alcohol.

4-((5-mercapto-4-phenyl-4H-1, 2, 4-triazol-3-yl)methyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (5a)

Yield: (0.90 g, 64%). mp: 210°C–216°C; IR (KBr) cm−1: 3,294 (N-H), 3,009 (Ar C-H), 1,724 (C=O), 1,585 (C=S); 1H NMR (DMSO-d6, δ ppm): 3.41 (s, 2H, CH2), 4.23 (s, 2H, CH2), 6.53–7.55 (m, 9H, ArH), 13.78(s, 1H, NH); 13C NMR (DMSO-d6, δ ppm): 38.9, 56.0, 124.5, 125.2, 125.3, 128.4, 129.0, 133.0, 133.9, 135.0, 135.7, 157.0, 166.7; LCMS m/z: 355.07 (M+); Anal. Calcd. for C17H14N4OS2: C, 57.61; H, 3.98; N, 15.81; O, 4.51; S, 18.09, found: C, 56.4; H, 3.66; N, 15.17.

4-((4-cyclohexyl-5-mercapto-4H-1,2,4-triazol-3-yl)methyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (5b)

Yield: (0.96 g, 67%). mp: 196°C–202°C; IR (KBr) cm−1: 3,340 (N-H), 2,852 (Aliphatic C-H), 1,724 (C=O), 1,585 (C=S); 1H NMR (DMSO-d6, δ ppm): 1.15–2.93 (m, 11H, cyclohexyl), 3.29 (s, 2H, CH2), 4.49 (s, 2H, CH2), 6.51–7.31 (m, 4H, ArH), 13.52 (s, 1H, NH); 13C NMR (DMSO-d6, δ ppm): 24.7, 25.7, 32.8, 39.2, 49.1, 56.5, 124.5, 125.2, 135, 135.7, 155.6, 162.3, 166.7; LCMS m/z: 361.4 (M+); Anal. calcd. for C17H20N4OS2: C, 56.23; H, 5.59; N, 15.54; O, 4.44; S, 16.68, found: C, 56.14; H, 5.11; N, 16.01.

4-((5-mercapto-4-(4-methoxyphenyl)-4H-1, 2, 4-triazol-3-yl) methyl)-2H-benzo[b][1, 4]thiazin-3(4H)-one (5c)

Yield:(0.95 g, 62%). mp: 234°C–238°C; IR (KBr) cm−1: 3,581 (N-H), 3,024 (Ar C-H), 1,716 (C=O), 1,591 (C=S); 1H NMR (DMSO-d6, δ ppm): 3.40 (s, 2H, CH2), 3.78 (s, 3H, CH3), 4.43 (s, 2H, CH2), 6.43–7.35 (m, 8H, ArH), 13.52 (s, 1H, NH); 13C NMR (DMSO-d6, δ ppm): 38.9, 55.8, 56.0, 114.6, 124.5, 125.2, 134.8, 135.7, 137.8, 151.6, 160.6, 166.7, 168.9; LCMS m/z: 385.84 (M+); Anal. calcd. for C18H10N4O2S2: C, 56.23; H, 4.19; N, 14.57; O, 8.32; S, 16.68, found: C, 56.33; H, 4.27; N, 13.88.

4-((5-mercapto-4-(naphthalen-1-yl)-4H-1,2,4-triazol-3-yl)methyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (5d)

Yield: (1.14 g, 71%). mp: 212°C–216°C; IR (KBr) cm−1: 3,358 (N-H), 3,053 (Ar C-H), 1,708 (C=O), 1,589 (C=S); 1H NMR (DMSO-d6, δ ppm): 3.43 (s, 2H, CH2), 4.05 (s, 2H, CH2), 6.33–7.66 (m, 11H, ArH), 13.82 (s, 1H, NH); 13C NMR (DMSO-d6, δ ppm): 39.2, 48.2, 123.9, 124.5, 125.2, 125.3, 126.3, 126.7, 128.3, 131.6, 133.7, 135.0, 135.7, 138.2, 151.6, 166.7, 168.9; LCMS m/z: 404.7 (M+); Anal. calcd. for C21H16N4OS2: C, 62.36; H, 3.99; N, 13.85; O, 3.96; S, 15.85, found: C, 61.97; H, 4.11; N, 13.71.

4-((4-(4-fluorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)methyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (5e)

Yield: (0.99 g, 67%). mp: 198°C–202°C; IR (KBr) cm−1: 3,320 (N-H), 3,119 (Ar C-H), 1,731 (C=O), 1,587 (C=S); 1H NMR (DMSO-d6, δ ppm): 3.64 (s, 2H, CH2), 4.15 (s, 2H, CH2), 6.41–7.45 (m, 9H, ArH), 13.52 (s, 1H, NH); 13C NMR (DMSO-d6, δ ppm): 39.2, 48.2, 115.5, 124.5, 125.2, 130.1, 135.0, 135.7, 141.1, 151.6, 162.9, 166.7, 168.9; LCMS m/z: 372.1 (M+); Anal. calcd. for C17H13FN4OS2: C, 54.82; H, 3.52; F, 5.10; N, 15.04; O, 4.30; S, 17.22, found: C, 55.11; H, 3.54; N, 15.17.

4-((5-mercapto-4-(p-tolyl)-4H-1,2,4-triazol-3-yl)methyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (5f)

Yield: (0.92 g, 63%). mp: 220°C–226°C; IR (KBr) cm−1: 3,298 (N-H), 3,047 (Ar C-H), 1,754 (C=O), 1,589 (C=S); 1H NMR (DMSO-d6, δ ppm): 2.25 (s, 3H, CH3), 3.37 (s, 2H, CH2), 4.50 (s, 2H, CH2), 6.52–7.31 (m, 8H, ArH), 13.48 (s, 1H, NH); 13C NMR (DMSO-d6, δ ppm): 21.3, 39.2, 48.4, 124.5, 125.2, 129.0, 131.0, 135, 135.7, 138.4, 142.5, 151.6, 166.7, 168.9; LCMS m/z: 368.3 (M+); Anal. calcd. for C18H16N4OS2: C, 58.67; H, 4.38; N, 15.21; O, 4.34; S, 17.40.

Synthesis of S-(5-((3-oxo-2,3-dihydro-4H-benzo[b][1,4]thiazin-4-yl)methyl)-4-aryl/alkyl-4H-1,2,4-triazol-3-yl) 4-nitrobenzothioates (6a–d)

An equimolar quantity (0.002 M mol) of 4-nitrobenzoyl chloride and appropriate 1,2,4-triazoles (5a–c, 5e) were added to dry acetone in the presence of Cs2CO3 (0.002 M mol), and the reaction content was refluxed for 14 hours. The reaction progress was observed by TLC using chloroform and methanol as a mobile phase (3:1). Once the reaction was completed, the reaction mixture was filtered, and the collected filtrate was evaporated using rotary vacuum evaporator to get the desired compound.

S-(5-((3-oxo-2,3-dihydro-4H-benzo[b][1, 4]thiazin-4-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-yl) 4-nitrobenzothioate (6a)

Yield: (0.76 g, 76%). mp: 236°C–240°C; IR (KBr) cm−1: 3,298 (N-H), 3,024 (Ar C-H), 1,708, 1,724 (C=O), 1,606 (C=S), 1,501(N-O); 1H NMR (DMSO-d6, δ ppm): 3.42 (s, 2H, CH2), 4.23 (s, 2H, CH2), 6.91–8.03 (m, 9H, ArH), 8.26–8.38 (m, 4H, ArH); 13C NMR (DMSO-d6, δ ppm): 39.3, 49.1, 124.1, 124.5, 129.0, 129.6, 135.0, 135.7, 140.8, 145.5, 151.6, 153.3, 159.7, 166.7, 187.6, 187.7; LCMS m/z: 504.43 (M+); Anal. Calcd. for C24H17N5O4S2: C, 57.25; H, 3.40; N, 13.91; O, 12.71; S, 12.73, found: C, 56.84; H, 3.46; N, 13.27.

S-(4-cyclohexyl-5-((3-oxo-2,3-dihydro-4H-benzo[b][1,4]thiazin-4-yl)methyl)-4H-1,2,4-triazol-3-yl) 4-nitrobenzothioate (6b)

Yield: (074 g, 73%). mp: 228°C–232°C; IR (KBr) cm−1: 3,140 (Ar C-H), 1,718, 1,729 (C=O), 1,599 (C=S), 1,475 (N-O); 1H NMR (DMSO-d6, δ ppm): 1.15–2.93 (m, 11H, cyclohexyl), 3.30 (s, 2H, CH2), 4.49 (s, 2H, CH2), 6.89–8.01 (m, 4H, ArH), 8.26–8.32 (m, 4H, ArH); 13C NMR (DMSO-d6, δ ppm): 24.7, 25.7, 33.2, 39.5, 39.3, 49.1, 57.1, 124.1, 124.5, 129.0, 129.6, 135.0, 135.7, 140.8, 145.5, 151.6, 153.3, 159.7, 166.7, 187.6, 187.7; LCMS m/z: 510.78 (M+); Anal. calcd. for C24H23N5O4S2: C, 56.57; H, 4.55; N, 13.74; O, 12.56; S, 12.58, found: C, 57.01: H, 4.54; N, 13.11.

S-(4-(4-methoxyphenyl)-5-((3-oxo-2,3-dihydro-4H-benzo[b][1,4]thiazin-4-yl)methyl)-4H-1,2,4-triazol-3-yl) 4-nitrobenzothioate (6c)

Yield: (0.72 g, 68%). mp: 216°C–220°C; IR (KBr) cm−1: 3,162 (Ar C-H), 1,713, 1,739 (C=O), 1,579 (C=S), 1,535 (N-O); 1H NMR (DMSO-d6, δ ppm): 3.40 (s, 2H, CH2), 3.78 (s, 3H, CH3), 4.42 (s, 2H, CH2), 6.72–8.03 (m, 8H, ArH), 8.22–8.30 (m, 4H, ArH); 13C NMR (DMSO-d6, δ ppm): 39.3, 49.1, 56.1, 114.3, 124.1, 124.5, 129.0, 129.6, 134.8, 135.0, 140.8, 145.5, 151.6, 153.3, 159.7, 166.7, 187.6, 187.7; LCMS m/z: 533.0 (M+); Anal. calcd. for C25H19N5O5S2: C, 56.28; H, 3.59; N, 13.13; O, 14.99; S, 12.02, found: C, 57.01: H, 3.44; N, 12.93.

S-(4-(4-fluorophenyl)-5-((3-oxo-2,3-dihydro-4H-benzo[b][1,4]thiazin-4-yl)methyl)-4H-1,2,4-triazol-3-yl) 4-nitrobenzothioate (6d)

Yield: (0.66 g, 64%). mp: 231°C–235°C; IR (KBr) cm−1: 3,103 (Ar C-H), 1,699, 1,731 (C=O), 1,563 (C=S), 1,526 (N-O); 1H NMR (DMSO-d6, δ ppm): 3.40 (s, 2H, CH2), 4.42 (s, 2H, CH2), 6.79–8.01(m, 8H, ArH), 8.20–8.33 (m, 4H, ArH); 13C NMR (DMSO-d6, δ ppm): 39.2, 48.8, 115.5, 124.1, 124.5, 129.0, 130.1, 135.0, 135.7, 140.8, 141.1, 151.6, 153.3, 159.7, 162.7, 166.7, 187.6; LCMS m/z: 521.5 (M+); Anal. calcd. for C24 H16FN5O4S2: C, 55.27; H, 3.09; F, 3.64; N, 13.43; O, 12.27; S, 12.29, found: C, 55.31; H, 2.94; N, 13.32.

In vitro antitubercular activity

In vitro antitubercular activity of the synthesized molecules was tested by the MABA method (Coxon et al., 2012; Leonard et al., 2008) against M. tuberculosis H37Rv strain (ATCC 27294). Mtb suspension stock culture from Middlebrook 7H9-S was vortexed and adjusted to 1 McFarland standard (3 ×108 CFU/ml (Colony Forming Unit/ml)). The culture was further diluted to 2 × 105 CFU/ml concentration and used as inoculum. Test samples and standard drug stock solutions of 20,000 μg/ml concentration was prepared with DMSO. Serially the stock solutions (4×) were diluted using media into a 96-well plate to achieve working solutions. Twofold serial dilutions of the test compounds were prepared on the well plate, and 100 μl of inoculum (2 × 105 CFU/ml) and 100 μl media were added to each well of the 96-well plate to obtain a concentration of 200 μl. The final concentrations of test compounds and standard drugs (pyrazinamide, ciprofloxacin, and streptomycin) were between 0.8 and 100 μg/ml. Positive control (inoculum), DMSO as a blank, and negative control (media) in the plate were used to lessen the error. Then, the plates under aeration were incubated at 37°C. alamarBlue reagent (20 μl) was added on day 7 of incubation to each well plate and further incubated for 24 hours at 37°C. The color of the media changing from blue to pink was considered as the Mycobacterium’s growth at a particular drug concentration. The test compound’s well plate color matched with the color of the growth control well plate to understand the results better. The lowest drug concentration that inhibited bacterial growth was referred to as minimum inhibitory concentration (MIC).

In vitro cytotoxicity (CC50)

The synthesized molecules were assessed for in vitro CC50 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) (Chang et al., 2010) involving human embryonic kidney 293 cells (HEK293T). The test compound’s 300–25 μg/ml concentrations were prepared in the 96-well plate containing the minimum essential medium without the fetal bovine serum. The well plate was then upturned onto a filter paper so that the resilient media could be separated and washed with PBS (Phosphate Buffered Saline) gently. The outer perimeter walls were treated with 100 μL of sterile water. Then, the prepared dilutions were added (100 μl) into the wells. The plates were incubated with 5% CO2 considering 72 hours for HEK293T cells at 37°C. This was succeeded by supernatant media removal by following the procedure mentioned earlier, and then it was washed with PBS. The MTT solution (50 μl) was supplemented to each well in a place devoid of light and was again subjected to incubation for 3 hours. The MTT solution was removed by inversion method, and DMSO was poured into every well and was placed aside in the dark for nearly 1–2 hours. ELISA (Enzyme-linked Immunosorbent Assay) reader was employed to record the optical density readings using a wavelength of 540 nm. CC50 was determined by deducing a graph obtained by taking test compound concentration and % of cell inhibition on the x-axis and y-axis, respectively.

% Cytotoxicity = Control Absorbance Test Absorbance Control absorbance × 100

In silico absorption, distribution, metabolism, elimination, and toxicity (ADMET) prediction

Pharmacokinetic properties such as ADMET of compound structures were checked using the ADMETSAR web server and the Discover Studio 19.0 software package. Molecules were prepared accordingly via ligand preparation tools and subjected to ADMET predictions. The lipophilicity (clogP) of the compounds was predicted using Chemdraw software.

Molecular docking study

Docking study is like a lock and key concept, executable by implementing various algorithms (Inturi et al., 2016). LIBDOCK algorithm was implemented in the present study by CHARMm force field (Biovia Discovery Studio 19.0 software). The preamble to docking procedure, protein’s 3D structure (PDB ID-4PFD) was downloaded from the Protein databank (PDB) (http://www.rcsb.org). The x-ray crystallographic form of DprE1 with a resolution of 2.30 Å was used to execute a protein preparation protocol to correct the side chains and loops region, followed by energy minimization. Consecutively, the water molecules and co-crystallized ligand were removed. Then, ligands were subjected further for duplicate removal and to fix their chemical valences. So, the prepared DprE1 protein and ligands were taken for the docking protocol. The binding site within the protein was recognized “from the receptor cavity” tool site search by means of flood-filling algorithm. Among other designated sites, the prime site 1 was found to be a potential one with a volume of 1,231 Ǻ and a point count of 7,824 in equal grid spacing of 0.5 in three coordinate axes. The designed ligands and reference molecule were subjected to docking by default simulation annealing and fast conformation generation with a high precision mode that generates random conformations with 1,000 steps of dynamics. The top pose results were taken for the interactions study.


RESULTS AND DISCUSSION

Chemistry

The synthesis of intermediates and final compound structures was achieved as per the structure shown in Scheme 1. 2-aminothiophenol was reacted with 2-chloroacetic acid in the presence of sodium acetate to obtain 2H-benzo[b][1, 4] thiazin-3(4H)-one (1) (Shaikh et al., 2016; Rajender et al., 2019). N-alkylation of compound 1 was achieved through ethyl bromoacetate to offer ethyl 4-( benzo[1,4]thiazin-3-one)acetate (2). The 4-(benzo[1,4]thiazin-3-one) acetohydrazide (3) (Krishna et al., 2014) was prepared by a reaction between compound 2 and hydrazine hydrate in ethanol at an ambient temperature on overnight stirring. The reaction of acetohydrazide (3) with different alkyl/aryl isothiocyanates yields 4-(benzo[1,4]thiazin-3-one)carbothioamides (4a–f). The 4-((4-aryl/cycloalkyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)-2H-benzo[b][1,4]thiazin-3(4H)-ones (5a–f) were conveniently synthesized from carbothioamides (4a–f) by reflexing under the strong alkaline (2N NaOH) condition, followed by acidification with 2N HCl (Karabanovich et al., 2019). The target compounds, S-(5-((3-oxo-2,3-dihydro-4H-benzo[b][1,4]thiazin-4-yl)methyl)-4-aryl/alkyl-4H-1,2,4-triazol-3-yl) 4-nitrobenzothioates (Rabie et al., 2017) (6a–d) were synthesized by S-alkylation of appropriate compounds (5a–c and 5e) with 4-nitrobenzoyl chloride in a solvent of dry acetone and cesium carbonate (Cs2CO3). All the intermediates and final compounds were synthesized in good yield (69%–75%). The reaction’s progress was checked by TLC through an appropriate solvent system in each reaction step. The compounds’ structures were characterized by a spectral study and are consistent with the proposed structures for intermediates and final compounds.

Scheme 1. Synthesis of benzothiazinone linked 1,2,4-triazoles.

[Click here to view]

The IR spectrum of 2H-benzo[b][1, 4]thiazin-3(4H)-one (1) exhibits a characteristic peak at 3,359 cm−1 for NH, 1,702 cm−1 for C=O, and at 3,024 cm−1 for aromatic C-H group. The 1H NMR spectrum of compound 1 integrates for seven protons. The multiplet peaks at 6.80–7.31 ppm integrate for aromatic protons, a singlet at 8.61 ppm for the proton of NH, and a singlet at 3.42 ppm for the CH2 protons of benzothiazinone indicates that compound 1 was formed. The IR spectral analysis of compound 2 exhibits two characteristic carbonyl peaks in the regions of 1,654, 1,702, and 3,026 cm−1 for the aromatic C-H group. The 1H NMR spectrum of compound 2 integrates for 13 protons. The multiplet at 6.92–7.30 ppm integrates for aromatic protons. A triplet resonates at 1.46 ppm for CH3 protons of ester. A multiplet of OCH2 protons at 4.23 ppm and a singlet at 4.43 ppm for CH2 protons confirms the formation of N-alkylation and compound 2. The IR spectrum of compound 3 exhibits two characteristic carbonyl peaks in the regions of 1,654 and 1,765 cm−1, broad peaks at 3,365 and 3,630 cm−1 of the primary and secondary amine of hydrazide and 3,024 cm−1 for aromatic C-H group. The 1H NMR spectrum of compound 3 integrates for 11 protons. A multiplet at 7.1–7.3 ppm integrates four aromatic protons, and a singlet resonates at 4.43 ppm for CH2 protons of benzothiazinone. The absence of ester peaks and the presence of hydrazide (NHNH2) protons as two singlets at 9.63 ppm and 10.30 ppm, respectively, confirm compound 3. The IR spectrum of compounds 4a–4f exhibits two characteristic carbonyl peaks in the regions of 1,686 and 1,731 cm−1, a broad peak at 3,351 cm−1 N-H, at 3,039 cm−1 for aromatic C-H group, and a peak at 1,585 cm−1 for C=S. The IR spectrum of 5a exhibits a characteristic carbonyl group peak in the region of 1,724cm−1, a broad peak at 3,340 cm−1 for N-H, a peak at 3,024 cm−1 for aromatic C-H group, and a peak at 1,585 cm1 for C=S group. The 1H NMR spectrum of compound 5a integrates for 14 protons. A multiplet at 6.53–7.55 ppm integrates for aromatic protons, a singlet at 3.41 ppm for CH2 protons and a singlet at 4.23 ppm for CH2 protons of benzothiazinone. A singlet peak at 13.78 ppm of NH proton of triazole (NH-C=S) confirms the formation of compound 5a. The IR spectrum of 6a exhibited a characteristic carbonyl group peak in the region of 1,724 cm−1 and at3,024 cm−1 for aromatic C-H group, and a peak at 1,578 cm−1 for the N-O group. The 1H NMR spectrum of compound 6a integrates for 17 protons. A multiplet peak at 6.91–8.03 ppm integrates for aromatic protons and protons of nitroaromatic appears as a multiplet at 8.26–8.38 ppm, a singlet at 3.41 ppm for CH2 protons and a singlet at 4.23 ppm for CH2 protons of benzothiazinone confirms the formation of the compound 6a.

In-vitro antitubercular activity and CC50

Synthesized benzothiazinone encompassed 3,5 disubstituted-1,2,4-triazoles (5a–f and 6a–d) were tested for in vitro anti-TB activity against the Mtb strain of H37Rv. The antitubercular activity of the synthesized molecules was tested at 100–0.8 μg/ml concentrations by Microplate Alamar Blue Assay using antitubercular drugs, namely pyrazinamide, ciprofloxacin, streptomycin as the reference standard. The antitubercular activity data are depicted in Table 1. Synthesized compounds have antitubercular activity in the range of 12.5–100 μg/ml concentration. The data indicate that the compounds 5c, 5e, and 6c were promising candidates during the study and demonstrated MIC at 12.5 μg/ml. Molecule 6d has an MIC at 25 μg/ml, while 5a, 5b, 5d, 5f, and 6a have shown modest activity (MIC = 50 μg/ml). The compound 6b showed the least antitubercular activity at 100 μg/ml concentration. It was observed from the data that antitubercular activity depends on substituent groups on the phenyl group at fourth position of the 1, 2, 4-triazole ring. The electron-withdrawing group (OCH3) on phenyl group in 5c and an electronegative atom in 5e on phenyl ring exhibited good activity among the 5 series compounds. The substitution of 4-nitrobenzoyl group by thiol hydrogen of the 5 series did not result in better antitubercular activity. Only compound 6c retained activity among the 6 series compounds. The CC50 data of the synthesized compounds against HEK293T reveal that the compounds require >132.26 μg/ml concentration to kill 50% of the viable cells. This indicates that the anti-TB activity of the synthesized molecules is not caused by the cytotoxic effect. Furthermore, it was observed that the introduction of 4-nitrobenzoyl group at the 3-thiol of triazole ring in the final compounds did not form covalent interactions with DprE1, which is evident from the molecular docking study and further exhibited CC50 slightly more (lower CC50 values) than the 5 series compounds. However, the 4-nitrobenzoyl group did not pay to the enhancement of antitubercular action.

In silico ADMET prediction

The ADMET properties of designed molecules were measured through ADMETSAR and Discovery Studio 19.0. The designed molecules showed suitable value for the evaluated in silico parameters (Table 2). The predicted ADMET data show that the molecules have considerable oral bioavailability and moderate acute oral toxicity. The predicted oral availability human intestinal absorption (HIA) was excellent, which displayed a calculated probability of 0.85–0.98. The predicted blood–brain barrier (BBB) penetration values suggest good penetration in the range of 0.74–0.98. The compound also exhibited moderate Caco2 cell permeability, ranging from 0.5 to 0.59 and acute oral toxicity within the range of 0.56–0.61. The literature evidence suggests that the control of lipophilicity within a particular range can increase compound features and the chance of therapeutic achievement (Arnott et al., 2012; Shaikh et al., 2016). The R in the final compounds is substituted with lipophilic modifiers to increase lipophilicity (no defilement of rule of five) values of the designed compounds and introduction of nitroaromatic functionality to realize the antitubercular potentials in additions to the final core heterocyclic moieties. The predicted clogP data (Table 2) substantiate the increase of lipophilicity of the final molecules.

Table 1. Molecular docking and in vitro biological activity data of structures synthesized.



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Table 2. In-silico ADMET and clogP data of synthesized compounds.



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Molecular docking study

In silico prediction of ligand interactions to the target DprE1 receptor’s binding site is understood through molecular docking. The application populates individual ligands’ respective scores and the best conformation of different ligands (Fig. 2) to the active site. The adopted docking protocol indicates the binding affinities of designed and synthesized ligands into the binding site of the DprE1. The validation of docking was conducted by re-docking the co-crystalized ligand and evaluating the with RMSD (Root-Mean-Square Deviation) analysis. It was found that the co-crystallized ligand interacted identically to the binding site, as mentioned in the PDB. The binding affinities for respective ligands are indicated in Table 1, which indicates the individual affinities to the binding site. The compounds 6ad were found to be the best from the docking score. Most interacting amino acids and cofactor are LYS418, LYS134, GLN336, ASN385, PHE332, and LEU317 with FAD501, shared among all the ligands in a bound form to the DprE1 active site. Validation of docking protocol was confirmed with superimposing pre- and post-dock structures of co-crystalized ligand, having an RMSD value of 1.85 Å. Analysis of docking poses reveals the presence of hydrogen bonds and other hydrophobic interactions.

Figure 2. Docking interaction and binding mode of (a,b) 6c in 3D and 2D forms at the binding site of DPrE1, (c,d) co-crystallized ligand in 3D and 2D forms at the binding site of DPrE1, (e) superposition of pre-and post-dock confirmation of co-crystalized ligand, where the cyan and purple colors represent the confirmations, respectively.

[Click here to view]

Figure 3. 2D interaction analysis of the nitroso derivative of 6c and 4PFD.

[Click here to view]

The 2D interaction analysis suggests that both compound 6c and the non-covalent co-crystallized ligand cBT ([4-(2-methoxyethyl)piperazin-1-yl][6-methyl-7-nitro-5-(trifluoromethyl)-1, 3-benzothiazol-2-yl]methanone) forms similar π-sulfur stacking interactions between the sulfur group of CYS387 and π electrons of the phenyl ring and benzothiazole ring, respectively, at the DprE1 active site. In the known non-covalent inhibitors (BTZ 043 and PBTZ 169) of DprE1, the nitro group is present in the structure’s core nucleus (benzothiazinone), while in our title compounds, the nitro group is in the side chain. Furthermore, amino acid residue CYS387 and nitro of BTZ043 is a mechanism-based covalent inhibition.

The change of the nitro group of BTZ043 to the active nitroso form requires the protein’s enzymatic activity with the substrate (Piton et al., 2017). For further clarification, we docked our active moiety with DprE1 (4PFD) by slightly changing our compound 6c from the nitro to nitroso group to reveal any possible interaction that may occur with nitrogen in the nitroso group and amino acid residue CYS387. The 2D interaction analysis showed (Fig. 3) that CYS387 does not form any electrostatic interaction with the nitroso group. We suggest that the title compounds may interact with a binding cavity of DprE1 in a non-covalent fashion from the above discussion. It was observed that the introduction of the nitro benzoyl moiety in the final compounds did not form covalent interactions with DprE1, hence less contributed to the overall toxicity of the final compounds. The docking protocol so adopted is in good correlation to the in vitro antitubercular activity.


CONCLUSION

New benzothiazinone linked 1,2,4-triazoles (5a–5f, 6a–d) were synthesized conveniently and characterized with the help of spectral studies. The in silico ADMET properties data provided the desired draggability of the synthesized compounds. Compounds 5c, 5e, and 6c were found to be active antitubercular agents among the reported derivatives. It was observed from the data that antitubercular activity depends on substituent groups on phenyl moiety at fourth position of 1, 2, 4-triazole ring. The electron-withdrawing group (OCH3) on the phenyl group in 5c and an electronegative atom in 5e on the phenyl ring exhibited good activity. The substitution of the 4-nitrobenzoyl group by thiol hydrogen of the 5 series did not result in a better antitubercular activity. Only the compound 6c retained activity among the 6 series compounds. The CC50 data against HEK293T reveal that the title compounds’ antitubercular activity is not due to CC50. The compounds 6a–d showed good molecular docking scores, and their interactions with amino acids with active site pockets of the DprE1 are in line with the reference ligand. Hence, the study’s data may provide the basis for the development of benzo[1, 4]thiazin-3(4H)-one encompassed 1,2,4-triazole scaffolds as potential DprE1 inhibitors.


ACKNOWLEDGMENTS

The authors are thankful to the Principal, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysore, India, for providing necessary facilities. The authors express their thanks to the Institution of Excellence, Vijnana Bhavan, Manasagangotri, Mysuru-570006, for spectral data. They also express their thanks to the Department of Microbiology and Molecular Biology, Maratha Mandal’s NGH Institute of Dental Sciences and Research Centre, Belgaum-590 010, Karnataka, for antitubercular studies.


AUTHOR CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.


FUNDING

There is no external funding to report.


CONFLICTS OF INTEREST

The authors report no financial or any other conflicts of interest in this work.


ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.


PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.


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