Synthesis, biological evaluation and computational study of novel isoniazid containing 4H-Pyrimido[2,1-b]benzothiazoles derivatives

European Journal of Medicinal Chemistry 177 (2019) 12e31

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis, biological evaluation and computational study of novel isoniazid containing 4H-Pyrimido[2,1-b]benzothiazoles derivatives Manoj N. Bhoi a, *, Mayuri A. Borad a, Divya J. Jethava a, Prachi T. Acharya a, Edwin A. Pithawala b, Chirag N. Patel c, Himanshu A. Pandya c, Hitesh D. Patel a, ** a b c

Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, India Department of Life Sciences, School of Sciences, Gujarat University, Ahmedabad, India Department of Bioinformatics, Applied Botany Centre (ABC), School of Sciences, Gujarat University, Ahmedabad, 380009, Gujarat, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 December 2017 Received in revised form 23 March 2019 Accepted 7 May 2019 Available online 11 May 2019

Synthesis of novel and potent hit molecules has an eternal demand. It is our continuous study to search novel bioactive hit molecules and as a part of this, a series of novel N0 -isonicotinoyl-2-methyl-4-(pyridin2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide analogs (5a-5n) were synthesized with good yields by the conventional method. The various novel compounds have been characterized and identified by many analytical technique such as IR, 1H NMR, 13C NMR, mass spectral analysis, and elemental analysis. All the synthetic analogs (5a-5n) are evaluated for their in vitro antibacterial and antimycobacterial activities against different bacterial strains. Molecular docking and Molecular dynamics studies were helped in revealing the mode of action of these compounds through their interactions with the active site of the Mycobacterium tuberculosis enoyl reductase (InhA) enzyme. The calculated ADMET descriptors for the synthesized compounds validated good pharmacokinetic properties, confirming that these compounds could be used as templates for the development of new Anti-mycobacterial agents. © 2019 Published by Elsevier Masson SAS.

Keywords: ADMET Antibacterial Anti-mycobacterial Isoniazid Molecular docking Molecular dynamics Pyrimido-benzothiazoles Tuberculosis

1. Introduction Tuberculosis (TB) is a one of the most predominant and lethal airborne communicable diseases which are a long-lasting disorder caused by five closely related mycobacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canetti. Amongst them, Mycobacterium tuberculosis (MTB) is one of the most responsible causative agent for TB which is in either latent or active form. Almost, one-third of the world's population is infected with M. tuberculosis, and 5e10% of infected individuals are prone to develop active TB disease in their lifetime. As a result of this, around 9 million new cases of active TB disease and 1.5 million death noted every year. According to the World Health Organization (WHO, Global Tuberculosis Report 2015), 9.6 million people are estimated

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] rediffmail.com (H.D. Patel).

(M.N.

https://doi.org/10.1016/j.ejmech.2019.05.028 0223-5234/© 2019 Published by Elsevier Masson SAS.

Bhoi),

hitesh13chem@

to have TB (5.4 million men, 3.2 million women and 1.0 million children) and 1.5 million (1.1 million HIV-negative, and 0.4 million HIV-positive) death per year was reported in 2014 [1e3]. Mostly, TB is caused by acid-fast bacillus M. tuberculosis that mainly infects the lungs (pulmonary TB), and other organs in the body (extra pulmonary TB) including the kidney, liver, and brain to some extent. The traditional first-line treatment of drug-sensitive TB infections includes four-drug regimen namely isoniazid, rifampin, pyrazinamide, and ethambutol [4,5]. The existence of MTB in diverse physiological conditions and its pathogenesis with complex biology poses specific challenges to drug discovery against TB. It is estimated that one billion people will be newly infected with around 125 million people in receipt of sickness, and 14 million dead in the next ten years [6e12]. Therefore, there is huge scope for the development of the new effective chemotherapeutic drug with less complexity, cheaper and minimum side effects. The result of inadequate therapy and poor compliance also contribute significantly for the emergence of resistant Multi-Drug Resistant (MDR) to isoniazid and rifampicin, and extensively drug-resistant (XDR) strains, resistant to a fluoroquinolone [12]. Thus, There is an urgent need for the development of novel anti-TB drug effective against

M.N. Bhoi et al. / European Journal of Medicinal Chemistry 177 (2019) 12e31

both drug sensitive and resistant M. tuberculosis (MTB). [13]. In view of this, a great deal of research work is being dedicated to find novel molecular entities which are active against the bacterial strains. The most important molecular drug design strategies in this direction are (i) computer-aided drug design (CADD) for ligand-protein interaction [14] (ii) two active pharmacophore units into a single molecular framework using molecular hybridization [15,16] (iii) structural modification of well-known marketed drug molecules [17] and (iv) the random screening of different structural units and proceeding in an observed window of anti-Tuberculosis activity (blind study) [18,19], all these above approaches are considered as promising for the development of effective drugs [20,21]. Numerous anti-tubercular drugs were developed over the past decades (Fig. 1), however, known drug-resistance issue has not been solved satisfactorily as yet. Thus, there is an urgent need to develop new anti-tubercular drugs that are active against acute as well as chronic growth phases of Mycobacterium to stop all form into drug resistant-TB [22,23]. However, a number of new known drug candidates or repurposed drugs namely gatifloxacin, moxifloxacin, rifapentine, TMC207, OPC67683, PA824, linezolid, PNU100480, AZD5847, SQ109, etc. have been developed in recent years, out of which some are also in the advanced stages of clinical trials for the treatment of tuberculosis [24]. Bedaquiline is only one

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of the newly approved drug by the Food and Drug Administration (FDA) for drug-resistant tuberculosis (Fig. 2) [25,26]. In this point of view, many researcher have worked for drug discovery on targeting the cell wall of mycobacteria. Biosynthesis of mycolic acid has been carried out [27] by numerous successive enzymatic cycles equivalent of Fatty Acid Synthase (FAS) systems viz., FAS I and II. Mycolic acid is a unique fatty acid, which is a core integral part of the mycobacterial cell wall present in the fatty acid synthase system of M. tuberculosis (MTB). InhA, the enoyl-acyl carrier protein reductase (ENR) from M. tuberculosis is the key enzyme for type II fatty acid synthesis [28] (FAS II), which catalyses NADH-dependent reduction of 2-transenoyl-ACP (acyl carrier protein) to yield NADþ and reduced enoyl thioester-ACP substrate, which in turn, helps the synthesis of mycolic acid [29,30]. Isoniazid (INH) has been widely used as a frontline antitubercular drug for the past 40 years. It is a prodrug and must be activated by bacterial catalase [31,32]. It is activated by the mycobacterial catalase-peroxidase enzyme known as KatG. The activated form reacts with the coenzyme NADH to form an isonicotinic acylNADH complex [29,30] that binds with the enoyl-acyl carrier protein (ACP) reductase InhA, which involves in the elongation of fatty acids during the mycolic acid synthesis [33]. Thus, isoniazid inhibits the synthesis of mycolic acid which required for the mycobacterial

Fig. 1. Milestone in TB Drug research.

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Fig. 2. Mechanism of action of the anti-tuberculosis drug. The drug already in use shown in blue, the newly approved drug are shown in pink & drug undergoing clinical trial are shown in red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

cell wall. INH is metabolized in the liver and forms hydrazine, which is toxic for the central nervous system and other organs [34,35]. Since INH is an important drug in the therapeutic arsenal for TB treatment. Many efforts are being made to develop new INH derivatives with good advantages such as greater activity, lower toxicity and less side effects than INH [36,37]. Several recent reports indicate that the combination of hydrophobic moieties into the basic structure of INH can enhance penetration of the drug into the highly lipophilic bacterium cell wall. In addition, by functioning property of the hydrazine group of isoniazid and retaining its activity, toxicity and other severe problems related to the inactivation of isoniazid by the enzyme N-acetyltransferase-2 (NAT2) can be avoided [24]. The pyridine scaffold is the most common N-hetero aromatics which combined with the structure of various therapeutic agents to get good biological activity. Many naturally occurring and synthetic compounds contain pyridine scaffold which possess a broad spectrum of biological activities [38]. On the other hand, benzothiazole containing alkaloids are relatively rare in nature but compounds containing benzothiazole ring displays a wide-ranging spectrum of various biological activities and used in material science and in other industrial applications [39,40]. Encouraged by the previous studies [39,40] and our continuous efforts toward the synthesis of new anti-tuberculosis agents, we have carried out an amalgamation of three biologically versatile heterocyclic scaffolds like Isoniazid, pyridine, and pyrimido[2,1-b]

benzothiazoles in the single molecular platform. We have attached isoniazid linkage to our previous reported various derivatives of Ethyl-2-methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a] pyrimidine-3-carboxylate to form N0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4H-benzo [4,5] thiazolo[3,2-a]pyrimidine-3carbohydrazide derivatives by using the conventional method. The synthesized compounds were screened for their in vitro antibacterial activity against Gram-positive and Gram-negative bacterial strains, and also screened in vitro anti-mycobacterial activity against M. tuberculosis H37Rv. We have carried out molecular docking study correlating with the docking score and biological activity for novel synthesized compounds. We have also carried out molecular dynamics and in silico ADMET prediction of synthesized compounds.

2. Materials and methods 2.1. Chemistry All the required chemicals and solvents were purchased from commercial sources (sigma Aldrich and Merck chemical company). The progress of reactions were monitored by thin layer chromatography (TLC) and judged by the consumption of starting material. TLC was visualized by UV radiation, exposure to iodine vapor and various spray reagent. Pre-coated aluminum sheets (Silica gel G 60 F254, Merck Germany) were employed for thin layer

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chromatography (TLC). Column chromatography was performed using silica gel 60 (60e120 mesh size). The melting points recorded on optimelt automated melting point system which was uncorrected. IR spectra were recorded on a PerkinElmer 377 spectrophotometer in KBr with absorption in cm1. Elemental analysis was performed on vario MICRO cube, elementar CHNS analyser serial no.: 15084053. 1H- NMR and 13C NMR spectra were recorded on Bruker AV 400 and 100 MHz using DMSO‑d6 as solvent and TMS as an internal standard. Splitting patterns are designated as follows; s, singlet; d, doublet; m, multiplet dd, double doublet, etc … Chemical shift values are given in ppm. Mass spectra were recorded on Advion Expression CMS, USA, using Methanol: Water: Formic acid (80: 20: 0.1) as the mobile phase. 2.2. General process of the synthesis of N0 -isonicotinoyl-2-methyl4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine-3carbohydrazide derivatives (5a-5n) To a stirred solution of previously synthesized derivatives (4a4n) (1.422 mmol, 1 eq.) in methanol (10 mL) was dropwise added hydrazine hydrate solution (80% w/v, 3.555 mmol, 2.5 eq.) in the presence of catalytic amount of con. H2SO4. The resulting reaction mixture was refluxed for 5 h-7h. The completion of the reaction was monitored on TLC using 30% Ethyl acetate: hexane as a solvent system. After completion of the reaction, the reaction mixture was poured in ice-water to afford precipitates out in good practical yields which was then filtered, dried using vacuum to get crude intermediate hydrazine hydrate derivative of Ethyl-2-methyl-4(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine-3carboxylate. Then intermediate hydrazine hydrate derivative (1.481 mmol, 1 eq.) in Dichloromethane was added triethylamine (3.702 mmol, 2.5 eq.) and followed by hydrochloride salt of isonicotinoyl chloride (1.481 mmol, 1 eq.) at 0  C temperature. The resulting reaction mixture was refluxed for 6 h. The progress of the reaction was monitored by TLC. After competition of the reaction, the reaction mixture was diluted with water (25 mL), extracted with Dichloromethane (3  10 mL), the combined organic layer was washed with brine solution (20 mL) and dried over Na2SO4, filtered and evaporated. The crude material was further purified by column chromatography using 15e25% ethyl acetate in hexane as eluent to afford the targeted product (5a-5n) with good practical (65e80%) yields. 2.3. Characterizations data 2.3.1. N0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4H-benzo [4,5] thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5a) Solid, mp 123e124  C; Anal. Calcd for C23H18N6O2S: C, 62.43; H, 4.10; N, 18.99; O, 7.23; S, 7.25%; found C, 62.56; H, 4.23; N, 18.85; S, 7.36%; IR (KBr) (ymax, cm1): 3045 (C-HAro.str), 2945 (C-Hstr), 1662 (C¼Ostr), 1587 (C¼Nstr) 1512 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 11.94 (s, 1H), 9.66 (s, 1H), 8.97 (d, J ¼ 7.4 Hz, 2H), 8.67 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.95 (d, J ¼ 7.4 Hz, 2H), 7.81 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.44e7.31 (m, 2H), 7.07 (td, J ¼ 7.6, 1.6 Hz, 1H), 6.81e6.69 (m, 2H), 5.95 (s, 1H), 2.34 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.91, 166.43, 158.56, 157.23, 150.99, 150.69, 146.83, 141.39, 139.99, 139.47, 127.91, 127.43, 127.15, 124.58, 123.03, 121.61, 114.02, 106.79, 61.06, 18.09; ESI-MS: m/z Calculated 442.49, found [MþH] 443.4. 2.3.2. 8-Bromo-n0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5b) Solid, mp 145e146  C; Anal. Calcd for C23H17BrN6O2S: C, 52.98; H, 3.29; Br, 15.33; N, 16.12; O, 6.14; S, 6.15%; found C, 53.10; H, 3.18; N, 16.26; S, 6.28%; IR (KBr) (ymax, cm1): 3054 (C-HAro.str), 2950 (C-

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Hstr), 1653 (C¼Ostr), 1545 (C¼Nstr), 1470 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 9.77 (s, 1H), 9.68 (s, 1H), 8.98 (d, J ¼ 7.2 Hz, 2H), 8.73 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.93 (d, J ¼ 7.4 Hz, 2H), 7.81 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.68 (d, J ¼ 1.4 Hz, 1H), 7.61 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.37 (td, J ¼ 7.4, 1.5 Hz, 1H), 7.23 (dd, J ¼ 7.4, 1.5 Hz, 1H), 6.59 (d, J ¼ 7.5 Hz, 1H), 5.92 (s, 1H), 2.24 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.22, 150.99, 150.68, 146.82, 143.48, 139.99, 139.47, 128.81, 127.90, 126.09, 124.22, 123.54, 123.03, 121.55, 115.44, 106.80, 61.07, 18.11; ESI-MS: m/z Calculated 521.39, found [MþH] 522.3. 2.3.3. N0 -isonicotinoyl-2,8-dimethyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5c) Solid, mp 139e140  C; Anal. Calcd for C24H20N6O2S: C, 63.14; H, 4.42; N, 18.41; O, 7.01; S, 7.02%; found C, 63.26; H, 4.56; N, 18.39; S, 7.12; IR (KBr) (ymax, cm1): 3010 (C-HAro.str), 2860 (C-Hstr), 1687 (C¼Ostr), 1540 (C¼Nstr), 1452 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 12.35 (s, 1H), 9.05 (s, 1H), 8.98 (d, J ¼ 7.4 Hz, 2H), 8.66 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.93 (d, J ¼ 7.4 Hz, 2H), 7.79 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.56 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.39e7.30 (m, 2H), 6.92 (dd, J ¼ 7.5, 1.4 Hz, 1H), 6.66 (d, J ¼ 7.5 Hz, 1H), 5.85 (s, 1H), 2.24 (s, 3H), 2.12 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.22, 150.99, 150.68, 146.82, 140.74, 139.99, 139.47, 135.26, 128.24, 127.90, 124.00, 123.03, 122.43, 121.55, 112.72, 106.80, 61.07, 21.20, 18.11; ESI-MS: m/ z Calculated 456.52, found [MþH] 457.5. 2.3.4. N0 -isonicotinoyl-2,6-dimethyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5d) Solid, mp 140e141  C; Anal. Calcd for C24H20N6O2S: C, 63.14; H, 4.42; N, 18.41; O, 7.01; S, 7.02%; found C, 63.39; H, 4.59; N, 18.46; S, 7.21; IR (KBr) (ymax, cm1): 3089 (C-HAro.str), 2973 (C-Hstr), 1685 (C¼Ostr), 1575 (C¼Nstr), 1485 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 9.45 (s, 1H), 9.28 (s, 1H), 9.00 (d, J ¼ 7.4 Hz, 2H), 8.60 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.95 (d, J ¼ 7.4 Hz, 2H), 7.83 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.72 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.38e7.26 (m, 2H), 6.91 (dd, J ¼ 7.5, 1.4 Hz, 1H), 6.71 (t, J ¼ 7.4 Hz, 1H), 5.45 (s, 1H), 2.35 (d, J ¼ 5.5 Hz, 6H); 13C NMR (100 MHz, DMSO) d 166.90, 166.09, 158.56, 157.22, 150.99, 150.68, 146.82, 139.99, 139.50, 130.19, 128.61, 127.90, 127.01, 123.03, 121.55, 118.62, 118.08, 106.80, 60.74, 18.06; ESI-MS: m/z Calculated 456.52, found [MþH] 457.5. 2.3.5. N0 -isonicotinoyl-2-methyl-8-nitro-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo[3,2-a] pyrimidine-3-carbohydrazide (5e) Solid, mp 162e163  C; Anal. Calcd for C23H17N7O4S: C, 56.67; H, 3.52; N, 20.11; O, 13.13; S, 6.58%; found C, 56.75; H, 3.62; N, 20.26; S, 6.62; IR (KBr) (ymax, cm1): 3075 (C-HAro.str), 2955 (C-Hstr), 1655 (C¼Ostr), 1552 (C¼Nstr), 1545 and 1365 (N-Ostr), 1505 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 12.37 (s, 1H), 9.88 (s, 1H), 8.97 (d, J ¼ 7.4 Hz, 2H), 8.67 (dd, J ¼ 7.4, 1.5 Hz, 1H), 8.43 (d, J ¼ 1.4 Hz, 1H), 8.01 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.94 (d, J ¼ 7.4 Hz, 2H), 7.82 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.59 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.36 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.03 (d, J ¼ 7.5 Hz, 1H), 5.94 (s, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.91, 166.43, 158.56, 157.23, 150.99, 150.69, 147.15, 146.83, 145.83, 139.99, 139.47, 133.29, 127.91, 123.62, 123.03, 121.55, 115.54, 112.10, 106.79, 61.06, 18.09; ESI-MS: m/z Calculated 487.49, found [MþH] 488.4. 2.3.6. 8-Chloro-n0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo [3,2-a] pyrimidine-3-carbohydrazide (5f) Solid, mp 160e161  C; Anal. Calcd for C23H17ClN6O2S: C, 57.92; H, 3.59; Cl, 7.43; N, 17.62; O, 6.71; S, 6.72%; found C, 58.09; H, 3.69; N, 17.75; S, 6.84; IR (KBr) (ymax, cm1): 3090 (C-HAro.str), 2890 (CHstr), 1670 (C¼Ostr), 1576 (C¼Nstr), 1481 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 9.96 (s, 1H), 9.79 (s, 1H), 8.99 (d, J ¼ 7.4 Hz, 2H), 8.68 (dd, J ¼ 7.4, 1.5 Hz, 1H), 7.95 (d, J ¼ 7.4 Hz, 2H), 7.81 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.51 (d, J ¼ 1.4 Hz, 1H), 7.36

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(td, J ¼ 7.5, 1.5 Hz, 1H), 7.07 (dd, J ¼ 7.5, 1.4 Hz, 1H), 6.65 (d, J ¼ 7.5 Hz, 1H), 5.91 (s, 1H), 2.28 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.22, 150.99, 150.68, 146.82, 141.00, 139.99, 139.47, 132.26, 129.15, 127.90, 127.13, 123.03, 121.55, 120.29, 117.30, 106.80, 61.07, 18.11; ESI-MS: m/z Calculated 476.94, found [MþH] 477.9. 2.3.7. 6-Chloro-n0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5g) Solid, mp 170e171  C; Anal. Calcd for C23H17ClN6O2S: C, 57.92; H, 3.59; Cl, 7.43; N, 17.62; O, 6.71; S, 6.72%; found C, 58.02; H, 3.61; N, 17.72; S, 6.83; IR (KBr) (ymax, cm1): 3072 (C-HAro.str), 2952 (CHstr), 1666 (C¼Ostr), 1545 (C¼Nstr), 1495 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 11.13 (s, 1H), 9.01e8.91 (m, 3H), 8.60 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.96 (d, J ¼ 7.4 Hz, 2H), 7.84 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.68 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.53e7.26 (m, 2H), 7.05 (dd, J ¼ 7.5, 1.4 Hz, 1H), 6.70 (t, J ¼ 7.4 Hz, 1H), 5.74 (s, 1H), 2.33 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.09, 158.56, 157.22, 150.99, 150.68, 146.82, 139.99, 139.47, 137.37, 129.08, 128.21, 127.90, 125.88, 123.71, 123.03, 121.55, 121.02, 106.80, 60.74, 18.11; ESI-MS: m/z Calculated 476.94, found [MþH] 477.9. 2.3.8. 8-Fluoro-n0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5h) Solid, mp 150e151  C; Anal. Calcd for C23H17FN6O2S: C, 59.99; H, 3.72; F, 4.13; N, 18.25; O, 6.95; S, 6.96%; found C, 60.19; H, 3.86; N, 18.36; S, 6.85; IR (KBr) (ymax, cm1): 3050 (C-HAro.str), 2850 (C-Hstr), 1656 (C¼Ostr), 1565 (C¼Nstr), 1466 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 10.99 (s, 1H), 9.06e8.94 (m, 3H), 8.66 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.93 (d, J ¼ 7.4 Hz, 2H), 7.79 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.56 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.35 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.25 (dd, J ¼ 8.0, 1.4 Hz, 1H), 6.86e6.69 (m, 2H), 5.91 (s, 1H), 2.14 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 160.78, 158.56, 158.16, 157.22, 150.99, 150.68, 146.82, 139.99, 139.47, 138.13, 127.90, 123.03, 121.55, 118.12, 117.85, 115.88, 110.15, 106.80, 61.07, 18.11; ESI-MS: m/z Calculated 460.49, found [MþH] 461.4. 2.3.9. N0 -isonicotinoyl-8-methoxy-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5i) Solid, mp 142e143  C; Anal. Calcd for C24H20N6O2S: C, 61.00; H, 4.27; N, 17.79; O, 10.16; S, 6.79%; found C, 61.19; H, 4.35; N, 17.85; S, 6.79; IR (KBr) (ymax, cm1): 3012 (C-HAro.str), 2970 (C-Hstr), 1669 (C¼Ostr), 1580 (C¼Cstr), 1287 (C-Nstr); 1H NMR (400 MHz, DMSO) d 11.95 (s, 1H), 9.67 (s, 1H), 8.97 (d, J ¼ 7.4 Hz, 2H), 8.67 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.95 (d, J ¼ 7.4 Hz, 2H), 7.81 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.36 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.09 (d, J ¼ 1.4 Hz, 1H), 6.67 (dt, J ¼ 7.5, 4.3 Hz, 2H), 5.95 (s, 1H), 3.79 (s, 3H), 2.34 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.59, 157.22, 150.99, 150.68, 146.82, 139.99, 139.47, 136.71, 127.90, 127.48, 123.03, 121.55, 118.44, 114.13, 111.14, 106.80, 61.07, 56.03, 18.11; ESIMS: m/z Calculated 472.52, found [MþH] 473.5. 2.3.10. 8-Ethoxy-n0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo[3,2-a] pyrimidine-3-carbohydrazide (5j) Solid, mp 175e176  C; Anal. Calcd for C25H22N6O3S: C, 61.72; H, 4.56; N, 17.27; O, 9.86; S, 6.59%; found C, 61.85; H, 4.63; N, 17.42; S, 6.67; IR (KBr) (ymax, cm1): 3034 (C-HAro.str), 2876 (C-Hstr), 1672 (C¼Ostr), 1573 (C¼Nstr), 1456 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 11.95 (s, 1H), 9.65 (s, 1H), 8.97 (d, J ¼ 7.4 Hz, 2H), 8.67 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.95 (d, J ¼ 7.4 Hz, 2H), 7.81 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.35 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.11 (d, J ¼ 1.4 Hz, 1H), 6.72e6.62 (m, 2H), 5.95 (s, 1H), 4.03 (q, J ¼ 5.9 Hz, 2H), 2.34 (s, 3H), 1.39 (t, J ¼ 5.9 Hz, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.29, 150.99, 150.68, 146.82, 139.99, 139.47, 136.45, 127.90, 127.10, 123.03, 121.55, 118.40, 115.69, 112.51, 106.80, 63.98, 61.07, 18.11, 13.82; ESI-MS: m/z Calculated 486.55, found [MþH]

487.5. 2.3.11. N0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-8(trifluoromethoxy)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine-3carbohydrazide (5k) Solid, mp 182e185  C; Anal. Calcd for C24H17F3N6O3S: C, 54.75; H, 3.25; F, 10.83; N, 15.96; O, 9.12; S, 6.09%; found C, 54.86; H, 3.50; N, 15.89; S, 6.21; IR (KBr) (ymax, cm1): 3070 (C-HAro.str), 2957 (CHstr), 1676 (C¼Ostr), 1564 (C¼Nstr), 1448 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 11.96 (s, 1H), 9.67 (s, 1H), 8.97 (d, J ¼ 7.4 Hz, 2H), 8.67 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.95 (d, J ¼ 7.4 Hz, 2H), 7.81 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.36 (td, J ¼ 7.5, 1.6 Hz, 1H), 7.20 (t, J ¼ 4.9 Hz, 1H), 6.74e6.64 (m, 2H), 5.95 (s, 1H), 2.34 (s, 3H); 13 C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.22, 150.99, 150.68, 149.85, 146.82, 139.99, 139.47, 131.48, 127.90, 125.81, 125.36, 123.11, 122.75, 122.34, 121.55, 120.57, 117.95, 114.15, 106.80, 61.07,18.11; ESI-MS: m/z Calculated 526.49, found [MþH] 527.4. 2.3.12. 8-Hydroxy-n0 -isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo [3,2-a]pyrimidine-3-carbohydrazide (5l) Solid, mp 133e134  C; Anal. Calcd for C23H18N6O3S: C, 60.25; H, 3.96; N, 18.33; O, 10.47; S, 6.99%; found C, 60.39; H, 3.89; N, 18.51; S, 7.12; IR (KBr) (ymax, cm1): 3041 (C-HAro.str), 2850 (C-Hstr), 1688 (C¼Ostr), 1559 (C¼Nstr), 1472 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 9.71 (s, 1H), 9.58 (s, 1H), 8.98 (d, J ¼ 7.4 Hz, 2H), 8.70 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.93 (d, J ¼ 7.4 Hz, 2H), 7.80 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.59 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.37 (td, J ¼ 7.5, 1.4 Hz, 1H), 6.98 (d, J ¼ 1.3 Hz, 1H), 6.55 (dt, J ¼ 14.6, 4.4 Hz, 2H), 5.84 (s, 1H), 4.81 (s, 1H), 2.12 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.22, 154.78, 150.99, 150.68, 146.82, 139.99, 139.47, 134.83, 127.90, 126.04, 123.03, 121.55, 118.57, 113.54, 111.39, 106.80, 61.07, 18.11, 8.11e7.91; ESI-MS: m/z Calculated 458.50, found [MþH] 459.5. 2.3.13. N0 -isonicotinoyl-6-methoxy-2-methyl-4-(pyridin-2-yl)-4Hbenzo [4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide (5m) Solid, mp 145e146  C; Anal. Calcd for C24H20N6O3S: C, 61.01; H, 4.27; N, 17.79; O, 10.16; S, 6.78%; found C, 61.26; H, 4.35; N, 17.85; S, 6.86; IR (KBr) (ymax, cm1): 3025 (C-HAro.str), 2937 (C-Hstr), 1597 (C¼Ostr), 1562 (C¼Nstr), 1516 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 10.87 (s, 1H), 8.98 (d, J ¼ 7.4 Hz, 2H), 8.79 (s, 1H), 8.60 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.94 (d, J ¼ 7.4 Hz, 2H), 7.83 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.69 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.34 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.07 (dd, J ¼ 7.5, 1.6 Hz, 1H), 6.72 (t, J ¼ 7.5 Hz, 1H), 6.64 (dd, J ¼ 7.5, 1.4 Hz, 1H), 5.95 (s, 1H), 3.80 (s, 3H), 2.40 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.09, 158.56, 157.22, 150.99, 150.68, 149.39, 146.82, 139.99, 139.47, 131.25, 127.90, 123.03, 121.55, 119.01, 115.61, 114.84, 106.80, 60.74, 56.78, 18.11; ESI-MS: m/z Calculated 472.52, found [MþH] 473.5. 2.3.14. N0 -isonicotinoyl-2,7,8-trimethyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a] pyrimidine-3-carbohydrazide (5n) Solid, mp 152e153  C; Anal. Calcd for C25H22N6O2S: C, 63.81; H, 4.71; N, 17.86; O, 6.80; S, 6.81%; found C, 63.89; H, 4.82; N, 17.96; S, 6.76; IR (KBr) (ymax, cm1): 3056 (C-HAro.str), 2925 (C-Hstr), 1663 (C¼Ostr), 1564 (C¼Nstr), 1480 (C¼Cstr); 1H NMR (400 MHz, DMSO) d 12.35 (s, 1H), 9.05 (s, 1H), 8.98 (d, J ¼ 7.4 Hz, 2H), 8.66 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.93 (d, J ¼ 7.4 Hz, 2H), 7.79 (td, J ¼ 7.4, 1.5 Hz, 1H), 7.56 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.39e7.29 (m, 2H), 6.62 (s, 1H), 5.86 (s, 1H), 2.24 (d, J ¼ 9.5 Hz, 6H), 2.12 (s, 3H); 13C NMR (100 MHz, DMSO) d 166.90, 166.42, 158.56, 157.22, 150.99, 150.68, 146.82, 142.37, 139.99, 139.47, 135.68, 130.88, 127.90, 123.03, 121.55, 120.44, 119.48, 106.80, 61.07, 20.36, 18.11; ESI-MS: m/z Calculated 470.55, found [MþH] 471.5.

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Hed

Cc  Ct X 100 Cc

where, c ¼ control, t ¼ test. CYc

2.5.2. Method 2: LowensteineJensen (LJ) method for MIC value Drug susceptibility and determination of Minimum inhibitory concentration (MIC) of the test compounds (5a-5n) against Mycobacterium tuberculosis H37Rv were performed by LowensteinJensen (L-J) MIC method [40e42]

3 4 5 Yes, Yes, No, too good optimal soluble 2 Yes, low 0 1 Extremely No, very low, low but possible 2 3 Low Very low absorption absorption

solubility HIAa

Table 1 ADMET descriptors and their rules/keys.

In silico Absorption, distribution, metabolism, elimination, and toxicity (ADMET) properties were predicted using ADMET descriptors in Accelrys Discovery Studio 2.1. Structures of the compounds were saved for the ‘mol2’ format using Marvin suite software. The six mathematical models were used for the module, to quantitatively forecast properties by a set of rules/keys (see Table 1) that specifies threshold ADMET feature for the chemical structure of the molecules based on the available drug information. ADMET absorption predicts human intestinal absorption (HIA) after oral administration. The model was developed using 199 compounds in training set based on the calculations AlogP (ADMET_AlogP98) and 2D polar surface area (PSA_2D). The absorption levels of HIA model know like 95% and 99% confidence ellipses in the ADMET_PSA_2D, ADMET_AlogP98 plane [43]. These are ellipses regions somewhere well-absorbed compounds are expected to be found. At 95% and 99% confidence ellipsoid, the upper limit of PSA_2D value around 131.62, 148.12 respectively. The model for calculation of ADMET aqueous solubility of each compound at 25 C temperature in water is based on genetic partial least squares method that contains 784 compounds in training set with experimentally measured solubility [44]. After oral administration, ADMET blood-brain barrier model prognosticate blood-brain penetration (blood-brain barrier, BBB) of a molecule that was derived from a quantitative linear regression model for the forecast of blood-brain penetration with 95% and 99% confidence ellipses. BBB was resulting from 800 compounds that are known to enter the CNS after oral administration [45]. A model used in which a compound is likely to be highly bound to carrier proteins in the blood for the prediction of ADMET plasma protein binding. Two sets of “marker” molecules used for predictions based on 1D and AlogP98 similarities. Two sets of markers were used to flag binding at a level of 90% and 95% or greater. According to conditions on calculated logP value, the marker similarities are modified for binding level prediction [46]. ADMET CYP2D6 binding predicts cytochrome P450 2D6 enzyme inhibition using the 2D chemical structure as input as well as a ̊ probability estimate at the prediction. Predictions are based on a

BBBb

2.6. In silico ADMET/5 rule of Lipinsky rule

Level 0 1 Description Good Moderate absorption absorption

% inhibition ¼

PPBe

2.5.1. Method 1: LowensteineJensen (LJ) method for Percentage inhibition In vitro anti-mycobacterial activity of all synthesized compound was performed. Antimicrobial assay was performed in Lowenstein Jensen (L-J) medium and Colony forming units (c.f.u) on Lowenstein-Jensen (L-J) were determined [40].

*a ¼ human intestinal absorption, b ¼ blood brain barrier, c ¼ cytochrome P450 2D6 enzyme inhibition, d ¼ human hepatotoxicity, e ¼ plasma protein binding property.

2.5. In vitro anti-mycobacterial activity

0 Very High

Antibacterial activities of 5a-5n were carried out in Nutrientagar plates by well diffusion assay as reported by bhoi et al. [40].

1 2 3 0 1 0 1 0 1 High Medium Low Non Inhibitor Nontoxic Toxic Binding is Binding inhibitor <90% is  90%

2 Binding is  95%

2.4. In vitro antibacterial activity

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training set of 100 compounds with known CYP2D6 inhibitions [47]. ADMET hepatotoxicity predicts the potential human hepatotoxicity for a wide range of structurally diverse compounds. Predictions are based on an ensemble recursive partitioning model of 382 training compounds known to exhibit liver toxicity (i.e., positive dose-dependent cholesteric, hepatocellular, neoplastic, etc.) or to trigger dose-related elevated aminotransferase levels in more than 10% of the human population [44]. 2.7. Molecular docking study Molecular docking and Molecular dynamics were performed using the YASARA (Yet Another Scientific Artificial Reality Application) commercial package. Docking analysis is a significant step for the selection of potential hits in virtual screening. The YASARA energy parameter are assembled through docking poses, docking energy (kcal/mol) and root mean squared deviation (RMSD). The optimum docked pose identified with relative lower binding energy for further analysis. Better binding of ligands shows the better binding affinity through higher positive energies whereas no binding recognized by negative energies mean. The docking energy was calculated by the following equation [48].

DG ¼ DGvdW þ DGHbond þ DGelec þ DGtor þ DGdesolv Where.

DGvdW ¼ van der Waals term for docking energy DGHbond ¼ H bonding term for docking energy DGelec ¼ electrostatic term for docking energy DGtor ¼ torsional free energy term for ligand when the ligand transits from unbounded to bounded state

docked flexibly into the enzyme structures using with an extra precision scoring function to estimate the protein-ligand binding affinities. The active side of amino acid residues was determined which is being used to dock molecules for automated docking and scoring. 2.8. Molecular dynamics simulations We have carried out Molecular dynamics simulations for the conformational changes and binding stability of the designed ligands in complex with Crystal structure of Mycobacterium tuberculosis enoyl reductase (INHA) complexed with N-(3bromophenyl)-1-cyclohexyl-5-oxopyrrolidine-3-carboxamide protein. We have selected best docked ligand for the molecular dynamics simulation studies in YASARA Structure software [49,50]. The removal of water molecules and optimization was performed using (Y) AMBER force field [53e55], acid dissociation constant (pKa), density 0.997 g l1, density 0.997 g l1 set as per the YASARA Structure software to neutralize the system and subjected to energy minimization using steepest gradient approach (100 cycles). As per the software parameters, force constant has been kept at 1000 kJ mol1 nm2), while number of atoms N, pressure P, and temperature T were stored to standard level including temperature of 298 K (physiological condition, pH ¼ 7.4), pressure of 1 bar using Berendsen thermostat and barostat [48], respectively. The selected complex was simulated for 10000 ps (production period) with frame capture at every 2500 ps step to analyze the trajectory by various evaluative quantities including root mean squared deviation (RMSD) and root mean square fluctuation (RMSF). The proteinligand interaction patterns obtained from the averaged conformations were graphically illustrated using Protein-Ligand Interaction Profiler 1.2.0 program [55].

DGdesolv ¼ desolvation term for docking energy 3. Result and discussion 2.7.1. Preparation of ligands The 3D structures of the pyrimido[2,1-b]benzothiazoles derivatives (5a-5n) were sketched using Marvin Suite applications (Marvin Sketch) followed by adding explicit hydrogen with cleaning, energy minimization, and alignment process. The generated structures were saved as mol2/sdf files by energy minimization using the MMFF94 force field. 2.7.2. Preparation of receptors We have carried out docking studies using Autodock Vina 1.0 algorithm [49] configured in YASARA [50] molecular modeling package. The crystal structure of Mycobacterium tuberculosis fatty acyl CoA synthetase complexed with its inhibitor was retrieved from the protein data bank (PDB) (PDB code: 3R44). As the protein PDB file was not suitable for use in molecular docking studies, the optimization and minimization of the structure were performed using protein preparation wizard tool. This involved deleting the crystallographically observed water molecules (water without H bonds) as no water molecule was found to be conserved, assigning the correct bond orders, adding hydrogen atoms corresponding to pH 7.0 considering the appropriate ionization states for the acidic as well as basic amino acid residues corresponding to pH 7.0. After assigning Kollman charge [51] and protonation state finally energy minimization was performed using Amber03 force field [52] and energy minimized using steepest descent technique (100 iterations) in YASARA Structure program [50]. The shape and properties of the binding site of InhA enzyme were characterized and setup for docking using the receptor grid generation panel. This grid was defined by a box having dimensions of 10  10  10 Å box centered on the native ligand in the crystal complex. The ligands were

3.1. Design and synthesis Molecular hybridization is a rational drug design strategy based on the identification of active pharmacophoric sub-units, which facilitate the formation of molecular hybrids of two or more known bioactive inhibitors by fusion of sub-units while retaining desired characteristics of the original templates. The main aim is to design the hybrid analog and to develop compounds with improved potency towards the target minimizing the side effects. Many scientists have explored the hybrid design of analogs as potential candidates for biological evaluation. The structural modification of the known drug molecule is one of the way to identify novel molecular entities which are active against the bacterial strain [56]. In our previous study, we have reported microwave-assisted open vessel microwave assisted technique for the synthesis of Ethyl-2-methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate derivatives (4a-4l) [40]. We have also described the antibacterial activity and In vitro anti-mycobacterial activity of all synthesized compounds. In continuous our efforts, we further attempted to design novel anti-mycobacterial agent by hybridizing isoniazid linker with the various derivative of Ethyl-2methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine3-carboxylate derivatives (4a-4n) to get desired compounds (5a5n). Thus, designed ligands possessed two pharmacophoric units, Isoniazid, a linker stacked between a left pyrimido[2,1-b]benzothiazole, core and a substituted heterocyclic moiety (Isoniazid linkage) at the right side as shown in Fig. 3. The titled compounds (5a-5n) were synthesized by a sequence of reactions illustrated in Scheme 1. In the first step, we have reported an efficient, one pot three-component synthesis of ethyl 2-

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19

Fig. 3. Some representative isoniazid containing patent anti-TB analogs. The target isoniazid analogs 5 k exhibited the highest anti-TB activity among the synthesized compounds.

Scheme 1. Synthetic protocol for the synthesis of title compounds (5a-5n).

methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine3-carboxylate derivatives (4a-4n) by reaction between Pyridine 2aldehyde 1 and Ethyl acetoacetate 2 and various derivatives of 2amino-benzothiazole (3a-3n) using PdCl2 as an efficient catalyst under microwave-assisted solvent-free conditions, with better yield and short reaction time in our previous work [40]. Here, Compounds (4a-4n) was further reacted with hydrazine hydrate in the presence of catalytic amount of Con. H2SO4 to provide Ethyl-2methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine3-carboxylate as an intermediate product. In the next step, intermediate hydrazine hydrate derivative dissolved in dichloromethane and reacted with hydrochloride salt of isonicotinoyl

chloride using triethylamine as a base to yield N0 -isonicotinoyl-2methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo[3,2-a]pyrimidine3-carbohydrazide derivatives (5a-5n) with good (65e80%) practical yield. Various chemical structure and % yield of synthesized compounds (5a-5n) are abridged in Table 2. All the newly synthesized compounds (5a-5n) were characterized by different spectroscopic techniques such as 1H NMR, 13C NMR, mass spectrometry, IR, and elemental analysis were in full agreement with proposed structures before evaluating for biological activity such as In vitro Antibacterial Activity and anti-mycobacterial activity. The IR spectrum of compounds 5a shows absorption bands at 3045 cm1 and 2945 cm1 corresponding to

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CeH aromatic stretching and CeH alkane type stretching correspondingly. Strongly intense absorption bands were observed around 1662, 1587 and 1512 cm1 due to stretching vibrations of eC¼Ostr, eC¼Nstr, eC¼Cstr groups, respectively. 1H NMR spectroscopy analysis carried out for compound 5a in DMSO‑d6 at 400 MHz revealed the presence of a singlet at d value 5.949 ppm corresponding to the chiral center of the molecule. Pyridine ring protons (Py-H) attached with the chiral center shows peaks at 8.67 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.81 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.6 Hz, 1H), and 7.44e7.31 (m, 1H) d value. Methyl group displays a peak at 2.34 (s, 3H) d value. Side chain contained pyridine ring's proton resonates at 8.97 (d, J ¼ 7.4 Hz, 2H), 7.95 (d, J ¼ 7.4 Hz, 2H) d ppm. Also aromatic ring hydrogens (AreH) of fused ring resonates

Table 2 Chemical structure and % yield of synthesized compound (5a-5n).

at 7.40 (m, 1H), 7.07 (td, J ¼ 7.6, 1.6 Hz, 1H), 6.81e6.69 (m, 2H), 5.95 (s, 1H) d value. The 13C NMR spectrum also in good agreement with the structure assigned. Characteristic peaks at d value 166.91 and 158.56 ppm which was the most deshielded signal in 13C NMR confirmed the presence of >C¼O in amide-type ketones in the side chain. The mass spectrum of 5a revealed that observed molecular ion peaks were in agreement with the molecular weight of respective compounds. 3.2. In vitro antibacterial activity All the synthesized compounds (5a-5n) were screened for their In vitro Antibacterial activity using Streptomycin as standard drugs.

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The evaluation of antimicrobial activity was carried out by using Nutrient-agar plates through well-diffusion assay. The results are summarized in Table 3 and Fig. 4. The activity index of the probiotic culture was calculated as:

coli). Compounds 5a, 5d, 5i and 5j displayed good activity against Micrococcus luteus stain. Compound 5a, 5d, 5e, and 5l demonstrated good activity against Gram-positive Bacillus cereus strain.

Activity index ðA:I:Þ

3.3. In vitro anti-mycobacterial activity

¼

Mean of zone of inhibition of derivative Zone of inhibition obtained for standard antibiotic drug

The Antibacterial activity data revealed that almost all of the compounds (5a-5n) exhibited promising antibacterial activity against test culture compared to streptomycin as a standard drug. As from the bioassay results, Compound 5j and 5m demonstrated highest antibacterial efficacy against Gram-negative bacteria Enterobacter aerogens and Gram-positive bacteria Bacillus cereus respectively as compare to standard drug. Six of synthesized compounds i.e. 5d, 5i, and 5j-5m were found to exhibit better activity compared to streptomycin against Enterobacter aerogens strain. Compounds 5c, 5g, 5h, and 5l showed comparable activity to streptomycin against Gram-negative bacterial strain (Escherichia

Owing to the increased resistance diversity of tuberculosis against standard drugs, there is a requirement to development of novel drug-like moiety that could susceptibly kill Mycobacterium species. This approach was laid for the development of such compound with increased efficiency in terms of lower MIC values to kill H37RV with maximum percent inhibition. In a standard primary screen, all the newly synthesized compounds 5a-5n were evaluated for their in vitro anti-mycobacterial activity against M. tuberculosis H37Rv using a well-known Lowenstein-Jensen (L-J) method. The results of anti-mycobacterial activity indicated that most of the synthesized compounds exhibited low to good activity against M. tuberculosis strains in vitro. As seen in Table 4 (Fig. 5), the tested synthesized derivatives showed diverse tuberculostatic activity.

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Table 3 in vitro Antibacterial Activity of synthesized compounds (5a-5n). Sample code

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n Std. a

Enterobacter aerogens MTCC No. 8558

Escherichia coli MTCC No. 1610

Micrococcus luteus MTCC No. 11948

Bacillus cereus MTCC No. 8558

M.Z.Ia (mm)

A.I.a

M.Z.Ia (mm)

A.I.a

M.Z.Ia (mm)

A.I.a

M.Z.Ia (mm)

A.I.a

19 ± 1.145 20 ± 1.121 15 ± 1.132 23 ± 1.141 17 ± 1.049 16 ± 1.112 18 ± 1.109 19 ± 1.113 22 ± 1.100 25 ± 1.115 22 ± 1.119 20 ± 1.123 23 ± 1.112 19 ± 1.131 24

0.792 0.833 0.625 0.958 0.708 0.667 0.750 0.792 0.917 1.042 0.917 0.833 0.958 0.792 e

13 ± 1.047 19 ± 1.101 21 ± 1.123 17 ± 1.054 16 ± 1.012 18 ± 1.026 22 ± 1.074 24 ± 1.026 15 ± 1.029 13 ± 1.031 19 ± 1.075 24 ± 1.012 17 ± 1.019 15 ± 1.100 24

0.542 0.792 0.875 0.708 0.667 0.750 0.917 1.000 0.625 0.542 0.792 1.000 0.708 0.625 e

20 ± 1.062 15 ± 1.012 19 ± 1.056 23 ± 1.057 14 ± 1.062 16 ± 1.042 17 ± 1.033 19 ± 1.074 22 ± 1.026 23 ± 1.029 18 ± 1.025 16 ± 1.017 17 ± 1.021 19 ± 1.059 24

0.833 0.625 0.792 0.958 0.583 0.667 0.708 0.792 0.917 0.958 0.750 0.667 0.708 0.792 e

22 ± 1.025 18 ± 1.025 19 ± 1.025 23 ± 1.025 24 ± 1.025 17 ± 1.025 16 ± 1.025 12 ± 1.025 15 ± 1.025 18 ± 1.025 19 ± 1.025 24 ± 1.025 26 ± 1.025 19 ± 1.025 24

0.917 0.750 0.792 0.958 1.000 0.708 0.667 0.500 0.625 0.750 0.792 1.000 1.083 0.792 e

M.Z.I. ¼ Mean value for Zone of Inhibition (mm), A.I. ¼ Activity Index.

through the biological membrane of the micro-organism thereby inhibiting their growth. From biological assay, the result revealed that synthesized isoniazid based pyrimido[2,1-b]benzothiazole showed significant anti-tubercular activity against test culture. 3.4. In silico ADMET/5 rule of Lipinsky rule

Fig. 4. In vitro antibacterial activity of synthesized compounds (5a-5n).

From our research study, out of 14 synthesized compounds, the 5k, 5n, 5h, 5g, 5b, 5l, 5a, 5f, 5m, 5c, 5i, and 5d were with ascending MIC values against test culture. Compound 5k which have trifluoromethoxy (-OCF3) group at 8th position found to be more potent anti-mycobacterial agent (% inhibition value 79.67) with MIC value 6.25 mg mL1. While compound 5n have dimethyl group at 7th and 8th position displayed good MIC value 12.5 mg mL1 and highest % inhibition value 82.11. In the case of compound 5g which have chloro (-Cl) group at 6th position showed less MIC value 50 mg mL1 (% inhibition value 81.30) as compared with compound 5h which possessed Fluoro (-F) at 8th position (% inhibition 80.48 and MIC value 25 mg mL1) due to high electronegativity of fluorine atom. Compound 5a (% inhibition value 69.91), 5f (% inhibition value 71.54), 5m (% inhibition value 50.40) were showed same MIC value (125 mg mL1) with slight variation in percent inhibition values of the targeted pathogenic strain of Mycobacterium. Compound 5b and 5j presented anti-tuberculosis activity MIC value 62.5 mg mL1. The biological significant heterocyclic nucleus such as Isoniazid, pyridine, and pyrimido[2,1-b]benzothiazole presented in one molecule as pharmacophores and increased in the lipophilic character of the molecule due to the various electron donating and electron withdrawing group at position 6th, 7th, 8th on pyrimido [2,1-b]benzothiazole fused ring system. This facilitated the crossing

3.4.1. Lipinsky rule All the synthesized ligands were checked for amenability to the Lipinsky rule of five, and the results are summarized in Table 5. The rule states that a molecule probable to be developed as an orally active drug candidate should show no more than one violation of the following four criteria: [i] It should not have a molecular weight greater than 500 Da [ii] It should not have more than five hydrogen bond donors, [iii] it should not have more than 10 hydrogen bond acceptors, and [iv] it should not have an octanol-water partition coefficient greater than 5. Molecular properties of all ligands were calculated by DruLIto software. Lipophilicity is a property that has a major effect not only on the absorption, distribution, metabolism, excretion and toxicity properties but also pharmacological activity since many drugs cross biological membranes through passive transport, which strongly depends on their lipophilicity. Lipophilicity has been studied and applied as an important drug property for eras. Compounds 5a showed the lowest lipophilicity, while compounds 5k demonstrated the highest lipophilicity. It was found that all the ligands followed the Lipinsky rule with maximum ligands (except 5b and 5k) showed no violation of the above criteria which is mentioned in Table 5. Therefore, these ligands have good potential for ensuring development as an oral agent and also potentially active drug candidates. 3.4.2. In silico ADMET profile On account of undesired pharmacokinetics and toxicity properties of compound is a major reason for drug candidate failures in drug discovery at early and late pipeline stage. If these issues could be resolved early, it would be tremendously beneficial for the drug discovery. In light of these, there is a great interest of scientists in developing predictive in silico models to predict ADMET properties which may be used in the initial stage of the drug candidate development [57]. As the first step in this way to analyze the novel chemical entities to avert wasting time on lead candidates that would be toxic or metabolized by the body into an inactive form and unable to cross membranes. The results of such type of analysis are reported in Table 6. The pharmacokinetic outline of all the

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23

Table 4 In vitro Anti-mycobacterial activity of synthesized compounds (5a-5n). LowensteineJensen (LJ) method (Culture: H37RV) Sample code

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n Isoniazid

MIC value (mg/ml)

Mean Colony forming unit (c.f.u.) on media Control

Treatment concentration (1000 mg/ml)

Percentage inhibition (%)

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

37 33 56 65 53 35 23 24 62 45 25 39 61 22 1

69.91 73.17 54.47 47.15 56.91 71.54 81.30 80.48 49.59 63.41 79.67 68.29 50.40 82.11 99.18

Fig. 5. In vitro anti-mycobacterial activity of synthesized compounds (5a-5n).

125 62.5 500 1000 250 125 50 25 500 62.5 6.25 100 125 12.5 0.20

the pharmaceutically relevant properties to evaluate the druglikeness and pharmacokinetic properties. The biplot displayed the two analogous 95% and 99% confidence ellipses to HIA and BBB models respectively. PSA has an inverse relationship with cell wall permeability and percent human intestinal absorption [58]. A relationship of PSA with permeability has been demonstrated, however, the models usually do not take into account the effects of other descriptors. The fluid mosaic model of cell membrane proposed that hydrophobic and hydrophilic interactions are possible due to phospholipid bilayer of the membrane. Henceforth, lipophilicity is also deliberated as an essential property for drug design. Lipophilicity could be assessed as the log of the partition coefficient between n-octanol and water (logP). Although logP is generally used to estimate a compound's lipophilicity. logP is a ratio which raise a concern about the use of log P to estimate hydrophilicity and hydrophobicity. Therefore, the information about H-bonding physiognomies as obtained by calculating PSA could be taken into consideration along with logP calculation [43]. Thus, a model with two descriptors not only AlogP98 but also PSA_2D with a biplot comprising 95% and 99% confidence ellipses

Table 5 Prediction of Lipinski's ‘Rule of 5’ for the active test compounds. Code No.

Mol_MW (<500)

Donor HB (<5)

Accpt. HB (<10)

logPo/w (<5)

Rule of Five (<4)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n

442.49 521.39 456.52 456.52 487.49 476.94 476.94 460.48 472.52 486.55 526.49 458.49 472.52 470.55

2 2 2 2 2 2 2 2 2 2 2 3 2 2

8 8 8 8 8 8 8 8 9 9 9 9 9 8

0.117 0.870 0.509 0.298 0.334 0.694 0.694 0.233 0.357 0.780 1.609 0.036 0.357 0.901

0 1 0 0 0 0 0 0 0 0 1 0 0 0

synthesized compound (5a-5n) under examination was predicted by way of six pre-calculated ADMET models delivered by the Discovery Studio 2.1 software. We have predicted the ADMET properties of test compounds with reference compound isoniazid for

were considered for the accurate prediction for the cell permeability of compounds. The chemical space region where expected to find well-absorbed compounds (90%) 95 out of 100 times was known as 95% confidence ellipse. Whereas 99% is a confidence

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.266 3.015 2.753 2.753 2.161 2.931 2.931 2.472 2.250 2.599 4.386 2.024 2.250 3.239 0.811 0.306 0.178 0.316 0.386 0.346 0.237 0.247 0.297 0.376 0.415 0.198 0.287 0.514 0.306 2.9e002 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0

2 2 2 2 2 2 2 2 2 2 1 2 2 2 1

ADMET_ unknown_ALogp98 ADMET_ AlogP98 ADMET_ CYP2D6_ Probability ADMET_ CYP2D6

ADMET_ PPB_level

97.419 97.419 97.419 97.419 140.242 97.419 97.419 97.419 106.349 118.235 106.349 118.235 106.349 97.419 67.912

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ADMET_PSA_2D

24

ellipse signified the chemical space region with compounds having excellent absorption through the cell membrane. As per the model for a compound to have an ideal cell permeability, it should follow PSA <140 Å2 and AlogP98 < 5 [43]. All the compounds showed polar surface area (PSA) < 140 Å2) except compound 5e (PSA < 140.242 Å2). As per the AlogP98 criteria, all the synthesized compound (5a-5n) had AlogP98 value < 5. As per the result is shown in Table 6, the synthesized compound (5a-5n) have low (5a-5i, 5m and 5n) or undefined (5j, 5k, 5l) values for BBB penetration levels (levels 3 and 4 as stated in Table 1). The aqueous solubility of drug candidate plays a critical role in the bioavailability of drugs, as the exception of compound 5k (6.726) having low aqueous solubility level 1 as referred in Table 1, all other compounds showed respectable aqueous solubility levels (level 2). Furthermore, all compounds have been predicted to have hepatotoxicity level of 1. Similarly, all the synthesized compounds (5a-5n) were shown the satisfactory result with respect to CYP2D6 liver (with reference to Table 1), signifying that PA is non-inhibitors of CYP2D6 (Table 6) except compound 5m (level 1 and probability 0.514). This indicates that the synthesized compounds (5a-5n) were well metabolized in Phase-I metabolism. Lastly, the ADMET plasma protein binding property prediction denoted that all the synthesized compound (5a-5n) have binding 95%, with an exception of compounds 5k (have binding 90%) (Referred to Table 1), noticeably suggesting that most have good bioavailability and not likely to be highly bound to carrier proteins in the blood.

0.847 0.927 0.927 0.880 0.927 0.920 0.867 0.927 0.933 0.927 0.947 0.927 0.854 0.933 0.433 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 2 2 2 2 2 2 2 2 2 2 1 2 2 2 4 4.407 5.240 4.842 4.864 4.605 5.166 5.189 4.766 4.426 4.568 6.726 4.197 4.471 5.285 3.3e002 0 0 0 0 2 0 0 0 0 0 1 0 0 0 0 0.995 0.764 0.845 0.845 e 0.79 0.79 0.931 1.141 e e e 1.141 0.694 1.479 3 3 3 3 4 3 3 3 3 4 4 4 3 3 3 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n isoniazid

ADMET_ Absorption_ level ADMET_BBB ADMET_BBB_ level Com. code

Table 6 Calculated ADMET properties of compound (5a-5n).

ADMET_ Solubility

ADMET_ Solubility_ level

ADMET_ hepatotoxicity

ADMET_ Hepatotoxicity_ Probability

3.5. Molecular docking study The promising biological results for the anti-tubercular activity could not lead to the final conclusion of the exact binding mode of pyrimido[2,1-b]benzothiazole derivatives into the mycobacterial target and the types of main binding forces governing the variation in observed binding affinity. With this purpose, we have selected in silico computational chemistry approaches like molecular docking protocol to find out the interaction mechanism of most active pyrimido[2,1-b]benzothiazole-isoniazid derivatives with Mycobacterium tuberculosis fatty acyl CoA synthetase (PDB code: 3R44). The pyrimido[2,1-b]benzothiazole derivatives were reported to inhibit mycobacterial enoyl reductase (InhA) [59]. The enoyl-ACP (CoA) reductase is an important enzyme in the mycobacterial type II fatty acid biosynthesis pathway. In silico docking studies were carried out with the crystal structure of Mycobacterium tuberculosis fatty acyl CoA synthetase from Mycobacterium tuberculosis (PDB entry: 3R44) with two small native ligands HIS and MLI (Fig. 6 (a)). All the synthesized compounds were docked into active sites of Mycobacterium tuberculosis fatty acyl CoA synthetase enzyme has been reported as the target receptors for docking studies in finding the suitable drug candidates against the Mycobacterium bacteria. A necessity to any successful experiment is the validation step. Accurate generation and scoring of known ligand binding pose by a given procedure should be investigated [60,61]. Docking success is usually observed when the top scoring pose was approximately 2.0e2.5 Å heavy atom root-mean-square deviation (RMSD) of the crystal ligand [60e62]. A top-scoring pose not within 2.5 Å is defined as a scoring failure. We have done re-docking of cocrystallized native ligand (HIS) into the active site of 3R44 using YASARA software for the validation of the docking process. The binding energy of this re-docked analysis was 4.782 kcal/mol which displayed hydrogen bonding, hydrophobic, van der Waals, and p-p interactions were observed with responsible key amino acids (peptides) such as Pro83, Ile84, Asn85, Leu88, Ile96, Met162, Tyr163, Thr164, Ser165, Pro171, and Phe207. The docked position was compared to the crystal structure position by calculating RMSD

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values 0.5448 Å. In this study, RMSD values were within 2.0 Å, indicating docking methods are valid for the given structures and YASARA software, therefore considered reliable for docking of pyrimido[2,1-b]benzothiazole derivatives (5a-5n) into the inhibitor binding cavity of 3R44. The stereochemistry was measured for the protein 3R44 using Ramachandran plot [63,64] implemented in RAMPAGE server exhibited 98.8% in j-f core areas (Fig. 6 (b)). The tenancy of residues in most favored, additional allowed and generally allowed regions with an exception of only one amino acids excluding, Glycine and Proline occupied in disallowed regions. While relatively consistent interactions were observed for all the molecules (5a-5n) in regard to the pyrimido[2,1-b]benzothiazole ring, it was the interaction of the pyridine, pyridine in side chain and mid of side chain (eCOeNHeNHeCO-) with the surrounding amino acid residues that caused the variation in the observed binding affinity for these derivatives. The best-docked poses were chosen on the basis of scoring function and their binding characteristics compared with the reference ligand. Molecular docking results of the docked ligands with interacting amino acids into the binding site of the protein were mentioned in Table 7. The docking result for compound 5k was showed in Fig. 7 with the highest binding energy 9.077 kcal/mol. The highest energy docking pose of 5k revealed the presence of four decisive hydrogen bond interactions with Thr164 (3.74 Å), Thr164 (3.92 Å), His208 (4.06 Å), and Thr304 (4.05 Å) through pyrimidine ring and side chain of two eNH atom of pyridine ring respectively (Fig. 7) and also showed three hydrophobic interaction with Asn85 (3.88 Å), Leu88 (3.95 Å) and Leu303 (3.67 Å). The compound 5k was observed to be stabilized within the active site through strong van der Waals interactions, weak p-alkyl, p-sigma interaction observed with peptides such as Glu92, Ile96, Met162, Tyr163, Thr164, Ser165, Gly169, Pro171, Phe207, His 208, Val209, Ala210, Thr304, Glu305, Thr380, Asp382, Arg397, Leu398, and Lys399. The docking result of 5h into the active site of targeted protein showed the similar chain interactions in the protein. Compound 5h formed two strong hydrogen bond interaction with Thr164 at a distance of 3.78, 3.96 Å through the side chain of two eNH atom of pyridine ring. It showed two hydrophobic interaction with Leu88 (3.73 Å), and Leu303 (3.77 Å). The compound 5h was stabilized within the active site through strong van der Waals interactions, electrostatic interactions, weak p-alkyl, p-sigma and other

25

interactions with amino acid residues like Asn85, Leu88, Ile96, Met162, Tyr163, Thr164, Ser165, Pro171, His208, Val209, Leu303, Thr304, Glu305, Thr380, Asp382, Arg397, Leu398, and Lys399. The per-residue interaction analysis revealed that the primary driving forces for mechanical interlocking between isoniazid-pyrimido[2,1-b]benzothiazole derivatives and the active site of the Mycobacterium tuberculosis fatty acyl CoA synthetase were the steric and electrostatic complementarities. Also, it could provide quantitative insight into various non-bonded interactions (Steric and electrostatic) responsible for the variation in the binding affinity of these molecules which can guide site-specific modification in these molecules to arrive at potent anti-mycobacterial candidates. 3.6. Molecular dynamics simulations Docking outcomes were not considered decisive due to in vivo binding of the inhibitor to a protein is a lively process. Docking result could be the immediate state and stable binding mode of ligand is suitable for advanced studies. In order to measure the structural integrity and stability of the complex, we performed Molecular dynamics (MD) simulations (10 ns (ns)) of the docked inclusion (enoyl reductase (INHA) complexed with compound 5k) in a water solvent system. The simulations were executed with the constant shape and volume at room temperature to study the structural features and behavior of the protein-ligand inclusion system with the aim to reveal its ability to penetrate through the bio-membrane. Afterward, a resultant solvated low energy inclusion complex was created from an energy minimization technique which was analyzed during the simulation trajectory by computing especially Root mean square deviation (RMSD), energy parameters, the total number of intra and intermolecular hydrogen bonds with steepest descent function of MD simulation. A molecule is expected to possess different conformations if its initial conformation is varied from its final by RMSD >4 Å [65]. In our case, The inclusion complex displayed negligible RMSD fluctuations (a maximum of 2.49 Å observed) throughout the trajectory, which supported the compatibility of the developed inclusion structure. The time dependent fluctuations of the RMSD based on the backbone atoms during the complete 10 ns simulation period was graphically portrayed in Fig. 8. As we have analyzed the statistically significant results of the

Fig. 6. (a) Crystal structure of Mycobacterium tuberculosis fatty acyl CoA synthetase (PDB code: 3R44) (b) Ramachandran plot of 2IE0 showed 98.8% in core regions.

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Table 7 Molecular docking results of the docked ligands with interacting amino acids into the binding site of the protein (PDB: 3R44). Code No.

Bind. Energy [kcal/mol]

H-bond

Amino acid

L. atom

H-Bond Length (Ao)

Hydrophobic interaction

5a

8.249

3

8.101

2

5c

8.352

2

5d

8.141

3

5e

7.964

2

5f

8.137

3

5g

8.100

3

5h

8.533

2

5i

8.170

3

5j

8.418

4

5k

9.077

4

5l

8.136

6

5m

7.974

5

5n

8.348

3

N O N N O N O N O N N N N N N N N N N O N O N N O N N N O N N O N N N N O N O N N N N N N

3.57 3.78 4.08 3.06 3.28 3.13 3.31 3.94 4.08 3.85 3.15 3.43 3.14 3.89 4.06 3.95 3.44 4.00 3.78 3.96 3.59 3.78 4.04 3.76 3.96 3.83 4.09 3.74 3.92 4.06 4.05 3.26 3.26 3.74 4.07 3.04 3.73 3.89 4.00 3.35 3.91 4.04 3.68 3.01 3.85

Asn85, Leu88 Leu303

5b

Thr164 Thr164 His208 Thr164 Thr164 Thr164 Thr164 Thr164 Thr164 His208 Thr164 Arg397 Thr164 Arg397 Leu398 Thr164 Arg397 Leu398 Thr164 Thr164 Thr164 Thr164 His208 Thr164 Thr164 His208 Thr304 Thr164 Thr164 His208 Thr304 Thr164 Thr164 Lys172 His208 Ala210 Tyr362 Thr164 Thr164 Ser165 His208 Thr304 His208 Thr304 Thr304

simulation presented at various time intervals periods (such as 0, 2.5, 5, 7.5, and 10 ns) which was illustrated in Table 8 and Fig. 9. The average energy of the protein-ligand complex calculated over the simulation trajectory showed that PDB 3R44 developed an effective interactions with the complete ligand dataset as their energies were in the range of 398652.432 kJ mol1 1 to 303326.185 kJ mol . Various energy results were measured during molecular dynamics simulations, 398652.432 mol-1 retrieved at initial start, while 303326.185 kJ mol1 at 10 ns time trajectory and the average energy has been recognized with 303558.352 kJ mol1. The protein-ligand interaction maps generated using Accelry's Discovery Studio Visualizer 2016 showed the dominance of residues Val82, Leu88, Ile96, Tyr163, Thr164, Ser165, Phe207, Leu398, Lys399 for the ligand to bind throughout the MD simulation. The diverse pattern of unfavorable donor interaction with Lys399 was seen only at 2.5 ns simulation which showed van der Waals interaction after the completion. van der Waals interaction of Val82, Ile84, Arg87, Ile96, Gly169, His208, Val209, Leu303, and Thr304 were additionally obtained after the completion of the 10 ns target trajectories.

Leu88, Leu303 Leu88, Ile96 Leu303 Asn85, Leu88, Val209, Leu303

Leu88, Leu303, Lys399 Leu88, Leu303, Lys399 Asn85, Leu88 Leu303, Lys399 Leu88 Leu303, Asn85, Leu88 Leu303, Asn85, Leu88, leu88, Glu92, Pro171, Leu303,

Asn85, Leu88 Leu303, Thr164, Val209, Leu398, Lys399

Asn85, Leu88, Val209, Leu303 Leu88 Val209, Leu303

The simulation analyzed from the initial (0 ns) to final (10 ns) conformations. RMSD of common target trajectories highlighted the importance of van der Waals interactions, Conventional HBonds, Alkyl, p-Alkyl, CeH bond and Halogen (Fluorine). All the four types of interactions have been measured at the initiation (0 ns) of molecular dynamics simulation, which includes three conventional hydrogen bonds, two Alkyl, one p-Alkyl, one attractive Charge and one Van Der Waals interaction with different type of residues such as His208, Thr304, Val209, His208, Leu303, Glu305 and Asp382 with 3.91, 3.82, 3.91, 4.49, 4.55, 4.80, 7.06 and 4.28 Å respectively. After the time period of 2.5 ns, conventional hydrogen bonds replaced by Asn85, Thr 164 and Lys399 with distance 3.90, 4.16, 5.97, 3.50 Å respectively, whereas Alkyl group has totally missing during this time interval. However, three p-Alkyl interaction have been found with Met162 and Lys399 with the 5.91, 4.72 and 4.25 Å value. There was two exceptional interaction Halogen (Fluorine) developed at Pro83 with 4.64 and 5.23 Å. In addition, two Unfavorable Positive-Negative interaction has been notified with amino acid Lys399 (5.08 and 5.97 Å correspondingly), while p-Sulfur and Sulfur-X found at Phe207 and His208 with 5.34 and 4.67 Å and one

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27

Fig. 7. Different orientation of the ligand 5 k into the binding pocket of the 3R44 protein.

Fig. 8. a) Energy and b) RMSD plots produced from MD trajectories of prioritized target: Mycobacterium tuberculosis enoyl reductase (INHA)e5 K complex.

Carbon hydrogen and one van der Waals interaction have been made with Lys399 and Ile84 at 3.74 and 3.56 Å respectively. At the end of 5 ns, the trajectory of amino acids interaction have been measured which included three van der Waals interactions, one conventional hydrogen bond, two CeH bonds, three Halogen (Fluorine), one p-Donor Hydrogen Bond and three p-Alkyl

interaction with Ile84 and Met162 with 3.90, 4.84 and 3.47 Å, conventional hydrogen bond at Asn85 with 5.22 Å, CeH bond at Tyr163 and Thr164 with 4.69 and 7.16 Å, Halogen (Fluorine) at Pro83 and Ile 161 with 3.90, 4.55 and 4.87 Å, p-Donor Hydrogen Bond Tyr163 with 4.16 Å, p-Alkyl at Leu88 and Met162 with 6.53, 3.47, and 4.97 Å.

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Table 8 Protein-Ligand interaction profiler results of different time intervals of MD simulation of Mycobacterium tuberculosis enoyl reductase (INHA) e5 k complex. Time (ns) Van der Waals Interactions

Conventional H-Bonds Alkyl

0

His 208, Thr 304

2.5 5 7.5 10

Asn 85, Leu 88, Glu 92, Ile 96, Met 162, Tyr 163, Thr 164, Ser 165, Pro 171, Phe 207, Ala 210, Tyr 362, Phe 378 Thr 380, Arg 397, Leu 398 Val 82, Leu 88, Ile 84, Ile 96, Ile 161, Tyr 163, Ser 165, Val 209, Leu 303, Thr 304, Leu 398 Val 82, Arg 87, Ile 96, Ser 165, Phe 207, His 208, Leu 398, Lys 399 Val 82, Leu 88, Ile 96, Ser 165, Lys 172, His 208, Val 209, Leu 303, Thr 304, Leu 398, Lys 399 Val 82, Ile 84, Arg 87, Leu 88, Ile 96, Tyr 163, Thr 164, Ser 165, Gly 169, Phe 207, His 208, Val 209, Leu 303, Thr 304

p-Alkyl

His 208, Val 209 Leu 303

Asn 85, Thr 164, Lys 399 Asn 85

Leu 88, Met 162

Asn 85, Tyr 163

e

CeH bond

Halogen (F)

Asp 382

e

Met 162, Lys 399

Pro 83 Ile 84, Tyr 163 Ile 84, Met 162

Pro 83, Ile 161 Pro 83, Ile 161

Met 162, Lys 399 Tyr 163, Lys 399 Ile 161

Fig. 9. The protein-ligand interaction maps developed from 0 ns to 10 ns of molecular dynamics (MD) conformations of prioritized Protein-5k (van der Waals interaction-Green, Salt Bridge- Yellow, Pi-cation- Dark yellow, Pi-Alkyl-Light Pink, p-Don H-bonds-Light Green, Carbon H-bonds- Dull Green, Conventional H-bonds- Bright Green, Unfavorable Donordonor-Red, Alkyl- Dull Pink, Pi-Sulfur- Yellow, Halogen (fluorine) - Sky blue, Unfavorable Positive-Negative- Red, Attractive Charge- Dark Yellow). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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be active against Gram-positive and Gram-negative bacterial strains and also demonstrated good antibacterial activity. We have also done in vitro anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv strain. These compounds exhibited good minimum inhibitory concentration between 6.25 and 62.5 mg/mL. Among them, compounds 5k found to be most potent compound with MIC value 6.25 mg/mL, while compounds 5n (MIC 12.5 mg/mL) showed good anti-mycobacterial activity. Compounds 5b, 5g, 5h, and 5j were exhibited good to moderate anti-mycobacterial activity. On the base of docking results, the top scoring molecule 5k possessed binding energy of 9.077 kcal/mol. It formed four hydrogen bonding interactions and three hydrophobic interaction with targeted protein. Compound 5k was practically stabilized within the active site through strong van der Waals interactions, weak p-alkyl, p-sigma observed with peptides. This study suggested that synthesized analog 5k and 5h have great potential for the promising candidate in developing anti-tubercular agents. On the basis of best scoring ligands from docking study, we have performed molecular dynamics study between top scoring molecule 5k and targeted protein for measure the binding stability of the ligand from 0 to 10 ns? The calculated ADMET parameters validated good pharmacokinetic properties of synthesized compounds. Fig. 10. Superimposed view of docked pose and equilibrated structure, the variations of total energy at 0 ns (cyan-Blue color) and over 10 ns (Gray-maganta color) of MD trajectory. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Likewise, two van der Waals interactions, two conventional hydrogen bonds, one CarboneHydrogen bond, three Halogen (Fluorine), 1 p-Sulfur and two p-Alkyl interactions have been observed after the completion of 7.5 ns? They were described different interactions such as van der Waals interactions at Ile84 and Met162 with 3.56 and 4.16 Å, conventional hydrogen bonds at Asn85 and Tyr163 with 4.16 and 4.73 Å, Thr164 with 4.35 Å, Halogen (Fluorine) at Pro83 and Ile161 with 4.72, 4.76 and 6.25 Å, p-Sulfur at Phe207 with 4.88 Å, and p-Alkyl interactions at Met162 with 4.54 and 5.65 Å respectively. After completion of 10 ns time interval of molecular dynamics simulation, two van der Waals interactions, one conventional hydrogen bond, two CarboneHydrogen bonds, four Halogen (Fluorine), one p-donor hydrogen bonds and two p-alkyl interactions remained as it is. however, their values have been changed as van der Waals interactions at Pro83 and Ile84 with 3.73 and 3.69 Å, conventional hydrogen bond at Asn85 with 4.60 Å, CeH bond at Tyr163 and Lys399 with 7.25 and 5.37 Å, Halogen (Fluorine) at Pro83, Ile84 and Ile161 with 3.73, 4.56, 3.69 and 5.98 Å, p-donor hydrogen bonds at Tyr163 with 4.97 Å and p-Alkyl interactions at Met162 and Lys399 with 3.84, 5.10 and 4.80 Å respectively. The superimposition of the protein-ligand complex of 0 ns and 10 ns?has been done and the diversity in the docked pose of the ligand is illustrated in Fig. 10. Blue colored ligand is obtained at 0 ns and Magenta colored ligand is obtained at 10 ns which shows the clear idea of the conformational change in the position of the ligand binding area with the RMSD value of 2.6720 Å. 4. Conclusion The aim of the present study is to design, synthesize and investigate the biological profile of the novel 4H-Pyrimido[2,1-b] Benzothiazoles scaffold having Isoniazid nucleus. The synthesis was completed using the conventional method with the hope of discovering new analog leads capable of serving as an antimycobacterial agent. Most of the synthetic analogs were found to

Conflicts of interest Authors have declared that there is not any conflict of interest. Acknowledgement We are thankful to UGC-Info net and INFLIBNET Gujarat University for providing e-source facilities and also thankful to the department of the chemistry for providing necessary facility during work. Both Manoj N. Bhoi and Mayuri A. Borad were thankful to UGC-BSR fellowship for financial assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.05.028. References [1] W. WHO, Global Tuberculosis Report 2012, World Health Organization, Geneva, 2012. http://apps.Who.Int/iris/bitstream/10665/91355/1/ 9789241564656_eng.pdf. [2] W. H. Organization, Global Tuberculosis Report 2013, World Health Organization, 2013. [3] W. H. Organization, Global Tuberculosis Report 2015, World Health Organization, 2015. [4] Y.L. Janin, Antituberculosis drugs: ten years of research, Bioorg. Med. Chem. 15 (2007) 2479e2513. [5] L.G. Dover, G.D. Coxon, Current status and research strategies in tuberculosis drug development: miniperspective, J. Med. Chem. 54 (2011) 6157e6165. [6] J.C. Sacchettini, E.J. Rubin, J.S. Freundlich, Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis, Nat. Rev. Microbiol. 6 (2008) 41e52. [7] H.H. Showalter, W.A. Denny, A roadmap for drug discovery and its translation to small molecule agents in clinical development for tuberculosis treatment, Tuberculosis 88 (2008) S3eS17. €nnroth, E. Jaramillo, J. Weiss, M. Uplekar, J. Porter, [8] D. Boccia, J. Hargreaves, K. Lo C. Evans, Cash transfer and microfinance interventions for tuberculosis control: review of the impact evidence and policy implications, Int. J. Tuberc. Lung Dis. 15 (2011) S37eS49. [9] J. Maurice, WHO framework targets tuberculosisediabetes link, Lancet 378 (2011) 1209e1210. [10] S. Loewenberg, India reports cases of totally drug-resistant tuberculosis, Lancet 379 (2012) 205. [11] S.R. Patpi, L. Pulipati, P. Yogeeswari, D. Sriram, N. Jain, B. Sridhar, R. Murthy, S.V. Kalivendi, S. Kantevari, Design, synthesis, and structureeactivity correlations of novel dibenzo [b, d] furan, dibenzo [b, d] thiophene, and N-methylcarbazole clubbed 1, 2, 3-triazoles as potent inhibitors of mycobacterium

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Abbreviations ADMET: Absorption, distribution, metabolism, elimination, and toxicity BBB: blood brain barrier CADD: computer-aided drug design FAS: Fatty Acid Synthase FDA: Food and Drug Administration INH: Isoniazid MD: Molecular dynamics

MDR: Multi-drug-resistant MIC: Minimum inhibitory concentration MTB: Mycobacterium tuberculosis NMR: Nuclear magnetic resonance PDB: Protein data bank PSA: Polar surface area RMSD: Root mean squared deviation RMSF: Root mean squared fluctuation TB: Tuberculosis TLC: Thin layer chromatography WHO: World Health Organization XDR: Extensively drug-resistant YASARA: Yet another Scientific Artificial Reality Application

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