Synthesis, antimicrobial, and antitubercular evaluation of new Schiff bases with in silico ADMET and molecular docking studies European Journal of Chemistry 13 (1) (2022) 109-116 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2022 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.13.1.109-116.2216 European Journal of Chemistry View Journal Online View Article Online Synthesis, antimicrobial, and antitubercular evaluation of new Schiff bases with in silico ADMET and molecular docking studies Sakshith Raghavendra Prasad 1,2, Nayak Devappa Satyanarayan 2,*, Avarse Satish Kumar Shetty 1 and Basaiah Thippeswamy 3 1 Department of Pharmaceutical Chemistry, National College of Pharmacy, Balaraj Urs Road, Shivamogga-577201, Karnataka, India 2 Department of Pharmaceutical Chemistry, Kuvempu University, Post-Graduate Centre, Kadur-577548, Karnataka, India 3 Department of Post-Graduation Studies and Research in Microbiology, Jnanasahyadri, Kuvempu University, Shankaraghatta-577451, India * Corresponding author at: Department of Pharmaceutical Chemistry, Kuvempu University, Post-Graduate Centre, Kadur-577548, Karnataka, India. e-mail: satya1782005@gmail.com (N.D. Satyanarayan). 10.5155/eurjchem.13.1.109-116.2216 Received: 18 November 2021 Received in revised form: 31 December 2021 Accepted: 03 January 2022 Published online: 31 March 2022 Printed: 31 March 2022 Schiff bases are a proven moiety in antitubercular drug discovery and the antitubercular drug development. Drug discovery is a never-ending process due to evolving drug resistance by the bacteria, as a result, there is a need of developing new antitubercular drugs. In this continuous process of antitubercular drug discovery, new series of Schiff bases are synthesized using quinoline carbohydrazide upon coupling with different aldehydes in ethanolic media through multistep synthesis. These synthesized compounds were purified and characterized by different spectroscopic techniques. The molecules were in vitro screened for antifungal and antibacterial potential by Agar well diffusion assay, antitubercular activity by using microplate Alamar blue assay, and an attempt has been made to study the in-silico relationship between new Schiff base derivatives 4a-f and the crystal structure of M. tuberculosis (5V3Y) protein by molecular docking studies. Synthesized compounds 4a-f show good interaction with the crystal structure of M. tuberculosis protein (5V3Y) and fulfill ADMET characteristics in silico experiments. Among the compounds tested, compound 4d was found to be active against bacteria and fungi. Compound 4b was found to be sensitive against M. tuberculosis at 50 µg/mL concentration. 5V3Y MABA In silico Antifungal activity Multistep synthesis Antibacterial activity Cite this: Eur. J. Chem. 2022, 13(1), 109-116 Journal website: www.eurjchem.com 1. Introduction Mycobacterium tuberculosis is a highly infectious airborne bacterium that causes tuberculosis (TB) disease and infects about 10 million people and kills over 1 million people each year [1]. Antitubercular drug design and development is one of the challenging research areas in the scientific forum, because of the limited efficacy and insufficiency of the drug options with their drawn-out duration of therapy, toxicity, high cost, and resistivity [2] in the current antitubercular treatment. Schiff bases are molecules with a –C=N– group that are synthesized via the condensation of a primary amine and an aldehyde [3]. Schiff bases ligands are a type of molecule that have biological and pharmacological properties such as antibacterial, antifungal, and antitubercular properties [4,5]. Many studies on Schiff bases have been investigated as can be seen from the literature [6,7]. However, no work on this specific type of Schiff base has been envisaged. In search of bioactive structures, we investigated quinoline and its derivatives, which turned out as potent antimicrobial and, antitubercular moiety [8,9]. Quinoline derivatives containing Schiff bases have received attention due to their significant applications in medicine [10,11]. Considering the important features and facts of quinoline with Schiff base moiety, we welcomed and directed our research towards developing new compounds with similar structural features. In this work, the designed Schiff bases were synthesized from the condensation of quinoline carbohyd- razide with different aldehydes and subsequently purified and characterized by different spectroscopic techniques. The molecules were in vitro screened for antifungal and antibacte- rial potential by Agar well diffusion assay, antitubercular activity by using microplate Alamar blue assay, and also an attempt has been made to study an in-silico relationship between new Schiff base derivatives 4a-f and crystal structure of M. tuberculosis (5V3Y) protein by molecular docking studies. 2. Experimental 2.1. Material 2D structural models were drawn in ACD/ChemSketch software [12] and SMILES were generated for the molecules. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.13.1.109-116.2216 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.13.1.109-116.2216 mailto:satya1782005@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.13.1.109-116.2216&domain=pdf&date_stamp=2022-03-31 110 Prasad et al. / European Journal of Chemistry 13 (1) (2022) 109-116 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.109-116.2216 N O OH S Cl + Reflux, 75-80 oC N O O S Cl H2N NH2.H2O C2H5-OH N O H N S NH2 Cl SOCl2 R O H N O H N S N Cl R R = O H OH O F HO H N O O H 1 2 34(a-f) HO O N O H C2H5-OH Reflux, 75-80 oC C2H5-OH Reflux, 75-80 oC Scheme 1. Synthesis of 6-chloro-N'-methylidene-2-(thiophen-2-yl)quinoline-4-carbohydrazide derivatives (4a-f). Individual ADME, bioactive, and drug-likeness scores were predicted for the designed molecules by online tools, admetSAR and molinspiration cheminformatics. Preparations of ligands were carried out by the Chimera docking tool [13]. The docking was done by the PyRx docking tool [14]. Visualization of the docked ligand and target protein interaction was carried out using Discovery studies 2020 [15]. Chemicals used for synthesis were from Sigma Aldrich, Spectrochem Pvt, Ltd. and Alfa Aesar. The solvents used for synthesis were distilled, and they were of reagent quality. Shimadzu LC-MS was used for mass spectros- copic analysis. TLC analysis was carried out on Merck 0.25 mm pre-coated silica gel 60F254 plates, and the spots were seen under UV light. The infrared data was obtained by Bruker spectrophotometer using the KBr pellet method. Bruker spectrometer were used with deuterated DMSO solvent and internal standard TMS to study 1H and 13C NMR spectra. For column chromatographic purification, Merck silica gel (100- 200) mesh was utilised. 2.2. Synthesis 2.2.1. The starting material 2-thiophene quinoline-4- carboxylic acid (1) To the mixture of 0.01 mol of 5-chloro isatin in 10 mL of ethanol and 10 mL of 33% KOH solution, 0.01 mol of 3-acetyl thiophene was added and kept for reflux for about 8 hours at 75-80 °C with a monitor of progress by TLC. After complete reflux, allowed to cool and pour onto the crushed ice slowly and neutralize by HCl solution. The separated solid was filtered, dried and recrystallized to yield compound 1 (Scheme 1) [16]. 2.2.2. Synthesis of ethyl 6-chloro-2-(thiophen-2-yl) quinoline-4-carboxylate (2) To the ethanol taken in a round bottom flask, thionyl chloride was added dropwise with constant stirring at 0 °C and followed by the addition of quinoline 4-carboxylic acid (1) and kept for reflux for about 8 hours at 75-80 °C with a monitor of progress by TLC. After complete reflux, allowed to cool and pour onto crushed ice slowly, neutralize by NaHCO3 solution and the solid obtained was collected and purified by column chromatographic method to yield compound 2 (Scheme 1). 2.2.3. Synthesis of 6-chloro-2-(thiophen-2-yl)quinoline-4- carbohydrazide (3) To the mixture of 0.01 mol of ethyl 6-chloro-2-(thiophen-2- yl)quinoline-4-carboxylate (2) in 10 mL of ethanol, 0.06 mol of hydrazine hydrate was added and kept for reflux for about 8 hours at 75-80 °C with a monitor of progress by TLC. After complete reflux, allow to cool and pour onto the crushed ice slowly and neutralize by HCl solution. The separated solid was filtered, dried and washed with diethyl ether to yield compound 3 (Scheme 1). 2.2.4. Synthesis of 6-chloro-N'-methylidene-2-(thiophen-2- yl)quinoline-4-carbohydrazides (4a-f) 6-Chloro-2-(thiophen-2-yl)quinoline-4-carbohydrazide (3) (0.02 mol) is dissolved in 10 mL of ethanol, to this 0.02 mol of different carboxylic aldehydes was added and followed by the addition of acetic acid in a catalytic amount, kept for reflux for 3 hours at 75-80 °C with a monitor of progress by TLC. After complete reflux, allowed to cool and pour onto crushed ice slowly and the solid obtained was collected and purified by column chromatographic method to yield compounds 4a-f (Scheme 1). 6-Chloro-N'-{(E)-[4-(dimethylamino)phenyl]methylidene} -2-(thiophen-2-yl)quinoline-4-carbohydrazide (4a): Color: Yellow amorphous. Yield: 82%. M.p.: 164-166 °C. FT-IR (KBr, ν, cm-1): 3450 (N-H), 1650 (C=N), 1600 (C=C), 1550 (C=O), 1370 (CH3), 750 (C-S), 600 (C-Cl). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 3.150 (s, 6H, N-(CH3)2), 6.316 (m, 2H, J = 7 Hz, Ar-H), Prasad et al. / European Journal of Chemistry 13 (1) (2022) 109-116 111 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.109-116.2216 6.632 (d, 1H, J = 9.2 Hz, Ar-H), 6.817 (t, 1H, J = 8 Hz, Ar-H), 7.042 (m, 2H, J = 8.5 Hz, Ar-H), 7.204 (d, 1H, J = 8.2 Hz, Ar-H), 7.577 (t, 1H, J = 10 Hz, Ar-H), 7.813 (d, 1H, J = 9.5 Hz, Ar-H), 8.212 (s, 1H, N=CH), 8.525 (s, 1H, Ar-H), 11.225 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 37.66 ((-CH3)2), 111.50, 119.20, 126.08, 126.57, 127.24, 128.04, 128.59, 128.74, 129.21, 129.40, 130.79, 132.02, 134.24, 142.50, 144.82, 145.65, 151.45, 152.90 (Ar-C), 162.50 (C=O). MS (EI, m/z (%)): 435.09 (M+1). 6-Chloro-N'-[(E)-(furan-2-yl) methylidene]-2-(thiophen-2- yl)quinoline-4-carbohydrazide (4b): Color: Pale green amorphous. Yield: 83%. M.p.: 180-182 °C. FT-IR (KBr, ν, cm-1): 3460 (N-H), 1710 (C=N), 1620 (C=C), 1520 (C=O), 1100 (C-O- C), 745 (C-S), 600 (C-Cl). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 6.324 (t, 1H, J = 3.4 Hz, Ar-H), 6.632(d, 1H, J = 4 Hz, Ar-H), 6.817 (t, 1H, J = 7.5 Hz, Ar-H), 7.034 (d, 1H, J = 4 Hz, Ar-H), 7.068 (d, 1H, J = 8 Hz, Ar-H), 7.204 (d, 1H, J = 8 Hz, Ar-H), 7.572 (t, 1H, J = 7 Hz, Ar-H), 7.601 (s, 1H, Ar-H), 7.813 (d, 1H, J = 9.5 Hz, Ar-H), 8.212 (s, 1H, N=CH), 8.525 (s, 1H, Ar-H), 11.225 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 109.50, 112.48, 119.22, 126.02, 126.54, 127.26, 128.02, 128.51, 128.72, 129.25, 130.77, 132.03, 134.87, 139.33, 143.24, 145.56, 147.82, 152.94 (Ar-C), 161.10 (C=O). MS (EI, m/z (%)): 382.03 (M+1). 6-Chloro-N'-[(E)-(4-fluorophenyl) methylidene]-2-(thio phen-2-yl)quinoline-4-carbohydrazide (4c): Color: Pale yellow amorphous. Yield: 78%. M.p.: 150-152 °C. FT-IR (KBr, ν, cm-1): 3480 (N-H), 1690 (C=C), 1600 (C=N), 1500 (C=O), 1490 (C-F), 749 (C-S), 600 (C-Cl). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 6.328 (m, 2H, J = 8 Hz, Ar-H), 6.751 (d, 1H, J = 9 Hz, Ar-H), 6.817 (t, 1H, J = 7.4 Hz, Ar-H), 7.061 (m, 2H, J = 8 Hz, Ar-H) 7.207 (d, 1H, J = 4 Hz, Ar-H) 7.565 (d, 1H, J = 7.5 Hz, Ar-H), 7.601 (s, 1H, Ar-H), 7.816 (d, 1H, J = 4 Hz, Ar-H), 8.252 (s, 1H, N=CH), 8.555 (s, 1H, Ar-H), 11.204 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 116.70, 120.38, 127.28, 128.34, 128.91, 129.43, 129.89, 130.40, 131.48, 131.80, 133.10, 135.95, 140.63, 145.47, 147.10, 146.83, 152.77, 155.80, 156.03, 156.10 (Ar-C), 166.24 (C=O). MS (EI, m/z (%)): 410.04 (M+1). 6-Chloro-N'-[(E)-(3-ethoxyphenyl) methylidene]-2-(thio phen-2-yl)quinoline-4-carbohydrazide (4d): Color: Off white amorphous. Yield: 77%. M.p.: 168-170 °C. FT-IR (KBr, ν, cm-1): 3400 (N-H), 1680 (C=C), 1640 (C=N), 1550 (C=O), 1200 (C-O- C), 720 (C-S), 610 (C-Cl). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 2.478 (s, 3H, -CH3), 4.449 (m, 2H, J = 6.5 Hz, O-CH2-), 7.107 (t, 2H, J = 6 Hz, Ar-H), 7.8188 (d, 1H, J = 6.5 Hz, Ar-H), 7.899 (d, 2H, J = 6.5 Hz, Ar-H), 8.000 (d, 2H, J = 12.5 Hz, Ar-H), 8.130 (d, 1H, J = 5 Hz, Ar-H), 8.216 (s, 1H, N=CH), 8.322 (s, 1H, Ar-H), 8.394 (s, 1H, Ar-H), 8.688 (s, 1H, Ar-H), 11.401 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.89 (-CH3), 65.12 (O-CH2-), 115.18, 116.94, 122.56, 124.93, 125.67, 127.28, 127.54, 127.86, 129.72, 130.22, 131.87, 133.45, 139.56, 141.56, 144.83, 152.94, 158.51 (Ar-C), 161.93 (C=O). MS (EI, m/z (%)): 436.07 (M+1). 6-Chloro-N'-[(E)-(4-hydroxyphenyl) methylidene]-2-(thio phen-2-yl)quinoline-4-carbohydrazide (4e): Color: Pale yellow amorphous. Yield: 68%. M.p.: 160-162 °C. FT-IR (KBr, ν, cm-1): 3600 (-OH), 3400 (N-H), 1660 (C=C), 1622 (C=N), 1510 (C=O), 730 (C-S), 670 (C-Cl). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 5.019 (s, 1H, -OH), 6.329 (t, 2H, J = 8 Hz, Ar-H), 6.636 (d, 1H, J = 7.5 Hz, Ar-H), 6.817 (t, 1H, J = 8.3 Hz, Ar-H), 7.034 (m, 2H, J = 7.5 Hz, Ar-H), 7.566 (d, 1H, J = 4Hz, Ar-H), 7.601 (s, 1H, Ar-H), 7.814 (d, 1H, J = 8.4 Hz, Ar-H), 8.252 (s, 1H, N=CH), 8.523 (s, 1H, Ar- H), 11.274 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 115.68, 117.63, 118.06, 121.94, 125.81, 126.58, 127.45, 127.66, 127.82, 129.06, 129.84, 131.12, 131.64, 134.91, 138.83, 141.75, 144.86, 152.67, 154.21 (Ar-C), 164.25 (C=O). MS (EI, m/z (%)): 408.05 (M+1). 6-Chloro-N'-[(E)-(pyridin-4-yl) methylidene]-2-(thiophen- 2-yl)quinoline-4-carbohydrazide (4f): Color: Pale yellow amorphous. Yield: 71%. M.p.: 176-178 °C. FT-IR (KBr, ν, cm-1): 3490 (N-H), 1690 (C=N), 1600 (C=C), 1520 (C=O), 730 (C-S), 690 (C-Cl). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 7.001 (d, 2H, J = 9.5 Hz, Ar-H), 7.120 (t, 1H, J = 8 Hz, Ar-H), 7.301 (d, 2H, J = 8 Hz, Ar-H), 7.600 (d, 1H, J = 9 Hz, Ar-H), 7.775 (d, 1H, J = 6 Hz, Ar- H), 7.947 (d, 1H, J = 6.6 Hz, Ar-H), 8.206 (d, 1H, J = 8.5 Hz, Ar-H), 8.266 (s, 1H, N=CH), 8.378 (s, 1H, Ar-H), 8.412 (s, 1H, Ar-H), 8.868 (s, 1H, Ar-H), 11.351 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 116.54, 121.78, 121.21, 125.37, 126.53, 127.27, 127.88, 128.18, 129.06, 130.24, 131.89, 134.15, 139.87, 141.53, 144.81, 149.62, 152.49 (Ar-C), 162.85 (C=O). MS (EI, m/z (%)): 393.05 (M+1). 2.3. Biological activity 2.3.1. Molecular docking The molecules were drawn and the corresponding smile notations were generated using ACD-ChemSketch freeware software [12], and these smile notations were used to prepare the ligands for docking studies by USCF chimera tool [13]. The crystal structure of M. tuberculosis (PDB ID:5V3Y) was taken as macromolecule/protein for docking was obtained from online data server Protein Data Bank [17], prepared and converted to PDB format using USCF chimera tool. These prepared ligands were docked against protein using PyRX workstation’s Autodock Vina [14] where the grid box is placed around the active site of the macromolecule and this active site was identified by the Uniport chimera tool. Using Schrödinger PyMol [18], the docked protein and ligands were saved in PDB format, and interactions were visualized using Biovia Discovery studios [19]. 2.3.2. ADMET studies The in silico pharmacokinetic parameters of the compounds were predicted by the online tool, admetSAR [20] and the drug likeness and bioactive scores were predicted by the online tool, Molinspiration cheminformatics [21]. The 2D structural models of the designed molecules were drawn on ACD/ChemSketch software and SMILES were generated for the molecules along with standard drugs, these smiles notations were used for predicting the individual ADMET, bioactive and drug likeness scores. AdmetSAR gives data for the evaluation of active molecules and also for the removal of biologically defective major molecules with unwanted functional groups. The overall analysis of the significant molecules involves geometry, surface area, and fingerprint properties, which determine the biological significance of the region in a molecule. CACO-2 cell perme- ability, intestinal absorption, water solubility, hepatotoxicity, and blood-brain barrier penetration were the other parameters that helps to understand the metabolic drug mechanism of the designed molecules. 2.3.3. Antibacterial activity The potential of the synthesized compounds 4a-f to inhibit the pathogenic bacteria Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus, Salmonella typhimurium were determined by Agar well diffusion assay [22,23]. The test bacteria were aseptically injected into sterile nutrient broth tubes and cultured at 38 °C for 24 hours in this technique. The 24 hours old liquid bacterial cultures were swab inoculated on sterile nutrient agar plates. In the inoculated plates, 6mm diameter wells were punched using sterile gel borer. Standard antibiotic (Chloramphenicol, 5 mg/mL of sterile distilled water (positive control)) prepared. A 100 µL of compound solutions and standard antibiotic solution were transferred aseptically into labelled wells. Sample loaded plates were not disturbed for 30 min and then incubated in the upright position for 24 hours at 38 °C. The inhibited zones developed around the wells were measured using a zone scale or ruler. 112 Prasad et al. / European Journal of Chemistry 13 (1) (2022) 109-116 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.109-116.2216 Table 1. Docking results of synthesized compounds 4a-f Schiff bases with 5V3Y. Compound Hydrogen bond interactions Hydrogen Bond’s length in Å Binding affinity in kcal/mol Electrostatic interactions Other interactions 4a PRO1595 ALA1596 3.96 5.21 -9.1 PRO1598, ILE1594, VAL1614, TRF1579, VAL1611, TYR1582, PHE1585, PHE1670 PHE1613, LEU1602, ALA1583, ILE1597, ALA1586, TYR1637 4b - - -8.6 PHE1585, MET1669, TYR1637, TRP1579, ALA1583, ILE1594, ALA1586 PHE1670, VAL1611, VAL1614, ILE1597, TYR1582 4c PRO1598 4.70 -9.2 PHE1585, PHE1670, TYR1582, VAL1611, TRP1579, VAL1614 PRO1595, LEU1602, ALA1583, ILE1594, ALA1586, TYR1637 4d - - -9.0 PHE1585, PHE1670, TYR1637, VAL1614, ILE1594, ALA1583 THR1589, LYS1588, PHE1590, VAL1611, TRP1579, ILE1597 4e TYR1582 5.01 -9.5 PHE1585, MET1669, PHE1670, TRP1579, ILE1597, ALA1583, VAL1614 VAL1618, ALA1586, LEU1615, ILE1625 4f - - -8.7 VAL1614, TRP1579, VAL1611, TYR1637, PHE1585, PHE1670 ILE1597, ALA1583, ILE1594, ALA1586, TYR1637, PRO1598, LEU1602 The size of the zone indicates the effectiveness of the compound towards pathogenic bacteria. 2.3.4. Anti-fungal activity The potential of the synthesized compounds 4a-f to inhibit the pathogenic fungi Aspergillus niger and Aspergillus flavus were determined by Agar well diffusion assay [24,25]. In this assay, the fungal spore suspension was prepared by inoculating loop full of fungal culture was inoculated into the 5 mL sterile water taken in test tube amended with 2 drops of an emulsifying agent; between 80. Test fungal spore suspensions were swabs inoculated on sterile Rose Bengal agar plates, and 6mm wells were punched in the inoculation plates with a sterile gel borer. Standard antifungal (Fluconazole, 10 mg/mL in sterile distilled water (positive control)) prepared. A 100 µL of 10 mg/mL concentration compound solution and standard antifungal solution were transferred aseptically into labeled wells. Sample loaded plates were not disturbed for 30 min and then incubated in the upright position for 72 hours at 28 °C. The inhibited zones developed around the wells were measured using a zone scale or ruler. The size of the zone indicates the effectiveness of the compound towards pathogenic fungi. 2.3.5. Anti-tubercular activity The thermally stable and nontoxic microplate Alamar blue assay (MABA) method [26] was adopted to screen the title compounds 4a-f against M. tuberculosis H37RV strain at a concentration from 0.8 to 100 μg/mL in a sterile 96 well plate. Deionized sterile water (200 μL) was added to the outer perimeter well to avoid evaporation. 100 μL of Middle brook 7H9 broth and serial dilutions of the compounds were introduced to the 96 wells plate and incubated at 36 °C for 120 hours. The sensitive compounds exhibited blue colour after 24 hours incubation against M. tuberculosis H37RV strain and the concentrations were recorded. 3. Results and discussion 3.1. Chemistry The most efficient method for the synthesis of compounds 4a-f was performed, 2-thiophen quinoline 4-carbohydrazide reacted with different aldehydes (4-dimetyl amine benzal- dehyde, furfuran, 4-fluro benzaldehyde, 4-ethoxy benzadehyde, 4-hydroxy benzaldehyde and 4-carboxyldehyde pyridine) in ethanol as solvent and catalytic amount of acetic acid. The results are summarized in Scheme 1. The synthesized molecu- les were purified by column chrmotagraphy using twenty percent of ethyl acetate in n-hexane as eluent. Analytical TLCs were performed using twenty percent ethyl acetate in n-hexane as the eluent on precoated Merck 0.25 mm silica gel 60F254 plates, and the spots on developed TLC plates were identified under UV light. The structure of the molecules was characterized by IR, 1H NMR, 13C NMR, and mass spectroscopic techniques. The synthesized compounds 4a-f were showed absorption bands ranging from 600-690 cm-1 for C-Cl stretching, 720-750 cm-1for C-S stretching, 1500-1550 cm-1 for C=O stretching, 1600-1700 cm-1 for C=N stretching, and C=C aromatic stretching, 3400-3500 cm-1 for N-H stretching. In 1H NMR spectra, the aliphatic protons appeared in the range between δ 2.1 and 5.5 ppm, aromatic protons appeared in the range of δ 6.5-8.8 ppm and NH protons appeared in the range of δ 11.2-11.4 ppm, in 13C NMR all the aromatic carbon appeared in a range of δ 100-170 ppm. 3.2. Molecular docking In silico molecular docking studies of the synthesized compounds against the crystal structure of M. tuberculosis (5V3Y) protein. The binding affinity of ligands and protein varies from -8.6 to -9.5 Kcal/mol, compound 4e shows a greater binding affinity of -9.5 Kcal/mol and others also with good binding affinity varies from -8.6 to -9.2 Kcal/mol, compounds 4a, 4c, and 4e show H-bond interaction with PRO1595, ALA1596, PRO1598, and TYR1582. The synthesized compounds show electrostatic interactions with PHE1585, PHE1670, TYR1582, VAL1611, TRP1579, and VAL1614. Binding modes and visual interaction are shown in Figure 1 and docking results are discussed in Table 1. 3.3. In-silico pharmacokinetic properties 3.3.1. ADMET studies Pharmacokinetic ADME properties are one of the main descriptors in drug discovery for human therapeutic use. These ADME descriptor properties were calculated for the molecules with biological importance and are compared with the ranges acquired for standard drugs. The synthesized compounds 4a-f do possess a considerable degree of hydrogen bond donors and acceptors as recorded through in silico data [27]. The deriva- tives were designed in such a way to acquire an increase in the binding capacity with that of the receptors through hydrogen bonding. The molecules synthesized accept Lipinski’s rule 5 for oral bioavailability and character which enhances their chance to be considered as future drug candidates. The estimated Caco- 2 permeability and BBB coefficient (log BB) were used to determine the total distribution of compounds in the human body, and the calculated BBB values were found to be within acceptable limits. The ADMET parameters of the synthesized compounds are shown in Table 2. The toxicity of compounds 4a-f is estimated based on lethal doses and their functioning ranges on various organs/tissues. Table 3 shows the probability of health consequences and the LD50 values that were predicted. Prasad et al. / European Journal of Chemistry 13 (1) (2022) 109-116 113 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.109-116.2216 Compound Ligand protein interaction 3D Interaction 2D Interaction 4a 4b 4c 4d 4e 4f Figure 1. Interaction of synthesized compounds 4a-f Schiff bases with 5V3Y. 114 Prasad et al. / European Journal of Chemistry 13 (1) (2022) 109-116 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.109-116.2216 Table 2. Predicted ADME and pharmacological characteristics of the synthesized compounds 4a-f. Compounds Log BB Log HIA Caco-2 Substrate log pGI Non-inhibitor log PGI Log S Log papp 4a 0.8974 0.9912 0.5439 0.8215 0.8492 -3.6424 1.1402 4b 0.9524 1.0000 0.5783 0.7306 0.9343 -3.8535 0.8974 4c 0.9806 1.0000 0.5307 0.7924 0.8376 -3.7333 1.0494 4d 0.9848 1.0000 0.5407 0.7916 0.7742 -3.6350 0.9936 4e 0.8940 1.0000 0.5571 0.7572 0.9149 -4.1081 1.1227 4f 0.9114 1.0000 0.5000 0.6685 0.6090 -3.4684 1.0327 Chloramphenicol 0.9382 0.9871 0.7250 0.7313 0.9019 -2.1694 0.9184 Fluconazole 0.9382 0.9894 0.8867 0.6008 0.8782 -1.8626 1.3598 Isoniazid 0.9895 0.9892 0.6959 0.8315 0.9778 -0.0521 1.2413 Table 3. TOX parameters for the synthesized ligands. TOX parameters LD50 Human hepatotoxicity Mutagenicity Skin sensitization 4a 1353.201 Yes Yes Yes 4b 1424.081 Yes No No 4c 1258.183 Yes No No 4d 1269.549 Yes No No 4e 1481.643 Yes No No 4f 1237.204 Yes No No Table 4. Drug likeness score for the synthesized ligands. Compounds MW mi LogP TPSA Number of atoms Number of ON Number of OHNH Violations Rotatable bonds 4a 434.95 5.76 57.59 30 5 1 1 5 4b 381.84 4.92 67.49 26 5 1 0 4 4c 409.87 5.83 54.35 28 4 1 1 4 4d 435.94 6.10 63.59 30 5 1 1 6 4e 405.91 6.09 54.35 28 4 1 1 4 4f 392.87 4.37 67.25 27 5 1 0 4 Chloramphenicol 323.13 0.73 115.38 20 7 3 0 6 Fluconazole 306.28 -0.12 81.66 22 7 1 0 5 Isoniazid 137.14 -0.97 68.01 10 4 3 0 1 Table 5. Bioactive score for the synthesized ligands. Compounds GPCR Ion channel modulators Kinase inhibitors Nuclear receptors Protease inhibitors Enzyme inhibitor 4a -0.51 -0.71 -0.37 -0.56 -0.63 -0.47 4b -0.32 -0.66 -0.17 -0.43 -0.52 -0.28 4c -0.36 -0.69 -0.20 -0.55 -0.53 -0.33 4d -0.35 -0.70 -0.18 -0.53 -0.55 -0.34 4e -0.33 -0.71 -0.19 -0.46 -0.47 -0.29 4f -0.34 -0.67 -0.16 -0.51 -0.53 -0.33 Chloramphenicol -0.22 -0.28 -0.38 -0.41 -0.21 -0.00 Fluconazole 0.04 0.01 -0.09 -0.23 -0.09 0.03 Isoniazid -1.39 -1.45 -1.05 -2.33 -1.23 -0.66 Table 6. Inhibited zone’s diameter in mm against bacteria. Compounds Escherichia coli Klebsiella pneumoniae Staphylococcus aureus Salmonella typhimurium 4a 3 5 2 3 4b 4 4 4 4 4c 5 3 4 4 4d 6 7 5 4 4e 2 3 1 2.5 4f 3 5 2 3 Chloramphenicol 14 17 18 15 DMSO 0 0 0 0 3.3.2. Bioactivity and drug-likeness scores of synthesized compounds 4a-f The five rules of Lipinski’s are used predominantly by drug discovery teams in designing a drug and their development for their oral bioavailability. The rule insists that a drug candidate is to be orally active: i) Molecular weight should be less than 500 g/mol, ii) Hydrogen bond acceptors should be less than ten, iii) The partition coefficient should be less than five, iv) TPSA should be less than 160 Å and, v) Hydrogen bond donors should be less than five. None of the analogs have violated any rules of Lipinski and can be expected to be active orally. The molecular weights of 4a-f are below 500 and are expected to be transport- ted, diffused and absorbed across the membranes pretty easily than the macromolecules. The synthesized compounds 4a-f correspond to the Lipinski rule. TPSA data of the molecules which are correlated with hydrogen bonding and are an indicator for bioavailability orally as the data is in the range of 64.70 to 110.52 Å well below 160 Å the limit (Table 4). Molinspiration cheminformatics is the software approach to predict the bioactivity score of the synthesized compounds 4a-f for drug targets and is represented in Table 5. Different pathways, such as interactions with inhibiting protease, nuclear receptor ligands, GPCR ligands, and other enzymes, are invol- ved in the physiological functions obtained from synthesized molecules. The data also show that there is substantial interaction between the molecules and the drug targets. Molecules have shown a strong bioactivity score. 3.4. Antibacterial activity The potential of synthesized compounds 4a-f to inhibit the pathogenic bacteria was determined by Agar well diffusion assay (Table 6). In this study, Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus, Salmonella typhimurium were selected because of their infectious nature. The study found that among the compounds tested, all the synthesized compounds 4a-f, concerning antibacterial activities, compound 4d shows potency against E. coli and K. pneumonia. Prasad et al. / European Journal of Chemistry 13 (1) (2022) 109-116 115 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.109-116.2216 Table 7. Inhibited zone’s diameter in mm against fungi. Compounds Aspergillus niger Aspergillus flavus 4a 8 8.5 4b 8 10.5 4c 9 9 4d 10 10 4e 9.5 9.5 4f 10 10 Fluconazole 19.5 25 DMSO 0 0 Table 8. The antitubercular MIC values are expressed in µg/mL. Samples MIC (µg/mL) 4a 100±0.5 4b 50±0.5 4c 100±0.5 4d 100±0.5 4e 100±0.5 Pyrazinamide 3.125±0.20 Ciprofloxacin 3.125±0.20 Streptomycin 6.250±0.12 3.5. Antifungal activity The potential of synthesized compounds 4a-f to inhibit the pathogenic fungi Aspergillus niger and Aspergillus flavus were determined by Agar well diffusion assay. The study found that among the compounds tested, compound 4d shows good activity against the fungi Aspergillus niger and all other compounds show considering activity against Aspergillus niger and Aspergillus flavus. The antifungal activity of compounds 4a- f is shown in Table 7. 3.6. Antitubercular activity The synthesized compounds 4a-f were screened against M. tuberculosis using microplate Alamar Blue assay (MABA). The study found that among the compounds tested, concerning antitubercular activities, the synthesized compounds 4a-f were found to be sensitive against M. tuberculosis at a concentration of 100 µg/mL concentration, and compound 4b shows sensiti- vity at 50 µg/mL concentration. The antitubercular activity of the compounds is shown in Table 8. 4. Conclusion A new series of Schiff bases were synthesized in the most efficient multistep route with good yield and screened for in vitro antimicrobial activity along with the antitubercular activity. The title compounds in silico studies have been determined to comply with ADME studies and Lipinski's five requirements. The docked ligands against the protein 5V3Y vary a binding affinity from -8.6 to -9.5 Kcal/mol, compound 4e shows a greater binding affinity of -9.5 Kcal/mol among all other also compounds. The antimicrobial study reveals that among the compounds 4a-f tested, compound 4d was found to be active against test bacteria and fungi with a greater zone of inhibition but comparatively lower to standard drug. The antitubercular activity reveals that among the tested compounds 4a-f, compound 4b shows sensitivity at 50 µg/mL concentration but not as good as standard. The compounds 4b and 4d can be taken as lead molecules where they can be further modified and developed to achieve the good activity. Acknowledgements The authors are grateful to the officials of Kuvempu University that they have provided the necessary facilities to carry out the present work. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Nayak Devappa Satyanarayan; Methodology: Sakshith Raghavendra Prasad; Validation: Avarse Satish Kumar Shetty; Data Curation: Basaiah Thippeswamy; Writing - Original Draft: Sakshith Raghavendra Prasad; Writing - Review and Editing: Nayak Devappa Satyanarayan; Supervision: Nayak Devappa Satyanarayan, Avarse Satish Kumar Shetty. ORCID and Email Sakshith Raghavendra Prasad prasadsakshith@gmail.com https://orcid.org/0000-0003-4700-1679 Nayak Devappa Satyanarayan satya1782005@gmail.com https://orcid.org/0000-0003-4511-3749 Avarse Satish Kumar Shetty skshettyncp@gmail.com https://orcid.org/0000-0003-2903-7678 Basaiah Thippeswamy thippeswamyb205@gmail.com https://orcid.org/0000-0002-6436-0289 References [1]. Alsayed, S. S. R.; Lun, S.; Bailey, A. W.; Suri, A.; Huang, C.-C.; Mocerino, M.; Payne, A.; Sredni, S. T.; Bishai, W. R.; Gunosewoyo, H. 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Evaluation of anti- tubercular activity of nicotinic and isoniazid analogues. ARKIVOC 2007, 2007, 181–191. [27]. Lipinski, C. A. Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov. Today Technol. 2004, 1, 337–341. Copyright © 2022 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.pymol.org/pymol http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Material 2.2. Synthesis 2.2.1. The starting material 2-thiophene quinoline-4-carboxylic acid (1) 2.2.2. Synthesis of ethyl 6-chloro-2-(thiophen-2-yl) quinoline-4-carboxylate (2) 2.2.3. Synthesis of 6-chloro-2-(thiophen-2-yl)quinoline-4-carbohydrazide (3) 2.2.4. Synthesis of 6-chloro-N'-methylidene-2-(thiophen-2-yl)quinoline-4-carbohydrazides (4a-f) 2.3. Biological activity 2.3.1. Molecular docking 2.3.2. ADMET studies 2.3.3. Antibacterial activity 2.3.4. Anti-fungal activity 2.3.5. Anti-tubercular activity 3. Results and discussion 3.1. Chemistry 3.2. Molecular docking 3.3. In-silico pharmacokinetic properties 3.3.1. ADMET studies 3.3.2. Bioactivity and drug-likeness scores of synthesized compounds 4a-f 3.4. Antibacterial activity 3.5. Antifungal activity 3.6. Antitubercular activity 4. Conclusion Acknowledgements Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: