Bangladesh Journal of Pharmacology Research Article Design, docking, synthesis and anti E. coli screening of novel thiadiazolo thiourea derivatives as possible in- hibitors of Enoyl ACP reductase BJP Introduction The major drawback in the antimicrobial drug therapy is the resistance offered by the microbes even to the established entities. These shortcomings are owing to the mutations associated, noncompliance in the drug therapy etc. The discovery of new drugs is now focuss- ed on drug targets like enzymes or receptors. FabI, the enoyl acyl carrier protein reductase of the Escherichia coli species is one of the attractive targets in E. coli associated diseases (Helmut et al., 1994). Enoyl ACP reductase is involved in the elongation of fatty acid which serves as the precursor for mycolic acid biosyn- thesis (Richard et al., 1995). The biocidal activity possessed by triclosan is identified recently due to its interactions with the enoyl ACP reductase (Roujeinikova et al., 1999). Other moieties like oxadiazoles, thiadiazoles, pyrazoles etc are also repor- ted to possess inhibition of this enzyme target (Sonia et al., 2012; Sonia et al., 2011; Kuo et al., 2003). Thiourea derivatives are reported to possess excellent antibacte- rial potential (Arslan et al., 2009). Owing to the above facts and in continuation of the research on enoyl ACP reductase inhibitors, an attempt is made in the present research to design and develop new anti E. coli agents possessing enoyl ACP reductase inhibition. The antibac -terial effect of the incorporation of phenyl thiourea to the thiadiazoles was predicted by the PASS computa- tional approach. Materials and methods Melting points were determined by using melting point apparatus MR-VIS (MR08190508). IR spectra were recorded on JASCO FT/IR-410 spectrometer on KBr pellets. 1HNMR and 13C NMR spectra were recorded on a Bruker 300 MHz NMR spectrometer. Mass spectra data was obtained from Shimadzu, LCMS 2010 EV. Purity of all the compounds were checked by thin layer chromatography using silica gel-G as adsorbent and A Journal of the Bangladesh Pharmacological Society (BDPS); www.bdps.info Bangladesh J Pharmacol 2014; 9: 49-53 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract A series of 1-phenyl-[5-substitutedphenyl)-1,3,4-thiadiazol-2-yl]-3-thiourea (3a-h) were synthesized and screened for their anti Escherichia coli potential. The characterization of the newly synthesized compounds was based upon their spectral data. The design of the title compounds was done utilizing the in silico methodology. Virtual screening technique was utilized for the identification of the lead, thiadiazole. The pharmacokinetic behavior was predicted by lead optimization and docking studies helped to analyze the binding interactions. Antibacterial activity of the title compounds were predicted by the PASS prediction software. The anti E. coli screening results showed that the derivatives, 3b and 3h possessed significant activity. Article Info Received: 13 November 2013 Accepted: 24 December 2013 Available Online: 31 January 2014 DOI: 10.3329/bjp.v9i1.16992 Cite this article: George S, Basheer RM, Ram SV, Sel- varaj SK, Rajan S. Ravi TK. Design, docking, synthesis and anti E. coli screening of novel thiadiazolo thiou- rea derivatives as possible inhibitors of Enoyl ACP reductase (FabI) en- zyme. Bangladesh J Pharmacol. 2014; 9: 49-53. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Design, docking, synthesis and anti E. coli screening of novel thiadiazolo thiourea derivatives as possible inhibitors of Enoyl ACP reductase (FabI) enzyme Sonia George, Ramzeena Mohammed Basheer, Sayee Vignesh Ram, Senthil Kumar Selvaraj, Shinu Rajan and Thengungal Kochupappy Ravi Department of Pharmaceutical Chemistry, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, Tamilnadu, India iodine vapour as detecting agent. The softwares used for in silico works were iGEMDOCK v.2, Accelerys Accord for Excel and AutoDock4.2. Virtual Screening: Virtual screening technique is utilized for the selection of target and the ligand. Virtual screening was performed utilizing iGEMDOCK v.2 (http: //gemdock.life.nctu.edu.tw/ dock/). The hits were selected from the ZINC database (http:// zinc.docking.org/), a database of thirteen million compounds in purchasable format. Selection of the target and lead: The target enzyme selected was enoyl-acyl protein reductase (FabI) and the hits were obtained from the zinc database. The selection of lead from the hits was based on the virtual screening technique using iGEMDOCK v.2. A small molecular library of 10,000 hits was constructed utilizing ZINC database and was docked into the enzyme and the potential leads were discovered based on the binding affinity. Thiadiazole was selected as the potential lead. The binding alignment of the selected moiety is depicted in Figure 1. Lead optimization: The selected lead was substituted with various substituent and the in silico lead optimization was done to evaluate its pharmacokinetic profile (Table I). The optimization of the lead was done utilizing Accelerys Accord for Excel (Accelerys Software Inc., San Diego). Molecular docking studies: AutoDock4.2 was the software used for docking (http://autodock. scripps.edu/). Enzyme and ligand preparation: The X-ray crystal structure of the enzyme enoyl ACP reductase (FabI) of E. coli (PDB entry: 1C14) was obtained from Protein Data Bank (http://www.rcsb.pdb.org). The enzyme was refined to remove the hetero atoms and water and Kollmann charges were added. The optimized ligand structures were energy minimized, Gasteiger charges and polar hydrogens were added and torsion was set. Docking simulations: The energy minimized ligands were subjected to docking studies in order to predict its interaction with the key binding sites on the enzyme. Initially Grid map was set with 60 points and a spacing of 0.375 A° was set. Lamarckian genetic algorithm was used, with a maximum number of 25,000,000 energy evaluations and a maximum number of 5,000 generations, for each run and 150 docking runs were performed. The binding energies obtained through in silico studies are depicted in Table I. The snap shots of the docked structures are shown in Figure 2. PASS prediction: PASS prediction is based on SAR analysis of the training set containing more than 35,000 compounds which have more than 500 kinds of biological activity. This in silico method is used to predict the activity of the chemical entity before its synthesis. The compounds subjected to docking studies in the present study were subjected to PASS prediction to confirm its potential as anti E. coli agents. If Pa>0.7 the chance to find the activity in experiment is high, but in many cases the compound may occur to be the close analogue of known pharmaceutical agents. If 50 Bangladesh J Pharmacol 2014; 9: 49-53 Figure 1: Binding alignment of thiadiazole moieties with the enzyme in virtual screening Table I Data of in silico screening SL No. Compound code Pharmacokinetic parameters PASS prediction Docking parameters ΔG (kcal/mol) mLog p HIA PBL HTL Pa Pi 1 3a 4.321 1 3 0 0.710 0.004 -7.34 2 3b 3.987 1 4 0 0.790 0.004 -8.33 3 3c 4.324 1 3 0 0.687 0.004 -7.48 4 3d 4.562 1 4 0 0.667 0.004 -7.56 5 3e 4.128 1 4 0 0.742 0.003 -6.23 6 3f 4.387 1 2 0 0.674 0.003 -7.46 7 3g 3.888 1 4 0 0.732 0.003 -7.01 8 3h 4.012 1 3 0 0.810 0.003 -8.74 9 Triclosan - - - - - - -6.78 HIA- human intestinal absorption (<2); PBL- protein binding level (>2); HTL- hepato toxicity level (0- no probability) 0.53b>3e>3g>3a>3c>3d>3f order. The prediction showed that compound 3h has the highest probability and 3f has the least probability of antibacterial activity. Most of the derivatives possessed a correlation with the PASS prediction. The derivatives obtained through computational tools were synthesized. The title compounds, 1phenyl-[5- substituted phenyl)-1,3,4-thiadiazol-2-yl]-3-thiourea (3a -h) were synthesized by the reaction of 2-amino-5- substituted phenyl -1, 3, 4-thiadiazole (2a-h) with phenyl isothiocyanate and ethanolic potassium hydro- xide. The amines, 2a-h were obtained by the oxidative cyclization of thiosemicarbazones with FeCl3 in presence of citric acid. The synthetic strategy is given in the Scheme 1. The spectral investigations revealed, the successful formation of the title compounds. The 52 Bangladesh J Pharmacol 2014; 9: 49-53 Table II Antibacterial screening data SL No. Compound code Zone of inhibition (mm) 1 3a 22 2 3b 28 3 3c 22 4 3d 24 5 3e 20 6 3f 23 7 3g 21 8 3h 28 9 Ciprofloxacin 30 Figure 2: Snap shot representing the binding interactions of 3 hours binding with enoyl ACP reductase fab1 with the active residue GLY93 with two hydrogen bonds presence of IR absorption bands at 3262.134 and 1591.32 corresponded to NH and thiadiazolyl C=N respec- tively. Also, in the 1HNMR spectrum, broad singlets at 10.491 and 9.241 corresponded for the NH groups in 3a. The multiplets ranging from 7.653-7.936 revealed the presence of aryl protons. The signals at 58.765, 121.234- 127.324, 162.375 and 172.310 obtained in 13CNMR also revealed the successful formation of the title com- pounds. The molecular ion peak at 312 was corres- ponding with the molecular formula, C15H12N4S2 of 3a. The antibacterial results revealed that the derivatives, 3b and 3h showed a zone of inhibition of 28 mm when compared to the standard, ciprofloxacin (30 mm) at 250 µg/mL. Other derivatives also showed significant activity as predicted by the in silico studies. It can be assumed that the significant antibacterial potential exihibited by the above derivatives are due to the presence of electron with drawing groups like chloro and nitro groups. To conclude with, the present research focussed on the design of enoyl ACP reductase inhibitors of E. coli has resulted in the development of novel thiourea linked 1,3,4-thiadiazole derivatives. The activity profile of the designed compounds indicated that there existed a significant correlation with the computational data. Thus the new 1,3,4-thiadiazolyl thiourea derivatives are excellent candidates in antibacterial drug discovery. References Arslan H, Duran N, Borekci G, Koray Ozer C, Akbay C. 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Author Info Sonia George (Principal contact) e-mail: soniashenjeev@gmail.com Bangladesh J Pharmacol 2014; 9: 49-53 53 H O R + NH2 NH NH2 S N NH NH2 S R N N S NH2 R thiosemicarbazidearomatic aldehyde 1a-h 2a-h N N S NH R NH S 3a-h R = unsubstituted, p-chloro, p-flouro, p-methoxy, 2,3,4-trimethoxy, p-isopropyl, 4-hydroxy, m-nitro I II Scheme 1: Synthesis of thiadiazolyl thiourea Reagents & conditions: I: FeCl3, 80-90°C, 45 min; II: Phenyl isothiocyanate, KOH, 2h, reflux DatePrinted: This article was downloaded by you on: Sep 15, 2018