Ba gladesh Jour al of Phar acology Resear h Arti le Design of 1-(furan-2-yl)-N-(5- substituted phenyl-1, 3, 4- thiadiazol-2-yl) methanimine deriv- atives as Enoyl-ACP reductase inhib- itors: Synthesis, molecular docking studies and anti-tubercular activity BJP Introduction Tuberculosis (TB), a lung infection is one of the contagious and deadly diseases which have added to the woes of the mankind. It is the most dreadful infectious disease caused by Mycobacterium tuberculosis. The widespread of this disease is primarily due to the population growth, emergence of multi-drug resistant TB strains, financial burden in the developing countries and unsuccessful attempt to synthesize a new drug with novel mechanism of action (Thomas et al., 2011). Mycolic acid biosynthesis is essential for the building up of cell wall in mycobacterial and related species (Kolattukudy et al., 1997). InhA, the enoyl acyl carrier protein reductase from Mycobacterium tuberculosis is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target (Banerjee et al., 1994). A series of pyrrolidine carboxamides have been recog- nized as potent direct class of InhA inhibitors (He et al., 2006). Recently Oxadiazolo pyrrolidine carboxamides were designed as enol-ACP reductase inhibitors (Sonia et al., 2012). Considering the structural features of the inhibitor of enol-ACP reductase enzyme, we had under- taken the design a novel class of 1,3,4-thiadiazole clubb- ed with the furan moiety by means an azomethine linkage (Figure 1). The Schiff base associated with the 2-furfural exhibit anti-microbial and anti-proliferative properties were already reported (Gaballa et al., 2007; Rajendran et al., 2010; Hranjec e t al., 2010). 1,3,4-thiadiazole pharmaco- phore displayed a wide range of pharmacological acti- vities such as anti-inflammatory, CNS depressant acti- vity and mucomembranous protector (Varandas et al., 2005; Jatav et al., 2008; Mathew et al., 2013). It has been predicted by the PASS computational approach, that the combination of above these pharmacophore in the A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2013; 8: 242-248 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 Design of 1-(furan-2-yl)-N-(5-substituted phenyl-1, 3, 4-thiadiazol -2-yl) methanimine derivatives as Enoyl-ACP reductase inhibitors: Synthesis, molecular docking studies and anti-tubercular activity Bijo Mathew1, Githa Elizabeth Mathew1, George Sonia2, Anila Kumar1, Neethu P. Charles1 and Palanisamy Kumar1 1Department of Pharmaceutical Chemistry, Grace College of Pharmacy, Palakkad, Kerala, India; 2College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, Tamil Nadu, India. Abstract A series of 5-phenylsubstiuted 1, 3, 4 thiadiazoles clubbed with furan moiety (Fa-Fe) by means of azomethine linkage have been synthesized. All the newly synthesized compounds were characterized by IR,1HNMR and Mass analyses. All the synthesized molecules have been predicted as anti- tubercular in nature by PASS in silico approach. In vitro anti-tubercular screening was performed by alamar blue assay method on Mycobacterium tuberculosis H37Rv strain. Among the synthesized derivatives Fb and Fe were active at 3.1 µg/mL against Mycobacterium tuberculosis H37Rv strain. The mechanism of action of the active compounds was carried out by docking of receptor enoyl-ACP reductase. It has been concluded that both Fb and Fe posses a significant interaction of hydrogen bonding and electrostatic attraction with Tyr 158 and Met103 in the active site of enzyme. Article Info Received: 26 April 2013 Accepted: 8 May 2013 Available Online: 16 May 2013 DOI: 10.3329/bjp.v8i3.14778 Cite this article: Mathew B, Mathew GE, Sonia G, Kumar A, Charles NP, Kumar P. De- sign of 1-(furan-2-yl)-N-(5-substituted phenyl-1, 3, 4-thiadiazol-2-yl) methanimine derivatives as Enoyl- ACP reductase inhibitors: Synthesis, molecular docking studies and anti- tubercular activity. Bangladesh J Pharmacol. 2013; 8: 242-48. This ork is li e sed u der a Creati e Co o s Attri utio 3.0 Li e se. You are free to opy, distri ute a d perfor the ork. You ust attri ute the ork i the a er spe ified y the author or li e sor. designed scaffold would exhibit better anti-tubercular nature. Materials and Methods Melting points were determined by using open capillary tube method and the values were uncorrected. IR spectra were recorded on JASCO FT/IR-140 spectrophotometer by using KBr pellets technique. PMR spectra were recorded using BRUCKER FT-NMR- 500 MHz spectrophotometer by using DMSO as solvent and TMS as internal standard. The chemical shift was expressed in  ppm. Mass spectra were recorded on a JEOL GCmate mass spectrometer. Synthesis of thiosemicarbazones Aromatic aldehyde (0.2 M) in warm alcohol (300 mL) and thiosemicarbazide (0.2 M) in warm water (300 mL) were mixed slowly with continuous stirring. The product separated immediately on cooling which was filtered with suction, dried and recrystallized in etha- nol: water (7:3). Synthesis of 5-substituted phenyl-1, 3, 4-thiadiazol-2- amines Thiosemicarbazone (0.05 M) was suspended in 300 mL warm water, FeCl3 (0.15 M) in 300 mL water was added quantitatively, slowly with constant stirring. The contents were heated at 80-90°C for 45 min. Solution was filtered hot and then citric acid (0.11 M) and sodium citrate (0.05 M) were added. The resulting mixture was divided into 4 parts and each part was neutralized separately with ammonia (10%). The requir- ed amine separated out, filtered with suction, dried and recrystallized in ethanol:water (5:5). Synthesis of 1-(furan-2-yl)-N-(5-substiuted-phenyl-1, 3, 4-thiadiazol-2-yl) methanimine 5- phenyl-1, 3, 4-thiadiazol-2-amine (0.01M) was suspended in DMF and add furan-aldehyde (0.015 M) with 2-3 drops of Conc. H2SO4.The reaction mixture was then refluxed for 6-7 hours. The reaction progress was monitored by TLC (chloroform:ethanol 4:1). The resultant contents were poured into crushed ice. The crude product was filtered, washed with water until it is free from acidic catalyst, dried and recrystallized with ethanol. 1-(furan-2-yl)-N-(5-phenyl-1, 3, 4-thiadiazol-2-yl) methanimine (Fa): M.p-175-178, Rf: 0.65 IR: vmax /cm- 13160(-CH aryl), 1615(C=N), 732(C-S-C). 1HNMR (DMSO-d6/TMS): 8.46(s, 1H, N=CH), 7.21-7.96(m, 8H, aryl protons).MS: m/z (M+)+255. N-[5-(4-chlorophenyl)-1, 3, 4-thiadiazol-2-yl]-1-(furan-2-yl) methanimine (Fb): M.p-170-172, Rf: 0.72, IR: vmax /cm- 13010(-CH aryl), 1588(C=N), 727(C-S-C). 1HNMR (DMSO-d6/TMS):8.23(s, 1H, N=CH), 7.19-7.78(m, 7H, aryl protons). MS: m/z (M+2)+289. 1-(furan-2-yl)-N-[5-(4-methoxyphenyl)-1, 3, 4-thiadiazol-2- yl] methanimine (Fc): M.p-183-186, Rf: 0.81, IR: vmax /cm- 13150(-CH aryl), 1595(C=N), 742(C-S-C). ). 1HNMR (DMSO-d6/TMS):8.55(s, 1H, N=CH), 7.01-7.90(m, 7H, aryl protons), 3.55(s, 3H, OCH3). MS: m/z (M+)+285. 1-(furan-2-yl)-N-[5-(2-nitrophenyl)-1, 3, 4-thiadiazol-2-yl] methanimine (Fd): M.p-165-168 Rf: 0.75, IR: vmax /cm- 13133(-CH aryl), 1625(C=N), 728(C-S-C). IR: vmax /cm- 13176(-CH aryl), 1610(C=N), 735(C-S-C). 1HNMR (DMSO-d6/TMS):8.87(s, 1H, N=CH), 7.2-7.95(m, 7H, aryl protons) MS: m/z (M+1)+300. 1-(furan-2-yl)-N-[5-(3-nitrophenyl)-1, 3, 4-thiadiazol-2-yl] methanimine (Fe): M.p-165-168 Rf: 0.75, IR: vmax /cm- 13165(-CH aryl), 1623(C=N), 721(C-S-C). 1HNMR (DMSO-d6/TMS):8.74(s, 1H, N=CH), 7.1-8.01(m, 7H, aryl protons) MS: m/z (M+1)+300. Computational studies PASS prediction Prediction of this spectrum by PASS is based on SAR analysis of the training set containing more than 35,000 compounds which have more than 500 kinds of biological activity. 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 0.5Fa>Fd >Fb>Fc. The PASS program significantly predicted the Fe compound has the highest probability and was most active in the in vitro anti-tubercular studies, while Fc with lowest Pa value in the series was practically less active. It has been noted that the PASS program predicts Fb with a low Pa value of 0.691. This low score cannot favours its activity ratio because it showed significant activity with MIC of 3.3 µg/mL. The active pocket was considered to be the site where 1- cyclohexyl-N-(3, 5- dichlorophenyl)-5-oxopyrrolidine-3- carboxamide (pyrrolidine carboxamides) complexed with enoyl-ACP reductase in 2H7M. Hydrogen bond- ing network of ligand and Tyr158 seems to be a promi- nent feature among all the InhA-inhibitor complexes identified so far (Kuo et al., 2003). All the newly designed molecules showed good binding interaction towards the active site of enoyl-ACP reductase. Molecular docking study suggested that compound Fb and Fe showed the best docking score of-9.2778 and 9.2311 respectively. The docking pose of Fe showed a Bangladesh J Pharmacol 2013; 8: 242-248 245 R Fa: phenyl Fb: 4- chloro phenyl Fc: 4- methoxy phenyl Fd: 2- nito phenyl Fe: 3- nitro phenyl R-CHO + H2NNHCSNH RCH=NNHCSNH 2 2 S NN R NH2 S N N R N O a b Synthesis of (Fa-Ff).Reagents and conditions a: I-Heat 45 min with FeCl3 sol. II- citric acid(0.11M)&sodium citrate(0.05M) III-neutralization with 10% ammonia b: Furfural,conc.H2SO4,DMF,7h reflux Figure 3: Synthetic route of the titled compounds crucial π-π stacking interaction between the azomethine unit and phenyl system of Tyr158 (Figure 5). The in silico study of the Fb and Fe revealed that its binding affinity towards the enzyme is due to the hydrogen bonding interaction with Tyr 158.The ligand map of Fb (Figure 6) suggested that a steric interaction of furan part in the scaffold to the Met 103 also contribute an interesting result in the activity ratio. Tyr 158 and Met 103 are the major interacting residues of the known inhibitor of 1-cyclohexyl-N-(3,5- dichlorophenyl)-5-oxopyrrolidine-3-carboxamide towards enoyl-ACP reductase. Another interesting factor raised from our finding is that the poor affinity and MIC of Fc is due to its lack of hydrogen bonding between Tyr158. Estimated binding energy (Kcal/mol) of all the designed molecules and the amino acid residues enveloped by the designed molecules was represented in Table II. Anti-tubercular screening Compounds Fe and Fb showed significant activity with MIC of 3.1 µg/mL. It is interesting to note that the activity ratio is decreased in the designed structures when the presence of electron donating group such as methoxy group in the phenyl system. The promising activity of the designed molecules is mainly attributed with the presence of electron withdrawing group such as nitro and chloro in the phenyl system.2-nitro (Fd) also showed a comparable MIC of 12.5 µg/mL. The present study established the design and synthesis of some new phenyl substituted 1,3,4-thiadiazole clubbed with furan moiety as a potent inhibitor of enoyl -ACP reductase. The anti-tubercular activity of the designed scaffold was predicted by PASS in silico approach. Molecular docking study revealed that the structural features of the derivatives can make a significant interaction towards the active site of the enzyme which is necessary for the development of 246 Bangladesh J Pharmacol 2013; 8: 242-248 Figure 4: Mass fragmentation pattern of Fe Table I PASS prediction of Fa-Fe Anti-tubercular activity Anti-tubercular activity Compound code Pa Pi Fa 0.710 0.004 Fb 0.691 0.004 Fc 0.689 0.004 Fd 0.709 0.004 Fe 0.804 0.003 mycolic acid. In the light of our interesting results of some of the derivative’s binding mode, a new design has been developed for the inhibition of enoyl-ACP reductase. Acknowledgement The authors are highly thankful to Maratha Mandal’s Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre; Belgaum for anti-tubercular activity. Our sincere thanks goes to Grace College of Pharmacy, Kerala, India, for carrying out the synthetic work for the present study. Authors also acknowledge the, SAIF IIT, Chennai, for carrying out the spectral analysis. References Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson T, Collins D, de Lisle G, Jacobs WR Jr. InhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994; 263: 227-30. Collins L, Franzblau SG. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother. 1997; 41: 1004-09. Gaballa AS, Asker MS, Barakat AS, Teleb SM. Synthesis, characterization and biological activity of some platinum (II) complexes with Schiff bases derived from salicylaldehyde, 2 -furaldehyde and phenylenediamine. 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Lett Drug Des Discov. 2005; 2: 62-67. 248 Bangladesh J Pharmacol 2013; 8: 242-248 Author Info Bijo Mathew (Principal contact) e-mail: bijovilaventgu@gmail.com DatePrinted: This article was downloaded by you on: Jul 04, 2018