Design, Synthesis and Characterization of Thiophene Substituted Chalcones for Possible Biological Evaluation

Development of new antimicrobial agents is a better solution to rectify drug resistance problems in society. In this circumstances new functionalized sulphur bearing heterocyclic moiety were designed, synthesized and evaluated for their in vitro antibacterial activity. The present work encompasses the designing novel series of thiophene substituted analogous linked to para amino acetophenone and different aldehydes were successfully synthesized and biological activity was predicted using various computational software’s such as Chemsketch, Molinspiration, and admetSAR. Among the synthesized thiophene substituted chalcones T-IV-I and thiophene T-IV-B displayed significant activity against Streptococcus auresis. Compounds T-IV-J, T-IV-H and T-IV-C bearing sulphur moiety possess better activity against Staphylococcus aureus. Moreover T-IV-C and T-IV-J exhibits good antibacterial activity against E. coli and Pseudomonas aeruginosa. In general, most of the synthesized compounds exhibited remarkable antibacterial activity due to the presence of sulphur atom in the heterocyclic moieties as well as its lipophilic characters. Molecular docking studies indicated that the synthesized compounds are potent inhibitor of microsomal enzyme Glutathione-S-transferases (PDB ID: 1GNW) also find the different interacting residues, Original Research Article Joseph and Alaxander; JPRI, 33(49B): 55-79, 2021; Article no.JPRI.76759 56 bond distanceand nature of bondingbetween the target and the ligand molecules. The results provide important information for the future design of more effective antibacterial agents.


INTRODUCTION
In sulphur containing heterocyclic, thiophene substituted chalcone derivatives are at the focus as these candidates have structural similarities with active compounds to develop new potent lead molecules in drug design. Thiophene scaffold is one of the privileged structures in drug discovery as this core exhibits various biological activities allowing them to act as antimicrobial, antioxidant, antitubercular, antifungal [1] actions. Further, numerous thiophene -based compounds as clinical drugs have been extensively used to treat various types of diseases with high therapeutic potency, which has lead to their extensive developments. Due to the wide range of biological activities of substituted thiophenes, their structure activity relationships (SAR) have generated interest among medicinal chemists, and this has culminated in the discovery of several lead molecules against numerous diseases [2]. The present review is endeavouring to highlight the progress in the various pharmacological activities of thiophene substituted chalcone derivatives [3]. Also biological studies that highlight the chemical groups responsible for evoking the pharmacological activities of synthesized derivatives are studied and compared. Design of Insilico filters to eliminate compounds with undesirable properties poor activity or poor Absorption, Distribution, Metabolism, Excretion and Toxicity, (ADMET)) and select the most promising candidate. Fast expansion in this area has been made possible by advances in software and hardware computational power and sophistication. Identification of molecular targets and an increasing database of publicly available target protein structures like the protein data bank www.pdb.org. CADD is being utilized to identify this active drug candidates, select lead compounds (most likely candidates for further evaluation), and optimize leads compounds i.e. transform biologically active compounds into suitable drugs by improving their physicochemical, pharmaceutical, ADMET/PK (pharmacokinetic) properties. Virtual screening is used to discover new drug candidates from different chemical scaffolds by searching commercial, public, or private 3-dimensional chemical structure databases [4].
In-silico technique is reducing the number of molecules synthesized and helping researchers in the process of drug development. Tools and models available are used to estimate the ADMET properties, and structure-based molecular docking, helps in predicting the possible interactions with the target under study. Major information whether the compound under study can work as a drug at an early stage of development is provided by in-silico physico chemical properties such as saturation, size, lipophilicity, solubility, polarity, and flexibility [5].
The purpose of the current study was to perform simulated screening of molecules through molecular docking strategy and identify possible lead molecules which could serve as a template for designing new proposed molecules with improved binding affinities, and better molecular interactions with the receptor. Additionally in-silicoADME and drug likeness properties of the designed compounds were also evaluated for oral bioavailability and safety of the compound. Discovery studio 2020 prediction was performed on selected compounds to assess the probability of antimicrobial activity.

Protein Target
3D Structures of protein were procured from PDB. The protein structures were cleaned (water molecules and other hetero atoms removed), prepared and minimized before docking.

Docking
Docking module LibDock using Discovery Studio 2020 was used to study interaction between the Protein and ligand molecules. The binding site of the protein defined and the docking performed. The LibDock scores, nature of bonding and bond length of the docked ligands were estimated.

Protein Preparation
Prepare the protein structure before docking because, In general PDB structures contain water molecules, all water molecules are removed except the important ones in protein preparation. Hydrogen atoms will be missing in PDB structure; many docking programs need the protein to have explicit hydrogen. Hydrogen can be added unambiguously except in the case of acid/ basic side chains through protein preparation. The PDB structure can be incorrect in some protein side chains. The crystallographic structure gives electron density, not molecular structure. Click on, Macromolecule then prepare protein then automatic preparation followed by prepare protein give input protein (select the saved protein structure) apply run. Then save the resultant prepared structure in a new file.

Ligand Preparation
Preparation of ligand is also done because of some reasons; a reasonable 3D structure is needed as starting point. Protonation state and tautomeric form of a particular ligand could influence its hydrogen bonding ability. Small molecule Prepare then alter-ligands then prepare ligand add input ligand (select the saved ligand structure) finally run. The resultant prepared structures of ligands are saved in new file

Define Binding Site
After the protein and ligand preparation, next step is to define binding site for docking.

Docking
Click on Receptor ligand interaction open dock Ligands click LibDockwhich was used for docking, because a target needs to dock with multiple ligands. After docking each and every poses of dock result are analysed in detail. Then the result was screened based on presence of Hydrogen-bond interaction and Libdock score, and are listed out. The listed ligand poses are screened based on the presence of H-bond interaction at GLU230 residue and the molecular properties of these ligands are calculated in DS by ADMET descriptors and toxicity prediction. The binding energy of the ligands was also calculated.

In silico Molecular Studies
In silico molecular modeling studies were carried out for different derivatives using different softwares like ACD Lab Chemsketch, Molinspiration, admet SAR and Biovia Discovery Studio 2020. Analysis of Lipinski Rule of Five was carried out for the proposed analogues using Molinspiration software. Insilicomolecular modeling using ACD Lab Chemsketch software was carried out as ACD Lab Chem sketch is a chemically intelligent drawing interface that allows drawing almost all chemical structure including organics, organo metallics, polymers and Markush structures. Use it to produce professional looking structures and diagrams for reports and publications. Determination of drug likeness and Lipinski rule of five using Molinspiration software [6] indicated in Table 1.
Determination of drug likeness is an important aspect of the drug design. These properties mainly electronic distribution, hydrophobicity, molecular size, hydrogen bonding characteristic, flexibility and presence of various pharmacophoric features influence the behaviour of molecule in a living organism, including bioavailability, transport properties, affinity to proteins, reactivity metabolic stability, toxicity and many others. Drug likeness score is calculated by Mol inspiration software [7] mentioned in Table 2.
The Lipinski Rule of five provides a measure for determining the oral bioavailability of a compound specified in Table 3.

Data's Computed from Software
Antimicrobial activity was predicted using Discovery Studio 2020 software

Determination of ADMET Profile Using AdmetSAR
In total, 22 highly predictive qualitative classification models were implemented in admetSAR software. These models includes human intestinal absorption, blood-brain barrier penetration, Caco-2 permeability, P-glycoprotein substrate and inhibitor, CYP450 substrate and inhibitor (CYP1A2, 2C9, 2D6, 2C19, and 3A4), hERG inhibitors, AMES mutagenicity, carcinogens, fathead minnow toxicity, honey bee toxicity, and tetrahymenapyriformis toxicity. In addition, all classification models were given a probability output instead of simple binary output. In scientific community of ADMET prediction, quantitative predictions are more useful. The report was summarized in

EXPERIMENTAL
A mixture of ketone like 4-(acetyl phenyl) thiophene 2-carboxamide and different ortho, meta, para substituted benzaldehyde ((0.01m) and 40% aqueous potassium hydroxide is added to 30 ml of ethanol and was stirred at room temperature for about 2-6 hrs. The resulting products was keep overnight in refrigerator. The solid separated out was filtered, washed with water and recrystallized from ethanol yields pure crystalline products. After drying in an over at about 70 0 C yield was 68%. Melting point was found to be 132 0 C. TLC was checked by n-hexane and chloroform (9:1) ratio as an eluent. All compounds were prepared by same method. Chemical characterization in synthesized derivatives summarized in Table 6.

N-(4-(3-(Furan-3-yl)Acryloyl)Phenyl) Thiophene-2-Carboxamide(T-IV-J)
Yield, (70%).IR-vales.(cm The three-dimensional structure of Glutathione S-Transferase was downloaded from PDB database with PDB ID: 1GNW with crystallographic resolution 2.20 A 0 (Fig. 1). The protein chain consists of two polypeptide chain A and B with total 211 amino acids and has a molecular weight of 47860.6 Daltons. The active site of protein interacting with the standardised ligand molecules was selected as the binding site.

Fig. 12. Spectral images of all synthesized compounds
The result showed that T-IV-I have high binding affinity to target compared to other ligands. All the docked complexes were analysed to study non-bond interactions between the target and the ligand molecule. The results are summarized in the Table 10. The results revealed that all the ligands bind the same active site.

In vitro Antibacterial Activity
The selected synthesized thiophene derivatives of the present investigation were screened for their anti-bacterial activity by subjecting the compounds to standard procedures [8]. The antibacterial activity was performed by cup and plate method (diffusion technique). The fresh culture of bacteria was obtained by inoculating bacteria in nutrient broth media and incubated at 37 ± 2 o C for 18 -24 hrs. This fresh culture was mixed with nutrient agar media and poured into sterile petri-plates by pour plate method, by following aseptic technique [9]. After solidification of the media, six bores were made at equal distance by using sterile steel cork borer (8 mm diameter). Into this cup 100µg/ml, 200µg/ml concentration solution of standard drug and synthesized compounds were introduced. Dimethyl formamide [10] was used as a control. After introduction of standard drugs and synthesized compounds, the plates were placed for proper diffusion of drug into the media for about 2 hrs. After 2hrs. The plates were incubated in BOD incubator and maintained at 37 ± 0.5 o C for 18-24 hrs. After the incubation period, the plates were observed for zone of inhibition [11] by using Hi-antibiotic zone reader. Results were evaluated by comparing the zone of inhibition shown by synthesized compounds with the standard drug. [12] the results were the average value of zone of inhibition measured in millimeter of three sets. The standard drug was dissolved in distilled water and the synthesized compounds were dissolved [13] in minimum quantity of DMF and diluted with water to get desired concentrations [14]. All data's were summarized in Table 12.  ) and Streptomycin was used against Escherichia coli and. P.aeruginosa [15] (gram-ve organism).

DISCUSSION
The present study can be summarized as the designing of novel thiophene substituted chalcones as Glutathione-S-Transferase (PDB ID: 1GNW) selective inhibitors and analysis of the compounds through ADMET filters and molecular docking studies. From a library of designed compounds were chosen which had binding energy more could serve as lead compound for the development of newer potent anti-bacterial agents. In vitro study of synthesized compounds T-IV B and T-IV-H shows good antibacterial activity against Streptococcus auresis compared to docking result because docking is a hypothetical methodology. Sometimes experimental result may change with docking report.  T-IV-B  15  12  11  11  T-IV-C  13  13  15  14  T-IV-F  11  12  13  12  T-IV-H  14  13  11  12  T-IV-I  15  10  14  15  T-IV-J  12  13  15  14  Procaine penicillin,  18  19  --Streptomycin  --19 20 Control(DMF) 00 00 00 00

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.