Synthesis and in silico study of 2-furyl ( 4-{ 4-[ ( substituted ) sulfonyl ] benzyl }-1-piperazinyl ) methanone derivatives as suitable therapeutic agents

1Department of Chemistry, Government College University, Lahore, Pakistan,2Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia, 3Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia, 4Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur, Pakistan, 5Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur, Pakistan. 6Department of Biochemistry, Abdul Wali Khan University, Mardan, Pakistan, 7Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan


INTRODUCTION
Sulfonamides have received a lot of attention in the literature because of their exciting biological properties and their role as pharmacophores of considerable historical importance (Abbasi et al., 2016a, b).Heterocyclic sulfonamides have been found to be carbonic anhydrase inhibitors (Surpuran et al., 1998;Di Fiore et al., 2010;Smaine et al., 2008) and antibacterial (Gadad et al., 2000) and anticancer, antiinflammatory and analgesic (Sondhi et al., 2000) agents.Furthermore, the piperazine core has displayed a wide spectrum of pharmacological activities and is part of a number of drugs that are preclinical and clinical candidates (Welsch, Snyder, Stockwell, 2010;Hussain et al., 2016).
Cholinesterases (acetylcholinesterase, AChE (EC 3.1.1.7)),butyrylcholinesterase, BChE (EC 3.1.1.8)),belong to the class of serine hydrolases and are responsible for the inactivation of acetylcholine at cholinergic synapses.The main effect of AChE and BChE is to catalyze the hydrolysis of the neurotransmitter acetylcholine and termination of the nerve impulse at cholinergic synapses (Cygler et al., 1993).BChE is extensively present in Alzheimer's plaques (Gauthier, 2001).Cholinesterases play an important role in Alzheimer's disease, and so their inhibitors are of great importance for the treatment of such diseases.
The bioactivity of piperazine and sulfamoyl moieties prompted us to synthesize some new molecules bearing these moieties together.The compounds synthesized were screened to explore their enzyme inhibitory and antibacterial potential.Moreover, we also carried out cytotoxicity and molecular docking studies to determine their utility as possible therapeutic agents in drug development programs.

General
Chemicals were purchased from Sigma Aldrich and Alfa Aesar (Germany), and solvents of analytical grade were from local suppliers.Melting points were taken uncorrected on a Griffin and George apparatus using the open capillary tube method.Thin layer chromatography (TLC) using ethylacetate:n-hexane (30:70) as mobile phase, with detection at 254 nm, was used to determine the initial purity of the compounds.IR spectra were recorded on a Jasco-320-A spectrometer by using the KBr pellet method. 1 H-NMR spectra were recorded at 500 MHz in CDCl 3 using a Bruker spectrometer.EIMS spectra were recorded with a JMS-HX-110 spectrometer.

Cholinesterase assays
The AChE and BChE inhibition study was performed according to an established method (Ellman et al., 1961).The percent inhibition was calculated by the following equation: The IC 50 (concentration at which there is 50% enzyme inhibition) of compounds was calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).

Antibacterial activity
The antibacterial activity test was performed in sterile 96-wells microplates under aseptic environments.The method is rooted in the principle that microbial cell number increases as the microbial growth proceeds in a log phase of growth, which results in increased absorbance of broth medium (Kaspady et al., 2009;Yang et al., 2006).Three Gram-negative (Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) were included in the study.The organisms were maintained on stock culture agar medium.The test samples with suitable solvents and dilutions were pipette into wells (20 µg/well).Overnight maintained fresh bacterial culture after suitable dilution with fresh nutrient broth was poured into wells (180 µL).The initial absorbance of the culture was strictly maintained between 0.12-0.19 at 540 nm.The total volume in each well was kept to 200 µL.The incubation was done at 37 o C for 16-24 hours with lid on the microplate.The absorbance was measured, before and after incubation and the difference was noted as an index of bacterial growth at 540 nm by using microplate reader.The percent inhibition was calculated by using the formula: where X is absorbance in control with bacterial culture and Y is absorbance in test sample.Results are mean of triplicate (n=3, ± SEM).Ciprofloxacin was taken as standard.

Statistical analysis
The results are written as mean ± SEM after performance in three-folds and statistical analysis by Microsoft Excel 2010.Minimum inhibitory concentration (MIC) was calculated by using different dilutions (ranging 5-30 μg/well) and EZFit Perrella Scientific Inc. Amherst USA software.

Hemolytic activity
Hemolytic activity was evaluated by a previously reported method (Sharma, Sharma, 2001;Powell, Catranis, Maynard, 2000).Human blood was obtained from volunteers according to the Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan.After centrifugation, separation and washing, the % RBCs lysis was computed by noting the absorbance.

Molecular docking
To predict the bioactive conformations, various compounds (ligands) were docked into the binding pockets of the selected proteins (enzymes) by using the default parameters of MOE-Dock program.

Ligand preparation
The three-dimensional (3D) structures of the compounds synthesized were modeled by using the Build program of MOE 2009-10.The energies of the compounds were then minimized by using the default parameter of MOE energy minimization algorithm (gradients: 0.05, force field: MMFF94X).Database was created in which all the compounds (3D structures) were saved in the mdb file format for the next step of docking.

Receptor protein preparation
The 3D structures of receptor protein molecules of AChE (PDB ID code: 1Zi3; Resolution: 1.69Å) and BChE (PDB ID code: 1POP; Resolution: 2.30Å) were downloaded from Protein Data Bank.All water molecules were removed from the receptor proteins and 3D protonation was carried out by using Protonate 3D Option.Protein molecules were energy minimized by using the default parameters of MOE 2009-10 energy minimization algorithm (gradient: 0.05, Force Field: MMFF94X).By using default parameters of MOE-Dock Program, all the ligands were docked into binding sites of the above proteins.Re-docking procedure was also used to increase the validity of docking protocol (Bostro, Greenwood, Gottfries, 2003).

Chemistry
The structural analysis of one of the compounds is discussed here in detail for the benefit of the reader.3, respectively.These spectral data confirmed thev structure of this molecule as 2-furyl{4- [4-(1-piperidinylsulfonyl)benzyl]-1-piperazinyl}methanone.Similarly, the structures of all the synthesized molecules, 5a-h, were characterized by their IR, 1 H-NMR and EI-MS spectral analysis.

Cholinesterase assays
The results of screening the compounds against cholinesterase are given as % inhibition and IC 50 in Table I.

Antibacterial activity (in vitro)
All the compounds synthesized were screened against various Gram-positive and Gram-negative bacterial strains, and most of them were found to be potent inhibitors.The results are tabulated as % inhibition and MIC values in Tables II and III

Hemolytic activity
The highest hemolytic activity was shown by 5g (35.98%), higher than the positive control (Triton X-100).The lowest activity was shown by 5e (1.66%) but higher than the negative control (PBS), as shown in Table I.Some of these molecules might be further tested for their application in drug designing programs because of moderate toxicity.

Computational Docking
It is clear from Figure 2 (2D and 3D) that compound 5h was deeply bound in the binding pockets of AChE by making two important interactions with the amino acid residues Asp211 and Trp300.Asp211 interacted strongly with the nitrogen of the piperazine ring of the ligand through Mn ++ , giving a bond length of 2.19 Å.A second arene-arene interaction was made between Trp300 and furyl rings with a bond distance of 3.60 Å. Val210, Asp302, Glu303 and Phe121 were also present in the nearby vicinity.
Similarly, molecule 5f showed two interactions with this enzyme.Lys125 interacted strongly with the sulfonyl oxygen of the ligand through side chain donor interaction.The bond length calculated was 2.57 Å. Tyr126 made an arene-arene interaction with the benzyl ring of the compound, giving a bond distance of 3.77 Å, as shown in Figure 3 (2D and 3D).
The in silico study with butyrylcholinesterase (BChE) revealed that 5h also and another compound, 5e, exhibited considerable interaction.It was inferred that  5h exhibited two interactions.The first side chain donor interaction was between Thr120 and the sulfonyl oxygen with a distance of 3.53 Å, while the second arene-cation interaction was between His438 and the furoyl ring of the ligand with a bond length of 3.8 9Å (Figure 4; 2D and 3D).
In the same way, compound 5e had two interactions with the active site residues Thr120 and Trp82.Thr120 had a strong side chain donor interaction with the sulfonyl oxygen, showing a bond distance of 3.56 Å, but Trp82 produced an arene-arene interaction with the furoyl ring, showing a bond length of 4.06Å (Figure 5; 2D and 3D).Met437, Gly116 and His438 were also present very close to the ligand.

CONCLUSION
The synthesized compounds were confirmed by spectral data.It was evident from enzyme inhibition analysis that compound 5h exhibited potent inhibitory activity against AChE and BChE, with IC 50 of 2.91 ± 0.001 and 4.35±0.004μM, respectively, as compared to the standard eserine, with IC 50 of 0.04 ± 0.0001 and 0.85 ± 0.001 μM, which could be attributed to the presence of the 3,5-dimethyl-1-piperidinyl group in this molecule.These results were fully supported by their in silico study.Putting S. aureus aside, all of the synthesized molecules were active against the Gram-positive and Gram-negative bacterial strains tested, except compound 5e, which was inactive against P. aeroginosa.The hemolytic study was also carried out to evaluate the cytotoxicity profile of the synthesized molecules.It was inferred from the results that most of molecules were moderately toxic.Hence, these molecules could be recommended as suitable therapeutic entrants in a drug development program for the pharmaceutical industry.

F
I G U R E 4 -T h e 2 D a n d 3 D i n t e r a c t i o n a n a l y s i s o f 4 -{ 4 -[ ( 3 , 5 -d i m e t h y l -1 -p i p e r i d i n y l ) s u l f o n y l ] benzyl}-1-piperazinyl)(2-furyl)methanone (5h) against butrylcholinesterase. FIGURE 5 -The 2D and 3D interaction analysis of 2-furyl(4-{4-[(3-methyl-1-piperidinyl)sulfonyl]benzyl}-1-piperazinyl) methanone (5e) with butrylcholinesterase.

TABLE III -
Antibacterial