Convergent synthesis of new N-substituted 2-{ [ 5-( 1 H-indol-3-ylmethyl )-1 , 3 , 4-oxadiazol-2-yl ] sulfanyl } acetamides as suitable therapeutic agents

1Department of Chemistry, Government College University, Lahore, Pakistan, 2Department of Biochemistry and Biotechnology, 3Department of Pharmacy; The Islamia University of Bahawalpur, Bahawalpur, Pakistan, 4Department of Biochemistry, Abdul Wali Khan University, Mardan, Pakistan, 5Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan, 6Faculty of Industrial Sciences & Technology, University Malaysia Pahang, Leburaya TunRazak, Kuantan Pahang, Malaysi.


INTRODUCTION
Oxadiazoles comprise four different classes, where 1,3,4-oxadiazoles are more important and are present in a number of biologically active molecules.(Renukadevi, Birada, 1999).Due to the propensity of the 1,3,4-oxadiazole ring to undergo a variety of electrophilic and nucleophilic substitution reactions, a large number of derivatives have been synthesized that have remarkable pharmacological, medicinal, and biological activities such as antimicrobial (Rakesh et al., 2010;Zuhair et al., 2008), anti-inflammatory (Dewangan et al., 2010;Omar, Mahfouz, Rahman, 1996), cytotoxic and hypoglycemic (Hasan et al., 2011), anticancer (Bhatnacar et al., 1986), anticonvulsive (Yar, Akhter, 2009), antitubercular (Ali, Shaharyar, 2007), and fungicidal and insecticidal (Zou, Zhang, Jin, 2002;Holla et al., 2004).Indole derivatives display a wide range of biological activities.The indole ring is found in the hormones serotonin and melatonin, amino acid tryptophan, the anti-inflammatory drug indomethacin, the psychotropic drug LSD and the antitumor agents vinblastine and vincristine (Murphy et al., 1997).Coronaridine and voacangine are indole alkaloids that have antileishmanial, antitumor, diuretic and hypoglycemic activities (Camila, Paulo, Meireles, 2007).The indole moiety has been incorporated as a core skeleton to access active sites, implicated in enzyme inhibitions (Lednicer, 2007).Some indole derivatives are also identified as good inhibitors of 5-lipoxygenase (LOX) (Hutchinson et al., 1995;Yar et al., 2014).LOX is a lipidperoxidizing enzyme, which shows an essential role in leukotriene biosynthesis and mediates the pathophysiology of allergic disorders, inflammatory reactions and neoangiogenesis (Bhattacharjee, 2005;Jampilek et al., 2006;Pommery et al., 2004).Cholinesterases are wellknown enzymes that are present in cholinergic and noncholinergic tissues and in plasma and other body fluids of animals (Ryhanen, 1983).Butrylcholinesterase (BChE) is present in intestine, liver, heart, kidneys and lungs (Dave et al., 2000).BChE hydrolyzes ester-containing drugs and scavenges cholinesterase inhibitors, including potent organophosphorus nerve agents before reaching the synaptic targets (Silver, 1974).Inhibitors of the enzyme α-glucosidase are molecules that are used as oral antidiabetic drugs in the treatment of type-2 diabetes mellitus.The inhibitors of this enzyme can delay the release of D-glucose from disaccharides and oligosaccharides from dietary carbohydrates and slow down glucose absorption, resulting in reduced postprandial hyperglycemia (Lebovitz, 1997).Therefore, inhibition of α-glucosidase is considered important in managing type-2 diabetes.Cytotoxicity of synthesized compounds in terms of hemolytic activity may be studied to assess the possible toxicity profile of the most active compounds.Shirinzadeh et al. (2010) studied indole hydrazide/hydrazone derivatives for their inhibitory effect on AAPH-induced hemolysis of human erythrocytes and found most of the compounds had significantly high antioxidant activity, when compared to melatonin.A new series of 2-[ [5-alkyl/aralkyl-1,3,4-oxadiazol-2-yl]thio]-N- [4-(4-morpholinyl)phenyl]acetamides was evaluated by Akhtar et al. (2014) for antimicrobial and hemolytic activity; most of them were found to be active against the selected microbial species to a variable extent relative to reference standards, and showed less toxicity as well.
The aim of present study was to design and synthesize some novel poly-functional compounds having 1,3,4-oxadiaozole, indole and substituted acetamide moieties together in one molecule, which may prove to be potent antibacterial and anti-enzymatic agents and which could be studied further for pharmacological purposes.

General
The chemicals employed in this study were of analytical grade and purchased from Merck and Alfa Aesar.All solvents were distilled before use.Melting points of the synthesized compounds were recorded on a Gallenkamp melting point apparatus by open capillary tube and were uncorrected.To observe the progress of the reaction, thin layer chromatography (TLC) was carried out on pre-coated silica gel G-25-UV 254 plates using ethyl acetate and n-hexane in various proportions as mobile phase.Spots were visualized at 254 nm under a UV lamp.IR spectra were recorded using the KBr pellet method with a HYPER IR spectrometer, and wave number was given in cm -1 .Mass spectra (EI-MS) were recorded using a JOEL JMS 600H spectrometer, with a data system.Nuclear magnetic resonance spectra were recorded in DMSO-d 6 on a Bruker spectrometer operating at 300-500 MHz, and chemical shifts (δ) were given in ppm.

Synthesis
Preparation of ethyl 2-(1H-indol-3-yl)acetate (2) 2-(1H-Indol-3-yl)acetic acid (20.0 g; 0.11 mol; 1) in absolute ethanol (60 mL) and catalytic amount of concentrated sulfuric acid (10 mL; 0.18 mol) were added to a 500-mL round-bottomed flask (RB), which was refluxed for 8 h until completion of the reaction.The reaction mixture was neutralized with 20 mL of 10% aqueous sodium carbonate.The product was isolated by the solvent extraction technique using chloroform (50x3 mL).The organic phase containing the ester was decanted off and washed a number of times with distilled water and dried using anhydrous MgSO 4 .

Antibacterial activity
The antibacterial activity was performed in sterile 96-wells micro plates under aseptic environments.The method is based on 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).

Hemolytic activity assay
Hemolytic activity of the compounds was performed by the reported methods (Shahid et al., 2013;Zuber et al., 2014).

Statistical analysis
All the measurements were carried out in triplicate and statistical analysis was performed by Microsoft Excel 2010.The results are presented as mean ± SEM with 90% CL.

Molecular docking methodology
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.
Prior to docking, the protein molecules of α-glucosidase, lipoxygenase and butyrylcholinesterase were retrieved from Protein Data Bank having PDB ID codes of 3NO4 (Resolution: 2.02Å), 1IK3 (Resolution: 2.0Å) and 1POP (Resolution: 2.0Å) respectively.All the water molecules were removed from receptor proteins and 3D protonation was carried out by using the Protonate 3D Option.The energies of protein molecules were minimized by using the default parameters of MOE 2009-10 energy minimization algorithm (gradient: 0.05, Force Field: MMFF94X).Then all the ligands were docked into the binding pockets (selective residues/amino acids as shown in the docking images) of the above mentioned proteins using Triangular Matching docking method.Re-docking procedure was also applied to validate the docking protocol.Each complex was analyzed for the type of interactions; bond distances and their 3D images were taken.

RESULTS AND DISCUSSION
We report herein the synthesis of some new N-substituted derivatives of 2-{ [5-(1H-indol-3-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetamides.These synthesized compounds were evaluated for their antibacterial and hemolytic activity.Enzyme inhibition activity was well supported by molecular docking results.The reaction sequence leading to the preparation of the desired heterocyclic compounds is outlined in Scheme 1. 5-(1H-Indole-3-yl-methyl)-1,3,4-oxadiazole-2-thiol was prepared by converting 2-(1H-Indol-3-yl)acetic acid (1) to its corresponding ester 2 in ethanol, which was used in excess as solvent to shift this reversible reaction in the forward direction, where H 2 SO 4 was used as catalyst.Aqueous Na 2 CO 3 was used to neutralize the catalyst and unreacted acid (1).Ester 2, on further reaction with hydrazine monohydrate, yielded acid hydrazide 3 after 3 h stirring at room temperature.This reaction readily occurred, otherwise a few drops of glacial acetic acid was added to speed up this reaction.The hydrazide (3) was cyclized to 5-(1H-indole-3yl-methyl)-1,3,4-oxadiazole-2-thiol ( 4) in the presence of CS 2 and alcoholic KOH with refluxing for 6 h .CS 2 is volatile and is used in double quantity compared to 4 to favor product formation.KOH was used to facilitate the delocalization of negative charge during intramolecular cyclization to form 4. A series of N-substituted 2-bromoacetmaide derivatives were also synthesized by manually shaking equimolar amounts of bromoacetyl bromide (VI) with different substituted amines (Va-w) in 10% aqueous Na 2 CO 3 (pH 8-9).Amines with electron-donating groups were more active in nucleophilic reactions than those with electron-withdrawing groups.These N-substituted 2-bromoacetmaide derivatives (7a-w) were reacted further with 5-(1H-indole-3-yl-methyl)-1,3,4oxadiazole-2-thiol (4) in NaH as weak base and DMF as aprotic medium to produce 2-{[5-(1H-indol-3-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetamides (8a-w), where complete conversion was achieved within almost 8 h by stirring with 68-98% yields, as outlined in Scheme 1 (Sabahat et al., 2013).The products were isolated by adding ice-cold water to the reaction mixture and filtering off the precipitated solid.In some cases, the compound was obtained with the solvent extraction method using chloroform/ethyl acetate.The structure of the parent compound and its derivatives were confirmed by IR, 1 H-NMR, and EI-MS data, as described in the experimental section.
Compound 8g was obtained as a light-brown amorphous powder with 88% yield and m.p. 142 °C.The molecular formula C 21 H 20 N 4 O 2 S was ascertained by EI-MS data which revealed an [M] + ion peak at m/z 378 and by the assigned protons in the 1 H-NMR spectrum.IR spectrum further revealed characteristic peaks at 3339 cm -1 for N-H stretch of the indole moiety, 3085 cm -1 for aromatic C-H stretching, 1667 cm -1 for C=N stretching due to the presence of the oxadiazole ring, 1560 cm -1 for stretching of C=C phenyl rings of indole and N-substituted aromatic ring, 1655 cm -1 for stretching of C=O, 1065 cm -1 for C-O-C bond stretching of oxadiazole ring and 637 cm -1 for C-S stretching.EIMS data further supported the structure by revealing a base peak at m/z 130 for (C 9 H 8 N) + of the indole moiety and of major fragments given in Figure 2. In   7.37 (br.d,J = 8.5 Hz,1H,, and two ortho coupled triplets with integration of one proton each appeared at δ 7.09 for H-5' and δ H-6', respectively, In the aromatic region, a broad singlet appeared at δ 7.32 of H-2', and the last signal for the indole moiety appeared in the aliphatic region with integration of two protons at δ 4.30 of H-10'. The acetamide part of the molecule gave a deshielded singlet of -NH of amide at δ 10.24 for NH-1'''', doublet in aromatic region at δ 7.48 of H-2''' & H-6''' and two other protons of this ring gave a doublet at δ 7.12 of H-3''' & H-5'''.Two more singlets appeared in the aliphatic region with integration of two protons at δ 4.19 of H-2" and with three protons at δ 2.25 for CH 3 -4"' (Figure 3).On the basis of this spectral evidence, the structure was determined as N-(4-methylphenyl)-2-{ [5-(1H-indol-3-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetamide (8g).Similarly, the structures of other derivatives of 5-(1H-indole-3ylmethyl)-1,3,4-oxadiazole-2-thiol were elucidated in the same way, as described in the experimental section.

Hemolytic activity
The lowest hemolytic activity (Table IV) was exhibited by 8p (1.67%), 8t (2.16%) and 8u (2.93%).The highest hemolytic activity was shown by 8j (48.66%) which was lower than the positive control (Triton-X-100).Other compounds mostly showed low hemolytic activity.PBS was used as negative control.On the basis of the antibacterial and enzyme inhibition activities observed, we infer that the compounds synthesized, with mostly little toxicity, may prove to be suitable drug candidates for further improvements to address different targets.

Computational docking
To determine the validity of accuracy, the cocrystallized ligands of the following enzymes were extracted and then re-docked into the binding pockets of the receptors.In all these cases, RMSD values between docked and co-crystallized ligands were less than 2 A°, indicating the reliability of the docking method, and thus, our protocol can be used for further studies.
Almost all the derivatives synthesized were computationally docked against α-glucosidase, BChE and LOX to explore the binding modes of all ligands (Figure 4).The results were very much in favor of the experimental work.For example, compound 8q showed interactions with three active site amino acid residues of the target protein.Amino acid Asp197 showed a strong hydrogen bond acceptor (polar) interaction with the five-member ring of the indole moiety residue, with a bond distance of 2.23 Å, while Trp271 showed a strong hydrogen bond donor interaction with the nitrogen atom of the oxadiazole ring, with a bond distance of 2.28 Å. Arg404 showed an arene cation interaction with the oxadiazole ring, with a bond length of 3.92 Å.The other residues in the nearby vicinity of the ligand were Asp73,Trp169,Trp417 and Ala480 (Figure 4,a).Two amino acid residues interacted with compound 8g; His438 interacted strongly with the indole moiety through a couple of arene cation interactions showing bond lengths of 4.62 and 4.83 Å, while Trp82 showed arene-arene interaction with the oxadiazole ring, with a bond distance of 3.33 Å.Other residues that were in close contact with the ligand were Ser79, Met437, Gly116, Gln119 etc. (Figure 4,b).Strong metallic interactions were shown by compound 8b, with amino acid residues Ile857 and Asn713, via one nitrogen atom of the oxadiazole ring through the iron atom, giving bond distances of 1.81 and 2.07 Å, respectively, while His518 showed a hydrogen bond donor interaction with another nitrogen atom of the oxadiazole ring, with a bond length of 2.93 Å.Other residues that existed in the near most region of the ligand were Gln716, Ile557 and Leu773 (Figure 4, c).

CONCLUSION
In conclusion, this paper reports the synthesis of N-substituted 2-{ [5-(1H-indol-3-ylmethyl)-1,3,4oxadiazol-2-yl]sulfanyl}acetamides (8a-w) as potential antibacterial agents, in view of MIC values.Compounds 8b (9.84 ± 2.99 µM against S. typhi), 8c (9.75 ± 3.57 µM against S. typhi and 9.15 ± 4.12 µM against S. aureus), and 8l (9.96 ± 3.58 µM against S. aureus) showed strong antibiotic activity, which was very close to that of the reference standard.These results indicate that our synthesized molecules can be considered as suitable drug candidates.Moreover, the enzyme inhibition analysis was also supported by computational docking, and there was only low to moderate hemolytic activity.Compound 8q showed comparatively good inhibitory potential against α-glucosidase with an IC 50 of 49.71 ± 0.19 µM, and 8g showed moderate inhibition of BChE, with an IC 50 of 31.62 ± 0.16 µM, while all other compounds synthesized showed weak or no inhibition of LOX.In this regard, some of the compounds synthesized are potential candidates for future drug development studies.

TABLE - II
Enzyme Bacillus subtilis and Staphylococcus aureus, and three Gram-negative strains, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa.