Biological screening and docking studies of unique hybrids synthesized by conventional versus microwave- assisted techniques

Purpose: To carry out the synthesis of various hybrids of 1,2,4-triazole in search of potential therapeutic enzyme inhibitory agents, and carry out docking and bovine serum albumin (BSA) binding studies on docking and bovine serum albumin (BSA) binding studies on the hybrids. Methods: The target compounds were synthesized by following a multistep protocol. Compound 1 was synthesized from 4-methoxybenzenesulfonyl chloride (a) and ethyl isonipecotate (b). Compound 1 was refluxed with hydrazine to synthesize compound 2, which was converted to compound 3 through two consecutive steps. Compound 4 and different amines (5a-5i), were utilized to synthesize an array of electrophiles (6a-6i). A series of 1,2,4-triazole hybrids (7a-7i) were synthesized at room temperature by stirring together 3 and 6a-6i. The final structures of 7a-7i were elucidated through H-NMR, C-NMR and EI-MS spectroscopy. The BSA binding studies were performed by fluorometric titration. Furthermore, antioxidant and enzyme inhibition activities were determined colorimetrically. Results: Compound 7d was the most active antioxidant agent, compared to butylated hydroxyanisole (BHA), while compounds 7d, 7e, 7f, 7g and 7i proved to be potent urease inhibitors with half-maximal inhibitory concentration (IC50) values of 19.5 ± 0.12, 21.1 ± 0.68, 18.2 ± 0.78, 19.9 ± 0.77 and 17.9 ± 0.10 μM, respectively, compared to thiourea with an IC50 of 24.3 ± 0.24 μM. Compounds 7a, 7b, 7d, and 7e exhibited high butyrylcholinesterase inhibition potential, compared to eserine. Conclusion: The synthesized compounds require studies further as potential therapeutic enzyme inhibitory agents in view of their urease inhibition as well as antioxidant activity.


INTRODUCTION
Nitrogen-based heterocyclic compounds are pharmacologically active agents [1]. These moieties include piperidine [2][3][4][5] and 1,2,4triazole [4,[6][7][8][9], which show different biological activities. On the basis of their biological potential, we designed the current study to combine piperidine and 1,2,4-triazole in a single compound to explore their biological potential. Both conventional and microwave-assisted synthetic protocols were utilized. Microwaveassisted synthesis is found to be more suitable in term of purity, time of synthesis and yield [10]. The designed series of compounds were screened for antioxidant and enzyme-inhibitory activity [13], followed by docking studies [11,12]. The docking studies helped elucidate the interaction of the most active synthesized compounds with a specific protein. The BSA binding studies provided more information about the protein interaction of the synthesized compounds.

EXPERIMENTAL General
The 1 H -NMR and 13 C -NMR spectra were obtained with a Bruker spectrometer operating at 600 and 150 MHz, respectively. A Jasco -320 spectrophotometer was used to collect the IR spectra. Thin layer chromatography helped to determine the completion of reactions and purity of the synthesized compounds. Both Alfa Aeser and Sigma Aldrich provided analytical grade chemicals.

Synthesis of 1-[(4-methoxyphenyl)sulfonyl] piperidine-4-carbohydrazide (2)
Compound 2 was synthesized by refluxing an equimolar mixture of compound 1 and hydrazine hydrate for 3.5 h in EtOH. 2 was precipitated by the addition of chilled distilled water, filtered out, washed with distilled water and dried at room temperature.
Microwave-assisted method: Compound 3 was stirred with an equimolar amount of NaH as a catalyst in DMF for 5 s. Equimolar electrophile (6a-6i) was added and the reaction mixture stirred for 34 -73 s. The precipitate of 7a-7i appeared upon addition of chilled distilled water and was filtered out, washed with distilled water and dried at room temperature.
Antioxidant assay (DPPH assay) The protocol described by Koleva and coworkers was used with some modifications to measure antioxidant activity [14]. Butylated hydroxyanisole (BHA) was the reference standard. The change in absorbance in the presence of test compound was noted and compared to that of control.

Butyrylcholinesterase (BChE) inhibition assay
The protocol described by Ellman et al was used with modifications to measure butyrylcholinesterase (BChE) inhibitory activity [15]. Eserine was the reference standard. The change in absorbance in the presence of test compound was noted and compared to that of control.

Urease inhibition assay
Mobley and coworkers reported a method to measure urease inhibition activity [16] which we used with some modifications. Thiourea was used as the reference standard. The change in absorbance in the presence of test compound was noted and compared to that of control.

Docking studies
The urease protein structure was obtained from the Protein Data Bank (PDB) ID: 3LA4 with a resolution of 2.05 Å [17]. The calculations of docked molecules were obtained using OpenEye Scientific Software. The different conformations were obtained through OMEGA 3.0.0 (OMEGA, version 2.4.6, 2013), and the binding interactions were visualized using Discovery Studio Client v16.1.0 (BIOVIA, 2017).

BSA binding studies
BSA binding interactions were determined using the method used by Chmara and coworkers [18], with some modifications. Buffer and BSA were purchased from Sigma-Aldrich. Fluorescence was measured with an LS 55 Perkin Elmer fluorescence spectrophotometer [19]. Fluorescence of BSA in the presence of synthesized compounds was recorded and compared with that of BSA alone.

Statistical analysis
Each experiment was performed in triplicate, and the results are presented as mean ± SEM. MS Excel 2010 was used to perform statistical analysis with 85 %CI. The results of enzyme inhibition assays are expressed as IC 50 (concentration for 50 % inhibition) using EZ-Fitz software (Perrella Scientific Inc, USA).

RESULTS
Hybrids bearing 1,2,4-triazole and piperidine were synthesized according to Scheme 1. The complete structures of the synthesized compounds are listed in Table 1. Comparison of conventional and microwave-assisted methods is given in Table 2 with regard to time and yield.
BChE was inhibited by six of the nine synthesized compounds. The most active were 7a, 7b, 7d and 7e with low IC 50 values. The highest potential was shown by 7d with the lowest IC 50 value, 15.5 ± 0.77 µM, which could have been due to 2-chloro-5-methoxyphenyl as the amide aromatic ring.
All the synthesized compounds were analyzed by molecular docking studies to determine the orientation in the active site of the target protein. Figure 1 shows the superimposition of all the docked compounds at the active site of urease. Binding interaction analysis of the most active compounds 7f ( Figure 2) and 7i (Figure 3) indicated hydrogen bonding with Arg439 and CME592 through triazole and oxygen of acetamoyl moieties. Hydrophobic interactions with amino acid residues Ala440, Ala636 and Arg639 were also found. Hydrogen bonding is shown with green dotted line while π-alkyl interactions are shown with light pink dotted line.
The fluorescence emission spectral data (at an excitation of 295 nm) of BSA was recorded ( Table 4). The strong emission by BSA appeared at λ max = 336 nm. Fluorescence decreased on binding of the synthesized compounds with BSA. The Stern-Volmer equation (Equation 1) helped to determine the nature of this fluorescence quenching, that is, static or dynamic. where F is fluorescence intensity of BSA in the presence of compounds, F 0 is fluorescence intensity of BSA in the absence of compounds, K q is apparent bimolecular quenching rate constant, [Q] is concentration of synthesized compounds (quencher), K SV is Stern-Volmer quenching constant, and τ 0 is the average life time of biomolecule without synthesized compound (10 −8 s) [20]. The quenching of BSA was found to be static as indicated by the larger values of k q than maximum scattering collision quenching rate constant (2 × 10 10 M -1 s −1 ).

CONCLUSION
Microwave-assisted protocol is superior to the conventional method in terms of high yield and short time. Biological screening along with docking and BSA binding studies indicate that compounds 7d, 7e, 7f, 7g, and 7i are potent antiurease agents. Thus, the current synthesis might be a good addition in the pharmaceutical industry after further biological evaluation.