Synthesis, bioevaluation and molecular docking study of new piperazine and amide linked dimeric 1,2,3-triazoles

Abstract In search of more potent new antitubercular agents, a library of novel piperazine tethered dimeric 1,2,3-triazoles were designed by assembling 1,2,3-triazoles and piperazine in a single molecular architectural framework. The titled compounds (3a–m) were synthesized by 1,3-dipolar cycloaddition of 1,4-di(prop-2-yn-1-yl)piperazine (1) and various azides (2a–m) using click chemistry approach with good yields. All the synthesized compounds (3a–m) have been screened for their in vitro antitubercular, antifungal and antioxidant activities against their respective strains. Among them, 3b, 3d, and 3i have revealed promising antitubercular activity against Mycobacterium tuberculosis (Mtb) H37Rv with MIC 12.5 µg/mL. Molecular docking results provided well-clustered solutions to the mode of binding for these molecules into the active site of Mtb enoyl reductase (InhA). In addition to this, most of synthesized compounds were found to have potential antifungal as well as antioxidant activity. Graphical Abstract

2 reaction mixture drop wise with continuous stirring on magnetic stirrer. After the addition, the ice bath was removed and the reaction mixture was stirred at room temperature for 4 h.
The progress of the reaction was monitored by TLC using ethyl acetate: hexane (1:9) as a solvent system. After completion of the reaction, the reaction mixture was poured on crushed ice and neutralised by adding acetic acid to it. The obtained solid intermediates of respective amines were filtered, dried and crystallised in ethanol.

b) Synthesis of azides (2a-r)
The freshly synthesized above intermediates were heated at 100 °C with sodium azide (0.1 mol) in DMF-H2O for 6 h. The progress of the reaction was monitored by TLC using ethyl acetate: hexane (1:9) as a solvent system. The reaction mixture was poured on crushed ice.
Ethyl acetate was added to the mixture and the organic layer was separated. The organic layers were dried over MgSO4 and solvent was removed under reduced pressure. All the azides (2a-r) were synthesized from respective amines, were obtained as solid compounds except azide (2e) obtained from o-anisidine in liquid state. This azide (2e) was extracted in organic layer using ethyl acetate and solvent is removed under reduced pressure. After this it was used for further reaction without any purification. All the newly synthesized solid azides were crystallised in ethanol and then used for further reaction. [2] Synthesis of 2,2'-(4,4'-(piperazine-1,4-diylbis(methylene))bis(1H-1,2,3-triazole-4,1-

In vitro Mtb MABA assay
The antitubercular activity of newly synthesized Compounds (3a-r) have been screened for their in vitro effects against Mtb H37Rv (ATCC 27294) by using microplate Almar Blue assay (MABA) [3] for determination of MIC in triplicates. The MIC (in µg/mL) was recorded as the lowest concentration/highest dilution of the compounds/control drugs that completely inhibited the growth of Mtb cultures. The MIC values of compounds (3a-r) have been compared with standard drugs (Rifampicin, Isoniazid, Ethambutol and Ciprofoxacin). The experimental method for antitubercular activity is briefly described as follows-Initially, the inoculum was prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase OADC), adjusted to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution was thawed and diluted in 7H9-S at four-fold the final highest concentration tested. Serial two-fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100µl 7H9-S. A growth control containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was added to all perimeter wells to avoid evaporation during the incubation. The plate was covered, sealed in plastic bags and incubated at 37 o C in normal atmosphere. After 7 days incubation, 30 µl of alamar blue solution was added to each well, and the plate was re-incubated overnight. A change in colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest concentration of drug that prevented this change in colour.

In vitro antifungal activity
Antifungal activity was determined by standard agar dilution method as per CLSI (formerly, NCCLS) guidelines [4] These newly synthesized compounds were screened for their in vitro

General procedure for antioxidant activity
The antioxidant activity of all the synthesized compounds have been assessed in vitro by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay [5] and the results were compared with standard synthetic antioxidant BHT (Butylated Hydroxy Toluene). The hydrogen atom or electron donation ability of the compounds were measured from the bleaching of the purple coloured methanol solution of 1,1-diphenyl-1-picrylhydrazyl (DPPH).
The spectrophotometric assay uses the stable radical DPPH as a reagent. Where, Acontrol is the absorbance of the control (DPPH radical without test sample) Asample is the absorbance of the test sample (DPPH radical with test sample). The control contains all reagents except the test samples.

Molecular docking study
Molecular docking studies were performed using with Glide (Grid-Based Ligand Docking With Energetics) program [6] integrated in the Schrodinger molecular modeling package (Schrodinger, LLC, New York, NY, 2015). With this purpose, the crystal structure of mycobacterial enoyl-ACP reductase (InhA) complexed with its inhibitor was retrieved from the protein data bank (PDB) (pdb code: 4TZK) and refined using the protein preparation wizard .The 3D structures of the triazole derivatives to be docked were sketched using the build panel in Maestro and their geometries were optimized using LigPrep module. Both the enzyme as well as ligand structures were subjected to energy minimization, before submitting the docking simulations, until their average RMSD reached 0.01Å. The active site of InhA was defined using the receptor grid generation panel. This active site grid was defined by a box having a dimension of 10X10X10Å centered on the co-crystallized ligand in the crystal complex. The co-ordinates of native ligand served as the reference to define the active site of a ligand with respect to the target. Following this, the ligands were subjected to flexible docking against the InhA structure using with extra precision (i.e., with GlideXP) scoring