Design, synthesis, in silico and in vitro antimicrobial screenings of novel 1,2,4-triazoles carrying 1,2,3-triazole scaffold with lipophilic side chain tether

Background 1,2,4-Triazoles and 1,2,3-triazoles have gained significant importance in medicinal chemistry. Results This study describes a green, efficient and quick solvent free click synthesis of new 1,2,3-triazole-4,5-diesters carrying a lipophilic side chain via 1,3-dipolar cycloaddition of diethylacetylene dicarboxylate with different surfactant azides. Further structural modifications of the resulting 1,2,3-triazole diesters to their corresponding 1,2,4-triazole-3-thiones via multi-step synthesis has been also investigated. The structures of the newly designed triazoles have been elucidated based on their analytical and spectral data. These compounds were evaluated for their antimicrobial activities. Relative to the standard antimicrobial agents, derivatives of 1,2,3-triazole-bis-4-amino-1,2,4-triazole-3-thiones were the most potent antimicrobial agents with compound 7d demonstrating comparable antibacterial and antifungal activities against all tested microorganisms. Further, the selected compounds were studied for docking using the enzyme, Glucosamine-6-phosphate synthase. Conclusions The in silico study reveals that all the synthesized compounds had shown good binding energy toward the target protein ranging from − 10.49 to − 5.72 kJ mol−1 and have good affinity toward the active pocket, thus, they may be considered as good inhibitors of GlcN-6-P synthase.

Recent research in drug discovery aimed to introduce the 1,2,3-triazole moiety as a connecting unit to link together two or more pharmacophores for the design of novel bioactive molecules. Thus, it was hypothesized that the chemical combination of 1,2,4-triazole, 1,2,3-triazole and surfactants side chain in one scaffold may prove to be a breakthrough for chemical and biological activity as continuation of our effort in the designing of novel polyheterocyclic bioactive molecules [20][21][22][23][24].
In modern drug designing, molecular docking is routinely used for understanding drug-receptor interaction. Molecular docking provides useful information about drug receptor interactions and is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to predict the affinity and activity of the small molecule [25]. When designing novel antimicrobial agents, enzymes involved in the biosynthesis of microbial cell walls are generally good targets. In this regard, the enzyme glucosamine-6-phosphate synthase (GlmS, GlcN-6-P synthase, l-glutamine: d-fructose-6P amido-transferase, EC 2.6.1. 16) is particularly attractive [26]. It is involved in the first step of the formation of the core amino-sugar, N-acetyl Glucosamine which is an essential building block of bacterial and fungal cell walls [27,28]. Accordingly, GlcN-6-P serves as a promising target for antibacterial and antifungal drug discovery. Structural differences between prokaryotic and human enzymes may be exploited to design specific inhibitors, which may serve as prototypes of anti-fungal and anti-bacterial drugs [28]. Triazole type units have been reported to be good inhibitors of GlcN-6-P synthase [29][30][31]. Moreover, ciprofloxacin, the standard drug used for in vitro screenings in our studies, has been reported to be a good inhibitor of GlcN-6-P synthase [31][32][33][34]. Therefore, it was thought worthwhile to select GlcN-6-P synthase as the target for the synthesized triazole compounds.

Results and discussion
Chemistry An optimized eco-friendly click procedure has been previously developed in our laboratory for the construction of a series of novel 4,5-disubstituted 1,2,3-triazoles via 1,3-dipolar cycloaddition of dimethylacetylene dicarboxylate with different aromatic azides under solvent-free conditions. In the present work, we have investigated the applicability of the solvent-free conditions as a green procedure for the synthesis of novel non-ionic surfactants carrying 1,2,3-triazole and 1,2,4-triazole moieties. Thus, 1,3-dipolar cycloaddition of diethylacetylene dicarboxylate (1) with different surfactant azides 2a-d under solvent free conditions, furnished the targeted non-ionic surfactants based 1,2,3-triazole-4,5-disesters 3a-d in 95-98% yields (Scheme 1). The reaction required heating in a water bath for 3 min.
The diacid hydrazides 4a-d have been prepared successfully by stirring an ethanolic solution of the synthesized di-esters 3a-d with hydrazine hydrate for 4 h at room temperature (Scheme 2). Thus, the condensation of the diacid hydrazides 4a-d with phenyl isothiocyanate, in refluxing ethanol for 6 h, furnished the targeted phenylthiosemicarbazide derivatives 5a-d in good yields (82-87%) (Scheme 2).
The synthesis of 4-amino-1,2,4-triazole-3-thione derivatives 7a-d pass first through the formation of the appropriate potassium dithiocarbazinate salt through the reaction of the acid hydrazides 4a-d with carbon disulphide in ethanolic potassium hydroxide solution (Scheme 3). The resulting potassium salts were then subjected to intramolecular ring closure, in the presence of hydrazine hydrate under reflux for 6 h, to afford 80-84% yields of the desired 4-amino-1,2,4-triazole-3-thiones 7a-d.
The newly synthesized compounds were fully characterized based on their IR, 1 H NMR and 13 C NMR spectra. The IR spectra of the 1,2,3-triazole di-esters 3a-d revealed the presence of strong absorption bands at 1738-1745 cm −1 assigned to the ester C=O groups. The 1 H NMR spectrum of compound 3c showed a quartet at δ H 4.27-4.32 ppm and a multiplet at δ H 4.40-4.48 ppm characteristic for the two non-equivalent ester methylene groups. The two ester methyl protons were recorded as a triplet integrated for six protons at δ H 1.41 ppm. The proton spectral analysis also showed the surfactant proton signals on their appropriate aliphatic region (see "Experimental"). Its 13 C NMR spectrum revealed no signals on the sp-carbon regions confirming the success of the cycloaddition reaction, and two characteristic signals appeared at δ C 158.72 and 160.33 ppm attributed to Scheme 2 Synthesis of 1,2,3-triazole bis-1,2,4-triazole-3-thiones 6a-d Scheme 3 Synthesis of 1,2,3-triazole bis-4-amino-1,2,4-triazole-3-thiones 7a-d the two ester carbonyl carbons (C=O). The surfactant side chain carbons appeared in their expected aliphatic region.
The success of the hydrazinolysis reaction was confirmed by the spectral data analysis of the diacid hydrazides 4a-d. Their IR spectra showed characteristic NH and NH 2 bands of the hydrazide functionalities near 3246-3367 cm −1 . The 1 H NMR spectrum of the diacid hydrazide 4b was taken as example to confirm the success of the reaction. It showed the disappearance of the ethyl ester protons (CH 2 CH 3 ) and the appearance of new multiplet at δ H 4.74-4.79 ppm assignable to the NH 2 and NCH 2 groups. The two non-equivalent NH amide protons were assigned to two singlets at δ H 10.42 and 11.83 ppm. The 13 C NMR spectrum also confirmed the success of the hydrazinolysis reaction through, first the absence of the two ethoxy signals from their chemical shift regions, second the appearance of the two carbonyl hydrazide moieties at lower frequencies (δ C 155.46 and 159.23 ppm) compared to their ester precursors (δ C 158.72 and 160.33 ppm).
The IR spectra of the thiosemicarbazides 5a-d revealed the presence of the thiocarbonyl groups (C=S) by the appearance of new absorption bands at 1289-1298 cm −1 . The 1 H NMR spectrum of compound 5a was characterized by the disappearance of the NH 2 signals and appearance of ten aromatic protons of the two phenyl rings at δ H 7.12-7.74 ppm, confirmed the success of the condensation reaction. The two NH-protons bonded to the two phenyl groups appeared as two singlets at δ H 9.64 and 9.67 ppm. The 1 H NMR also showed four singlets at δ H 9.90, 10.08, 11.23 and 11.55 ppm integrated for four protons related to the NH amidic (NHCO) and NH thioamidic (NHCS) protons of the two thiosemicarbazide moieties. The 13 C NMR spectrum also approve the formation of the expected thiosemicarbazide product 5a through the appearance of the aromatic carbons at δ C 124.04-138.90 ppm and the presence of two characteristic signals at δ C 180.18 and 181.07 ppm attributed to the two thiocarbonyl groups (C=S). Additionally, the spectrum revealed the aliphatic carbons for the surfactant side chain on their expected chemical shifts.
In the IR spectra of compounds 6a-d, the absence of the carbonyl (C=O) and thiocarbonyl (C=S) absorption bands and the presence of new absorption band near 1608-1615 cm −1 characteristic for the C=N groups confirmed the success of the intramolecular ring closure to form 1,2,4-triazole-3-thione. In addition, the exhibited chemical shifts obtained from their 1 H NMR, 13 C NMR and spectra were all supported the proposed structures of 6a-d. The 1 H NMR spectrum of compound 6d revealed the appearance of a diagnostic broad singlet at δ C 10.60 ppm assignable to the NH's of the thione isomer.
The phenyl protons resonated as a multiplet at δ H 7.02-7.49 ppm. In the 13 C NMR spectrum of compound 6d, the C=S signals appeared at 187.84 ppm confirming the predominance of the thione isomer. Furthermore, the aromatic carbons and the surfactant side chain carbons were observed on their appropriate chemical shifts.
The structures of the aminotriazoles 7a-d have been also deduced from their elemental and spectral data. In their IR spectra, the presence of strong absorption bands at 1288-1296 and 3275-3380 cm −1 attributed to the C=S, NH and NH 2 functional groups confirmed the formation of the 1,2,4-triazole ring. The 1 H-NMR analysis revealed the presence of two diagnostic singlets at δ H 5.19-5.27 ppm (NH 2 ) and 9.21-9.31 ppm (NH), confirming the presence of the triazole ring in its thione form.
In their 13 C-NMR spectra, the presence of signals at δ C 187.60-187.68 ppm attributed to the thiocarbonyl carbons (C=S), which were not observed on their corresponding starting hydrazides 4a-d is another support for the predominance of the thione form.

Antimicrobial evaluation
Antimicrobial activities of the newly synthesized compounds were evaluated against a panel of pathogenic microorganisms including Gram-positive bacteria, Gram-negative bacteria, and fungi. Antimicrobial activities were expressed as the Minimum Inhibitory Concentration (MIC) that is defined as the least concentration of the examined compound resulted in more than 80% growth inhibition of the microorganism [35,36]. Bacillus cereus, Enterococcus faecalis and Staphylococcus aureus were used as model microorganisms representing Gram positive bacteria while Proteus mirabilis, Escherichia coli and Pseudomonas aeruginosa were used as representative of the Gram negative bacteria. On the other hand, Candida albicans and Aspergillus brasiliensis were chosen to study the antifungal activities of the synthesized compounds under examination (Table 1).
Consistent with previous reports [20], and on the basis of the observed MIC values for the examined compounds, it was concluded that 1,2,4-triazole derivatives with elongated chain substitution at the 1,2,3-triazole N-1 position likely exhibit enhanced antibacterial and antifungal activities over analogous 1,2,4-triazole derivatives.

In-silico screenings (molecular docking)
In correlation to in vitro antimicrobial activity, it was thought worthy to perform molecular docking studies, hence screening the compounds, inculcating both in silico and in vitro results. The amino sugars are the significant building blocks of polysaccharides found in the cell wall of most human pathogenic microorganisms. Therefore not surprising that a number of GlcN-6-P synthase inhibitors of natural or synthetic origin display bactericidal or fungicidal properties [37]. Considering GlcN-6-P synthase as the target receptor, comparative and automated docking studies with newly synthesized candidate lead compounds was performed to determine the best in silico conformation. The molecular docking of the synthesized compounds with GlcN-6-P synthase revealed that all tested compounds have shown the bonding with one or the other amino acids in the active pockets. Figure 1 shows the docked images of selected candidate ligands including the considered standard drug i.e. Ciprofloxacin. Table 2 shows the binding energy and inhibition constant of the tested compounds including the standard. In-silico studies revealed all the synthesized molecules showed good binding energy toward the target protein ranging from − 5.72 to − 10.49 kJ mol −1 .

General chemistry
Melting points were recorded on a Stuart Scientific SMP1 apparatus and are uncorrected. The IR spectra were measured using an FTIR-8400 s-Fourier transform infrared spectrophotometer-Shimadzu. The NMR spectra were determined on Advance Bruker NMR spectrometer at 400 MHz with TMS as internal standard. The ESI mass spectra were measured by a Finnigan LCQ spectrometer.

Synthesis and characterization of 1,2,3-triazole di-esters 3a-d
Diethyl acetylenedicarboxylate 1 (15 mmol) and the appropriate surfactant azide 2a-d (20 mmol) were heated on a water bath for 3 min. The reaction mixture was cooled and then ether was added to precipitate the product. The solid was filtered and washed with hexane.    13

Synthesis and characterization of 1,2,3-triazole di-acid hydrazides 4a-d
A mixture of compound 3a-d (10 mmol) and hydrazine hydrate (20 mmol) in ethanol (50 mL) was stirred for 5-15 min at rt. Ethanol was removed under reduced pressure, and the product formed was recrystallized from ethanol to give the titled compounds 4a-d.

Synthesis and characterization of 1,2,3-triazole bis-1,2,4-triazole-3-thiones 6a-d
A mixture of compound 5a-d (10 mmol) and 10% aqueous sodium hydroxide solution (200 mL) was refluxed for 6 h. The mixture was then cooled to room temperature and filtered. The filtrate was acidified by the addition of hydrochloric acid. The resulting solid was collected by filtration, washed with water and recrystallized from ethanol to give compound 6a-d.

Synthesis and characterization of 1,2,3-triazole bis-4-amino-1,2,4-triazole-3-thiones 7a-d
Step 1 Carbon disulfide (30 mmol) was added dropwise to a stirred solution of compound  Switzerland). An inoculum size of 1 × 10 5 CFU mL −1 of each microorganism was inoculated in each microtiter plate well. Test wells were filled with 100 μL nutrient broth and a series of dilutions of each examined compound dissolved in DMSO (1-500 mg mL −1 ). Positive control wells consisted of the individual microorganism under investigation inoculated in 100 μL nutrient broth while negative control wells contained DMSO at the same concentration present in the test wells.
Plates were incubated for 24 h at 37 °C, with shaking. To evaluate microbial growth, optical densities were measured at 600 nm (OD600) using a Microplate Reader (Palo Alto, CA, USA). The MIC value was designated as the least concentration at which more than 80% of the microbial growth is inhibited. MIC assessment was carried out in triplicates and repeated three times for each microorganism.

In-silico molecular docking studies
The compounds synthesized in the present investigation were subjected for molecular docking studies using Auto Dock (version 4.0) with Lamarckian genetic algorithm [38]. We have considered using Lamarckian genetic algorithm over Monte Carlo simulated annealing and traditional genetic algorithm. The previous method can handle ligands with more degrees of freedom than the Monte Carlo method used in earlier versions of AUTO-DOCK. The Lamarckian genetic algorithm is the most efficient, reliable, and successful. AutoDock 4.0, combines energy evaluation through grids of affinity potential employing various search algorithms to find the suitable binding position for a ligand on a given protein.
The ligands were drawn in ChemSketch. Energy of molecule was minimized using by PRODRG server [39]. In the present study, the binding site was selected based on the amino acid residues, which are involved in binding with glucosamine-6-phosphate of GlcN-6-P synthase as obtained from Protein Data Bank (http://www.pdb.org/ pdb/home/home.do) with the PDB ID 2VF5 which would be considered as the best accurate active region as it is solved by experimental crystallographic data [40]. It was then edited by removing the heteroatoms, adding the C-terminal oxygen, rotating all the torsions during docking. Steepest Descent methods were applied for minimization by considering the default parameters. Polar hydrogen's were added to ligands using the hydrogen's module in Autodock tool and thereafter assigning Kollman united atom partial charges. Docking to ligands was carried out with standard docking protocol on the basis a population size of 150 randomly placed individuals; a maximum number of 2.5*107 energy evaluations, a mutation rate of 0.02, a crossover rate of 0.80 and an elitism value of 1. Fifteen independent docking runs were carried out for ligands. The grid was centered at the region including all the 12 amino acid residues (Ala602, Val399, Ala400, Gly301, Thr302, Ser303, Cys300, Gln348, Ser349, Thr352, Ser347 and Lys603). The grid box size was set at 70, 64, and 56 Å̊ for x, y and z respectively, and the grid center was set to 30.59, 15.822 and 3.497 for x, y and z respectively, which covered all the 12 amino acid residues in the considered active pocket. The spacing between grid points was 0.375 angstroms. The docking results were interpreted according to the.pdb file. Using the rmsd table created in the.dlg file, we have determined the co-ordinates of the minimum energy run. UCSF chimera was used to visualize the coordinate of the docked protein along with targeted compounds within 6.5 Ǻ region.

Conclusions
A series of novel 1,2,3-triazole-1,2,4-triazole hybrids carrying variant lipophilic side chain were synthesized and screened for antibacterial and antifungal activity. Finally, the synthesized compounds were docked inside the active site of Glucosamine-6-phosphate synthase, the potential target for antimicrobial and antifungal agents and the results of such studies were reported. Insilico studies revealed that all the synthesized compounds 3a, c, d, 4a-d, 5a-c, 6a-d, 7a-d have relatively less binding energy as compared to the standard drug and may be considered as a good inhibitors of GlcN-6-P. The binding energy toward the target protein ranged from − 5.72 to − 10.49 kJ mol −1 . The high-ranking binding energy of the synthesized compound, 6d was − 10.49 kcal/ mL. Consistent with the in silico studies, all synthesized compounds demonstrated fair to excellent antimicrobial activities relative to standard potent antibacterial and antifungal agents, with remarkably enhanced antimicrobial activities associated with the 1,2,4-triazole derivatives tailoring elongated chain substitution at the 1,2,3-triazole N-1 position.