Novel tetrazole derivatives: synthesis, anticholinesterase activity and cytotoxicity evaluation Yeni tetrazol türevleri: sentezleri, antikolinesteraz ve sitotoksisite

Objective(s): The synthesis of new N ′ -arylidene-4-[(1-phenyl-1 H -tetrazole-5-yl)thio]butanoylhydrazide derivatives ( 1–26 ) and investigation of their potential anticholinesterase (AChE), butyrylcholinesterase (BuChE) enzyme inhibition activities and also cytotoxic properties on mouse embryonic fibroblast cells (NIH/3T3) were aimed in this work. Materials and methods: The target compounds were prepared by a three step synthetic procedure using 1-phenyl-1 H -tetrazole-5-thiol and ethyl 4-chlorobutanoate as start-ing materials. The structures of the obtained compounds were elucidated by IR, 1 H-NMR, 13 C-NMR spectra and elemental analysis data. The enzyme inhibition and cytotoxic activities were determined according to Ellman and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] methods, respectively. Results: Compounds 14 , 15 and compound 18 exhibited the highest inhibitory activity on AChE and BuChE enzymes. Additionally, compounds 4, 5, 8 and 16 exhibited the lowest cytotoxicity against NIH/3T3 cells. Conclusion: Compounds 14, 15 and 18 bearing 2-nitro, 3-nitro and 3-hydroxy substituents have showed selective enzyme inhibitory activities.


Introduction
Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disorder of the central nervous system and primarily older people suffer from AD [1]. The disease is characterized by cognitive dysfunction which is associated with a reduction in cholinergic transmission in cortical and hippocampal neurons [2]. Cholinergic neurotransmission is mediated by the neurotransmitter acetylcholine (ACh) which is rapidly hydrolyzed to choline and acetate after its presynaptic release by the enzyme acetylcholinesterase [3,4]. In this way, AD therapy is mainly managed with acetylcholinesterase inhibitors (AChEIs) like donepezil, rivastigmine, galantamine and also the N-methyl-D-aspartate-receptor (NMDA) antagonist memantine, currently [5,6]. Tetrazole ring is an important pharmacophoric structure which can serve as a bioisosteric moiety of carboxylic group in biologically active molecules, because both groups possess comparable acidities and sizes [7][8][9][10][11]. In particular, 5-substituted thiotetrazoles and 1,5-disubstituted tetrazoles have been used in the synthesis of pharmacologically active drugs [12]. A lot of studies dealing with the synthesis of new tetrazole derivatives exhibiting diverse biological activities as hypotensive, antimicrobial, antiviral, antiallergic, cytostatic, nootropic are published [13][14][15]. Tetrazole bearing compounds were also reported as anticholinesterase inhibitors in the literature [16][17][18]. The approach of replacing the ester group with five-membered rings such as tetrazoles, triazoles to discover more potent and metabolically stable cholinomimetic ligands could be confirmative [19].
Hydrazones also constitute an important class of biologically active drug molecules for new drug development [20]. They has attracted the attention of medicinal chemists due to their wide range of pharmacological properties such as antimicrobial, antimycobacteria, anticonvulsant, analgesic, antiinflammatory, antiplatelet, antitubercular, and antitumoral [21,22]. Moreover, hydrazone bearing molecules with cholinesterase inhibition activity have also been reported in recent literature [23][24][25].
In the view of the above literature findings and as an extension of our previous study [26] we have synthesized new N′-arylidene-2-[(tetrazol-5-yl)thio]butanohydrazide derivatives (1-26) and evaluated their inhibitor activity on AChE and BuChE enzymes and their cytotoxic profiles against NIH/3T3 cell line.

Materials and methods Chemistry
All compounds and chemicals were purchased from Sigma-Aldrich Chemical Co (Sigma-Aldrich Corp., St. Louis, MO, USA) and Merck (Darmstadt, Germany) if not otherwise indicated. Melting points were determined using an Electrothermal 9300 digital melting point apparatus (Electrothermal, Essex, UK) and were uncorrected. All the reactions were monitored by thinlayer chromatography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany). Spectroscopic data were recorded with the following instruments: FTIR, Perkin Elmer Spectrum 100 (Perkin Elmer Inc., Waltham, MA, USA); 1 H-NMR, Bruker DPX 500 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA); 13

Biochemistry AChE and BuChE inhibitory activity
AChE and BuChE inhibitory activity was determined by Ellman's [27] method with minor modifications.
Test compounds were dissolved in dimethyl sulfoxide (DMSO) and tested at maximum final concentration 80 μg/mL. Twenty microliter of enzyme (electric eel AChE or equine serum BuChE 1 U/mL), 10 μL sample were added to 2.4 mL buffer and the mixture was incubated at 37°C for 15 min. After 15 min incubation, 50 μL of 0.01 M 5.5′-dithio-bis(2-nitrobenzoic acid) (DTNB) and 20 μL of 75 mM acetylthiocholine iodide (ATCI) or 25 mM butyrylthiocholine iodide (BTCI) were added and the final mixture was incubated at room temperature for 30 min. Blank was prepared using 10 μL of DMSO instead of the test sample with all other procedures similar to those used in the case of the sample mixture. Absorbances were measured at 412 nm and 37°C using polystyrene cuvettes with spectrophotometer (Shimadzu UV-1700). Experiment was done in triplicate. Data are expressed as mean ± standart deviation (SD). Donepezil and galanthamine were used as standard drugs. The inhibition (percent) of AChE or BuChE was calculated using the following equation.

Cytotoxicity
In vitro cytotoxicity of the synthesized compounds was assessed by using standard MTT bioassay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] against NIH/3T3 mouse embryonic fibroblast cells at 24 h of drug administration [28]. This time interval was determined by our preliminary studies [29]. NIH/3T3 cells were cultured in 96-well flat-bottom plates at 37°C for 24 h (2 × 10 4 cells per well). All of the compounds were DMSO individually and added to culture wells at varying concentrations (50-200 µg/mL), the highest final DMSO concentration was under 0.1% which had no effect on the cell viability [26]. After 24 h drug incubation at 37°C, 20 µL MTT solution (5 mg/mL MTT powder in PBS) was added to each well. Than 3 h incubation period was maintained in the same conditions. Purple formazan was occurred at the end of the process which is the reduction product of MTT agent by the mitochondrial dehydrogenase enzyme of intact cells. Formazan crystals were dissolved in 100 µL DMSO and the absorbance was read by ELISA reader (OD570 nm). The percentage of viable cells was calculated based on the medium control. Every concentration was repeated in three wells and IC50 values were defined as the drug concentrations that reduced absorbance to 50% of control values by using logarithmic graphics.

Chemistry
In the present study, some N′-arylidene-4-[(1-phenyl-1Htetrazol-5-yl)thio]butanohydrazide derivatives (1-26) were synthesized and some characteristics of the compounds were presented in Table 1. Target compounds were obtained at three steps as can be seen from Scheme 1. Initially, 1-phenyl-1H-tetrazole-5-thiol and ethyl 4-chlorobutanoate were refluxed with potassium carbonate in acetone to give ethyl 4-[(1-phenyl-1H-tetrazole-5-yl)thio] butanoate compound (A). Then, this ester compound was reacted with hydrazine hydrate to reach its corresponding hydrazide (B). To obtain final hydrazone compounds, compound B was reacted with substituted benzaldehydes. Structure elucidations of the final compounds were performed with IR, 1 H NMR, 13 C-NMR and MS spectroscopic methods and elemental analyses. In the IR spectra of all compounds characteristic nitrogen-hydrogen, carbonoxygen bonds were observed at about 1668-1674 cm − 1 region due to amide function. Also bands belong to amino group were seen at 3336-3341 cm −1 region compounds as expected. In the 1 H-NMR spectra of the compounds, due to E/Z isomerization, most of the protons were resonated two different regions of the spectrum. The proton of the azomethine group was observed as two singlet peaks at about 7.84-8.54 ppm. Similarly, the signal belongs to the amine proton of this group was seen as two singlets at 11.10-11.76 ppm in the lowest field of the spectrum. The alkyl protons were also affected by the isomerization and mostly they were seen as multiplet peaks. -SCH 2 -protons were resonated at 3.33-3.46 ppm as two triplets or a multiplet peak. -COCH 2 -protons were observed as two triplets and at about 2.16-2.83 ppm. -C-CH 2 C-protons were seen as multiplet peak due to vicinal protons and also isomeric feature of the molecules. All other aromatic protons owing to phenyl ring and alkyl protons owing to methyl, methoxy substituents of the ring were determined at expected areas of the spectrum. In 13 C-NMR spectra of the compounds, because of isomerism signals belong to alkyl carbons were resonated as two different peaks. The signals which were detected at highest value range 168.4-175.4 ppm were assigned for carbonyl carbon. All compounds demonstrated satisfactory elemental analyses results within ± 0.4%. In the MS spectra, the electron spraying technique with positive polarity mode was applied and M + 1 peaks were detected as base peak in agreement with the molecular weights.

Biochemistry
All of the compounds were tested to determine their anticholinesterase activity and cytotoxicity. The anticholinesterase activity of the compounds for AChE and BuChE enzymes were determined using, donepezil and galanthamine as standard drugs ( The cytotoxicity of the compounds were determined against NIH/3T3 normal cells and IC 50 values were detected as the half maximal inhibitory concentration of the compounds to inhibit 50% of the mouse fibroblast cell proliferations and results were given in Table 2. Accordingly, compounds 4, 5, 8 and 16 exhibited the highest potential showing with the lowest cytotoxicity against NIH/3T3 cells. These compounds did not inhibited 50% of the cells on the concentrations used and the IC 50 values could not be calculated. The IC 50 values for the compounds 2, 7, 9, 10, 13, 18, 19, 21, 23, 24 and 26 have been determined higher than 100 µg/mL which can be interpreted with moderate cytotoxicity. Furthermore when we evaluate the most active compounds, the IC 50 values of the compounds 14 and 15 on AChE enzyme and compound 18 on BuChE were found lower than cytotoxic dose.

Discussion
The title tetrazole-hydrazone compounds (1-26) were obtained from various aldehydes and the main structure of the molecules differs by benzaldehyde moiety possessing methyl, methoxy, bromo, nitro, fluoro, hydroxy, benzyloxy and isopropyl substitutions. Regarding with substituent effect and position, 2-nitro and 3-nitro groups have positively contributed anticholinesterase activity of the compounds (Compound 14 and 15). A similar finding was observed in our previous study [30] that 3-nitro substituent caused an obvious increase of AChE enzyme inhibitory activity. However, there was not seen same situation for 3-methyl and 3-chloro substituents reported in the mentioned paper. In another reported literature, fluoro substituted derivatives showed higher AChE enzyme inhibitory activity than methyl substituted derivatives as in our study [31]. Mostly, methyl and methoxy substituents caused activity increase [32,33], for our compounds this information has not been valid.
Considering BuChE inhibitory activity, compound 18 with 3-hydroxy substituent showed the highest enzyme inhibition activity differently from the literatures [17,34]. In these studies, the positive effect of fluoro substituent was focused supporting the percentage inhibitions finding at 80 µg/mL of BuChE enzyme. Also, benzyl moiety which presents in the structure of the anticholinesterase drug donepezil contributed enzyme inhibitory activity on both enzymes [35].
Compounds 4, 5, 8 and 16 were determined as the least cytotoxic derivatives which possessed 4-methyl, 2-methoxy, 2-bromo and 4-nitro functions, respectively. Similar finding was seen in the previous study that nitro function caused a decrease in cytotoxic activity against healthy cell line NIH/3T3 [36]. The comparative interpretation of the cytotoxic activity and enzyme inhibitory activity of the compounds could not be done due to unlike and incompatible activity results.