Synthesis of New Triazole-Based Thiosemicarbazone Derivatives as Anti-Alzheimer’s Disease Candidates: Evidence-Based In Vitro Study

Triazole-based thiosemicarbazone derivatives (6a–u) were synthesized then characterized by spectroscopic techniques, such as 1HNMR and 13CNMR and HRMS (ESI). Newly synthesized derivatives were screened in vitro for inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. All derivatives (except 6c and 6d, which were found to be completely inactive) demonstrated moderate to good inhibitory effects ranging from 0.10 ± 0.050 to 12.20 ± 0.30 µM (for AChE) and 0.20 ± 0.10 to 14.10 ± 0.40 µM (for BuChE). The analogue 6i (IC50 = 0.10 ± 0.050 for AChE and IC50 = 0.20 ± 0.050 µM for BuChE), which had di-substitutions (2-nitro, 3-hydroxy groups) at ring B and tri-substitutions (2-nitro, 4,5-dichloro groups) at ring C, and analogue 6b (IC50 = 0.20 ± 0.10 µM for AChE and IC50 = 0.30 ± 0.10 µM for BuChE), which had di-Cl at 4,5, -NO2 groups at 2-position of phenyl ring B and hydroxy group at ortho-position of phenyl ring C, emerged as the most potent inhibitors of both targeted enzymes (AChE and BuChE) among the current series. A structure-activity relationship (SAR) was developed based on nature, position, number, electron donating/withdrawing effects of substitution/s on phenyl rings. Molecular docking studies were used to describe binding interactions of the most active inhibitors with active sites of AChE and BuChE.


Introduction
Alzheimer's disease (AD) is an irreversible, neurodegenerative and progressive disorder of the brain that diminishes the cholinergic system and results in disorientation, memory loss, impaired ability to solve problems and impaired cognition [1][2][3]. AD is the major cause of dementia in aging populations. The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes cause apoptosis of neuronal cells by plaques formed by aggregation of neurotoxic beta amyloid. They are involved in hydrolysis of acetylcholine to generate acetic acid and choline, leading to shortening of duration of acetylcholine in the hippocampus and cortex of the brain and thus facilitating normal regeneration of synapses and functioning. Therefore, targeting both AChE and BuChE enzymes is one approach for treatment of AD [4][5][6][7]. Two binding sites are present in AChE: the peripheral site, which is responsible for beta amyloid interaction, and the catalytic site, which causes hydrolysis of acetylcholine. Interactions of beta amyloid protein (Aβ) with AChE access the formation of beta amyloid protein-acetylcholinesterase (Aβ-AChE) complex and thus result in neurotoxicity. BuChE is found in liver, intestine, heart, kidney, serum and lungs, while AChE is present in cholinergic neurons, brain and muscle [8,9]. The cholinesterase enzymes perform key roles in the breakdown of compounds, having ester moieties in their core structures. Generally, AChE is dominant in brain, while BuChE functions when acetylcholine gradually decreases its function in the brain of AD patients. Hence, synthesis of drugs that function as inhibitors of both AChE and BuChE enzymes should be effective treatments of AD [10]. Several drugs have been approved by the Food and Drug Administration (FDA) for treatment of AD. These include galantamine and donepezil which are selective for AChE, while rivastigmine and tacrine inhibit both BuChE and AChE ( Figure  1) [11].  Triazole analogues are reported to have therapeutic and biological activities, such as anticonvulsant [12], anti-inflammatory [13], antifungal [14], insecticidal [15] and plant growth regulation [16]. There are some important drugs, such as letrozole (anticancer), tazobactam (antibacterial), isavuconazole (antifungal), sitagliptin (antidiabetic), ribavirin (antiviral), and rufinamide (seizure disorder), that contain triazole moieties in their core structures ( Figure 2) [17][18][19][20][21][22][23]. Triazole analogues are reported to have therapeutic and biological activities, such as anticonvulsant [12], anti-inflammatory [13], antifungal [14], insecticidal [15] and plant growth regulation [16]. There are some important drugs, such as letrozole (anticancer), tazobactam (antibacterial), isavuconazole (antifungal), sitagliptin (antidiabetic), ribavirin (antiviral), and rufinamide (seizure disorder), that contain triazole moieties in their core structures ( Figure 2) [17][18][19][20][21][22][23].
Recently, several classes of heterocyclic compounds have been reported to be potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) [24][25][26][27][28]. Based on the biological importance of thiosemicarbazone [29,30] and triazole [31,32] compounds (Figure 3), it was decided to synthesize hybrid analogues based on triazole bearing thiosemicarbazone moiety as effective inhibitors of cholinesterase enzymes, such as AChE and BuChE, that could be effective treatments for AD. Molecules 2023, 28 Recently, several classes of heterocyclic compounds have been reported to be potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) [24][25][26][27][28]. Based on the biological importance of thiosemicarbazone [29,30] and triazole [31,32] compounds (Figure 3), it was decided to synthesize hybrid analogues based on triazole bearing thiosemicarbazone moiety as effective inhibitors of cholinesterase enzymes, such as AChE and BuChE, that could be effective treatments for AD.     Recently, several classes of heterocyclic compounds have been reported to be po inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) [24 Based on the biological importance of thiosemicarbazone [29,30] and triazole [31,32] c pounds ( Figure 3), it was decided to synthesize hybrid analogues based on triazole b ing thiosemicarbazone moiety as effective inhibitors of cholinesterase enzymes, suc AChE and BuChE, that could be effective treatments for AD.

Chemistry
Thiosemicarbazide (1) was treated with 4-nitrobenzoyl chloride in DMF in the presence of triethylamine and refluxed for 3 h to yield 2-(4-nitrobenzoyl)hydrazine-1-carbothioamide as the first intermediate (2), which further underwent cyclization during stirring overnight in 2% aqueous solution of sodium hydroxide followed by neutralization with dil. HCl to yield 1,2,4-triazole-3-thiole (3) as the second intermediate product. Intermediate (3) was then reacted with different substituted phenacyl bromide in ethanol in the presence of triethylamine and refluxed for 3 h to obtain the third intermediate (4). Intermediate (4) was then mixed with hydrazine hydrate in methanol in the presence of a few drops of glacial acetic acid to form intermediate (5). Finally, intermediate (5) was treated with different substituted isothiocyanate in tetrahydrofuran in the presence of triethylamine, with the resulting mixture stirred under reflux until conversion had been completed, as monitored by TLC during refluxing for 6-8 h. After being cooled to room temperature, the product was reacted with 5% Na 2 S 2 O 3 and extracted with CH 2 Cl 2 /MeOH (10:1, 10 mL × 4). The combined organic layer was dried over anhydrous sodium sulfite and concentrated. The resulting residue was purified through silica gel column chromatography using a mixture of petroleum ether and EtOAc as eluent to yield the desired triazole-based thiosemicarbazone derivatives (6a-u) as the final product (Scheme 1, Table 1). Primary confirmation of the product was done using thin layer chromatography (TLC) and further confirmed by with nuclear magnetic resonance (NMR).

Chemistry
Thiosemicarbazide (1) was treated with 4-nitrobenzoyl chloride in DMF in the presence of triethylamine and refluxed for 3 h to yield 2-(4-nitrobenzoyl)hydrazine-1-carbothioamide as the first intermediate (2), which further underwent cyclization during stirring overnight in 2% aqueous solution of sodium hydroxide followed by neutralization with dil. HCl to yield 1,2,4-triazole-3-thiole (3) as the second intermediate product. Intermediate (3) was then reacted with different substituted phenacyl bromide in ethanol in the presence of triethylamine and refluxed for 3 h to obtain the third intermediate (4). Intermediate (4) was then mixed with hydrazine hydrate in methanol in the presence of a few drops of glacial acetic acid to form intermediate (5). Finally, intermediate (5) was treated with different substituted isothiocyanate in tetrahydrofuran in the presence of triethylamine, with the resulting mixture stirred under reflux until conversion had been completed, as monitored by TLC during refluxing for 6-8 h. After being cooled to room temperature, the product was reacted with 5% Na2S2O3 and extracted with CH2Cl2/MeOH (10:1, 10 mL × 4). The combined organic layer was dried over anhydrous sodium sulfite and concentrated. The resulting residue was purified through silica gel column chromatography using a mixture of petroleum ether and EtOAc as eluent to yield the desired triazole-based thiosemicarbazone derivatives (6a-u) as the final product (Scheme 1, Table  1). Primary confirmation of the product was done using thin layer chromatography (TLC) and further confirmed by with nuclear magnetic resonance (NMR). (1) Scheme 1. Synthesis of triazole-based thiosemicarbazone derivatives (6a-u). Scheme 1. Synthesis of triazole-based thiosemicarbazone derivatives (6a-u).

In Vitro Inhibition of Acetylcholinesterase and Butyrylcholinesterase Activities
All the newly synthesized derivatives of triazole-based thiosemicarbazone (6a-u) were screened in vitro for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities. All the newly afforded derivatives, except 6c and 6d, which are found to be inactive, displayed good to moderate inhibition, with IC50 ranging from 0.10 ± 0.050 to 12.20 ± 0.30 µM against AChE and 0.20 ± 0.050 µM to 14.10 ± 0.40 µM against BuChE compared to the standard drug donepezil, which exhibited IC50 of 2.16 ± 0.12 and 4.5 ± 0.11 µM against AChE and BuChE, respectively (Table 1). A structure-activity relationship (SAR) based on substituent/s and electron donating/withdrawing effects on phenyl rings B and C was developed. The compounds were divided into five major parts: triazole moiety, ring A, thiosemicarbazone moiety, 6s 1.

In Vitro Inhibition of Acetylcholinesterase and Butyrylcholinesterase Activities
All the newly synthesized derivatives of triazole-based thiosemicarbazone (6a-u) were screened in vitro for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities. All the newly afforded derivatives, except 6c and 6d, which are found to be inactive, displayed good to moderate inhibition, with IC50 ranging from 0.10 ± 0.050 to 12.20 ± 0.30 µM against AChE and 0.20 ± 0.050 µM to 14.10 ± 0.40 µM against BuChE compared to the standard drug donepezil, which exhibited IC50 of 2.16 ± 0.12 and 4.5 ± 0.11 µM against AChE and BuChE, respectively (Table 1). A structure-activity relationship (SAR) based on substituent/s and electron donating/withdrawing effects on phenyl rings B and C was developed. The compounds were divided into five major parts: triazole moiety, ring A, thiosemicarbazone moiety,

In Vitro Inhibition of Acetylcholinesterase and Butyrylcholinesterase Activities
All the newly synthesized derivatives of triazole-based thiosemicarbazone (6a-u) were screened in vitro for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities. All the newly afforded derivatives, except 6c and 6d, which are found to be inactive, displayed good to moderate inhibition, with IC 50 ranging from 0.10 ± 0.050 to 12.20 ± 0.30 µM against AChE and 0.20 ± 0.050 µM to 14.10 ± 0.40 µM against BuChE compared to the standard drug donepezil, which exhibited IC 50 of 2.16 ± 0.12 and 4.5 ± 0.11 µM against AChE and BuChE, respectively (Table 1). A structure-activity relationship (SAR) based on substituent/s and electron donating/withdrawing effects on phenyl rings B and C was developed. The compounds were divided into five major parts: triazole moiety, ring A, thiosemicarbazone moiety, ring B, and ring C. Each part of the synthesized compounds was found to be actively participating in inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Furthermore, it was determined that by keeping the triazole, ring A, and thiosemicarbazone moieties constant, the variation in inhibitory potentials was determined by attachment of substituents of diverse nature at various positions in different number/s around both rings B and C ( Figure 4, Table 1).

Structure-Activity Relationship (SAR) for Inhibition of Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE)
Analogue 6i with IC 50 = 0.10 ± 0.050 (for AChE) and IC 50 = 0.20 ± 0.050 µM (for BuChE) having di-substitutions (2-nitro, 3-hydroxy groups) at ring B and tri-substitutions (2-nitro, 4,5-dichloro groups) at ring C emerged as the most active inhibitor of targeted AChE and BuChE enzymes, whereas analogue 6b (IC 50 = 0.20 ± 0.10 µM for AChE) (IC 50 = 0.30 ± 0.10 µM for BuChE) having di-Cl at 4,5-and-NO 2 groups at 2-position of phenyl ring B and hydroxy group at ortho-position of phenyl ring C was recognized as the second-most active among the current synthesized series (Table 1). The greater number of attached electron-withdrawing groups, such as di-Cl and -NO 2 groups around ring C, as well as the presence of substituents (-OH) capable of forming hydrogen bonds with the active residue of amino acids of these analogues were responsible for enhanced inhibitory potentials for both targeted AChE and BuChE. The majority of the electronic density is removed from Ph-ring B and C by these di-Cl and -NO 2 groups, making it electron-deficient and further regaining stability through interactions with the active sites of targeted AChE and BuChE enzymes. The derivative 6k, which has a para-bromo substitution on ring B and a para-tolyl group at 4-position of aryl ring C, was shown to have the least inhibitory activity against AChE and BuChE enzymes with IC 50 values of 12.20 ± 0.30 and 14.10 ± 0.40µM. This reduced the potency of derivative 6k, caused by the greater size of the attached substituent(s), which increased the crowdedness around both rings B and C and thus reduced the chance of interactions with catalytic residues of targeted enzymes (Table 1). synthesized compounds was found to be actively participating in inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Furthermore, it was determined that by keeping the triazole, ring A, and thiosemicarbazone moieties constant, the variation in inhibitory potentials was determined by attachment of substituents of diverse nature at various positions in different number/s around both rings B and C (Figure 4, Table 1

Structure-Activity Relationship (SAR) for Inhibition of Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE)
Analogue 6i with IC50 = 0.10 ± 0.050 (for AChE) and IC50 = 0.20 ± 0.050 µM (for BuChE) having di-substitutions (2-nitro, 3-hydroxy groups) at ring B and tri-substitutions (2-nitro, 4,5-dichloro groups) at ring C emerged as the most active inhibitor of targeted AChE and BuChE enzymes, whereas analogue 6b (IC50 = 0.20 ± 0.10 µM for AChE) (IC50 = 0.30 ± 0.10 µM for BuChE) having di-Cl at 4,5-and-NO2 groups at 2-position of phenyl ring B and hydroxy group at ortho-position of phenyl ring C was recognized as the second-most active among the current synthesized series (Table 1). The greater number of attached electron-withdrawing groups, such as di-Cl and -NO2 groups around ring C, as well as the presence of substituents (-OH) capable of forming hydrogen bonds with the active residue of amino acids of these analogues were responsible for enhanced inhibitory potentials for both targeted AChE and BuChE. The majority of the electronic density is removed from Ph-ring B and C by these di-Cl and -NO2 groups, making it electron-deficient and further regaining stability through interactions with the active sites of targeted AChE and BuChE enzymes. The derivative 6k, which has a para-bromo substitution on ring B and a paratolyl group at 4-position of aryl ring C, was shown to have the least inhibitory activity against AChE and BuChE enzymes with IC50 values of 12.20 ± 0.30 and 14.10 ± 0.40µM. This reduced the potency of derivative 6k, caused by the greater size of the attached substituent(s), which increased the crowdedness around both rings B and C and thus reduced the chance of interactions with catalytic residues of targeted enzymes (Table 1). Derivatives 6a, 6b and 6l containing di-Cl groups at meta-and para-position and nitro group at ortho-position of ring C and a variety of other groups, including Br, OH, NO 2 and CH 3 , at various position of ring B, improved inhibition of activities of both AChE and BuChE enzymes. Among these three derivatives, derivative 6b (IC 50 = 0.20 ± 0.10 and 0.30 ± 0.10 µM) with hydroxy group at ortho-position on ring B along with tri-substitutions (2-nitro and 3,4-di-Cl groups) at aryl ring C displayed superior inhibition of AChE and BuChE, compared to derivative 6a (IC 50 = 5.10 ± 0.20 and 6.40 ± 0.20 µM), which had a bromo group at ortho-position of ring B and derivative 6l (IC 50 = 4.60 ± 0.010 and 5.90 ± 0.10 µM), bearing NO 2 at ortho and CH 3 groups at para on ring B along with di-Cl groups at meta-and para-position and nitro group at ortho-position of ring C (Table 1). These three derivatives contain tri-substitutions (2-nitro, 3,4-dichloro groups) at ring C, but have different substituents (Br, OH, CH3 and NO 2 groups) around ring B. This diverse nature of substituents around ring B have different tendencies to interact with active site of targeted enzymes and hence cause variation in inhibitory potentials of these three derivatives (Table 1). Moreover, derivative 6a which contains a bromo at ortho on ring B and di-Cl groups at meta-and para-position and nitro group at ortho-position of ring C, exhibits better inhibition of AChE and BuChE activities than derivative 6h, which has a bromo moiety at para on ring B and ortho-nitro and para-methyl substitutions on ring C, might be due to di-Cl groups on ring, as well as different position of bromo moiety around ring B (Table 1).
Derivative 6g (IC 50 = 2.10 ± 0.10 and 4.30 ± 0.10 µM), containing two methoxy groups at ortho-and meta-position of ring B and a nitro group at the para-position of the phenyl ring C, exhibited better inhibition of AChE and BuChE activities, compared to derivative 6f (IC 50 = 2.40 ± 0.10 and 4.70 ± 0.10 µM), which had two methoxy groups at the ortho-and meta-positions of ring B and ortho-nitro and para-methyl groups on phenyl ring C (Table 1). This enhanced inhibition of AChE and BuChE activities of derivative 6g might be due to the stronger electron withdrawing nature of nitro group, making the ring C partially positive, which further established a pi-cation interaction with the active enzyme site. Alternatively, derivative 6f had both electron-donating (CH 3 ) and electron-withdrawing (NO 2 ) groups on ring C, which did not create charge on ring C and hence resulted in lesser activities of AChE and BuChE (Table 1).
Comparing derivative 6h (IC 50 = 9.10 ± 0.20 and 11.20 ± 0.30 µM), which has a bromo group at the para position of the phenyl ring B and a nitro group at orthoposition and a methyl group at the para-position of the phenyl ring C, with derivatives 6m (IC 50 = 2.70 ± 0.10 and 3.80 ± 0.10 µM) having di-Cl groups at meta-and para-positions of ring B and nitro group at ortho-position and methyl group at para-position on phenyl ring C and 6r (IC 50 = 1.30 ± 0.050 and 2.20 ± 0.10 µM) having nitro group at ortho on ring B and nitro group at ortho-position and methyl group at para-position on phenyl ring C and 6t (IC 50 = 1.90 ± 0.10 and 2.50 ± 0.10 µM) with a methoxy group at meta-position of ring B and the nitro group at ortho-position and the methyl group at para-position on phenyl ring C (Table 1). The small difference in the inhibitory activities (AChE and BuChE) of all derivatives might be due to the different nature and position of the substituent/s on phenyl ring B (Table 1).
Derivative 6n (IC 50 = 0.70 ± 0. 05 and 1.70 ± 0.050 µM), which has di-chloro groups at meta-and para-positions on ring B and a nitro group at para-position on ring C, with derivative 6s (IC 50 = 1.40 ± 0.050 and 2.30 ± 0.10µM), which has a nitro group at ortho position on ring B and nitro moiety at para position on ring C, and derivative 6u (IC 50 = 2.90 ± 0.10 and 3.70 ± 0.10 µM), with a methoxy group at the meta-position on ring B and nitro moiety at the para position on ring C were compared (Table 1). Difference in the inhibitory activities (AChE and BuChE) of these derivatives might be due to the different nature and position of substituent/s on ring B (Table 1). Number, nature, position and electron-donating/withdrawing nature of substituents considerably influenced inhibition of activities.

Docking Study
Molecular docking was analyzed in order to gain an understanding of the binding mechanism of synthesized compounds against both the targeted enzymes. The optimized compounds were docked based on the co-crystal of each crystallographic structure. Each compound was assigned a total of 30 conformations prior to the docking process. For further investigation, the top-ranked conformations of potent compounds were chosen. The docking results revealed that all the compounds were well oriented in the active site of both enzymes. In general, we found that all of the compounds in the series-with different substituted groups at all three ends of the compound (according to the scheme), where one end has a nitro group, the second end has a halogen group, and the most important end (third) has a different substituted group-had inhibitory potential against the target. These typically belong to electron-withdrawing or electron-donating groups.

Experimental
All chemicals and solvents were purchased from Sigma Aldrich (St. Louis, MO, USA) with a purity of 97 up to 99%. Thiosemicarbazide (1, 0.5 mmol) was treated with 4-nitrobenzoyl chloride (0.5 mmol) in DMF (10 mL) in the presence of triethylamine (0.5 mL) and refluxed for 3 h to yield 2-(4-nitrobenzoyl)hydrazine-1-carbothioamide as first intermediate (2), which further undergoes cyclization on stirring overnight in 2% aqueous solution of sodium hydroxide (10 mL followed by neutralization with dilute HCl (5 mL) to yield 1,2,4-triazole-3-thiole (3) as the second intermediate product. Intermediate (3) was then reacted with equivalent different substituted phenacyl bromide in ethanol (10 mL) in the presence of triethylamine (0.5 mL) and refluxed for 3 h to obtain third intermediate (4). Intermediate (4) was then mixed with hydrazine hydrate (5 mL) in methanol (10 mL) in the presence of a few drops of glacial acetic acid to give fourth intermediate (5). Finally intermediate (5) was treated with equivalent different substituted isothiocyanate in tetrahydrofuran (10 mL) in the presence of triethylamine (0.5 mL) and the resulting mixture was stirred under reflux until the conversion was completed (monitored by TLC, reflux 6-8 h). After being cooled to room temperature, it was reacted with 5% Na 2 S 2 O 3 (20 mL) and extracted with CH 2 Cl 2 /MeOH (10:1, 10 mL × 4). The combined organic layer was dried over anhydrous sodium sulfate and concentrated. The given residue was purified through silica gel column chromatography using a mixture of petroleum ether and EtOAc as eluent to yield the desired triazole-based thiosemicarbazone derivatives (6a-u).

Spectral Analysis
All the proton NMR spectra are shown in Supplementary Materials.

Molecular Docking Protocol
Molecular docking was studied using MOE software to understand the binding mode of synthesized compounds against both the targeted enzymes in order to triangulate in vitro and in silico results well. The crystal structures of both targets were retrieved from the RCSB protein databank using the PDB codes 1ACL for AChE and 1P0P for BuChE. Using the default MOE-Dock module parameters, the crystallographic structures and all synthesized compounds were protonated and energy was minimized, resulting in optimized enzyme and compound structures. After that, the optimized enzyme and compound structures were used in a docking study. Comprehensive details of the docking protocol are included in our previous investigations [33,34].

Acetylcholinesterase Activity Assay Protocol
Based on previously described methods, in vitro study of AChE inhibitory profile was calculated [35,36]. In order to make a stock solution, compounds being analyzed were dissolved in DMSO (1 mg/mL). In addition, the working solutions were also prepared by using serial dilution (1-100 µg/mL). The solution of AChE enzyme (20 µL; 0.1 U/mL), analogues being tested with different concentration and buffer of sodium phosphate (150 µL; pH 8.0; 0.1 M) were preincubated appropriately at 25 • C. The process was initiated as DTNB (10 mM; 10 µL) and AChEI (14 mM; 10 µL) were accumulated. The resulting residue was mixed (by using cyclomixer) and put on incubation for 10 min at 25 • C. Instead of compounds being tested using 10 µL DMSO, the absorbance against blank reading was calculated with a microplate reader at 410 nm. Using the given formula as given, inhibition and IC 50 values were measured in comparison to donepezil (0.01-100 µg/mL) as the reference standard (Equation (1)). %Inhibition = Absorbance of control − Absorbance of compound × 100 Acontrol (1) By plotting a nonlinear graph between inhibition and concentration, IC 50 was calculated using GraphPad Prism 5.3.

Butyrylcholinesterase Activity Assay Protocol
In order to explore the inhibition profile of in vitro BuChE enzyme, a similar procedure was adopted. For measurement of BuChE activity, the solution containing BuChE enzyme was used [35,36].

Conclusions
Triazole-based thiosemicarbazone derivatives (6a-u) were synthesized and screened for potential to inhibit activities of acetylcholinesterase and butyrylcholinesterase enzymes. All the synthetic derivatives (except compounds 6c and 6d, which were found to be completely inactive) displayed moderate to good inhibitory activities having an IC 50 values ranging from 0.20 ± 0.050 to 12.20 ± 0.30 µM (against AChE) and 0.40 ± 0.050 to 14.10 ± 0.40 µM (against BuChE) compared to the standard drug donepezil (IC 50 = 2.16 ± 0.12 (AChE) and 4.5 ± 0.11 µM (BuChE)). Among the series, derivative 6q (IC 50 = 0.20 ± 0.050 µM) was the most potent inhibitor of acetylcholinesterase enzyme, while derivative 6o (IC 50 = 0.40 ± 0.050 µM) was the most active inhibitor of butyrylcholinesterase enzyme. The binding interactions of most active compounds with the active site of enzymes were established with the help of molecular docking studies.