A biochemistry‐oriented drug design: synthesis, anticancer activity, enzymes inhibition, molecular docking studies of novel 1,2,4-triazole derivatives

Abstract In this study, we researched the reactions of 5-(5-bromofuran-2-yl)-4-methyl-1,2,4-triazole-3-thiol and 5-thiophene-(3-ylmethyl)-4R-1,2,4-triazole-3-thiols with some halogen-containing compounds, a number of new compounds were synthesized (1.1–1.5 and 2.1–2.8). These compounds showed excellent to good inhibitory activities on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. For obtaining the effects of these compounds on AChE and BChE enzymes were determined spectrophotometrically according to Ellman. IC50 values of these enzymes were ranging between 1.63 and 17.68 nM for AChE and 8.71 and 84.02 nM for BChE. After, prostate cancer is the second leading cause of cancer-related mortality for men over the age of 65 in developed countries. Current treatment options remain limited in the treatment of advanced-stage prostate cancer leading to biochemical recurrence in almost 40% of the patients. Therefore, there is an urgent need for development of novel therapeutic tools for treatment of prostate cancer patients. In this study, we aimed at analyzing the potential of all compounds against prostate cancer cells. We found that, of the tested compounds, 2.1, 2.2 and 2.3 showed significant cytotoxic activities against PC3 prostate cancer cells, although their effect on the viability of normal prostate cells was limited. These findings suggest their selective targeting potential for prostate cancer cells and offer them as candidate therapeutic agents against prostate cancer. The inhibitory activities of some chemical compounds, such as (1.1–1.5 and 2.1–2.8) were assessed by performing the molecular docking study in the presence of AChE, BChE and prostate cancer protein. MM/GBSA methods are calculated binding free energy. Finally, ADME/T analysis was performed to examine the drug properties of the 13 studied molecules. Communicated by Ramaswamy H. Sarma


Introduction
Heterocyclic compounds are widely represented in nature; they are components of various biologically active molecules, effective modern drugs and plant protection products (Ivansky, 1978).In the last ten years, the synthesis of heterocyclic systems with a high nitrogen content has become very popular.The class of compounds based on 1,2,4-triazole is widely represented and studied.Today we know many compounds that have various types of biological action.Other compounds are characterized by the properties of photopolymers, plasticizers of plastics and anticorrosive agents.For example, antifungal drugs are well known, the active substances of which are 1,2,4-triazole derivatives: Itraconazole, Fluconazole, Voriconazole and Ravuconazole (Ezabadi et al., 2008;Guillon et al., 2009).Some of the presented compounds are used as crop protection agents.
The synthesis of 1,2,4-triazole derivatives containing heterocyclic fragments is very popular due to the possibility of their various applications.Among them were found compounds with antibacterial, antidepressant, antiviral, antitumor and anti-inflammatory activity (Bayrak et al., 2009;Parchenko, 2014).As we can see from previously published studies, the team of scientists focused on the development of new compounds with analgesic and anti-inflammatory effects among 1,2,4-triazole derivatives (Sarigol et al., 2015;Tozkoparan, Aytac¸, G€ ursoy, Aktay, 2012;Tozkoparan, Aytac, Gursoy, Gunal, et al., 2012;Uzg€ oren-Baran et al., 2012).Other substances in this class have pharmacological parameters with a wide range of therapeutic applications (Sert-Ozgur et al., 2017;Tozkoparan et al., 2005).Therefore, as part of our studies in the chemistry of 1,2,4-triazoles as well as taking into account the structural analogy with the mentioned drugs in Figure 1, herein, we disclose the results of biological activity assessment of the synthesized products 1.1-1.5 and 2.1-2.8 in Figure 2 (Khilkovets, 2021;Zazharskiy et al., 2020).
Prostate cancer is one of the most commonly diagnosed malignancies and the second leading cause of cancer-related mortality for men over the age of 65 in developed countries (Khilkovets, 2021;Pashaei et al., 2017).Approximately, 1.6 million men with a diagnosis of prostate cancer and > 300,000 deaths/year are estimated annually worldwide (Jiang et al., 2019;Pashaei et al., 2017).Several factors including advanced age, lifestyle, race, diet and genetic factors are considered to be associated with the development and progression of prostate cancer (Abidi et al., 2018;Pernar et al., 2018;Xing et al., 2020).In recent years, surgical resection, radiation therapy, brachytherapy, chemotherapy, androgen deprivation therapy  and immunotherapy have been the major methods used to treat local prostate cancer and to improve the survival rates of patients (Barlak et al., 2021;Sur et al., 2019;Yan et al., 2018).Current treatment options, although effective, do not provide enhanced survival rates and remain limited in the treatment of advanced-stage prostate cancer, which results in experiencing a biochemical recurrence in almost 40% of the patients (Khilkovets, 2021;Liu et al., 2016).On the other hand, these treatment options possess severe side effects that impact the patient's life quality (Gheewala et al., 2017).Therefore, there is an urgent need for development of novel therapeutic tools for treatment of prostate cancer patients.
In non-cholinergic and cholinergic tissue cells, including plasma and some bodily fluids, cholinesterases are enzymes that are widely distributed.In addition to helping cells differentiate and regenerate, cholinesterases also help cells respond to stress brought on by a variety of factors.There are two categories for cholinesterases.According to their substrate specificity, how they behaved when the substrate was saturated, and how well they handled inhibitors, the two groups were created.These two definitions are butyrylcholinesterase (BChE), a non-specific cholinesterase and genuine cholinesterase, AChE.AChE is present in the brain, nerve cells, muscle and erythrocytes in high concentrations, whereas BChE is present in the serum, pancreas, liver and central nervous system.BChE is particularly common in the animal kingdom (Bilgicli et al., 2019;€ Okten et al., 2019).Theoretical calculations are one of the fastest methods used to compare the activities of molecules (Yavuz et al., 2021).Theoretical calculations are an important method in designing more effective and active molecules by quickly determining the active sites of molecules before experimental processes (Al-Janabi et al., 2022).In this study, calculations were made to compare the chemical properties of the molecules at the B3LYP, HF and M062X (Becke, 1992;Hohenstein et al., 2008;Vautherin & Brink, 1972) levels with the 6-31þþg(d,p) basis set.Afterward, the docking calculations of the molecules were made and their biological activities were compared.In the molecular docking calculations, prostate cancer protein (PDB ID: 3RUK and 6XXP) (DeVore & Scott, 2012;Rosenfeld et al., 2020), the structure of acetylcholinesterase (AChE) (PDB ID: 4M0E) (Cheung et al., 2013) and BChE (PDB ID: 5NN0) (Ko� sak et al., 2018) for Alzheimer's disease (AD), were used to compare the molecular activities.MM/GBSA methods are calculated binding free energy.ADME/T calculations of molecules were made and their drug properties were investigated.

General
Kofler apparatus was used to determine melting points.ElementarVario L cube (CHNS) (standard -sulfanilamide) was used to determine the elemental composition of novel compounds.The 1 H NMR spectra were recorded in DMSO-d

Cell viability assay
A Cell Viability Detection Kit-8 (CVDK-8; EcoTech Biotechnology, Erzurum, Turkey) was used to detect cell viability according to the manufacturer's instructions.Briefly, PC3 (2.5 � 103) and PNT1a (3 � 103) cells were seeded in a 96-well culture plate.After attachment, cells were treated with compounds at 10, 50 and 200 lM concentrations and were incubated at 37 � C with 5% CO 2 for 24 h.Then, diluted CVDK-8 reagent in RPMI medium was added to each well and cells were incubated at 37 � C for 3 h protected from light.The percent of viable cells were calculated using the optical density (OD) values recorded at 450 nm using a microplate reader.

Theoretical methods
The chemical and biological characteristics of molecules can be learned a lot from theoretical calculations.Theoretical computations are used to determine a number of quantum chemical characteristics.The estimated parameters are employed to provide an explanation for the molecules' chemical behaviors.Molecule calculations are done using a variety of programs.These programs are Gaussian version 09 RevD.01 (Wallingford, CT, USA) and GaussView version 6.0 (Wallingford, CT, USA) (Dennington et al., 2016;Frisch et al., 2009).By using these programs, calculations were made in B3LYP, HF and M06-2x (Becke, 1992;Hohenstein et al., 2008;Vautherin and Brink, 1972) (Khalilov et al., 2021;K€ okbudak et al., 2022;Rezaeivala et al., 2022a).
To compare a molecule's biological activity to a biological substance, molecular docking calculations are used.For the computations of molecular docking, Schr€ odinger's Maestro Molecular modeling platform version 12.8 was employed (Schr€ odinger Release 2021-3, 2021a).Numerous processes go into calculations.Every stage is carried out uniquely.The protein preparation module (Schr€ odinger Release 2021-3, 2021b) was utilized to prepare proteins in the first step.The active sites of the proteins were identified in this module.The examined molecules are synthesized in the following step.The Gaussian software program optimizes the molecules first, and then the LigPrep module (Schr€ odinger Release 2021-3, 2021c) is ready for calculations with optimized structures.After preparation, the interactions between the compounds and the cancer protein were looked at using the Glide ligand docking program (El Faydy et al., 2022).All computations were performed using the OPLS3e method.In order to assess the pharmacological potential of the investigated compounds, an ADME/T study (absorption, distribution, metabolism, excretion and toxicity) will be carried out.The Schr€ odinger software's Qik-prop module was used to forecast the effects and reactions of chemicals on human metabolism (Schr€ odinger Release 2021-3, 2021d).

MM-GBSA calculation
The binding free energy of ligand-protein complexes was found using the MM-GBSA method of the Prime module from Schrodinger (Schr€ odinger Release 2021-3, 2021a).Other parameters were set by default (Shekhar et al., 2019).During the calculation, the OPLS3e force field, VSGB solvent model, and rotamer search algorithms were applied to define the bonding free energy (Wang et al., 2018).Here, we performed the binding free energy calculations of all complexes with the following equation: where DG bind is the binding free energy, G complex ligand-protein complexes are the free energy value, G protein is the target protein's free energy value and G ligand is the free energy value of the ligand.

Enzymes study
For obtaining the effects of novel compounds (1.1-1.5 and 2.1-2.8) on AChE and BuChE enzymes were determined spectrophotometrically according to the Ellman et al. (1961) method.The substrate for the Ellman technique is the thiol ester acetylthiocholine rather than the oxy ester acetylcholine.The Ellman method's basic idea is that AChE hydrolyzes acetylthiocholine, and the thiocholine that is generated as a result of this reaction combines with the Ellman reagent, 5,5 0 -dithio-bis-(2-nitrobenzoic acid) (DTNB).The reaction results in the formation of the yellow chromophore 5-thio-2nitrobenzoic acid (TNB).The absorbance at 412 nm is used to calculate the rate of production (color intensity) of the yellow chemical that results from the reaction.The AChE/BuChE enzyme activity is directly inversely correlated with the intensity of this yellow color.IC50 values were determined from the equation of the curve for a few natural compounds after their activity was evaluated at various inhibitor doses and shown as %activity À [I].(Bilgicli et al., 2019;Koc¸yi� git et al., 2022).

Results and discussion
b-Amino alcohols were resynthesized according to the literature (Khilkovets, 2021;Zazharskiy et al., 2020).The formulas of the compounds used in the study are given in Figure 2.
The cytotoxicity of the synthesized chemical compounds against PC3 human prostate cancer cells and PNT1a, immortalized normal prostate epithelial cell line serving as healthy control, were evaluated using CVDK-8 assay.The percent viabilities of PC3 and PNT1a cells compared to DMSO control are shown in Figure 3.
2.1, 2.2 and 2.3 compounds showed significant cytotoxic activities against PC3 cancer cells in Figure 3(A), although their effect on the viability of normal prostate cells was limited in Figure 3(B), pointing their selective targeting potential for cancer cells in Table 1. The effects of 2.1, 2.2 and 2.3 compounds on PC3 and PNT1a cells were also evaluated by morphological assessment.PC3 and PNT1a cells were treated  with 2.1, 2.2 and 2.3 compounds at corresponding IC 50 doses of PC3 cells for 24 h.
As shown in Figure 4, slight alterations in terms of morphology and cell number were observed in PNT1a cells while much more destructive effects were seen in PC3 cells treated with IC 50 doses of the compounds.These findings suggest that these compounds exhibit significant selectivity toward cancer cells.
2.1, 2.2 and 2.3 have similar molecular structures.The difference is the alkyl chains attached at the terminal sulfur atom.It seems that the shorter the alkyl chain attached at the terminal sulfur atom, molecules exert higher cytotoxic effects since 2.1 has the highest potential to kill the cancer cells.Although there is no consensus for the effect of the length of the alkyl chains attached to a molecule in terms of its cytotoxic potential, there are studies reporting that  Because they reveal a lot about the active sites and activities of the compounds before experimental processes, theoretical calculations provide a quick and effective way to compare the activities of molecules.In the context of this study, a Gaussian software program for molecules is used to determine several quantum chemical characteristics of molecules that explain their chemical properties.Two of these parameters -HOMO and LUMO -are frequently employed to explain the activities of molecules.The ability of molecules to give electrons to other molecules is demonstrated by the HOMO parameter, where the most positive numerical value of this parameter has the highest activity (Alici et al., 2022).In contrast, the LUMO parameter, which measures the activity of the molecule with the parameter's highest negative numerical value, demonstrates molecules' capacity to absorb electrons from other molecules (Lakhrissi et al., 2022).These two factors are frequently compared to determine how active various compounds are.Table 2 includes all calculated parameters aside from these ones.
There are more parameters in addition to these two.The ability of atoms in a molecule to draw bond electrons is measured by a characteristic called electronegativity (Rbaa et al., 2022).The molecular atoms draw more bond electrons to themselves as this parameter's numerical value rises, which reduces the activity of the molecule.However, the DE (E HOMO À E LUMO ) value of the molecules is another parameter that determines the activities of the molecules, and the molecule with the smallest numerical value of this parameter is considered to have the highest activity (Rezaeivala et al., 2022b).
As a result of the DFT calculations, it is seen that many parameters of the molecules are calculated.At the B3LYP and M062X levels, the two molecules with the most negative E LUMO energy of the molecules are 2.7 and 2.8.E LUMO energy value of molecule 2.7 is À 2.4047, while E LUMO energy value of molecule 2.8 is À 2.2112.However, the molecule of 2.8 has the most negative value at À 1.2681 at the M062x level.On the other hand, it is seen that 2.7 and 2.8 are among the lowest DE energy values of molecules at B3LYP and M062X levels.Molecule 2.7 has a DE energy value of 3.6151, which is less than other molecules, and molecule 2.8 is 4.1966.
Figure 5 shows the visual representation of a few molecular properties following calculations.In this manner, there are 4 images.The optimized structure can be seen in the first of these photos.The HOMO and LUMO orbitals of the molecule are depicted on which atoms in the second and third figures.The electrostatic potentials of the molecules are displayed in the final image.This picture features a variety of hues, from red to blue.However, the red hue is where the molecule's electron density is greatest.On the other hand, the areas of the molecule that are blue in hue have the fewest electrons.The parts of the molecule that are red and blue have the highest activity levels.The molecule develops an association by donating electrons from this area when the electron density is high.Conversely, areas of the molecule with low electron density interact and take electrons (Bilgic¸li et al., 2021;Genc ¸ Bilgic ¸li et al.,2020).
With the theoretical calculations made, not only the activity calculations are made, it is possible to compare the chemical shift values found experimentally by finding the 1 H and 13 C chemical shift values of the molecules for the characterization of the molecules.Nuclear magnetic resonance (NMR) spectroscopy is among the important methods used to determine the structure of the molecule in general.NMR spectroscopy is an important characterization method based on the measurement of atoms in the molecule excited to rotational energy levels by the absorption of electromagnetic radiation in the radio frequency range (Akin et al., 2020;Bilgicli et al., 2020).Experimental and theoretical NMR values of carbon and hydrogen atoms of the studied molecules are examined and given in Tables S1-S13.In the theoretical calculations, the chemical shift values of the molecules were made using the gauge-independent atomic orbital (GIAO) (G€ unsel et al., 2020).The chemical shift values of the studied molecules are determined by the atoms and atomic groups around the atoms.Theoretically, 1 H and 13 C chemical shift values of the molecules HF/6-31þþg(d,p) were used.The spectrum data of the 1 H chemical shift values of the molecules in the experimental processes are given in Figures S1-S13.
It is seen that the experimental and theoretical NMR results are in great agreement with each other.When the chemical shift values obtained in general were examined, it was seen that the chemical shift values of aliphatic carbon atoms were between 16.94 and 35.12.On the other hand, the chemical shift values of the aromatic carbon atoms were found to be between 108.86 and 169.42.On the other hand, when the chemical shift values of the hydrogen atoms obtained are examined, it is seen that the chemical shift values of the aliphatic hydrogen atoms are between 1.30 and 5.45, while the chemical shift values of the aromatic hydrogen atoms are between 7.07 and 9.26.
Molecular docking is a popular technique for comparing a molecule's activity to that of biological components.Numerous parameters are generated as a result of molecular docking calculations, and the activities of the molecules are contrasted.These computations produce a prediction of the molecules' active locations.By maintaining these active sites, altering other sites or adding groups, it is attempted to increase activity while creating new compounds (G€ unsel, Bilgic¸li, Pis ¸kin, T€ uz€ un, Delibas ¸, et al., 2019).The method is also time-consuming and expensive to synthesize.For this reason, applying theoretical methods offers a lot of convenience.The docking score parameter, which is one of the calculated parameters, is the one that has the greatest impact on how the activities of the molecules are determined.According to G€ unsel, Bilgic ¸li, Pis ¸kin, T€ uz€ un, Yarasir, et al. ( 2019), the activity of the molecule with the most negative numerical value of this parameter is the highest.The contacts between molecules and proteins, namely hydrogen bonds, polar and hydrophobic interactions, p-p and halogen interactions, are crucial in determining how molecules behave (Poustforoosh et al., 2021).It should be well known that as this interaction increases; the activity of the molecules increases.
In Table 3, all parameters derived from molecular docking calculations are listed.The estimated parameters, such as Glide ligand efficiency, Glide hbond, Glide evdw and Glide ecoul, when taken into account with the parameters listed in this table, provide information on the effectiveness of the molecule as well as the chemical interactions that take place between the molecule and the proteins (T€ urkan et al., 2021).The other parameter group, on the other hand, provides numerical values for the interaction posture between molecules and proteins through parameters like Glide emodel, Glide energy, Glide einternal and Glide posenum (Gedikli et al., 2021).
In the docking calculations, besides the studied molecules, the tacrine molecule was used as a reference molecule.It is seen both experimentally and theoretically that the reference molecule does not have better activity than the studied molecule series.
When the interactions between molecules and proteins are examined in detail, it is seen in Figure 6 that in the interaction between molecules 2.1 and 3RUK protein, hydrophobic, metal and polar interactions occur between molecule 2.1 and protein.When the interaction between molecules 2.8 and 6XXP protein in Figure 7 is examined, it is seen that Pi-Pi stacking interaction occurs between the thiophene ring and the ARG 45 protein and the pi-cation and the TYR 95 protein.It is observed that hydrogen bonding occurs between the oxygen atom attached to the carbonyl carbon in the center of the molecule and the LEU 4 protein.It is observed that hydrogen bonding occurs between the nitrogen atom in the 1,2,4-triazolidine ring in the center of the molecule and the GLY 115 protein.In addition, when the interaction of molecules 2.8 with AChE proteins is examined in Figure 8, it is seen that hydrogen bonding occurs between the nitrogen It is seen that Pi-Pi stacking interaction occurs between the F-benzene ring in the molecule and the PHE 338 and TYR 337 proteins.However, in the interaction between molecules 2.8 and BChE proteins in Figure 9, Pi-Pi stacking interaction occurs between the 1,2,4triazolidine ring in the center of molecule 2.8 and the PHE 398 protein.It is observed that Pi-Pi stacking interaction occurs between the benzene ring attached to the 1,2,4-triazolidine ring in the molecule and the PHE 329 and TRP 231 proteins.
Considering the results of the molecular docking calculations, it is known that the docking score parameters of the molecules are a parameter used to explain the biological activities of the molecules.When the activities of molecules against four different proteins were compared, it was seen that the activity of molecule 2.8 was generally higher than all other molecules.
As a result of the MM-GBSA calculations, the binding free energy values of some molecules were calculated.As a result of the calculations, the energy values of some molecules are given in Table 4.These calculations are the MM-GBSA values   that occur from the interaction of some molecules with the protein 3RUK.These selected molecules have been chosen because some parameters are better in theoretical calculations.As a result of the calculations, the molecule with the most negative value among the binding free energy values is 2.1 with a value of À 9.79.However, there are many interactions between molecules and proteins.these interactions are coulomb, covalent, Hbond, lipophilic, packing, SolvGB and vdW interactions (Chinnasamy et al., 2020;Gao et al., 2020) Each interaction was found to be of greater importance in different molecules.For example, for molecule 2.1, the Lipo (lipophilic) and vdw (van der Waals) interactions seem to be of greater importance.Chemicals with high activity were identified after looking at how chemicals and proteins interact.High molecular activity is insufficient on its own.Additionally, the effects and interactions of chemicals that are introduced into the human metabolism as pharmacological molecules need to be foreseen.These compounds' ADME/T characteristics should be investigated.(Riaz et al., 2021).On the other hand, some biological features in human metabolism are parameters, such as Predicted IC 50 value for blockage of HERG K þ channels   (QPlogHERG), Predicted apparent Caco-2 cell permeability (QPPCaco), Predicted brain/blood partition coefficient (QPlogBB) (Aktas et al., 2020).Apart from this, there are generally two important parameters for molecules to be drugs, the first is RuleOfFive (Lipinski, 2004;Lipinski et al., 1997), number of violations of Lipinski's rule of five, and the second is RuleOfThree (Jorgensen & Duffy, 2002), Number of violations of Jorgensen's rule of three.The numerical value of these two parameters is required to be zero to be a drug.ADME/T calculations of the studied molecules were made using the Qik-prop module in the Maestro Molecular modeling platform by Schr€ odinger program.The mol_MW parameter of molecules shows the moles of molecules, all molecules are within the desired value range.On the other hand, it is seen that the numerical value of the Percent Human Oral Absorption parameter of the molecules is quite high.In this case, it is seen that the RuleOfFive and RuleOfThree parameters of the molecules are generally zero, which is in the desired value range.The numerical value of the QPPMDCK parameter of the molecules is expected to be in the range < 25 is poor and > 500 is great.However, some molecules appear to be too high in this value line.However, it is seen that molecule 2.8, which has a high activity, meets this condition.Therefore, this molecule can be used for both prostate cancer and AD disease.When the ADME/T properties of working molecules are examined, it is seen that they meet the conditions for being a drug.With Ellman's (Ellman et al., 1961) method, anti-cholinesterase activities of novel group 1.1-15 and 2.1-2.8 and tacrine as the positive control were evaluated.Inhibitory activities of the newly synthesized compounds against AChE and BChE are presented by IC 50 and K i values in Table 6.As observed in Table 6, 1.1-15 and 2.1-2.8new synthesized compounds were more potent than tacrine against AChE and BChE.
The K i values of the test compounds demonstrated that the most active compound against AChE and BChE was complex 2.6 and 2.8 (AChE, K i ¼ 1.08 ± 0.14 nM; BChE, K i ¼ 7.54 ± 0.96 nM), respectively.The second and third most potent compounds among the synthesized derivatives against AChE were derivatives 2.8 and 2.7 with Ki: 2.38 ± 0.28 and 2.81 ± 0.18 nM.Alzheimer's disease (AD) is a neurological illness that causes dementia and has recently become more common.Memory dysfunction has been described as the main symptom of this illness.The largest molecular alteration in AD is a decrease in acetylcholine levels in the brain (Taslimi & Gulc¸in, 2018).The condition is a form of dementia.Dementia disease; Memory, speaking ability, perception and judgment capacity, abstract thinking and problem-solving abilities are impaired as a result of the decline in social and learning functions.AD cannot be permanently cured.The current therapies work to get rid of the disease's symptoms.There is no cure that will get rid of it.Usually, AChE inhibitors like Donepezil and Rivastigmine are used for this.However, hepatotoxicity and gastrointestinal issues have reportedly been linked to these medications' negative effects (Bursal et al., 2019).As a result, natural AChE inhibitors that are secure and efficient have recently become more crucial.The majority of applications for reversible competitive and noncompetitive AChE inhibitors are therapeutic.Physostigmine, carbamates, neostigmine, pyridostigmine, demecarium, phenanthrene derivatives, galantamine, ambenonium, piperidines, caffeine, donepezil, tacrine (tetrahydroaminocridine, THA), rivastigmine, huperophonymine, edrophonium and lalactuungados are some examples of these inhibitors (Sujayev et al., 2016).In many different fields, AChE enzyme inhibitors are used.Here is a list of these areas.(a) Venom from both plants and animals naturally inhibits the AChE enzyme.(b) Insecticides contain them.(c) It is used medically to treat glaucoma, myasthenia gravis, anticholinergic poisoning, to reverse the effects of non-storing muscle relaxants, to treat neuropsychiatric symptoms of diseases like Alzheimer's, particularly unresponsiveness, to treat Lewy Body Dementia, to treat Parkinson's and Parkinson's disease (Ozgun et al., 2016).Similar to AChE inhibitors, buChE enzyme inhibitors have recently discovered significant applications in the treatment of AD.In this work, the second and third most potent compounds among the synthesized derivatives against BChE were derivatives 2.7 and 2.6 with Ki: 10.25 ± 1.71 and 15.33 ± 2.13 nM.Additionally, the weak inhibitors for both enzymes were 1.2, 1.3 and 1.4 compounds with Ki values of 12.07 ± 1.24, 8.50 ± 1.55 and 10.56 ± 1.22 nM against AChE, and 64.47 ± 8.61, 74.16 ± 7.53 and 87.45 ± 12.28 nM against BChE, respectively, in Figure 10.

Conclusion
In this study, the activities of 13 molecules obtained from 1,2,4-triazole derivatives (1.1-1.5 and 2.1-2.8)were compared against both chemical and biological materials.As a result of these comparisons, cell culture of molecules was studied first.In cell culture studies, the biological activity of molecular 2.1 with 58.36 ± 6.30 lM was found to have higher activity against prostate cancer.However, it was observed that molecule 2.8 had a higher activity with 8.71 nM against BChE enzyme and molecule 2.6 with 1.63 nM against AChE enzyme than other molecules.As a result of the theoretical calculations made, it has been seen that molecule 2.8 has higher activity than both theoretical and experimental results.In the theories made, at the B3LYP and M062X levels, molecules with the most negative E LUMO energy of the molecules are molecule 2.8.E LUMO energy value of molecule 2.8 is À 2.2112.However, the molecule of 2.8 has the most negative value at À 1.2681 at the M062x level.On the other hand, it is seen that 2.8 are among the lowest DE energy values of molecules at B3LYP and M062X levels.Theoretical calculations have been made to support the experimental studies.The results of these calculations were found to support the experimental results.In addition, ADME/T calculations have been made so that the molecules can be used as drug molecules in the future.When the results of these calculations were examined, it was seen that there was no harm in being a drug.It should be known that these studies will be an important guide for future in vivo studies.

Figure 3 .
Figure 3.The effects of compounds(1.1-1.5 and 2.1-2.8) on the viability of PC3 prostate cancer and PNT1a normal prostate cells.

Figure 4 .
Figure 4. Morphology of PC3 and PNT1a cells treated with the IC 50 doses of the compounds of 2.1, 2.2 and 2.3 for PC3 cells.Scale bars represent 200 lm.

Figure 5 .
Figure 5. HOMO, LUMO and ESP representations of all molecules.
Inc.); chemical shifts were reported in ppm (d scale) down field with residual protons of the solvent (DMSO-d 6 , d ¼ 2.49 ppm) as internal standard.
6 at 400 MHz by a Varian MR-400 spectrometer and analyzed with ADVASP TM Analyzer program (USA) (Umatek International
(Villa- Pulgarin et al., 2020)the length of the substituted alkyl chain might decrease the antitumor activity against cancer cells(Villa- Pulgarin et al., 2020).

Table 2 .
The calculated quantum chemical parameters of molecules.

Table 3 .
Numerical values of the docking parameters of molecule against enzymes.Docking score Glide ligand efficiency Glide hbond Glide evdw Glide ecoul Glide emodel Glide energy Glide einternal Glide posenum Table 5 lists the outcomes of the AMDE/T analysis performed on the compounds used in this study.The first parameters in this table look at a molecule's chemical makeup.

Table 4 .
MM-GBSA parameter of all molecules.

Table 6 .
The Tacrine values take from these references.