Click-designed vanilloid-triazole conjugates as dual inhibitors of AChE and Aβ aggregation

Based on their reported neuroprotective properties, vanilloids provide a good starting point for the synthesis of anti-Alzheimer's disease (AD) agents. In this context, nine new 1,2,3-triazole conjugates of vanilloids were synthesized via click chemistry. The compounds were tested for their effect on acetylcholine esterase (AChE) and amyloid-beta peptide (Aβ) aggregation. The triazole esters (E)-(1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl 3-(4-hydroxy-3 methoxyphenyl)acrylate 9 and (1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl-4-hydroxy-3-methoxybenzoate 8 displayed dual inhibitory activity for AChE and Aβ aggregation with IC50 values of 0.47/0.31 μM and 1.2/0.95 μM, respectively, as compared to donepezil (0.27 μM) and tacrine (0.41 μM), respectively. The results showed that the triazole ester moiety is more favorable for the activity than the triazole ether moiety. This could be attributed to the longer length of the spacer between the two vanillyl moieties in the triazole esters. Furthermore, the binding affinities and modes of the triazole esters 9 and 8 were examined against AChE and Aβ utilizing a combination of docking predictions and molecular dynamics (MD) simulations. Docking computations revealed promising binding affinity of triazole esters 9 and 8 as potential AChE, Aβ40, and Aβ42 inhibitors with docking scores of −10.4 and −9.4 kcal mol−1, −5.8 and −4.7 kcal mol−1, and −3.3 and −2.9 kcal mol−1, respectively. The stability and binding energies of triazole esters 9 and 8 complexed with AChE, Aβ40, and Aβ42 were measured and compared to donepezil and tacrine over 100 ns MD simulations. According to the estimated binding energies, compounds 9 and 8 displayed good binding affinities with AChE, Aβ42, and Aβ40 with average ΔGbinding values of −32.9 and −31.8 kcal mol−1, −12.0 and −10.5 kcal mol−1, and −20.4 and −16.6 kcal mol−1, respectively. Post-MD analyses demonstrated high steadiness for compounds 9 and 8 with AChE and Aβ during the 100 ns MD course. This work suggests the triazole conjugate of vanilloids as a promising skeleton for developing multi-target potential AD therapeutics.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most prevalent cause of dementia. 1 It affects more than 40 million people worldwide. 2 Even though the pathophysiology of AD has not been completely elucidated, cholinergic deciency and amyloid-b peptide (Ab) deposition are widely accepted as important features of the pathophysiology of AD, 3 the pathological aggregates of Ab deposits being known as senile plaques. 4 Acetylcholine esterase (AChE) has been reported to consistently co-localize with Ab deposits and induce their assembly by forming a complex with the growing brils. 4 The enzyme AChE is involved in the hydrolysis of the neurotransmitter acetylcholine (ACh). The profound loss of forebrain cholinergic neurons during the progression of AD, results in a progressive decline in acetylcholine. Current therapies are mostly based on AChE inhibitors (AChEI) to reverse the cholinergic decit. 5 Dual targeted inhibitors of AChE and Ab aggregation are the main focus of AD paradigm. 6 These drugs can be synthesized or harvested from nature, the advantage of the latter being the potential for chemical diversity, biological selectivity and favorable properties. Natural products and their derivatives represent more than 50% of the market pharmaceutics. 7 Vanilloids are a group of natural products that are characterized by the presence of a vanillyl group. They include vanillin, vanillic acid, eugenol, capsaicin etc. Vanilloids attracted the attention of the authors because of their reported neuroprotective properties. Vanillin is reported to inhibit both Ab aggregation and AChE and its profound antioxidant activity in neuroblastoma cells. 8 Eugenol, 9 vanillic acid, 10 ferulic acid, 11

Chemistry
Based on the reported anti-Alzheimer properties of both of vanilloids and triazole-based compounds, the authors set a rationale for synthesis of triazole-conjugates of vanilloids via click chemistry. Nine new 1,2,3-triazole-conjugates of vanilloids ( Fig. 1) were prepared via click chemistry (Scheme 1).
For azide, vanillyl alcohol were purchased from Sigma-Aldrich. For alkynes, ferulic acid, and vanillin were purchased from Sigma-Aldrich. Coniferaldehyde and vanillic acid were previously isolated from Cocos nucifera L. 26 Eugenol was extracted and puried from clove oil using 30% aqueous KOH, according to the literature. 27 Vanillyl alcohol was converted to vanillyl azide and reacted with different alkynes. The monoalkynes were prepared from natural vanilloids, as will be described in the experimental part.
Compounds 1-3 were obtained via click reaction of vanillyl azide with the respective mono-alkyne ether. Compounds 4 and 6 were obtained via click reaction of vanillyl azide with the respective mono-alkyne ether aer masking the carboxylic moiety with an ester. Compounds 5 and 7 were obtained from the ester hydrolysis of compounds 4 and 6, respectively. Compounds 8 and 9 were obtained via click reaction of vanillyl azide with the mono-alkyne ester aer masking the hydroxyl group.
Compound 5 was obtained from the ester hydrolysis of compound 4, as white amorphous powder, yielding 95.2%. Compounds 5 and its corresponding ethyl ester 4 showed the same NMR data except for the presence of the characteristic signals of the ethyl moiety at d H 4.27 (2H, q) and 1.3 (2H, t). For the APT spectrum, the free carboxylic group in compound 5 resonated at a higher eld at d C 167.1 compared to the corresponding ethyl ester 4, where it resonated at d C 165.5. The same pattern was observed for compound 7 and its corresponding ethyl ester 6. Compound 5 was identied as 4-((1-(3-methoxy-4hydroxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-3methoxybenzoic acid.
The isomeric compounds 5 and 8 were assigned to the molecular formula C 19 H 19 N 3 O 6 based on the molecular ion peak at 408.1170 and 408.1178, respectively. The triazole ester 8 showed a distinct upeld shi in C-6 ′ (d C 57.7) and C-7 ′′ (d C The rest of compounds are recorded in DMSO-d 6 . 166.3) as compared to the triazole ether 5 (d C 61.6) and (d C 167.1). Also, the chemical shi of the vanillic acid moiety was slightly different between the two compounds. The same pattern was observed for the isomeric compounds 7 and 9, where C-6 ′ and C-7 ′′ resonated at d C 61.6/57.0 and 168.0/166.4, respectively. It was named (1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-hydroxy-3-methoxy benzoate. Similarly, compounds 6 and 9 were obtained via click reaction of vanillyl azide with the mono-alkyne ether of ferulic acid ester and the mono-alkyne ester of acetyl ferulic acid, respectively, as a white powder, yielding 56.8%; and white powder yielded 68.0%.
Compound 6 was assigned to the molecular formula C 23 Compound 7 was obtained from the ester hydrolysis of compound 6, as white amorphous powder, yielding 43.4%. Both compounds displayed the same NMR data except for the presence of the characteristic signals of the ethyl moiety at d H 4.17 (2H, q), 1.2 (2H, t), and d C 59.9, 14.0. For the APT spectrum, the free carboxylic group in compound 7 resonated at a higher eld at d C 168.0 as compared to the corresponding ethyl ester 6, where it resonated at d C 166.6. It was named as 4- The isomeric compounds 7 and 9 were assigned to the molecular formula C 21 H 21 N 3 O 6 based on the molecular ion peak at 410.1438 and 410.1361 (calc. 410.1352), respectively. The triazole ester 9 showed a distinct upeld shi in C-6 ′ (d C 57.0) and C-9 ′ (d C 166.4) as compared to the triazole ether 7 (d C 61.6) and C-9 ′′ (d C 168.0). It was named as (E)-(1-(4-hydroxy-3methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl 3-(4-hydroxy-3 methoxyphenyl)acrylate.

Biological evaluation
The semi-synthetic compounds (1-9) and curcumin were evaluated for their inhibitory effect on acetylcholine esterase and bamyloid aggregation Table 3. Curcumin was chosen because it is a natural vanilloid analogue with reported AChE and Ab aggregation inhibition activity.
Compound 9 was the most active, showing an IC 50 value of 0.47 ± 0.02 mM, which is about two times that of the standard donepezil, the IC 50 value of 0.27 ± 0.01 mM. It is worth noting that its isomeric compound 7 was much less active, the IC 50 value of 10.55 ± 0.53 mM. This may suggest that the triazole ester moiety is more favorable to the activity than the triazole ether moiety. This may be also conrmed by observing the IC 50 values of the isomeric compounds 5 and 8; the triazole ester 8 displayed about half of the IC 50 value of its isomeric triazole ether 5, IC 50 value 1.2 ± 0.06 and 2.11 ± 0.11 mM, respectively.
Compound 4 showed nearly similar activity to compound 5, suggesting that a free or conjugated carboxylic group may have not impact on the activity. However, this pattern was not observed for 7 and its ethyl ester 6 compound 4 showed nearly similar activity to compound 5, suggesting that a free or conjugated carboxylic group may have no impact on the activity. However, this pattern was not observed for 7 and its ethyl ester For the amyloid-b aggregation assay, compound 9 was more active than tacrine; their IC 50 values were 0.31 ± 0.02 and 0.41 ± 0.02 mM, respectively. Compound 8 was next in activity with the IC 50 values of 0.95 ± 0.05 mM. It is worth noting that the triazole esters 9 and 8 were much more active than their isomeric triazole ethers 7 and 5, respectively. This may suggest that the triazole ester moiety is more favorable to the activity. Compound 5 and its ethyl ester 4 also showed comparable IC 50 values of 1.48 ± 0.07 and 1.59 ± 0.08 mM, respectively. For compound 7 and its ethyl ester 6, they showed nearly similar IC 50 values of 4.48 ± 0.22 and 3.06 ± 0.15 mM, respectively. Next in activity to compounds 5 and 4 was compound 3, showing the IC 50 value of 2.86 ± 0.14 mM. All compounds except for compound 2 were more active than curcumin, the IC 50 value of 22.74 ± 1.14 and 13.39 ± 0.67 mM, respectively. The hybrid containing the eugenol moiety was the least active.
From the above results, it can be concluded that compounds 9 and 8 could act as dual inhibitors for AChE and Ab aggregation with IC 50 values 0.47/1.2 and 0.31/0.95 mM, respectively. Their promising activity over compounds 1-7, could be attributed to the longer length of the spacer between the two vanillyl moieties. Therefore, they hold a particular interest in developing new anti-Alzheimer drugs. The skeleton of 9 and 8 may offer some structural features for the development of dual inhibitors. Hence, they should be subjected to further investigation for designing novel anti-Alzheimer drugs. The results provides a preliminary idea about the anti-AD potential of vanilloid-triazole conjugates. However, a further extensive study is required to investigate their activity in vitro and in vivo, including the morphology of the Ab-oligomers.

Molecular docking
The docking scores and poses of triazole esters 9 and 8 with AChE and Ab40/42 were predicted using AutoDock4.2.6 soware. The protended binding features and docking scores are shown in Fig. 2. As depicted in Fig. 2, triazole esters 9 and 8 unveiled good docking scores towards AChE and Ab40 with values of −10.4 and −5.8 kcal mol −1 and −9.4 and −4.7 kcal mol −1 , respectively. Triazole esters 9 and 8 were also investigated against the Ab42 protein. For Ab42, the docking scores were not promising compared to those against Ab40, with values of −3.3 and −2.9 kcal mol −1 of compounds 9 and 8, respectively (Fig. 2). The good potentiality of compounds 9 and 8 may be imputed to their ability to form a variation of H-bonds, p-based, and other interactions with the key residues within the binding sites of AChE and Ab40. More precisely, compound 9 demonstrated two hydrogen bonds with THR83 (2.27Å) and ARG296 (1.86Å) inside the binding site of AChE (Fig. 2). For Ab40, compound 9 formed three hydrogen bonds with ASP1 (2.07, 2.80Å) and LYS16 (1.76Å). Compound 8 exhibited ve hydrogen bonds with ASP1 (2.13Å), GLU3 (3.06Å), ASP7 (1.94, 2.14Å), and GLY9 (2.84Å) (Fig. 2). Although compound 8 could not form any hydrogen bond within the binding site of AChE, other noncovalent interactions were noticed, involving p-p stacking interactions with PHE297, TYR337, TYR341, and TRP286 (Fig. 2). Compared with compounds 9 and 8, donepezil displayed a similar docking score towards AChE with a value of −11.1 kcal mol −1 , forming only one hydrogen bond with PHE295 and p-p stacking interactions with TRP86 and TRP286 (Fig. 2). On the other hand, tacrine exhibited one and two hydrogen bonds with GLN15 (1.85Å) and ASP1 (2.08Å) and GLY29 (1.92Å) with the Ab42 and Ab40, respectively (Fig. 3).

Molecular dynamics simulations
MD simulations were utilized to puzzle out the stabilization of the ligand-target complex, structural specics, conformational elasticities, and the trustworthiness of ligand-target binding energy. 31,32 Consequently, the inspected triazole esters 9 and 8 in complex with AChE and Ab40/42 were submitted to MD simulations over 100 ns, followed by binding affinity evaluations. The estimated MM-GBSA binding affinities over 100 ns MD simulations are depicted in Fig. 3. As illustrated in Fig. 3, compounds 9 and 8 complexed with AChE exposed competitive binding affinities with an average DG binding values of −32.9 and −31.8 kcal mol −1 , respectively, compared to donepezil in complex with AChE (calc. −35.2 kcal mol −1 ). However,  (Fig. 2). A comparison of DG binding values of compounds 9 and 8 complexed with Ab42 and those with Ab40 revealed the higher potency of compounds 9 and 8 with Ab40 over Ab42 with DG binding values of −20.4 and −16.6 kcal mol −1 , respectively (Fig. 3). The calculated MM-GBSA binding energies were in line with the IC 50 values.
To determine the most crucial interactions between ligand and target, binding affinities of the studied ligands in complex with AChE and Ab40/42 were decomposed and illustrated in Fig. 3. As shown in Fig. 3, the binding energies of compounds 9 and 8 and donepezil complexed with AChE were dominated by E vdw interactions with values of −51.8, −46.7, and −49.5 kcal mol −1 , respectively. E ele interactions were appropriate with values of −20.9, −30.6, and −14.8 kcal mol −1 for compounds 9 and 8 and donepezil complexed with AChE, respectively (Fig. 3).

Post-MD analyses
To conrm the steadiness of compounds 9 and 8 in complex with AChE and Ab40/42, the complexes were investigated structurally and energetically during a period of 100 ns MD, and the results were compared to those of controls (i.e., donepezil and tacrine).
2.5.1. Binding affinity analysis. Gauging the correlation between binding affinity per trajectory and time was used to investigate the comprehensive structural steadiness of compounds 9 and 8 complexed with AChE and Ab40/42 over the 100 ns MD simulations. Overall stability for compounds 9 and 8 was observed with AChE, Ab42, and Ab40 with average DG binding values of −32.9 and −31.8 kcal mol −1 , −12.0 and −10.5 kcal mol −1 , and −20.4 and −16.6 kcal mol −1 , respectively (Fig. 4). Comparing DG binding values of 9 and 8 with donepezil and tacrine, it can be seen that these compounds are competitive inhibitors compared to controls (Fig. 4). According to binding affinity analysis, all inspected systems preserved stabilization over the 100 ns MD.
2.5.2. Root-mean-square deviation. The RMSD of the backbone atoms of the whole system was inspected to check the conformational stability of 9, 8, and controls complexed with AChE and Ab40/42 (Fig. 5). As depicted in Fig. 5, the estimated RMSD values for the investigated complexes continued below 0.3 nm during a period of 100 ns MD. The inspected complexes achieved the stabilization state in the rst 15 ns MD and preserved their steady till the termination of the simulation. The RMSD data proved that compounds 9 and 8 are tightly bound and do not impact the global topology of AChE and Ab40/42.
2.5.3. Root-mean-square uctuation. To inspect the conformational change and steadiness of the backbone of the apo AChE/Ab, compounds 9, 8, and controls complexed with AChE and Ab40/42, the root-mean-square uctuation (RMSF) of alpha carbon was measured and depicted in Fig. 6. As illustrated in Fig. 6, the amino acids were found stable in compounds 9, 8, and controls complexed with AChE and Ab40/ 42 during the 100 ns MD course.

General experimental procedures
The progress of reactions and the purity of nal products were monitored by thin layer chromatography (TLC), carried out using Merck precoated silica gel F254 plates (E-Merck, Germany) and using vanillin-sulfuric acid spray reagent. Column chromatography was carried out using silica gel G 60-230 (Merck, Germany). The solvents used included n-hexane, methylene chloride (CH 2 Cl 2 ), and ethyl acetate (EtOAc) used were of reagent grade (El-Nasr Co., Abu Zaabal -Kalyoubia, Cairo, Egypt). 1 H and APT spectra were measured in CDCl 3 and DMSO-d 6 using Bruker Avance III HD-400 spectrometer at 400 MHz for 1 H and 100 MHz for APT in NMR unit, Faculty of Pharmacy, Mansoura, Egypt. Chemical shis (d) are expressed in ppm with reference to the residual solvent signal. Coupling constants (J values) are given in Hz. Melting points were determined on Stuart® melting point apparatus model SMP10 and are uncorrected. High-resolution mass (HR-ESI-MS) was measured using a Bruker microTOF mass spectrometer (Shimadzu, Tokyo, Japan). IR spectra were obtained using a Thermo Scientic Nicolet™ iS™ 10 FT-IR spectrometer instrument.

Chemicals
Propargyl bromide, propargyl alcohol, vanillyl alcohol, ferulic acid, clove oil, sodium azide, N,N ′ -dicyclohexylcarbodiimide solution (DCC) and 4-(dimethylamino)pyridine (DMAP) and were purchased from Sigma-Aldrich St. Louis, USA. Vanillin (100% purity) was purchased from Eternal Pearl (The Zhonghua Chemical Factory, Zhejiang, China). Coniferaldehyde and vanillic acid were previously isolated from Cocos nucifera L., as reported. 26 Eugenol was extracted and puried from clove oil using 30% aqueous KOH, according to the literature. 27 3.2.1. General procedure for preparation of vanillyl azide. Vanillyl alcohol (32 mmol) was dissolved in dry CH 2 Cl 2 containing catalytic drops of DMF, then thionyl chloride (SOCl 2 ) (96 mmol) was added dropwise. The reaction was reuxed and monitored by TLC till the complete reaction. 33,34 The solvent was evaporated under reduced pressure to give a brown amorphous residue. The oily residue was used as such without any further purication.
Vanillyl chloride (24.15 mmol) was dissolved in 5 ml DMSO, then sodium azide (48.3 mmol) was added. The reaction was stirred at room temperature and monitored by TLC. Aer completion, the reaction was quenched with water. The crude was extracted with ethyl acetate, dried over anhydrous sodium sulphate and concentrated under a vacuum. 35 The azide was puried by silica gel column chromatography (2 cm × 28 cm, 35 g) using a gradient elution of ethyl acetate in hexane. The effluents, 50 ml each were collected, concentrated, and screened by TLC. Fractions with the same chromatographic pattern were pooled together. Fractions (7)(8)(9)(10)(11)(12)(13)(14)(15), eluted with 5-7% ethyl acetate in hexane, afforded vanillyl azide as a pure compound.
3.2.2. Preparation of alkynes. Five natural vanilloid precursors were used for the preparation of the seven readily clickable propargyl ethers and propargyl esters. Vanillin, eugenol and coniferaldehyde were converted for their respective propargyl ethers using propargyl bromide. Vanillic and ferulic acid were converted to respective propargyl ethers and esters using propargyl bromide and propargyl alcohol, respectively.
3.2.2.1. General procedure for preparation of alkynes by propargylic etherication. The start vanilloid was stirred with K 2 CO 3 (3.5 equivalent) in dry acetone at room temperature for 2 hours. Propargyl bromide (1.3 equivalents) was added to the mixture was stirred under reux at 80°C for 10 h. 36 Aer the completion of the reaction conrmed by TLC, the reaction was stopped by the addition of water. The solid salts were separated by ltration, and the product was extracted with ethyl acetate (3 × 50 ml). The ethyl acetate was concentrated under reduced pressure to give corresponding alkynes.
Acetyl vanillic acid (360 mg, 1.7 mmol) or acetyl ferulic acid (415 mg, 1.75 mmol) and propargyl alcohol (1.7 mmol) were dissolved in dry CH 2 Cl 2 . To the stirred mixture, DCC (3.4 mmol) and DMAP (0.17 mmol) were added dropwise. The reaction mixture was monitored by TLC for completion. Aer 24 h stirring at room temperature, the reaction mixture was ltered over silica and the solution was washed with CH 2 Cl 2 and concentrated under reduced pressure.
3.2.3. General procedure for preparation of triazole derivatives by click reaction. Equimolar amounts of the alkynes (1-7a) and vanillyl azide were dissolved in 2 ml CH 2 Cl 2 : H 2 O (1 : 1). To this mixture, CuSO 4 $5H 2 O (0.15 eq.) and ascorbic acid (0.45 eq.) were added and stirred at room temperature. The reaction was monitored by TLC till completion. The reaction was quenched with distilled water and extracted with ethyl acetate, dried over anhydrous Na 2 SO 4 and concentrated under a vacuum. 39 The product was puried by silica gel column chromatography using gradient elution of ethyl acetate in hexane as to give 1,2,3-triazole derivatives.
3.2.4. Hydrolysis of triazole esters into the corresponding alcohol. The triazole esters 4 and 5 were dissolved in 1 M aqueous NaOH and stirred at room temperature. Aer reaction completion, 1 N HCl was added dropwise till neutral pH. The product was extracted using ethyl acetate. 42)) were obtained and utilized as templates for all in silico computations. For the AChE and Ab40/42 preparation, all crystallographic water molecules, ligands, ions, and heteroatoms were eliminated. The empirical program PropKa was utilized to determine the protonation state of titratable residues of the investigated targets. 43 3.4.2. Ligand preparation. The chemical structures of triazole esters 9 and 8 were manually created. Omega2 soware was applied to convert two-dimensional formats into threedimensional structures. 44,45 The compounds were then minimized using an MMFF94S force eld within SZYBKI soware. 46,47 The Gasteiger-Marsili method was used to assign the atomic charges of these compounds. 48 3.4.3. Molecular docking. All docking predictions were executed using AutoDock4.2.6 soware. 49 A maximum of 25 000 000 energy evaluations and 250 independent runs were employed for docking computations. The remaining docking parameters were kept at the default settings. The AutoGrid program was employed to construct the grid maps. A grid box with dimensions 50Å × 50Å × 50Å (x, y, z directions) was set around the binding pocket of AChE. The grid spacing value of 0.375Å was utilized. The grid of AChE was positioned at the coordinates X = 11.367, Y = −56.25, and Z = −22.605. For Ab40/ 42 targets, the AutoDock-based blind docking strategy was utilized in the current study.
3.4.4. MD simulations. Molecular dynamics (MD) simulations were conducted using AMBER16 soware for the studied ligands in complex with AChE and Ab40/42 at a time scale of 100 ns. 50 The details of the employed MD simulations are described elsewhere. [51][52][53] Briey, the parameters of the investigated ligands were generated utilizing the general AMBER force eld (GAFF2). 54 AMBER force eld 14SB was employed for AChE and Ab40/42 parametrization. 55 The geometry optimization was executed at the HF/6-31G* level of theory using Gaussian 09 soware. 56 Thereaer, the restrained electrostatic potential (RESP) approach was utilized to compute the atomic charges of the investigated compounds. 57 An octahedron box with a distance of 1.2 nm was used to solvate the inspected target-inhibitor complexes using the TIP3P water model. The inspected complexes were neutralized using Na + and Cl − ions. 58 The solvated complexes were minimized for 5000 cycles. The minimized complexes were smoothly heated up to 300 K during a period of 50 ps. Subsequently, equilibration of the systems was performed for 10 ns, followed by a production stage of 100 ns. All MD simulations were conducted with the pmemd.cuda module implemented within AMBER16 soware on the CompChem GPU/CPU cluster (https:// hpc.compchem.net). The BIOVIA Discovery Studio Visualizer 2020 was employed to depict the molecular interactions. 59 3.4.5. Binding energy estimation. The molecular mechanical/generalized born surface area (MM/GBSA) approach was utilized to compute the binding energies of the inspected ligands in complex with AChE and Ab40/42. 60 Uncorrelated trajectories were gathered every 10 ps during the production run for the MM/GBSA computations. The binding energy (DG binding ) was evaluated using the following equation:

Conclusion
Based on their reported neuroprotective properties, the authors have selected the vanilloid pharmacophore to design potential anti-AD drugs. Nine new vanilloids hybrids (1-9) were semisynthetized via click reactions of vanillyl azide and several vanilloid monoalkynes. Compounds 9 and 8 showed remarkable ACE and Ab aggregation inhibition. The results suggested that the triazole ester moiety may be favorable for the activity over the triazole ether moiety. The results showed that compounds 9 and 8 are promising dual AChE/Ab aggregation inhibitors. They may serve as potential leads for designing novel anti-Alzheimer agents.

Conflicts of interest
There are no conicts to declare.