Design and Development of Multi-target Directed 1,2,3-rriazole-dimethylaminoacryloyl-chromenone Derivatives with Potential use in Alzheimer's Disease

To discover multifunctional agents for the treatment of Alzheimer's disease (AD), a new series of 1,2,3-triazole-chromenone derivatives were designed and synthesized based on the multi target-directed ligands approach. The in vitro biological activities were evaluated including acetylcholinesterase (AChE), butylcholinesterase (BuChE), and Aβ 1−42 aggregation inhibition as well as neuroprotective effects and metal-chelating properties. The results indicated highly selective BuChE inhibitory activity with IC 50 values of 21.71 µM for compound 10 h as the most potent compound. Besides, compound 10 h could inhibit self-induced Aβ 1−42 aggregation and AChE-induced Aβ aggregation with 32.6% and 29.4% inhibition value, respectively. A Lineweaver–Burk plot and molecular modeling study also showed that compound 10 h targeted both the catalytic active site (CAS) and peripheral anionic site (PAS) of BuChE. It should be noted that compound 10 h was potent as a selective Cu 2+ chelator. Thus, the designed scaffold could be considered as multifunctional agents for AD drug discovery developments. the potent BuChE The inhibition kinetic analysis revealed a mixed-type inhibition The molecular modeling study of for indicated that both and PAS signicant and aggregation capacity. as selective metal chelator chelating Cu 2+


Introduction
Dementia is one of the noteworthy problems in public health management as over 80% of dementia cases are suffering from Alzheimer's disease (AD). Currently, available therapies provide temporary symptomatic relief but do not target the distractive neuropathology. Therefore, a new treatment to delay or halt disease progression has remained an urgent medical need.
The pathophysiological processes of AD remain still unclear to this day. However, alongside of its complexity, several neurodegenerative process during AD could be identi ed including (I) aggregation of insoluble β-amyloid (Aβ) plaques mostly trigger from sequential cleavage of amyloid precursor protein (APP) by the aspartyl protease β-site APP cleaving enzyme-1 (BACE1) and γ-secretase, (II) neuro brillary tangles (NFTs) form through hyperphosphorylation of tau proteins, (III) biometals dysfunction, and (IV) oxidative stress which in return results in synapse loss and death of neuronal cells in the brain. Different hallmarks also have been recognized including the loss of cholinergic neurons, reduction of the neurotransmitter acetylcholine and increased expression of in ammatory mediators [1][2][3].
Based on the approved theory for AD, the loss of cholinergic neurons results in the reduction of the neurotransmitter acetylcholine (ACh). As a result, inhibition of the AChE raises the level of ACh and improves cognitive performance at the early stage of AD. The critical point is that AChE level decreases with the progression of AD, subsequently, AChE inhibition seems to be ineffective during the progression of AD [4,5]. Interestingly, the level of the butyrylcholinesterase (BuChE) remains unchanged or even increases at the late stage of disease [6]. BuChE can hydrolyze ACh and, thereby, compensate the reduction of AChE activity [7]. An experiment with AChE knockout mice supported this hypothesis [8].
Results from further studies were in accordance with the role of BuChE in AD brains and showed a positive correlation between selective BuChE inhibition and improved cognitive performance and memory [9,10].
Moreover, produced Aβ peptides can aggregate into Aβ plaques which initiate pathogenic cascade and lead to neuronal loss and dementia. Inhibition of the accumulation of Aβ peptide in the brain could be another therapeutic strategy against the development of AD [1]. Metal chelatory potential of compounds has been also demonstrated to exert bene cial effects via decreasing the plaque aggregation [11,12].

Design
Because of multifactorial and sporadic nature of AD, the modern approach "multi-target − one disease" could be effective to develop an effevtive drug to simultaneously acting at different targets. Selective BuChE inhibitors could be a promising target for the treatment of AD at the moderate and advanced stages of the disease [13]. Closer looks at X-ray crystallography of hBuChE depicted that it usually tolerates bigger scaffolds than hAChE as its active site of hBuChE is approximately 200 Å larger than hAChE. Analysis of the top potent hBuChE revealed that N-aromatic ring group is necessary for interactions with ChE active site ( Fig. 1, A) [14]; however, as appeared in compounds B and C increasing bulkiness and length of drug candidate could increase the selectivity of BuChE over hAChE. Besides, this part could also exhibit metal-chelating potential [15,16]. Moreover, introducing a relatively spacious di methylamino propenone entity into our system would be a good strategy to increase selectivity toward BuChE.
In the case of anti-Aβ plaques aggregation, it is important to keep the potential moiety in the structure to inhibit the aggregation of the peptide.
Coumarin structures as active natural compounds may simultaneously possess anti-oxidative [1,17], neuroprotective [18] anti-ChE [14] and anti-Aβ aggregative properties [19,20]. Coumarins pharmacophore owing to the presence of polar elements in the structure (Fig. 1, compound D and E) might help to inhibit amyloid bril formation through interaction with the polar surface of Aβ [21][22][23]. Hence, coumarins could serve as a rational framework for the prevention of Aβ 1−42 aggregation [24]. In addition to the inhibition of the BuChE and Aβ plaques aggregation, an inhibitor that can tackle toxicity of AB peptide, ROS and RNS could be effective for a longer period of AD progression. Recently, iminochromene was characterized as potent neuroprotective agents. The iminochromene groups of compound F were bioisosterically replaced with chromenone moieties to evaluate the possible neuroprotectivity [25].
Hence, in the present work, a molecular hybridization and bioisosteric replacement approach were used to design multi-target agents with BuChE and Aβ aggregation inhibition, neuroprotective and metal chelating properties.

AChE And BuChE Inhibitory Activity
In vitro anti-AChE and anti-BuChE activity of synthesized compounds, 10a-m were performed based on the modi ed Ellman's method [28] comparing with donepezil as the reference drug.
Compounds were initially screened in vitro against AChE, and none of them exhibited inhibitory properties against the AChE enzymes. Interestingly, all the 1,2,3-triazolechromenone derivatives showed remarkable selectivity and potency towards BuChE, which exerted a more prominent role at later stages of the disease [29]. As can be seen in Table 1, the inhibitory activity directly depended on the electronic property of substituents and their positions on the benzyl moiety connected to 1,2,3-triazole ring.
Results showed that compound 10 h possessing 3,4-diF on the aryl ring induced the best BuChEI activity (IC 50 = 21.71 µM); however, the elimination of 3-F of the substituents completely changed the activity in such a manner that compound 10 g did not show any potency. meta-Fluorinated derivative 10f was found to be a moderate inhibitor as calculated IC 50 was 59.58 µM and parauorinated derivative 10 g showed no inhibitory activity toward BuChE (IC 50 > 100 µM).
Considering the inhibitory activity of other halogenated derivatives 10i-m depicted that chlorinated compounds 10i and 10j showed no activity (IC 50 > 100 µM). In the case of brominated derivatives 10 k-m, compounds 10 l possessing Br at 3-position of aryl ring showed moderate activity with IC 50 = 65.96 µM. Finally, the absence of substituent on the aryl ring (compound 10a) also depicted relatively good activity (IC 50 = 34.41 µM).
The in vitro anti-cholinesterase assay showed that the unsubstituted benzyl derivative (10a) along with the metasubstituted analogues (10d, 10f, and 10 l) had signi cant anti-BuChE activity. Adding extra small-size halogen atoms such as uorine (compound 10 h) resulted in the most potent activity with an IC 50 value of 21.71 µM.

Kinetic Study Of Buche Inhibition
The kinetic study was performed to investigate the mechanism of inhibition by compound 10 h against BuChE. Graphical analysis of the reciprocal Lineweaver-Burk plot of compound 10 h described a mixedtype inhibition pattern (Fig. 2, A.) in which compound 10 h may bound to the BuChE whether it already bound to the substrate. In addition, the Ki value was calculated using the secondary plot as 28.2 µM (Fig. 2, B.).

Inhibition of AChE-induced and self-induced Aβ aggregation
Aβ peptide is the major constituent of senile plaques in the brains of patients with AD. In this respect, the effect of the most potent compound 10 h was assessed for the inhibition against Aβ 1−42 aggregations and AChE-induced Aβ 1−40 peptide aggregation using the Thio avin T (ThT) assay. Comparing with donepezil and tacrine as the reference compounds, 10 h was more potent than both controls in inhibiting Aβ 1−42 self-aggregation, as depicted 32.6% values at 10 µM (Table 2.). Furthermore, compound 10 h inhibited AChE-induced Aβ aggregation by 29.4% at 100 µM.

Metal Chelating
Compound 10 h was tested for its metal chelating ability towards Fe 2+ , Cu 2+ , and Zn 2+ ions (Fig. 3.) The UV spectrum of methanolic solution (20 µM) of that compound showed two characteristic absorption peaks at 309.9 and 386.7 nm. After the interaction of compound 10 h with the abovementioned ions for 30 min, red and blue shifts observed in the spectra con rmed desired interactions of that compound with biometals. Interaction of compound 10 h with Zn 2+ ions demonstrated two absorption peaks at 299.2 and 388.7 nm which demonstrated red and blue shifts, respectively. Similar changes were observed in the case of Fe 2+ ions and those absorptions were observed at 303.5 and 390.9 nm. Also, another absorption peak was obtained at 209.6 nm. When compound 10 h was treated with Cu 2+ ions, blue shifts were observed at 303.5 and 382.4 nm and a peak at 205.3 was also observed.
The stoichiometry of complex 10 h-Cu 2+ was also studied (Fig. 4.). The concentration of the test compound 10 h was 20 µM and the nal concentration of Cu 2+ ranged from 0-20 µM with 4 µM intervals at 205.3 nm. The plot was obtained by the corresponding absorption against the mole fraction of Cu 2+ to ligand 10 h. According to the plot, the ratio 1:1 complexation ration of 10 h-Cu 2+ can be seen at the fracture point of the plot with the mole fraction of 0.6.

Docking Study Of Buche
The volume of the BuChE active site gorge is considerably higher than the one found in AChE, so BuChE can accommodate bulkier inhibitors, and this may constitute the basis for the selectivity of these derivatives. An overlay of the best pose for 10 h with BuChE was depicted in Fig. 5, chromenenone core demonstrated π-π stacking interactions with Trp82, Gly117, and Phe329.
Carbonyl group of the mentioned ring allowed the oxygen to form a hydrogen bond with the hydroxyl on Ser198 of catalytic triads at 2.27 Å while 1,2,3-triazole ring formed a second hydrogen bond to Trp82 of anionic subside. The nitrogen of the dimethylamino acryloyl chromenone interacted with Pro285 via van der Waals interactions. Further π-π stacking interaction was constructed between 1,2,3-triazole moiety and Phe329 and Met437 with the PAS residue. The parauoro benzyl ring also showed π -aryl interaction with Ala328.

In silico ADME evaluation
The synthesized compounds were further assessed in terms of physiochemical parameters and pharmacokinetic properties using http://lmmd.ecust.edu.cn/admetsar2/ and http://preadmet.bmdrc.kr).  Table 3. most of compounds showed drug-like characteristics based on Lipinski's rule of ve (MW < 500, C-log P < 5, HB donor ≤ 5, HB acceptor ≤ 10). Our results indicated that lipophilicity and solubility of the derivatives were drug-like. Furthermore molecular weight, C-logP, and blood-brain barrier were well within the standard ranges.

Conclusion
In summary, a series of 1,2,3-triazole-dimethylaminoacryloyl-chromenone derivatives were designed and synthesized as multifunctional anti-Alzheimer's agents. All the target compounds were synthesized and screened as AChE/BuChE inhibition. The most active compound in this series was further evaluated by multiple biological activities including, Aβ Synthesis of 2-hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde (3) Compound 3 was prepared from the reaction of 2,4-dihydroxybenzaldehyde 1 and propargyl bromide 2 in the presence of potassium carbonate (K 2 CO 3 ) and potassium iodide (KI) in acetone at 50 ºC, according to the literature [26].
Synthesis of 3-acetyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (5) A few drops of piperidine was added to the mixture of compound 3 (1 mmol) and ethyl acetoacetate (2.5 mmol) 4 in ethanol (10 mL) and it was stirred overnight at room temperature to obtain yellow precipitates. After completion of reaction (checked by TLC), they were ltered off and used for the next step with no further puri cation.
Synthesis of 1,2,3-triazole-dimethylaminoacryloyl-chromenone hybrids 10a-m The nal step was performed by the click reaction of compound 7 and is suite prepared azides 9. For this purpose, a solution of benzyl chloride/bromide derivative 8 (1.1 mmol), sodium azide (0.06 g, 0.9 mmol), and trimethylamine (0.13 g, 1.3 mmol) in water (4 mL) and tert-butyl alcohol (4 mL) was stirred at room temperature for 30 min. Then, compound 7 (0.5 mmol) and CuSO 4 .5H 2 O (7 mol%) were added to the mixture and it was continued for 24 h. Upon completion of the reaction checked TLC), the mixture was diluted with water, extracted with chloroform, and dried over anhydrous Na 2 SO 4 . After evaporation of solvent, the residue was recrystallized from ethyl acetate and petroleum ether to give pure product 10. In the case of some compounds, they were puri ed using plate chromatography with ethyl acetate as eluent.

Inhibitory activities against AChE and BuChE
All enzymes and reagent requited for the assay was obtained from Aldrich. The in vitro anticholinesterase activity of all synthesized compounds 10a-m was assayed using modi ed Ellman's method using a 96well plate reader (BioTek ELx808) according to the literature [28,30]

Metal chelation studies
To study the metal chelating ability, the solutions of compound 10h and Fe 2+ , Cu 2+ , and Zn 2+ ions (from FeSO 4 , CuCl 2 , and ZnCl 2 ) were prepared in methanol. The mixture of compound 10h (1 mL) and the test ion solutions (1 mL) with the same nal concentration of 20 µM in a 1 cm quartz cuvette was incubated at room temperature for 30 min. Then, the absorption spectra were recorded with wavelength ranging from 200-600 nm. The stoichiometry of complex 10h-Cu 2+ was also studied using the molar ratio method [11,34]. The concentration of compound 10h was 20 μM and the nal concentration of Cu 2+ ranged from 0-20 μM with 4 μM intervals at 205.3 nm. The plot was obtained by the corresponding absorption versus mole fraction of Cu 2+ to ligand 10h.

Molecular docking
The molecular docking studies of the most potential ligand was performed on BuChE (PDB code: 4BDS) to observe the binding orientation and consensual binding interactions using AutoDock 4.2. The X-ray crystal structure of receptor was downloaded from the PDB database. All water and ligand molecules were removed from the structure, and the protein was prepared for docking. The co-crystallized ligand within the pdb structures was de ned as a center of the binding site. All ligands were created using Chem3D Ultra software, and energy minimizations were done by the semiempirical MM + [35]. The compounds were docked into the active site of proteins using default parameters for each ligand with 100 runs and 27,000 as maximum number of generations. The grid boxes were set with 60, 60 and 60 points in the x, y and z directions, respectively. All other options were set as default. The calculated geometries were ranked in terms of free energy of binding and the best pose was selected for further analysis. Molecular visualizations were performed by Discovery Studio 4.0 client software [14].

Figure 1
Designed hybrids used to investigate as selective BuChE with Aβ aggregation inhibition, neuroprotective and metal chelating properties.   Determination of the stoichiometry of complex 10h-Cu+2 using molar ratio method.