key: cord-0030837-kvzh6zl8
authors: Rana, Monika; Pareek, Abhishek; Bhardwaj, Shivani; Arya, Geeta; Nimesh, Surendra; Arya, Hemant; Bhatt, Tarun K.; Yaragorla, Srinivasarao; Sharma, Anuj K.
title: Aryldiazoquinoline based multifunctional small molecules for modulating Aβ(42) aggregation and cholinesterase activity related to Alzheimer's disease
date: 2020-08-04
journal: nan
DOI: 10.1039/d0ra05172a
sha: ff8bd40242697709c07a4ee1a80017bbc4e9c7d4
doc_id: 30837
cord_uid: kvzh6zl8

Research continues to find a breakthrough for the treatment of Alzheimer's Disease (AD) due to its complicated pathology. Presented herein is a novel series of arydiazoquinoline molecules investigated for their multifunctional properties against the factors contributing to Alzheimer's disease (AD). The inhibitory properties of fourteen closely related aryldiazoquinoline derivatives have been evaluated for their inhibitory effect on Aβ(42) peptide aggregation. Most of these molecules inhibited Aβ(42) fibrillation by 50–80%. Selected molecules were also investigated for their binding behaviour to preformed Aβ(40) aggregates indicating a nanomolar affinity. In addition, these compounds were further investigated as cholinesterase inhibitors. Interestingly, some of the compounds turned out to be moderate in vitro inhibitors for AChE activity with IC(50) values in low micro molar range. The highest anti-AChE activity was shown by compound labelled as 2a with an IC(50) value of 6.2 μM followed by 2b with IC(50) value of 7.0 μM. In order to understand the inhibitory effect, binding of selected molecules to AChE enzyme was studied using molecular docking. In addition, cell toxicity studies using Neuro2a cells were performed to assess their effect on neuronal cell viability which suggests that these molecules possess a non-toxic molecular framework. Overall, the study identifies a family of molecules that show good in vitro anti-Aβ-aggregation properties and moderately inhibit cholinesterase activity.

Alzheimer's disease (AD), the most common cause of dementia, affects around 50 million people worldwide. Its prevalence is expected to double every 20 years, rising to more than 130 million by 2050. Impact in countries varies with the proportion of elderly individuals in the population. 1 In the absence of an effective treatment for this, AD remains one of the most feared consequences of aging and requires better diagnostic tools, management and effective therapies. [2] [3] [4] [5] The aetiology of AD is very complicated. Several pathological factors such as loss of acetylcholine (ACh) and butyrylcholine (BuCh) neurotransmitters, amyloid beta (Ab) aggregation into toxic oligomers and plaques, metal ion dyshomeostasis and oxidative stress etc. are suggested for AD pathogenesis. [5] [6] [7] [8] [9] [10] [11] [12] The classical amyloid cascade hypothesis suggests that the most important contributor to AD is amyloid plaques which are deposits of Ab peptide aggregates. [13] [14] [15] However, recent studies suggest that in addition to the plaques, smaller soluble aggregates of Ab peptides are much more neurotoxic. [16] [17] [18] [19] Molecules that reduces the aggregation of Ab peptides and formation of such neurotoxic species are in demand. 7 Several inhibitors of Ab aggregation are reported in literature; some of these inhibitors lead to soluble neurotoxic oligomers and some to non-toxic aggregates. 2, 18, [20] [21] [22] On the other hand, inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) to protect acetylcholine in AD patients has been the most attractive therapeutic strategy. The cholinesterase (ChE) enzymes AChE and BuChE are responsible for the hydrolysis of acetylcholine. 23, 24 Tacrine and donepezil based scaffolds are so far the most potent type of AChE and BuChE inhibitors. 25, 26 However, alternative molecular scaffolds (e.g., coumarins, quinolines, xanthones etc.) are also known to inhibit AChE and BuChE, albeit with lower potency. [27] [28] [29] AD is truly a multifaceted disease and various groups have been working on developing suitable multitargeting molecules for its treatment. [20] [21] [22] [30] [31] [32] [33] [34] [35] For example, quinolines are natural alkaloids and their derivatives possess a number of biological and medical applications including antioxidation, antiosteoporosis, anti-inuenza, antimalarial and anticancer activities. [36] [37] [38] Hydroxychloroquine is getting popularity again for its possible role in COVID-19 treatment. Quinoline moiety has been widely explored for its application in treatment for AD. The molecular framework of quinoline leads to the discovery of a small lipophilic molecule clioquinolo (5-chloro-7-iodo-8hydroxyquinoline, CQ). 20, 39, 40 Recent studies reported the utility of quinoline derivatives as AChE and monoamineoxidase inhibitors. [41] [42] [43] Fu et al. explored a series of quinoline derivatives with dithiocarbamate moiety and reported their strong inhibition to AChE as well as Ab aggregation. Sang and co-workers have determined that quinoline derivatives effectively inhibit cholinesterse and monoamineoxidase. 41, 44 Considering their wide range of biological and medical importance, our group also recently only reported an attractive synthetic strategy of a series of 3-iodo-6-(aryldiazenyl)quinolines. 45 In the current work, we report the potential application of selected aryldiazoquinoline derivatives towards the factors contributing to AD. The inhibitory effect of these compounds was investigated for Ab aggregation using Ab 42 as a model peptide. In addition, these molecules were also investigated for inhibition of cholinergic enzymes AChE and BuChE. The molecular docking studies were carried out to get detailed insights for understanding underlying mechanisms for the observed in vitro inhibitory effect of these compounds on AChE. The cell viability studies show that these molecules are nontoxic in nature and can be used in vivo for further applications. The free radical scavenging assay suggested that these compounds do not enhance free radical species and can be safely used in biological settings. Overall, these aryldiazoquinoline derivatives possess a nontoxic molecular framework, effective cholinesterase inhibition together with antiaggregation properties towards Ab 42 peptide suggesting these as promising multifunctional agents for AD.

The design strategy for these compounds consists of a hybrid of quinoline molecule with the diazoaryl-group and to vary the substituents to equip compounds with better intercalating ability to inhibit the Ab aggregation and interact with cholinesterase enzymes. The design is novel and partly inspired by the properties of Congo Red (CR), an azo-dye known to stain ex vivo amyloid plaques. 31, 46 Half CR type molecules, resveratrolstilbene framework have been adopted since these are known for their amyloid binding affinities along with antioxidant properties. [46] [47] [48] On the other hand, in recent studies, quinolines have demonstrated potential anti-AD activity, in terms of inhibitors to peptide aggregation along with AChE and monoamineoxidase inhibitors and molecular docking studies indicated that it can serve as PAS binding moiety through p-p stacking interaction. 36, 41, 42 Unlike tacrine and donepezil, quinoline moiety is still less explored as cholinesterase and aggregation inhibitor. The design of the molecules used in the current studies, is an extension of our efforts in this direction. 31, 45 A total of >50 molecules were synthesized (Fig S3 and  S4 †), using all possible substitutions and combinations, but we  have chosen only selective 14 compounds for the current studies to understand their structure-activity relationship from prepared compounds which represent all types of substitution patterns and functional groups (Fig. 1) .

Most of these compounds are deep red in color and to understand their conjugation and aromatic properties, the electronic spectra for all the compounds were recorded at room temperature in methanol from 200 to 800 nm range (ESI Fig. S2 †) . All the compounds show mainly two intense absorption bands with high molar absorptivity values ($10 4 M À1 cm À1 ). One absorption band is observed in the range of 330 to 360 nm which may be attributed to n / p* transition. In most of the compounds, another absorption band is also present in the range of 240 to 300 nm which can be assigned to p / p* transition. When these molecules were excited at their respective l max values, no uorescence was observed. The non-uorescent nature may be due to the presence of lone pairs of azo-group quenching the uorescence. In addition, rotation about the double bond may be another factor contributing to non-uorescent nature of these molecules.

To consider a compound as an anti-AD drug, good permeability through blood-brain barrier (BBB) and its ability to reach the central nervous system (CNS) is an essential criterion. 49 The verication of Lipinski's rule of ve which includes parameters such as calculated octanol-water partition coefficient (clog P), number of hydrogen bond donor-acceptors, polar surface area provides a good estimate of the BBB permeability of any compound. 50 The log BB value is a common measure of the degree of BBB penetration and can be calculated using eqn (1). 51 log BB ¼ À0.0148(TPSA) + 0.152(clog P) + 0.139 (1) Drug likeness properties were calculated using program molinspiration property engine v2014.11 (available on http:// www.molinspiration.com/cgi-bin/properties).

Overall the calculated log BB parameter for all the compounds has the values within the acceptable range (À1.0 to 0.3), 52 albeit some individual parameters are found to be on a higher or lower side for some of these compounds. A table of such parameters has been now included in ESI (Table S2 †).

The studies on the effect of the compounds in the inhibition of peptide aggregation were carried out upon incubating Ab 42 in the presence and absence of the compounds. The affinity of the compounds toward Ab brillation inhibition was investigated by uorescence spectroscopy. The model peptide used for brillation was Ab 42 which is believed to play most important role in the AD pathogenesis. Thioavin T (ThT) is used as the most common probe for bril formation. 53, 54 When ThT binds to beta sheet-rich structures, more than 20-fold enhancement in its uorescence intensity is observed which is used for detecting the presence of brils. 55 In our experimental settings, Ab brillation (in absence of any aryldiazoquinoline additives) starts showing increase in ThT uorescence within 2-4 hours of incubation. The inhibitory effect of aryldiazoquinoline molecules was observed to be very prominent from beginning itself. The increase in ThT uorescence occurred aer 6-10 hours of incubation in presence of aryldiazoquinoline molecules and remained the same aer. Results of ThT uorescence inhibition assay are presented in Fig. 2 and S33. † Analysis of the result summarized in Table 1 show that all the compounds induced a decrease in ThT uorescence indicating the inhibition of Ab brillation in a range of 50 to 77%. Compounds 1d, 1e, 2d and 5 are the most effective inhibitors with $75% inhibition. In general, the inhibitory effect of these molecules can be attributed to their capability to intercalate and act as b-sheet disruptors. Presence of certain functional groups like substitution of an unsaturated ester functionality at 3-position of quinoline ring in molecule 5 are expected to increase the bioavailability and binding interactions of these molecules with Ab peptide and likely the reason for observed inhibitory effect.

In order to gain further insights for inhibition of Ab 42 aggregation, transmission electron microscopy (TEM) technique was employed for selected molecules. One inhibitor among each series was selected as representative compound which showed best IC 50 values (1e, 2a, 3a, 4a and 5). TEM images show the presence of dense brillar aggregates of Ab (Fig. 3, panel 1) . A change in morphology of these aggregates was observed in the presence of inhibitors (Fig. 3 , panel 2-6). Fewer Ab brils were detected as compared to Ab alone when compounds 1e and 5 (Fig. 3 , panel 2 & 6) were incubated with the sample, whereas 2a, 3a and 4a (Fig. 3 , panel 3-5) demonstrated larger amorphous aggregates. These results were consistent with the ThT assay showing a good inhibition of selfinduced Ab aggregation.

In order to measure the binding affinity of the molecular framework of the compounds studied here, ThT uorescence competition assays were performed. Since, these molecules consist of azo-stilbene type fragments and likely to intercalate in b-sheets and may replace ThT. The binding affinity of the selected three compounds (1e, 3a and 5) toward Ab brils was investigated. For binding studies, Ab 40 was used since it forms well dened amyloid brils. 21, 56 For the experiment a solution of compound under study was added sequentially in nanomolar amounts to a solution of xed concentration of Ab 40 brils (5 mM) in presence of ThT (2 mM). 57 A signicant decrease in ThT uorescence intensity was observed upon the addition which indicates the replacement of pre-bound ThT to Ab brils through competitive binding. One site competitive-binding model was used for data tting and yields a K i value of 204 AE 19 nM, 177 AE 15 nm and 168 AE 17 nm respectively for 1e, 3a and 5 (Fig. 4) . Overall the result suggests that this molecular framework exhibits a strong affinity for the Ab brils. The strong binding of these molecules also indirectly support the inhibitory effect on Ab bril formation as observed above.

The inhibitory effect of the designed compounds were analyzed in vitro for AChE using modied Ellman's method. 58 The experimental results were presented as IC 50 in Table 1 . In comparison to the known AChE inhibitors donepezil (DZ) and rivastigmine (RTG), the IC 50 values (Table 1) show a moderate AChE inhibitory activity of compounds studied here. Out of the total fourteen compounds, good anti-AChE activity was exhibited by compounds 2a, 2b, 1d and 1e with IC 50 values in the low micromolar range (6.2, 7.0, 9.3 and 8.4 mM, respectively). It is worth noting that 2a which has highest AChE activity, contains a chloro group at the para position of to diazoaryl group in This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 28827-28837 | 28829 combination with an aliphatic group (C 4 H 9 ) at 2-position of quinoline ring with iodo substitution at 3-position. Interestingly, the presence of these functional groups in other molecules but in different combinations does not exhibit similar activity. Even replacement of chloro group by a bromo group in the aryldiazo moiety decreased the AChE inhibitory activity. The compounds 2c and 2d, where C 4 H 9 group is replaced by C 5 H 11 group exhibited remarkable decline in inhibitory activity. The compounds with best AChE activity belong to series 1 and 2 containing iodo group at 3-position of quinoline ring. In general, molecules without a donepezil molecular framework show IC 50 values in very high micro molar range. Therefore, it is interesting to see that the chosen set of compounds showed good AChE inhibitory activity, which is comparable to reference inhibitor RTG in the same experimental settings (Table 1) .

These compounds were further tested for their inhibitory activity against BuChE following the similar protocol. Inspired by good AChE results; rst, the activity for these compounds were checked at low concentration range (2-100 mM). The compounds were found to be very less active in these settings for BuChE inhibition. Studies at their higher concentrations (up to 300 mM) showed moderate inhibition, but a regular pattern of inhibition could not be observed. It is likely due to the inherent absorption by the compounds themselves in the monitoring absorption wavelength range ($405 nm). For BuChE inhibition, compound 4a exhibited comparatively well with more than 65% inhibition at 100 mM concentration. As a regular pattern of inhibition was not observed, IC 50 values were not calculated from data. The volume of the catalytic site in BuChE is much larger than that of AChE and also, BuChE does not have a functional peripheral site, these structural differences might contribute towards the different activity of a molecule against AChE and BuChE. [59] [60] [61] [62] Overall, these results suggest that selected compounds from the series are good inhibitors of AChE activity but not for BuChE activity.

Molecular docking is a powerful bioinformatics approach to predict the binding mode of a ligand molecule with the active site of a target protein. Molecular docking provides information such as binding pattern, binding orientation, hydrophobic interaction, hydrogen bond, van der Waals force, electrostatic interaction between the ligand and active site of the target protein. 63, 64 In this study, the acetylcholinesterase enzyme of Tetronarce californica (TcAChE) and human (hAChE) was used to dock with the designed molecules to understand the binding mechanism between them. 65 The active site of TcAChE is almost identical to the hAChE. The only minor difference lies in the mutation of tyr337 in human to phe330 in the TcAChE. 66, 67 The crystal structure of TcAChE was superimposed with the hAChE and it is observed that these are structurally similar with 0.6Å RMSD between them (Fig. S34 †) . Structurally, AChE possesses a catalytic active site (CAS) and a peripheral anionic site (PAS), the commonly understood two binding sites. Molecular docking studies were performed with Maestro v11.9 of Schrödinger soware. The X-ray crystal structure of torpedo AChE (TcAChE) in complex with tacrine (PDB ID: 1ACJ) was downloaded from PDB and docked with the designed compounds (1a, 1b, 1c, 2a, 2b, 2c and 3a). Three compounds (2a, 2b and 3a) were able to interact with target protein TcAChE. Molecular docking results revealed that these compounds were able to interact via hydrophobic interaction (within the range of 5Å) with the CAS and PAS residues of the TcAChE. 68 In general, these compounds were enclosed by the active site residues -Asp72, Trp84, Tyr121, Tyr334, Trp279, His440 and Tyr442, which are similar to the native crystal structure interactions. Compound 3a was having a docking score of À10.20, glide energy of À37.93 kcal mol À1 and binding free energy of À30.36 kcal mol À1 and was making p-p interaction with Trp84, Trp279, Phe330 and Tyr334 (Fig. S35 †) . Similarly, compound 2b was having a docking score of À9.35, glide energy of À46.23 kcal mol À1 and binding free energy of À41.83 kcal mol À1 and was making three p-p interaction with Trp84, Trp279 and Phe330 (Table S3 † and Fig. S35 †) .

Compound 2a was docked in the active site the TcAChE with a docking score of À9.34, glide energy of À46.65 kcal mol À1 and binding free energy of À42.29 kcal mol À1 (Fig. 5 and S35 †) . The quinoline ring, chlorophenyl and phenyl group of 2a compound was interacted via p-p interactions with Trp84 and Phe330 residue of CAS and Trp279 (of PAS), respectively ( Fig. 5 and S35 †). Rivastigmine, an FDA approved drug is used as control for the in silico study. It was observed that rivastigmine also interacts with active site residues of TcAChE via hydrophobic interaction (Trp84 and Phe330) and one halogen bond interaction (Asp72) with a docking score of À5.59, glide energy of À44.27 kcal mol À1 and binding free energy of À40.51 kcal mol À1 (Table S3 † compared to the other designed compounds and the FDA approved drug rivastigmine which is in good agreement with experimental results (IC 50 of 2a 6.2 AE 1.2 mM). Further, 2a and rivastigmine (as control) was also docked into the active site of human acetylcholinesterase (hAChE, PDB ID: 6F25) using Maestro v11.9 since AChE of human and torpedo are structurally similar. On a comparative note, a docking score of À8.63, À4.64, glide energy of À71.75 kcal mol À1 , À47.71 kcal mol À1 and binding free energy of À70.74 kcal mol À1 , -52.22 kcal mol À1 , were observed for compound 2a and rivastigmine, respectively. Compound 2a forms one hydrogen bond with Gly82 and p-p stacking with Trp286 and Tyr341 (Fig. 6) , whereas rivastigmine made two hydrogen bonds with Phe295 and Arg296 (Fig. S35 †) . These studies revealed that 2a could be a better AChE inhibitor with hAChE than rivastigmine as it is capable to interact simultaneously with residues of both PAS and CAS of target enzyme.

As previously mentioned, cholinesterase enzyme holds a pivotal role in AD research and these enzymes work in controlling the levels of neurotransmitters in brain. AChE is also known to play a key role in accelerating Ab aggregation by interaction through PAS of enzyme to form stable AChE-Ab complex along with presence of BuChE also. 69,70 Therefore, inhibitors with dual interactions with both PAS and CAS simultaneously appears to be a better therapeutic strategy as these can combat cognitive dysfunctions as well as prevent complications related to Ab aggregation. 10, 29, [71] [72] [73] To further understand the role of these compounds as dual inhibitors as indicated by the chosen scaffold and also supported by docking results, AChE induced Ab 42 aggregation inhibitory activity was examined using the ThT uorometric assay. For this study, again same compound from each sub-series was selected (1e, 2a, 3a, 4a and 5) and results of the assay is presented in Fig. 7 . Results indicate that these compounds are capable of inhibiting the Ab 42 aggregation in presence of AChE also (inhibition in the range from 40 to 70%). Molecule 5 was the most efficient one in inhibiting the Ab brillation as well as AChE-induced Ab aggregation. As discussed above also, the ester functionality is expected to increase the bioavailability and binding interactions of molecule 5 with Ab (and may be AChE) and likely reason for observed inhibitory effect. These results validate the fact that the compound which binds both CAS and PAS of AChE simultaneously could inhibit AChE induced Ab aggregation.

Overall, the study identies a family of fourteen aryldiazoquinoline molecules that modestly inhibit cholinesterase activity as well as show in vitro anti-aggregation properties. These extended polyaromatic compounds are expected to intercalate in a hydrophobic binding pocket to disrupt aggregation pathway. These probes may be useful for studying the consequences of cholinesterase inhibition for AD progression. Molecular docking results and inhibition of AChE-induced Ab aggregation demonstrates their dual-site binding with both CAS and PAS which can simultaneously improve cognition and slow the rate of Ab-elicited neurodegeneration and could be used in future to design potential therapeutics for AD considering its multifactorial nature.

Cell viability can be greatly affected by the subtle changes in the structures of molecules; so studying all the fourteen molecules for their effect on cell survival was attempted. Cell viability studies were performed using Alamar blue assay using Neuro2A cells. Toxicity effects were investigated in a range of 2-20 mM concentration. Interestingly; almost all the molecules exhibit very low cell toxicity. Only two compounds, 3a and 4a showed slight toxicity at higher concentration (20 mM) ( Fig. 8 and S36 †) , all other compounds exhibited a cell survival of >90%. Overall, the cell studies suggested that the series of compounds studied here are almost nontoxic in nature towards Neuro2a cells. Since the molecules studied here possess a redox-active azofunction, it may enhance the levels of neurotoxic radical species. Proper management of reactive oxygen/nitrogen species and disposal of damaged cellular components is essential for maintaining regular neuronal function. 74, 75 To evaluate the antioxidant activities of compounds, DPPH and ABTS assay were performed. Trolox, a vitamin E analogue, is used as a standard in assay protocol. Analysis of results from the DPPH and ABTS assay suggested that these molecules did not lead to any enhancement of free radicals. Overall, the studies suggest that molecular framework may be expected to be used safely in biological settings.

A series of fourteen aryldiazoquinoline compounds have been evaluated for their biological potential particularly relevant to Alzheimer's pathology. Interestingly, the molecular framework used here show good inhibition of Ab aggregation as observed by ThT uorescence assay and TEM analysis. It was interesting that these molecules are effective inhibitors to brillation process, a major key player in AD development. Most of molecules possess a central aryldiazoquinoline moiety, likely responsible for intercalation and inhibition to aggregation. It was also very interesting to observe that most of the molecules exhibited an inhibitory effect on the esterase activity. These compounds inhibited AChE with a moderate activity in low micro molar range. Compound 2a was most potent with IC 50 value of 6.2 mM which is remarkable for a non-donepezil or tacrine molecular framework. Variation of substituents suggested that minor change in structure of the molecules could enhance or decrease the AChE activity or Ab inhibition. It was also satisfying that compounds are quite nontoxic in nature against neuronal cell line and suitable for further investigation in AD research.

All reagents were purchased from commercial sources and used as received unless stated otherwise. Solvents were puried by distillation prior to use. All aqueous solutions and buffers were prepared using commercially available distilled water. The UVvisible spectra were recorded on a Carry-60 UV-Visible Spectrophotometer and data are reported as l max (nm) absorbance. For the preparation of brils and inhibition studies, a laboratory thermomixer from Genetix Biotech Asia was used. All the AChE, BuChE, DPPH and ABTS studies were performed on Elisa reader from ThermoFisher Scientic (MultiSkan Go). Fluorescence measurements were done on Cary Eclipse Fluorescence Spectrophotometer from Agilent. TEM Images were captured using a FEI G2 Spirit Twin, MNIT, Jaipur. Ca(OTf) 2 and Bu 4 NPF 6 catalyst were obtained from Sigma-Aldrich and used without further purication. Reactions were performed in ame-dried or oven-dried glassware with magnetic stirring. Reactions were monitored using thin-layer chromatography (TLC) with aluminium sheets silica gel 60 F 254 from Merck. TLC plates were visualized with UV light (254 nm), iodine treatment or using ninhydrin stain. Column chromatography was carried out using silica gel 60-120 mesh as stationary phase. NMR spectra were recorded at 500 MHz and 400 MHz (H) and at 125 MHz and 100 MHz (C), respectively on Avance Bruker spectrometer. Chemical shis (d) are reported in ppm, using the residual solvent peak in CDCl 3 (H: d ¼ 7.26 and C: d ¼ 77.0 ppm) as internal standard, and coupling constants (J) are given in Hz.

A mixture of phenylacetylene IIa (510 mg, 5.1 mmol), t-BuOK (456 mg, 4.08 mmol) and 2-amino-benzophenone Ia (669 mg, 3.4 mmol) was placed into a reaction ask at room temperature under nitrogen atmosphere and the mixture was stirred for 2 h. The progress of reaction was monitored by TLC. Aer completion of the reaction, the resulting mixture was quenched with water and extracted into ethyl acetate (thrice). Combined organic layers were washed with brine solution and dried over anhydrous Na 2 SO 4 and the solvent was evaporated to obtain the pure compound IIIa.

(E)-3-Iodo-6-((4-nitrophenyl)diazenyl)-2,4-diphenylquinoline (1d). A mixture of 1-(2-aminophenyl)-1,3-diphenylprop-2-yn-1-ol (150 mg, 0.50 mmol) and (E)-1-(4-nitrophenyl)-2-(tetrauoro-l5boranyl)diazene (175 mg, 0.75 mmol) stirred in 1,2-dichloroethane (1.5 mL) at room temperature for 15 min then I 2 (194 mg, 0.75 mmol) was added to the reaction mixture and stirred till completion of the reaction (monitored by TLC). Aer completion of the reaction, the resulting mixture was quenched with aq. saturated solution of Na 2 S 2 O 3 (10 mL) and extracted into dichloromethane (15 mL, thrice). Combined organic layers were washed with brine solution and dried over anhydrous Na 2 SO 4 , and the solvent was evaporated under reduced pressure, and the residue was puried by column chromatography using EA/PE (5 : 95, v/v) to obtain the desired product 1d in 67% yield.

Mouse neuroblastoma Neuro2a (N2A) cell lines were purchased from the American Type Culture Collection (ATCC). Cells were This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 28827-28837 | 28833 grown in Dulbecco's Modied Eagle's medium (DMEM)/10% FBS, which is the regular growth media for N2A cells. N2A cells were plated to each well of a 96 well plate with DMEM/10% FBS. The media was changed to serum-free medium. Aer 1 h, the compounds were added into each well (nal volume: 100 mL) with different concentrations (2, 5, 10 and 20 mM), followed by incubation for 24 h at 37 C. Due to the poor solubility of compounds in water or media, their stock solutions were prepared in DMSO and diluted in media. The nal DMSO concentration in this way was less than 1%. Our control experiments suggest that there is no toxicity caused by 2% DMSO. At last, each well was treated with 10 mL of Alamar blue reagent and the cells were incubated for 1 h. The uorescence (excitation: 560 nm, emission: 590 nm) was measured using an Elisa Reader.

Acetylcholinesterase (AChE, Type V-S, lyophilized powder, from electric eel, 1000 unit), acetylthiocholine iodide (ATCI), and 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich. Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were obtained from Fisher Scientic. To measure, AChE activity, modied Ellman's method was performed. 58 The assay was performed using a 96 well plate reader (Thermo Scientic, MultiSkan Go) at 25 C. The assay was carried out in 0. Enzyme and samples were incubated rst for 15 minutes, followed by addition of DTNB and ATCI and the nal volume was reached by the addition of buffer and incubated for 20 minutes before the absorbance was recorded. The data was analyzed and IC 50 value was calculated using Graph Pad Prism soware. Multiple assays were performed and results reported are at least from the data collected in triplicate. BuChE activity was also determined following the same protocol where BuChE enzyme was used in place of AChE.

The chemical structure of the designed molecules was drawn using Marvin Sketch v19.20 (https://chemaxon.com/products/marvin) computational tool and the 3D orientations were saved in structure-data le (.sdf) le format. "LigPrep" module of Maestro v11.9 of Schrödinger soware v2019 was used to prepare the ligand library. 76 The ligands were prepared at pH 7.0 AE 0.2 using Optimized Potentials for Liquid Simulations (OPLS) force eld.

Next, the crystal structures of the target protein acetylcholinesterase of Tetronarce californica (TcAChE, PDB ID: 1ACJ) 77 and human (hAChE, PDB ID: 6F25) 8 were downloaded from the Protein Data Bank (PDB). The downloaded proteins were prepared individually by the multilevel process by adding hydrogen atoms, assigning bond orders, adding missing amino acids and removing water molecule using the "Protein preparation wizard" in the Prime module of Maestro v11.9. 78 The protein structures were optimized and minimized with OPLS force led at neutral pH. Molecular docking of the designed compounds with TcAChE and hAChE were carried out using Glide extra precision (XP) approach implemented in Maestro v11.9. 79 For this purpose, the native ligands (tacrine in TcAChE and C-35 in hAChE), which were present in the active site of the target proteins, were used to generate a receptor grid box around the active site using "Receptor Grid Generation" wizard of Maestro v11.9. Next, the designed compounds were allowed to dock in the dened active site of the target protein with the Glide XP default parameter. Later, the docked le was subjected to calculate binding free energy (DG) using Prime MM-GBSA. 78 The top-ranked protein-ligand complexes were analyzed carefully by studying docking score, glide energy, MM-GBSA score, hydrogen and hydrophobic interactions.

Ab monomeric lms were prepared by dissolving commercial Ab 42 peptide (Anaspec, USA) in HFIP and incubating for 1 h at room temperature. The solution was then aliquoted out and allowed it to evaporate at room temperature. The aliquots were vacuum centrifuged and the resulting monomeric lms stored at À80 C. Ab brils were generated by dissolving monomeric Ab lms in DMSO, diluting into the PBS buffer (pH 7.4), and incubating for 24 h at 37 C with continuous agitation of 400 rpm (nal DMSO concentration was <2%). For inhibition studies, compounds (DMSO stock solutions) 25 mM (nal concentration) were added to Ab solutions (25 mM) along with reference (Ab alone) and incubated for 24 h at 37 C with constant agitation of 400 rpm. The percent inhibition of the Ab aggregation due to the presence of the inhibitor was calculated by the following formula: 100 À (IF i /IF o  100), where IF i and IF o are the uorescence intensities obtained for Ab in the presence and in the absence of the inhibitor, respectively, minus the uorescence intensities due to the respective blanks.

Inhibition of AChE-induced Ab aggregation was measured employing a ThT assay. Monomeric Ab 42 lm was dissolved in DMSO and diluted into the PBS buffer (pH 7) to a nal concentration of 25 mM. For co-incubation experiments, 25 mM solution of Ab 42 , and AChE (20 mL, 1 U mL À1 , nal concentration) in presence and absence of inhibitors (4 mL, 25 mM, nal concentration) were incubated for 24 h at 37 C with continuous agitation of 400 rpm (nal DMSO concentration was <2%). Each inhibitor was examined in triplicate. Fluorescence intensities of respective blanks were subtracted in inhibition plot.

All uorescence measurements were performed using Agilent Cary Eclipse Fluorescence Spectrophotometer. For ThT uorescence studies, samples were diluted to 2.5 mM Ab in PBS containing 10 mM ThT and the uorescence measured at 485 nm (l ex ¼ 435 nm).

The DPPH assay is a well-known, simple and sensitive technique for determination of radical-scavenging ability. Antioxidant activity of compounds were evaluated using DPPH (1,1diphenyl-2-picrylhydrazyl) assay. Different concentrations of test compounds in DMSO were prepared and the compound solution was added to the methanolic DPPH solution (0.1 mM). The mixture was kept in the dark for 30 min. Then, the absorbance at 517 nm was measured using a microplate UV/Visible spectrophotometer (ThermoFisher, MultiScan Go).

ABTS (2,2 0 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), potassium persulfate (di-potassium peroxodisulfate), Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2carboxylic acid), and ascorbic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Aqueous solutions were prepared using deionized water obtained from Central Drug House Pvt Ltd. New Delhi, India. Trolox was used as the antioxidant standard. A 4 mM solution of Trolox was prepared in deionized water. ABTS was dissolved in deionized water to a 7 mM concentration. The solution of ABTS radical cation (ABTSc + ) was produced by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate aqueous solution in equal quantities and allowing them to react for 12-16 h at room temperature in the dark. For the working solution ABTSc + solution was diluted to get nal absorbance around 1 at l max 734 nm. Fresh working ABTSc + solution was prepared for each assay. The radical scavenging capacity of the compounds was analyzed by mixing 20 mL of compounds (different concentrations, ranging from 2 to 500 mm) with 80 mL of working solution of ABTSc + . The decrease in absorbance was measured spectrophotometrically at 734 nm aer 1 h of mixing the solutions using a microplate UV/VIS spectrophotometer (ThermoFisher, MultiScan Go).

Glow-discharged grids (Formar/Carbon 300-mesh, Electron Microscopy Sciences) were treated with preformed Ab aggregates in absence and presence of compounds for 30 min at room temperature. Excess solution was removed using lter paper and grids were rinsed with distilled H 2 O. Grids were stained with ammonium molybdate (1% w/v, H 2 O) for 1 min, blotted with lter paper, and dried overnight at room temperature. Images were captured using a FEI G2 Spirit Twin microscope (200 kV). TEM analysis was performed at the Material Research Centre at MNIT, Jaipur.

Ab 40 (Anaspec, USA) was used for Ab-bril-binding studies. ThT competition assay was followed. For ThT competition assays, a 5 mM Ab bril solution with 2 mM ThT was titrated with small amounts of compound and the ThT uorescence measured (l ex / l em ¼ 435/485 nm). For calculation of K i values, a K d value of 1.17 mM was used for the binding of ThT to Ab brils.

UV-Vis spectra for all compounds were recorded on Cary-60 UV-Vis spectrophotometer. The solution for the compounds were prepared from the stock solution of DMSO (2 mM) and diluted in distilled methanol.

Authors declare no conicts of interest.

Alzheimer's Res. Ther

Drug Discovery Today

Schrödinger Release 2019-4, LigPrep, Schrödinger, LLC

Schrödinger Release 2019-4: Prime, Schrödinger, LLC

Schrödinger Release 2019-4: Glide, Schrödinger, LLC

Notes and references