Indene-Derived Hydrazides Targeting Acetylcholinesterase Enzyme in Alzheimer’s: Design, Synthesis, and Biological Evaluation

As acetylcholinesterase (AChE) plays a crucial role in advancing Alzheimer’s disease (AD), its inhibition is a promising approach for treating AD. Sulindac is an NSAID of the aryl alkanoic acid class, consisting of a indene moiety, which showed neuroprotective behavior in recent studies. In this study, newer Indene analogs were synthesized and evaluated for their in vitro AChE inhibition. Additionally, compared with donepezil as the standard drug, these Indene analogs were accessed for their cell line-based toxicity study on SH-SY5Y cell line. The molecule SD-30, having hydrogen bond donor (HBD) at para-position, showed maximum AChE inhibition potential (IC50 13.86 ± 0.163 µM) in the indene series. Further, the SD-30 showed maximum BuChE inhibition potential (IC50 = 48.55 ± 0.136 µM) with a selectivity ratio of 3.50 and reasonable antioxidant properties compared to ascorbic acid (using DPPH assay). SD-30 (at a dose level: of 10 µM, 20 µM) effectively inhibited AChE-induced Aβ aggregation and showed no significant toxicity up to 30 mM against SH-SY5Y cell lines.


Introduction
The synthesis of fused aromatic ring scaffolds as a pharmacophore has immensely interested organic medicinal chemists in targeting intracellular and extracellular proteins [1,2]. The conjugation of the fused ring scaffolds with other aromatic systems or polyaromatic systems is commonly practiced by the medicinal chemist to bring photochemical control [3], improving physicochemical parameters [4] and reducing on-target toxicity [5]. Although fused ring scaffolds are widely implemented in various diseases, their implementation in neurodegenerative disease is still lower because of their limited physicochemical properties. One such neurodegenerative disease is Alzheimer's dementia (AD), which is clinically manifested with lowered levels of acetylcholine. Acetylcholine is a neurotransmitter; any abruption in its levels also leads to other diseases, such as Lambert-Eaton myasthenic syndrome (LEMS) and myasthenia gravis (MG). Numerous neurons, namely forebrain cholinergic neurons, become damaged due to the formation of serine hydrolase enzymes, which is linked to aberrant acetylcholine metabolism and impaired IQ. Based on epidemiology studies, 44.4 million individuals worldwide had dementia as of 2013, and it is expected to reach 75.6 million globally by 2030 and 135.5 million worldwide by 2050, especially in developing nations. Approximately 62% of people who have dementia reside in third-world countries, which will become 71 percent by 2050 [6]. Although the essential etiological factor for AD has not yet been identified, the hallmarks of this disorder are recognized abnormal brain diseases, such as the over-expression of Acetylcholinesterase (AChE) and the extracellular deposition of "mystery"-amyloid plaques. In the brain, the cholinergic neurons engage in cortical activity, cerebral blood flow, memory-and learning-oriented processes, and the modulation of cognition [7]. The choline acetyltransferase enzyme (AChE) is depleted explicitly in the brains of AD patients, which lowers acetylcholine synthesis and affects cortical cholinergic functions [7]. Therefore, inhibiting the cholinesterase enzymatic activity in the synaptic cleft was found to be a promising strategy to improve AD symptoms. Butyrylcholinesterase (BChE) is the other cholinesterase (ChE), which makes 20% of the total brain ChE activity. Clinical studies showed elevated levels of BChE during AD onset, making it a potential target in AD treatment [8]. Unlike AChE, BChE is not expressed in the peripheral and autonomous nervous systems; therefore, selective BChE inhibitors would be devoid of the adverse side effects as what is commonly seen with AChE-specific inhibitors, which would reduce the threshold dosing. In the context of AD, cymserine analogue selective targeting of BChE alleviated the secretion of amyloid β (Aβ) peptide in a human neuronal cell line, and enhances cognitive performance in aged rats [9].
To date, there is no cure for AD, and numerous reversible AChE inhibitors are in clinical trials. One of the major challenges that the drug designer commonly faces during AD drug development is to find a suitable molecular scaffold that can comply with the distinctive cellular physiochemical properties (specifically, the blood-brain barrier) in the brain. Therefore, molecules with higher AChE potency do not warrant their higher bloodbrain barrier (BBB) crossing. However, in some instances, an increase in dosage showed a marginal enhancement in BBB crossing but was flawed in producing on-target toxicities on smooth muscles. Therefore, the rational designing of molecular structure targeting AChE should display preferential cholinesterase selectivity to mitigate the on-target toxicity in the peripheral cellular system and typical physicochemical characteristic (ADMET) to cross BBB. To an extent, donepezil, rivastigmine, and galantamine were clinically developed as cholinesterase inhibitors (as shown in Figure 1A), which enhance cholinergic transmission and provide symptomatic relief for a portion of dementia patients [10]. Interestingly, donepezil is an indene-1-one analog, which shows preferential selectivity towards AChE than BChE. However, indene scaffolds are also devised to develop Mcl-1 inhibitors [11], estrogen-targeting compounds [12,13], and naturally derived fungal metabolites [12], as shown in Figure 1B. In our previously reported study, we performed virtual screening on 44 indene analogs, using flexible molecular docking to identify the binding mechanism of these indenecontaining ligands to AChE [14]. First, the resulting indene analogs (as hits) were synthesized, followed by biochemical assays performed (AChE-induced A1-42 aggregation assay, in vitro AChE, and BuChE inhibition assays, the antioxidant activity DPPH assay, a  In our previously reported study, we performed virtual screening on 44 indene analogs, using flexible molecular docking to identify the binding mechanism of these indene-containing ligands to AChE [14]. First, the resulting indene analogs (as hits) were synthesized, followed by biochemical assays performed (AChE-induced A1-42 aggregation assay, in vitro AChE, and BuChE inhibition assays, the antioxidant activity DPPH assay, a cell line-based toxicity investigation on the SH-SY5Y cell line).

General Materials and Methods
All chemicals, reagents, and solvents for the synthesis of indene analogs were purchased from commercial sources and were used without further purification. Indene analogs were synthesized starting from sulindac using a slightly modified protocol as previously described by Bhat et al. [15]. The reactions were monitored using thin-layer chromatography on silica gel aluminum sheets and visualized with a UV lamp. NMR spectra were recorded on Bruker AV 600 MHz and 300 MHz spectrometers, operating at 150.92 or 75.47 MHz for 13 C and 600.13 or 300.13 MHz for 1 H nuclei. Chemical shifts are quoted in ppm and are referenced to SiMe4 as an internal standard unless stated otherwise. Multiplets are abbreviated as follows: br-broad; s-singlet; d-doublet; t-triplet; q-quartet; m-multiplet. Mass spectra (ESI-MS) were obtained on Shimadzu LCMS 2010-A spectrometer, Japan. Compounds were purified using column chromatography with silica gel as the stationary phase and ethyl acetate/Petroleum acetate mixtures (5:5) as the eluent system.

General Procedure for the Synthesis of Indene Ester
A mixture consisting of 1 g (0.0028 mol) of sulindac, 11.89 g (15 mL, 2.7 mol) of absolute methanol, and 10 drops of concentrated sulphuric acid was placed in a roundbottomed flask with a capacity of 100 mL. After attaching a reflux condenser, the mixture was heated over low heat for a period of five hours. TLC was used to monitor the reaction in different polarity of binary solvent systems using ethyl acetate (EA) and petroleum ether (PE) (EA:PE = 20, 50, and 80%), respectively. The reaction solvent was removed under reduced pressure. The solid residue was dissolved in ethyl acetate and washed with fresh water using a 100 mL separating funnel. The mixture was then vigorously shaken in the separating funnel to separate the components of the mixture. After being allowed to stand, two distinct layers emerged and were collected individually. The organic layer (ethyl acetate layer) was collected, the remaining water layer was poured back into the separating funnel, and fresh ethyl acetate was added. The collected ethyl acetate fraction was washed with fresh water, then dried over 5 g of anhydrous magnesium/sodium sulfate. This process was repeated until no further effervescences of carbon dioxide were observed. The flask was given vigorous shaking for approximately five minutes before being left to rest for at least half an hour while being shaken intermittently. Following filtration of the ethyl acetate solution with a small, fluted filter paper directly into an RBF, the solution was then subjected to rotary evaporation to remove the solvent. After collecting the sulindac methyl ester as a yellow, viscous liquid, the methyl ester was then recrystallized from ethyl ether to give a yellow solid in quantitative yield [16].

General Procedure for the Synthesis of Hydrazide Analogs of Indene
Sulindac methyl ester (0.01 mol) and hydrazine hydrate (0.2 mol) were refluxed in ethanol (50 mL) for 30 h. TLC was used to monitor the reaction, and the reaction solvent (ethanol) was removed under reduced pressure. Later, the semi-solid residue was dissolved in crushed ice with continuous stirring, and the precipitates were filtered and washed with fresh water. These precipitates were collected and crystallized in ethanol, which yielded yellow colored solid with Yield (70%) and m.p. 120-122 • C.

General Procedure for the Synthesis of Indene Derivatives
A solution of Sulindac hydrazide (1 mmol) in ethanol (15 mL) containing appropriately substituted benzaldehydes (1.1 mmol) and a catalytic amount of glacial CH 3 COOH was heated under reflux for 3 h. The reaction mixture was added to ice-cold water in a beaker. The product was precipitated, filtered by vacuum filtration, and washed several times with cold water. The solid was recrystallized from ethanol (NMR spectra are shown in Supplementary Information).

Cholineseterase Inhibition
Enzyme activities were determined using the Ellman spectrophotometric method [17,18]. Enzyme substrates acetylthiocholine iodide (ATCh) and propionylthiocholine iodide (PTCh) were purchased from Sigma-Aldrich, Steinheim, Germany, and thiole reagent 5.5 -dithiobis-2-nitrobenzoic acid (DTNB) from Sigma-Aldrich, St. Louis, MO, USA. Donpezil and were used as reference compounds (purchased from sigma); In initial state, ATCh (derived from human erythrocytes) and PTCh (derived from human plasma) were dissolved in water and DTNB in 0.1 M sodium phosphate buffer (pH 7.4). Sulindac analogues were dissolved in water, and all further dilutions were made in water. Final concentrations of synthetics were in the range of 0.01-200 µM, while substrates were 1.0 mM and 4.0 mM for ATCh and PTCh, respectively. The final dilution of AChE and BChE was 500 and 300 times, respectively. Briefly, 50 µL of AChE (1.00 U mL −1 ) or 50 µL of BuChE (0.6 U mL −1 ) and 20 µL of the test or standard compounds were incubated in 96 well plates for 30 min at room temperature. Further, 100 µL (1.5 mM) of DTNB was added to the above solution. The substrate i.e., ATCI (15 mM, 10 µL) or BTCI (30 mM, 10 µL) was added to it, and absorbance was recorded immediately at 415 nm for 20 min at 1 min interval using Synergy HTX multi-mode reader (BioTek, USA) [19,20]. The IC 50 values were calculated using absorbance obtained from the test and standard compounds. The assays were performed in triplicate with three independent runs.

Cell Line-Based Toxicity Study
The toxicity of compound SD-30 was evaluated against SH-SY5Y neuroblastoma cell lines by the MTT assay according to the literature procedure with minor modifications [20]. Briefly, the cell lines (density 1 × 10 5 cells/wells) were plated in 96 well plates and incubated for 24 h at 37 • C in CO2 incubator. Various concentrations of SD-30 (10,20,40, and 50 mM) were added, and cells were incubated for 72 h. After that, 20 mL of MTT reagent was added, and the cells were incubated for an additional 2 h. The obtained purple colored formazan crystals were solubilized in 100 mL DMSO. The absorbance was measured at 570 nm, and the % cell viability was calculated. Each treatment was executed in triplicate and data are presented as percentage of the control.

Antioxidant Assay
The assay was performed by previously mentioned protocol [19,21]. Briefly, 10 and 20 µM (10 µL) of the synthesized compounds (Tris-HCl buffer, pH 7.4) were mixed with 20 µL, 10 mM (stock in methanol) of DPPH (Hi-Media) in 96 well plate. The volume of solution was adjusted to 200 µL using methanol. The 96-well plate was incubated at 37 • C for 25 min on shaking water bath with moderate shaking. The absorbance of reaction mixture was measured at 520 nm wavelength. The reducing percentage (RP) of the DPPH was determined by the equation: where A 0 was the control or untreated DPPH absorbance, and A c was the test treated DPPH absorbance. Ascorbic acid was used as the standard for the DPPH assay. The assay was performed in triplicate.

Statical Analysis
Data were analyzed by GraphPad Prism 9.4.0.673. One Way ANOVA with multiple comparisons was used in the cell toxicity determination. One-way ANOVA followed by one-way analysis of variance *** p < 0.0001 and standard deviation (SD) was calculated in the antioxidant study.

Design and Synthesis of Compounds
The ligand library with 44 methylated indene derivatives was designed by substituting various functionalities with diverse electronic features like electron donor and acceptor functional groups at positions R 1 and R 2 . The compounds which showed potent AChE binding affinity were synthesized (as shown in Scheme 1) through a three-step process, which includes esterification, the nucleophilic addition of hydrazide derivatives, and imine-coupling with aromatic aldehydes [22]. Later, the reaction solvent was evaporated under reduced pressure, and the resulting solid residue was then purified by column chromatography. Finally, FTIR, 1 H-NMR, 13 C-NMR, and HRMS spectral techniques were used for molecular characterization.

Design and Synthesis of Compounds
The ligand library with 44 methylated indene derivatives was designed by substituting various functionalities with diverse electronic features like electron donor and acceptor functional groups at positions R1 and R2. The compounds which showed potent AChE binding affinity were synthesized (as shown in Scheme 1) through a three-step process, which includes esterification, the nucleophilic addition of hydrazide derivatives, and imine-coupling with aromatic aldehydes [22]. Later, the reaction solvent was evaporated under reduced pressure, and the resulting solid residue was then purified by column chromatography. Finally, FTIR, 1 H-NMR, 13 C-NMR, and HRMS spectral techniques were used for molecular characterization.

AChE and BuChE Inhibition Assay
The inhibition potency of the synthesized indene analogs were evaluated for both BchE and AchE. All compounds have exhibited a time-dependent inhibition on both BchE and AchE, demonstrating first-order kinetics (Table 1).
Since AD is a complex disease regulated via multiple factors, we have developed multitargeting molecules to counter the disease. The first goal was to assess the effect of hydroxy and methoxy groups on the in vitro enzyme inhibition. These groups were substituted at the benzylidene ortho/meta and para-positions. The involvement of hydrogen bonds in the drug interaction with the macromolecular targets was discovered by inserting hydrogenbond donor (HBD) groups at the meta/para-position of the indene series of compounds. Molecule SD-30 having HBD at para position, showed maximum AChE inhibition potential (IC 50 = 13.86 ± 0.163 µM) in the indene series. Further, the same molecule showed maximum BuChE inhibition potential (IC 50 = 48.55 ± 0.136). Furthermore, the molecule SD-30 was found to be selective for AChE (3.50). Shifting the position of the OH group to the meta-position (SD-24, hAChE IC 50 = 40.43 ± 0.067 µM; hBuChE IC 50 = 92.86 ± 0.066 µM) leads to a decrease in inhibition potential on both targets. Further, the effect of the dihydroxy group was assessed. Two hydroxy groups containing compounds SD-40 (2,6-OH) and SD-42 (2,3-OH) showed furthermore decrease in inhibition potential on hAChE and hBuChE (hAChE IC 50 = 108.86 ± 0.142 µM, hBuChE IC 50 = 286.01 ± 0.143 µM and hAChE IC 50 = 92.44 ± 0.106 µM, hBuChE IC 50 = 264.33 ± 0.068 µM respectively). However, the substitution of the benzylidene ring with a methoxy group at ortho and meta positions

Evaluation of Cell Line-Based Toxicity Study on SH-SY5Y Cell Line
Generally, in vitro cell line studies have been routinely used as these can be helpful in primary optimization and mechanistic investigations before animal-based toxicology studies [23]. The evaluation of the neuroprotection effect of compound SD-30 (10,20,40, and 50 mM) on human neuroblastoma SH-SY5Y cell lines was assessed through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [23,24] (as shown in Figure 2). This cell line is often used in AD due to its human origin and ease of maintenance [25].

AChE-Induced Aβ1-42 Aggregation Assay
AChE enzyme is reported to facilitate the aggregation of Aβ. Thus, the potent compound SD-30 was evaluated for its ability to inhibit AChE induced Aβ aggregation [26,27]. Aβ1-42 and AChE treated Aβ1-42 are 100% aggregated after the incubation of 48 h. The marketed compound Donapezil showed a significant decrease in the Aβ1-42 aggregation at 10 and 20 µM. Further, the compound SD-30 was also found to be effective on aggregation at doses of 10 and 20 µM (Figure 3).

AChE-Induced Aβ1-42 Aggregation Assay
AChE enzyme is reported to facilitate the aggregation of Aβ. Thus, the potent compound SD-30 was evaluated for its ability to inhibit AChE induced Aβ aggregation [26,27]. Aβ 1-42 and AChE treated Aβ 1-42 are 100% aggregated after the incubation of 48 h. The marketed compound Donapezil showed a significant decrease in the Aβ 1-42 aggregation at 10 and 20 µM. Further, the compound SD-30 was also found to be effective on aggregation at doses of 10 and 20 µM (Figure 3).

AChE-Induced Aβ1-42 Aggregation Assay
AChE enzyme is reported to facilitate the aggregation of Aβ. Thus, the potent com pound SD-30 was evaluated for its ability to inhibit AChE induced Aβ aggregation [26,27]. Aβ1-42 and AChE treated Aβ1-42 are 100% aggregated after the incubation of 48 h The marketed compound Donapezil showed a significant decrease in the Aβ1-42 aggrega tion at 10 and 20 µM. Further, the compound SD-30 was also found to be effective on aggregation at doses of 10 and 20 µM (Figure 3).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author (Arvind Negi).