Synthesis and investigation of the cholinesterase inhibitory and antioxidant capacities of some novel N'‐(quinolin‐4‐ylmethylene)propanehydrazides against Alzheimer's disease

One of the worst long‐term health issues of the past few decades is Alzheimer's disease (AD). Unfortunately, there are currently insufficient choices for treating and caring for AD, which makes it a popular subject for drug development research. Studies on the development of drugs for AD have primarily concentrated on the use of multitarget directed ligands. Following this strategy, we designed new ChE inhibitors with additional antioxidant and metal chelator effects. In this research, eight novel N’‐(quinolin‐4‐ylmethylene)propanehydrazide derivatives were synthesized and characterized. We then evaluated the inhibition potency of all the final compounds for cholinesterase enzymes. Among them, 4e (IC50 acetylcholinesterase [AChE] = 0.69 µM and butyrylcholinesterase [BChE]= 26.00 µM) and 4h (IC50's AChE= 7.04 µM and BChE= 16.06 µM) were found to be the most potent AChE and BChE inhibitors, respectively.


| INTRODUCTION
Alzheimer's disease (AD) is a progressive and debilitating neurodegenerative disorder that is one of today's most pressing public health issues (Prince et al., 2016).With an aging global population, the prevalence of AD continues to rise, and the burden it places on individuals, families, and healthcare systems is increasing exponentially (Jia et al., 2018).Characterized by cognitive decline, memory loss, and a range of behavioral and psychological symptoms, AD robs individuals of their independence and quality of life (Cummings et al., 2014).The societal impact of AD is profound, encompassing not only the emotional and economic burdens on families but also straining healthcare resources worldwide (Wimo et al., 2017).
There are various pathophysiological hallmarks of AD have all been identified.Such as acetylcholine (ACh) deficiency, neuroinflammation, oxidative stress (OS), metal ion dyshomeostasis, and protein dysregulations (amyloid-β [Aβ] aggregation and tau hyperphosphorylation) (Blaikie et al., 2019;Deture & Dickson, 2019;Kumar et al., 2015).The mechanisms underpinning these pathological changes remain a subject of intense investigation.While the exact etiology of AD is still not fully understood, it is clear that a multifactorial interplay of genetic, environmental, and lifestyle factors contributes to disease development (Querfurth et al., 2010).Genetic studies have highlighted the importance of genes such as amyloid precursor protein, presenilin 1 (PSEN1), and presenilin 2 (PSEN2) in familial forms of the disease, while sporadic AD is associated with a more complex genetic landscape (Guerreiro et al., 2014).Medicinal chemistry plays a crucial role in the quest for new AD therapies.It involves the design, synthesis, and evaluation of small molecules with the potential to modulate specific biological targets involved in AD pathogenesis.Recent advances in our understanding of the molecular and cellular mechanisms underpinning AD have provided a wealth of potential drug targets, including Aβ aggregation, tau hyperphosphorylation, neuroinflammation, and OS (Selkoe & Hardy, 2016).
Current therapeutic strategies for AD primarily focus on symptom management and temporary relief of cognitive impairment, typically involving the use of cholinesterase (ChE) inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists (Birks, 2006).
These medications, although valuable in some cases, do not address the fundamental disease processes, and their efficacy is limited (Reisberg et al., 2003).Therefore, there is an urgent need for innovative and disease-modifying treatments to halt or slow the progression of AD (Cummings et al., 2019).
In recent years, AD drug development studies have shifted toward the MTDL (multitarget-directed ligands) strategy, moving away from the "one-target, one-drug" approach (Cavalli et al., 2008).(Chen et al., 2017;Li et al., 2020).As a result, BChE inhibition has received increased attention in recent AD drug development studies (Jing et al., 2019;Panek et al., 2023).Therefore, ChE continues to be the principal biological target for the development of MTDLs, typically combining ChE inhibitory activity with activity against disease-modifying or neuroprotective targets (Wichur et al., 2020).
On the other hand, OS has been identified as one of the early and triggering pathophysiology of AD.It also contributes significantly to neurodegeneration by causing damage to biological macromolecules.As a result, OS is a key player in the progression of AD and is closely linked to other pathophysiological pathways (Singh et al., 2019;Zhao & Zhao, 2013).As a result, compounds with antioxidant activity are thought to be useful in the development of MTDLs against AD.
An increasing amount of data points to the involvement of metal dysregulation in the intricate pathogenic network linked to 's AD.Fe, Cu, and Zn ions are significant, according to the metal hypothesis, because these three transition metals have been connected to the OS, Aβ, and tau pathologies in AD (Kenche et al., 2011).Metal chelators are also considered valuable for drug development studies due to their synergistic effects on various mechanisms of AD.
Recognized as a classic privileged and versatile structure for drug design and synthesis, recent studies have highlighted the potent effects of certain quinoline derivatives, including ChE inhibition, antioxidant properties, anticonvulsant, anti-inflammatory, and central nervous system (CNS) effects (Li et al., 2023;Matada et al., 2021).
Furthermore, molecular docking studies suggested that the quinoline moiety can bind to the PAS of AChE via π−π stacking interactions (Li et al., 2023).Notably, there are established anti-Alzheimer agents containing the quinoline moiety, such as Tacrine and Clioquinol.
Consequently, we also anticipated an increase in metal-chelating activity with the use of the quinoline scaffold.The quinoline scaffold, connected with tertiary nitrogen-bearing structures known for their ability to interact with the catalytic site of ChE enzymes (Bajda et al., 2013), was utilized by employing a propanehydrazide bridge.
This design choice could contribute to both antioxidant and metal chelator effects (Abouel-Enein et al., 2023).By considering the above-mentioned knowledge and design strategy, we aim to reach possible hit MTDLs for AD in the current study.Following this explained design strategy (Figure 1), we successfully synthesized eight novel N'-(quinolin-4-ylmethylene) propanehydrazide derivatives.

| Chemistry
Eight final compounds were synthesized in this study.In Scheme 1, the route for the synthesis of intermediates and final compounds is illustrated.As a start, at step (i), from the Michael addition reaction of methyl acrylate and appropriate amine derivative 1(a−h), methyl 3-(substitutedamino)propanoate intermediates 2(a-h) were prepared.Subsequently, in step (ii), hydrazide key intermediates 3(a−h) were prepared through hydrazinolysis of the methyl 3-(substitutedamino) propanoate intermediates 2(a−h).Final compounds 4(a−h) were synthesized from the nucleophilic addition of key intermediate 3(a−h) to quinoline-4-carbaldehyde (4).
1 H NMR, 13 C NMR, and high-resolution mass spectra (HRMS) were used for the chemical structure verification of the compounds.conformations is unlikely because of steric crowding of the acyl group and quinoline, as well as coplanarity and eclipsing of the acyl and amide bonds (Munir et al., 2021).Additionally, in the solid form, N-acyl hydrazones of aromatic aldehydes typically adopt the E configuration, and the less hindered E conformation is also favored in solution.However, in less polar solvents like chloroform, the Z isomer can be detected (Palla et al., 1986).Geometric isomers display F I G U R E 1 Design strategy and general structures of the final compounds.
By using modified Ellman's method (Ellman et al., 1961), inhibition percentages of compounds on ChE were evaluated at 10 and 100 μM.
Subsequently, the IC 50 s of the final compounds were assayed and calculated.All the ChE inhibitory activity results were presented in

| In vitro antioxidant activity assays
Compounds were tested for investigating radical scavenging (DPPH assay) as well as oxygen radical absorbance (ORAC-FL assay) capacities.
Results are presented in Unfortunately, compounds that have ChE inhibition (4d−h), showed lower ORAC-FL values compared to inactive compounds (4a−c).

| Metal binding studies
The absorptions in the 230−500 nm range of the compounds were screened to evaluate their Cu(II), Fe(II), and Zn(II) binding capacities.
Spectra of all compounds were presented in Supporting Information Material.Based on the UV-vis spectrophotometry method, any kind of variation in the spectra of the metal-treated ligand when compared to the spectra of the ligand alone is attributed to complexation (Bortolami et al., 2020).
The "difference UV-vis spectra" were replotted and presented in QPPCaco >22 nm/s, # metab <7).For both compounds, QPlogS (4d; −5.835, 4h; −6.198) is in the recommended range by the Qikprop manual.However, according to Jorgensen's rule of three QPlogS should be higher than −5.7.In a similar manner, the QlogPo/w value of compound 4h (5.057) is appropriate according to the Qikprop manual, but it violates Lipinski's rule of five (<5.000) with a minor difference.As a result, in silico ADME predictions revealed that the compounds generally have acceptable drug-likeness as well as significant BBB permeation capacity.Unless, as a potential lead compound, water solubility of 4d and 4h may need to be improved.

| CONCLUSION
In this study, designed N'-(quinolin-4-ylmethylene)propanehydrazide derivatives were synthesized and investigated for their anti-ChE, antioxidant potential with the aim of obtaining new hit compound.were dissolved in EtOH (20 mL).The mixture was refluxed for 4 h.After completion, the mixture was concentrated under reduced pressure, then diethyl ether-petroleum ether was added to the flask, precipitate was filtered.The filtered solid was recrystallized from the appropriate solvent.

| ChE inhibition assay
AChE (electric eel) and BChE (equine serum) from Sigma-Aldrich were employed in the assays, following the method previously reported by us (Bardakkaya et al., 2023;Kilic et al., 2023).To determine the IC 50 values, GraphPad Prism software (Version 9.0) was used.Doseresponse curves of the compounds were presented in Supporting Information Material.

| In vitro antioxidant activity assays (DPPH and ORAC-fluorescein)
DPPH and ORAC-FL assays were conducted using our previously reported procedures (Bardakkaya et al., 2023;Kilic et al., 2023).In the DPPH assay, test samples were assayed at 100 μM for a 30 min incubation time.ORAC-FL data are expressed as μmol of Trolox equivalent/ μmol of tested compounds.The assay was carried out in triplicate, and the mean ± SD was computed.

| Metal binding studies
Studies for the ligand-metal binding evaluation was carried out in accordance with our previously reported methodology (Bardakkaya et al., 2023;Kilic et al., 2023).The overlapping spectra of the metaltreated ligand and the control solution of the ligand were visualized, and the resulting wavelength (nm) versus absorbance graphs were included in the Supporting Information Data.

| Evaluation of in silico physicochemical parameters
To obtain low energy conformations of the ligands as well as potential ionization states for pH 7.0 ± 2.0, the LigPrep module was used.Table 3 shows the Qikprop predictions for the top-scoring states of each compound.
T A B L E 3 Predicted properties of compounds.
Based on current knowledge of AD, ChE inhibition is regarded as a critical and practical target for MTDL design.Cognitive impairment in AD is linked to decreased ACh levels.Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors are always required to overcome clinical problems caused by ACh deficiency.Previously, AChE received more attention due to its additional noncatalytic feature.AChE has a more hydrophobic peripheral aromatic site (PAS) than BChE, allowing it to interact with peptides and thus accelerate Aβ pathophysiology.Nonetheless, recent research indicates that AChE is selectively and primarily responsible for ACh hydrolysis in the healthy brain or in the early stages of AD.As the disease progresses in the AD brain, BChE activity increases and eventually replaces AChE

13C
NMR data of the (4a) was presented as a representative of the series.Additionally, to simplify the evaluation of spectral data, 1 H NMR in DMSO-d 6 and DMSO-D 2 O of (4c) was obtained.The data obtained from all analyses of the compounds matched the proposed structures.Common NMR peaks of N'-(quinolin-4-ylmethylene) propanehydrazide derivatives were presented in Figure 2. Acylhydrazone structure, (-C(O)-N-N=C<), can potentially have E/Z geometrical isomers (C=N), cis/trans amide conformers (C(O)-NH), and rotational isomers (N-N).However, the presence of Z (N-N) considerably distinct Rf (retention factor) values.During the characterization of title compounds with TLC and HRMS techniques, only the presence of E isomers was confirmed.But, as a result of the diastereomeric nature of the compounds (E/Z, synperiplanar, and antiperiplanar), explicit sets of certain protons were observed in 1 H NMR spectra with a similar pattern for all compounds.A comparison of CDCl 3 and DMSO-d 6 spectra revealed that the paired NMR peaks were mainly caused by synperiplanar (sp) and antiperiplanar (ap) conformers.In CDCl 3 , the emergence of the Z isomer caused additional multiplicity of some paired peaks, especially aromatic ones. 13C NMR spectra of the (4a) also exhibited duplicated signal sets because of the same reason.

(
4e−h) were found to be more active compared to phenyl-substituted tertiary amine derivatives (4a−d).Exceptionally, 4d (with 4-phenyl 1 H NMR 13 C NMR F I G U R E 2 Common NMR-CDCl 3 peaks of N'-(quinolin-4-ylmethylene)propanehydrazide derivatives.T A B L E 1 Cholinesterase inhibitory activity results of the synthesized compounds.
piperidine) exhibited ChE inhibitory activity among the phenyl-substituted tertiary amine derivatives.According to these findings, the basicity of the tertiary amine atom at the third position of the propane hydrazide chain (piperazine N1 or piperidine N) may be responsible for ChE inhibition.Besides electronic effects, the inclusion of a methylene bridge while changing phenyl-to benzyl-substituted tertiary amine derivatives enables the rotational relaxation of compounds.The activity increase over 100-fold between these two series may be attributed to the rotational freedom of the compounds at the enzyme active site.However, for the benzyl derivatives, the p-methoxy substitution at the phenyl ring (4g) caused the disappearance of the BChE inhibitor activity.Possibly, the loss of activity resulted from the steric hindrance of the methoxy substituent.In terms of ChE inhibitory selectivity, active compounds were found to possess dual ChE inhibitory activity.Additionally, their AChE selectivity was relatively higher compared to BChE.The most potent AChE inhibitor (4e) and the most potent BChE inhibitor (4h) were chosen for the kinetic study to determine their inhibition mechanism.The reciprocal Lineweaver-Burk plots were presented in Figure3.As we can see from the figure, with the concentrations of the compounds increasing, both the slopes (decreased V max ) and intercepts (higher Km) were increased, and these straight lines intersected at the second quadrant.This pattern indicated a mixed-type inhibitory behavior of both 4e and 4h.Additionally, Ki values for them were calculated from Lineweaver-Burk secondary plots as 0.685 and 2.579 μM, respectively.

Figure 4 .
Figure 4. To obtain "difference UV-vis spectra," the absorbances of the metal and the ligand were individually subtracted from the absorbance of the metal-ligand mixture.We discovered that all compounds

Table 2
. Compounds did not exhibit radical scavenging activity (≤5%) at 100 μM.However, they demonstrated good ORAC-FL values ranging from 0.359 to 6.257 Trolox equivalents.F I G U R E 3 Lineweaver-Burk plots of 4e for eeAChE (a) and 4h for eqBChE (c) and slope versus concentration replots of 4e (b) and 4h (d).AChE, acetylcholinesterase; BChE, butyrylcholinesterase.T A B L E 2 Antioxidant activity results of the compounds.