Palladium-catalyzed synthesis and anti-AD biological activity evaluation of N-aryl-debenzeyldonepezil analogues

A series of novel N-aryl-debenzeyldonepezil derivatives (1–26) were designed and synthesized as cholinesterase inhibitors by the modification of anti-Alzheimer’s disease drug donepezil, using Palladium catalyzed Buchwald-Hartwig cross-coupling reaction as a key chemical synthesis strategy. In vitro cholinesterase inhibition studies demonstrated that the majority of synthesized compounds exhibited high selective inhibition of AChE. Among them, analogue 13 possessing a quinoline functional group showed the most potent AChE inhibition effect and significant neuroprotective effect against H2O2-induced injury in SH-SY5Y cells. Furthermore, Compound 13 did not show significant cytotoxicity on SH-SY5Y. These results suggest that 13 is a potential multifunctional active molecule for treating Alzheimer’s disease.


Introduction
Alzheimer's disease (AD) is a common age-related neurodegenerative brain disorder characterized by progressive cognitive dysfunction, memory loss, and restrictions in daily activities (Ross and Poirier, 2004).According to the World Health Organization (WHO), approximately 50 million people suffer from AD disease around the world, and the number of patients will reach more than 150 million in 2050 as the population ages (Du et al., 2022).As one of the neurodegenerative disorders, the exact pathogenesis of AD is not fully known.Many studies have revealed that AD might involve abnormality of multiple neurochemical factors, such as low levels of acetylcholine (ACh), the amplitude of unusual deposits of βamyloid (Aβ) peptide, hyperphosphorylated tau protein, neuronal apoptosis, and so on (Li et al., 2020;Perry et al., 2002;Lee et al., 2018;Mozaffarnia et al., 2020).Many hypotheses have been proposed based on these pathogenic factors to treat AD.The typical cholinergic hypothesis suggests that the selective loss of cholinergic neurons in the brain results in cognitive impairment in AD, whereas improvement of the central cholinergic transmission is an effective treatment strategy (Du et al., 2018).Among the several approaches attempted to enhance ACh levels in the brain, acetylcholinesterase inhibitors (AChEIs) have been proven in patients with mild or moderate AD (Anand and Singh, 2013).Up to now, donepezil, galantamine, rivastigmine, and tacrine as AChEIs have been approved by FDA used in AD treatment (Figure 1) (Pardo-Moreno et al., 2022).Although tacrine regrettably had to be withdrawn from the market in 2005 due to its hepatotoxicity (Summers et al., 1989), all four approved AChEIs possess a basic nitrogen atom.Notably, the basic nitrogen is protonated at physiological pH to provide a temporary positive charge and interacts with the active site of AChE, generating an impact on the overall affinity of AChE for the ligands (Peauger et al., 2017).Therefore, the incorporation of a nitrogen atom in bioactive compounds to recreate their unique properties has been regarded as an effective AChEIs medicinal development route.
Donepezil, approved by the FDA in 1996, is a second-generation specific reversible AChEI for the treatment of mild and moderate AD, showing good patient tolerance and mild adverse effects (Seltzer, 2007).The crystal structure of donepezil-rhAChE complex showed benzyl moiety (Unit A) in donepezil as an important site forms π-π interactions with residues of the active site (Akhoon et al., 2020).Furthermore, the piperidine ring (Unit B) and indanone (Unit C) reach out to π-π stacking interactions with peripheral anionic and catalytic active sites of AChE (Cheung et al., 2012).These findings contribute to the cognition of potential pharmacophores interacting with the residues of the active site.With increased research enthusiasm in terms of functional AChEI molecule design, using donepezil's key structural moieties to develop novel AChEIs has drawn immense attention.For instance, fluorine-containing donepezil analogues fluoropezil possessing a longer drug-target residence time has been developed, which is preparing to enter phase II trial (Zhou et al., 2021;Qian et al., 2023).In our previous study, various nitrogen-containing functional groups were introduced into Unit C to provide a series of α-aryl donepezil analogues owning high AChE inhibition (Wan et al., 2021a).In order to find better anti-AD compounds, considering that the aromatic ring in Unit A is a vital binding site, we envisaged using aromatic nitrogen heterocycles in place of the phenyl group might form the additional bonding force to strengthen the AChE inhibitory activity.Furthermore, the Palladiumcatalyzed Buchwald-Hartwig cross-coupling reaction is one of the powerful approaches for constructing abundant structures libraries via the formation of C-N bonds and provides an important chemical ligation strategy for the present study (Dorel et al., 2019).
Therefore, as part of our ongoing research program toward exploiting novel anti-AD bioactive molecules (Wan et al., 2021a;Wan et al., 2021b;Wan et al., 2021c;Miao et al., 2021;Xu et al., 2023;Zhang et al., 2023), the present work pay attention to the AChE inhibitory activity affected by the benzyl moiety in donepezil, and proposed the structural modification of donepezil for efficiently introducing aromatic nitrogen heterocycles or aryl functional groups located at N position via Palladium-catalyzed Buchwald-Hartwig reaction, hoping to find the potential bioactive molecules.Herein, the synthesis and pharmacological evaluation of donepezil analogues are reported.

Chemistry
To conduct the biological activity evaluation of a new class of N-aryl-debenzeyldonepezil derivates, a set of target compound 1-26 were prepared by employing debenzeyldonepezil as a key intermediate and introducing an aryl functional group attached to the nitrogens of piperidine ring via Buchwald-Hartwig C-N cross-coupling.At the beginning of this study, donepezil was reacted with 10% Pd/C in methanol under a hydrogen atmosphere at room temperature for 4 h resulting in the generation of debenzeyldonepezil in 90% yield (Scheme 1).Next, debenzeyldonepezil and 2-bromopyridine were chosen as model substrates to explore the optimum Buchwald-Hartwig reaction conditions for the preparation of aiming N-aryldebenzeyldonepezil.According to previous reports (Heravi et al., 2018;Dorel et al., 2019), after examination of ligands, bases, and solvents, we found that the ligand played a vital role in this C-N crosscoupling process.As shown in Table 1, the monophosphine ligands PPh 3 and PCy 3 afforded little product 1 (entries 1 and 2).The yield of the derived product was dramatically improved when using Mephos as a ligand.Therefore, the N-arylated product 1 could be recieved in as high as 89% yield in the presence of Pd(OAc) 2 (10 mol%), Mephos (20 mol%), and t-BuOK (3 equiv.) in 1,4-dioxane at 90 °C for 17 h (entry 3).Then, the influence of different bases and solvents on the reaction process was investigated.When other bases such as t-BuONa, K 2 CO 3 , and KF were used instead of t-BuOK, the desired crosscoupling product 1 was obtained in unsatisfactory yields (entries 4-6).Similarly, replacing 1,4-dioxane with DME and THF provided a lower yield of 37% and 23%, respectively (entries 7 and 8).
Subsequently, Palladium-catalyzed Buchwald-Hartwig reactions between debenzeyldonepezil and various aryl bromides were conducted under standard reaction conditions.As exemplified in Scheme 2, the investigated N-heteroaryl bromides smoothly provided the corresponding N-arylated debenzeyldonepezil products 1-13 in excellent yields (65%-89%).Both electrondonating groups (methyl and methoxy) and halogen fluorine at meta-, para-or ortho-position of 2-bromopyridine were well tolerated.The bromide substrates bearing a multisubstituted ring were tested and afforded the expected products 3 and 5 in 77% and 69% yields, respectively.Meanwhile, quinoline groups were suitable as well in this process to furnish corresponding products in yields of 72% (11), 75% (12), and 68% (13).In addition to pyridine and quinoline bromides, the substrates carrying phenyl rings were examined and provided the conjugate products in 50-92% yield (14-26).The unsubstituted benzene-bromide furnished the derivative 14 in 89% yield.Similarly, the phenyl bromides bearing electron-donating groups (methyl, methoxy, and tertiary butyl) (15-18) worked efficiently.The substrates carrying electronwithdrawing groups (fluorine and trifluoromethyl) (19-23) were in lower yields in comparison to that of 15-18.Compounds 24 and 25 equipped biphenyl groups were successfully prepared in yields of 79% and 69%, respectively.Furthermore, the reaction of debenzeyldonepezil with 1-bromo-4-methoxynaphthalene gave compound 26 an 80% yield.Structures of these N-aryldebenzeyldonepezil analogues were characterized by NMR and HRESIMS spectra.

In vitro cholinesterase inhibition studies
The inhibitory activity of N-aryl-debenzeyldonepezil analogues  against AChE (from electric eel) was initially evaluated by means of Ellman 's spectrophotometric method (Ellman et al., 1961), employing donepezil as the positive control.The screening test was conducted at the concentrations of 100 and 50 μM, and the ability of these molecules to inhibit the enzymes is reported in Table 2.When introducing methylpyridine-provided compounds 2-5, the AChE inhibition activity of them was obviously lower than that of pyridine-substituted analogue 1.For the position of the methoxyl substituent group, compounds 8 with 4-methoxy pyridine and 9 with 3-methoxypyridine obtained maintenance in activity, while the activity of 6 (6-methoxypyridine) and 7 (5-methoxypyridine) decreased.The only compound 10 (EeAChE inhibitory rate = 44.1 ± 3.0% at 50 μM) in the series bearing a strong electronwithdrawing group also resulted in a larger decrease of AChE inhibitory activity than analogue 1.The above results implied that the steric effects of these substituent groups possibly combining with electronic effects led to an adverse impact on the inhibition potency for this set of analogues.In short, the N-aryldebenzeyldonepezils 1-10 equipping pyridine ring showed slightly decreased AChE inhibitory capacity, compared to that of donepezil, which revealed that the introduction of pyridine is at a significant disadvantage.
On the other hand, for the analogues possessing the quinoline fragments, compounds 11-13 exhibited favorable inhibitory activities, implying that the presence of this basic atomic group could increase interactions with the active sites of the enzymes.Compound 13 showed the best inhibitory potency, which needs an in-depth investigation.Many studies of AChE crystal structures have confirmed that the active site gorge is located in the deep and narrow "canyon" (Cheung et al., 2012;Liu et al., 2022), which implied that the larger group 2methylquinoline might contribute to occupying the active site and obtaining compound 13 with high activity against AChE.Moreover, most compounds of the phenyl series (14-23) showed moderate inhibition effect against AChE.Introducing electron-withdrawing groups into the benzene ring, as shown in compounds 19-23, improved activity more than that of analogues with electrondonating groups (15-18), probably due to electronic effects.However, two diphenyl analogues (24 and 25) among this subset of compounds seem to be noneffective, showing an EeAChE inhibitory rate of only about 50% at 50 µM.Then, the EqBuChE inhibition effects of these N-aryl-debenzeyldonepezil derivatives were also explored.As expected, all of them showed low inhibitory activity against eqBuChE.

Molecular modelling studies
To explore the binding mechanisms of compound 13 on AChE (PDB ID code: 1acj), a docking study to reveal the sites of binding and relative binging energy was performed.The highest-scored pose of 13 with a binding energy of −6.9 kcal/mol is depicted in Figure 2. Molecular docking results indicated that analogue 13 was suitably filled in the active site of AChE.Two π-alkyl hydrophobic interactions were formed by the quinoline fragment with Tyr 70 and Tyr 121, respectively.The benzene ring from quinoline established a π-anion bond with Asp 72.Moreover, attempt 334 was in a favorable position of stacking with the piperidine ring and quinoline's benzene ring, forming a π-π and a π-alkyl interactions.Comparatively, in the active cavity of BuChE (PDB ID code: 6ASM), only the benzene ring from indanone interacted with Val 348 by π-alkyl interaction, and the carbonyl showed a conventional hydrogen bond with residues Arg 132.The highestscored pose of 13 presented a binding energy of −5.6 kcal/mol (Figure 3).The above results could explain the compound 13 possessed the selective AChE inhibitory activity, which verified the in vitro enzyme inhibitory assay.

Neuroprotection effects against H 2 O 2induced injury in SH-SY5Y cells
The death of neuronal cells is the central abnormality occurring in brains suffering from AD (Niikura et al., 2002).Neuroprotection is a useful way to protect the neuronal cells from damage, which potentially slows down or prevents the progression of AD (Coleman et al., 2004).To explore the therapeutic potential of synthesized analogues 1-26, the neuroprotective effects of 1-26 against H 2 O 2 -induced injury in SH-SY5Y cells were performed.Firstly, the neurotoxicity of compounds 1-26 on SH-SY5Y cell viability was determined using the MTT assay method.As shown in

BBB prediction
Penetrating the blood-brain barrier (BBB) is extremely significant for anti-AD medicines, so the BBB penetration of compounds 1-26 was predicted by using Calculate Molecular Properties (Discovery Studio 2020 Client).The predicted data are listed in Table 5.The level "0" shows that the Brain-Blood ratio is greater than 5:1, the value "1" expresses that the Brain-Blood ratio is between 1:1 and 5:1, and the value "2" expresses that the Brain-Blood ratio is between 0.3:1 and 1:1.Besides compound 11, most of the synthesized compounds are BBB permeable.

Conclusion
In summary, a series of new N-aryl-debenzeyldonepezil analogues  were efficiently synthesized in modification of donepezil by using Palladium-catalyzed Suzuki-Miyaura crosscoupling reaction as a key synthetic method.The cholinesterase inhibition and neuroprotective effects of these analogues were evaluated to develop potential candidate molecules for the treatment of AD.Among them, compound 13 exhibited good a Percent inhibition data are the mean ± SD, of three independent experiments each performed in duplicate.
Frontiers in Chemistry frontiersin.org06 Xu et al. 10.3389/fchem.2023.1282978selective inhibitory activities against AChE, which might be attributed to the introduction of nitrogen-containing aromatic heterocycle.Molecular modeling was performed to explain the possible interaction between compound 13 and AChE.The prediction of bioavailability disclosed that analogue 13 had a favorable capacity to pass through the blood-brain barrier (BBB).Furthermore, further study showed that active compound 13 displayed non-neurotoxicity and good neuroprotective potency against H 2 O 2 -induced injury in SH-SY5Y cells.These results showed that compound 13 could be developed as an efficient multifunctional potential active molecule for the treatment of AD.

Materials and methods
All reactions were conducted under an inert atmosphere of dry argon.Anhydrous THF was purchased from Aladdin and used without further purification.Unless otherwise noted, the reagents and solvents used in this article were all commercially available analytical or chemical grades and used directly without any purification.Reactions were monitored by thin layer chromatography (TLC) on silica gel plates (GF 254) using UV light to visualize the course of the reactions.Silica gel H (Qingdao Sea Chemical Factory, Qingdao, PR China) was used for column chromatography.NMR spectra were recorded by using a Bruker AV 400 or 600 nuclear magnetic resonance instrument.Chemical shifts (δ) were recorded in ppm relative to TMS as the internal standard.HRESIMS spectra were recorded on a Waters Acquity UPLC/Q-TOF micro mass spectrometer.

Synthesis of N-aryl-debenzeyldonepezil analogues 1-26
The suspension of Pd(OAc) 2 (5 mol%) and Mephos (10 mol%) in anhydrous 1,4-dioxane (3 mL) was stirred at 30 °C under an argon atmosphere for 2 h to obtain a brown solution.The brown solution   a Data are the means ± SD, of at least three determinations.
Frontiers in Chemistry frontiersin.orgwas added to a 10 mL sealed dry reaction vial containing debenzeyldonepezil (1 mmol), naphthalenyl, bromides (1.5 mmol), and t-BuOK (3 mmol) via a syringe.The reaction mixture was stirred for 17 h at 90 °C before quenching with two drops of H 2 O.Then, the mixture was diluted with ethyl acetate (3 mL), and filtered over a pad of MgSO 4 .The pad was rinsed with ethyl acetate, and the mixture was concentrated in vacuo.The reaction crude product was purified by flash column chromatography using PE-EA (2:1-1:2) to obtain the N-aryldebenzeyldonepezil analogues 1-26 (50%-89% yields).

Cell culture and treatment
The neuroprotective activity was evaluated in human neuroblastoma SH-SY5Y cells which were treated with H 2 O 2 .Human SH-SY5Y neuroblastoma cells were obtained from ATCC (Manassas, VA, United States) and were cultured at 37 °C in a humidified atmosphere of 5% CO 2 , in Gibcotm Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heatinactivated fetal bovine serum.The viability of the cells was determined by MTT assay.MTT was purchased from Sigma-Aldrich (St. Louis, MO, United States).SH-SY5Y cells were seeded in 96-well plates at a density of 1 × 10 5 cells/well for 12 h.Cells were pretreated with 50 μM of each test compound.After 24 h of incubation, hydrogen peroxide solution (H 2 O 2 , 700 μM) was added and incubated for 4 h.Then, MTT (5 mg/mL) was added to each well.After a 4 h treatment, the supernatant was removed, and DMSO (150 μL/well) was added to dissolve the insoluble formazan crystals.Then, the plate was vibrated, and the absorbance was measured at 490 nm using a microplate reader.

Molecular docking analysis
The ligand-protein docking was guided by Discovery Studio 2020 Client and PyRx software to predict the binding poses of the ligand in the active site of AChE.The 3D models of AChE (PDB ID: 1acj) and BuChE (PDB ID: 6ASM) were obtained from Protein Data Bank (http://www.rcsb.org/).The 3D models of the protein of AchE and BuChE were further optimized by Discovery Studio 2020 Client to minimize energy.The AutoDock Vina was employed for docking calculations using the default parameters.The blood-brain barrier (BBB) penetration was predicted by the Discovery Studio 2020 Client.

FIGURE 2
FIGURE 2 Molecular modeling of 13 in the active cavity of AChE (PDB ID: 1acj).(A) Residues of the active site involved in docking, and types of bonds involved in docking: π-π bond is shown as pick dashed lines, π-Alkyl bonds are shown as yellow dashed lines, and π-Anion bonds are shown as purple dashed lines; (B) Best docking positioning of compound 13 with AChE.

FIGURE 3
FIGURE 3 Molecular modeling of 13 in the active cavity of BuChE (PDB ID: 6ASM).(A) Residues of the active site involved in docking, and types of bonds involved in docking: π-π bond is shown as a pink dashed line and conventional hydrogen bonds are shown as green dashed lines; (B) Best docking positioning of compound 13 with BuChE.

TABLE 1
Optimization of the N-arylation reaction conditions a .

Table 3
, N-aryldebenzeyldonepezils 6-8, 17-22, and 26 did not affect the cell viability of SH-SY5Y cells even at concentrations up to 50 μM, indicating that they were not neurotoxic.Then, non-cytotoxic compounds(6-8, 17-22, and 26) were examined for their potential neuroprotective activity on damaged SH-SY5Y cellular models induced by 700 μM H 2 O 2 .In the model group, 700 μM H 2 O 2 significantly reduced cell viability and decreased to 61% compared to the control group.The protective effect of analogues

TABLE 4
Neuroprotective effect of non-cytotoxic compounds against H 2 O 2 -induced injury in SH-SY5Y cells a .Neuroprotective effect of compounds (50 μM) on H 2 O 2 -induced (700 μM) neurotoxicity in SH-SY5Y, cells.The cell viability in the control was taken as 100%, and the average value of cell viability under H 2 O 2 exposure in the model group was 61.2% ± 1.3% (n = 3).
a b Data are the means ± SD, of at least three determinations.

TABLE 5
The BBB prediction of compounds by Discovery Studio 2020 Client.