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. 2021 Apr 2;6(14):9960–9972. doi: 10.1021/acsomega.1c01404

Late-Stage Modification of Medicine: Pd-Catalyzed Direct Synthesis and Biological Evaluation of N-Aryltacrine Derivatives

Lin-Xi Wan 1, Yong-Qi Zhen 1, Zhen-Xiang He 1, Yang Zhang 1, Lan Zhang 1, Xiaohuan Li 1, Feng Gao 1,*, Xian-Li Zhou 1,*
PMCID: PMC8047743  PMID: 33869976

Abstract

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A new series of N-aryltacrine derivatives were designed and synthesized as cholinesterase inhibitors by the late-stage modification of tacrine, using the palladium-catalyzed Buchwald–Hartwig cross-coupling reaction. In vitro inhibition assay against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) demonstrated that most of the synthesized compounds had potent AChE inhibitory activity with negative inhibition of BuChE. Among them, N-(4-(trifluoromethyl)phenyl)-tacrine (3g) and N-(4-methoxypyridin-2-yl)-tacrine (3o) showed the most potent activity against AChE (IC50 values of 1.77 and 1.48 μM, respectively). The anti-AChE activity of 3g and 3o was 3.5 times more than that of tacrine (IC50 value of 5.16 μM). Compound 3o also displayed anti-BuChE activity with an IC50 value of 19.00 μM. Cell-based assays against HepG2 and SH-SY5Y cell lines revealed that 3o had significantly lower hepatotoxicity compared to tacrine, with additional neuroprotective activity against H2O2-induced damage in SH-SY5Y cells. The advantages including synthetic accessibility, high potency, low toxicity, and adjunctive neuroprotective activity make compound 3o a new promising multifunctional candidate for the treatment of Alzheimer’s disease.

Introduction

Alzheimer’s disease (AD), a complex neurodegenerative disorder of the brain, is the most common cause of dementia and death in aging people, affecting a large number of elderly populations.1,2 The Alzheimer’s Disease International (ADI) estimated that there are about 50 million people suffering from AD worldwide. It also predicted that the incidence of AD will significantly rise to 152 million by 2050, which makes AD a health issue.3 The exact underlying cause of AD is obscure due to its complexity, and diverse factors are believed to play an important role in the onset and progression of the disease.4 Due to the unclear cause of this disease, no drug is able to stop or reverse the progression of AD. The approved drugs in clinics only improve the symptoms of AD. Thus, finding better strategies for the treatment of AD has become a worldwide endeavor.

The most promising approach to the symptomatic treatment of AD is the inhibition of acetylcholinesterase (AChE), which catalyzes the hydrolysis of acetylcholine to choline and acetate.4 Among the total five approved drugs for the clinical management of AD, four are AChE inhibitors (Figure 1). Tacrine was the first AChE inhibitor approved for the treatment of AD, which blocks the degradation of cholinergic nerves in the cerebral cortex and hippocampus to increase the cholinergic transmission.5,6 However, although tacrine has positive therapeutic effects, it has been demonstrated to increase serum alanine aminotransferase levels in about 30% of patients, instigating hepatotoxicity.7 This shortcoming seriously limited the use of tacrine in therapeutic applications. Thus, the development of tacrine derivatives with higher inhibitory activity and nonhepatotoxicity has drawn immense attention because of their affinity for both the active and peripheral sites of AChE.4,8−13

Figure 1.

Figure 1

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Approved drugs for the treatment of AD.

Over the past years, numerous tacrine derivatives have been synthesized to find better anti-AD lead compounds with lower hepatotoxicity. Some methods for the modification of tacrine focused on tacrine scaffolds such as benzene rings or hexane rings.2,14,15 In addition to this, multifunctional tacrine hybrids have been the most widely studied tacrine derivatives.11 Tacrine was conjugated with different moieties such as lophine, melatonin, 8-hydroxyquinoline, chromenes, coumarin, NO donor groups, caffeic acid, propargylamine, β-carboline, vilazodone, and carbohydrates to provide the effective MTDL strategy for the improvement of the therapeutic application of tacrine.16−24 The representative hybrids were bis(n)-tacrine linked by an alkylene chain, as reported by Pang et al., which were up to 1000-fold more potent than tacrine since they have double interactions in the cholinesterase with both CAS and PAS.8 Since then, the 9-NH2 group has been a key site for synthesizing tacrine analogues.24 It has been reported that the 1,2,3-triazole moiety in tacrine–coumarin hybrids linked to 1,2,3-triazoleas could bind to Asp 72 in AChE to enhance the interaction of hybrids and enzymes.25 Aromatic groups such as phenyl, pyridinyl, and quinolinyl are important pharmacophores in the field of medicinal chemistry. We believe that they could play the same role as 1,2,3-triazoleas to bind with Asp 72 in AChE. Encouraged by these findings, we designed and synthesized a series of novel N-aryltacrine derivatives, hoping to find better anti-AD lead compounds.

From another view point, all synthesis strategies mentioned above started from intermediates. Up to now, only a few literature studies have reported the direct modification of tacrine, which should be a highly efficient way to obtain tacrine derivatives.26−30 Amidation and nucleophilic substitution of halogens are the only reported methods to modify the 9-NH2 site of tacrine so far. Only one literature study reported the direct conjugation of 9-NH2 of tacrine with the aromatic group.31 Palladium-catalyzed C–H activation is a highly efficient method to form C–C, C–N, or C–O bonds.32−35 On these bases, we proposed the late-stage modification of tacrine, hoping to the direct conjugation of 9-NH2 in tacrine with aromatic groups using the palladium-catalyzed Buchwald–Hartwig cross-coupling reaction. Herein, we designed and developed 34 new N-aryltacrine derivatives (3a–t, 5a–n). Their synthesis and pharmacological evaluation are reported in this study, including their AChE and BuChE inhibition activity, cytotoxicity on HepG2 cells, and neuroprotection study in vitro. Moreover, molecular docking studies along with the prediction of blood–brain barrier (BBB) penetration of the most potential anti-AD compound 3o were also investigated.

Results and Discussion

Chemistry

The target compounds were synthesized utilizing the Buchwald–Hartwig cross-coupling reaction, as illustrated in Schemes 1 and 2. Tacrine was first prepared according to the literature with a total yield of 70%.36 It was then treated with different aryl bromides or heterocyclic aryl bromides in the presence of Pd(OAc)2, MePhos, and t-BuOK in the solution of dioxane at 100 °C for 12 h to obtain the desired tacrine derivatives. The observation of good in vitro inhibition activity on AChE and BuChE of some heterocyclic aryl tacrine derivatives (3o, 3r) encouraged us to synthesize more tacrine mimics containing other modified tacrine cores to substitute at the 9-position of pyridine or quinoline derivatives. Thus, the modified tacrine cores 2-methyl-1,2,3,4-tetrahydroacridin-9-amine (4A), 7-chloro-1,2,3,4-tetrahydroacridin-9-amine (4B), and 6-methyl-1,2,3,4-tetrahydroacridin-9-amine (4C) were synthesized in the same manner as previously described for the synthesis of tacrine. The same conditions for the Buchwald–Hartwig cross-coupling reaction of these three compounds with 2-bromopyridine or quinoline derivatives afforded required compounds 5a–n.

Scheme 1. Synthesis of Compounds 3a–t.

Scheme 1

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Scheme 2. Synthesis of Compounds 5a–n.

Scheme 2

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Biological Activity

Cholinesterase Inhibition Assay in Vitro

The synthesized tacrine analogues (3a–u, 5a–n) were evaluated in vitro for their anticholinesterase activity against AChE and BuChE in comparison with tacrine as a positive reference drug to determine the potential of these target compounds for the treatment of AD. The results revealed that most tested compounds are potent inhibitors of AChE with dose-dependence (Table 1). In particular, N-(4-(trifluoromethyl)phenyl)-tacrine (3g), N-(4-methoxypyridin-2-yl)-tacrine (3o), 2-methyl-N-(3-methylpyridin-2-yl)-tacrine (5f), 7-chloro-N-(5-methoxypyridin-2-yl)-tacrine (5i), 7-chloro-N-(quinolin-2-yl)-tacrine (5j), and N-(4-methoxypyridin-2-yl)-6-methyl-tacrine (5l) displayed excellent activity with inhibition rates of >90.00%, thus higher than the reference drug tacrine. Comparable to N-(4-(trifluoromethyl)phenyl)-tacrine (3g), N-(3-(trifluoromethyl)phenyl)-tacrine (3i) showed a lower inhibitory activity with the inhibition rate less than 50% at a concentration of 50 μM. Except for compound 3g, no matter the phenyl group was further substituted by o-electron-withdrawing or -donating groups, the N-phenyl (3a–f, 3h) and N-naphthyl (3j, 3k) substituents all exhibited lower activity than tacrine, which indicated that the o-trifluoromethyl group might play an important role in the anti-AChE activity. In general, the N-(pyridin-2-yl)- or N-(quinolin-2-yl)-tacrine derivatives showed better activity than N-phenyl or N-naphthyl tacrine analogs, especially when the 4-position of the pyridinyl group was substituted by the methoxy group (3o, inhibition rate of 96.21%). In addition, N-(quinolin-2-yl)-tacrine (3r) also had an identical inhibition rate with tacrine at a concentration of 100 μM but showed lower activity at 50 μM. The results of the inhibition rates of 5a–m indicated that N-substituents in tacrine had a stronger effect than the modified tacrine cores on anti-AChE activity.

Table 1. Inhibitory Activity toward AChE and BuChE by 3a–t, 5a–n, and Reference Compound.
  AChE inhibition (%)a
 BuChE inhibition [%]a
Compounds [I] = 100 μM [I] = 50 μM [I] = 100 μM [I] = 50 μM
3a 52.21 ± 2.16 12.72 ± 0.13 –31.14 ± 0.50 –59.10 ± 1.76
3bs 72.24 ± 5.21 15.95 ± 0.30 –36.08 ± 0.51 –1.24 ± 0.12
3c 65.00 ± 3.48 10.86 ± 0.27 70.11 ± 1.22 20.00 ± 1.35
3d 50.33 ± 0.70 24.01 ± 0.15 –105.98 ± 1.92 –233.79 ± 1.00
3e 67.41 ± 4.25 38.98 ± 1.33 –108.41 ± 3.26 –159.00 ± 3.63
3f 59.57 ± 1.92 44.35 ± 3.58 –54.26 ± 0.68 –34.15 ± 1.00
3g 94.46 ± 1.71 90.74 ± 5.15 –23.48 ± 0.65 –171.31 ± 7.17
3h 31.89 ± 1.32 10. 88 ± 0.41 –35.00 ± 1.14 –115.21 ± 0.40
3i 67.53 ± 1.39 44.53 ± 0.11 –60.90 ± 2.63 –131.54 ± 3.25
3j 65.11 ± 2.34 56.89 ± 0.19 79.84 ± 5.43 60.13 ± 1.76
3k 70.41 ± 4.41 57.72 ± 1.83 82.62 ± 1.45 46.33 ± 3.58
3l 44.66 ± 3.90 41.31 ± 0.17 –167.99 ± 2.48 –268.07 ± 6.03
3m 65.72 ± 1.06 52.46 ± 0.93 62.44 ± 3.33 5.56 ± 0.14
3n 69.88 ± 1.83 13.05 ± 0.49 –73.42 ± 2.94 –222.00 ± 0.15
3o 96.21 ± 4.11 85.19 ± 3.22 110.21 ± 2.36 78.02 ± 4.22
3p 54.32 ± 3.57 66.49 ± 3.00 65.68 ± 2.27 23.04 ± 0.85
3q 52.75 ± 1.00 46.42 ± 1.91 84.23 ± 7.48 85.75 ± 0.56
3r 83.52 ± 2.31 72.62 ± 0.16 87.95 ± 0.61 105.13 ± 1.57
3s 48.27 ± 1.30 34.71 ± 0.95 –44.53 ± 0.30 –169.43 ± 4.25
3t 46.00 ± 2.00 20.96 ± 0.35 –23.27 ± 0.11 –107.11 ± 2.45
5a 77.41 ± 3.11 79.47 ± 5.32 –117.41 ± 2.00 –218.93 ± 5.73
5b 66.60 ± 5.91 49.88 ± 1.07 –61.39 ± 0.54 –113.05 ± 1.74
5c 23.75 ± 0.60 22.22 ± 0.85 –55.11 ± 0.52 –112.36 ± 0.17
5d 38.88 ± 1.56 27.00 ± 1.80 –135.11 ± 1.21 –23.45 ± 1.34
5e 15.21 ± 1.42 33.62 ± 0.41 –38.76 ± 1.96 –175.23 ± 4.04
5f 91.11 ± 5.92 89.77 ± 1.04 –139.40 ± 3.23 –291.62 ± 5.65
5g 67.72 ± 2.46 39.28 ± 1.10 97.11 ± 0.68 53.73 ± 1.06
5h 37.60 ± 2.13 33.64 ± 0.60 –130.67 ± 0.62 –234.24± 6.16
5i 97.62 ± 3.92 72.49 ± 4.03 –47.14 ± 1.22 –63.77 ± 0.45
5j 89.87 ± 2.00 72.82 ± 1.04 –55.98 ± 2.66 –136.74 ± 3.24
5k 59.34 ± 1.53 35.91 ± 1.26 –26.64 ± 5.43 –251.53 ± 1.72
5l 98.50 ± 3.77 75.98 ± 0.14 –91.92 ± 1.47 –210.92 ± 3.5t
5m 62.10 ± 4.28 64.11 ± 3.08 –39.68 ± 2.43 –78.17 ± 6.08
5n 44.10 ± 2.00 25.26 ± 0.41 –49.24 ± 3.35 –88.55 ± 0.12
tacrine 84.32 ± 2.52 90.18 ± 4.42 107.12 ± 2.97 104.33 ± 0.13
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a

AChE from Electrophorus electricus and BuChE from equine serum were used. Percent inhibition data are the mean ± SD of three independent experiments each performed in duplicate.

Most of the tested compounds had negative effects on anti-BuChE activity. The inhibition rates of some analogues are even lower to −200% (3d, 5a, 5h, 5k, and 5l). N-(4-(Trifluoromethyl)phenyl)-tacrine (3g) presented a higher inhibition rate than tacrine on AChE but had a negative effect on BuChE with an inhibition rate of −171.3%, while the inhibition rate of the reference drug was 104.33% at a concentration of 50 μM. Four analogues (3c, 3j, 3m, and 3p) had moderate anti-BuChE activity, while compounds 3k, 3q, and 3r exhibited good activity with inhibition rates >80.00%. The inhibitory activity of N-(5-chloro-4-methylpyridin-2-yl)-tacrine (5g) on BuChE was comparable to tacrine at a concentration of 100 μM. Interestingly, N-(quinolin-2-yl)-tacrine (3r) showed better anti-BuChE activity with an inhibition rate of 105.13% at 50 μM than at 100 μM (87.95%). It was worth noting that 3o exhibited excellent inhibition activities on both AChE and BuChE simultaneously, compared with tacrine.

To explore the anti-ChE potential in more detail, the more active compounds with inhibition rates >80.00% listed in Table 1 were further determined for median inhibition concentrations (IC50) on both AChE and BuChE. The results are shown in Table 2. Gratifyingly, most tested compounds appeared as excellent inhibitors of AChE, exhibiting inhibitory activity much higher than the reference drug (tacrine). In particular, N-(4-(trifluoromethyl)phenyl)-tacrine (3g) and N-(4-methoxypyridin-2-yl)-tacrine (3o) with IC50 values of 1.77 and 1.481 μM, respectively, were found to be the most potent compounds against AChE. These two compounds were both 3.5-fold more potent than the reference drug tacrine. The anti-AChE activity of compounds 3r, 5f, and 5i was comparable to that of tacrine. Along with the result of compound 3o, it was demonstrated that the introduction of the N-(pyridin-2-yl)- or N-(quinolin-2-yl)- substituent at the nitrogen of C9 position in tacrine or tacrine mimics might improve the anti-AChE activity. Based on the IC50 values listed in Table 2, the tested compounds showed a weaker inhibitory activity for BuChE when compared to the reference drug tacrine. Because of the negative inhibitory activity against BuChE, the IC50 value of compound 3g was not determined. Interestingly, the most potent compound against AChE (compound 3o) displayed the highest activity toward BuChE.

Table 2. IC50 Values of Compounds against AChE and BuChE in Comparison with Tacrine.

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a

AChE from electric eel and BuChE from equine serum were used. IC50, inhibitor concentration (means ± SD of three experiments) for 50% inactivation of AChE or BuChE.

b

n.d. = not determined.

Cytotoxicity on HepG2 Cells

Tacrine was limited in the clinic since the serious hepatotoxicity.7 The goal of the present work was to provide new tacrine-like candidates with potent anti-AD activity and lower toxicity. Thus, the in vitro cytotoxicity of the synthesized tacrine-derived compounds on the liver hepatocellular cell line (HepG2) by MTT assay at a concentration of 50 μM was examined to verify the hepatotoxicity of these compounds.30,31 Tacrine was used as the positive control. The inhibitory rates of the tested compounds on HepG2 cells are outlined in Table 3. The obtained results showed that most of the tested compounds displayed significantly less toxicity than tacrine. Encouragingly, the most potential anti-AD compounds 3g and 3o also exhibited lower hepatotoxicity than tacrine.

Table 3. Cytotoxicity of Compounds 3a–t and 5a–n on HepG2 Cellsa.
compounds inhibitory rate (%) (50 μM) compounds inhibitory rate (%) (50 μM) compounds inhibitory rate (%) (50 μM)
3a 5.00 ± 0.30 3m 2.51 ± 0.11 5d 77.33 ± 2.53
3b 59.65 ± 2.44 3n 10.43 ± 0.24 5e –29.24 ± 0.33
3c –30.91 ± 1.54 3o 4.25 ± 0.26 5f –10.12 ± 0.34
3d –31.53 ± 0.45 3p 11.57 ± 0.28 5g –22.92 ± 0.74
3e –1.23 ± 0.16 3q 27.49 ± 1.06 5h –7.85 ± 0.36
3f 18.73 ± 0.64 3r 0.83 ± 0.12 5i –3.87 ± 0.18
3g –0.52 ± 0.16 3s 16.72 ± 0.65 5j 4.32 ± 0.14
3h 34.97 ± 1.54 3t 9.55 ± 0.56 5k –30.95 ± 1.16
3i 34.43 ± 1.72 5a 4.88 ± 0.27 5l –9.57 ± 0.28
3j 66.36 ± 2.85 5b 6.08 ± 0.10 5m –11.40 ± 0.57
3k 84.16 ± 2.27 5c –10.95 ± 0.34 5n –10.10 ± 0.50
3l 6.14 ± 0.23        
tacrine     49.91 ± 0.61    
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a

The cell viability in the control was taken as 100%, and the average value of cell viability under H2O2 exposure was 48.7 ± 2.0% (n = 5).

Neuroprotective Activity of the Synthesized Compounds against H2O2-Induced Cell Death in SH-SY5Y Cells

It has been reported that the protection against oxidative stress may be useful in the management of AD.28 Thus, the neuroprotective potential of the synthesized compounds 3a–t and 5a–n against H2O2-induced cell death in SH-SY5Y cells was evaluated in comparison with tacrine. The SH-SY5Y cells were pretreated with the compounds (50 μM) for 1 h, before treatment with H2O2 (50 μM). The cell viability was measured using MTT assay. As seen in Table 4, half of the tested compounds displayed neuroprotective activity on H2O2-induced SH-SY5Y cell damage. In particular, the percentage of surviving SH-SY5Y cells decreased to 48.7% after treatment with H2O2 (500 μM) but increased to 70.33% and 72.51% following the treatment with 50 μM 3l and 3m, respectively, while the cell viability of tacrine was 44.00%. One of the most potential inhibitors of AChE (3g) did not show any neuroprotective activity with a cell viability of 47.6%, which was comparable to the model group (48.7%). Compounds 3j and 3k with N-(naphthalenyl) along with N-(quinolin-5-yl)-tacrine (3t) even increased the apoptosis of SH-SY5Y cells with the cell viability of 37.31% and 31.59%, respectively. Compounds 5g–j with 7-chloro-tacrine cores and compounds 5k-m with 6-methyl-tacrine cores also showed no neuroprotective activity, and 5h was a little harmful to SH-SY5Y cells. However, it was notable that all N-(pyridin-2-yl)-tacrines (3l–q) possessed good neuroprotective activity, with cell viability ranging from 61.45% to 72.51%. Compared to tacrine, the result indicated that the most potential anti-AChE compound 3o not only displayed low hepatotoxicity but also exhibited neuroprotective activity.

Table 4. Neuroprotective Effects of Compounds 3a–t and 5a–n against H2O2-Induced Neurotoxicity in SH-SY5Y Cells.
compounds cell viabilitya (%) (50 μM) compounds cell viabilitya (%) (50 μM) compounds cell viabilitya (%) (50 μM)
3a 60.42 ± 2.73 3m 72.51 ± 3.23 5d 59.54 ± 2.56
3b 25.83 ± 1.25 3n 61.84 ± 1.55 5e 67.77 ± 5.28
3c 63.14 ± 0.44 3o 63.24 ± 2.93 5f 56.95 ± 2.66
3d 47.74 ± 2.63 3p 69.03 ± 2.55 5g 48.04 ± 4.33
3e 50.47 ± 4.32 3q 61.45 ± 0.64 5h 39.13 ± 1.43
3f 39.26 ± 1.66 3r 62.84 ± 3.37 5i 46.75 ± 2.66
3g 47.64 ± 1.27 3s 52.04 ± 2.26 5j 48.18 ± 1.57
3h 59.76 ± 2.58 3t 29.27 ± 0.98 5k 38.49 ± 1.87
3i 45.25 ± 2.74 5a 58.05 ± 4.35 5l 42.55 ± 2.94
3j 37.31 ± 4.23 5b 41.22 ± 3.32 5m 44.05 ± 3.20
3k 31.59 ± 4.02 5c 56.81 ± 5.51 5n 57.54 ± 4.36
3l 70.33 ± 4.51        
tacrine     44.00 ± 3.2    
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Molecular Docking Study

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 coordinate of the AChE (PDB ID:1acj) was retrieved from the Protein Date Bank at http://www.rcsb.org/. All of the nonprotein atoms were removed by Discovery Studio 2020 Client and then saved as a PDB file. The dehydrated protein file was uploaded using PyRx software and converted into a pdbqt format file to minimize the energy. Finally, Vina was used for docking.

As seen in Figure 2 (all figures were prepared using the Discovery Studio Visualizer), the molecular docking results for the representative compound 3o showed that the ligand was well accommodated in the active site with a binding energy of −11.50 kcal/mol. The ligand was positioned at the bottom of the active site gorge near the catalytic triad. The central pyridine ring and benzene ring were stacked between two aromatic residues Trp84 and Phe330 by π–π stacking interactions. It has been reported that the π–π interactions with Typ84 and Phe330 restrict the entry of substrates into the acyl pocket. Therefore, the inhibition of AchE with compound 3o was mediated by a similar interaction with Typ84 and Phe330. This orientation of the ligand enables the cyclohexyl ring to make π–alkyl interactions with Trp84. In addition, the 4-methoxypyridine ring created anion−π interactions with Asp72 and as a hydrogen acceptor it made interaction with Tyr121. The benzene ring and 4-methoxypyridine ring were well fitted in the hydrophobic pocket that is located in the vicinity of the catalytic triad.

Figure 2.

Figure 2

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(a) Best pose of compound 3o in the active site of AChE. (b) Residues of the active site involved in ligand binding.

The 3D coordinate of BuChE (PDB ID: 6ASM) was retrieved from the PDB. Discovery Studio 2020 Client and Pyrx software were used to prepare proteins and compounds. After selecting the best docking pose for further analysis, docking results showed that the binding energy of compound 3o docked with BuChE was −9.71 kcal/mol. As depicted in Figure 3, the central tacrine core was located in the hydrophobic cavity interacting with the amino acid residue Trp82. The core made a remarkable π–π interaction with the Trp82 of the CAS. Moreover, this orientation of the ligand enables the cyclohexyl ring to make π–alkyl interactions with Trp82, Ala328, TYR 332, and TRP 430. The 4-methoxypyridine ring as a hydrogen acceptor made interaction with Thr120.

Figure 3.

Figure 3

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(a) Best pose of compound 3o in the active site of BuChE. (b) Residues of the active site involved in ligand binding.

Prediction of BBB Penetration

Penetration across the BBB is an essential property for compounds targeting the central nervous system. To predict the BBB permeability of the target compounds 3a–t and 5a–n, the Calculate Molecular Properties (Discovery Studio 2020 Client) was used. The predicted data are listed in Table 5. The value of level “0” showed that the brain–blood ratios were greater than 5:1, and the value of level “1” expressed that the brain–blood ratios were between 1:1 and 5:1. As seen, most of the tested compounds are BBB permeable.

Table 5. BBB Prediction of Compounds by Discovery Studio 2020 Client.
compounds BBB_LEVEL ADMET_BBB compounds BBB_LEVEL ADMET_BBB
3a 0 1.470 3r 0 1.083
3b 0 1.037 3s 0 0.917
3c 0 1.187 3t 0 0.917
3d 0 0.891 5a 0 1.026
3e 0 1.243 5b 0 0.729
3f 0 1.101 5c 0 0.729
3g 0 1.328 5d 0 1.289
3h 0 1.328 5e 0 0.971
3i 0 0.917 5f 1 0.674
3j 0 1.318 5g 1 0.674
3k 0 1.172 5h 0 1.233
3l 0 0.757 5i 0 0.898
3m 0 0.820 5j 0 0.835
3n 0 0.820 5k 0 0.898
3o 1 0.524 5l 1 0.601
3p 1 0.524 5m 1 0.601
3q 1 0.503 5n 0 1.161
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Conclusions

In conclusion, to find new potential entities for the treatment of AD, we have designed and synthesized 34 novel N-aryltacrines, including phenyl, naphthalenyl, pyridinyl, and quinolinyl tacrines, targeting AChE and BuChE in the field of AD therapy by the direct late-stage modification of tacrine using the palladium-catalyzed Buchwald–Hartwig cross-coupling reaction. Most of compounds showed potent activity against AChE and negative inhibitory activity toward BuChE. Representatively, compounds 3g and 3o displayed inhibition against AChE (IC50 values of 1.77 and 1.48 μM, respectively), being more potent than the reference drug tacrine. Moreover, compound 3o with an IC50 value of 19.00 μM had potency against BuChE. Hepatotoxicity assays in vitro suggested that 3o had no obvious hepatotoxicity and was much safer than tacrine. In addition, the promising compound 3o had strong antioxidation activity, which could protect SH-SY5Y cells from H2O2-induced damage at a concentration of 50 μM. Taken together, the results show that the new compound N-(4-methoxypyridin-2-yl)-tacrine (3o) can be considered as a very promising lead compound or candidate for the treatment of AD.

Experimental Section

Chemistry

Materials and General Method

All reactions were conducted under an inert atmosphere of dry argon. Anhydrous 1,4-dioxane 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. The 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, People’s Republic of China) was used for column chromatography.

1H NMR spectra were recorded on a Bruker AV 400 nuclear magnetic resonance instrument (400 MHz). Chemical shifts were recorded in ppm relative to tetramethylsilane as the internal standard. Data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet-doublet, dt = doublet-triplet, m = multiplet, br = broad), coupling constants (Hz), and integration. 13C NMR data were collected on a Bruker AV 400 nuclear magnetic resonance instrument (100 MHz) with complete proton decoupling. Chemical shifts were reported in ppm, with tetramethylsilane as the internal standard. The high resolution electrospray ionization mass spectroscopy (HRESIMS) spectrum was determined using a Waters ACQUITY UPLC/Xevo G2-S QTOF mass spectrometer.

Preparation of Compounds 1 and 4A–C

The synthesis of tacrine and its core modified analogues followed the previously reported method with modification. AlCl3 (10.0 mmol) was added to a solution of 2-aminobenzonitrile derivatives (10.0 mmol) and cyclohexanone (10.0 mmol) or 3-methylcyclohexanone (10.0 mmol) in toluene (50 mL). The reaction mixture was refluxed for 8–12 h with a Dean–Stark water separator until TLC showed that 2-aminobenzonitrile derivatives were completely consumed. After concentration, the solid residue was dissolved in 10% aq NOH and then filtered. The filtrate was extracted with EtOAc, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The products were purified by silica gel flash column chromatography, using CH2Cl2–MeOH (30:1 to 20:1) as an eluent to yield tacrine 1 and its analogues 4A–C in 45–73% yields.

Tacrine (1), yield 70%: 1H NMR (400 MHz, DMSO-d6) δ 7.67 (1H, d, J = 8.4 Hz), 7.08 (1H, d, J = 8.4 Hz), 6.97 (1H, t, J = 7.6 Hz), 6.69 (1H, t, J = 7.6 Hz), 2.11 (2H, s), 1.68–1.62 (2H, m), 0.97 (4H, s); HRESIMS m/z 199.1235 [M + H]+ (calcd for C13H15N2, 199.1223).

1,2,3,4-Tetrahydro-2-methyl-9-acridinamine (4A), yield 73.0%: 1H NMR (400 MHz, CDCl3) δ 7.88 (1H, d, J = 8.5 Hz), 7.68 (1H, d, J = 8.4 Hz), 7.55 (1H, s), 7.34 (1H, s), 4.68 (2H, s), 3.17–2.92 (2H, m), 2.76–2.45 (2H, m), 2.19–1.98 (2H, m), 1.52 (1H, qd, J = 11.7, 5.3 Hz), 1.16 (3H, d, J = 6.4 Hz); HRESIMS m/z 213.1392 [M + H]+ (calcd for C14H17N2, 213.1383).

7-Chloro-1,2,3,4-tetrahydro-9-acridinamine (4B), yield 45%: 1H NMR (400 MHz, CDCl3) δ 7.82 (1H, d, J = 9.0 Hz), 7.68 (1H, d, J = 2.1 Hz), 7.49 (1H, dd, J = 9.0, 2.2 Hz), 4.68 (2H, s), 3.01 (2H, t, J = 5.9 Hz), 2.60 (2H, t, J = 6.0 Hz), 1.94–1.92 (4H, m); HRESIMS m/z 233.0846 [M + H]+ (calcd for C13H14ClN2, 233.0832).

1,2,3,4-Tetrahydro-6-methyl-9-acridinamine (4C), yield 72%: 1H NMR (400 MHz, CDCl3) δ 7.67 (1H, s), 7.58 (1H, d, J = 8.5 Hz), 7.19 (1H, d, J = 8.4 Hz), 4.67 (2H, s), 3.00 (2H, t, J = 5.9 Hz), 2.59 (2H, t, J = 6.0 Hz), 2.49 (3H, s), 1.93 (4H, dd, J = 7.3, 4.5 Hz); HRESIMS m/z 213.1392 [M + H]+ (calcd for C14H17N2, 213.1382).

General Procedure for Pd-Catalyzed N-Arylation of Tacrine and Its Core Modified Analogues

The suspension of 5 mol% Pd(OAc)2/10 mol% Mephos in anhydrous 1,4-dioxane (3.0 mL) was stirred at 45 °C under an argon atmosphere for 1 h to turn into a dark brown solution. Then, the dark brown solution was added to a 10 mL sealed dry reaction vial containing tacrine 1 (1.0 mmol) or its analogues 4A–C (1.0 mmol); different aryl, naphthalenyl, pyridinyl, or quinolinyl bromides (1.5 mmol); and t-BuOK (3.0 mmol) via a syringe. The reaction mixture was stirred for 12 h at 100 °C before quenching with two drops of H2O. The mixture was then diluted with ethyl acetate (3 mL) and filtered over a pad of MgSO4. The pad was rinsed with additional ethyl acetate, and the solution was concentrated in vacuo. The crude residue was loaded onto a silica gel column and purified by flash chromatography, using petroleum ether–EtOAc (2:1 to 1:2) as an eluent.

Compound 3a, yield 70.2%: 1H NMR (400 MHz, CDCl3) δ 8.00 (1H, d, J = 8.4 Hz), 7.78 (1H, d, J = 8.4 Hz), 7.60 (1H, dd, J = 11.2, 4.1 Hz), 7.33 (1H, d, J = 7.3 Hz), 7.19 (2H, d, J = 8.1 Hz), 6.90 (1H, t, J = 7.4 Hz), 6.70 (2H, d, J = 7.7 Hz), 5.91 (1H, s), 3.16 (2H, t, J = 6.4 Hz), 2.74 (2H, t, J = 6.4 Hz), 1.98–1.92 (2H, m), 1.87–1.81 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.0, 147.4, 144.7, 143.5, 129.5, 129.0, 128.8, 125.2, 123.5, 123.4, 123.2, 120.9, 116.8, 34.1, 25.6, 23.0, 22.9; HRESIMS m/z 275.1548 [M + H]+ (calcd for C19H19N2, 275.1530).

Compound 3b, yield 68.8%: 1H NMR (400 MHz, CDCl3) δ 7.98 (1H, d, J = 8.4 Hz), 7.76 (1H, d, J = 8.3 Hz), 7.58 (1H, t, J = 7.6 Hz), 7.30 (1H, t, J = 7.6 Hz), 7.01 (2H, d, J = 8.2 Hz), 6.63 (2H, d, J = 8.3 Hz), 5.83 (1H, s), 3.14 (2H, t, J = 6.4 Hz), 2.72 (2H, t, J = 6.3 Hz), 2.28 (3H, s), 2.00–1.91 (2H, m), 1.90–1.82 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.0, 147.6, 143.8, 142.2, 130.6, 130.0, 129.0, 128.80, 125.0, 123.4, 122.9, 122.7, 117.3, 34.3, 25.6, 23.1, 22.9, 20.8; HRESIMS m/z 289.1705 [M + H]+ (calcd for C20H21N2, 289.1700).

Compound 3c, yield 69.1%: 1H NMR (400 MHz, CDCl3) δ 7.99 (1H, d, J = 8.4 Hz), 7.78 (1H, d, J = 8.1 Hz), 7.60–7.55 (1H, m), 7.30 (1H, t, J = 7.3 Hz), 7.21 (2H, d, J = 8.6 Hz), 6.65 (2H, d, J = 8.6 Hz), 5.88 (1H, s), 3.15 (2H, t, J = 6.5 Hz), 2.72 (2H, t, J = 6.4 Hz), 1.98–1.90 (2H, m), 1.87–1.81 (2H, m), 1.29 (9H, s); 13C NMR (100 MHz, CDCl3) δ 159.9, 147.5, 143.9, 142.1, 128.8, 128.8, 126.2, 126.1 124.9, 123.5, 123.0, 122.9, 116.7, 34.3, 34.2, 31.6, 25.5, 23.0, 22.9; HRESIMS m/z 331.2174 [M + H]+ (calcd for C23H27N2, 331.2164).

Compound 3d, yield 59.3%: 1H NMR (400 MHz, CDCl3) δ 8.00 (1H, d, J = 8.4 Hz), 7.71 (1H, d, J = 8.4 Hz), 7.57 (1H, t, J = 7.6 Hz), 7.29 (1H, s), 6.77 (4H, q, J = 9.1 Hz), 5.91 (1H, s), 3.78 (3H, s), 3.15 (2H, t, J = 6.4 Hz), 2.68 (2H, t, J = 6.3 Hz), 1.96–1.92 (2H, m), 1.88–1.85 (2H, m); 13C NMR (100 MHz, CDCl3) δ 159.3, 155.1, 146.7, 145.3, 137.7, 129.0, 128.1, 124.8, 123.5, 121.8, 120.7, 120.2, 114.7, 55.8, 25.5, 22.9, 22.8, 20.7; HRESIMS m/z 305.1654 [M + H]+ (calcd for C20H21N2O, 305.1641).

Compound 3e, yield 44.4%: 1H NMR (400 MHz, CDCl3) δ 7.99 (1H, d, J = 8.4 Hz), 7.72 (1H, d, J = 8.1 Hz), 7.59 (1H, dd, J = 11.2, 4.1 Hz), 7.31 (1H, t, J = 7.6 Hz), 6.95–6.85 (2H, m), 6.72–6.64 (2H, m), 5.86 (1H, s), 3.15 (2H, t, J = 6.5 Hz), 2.71 (2H, t, J = 6.4 Hz), 2.00–1.92 (2H, m), 1.89–1.82 (2H, m); 13C NMR (100 MHz, CDCl3) δ 159.8 (d, J = 250.0 Hz), 158.9, 156.5, 147.3, 143.6, 140.6, 128.7, 124.9, 123.0, 122.5, 122.5, 118.4, 115.9, 34.0, 25.4, 22.8, 22.7; HRESIMS m/z 293.1454 [M + H]+ (calcd for C19H18FN2, 293.1440).

Compound 3f, yield 40.5%: 1H NMR (400 MHz, CDCl3) δ 7.99 (1H, d, J = 8.4 Hz), 7.73 (1H, d, J = 8.3 Hz), 7.60 (1H, dd, J = 11.2, 4.0 Hz), 7.34 (1H, t, J = 7.6 Hz), 7.16–7.11 (2H, m), 6.62–6.57 (2H, m), 5.85 (1H, s), 3.14 (2H, t, J = 6.5 Hz), 2.72 (2H, t, J = 6.4 Hz), 1.98–1.92 (2H, m), 1.88–1.81 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.2, 147.6, 143.4, 142.8, 129.4, 129.1, 129.0, 125.5, 125.4, 124.0, 123.2, 123.1, 117.6, 34.2, 25.6, 23.0, 22.9; HRESIMS m/z 309.1159 [M + H]+ (calcd for C19H18ClN2, 309.1142).

Compound 3g, yield 31.2%: 1H NMR (400 MHz, CDCl3) δ 8.03 (1H, d, J = 8.5 Hz), 7.83 (2H, d, J = 8.7 Hz), 7.77 (1H, d, J = 8.3 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.39–7.34 (1H, m), 6.61 (2H, d, J = 8.6 Hz), 6.05 (1H, s), 3.17 (2H, t, J = 6.4 Hz), 2.77 (2H, dd, J = 15.0, 8.6 Hz), 2.03–1.93 (2H, m), 1.84 (2H, dd, J = 11.7, 6.2 Hz); 13C NMR (100 MHz, CDCl3) δ 160.3, 148.5, 147.3, 142.1, 131.4, 130.1, 129.3, 128.9, 125.8, 125.5, 123.8, 123.7 (q, J = 280.5 Hz), 123.1, 114.7, 34.0, 25.7, 22.9, 22.8; HRESIMS m/z 343.1422 [M + H]+ (calcd for C20H18F3N2, 343.1412).

Compound 3h, yield 65.8%: 1H NMR (400 MHz, CDCl3) δ 8.02 (1H, d, J = 8.4 Hz), 7.90 (2H, d, J = 8.7 Hz), 7.78 (1H, d, J = 8.3 Hz), 7.63 (1H, t, J = 7.3 Hz), 7.37 (1H, t, J = 7.6 Hz), 6.64 (2H, d, J = 8.7 Hz), 6.17 (1H, s), 3.17 (2H, t, J = 6.5 Hz), 2.77 (2H, t, J = 6.4 Hz), 2.63–2.53 (1H, m), 2.00–1.93 (2H, m), 1.88–1.79 (2H, m), 1.18 (2H, dt, J = 7.5, 3.7 Hz), 0.95 (2H, td, J = 7.0, 3.5 Hz); 13C NMR (100 MHz, CDCl3) δ 198.8, 160.4, 149.1, 147.6, 141.7, 130.4, 130.0, 129.2, 129.1, 126.1, 125.9, 124.0, 123.1, 114.5, 34.2, 25.7, 23.0, 22.8, 16.6, 11.2; HRESIMS m/z 343.1810 [M + H]+ (calcd for C23H23N2O, 343.1800).

Compound 3i, yield 55.6%: 1H NMR (400 MHz, CDCl3) δ 8.02 (1H, d, J = 8.4 Hz), 7.75 (1H, d, J = 8.4 Hz), 7.62 (1H, t, J = 7.6 Hz), 7.37 (1H, t, J = 7.6 Hz), 7.28 (1H, d, J = 8.1 Hz), 7.11 (1H, d, J = 7.7 Hz), 6.95 (1H, s), 6.73 (1H, d, J = 8.1 Hz), 5.98 (1H, s), 3.17 (2H, t, J = 6.5 Hz), 2.74 (2H, t, J = 6.4 Hz), 2.03–1.93 (2H, m), 1.89–1.83 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.4, 147.6, 145.2, 142.1, 132.1, 131.8, 130.0, 129.2, 125.7, 125.6, 124.9, 123.5 (q, J = 280.2 Hz), 122.9, 118.7, 116.9, 112.6, 34.2, 25.7, 23.0, 22.8; HRESIMS m/z 343.1422 [M + H]+ (calcd for C20H18F3N2, 3343.1410).

Compound 3j, yield 57.2%: 1H NMR (400 MHz, CDCl3) δ 8.26 (1H, d, J = 7.6 Hz), 8.03 (1H, d, J = 8.4 Hz), 7.97–7.86 (1H, m), 7.72 (1H, d, J = 8.3 Hz), 7.64–7.53 (3H, m), 7.49 (1H, d, J = 8.2 Hz), 7.28 (1H, s), 7.21 (1H, t, J = 7.9 Hz), 6.44 (1H, d, J = 7.5 Hz), 6.22 (1H, s), 3.19 (2H, t, J = 6.4 Hz), 2.71 (2H, s), 1.96 (2H, dt, J = 12.6, 6.4 Hz), 1.89–1.77 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.1, 147.7, 144.4, 140.2, 134.8, 129.1, 129.0, 129.0, 126.6, 126.2, 126.2, 125.9, 125.4, 125.3, 123.1, 122.9, 122.0, 121.3, 112.9, 34.4, 25.5, 23.2, 23.0; HRESIMS m/z 325.1705 [M + H]+ (calcd for C23H21N2, 325.1690).

Compound 3k, yield 74.2%: 1H NMR (400 MHz, CDCl3) δ 8.02 (1H, d, J = 8.4 Hz), 7.80 (1H, d, J = 8.4 Hz), 7.60 (2H, dd, J = 19.0, 8.4 Hz), 7.43 (1H, d, J = 9.3 Hz), 7.28 (1H, d, J = 7.4 Hz), 7.11–6.99 (3H, m), 6.86 (1H, d, J = 1.8 Hz), 6.00 (1H, s), 3.88 (3H, s), 3.17 (2H, t, J = 6.5 Hz), 2.74 (2H, t, J = 6.4 Hz), 2.00–1.91 (2H, m), 1.88–1.78 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.1, 156.4, 147.6, 143.7, 140.6, 130.1, 129.8, 129.0, 128.9, 128.2, 128.1, 125.1, 123.4, 123.1, 123.1, 119.8, 119.4, 112.1, 106.2, 55.5, 34.3, 25.6, 23.0, 22.9; HRESIMS m/z 355.1810 [M + H]+ (calcd for C24H23N2O, 355.1800).

Compound 3l, yield 48.9%: 1H NMR (400 MHz, CDCl3) δ 8.01 (1H, d, J = 8.4 Hz), 7.85 (1H, d, J = 8.4 Hz), 7.61 (1H, dd, J = 11.2, 4.0 Hz), 7.38 (1H, t, J = 7.6 Hz), 7.28 (1H, s), 6.61 (2H, d, J = 7.3 Hz), 5.85 (1H, d, J = 8.2 Hz), 3.16 (2H, t, J = 6.5 Hz), 2.82 (2H, t, J = 6.4 Hz), 2.45 (3H, s), 1.99–1.93 (2H, m), 1.89–1.81 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.5, 157.6, 156.3, 147.6, 141.4, 138.4, 129.1, 129.1, 126.5, 125.8, 124.4, 123.3, 114.8, 104.5, 34.3, 25.7, 24.4, 23.0, 22.8; HRESIMS m/z 290.1657 [M + H]+ (calcd for C19H20N3, 290.1637).

Compound 3m, yield 71.1%: 1H NMR (400 MHz, CDCl3) δ 7.99 (1H, d, J = 8.4 Hz), 7.95 (1H, d, J = 4.9 Hz), 7.73 (1H, d, J = 8.2 Hz), 7.58 (1H, t, J = 7.6 Hz), 7.43 (1H, d, J = 7.2 Hz), 7.35 (1H, t, J = 7.3 Hz), 6.71 (1H, dd, J = 7.2, 5.0 Hz), 6.01 (1H, s), 3.16 (2H, t, J = 6.5 Hz), 2.74 (2H, t, J = 6.4 Hz), 2.37 (3H, s), 2.01–1.89 (2H, m), 1.89–1.79 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.0, 154.9, 147.4, 146.1, 142.3, 138.3, 129.0, 128.7, 126.1, 125.4, 124.5, 122.8, 118.2, 115.8, 34.2, 26.0, 23.0, 22.8, 17.7; HRESIMS m/z 290.1657 [M + H]+ (calcd for C19H20N3, 290.1645).

Compound 3n, yield 47.8%: 1H NMR (400 MHz, CDCl3) δ 8.05–7.97 (2H, m), 7.88 (1H, d, J = 8.3 Hz), 7.63 (1H, t, J = 7.2 Hz), 7.39 (1H, t, J = 7.5 Hz), 7.08 (1H, s), 6.55 (1H, d, J = 5.1 Hz), 5.92 (1H, s), 3.17 (2H, t, J = 6.5 Hz), 2.82 (2H, t, J = 6.4 Hz), 2.11 (3H, s), 2.01–1.93 (2H, m), 1.88–1.79 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.5, 157.1, 149.4, 148.2, 147.6, 141.6, 129.1, 129.0, 126.8, 125.8, 124.6, 123.3, 116.7, 108.1, 34.3, 25.8, 23.1, 22.8, 21.4; HRESIMS m/z 290.1657 [M + H]+ (calcd for C19H20N3, 290.1643).

Compound 3o, yield 73.6%: 1H NMR (400 MHz, CDCl3) δ 8.01 (1H, d, J = 8.4 Hz), 7.92 (2H, t, J = 7.3 Hz), 7.82 (1H, s), 7.62 (1H, t, J = 7.5 Hz), 7.40 (1H, t, J = 7.5 Hz), 6.27 (1H, d, J = 4.2 Hz), 5.54 (1H, s), 3.57 (3H, s), 3.16 (2H, t, J = 6.4 Hz), 2.84 (2H, t, J = 6.3 Hz), 2.02–1.91 (2H, m), 1.90–1.78 (2H, m); 13C NMR (100 MHz, CDCl3) δ 167.7, 160.5, 158.9, 149.5, 147.6, 141.5, 129.1, 129.0, 126.9, 125.8, 124.6, 123.4, 102.8, 92.3, 55.2, 34.2, 25.8, 23.1, 22.8; HRESIMS m/z 306.1606 [M + H]+ (calcd for 19H20N3O, 306.1590).

Compound 3p, yield 75.7%: 1H NMR (400 MHz, CDCl3) δ 7.99 (1H, d, J = 8.4 Hz), 7.88 (1H, d, J = 2.8 Hz), 7.83 (1H, d, J = 8.3 Hz), 7.59 (1H, t, J = 7.2 Hz), 7.35 (1H, t, J = 7.5 Hz), 7.01 (1H, dd, J = 8.9, 3.0 Hz), 6.94 (1H, s), 6.14 (1H, d, J = 8.9 Hz), 3.75 (3H, s), 3.14 (2H, t, J = 6.5 Hz), 2.77 (2H, t, J = 6.4 Hz), 1.98–1.89 (2H, m), 1.87–1.73 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.3, 151.3, 150.4, 147.6, 142.2, 134.3, 129.0, 128.9, 125.5, 125.4, 125.0, 124.0, 123.3, 108.9, 56.4, 34.2, 25.7, 23.0, 22.8; HRESIMS m/z 306.1606 [M + H]+ (calcd for C19H20N3O, 306.1595).

Compound 3q, yield 55.2%: 1H NMR (400 MHz, CDCl3) δ 8.20 (1H, d, J = 2.4 Hz), 8.12 (1H, d, J = 4.5 Hz), 8.01 (1H, d, J = 8.4 Hz), 7.75 (1H, d, J = 8.3 Hz), 7.61 (1H, t, J = 7.6 Hz), 7.35 (1H, t, J = 7.6 Hz), 7.07 (1H, dd, J = 8.2, 4.8 Hz), 6.80 (1H, d, J = 8.2 Hz), 6.11 (1H, s), 3.15 (2H, t, J = 6.4 Hz), 2.74 (2H, t, J = 6.3 Hz), 1.98–1.92 (2H, m), 1.85 (2H, d, J = 5.8 Hz); 13C NMR (100 MHz, CDCl3) δ 160.0, 147.1, 141.7, 140.9, 138.8, 129.1, 128.8, 125.6, 125.0, 124.1, 123.7, 122.9, 122.7, 122.3, 29.7, 25.5, 22.7, 22.6; HRESIMS m/z 276.1501 [M + H]+ (calcd for C18H18N3, 276.1491).

Compound 3r, yield 60.2%: 1H NMR (400 MHz, CDCl3) δ 8.04 (1H, d, J = 8.4 Hz), 7.89 (1H, d, J = 8.3 Hz), 7.82 (1H, d, J = 8.9 Hz), 7.70 (1H, d, J = 8.4 Hz), 7.64 (2H, t, J = 6.9 Hz), 7.58 (1H, t, J = 7.6 Hz), 7.37 (1H, t, J = 7.5 Hz), 7.29 (1H, t, J = 7.5 Hz), 6.39 (1H, d, J = 8.9 Hz), 3.19 (2H, t, J = 6.5 Hz), 2.87 (2H, t, J = 6.4 Hz), 1.97 (2H, dt, J = 9.1, 6.4 Hz), 1.87–1.79 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.5, 155.7, 147.7, 141.0, 141.0, 138.5, 130.3, 129.2, 129.1, 127.8, 127.3, 126.2, 126.0, 124.8, 124.3, 123.4, 123.2, 110.4, 34.3, 25.9, 23.0, 22.8; HRESIMS m/z 326.1657 [M + H]+ (calcd for C22H20N3, 326.1645).

Compound 3s, yield 52.3%: 1H NMR (400 MHz, CDCl3) δ 8.73 (1H, d, J = 2.7 Hz), 8.03 (2H, dd, J = 18.3, 8.4 Hz), 7.79 (1H, d, J = 8.4 Hz), 7.63 (1H, t, J = 7.6 Hz), 7.55–7.44 (2H, m), 7.44–7.38 (1H, m), 7.35 (1H, t, J = 7.6 Hz), 6.92 (1H, d, J = 2.2 Hz), 6.19 (1H, s), 3.19 (2H, t, J = 6.5 Hz), 2.78 (2H, t, J = 6.3 Hz), 1.97 (2H, dt, J = 12.4, 6.3 Hz), 1.86 (2H, dd, J = 10.8, 4.8 Hz); 13C NMR (100 MHz, CDCl3) δ 160.4, 147.5, 143.8, 143.6, 142.2, 138.4, 129.3, 129.3, 129.1, 128.8, 127.4, 126.7, 126.6, 125.9, 124.7, 123.2, 123.0, 116.3, 29.9, 25.8, 23.0, 22.8; HRESIMS m/z 326.1657 [M + H]+ (calcd for C22H20N, 326.1646).

Compound 3t, yield 55.9%: 1H NMR (400 MHz, CDCl3) δ 9.00 (1H, s), 8.60 (1H, d, J = 8.3 Hz), 8.02 (1H, d, J = 8.3 Hz), 7.75 (1H, d, J = 8.2 Hz), 7.68 (1H, d, J = 8.2 Hz), 7.60 (1H, t, J = 7.4 Hz), 7.54–7.36 (2H, m), 7.30 (1H, d, J = 7.5 Hz), 6.49 (1H, d, J = 7.2 Hz), 6.22 (1H, s), 3.18 (2H, s), 2.71 (2H, s), 1.90 (4H, d, J = 51.0 Hz); 13C NMR (100 MHz, CDCl3) δ 160.1, 150.8, 149.5, 147.6, 143.9, 140.3, 129.9, 129.9, 129.2, 129.1, 125.5, 123.3, 123.2, 122.7, 121.9, 121.1, 120.8, 113.3, 34.3, 25.5, 23.0, 22.9; HRESIMS m/z 326.1657 [M + H]+ (calcd for C22H20N3, 326.1644).

Compound 5a, yield 54.7%: 1H NMR (400 MHz, CDCl3) δ 8.03 (2H, dd, J = 11.2, 6.8 Hz), 7.85 (1H, d, J = 8.3 Hz), 7.63 (1H, t, J = 7.6 Hz), 7.39 (1H, t, J = 7.6 Hz), 6.82 (1H, s), 6.57 (1H, d, J = 5.1 Hz), 5.91 (1H, s), 3.31–3.24 (1H, m), 3.20–3.11 (1H, m), 3.00 (1H, dd, J = 17.0, 3.3 Hz), 2.33 (1H, dd, J = 16.9, 10.6 Hz), 2.11 (3H, s), 2.05 (1H, dd, J = 6.5, 3.7 Hz), 1.64–1.54 (1H, m), 1.09 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) δ 160.2, 157.1, 149.5, 148.3, 147.7, 141.5, 129.1, 129.0, 126.4, 125.8, 124.5, 123.4, 116.9, 108.1, 34.1, 33.9, 31.2, 29.2, 22.0, 21.4; HRESIMS m/z 304.1814 [M + H]+ (calcd for C20H22N3, 304.1804).

Compound 5b, yield 49.9%: 1H NMR (400 MHz, CDCl3) δ 8.00 (2H, dd, J = 10.5, 7.2 Hz), 7.88 (1H, d, J = 8.3 Hz), 7.62 (1H, t, J = 7.6 Hz), 7.40 (1H, t, J = 7.6 Hz), 7.26 (1H, s), 6.32 (1H, dd, J = 5.9, 2.2 Hz), 5.54 (1H, d, J = 2.0 Hz), 3.58 (3H, s), 3.30–3.23 (1H, m), 3.19–3.10 (1H, m), 3.03 (1H, dd, J = 17.0, 3.3 Hz), 2.35 (1H, dd, J = 17.0, 10.7 Hz), 2.15–1.84 (3H, m), 1.63–1.53 (1H, m), 1.09 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) δ 167.7, 160.2, 158.8, 149.6, 147.7, 1414, 129.1, 129.0, 126.5, 125.8, 124.4, 123.4, 103.0, 92.4, 55.2, 34.1, 33.9, 31.3, 29.2, 22.0; HRESIMS m/z 320.1763 [M + H]+ (calcd for C20H22N3O, 320.1751).

Compound 5c, yield 65.7%: 1H NMR (400 MHz, CDCl3) δ 7.99 (1H, d, J = 8.4 Hz), 7.91 (1H, d, J = 2.9 Hz), 7.80 (1H, d, J = 8.4 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.35 (1H, t, J = 7.6 Hz), 7.02 (1H, dd, J = 8.9, 3.0 Hz), 6.73 (1H, s), 6.13 (1H, d, J = 8.9 Hz), 3.77 (3H, s), 3.28–3.21 (1H, m), 3.17–3.08 (1H, m), 2.95 (1H, dd, J = 16.3, 4.2 Hz), 2.29 (2H, dd, J = 16.8, 10.6 Hz), 2.09–1.98 (1H, m), 1.61–1.51 (1H, m), 1.07 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) δ 160.1, 151.3, 150.5, 147.6, 142.2, 134.4, 129.1, 129.0, 129.0, 125.5, 125.1, 123.9, 123.3, 108.8, 56.4, 34.1, 33.9, 31.2, 29.2, 22.0; HRESIMS m/z 320.1763 [M + H]+ (calcd for C20H22N3O, 320.1753).

Compound 5d, yield 60.0%: 1H NMR (400 MHz, CDCl3) δ 8.03 (1H, d, J = 8.4 Hz), 7.88 (1H, d, J = 8.3 Hz), 7.80 (1H, d, J = 8.9 Hz), 7.67 (1H, d, J = 8.4 Hz), 7.62 (2H, t, J = 6.8 Hz), 7.55 (1H, t, J = 7.3 Hz), 7.35 (1H, t, J = 7.5 Hz), 7.28 (1H, d, J = 7.4 Hz), 6.35 (1H, d, J = 8.9 Hz), 3.30–3.24 (1H, m), 3.19–3.10 (1H, m), 3.04 (1H, dd, J = 17.0, 3.6 Hz), 2.33 (1H, dd, J = 17.0, 10.7 Hz), 2.03 (1H, dd, J = 12.2, 3.3 Hz), 1.86 (1H, s), 1.60–1.49 (1H, m), 1.01 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) δ 160.2, 155.8, 147.6, 141.1, 138.6, 130.3, 129.2, 129.0, 127.8, 126.9, 126.0, 124.7, 124.1, 123.3, 123.3, 121.8, 116.0, 110.5, 34.2, 33.8, 31.1, 29.1, 21.9; HRESIMS m/z 340.1814 [M + H]+ (calcd for C23H22N3, 340.1800).

Compound 5e, yield 35.5%: 1H NMR (400 MHz, CDCl3) δ 8.00 (1H, d, J = 8.4 Hz), 7.83 (1H, d, J = 8.3 Hz), 7.61 (1H, t, J = 7.3 Hz), 7.37 (1H, t, J = 7.6 Hz), 7.24 (1H, d, J = 7.8 Hz), 6.72 (1H, s,), 6.60 (1H, d, J = 7.3 Hz), 5.84 (1H, d, J = 8.2 Hz), 3.29–3.22 (1H, m), 3.18–3.09 (1H, m), 3.00 (1H, dd, J = 17.0, 3.4 Hz), 2.45 (3H, s), 2.33 (1H, dd, J = 17.0, 10.7 Hz), 1.64–1.53 (1H, m), 1.35–1.23 (1H, m), 1.08 (3H, d, J = 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 160.3, 157.7, 156.5, 147.7, 141.5, 138.5, 129.2, 129.1, 126.3, 125.8, 124.4, 123.5, 114.9, 104.5, 34.2, 34.0, 31.3, 29.2, 24.5, 22.1; HRESIMS m/z 304.1814 [M + H]+ (calcd for C20H22N3, 304.1800).

Compound 5f, yield 67.0%: 1H NMR (400 MHz, CDCl3) δ 7.98 (1H, d, J = 8.4 Hz), 7.93 (1H, d, J = 4.3 Hz), 7.69 (1H, d, J = 8.3 Hz), 7.57 (1H, t, J = 7.5 Hz), 7.43 (1H, d, J = 7.0 Hz), 7.33 (1H, t, J = 7.6 Hz), 6.71 (1H, dd, J = 7.1, 5.1 Hz), 5.99 (1H, s), 3.29–3.22 (1H, m), 3.18–3.09 (1H, m), 2.86 (1H, dd, J = 16.7, 3.4 Hz), 2.37 (3H, s), 2.31 (2H, dd, J = 16.8, 10.8 Hz), 2.10–1.98 (1H, m), 1.64–1.54 (1H, m), 1.05 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) δ 159.7, 154.9, 147.4, 146.1, 142.3, 138.4, 128.9, 128.7, 125.8, 125.3, 124.4, 122.9, 118.2, 115.8, 34.3, 33.8, 31.2, 29.1, 22.1, 17.7; HRESIMS m/z 304.1814 [M + H]+ (calcd for C20H22N3, 304.1802).

Compound 5g, yield 32.1%: 1H NMR (400 MHz, CDCl3) δ 8.17–7.81 (3H, m), 7.55 (1H, dd, J = 8.9, 1.9 Hz), 6.57 (1H, d, J = 5.0 Hz), 5.93 (1H, s), 3.15 (2H, t, J = 6.5 Hz), 2.79 (2H, t, J = 6.3 Hz), 2.14 (3H, s), 2.03–1.92 (2H, m), 1.89–1.68 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.9, 156.9, 149.7, 148.2, 146.0, 140.9, 131.7, 130.6, 130.0, 128.0, 125.6, 122.3, 117.0, 108.1, 34.2, 25.9, 22.9, 22.7, 21.4; HRESIMS m/z 324.1268 [M + H]+ (calcd for C19H19ClN3, 324.1257).

Compound 5h, yield 44.4%: 1H NMR (400 MHz, CDCl3) δ 7.99 (1H, d, J = 5.9 Hz), 7.94 (1H, d, J = 9.0 Hz), 7.89 (1H, s), 7.56 (1H, dd, J = 8.9, 1.9 Hz), 6.35 (1H, d, J = 5.7 Hz), 5.57 (1H, s), 3.64 (3H, s), 3.14 (2H, t, J = 6.5 Hz), 2.82 (2H, t, J = 6.3 Hz), 1.99–1.93 (2H, m), 1.89–1.82 (2H, m); 13C NMR (100 MHz, CDCl3) δ 167.9, 160.9, 158.4, 149.5, 146.0, 140.6, 131.8, 130.7, 130.1, 128.1, 125.5, 122.3, 103.2, 92.6, 55.3, 34.2, 25.8, 23.0, 22.7; HRESIMS m/z 340.1217 [M + H]+ (calcd for C19H19ClN3O, 340.1200).

Compound 5i, yield 39.6%: 1H NMR (400 MHz, CDCl3) δ 7.92 (2H, dd, J = 10.2, 5.9 Hz), 7.80 (1H, d, J = 2.1 Hz), 7.54 (1H, dd, J = 8.9, 2.2 Hz), 7.08 (1H, dd, J = 8.9, 2.9 Hz), 6.18 (1H, d, J = 8.9 Hz), 3.81 (3H, s), 3.13 (2H, t, J = 6.4 Hz), 2.77 (2H, t, J = 6.4 Hz), 1.95 (2H, dd, J = 11.9, 5.9 Hz), 1.85 (2H, dd, J = 11.7, 5.8 Hz); 13C NMR (100 MHz, CDCl3) δ 160.7, 150.8, 150.7, 141.6, 140.7, 134.0, 131.5, 130.6, 130.0, 126.5, 125.5, 124.9, 122.3, 109.1, 56.4, 29.9, 25.8, 22.9, 22.7; HRESIMS m/z 340.1217 [M + H]+ (calcd for C19H19ClN3O, 340.1207).

Compound 5j, yield 28.4%: 1H NMR (400 MHz, CDCl3) δ 7.96 (1H, d, J = 9.0 Hz), 7.87 (2H, dd, J = 7.9, 5.7 Hz), 7.66 (2H, dd, J = 15.8, 8.2 Hz), 7.61–7.53 (2H, m), 7.31 (1H, t, J = 7.3 Hz), 6.40 (1H, d, J = 8.9 Hz), 3.16 (2H, t, J = 6.5 Hz), 2.84 (2H, t, J = 6.4 Hz), 1.96 (2H, dt, J = 12.5, 6.4 Hz), 1.86–1.77 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.9, 155.3, 155.2, 145.9, 138.9, 132.0, 130.7, 130.5, 130.1, 128.3, 127.9, 125.7, 124.2, 123.6, 122.1, 110.5, 110.4, 100.2, 34.2, 26.0, 22.9, 22.6; HRESIMS m/z 360.1268 [M + H]+ (calcd for C22H19ClN3, 360.1255).

Compound 5k, yield 67.1%: 1H NMR (400 MHz, CDCl3) δ 8.00 (1H, d, J = 4.9 Hz), 7.81–7.73 (2H, m), 7.22 (1H, d, J = 8.5 Hz), 6.54 (1H, d, J = 5.0 Hz), 5.91 (1H, s), 3.15 (2H, t, J = 6.5 Hz), 2.79 (2H, t, J = 6.4 Hz), 2.52 (3H, s), 2.10 (3H, s), 1.95 (2H, dd, J = 11.9, 5.9 Hz), 1.83 (2H, dd, J = 11.5, 6.1 Hz); 13C NMR (100 MHz, CDCl3) δ 160.3, 157.1, 149.5, 148.0, 147.8, 141.5, 139.2, 128.1, 128.0, 125.8, 123.0, 122.5, 116.7, 108.2, 34.2, 29.9, 25.6, 23.1, 21.9, 21.4; HRESIMS m/z 304.1814 [M + H]+ (calcd for C20H22N3, 304.1800).

Compound 5l, yield 69.0%: 1H NMR (400 MHz, CDCl3) δ 7.95 (1H, d, J = 5.9 Hz), 7.85–7.71 (2H, m), 7.23 (1H, d, J = 8.7 Hz), 6.29 (1H, dd, J = 5.9, 1.9 Hz), 5.54 (1H, d, J = 1.8 Hz), 3.58 (3H, s), 3.14 (2H, t, J = 6.5 Hz), 2.82 (2H, t, J = 6.3 Hz), 2.51 (3H, s), 1.95 (2H, dt, J = 12.3, 6.3 Hz), 1.89–1.79 (2H, m); 13C NMR (100 MHz, CDCl3) δ 167.8, 160.3, 158.8, 149.3, 147.9, 141.3, 139.3, 128.1, 128.0, 125.9, 123.1, 122.5, 102.9, 92.4, 55.2, 34.2, 29.9, 25.6, 23.1, 21.9; HRESIMS m/z 320.1763 [M + H]+ (calcd for C20H22N3O, 320.1752).

Compound 5m, yield 25.5%: 1H NMR (400 MHz, CDCl3) δ 7.90 (1H, d, J = 2.9 Hz), 7.77 (1H, s), 7.68 (1H, d, J = 8.5 Hz), 7.19 (1H, d, J = 8.5 Hz), 7.02 (1H, dd, J = 9.0, 3.0 Hz), 6.70 (1H, s), 6.14 (1H, d, J = 8.9 Hz), 3.78 (3H, s), 3.13 (2H, t, J = 6.5 Hz), 2.76 (2H, t, J = 6.4 Hz), 2.50 (3H, s), 1.97–1.90 (2H, m), 1.89–1.80 (2H, m); 13C NMR (100 MHz, CDCl3) δ 160.2, 151.3, 150.4, 147.8, 142.2, 139.2, 134.2, 128.0, 127.8, 125.2, 124.4, 123.0, 121.9, 109.0, 56.4, 34.2, 25.6, 23.0, 22.9, 21.9; HRESIMS m/z 320.1763 [M + H]+ (calcd for C20H22N3O, 320.1750).

Compound 5n, yield 59.9%: 1H NMR (400 MHz, CDCl3) δ 7.84–7.80 (2H, m), 7.75 (2H, d, J = 8.3 Hz), 7.62 (2H, dd, J = 11.9, 6.7 Hz), 7.32 (1H, d, J = 7.3 Hz), 7.22 (1H, d, J = 8.4 Hz), 6.36 (1H, d, J = 8.9 Hz), 3.16 (2H, t, J = 6.4 Hz), 2.85 (2H, t, J = 6.3 Hz), 2.52 (3H, s), 1.99–1.92 (2H, m), 1.84 (2H, d, J = 5.9 Hz); 13C NMR (100 MHz, CDCl3) δ 178.4, 160.3, 155.7, 147.7, 146.4, 139.5, 139.1, 130.7, 128.4, 128.0, 127.9, 126.3, 125.0, 123.9, 123.6, 122.9, 122.6, 110.7, 34.1, 29.9, 25.7, 23.0, 22.8, 21.9; HRESIMS m/z 340.1814 [M + H]+ (calcd for C23H22N3, 340.1800).

Biological Materials and Methods

Cholinesterase Inhibition Assay

AChE from Electrophorus electricus (electric eel), 5,5′-dithiobis-2-nitrobenzoic acid (Ellman’s reagent, DTNB), and acetylthiocholine chloride (ATC) were purchased from Macklin. BuChE from Equine Serum and butylthiocholine chloride were purchased from Sigma-Aldrich. The inhibitory activity of test compounds 3a–t and 5a–n against AChE/BuChE was assessed by Ellman’s method. Tacrine and the synthesized compounds were dissolved in DMSO to 100 or 50 μM concentration. All the assays were under 0.2 M NaH2PO4/Na2HPO4 buffer (pH 6.7), using a spectraMax absorbance reader instrument. Enzyme solutions were prepared using 2.5 mg (0.5U/mL) of AChE in pH 8.0 buffer (1 mL). The assay medium contained phosphate buffer (pH 6.7, 140 μL), tacrine derivatives (I = 100 μM or 50 μM, 10μL), and 0.5 U/mL of enzyme (10 μL). After 20 min of incubation time, DTNB (0.75 mM, 10μL) and ATC (1.5 mM, 10μL) were added for incubation for another 20 min. The inhibitory activity was determined by measuring the increase in absorbance at 405 nm. Each concentration was assayed in triplicate. Enzyme inhibitory activity (%) = [1 – (A sample-blank/A control-blank)] × 100%. IC50 values were evaluated by using the software package SPSS. In vitro BuChE assays were carried out using a similar method, as described above.

In Vitro Cytotoxicity Assay against HepG2 Cell Line

MTT (M2128) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and incubated with 5% CO2.

The HepG2 cells were seeded at a density of 5 × 103/well in a 96-well plate for 24 h and then treated with different tested compounds dissolved in 100% DMSO for 24 h, with the final DMSO concentrations lower than 0.1%. Control cells were treated with tacrine containing 0.1% DMSO. DMSO served as a negative control. Then, 10 μL of MTT (5 mg/mL) was added into each well and incubated for another 4 h. The purple formazan crystals were solved in 100 μL DMSO, and the absorbance was detected at 570 nm using a microplate reader (Thermo MK3, USA).

H2O2-Induced Cell Death in SH-SY5Y Cells

The neuroprotective activity was assessed using the MTT assay. The human SH-SY5Y neuroblastoma cells were cultured at 37 °C in a humidified atmosphere of 5% CO2, in Gibco DMEM supplemented with 10% heat-inactivated fetal bovine serum. Briefly, the SH-SY5Y cells were seeded in 96-well plates and then pretreated with 50 μM tested compounds. After 1 h of incubation, hydrogen peroxide solution (H2O2, 500 μM) was added into cells and incubated for 4 h. Then, MTT (5 mg/mL) was added into 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.

Acknowledgments

This work was financially supported by the NSF of China (81773605 and 82003634) and the Science and Technology Program of Sichuan, China (2018JY0077).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c01404.

  • 1H and 13C NMR spectra for all the new compounds (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao1c01404_si_001.pdf (4.3MB, pdf)

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