Design and Synthesis of Tacrine–Coumarin Hybrids via Click Chemistry as Multifunctional Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease

Abstract

A new series of tacrine–coumarin hybrids (compounds 15a–18b) linked by 1,2,3-triazole had been designed and synthesized as multifunctional ligands for the treatment of Alzheimer’s disease (AD). The inhibitory effects of the synthesized compounds on AChE and BuChE, their ability to inhibit Aβ aggregation, and their MAO inhibitory activities were evaluated. In vitro studies showed that some of the hybrids (compounds 17a–18b) exhibited significant abilities to inhibit both AChE and BuChE, self-induced Aβ aggregation, and MAO-B. In particular, compound 17d showed a well-balanced inhibitory profile against AChE and BuChE (IC₅₀ = 0.080 ± 0.007 μM for AChE, IC₅₀ = 0.044 ± 0.004 μM for BuChE), self-induced Aβ aggregation (58.4% ± 2.1% at 20 μM), and MAO-B (IC₅₀ = 0.18 ± 0.01 μM), suggesting that 17d might be an excellent multifunctional agent for AD treatment. In addition, compounds 15a and 15b were identified as selective inhibitors of BuChE at micromolar concentrations.

1. Introduction

Alzheimer’s disease (AD) is an age-related neurodegenerative process. Clinically, Alzheimer’s disease is mainly characterized by comprehensive dementia, including memory disorder, cognitive disorder, executive dysfunction, and personality and behavior changes, and is accompanied by mental disorder symptoms in most patients [1]. It has been reported that the global number of people with dementia increased to 43.8 million in 2016, an increase of 117% compared with 20.3 million in 1990, costing more than a trillion US dollars per year [2,3]. In addition, most patients have one or more family caregivers who devote time and effort to unpaid care, resulting in psychological morbidity, social isolation, physical ill health, and financial hardship. It is estimated that, by 2050, there will be 152 million people with Alzheimer’s disease and other dementias; the large number of patients will place an increasing burden on society [4].

Despite several involved factors have been identified and found consistent with its onset in recent decades, such as amyloid-β (Aβ) deposits [5], high levels of metal ion and related oxidative stress [6], or low levels of choline [7] are thought to play significant roles in the pathology of the disease, and, therefore, amyloid-β hypothesis [8], metal hypothesis [9], and cholinergic hypothesis [10] were suggested. However, the exact etiopathogenesis still remains unknown. The primary therapeutic strategy for Alzheimer’s disease (AD) over the past decades has been grounded in the cholinergic hypothesis [11]. This hypothesis posits that the selective degeneration of cholinergic neurons in AD leads to a deficit of acetylcholine (ACh) in brain regions critical for learning and memory. Under pathological conditions, elevated activity of acetylcholinesterase (AChE) reduces ACh concentration in the synaptic cleft, thereby impairing the transmission of cholinergic signals between neurons. Additionally, butyrylcholinesterase (BuChE) provides compensatory metabolic support when acetylcholinesterase (AChE) is deficient, working together to maintain the cholinergic system’s dynamic balance [12]. Therefore, inhibiting the activities of both AChE and BuChE to prevent the breakdown of acetylcholine (ACh) in synapses, thereby sustaining or restoring ACh levels, can help alleviate symptoms of Alzheimer’s disease (AD) [13,14]. As a result, four AChE inhibitors—tacrine, donepezil, rivastigmine, and galantamine—have been approved by the US Food and Drug Administration (FDA) for commercial use, with three of these still commonly used in clinical practice today [15].

Crystallographic studies demonstrate that AChE has a nearly 20 deep narrow gorge, which consists of two binding sites: the catalytic active site (CAS) located at the bottom of the gorge, and the peripheral anionic site (PAS) at the entrance [16,17]. Typically, inhibitors that target either site can block AChE activity. However, studies have revealed that the PAS plays a crucial role in promoting the aggregation of amyloid-beta (Aβ), which is involved in the complex molecular mechanisms associated with Alzheimer’s disease (AD) [18]. Moreover, as AD progresses, AChE activity gradually declines, while butyrylcholinesterase (BuChE) activity significantly increases (by 40–90%) in the hippocampus and temporal cortex. Elevated BuChE levels have been linked to Aβ aggregation during the early stages of senile plaque formation [19]. Therefore, simultaneously inhibiting both AChE (targeting both CAS and PAS) and BuChE may offer enhanced therapeutic benefits for AD treatment.

Research also indicates that monoamine oxidases (MAOs) are implicated in the etiology of Alzheimer’s disease [20,21]. MAOs are flavin adenine dinucleotide (FAD) containing enzymes, which are widely distributed in the central nervous system, and present in two main isoforms, MAO-A and MAO-B. They are classified based on inhibitor sensitivity and substrate specificity [22,23,24]. In the central nervous system, the MAO-A isoform appears to be present mainly in catecholaminergic neurons, whereas the MAO-B form is primarily located in the glia and in serotonergic neurons [25]. The activity of MAO-B increases with age due to an increased concentration of MAO-B, leading to an enhanced metabolism of dopamine and to the production of large amounts of hydrogen peroxide, which ultimately gives rise to neuronal damage [26]. Therefore, selective inhibition of MAO-B provides another therapeutic approach in treating AD. Selective MAO-B inhibitors such as selegiline and rasagiline have been demonstrated to retard further neurodegeneration [27,28] and can be used to treat the neurodegenerative disorders such as AD, while selective inhibitors for MAO-A are usually used as antidepressants.

The intricate and multifaceted nature of Alzheimer’s disease (AD) is believed to be the main factor behind the lack of effective treatments. Consequently, the multitarget-directed ligand strategy, which entails creating a single chemical compound that targets two or more AD-related factors, has gained growing attention. Multiple research groups have shared encouraging results employing this approach [29,30,31,32]. Tacrine was the first AChE inhibitor approved by the FDA. Though its clinical use has been limited by hepatotoxicity, tacrine remains an important starting point in research towards developing new drugs for AD due to its straightforward synthesis and comparatively low cost. In fact, tacrine hybrids, bifunctional molecules where tacrine has been chemically linked to another molecule, always act as multi-targeted compounds against AD, and have been well characterized in the literature. And also, coumarin analogs, which are widely used in medicinal chemistry, have found to exhibit potent AChE inhibitory activity and Aβ anti-aggregation by interacting with PAS of AChE [33]. In addition, coumarin derivatives are known to have potent activity to inhibit MAOs, with some natural and synthetic coumarin compounds identified as MAO inhibitors [34,35]. Especially, 7-substituted coumarin derivatives can significantly improve inhibitory activity and selectivity toward MAO-B [36]. Several tacrine–coumarin hybrids have been reported as dual or multiple inhibitors for AD [37,38,39,40].

Based on the pharmacophoric role of both tacrine and coumarin, it was desirable to design tacrine–coumarin hybrids derivatives by linking different linkers for pharmacological studies for AD therapy. As the mid-gorge of AChE that is lined with aromatic residues could establish cation-π or π-π interaction, the role of the linker in inhibitory activity for AChE is also very important, and it attracted more attention in recent years [41]. Triazoles moiety, which can be easily synthesized by using click chemistry, has found increasing applications in all aspects of drug development and discovery. Several triazole-containing hybrids evaluated as AChE inhibitors have been reported (Figure 1) [42,43]. The X-ray crystallographic studies of 1,2,3-triazole-containing AChE inhibitors and mouse AChE suggested that the 1,2,3-triazole moiety of these compounds was involved in van der Waals contact with the Phe297 and Tyr341 side chains [44], thus contributing to the energetics of inhibitor binding to AChE.

Figure 1.

Chemical structure of triazole-containing hybrids: (a) 9H-Carbazole derivatives, (b) acridone derivatives, (c) berberine derivatives.

Motivated by these studies, we designed and synthesized a series of tacrine–coumarin hybrid molecules connected through 1,2,3-triazole linkers to identify potent candidate compounds exhibiting cholinergic activity, Aβ reduction, and MAO-B inhibition for Alzheimer’s disease treatment. Herein, we report our preliminary results on the synthesis and evaluation of these tacrine–coumarin hybrids linked by 1,2,3-triazole with Aβ reducing properties, MAO-B inhibition properties, and dual inhibition of AChE and BuChE as multitarget-directed ligands against AD.

2. Result and Discussion

2.1. Design

Studies have shown that tacrine fragments have a high affinity for CAS. In order to develop dual AChE inhibitors binding to both CAS and PAS, 7-hydroxy coumarin derivatives were chosen to attach to tacrine through 1,2,3-triazole linkers, with the expectation of allowing the inhibitors to occupy both the CAS and the PAS of AChE, while possessing Aβ reducing properties and MAO inhibitory activities. The 1,2,3-triazole moiety of the linker could establish a cation-π interaction with the mid-gorge of AChE, which would enhance the inhibition of AChE. Based on this idea, the azide intermediates were prepared from tacrine, and the terminal alkyne intermediates were prepared from coumarin. And the final products of the tacrine–coumarin hybrids (compounds 15–18) were prepared from these two intermediates via click chemistry (Figure 2).

Figure 2.

Design of tacrine–coumarin hybrids as dual AChE inhibitors.

2.2. Synthetic Methodology

The triazole-linked tacrine–coumarin hybrids (15, 16, 17, 18) were synthesized as shown in Scheme 1. First, terminal alkyne intermediates 2 and 3 were prepared by acylation of alkynol with bromo-substituted acyl chloride or methanesulfonyl chloride, then reacted with 7-hydroxy-2H-chromen-2-ones in the presence of cesium carbonate to provide intermediates 5 and 6. Tacrine 8 and 9-chloro-1,2,3,4-tetrahydroacridine 12 were prepared from commercially available reagents 2-aminobenzonitrile or 2-aminobenzoic acid according to the reported literature [45,46]. By acylation of tacrine with chloroacetyl chloride, intermediate 9 was obtained, and then it was treated with sodium azide in DMF to give intermediate 13. Alternatively, intermediate 14 was produced by refluxing compound 12 with 2-azidoethylamine in phenol. Finally, compounds 13 and 14 were reacted with compounds 5 and 6 under click-chemistry conditions, using catalytic amounts of copper sulfate and sodium ascorbate in a mixture of n-C₄H₉OH/DCM/H₂O (v/v/v, 1:1:0.1), yielding the desired products 15, 16, 17, and 18.

Scheme 1.

Synthesis of triazole-linked tacrine–coumarin hybrids 15, 16, 17, 18. Reagents and conditions: (a) Br(CH₂)_nCOCl, DCM, 0 °C, 2 h; (b) MsCl, DCM, 0 °C, 2 h; (c) NaI, acetone/THF = 1/1, reflux, 12 h; (d) (i) cyclohexanone, BF₃·Et₂O, toluene, reflux, 12 h; (ii) KOH (50%), dioxane, reflux, 3 h; (e) chloroacetyl chloride, Et₃N, reflux, 5 h; (f) NaN₃, DMF, 80 °C, 12 h; (g) cyclohexanone, POCl₃, reflux, 3 h; (h) 2-azidoethylamine hydrochloride, PhOH, 130 °C, 12 h; (i) CuSO₄, NaAsc, n-C₄H₉OH/DCM/H₂O = 1/1/0.1, r.t, overnight.

2.3. Cholinesterase Inhibitory Activity

The inhibitory activities of hybrids 15–18 against AChE (from electric eel) and BuChE (from horse serum) were measured according to the spectrophotometric method of Ellman et al. [47], using tacrine as a reference compound. The IC₅₀ values of all test compounds and their selectivity index for AChE over BuChE are summarized in Table 1. It could be seen from Table 1 that the novel target compounds 17 and 18 showed strong inhibitory activities against both AChE and BuChE, whereas compounds 15 and 16 displayed little to no inhibitory activity. Earlier studies indicated that acylating the 9-amino group of tacrine might enhance choline uptake in rat hippocampus synaptosomes and reduce tacrine’s toxicity [48]. Based on this, we initially designed target compound 15 by acylating its 9-amino group with acetyl chloride. However, compounds 15a–15h all exhibited little to no inhibitory effect on AChE. The length of the linkers and the substituent groups at the 4-position of the coumarin ring appeared to have little impact on the inhibitory activity against AChE (compounds 15a–15h). Nevertheless, compounds 15a and 15b demonstrated selective inhibitory activity toward BuChE (15a: IC₅₀ > 100 μM for AChE, IC₅₀ = 0.60 μM for BuChE, SI > 166.7; 15b: IC₅₀ > 100 μM for AChE, IC₅₀ = 1.26 μM, SI > 79.4). The bulky group (Ph) introduced to the 4-position of the coumarin ring significantly reduced the inhibitory activity for BuChE (15c, IC₅₀ = 15.65 μM), and the increased length of the linker also reduced the inhibitory effect on BuChE significantly. Then, the influence of the ester group on the linker was investigated. Compounds 16 were synthesized by removing the ester group on the linker, but also produced a disappointing result on the inhibitory activity against both AChE and BuChE, and 16a and 16b showed almost no inhibitory activity on both AChE and BuChE. At last, compounds 17 and 18, in which the 9-amino group remained unacylated, were synthesized and evaluated. The result indicated that the inhibitory activities against both AChE and BuChE increased significantly; all six compounds (17a–17d, 18a–18b) showed better inhibitory activity (2–6 times stronger) against AChE than the reference compound tacrine and almost comparable inhibitory activity against BuChE. No clear trend was observed when the linker length was changed in these compounds. And among all the target compounds, 18b showed the highest inhibitory activity against AChE and BuChE (IC₅₀ = 0.027 μM for AChE, IC₅₀ = 0.024 μM for BuChE, SI = 1.13).

Table 1.

Inhibition of ChEs activity, selectivity index, and self-induced Aβ_1–42 aggregation.

Compd.	X, R, n	IC50 (μM) a		Selectivity Index b	Aβ Aggregation Inhibition (%) c
		AChE Inhibition	BuChE Inhibition
15a	m = 1, n = 1, R = H	>100	0.60 ± 0.07	>166.7	24.6 ± 3.2
15b	m = 1, n = 1, R = Me	>100	1.26 ± 0.12	>79.4	N.D d
15c	m = 1, n = 1, R = Ph	79.73 ± 2.10	15.65 ± 2.03	5.09	26.2 ± 4.5
15d	m = 2, n = 1, R = H	>100	13.02 ± 1.01	>7.68	14.6 ± 1.4
15e	m = 2, n = 1, R = Me	58.26 ± 2.42	10.23 ± 0.85	5.70	21.1 ± 2.2
15f	m = 2, n = 3, R = H	>100	>100	–	N.D
15g	m = 2, n = 4, R = H	83.73 ± 5.12	20.88 ± 1.02	4.01	23.0 ± 1.9
15h	m = 2, n = 4, R = Me	>100	>100	–	N.D
16a	m = 0, R = H	>100	>100	–	N.D
16b	m = 0, R = Me	>100	75.62 ± 4.31	>1.32	33.2 ± 2.8
17a	m = 1, n = 1, R = H	0.034 ± 0.002	0.045 ± 0.005	0.76	32.2 ± 1.7
17b	m = 1, n = 1, R = Me	0.045 ± 0.004	0.147 ± 0.005	0.31	32.1 ± 1.3
17c	m = 2, n = 1, R = H	0.036 ± 0.003	0.060 ± 0.003	0.6	55.0 ± 2.2
17d	m = 2, n = 3, R = H	0.080 ± 0.007	0.044 ± 0.004	1.82	58.4 ± 2.1
18a	m = 0, R = H	0.067 ± 0.007	0.026 ± 0.003	2.58	66.4 ± 1.6
18b	m = 1, R = H	0.027 ± 0.005	0.024 ± 0.002	1.13	50.7 ± 2.0
tacrine		0.17 ± 0.04	0.029 ± 0.005	5.86	–
curcumin		–	–		46.6 ± 2.5

^a Data are expressed as means ± SEM of at least three experiments. ^b Selectivity index = IC₅₀ (AChE)/IC₅₀ (BuChE). ^c Inhibition of self-induced Aβ_1–42 aggregation, the thioflavin-T fluorescence method was used, the mean ± SD of at least three independent experiments, and the measurements were carried out in the presence of 20 μM compounds. ^d. N.D = Not detected.

2.4. Kinetic Study of AChE Inhibition

In order to explore the inhibition mechanism of compounds 17 and 18 with AChE, an enzyme kinetic study was performed by using eeAChE. Compound 18b was chosen as the model compound, and the reciprocal plots of 1/velocity versus 1/[substrate] were constructed at different concentrations of the substrate acetylthiocholine (0.05–1 mM) by using Ellman’s method. Graphical analysis of the Lineweaver–Burk reciprocal plots (Figure 3) indicated that both increasing slopes and intercepts with increasing inhibitor concentrations. This pattern suggested a mixed-type of inhibition, which implied that 18b could interact simultaneously with dual sites (PAS and CAS) of AChE.

Figure 3.

Lineweaver–Burk plot for the kinetic study of eeAChE inhibition by compound 18b.

2.5. Self-Induced Aβ_1–42 Aggregation

All compounds tested for both ChEs inhibition were also evaluated for their ability to inhibit self-induced Aβ_1–42 aggregation through a thioflavin T-based fluorometric assay [49]. Curcumin was used as a reference compound. From the results summarized in Table 1, it can be observed that the results are similar to those of cholinesterase inhibitory activity. Compounds 15 and 16 exhibited little to no inhibition of self-induced Aβ_1–42 aggregation, with compounds 15b, 15f, 16a, and 16b showing no inhibition at all. Other compounds demonstrated weak inhibition, with inhibition percentages around 20% at 20 μM. Compounds 17 and 18 showed better results, with inhibition percentages ranging from 32.1% to 66.4%. Additionally, compounds 17c–18b exhibited better potencies than the reference compound curcumin.

2.6. MAOs Inhibitory Activity

Through screening for cholinesterase inhibitory activity, six compounds (17a–18b) demonstrated strong inhibition against both AChE and BuChE, while two compounds (15a, 15b) showed selective inhibition against BuChE. These eight compounds were then tested for their inhibitory effects on human MAOs using a spectrophotometric method, with iproniazide, a non-selective MAO inhibitor, serving as the reference compound [50,51]. As shown in Table 2, except for 15a, 15b, and 18a, the other five compounds exhibited selective inhibition of MAO-B, with potency ranging from micromolar to sub-micromolar levels. It seemed that the presence of an acyl group on the amino group and an ester group in the linker significantly affected MAO-B activity. Compounds 15a and 15b, which contain an acyl group on the amino group, showed no inhibition of either MAO-A or MAO-B, whereas compounds 17a–17d, lacking the acyl group on the amino group, displayed stronger inhibition of MAO-B (IC₅₀ values between 0.18 and 1.68 μM) compared to the reference. Additionally, the ester group in the linker played a crucial role in MAO-B inhibition; removal of this ester group led to a sharp decline in activity, with 18a showing no significant inhibition and 18b exhibiting reduced inhibition (IC₅₀ = 12.98 μM). The inhibitory activity of 7-hydroxy coumarin on MAOs was also evaluated, revealing no significant inhibition of either MAO-A or MAO-B, indicating that the inhibitory effects of the target compounds are not solely dependent on the coumarin structure.

Table 2.

Inhibition of MAO activity, selectivity index.

Comp	X, R, n	IC50 (μM) a		Selectivity Index b
		MAO-A	MAO-B
15a	m = 1, n = 1, R = H	>100	>100	-
15b	m = 1, n = 1, R = Me	>100	>100	-
17a	m = 1, n = 1, R = H	>100	1.01 ± 0.09	>99.01
17b	m = 1, n = 1, R = Me	>100	0.94 ± 0.09	>106.38
17c	m = 2, n = 1, R = H	>100	1.68 ± 0.11	>59.52
17d	m = 2, n = 3, R = H	>100	0.18 ± 0.01	>555.56
18a	m = 0, R = H	>100	>100	-
18b	m = 1, R = H	>100	12.98 ± 1.02	>7.70
coumarin		>100	>100	-
iproniazide		2.83 ± 0.14	3.96 ± 0.35	0.71

^a Data are expressed as means ± SEM of at least three experiments. ^b Selectivity index = IC₅₀ (MAO-A)/IC₅₀ (MAO-B).

2.7. Molecular Docking into Cholinesterases

To understand how the most effective compounds bind to AChE, molecular docking simulations were conducted using the crystal structures of human AChE (PDB ID: 4EY7) and BuChE (PDB ID: 1POI) [52]. These docking studies were performed with Schrödinger 2023-3 software [53], and the resulting complexes were visualized using PyMOL version 3.1.6.1. Compounds 15a and 17d were chosen as representative ligands due to their strong ChE inhibitory activity.

As shown in Figure 4, molecular docking studies revealed that compound 15a is well accommodated in the BuChE active site, forming key hydrogen-bond interactions with Lys142, Asp149, and Gln172, together with favorable hydrophobic contacts. Its preferential occupation of the catalytically active site provides a structural explanation for its selective BuChE inhibitory activity. In contrast, compound 17d binds deeply within the AChE active-site gorge, spanning both the catalytic active site and the peripheral anionic site, and forms stabilizing interactions with Glu202, Tyr124, and Phe295. This dual-site binding mode supports its potent AChE inhibition and Aβ aggregation inhibitory activity.

Figure 4.

Molecular docking analysis of the target compounds. (A) The predicted 3D binding mode of compound 15a within the active site of BuChE (PDB ID: 1POI). Key residues forming interactions (Lys142, Asp149, and Gln172) are shown as sticks. (B) The 2D interaction diagram of compound 15a with BuChE residues. (C) The predicted 3D binding mode of compound 17d within the active site of AChE (PDB ID: 4EY7). Key residues (Glu202, Tyr124, and Phe295) are shown as sticks. (D) The 2D interaction diagram of compound 17d with AChE residues. The ligands are rendered in orange sticks, and the protein backbone is shown as a grey cartoon. Hydrogen bonds are indicated by yellow dashed lines.

3. Experimental Section

3.1. Chemistry

Analytical grade reagents were purchased from Sigma-Aldrich, Energy (Shanghai, China), and Aladdin (Shenzhen, China) without further purification. Reaction progress was monitored using analytical thin-layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) plates, and the spots were detected under UV light (254 nm). The melting point was measured on an XT-4 micromelting point instrument and uncorrected. ¹H NMR spectra (400 MHz) and ¹³C NMR spectra (100 MHz) were measured on a Bruker AVANCE III at 25 °C and referenced to TMS. Elemental analyses were carried out by a Perkin Elmer Series II elemental analyzer (some of the samples included partial solvent/hydrate, which may not be fully purified). Compounds 8, 9, 12, 13, 14 were prepared as previously described [45,46,50]. (Caution: during the preparation of compound 14, do not use an iron medicine spoon when measuring the potentially explosive reagent 2-azidoethylamine hydrochloride).

3.1.1. General Procedures for the Preparation of Compounds 15–16

Compounds 15 and 16 were prepared as previously described [54]. In a 25 mL round-bottom flask, a mixture of 5/6 (1.2 mmol), 13 (1 mmol), CuSO₄·5H₂O (0.1 mmol), and sodium ascorbate (0.2 mmol) was combined in a solvent mixture of n-butanol and dichloromethane (4 mL, 1:1). Then, water (0.2 mL) was added, causing the solution to turn black immediately. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, as confirmed by TLC, water (10 mL) was added, and the mixture was extracted three times with 15 mL portions of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel (300–400 mesh) to yield compounds 15 and 16.

• (1-(2-Oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl

• 2-((2-oxo-2H-chromen-7-yl)oxy)acetate (15a). Yield 76%, white solid, mp 226–228 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.50 (s, 1H), 8.27 (s, 1H), 7.95 (d, J = 13.2 Hz, 2H), 7.88 (d, J = 10.8 Hz, 1H), 7.69–7.63 (m, 1H), 7.60 (d, J = 10.8 Hz, 1H), 7.55–7.49 (m, 1H), 7.00 (d, J = 3.2 Hz, 1H), 6.95 (dd, J₁ = 10.8 Hz, J₂ = 3.2 Hz, 1H), 6.29 (d, J = 12.8 Hz, 1H), 5.59 (s, 2H), 5.30 (s, 2H), 4.97 (s, 2H), 3.04 (t, J = 8.4 Hz, 2H), 2.77 (t, J = 8.4 Hz, 2H), 1.88–1.79 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.5, 165.0, 161.1, 160.6, 159.9, 155.6, 146.8, 144.6, 141.7, 139.0, 130.0, 129.2, 128.7, 127.9, 127.3, 126.1, 125.4, 124.2, 123.7, 113.4, 113.1, 102.1, 65.4, 58.4, 52.2, 33.9, 25.3, 22.8, 22.4 ppm; elemental analysis calcd (%) for C₂₉H₂₅N₅O₆·1.5H₂O: C 61.48, H 4.98, N 12.36; found: C 61.86, H 4.78, N 12.65.

• (1-(2-Oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl

• 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate (15b). Yield 71%, pale yellow solid, mp 252–254 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.51 (s, 1H), 8.23 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.67–7.64 (m, 2H), 7.53 (t, J = 7.2 Hz, 1H), 6.99 (d, J = 6.4 Hz, 1H), 6.96 (dd, J₁ = 8.8 Hz, J₂ = 2.4 Hz, 1H), 6.21 (d, J = 1.2 Hz, 1H), 5.61 (s, 2H), 5.31 (s, 2H), 4.98 (s, 2H), 3.04 (t, J = 6.4 Hz, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.34 (s, 3H), 1.89–1.78 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.5, 165.0, 161.0, 160.5, 159.9, 155.0, 153.7, 146.8, 141.7, 139.0, 129.2, 128.7, 127.9, 127.3, 127.0, 126.1, 124.2, 123.7, 114.2, 112.7, 112.0, 102.2, 65.4, 58.3, 52.2, 33.9, 25.3, 22.8, 22.4, 18.5 ppm; elemental analysis calcd (%) for C₃₀H₂₇N₅O₆·0.1H₂O: C 64.88, H 4.94, N 12.61; found: C 64.83, H 5.11, N 12.31.

• (1-(2-Oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl

• 2-((2-oxo-4-phenyl-2H-chromen-7-yl)oxy)acetate (15c). Yield 65%, white solid, mp 255–257 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.51 (s, 10.52, 1H), 8.29 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 7.2 Hz, 1H), 7.56–7.49 (m, 6H), 7.34 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 6.96 (dd, J₁ = 9.2 Hz, J₂ = 2.4 Hz, 1H), 6.25 (s, 1H), 5.61 (s, 2H), 5.32 (s, 2H), 4.99 (s, 2H), 3.05 (t, J = 6.4 Hz, 2H), 2.78 (t, J = 6.4 Hz, 2H), 1.89–1.78 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.4, 165.0, 161.2, 160.4, 159.9, 155.7, 155.5, 146.7, 141.7, 139.1, 135.4, 130.2, 129.33, 129.28, 128.9, 128.6, 128.4, 127.9, 127.3, 126.1, 124.2, 123.7, 113.2, 112.9, 112.2, 102.6, 65.5, 58.4, 52.2, 33.8, 25.3, 22.8, 22.4 ppm; elemental analysis calcd (%) for C₃₅H₂₉N₅O₆·H₂O: C 66.34, H 4.93, N 11.05; found: C 66.42, H 5.15, N 11.33.

• 2-(1-(2-Oxo-2-((1,2,3,4-tetrahydroanthracen-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)ethyl

• 2-((2-oxo-2H-chromen-7-yl)oxy)acetate (15d). Yield 74%, white solid, mp 215–217 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.47 (s, 1H), 8.06 (s, 1H), 7.95 (d, J = 9.2 Hz, 2H), 7.89 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.52 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 2.0 Hz, 1H), 6.94 (dd, J₁ = 8.4 Hz, J₂ = 2.0 Hz, 1H), 6.30 (d, J = 9.6 Hz, 1H), 5.57 (s, 2H), 4.92 (s, 2H), 4.42 (t, J = 6.4 Hz, 2H), 3.06–3.01 (m, 4H), 2.76 (t, J = 6.4 Hz, 2H), 1.86–1.77 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.6, 165.2, 161.1, 160.7, 159.9, 155.6, 146.8, 144.6, 143.3, 139.1, 130.0, 129.2, 128.7, 127.9, 126.1, 125.0, 124.3, 123.7, 113.4, 113.4, 113.0, 102.1, 65.4, 64.1, 52.2, 33.9, 25.31, 25.28, 22.8, 22.4 ppm; elemental analysis calcd (%) for C₃₀H₂₇N₅O₆·0.8H₂O: C 63.44, H 5.08, N 12.33; found: C 63.52, H 5.35, N 12.58.

• 2-(1-(2-Oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)ethyl

• 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate (15e). Yield 69%, white solid, mp 160–162 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.51 (s, 10.47, 1H), 8.06 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.67–7.61 (m, 2H), 7.54 (t, J = 7.6 Hz, 1H), 6.96–6.93 (m, 2H), 6.21 (s, 1H), 5.56 (s, 2H), 4.92 (s, 2H), 4.42 (t, J = 6.4 Hz, 2H), 3.07–3.01 (m, 4H), 2.77 (t, J = 6.4 Hz, 2H), 2.36 (s, 3H), 1.86–1.76 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.6, 165.2, 161.0, 160.5, 159.9, 155.0, 153.7, 146.7, 143.3, 139.1, 129.2, 128.6, 127.9, 127.0, 126.1, 125.0, 124.3, 123.7, 114.2, 112.7, 112.0, 102.1, 65.4, 64.1, 52.2, 33.8, 25.32, 25.30, 22.8, 22.4, 18.5 ppm; elemental analysis calcd (%) for C₃₁H₂₉N₅O₆·1.5H₂O: C 62.62, H 5.42, N 11.78; found: C 62.83, H 5.56, N 11.92.

• 3-(1-(2-Oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)propyl

• 3-((2-oxo-2H-chromen-7-yl)oxy)propanoate (15f). Yield 63%, pale yellow solid, mp 144–146 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.47 (s, 1H), 8.04 (s, 1H), 7.95 (d, J = 9.6 Hz, 2H), 7.89 (d, J = 8.4 Hz, 1H), 7.68–7.64 (m, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.54–7.50 (m, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.88 (dd, J₁ = 8.4 Hz, J₂ = 2.4 Hz, 1H), 6.27 (d, J = 9.6 Hz, 1H), 5.55 (s, 2H), 4.31 (t, J = 6.8 Hz, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.04–2.99 (m, 4H), 2.76 (t, J = 6.4 Hz, 2H), 2.46 (t, J = 6.4 Hz, 2H), 1.99–1.95 (m, 2H), 1.87–1.77 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.9, 165.2, 162.1, 160.8, 159.9, 155.9, 146.7, 144.7, 143.5, 139.1, 129.9, 129.2, 128.6, 127.9, 126.1, 124.9, 124.3, 123.7, 113.1, 112.9, 112.8, 101.7, 67.8, 63.4, 52.2, 33.8, 30.5, 25.4, 25.3, 24.4, 22.8, 22.4 ppm; elemental analysis calcd (%) for C₃₂H₃₁N₅O₆·0.6H₂O: C 64.88, H 5.48, N 11.82; found: C 64.88, H 5.28, N 11.66.

• (1-(2-Oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl

• 5-((2-oxo-2H-chromen-7-yl)oxy)pentanoate (15g). Yield 71%, pale yellow solid, mp 214–216 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.51 (s,1H), 8.24 (s, 1H), 7.97–7.94 (m, 2H), 7.90 (d, J = 8.0 Hz, 1H), 7.70–7.65 (m, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.56–7.52 (m, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.90 (dd, J₁ = 8.8 Hz, J₂ = 2.0 Hz, 1H), 6.27 (d, J = 7.2 Hz, 1H), 5.60 (s, 2H), 5.18 (s, 2H), 4.05 (t, J = 6.4 Hz, 2H), 3.04 (t, J = 6.4 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H), 2.41 (t, J = 6.4 Hz, 2H), 1.88–1.68 ppm (m, 8H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.0, 165.1, 162.3, 160.8, 159.9, 155.8, 144.8, 142.3, 139.2, 129.9, 129.3, 128.5, 127.9, 126.9, 126.2, 124.2, 123.7, 119.9, 113.2, 112.9, 112.8, 101.6, 68.4, 57.6, 52.2, 33.7, 33.4, 28.2, 27.6, 25.3, 22.8, 22.4, 21.5 ppm; elemental analysis calcd (%) for C₃₂H₃₁N₅O₆·0.2H₂O: C 65.68, H 5.41, N 11.97; found: C 65.63, H 5.66, N 12.28.

• (1-(2-Oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)methyl

• 5-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)pentanoate (15h). Yield 69%, pale yellow solid, mp 212–214 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.50 (s,1H), 8.25 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.69–7.65 (m, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.55–7.51 (m, 1H), 6.93–6.89 (m, 2H), 6.18 (d, J = 1.2 Hz, 1H), 5.60 (s, 2H), 5.18 (s, 2H), 4.05 (t, J = 6.0 Hz, 2H), 3.03 (t, J = 6.0 Hz, 2H), 2.77 (t, J = 6.0 Hz, 2H), 2.41 (t, J = 6.4 Hz, 2H), 2.36 (d, J = 0.8 Hz, 3H), 1.88–1.67 ppm (m, 8H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.0, 165.1, 162.1, 160.6, 159.9, 155.2, 153.8, 146.8, 142.3, 139.1, 129.2, 128.6, 127.8, 126.9, 126.8, 126.1, 124.2, 123.7, 113.5, 112.9, 111.5, 101.6, 68.3, 57.6, 52.2, 33.8, 33.4, 28.2, 25.3, 22.8, 22.4, 21.6, 18.6 ppm; elemental analysis calcd (%) for C₃₃H₃₃N₅O₆: C 66.54, H 5.58, N 11.76; found: C 66.45, H 5.68, N 11.78.

• 2-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(1,2,3,4-tetrahydroacridin-9-yl)acetamide (16a). Yield 65%, white solid, mp 259–261 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.52 (s, 1H), 8.37 (s, 1H), 8.01–7.96 (m, 2H), 7.90 (d, J = 8.4 Hz, 1H), 7.69–7.63 (m, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 2.0 Hz, 1H), 7.05–7.02 (m, 1H), 6.31 (d, J = 9.2 Hz, 1H), 5.62 (s, 2H), 5.31 (s, 2H), 3.04 (d, J = 6.4 Hz, 2H), 2.77 (d, J = 6.4 Hz, 2H), 1.90–1.87 (m, 2H), 1.81–1.80 ppm (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.1, 161.6, 160.8, 159.9, 155.8, 146.8, 144.8, 142.3, 139.0, 130.0, 129.3, 128.7, 127.8, 127.2, 126.1, 124.2, 123.7, 113.4, 113.1, 113.0, 102.0, 62.0, 52.2, 33.8, 25.3, 22.9, 22.4 ppm; elemental analysis calcd (%) for C₂₇H₂₃N₅O₄·0.1H₂O: C 67.10, H 4.84, N 14.49; found: C 67.35, H 5.11, N 14.24.

• 2-(4-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)-N-(1,2,3,4-tetrahydroacridin-9-yl)acetamide (16b). Yield 66%, white solid, mp 253–255 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.48 (s, 1H), 8.09 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.69–7.65 (m, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.02–6.97 (m, 2H), 6.21 (s, 1H), 5.56 (s, 2H), 4.37 (t, J = 6.4 Hz, 2H), 3.17 (t, J = 6.4 Hz, 2H), 3.04 (t, J = 6.0 Hz, 2H), 2.76 (t, J = 5.6 Hz, 2H), 2.34 (s, 3H), 1.88–1.86 (m, 2H), 1.80–1.79 ppm (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.2, 161.9, 160.6, 159.9, 155.2, 153.9, 146.8, 143.6, 139.1, 129.2, 128.7, 127.8, 126.9, 125.0, 124.2, 123.7, 113.7, 112.9, 111.7, 101.8, 67.8, 52.1, 33.8, 25.8, 25.3, 22.8, 22.4, 18.6 ppm; elemental analysis calcd (%) for C₂₉H₂₇N₅O₄·0.3H₂O: C 67.64, H 5.40, N 13.60; found: C 67.44, H 5.27, N 13.93.

3.1.2. General Procedures for the Preparation of Compounds 17–18

To a 25 mL round flask charged with a mixture of 5/6 (1.2 mmol), 13 (1 mmol), CuSO₄·5H₂O (0.1 mmol), and sodium ascorbate (0.2 mmol) in n-C₄H₉OH/DCM (4 mL, 1/1), water (0.2 mL) was added, and the solution changed black immediately. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, as confirmed by TLC, water (10 mL) was added, and the mixture was extracted three times with 15 mL portions of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was then purified by column chromatography on silica gel (300–400 mesh), after evaporation under reduced pressure to remove the solvents, HCl in ethanol (2.3 M, 2 mL) was added, and solid precipitates formed. After filtration and drying, the hydrochloride salts of compounds 17 and 18 were obtained.

• N-(2-(4-((2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-aminium chloride (17a). Yield 62%, pale yellow solid, mp 126–128 °C; ¹H NMR (400 MHz, CD₃OD) δ 8.13 (d, J = 8.8 Hz, 1H), 7.93 (s, 1H), 7.73–7.70 (m, 2H), 7.65–7.64 (m, 1H), 7.47 (t, J = 7.2 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 6.80–6.77 (m, 1H), 6.61 (d, J = 2.4 Hz, 1H), 6.10 (d, J = 9.2 Hz, 1H), 5.12 (s, 2H), 4.74–4.71 (m, 2H), 4.68 (s, 2H), 4.36 (t, J = 9.2 Hz, 2H), 2.95–2.85 (m, 2H), 2.59–2.46 (m, 2H), 1.88–1.77 ppm (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 168.2, 161.6, 161.1, 157.0, 155.4, 151.3, 144.1, 142.4, 138.0, 132.9, 129.2, 125.5, 125.2, 124.6, 118.8, 116.1, 113.3, 113.0, 112.8, 112.6, 101.1, 64.8, 57.4, 56.9, 49.9, 28.0, 23.7, 21.5, 20.3 ppm; elemental analysis calcd (%) for C₂₉H₂₈ClN₅O₅·0.5EtOH: C 61.59, H 5.34, N 11.97; found: C 61.68, H 5.61, N 12.13.

• N-(2-(4-((2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetoxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-aminium chloride (17b). Yield 61%, white solid, mp 120–122 °C; ¹H NMR (400 MHz, CD₃OD) δ 8.23 (d, J = 8.4 Hz, 1H), 8.06 (s, 1H), 7.84–7.80 (m, 1H), 7.76–7.74 (m, 1H), 7.62–7.57 (m, 2H), 6.93–6.90 (m, 1H), 6.69 (s, 1H), 6.09 (s, 1H), 5.23 (s, 2H), 4.84–4.79 (m, 2H), 4.78 (s, 2H), 4.51–4.42 (m, 2H), 3.10–2.94 (m, 2H), 2.70–2.58 (m, 2H), 2.38 (s, 3H), 1.99–1.86 ppm (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 168.2, 161.9, 161.7, 161.07, 160.95, 156.9, 154.7, 154.0, 151.3, 138.0, 132.8, 126.1, 125.6, 124.5, 118.8, 116.0, 114.0, 112.5, 112.3, 111.1, 101.2, 64.8, 57.4, 56.9, 50.0, 28.0, 23.7, 21.5, 20.3, 17.3 ppm; elemental analysis calcd (%) for C₃₀H₃₀ClN₅O₅·0.3EtOH: C 62.31, H 5.43, N 11.87; found: C 62.34, H 5.19, N 11.87.

• N-(2-(4-(2-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)ethyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-aminium chloride (17c). Yield 57%, pale yellow solid, mp 129–131 °C; ¹H NMR (400 MHz, CD₃OD) δ 8.32 (d, J = 8.4 Hz, 1H), 7.87–7.81 (m, 3H), 7.63 (t, J = 7.6 Hz, 1H), 7.55–7.52 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.84–6.83 (m, 1H), 6.25–6.21 (m, 1H), 4.97–4.96 (m, 2H), 4.84 (s, 2H), 4.55 (t, J = 5.2 Hz, 2H), 3.82–3.70 (m, 2H), 3.13–3.07 (m, 2H), 3.02–2.94 (m, 2H), 2.75–2.60 (m, 2H), 2.03–1.87 ppm (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 169.2, 168.7, 161.6, 161.2, 161.2, 156.7, 155.4, 151.7, 144.2, 137.9, 132.9, 129.2, 125.8, 124.4, 119.0, 116.2, 113.2, 113.1, 112.5, 112.4, 101.4, 64.8, 59.4, 57.0, 51.7, 28.1, 27.2, 24.1, 20.3 ppm; elemental analysis calcd (%) for C₃₀H₃₀ClN₅O₅·0.2EtOH: C 62.39, H 5.37, N 11.97; found: C 62.09, H 5.16, N 12.06.

• N-(2-(4-(2-((4-((2-oxo-2H-chromen-7-yl)oxy)butanoyl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-aminium chloride (17d). Yield 59%, white solid, mp 100–102 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 13.71 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 10.0 Hz, 1H), 7.87–7.81 (m, 3H), 7.71 (s, 1H), 7.61–7.57 (m, 1H), 6.93 (d, J = 2.4 Hz, 1H), 6.90 (dd, J₁ = 8.4 Hz, J₂ = 2.4 Hz, 1H), 6.28 (d, J = 9.6 Hz, 1H), 4.71 (t, J = 5.6 Hz, 2H), 4.34–4.30 (m, 2H), 4.15 (t, J = 6.8 Hz, 2H), 4.05 (t, J = 6.4Hz, 2H), 3.03–2.93 (m, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.54–2.52 (m, 2H), 2.42 (t, J = 7.2 Hz, 2H), 1.97–1.93 (m, 2H), 1.88–1.73 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.8, 162.0, 160.7, 156.7, 155.8, 151.8, 144.7, 143.6, 138.0, 133.3, 129.9, 125.8, 125.1, 123.7, 119.6, 116.4, 113.1, 113.0, 112.8, 101.6, 67.8, 63.3, 49.7, 47.8, 30.5, 28.4, 25.2, 24.4, 21.8, 20.7 ppm; elemental analysis calcd (%) for C₃₂H₃₄ClN₅O₅·0.7EtOH: C 63.04, H 6.05, N 11.01; found: C 63.32, H 5.86, N 11.19.

• N-(2-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-aminium chloride (18a). Yield 65%, white solid, mp 132–134 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 14.22 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.28 (s, 1H), 7.98 (m, 2H), 7.86–7.83 (m, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 6.96–6.93 (m, 1H), 6.30 (d, J = 9.6 Hz, 1H), 5.18 (s, 2H), 4.81 (t, J = 5.2 Hz, 2H), 4.36–4.35 (m, 2H), 3.05–2.95 (m, 2H), 2.59–2.51 (m, 2H), 1.83–1.68 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 161.5, 160.7, 156.6, 155.7, 151.8, 144.7, 142.5, 138.0, 133.2, 130.0, 125.9, 125.8, 125.1, 119.6, 116.4, 113.3, 113.2, 113.0, 112.7, 102.0, 62.0, 49.8, 47.7, 28.4, 24.5, 21.8, 20.6 ppm; elemental analysis calcd (%) for C₂₇H₂₆ClN₅O₃·0.7H₂O: C 62.78, H 5.35, N 13.56; found: C 62.58, H 5.56, N 13.28.

• N-(2-(4-(2-((2-oxo-2H-chromen-7-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-aminium chloride (18b). Yield 56%, white solid, mp 168–170 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 14.32 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.01–7.96 (m, 3H), 7.90 (brs, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 8.91 (s, 1H), 8.54 (d, J = 8.8 Hz, 1H), 6.30 (d, J = 9.2 Hz, 1H), 4.79–4.77 (m, 2H), 4.34–4.33 (m, 2H), 4.19 (t, J = 6.4 Hz, 2H), 3.03–2.99 (m, 4H), 2.61–2.55 (m, 2H), 1.83–1.68 ppm (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 161.8, 160.7, 156.5, 155.8, 151.7, 144.8, 143.8, 138.0, 133.0, 130.0, 125.7, 125.1, 124.0, 119.6, 116.4, 113.03, 112.97, 112.9, 112.6, 101.7, 67.7, 49.7, 47.8, 28.4, 25.6, 24.6, 21.8, 20.7 ppm; elemental analysis calcd (%) for C₂₈H2₈ClN₅O₃·0.3H₂O: C 64.25, H 5.51, N 13.38; found: C 64.07, H 5.23, N 13.17.

3.2. Biological Evaluation

3.2.1. Inhibition Experiments of ChEs

Cholinesterase (ChE) inhibitory activity was assessed using modified Ellman’s spectrophotometric method. The materials used included acetylcholinesterase (AChE) from electric eel, butyrylcholinesterase (BuChE) from equine serum, 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), S-butyrylthiocholine iodide (BTCI), acetylthiocholine iodide (ATCI), and tacrine hydrochloride, all obtained from Sigma-Aldrich. Tacrine hydrochloride served as the reference standard. All experiments were conducted in a 0.1 M KH₂PO₄/K₂HPO₄ buffer at pH 7.4. Stock solutions of the test compounds (10 mM) were prepared in DMSO (less than 0.5% v/v) and then diluted with phosphate buffer. The assay was carried out in 96-well plates by adding 25 μL of 1 mM acetylthiocholine iodide or butyrylthiocholine iodide as substrates, 125 μL of 1 mM DTNB, 25 μL of 0.1 M phosphate buffer (pH 7.4), 25 μL of the test compound at various concentrations, and 50 μL of 0.2 Units/mL AChE or BuChE, depending on the enzyme tested. At least five different concentrations of each test compound were evaluated. Absorbance changes at 405 nm were measured every 5 min over a 35 min period using a microplate reader. Enzyme activity and percentage inhibition were calculated, and the concentration of each compound required to inhibit 50% of ChE activity (IC50) was determined graphically from concentration–inhibition curves using GraphPad Prism 5.0.

3.2.2. Kinetic Analysis of AChE Inhibition

A kinetic analysis of AChE inhibition by compound 18b was conducted employing Lineweaver–Burk double reciprocal plots. The assay was carried out at relatively low substrate concentrations (0.05–1 μM ATC) using Ellman’s method (Lineweaver–Burk equation 1/v = (Km/Vmax)(1/[S]) + 1/Vmax). Inhibitor concentrations of 10, 50, and 100 nM were evaluated. Each reaction well contained a final mixture of 25 μL PBS (pH 7.4), 50 μL AChE, 25 μL of inhibitor solution, and 125 μL of 1.0 mM DTNB. Following the addition of 25 μL of ATC to initiate the reaction, the kinetic progress of the enzymatic hydrolysis was characterized by monitoring the absorbance at 415 nm at 3 min intervals in a microplate reader.

3.2.3. Inhibition of Self-Induced Aβ_1–42 Aggregation

Sample preparation: Aβ_1–42 was dissolved in 100% HFIP to a final concentration of 1 mg/mL. After overnight sonication in a water bath, the solution was aliquoted, vacuum-dried, and stored at –20 °C. Immediately before use, the peptide was reconstituted in DMSO to obtain the working stock solution.

Aggregation inhibition assay: The screening of compounds for inhibitory activity against Aβ_1–42 aggregation was conducted using a ThT fluorescence assay. A mixture containing 5 μL of compound (200 μM) and 5 μL of Aβ_1–42 (200 μM) in 40 μL of PBS (pH 7.4) was incubated at room temperature for 72 h on a thermostatic shaker. Subsequently, 100 μL of 5 μM ThT solution (in PBS, pH 7.4) was added. Fluorescence measurements were performed on a microplate reader with excitation at 435 nm and emission at 485 nm.

The Aβ_1–42 aggregation inhibition percent was calculated using the following formula. Inhibition percent = (F1 − F2)/(F1 − F0) × 100% (F0: fluorescence intensity of ThT solution, F1: fluorescence intensity of Aβ_1–42 and ThT solution, F2: fluorescence intensity of solution added tacrine–quinoline hybrids, Aβ_1–42 and ThT).

3.2.4. MAO Inhibitory Activity

MAO inhibitory activity was evaluated using a modified spectrophotometric method. The assay was based on the absorbance measurement of resorufin (k = 570 nm) produced by the stoichiometric interaction of 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent) in the presence of horseradish peroxidase with hydrogen peroxide (H₂O₂), one of the products generated during the oxidation of the MAO substrate (MAO-A: p-tyramine, MAO-B: benzylamine). Resorufin has absorption and fluorescence emission maxima of approximately 570 nm.

In our experiments, MAO activity was evaluated by using a modified procedure described in the literature [55]. The assays were performed in 96-well plates in a final volume of 200 μL. Briefly, recombinant human MAO-A and MAO-B enzymes (for MAO-A, 6 μL from a 5.0 mg/mL solution supplied by Sigma-Aldrich; for MAO-B, 50 μL from a 5.0 mg/mL solution supplied by Sigma-Aldrich) were diluted in aqueous 50 mM phosphate buffer (10 mL, pH 7.4). The enzyme solution (98 μL, final protein amounts: ~0.3 μg protein/well for MAO-A and ~2.5 μg protein/well for MAO-B) was treated with the test compound in DMSO (2 μL) and then incubated for 30 min at 25 °C. The resulting solution (100 μL) was treated with assay solution (100 μL), which is the mixed solution of 20 mM Amplex Red reagent (200 μL, final concentration: 0.2 mM), 100 mM substrate (200 μL, MAO-A: p-tyramine, MAO-B: benzylamine, final concentration: 1 mM), and 200 U/mL HRP (100 μL, final concentration: 1 U/mL) in aqueous 50 mM phosphate buffer (9.5 mL, pH 7.4). The production of resorufin was quantified at 37 °C, and the absorbance changes at 570 nm were detected every 5 min over a period of 60 min with a microplate reader. The enzyme activity and the percent inhibition were determined. The compound concentration producing 50% of MAO inhibition (IC₅₀) was calculated graphically from a concentration–inhibition curve for each compound using Graphpad Prism 5.0.

3.2.5. Molecular Docking Studies

Molecular docking studies were performed using the Schrödinger Release 2023-3. The crystal structures of human acetylcholinesterase (AChE, PDB ID: 4EY7) and human butyrylcholinesterase (BuChE, PDB ID: 1POI) were retrieved from the RCSB Protein Data Bank. Protein structures were prepared using the Protein Preparation Wizard in Maestro. This process included the assignment of bond orders, addition of hydrogen atoms, correction of missing side chains, and optimization of protonation states at physiological pH (7.0 ± 2.0) using Epik. Water molecules beyond 5 Å from the co-crystallized ligand were removed, while those involved in key bridges were retained. The protein structures were subsequently restrained-minimized using the OPLS4 force field until the root-mean-square deviation (RMSD) of heavy atoms converged to 0.3 Å.

Ligand structures (compounds 15a and 17d) were prepared using the LigPrep module to generate low-energy three-dimensional conformations with appropriate ionization states at pH 7.0 ± 2.0 using Epik. Geometry optimization was carried out using the OPLS4 force field. Receptor grids were generated using the Receptor Grid Generation module, centered on the centroid of the co-crystallized ligand in each protein structure to define the active site. Docking calculations were performed using the Glide module in Standard Precision (SP) mode with flexible ligand sampling. The final binding poses were selected based on GlideScore values, binding orientation, and visual inspection of key interactions within the active-site gorge. The resulting protein–ligand complexes were analyzed and visualized using PyMOL 3.1.6.1, and two-dimensional interaction diagrams were generated to identify hydrogen bonds, π–π interactions, and hydrophobic contacts between ligands and key amino acid residues.

4. Conclusions

In conclusion, a series of tacrine–coumarin hybrids have been designed, synthesized, and evaluated as novel multifunctional anti-AD agents. AChE and BuChE inhibitory activities, Aβ-reducing activity, and MAOs inhibitory activities were investigated, and the results showed that some of the compounds exhibited strong activities against both AChE and BuChE, good inhibition of Aβ aggregation, and effective MAO-B inhibitory activity. Within the four hybrid series, compounds 17 and 18, which possessed a free 9-amino group (unacylated), effectively inhibited AChE and BuChE in vitro at sub-micromolar levels. In particular, compound 18b demonstrated the most potent inhibition against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), being six times more effective than the reference compound tacrine for AChE and exhibiting comparable activity to tacrine for BuChE. Compound 18b can simultaneously bind to both the PASs and CASs of AChE. The fact that compounds 17 and 18 demonstrated strong inhibitory effects on Aβ aggregation also indicated that these compounds likely bind to the PAS of AChE to prevent Aβ aggregation. In addition, tests on MAO inhibition revealed that compound 17 exhibited good inhibition of MAO-B. Besides this, compounds 15a and 15b were found to selectively inhibit BuChE at the micromolar level. Overall, our preliminary findings indicated that the triazole-linked tacrine–coumarin hybrids, compounds 17 and 18, exhibit multifunctional activities against Alzheimer’s disease, including inhibition of ChEs, MAOs, and Aβ aggregation, making them promising candidates for anti-AD drugs. In particular, compound 17d showed a well-balanced inhibitory profile against AChE and BuChE, self-induced Aβ aggregation, and MAO-B. These results suggested 17d might be an excellent multifunctional agent for AD treatment, and further study on cells and even animals is ongoing in our lab.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31040595/s1, supplementary data of 1H NMR and 13C NMR spectra of all the products.

Author Contributions

Conceptualization, X.W.; Validation, X.W., X.L., W.J. and X.T.; Formal analysis, X.L. and X.T.; Investigation, X.W.; Resources, G.W.; Data curation, X.W., X.L. and W.J.; Writing—original draft, W.J. and X.T.; Writing—review & editing, G.W.; Visualization, X.W.; Supervision, X.W. and G.W.; Project administration, G.W.; Funding acquisition, X.W. and G.W. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This work was supported by the Excellent Young and Middle-aged Scientific and Technological Innovation Team Program of Hubei Province (No. T2023042), the Research and Development Fund of Wuchang University of Technology (No. X2024ZZ001), and the Innovation Foundation of the Synergy Innovation Center of Biological Peptide Antidiabetics of Hubei Province (Nos. HBSWDT202503Z and HBSWDT202505Z).