Synthesis and Biological Evaluation of Coumarin Triazoles as Dual Inhibitors of Cholinesterases and β-Secretase

Ankita Sharma; Sandip B Bharate

doi:10.1021/acsomega.2c07993

. 2023 Mar 16;8(12):11161–11176. doi: 10.1021/acsomega.2c07993

Synthesis and Biological Evaluation of Coumarin Triazoles as Dual Inhibitors of Cholinesterases and β-Secretase

Ankita Sharma ^†,^‡, Sandip B Bharate ^†,^‡,^*

PMCID: PMC10061512 PMID: 37008108

Abstract

Coumarin is a naturally occurring bioactive pharmacophore with wide occurrence among central nervous system (CNS)-active small molecules. 8-Acetylcoumarin, one of the natural coumarins, is a mild inhibitor of cholinesterases and β-secretase, which are vital targets of Alzheimer’s disease. Herein, we synthesized a series of coumarin–triazole hybrids as potential multitargeted drug ligands (MTDLs) with better activity profiles. The coumarin–triazole hybrids occupy the cholinesterase active site gorge from the peripheral to the catalytic anionic site. The most active analogue, 10b, belonging to the 8-acetylcoumarin core, inhibits acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase-1 (BACE-1) with IC₅₀ values of 2.57, 3.26, and 10.65 μM, respectively. The hybrid, 10b, crosses the blood–brain barrier via passive diffusion and inhibits the self-aggregation of amyloid-β monomers. The molecular dynamic simulation study reveals the strong interaction of 10b with three enzymes and forming stable complexes. Overall, the results warrant a detailed preclinical investigation of the coumarin–triazole hybrids.

1. Introduction

Alzheimer’s disease (AD) is a multifaceted progressive neurodegenerative disease with a time lag between the onset of a responsible biochemical process and the first found symptom. The former begins to cause neuronal damage many years advance when the latter is found. The impaired cholinergic transmission, accumulation of amyloid-β (Aβ) plaques, and neurofibrillary tangles in the brain are major hallmarks of AD that gradually lead to the death of nerve cells and brain atrophy.¹ Hampering of the cholinergic transmission results in memory and attention-deficit issues. This hampering is caused due to overactivity of acetylcholinesterase (AChE), which causes the breakdown of the somatic neurotransmitter acetylcholine (ACh).² Donepezil and galantamine are selective Food and Drug Administration (FDA)-approved AChE inhibitors. In the normal brain, the loss of ACh is compensated by butyrylcholine (BCh), but in AD, the levels of BCh are also depleted due to overexpression of the butyrylcholinesterase (BChE).³ This makes the dual inhibition of AChE and BChE important enzymatic targets. Rivastigmine is the only FDA-approved dual cholinesterase (ChE) inhibitor.⁴

The Aβ accumulation is considered a major culprit in AD pathology. The cleavage of Aβ precursor protein (APP) is responsible for forming the Aβ oligomers and plaques in the amyloidogenic pathway. In the nonamyloidogenic pathway, α-secretase cleaves the APP into water-soluble fragments, which could be eliminated from the body. In the amyloidogenic pathway, the cleavage of APP is caused by β-secretase (BACE-1 or Aβ precursor cleaving enzyme), which leads to the formation of insoluble waxy fragments called Aβs.⁵ This makes BACE-1 a vital target in AD drug development. Besides, the amyloid β 1–42 (Aβ_1–42) is a self-aggregatory protein;⁶ thus, inhibition of its spontaneous aggregation is also an important target.⁷ The recently approved aducanumab is an anti-amyloid monoclonal antibody, the only disease-modifying therapy for treating AD. In contrast, all other marketed drugs only provide temporary relief but do not cure or alter the underlying pathogenesis of the disease.⁸

Natural products (NPs) have been the basis of traditional medicines throughout history. NPs are recognized to have chemical diversity and biochemical specificity, rendering them promising leads as such or via vital synthetic modifications in drug discovery. The literature reveals that nearly 84% of the FDA-approved drugs for treating central nervous system (CNS) diseases belong to NPs.⁹ NPs with coumarin motifs have been extensively studied for drug discovery and development as it is a multitargeted ligand with minimal toxic side effects. Coumarins are the NPs containing benzopyrone nucleus, which occurs in over 1300 secondary metabolites from terrestrial and marine sources.¹⁰ The conjugated double-bond system of coumarin has been interestingly manifested in medicinal chemistry and chemical biology. Many synthetic coumarins exhibit a broad spectrum of biological activities for treating various ailments like cancer,¹¹ infectious diseases,¹² diabetes,¹³ and neurological disorders.¹⁴ The FDA-approved coumarin class of drugs comprises anticoagulants such as warfarin, acenocoumarin, and the antibiotic novobiocin.¹⁵ Coumarin pharmacophore has been documented with neuroprotective and anti-Alzheimer properties.¹⁶ Thus, its relative availability, low toxicity, and ethnopharmacological importance have made it prudent to explore new derivatives of coumarins for AD.¹⁷ The coumarin-based compounds show anti-cholinesterase activity. The 8-acetylcoumarin 1 is a moderate inhibitor of AChE and BACE-1. Its hybrid structure 2 with donepezil shows improvement in the AChE and BACE-1 inhibition activity.¹⁸ Similarly, Zhao’s group has recently reported another coumarin-donepezil hybrid, 3, with dual ChE/BACE-1 inhibition activity.¹⁹ In both these hybrid structures, incorporating benzyl-piperidine into the structure helps interact with the catalytic site of ChEs. Recently, we reported that the placement of benzyl triazole (e.g., compound 4) to the AChE pharmacophore also helps to gain the interaction at the catalytic site of ChEs.²⁰ Besides, the triazole-based compounds are known to show inhibition of ChEs,²¹ BACE-1,²² and Aβ-aggregation.²³ Thus, based on the literature precedence, we synthesized coumarin–triazole hybrids (general structure shown in Figure 1) as potential dual inhibitors of ChEs and BACE-1.

Chemical structures of coumarin derivatives 1–3 and triazole 4 that are reported to show dual ChE/BACE-1 inhibition. The proposed coumarin–triazole series is also shown with general structure A. Here, the coumarin may be connected to the triazole through ring A or B.

2. Results and Discussion

2.1. Synthesis and In Vitro Biological Evaluation

The first series of coumarin–triazole hybrids were synthesized with 4-hydroxycoumarin (5) as a precursor. The propargylation of 4-hydroxycoumarin yielded 4-O-propargylated coumarin 6. The click reaction of propargyl product with the benzyl bromide (7a) in the presence of sodium azide yielded the corresponding triazole, 8a, in 82% yield, as shown in Scheme 1. Various substituted benzyl bromides (7) also participated in the click reaction to produce corresponding triazoles, 8b–p, in good yields.

Scheme 1 — Reagents and conditions: (a) propargyl bromide, K₂CO₃, dimethylformamide (DMF), 70 °C, 24 h, reflux, 95%; (b) TEA, sodium ascorbate, CuSO₄·5H₂O, room temperature (rt), 24 h, 54–85%.

All prepared coumarin triazoles 8a–p were screened for inhibition of ChEs and BACE-1 using the Ellman and fluorescence resonance energy transfer (FRET) assay, and results are summarized in Table 1. Cryptolepine and donepezil were used as positive controls for ChEs. Cryptolepine is a potent dual inhibitor of ChEs reported by us.²⁴ Donepezil is an FDA-approved ChE inhibitor. The commercially available β-Secretase inhibitor IV was used as a positive control for BACE-1.²⁵ The 4-hydroxycoumarin 5 is a poor inhibitor of AChE, BChE, and BACE-1. The condensation of 5 with the benzyl triazole did not significantly boost the inhibition of ChEs; however, a marginal improvement in BACE-1 inhibition was observed (5vs8a). However, placing various electron-withdrawing groups on the benzyl ring has helped dramatically increase the activity. The careful analysis of results provided structure–activity relationship (SAR) patterns. The boost in ChE inhibition by 2–3-fold was noticed simply by placing −Cl substitution on the benzyl ring (analogues, 8b, 8c, 8d). The −F substitution also boosted ChE inhibition, but only at the ortho-position (analogue 8e). The ortho-substitution with a powerful electron-withdrawing group, trifluoromethyl, yielded a compound 8i that inhibits AChE, BChE, and BACE-1 with IC₅₀ values of 4.83, 7.04, and 10.17 μM, respectively. Similarly, the ortho-OCF₃ substitution significantly boosted the inhibition of all three enzymes (analogue 8k). However, the analogues with −CF₃ or −OCF₃ at para-position were less effective than ortho-substituted analogues. Methoxy substitution provided the best results with better activity, particularly by disubstituted analogues. The 3,5-dimethoxy-substituted analogue, 8o, is the most active, with IC₅₀ values of 2.76, 3.30, and 9.79 μM, respectively. Thus, it is clear that the substituents on the triazole benzyl ring play a vital role in the modulation of the activity of all three enzymes. The 2-trifluoromethyl-, 2-trifluoromethoxy-, 3-methoxy-, and 3,5-dimethoxy-substituted analogues were the most active in the coumarin A-ring connected triazole hybrid series.

Table 1. Cholinesterase and BACE-1 Inhibition Results for Coumarin A-Ring Connected Triazole Hybrids, 8a–p.

		IC₅₀ (μM) ± SD
entry	R	EeAChE	eqBChE	hBACE-1
5		138.56 ± 1.85	167.06 ± 5.63	168.63 ± 7.67
8a	H	150.70 ± 3.14	212.4 ± 2.55	72.90 ± 2.40
8b	2-Cl	68.71 ± 0.71	42.22 ± 0.78	186.6 ± 1.21
8c	3-Cl	62.64 ± 2.40	61.9 ± 3.66	24.82 ± 1.49
8d	2,6-Cl	59.41 ± 2.71	40.29 ± 0.93	105.83 ± 1.38
8e	2-F	89.23 ± 1.88	62.32 ± 1.20	111.23 ± 0.63
8f	3-F	232.76 ± 2.31	235.8 ± 3.60	22.32 ± 1.54
8g	4-F	193.6 ± 2.19	249.4 ± 3.1	24.22 ± 1.98
8h	2,6-F	73.89 ± 0.63	66.66 ± 1.37	134.53 ± 1.90
8i	2-CF₃	4.83 ± 0.11	7.04 ± 0.77	10.17 ± 0.79
8j	4-CF₃	23.26 ± 2.86	209.13 ± 2.11	19.54 ± 0.66
8k	2-OCF₃	9.13 ± 0.73	9.95 ± 0.89	17.98 ± 1.59
8l	4-OCF₃	45.39 ± 1.40	157.5 ± 1.98	132.9 ± 1.08
8m	2-CH₃	210.7 ± 1.83	171.6 ± 1.98	161.5 ± 1.85
8n	3-OCH₃	11.39 ± 1.16	139.7 ± 6.25	125.7 ± 4.18
8o	3,5-di-OCH₃	2.76 ± 0.04	3.30 ± 0.01	9.79 ± 1.36
8p	4-NO₂	200.4 ± 0.85	219.8 ± 3.04	156.03 ± 1.09
cryptolepine		0.33 ± 0.2	0.61 ± 0.3	nt^a
donepezil		0.025 ± 0.005	5.72 ± 0.59	nt
β-secretase inhibitor IV		nt	nt	0.019 ± 0.006

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nt: not tested.

Next, we synthesized a series of coumarin–triazole hybrids with triazole connected via the B-ring of coumarin. For this, we employed four different hydroxy coumarins, namely, 8-acetylcoumarin (1), 4-methyl-6,7-dihydroxycoumarin (11), 4-methyl-6-methoxy-7-hydroxycoumarin (14), and 7-hydroxycoumarin (umbelliferone, 17) as coumarin precursors. Based on the biological results of the 8a–p series, 2-trifluoromethyl, 2-trifluoromethoxy, 3-methoxy, and 3,5-dimethoxybenzyl substitutions were used for synthesizing coumarin B-ring connected triazole hybrids. Using a similar synthetic strategy, four series of coumarin–triazole hybrids, 10a–d, 13a–d, 16a–d, and 19a–d, were synthesized as depicted in Scheme 2.

Scheme 2 — Reagents and conditions: (a) propargyl bromide, K₂CO₃, DMF, 70 °C, 24 h, reflux, 75–91%; (b) TEA, NaN₃, sodium ascorbate, CuSO₄·5H₂O, rt, 24 h, 50–80%; (c) methyl iodide, NaH, DMF, 0 °C, 90%.

The biological screening results of coumarin B-ring-connected triazole hybrids are provided in Table 2. In the acetylcoumarin series, the hybrids with methoxy substitution were better than parent coumarin; however, trifluoromethyl and trifluoromethoxy substitutions were unfavorable as they resulted in the loss of activity against all three targets. The 4-methylcoumarin-based hybrids 13a–d and 16a–d were ineffective with all four benzylic substitutions. The unsubstituted coumarin-based hybrids, 19a–d, were better inhibitors of ChEs than 4-methyl-substituted coumarin analogues. The 3-methoxybenzyl-containing coumarin–triazole 19a displayed significant inhibition of ChEs with IC₅₀ values of 3.6 and 2.8 μM, respectively. However, its dimethoxy analogue 19b was not equally effective. Overall, the 3,5-dimethoxy analogue 10b from the acetylcoumarin series has potent inhibition of AChE and BChE and also BACE-1. However, the corresponding 3,5-dimethoxy analogues from other series, 13b, 16b, and 19b, were not active to the level of 10b. This probably indicates some role of the C8-acetyl group in the activity. Interestingly, compound 13b is a selective BChE inhibitor with IC₅₀ of 4.7 μM and shows poor activity against AChE and BACE-1. The dimethoxy substitution with the bis-triazole unit in 13b probably fits appropriately in a wider BChE active site gorge but not in AChE active site gorge, which is comparatively narrower.

Table 2. Cholinesterase and BACE-1 Inhibition Results for Coumarin B-Ring Connected Triazole Hybrids, 10a–d, 13a–d, 16a–d, and 19a–d.

		IC₅₀ (μM) ± SD
entry	R	EeAChE	eqBChE	hBACE-1
1		23.71 ± 3.21	155.03 ± 3.09	18.42 ± 1.04
11		68.14 ± 4.05	147.70 ± 5.46	104.8 ± 3.48
14		89.09 ± 3.51	146.07 ± 4.91	91.09 ± 2.41
17		164.46 ± 5.22	180.33 ± 4.58	174.53 ± 4.37
10a	3-OCH₃	8.36 ± 1.29	7.40 ± 0.94	118.8 ± 2.49
10b	3,5-di-OCH₃	2.57 ± 0.31	3.26 ± 0.13	10.65 ± 0.47
10c	2-CF₃	173.06 ± 1.89	44.60 ± 1.84	138.8 ± 1.47
10d	2-OCF₃	139.20 ± 1.64	170.10 ± 1.76	141.9 ± 1.59
13a	3-OCH₃	130.70 ± 0.45	51.70 ± 1.42	128.6 ± 1.49
13b	3,5-di-OCH₃	167.53 ± 6.70	4.7 ± 0.67	115.2 ± 3.86
13c	2-CF₃	154.70 ± 1.42	82.54 ± 1.41	139.4 ± 1.00
13d	2-OCF₃	176.0 ± 10.25	167.06 ± 5.63	20.72 ± 0.93
16a	3-OCH₃	164.2 ± 1.84	95.82 ± 1.12	138.4 ± 1.02
16b	3,5-di-OCH₃	150.96 ± 5.05	191.2 ± 1.13	9.06 ± 0.88
16c	2-CF₃	141.7 ± 0.89	57.45 ± 1.42	156.1 ± 1.42
16d	2-OCF₃	166.5 ± 2.45	141.03 ± 0.60	167.6 ± 1.40
19a	3-OCH₃	3.62 ± 0.33	2.80 ± 0.22	125.6 ± 3.92
19b	3,5-di-OCH₃	59.67 ± 0.62	63.24 ± 2.44	132.0 ± 2.02
19c	2-CF₃	20.64 ± 0.95	77.33 ± 6.38	17.71 ± 0.65
19d	2-OCF₃	52.54 ± 0.40	97.23 ± 1.29	172.8 ± 0.66
cryptolepine		0.33 ± 0.2	0.61 ± 0.3	nt^a
donepezil		0.025 ± 0.005	5.72 ± 0.59	nt
β-secretase inhibitor IV		nt	nt	0.019 ± 0.006

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nt, not tested.

2.2. Enzyme Kinetics and Molecular Modeling for 10b

The enzyme kinetics of 10b was carried out to assess the mode of inhibition for all three targets. The Lineweaver–Burk (LB) double-reciprocal plot for AChE indicates a noncompetitive mode of inhibition with a k_i value of 0.27 μM (Figure 2A). However, for BChE and BACE-1, the depicted mode of inhibition is a mixed type with k_i values of 3.5 and 7 μM, respectively (Figure 2C,E). Further, we studied the interaction pattern of 10b with each enzyme via molecular modeling studies. As depicted in Figure 2B, 10b interacts with peripheral anionic site (PAS) as well as catalytic anionic site (CAS) residues of AChE. The acetylcoumarin core stays at the entrance of the gorge, and benzyl triazole orients toward the bottom of the cavity. Both the rings of coumarin form π–π stacking with Trp 286 residue. Interestingly, the carbonyl oxygen of the acetyl group forms H-bonding with Arg 296 and Phe 296 residues, which could be the crucial factor for the superior activity of the acetylcoumarin series vs other series. The linker oxygen also forms H-bond with the Phe 295 residue. The triazole ring and aryl of coumarin form π–π stacking with the Tyr 341 residue. The benzyl ring forms a vital π–π stacking with Trp 86 residue of the anionic subsite of the CAS. The noncompetitive mode of inhibition, as seen in the kinetic study, could be because of the strong interaction of the 10b with PAS residues via multiple π–π stacking and H-bonds. The docking study of 10b with BChE (Figure 2D) shows π–π interactions of benzyl with Phe 329 but not with the Trp 82 of the anionic subsite. The triazole ring forms π–π stacking with Tyr 332. Like in the case of AChE, the acetyl group participated in vital H-bonding with the PAS residues of BChE. The carbonyl oxygen of acetyl forms H-bond with Ser 287, which is also H-bonded with a coumarin ring oxygen atom. Figure 2F shows the interactions of 10b with BACE-1 (PDB: 1W51). It shows H-bonding with Thr 232, Asn 233, Arg 235, Gly 13, and Trp 115 residues; however, the interaction with Asp 32 and Asp 228 was missing. The interaction with these aspartate residues is vital for the potent inhibition of BACE-1.²⁶ This could be the reason for its low activity level against the BACE-1 enzyme.

Enzyme kinetics and molecular modeling studies of **10b** with AChE, BChE, and BACE-1. (A) LB plot for inhibition of AChE by **10b**; (B) molecular docking of **10b** with AChE (PDB: 4EY7); (C) LB plot for inhibition of BChE by **10b**; (D) molecular docking of **10b** with BChE (PDB: 6EP4); (E) LB plot for inhibition of BACE-1 by **10b**; (F) molecular docking of **10b** with the BACE-1 (PDB: 1W51). The light blue dotted lines indicate π–π interactions, and the dark red dotted lines indicate H-bonding interactions.

To understand the superiority of 8-acetyl-7-hydroxycoumarin framework vs 4-hydroxycoumarin and 7-hydroxycoumarin, we analyzed the binding pattern of 10b, 8o, and 19b in the AChE active site (Figure 3). These three analogues differ only via the coumarin framework; thus, their different activity is related to the coumarin–triazole linkage and substitution on coumarins. Both rings of coumarin core in 10b and 8o offered π–π stacking with Trp 286 at PAS, whereas in 19b, this π–π interaction is missing. Molecule 19b is flipped by 180° in the AChE active site, with the coumarin core oriented toward the bottom of the AChE active site gorge. The 3,5-dimethoxybenzyl unit of 19b did not show π–π stacking with Trp 286 though it offered H-bonding with Phe 295 residue. In particular, the bidentate H-bonding offered by acetyl carbonyl oxygen of 10b with Phe 295 and Arg 296 was missing in 19b, which could also be accounting for the 20-fold difference in AChE inhibition activity of 10bvs19b.

Interaction pattern of 3,5-dimethoxybenzyl coumarin–triazole hybrids, **10b**, 8o, and **19b** with AChE (PDB: 4EY7).

The 50 ns molecular dynamic simulation was carried out for enzyme–inhibitor complexes to determine the stability. The complex of compound 10b with AChE is stable throughout the study, as indicated by the root mean square deviation (RMSD) value below 3 Å for both protein and ligand (Figure 4A). The root mean square fluctuation (RMSF) plot shown in Figure 4B,C further indicates minimal fluctuations in the residues of protein and atom of the ligand (RMSF is below 4 Å) during the entire period of study. The orientation map of compound 10b is depicted in Figure 4D,E. The hydrophobic interaction with Tyr 341 was sustained for >56% and Trp 86 for more than 79% of the simulation time. Tyr 124 shows H-bonding with the triazole ring for >51% of the simulation run. Trp 286 offers hydrophobic interactions for >33% of the simulation run. Phe 295 depicts H-bonding >95% persistently throughout the simulation study (Figure 4D,E).

MD simulation of **10b**–AChE complex for 50 ns. (A) Protein–ligand RMSD during simulation; (B) RMSF of AChE; (C) RMSF of compound **10b**; (D) interaction pattern of compound **10b** with AChE during MD simulation; (E) two-dimensional (2D) diagram for the interaction of compound **10b** with AChE.

As depicted in Figure 5A, the RMSD below 3 Å indicates the stability of the protein in the 10b–BChE complex throughout the study. The ligand slightly diffuses from the initial binding site; however, the overall orientation and interaction pattern is unaffected (Figure 5A). The RMSF depicts a value below the range of 4 Å, which indicates no significant conformational changes along the protein chain as the ligand fits in the protein (Figure 5B,C). The most preserved interactions during the simulation run were hydrophobic π–π stacking and H-bonding with Trp 82 for 39 and 36% of the run (Figure 5E).

MD simulation of **10b**–BChE complex for 50 ns. (A) Protein–ligand RMSD during simulation; (B) RMSF of BChE; (C) RMSF of compound **10b**; (D) interaction pattern of compound **10b** with BChE during MD simulation; (E) 2D diagram for compound **10b** interactions with BChE.

The MD simulation of the 10b–BACE-1 complex also indicated the stability of the complex. The RMSD for the protein is below 3 Å, which indicates stability. In contrast, for the ligand, RMSD is slightly above 3 Å, which is conclusive that the ligand diffuses away from the protein (Figure 6A). The RMSF for the protein and the ligand fit is below 3.5 and 2 Å, respectively (Figure 6B,C). The H-bonding between the acetyl group of coumarin with Thr 72 is sustained for >89% of the simulation time. The triazole ring also maintained hydrophobic interaction with the Tyr 71 residue for >72% of the simulation run. The hydrophobic interaction with Trp 115 residue is also retained for a significant time (Figure 6D,E).

MD simulation of **10b**–BACE-1 complex for 50 ns. (A) Protein–ligand RMSD during simulation; (B) RMSF of BACE-1; (C) RMSF of compound **10b**; (D) interaction pattern of compound **10b** with BACE-1 during MD simulation; (E) 2D diagram for compound **10b** interactions with BACE-1.

2.3. Inhibition and Aβ Self-Aggregation

AD’s main hallmark is forming Aβ plaques, which occur via the aggregation of Aβ monomers.²⁷ Thus, we evaluated the effect of compound 10b on the self-aggregation of Aβ_1–42 monomers, which is one way to form oligomers and plaques. Analogue 10b displayed a 37% inhibition of self-aggregation of Aβ_1–42 monomers, whereas the positive control, curcumin, showed 81% inhibition at 10 μM. The molecular docking of 10b with the Aβ-monomer revealed that this compound binds to the metal binding site residue His 14; however, it does not interact with the Asp 23, the key residue in forming a salt bridge between monomers during the aggregation process.²⁸ Curcumin forms H-bonding with the metal binding site and the salt-bridge forming residue (Figure 7).

Molecular docking of curcumin (A) and compound **10b** (B) with the Aβ monomer (PDB: 1Z0Q).

2.4. Parallel Artificial Membrane Permeability Assay (PAMPA)–Blood–Brain Permeability (BBB) Permeability and In Silico Absorption Distribution Metabolism Elimination (ADME) Properties of 10b

Along with the activity profile of the compound against various targets, CNS blood–brain permeability (BBB) is also an indispensable factor for a drug with CNS disease on target. The blood–brain barrier restricts molecules’ passage from the brain vasculature to the brain due to its high trans-endothelial electrical resistance and low paracellular and transcellular permeability. Thus, the BBB permeability of 10b was experimentally determined using a PAMPA assay (Table 3).

Table 3. BBB Permeability of 10b Determined Using PAMPA–BBB Assay.

compound	λ_max (nm)	P_e × 10^–6 cm/s ± SD	prediction of CNS permeation
10b	261	5.68 ± 1.1	CNS+
donepezil	270	13.32 ± 1.82	CNS+
theophylline	272	1.22 ± 1.38	CNS–

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The ADME properties determine whether a compound has druglike properties. It is included at a very early stage of drug discovery. QikProp module of the Schrodinger molecular modeling software computationally determines the number of descriptors for small molecules, predicting the ADME properties.²⁹ These properties include total solvent-accessible surface area (SASA), the hydrophobic component of the SASA (FOSA), the hydrophilic component of the SASA (FISA), and the estimated number of H-bond donors (donorHB) and acceptors (accptHB) in aqueous solution. Table 4 also shows that the predicted ranges for the calculated partition coefficients, which are octanol/water (QP log P_o/w), aqueous solubility (QP log S), and skin permeability (QP log Kp) fall within the acceptable range. The computed IC₅₀ for blockage of HERG K+ channels (QP log HERG), Caco-2 permeability, and Madin–Darby canine kidney (MDCK) permeability values are also within the normal limits. The BBB is also predicted computationally. The results show that QPlog BB also falls within the recommended range. This complies with the experimental results of the PAMPA assay, as shown in Table 3. ADME properties were also estimated for donepezil and β-secretase inhibitor IV and are listed in Table 4.

Table 4. ADME Properties for 10b and Positive Controls^a.

property	reference range	10b	donepezil	β-secretase inhibitor IV
molecular weight	300–1000	435.435	379.498	578.725
SASA	0–750	734.966	726.298	860.774
FOSA	7–330	324.954	410.311	391.518
FISA	0–6	139.219	58.288	123.351
donorHB	2–20	0	0	4
accptHB	2–20	9.25	5.5	12.7
QP log P_o/w	–2 to 6.5	2.77	4.436	3.543
QP log S	–6.5 to 0.5	–3.99	–4.904	–4.698
QP log HERG	concern below—5	–6.178	–6.868	–7.634
QPPCaco	<25 poor, >500 great	473.899	691.924	167.134
QP log BB	–3.0 to 1.2	–1.396	–0.021	–1.597
QPP MDCK	<25 poor, >500 great	220.701	367.58	40

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ADME properties were determined using the QikProp module of Schrodinger 10.2 software. Properties: total solvent-accessible surface area (SASA), the hydrophobic component of the SASA (FOSA), the hydrophilic component of the SASA (FISA), the estimated number of hydrogen-bond donor (donorHB) acceptor (accptHB) in aqueous solution. Also, QP log P_o/w is the octanol/water partition coefficient, QP log S is aqueous solubility (mol/L), QP log HERG is IC₅₀ value for blockage of HERG K⁺ channels, QPPCaco is Caco-2 permeability (nm/s), QP log BB is brain/blood partition coefficient, and QPPMDCK is apparent MDCK cell permeability (nm/s). MDCK cells are considered a good mimic for the blood–brain barrier.

3. Conclusions

We designed and synthesized coumarin–triazole hybrid 10b, displaying a multitargeted effect against AD targets, AChE, BChE, BACE-1, and Aβ-aggregation. It is predicted to cross the BBB based on the experimental PAMPA results and in silico QikProp prediction. The structure–activity relationship has been established for this scaffold, indicating that 3,5-dimethoxy substitution on the benzyl group along with the coumarin core with acetyl substitution is important for activity. The study also further validated the capability of triazole to interact at the CAS of the ChEs. The results presented herein warrant further investigation of 10b in preclinical studies.

4. Experimental Section

4.1. General

The local suppliers of Sigma-Aldrich, TCI chemicals, and CDH have provided the chemicals, solvents, reagents, and the required glassware and plasticware for the study. The enzymes, substrates, and positive controls for the biological studies are also procured from them. NMR spectra were recorded on a Bruker-Avance DPX FT-NMR 400 MHz instrument. IR spectra were recorded on a PerkinElmer IR spectrophotometer. High-resolution electrospray ionization mass spectra (HR-ESIMS) were obtained from an Agilent HR-ESIMS-6540-UHD machine. Absorbance and fluorescence readings were recorded on Molecular Devices and Biotage microplate readers, respectively. The absorbance readings in Ellman and PAMPA assays were recorded on SpectraMax Plus 384 plate reader. The fluorescence for the FRET assay was recorded on the Synergy H1 Hybrid Reader (Biotek). Melting points were recorded on a Buchi digital melting point apparatus.

4.2. Synthesis of 7-Hydroxy-6-methoxy-4-methyl-2H-chromen-2-one (14)³⁰

The mixture of compound 11 (0.52 mmol) and NaH (1.05 mmol) in 2 mL of DMF was stirred at 0 °C for half an hour. After half an hour, methyl iodide (0.52 mmol) was added, and the reaction was further stirred for 5 h at rt. After 5 h, the reaction mixture was added to 50 mL of ice-cold water to remove DMF efficiently. Extraction was done with EtOAc (3 × 50 mL). The EtOAc layer was concentrated on the vacuum evaporator to obtain 14 (1.2 g, yield: 90%). Brown powder; m.p. 130–131 °C; IR (CHCl₃) ν_max (cm^–1): 1710 (C=O), 1423 (C–O); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.09 (s, 1H), 6.83 (s, 1H), 6.17 (s, 1H), 3.97 (s, 3H), 2.36 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 162.90, 153.90, 151.29, 150.02, 143.95, 114.82, 114.03, 109.71, 100.82, 57.82, 20.22; ESIMS: m/z 207.1 [M + H]⁺; HR-ESIMS: m/z 207.0667 [M + H]⁺ calcd for C₁₁H₁₀O₄ + H⁺ (207.0657).

4.3. Propargylation of Coumarin Hydroxyl Group: Synthesis of 6, 9, 12, 15, and 18

The coumarin 1, 5, 11, 14, or 17 (1 equiv) was treated with potassium carbonate (4 equiv) in DMF for 30 min at 70 °C. After 30 min, propargyl bromide (1.5 equiv) was added, and the reaction was continued for 24 h. After 24 h, the reaction was cooled and poured into ice-cold water to remove the DMF. The extraction was done with EtOAc (3 × 50 mL). The combined ethyl acetate layer was concentrated on a rotary evaporator, and the residue was purified over silica gel (solvent system: 40% EtOAc in hexane) to obtain respective propargylated compounds, 6, 9, 12, 15, and 18.

4.3.1. 4-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (6)³¹

White fluffy powder; yield, 95%; m.p.153–154 °C; IR (CHCl₃) ν_max (cm^–1): 1719 (C=O), 1251 (C–O); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.85 (dd, J = 7.9, 1.5 Hz, 1H), 7.59–7.55 (m, 1H), 7.34–7.31 (m, 1H), 7.29 (d, J = 7.3 Hz, 1H), 5.84 (s, 1H), 4.88 (d, J = 16 Hz, 2H), 2.69 (t, J = 2.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.29, 162.66, 153.52, 132.61, 125.004, 123.27, 116.69, 91.72, 78.20, 75.58, 56.87; ESIMS: m/z 201 [M + H]⁺; HR-ESIMS: m/z 201.0557 [M + H]⁺ calcd for C₁₂H₈O₃ + H⁺ (201.0552).

4.3.2. 8-Acetyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (9)

White powder; yield, 79%; m.p. 155–157 °C; IR (CHCl₃) ν_max (cm^–1): 1722 (C=O), 1122 (C–O), 1324 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.67 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 8.6 Hz, 2H), 6.31 (d, J = 8.8 Hz, 1H), 4.82 (s, 2H), 2.62 (s, 3H), 2.58 (t, J = 2.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 198.92, 159.57, 156.19, 151.43, 143.37, 129.62, 120.47, 114.42, 113.41, 109.38, 56.56, 32.37; ESIMS: m/z 243.06 [M + H]⁺; HR-ESIMS: m/z 243.0657 [M + H]⁺ calcd for C₁₄H₁₁O₄ + H⁺ (243.0669).

4.3.3. 4-Methyl-6,7-bis(prop-2-yn-1-yloxy)-2H-chromen-2-one (12)

Light brown powder; yield, 77%; m.p. 183–184 °C; IR (CHCl₃) ν_max (cm^–1): 1714 (C=O), 1183 (C–O), ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.23 (s, 1H), 7.05 (s, 1H), 6.20 (s, 1H), 4.84 (d, J = 2.4 Hz, 2H), 4.82 (d, J = 2.3 Hz, 2H), 2.59 (t, J = 2.5 Hz, 1H), 2.57 (t, J = 2.4 Hz, 1H), 2.41 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.02, 152.53, 151.57, 149.80, 144.13, 113.58, 113.05, 110.11, 102.66, 78.08, 57.46, 56.50, 18.50; ESIMS: m/z 269.08 [M + H]⁺; HR-ESIMS: m/z 269.0830 [M + H]⁺ calcd for C₁₆H₁₂O₄ + H⁺ (269.0814).

4.3.4. 6-Methoxy-4-methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (15)

Light brown powder; m.p. 177–179 °C; IR (CHCl₃) ν_max (cm^–1): 1723 (C=O), 1159 (C–O); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.03 (s, 1H), 6.97 (s, 1H), 6.20 (s, 1H), 4.84 (d, J = 2.4 Hz, 2H), 3.94 (s, 3H), 2.60 (t, J = 2.4 Hz, 1H), 2.42 (d, J = 3.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.87, 152.51, 150.43, 149.11, 146.76, 113.79, 113.10, 106.03, 102.66, 56.92, 55.93, 18.52; ESIMS: m/z 245.2 [M + H]⁺; HR-ESIMS: m/z 245.0825 [M + H]⁺ calcd for C₁₄H₁₂O₄ + H⁺ (245.0814).

4.3.5. 7-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (18)

Yellowish brown powder; yield, 91%; m.p. 114–115 °C; IR (CHCl₃) ν_max (cm^–1): 1726 (C=O), 1673, 1160 (C–O), ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.67 (d, J = 14.7 Hz, 1H), 7.42 (d, J = 13.7 Hz, 1H), 6.94–6.90 (m, 2H), 6.29 (d, J = 12.4 Hz, 1H), 4.77 (d, J = 2.0 Hz, 2H), 2.59 (t, J = 2.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.18, 160.18, 155.81, 143.29, 128.85, 113.67, 113.20, 113.06, 102.35, 56.22; ESIMS: m/z 201.05 [M + H]⁺; HR-ESIMS: m/z 201.0561 [M + H]⁺ calcd for C₁₂H₈O₃ + H⁺ (201.0552).

4.4. General Procedure for the Synthesis of Triazoles 8a–o, 10a–d, 13a–d, 16a–d, and 19a–d

To a round-bottom flask, substituted benzyl bromide (7) (1.5 equiv), triethylamine (0.3 equiv), and sodium azide (1.5 equiv) were taken in tert-butanol/water (1:1). This mixture was stirred at room temperature for half an hour. After half an hour, propargylated coumarin 6, 9, 12, 15, or 18 (1.5 equiv) was added along with sodium ascorbate (0.2 equiv) and copper sulfate (0.1 equiv), and the reaction was stirred for 24 h. Then, 30 mL water was added and extraction was done with EtOAc (3 × 50 mL). The three layers are combined and concentrated on a vacuum rotary evaporator. Thus, the residue was purified by column chromatography (solvent system: 40% EtOAc in hexane for 8a–o and 19a–d and 70% EtOAc in hexane for 10a–d, 13a–d, and 16a–d) to obtain final triazoles.

4.4.1. 4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8a)

Off-white coarse powder; yield, 82%; m.p. 176–177 °C; IR (KBr) ν_max (cm^–1): 1715 (C=O), 1238 (C–O), 1376 (C–N); ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.78 (s, 2H), 7.76 (d, J = 13 Hz, 1H), 7.56–7.53 (m, 1H), 7.43–7.37 (m, 1H), 7.31 (dd, J = 8.4, 1.4 Hz, 1H), 7.24–7.22 (m, 1H), 7.03–6.99 (m, 3H), 5.85 (s, 1H), 5.69 (s, 2H), 5.30 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 165.03, 162.46, 159.39, 153.23, 141.61, 132.57, 131.73, 124.31, 123.79, 122.75, 117.85, 116.28, 112.94, 91.24, 62.39, 41.98. ESIMS: m/z 334.11 [M + H]⁺; HR-ESIMS: m/z 334.1197 [M + H]⁺ calcd for C₁₉H₁₅N₃O₃ + H⁺ (334.1192).

4.4.2. 4-((1-(2-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8b)

White powder; yield, 67%; m.p. 162–163 °C; IR (CHCl₃) ν_max (cm^–1): 1715 (C=O), 767 (C–Cl), 1187 (C–O), 1273 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.79 (s, 1H, triazole), 7.77 (d, J = 8.1 Hz, 1H), 7.55–7.51 (m, 1H), 7.45–7.44 (m, 1H), 7.37–7.33 (m, 1H), 7.31–7.29 (m, 3H), 7.25 (t, J = 4.3 Hz, 1H), 5.85 (s, 1H), 5.72 (s, 2H), 5.32 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.86, 162.65, 153.31, 142.01, 133.54, 132.40, 130.43, 129.86, 129.28, 127.32, 123.93, 123.65, 123.09, 117.13, 115.17, 91.16, 62.86, 51.87; ESIMS: m/z 368.08 [M + H]⁺; HR-ESIMS: m/z 368.0810 [M + H]⁺ calcd for C₁₉H₁₄ClN₃O₃ + H⁺ (368.0802).

4.4.3. 4-((1-(3-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8c)

White powder; yield, 71%; m.p. 198–199 °C; IR (CHCl₃) ν_max (cm^–1): 1716 (C=O), 761 (C–Cl), 1214 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.78 (dd, J = 8.0, 4.0 Hz, 1H), 7.69 (s, 1H, triazole), 7.56–7.52 (m, 1H), 7.37–7.34 (m, 2H), 7.32–7.29 (m, 2H), 7.25 (d, J = 6.8 Hz, 1H), 7.21 (d, J = 6.5 Hz, 1H), 5.85 (s, 1H), 5.57 (s, 2H), 5.33 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.85, 162.45, 153.25, 142.17, 132.60, 130.59, 129.26, 128.25, 126.24, 123.95, 123.42, 123.17, 116.91, 115.46, 91.46, 62.60, 53.51; ESIMS: m/z 368.08 [M + H]⁺; HR-ESIMS: m/z 368.0809 [M + H]⁺ calcd for C₁₉H₁₄ClN₃O₃ + H⁺ (368.0802).

4.4.4. 4-((1-(2,6-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8d)

Yellowish orange powder; yield, 74%; m.p. 174–175 °C; IR (CHCl₃) ν_max (cm^–1): 1716 (C=O), 761 (C–Cl), 1215 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.77 (dd, J = 8.0, 4.0 Hz, 1H), 7.72 (s, 1H, triazole), 7.54–7.52 (m, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.43 (s, 1H), 7.35–7.30 (m, 2H), 7.25–7.21 (m, 1H), 5.92 (s, 1H), 5.85 (s, 2H), 5.29 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.92, 162.40, 153.83, 141.31, 136.83, 132.29, 131.05, 129.79, 129.53, 124.30, 123.46, 123.23, 122.99, 117.00, 115.47, 91.21, 62.81, 49.25; ESIMS: m/z 402.04 [M + H]⁺; HR-ESIMS: m/z 402.0415 [M + H]⁺ calcd for C₁₉H₁₃Cl₂N₃O₃ + H⁺ (402.0412).

4.4.5. 4-((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8e)

Pale white powder; yield, 65%; m.p. 182–183 °C; IR (CHCl₃) ν_max (cm^–1): 1713 (C=O), 1452 (C–F), 1215 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.77 (d, J = 8 Hz, 1H), 7.63 (s, 1H, triazole), 7.56 (t, J = 4 Hz, 1H), 7.34–7.32 (m, 3H), 7.24 (d, J = 8 Hz, 1H), 7.12 (t, J = 7.4 Hz, 2H), 5.84 (s, 1H), 5.56 (s, 2H), 5.32 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.97, 164.25 (d, ¹J_C–F = 279 Hz), 162.66, 153.35, 141.96, 132.61, 130.20 (d, ³J_C–F = 9 Hz), 129.99, 123.94, 123.16, 116.82, 116.46 (d, ²J_C–F = 21 Hz), 116.24, 115.41, 91.20, 62.61, 53.71; ESIMS: m/z 352.11 [M + H]⁺; HR-ESIMS: m/z 352.1107 [M + H]⁺ calcd for C₁₉H₁₄FN₃O₃ + H⁺ (352.1097).

4.4.6. 4-((1-(3-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8f)

White powder; yield, 83%; m.p. 173–175 °C; IR (CHCl₃) ν_max (cm^–1): 1714 (C=O), 1761 (C–F), 1266 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.77 (dd, J = 8.0, 4.4 Hz, 1H), 7.63 (s, 1H, triazole), 7.57–7.53 (m, 3H), 7.34–7.30 (m, 1H), 7.12–7.09 (m, 2H), 5.84 (s, 1H), 5.56 (s, 2H), 5.32 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.97, 164.24 (d, ¹J_C–F = 247 Hz), 162.66, 152.33, 141.96, 132.61, 130.20 (d, ³J_C–F = 8 Hz), 130.03 (d, ⁴J_C–F = 3 Hz), 123.94, 123.20, 123.15, 116.81, 116.45 (d, ²J_C–F = 21 Hz), 115.41, 91.19, 62.61, 53.71; ESIMS: m/z 352.11 [M + H]⁺; HR-ESIMS: m/z 352.1103 [M + H]⁺ calcd for C₁₉H₁₄FN₃O₃ + H⁺ (352.1097).

4.4.7. 4-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8g)

White powder; yield, 80%; m.p. 179–180 °C; IR (CHCl₃) ν_max (cm^–1): 1714 (C=O), 761 (C–F), 1188 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.77 (d, J = 4.7 Hz, 1H), 7.62 (s, 1H, triazole), 7.57–7.53 (m, 1H), 7.34–7.29 (m, 3H), 7.23–7.21 (m, 1H), 7.12–7.10 (m, 2H), 5.84 (s, 1H), 5.56 (s, 2H), 5.32 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.99, 164.23 (d, ¹J_C–F = 247 Hz), 162.70, 153.24, 141.93, 132.61, 130.20 (d, ³J_C–F = 8 Hz), 130.05 (d, ⁴J_C–F = 4 Hz), 123.96, 123.27, 123.16, 116.78, 116.43 (d, ²J_C–F = 8 Hz), 91.64, 62.60, 53.60; ESIMS: m/z 352.11 [M + H]⁺; HR-ESIMS: m/z 352.1103 [M + H]⁺ calcd for C₁₉H₁₄FN₃O₃ + H⁺ (352.1097).

4.4.8. 4-((1-(2,6-Difluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8h)

White powder; yield, 70%; m.p. 176–178 °C; IR (CHCl₃) ν_max (cm^–1): 1715 (C=O), 1452 (C–F), 1272 (C–O), 1376 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.77 (dd, J = 8.0, 1.5 Hz, 1H), 7.64 (s, 1H), 7.56–7.52 (m, 1H), 7.41–7.38 (m, 2H), 7.33–7.30 (m, 2H), 7.24 (t, J = 7.6 Hz, 1H), 5.85 (s, 1H), 5.59 (s, 2H), 5.31 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 165.04, 162.76, 160.58 (d, ¹J_C–F = 243 Hz), 153.32, 141.81, 134.12, 132.60, 129.70, 129.07, 128.48,123.35, 123.16, 116.72, 115.42, 91.43, 62.64, 54.78; ESIMS: m/z 370.10 [M + H]⁺; HR-ESIMS: m/z 370.1010 [M + H]⁺ calcd for C₁₉H₁₃F₂N₃O₃ + H⁺ (370.1003).

4.4.9. 4-((1-(2-(Trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8i)

Cream powder; yield, 78%; m.p. 185–186 °C; IR (CHCl₃) ν_max (cm^–1): 1725 (C=O), 1454 (C–F), 1175 (C–O), 1334 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.76 (s, 1H, triazole), 7.74 (s, 2H), 7.60–7.56 (m, 1H), 7.54–7.48 (m, 2H), 7.31–7.28 (m, 2H), 7.25–7.19 (m, 1H), 5.86 (s, 1H), 5.80 (s, 2H), 5.33 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.96, 162.60, 153.34, 149.20, 138.10, 132.63, 131.48 (d, ³J_C–F = 32 Hz), 128.38, 126.34 (q, ⁴J_C–F = 7, 4 Hz), 125.06 (d, ¹J_C–F = 270 Hz), 123.95 (d, ²J_C–F = 83 Hz), 123.44, 116.82, 115.39, 91.20, 62.59, 53.56; ESIMS: m/z 402.1 [M + H]⁺; HR-ESIMS: m/z 402.1073 [M + H]⁺ calcd for C₂₀H₁₄F₃N₃O₃ + H⁺ (402.1066).

4.4.10. 4-((1-(4-(Trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8j)

White powder; yield, 69%; m.p. 188–189 °C; IR (CHCl₃) ν_max (cm^–1): 1714 (C=O), 761 (C–F), 1280 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.77 (d, J = 8.4 Hz, 1H), 7.63 (s, 1H, triazole), 7.57–7.53 (m, 1H), 7.34–7.30 (m, 3H), 7.24–7.21 (m, 1H), 7.13–7.07 (m, 2H), 5.84 (s, 1H), 5.56 (s, 2H), 5.32 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.98, 162.67, 161.78, 153.34, 141.95, 132.62, 130.20 (d, ⁴J_C–F = 8 Hz), 129.99, 123.94, 123.17, 123.15, 116.82 (d, ²J_C–F = 36 Hz), 116.24, 115.41, 91.20, 62.61, 53.71; ESIMS: m/z 402.10 [M + H]⁺; HR-ESIMS: m/z 402.1071 [M + H]⁺ calcd for C₂₀H₁₄F₃N₃O₃ + H⁺ (402.1066).

4.4.11. 4-((1-(2-(Trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8k)

Light yellow powder; yield, 75%; m.p. 154–155 °C; IR (CHCl₃) ν_max (cm^–1): 1726 (C=O), 1458 (C–F), 1248 (C–O), 1275 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.77 (d, J = 9.2 Hz, 1H), 7.72 (s, 1H, triazole), 7.57–7.52 (m, 1H), 7.47–7.43 (m, 1H), 7.35–7.30 (t, J = 7.4 Hz, 4H), 7.25 (t, J = 6.9 Hz, 1H), 5.85 (s, 1H), 5.68 (s, 2H), 5.32 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.96, 162.63, 153.33, 149.61, 142.07, 132.87, 132.63, 129.79, 130.79 (d, ³J_C–F = 36 Hz), 129.72, 123.95 (d, ²J_C–F = 81 Hz), 123.36, 121.70, 121.63 (d, ¹J_C–F = 256 Hz), 116.81, 115.39, 91.20, 62.59, 53.56; ESIMS: m/z 418.10 [M + H]⁺; HR-ESIMS: m/z 418.1021 [M + H]⁺ calcd for C₂₀H₁₄F₃N₃O₄ + H⁺ (418.1015).

4.4.12. 4-((1-(4-(Trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8l)

White powder; yield, 78%; m.p. 165–167 °C; IR (CHCl₃) ν_max (cm^–1): 1727 (C=O), 1452 (C–F), 1243 (C–O), 1278 (C–N); ¹H NMR (400 MHz, dimethyl sulfoxide (DMSO)): δ (ppm) δ 8.49 (s, 1H, triazole), 7.74 (dd, J = 9.5, 4.5 Hz, 1H), 7.66–7.62 (m, 1H), 7.50–7.47 (m, 2H), 7.40–7.38 (m, 3H), 7.34–7.30 (m, 1H), 6.16 (s, 1H), 5.71 (s, 2H), 5.43 (s, 2H); ¹³C NMR (100 MHz, DMSO): δ (ppm) 164.81, 162.04, 153.21, 148.59, 148.57, 141.83, 135.80, 133.27, 130.54, 125.98, 124.66, 123.32, 121.86, 121.75 (d, ¹J_C–F = 255 Hz), 116.91, 115.50, 91.78, 63.20, 52.50; ESIMS: m/z 418.1 [M + H]⁺; HR-ESIMS: m/z 418.1015 [M + H]⁺ calcd for C₂₀H₁₅F₃N₃O₄F₃ + H⁺ (418.1015).

4.4.13. 4-((1-(2-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8m)

Yellow powder; yield, 76%; m.p. 155–156 °C; IR (CHCl₃) ν_max (cm^–1): 1715 (C=O), 1261 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.74 (d, J = 6.7 Hz, 1H), 7.59 (s, 1H, triazole), 7.54–7.50 (m, 1H), 7.32–7.29 (m, 2H), 7.25–7.20 (m, 4H), 5.83 (s, 1H), 5.60 (s, 2H), 5.28 (s, 2H), 2.31 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 165.03, 162.72, 153.25, 141.55, 136.98, 132.56, 132.13, 131.17, 129.55, 129.39, 126.80, 123.94, 123.43, 123.19, 116.70, 115.40, 91.09, 62.64, 52.58, 19.05, 18.83; ESIMS: m/z 348.1 [M + H]⁺; HR-ESIMS: m/z 348.1345 [M + H]⁺ calcd for C₂₀H₁₇N₃O₃ + H⁺ (348.1348).

4.4.14. 4-((1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8n)

White powder; yield, 60%; m.p. 127–129 °C; IR (CHCl₃) ν_max (cm^–1): 1717 (C=O), 1159 (C–O), 1375 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.78 (dd, J = 7.4, 4.5 Hz, 1H), 7.64 (s, 1H), 7.56–7.52 (m, 1H), 7.34–7.30 (m, 2H), 7.27–7.21 (m, 1H), 6.92–6.88 (m, 2H), 6.84–6.83 (m, 1H), 5.84 (s, 1H), 5.55 (s, 2H), 5.31 (s, 2H), 3.80 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.95, 162.58, 159.88, 153.50, 142.37, 135.69, 132.57, 130.93, 124.23, 122.85, 120.17, 116.48, 115.08, 114.47, 114.09, 90.58, 62.66, 55.25, 54.24; ESIMS: m/z 364.1 [M + H]⁺; HR-ESIMS: m/z 364.1297 [M + H]⁺ calcd for C₂₀H₁₈FN₃O₄ + H⁺ (364.1303).

4.4.15. 4-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8o)

Off-white powder; yield, 72%; m.p. 159–160 °C; IR (CHCl₃) ν_max (cm^–1): 1713 (C=O), 1226 (C–O), 1369 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.78 (d, J = 8.0 Hz, 1H), 7.65 (s, 1H, triazole), 7.57–7.53 (m, 1H), 7.32 (d, J = 5.0 Hz, 1H), 7.24 (d, J = 4.8 Hz, 1H), 6.45–6.43 (m, 3H), 5.84 (s, 1H), 5.50 (s, 2H), 5.33 (s, 2H), 3.78 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 165.01, 162.68, 161.41, 153.33, 141.83, 136.22, 132.58, 123.94, 123.38, 123.20, 116.79, 115.43, 106.25, 100.50, 91.16, 62.67, 55.48, 54.50; ESIMS: m/z 394.14 [M + H]⁺; HR-ESIMS: m/z 394.1414 [M + H]⁺ calcd for C₂₁H₁₉N₃O₅ + H⁺ (394.1403).

4.4.16. 4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8p)

Light yellow powder; yield, 75%; m.p. 193–194 °C; IR (CHCl₃) ν_max (cm^–1): 1731 (C=O), 1673, 1452 (NO₂), 1187 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.28 (d, J = 1.8 Hz, 2H), 7.75 (dd, J = 7.4, 4.5 Hz, 1H), 7.71 (s, 1H), 7.60–7.54 (m, 1H), 7.46 (d, J = 1.9, 2H), 7.34–7.33 (m, 1H), 7.21–7.19 (m, 1H), 5.86 (s, 1H), 5.73 (s, 2H), 5.34 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.57, 162.52, 154.64, 153.26, 142.51, 141.21, 133.07, 124.90, 123.90, 123.62, 123.36, 116.78, 115.71, 90.76, 62.70, 52.69; ESIMS: m/z 379.10 [M + H]⁺; HR-ESIMS: m/z 379.1046 [M + H]⁺ calcd for C₁₉H₁₄N₄O₅ + H⁺ (379.1042).

4.4.17. 8-Acetyl-7-((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10a)

Light orange powder; yield, 55%; m.p. 213–214 °C; IR (CHCl₃) ν_max (cm^–1): 1731 (C=O), 1141 (C–O), 1391 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.64 (d, J = 7.7 Hz, 1H), 7.57 (s, 1H, triazole), 7.47 (d, J = 7.7 Hz, 1H), 7.30 (d, J = 7.7 Hz, 1H), 7.11 (d, J = 4.0 Hz, 2H), 6.90–6.83 (m, 2H), 6.77 (s, 1H), 6.31 (d, J = 4.3 Hz, 1H), 5.49 (s, 2H), 5.31 (s, 2H), 3.78 (s, 3H), 2.55 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 198.91, 160.19, 159.88, 151.13, 143.03, 142.36, 135.31, 130.22, 129.60, 122.54, 119.86, 114.10, 113.10, 112.79, 109.41, 63.32, 55.28, 54.29, 32.04.; ESIMS: m/z 406.14 [M + H]⁺; HR-ESIMS: m/z 406.1406 [M + H]⁺ calcd for C₂₃H₂₀N₃O₅ + H⁺ (406.1403).

4.4.18. 8-Acetyl-7-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10b)

White powder; yield, 54%; m.p. 232–234 °C; IR (CHCl₃) ν_max (cm^–1): 1727 (C=O), 1142 (C–O), 1367 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.64 (d, J = 8.5 Hz, 1H), 7.58 (s, 1H, triazole), 7.45 (d, J = 8.7 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 6.43–6.41 (m, 1H), 6.38 (s, 2H), 6.30–6.27 (m, 1H), 5.42 (s, 2H), 5.29 (s, 2H), 3.76 (s, 6H), 2.56 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 199.32, 161.36, 159.73, 156.99, 151.56, 143.34, 142.82, 136.53, 129.71, 122.87, 120.15, 114.47, 113.34, 109.55, 106.24, 100.22, 62.87, 55.50, 54.66, 32.32.; ESIMS: m/z 436.15 [M + H]⁺; HR-ESIMS: m/z 436.1509 [M + H]⁺ calcd for C₂₃H₂₂N₃O₆ + H⁺ (436.1508).

4.4.19. 8-Acetyl-7-((1-(2-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10c)

Orange sticky powder; yield, 51%; IR (CHCl₃) ν_max (cm^–1): 1726 (C=O), 1185 (C–O), 1315 (C–N), 1255 (C–F); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.65 (d, J = 12 Hz, 2H), 7.46–7.41 (m, 2H), 7.33–7.30 (m, 2H), 7.28–7.27 (m, 1H), 7.11 (d, J = 12 Hz, 1H), 6.30 (d, J = 8 Hz, 1H), 5.62 (s, 2H), 5.32 (s, 2H), 2.56 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 198.93, 159.71, 157.17, 151.26, 143.38, 143.00, 130.91, 130.67, 130.49, 129.69, 128.82 (d, ¹J_C–F = 242 Hz), 127.49, 123.38, 120.63, 114.27, 113.37, 109.56, 63.16, 48.69, 32.43; ESIMS: m/z 444.1 [M + H]⁺; HR-ESIMS: m/z 444.1160 [M + H]⁺ calcd for C₂₂H₁₇N₃O₄F₃ + H⁺ (444.1171).

4.4.20. 8-Acetyl-7-((1-(2-(trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10d)

Reddish sticky powder; yield, 52%; IR (CHCl₃) ν_max (cm^–1): 1728 (C=O), 1187 (C–O), 1312 (C–N), 1124 (C–F); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.64–7.62 (m, 2H), 7.46–7.40 (m, 2H), 7.32–7.30 (m, 2H), 7.26–7.24 (m, 1H), 7.11 (d, J = 7.7 Hz, 1H), 6.30 (d, J = 9.6 Hz, 1H), 5.62 (s, 2H), 5.32 (s, 2H), 2.56 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 198.95, 159.73, 157.16, 151.25, 143.37, 143.01, 130.67 (d, ³J_C–F = 18 Hz), 129.69 (d, ¹J_C–F = 219 Hz), 126.39, 120.62, 120.01, 114.26, 113.37, 109.55, 63.15, 48.69, 32.44; ESIMS: m/z 460.1 [M + H]⁺; HR-ESIMS: m/z 460.1118 [M + H]⁺ calcd for C₂₂H₁₇N₃O₅F₃ + H⁺ (460.1120).

4.4.21. 6,7-Bis((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one (13a)

Yellowish oil; yield, 64%; m.p. 129–131 °C; IR (CHCl₃) ν_max (cm^–1): 1712 (C=O), (NO₂), 1162 (C–O), 1388 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) δ 7.55 (d, J = 9.8 Hz, 2H), 7.23–7.21 (m, 1H), 7.19 (s, 1H), 7.17 (s, 1H), 6.87 (s, 2H), 6.83–6.78 (m, 3H), 6.75–6.72 (m, 2H), 6.69–6.68 (m, 1H), 6.08 (s, 1H), 5.41 (d, J = 9.1 Hz, 4H), 5.18 (d, J = 13.2 Hz, 4H), 3.70 (s, 6H), 2.28 (s 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.27, 160.12, 152.45, 152.08, 149.72, 144.67, 144.01, 135.92, 135.78, 130.28, 130.22, 123.42, 123.27, 120.35, 120.24, 114.29, 114.09, 113.83, 113.80, 113.35, 112.66, 111.01, 102.24, 64.21, 63.00, 55.33, 54.23, 18.86; ESIMS: m/z 595.2 [M + H]⁺; HR-ESIMS: m/z 595.2297 [M + H]⁺ calcd for C₃₂H₃₁N₆O₆ + H⁺ (595.2305).

4.4.22. 6,7-Bis((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one (13b)

Light yellow powder; yield, 61%; m.p. 124–126 °C; IR (CHCl₃) ν_max (cm^–1): 1711 (C=O), 1159 (C–O), 1430 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.56 (d, J = 8.1 Hz, 2H), 7.17 (s,1H), 6.87 (s, 1H, triazole), 6.35–6.33 (m, 4H), 6.29 (d, J = 2.2 Hz, 2H), 6.08 (d, J = 1.1 Hz, 1H), 5.37 (d, J = 9.0 Hz, 4H), 5.19 (d, J = 11.5 Hz, 4H), 3.67 (s, 12H), 2.28 (d, J = 1.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.34, 161.31, 161.26, 152.44, 152.08, 149.72, 144.66, 144.02, 143.09, 136.60, 136.46, 123.43, 123.27, 113.34, 112.64, 110.97, 106.17, 106.12, 102.22, 100.48, 100.28, 64.21, 63.00, 55.44, 54.24, 18.85; ESIMS: m/z 655.2 [M + H]⁺; HR-ESIMS: m/z 655.2520 [M + H]⁺ calcd for C₃₄H₃₅N₆O₈ + H⁺ (655.2516).

4.4.23. 4-Methyl-6,7-bis((1-(2-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (13c)

Pale yellow powder; yield, 74%; m.p. 168–170 °C; IR (CHCl₃) ν_max (cm^–1): 1728 (C=O), 1673, 1452 (NO₂), 1163 (C–O), 1314 (C–N), 1062 (C–F); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.73 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.54–7.49 (m,2H), 7.46–7.44 (m, 3H), 7.24 (s, 1H), 7.20 (d, J = 1.2 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 6.96 (s, 1H), 6.16 (d, J = 1.2 Hz, 1H), 5.73 (d, J = 7.8 Hz, 4H), 5.28 (d, J = 9.8 Hz, 4H), 2.36 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.19, 152.34, 152.05, 149.75, 144.69, 144.16, 143.29, 132.79, 132.69, 130.27, 130.19, 128.89, 128.16 (d, ²J_C–F = 31 Hz), 126.32 (q, ⁴J_C–F = 6 Hz), 125.45 (d, ¹J_C–F = 272 Hz), 123.79 (d, ³J_C–F = 15 Hz), 113.41, 112.75, 110.99, 102.39, 64.08, 63.02, 50.26, 18.81. ESIMS: m/z 671.18 [M + H]⁺; HR-ESIMS: m/z 671.1832 [M + H]⁺ calcd for C₃₂H₂₅N₆O₄F₆ + H⁺ (671.1841).

4.4.24. 4-Methyl-6,7-bis((1-(2-(trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (13d)

Light brown powder; yield, 78%; m.p. 173–174 °C; IR (CHCl₃) ν_max (cm^–1): 1724 (C=O), 1184 (C–O), 1339 (C–N) 1070 (C–F); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.67 (d, J = 8.0 Hz, 2H), 7.44–7.38 (m, 2H), 7.33–7.31 (m, 2H), 7.29 (s, 1H), 7.25 (s, 2H), 7.24–7.23 (m, 1H), 7.20–7.10 (m, 1H), 6.96 (s, 1H), 6.16 (d, J = 1.2 Hz, 1H), 5.63 (d, J = 7.8 Hz, 4H), 5.28 (d, J = 9.8 Hz, 4H), 2.35 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.25, 152.40, 152.01, 149.71, 146.94, 144.67, 144.06, 130.58, 130.55, 130.50 (d, ³J_C–F = 11Hz), 130.39, 127.54 (d, ⁴J_C–F = 8 Hz), 127.12 (d, ⁴J_C–F = 8 Hz), 123.66 (d, ²J_C–F = 16 Hz), 120.63, 113.38, 112.71, 110.88, 102.34, 64.01, 62.98, 48.49, 18.82; ESIMS: m/z 703.1 [M + H]⁺; HR-ESIMS: m/z 703.1733 [M + H]⁺ calcd for C₃₂H₂₅N₆O₄F₆ + H⁺ (703.1740).

4.4.25. 6-Methoxy-7-((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one (16a)

Light yellow powder; yield, 70%; m.p. 159–161 °C; IR (CHCl₃) ν_max (cm^–1): 1713 (C=O), 1275 (C–O), 1381 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.61 (s, 1H, triazole), 7.31–7.27 (m, 1H), 7.00 (s, 1H), 6.93 (s, 1H), 6.91–6.85 (m, 2H), 6.80–6.78 (m, 1H), 6.17 (s, 1H), 5.50 (s, 2H), 5.28 (s, 2H), 3.89 (s, 3H), 3.78 (s, 3H), 2.40 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.38, 161.34, 152.19, 151.20, 149.07, 146.45, 143.17, 136.36, 123.13, 113.11, 112.68, 106.20, 105.59, 101.94, 100.55, 63.07, 56.47, 55.46, 54.39, 18.91; ESIMS: m/z 408.1 [M + H]⁺; HR-ESIMS: m/z 408.1566 [M + H]⁺ calcd for C₁₂H₂₁N₃O₅ + H⁺ (408.1559).

4.4.26. 7-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-6-methoxy-4-methyl-2H-chromen-2-one (16b)

White powder; yield, 71%; m.p. 151–152 °C; IR (CHCl₃) ν_max (cm^–1): 1728 (C=O), 1123 (C–O), 1350 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.61 (s, 1H, triazole), 7.03 (s, 1H), 6.93 (s, 1H), 6.43 (s, 1H), 6.37 (s, 2H), 6.18 (s, 1H), 5.45 (s, 2H), 5.29 (s, 2H), 3.89 (s, 3H), 3.76 (s, 6H), 2.40 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.23, 151.92, 151.16, 148.87, 146.62, 143.10, 136.03, 123.20, 113.07, 112.84, 106.56, 105.75, 101.49, 100.75, 63.17, 56.37, 55.63, 54.38, 19.08; ESIMS: m/z 438.1 [M + H]⁺; HR-ESIMS: m/z 438.1659 [M + H]⁺ calcd for C₂₃H₂₃N₃O₆ + H⁺ (438.1665).

4.4.27. 6-Methoxy-4-methyl-7-((1-(2-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (16c)

White powder; yield, 70%; m.p. 151–153 °C; IR (CHCl₃) ν_max (cm^–1): 1727 (C=O), 1273 (C–O), 1427 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.67 (d, J = 8.5 Hz, 1H), 7.57 (s, 1H, triazole), 7.49–7.46 (m, 1H), 7.42–7.38 (m, 1H), 7.17 (s, 1H), 6.95 (s, 1H), 6.87 (s, 1H), 6.12 (s, 1H), 5.68 (s, 2H), 5.24 (s, 2H), 3.83 (s, 3H), 2.34 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.37, 152.16, 151.16, 149.06, 146.48, 143.44, 132.82, 130.39, 128.91, 123.16 (d, ¹J_C–F = 274 Hz), 113.21, 112.76, 105.65, 102.11, 63.09, 56.49, 49.68, 18.91; ESIMS: m/z 446.1 [M + H]⁺; HR-ESIMS: m/z 446.1330 [M + H]⁺ calcd for C₂₂H₁₈N₃O₄F₃ + H⁺ (446.1328).

4.4.28. 6-Methoxy-4-methyl-7-((1-(2-(trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (16d)

White powder; yield, 79%; m.p. 164–166 °C; IR (CHCl₃) ν_max (cm^–1): 1718 (C=O), 1275 (C–O), 1385 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.66 (s, 1H, triazole), 7.44–7.40 (m, 1H), 7.33–7.27 (m, 3H), 7.02 (s, 1H), 6.94 (s, 1H), 6.18 (s, 1H), 5.63 (s, 2H), 5.30 (s, 2H), 3.90 (s, 3H), 2.41 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.39, 152.21, 151.16, 149.05, 146.93, 146.46, 143.28, 130.60, 127.56, 127.03, 123.45 (d, ¹J_C–F = 257 Hz), 120.63, 113.16, 112.70, 105.61, 102.00, 63.03, 56.48, 48.47, 18.92. ESIMS: m/z 462.1 [M + H]⁺; HR-ESIMS: m/z 462.1265 [M + H]⁺ calcd for C₂₂H₁₉N₃O₅F₃ + H⁺ (462.1277).

4.4.29. 7-((1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19a)

White powder; yield, 70%; m.p. 137–138 °C; IR (CHCl₃) ν_max (cm^–1): 1725 (C=O), 1156 (C–O), 1305 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.64 (d, J = 9.5 Hz, 1H), 7.60 (s, 1H, triazole), 7.38 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 7.5 Hz, 1H), 6.92–6.86 (m, 4H), 6.81 (t, J = 4.5 Hz, 1H), 6.26 (d, J = 9.5 Hz, 1H), 5.52 (s, 2H), 5.22 (s, 2H), 3.78 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.67, 160.95, 160.19, 155.93, 143.75, 135.76, 130.78, 129.01, 123.01, 120.08, 114.27, 113.83, 113.44, 112.98, 112.77, 101.59, 62.23, 55.51, 53.83. ESIMS: m/z 364.1 [M + H]⁺; HR-ESIMS: m/z 364.1303 [M + H]⁺ calcd for C₂₀H₁₇N₃O₄ + H⁺ (364.1297).

4.4.30. 7-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19b)

Light yellow powder; yield, 69%; m.p. 132–133 °C; IR (CHCl₃) ν_max (cm^–1): 1728 (C=O), 1120 (C–O), 1277 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.65 (d, J = 9.5 Hz, 1H), 7.59 (s, 1H, triazole), 7.39 (d, J = 7.9 Hz, 1H), 6.93–6.90 (m, 2H), 6.44–6.43 (m, 1H), 6.41 (d, J = 7.8 Hz, 1H), 6.28 (d, J = 7.4 Hz, 1H), 5.47 (s, 2H), 5.23 (s, 2H), 3.76 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.37, 161.30, 161.09, 155.50, 143.38, 136.30, 128.91, 122.85, 113.49, 113.00, 112,78, 106.20, 102.08, 100.47, 61.94, 55.25, 54.25. ESIMS: m/z 394.14 [M + H]⁺; HR-ESIMS: m/z 394.1406 [M + H]⁺ calcd for C₂₁H1₉N₃O₅ + H⁺ (394.1403).

4.4.31. 7-((1-(2-(Trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19c)

Dark brown powder; yield, 67%; m.p. 119–121 °C; IR (CHCl₃) ν_max (cm^–1): 1731 (C=O), 1673, 1452 (NO₂), 1187 (C–O), 1377 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.74 (d, J = 7.8 Hz, 1H), 7.68 (s, 1H, triazole), 7.65 (d, J = 9.5 Hz, 1H), 7.56–7.53 (m, 1H), 7.49–7.46 (m, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 6.93–6.91 (m, 1H), 6.89–6.88 (m, 1H), 6.26 (d, J = 9.5 Hz, 1H), 5.77 (s, 2H), 5.25 (s, 2H). ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.26, 161.05, 155.66, 143.51, 143.38, 132.79, 130.28, 128.95 (d,³J_C–F = 3 Hz), 128.13 (d, ²J_C–F = 30 Hz), 126.37 (q, ⁴J_C–F = 12, 6 Hz), 125.44 (d, ¹J_C–F = 272 Hz), 123.54, 113.41, 113.00, 112.77, 102.02, 62.25, 50.32. ESIMS: m/z 402.1 [M + H]⁺; HR-ESIMS: m/z 402.1076 [M + H]⁺ calcd for C₂₀H₁₄N₃O₃F₃ + H⁺ (402.1066).

4.4.32. 7-((1-(2-(Trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19d)

Pale white powder; yield, 62%; m.p. 123–124 °C; IR (CHCl₃) ν_max (cm^–1): 1723 (C=O), 1124 (C–O), 1274 (C–N); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.78 (d, J = 7.9 Hz, 1H), 7.74 (s, 1H, triazole), 7.57–7.53 (m, 1H), 7.48–7.43 (m, 1H), 7.37–7.34 (m, 3H), 7.33 (d, J = 7.5 Hz, 1H), 7.27–7.23 (m, 1H), 5.87 (s, 1H), 5.69 (s, 2H), 5.34 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.99, 162.64, 153.32, 147.04, 141.89, 132.57, 130.77 (d, ⁴J_C–F = 8.08 Hz), 127.60, 126.82, 123.93, 123.70, 123.14, 121.76 (d, ¹J_C–F = 259.76 Hz), 120.65, 116.76, 115.41, 91.17, 62.61, 48.70; ESIMS: m/z 418.1 [M + H]⁺; HR-ESIMS: m/z 418.1006 [M + H]⁺ calcd for C₂₀H₁₄N₃O₄F₃ + H⁺ (418.1015).

4.5. In Vitro AChE and BChE Inhibition Assay

The cholinesterase inhibition experiments, including the enzyme kinetics, were evaluated by Ellman assay as described earlier.³²

4.6. In Vitro BACE-1 Inhibition Assay

The fluorescence resonance energy transfer (FRET) assay was used to screen all synthesized compounds²⁴ for BACE-1 inhibition and its enzyme kinetics.

4.7. Molecular Modeling

The Schrodinger software was used to carry out the molecular docking study. The crystal structures of human AChE (PDB ID: 4EY7),³⁴ human BChE (PDB ID: 6EP4),³⁵ and human BACE-1 (PDB ID: 1W51)³⁶ were obtained from the RCSB Protein Data Bank. The protocols employed were as given under the default settings of glide. Further, MD simulation was carried out using Desmond software (v 3.8) for 50 ns by a similar protocol as used in the previous publication of our group.²⁰

4.8. Aβ-42 Self-Aggregation Inhibition Assay

The fluffy white solid obtained by pretreatment of amyloid β 1–42 peptide (PP69, Sigma) was reconstituted to produce a 200 mM solution in a mixture of ACN/Na₂CO₃/NaOH in a ratio of 48.4:48.4:3.2. The Aβ aliquots with and without test inhibitors were incubated for 24 h at 30 °C. thioflavin T (ThT) fluorescence method was used to assess the aggregates.³⁷

4.9. In Vitro PAMPA–BBB Assay

The samples were diluted with PBS (pH 7.4). The acceptor plate wells were filled with 300 μL of PBS. The 4 μL of 20 mg/mL porcine brain lipid (PBL) in dodecane was used to coat the permeable layer of the donor plate. The test compounds were added to the donor plate. The donor plate was kept on the acceptor plate like a “sandwich” and incubated at 25 °C for 18 h. UV absorbance was used to access the concentration of test samples in donor and acceptor wells. As described earlier, the absorbance was used to calculate the test compound’s effective permeability (P_e).³⁸

4.10. In Silico ADME Properties

The ADME properties of ligands were determined using the QikProp module of the Schrodinger software. The descriptors and properties for reference ranges are for 95% of known drugs.

Acknowledgments

The authors thank the analytical department, IIIM, for analytical support. The financial support from CSIR YSA Grant (P90807) is gratefully acknowledged. IIIM Publication Number. CSIR-IIIM/IPR/00518.

Glossary

Abbreviations.

AD: Alzheimer’s disease
ACh: acetylcholine
ADME: absorption distribution metabolism elimination
BCh: butyrylcholine
BBB: blood–brain barrier
CAS: catalytic anionic site
EeAChE: electric eel cholinesterase
eqBChE: equine serum butyrylcholinesterase
hBACE-1: human amyloid β precursor protein cleaving enzyme or β-secretase
MD: molecular dynamics
NP: natural product
PDB: Protein Data Bank
PAS: peripheral anionic site
PAMPA: parallel artificial membrane permeability assay
P_e: effective permeability coefficient
RMSD: root mean square deviation
SAR: structure–activity relationship
ThT: thioflavin T

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c07993.

Spectral data scans for all compounds (PDF)

Author Contributions

S.B.B. designed, executed, and coordinated this study. A.S. synthesized all compounds and performed biological assays and molecular modeling. A.S. and S.B.B. contributed to the manuscript writing.

The authors declare no competing financial interest.

Supplementary Material

ao2c07993_si_001.pdf^{(5.8MB, pdf)}

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Associated Data

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Supplementary Materials

ao2c07993_si_001.pdf^{(5.8MB, pdf)}

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PERMALINK

Synthesis and Biological Evaluation of Coumarin Triazoles as Dual Inhibitors of Cholinesterases and β-Secretase

Ankita Sharma

Sandip B Bharate

Abstract

1. Introduction

Figure 1.

2. Results and Discussion

2.1. Synthesis and In Vitro Biological Evaluation

Scheme 1. Synthesis of Coumarin A-Ring Connected Triazole Hybrids, 8a–p.

Table 1. Cholinesterase and BACE-1 Inhibition Results for Coumarin A-Ring Connected Triazole Hybrids, 8a–p.

Scheme 2. Synthesis of Coumarin B-Ring Connected Triazole Hybrids, 10a–d, 13a–d, 16a–d, and 19a–d.

Table 2. Cholinesterase and BACE-1 Inhibition Results for Coumarin B-Ring Connected Triazole Hybrids, 10a–d, 13a–d, 16a–d, and 19a–d.

2.2. Enzyme Kinetics and Molecular Modeling for 10b

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

2.3. Inhibition and Aβ Self-Aggregation

Figure 7.

2.4. Parallel Artificial Membrane Permeability Assay (PAMPA)–Blood–Brain Permeability (BBB) Permeability and In Silico Absorption Distribution Metabolism Elimination (ADME) Properties of 10b

Table 3. BBB Permeability of 10b Determined Using PAMPA–BBB Assay.

Table 4. ADME Properties for 10b and Positive Controlsa.

3. Conclusions

4. Experimental Section

4.1. General

4.2. Synthesis of 7-Hydroxy-6-methoxy-4-methyl-2H-chromen-2-one (14)30

4.3. Propargylation of Coumarin Hydroxyl Group: Synthesis of 6, 9, 12, 15, and 18

4.3.1. 4-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (6)31

4.3.2. 8-Acetyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (9)

4.3.3. 4-Methyl-6,7-bis(prop-2-yn-1-yloxy)-2H-chromen-2-one (12)

4.3.4. 6-Methoxy-4-methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (15)

4.3.5. 7-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (18)

4.4. General Procedure for the Synthesis of Triazoles 8a–o, 10a–d, 13a–d, 16a–d, and 19a–d

4.4.1. 4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8a)

4.4.2. 4-((1-(2-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8b)

4.4.3. 4-((1-(3-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8c)

4.4.4. 4-((1-(2,6-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8d)

4.4.5. 4-((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8e)

4.4.6. 4-((1-(3-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8f)

4.4.7. 4-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8g)

4.4.8. 4-((1-(2,6-Difluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8h)

4.4.9. 4-((1-(2-(Trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8i)

4.4.10. 4-((1-(4-(Trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8j)

4.4.11. 4-((1-(2-(Trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8k)

4.4.12. 4-((1-(4-(Trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8l)

4.4.13. 4-((1-(2-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8m)

4.4.14. 4-((1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8n)

4.4.15. 4-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8o)

4.4.16. 4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8p)

4.4.17. 8-Acetyl-7-((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10a)

4.4.18. 8-Acetyl-7-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10b)

4.4.19. 8-Acetyl-7-((1-(2-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10c)

4.4.20. 8-Acetyl-7-((1-(2-(trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (10d)

4.4.21. 6,7-Bis((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one (13a)

4.4.22. 6,7-Bis((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one (13b)

4.4.23. 4-Methyl-6,7-bis((1-(2-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (13c)

4.4.24. 4-Methyl-6,7-bis((1-(2-(trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (13d)

4.4.25. 6-Methoxy-7-((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one (16a)

4.4.26. 7-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-6-methoxy-4-methyl-2H-chromen-2-one (16b)

4.4.27. 6-Methoxy-4-methyl-7-((1-(2-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (16c)

4.4.28. 6-Methoxy-4-methyl-7-((1-(2-(trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (16d)

4.4.29. 7-((1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19a)

4.4.30. 7-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19b)

4.4.31. 7-((1-(2-(Trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19c)

4.4.32. 7-((1-(2-(Trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (19d)

4.5. In Vitro AChE and BChE Inhibition Assay

4.6. In Vitro BACE-1 Inhibition Assay

4.7. Molecular Modeling

4.8. Aβ-42 Self-Aggregation Inhibition Assay

4.9. In Vitro PAMPA–BBB Assay

4.10. In Silico ADME Properties

Acknowledgments

Glossary

Abbreviations.

Supporting Information Available

Author Contributions

Supplementary Material

References

Table 4. ADME Properties for 10b and Positive Controls^a.

4.2. Synthesis of 7-Hydroxy-6-methoxy-4-methyl-2H-chromen-2-one (14)³⁰

4.3.1. 4-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (6)³¹