Discovery of new acetylcholinesterase inhibitors derived from quinazolinones: synthesis, in vitro, and molecular docking study

Discovery of new acetylcholinesterase inhibitors derived from quinazolinones: synthesis, in vitro, and molecular docking study Ade Danova1, Angellyn Carnelian Christy1, Iqbal Musthapa2, Fera Kurniadewi3, Warinthorn Chavasiri4, Didin Mujahidin1, Robby Roswanda1, Elvira Hermawati1 1 Organic Chemistry Division, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung 40132, West Java, Indonesia 2 Chemistry Study Program, Faculty of Mathematics and Natural Science Education, Universitas Pendidikan Indonesia, Jl. Dr. Setiabudi No 229, Bandung 40154, Indonesia 3 Chemistry Study Program, Universitas Negeri Jakarta, Jakarta, 13220, Indonesia 4 Center of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand Corresponding authors: Ade Danova ([email protected]); Elvira Hermawati ([email protected]) Received 20 June 2025♦ Accepted 29 September 2025♦ Published 15 October 2025 Citation: Danova A, Carnelian Christy A, Musthapa I, Kurniadewi F, Chavasiri W, Mujahidin D, Roswanda R, Hermawati E (2025) Discovery of new acetylcholinesterase inhibitors derived from quinazolinones: synthesis, in vitro, and molecular docking study. Pharmacia 72: 1–10. https://doi.org/10.3897/pharmacia.72.e162676 Abstract In the present study, twelve quinazolinones (2, 3, 4a–4j) were successfully synthesized. Among them, five compounds (4c, 4d, 4f, 4h, 4i) were newly reported. Furthermore, twelve compounds were assessed against acetylcholinesterase (AChE). Seven compounds (4b, 4c, 4d, 4f, 4g, 4h, 4j) showed inhibitory activity above 70%, where compounds 4c and 4h had inhibitory activity with IC₅₀ values of 2.97 and 5.86 μM, respectively. The kinetic study showed that compounds 4c and 4h exhibited mixed-type inhibition. Molecular docking suggested that compounds 4c and 4h displayed binding energy values of −8.7 and −8.4 kcal/mol. Compound 4c showed H-bonds with four residues (Tyr124, Tyr337, Tyr341, and Glu202), but compound 4h formed H-bonds with three residues (Tyr124, Tyr133, and Glu202) in the binding pocket of AChE. Thus, quinazolinone derivatives with a nitro group at the C-6 and alkoxy groups at the C-7 position have the potential for further investigation as AChE inhibitors. Keywords acetylcholinesterase inhibitors, Alzheimer’s disease, IC₅₀, kinetic study, molecular docking, quinazolinone Introduction Alzheimer’s disease (AD) is a formidable challenge in modern medicine because of its complex pathophysiology and profound impact on cognitive function and quality of life (Seo and Holtzman 2024; Azam et al. 2025). It is the leading cause of dementia worldwide, accounting for approximately 60–80% of cases (Gandini et al. 2022; Better 2023). In 2020, over 55 million individuals were affected by dementia globally, with the associated care costs estimated at approximately USD 2.8 trillion by 2030. Alarmingly, projections indicate that the global prevalence of Copyright Danova A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pharmacia 72: 1–10 DOI 10.3897/pharmacia.72.e162676 Research Article Danova A et al.: Discovery of new acetylcholinesterase inhibitors derived from quinazolinones2 dementia will rise to 139 million by 2050 (ADl’S 2021). Given the multifaceted and ambiguous etiology of AD, several factors are implicated in its onset and progression, including acetylcholine (ACh) deficiency, β-amyloid (Aβ) accumulation, biometal dyshomeostasis, oxidative stress, and hyperphosphorylated τ-protein. Furthermore, monoclonal antibodies (MABs) are being tested in clinical trials for AD treatment, focusing on amyloid-β (Aβ), tau, and inflammation. First-generation anti-amyloid MABs in these trials did not show significant benefits, as they were unable to eliminate Aβ plaques in all patients. Recently, second-generation MABs, including aducanumab, lecanemab, and donanemab, have been associated with hyperintensity abnormalities on imaging scans–such as alterations in the cortical folds (Kim et al. 2025). To date, available pharmacological treatments for AD offer only temporary and modest cognitive improvements without significantly altering disease progression or ultimate prognosis (Genc Bilgicli et al. 2020; Işık et al. 2020; Lolak et al. 2020). Therefore, there is a critical need to develop innovative therapeutic approaches for AD. Acetylcholine (ACh) is an essential neurotransmitter that mediates synaptic transmission in the central and peripheral nervous systems. Disruption of central cholinergic transmission is associated with various disorders, including AD, Parkinson’s disease, schizophrenia, and epilepsy. In both the central and peripheral nervous systems, termination of impulse transmission is achieved through the rapid hydrolysis of ACh by acetylcholinesterase (AChE). AChE degrades ACh into choline and acetic acid, thereby restoring a cholinergic neuron to its resting state (Biçer et al. 2020; Gülçin et al. 2020; Hulya and İlhami 2020; Taslimi et al. 2020; Tokalı et al. 2021). Currently, the market provides a limited selection of FDA-approved acetylcholinesterase inhibitors–specifically, galantamine, tacrine, donepezil, and rivastigmine. However, these pharmaceuticals are linked to significant adverse effects, including hepatotoxicity and anxiety (Agrawal et al. 2018; Li et al. 2018; Bagrowska et al. 2024). Therefore, the primary goal in the field of medicinal chemistry is to discover novel AChE inhibitors that are stable, safe, and highly efficacious. Quinazolinone derivatives, including 4(3H)-quinazolinone (Fig. 1), constitute a significant class of fused heterocycles present in over 100 naturally occurring alkaloids (Mhaske and Argade 2006). The 4(3H)-quinazolinone framework demonstrates a diverse array of biological activities, such as anticancer, antiviral, anti-inflammatory, antimicrobial, cholinesterase, antifolate, antitumor, and protein kinase inhibition (Michael 2008). Previous research has indicated that the incorporation of alkyl and aromatic rings into the pyrimidone ring enhances AChE inhibition, as evidenced in compounds B, C, and D, except for compound A (Uraz et al. 2016; Dighe et al. 2020; Tokalı et al. 2021; Babatunde et al. 2023), as depicted in Fig. 1. To the best of our knowledge, the influence of hydrophobic tail substitution on the phenyl ring of quinazolinones has not been investigated in the context of AChE inhibition–this remains a promising area for further investigation. A hydrophobic tail was attached to the phenyl ring of quinazolinone via Williamson etherification under basic conditions using an alkyl alcohol. Subsequently, we assessed the AChE inhibitory activity of the modified quinazolinone derivatives by in vitro evaluation and molecular docking study. Experimental section Materials All chemicals were acquired from commercial suppliers (Sigma Aldrich and Merck) and utilized without any additional purification. All solvents were sourced from non-commercial sources and subsequently purified by distillation for furFigure 1. Quinazolinone derivatives as AChE inhibitors. N N OS N N NH N N O N CF3 N H N O Cl N N O S O NH H N O H N O F3C N NH O O O2N R Hydrophobic Tail Ring A = Phenyl Ring B = Pyrimidone AB IC 50 AChE= > 1 mM (A) IC 50 AChE= 38.94 nM (C)IC50 AChE= 23.08 µM (D) IC50 AChE= 6.10 µM (B) Our Currently Target: Pharmacia 72: 1–10 3 ther use. Silica gel for column chromatography (0.063–0.200 mm) was purchased from Merck Company. TLC was conducted on Merck TLC plates (0.23 mm thickness) and visualized by UV light. NMR measurements were performed using Agilent Varian DD2 500 MHz spectrometers. Mass spectrum (MS) was obtained on a Waters LCT Premier XE ESI-TOF-MS. The molecular docking study used a Lenovo Yoga 7 computer with AMD Ryzen AI 7-8840HS w/ Radeon 780M graphics, 3301 MHz, 8 cores, and 16.0 GB RAM. Methods Synthesis of 7-fluoroquinazolinone (2) and 7-fluoro-6-nitroquinazolinone (3) The product was synthesized according to a previous report (Maulana et al. 2025). 7-Fluoroquinazolin-4(3H)-one (2): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.30 (1H, s), 8.13 (1H, dd, JH-H = 8.8 Hz, JH-F = 6.4 Hz), 8.08 (1H, s), 7.39 (1H, dd, JH-F = 10.1 Hz, JH-H = 2.4 Hz), 7.33 (1H, td, JH-H = 8.8 Hz, JH-F = 2.5 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 165.5 (d, JC-F = 249.5 Hz), 160.0, 150.9 (d, JC-F = 12.3 Hz), 146.8, 129.0 (d, JC-F = 10.8 Hz), 119.6, 115.3 (d, JC-F = 23.4 Hz), 112.3 (d, JC-F = 21.5 Hz). HRESITOF-MS: m/z 165.0467 [M+H]+ (calcd. for C8H6FN2O: 165.0459). All data were consistent with literature (Maulana et al. 2025). 7-Fluoro-6-nitroquinazolin-4(3H)-one (3): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.78 (1H, s), 8.74 (1H, d, JH-F = 10.0 Hz), 8.31 (1H, s), 7.79 (1H, d, JH-F = 10.0 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 159.3, 157.6 (d, JC-F = 264.1 Hz), 154.0 (d, JC-F = 13.3 Hz), 150.0, 135.4 (d, JC-F = 9.9 Hz), 125.5, 119.3, 115.6 (d, JC-F = 21.3 Hz). HRESITOF-MS: m/z 210.0312 [M+H]+ (calcd. for C8H5FN3O3: 210.0309). All data were consistent with literature (Maulana et al. 2025). Synthesis of 7-alkoxy-6-nitroquinazolinones (4a-4j) The product was synthesized according to a previous report (Maulana et al. 2025). A mixture of 3 (1 eq.), alkyl alcohol (60 eq.), and 50% NaOH was added to the reaction mixture at room temperature. Subsequently, the reaction mixture was refluxed for 1 h and monitored using TLC. After cooling the reaction mixture to room temperature, it was poured into a sodium bicarbonate solution, yielding a precipitate that was separated by filtration, washed with water, dried to obtain compounds (4a-4j), and characterized using NMR spectroscopy and mass spectrometry. 7-Methoxy-6-nitroquinazolin-4(3H)-one (4a): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.48 (1H, s), 8.50 (1H, s), 8.21 (1H, s), 7.40 (1H, s), 4.03 (3H, s). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 160.0, 156.4, 153.5, 149.2, 138.7, 124.2, 115.6, 110.9, 57.8. HRESITOF-MS: m/z 220.0361 [M-H]- (calcd. for C9H6N3O4: 220.0364). All data were consistent with literature (Zhou et al. 2025). 7-Ethoxy-6-nitroquinazolin-4(3H)-one (4b): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.45 (1H, s), 8.49 (1H, s), 8.20 (1H, s), 7.38 (1H, s), 4.33 (2H, q, JH-H = 6.9 Hz), 1.37 (3H, t, JH-H = 6.9 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 159.7, 155.1, 153.0, 148.8, 138.5, 123.6, 115.0, 110.9, 65.8, 14.1. HRESITOF-MS: m/z 234.0524 [M-H]- (calcd. for C10H8N3O4: 234.0520). All data were consistent with literature (Li et al. 2022). 6-Nitro-7-propoxyquinazolin-4(3H)-one (4c): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 8.35 (1H, s), 8.06 (1H, s), 6.90 (1H, s), 4.03 (2H, t, JH-H = 6.2 Hz), 1.69 (2H, sext, JH-H = 7.4 Hz), 0.93 (3H, t, JH-H = 7.4 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 170.8, 161.4, 156.4, 153.4, 135.4, 124.9, 115.6, 107.8, 70.2, 21.8, 10.3. HRESITOF-MS: m/z 248.0677 [M-H]- (calcd. for C11H10N3O4: 248.0677). 7-Isopropoxy-6-nitroquinazolin-4(3H)-one (4d): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 8.45 (1H, s), 8.19 (1H, s), 7.40 (1H, s), 4.99 (1H, sept, JH-H = 5.0 Hz), 1.33 (6H, d, JH-H = 5.0 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 160.7, 154.4, 153.4, 149.7, 139.6, 124.0, 115.4, 112.0, 73.0, 21.8. HRESITOF-MS: m/z 248.0675 [M-H]- (calcd. for C11H10N3O4: 248.0677). 7-(2-Hydroxyethoxy)-6-nitroquinazolin-4(3H)-one (4e): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.48 (1H, s), 8.46 (1H, s), 8.17 (1H, s), 7.40 (1H, s), 4.97 (1H, s), 4.27 (2H, t, JH-H = 4.6 Hz), 3.71 (2H, t, JH-H = 4.6 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 159.7, 155.4, 152.9, 148.8, 138.6, 123.6, 115.1, 111.2, 71.9, 59.1. HRESITOF-MS: m/z 250.0468 [M-H]- (calcd. for C10H8N3O5: 250.0469). All data are consistent with literature (Sun et al. 2020). 7-(2-Mercaptoethoxy)-6-nitroquinazolin-4(3H)-one (4f): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.59 (1H, s), 8.76 (1H, s), 8.27 (1H, s), 7.73 (1H, s), 5.14 (1H, s), 3.72 (2H, t, JH-H = 6.7 Hz), 3.26 (2H, t, JH-H = 6.2 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 159.7, 151.4, 149.4, 143.4, 142.9, 124.6, 124.4, 118.6, 58.6, 34.8. HRESITOF-MS: m/z 268.0386 [M+H]+ (calcd. for C10H10N3O4S: 268.0387). 7-(2-Methoxyethoxy)-6-nitroquinazolin-4(3H)-one (4g): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 8.42 (1H, s), 8.14 (1H, s), 7.00 (1H, s), 4.27 (2H, t, JH-H = 4.5 Hz), 3.69 (2H, t, JH-H = 4.5 Hz), 3.32 (3H, s). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 170.8, 161.4, 156.6, 153.7, 136.0, 125.3, 116.2, 108.7, 70.4, 69.2, 58.8. HRESITOF-MS: m/z 264.0625 [M-H]- (calcd. for C11H10N3O5: 264.0626). All data were consistent with literature (Smaill et al. 2016; Mao et al. 2019). 7-(2-Ethoxyethoxy)-6-nitroquinazolin-4(3H)-one (4h): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.48 (1H, s), 8.49 (1H, s), 8.21 (1H, s), 7.44 (1H, s), 4.40 (2H, q, JH-H = 6.9), 3.75 (3H, t, JH-H = 6.9), 3.51 (2H, q, JH-H = 7.0), 1.11 (3H, t, JH-H = 7.0). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 160.0, 155.6, 153.4, 149.1, 139.0, 124.0, 115.7, 111.7, 70.3, 68.2, 66.2, 15.5. HRESITOF-MS: m/z 280.0931 [M+H]+ (calcd. for C12H14N3O5: 280.0928). 7-(2,3-Dihydroxypropoxy)-6-nitroquinazolin-4(3H)-one (4i): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.48 (1H, s), 8.50 (1H, s), 8.21 (1H, s), 7.42 (1H, s), 5.05 (1H, s), 4.71 (1H, s), 4.29 (1H, dd, JH-H = 10.3, 4.2 Hz), 4.21 (1H, dd, JH-H = 10.1, 5.5), 3.83 (1H, qi, JH-H = 5.4 Hz), 3.47 (1H, t, JH-H = 5.4 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 159.7, 155.5, 152.9, 148.7, Danova A et al.: Discovery of new acetylcholinesterase inhibitors derived from quinazolinones4 138.5, 123.7, 115.1, 111.1, 71.5, 69.5, 62.2. HRESITOF-MS: m/z 280.0579 [M-H]- (calcd. for C11H10N3O6: 280.0575). (S)-6-Nitro-7-((tetrahydrofuran-3-yl)oxy)quinazolin4(3H)-one (4j): 1H NMR (500 MHz, DMSO-d6) δH (ppm) 12.47 (1H, s), 8.50 (1H, s), 8.21 (1H, s), 7.41 (1H, s), 5.41 (1H, t, JH-H = 5.4), 3.95 (1H, dd¸ JH-H = 10.6, 4.4 Hz), 3.80 (3H, m), 2.31 (1H, sext, JH-H = 22.2, 14.2, 8.3 Hz), 2.03 (1H, qui, JH-H = 12.5, 5.7 Hz). 13C NMR (125 MHz, DMSO-d6) δC (ppm) 160.1, 154.3, 153.3, 149.3, 139.4, 124.3, 115.8, 112.2, 80.4, 72.4, 66.9, 32.8. HRESITOF-MS: m/z 276.0628 [M-H]- (calcd. for C12H10N3O5: 276.0626). All data were consistent with literature (Slobbe et al. 2014). Acetylcholinesterase (AChE) inhibition assay The cholinesterase inhibitory assay was performed using a microplate reader, modified from Ellman’s colorimetric method described by (Ingkaninan et al. 2006). Briefly, 25 μL of the sample, 50 μL of 50 mM Tris-HCl buffer, 125 μL of 3 mM DTNB, and 25 μL of 1.5 mM ATCI were added to the wells. Next, 25 μL of 0.3 U/mL AChE was added and incubated for 10 min at 25 °C. The reaction was measured at 405 nm using a BIOBASE microplate reader in triplicate. The percentage of AChE inhibition was calculated using the following equation: Inhibition (%) = 1-[Asample/Acontrol] × 100 Where Asample is the rate of the reaction of the samples, Acontrol is that of the negative control, and IC₅₀ was calculated using percentage inhibition versus logarithm of concentration with an online tool (https://www.aatbio. com/tools/ic50-calculator). Donepezil was used as a positive control. The IC₅₀ values were evaluated for significance using a one-way ANOVA in RStudio software, with a p-value below 0.05 considered statistically significant. A kinetic study was performed to determine the type of AChE inhibition by varying the concentrations of the substrate (0.01–0.15 mM) and inhibitors 4c (0, 1.5, 3.0 μM) and 4h (0, 3.5, 7.0 μM). The reaction time ran for 10 min, and the absorbance was read at 405 nm using a BIOBASE microplate reader in triplicate. The Km and Vmax values were calculated from reciprocal plots of 1/V versus 1/[S]. Molecular docking The molecular structures were drawn using ChemOffice Professional 15.0 and optimized with the Merck molecular force field (MMFF94). Due to the absence of the crystallographic structure of AChE (PDB ID: 4EY7) in the Protein Data Bank (https://www.rcsb.org/), AutoDock Vina in PyRx v.1.1 software was used for molecular docking (Trott and Olson 2010; Dallakyan and Olson 2015) with 32 for exhaustiveness and nine poses for each docked ligand. The binding site of AChE used a grid box with dimensions of 30 × 30 × 30 Å, located at x = −14.108, y = −43.833, and z = 27.669, as reported by (Nour et al. 2023). Docking validation was performed by redocking the native ligand, with an RMSD value less than 2.0 Å. The final step involved binding interaction analysis and visualizing the docking results in 2D and 3D using the BIOVIA Discovery Studio Visualizer. Results and discussion Synthesis of quinazolinones Our study successfully synthesized ten 7-alkoxy-6-nitroquinazolinones (4a–4j) from 4-fluoroanthranilic acid N NH O O2N ON NH O O2N ON NH O O2N ON NH O O2N O N NH O O2N O HO N NH O O2N O OH HO N NH O O2N O ON NH O O2N O O N NH O O2N O O N NH O O2N O HS 4a (83%) 4b (87%) 4c (67%) 4d (74%) 4e (92%) 4f (73%) 4g (96%) 4h (50%) 4i (72%) 4j (66%) N NH O O2N F 3 N NH O F 2 NH2 OH O F 1 ABC N NH O O2N RO 4a-j Scheme 1. A. formamidine acetate, C2H5OH, 80 °C, 16 h. B. H2SO4 98%, HNO3 65%, 110 °C, 2 h. C. Alcohol derivatives, NaOH, room temperature, or 80 °C for 2 h. Pharmacia 72: 1–10 5 through a three-step reaction (cyclic formation, nitration, and nucleophilic aromatic substitution), as presented in Scheme 1. In the first step, 4-fluoroanthranilic acid (1) was reacted with formamidine acetate in methanol at 80 °C for 16 h to form the cyclic ring of 7-fluoroquinazolinone (2). In the second step, 7-fluoroquinazolinone (2) was reacted with nitric acid in sulfuric acid at 110 °C for 2 h to yield 7-fluoro-6-nitroquinazolinone (3) as a precursor for the target compounds. In the last step, 7-fluoro-6-nitroquinazolinone (3) was reacted with several aliphatic alcohols by performing nucleophilic aromatic substitution between the fluor and alcohol groups, as shown in Scheme 1. Compounds 4a–4j were obtained with yields of 50–96%, including five new compounds (4c, 4d, 4f, 4h, 4i), as presented in Scheme 1. Compounds 4a–4d were synthesized from compound 3 and alcohol via nucleophilic aromatic substitution in the presence of NaOH as a base at room temperature for 2 h. However, compounds 4e–4j were produced from compound 3 with alcohol by heating at 80 °C for 2 h. This difference in conditions could be caused by the steric and nucleophilic properties of the alcohol derivatives. Biological activity All synthesized compounds (2, 3, 4a–4j) were tested against acetylcholinesterase (AChE) to calculate their inhibitory activity, as presented in Table 1. The results showed that seven compounds (4b, 4c, 4d, 4f, 4g, 4h, 4j) inhibited AChE by more than 70%. Furthermore, the IC₅₀ values of these seven compounds were measured to calculate the concentration required to inhibit 50% of the total AChE. Compounds 4c and 4h displayed strong inhibition with IC₅₀ values of 2.97 and 5.86 μM, respectively, compared with the other synthesized compounds, but all quinazolinones showed good inhibitory activity compared with donepezil as a drug standard. This finding suggested that compounds possessing hydrophobic moieties may strongly interact with residues in the binding pocket of AChE (Sugimoto 1999; Valasani et al. 2013). Hydrophobic interactions are important for the formation of enzyme– inhibitor complexes and affect the effectiveness of cholinesterase inhibition (Basova et al. 2013). Moreover, it has been reported that compounds containing hydrophobic aromatic rings have a high affinity for AChE (Mukhametgalieva et al. 2021). As shown in Table 1, compound 2 possessing a fluor atom at carbon 7 showed no inhibition against AChE, but the presence of a nitro group on compound 3 could inhibit AChE with an inhibition value of 40.5% at 50 μM. This finding showed that the presence of an electron-withdrawing group could be important to retain inhibitory activity. To further our investigation on compound 3, we initiated the introduction of the hydrophobic effect on the quinazolinone structure via nucleophilic aromatic substitution. Therefore, we synthesized compounds 4a–4j, as presented in Scheme 1. After evaluating compounds 4a– 4j, we found that hydrophobicity may significantly influence inhibitory activity against AChE. Compounds 4a–4c demonstrated that the longer the carbon chain used, the stronger the inhibitory activity against AChE, because of the more hydrophobic compounds. However, compound 4d had lower inhibitory activity than 4c. This result revealed that the propyl group is more hydrophobic than the isopropyl group. In addition, the isopropyl may provide steric hindrance, decreasing its inhibitory activity against AChE. Therefore, the presence of long carbon chains affects AChE inhibitory activity (Tharamak et al. 2023). Furthermore, compounds 4e and 4i, having hydroxy groups on the long chain, showed decreased inhibitory activity compared with 4b and 4c. In addition, the presence of thiol, methoxy, and ethoxy groups on compounds 4f, 4g, and 4h raised the inhibitory activity compared with 4e, because this was influenced by the hydrophobicity of each compound, as presented in Table 1. Compound 4j exhibited decreased activity compared with 4h due to the steric effect of the cyclic ether. Moreover, the introduction of a thiol group on compound 4f facilitates a disulfide bond with cysteine residues in the AChE enzyme (Sevier and Kaiser 2002). To further our investigation on the inhibition mechanism of the best inhibitors against acetylcholinesterase, a kinetic study was conducted on compounds 4c and 4h, as presented in Fig. 2. The inhibition mechanisms of acetylcholinesterase inhibitors were reported to act as competitive, noncompetitive, and mixed-type inhibitors (Heo et al. 2020; Zhang et al. 2023; Thi Duong et al. 2024). The results conveyed that compounds 4c and 4h inhibited acetylcholinesterase through a mixed-type mechanism that was a good competitive mode, with Ki values of 2.5 and 5.7 μM, respectively. This result demonstrated that compounds 4c and 4h could bind both to the free enzyme and to the enzyme–substrate complex (Shams and Ghazi 2019; Thi Duong et al. 2024). Moreover, a lower Ki value indicates a stronger binding affinity of the inhibitor to the enzyme (Ding et al. 2018; Chen et al. 2024). Thus, compound 4c demonstrated a stronger binding affinity compared to compound 4h. Table 1. Bbiological activity of compounds 2, 3, 4a-4j, and donepezil against acetylcholinesterase. Compound AChE inhibition (50 µM) IC50 (µM)aBinding affinity (kcal/mol) 2NIbNDc-7.3 340.5 ± 8.9 NDc-7.8 4a 57.8 ± 6.7 NDc-8.4 4b 84.5 ± 1.0 17.14 ± 1.63 -8.5 4c 90.5 ± 8.2 2.97 ± 0.02 -8.7 4d 86.7 ± 5.7 6.39 ± 0.24 -8.7 4e 59.7 ± 9.4 NDc-8.6 4f 79.2 ± 0.7 27.98 ± 3.29 -8.3 4g 71.2 ± 3.7 26.38 ± 3.72 -8.5 4h 89.2 ± 3.4 5.86 ± 0.44 -8.4 4i 46.3 ± 3.9 NDc-9.2 4j 75.5 ± 7.2 11.34 ± 1.36 -9.1 Donepezil 91.68 ± 4.92 0.02 ± 0.001 -12.2 aIC50 was averaged from the triplicate experiment. bNI = no inhibition. cND = not determined (inhibition less than 70%). Statistical significance: p-value < 0.05 compared with donepezil as a positive control. Danova A et al.: Discovery of new acetylcholinesterase inhibitors derived from quinazolinones6 A B Figure 2. Lineweaver–Burk plots and secondary plot (slope) of compounds 4c (A) and 4h (B). Molecular docking study Molecular docking was conducted to further evaluate the interaction profiles between the inhibitor and protein target. In this study, the crystal structure of the AChE protein in complex with donepezil was obtained from the Protein Data Bank (PDB ID: 4EY7; https:// www.rcsb.org/). Molecular docking was validated by redocking donepezil as a native ligand that fits the co-crystallized donepezil and its binding interaction, as presented in Fig. 3. Moreover, acetylcholinesterase has two types of binding sites, namely the catalytic anionic site (CAS) and the peripheral anionic site (PAS) (Dvir et al. 2010; Kermaninia et al. 2025). The catalytic anionic site (CAS) has three regions: the acyl pocket (Phe295 and Phe297); the choline (anionic) binding site (Trp86, Tyr133, Tyr337, and Phe338); the oxyanion hole (Gly120, Gly121, and Ala204); and the catalytic triad/esteratic site (Ser203, Glu334, and His447). Moreover, the peripheral anionic site (PAS) is surrounded by Tyr72, Asp74, Tyr124, Trp286, and Tyr341 (Asghar et al. 2024). The binding affinity of twelve synthesized compounds ranged from −7.3 to −9.2 kcal/mol, whereas donepezil showed a binding affinity of −12.2 kcal/mol. Moreover, this study showed that compounds with inhibition greater than 70% displayed binding affinities, such as −8.5 kcal/mol for 4b, −8.7 kcal/mol for 4c and 4d, −8.3 kcal/mol for 4f, −8.5 kcal/mol for 4g, −8.4 kcal/mol for 4h, and −8.4 kcal/mol for 4j, as presented in Table 1. The binding pose of donepezil showed interaction with several residues in the four locations of the binding sites of AChE, such as the peripheral anionic site (Tyr72, Trp286, and Tyr341), the acyl pocket (Phe295), the choline binding site (Trp86, Tyr337, and Tyr338), and the catalytic triad (His447). Compound 2 presented no inhibition at 50 μM (Table 1). This result was in line with its binding pose, as it interacted only with Tyr133 and Gly120 (see Suppl. material 1). Moreover, the incorporation of nitro and alkoxy groups on the phenyl ring appeared to confer inhibition at 50 μM, as in compounds 3, 4a–4d, 4f–4h, and 4j, except for compounds 4e and 4i (Table 1). According to the docking results, the binding poses of those active compounds (3, 4a–4d, 4f–4h, 4j) showed interaction with vital residues in the peripheral anionic site, choline binding site, and catalytic triad (see Suppl. material 1). However, compounds 4e and 4i were not active because of the polarity of their alkoxy groups, although both compounds interacted with residues in the binding pocket of AChE. Furthermore, as shown in Fig. 4, compound 4c displayed hydrogen bonds with four residues: Tyr124 with oxygen atoms (distance ~3.15 Å) in ether groups; Glu202 with hydrogen atoms in the NH group (distance ~3.09 Å); Tyr341 (distance ~2.96 and 3.13 Å); and Tyr337 (distance ~2.98 Å) with Pharmacia 72: 1–10 7 Figure 3. Validation by redocking donepezil. A. The co-crystallized ligand was modeled with a gray stick, and the redocked ligand was modeled with a yellow stick, with an RMSD value of 0.348 Å. B. Binding interaction of donepezil in complex with acetylcholinesterase (PDB ID: 4EY7). A B Figure 4. 2D and 3D interaction models of compounds 4c (1) and 4h (2) in the binding pocket of AChE (PDB ID: 4EY7). 1 2 Danova A et al.: Discovery of new acetylcholinesterase inhibitors derived from quinazolinones8 NO₂ groups. Compound 4h also formed three hydrogen bonds with three residues: Tyr124 with a NO₂ group (distance ~2.74 Å); Tyr133 with a C=O group (distance ~2.57 Å); and Glu202 with an NH group (distance ~2.15 Å). In addition, hydrophobic interactions were formed with four residues, namely Phe338, Trp86, Tyr341, and Tyr337. Furthermore, the interaction between a compound and the CAS component is crucial for achieving the inhibitory effect on AChE. Numerous studies have demonstrated that the PAS region of AChE can facilitate the polymerization of Aβ peptides, and inhibiting this region can disrupt the polymerization process of these peptides (De Ferrari et al. 2001; Pradhan et al. 2018). Thus, the interaction between ligand and residues in the CAS and PAS locations, as well as the hydrophobicity of the ligand, could be crucial to maintaining inhibitory activity against AChE. Conclusion Twelve synthesized compounds (2, 3, 4a–4j) were successfully obtained with moderate to high yields. Among these twelve compounds, two (4c, 4h) showed strong inhibition against acetylcholinesterase with IC₅₀ values of 3.2 and 3.8 μM and acted as mixed-type inhibitors with Ki values of 2.5 and 5.7 μM. Donepezil presented an IC₅₀ value of 0.02 μM. Molecular docking showed that compounds 4c and 4h had binding affinity scores of −8.7 and −8.4 kcal/mol, respectively. These compounds interacted with key residues in the CAS and PAS locations of AChE through hydrogen bonding and hydrophobic interactions. This finding revealed that quinazolinones possessing electron-withdrawing groups and hydrophobicity on the phenyl ring could be vital to facilitate inhibition of AChE in the CAS and PAS regions. Therefore, these quinazolinone derivatives are promising candidates as anti-acetylcholinesterase agents for further study. Acknowledgments The authors are grateful to the Ministry of Higher Education, Science, and Technology through contract number 338/IT1.B07.1/SPP-DRI/V/2025, 2 June 2025. We also thank the Integrated Chemistry Laboratory, Faculty of Mathematics and Natural Sciences, and Direktorat Penerapan Ilmu dan Teknologi Multidisiplin (DPITM), ITB, for instrumental support. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statements The authors declared that no clinical trials were used in the present study. The authors declared that no experiments on humans or human tissues were performed for the present study. The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study. The authors declared that no experiments on animals were performed for the present study. The authors declared that no commercially available immortalised human and animal cell lines were used in the present study. Use of AI No use of AI was reported. Funding This research was funded by the Ministry of Higher Education, Science, and Technology through contract number 338/IT1. B07.1/SPP-DRI/V/2025, 2 June 2025. Author contributions A.D.: writing – original draft, writing – review and editing; conceptualization, validation, investigation, methodology, formal analysis, software, resources, project administration, and data curation. A.C.C.: methodology, investigation, and formal analysis. I.M., F.K., D.M., and R.R.: writing – review and editing. E.H.: writing – review and editing; supervision, conceptualization, resources, software, project administration, and data curation. Author ORCIDs Ade Danova https://orcid.org/0000-0003-4716-1170 Angellyn Carnelian Christy https://orcid.org/0009-00014748-3543 Iqbal Musthapa https://orcid.org/0000-0002-0368-0270 Fera Kurniadewi https://orcid.org/0000-0002-3541-8103 Warinthorn Chavasiri https://orcid.org/0000-0001-5201-1324 Didin Mujahidin https://orcid.org/0000-0002-6903-8694 Robby Roswanda https://orcid.org/0000-0002-4160-7066 Elvira Hermawati https://orcid.org/0000-0002-5492-3316 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References ADl’S (2021) Dementia statistics. 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