ACCEPTED MANUSCRIPT This is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the Journal of the Serbian Chemical Society but has not yet been subjected to the editing process and publishing procedure applied by the JSCS Editorial Office. Please cite this article as O. Babatunde, S. Hameed, K. A. Mbachu, F. Saleem, S. Chigurupati, A. Wadood, A. Ur Rehman, V. Venugopal, K. M. Khan, M. Taha, O. Ekundayo, M A. Khan, J. Serb. Chem. Soc. (2023) https://doi.org/10.2298/JSC 211106005B This “raw” version of the manuscript is being provided to the authors and readers for their technical service. It must be stressed that the manuscript still has to be subjected to copyediting, typesetting, English grammar and syntax correc- tions, professional editing, and authors’ review of the galley proof before it is published in its final form. Please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the Journal. https://doi.org/10.2298/JSC211106005B https://doi.org/10.2298/JSC211106005B J. Serb. Chem. Soc.00(0)1-18 (2023) Original scientific paper JSCS–11370 Published DD MM, 2023 1 Evaluation of derivatives of 2,3-dihydroquinazolin-4(1H)-one as inhibitors of cholinesterases and their antioxidant activity: In vitro, in silico, and kinetics studies OLUWATOYIN BABATUNDE1, SHEHRYAR HAMEED1, KINGSLEY ADIBE MBACHU1,2, FAIZA SALEEM1, SRIDEVI CHIGURUPATI3, ABDUL WADOOD4, ASHFAQ UR REHMAN4, VIJAYAN VENUGOPAL5, KHALID MOHAMMED KHAN1,7* MUHAMMAD TAHA7, OLUSEGUN EKUNDAYO2 and MARIA AQEEL KHAN6 1H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan; 2Department of Chemistry, University of Ibadan, Nigeria; 3Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University, Buraydah-52571, Saudi Arabia; 4Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University, Mardan, Pakistan; 5Faculty of Pharmacy, AIMST University, Kedah-08100, Malaysia; 6Third World Center for Science and Technology, H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan and 7Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P. O. Box 1982, Dammam, 31441, Saudi Arabia (Received 6 November 2021; Revised 17 May 2022; Accepted 2 February 2023) Abstract: In search of potent inhibitors of cholinesterase enzymes and antioxidant agents, synthetic derivatives dihydroquinazolin-4(1H)-one 1-38 were evaluated as potential anti-Alzheimer agents through in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitions and radical (DPPH and ABTS) scavenging activities. The (SAR) was mainly based on the different substituents at the aryl part which showed a significant effect on the inhibitory potential of enzymes and radical scavenging activities. The kinetic studies of most active compounds showed a noncompetitive mode of inhibition for AChE and a competitive mode of inhibition for the BChE enzyme. Additionally, molecular modeling studies were carried out to investigate the possible binding interactions of quinazolinone derivatives with the active site of both enzymes. Keywords: quinazolinone; dual inhibitors; acetylcholinesterase; butyrylcholinesterase *Corresponding author E-mail: khalid.khan@iccs.edu, drkhalidhej@gmail.com; Tel.: +922134824910 https://doi.org/10.2298/JSC211106005B A cc ep te d m an us cr ip t mailto:khalid.khan@iccs.edu mailto:drkhalidhej@gmail.com https://doi.org/10.2298/JSC211106005B 2 BABATUNDE et al. INTRODUCTION In the central nervous system (CNS), one of the preeminent neurotransmitters is acetylcholine (ACh) which is related to memory and cognition. Insufficient ACh levels in the CNS can lead to diseases such as Alzheimer’s disease (AD).1 AD is the most common cause of dementia in elderly people and is characterized by several impaired cortical functions, including judgment, memory loss, comprehension, orientation, language deficit, and learning capacity.2 The predominant symptoms of all types of dementia are thought to be associated with the gradual decline of broad and compact cholinergic innervation of the human cerebral cortex. This decline contributes to the behavioral and cognitive deficits in AD and is also linked with the reduced levels of neurotransmitters, choline acetyltransferase, acetylcholinesterase (AChE), and ACh.3 AChE and butyrylcholinesterase (BChE) enzymes are hydrolytic enzymes that act on the neurotransmitter ACh by cleaving it into choline and acetate, thereby stopping their action in the synaptic cleft.4 Both enzymes are found in amyloid plaques and neurofibrillary tangles in the brain.5 AChE is the most important enzyme that regulates the level of acetylcholine in a healthy brain, while BChE plays an insignificant role. In AD patients, the AChE activity decreases, BChE activity increases and the ratio between AChE and BChE varies from normal to high levels (0.6-11) in the cortical regions of the brain that affect the disease.6,7 These observations lead to the concept of dual inhibition, and the most effective treatment approach has been suggested to increase ACh levels and limit cholinergic function by inhibiting AChE and BChE enzymes. Quinazolinones are extensively explored and are considered important as bioactive synthetic molecules for the development of novel therapeutic agents.8 Quinazolinone belongs to the N-containing fused heterocyclic compounds and is a quinazoline with a carbonyl group in the C4N2 ring. There are two isomers possible: 4-quinazolinone and 2-quinazolinone, however, the 4-quinazolinone isomer is more common.9 These compounds have raised universal concerns due to their broad and pronounced biopharmaceutical activities.10 Many substituted quinazolinones have a broad range of bioactivities such as antimicrobial, antimalarial, antifungal, antiprotozoal, anticancer, antiviral, anti-inflammatory, anti-tubercular, anticonvulsant, diuretic, acaricidal, muscle relaxant, antidepressant, weedicide, and many other biological activities.11 Quinazolinone compounds are also used in the syntheses of a variety of functional substances for synthetic chemistry and are also present in various drugs (Figure 1). 12 A cc ep te d m an us cr ip t SYNTHESIS OF 2,3-DIHYDROQUINAZOLIN-4(1H)-ONE AND THEIR DERIVATIVES 3 Figure 1. Pharmacological importance of quinazolinone-based drugs Antioxidant compounds exhibit an important part as a health protection factor.13 Free radicals are ions, atoms, or molecules possessing an unpaired electron such as hydroxyl, nitric oxide, and superoxide which are called reactive oxygen species (ROS) 14. ROS are generated in the human body and can damage DNA, proteins, and lipids thus may lead to different complications such as inflammation, toxicity, and carcinogenesis. Plants-derived antioxidants include carotenes, phytoestrogens, vitamin C, vitamin E, and phytates.15 Furthermore, chronic diseases which are life-limiting, such as diabetes, cancer, arteriosclerosis, AD, and aging, are developed by radical reactions.16 Natural or synthetic antioxidant compounds terminate the chain reactions by interacting with free radicals before essential molecules are damaged.17 Thus, the synthesis of new potent antioxidant compounds is of vital importance for rapidly quantifying the effectiveness of antioxidants in disease prevention. Our research group is continuously doing efforts in search of lead compounds for two decades to discover new enzyme inhibitors.18-21 previously, we have explored a large number of potent inhibitors based on quinazoline derivatives, including α-amylase, α-glucosidase,22,23 β-glucuronidase,24, and antileishmanial activities.25 These heterocycles are reported to possess various significant biological activities. Derivatives of dihydroquinazolin-4(1H)-one, in particular, has drawn more and more attention for synthesizing pharmaceuticals and in the field of agrochemicals. Herein we are going to report dihydroquinazolin-4(1H)- ones as a new class of inhibitors against acetylcholinesterase, butyrylcholinesterase enzymes, and with its antioxidant potential (Figure 2). A cc ep te d m an us cr ip t https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/heterocyclic-compound https://www.sciencedirect.com/topics/chemistry/agrochemical 4 BABATUNDE et al. Figure 2. Rationale of the current study In this study, dihydroquinazolin-4(1H)-ones 1-38 (Table I) have been reported as antioxidant agents and potent cholinesterase inhibitors which may improve clinical outcomes for developing anti-AD agents. Table I. In vitro acetylcholinesterase, butyrylcholinesterase activity, and antioxidant activity after using dihydroquinazolin-4(1H)-one derivatives 1-38 Comp. No. R AChE activity BChE activity DPPH radical activity ABTS radical activity IC50 ± SEMa / µM) 1 4-Cl (C6H4) 35.04 ± 0.20 37.13 ± 0.18 41.7 ± 0.06 42.97 ± 0.19 2 2-Cl (C6H4) 23.08 ± 0.03 26.08 ± 0.43 17.65 ± 0.23 19.47 ± 0.03 3 2,6-Cl (C6H3) 24.94 ± 0.12 27.13 ± 0.08 30.7 ± 0.06 32.97 ± 0.19 4 2,4-Cl (C6H3) 24.57 ± 0.07 27.57 ± 0.07 16.33 ± 0.02 18.01 ± 0.12 5 2-OH, 3,5-Cl (C6H2) 61.89 ± 0.12 67.91 ± 0.18 57.33 ± 0.02 58.01 ± 0.12 6 2-Cl, 6-NO2 (C6H3) NAb NAb 70.7± 0.06 71.97± 0.19 7 5-Cl, 2-OH (C6H3) 81.94 ± 0.12 82.13 ± 0.08 83.57 ± 0.17 83.68 ± 0.36 8 3,5-OCH3 (C6H3) NAb NAb 96.65 ± 0.03 94.47 ± 0.13 9 2,5-OCH3 (C6H3) 88.15 ± 0.12 87.15 ± 0.12 84.04 ± 0.02 85.99 ± 0.09 10 2,6-OCH3 (C6H3) 26.94 ± 0.12 27.99 ± 0.09 24.33 ± 0.02 25.01 ± 0.12 11 3,4-OCH3 (C6H3) 87.27 ± 0.18 86.08 ± 0.43 87.57 ± 0.08 89.27 ± 0.18 A cc ep te d m an us cr ip t SYNTHESIS OF 2,3-DIHYDROQUINAZOLIN-4(1H)-ONE AND THEIR DERIVATIVES 5 12 2-Br, 4,5-OCH3 (C6H3) 67.91 ± 0.18 69.02 ± 0.11 51.65 ± 0.03 52.47 ± 0.13 13 2,4-OCH3 (C6H3) 89.7 ± 0.16 85.97 ± 0.19 82.17 ± 0.14 82.01 ± 0.09 14 3,4,5-OCH3 (C6H2) Nab NAb 86.65 ± 0.23 87.47 ± 0.03 15 2,3,4-OCH3 (C6H2) NAb NAb 83.33 ± 0.02 85.01 ± 0.12 16 3-OC2H5, 4-OCH3 (C6H3) 27.57 ± 0.07 29.13 ± 0.18 30.04 ± 0.02 31.99 ± 0.09 17 3-OCH3, 4-OC2H5 NAb NAb 92.7± 0.06 94.97± 0.19 18 3,5-OCH3, 4-OH (C6H2) 87.27± 0.18 89.7± 0.16 83.46± 0.03 84.61± 0.11 19 4-Br, 3,5-OCH3 (C6H2) 83.08 ± 0.03 84.94 ± 0.12 76.33 ± 0.02 79.01 ± 0.12 20 4-F, 3-OCH3 (C6H3) 51.94 ± 0.12 53.33 ± 0.02 48.65 ± 0.23 49.47 ± 0.03 21 3-Br, 2-OCH3 (C6H3) 89.17 ± 0.16 88.33 ± 0.12 81.7 ± 0.06 85.97 ± 0.19 22 2-F, 4-OCH3 (C6H3) 27.91 ± 0.18 29.02 ± 0.11 31.33 ± 0.12 32.01 ± 0.12 23 2-Cl, 3-OCH3 (C6H3) 88.15 ± 0.12 87.13 ± 0.12 83.04 ± 0.02 84.99 ± 0.09 24 3-OC2H5, 2-OH (C6H3) 61.01 ± 0.17 64.57 ± 0.07 49.84 ± 0.03 52.71 ± 0.11 25 2-OCH2(C6H5) (C6H4) NAb NAb 72.7 ± 0.06 74.97 ± 0.19 26 3-OCH2(C6H5) 4-OCH3 (C6H3) NAb NAb 88.89 ± 0.10 89.09 ± 0.09 27 4-OCH2(C6H5) (C6H4) NAb NAb 84.89 ± 0.20 89.09 ± 0.19 28 4-Br (C6H4) 25.33 ± 0.02 26.27 ± 0.18 27.33 ± 0.02 28.01 ± 0.12 29 4-CF3 (C6H4) NAb NAb 92.13 ± 0.08 92.79 ± 0.17 30 2-Thiophene 43.08 ± 0.03 46.08 ± 0.43 47.65 ± 0.23 49.47 ± 0.03 31 3-Bromo, 4-OH (C6H3) 85.33 ± 0.02 87.47 ± 0.13 83.01 ± 0.07 83.11 ± 0.15 32 4-OCH3, 3-OH (C6H3) 77.27 ± 0.18 75.04 ± 0.52 71.7 ± 0.06 72.97 ± 0.19 33 3-OH (C6H4) 47.17 ± 0.15 48.15 ± 0.12 42.33 ± 0.12 43.01 ± 0.12 34 2-OH (C6H4) 27.57 ± 0.07 29.02 ± 0.11 28.46 ± 0.03 30.71 ± 0.11 35 4-OH (C6H4) 37.7 ± 0.16 38.94 ± 0.12 39.7 ± 0.16 40.97 ± 0.14 36 3,4-OH (C6H3) 45.04 ± 0.52 47.7 ± 0.16 48.46 ± 0.03 52.71 ± 0.11 37 2,5-OH (C6H3) 77.33 ± 0.02 79.7 ± 0.16 76.65 ± 0.03 77.47 ± 0.13 A cc ep te d m an us cr ip t 6 BABATUNDE et al. 38 2,3-OH (C6H3) 81.94 ± 0.12 83.33 ± 0.02 82.7 ± 0.06 84.97 ± 0.19 Standard= Asc. Acidc 15.08 ± 0.03 16.09 ± 0.17 Standard = Donepezild 15.08 ± 0.03 15.08 ± 0.03 SEMa (Standard error of the mean); NAb (Not Active); Ascorbic acidc (Standard for DPPH and ABTS activities); Donepezild (Standard for AChE and BChE inhibitions). EXPERIMENTAL Materials and methods All enzymes were purchased from Sigma-Aldrich and used without further purification. The acetylcholinesterase enzyme from Electrophorus electricus (electric eel) supplied by Sigma-Aldrich (GmbH, USA) whereas butyrylcholinesterase from equine serum procured from Sigma-Aldrich, SRE020, Missouri, USA); 5,5-dithio-bis-nitrobenzoic acid (DTNB), acetylthiocholine iodide 99 % (ATChI), donepezil hydrochloride was obtained from Sigma-Aldrich (United Kingdom). All reagents were purchased from Merck (Germany) and Sigma-Aldrich (USA). Thin-layer chromatography was carried out on precoated silica gel, GF-254 (Merck, Germany). Spots were visualized under ultraviolet light at 254, 366 nm or iodine vapors. EI- and HREI-MS spectra were recorded on MAT 312 and MAT 113D mass spectrometers. The 1H-, 13C-NMR were recorded on Bruker AM spectrometers, operating at 300 and 400 MHz. The chemical shift values are presented in ppm (δ), relative to tetramethylsilane (TMS) as an internal standard, and the coupling constant (J) is in Hz. Cholinesterase enzyme activity The in vitro AChE and BChE inhibitory activity were measured using the methods described earlier.26 Briefly, stock solutions (1 mg/mL) of test compounds were prepared using 0.01 % DMSO. Working solutions (0.01 – 100 μg/mL) were prepared by serial dilutions. The various concentrations of test compounds (10 μL) were pre-incubated with sodium phosphate buffer (0.1 M; pH 8.0; 150 μL); AChE solution/ BChE (0.1 U/mL; 20 μL) for 15 min at 25 ˚C and addition of DTNB (10 mM; 10 μL). The reaction was initiated by the addition of ATChI (14 mM; 10 μL). The reaction mixture was mixed using a cyclomixer and incubated for 10 min at room temperature. The absorbance was measured using a microplate reader at 410 nm wavelength against the blank reading containing 10 μL DMSO instead of the test compound. The inhibition was calculated using the formula described in Eq. (1) and the IC50 was calculated. Donepezil (0.01–100 μg/mL) was used as the positive control. Inhibition= ((1-absorbance sample)/absorbance control)100 (1) A cc ep te d m an us cr ip t SYNTHESIS OF 2,3-DIHYDROQUINAZOLIN-4(1H)-ONE AND THEIR DERIVATIVES 7 Kinetic study assay In derivatives of 2,3-dihydroquinazolin-4(1H)-one, seven compounds 2, 3, 4, 10, 16, 28 and 34 were selected for kinetic studies, based on their lower IC50 values (23.08 to 27.57 μM). In kinetic studies, we used acetyl thiocholine iodide (ATCI)/butyrylthiocholine iodide (BTCI) as a substrate at various concentrations (0.175, 0.35, 0.7, and 1.40 mM) and different concentrations of AChE/BChE inhibitors (0, 0.625, 1.25 and 2.5 μM) were used. Enzyme inhibition kinetic mechanisms were determined by using Sigma Plot 14.0 software. The rate of substrate and inhibitor reactions was calculated. Based on the rate of reactions, the software showed the type of enzyme kinetics mechanism. Kinetic studies have shown all the compounds followed as non-competitive type inhibitors (Table I). The types of inhibition of AChE/BChE were determined by Lineweaver Burk plots. The reciprocal of the rate of the reaction was plotted against the reciprocal of substrate concentration to monitor the effect of the inhibitor on both km (Michaelis constant the substrate concentration at which the reaction rate is 50% of the Vmax) and Vmax (In enzyme kinetics, Vmax is the maximum velocity of an enzymatically catalyzed reaction when the enzyme is saturated with its substrate. values. Radical scavenging assay DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical activity Preparation of the DPPH solution was adopted from Molyneux 27 and Blois 28 with minor modifications. All the test compounds were dissolved in 95 % ethanol. Briefly, 0.5 mL of test compounds were added (0 - blank control, 10, 25, 50, 100, 250, 500 and 1000 g/mL) to 0.5 mL of DPPH (2 µM in 95 % ethanol) and the mixture was incubated at room temperature for 30 min. The absorbance was measured at 517 nm,29 and the percentage inhibition of test compounds was calculated using the following equation using Microsoft Excel software (version 2010). Ascorbic acid was used as the positive control. Scavenging = ((1-absorbance sample)/absorbance control)100 (2) The IC50 (half maximal inhibitory concentration) was calculated by constructing a non-linear regression graph between inhibition vs. concentration, using Graph Pad Prism software (version 5).34 ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) free-radical cation activity The ABTS free radical cation scavenging ability of the synthesized compounds was determined according to the procedure described earlier.30 ABTS was dissolved in distilled water (7 mM) and potassium persulphate (2.45 mM) was added. This reaction mixture was left overnight (12 to 16 h) in the dark, at room temperature. Various concentrations of test substances (1000, 500, 250, 100, 50, 25, and 10 µg/mL) were incubated with the ABTS+ solution for 30 min. The absorbance was measured at 734 nm, the inhibition was calculated using the A cc ep te d m an us cr ip t 8 BABATUNDE et al. formula described in Eq. (1) and the IC50 was calculated. Ascorbic acid was used as the positive control. Molecular docking protocol Acetylcholinesterase and butyrylcholinesterase Molecular docking (MD) was performed using Molecular Operating Environment (MOE)31 to explore the binding mode of the synthetic compounds against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. First, the 3D structures for all the compounds were generated using the MOE- builder module. Next, the compounds were protonated, and energy was minimized using the default parameters of the MOE. The structural coordinates for AChE and BChE were retrieved from the protein databank (PDB code; 1acl and 1p0p). All the structure was subjected to MOE for preparation. Further, the protonation was done using the default parameters of the structure preparation module of MOE. Next, the energy was minimized for both coordinates to get minimal energy conformation. Finally, refined structures were used for the docking study using the default parameters of MOE. Before running the docking protocol, we selected a total of often conformations for each compound. The top-ranked conformations based on docking score (S) were selected for protein-ligand interaction (PL) analysis. RESULTS AND DISCUSSION Chemistry Dihydroquinazolin-4(1H)-ones 1-38 were synthesized by treating isatoic anhydride, substituted aldehyde, and aniline under reflux for 3 h. The reaction was carried out in acetic acid as a solvent at 80-90 °C in Scheme 1. After reaction completion, it was cooled to room temperature. The solution was added to ice water to form a precipitate. The mixture was filtered, and the crude product was washed continuously with an excess of water. The obtained crude product was washed with different solvents to remove impurities, on crystallization from ethanol gave the corresponding pure products having 60-85 % yields.23 Molecular structures of all compounds 1–38 were identified by EI-MS, HREI-MS, 1H-, and 13C-NMR. Scheme 1. Synthesis of dihydroquinazolin-4(1H)-ones 1-38 A cc ep te d m an us cr ip t SYNTHESIS OF 2,3-DIHYDROQUINAZOLIN-4(1H)-ONE AND THEIR DERIVATIVES 9 In vitro AChE, BChE inhibitions, and antioxidant activities: All synthetic dihydroquinazolin-4(1H)-ones 1-38 were screened for in vitro acetylcholinesterase and butyrylcholinesterase inhibitions, and antioxidant activities. All compounds exhibited good to moderate inhibitory activities in the range of IC50 values 23.08-89.7 and 26.01-89.7 µM against AChE, and BChE inhibitions and 16.33-96.65 and18.01-94.97 µM against DPPH and ATBS activities when compared to the donepezil (IC50 = 15.08 ± 0.07 µM) and ascorbic acid as the standards (IC50 = 15.08 ± 0.07 and 16.09 ± 0.17 µM), respectively (Table I). The structure-activity relationship proposed that all structural features such as benzene ring, carbonyl group, quinazoline moiety, phenyl ring, and aryl ring “R” are taking part in the activity, and due to the presence of different groups “R” at the aryl part significant fluctuation in the activity was observed (Figure S- 1 in Supplementary Material). SAR for AChE and BChE inhibitions and antioxidant activities Structure-activity relationship (SAR) was discussed for all synthetic compounds which were screened for in vitro acetylcholinesterase, butyrylcholinesterase inhibitions, and antioxidant (DPPH and ABTS) activities. Structure-activity relationship (SAR) for AChE and BChE inhibitory activities Compounds 1-7, 28, and 29 were halogen-substituted including F, Cl, and Br. These compounds displayed inconsistent inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Of these, compound 2 with the ortho-chloro substitution was found to be the most potent AChE and BChE inhibitor with IC50 values of 23.08 ± 0.03, and 26.08 ± 0.43 µM, respectively. A comparison of the inhibitory activities of compound 2 and compound 1 showed a positional effect on the inhibitory potential. Such in compound 1 the presence of chloro group at para-position reduces the inhibitory activity as shown by the IC50 values 35.04 ± 0.20 µM for AChE and 37.13 ± 0.18 µM for BChE enzymes. Correspondingly, in compounds 3 (IC50 = 24.94 ± 0.12, 27.13±0.08 µM) and 4 (IC50 = 24.57 ± 0.07, 27.57 ± 0.07µM), a slight decrease in the inhibitory potential was seen by the addition of chloro groups at the ortho-, para- and di-ortho-positions against AChE and BChE enzymes, respectively. However, the presence of chloro groups in compounds 5-7, along with other groups such as NO2 and OH, demonstrated lower potential against AChE and BChE enzymes. Para-Bromo substituted compound 28 (IC50 = 25.33 ± 0.02, 26.27 ± 0.18 µM), exhibited pronounced activity against both AChE and BChE enzymes, respectively. However, compound 29 with trifluoromethyl substitution was found to be inactive against both enzymes which indicates that the trifluoromethyl group is not actively involved in the binding interaction to the active site of the enzyme (Figure S-2). A cc ep te d m an us cr ip t 10 BABATUNDE et al. In a recently published quinazolinone derivative 33, cholinesterase inhibitors showed superior inhibitory activity compared to the standard drug tacrine. Among them, halogenated compounds showed potential activity against AChE and BChE enzymes. These results showed similarity with our work in which halogenated compounds showed potential activities as compared to the standard donepezil. It has been found that the incorporation of methoxy substitutions in compounds 8-17 at different positions of aryl moiety (R) has a varying degree of inhibition. Among them, ortho-dimethoxy substituted compound 10 was found significantly active with IC50 = 26.94 ± 0.12, 27.99 ± 0.09 µM for AChE and BChE enzymes as compared to its ortho, meta-dimethoxy derivative compound 9. Surprisingly, it was found that its meta-dimethoxy substituted positional isomer 8 was completely inactive. There might be a possibility that compound 8 attained such a conformation that does not fit well into the active site of the enzyme. However, when methoxy groups are present at the adjacent positions in compound 11 (IC50= 87.27 ± 0.02, 86.08 ± 0.43 μM) a noticeable decline in the activity was observed as compared to compound 12 (IC50= 67.91 ± 0.18, 69.02 ± 0.11 μM), where an additional Bromo group is present at ortho-position. The positional isomer of 11 i.e., compound 13 demonstrated weak inhibitory potential against both enzymes. In the case of trimethoxy substituted derivatives (compounds 14 and 15), a complete loss of activity was observed. This might be due to the steric hindrance and bulkiness of the groups. Compounds 16 with para-methoxy and meta-ethoxy substitutions displayed considerable inhibitory potential with IC50 = 27.57 ± 0.07; 29.13 ± 0.18 μM against acetylcholinesterase and butyrylcholinesterase enzymes, respectively. In contrast, compound 17 was found to be inactive against both enzymes (Figure-S-3). Compounds 18-24 and 32 with the combinations of ethoxy/methoxy and other substitutions such as OH, Cl, F, and Br, exhibited moderate inhibition activities against both enzymes. ortho-Fluoro and para-methoxy substituted compound 22 was found to have relatively good activity in comparison to its other positional analogs. Compound 18 displayed IC50 values of 27.91 ± 0.18, and 27.91 ± 0.18 µM against AChE and BChE enzymes, respectively. In contrast, its positional isomer (compound 20) exhibited low inhibitory potential with IC50 = 51.41 ± 0.12, 53.33 ± 0.02 µM against acetylcholinesterase and butyrylcholinesterase enzymes. The activity of the combination of Cl, Br, and OH with methoxy-substituted compounds 18, 19, 21, 23, 24, and 32, displayed moderate to weak inhibitory activities which indicates that these groups are creating steric hindrance and less binding interaction in the enzyme’s active site or their positive mesomeric effect is negatively contributing in the activity (Figure S-4). Surprisingly, ortho, meta, and para benzyloxy substituted derivatives 25-27 were found to be inactive against acetylcholinesterase and butyrylcholinesterase enzymes. It might be due to bulky groups that do not favorably fit in the active site A cc ep te d m an us cr ip t SYNTHESIS OF 2,3-DIHYDROQUINAZOLIN-4(1H)-ONE AND THEIR DERIVATIVES 11 of the enzyme, which displayed that the presence of hydrophobic groups on the aryl part more specifically the presence of the benzyloxy group, resulted in the loss of activity profile of compounds 25, 26 and 27, respectively. Exceptionally, thiophene-substituted analog 30 showed moderate activity against AChE and BChE enzymes with IC50 values of 43.08 ± 0.03 and 46.08 ± 0.43 µM, respectively (Figure S-5). Mono-hydroxyl substituted compounds 31-35 showed good to moderate results against acetylcholinesterase and butyrylcholinesterase enzymes. The activity of five hydroxy-substituted derivatives such as 31-35 was different from each other against both enzymes. However, the structure of all five derivatives is very similar to each other but differ only in the position of hydroxyl at aryl part “R”. Amongst them, compound 34 (IC50 = 27.57 ± 0.07, 29.02 ± 0.11 µM) has ortho-hydroxyl group exhibited better activity against AChE and BChE enzymes as compared with compounds 33 and 35, respectively, which indicate that groups and position displayed significant role in the enzyme inhibition. However, compounds 31 and 32 with the combination of Bromo and methoxy with a hydroxyl group, respectively, exhibited weak inhibitory activities against AChE and BChE enzymes. This activity pattern demonstrated the involvement of di- substituted hydroxy compounds 36-38, which also displayed moderate to weak inhibitory activities. Compound 36 (IC50 = 45.04 ± 0.52, 47.7±0.16 µM) with meta, para di-hydroxy substitution showed better activity as compared to compounds 37 and 38 against acetylcholinesterase and butyrylcholinesterase enzymes (Figure S- 6). SAR for DPPH and ABTS radical scavenging activities Based on (SAR), the variations observed in DPPH and ATBS activities of quinazolinones 1-38 were discussed and compared against standard ascorbic acid with IC50 = 15.08 ± 0.03 and 16.09 ± 0.17 µM, respectively. Dichloro-substituted compound 4 showed DPPH (IC50 = 16.33 ± 0.02 µM) and ABTS (IC50 =18.01 ± 0.12 µM) radical scavenging activities, respectively, and was found to be most active in the series. Its positional isomer (compound 3) displayed a decline in activity against both radicals. However, mono-substituted compound 2 having chloro group at meta position (IC50 = 17.65 ± 0.23, 19.47 ± 0.03 µM), showed better DPPH and ABTS radical scavenging activities as compared to its positional isomer 1. Antiradical activity depends on proton and electron transfer between the radical and the scavenging agent. Here 1,4 disubstituted chloro compounds seem to involve electron transfer and free radical scavenging compared to monosubstituted and 1,3 disubstituted chloro compounds. The addition of hydroxyl and nitro substitution at the aryl ring in compounds 5, 6, and 7, respectively, showed moderate to weak potential against DPPH and ABTS radical scavenging activities. The activity of di-methoxy substituted compounds 8, 9, and 11-13 showed a further decrease in the activity as compared to ortho-dimethoxy substituted compound 10 which showed enhanced DPPH and ABTS radical A cc ep te d m an us cr ip t 12 BABATUNDE et al. scavenging activities. The addition of the methoxy group in compounds 14 and 15 further reduced the activity (Figure S-7). In the case of compound 16 (IC50 = 30.04 ± 0.02, 31.99 ± 0.99 µM) para-methoxy and meta-ethoxy groups showed better activities as compared to compound 17 (Figure S-7). Another combination of methoxy with OH, Br, F, and Cl substitutions in compounds 18-23 showed weak potential against DPPH and ATBS activities. Compounds 25, 26, and 27 bearing benzyloxy substitution displayed decreased radical scavenging activities against DPPH and ATBS. The incorporation of the Bromo group as “R” in compound 28 with IC50 values 27.33 ± 0.02, 28.01 ± 0.12 µM, showed better potential than compound 31. Mono-hydroxy and di-hydroxy substituted compounds 32, 33, 35, and 38 demonstrated good potential against DPPH and ABTS radical scavenging activities as compared to compound 34. Compounds 23, 29, and 30 showed a further decline in the activities as compared to the standard ascorbic acid (Figure S-7). Kinetic studies on acetylcholinesterase inhibitors Kinetic studies on the most active AChE enzyme inhibitors (compounds 2-4, 10, 16, 28, and 34) were analyzed to interpret the enzyme inhibition mechanisms by using graph fitting analysis in the Sigma-Plot enzyme kinetic software (Figures S-8A-B). In 2,3-dihydroquinazolin-4(1H)-ones all the seven compounds (2, 3, 4, 10, 16, 28, and 34) Vmax and Km (Michelis-Menton constant) were in the range of 60.5 to 79.8 (µM/min)/mg and 3.0 to 3.6 mM respectively (Figure S-8A). The Ki (Dissociation constant) values were confirmed from the Dixon plot by plotting the reciprocal of the rate of reaction against different concentrations of compounds, where Ki values of all eight compounds were in the range of 5.0 to 5.9 µM (Figure S-8B). In the uncompetitive type of inhibition, only Vmax values are affected, and no changes in the Km value. The low Vmax and no effect in the Km value of these compounds indicated an uncompetitive type of inhibition (Table II). Table II. Kinetic studies of active compounds for acetylcholinesterase inhibition (uncompetitive type of inhibition) Compound No Vmax (µM/min)/mg Km / mM Ki / µM 2 79.8 ± 1.2 3.2 ± 0.01 5.2 ± 0.1 3 70.4 ± 1.0 3.6 ± 0.02 5.4 ± 0.2 4 60.5 ± 2.2 3.0 ± 0.01 5.5 ± 0.5 10 66.8 ± 1.8 3.3 ± 0.02 5.8 ± 0.1 16 71.0 ± 1.2 3.1 ± 0.01 5.0 ± 0.2 28 65.4 ± 1.0 3.2 ± 0.02 5.3 ± 0.1 34 53.2 ± 2.2 3.4 ± 0.01 5.9 ± 0.2 Donepezil 62.0 ± 1.0 3.0 ± 0.01 5.1 ± 0.1 A cc ep te d m an us cr ip t SYNTHESIS OF 2,3-DIHYDROQUINAZOLIN-4(1H)-ONE AND THEIR DERIVATIVES 13 Kinetic studies on butyrylcholinesterase inhibition Kinetic studies on the most active AChE enzyme inhibitors compounds 2-4, 10, 16, 28, and 34 were analyzed to interpret their inhibition mechanisms (Figure S-9). In 2,3-dihydroquinazolin-4(1H)-ones the Vmax and Km of all the seven com- pounds were in the range of 80.3 to 85.4 (µM/min)/mg and 3.1 to 31.8 mM respectively (Figure S-9A). The Ki values were confirmed from the Dixon plot by plotting the reciprocal of the rate of reaction against different concentrations of compounds, where Ki values of all the five compounds were in the range of 10.3 to 10.9 µM (Figure S-9B). In the competitive type of inhibition, only km values are affected and there are no changes in the Vmax value. The high km and no effect in the Vmax of these compounds indicated a competitive type of inhibition (Table III). Table III. Kinetic studies of active compounds for butyrylcholinesterase inhibition (competitive type of inhibition) Compound No Vmax (µM/min)/mg Km / mM Ki / µM 2 82.0 ± 2.2 3.1 ± 0.2 10.6 ± 0.5 3 80.3 ± 2.7 9.2 ± 0.1 10.4 ± 0.3 4 82.2 ± 5.3 20.2 ± 0.2 10.7 ± 0.2 10 85.4 ± 1.2 2.1 ± 0.1 10.6 ± 0.4 16 82.0 ± 1.4 3.7 ± 0.2 10.4 ± 0.1 28 84.1 ± 2.4 31.8 ± 0.1 10.3 ± 0.1 34 82.5 ± 2.9 4.7 ± 0.1 10.9 ± 02 Donepezil 80.1 ± 1.6 13.5 ± 0.1 10.2 ± 0.1 Molecular docking studies AChE and BChE molecular docking study MD was performed to explore the binding mode of the synthesized compounds against the targeted enzyme (AChE and BChE). MD results are in good agreement with experimental results. We have noticed that compounds bearing the electron- withdrawing groups (EWGs) showed the best inhibitory activity against both targets. Interestingly, as compared with the other activity (α-amylase and α-glucosidase),23 we have noted that the compounds bearing 1,3-dichlorobenzene showed high inhibitory potency as compared to 1-chlorobenzene. Similarly, the following compounds showed invert phenomena in the activity against both targets. Those compounds bearing 1-chlorobenzene/1-bromobenzene substitution were found to be active. The PLI profile was enlisted for all docked compounds in Tables S-I and S- II in the Supplementary Material. A cc ep te d m an us cr ip t 14 BABATUNDE et al. Acetylcholinesterase (AChE) molecular docking study The docking results for most active compound 2 against AChE revealed that the 3-methyl-tetrahydro pyrimidine-4(1H)-one moiety of the compound adopted several favorable interactions with catalytic residues (Figure 3A surface repre- sentation) including acidic residue Glu72, hydrophobic side chain Tyr334, Trp279, and Phe331, respectively (Figure 3B). Figure 3A-C. The PLI profile for synthesized compounds against the acetylcholinesterase (AChE) enzymes. (A) The surface representation of the enzyme, (B) The binding mode of the most potent compound 2 in the series, and (C) compound 4. A double-sided arrow represented the π-stacking The reason for high potency might include the high number of adopted favorable interactions with catalytic residues. In the case of the 2nd ranked active compound 4, where the substitution groups are the 2,3-dichloro, a similar interaction was observed. But the only difference so far found is: the active compound adopted π-stacking interaction with the 1-Chloro moiety, whereas it lacks in the 2nd active compound (Figure 3C). This might be one of the reasons for reduced activity in compound 4. The PLI profile was enlisted for all docked compounds in (Table S-II). Butyrylcholinesterase (BChE) molecular docking study In the case of the docking results for most active compounds against BChE (Figure 4A), activity revealed that the compound bearing electron-withdrawing groups (EWG), i.e. 1-chlorobenzene (Figure 4B) and 1-bromobenzene (Figure-4C), showed best inhibitory activity against the BChE enzyme. The protein-ligand interaction (PLI) profile for the most active compound 2 and 2nd ranked active compound 28 revealed an interesting observation that both the compound shared similar interaction with the hydrophobic residue Phe329. A cc ep te d m an us cr ip t SYNTHESIS OF 2,3-DIHYDROQUINAZOLIN-4(1H)-ONE AND THEIR DERIVATIVES 15 Figure 4A-C. The PLI profile for synthesized compounds against the butyrylcholinesterase (BChE) enzymes. (A) The surface representation of the enzyme, (B) The binding mode of the most potent compound 2 in the series, and (C) Compound 28. The π-stacking was represented by a double-sided arrow More interestingly, the most active compound 2 adopted interaction with the acidic residue Glu70 while compound 28 with Glu197, which suggested that might be these two residues play a vital role in enhancing the enzymatic activity. The hydrophobic residue Trp82, which is an active residue in the active site and plays a vital role in the enzymatic activity, adopted two π-stacking interactions with the substituted benzene ring while the compound 28 does not attempt to adapt interaction even though this residue is found in proximity with the 6-ring of the compound. Overall, these results describe that the compounds bearing the EWG either at ortho- or meta-position displayed good inhibitory potential against the enzyme while others bearing both ortho- and meta- or ortho- and para-positions showed less activity. The PLI profiles were enlisted for all docked compounds in (Table S-II). CONCLUSION In the present study, compounds showed moderate to good inhibition against AChE, BChE, and antioxidant activities as compared with the standards donepezil and ascorbic acid, respectively. A structure-activity relationship was also established. In silico modeling studies revealed the binding mode of the quinazolinone derivatives. The kinetic studies on the seven most active compounds 2, 3, 4, 10, 16, 28, and 34 were carried out. The compounds 2, 3, 4, 10, 16, 28, and 34 were found to have an uncompetitive mode for acetylcholinesterase enzyme and the compounds 2, 3, 4, 10, 16, 28, and 34 were found to be the competitive mode for butyrylcholinesterase enzymes. Acknowledgments: The authors acknowledge the financial support of the Sindh Higher Education Commission (SHEC), Pakistan vide letter No. NO.DD/SHEC/1-14/2014, Project Code SHEC/SRSP/Med-3/15/2021-21. A cc ep te d m an us cr ip t 16 BABATUNDE et al. SUPPLEMENTARY MATERIAL Additional data are available electronically at the pages of the journal website: https://www.shd-pub.org.rs/index.php/JSCS/article/view/11370, or from the corresponding author on request. ИЗВОД ИЗУЧАВАЊЕ ДЕРИВАТА 2,3-ДИХИДРОХИНАЗОЛИН-4(1H)-ОН КАО ИНХИБИТОРА ХОЛИНЕСТЕРАЗА И ЊИХОВЕ АНТИОКСИДАТИВНЕ АКТИВНОСТИАКТИВНОСТИ: In vitro, in silico, И КИНЕТИЧКА ИСПИТИВАЊА OLUWATOYIN BABATUNDE1, SHEHRYAR HAMEED1, KINGSLEY ADIBE MBACHU1,2, FAIZA SALEEM1, SRIDEVI CHIGURUPATI3, ABDUL WADOOD4, ASHFAQ UR REHMAN4, VIJAYAN VENUGOPAL5, KHALID MOHAMMED KHAN1,7, MUHAMMAD TAHA7, OLUSEGUN EKUNDAYO2 и MARIA AQEEL KHAN6 1H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan; 2Department of Chemistry, University of Ibadan, Nigeria; 3Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University, Buraydah-52571, Saudi Arabia; 4Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University, Mardan, Pakistan; 5Faculty of Pharmacy, AIMST University, Kedah-08100, Malaysia; 6Third World Center for Science and Technology, H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan и 7Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam, 31441, Saudi Arabia Током истраживања нових активних инхибитора холинестераза и антиоксидативних агенаса, испитивани су синтетички деривати дихидрохиназолин-4(1H)-он 1-38 као потен- цијални агенси за третман Алцхајмерове болести инхибицијом ацетилхолин естеразе (AChE), бутирлихолин естеразе (BChE) и као хватачи слободних радикала (DPPH и ABTS). Доминантан утицај на инхибицију ензима и способност хватања слободних радикала имају супституенти на ароматичном језгру. На основу резултата испитивања кинетике закључено је да једињења делују некопетентивним механизмом инхибиције. Молекулским моде- ловањем су испитане могуће интеракције током везивања киназолинских деривата у активним местима оба ензима. (Примљено 6. новембра 2021; ревидирано 17 маја 2022; прихваћено 2. фебруара 2023.) REFERENCES 1. S. Kumar, D. S. Brijeshlata, S. Dixit, Int. J. Pharm. a Bio. 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