New 3-O-substituted xanthone derivatives as promising acetylcholinesterase inhibitors

Abstract

A new series of 3-O-substituted xanthone derivatives were synthesised and evaluated for their anti-cholinergic activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The results indicated that the xanthone derivatives possessed good AChE inhibitory activity with eleven of them (5, 8, 11, 17, 19, 21-23, 26-28) exhibited significant effects with the IC₅₀ values ranged 0.88 to 1.28 µM. The AChE enzyme kinetic study of 3-(4-phenylbutoxy)-9H-xanthen-9-one (23) and ethyl 2-((9-oxo-9H-xanthen-3-yl)oxy)acetate (28) showed a mixed inhibition mechanism. Molecular docking study showed that 23 binds to the active site of AChE and interacts via extensive π–π stacking with the indole and phenol side chains of Trp86 and Tyr337, besides the hydrogen bonding with the hydration site and π–π interaction with the phenol side chain of Y72. This study revealed that 3-O-alkoxyl substituted xanthone derivatives are potential lead structures, especially 23 and 28 which can be further developed into potent AChE inhibitors.

Introduction

Dementia is a syndrome of deterioration in memory, thinking, orientation, comprehension, calculation, learning capacity, language, judgement, and the ability to perform daily activities. An estimated number of 50 million people worldwide are living with dementia and the number is speculated to rise to 75 million in 2030 and 131.5 million in 2050 due to an increase of the aged population¹. Furthermore, the current costs associated with dementia are tremendous with approximately 1 trillion US dollars and it is estimated to rise to 2 trillion US dollars by year 2030¹. Alzheimer’s disease (AD) was reported to account for 60–80% of all cases of dementia, besides Lewy body dementia, frontotemporal disorders and vascular dementia in 2019 Alzheimer’s disease facts and figures report². AD is commonly affecting the elderly as the occurrence of AD doubles every five years beyond the age of 65. It is an irreversible and progressing neurodegenerative brain disorder that leads to cognitive impairment and memory loss³. AD is a very burdensome disease to patients and their families, as well as informal caregivers, due to long term illness in terms of disability and dependence⁴. Therefore, AD becomes one of the biggest global health challenges for society.

The pathogenesis of AD is multifactor, including the loss of cholinergic neuron, extracellular deposit of fibrils, aggregation of amyloid beta-peptide, oxidative stress, neuroinflammation. The most well-received hypothesis is the deficit of an important neurotransmitter in the cholinergic neurotransmission, acetylcholine (ACh)^3,5–7. The deficit of ACh has profound effects on the signaling that involves learning ability and long-term memory^8–10. The cholinesterase enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are accountable for the degradation of ACh through hydrolysis. Thus, cholinesterase inhibitors are important drugs in treating AD by maintaining the level of ACh^10–12. AChE predominates in the brain of a healthy person and contributes to ACh hydrolysis in the brain for approximately 80%^7,13. Studies have shown that the inhibition of AChE could prevent the degradation of ACh and in turn sustaining its level and duration of action^14,15. Furthermore, AChE could facilitate the formation of amyloid fibril to obtain a stable AChE-β-amyloid complex, which is more toxic than single β-amyloid peptides⁵. Hence, targeting AChE could hinder the production of AChE-β-amyloid complexes. The current management of AD is focussing on the enhancement of the concentration of ACh in the synaptic cleft by inhibiting cholinesterase^16,17. Most of the U.S. Food and Drug Administration (FDA)-approved prescription drugs for AD are AChE inhibitors including Donepezil¹⁸, Galanthamine¹⁹, and the dual cholinesterase inhibitor Rivastigmine²⁰. Moreover, patients who received treatment with AChE inhibitor experienced a deceleration of the progression of the disease and an increase of attention span²¹. Therefore, research studies on the discovery and development of AChE inhibitors with higher potency and efficacy is essential since these inhibitors are the therapeutically potential as a principal approach of AD treatments. The adverse effects were associated with currently available drugs and worse still that there is no cure for AD up to date. Current approaches for the treatment of AD are solely focussing on maintaining the mental function, managing behavioural symptoms, and slowing down or delaying the symptoms of the disease²². Hence, this study aimed to search for lead compounds with significant cholinesterase inhibitory activities that are highly potential to be further developed into alternative drugs to treat AD more effectively.

To date, more than 50% of the marketed drugs were developed from natural products, especially secondary metabolites and their derivatives^23,24. Xanthones are one of these important secondary metabolites that possessed a broad spectrum of biological activities such as anti-cancer²⁵, antioxidant²⁶, antibacterial²⁷, anti-fungal²⁸, antimalarial²⁹, antidiabetic^30,31, anti-cholinergic^15,32 and anti-inflammatory activities^33–36. The biological activities of xanthones vary depending on their chemical structures, particularly the type of substituents and their respective positions on the two phenyl rings of the core structure of xanthone^37–39. However, xanthones obtained from the natural resources are limited in substituent variation available on the rings³⁷, as well as time-consuming on the extraction and purification processes⁴⁰. Hence, structural modification of xanthone-based skeleton is of interest to obtain various xanthone derivatives. In this study, a series of twenty-nine new xanthone derivatives (2–30) with seven types of side chains, alkyl, alkenyl, alkynyl, alkylphenyl, ether, ester, and hydroxyl substituents were synthesised. The chemical structures of the xanthone derivatives were elucidated by spectroscopic analyses, including mass spectrometry (MS), nuclear magnetic resonance (NMR), and Fourier-transform infrared (FTIR). Moreover, crystal structures for compounds 5 and 13 were determined through single-crystal X-ray diffraction analysis to further support the spectroscopy data. The xanthone derivatives were evaluated for their AChE and BChE inhibitory activities by using Ellman’s method with minor modification⁴¹. The enzyme kinetic study of the AChE inhibition by compounds 23 and 28 was reported in this paper. Furthermore, molecular docking of compound 23 was performed in order to elucidate its binding interactions with AChE.

Material and methods

The solvents (analytical grade), silica gel 60, and thin-layer chromatography (TLC) silica gel 60 F254 were obtained commercially from Merck (United States). The chemicals and reagents used in the synthesis reactions were obtained from Sigma Aldrich (Germany) with high purity (>95%). Acetylcholinesterase from Electrophorus electricus (500 UN), butyrylcholinesterase from Equine serum (1.2 KU), acetylthiocholine iodide, S-butyrylthiocholine chloride, and 5,5′-Dithiobis (2-nitro benzoic acid) were obtained from Sigma Aldrich. Tacrine (Purity >98%) was obtained from Cayman Chemical. The melting point of derivatives was determined by Electrothermal 9100 Series Apparatus. Mass spectra of derivatives were acquired from Gas Chromatography-Mass Spectrometry (GCMS) (Agilent J&W) equipped with GC column HP-5MS (30 m × 0.25 mm × 0.25 µm). ¹H and ¹³C NMR spectral data of derivatives were obtained from JEOL JNM-ECX 500 or JNM-ECZ 600 R NMR spectrometers. FTIR spectra of derivatives were recorded on Perkin Elmer Spectrum 100 (Perkin Elmer) equipped with attenuated total reflection (ATR). X-ray analysis was performed using Bruker APEX II DUO CCD diffractometer. Optically active xanthones were analysed using polarimeter Optika POL-1 bench polarimeter. The absorbance of the biological assay was measured with microplate reader BioTek Epoch 2.

Synthesis of xanthones

The synthesis of 3-hydroxyxanthone (1) was conducted by mixing the salicylic acid with resorcinol in the presence of Eaton’s reagent under reflux. The reaction mixture was poured into ice water, filtered, and subsequently washed with water. The dried powder was subjected to solvent extraction by using ethyl acetate and afforded 1⁴². Xanthone derivatives (2–30) were then synthesised by mixing 1 and the corresponding bromide with potassium carbonate in acetone under reflux⁴³. The reaction scheme is shown in Figure 1. The reaction mixture was continuously stirred under reflux for hours and monitored by TLC. The reaction mixture was mixed with water upon reaction completion and extracted twice by using chloroform. The organic part was washed successively with hydrochloric acid, sodium carbonate solution, and water. The product was then subjected to purification using column chromatography by eluting a series of solvents (hexane, chloroform, and methanol) with increasing polarity over the silica gel. The chemical structure of pure xanthone derivatives was characterised using MS, NMR and FTIR spectroscopic analyses. Single crystals were obtained for compounds 5 and 13, thus further analysed by using X-ray diffraction. The optical rotation of xanthone derivatives 7, 29, and 30 were measured using a polarimeter.

Figure 1.

Reaction scheme for the synthesis of xanthone derivatives.

3-hydroxyxanthone (1): M.P. 251–253 °C; m/z, C₁₃H₈O₃: 212, 184, 155, 128, 102, 92, 77, 63, 51; IR ν_max cm⁻¹: 3115, 2884, 1641, 1609, 1586, 1566, 1450, 1226; ¹H NMR (600 MHz, DMSO-d6): δ_H 10.93 (s, 1H, OH-3), 8.10 (dd, J = 2.1, 8.3 Hz, 1H, H-8), 7.99 (d, J = 9.0 Hz, 1H, H-1), 7.75 (td, J = 2.1, 7.6 Hz, 1H, H-6), 7.54 (d, J = 8.3 Hz, 1H, H-5), 7.38 (deformed t, J = 7.6, 8.3 Hz, 1H, H-7), 6.86 (dd, J = 2.1, 9.0 Hz, 1H, H-2), 6.82 (d, J = 2.1 Hz, 1H, H-4); ¹³C NMR (150 MHz, DMSO-d6): δ_C 175.3 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.1 (C-5a), 135.3 (C-6), 128.5 (C-1), 126.4 (C-8), 124.6 (C-7), 121.7 (C-8a), 118.4 (C-5), 114.7 (C-2), 114.5 (C-9a), 102.7 (C-4).

3-Propoxy-9H-xanthen-9-one (2): Yield: 92%; White amorphous solid; M.P. 120–122 °C; m/z, C₁₆H₁₄O_3: 254, 212, 195, 184, 155, 139, 128, 113, 102, 92, 77, 63, 51; IR ν_max cm⁻¹: 2874, 1654 1623, 1232; ¹H NMR (600 MHz, CDCl₃): δ_H 8.20 (d, J = 8.2 Hz, 1H, H-8), 8.10 (d, J = 8.2 Hz, 1H, H-1), 7.54 (deformed t, J = 6.8, 8.2 Hz, 1H, H-6), 7.28 (d, J = 8.2 Hz, 1H, H-5), 7.23 (deformed t, J = 6.8, 8.2 Hz, 1H, H-7), 6.78 (dd, J = 2.8, 8.2 Hz, 1H, H-2), 6.67 (d, J = 2.8, 1H, H-4), 3.88 (t, J = 6.9 Hz, 2H, H-1′), 1.77 (m, 2H, H-2′), 0.99 (t, J = 6.9 Hz, 3H, H-3′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.1 (C-9), 164.6 (C-3), 158.0 (C-4a), 156.1 (C-5a), 134.1 (C-6), 128.0 (C-1), 126.5 (C-8), 123.7 (C-7), 121.9 (C-8a), 117.7 (C-5), 115.5 (C-2), 113.6 (C-9a), 100.6 (C-4), 70.1 (C-1′), 22.4 (C-2′), 10.5 (C-3′).

3-Butoxy-9H-xanthen-9-one (3): Yield: 84%; White amorphous solid; M.P. 115–117 °C; m/z, C₁₇H₁₆O₃: 268, 212, 184, 155, 139, 121, 92, 63; IR ν_max cm⁻¹: 2874, 1647, 1618, 1239; ¹H NMR (600 MHz, CDCl₃): δ_H 8.26 (d, J = 8.2 Hz, 1H, H-8), 8.17 (d, J = 9.6 Hz, 1H, H-1), 7.62 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.37 (d, J = 8.2 Hz, 1H, H-5), 7.30 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.86 (dd, J = 2.7, 9.6 Hz, 1H, H-2), 6.78 (d, J = 2.7 Hz, 1H, H-4), 4.01 (t, J = 6.9 Hz, 2H, H-1′), 1.78 (m, 2H, H-2′), 1.49 (m, 2H, H-3′), 0.97 (t, J = 6.9 Hz, 3H, H-4′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.3 (C-9), 164.7 (C-3), 158.1 (C-4a), 156.2 (C-5a), 134.2 (C-6), 128.2 (C-1), 126.7 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7 (C-9a), 100.6 (C-4), 68.5 (C-1′), 31.1 (C-2′), 19.3 (C-3′), 13.9 (C-4′).

3-Isopropoxy-9H-xanthen-9-one (4): Yield: 9.1%; White amorphous solid; M.P. 76–78 °C; m/z, C₁₆H₁₄O₃: 254, 212, 184, 155, 139, 128, 102, 92, 77, 63, 51; IR ν_max cm⁻¹: 2923, 1659, 1623, 1280; ¹H NMR (600 MHz, CDCl₃): δ_H 8.31 (d, J = 8.2 Hz, 1H, H-8), 8.22 (d, J = 8.2 Hz, 1H, H-1), 7.67 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.43 (d, J = 8.2 Hz, 1H, H-5), 7.35 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.89 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.85 (d, J = 2.7 Hz, 1H, H-4), 4.68 (m, 1H, H-1′), 1.41 (d, J = 6.9 Hz, 6H, H-2′ and H-3′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.3 (C-9), 163.7 (C-3), 158.2 (C-4a), 156.3 (C-5a), 134.3 (C-6), 128.4 (C-1), 126.7 (C-8), 123.9 (C-7), 122.1 (C-8a), 117.7 (C-5), 115.6 (C-2), 114.3 (C-9a), 101.6 (C-4), 70.9 (C-1′), 21.9 (C-2′ and C-3′).

3-Isobutoxy-9H-xanthen-9-one (5): Yield: 29%; White crystalline solid; M.P. 108–110 °C; m/z, C₁₇H₁₆O₃: 268, 212, 184, 155, 139, 119, 102, 77, 57; IR ν_max cm⁻¹: 2899, 1659, 1623, 1280; ¹H NMR (500 MHz, CDCl₃): δ_H 8.32 (dd, J = 2.3, 8.0 Hz, 1H, H-8), 8.23 (d, J = 8.0 Hz, 1H, H-1), 7.68 (dt, J = 2.3, 6.9, 9.2 Hz, 1H, H-6), 7.44 (d, J = 9.2 Hz, 1H, H-5), 7.35 (deformed t, J = 6.9, 8.0 Hz, 1H, H-7), 6.94 (dd, J = 2.3, 8.0 Hz, 1H, H-2), 6.86 (d, J = 2.3 Hz, 1H, H-4), 3.84 (d, J = 6.9 Hz, 2H, H-1′), 2.15 (m, 1H, H-2′), 1.06 (d, J = 6.9 Hz, 6H, H-3′ and H-4′); ¹³C NMR (125 MHz, CDCl₃): δ_C 176.4 (C-9), 164.9 (C-3), 158.2 (C-4a), 156.3 (C-5a), 134.3 (C-6), 128.3 (C-1), 126.8 (C-8), 123.9 (C-7), 122.1 (C-8a), 117.8 (C-5), 115.7 C-2), 113.7 (C-9a), 100.7 (C-4), 75.1 (C-1′), 28.2 (C-2′), 19.3 (C-3′ and C-4′).

3-(Isopentyloxy)-9H-xanthen-9-one (6): Yield: 82%; White crystalline solid; M.P. 83–85 °C; m/z, C₁₈H₁₈O₃: 282, 212, 184, 155, 139, 92, 71, 55; IR ν_max cm⁻¹: 2958, 1651, 1623, 1256; ¹H NMR (500 MHz, CDCl₃): δ_H 8.24 (dd, J = 2.3, 8.0 Hz, 1H, H-8), 8.15 (d, J = 9.2 Hz, 1H, H-1), 7.58 (dt, J = 2.3, 6.9, 9.2 Hz, 1H, H-6), 7.33 (d, J = 9.2 Hz, 1H, H-5), 7.27 (deformed t, J = 6.9, 8.0 Hz, 1H, H-7), 6.83 (dd, J = 2.3, 9.2 Hz, 1H, H-2), 6.74 (d, J = 2.3 Hz, 1H, H-4), 4.01 (t, J = 6.9 Hz, 2H, H-1′), 1.81 (m, 1H, H-3′), 1.68 (m, 2H, H-2′), 0.95 (d, J = 6.9 Hz, 6H, H-4′ and H-5′); ¹³C NMR (125 MHz, CDCl₃): δ_C 176.2 (C-9), 164.7 (C-3), 158.0 (C-4a), 156.2 (C-5a), 134.2 (C-6), 128.1 (C-1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7 (C-9a), 100.6 (C-4), 67.2 (C-1′), 37.7 (C-2′), 25.1 (C-3′), 22.6 (C-4′ and C-5′).

(S)-3-(2-Methylbutoxy)-9H-xanthen-9-one (7): Yield: 32%; White amorphous solid; M.P. 46–48 °C; [α] 25/D + 16.5°, in methanol; m/z, C₁₈H₁₈O₃: 282, 212, 184, 155, 139, 92, 71, 55; IR ν_max cm⁻¹: 2969, 1654, 1616, 1256; ¹H NMR (500 MHz, CDCl₃): δ_H 8.28 (d, J = 8.0 Hz, 1H, H-8), 8.20 (d, J = 9.2 Hz, 1H, H-1), 7.63 (dt, J = 2.3, 6.9, 9.2 Hz, 1H, H-6), 7.39 (d, J = 9.2 Hz, 1H, H-5), 7.31 (deformed t, J = 6.9, 8.0 Hz, 1H, H-7), 6.89 (dd, J = 2.3, 9.2 Hz, 1H, H-2), 6.81 (d, J = 2.3 Hz, 1H, H-4), 3.89 (dd, J = 5.7, 9.2 Hz, 1H, H-1’a), 3.81 (dd, J = 6.9, 9.2 Hz, 1H, H-1’b), 1.90 (m, 1H, H-2′), 1.57 (m, 1H, H-3’a), 1.28 (m, 1H, H-3’b), 1.03 (d, J = 6.9 Hz, 3H, H-5′), 0.95 (deformed t, J = 6.9, 8.0 Hz, 3H, H-4′); ¹³C NMR (125 MHz, CDCl₃): δ_C 176.3 (C-9), 164.9 (C-3), 158.1 (C-4a), 156.3 (C-5a), 134.2 (C-6), 128.2 (C-1), 126.7 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7 (C-9a), 100.7 (C-4), 73.5 (C-1′), 34.6 (C-2′), 26.2 (C-3′), 16.5 (C-5′), 11.4 (C-4′).

3-(2-Ethylbutoxy)-9H-xanthen-9-one (8): Yield: 50%; Colourless oil; m/z, C₁₉H₂₀O₃: 296, 212, 184, 155, 139, 119, 102, 85, 55; IR ν_max cm⁻¹: 2958, 1659, 1623, 1256; ¹H NMR (600 MHz, CDCl₃): δ_H 8.26 (d, J = 8.2 Hz, 1H, H-8), 8.17 (d, J = 8.2 Hz, 1H, H-1), 7.60 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.35 (d, J = 8.2 Hz, 1H, H-5), 7.28 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.87 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.79 (d, J = 2.7 Hz, 1H, H-4), 3.90 (d, J = 6.9 Hz, 2H, H-1′), 1.68 (m, 1H, H-2′), 1.46 (m, 4H, H-3′ and H-5′), 0.92 (t, J = 6.9, 8.2 Hz, 6H, H-4′ and H-6′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 164.9 (C-3), 158.1 (C-4a), 156.2 (C-5a), 134.2 (C-6), 128.1 (C-1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7 (C-9a), 100.6 (C-4), 70.9 (C-1′), 40.8 (C-2′), 23.4 (C-3′ and C-5′), 11.2 (C-4′ and C-6′).

3-((4-Methylpentyl)oxy)-9H-xanthen-9-one (9): Yield: 83%; White crystalline solid; M.P. 66–68 °C; m/z, C₁₉H₂₀O₃: 296, 212, 184, 155, 139, 119, 102, 85, 69; IR ν_max cm⁻¹: 2958, 1654, 1623, 1261; ¹H NMR (600 MHz, CDCl₃): δ_H 8.24 (d, J = 8.2 Hz, 1H, H-8), 8.14 (d, J = 8.2 Hz, 1H, H-1), 7.58 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.33 (d, J = 8.2 Hz, 1H, H-5), 7.26 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.82 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.72 (s, 1H, H-4), 3.94 (t, J = 6.9 Hz, 2H, H-1′), 1.77 (m, 2H, H-2′), 1.58 (m, 1H, H-4′), 1.31 (m, 2H, H-3′), 0.90 (d, J = 6.9 Hz, 6H, H-5′ and H-6′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.1 (C-9), 164.7 (C-3), 158.0 (C-4a), 156.2 (C-5a), 134.2 (C-6), 128.1 (C-1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.6 (C-9a), 100.6 (C-4), 69.0 (C-1′), 35.1 (C-3′), 27.9 (C-4′), 27.0 (C-2′), 22.6 (C-5′ and C-6′).

3-(Cyclobutylmethoxy)-9H-xanthen-9-one (10): Yield: 14%; White amorphous solid; M.P. 95–97 °C; m/z, C₁₈H₁₆O₃: 280, 212, 184, 155, 127, 107, 92, 69, 53; IR ν_max cm⁻¹: 2958, 1659, 1623, 1280; ¹H NMR (600 MHz, CDCl₃): δ_H 8.31 (dd, J = 1.8, 7.3 Hz, 1H, H-8), 8.23 (d, J = 8.3 Hz, 1H, H-1), 7.67 (dt, J = 1.8, 8.3 Hz, 1H, H-6), 7.43 (d, J = 8.3 Hz, 1H, H-5), 7.35 (deformed t, J = 7.3, 8.3 Hz, 1H, H-7), 6.93 (dd, J = 2.8, 8.3 Hz, 1H, H-2), 6.86 (d, J = 2.8 Hz, 1H, H-4), 4.04 (d, J = 7.3 Hz, 2H, H-1′), 2.82 (m, 1H, H-2′), 2.17 (m, 2H, H-4′), 1.95 (m, 4H, H-3′ and H-5′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.4 (C-9), 164.9 (C-3), 158.2 (C-4a), 156.3 (C-5a), 134.3 (C-6), 128.3 (C-1), 126.7 (C-8), 123.9 (C-7), 122.1 (C-8a), 117.8 (C-5), 115.7 (C-2), 113.8 (C-9a), 100.8 (C-4), 72.7 (C-1′), 34.4 (C-2′), 24.9 (C-3′ and C-5′), 18.6 (C-4′).

3-(2-Cyclohexylethoxy)-9H-xanthen-9-one (11): Yield: 59%; White crystalline solid; M.P. 134–136 °C; m/z, C₂₁H₂₂O₃: 322, 239, 212, 184, 155, 139, 111, 95, 69, 53; IR ν_max cm⁻¹: 2923, 1652, 1623, 1256; ¹H NMR (600 MHz, CDCl₃): δ_H 8.28 (dd, J = 2.8, 8.2 Hz, 1H, H-8), 8.19 (d, J = 8.2 Hz, 1H, H-1), 7.63 (dd, J = 2.8, 8.2 Hz, 1H, H-6), 7.38 (d, J = 8.2 Hz, 1H, H-5), 7.31 (t, J = 8.2 Hz, 1H, H-7), 6.88 (dd, J = 2.8, 8.2 Hz, 1H, H-2), 6.80 (d, J = 2.8 Hz, 1H, H-4), 4.06 (t, J = 6.9 Hz, 2H, H-1′), 1.70 (m, 7H, H-2′, H-4′, H-7’a, H-8′), 1.50 (m, 1H, H-3′), 1.20 (m, 3H, H-5′ and H-7’b), 0.97 (m, 2H, H-6′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.3 (C-9), 164.7 (C-3), 158.1 (C-4a), 156.2 (C-5a), 134.2 (C-6), 128.2 (C-1), 126.7 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7 (C-9a), 100.7 (C-4), 66.8 (C-1′), 36.4 (C-2′), 34.6 (C-3′), 33.4 (C-4′ and C-8′), 26.6 (C-6′), 26.3 (C-5′ and C-7′).

3-(But-3-en-1-yloxy)-9H-xanthen-9-one (12): Yield: 46%; White amorphous solid; M.P. 85–87 °C; m/z, C₁₇H₁₄O₃: 266, 237, 212, 184, 155, 139, 121, 92, 75, 55; IR ν_max cm⁻¹: 2948, 1654, 1623, 1258; ¹H NMR (600 MHz, CDCl₃): δ_H 8.27 (dd, J = 2.1, 8.3 Hz, 1H, H-8), 8.18 (d, J = 9.0 Hz, 1H, H-1), 7.63 (dt, J = 2.1, 6.9, 8.3 Hz, 1H, H-6), 7.37 (d, J = 8.3 Hz, 1H, H-5), 7.31 (deformed t, J = 6.9, 8.3 Hz, 1H, H-7), 6.87 (dd, J = 2.8, 9.0 Hz, 1H, H-2), 6.79 (d, J = 2.8 Hz, 1H, H-4), 5.88 (m, 1H, H-3′), 5.18 (dd, J = 1.4, 17.2 Hz, 1H, H-4’a), 5.12 (dd, J = 1.4, 10.3 Hz, 1H, H-4’b), 4.07 (t, J = 6.9 Hz, 2H, H-1′), 2.57 (m, 2H, H-2′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.3 (C-9), 164.4 (C-3), 158.0 (C-4a), 156.2 (C-5a), 134.3 (C-6), 133.9 (C-3′), 128.3 (C-1), 126.7 (C-8), 123.9 (C-7), 122.0 (C-8a), 117.7 (C-5), 117.6 (C-4′), 115.8 (C-2), 113.6 (C-9a), 100.8 (C-4), 67.9 (C-1′), 33.4 (C-2′).

3-(Pent-4-en-1-yloxy)-9H-xanthen-9-one (13): Yield: 88%; White crystalline solid; M.P. 86–88 °C; m/z, C₁₈H₁₆O₃: 280, 239, 212, 184, 155, 139, 121, 92, 69, 53; IR ν_max cm⁻¹: 2958, 1647, 1618, 1258; ¹H NMR (600 MHz, CDCl₃): δ_H 8.26 (d, J = 8.2 Hz, 1H, H-8), 8.17 (d, J = 9.6 Hz, 1H, H-1), 7.61 (t, J = 6.9 Hz, 1H, H-6), 7.35 (d, J = 8.2 Hz, 1H, H-5), 7.29 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.85 (dd, J = 2.8, 9.6 Hz, 1H, H-2), 6.76 (d, J = 2.8 Hz, 1H, H-4), 5.83 (m, 1H, H-4′), 5.03 (m, 2H, H-5′), 4.01 (deformed t, J = 5.5, 6.9 Hz, 2H, H-1′), 2.23 (m, 2H, H-3′), 1.90 (m, 2H, H-2′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.2 (C-5a), 137.5 (C-4′), 134.2 (C-6), 128.2 (C-1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.7 (C-5′), 115.6 (C-2), 113.6 (C-9a), 100.7 (C-4), 67.9 (C-1′), 30.0 (C-3′), 28.2 (C-2′).

3-(Hex-5-en-1-yloxy)-9H-xanthen-9-one (14): Yield: 81%; White amorphous solid; M.P. 73–75 °C; m/z, C₁₉H₁₈O₃: 294, 251, 212, 184, 155, 139, 121, 102, 82, 55; IR ν_max cm⁻¹: 2850, 1647, 1623, 1256; ¹H NMR (600 MHz, CDCl₃): δ_H 8.26 (d, J = 8.2 Hz, 1H, H-8), 8.17 (d, J = 8.2 Hz, 1H, H-1), 7.61 (m, 1H, H-6), 7.36 (d, J = 8.2 Hz, 1H, H-5), 7.29 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.85 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.76 (d, J = 2.7 Hz, 1H, H-4), 5.81 (m, 1H, H-5′), 5.00 (m, 2H, H-6′), 4.00 (deformed t, J = 5.5, 6.9 Hz, 2H, H-1′), 2.12 (m, 2H, H-4′), 1.81 (m, 2H, H-2′), 1.56 (m, 2H, H-3′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.2 (C-5a), 138.3 (C-5′), 134.2 (C-6), 128.2 (C-1), 126.7 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.7 (C-2), 115.0 (C-6′), 113.6 (C-9a), 100.6 (C-4), 68.5 (C-1′), 33.4 (C-4′), 28.5 (C-2′), 25.3 (C-3′).

3-((2-Methylallyl)oxy)-9H-xanthen-9-one (15): Yield: 87%; White amorphous solid; M.P. 121–123 °C; m/z, C₁₇H₁₄O₃: 266, 251, 225, 197, 181, 165, 139, 115, 92, 77, 55; IR ν_max cm⁻¹: 2923, 1654, 1623, 1275; ¹H NMR (600 MHz, CDCl₃): δ_H 8.20 (d, J = 8.2 Hz, 1H, H-8), 8.12 (d, J = 8.2 Hz, 1H, H-1), 7.54 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.28 (d, J = 8.2 Hz, 1H, H-5), 7.23 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.82 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.71 (d, J = 2.7 Hz, 1H, H-4), 5.06 (s, 1H, H-3’a), 4.98 (s, 1H, H-3’b), 4.42 (s, 2H, H-1′), 1.79 (s, 3H, H-4′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.1 (C-9), 164.1 (C-3), 157.9 (C-4a), 156.1 (C-5a), 139.8 (C-2′), 134.2 (C-6), 128.1 (C-1), 126.6 (C-8), 123.8 (C-7), 121.9 (C-8a), 117.7 (C-5), 115.8 (C-2), 113.7 (C-9a), 113.5 (C-3′), 101.1 (C-4), 72.1 (C-1′), 19.4 (C-4′).

3-((3-Methylbut-2-en-1-yl)oxy)-9H-xanthen-9-one (16): Yield: 84%; White amorphous solid; M.P. 115–117 °C; m/z, C₁₈H₁₆O₃: 280, 212, 184, 155, 127, 92, 69, 53; IR ν_max cm⁻¹: 2958, 1654, 1625, 1287; ¹H NMR (600 MHz, CDCl₃): δ_H 8.25 (dd, J = 2.7, 8.2 Hz, 1H, H-8), 8.16 (d, J = 8.2 Hz, 1H, H-1), 7.60 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.35 (d, J = 8.2 Hz, 1H, H-5), 7.28 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.86 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.78 (s, 1H, H-4), 5.46 (t, J = 6.9 Hz, 1H, H-2′), 4.55 (d, J = 6.9 Hz, 2H, H-1′), 1.78 (s, 3H, H-4′), 1.74 (s, 3H, H-5′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 164.4 (C-3), 158.0 (C-4a), 156.2 (C-5a), 139.3 (C-3′), 134.2 (C-6), 128.2 (C-1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 118.7 (C-2′), 117.7 (C-5), 115.7 (C-2), 113.8 (C-9a), 100.9 (C-4), 65.5 (C-1′), 25.9 (C-4′), 18.4 (C-5′).

3-((4-Methylpent-3-en-1-yl)oxy)-9H-xanthen-9-one (17): Yield: 77%; White amorphous solid; M.P. 68–70 °C; m/z, C₁₉H₁₈O₃: 294, 213, 195, 155, 139, 119, 101, 83, 55; IR ν_max cm⁻¹: 2909, 1654, 1625, 1263; ¹H NMR (600 MHz, CDCl₃): δ_H 8.21 (d, J = 8.2 Hz, 1H, H-8), 8.11 (d, J = 9.6 Hz, 1H, H-1), 7.54 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.28 (d, J = 8.2 Hz, 1H, H-5), 7.23 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.79 (dd, J = 2.7, 9.6 Hz, 1H, H-2), 6.68 (d, J = 2.7 Hz, 1H, H-4), 5.15 (deformed t, J = 6.9, 8.2 Hz, 1H, H-3′), 3.91 (t, J = 6.9 Hz, 2H, H-1′), 2.45 (dd, J = 6.9, 8.2 Hz, 2H, H-2′), 1.68 (s, 3H, H-5′), 1.62 (s, 3H, H-6′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.1 (C-9), 164.5 (C-3), 157.9 (C-4a), 156.1 (C-5a), 134.9 (C-4′), 134.1 (C-6), 128.1 (C-1), 126.5 (C-8), 123.7 (C-7), 121.9 (C-8a), 119.1 (C-3′), 117.7 (C-5), 115.6 (C-2), 113.6 (C-9a), 100.6 (C-4), 68.3 (C-1′), 28.0 (C-2′), 25.8 (C-5′), 18.0 (C-6′).

3-(But-2-yn-1-yloxy)-9H-xanthen-9-one (18): Yield: 56%; White crystalline solid; M.P. 147–149 °C; m/z, C₁₇H₁₂O₃: 264, 249, 236, 212, 184, 155, 127, 92, 77, 53; IR ν_max cm⁻¹: 2983, 2238, 1654, 1618, 1251; ¹H NMR (600 MHz, CDCl₃): δ_H 8.30 (d, J = 8.2 Hz, 1H, H-8), 8.24 (d, J = 8.2 Hz, 1H, H-1), 7.68 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.44 (d, J = 8.2 Hz, 1H, H-5), 7.35 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.97 (d, J = 8.2 Hz, 1H, H-2), 6.97 (s, 1H, H-4), 4.76 (s, 2H, H-1′), 1.87 (d, J = 2.7 Hz, 3H, H-4′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.4 (C-9), 163.3 (C-3), 157.9 (C-4a), 156.3 (C-5a), 134.4 (C-6), 128.4 (C-1), 126.8 (C-8), 124.0 (C-7), 122.0 (C-8a), 117.8 (C-5), 116.3 (C-2), 113.7 (C-9a), 101.6 (C-4), 85.0 (C-2′), 73.0 (C-3′), 57.0 (C-1′), 3.8 (C-4′).

3-(Pent-2-yn-1-yloxy)-9H-xanthen-9-one (19): Yield: 83%; White amorphous solid; M.P. 119–121 °C; m/z, C₁₈H₁₄O₃: 278, 249, 212, 184, 155, 127, 92, 65; IR ν_max cm⁻¹: 2972, 2238, 1654, 1618, 1256; ¹H NMR (600 MHz, CDCl₃): δ_H 8.21 (d, J = 8.2 Hz, 1H, H-8), 8.14 (d, J = 8.2 Hz, 1H, H-1), 7.57 (t, J = 8.2 Hz, 1H, H-6), 7.32 (d, J = 8.2 Hz, 1H, H-5), 7.25 (t, J = 8.2 Hz, 1H, H-7), 6.87 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.84 (d, J = 2.7 Hz, 1H, H-4), 4.69 (t, J = 2.7 Hz, 2H, H-1′), 2.19 (m, 2H, H-4′), 1.08 (deformed t, J = 6.9, 8.2 Hz, 3H, H-5′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.1 (C-9), 163.2 (C-3), 157.8 (C-4a), 156.2 (C-5a), 134.3 (C-6), 128.2 (C-1), 126.6 (C-8), 123.8 (C-7), 121.9 (C-8a), 117.7 (C-5), 116.1 (C-2), 113.6 (C-9a), 101.5 (C-4), 90.7 (C-3′), 73.2 (C-2′), 57.0 (C-1′), 13.6 (C-5′), 12.5 (C-4′).

3-(But-3-yn-2-yloxy)-9H-xanthen-9-one (20): Yield: 73%; White amorphous solid; M.P. 170–172 °C; m/z, C₁₇H₁₂O₃: 264, 249, 207, 165, 139, 89, 63; IR ν_max cm⁻¹: 2927, 2112, 1637, 1618, 1251; ¹H NMR (600 MHz, CDCl₃): δ_H 8.30 (d, J = 8.2 Hz, 1H, H-8), 8.24 (d, J = 9.6 Hz, 1H, H-1), 7.67 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.44 (d, J = 8.2 Hz, 1H, H-5), 7.34 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 7.03 (d, J = 2.7 Hz, 1H, H-4), 6.99 (dd, J = 2.7, 9.6 Hz, 1H, H-2), 4.97 (m, 1H, H-1′), 2.56 (d, J = 2.7 Hz, 1H, H-3′), 1.72 (d, J = 6.9 Hz, 3H, H-4′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.4 (C-9), 162.7 (C-3), 157.9 (C-4a), 156.3 (C-5a), 134.4 (C-6), 128.3 (C-1), 126.7 (C-8), 124.0 (C-7), 122.0 (C-8a), 117.8 (C-5), 116.3 (C-2), 114.1 (C-9a), 102.3 (C-4), 81.8 (C-2′), 75.0 (C-3′), 64.1 (C-1′), 22.1 (C-4′).

3-(Benzyloxy)-9H-xanthen-9-one (21): Yield: 61%; White crystalline solid; M.P. 178–180 °C; m/z, C₂₀H₁₄O₃: 302, 183, 155, 127, 91, 65; IR ν_max cm⁻¹: 2958, 1637, 1618, 1251; ¹H NMR (500 MHz, CDCl₃): δ_H 8.31 (d, J = 6.9 Hz, 1H, H-8), 8.25 (d, J = 9.2 Hz, 1H, H-1), 7.67 (t, J = 6.9 Hz, 1H, H-6), 7.43 (q, J = 8.0 Hz, 5H, H-3′, H-4′, H-5′, H-6′, H-7′), 7.35 (q, J = 6.9 Hz, 2H, H-5 & H-7), 7.00 (dd, J = 2.3, 9.2 Hz, 1H, H-2), 6.94 (d, J = 2.3 Hz, 1H, H-4), 5.17 (s, 2H, H-1′); ¹³C NMR (125 MHz, CDCl₃): δ_C 176.3 (C-9), 164.2 (C-3), 158.1 (C-4a), 156.3 (C-5a), 135.8 (C-2′), 134.4 (C-6), 128.9 (C-4′ and C-6′), 128.5 (C-1), 128.4 (C-5′), 127.6 (C-3′ and C-7′), 126.7 (C-8), 124.0 (C-7), 122.1 (C-8a), 117.8 (C-5), 116.1 (C-2), 113.9 (C-9a), 101.4 (C-4), 70.6 (C-1′).

3-(3-Phenylpropoxy)-9H-xanthen-9-one (22): Yield: 94%; White amorphous solid; M.P. 100–102 °C; m/z, C₂₂H₁₈O₃: 330, 212, 184, 155, 139, 118, 91, 65; IR ν_max cm⁻¹: 2958, 1659, 1618, 1282; ¹H NMR (600 MHz, CDCl₃): δ_H 8.29 (dd, J = 2.7, 8.3 Hz, 1H, H-8), 8.19 (d, J = 8.2 Hz, 1H, H-1), 7.61 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.36 (d, J = 8.2 Hz, 1H, H-5), 7.30 (deformed t, J = 6.9, 8.2 Hz, 3H, H-7, H-5′, H-9′), 7.21 (deformed t, J = 6.9, 8.2 Hz, 3H, H-6′, H-7′, H-8′), 6.87 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.74 (d, J = 2.7 Hz, 1H, H-4), 3.99 (t, J = 6.9 Hz, 2H, H-1′), 2.82 (deformed t, J = 6.9, 8.2 Hz, 2H, H-3′), 2.14 (m, 2H, H-2′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 164.6 (C-3), 158.0 (C-4a), 156.2 (C-5a), 141.2 (C-4′), 134.3 (C-6), 128.6 (C-6′ and C-8′), 128.6 (C-5′ and C-9′), 128.2 (C-1), 126.7 (C-7′), 126.2 (C-8), 123.9 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.8 (C-2), 113.6 (C-9a), 100.7 (C-4), 67.6 (C-1′), 32.1 (C-3′), 30.6 (C-2′).

3-(4-Phenylbutoxy)-9H-xanthen-9-one (23): Yield: 90%; White amorphous solid; M.P. 88–90 °C; m/z, C₂₃H₂₀O₃: 344, 212, 184, 155, 133, 117, 91, 65; IR ν_max cm⁻¹: 2944, 1654, 1623, 1286; ¹H NMR (600 MHz, CDCl₃): δ_H 8.29 (dd, J = 2.7, 8.2 Hz, 1H, H-8), 8.19 (d, J = 8.2 Hz, 1H, H-1), 7.61 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.36 (d, J = 8.2 Hz, 1H, H-5), 7.30 (deformed t, J = 6.9, 8.2 Hz, 3H, H-7, H-6′ H-10′), 7.20 (t, J = 8.2 Hz, 3H, H-7′, H-8′, H-9′), 6.84 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.75 (d, J = 2.7 Hz, 1H, H-4), 3.99 (deformed t, J = 5.5, 6.9 Hz, 2H, H-1′), 2.70 (t, J = 6.9 Hz, 2H, H-4′), 1.83 (m, 4H, H-2′, H-3′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.2 (C-5a), 142.0 (C-5′), 134.3 (C-6), 128.5 (C-7′ and C-9′), 128.2 (C-6′ and C-10′), 126.7 (C-1), 126.0 (C-8 and C-8′), 123.9 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.7 (C-2), 113.6 (C-9a), 100.7 (C-4), 68.5 (C-1′), 35.6 (C-4′), 28.7 (C-2′), 27.8 (C-3′).

3-(3-Methoxypropoxy)-9H-xanthen-9-one (24): Yield: 72%; White amorphous solid; M.P. 82–83 °C; m/z, C₁₇H₁₆O₄: 284, 269, 252, 239, 226, 213, 197, 184, 155, 139, 73; IR ν_max cm⁻¹: 2934, 1661, 1563, 1282; ¹H NMR (600 MHz, CDCl₃): δ_H 8.29 (d, J = 8.2 Hz, 1H, H-8), 8.21 (d, J = 8.2 Hz, 1H, H-1), 7.65 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.41 (d, J = 8.2 Hz, 1H, H-5), 7.33 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.90 (d, J = 8.2 Hz, 1H, H-2), 6.85 (s, 1H, H-4), 4.15 (deformed t, J = 5.5, 6.9 Hz, 2H, H-1′), 3.56 (deformed t, J = 5.5, 6.9 Hz, 2H, H-3′), 3.35 (s, 3H, H-4′), 2.09 (m, 2H, H-2′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.3 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.3 (C-5a), 134.3 (C-6), 128.3 (C-1), 126.7 (C-8), 123.9 (C-7), 122.0 (C-8a), 117.8 (C-5), 115.8 (C-2), 113.6 (C-9a), 100.8 (C-4), 68.9 (C-3′), 65.6 (C-1′), 58.8 (C-4′), 29.5 (C-2′).

3-(2-(Methoxymethoxy)ethoxy)-9H-xanthen-9-one (25): Yield: 77%; White amorphous solid; M.P. 108–109 °C; m/z, C₁₇H₁₆O₅: 300, 269, 239, 212, 184, 155, 139, 89, 59; IR ν_max cm⁻¹: 2923, 1649, 1618, 1258; ¹H NMR (600 MHz, CDCl₃): δ_H 8.21 (d, J = 8.2 Hz, 1H, H-8), 8.13 (d, J = 8.2 Hz, 1H, H-1), 7.57 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.31 (d, J = 8.2 Hz, 1H, H-5), 7.25 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.85 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.76 (d, J = 2.7 Hz, 1H, H-4), 4.66 (s, 2H, H-3′), 4.16 (deformed t, J = 4.1, 5.5 Hz, 2H, H-1′), 3.87 (deformed t, J = 4.1, 5.5 Hz, 2H, H-2′), 3.34 (s, 3H, H-4′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 164.2 (C-3), 157.9 (C-4a), 156.2 (C-5a), 134.3 (C-6), 128.2 (C-1), 126.6 (C-8), 123.9 (C-7), 121.9 (C-8a), 117.7 (C-5), 115.9 (C-2), 113.5 (C-9a), 100.8 (C-4), 96.7 (C-3′), 67.9 (C-1′), 65.6 (C-2′), 55.4 (C-4′).

3-(2-(2-Methoxyethoxy)ethoxy)-9H-xanthen-9-one (26): Yield: 70%; White amorphous solid; M.P. 73–75 °C; m/z, C₁₈H₁₈O₅: 314, 282, 256, 239, 212, 184, 155, 139, 119, 103, 75, 59; IR ν_max cm⁻¹: 2899, 1623, 1469, 1256; ¹H NMR (600 MHz, CDCl₃): δ_H 8.17 (dd, J = 2.7, 8.2 Hz, 1H, H-8), 8.08 (d, J = 8.2 Hz, 1H, H-1), 7.54 (deformed t, J = 6.9, 8.2 Hz, 1H, H-6), 7.27 (d, J = 8.2 Hz, 1H, H-5), 7.22 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.81 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.71 (s, 1H, H-4), 4.12 (deformed t, J = 4.1, 5.5 Hz, 2H, H-1′), 3.80 (deformed t, J = 4.1, 5.5 Hz, 2H, H-2′), 3.64 (deformed t, J = 4.1, 5.5 Hz, 2H, H-4′), 3.49 (dd, J = 4.1, 5.5 Hz, 2H, H-3′), 3.30 (s, 3H, H-5′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.1 (C-9), 164.2 (C-3), 157.9 (C-4a), 156.1 (C-5a), 134.2 (C-6), 128.1 (C-1), 126.5 (C-8), 123.8 (C-7), 121.9 (C-8a), 117.7 (C-5), 115.8 (C-2), 113.6 (C-9a), 100.8 (C-4), 72.0 (C-4′), 70.9 (C-1′), 69.4 (C-3′), 68.0 (C-2′), 59.1 (C-5′).

Methyl 2-((9-oxo-9H-xanthen-3-yl)oxy)acetate (27): Yield: 92%; White amorphous solid; M.P. 151–152 °C; m/z, C₁₆H₁₂O₅: 284, 225, 195, 155, 139, 119, 92, 63; IR ν_max cm⁻¹: 2958, 1623, 1464, 1256; ¹H NMR (600 MHz, CDCl₃): δ_H 8.26 (d, J = 8.2 Hz, 1H, H-8), 8.22 (d, J = 9.6 Hz, 1H, H-1), 7.65 (t, J = 6.9 Hz, 1H, H-6), 7.39 (d, J = 6.9 Hz, 1H, H-5), 7.32 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.94 (dd, J = 2.7, 9.6 Hz, 1H, H-2), 6.81 (d, J = 2.7 Hz, 1H, H-4), 4.73 (s, 2H, H-1′), 3.81 (s, 3H, H-3′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.2 (C-9), 168.5 (C-2′), 163.0 (C-3), 157.8 (C-4a), 156.2 (C-5a), 134.5 (C-6), 128.7 (C-1), 126.7 (C-8), 124.1 (C-7), 121.9 (C-8a), 117.8 (C-5), 116.6 (C-2), 113.2 (C-9a), 101.4 (C-4), 65.3 (C-1′), 52.6 (C-3′).

Ethyl 2-((9-oxo-9H-xanthen-3-yl)oxy)acetate (28): Yield: 77%; White amorphous solid; M.P. 123–124 °C; m/z, C₁₇H₁₄O₅: 298, 225, 195, 155, 139, 119, 92, 63; IR ν_max cm⁻¹: 2983, 1618, 1464, 1207; ¹H NMR (500 MHz, CDCl₃): δ_H 8.28 (d, J = 8.0 Hz, 1H, H-8), 8.24 (d, J = 9.2 Hz, 1H, H-1), 7.66 (m, 1H, H-6), 7.41 (d, J = 8.0 Hz, 1H, H-5), 7.33 (t, J = 8.0 Hz, 1H, H-7), 6.95 (dd, J = 2.3, 8.0 Hz, 1H, H-2), 6.83 (d, J = 2.3 Hz, 1H, H-4), 4.72 (s, 2H, H-1′), 4.28 (q, J = 6.9 Hz, 2H, H-3′), 1.30 (t, J = 6.9 Hz, 3H, H-4′); ¹³C NMR (125 MHz, CDCl₃): δ_C 176.2 (C-9), 168.0 (C-2′), 163.1 (C-3), 157.8 (C-4a), 156.3 (C-5a), 134.5 (C-6), 128.6 (C-1), 126.7 (C-8), 124.0 (C-7), 122.0 (C-8a), 117.8 (C-5), 116.6 (C-2), 113.2 (C-9a), 101.4 (C-4), 65.5 (C-1′), 61.8 (C-3′), 14.2 (C-4′).

(R)-3-(3-Hydroxy-2-methylpropoxy)-9H-xanthen-9-one (29): Yield: 95%; White amorphous solid; M.P. 149–150 °C; [α] 25/D −54.5°, in methanol; m/z, C₁₇H₁₆O₄: 284, 212, 184, 155, 139, 55; IR ν_max cm⁻¹: 3451, 2923, 1618, 1464, 1256; ¹H NMR (600 MHz, CDCl₃): δ_H 8.29 (d, J = 8.2 Hz, 1H, H-8), 8.17 (d, J = 8.2 Hz, 1H, H-1), 7.66 (m, 1H, H-6), 7.40 (d, J = 8.2 Hz, 1H, H-5), 7.34 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.88 (dd, J = 2.7, 8.2 Hz, 1H, H-2), 6.82 (d, J = 2.7 Hz, 1H, H-4), 4.04 (m, 2H, H-1′), 3.73 (m, 1H, H-3′), 2.23 (m, 2H, H-2′), 1.08 (d, J = 6.9 Hz, 3H, H-4′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.4 (C-9), 164.6 (C-3), 158.0 (C-4a), 156.3 (C-5a), 134.4 (C-6), 128.3 (C-1), 126.7 (C-8), 123.9 (C-7), 122.0 (C-8a), 117.8 (C-5), 115.8 (C-2), 113.6 (C-9a), 100.8 (C-4), 70.9 (C-1′), 65.0 (C-3′), 35.7 (C-2′), 13.7 (C-4′).

(S)-3-(3-hydroxy-2-methylpropoxy)-9H-xanthen-9-one (30): Yield: 58%; White amorphous solid; M.P. 149–151 °C; [α] 25/D + 17.7°, in methanol; m/z, C₁₇H₁₆O₄: 284, 212, 184, 139, 92, 55; IR ν_max cm⁻¹: 3448, 2923, 1618, 1466, 1258; ¹H NMR (600 MHz, CDCl₃): δ_H 8.29 (d, J = 8.2 Hz, 1H, H-8), 8.17 (d, J = 9.6 Hz, 1H, H-1), 7.66 (m, 1H, H-6), 7.40 (d, J = 8.2 Hz, 1H, H-5), 7.34 (deformed t, J = 6.9, 8.2 Hz, 1H, H-7), 6.88 (dd, J = 2.7, 9.6 Hz, 1H, H-2), 6.82 (d, J = 2.7 Hz, 1H, H-4), 4.04 (m, 2H, H-1′), 3.73 (m, 2H, H-3′), 2.23 (m, 1H, H-2′),1.08 (d, J = 6.9 Hz, 3H, H-4′); ¹³C NMR (150 MHz, CDCl₃): δ_C 176.4 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.3 (C-5a), 134.4 (C-6), 128.3 (C-1), 126.7 (C-8), 123.9 (C-7), 122.0 (C-8a), 117.8 (C-5), 115.8 (C-2), 113.6 (C-9a), 100.8 (C-4), 71.0 (C-1′), 65.1 (C-3′), 35.7 (C-2′), 13.7 (C-4′).

X-ray diffraction analysis

X-ray analyses for compounds 5 and 13, which exist in the single crystal form, were performed using Bruker APEX II DUO CCD diffractometer, employing MoKα radiation (λ = 0.71073 Å) with φ and ω scans, at room temperature. Data reduction and absorption correction were performed using the SAINT and SADABS programs⁴⁴. Both structures were solved by direct methods and refined by full-matrix, least-squares techniques on F² using the SHELXTL software package^45,46. All H atoms were placed in geometrically idealised positions and constrained to ride on their parent atoms with C–H distance in the range of 0.93–0.97 Å and U_iso(H) = 1.2 U_eq(C), while U_iso(H) for methyl H atoms were set at 1.5 U_eq(C) and each group was allowed to rotate freely about its C–C bond. Crystallographic data for compounds 5 and 13 had been deposited at the Cambridge Crystallographic Data Centre (CCDC) with the deposition numbers of 1863942 and 1863946, respectively. Copies of available material can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223–336033 or e-mail: deposit@ccdc.cam.ac.uk). The ORTEP (Oak Ride Thermal Ellipsoid Plot Program) of 5 and 13 are shown in Figure 2 and X-ray crystallographic data are presented in Table 1.

Figure 2.

ORTEP diagram of compounds 5 and 13.

Table 1.

Crystal data and parameters for the structure refinement of 5 and 13.

Compound	5	13
CCDC number	1863942	1863946
Molecular formula	C17H16O3	C18H16O3
Molecular weight	268.30	280.31
Crystal system	Monoclinic	Triclinic
Space group	P21/c	$P\overline{1}$
a (Å)	12.1571 (13)	7.503 (6)
b (Å)	5.0697 (5)	11.297 (9)
c (Å)	21.793 (2)	17.769 (14)
α (°)	90	98.931 (10)
β (°)	98.156 (5)	95.355 (11)
γ (°)	90	103.272 (10)
V (Å3)	1329.6 (2)	1435 (2)
Z	4	4
Dcalc (Mg m−3)	1.340	1.297
Crystal dimensions (mm)	0.49 × 0.08 × 0.06	0.38 × 0.27 × 0.25
μ (mm−1)	0.09	0.09
Tmin/Tmax	0.747, 0.977	0.658, 0.952
Reflections measured	32949	18566
Ranges/indices (h, k, l)	−14→14,	−8→8,
	−6→6,	−13→13,
	−25→25	−21→21
θ limit (°)	2.4–29.8	2.4–21.9
Unique reflections	2325	5042
Observed reflections (I >  2σ(I))	1334	2531
Parameters	181	379
Goodness of fit on F2	1.03	1.08
R1, wR2 [I ≥  2σ(I)]	0.066, 0.172	0.096, 0.266
R1, wR2 [all data]	0.120, 0.216	0.165, 0.335

Biological assay

Cholinesterase inhibition activity assay

The assay was carried out according to Ellman’s method with slight modification by using AChE and BChE⁴¹. Tacrine was used as a positive control in this assay. Acetylcholinesterase (AChE) (source: Electrophorus electricus), butyrylcholinesterase (BChE) (source: Equine serum), acetylthiocholine iodide (ATCI), S-butyrylthiocholine chloride (BTCC) and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) were dissolved in sodium phosphate buffer (100 mM, pH 8.0). A stock solution of tacrine and samples were prepared in DMSO and further diluted using sodium phosphate buffer. Firstly, the samples were evaluated at the concentration of 1 and 4 µg/mL for AChE and BChE inhibition activities, respectively. The xanthone derivatives that showed more than 50% of enzyme inhibition were further evaluated in a series of concentration from 0.0625 to 1 μg/mL to determine their IC₅₀ values. Briefly, 200 µL of DTNB (0.5 mg/mL) was added into a 96 well plate for AChE assay while 150 µL of sodium phosphate buffer and 50 µL of DTNB (0.5 mg/mL) were added for BChE assay. Then, 20 µL of the compounds were added into the wells together with 20 µL of AChE solution (0.266 U/mL) or BChE solution (0.15 U/mL). The mixtures were incubated for 15 min in the dark at 37 °C. After that, 10 µL of ATCI (0.2052 mg/mL) or BTCC (0.5 mg/mL) were added into the wells to initiate the reaction. Immediately, the absorbance was measured at 412 nm for 10 times with 1-min interval. The percentage of enzyme inhibition was calculated using the following formula:

$$\text{Percentage inhibition}=\left({\text{Slope}}_{\text{control}}–{\text{Slope}}_{\text{drug}}/{\text{Slope}}_{\text{control}}\right)\times \text{1}00$$

AChE enzyme kinetic study

The enzyme inhibition modes of AChE by compounds 23 and 28 were evaluated by using the substrate ATCI in the range of concentrations (0.43, 0.86, 1.73, 3.45, and 6.90 mM) with the absence and presence of compounds. The concentration of the compound used were 2.91, 0.73, 0.36 and 0.18 μM for 23 and 3.36, 1.68, 0.84 and 0.42 μM for 28. The mode of inhibition was determined by using Lineweaver–Burk plot⁴⁷.

Computational studies

Flexible docking

The X-ray crystal structure of Electrophorus electricus acetylcholinesterase (PDB ID: 1C2O) was retrieved from the protein database bank (PDB) (http://www.rcsb.org). Although the resolution is 4.2 Å, visual inspection showed minimal difference in the position of the amino acid residues in the active site as compared with some high-resolution crystal structures available in the PDB. Thus, this protein crystal structure was deemed suitable to be used in our docking study. The ligand-binding site was identified based on the position of the inhibitor-binding site as reported by literature. Residues W108, E224, S225, Y313, Y355, F356 and Y359 were specifically selected as flexible based on the observation by⁴⁸, in which they superposed 68 AChE complex crystal structures over the crystal structure of human apo-AChE (PDB ID: 4PQE)⁴⁸. Additionally, several conserved water molecules (structural water molecules) were inserted manually to the binding site according to the protein crystal structure (PDB ID: 1ACJ) to create a more realistic environment of AChE binding site⁴⁹.

Both compound 23 and tacrine were then prepared and minimised before the docking procedure. The DS Flexible Docking module was adopted for the receptor-ligand docking. The ligand was docked to the active site of each receptor conformation using LibDock. The maximum number of compounds conformations generated were set at 225 numbers followed by clustering to remove similar ligand pose. The selected protein side-chains were refined in the presence of the rigid ligand using ChiRotor and the final ligand pose was optimised using CDOCKER.

Statistical analysis

The values were expressed as mean ± SD from three independent experiments. The statistical significance was calculated by one-way analysis of variance (ANOVA), followed by Tukey’s test. The percentage inhibition of AChE and BChEand Lineweaver-Burk plot were plotted using GraphPad Prism version 7.

Results and discussion

Characterisation of 3-hydroxyxanthone (1)

The structure of 3-hydroxyxanthone (1) was elucidated using MS, FTIR and NMR spectroscopies. The mass spectrum demonstrated a molecular ion peak at m/z 212, which corresponds to the molecular weight of 1. The FTIR spectrum displayed absorptions of free –OH at 3112 cm⁻¹, sp³ C–H at 2888 cm⁻¹, C=O at 1644 cm⁻¹, aromatic C=C at 1607 cm⁻¹, and C–O at 1228 cm⁻¹. The ¹H NMR spectrum showed the presence of a chelated hydroxyl group at position C-3, as indicated by a singlet peak at the downfield region, δ 10.93. The de-shielding effects of carbon nucleus by oxygen atom leads to the exposure of the higher external magnetic field, which in turn, requires higher frequency to achieve resonance and results in a peak in a downfield region. Besides, three doublets were detected at δ 7.99, 7.54, and 6.82 for H-1, H-5, and H-4, respectively, while a deformed triplet peak was spotted at δ 7.38 for H-7. Two sets of doublet of doublet were observed at δ 8.10 and 6.86 with respect to H-8 and H-2, while a triplet of doublet was detected at δ 7.75 for H-6. These signals revealed the presence of two aromatic rings in the main skeleton of xanthone. For ¹³C NMR, the highest chemical shift was given by the carbonyl carbon at C-9 (δ 175.3) and followed by the hydroxyl carbon at C-3 (δ 164.6). These peaks were attributed to the electron-withdrawing effect of oxygen atom. On the other hand, the aromatic carbon signals of xanthone skeleton were found in the range of δ 103 to 158 ppm. The ¹H and ¹³C NMR spectral data and melting point of 1 are in good agreement with the literature data^50,51 and were used as the standard spectra for the spectral data analyses of xanthone derivatives (2–30).

Characterisation of xanthone derivatives (2–30)

The mass spectra of the xanthone derivatives demonstrated molecular ion peaks that correspond to the molecular weight of the respective compounds. The ¹H NMR spectral data of the xanthone derivatives (2–30) revealed an absence of the chelated hydroxyl singlet peak at the chemical shift region near δ 11 ppm. Moreover, ¹H NMR spectra of the xanthone derivatives showed additional peaks when compared to that of 1. For xanthone derivatives 2–20, 24–30, additional peaks appeared in different regions of chemical shifts from those represented the main skeleton (δ 6.8–8.3), which was seen in the spectrum of 1. Xanthone derivatives 21–23, which bear a phenyl substituent have additional peaks in their ¹H NMR spectra that are overlapped with the chemical shift region of the main skeleton of xanthone (1). Analyses of these additional peaks were found to be consistent with the proposed structures. Based on the integration values in the spectra, the number of protons are in agreement with that in the structures.

Similarly, additional peaks were observed in the ¹³C NMR spectra for the xanthone derivatives (2–30). Besides, the absence of a broad hydroxyl (–OH) peak at the region of 3100–3500 cm⁻¹ in the FTIR spectra of xanthone derivatives 2–28 further confirmed the successful attachment of the substituent groups at C-3 position of 1. The results showed that the hydroxyl group at position C-3 was successfully substituted by the respective hydrocarbon, ether, ester, and hydroxyl substituent groups.

AChE and BChE inhibitory activities

The parent compound, 3-hydroxyxanthone, 1, together with its derivatives 2–30 were evaluated for their cholinesterase inhibitory activities using the Ellman’s method with minor modifications. Tacrine was used as a positive control throughout the experiment. To begin with, compounds 1–30 were evaluated with AChE and BChE inhibition activities at the concentration of 1 µg/mL. Remarkably, all compounds exhibited good to moderate inhibition activity against AChE but not BChE. The results strongly indicate that the compounds were more selective towards AChE over BChE. A previous molecular docking study focussed on 3,5-dimethoxy-N-methylenebenzenamine derivatives showed that a small compound is unable to bind efficiently to the BChE binding site which has a larger volume of the bottom gorge. Thus, BChE binding site is ideal in accommodating bulkier substrate. As a result, all the derivatives with small substituent groups such as methoxy, hydroxy, and nitro groups, showed potent AChE inhibition effect and highly selective towards AChE over BChE⁵². Moreover, a recent study reported an oxygenated xanthone with bulky substituents of geranyl unit at C-8 and acetate at C-14 as a potent and selective BChE inhibitor. Besides two hydrogen bonding between the xanthone core and Tyr128 and Asn68 from BChE active pocket, hydrophobic interactions were observed between the geranyl and Trp82 from the choline-binding site, and His438 and Trp430 from the catalytic active site⁵³, deducing that a more bulky substituent is needed for BChE inhibitory effect. The synthesised xanthone derivatives in this study are considered as smaller compounds and thus exhibiting lower inhibitory effect towards BChE, if compared to AChE.

Overall, most of the compounds are able to reduce 50% of AChE activity at the concentration of 1 µg/mL, except for 13, 14, 18, 24, 29, and 30. The active compounds were then determined for their IC₅₀ values and the result was tabulated in Table 2. Compounds 23 and 28 are the most potent xanthone derivatives with the same IC₅₀ values of 0.88 µM. Conversely, the xanthone derivatives exhibited weak BChE inhibition even a higher concentration of 4 μg/mL. Thus, the IC₅₀ values were not determined for BChE inhibition activities (Figures S1 and S2).

Table 2.

AChE and BChE inhibitory activities of compounds 1–30.

Compound	Substituent, R group	AChE IC50 (µM)	BChE Percentage of inhibition at 4 µg/mL (%)
1	H	2.39 ± 0.11a	6.55 ± 0.82
2	Propyl	1.86 ± 0.04bcd	11.94 ± 1.74
3	Butyl	1.40 ± 0.14efgh	17.58 ± 1.89
4	Isopropyl	1.41 ± 0.13efgh	20.68 ± 1.75
5	Isobutyl	1.18 ± 0.11ghij	9.78 ± 1.22
6	Isopentyl	1.84 ± 0.21bcd	5.47 ± 0.61
7	(S)-2-Methylbutyl	1.74 ± 0.20cde	24.78 ± 1.96
8	2-Ethylbutyl	1.15 ± 0.19hij	12.62 ± 1.26
9	2-Methylpentyl	2.07 ± 0.06abc	11.76 ± 0.98
10	Methylcyclobutyl	1.61 ± 0.11def	7.01 ± 1.16
11	Ethylcyclohexyl	1.27 ± 0.13fghij	24.33 ± 2.29
12	Buten-3-yl	2.09 ± 0.21abc	5.33 ± 0.17
13	Penten-4-yl	>3.57	20.89 ± 3.07
14	Hexen-5-yl	>3.40	16.35 ± 1.34
15	2-Methylpropenyl	1.60 ± 0.15defg	2.33 ± 0.30
16	2-Methylbuten-2-yl	1.86 ± 0.15bcd	7.96 ± 1.22
17	2-Methylpenten-2-yl	1.06 ± 0.12hij	20.94 ± 3.03
18	2-Butynyl	>3.79	4.53 ± 0.88
19	2-Pentynyl	1.21 ± 0.05fghij	5.24 ± 0.25
20	3-Methyl-propynyl	2.24 ± 0.17ab	5.00 ± 0.74
21	Benzyl	0.99 ± 0.19hij	5.04 ± 0.70
22	Phenylpropyl	1.22 ± 0.12fghij	15.50 ± 1.93
23	Phenylbutyl	0.88 ± 0.04j	9.19 ± 0.55
24	Methoxypropyl	>3.52	11.00 ± 1.38
25	Methoxymethoxyethyl	1.37 ± 0.12efghi	10.26 ± 1.24
26	Methoxyethoxyethyl	0.97 ± 0.06ij	17.76 ± 1.60
27	Methyl acetate	1.28 ± 0.13fghij	7.69 ± 0.34
28	Ethyl acetate	0.88 ± 0.15j	4.11 ± 0.35
29	(R)-Hydroxyl-2-methylpropyl	>3.52	13.94 ± 0.96
30	(S)-Hydroxyl-2-methylpropyl	>3.52	5.48 ± 0.31
Tacrine		0.06 ± 0.00k	96.55 ± 0.18*

Data represents the mean ± SD of three independent experiments; Different letters indicated significantly different at p < 0.05 between sample groups among AChE assay.

*The concentration of tacrine (positive control) used in the experiment was 1 µg/mL.

The result showed that twenty-three synthesised xanthones exhibited significant AChE inhibition with low IC₅₀ values ranging from 0.88 to 2.39 µM although their activities are weaker than the positive control, tacrine. The results are in good agreement with previous studies that xanthones are potential AChE inhibitors. By comparing their structural features, the reported xanthones are also bearing different substituents, particularly methoxyl, prenyl, benzyl, pyranyl, or hydroxyl, which were attached at different positions on the xanthone main skeleton^{15,32,54–57}.

Overall, all the xanthone derivatives exhibited stronger AChE inhibitory effect than the parent compound (1) except for 9, 12, and 20 that exhibited comparable effects with 1. The results are consistent with a previous study that etherified xanthones with alkyl groups, particularly methoxyl, prenylated oxyl, and allyl oxyl group at the position 3, possessed stronger anti-AChE activity than those with a hydroxyl group⁵⁸. SAR analysis revealed that hydrophobic interaction at position 3 of xanthone contributes positive effects to the AChE inhibition. By comparing the substituent groups attached to the C-3 position of the xanthone, the derivatives (24–28) with ether (ROR’) and ester (RCOOR’) groups demonstrated good AChE inhibition activities (except 24). The results indicated that the hydrogen bonding, contributed by –C–O–C from the substituent groups, play a vital role in the inhibitory activities, in addition to the hydrophobic interactions. The finding is supported by a previous molecular docking study where potent AChE inhibition effects of three xanthones, namely α-mangostin, γ-mangostin, and garcinone C were suggested to be contributed by the strong hydrogen bonding and π–σ interaction with several critical amino acid residues in the active sites of AChE¹⁵. Hydrogen bond was formed in between hydroxylated prenyl group at C-8 of garcinone C and His 440 of the AChE catalytic site. A π–σ interaction, on the other hand, formed between hydroxyl group at C-6 of garcinone C and Trp 84 from the choline binding site. Likewise, Trp 84 at the choline-binding site forms favourable π–π interactions with xanthone skeleton of α-mangostin and γ-mangostin¹⁵. Furthermore, another study reported that two oxygen atom at position 5 and 6 from macluraxanthone binds tightly to AChE through hydrogen bondings with the OH of Tyr 124 and O of Tyr 72, as well as hydrophobic interactions between hydrocarbons of macluraxanthone with several subsites, including PAS, anionic subsite (AS) and acyl-binding pocket (ABP) of AChE⁵⁵.

The results obtained from the AChE inhibition of xanthone derivatives showed that the chain length of hydrocarbons present in the C-3 side chain of xanthone influences the level of activities. Compound 3, which bears a 4-carbons chain, exhibited stronger inhibition activity if compared to 2 (3-carbons chain). A similar trend was observed for compounds 12–14 that bear an unsaturated 4- to 6-carbons chain. Compound 12 that has a butenyl substituent group showed an enhanced AChE inhibition activity than compounds 13 and 14. Among the linear type of substituents, our results suggest that a 4-carbons chain length is favourable to AChE inhibition effect either with or without the unsaturated bond. A previous study focussed on the amine side chains of the xanthone reported that elongation of the hydrocarbon chain attached to the xanthone skeleton units resulted in a drastic drop of AChE inhibition activities. The authors reported that three methylene units as the linker carbon between the xanthone skeleton and the amino side chain exhibited the most potent inhibition effect with an IC₅₀ value of 2.68 µM⁵⁹, which is consistent with our study where compounds 3 and 12 that bear a three methylene unit possessed strong effect with IC₅₀ values of 1.40 and 2.09 µM, respectively.

By comparing linear substituent groups of xanthone derivatives with the same carbon chain, the AChE inhibition effects were observed to have an increasing trend from alkynyl, alkenyl to alkyl substituents. The evidence is that the lowest IC₅₀ values were found to be exhibited by 3 (butyl), followed by 12 (butenyl) and 18 (butynyl). This might be due to the structural flexibility of 3 that bind sterically to AChE as the inhibition activity are correlated with hydrophobicity, electronic, inductive, or polar properties, and steric effects⁶⁰. However, the introduction of an unsaturated bond into the substituent groups that made up of branched hydrocarbon chain has a different influence on activity, depending on the length of the carbon chain. Xanthone derivative 17 (IC₅₀ = 1.06 μM), which has a substituent group of 5-carbons in chain length with an unsaturated bond (2-methylpenten-2-yl) possessed 2-folds stronger inhibitory activities than 9 (IC₅₀ = 2.07 μM) that bears a substituent group with the same chain length but without any unsaturated bond (2-methylpentyl). There is no significant difference in AChE inhibition being observed for 5 (isobutyl; IC₅₀ = 1.18 μM) and 15 (2-methylpropenyl; IC₅₀ = 1.60 μM) and between 6 (isopentyl; IC₅₀ = 1.84 μM) and 16 (isopentenyl; IC₅₀ = 1.86 μM). Moreover, compound 20 (IC₅₀ = 2.24 μM) that consisted of a branched substituent with 3-carbons chain length and a triple bond experienced a drop in activity when compared to compound 5. Therefore, we anticipated that the xanthone derivatives with branched substituents, the presence of unsaturated bond would improve the AChE inhibition activity for those substituent groups consisting of more than 5-carbons chain length. The finding is further supported by the xanthone derivatives with phenyl substituent, including compounds 21–23 that showed good inhibitory effects with IC₅₀ values ranging from 0.88 to 1.22 µM. The inhibition potency of phenyl substituted xanthone derivatives could be explained by potential hydrophobic interaction and π–π stacking with the aromatic residues of the enzyme gorge in AChE⁶¹. A high degree of lipid solubility compound favours the crossing of the blood–brain barrier (BBB) by transmembrane diffusion and also the uptake by the peripheral tissues⁶². Hence, small molecule drugs with molecular weight less than 400 Da and lipid solubility such as the xanthone derivatives synthesised and presented in this study, could potentially cross the BBB and preferred in the drug development for brain diseases⁶³. As a summary, eleven xanthone derivatives (5, 8, 11, 17, 19, 21–23, 26–28) possessed potent AChE inhibition activity. The overall results deduced that 3-O-substituted xanthones that carry a saturated linear hydrocarbon side chain of 4-carbons in chain length with an addition of phenyl group, or diether or ester are beneficial in AChE inhibition activity.

AChE enzyme kinetic analysis

The enzyme inhibition mode of two promising compounds, 23 and 28 against AChE was analysed by a double-reciprocal Lineweaver–Burk plot as shown in Figures 3 and 4. Both the maximal velocity of the AChE enzyme-substrate compound reaction (V_max) and the affinity (K_m) were affected by the compound concentration as shown in the kinetic parameters calculated from the double reciprocal trend lines, hence indicating a mixed-mode inhibition. The Michaelis–Menten parameters are tabulated in Table S1. In a mixed-mode inhibition, the inhibitors can bind to the free enzyme or the enzyme-substrate complex. The binding of inhibitors to the enzyme-substrate complex resulted in a reduction in the complex affinity towards substrates, thus explaining the increase in K_m.

Figure 3.

Lineweaver–Burk plot of 3-(4-phenylbutoxy)-9H-xanthen-9-one (23) against AChE. Bar indicates the standard deviation.

Figure 4.

Lineweaver–Burk plot of ethyl 2-((9-oxo-9H-xanthen-3-yl)oxy)acetate (28) against AChE. Bar indicates the standard deviation.

Binding interactions of 3-(4-phenylbutoxy)-9H-xanthen-9-one (23) with AChE

It is known that the amino acid residues surrounding the active site of Electrophorus electricus AChE are highly identical to the human AChE^64,65. In general, the active sites of AChE contain anionic catalytic sites which are located at the bottom of the gorge while peripheral anionic binding sites at the entrance (mouth) of the gorge. Inhibitors such as tacrine which is comprised of a small volume group could enter easily into the gorge and interact with the residing active catalytic site Trp86 and Tyr337 residues, which is located at the bottom of the gorge, as observed in the crystal structure and molecular docking study⁴⁹. Similarly, we observed that the xanthone ring could protrude deeply into the active site and interact via extensive π–π stacking with the indole and phenol side chains of Trp86 and Tyr337 (Figure 5). Apart from that, the planar geometry form of the xanthone ring as supported by the XRD analysis could also prove to be an important factor in aiding the compound’s binding process. Besides, the position of the carbonyl group of compound 23 is also similar to the amino group of tacrine, which is directed towards the hydration site comprised of D74, T83, W86, N87, and S125 residues. Since these water molecules are highly buried, they could possibly involve in a complex hydrogen bonding interaction with the inhibitors. It is likely that the carbonyl group could form hydrogen-bonding interactions with these buried water molecules thus enforcing its binding in the active site. In addition, the substituted alkyl benzene ring could interact with the phenol side chain of Y72 via π–π interaction further strengthening its binding interactions in the AChE active-site gorge.

Figure 5.

Binding interactions of tacrine and 3-(4-phenylbutoxy)-9H-xanthen-9-one (23) (carbon atoms are coloured green) with the adjacent amino residues (carbons atoms are purple) in AChE active site. Other atoms are coloured according to elements. (a) and (b) represent the docking results of the ligands to Electrophorus electricus AChE (PDB ID: 1C2O) while (c) and (d) represent the docking results of the ligands to human AChE (PDB ID: 4PQE).

Attempts to correlate the predicted CDOCKER binding energy with the AChE inhibitory activity were however unsuccessful. Thus, several scoring functions including LigScore1, LigScore2, PLP1, PLP2, Jain, PMF, PMF04 and Libdock score were explored further to quantify the interactions of compound 23 and tacrine with Electrophorus electricus AChE. Among the scoring functions, only the Jain score managed to give a positive correlation. The Jain score is a sum of five interaction terms including lipophilic interactions, polar attractive interactions, polar repulsive interactions, solvation of the protein and ligand, and an entropy term of the ligand⁶⁶. According to the predicted Jain score, compound 23 is predicted to bind favourable to the human AChE. This suggests that this compound could potentially serve as a lead AChE inhibitor and may be further tested in the human enzyme model.

Conclusions

In summary, 3-hydroxyxanthone (1) and a series of twenty-nine new xanthone derivatives (2–30) were synthesised successfully by reacting 1 with seven different types of hydrocarbon substituents, including alkyl, alkenyl, alkynyl, alkylphenyl, ether, ester, and hydroxyl groups. These xanthones possessed stronger anticholinergic activities towards AChE than BChE. Eleven xanthone derivatives were found to exhibit potent AChE inhibition effect with the IC₅₀ values lower than 1.28 µM. SAR study revealed that hydrophobic interaction and hydrogen bonding of the substituent groups contribute to the inhibition effects, particularly the substituent group that is made up of saturated linear hydrocarbon with 4-carbons in the chain and preferably with an addition of phenyl or oxygenated group. Molecular docking study on 23 further confirmed the importance of the hydrophobic group as the side chain of these derivatives in the anti-AChE activities through π-π interactions in the active site, in addition to π–π stacking and hydrogen-bonding contributed by the xanthone main skeleton. The present work revealed that the new xanthone derivatives, particularly 23 and 28 are potent and selective AChE inhibitors that are potential lead compounds to be developed into anti-Alzheimer drug.

Funding Statement

The present work was financially supported by the Fundamental Research Grant Scheme (FRGS) [FRGS/1/2019/STG01/TAYLOR/02/1] from the Ministry of Education (MOE).

Disclosure statement

No potential conflict of interest was reported by the author(s).