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

Article abstract of DOI:10.1080/14756366.2021.1882452

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.

Full text of DOI:10.1080/14756366.2021.1882452

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
New 3-O-substituted xanthone derivatives as
promising acetylcholinesterase inhibitors
Zi Han Loh, Huey Chong Kwong, Kok Wai Lam, Soek Sin Teh, Gwendoline
Cheng Lian Ee, Ching Kheng Quah, Anthony Siong Hock Ho & Siau Hui Mah
To cite this article: Zi Han Loh, Huey Chong Kwong, Kok Wai Lam, Soek Sin Teh, Gwendoline
Cheng Lian Ee, Ching Kheng Quah, Anthony Siong Hock Ho & Siau Hui Mah (2021) New 3-O-
substituted xanthone derivatives as promising acetylcholinesterase inhibitors, Journal of Enzyme
Inhibition and Medicinal Chemistry, 36:1, 627-639, DOI: 10.1080/14756366.2021.1882452
To link to this article: https://doi.org/10.1080/14756366.2021.1882452
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 627–639
https://doi.org/10.1080/14756366.2021.1882452
RESEARCH PAPER
New 3-O-substituted xanthone derivatives as promising
acetylcholinesterase inhibitors
Zi Han Loha, Huey Chong Kwongb, Kok Wai Lamc, Soek Sin Tehd, Gwendoline Cheng Lian Eee, Ching Kheng
Quahf, Anthony Siong Hock Hoa and Siau Hui Maha,g
b
aSchool of Biosciences, Taylor’s University, Lakeside Campus, Subang Jaya, Malaysia; School of Chemical Sciences, Universiti Sains Malaysia,
c
George Town, Malaysia; Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia;
e
dEnergy and Environment Unit, Engineering and Processing Division, Malaysian Palm Oil Board, Bandar Baru Bangi, Malaysia; Department of
f
Chemistry, Faculty of Science, University Putra Malaysia, Serdang, Malaysia; X-ray Crystallography Unit, School of Physics, Universiti Sains
g
Malaysia, George Town, Malaysia; Centre for Drug Discovery and Molecular Pharmacology, Faculty of Health and Medical Sciences, Taylor's
University, Lakeside Campus
ABSTRACT
ARTICLE HISTORY
Received 21 September 2020
Revised 16 December 2020
Accepted 20 January 2021
A new series of 3-O-substituted xanthone derivatives were synthesised and evaluated for their anti-cholin-
ergic 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 mM. The AChE
enzyme kinetic study of 3-(4-phenylbutoxy)-9H-xanthen-9-one (23) and ethyl 2-((9-oxo-9H-xanthen-3-yl)ox-
y)acetate (28) showed a mixed inhibition mechanism. Molecular docking study showed that 23 binds to
the active site of AChE and interacts via extensive p–p stacking with the indole and phenol side chains of
Trp86 and Tyr337, besides the hydrogen bonding with the hydration site and p–p interaction with the
phenol side chain of Y72. This study revealed that 3-O-alkoxyl substituted xanthone derivatives are poten-
tial lead structures, especially 23 and 28 which can be further developed into potent AChE inhibitors.
KEYWORDS
Alzheimer’s disease; enzyme
kinetic study; molecular
docking; synthesis;
structure–activity relation-
ship study
Introduction
neurotransmitter in the cholinergic neurotransmission, acetylcho-
line (ACh)3,5–7. The deficit of ACh has profound effects on the sig-
Dementia is a syndrome of deterioration in memory, thinking,
orientation, comprehension, calculation, learning capacity, lan-
guage, 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, frontotempo-
ral 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.
naling that involves learning ability and long-term memory8–10
.
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
ACh10–12. 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 action14,15. Furthermore, AChE could facilitate the
formation of amyloid fibril to obtain a stable AChE-b-amyloid
complex, which is more toxic than single b-amyloid peptides5.
Hence, targeting AChE could hinder the production of AChE-
b-amyloid complexes. The current management of AD is focussing
on the enhancement of the concentration of ACh in the synaptic
cleft by inhibiting cholinesterase16,17. 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 decel-
eration of the progression of the disease and an increase of atten-
tion span21. Therefore, research studies on the discovery and
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 development of AChE inhibitors with higher potency and efficacy
CONTACT Siau Hui Mah
siauhui.mah@taylors.edu.my School of Biosciences, Taylor’s University, Lakeside Campus, 1, Jalan Taylor’s, 47500 Subang Jaya,
Selangor, Malaysia
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

628
Z. H. LOH ET AL.
Figure 1. Reaction scheme for the synthesis of xanthone derivatives.
is essential since these inhibitors are the therapeutically potential Electrothermal 9100 Series Apparatus. Mass spectra of derivatives
as a principal approach of AD treatments. The adverse effects were acquired from Gas Chromatography-Mass Spectrometry
were associated with currently available drugs and worse still that (GCMS) (Agilent J&W) equipped with GC column HP-5MS (30 m ꢀ
there is no cure for AD up to date. Current approaches for the 0.25 mm ꢀ 0.25 mm). 1H and 13C NMR spectral data of derivatives
treatment of AD are solely focussing on maintaining the mental were obtained from JEOL JNM-ECX 500 or JNM-ECZ 600 R NMR
function, managing behavioural symptoms, and slowing down or spectrometers. FTIR spectra of derivatives were recorded on Perkin
delaying the symptoms of the disease22. Hence, this study aimed Elmer Spectrum 100 (Perkin Elmer) equipped with attenuated total
to search for lead compounds with significant cholinesterase reflection (ATR). X-ray analysis was performed using Bruker APEX II
inhibitory activities that are highly potential to be further devel- DUO CCD diffractometer. Optically active xanthones were analysed
oped into alternative drugs to treat AD more effectively.
using polarimeter Optika POL-1 bench polarimeter. The absorb-
ance of the biological assay was measured with microplate reader
BioTek Epoch 2.
To date, more than 50% of the marketed drugs were devel-
oped from natural products, especially secondary metabolites and
their derivatives23,24. Xanthones are one of these important sec-
ondary metabolites that possessed a broad spectrum of biological
activities such as anti-cancer25, antioxidant26, antibacterial27, anti-
fungal28, antimalarial29, antidiabetic30,31, anti-cholinergic15,32 and
anti-inflammatory activities33–36. The biological activities of xan-
thones 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 xanthone37–39. However, xan-
thones obtained from the natural resources are limited in substitu-
ent 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 sub-
stituents were synthesised. The chemical structures of the xan-
thone 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 sin-
gle-crystal X-ray diffraction analysis to further support the spec-
troscopy 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.
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, fil-
tered, 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 car-
bonate 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 chroma-
tography 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.
3-hydroxyxanthone (1): M.P. 251–253 ꢁC; m/z, C13H8O3: 212,
184, 155, 128, 102, 92, 77, 63, 51; IR ꢀmax cmꢂ1: 3115, 2884, 1641,
1609, 1586, 1566, 1450, 1226; 1H NMR (600 MHz, DMSO-d6): dH
10.93 (s, 1H, OH-3), 8.10 (dd, J ¼ 2.1, 8.3 Hz, 1H, H-8), 7.99 (d,
J ¼ 9.0 Hz, 1H, H-1), 7.75 (td, J ¼ 2.1, 7.6 Hz, 1H, H-6), 7.54 (d,
J ¼ 8.3 Hz, 1H, H-5), 7.38 (deformed t, J ¼ 7.6, 8.3 Hz, 1H, H-7), 6.86
(dd, J ¼ 2.1, 9.0 Hz, 1H, H-2), 6.82 (d, J ¼ 2.1 Hz, 1H, H-4); 13C NMR
(150 MHz, DMSO-d6): dC 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).
Material and methods
The solvents (analytical grade), silica gel 60, and thin-layer chro-
matography (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
3-Propoxy-9H-xanthen-9-one (2): Yield: 92%; White amorphous
Electrophorus electricus (500 UN), butyrylcholinesterase from Equine solid; M.P. 120–122 ꢁC; m/z, C16H14O3: 254, 212, 195, 184, 155, 139,
serum (1.2 KU), acetylthiocholine iodide, S-butyrylthiocholine chlor- 128, 113, 102, 92, 77, 63, 51; IR ꢀmax cmꢂ1: 2874, 1654 1623, 1232;
ide, and 5,50-Dithiobis (2-nitro benzoic acid) were obtained from 1H NMR (600 MHz, CDCl3): dH 8.20 (d, J ¼ 8.2 Hz, 1H, H-8), 8.10 (d,
Sigma Aldrich. Tacrine (Purity >98%) was obtained from Cayman J ¼ 8.2 Hz, 1H, H-1), 7.54 (deformed t, J ¼ 6.8, 8.2 Hz, 1H, H-6), 7.28
Chemical. The melting point of derivatives was determined by (d, J ¼ 8.2 Hz, 1H, H-5), 7.23 (deformed t, J ¼ 6.8, 8.2 Hz, 1H, H-7),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
629
6.78 (dd, J ¼ 2.8, 8.2 Hz, 1H, H-2), 6.67 (d, J ¼ 2.8, 1H, H-4), 3.88 (t, J ¼ 6.9, 9.2 Hz, 1H, H-1’b), 1.90 (m, 1H, H-20), 1.57 (m, 1H, H-3’a),
J ¼ 6.9 Hz, 2H, H-10), 1.77 (m, 2H, H-20), 0.99 (t, J ¼ 6.9 Hz, 3H, H-30); 1.28 (m, 1H, H-3’b), 1.03 (d, J ¼ 6.9 Hz, 3H, H-50), 0.95 (deformed t,
13C NMR (150 MHz, CDCl3): dC 176.1 (C-9), 164.6 (C-3), 158.0 (C-4a), J ¼ 6.9, 8.0 Hz, 3H, H-40); 13C NMR (125 MHz, CDCl3): dC 176.3 (C-9),
156.1 (C-5a), 134.1 (C-6), 128.0 (C-1), 126.5 (C-8), 123.7 (C-7), 121.9 164.9 (C-3), 158.1 (C-4a), 156.3 (C-5a), 134.2 (C-6), 128.2 (C-1),
(C-8a), 117.7 (C-5), 115.5 (C-2), 113.6 (C-9a), 100.6 (C-4), 70.1 (C-10), 126.7 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7
22.4 (C-20), 10.5 (C-30).
(C-9a), 100.7 (C-4), 73.5 (C-10), 34.6 (C-20), 26.2 (C-30), 16.5 (C-50),
11.4 (C-40).
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,
3-(2-Ethylbutoxy)-9H-xanthen-9-one (8): Yield: 50%; Colourless
92, 63; IR ꢀmax cmꢂ1: 2874, 1647, 1618, 1239; 1H NMR (600 MHz, oil; m/z, C19H20O3: 296, 212, 184, 155, 139, 119, 102, 85, 55; IR
CDCl3): dH 8.26 (d, J ¼ 8.2 Hz, 1H, H-8), 8.17 (d, J ¼ 9.6 Hz, 1H, H-1), ꢀmax cmꢂ1: 2958, 1659, 1623, 1256; 1H NMR (600 MHz, CDCl3): dH
7.62 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.37 (d, J ¼ 8.2 Hz, 1H, H- 8.26 (d, J ¼ 8.2 Hz, 1H, H-8), 8.17 (d, J ¼ 8.2 Hz, 1H, H-1), 7.60
5), 7.30 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7), 6.86 (dd, J ¼ 2.7, (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.35 (d, J ¼ 8.2 Hz, 1H, H-5),
9.6 Hz, 1H, H-2), 6.78 (d, J ¼ 2.7 Hz, 1H, H-4), 4.01 (t, J ¼ 6.9 Hz, 2H, 7.28 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7), 6.87 (dd, J ¼ 2.7, 8.2 Hz,
H-10), 1.78 (m, 2H, H-20), 1.49 (m, 2H, H-30), 0.97 (t, J ¼ 6.9 Hz, 3H, 1H, H-2), 6.79 (d, J ¼ 2.7 Hz, 1H, H-4), 3.90 (d, J ¼ 6.9 Hz, 2H, H-10),
H-40); 13C NMR (150 MHz, CDCl3): dC 176.3 (C-9), 164.7 (C-3), 158.1 1.68 (m, 1H, H-20), 1.46 (m, 4H, H-30 and H-50), 0.92 (t, J ¼ 6.9,
(C-4a), 156.2 (C-5a), 134.2 (C-6), 128.2 (C-1), 126.7 (C-8), 123.8 (C-7), 8.2 Hz, 6H, H-40 and H-60); 13C NMR (150 MHz, CDCl3): dC 176.2 (C-
122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7 (C-9a), 100.6 (C-4), 68.5 9), 164.9 (C-3), 158.1 (C-4a), 156.2 (C-5a), 134.2 (C-6), 128.1 (C-1),
(C-10), 31.1 (C-20), 19.3 (C-30), 13.9 (C-40).
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-10), 40.8 (C-20), 23.4 (C-30 and C-50), 11.2
3-Isopropoxy-9H-xanthen-9-one (4): Yield: 9.1%; White amorph-
ous solid; M.P. 76–78 ꢁC; m/z, C16H14O3: 254, 212, 184, 155, 139, (C-40 and C-60).
128, 102, 92, 77, 63, 51; IR ꢀmax cmꢂ1: 2923, 1659, 1623, 1280; H
3-((4-Methylpentyl)oxy)-9H-xanthen-9-one (9): Yield: 83%; White
1
NMR (600 MHz, CDCl3): dH 8.31 (d, J ¼ 8.2 Hz, 1H, H-8), 8.22 (d, crystalline solid; M.P. 66–68 ꢁC; m/z, C19H20O3: 296, 212, 184, 155,
J ¼ 8.2 Hz, 1H, H-1), 7.67 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.43 139, 119, 102, 85, 69; IR ꢀmax cmꢂ1: 2958, 1654, 1623, 1261; 1H
(d, J ¼ 8.2 Hz, 1H, H-5), 7.35 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7), NMR (600 MHz, CDCl3): dH 8.24 (d, J ¼ 8.2 Hz, 1H, H-8), 8.14 (d,
6.89 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-2), 6.85 (d, J ¼ 2.7 Hz, 1H, H-4), 4.68 J ¼ 8.2 Hz, 1H, H-1), 7.58 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.33
(m, 1H, H-10), 1.41 (d, J ¼ 6.9 Hz, 6H, H-20 and H-30); 13C NMR (d, J ¼ 8.2 Hz, 1H, H-5), 7.26 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7),
(150 MHz, CDCl3): dC 176.3 (C-9), 163.7 (C-3), 158.2 (C-4a), 156.3 (C- 6.82 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-2), 6.72 (s, 1H, H-4), 3.94 (t,
5a), 134.3 (C-6), 128.4 (C-1), 126.7 (C-8), 123.9 (C-7), 122.1 (C-8a), J ¼ 6.9 Hz, 2H, H-10), 1.77 (m, 2H, H-20), 1.58 (m, 1H, H-40), 1.31 (m,
117.7 (C-5), 115.6 (C-2), 114.3 (C-9a), 101.6 (C-4), 70.9 (C-10), 21.9 2H, H-30), 0.90 (d, J ¼ 6.9 Hz, 6H, H-50 and H-60); 13C NMR (150 MHz,
(C-20 and C-30).
CDCl3): dC 176.1 (C-9), 164.7 (C-3), 158.0 (C-4a), 156.2 (C-5a), 134.2
3-Isobutoxy-9H-xanthen-9-one (5): Yield: 29%; White crystalline
(C-6), 128.1 (C-1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5),
solid; M.P. 108–110 ꢁC; m/z, C17H16O3: 268, 212, 184, 155, 139, 119, 115.6 (C-2), 113.6 (C-9a), 100.6 (C-4), 69.0 (C-10), 35.1 (C-30), 27.9
102, 77, 57; IR ꢀmax cmꢂ1: 2899, 1659, 1623, 1280; 1H NMR
(500 MHz, CDCl3): dH 8.32 (dd, J ¼ 2.3, 8.0 Hz, 1H, H-8), 8.23 (d,
(C-40), 27.0 (C-20), 22.6 (C-50 and C-60).
3-(Cyclobutylmethoxy)-9H-xanthen-9-one (10): Yield: 14%;
J ¼ 8.0 Hz, 1H, H-1), 7.68 (dt, J ¼ 2.3, 6.9, 9.2 Hz, 1H, H-6), 7.44 (d, White amorphous solid; M.P. 95–97 ꢁC; m/z, C18H16O3: 280, 212,
J ¼ 9.2 Hz, 1H, H-5), 7.35 (deformed t, J ¼ 6.9, 8.0 Hz, 1H, H-7), 6.94 184, 155, 127, 107, 92, 69, 53; IR ꢀmax cmꢂ1: 2958, 1659, 1623,
1
(dd, J ¼ 2.3, 8.0 Hz, 1H, H-2), 6.86 (d, J ¼ 2.3 Hz, 1H, H-4), 3.84 (d, 1280; H NMR (600 MHz, CDCl3): dH 8.31 (dd, J ¼ 1.8, 7.3 Hz, 1H, H-
J ¼ 6.9 Hz, 2H, H-10), 2.15 (m, 1H, H-20), 1.06 (d, J ¼ 6.9 Hz, 6H, H-30 8), 8.23 (d, J ¼ 8.3 Hz, 1H, H-1), 7.67 (dt, J ¼ 1.8, 8.3 Hz, 1H, H-6),
and H-40); 13C NMR (125 MHz, CDCl3): dC 176.4 (C-9), 164.9 (C-3), 7.43 (d, J ¼ 8.3 Hz, 1H, H-5), 7.35 (deformed t, J ¼ 7.3, 8.3 Hz, 1H, H-
158.2 (C-4a), 156.3 (C-5a), 134.3 (C-6), 128.3 (C-1), 126.8 (C-8), 7), 6.93 (dd, J ¼ 2.8, 8.3 Hz, 1H, H-2), 6.86 (d, J ¼ 2.8 Hz, 1H, H-4),
123.9 (C-7), 122.1 (C-8a), 117.8 (C-5), 115.7 C-2), 113.7 (C-9a), 100.7 4.04 (d, J ¼ 7.3 Hz, 2H, H-10), 2.82 (m, 1H, H-20), 2.17 (m, 2H, H-40),
(C-4), 75.1 (C-10), 28.2 (C-20), 19.3 (C-30 and C-40).
1.95 (m, 4H, H-30 and H-50); 13C NMR (150 MHz, CDCl3): dC 176.4
3-(Isopentyloxy)-9H-xanthen-9-one (6): Yield: 82%; White crys- (C-9), 164.9 (C-3), 158.2 (C-4a), 156.3 (C-5a), 134.3 (C-6), 128.3 (C-1),
talline solid; M.P. 83–85 ꢁC; m/z, C18H18O3: 282, 212, 184, 155, 139,
126.7 (C-8), 123.9 (C-7), 122.1 (C-8a), 117.8 (C-5), 115.7 (C-2), 113.8
92, 71, 55; IR ꢀmax cmꢂ1: 2958, 1651, 1623, 1256; 1H NMR
(C-9a), 100.8 (C-4), 72.7 (C-10), 34.4 (C-20), 24.9 (C-30 and C-50), 18.6
(500 MHz, CDCl3): dH 8.24 (dd, J ¼ 2.3, 8.0 Hz, 1H, H-8), 8.15 (d, (C-40).
J ¼ 9.2 Hz, 1H, H-1), 7.58 (dt, J ¼ 2.3, 6.9, 9.2 Hz, 1H, H-6), 7.33 (d,
3-(2-Cyclohexylethoxy)-9H-xanthen-9-one (11): Yield: 59%;
J ¼ 9.2 Hz, 1H, H-5), 7.27 (deformed t, J ¼ 6.9, 8.0 Hz, 1H, H-7), 6.83 White crystalline solid; M.P. 134–136 ꢁC; m/z, C21H22O3: 322, 239,
(dd, J ¼ 2.3, 9.2 Hz, 1H, H-2), 6.74 (d, J ¼ 2.3 Hz, 1H, H-4), 4.01 (t, 212, 184, 155, 139, 111, 95, 69, 53; IR ꢀmax cmꢂ1: 2923, 1652, 1623,
1
J ¼ 6.9 Hz, 2H, H-10), 1.81 (m, 1H, H-30), 1.68 (m, 2H, H-20), 0.95 (d, 1256; H NMR (600 MHz, CDCl3): dH 8.28 (dd, J ¼ 2.8, 8.2 Hz, 1H, H-
J ¼ 6.9 Hz, 6H, H-40 and H-50); 13C NMR (125 MHz, CDCl3): dC 176.2 8), 8.19 (d, J ¼ 8.2 Hz, 1H, H-1), 7.63 (dd, J ¼ 2.8, 8.2 Hz, 1H, H-6),
(C-9), 164.7 (C-3), 158.0 (C-4a), 156.2 (C-5a), 134.2 (C-6), 128.1 (C-1), 7.38 (d, J ¼ 8.2 Hz, 1H, H-5), 7.31 (t, J ¼ 8.2 Hz, 1H, H-7), 6.88 (dd,
126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.6 (C-2), 113.7 J ¼ 2.8, 8.2 Hz, 1H, H-2), 6.80 (d, J ¼ 2.8 Hz, 1H, H-4), 4.06 (t,
(C-9a), 100.6 (C-4), 67.2 (C-10), 37.7 (C-20), 25.1 (C-30), 22.6 (C-40 and J ¼ 6.9 Hz, 2H, H-10), 1.70 (m, 7H, H-20, H-40, H-7’a, H-80), 1.50 (m,
C-50).
1H, H-30), 1.20 (m, 3H, H-50 and H-7’b), 0.97 (m, 2H, H-60); 13C NMR
(150 MHz, CDCl3): dC 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-10), 36.4
(C-20), 34.6 (C-30), 33.4 (C-40 and C-80), 26.6 (C-60), 26.3 (C-50 and
(S)-3-(2-Methylbutoxy)-9H-xanthen-9-one (7): Yield: 32%; White
amorphous solid; M.P. 46–48 ꢁC; [a] 25/D þ 16.5ꢁ, in methanol; m/
z, C18H18O3: 282, 212, 184, 155, 139, 92, 71, 55; IR ꢀmax cmꢂ1
:
2969, 1654, 1616, 1256; 1H NMR (500 MHz, CDCl3): dH 8.28 (d,
J ¼ 8.0 Hz, 1H, H-8), 8.20 (d, J ¼ 9.2 Hz, 1H, H-1), 7.63 (dt, J ¼ 2.3, C-70).
6.9, 9.2 Hz, 1H, H-6), 7.39 (d, J ¼ 9.2 Hz, 1H, H-5), 7.31 (deformed t,
3-(But-3-en-1-yloxy)-9H-xanthen-9-one (12): Yield: 46%; White
J ¼ 6.9, 8.0 Hz, 1H, H-7), 6.89 (dd, J ¼ 2.3, 9.2 Hz, 1H, H-2), 6.81 (d, amorphous solid; M.P. 85–87 ꢁC; m/z, C17H14O3: 266, 237, 212, 184,
J ¼ 2.3 Hz, 1H, H-4), 3.89 (dd, J ¼ 5.7, 9.2 Hz, 1H, H-1’a), 3.81 (dd, 155, 139, 121, 92, 75, 55; IR ꢀmax cmꢂ1: 2948, 1654, 1623, 1258; H
1

630
Z. H. LOH ET AL.
NMR (600 MHz, CDCl3): dH 8.27 (dd, J ¼ 2.1, 8.3 Hz, 1H, H-8), 8.18 (d, 213, 195, 155, 139, 119, 101, 83, 55; IR ꢀmax cmꢂ1: 2909, 1654,
1
J ¼ 9.0 Hz, 1H, H-1), 7.63 (dt, J ¼ 2.1, 6.9, 8.3 Hz, 1H, H-6), 7.37 (d, 1625, 1263; H NMR (600 MHz, CDCl3): dH 8.21 (d, J ¼ 8.2 Hz, 1H, H-
J ¼ 8.3 Hz, 1H, H-5), 7.31 (deformed t, J ¼ 6.9, 8.3 Hz, 1H, H-7), 6.87 8), 8.11 (d, J ¼ 9.6 Hz, 1H, H-1), 7.54 (deformed t, J ¼ 6.9, 8.2 Hz, 1H,
(dd, J ¼ 2.8, 9.0 Hz, 1H, H-2), 6.79 (d, J ¼ 2.8 Hz, 1H, H-4), 5.88 (m, H-6), 7.28 (d, J ¼ 8.2 Hz, 1H, H-5), 7.23 (deformed t, J ¼ 6.9, 8.2 Hz,
1H, H-30), 5.18 (dd, J ¼ 1.4, 17.2 Hz, 1H, H-4’a), 5.12 (dd, J ¼ 1.4, 1H, H-7), 6.79 (dd, J ¼ 2.7, 9.6 Hz, 1H, H-2), 6.68 (d, J ¼ 2.7 Hz, 1H,
10.3 Hz, 1H, H-4’b), 4.07 (t, J ¼ 6.9 Hz, 2H, H-10), 2.57 (m, 2H, H-20); H-4), 5.15 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-30), 3.91 (t, J ¼ 6.9 Hz,
13C NMR (150 MHz, CDCl3): dC 176.3 (C-9), 164.4 (C-3), 158.0 (C-4a), 2H, H-10), 2.45 (dd, J ¼ 6.9, 8.2 Hz, 2H, H-20), 1.68 (s, 3H, H-50), 1.62
156.2 (C-5a), 134.3 (C-6), 133.9 (C-30), 128.3 (C-1), 126.7 (C-8), 123.9 (s, 3H, H-60); 13C NMR (150 MHz, CDCl3): dC 176.1 (C-9), 164.5 (C-3),
(C-7), 122.0 (C-8a), 117.7 (C-5), 117.6 (C-40), 115.8 (C-2), 113.6 (C- 157.9 (C-4a), 156.1 (C-5a), 134.9 (C-40), 134.1 (C-6), 128.1 (C-1),
9a), 100.8 (C-4), 67.9 (C-10), 33.4 (C-20).
126.5 (C-8), 123.7 (C-7), 121.9 (C-8a), 119.1 (C-30), 117.7 (C-5), 115.6
3-(Pent-4-en-1-yloxy)-9H-xanthen-9-one (13): Yield: 88%; White (C-2), 113.6 (C-9a), 100.6 (C-4), 68.3 (C-10), 28.0 (C-20), 25.8 (C-50),
crystalline solid; M.P. 86–88 ꢁC; m/z, C18H16O3: 280, 239, 212, 184,
18.0 (C-60).
3-(But-2-yn-1-yloxy)-9H-xanthen-9-one (18): Yield: 56%; White
155, 139, 121, 92, 69, 53; IR ꢀmax cmꢂ1: 2958, 1647, 1618, 1258; H
1
NMR (600 MHz, CDCl3): dH 8.26 (d, J ¼ 8.2 Hz, 1H, H-8), 8.17 (d, crystalline solid; M.P. 147–149 ꢁC; m/z, C17H12O3: 264, 249, 236,
J ¼ 9.6 Hz, 1H, H-1), 7.61 (t, J ¼ 6.9 Hz, 1H, H-6), 7.35 (d, J ¼ 8.2 Hz, 212, 184, 155, 127, 92, 77, 53; IR ꢀmax cmꢂ1: 2983, 2238, 1654,
1
1H, H-5), 7.29 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7), 6.85 (dd, 1618, 1251; H NMR (600 MHz, CDCl3): dH 8.30 (d, J ¼ 8.2 Hz, 1H, H-
J ¼ 2.8, 9.6 Hz, 1H, H-2), 6.76 (d, J ¼ 2.8 Hz, 1H, H-4), 5.83 (m, 1H, H- 8), 8.24 (d, J ¼ 8.2 Hz, 1H, H-1), 7.68 (deformed t, J ¼ 6.9, 8.2 Hz, 1H,
40), 5.03 (m, 2H, H-50), 4.01 (deformed t, J ¼ 5.5, 6.9 Hz, 2H, H-10), H-6), 7.44 (d, J ¼ 8.2 Hz, 1H, H-5), 7.35 (deformed t, J ¼ 6.9, 8.2 Hz,
2.23 (m, 2H, H-30), 1.90 (m, 2H, H-20); 13C NMR (150 MHz, CDCl3): dC 1H, H-7), 6.97 (d, J ¼ 8.2 Hz, 1H, H-2), 6.97 (s, 1H, H-4), 4.76 (s, 2H,
176.2 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.2 (C-5a), 137.5 (C-40), H-10), 1.87 (d, J ¼ 2.7 Hz, 3H, H-40); 13C NMR (150 MHz, CDCl3): dC
134.2 (C-6), 128.2 (C-1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 117.7 176.4 (C-9), 163.3 (C-3), 157.9 (C-4a), 156.3 (C-5a), 134.4 (C-6),
(C-5), 115.7 (C-50), 115.6 (C-2), 113.6 (C-9a), 100.7 (C-4), 67.9 (C-10), 128.4 (C-1), 126.8 (C-8), 124.0 (C-7), 122.0 (C-8a), 117.8 (C-5), 116.3
30.0 (C-30), 28.2 (C-20).
(C-2), 113.7 (C-9a), 101.6 (C-4), 85.0 (C-20), 73.0 (C-30), 57.0 (C-10),
3.8 (C-40).
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,
3-(Pent-2-yn-1-yloxy)-9H-xanthen-9-one (19): Yield: 83%; White
155, 139, 121, 102, 82, 55; IR ꢀmax cmꢂ1: 2850, 1647, 1623, 1256; amorphous solid; M.P. 119–121 ꢁC; m/z, C18H14O3: 278, 249, 212,
1H NMR (600 MHz, CDCl3): dH 8.26 (d, J ¼ 8.2 Hz, 1H, H-8), 8.17 (d, 184, 155, 127, 92, 65; IR ꢀmax cmꢂ1: 2972, 2238, 1654, 1618, 1256;
J ¼ 8.2 Hz, 1H, H-1), 7.61 (m, 1H, H-6), 7.36 (d, J ¼ 8.2 Hz, 1H, H-5), 1H NMR (600 MHz, CDCl3): dH 8.21 (d, J ¼ 8.2 Hz, 1H, H-8), 8.14 (d,
7.29 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7), 6.85 (dd, J ¼ 2.7, 8.2 Hz, J ¼ 8.2 Hz, 1H, H-1), 7.57 (t, J ¼ 8.2 Hz, 1H, H-6), 7.32 (d, J ¼ 8.2 Hz,
1H, H-2), 6.76 (d, J ¼ 2.7 Hz, 1H, H-4), 5.81 (m, 1H, H-50), 5.00 (m, 1H, H-5), 7.25 (t, J ¼ 8.2 Hz, 1H, H-7), 6.87 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-
2H, H-60), 4.00 (deformed t, J ¼ 5.5, 6.9 Hz, 2H, H-10), 2.12 (m, 2H, 2), 6.84 (d, J ¼ 2.7 Hz, 1H, H-4), 4.69 (t, J ¼ 2.7 Hz, 2H, H-10), 2.19 (m,
H-40), 1.81 (m, 2H, H-20), 1.56 (m, 2H, H-30); 13C NMR (150 MHz, 2H, H-40), 1.08 (deformed t, J ¼ 6.9, 8.2 Hz, 3H, H-50); 13C NMR
CDCl3): dC 176.2 (C-9), 164.6 (C-3), 158.1 (C-4a), 156.2 (C-5a), 138.3 (150 MHz, CDCl3): dC 176.1 (C-9), 163.2 (C-3), 157.8 (C-4a), 156.2 (C-
(C-50), 134.2 (C-6), 128.2 (C-1), 126.7 (C-8), 123.8 (C-7), 122.0 (C-8a), 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), 115.7 (C-2), 115.0 (C-60), 113.6 (C-9a), 100.6 (C-4), 68.5 117.7 (C-5), 116.1 (C-2), 113.6 (C-9a), 101.5 (C-4), 90.7 (C-30), 73.2
(C-10), 33.4 (C-40), 28.5 (C-20), 25.3 (C-30).
(C-20), 57.0 (C-10), 13.6 (C-50), 12.5 (C-40).
3-((2-Methylallyl)oxy)-9H-xanthen-9-one (15): Yield: 87%; White
3-(But-3-yn-2-yloxy)-9H-xanthen-9-one (20): Yield: 73%; White
amorphous solid; M.P. 121–123 ꢁC; m/z, C17H14O3: 266, 251, 225, amorphous solid; M.P. 170–172 ꢁC; m/z, C17H12O3: 264, 249, 207,
197, 181, 165, 139, 115, 92, 77, 55; IR ꢀmax cmꢂ1: 2923, 1654, 1623, 165, 139, 89, 63; IR ꢀmax cmꢂ1: 2927, 2112, 1637, 1618, 1251; 1H
1275; 1H NMR (600 MHz, CDCl3): dH 8.20 (d, J ¼ 8.2 Hz, 1H, H-8), NMR (600 MHz, CDCl3): dH 8.30 (d, J ¼ 8.2 Hz, 1H, H-8), 8.24 (d,
8.12 (d, J ¼ 8.2 Hz, 1H, H-1), 7.54 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H- J ¼ 9.6 Hz, 1H, H-1), 7.67 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.44
6), 7.28 (d, J ¼ 8.2 Hz, 1H, H-5), 7.23 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, (d, J ¼ 8.2 Hz, 1H, H-5), 7.34 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7),
H-7), 6.82 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-2), 6.71 (d, J ¼ 2.7 Hz, 1H, H-4), 7.03 (d, J ¼ 2.7 Hz, 1H, H-4), 6.99 (dd, J ¼ 2.7, 9.6 Hz, 1H, H-2), 4.97
5.06 (s, 1H, H-3’a), 4.98 (s, 1H, H-3’b), 4.42 (s, 2H, H-10), 1.79 (s, 3H, (m, 1H, H-10), 2.56 (d, J ¼ 2.7 Hz, 1H, H-30), 1.72 (d, J ¼ 6.9 Hz, 3H, H-
H-40); 13C NMR (150 MHz, CDCl3): dC 176.1 (C-9), 164.1 (C-3), 157.9 40); 13C NMR (150 MHz, CDCl3): dC 176.4 (C-9), 162.7 (C-3), 157.9 (C-
(C-4a), 156.1 (C-5a), 139.8 (C-20), 134.2 (C-6), 128.1 (C-1), 126.6 (C- 4a), 156.3 (C-5a), 134.4 (C-6), 128.3 (C-1), 126.7 (C-8), 124.0 (C-7),
8), 123.8 (C-7), 121.9 (C-8a), 117.7 (C-5), 115.8 (C-2), 113.7 (C-9a), 122.0 (C-8a), 117.8 (C-5), 116.3 (C-2), 114.1 (C-9a), 102.3 (C-4), 81.8
113.5 (C-30), 101.1 (C-4), 72.1 (C-10), 19.4 (C-40).
(C-20), 75.0 (C-30), 64.1 (C-10), 22.1 (C-40).
3-((3-Methylbut-2-en-1-yl)oxy)-9H-xanthen-9-one (16): Yield:
3-(Benzyloxy)-9H-xanthen-9-one (21): Yield: 61%; White crystal-
84%; White amorphous solid; M.P. 115–117 ꢁC; m/z, C18H16O3: 280, line solid; M.P. 178–180 ꢁC; m/z, C20H14O3: 302, 183, 155, 127, 91,
212, 184, 155, 127, 92, 69, 53; IR ꢀmax cmꢂ1: 2958, 1654, 1625, 65; IR ꢀmax cmꢂ1: 2958, 1637, 1618, 1251; 1H NMR (500 MHz,
1
1287; H NMR (600 MHz, CDCl3): dH 8.25 (dd, J ¼ 2.7, 8.2 Hz, 1H, H- CDCl3): dH 8.31 (d, J ¼ 6.9 Hz, 1H, H-8), 8.25 (d, J ¼ 9.2 Hz, 1H, H-1),
8), 8.16 (d, J ¼ 8.2 Hz, 1H, H-1), 7.60 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, 7.67 (t, J ¼ 6.9 Hz, 1H, H-6), 7.43 (q, J ¼ 8.0 Hz, 5H, H-30, H-40, H-50,
H-6), 7.35 (d, J ¼ 8.2 Hz, 1H, H-5), 7.28 (deformed t, J ¼ 6.9, 8.2 Hz, H-60, H-70), 7.35 (q, J ¼ 6.9 Hz, 2H, H-5 & H-7), 7.00 (dd, J ¼ 2.3,
1H, H-7), 6.86 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-2), 6.78 (s, 1H, H-4), 5.46 (t, 9.2 Hz, 1H, H-2), 6.94 (d, J ¼ 2.3 Hz, 1H, H-4), 5.17 (s, 2H, H-10); 13C
J ¼ 6.9 Hz, 1H, H-20), 4.55 (d, J ¼ 6.9 Hz, 2H, H-10), 1.78 (s, 3H, H-40), NMR (125 MHz, CDCl3): dC 176.3 (C-9), 164.2 (C-3), 158.1 (C-4a),
1.74 (s, 3H, H-50); 13C NMR (150 MHz, CDCl3): dC 176.2 (C-9), 164.4 156.3 (C-5a), 135.8 (C-20), 134.4 (C-6), 128.9 (C-40 and C-60), 128.5
(C-3), 158.0 (C-4a), 156.2 (C-5a), 139.3 (C-30), 134.2 (C-6), 128.2 (C- (C-1), 128.4 (C-50), 127.6 (C-30 and C-70), 126.7 (C-8), 124.0 (C-7),
1), 126.6 (C-8), 123.8 (C-7), 122.0 (C-8a), 118.7 (C-20), 117.7 (C-5), 122.1 (C-8a), 117.8 (C-5), 116.1 (C-2), 113.9 (C-9a), 101.4 (C-4), 70.6
115.7 (C-2), 113.8 (C-9a), 100.9 (C-4), 65.5 (C-10), 25.9 (C-40), 18.4 (C-10).
(C-50).
3-(3-Phenylpropoxy)-9H-xanthen-9-one (22): Yield: 94%; White
3-((4-Methylpent-3-en-1-yl)oxy)-9H-xanthen-9-one (17): Yield: amorphous solid; M.P. 100–102 ꢁC; m/z, C22H18O3: 330, 212, 184,
77%; White amorphous solid; M.P. 68–70 ꢁC; m/z, C19H18O3: 294, 155, 139, 118, 91, 65; IR ꢀmax cmꢂ1: 2958, 1659, 1618, 1282; 1H

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
631
NMR (600 MHz, CDCl3): dH 8.29 (dd, J ¼ 2.7, 8.3 Hz, 1H, H-8), 8.19 (d, 123.8 (C-7), 121.9 (C-8a), 117.7 (C-5), 115.8 (C-2), 113.6 (C-9a),
J ¼ 8.2 Hz, 1H, H-1), 7.61 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.36 100.8 (C-4), 72.0 (C-40), 70.9 (C-10), 69.4 (C-30), 68.0 (C-20), 59.1
(d, J ¼ 8.2 Hz, 1H, H-5), 7.30 (deformed t, J ¼ 6.9, 8.2 Hz, 3H, H-7, H- (C-50).
50, H-90), 7.21 (deformed t, J ¼ 6.9, 8.2 Hz, 3H, H-60, H-70, H-80), 6.87
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,
(dd, J ¼ 2.7, 8.2 Hz, 1H, H-2), 6.74 (d, J ¼ 2.7 Hz, 1H, H-4), 3.99 (t,
195, 155, 139, 119, 92, 63; IR ꢀmax cmꢂ1: 2958, 1623, 1464, 1256;
J ¼ 6.9 Hz, 2H, H-10), 2.82 (deformed t, J ¼ 6.9, 8.2 Hz, 2H, H-30),
1H NMR (600 MHz, CDCl3): dH 8.26 (d, J ¼ 8.2 Hz, 1H, H-8), 8.22 (d,
2.14 (m, 2H, H-20); 13C NMR (150 MHz, CDCl3): dC 176.2 (C-9), 164.6
(C-3), 158.0 (C-4a), 156.2 (C-5a), 141.2 (C-40), 134.3 (C-6), 128.6 (C-60
J ¼ 9.6 Hz, 1H, H-1), 7.65 (t, J ¼ 6.9 Hz, 1H, H-6), 7.39 (d, J ¼ 6.9 Hz,
and C-80), 128.6 (C-50 and C-90), 128.2 (C-1), 126.7 (C-70), 126.2 (C-
1H, H-5), 7.32 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7), 6.94 (dd,
J ¼ 2.7, 9.6 Hz, 1H, H-2), 6.81 (d, J ¼ 2.7 Hz, 1H, H-4), 4.73 (s, 2H, H-
8), 123.9 (C-7), 122.0 (C-8a), 117.7 (C-5), 115.8 (C-2), 113.6 (C-9a),
10), 3.81 (s, 3H, H-30); 13C NMR (150 MHz, CDCl3): dC 176.2 (C-9),
168.5 (C-20), 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-10), 52.6 (C-30).
100.7 (C-4), 67.6 (C-10), 32.1 (C-30), 30.6 (C-20).
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 ꢀmax cmꢂ1: 2944, 1654, 1623, 1286; 1H NMR
(600 MHz, CDCl3): dH 8.29 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-8), 8.19 (d,
J ¼ 8.2 Hz, 1H, H-1), 7.61 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.36
(d, J ¼ 8.2 Hz, 1H, H-5), 7.30 (deformed t, J ¼ 6.9, 8.2 Hz, 3H, H-7, H-
60 H-100), 7.20 (t, J ¼ 8.2 Hz, 3H, H-70, H-80, H-90), 6.84 (dd, J ¼ 2.7,
8.2 Hz, 1H, H-2), 6.75 (d, J ¼ 2.7 Hz, 1H, H-4), 3.99 (deformed t,
J ¼ 5.5, 6.9 Hz, 2H, H-10), 2.70 (t, J ¼ 6.9 Hz, 2H, H-40), 1.83 (m, 4H,
H-20, H-30); 13C NMR (150 MHz, CDCl3): dC 176.2 (C-9), 164.6 (C-3),
158.1 (C-4a), 156.2 (C-5a), 142.0 (C-50), 134.3 (C-6), 128.5 (C-70 and
C-90), 128.2 (C-60 and C-100), 126.7 (C-1), 126.0 (C-8 and C-80), 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-10), 35.6 (C-40), 28.7 (C-20), 27.8 (C-30).
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 ꢀmax cmꢂ1: 2934, 1661, 1563,
1282; 1H NMR (600 MHz, CDCl3): dH 8.29 (d, J ¼ 8.2 Hz, 1H, H-8),
8.21 (d, J ¼ 8.2 Hz, 1H, H-1), 7.65 (deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-
6), 7.41 (d, J ¼ 8.2 Hz, 1H, H-5), 7.33 (deformed t, J ¼ 6.9, 8.2 Hz, 1H,
H-7), 6.90 (d, J ¼ 8.2 Hz, 1H, H-2), 6.85 (s, 1H, H-4), 4.15 (deformed
t, J ¼ 5.5, 6.9 Hz, 2H, H-10), 3.56 (deformed t, J ¼ 5.5, 6.9 Hz, 2H, H-
30), 3.35 (s, 3H, H-40), 2.09 (m, 2H, H-20); 13C NMR (150 MHz, CDCl3):
dC 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-30), 65.6 (C-10), 58.8 (C-40),
29.5 (C-20).
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 ꢀmax cmꢂ1: 2983, 1618, 1464, 1207;
1H NMR (500 MHz, CDCl3): dH 8.28 (d, J ¼ 8.0 Hz, 1H, H-8), 8.24 (d,
J ¼ 9.2 Hz, 1H, H-1), 7.66 (m, 1H, H-6), 7.41 (d, J ¼ 8.0 Hz, 1H, H-5),
7.33 (t, J ¼ 8.0 Hz, 1H, H-7), 6.95 (dd, J ¼ 2.3, 8.0 Hz, 1H, H-2), 6.83
(d, J ¼ 2.3 Hz, 1H, H-4), 4.72 (s, 2H, H-10), 4.28 (q, J ¼ 6.9 Hz, 2H, H-
30), 1.30 (t, J ¼ 6.9 Hz, 3H, H-40); 13C NMR (125 MHz, CDCl3): dC
176.2 (C-9), 168.0 (C-20), 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-10), 61.8 (C-30),
14.2 (C-40).
(R)-3-(3-Hydroxy-2-methylpropoxy)-9H-xanthen-9-one
(29):
Yield: 95%; White amorphous solid; M.P. 149–150 ꢁC; [a] 25/D
ꢂ54.5ꢁ, in methanol; m/z, C17H16O4: 284, 212, 184, 155, 139, 55; IR
ꢀmax cmꢂ1: 3451, 2923, 1618, 1464, 1256; 1H NMR (600 MHz,
CDCl3): dH 8.29 (d, J ¼ 8.2 Hz, 1H, H-8), 8.17 (d, J ¼ 8.2 Hz, 1H, H-1),
7.66 (m, 1H, H-6), 7.40 (d, J ¼ 8.2 Hz, 1H, H-5), 7.34 (deformed t,
J ¼ 6.9, 8.2 Hz, 1H, H-7), 6.88 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-2), 6.82 (d,
J ¼ 2.7 Hz, 1H, H-4), 4.04 (m, 2H, H-10), 3.73 (m, 1H, H-30), 2.23 (m,
2H, H-20), 1.08 (d, J ¼ 6.9 Hz, 3H, H-40); 13C NMR (150 MHz, CDCl3):
dC 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-10), 65.0 (C-30), 35.7 (C-20),
13.7 (C-40).
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 ꢀmax cmꢂ1: 2923, 1649,
(S)-3-(3-hydroxy-2-methylpropoxy)-9H-xanthen-9-one (30): Yield:
58%; White amorphous solid; M.P. 149–151 ꢁC; [a] 25/D þ 17.7ꢁ, in
methanol; m/z, C17H16O4: 284, 212, 184, 139, 92, 55; IR ꢀmax cmꢂ1
:
1
1618, 1258; H NMR (600 MHz, CDCl3): dH 8.21 (d, J ¼ 8.2 Hz, 1H, H-
3448, 2923, 1618, 1466, 1258; 1H NMR (600 MHz, CDCl3): dH 8.29
(d, J ¼ 8.2 Hz, 1H, H-8), 8.17 (d, J ¼ 9.6 Hz, 1H, H-1), 7.66 (m, 1H, H-
6), 7.40 (d, J ¼ 8.2 Hz, 1H, H-5), 7.34 (deformed t, J ¼ 6.9, 8.2 Hz, 1H,
H-7), 6.88 (dd, J ¼ 2.7, 9.6 Hz, 1H, H-2), 6.82 (d, J ¼ 2.7 Hz, 1H, H-4),
4.04 (m, 2H, H-10), 3.73 (m, 2H, H-30), 2.23 (m, 1H, H-20),1.08 (d,
J ¼ 6.9 Hz, 3H, H-40); 13C NMR (150 MHz, CDCl3): dC 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-10), 65.1 (C-30), 35.7 (C-20), 13.7 (C-40).
8), 8.13 (d, J ¼ 8.2 Hz, 1H, H-1), 7.57 (deformed t, J ¼ 6.9, 8.2 Hz, 1H,
H-6), 7.31 (d, J ¼ 8.2 Hz, 1H, H-5), 7.25 (deformed t, J ¼ 6.9, 8.2 Hz,
1H, H-7), 6.85 (dd, J ¼ 2.7, 8.2 Hz, 1H, H-2), 6.76 (d, J ¼ 2.7 Hz, 1H,
H-4), 4.66 (s, 2H, H-30), 4.16 (deformed t, J ¼ 4.1, 5.5 Hz, 2H, H-10),
3.87 (deformed t, J ¼ 4.1, 5.5 Hz, 2H, H-20), 3.34 (s, 3H, H-40); 13C
NMR (150 MHz, CDCl3): dC 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-30),
67.9 (C-10), 65.6 (C-20), 55.4 (C-40).
3-(2-(2-Methoxyethoxy)ethoxy)-9H-xanthen-9-one (26): Yield:
70%; White amorphous solid; M.P. 73–75 ꢁC; m/z, C18H18O5: 314,
X-ray diffraction analysis
282, 256, 239, 212, 184, 155, 139, 119, 103, 75, 59; IR ꢀmax cmꢂ1
:
2899, 1623, 1469, 1256; 1H NMR (600 MHz, CDCl3): dH 8.17 (dd,
J ¼ 2.7, 8.2 Hz, 1H, H-8), 8.08 (d, J ¼ 8.2 Hz, 1H, H-1), 7.54 (deformed
t, J ¼ 6.9, 8.2 Hz, 1H, H-6), 7.27 (d, J ¼ 8.2 Hz, 1H, H-5), 7.22
(deformed t, J ¼ 6.9, 8.2 Hz, 1H, H-7), 6.81 (dd, J ¼ 2.7, 8.2 Hz, 1H,
H-2), 6.71 (s, 1H, H-4), 4.12 (deformed t, J ¼ 4.1, 5.5 Hz, 2H, H-10),
X-ray analyses for compounds 5 and 13, which exist in the single
crystal form, were performed using Bruker APEX II DUO CCD dif-
fractometer, employing MoKa radiation (k ¼ 0.71073 Å) with u and
x scans, at room temperature. Data reduction and absorption cor-
rection were performed using the SAINT and SADABS programs44.
3.80 (deformed t, J ¼ 4.1, 5.5 Hz, 2H, H-20), 3.64 (deformed t, Both structures were solved by direct methods and refined by
J ¼ 4.1, 5.5 Hz, 2H, H-40), 3.49 (dd, J ¼ 4.1, 5.5 Hz, 2H, H-30), 3.30 (s, full-matrix, least-squares techniques on F2 using the SHELXTL soft-
3H, H-50); 13C NMR (150 MHz, CDCl3): dC 176.1 (C-9), 164.2 (C-3), ware package45,46. All H atoms were placed in geometrically ideal-
157.9 (C-4a), 156.1 (C-5a), 134.2 (C-6), 128.1 (C-1), 126.5 (C-8), ised positions and constrained to ride on their parent atoms with

632
Z. H. LOH ET AL.
Figure 2. ORTEP diagram of compounds 5 and 13.
Table 1. Crystal data and parameters for the structure refinement of 5 and 13.
Biological assay
Compound
5
13
1863946
C18H16O3
280.31
Cholinesterase inhibition activity assay
CCDC number
Molecular formula
Molecular weight
Crystal system
Space group
a (Å)
1863942
C17H16O3
268.30
Monoclinic
P21/c
12.1571 (13)
5.0697 (5)
21.793 (2)
90
98.156 (5)
90
1329.6 (2)
4
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 5,50-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 mg/mL for AChE
and BChE inhibition activities, respectively. The xanthone deriva-
tives that showed more than 50% of enzyme inhibition were fur-
ther evaluated in a series of concentration from 0.0625 to 1 lg/mL
to determine their IC50 values. Briefly, 200 mL of DTNB (0.5 mg/mL)
was added into a 96 well plate for AChE assay while 150 mL of
sodium phosphate buffer and 50 mL of DTNB (0.5 mg/mL) were
added for BChE assay. Then, 20 mL of the compounds were added
into the wells together with 20 mL 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 mL of ATCI (0.2052 mg/mL) or
BTCC (0.5 mg/mL) were added into the wells to initiate the reac-
tion. 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:
Triclinic
ꢀ
P1
7.503 (6)
11.297 (9)
17.769 (14)
98.931 (10)
95.355 (11)
103.272 (10)
1435 (2)
4
b (Å)
c (Å)
a (ꢁ)
b (ꢁ)
c (ꢁ)
V (Å3)
Z
Dcalc (Mg mꢂ3
)
1.340
1.297
Crystal dimensions (mm)
0.49 ꢀ 0.08 ꢀ 0.06
0.38 ꢀ 0.27 ꢀ 0.25
l (mmꢂ1
Tmin/Tmax
)
0.09
0.09
0.747, 0.977
32949
ꢂ14!14,
ꢂ6!6,
ꢂ25!25
2.4–29.8
2325
0.658, 0.952
18566
Reflections measured
Ranges/indices (h, k, l)
ꢂ8!8,
ꢂ13!13,
ꢂ21!21
2.4–21.9
5042
h limit (ꢁ)
Unique reflections
Observed reflections (I > 2r(I))
Parameters
1334
181
1.03
2531
379
1.08
0.096, 0.266
Goodness of fit on F2
R1, wR2 [I ꢃ 2r(I)]
R1, wR2 [all data]
0.066, 0.172
0.120, 0.216
0.165, 0.335
À
Á
C–H distance in the range of 0.93–0.97 Å and Uiso(H) ¼ 1.2 Ueq(C),
while Uiso(H) for methyl H atoms were set at 1.5 Ueq(C) and each
group was allowed to rotate freely about its C–C bond.
Crystallographic data for compounds 5 and 13 had been depos-
ited 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: þ44-(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.
Percentage inhibition ¼ Slopecontrol–Slopedrug=Slopecontrol ꢀ 100
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 con-
centrations (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 lM for 23 and 3.36, 1.68, 0.84
and 0.42 lM for 28. The mode of inhibition was determined by
using Lineweaver–Burk plot47.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
633
Computational studies
carbon signals of xanthone skeleton were found in the range of d
103 to 158 ppm. The 1H and 13C NMR spectral data and melting
point of 1 are in good agreement with the literature data50,51 and
were used as the standard spectra for the spectral data analyses
of xanthone derivatives (2–30).
Flexible docking
The X-ray crystal structure of Electrophorus electricus acetylcholin-
esterase (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 spe-
cifically selected as flexible based on the observation by48, in
which they superposed 68 AChE complex crystal structures over
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 xan-
thone derivatives (2–30) revealed an absence of the chelated
hydroxyl singlet peak at the chemical shift region near d 11 ppm.
Moreover, 1H NMR spectra of the xanthone derivatives showed
additional peaks when compared to that of 1. For xanthone deriv-
atives 2–20, 24–30, additional peaks appeared in different regions
of chemical shifts from those represented the main skeleton (d
6.8–8.3), which was seen in the spectrum of 1. Xanthone deriva-
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.
tives 21–23, which bear a phenyl substituent have additional
1
Both compound 23 and tacrine were then prepared and mini- peaks in their H NMR spectra that are overlapped with the chem-
mised before the docking procedure. The DS Flexible Docking ical shift region of the main skeleton of xanthone (1). Analyses of
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.
these additional peaks were found to be consistent with the pro-
posed 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 spec-
tra for the xanthone derivatives (2–30). Besides, the absence of a
broad hydroxyl (–OH) peak at the region of 3100–3500 cmꢂ1 in
the FTIR spectra of xanthone derivatives 2–28 further confirmed
the successful attachment of the substituent groups at C-3 pos-
ition 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.
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.
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 mg/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-dime-
thoxy-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 bind-
ing 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 sub-
stituents 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 syn-
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 molecu-
lar weight of 1. The FTIR spectrum displayed absorptions of free
–OH at 3112 cmꢂ1, sp3 C–H at 2888 cmꢂ1, C¼O at 1644 cmꢂ1, aro-
matic C¼C at 1607 cmꢂ1, and C–O at 1228 cmꢂ1. 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, d 10.93. The de-shielding effects of carbon nucleus by oxy-
gen atom leads to the exposure of the higher external magnetic
field, which in turn, requires higher frequency to achieve reson-
ance and results in a peak in a downfield region. Besides, three
doublets were detected at d 7.99, 7.54, and 6.82 for H-1, H-5, and
H-4, respectively, while a deformed triplet peak was spotted at d
7.38 for H-7. Two sets of doublet of doublet were observed at d
8.10 and 6.86 with respect to H-8 and H-2, while a triplet of doub-
let was detected at d 7.75 for H-6. These signals revealed the pres-
ence 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 (d 175.3) and followed by the hydroxyl carbon at C-
3 (d 164.6). These peaks were attributed to the electron-withdraw-
ing effect of oxygen atom. On the other hand, the aromatic thesised xanthone derivatives in this study are considered as

634
Z. H. LOH ET AL.
Table 2. AChE and BChE inhibitory activities of compounds 1–30.
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 substitu-
ent 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
a-mangostin, c-mangostin, and garcinone C were suggested to be
contributed by the strong hydrogen bonding and p–r 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 cata-
lytic site. A p–r 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 p–p interactions with xanthone skeleton of a-mangos-
tin and c-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 unsatur-
ated 4- to 6-carbons chain. Compound 12 that has a butenyl sub-
stituent 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 skel-
eton and the amino side chain exhibited the most potent inhib-
ition effect with an IC50 value of 2.68 mM59, 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 mM, respectively.
BChE Percentage
Substituent,
R group
AChE IC50
(mM)
of inhibition at
Compound
4 mg/mL (%)
1
2
3
4
5
6
7
8
H
2.39 0.11a
1.86 0.04bcd
1.40 0.14efgh
1.41 0.13efgh
1.18 0.11ghij
1.84 0.21bcd
1.74 0.20cde
1.15 0.19hij
2.07 0.06abc
1.61 0.11def
1.27 0.13fghij
2.09 0.21abc
>3.57
6.55 0.82
11.94 1.74
17.58 1.89
20.68 1.75
9.78 1.22
5.47 0.61
24.78 1.96
12.62 1.26
11.76 0.98
7.01 1.16
24.33 2.29
5.33 0.17
20.89 3.07
16.35 1.34
2.33 0.30
7.96 1.22
20.94 3.03
4.53 0.88
5.24 0.25
5.00 0.74
5.04 0.70
15.50 1.93
9.19 0.55
11.00 1.38
10.26 1.24
17.76 1.60
7.69 0.34
4.11 0.35
13.94 0.96
5.48 0.31
Propyl
Butyl
Isopropyl
Isobutyl
Isopentyl
(S)-2-Methylbutyl
2-Ethylbutyl
2-Methylpentyl
Methylcyclobutyl
Ethylcyclohexyl
Buten-3-yl
Penten-4-yl
Hexen-5-yl
2-Methylpropenyl
2-Methylbuten-2-yl
2-Methylpenten-2-yl
2-Butynyl
2-Pentynyl
3-Methyl-propynyl
Benzyl
Phenylpropyl
Phenylbutyl
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Tacrine
>3.40
1.60 0.15defg
1.86 0.15bcd
1.06 0.12hij
>3.79
1.21 0.05fghij
2.24 0.17ab
0.99 0.19hij
1.22 0.12fghij
0.88 0.04j
>3.52
Methoxypropyl
Methoxymethoxyethyl
Methoxyethoxyethyl
Methyl acetate
Ethyl acetate
(R)-Hydroxyl-2-methylpropyl
(S)-Hydroxyl-2-methylpropyl
1.37 0.12efghi
0.97 0.06ij
1.28 0.13fghij
0.88 0.15j
>3.52
>3.52
0.06 0.00k
96.55 0.18
ꢄ
Data represents the mean SD of three independent experiments; Different let-
ters indicated significantly different at p < 0.05 between sample groups among
AChE assay.
ꢄ
The concentration of tacrine (positive control) used in the experiment was
1 mg/mL.
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 mg/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 mM. Conversely, the xanthone
derivatives exhibited weak BChE inhibition even a higher concen-
tration of 4 lg/mL. Thus, the IC50 values were not determined for
BChE inhibition activities (Figures S1 and S2).
The result showed that twenty-three synthesised xanthones
By comparing linear substituent groups of xanthone derivatives
exhibited significant AChE inhibition with low IC50 values ranging with the same carbon chain, the AChE inhibition effects were
from 0.88 to 2.39 mM although their activities are weaker than the observed to have an increasing trend from alkynyl, alkenyl to alkyl
positive control, tacrine. The results are in good agreement with substituents. The evidence is that the lowest IC50 values were
previous studies that xanthones are potential AChE inhibitors. By found to be exhibited by 3 (butyl), followed by 12 (butenyl) and
comparing their structural features, the reported xanthones are 18 (butynyl). This might be due to the structural flexibility of 3
also bearing different substituents, particularly methoxyl, prenyl, that bind sterically to AChE as the inhibition activity are correlated
benzyl, pyranyl, or hydroxyl, which were attached at different posi- with hydrophobicity, electronic, inductive, or polar properties, and
tions on the xanthone main skeleton15,32,54–57
.
steric effects60. However, the introduction of an unsaturated bond
Overall, all the xanthone derivatives exhibited stronger AChE into the substituent groups that made up of branched hydrocar-
inhibitory effect than the parent compound (1) except for 9, 12, bon chain has a different influence on activity, depending on the
and 20 that exhibited comparable effects with 1. The results are length of the carbon chain. Xanthone derivative 17 (IC50
¼

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
635
Figure 3. Lineweaver–Burk plot of 3-(4-phenylbutoxy)-9H-xanthen-9-one (23) against AChE. Bar indicates the standard deviation.
1.06 lM), which has a substituent group of 5-carbons in chain (Vmax) and the affinity (Km) were affected by the compound con-
length with an unsaturated bond (2-methylpenten-2-yl) possessed
2-folds stronger inhibitory activities than 9 (IC50 ¼ 2.07 lM) that
bears a substituent group with the same chain length but without
any unsaturated bond (2-methylpentyl). There is no significant dif-
centration 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 Km.
ference in AChE inhibition being observed for 5 (isobutyl; IC50
¼
1.18 lM) and 15 (2-methylpropenyl; IC50 ¼ 1.60 lM) and between
6 (isopentyl; IC50 ¼ 1.84 lM) and 16 (isopentenyl; IC50 ¼ 1.86 lM).
Moreover, compound 20 (IC50 ¼ 2.24 lM) that consisted of a
branched substituent with 3-carbons chain length and a triple
bond experienced a drop in activity when compared to com-
pound 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 find-
ing 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 mM.
The inhibition potency of phenyl substituted xanthone derivatives
could be explained by potential hydrophobic interaction and p–p
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 diffu-
sion 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 pre-
sented 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) pos-
sessed potent AChE inhibition activity. The overall results deduced
that 3-O-substituted xanthones that carry a saturated linear hydro-
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 AChE64,65. 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 com-
prised 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 p–p stacking
with the indole and phenol side chains of Trp86 and Tyr337
(Figure 5). Apart from that, the planar geometry form of the xan-
thone 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
carbon side chain of 4-carbons in chain length with an addition of also similar to the amino group of tacrine, which is directed
phenyl group, or diether or ester are beneficial in AChE inhib- towards the hydration site comprised of D74, T83, W86, N87, and
ition activity.
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 substi-
tuted alkyl benzene ring could interact with the phenol side chain
of Y72 via p–p interaction further strengthening its binding inter-
AChE enzyme kinetic analysis
The enzyme inhibition mode of two promising compounds, 23
and 28 against AChE was analysed by
Lineweaver–Burk plot as shown in Figures 3 and 4. Both the max-
a double-reciprocal
imal velocity of the AChE enzyme-substrate compound reaction actions in the AChE active-site gorge.

636
Z. H. LOH ET AL.
Figure 4. Lineweaver–Burk plot of ethyl 2-((9-oxo-9H-xanthen-3-yl)oxy)acetate (28) against AChE. Bar indicates the standard deviation.
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 electri-
cus 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 is predicted to bind favourable to the human AChE. This suggests
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 interac-
tions, solvation of the protein and ligand, and an entropy term of
that this compound could potentially serve as a lead AChE inhibi-
tor 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,
the ligand66. According to the predicted Jain score, compound 23 including alkyl, alkenyl, alkynyl, alkylphenyl, ether, ester, and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
637
hydroxyl groups. These xanthones possessed stronger anticholin- 10. Williams A, Zhou S, Zhan C-G. Discovery of potent and
ergic activities towards AChE than BChE. Eleven xanthone deriva-
tives were found to exhibit potent AChE inhibition effect with the
IC50 values lower than 1.28 mM. SAR study revealed that hydropho-
bic 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-car-
bons in the chain and preferably with an addition of phenyl or
oxygenated group. Molecular docking study on 23 further con-
firmed the importance of the hydrophobic group as the side chain
of these derivatives in the anti-AChE activities through p-p interac-
tions in the active site, in addition to p–p 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.
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Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
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).
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