Blood-brain barrier permeable anticholinesterase aurones: Synthesis, structure-activity relationship, and drug-like properties

Article abstract of DOI:10.1016/j.ejmech.2015.02.055

A series of novel aurones bearing amine and carbamate functionalities at various positions (rings A and/or B) of the scaffold was synthesized and evaluated for their acetylcholinesterase and butyrylcholinesterase inhibitory activities. Structure-activity relationship study disclosed several potent submicromolar acetylcholinesterase inhibitors (AChEIs) particularly aurones bearing piperidine and pyrrolidine moieties at ring A or ring B. Bulky groups particularly methoxyls, and carbamate to a lesser extent, at either rings were also prominently featured in these AChEI aurones as exemplified by the trimethoxyaurone 4-3. The active aurones exhibited a lower butyrylcholinesterase inhibition. A 3g€2-chloroaurone 6-3 originally designed to improve the metabolic stability of the scaffold was the most potent of the series. Molecular docking simulations showed these AChEI aurones to adopt favourable binding modes within the active site gorge of the Torpedo californica AChE (TcAChE) including an unusual chlorine-π interaction by the chlorine of 6-3 to establish additional bondings to hydrophobic residues of TcAChE. Evaluation of the potent aurones for their blood-brain barrier (BBB) permeability and metabolic stability using PAMPA-BBB assay and in vitro rat liver microsomes (RLM) identified 4-3 as an aurone with an optimal combination of high passive BBB permeability and moderate CYP450 metabolic stability. LC-MS identification of a mono-hydroxylated metabolite found in the RLM incubation of 4-3 provided an impetus for further improvement of the compound. Thus, 4-3, discovered within this present series is a promising, drug-like lead for the development of the aurones as potential multipotent agents for Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2015.02.055

Accepted Manuscript
Blood-brain barrier permeable anticholinesterase aurones: Synthesis, structure-
activity relationship, and drug-like properties
Kok-Fui Liew, Kit-Lam Chan, Chong-Yew Lee
PII:
S0223-5234(15)00151-8
10.1016/j.ejmech.2015.02.055
EJMECH 7737
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 January 2015
Revised Date: 26 February 2015
Accepted Date: 27 February 2015
Please cite this article as: K.-F. Liew, K.-L. Chan, C.-Y. Lee, Blood-brain barrier permeable
anticholinesterase aurones: Synthesis, structure-activity relationship, and drug-like properties, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.055.
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ACCEPTED MANUSCRIPT
Graphical Abstract

1
ACCEPTED MANUSCRIPT
Blood-brain barrier permeable anticholinesterase aurones: Synthesis, structure-activity
relationship, and drug-like properties
1
1
1
Kok-Fui Liew , Kit-Lam Chan , and Chong-Yew Lee *.
1
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia.
*
Corresponding author
Chong-Yew Lee
School of Pharmaceutical Sciences
Universiti Sains Malaysia
11800 Penang, Malaysia.
Tel: +604-6532793
Email: chongyew@usm.my, chongyew79@gmail.com
Abstract
A series of novel aurones bearing amine and carbamate functionalities at various positions
(
rings A and/or B) of the scaffold was synthesized and evaluated for their
acetylcholinesterase and butyrylcholinesterase inhibitory activities. Structure-activity
relationship study disclosed several potent submicromolar acetylcholinesterase inhibitors
(
AChEIs) particularly aurones bearing piperidine and pyrrolidine moieties at ring A or ring B.
Bulky groups particularly methoxyls, and carbamate to a lesser extent, at either rings were
also prominently featured in these AChEI aurones as exemplified by the trimethoxyaurone 4-
3. The active aurones exhibited a lower butyrylcholinesterase inhibition. A 3'-chloroaurone 6-
3
originally designed to improve the metabolic stability of the scaffold was the most potent of
the series. Molecular docking simulations showed these AChEI aurones to adopt favourable
binding modes within the active site gorge of the Torpedo californica AChE (TcAChE)
including an unusual chlorine-π interaction by the chlorine of 6-3 to establish additional
bondings to hydrophobic residues of TcAChE. Evaluation of the potent aurones for their
blood-brain barrier (BBB) permeability and metabolic stability using PAMPA-BBB assay and
in vitro rat liver microsomes (RLM) identified 4-3 as an aurone with an optimal combination of
high passive BBB permeability and moderate CYP450 metabolic stability. LC-MS
identification of a mono-hydroxylated metabolite found in the RLM incubation of 4-3 provided
an impetus for further improvement of the compound. Thus, 4-3, discovered within this
present series is a promising, drug-like lead for the development of the aurones as potential
multipotent agents for Alzheimer’s disease.
Keywords: Aurones; acetylcholinesterase inhibitors; structure-activity relationship; blood-
brain barrier permeability; metabolic stability; drug-like properties.
1

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1.
Introduction
Alzheimer’s disease, the most common cause of dementia in the aging population is a
chronic, progressive neurodegenerative disorder. This syndrome is characterized by synapse
dysfunction, neurofibrillary tangles, formation of extracellular beta-amyloid aggregates, and
neuronal death, leading to impairment in cognitive function and death. Centrally acting
anticholinesterases particularly acetylcholinesterase inhibitors (AChEIs) remain the
mainstream, first-line treatment for Alzheimer’s disease. These AChEIs (tacrine,
galantamine, donepezil, and rivastigmine) provide symptomatic control of the disease by
restoring the cholinergic function in Alzheimer sufferers based on the postulation that
extensive loss of cholinergic neurons in certain regions of the brain leads to the cognitive
decline [1, 2 ]. To improve upon the current available treatment modalities, efforts for the past
decade have been invested in disease-modifying agents that target the other factors
associated with Alzheimer’s (beta-amyloid inhibitors, tau protein inhibitors, anti-inflammatory
agents, neurotrophic agents, and antioxidants) [3, 4, 5]. This has led to the idea of
multipotent agents designed to combine functionalities needed to hit several targets in one
molecule [6, 7, 8]. These multitargeting agents were typically bulky, had high molecular
weights (> 500 Da), and were tethered hybrids of two drug molecules, features which may
compromise their drug-likeness and ability to penetrate the brain [9].
In the search for novel multipotent AChEIs with sustained drug-like properties
(
metabolic stability and blood-brain barrier permeability), we revisited the aurones (2-
benzylidenebenzofuran-3(2H)-ones) as a lead scaffold. Aurones are a class of flavonoids
naturally found in ornamental plants (Cosmos, Antirrhinium) and fruits as bright yellow
pigments [10]. It has been considered a privileged structure due to its promising utility in
many medicinal chemistry projects [11-15]. Our particular interest in this scaffold stems from
the structural resemblance of its benzofuranone core to the indanone of donepezil, one of the
current AChEIs used in Alzheimer (Figure 1). Aurones have also been reported to show high
affinity for beta-amyloid aggregates [16], a desirable feature in a multitargeting strategy. We
reasoned that the aurone scaffold with a tenable molecular weight (222.24 Da) and a small
2
topological polar surface area (26.3Å ) [17] would provide a suitable starting point for the
introduction of key functionalities from donepezil (various amines) and from another clinically
used AChEI, rivastigmine (carbamate) while maintaining reasonably small molecular weight
and polar surface area.
Despite its recurrent use as a scaffold in drug discovery, the drug-like properties of
aurones have not received much attention. Particularly, the metabolic stability and the ability
to permeate the blood-brain barrier (BBB) of compounds derived from this scaffold via an
early, in vitro assessment would be beneficial during the design and structure-activity
elucidation stage [18]. Little is known about the stability of aurones when exposed to liver
metabolism, particularly the Phase 1 cytochrome P450 (CYP450) enzymes. Early
assessment of metabolic stability of novel compounds is useful in determining adequate
availability of the compound to the brain [19]. Similarly, the blood-brain barrier permeability of
aurones has not been well studied when compared to other flavonoids found in the diet [20,
21]. The penetration of centrally active drugs such as alkaloids and basic nitrogen-containing
drugs into the blood-brain barrier mainly involves passive diffusion across the cell
membranes of the brain endothelial cells which hinges upon a suitable balance of lipophilicity
and pKa of the ionisable groups in the molecule [22, 23]. The present aurones were
2

3
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designed to explore the role of such basic nitrogen moieties as well carbamate on their drug-
like potential.
We report here the synthesis of a series of amine- and carbamate-bearing aurones
and the evaluation of their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
inhibitory activities. The drug-like properties of the more potent aurone inhibitors were
evaluated using parallel artificial membrane permeability assay for blood-brain barrier
(
PAMPA-BBB) and in vitro stability assay in rat liver microsomes (RLM) in order to identify an
aurone derivative with an optimal combination of cholinesterase inhibitory activity, metabolic
stability, and BBB permeability.
2.
Chemical considerations
2.1 Design of target compounds
Observations of the structures of centrally active AChEIs (galanthamine, donepezil,
huperzine A) and recently synthesized derivatives [24, 25] suggest that basic, ionizable
nitrogen-containing moieties contribute to their activity and pharmacokinetic property.
Carbamate is another classic group frequently incorporated into the design of AChEIs such
as rivastigmine and xanthostigmine derivatives [26]. Following these leads, we synthesized
six series of aurones that carry various basic amines (dimethylamino-, piperidine, and
pyrrolidine alkyls) and diethylcarbamate at rings A and B of the scaffold (Table 1). Series 1
consists of 6-hydroxyaurones with the aforementioned functionalities at ring B in addition to
several weakly basic nitrogen-containing moieties such as dimethylamino (1-4), diethylamino
(
1-5) and pyridinyl (1-6) groups. Series 2 which are 6-methoxyaurones with similar
functionalities at ring B represents a more lipophilic group of compounds than Series 1.
Series 3 aurones are the “reversed” functionalized equivalents of Series 2 in which the
various functionalities are placed at ring A while the methoxyl group is at the 4ˈ-position of
ring B. They provide suitable comparisons with Series 2 and Series 5 by which to explore the
placement of the amine or carbamate groups at the other end of the scaffold. Series 4
aurones are akin to Series 3 with the functionalization at ring A but with a 3ˈ,4ˈ,5ˈ-trimethoxy
motif at ring B. This modification was inspired by a report on a series of multipotent AChEIs
in which the use of this motif gave rise to potent inhibitors [27]. Series 5 are 6-
diethylcarbamoylaurones with the amine functionalities at ring B to test the effect of having
the carbamate moiety at ring A. Series 6 aurones are chlorinated equivalents of Series 2
where one chlorine is placed at positions ortho and meta to the amine functionality at ring B.
This series serves to test whether the placement of electron-withdrawing groups (such as
chloro) near a potentially labile site can increase the metabolic stability of the compounds.
3

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2.2 Synthetic approaches
The synthesis of the present aurones involved the construction of the 6-hydroxybenzofuran-
(2H)-one core (1-12) following methods reported earlier [12, 28] (Scheme 2). Resorcinol
3
was acylated with bromoacetonitrile in an acid-catalysed Houben-Hoesch condition to form
alpha-bromoacetophenone 1-10 which was subsequently cyclised to 1-12. A series of 4-O-
functionalized benzaldehydes (dimethylaminoethyl, pyrrolidine ethyl, piperidine ethyl,
dimethylaminopropyl, and diethylcarbamate) were prepared by base-catalysed alkylation of
4-hydroxybenzaldehyde (Scheme 1). These functionalized benzaldehydes as well as several
commercially obtained non-basic benzaldehydes (pyridine 4-carboxaldehyde, 4-
dimethylaminobenzaldehyde, 4-diethylaminobenzaldehyde, and 4-methoxybenzaldehyde)
were coupled to benzofuranone 1-12 via aldol condensation to yield the 6-hydroxyaurones of
Series 1. Selected aurones from Series 1 were converted to 6-carbamoylaurones (Series 5)
via a base (potassium carbonate)-catalysed carbamylation with diethylcarbamyl chloride
(
Scheme 2).
The 6-methoxyaurones (Series 2) were synthesized firstly by O-methylation of
benzofuranone 1-12 using dimethyl sulfate to obtain 6-methoxybenzofuran-3(2H)-one 2-12
(
(
Scheme 3). 2-12 was coupled to the various functionalized benzaldehydes via a base
potassium hydroxide)-catalysed aldol condensation to yield the final aurones 2-1 to 2-11
with the exception of aurones 2-6 (pyridin-4ˈ-ylmethylene) and 2-9 (4ˈ-hydroxybenzylidene).
Base-catalysed condensations to prepare these two compounds gave poor yields and side
products. Hydrochloric acid-catalysed condition was instead employed to give satisfactory
yields of 2-6 (38%) and 2-9 (98%).
Series 3 (4ˈ-methoxyl at ring B) and Series 4 (3ˈ,4ˈ,5ˈ-trimethoxyls at ring B) aurones
were
prepared
by
aldol
coupling
of
4-methoxybenzaldehyde
and
3,4,5-
trimethoxybenzaldehyde with benzofuranone 1-12 respectively (Scheme 4). The resulting
aurones were then O-alkylated with four variants of aminoalkyl chlorides
(
dimethylaminoethyl, dimethylaminopropyl, pyrrolidine ethyl, and piperidine ethyl chlorides) to
yield the final compounds. Similarly, the ring B-chlorinated aurones of Series 6 were
synthesized by first constructing the aurone scaffold by acid-catalysed aldol condensation of
2-12 with appropriately substituted benzaldehydes (Scheme 5) followed by O-alkylation of
the ring B 4-hydroxyl with the same aminoalkyl chlorides as used for Series 4 and 5.
3. Results and discussion
3.1 AChE and BuChE inhibitory activities
The newly synthesized aurones were evaluated for their inhibitory activities in two
cholinesterases, AChE from the electric eel (Electrophorus electricus) and BuChE from
equine serum (Table 2). Inhibition towards BuChE was done to measure the relative
selectivity of the compounds in inhibiting AChE over BuChE. Donepezil and rivastigmine
were used as positive controls to ensure the validity of the in vitro assay by comparing our
inhibitory concentrations (IC ) with reported values [29]. All aurones were tested at
50
concentrations to a maximum of 100 ꢀM at which point test of some compounds were limited
by their solubility.
4

5
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The AChE inhibitory activities of these aurone derivatives revealed several interesting
structure-activity correlations. Most notable is the prevalence of aurones having cyclic amine
moieties (piperidine and pyrrolidine) exhibiting the highest AChE inhibition at submicromolar
IC (0.16 to 0.88 ꢀM) across the six series. Compound 6-3 (3ˈ-chloro, 4ˈ-O-ethyl piperidine
50
at ring B) was most potent (IC = 0.16 ꢀM). Compounds bearing piperidine (2-3, 3-3, 4-3, 5-
50
3
) and pyrrolidine (2-2, 3-2, 4-2, 5-2, 6-2) exhibited comparably potent IC s. In the 6-
50
hydroxyaurones (Series 1) and 6-methoxyaurones (Series 2) an interesting trend can be
seen regarding the nature of the nitrogen moieties needed for activity. Compounds with
weakly basic nitrogen moieties such as pyridinyl (1-6, 2-6) and aniline (1-4, 1-5, 2-4, 2-5) and
without any nitrogen (1-8, 2-8, 2-9) were devoid of activities (IC s > 100 ꢀM) indicating the
50
requirement for a basic (pKa 8 to 10) amine in the scaffold for AChE inhibition. Non-cyclic
amine-bearing compounds (2-1, 2-7) were also weaker AChEIs than their piperidine and
pyrrolidine counterparts. Changing the position of the piperidine/pyrrolidine moiety at ring B
(
such as 2-3) to ring A did not affect significantly their activities as 3-3, the “reversed”
equivalent of 2-3, and 4-3 were similarly potent.
Evident also is the need for bulky substituents (such as methoxyl) at position 6 (of
ring A) of the benzofuranone core for potent activity. The better 6-hydroxyaurones inhibitors
(
(
1-2, IC 2.2 ꢀM; 1-3 IC 3.2 ꢀM) were relatively weaker than the other 6-O-functionalized
50 50
6-methoxyl and 6-carbamoyl) aurones. This finding is consistent with that in other
heterocyclic scaffolds including donepezil in which bulky, hydrogen acceptor groups placed
at their aryl heterocyclic cores are favourable for AChE inhibition [24, 30]. The presence of
these methoxyl groups has been shown to enhance the interaction of the indanone core of
donepezil with hydrophobic residues along the gorge leading to the active site of the AChE
[
31]. It is tempting to suggest that in compound 4-3 (6-O-ethyl piperidine at ring A, 3ˈ,4ˈ,5’-
trimethoxyls at ring B), one of the better AChEIs (IC50 0.43 ꢀM), the pendant
trimethoxybenzylidene part of the compound may play the role of the indanone of donepezil
by having three methoxyl (bulky) groups and interacting with the outer part of active site
gorge.
In contrast to the prominent influence of the piperidine/pyrrolidine moiety on AChE
inhibition, it is surprising to find that incorporating a carbamate to the aurone scaffold (Series
5) did not lead to additional improvement in activity. The potencies of 5-2 and 5-3 were
clearly more attributed to the pyrrolidine and piperidine placed at ring B than to the
carbamate at ring A. Without these amine functionalities, the carbamoylaurones (5-4, 5-5, 5-
8) were inactive (IC50 > 100 ꢀM). However, placing the carbamate at ring B (2-11) gave
moderate potency (10.9 ꢀM) underscoring the importance of the position of the carbamate on
the scaffold to exert its activity. It could not be determined however whether 2-11’s mode of
action was via the carbamylation to the serine residue of the catalytic triad as is seen in
classical carbamate AChE inhibitors.
In general, the aurones were found to be moderate BuChE inhibitors (the best IC s
50
being between 6.15 to 25 ꢀM). BuChE inhibition was found among Series 2 (6-
methoxyaurones), Series 3 (“reversed” functionalized versions of Series 2), Series 4 (3’,4’,5’-
trimethoxyls at ring B), Series 5 (6-carbamoylaurones), and Series 6 (2ˈ- and 3ˈ-chlorinated
6-methoxyaurones). The 6-hydroxyaurones (Series 1) were inactive in BuChE inhibition. The
most potent BuChEI was 4-3 (IC 6.15 ꢀM) suggesting the need for bulky groups (the ring B
50
3’,4’,5’-trimethoxybenzylidene) for better fit into the larger cavity of the BuChE active site.
Interestingly, within 3ˈ,4ˈ,5ˈ-trimethoxybenzylidene compounds (Series 4), 4-7 bearing a 6-O-
5

6
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dimethylaminopropyl (a longer alkyl moiety) was inactive suggesting that there is an optimal
size allowance for binding to BuChE. As in the structure-activity relationship for AChE, the
prevalence of cyclic amine moieties such as piperidine (2-3, 3-3, 4-3, 5-3, 6-3) among the
active BuChEIs was clearly noted. The carbamate 2-11 (without amine functionality) which
was active in AChE inhibition, did not inhibit BuChE (IC > 100 ꢀM) accentuated this finding.
50
In summary, these active aurones were potent AChEIs (submicromolar IC s) with a lower
50
propensity towards BuChE inhibition.
3.2 Molecular docking of selected aurones with AChE and BuChE
To rationalize the structure-activity relationship of the present compounds, a molecular
docking study was conducted on selected potent aurones 1-3, 2-3, 3-3, 4-3, and 6-3 to model
the possible binding interactions that these compounds may assume with AChE. The AChEI
donepezil was included as a comparison. The compounds were docked to a Torpedo
californica AChE structure (TcAChE, PDB ID: 1EVE) originally having donepezil bound to its
active site. The donepezil ligand was removed using Discovery Studio 4.1 and a search grid
was built encompassing the vacant active site cavity using Autodock 4.2.3. Docking
simulations were performed following a reported procedure [32]. The binding interactions,
sites of binding, and estimated binding free energies for each compound are listed in Table
3.
2
-3, 3-3, and 6-3, the more potent AChEIs (submicromolar IC ) stood out as ligands
50
with higher binding free energies (absolute magnitudes 10.07 to 10.83 kcal) indicating
favourable interactions and orientation within the active site gorge. The 6-hydroxyaurone 1-3
had a somewhat lower binding energy (absolute magnitude 9.96 kcal) cohering to its lower
IC (2.23 ꢀM). Examining the types of interactions and the modes assumed by the aurones
50
(
Figure 2) and donepezil (re-docked to TcAChE), we found the aurones to establish multiple,
mostly hydrophobic sigma-π, alkyl-π and π-π stacking interactions with the hydrophobic
residues (Trp84, Phe330, Tyr334, Trp279) particularly in the choline binding pocket and the
peripheral anionic site (PAS). The interactions were made using their piperidine moiety
(
positioned at ring B for 1-3, 2-3, and 6-3, at ring A for 3-3 and 4-3), the benzofuranone (ring
A) as well as the ring B benzylidene appendage in a manner similar to the mode assumed by
donepezil (Figure 2D). But clearly there were subtle differences in the additional interactions
made and the orientation adopted by the aurones which may explain their potent IC .
50
The ring B trimethoxyaurone 4-3 (Figure 2B) had its benzofuranone core orientated
“
head-first” into the inner part of the active site gorge (the choline binding site) and the
3ˈ,4ˈ,5ˈ-trimethoxybenzylidene (ring B) pendant interacting with the outer PAS region. This
“
reversed” orientation to those of 2-3 (Figure 2A) and 6-3 (Figure 2C) and the multiple, bulky
methoxyls on ring B led to additional interactions made by its 3ˈ-methoxyl group via sigma-π
and H-bonding to residues Trp279 and Tyr70 respectively while the benzofuranone core and
piperidine made two additional π-π/ alkyl-π interactions with Trp84 and Phe330 in the inner
side of the gorge.
An interesting binding mode with the TcAChE was seen with the 3ˈ-chloroaurone 6-3
(
Figure 2C), the most potent of this series. While 6-3 assumed a similar ring B “head-first”
orientation within the TcAChE active site gorge as that of 2-3, the presence of the 3ˈ-chlorine
at ring B of 6-3 affected its orientation in a way which enabled the ligand to form additional
6

7
ACCEPTED MANUSCRIPT
interactions at the choline binding site, the PAS (π-π stackings by the benzofuranone core
with Trp279 and Tyr121) as well as the acyl binding region (H-bonding by the 6-methoxyl
group with Arg289). The estimated free binding energy accounted for by these additional
interactions by 6-3 was the highest (absolute magnitude 10.89 kcal). Remarkably, the
chlorine atom itself was seen to participate in interactions with the aromatic residues (Trp84
and Phe330, with interatomic distances of ~ 4Å apart) of the PAS region. The docking
program however was unable to discern this chlorine-π bonding as it was labeled as an
“
alkyl-π” interaction. To verify our finding, a literature search regarding halogen bonding to π
systems was made. We found that the role of such chlorine-π interactions in protein-ligand
complexes are increasingly recognized [33-37]. A systematic reexamination of protein-ligand
complexes in the RCSB Protein Data Bank by Imai et al [33] has identified short distance (3
to 4 Å) positioning of chlorine atoms (of ligands) to aromatic protein residues suggestive of
such interactions. They are commonly found in ligand-protein complexes of serine proteases
[
38] and protein kinases [39] but in the case of AChE, we found no reports of such
interactions to date. Aside from these current limitations, we surmised that this unusual mode
may explain the high potency exhibited by 6-3.
We also performed a docking simulation of 4-3 which was the most potent BuChEI
(
6.15 ꢀM) of the series on a human BuChE structure (hBuChE, PDB ID: 2WIJ) using the
same procedure (Figure 3). The hBuChE which has a larger active site gorge allowed the
compound to make interactions with residues and sites that were further distributed. 4-3 was
able to interact with the catalytic triad (His438), as well as the choline binding pocket (Trp82),
and the peripheral anionic site (Asp70, Tyr332) using its piperidine moiety, benzofuranone
core, its bulky trimethoxy ring B, and a methoxyl group at ring B. Compared to its binding
with TcAChE, 4-3 assumed a notably “L-shaped” pose with the pendant piperidine ethyl
sharply bent to accommodate the inner side of the gorge. These may indicate that 4-3 was
bound at a position deeper into the hBuChE active site gorge (making contact with the
catalytic site itself) than when it was docked to the TcAChE, which may account for its
inhibitory activity in BuChE.
3.3 In vitro blood-brain barrier permeability using PAMPA-BBB
Having identified several active aurone AChEIs, these compounds were next evaluated for
their permeability in the parallel artificial membrane permeability assay (PAMPA) using
porcine brain lipid to mimic the lipid membrane of the tight junction-linked endothelial cells of
the brain. This assay, also referred to as PAMPA-BBB, involved measuring the rate at which
a compound passively diffuses across the lipid barrier separating a donor compartment and
an acceptor compartment filled with phosphate buffer (pH 7.4). The concentrations of the
compound in both compartments were then measured to obtain the effective permeability
rate (P ). The PAMPA-BBB method has been shown [40, 41] to give reasonably good
e
predictions on the passive BBB permeability of commercial centrally active drugs except for
compounds which involved active uptake/efflux transport or were heavily metabolized in vivo.
Theophylline, caffeine, and donepezil were used as validity controls to monitor the
consistency of the assay by cross-referencing with reported P values (Table 4). Caffeine
e
and theophylline are representatives of low permeability drugs (“CNS –”; they are CNS active
drugs requiring active transport [41] into the brain) while the Alzheimer drug donepezil
represents a high permeability compound (“CNS +”). The P of theophylline and caffeine
e
7

8
ACCEPTED MANUSCRIPT
compared favourably with values reported by Di et al. [42]. Donepezil in our hands
(
determined in three independent experiments) showed a higher P than the reported value
e
[
35] (Table 4), possibly due to slight differences in the assay condition such as inter-batch
variation in the porcine lipid. An inactive AChEI, 2-8 (6,4ˈ-dimethoxyaurone) was included in
this assay as a representative of an unfunctionalized aurone.
The permeability rates (P ) of aurones tested in the assay are listed in Table 5. All
e
-
6
aurones were highly permeable (P 9.8 to 24.7 × 10 cm/s) across the lipid barrier and were
e
-6
deemed “CNS +” (> 4.0 × 10 cm/s) under the assay classification [36]. 6-methoxyaurone
bearing a pyrrolidine at ring B 2-2 showed the highest P (24.7 × 10 cm/s) while the other
-6
e
methoxyaurones sharing in common a piperidine moiety (2-3, 3-3, 4-3) have similarly high
-6
permeabilities (~ 20 to 21 × 10 cm/s). The 6-carbamoylaurone 5-3 and 3ˈ-chloroaurone 6-3,
both bearing piperidine at ring B were less permeable than 2-3 (6-methoxyaurone with
piperidine at ring B). The 6-hydroxyaurone with piperidine at ring B, 1-3 also showed a lower
permeability than the methoxyaurones with piperidine/pyrrolidine. 6,4ˈ-dimethoxyaurone 2-8
similarly had a lower (9.8 × 10-6 cm/s) permeability than the rest of the tested aurones.
Examining the calculated lipophilicity log D of the compounds, we found that they fell within
7.4
the optimal log D range (1 to 3) often reported by others for compounds with good brain
penetration in vitro and in vivo [44, 45]. For our limited set of eight compounds, this optimal
log D7.4 for high permeability resided between ~1.7 to 2.8. The piperidine and pyrrolidine
bearing 6-methoxyaurones (2-2, 2-3) and 3ˈ,4ˈ,5ˈ-trimethoxyaurones 4-3 represented
compounds with log D within this range. More lipophilic (log D > 2.8) compounds such as the
3
ˈ-chloroaurone 6-3 and the carbamate-bearing 5-3 had lower P with the exception of 3-3
e
(
(
log D = 3.16). At a casual examination, it would seem that compounds that are more polar
7.4
lower log D) have higher permeability. But bivariate correlation analysis did not show
significant inverse correlation (Spearman rho -0.335, p > 0.1, n = 8) between the log D
values and P of the compounds. We concluded that lipophilicity played a significant, if not
e
absolute role in the passive permeability of the aurones across the lipid barrier.
3.4 In vitro metabolic stability in rat liver microsomes (RLM)
Besides the ability to permeate the blood-brain barrier, a centrally active drug needs to be
metabolically stable to deliver to the brain in adequate concentrations [19]. Selected active
AChEI aurones (2-2, 2-3, 3-3, 4-3, 5-3, 6-3) and an inactive AChEI, 2-8 (6,4’-
dimethoxyaurone) were subjected to in vitro incubations with rat liver microsomes (RLM) in
buffered media (potassium phosphate, pH 7.4) containing NADPH regenerating system.
Commercially obtained RLM (pooled from the livers of several Sprague-Dawley rats)
containing major CYP450 enzymes were incubated with the compounds to measure their
stability when exposed to phase 1 (oxidative) liver clearance. The percentage remaining and
half-life (T ) of each compound was determined by LC-MS following reported procedures
1/2
[
45-49]. Donepezil was used to validate the assay and as a drug-like comparator to the
aurones. Testosterone was used as a representative of a short half-life compound.
In general, the aurones were moderately stable with T1/2 ranging from 5 to 44 minutes
(
(
Table 6). The 3ˈ,4ˈ,5ˈ-trimethoxybenzylidene aurone 4-3 was the most metabolically stable
T1/2 = 44.3 minutes) among the aurones. The pyrrolidine-bearing 6-methoxyaurone
appeared to be the least stable while 2-8 (6,4’-dimethoxyaurone) and 6-carbamoylaurone 6-3
8

9
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also presented lower T1/2 (~ 8 minutes). By comparison, a piperidine moiety (2-3) fared better
in withstanding CYP450 metabolism but whether the pyrrolidine moiety was by itself
contributing to the lability could not be ascertained with this set of compounds. Nevertheless,
the piperidine moiety seemed to confer stability to the scaffold as compounds bearing this
amine moiety (except for carbamoylaurone 5-3) had T1/2 of more than 10 minutes. The 3’-
chloroaurone 6-3 which was originally designed to improve the metabolic stability of a
potentially labile group (the O-ethyl piperidine) by having an adjacently placed chlorine was
only moderately stable (T = 13 minutes). In comparison to the drug donepezil which had an
½
optimized T1/2 (> 60 minutes), the rather moderate T1/2 displayed by these aurones suggest
that there is room for further optimization of the scaffold. Nevertheless, it is encouraging to
find that a suitably functionalized aurone 4-3 was uncovered among these active AChEIs that
had better CYP450 metabolic stability than the common, unfunctionalized aurone 2-8.
It seemed that the placement of the amine moiety whether on ring B (2-3, 3-3, 5-3, 6-
3) and ring A (4-3) as well as the substitution patterns (by methoxyls) on both rings
influenced their stability against CYP450. Considering CYP450 metabolism involves to a
large extent hydroxylations on aryl and heteroaryl rings, it is reasonable to assume that there
are preferred positions at ring A and/or ring B liable to hydroxylation. Inspection of the LC-MS
spectra of the RLM incubation mixtures of each aurone derivative confirmed this as each
spectrum showed the presence of a prominent monohydroxylated metabolite (m/z M + 16),
represented here by 4-3 and 2-2 (Figure 4). The appearance of this monohydroxylated
metabolite coincided with the diminishment of the parent compound across the time period of
incubation (10 to 30 minutes). Seeing as most of the aurones contained methoxyl groups, O-
demethylated metabolites would be expected but only a mono-demethylated peak (m/z M –
14) was seen as a minor metabolite for 4-3. Hydroxylation and demethylation at more than
one position and other oxidative alterations were not found in all aurones as well as
donepezil, possibly due to the short period of incubations. However, based on the present
finding that 4-3 was the most metabolically stable aurone among the selected promising
derivatives, one may surmise that from the substitution patterns of both rings A (6-O-
functionalized) and B (3',4',5'-trimethoxy) shown by 4-3, a likely position of aryl
monohydroxylation may be the ring A (the benzofuranone core), possibly at position 4 or 5,.
If this is true, then blocking this position (with an electron-withdrawing halogen, for example)
may give rise to improved CYP450 metabolic stability. A precise elucidation of the structure
of the mono-hydroxylated metabolite to locate the site of hydroxylation on the scaffold using
tandem MS/MS or NMR would confirm the feasibility of this proposal for further optimization
of the compound.
4. Conclusion
Taken together, the present study has uncovered several promising AChE-inhibiting aurones
that bear amine functionalities with some propensity for BuChE inhibition. Particularly, the
piperidine and pyrrolidine moieties confer activity to the scaffold to a significant degree. The
placement of a chlorine ortho to this moiety (6-3) leads to the most potent AChEI of the
series. Using molecular docking, we explain this unexpected potency by showing an
enhanced albeit unusual binding mode of the compound within the TcAChE active site.
Besides subtly altering the binding orientation of the molecule, this ortho chlorine establishes
additional interactions with aromatic residues in the active site. Such chlorine-π contacts
9

10
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between ligand and protein as exhibited by 6-3 may well be a special example where
halogens (usually employed in medicinal chemistry for physicochemical modifications and
improvement of metabolic stability) play an important role in a compound’s affinity for its
target protein. Bulky characteristics as exemplified by the ring B trimethoxyaurone 4-3 also
contribute to favourable interactions with AChE as well as BuChE which may explain its
potency for both cholinesterases as shown in the docking study. We also show for the first
time the passive blood-brain barrier permeability of the aurones by means of PAMPA. The
aurones are found to have log D values favourable for CNS-active, BBB permeable drugs
-6
which coincide with their high passive permeability (P > 20 × 10 cm/s) status. Evaluation of
e
drug-like properties (metabolic stability, BBB permeability) of the more potent AChEI aurones
identifies 4-3 as the optimal AChEI with high BBB permeability and satisfactory metabolic
stability. Examination of the LC-MS of the liver microsome incubation of 4-3 reveals a
prominent hydroxylated metabolite that may provide clues for future improvement on
metabolic stability of the compound. Hence, within this present series, 4-3 is a promising lead
for further optimization and the development of the aurone scaffold as a potential multipotent
agent for Alzheimer’s disease.
5. Experimental
1.1 General details
Reagents (synthetic grade or better) were obtained from Sigma-Aldrich Chemical Company
1
Inc (Singapore) and Acros Organics (Geel, Belgium) and used without further purification. H
13
NMR and C NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer
Bruker Biospin AG; Fallanden, Switzerland). Chemical shifts were reported in δ (ppm)
(
relative to TMS and the coupling constants in Hz. Electrospray Ionization Mass Spectra (ESI-
MS) were obtained using a Waters Xevo G2 Q-Tof mass spectrometer (Waters Corporation,
Milford, MA, USA) and accurate m/z for molecular ions were reported. Melting points of
individual compounds were determined with Stuart SMP10 melting point apparatus
(
Staffordshire, United Kingdom). Flash column chromatography was performed using silica
gel no.9385 (Merck; Darmstadt, Germany) and amino-functionalized silica gel, spherical 40-
5 ꢀm (Sigma; MO, USA). All reactions were monitored by thin layer chromatography on
7
silica gel 60 F254 plates (Merck; Darmstadt, Germany). The purity of compounds (peak area
no less than 95% of the total peak area) were confirmed by high pressure liquid
chromatography on a Waters HPLC 600 system (Waters Corporation, Milford, MA, USA)
using a Puropher STAR RP-18e column (4.6 x 100 mm, 5 µm) at 1 ml/min on two different
1
13
solvent systems. H NMR, C NMR, mass spectra, melting points, and HPLC purity of final
compounds are given in Supplementary Section.
1.2. General procedure for the synthesis of Series 1
1.2.1. 2ˈ,4ˈ-Dihydroxy-2-bromoacetophenone (1-10)
To a mixture of resorcinol (5 g, 45.4 mmol) and bromoacetonitrile (3.16 mL, 45.4 mmol) in
diethyl ether (50 ml) was added ZnCl (4.54 mmol). The reaction mixture was cooled to 0°C
2
and dry HCl gas was bubbled through the reaction mixture for 15 minutes. The solution was
10

11
ACCEPTED MANUSCRIPT
left stirring at 0 °C with intermittent introduction of dry HCl gas (15 min each time) until the
appearance of pale pink precipitates (after 4 h). The precipitated imine salt was filtered off
and washed three times with diethyl ether. The imine was dissolved in 60 ml of 1M HCl and
heated at 100 °C for 1 h and subsequently left to st and at 0 °C overnight. The solid was
filtered off, washed with water, and dried to yield pure acetophenone 1-10 (3.66 g) as a pale
1
brown solid which was used without further purification, 41% yield. M.p. 123-126 °C ; H NMR
(
(
MeOD, 500 MHz): δ 7.71 (d, J = 8.9 Hz, H6ˈ), 6.40 (dd, J = 2.4 Hz, J = 8.9 Hz, H5ˈ), 6.30
1 2
d, J = 2.4 Hz, H3ˈ), 4.78 (s, 2H).
1.2.2. 6-hydroxybenzofuran-3(2H)-one (1-12)
Compound 1-10 (3.66 g, 15.8 mmol) in methanol (50 ml) was added sodium acetate (6.5 g,
9.2 mmol) and the mixture was stirred with heating at 70 °C for 2 h. The reaction mixture
7
was concentrated in vacuo and diluted with 1M HCl. The aqueous layer was extracted with
ethyl acetate (3 × 50 ml), dried with Na SO , and evaporated to dryness to yield 6-
2
3
hydroxybenzofuran-3(2H)-one 1-12 (1.81 g) as a pale brown solid which was used without
°
1
further purification, 76.3 % yield. M.p. 245-247 ºC (Lit. mp 245 C; H NMR (MeOD, 500 MHz):
δ 7.49 (d, J = 8.55 Hz, H4), 6.58 (dd, J1 = 1.95 Hz, J2 = 8.55 Hz, H5), 6.46 (d, J = 1.96, H7),
1
3
4
7
.64 (s, 2H); C NMR (MeOD, 125 MHz): δ 200.7, 178.5, 169.2, 126.5, 114.3, 113.5, 99.5,
+
+
6.8; HRMS (TOF-ES) calcd for C H O ([M+1] ): 151.0395, found 151.0392
8
7
3
1.2.3. O-alkylation of 4-hydroxybenzaldehydes
To a solution of 4-hydroxybenzaldehyde (0.4 g, 3.275 mmol) in acetone (13 mL) was added
K CO (1.38 g, 10 mmol). The reaction mixture was vigourously stirred with heating at 60°C
2
3
for 2 hours. The reaction mixture was then cooled to room temperature, followed by the
addition of appropriate chlorides (4.585 mmol) dissolved in acetone. The reaction mixture
was allowed to stir again with heating at 60 °C for 2 hours. The reaction mixture was then
filtered to remove K CO . The filtrate was evaporated to dryness to yield crude products
2
3
which were subjected column chromatography to obtain pure 4-O-alkylated benzaldehydes.
1
4
-(2-(dimethylamino)ethoxy)benzaldehyde: Yield: 75.9 %, colourless oil; H NMR (CD OD,
3
500 Mz): δ 9.84 (s, CHO), 7.87 (d, J = 8.8 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H), 4.22 (t, J = 5.4
Hz, 2H), 2.81 (t, J = 5.4 Hz, 2H), 2.35 (s, 6H).
1
4
-(2-(piperidin-1-yl)ethoxy)benzaldehyde: Yield: 78.6 %, colourless oil; H NMR (CD OD, 500
3
MHz): δ 9.83 (s, CHO), 7.87 (d, J = 8.8 Hz, 2H), 7.1 (d, J = 8.8 Hz, 2H), 4.24 (t, J = 5.63 Hz,
2H), 2.81 (t, J = 5.63 Hz, 2H), 2.56 (brs, 4H), 1.61-1.66 (m, 4H), 1.46-1.51 (m, 2H).
1
4
-(2-(pyrrolidin-1-yl)ethoxy)benzaldehyde: Yield: 92.2 %, colourless oil; H NMR (CD OD,
3
500 Mz): δ 9.84 (s, CHO), 7.88 (d, J = 8.9 Hz, 2H), 7.12 (d, J = 8.75 Hz, 2H), 4.24 (t, J = 5.55
Hz, 2H), 2.96 (t, J = 5.55 Hz, 2H), 2.67-2.7 (m, 4H), 1.83-1.85 (m, 4H).
1
4
-(3-(dimethylamino)propoxy)benzaldehyde: Yield: 46 %, colourless oil; H NMR (CD OD,
3
500 Mz): δ 9.83 (s, CHO), 7.86 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 4.14 (t, J = 6.18
Hz, 2H), 2.55-2.58 (m, 2H), 2.31 (s, 6H), 1.99-2.04 (m, 2H).
11

12
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1
Diethyl-carbamic acid 4-formyl-phenyl ester: Yield: 22.1%, colourless oil; H NMR (CD OD,
3
500 Mz): δ 9.96 (s, CHO), 7.96 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 3.48-3.52 (m,
2H), 3.38-3.42 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H).
1.2.4. Condensation of 1-12 with functionalized benzaldehydes
To a solution of 1-12 in methanol (100 mg, 0.67 mmol, 10 ml/mmol) was added the suitably
functionalized benzaldehydes (0.67 mmol), followed by a solution of KOH 50% in water (1.5
ml/mmol). The solution is heated at 50-60 °C for 2 t o 3 h. The reaction mixture was
concentrated in vacuo and diluted with distilled water. The reaction mixture was adjusted to
pH 7 with 1N HCl. The aqueous layer was extracted with ethyl acetate and dried (Na SO ).
2
3
The organic phase was evaporated in vacuo to yield crude products which were subjected to
column chromatography by using chloroform: methanol (8:2) to yield pure Series 1 aurones.
1.3. General procedure for the synthesis of Series 2
1.3.1. 6-methoxybenzofuran-3(2H)-one (2-12)
To a solution of 1-12 (1.5 g, 10 mmol) in acetone (7 ml) was added K CO (2.073 g, 15
2
3
mmol), followed by the addition of dimethyl sulfate (1.387 g, 11 mmol). The mixture was
stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo and
diluted with water. The aqueous layer was extracted with dichloromethane (3 × 50 ml), dried
with Na SO , and evaporated to dryness to yield 2-12 as yellow solid which was used without
2
3
°
1
further purification, 72.84 % yield. M.p. 116-118 °C (Lit. m.p. 102-103 C); H NMR (CDCl ,
3
5
00 MHz): δ 7.57 (d, J = 8.6 Hz, H4), 6.65 (dd, J1 = 2 Hz, J2 = 8.6 Hz, H5), 6.55 (d, J = 2 Hz,
+
+
H7), 4.63 (s, 2H), 3.89 (s, 3H). HRMS (TOF-ES) calcd for C H O ([M+1] ): 165.0552, found
9
9
3
165.0535.
1.3.2. Condensation of 2-12 with functionalized benzaldehydes
To a solution of 2-12 in ethanol (100 mg, 0.62 mmol, 13 ml/mmol) was added the suitably
functionalized benzaldehydes (0.62 mmol), followed by a solution of KOH 20% in water (0.8
ml/mmol). The solution is stirred at room temperature for 2 h. The reaction mixture was
concentrated in vacuo and diluted with distilled water. The aqueous layer was extracted with
ethyl acetate and dried (Na SO ). The organic phase was evaporated in vacuo to yield crude
2
3
products which were subjected to column chromatography by using amino silica gel as
adsorbent and solvent system of hexane: ethyl acetate (7:3) followed by silica gel column
chromatography by using chloroform: methanol (9.5:0.5) to yield Series 2 derivatives except
for 2-6 and 2-9.
1.3.3. (Z)-6-methoxy-2-pyridin-4-ylmethylene-benzofuran-3(2H)-one (2-6)
To a solution of 2-12 (100 mg, 0.62 mmol) in anhydrous acetic acid (4.9 ml/mmol) was added
the pyridine-4-carbaldehyde (0.62 mmol), followed by dropwise addition of concentrated HCl
12

13
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(
0.3 ml/ mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction
mixture was diluted with distilled water (100 ml) and adjusted to pH 9 with 50% KOH. The
mixture was stirred for 15 minutes. The precipitate formed was filtered under suction to yield
orange solid of 2-6 which was purified by column chromatography by using hexane:ethyl
acetate (6:4). Yield: 38.2%.
1.3.4. (Z)-2-(4-hydoxybenzylidene)-6-methoxybenzofuran-3(2H)-one (2-9)
To a solution of 2-12 (100 mg, 0.62 mmol) in anhydrous acetic acid (4.9 ml/mmol) was added
the 4-hydroxybenzaldehyde (0.62 mmol), followed by dropwise addition of concentrated HCl
(
0.3 ml/ mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction
mixture was diluted with distilled water (100 ml) and stirred for 15 minutes. The precipitate
formed was filtered under suction to yield yellow solid of 2-9 which was purified by column
chromatography by using chloroform: methanol (9:1). Yield: 97.9%.
1
1
1
.4. General procedure for the synthesis of Series 3
.4.1. (Z)-6-Hydroxy-2-(4-methoxy-benzylidene)-benzofuran-3-one (1-8)
-12 was reacted with commercially purchased 4-methoxy benzaldehyde (purity 98%) as
described in Section 1.2.4. to yield 1-8 as yellow solid (0.93 g), 69.4% yield. M.p. 273-275
1
°
C, H NMR (DMSO-d , 500 MHz): δ 7.92 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.45 Hz, H4), 7.06
6
(
d, J = 8.8 Hz, 2H), 6.79 (d, J = 1.9 Hz, H7), 6.77 (s, 1H), 6.7 (dd, J1 = 1.9 Hz, J2 = 8.45 Hz,
H5), 3.83 (s, 3H).
1.4.2 O-alkylation of 1-8.
To a solution of 1-8 in acetone (200 mg, 0.746 mmol, 15 ml/mmol) was added K CO (315
2
3
mg, 2.28 mmol). The reaction mixture was vigourously stirred with heating at 60 °C for 2
hours. The reaction mixture was then cooled to room temperature, followed by the addition of
appropriate chlorides (1.5 mmol) dissolved in acetone. The reaction mixture was allowed to
stir again with heating at 60 °C for 10 hours. The r eaction mixture was then filtered to remove
K CO . The filtrate was evaporated to dryness to yield crude products which were subjected
2
3
column chromatography by using amino silica gel as adsorbent and solvent system of
hexane: ethyl acetate (7:3) followed by silica gel column chromatography by using
chloroform: methanol (9.5:0.5) to yield Series 3 derivatives.
13

14
ACCEPTED MANUSCRIPT
1
1
1
.5. General procedure for the synthesis of Series 4
.5.1. (Z)-6-Hydroxy-2-(3,4,5-trimethoxy-benzylidene)-benzofuran-3-one (4-8)
-12 was reacted with commercially purchased 3,4,5-trimethoxy benzaldehyde (purity 98%)
1
as described in Section 1.2.4. to yield 4-8 as yellow solid, 88.24% yield. M.p. 254-256 °C , H
NMR (DMSO-d , 500 MHz): δ 7.62 (d, J = 8.45 Hz, H4), 7.32 (s, 2H), 6.83 (d, J = 1.9 Hz,
6
H7), 6.76 (s, 1H), 6.73 (dd, J1 = 1.9 Hz, J2 = 8.45 Hz, H5), 3.85 (s, 6H), 3.73 (s, 3H).
1.5.2 O-alkylation of 4-8.
To a solution of 4-8 in acetone (200 mg, 0.610 mmol, 15 ml/mmol) was added K CO (256
2
3
mg, 1.86 mmol). The reaction mixture was vigourously stirred with heating at 60 °C for 2
hours. The reaction mixture was then cooled to room temperature, followed by the addition of
appropriate chlorides (1.5 mmol) dissolved in acetone. The reaction mixture was allowed to
stir again with heating at 60 °C for 10 hours. The r eaction mixture was then filtered to remove
K CO . The filtrate was evaporated to dryness to yield crude products which were subjected
2
3
column chromatography by using amino silica gel as adsorbent and solvent system of
hexane: ethyl acetate (7:3) followed by silica gel column chromatography by using
chloroform: methanol (9.5:0.5) to yield Series 4 derivatives.
1.6. General procedure for the synthesis of Series 5
1.6.1 Carbamylation of Series 1 derivatives (1-1 to 1-8 except 1-6)
To a solution of Series 1 derivatives (0.5 mmol) in acetone (15 mL/ mmol) was added K CO3
2
(
1.5 mmol), followed by the addition of diethylcarbamyl chloride (1 mmol). The reaction
mixture was allowed to stir with at 50 °C for 10 hou rs. The reaction mixture was then filtered
to remove K CO . The filtrate was evaporated to dryness to yield crude products which were
2
3
subjected column chromatography by using amino silica gel as adsorbent and solvent
system of hexane: ethyl acetate (7:3) followed by silica gel column chromatography by using
chloroform: ethanol (9:1) to yield Series 5 derivatives.
1.7. General procedure for the synthesis of Series 6
1.7.1. Condensation of 2-12 with 3-chloro-4-hydroxybenzaldehyde
To a solution of 2-12 (450 mg, 2.74 mmol) in anhydrous acetic acid (4.9 ml/mmol) was added
the 3-chloro-4-hydroxybenzaldehyde (2.74 mmol), followed by dropwise addition of
concentrated HCl (0.3 ml/ mmol). The reaction mixture was stirred at room temperature for 2
h. The reaction mixture was diluted with distilled water (100 ml) and stirred for 15 minutes.
The precipitate formed was filtered under suction to yield aurone 6-12 which was used
1
without further purification, yellow solid, yield 91.6%. M.p. 210-212 °C , H NMR (DMSO-d ,
6
5
00 MHz): δ 8.0 (d, J = 2 Hz, 1H), δ 7.79 (dd, J1 = 2 Hz, J2 = 8.6 Hz, 1H), 7.68 (d, J = 8.6
Hz, 1H), 7.17 (d, J = 2 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 6.85 (dd, J1 = 2.1, J2 = 8.5 Hz, 1H),
.79 (s, 1H), 3.93 (s, 3H).
6
14

15
ACCEPTED MANUSCRIPT
1.7.2. O-alkylation of 6-12
To a solution of 6-12 in acetone (200 mg, 0.661 mmol, 15 ml/mmol) was added K CO (278.5
2
3
mg, 2.02 mmol). The reaction mixture was vigourously stirred with heating at 60 °C for 2
hours. The reaction mixture was then cooled to room temperature, followed by the addition of
appropriate chlorides (1.5 mmol) dissolved in acetone. The reaction mixture was allowed to
stir again with heating at 60 °C for 10 hours. The r eaction mixture was then filtered to remove
K CO . The filtrate was evaporated to dryness to yield crude products which were subjected
2
3
column chromatography by using amino silica gel as adsorbent and solvent system of
hexane: ethyl acetate (7:3) followed by silica gel column chromatography by using
chloroform: methanol (9.5:0.5) to yield compounds 6-1, 6-2, 6-3, and 6-7.
1.7.3. Condensation of 2-12 with 2-chloro-4-hydroxybenzaldehyde
To a solution of 2-12 (450 mg, 2.74 mmol) in anhydrous acetic acid (4.9 ml/mmol) was added
the 2-chloro-4-hydroxybenzaldehyde (2.74 mmol), followed by dropwise addition of
concentrated HCl (0.3 ml/ mmol). The reaction mixture was stirred at room temperature for 2
h. The reaction mixture was diluted with distilled water (100 ml) and the precipitate formed
was filtered under suction to yield aurone 6-14 which was used without further purification.
1
Yellow solid, yield 88.9%. M.p. 268-270 °C , H NMR (DMSO-d , 500 MHz): δ 8.2 (d, J = 8.8
6
Hz, 1H), 7.7 (d, J = 8.6 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H), 6.98 (d, J = 2.5 Hz, 1H), 6.95 (s,
1
3
H), 6.93 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 6.86 (dd, J1 = 2.1, J2 = 8.6 Hz, 1H) , 3.92 (s,
H).
1.7.4. O-alkylation of 6-14
To a solution of 6-14 in acetone (200 mg, 0.661 mmol, 15 ml/mmol) was added K CO (278.5
2
3
mg, 2.02 mmol). The reaction mixture was vigourously stirred with heating at 60 °C for 2
hours. The reaction mixture was then cooled to room temperature, followed by the addition of
appropriate chlorides (1.5 mmol) dissolved in acetone. The reaction mixture was allowed to
stir again with heating at 60 °C for 10 hours. The r eaction mixture was then filtered to remove
K CO . The filtrate was evaporated to dryness to yield crude products which were subjected
2
3
column chromatography by using amino silica gel as adsorbent and solvent system of
hexane: ethyl acetate (7:3) followed by silica gel column chromatography by using
chloroform: methanol (9.5:0.5) to yield compounds 6-8 to 6-11.
1.8 In vitro inhibition studies on cholinesterase enzymes
Acetylcholinesterase from electric eel, acetylcholine iodide, butyrylcholine esterase from
equine serum, S-butyrylthiocholine chloride, 5,5ˈ-dithiobis(2-nitrobenzoic acid) (DTNB), and
PEG-400 were purchased from Sigma-Aldrich (St Louis, MO). Donepezil hydrochloride and
rivastigmine tartrate were purchased from Santa-Cruz Biotechnology Inc. (Santa Cruz, CA,
USA). Sodium dihydrogen phosphate anhydrous was purchased from R&M chemicals
15

16
ACCEPTED MANUSCRIPT
(
Essex, UK), disodium hydrogen phosphate anhydrous from Merck (Darmstadt, Germany),
96-well microplate from Greiner Bio-one (Frickenhausen, Germany). All the compounds are
tested at the concentration below 100 ꢀM. Stock solutions of test compounds were prepared
using a solvent mixture (1:1) of PEG-400 and 0.1 M potassium phosphate buffer (pH 7.8)
These were diluted with the reaction medium to the final concentrations during the assay.
PEG-400 was diluted to a concentration of less than 1% and no inhibitory activity on either
AChE or BuChE was detected with this PEG content.
The anti-cholinesterase activity of the aurones was evaluated by the colorimetric
Ellman’s method [48] with modifications. The solution temperature was adjusted to room
temperature prior to use. Potassium phosphate buffer (pH 7.8, 140 ꢀL) was added into 96
wells plate followed by sample solution (20 ꢀL), and AChE or BChE enzyme (0.2 U/ml, 20
uL). The mixture was incubated at room temperature for 15 minutes. DTNB (10 mM, 10 ꢀL)
was then added into the mixture, followed by the addition of acetylthiocholine iodide or
butyrylthiocholine iodide (14 mM, 10 ꢀL).The rate of absorbance change was measured at
412 nm for 30 minutes with a Multiskan FC Microplate Photometer (Thermo Fisher Scientific,
MA, US). Each assay was carried out with donepezil or rivastigmine tartrate as positive
controls. The reactions were performed in three independent runs. Each run of a sample was
performed in triplicates and the IC50 values were determined from the inhibition versus
concentration curves plotted using GraphPad Prism 4.0 (La Jolla, CA, USA).
1.9 Molecular docking
Molecular docking of selected aurones was performed using Autodock 4.2.3 along with
AutoDockTools (ADT) following the procedure of Morris et al [32]. The structures of
compounds were drawn using ChemBioDraw Ultra 13.0 (CambridgeSoft, Perkin Elmer Inc,
MA, USA) and minimization was performed with the CHARMm force field by Acceryls
Discovery Studio 4.1 (Accelrys Inc, San Diego, CA, USA) to refine the ligand poses and the
resulting structures were saved in pdb format for docking. The three dimensional structure of
AChE from Torpedo california in complex with donepezil (PDB ID: 1EVE) and BChE from
Homo sapiens in complex with tabun analogue (PDB ID: 2WIJ) were obtained from the
Brookhaven Protein Database (RCSB) (http://www.rcsb.org). Water molecules and
heteroatom groups were removed from the receptor beyond the radius of 4 Å of the
reference ligand (donepezil or Tabun) and CHARMm force field was assigned to all atoms
using Discovery Studio 4.1. Subsequently, both proteins were edited using ADT to add the
polar hydrogen atoms and Kollman charges as means to rigidify the protein structures. A
grid box of 40 × 40 × 40 points, with a spacing of 0.375 Å was positioned at the center of the
active site gorge. Docking simulations were carried out with Lamarckian Genetic Algorithm
employed as the docking algorithm and the lowest docked energy conformation between the
receptor and ligand was selected. Analysis and visualization of the docking results was done
using Acceryls Discovery Studio 4.1.
1.10 In vitro blood-brain barrier permeation assay.
Porcine brain lipid (PBL) was purchased from Avanti Polar Lipids (Alabaster, AL). Caffeine,
theophylline and dodecane were purchased from Sigma-Aldrich (St Louis, MO). The 96-well
16

17
ACCEPTED MANUSCRIPT
donor plate microplate (PVDF membrane, pore size 0.45 ꢀm) and the acceptor microplate
were both from Merck Millipore Bioscience (Bredford, USA).
The method described by Di et al. [42] was followed for the evaluation of the blood-
brain barrier permeation assay with some modifications. Test compounds were dissolved in
DMSO at 2 mg/mL and diluted in PBS to achieve a concentration of 30 ꢀM, 300 ꢀL of which
was added to the donor wells. The filter membrane of the acceptor microplate was
impregnated with 4 ꢀL of PBL in dodecane (20mg/mL) and each of the wells was filled with
200 ꢀL of PBS. The acceptor filter plate was carefully placed on the donor plate to form a
sandwich and left undisturbed for 16 h at room temperature. After incubation, the donor plate
was removed and the concentration of the compounds in the acceptor and donor wells was
determined using Waters HPLC 600 system with fluorescence detection ( for donepezil, 1-3,
2-2, 2-3, 2-8, 3-3, 4-3, 5-3, and 6-3) and UV detection (theophylline and caffeine). The HPLC
conditions for the assay of each compound are given in the Supplementary Section.
Permeability rates (P ) and membrane retention (R) were calculated using the following
e
equation:
2
.303ꢂꢃ ꢂꢂꢂ
1
1 + ꢈꢎꢏ
ꢑꢄꢂ(ꢆ)
1 − ꢐ ꢑꢒꢂ(ꢆ = ꢂ0)
ꢄ
ꢀꢁ =ꢂ−
ꢂꢇ
ꢉ ⋅ ꢊꢋꢌ ꢍ
∙
ꢂꢓ
ꢅꢂ ∙ ꢆ
1 + ꢈ
ꢃ ꢑ (ꢆ) + ꢃ ꢑ (ꢆ)
ꢄ ꢄ
ꢒꢂ ꢒ
ꢐ = 1 −
ꢃ ꢑ (ꢆ = 0)
ꢒ
ꢒ
2
where A is the active surface area of the membrane (0.24 cm ), t is the incubation time (in s),
3
V and V are the volumes (cm ) of the acceptor and donor compartments, r = V /V , C and
A
D
D
A
A
3
C are drug concentrations (mol/cm ) in the acceptor and donor compartments. Theophylline,
D
caffeine and donepezil were used as validity controls of known BBB permeability to monitor
the consistency of each experiment. Every sample was analyzed in triplicates in three
independent runs, and P were given as the mean ± standard error mean.
e
1.11 In vitro Metabolic Stability by Rat Liver Microsomes
The rat liver microsomal stability of selected compounds was evaluated using a single time-
point microsomal stability assay as described by Di et al [49]. All the reagents used were of
HPLC grade or better. Testosterone, β-Nicotinamide adenine dinucleotide phosphate sodium
salt, glucose-6-phospate dehydrogenase from baker’s yeast, and D-glucose-6-phosphate
sodium salt were purchased from Sigma-Aldrich (St Louis, MO). Male Sprague-Dawley rat
liver microsomes were purchased from BD Biosciences (San Jose, CA). Sample analysis
was carried out using a Waters Xevo G2 Q-Tof mass spectrometer.
Rat liver microsomes (0.5 mg/mL) were pre-incubated with NADPH regenerating system (1.3
mM NADP, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase,
and 3.3 mM magnesium chloride) at 37 °C for 10 minut es. Test compounds were dissolved in
DMSO at a concentration of 2 mg/mL and diluted with phosphate buffer pH 7.4 to the desired
concentration. The reaction was initiated by the addition of 100 ꢀL of the diluted sample into
the mixture and incubated at 37 °C . Final concentrat ion of the sample was 2 ꢀM. At time 0, 60
ꢀ
L of the reaction mixture was sampled and quenched with 60 ꢀL of ice-cold acetonitrile with
acetaminophen as an internal standard. The microsomal reactions were terminated after 10
17

18
ACCEPTED MANUSCRIPT
minutes by adding equal amount of ice-cold acetonitrile to the reaction mixture with the
exception of donepezil and 4-3, which were terminated at 30 minutes. The quenched
samples were vigorously vortexed and centrifuged 10 000 rpm for 10 min. Supernatant was
collected and quantitated by LC-MS using MassLynx 4.0 (Waters Corporation, Milford, MA).
Results were expressed as percentage of compound remaining (%) and single time-point
half-life (T ) in vitro.
1/2
Appendix - Supplementary Data
1
13
H NMR, C NMR, melting points, MS, and HPLC purity of the final compounds are given as
supplementary data. Also included are the HPLC conditions for the determination of
compound concentrations (PAMPA-BBB) and LC-MS conditions (in vitro metabolic stability).
Acknowledgements
The authors gratefully acknowledged support by the Fundamental Research Grant Scheme
(
FRGS) Phase 1/2013 (203/PFARMASI/6711305) awarded (to CKL and LCY) by the Ministry
of Higher Education (MOHE) of Malaysia and Universiti Sains Malaysia. LKF was supported
by the MyPhD Scholarship from the Ministry of Education.
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List of figures and schemes
Figure 1. Structures of AChEIs donepezil and rivastigmine and their key functionalities.
Figure 2. Binding modes of aurones 2-3 (A), 4-3 (B), and 6-3 (C) docked to TcAChE.
Donepezil was re-docked to TcAChE (D).
Figure 3. Binding mode of 4-3 docked to hBuChE.
Figure 4. (A) LC-MS of 4-3 (m/z 440.2848) and its mono-hydroxylated (m/z 456.2846) and
demethylated (m/z 426.2592) metabolites in RLM incubation at 30 minutes. (B) LC-MS of 2-2
(
m/z 366.220) and its monohydroxylated metabolite (m/z 382.2161) in RLM incubation at 10
minutes.
Scheme 1. Reagents and conditions: (a) R-Cl, K CO , acetone, 60º C, 2 h.
2
3
Scheme 2. Reagents and conditions: (a) Bromoacetonitrile, ZnCl , HCl, diethyl ether, 0º C,
2
followed by HCl 1N, 100º C. (b) Sodium acetate, MeOH, 70º C, 2 h. (c) O-functionalized
benzaldehydes or substituted benzaldehydes, KOH, MeOH, 50º C, 2-3 h. (d) Diethylcarbamyl
chloride, K CO , acetone, 50 °C , 10 h.
2
3
Scheme 3. Reagents and conditions: (a) Dimethyl sulfate, K CO , acetone, rt, 2 h. (b)
2
3
Method A: O-functionalized benzaldehydes, KOH, MeOH, 50º C, 2-3 h or Method B: HCl,
acetic acid, rt, 2 h (for 2-6 and 2-9).
Scheme 4. Reagents and conditions: (a) 6-methoxybenzaldehyde, KOH, MeOH, 50º C, 2 h.
(
b) R-Cl, K CO , acetone, 60º C, 2 h. (c) 3,4,5-trimethoxybenzaldehyde, KOH, MeOH, 50º C,
2 3
2
h. (d) R-Cl, K CO , acetone, 60º C, 2 h.
2 3
22

23
ACCEPTED MANUSCRIPT
Scheme 5. Reagents and conditions: (a) HCl, acetic acid, rt, 2 h. (b) R-Cl, K CO , acetone,
2
3
60º C, 2 h.
List of tables
Table 1. Structures of compounds in Series 1 to 6.
Table 2. AChE and BuChE inhibitory activities of the aurone derivatives.
Table 3. Binding interactions, sites of interactions, and binding energy for selected aurones
docked into active site gorge of TcAChE and hBChE.
Table 4. Permeability of controls in the PAMPA-BBB assay.
Table 5. PAMPA-BBB permeability of selected active aurones.
Table 6. Rat liver microsomal stability of selected active aurones.
23

ACCEPTED MANUSCRIPT
Table 1. Structures of compounds in Series 1 to 6.
1
Compound
Series 1
R
% Yield
1-1
63
1-2
1-3
1-4
64
59
99
1-5
81
1
-6
-7
53
84
1
1-8
68
1
Compound
Series 2
R
% Yield
R1
H CO
3
O
6
A
O
2-1
53

ACCEPTED MANUSCRIPT
2-2
2-3
2-4
26
19
42
2-5
47
2
-6
-7
38
33
2
2
2
2
-8
36
98
20
-9
-11
1
Compound
Series 3
R
% Yield
3
-1
-2
46
3
40

ACCEPTED MANUSCRIPT
3
-3
-7
46
35
3
1
Compound
Series 4
R
% Yield
4-1
42
4
-2
-3
59
70
4
4-7
39
1
Compound
Series 5
R
% Yield
5-1
58
5-2
5-3
5-4
68
47
76

ACCEPTED MANUSCRIPT
5-5
75
5
-6
-7
55
63
5
1
Compound
Series 6
R
% Yield
R1
H CO
3
O
6
A
O
6-1
Cl
'
60
3
O
CH3
N
B
CH3
6-2
6-3
6-7
32
38
29
6-8
52
6
-9
39
33
6-10

ACCEPTED MANUSCRIPT
6-11
29