Synthesis and evaluation of methoxy substituted 2-benzoyl-1-benzofuran derivatives as lead compounds for the development adenosine A1 and/or A2A receptor antagonists

Article abstract of DOI:10.1016/j.bioorg.2019.103459

A series of fourteen methoxy substituted 2-benzoyl-1-benzofuran derivatives were synthesised and their affinities determined for adenosine A₁ and A_2A receptors via radioligand binding assays to establish the structure activity relationships pertinent for A₁ and A_2A affinity. Compound 3j (6,7-dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone exhibited A₁ affinity (A₁K_i (rat) = 6.880 μM) as well as A_2A affinity (A_2AK_i (rat) = 0.5161 μM). Compounds 3a–b & 3i–k exhibited selective affinity towards A₁ with K_i values below 10 μM. The results indicate that C6,7-diOCH₃ substitution on ring A in combination with meta (C3′)–OCH₃ substitution on ring B is beneficial for A₁ and A_2A affinity and activity. Compounds 3a–b & 3j–k showed low cytotoxicity. Upon in vitro and in silico evaluation, compound 3j may be considered lead-like (i.e. a molecular entity suitable for optimization) and, thus, of value in the design of novel, potent and selective adenosine A₁ and A_2A receptor antagonists.

Full text of DOI:10.1016/j.bioorg.2019.103459

Journal Pre-proofs
Synthesis and evaluation of methoxy substituted 2-benzoyl-1-benzofuran de-
rivatives as lead compounds for the development adenosine A1 and/or A2A re-
ceptor antagonists
Helena D. Janse van Rensburg, Lesetja J. Legoabe, Gisella Terre'Blanche,
Janine Aucamp
PII:
S0045-2068(19)31436-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.103459
YBIOO 103459
Reference:
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Bioorganic Chemistry
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Accepted Date:
31 August 2019
16 November 2019
19 November 2019
Please cite this article as: H.D. Janse van Rensburg, L.J. Legoabe, G. Terre'Blanche, J. Aucamp, Synthesis and
evaluation of methoxy substituted 2-benzoyl-1-benzofuran derivatives as lead compounds for the development
adenosine A1 and/or A2A receptor antagonists, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103459
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Synthesis and evaluation of methoxy substituted 2-benzoyl-1-benzofuran derivatives as
lead compounds for the development adenosine A1 and/or A2A receptor antagonists
Helena D. Janse van Rensburga, Lesetja J. Legoabea*, Gisella Terre’Blanchea,b, Janine Aucampa
a
Centre of Excellence for Pharmaceutical Sciences,
School of Pharmacy, North-West University,
Private Bag X6001, Potchefstroom, 2520, South Africa
b
Pharmaceutical Chemistry,
School of Pharmacy,
North-West University,
Private Bag X6001, Potchefstroom, 2520, South Africa
*Corresponding authors at:
Centre of Excellence for Pharmaceutical Sciences,
School of Pharmacy, North-West University,
Private Bag X6001, Potchefstroom, 2520, South Africa
E-mail address: Lesetja.Legoabe@nwu.ac.za (L.J. Legoabe)
1

Abstract
A series of fourteen methoxy substituted 2-benzoyl-1-benzofuran derivatives were synthesised
and their affinities determined for adenosine A1 and A2A receptors via radioligand binding assays
to establish the structure activity relationships pertinent for A1 and A2A affinity. Compound 3j (6,7-
dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone exhibited A1 affinity (A1Ki (rat) = 6.880
µM) as well as A2A affinity (A2AKi (rat) = 0.5161 µM). Compounds 3a–b & 3i–k exhibited selective
affinity towards A1 with Ki values below 10 µM. The results indicate that C6,7-diOCH3 substitution
on ring A in combination with meta (C3’)-OCH3 substitution on ring B is beneficial for A1 and A2A
affinity and activity. Compounds 3a–b & 3j–k showed low cytotoxicity. Upon in vitro and in silico
evaluation, compound 3j may be considered lead-like (i.e. a molecular entity suitable for
optimization) and, thus, of value in the design of novel, potent and selective adenosine A1 and
A2A receptor antagonists.
Keywords
2-Benzoyl-1-benzofuran derivatives
Rap-Stoermer condensation reaction
Adenosine A1 and/or A2A receptor antagonists
Parkinson’s disease
Alzheimer’s disease
2

1. Introduction
Neurological conditions are now the leading cause of disability worldwide and, of these conditions;
Parkinson’s disease (PD) is the fastest growing – even surpassing Alzheimer’s disease (AD).[1,
2] PD is a neurodegenerative disorder characterized by both motor and non-motor symptoms.[3]
The pharmacological treatment of PD is as dependent on levodopa today, as it was 50 years
ago.[4] Although other drugs have been introduced since then, levodopa remains the most
effective drug for the treatment of parkinsonian motor symptoms.[3] Despite the clear benefits of
levodopa treatment, there are many limitations associated with initial levodopa treatment (e.g.
nausea, drowsiness and orthostatic lightheadedness) as well as chronic levodopa treatment (e.g.
motor fluctuations, dyskinesias and hallucinations).[3] Additionally, the non-motor symptoms of
PD do not respond well to dopaminergic treatment and forms a major challenge in the
pharmacological treatment of the disease.[5] Disease progression or neurodegeneration also
remains untreated, with no drugs currently approved for use as neuroprotective agents.[6]
Because of the aforementioned limitations of dopaminergic treatment, non-dopaminergic
strategies have been explored for the potential treatment of PD. One of these strategies includes
the first in class selective A2A AR antagonist, istradefylline (1a). This xanthine derivative was
developed by the Japanese company Kyowa Hakko Kirin and has been approved for
manufacturing and marketing in Japan since 2013 as NOURIAST®; a novel antiparkinsonian
agent for the wearing-off phenomena associated with levodopa treatment.[7] A post-marketing
surveillance study conducted in Japan proved istradefylline (1a) safe and effective; it is generally
well tolerated, despite dyskinesias and hallucinations as common adverse effects.[8]
Recently, the United States Food and Drug Administration (FDA) approved istradefylline (1a) as
an add-on drug to treat off episodes in adults with PD.[9] Istradefylline (1a) not only improves
motor fluctuations but also some non-motor symptoms of PD; such as the mood disorder
depression – the most common non-motor symptom of PD.[10, 11] However; this is yet to be
confirmed by a double-blind placebo-controlled trial.[11] Additionally, istradefylline (1a) improves
daytime sleepiness and bladder dysfunction.[12, 13] It is, unfortunately, difficult to demonstrate
neuroprotection by istradefylline (1a) in humans; as, to date, there is no way to assess
neurodegeneration or neuroprotection in humans.[6] Yet, in animal models of PD istradefylline
(1a) protected against dopaminergic neurodegeneration.[14, 15]
The adenosine-dopamine receptor-receptor interaction on striatopallidal output neurons in the
striatum,[16] where A2A ARs are abundantly expressed,[17] prompted the use of A2A AR
antagonists for the treatment of PD. These two receptors exert opposite effects on motor
behaviour.[16] For example, stimulation of dopamine D2 receptors enhances motor activity,
whereas activation of A2A ARs reduces this effect by inhibiting dopamine D2 receptor
3

signalling.[18] Therefore, antagonism of A2A ARs enhances dopamine D2 receptor dependant
signalling and, thus, reduces motor impairment.[19] Additionally, blockade of A2A ARs modulate
cholinergic, glutamatergic and GABAergic functions in the central nervous system (CNS).[20]
A1 ARs are localized presynaptically to dopamine axon terminals in the striatum
where these receptors inhibit dopamine release.[21]Consequently, antagonism of both A2A and
A1 ARs will be synergistic; inhibition of the A1 AR will facilitate dopamine release, while inhibition
of the A2A AR will enhance postsynaptic responses to dopamine.[22]
The A1 AR is also localized in the neocortical and limbic system structures that are associated
with cognitive function[22]. Antagonism of the A1 AR enhances neurotransmitter release in the
hippocampus and enhances performance in animal models of learning and memory.[23, 24]
It is not clear yet which is the better approach towards treating the symptoms of PD: a selective
A2A AR antagonist or a dual A2A and A1 AR antagonist.[19] Based on published preclinical and
clinical data it seems that most researchers are searching for selective A2A AR antagonists in
order to minimize possible cardiovascular effects caused by interaction with the A1 AR.[25] Other
researchers consider A1 AR antagonism as a desirable feature to address unmet needs in PD
treatment.[26, 27] For example, the dual A2A and A1 AR antagonists ASP5854 (1b) and JNJ-
40255293 (1c) with inhibition constant (Ki) values in the nanomolar-range (Figure 1) showed
promising effects in animal models of PD neurodegeneration and motor symptoms, as well as
cognition.[28]
O
O
O
O
N
N
N
N
N
N
O
N
O
O
N
O
N
H2N
N
NH2
F
1a
1b
1c
Istradefylline (Nouriast®)
A1Ki (h) = 841 nM
ASP5854
A1Ki (h) = 9.03 nM
2AKi (h) = 1.76 nM
JNJ-40255293
A1Ki (h) = 48 nM
A2AKi (h) = 6.50 nM
A2AKi (h) = 12 nM
A
Figure 1
Structures and Ki values of istradefylline (1a), ASP5854 (1b) and JNJ-40255293 (1c) against
human (h) A1 and A2A ARs.
However, due to the multifactorial etiology of the neurodegenerative disorder PD a robust
multitarget approach may possibly be necessary. This will involve the use of dual-acting
compounds that inhibit not only A2A and A1 ARs, but also mono-amine oxidases (MAO) and/or
cholinesterases.[20] For example, the A2A AR antagonist 8-(3-chlorostyryl)-caffeine (CSC) (A2AKi
(r)): 0.054 µM)[29] is also a reversible inhibitor of MAO-B (IC50: 146 nM).[30] It has been
4

suggested that antagonism of the A2A ARs and the selective inhibition of MAO-B may be
neuroprotective.[20]
The majority of AR antagonists are xanthine derivatives, for example istradefylline (1a). Xanthine
derivatives, however, have limitations such as poor water solubility.[31] In addition rapid photo
isomerization of the acyclic alkene of istradefylline (1a) after exposure to daylight in dilute
solutions has been observed.[32] Due to these undesirable physiochemical properties of xanthine
derivatives, the focus has shifted from xanthine to non-xanthine based heterocycles as A2A AR
antagonists.[19]
Heterocycles occupy a central position in medicinal chemistry and a number of both approved
and potential drugs contain at least one heterocyclic nucleus.[33, 34]
For example, the non-xanthine 7-OCH3 substituted benzofuran derivatives 1d–g (Figure 2) – also
from the Japanese company Kyowa Hakko Kirin that developed istradefylline (1a) – are potent
and selective A2A AR antagonists that show promise in animal models of PD.[35, 36] These
derivatives are heterocycles with a core consisting of fused 6- and 5-membered rings, where the
former is a benzene ring and the latter a furan ring.[19]
O
Cl
N
O
H
N
H
N
H
N
H
N
N
O
N
N
O
O
O
O
O
Cl
O
O
O
O
O
O
O
1d
1e
1f
1g
A
2A AR percentage inhibition at 10 nM = 39%
A
2A AR percentage inhibition at 10 nM = 38%
A
2A AR percentage inhibition at 10 nM = 28%
A
2A AR percentage inhibition at 10 nM = 30%
A2A AR percentage inhibition at 100 nM = 81%
A2A AR percentage inhibition at 100 nM = 101%
A2A AR percentage inhibition at 100 nM = 85%
A2A AR percentage inhibition at 100 nM = 73%
Figure 2
Structures and A2A AR percentage inhibition at 10 nM and 100 nM of compounds 1d–g.
The benzoyl functional group is also present in compounds with either A2A or A1AR affinity; such
as compounds 1h–i that are selective A2A AR antagonists and compound 1j that is a selective A1
AR antagonist (Figure 3).[37, 38]
5

O
N
S
H
N
N
S
O
S
O
H
N
O
S
O
N
N
O
O
1h
1i
1j
A1Ki (h) = >6 700 nM
2AKi (h) = 12 nM
A1Ki (h) = >10 000 nM
2AKi (h) = 88 nM
A1Ki (r) = 4.83 nM
2AKi (r) = >1 000 nM
A1Ki (h) = 57.4 nM
A
A
A
A2AKi (h) = 6250 nM
Figure 3
Structures and Ki values of compounds 1h–j against human (h) and/or rat (r) A1 and A2A ARs.
Coupling of the benzofuran and benzoyl moieties lead to 1-benzofuran-1-yl(phenyl)methanone
(2-benzoyl-1-benzofuran), a regioisomer of 2-benzylidene-benzofuran-3(2H)-one (aurone)
(Figure 4).
O
3
4'
5'
4
A
7
6'
B
3'
5
6
2
4
A
7
C
3
1' 2'
O
1'
5
6
2'
4'
O
2
C
1
6'
B
3'
O
1
5'
15H10O2
C
15H10O2
C
1-Benzofuran-2-yl(phenyl)methanone
2-Benzylidene-benzofuran-3(2H)-one
Figure 4
The regioisomers 2-benzylidene-benzofuran-3(2H)-one (aurone) and 1-benzofuran-1-
yl(phenyl)methanone (2-benzoyl-1-benzofuran).
Some aurone derivatives also possess A1 and/or A2A AR affinity (such as, hispidol (1k) and
maritimetin (1l)[39] (Figure 5)) and served as inspiration for the structurally related 2-benzylidene-
1-tetralone[40] and 2-benzylidene-1-indanone derivatives.[41, 42] This also lead to compounds
with affinity for both the A1 and A2A AR in the nanomolar range, including 4-OCH3 substituted 2-
benzylidene-1-indanone derivatives (E)-2-(3-Hydroxybenzylidene)-4-methoxy-2,3-dihydro-1H-
inden-1-one (1m)[41] and (E)-2-(3,4-Dihydroxybenzylidene)-4-methoxy-2,3-dihydro-1H-inden-1-
one (1n)[41] (Figure 5).
O
O
O
O
O
O
HO
HO
OH
OH
HO
O
OH
O
OH
OH
OH
1k
1l
Maritimetin
1m
1n
A1Ki (r) = 0.041 µM
A2AKi (r) = 0.097 µM
A1Ki (r) = 0.042 µM
A2AKi (r) = 0.078 µM
Hispidol
A1Ki (r) = 0.352 µM
A1Ki (r) = 3.47 µM
A2AKi (r) = >100 µM
A2AKi (r) = 9.35 µM
6

Figure 5
Structures and Ki values of compounds 1k–n against rat (r) A1 and A2A ARs.
The 2-benzoyl-1-benzofuran scaffold also bears resemblance to the simple, nonetheless
privileged, medicinal chemistry structure of 1,3-diphenylprop-2-en-1-one (chalcone) (Figure
6).[43] Chalcones and chalcone-based compounds have attracted a lot of attention from
both chemical (synthesis and biosynthesis) and, specifically, biological standpoints; due
to the wide-ranging biological activities exhibited by these compounds.[43, 44] These
activities include anticancer, antileishmanial, antimalarial, antimicrobial, antiviral,
antifungal, antioxidant, antihypertensive, antidiabetic biological activities – to name a
few.[45-49] Additionally, chalcones show potential in the treatment of neurological
conditions by acting as antidepressants and anxiolytics, as well as the modulation of
GABA receptors and the inhibition of acetylcholinesterase (AChE), butyrylcholinesterase
(BChE) and MAO.[44] Of note, the chalcone-coumarin hybrid (1o) possesses selective
A3 AR affinity (Figure 6),[50] the blockade of A3 ARs may be advantageous in brain
ischemia.[51]
4'
4'
5'
5'
O
6'
B
3'
6'
B
3'
4
A
7
4
A
7
3
3
1' 2'
O
1' 2'
O
5
6
5
6
2
2
C
S
O
O
O
1
1o
C15H12O
C15H10O2
A3Ki (h) = 5.16 µM
1,3-diphenylprop-2-en-1-one
1-Benzofuran-2-yl(phenyl)methanone
Figure 6
The structurally related 1,3-diphenylprop-2-en-1-one and 2-benzoyl-1-benzofuran scaffolds, as
well as the structure and Ki value of compound 1o against the human (h) A3 AR.
However, the chalcones (1o) and structurally related compounds, such as the aurone derivatives
(1k–l) as well as 2-benzylidene-1-tetralone and -indanone derivatives (1m–n) contain reactive
compound substructures. These substructures can be a risk factor in early drug discovery and
development; as the results of biological assays may be subjected to interference from these
reactive moieties.[52, 53] The said compounds all possess an α,β-unsaturated ketone group
perceived as a potential Michael acceptor[43, 54] and, unfortunately, Michael acceptors are
notoriously reactive compound substructures.[52, 55] As stated, the selective A2A AR antagonist
istradefylline (1a) also possess an exocyclic alkene (as seen in Figure 1) that has been
troublesome.[32] Compounds containing a catechol group, such as 1n are widespread in the
literature as potential starting points to further explore structure activity relationships. Yet,
compounds that contain known reactive moieties, such as a catechol group, are also considered
7

a liability; as catechols are potentially chelators, redox-active and oxidizes to form protein-reactive
quinones.[52, 53, 55]
Based on the above and in continuation of the efforts to obtain novel, potent and selective
heterocycles as A1 and A2A AR antagonists, a series of simple methoxy substituted 2-benzoyl-1-
benzofuran derivatives (a combination of the bioactive benzofuran and benzoyl moieties without
any reactive compound substructures) was designed (Figure 7), synthesised and evaluated to
ascertain the structure activity relationships that govern A1 and A2A AR affinity and activity.
2-Benzoyl
4'
5'
1-Benzofuran
6'
B
3'
4
3
1' 2'
O
5
6
2
A
7
C
O
1
1-Benzofuran-2-yl(phenyl)methanone
Figure 7
Scaffold of 1-benzofuran-1-yl(phenyl)methanone, highlighting the benzofuran and benzoyl
moieties.
2. Materials and Methods
2.1 Chemistry
Unless otherwise noted, all starting materials and solvents were procured from Sigma-Aldrich and
used without further purification. Thin layer chromatography on TLC silica gel 60 F254 aluminium
sheets from Merck was used to monitor reaction progress. Melting points were measured with a
Buchi B545 melting point apparatus and are uncorrected. Proton (1H) and carbon (13C) nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 600 spectrometer at
frequencies of 600 MHz and 151 MHz, respectively, using either CDCl3 or DMSO-d6 as solvent
and TMS as reference. Chemical shifts are reported in parts per million (δ) in relation to the solvent
1
peak (CDCl3: residual CH at 7.26 ppm and DMSO-d6: residual CH3 at 2.50 ppm for H NMR.)
Spin multiplicities are indicated as follow: singlet (s), doublet (d), dd (doublet of doublets), triplet
(t) and multiplet (m). J values are reported in Hz. High resolution mass spectra (HRMS) were
recorded on a Bruker micrOTOF-Q II mass spectrometer in atmospheric chemical ionisation
(APCI) mode. High performance liquid chromatography (HPLC) analyses were done on an Agilent
1100 HPLC system.
2.1.1 General procedure for the synthesis of 2a–d
2-Hydroxy-4,6-dimethoxybenzaldehyde (2a)
8

BBr3 (3.83 g, 15.29 mmol) was added dropwise to a solution of 2,4,6-trimethoxybenzaldehyde
(3.00 g, 15.29 mmol) in DCM (20 mL) cooled to 0°C. The ensuing reaction mixture was
mechanically stirred at 0°C for approximately 15 min and then at room temperature, the progress
of the reaction was continually monitored by TLC. Upon completion, the reaction mixture was
again cooled to 0°C and mechanically stirred while HCl (32%, 15 mL) was added slowly to the
reaction mixture. The resulting precipitate was filtered, dried (30°C) and used without further
purification. Additionally, the filtrate was extracted with DCM (5 x 20 mL), the combined organic
extracts were dried (MgSO4), filtered and concentrated. The dried precipitate and concentrated
combined organic extracts were pooled to yield compound 2a as a red solid (7.45 g, 89%), which
1
was used without further purification: Rf: 0.60 (PE/EtOAc 2:1); mp: 63.0–70.0°C; H NMR (600
MHz, CDCl3) δ 12.51 (s, 1H), 10.09 (s, 1H), 6.01 (d, J = 1.3 Hz, 1H), 5.91 (d, J = 1.6 Hz, 1H), 3.84
(d, J = 9.7 Hz, 6H); 13C NMR (151 MHz, CDCl3) δ 191.99, 168.26, 166.50, 163.69, 106.16, 93.04,
90.74, 55.91, 55.89. APCI-HRMS m/z calculated for C9H10O4 (MH+): 183.0652, found: 183.0662.
Purity (HPLC, Sig = 205): 87.8254%.
2-Hydroxy-4,5-dimethoxybenzaldehyde (2b)
Prepared as for 2a from 2,4,5-trimethoxybenzaldehyde (3.00 g, 15.29 mmol) and BBr3 (3.83 g,
15.29 mmol) to yield compound 2b as dark green solid (2.22 g, 80%), which was used without
1
further purification: Rf: 0.62 (PE/EtOAc 1:1); mp: 100.1–102.4°C; H NMR (600 MHz, CDCl3) δ
11.40 (s, 1H), 9.69 (s, 1H), 6.89 (s, 1H), 6.47 (s, 1H), 3.93 (s, 3H), 3.87 (s, 3H); 13C NMR (151
MHz, CDCl3) δ 194.12, 159.49, 157.37, 143.08, 113.29, 112.99, 100.28, 77.16, 56.58, 56.49.
APCI-HRMS m/z calculated for C9H10O4 (MH+): 183.0652, found: 183.0667. Purity (HPLC, Sig =
205): 91.1311%.
2-Hydroxy-3,4-dimethoxybenzaldehyde (2c)
A mixture of 2,3,4-trimethoxybenzaldehyde (3.00 g, 15.29 mmol) and AlCl3 (2.24 g, 16.80 mmol)
in PhH (60 mL) was mechanically stirred at room temperature for 5 min and then at 75°C under
reflux for 24h, the progress of the reaction was continually monitored by TLC. Upon completion,
the reaction mixture was quenched with H2O (25 mL) and HCl (32%, 10 mL). Next, the reaction
mixture was extracted with EtOAc (4 x 25 mL), the combined organic extracts were dried (MgSO4),
filtered, concentrated to yield compound 2c as beige crystals (0.57 g, 21%), which was used
without further purification: Rf: 0.29 (PE/EtAc 4:1); mp: 69.8–70.2°C; 1H NMR (600 MHz, CDCl3)
δ 11.20 – 11.17 (m, 2H), 9.74 (s, 2H), 7.28 (d, J = 8.7 Hz, 2H), 6.60 (d, J = 8.7 Hz, 2H), 3.94 (s,
7H), 3.90 (s, 7H); 13C NMR (151 MHz, CDCl3) δ 195.03, 159.52, 155.89, 136.32, 130.35, 116.70,
104.17, 60.89, 56.40. APCI-HRMS m/z calculated for C9H10O4 (MH+): 183.0652, found: 183.0660.
Purity (HPLC, Sig = 205): 35.6459%.
9

2-Bromo-3’-methoxyacetophenone (2d)
To a colourless solution of 3’-methoxyacetophenone (5.00 g, 33.29 mmol) in DCM (75 mL) and
EtOAc (75 mL) (1:1), CuBr2 (14.88 g, 66.62 mmol) was added and the reaction mixture was stirred
at 85°C under reflux for 24 h, the progress of the reaction was continually monitored by TLC.
Upon completion, the reaction mixture was filtered over a bed of SiO2 and the filtrate was
concentrated to yield the title compound 2a as a green solid (6.98 g, 91%), which was used
without further purification: Rf: 0.78 (DCM/EtOAc 10:1); mp: 53.7–54.9°C;1H NMR (600 MHz,
DMSO) δ 7.60 (d, J = 7.8 Hz, 1H), 7.50 – 7.45 (m, 2H), 7.27 – 7.24 (m, 1H), 4.95 (s, 2H), 3.82 (s,
3H); 13C NMR (151 MHz, DMSO) δ 191.57, 159.44, 135.35, 130.04, 121.18, 119.84, 113.28,
55.42, 34.36. APCI–HRMS m/z calculated for C9H9BrO2 (MH+): 228.9859, found: 228.9859. Purity
(HPLC, Sig = 205): 56.0755%.
2.1.2 General procedure for the synthesis of 3a–n
(6-Methoxybenzofuran-2-yl)(3-methoxyphenyl)methanone (3a)
To commercially available 2-hydroxy-4-methoxybenzaldehyde (0.50 g, 3.29 mmol) and
synthesised 2-bromo-3’-methoxyacetophenone (2d) (0.75 g, 3.29 mmol) in PEG-400 (5 mL),
anhydrous K2CO3 (1.36 g, 9.86 mmol) was added and the ensuing reaction mixture was
mechanically stirred at 100°C, the progress of the reaction was continually monitored by TLC.
Upon completion, the reaction mixture was cooled down to room temperature, quenched with H2O
(20 mL) and the resulting precipitate was filtered, dried (30°C) and recrystallized from a suitable
solvent (either MeOH (3a–b & f–n) or EtOH (3d–e)) to yield the title compound 3a as a light grey
solid (0.48 g, 52%): Rf: 0.67 (PE/EtAc 4:1); mp: 81.4–81.5°C; 1H NMR (600 MHz, CDCl3) δ 7.58
(dd, J = 14.9, 8.1 Hz, 2H), 7.50 (s, 1H), 7.47 (s, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.18 – 7.14 (m, 1H),
7.10 (s, 1H), 6.96 (dd, J = 8.6, 2.0 Hz, 1H), 3.89 (d, J = 1.5 Hz, 6H); 13C NMR (151 MHz, CDCl3)
δ 183.77, 161.43, 159.81, 157.84, 151.93, 138.96, 129.61, 123.80, 121.99, 120.50, 119.09,
117.54, 114.70, 114.01, 95.79, 55.90, 55.66. APCI-HRMS m/z calculated for C17H14O4 (MH+):
283.0965, found: 283.0946. Purity (HPLC, Sig = 254,4): 99.4029%.
(7-Methoxybenzofuran-2-yl)(3-methoxyphenyl)methanone (3b)
Prepared as for 3a from 2-hydroxy-3-methoxybenzaldehyde (0.50 g, 3.29 mmol) and compound
2d (0.75 g, 3.29 mmol) with anhydrous K2CO3 (1.36 g, 9.86 mmol) to yield compound 3b as a
brown solid (0.43 g, 46%): Rf: 0.32 (PE/EtOAc 4:1); mp: 80.1–86.8°C; 1H NMR (600 MHz, CDCl3)
δ 7.81 – 7.73 (m, 1H), 7.66 (d, J = 0.8 Hz, 1H), 7.62 (t, J = 6.7 Hz, 1H), 7.57 – 7.48 (m, 1H), 7.44
– 7.29 (m, 3H), 7.05 (dd, J = 13.0, 7.0 Hz, 1H), 4.12 (t, J = 6.7 Hz, 3H), 3.97 (t, J = 6.7 Hz, 3H);
13C NMR (151 MHz, CDCl3) δ 183.83, 159.83, 152.65, 146.29, 145.92, 138.53, 129.66, 128.76,
10

124.78, 122.35, 119.61, 116.56, 115.14, 114.03, 109.83, 56.30, 55.64. APCI-HRMS m/z
calculated for C17H14O4 (MH+): 283.0965, found: 283.0970. Purity (HPLC, Sig = 254,4): 98.0275%.
(4,6-Dimethoxybenzofuran-2-yl)(phenyl)methanone (3c)
Prepared as for 3a from 2-hydroxy-4,6-dimethoxybenzaldehyde (2a) (0.30 g, 1.65 mmol) and 2-
bromoacetophenone (0.33 g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield
compound 3c as brown crystals (0.15 g, 32%): Rf: 0.32 (PE/EtOAc 6:1); mp: 138.9–142.5°C; 1H
NMR (600 MHz, DMSO) δ 7.95 (d, J = 7.7 Hz, 2H), 7.69 (t, J = 7.4 Hz, 1H), 7.57 (dd, J = 18.4,
10.7 Hz, 3H), 6.95 (s, 1H), 6.50 (d, J = 1.5 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H); 13C NMR (151 MHz,
DMSO) δ 182.29, 162.69, 157.68, 154.91, 149.95, 137.07, 132.68, 128.91, 128.64, 114.86,
111.27, 95.56, 88.37, 56.00, 55.86. APCI-HRMS m/z calculated for C17H14O4 (MH+): 283.0965,
found: 283.0960. Purity (HPLC, Sig = 254,4): 99.9240%.
(4,6-Dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone (3d)
Prepared as for 3a from 2-hydroxy-4,6-dimethoxybenzaldehyde (2a) (0.30 g, 1.65 mmol) and
compound 2d (0.38 g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield compound
3d as a brown solid (0.24 g, 47%): Rf: 0.27 (PE/EtOAc 6:1); mp: 117.7–117.8°C; 1H NMR (600
MHz, CDCl3) δ 7.57 (d, J = 7.4 Hz, 1H), 7.53 (s, 1H), 7.48 (s, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.14
13
(dd, J = 7.8, 2.0 Hz, 1H), 6.71 (s, 1H), 6.33 (s, 1H), 3.91 (s, 3H), 3.88 (d, J = 4.3 Hz, 6H); C
NMR (151 MHz, CDCl3) δ 183.46, 162.95, 159.79, 158.59, 155.49, 150.66, 139.12, 129.57,
121.91, 118.93, 115.80, 113.92, 112.38, 95.46, 88.20, 56.03, 55.82, 55.66. APCI-HRMS m/z
calculated for C18H16O5 (MH+): 313.1071, found: 131.1073. Purity (HPLC, Sig = 254,4): 98.3448%.
(4,6-Dimethoxybenzofuran-2-yl)(4-methoxyphenyl)methanone (3e)
Prepared as for 3a from compound 2a (0.30 g, 1.65 mmol) and 2-bromo-4’-
methoxyacetophenone (0.38 g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield
compound 3e as golden crystals (0.35 g, 68%): Rf: 0.17 (PE/EtOAc 6:1); mp: 138.5–139.0°C; 1H
NMR (600 MHz, CDCl3) δ 8.03 (d, J = 8.6 Hz, 2H), 7.51 (s, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.71 (s,
1H), 6.33 (d, J = 1.3 Hz, 1H), 3.91 (d, J = 9.0 Hz, 6H), 3.87 (s, 3H); 13C NMR (151 MHz, CDCl3)
δ 182.33, 163.38, 162.61, 158.31, 155.34, 151.05, 131.74, 130.43, 114.58, 113.89, 112.33,
95.36, 88.23, 56.01, 55.82, 55.64. APCI-HRMS m/z calculated for C18H16O5 (MH+): 313.1071,
found: 313.1078. Purity (HPLC, Sig = 254,4): 98.8316%.
(4-Chlorophenyl)(4,6-dimethoxybenzofuran-2-yl)methanone (3f)
11

Prepared as for 3a from compound 2a (0.30 g, 1.65 mmol) and 2,4’-dichloroacetophenone (0.31
g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield compound 3f as mustard
crystals (0.31 g, 59%): Rf: 0.77 (PE/EtOAc 1:1); mp: 159.6–160.5°C; 1H NMR (600 MHz, CDCl3)
δ 7.94 (d, J = 8.2 Hz, 2H), 7.57 – 7.43 (m, 3H), 6.69 (s, 1H), 6.33 (s, 1H), 3.89 (d, J = 20.4 Hz,
6H); 13C NMR (151 MHz, CDCl3) δ 182.24, 163.10, 158.61, 155.48, 150.50, 139.01, 136.06,
130.79, 128.92, 115.69, 112.35, 95.55, 88.15, 56.04, 55.85. APCI-HRMS m/z calculated for
C17H13ClO4 (MH+): 317.0575, found: 317.0574. Purity (HPLC, Sig = 254,4): 97.7040%.
(2,4-Dichlorophenyl)(4,6-dimethoxybenzofuran-2-yl)methanone (3g)
Prepared as for 3a from compound 2a (0.30 g, 1.65 mmol) and 2,2’,4’-trichloroacetophenone
(0.37 g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield compound 3g as a yellow
solid (0.51 g, 88%): Rf: 0.76 (PE/EtOAc 1:1); mp: 131.7–132.8°C; 1H NMR (600 MHz, CDCl3) δ
7.50 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.36 (dd, J = 8.2, 1.9 Hz, 1H), 7.30 (s, 1H), 6.66
(s, 1H), 6.31 (d, J = 1.6 Hz, 1H), 3.87 (d, J = 5.2 Hz, 6H); 13C NMR (151 MHz, CDCl3) δ 181.56,
163.58, 159.11, 155.58, 150.25, 137.08, 136.15, 132.98, 130.35, 127.08, 117.23, 112.44, 95.65,
88.08, 56.07, 55.83. APCI-HRMS m/z calculated for C17H12Cl2O4 (MH+): 351.0185, found:
351.0207. Purity (HPLC, Sig = 254,4): 98.6778%.
(4-Bromophenyl)(4,6-dimethoxybenzofuran-2-yl)methanone (3h)
Prepared as for 3a from compound 2a (0.30 g, 1.65 mmol) and 2,4’-dibromoacetophenone (0.46
g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield compound 3h as a brown solid
(0.35 g, 59%): Rf: 0.62 (PE/EtOAc 1:1); mp: 176.4–179.4°C; 1H NMR (600 MHz, CDCl3) δ 7.87
(d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.52 (s, 1H), 6.69 (s, 1H), 6.33 (s, 1H), 3.90 (d, J =
20.1 Hz, 6H); 13C NMR (151 MHz, CDCl3) δ 182.39, 163.13, 158.64, 155.50, 150.47, 136.52,
131.91, 130.90, 127.60, 115.75, 112.36, 95.57, 88.16, 56.05, 55.86. APCI-HRMS m/z calculated
for C17H13BrO4 (MH+): 361.0070, found: 361.0068. Purity (HPLC, Sig = 254,4): 97.5698%.
(5,6-Dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone (3i)
Prepared as for 3a from 2-hydroxy-4,5-dimethoxybenzaldehyde (2b) (0.40 g, 2.18 mmol) and
compound 2d (0.50 g, 2.18 mmol) with anhydrous K2CO3 (0.91 g, 6.55 mmol) to yield compound
1
3i as a light yellow solid (0.58 g, 86%): Rf: 0.38 (PE/EtOAc 1:1); mp: 103.8–105.2°C; H NMR
(600 MHz, CDCl3) δ 7.62 – 7.58 (m, 1H), 7.51 (dd, J = 2.3, 1.5 Hz, 1H), 7.46 (d, J = 0.7 Hz, 1H),
7.42 (t, J = 7.9 Hz, 1H), 7.17 – 7.13 (m, 1H), 7.12 (s, 1H), 7.06 (s, 1H), 3.95 (d, J = 21.3 Hz, 6H),
3.89 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 183.40, 159.79, 152.09, 152.03, 152.01, 147.99,
139.02, 129.56, 122.02, 119.26, 119.00, 117.60, 114.06, 102.93, 95.34, 77.16, 56.48, 56.47,
55.66. APCI-HRMS m/z calculated for C18H16O5 (MH+): 313.1071, found: 313.1064. Purity (HPLC,
Sig = 254,4): 100%.
12

(6,7-Dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone (3j)
Prepared as for 3a from 2-hydroxy-3,4-dimethoxybenzaldehyde (2c) (0.50 g, 2.74 mmol) and
compound 2d (0.63 g, 2.74 mmol) with anhydrous K2CO3 (1.14 g, 8.23 mmol) to yield compound
3j as a brown solid (0.47 g, 55%): Rf: 0.52 (PE/EtOAc 1:1); mp: 79.8–80.3°C; 1H NMR (600 MHz,
CDCl3) δ 7.63 (d, J = 7.3 Hz, 1H), 7.54 (s, 1H), 7.49 (s, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.32 (d, J =
8.5 Hz, 1H), 7.16 (dd, J = 8.1, 2.3 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 4.23 (s, 3H), 3.93 (d, J = 47.9
Hz, 6H); 13C NMR (151 MHz, CDCl3) δ 183.57, 159.82, 152.61, 151.70, 148.50, 138.79, 134.87,
129.61, 123.15, 122.08, 119.29, 117.16, 116.78, 114.03, 111.32, 61.20, 57.31, 55.64. APCI-
HRMS m/z calculated for C18H16O5 (MH+): 313.1071, found: 313.1069. Purity (HPLC, Sig = 254,4):
97.0823%.
(6,7-Dimethoxybenzofuran-2-yl)(4-methoxyphenyl)methanone (3k)
Prepared as for 3a from compound 2c (0.20 g, 1.10 mmol) and 2-bromo-4’-
methoxybacetophenone (0.25 g, 1.10 mmol) with anhydrous K2CO3 (0.46 g, 3.29 mmol) to yield
compound 3k as a beige solid (0.19 g, 55%): Rf: 0.17 (PE/EtOAc 4:1); mp: 86.1-87.3°C; 1H NMR
(600 MHz, CDCl3) δ 8.11 (d, J = 8.7 Hz, 2H), 7.48 (s, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.01 (dd, J =
8.5, 2.2 Hz, 3H), 4.23 (s, 3H), 3.94 (d, J = 34.2 Hz, 6H); 13C NMR (151 MHz, CDCl3) δ 182.35,
163.62, 153.19, 151.40, 148.26, 134.88, 132.02, 130.10, 123.24, 116.56, 115.96, 113.94, 111.22,
61.18, 57.33, 55.67. APCI-HRMS m/z calculated for C18H16O5 (MH+): 313.1071, found: 313.1075.
Purity (HPLC, Sig = 254,4): 98.9813%.
(4-Chlorophenyl)(6,7-dimethoxybenzofuran-2-yl)methanone (3l)
Prepared as for 3a from compound 2c (0.30 g, 1.65 mmol) and 2,4’-dichloroacetophenone (0.31
g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield compound 3l as yellow crystals
(0.12 g, 23%): Rf: 0.86 (PE/EtAc 1:1); mp: 106.6–107.6°C; 1H NMR (600 MHz, CDCl3) δ 8.01 (d,
J = 8.4 Hz, 2H), 7.51 (d, J = 8.3 Hz, 3H), 7.34 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.22
(s, 3H), 3.97 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 182.48, 152.45, 151.81, 148.51, 139.38,
135.70, 134.75, 130.98, 128.98, 122.99, 117.09, 116.88, 111.32, 61.21, 57.26. APCI-HRMS m/z
calculated for C17H13ClO4 (MH+): 317.0575, found: 317.0575. Purity (HPLC, Sig = 254,4):
96.7780%.
(4-Bromophenyl)(6,7-dimethoxybenzofuran-2-yl)methanone (3m)
Prepared as for 3a from compound 2c (0.30 g, 1.65 mmol) and 2,4’-dibromoacetophenone (0.46
g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield compound 3m as bright yellow
crystals (0.92 g, 49%): Rf: 0.77 (PE/EtOAc 1:1); mp: 124.1–124.4°C; 1H NMR (600 MHz, CDCl3)
δ 7.93 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.50 (s, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.02 (d,
13

J = 8.6 Hz, 1H), 4.21 (s, 3H), 3.97 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 182.58, 152.47, 151.86,
148.56, 136.16, 134.84, 131.96, 131.08, 128.01, 123.02, 117.07, 116.88, 111.45, 61.20, 57.30.
APCI-HRMS m/z calculated for C17H13BrO4 (MH+): 361.0067, found: 361.0077. Purity (HPLC, Sig
= 254,4): 98.1484%.
4-(6,7-Dimethoxybenzofuran-2-carbonyl)benzonitrile (3n)
Prepared as for 3a from compound 2c (0.30 g, 1.65 mmol) and 2-bromo-4’-cyanoacetophenone
(0.37 g, 1.65 mmol) with anhydrous K2CO3 (0.68 g, 4.94 mmol) to yield compound 3n as mustard
crystals (0.43 g, 85%): Rf: 0.58 (PE/EtOAc 1:1); mp: 127.2–127.9°C; 1H NMR (600 MHz, CDCl3)
δ 8.13 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.53 (s, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.04 (d,
J = 8.5 Hz, 1H), 4.20 (s, 3H), 3.97 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 182.00, 152.29, 152.02,
148.85, 140.87, 134.78, 132.45, 129.93, 129.73, 127.66, 122.83, 118.08, 117.92, 117.18, 116.14,
111.66, 61.22, 57.27. APCI-HRMS m/z calculated for C18H13NO4 (MH+): 308.0917, found:
308.0917. Purity (HPLC, Sig = 254,4): 98.9628%.
2.2 Biology
2.2.1 In vitro evaluation: Radioligand binding studies
All reagents were commercially available and purchased from various manufacturers.
Radioligands [3H]DPCPX (specific activity 120 Ci/mmol) and [3H]NECA (specific activity 21.1
Ci/mmol) as well as filter-count from PerkinElmer and Whatman GF/B 25 mm diameter filters were
obtained from Merck. Radio activity was counted by a Packard Tri-CARB 2810 liquid scintillation
counter.
2.2.1.1 Membrane preparations
The collection of tissue samples for the A1 and A2A radioligand binding assays were approved by
the Research Ethics Committee of the North-West University (NWU) (application number NWU -
00260-17-A5). Male Sprague-Dawley rats were dissected to attain rat whole brain membranes
(excluding cerebellum and brain stem) and rat striatal membranes for the A1 and A2A AR
radioligand binding assays, respectively. Tissue samples were prepared and stored as described
in literature.[56] Protein concentrations were determined according to the Bradford assay, using
bovine serum albumin as reference standard.[57]
2.2.1.2 Adenosine A1 and A2A receptor radioligand binding assays
The A1 AR radioligand binding assay used rat whole brain membranes (expressing A1 ARs) and
1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) (selective A1 AR antagonist) as
radioligand[58] and, in turn, the A2A AR radioligand binding assay used rat striatal membranes
14

(expressing A2A ARs) and 5’-N-ethylcarboxamido[3H]adenosine ([3H]NECA) (non-selective A1
and A2A AR antagonist) as radioligand.[59] Each incubation of the A1 AR assay consisted of (in
order of additions): (i) test compound (10 µL), (ii) 0.1 nM [3H]DPCPX (radioligand solution, 100
µL) and (iii) 120 µg rat whole brain membranes and 0.1 units/mL adenosine deaminase
(membrane suspension, 890 µL).[56, 58] Whereas, every incubation of the A2A AR assay
consisted of (in order of additions): (i) 120 µg rat striatal membranes and 0.2 units/mL adenosine
deaminase (membrane suspension, 790 µL), (ii) test compound (10 µL), (iii) CPA (100 µL) and
(iv) 4 nM [3H]NECA (radioligand solution, 100 µL).[56, 59] The agonist radioligand [3H]NECA) is
used in A2A AR binding assays; however, [3H]NECA is known to bind to both the A1 and A2A AR
subtypes in rat striatal membranes.[59] To prevent [3H]NECA from compromising the validity of
the A2A AR radioligand binding assay N6-cyclopentyladenosine (CPA) was also added to
selectively eliminate the A1 component of binding; CPA is a potent A1 AR agonist with a higher
affinity toward the A1 ARs than [3H]NECA[59] and as a result, the presence of CPA increases
[3H]NECA’s selectivity for the A2A ARs.[59] MgCl2 is also added to an A2A AR binding assay with
[3H]NECA as radioligand; as it increases radioligand binding and decreases non-specific
binding.[59] The final volume of all incubations also contained 1 mL of 50 mM Tris.HCl buffer and
1% DMSO.[56] Non-specific binding of [3H]DPCPX and [3H]NECA for the A1 and A2A AR assay,
respectively was defined as binding in the presence of 100 µM CPA.[56, 59] Specific binding was
defined as the total binding minus the non-specific binding.[56]
2.2.1.3 GTP shift assay
A GTP shift assay resembles the A1 AR radioligand binding assay. However, it requires the
addition of 100 µM [35S]guasine-5’-O-(3-thio)triphosphate (GTP). Non-specific binding was
defined as binding in the presence of 10 µM DPCPX.[60, 61]
2.2.1.4 Data analysis
In short, all statistical data analyses were done using Microsoft Excel and GraphPad Prism
Software..
Sigmoidal dose response curves, from which IC50 values were calculated, were obtained by
plotting the specific binding against the logarithm of the test compounds’ concentrations.
Subsequently, the IC50 values were used to calculate the Ki values for the competitive inhibition
of [3H]DPCPX (Kd = 0.36 nM) agains rat whole brain membranes and [3H]NECA (Kd = 15.3 nM)
against rat striatal membranes by the test compounds by means of the Cheng-Prusoff equation.
All calculated Ki values are determined in triplicate and given as mean ± standard error of the
mean (SEM). SI index values were calculated as a ratio of the A1 and A2A AR Ki values of test
15

compounds. GTP shifts were calculated by dividing the Ki values of compounds reported in the
presence of GTP by the Ki values obtained in the absence of GTP
2.2.2 In vitro evaluation: Cytotoxicity
All reagents were commercially available and purchased from various manufacturers: Hyclone
Vero cells from Cellonex, South Africa. Dulbecco’s modified Eagle’s medium with high glucose
from Separations, 10% fetal bovine serum from Thermofisher Scientific and 1% Lglutamine,
penicillin-streptomycin, amphotericin B and non-essential amino acids all from Lonza. Emetine
dihydrochloride and resazurin sodium salt were purchased from Sigma Aldrich. Absorbance was
measured using the Thermofisher Scientific GO Multiscan plate reader.
2.2.2.1 Resazurin assay (AlamarBlue® assay)
To gain an indication of potential cytotoxicity of the 2-benzoyl-1-benzofuran derivatives that
possess A1 or A2A AR affinity, namely compounds 3a–b and 3i–k, the effect of these compounds
on the viability of cultured Vero cells were measured. For this purpose, the resazurin assay, also
known as the AlamarBlue® assay was used. The assay involves the irreversible enzymatic
reduction of oxidized blue resazurin dye to pink, highly fluorescent resorufin by viable cells.[62]
This non-toxic reagent serves as an effective tool for assessing cell proliferation and drug toxicity.
Vero cells (Cellonex, South Africa passage 9–11) were cultured in Hyclone Dulbecco’s modified
Eagle’s medium with high glucose supplemented with 10% fetal bovine serum and 1%
Lglutamine, penicillin-streptomycin solution, amphotericin B and non-essential amino acids. The
cells were maintained in a humidified atmosphere at 37°C and 5% CO2. For the resazurin assay,
96 well plates were prepared with 200 μL of cell suspension (30 000 cells/mL) and incubated for
24 h. The cells were then treated with: (i) 100 μL of emetine dihydrochloride solution diluted with
growth medium to the necessary concentrations (positive control); (ii) 80 μL of growth medium
and 20 μL of 1% DMSO (negative control to compensate for possible solvent effects); (iii) 80 μL
of growth medium and 20 μL of experimental compound solutions in 1% DMSO (the final DMSO
concentration when added to well was 0.2%). Compounds were dissolved in 100% DMSO, but
precipitated once the DMSO concentration was diluted to 1%. A serial two-fold dilution with a
starting concentration of 100 μM were done to obtain the final well concentration. Blanks
contained growth medium without cells. The treated plates were incubated for 48 h. Blank and
positive control results were obtained in triplicate, whereas the test compounds’ results were
obtained in duplicate. The Z’-prerequisite is >0.7. To initiate the resazurin assay, 50 μL of sterile-
filtered resazurin sodium salt solution (0.01 % in PBS) was added and the plates incubated for 2
h. Absorbance was measured at 570 and 600 nm.
2.2.2.3 Data analysis
16

Data analysis was performed for each biological replicate using SkanIt 4.0 Research Edition
software. Background resazurinabsorbance (600 nm) was subtracted from resorufin absorbance
values (570 nm), the mean absorbance calculated and the percentage cell viability was
determined by the following equation:
Cell viability % = (Δ Abs sample – Δ Abs blank) / (Δ Abs neg control – Δ Abs blank) X 100
The IC50 and Z-score were determined for each compound’s biological replicate using GraphPad
Prism. For the final IC50 of each compound, the mean IC50 of the biological replicates were
calculated with standard error of the mean (SEM).
2.3 In silico evaluation
SwissADME (http://www.swissadme.ch), a free web tool, was used to evaluate key parameters
of small molecules; such as pharmacokinetics, drug-likeness and medicinal chemistry
friendliness. Pharmacokinetic parameters (absorption, distribution, metabolism and excretion),
among others, are predicted from molecular structures using the most relevant computational
methods – and, thus, do not focus on just one specific property or model.[63]
3. Results and discussion
3.1 Chemistry
But for reagents (2a–d), all starting materials were commercially available and used without
further purification.
The synthesis of reagents (2a–d) and test compounds (3a–n) are depicted in Scheme 1.
Reagents 2a–c were obtained by ortho-demethylation of trimethoxy substituted benzaldehydes
(namely, 2,4,6-trimethoxy- (2a), 2,4,5-trimethoxy- (2b) and 2,3,4-trimethoxybenzaldehyde (2c))
using BBr3 (2a–b)[64] and AlCl3 (2c)[65] as demethylation agents in DCM and PhH, respectively.
This selective ortho-demethylation is due to the ortho-directing influence of the carbonyl
substituent of the respective benzaldehydes.[66]
The heterogeneous bromination of 3’-methoxyacetophenone using CuBr2 in DCM/EtOAc (1:1)
yielded 2-bromo-3’-methoxyacetophenone (2d).[67] Note, only selective α-bromination occurred
and polar solvents, such as DCM and EtOAc, are ideal for the present reaction system.[68]
A
total
of
novel
fourteen
test
compounds
(3a–n),
3i–n),
including
nine
2-benzoyl-1-benzofurans
(3d–e
&
were prepared by a base-mediated Rap-Stoermer condensation reaction.[69]
17

O
O
2
O
OH
2
O
O
OH
2
O
O
O
2
1
6
1
6
3
4
3
1
6
3
1
6
3
a
a
4
O
O
4
4
O
5
5
O
5
5
O
O
2a
2b
O
2
O
O
OH
2
O
O
O
O
O
O
1
6
1
6
3
4
2'
2'
3
O
Br
O
3'
b
c
1'
3'
1'

2
4
5
5
2d
2c
R3'
3'
R1'
2'
O
OH
2
O
4'
R1
R4
R3
R1''
R2'
R3'
R2'
B
1
6
3
4
1'
3'
4'
R2
R3
4
d
3
2
2'
R1'
+
5
6
1'
O
2
R1
A
C
5
R2
O
1
7
R4
3a-e, 3h-l & 3n: R'' = Br
3f-g & 3m: R'' = Cl
3a-n
Scheme 1
Synthesis of 2a–d & 3a–n. Reagents and conditions: a) BBr3, DCM, 0°C (15 min), room
temperature (1h); b) AlCl3, PhH, room temperature (5 min), 75°C (24 h); c) CuBr2, DCM/EtOAc
(1:1) 85°C (24 h); d) K2CO3, PEG-400, 100°C (24 h).
A plausible reaction mechanism toward the synthesis of test compounds 3a–n is depicted in
Scheme 2. Initially, 2-hydroxybenzaldehyde (I) and the base K2CO3 (II) formed the potassium salt
III that, subsequently, reacted with 2-halogen substituted acetophenone (IV), yielding the
intermediate 2-(2-oxo-2-phenylethoxy)benzaldehyde (V).[70] Intramolecular cyclisation of the
carbanion (VI) and dehydration of the ketoalcohol (VII) afforded 2-benzoyl-1-benzofuran (VIII).[70,
71] The cis, rather than trans, conformation of VII is suggested; as there is no serious repulsion
between the formyl hydrogen and the hydrogen binding to the carbanion during the cyclisation of
VI.[70]
O
R1'
R2'
R3'
H
O
H
O
OH
O
OK
O
R1'
II
R1
R2
R1
R2
R1
R2'
R3'
O
-R1K
H
K2CO3
H
R1
R2
R4
R4
H
R3
R3
R4
R3
I
III
IV
V
-H+
O
R1'
R3'
R3'
R2'
R3'
H
O
R1
R1
R2'
OH
R2'
O
-H+
-H2O
R2
R3
R2
R3
R1
R2
H
R1'
R1'
H
O
O
H
O
O
R4
R4
R4
R3
VIII
(3a-n)
VII
VI
18

Scheme 2
Plausible reaction mechanism toward the synthesis of 2-benzoyl-1-benzofuran derivatives by a
base-mediated Rap-Stoermer condensation reaction.
Generally, this reaction is carried out under basic conditions in organic solvents. However, Zhao
and colleagues recommend PEG-400 as alternative solvent for the basic assisted condensation
of 2-hydroxybenzaldehydes and 2-halogen substituted acetophenones.[69, 72] This
recommendation is made due to the hydrophilicity and thermal stability of PEG-400.[69] PEG-400
mediated reactions are useful from both economical and environmental standpoints; it is not only
inexpensive, but also non-toxic, bio-compatible and bio-degradable.[69, 73]
All synthesized reagents (2a–d) and test compounds (3a–n) were obtained in fair to poor yields,
purified by recrystallization from a suitable solvent (either MeOH or EtOH) and, in each instance,
1
13
the structure, molecular mass and/or purity of these compounds were verified by H NMR, C
NMR, MS and/or HPLC.
3.2 Biology
3.2.1 In vitro evaluation: Radioligand binding studies
3.2.1.1 Adenosine A1 and A2A receptor radioligand binding assays
The degree of binding affinity that the test compounds (3a–n) possesses towards rat A1 and A2A
ARs was determined via radioligand binding assays in either duplicate (specific binding (%)) or
triplicate (inhibition constant (Ki, µM)) and expressed as mean ± standard error of the mean
(SEM). Only compounds 3a–b and 3i–k – that displayed specific binding values of <20% at a
maximum tested concentration of 100 µM – justified the determination of inhibition constant
values (Ki, µM), unlike compounds 3c–h and 3m–n (specific binding values of >20%).
Furthermore, concurrent radioligand binding assays allow for the calculation of the degree of
selectivity the test compounds (3a–n) possesses towards either rat (r) A1 or A2A ARs (selectivity
index (SI)). The A1 and A2A AR radioligand binding assays were validated with CPA (A1 agonist),
DPCPX (A1 antagonist) and istradefylline (1a) (A2A antagonist) as reference compounds and
results were in accordance with literature values. These results, expressed as Ki values (µM), are
summarized in Table 1 and 2.
As seen in Table 3, of the 15 compounds (3a–n) evaluated, 6 of these compounds (3a–b, 3i–k)
have A1 AR affinity and only 1 compound (3j) has A2A AR affinity (A2AKi (r) = 0.5161 µM) – making
compound 3j the only dual A1 and A2A AR antagonist in this series. In general, however,
compound 3a had the highest A1 AR affinity (A1Ki (r) = 5.525 µM), closely followed by compounds
19

3j (A1Ki (r) = 6.880 µM) and 3k (A1Ki (r) = 6.858 µM). Nonetheless, the standout compound of the
present series remains 3j.
Structure activity relationships (SAR) based on Ki values (µM) of the methoxy substituted 2-
benzoyl-1-benzofuran derivatives showed a coherent relationship between these structures and
A1 and A2A AR affinity.
Optimal position for OCH3 group on ring A:
The affinities of compounds 3a, 3b, 3d, 3i and 3j, that possess meta (C3’)-OCH3 group
substitution on ring B, were compared and it was found that that the highest A1 AR affinity of the
series was attained by C6-OCH3 substitution on ring A (3a) – closely followed by C6, C7-diOCH3
(3j–k) and then C5,6-diOCH3 (3i) and C7-OCH3 (3b) in decreasing order of affinity. Comparison
of 3d to 3j and 3e to 3k, respectively, corroborates this; as compound 3d and 3e with C4,6-
diOCH3 substitution on ring A lacks affinity (A1Ki (r) = >100 µM) and compound 3j and 3k with
C6,7-diOCH3 substitution on ring A retains A1 AR affinity (3j: A1Ki (r) = 6.880 µM; 3k: A1Ki (r) =
6.858 µM).
Other compounds with corresponding ring B substitutions included 3f and 3l (para (C4’)-Cl) as
well as 3h and 3m (para (C4’)-Br). These compounds, unfortunately, lacked both A1 and A2A
affinity (A1 and A2A Ki (r) = >100 µM).
Interestingly, for the benzofuran derivatives 1d–g (Figure 2) that bear a 7-OCH3 substituent on
ring A, the same pattern of affinity was observed. The same is observed with compound 3b and
3j–k.
Optimal group and position of substituent on ring B:
A1 or A2A AR ligands 1h–i (Figure 2) all contained unsubstituted benzoyl groups. Yet, compound
3c with an unsubstituted benzoyl group does not have either A1 or A2A affinity.
Comparison of compounds 3j and 3k to compounds 3l–n (all with C6,7-diOCH3 substitution on
ring A) demonstrates that, OCH3 group substitution on ring B is beneficial for either dual A1 and
A2A AR affinity (3j) or selective A1 AR affinity (3k) in contrast to electron withdrawing groups such
as Cl (3l: A1 and A2A Ki (r) = >100 µM), Br (3m: A1 and A2A Ki (r) = >100 µM) and CN (3n: A1 and
A2A Ki (r) = >100 µM). It may be assumed that OCH3-group substitution on ring B is preferred to
halogen and nitrile substituents and that the meta (C3’)-OCH3 substitution is preferred to para
(C4’)-OCH3 substitution to attain dual A1 and A2A AR affinity.
These results highlight the important roles both ring A and ring B substitutions and the
combination of such substitutions play in gaining or even losing A1 and A2A AR affinity. When ring
20

A bears C4,6-diOCH3 substitution, ring B prefers electron withdrawing groups as well as H- and
OCH3-groups. Yet, when ring A bears C6,7-diOCH3 substitution, ring B prefers either meta (C3’)
or para (C4’)-OCH3-group substitution.
Although, compound 3j does not compare well to benzofuran derivatives (1d–g) and benzoyl
derivatives (1h-j) with regards to A1 and A2A AR affinity, this methoxy substituted 2-benzoyl-1-
benzofuran derivative does possess a Ki value higher than both hispidol (1k: A2AKi (r) = >100 µM)
and maritimetin (1l: A2AKi (r) = 9.35 µM) for the A2A AR of 0.5161 µM with selectivity in favour of
the A1 AR. Additionally, compound 3j does not contain reactive compound substructures, such
as compounds 1k–m.
Structural requirements of the 2-benzoyl-1-benzofuran scaffold for A1 and/or A2A AR affinity are
summarized in Figure 8.
Table 1
The Ki values for the binding of reference compounds at rat A1 and A2A ARs
Ki ± SEM (µM)a
SId
A1/A2A
A1b vs
A2Ab vs
A1b + GTPc vs
[3H]DPCPX
#
GTP shifte
[3H]DPCPX
[3H]NECA
CPA
(A1 agonist)
0.0057 ± 0.0015a
(0.0068)f
0.4004 ± 0.1715a
(0.163)f
0.0990 ± 0.0150a
(0.099)f
0.0142d
17.36e
(15)f
(0.0417)d
(0.0453)d
(0.015)g
(0.331)g
(0.099)g
(14)g
(0.0079)h
DPCPX
(A1 antagonist)
0.0005 ±
0.2338 ± 0.0328a
(0.545)f
0.0006 ±
0.00003a
(0.0004)f
(0.0004)g
0.0021d
1.2e
0.00003a
(0.0007)d
(0.0009)d
(0.0008)d
(1)f
(0.0004)f
(0.530)g
(1.25)g
(0.0005)g
(0.340)i
(0.0003)i
Istradefylline (1a)
(A2A antagonist)
0.1924 ± 0.0131a
(0.2300)j
0.0014 ± 0.0003a
(0.0022)j
0.1514 ± 0.0178a
137.4d
0.7869e
(104.5)d
a All inhibition constant (Ki) values were determined in triplicate and expressed as mean ± standard error of the mean
(SEM) in µM.
b Rat receptors were used (A1: rat whole brain membranes; A2A: rat striatal membranes).
c Addition of 100 µM GTP to A1 AR radioligand binding assay.
d Selectivity index (SI) for the A2A AR isoform calculated as a ratio of A1Ki/A2AKi.
e GTP shift calculated by dividing Ki value in the presence of 100 µM GTP by Ki value in the absence of 100 µM GTP.
f Literature value obtained from [74]
g Literature value obtained from [56]
h Literature value obtained from [58]
i Literature value obtained from [75]
j Literature value obtained from [76]
Table 2
The Ki values for the binding of 2-benzoyl-1-benzofuran derivatives at rat A1 and A2A ARs
R3'
4'
R1
R2'
3'
B
R2
R3
4
3
2'
R1'
5
6
1'
O
2
A
C
O
1
7
R4
3a-n
#
Ring A
Ring B
Ki ± SEM (µM)a
SId
21

(Specific binding (%))b
4
5
6
7
2’
R1’
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
3’
R2’
OCH3
OCH3
H
OCH3
H
H
H
H
OCH3
OCH3
H
4’
A1c vs [3H]DPCPX A2Ac vs [3H]NECA A1/A2A
R1
R2
R3
R4
R3’
H
H
H
H
3a
3b
3c
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
5.525 ± 0.8452a
8.854 ± 0.2542a
(57 ± 3)b
(51 ± 5)b
-
-
-
-
-
-
-
OCH3
H
(26 ± 3)b
OCH3
3d OCH3
OCH3
OCH3
3g OCH3
3h OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
(149 ± 53.5)b
(90 ± 11)b
(98 ± 9)b
H
H
H
H
H
H
(44 ± 0)b
3e
3f
OCH3 (84 ± 1)b
Cl
Cl
Br
H
(96 ± 10)b
(77 ± 2)b
(113 ± 11.5)b
8.478 ± 0.7356a
6.880 ± 0.6612a
(81 ± 1)b
(114 ± 15.5)b
(126 ± 18)b
(43 ± 7.5)b
0.5161 ± 0.0578a
(33 ± 5)b
-
-
3i
3j
3k
3l
3m
3n
H
H
H
H
H
H
OCH3 OCH3
H
H
H
H
H
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
H
13.33d
OCH3 6.858 ± 2.859a
Cl
Br
CN
-
-
-
-
H
H
H
(44 ± 0)b
(68 ± 3)b
(30 + 1)b
(86 ± 15)b
(100 ± 3)b
(48 ± 4)b
a Inhibition constant (Ki) values were determined in triplicate and expressed as mean ± standard error of the mean
(SEM) in µM.
b Specific binding (%) values of the radioligand at a maximum tested concentration of 100 µM were determined in
duplicate and expressed as mean ± standard error of the mean (SEM) in %.
c Rat receptors were used (A1: rat whole brain membranes; A2A: rat striatal membranes).
d Selectivity index (SI) for the A2A AR isoform calculated as a ratio of A1Ki/A2AKi.
R3'
Ring A
5'
Ring B
R1
4
R2'
6'
B
Best A1 AR affinity of series:
R1 = R2 = R4 = H, R3 = OCH3
Best A1 AR affinity:
R1' = R3' = H, R2' = OCH3
R2
R3
3
5
6
1'
R1'
2
A
C
Best A2A AR affinity of series:
R1 = R2 = H, R3 = R4 = OCH3
Best A2A AR affinity:
R1' = R3' = H, R2' = OCH3
O
O
1
7
R4
Figure 8
Structural requirements of the 2-benzoyl-1-benzofuran scaffold for A1 and/or A2A AR affinity.
3.2.1.2 GTP shift assay
The type of binding affinity that a test compound (3a) exhibits at the rat A1 AR was determined
via a GTP shift assay and the results are summarized in Table 3. Compound 3a, possessing the
highest A1 AR affinity and selectivity, was selected for this purpose. GTP may possibly exert its
effects by uncoupling the A1 AR receptor from its G-protein, leading to a shift from the high to the
low affinity state for agonists.[77] Therefore, by comparing a test compound’s binding curves in
the presence and absence of GTP it is possible to determine if a compound will act as an agonist,
a partial agonist or an antagonist.[56] An antagonist’s binding curve in the presence of GTP is
unaffected and a test compound with a calculated GTP shift of approximately 1 may be
considered an antagonist.[60, 78] The results suggest that compound 3a acts as an A1 AR
antagonist; as no significant rightward shift of the binding curve was observed in the presence of
GTP and compound 3a has a calculated GTP shift of 1.186 (Figure 9). On account of the
structural similarity of the test compounds (3a–n), it may be proposed that these 2-benzoyl-1-
benzofuran derivatives are all A1 AR antagonists. Interestingly, human and rat proteins
22

demonstrate similar regulation of GTP and endogenous adenosine binding interactions in A1 and
A2A AR binding studies.[77] The GTP shift assays were also validated with CPA (A1 agonist),
DPCPX (A1 antagonist) and istradefylline (1a) (A2A antagonist) as reference compounds and
results were in accordance with literature values. These results are also summarized in Table 2.
Table 3
The A1Ki values (in the absence and presence of GTP) and calculated GTP shifts of 3a
Ki ± SEM (µM)a
#
GTP shiftd
A1b vs [3H]DPCPX
5.525 ± 0.8452a
A1b + GTPc vs [3H]DPCPX
6.551 ± 0.1457a
3a
1.186d
a All inhibition constant (Ki) values were determined in triplicate and expressed as mean ± standard error of the mean
(SEM) in µM.
b Rat receptors were used (A1: rat whole brain membranes).
c Addition of 100 µM GTP to A1 AR radioligand binding assay.
d GTP shift calculated by dividing Ki value in the presence of 100 µM GTP by Ki value in the absence of 100 µM GTP.
150
+ GTP (100 µM)
- GTP
100
50
-2
-1
0
1
2
3
Log[3a] (µM)
Figure 9
The binding curve of compound 3a is an example of A1 AR antagonistic action determined via a
GTP shift assay (with and without 100 μM GTP) in rat whole brain membranes expressing A1
ARs with [3H]DPCPX as radioligand. GTP shift of 1.186 calculated for compound 3a.
3.2.2 In vitro evaluation: Cytotoxicity
The effect of selected compounds on the viability of cultured Vero cells were assessed and
preliminary results indicate low cytotoxicity (IC50 > 100 µM) for compounds 3a–b and 3j–k,[79,
80] with the exception of compound 3i (IC50 = 41.40 ± 5.64 µM) that resulted in mild to moderate
cytotoxicity (10 µM < IC50 < 50 µM).[81, 82] The positive control, Emetine, showed significant to
high cytotoxicity (IC50 <10 µM) with an IC50 value of 0.05 ± 0.004 µM, as anticipated.[81-83]
3.2.2 In silico evaluation
The physiochemical, pharmacokinetic as well as drug-likeness and medicinal chemistry
friendliness of compounds 3a–b and 3i–k were computed via SwissADME and these results are
23

summarized in Table 5, 6 and 7, respectively. (The results of compounds 3a–n are summarized
in Table 1, 2 and 3 in the Supplementary material.) For the purpose of this discussion, focus
was placed on compound 3j because it is the only compound in the series of methoxy substituted
2-benzoyl-1-benzofuran derivatives (3a–n) that possesses dual A1 and A2A AR affinity.
The bioavailability radar (Figure 10) gives a first glance at the drug-likeness (the promise a
molecule holds to be an oral drug based on bioavailability) of compound 3j. The pink area (Figure
10) represents the optimal range for the parameters lipophilicity (-0.7<XLOGP3<+5.0), size
(150<MW<500), polarity (20<TPSA<130), solubility (logS<6), saturation (fraction Csp3>0.25) and
flexibility (num. rotatable bonds<9).[63] Compound 3j falls within the optimal range for the
parameters lipophilicity (3j: 3.29), size (3j: 314.33 g/mol), polarity (3j: 53.99 Å2), solubility (3j: -
4.44), saturation (3j: 0.28) and flexibility (3j: 5 rotatable bonds); therefore, it is predicted that
compound 3j will be orally bioavailable.
Compound 3j is soluble to moderately soluble in water. This is encouraging finding as a soluble
molecule will facilitate drug development.[84] Also, solubility is a major property that influences
GI absorption.[85]
The predictions for passive human gastrointestinal absorption (HIA) and blood-brain-barrier
(BBB) permeation may be visualized as a function of the position of 3j in the WLOGP-versus-
TPSA referential – also known as the BOILED-Egg model (Figure 11).[86] Compound 3j that is
predicted to have high GI absorption and to permeate the BBB. Additionally, compound 3j is not
a substrate for the permeability glycoprotein (Pgp) that is responsible for efflux through biological
membranes, for instance from the gastrointestinal wall to the lumen or from the brain.[87] Pgp
also protects the CNS from xenobiotics.[88] Whether a compound interacts with cytochromes
P450 (CYP) is also of note given that this superfamily of isoenzymes is important in drug
elimination through metabolic transformation.[89] Compound 3j is predicted to inhibit the major
CYP isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4). Inhibition of these CYP
isoforms is, undoubtedly, a main cause of pharmacokinetics-related drug-drug interactions.[90,
91] Inhibition of CYP isoforms may lead to adverse or even toxic effects due to the lower clearance
and accumulation of the drug or its metabolites.[92] It is essential for drug discovery to predict the
propensity with which a molecule will cause significant drug interactions through inhibition of CYP
isoforms and to determine which isoforms are affected.[63]
The drug-likeness of compound 3j was assessed by rule-based filters from Lipinski,[93]
Ghose,[94] Veber,[95] Egan [96] and Muegge[97], of which none were violated. Additionally,
compound 3j has a bioavailability score (the probability that a compound will have >10%
bioavailability in rat or measurable Caco-2 permeability) of 0.55 – meaning it passes the rule-of-
five.[98]
24

With regards to medicinal chemistry; two methods allow for the identification of potentially
problematic fragments namely PAINS and Brenk violations. The pan assay interference
compounds, also known as frequent hitters or promiscuous compounds, refers to molecules
containing reactive compound substructures showing potent response in assays irrespective of
the protein target[53] and Brenk violations refer to 105 fragments identified by Brenk and
colleagues that may be putatively toxic, chemically reactive, metabolically unstable or bear
properties responsible for poor pharmacokinetics.[99] Compound 3j contains no PAINS or Brenk
violations. As stated, the aurone as well as 2-benzylidene-1-tetralone and -indanone derivatives
contain reactive compound substructures, such as the α,β-unsaturated ketone group (present in
compounds 1k–n) or the catechol group (present in 1l & 1n). Failure to identify these moieties
may lead to wasted resources, as such, compounds have poor development potential.Therefore,
a lack of reactivity interference, must be demonstratedbefore further development.
As 3j does not have outstanding affinity for the A1 and A2A ARs, the present structure needs to be
optimised and, here, lead-likeness plays a role. By applying the above and other physiochemical
filters to design screening libraries, Teague[100] observed that compounds with a molecular
weight between 250 and 350 g/mol (3j: 314.33), a XLOGP3 value of <3.5 (3j: 3.29) and <7
rotatable bonds (3j: 5 rotatable bonds) satisfy criteria for lead-likeness. Accordingly, compound
3j may be considered lead-like. This concept is similar to drug-likeness, but focus on
physiochemical boundaries defining a good lead, i.e. molecular entity suitable for optimization. By
definition, leads are subjected to chemical modifications that will most likely increase size and
lipophilicity.[101] As a consequence leads are required to be smaller and less hydrophobic than
drug-like molecules.
Table 5
Physiochemical properties of compounds 3a–b and 3i–k
Physiochemical properties
Lipophilicity Water solubility
#
3a
3b
3i
3j
3k
C17H16O4
C17H16O4
C18H18O5
C18H18O5
C18H18O5
284.31 0.24
284.31 0.24
314.33 0.28
314.33 0.28
314.33 0.28
4
4
5
5
5
4
4
5
5
5
0
0
0
0
0
78.49 44.76
78.49 44.76
84.98 53.99
84.98 53.99
84.98 53.99
2.88
2.82
2.87
2.81
2.81
-4.32
-4.32
-4.44
-4.44
-4.44
Table 6
Pharmacokinetic properties of compounds 3a–b and 3i–k
25

Pharmacokinetic properties
#
GI
BBB
Pgp
substrate
No
CYP1A2
inhibitor
Yes
CYP2C19
inhibitor
Yes
CYP2C9
inhibitor
Yes
CYP2D6
inhibitor
Yes
CYP3A4
inhibitor
Yes
absorption permeation
3a
3b
3i
3j
3k
High
High
High
High
High
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Table 7
Drug-likeness and medicinal chemistry friendliness of compounds 3a–b and 3i–l
Drug-likeness
Num. violations
Medicinal chemistry
Num. violations
#
Bioavailability
scoref
0.55
Lead-
Lipinskia
Ghoseb
Veberc
Egand
Mueggee
PAINSg
Brenkh
likenessi
3a
3b
3i
3j
3k
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.55
0.55
0.55
0.55
a Lipinski: MW<500, MLOGP<4.15, N or O <10, NH or OH <5.[93]
b Ghose: 160<MW<480, -04<WLOGP<5.6, 40<MR<130, 20<atoms<70.[94]
c Veber: Num. rotatable bonds<10, TPSA<140.[95]
d Egan: WLOGP<5.88, TPSA<131.6.[96]
e
Muegge: 200<MW<600, -2<XLOGP<5, TPSA<150, num. rings<7, num. carbon>4, num. heteroatoms>1, num.
rotatable bonds<15, num. H-bond acceptors<10, num. H-bond donors <5.[97]
f The probability that a compound will have >10% bioavailability in rat or measurable Caco-2 permeability.[98]
g PAINS: Pan Assay Interference Structures.[53]
h Brenk: Structural Alert.[99]
i Lead-likeness: 250<MW<350, XLOGP3<3.5, num. rotatable bonds <7.[100]
Figure 10
The pink area represents the optimal range for lipophilcity (LIPO), size (SIZE), polarity
(POLAR), solubility (INSOLU), saturation (INSATU) and flexibility (FLEX) and the red lines
represent these parameters of compound 3j.
26

Figure 11
Compound 3j, which is not a substrate for Pgp (PGP-), is represented by the red circle in the
yellow region. The white region is for high probability of passive absorption by the
gastrointestinal tract (HIA), and the yellow region (yolk) is for high probability of brain
penetration (BBB). White and yellow (yolk) regions are not mutually exclusive.
5. Conclusion
The difficulties encountered in the treatment of neurological conditions such as PD demonstrate
the importance of the design, synthesis and evaluation of novel compounds with potential for
further development as agents for the treatment of PD. Herein, a series of fourteen 2-benzoyl-1-
benzofuran derivatives were synthesised and biologically evaluated with five compounds showing
A1 AR affinity below 10 µM and one compound (3j) possessing A2A AR affinity below 1 µM (with
A2A AR selectivity). Compound 3j is the only compound that showed dual A1 and A2A AR affinity.
This compound may be used as a lead compound for design of A1 and A2A ligands with potential
application in the treatment of neurological conditions such as PD; particularly due to its low
cytotoxicity and lead-like properties, as assessed by in vitro and in silico screening, respectively.
Acknowledgements
This work is based on the research supported in part by the National Research Foundation (NRF)
of South Africa (grant number 111814) and the North-West University (NWU). The authors wish
27

to thank Dr D. Otto for NMR analyses and Dr J. Jordaan for MS analyses both of Chemical
Research Beneficiation, NWU, as well as Prof F. Van der Kooy for HPLC analyses and Ms S.
Lowe for assistance with biological assays both of the Centre of Excellence for Pharmaceutical
Sciences (Pharmacen), NWU. Additionally, the authors are grateful to Prof. D. N’Da (Pharmacen)
for the use of his facilities and resources with regard to the cytotoxicity assays.
Conflict of Interest: The authors declare that they have no conflict of interest.
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