Exploration of benzofuran-based compounds as potent and selective Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104839

Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) has been identified as a potential target for the development of novel drugs against multi-drug resistant malaria. A series of benzofuran-based compounds was synthesised and evaluated as inhibitors of recombinantly expressed and purified PfGSK-3 and human glycogen synthase kinase-3 beta (HsGSK-3β). Of this series, five compounds (5k, 5m, 5p, 5r, 5s) preferentially inhibited PfGSK-3, with four of these compounds exhibiting IC₅₀ values in the sub-micromolar range (0.00048–0.440 μM). Evaluation of the structure-activity relationships required for PfGSK-3 selective inhibition indicated that a C6-OCH₃ substitution on ring A is preferred, while the effect of the ring B substituent on activity, in decreasing order is: C4′-CN > C4′-F > C3′-OCH₃ > C3′,4′-diCl. To date, development of PfGSK-3 inhibitors has been limited to the 4-phenylthieno[2,3-b]pyridine class. Chalcone-based scaffolds, such as the benzofurans described herein, are promising new hits which can be explored for future design of PfGSK-3 selective inhibitors.

Full text of DOI:10.1016/j.bioorg.2021.104839

Bioorganic Chemistry 112 (2021) 104839
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Exploration of benzofuran-based compounds as potent and selective
Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) inhibitors
,
*
Chantalle Moolmana, Rencia van der Sluisb, Richard M. Betecka, Lesetja J. Legoabea
a Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
b Focus Area for Human Metabolomics, Biochemistry, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords:
Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) has been identified as a potential target for the
development of novel drugs against multi-drug resistant malaria. A series of benzofuran-based compounds was
synthesised and evaluated as inhibitors of recombinantly expressed and purified PfGSK-3 and human glycogen
synthase kinase-3 beta (HsGSK-3β). Of this series, five compounds (5k, 5m, 5p, 5r, 5s) preferentially inhibited
PfGSK-3, with four of these compounds exhibiting IC50 values in the sub-micromolar range (0.00048–0.440 µM).
Evaluation of the structure-activity relationships required for PfGSK-3 selective inhibition indicated that a C6-
OCH3 substitution on ring A is preferred, while the effect of the ring B substituent on activity, in decreasing order
is: C4′-CN > C4′-F > C3′-OCH3 > C3′,4′-diCl. To date, development of PfGSK-3 inhibitors has been limited to the
4-phenylthieno[2,3-b]pyridine class. Chalcone-based scaffolds, such as the benzofurans described herein, are
promising new hits which can be explored for future design of PfGSK-3 selective inhibitors.
Glycogen synthase kinase-3
Plasmodium falciparum
PfGSK-3
Malaria
Protein kinase
Structure-activity relationship
Benzofurans
1. Introduction
children have not yet acquired partial immunity against the disease. In
2018 alone, a staggering 272 000 children under the age of five, died of
Protein kinases have been exploited as targets for the design and
development of small molecules to treat various types of cancers for
decades [1,2]. Inspired by the success achieved in oncology, protein
kinases are currently being exploited as viable drug targets in other
diseases; including diabetes, arthritis and immune disorders [3–5]. The
completion of the Plasmodium falciparum genome sequencing project in
2002 [6] further stimulated an interest in exploiting the parasite’s
protein kinases for the development of novel drugs against multi-drug
resistant malaria [7–11].
this disease [12].
Among the five Plasmodium species affecting humans, P. falciparum is
responsible for the most lethal form of malaria and is also the most
prevalent species in sub-Saharan Africa [12]. P. falciparum is resistant to
several antimalarial drugs including current first-line artemisinin-based
combination therapies (ACTs) [14]. Artemisinin resistance is prevalent
across Southeast Asia, however, resistance has now also emerged in
Rwanda which can significantly compromise malaria control in sub-
Saharan Africa [15]. In order to combat multi-drug resistant malaria
an urgent need exists to develop new antimalarial drugs with novel
modes of action.
Despite significant progress made in reducing malaria morbidity and
mortality over the last decade, this infectious disease continues affecting
people worldwide. In 2018, an estimated 228 million malaria cases and
405 000 malaria-related deaths were reported globally [12]. Developing
countries, such as sub-Saharan Africa, are more severely affected by
malaria due to poverty, limited access to adequate health care and
preventative measures, and recurring resistance to insecticides and
antimalarial drugs [13]. Sub-Saharan Africa accounted for 93% of all
malaria cases and 94% of deaths reported for 2018 [12]. Pregnant
women and young children are particularly vulnerable to infection and
developing severe malaria, as pregnancy reduces immunity and young
Plasmodium kinases are attractive targets for new antimalarial drugs,
as they are involved in key signalling pathways throughout the life cycle
of the parasite [7]. Of the 65 Plasmodium kinases that cluster within
known eukaryotic protein kinase families, 36 are considered to be
essential for the erythrocytic stages of the parasite [16,17]. P. falciparum
glycogen synthase kinase-3 (PfGSK-3) is one such essential kinase and
has been suggested as a potential drug target [6,18]. PfGSK-3 was
identified as one of three GSK-3-related kinases in the P. falciparum
parasite [17], and shares a high degree of sequence homology with the
* Corresponding author.
E-mail address: Lesetja.Legoabe@nwu.ac.za (L.J. Legoabe).
https://doi.org/10.1016/j.bioorg.2021.104839
Received 15 May 2020; Received in revised form 12 February 2021; Accepted 17 March 2021
Available online 22 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
two human GSK-3 isoforms (HsGSK-3
α
and HsGSK-3β) [18]. As the
to gain further insight into the structure-activity relationship around this
crystal structure of PfGSK-3 is yet to be determined, a tridimensional
structure model generated from the PfGSK-3 sequence and the crystal
structure of HsGSK-3β [19], showed a high degree of structural simi-
larity between the two enzymes [18].
scaffold for the design of potent and selective PfGSK-3 inhibitors.
2. Experimental section
As HsGSK-3 is involved in numerous cellular processes, the extent to
which HsGSK-3 inhibition can be tolerated without presenting un-
wanted side effects, is still debated on [20–23]. Therefore, inhibitors
targeting the plasmodial enzyme would have to be highly selective in
order to prevent simultaneous inhibition of HsGSK-3 and the potential
side effects this may cause. Previous studies have tested various estab-
lished kinase inhibitors such as staurosporine, indirubin-3′-monoxime,
hymenialdisine and its derivatives, and members of the paullone family
against both enzymes to determine whether PfGSK-3 selective inhibition
would be possible [18,24–26]. The majority of these inhibitors (staur-
osporine, indirubin-3′-monoxime and hymenialdisine derivatives) were
unselective, inhibiting both PfGSK-3 and mammalian GSK-3β in the
nanomolar range [18,24–26]. However, the paullone inhibitors were
more selective towards mammalian GSK-3β which indicated that species
selectivity could be achieved [18,25,26]. Furthermore, certain key
structural differences were identified in the active sites of these two
enzymes that can be exploited for selective drug design [27,28].
Fugel and co-workers [25] were the first to design a series of novel
3,6-diamino-4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridine-5-car-
bonitrile derivatives that were highly selective towards PfGSK-3. The
most active compound of this series (1, Fig. 1) inhibited PfGSK-3 at a
sub-micromolar concentration (IC50: 0.48 µM) and demonstrated in vitro
antiplasmodial activity (EC50: 5.5 µM) when tested against the eryth-
rocytic stages of P. falciparum. Masch and co-workers [29] further
optimised compound 1 by attaching various substituents to the para
position of the 4-phenyl ring. Compound 2 (Fig. 1) was the most
2.1. Chemistry
All starting materials for chemical synthesis were purchased from
Sigma-Aldrich and used without further purification. Progress of re-
actions were monitored by thin layer chromatography (TLC) using
Silica gel 60F254 aluminium sheets (Merck) and a UV254 fluorescent
indicator. The 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. Either deuterated
chloroform (CDCl3) or dimethylsulfoxide (DMSO‑d6) was used as solvent
and tetramethylsilane (TMS) as internal reference for NMR analysis. All
chemical shifts (δ) are reported in parts per million (ppm) in relation to
the solvent signal (CDCl3 residual peak at 7.26 ppm and DMSO‑d6 re-
sidual peak at 2.50 ppm for 1H NMR). Spin multiplicities are reported as
follows: s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of
doublet of doublets), t (triplet) and m (multiplet). Coupling constants (J)
are reported in Hz. High resolution mass spectra (HRMS) were recorded
on a Bruker micrOTOF-Q II mass spectrometer using atmospheric pres-
sure chemical ionisation (APCI) in positive mode. High performance
liquid chromatography (HPLC) analyses were done using an Agilent
1100 HPLC system. Melting points (mp) were determined using a Buchi
B545 melting point apparatus and values are reported as obtained.
Characterisation data for all compounds are available in the Supple-
mentary material.
2.1.1. Synthesis of compounds 3a and 4a
promising of the series as it retained PfGSK-3 selectivity (PfGSK-3 IC50
:
0.72 µM; HsGSK-3β IC50: 40.2 µM), and showed improved aqueous sol-
ubility (4.8 µM) and in vitro antiplasmodial activity (EC50: 1.2 µM)
compared to compound 1 (solubility: 1.5 µM; EC50: 5.5 µM). The
structural core of these inhibitors, 3-amino-4-arylthieno[2,3-b]pyridine
motif, is based on a well-known chalcone scaffold (highlighted in blue,
1, Fig. 1). This suggested the potential of using chalcone-like scaffolds in
the design of new PfGSK-3 inhibitors.
2.1.1.1. 2-hydroxy-4,5-dimethoxybenzaldehyde (3a). 2,4,5-Trimethox-
ybenzaldehyde (15.0 g, 76 mmol) was added to DCM (100 ml) and the
mixture was cooled on ice. BBr3 (19.2 g, 76 mmol) was then added
dropwise and the solution was stirred while on ice for approximately 10
min before stirring at room temperature for 5 h. Upon reaction
completion as suggested by TLC analysis, the reaction mixture was again
cooled on ice and HCl (32%, 40 ml) was slowly added to the mixture
while stirring. The resulting precipitate was collected by filtration. The
filtrate was extracted with DCM (3x 100 ml), the combined organic
extracts were dried over MgSO4 and solvent removed in vacuo. The
resultant crude from the organic extracts were pooled with the dried
precipitate to yield compound 2a as a dark green solid (2.6 g, 91%),
which was used without further purification. mp: 99.8–102.3 ◦C; 1H
Herein we explore an alternative scaffold, benzofurans (3, Fig. 1),
which is somewhat structurally similar to the thieno[2,3-b]pyridine
motif. A series of benzofuran-based compounds (5a-v) with a variety of
polar and lipophilic substituents on both ring A (R1, R2, R3) and ring B
′
′
(R1 , R2 ), were synthesised and evaluated as potential inhibitors of
recombinantly expressed and purified PfGSK-3 and HsGSK-3β, in order
Fig. 1. PfGSK-3 selective inhibitors (1 and 2)
belonging to the 4-phenylthieno[2,3-b]pyri-
dine class, synthesised by Fugel et al. [25]
and Masch et al. [29]. The structural core of
these compounds is based on a well-known
chalcone scaffold (1, highlighted in blue).
Another chalcone-based scaffold, benzofu-
rans (3), was explored for the design of
potent and selective PfGSK-3 inhibitors dur-
ing the present study. IC50: half-maximal
inhibitory concentration; PfNF54-Luc: trans-
genic Plasmodium falciparum NF54 strain
expressing the luciferase gene; EC50: half-
maximal effective concentration. (For inter-
pretation of the references to colour in this
figure legend, the reader is referred to the
web version of this article.)
2

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
NMR (600 MHz, CDCl3) δ 11.41 (s, 1H), 9.70 (s, 1H), 6.91 (s, 1H), 6.47
(s, 1H), 3.94 (s, 3H), 3.88 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 194.01,
159.38, 157.26, 142.97, 113.18, 112.88, 100.17, 56.46, 56.38. APCI-
HRMS m/z calculated for C9H11O4 (MH+): 183.0652, found:
183.0667. Purity (HPLC): 90.81%.
and 5-chlorosalicylaldehyde (0.25 g, 1.59 mmol) with anhydrous K2CO3
(0.66 g, 4.76 mmol) to yield compound 5d as white crystals (66%): mp:
191.3–191.8 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.03–7.96 (m, 2H), 7.69
(d, J = 2.0 Hz, 1H), 7.57–7.41 (m, 5H); 13C NMR (151 MHz, CDCl3) δ
182.64, 154.21, 153.18, 139.74, 134.99, 130.93, 129.76, 128.97,
128.84, 128.09, 122.63, 115.38, 113.64. APCI-HRMS m/z calculated for
C
15H9Cl2O2 (MH+): 290.9974, found: 290.9963. Purity (HPLC): 100%.
2.1.1.2. 2-bromo-3′-methoxyacetophenone (4a). CuBr2 (14.88 g, 66.62
mmol) was added to a solution of 3′-methoxyacetophenone (5.00 g,
33.29 mmol) in ethyl acetate (75 ml) and DCM (75 ml). The reaction
mixture was stirred at 85 ◦C for 24 h. Upon completion as suggested by
TLC analysis, the reaction mixture was cooled to room temperature and
then filtered through a bed of silica. The filtrate was concentrated to
yield compound 2b as a green solid (6.98 g, 91%), which was used
without further purification. 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 C9H10BrO2 (MH+): 228.9859, found:
228.9862. Purity (HPLC): 56.08%.
2.1.2.5. (7-bromo-5-chloro-1-benzofuran-2-yl)(4-chlorophenyl)meth-
anone (5e). Prepared as for 5a from 2,4′-dichloroacetophenone (0.30 g,
1.59 mmol) and 3-bromo-5-chlorosalicylaldehyde (0.37 g, 1.59 mmol)
with anhydrous K2CO3 (0.66 g, 4.76 mmol) to yield compound 5e as
beige crystals (42%): mp: 164.9–165.0 ◦C; 1H NMR (600 MHz, CDCl3) δ
8.12–8.07 (m, 2H), 7.64 (q, J = 1.8 Hz, 2H), 7.57 (s, 1H), 7.52 (d, J =
8.5 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ 181.81, 153.99, 151.69,
140.08, 134.51, 131.19, 130.97, 130.20, 129.04, 128.55, 121.73,
115.26, 105.58. APCI-HRMS m/z calculated for C15H8BrCl2O2 (MH+):
368.9079, found: 368.9052. Purity (HPLC): 99.76%.
2.1.2.6. 1-benzofuran-2-yl(4-chlorophenyl)methanone (5f). Prepared as
for 5a from 2,4′-dichloroacetophenone (0.30 g, 1.59 mmol) and sali-
cylaldehyde (0.17 ml, 1.59 mmol) with anhydrous K2CO3 (0.66 g, 4.76
mmol) to yield compound 5f as beige crystals (65%): mp:
150.0–151.4 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.10–7.98 (m, 2H), 7.72
(d, J = 7.7 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.60–7.46 (m, 4H), 7.33 (t,
J = 7.4 Hz, 1H); 13C NMR (151 MHz, CDCl3) δ 182.94, 155.99, 152.04,
139.42, 135.36, 130.91, 128.87, 128.55, 126.87, 124.09, 123.34,
116.48, 112.54. APCI-HRMS m/z calculated for C15H10ClO2 (MH+):
257.0364, found: 257.0339. Purity (HPLC): 100%.
2.1.2. Synthesis of compounds 5a-r
2.1.2.1. 1-benzofuran-2-yl(phenyl)methanone (5a). Anhydrous K2CO3
(1.70 g, 12.28 mmol) was added to a mixture of 2-bromoacetophenone
(0.82 g, 4.09 mmol) and salicylaldehyde (0.44 ml, 4.09 mmol) in PEG-
400 (5 ml) and the mixture was stirred at 100 ◦C. Upon reaction
completion as indicated by TLC analysis, the reaction mixture was
cooled to room temperature and quenched with deionised water (10 ml).
In cases where a precipitate was formed, it was collected by filtration.
When no precipitate formed upon addition of water, the mixture was
extracted with ethyl acetate (3x 20 ml). The combined organic extracts
were washed with brine (20 ml), dried over MgSO4, concentrated and
recrystallized from a suitable solvent [either ethanol (5a-q) or methanol
(5r-s)] to yield the title compound 5a as a yellow solid (67%): mp:
90.3–91.8 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.08–8.02 (m, 2H), 7.73 (d, J
= 7.8 Hz, 1H), 7.68–7.61 (m, 2H), 7.58–7.47 (m, 4H), 7.37–7.31 (m,
1H); 13C NMR (151 MHz, CDCl3) δ 184.44, 156.02, 152.22, 137.25,
132.92, 129.46, 128.56, 128.41, 127.02, 124.01, 123.34, 116.60,
112.60. APCI-HRMS m/z calculated for C15H11O2 (MH+): 223.0754,
found: 223.0763. Purity (HPLC): 95.88%.
2.1.2.7. [6-(diethylamino)benzofuran-2-yl](phenyl)methanone
hydro-
chloride (5g). [6-(diethylamino)benzofuran-2-yl](phenyl)methanone
was prepared as for 5a from 2-bromoacetophenone (0.52 g, 2.59 mmol)
and 4-(diethylamino)salicylaldehyde (0.5 g, 2.59 mmol) with anhydrous
K2CO3 (1.07 g, 7.76 mmol) to yield a yellow oil. The oil was dissolved in
ethanol (10 ml), and acetyl chloride (5 ml) was added dropwise to the
mixture while stirring at room temperature. The title compound (5g)
was collected by filtration as a yellow solid (50%): mp: 215.4–217.1 ◦C;
1H NMR (600 MHz, CDCl3) δ 8.26 (s, 1H), 8.12–8.07 (m, 2H), 7.93 (d, J
= 8.5 Hz, 1H), 7.81 (dd, J = 8.4, 1.1 Hz, 1H), 7.68 (dd, J = 10.6, 4.3 Hz,
1H), 7.63 (s, 1H), 7.57 (t, J = 7.8 Hz, 2H), 3.63 (d, J = 7.2 Hz, 4H), 1.32
(t, J = 7.2 Hz, 6H); 13C NMR (151 MHz, CDCl3) δ 183.58, 155.20,
154.82, 136.95, 136.30, 133.59, 129.65, 128.76, 128.42, 125.17,
118.63, 114.90, 108.29, 54.07, 10.44. APCI-HRMS m/z calculated for
2.1.2.2. (5-chloro-1-benzofuran-2-yl)(phenyl)methanone (5b). Prepared
as for 5a from 2-bromoacetophenone (0.30 g, 1.51 mmol) and 5-chlor-
osalicylaldehyde (0.24 g, 1.51 mmol) with anhydrous K2CO3 (0.63 g,
4.25 mmol) to yield compound 5b as white crystals (66%): mp:
142.8–143.3 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.04–7.99 (m, 2H),
7.70–7.60 (m, 2H), 7.58–7.49 (m, 3H), 7.46–7.41 (m, 2H); 13C NMR
(151 MHz, CDCl3) δ 184.10, 154.21, 153.30, 136.84, 133.16, 129.60,
129.45, 128.65, 128.61, 128.19, 122.59, 115.45, 113.65. APCI-HRMS
m/z calculated for C15H10ClO2 (MH+): 257.0364, found: 257.0364.
Purity (HPLC): 99.1%.
C
19H20NO2 (MH+): 294.1489, found: 294.1517. Purity (HPLC): 100%.
2.1.2.8. (4-chlorophenyl)(7-methoxy-1-benzofuran-2-yl)methanone
(5h). Prepared as for 5a from 2,4′-dichloroacetophenone (0.50 g, 2.64
mmol) and o-vanillin (0.40 g, 2.64 mmol) with anhydrous K2CO3 (1.10
g, 7.93 mmol) to yield compound 5h as beige crystals (82%): mp:
189.9–190.5 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.08–8.03 (m, 2H), 7.55
(s, 1H), 7.53–7.48 (m, 2H), 7.27 (ddd, J = 15.7, 10.5, 4.4 Hz, 2H), 6.96
(dd, J = 7.8, 0.8 Hz, 1H), 4.02 (s, 3H); 13C NMR (151 MHz, CDCl3) δ
182.56, 152.45, 146.08, 145.72, 139.42, 135.26, 131.06, 128.87,
128.49, 124.75, 116.27, 114.97, 109.63, 56.07. APCI-HRMS m/z
calculated for C16H12ClO3 (MH+): 287.0469, found: 287.0473. Purity
(HPLC): 100%.
2.1.2.3. (7-bromo-5-chloro-1-benzofuran-2-yl)(phenyl)methanone (5c).
Prepared as for 5a from 2-bromoacetophenone (0.30 g, 1.50 mmol) and
3-bromo-5-chlorosalicylaldehyde (0.36 g, 1.50 mmol) with anhydrous
K2CO3 (0.63 g, 4.25 mmol) to yield compound 5c as dark beige crystals
(43%): mp: 143.4–144.5 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.12–8.07 (m,
2H), 7.67–7.59 (m, 3H), 7.57–7.51 (m, 3H); 13C NMR (151 MHz, CDCl3)
δ 183.25, 154.10, 151.71, 136.37, 133.42, 130.82, 130.04, 129.69,
129.46, 128.66, 121.69, 115.30, 105.61. APCI-HRMS m/z calculated for
2.1.2.9. (4-chlorophenyl)(6-methoxy-1-benzofuran-2-yl)methanone (5i).
Prepared as for 5a from 2,4′-dichloroacetophenone (0.50 g, 2.64 mmol)
and 2-hydroxy-4′-methoxybenzaldehyde (0.40 g, 2.64 mmol) with
anhydrous K2CO3 (1.10 g, 7.93 mmol) to yield compound 5i as pink
crystals (54%): mp: 186.7–187.4 ◦C; 1H NMR (600 MHz, CDCl3) δ
7.98–7.93 (m, 2H), 7.56 (d, J = 8.7 Hz, 1H), 7.50–7.44 (m, 3H), 7.07 (d,
J = 1.9 Hz, 1H), 6.95 (dd, J = 8.7, 2.2 Hz, 1H), 3.87 (s, 3H); 13C NMR
C
15H9BrClO2 (MH+): 334.9469, found: 334.9465. Purity (HPLC):
97.35%.
2.1.2.4. (5-chloro-1-benzofuran-2-yl)(4-chlorophenyl)methanone (5d).
Prepared as for 5a from 2,4′-dichloroacetophenone (0.30 g, 1.59 mmol)
3

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
(151 MHz, CDCl3) δ 182.42, 161.37, 157.68, 151.62, 139.05, 135.71,
130.74, 128.80, 123.67, 120.24, 117.26, 114.70, 95.54, 55.74. APCI-
HRMS m/z calculated for C16H12ClO3 (MH+): 287.0469, found:
287.0498. Purity (HPLC): 100%.
96.52%.
2.1.2.15. (6-methoxy-1-benzofuran-2-yl)(4-methoxyphenyl)methanone
(5o). Prepared as for 5a from 2-bromo-4′-methoxyacetophenone (0.75
g, 3.29 mmol) and 2-hydroxy-4′-methoxybenzaldehyde (0.50 g, 3.29
mmol) with anhydrous K2CO3 (1.36 g, 9.86 mmol) to yield compound
5o as a beige solid (86%): mp: 151.0–151.3 ◦C; 1H NMR (600 MHz,
CDCl3) δ 8.12–8.03 (m, 2H), 7.57 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 0.7 Hz,
1H), 7.10 (d, J = 1.9 Hz, 1H), 7.04–6.93 (m, 3H), 3.90 (d, J = 9.5 Hz,
6H); 13C NMR (151 MHz, CDCl3) δ 182.46, 163.38, 160.97, 157.42,
152.25, 131.76, 130.13, 123.48, 120.42, 116.25, 114.34, 113.79, 95.67,
55.75, 55.53. APCI-HRMS m/z calculated for C17H15O4 (MH+):
283.0965, found: 283.0974. Purity (HPLC): 100%.
2.1.2.10. (4-chlorophenyl)(5-methoxy-1-benzofuran-2-yl)methanone
(5j). Prepared as for 5a from 2,4′-dichloroacetophenone (0.30 g, 1.59
mmol) and 2-hydroxy-5′-methoxybenzaldehyde (0.20 ml, 1.59 mmol)
with anhydrous K2CO3 (0.66 g, 4.76 mmol) to yield compound 5j as pink
crystals (65%): mp: 190.6–191.1 ◦C; 1H NMR (600 MHz, CDCl3) δ
8.04–7.97 (m, 2H), 7.53–7.46 (m, 4H), 7.14–7.07 (m, 2H), 3.84 (s, 3H);
13C NMR (151 MHz, CDCl3) δ 182.78, 156.71, 152.78, 151.22, 139.36,
135.38, 130.94, 128.85, 127.39, 118.78, 116.45, 113.22, 103.83, 55.84.
APCI-HRMS m/z calculated for C16H12ClO3 (MH+): 287.0469, found:
287.0499. Purity (HPLC): 100%.
2.1.2.16. 4-[(6-methoxy-1-benzofuran-2-yl)carbonyl]benzonitrile (5p).
Prepared as for 5a from 2-bromo-4′-cyanoacetophenone (0.74 g, 3.29
mmol) and 2-hydroxy-4′-methoxybenzaldehyde (0.50 g, 3.29 mmol)
with anhydrous K2CO3 (1.36 g, 9.86 mmol) to yield compound 5p as a
2.1.2.11. (3,4-dichlorophenyl)(6-methoxy-1-benzofuran-2-yl)methanone
(5k). Prepared as for 5a from 2-bromo-3′,4′-dichloroacetophenone
(0.88 g, 3.29 mmol) and 2-hydroxy-4′-methoxybenzaldehyde (0.50 g,
3.29 mmol) with anhydrous K2CO3 (1.36 g, 9.86 mmol) to yield com-
pound 5k as brown crystals (52%): mp: 162.5–163.7 ◦C; 1H NMR (600
MHz, DMSO) δ 8.21 (d, J = 2.0 Hz, 1H), 8.00 (dd, J = 8.3, 2.0 Hz, 1H),
7.93 (d, J = 8.3 Hz, 1H), 7.88 (s, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.44 (d, J
= 1.6 Hz, 1H), 7.09 (dd, J = 8.7, 1.6 Hz, 1H), 3.93 (s, 3H); 13C NMR
(151 MHz, DMSO) δ 180.89, 161.78, 157.80, 150.95, 137.77, 136.00,
132.14, 131.50, 131.23, 129.58, 124.91, 120.51, 119.20, 115.19, 96.23,
56.39. APCI-HRMS m/z calculated for C16H11Cl2O3 (MH+): 321.0080,
found: 321.0062. Purity (HPLC): 100%.
1
◦
beige solid (80%): mp: 183.1–186.7 C; H NMR (600 MHz, CDCl3) δ
8.14–8.08 (m, 2H), 7.86–7.80 (m, 2H), 7.60 (d, J = 8.7 Hz, 1H), 7.52 (d,
J = 0.9 Hz, 1H), 7.09 (d, J = 1.9 Hz, 1H), 6.99 (dd, J = 8.7, 2.2 Hz, 1H),
3.91 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 181.93, 161.81, 158.00,
151.27, 140.93, 132.32, 129.76, 123.91, 120.18, 118.09, 118.02,
115.88, 115.12, 95.53, 55.82. APCI-HRMS m/z calculated for
C
17H12NO3 (MH+): 278.0812, found: 278.0801. Purity (HPLC): 98.24%.
2.1.2.17. (4-chlorophenyl)(5,6-dimethoxy-1-benzofuran-2-yl)methanone
(5q). Prepared as for 5a from 2,4′-dichloroacetophenone (0.52 g, 2.74
mmol) and compound 3a (0.50 g, 2.74 mmol) with anhydrous K2CO3
(1.14 g, 8.23 mmol) to yield compound 5q as white crystals (85%): mp:
183.8–184.0 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.01–7.97 (m, 2H),
7.54–7.46 (m, 3H), 7.11 (s, 1H), 7.07 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H);
13C NMR (151 MHz, CDCl3) δ 182.06, 152.03, 151.98, 151.78, 147.94,
139.00, 135.81, 130.82, 128.79, 119.09, 117.34, 102.69, 95.13, 56.36,
56.35. APCI-HRMS m/z calculated for C17H14ClO4 (MH+): 317.0575,
found: 317.0574. Purity (HPLC): 100%.
2.1.2.12. (6-methoxy-1-benzofuran-2-yl)(phenyl)methanone (5l). Pre-
pared as for 5a from 2-bromoacetophenone (0.65 g, 3.29 mmol) and 2-
hydroxy-4′-methoxybenzaldehyde (0.50 g, 3.29 mmol) with anhydrous
K2CO3 (1.36 g, 9.86 mmol) to yield compound 5l as beige crystals
(57%): mp: 103.8–104.6 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.00–7.96 (m,
2H), 7.62–7.47 (m, 4H), 7.44 (d, J = 0.7 Hz, 1H), 7.08 (d, J = 1.9 Hz,
1H), 6.94 (dd, J = 8.7, 2.2 Hz, 1H), 3.87 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 183.90, 161.23, 157.65, 151.80, 137.53, 132.58, 129.26,
128.46, 123.63, 120.34, 117.33, 114.53, 95.59, 55.72. APCI-HRMS m/z
calculated for C16H13O3 (MH+): 253.0859, found: 253.0861. Purity
(HPLC): 100%.
2.1.2.18. (6-Methoxybenzofuran-2-yl)(3-methoxyphenyl)methanone
(5r). Prepared as for 5a from compound 4a (0.75 g, 3.29 mmol) and 2-
hydroxy-4-methoxybenzaldehyde (0.50 g, 3.29 mmol) with anhydrous
K2CO3 (1.36 g, 9.86 mmol) to yield compound 5r as a light grey solid
(52%): 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 (m,
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
2.1.2.13. (4-fluorophenyl)(6-methoxy-1-benzofuran-2-yl)methanone
(5m). Prepared as for 5a from 2-bromo-4′-fluoroacetophenone (0.71 g,
3.29 mmol) and 2-hydroxy-4′-methoxybenzaldehyde (0.50 g, 3.29
mmol) with anhydrous K2CO3 (1.36 g, 9.86 mmol) to yield compound
1
◦
5m as a light brown solid (90%): mp: 161.1–163.1 C; H NMR (600
MHz, CDCl3) δ 8.11–8.05 (m, 2H), 7.58 (d, J = 8.7 Hz, 1H), 7.48 (d, J =
0.9 Hz, 1H), 7.24–7.18 (m, 2H), 7.10 (d, J = 1.9 Hz, 1H), 6.97 (dd, J =
8.7, 2.2 Hz, 1H), 3.90 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 182.22,
165.52 (d, J = 254.3 Hz), 161.32, 157.66, 151.79, 133.70 (d, J = 3.1
Hz), 131.97 (d, J = 9.1 Hz), 123.66, 120.31, 117.08, 115.68 (d, J = 21.8
Hz), 114.67, 95.61, 55.77. APCI-HRMS m/z calculated for C16H12FO3
(MH+): 271.0765, found: 271.0743. Purity (HPLC): 98.28%.
C
17H15O4 (MH+): 283.0965, found: 283.0946. Purity (HPLC): 99.40%.
2.1.2.19. (7-Methoxybenzofuran-2-yl)(3-methoxyphenyl)methanone
(5s). Prepared as for 5a from 2-hydroxy-3-methoxybenzaldehyde (0.50
g, 3.29 mmol) and compound 4a (0.75 g, 3.29 mmol) with anhydrous
K2CO3 (1.36 g, 9.86 mmol) to yield compound 5s as a brown solid
(46%): 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 (s, 3H),
3.97 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 183.83, 159.83, 152.65,
146.29, 145.92, 138.53, 129.66, 128.76, 124.78, 122.35, 119.61,
116.56, 115.14, 114.03, 109.83, 56.30, 55.64. APCI-HRMS m/z calcu-
lated for C17H15O4 (MH+): 283.0965, found: 283.0970. Purity (HPLC):
98.03%.
2.1.2.14. (4-bromophenyl)(6-methoxy-1-benzofuran-2-yl)methanone
(5n). Prepared as for 5a from 2,4′-dibromoacetophenone (0.91 g, 3.29
mmol) and 2-hydroxy-4′-methoxybenzaldehyde (0.50 g, 3.29 mmol)
with anhydrous K2CO3 (1.36 g, 9.86 mmol) to yield compound 5n as a
light brown solid (85%): mp: 200.9–202.4 ◦C; 1H NMR (600 MHz,
CDCl3) δ 7.94–7.87 (m, 2H), 7.71–7.61 (m, 2H), 7.59 (d, J = 8.7 Hz, 1H),
7.48 (d, J = 0.9 Hz, 1H), 7.10 (d, J = 1.9 Hz, 1H), 6.98 (dd, J = 8.7, 2.2
Hz, 1H), 3.90 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 182.60, 161.43,
157.74, 151.63, 136.19, 131.82, 130.88, 127.71, 123.72, 120.28,
117.34, 114.76, 95.59, 55.79. APCI-HRMS m/z calculated for
2.1.3. Synthesis of compounds 5t-v
C
16H12BrO3 (MH+): 330.9964, found: 330.9965. Purity (HPLC):
2.1.3.1. (4-chlorophenyl)(7-hydroxy-1-benzofuran-2-yl)methanone (5t).
4

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
AlCl3 (0.87 g, 6.49 mmol) was added to a 100 ml round bottom flask
containing 5h (0.62 g, 2.17 mmol) and toluene (5 ml). The mixture was
stirred at 100 ◦C. Upon reaction completion as suggested by TLC anal-
ysis, the mixture was cooled to room temperature and quenched with
deionised water (10 ml). The mixture was then extracted with ethyl
acetate (3x 20 ml). The combined organic extracts were washed with
brine (20 ml), dried over MgSO4, concentrated and purified using silica
gel column chromatography (petroleum ether: ethyl acetate, 5:1) to
yield compound 5t as a beige solid (29%): mp: 213.4–214.5 ◦C; 1H NMR
(600 MHz, DMSO) δ 10.42 (s, 1H), 8.06–7.99 (m, 2H), 7.76 (s, 1H),
7.70–7.63 (m, 2H), 7.24 (dd, J = 7.8, 1.0 Hz, 1H), 7.16 (t, J = 7.8 Hz,
1H), 6.97 (dd, J = 7.7, 1.0 Hz, 1H); 13C NMR (151 MHz, DMSO) δ
182.29, 151.05, 144.88, 143.32, 137.97, 135.45, 131.13, 128.86,
128.61, 124.96, 117.75, 114.02, 113.81. APCI-HRMS m/z calculated for
2.2.2. Expression of recombinant HsGSK-3β and PfGSK-3
A single colony of Origami™ cells co-transformed with the pGro7
and pET32a(+)-HsGSK-3β plasmids, was inoculated into 5 ml of LB
medium with carbenicillin (50 µg/ml) and chloramphenicol (20 µg/ml).
The culture was incubated overnight at 37 ◦C and 225 rpm. The over-
night culture was transferred into 100 ml of LB medium with carbeni-
cillin (50 µg/ml), chloramphenicol (20 µg/ml), and 0.5 mg/ml L-
arabinose (for expression of the pGro7 chaperone proteins). The culture
was incubated at 37 ◦C and 225 rpm, until an OD600 of ±0.5 was
reached. Expression of the HsGSK-3β protein was then induced with 1
mM IPTG for 4 h at 30 ◦C and 225 rpm.
A single colony of TOP10 E. coli transformed with pBAD/Thio-
PfGSK-3 was inoculated into 5 ml of LB medium containing carbenicillin
(100 µg/ml). The culture was incubated overnight at 37 ◦C and 225 rpm.
The overnight culture was transferred into 500 ml of LB medium with
carbenicillin (100 µg/ml) and cultured at 37 ◦C and 225 rpm, until an
OD600 of ±0.5 was reached. Expression was then induced with 0.02% L-
arabinose for 3 h at 37 ◦C and 225 rpm.
C
15H10ClO3 (MH+): 273.0313, found: 273.0304. Purity (HPLC):
98.58%.
2.1.3.2. (4-chlorophenyl)(6-hydroxy-1-benzofuran-2-yl)methanone
(5u). Prepared as for 5s from compound 5i (0.41 g, 1.43 mmol) with
AlCl3 (0.57 g, 4.28 mmol) to yield compound 5u as a beige solid (27%):
mp: 212.2–212.9 ◦C; 1H NMR (600 MHz, DMSO) δ 10.27 (s, 1H),
7.99–7.95 (m, 2H), 7.71 (d, J = 0.8 Hz, 1H), 7.68–7.62 (m, 3H),
7.06–7.02 (m, 1H), 6.91 (dd, J = 8.6, 2.1 Hz, 1H); 13C NMR (151 MHz,
DMSO) δ 181.98, 160.02, 157.78, 150.73, 138.01, 136.33, 131.32,
129.25, 124.95, 119.49, 118.99, 115.23, 97.95. APCI-HRMS m/z
calculated for C15H10ClO3 (MH+): 273.0313, found: 273.0326. Purity
(HPLC): 100%.
2.2.3. Extraction and purification of recombinant HsGSK-3β and PfGSK-3
Bacterial cells were harvested by centrifugation (5000 xg, 10 min,
room temperature) and the proteins were extracted using B-PER™
Complete Bacterial Protein Extraction Reagent (Thermo Fisher Scienti-
fic, Waltham, MA) according to the supplier’s procedure. Briefly, the
bacterial pellet was resuspended in the required amount of B-PER™
Reagent (with added protease inhibitors; Complete Ultra Protease In-
hibitor Mini Tablets; Merck, Darmstadt, Germany), and incubated at
room temperature with gentle rocking for 15 min. The lysate was then
centrifuged (16 000 xg, 20 min, 4 ◦C) and the soluble fraction collected
for purification. The recombinant His-tag proteins were then purified by
immobilized metal affinity chromatography using Protino® Ni-TED
2000 Packed Columns (Macherey-Nagel Inc., Düren, Germany).
Briefly, the entire soluble protein fraction was loaded onto a pre-
equilibrated column. For the HsGSK-3β protein, the column was
washed twice with LEW buffer, before eluting the protein with Elution
buffer (250 mM imidazole). For the PfGSK-3 protein, the column was
washed once with LEW buffer and then three times with diluted Elution
buffer (5 mM imidazole), before eluting the protein with Elution buffer
(250 mM imidazole, 5% glycerol).
2.1.3.3. (4-chlorophenyl)(5-hydroxy-1-benzofuran-2-yl)methanone
(5v). Prepared as for 5s from compound 5j (0.28 g, 0.96 mmol) with
AlCl3 (0.39 g, 2.89 mmol) to yield compound 5v as a yellow solid (29%):
mp: 215.8–217.1 ◦C; 1H NMR (600 MHz, DMSO) δ 9.57 (s, 1H),
8.04–7.98 (m, 2H), 7.71–7.64 (m, 3H), 7.58 (d, J = 8.9 Hz, 1H), 7.12 (d,
J = 2.4 Hz, 1H), 7.04 (dd, J = 8.9, 2.5 Hz, 1H); 13C NMR (151 MHz,
DMSO) δ 182.65, 154.64, 152.16, 150.32, 138.34, 135.99, 131.51,
129.30, 128.08, 119.11, 117.69, 113.21, 107.23. APCI-HRMS m/z
calculated for C15H10ClO3 (MH+): 273.0313, found: 273.0309. Purity
(HPLC): 95.49%.
Amicon® Ultra-15 centrifugal devices with a 50 kDa molecular
weight cut-off (Merck, Darmstadt, Germany), were used for further
purification and concentration of both recombinant proteins, as well as
for exchanging the elution buffer with storage buffer. Storage buffer
consisted of 50 mM Tris-HCl pH 8.0 (with added protease inhibitors,
Complete Ultra Protease Inhibitor Mini Tablets, Roche); 150 mM NaCl;
0.5 mM EDTA; 0.25 mM DTT; 25% glycerol. SDS-PAGE analysis with
Coomassie brilliant blue staining was used to monitor the expression and
purification procedures. To confirm the presence of the His-tag proteins,
western blot analysis was performed using a 6x His-tag monoclonal
antibody (Thermo Fisher Scientific, Madison, WI). Protein concentra-
tions were determined using the Qubit® protein assay kit (Life Tech-
nologies, Carlsbad, CA) with the Qubit® 2.0 fluorometer. Both
recombinant proteins were stored in aliquots at ꢀ 80 ◦C.
2.2. Biology
2.2.1. Cloning of HsGSK-3β and PfGSK-3
The full length open reading frame of HsGSK-3β (1263 bp; Ensembl
Genome ID: ENST00000264235.12) and PfGSK-3 (1323 bp; Ensembl
Genome ID: PF3D7_0312400) gene sequences were purchased from
GeneArt (Thermo Scientific, Waltham, MA). The HsGSK-3β and PfGSK-3
sequences are available in the Supplementary material (Figure A and B).
The HsGSK-3β gene was subcloned into the pET32a(+) expression
vector using restriction enzyme directional cloning and verified by
Sanger sequencing. Purified plasmid DNA was used to transform
Origami™ cells (Novagen, Madison, WI) already containing the pGro7
chaperone expression plasmid (Takara, Madison, WI).
The PfGSK-3 gene was amplified by PCR using 5′-ATG AAA AAT TGG
CCT-3′ as the forward primer and 5′-ACT TTC TAT GAT AAC GTG CGT-
3′ as the reverse primer (IDT, Coralville, IA). Amplification reactions
were prepared according to the Platinum™ Taq DNA polymerase
(Invitrogen, Carlsbad, CA) instruction manual (annealing step: 51.6 ◦C
for 30 s; total of 30 PCR cycles). The PfGSK-3 gene was then cloned into
the pBAD/Thio-TOPO® expression vector (pBAD/TOPO® ThioFusion™
Expression Kit; Invitrogen, Carlsbad, CA), by direct insertion of the PCR
product. The cloning reaction was transformed into chemically compe-
tent TOP10 One Shot® E. coli cells included in the kit. The orientation of
the inserts and the fidelity of the constructs were verified by Sanger
sequencing.
2.2.4. ADP-Glo™ kinase assay
Activity of the recombinant HsGSK-3β and PfGSK-3 were measured
using the ADP-Glo™ Kinase Assay Kit (Promega, Madison, WI). The
assay was optimised by determining the optimal substrate (GS-1 pep-
tide: YRRAAVPPSPSLSRHSSPHQpSEDEEE, where pS is a phosphory-
lated serine; GenScript, Piscataway, NJ) and enzyme concentrations to
use for each reaction. To determine the optimal substrate concentration,
serial twofold dilutions of substrate in kinase reaction buffer (40 mM
Tris-HCl pH 8.0; 20 mM MgCl2; 0.1 mg/ml BSA; 50 µM DTT) were made,
and the reactions were performed using 30 ng of enzyme and 10 µM of
ATP per 30 µl reaction. The control reactions consisted of the same serial
dilutions of substrate but without any added enzyme. The optimal
5

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
substrate concentrations were calculated to be 0.8 mg/ml for HsGSK-3β
and 0.6 mg/ml for PfGSK-3, as these concentrations resulted in the
largest difference in luminescence between the reaction wells and the
control wells. To determine the optimal enzyme concentration, serial
twofold dilutions of enzyme in kinase reaction buffer were made, and
the reactions were performed using the optimal concentration of sub-
strate and 10 µM of ATP per 20 µl reaction. The optimal enzyme con-
centration was within the linear range of the kinase titration curve
(luminescence vs enzyme concentration) and generated a signal-to-
background ratio of 10 (luminescence of reaction/ luminescence of re-
action without kinase). The optimal enzyme concentrations were 8 ng
and 32 ng for HsGSK-3β and PfGSK-3, respectively.
accompanying SoftMax Pro 7 software (Molecular Devices, San Jose,
CA).
For compounds showing inhibitory activity at 10 µM, dose-response
curves were performed. Using Graphpad Prism Software, the sigmoidal
dose-response curves were obtained by plotting luminescence against
the logarithm of the compounds’ concentrations. The IC50 values were
then calculated using nonlinear regression. All reactions were performed
in triplicate, and data was normalised to a negative control (reaction
without kinase). Indirubin-3′-monoxime, a known inhibitor of both
HsGSK-3β and PfGSK-3 [18,25], was included as a positive control. The
intra- and inter-assay CV values are available in the Supplementary
material (Table A).
All test compounds were screened at a concentration of 10 µM (1%
final DMSO concentration). The compounds were dissolved in 100%
DMSO, diluted with kinase reaction buffer, and incubated with the ki-
nase for 10 min at room temperature (23 ◦C), before adding the substrate
and ATP to the reaction. The 20 µl kinase reaction consisted of the
following: 4 µl of test compound (10 µM, 1% DMSO), 8 µl of enzyme (8
ng HsGSK-3β or 32 ng PfGSK-3) and 8 µl of ATP/substrate mix (ATP: 10
µM; substrate: 0.8 mg/ml for HsGSK-3β or 0.6 mg/ml for PfGSK-3) in
kinase reaction buffer. The reaction was incubated at 30 ◦C for 30 min
and then equilibrated to room temperature for 30 min. 5 µl of the kinase
reaction was then transferred to a solid white 384-well plate (Brand-
plates®; Merck, Darmstadt, Germany), 5 µl of ADP-Glo™ Reagent was
added, and incubated for 40 min at room temperature. 10 µl of Kinase
Detection Reagent was then added and incubated for 30 min at room
temperature. Luminescence was measured using a SpectraMax® iD3
multi-mode microplate reader (integration time: 0.5 s per well), and the
3. Results and discussion
3.1. Chemistry
Except for reagents 3a and 4a, all other reagents were purchased
from commercial suppliers and were used as supplied. The reagents (3a
& 4a) and target compounds (5a-v) were synthesised according to the
reactions shown in Scheme 1. 2-Hydroxy-4,5-dimethoxybenzaldehyde
(3a) was obtained by ortho-demethylation of 2,4,5-trimethoxybenzalde-
hyde using BBr3 as demethylation agent in DCM [30]. The selective
demethylation at the ortho-position is a result of the ortho-directing ef-
fect of the carbonyl group [31]. The selective
α
-bromination of 3′-
methoxyacetophenone was achieved using CuBr2 in ethyl acetate/DCM
(1:1) to yield 2-bromo-3′-methoxyacetophenone (4a) [32,33]. Com-
pounds 5a-v were prepared using Rap-Stoermer condensation reaction
Scheme 1. Synthesis of reagents 3a & 4a, and compounds 5a-v. Reagents and conditions: i) BBr3, ice bath (10 min), room temperature (5 h); ii) CuBr2, ethyl acetate/
DCM (1:1), 85 ◦C (24 h); iii) K2CO3, PEG-400, 100 ◦C (1–2 h); iv) AlCl3, toluene, 100 ◦C (2–3 h).
6

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
between salicylaldehydes and
α
-haloketones in PEG-400 [34]. Com-
smaller than 50 kDa. Despite the additional measures to purify the
PfGSK-3 protein, an additional protein (>50 kDa) still remained.
Droucheau and co-workers [18] also reported the presence of another
protein after PfGSK-3 purification. They concluded that this additional
protein was likely a proteolytic degradation product of PfGSK-3, and
used the PfGSK-3 protein without further purification.
pounds 5t-v were prepared by demethylation of compounds 5h-j with
AlCl3 in toluene [35].
Reagents and compounds were obtained in fair to good yields
(42–91%), except for compounds 5t-v which had poor yields (27–29%).
The structures and purity of the target compounds were verified by 1H
NMR, 13C NMR, MS, melting point and HPLC analyses.
3.3. ADP-Glo™ kinase assay
3.2. Expression and purification of recombinant HsGSK-3β and PfGSK-3
A series of benzofuran derivatives (5a-v) was screened against re-
combinant PfGSK-3 and HsGSK-3β using the ADP-Glo™ Kinase Assay.
The compounds that showed inhibitory activity at a concentration of 10
µM, were evaluated further by determining the IC50 values (Table 1). Of
the seven compounds that showed inhibitory activity during the initial
screening, one compound was selective towards HsGSK-3β (5u), one
compound was unselective inhibiting both PfGSK-3 and HsGSK-3β (5v),
and five compounds were selective towards PfGSK-3 (5k, 5m, 5p, 5r,
5s).
Recombinant HsGSK-3β was expressed from pET32a(+) as a
thioredoxin-histidine-fusion of approximately 64 kDa. Co-expression of
the pGro7 chaperone proteins (groES and groEL) with recombinant
HsGSK-3β, facilitated the protein folding process and thereby increased
the recovery of soluble HsGSK-3β. The C-terminal histidine-tag allowed
for easy detection and purification using nickel-affinity
chromatography.
PfGSK-3 was expressed using a similar method to the one described
by Droucheau and co-workers [18], however, the present study
expressed the full length PfGSK-3 protein instead of a truncated version
were the N-terminal is omitted. The pBAD/Thio-TOPO® system was
used as it tightly regulates expression, thus preventing “leaky” expres-
sion of potentially toxic proteins such as PfGSK-3. The recombinant
PfGSK-3 protein (approximately 68 kDa) was expressed with an N-ter-
minal thioredoxin fusion protein for increased solubility, and a C-ter-
minal histidine-tag for purification with nickel-affinity chromatography.
The PfGSK-3 protein required additional purification due to the
presence of several other proteins that co-eluted with PfGSK-3 (Fig. 2).
The washing protocol of the Protino® Ni-TED column was adapted as
follows: instead of washing only with LEW buffer, the column was also
washed with diluted Elution buffer (containing only 5% imidazole). As a
result, some of the previously co-eluted proteins could be washed away
before eluting the PfGSK-3 protein. As a second purification step, the
Amicon® Ultra-15 centrifugal device was used to remove all proteins
Compound 5p was the most active compound of the series with an
IC50 value of 0.00049 µM and a 316-fold selectivity for PfGSK-3 versus
HsGSK-3β. This was closely followed by the activity of compound 5m
which exhibits an IC50 value of 0.00168 µM with a 175-fold selectivity
towards PfGSK-3. Compared to the reference inhibitor, indirubin-3′-
monoxime (IC50: 0.250 µM), compound 5p and 5m are approximately
510-fold and 148-fold more potent, respectively. The most selective
PfGSK-3 inhibitor of the series was compound 5r, with a 559-fold
selectivity for PfGSK-3 versus HsGSK-3β. As mentioned previously,
PfGSK-3 inhibitors would have to be very selective in order to prevent
simultaneous inhibition of HsGSK-3β and the possible ensuing side ef-
fects [21,22].
Comparing the activity of compound 5p and 5m to that of the 4-
phenylthieno[2,3-b]pyridine inhibitors (1 and 2, Fig. 1), the benzofu-
rans developed in this study were more potent PfGSK-3 inhibitors,
demonstrating lower IC50 values than the 4-phenylthieno[2,3-b]
Fig. 2. 10% SDS-PAGE analysis of the different fractions collected during purification of recombinant HsGSK-3β (a) and PfGSK-3 (b). For HsGSK-3β (a): Lane 1:
protein ladder (kDa); SF: soluble fraction loaded onto the Protino® Ni-TED column; W1-W2: column wash with LEW buffer; E: column elution fraction. For PfGSK-3
(b): Lane 1: protein ladder (kDa); SF: soluble fraction loaded onto the Protino® Ni-TED column; W1: column wash with LEW buffer; W2-W4: column wash with 5%
imidazole buffer; E: column elution fraction; AC: concentrate collected from the Amicon® centrifugal device. Western blot analysis (c) of the column elution fractions
of HsGSK-3β and PfGSK-3.
7

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
Table 1
Structures and IC50 values of benzofuran derivatives assayed against recombinant HsGSK-3β and PfGSK-3.
#
Ring A
Ring B
IC50 SD (µM) a
HsGSK-3β
SI b
5
6
7
3′
4′
PfGSK-3
R1
R2
R3
R1′
R2′
5a
5b
5c
5d
5e
5f
H
H
H
H
H
–
–
–
Cl
Cl
Cl
Cl
H
H
H
H
H
–
–
–
H
Br
H
H
H
–
–
–
H
H
Cl
Cl
Cl
H
–
–
–
H
Br
H
H
–
–
–
H
H
–
–
–
5g
5h
5i
H
Diethylamino
H
H
H
–
–
–
H
OCH3
H
H
Cl
Cl
Cl
Cl
H
–
–
–
H
OCH3
H
H
–
–
–
5j
OCH3
H
H
H
–
–
–
5k
5l
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
Cl
H
25.817 ± 0.354
1.692 ± 0.850
15
–
H
H
–
–
5m
5n
5o
5p
5q
5r
H
H
H
F
0.294 ± 0.014
0.00168 ± 0.00005
175
–
H
H
H
Br
OCH3
CN
Cl
H
–
–
–
–
H
H
H
–
H
H
H
0.155 ± 0.005
0.00049 ± 0.00010
316
–
OCH3
H
H
H
–
–
H
OCH3
OCH3
H
70.997 ± 0.506
33.960 ± 1.054
–
0.127 ± 0.010
0.440 ± 0.113
–
559
77
–
5s
5t
H
OCH3
OH
H
H
H
H
Cl
Cl
Cl
5u
5v
H
OH
H
0.137 ± 0.002
5.449 ± 0.170
0.005 ± 0.001
(0.005–0.05) c
12.560 ± 0.352
5.103 ± 0.580
0.250 ± 0. 080
(0.065) d
0
OH
H
H
H
1
Indirubin-3′-monoxime
–
a
The IC50 values were determined in triplicate and expressed as the mean ± standard deviation (SD) in µM. IC50 values were only determined for those compounds
that exhibited inhibitory activity during the initial screening at 10 µM concentration.
b
The selectivity index (SI) is the selectivity for PfGSK-3 and is given as the ratio of (HsGSK-3β IC50)/ (PfGSK-3 IC50).
c
Literature value obtained from [37] for recombinant HsGSK-3β.
d
Literature value obtained from [18] for a truncated form of recombinant PfGSK-3.
pyridines. However, as Masch and co-workers [29] pointed out, com-
parison of IC50 values is only relevant when the same assay is used to
generate the data. Fugel and co-workers [25] used a radiometric kinase
assay, while Masch and co-workers [29] compared a radiometric assay
to the Kinase-Glo® Plus Luminescent Assay (Promega, Madison, WI).
Although, the ADP-Glo™ Kinase Assay used during this study is similar
to the Kinase-Glo® Plus Assay, the two assays differ in that they quantify
different aspects of the kinase reaction. The Kinase-Glo® Plus Assay
quantifies the remaining ATP after the kinase reaction has taken place,
and the resulting luminescent signal is inversely proportional to the
kinase activity. The ADP-Glo™ Kinase Assay quantifies ADP generated
during the kinase reaction, and the luminescent signal is directly pro-
portional to the kinase activity. An advantage of the ADP-Glo™ Kinase
Assay used in this study is that much lower levels of ADP formation can
be detected, which is beneficial when working with low enzyme con-
centrations or kinases with a low enzyme turnover rate [36]. The choice
of kinase assay is therefore an important aspect that should be carefully
considered and standardised to enable comparison of the IC50 values in
future studies.
loses its activity. Likewise, when substituents other than OCH3 are
present at position C6 of ring A, the compound either loses its activity
(5g: C6-diethylamino group), or becomes more selective towards
HsGSK-3β (5u: C6-OH). A loss of activity is also observed when ring A is
substituted with Cl and Br groups (5b & 5d: C5-Cl; 5c & 5e: C5-Cl, C7-
Br), or when ring A is unsubstituted (5a & 5f). Substituting ring A with
an OH group results in unselective activity (5v: C5-OH) or loss of activity
(5t: C7-OH).
The activity of the compounds also seems to be influenced by the
substitution pattern of ring B. The compounds with C6-OCH3 substitu-
tion on ring A are active and selective towards PfGSK-3 when ring B is
substituted with a C3′,4′-diCl (5k); C4′-F (5m); C4′-CN (5p); or C3′-
OCH3 (5r) group. However, when ring B is unsubstituted (5l) or
substituted with a Cl (5i), Br (5n) or OCH3 (5o) group at position C4′,
the compounds were inactive. Likewise, the activity of compounds with
C7-OCH3 substitution on ring A is also influenced by the substitution
pattern of ring B. This is clearly demonstrated when comparing com-
pounds 5s and 5h. The PfGSK-3 selective compound 5s is substituted at
position C3′ (OCH3 group) of ring B, while the inactive compound 5h is
substituted at position C4′ (Cl group). The effect of substituting ring B
with other types of substituents at position C3′ and C4′ still has to be
determined.
Analysing the structure-activity relationships (SARs) of the benzo-
furan derivatives with both PfGSK-3 and HsGSK-3β, revealed some
interesting trends. The PfGSK-3 selective compounds (5k, 5m, 5p, 5r,
5s) all have a C6-OCH3 substitution on ring A, except for compound 5s
which has a C7-OCH3 substitution. Active compounds seem to favour the
substitution of ring A with a single OCH3 group at position C6, for when
the position of the OCH3 group is changed (5j: C5-OCH3) or an addi-
tional OCH3 group is added to ring A (5q: C5,6-diOCH3), the compound
4. Conclusion
With the continuing threat of multi-drug resistant malaria, an urgent
need exists to develop novel antimalarial drugs. Herein, a series of
8

C. Moolman et al.
Bioorganic Chemistry 112 (2021) 104839
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Acknowledgements
Financial support for this study was provided by the North-West
University and the National Research Foundation (NRF) of South Af-
rica (Grant number: 112535). The authors wish to thank Dr. D. Otto and
Dr. J. Jordaan of Chemical Research Beneficiation (NWU) for NMR and
MS analyses, respectively. We also wish to thank Prof. J. du Preez for his
assistance with the HPLC analyses, and Prof. O. Thekisoe for use of the
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