Synthesis and Structure Activity Relationships of Chalcone based Benzocycloalkanone Derivatives as Adenosine A 1 and/or A 2A Receptor Antagonists

Article abstract of DOI:10.1055/a-1146-2996

Adenosine A ₁ and/or A _2A receptor antagonists hold promise for the potential treatment of neurological conditions, such as Parkinson's disease. Herein, a total of seventeen benzocycloalkanone derivatives were synthesised and evaluat

Full text of DOI:10.1055/a-1146-2996

Published online: 2020-04-29
Thieme
Original Article
Synthesis and Structure Activity Relationships of Chalcone based
Benzocycloalkanone Derivatives as Adenosine A1 and/or A2A
Receptor Antagonists
Authors
Helena D. Janse van Rensburg1, Lesetja J. Legoabe1, Gisella Terre’Blanche1, 2
Affiliations
Supporting Information for this article is available
1
Centre of Excellence for Pharmaceutical Sciences,
online at http://www.thieme-connect.de/products.
North-West University, Potchefstroom, South Africa
Pharmaceutical Chemistry, School of Pharmacy,
North-West University, Potchefstroom, South Africa
2
ABSTRAcT
Adenosine A1 and/or A2A receptor antagonists hold promise for
the potential treatment of neurological conditions, such as
Parkinson’s disease. Herein, a total of seventeen benzocycloal-
kanone derivatives were synthesised and evaluated for affinity
towards adenosine receptors (A1 and A2A AR). The obtained
results allowed for the conclusion that affinity and/or selectiv-
ity of the 2-benzylidene-1-indanone and -tetralone derivatives
toward A1 and/or A2A ARs may be modulated by the nature of
the substituents (either -OH, -OCH3 or morpholine) attached
at position C4 of the 1-indanone core and C5 of the 1-tetralone
core as well as the meta (C3’) and/or para (C4’) position(s) on
ring B. Several compounds (2a–b, 3b–c and 4a–b) possessed
affinity for the A1 and/or A2A AR below 10 µM. Additionally,
compounds 2a, 3b and 4a were A1 AR antagonists. These re-
sults, once again, confirmed the importance of C4 methoxy-
group substitution on ring A in combination with meta (C3’)
and/or para (C4’) hydroxyl-group substitution ring B of the
2-benzylidene-1-indanone scaffold leading to drug-like com-
pounds 1h and 1j with affinity in the nanomolar-range.
Key words
2-Benzylidene-1-tetralone derivatives, 2-Benzylidene-1-
indanone derivatives, acid catalysed aldol condensation
reaction, neurological conditions
received
accepted
02.03.2020
23.03.2020
Bibliography
DOI https://doi.org/10.1055/a-1146-2996
Published online: 29.4.2020
Drug Res 2020; 70: 243–256
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
Lesetja J. Legoabe
Centre of Excellence for Pharmaceutical Sciences,
North-West University
2520 Potchefstroom
South Africa
Tel.: + 27 18 299 2182, Fax: + 27 18 299 4243
lesetja.legoabe@nwu.ac.za
Introduction
marketing as Nouriast® in Japan only since 2013 as a novel
antiparkinsonian agent for wearing-off phenomena associated with
levodopa treatment [2]. 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 [3]. Recently, the USFDA approved
istradefylline (1a) as add-on drug to treat off episodes in adults with
Parkinson's disease (PD) [4].
Ensuing almost 100 years of research on adenosine and its receptors
(A1, A2A, A2B and A3) as well as the ligands that bind to these
adenosine receptors (ARs) and, subsequently, modulate the
adenosine system, the potential of the said system as a drug
discovery target is evident [1].
Despite the amount of research on adenosine and its receptors,
there are only two United States Food and Drug Administration
(USFDA) approved AR agonist drugs, namely, adenosine and re-
gadenoson. Furthermore, the first in class selective A2A AR antago-
nist, istradefylline (1a) has been approved for manufacturing and
The xanthine derivative istradefylline (1a) not only improves
wearing-off phenomena but also some non-motor symptoms of
PD, such as the common mood disorder in PD, namely depression
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
243

Thieme
Original Article
[5, 6]. This is, however, yet to be confirmed by a double-blind pla-
cebo-controlled trial [6]. Furthermore, istradefylline (1a) also im-
proves daytime sleepiness and bladder dysfunction, also non-mo-
tor symptoms associated with PD [7,8]. It is, unfortunately, difficult
to demonstrate neuroprotection by istradefylline (1a) in humans,
because there is, to date, no way to assess neurodegeneration or
neuroprotection in humans [9]. Although, in animal models of PD
istradefylline (1a) protected against dopaminergic neurodegen-
eration [10, 11].
Consequently, no approved drug is available for the prevention
of neuronal cell loss in patients suffering from PD; although, the
multifactorial aetiology of PD and, thus, the underlying molecular
causes of the disease are more clear – yet, the approach to drug
discovery is not multifaceted [12]. For that reason, it may be said
that the idea of “one protein, one target” is outdated [12]. In con-
trast, a robust (and, indeed, a multi-target approach) may be the
restoration of neurotransmitter levels by means of a combined in-
hibition of cholinesterases, monoamine oxidases and, of note, A2A
and A1 ARs [12].
It is not yet clear which is the better approach towards treating
the symptoms of PD between treatment with a selective A2A AR an-
tagonist or a dual A2A and A1 AR antagonist [1]. Based on published
preclinical and clinical data, it seems that most researchers are
searching for selective A2A AR antagonists in order to minimize pos-
sible cardiovascular effects caused by interaction with the A1 AR
[13]. Other researchers consider A1 AR antagonism a desirable fea-
ture to address unmet needs in PD treatment (such as a decline in
cognitive function) [14, 15]. For example, the dual A2A and A1 AR
antagonists ASP5854 (1b) and JNJ-40255293 (1c), with Ki values in
the nanomolar-range (▶Fig. 1), showed promising effects in ani-
mal models of PD neurodegeneration (A2A) and motor symptoms
(A2A), as well as cognition (A1) [16].
Of interest to the current study is dual A2A and A1 AR antagonist
compound 1c (▶Fig. 1)[16]; which contains the 1-indanone moi-
ety. The 1-indanone moiety is associated with wide-ranging bio-
logical activity and considered a privileged scaffold in medicinal
chemistry [17, 18]. In fact, it is present in therapeutic agents, such
as the acetylcholinesterase inhibitor donepezil (Aricept®) (1d)
(▶Fig. 2), indicated for the symptomatic treatment of mild to mod-
erate dementia in Alzheimer’s disease (AD) [19, 20].
namely ring A and ring C), as well as phenyl ring B, linked to the
1-indanone core by an exocyclic double bond. This leads to a pla-
nar chemical structure that permits transmission of electron do-
nating substituent effects through the exocyclic double bond onto
the carbonyl functional group of the 1-indanone core [21, 22]. Nu-
merous 2-benzylidene-1-indanone derivatives (1e–j, ▶Fig. 3) were
previously synthesised and evaluated as A2A and A1 AR antagonists
[23, 24]. (Other ring A and benzylidene ring B substitutions such as
halogens were also previously explored.) The methoxy substituted
2-benzylidene-1-indanone derivatives 1h and 1j (▶Fig. 3) proved
to be potent dual A2A and A1 AR antagonists with affinity in the na-
nomolar-range. However, the presence of liable reactive function-
al groups in compounds 1h and 1j (namely α,β-unsaturated ketone
(1h & 1j) and catechol (1j)) have raised questions [23]. A series of
the structurally related 2-benzylidene-1-tetralone derivatives (1k–
q, ▶Fig. 3)) were also previously synthesised and evaluated as A2A
and A1 AR antagonists [25, 26].
The term chalcone generally refers to chemicals with an α,ß-
unsaturated carbonyl system; thus, the chalcone family has exten-
sive structural diversity [27, 28]; as seen with the aforesaid com-
pounds, that may be considered hybrid chalcones.
In order to further investigate the structure activity relation-
ships (SARs) of this class of compounds (benzocycloalkanones) that
govern A1 and A2A AR affinity, two series of arylidene indanone and
one series of arylidene tetralone derivatives were designed.
Materials and Methods
Chemistry
Unless otherwise noted, all starting materials and solvents were
procured from Sigma-Aldrich and used without further purifica-
tion. TLC silica gel 60 F254 aluminium sheets from Merck was used
to monitor reaction progress. Melting point range was measured
with a Buchi B545 m.p. apparatus and are uncorrected. Proton (1H)
and carbon (13C) NMR spectra were recorded on a Bruker Avance
III 600 spectrometer at frequencies of 600 MHz and 151 MHz, re-
spectively, using either CDCl3 or DMSO-d6 as solvent and TMS as
reference. Chemical shifts were reported in parts per million (δ) in
relation to the solvent peak (CDCl3: residual CH at 7.26 ppm and
DMSO-d6: residual CH3 at 2.50 ppm for 1H NMR.) Spin multiplici-
ties were indicated as follows: singlet (s), doublet (d), dd (doublet
The 2-benzylidene-1-indanone (or arylidene indanone) scaffold
consists of 1-indanone core (fused 6- and 5-membered rings,
▶Fig. 1 The structure and Ki values for the binding affinity of 1a–c against human (h) A1 and A2A ARs.
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
244

(2E)-4-hydroxy-2-(4-hydroxybenzylidene)-2,3-dihydro-1H-
inden-1-one (2b)
of doublets), triplet (t) and multiplet (m). J values were reported in
Hz. High resolution mass spectra (HRMS) were recorded on a Bruk-
er micrOTOF-Q II mass spectrometer in atmospheric chemical ion-
isation (APCI) mode. HPLC analyses were done on an Agilent 1100
HPLC system.
Prepared as for 2a from 4-hydroxy-1-indanone (0.500 g, 3.375
mmol) and 4-hydroxybenzaldehyde (0.412 g, 3.375 mmol) to yield
2b as brown powder (0.690 g, 81 %): Rf: 0.13 (DCM/EtOAc 10:1);
mp: 276.3–278.6 °C; 1H NMR (600 MHz, DMSO) δ 10.23 (s, 1H),
10.15 (s, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.44 (s, 1H), 7.29 (t, J = 7.6
Hz, 1H), 7.22 (d, J = 7.3 Hz, 1H), 7.07 (dd, J = 7.8, 0.7 Hz, 1H), 6.90
(t, J = 5.7 Hz, 2H), 3.85 (s, 2H); 13C NMR (151 MHz, DMSO) δ
193.82, 159.56, 154.88, 139.42, 136.23, 133.60, 133.16, 131.71,
129.19, 126.20, 120.32, 116.27, 114.26, 29.28. APCI-HRMS m/z
calcd. for C16H12O3 (MH + ): 253.0859, found: 252.0793. Purity
(HPLC): 99.5 %.
General procedure for the synthesis of 2a–h
(2E)-4-hydroxy-2-(3-hydroxybenzylidene)-2,3-dihydro-1H-
inden-1-one (2a)
4-Hydroxy-1-indanone (0.500 g, 3.375 mmol) and 3-hydroxyben-
zaldehyde (0.412 g, 3.375 mmol) were suspended in HCl (32 %,
6 mL) and MeOH (4 mL) and mechanically stirred at 120 °C under
reflux for 24 h. Upon completion, the reaction mixture was cooled
down to room temperature, quenched with ice (20 g), filtered,
dried (30°C) and recrystallized from a suitable solvent (either EtOH
or MeOH) to yield 2a as dark brown powder (0.390 g, 46%): Rf: 0.09
(DCM/EtOAc 10:1); mp: 280.2–281.5°C; 1H NMR (600 MHz, DMSO)
δ 10.14 (s, 1H), 9.72 (s, 1H), 7.42 (s, 1H), 7.31 (dd, J = 7.8, 7.3 Hz,
2H), 7.23 (dd, J = 23.1, 7.0 Hz, 3H), 7.10 (d, J = 7.7 Hz, 1H), 6.87
(dd, J = 7.4, 1.7 Hz, 1H), 3.90 (s, 2H); 13C NMR (151 MHz, DMSO)
δ 193.64, 157.72, 154.83, 138.92, 136.35, 136.10, 134.85, 133.04,
130.05, 129.10, 122.25, 120.39, 117.14, 116.73, 114.15, 29.17.
APCI-HRMS m/z calcd. for C16H12O3 (MH + ): 253.0859, found:
253.0866. Purity (HPLC): 100 %.
(2E)-4-hydroxy-2-(3-methoxybenzylidene)-2,3-dihydro-
1H-inden-1-one (2c)
Prepared as for 2a from 4-hydroxy-1-indanone (0.300 g, 2.025
mmol) and 3-methoxybenzaldehyde (0.276 g, 2.025 mmol) to yield
2c as black powder (0.350 g, 65%): Rf: 0.60 (DCM/EtOAc 10:1); mp:
226.4–239.4 °C; 1H NMR (600 MHz, DMSO) δ 10.10 (s, 1H), 7.50
(t, J = 1.8 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.40–7.28 (m, 3H), 7.25
(d, J = 7.1 Hz, 1H), 7.10 (dd, J = 7.8, 0.7 Hz, 1H), 7.05 (dd, J = 8.0,
2.1 Hz, 1H), 3.94 (d, J = 1.3 Hz, 2H), 3.83 (s, 3H); 13C NMR (151
MHz, DMSO) δ 193.62, 159.55, 154.82, 138.85, 136.33, 136.26,
135.34, 132.81, 130.09, 129.12, 123.04, 120.53, 116.05, 115.55,
114.20, 55.25, 29.06. APCI-HRMS m/z calcd. for C17H14O3 (MH + ):
267.1016, found: 267.1018. Purity (HPLC): 99.5 %.
(2E)-4-hydroxy-2-(4-methoxybenzylidene)-2,3-dihydro-
1H-inden-1-one (2d)
Prepared as for 2a from 4-hydroxy-1-indanone (0.500 g, 3.375
mmol) and 4-methoxybenzaldehyde (0.460 g, 3.375 mmol) to yield
2d as brown powder (0.600 g, 67 %): Rf: 0.44 (DCM/EtOAc 10:1);
mp: 251.7–271.8 °C; 1H NMR (600 MHz, DMSO) δ 10.09 (s, 1H),
7.75 (d, J = 8.6 Hz, 2H), 7.49 (s, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.23
(d, J = 7.4 Hz, 1H), 7.08 (dd, J = 8.1, 4.6 Hz, 3H), 3.88 (s, 2H), 3.83
(s, 3H); 13C NMR (151 MHz, DMSO) δ 193.57, 160.66, 154.81,
139.18, 136.15, 132.87, 132.72, 132.60, 129.04, 127.58, 120.23,
114.65, 114.10, 55.43, 29.11. APCI-HRMS m/z calcd. for C17H14O3
(MH + ): 267.1016, found: 267.1016. Purity (HPLC): 97.8 %.
▶Fig. 2 The structure of Donepezil (1d).
▶Fig. 3 The structures and Ki values for the binding affinity of 1e–q against rat (r) A1 and A2A ARs.
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
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General procedure for the synthesis of 3a–f
4-[2-(morpholin-4-yl)ethoxy]-2,3-dihydro-1H-inden-1-one
hydrochloride (3a)
(2E)-2-(3,4-dimethoxybenzylidene)-4-hydroxy-2,3-
dihydro-1H-inden-1-one (2e)
Prepared as for 2a from 4-hydroxy-1-indanone (0.500 g, 3.375
mmol) and 3,4-dimethoxybenzaldehyde (0.561 g, 3.375 mmol) to
yield 2e as brown powder (0.630 g, 63 %): Rf: 0.53 (PE/EtOAc 1:1);
mp: 224.6–226.2 °C; 1H NMR (600 MHz, DMSO) δ 10.06 (s, 1H),
7.50 (s, 1H), 7.42–7.35 (m, 2H), 7.30 (t, J = 7.6 Hz, 1H), 7.24 (d,
J = 7.1 Hz, 1H), 7.12–7.07 (m, 2H), 3.92 (d, J = 1.0 Hz, 2H), 3.85
(d, J = 16.9 Hz, 6H); 13C NMR (151 MHz, DMSO) δ 193.49, 154.76,
150.49, 148.81, 139.16, 136.05, 133.33, 132.63, 129.01, 127.72,
124.59, 120.26, 114.08, 113.98, 111.92, 55.64, 55.60, 28.93. AP-
CI-HRMS m/z calcd. for C18H16O4 (MH + ): 297.1121, found:
297.1121. Purity (HPLC): 99.6 %.
A mixture of 4-hydroxy-1-indanone (1.21 g, 8.17 mmol), 4-(2-chlo-
roethyl)morpholine hydrochloride (1.22 g, 8.17 mmol) and K2CO3
(2.53g, 40.85 mmol) in acetone (40 mL) were mechanically stirred
at 120°C under reflux for approximately 24 h. The reaction mixture
was then cooled down to room temperature, filtered over SiO2 and
the filtrate conc. to yield a dark brown oil. The said oil was dissolved
in EtOAc (20 mL) and acetyl chloride (6 mL) was added to the solu-
tion and mechanically stirred at room temperature until a precipi-
tate formed. The resulting precipitate was filtered and dried at 30°C
to yield 3a as a beige powder (0.92 g, 43%): Rf: 0.17 (PE/EtOAc 3:1);
mp: 223.2–223.6°C; 1H NMR (600 MHz, DMSO) δ 11.77 (d, J = 1.9
Hz, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.28 (dd, J = 21.2, 7.7 Hz, 2H), 4.58
(s, 2H), 4.00–3.82 (m, 4H), 3.62–3.46 (m, 4H), 3.24 (dd, J = 3.6,
2.3 Hz, 2H), 3.07–2.97 (m, 2H), 2.68–2.59 (m, 2H); 13C NMR (151
MHz, DMSO) δ 206.22, 155.18, 143.81, 138.36, 129.05, 116.53,
115.25, 63.20, 62.82, 54.81, 51.76, 39.52, 35.73, 22.35. APCI-
HRMS m/z calcd. for C15H19NO3 · HCl (MH + ): 262.1438, found:
262.1458. Purity (HPLC): 74.4 %.
(2E)-4-methoxy-2-(3-methoxybenzylidene)-2,3-dihydro-
1H-inden-1-one (2f)
Prepared as for 2a from 4-methoxy-1-indanone (0.500 g, 3.083
mmol) and 3-methoxybenzaldehyde (0.420 g, 3.083 mmol) to yield
2f as light brown powder (0.640 g, 74%): Rf: 0.39 (PE/EtOAc 4:1);
mp: 125.9–299.1 °C; 1H NMR (600 MHz, CDCl3) δ 7.63 (t, J = 2.0
Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.39 (dt, J = 11.1, 7.9 Hz, 2H), 7.32
(d, J = 7.7 Hz, 1H), 7.24–7.20 (m, 1H), 7.08 (d, J = 7.9 Hz, 1H), 6.96
(dd, J = 8.1, 2.0 Hz, 1H), 3.96–3.91 (m, 5H), 3.87 (s, 3H); 13C NMR
(151 MHz, CDCl3) δ 194.66, 159.92, 156.79, 139.52, 138.52,
136.80, 135.05, 134.09, 130.01, 129.27, 123.38, 116.43, 116.20,
115.25, 115.16, 55.64, 55.51, 29.44. APCI-HRMS m/z calcd. for
C18H16O3 (MH + ): 281.1172, found: 281.1182. Purity (HPLC):
98.1 %.
(2E)-2-(3-hydroxybenzylidene)-4-[2-(morpholin-4-yl)
ethoxy]-2,3-dihydro-1H-inden-1-one (3b)
Prepared as for 2a from 3a (0.300 g, 1.007 mmol) and 3-hydroxy-
benzaldehyde (0.123 g, 1.007 mmol) to yield 3b as black powder
(0.030 g, 8 %): Rf: 0.11 (PE/EtOAc/MeoH 5:1:1); mp: 213.1–
214.5 °C; 1H NMR (600 MHz, DMSO) δ 9.74 (s, 1H), 7.47–7.43 (m,
2H), 7.38–7.30 (m, 3H), 7.21–7.16 (m, 2H), 6.88 (d, J = 7.9 Hz,
1H), 4.28 (t, J = 5.9 Hz, 2H), 4.11 (dd, J = 10.3, 5.1 Hz, 1H), 3.90
(s, 2H), 3.60–3.55 (m, 4H), 3.17 (d, J = 5.1 Hz, 2H), 2.78 (t, J = 5.9
Hz, 2H); 13C NMR (151 MHz, DMSO) δ 193.31, 157.69, 155.78,
138.72, 138.30, 136.00, 134.50, 133.32, 130.06, 129.41, 122.14,
117.37, 117.19, 116.72, 115.39, 66.23, 56.84, 53.67, 48.60,
28.93. APCI-HRMS m/z calcd. for C22H23NO4 ·HCl (MH+): 366.1700,
found: 366.1731. Purity (HPLC): 99.5 %.
(2E)-4-methoxy-2-(4-methoxybenzylidene)-2,3-dihydro-
1H-inden-1-one (2g)
Prepared as for 2a from 4-methoxy-1-indanone (0.500 g, 3.083
mmol) and 4-methoxybenzaldehyde (0.420 g, 3.083 mmol) to yield
2g as golden brown powder (0.380 g, 44 %): Rf: 0.77 (DCM/EtOAc
10:1); mp: 117.1–296.0°C; 1H NMR (600 MHz, CDCl3) δ 7.67 (d, J =
8.5 Hz, 2H), 7.63 (s, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.7
Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 3.94 (s,
3H), 3.89 (s, 2H), 3.86 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 194.66,
160.97, 156.73, 139.82, 138.28, 134.03, 132.76, 132.42, 129.16,
128.26, 116.13, 114.90, 114.56, 55.65, 55.52, 29.46. APCI-HRMS
m/z calcd. for C18H16O3 (MH + ): 281.1172, found: 281.1172. Puri-
ty (HPLC): 97.3 %.
(2E)-2-(3,4-dihydroxybenzylidene)-4-[2-(morpholin-4-yl)
ethoxy]-2,3-dihydro-1H-inden-1-one (3b)
Prepared as for 2a from 3a (0.300 g, 1.007 mmol) and 3,4-dihy-
droxybenzaldehyde (0.139 g, 1.007 mmol) to yield compound 3c
as dark green powder (0.070 g, 18%): Rf: 0.15 (EtOAc 100 %); mp:
161.4–184.4 °C; 1H NMR (600 MHz, DMSO) δ 11.20 (d, J = 9.2 Hz,
1H), 9.78 (s, 1H), 9.32 (s, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.40 (d, J =
7.8 Hz, 2H), 7.34 (dd, J = 9.1, 4.9 Hz, 2H), 7.14 (dd, J = 8.3, 1.9 Hz,
1H), 6.88 (d, J = 8.2 Hz, 1H), 4.59 (s, 2H), 4.03–3.98 (m, 2H), 3.95
(s, 2H), 3.88 (t, J = 11.8 Hz, 2H), 3.65 (s, 2H), 3.58 (d, J = 11.3 Hz,
2H), 3.29 (d, J = 7.6 Hz, 1H); 13C NMR (151 MHz, DMSO) δ 193.09,
154.85, 148.18, 145.67, 139.30, 138.23, 134.19, 130.98, 129.29,
126.30, 124.43, 117.68, 116.98, 116.12, 115.95, 63.25, 62.69,
54.83, 51.77, 29.23. APCI-HRMS m/z calcd. for C22H23NO5 · HCl
(MH + ): 382.1649, found: 382.1680. Purity (HPLC): 74.7 %.
(2E)-2-(3,4-dimethoxybenzylidene)-4-methoxy-2,3-
dihydro-1H-inden-1-one (2h)
Prepared as for 2a from 4-methoxy-1-indanone (0.500 g, 3.083
mmol) and 3,4-dimethoxybenzaldehyde (0.512 g, 3.083 mmol) to
yield 2h as brown powder (0.520 g, 54%): Rf: 0.20 (PE/EtOAc 4:1);
mp: 173.8–175.1°C; 1H NMR (600 MHz, CDCl3) δ 7.61 (t, J = 1.8 Hz,
1H), 7.50 (d, J = 7.5 Hz, 1H), 7.42–7.33 (m, 2H), 7.20 (d, J = 1.9 Hz,
1H), 7.07 (d, J = 7.9 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 3.98–3.92 (m,
9H), 3.91 (d, J = 1.4 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ 194.58,
156.72, 150.69, 149.16, 139.77, 138.22, 134.32, 132.71, 129.22,
128.53, 124.64, 116.14, 114.95, 113.82, 111.38, 56.16, 56.12,
55.64, 29.33. APCI-HRMS m/z calcd. for C19H18O4 (MH + ):
311.1278, found: 311.1278. Purity (HPLC): 100 %.
5-[2-(morpholin-4-yl)ethoxy]-2,3-dihydro-1H-inden-1-one
hydrochloride (3d)
Prepared as for 2a from 5-hydroxy-1-indanone (1.000 g, 6.75
mmol) and 4-(2-chloroethyl)morpholine hydrochloride (1.260 g,
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
246

6.75 mmol) to yield compound 3d as light brown powder (0.930
g, 53%): Rf: 0.11 (PE/EtOAc 3:1); mp: 199.9–200.8°C; 1H NMR (600
MHz, DMSO) δ 11.73 (d, J = 1.1 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H),
7.17 (d, J = 1.6 Hz, 1H), 7.03 (dd, J = 8.5, 2.1 Hz, 1H), 4.60–4.54
(m, 2H), 3.98–3.83 (m, 5H), 3.57 (s, 2H), 3.48 (d, J = 12.0 Hz, 2H),
2.5 Hz, 1H), 6.06 (d, J = 6.0 Hz, 1H), 3.84 (s, 3H), 3.11 (td, J = 6.6,
1.6 Hz, 2H), 2.93 (t, J = 6.5 Hz, 2H); 13C NMR (151 MHz, CDCl3)
δ 188.63, 159.60, 152.92, 137.23, 136.85, 135.75, 134.79, 130.33,
129.61, 127.37, 122.46, 120.61, 119.84, 115.48, 114.27, 55.46,
26.65, 21.71. APCI-HRMS m/z calcd. for C18H16O3 (MH + ):
281.1172, found: 281.1181. Purity (HPLC): 99.5 %.
3.20 (d, J = 9.1 Hz, 2H), 3.08–3.04 (m, 2H), 2.62–2.58 (m, 2H); 13
C
NMR (151 MHz, DMSO) δ 204.35, 162.88, 158.19, 130.44, 124.63,
115.74, 111.02, 63.12, 62.69, 54.59, 51.61, 36.05, 25.49. APCI-
HRMS m/z calcd. for C15H19NO3 · HCl (MH + ): 262.1438, found:
262.1456. Purity (HPLC): 100 %.
(2E)-2-(3,4-dimethoxybenzylidene)-5-hydroxy-3,4-
dihydronaphthalen-1(2H)-one (4b)
Prepared as for 4a from 5-hydroxy-1-tetralone (0.500 g, 3.083
mmol) and 3,4-dimethoxybenzaldehyde (0.512 g, 3.083 mmol) to
yield 4b as dark brown powder (0.670 g, 70%): Rf: 0.57 (PE/EtOAc
1:1); mp: 252.5–253.0°C; 1H NMR (600 MHz, CDCl3) δ 7.81 (s, 1H),
7.73 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 7.9 Hz, 1H), 7.11–7.01 (m, 2H),
6.98 (s, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.04 (d, J = 0.4 Hz, 1H), 3.91
(2E)-2-(3-hydroxybenzylidene)-5-[2-(morpholin-4-yl)
ethoxy]-2,3-dihydro-1H-inden-1-one (3e)
Prepared as for 2a from 3d (0.300 g, 1.007 mmol) and 3-hydroxy-
benzaldehyde (0.123 g, 1.007 mmol) to yield 3e as dark brown
powder (0.150 g, 41 %): Rf: 0.07 (PE/EtOAc/MeoH 5:1:1); mp:
150.6–153.0°C; 1H NMR (600 MHz, DMSO) δ 9.69 (s, 1H), 7.71 (d,
J = 8.4 Hz, 1H), 7.35 (s, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.19 (s, 1H),
7.17 (d, J = 7.8 Hz, 1H), 7.14 (s, 1H), 7.04 (d, J = 8.1 Hz, 1H), 6.86
(dd, J = 8.0, 1.9 Hz, 1H), 4.34–4.16 (m, 3H), 4.05–3.99 (m, 3H),
3.60 (s, 6H), 2.73 (d, J = 16.5 Hz, 2H); 13C NMR (151 MHz, DMSO)
δ 191.57, 157.69, 152.85, 136.20, 135.39, 131.75, 129.94, 128.65,
125.45, 124.53, 121.74, 116.82, 115.72, 115.64, 110.88, 67.40,
66.04, 59.75, 53.45, 32.04. APCI-HRMS m/z calcd. for
C22H23NO4 ·HCl (MH+): 366.1700, found: 366.1718. Purity (HPLC):
95.5 %.
(d, J = 12.6 Hz, 6H), 3.18–3.05 (m, 2H), 2.94 (t, J = 6.5 Hz, 2H); 13
C
NMR (151 MHz, CDCl3) δ 188.43, 152.86, 149.71, 148.82, 137.19,
134.97, 133.81, 130.05, 128.74, 127.32, 123.47, 120.54, 119.68,
113.38, 111.04, 56.08, 56.07, 26.70, 21.55. APCI-HRMS m/z calcd.
for C19H18O4 (MH + ): 311.1278, found: 311.1293. Purity (HPLC):
91.6 %.
(2E)-5-methoxy-2-(3-methoxybenzylidene)-3,4-
dihydronaphthalen-1(2H)-one (4c)
Prepared as for 4a from 5-methoxy-1-tetralone (0.500 g, 2.806
mmol) and 3-methoxybenzaldehyde (0.382 g, 2.806 mmol) to yield
4c as golden brown powder (0.730 g, 88%): Rf: 0.67 (PE/EtOAc 1:1);
mp: 114.1–143°C; 1H NMR (600 MHz, CDCl3) δ 7.82–7.74 (m, 2H),
7.37–7.29 (m, 2H), 7.04 (dd, J = 10.6, 3.5 Hz, 2H), 6.97 (d, J = 1.9
Hz, 1H), 6.91 (dd, J = 8.1, 2.4 Hz, 1H), 3.86 (d, J = 18.1 Hz, 6H),
3.09 (td, J = 6.5, 1.8 Hz, 2H), 2.92 (t, J = 6.5 Hz, 2H); 13C NMR (151
MHz, CDCl3) δ 188.29, 159.62, 156.43, 137.37, 136.32, 135.89,
134.55, 132.49, 129.57, 127.31, 122.43, 120.02, 115.45, 114.46,
114.15, 55.87, 55.44, 26.72, 21.66. APCI-HRMS m/z calcd. for
C19H18O3 (MH + ): 295.1329, found: 295.1333. Purity (HPLC):
69.4 %.
(2E)-2-(3,4-dihydroxybenzylidene)-5-[2-(morpholin-4-yl)
ethoxy]-2,3-dihydro-1H-inden-1-one (3f)
Prepared as for 2a from 3d (0.089 g, 0.300 mmol) and 3,4-dihy-
droxybenzaldehyde (0.041 g, 0.300 mmol) to yield compound 3f
as brown powder (0.100 g, 88 %): Rf: 0.17 (EtOAc 100 %); mp:
151.8–151.9 °C; 1H NMR (600 MHz, DMSO) δ 11.21 (s, 1H), 9.72
(s, 1H), 9.28 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.24 (d,
J = 1.0 Hz, 1H), 7.20 (d, J = 1.9 Hz, 1H), 7.11–7.07 (m, 2H), 6.87
(d, J = 8.2 Hz, 1H), 4.58 (s, 2H), 3.99 (s, 2H), 3.96 (s, 1H), 3.83 (t,
J = 12.0 Hz, 2H), 3.61 (d, J = 1.2 Hz, 2H), 3.52 (d, J = 12.2 Hz, 2H),
3.22 (dd, J = 9.7, 1.3 Hz, 2H); 13C NMR (151 MHz, DMSO) δ 191.58,
162.70, 152.46, 147.85, 145.61, 132.74, 131.71, 131.50, 126.47,
125.25, 123.89, 117.55, 116.06, 115.63, 111.10, 63.16, 62.61,
54.75, 51.66, 32.09. APCI-HRMS m/z calcd. for C22H23NO5 · HCl
(MH + ): 382.1649, found: 382.1674. Purity (HPLC): 75.5 %.
(2E)-5-methoxy-2-(4-methoxybenzylidene)-3,4-
dihydronaphthalen-1(2H)-one (4d)
Prepared as for 4a from 5-methoxy-1-tetralone (0.500 g, 2.806
mmol) and 4-methoxybenzaldehyde (0.382 g, 2.806 mmol) to yield
4d as dark brown powder (0.810 g, 98 %): Rf: 0.77 (PE/EtOAc 1:1);
mp: 99.4–185.4 °C; 1H NMR (600 MHz, CDCl3) δ 7.81 (s, 1H), 7.75
(dd, J = 7.8, 0.9 Hz, 1H), 7.47–7.34 (m, 2H), 7.31 (t, J = 8.0 Hz, 1H),
7.04 (dd, J = 8.1, 0.8 Hz, 1H), 6.97–6.86 (m, 2H), 3.89–3.84 (m,
6H), 3.10 (td, J = 6.6, 1.7 Hz, 2H), 2.92 (t, J = 6.6 Hz, 2H); 13C NMR
(151 MHz, CDCl3) δ 188.24, 160.02, 156.36, 136.51, 134.74,
133.67, 132.24, 131.88, 128.59, 127.23, 120.00, 114.31, 114.06,
55.87, 55.48, 26.67, 21.48. APCI-HRMS m/z calcd. for C19H18O3
(MH + ): 295.1329, found: 295.1329. Purity (HPLC): 83.4 %.
General procedure for the synthesis of 4a–e
(2E)-5-hydroxy-2-(3-methoxybenzylidene)-3,4-
dihydronaphthalen-1(2H)-one (4a)
5-Hydroxy-1-tetralone (0.500 g, 3.083 mmol) and 3-methoxyben-
zaldehyde (0.420 g, 3.083 mmol) were suspended in HCl (32 %, 6
mL) and MeOH (4 mL) and mechanically stirred at 120°C under re-
flux for 24 h. Upon completion, the reaction mixture was cooled
down to room temperature, extracted with EtOAc (3 × 100 mL),
combined organic extracts dried (MgSO4), filtered and conc. to
yield 4a as dark brown powder (0.540g, 62%): Rf: 0.88 (PE/EtOAc
1:1); mp: 328.2–328.3°C; 1H NMR (600 MHz, CDCl3) δ 7.81 (s, 1H),
7.74 (d, J = 7.8 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.21 (t, J = 7.9 Hz,
1H), 7.04 (dd, J = 10.8, 3.9 Hz, 2H), 6.96 (s, 1H), 6.91 (dd, J = 8.3,
(2E)-2-(3,4-dimethoxybenzylidene)-5-methoxy-3,4-
dihydronaphthalen-1(2H)-one (4e)
Prepared as for 4a from 5-methoxy-1-tetralone (0.500 g, 2.806
mmol) and 3,4-dimethoxybenzaldehyde (0.466 g, 2.806 mmol) to
yield 4e as dark brown powder (0.750 g, 82%): Rf: 0.39 (PE/EtOAc
1:1); mp: 117.7–154.6°C; 1H NMR (600 MHz, CDCl3) δ 7.79 (s, 1H),
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
247

Thieme
Original Article
7.77–7.72 (m, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.07 (ddd, J = 24.4, 6.4,
1.1 Hz, 2H), 6.99 (d, J = 2.0 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 3.92
(d, J = 10.4 Hz, 6H), 3.87 (s, 3H), 3.12 (td, J = 6.6, 1.7 Hz, 2H), 2.93
(t, J = 6.5 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ 188.18, 156.36,
149.64, 148.83, 136.68, 134.71, 133.96, 132.23, 128.85, 127.27,
123.39, 120.00, 114.35, 113.36, 111.02, 56.08, 56.06, 55.88,
26.74, 21.48. APCI-HRMS m/z calcd. for C20H20O4 (MH + ):
325.1434, found: 325.1457. Purity (HPLC): 79.9 %.
the competitive inhibition of [3H]DPCPX (Kd = 0.36 nM) against rat
whole brain membranes (expressing the A1 AR) and [3H]NECA (Kd =
15.3 nM) against rat striatal membranes (expressing the A2A AR) by
the test compounds by means of the Cheng-Prusoff equation. Also,
concurrent radioligand binding assays allow for the calculation of
the degree of selectivity that the test compounds (2a–h, 3a–f &
4a–e) possess towards either rat (r) A1 or A2A ARs (selectivity index
(SI)). These results, expressed as Ki values (µM), are summarized in
▶Tables 1–ꢀ3.
Biology
GTP shift assay
All reagents were commercially available and purchased from var-
ious 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 from Merck. Radio activity was counted by a Packard Tri-
CARB 2810 liquid scintillation counter.
The type of binding affinity that test compounds 2a, 3b and 4a ex-
hibited at the rat A1 AR was determined via a GTP shift assay, as de-
scribed previously [31, 33, 34]. GTP shifts were calculated by divid-
ing the Ki values of compounds reported in the presence of GTP by
the Ki values obtained in the absence of GTP and the results are
summarized in ▶Table 4.
Adenosine A1 and A2A receptor radioligand binding
assays
SwissADME
The degree of binding affinity that the test compounds (2a–h, 3a–
f & 4a–e) possess towards rat A1 and A2A ARs were determined via
radioligand binding assays, as described previously [29–32]. The
collection of tissue samples for the A1 and A2A radioligand binding
assays was approved by the Research Ethics Committee of the
North-West University (NWU) (application number NWU - 00260–
17-A5). 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. Sub-
sequently, the IC50 values were used to calculate the Ki values for
SwissADME (http://www.swissadme.ch), a free web tool, was used
to evaluate key parameters of small molecules, such as pharma-
cokinetics, drug-likeness and medicinal chemistry friendliness [35].
Results, Discussion
Chemistry
A total of seventeen test compounds (2a–h, 3b–c, 3e–f & 4a–e),
of which eleven are either novel 2-benzylidene-1-indanone (2f–h,
▶Table 1 Ki values for the binding affinity of 2-benzylidene-1-indanone derivatives against rat A1 and A2A ARs.
2-Benzylidene-1-indanone derivatives
O
A
C
4
R1'
3'
R1
B
4'
R2'
2a-h
Ring A
4
Ring B
4’
Ki ±SEM (µM)a (Specific binding (%))b
SId
#
3’
A1c vs [3H]DPcPX
A2Ac vs [3H]NEcA
A1/A2A
R1
R1’
R2’
H
2a
2b
2c
2d
2e
2f
OH
OH
0.7921 ± 0.0301a
2.897 ± 0.2126a
(55.5)b
0.4345 ± 0.0171a
1.051 ± 0.2480a
(23)b
1.823
OH
H
OH
H
2.756
OH
OCH3
H
-
-
-
-
-
-
OH
OCH3
OCH3
H
(44.5)b
(30.5)b
OH
OCH3
OCH3
H
(42)b
(26)b
OCH3
OCH3
OCH3
(29)b
(46.5)b
2g
2h
OCH3
OCH3
(79.5)b
(45.5)b
OCH3
(27)b
(23.5)b
a All Ki values were determined in triplicate and expressed as mean± standard error of the mean (SEM) in µM. b Specific binding ( %) of the radioligand
at a maximum tested concentration of 100 µM were determined in duplicate and expressed as the mean in %. c Rat receptors were used (A1: rat
whole brain membranes; A2A: rat striatal membranes). d Selectivity index (SI) for the A2A AR isoform calcd. as a ratio of A1Ki/A2AKi.
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
248

▶Table 2 Ki values for the binding affinity of 2-benzylidene-1-indanone derivatives against rat A1 and A2A ARs.
2-Benzylidene-1-indanone derivatives
Ring A
Ring B
Ki ±SEM (µM)a (Specific binding (%))b
SId
4
5
3’
4’
A1c vs [3H]DPcPX
A2Ac vs [3H]
NEcA
A1/A2A
#
R1
R2
H
R1’
-
R2’
-
3a
3b
3c
3d
3e
3f
(91)b
(99)b
(51.5)b
(55)b
-
-
-
-
-
-
H
OH
OH
-
H
6.398 ± 0.2344a
12.35 ± 1.058a
(98)b
H
OH
-
H
H
H
(91)b
OH
OH
H
(29.5)b
(35.5)b
(83)b
OH
(56.5)b
a All Ki values were determined in triplicate and expressed as mean± standard error of the mean (SEM) in µM. b Specific binding ( %) of the radioligand
at a maximum tested concentration of 100 µM were determined in duplicate and expressed as the mean in %. c Rat receptors were used (A1: rat
whole brain membranes; A2A: rat striatal membranes). d Selectivity index (SI) for the A2A AR isoform calcd. as a ratio of A1Ki/A2AKi.
▶Table 3 Ki values for the binding affinity of 2-benzylidene-1-tetrealone derivatives against rat A1 and A2A ARs.
2-Benzylidene-1-tetralone derivatives
O
R1'
3'
C
B
A
R2'
4'
5
R1
4a-e
Ring A
5
Ring B
3’
Ki ±SEM (µM)a (Specific binding (%))b
SId
#
4’
A1c vs [3H]DPcPX
A2Ac vs [3H]NEcA
A1/A2A
R1
R1’
R2’
4a
4b
4c
4d
4e
OH
OCH3
OCH3
OCH3
H
H
4.343 ± 0.4174a
6.843 ± 1.118a
(38)b
(39)b
7.054 ± 2.752 a
(45.5)b
-
OH
OCH3
H
0.9701
OCH3
OCH3
OCH3
-
-
-
OCH3
OCH3
(32)b
(54)b
OCH3
(29)b
(32.5)b
a All Ki values were determined in triplicate and expressed as mean± standard error of the mean (SEM) in µM. b Specific binding ( %) of the radioligand
at a maximum tested concentration of 100 µM were determined in duplicate and expressed as the mean in %. c Rat receptors were used (A1: rat
whole brain membranes; A2A: rat striatal membranes). d Selectivity index (SI) for the A2A AR isoform calcd. as a ratio of A1Ki/A2AKi.
3b–c & 3e–f) or –tetralone derivatives (4a–c & 4e), were synthe-
sised via acid-catalysed aldol condensation reactions, as described
in literature and outlined in ▶Fig. 4b, [23, 25, 26]. As previously
described, the key starting material for 2a–e was prepared by re-
arrangement of 3,4-dihydrocoumarin to yield 4-hydroxy-1-in-
danone [24] and the key starting material for 2f–h by methylation
of 4-hydroxy-1-indanone to yield 4-methoxy-1-indanone [23].
Compound 3a, the key starting material for 3b–c, was prepared by
reacting the previously synthesised phenol 4-hydroxy-1-indanone
[24] with 4-(2-chloroethyl)morpholine in the presence of K2CO3 in
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
249

Thieme
Original Article
acetone (similar to a method used by [36]) to give the ethers 3b–c
(▶Fig. 4a). Demethylation of 5-methoxy-1-indanone to yield 5-hy-
droxy-1-indanone provided the starting material for compound 3d
[36]. Compound 3d, the key starting material for 3e–f, was pre-
pared as for compound 3a. All synthesized reagents and test com-
pounds (2a–h, 3b–c, 3e–f & 4a–e) were obtained in fair to poor
yields, purified by recrystallization (except synthesized reagents)
from a suitable solvent (either MeOH or EtOH) and, in each in-
stance, the structure, molecular mass and/or purity of these
compounds were verified by 1H NMR, 13C NMR, MS and/or HPLC.
The 2-benzylidene-1-indanone (2a–h, 3b–c & 3e–f) and –te-
tralone derivatives (4a–e) were always obtained in the (E)-config-
uration, as seen in the 1H NMR spectra. On the 1H NMR spectra of
the said derivatives the signal of the vinyl proton of the α,β-
unsaturated carbonyl group was observed at approximately 7.42–
7.81 ppm. The same trend was previously observed with the char-
acterization of benzylidene indanones [23, 24] and tetralones
[25, 26, 37, 38].Theoretically, these derivatives may be either (E)-
or (Z)-isomers [39]. The (E)-configuration is favourable for thermo-
dynamic reasons, due to steric interaction between the carbonyl
and aryl groups in the case of (Z)-isomers [34,39]. As a result of the
diamagnetic anisotropy (deshielding of proton due to local diamag-
netic current) of the carbonyl functional group, the vinyl proton of
the (E)-isomer gives a signal at a greater chemical shift than the
vinyl proton of the (Z)-isomer, as the latter is more remote from
the carbonyl functional group [40].
▶Table 4 A1Ki values (in the absence and presence of GTP) and calcd. GTP
shifts of 2a, 3b & 4a.
#
Ki ±SEM (µM)a
GTP
shiftd
A1b vs [3H]DPcPX
A1 + GTPc vs [3H]DPcPX
0.9467 ± 0.0998
b
2a
3b
4a
0.7921 ± 0.0301a
6.398 ± 0.2344a
4.343 ± 0.4174a
1.195
0.9293
1.281
5.945 ± 0.5055a
The 1H NMR spectra of the C4 and C5 morpholine substituted
2-benzylidene-1-indanone derivatives 3a–f showed that the pro-
tons present in the morpholine moiety correspond to previously
synthesised fluorinated heteroaryl chalcones containing a morpho-
line moiety [41].
Also, the 13C NMR spectra displayed a prominent signal for the
carbonyl group at approximately 191.57–194.66 ppm (2a–h, 3b–
5.561 ± 0.8727a
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 calcd. by
dividing Ki value in the presence of 100 µM GTP by Ki value in the
absence of 100 µM GTP.
▶Fig. 4 Synthesis 3a & 3d. Reagents and conditions: a) K2CO3, Acetone (40 mL), 120°C (24 h); Synthesis of 2a–h, 3b–c, 3e–f & 4a–e. b) HCl (32%,
6 mL), MeOH (4 mL), 120°C (24 h)
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
250

▶Fig. 5 Structural requirements of the 2-benzylidene-1-indanone scaffold for A1 and/or A2A AR affinity.
▶Fig. 6 Structural requirements of the 2-benzylidene-1-tetralone scaffold for A1 and/or A2A AR affinity.
▶Fig. 7 The binding curves of compounds 2a (A), 3b (B) and 4a (C) are examples 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. Calcd. GTP shift of: 1.195 (2a),
0.9293 (3b) 1.281(4a).
c, 3e–f), 204.22–206.22 ppm (3a & 3d) and 188.18–188.63 ppm
(4a–e). Signals for the CH2-group present in the 1-indanone core
Biology
In vitro evaluation
were observed at 28.93–29.46 ppm (2a–h, 3b–c) and 32.04–32.09
The SAR studies of the 2-benzylidene-1-indanone derivatives (2a–
(3e–f), whereas the (CH2)2-groups present in the compounds 3a &
h) for the A1 and A2A ARs, summarized in ▶Table 1 (▶Fig. 5), illus-
3d (starting material for 3b–c & 3e–f, respectively) were observed
trated that the two compounds with both A1 and A2A AR affinity,
at 35.73–36.05 ppm and 22.35–25.49 ppm. The (CH2)2-groups
with slight selectivity toward the A2A AR, contain only strong elec-
present in the 1-tetralone core (4a–e) were observed at 26.65–
tron donating OH-groups on both ring A and phenyl ring B.
26.74 ppm and 21.48–21.71 ppm.
Comparison of compound 2a to the previously synthesised and
Mass spectra of all chalcone based benzocycloalkanone deriva-
evaluated unsubstituted compound 1e showed that meta (C3’) OH-
tives showed intensive molecular ions in accordance with the for-
group substitution on ring B led to A1 and A2A AR affinity below
mulations depicted.
1 µM. Compound 1e possessed selective A2A AR affinity (A2AKi (r) =
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
251

Thieme
Original Article
▶Table 5 Ki values for the binding affinity of reference compounds against rat A1 and A2A ARs.
#
Ki ±SEM (µM)a
SId A1/A2A
GTP shifte
A1b vs [3H]DPcPX
A2Ab vs [3H]NEcA
A1 + GTPc vs [3H]DPcPX
b
CPA
(A1 agonist)
0.0057 ± 0.0015a
(0.0068)f
0.4004 ± 0.1715a
(0.163)f
(0.331)g
0.0990 ± 0.0150a
(0.099)f
(0.099)g
0.0142d
(0.0417)d
(0.0453)d
17.36e
(15)f
(14)g
(0.015)g
(0.0079)h
DPCPX
(A1 antagonist)
0.0005 ± 0.00003a
(0.0004)f
0.2338 ± 0.0328a
(0.545)f
0.0006 ± 0.00003a
(0.0004)f
(0.0004)g
0.0021d
1.2e
(0.0007)d
(0.0009)d
(0.0008)d
(1)f
(0.0005)g
(0.530)g
(1.25)g
(0.0003)i
(0.340)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
(104.5)d
0.7869e
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 calcd. as a ratio of A1Ki/A2AKi. e GTP shift calcd. 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 [25]g Literature value obtained from [28]h Literature value obtained from [26]i Literature
value obtained from [30]j Literature value obtained from [46].
▶Table 6 Physiochemical properties of compounds ▶2a–b, 3b–c & 4a–b
#
Physiochemical properties
Lipophi-
licity
Water
solubility
Molecular
formula
Molecular
weight
(g/mol)
Frac-
tion
csp3
Num.
rotatable
bonds
Num.
H-bond
acceptors
Num.
H-bond
donors
Molar
refrac-
tivity
TPSA
(Å2)
consen-
sus
logPo/w
consen-
sus logS
2a
2b
3b
3c
4a
4b
C16H12O3
252.26
252.26
365.42
381.42
280.32
310.34
0.06
0.06
0.32
0.32
0.17
0.21
1
1
5
5
2
3
3
3
5
6
3
4
2
2
1
2
1
1
73.15
73.15
107.43
109.46
82.42
88.91
57.53
57.53
59.00
79.23
46.53
55.76
2.62
2.64
2.87
2.58
3.38
3.27
−4.08
−4.08
−4.60
−4.37
−4.69
−4.80
C
C
C
C
C
16H12O3
22H23NO4
22H23NO5
18H16O3
19H18O4
▶Table 7 Drug-likeness and medicinal chemistry friendliness of compounds 2a–b, 3b–c & 4a–b
#
Drug-likeness
Lead-likeness
Num. violations
Lipinskia
Ghoseb
Veberc
Egand
Mueggee
Teaguef
2a
2b
3b
3c
4a
4b
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
1
1
1
1
a Lipinski: MW < 500, MLOGP < 4.15, N or O < 10, NH or OH < 5 [39]b Ghose: 160 < MW < 480, -04 < WLOGP < 5.6, 40 < MR < 130, 20 < atoms < 70 [40]c
Veber: Num. rotatable bonds < 10, TPSA < 140 [41]d Egan: WLOGP < 5.88, TPSA < 131.6 [42]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 [43]f
Teague: 250 < MW < 350, XLOGP3 < 3.5, num. rotatable bonds < 7 [44].
1.55 µM), whereas 2b gained A1 (A1Ki (r) = 0.7921 µM) and in-
creased A2A (A2AKi (r) = 0.4345 µM) AR affinity and, thus, attained
dual A1 and A2A AR affinity. Meta (C3’) OH-group substitution on
ring B was preferred to para (C4’) substitution, as seen upon com-
parison of compound 2a with 2b (A1Ki (r) = 2.897 µM; A2AKi (r) =
1.051 µM). Yet, meta (C3’) and para (C4’) diOH-group substitution
on ring B was favoured over either meta (C3’) or para (C4’) substi-
tution regarding the A1 AR (1f: A1Ki (r) = 0.435 µM > 2a: A1Ki (r) =
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
252

0.7921 µM > 2b: A1Ki (r) = 2.897 µM). The almost exact opposite
is true regarding the A2A AR, as meta (C3’) OH-group substitution
was favoured over meta (C3’) and para (C4’) disubstitution (2a:
A2AKi (r) = 0.4345 µM > 1f: A2AKi (r) = 0.903 µM > 2b: A2A1Ki (r) =
1.051 µM).
ilar A1Ki values (for example 1h (A1Ki (r) = 0.041 µM) and 1j (A1Ki
(r) = 0.042 µM)). Now, meta monosubstitution (3b) is favoured
over meta and para disubstitution (3b) (3b: A1Ki (r) = 6.398 µM ver-
sus 3c: A1Ki (r) = 12.35 µM). Additionally, the C5 substituted com-
pounds 3e–f did not retain either A1 or A2A AR affinity.
Compounds 2c–h lack A1 and A2A AR affinity and meta (C3’) and/
or para (C4’) OCH3-group substitution on ring B may well be detri-
mental to AR affinity (2c–h: A1 & A2AKi (r) >100 µM versus 2a–b, 1f
& 1h–j). This was, however, specific to 2-benzylidene-1-indanone
derivatives, as the first in class selective A2A AR antagonist istrade-
fylline (1a) also possessed meta (C3’) and para (C4’) diOCH3-group
substitution on the phenyl ring.
Comparison of 3b–c to 3e–f, respectively, again demonstrates
that the C4 position is preferred to the C5 position on ring A [23].
(For selective A1 AR affinity: C4 morpholine substitution > C5 mor-
pholine substitution.)
Fortunately, all was not lost as another observation could be
made. Comparison of compounds 3b–c with 1h and 1j, respective-
ly, highlighted the importance of C4 OCH3-group substitution on
ring A in order to attain dual A1 and A2A AR affinity. Moreover, the
aforementioned catechol group (previously considered to be prob-
lematic) is now in the clear (3c versus 1j); because it may now be
said that meta (C3’) and para (C4’) diOH-group substitution on ring
B is not solely responsible for the nanomolar dual A1 and A2A AR af-
finity possessed by compound 1j.The structurally related 2-ben-
zylidene-1-tetralone derivatives (4a–e, ▶Table 3) were also evalu-
ated in accordance with previous research (▶Fig. 6) [25, 26].
The A1 and A2A ARs did not tolerate C5 OCH3-group substitution
on ring A, as seen with compound 4c (A1Ki (r) >100 µM) as opposed
to 4a (A1Ki (r) = 4.343 µM) and 4e (A1 & A2AKi (r) > 100 µM) as op-
posed to 4b (A1Ki (r) = 6.843 µM; A2AKi (r) = 7.054 µM). A prior
study came to the same conclusion (1o–q: A1 & A2AKi (r) >100 µM).
Therefore, regardless of ring B substitution (either -H (1o), -OH
(1p–q) or -OCH3 (4c–e)), C5 OCH3-group substitution on ring A of
the 2-benzylidene-1-tetralone derivatives is detrimental to A1 and
A2A AR affinity.
Interestingly, the structurally related 2-benzylidene-1-indanone
derivatives generally preferred OCH3- to OH-group substitution on
ring A and OH- to OCH3-group substitution on ring B – contradic-
tory to the 2-benzylidene-1-tetralone derivatives [23, 24, 26].
Comparison of meta (C3’) OCH3-group substituted 4a to meta
(C3’) OH-group substituted 1l showed a more than 2.5-fold de-
crease in A1 AR affinity (4a: A1Ki (r) = 4.343 µM versus 1l: A1Ki (r) =
1.62 µM), whereas A2A AR affinity was completely lost (4a: A1Ki
(r) > 100 µM). Compound 4b possessed dual A1 and A2A AR affinity
below 10 µM, with a SI of approximately 1. Interestingly, the ben-
zylidene tetralones 4a and 4b contrasted with the benzylidene in-
danones 2c and 2e, respectively, revealing that (unlike the ben-
zylidene indanones 2c–h) meta (C3’) and para (C4’) OCH3-group
substitution on ring B of the 2-benzylidene-1-tetralone scaffold is
not disadvantageous to A1 and A2A AR affinity.
Interestingly, para (C4’) OH- or OCH3- group substitution on
phenyl ring B appears to diminish both A1 and A2A AR affinity, e. g.
compounds 2d and 1i - except for 2b. Although 2b has A1 and A2A
AR affinity, these affinities are much lower than 2b’s meta (C3’) and/
or para (C4’) substituted counterparts that retain C4 OH-group
substitution on ring A (2a & 1f). The para substituted compounds
possess lower affinity than the meta and/or para counterparts.
2-Benzylidene-1-indanone derivatives holding meta (C3’) and/
or para (C4’) OCH3-group substitution on ring B were specifically
designed to point out the importance of hydroxyl group substitu-
tion, e. g. compounds 2c, 2e, 2f & 2h versus 2a, 1f, 1h & 1j. A pre-
vious study identified compounds 1h and 1j as potent A1 and A2A
AR antagonists, but questions were raised due to the presence of
a catechol group in compound 1j. A catechol group, a known reac-
tive functional group, is considered a liability, although widespread
in literature as a potential starting point to further explore SARs.
The absence of a catechol group, unfortunately, also led to the ab-
sence of A1 and A2A AR affinity; 2e: A1 & A2AKi (r) > 100 µM versus
1f: A1Ki (r) = 0.435 µM; A2AKi (r) = 0.903 µM and 2h: A1 & A2AKi
(r) > 100 µM versus 1j: A1Ki (r) = 0.042 µM; A2AKi (r) = 0.078 µM.
Further analysis points out that affinity is not only due to hydroxyl
group substitution, as insinuated, but that the methoxy group at
position C4 on ring A is pivotal to A1 and A2A AR affinity. Therefore,
it may be said that the A1 and A2A ARs desire either C4 OH- or OCH3-
group substitution on ring A in combination with either meta (C3’)
or meta (C3’) and para (C4’) OH-group substitution on phenyl ring
B (as exemplified with compounds 2a–b, 1f, 1g, 1h & 1j). The pres-
ence of a OCH3-group on phenyl ring B, no matter the substitution
pattern on ring A, led to a loss of both A1 and A2A AR affinity (as ex-
emplified with compounds 2c–h).
Furthermore, compounds 3b–c and 3e–f were designed (see
▶Table 2) – the morpholine analogues of the 2-benzylidene-1-in-
danone derivatives 1h and 1i, respectively. Inclusion of the mor-
pholine moiety was inspired by JNJ-40255293 (1c), as it gave opti-
mal in vitro as well as in vivo activity and had an ED50 value of < 0.1
mg/kg in the mouse catalepsy model [42].
The parent scaffolds of the present series 3a and 3d (unsubsti-
tuted at position C2 of the 1-indanone core) lack both A1 and A2A
AR affinity. The radioligand binding assay results of compounds
3b–c and 3e–f were also disappointing; compounds 3b–c showed
more than 155- and 290-fold decrease in A1 AR affinity, respective-
ly, and a complete loss of A2A AR affinity upon comparison to the
C4 OCH3-group substituted compounds 1h and 1j. Before, meta
(C3’) and/or para (C4’) OH-group substitution on ring B gave sim-
The influence of ring C on A1 and A2A AR affinity is inconclusive.
Comparison of benzylidene tetralone 4a to benzylidene indanone
2c, as well as 4b–2e, 4c–2f, 4d–2g and 4e–2h, respectively, led to
mixed results. On the one hand, compounds 4a and 4b (6-mem-
bered ring C) possess A1 and/or A2A AR affinity and compounds 2c
and 2e (5-membered ring C) are without A1 and/or A2A AR affinity,
leading to the assumption that a 6-membered ring C (tetralone) is
favoured over a 5-membered ring C (indanone). On the other hand,
neither 4c–e (tetralone) nor 2f–h (indanone) possess A1 and/or A2A
AR affinity. Moreover, the 2-benzylidene-1-tetralone derivative 1p
had only micromolar affinity whereas its counterpart, the 2-ben-
zylidene-1-indanone derivative 1h, had nanomolar affinity.
Janse van Rensburg HD et al. A1/A2A Affinity of Chalcone Analogues … Drug Res 2020; 70: 243–256
253

Thieme
Original Article
The type of binding affinity that test compounds 2a, 3b and 4a
exhibited at the rat A1 AR was determined via a GTP shift assay, as
described previously [31, 33, 34]. GTP shifts were calculated by di-
viding the Ki values of compounds reported in the presence of GTP
by the Ki values obtained in the absence of GTP and the results are
summarized in ▶Table 4. Test compounds 2a, 3b and 4a were se-
lected as they possess the highest A1 AR affinity in the three respec-
tive series (▶Table 1, ▶2, ▶3, ▶4). The results suggest that com-
pounds 2a, 3b and 4a act as A1 AR antagonists, as the binding
curves in the presence of GTP are almost unaffected and the calcu-
lated GTP shifts are approximately 1 (▶Table 4 and ▶Fig. 7)
[43, 44]. On account of the structural similarity of the test com-
pounds (2a–h, 3a–f & 4a–e), it may be supposed that these 2-ben-
zylidene-1-indanone and –tetralone derivatives are all A1 AR an-
tagonists.
C4 on ring A of the 2-benzylidene-1-indanone derivative 1j also
contributed to A1 and A2A AR affinity – and not only the catechol
functional group on ring B. In the future the concerns raised about
the α,β-unsaturated ketone system (perceived as a potential Mi-
chael acceptor) present in the 2-benzylidene-1-indanone and -te-
tralone scaffold may be eased by converting the lead-like com-
pounds (2a–b) into either isoxazole or pyrazole derivatives (thus
eliminating the said reactive functional group, while retaining the
necessary substituents on ring A and phenyl ring B).
Acknowledgements
The authors wish 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 Ex-
cellence for Pharmaceutical Sciences (Pharmacen), NWU.
The A1 and A2A AR radioligand binding assays, as well as GTP shift
assays were validated with CPA (A1 agonist), DPCPX (A1 antagonist)
and istradefylline (1a) (A2A antagonist) as reference compounds
and results are in accordance with literature values. These results,
expressed as Ki values (µM), are summarized in ▶Table 5.
Funding
This work is based on the research supported in part by the Nation-
al Research Foundation (NRF) of South Africa (grant number
111814) and the North-West University (NWU).
In silico evaluation
The physical-chemical properties (▶Table 6), as well as drug-like-
ness and medicinal chemistry friendliness (▶Table 7) of com-
pounds 2a–b, 3b–c and 4a–b (which showed A1 and/or A2A AR af-
finity) indicate that compounds 2a–b, 3b–c and 4a–b are drug-like
according to rule-based filters [45–49], despite their low A1 and/or
A2A AR affinity. However, only compounds 2a–b are lead-like (i. e.
a molecular entity suitable for optimization), as compounds 3b–c
and 4a–b do not satisfy the criteria set out (3b–c: MW > 350 & 4a–
b: XLOGP3 > 3.5) [50]. A lead compound is subject to chemical
modifications that will, in all likelihood, increase size (MW) and li-
pophilicity (XLOGP3); therefore, a lead-like compound is required
to be smaller and less hydrophobic than a drug-like compound [51].
Conflict of Interest
The authors have no conflict of interest to declare.
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