Evaluation of 2-benzylidene-1-tetralone derivatives as antagonists of A1 and A2A adenosine receptors

Article abstract of DOI:10.1111/cbdd.13074

Antagonists of the adenosine receptors (A₁ and A_2A) are thought to be beneficial in neurological disorders, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore 2-benzylidene-1-tetralone derivatives as antagonists of A₁ and/or A_2A adenosine receptors. In general, the test compounds were found to be selective for the A₁ adenosine receptor, with only three test compounds possessing affinity for both the A₁ and A_2A adenosine receptor. The 2-benzylidene-1-tetralones bearing a hydroxyl substituent at either position C5, C6 or C7 of ring A displayed favourable adenosine A₁ receptor binding, while C5 hydroxy substitution led to favourable A_2A adenosine receptor affinity. Interestingly, para-hydroxy substitution on ring B in combination with ring A bearing a hydroxy at position C6 or C7 provided the 2-benzylidene-1-tetralones with both A₁ and A_2A adenosine receptor affinity. Compounds 4 and 8 displayed the highest A₁ and A_2A adenosine receptor affinity with values below 7?μm. Both these compounds behaved as A₁ adenosine receptor antagonists in the performed GTP shift assays. In conclusion, the 2-benzylidene-1-tetralone derivatives can be considered as lead compounds to design a new class of dual acting adenosine A₁/A_2A receptor antagonists that may have potential in treating both dementia and locomotor deficits in Parkinson's disease.

Full text of DOI:10.1111/cbdd.13074

PROFESSOR LESETJA J LEGOABE (Orcid ID : 0000-0003-2440-4993)
Article type : Research Article
Evaluation of 2-benzylidene-1-tetralone derivatives as antagonists of A1 and
A2A adenosine receptors
Lesetja J. Legoabe1,*, Mietha M. Van der Walt1 and Gisella Terre'Blanche1,2
1. Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001,
Potchefstroom 2520, South Africa
2. Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001,
Potchefstroom 2520, South Africa
Antagonists of the adenosine receptors (A1 and A2A) are thought to be beneficial in neurological
disorders, such as Alzheimer’s and Parkinson’s disease. The aim of this study was to explore 2-
benzylidene-1-tetralone derivatives as antagonists of A1 and/or A2A adenosine receptors. In general,
the test compounds were found to be selective for the A1 adenosine receptor, with only three test
compounds possessing affinity for both the A1 and A2A adenosine receptor. The 2-benzylidene-1-
tetralones bearing a hydroxyl substituent at either position C5, C6 or C7 of ring A displayed favorable
adenosine A1 receptor binding, while C5 hydroxy substitution led to favorable A2A adenosine
receptor affinity. Interestingly, para-hydroxy substitution on ring B in combination with ring A
bearing a hydroxy at position C6 or C7 provided the 2-benzylidene-1-tetralones with both A1 and A2A
adenosine receptor affinity. Compounds 4 and 8 displayed the highest A1 and A2A adenosine receptor
affinity with values below 7µM. Both these compounds behaved as A1 adenosine receptor
antagonists in the performed GTP shift assays. In conclusion, the 2-benzylidene-1-tetralone
derivatives can be considered as lead compounds to design a new class of dual acting adenosine
A1/A2A receptor antagonists that may have potential in treating both dementia and locomotor
deficits in Parkinson’s disease.
KEYWORDS
2-Benzylidene-1-tetralone derivatives, A1 adenosine receptor antagonists, A2A adenosine receptor
antagonists
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13074
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Correspondence
Lesetja J Legoabe, Centre of Excellence for Pharmaceutical Sciences, North-West University, Private
Bag X6001, Potchefstroom 2520, South Africa, Tel +27 18 299 2182, Fax +27 18 299 4243, email
lesetja.legoabe@nwu.ac.za.
Parkinson’s disease (PD) is a common but complex neurodegenerative disease characterized by a
loss of dopaminergic neurons in the substantia nigra pars compacta[1] and abnormal aggregates of
α-synuclein protein called Lewy bodies and Lewy neurites.[2] The decrease of dopamine in the basal
ganglia leads to classical motor symptoms associated with PD, which include bradykinesia, rigidity,
resting tremor and postural instability.[3] Although these four cardinal signs generally define PD, it is
important not to dismiss the non-motor symptoms associated with PD.[4] Non-motor symptoms
range from dribbling saliva, constipation, depression, sleeping disorders, apathy, hallucinations, and
dementia.[5]
Current treatment of PD is symptomatic and consists of drugs that restore dopamine concentrations
and/or effects.[6] L-3,4-dihydroxyphenylalanine (L-dopa), dopamine’s immediate precursor, is
considered the most effective drug for the treatment of the motor symptoms of PD.[7] Though L-
dopa provides the greatest symptomatic relief, its adverse effects include motor fluctuations
(“wearing-off phenomenon” and “on-off phenomenon”), non-motor fluctuations, dyskinesia, and
drug-induced psychosis.[8] Other drugs used for the treatment of PD are dopamine agonists,
catechol-O-methyltransferase inhibitors, monoamine oxidase inhibitors, amantadine, and anti-
cholinergic drugs.[9] These drugs are focused on either replacing the concentrations and/or effects of
dopamine in the brain and only address motor symptoms and not non-motor symptoms.[10]
Therefore an ideal drug would address motor symptoms and non-motor symptoms, be disease
modifying, and neuroprotective. Adenosine receptor (AR) antagonists may address the
aforementioned problems.[11]
Adenosine is a neuro- and homeostatic modulator with wide ranging effects throughout the human
body.[12, 13] Currently four AR subtypes, namely A1, A2A, A2B and A3, are known[14] and chemical
compounds acting on them such as adenosine analogues and conjugates exhibited therapeutic
[15, 16]
potential.
The A1 ARs are highly expressed throughout the brain, while A2A AR expression is
limited to the striatum, nucleus accumbens and the olfactory tubercle.[17] The A1 and A2A ARs have
been identified as drug targets for the treatment of PD and other neurological conditions.[18, 19]
Adenosine plays a role opposite to dopamine in the brain.[20] Agonists and antagonists of ARs
produce behavioural effects similar to antagonists and agonists of dopamine receptors,
respectively.[11] Therefore, the antagonism of A2A ARs in striatopallidal neurons reduces postsynaptic
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effects of dopamine depletion, and sequentially reduces motor symptoms of PD.[21] Furthermore, A2A
AR antagonists might protect dopaminergic neurons from deterioration and thus be
neuroprotective.[22] The combination of A2A AR antagonists and L-dopa could reduce the risk of
developing dyskinesia associated with long term L-dopa treatment.[23] In addition cognitive
impairment associated with PD may potentially improve through A1 AR antagonism.[19] Evidence for
the improvement of cognitive impairment associated with PD through the antagonism of the A1 AR
came from a study where a dual A1/A2A receptor antagonist, ASP-5854 (Figure 1), reversed
scopolamine-induced memory deficits in rats, whereas a specific A2A AR antagonist, KW-6002 (Figure
1) did not.[19]
It is plausible that a dual-target A1/A2A AR antagonist may have significance in the treatment of PD as
it reduces motor symptoms, is neuroprotective through the antagonism of A2A ARs, and improves
cognitive impairment through the antagonism of A1 ARs.[19] Furthermore a dual-target A1/A2A AR
antagonist may have a synergistic motor activating effect; antagonism of the A1 AR facilitates
presynaptic dopamine release and antagonism of the A2A AR facilitates postsynaptic dopamine
release.[24] For example, the motor activating effect of caffeine (Figure 1) may be due to the
synergistic activity of antagonising both the A1 and A2A ARs.[25]
Affinity for both the A1 and A2A ARs were found for selected aurone derivatives.[26] An aurone (2-
benzylidene-1-benzofuran-3-one) is a heterocyclic chemical compound (fused 6- and 5-membered
rings) and a type of flavonoid.[27] Hispidol , an aurone derivative, was found to be a selective A1 AR
antagonist with Ki values of 0.352 µM and 52.7 µM for the A1 and A2A ARs, respectively.[26]
Maritimetin, another aurone derivative, was found to possess affinity for both the A1 (Ki=3.47 µM)
and A2A (Ki=9.35 µM) ARs.[26] It seems that single hydroxy substitution (hispidol) in ring A (position 6)
and ring B (para position) is well tolerated for both A1 and A2A AR affinity. In addition, di-substitution
of hydroxy on ring A (position 6 and 7) and ring B (para and meta position) lead to a 10-fold decrease
in A1 AR affinity but surprisingly a 5-fold increase in A2A AR affinity (hispidol vs maritimetin) was
obtained. However, in the case of Auresidin, where the di-hydroxy-substitution on ring A is in
position 4 and 6 and on ring B in the para and meta (3’ and 4’) position, it is found that A1 AR affinity
(Ki=13.2 µM) is retained, but A2A AR affinity is diminished (Ki > 100 µM ). Sulfuretin bearing a single
hydroxy substitutent on ring A (position 6) and di-hydroxy-substitution on ring B (3’ and 4’) retained
both A1 (Ki=4.44 µM) and A2A (Ki=28.1 µM) AR affinity, thereby making the assumption that hydroxy
substitution at position 4 of ring A is detrimental for A2A AR affinity (Figure 2).
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In addition, the isoflavones (heterocyclic fused 6- and 6-membered rings) were also evaluated for
their affinity at A1 ARs.[26] Overall, hydroxy substitution at position 5 and 7 of ring A and para position
of ring B (4’) were well tolerated for A1 AR affinity. Methoxy substitution in the para (4’) position of
ring B (Biochanin) slightly decreased A1 AR affinity, while methoxy substitution at position 7
(Prunetin) of ring A diminished A1 AR affinity (Figure 2).
2-Benzylidene-1-tetralones are bicyclic benzofused ring systems and are structurally related to
aurones and isoflavones (Figure 2). In addition, 2-benzylidene-1-tetralone derivatives are also known
for their antitumor,[28] antifungal[29] and anti-microbial properties.[30] The 2-benzylidene-1-tetralone
has not previously been evaluated as a possible scaffold for developing AR antagonists. Based on the
aforementioned literature findings that the aurone derivatives possess affinity for the A1 and A2A
ARs, as well as the observation that isoflavones exert A1 AR binding, we conducted a pilot study to
investigate the potential of several structurally related 2-benzylidene-1-tetralones to possess affinity
for the A1 and A2A ARs. Thus, in order to find new insights into the structural requirements for AR
binding by 2-benzylidene-1-tetralone based analogues, various modifications to both ring A and B
was explored.
1. METHODS AND MATERIALS
1.1 Materials and general procedures
Unless otherwise noted, all starting materials for the synthesis were obtained from Sigma-Aldrich
and were used without further purification. Proton (1H) and carbon (13C) NMR spectra were recorded
on a Bruker Avance III 600 spectrometer at frequencies of 600 MHz and 151 MHz, respectively. NMR
measurements were carried out in CDCl3 or DMSO-d6 as NMR solvent. All chemical shifts are
reported in parts per million (∂) downfield from the signal of Si(CH3)4. Spin multiplicities are given as
s (singlet), d (doublet), dd (doublet of doublets), t (triplet), and m (multiplet). The melting points
(mp) were determined with a Buchi B-545 melting point apparatus and are uncorrected. Thin layer
chromatography (TLC) was carried out using silica gel 60 (Merck). High resolution mass spectra
(HRMS) were recorded on a Bruker micrOTOF-Q II mass spectrometer in atmospheric-pressure
chemical ionisation (APCI) mode. The affinities of 2-benzylidene-1-tetralones for rat adenosine A1
and A2A receptor subtypes were determined with radioligand competition experiments as described
previously.[31] All commercially available reagents were obtained from various manufacturers
(Sigma–Aldrich, Ascent Scientific and Merck). The radioligands [3H]NECA (specific activity 25
Ci/mmol) and [3H]DPCPX (specific activity 120 Ci/mmol) were obtained from Amersham Biosciences
and PerkinElmer, respectively. Filter-count was purchased from PerkinElmer, while Whatman GF/B
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25 mm diameter filters was obtained from Merck. Counting of radio activities were performed using
a Packard Tri-CARB 2810 TR liquid scintillation counter.
1.2 General method for preparation of 2-benzylidene-3,4-dihydronaphthalen-1(2H)-one
analogues (1–22)
The target compounds were prepared by employing an acid-catalysed Claisen-Schmidt condensation
method.[32] This chemical transformation involves the nucleophilic addition of a ketone enolate to an
aldehyde to form a β‐hydroxyaldehyde or β‐hydroxyketone, before dehydration to give a conjugated
[33]
enone.
Briefly, the appropriate substituted tetralone derivatives (2 mmol) were reacted with
appropriate benzaldehydes (2 mmol) in a mixture of methanol (15 mL) and hydrochloric acid (32%;
22.5 mL) and were reflux for 1–6 h. The reaction progress was monitored using silica gel TLC. Upon
completion of the reaction, ice-cold water (20 mL) was added and the resulting precipitate was
collected by filtration, dried and purified by recrystallization from a suitable solvent (mainly ethanol)
(Scheme 1).
All the final products in this study possessed E-configuration. Although in principle chalcones can
exist as E- or Z-isomers; previous studies found that the E-isomer is the most thermodynamically
stable form.[34] It was further demonstrated that the Z-configuration is highly unfavourable due to
strong steric interaction between the aryl and carbonyl groups. [35]
1.2.1 (2E)-2-benzylidene-3,4-dihydronaphthalen-1(2H)-one (1)
The title compound is a product of 3,4-dihydronaphthalen-1(2H)-one and benzaldehyde in a yield of
o
67.4%; clear yellow crystals; mp: 106.0–107.3 C (ethanol). 1H NMR (600 MHz, Chloroform-d) δ 8.12
(d, J = 7.7 Hz, 1H), 7.86 (s, 1H), 7.50 – 7.31 (m, 7H), 7.23 (d, J = 7.2 Hz, 1H), 3.12 (t, J = 6.4 Hz, 2H),
13
2.93 (t, J = 6.5 Hz, 2H); C NMR (151 MHz, CDCl3) δ 187.88, 143.20, 136.63, 135.80, 135.42, 133.43,
133.25, 129.85, 128.51, 128.41, 128.19, 128.15, 126.99, 28.85, 27.16. APCI-HRMS m/z: calculated for
C17H14O: 234.292 found: 235.1121 [M+1]+.
1.2.2 (2E)-2-benzylidene-7-hydroxy-3,4-dihydronaphthalen-1(2H)-one (2)
The title compound is a product of 7-hydroxy-3,4-dihydronaphthalen-1(2H)-one and
benzaldehyde in a yield of 71.5%: mp 151.2–151.9 oC (ethanol). 1H NMR (600 MHz, CDCl3) δ
7.90 – 7.84 (m, 2H), 7.41 (dt, J = 15.2, 7.4 Hz, 5H), 7.35 (d, J = 7.0 Hz, 1H), 7.16 – 7.06 (m,
2H), 3.09 (dd, J = 9.1, 3.7 Hz, 2H), 2.86 (t, J = 6.4 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ 188.82,
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155.40, 137.55, 135.71, 135.67, 135.36, 134.03, 129.94, 129.67, 128.68, 128.42, 121.82,
113.99, 27.96, 27.42; APCI-HRMS m/z: calculated for C17H14O2: 250.29186, found: 251.1078
[M+1]+.
1.2.3 (2E)-2-benzylidene-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (3)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and benzaldehyde in
o
1
a yield of 76 %; purple crystal. mp 199.8–200.8 C (ethanol). H NMR(DMSO-d6, 600 MHz): δ (ppm)
10.44 (s, 1H), 7.85 (d, J=8.6 Hz, 1H), 7.62 (s, 1H), 7.48 (d, J=7.4 Hz, 2H), 7.44 (t, J=7.6 Hz, 2H), 7.37 (t,
J=7.3 Hz, 1H), 6.77 (dd, J=8.6, 2.3 Hz, 1H), 6.67 (s, 1H), 3.01 (t, J=5.8 Hz, 2H), 2.82 (t, J=6.4 Hz, 2H); 13C
NMR (151 MHz, DMSO) δ 185.25, 162.29, 146.06, 135.88, 135.47, 134.48, 130.30, 129.77, 128.54,
128.51, 125.08, 114.77, 113.94, 28.24, 26.75; APCI-HRMS m/z: calculated for C17H14O2: 250.292,
found: 251.1047 [M+1]+.
1.2.4 (2E)-2-benzylidene-5-hydroxy-3,4-dihydronaphthalen-1(2H)-one (4)
The title compound is a product of 5-hydroxy-3,4-dihydronaphthalen-1(2H)-one and
benzaldehyde in a yield of 88%: mp 174.2–175.3 oC (ethanol). 1H NMR(DMSO-d6, 600 MHz):
δ (ppm) 9.85 (s, 1H), 7.65 (s, 1H), 7.51 (d, J=7.4 Hz, 2H), 7.45 (t, J=7.3 Hz, 3H), 7.38 (t, J=7.3
Hz, 1H), 7.20 (t, J=7.9 Hz, 1H), 7.07 (d, J=8.0 Hz, 1H), 3.03 (t, J=5.8 Hz, 2H), 2.81 (t, J=6.5 Hz,
13
2H); C NMR (151 MHz, DMSO) δ 187.07, 154.35, 135.61, 135.30, 135.19, 134.02, 130.11,
129.88, 128.68, 128.58, 127.06, 119.27, 117.97, 26.10, 21.13; APCI-HRMS m/z: calculated
for C17H14O2: 250.29186, found: 251.1049 [M+1]+
1.2.5 (2E)-2-benzylidene-7-methoxy-3,4-dihydronaphthalen-1(2H)-one (5)
The title compound is a product of 7-methoxy-3,4-dihydronaphthalen-1(2H)-one and benzaldehyde
o
1
in a yield of 51.2%; pale yellow crystals; mp: 121.5–122.7 C (dichloromethane). H NMR (600 MHz,
CDCl3) δ 7.85 (s, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.41 (dt, J = 15.2, 7.4 Hz, 4H), 7.33 (t, J = 7.0 Hz, 1H),
7.15 (d, J = 8.3 Hz, 1H), 7.06 (dd, J = 8.3, 2.8 Hz, 1H), 3.85 (s, 3H), 3.09 (td, J = 6.7, 1.6 Hz, 2H), 2.90 –
13
2.84 (m, 2H); C NMR (151 MHz, CDCl3) δ 187.80, 158.62, 136.68, 135.92, 135.85, 135.43, 134.23,
129.84, 129.42, 128.49, 128.40, 121.50, 110.26, 55.52, 28.02, 27.34; APCI-HRMS m/z: calculated for
C18H16O2: 264.318, found: 265.1212 [M+1]+.
1.2.6 (2E)-2-benzylidene-6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one (6)
The title compound is a product of 6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one and
o
benzaldehyde in a yield of 91%: mp 135.1–135.8 C. 1H NMR(DMSO-d6, 600 MHz): δ (ppm) 7.64 (s,
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1H), 7.47 (dd, J=34.3, 8.3 Hz, 5H), 7.38 (t, J=7.3 Hz, 1H), 6.92 (s, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.04 (t,
13
J=5.8 Hz, 2H), 2.86 (t, J=6.4 Hz, 2H); C NMR (151 MHz, DMSO) δ 185.33, 153.47, 147.86, 138.35,
135.55, 135.39, 134.65, 129.76, 128.52, 125.63, 110.66, 108.95, 55.79, 55.46, 27.63, 26.95; APCI-
HRMS m/z: calculated for C19H18O3: 294.34442, found: 295.1352 [M+1]+.
1.2.7 (2E)-6-amino-2-benzylidene-3,4-dihydronaphthalen-1(2H)-one (7)
The title compound is a product of 6-amino-3,4-dihydronaphthalen-1(2H)-one and benzaldehyde in a
o
1
yield of 51.3%; brown shinny crystals; mp: 230.0–233.6 C (ethanol). H NMR (600 MHz, DMSO) δ
7.81 (d, J = 8.5 Hz, 1H), 7.60 (s, 1H), 7.50 – 7.39 (m, 4H), 7.36 (d, J = 7.2 Hz, 1H), 6.80 (dd, J = 8.5, 1.7
13
Hz, 1H), 6.67 (s, 1H), 4.92 (s, br, 3H), 2.99 (t, J = 5.8 Hz, 2H), 2.79 (t, J = 6.4 Hz, 2H); C NMR (151
MHz, DMSO) δ 184.81, 148.33, 145.49, 136.10, 135.60, 134.24, 129.92, 129.80, 128.59, 128.50,
124.98, 115.69, 114.58, 28.32, 26.74; APCI-HRMS m/z: calculated for C17H15NO: 249.3071, found:
250.1226 [M+1]+.
1.2.8 (2E)-7-hydroxy-2-(4-hydroxybenzylidene)-3,4-dihydronaphthalen-1(2H)-on (8)
The title compound is a product of 7-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
o
hydroxybenzaldehyde in a yield of 39.2%; brown crystals, mp: 249.5–249.7 C (ethyl acetate).1H
NMR (600 MHz, DMSO) δ 9.92 (s, 1H), 9.60 (s, 1H), 7.61 (s, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.31 (d, J =
2.6 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 6.96 (dd, J = 8.2, 2.7 Hz, 1H), 6.84 (d, J = 8.6 Hz, 2H), 3.02 (t, J =
13
5.9 Hz, 2H), 2.79 (t, J = 6.4 Hz, 2H); C NMR (151 MHz, DMSO) δ 186.61, 158.35, 156.17, 136.17,
133.96, 133.82, 132.49, 132.08, 129.57, 126.16, 121.04, 115.51, 112.58, 27.96, 27.01; APCI-HRMS
m/z: calculated for C17H14O3: 266.291, found: 267.1027 [M+1]+.
1.2.9 (2E)-6-hydroxy-2-(4’-hydroxybenzylidene)-3,4-dihydronaphthalen-1(2H)-one (9)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
hydroxybenzaldehyde in a yield of 72.3%: mp 279.4–280.9 oC (ethanol).1H NMR(DMSO-d6, 600 MHz):
δ (ppm) 10.37 (s, 1H), 9.89 (s, 1H), 7.82 (d, J=8.6 Hz, 1H), 7.56 (s, 1H), 7.36 (d, J=8.5 Hz, 2H), 6.83 (d,
J=8.6 Hz, 2H), 6.75 (dd, J=8.6, 2.3 Hz, 1H), 6.65 (s, 1H), 3.01 (t, J=6.0 Hz, 2H), 2.81 (t, J=6.5 Hz, 2H);13C
NMR (151 MHz, DMSO) δ 185.25, 162.05, 158.13, 145.78, 135.15, 132.81, 131.90, 130.16, 126.33,
125.34, 115.49, 114.63, 113.87, 28.20, 26.79; APCI-HRMS m/z: calculated for C17H14O3: 266.29126,
found: 267.0988 [M+1]+.
1.2.10 (2E)-6-hydroxy-2-(3’-methoxybenzylidene)-3,4-dihydronaphthalen-1(2H)-one (10)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 3-
o
methoxybenzaldehyde in a yield of 90%: mp 287.5–289.5 C (ethanol).1H NMR(DMSO-d6, 600 MHz):
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δ (ppm) 10.45 (s, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.59 (s, 1H), 7.35 (t, J=7.9 Hz, 1H), 7.07 – 7.00 (m, 2H),
6.94 (dd, J=8.2, 2.2 Hz, 1H), 6.77 (dd, J=8.6, 2.3 Hz, 1H), 6.66 (d, J=2.1 Hz, 1H), 3.77 (s, 3H), 3.01 (t,
13
J=5.8 Hz, 2H), 2.82 (t, J=6.4 Hz, 2H); C NMR (151 MHz, DMSO) δ 185.27, 162.32, 159.26, 146.13,
136.85, 136.15, 134.47, 130.33, 129.62, 125.09, 121.98, 115.13, 114.80, 114.25, 113.97, 55.17,
28.26, 26.85; APCI-HRMS m/z: calculated for C18H16O3: 280.31784, found: 281.1147 [M+1]+.
1.2.11 (2E)-2-(3’,4’-dichlorobenzylidene)-7-hydroxy-3,4-dihydronaphthalen-1(2H)-one (11)
The title compound is a product of 7-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 3,4-
dichlorobenzaldehyde in a yield of 83%: mp 183.3–184.1 oC (ethanol).1H NMR(DMSO-d6, 600 MHz): δ
(ppm) 9.69 (s, 1H), 7.76 (s, 1H), 7.68 (d, J=8.3 Hz, 1H), 7.59 (s, 1H), 7.48 (d, J=8.3 Hz, 1H), 7.33 (d,
J=2.6 Hz, 1H), 7.18 (d, J=8.3 Hz, 1H), 6.99 (dd, J=8.2, 2.7 Hz, 1H), 2.98 (t, J=5.8 Hz, 2H), 2.81 (t, J=6.4
Hz, 2H); 13C NMR (151 MHz, DMSO) δ 186.48, 156.28, 137.42, 136.11, 134.23, 133.47, 132.79,
131.52, 131.34, 131.11, 130.66, 129.88, 129.83, 121.67, 112.58, 26.98, 26.95; APCI-HRMS m/z:
calculated for C17H12Cl2O2: 319.18198, found: 319.0296 [M]+
1.2.12 (2E)-2-(3’,4’-dichlorobenzylidene)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (12)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 3,4-
o
1
dichlorobenzaldehyde in a yield of 88%: mp 214.2–215.5 C (ethanol). H NMR(DMSO-d6, 600 MHz):
δ (ppm) 10.49 (s, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.74 (s, 1H), 7.68 (d, J=8.3 Hz, 1H), 7.55 (s, 1H), 7.47 (d,
J=8.3 Hz, 1H), 6.77 (dd, J=8.6, 2.2 Hz, 1H), 6.66 (s, 1H), 2.97 (t, J=5.8 Hz, 2H), 2.83 (t, J=6.4 Hz, 2H),
13
2.49; C NMR (151 MHz, DMSO) δ 184.90, 162.48, 146.17, 137.72, 136.31, 131.85, 131.43, 131.30,
130.90, 130.63, 130.40, 129.83, 124.89, 114.90, 114.00, 28.09, 26.70; APCI-HRMS m/z: calculated for
C17H12Cl2O2: 319.18198, found: 319.0306 [M]+.
1.2.13 (2E)-2-(3’,4’-dichlorobenzylidene)-5-hydroxy-3,4-dihydronaphthalen-1(2H)-one (13)
The title compound is a product of 5-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 3,4-
o
1
dichlorobenzaldehyde in a yield of 78%: mp 398.6–398.7 C. H NMR(DMSO-d6, 600 MHz): δ (ppm)
9.89 (s, 1H), 7.77 (s, 1H), 7.69 (d, J=8.3 Hz, 1H), 7.57 (s, 1H), 7.49 (d, J=8.3 Hz, 1H), 7.44 (d, J=7.2 Hz,
1H), 7.20 (t, J=7.9 Hz, 1H), 7.07 (d, J=7.9 Hz, 1H), 2.99 (t, J=5.9 Hz, 2H), 2.81 (t, J=6.4 Hz, 2H);13C NMR
(151 MHz, DMSO) δ 186.82, 154.41, 137.43, 136.12, 133.81, 132.53, 131.54, 131.35, 131.09, 130.68,
130.20, 129.89, 127.16, 119.46, 118.02, 26.06, 21.06; APCI-HRMS m/z: calculated for C17H12Cl2O2:
319.18198, found: 319.0301 [M]+
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1.2.14 (2E)-2-(4’-fluorobenzylidene)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (14)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
o
fluorobenzaldehyde in a yield of 38.1%: mp 186.3–186.6 C (ethanol).1H NMR(DMSO-d6, 600 MHz):
δ (ppm) 10.46 (s, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.60 (s, 1H), 7.54 (dd, J=8.4, 5.7 Hz, 2H), 7.27 (t, J=8.8
Hz, 2H), 6.77 (dd, J=8.6, 2.3 Hz, 1H), 6.66 (d, J=2.1 Hz, 1H), 2.99 (t, J=5.9 Hz, 2H), 2.82 (t, J=6.5 Hz,
2H).13C NMR (151 MHz, DMSO) δ 185.19, 162.75, 162.33, 161.11, 146.06, 135.75, 133.45, 132.10,
132.04, 131.98, 131.96, 130.33, 125.07, 115.62, 115.48, 114.81, 113.96, 28.18, 26.67; APCI-HRMS
m/z: calculated for C17H13FO2: 268.2823232, found: 269.0954 [M+1]+.
1.2.15 (2E)-2-(4’-chlorobenzylidene)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (15)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
o
chlorobenzaldehyde in yield of 52.6 %; mp: 209.1–210.2 C .1H NMR(DMSO-d6, 600 MHz): δ (ppm)
10.46 (s, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.59 (s, 1H), 7.54 – 7.47 (m, 4H), 6.66 (d, J=2.1 Hz, 1H), 2.98 (t,
J=6.4 Hz, 2H), 2.82 (t, J=6.5 Hz, 2H);13C NMR (151 MHz, DMSO) δ 185.05, 162.37, 146.07, 136.54,
134.35, 133.12, 133.06, 131.56, 130.33, 128.56, 124.97, 114.81, 113.95, 28.14, 26.70; APCI-HRMS
m/z: calculated for C17H13ClO2: 284.73692, found: 285.0694 [M+1]+.
1.2.16 (2E)-2-(4’-bromobenzylidene)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (16)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
bromobenzaldehyde in yield of 71.6 %: mp 202.0–203.4 oC (ethanol).1H NMR(DMSO-d6, 600
MHz): δ (ppm) 10.47 (s, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.62 (d, J=8.4 Hz, 2H), 7.56 (s, 1H), 7.43
(d, J=8.4 Hz, 2H), 6.79 – 6.74 (m, 1H), 6.66 (d, J=2.1 Hz, 1H), 2.97 (t, J=5.8 Hz, 2H), 2.82 (t,
J=6.4 Hz, 2H);13C NMR (151 MHz, DMSO) δ 185.10, 162.39, 146.11, 136.62, 134.72, 133.21,
131.82, 131.51, 130.36, 125.00, 121.81, 114.84, 113.98, 28.16, 26.73; APCI-HRMS m/z:
calculated for C17H13BrO2: 329.18792, found: 329.0176 [M]+.
1.2.17 3’-[(E)-(6-hydroxy-1-oxo-3,4-dihydronaphthalen-2(1H)-ylidene)methyl]benzonitrile (17)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 3-
formylbenzonitrile in yield of 85 %: mp 51.3–51.4 oC (ethanol). 1H NMR (600 MHz, DMSO) δ 10.49 (s,
1H), 7.95 (s, 1H), 7.88 – 7.77 (m, 3H), 7.63 (dd, J = 19.0, 11.2 Hz, 2H), 6.77 (dd, J = 8.6, 2.2 Hz, 1H),
6.67 (d, J = 2.0 Hz, 1H), 2.99 (t, J = 5.9 Hz, 2H), 2.84 (t, J = 6.4 Hz, 2H).;13C NMR (151 MHz, DMSO) δ
184.95, 162.51, 146.21, 137.86, 136.78, 134.31, 133.02, 132.18, 131.86, 130.41, 129.75, 124.87,
118.60, 114.90, 114.00, 111.76, 28.10, 26.64; APCI-HRMS m/z: calculated for C18H13NO2: 275.30132,
found: 276.0992 [M+1]+.
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1.2.18 4’-[(E)-(6-hydroxy-1-oxo-3,4-dihydronaphthalen-2(1H)-ylidene)methyl]benzonitrile (18)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
o
formylbenzonitrile in yield of 56.0 %: mp 231.4–233.3 C (ethanol).1H NMR (600 MHz, DMSO) δ
10.50 (s, 5H), 7.87 (dd, J = 19.8, 8.4 Hz, 14H), 7.69 – 7.58 (m, 15H), 6.77 (dd, J = 8.6, 2.3 Hz, 5H), 6.67
13
(d, J = 2.1 Hz, 5H), 2.98 (t, J = 5.8 Hz, 11H), 2.83 (t, J = 6.4 Hz, 11H); C NMR (151 MHz, DMSO) δ
184.93, 162.55, 146.23, 140.41, 138.51, 132.54, 132.35, 130.48, 130.46, 124.87, 118.77, 114.94,
114.02, 110.61, 28.11, 26.77; APCI-HRMS m/z: calculated for C18H13NO2: 275.30132, found: 276.1025
[M+1]+.
1.2.19 (2E)-6-hydroxy-2-(3’-nitrobenzylidene)-3,4-dihydronaphthalen-1(2H)-one (19)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 3-
nitrobenzaldehyde in yield of 69.4 %: mp 51.3–51.4 oC (ethanol). 1H NMR (600 MHz, DMSO) δ 10.47
(s, 1H), 8.26 (d, J = 8.7 Hz, 4H), 7.86 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.6 Hz, 4H), 7.66 (s, 2H), 6.78 (dd, J
= 8.6, 2.3 Hz, 2H), 6.67 (d, J = 2.1 Hz, 2H), 3.00 (t, J = 5.7 Hz, 4H), 2.85 (t, J = 6.4 Hz, 4H);13C NMR (151
MHz, DMSO) δ 184.87, 162.62, 146.74, 146.27, 142.44, 139.08, 132.05, 130.87, 130.49, 124.82,
123.60, 114.98, 114.04, 28.10, 26.82; APCI-HRMS m/z: calculated for C17H13NO4: 295.28942, found:
296.0900 [M+1]+.
1.2.20 (2E)-6-hydroxy-2-(4’-nitrobenzylidene)-3,4-dihydronaphthalen-1(2H)-one (20)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
nitrobenzaldehyde in yield of 55.7 %: mp 51.5–51.6 oC (ethanol). 1H NMR (600 MHz, DMSO) δ 10.50
(s, 1H), 8.26 (s, 1H), 8.20 (dd, J = 8.2, 1.4 Hz, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.72
(t, J = 8.0 Hz, 1H), 7.67 (s, 1H), 6.77 (dd, J = 8.6, 2.3 Hz, 1H), 6.67 (d, J = 2.1 Hz, 1H), 3.01 (t, J = 5.8 Hz,
13
2H), 2.84 (t, J = 6.4 Hz, 2H); C NMR (151 MHz, DMSO) δ 184.91, 162.54, 147.92, 146.18, 138.21,
137.20, 136.08, 132.00, 130.43, 130.07, 124.86, 123.98, 122.97, 114.93, 114.02, 28.11, 26.65; APCI-
HRMS m/z: calculated for C17H13NO4: 295.28942, found: 296.0908 [M+1]+.
1.2.21 (2E)-2-[4’-(dimethylamino)benzylidene]-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (21)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and 4-
o
1
(dimethylamino)benzaldehyde in yield of 62.8 %: mp 219.5–240.7 C (ethanol). H NMR (600 MHz,
DMSO) δ 7.81 (d, J = 8.5 Hz, 1H), 7.58 (s, 1H), 7.44 (d, J = 8.6 Hz, 2H), 6.96 (s, 2H), 6.76 (dd, J = 8.5,
2.3 Hz, 1H), 6.66 (d, J = 2.1 Hz, 1H), 3.80 (s, 1H), 3.02 (t, J = 6.1 Hz, 2H), 2.99 (s, 6H), 2.80 (t, J = 6.5 Hz,
13
2H); C NMR (151 MHz, DMSO) δ 185.10, 162.03, 145.66, 134.78, 131.65, 130.09, 125.41, 114.62,
113.85, 40.74, 28.18, 26.86; APCI-HRMS m/z: calculated for C19H19NO2: 293.35966, found: 294.1480
[M+1]+.
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1.2.22 (2E)-2-[(5'-chlorothiophen-2’-yl)methylidene]-6-hydroxy-3,4-dihydronaphthalen-1(2H
)-one (22)
The title compound is a product of 6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and
5-
chlorothiophene-2-carbaldehyde in yield of 64.0 %: mp 234.2–235.5 oC (ethanol). 1H NMR (600 MHz,
DMSO) δ 10.46 (s, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.72 (s, 1H), 7.45 (d, J = 4.0 Hz, 1H), 7.24 (d, J = 4.0 Hz,
1H), 6.76 (dd, J = 8.6, 2.3 Hz, 1H), 6.68 (d, J = 2.1 Hz, 1H), 2.99 (dd, J = 9.4, 3.8 Hz, 2H), 2.90 (t, J = 6.5
Hz, 2H); 13C NMR (151 MHz, DMSO) δ 184.31, 162.29, 145.85, 137.56, 133.36, 132.68, 131.95,
130.28, 127.71, 127.00, 125.08, 114.81, 113.96, 27.47, 26.72; APCI-HRMS m/z: calculated for
C15H11ClO2S: 290.76464, found: 291.0266 [M+1]+.
1.3 Radioligand binding assays
The A1 and A2A AR binding affinity of the test compounds (1–22) were determined with radioligand
competition experiments described previously.[31] The A1 AR binding affinity was performed with rat
whole brain membranes in the presence of the radioligand [3H]-8-cylcopentyl-1,3-dipropylxanthine
([3H]DPCPX).[31, 36] In turn, the A2A AR binding affinity was measured at rat striatal membranes with 5’-
37]
N-[3H]-ethylcarboxamideadenosine ([3H]NECA) as radioligand.[31,
In addition, N6-
cyclopentyladenosine (CPA) was added to the A2A AR binding studies to minimize the binding of the
radioligand [3H]NECA to A1 ARs.[31, 37] Nonspecific binding was defined by the addition of 100 μM
CPA.[31] The adenosine A1 agonist CPA and adenosine A2A antagonist ZM-241385 was chosen to
validate the radioligand binding assays of the current study.
1.4 Functional characterization of compounds 4 and 8
In order to determine if compounds 4 and 8 function as agonists or as antagonists, the competition
curves of the latter compounds were evaluated via a GTP shift assay reported previously. [29] The
GTP shift assays were carried out with rat whole brain membranes in the absence and presence of
[31]
0.1 mM GTP. Nonspecific binding was defined by the addition of 10 μM DPCPX (unlabelled).
The
adenosine A1 agonist CPA and adenosine A1 antagonist DPCPX was chosen as reference compounds
of the current study.
1.5 Data analysis
The competition curves were obtained by using the Prism software package (GraphPad Software
Inc.). The results of the competition experiments were expressed as dissociation constant values (Ki)
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of triplicate determinations and are expressed as mean ± standard error of mean (SEM). The Ki
values were calculated by using the Kd values of 0.36 nM for [3H]DPCPX at rat whole brain
membranes,[31, 36] while 15.3 nM for [3H]NECA at rat striata membrane[31, 37] were used. Furthermore,
GTP shifts are calculated by dividing the Ki value of a compound reported in the presence of GTP by
the Ki value obtained in the absence of GTP.[38] A compound with a calculated GTP shift of
approximately 1 is considered an antagonist; in turn the presence of GTP affects the competition
curves of an agonist and shifts the curve to the right.[31, 39]
2. RESULTS
2.1 Radioligand binding assays
The results of the radioligand binding experiments for the reference (CPA and ZM-241385) and test
compounds (4 to 8) are summarized in Table 1. Furthermore, the experimental results of CPA and
ZM-241385 were found in accordance with literature values (Table 1).
2.2 Functional characterization of compounds 4 and 8
The affinities of the reference (CPA and DPCPX) and test compounds (4 and 8) were determined in
the absence and presence of 0.1 mM GTP and are reported with the calculated GTP shifts in Table 2.
The calculated GTP shift results for CPA and DPCPX (Table 2) was found to correspond with literature
values, where CPA act as an agonist and DPCPX as an antagonist (Figure 3).
3. DISCUSSION
3.1 Radioligand binding assays – A1 and A2A AR affinity
Structural modifications on ring A: First of all, in analogy with the aforementioned aurones, the
impact of hydroxy substitution at either the C5-, C6- or C7-position of ring A was evaluated by
comparing the dissociation constant (Ki) values of these compounds (2–4) to their unsubstituted
counterpart compound 1 (A1Ki = 22.78 µM). Similarly to the aurones, the position of the hydroxy
group on ring A was found to be important to modulate both the A1 and A2A AR affinity. For instance,
the hydroxy substitution at C7 (2; A1Ki = 11.75 µM) increased the A1 AR binding affinity
approximately two-fold compared to compound 1. In turn, hydroxy substitution at C6 (3; A1Ki = 25.42
µM) resulted in a similar Ki value as compound 1. Of note, compound 4 (C5-OH) exhibited the second
highest A1 AR affinity (A1Ki = 5.93 µM) and highest A2A AR binding (A2AKi = 2.90 µM) among
compounds 1, 2 and 3. Thus, substitution with a hydroxy group at position C5 of ring A may
modulate the AR affinity of 2-benzylidene-1-tetralones to such an extent that both A1 and A2A AR
affinity are obtained. Thus, A1 AR affinity may be governed by hydroxy substitution at position C5, C6
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and C7 of ring A, with the order of increased binding affinity documented as follow: 4 (C5-OH) > 2
(C7-OH) > 3 (C6-OH). Furthermore, in agreement with the abovementioned literature findings of the
isoflavone derivatives, the investigated 2-benzylidene-1-tetralones seems to favour hydroxy
substitution of ring A over substitution with a methoxy group. For example, compound 2 (C7-OH)
exhibited a four-fold enhanced affinity for A1 ARs compared to compound 5 that bear a methoxy
group at C7 of ring A. In addition, compound 6 was synthesized containing a di-methoxy substitution
at position C6 and C7 of ring A. However, this structural modification resulted in a diminished affinity
for the A1 AR. Compound 7 (C6-NH2) exhibited an approximate three-fold reduced A1 affinity
compared to the unsubstituted compound 1 and C6-hydroxy substituted compound 3.
Consequently, optimization of ring A, of the 2-benzylidene-1-tetralones, towards A1 AR affinity
seems to favour hydroxy substitution (at C5, C6 or C7) over methoxy or amino substitution. With the
exception of compound 4, the structural modifications to ring A (without changes to ring B) did not
result in A2A AR affinity (at a maximum tested concentration of 100 µM) for compounds 1 to 7.
Structural modifications on ring B: Notably, C4’-hydroxy substitution of ring B led to an increase of
the A1 AR binding and significantly yielded gained A2A AR affinity. For example, both the C6- and C7-
hydroxy substituted derivatives (8 and 9) exerted binding affinity for the A1 and A2A ARs. Compound
8 possessed Ki values of 5.39 µM and 6.60 µM for the A1 and A2A ARs, respectively. Furthermore,
compound 8 displayed an approximate three-fold higher affinity towards the A1 and A2A ARs
compared to compound 9 (A1Ki = 16.15 µM; A2AKi = 19.14 µM). Interestingly, both of the latter
compounds (8 and 9) exhibited a non-selective activity towards the A1 and A2A ARs. Similar to the
above findings, the compound bearing C7-hydroxy substitution (8) is preferred over the compound
possessing a C6-hydroxy substituent (9). Introduction of a 3’-methoxy group (compound 3 vs
compound 10) did not increase A1 AR affinity but A1 AR activity was retained (10 A1Ki = 28.59 µM vs 3
A1Ki = 25.42 µM ). Furthermore, affinity at the A2A AR subtype remained elusive. The influence of
halogen substitution on ring B was also investigated. Di-substitution with chlorine at positions 3’ and
4’ of ring B led to compounds 11 (A1Ki = 37.10 µM), 12 (A1Ki = 39.29 µM) and 13 (A1Ki = 23.29 µM)
displaying A1 AR affinity. Although a decrease in A1 AR affinity was observed compared to their
corresponding counterparts compounds 2 (C7-OH), 3 (C6-OH) and 4 (C5-OH), compound 13,
possessing a C5-hydroxy substituent on ring A, exhibited the highest A1 AR affinity among
compounds 11 (C7-OH), 12 (C6-OH) and 13 (C5-OH), thus showing the same trend as their
corresponding homologues (e.g. 13 > 11 > 12 vs 4 > 2 > 3) where compound 4 possess a C5-hydroxy
substitute on ring A. Furthermore, compounds 11, 12 and 13 were deemed inactive towards the A2A
AR. As part of the pilot study, various substitutions on ring B of compound 3 (C6-OH) was
additionally explored to gain further insight into compound 3’s structural requirements. For
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instance, the presence of C4’-fluorine (14; A1Ki = 24.85 µM) substitution on ring B displayed a similar
binding affinity towards the A1 AR for compound 14 compared to compound 3 (unsubstituted ring
B), while C4’-bromine (16; A1Ki = 34.57 µM) substitution on ring B resulted in a slight decrease in A1
AR affinity in comparison with compound 3. Both of these compounds (14 and 16) showed no
affinity towards the A2A AR subtype (A2AKi > 100 µM). Surprisingly C4’-chlorine substitution (15) on
ring B resulted in a loss of A1 AR affinity in comparison to compounds 3, 14 and 16. Further
substitution of compound 3 with electron withdrawing groups (CN, NO2) at either position C3’ or C4’
(17–20) of ring B did not govern A1 or A2A AR binding. By replacing the chlorine substituted phenyl
ring of compound 15 with a chlorine substituted thiophene ring (22), A1 AR affinity (A1Ki = 42.87 µM)
was gained as compound 15 displayed no affinity towards the A1 ARs. Compound 22 also showed no
affinity towards the A2A ARs (A2AKi > 100 µM). It could be assumed that by replacing phenyl ring B
with another ring system (e.g. thiophene) that there would be no significant impact on the A1 AR
activity and future research with other ring systems are necessary to gain more insight. In general,
the test compounds (11–16 and 22) possessing halogen substitution on ring B exerted a more
favourable effect on the A1 AR, over the A2A AR subtype, with the only exception being compound 15
(C4’-Cl) that exhibited no affinity towards either one of the AR subtypes. Overall, both A1 and A2A AR
binding affinity is favoured by ring B bearing a C4’-hydroxy group (8 and 9), whereas other
substituents on ring B led to a decreased or loss in A1 AR affinity with A2A AR binding remaining
elusive.
3.2 Functional characterization of compounds 4 and 8
In order to determine if the test compounds (1–22) act as agonists or antagonists, GTP shift
experiments were carried out. For this purpose compounds 4 and 8 were chosen as they exhibited
the highest A1 AR binding affinity among the investigated compounds. Generally, a rightward shift of
the binding curve in the presence of GTP (due to an uncoupling of the A1 AR from its Gi protein) is
expected for an A1 AR agonist.[31, 39] In the case of an A1 AR antagonist no significant shift is
[31, 39]
anticipated in the presence of GTP.
The results suggest that compounds 4 and 8 act as A1 AR
antagonists as no significant rightward shift of the binding curves were observed in the presence of
GTP (Figure 3).
4. CONCLUSION
In conclusion, the pilot study indicated the importance of structural modifications at both rings A
and B of the 2-benzylidene-1-tetralone scaffold for gaining and even losing A1 and/or A2A AR affinity.
Overall, the test compounds (1–22) were found to be selective for the A1 AR, with only three test
compounds (4, 8 and 9) possessing affinity for the A2A AR subtype. In general, substitution with a
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hydroxy group on ring A and/or B play a key role in modulating the binding affinity and selectivity at
A1 and A2A ARs. Specifically, C5-hydroxy substitution on ring A was preferred over the C6- and C7-
positions (i.e. 4 vs 2&3; 13 vs 11&12) for A1 affinity, while C4’-hydroxy substitution (8 and 9) on ring
B favored both A1 and A2A AR affinity. Compounds 4 and 8 are two promising drug candidates that
both act as A1 AR antagonists in vitro with Ki values below 7 µM. Compound 8 was found to possess
the highest A1 AR affinity (A1Ki = 5.39 µM) and the second highest A2A AR affinity (A2AKi = 6.60 µM)
among the test compounds (1–22) and acted non-selectively. Furthermore, compound 4 was
reported with affinity for both the A1 (A1Ki =5.93 µM) and A2A (A2AKi =2.90 µM) ARs and displayed
selectivity towards the A2A AR. Therefore, compounds 4 and 8 are ideal drug candidates for future in
vivo investigation of the 2-benzylidene-1-tetralone class of compounds as adenosine receptor
antagonists in neurodegenerative disorders.
ACKNOWLEDGEMENTS
We are grateful to Dr. J. Jordaan and Mr. A. Joubert of the SASOL Centre for Chemistry, North-West
University, for recording the NMR and MS spectra, respectively. We are also thankful to Prof. J. du
Preez for HPLC analysis. Financial support for this work was provided by the North-West University,
the National Research Foundation (Grant No: 96135) and the Medical Research Council, South Africa.
CONFLICT OF INTEREST
The authors declared that they have no competing financial interest.
Figure legends section
FIGURE 1 The structures of caffeine, KW-6002 and ASP-5854.
FIGURE 2 The structures of various aurones and isoflavones previously documented to possess affinity for the
adenosine receptor and the general structure of the target 2-benzylidene-1-tetralone scaffold being explored
in the current study.
SCHEME 1
General synthetic route for preparation of target substituted (2E)-2-benzylidene-3,4-
dihydronaphthalen-1(2H)-one derivatives
FIGURE 3 The binding curves of CPA (reference compound) and compounds 4 and 8, indicating their A1 AR
agonist/antagonistic action as determined via GTP shift assays (with and without 0.1 mM GTP) in rat whole
brain membranes expressing A1 ARs with [3H]DPCPX as radioligand. (A) GTP shift of 1 calculated for the A1 AR
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antagonist compound 4, (B) GTP shift of 1 calculated for the A1 AR antagonist compound 8 and (C) GTP shift of
6 calculated for the A1 AR agonist CPA.
TABLE 1 The dissociation constant values (Ki values) for the binding of the 2-benzylidene-1-
tetralones to rat adenosine A1 and A2A receptors.
TABLE 2 The adenosine A1 affinities (in the absence and presence of GTP) and GTP shifts of selected
test compounds (4 and 8) and reference compounds.
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O
O
O
Aryl R2
MeOH, HCl
R1
+
R1
Aryl R2
SCHEME 1
General synthetic route for preparation of target substituted (2E)-2-benzylidene-3,4-
dihydronaphthalen-1(2H)-one derivatives
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TABLE 1 The dissociation constant values (Ki values) for the binding of the 2-benzylidene-1-
tetralones to rat adenosine A1 and A2A receptors.
O
O
S
B
3'
4'
7
Cl
A
A
5
B
6
6
HO
compounds: 22
compounds: 1-21
Ring A
Ring B
Ki ± SEM (µM)a
SIc
b
b
A1 vs
A2A vs
(A2A/A1)
Compd
7
6
5
3’
4’
[3H]DPCPX
[3H]NECA
Structural modifications of ring A
1
2
3
4
5
6
7
-H
-OH
-H
-H
-H
-OH
-H
-H
-H
-H
-OH
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
22.78 ± 1.28
11.75 ± 0.615
25.42 ± 0.94
5.93 ± 0.45
48.16 ± 5.18
> 100
> 100
> 100
> 100
2.90 ± 0.66
> 100
-
-
-
0.5
-
-
-
-H
-OCH3 -H
-OCH3 -OCH3
-H
-H
-H
> 100
> 100
-NH2
65.99 ± 4.85
Structural modifications of ring B
8
9
-OH
-H
-H
-OH
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-OH
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-OH
-OH
5.39 ± 0.29
16.15 ± 1.23
28.59 ± 2.59
37.10 ± 3.60
39.29 ± 2.26
23.29 ± 0.74
24.85± 1.65
> 100
34.57 ± 4.52
>100
>100
>100
>100
6.60 ± 0.75
19.14 ± 7.00
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-OH
-OH
-H
-OH
-H
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
10
11
12
13
14
15
16
17
18
19
20
21
22
- OCH3 -H
-Cl
-Cl
-Cl
-H
-H
-H
-CN
-H
-NO2
-H
-H
-
-Cl
-Cl
-Cl
-F
-Cl
-Br
-H
-CN
-H
-NO2
-N(CH3)2 >100
42.87 ± 4.22
-
Reference compounds
CPA (A1 agonist)
0.010 ± 0.002
-
-
-
(0.015)d
-
ZM-241385 (A2A antagonist)
0.002 ± 0.001
(0.002)e
a All Ki values determined in triplicate and expressed as mean ± SEM.
b Rat receptors were used (A1: rat whole brain membranes; A2A: rat striatal membranes).
c Selectivity index (SI) for the A1 receptor isoform calculated as the ratio of Ki (A2A)/Ki (A1).
d Literature value obtained from reference.[31]
e Literature value obtained from reference.[40]
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TABLE 2 The adenosine A1 affinities (in the absence and presence of GTP) and GTP shifts of selected
test compounds (4 and 8) and reference compounds.
O
3'
7
A
5
B
6
4'
Ring A
6
Ring B
Ki ± SEMa
A1 vs
A1 + GTPb vs
GTP
Compd
7
5
3’
4’
[3H]DPCPX
[3H]DPCPX
Shiftc
4
8
-H
-OH
-H
-H
-OH
-H
-H
-H
-H
-OH
5.93 ± 0.45 µM
5.39 ± 0.29 µM
6.92 ± 0.81 µM
6.91 ± 1.19 µM
1
1
Reference compounds
CPA (A1 agonist)
10.9 ± 2.34 nM
0.5 ± 0.10 nM
62.17 ± 3.2 nM
0.5 ± 0.05 nM
6
(6.48)d
1
DPCPX (A2A antagonist)
(1)d
a All Ki values determined in triplicate and expressed as mean ± SEM.
b GTP shift assay, where the 0.1 mM GTP was added to the A1 AR radioligand binding assay.
c GTP shifts calculated by dividing the Ki in the presence of GTP by the Ki in the absence of GTP.
d Literature values obtained from reference.[31]
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.