2–Benzylidene–1–Indanone Analogues as Dual Adenosine A₁/A_2a Receptor Antagonists for the Potential Treatment of Neurological Conditions

Abstract

Previous studies explored 2-benzylidine-1-tetralone derivatives as innovative adenosine A₁ and A_2A receptor antagonists for alternative non-dopaminergic treatment of Parkinson’s disease. This study’s aim is to investigate structurally related 2-benzylidene-1-indanones with substitutions on ring A and B as novel, potent and selective adenosine A₁ and A_2A receptor blockers. 2-Benzylidene-1-indanone derivatives were synthesised via acid catalysed aldol condensation reactions and evaluated via radioligand binding assays to ascertain structure activity relationships to govern A₁ and A_2A AR affinity. The results indicated that hydroxy substitution at C4 of ring A and meta (3’), or para (4’) substitution on ring B of the 2-benzylidene-1-indanone scaffold (2c) is preferred over substitution at C5 (2d) or C6 (2e) of ring A for adenosine A₁ receptor activity and selectivity in the micromolar range. Furthermore, substitution at the meta (3’) position of ring B with chlorine lead to the highly potent and selective adenosine A_2A receptor antagonist, compound 2 h. Compound 2c and the 2q behaved as adenosine A₁ receptor antagonists in the performed GTP shift assays. In view of these findings, compounds 2c, 2 h, 2q and 2p are potent and selective adenosine A₁ and A_2A receptor antagonists for the potential treatment of neurological conditions.

Key words

parkinson’s disease - alzheimer’s disease - 4–hydroxy–2–benzylidene–1–indanones - adenosine antagonists

Introduction

Existing treatment for Parkinson’s disease (PD) is controversial; on the one hand the gold standard of PD treatment L-3,4-dihydroxyphenylalanine (Levo-dopa/L-dopa) effectively relieves motor symptoms, yet, on the other hand, its adverse effects comprise of motor and non-motor complications [1]. Additionally, it does not address non-motor symptoms or neurodegeneration [2]. Justly, a novel drug that addresses all said problems is needed, seeing as non-dopaminergic treatment may possibly improve PD patients’ quality of life.

Adenosine receptor (AR) antagonists may be the solution to the PD–conundrum; as an epidemiological study has established an association between the consumption of coffee or caffeine and a reduced risk of developing PD. Caffeine is a xanthine derivative and non–selective A₁ and A_2A AR antagonist [3]. An A₁ AR antagonist could alleviate cognitive deficits in PD, a non-motor symptom of the disease [4]. This is substantiated by the A₁ AR’s distribution and expression in the prefrontal cortex and hippocampus [5], which are areas linked to cognition [6]. Moreover, blockade of A₁ AR’s increase acetylcholine — a neurotransmitter associated with learning and memory [7]. Another non–motor symptom of PD, namely depression, may be addressed by A_2A AR antagonists; evidenced by a decrease in immobility time during the forced swim test and tail suspension test in rodents when KW-6002 (selective A_2A AR antagonist) was administered [8] [9]. A_2A AR antagonists are also relevant to motor control — which is affected in PD [1]. Firstly, these receptors are abundantly expressed in the striatum [5], a brain area associated with motor control [6]. Secondly, adenosine A_2A and dopamine D₂ receptors are co-expressed on the striatopallidal (inhibitory) pathway, inhibition of A_2A AR’s compensate for hypolocomotion as a result of dwindling stimulation of striatopallidal dopamine D₂ receptors by dopamine [1]. Adverse effects of L–dopa (most effective drug for treatment of motor symptoms), such as dyskinesias, may be lessened by concomitant administration of an A_2A AR antagonist to L-dopa [10]. Blockade of both A₁ and A_2A AR’s synergistically improve motor control by increasing presynaptic dopamine release via A₁ AR inhibition and postsynaptic dopamine response via A_2A AR inhibition [11]. Additionally, blockade of the A_2A AR may possibly facilitate neuroprotection in neurodegenerative disorders, like PD [12].

Recently, 2-benzylidene-1-tetralone analogues were explored as A₁ and/or A_2A AR antagonists [13] [14]. The 2-benzylidene-1-tetralone derivative (1a), possessed both A₁ and A_2A AR affinity (A₁ K _i=5.93 μM; and A_2A K _i+2.90 μM) with a selectivity index of 2 towards the A_2A AR. Compound 1a has a basic benzylidene tetralone backbone (fused 6- and 6-membered rings, namely ring A and ring C), where ring C bears a C2-phenyl substituted side-chain (ring B). It was found that C5–hydroxy substitution on ring A is optimal for A₁ and A_2A AR binding and that the 2-benzylidene side-chain, ring B, also governs AR binding. Similar to these bicyclic benzofused ring systems are the heterocyclic aurones — fused 6- and 5-membered rings — which also possess A₁ and/or A_2A AR affinity. Hispidol (1b) and maritimetin (1c) are examples of aurones with AR affinity [15].

In analogy to the structure of compound 1a and the aurones (1b-c) ([Fig. 1]), the present study investigates the structurally related 2-benzylidene-1-indanone scaffold as potent A₁ and A_2A AR antagonists.

Fig. 1 Structural and heterocyclic ring changes to compound 1a, hispidol and maritimetin to determine features essential for dual A1/A2A antagonistic activity.

The 2-benzylidene-1-indanone scaffold will be modified to include changes to ring A and ring B ([Fig. 1]) and, subsequently, evaluated to ascertain which structure activity relationships govern A₁ and A_2A AR affinity.

Materials and Methods

Chemistry

Unless otherwise noted, all starting materials and solvents were procured from Sigma–Aldrich and used without further purification. Proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 600 spectrometer at frequencies of 600 MHz and 151 MHz, respectively, with deuterated dimethylsulfoxide (DMSO–d6) as solvent. Chemical shifts are reported in parts per million (δ) in relation to the signal of tetramethylsilane (Si(CH₃)₄). High resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF–Q II mass spectrometer in atmospheric pressure chemical ionisation (APCI) mode. High performance liquid chromatography (HPLC) analyses were determined on an Agilent 1100 HPLC system. Melting points (mp) were measured with a Buchi B545 melting point apparatus and are uncorrected.

Fig. 2 Synthesis of 2a, starting material for 2d and 2b–q.Reagents and conditions: a) AlCl3, NaCl, 120–150°C, 3,4-dihydrocoumarin, 200°C (1 h 30 min), ice, HCl, rt (2 h); b) AlCl3, toluene, 120°C (1 h); c) MeOH, HCl (32%), 120°C (24 h).

Fig. 3 A broad overview of ring a and b substitutions on 2-benzylidene-1-indanone core’s influence on A1 and A2A AR affinity.

Synthesis of 4-hydroxy-2,3-dihydro-1H–inden-1-one (2 a)

AlCl₃ (148 mmol) and NaCl (86 mmol) were mixed and mechanically stirred at 120–150°C. At 150°C, 3,4-dihydrocoumarin (27 mmol) was slowly added to the AlCl₃ and NaCl mixture. Subsequently, the temperature was raised to 200°C and the reaction mixture mechanically stirred under reflux for 1h30 min. Ice (103 g) and HCl (32%; 53 mL) were added to the reaction mixture and mechanically stirred at room temperature for 2 h. The resulting grey solid was washed with water, filtered and dried to yield 2a as a grey powder, used without further purification: Yield 85%; mp 231.4–232.5 °C; ¹H NMR (600 MHz, DMSO) δ 10.00 (s, 1 H), 7.23 (t, J=7.6 Hz, 1 H), 7.08 (d, J=7.4 Hz, 1 H), 7.05 (d, J=7.3 Hz, 1 H), 2.92 (t, J=5.7 Hz, 2 H), 2.59 (t, J=5.7 Hz, 2 H); ¹³C NMR (151 MHz, DMSO) δ 206.68, 155.17, 141.90, 138.43, 128.66, 119.82, 113.34, 35.81, 22.33. APCI–HRMS m/z calculated for C₉H₉O₂ (MH⁺): 149.0597, found: 149.0597. Purity (HPLC): 97.2%.

General procedure for synthesis of 2-benzylidene-1-indanone analogues (2b–q)

Firstly, 4-hydroxy-2,3-dihydro-1H-inden-1-one (2a) (2.025 mmol), 5-hydroxy-2,3-dihydro-1H-inden-1-one (2.025 mmol) or 6-hydroxy-2,3-dihydro-1H-inden-1-one (2.025 mmol) was added to an empty flat bottomed flask and, secondly, the appropriate benzaldehyde (2.025 mmol) (as stated in b–q), thereafter MeOH (4 mL) was added to the contents of the flat bottomed flask, followed by HCl (32%; 190.9 mmol, 6 mL). The subsequent suspension was mechanically stirred at 120 °C under reflux for 24 h. Thereafter, the reaction mixture was cooled to room temperature, ice (20 g) was added and the resulting precipitate was filtered, dried and recrystallized from a suitable solvent to yield compounds 2b–q.

(E)-2-benzylidene-4-hydroxy-2,3-dihydro-1H-inden-1-one (2 b)

The title compound (light brown powder) was prepared in a yield of 24% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and benzaldehyde: mp 327.2–327.5 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.11 (s, 1 H), 7.79 (d, J=7.5 Hz, 2 H), 7.52 (dd, J=9.7, 5.1 Hz, 3 H), 7.46 (t, J=7.3 Hz, 1 H), 7.31 (t, J=7.6 Hz, 1 H), 7.25 (d, J=7.4 Hz, 1 H), 7.10 (d, J=7.7 Hz, 1 H), 3.94 (d, J=1.0 Hz, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.60, 154.83, 138.87, 136.37, 135.11, 134.93, 132.80, 130.75, 129.81, 129.08, 129.04, 120.44, 114.17, 29.08. APCI–HRMS m/z calculated for C₁₆H₁₃O₂ (MH⁺): 237.0910, found: 237.0909. Purity (HPLC): 100%.

(E)-2-(3,4-dihydroxybenzylidene)-4-hydroxy-2,3-dihydro-2,3-dihydro-1H-inden-1-one (2 c)

The title (green powder) compound was prepared in a yield of 55% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 3,4-dihydroxybenzaldehyde: 25.8–25.9 °C; ¹H NMR (600 MHz, DMSO) δ 10.10 (s, 1 H), 9.67 (s, 1 H), 9.33 (s, 1 H), 7.36 (s, 1 H), 7.33–7.24 (m, 2 H), 7.22 (d, J=7.4 Hz, 1 H), 7.14–7.05 (m, 2 H), 6.86 (d, J=8.2 Hz, 1 H), 3.83 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.47, 154.75, 148.05, 145.65, 139.35, 136.03, 133.76, 131.34, 128.94, 126.46, 124.49, 119.98, 117.24, 116.06, 113.97, 29.17. APCI–HRMS m/z calculated for C₁₆H₁₃O₄ (MH⁺): 269.0808, found 269.0810. Purity (HPLC): 98.7%.

(E)-2-(3,4-dihydroxybenzylidene)-5-hydroxy-2,3-dihydro-1H-inden-1-one (2 d)

The title compound (beige powder) was prepared in a yield of 48% from 5-hydroxy-2,3-dihydro-1H-inden-1-one and 3,4-dihydroxybenzaldehyde: 280.8–287.2°C; ¹H NMR (600 MHz, DMSO) δ 10.55 (s, 1 H), 9.59 (s, 1 H), 9.22 (s, 1 H), 7.61 (d, J=8.3 Hz, 1 H), 7.25 (t, J=1.7 Hz, 1 H), 7.18 (d, J=2.0 Hz, 1 H), 7.07 (dd, J=8.3, 2.0 Hz, 1 H), 6.96 (d, J=1.8 Hz, 1 H), 6.87–6.82 (m, 2 H), 3.92 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 191.43, 163.55, 152.67, 147.58, 145.55, 132.17, 131.89, 129.64, 126.66, 125.53, 123.76, 117.33, 115.99, 111.92, 39.52, 31.90. APCI–HRMS m/z calculated for C₁₆H₁₃O₄ (MH⁺): 269.0808, found 269.0769. Purity (HPLC): 92%.

(E)-2-(3,4-dihydroxybenzylidene)-6-hydroxy-2,3-dihydro-1H-inden-1-one (2 e)

The title compound (dark brown powder) was prepared in a yield of 11% from 6-hydroxy-2,3-dihydro-1H-inden-1-one and 3,4-dihydroxybenzaldehyde: 26.1–26.2°C; ¹H NMR (600 MHz, DMSO) δ 9.81 (s, 1 H), 9.67 (s, 1 H), 9.27 (s, 1 H), 7.46 (d, J=8.2 Hz, 1 H), 7.33 (s, 1 H), 7.20 (d, J=1.9 Hz, 1 H), 7.14–7.04 (m, 3 H), 6.85 (d, J=8.2 Hz, 1 H), 3.89 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.20, 157.11, 147.97, 145.61, 140.43, 138.92, 133.40, 127.34, 126.50, 124.21, 122.92, 117.44, 116.05, 108.09, 39.52, 31.29. APCI–HRMS m/z calculated for C₁₆H₁₃O₄ (MH⁺): 269.0808, found 268.0730. Purity (HPLC): 96.3%.

(E)-2-(3-fluorobenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (2 f)

The title compound (light brown crystals) was prepared in a yield of 58% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 3-fluorobenzaldehyde: mp 30.0–30.1 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.12 (d, J=1.2 Hz, 1 H), 7.62 (t, J=10.2 Hz, 2 H), 7.59–7.49 (m, 2 H), 7.34–7.23 (m, 3 H), 7.10 (dd, J=7.7, 0.7 Hz, 1 H), 3.95 (d, J=1.4 Hz, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.55 (s), 163.16 (s), 161.54 (s), 154.86 (s), 138.69 (s), 137.34 (d, J=8.0 Hz), 136.44 (d, J=10.0 Hz), 131.40 (d, J=2.4 Hz), 130.95 (d, J=8.4 Hz), 129.17 (s), 126.95 (d, J=2.5 Hz), 120.63 (s), 116.83 (d, J=21.9 Hz), 116.54 (d, J=21.2 Hz), 114.24 (s), 28.96 (s). APCI–HRMS m/z calculated for C₁₆H₁₂FO₂ (MH⁺): 255.0816, found: 255.0816. Purity (HPLC): 100%.

(E)-2-(4-fluorobenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (2 g)

The title compound (light brown crystals) was prepared in a yield of 44% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 4-fluorobenzaldehyde: mp 51.5–51.6 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.12 (s, 1 H), 7.86 (dd, J=8.4, 5.7 Hz, 2 H), 7.52 (s, 1 H), 7.33 (dt, J=15.2, 8.2 Hz, 3 H), 7.25 (d, J=7.4 Hz, 1 H), 7.10 (d, J=7.7 Hz, 1 H), 3.91 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.55, 163.56, 161.91, 154.82, 138.83, 136.33, 134.78 (d, J=2.2 Hz), 133.06 (d, J=8.6 Hz), 131.64, 131.59 (d, J=3.1 Hz), 129.09, 120.42, 116.15, 116.01, 114.15, 28.90. APCI–HRMS m/z calculated for C₁₆H₁₂FO₂ (MH⁺): 255.0816, found: 255.0816. Purity (HPLC): 98.1%.

(E)-2-(3-chlorobenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (2 h)

The title compound (beige powder) was prepared in a yield of 75% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 3-chlorobenzaldehyde: mp 386.7–386.8 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.14 (s, 1 H), 7.85 (s, 1 H), 7.77 (d, J=7.4 Hz, 1 H), 7.58–7.48 (m, 3 H), 7.32 (t, J=7.6 Hz, 1 H), 7.26 (d, J=6.9 Hz, 1 H), 7.11 (dd, J=7.8, 0.8 Hz, 1 H), 3.95 (d, J=1.5 Hz, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.46, 154.82, 138.65, 137.10, 136.60, 136.38, 133.68, 131.15, 130.79, 129.95, 129.41, 129.24, 129.15, 120.63, 114.23, 39.52, 28.91. APCI–HRMS m/z calculated for C₁₆H₁₂ClO₂ (MH⁺): 271.0520, found: 271.0520. Purity (HPLC): 100%.

(E)-2-(4-chlorobenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (2 i)

The title compound (gold crystals) was prepared in a yield of 61% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 4-chlorobenzaldehyde: mp 30.9–31.0 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.14 (d, J=10.6 Hz, 1 H), 7.81 (d, J=8.4 Hz, 2 H), 7.57 (d, J=8.4 Hz, 2 H), 7.50 (s, 1 H), 7.30 (t, J=7.6 Hz, 1 H), 7.24 (d, J=7.4 Hz, 1 H), 7.10 (d, J=7.8 Hz, 1 H), 3.91 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.53, 154.86, 138.77, 136.33, 135.78, 134.42, 133.85, 132.38, 131.42, 129.14, 129.07, 120.53, 114.19, 28.98. APCI–HRMS m/z calculated for C₁₆H₁₂ClO₂ (MH⁺): 271.0520, found: 271.0520. Purity (HPLC): 100%.

(E)-2-(3-bromobenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1–one (2 j)

The title compound (beige powder) was prepared in a yield of 68% from 4-hydroxy-2,3–dihydro-1H-inden-1-one and 3-bromobenzaldehyde: mp 30.8–30.9 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.15 (d, J=4.8 Hz, 1 H), 7.97 (s, 1 H), 7.80 (d, J=7.8 Hz, 1 H), 7.64 (dd, J=7.9, 1.1 Hz, 1 H), 7.53–7.44 (m, 2 H), 7.31 (t, J=7.6 Hz, 1 H), 7.25 (d, J=7.3 Hz, 1 H), 7.13–7.08 (m, 1 H), 3.93 (d, J=1.3 Hz, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.48, 154.86, 138.68, 137.40, 136.59, 136.40, 132.86, 132.33, 131.13, 131.06, 129.60, 129.17, 122.31, 120.66, 114.24, 28.91. APCI–HRMS m/z calculated for C₁₆H₁₂BrO₂ (MH⁺): 315.0015, found: 315.0015. Purity (HPLC): 100%.

(E)-2-(4-bromobenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (2 k)

The title compound (gold crystals) was prepared in a yield of 49% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 4-bromobenzaldehyde: mp 30.9–31.0 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.14 (d, J=13.7 Hz, 1 H), 7.72 (qd, J=8.4, 2.6 Hz, 4 H), 7.49 (d, J=1.8 Hz, 1 H), 7.31 (td, J=7.7, 1.5 Hz, 1 H), 7.25 (d, J=7.5 Hz, 1 H), 7.10 (d, J=7.8 Hz, 1 H), 3.91 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.53, 154.85, 138.76, 136.33, 135.90, 134.17, 132.58, 132.01, 131.51, 129.15, 123.31, 120.54, 114.19, 28.99. APCI–HRMS m/z calculated for C₁₆H₁₂BrO₂ (MH⁺): 315.0015, found: 315.0015. Purity (HPLC): 100%.

(E)-4–hydroxy-2-(4-(trifluoromethyl)benzylidene)-2,3-dihydro-1H-inden-1–one (2 l)

The title compound (green crystals) was prepared in a yield of 39% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 4-(trifluoromethyl)benzaldehyde: mp 317.3–397.4 °C (EtOH); ¹H NMR (600 MHz, DMSO) δ 10.18 (d, J=4.9 Hz, 1 H), 7.99 (d, J=8.1 Hz, 2 H), 7.85 (d, J=8.2 Hz, 2 H), 7.57 (s, 1 H), 7.31 (t, J=7.6 Hz, 1 H), 7.26 (d, J=7.4 Hz, 1 H), 7.11 (d, J=7.7 Hz, 1 H), 3.96 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.50, 154.90, 138.94, 138.61, 137.66, 136.50, 131.19, 130.92, 129.36, 129.22, 129.15, 125.75 (dd, J=7.3, 3.5 Hz), 125.00, 123.20, 120.69, 114.27, 28.96. APCI–HRMS m/z calculated for C₁₇H₁₂F₃O₂ (MH⁺): 305.0784, found: 305.0807. Purity (HPLC): 98.4%.

(E)-4-((4-hydroxy-1-oxo-1H-inden-2(3 H)-ylidene)methyl)benzonitrile (2 m)

The title compound (green crystals) was prepared in a yield of 53% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 4-formylbenzonitrile: mp 304.5–307.4 °C (MeOH); ¹H NMR (600 MHz, DMSO) δ 10.17 (s, 1 H), 7.95 (s, 4 H), 7.54 (d, J=1.8 Hz, 1 H), 7.31 (t, J=7.6 Hz, 1 H), 7.25 (d, J=7.4 Hz, 1 H), 7.11 (d, J=7.6 Hz, 1 H), 3.95 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.40, 154.86, 139.48, 138.51, 138.25, 136.43, 132.69, 131.13, 130.70, 129.21, 120.74, 118.66, 114.26, 111.52, 29.00. APCI–HRMS m/z calculated for C₁₇H₁₂NO₂ (MH⁺): 262.0863, found: 262.0857. Purity (HPLC): 96.6%.

(E)-2((2-aminopyrimidine-5-yl)methylene)-4-hydroxy-2,3-dihydro-1H–inden-1-one (2 n)

The title compound (beige powder) was prepared in a yield of 10% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and 2-aminopyrimidine-5-carbaldehyde: mp 398.5–398.8 °C (MeOH); ¹H NMR (600 MHz, DMSO) δ 10.05 (s, 1 H), 8.67 (s, 2 H), 7.38–7.26 (m, 4 H), 7.22 (d, J=7.3 Hz, 1 H), 7.08 (d, J=7.7 Hz, 1 H), 3.87 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.09, 162.95, 160.38, 154.74, 139.21, 135.83, 131.74, 129.00, 128.27, 120.20, 117.94, 114.07, 39.52, 29.19. APCI–HRMS m/z calculated for C₁₄H₁₂N₃O₂ (MH⁺):254.0924, found: 254.0924. Purity (HPLC): 76%.

(E)-4-hydroxy-2-(pyridin-2-ylmethylene)-2,3-dihydro-1H-inden-1-one (2 o)

The title compound (green powder) was prepared in a yield of 71% from 4-hydroxy-2,3-dihydro–1H-inden-1-one and picolinaldehyde: mp 30.8–30.9 °C (H₂O); ¹H NMR (600 MHz, DMSO) δ 10.28 (s, 1 H), 8.85 (dd, J=4.9, 0.8 Hz, 1 H), 8.16 (t, J=7.4 Hz, 1 H), 8.04 (d, J=7.9 Hz, 1 H), 7.66–7.56 (m, 2 H), 7.35–7.24 (m, 2 H), 7.18–7.13 (m, 1 H), 4.09 (d, J=1.6 Hz, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.64, 154.98, 151.92, 147.81, 139.84, 138.52, 137.21, 129.17, 128.07, 127.49, 124.62, 120.95, 115.73, 114.31, 29.80. APCI–HRMS m/z calculated for C₁₅H₁₂NO₂ (MH⁺): 238.0863, found: 238.0879. Purity (HPLC): 100%.

(E)-4-hydroxy-2-(pyridin-3-ylmethylene)-2,3-dihydro-1H-inden-1-one (2 p)

The title compound (green powder) was prepared in a yield of 50% from 4-hydroxy-2,3-dihydro–1H-inden-1-one and nicotinaldehyde: mp 30.9–31.0 °C (MeOH); ¹H NMR (600 MHz, DMSO) δ 10.32 (s, 1 H), 9.18 (d, J=1.7 Hz, 1 H), 8.83 (dd, J=5.3, 1.2 Hz, 1 H), 8.70 (d, J=8.2 Hz, 1 H), 7.98 (dd, J=8.1, 5.4 Hz, 1 H), 7.63 (t, J=2.0 Hz, 1 H), 7.33 (t, J=7.6 Hz, 1 H), 7.28 (d, J=7.0 Hz, 1 H), 7.17 (dd, J=7.8, 0.8 Hz, 1 H), 4.04 (d, J=1.6 Hz, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.16, 154.94, 146.01, 144.27, 142.73, 139.56, 138.33, 136.52, 133.15, 129.30, 127.14, 126.22, 120.94, 114.34, 28.76. APCI–HRMS m/z calculated for C₁₅H₁₂NO₂ (MH⁺): 238.0863, found: 238.0876. Purity (HPLC): 100%.

(E)-4-hydroxy-2-(pyridin-4-ylmethylene)-2,3-dihydro-1H-inden-1-one (2 q)

The title (green powder) compound was prepared in a yield of 52% from 4-hydroxy-2,3-dihydro-1H-inden-1-one and isonicotinaldehyde: mp 30.9–31.0 °C (MeOH); ¹H NMR (600 MHz, DMSO) δ 10.29 (d, J=0.7 Hz, 1 H), 8.86 (d, J=5.6 Hz, 2 H), 8.07 (d, J=4.6 Hz, 2 H), 7.58 (s, 1 H), 7.38–7.25 (m, 2 H), 7.17 (d, J=7.6 Hz, 1 H), 4.05 (s, 2 H); ¹³C NMR (151 MHz, DMSO) δ 193.20, 154.91, 146.02, 138.50, 138.20, 136.59, 135.16, 129.38, 128.42, 125.79, 121.10, 118.17, 114.41, 28.92. APCI–HRMS m/z calculated for C₁₅H₁₂NO₂ (MH⁺): 238.0863, found: 238.0879. Purity (HPLC): 100%.

Biology

All commercially available reagents were obtained from various manufacturers: radioligands [³H]NECA (specific activity 27.1 Ci/mmol) procured from PerkinElmer and [³H]DPCPX (specific activity 120 Ci/mmol) from Amersham Biosciences, filter-count from PerkinElmer and Whatman GF/B 25 mm diameter filters from Merck. Radio activity was calculated by a Packard Tri-CARB 2810 TR liquid scintillation counter.

Radioligand binding assays

The collection of tissue samples for the A₁ and A_2A AR binding studies were approved by the Research Ethics Committee of the North-West University (application number NWU-0035–10-A5). The rat whole brains (expressing A₁ AR’s) and rat striata (expressing A_2A AR’s ) were prepared according to the protocol described in literature [16] [17].

The competition experiments were carried out in the presence of the radioligands [³H]-8-cylcopentyl-1,3-dipropylxanthine ([³H]DPCPX; 0.1 nM; K_d=0.36 nM) and 5’-N-[³H]-ethylcarboxamideadenosine ([³H]NECA; 4 nM; K_d=15.3 nM) for the A₁ and A_2A AR radioligand binding assays, respectively [16] [17]. In addition, the A_2A AR binding studies were determined in the presence of N⁶-cyclopentyladenosine (CPA) to minimize the binding of [³H]NECA to A₁ AR’s. Non-specific binding was defined by the addition of a final concentration of 100 μM CPA. The sigmoidal-dose response curves, via Graphpad Software Inc. package, were obtained by plotting the specific binding vs. the logarithm of the test compound’s concentrations. Subsequently, the K _i values were obtained by using the IC₅₀ values that were determined from sigmoidal–dose response curves. All incubations were carried out in triplicate and the dissociation constants (K _i values) are expressed as the mean±standard error of mean (SEM). CPA and DPCPX (unlabelled) were used as reference compounds and their assay results confirmed validity of the radioligand binding assays.

GTP shift assays

In addition, compounds 2c and 2q were explored via a GTP shift assay to determine the agonistic or antagonistic functionality of the investigated 2-benzylidene-1-indanones towards the A₁ AR. The GTP shift assay was performed as described previously with rat whole brain membranes and [³H]DPCPX (0.1 nM; K_d=0.36 nM) in the absence and presence of a final concentration of 100 μM GTP [16] [18]. Non–specific binding was defined by the addition of 10 μM DPCPX (unlabeled). If a calculated GTP shift of approximately 1 is obtained, that compound is considered to function as an antagonist. On the other hand, the presence of GTP affects the competition curve of an agonist and shifts the curve to the right, as previously demonstrated by the A₁ AR agonist CPA [16] [18]. The sigmoidal-dose response curves were obtained via the Graphpad Software Inc. package and the K _i values determined as described above. The GTP shift was calculated by dividing the K _i value of a compound reported in the presence of GTP by the K _i value obtained in the absence of GTP [16] [18].

Results and Discussion

Chemistry

([Fig. 2]) The key starting material 4-hydroxy-2,3-dihydro-1H-inden-1-one (2a) was synthesised in a good yield by a rearrangement reaction. The crude product was used without further purification to synthesise compounds 2b–c and 2f–q. The starting material for compound 2d, was synthesised via demethylation of 5-methoxy-2,3-dihydro-1H-inden-1-one using AlCl₃ to obtain 5-hydroxy-2,3-dihydro-1H-inden-1-one. Conversely, starting material for compound 2e, namely 6-hydroxy-2,3-dihydro-1H-inden-1-one was commercially available and procured from Sigma–Aldrich. The target 2-benzylidene-1-indanones were synthesised through an acid catalysed aldol condensation reaction. The novel compounds 2b–q were purified by recrystallization from a suitable solvent (yields of 10–85%) and, in each instance, the structures and purity of these compounds were verified by ¹H-NMR, ¹³C-NMR, mass spectrometry and HPLC analysis.

The novel synthesized compounds (2b–q) possess E-configuration, similar to the 2-benzylidene-1-tetralone analogues synthesized by Legoabe and co–workers [13] and Janse van Rensburg and co–workers [14].

Biology

The degree and type of binding affinity of the 2–benzylidene–1–indanone analogues (2a–q) at rat A₁ and A_2A AR’s were determined by radioligand binding assays and GTP shift assays, respectively, and results are summarized in [Table 1]. Two reference compounds, namely CPA and DPCPX, were included in the study and the results are in accordance with literature values.

Table 1 The dissociation constant values (K _i values) for the binding of the 2-benzylidene-1-indanones at rat A₁ and A_2A AR’s.

	Ring A			Ring B		K i±SEM (µM)a (% displacement)b		SId	K i±SEM (µM)e	GTP Shift f
#	4	5	6	3’	4’	A1 c vs [3H]DPCPX	A2A c vs [3H]NECA	(A1/A2A)	A1 c+GTP vs [3H]DPCPX
Tetralones
1a	H	OH	H	H	H	5.93±0.45a,g	2.90 ±0.66a,g	13d	6.92±0.81a,g	1 f
Indanones
2a	OH	H	H	–		>100 (58%)b	>100 (68%)b	–	–	–
2b	OH	H	H	H	H	>100 (29%)b	1.55±0.28a	–	–	–
2c	OH	H	H	OH	OH	0.435±0.050a	0.903±0.081a	0.5d	0.339±0.071e	0.9 f
2d	H	OH	H	OH	OH	5.31±0.50a	>100 (95%)b	–	–	–
2e	H	H	OH	OH	OH	4.01±0.30a	2.12±0.38a	1.9d	–	–
2 f	OH	H	H	F	H	>100 (85%)b	>100 (26%)b	–	–	–
2 g	OH	H	H	H	F	>100 (26%)b	>100 (24%)b	–	–	–
2 h	OH	H	H	Cl	H	>100 (75%)b	0.512±0.051a	–	–	–
2i	OH	H	H	H	Cl	>100 (40%)b	2.73±0.28a	–	–	–
2j	OH	H	H	Br	H	>100 (69%)b	1.04±0.18a	–	–	–
2k	OH	H	H	H	Br	>100 (51%)b	>100 (46%)b	–	–	–
2 l	OH	H	H	H	CF3	>100 (88%)b	>100 (56%)b	–	–	–
2 m	OH	H	H	H	CN	>100 (49%)b	>100 (66%)b	–	–	–
2n	OH	H	H	–	–	>100 (57%)b	>100 (77%)b	–	–	–
2o	OH	H	H	–	–	4.71±0.58a	1.79±0.13a	2.6d	–	–
2p	OH	H	H	–	–	6.58±0.68a	1.61±0.17a	4.1d	–	–
2q	OH	H	H	–	–	1.69±0.13a	3.37±0.90a	0.5d	1.83±0.09e	1 f
Reference compounds
CPA (A 1 agonist)						0.0068±0.0001a (0.0079)h; (0.015)i	0.163±0.001a (0.331)i	24d (22)i	0.099±0.015a (0.099)i	15 f (14)i
DPCPX (A 1 antagonist)						0.0004±0.0002a (0.0005)i; (0.0003)j	0.545±0.204a (0.530)i; (0.340)j	1363d (958)i; (1130)j	0.0004±0.0002a (0.0004)i	1.0 f

^aAll K _i values determined in triplicate and expressed as mean±SEM; ^bPercentage displacement of the radioligand at a maximum tested concentration (100 µM); ^cRat receptors were used (A₁: rat whole brain membranes; A_2A: rat striatal membranes); ^dSelectivity index (SI) for the A_2A receptor isoform calculated as the ratio of K _i (A₁)/K _i (A_2A); ^eGTP shift assay, where the 100 µM GTP was added to the A₁ AR radioligand binding assay; ^fGTP shifts calculated by dividing the K _i in the presence of GTP by the K _i in the absence of GTP; ^gLiterature value obtained from reference [13]; ^hLiterature value obtained from reference [19]; ⁱLiterature value obtained from reference [16]; ^jLiterature value obtained from reference [20].

Previous studies identified the 5-hydroxy substituted 2-benzylidene-1-tetralone derivative, 1a, as a lead compound to design novel and potent A₁ and A_2A AR antagonists [13]. The 4-hydroxy substituted compound 2a, the parent scaffold of this study, is unsubstituted at position 2 and lacked A₁ and/or A_2A AR activity.

Structural modifications to ring A

In analogy to previous studies of the 2-benzylidene-1-tetralones which determined optimal ring A substitution [13] [14], the impact of OH-substitution at position 4, 5 or 6 of ring A and meta (3’) and para (4’) substitution on ring B of the 2-benzylidene-1-indanones were evaluated by comparing the dissociation constant values (K _i values) of these compounds (2c–2e) to each other. Similar to the 2-benzylidene-1-tetralones, the position of the OH-group on ring A, together with meta (3’) and para (4’) substitution on ring B, of the 2-benzylidene-1-indanones modulates A₁ and A_2A AR binding affinity and C4-OH substitution (2c; A₁ K _i=0.435 µM and A_2A K _i=0.903 µM) on ring A is preferred over C6- (2e; A₁ K _i=4.01 µM and A_2A K _i=2.12 µM) and C5-OH substitution (2d; A₁ K _i=5.31 µM and A_2A K _i=>100 µM).

Structural modifications to ring B

Comparison of compound 2a to 2b showed that the 2-benzylidene side chain increases both A₁ and A_2A AR affinity — conveying the necessity of C2 substitution on ring C. The A_2A K _i value of compound 2b (1.55 µM) suggests that phenyl ring B is valuable to A_2A AR affinity.

In correlation to previous studies, A₁ and A_2A AR binding affinity favour OH-group substitution on meta (3’) and para (4’) positions of ring B. For example, compound 2c possessed a 1.7 fold increase in A_2A AR affinity compared to its unsubstituted counterpart 2b (A₁ K _i=>100 µM and A_2A K _i=1.55 µM).

Further investigation of halide substituents on C3’ or C4’ of ring B (retaining C4-OH ring A) provided results similar to research by Legoabe and co-workers [13], as well as Janse van Rensburg and colleagues [14]. Generally, halogen substitution at either the meta (3’) or para (4’) position of ring B proved detrimental to both A₁ and A_2A AR binding affinity (K _i values=>100 µM) when compared to compound 2b. While, halogen substitution with Cl at the meta (3’) or para (4’) position (2 h & 2i) and Br at the meta (3’) position (2j) is detrimental to A₁ AR affinity (K _i values=>100 µM), it was beneficial to A_2A AR affinity. Additionally, comparison of 2 h and 2i indicated that C3’-Cl substitution (2 h; A_2A K _i=0.512 µM) is preferred over C4’-Cl substitution (2i; A_2A K _i=2.73 µM), as 2 h shows a 5.3 fold increase in A_2A AR affinity.

As with previous studies [13] [14], other ring systems were also explored by replacing phenyl ring B with either a pyridine ring or 2-aminopyrimidine ring. Pyridine ring substitution proved advantageous to both A₁ and A_2A AR binding affinity; with compounds 2o–2q exhibiting, in decreasing order of affinity, A₁ 2q; N4’ K _i=1.69 µM>2o; N2’ K _i=4.71 µM>2p N3’ K _i=6.58 µM and A_2A 2p; N3’ K _i=1.61 µM>2o; N2’ K _i=1.79 µM>2q; N4’ K _i=3.37 µM AR affinity. It 7seems that A₁ AR binding favours N4’-substitution, whereas A_2A AR binding prefers N3’-substitution. Compound 2n, containing a 2-aminopyrimidine ring, was devoid of A₁ and A_2A AR binding affinity (A₁&A_2A K _i value=>100 µM).

Structural modifications to ring C

Evaluation of compound 2b in relation to compound 1a showed that reduction of ring C from a 6 membered ring (tetralone) to a 5-membered ring (indanone) decreased A₁ AR affinity and increased A_2A AR binding affinity approximately 2 fold (2b, A_2A K _i=1.55 µM vs 1a; A_2A K _i=2.90 µM).

Of the 2-benzylidene-1-indanones, 3’,4’-diOH substituted compound 2c shows the best A₁ AR affinity and the second best A_2A AR affinity, while 2-’Cl substituted compound 2 h possessed the highest A_2A AR affinity and no A₁ AR affinity. Other compounds exhibiting relatively good A₁ and/or A_2A AR affinity are: 6-OH substituted 2e and pyridine ring substituted compounds 2o–q ([Fig. 3]).

The GTP shift assay results suggest that compounds 2c and 2q act as A₁ AR antagonists – as no significant rightward shift of the binding curves were observed in the presence of GTP ([Fig. 4]).

Fig. 4 The binding curves of compounds 2c and 2q are examples of A1 AR antagonistic action determined via GTP shift assays (with and without 100 μM GTP) in rat whole brain membranes expressing A1 ARs with [3 H]DPCPX as radioligand. a GTP shift of 0.9 calculated for compound 2c, b GTP shift of 1.0 calculated for compound 2q.

Conclusions

In summary, this study involved the synthesis, characterization and evaluation of novel 2-benzylidene-1-indanone analogues to understand the importance of structural modifications to ring A, B and C of the 2-benzylidene-1-tetralone scaffold in gaining or even losing A₁ and/or A_2A AR affinity. Upon analysis, it was found that C4-OH substitution on ring A and 3’- and 4’-OH substitution on ring B (2c) is complimentary to A₁ and A_2A AR affinity, affording this non-selective compound K _i values below 1 µM for both the A₁ and A_2A AR. C3’-Cl substitution on ring B (retaining C4-OH ring A) provided compound 2 h with the highest A_2A AR affinity (K _i=0.512 µM) and selectivity. Replacing phenyl ring B with a pyridine ring increased A₁ AR affinity and slightly decreased A_2A AR affinity (2o–q), in comparison to 2b — yet compounds 2o–q still possess relatively good A₁ and A_2A AR affinity. Additionally, it seems that A₁ AR binding favours N4’-substitution (2q), whereas A_2A AR binding favours N3’–substitution (2p). In general, conversion from fused 6– and 6–membered rings (2-benzylidene-1-tetralones) to fused 6- and 5-membered rings (2-benzylidene-1-indanones) in combination with ring B substitutions improved A₁ and A_2A AR affinity. In view of these findings, compounds 2c, 2 h, 2q and 2p are worthy candidates to further explore as potent and selective A₁ and A_2A AR antagonists for the potential treatment of neurological conditions, achieved by optimization of the 2-benzylidene-1-indanone scaffold.

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgements

Financial support for this work was provided by the North-West University (NWU), the National Research Foundation (96135) and the Medical Research Council, South Africa. We are grateful to Dr. J. Jordaan of Chemical Research Beneficiation, NWU for NMR and MS analysis, as well as Prof. J. Du Preez of Pharmaceutics, School of Pharmacy, NWU for HPLC analysis.

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