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A2A receptor affinity and selectivity of caffeine
Accepted Manuscript Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine M.M. van der Walt, G. Terre’Blanche PII:
S0223-5234(16)30808-X
DOI:
10.1016/j.ejmech.2016.09.072
Reference:
EJMECH 8938
To appear in:
European Journal of Medicinal Chemistry
Received Date: 11 August 2016 Revised Date:
20 September 2016
Accepted Date: 21 September 2016
Please cite this article as: M.M. van der Walt, G. Terre’Blanche, Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.072. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical abstract
caffeine core O
N
increase A1 affinity decrease A2A affinity
(X) (Y)
R
R: Cl =
decrease A1 affinity increase A2A affinity
decrease A1 affinity increase A2A affinity
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Y: O,S =
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O
N
8
X: O,S =
RI PT
N
N
linker
ACCEPTED MANUSCRIPT
MM van der Walt a* and G Terre’Blanche a,b
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Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine a Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa b
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Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
Abstract
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Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 µM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 µM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl
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linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating
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neurological disorders.
Keywords
Keywords: Caffeine; Adenosine A2A receptor antagonist; Adenosine A1 receptor antagonist; 1,3,7-trimethylxanthine; Parkinson's disease
*Corresponding author. Present Address: Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa. E-mail addresses: [email protected]; [email protected] (M.M van der Walt). Abbreviations: PD, Parkinson’s disease; AD, Alzheimer’s disease; AR, adenosine receptor; GTP, guanosine triphosphate; [3H]DPCPX, [3H]-dipropyl-8-cyclopentylxanthine; [3H]NECA, N-[3H]-ethyladenosin-5’-uronamide; CPA, N6-cyclopentyladenosine.
ACCEPTED MANUSCRIPT 1. Introduction Caffeine is a well-known 1,3,7-trimethyl-substituted xanthine derivative that occurs naturally in coffee and tea [1] or may be prepared via organic synthesis [2]. Worldwide, caffeine is consumed daily by a vast number of people due to its perceived central nervous system stimulant properties [3,4]. It is thought that caffeine mainly exhibits its action through its non-selective antagonistic
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properties of the A1 and A2A adenosine receptor (AR) subtypes [5]. According to Daly and colleagues [6], caffeine displays dissociation constant (Ki) values of 55 µM and 50 µM for the A1 and A2A ARs, respectively. Currently, four AR subtypes exist, namely the A1, A2A, A2B and A3 [7], although only the A1 and A2A AR subtypes have been identified as drug targets for the treatment of neurological
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conditions [8,9].
Numerous biological effects of caffeine have been extensively studied in the past. It is suggested that caffeine possesses cognition-enhancing properties based on the finding that it may counteract age-
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related cognitive decline [3], as well as prevent sporadic dementia associated with Alzheimer’s disease [10]. The cognitive effects of caffeine may be ascribed to its ability to antagonise the A1 AR subtype in the hippocampus and prefrontal cortex [5,11]. Furthermore, evidence from epidemiological studies indicates a strong direct relationship between coffee drinking and a reduced risk of Parkinson’s disease (PD) within many populations [12,13]. The latter finding was further supported by Qi and Li [14], who demonstrated a linear relationship between the consumption of caffeine and a
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reduced risk of developing PD from a dose–response meta-analysis on caffeinated beverages. PD is clinically manifested by a triad of cardinal motor symptoms [15] related to dopamine deficiency. However, non-motor symptoms, not related to dopamine, are sometimes present before diagnosis; by contrast with the motor symptoms of the disease, for which treatment is available, these
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non-motor symptoms are often poorly recognised and inadequately treated [16,17]. Some of the nonmotor symptoms include depression and cognitive impairment [18]. The latter linked to PD may
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potentially improve via antagonism of the A1 AR subtype [9]; in turn, antagonism of the A2A AR may enhance motor activity with a reduced risk of developing dyskinesia [19]. The motor activating effect of caffeine may be due to the synergistic activity of antagonising both the A1 and A2A ARs [20], where antagonism of the A1 AR facilitates presynaptic dopamine release and antagonism of the A2A AR facilitates postsynaptic dopamine release [21]. In addition, neuroprotective properties of caffeine have been demonstrated in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD [22]. Even though evidence suggests that caffeine exerts beneficial effects in PD, its low AR affinity makes it relatively unattractive as a treatment option. However, caffeine (1a) is considered a promising lead compound for designing favourable dual A1/A2A AR antagonists [11]. Recently, we reported on the A1 and A2A AR affinities of the caffeine-based derivatives 8-(2-phenylethyl)-1,3,7-trimethylxanthine (1b)
ACCEPTED MANUSCRIPT and 8-(phenoxymethyl)-1,3,7-trimethylxanthine (1c) [23]. The latter study further supported the theory that caffeine (1a) is an ideal scaffold for designing compounds with enhanced AR affinity.
caffeine core O
O
N
N
8
X: CH2, O, S, NH Y: CH2, O, S R: H, Cl
(X) (Y)
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N
N
linker
R
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Figure 1. Structural modifications to the caffeine core. Various two-chain-length linkers (bearing either C, O, S or N at positions X or Y), as well as the influence of para-chlorine (R) substitution on the phenyl ring, were investigated for their ability to govern A1 and A2A binding affinity and selectivity.
By analogy with compound 1c, we investigated a broader range of C8 two-chain-length linker moieties to the phenyl ring where an oxygen or a sulphur was either attached directly to the phenyl ring or to the C8 of the caffeine scaffold (Figure 1). These compounds include the phenylethyl (1b), phenoxymethyl (1c and 1d), benzyloxy (1e and 1f), (phenylsulfanyl)methyl (1g and 1h) and benzylsulfanyl (1i and 1j) side-chains. In addition, the benzylamide (1k) side-chain and para-chlorine
Materials and methods
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substitution on the phenyl ring of selected side-chains (1d, 1f, 1h, 1j) were also evaluated.
The test compounds were prepared as previously described: 1b, 1c [23]; 1d, 1g, 1h [24]; 1e, 1f [25];
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1i, 1j, 1k [26]. See the references indicated for a detailed discussion of the syntheses. All other reagents, which were obtained from standard commercial sources, were of analytical grade.
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Radioligand binding assay
The A1 and A2A AR affinities of compounds 1a–1k were determined with radioligand binding assays as described previously [23]. The collection of tissue samples for the A1 and A2A AR binding studies was approved by the Research Ethics Committee of North-West University (application number NWU-0035-10-A5). In short, the A1 AR affinity assay was performed with rat whole brain membranes in the presence of the radioligand [3H]-8-cylcopentyl-1,3-dipropylxanthine ([3H]DPCPX) [23,27]. In turn, the A2A AR affinity was measured at rat striatal membranes with 5’-N-[3H]ethylcarboxamideadenosine ([3H]NECA) as radioligand [23,28]. In addition, N6-cyclopentyladenosine (CPA) was added to the A2A AR competition experiments to minimize the binding of the radioligand [3H]NECA to A1 AR [23,28]. Nonspecific binding was defined by the addition of 100 µM CPA [23].
ACCEPTED MANUSCRIPT GTP shift assay The agonistic or antagonistic functionality of compounds 1c and 1e was determined by evaluating the competition curves in a GTP (guanosine triphosphate) shift assay as reported previously [23]. The GTP shift assays were carried out with rat whole brain membranes in the absence and presence of 0.1
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mM GTP. Nonspecific binding was defined by the addition of 10 µM DPCPX (unlabelled) [23]. Data analysis
The competition curves were obtained by using the Prism software package (GraphPad Software Inc.). The competition experiments were carried out in triplicate; the values of the dissociation
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constant (Ki) are expressed as mean ± standard error of the mean (SEM). The Ki values were calculated by using the Kd values of 0.36 nM for [3H]DPCPX at rat whole brain membranes [23,27],
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whereas 15.3 nM for [3H]NECA at rat striata membrane [23,28] was used. Furthermore, GTP shifts were calculated by dividing the Ki value of a compound reported in the presence of GTP by the corresponding value obtained in the absence of GTP [29]. A compound with a calculated GTP shift of approximately 1 is considered an antagonist; the presence of GTP affects the competition curves of an agonist and shifts the curve to the right [23,30].
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Results and discussion
The influence of C8 substitution on governing A1 and A2A AR affinities of the 1,3,7-trimethylxanthine core was investigated by comparing the Ki values of caffeine (1a) with that of the selected caffeinebased analogues (1b–1k) (Table 1). Comparing previously synthesised [23] 8-(2-phenylethyl)-1,3,7trimethylxanthine (1b) and 8-(phenoxymethyl)-1,3,7-trimethylxanthine (1c) with caffeine (1a)
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showed improved A1 and A2A AR affinities for both compounds; their corresponding dissociation constants were A1Ki = 5.74 µM; A2AKi = 4.95 µM and A1Ki = 3.09 µM; A2AKi = 2.37 µM, respectively
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[23]. Thus, introducing a two-chain-length side-chain at the C8 position of caffeine, increased the affinity for both A1 and A2A ARs.
ACCEPTED MANUSCRIPT Table 1 The dissociation constants (Ki values) for the binding of the test compounds to rat adenosine A1 and A2A receptors. O N
N
R8
O
N
N
Ki ± SEM (µM)a
-H
Phenylethyl linker -(CH2)2C6H5 1b Phenoxymethyl linker -CH2-O-C6H5 1c -CH2-O-[4-Cl-(C6H5)] 1d Benzyloxy linker -O-CH2-C6H5 1e -O-CH2-[4-Cl-(C6H5)] 1f (Phenylsulfanyl)methyl linker -CH2-S-C6H5 1g -CH2-S-[4-Cl-(C6H5)] 1h Benzylsulfanyl linker -S-CH2-C6H5 1i -S-CH2-[4-Cl-(C6H5)] 1j Benzylamide linker -NH-CH2-C6H5 1k
A1 + GTPb vs [3H]DPCPX -
GTP shiftc -
SId (A2A/A1) 1.1
SIe (A1/A2A) 0.9
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3
5.74 ± 1.19h
4.95 ± 0.19h
-
-
0.9
1.2
3.09 ± 0.40h 7.60 ± 0.95
2.37 ± 0.42h 1.33 ± 0.20
4.01 ± 0.06 -
1.3 -
0.8 0.2
1.3 5.7
1.52 ± 0.01 17.38 ± 1.85
4.30 ± 1.04 3.31 ± 0.11
1.93 ± 0.10 -
1.2 -
2.8 0.2
0.4 5.3
4.85 ± 0.01 20.84 ± 3.76
3.33 ± 0.19 1.50 ± 0.56
-
-
0.8 0.1
1.5 13.9
4.32 ± 0.76 90.00 ± 6.00
4.64 ± 1.49 51.43 ± 3.82
-
-
1.1 0.6
0.9 1.8
7.95 ± 0.82
4.50 ± 1.82
-
-
0.6
1.8
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1a
A2A vs A1 vs [ H]DPCPX [3H]NECA 43.9 ± 3.15 47.2 ± 15.1 41f; 26 f; 55g 43f; 22 f; 50g
R8
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Compd
a
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All Ki values were determined in triplicate and expressed as mean ± SEM. b GTP shift assay; 0.1 mM GTP was added in the A1 AR radioligand binding assay. c GTP shifts calculated by dividing the Ki value in the presence of GTP by the corresponding value in the absence of GTP. d Selectivity index (SI) for the A1 receptor isoform calculated as the ratio Ki (A2A)/Ki (A1). e Selectivity index (SI) for the A2A receptor isoform calculated as the ratio Ki (A1)/Ki (A2A). f Literature values obtained from reference [31]. g Literature values obtained from reference [6]. h Literature values obtained from reference [23].
By analogy with compound 1c, we investigated other two-chain-length linkers, namely: phenylethyl (1b), phenoxymethyl (1c), benzyloxy (1e), (phenylsulfanyl)methyl (1g), benzylsulfanyl (1i) and benzylamide (1k). Furthermore, p-chlorine substitution on the phenyl ring of selected side-chains (1d, 1f, 1h, 1j) was also evaluated. Table 1 summarizes the observed affinities of the selected caffeine analogues investigated in radioligand binding assays at rat brain membrane A1 and A2A ARs. Overall, the introduction of an oxygen (1c, 1e) or a sulphur (1g, 1i) to the unsubstituted phenylethyl (1b) side-chain led to an increase in A1 and A2A AR affinity. When comparing the position of the oxygen (phenoxymethyl 1c vs benzyloxy 1e) and sulphur ((phenylsulfanyl)methyl 1g vs benzylsulfanyl 1i), we found that moving the oxygen from the position next to the phenyl ring (1c
ACCEPTED MANUSCRIPT A1Ki = 3.09 µM; A2AKi = 2.37 µM ) to that next to C8 of the caffeine core — separating the phenyl ring and the oxygen with a methyl (1e A1Ki = 1.52 µM; A2AKi = 4.3 µM) — increased the A1 AR affinity twofold. In turn, the latter rearrangement (phenoxymethyl vs benzyloxy) decreased the A2A AR affinity twofold. The position of the sulphur had less effect on A1 and A2A AR affinity but the same trend was observed. For example, a slight increase in A1 AR affinity and a slight decrease in A2A AR affinity were found when moving the sulphur from the position next to the ring (1g A1Ki = 4.85
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µM; A2AKi = 3.33 µM ) to that next to C8, separating the aromatic ring and the sulphur with a methyl group (1i A1Ki = 4.32 µM; A2AKi = 4.64 µM ).
Furthermore, introduction of a chlorine in the para position of the phenyl ring (1c vs 1d; 1e vs 1f; 1g vs 1h; 1i vs 1j) generally resulted in a decrease in A1 AR affinity compared to the unsubstituted
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phenylethyl compound (1b). However, selected compounds (1d, 1f, 1h) still showed greater A1 AR affinity (of between 2 and 12 times) compared to caffeine (1a). Para-chloro substitution decreased the
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A1 AR binding affinity as follow: 2.45-fold (1c vs 1d); 11.43-fold (1e vs 1f); 4.29-fold (1g vs 1h); 20.83-fold (1i vs 1j). The most substantial decrease in A1 AR affinity, with the introduction of a parachloro, was found where the heteroatom (O or S) was directly linked to the C8 of the xanthine core (1e vs 1f; 1i vs 1j). In general, affinity for the A2A AR increased by twofold with para-chloro substitution on the phenyl ring for the phenoxymethyl, benzyloxy and (phenylsulfanyl)methyl linkers (1c vs 1d; 1e vs 1f; 1g vs 1h), with the exception of the benzylsulfanyl linker (1i vs 1j), for which a significant 11-fold decrease was observed. Therefore, the introduction of a para-chloro on the
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benzylsulfanyl (1j) resulted in the weakest A1 and A2A AR affinity among the test compounds (1a–k). An overall decrease in A1 and A2A AR affinity was found when comparing the position of the oxygen and the sulphur in the para-chloro-substituted derivatives (phenoxymethyl 1d vs benzyloxy 1f and
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(phenylsulfanyl)methyl 1h vs benzylsulfanyl 1j). Interestingly, the sulphur-containing linkers displayed the most significant decrease (34-fold) in the A2A AR affinity when the sulphur moved from the position next to the phenyl ring [(phenylsulfanyl)methyl] to that next to C8 (benzylsulfanyl) of the
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caffeine scaffold (1h A2AKi = 1.5 µM vs 1j A2AKi = 51.43 µM). In addition, the introduction of nitrogen was explored by comparing compound 1k, that bore a benzylamide side-chain, with the unsubstituted phenylethyl (1b), benzyloxy (1e) and benzylsulfanyl (1i) linkers. The influence of the different atoms (C, N, O, S) on the A2A AR affinity was not significant and the corresponding affinities were in the same range: O > N > S > C (1e 4.30 µM > 1k 4.50 µM > 1i 4.64 µM > 1b 4.95 µM). The A1 AR affinity showed a larger variation in Ki values such that the oxygen exhibited a 5-fold increase compared to the nitrogen: O > S > C > N (1e 1.52 µM > 1i 4.32 µM > 1b 5.74 µM > 1k 7.95 µM).
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Compounds 1b, 1c and 1i were found to act non-selectively in respect of the A1 and A2A ARs (Table 1). The position of the oxygen, phenoxymethyl vs benzyloxy, favoured A1 AR selectivity (1c SI = 0.86 vs 1e SI = 2.83); introducing a para-chloro substituent achieved A2A AR selectivity (1c SI = 1.3 vs 1d SI = 5.71, 1g SI = 1.5 vs 1h SI = 13.89). Even though the test compounds exhibit values in the
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low micromolar range, the structural modifications investigated in this study might contribute significantly to increased A1 and A2A AR affinity and selectivity in caffeine derivatives.
Compounds 1c and 1e were identified as possessing the highest affinity for the A1 AR and so were selected for functional GTP shift assays to determine if these compounds acted as agonists or
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antagonists (as expected). The binding of an agonist to the A1 ARs is reduced in the presence of GTP due to the A1 adenosine receptor being shifted from a high- to a low-affinity state [32]. For this reason, an A1 AR agonist results in a corresponding rightward shift of the binding curve in the
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presence of GTP, whereas no shift of the curve would be observed in the presence of an A1 AR antagonist. Compounds 1c and 1e showed no significant shifts (Table1, Figure 2) of the binding curves in the presence of GTP and may therefore be considered as antagonists for the A1 ARs.
100
+ GTP (0.1 mM) - GTP
50
0 -1
0
1
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-2
Log [1c]
2
3
specific binding (%)
B.
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Adenosine A1 binding
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specific binding (%)
A.
Adenosine A1 binding
100
+ GTP (0.1 mM) - GTP
50
0 -3
-2
-1
0
1
2
3
Log [1e]
Figure 2. The binding curves of compounds 1c and 1e, indicating their A1 AR antagonistic action as determined via GTP shift assays (with and without 0.1 mM GTP) in rat whole brain membranes, using [3H]DPCPX as radioligand. (A) GTP shift of 1.3 calculated for the A1 AR antagonist 1c. (B) GTP shift of 1.2 calculated for the A1 AR antagonist 1e.
ACCEPTED MANUSCRIPT Conclusion The caffeine analogues investigated (1b–1k) demonstrated their potential as A1 and A2A AR antagonists with Ki values in the micromolar range (A1Ki = 1.52–90.0 µM; A2AKi = 1.33–51.43 µM). In general, the caffeine analogues (1b–1k) displayed improved A1 and A2A AR affinity compared to caffeine (1a), with the only exception being compound 1j.
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Overall, the introduction of an oxygen in the two-chain-length linker increased both A1 and A2A AR affinity, with the unsubstituted benzyloxy linker (1e A1Ki = 1.52 µM) documented with the greatest affinity and selectivity (SI = 2.8) for the A1 AR. The introduction of a para-chloro substituent decreased A1 AR affinity, but significantly increased A2A AR affinity with the phenoxymethyl (1d)
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and (phenylsulfanyl)methyl (1h) compounds displaying the greatest A2A AR affinity among the parachloro-substituted analogues (1d A2AKi = 1.33 µM; 1h A2AKi = 1.50 µM).
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For these reasons, the 8-phenoxymethyl and 8-(phenylsulfanyl)methyl side-chains should not be disregarded when designing compounds with selectivity and affinity for the A2A ARs. The various C8 side-chains investigated in this study are thus important to understand the structure–activity relationships of caffeine analogues and we therefore conclude that suitable selection of a C8 sidechain may lead to the development of improved A1 and A2A AR affinity and selectivity for caffeine-
Author contributions
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based AR antagonists.
The manuscript was written by GT and MMvdW. The biological evaluations were performed by MMvdW and the interpretation of the data by GT and MMvdW. Both authors give their approval of
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this version of the manuscript. Acknowledgements
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We are grateful to Prof. J.P. Petzer of the North-West University for donating test compounds (1d– 1k). The financial assistance of the North-West University, the South African Medical Research Council and the National Research Foundation towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the authors and are not necessarily to be attributed to the NRF.
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[25] B. Strydom, J.J. Bergh, J.P. Petzer, 8-Aryl- and alkyloxycaffeine analogues as inhibitors of monoamine oxidase, Eur. J. Med. Chem. 46 (2011) 3474-85.
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[26] H.P. Booysen, C. Moraal, G. Terre'Blanche, A. Petzer, J.J. Bergh, J.P. Petzer, Thio- and aminocaffeine analogues as inhibitors of human monoamine oxidase, Bioorg. Med. Chem. 19 (2011) 7507-18 [27] R.F. Bruns, J.H. Fergus, E.W. Badger, J.A. Bristol, L.A. Santay, J.D. Hartman, S.J. Hays, C.C. Huang, Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes, Naunyn-Schmiedeberg's Arch. Pharmacol.335 (1987) 59-63. [28] R.F. Bruns, G.H. Lu, T.A. Pugsley, Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes, Mol. Pharmacol. 29 (1986) 331-346. [29] E.M. Van der Wenden, H.R. Hartog-Witte, H.C.P.F. Roelen, J.K. Von Frijtag Drabbe Kunzel, I.M. Pirovano, R.A.A. Mathot, M. Danhof, A. Van Aerschot, J.M. Lidaks, A.P. Ijzerman, W. Soudijn, 8-Substituted adenosine and theophylline-7-riboside analogues as potential partial agonists for the adenosine A1 receptor, Eur. J. Pharmacol-Mol. Pharmacol. Sect. 290 (1995) 189–199.
ACCEPTED MANUSCRIPT [30] M. Gutschow, M. Schlenk, J. Gab, M. Paskaleva, M. Wassam Alnouri, S. Scolari, J. Iqbal, C.E. Muller, Benzothiazinones: a novel class of adenosine receptor antagonists structurally unrelated to xanthine and adenine derivatives, J.Med. Chem. 55 (2012) 3331–3341. [31] C.E. Muller, U. Geis, J. Hipp, U. Schobert, W. Frobenius, M. Pawłowski, F. Suzuki, J. SandovalRamirez, Synthesis and structure-activity relationships of 3,7-dimethyl-1-propargylxanthine derivatives, A2A-selective adenosine receptor antagonists, J. Med. Chem. 40 (1997) 4396–4405.
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[32] R.R. Goodman, M.J. Cooper, M. Gavish, S.H. Snyder, Guanine nucleotide and cation regulation of the binding of [3H]cyclohexyladenosine and [3H]diethylphenylxanthine to adenosine A1 receptors in brain membranes, Mol. Pharmacol. 21 (1982) 329-335.
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Highlights: Selected C8 two-chain linkers yield enhanced adenosine receptor affinity.
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A1 receptor antagonists are considered agents for Parkinson’s disease therapy.
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A2A receptor antagonists are considered agents for Parkinson’s disease therapy.
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Introduction of a para-chloro substitution govern A2A receptor selectivity.
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S0223-5234(16)30808-X
DOI:
10.1016/j.ejmech.2016.09.072
Reference:
EJMECH 8938
To appear in:
European Journal of Medicinal Chemistry
Received Date: 11 August 2016 Revised Date:
20 September 2016
Accepted Date: 21 September 2016
Please cite this article as: M.M. van der Walt, G. Terre’Blanche, Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.072. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical abstract
caffeine core O
N
increase A1 affinity decrease A2A affinity
(X) (Y)
R
R: Cl =
decrease A1 affinity increase A2A affinity
decrease A1 affinity increase A2A affinity
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Y: O,S =
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O
N
8
X: O,S =
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N
N
linker
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MM van der Walt a* and G Terre’Blanche a,b
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Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine a Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa b
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Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
Abstract
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Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 µM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 µM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl
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linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating
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neurological disorders.
Keywords
Keywords: Caffeine; Adenosine A2A receptor antagonist; Adenosine A1 receptor antagonist; 1,3,7-trimethylxanthine; Parkinson's disease
*Corresponding author. Present Address: Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa. E-mail addresses: [email protected]; [email protected] (M.M van der Walt). Abbreviations: PD, Parkinson’s disease; AD, Alzheimer’s disease; AR, adenosine receptor; GTP, guanosine triphosphate; [3H]DPCPX, [3H]-dipropyl-8-cyclopentylxanthine; [3H]NECA, N-[3H]-ethyladenosin-5’-uronamide; CPA, N6-cyclopentyladenosine.
ACCEPTED MANUSCRIPT 1. Introduction Caffeine is a well-known 1,3,7-trimethyl-substituted xanthine derivative that occurs naturally in coffee and tea [1] or may be prepared via organic synthesis [2]. Worldwide, caffeine is consumed daily by a vast number of people due to its perceived central nervous system stimulant properties [3,4]. It is thought that caffeine mainly exhibits its action through its non-selective antagonistic
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properties of the A1 and A2A adenosine receptor (AR) subtypes [5]. According to Daly and colleagues [6], caffeine displays dissociation constant (Ki) values of 55 µM and 50 µM for the A1 and A2A ARs, respectively. Currently, four AR subtypes exist, namely the A1, A2A, A2B and A3 [7], although only the A1 and A2A AR subtypes have been identified as drug targets for the treatment of neurological
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conditions [8,9].
Numerous biological effects of caffeine have been extensively studied in the past. It is suggested that caffeine possesses cognition-enhancing properties based on the finding that it may counteract age-
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related cognitive decline [3], as well as prevent sporadic dementia associated with Alzheimer’s disease [10]. The cognitive effects of caffeine may be ascribed to its ability to antagonise the A1 AR subtype in the hippocampus and prefrontal cortex [5,11]. Furthermore, evidence from epidemiological studies indicates a strong direct relationship between coffee drinking and a reduced risk of Parkinson’s disease (PD) within many populations [12,13]. The latter finding was further supported by Qi and Li [14], who demonstrated a linear relationship between the consumption of caffeine and a
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reduced risk of developing PD from a dose–response meta-analysis on caffeinated beverages. PD is clinically manifested by a triad of cardinal motor symptoms [15] related to dopamine deficiency. However, non-motor symptoms, not related to dopamine, are sometimes present before diagnosis; by contrast with the motor symptoms of the disease, for which treatment is available, these
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non-motor symptoms are often poorly recognised and inadequately treated [16,17]. Some of the nonmotor symptoms include depression and cognitive impairment [18]. The latter linked to PD may
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potentially improve via antagonism of the A1 AR subtype [9]; in turn, antagonism of the A2A AR may enhance motor activity with a reduced risk of developing dyskinesia [19]. The motor activating effect of caffeine may be due to the synergistic activity of antagonising both the A1 and A2A ARs [20], where antagonism of the A1 AR facilitates presynaptic dopamine release and antagonism of the A2A AR facilitates postsynaptic dopamine release [21]. In addition, neuroprotective properties of caffeine have been demonstrated in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD [22]. Even though evidence suggests that caffeine exerts beneficial effects in PD, its low AR affinity makes it relatively unattractive as a treatment option. However, caffeine (1a) is considered a promising lead compound for designing favourable dual A1/A2A AR antagonists [11]. Recently, we reported on the A1 and A2A AR affinities of the caffeine-based derivatives 8-(2-phenylethyl)-1,3,7-trimethylxanthine (1b)
ACCEPTED MANUSCRIPT and 8-(phenoxymethyl)-1,3,7-trimethylxanthine (1c) [23]. The latter study further supported the theory that caffeine (1a) is an ideal scaffold for designing compounds with enhanced AR affinity.
caffeine core O
O
N
N
8
X: CH2, O, S, NH Y: CH2, O, S R: H, Cl
(X) (Y)
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N
linker
R
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Figure 1. Structural modifications to the caffeine core. Various two-chain-length linkers (bearing either C, O, S or N at positions X or Y), as well as the influence of para-chlorine (R) substitution on the phenyl ring, were investigated for their ability to govern A1 and A2A binding affinity and selectivity.
By analogy with compound 1c, we investigated a broader range of C8 two-chain-length linker moieties to the phenyl ring where an oxygen or a sulphur was either attached directly to the phenyl ring or to the C8 of the caffeine scaffold (Figure 1). These compounds include the phenylethyl (1b), phenoxymethyl (1c and 1d), benzyloxy (1e and 1f), (phenylsulfanyl)methyl (1g and 1h) and benzylsulfanyl (1i and 1j) side-chains. In addition, the benzylamide (1k) side-chain and para-chlorine
Materials and methods
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substitution on the phenyl ring of selected side-chains (1d, 1f, 1h, 1j) were also evaluated.
The test compounds were prepared as previously described: 1b, 1c [23]; 1d, 1g, 1h [24]; 1e, 1f [25];
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1i, 1j, 1k [26]. See the references indicated for a detailed discussion of the syntheses. All other reagents, which were obtained from standard commercial sources, were of analytical grade.
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Radioligand binding assay
The A1 and A2A AR affinities of compounds 1a–1k were determined with radioligand binding assays as described previously [23]. The collection of tissue samples for the A1 and A2A AR binding studies was approved by the Research Ethics Committee of North-West University (application number NWU-0035-10-A5). In short, the A1 AR affinity assay was performed with rat whole brain membranes in the presence of the radioligand [3H]-8-cylcopentyl-1,3-dipropylxanthine ([3H]DPCPX) [23,27]. In turn, the A2A AR affinity was measured at rat striatal membranes with 5’-N-[3H]ethylcarboxamideadenosine ([3H]NECA) as radioligand [23,28]. In addition, N6-cyclopentyladenosine (CPA) was added to the A2A AR competition experiments to minimize the binding of the radioligand [3H]NECA to A1 AR [23,28]. Nonspecific binding was defined by the addition of 100 µM CPA [23].
ACCEPTED MANUSCRIPT GTP shift assay The agonistic or antagonistic functionality of compounds 1c and 1e was determined by evaluating the competition curves in a GTP (guanosine triphosphate) shift assay as reported previously [23]. The GTP shift assays were carried out with rat whole brain membranes in the absence and presence of 0.1
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mM GTP. Nonspecific binding was defined by the addition of 10 µM DPCPX (unlabelled) [23]. Data analysis
The competition curves were obtained by using the Prism software package (GraphPad Software Inc.). The competition experiments were carried out in triplicate; the values of the dissociation
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constant (Ki) are expressed as mean ± standard error of the mean (SEM). The Ki values were calculated by using the Kd values of 0.36 nM for [3H]DPCPX at rat whole brain membranes [23,27],
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whereas 15.3 nM for [3H]NECA at rat striata membrane [23,28] was used. Furthermore, GTP shifts were calculated by dividing the Ki value of a compound reported in the presence of GTP by the corresponding value obtained in the absence of GTP [29]. A compound with a calculated GTP shift of approximately 1 is considered an antagonist; the presence of GTP affects the competition curves of an agonist and shifts the curve to the right [23,30].
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Results and discussion
The influence of C8 substitution on governing A1 and A2A AR affinities of the 1,3,7-trimethylxanthine core was investigated by comparing the Ki values of caffeine (1a) with that of the selected caffeinebased analogues (1b–1k) (Table 1). Comparing previously synthesised [23] 8-(2-phenylethyl)-1,3,7trimethylxanthine (1b) and 8-(phenoxymethyl)-1,3,7-trimethylxanthine (1c) with caffeine (1a)
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showed improved A1 and A2A AR affinities for both compounds; their corresponding dissociation constants were A1Ki = 5.74 µM; A2AKi = 4.95 µM and A1Ki = 3.09 µM; A2AKi = 2.37 µM, respectively
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[23]. Thus, introducing a two-chain-length side-chain at the C8 position of caffeine, increased the affinity for both A1 and A2A ARs.
ACCEPTED MANUSCRIPT Table 1 The dissociation constants (Ki values) for the binding of the test compounds to rat adenosine A1 and A2A receptors. O N
N
R8
O
N
N
Ki ± SEM (µM)a
-H
Phenylethyl linker -(CH2)2C6H5 1b Phenoxymethyl linker -CH2-O-C6H5 1c -CH2-O-[4-Cl-(C6H5)] 1d Benzyloxy linker -O-CH2-C6H5 1e -O-CH2-[4-Cl-(C6H5)] 1f (Phenylsulfanyl)methyl linker -CH2-S-C6H5 1g -CH2-S-[4-Cl-(C6H5)] 1h Benzylsulfanyl linker -S-CH2-C6H5 1i -S-CH2-[4-Cl-(C6H5)] 1j Benzylamide linker -NH-CH2-C6H5 1k
A1 + GTPb vs [3H]DPCPX -
GTP shiftc -
SId (A2A/A1) 1.1
SIe (A1/A2A) 0.9
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5.74 ± 1.19h
4.95 ± 0.19h
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-
0.9
1.2
3.09 ± 0.40h 7.60 ± 0.95
2.37 ± 0.42h 1.33 ± 0.20
4.01 ± 0.06 -
1.3 -
0.8 0.2
1.3 5.7
1.52 ± 0.01 17.38 ± 1.85
4.30 ± 1.04 3.31 ± 0.11
1.93 ± 0.10 -
1.2 -
2.8 0.2
0.4 5.3
4.85 ± 0.01 20.84 ± 3.76
3.33 ± 0.19 1.50 ± 0.56
-
-
0.8 0.1
1.5 13.9
4.32 ± 0.76 90.00 ± 6.00
4.64 ± 1.49 51.43 ± 3.82
-
-
1.1 0.6
0.9 1.8
7.95 ± 0.82
4.50 ± 1.82
-
-
0.6
1.8
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1a
A2A vs A1 vs [ H]DPCPX [3H]NECA 43.9 ± 3.15 47.2 ± 15.1 41f; 26 f; 55g 43f; 22 f; 50g
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All Ki values were determined in triplicate and expressed as mean ± SEM. b GTP shift assay; 0.1 mM GTP was added in the A1 AR radioligand binding assay. c GTP shifts calculated by dividing the Ki value in the presence of GTP by the corresponding value in the absence of GTP. d Selectivity index (SI) for the A1 receptor isoform calculated as the ratio Ki (A2A)/Ki (A1). e Selectivity index (SI) for the A2A receptor isoform calculated as the ratio Ki (A1)/Ki (A2A). f Literature values obtained from reference [31]. g Literature values obtained from reference [6]. h Literature values obtained from reference [23].
By analogy with compound 1c, we investigated other two-chain-length linkers, namely: phenylethyl (1b), phenoxymethyl (1c), benzyloxy (1e), (phenylsulfanyl)methyl (1g), benzylsulfanyl (1i) and benzylamide (1k). Furthermore, p-chlorine substitution on the phenyl ring of selected side-chains (1d, 1f, 1h, 1j) was also evaluated. Table 1 summarizes the observed affinities of the selected caffeine analogues investigated in radioligand binding assays at rat brain membrane A1 and A2A ARs. Overall, the introduction of an oxygen (1c, 1e) or a sulphur (1g, 1i) to the unsubstituted phenylethyl (1b) side-chain led to an increase in A1 and A2A AR affinity. When comparing the position of the oxygen (phenoxymethyl 1c vs benzyloxy 1e) and sulphur ((phenylsulfanyl)methyl 1g vs benzylsulfanyl 1i), we found that moving the oxygen from the position next to the phenyl ring (1c
ACCEPTED MANUSCRIPT A1Ki = 3.09 µM; A2AKi = 2.37 µM ) to that next to C8 of the caffeine core — separating the phenyl ring and the oxygen with a methyl (1e A1Ki = 1.52 µM; A2AKi = 4.3 µM) — increased the A1 AR affinity twofold. In turn, the latter rearrangement (phenoxymethyl vs benzyloxy) decreased the A2A AR affinity twofold. The position of the sulphur had less effect on A1 and A2A AR affinity but the same trend was observed. For example, a slight increase in A1 AR affinity and a slight decrease in A2A AR affinity were found when moving the sulphur from the position next to the ring (1g A1Ki = 4.85
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µM; A2AKi = 3.33 µM ) to that next to C8, separating the aromatic ring and the sulphur with a methyl group (1i A1Ki = 4.32 µM; A2AKi = 4.64 µM ).
Furthermore, introduction of a chlorine in the para position of the phenyl ring (1c vs 1d; 1e vs 1f; 1g vs 1h; 1i vs 1j) generally resulted in a decrease in A1 AR affinity compared to the unsubstituted
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phenylethyl compound (1b). However, selected compounds (1d, 1f, 1h) still showed greater A1 AR affinity (of between 2 and 12 times) compared to caffeine (1a). Para-chloro substitution decreased the
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A1 AR binding affinity as follow: 2.45-fold (1c vs 1d); 11.43-fold (1e vs 1f); 4.29-fold (1g vs 1h); 20.83-fold (1i vs 1j). The most substantial decrease in A1 AR affinity, with the introduction of a parachloro, was found where the heteroatom (O or S) was directly linked to the C8 of the xanthine core (1e vs 1f; 1i vs 1j). In general, affinity for the A2A AR increased by twofold with para-chloro substitution on the phenyl ring for the phenoxymethyl, benzyloxy and (phenylsulfanyl)methyl linkers (1c vs 1d; 1e vs 1f; 1g vs 1h), with the exception of the benzylsulfanyl linker (1i vs 1j), for which a significant 11-fold decrease was observed. Therefore, the introduction of a para-chloro on the
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benzylsulfanyl (1j) resulted in the weakest A1 and A2A AR affinity among the test compounds (1a–k). An overall decrease in A1 and A2A AR affinity was found when comparing the position of the oxygen and the sulphur in the para-chloro-substituted derivatives (phenoxymethyl 1d vs benzyloxy 1f and
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(phenylsulfanyl)methyl 1h vs benzylsulfanyl 1j). Interestingly, the sulphur-containing linkers displayed the most significant decrease (34-fold) in the A2A AR affinity when the sulphur moved from the position next to the phenyl ring [(phenylsulfanyl)methyl] to that next to C8 (benzylsulfanyl) of the
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caffeine scaffold (1h A2AKi = 1.5 µM vs 1j A2AKi = 51.43 µM). In addition, the introduction of nitrogen was explored by comparing compound 1k, that bore a benzylamide side-chain, with the unsubstituted phenylethyl (1b), benzyloxy (1e) and benzylsulfanyl (1i) linkers. The influence of the different atoms (C, N, O, S) on the A2A AR affinity was not significant and the corresponding affinities were in the same range: O > N > S > C (1e 4.30 µM > 1k 4.50 µM > 1i 4.64 µM > 1b 4.95 µM). The A1 AR affinity showed a larger variation in Ki values such that the oxygen exhibited a 5-fold increase compared to the nitrogen: O > S > C > N (1e 1.52 µM > 1i 4.32 µM > 1b 5.74 µM > 1k 7.95 µM).
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Compounds 1b, 1c and 1i were found to act non-selectively in respect of the A1 and A2A ARs (Table 1). The position of the oxygen, phenoxymethyl vs benzyloxy, favoured A1 AR selectivity (1c SI = 0.86 vs 1e SI = 2.83); introducing a para-chloro substituent achieved A2A AR selectivity (1c SI = 1.3 vs 1d SI = 5.71, 1g SI = 1.5 vs 1h SI = 13.89). Even though the test compounds exhibit values in the
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low micromolar range, the structural modifications investigated in this study might contribute significantly to increased A1 and A2A AR affinity and selectivity in caffeine derivatives.
Compounds 1c and 1e were identified as possessing the highest affinity for the A1 AR and so were selected for functional GTP shift assays to determine if these compounds acted as agonists or
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antagonists (as expected). The binding of an agonist to the A1 ARs is reduced in the presence of GTP due to the A1 adenosine receptor being shifted from a high- to a low-affinity state [32]. For this reason, an A1 AR agonist results in a corresponding rightward shift of the binding curve in the
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presence of GTP, whereas no shift of the curve would be observed in the presence of an A1 AR antagonist. Compounds 1c and 1e showed no significant shifts (Table1, Figure 2) of the binding curves in the presence of GTP and may therefore be considered as antagonists for the A1 ARs.
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Adenosine A1 binding
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Adenosine A1 binding
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Figure 2. The binding curves of compounds 1c and 1e, indicating their A1 AR antagonistic action as determined via GTP shift assays (with and without 0.1 mM GTP) in rat whole brain membranes, using [3H]DPCPX as radioligand. (A) GTP shift of 1.3 calculated for the A1 AR antagonist 1c. (B) GTP shift of 1.2 calculated for the A1 AR antagonist 1e.
ACCEPTED MANUSCRIPT Conclusion The caffeine analogues investigated (1b–1k) demonstrated their potential as A1 and A2A AR antagonists with Ki values in the micromolar range (A1Ki = 1.52–90.0 µM; A2AKi = 1.33–51.43 µM). In general, the caffeine analogues (1b–1k) displayed improved A1 and A2A AR affinity compared to caffeine (1a), with the only exception being compound 1j.
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Overall, the introduction of an oxygen in the two-chain-length linker increased both A1 and A2A AR affinity, with the unsubstituted benzyloxy linker (1e A1Ki = 1.52 µM) documented with the greatest affinity and selectivity (SI = 2.8) for the A1 AR. The introduction of a para-chloro substituent decreased A1 AR affinity, but significantly increased A2A AR affinity with the phenoxymethyl (1d)
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and (phenylsulfanyl)methyl (1h) compounds displaying the greatest A2A AR affinity among the parachloro-substituted analogues (1d A2AKi = 1.33 µM; 1h A2AKi = 1.50 µM).
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For these reasons, the 8-phenoxymethyl and 8-(phenylsulfanyl)methyl side-chains should not be disregarded when designing compounds with selectivity and affinity for the A2A ARs. The various C8 side-chains investigated in this study are thus important to understand the structure–activity relationships of caffeine analogues and we therefore conclude that suitable selection of a C8 sidechain may lead to the development of improved A1 and A2A AR affinity and selectivity for caffeine-
Author contributions
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based AR antagonists.
The manuscript was written by GT and MMvdW. The biological evaluations were performed by MMvdW and the interpretation of the data by GT and MMvdW. Both authors give their approval of
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this version of the manuscript. Acknowledgements
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We are grateful to Prof. J.P. Petzer of the North-West University for donating test compounds (1d– 1k). The financial assistance of the North-West University, the South African Medical Research Council and the National Research Foundation towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the authors and are not necessarily to be attributed to the NRF.
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[8] P.J. Richardson, H. Kase, P.G. Jenner, Adenosine A2A receptor antagonists as new agents for the treatment of Parkinson’s disease, Trends Pharmacol. Sci. 18 (1997) 338–344.
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[9] T. Mihara, K. Mihara, J. Yarimizu, Y. Mitani, R. Matsuda, H. Yamamoto, S. Aoki, A. Akahane, A. Iwashita, N. Matsuoka, Pharmacological characterization of a novel, potent adenosine A1 and A2A receptor dual antagonist, 5-[5-Amino-3-(4-fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), in models of Parkinson’s disease and cognition, J. Pharmacol. Exp. Ther. 323 (2007) 708–719.
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Highlights: Selected C8 two-chain linkers yield enhanced adenosine receptor affinity.
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A1 receptor antagonists are considered agents for Parkinson’s disease therapy.
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A2A receptor antagonists are considered agents for Parkinson’s disease therapy.
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Introduction of a para-chloro substitution govern A2A receptor selectivity.
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