European Journal of Medicinal Chemistry 70 (2013) 525e535
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
8-(2-Furyl)adenine derivatives as A2A adenosine receptor ligands Diego Dal Ben a, Michela Buccioni a, Catia Lambertucci a, Ajiroghene Thomas a, Karl-Norbert Klotz b, Stephanie Federico c, Barbara Cacciari d, Giampiero Spalluto c, Rosaria Volpini a, * a
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, I-62032 Camerino, MC, Italy Institut für Pharmakologie und Toxikologie, University of Würzburg, D-97078 Würzburg, Germany c Dipartimento di Scienze Chimiche e Farmaceutiche, University of Trieste, Piazzale Europa 1, I-34127 Trieste, Italy d Dipartimento di Scienze Chimiche e Farmaceutiche, University of Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 July 2013 Accepted 3 October 2013 Available online 17 October 2013
Selective adenosine receptor modulators are potential tools for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases. In this work, the synthesis and biological evaluation at the four human adenosine receptor subtypes of a series of 9-substituted 8-(2furyl)adenine derivatives are reported. Results show that 8-(2-furyl)-9-methyladenine is endowed with high affinity at the A2A subtype. Further modification of this compound with introduction of arylacetyl or arylcarbamoyl groups in N6-position takes to different effects on the A2A affinity and in particular on the selectivity versus the other three adenosine receptor subtypes. A molecular modelling analysis at three different A2A receptor crystal structures provides an interpretation of the obtained biological results. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Purinergic receptors Adenosine receptors Adenosine receptor antagonists Adenine derivatives Purine derivatives Molecular modelling
1. Introduction Agonism and antagonism of adenosine receptors (ARs) are responsible of several effects in different organ systems suggesting that regulation of ARs has substantial therapeutic potential [1]. All ARs are G protein-coupled receptors (GPCRs), with four subtypes classified as A1, A2A, A2B, and A3 ARs [2e4], which exert their physiological role by activation or inhibition of different second messenger systems. In particular, the modulation of adenylyl cyclase activity could be considered as the principal signal mediated by these receptors [5,6]. Furthermore, ARs can be distinguished on the base of their tissue distribution and unique pharmacological profiles. In fact, a variety of physiological actions can be referred to adenosine, including effects on heart rate and atrial contractility, vascular smooth muscle tone, release of
Abbreviations: AR, adenosine receptor; GPCR, G protein-coupled receptor; CNS, central nervous system; DMF, dimethylformamide; NBS, N-bromosuccinimide; CHO, Chinese hamster ovary; pdb, protein data bank; EL, extracellular loop; IL, intracellular loop; MOE, molecular operating environment; RMSD, root mean square deviation; TM, transmembrane; TLC, thin-layer chromatography; IR, infrared; THF, tetrahydrofuran. * Corresponding author. Tel.: þ39 0737 402278; fax: þ39 0737 402295. E-mail address:
[email protected] (R. Volpini). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.10.006
neurotransmitters, lipolysis, as well as renal, platelet, and white blood cell functions [7]. Hence, selective AR modulators are promising for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases [1]. The prototypical AR antagonists are the naturally occurring methylxanthines, whose modifications led to derivatives endowed with marked potency and selectivity for specific AR subtypes [8e 10]. However, only a few xanthine antagonists such as caffeine and theophylline have been approved as drugs for their central nervous system (CNS) stimulating, diuretic, and bronchodilating effects [10,11]. Very recently, the A2AAR antagonist istradefylline (KW-6002) has been approved for manufacturing and marketing in Japan for the treatment of Parkinson’s disease [12]. Many different classes of compounds have been proposed as AR antagonists, with both good affinity and selectivity [13,14]. Among them, a series of AR antagonists have been obtained by removing the ribose moiety from the natural ligand adenosine and by modifying the adenine core. In fact, the introduction of different substituents in 2-, 8-, and 9-position of adenine resulted in highaffinity antagonists with distinct receptor selectivity profile [15e 26]. In particular, some 8-substituted-9-ethyladenine derivatives were reported to ameliorate motor deficits in rat models of Parkinson’s disease, suggesting potential therapeutic application for these compounds [27]. Among them, the 8-bromo-9-ethyladenine
526
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
(1; Fig. 1, Table 1) showed good affinity (Ki A2AAR ¼ 52 nM) and moderate selectivity at A2AAR especially vs the A1 receptor subtype (selectivity A1/A2A ¼ 5). Substitution of the bromine atom with a 2-furyl ring in 8-position of this molecule led to 9-ethyl-8-furyl adenine (2), which showed increased affinity at all ARs and in particular at the A2A subtype, but with still low selectivity vs the A1AR (2; Ki A2AAR ¼ 3.7 nM, selectivity A1/A2A ¼ 6; Fig. 1, Table 1) [28]. In this work, aimed at modulating the affinity and selectivity at the A2A subtype of compound 2, its 9-ethyl group was replaced by small alkyl or hydroxyalkyl chains (12e15; Fig. 1, Scheme 1). The choice of the 9-substituents was made on the base of previously reported results indicating that the introduction of small alkyl or hydroxyalkyl chains in 9-position of 8-bromoadenine (8e 11, Scheme 1, Table 1) leads to A2AAR ligands endowed with high affinity for the same subtype [22]. Among the newly synthesized 8-furyl derivatives 12e15, the 8-(2-furyl)-9-methyladenine (12) was selected for further investigations since it showed the best A2AAR selectivity profile (Scheme 1, Table 1). Very recently, a trisubstituted adenosine derivative named UK-432097 (Fig. 1), bearing a large and lipophilic group at N6-position, has been reported as a potent and selective A2AAR agonist [29]. Starting from this observation, in a second phase of the present work, large and lipophilic arylacetyl and arylcarbamoyl substituents, which are
more flexible respect to the 2,2-diphenylethyl group present in UK-432097, were introduced at N6-position of the selected compound 12 to give the newly trisubstituted adenine derivatives 16e 22 (Fig. 1, Scheme 2). 2. Results and discussion 2.1. Chemistry Desired compounds 12e22 were obtained as summarized in Schemes 1 and 2. Alkylation of commercially available adenine (3) with the suitable alkyl halide in dry dimethylformamide (DMF), employing potassium carbonate as base, led to a mixture (ratio 3:1) of the N9- and N7-isomers, 4e7 and 4ae7a, respectively, which were separated by chromatography (Scheme 1). Treatment of the N9 isomers 4e7 with N-bromosuccinimide (NBS) led to the 8-bromo derivatives 8e11 [22] that, after reaction with tributylstannylfurane in the presence of (PPh3)2PdCl2 as a catalyst, gave the desired 8-furyl-9-substituted adenine derivatives 12e15. N6-Substituted adenines 16e22 were prepared by reacting 12 with the appropriate isocyanate or acyl chloride in dioxane at reflux for 1e2 h in the presence of triethylamine (Scheme 2).
Fig. 1. Structure of known and newly synthesized AR ligands.
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
527
Table 1 Affinity (Ki, nM) of compounds 1, 2, 8e15 in radioligand binding assays at human A1, A2A, and A3 ARs and NECA-stimulated adenylyl cyclase assays at membranes from stably transfected CHO cells expressing human A2BAR subtype.
Scheme 2. Reagents: i: RCOCl, Et3N, dioxane, reflux, 2 h or ReN]C]O, dioxane, reflux, 1 h.
Cpd
R
X
Ki A1ARa Ki A2AARb Ki A2BARc Ki A3ARd
1 [28] 2 [28] 8 [28] 12 9 [28] 13 10 [28] 14 11 [28] 15
CH2CH3 CH2CH3 CH3 CH3 CH2CH2OH CH2CH2OH CH2CH2CH3 CH2CH2CH3 CH2CH2CH2OH CH2CH2CH2OH
Br 2-Furyl Br 2-Furyl Br 2-Furyl Br 2-Furyl Br 2-Furyl
280 24 570 62 1500 69 1100 19 1000 60
52 3.7 120 7.5 620 33 300 18 85 15
840 375 720 786 2800 470 200 250 5100 350
27,800 4670 >100,000 6419 >100,000 3900 100,000 790 3200 4300
a Displacement of specific [3H]CCPA binding in CHO cells transfected with human recombinant A1AR. b Displacement of specific [3H]NECA binding in CHO cells transfected with human recombinant A2AAR. c Ki values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing human A2BAR. d Displacement of specific [3H]NECA binding in CHO cells transfected with human recombinant A3AR.
2.2. Biological results and structureeactivity relationships Newly synthesized compounds 12e22 were evaluated at the human recombinant adenosine receptors, stably transfected into Chinese hamster ovary (CHO) cells, utilizing radioligand binding studies (A1, A2A, and A3 ARs) or adenylyl cyclase activity assay (A2BAR). Receptor binding affinity was determined using [3H]CCPA as radioligand for A1AR, whereas [3H]NECA was used for the A2A and A3 subtypes. In the case of A2BAR, Ki values were calculated from IC50 values determined by inhibition of NECA-stimulated adenylyl cyclase activity [30]. The binding and functional data of the new compounds are shown in Tables 1 and 2. Table 1 shows
Scheme 1. Reagents: i: ReX, DMF, K2CO3, r.t.; ii: NBS, MeCN, MeOH, r.t.; iii: 2tributylstannylfurane, (Ph3P)2PdCl2, THF, 70 C.
data of the new molecules 12e15; the adenine derivatives 1, 2 and 8e11 [28] are reported as reference compounds. Biological results show that the newly synthesised compounds 12e15 are endowed with AR affinity at A1 and A2A subtypes in the nanomolar range, while they bind the A3 ARs at higher concentrations. The analysis of the affinity data of these compounds with respect to the binding data of the 8-bromo analogues 8e11 [22] shows that the substitution of a bromine atom with a 2-furyl ring in 8-position significantly increases the affinity at all AR subtypes, with low nanomolar binding data obtained at A2AAR (12: Ki A2AAR ¼ 7.5 nM vs 8: Ki A2AAR ¼ 120 nM; 13: Ki A2AAR ¼ 33 nM vs 9: Ki A2AAR ¼ 620 nM; 14: Ki A2AAR ¼ 18 nM vs 10: Ki A2AAR ¼ 300 nM; 15: Ki A2AAR ¼ 15 nM vs 11: Ki A2AAR ¼ 85 nM). An exception to this trend is given by compound 15 that shows an A3AR affinity comparable to that of the corresponding 8-bromo derivative 11 (Ki of 4300 and 3200 nM, respectively). These data confirm our previous observation about the helpful role for AR affinity of 2-furyl ring in 8-position of adenine derivatives already detected in the case of compound 2 in comparison with 1. Comparing the binding data of compounds 12e15 at the other ARs versus the A2AAR, it can be observed that the affinity of these compounds at A3AR results at low micromolar levels, leading to compound selectivity at A2AAR respect to these AR subtype, in particular in the case of molecule 12 (A3/A2A ¼ 856). On the other hand, it must be noted that the selectivity versus A1AR represents the most critical issue related to 8-furyl adenine derivatives. In fact, it can be observed that these molecules present good affinity for A1AR, with compound 14 being the best ligand of the series for this AR subtype (Ki A1AR ¼ 19 nM). This compound is endowed with almost identical A1AR and A2AAR affinity values. In general, the selectivity versus A1AR is low, with the best result obtained with the 8-furyl-9-methyladenine (12; A1/A2A ¼ 8). Although this compound shows a slight decrease of A2AAR affinity respect to the previously synthesized 8-furyl derivative 2 (12: Ki A2AAR ¼ 7.5 vs 2: Ki A2AAR ¼ 3.7), its A2AAR vs A1AR selectivity is slightly improved (12; A1/A2A ¼ 8 vs 2: A1/A2A ¼ 6). The potency of compounds 12e15 at A2BAR is at high nanomolar concentrations and even in this case the substitution of 8-bromine with a 2-furyl group leads to an improvement of potency a part in the case of compounds 12 and 14 (Ki of 786 and 250 nM, respectively) respect to the corresponding 8bromo analogues 8 and 10 (Ki of 720 and 200 nM, respectively). In general the selectivity for A2AAR versus A2BAR (please note that Ki A2AAR values are related to displacement of specific [3H]NECA binding while Ki A2BAR values are referred to the inhibition of NECA-stimulated adenylyl cyclase activity) results improved respect to the 8-bromo analogues, in particular in the case of molecule 12 (A2B/A2A ¼ 105). Hence, taking in account for each synthesised molecule the combination of its A2AAR affinity and selectivity versus the other AR subtypes, compound 12 was selected as starting point for the preparation of a second series of derivatives bearing a 2-furyl ring in 8-position, a methyl group in 9-position, and different arylacetyl
528
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
Table 2 Affinity (Ki, nM) of compounds 16e22 in radioligand binding assays at human A1, A2A, and A3 ARs. NECA-stimulated adenylyl cyclase assays at membranes from stably transfected CHO cells expressing human A2BAR subtype.
Cpd
R
Ki A1ARa
Ki A2AARb
Ki A2BARc
Ki A3ARd
12
H
62.3
7.5
786
6419
16
583
110
2510
1952
17
652
1270
>30,000
20,285
18
3000
59
792
193
19
438
50
5070
5233
20
922
89
2780
906
21
496
57
6250
23,307
22
756
106
>30,000
37,957
a b c d
Displacement of specific [3H]CCPA binding in CHO cells transfected with human recombinant A1AR. Displacement of specific [3H]NECA binding in CHO cells transfected with human recombinant A2AAR. Ki values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing human A2BAR. Displacement of specific [3H]NECA binding in CHO cells transfected with human recombinant A3AR.
or arylcarbamoyl moieties in N6-position. The obtained compounds 16e22 were then tested at the four human ARs as described above (Table 2). In general the introduction of an acyl moiety at the N6-position of 8-furyl-9-methyladenine (12) led to a decrease of affinity at all the four ARs with some exceptions at A3 subtype. In particular, the introduction of a phenylacetyl group in N6-position led to a decrease (3e15 fold) of A1 and A2AAR affinity with an improvement of A3AR binding (16: Ki A1AR ¼ 583 nM; Ki A2AAR ¼ 110 nM; Ki
A3AR ¼ 1952 nM), while the introduction of a biphenylcarbonyl moiety (17) in the same position led to a general drop of AR affinity, especially in the case of A2AAR (170 fold). Introduction of a second phenyl ring in the benzylic carbon atom of the N6-substituent of compound 16 led to compound 18 endowed with the highest selectivity versus the A1AR but, at the same time, with the lowest selectivity versus A3AR (18: Ki A1AR ¼ 3000 nM; Ki A2AAR ¼ 59 nM; Ki A3AR ¼ 193 nM). Further modification of compound 16 by insertion of a benzyloxy group in para position of the N6-
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
529
substituent led to compound 19 presenting the highest A2AAR affinity of this second series (Ki A2AAR ¼ 50 nM) and a comparable selectivity versus A1AR respect to the N6-unsubstituted compound 12. The introduction of a methyl group at the para position of benzyloxy moiety (20) led to a reduction of potency at the A1 and A2AARs but an increased affinity for the A3AR. The presence of an arylcarbamoyl moiety (compounds 21 and 22) reduced the affinity at the four ARs (5e30 fold) respect to the unsubstituted compound 12. On the other hand compound 21 showed a good affinity at A2AAR (21: Ki A2AAR ¼ 57 nM) and selectivity versus the other AR subtypes, with a slight improvement respect to compound 16 in particular versus A1AR and A2BAR. The introduction of a methyl group at the para position of arylcarbamoyl moiety (22) increased the selectivity versus A2BAR (even in this case, please note that Ki A2AAR values are related to displacement of specific [3H]NECA binding while Ki A2BAR values are referred to the inhibition of NECA-stimulated adenylyl cyclase activity) maintaining at the same time comparable selectivity versus A1AR and A3AR respect to compound 21. Considering the antagonist potency at A2BAR, the introduction of a phenylacetyl group in N6-position led to a decrease of activity at this AR subtype (16: Ki A2BAR ¼ 2510 nM), while the presence of a second phenyl ring in the benzylic carbon atom of the N6-substituent of compound 16 led to a compound with the best potency data of this second group of derivatives (18: Ki A2BAR ¼ 792 nM). Insertion of a benzyloxy group or a paramethylbenzyloxy group in para position of the N6-substituent of 16 led to compounds 19 and 20, respectively, presenting partially restored potency at A2BAR. The introduction of an arylcarbamoyl moiety (compounds 21 and 22) did not improved the A2BAR potency leading to compounds selective for A2AAR versus this AR subtype. 2.3. Molecular modelling A molecular modelling analysis was performed to better understand the different interaction of synthesised molecules with A2AAR. In particular, molecular docking methods were employed to simulate the interaction of compounds 12e22 with the binding site of the three human A2AAR crystal structures solved in complex with ZM241385 (Fig. 1) antagonist (Protein Data bank e pdb-code: 3EML; 2.6- A resolution [31]; pdb code: 3PWH; 3.3- A resolution [32]; pdb code: 3VG9; 2.7- A resolution [33]). Compound 1 was also subjected to this analysis. The choice of the A2AAR structures to be used for this analysis was based on the fact that the analysed compounds present several structural analogies with respect to the co-crystallized ZM241385 antagonist (which can be taken as “comparison term” in our analysis), like a similar scaffold and the presence of an exocyclic amino group (in particular considering compounds 2, 12e15) and of a 2-furyl ring in analogue scaffold positions. Furthermore, the three A2AAR structures present highly similar binding sites by considering both pocket volumes and receptor residues orientation. Nevertheless, rearrangements of some flexible residues and conformational variability at extracellular loops (EL) in peripheral regions of binding site are observed. The most evident residue rearrangement is a different orientation of E169 side chain that interacts with the ZM241385 exocyclic amino group in 3EML and 3VG9 structures while it is externally oriented in 3PWH X-ray data (hence interacting with the “tail segment” of the same antagonist) (Fig. 2). A first consequence of this variation is a different conformation of the “tail segment” (the amino-ethylphenol group) of the ZM241385 antagonist, while its triazolotriazine scaffold is located and oriented in similar way in the three crystal structures. A second consequence of E169 rearrangement is the formation (in the case of 3EML and 3VG9 crystal structures) or the loss (in the case of 3PWH X-ray) of an H-bond interaction with
Fig. 2. A. Comparison of binding mode of compound ZM241385 to A2AAR as observed in two published crystal structures (pdb code 3EML in dark, pdb code 3PWH in light); the different orientation of residues E169 and H264 is highlighted. BeC. Surface representation of the A2AAR binding site as observed from the extracellular environment; 3EML and 3VG9 crystal structures (B) are represented in dark and light, respectively, while 3PWH crystal structure (C) is coloured light; the different orientation of the above cited E169 and H264 residues allows a different exposure of ligand to extracellular environment.
H264 residue (EL3). In the first case the top of the binding site results partially obstructed, with the phenol group of ZM241385 antagonist as the sole ligand moiety exposed to the external environment (Fig. 2B). In the second case, even the region in proximity of ZM241385 free amino group is externally exposed (Fig. 2C). On this base, we decided to employ all three A2AAR-ZM241385 crystal structures even to verify the effect of this binding site variation on the binding mode of synthesised molecules. Each crystal structure was re-modelled by firstly removing eventual external segments
530
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
(i.e. the T4L segment present in 3EML crystal structure) and secondly by performing a building of eventual missing receptor regions (i.e. missing sections of EL2 or intracellular 3-IL3-domains). This procedure allowed also the rebuilding of wild type receptors from mutated A2AAR structures (i.e. 3PWH crystal structure). The obtained A2AAR models were checked by using the Protein Geometry Monitor application within Molecular Operating Environment (MOE, version 2010.10) suite [34], which provides a variety of stereochemical measurements for structural quality inspection in a given protein, such as backbone bond lengths, angles and dihedrals, Ramachandran 4ej dihedral plots, and side chain rotamer and nonbonded contact quality. A2AAR structures were then used as target for the docking analysis of synthesised derivatives. All ligand structures were optimized using RHF/AM1 semi-empirical calculations and the MOPAC software package implemented in MOE was utilized for these calculations [35]. The compounds were then docked into the binding site of the A2AAR models by using the MOE Dock tool. Docking poses of each compound were subjected to energy minimization and then rescored using three available methods implemented in MOE: the London dG scoring function that estimates the free energy of binding of the ligand from a given pose; the Affinity dG scoring tool that estimates the enthalpic contribution to the free energy of binding; the dock-pKi predictor that uses the MOE scoring.svl script to estimate for each ligand a pKi value, which is described by the Hbonds, transition metal interactions, and hydrophobic interactions energy. For each compound, the top-score docking pose at each A2AAR model, according to at least two out of three scoring functions, was selected for final ligandetarget interaction analysis. The docking methodology was firstly tested by comparing the predicted binding mode of ZM241385 within A2AAR pocket respect to the X-ray data. The docking method showed good ability in reproducing the ZM241385 binding mode observed in the experimental data. In particular, the Root Mean Square Deviation (RMSD) from the comparison of the crystal structure and the docking conformation was calculated as 1.45, 1.33, and 2.73 A (for the 3EML, 3PWH, and 3VG9 crystal structure, respectively), with the best matching being given by the 2-(furan-2-yl)-[1,2,4]triazolo[1,5-a] [1,3,5]triazin-7-amine moiety and the highest deviation being given by a slightly different orientation of the 4-(2-aminoethyl) phenol group. Considering the general binding mode of N6-unsubstituted adenine derivatives (2, 12e15), docking results show that the compounds share a similar binding motif inside the transmembrane (TM) region of the A2AAR models with the adenine pyrimidine ring located between TM3 and TM7 domains and the imidazole ring located between TM3 and TM6 helices. This scaffold orientation makes the 2-furyl ring in 8-position to be oriented towards the central TM core. This binding mode makes the adenine moiety location and orientation quite similar with respect to compound ZM241385 scaffold as observed from the X-ray data (Fig. 3A). The synthesised compounds show also analogue interaction with the receptor respect to the co-crystallised ligand, as the key receptor residues for the interaction with these derivatives are in all cases F168 and N253. F168 phenyl ring and adenine scaffold form a p-stacking interaction, while N253 amide function is involved in H-bonding with adenine N6 amine and in an additional polar interaction with adenine scaffold N7 atom. The N6-amino group makes also an additional H-bond interaction with E169 residue, but this interaction is lost when docking simulations at 3PWH-based A2AAR model are considered. As described above, this residue has a different orientation in 3PWH crystal structure making impossible a polar interaction with the free amino group of adenine derivatives 2, 12e15.
Fig. 3. A. Comparison of docking pose of compound 12 (ball and stick style) at 3EMLbased A2AAR structure with the binding mode of compound ZM241385 (stick style) at the same X-ray. B. Docking pose of compound 15 at 3EML-A2AAR structure.
Considering the 9-substituents, docking results show that these groups are located in a narrow and mainly hydrophobic region. This data can explain why the methyl and ethyl substituents (2, 12) provide good affinity at the A2AAR and why the introduction of a hydroxyl group (13) causes a significant decrease of potency. When a hydroxypropyl substituent is present in 9-position (15), the A2AAR affinity is partially restored and this can be explained by docking results by the observation that the longer chain than compound 13 allows the hydroxyl function to be located in proximity of two polar regions given by the H278 side chain and the A59 and A81 carbonyl groups, at the bottom of the binding site (Fig. 3B). Results of docking simulations of compounds 16e22 highlight the effect of E169 rearrangement on ligand binding mode and on receptoreligand interaction. As described above, the presence or the loss of a H-bond between E169 and H264 causes a different availability of space for N6-substituents and this has an evident effect when bulky moieties are introduced at the adenine amino group. As example, docking conformations of compound 18 at 3EML and 3VG9 A2AAR structures present the N6-arylacetyl group in an approximately identical position to the “tail segment” of ZM241385 reference compound (Fig. 4A). This binding mode causes a disruption of the H-bonding between N253 and N7 and N6 amino group of adenine derivative. Docking experiments of the same
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
531
Fig. 4. A-B. Docking pose of compound 18 at 3EML (A) and 3PWH (B) A2AAR structures. The different amount of space available for N6-substituent is at the base of a slightly different binding mode and receptor interaction of the compound. CeD. Docking pose of compound 19 at 3EML (C) and 3PWH (D) A2AAR structures. The compound presents similar conformations but slightly different locations at the two binding sites due to the different orientations of E169 residues. EeF. Docking pose of compound 21 at 3EML (E) and 3PWH (F) A2AAR structures. The presence of an arylcarbamoyl moiety in N6-position takes to the formation of a H-bond between the amino group of the substituent and the N1 atom of adenine scaffold.
molecule at 3PWH A2AAR structure show a conservation of this Hbonding interaction (Fig. 4B). Interestingly, in this case the N6arylacetyl group results similarly located and oriented respect to the analogue 2,2-diphenylethyl group of compound UK-432097 as observed from another recently reported A2AAR crystal structure [36] that presents a E169 side chain orientation similar to 3PWH X-ray. As expected, the difference in binding mode to the three A2AAR structures is related also to the rigidity of the N6-substituent. Compounds 16, 19, 20 present similar docking conformations at the three receptor pockets due to some flexibility of the arylacetyl substituent, even if a slightly different position of compounds is observed if the respective binding modes at 3EML/3VG9 and 3PWH
A2AAR structures are compared (Fig. 4CeD). On the other hand, compounds 17, 21, 22 contain rigid groups that cause different behaviour during docking experiments. In particular the presence of an arylcarbamoyl moiety in N6-position (i.e. compound 21, Fig. 4) takes to the formation of a H-bond between the amino group of the substituent and the N1 atom of adenine scaffold, hence providing higher rigidity respect to the analogue arylacetyl substituted compounds. The rigidity of N6-substituent makes even more evident the different binding mode of these compounds at the 3EML/3VG9 and 3PWH A2AAR pockets. At the same time, the higher hydrophilicity of arylcarbamoyl substituents respect to the analogue arylacetyl group and the fact that the residue E169 (located in close proximity to the compound N6-position) is
532
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
substituted in A3AR by a valine could explain the higher selectivity versus A3AR of the arylcarbamoyl substituted molecules. The results of these docking experiments suggest that there can be some flexibility at A2AAR binding site that cause ligand rearrangement on the basis of different orientation of binding site residues. The docking simulation at different receptor conformations can help to interpret the ligand ability to adapt itself to different conformations of the binding pocket and hence the stability of ligandereceptor interaction.
4.2. General procedure for the preparation of compounds 12e15 To a solution of the appropriate bromine derivative 8e11 (1 mmol) in dry tetrahydrofuran (THF) (6 mL), (PhP3)2PdCl2 (0.06 mmol) and 2-tributylstannylfuran (5 mmol) were added and the resulting mixtures were stirred under reaction conditions described for each compound. Then the solvent was removed under reduced pressure and the crude residue purified by chromatography with the suitable mixture of solvents to obtain the desired compounds (12e15) as solids.
3. Conclusions Results from the biological assays reported in Tables 1 and 2 show that the synthesised 9-substituted 8-(2-furyl)-adenine derivatives are endowed with good affinity at the human A2AAR and with higher potency respect to the corresponding 8-bromo derivatives previously synthesised and reported in literature. In particular, 8-(2-furyl)-9-methyladenine (12) shows low nanomolar affinity at this AR subtype and high selectivity in particular versus A2B and A3 ARs, with an improvement of selectivity versus A1AR respect to the previously reported 9-ethyl analogue 1. Further modification of compound 12 with introduction of arylacetyl or arylcarbamoyl groups in N6-position did not take to an improvement of A2AAR affinity and had different impacts on the selectivity versus the other three adenosine receptor subtypes. In particular, the presence of an acyl moiety at the N6-position led to a general decrease of affinity at the four ARs with respect to the unsubstituted derivative 12 with the exception of compounds 18 and 20 that present a significant improvement of A3AR binding data. On the other hand, the introduction of an arylcarbamoyl moiety in N6 position of 12 (compounds 21 and 22) restored the A2AAR selectivity profile observed for the N6-unsubstituted compound. The comparative analysis of docking studies at the three different A2AAR crystal structures suggests that slight rearrangements are possible at the receptor binding site and that the ability of ligand to adapt its conformation to the binding site residues could be related to the stability of ligandereceptor interaction and hence to the binding affinity. In this sense, while the general binding mode of synthesised compounds at the receptor pocket is conserved at the three receptor structures, on the other hand the impact of the different N6-substituents can be more easily interpreted by comparing the results at the different A2AAR binding sites. 4. Experimental section
4.2.1. 8-(Furan-2-yl)-9-methyl-9H-purin-6-amine (12) Compound 12 was obtained from 8 by reaction at 60 C, overnight. The crude mixture has been purified through silica gel normal column chromatography eluting with CHCl3ecC6H12e MeOH (65:35:5, v/v). Yield 65%; white solid, m.p.: 220 C (dec). IR (KBr): 3285e2960, 1610, 1590, 1500, 1410 cm1; 1H NMR (DMSOd6): d 3.89 (s, 3H, CH3), 6.75 (t, J ¼ 1.6 Hz, 1H, H-40 ), 7.18 (dd, J ¼ 0.8 and 3.6 Hz, 1H, H-30 ), 7.32 (bs, 2H, NH2), 7.96 (m, 1H, H-50 ), 8.14 (s, 1H, H-2). Anal. Calcd. for C10H9N5O (M.W.: 215.21): C, 55.81; H, 4.22; N, 32.54. Found: C, 56.01; H, 4.24; N, 32.66. 4.2.2. 2-(6-Amino-8-(furan-2-yl)-9H-purin-9-yl)ethanol (13) Compound 13 was obtained from 9 by reaction at 60 C for 20 h. The crude mixture has been purified through silica gel normal column chromatography eluting with CHCl3ecC6H12eMeOH (70:25:5, v/v). Yield 87%; white solid (CHCl3ecC6H12eMeOH) m.p.: dec. 245 C. IR (KBr): 3281e2924, 1667, 1615, 1589, 1572, 1506, 1466 cm1; 1H NMR (DMSO-d6) d 3.74 (m, 2H, CH2eO), 4.46 (t, J ¼ 6.0 Hz, 2H, CH2eN), 5.01 (t, J ¼ 5.7 Hz, 1H, OH), 6.76 (m, 1H, H40 ), 7.22 (m, 1H, H-30 ), 7.35 (bs, 2H, NH2), 7.97 (m, 1H, H-50 ), 8.16 (s, 1H, H-2). Anal. Calcd. for C11H11N5O2 (M.W.: 245.24): C, 53.87; H, 4.52; N, 28.56. Found: C, 53.99; H, 4.45; N, 28.66. 4.2.3. 8-(Furan-2-yl)-9-propyl-9H-purin-6-amine (14) Compound 14 was obtained from 10 by reaction at 60 C for 30 min. The crude mixture has been purified through silica gel normal column chromatography eluting with cC6H12eCHCl3e MeOH (60:35:5, v/v) to give final compound 14. Yield 50%; white solid (MeOH) m.p.:193e195 C; IR (KBr): 3308, 3122, 2923, 1661, 1622, 1589, 1570, 1518, 1469 cm1; 1H NMR (DMSO-d6): d 0.86 (t, J ¼ 7.5, 3H, CH3), 1.76 (m, 2H, NCH2CH2), 4.39 (t, J ¼ 7.3, 2H NCH2), 6.77 (m, 1H, H-40 ), 7.17 (d, J ¼ 3.7, 1H, H-30 ), 7.35 (bs, 2H, NH2), 7.99 (d, J ¼ 1.0, 1H, H-50 ), 8.17 (s, 1H, H-2). Anal. Calcd. for C12H13N5O (M.W.: 243.26): C, 59.25; H, 5.39; N, 28.79.Found: C, 59.33; H, 5.24; N, 28.85.
4.1. Chemistry General: Reactions were routinely monitored by thin-layer chromatography (TLC) on silica gel (precoated F254 Merck plates) and products visualized with UV light l ¼ 254 nm and iodine or potassium permanganate solution. Infrared (IR) spectra were measured on a Perkin Elmer 257 instrument. 1H NMR were determined in DMSO-d6 solutions with a Bruker AC 200 spectrometer, peaks positions are given in parts per million (d) downfield from tetramethylsilane as internal standard, and J values are given in Hz. Light petroleum ether refers to the fractions boiling at 40e60 C. Melting points were determined on a Buchi-Tottoli instrument and are uncorrected. Chromatographies were performed using Merck 60e200 mesh silica gel. All reported products showed IR and 1H NMR spectra in agreement with the assigned structures. Organic solutions were dried over anhydrous magnesium sulphate. Elemental analyses were performed by the microanalytical laboratory of Department of Chemical and Pharmaceutical Sciences, University of Trieste, and they were within 0.4% of the theoretical values for C, H, and N.
4.2.4. 3-(6-Amino-8-(furan-2-yl)-9H-purin-9-yl)propan-1-ol (15) Compound 15 was obtained from 11 by reaction at r.t. overnight. The crude mixture has been purified through silica gel normal column chromatography eluting with CHCl3eMeOH (96:4, v/v) to give final compound 15. Yield 85%; white solid (MeOH) m.p.: 203e 205 C; IR (KBr): 3320e2924, 1662, 1614, 1594, 1574, 1499, 1458 cm1; 1H NMR (DMSO-d6): d 0.86 (t, J ¼ 7.5 Hz, 3H, CH3), 1.90 (m, 2H, CH2CH2CH2), 3.46 (m, 2H, CH2O), 4.45 (t, J ¼ 7.2 Hz, 2H Ne CH2), 4.72 (t, J ¼ 5.4 Hz, 1H, OH), 6.77 (m, 1H, H-40 ), 7.21 (d, J ¼ 3.3 Hz, 1H, H-30 ), 7.39 (bs, 2H, NH2), 7.98 (d, J ¼ 1.2 Hz, 1H, H-50 ), 8.17 (s, 1H, H-2). Anal. Calcd. for C12H13N5O2 (M.W.: 259.26): C, 55.59; H, 5.05; N, 27.01. Found: C, 56.11; H, 4.94; N, 27.03. 4.3. General procedure for the preparation of compounds 16e20 To a solution of amino compound 12 (50 mg, 0.232 mmol) in dry dioxane (5 mL) the appropriate acyl chloride (1.39 mmol) and triethylamine (32 mL, 0.232 mmol) were added and stirred at reflux for 2 h. Then the solvent was removed in vacuo and the residue
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
purified by flash chromatography eluting with EtOAcelight petroleum (7:3, v/v) to afford the desired compounds (16e20) as a solid. 4.3.1. N-(8-(Furan-2-yl)-9-methyl-9H-purin-6-yl)-2phenylacetamide (16) Compound 16 has been prepared by reaction of 12 with 2phenylacetyl chloride. Yield 73%; pale yellow solid (EtOAc-light petroleum) m.p.: 185 C; IR (KBr): 3325e2965, 1680, 1600, 1505, 1415 cm1; 1H NMR (DMSO-d6): d 3.92 (s, 2H, CH2), 4.01 (s, 3H, CH3), 6.83 (dd, 1H, J ¼ 1.6 Hz, J ¼ 3.4 Hz, H-40 ), 7.26e7.40 (m, 6H, H30 , H-Ph), 8.07 (d, 1H, J ¼ 1.6 Hz, H-50 ), 8.64 (s, 1H, H-2), 10.99 (s, 1H, NH). Anal. Calcd. for C18H15N5O2 (M.W.: 333.34): C, 64.86; H, 4.54; N, 21.01. Found: C, 64.70; H, 4.47; N, 21.08. 4.3.2. N-(8-(Furan-2-yl)-9-methyl-9H-purin-6-yl)-[1,10 -biphenyl]4-carboxamide (17) Compound 17 has been prepared by reaction of 12 with [1,10 biphenyl]-4-carbonyl chloride. Yield 64%; dark yellow solid (EtOAce light petroleum) m.p.: 205 C; IR (KBr): 3335e2980, 1680, 1615, 1510, 1400 cm1; 1H NMR (DMSO-d6): d 4.05 (s, 3H, CH3), 6.82 (dd, 1H, J ¼ 1.6 Hz, J ¼ 3.4 Hz, H-40 ), 7.40 (d, 1H, J ¼ 3.4 Hz, H-30 ), 7.44e7.56 (m, 3H, H-Ph), 7.80 (d, 2H, J ¼ 6.8 Hz, H-Ph), 7.88 (d, 2H, J ¼ 8.2 Hz, HPh), 8.07 (d, 1H, J ¼ 1.6, H-50 ), 8.17 (d, 2H, J ¼ 8.2, H-Ph), 8.76 (s, 1H, H2), 11.26 (s, 1H, NH). Anal. Calcd. for C23H17N5O2 (M.W.: 395.41): C, 69.86; H, 4.33; N, 17.71. Found: C, 69.57; H, 4.40; N, 17.78. 4.3.3. N-(8-(Furan-2-yl)-9-methyl-9H-purin-6-yl)-2,2diphenylacetamide (18) Compound 18 has been prepared by reaction of 12 with 2,2diphenylacetyl chloride. Yield 70%; yellow solid (EtOAcelight petroleum) m.p.: 130 C; IR (KBr): 3330e2975, 1685, 1610, 1515, 1410 cm1; 1H NMR (DMSO-d6): d 4.01 (s, 3H, CH3), 5.67 (s, 1H, COCH), 6.83 (dd, 1H, J ¼ 1.8 Hz, J ¼ 3.4 Hz, H-40 ), 7.26e7.39 (m, 11H, H-30 , H-Ph), 8.08 (d, 1H, J ¼ 1.8 Hz, H-50 ), 8.65 (s, 1H, H-2), 11.25 (s, 1H, NH). Anal. Calcd. for C24H19N5O2 (M.W.: 409.44): C, 70.40; H, 4.68; N, 17.10. Found: C, 70.26; H, 4.54; N, 17.19. 4.3.4. 2-(4-(Benzyloxy)phenyl)-N-(8-(furan-2-yl)-9-methyl-9Hpurin-6-yl)acetamide (19) Compound 19 has been prepared by reaction of 12 with 2-(4(benzyloxy)phenyl)acetyl chloride. Yield 72%; yellow solid (EtOAce light petroleum) m.p.: ¼ 110 C; IR (KBr): 3330e2970, 1680, 1625, 1530, 1420 cm1; 1H NMR (DMSO-d6): d 3.85 (s, 2H, CH2), 4.03 (s, 3H, CH3), 5.16 (s, 2H, OCH2), 6.85 (dd, 1H, J ¼ 1.8 Hz, J ¼ 3.4 Hz, H-40 ), 7.12 (d, 2H, J ¼ 8.6 Hz, H-Ph), 7.35e7.44 (m, 8H, H-30 , H-Ph), 8.10 (d, 1H, J ¼ 1.8 Hz, H-50 ), 8.64 (s, 1H, H-2), 10.91 (s, 1H, NH). Anal. Calcd. for C25H21N5O3 (M.W.: 439.47): C, 68.33; H, 4.82; N, 15.94. Found: C, 68.54; H, 4.87; N, 15.88. 4.3.5. N-(8-(Furan-2-yl)-9-methyl-9H-purin-6-yl)-2-(4-((4methylbenzyl)oxy)phenyl)acetamide (20) Compound 20 has been prepared by reaction of 12 with 2-(4-((4methylbenzyl)oxy)phenyl)acetyl chloride. Yield 76%; pale yellow solid (EtOAc-light petroleum) m.p.: 100 C; IR (KBr): 3320e2975, 1683, 1620, 1520, 1410 cm1; 1H NMR (DMSO-d6): d 2.29 (s, 3H, CH3), 3.83 (s, 2H, COCH2), 4.01 (s, 3H, NCH3), 5.03 (s, 2H, OCH2), 6.82 (dd,1H, J ¼ 1.8 Hz, J ¼ 3.6 Hz, H-40 ), 6.95 (d, 2H, J ¼ 8.6 Hz, H-Ph), 7.16e7.34 (m, 6H, H-Ph), 7.38 (d, 1H, J ¼ 3.6 Hz, H-30 ), 8.07 (d, 1H, J ¼ 1.8 Hz, H-50 ), 8.63 (s, 1H, H-2), 10.94 (s, 1H, NH). Anal. Calcd. for C26H23N5O3 (M.W.: 453.49): C, 68.86; H, 5.11; N, 15.44. Found: C, 68.71; H, 5.14; N, 15.29. 4.4. General procedure for the preparation of compounds 21 and 22 A solution of compound 12 (50 mg, 0.232 mmol) in dry dioxane (5 mL) and the appropriate isocyanate (0.697 mmol, 3 eq) was
533
stirred at reflux for 1 h. Then the solvent was removed in vacuum and the residue purified by flash chromatography eluting with EtOAcelight petroleum (7:3, v/v) to afford the desired compound 21 or 22 as a solid. 4.4.1. 1-(8-(Furan-2-yl)-9-methyl-9H-purin-6-yl)-3-phenylurea (21) Compound 21 has been prepared by reaction of 12 with phenyl isocyanate. Yield 65%; white solid (EtOAcelight petroleum) m.p.: 220 C; IR (KBr): 3335e2940, 1690, 1610, 1520, 1430 cm1; 1H NMR (DMSO-d6): d 4.02 (s, 3H, CH3), 6.84 (dd, 1H, J ¼ 1.8 Hz, J ¼ 3.6 Hz, H40 ), 7.08 (t, 1H, J ¼ 7.6 Hz, H-Ph), 7.32e7.40 (m, 3H, H-30 , H-Ph), 7.63 (d, 2H, J ¼ 8.4 Hz, H-Ph), 8.09 (d, 1H, J ¼ 1.8 Hz, H-50 ), 8.68 (s, 1H, H2), 10.09 (s, 1H, NH), 11.78 (s, 1H, NH). Anal. Calcd. for C17H14N6O2 (M.W.: 334.33): C, 61.07; H, 4.22; N, 25.14. Found: C, 60.87; H, 4.05; N, 25.27. 4.4.2. 1-(8-(Furan-2-yl)-9-methyl-9H-purin-6-yl)-3-(p-tolyl)urea (22) Compound 22 has been prepared by reaction of 12 with p-tolyl isocyanate. Yield 71%; white solid (EtOAcelight petroleum) m.p.: 212 C; IR (KBr): 3330e2945, 1690, 1620, 1515, 1410 cm1; 1H NMR (DMSO-d6): d 2.28 (s, 3H, CH3), 4.01 (s, 3H, NCH3), 6.82 (dd, 1H, J ¼ 1.6 Hz, J ¼ 3.4 Hz, H-40 ), 7.16 (d, 2H, J ¼ 8.2 Hz, H-Ph), 7.37 (d, 1H, J ¼ 3.4 Hz, H-30 ), 7.51 (d, 2H, J ¼ 8.2 Hz, H-Ph), 8.07 (d, 1H, J ¼ 1.6 Hz, H-50 ), 8.66 (s, 1H, H-2), 9.99 (s, 1H, NH), 11.70 (s, 1H, NH). Anal. Calcd. for C18H16N6O2 (M.W.: 348.36): C, 62.06; H, 4.63; N, 24.12. Found: C, 62.28; H, 4.55; N, 24.15. 4.5. Biological assay All pharmacological methods followed the procedures as described earlier [30]. In brief, membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtypes in a two-step procedure. In a first lowspeed step (1000g) cell fragments and nuclei were removed. The crude membrane fraction was sedimented from the supernatant at 100,000g. The membrane pellet was resuspended in the buffer used for the respective binding experiments, frozen in liquid nitrogen and stored at 80 C. For the measurement of adenylyl cyclase activity only one high speed centrifugation of the homogenate was used. The resulting crude membrane pellet was resuspended in 50 mM Tris/HCl, pH 7.4 and immediately used for the cyclase assay. For radioligand binding at A1AR 1 nM [3H]CCPA was used, whereas 30 and 10 nM [3H]NECA were used for A2A and A3 ARs, respectively. Non-specific binding of [3H]CCPA was determined in the presence of 1 mM theophylline, in the case of [3H]NECA 100 mM R-PIA was used. Ki-values from competition experiments were calculated with the program SCTFIT [37]. Inhibition of NECA-stimulated adenylyl cyclase activity was determined as a measurement of affinity of compounds. EC50values from these experiments were converted to Ki-values with the Cheng and Prusoff equation [38]. 4.6. Molecular modelling All molecular modelling studies were performed on a 2 CPU (PIV 2.0e3.0 GHz) Linux PC. Homology modelling, energy minimization, and docking studies were carried out using MOE (version 2010.10) suite [34]. All ligand structures were optimized using RHF/AM1 semiempirical calculations and the software package MOPAC implemented in MOE was utilized for these calculations [35].
534
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
4.6.1. Refinement of the human A2AAR structures The crystal structures of the hA2AAR in complex with ZM241385 were retrieved from the Protein Data Bank (http://www.rcsb.org; pdb code: 3EML; 2.6- A resolution [31]; pdb code: 3PWH; 3.3- A resolution [32]; pdb code: 3VG9; 2.7- A resolution [33]). Each structure was re-modelled by firstly removing eventual external segments (i.e. the T4L segment present in 3EML crystal structure) and secondly by performing a building of eventual missing receptor regions (i.e. missing sections of EL2 or IL3 domains). This procedure allowed also the rebuilding of wild type receptors from mutated hA2AAR structures (i.e. 3PWH crystal structure). The Homology Modelling tool of MOE was employed. In details, for each hA2AAR model, the boundaries identified from the used hA2AAR X-ray crystal structure were applied. The missing loop domains were built by the loop search method implemented in MOE. Once the heavy atoms were modelled, all hydrogen atoms were added, and the protein coordinates were then minimized with MOE using the AMBER99 force field [39]. The minimizations were performed by 1000 steps of steepest descent followed by conjugate gradient minimization until the RMS gradient of the potential energy was less than 0.05 kJ mol1 A1. Reliability and quality of these models were checked using the Protein Geometry Monitor application within MOE, which provides a variety of stereochemical measurements for inspection of the structural quality in a given protein, like backbone bond lengths, angles and dihedrals, Ramachandran 4ej dihedral plots, and sidechain rotamer and nonbonded contact quality. 4.6.2. Molecular docking analysis All compound structures were docked into the binding site of the three hA2AAR models using the MOE Dock tool. This method is divided into a number of stages: Conformational analysis of ligands. The algorithm generated conformations from a single 3D conformation by conducting a systematic search. In this way, all combinations of angles were created for each ligand. Placement. A collection of poses was generated from the pool of ligand conformations using Triangle Matcher placement method. Poses were generated by superposition of ligand atom triplets and triplet points in the receptor binding site. The receptor site points are alpha sphere centres which represent locations of tight packing. At each iteration a random conformation was selected, a random triplet of ligand atoms and a random triplet of alpha sphere centres were used to determine the pose. Scoring. Poses generated by the placement methodology were scored using two available methods implemented in MOE, the London dG scoring function which estimates the free energy of binding of the ligand from a given pose, and Affinity dG scoring which estimates the enthalpy contribution to the free energy of binding. The top 30 poses for each ligand were output in a MOE database. 4.6.3. Post docking analysis The docking poses of each compound were then subjected to AMBER99 force field energy minimization until the RMS gradient of the potential energy was less than 0.05 kJ mol1 A1. Receptor residues within 6 A distance from the ligand were left free to move, while the remaining receptor coordinates were kept fixed. AMBER99 partial charges of receptor and MOPAC output partial charges of ligands were utilized. Once the compound-binding site energy minimization was completed, receptor coordinates were fixed and a second energy minimization stage was performed leaving free to move only compound atoms. MMFF94 force field [40e46] was applied. For each compound, the minimized docking poses were then rescored using London dG and Affinity dG scoring functions and the dock-pKi predictor. The latter tool allows estimating the pKi for each ligand using the “scoring.svl” script
retrievable at the SVL exchange service (Chemical Computing Group, Inc. SVL exchange: http://svl.chemcomp.com). The algorithm is based on an empirical scoring function consisting of a directional hydrogen-bonding term, a directional hydrophobic interaction term, and an entropic term (ligand rotatable bonds immobilized in binding). The obtained pKi values must be considered as docking scores and not as prediction of binding affinity. For each compound, the three top-score docking poses according to at least two out of three scoring functions were selected for final ligandetarget interaction analysis. Acknowledgements This work was supported by Fondo di Ricerca di Ateneo (University of Camerino) and by a grant of the Italian Ministry for University and Research (PRIN2010-11 n 20103W4779_003). References [1] K.A. Jacobson, Z.G. Gao, Adenosine receptors as therapeutic targets, Nat. Rev. Drug Discov. 5 (2006) 247e264. [2] A.S. Robeva, R.L. Woodard, X. Jin, Z. Gao, S. Bhattacharya, H.E. Taylor, D.L. Rosin, J. Linden, Molecular characterization of recombinant human adenosine receptors, Drug Dev. Res. 39 (1996) 243e252. [3] B.B. Fredholm, G. Arslan, L. Halldner, B. Kull, G. Schulte, W. Wasserman, Structure and function of adenosine receptors and their genes, NaunynSchmiedeberg’s Arch. Pharmacol. 362 (2000) 364e374. [4] B.B. Fredholm, A.P. IJzerman, K.A. Jacobson, J. Linden, C.E. Muller, International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptorsean update, Pharmacol. Rev. 63 (2011) 1e34. [5] J.M. Downey, M.V. Cohen, K. Ytrehus, Y. Liu, Cellular mechanisms in ischemic preconditioning: the role of adenosine and protein kinase C, Ann. N.Y. Acad. Sci. 723 (1994) 82e98. [6] J.A. Auchampach, X. Jin, T.C. Wan, G.H. Caughey, J. Linden, Canine mast cell adenosine receptors: cloning and expression of the A3 receptor and evidence that degranulation is mediated by the A2B receptor, Mol. Pharmacol. 52 (1997) 846e860. [7] G. Cristalli, R. Volpini, Adenosine receptors: chemistry and pharmacology, Curr. Top. Med. Chem. 3 (2003) 355e469. [8] K.-N. Klotz, Adenosine receptors and their ligands, Naunyn-Schmiedeberg’s Arch. Pharmacol. 362 (2000) 382e391. [9] C.E. Muller, B. Stein, Adenosine receptor antagonists: structures and potential therapeutic applications, Curr. Pharm. Des. 2 (1996) 501e530. [10] S.A. Poulsen, R.J. Quinn, Adenosine receptors: new opportunities for future drugs, Bioorg. Med. Chem. 6 (1998) 619e641. [11] S. Hess, Recent advances in adenosine receptor antagonist research, Expert Opin. Ther. Pat. 11 (2001) 1533e1561. [12] Kyowa Hakko Kirin, Approval for Manufacturing and Marketing of NOURIASTÒ Tablets 20 mg, A Novel Antiparkinsonian Agent, 2013. http:// www.kyowa-kirin.com/news_releases/2013/e20130325_04.html. [13] S.L. Cheong, G. Venkatesan, P. Paira, R. Jothibasu, A.L. Mandel, S. Federico, G. Spalluto, G. Pastorin, Pyrazolo derivatives as potent adenosine receptor antagonists: an overview on the structureeactivity relationships, Int. J. Med. Chem. 2011 (2011). ID 480652. [14] S. Moro, Z.G. Gao, K.A. Jacobson, G. Spalluto, Progress in the pursuit of therapeutic adenosine receptor antagonists, Med. Res. Rev. 26 (2006) 131e159. [15] G. Cristalli, E. Camaioni, S. Costanzi, S. Vittori, R. Volpini, K.-N. Klotz, Characterization of potent ligands at human recombinant adenosine receptors, Drug Dev. Res. 45 (1998) 176e181. [16] E. Camaioni, S. Costanzi, S. Vittori, R. Volpini, K.-N. Klotz, G. Cristalli, New substituted 9-alkylpurines as adenosine receptor ligands, Bioorg. Med. Chem. 6 (1998) 523e533. [17] K.-N. Klotz, S. Kachler, C. Lambertucci, S. Vittori, R. Volpini, G. Cristalli, 9Ethyladenine derivatives as adenosine receptor antagonists: 2- and 8substitution results in distinct selectivities, Naunyn-Schmiedeberg’s Arch. Pharmacol. 367 (2003) 629e634. [18] R. Volpini, S. Costanzi, S. Vittori, G. Cristalli, K.-N. Klotz, Medicinal chemistry and pharmacology of A2B adenosine receptors, Curr. Top. Med. Chem. 3 (2003) 427e443. [19] R. Volpini, S. Costanzi, C. Lambertucci, S. Vittori, C. Martini, M.L. Trincavelli, K.N. Klotz, G. Cristalli, 2- and 8-alkynyl-9-ethyladenines: synthesis and biological activity at human and rat adenosine receptors, Purinergic Signal. 1 (2005) 173e181. [20] C. Lambertucci, S. Vittori, R.C. Mishra, D. Dal Ben, K.-N. Klotz, R. Volpini, G. Cristalli, Synthesis and biological activity of trisubstituted adenines as A2A adenosine receptor antagonists, Nucleosides Nucleotides Nucleic Acids 26 (2007) 1443e1446. [21] C. Lambertucci, G. Cristalli, D. Dal Ben, D.D. Kachare, C. Bolcato, K.-N. Klotz, G. Spalluto, R. Volpini, New 2,6,9-trisubstituted adenines as adenosine
D. Dal Ben et al. / European Journal of Medicinal Chemistry 70 (2013) 525e535
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
receptor antagonists: a preliminary SAR profile, Purinergic Signal. 3 (2007) 339e346. C. Lambertucci, I. Antonini, M. Buccioni, D. Dal Ben, D.D. Kachare, R. Volpini, K.-N. Klotz, G. Cristalli, 8-Bromo-9-alkyl adenine derivatives as tools for developing new adenosine A2A and A2B receptors ligands, Bioorg. Med. Chem. 17 (2009) 2812e2822. H. Harada, O. Asano, Y. Hoshino, S. Yoshikawa, M. Matsukura, Y. Kabasawa, J. Niijima, Y. Kotake, N. Watanabe, T. Kawata, T. Inoue, T. Horizoe, N. Yasuda, H. Minami, K. Nagata, M. Murakami, J. Nagaoka, S. Kobayashi, I. Tanaka, S. Abe, 2-Alkynyl-8-aryl-9-methyladenines as novel adenosine receptor antagonists: their synthesis and structureeactivity relationships toward hepatic glucose production induced via agonism of the A2B receptor, J. Med. Chem. 44 (2001) 170e179. R.A. de Ligt, P.A. van der Klein, J.K. von Frijtag Drabbe Kunzel, A. Lorenzen, F. Ait El Maate, S. Fujikawa, R. van Westhoven, T. van den Hoven, J. Brussee, A.P. IJzerman, Synthesis and biological evaluation of disubstituted N6-cyclopentyladenine analogues: the search for a neutral antagonist with high affinity for the adenosine A1 receptor, Bioorg. Med. Chem. 12 (2004) 139e149. M. Perreira, J.K. Jiang, A.M. Klutz, Z.G. Gao, A. Shainberg, C. Lu, C.J. Thomas, K.A. Jacobson, “Reversine” and its 2-substituted adenine derivatives as potent and selective A3 adenosine receptor antagonists, J. Med. Chem. 48 (2005) 4910e4918. P. Minetti, M.O. Tinti, P. Carminati, M. Castorina, M.A. Di Cesare, S. Di Serio, G. Gallo, O. Ghirardi, F. Giorgi, L. Giorgi, G. Piersanti, F. Bartoccini, G. Tarzia, 2n-Butyl-9-methyl-8-[1,2,3]triazol-2-yl-9H-purin-6-ylamine and analogues as A2A adenosine receptor antagonists. Design, synthesis, and pharmacological characterization, J. Med. Chem. 48 (2005) 6887e6896. A. Pinna, R. Volpini, G. Cristalli, M. Morelli, New adenosine A2A receptor antagonists: actions on Parkinson’s disease models, Eur. J. Pharmacol. 512 (2005) 157e164. R. Volpini, D. Dal Ben, C. Lambertucci, G. Marucci, R.C. Mishra, A.T. Ramadori, K.-N. Klotz, M.L. Trincavelli, C. Martini, G. Cristalli, Adenosine A2A receptor antagonists: new 8-substituted 9-ethyladenines as tools for in vivo rat models of Parkinson’s disease, Chem. Med. Chem. 4 (2009) 1010e1019. C.E. Muller, K.A. Jacobson, Recent developments in adenosine receptor ligands and their potential as novel drugs, Biochim. Biophys. Acta 1808 (2011) 1290e 1308. K.-N. Klotz, J. Hessling, J. Hegler, C. Owman, B. Kull, B.B. Fredholm, M.J. Lohse, Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells, Naunyn-Schmiedeberg’s Arch. Pharmacol. 357 (1998) 1e9. V.P. Jaakola, M.T. Griffith, M.A. Hanson, V. Cherezov, E.Y. Chien, J.R. Lane, A.P. Ijzerman, R.C. Stevens, The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist, Science 322 (2008) 1211e1217.
View publication stats
535
[32] A.S. Doré, N. Robertson, J.C. Errey, I. Ng, K. Hollenstein, B. Tehan, E. Hurrell, K. Bennett, M. Congreve, F. Magnani, C.G. Tate, M. Weir, F.H. Marshall, Structure of the adenosine A2A receptor in complex with ZM241385 and the xanthines XAC and Caffeine, Structure 19 (2011) 1283e1293. [33] T. Hino, T. Arakawa, H. Iwanari, T. Yurugi-Kobayashi, C. Ikeda-Suno, Y. Nakada-Nakura, O. Kusano-Arai, S. Weyand, T. Shimamura, N. Nomura, A.D. Cameron, T. Kobayashi, T. Hamakubo, S. Iwata, T. Murata, G-proteincoupled receptor inactivation by an allosteric inverse-agonist antibody, Nature 482 (2012) 237e240. [34] Molecular Operating Environment, C.C.G., Inc., 1255 University St., Suite 1600, Montreal, Quebec, Canada, H3B 3X3. [35] J.J. Stewart, MOPAC: a semiempirical molecular orbital program, J. Comput. Aided Mol. Des. 4 (1990) 1e105. [36] F. Xu, H. Wu, V. Katritch, G.W. Han, K.A. Jacobson, Z.G. Gao, V. Cherezov, R.C. Stevens, Structure of an agonist-bound human A2A adenosine receptor, Science 332 (2011) 322e327. [37] A. De Lean, A.A. Hancock, R.J. Lefkowitz, Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes, Mol. Pharmacol. 21 (1982) 5e16. [38] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108. [39] W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz, D.M. Ferguson, D.C. Spellmeyer, T. Fox, J.W. Caldwell, P.A. Kollman, A second generation force field for the simulation of proteins, nucleic acids, and organic molecules, J. Am. Chem. Soc. 117 (1995) 5179e5197. [40] T.A. Halgren, Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94, J. Comput. Chem. 17 (1996) 490e519. [41] T.A. Halgren, Merck molecular force field. II. MMFF94 van der Waals and electrostatic parameters for intermolecular interactions, J. Comput. Chem. 17 (1996) 520e552. [42] T.A. Halgren, Merck molecular force field. III. Molecular geometries and vibrational frequencies for MMFF94, J. Comput. Chem. 17 (1996) 553e586. [43] T.A. Halgren, Merck molecular force field. IV. conformational energies and geometries for MMFF94, J. Comput. Chem. 17 (1996) 587e615. [44] T.A. Halgren, R. Nachbar, Merck molecular force field. V. Extension of MMFF94 using experimental data, additional computational data, and empirical rules, J. Comput. Chem. 17 (1996) 616e641. [45] T.A. Halgren, MMFF VI. MMFF94s option for energy minimization studies, J. Comput. Chem. 20 (1999) 720e729. [46] T.A. Halgren, MMFF VII. Characterization of MMFF94, MMFF94s, and other widely available force fields for conformational energies and for intermolecular-interaction energies and geometries, J. Comput. Chem. 20 (1999) 730e748.
