Enantiospecific Recognition at the A2B Adenosine Receptor by Alkyl 2

Article pubs.acs.org/jmc

Enantiospecific Recognition at the A2B Adenosine Receptor by Alkyl 2‑Cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine5-carboxylates Carlos Carbajales,†,‡ Jhonny Azuaje,†,‡ Ana Oliveira,∥ María I. Loza,§ José Brea,§ María I. Cadavid,§ Christian F. Masaguer,‡ Xerardo García-Mera,‡ Hugo Gutiérrez-de-Terán,*,∥ and Eddy Sotelo*,†,‡ †

Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), ‡Departamento de Química Orgánica, Facultade de FarmaciaUniversidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain § Drug Screening Platform/Biofarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain ∥ Department of Cell and Molecular Biology, Uppsala University, Uppsala SE-75124, Sweden S Supporting Information *

ABSTRACT: A novel family of structurally simple, potent, and selective nonxanthine A2BAR ligands was identified, and its antagonistic behavior confirmed through functional experiments. The reported alkyl 2-cyanoimino-4-substituted-6-methyl1,2,3,4-tetrahy-dropyrimidine-5-carboxylates (16) were designed by bioisosteric replacement of the carbonyl group at position 2 in a series of 3,4-dihydropyrimidin-2-ones. The scaffold (16) documented herein contains a chiral center at the heterocycle. Accordingly, the most attractive ligand of the series [(±)16b, Ki = 24.3 nM] was resolved into its two enantiomers by chiral HPLC, and the absolute configuration was established by circular dichroism. The biological evaluation of both enantiomers demonstrated enantiospecific recognition at A2BAR, with the (S)-16b enantiomer retaining all the affinity (Ki = 15.1 nM), as predicted earlier by molecular modeling. This constitutes the first example of enantiospecific recognition at the A2B adenosine receptor and opens new possibilities in ligand design for this receptor.

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generally defined as a low affinity receptor12 that remains silent under physiological conditions but is rapidly activated during chronic highly oxidative stress conditions (e.g., hyperglycemia or mast cell activation). Recent evidence supports the situation where the A2BAR is transcriptionally regulated by factors implicated in inflammatory hypoxia,12 thus participating in key biological processes (e.g., vascular tone, glucose homeostasis, pulmonary inflammation, inflammatory response, or pain). Nevertheless, from a pharmacological perspective, A2BAR is the least well characterized of the four AR subtypes due to its general low affinity toward prototypic AR ligands.13,14 Accordingly, novel potent and selective A2BAR antagonists are required for drug development or as pharmacological tools to

INTRODUCTION A set of specific receptors, classified as A1, A2A, A2B, and A3 adenosine receptors (ARs), mediate the plethora of physiological processes regulated by the purine nucleoside adenosine.1,2 Accordingly, since their discovery, ARs have been considered to be attractive targets in drug discovery.3,4 ARs belong to the superfamily of the G-protein coupled receptors (GPCRs), and they therefore exert their physiological roles by activation or inhibition of different secondary messenger systems.2,3 Our improved understanding of the physiology, pharmacology, and structural and molecular biology of adenosine signaling pathways supports the development of potent and selective ligands, ultimately leading to the introduction of novel therapeutic strategies to address significant unmet medical needs.5−7 The A2BAR is ubiquitously expressed in the body, and it regulates a wide range of physiopathological events.8−11 It is © 2017 American Chemical Society

Received: January 26, 2017 Published: April 3, 2017 3372

DOI: 10.1021/acs.jmedchem.7b00138 J. Med. Chem. 2017, 60, 3372−3382

Journal of Medicinal Chemistry

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Figure 1. Structures of representative A2BAR antagonists.15−26

Figure 2. Design strategy employed for the target structures (16).

of this scaffold, which is readily assembled through a Biginelli multicomponent reaction, enabled the rapid assessment of the most prominent features of the structure−activity and selectivity relationships in this series. In an effort to explore new chemotypes exhibiting high A2BAR affinity, we then reported the pharmacological properties of three series of analogues, namely 3-deaza[pyridin-2(1H)-ones] and bicyclic and tricyclic derivatives fused at positions 2,3 or 5,6 of the heterocyclic framework.26 That study revealed the potential of structural diversification at face 2 of the diazinone scaffold and highlighted some promising bicyclic (13, 14) and tricyclic (15) ligands (Figure 1).26 We report here the discovery of a novel family of potent and highly selective A2BAR antagonists based on the 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate scaffold. The two enantiomers of the most attractive ligand (16b) were separated by chiral HPLC and their absolute configurations established by circular dichroism. The biological evaluation of the two enantiomers demonstrated that the affinity is exclusively due to the (S)-16b enantiomer, in line with our previous25,26 and current molecular

validate novel therapeutic applications (e.g., asthma, colitis, diabetic retinopathy, or cancer).8−11 The search for A2BAR antagonists5−7 has been traditionally focused on naturally occurring xanthines (Figure 1, compounds 1−3),15,16 which have evolved into several high-affinity and selective ligands (Figure 1, compounds 4−7),17−20 including xanthine-like compounds (Figure 1, compound 8).21 In contrast, only a few examples of potent and selective nonxanthine A2BAR antagonists have been described (Figure 1, compounds 9−15).22−26 In common with all AR ligands, all known A2BAR antagonists contain a planar central scaffold (Figure 1, compounds 1−11), which prevents the establishment of stereoselective ligand−target interactions. Hence, the assessment of novel chemotypes that have stereodiverse topologies provides solid foundations to explore alternative and stereospecific binding modes, thus reconciling molecular simplicity with optimal affinity and selectivity profiles. We recently documented the first family of nonplanar, structurally simple, selective, and high-affinity A2BAR antagonists based on the 3,4-dihydropyrimidin-2(1H)-one scaffold (Figure 1, compound 12).25 The excellent synthetic feasibility 3373



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Scheme 1. Synthesis and Molecular Properties of the 2-Cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine-5carboxylates 16

Figure 3. Structural analysis of 2-cyanoimino-4-(furan-2-yl)-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylates (16). (A) Possible tautomeric forms of 16. (B) ORTEP diagram of racemic 16b (CCDC1475413). (C) NOE experiments employing 16b. Lower trace: 300 MHz 1H NMR of 16b (DMSO-d6). Middle trace: NOE difference spectrum of 16b; irradiation of proton at position 4. Upper trace: NOE difference spectrum of 16b; irradiation of the methyl group at position 6.

models, constituting the first example of enantiospecific recognition at A2BAR.

tional, electronic, steric, and lipophilic characteristics of the scaffold, with a potential impact on the pharmacological profile for the ARs. On the basis of the SAR observed for the previous series (17−19), our hA2BAR model,25,26 and the prospective modeling study performed in this work (see below), the most advantageous substituents at positions 4 and 5 of the pyrimidine scaffold were retained. Thus, once the new series of compounds had been synthesized and pharmacologically evaluated, we performed a comparative analysis between the most similar scaffolds (16 and 17). Chemistry. In the same way as the 3,4-dihydropyrimidin2(1H)-ones (17) that inspired this work,25 the compounds documented here (16) were assembled by employing a

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RESULTS AND DISCUSSION The design of the 2-cyanoimino-4-substituted-6-methyl-1,2,3,4tetrahydro-pyrimidine-5-carboxylates (Figure 2) was based on the bioisosteric replacement of the carbonyl group at position 2 of the 3,4-dihydropyrimidin-2(1H)-one scaffold (17),25 or the imino group present at the azole ring fused to the pyrimidine core in bicyclic derivatives (18 and 19),26 by an exocyclic cyanoimino group (Figure 2). Such an apparently subtle structural modification should, however, affect the conforma3374



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Table 1. Structure and Affinity Binding Data for the 2-Cyanamido-4-substituted-1,4-dihydropyrimidine-5-carboxylates 16 at the Human Adenosine Receptors

Ki (nM) or % at 1 μMa b

compd

R4

R5

hA1

16a 16b (SY1K024) 16c 16d (SY1K025) 16e (SY1K022) 16f 16g 16h 16i 16j 16k 16l DPCPX ZM241385 (23)

2-furyl 2-furyl 3-furyl 3-furyl 2-thienyl 2-thienyl 3-thienyl 3-thienyl phenyl phenyl cyclopentyl cyclopentyl

Et i-Pr Et i-Pr Et i-Pr Et i-Pr Et i-Pr Et i-Pr

27% 21% 28% 34% 26% 31% 29% 11% 18% 20% 11% 3% 2.2 ± 0.2 683 ± 4.1

hA2Ac

hA2Bd

hA3e

20% 19% 31% 25% 33% 16% 30% 18% 13% 12% 10% 5% 157 ± 2.9 1.90 ± 0.1

900 ± 6 24.3 ± 0.9 102 ± 3 59.4 ± 1.8 51.0 ± 2.1 349.1 ± 3 461 ± 2 647 ± 4 25% 32% 19% 11% 73.2 ± 2.0 65.7 ± 1.7

20% 4% 14% 41% 3% 27% 23% 13% 13% 6% 4% 3% 1722 ± 11 863 ± 4.0

a

n = 3 for Ki values, or n = 2 for percentage displacement of specific binding. bDisplacement of specific [3H]DPCPX binding in adenosine A1 receptors expressed in human CHO cells. cDisplacement of specific [3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (23) binding in adenosine A2A receptors expressed in human HeLa cells. dDisplacement of specific [3H]DPCPX binding in human HEK-293 cells. eDisplacement of specific [3H]NECA binding in adenosine A3 receptors expressed in human HeLa cells.

Table 2. Structure and Affinity Binding Data for the 3,4-Dihydropyrimidin-2(1H)-ones 17 at the Human Adenosine Receptors26

Ki (nM) or % at 1 μMa b

compd

R4

R

hA1

17a 17b 17c 17d 17e 17f 12g 17h 17i 17j 17k 17l DPCPX (23)

2-furyl 2-furyl 3-furyl 3-furyl 2-thienyl 2-thienyl 3-thienyl 3-thienyl phenyl phenyl cyclopentyl cyclopentyl

Et i-Pr Et i-Pr Et i-Pr Et i-Pr Et i-Pr Et i-Pr

18% 20% 21% 25% 1% 37% 22% 26% 1% 2% 3% 1% 2.2 ± 0.2 683 ± 4.1

hA2Ac

hA2Bd

hA3e

41% 30% 39% 26% 33% 19% 39% 25% 10% 1% 1% 1% 157 ± 2.9 1.90 ± 0.1

585.5 ± 61 40.8 ± 3.1 39.6 ± 2.9 1486 ± 41 44% 44% 23.6 ± 1.3 56.7 ± 2.9 2% 2% 2% 3% 73.2 ± 2.0 65.7 ± 1.7

2% 1% 1% 3% 1% 14% 24% 1% 2% 2% 18% 7% 1722 ± 11 863 ± 4.0

a

n = 3 for Ki values, or n = 2 for percentage displacement of specific binding. bDisplacement of specific [3H]DPCPX binding in adenosine A1 receptors expressed in human CHO cells. cDisplacement of specific [3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (23) binding in adenosine A2A receptors expressed in human HeLa cells. dDisplacement of specific [3H]DPCPX binding in human HEK-293 cells. eDisplacement of specific [3H]NECA binding in adenosine A3 receptors expressed in human HeLa cells.

multicomponent reaction approach (MCR).27 The Biginelliinspired reaction behind our synthetic strategy is depicted in Scheme 1. Following a previously described procedure,28 cyanamide (20), a β-ketoester (21a−b), and the corresponding carbaldehyde (22a−d) were heated (80 °C) in ethanol using HCl/NaOAc as catalyst in a sealed tube for 4−6 h. The target 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydro-pyrimi-

dine-5-carboxylates (16a−j) were isolated in moderate to excellent yields (68−93%) after purification by either column chromatography or recrystallization (2-propanol). The reaction sequence started with the autocondensation of cyanamide in situ to afford the cyanoguanidine intermediate, which was then combined with either the aldehyde or the β-ketoester following any of the alternative mechanistic pathways proposed for the 3375



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Biginelli heterocyclization.28 An exhaustive description of the synthetic method and the complete structural and spectroscopic data for all compounds are provided in the Experimental Section. Assuming the superposition of the scaffolds discussed above, a collection of compounds was therefore prepared in which, according to the SAR trends of chemotype 17,25 we maintained the substituents at positions 4 (2-furyl, 3-furyl, 2thienyl, and 3-thienyl) and 5 (ethoxycarbonyl or isopropyloxycarbonyl) of the heterocycle (16a−h). Addtionally, phenyl and cyclopentyl groups were introduced at position 4 (compounds 16i−l) to evaluate the effect of removing the heterocyclic core. As stated above (Figure 2), the exocyclic cyanoimino group at position 2 in 16 was conceived as a bioisoster of the carbonyl group in 17 in such a way that the NH groups remaining at positions 1 and 3 would play equivalent roles in the two scaffolds. However, as depicted in Figure 3, there are three possible tautomeric forms for scaffold 16, which due to the different H-bond patterns would potentially lead to distinct binding modes between tautomers. To determine the prevalent tautomeric form, both in the solid state and in solution, X-ray crystallography and NMR spectroscopy (1D NOE difference experiments) were carried out and provided full information on the structure of a representative compound in the series, namely isopropyl 2-cyanoimino-4-(furan-2-yl)-6-methyl-1,2,3,4tetrahydropyrimidine-5-carboxylate (16b). The crystallographic data of a monocrystal of 16b confirmed the prevalence of tautomer 16-I in the solid state (Figure 3B), additionally providing further evidence for the pseudo boat conformation adopted by the heterocyclic ring. This is a common feature assumed for the parent series (17−19)25,26 and constitutes a major structural difference from all known A2B antagonists,6,7,13,14 which are planar structures. The one-dimensional (1D) NOE difference experiments on 16b (Figure 3C) unambiguously confirmed that the predominant tautomer is indeed the one initially considered (Figure 3, 16-I), at least in DMSO-d6 solution. Accordingly, the new heterocyclic core displays optimal overlay of chemical groups with the model scaffold 17 (Figure 2). Biological Evaluation. The affinities of the new ligands (16) for the four (A1, A2A, A2B, and A3) human adenosine receptor subtypes were determined in vitro with radioligand binding assays.29 Human adenosine receptors expressed in transfected CHO (A1AR), HeLa (A2AAR and A3AR), and HEK293 (A2BAR) cells were employed. The data obtained are provided in Table 1. As in previous studies,25,26 all compounds were initially evaluated as racemic mixtures. The biological data are expressed as Ki ± SEM (nM, n = 3) or as percentage inhibition of specific binding at 1 μM (n = 2, average) for those compounds that did not fully displace radioligand binding. For the sake of comparison, the biological data obtained for the 3,4dihydropyrimidin-2(1H)-one parent series (17) are also included (Table 2). Ki values were obtained by fitting the data with nonlinear regression using Prism 2.1 software (GraphPad, San Diego, CA). For those compounds that showed either little affinity or poor solubility, a percentage inhibition of specific binding is reported. Results are the mean of three experiments (n = 3), each performed in duplicate. Functional Experiments. cAMP Assays. As part of the pharmacological characterization of this novel series of selective A2BAR ligands, the most appealing compound of the series (16b) was tested in cAMP assays in order to evaluate its ability to inhibit NECA-stimulated (10 μM) cAMP production. These

experiments (see Supporting Information) confirmed that 16b inhibits cAMP accumulation, which validates the antagonistic behavior at A2BAR of the novel chemotype described here. A comparison of the Ki and KB values reveals a good correlation between data obtained from the binding (Ki = 24.3 nM) and functional assays (KB = 116.0 nM). Structure−Activity Relationships and Molecular Modeling. In the following section the structure−affinity and structure−selectivity relationships established from the pharmacological data are described. The trends are further supported and explained in the context of a computational 3D model of the hA2BAR. For the sake of comparison, Table 2 contains the pharmacological data obtained for the parent pyrimidin-2(1H)-one series 17,26 evaluated as racemic mixtures, with the addition of four novel compounds that explore cyclopentyl and phenyl substituents at position 4. The pharmacological data obtained for the new series (16), also evaluated as racemic mixtures, are listed in Table 1. Several interesting hA2BAR ligands can be identified that have high potency (Ki < 60 nM) and, most importantly, excellent subtype selectivity, with most ligands presenting low percentage inhibition at the remaining ARs (100-fold for the most potent ligands. Ligand 16b exhibits the most attractive pharmacological profile of the series as it elicits affinity in the low nanomolar range (Ki = 24.3 nM) and has outstanding selectivity toward hA1, hA2A, and hA3 ARs. The subseries bearing (2- or 3-) furyl rings at position 4 of the heterocyclic core generally elicited superior affinity at the hA2BAR than their (2- or 3-) thienyl congeners, with the 2-furyl derivative 16a being the only exception to this trend (Ki = 900 nM). More precisely, the combination of the furyl group at position 4 and an isopropyl residue at the ester moiety systematically provides better affinity (cf. pairs 16a/16b and, to a lesser extent, 16c/16d). Interestingly, the opposite effect was observed for compounds containing a (2- or 3-) thienyl group, for which the ethyl esters exhibited superior affinity (cf. pairs 16e/16f and, to a lesser extent, 16g/16h). As compared to the parent 3,4-dihydropyrimidin-2(1H)-one series (Table 2 and Supporting Information, Figure S2), the significant increase in affinity observed for ligands bearing a 2-thienyl residue at position 4 (cf. pairs 16e/17e and 16f/17f) is remarkable. Thus, while their 3,4-dihydropyrimidin-2(1H)-one congeners are practically inactive (44% at 1 μM), 16e and 16f have attractive Ki values (51.02 and 349 nM, respectively). A similar trend is observed for the aforementioned combination of furyl at position 4 (in particular 3-furyl) and an isopropyl ester (i.e., 16d/17d, where there is a 25-fold Ki difference). Curiously, a slight opposite effect is observed for the equivalent ligands containing ethyl ester residues, where substitution of the carbonyl at position 2 by a cyanoimino group is detrimental for the affinity (cf. pairs 17a/16a, 17c/16c). In contrast, 2cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylates bearing a 3-thienyl framework at position 4 (16g−h) exhibit attenuated affinities (Ki = 533 and 647 nM, respectively) when compared to their 3,4-dihydropyrimidin2(1H)-one congeners 17g−h (Ki = 23.6 and 56.7 nM, respectively). Finally, the compounds bearing phenyl (16i−j) or cyclopentyl (16k−l) rings at position 4 are deprived of affinity at any of the four ARs. This behavior, which was reproduced by the introduction of these substituents in the parent scaffold 17 (Table 2, compounds 17i−l), clearly suggests that the requirements of the binding cavity of the 3376



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group is oriented toward the interior of the binding site in the region defined by Val2506.51 so that the hydrogen bond with the amino side chain of Asn2546.55 is established with the exocyclic imino nitrogen. This pose is comparable to that previously reported for the parent series 1225 as well as for the bicyclic and tricyclic derivatives 18−19.26 Remarkably, the two water molecules predicted in that study26 also play an important role in ligand−receptor interactions for the present series 16. One of these water molecules mediates an H-bond network between Asn2546.55, Glu174EL2, and one of the NH groups in the dihydropyrimidine ring (N1 in pose A, N3 in pose B, see Figure 4). The second water molecule is equivalent to a structural water observed in the crystal structures of hA2AAR with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (23).30,31 In pose A, this water molecule mediates an internal hydrogen bond between the alkoxy substituent and the furan/thienyl ring, as previously described for series 18−19.26 In the alternative binding pose B, the cyanoimino substituent points toward the side chain of Asn2546.55 and the H-bond with this residue is achieved in this case through the cyano group. This second (inner) water molecule stabilizes the NH at position 3 of the ligand scaffold and could potentially bridge an H-bonding interaction with His2787.43. From the SAR study, it emerged that cyclopentyl and phenyl substituents (compounds 16i−l) are detrimental for the affinity, suggesting that these scaffolds cannot be easily accommodated in the binding site. These results are consistent with binding mode A, where this substituent points to the inner pocket in a tight and regioselective cavity presided by the A2BAR specific residue Val2506.51. The phenyl and cyclopentyl substituents would not fit optimally here and, furthermore, they would displace the second water molecule consistently observed in A2AAR crystal structures,31,32 which mediates interactions with the receptor for this and similar scaffolds.26 In contrast, in binding mode B the aromatic substituents in position 4 are located in the extracellular region. This is a region of intrinsic flexibility that could in principle readjust to accommodate more bulky substituents. However, it could be argued that binding mode B is less feasible to explain the SAR of the aromatic substitution but the lower reliability of the homology models of the loops does not rule out the possibility that this region may contain a stereoselective pocket. As occurred with the previous series (17−19, Figure 1), the present A2BAR selective antagonists (16) were obtained and initially tested as racemates. It is noteworthy that our molecular models predict that there is only one enantiomer [(S)-16b and topologically equivalent enantiomers, see Supporting Information, Figure S3] that should bind the active site of the A2BAR. This model of an enantioselective interaction with the A2BAR binding pocket, which was initially proposed for the previous series of antagonists,26 was reinforced by the observation that aromatization of the central heterocycle in series 12 abolished the A2BAR affinity.25 In an effort to confirm this enantioselective model, it was decided to separate the most attractive ligand identified [(±)-16b] into its respective enantiomers. A combination of chiral HPLC and circular dichroism (CD) spectroscopy was employed to achieve this aim, in the same way as described for previous examples of the pharmacologically active 3,4-dihydropyrimidin-2-ones.33−35 Semipreparative HPLC separation of the racemic 1,2,3,4-tetrahydropyrimidin-2one (±)-16b on a chiral stationary phase (see Experimental Section) provided each enantiomer with excellent stereo-

substituent at position 4 of the two scaffolds are common and highly specific within the hA2BAR binding pocket. To project the observed SAR onto receptor−ligand interactions and to assess the further growth of these series, the binding mode of the new antagonists to the hA2BAR was explored with molecular docking using our previously refined computational model of this receptor.25 Each molecule with a measured Ki for the hA2BAR (i.e., 16a−h) was subject to four parallel dockings: with or without the presence of predicted water molecules in the binding site, as previously identified,26 and considering the two possible enantiomers in each case. The results were then pulled together to elucidate the possible binding mode(s) for equivalent enantiomers in the series. It should be noted that, due to the different substituents on the scaffold, the R/S nomenclature does not necessarily correspond with the topological equivalences (see Supporting Information, Figure S3). In line with our previous report,26 analysis of the docking poses provided clear evidence that the topologically equivalent isomer to (S)-16b was the only one that could achieve any convergent solutions in the series. Indeed, two putative binding modes were identified only for this stereoisomer, namely pose A and pose B (Figure 4). In both cases,

Figure 4. Two possible binding modes, A (top) and B (bottom), proposed for compounds in series 16. In thin sticks, compounds with a measured have Ki value at the hA2BAR. The receptor is shown in gray ribbons, with residues that contribute to ligand binding in sticks and labeled. The two water molecules that mediate the binding stability are shown, and dashed lines depict H-bond interactions.

the heterocyclic ring is stabilized by aromatic interactions with the side chain of Phe173EL2 in such a way that one of the N atoms within the cyanoimino group contribute to the key hydrogen-bonding with the side chain of Asn2546.55, a totally conserved residue in ARs that interacts with all ligands crystallized to date.30 In pose A (Figure 4, top), the cyanoimino 3377



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Figure 5. Chiral HPLC traces, circular dichroism spectra, absolute configuration, and biological data for (R)-16b and (S)-16b.

binding mode of the two series was also consistent, with a stereospecific interaction proposed between the isomer (S)-16b and analogues and the receptor. The hypothesis of a stereospecific interaction with the binding site at the A2BAR was confirmed experimentally for the first time, with the eutomer [(S)-16] exhibiting remarkable potency (Ki value of 15.1 nM) while retaining the full selectivity profile at the four ARs. This study constitutes the first example of enantiospecific recognition at the A2BAR and opens new possibilities in ligand design for this receptor.

chemical purity (>99%). The characteristic CD activity of the enamide chromophore at around 300 nm allowed the unambiguous assignment of the absolute configuration of each enantiomer (Figure 5)33−35 by comparison with the reported CD data for enantiopure 3,4-dihydropyrimidin-2(1H)ones of known configuration.34,35 At this wavelength, the enantiomer that showed a negative Cotton effect (blue line) was identified as (R)-16b, while the stereoisomer that gave positive Cotton effect (red line) was assigned as (S)-16b. The pharmacological profiles of the two enantiomers [(R)16b and (S)-16b] at the four human adenosine receptors were then examined (Figure 5). The results confirmed that the observed affinity at the A2BAR is due to the (S)-16b enantiomer (Ki = 15.10 nM), which is almost twice as potent as the racemic mixture [16b, (Ki = 24.3 nM)], whereas (R)-16b is inactive on the four ARs (Figure 5). To the best of our knowledge, this is the first example of an A2BAR antagonist that has an enantiospecific recognition profile. Moreover, this result confirms the binding mode hypothesis for this series (Figure 4), which is in agreement with our previous proposal for the analogous bioisosteres 17−19.26

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EXPERIMENTAL SECTION

Chemistry. Commercially available starting materials and reagents were purchased and used without further purification from freshly opened containers. All solvents were purified and dried by standard methods. Organic extracts were dried with anhydrous Na2SO4. The reactions were monitored by TLC, and purified compounds each showed a single spot. Unless stated otherwise, UV light and/or iodine vapor were used for the detection of compounds. The synthesis and purification of all compounds were accomplished using the equipment routinely available in organic chemistry laboratories. Most of the preparative experiments were performed in coated vials on an organic synthesizer with orbital stirring. Purification of isolated products was carried out by column chromatography. Compounds were routinely characterized by spectroscopic and analytical methods. Melting points were determined on a melting point apparatus and are uncorrected. The chemical structures of the compounds were determined by nuclear magnetic resonance spectroscopy (1H and 13C NMR) and high-resolution mass spectroscopy (HRMS). Unless otherwise stated NMR spectra were recorded in CDCl3. Chemical shifts are given as δ values against tetramethylsilane as internal standard, and J values are given in Hz. Analytical HPLC was performed on an Agilent 1100 system using an Agilent Zorbax SB-Phenyl, 2.1 mm × 150 mm, 5 μm column with gradient elution using the mobile phases (A) H2O

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CONCLUSION A novel family of structurally simple, potent, and highly selective A2BAR antagonists based on the 2-cyanoimino-4substituted-6-methyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate chemotype has been studied. The biological data and the SAR obtained for this scaffold are consistent with those obtained for a previous series [i.e., the 3,4-dihydropyrimidin2(1H)-one scaffold (17)25], thus validating our design based on bioisosteric replacement of the carbonyl by a cyano group. The 3378



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containing 0.1% CF3COOH and (B) MeCN and a flow rate of 1 mL/ min. The purity of all tested compounds was determined to be >95%. The NOE experiments were performed employing the model compound 16b. Irradiation of the methyl group at position 6 (δ = 2.26 ppm) led to an NOE effect (2.6%) at the signal corresponding to the NH group at position 1 of the heterocycle (δ = 10.16 ppm); similarly, irradiation at position 1 (NH, δ = 10.16 ppm) led to an NOE effect (8.60%) at the signal corresponding to the methyl group at position 6 (δ = 2.26 ppm). Irradiation of the proton at position 4 of the heterocyclic core (δ = 5.29 ppm) led to an NOE effect (5.34%), with the signal corresponding to the proton of the NH present at in position 3 of the ring (δH = 9.09 ppm), and a similar experiment irradiating the proton at position 3 (NH, δ = 9.09 ppm) led to an NOE effect (12.26%), with the proton at position 4 of the heterocycle. The chiral resolution of 16b was performed using a Hitachi ELITE chromatograph (L-2130 pump, L-2350 column oven, and a L-2455 diode array detector) with a 250 mm × 4.6 mm Chiralcel OD-H column (DAICEL) equilibrated with n-hexane/i-propanol = 90/10 at 25 °C. Both enantiomers [(S)-16 (1.20 mg, retention time 13.4 min) and (R)-16 (1.05 mg, retention time 16.8 min)] were isolated and their stereochemical purity analyzed by chiral HPLC (>99% for each enantiomer) and then characterized by NMR in CDCl3. Circular dichroism spectra were recorded on a Jasco-815 system equipped with a Peltier-type thermostatic accessory (CDF-426S, Jasco). Measurements were carried out at 20 °C using a 1 mm quartz cell in a volume of 300−350 mL. Compounds (0.1 mg) were dissolved in MeOH (1.0 mL). The instrument settings were: bandwidth, 1.0 nm; data pitch, 1.0 nm; speed, 500 nm/min; accumulation, 10; wavelengths, 400−190 nm. General Procedure for the Synthesis of Derivatives 16.28 A mixture of cyanamide 20 (2 mmol), the corresponding aldehyde 21a− d (1 mmol), the 1,3-dicarbonyl compound 22a−b (1 mmol), sodium acetate (1 mmol), and concentrated hydrochloric acid (0.5 mL) in 7 mL of ethanol in a coated Kimble vial was stirred by orbital stirring at 80 °C for 8 h. After completion of the reaction, as indicated by TLC, the reaction mixture was poured onto crushed ice and stirred for 10 min. The solid was filtered off under reduced pressure, washed with ice-cold water (20 mL), and then purified either by recrystallization or column chromatography on silica gel. (±)-Ethyl 2-Cyanoimino-4-(furan-2-yl)-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16a). Purified by flash chromatography (hexane/EtOAc 5:3) to afford 16a as a pale-yellow solid, 76%; mp 244−245 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.26 (bs, 1H), 9.27 (bs, 1H), 7.41 (d, J = 3.8 Hz, 1H), 6.95 (t, J = 3.8 Hz, 1H), 6.91 (bs, 1H), 5.51 (d, J = 3.9 1H), 4.07 (q, J = 5.4, 2H), 2.27 (s, 3H), 1.14 (t, J = 5.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.9, 155.5, 147.2, 146.9, 127.4, 125.9, 124.8, 116.7, 102.2, 60.3, 49.0, 17.7, 14.5. HRMS (EI) m/z: calcd for C13H14N4O3 [M]+ 274.1066, found 274.1069. (±)-Isopropyl 2-Cyanoimino-4-(furan-2-yl)-6-methyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (16b). Purified by flash chromatography (hexane/EtOAc 2:1) to afford 16b as a white solid, 82%; mp 212−214 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.16 (bs, 1H), 9.09 (bs, 1H), 7.58 (d, J = 2.1 Hz, 1H), 6.38 (t, J = 2.2, 1H), 6.16 (d, J = 3.3 Hz, 1H), 5.29 (d, J = 3.9 Hz, 1H), 4.87 (p, J = 6.2 Hz, 1H), 2.26 (s, 3H), 1.17 (d, J = 6.1 Hz, 3H), 1.07 (d, J = 6.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.3, 155.7, 154.8, 147.2, 143.2, 116.7, 111.0, 106.9, 99.6, 67.5, 47.7, 22.1, 21.9, 17.7. HRMS (EI) m/z: calcd for C14H16N4O3 [M]+ 288.1222, found 288.1225. Isopropyl (S)-2-Cyanoimino-4-(furan-2-yl)-6-methyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate ((S)-16b). A 250 mm × 4.6 mm Chiralcel OD-H column (DAICEL) was equilibrated with n-hexane/ipropanol = 90/10, at 25 °C, 1.20 mg, retention time 13.4 min ee >99%. Isopropyl (R)-2-Cyanoimino-4-(furan-2-yl)-6-methyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate ((R)-16b). A 250 mm × 4.6 mm Chiralcel OD-H column (DAICEL) wasequilibrated with n-hexane/ipropanol = 90/10, at 25 °C, 1.05 mg, retention time 16.8 min ee >99%. (±)-Ethyl 2-Cyanoimino-4-(furan-3-yl)-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16c). Purified by flash chromatography

(hexane/EtOAc 2:1) to afford 16c as a pale-yellow solid, 62%; mp 241−243 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.11 (bs, 1H), 9.02 (bs, 1H), 7.57 (bs, 1H), 7.44 (bs, 1H), 6.34 (bs, 1H), 5.20 (bs, 1H), 4.06 (q, J = 6.2, 2H), 2.25 (s, 3H), 1.14 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.0, 155.9, 146.9, 144.4, 139.7, 127.9, 116.9, 109.4, 100.3, 60.2, 45.8, 17.7, 14.6. HRMS (EI) m/z: calcd for C13H14N4O3 [M]+ 274.1066, found 274.1067. (±)-Isopropyl 2-Cyanoimino-4-(furan-3-yl)-6-methyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (16d). Purified by flash chromatography (hexane/EtOAc 2:1) to afford 16d as a pale-yellow solid, 73%; mp 220−221 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.08 (bs, 1H), 9.00 (bs, 1H), 7.58 (d J = 1.7 Hz, 1H), 7.30 (s, 1H), 6.33 (d, J = 1.7 Hz, 1H), 5.18 (d, J = 3.9 Hz, 1H), 4.89 (p, J = 6.1 Hz, 1H), 2.24 (s, 3H), 1.18 (d, J = 6.2 Hz, 3H), 1.10 (d, J = 6.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.5, 155.9, 146.6, 144.4, 139.7, 128.0, 116.9, 109.4, 101.6, 67.5, 45.8, 22.2, 22.0, 17.7. HRMS (EI) m/z: calcd for C14H16N4O3 [M]+ 288.1222, found 288.1224. (±)-Ethyl 2-Cyanoimino-6-methyl-4-(thiophen-2-yl)-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (16e). Purified by flash chromatography (hexane/EtOAc 5:3) to afford 16e as a yellow solid, 81%; mp 243−244 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.19 (bs, 1H), 9.12 (bs, 1H), 7.59 (bs, 1H), 6.38 (bs, 1H), 6.17 (d, J = 2.8 Hz, 1H), 5.31 (d, J = 2.9 Hz, 1H), 4.05 (q, J = 6.9, Hz, 2H), 2.26 (s, 3H), 1.13 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.8, 155.7, 154.6, 147.4, 143.3, 116.7, 111.0, 106.8, 99.3, 60.2, 47.5, 17.7, 14.5. HRMS (EI) m/z: calcd for C13H14N4O2S [M]+ 290.0837, found 290.0840. (±)-Isopropyl 2-Cyanoimino-6-methyl-4-(thiophen-2-yl)-1,2,3,4tetrahydro-pyrimidine-5-carboxylate (16f). Purified by flash chromatography (hexane/EtOAc 2:1) to afford 16f as a white solid, 83%; mp 219−221 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.23 (bs, 1H), 9.25 (bs, 1H), 7.41 (d, J = 5.0 Hz, 1H), 6.96 (t, J = 4.2 Hz, 2H), 6.91 (bs, 1H), 5.49 (s, 1H), 4.89 (p, J = 6.0 Hz, 1H), 2.26 (s, 3H), 1.18 (d, J = 6.0 Hz, 3H), 1.08 (d, J = 6.0 Hz, 3H). 13C NMR (75 MHz, DMSOd6) δ 164.4, 155.5, 147.3, 146.6, 127.4, 125.9, 124.8, 116.7, 102.4, 67.7, 49.1, 22.2, 21.9, 17.6. HRMS (EI) m/z: calcd for C14H16N4O2S [M]+ 304.0994, found 304.0997. (±)-Ethyl 2-Cyanoimino-6-methyl-4-(thiophen-3-yl)-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (16g). Purified by flash chromatography (hexane/EtOAc 5:3) to afford 16g as an intense yellow solid, 72%; mp 192−194 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.12 (bs, 1H), 9.11 (bs, 1H), 7.49 (d, J = 4.9 Hz, 1H), 7.21 (s, 1H), 6.98 (d, J = 4.5 Hz, 1H), 5.31 (d, J = 3.2 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 2.26 (s, 3H), 1.13 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.1, 155.7, 146.6, 144.3, 127.6, 126.4, 122.3, 116.9, 101.9, 60.2, 49.2, 17.7, 14.5. HRMS (EI) m/z: calcd for C13H14N4O2S [M]+ 290.0837, found 290.0839. (±)-Isopropyl 2-Cyanoimino-6-methyl-4-(thiophen-3-yl)-1,2,3,4tetrahydropyri-midine-5-carboxylate (16h). Purified by flash chromatography (hexane/EtOAc 2:1) to afford 16h as a pale-yellow solid, 89%; mp 209−211 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.09 (bs, 1H), 9.09 (bs, 1H), 7.49 (d, J = 5.0 Hz, 1H), 7.20 (s, 1H), 6.97 (d, J = 5.0, 1H), 5.29 (s, 1H), 4.87 (p, J = 6.1 Hz, 1H), 2.25 (s, 3H), 1.17 (d, J = 6.2 Hz, 3H), 1.06 (d, J = 6.2 Hz, 3H). 13C NMR (75 MHz, DMSOd6) δ 164.7, 155.6, 146.3, 144.5, 127.6, 126.4, 122.3, 116.9, 102.1, 67.5, 49.3, 22.2, 21.9, 17.7. HRMS (EI) m/z: calcd for C14H16N4O2S [M]+ 304.0994, found 304.0993. (±)-Ethyl 2-Cyanoimino-6-methyl-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16i). Purified by flash chromatography (hexane/EtOAc 5:3) to afford 16i as a white solid, 85%; mp 248−249 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.12 (bs, 1H), 9.09 (bs, 1H), 7.42−7.16 (m, 5H), 5.24 (d, J = 3.8 Hz, 1H), 4.09 (q, J = 7 Hz, 2H), 2.29 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.1, 155.3, 146.4, 143.7, 129.1, 128.3, 126.9, 116.9, 101.7, 60.1, 53.7, 17.7, 14.4. HRMS (EI) m/z: calcd for C15H16N4O2 [M]+ 284.1273, found 284.1270. (±)-Isopropyl 2-Cyanoimino-6-methyl-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16j). Purified by flash chromatography (hexane/EtOAc 2:1) to afford 16j as a white solid, 62%; mp 214−216 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.08 (bs, 1H), 9.06 (bs,1H), 7.46−7.17 (m, 5H), 5.22 (d, J = 3.6 Hz, 1H), 4.81 (p, J = 6.3 Hz, 1H), 3379



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2.28 (s, 3H), 1.14 (d, J = 6.2 Hz, 3H), 0.96 (d, J = 6.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.6, 155.2, 146.2, 143.8, 129.0, 128.3, 127.0, 116.9, 101.9, 67.5, 53.9, 22.1, 21.8, 17.7. HRMS (EI) m/z: calcd for C16H18N4O2[M]+ 298.1430, found 298.1429. (±)-Ethyl 2-Cyanoimino-4-cyclopentyl-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16k). Purified by flash chromatography (hexane/EtOAc 5:3) to afford 16k as a white solid 85%; mp 219−221 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.92 (bs, 1H), 8.68 (bs, 1H), 4.10 (q, J = 6.9 Hz, 2H), 3.31 (s, 1H), 2.19 (s, 3H), 1.93 (bs, 1H), 1.63−1.25 (m, 8H), 1.18 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 170.40, 161.14, 151.08, 121.93, 106.91, 64.81, 57.47, 51.73, 32.57, 32.40, 29.61, 29.36, 22.43, 19.35. HRMS (EI) m/z: calcd for C15H16N4O2 [M]+ 276.1586, found 276.1589. (±)-Isopropyl 2-Cyanoimino-4-cyclopentyl-6-methyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (16l). Purified by flash chromatography (hexane/EtOAc 2:1) to afford 16l as a white solid, 62%; mp 222−223 °C. 1H NMR (300 MHz, ClCD3) δ 8.56 (bs, 1H), 7.26 (bs, 1H), 5.08 (hept, J = 6.2 Hz, 1H), 4.41 (d, J = 4.2 Hz, 1H), 2.34 (s, 3H), 2.26−1.98 (m, 1H), 1.91−1.40 (m, 8H), 1.27 (d, J = 6.2 Hz, 6H). 13C NMR (75 MHz, ClCD3) δ 164.94, 156.93, 143.46, 116.57, 104.34, 68.05, 53.95, 46.96, 28.03, 27.71, 24.84, 24.53, 21.96, 21.82, 18.06. HRMS (EI) m/z: calcd for C16H18N4O2[M]+ 290.1743, found 290.1746. Pharmacology. Radioligand binding competition assays were performed in vitro using human receptors expressed in transfected CHO (hA1), HeLa (hA2A and hA1), or HEK-293 (hA2B) cells as described previously.25,26,35 (3H)-1,3-Dipropyl-8-cyclopentylxanthine ([3H]DPCPX) for A1AR and A2BAR, (3H)-4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol ([3H]-23) for A2AAR, and (3H)-1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-Dribofuranuronamide ([3H]NECA) for A3AR were employed as radioligands in binding assays. A brief description is given below. Adenosine A1 receptor competition binding experiments were carried out in membranes made from CHO-A1 cells labeled with 2 nM [3H]DPCPX. Nonspecific binding was determined in the presence of 10 μM R-PIA. The reaction mixture was incubated at 25 °C for 60 min. Adenosine A2A receptor competition binding experiments were carried out in membranes from HeLa-A2A cells labeled with 3 nM [3H]-23. Nonspecific binding was determined in the presence of 50 μM NECA. The reaction mixture was incubated at 25 °C for 30 min. Adenosine A2B receptor competition binding experiments were carried out in membranes from HEK-293-A2B cells (Euroscreen, Gosselies, Belgium) labeled with 35 nM [3H]DPCPX. Nonspecific binding was determined in the presence of 400 μM NECA. The reaction mixture was incubated at 25 °C for 30 min. Adenosine A3 receptor competition binding experiments were carried out in membranes from HeLa-A3 cells labeled with 30 nM [3H]NECA. Nonspecific binding was determined in the presence of 100 μM R-PIA. The reaction mixture was incubated at 25 °C for 180 min. Functional Experiments. cAMP Assays. These assays were performed at adenosine receptors transfected using a cAMP enzyme immunoassay kit (Amersham Biosciences) following previously described protocols.25,26 HEK-293 cells were seeded (10000 cells/ well) in 96-well culture plates and incubated at 37 °C in an atmosphere with 5% CO2 in Eagle’s Medium Nutrient Mixture F-12 (EMEM F-12), containing 10% fetal calf serum (FCS) and 1% Lglutamine. Cells were washed three times with 200 μL of assay medium (EMEM-F12 and 25 mM HEPES pH = 7.4) and preincubated with assay medium containing 30 μM rolipram and test compounds at 37 °C for 15 min. Then 10 μM NECA was incubated for 15 min at 37 °C (total incubation time 30 min). Reaction was stopped with lysis buffer supplied in the kit, and the enzyme immunoassay was carried out for the detection of intracellular cAMP at 450 nm in an Ultra Evolution detector (Tecan). Data Analysis. IC50 values were obtained by fitting the data by a nonlinear regression using Prism 2.1 software (GraphPad, San Diego, CA). Ki values were obtained from Cheng−Prusoff equation.36 For those compounds that showed either little affinity or poor solubility, a percentage inhibition of specific binding is reported. Results are the mean of three experiments (n = 3) each performed in duplicate.

Functional KB values were calculated from IC50 values by using the method of Leff and Dougall.37 Computational Modeling. Our computational strategy for the structure-based design of adenosine receptor ligands has recently been reviewed,38 and it involves a combination of homology modeling and ligand−receptor docking. Homology Modeling of the hA2BAR. The generation of the inactive 3D structure of this receptor, used for the docking of antagonists, consisted of the following sequential steps: (i) Manual curation of the sequence alignment with the template A2AAR (PDB 3EML),31 (ii) generation and selection of homology models and loop refinement procedures with Modeler,39 (iii) assessment of Asn/Gln/His rotamers and side chain protonation states with the Molprobity web server (http://molprobity.biochem.duke.edu/), and (iv) use of tools from the Schrödinger Suite for energetic structural refinements.40 Ligand Docking. Both the R and the S stereoisomers for each ligand were built and optimized in 3D using the Maestro graphical interface and the LigPrep utility from the Schrödinger suite.40 Each ligand was docked 20 times with default (high accuracy) genetic algorithm (GA) search parameters, using the scoring function Chemscore as implemented in GOLD41 and allowing full flexibility for the ligand, including flipping of amide bonds. The search sphere was centered on the side chain (CD1) of Ile7.39 and expanded with a radius of 15 Å, thus ensuring a generous enough search space comprising the antagonist binding site experimentally determined for adenosine receptors. Two water molecules were considered in the binding cavity, as proposed42 previously and confirmed here by MD simulations (see below). The potential water contribution for the binding is only included in the final docking pose if it produces an increase in the predicted scoring, and the hydrogen bond network is optimized (option toogle trans_spin 2 in GOLD). Additionally, the two rotamers of Asn6.55 were also taken into consideration using the assemble structure option in GOLD. The criterion for the selection of docking poses was based on a combination of the Chemscore ranking and the population (convergence) of the solutions according to a clustering criteria of 1 Å.

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* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00138. Experimental details, biological data and curves obtained during functional assays (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*For E.S.: phone, +34 881815732; fax, +34-881815702; E-mail, [email protected]. *For H.G.d.-T.: phone, +46184715056; E-mail, hugo. [email protected]. ORCID

Eddy Sotelo: 0000-0001-5571-2812 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Conselleriá de Cultura, Educación e Ordenación Universitaria of the Galician Government (grant GPC2014/03), Centro Singular de Investigación de Galicia accreditation 2016−2019 (ED431G/ 09) and the European Regional Development Fund (ERDF), the Swedish Research Council (grant 521-2014-2118), and by the Swedish strategic research program eSSENCE. The 3380



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(16) Robeva, A. S.; Woodard, R.; Jin, X.; Gao, Z.; Bhattacharya, S.; Taylor, H. E.; Rosin, D. L.; Linden, J. Molecular characterization of recombinant human adenosine receptors. Drug Dev. Res. 1996, 39, 243−252. (17) Hayallah, A. M.; Sandoval-Ramirez, J.; Reith, U.; Schobert, U.; Preiss, B.; Schumacher, B.; Daly, J. W.; Müller, C. E. 1,8-Disubstituted xanthine derivatives: synthesis of potent A2B selective adenosine receptor antagonists. J. Med. Chem. 2002, 45, 1500−1510. (18) Kim, Y. C.; Ji, X.; Melman, N.; Linden, J.; Jacobson, K. A. Anilide derivatives of an 8-phenylxanthine carboxylic congener are highly potent and selective antagonists at human A2B adenosine receptors. J. Med. Chem. 2000, 43, 1165−1172. (19) Kalla, R. V.; Elzein, E.; Perry, T.; Li, X.; Gimbel, A.; Yang, M.; Zeng, D.; Zablocki, J. Selective, high affinity A2B adenosine receptor antagonists: N-1 monosubstituted 8-(pyrazol-4-yl)xanthines. Bioorg. Med. Chem. Lett. 2008, 18, 1397−1401. (20) Carotti, A.; Cadavid, M. I.; Centeno, N. B.; Esteve, C.; Loza, M. I.; Martínez, A.; Nieto, R.; Ravina, E.; Sanz, F.; Segarra, V.; Sotelo, E.; Stefanachi, A.; Vidal, B. Design, synthesis, and structure-activity relationships of 1-,3-,8-, and 9-substituted-9-deazaxanthines at the human A2B adenosine receptor. J. Med. Chem. 2006, 49, 282−299. (21) Stefanachi, A.; Brea, J. M.; Cadavid, M. I.; Centeno, N. B.; Esteve, C.; Loza, M. I.; Martínez, A.; Nieto, R.; Ravina, E.; Sanz, F.; Segarra, V.; Sotelo, E.; Vidal, B.; Carotti, A. 1-, 3- and 8-substituted-9deazaxanthines as potent and selective antagonists at the human A2B adenosine receptor. Bioorg. Med. Chem. 2008, 16, 2852−2869. (22) Vidal, B.; Nueda, A.; Esteve, C.; Domenech, T.; Benito, S.; Reinoso, R. F.; Pont, M.; Calbet, M.; López, R.; Cadavid, M. I.; Loza, M. I.; Cardenas, A.; Godessart, N.; Beleta, J.; Warrellow, G.; Ryder, H. Discovery and characterization of 4′-(2-Furyl)-N-pyridin-3-yl-4,5′bipyrimidin-2′-amine (LAS38096), a potent, selective, and efficacious A2B adenosine receptor antagonist. J. Med. Chem. 2007, 50, 2732− 2736. (23) Stewart, M.; Steinig, A. G.; Ma, C.; Song, J. P.; McKibben, B.; Castelhano, A. L.; MacLennan, S. J. [3H]OSIP339391, A selective, novel, and high affinity antagonist radioligand for adenosine A2B receptors. Biochem. Pharmacol. 2004, 68, 305−312. (24) Pastorin, G.; Da Ros, T.; Spalluto, G.; Deflorian, F.; Moro, S.; Cacciari, B.; Baraldi, P. G.; Gessi, S.; Varani, K.; Borea, P. A. Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as adenosine receptor antagonists. Influence of the N5 substituent on the affinity at the human A3 and A2B adenosine receptor subtypes: A molecular modeling investigation. J. Med. Chem. 2003, 46, 4287−4296. (25) Crespo, A.; El Maatougui, A.; Biagini, P.; Azuaje, J.; Coelho, A.; Brea, J.; Loza, M. I.; Cadavid, M. I.; García-Mera, X.; Gutiérrez-deTerán, H.; Sotelo, E. Discovery of 3,4-dihydropyrimidin-2(1H)-ones as a novel class of potent and selective A2B adenosine receptor antagonists. ACS Med. Chem. Lett. 2013, 4, 1031−1036. (26) El Maatougui, A.; Azuaje, J.; González-Gómez, M.; Miguez, G.; Crespo, A.; Carbajales, C.; Escalante, L.; García-Mera, X.; Gutiérrez de Terán, H.; Sotelo, E. Discovery of potent and highly selective A2B adenosine receptor antagonist chemotypes. J. Med. Chem. 2016, 59, 1967−1983. (27) Multicomponent Reactions; Zhu, J., Bienayme, H., Eds.; WileyVCH: Weinheim, 2005. (28) Hulme, R.; Zamora, O. D. P.; Mota, E. J.; Pastén, M. A.; Contreras-Rojas, R.; Miranda, R.; Valencia-Hernández, J.; CorreaBasurto, J.; Trujillo-Ferrara, J.; Delgado, F. Cyanamide: a convenient building block to synthesize 4-aryl-2-cyanoimino-3,4-dihydro-1Hpyrimidine systems via a multicomponent reaction. Tetrahedron 2008, 64, 3372−3380. (29) Yaziji, V.; Rodríguez, D.; Gutiérrez-de-Terán, H.; Coelho, A.; Caamaño, O.; García-Mera, X.; Brea, J.; Loza, M. I.; Cadavid, M. I.; Sotelo, E. Pyrimidine derivatives as potent and selective A3 adenosine antagonists. J. Med. Chem. 2011, 54, 457−471. (30) Gutiérrez-de-Terán, H.; Sallander, J.; Sotelo, E. Structure-based rational design of adenosine receptor ligands. Curr. Top. Med. Chem. 2017, 17, 40−58.

computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC). Our laboratories are part of the European COST Action CM1207 (GLISTEN).

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DEDICATION Dedicated to the memory of Professor Antonio Espinosa Ú beda (Granada University, Spain). ABBREVIATIONS USED ARs, adenosine receptors; GPCRs, G protein-coupled receptors; A2BR, human A2B adenosine receptors; A2AR, human A2A adenosine receptors; SAR, structure−activity relationships; MCR, multicomponent reaction; MD, molecular dynamics; CHO cells, Chinese hamster ovary cells; c-AMP, cyclic adenosine monophosphate; SEM, standard error of the mean; HPLC, high performance liquid chromatography; NOE, nuclear Overhauser effect; CD, circular dichroism; PDB, Protein Data Bank; RMSD, root mean square deviation

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