Exploring the Effect of Halogenation in a Series of Potent and Selective A2B Adenosine Receptor Antagonists

Exploring the Effect of Halogenation in a Series of Potent and Selective A2B Adenosine Receptor Antagonists Rubén Prieto-Díaz, Manuel González-Gómez, Hugo Fojo-Carballo, Jhonny Azuaje, Abdelaziz El Maatougui, Maria Majellaro, María I. Loza, JoséBrea,*Víctor Fernández-Duenas, M. Rita Paleo, Alejandro Díaz-Holguín, Beatriz Garcia-Pinel, Ana Mallo-Abreu, Juan C. Estévez, Antonio Andujar-Arias, Xerardo García-Mera, Iria Gomez-Tourino, Francisco Ciruela, Cristian O. Salas, Hugo Gutiérrez-de-Terán,*and Eddy Sotelo* Cite This: J. Med. Chem. 2023, 66, 890−912 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The modulation of the A2B adenosine receptor is a promising strategy in cancer (immuno) therapy, with A2BAR antagonists emerging as immune checkpoint inhibitors. Herein, we report a systematic assessment of the impact of (diand mono- )halogenation at positions 7 and/or 8 on both A2BAR affinity and pharmacokinetic properties of a collection of A2BAR antagonists and its study with structure-based free energy perturbation simulations. Monohalogenation at position 8 produced potent A2BAR ligands irrespective of the nature of the halogen. In contrast, halogenation at position 7 and dihalogenation produced a halogensize-dependent decay in affinity. Eight novel A2BAR ligands exhibited remarkable affinity (Ki< 10 nM), exquisite subtype selectivity, and enantioselective recognition, with some eutomers eliciting sub-nanomolar affinity. The pharmacokinetic profile of representative derivatives showed enhanced solubility and microsomal stability. Finally, two compounds showed the capacity of reversing the antiproliferative effect of adenosine in activated primary human peripheral blood mononuclear cells. ■INTRODUCTION Adenosine (Ado) is an essential signaling nucleoside that is either released from cells or extracellularly generated, by sequential hydrolysis of adenosine 5′-triphosphate and adenosine 5′-monophosphate by the ectonucleotidases CD39 and CD73. 1 Ado is ubiquitous in mammalian cells, being a key intermediate metabolite that modulates several biochemical processes, 2,3 ranging from energy transfer, signal transduction, sleep−wake cycle, 4 inflammation, 5 to immunity. 6,7 In healthy tissues, the extracellular concentration of Ado is generally low, although it can increase rapidly from nanomolar to micromolar, 1 which contributes to the protection of tissues against damage caused by stress, injury, hypoxia, or inflammation. 3,8,9 Four rhodopsin-like G protein-coupled receptors (GPCRs) sense the extracellular Ado levels, constituting the class 1 purinergic receptors (P1) A1AR, A2AAR, A2BAR, and A3AR. Each adenosine receptor (AR) subtype has unique sequence homology, tissue distribution, second messenger coupling, and pharmacology. 8 The physiology of Ado signaling through these receptors and its pathophysiological implications in human diseases have been recently reviewed. 1 A2BAR remains the most puzzling and poorest-characterized AR subtype. 10 Ubiquitously expressed but usually at low levels (except at human cecum, large intestine, mast cells, and hematopoietic cells), A2BAR displays the lowest affinity for Ado among all four subtypes (30−300 nM), remaining silent in healthy conditions. 1 Such a behavior, together with its high structural homology with the well-characterized A2AAR, led to the initial perception that A2BAR would have only minor physiological relevance. However, it is now well documented that A2BAR becomes activated under particular pathological conditions such as hypoxia, inflammation, infection, and cancer, where extracellular Ado levels are increased up to micromolar concentrations, consequently increasing attention to the therapeutic potential of A2BAR as a drug target. 11 A2BAR antagonists have been recently proposed as potential drugs for Received: October 31, 2022 Published: December 14, 2022 Articlepubs.acs.org/jmc © 2022 The Authors. Published by American Chemical Society 890 https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 Downloaded via UNIV DE SANTIAGO DE COMPOSTELA on February 2, 2024 at 08:38:52 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. the treatment of inflammation, diabetes, pain, asthma, and Alzheimer disease. 12−15 More recent is the identification of A2BAR as an important player to different facets of cancer progression, e.g., tumor growth, metastasis, and angiogenesis. 16,17 Advances in cancer immunotherapy highlight the importance of Ado as a key metabolite that suppresses antitumor immune response by T and NK cells in the tumor microenvironment, 18 this immune role being specifically mediated by A2A and A2B ARs. 19−21 Indeed, several A2AAR antagonists are under clinical trials as immune checkpoint inhibitors for the treatment of different cancer types. 22 A2AAR and A2BAR exhibit high homology and are often co-expressed on cells. 23 Recent studies suggested formation of heteromeric A2AAR−A2BAR complexes and revealed that ligand recognition, signaling, and pharmacology of A2AAR are blocked by A2BAR. 24 According to all this evidence, A2BAR antagonists and dual A2AAR-A2BAR antagonists are emerging as effective cancer (immuno)therapeutics, where the consequent reactivation of the immune system results in antiproliferative, antiangiogenic, and antimetastatic effects. 25,26 The therapeutic opportunities arising from A2BAR modulation have driven the development of A2BAR antagonists. The naturally occurring xanthine derivatives (e.g., caffeine and theophylline) early inspired the discovery and optimization of xanthine congeners and structurally related deaza analogues and purine derivatives. 27 Xanthines have been intensively studied, allowing the identification of derivatives eliciting optimal affinity−selectivity profiles (Figure 1, Cmpds 1−4), being so far the most widely explored A2BAR antagonists. 28 However, their challenging physicochemical features and pharmacokinetic (PK) profiles remain as the major drawback of this chemotype to advance further on the drug discovery pipeline. Consequently, recent efforts have been focused on the development of non-xanthinic A2BAR antagonists (Figure 1, Cmpds 5−10). 29−32 In this context, we have reported novel series of pyrimidine-based ligands (Figure 1, Cmpds 7−10), which encompass structural novelty, exquisite affinity and selectivity, and excellent synthetic feasibility. 29−31 A hybrid approach, combining scaffold hopping and thorough SAR investigation, have guided the expansion of these series. Importantly, these compounds contain a chiral center within the heterocyclic core (Figure 1, Cmpds 7−10), offering a novel structural element as compared to classical, planar A2BAR antagonists. Consequent racemate separation provided the first examples of antagonists with enantiospecific A2BAR recognition. 25,31,33−35 In the frame of an optimization program of non-xanthine A2BAR antagonists as promising cancer (immuno)- therapeutics, 25,36 we determined to analyze the impact of diand mono-halogenation at positions 7 and 8 of the tricyclic core of prototypical A2BAR antagonists (Figure 1, Cmpds 8− 10). 30,31 The main goal was to improve the pharmacodynamic and PK profiles of this chemotype, while maintaining (or even improving) the affinity and selectivity profiles. The phenyl ring of the tricyclic system is the most easily metabolizable part, only next to the exocyclic pentagonal ring that was already optimized in a previous work, 31 and halogenation arises as a strategy to reduce the probability of intensive metabolization. At the same time, the size variability of halogen substitution would allow a deeper exploration of the interactions proposed by earlier modeling studies in the internal cavity of A2BAR. 29−31 The synthesis and pharmacological characterization of a collection of 80 novel tricyclic ligands is here assessed by free energy perturbation (FEP) simulations, evidencing the superior profile of 8-halo derivatives by optimizing interactions with the modeled binding site. This study further confirms the previously modeled enantiospecific A2BAR recognition of the pentagonal ring at position 4, and the antagonist profile of the most promising compounds. In addition, examination of the ADMET profile of representative antagonists confirmed that the introduction of a fluorine atom improves solubility and microsomal stability. Two of the ligands here optimized were selected to investigate the effect of A2BAR antagonism on immune cell proliferation, demonstrating that the ligands reverse the reduction of Ado-related proliferation in human primary immune cells. ■RESULTS AND DISCUSSION Design. These novel series were conceived in the context of our ongoing projects to develop multi-target cancer (immuno)therapeutics and PET tracers for A2BAR, starting from the scaffold of ISAM-140 (Figure 1) as a prototype ligand. Introduction of functional groups at positions 7 and 8 of the heterotricyclic core evidenced conspicuous effects on the affinity and selectivity profile, thus inspiring the design of the present series. It was anticipated that halogenation at positions 7 and 8 of the phenyl ring within the tricyclic core would not only result in compounds with improved pharmacodynamic Figure 1. Structure of representative potent and selective A2B antagonists. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 891 and PK profiles but also provide valuable data to understand the molecular basis behind the observed SAR trends. Thus, we envisioned the development of the 80 new ligands (series I−IV) presented in Figure 2. Being aware that the impact of halogenation on binding affinity and selectivity might be position-dependent and that it can heavily rely on the nature of the halogen, we systematically introduced F, Cl, and Br atoms at positions 7 and 8 of the tricyclic scaffold (Figure 2). To preserve consistency from the early series and facilitate the comparative SAR, the herein obtained ligands (series I− IV) retained the structural elements that provided optimal A2BAR affinity at positions 3 and 4 (Figure 2). Thus, for R4, we initially considered a set of four pentagonal heteroaryl groups (2-furyl, 3-furyl, 2-thienyl, 3-thienyl), while the alkoxy residues of the ester moiety (R 3 ) consist of either ethyl or isopropyl groups. Three series were subsequently conceived, each consisting of 24 derivatives (Figure 2 and Tables 2−4). Series Icontain two identical halogen atoms at positions 7 and 8 (compounds 14a−x), whereas in series II and III (monohalogenated), the halogen atom is present in position 7 or 8 respectively (compounds 15a−xand 16a−x). Finally, a focused series consisted of eight 7-halo or 8-halo derivatives bearing a 4-oxazolyl group (Table 5), which we have recently identified as an optimal non-furan pentagonal nucleus for position 4. 31 This limited series was obtained by prioritizing two halogens (F and Cl) and the substitution patterns that provided better affinity and selectivity profiles in series I−III. Chemistry. The halogenated alkyl 4-heteroaryl-2-methyl1,4-dihydrobenzo[4,5]-imidazo[1,2-a]pyrimidine-3-carboxylates (14−18) were obtained as depicted in Scheme 1. A threecomponent synthesis, based on the robust and efficient Biginelli reaction (i.e., the three-component reaction of an aldehyde, a β-ketoester, and 1,3-dinucleophiles), enabled a timeand cost-effective assembly of the large collection obtained. A set of pentagonal carbaldehydes (12a−e) and βketoesters (13a,b) providing optimal substituents for positions 4 and 3 in combination with six halogenated 2-aminobenzimidazoles (11a−f) were employed as precursors. The starting materials, dissolved in THF and containing a catalytic amount of ZnCl2(or acetic acid), were submitted to orbital stirring at 90 °C (12 h) or microwave radiation at 80 °C (90 min). The targeted tricyclic derivatives were obtained in moderate to satisfactory yields (30−85%). For the synthesis of a series of monohalogenated (7-halo or 8-halo) derivatives (Scheme 1, Cmpds 15−18), the corresponding 2-amino-5halobenzimidazoles (11d−f) were employed as precursors. The phenomenon of tautomerization enabled to directly obtain the targeted (7or 8-)halogenated alkyl 4-heteroaryl2-methyl-1,4-dihydrobenzo-[4,5]imidazo[1,2-a]pyrimidine-3carboxylates as regioisomeric mixtures in a 1:1 ratio. Both regioisomers (series II and III) were isolated in pure forms after chromatographic separation. All reactions were monitored by thin-layer chromatography (TLC). After completion of the reaction, the solvent was evaporated to dryness and the isolated solid was purified by column chromatography on silica gel. A detailed description of the synthetic methods and the complete structural, spectroscopic, and analytical data for all compounds are provided in the experimental part. Unambiguous structural characterization of each regioisomer has been carried out with a combination of X-ray crystallography and 2D-NMR experiments (Scheme 1). The monohalogenated compounds (series II−IV) differ in the position of the halogen atom located at either C7 or C8. Both regioisomers present an ABX aromatic substitution pattern in the 1H NMR (Scheme 1). X-ray diffraction analysis of 16s (see the Supporting Information) shows that protons H4 (alpha hydrogen to the Figure 2. Design strategy and diversity elements explored during the study. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 892 five-membered ring) and H6 (aromatic proton) are 2.7 Å apart, allowing us to perform NOE experiments (Supplementary Figure S1). The 2D-ROESY NMR spectrum of 16s (Supplementary Figure S1A) showed a correlation of H4 (singlet, δ6.60 ppm) and the doublet (J= 8.4 Hz) at δ7.41 ppm corresponding to H6. The coupling constant value is typical of H−Hortho coupling and indicates the presence of a hydrogen in C7 and therefore of the halogen at C8. However, the 2D-ROESY NMR spectrum of 15s (Supplementary Figure S1B) showed a correlation between H4 (singlet at δ6.53 ppm) and H6 (doublet at δ7.70 ppm). The coupling constant value, J= 1.9 Hz, indicates a H−Hmeta coupling, and thus the halogen is in the ortho position (C7). Similar correlations were observed for other pairs of regioisomers (Supplementary Figure S2). As in previous series (Figure 1 and Table 1, compounds 7−10), all ligands obtained in this study contain one stereocenter at position 4 of the heterocyclic core and were isolated and evaluated as racemic mixtures. Six ligands eliciting an attractive A2BAR affinity/selectivity profile were submitted to chiral resolution to isolate its corresponding enantiomer pairs. Biological Evaluation. The adenosinergic profile (affinity and selectivity) of the 80 novel halogenated alkyl 1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxy-lates was evaluated in vitro using radioligand binding assays at the four human AR subtypes. 30,31,33,34 Tables 2−5contain the binding data obtained, while, to facilitate the comparative assessment, Table 1 shows the binding data for the prototype series. 30 All ligands reported in Tables 1−5were obtained and tested as racemic mixtures. The whole set was evaluated in silico with a Scheme 1. Biginelli-Based Synthesis. Method (A) ZnCl2, THF, 90 °C, 12 h. Method (B) AcOH, THF/DMF (2:1), 80 °C, MW, 90 min; and Regioisomer Characterization Strategy (in Red: Signal with NOE Effect Irradiating H4) Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 893 combination of a Rdkit 37 PAINS filter and Instant JChem from Chemaxon (https://www.chemaxon.com), to rule out the possibility that these ligands can be promiscuous pan-assay interference compounds (PAINS) and to assess their bioavailability, respectively. Human ARs were expressed in transfected cell lines [e.g., Chinese hamster ovary (CHO) cells (A1AR), human epithelial carcinoma (HeLa) cells (A2AAR and A3AR), and human embryonic kidney (HEK-293) cells (A2BAR)]. [3H]DPCPX for A1AR and A2BAR, [3H]NECA for A3AR, and [3H]ZM241385 for A2AAR were employed as radioligands. The binding data is presented as Ki±SEM (nM, n= 3) obtained by fitting the data by non-linear regression using Prism 5.0 software (GraphPad, San Diego, California), or as specific binding inhibition percentage at 1 μM (n= 2, average) for those compounds that did not completely displace the radioligand due to either little affinity or poor solubility. The binding affinity of reference AR ligands (ISAM-140, DPCPX, NECA, and ZM241385) was assessed using our experimental protocols and reported in Tables 1−5for comparison. The stereoisomers of selected compounds were obtained by chiral resolution and tested at the four human AR subtypes in their enantiopure forms (Table 6). This data was employed to complement the SAR study and to evaluate the importance of the configuration of the stereogenic center on the affinity. Intrinsic Activity Assays. Four representative ligands (16b,16j,16r, and 16s) were selected to gain insight into the functional effect of the obtained series. To assess their ability to block A2BAR agonist intrinsic activity, the effect on A2BARmediated cAMP accumulation and cellular impedance was evaluated. To this end, we used HEK-293 cells permanently expressing the A2BARSNAP construct, which can be visualized upon staining the cells with a SNAP-fluorescent substrate. As shown in Figure 3A, A2BARSNAP nicely decorated the cell surface of A2BARSNAP cells, indicating that the receptor was readily expressed at the plasma membrane. Subsequently, we evaluated the effect of 16b,16j,16r, and 16s in A2BAR signaling by monitoring cAMP accumulation in A2BARSNAP Figure 3. Blockade of A2BAR-mediated cAMP accumulation. (A) HEK-293 cells permanently expressing the A2BARSNAP construct were stained with the fluorescent SNAP-488 substrate and visualized using a confocal microscope. 38 Scale bar: 10 μM. (B) Determination of NECA-mediated cAMP accumulation in A2BARSNAP cells. Cells were incubated in the absence or presence of increasing concentrations of NECA, and the cAMP accumulation determined as described in Experimental Section. The straight TR-FRET signal (ratio 665 nm/620 nm) of the assay is shown. (C) cAMP accumulation in A2BARSNAP cells stimulated with NECA (1 μM) in the absence or presence of increasing concentrations of 16b,16j,16r, and 16s. Data are expressed as mean ±SEM (n= 4). Figure 4. Blockade of A2BAR-mediated whole-cell label-free responses. (A) Representative example of the impedance signal obtained over the time upon incubation with A2BAR ligands. Cells were stimulated with vehicle or NECA (1 μM) in the absence or presence of 16b,16j,16r, and 16s (1 μM), 18 h after seeding, and the impedance signal recorded in real-time during 45 min. (B) The area under the curve (AUC) derived from the normalized cell index (NCI) shown in (A) was calculated for each condition. Data are expressed as mean ±SEM (n= 4). ***P< 0.001 and **P< 0.01, one-way ANOVA with a Dunnett’s multiple-comparison test when compared to vehicle (Veh). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 894 cells treated with NECA, a non-selective Ado receptor agonist. NECA induced a concentration-dependent cAMP accumulation in A2BARSNAP cells with an EC50 of 257 ±94 nM (Figure 3B). Subsequently, A2BARSNAP cells were evaluated with a fixed concentration of NECA (1 μM) in the absence or presence of increasing concentrations of 16b,16j,16r, and 16s and the KB value for each compound was calculated, namely, 0.8 ±0.2, 0.6 ±0.2, 1.7 ±0.6, and 1.5 ±0.5 nM, respectively (Figure 3C). Overall, 16b,16j,16r, and 16s displayed low nanomolar potency while blocking A2BAR-mediated cAMP accumulation in A2BARSNAP cells, thus indicating an antagonist intrinsic activity nature. Comparative KBand Kidata for selected compounds is shown in Table 7. It should be noticed that two these selective A2BAR antagonists exhibit sub-nanomolar KB data. To further evaluate the ability of 16b,16j,16r, and 16s to preclude A2BAR function in living cells, we took advantage of the cellular impedance label-free method, a widely accepted morphological and functional biosensor of cell status. 39 Accordingly, whole-cell NECA-mediated impedance responses of A2BARSNAP cells were monitored in real time in the absence or presence of 16b,16j,16r, and 16s. Indeed, NECA (1 μM) induced a significant increase in A2BARSNAP cell impedance recordings, thus reflecting changes in cell morphology upon agonist challenge (Figure 4A). Importantly, 16b,16j,16r, and 16s partially blocked the NECA-induced impedance increase in A2BARSNAP cells (Figure 4A). When calculating the area under the curve (AUC) for the different experimental conditions, we can observe that 16b,16j,16r, and 16s significantly blocked NECA-induced changes in cellular impedance (Figure 4B). These results from an alternative cellular functional assay further confirmed that 16b,16j,16r, and 16s were able to block A2BAR agonist-mediated intrinsic activity. Interestingly, the absolute 16b,16j,16r, and 16s antagonist potencies in the cAMP accumulation assay were greater (∼10fold) than those determined in the radioligand binding assays, a fact that has been previously reported in HEK-293 cells stably overexpressing A2BAR. 40 Indeed, this notion can be supported by the spare receptor theory defining that under some circumstances, the potency of a named agonist in functional assays is proportional to the receptor density, thus producing a discontinuity between drug occupancy and cell response. 41 Our label-free experiments revealed a significant, but partial, blockade of a NECA-induced impedance increase in A2BARSNAP cells. Indeed, HEK-293 cells express endogenous Ado receptors as revealed by microarray analysis. 42 Thus, while A3AR was not detected, the remaining A2BAR, A2AAR, and A1AR were identified in HEK-293 cells. 42 Since NECA is a non-selective Ado receptor agonist showing affinity for A2AAR and A1AR (Kivalues of 20 and 14 nM, respectively), 43 it can be speculated that the remaining impedance increase in NECAtreated A2BARSNAP cells in the presence of 16b,16j,16r, and 16s can be mediated by endogenous A2A and A1ARs. These results might be interpreted as indirect evidence of selectivity for the A2BAR for compounds 16b,16j,16r, and 16s. Structure−Activity Relationship Analyses and Molecular Modeling. In this section, we examine the pharmacological data obtained for the newly synthesized series (Tables 2−6) and analyze the effect of halogenation in the structure− affinity (SAR) and structure−selectivity (SSR) relationships. All ligands were evaluated as racemic mixtures and comparative studies used as a reference of the parent series (Table 1). The SAR here outlined is then further assessed by FEP calculations in the context of a refined computational 3D model of hA2BAR. The adenosinergic affinity profiles of the 80 alkyl 7/8-halo-4heteroaryl-1,4-dihydro-benzo[4,5]imidazo[1,2-a]pyrimidine-3carboxylates are presented in Tables 2−6. Up to 25 ligands Table 1. Structure and Adenosine Receptor Affinities of Alkyl 1,4-Dihydrobenzo [4,5]imidazo[1,2-a]pyrimidine-3-carboxylates (Prototype Series) 30,31 Ki(nM) or % at 1 μM compound R4R3hA1 a hA2A b hA2B c hA3 d I(ISAM-134) 2-furyl Et 5% 14% 12.0 ±0.7 1% II (ISAM-140) 2-furyl i-Pr 20% 28% 3.49 ±0.2 2% III (ISAM-141) 3-furyl Et 7% 11% 20.6 ±1.1 1% IV (ISAM-142) 3-furyl i-Pr 12% 23% 11.4 ±0.5 2% V2-thienyl Et 8% 16% 484 ±3 1% VI 2-thienyl i-Pr 1% 17% 371 ±5 3% VII 3-thienyl Et 3% 10% 29.7 ±1.2 2% VIII 3-thienyl i-Pr 11% 3% 29.34 ±1.1 21% IX (ISAM-C032) 4-oxazolyl Et 5% 11% 8.10 ±0.5 7% X4-oxazolyl i-Pr 7% 19% 43.4 ±1.3 16% DPCPX 2.20 ±0.2 157 ±3 73.2 ±1.4 1722 ±11 ZM241385 683 ±4 1.9 ±0.1 65.7 ±1.1 863 ±4 NECA 14.0 ±1 20.0 ±3 2400 ±35 6.20 ±0.9 a Displacement of specific [3H]DPCPX binding in human CHO cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). b Displacement of specific [3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol binding in human HeLa cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). c Displacement of specific [3H]DPCPX binding in human HEK-293 cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). d Displacement of specific [3H]NECA binding in human HeLa cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 895 combine attractive A2BAR affinity (Ki<50 nM) and exquisite subtype selectivity (>1000-fold), from which 14 ligands exhibit outstanding A2BAR affinity (Ki<10 nM) [e.g., compounds 16a, 16b,16d,16i,16j,16k,16l,16r, 16s,16t (Table 4), 17b,18b, 18c, and 18d (Table 5)]. The available data reveal interesting and previously unexplored SAR trends, with derivatives bearing a halogen atom at position 8 of the heterotricyclic core collectively emerging as the most appealing A2BAR antagonists in these series (series III and IV,Tables 4 and 5). Compared to the congeners of the parent series (Table 1), 10 of the ligands here identified present improved pharmacodynamic and PK profiles, with ligands 16b,16j,16r, and 16s deserving particular attention, as they exhibit one-digit Kivalues (3−5 nM) and excellent subtype selectivity, thus justifying the selection of these compounds for further intrinsic activity assays (see the previous section). A comparative analysis of the binding data obtained for the parent series (Table 1) and ligands of series I(Table 2) evidenced that a simultaneous introduction of halogens at positions 7 and 8 has deleterious impact on A2BAR affinity. Moreover, it can be observed that this reduction in affinity is halogen dependent, with better affinity profiles observed for some 7,8-difluoro derivatives (Table 2, Cmpds 14a−d) and the affinity decay being proportional to the atomic radius of the halogen (Table 2 and Supplementary Figure S3). Ligands bearing a thienyl ring at position 4 of the heterotricyclic scaffold are inactive with the only exception of the low-affinity 14p. For the case of ligands containing a furyl group at R4, the A2BAR affinity seems to be highly dependent of the halogens present (Table 2). Thus, all ligands containing a furyl group of the 7,8-difluorinated scaffold (Cmpds 14a−d) elicit potent A2BAR affinity (Ki= 11.9−52.9 nM) irrespective of the substitution pattern at the furan ring (2-furyl or 3-furyl) or the alkoxy residue in the ester moiety (R3). Although potent, the difluorinated derivatives 14a−dshow lower affinity (threefold) than their parent series analogues (Table 1). Similarly, in the subsets bearing chlorine atoms (Table 2, Cmpds 14i−p), only the derivatives incorporating an optimal substituent at R4and R3(14j and 14k) show moderate affinity. Following this trend, the only derivative retaining A2BAR affinity in the 7,8-dibromo Table 2. Structure and Adenosine Receptor Affinities of Series I: Alkyl 7,8-Dihalo-1,4-dihydrobenzo[4,5]imidazo[1,2a]pyrimidine-3-carboxylates (Cmpds 14a−x) Ki(nM) or % at 1 μM compound R4R3X hA1 a hA2A b hA2B c hA3 d 14a 2-furyl Et F 1% 2% 44.1 ±1.7 2% 14b 2-furyl i-Pr F 13% 22% 11.9 ±1.1 14% 14c 3-furyl Et F 22% 36% 28.2 ±0.8 17% 14d 3-furyl i-Pr F 17% 16% 52.9 ±1.5 20% 14e 2-thienyl Et F 2% 3% 25% 5% 14f 2-thienyl i-Pr F 1% 1% 1% 10% 14g 3-thienyl Et F 1% 1% 519 ±7 5% 14h 3-thienyl i-Pr F 1% 3% 43% 4% 14i 2-furyl Et Cl 2% 5% 9% 1% 14j 2-furyl i-Pr Cl 43% 12% 117 ±5 7% 14k 3-furyl Et Cl 33% 34% 54.7 ±3.2 6% 14l 3-furyl i-Pr Cl 4% 31% 36% 8% 14m 2-thienyl Et Cl 2% 1% 9% 11% 14n 2-thienyl i-Pr Cl 1% 4% 1% 9% 14o 3-thienyl Et Cl 5% 1% 9% 1% 14p 3-thienyl i-Pr Cl 2% 1% 359 ±4 6% 14q 2-furyl Et Br 1% 17% 37% 2% 14r 2-furyl i-Pr Br 11% 28% 62.6 ±1.9 21% 14s 3-furyl Et Br 2% 2% 7% 10% 14t 3-furyl i-Pr Br 1% 1% 32% 9% 14u 2-thienyl Et Br 2% 7% 12% 5% 14v 2-thienyl i-Pr Br 1% 1% 9% 3% 14w 3-thienyl Et Br 2% 7% 47% 4% 14x 3-thienyl i-Pr Br 4% 1% 9% 8% DPCPX 2.20 ±0.2 157 ±3 73.2 ±1.4 1722 ±11 ZM241385 683 ±4 1.9 ±0.1 65.7 ±1.1 863 ±4 NECA 14.0 ±1 20.0 ±3 2400 ±35 6.20 ±0.9 a Displacement of specific [3H]DPCPX binding in human CHO cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). b Displacement of specific [3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol binding in human HeLa cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). c Displacement of specific [3H]DPCPX binding in human HEK-293 cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). d Displacement of specific [3H]NECA binding in human HeLa cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 896 subset is 14r (Ki= 62.6 nM), containing at R4and R3the same substitution pattern of the lead compound ISAM-140, albeit with a 20-fold decrease in affinity. The adenosinergic profile obtained for series II is presented in Table 3. Here, introduction of a halogen at position 7 reproduced some of the SAR trends detected in series I(Table 2). While some attractive ligands can be identified (e.g., 15b and 15c,Ki= 12.2 and 25.0 nM, respectively), a comparison with the parent series (Table 1) revealed that halogenation at position 7 is deleterious. As for series I, the effect of halogenation relied on the nature of the halogen, with fluor being the best tolerated (Table 3, Cmpds 15a−d). Similarly, most derivatives bearing thienyl groups remain inactive. For series bearing chloro and bromo atoms at position 7, only optimal pair combinations at R4and R3(e.g., 2-furyl and isopropyl, 3-furyl and ethyl, respectively) produced ligands with moderate (Ki∼45 nM) affinity (Table 3, Cmpds 15j, 15k,15r, and 15s). This trend is also observed for 7-fluoro derivatives (Table 3, Cmpds 15a−15d), while in this subset, all furyl-containing derivatives exhibited attractive A2BAR affinity (Ki= 12.2−136 nM). An evaluation of series III (Table 4) enabled the identification of the most potent (Ki< 10 nM) A2BAR antagonists described in this study. It also allowed to envision the significant effect of halogen substitution at position 8 and its rationalization from a molecular modeling perspective. In contrast to previous series, halogenation at position 8 systematically improved the A2BAR affinity irrespective of the nature of the halogen atom (Table 4 and Figure 5). Indeed, most 8-halo derivatives have measurable A2BAR affinity values (Table 4). All 8-halogenated derivatives bearing a furan ring at position 4 exhibit excellent A2BAR affinity (Ki= 3.05−26.9 nM), indeed generally superior by 4−10-fold to their analogues in the parent series (Figure 5). A similar trend, but with attenuated affinity, is observed for thiophene-based analogues (Ki= 61.1−911 nM). It should also be noticed that for thienyl derivatives, the introduction of a halogen at position 8 does not improve A2BAR affinities as compared to the nonhalogenated parent compounds (Table 1). A direct comparTable 3. Structure and Adenosine Receptor Affinities of Series II: Alkyl 7-Halo-1,4-dihydrobenzo[4,5]imidazo[1,2a]pyrimidine-3-carboxylates (Cmpds 15a−x) Ki(nM) or % at 1 μM compound R4R3X hA1 a hA2A b hA2B c hA3 d 15a 2-furyl Et F 16% 2% 67.5 ±3.1 10% 15b 2-furyl i-Pr F 32% 3% 12.2 ±1.2 5% 15c 3-furyl Et F 26% 21% 25.0 ±1.5 5% 15d 3-furyl i-Pr F 25% 11% 136 ±7 6% 15e 2-thienyl Et F 25% 1% 37% 11% 15f 2-thienyl i-Pr F 11% 2% 23% 6% 15g 3-thienyl Et F 7% 1% 1031 ±95 3% 15h 3-thienyl i-Pr F 1% 1% 764 ±11 1% 15i 2-furyl Et Cl 26% 2% 18% 4% 15j 2-furyl i-Pr Cl 667 ±12 17% 44.3 ±1.4 5% 15k 3-furyl Et Cl 22% 30% 47.3 ±1.9 3% 15l 3-furyl i-Pr Cl 6% 4% 50% 3% 15m 2-thienyl Et Cl 2% 3% 1% 1% 15n 2-thienyl i-Pr Cl 1% 3% 7% 1% 15o 3-thienyl Et Cl 4% 2% 50% 3% 15p 3-thienyl i-Pr Cl 7% 9% 48% 3% 15q 2-furyl Et Br 22% 21% 48% 5% 15r 2-furyl i-Pr Br 46% 1% 273 ±4 6% 15s 3-furyl Et Br 10% 1% 318 ±6 19% 15t 3-furyl i-Pr Br 11% 3% 26% 12% 15u 2-thienyl Et Br 1% 5% 1% 4% 15v 2-thienyl i-Pr Br 1% 6% 11% 8% 15w 3-thienyl Et Br 4% 3% 43% 3% 15x 3-thienyl i-Pr Br 1% 1% 10% 1% DPCPX 2.20 ±0.2 157 ±3 73.2 ±1.4 1722 ±11 ZM241385 683 ±4 1.9 ±0.1 65.7 ±1.1 863 ±4 NECA 14.0 ±1 20.0 ±3 2400 ±35 6.20 ±0.9 a Displacement of specific [3H]DPCPX binding in human CHO cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). b Displacement of specific [3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol binding in human HeLa cells expressed as Kiin nM (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). c Displacement of specific [3H]DPCPX binding in human HEK-293 cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). d Displacement of specific [3H]NECA binding in human HeLa cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 897 ison of the A2BAR affinity profile (pKi) of selected derivatives of different series is presented in Figure 5. The pharmacological data obtained for the exploratory derivatives bearing a 4-oxazolyl residue at position 4 of the tricyclic core (series IV,Table 5) introduced new SAR perspectives. Although in general this series reproduced the most prominent SAR trends discussed for the previous series, the effect of halogenation seems to be less marked if compared to their parent compounds (Table 1>, Cmpds IX and X). Collectively, 8-halo derivatives in this series (Table 5, Cmpds 18a−18d) exhibit excellent A2BAR affinity (Ki6.10−14.7 nM). A comparative analysis reveals that the affinity data in this series is lightly superior to the 7-halo analogues (Table 5, Cmpds 17a−17d) or the parent compounds in the prototype series (Table 1, Cmpds IX and X). These data highlight the interest of the 4-oxazolyl moiety as a privileged non-furane core at A2BAR, which, in addition to a superior metabolic stability profile, can capture the two productive interactions predicted for 2-furyl and 3-furyl rings within the A2BAR orthosteric binding site. This allows for better accommodation of the compounds at A2BAR (Supplementary Figure S4), even allowing a successful fitting of the 7-halo derivatives (Table 5). Table 4. Structure and Adenosine Receptor Affinities of Series III: Alkyl 8-Halo-1,4-dihydrobenzo[4,5]imidazo[1,2a]pyrimidine-3-carboxylates (Cmpds 16a−x) Ki(nM) or % at 1 μM compound R4R3X hA1 a hA2A b hA2B c hA3 d 16a 2-furyl Et F 10% 29% 6.60 ±1.0 2% 16b (ISAM-163) 2-furyl i-Pr F 11% 2% 3.05 ±0.7 3% 16c 3-furyl Et F 29% 21% 13.7 ±0.9 2% 16d 3-furyl i-Pr F 14% 31% 8.60 ±0.5 4% 16e 2-thienyl Et F 1% 3% 724 ±8 3% 16f 2-thienyl i-Pr F 2% 1% 668 ±6 7% 16g 3-thienyl Et F 1% 1% 187 ±2 3% 16h 3-thienyl i-Pr F 2% 1% 75.5 ±3.2 5% 16i 2-furyl Et Cl 23% 10% 8.42 ±1.4 4% 16j (ISAM-161) 2-furyl i-Pr Cl 35% 24% 5.03 ±0.3 2% 16k 3-furyl Et Cl 31% 43% 7.21 ±0.4 4% 16l (ISAM-M89A) 3-furyl i-Pr Cl 27% 176 ±4 6.10 ±0.7 1% 16m 2-thienyl Et Cl 3% 2% 41% 1% 16n 2-thienyl i-Pr Cl 2% 2% 837 ±11 1% 16o 3-thienyl Et Cl 2% 7% 911 ±14 8% 16p 3-thienyl i-Pr Cl 7% 7% 165 ±9 4% 16q 2-furyl Et Br 18% 29% 26.9 ±0.8 33% 16r (ISAM-157) 2-furyl i-Pr Br 25% 2% 5.23 ±0.4 10% 16s (ISAM-M114A) 3-furyl Et Br 1% 37% 3.32 ±0.4 25% 16t 3-furyl i-Pr Br 2% 509 ±6 8.20 ±1.1 2% 16u 2-thienyl Et Br 6% 3% 55% 6% 16v 2-thienyl i-Pr Br 1% 1% 115 ±3 1% 16w 3-thienyl Et Br 1% 9% 537 ±8 1% 16x 3-thienyl i-Pr Br 2% 1% 61.1 ±1.8 4% DPCPX 2.20 ±0.2 157 ±3 73.2 ±1.4 1722 ±11 ZM241385 683 ±4 1.9 ±0.1 65.7 ±1.1 863 ±4 NECA 14.0 ±1 20.0 ±3 2400 ±35 6.20 ±0.9 a Displacement of specific [3H]DPCPX binding in human CHO cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n = 2). b Displacement of specific [3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol binding in human HeLa cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). c Displacement of specific [3H]DPCPX binding in human HEK-293 cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). d Displacement of specific [3H]NECA binding in human HeLa cells expressed as Kiin nanomolars (n= 3) or percentage displacement of specific binding at a concentration of 1 μM (n= 2). Figure 5. Interleaved bar chart showing A2BAR pKi[−log(Ki)] for the halogenation of furyl and oxazolyl scaffolds (represented at the x-axis as I−IV and IX−X). Black: non-halogenated, green: fluorinated, cyan: chlorinated, and orange: brominated compounds. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 898 columns followed by recrystallization. Melting points were determined on a Stuart Scientific melting point apparatus and are uncorrected. The NMR spectra were recorded on Bruker AM300 and XM500 spectrometers. Chemical shifts are given as δvalues against tetramethylsilane as internal standard, and Jvalues are given in Hertz. Mass spectra were obtained on a Varian MAT 711 instrument. For regioisomer differentiation experiments, 1H NMR, 2D-ROESY, and 2D-NOESY were recorded on an AV NEO 750 MHz instrument. High-resolution mass spectra were obtained on an AutoSpec Micromass spectrometer. Analytical HPLC was performed on a Water Breeze 2 system (binary pump 1525, detector UV/Visible 2489, 7725i Manual Injector Kit 1500 Series) using a Luna 5 μm Silica (2) 100 Å, LC Column 150 ×4.6 mm column with gradient elution using the mobile phases dichloromethane, isopropanol, and a flow rate of 1 mL/min. The purity of all tested compounds was determined to be >95%. The chiral resolution was performed using a Water Breeze 2 (binary pump 1525, detector UV/Visible 2489, 7725i Manual Injector Kit 1500 Series). Compound 16b enantiomers were separated using a 250 mm ×10 mm Lux 5 μm Amylose-2 (Phenomenex) while ISAM140,16j,16l,16r,16s, and 18c enantiomers were separated using a 250 mm ×20 mm CHIRALPAK 5 μm IE-3 (DAICEL). A detailed description of the experimental protocols and relevant parameters (retention times, stereochemical purities) is provided in the Supporting Information. All single stereoisomers were isolated and their stereochemical purity analyzed by chiral HPLC (>97% for each stereoisomer) and then characterized by NMR in CDCl3. CD 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; and wavelengths, 400−190 nm. A detailed description of the synthesis and structural and spectroscopic data obtained for all compounds described is provided in the Supporting Information. General Procedure for the Synthesis of Alkyl 7,8-dihalobenzo- [4,5]imidazo[1,2-a]pyrimidine-3-carboxylates 14a−x.(Method A): A mixture of the corresponding 5,6-dihalo-2-aminobenzimidazole 11a−c(1.5 equiv), aldehyde 12a−d(1 equiv), β-ketoester 13a,b(1 equiv), and ZnCl2(0.1 equiv) in THF (2.5 mL) was stirred with orbital stirring at 90 °C for 12 h. After completion of the reaction, as indicated by TLC, the solvent was removed in vacuum and the obtained oily residue was purified by column chromatography on silica gel, obtaining the corresponding alkyl 7,8-dihalobenzo[4,5]- imidazo[1,2-a]pyrimidine-3-carboxylates (14a−x). General Procedure for the Synthesis of Alkyl 7-halobenzo[4,5]- imidazo[1,2-a]pyrimidine-3-carboxylates 15a−xand 8Halobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylates 16a−x. (Method A): A mixture of 2-amino-5-halobenzimidazole 11d−f (1.5 equiv), aldehyde 12a−d(1 equiv), β-ketoester 13a,b(1 equiv), and ZnCl2(0.1 equiv) in THF (2.5 mL) was stirred with orbital stirring at 90 °C for 12 h. After completion of the reaction, as indicated by TLC, the solvent was removed in vacuum and the obtained oily residue was purified by column chromatography on Table 8. Preliminary ADME and Binding Data of Novel and Previously Reported A2BAR Antagonists microsomal stability CYP3A4 IC50 (μM) e or % inh. at 10 μM f compound solubility (μM) a percent b t1/2 c Clint d DBF 7-BFC hA2BKi(nM) II (ISAM-140) 30 25.2 2.90 ±0.37 3.49 ±0.2 14b 62.0 20.97 28.93 21.65 >10 (39%) >10 (58%) 11.9 ±1.1 16b (ISAM-163) 47.5 19.83 27.40 20.07 >10 (40%) >10 (63%) 3.05 ±0.7 16j (ISAM-161) 23.7 22.75 28.14 18.60 3.68 ±0.47 >10 (54%) 5.03 ±0.3 16r (ISAM-157) 70.7 32.13 36.79 14.23 2.26 ±0.30 >10 (52%) 5.23 ±0.4 IV (ISAM-142) 30 26.1 2.68 ±0.53 11.4 ±0.5 15j 58.2 16.83 21.13 26.44 >10 (47%) >10 (11%) 44.3 ±1.4 16l (ISAM-M89A) 21.9 41.02 44.80 11.68 6.36 ±1.02 >10 (44%) 6.10 ±0.7 (A2B) 176 ±4 (A2A) X 31 39.7 5.92 ±0.68 43.4 ±1.3 18b (ISAM-R324A) 64.6 35.15 40.8 12.83 >10 (31%) >10 (52%) 6.10 ±0.3 PSB-603 28 0.2 0.553 progesterone 6.7 prazosin 31.3 testosterone 7.11 16.13 32.44 ketoconazole 0.008 μM 0.027 μM a Solubility in 1:99 DMSO/PSB buffer. b Percentage remanent (sampling time 60 min). c t1/2 in min. d Intrinsic clearance in microliters per minute per milligram of protein. e IC50 value obtained by extrapolation with analysis software. f Due the low activity shown at CYP3A4, the percentage of inhibition at 10 μΜis reported. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 905 silica gel, obtaining the corresponding alkyl 7and 8-halobenzo[4,5]- imidazo[1,2-a]pyrimidine-3-carboxylates. General Procedure for the Synthesis of Alkyl 7-Halo-4-(oxazol-4yl)-benzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylates 17a−dand 8-Halo-4-(oxazol-4-yl)-benzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylates 18a−d.(Method B): A mixture of 2-amino-5-halobenzimidazole 11d,e(1.5 equiv), oxazole-4-carbaldehyde 12e (1 equiv), βketoester 13a,b(1 equiv), and AcOH (three drops) in THF (2 mL) and DMF (1 mL) was stirred under microwave irradiation at 80 °C for 90 min. After completion of the reaction, as indicated by TLC, the solvent was removed in vacuum and the obtained oily residue was purified by column chromatography on silica gel, obtaining the corresponding alkyl 7and 8-halo-4-(oxazol-4-yl)-benzo[4,5]imidazo- [1,2-a]pyrimidine-3-carboxylates (17a−dand 18a−d). Pharmacology Binding Assays. Radioligand binding competition assays were performed in vitro using human ARs expressed in transfected HeLa [hA2AAR (9 pmol/mg protein) and hA3AR (3 pmol/mg protein)], HEK-293 [hA2BAR (1.5 pmol/mg protein)], and CHO [hA1AR (1.5 pmol/mg protein)] cells as described previously. 25,31,33 A brief description is given below. A1AR competition binding experiments were carried out in membranes from CHO-A1cells labeled with 1 nM [3H]DPCPX (KD= 0.7 nM). Non-specific binding was determined in the presence of 10 μM RPIA. The reaction mixture was incubated at 25 °C for 60 min. A2AAR competition binding experiments were carried out in membranes from HeLa-A2A cells labeled with 3 nM [3H]ZM241385 (KD= 2 nM). Non-specific binding was determined in the presence of 50 μM NECA. The reaction mixture was incubated at 25 °C for 30 min. A2BAR competition binding experiments were carried out in membranes from HEK-293-A2B cells (Euroscreen, Gosselies, Belgium) labeled with 25 nM [3H]DPCPX (KD= 21 nM). Nonspecific binding was determined in the presence of 400 μM NECA. The reaction mixture was incubated at 25 °C for 30 min. A3AR competition binding experiments were carried out in membranes from HeLa-A3cells labeled with 10 nM [3H]NECA (KD= 8.7 nM). Nonspecific binding was determined in the presence of 100 μM R-PIA. The reaction mixture was incubated at 25 °C for 180 min. After the incubation time, membranes were washed and filtered and radioactivity was detected in a MicroBeta TriLux reader (PerkinElmer). Pharmacology Functional Experiments. Reagents. The following reagents were used: adenosine deaminase (ADA, Roche Diagnostics, Mannheim, Germany), 5′-N-ethylcarboxamidoadenosine (NECA, Tocris, Bristol, UK), zardaverine (Calbiochem, San Diego, California, USA), SNAP-Surface Alexa Fluor 488 (New England BioLabs, Ipswich, Massachusetts, USA). Cloning. The cDNA encoding the human A2BAR was amplified by polymerase chain reaction using the primers FA2BREcoRV (5′- tcgagGATATCCTGCTGGAGACACAGGACGC-3′) and RA2BRHindIII (5′-cgagAAGCTTTCATAGGCCCACACCGAGAGC-3′) and subcloned into the EcoRV/HindIII restriction sites of the pRK5-A1RSNAP vector (kindly provided by Dr. J. P. Pin, Universite de Montpellier and Institut de Genomique Fonctionnelle, Montpellier, France) by replacing the Ado A1receptor. The resulting construct encoded the human A2BAR tagged with SNAP, a 20 kDa Figure 10. Effect of compounds 16b and 16j on the proliferation of PBMCs stimulated with antiCD3/CD28 + IL-2 in the presence of adenosine (ADO). PBMCs were CFSE-labeled, stimulated with anti-CD3/CD28 antibodies and IL-2, and cultured with the compounds of interest in the presence of adenosine for 3 days. The frequency of proliferating cells (CFSEdim) as well as their median fluorescence intensities were quantified and ratios calculated against the values of stimulated PBMCs with no further treatment. (A) Frequency of proliferating cells. (B) MFI of proliferating cells. (C−F) Representative dots plots of the proliferation of CFSE-stained PBMCs treated with anti-CD3/CD28 + IL-2 + ADO (C), anti-CD3/ CD28 + IL-2 + ADO +16b (D), anti-CD3/CD28 + IL-2 + ADO +16j (E), and anti-CD3/CD28 + IL-2 + ADO + DMSO (F). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 906 mutant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), at its N-terminus (pRK5-A2BARSNAP). Cell Culture and Stable Transfection. Human embryonic kidney (HEK)-293 T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, Missouri, USA) supplemented with 1 mM sodium pyruvate (Biowest, Nuaille, France), 2 mM L-glutamine (Biowest), 100 U/mL streptomycin (Biowest), 100 mg/mL penicillin (Biowest), and 5% (v/v) fetal bovine serum (Invitrogen, Carlsbad, California, USA) at 37 °C and in an atmosphere of 5% CO2. HEK-293 cells growing in 60 cm2plates were transfected with pRK5-A2BARSNAP using the polyethylenimine (PEI) method. 53 After 48 h of transfection, cells were stained using SNAP-Surface Alexa Fluor 488. 38 Fluorescent cells were selected every 2 weeks for 2 months using a Cell sorter (MoFlo Astrios, Beckman Coulter) to enrich the percentage of cells that express the receptor, thus ensuring its permanent expression. 54 cAMP Accumulation Assay. cAMP accumulation was measured using the LANCE Ultra cAMP kit (PerkinElmer, Waltham, Massachusetts, USA) as previously described. 55 In brief, HEK-293 T cells permanently expressing the A2BARSNAP construct (A2BARSNAP cells) were detached with Accutase (Sigma-Aldrich) and incubated for 1 h at 22 °C in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 0.1% BSA, ADA (0.5 U/mL), and zardaverine (100 μM). A2BARSNAP cells (1 ×104cells/200 μL) were incubated with NECA in the presence/absence of increasing concentrations of 16b,16j,16r, and 16s, during 30 min at 22 °C. EucAMP tracer and ULight-anti-cAMP reagents were prepared and added to the sample following the manufacturer’s instructions. The 384-well plate was incubated 1 h at 22 °C in the dark and was then read on a CLARIOstar Microplate Reader (BMG Labtech, Durham, North Carolina, USA). Measurements at 620 and 665 nm were used to detect the TR-FRET signal, and the concomitant cAMP levels were calculated following the manufacturer’s instructions. Data were fitted by non-linear regression using GraphPad Prism 9 (San Diego, California, USA). Concentration−response curves were carried out by assaying different 16b,16j,16r, and 16s concentrations ranging between 10 nM and 30 μM. Data was expressed as KBby following the formula reported by Leff and Dougall (eq 1): 56 = +[ ] [ ] i k j j jy { z z z ( ) KIC 2A A nn B 50 1/ 50 (1) Correction offset value for all the ABFE estimates. 56 where IC50 is the concentration of compound that inhibits NECA effect by 50%, [A] is the concentration of NECA employed in the assay, [A50] is the NECA EC50 value, and nis the Hill slope of the curve. Cellular Impedance Label-Free Assay. The xCELLigence RTCA system (Roche) was employed to assess the impact of blocking A2BAR-mediated effects in cellular impedance upon receptor activation, as previously described. 57−60 To this end, A2BARSNAP cells were growth in 16-well E-plates (Roche), using DMEM supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL streptomycin, 100 mg/mL penicillin, and 1.5% (v/v) fetal bovine serum in the presence of 0.5 U/mL of ADA. Of note, wells were previously coated with 50 μL fibronectin (10 μg/mL; Sigma-Aldrich) and the background index for each well was determined with supplemented DMEM (90 μL) in the absence of cells. Subsequently, A2BARSNAP cells (90 μL) were plated at a cell density of 10,000 cells/ well and grown for 18 h in the RTCA SP device station (Roche) at 37 °C in an atmosphere of 5% CO2. Then, before ligand addition, cell index values were normalized to the same time point using the RTCA software, providing the normalized cell index (NCI). After ligand stimulation, NCI was recorded every 15 s for a total time of at least 45 min. The area under the curve (AUC) for every condition was calculated using GraphPad Prism 9. Data and Statistical Analysis. Data are represented as mean ± standard error of mean (SEM) with statistical significance set at P< 0.05. The number of samples (n) in each experimental condition is indicated in the corresponding figure legend. Outliers were assessed by the ROUT method; 61 thus, any sample was excluded assuming a Q value of 1% in GraphPad Prism 9 (San Diego, California, USA). Comparisons among experimental groups were performed by the Student ttest or one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison post hoc test using GraphPad Prism 9, as indicated. CYP3A4 Inhibition. The inhibitory activity of selected compounds was evaluated by following a previously described method. 31 Incubations were conducted in a 200 μL volume in 96-well microtiter plates (Costar 3915). Addition of a cofactor−buffer mixture (KH2PO4buffer, 1.3 mM NADP, 3.3 mM MgCl2, 3.3 mM glucose6-phosphate, and 0.4 U/mL glucose-6-phosphate dehydrogenase), supersome control, standard inhibitor (ketoconazole from SigmaAldrich) previously diluted, and compounds to plates were carried out by a liquid handling station (Zephyr Caliper). The plate was then preincubated at 37 °C for 5 min and the reaction initiated by the addition of a pre-warmed enzyme/substrate (E/S) mix. The E/S mix contained buffer (KH2PO4), c-DNA-expressed P450 in insect cell microsomes, substrate (DBF: dibenzylfluorescein or 7-BFC: 7benzyloxy-4-trifluoromethyl-coumarin) and other components to give the final assay concentrations in a reaction volume of 200 μL. Reactions were terminated after various times (a specific time for each cytochrome) by addition of STOP solution (ACN/Tris−HCl 0.5 M 80:20, and NaOH 2 N for CYP3A4). Fluorescence per well was measured using a fluorescence plate reader (Tecan Infinite M1000 PRO), and percentage of inhibition was calculated. In silico predictions were performed using free web-based models (FAME 3, SmartCYP, and Xenosite). 62−64 Human Microsomal Stability. The human microsomal stability of selected compounds was evaluated by following a previously described method. 31 The human microsomes employed were purchased from Tebu-Xenotech. The compound was incubated with the microsomes at 37 °C in a 50 mM phosphate buffer (pH = 7.4) containing 3 mM MgCl2, 1 mM NADP, 10 mM glucose-6-phosphate, and 1 U/mL glucose-6-phosphate-dehydrogenase. Samples (75 μL) were taken from each well at 0, 10, 20, 40, and 60 min and transferred to a plate containing 4 °C 75 μL acetonitrile and 30 μL of 0.5% formic acid in water was added for improving the chromatographic conditions. The plate was centrifuged (46,000 g, 30 min), and supernatants were taken and analyzed in a UPLC-MS/MS (Xevo TQD, Waters) by employing a BEH C18 column and an isocratic gradient of 0.1% formic acid in water:0.1% formic acid acetonitrile (60:40). The metabolic stability of the compounds was calculated from the logarithm of the remaining compounds at each of the time point studied. Solubility Determinations. The solubility of selected compounds was evaluated by following a previously described method. 31 A 10 mM stock solution of the compound was serially diluted in 100% DMSO, and 2.5 μL of this solution was added to a 384-well UVtransparent plate (Greiner) containing 47.5 μL of PBS (pH = 7). The plate was incubated at 37 °C for 4 h, and the light scattering was measured in a NEPHELOstar Plus reader (BMG LABTECH). The data was fitted to a segmented linear regression for measuring the compound solubility. System Preparation and MD/FEP Simulations. The structural model for the A2BAR receptor derives from a homology model previously reported 31,33 and here processed using the Protein Preparation Wizard pipeline in Maestro (Schrodinger ver. 2021-3). Subsequently, possible 3D tautomers and protomers at pH 7 ±2 were generated for each compound using the OPLS4 force field 65 and Epik, the lowest energy conformer was chosen for molecular docking. A receptor grid was generated with the default Van der Waals radius scaling settings, and it was positioned in the center of geometry of the binding site. Thereafter, Glide SP docking was performed leading to the protein−ligand complexes, which were used as the starting point for the MD/FEP simulations carried out with the software package Q. 66 An FEP network was generated for each of chemical series using ECFP4 as a measure of the chemical similarity between ligand pairs, Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 907 ensuring smooth alchemical transformations while covering the entire dataset. For each vertex comparing a pair of compounds part of the FEP network, the QligFEP 67 pipeline was used for generating the MD/FEP input files and its posterior analysis. The MD simulations were carried out under spherical boundary conditions (SBC) with a sphere size of 25 Å, with solvent atoms lying in the outer shell of the sphere (22−25 Å) subject to radial and polarization restrains using the surface-constrained all-atom solvent (SCAAS). 68,69 All titratable residues outside the sphere were neutralized, and histidine protonation states were set by the Protein Preparation Wizard. Atoms outside the simulation sphere were excluded from the calculation of the nonbonded interactions and tightly constrained (200 kcal/mol·Å 2 ) to its position. Long-range electrostatic interactions beyond a 10 Å threshold were evaluated using the local reaction field method, 68 excluding the atoms undergoing the FEP transformation where no cut-off is applied. The OPLS-AA/M force field 70 was used for the protein and solvent (TIP3P) parameters, while OPLS2005 ligand parameters were generated using the ffld_server; 71 solvent bonds and angles were constrained using the SHAKE algorithm. 72 The MD/FEP simulations are preceded by a heating phase of the sphere from 0.1 to 298 K during a short 31 ps stage where a positional restraint of 10 kcal/mol·Å2is progressively released. This is followed by a 100 ps unbiased and unrestrained equilibration, and subsequently, MD sampling is performed across a predefined sigmoidal λschedule, consisting of 101 λwindows, each consisting of 10 ps sampling using a 2 fs timestep. To get initial relative binding free energy estimates (RBFE), the thermodynamic cycles are closed for each ligand pair defined in the FEP network by performing the corresponding MD/FEP simulation in a water sphere, and the free energies are calculated with the Bennet acceptance ratio (BAR) method. 66 The cycle closure correction framework 73 was utilized for assessing the convergence of the initial RBFE estimates, and additionally, it yielded corrected RBFE values, which eliminate the hysteresis along all the thermodynamic cycles encompassed by the FEP network. To get the ABFE for all the compounds of interest, the corrected RBFE values together with the FEP networks and the experimental binding free energy for a reference compound per network are used. The Bellman−Ford implementation from NetworkX 74 was used for finding the shortest path connecting a reference with the remaining target compounds, and, for each target compound, eq 1 was used to get the ABFE estimates. Finally, a correction offset value for all the ABFE estimates was calculated as described by Wang et al. 75 in order to minimize the experimental error introduced to the final estimates (eq 2). = + = G G G i N i pred Targ.comp. exp Ref.comp. 1 pred FEP, (2) Correction offset value for all the ABFE estimates. 75 In Vitro Evaluation of the Immunostimulatory Effect. PBMCs were isolated from a healthy donor, stained with CFSE (Abcam) following the manufacturer’s protocol, and cultured in duplicates in U-bottom 96-well plates at a density of 1.5 ×105cells/ well in RPMI 1640 medium (Gibco) supplemented with 10% AB Human Serum (Sigma-Aldrich) and 1% penicillin−streptomycin (Gibco). PBMCs were stimulated with ImmunoCult Human CD3/ CD28 T Cell Activator (STEMCELL Technologies) and 50 U/mL Interleukin 2 (Gibco). Ado was then added at a final concentration of 0.1 mM and compounds 16b and 16j at 15 μM. DMSO was used as vehicle control. PBMCs were kept in culture with the different treatments for 3 days in an incubator at 37 °C and 5% CO2. After the culture period, cells were washed twice with DPBS (Gibco) and CFSE fluorescence measured in a BD Accuri flow cytometer. The data was analyzed using FlowJo software (BD): the percentage of CFSE+cells, and their median fluorescence intensity (MFI) was quantified for each treatment condition, and a ratio against the stimulated condition without Ado was calculated for each treatment. Positive values indicate increases in the variable compared to the stimulated control, while negative values indicate decreases in the measured variable. ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01768. Molecular formula strings (CSV) Atomic coordinates and experimental data upon article publication (ZIP) Spectroscopic and analytical data for all compounds described, HPLC enantiomeric separations, X-ray crystallography assays, supplementary figures, supplementary tables, and HPLC traces for lead compounds (PDF) ■AUTHOR INFORMATION Corresponding Authors José Brea −Center for Research in Molecular Medicine and Chronic Diseases (CiMUS) and Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/00000002-5523-1979; Phone: +34 881815459; Email: [email protected]; Fax: +34-8818115474 Hugo Gutiérrez-de-Terán −Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden; orcid.org/0000-0003-0459-3491; Phone: +46 18 471 5056; Email: [email protected]; Fax: +46 18 536971 Eddy Sotelo −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0001-5571-2812; Phone: +34 881815732; Email: [email protected]; Fax: +34-881815704 Authors Rubén Prieto-Díaz −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden; orcid.org/0000-0003-2539-3106 Manuel González-Gómez −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Hugo Fojo-Carballo −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Jhonny Azuaje −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Abdelaziz El Maatougui −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01768 J. Med. Chem. 2023, 66, 890−912 908 Maria Majellaro −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain María I. Loza −Center for Research in Molecular Medicine and Chronic Diseases (CiMUS) and Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/00000003-4730-0863 Víctor Fernández-Duenas −Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neuroscience, University of Barcelona, 08907 L’Hospitalet de Llobregat, Spain; Neuropharmacology and Pain Group, Neuroscience Program, Institut d’InvestigacióBiomedica de Bellvitge, IDIBELL, 08907 L’Hospitalet de Llobregat, Spain M. Rita Paleo −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0002-0095-3931 Alejandro Díaz-Holguín −Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden Beatriz Garcia-Pinel −Center for Research in Molecular Medicine and Chronic Diseases (CiMUS) and Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0002-4473-7659 Ana Mallo-Abreu −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0002-5092-3538 Juan C. Estévez −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0001-9468-9045 Antonio Andujar-Arias −Center for Research in Biological Chemistry and Molecular Materials (CIQUS) and Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0001-9922-3543 Xerardo García-Mera −Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Iria Gomez-Tourino −Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Francisco Ciruela −Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neuroscience, University of Barcelona, 08907 L’Hospitalet de Llobregat, Spain; Neuropharmacology and Pain Group, Neuroscience Program, Institut d’InvestigacióBiomedica de Bellvitge, IDIBELL, 08907 L’Hospitalet de Llobregat, Spain; orcid.org/0000-0003-0832-3739 Cristian O. Salas −Department of Organic Chemistry, Faculty of Chemistry and Pharmacy, Pontificia Universidad Católica de Chile, Santiago 7820436, Chile Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.2c01768 Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS This work was financially supported by the Consellería de Cultura, Educación e Ordenación Universitaria of the Galician Government (grant: ED431B 2020/43), Centro Singular de Investigación de Galicia accreditation 2019-2022 (ED431G 2019/03), the European Regional Development Fund (ERDF), Ministerio de Ciencia e Innovación −Agencia Estatal de Investigación-FEDER-UE (PID2020-118511RB-I00 and PID2021-124010OB-100). Additional support from the Swedish strategic research program eSSENCE, the Swedish Cancer Society (#CAN 2018/451), and The Cancer Research Fundations of Radiumhemmet (#181183) is acknowledged. The computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC). We thank Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya for IDIBELL institutional support, Xunta de Galicia (ED431C 2018/21 and ED431G 2019/02), and European Regional Development Fund (ERDF) in the frame of the Recovery Assistance for Cohesion and the Territories of Europe (REACT-EU) funds. This research program has been developed in the frame of the European COST action ERNEST (CA 18133). ■ABBREVIATIONS ABFE, absolute binding free energies; ADMET, absorption, distribution, metabolism, excretion, and toxicity; Ado, adenosine; AGT, O6-alkylguanine-DNA alkyltransferase; AR, adenosine receptor; AUC, area under curve; cAMP, cyclic adenosine monophosphate; CD, circular dichroism; CFSE, carboxyfluorescein succinimidyl ester; CHO, Chinese hamster ovary; Cmpd, compound; Cmpds, compounds; DMEM, Dulbecco’s modified Eagle’s medium; DMF, dimethylformamide; DPCPX, dipropylcyclopentyl-xanthine; FEP, free energy perturbation; GPCRs, G protein-coupled receptors; MD, molecular dynamics; MFI, median fluorescence intensities; MPLC, medium pressure liquid chromatography; NECA, Nethoxycarbonyl adenosine; NMR, nuclear magnetic resonance; P1, class 1 purinergic receptors; PAINS, pan-assay interference compounds; PBMCs, peripheral blood mononuclear cells; RPIA, R-phenylisopropyl-adenosine; RBFE, relative binding free energies; ROESY, rotating frame Overhauser effect spectroscopy; SAR, structure−activity relationships; SBC, spherical boundary conditions; SCAAS, surface-constrained all-atom solvent; SSR, structure−selectivity relationships; THF, tetrahydrofuran; Veh, vehicle. ■REFERENCES (1) Borea, P. 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