Adenosine Receptor Ligands: Coumarin–Chalcone Hybrids as Modulating Agents on the Activity of hARs

Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer’s disease, Parkinson’s disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin–chalcone hybrids were synthesized (compounds 1–8) and screened in radioligand binding (hA1, hA2A and hA3) and adenylyl cyclase (hA2B) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin–chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA1 or hA3 subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA1, while the methoxy counterparts were selective for the hA3. The most potent hA1 ligand was compound 7 (Ki = 17.7 µM), whereas compound 4 was the most potent ligand for hA3 (Ki = 2.49 µM). In addition, docking studies with hA1 and hA3 homology models were established to analyze the structure–function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.


Introduction
Adenosine receptors (ARs) are cell membrane receptors, belonging to the G protein-coupled receptor (GPCRs) superfamily. ARs comprised of four different subtypes: A 1 , A 2A , A 2B and A 3 [1]. Adenosine is a purine nucleoside and an endogenous modulator of several physiological processes [1][2][3][4]. Extracellular adenosine activates the G i -coupled receptors of the A 1 and A 3 subtypes, depressing the action of the brain, heart, kidneys, and the immune system, amongst other systems, as a consequence of the inhibition of adenylyl cyclase [5]. The A 3 subtype of AR has been cloned [6,7], making it possible to establish its pharmacological [8][9][10][11] and regulatory features [12].
Due to their widespread presence in cells, ARs proved to be promising targets in drug discovery. During the last decade, the search for selective ligands has been raised [13][14][15]. Several AR antagonists appeared as promising drug candidates for different pathological processes such as inflammation (A 3 ) [14], heart and renal failure (A 1 ) [16], or neurological disorders including Parkinson [17,18] and Alzheimer's diseases (A 2A and/or A 1 ) [19]. ARs can work as targets for various diseases and can open a new window for new therapeutic approaches.
In particular, A 1 antagonists are effective as diuretic agents [20,21] and also show neuroprotective activity in animal models of in vivo ischemia [22]. On the other hand, A 3 antagonists are being investigated as potential agents against renal injury [23] and also as neuroprotective agents [24,25], while A 3 agonists are also under consideration for treating conditions of the central nervous system (CNS) and peripheral nervous system [26,27].
From the arsenal of molecules presenting high potency and selectivity on ARs, the xanthine scaffold was the first to be used to develop the so-called classical AR antagonists [28,29]. In the search for non-xanthine AR ligands, numerous structures were discovered over the years. Flavones and isoflavones have played a remarkable role. As an example, genistein, was described as a competitive antagonist at A 1 in FRTL (thyroid) cells [30], and galangin was found to bind to the three subtypes of ARs displaying micromolar affinity for the A 3 [31]. The affinity of flavonoids and other phytochemicals to ARs brings about the hypothesis that probably other types of natural substances, namely those present in the diet, can interact with this type of receptor.
Coumarins (chromone isosteres) and chalcones (a flavonoid precursor) are naturally occurring benzopyran-related molecules presenting a variety of pharmacological activities [32][33][34]. Having in mind that both the coumarin and chalcone nuclei are structurally close to flavonoids, the design of novel AR ligands based on their scaffolds emerged as an interesting idea. Our study was also motivated by the structural similarity between the coumarin and the chromone scaffolds, which were previously described as AR ligands [35,36], and by the similarities with some coumarin derivatives previously described in our group [37][38][39][40][41][42]. In this context, we focused our attention on the 3-benzoylcoumarin core, considered as a hybrid scaffold in which the chalcone is fixed in a trans conformation through the double bond of the pyrone ring of the coumarin skeleton (Figure 1), presenting a more restricted conformation compared to the previously described coumarin-chalcone hybrids [36]. Therefore, based on the structural similarities between flavones, chalcones and coumarins, we decided to design and synthesize a novel family of coumarin-chalcone hybrid derivatives and study their activity on the different subtypes of human AR. Therefore, based on the structural similarities between flavones, chalcones and coumarins, we decided to design and synthesize a novel family of coumarin-chalcone hybrid derivatives and study their activity on the different subtypes of human AR.

Adenosine Receptor Binding Affinity Assays
The adenosine binding affinity of derivatives 1-8 for the human AR subtypes hA1, hA2A and hA3, expressed in Chinese Hamster Ovary (CHO) cells, was determined in radioligand competition experiments [43,44]. In the binding affinity assay, it is measured the competition of ligands for specific binding of [

Adenosine Receptor Binding Affinity Assays
The adenosine binding affinity of derivatives 1-8 for the human AR subtypes hA 1 , hA 2A and hA 3 , expressed in Chinese Hamster Ovary (CHO) cells, was determined in radioligand competition experiments [43,44]. In the binding affinity assay, it is measured the competition of ligands for specific binding of [ 3 H]CCPA (2-chloro-N 6 -cyclopentyladenosine) to hA 1 ; specific binding of [ 3 H]NECA (5 -N-ethylcarboxamidoadenosine) to hA 2A ; and specific binding of [ 3 H]HEMADO (2-(1-hexynyl)-N 6 -methyladenosine) to hA 3 . The results are expressed as K i (dissociation constants), which were calculated with the program SCTFIT, and given as geometric means of at least three experiments, including 95% confidence intervals. Due to the lack of a suitable radioligand for the hA 2B receptor, the potency of antagonists at the hA 2B receptor was determined by inhibition of NECA-stimulated adenylyl cyclase activity with increasing concentrations of antagonist [43,44]. As a result, cAMP (cyclic adenosine monophosphate) production was inhibited in a concentration-dependent fashion, and K i values were calculated from the measured IC 50 values [45].
Derivatives 1-8 were efficiently synthesized and their in vitro binding affinity for human AR subtypes hA 1 , hA 2A , hA 2B and hA 3 , expressed in CHO cells, was evaluated. In the present communication, the studies were focused on the inspection of the effect on the binding affinity of different number and position of methoxy or hydroxy substituents on the 3-benzoylcoumarin scaffold. Data obtained for the binding affinity for hA 1 and hA 3 is summarized in Table 1. For all the tested compounds, no significant affinity was detected for the hA 2A (Ki > 100 µM) or hA 2B (K i > 10 µM). The binding affinity results show that derivatives 1 and 2, without substitutions on the coumarin scaffold or with a single methoxy group at the position 6 of the coumarin core, respectively, display no detectable binding affinity for the evaluated receptors (K i > 100 µM). However, the presence of two methoxy groups at positions 5 and 7 (compounds 3 and 4, respectively) lead to an increment on both the potency and selectivity for the hA 3 . Compound 3, presenting three methoxy groups at positions 5, 7 and 4 proved to be hA 3 selective, displaying a K i = 9.03 µM, whereas compound 4, presenting an extra methoxy groups at position 3 is not only selective for hA 3 , but also displays a increase in potency (K i = 2.49 µM). Compared to theophylline, classically used as a reference compound, we would like to highlight that both compounds 3 and 4 are more potent and hA 3 selective molecules.
Based on this data, it can be concluded that both nature and position of the substitution patterns on the coumarin-chalcone scaffold play a key role in the interaction with the hA 3 . It can be highlighted that positions 5 and 7 of the studied scaffold seem to be relevant for the observed selectivity and potency. Analyzing the methoxylated derivatives 1-4, only the molecules presenting substituents at these two positions (compounds 3 and 4) are hA 3 active and selective ligands.
Interestingly, a similar tendency was observed for hA 1 binding of the hydroxylated derivatives (5-8), which bear hydroxy groups instead of methoxy groups at positions 5 and 7 (compounds 7 and 8). Derivatives 7 and 8 display the highest potency and selectivity of the studied series towards hA 1 , but their activity towards this receptor is still low with K i = 17.7 µM and K i = 29.1 µM, respectively.

Theoretical Evaluation of ADME Properties
In order to explore the drug-like properties of compounds 1-8, the lipophilicity, expressed as the octanol/water partition coefficient and herein named clogP, as well as other theoretical calculations such as number of hydrogen acceptors and number of hydrogen bond donors, and topological polar surface area (TPSA), were calculated using the Molinspiration software [46]. Theoretical prediction of absorption, distribution, metabolism and excretion (ADME) properties of all derivatives is summarized in Table 2. Based on this theoretical data, it can be concluded that the study molecules 1-8 do not violate any of Lipinski's rules (namely molecular weight, clogP, number of hydrogen donors and acceptors). In addition, TPSA, described as an indicator of membrane permeability, was favorable for the studied compounds.

Molecular
Modeling hA 1 and hA 3 homology models were successfully constructed (Materials and methods section). A selection of models obtained from Induce Fit calculations were tested based on their ability to discriminate between known ligands, decoys and between subtype-selective compounds. The models selected for the docking calculations showed excellent results in both tests. A dataset of 200 randomly selected decoys from the ZINC database [47] were mixed up with 22 known ligands of each adenosine receptor subtype [48] Glide SP precision was used to dock the database to the hA 1 and hA 3 models [49]. Table 3 presents the area under the receiver operating characteristic (ROC) curve (AUROC) for both systems. To differentiate between subtype-selective ligands, a second and more challenging test was performed. As in a previous study [48], 66 subtype-selective molecules ( [50] and proved that the developed homology models are able to discriminate between subtype-selective compounds (Table 3). Glide SP molecular docking simulations were run with our data using the hA 1 and hA 3 selected homology models as protein structures to detect the hypothetical binding mode of the new synthesized compounds [51]. The Prime module was used to optimize the protein structure for each binding mode [52]. Molecular docking simulations are represented in Figure 2. Docking calculations and the established homology models for the hA1 and hA3 identified the hypothetical binding mode and rationalized the interaction of these derivatives with their respective ARs binding sites. The calculations showed a high level of variability since all the synthetized derivatives yielded different possible binding modes inside the pockets. Selection of the hypothetical binding pose was accomplished considering the number of similar poses extracted from the simulations and geometrical correspondence to crystallized ligands in the hA2A (Figure 2a).
Docking results disclosed important data about the binding mode: the oxygens presented in the benzopyrone system are oriented towards the Asn250 residue and the benzoyl moiety was buried in the hA3 pocket. This hypothetical binding mode corroborates the conformations shown by the cocrystallized ligands in the hA2A (PDB: 3EML and 3UZC) [48,53] (Figure 2a,b). The pose of compound 3 produced effective hydrogen bonds with Gln167, Asn250 and His272 residues.
Interestingly, when methoxy substituents were demethylated and changed into hydroxy equivalents (compounds 5-8) a modification in the profile of the studied derivatives was noticed: a loss of affinity for hA3 and a tendency for interaction with hA1. The only compound that discloses some affinity for both receptors was compound 5 (hA1 Ki = 39.5 and hA3 Ki = 34.5 μM), which presents a catechol at positions 3′ and 4′ and no substitutions in the coumarin fragment. The hypothetical binding mode for compound 5 in the hA3 pocket is represented in Figure 2c. The compound can establish hydrogen bonds with Ala69, Asn250 and His272 residues. As observed in the hA2A crystallized structure and previously published studies [54,55], the corresponding Asn250 residue seems to play an important role in ligand recognition. The compound 5 pose inside the hA1 pocket is likewise the described pose in the hA3 one. However, the position was slightly shifted, and calculations were not able to retrieve a hydrogen bond with the Asn250 residue. The introduction of an additional hydroxy group at position 6 of the coumarin scaffold (compound 6), resulted in a loss Docking calculations and the established homology models for the hA 1 and hA 3 identified the hypothetical binding mode and rationalized the interaction of these derivatives with their respective ARs binding sites. The calculations showed a high level of variability since all the synthetized derivatives yielded different possible binding modes inside the pockets. Selection of the hypothetical binding pose was accomplished considering the number of similar poses extracted from the simulations and geometrical correspondence to crystallized ligands in the hA 2A (Figure 2a).
Docking results disclosed important data about the binding mode: the oxygens presented in the benzopyrone system are oriented towards the Asn250 residue and the benzoyl moiety was buried in the hA 3 pocket. This hypothetical binding mode corroborates the conformations shown by the co-crystallized ligands in the hA 2A (PDB: 3EML and 3UZC) [48,53] (Figure 2a,b). The pose of compound 3 produced effective hydrogen bonds with Gln167, Asn250 and His272 residues.
Interestingly, when methoxy substituents were demethylated and changed into hydroxy equivalents (compounds 5-8) a modification in the profile of the studied derivatives was noticed: a loss of affinity for hA 3 and a tendency for interaction with hA 1 . The only compound that discloses some affinity for both receptors was compound 5 (hA 1 K i = 39.5 and hA 3 K i = 34.5 µM), which presents a catechol at positions 3 and 4 and no substitutions in the coumarin fragment. The hypothetical binding mode for compound 5 in the hA 3 pocket is represented in Figure 2c. The compound can establish hydrogen bonds with Ala69, Asn250 and His272 residues. As observed in the hA 2A crystallized structure and previously published studies [54,55], the corresponding Asn250 residue seems to play an important role in ligand recognition. The compound 5 pose inside the hA 1 pocket is likewise the described pose in the hA 3 one. However, the position was slightly shifted, and calculations were not able to retrieve a hydrogen bond with the Asn250 residue. The introduction of an additional hydroxy group at position 6 of the coumarin scaffold (compound 6), resulted in a loss of measurable hA 3 binding affinity. The most noticeable binding affinities were found for derivatives with hydroxy substitutions at positions 5 and 7 of the coumarin core, as stated for methoxy equivalents. Thereby, compound 7, with the same substitution pattern as quercetin (Figure 1), that is, hydroxy groups at positions 5, 7, 3 and 4 , displays hA 1 selectivity, and the best binding affinity (K i = 17.7 µM). Compound 8, with the same substitution pattern as genistein (Figure 1, hydroxy substituents at positions 5, 7 and 4 ) shows a similar hA 1 selectivity (K i = 29.1 µM). The pose obtained through docking calculations for compound 7 in the hA 1 protein pocket showed the possibility of establishment of hydrogen bonds with Glu172, Asn254 and Thr277 residues (Figure 2d).
Moreover, we calculated the interaction energy contributions of the residues in hA 3 and hA 1 pockets with compounds 3 and 7, respectively ( Figure 3). The sum of different individual contributions, such as Coulomb, van der Waals and hydrogen bond energies, was taken into account in the calculation of the interaction energies for each residue.  In addition, Figure 4 shows the molecular surface around the two residues in the hA1 and hA3 that could be responsible for the observed selectivity. In addition, Figure 4 shows the molecular surface around the two residues in the hA 1 and hA 3 that could be responsible for the observed selectivity.
Regarding the interaction energy contributions (Figure 4), calculations showed that the molecular surface around the two residues in the hA 1 and hA 3 could be responsible for the observed selectivity. Phe168, Asn250, Ile268 and His272 are important residues in the interaction between compound 3 and the hA 3 . Residues with important contributions in the stabilization of compound 7 inside the hA 1 are Phe171, Gln172, Asn254, Ile274 and Thr277. Regarding the interaction energy contributions (Figure 4), calculations showed that the molecular surface around the two residues in the hA1 and hA3 could be responsible for the observed selectivity. Phe168, Asn250, Ile268 and His272 are important residues in the interaction between compound 3 and the hA3. Residues with important contributions in the stabilization of compound 7 inside the hA1 are Phe171, Gln172, Asn254, Ile274 and Thr277.
There are different residues in both hA1 and hA3 with different hydrophobic/hydrophilic characteristics, which may be important to understand the observed selectivity. Hydrophobic residues in the hA3, such as Val169 and Leu264, could establish hydrophobic interactions and contribute towards stabilizing the ligand when the derivatives present hydrophobic substituents, like methoxy groups (i.e., 3 and 4) (Figure 4). However, in the case of hA1, the corresponding residues are Glu172 and Thr270. They have hydrophilic characteristics and so can stabilize the binding of derivatives with polar substituents, such as the hybrids with hydroxy groups (compounds 6-8). Yet, compound 5, with no substituents in the coumarin ring, can be stabilized in the pocket of both proteins.

General Methods
Starting materials and reagents were obtained from commercial suppliers and were used without purification. Melting points (mp) were determined using a Reichert Kofler thermopan or in capillary tubes on a Büchi 510 (Flawil, Switzerland) apparatus and were uncorrected. 1 H-NMR (300 MHz) and 13  There are different residues in both hA 1 and hA 3 with different hydrophobic/hydrophilic characteristics, which may be important to understand the observed selectivity. Hydrophobic residues in the hA 3 , such as Val169 and Leu264, could establish hydrophobic interactions and contribute towards stabilizing the ligand when the derivatives present hydrophobic substituents, like methoxy groups (i.e., 3 and 4) (Figure 4). However, in the case of hA 1 , the corresponding residues are Glu172 and Thr270. They have hydrophilic characteristics and so can stabilize the binding of derivatives with polar substituents, such as the hybrids with hydroxy groups (compounds 6-8). Yet, compound 5, with no substituents in the coumarin ring, can be stabilized in the pocket of both proteins.

General Methods
Starting materials and reagents were obtained from commercial suppliers and were used without purification. Melting points (mp) were determined using a Reichert Kofler thermopan or in capillary tubes on a Büchi 510 (Flawil, Switzerland) apparatus and were uncorrected. 1 H-NMR (300 MHz) and 13 C-NMR (75.4 MHz) spectra were recorded with a Bruker AMX spectrometer (Bruker Daltonics Inc., Fremont, CA, USA) using DMSO-d 6 or CDCl 3 as solvent. Chemical shifts (δ) are expressed in parts per million (ppm) using TMS as an internal standard. Coupling constants J are expressed in hertz (Hz). Spin multiplicities are given as s (singlet), bs (broad singlet), d (doublet), dd (doublet of doublets) and m (multiplet). Mass spectrometry was carried out with a Kratos MS-50 or a Varian MAT-711 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Elemental analyses were performed by a Perkin-Elmer 240B microanalyzer (Thermo Fisher Scientific, Waltham, MA, USA) and were within ±0.4% of the calculated values in all cases. The analytical results were ≥95% purity for all compounds. Flash Chromatography (FC) was performed on silica gel (Merck 60, 230-400 mesh, Kenilworth, NJ, USA) and analytical TLC on precoated silica gel plates (Merck 60 F254, Kenilworth, NJ, USA). Organic solutions were dried over anhydrous sodium sulfate. Concentration and evaporation of the solvent after reaction or extraction was carried out on a Büchi rotavapor (BÜCHI Labortechnik AG, Switzerland) operating at reduced pressure. The purity of compounds was assessed by high performance liquid chromatography (HPLC) coupled at diode array detector (DAD) on a Thermo Quest Spectrasystem (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a P4000 pump, an UV6000 UV-Vis diode array detector, and a SN4000 interface to be operated via a personal computer. The instrument software ChromQuest 5.0 (Thermo Fisher Scientific, Waltham, MA, USA) was used for data acquisition. Different analytical columns and mobile phases (all solvents were HPLC grade) were tested. The mobile phase was H 2 O:CH 3 CN = 70:30 and an Eclipse xdb C18 column (5 µm particle size, 0.46 mm i.d., 25 cm length; Agilent Technologies, CA, USA) was used. The purity of the compounds was found to be higher than 95%.

Synthetic Protocol to Obtain the Methoxy-3-benzoylcoumarins 1-4
To a solution of the appropriate β-ketoester (1 mmol) and the corresponding salicylaldehyde (1 mmol) in ethanol (5 mL) piperidine in catalytic amount (0.10 mL) was added. The reaction mixture was refluxed for 2-6 h and, after completion (followed by TLC), the reaction was cooled, and the precipitate was filtered and washed with cold ethanol and ether. The obtained solid was recrystallized in DCM to afford the corresponding methoxy-3-benzoylcoumarin compounds.

Synthetic Protocol to Obtain the Hydroxy-3-benzoylcoumarins 5-8
In a Schlenk tube, the appropriate methoxy derivative compound 1-4 (1 mmol) was dissolved in DCM (1 mL), and BBr 3 (20 mmol, 1M) was added dropwise. The tube was sealed, and the reaction mixture was heated at 80 • C for 48 h. The resulting crude product was treated with MeOH and rotated to dryness. The obtained crude solid was recrystallized in MeOH or purified by flash chromatography using hexane/ethyl acetate mixtures as eluent, to afford the desired hydroxy derivatives.

Binding Affinity Assays
The binding affinity for hA 1 , hA 2A , hA 3 of the synthetized compounds was evaluated using radioligand competition experiments in CHO cells that were stably transfected with the individual receptor subtypes [44,45]. The radioligands used were 1 nM Due to the lack of a suitable radioligand for the hA 2B receptor, the potency of antagonists at the hA 2B receptor (expressed on CHO cells) was determined by inhibition of NECA-stimulated adenylyl cyclase activity [44,45]. The IC 50 for inhibition of cAMP (cyclic adenosine monophosphate) production was determined and converted to K i values using the Cheng and Prusoff equation [56]. For all the tested compounds, no measurable activity for the hA 2B (K i > 10 µM) was detected.

Statistical Methods
K i values (dissociation constants) were determined in radioligand competition experiments with 7-8 different concentrations of test compound and each concentration was tested in duplicate. K i values are given as geometric means of three independent experiments with 95% confidence intervals. The program Prism 6 (GraphPad Software) was used for the analysis of the competition curves.

Theoretical Evaluation of ADME Properties
cLogP was calculated by the methodology developed by Molinspiration as a sum of fragment-based contributions and correction factors. Topological Polar Surface Area (TPSA) was calculated based on the methodology published by Ertl et al. as a sum of fragment contributions [57]. Oxygen-and nitrogen-centered polar fragments are considered. TPSA has been shown to be a very good descriptor characterizing drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and blood-brain barrier penetration. The method for calculation of molecule volume developed at Molinspiration is based on group contributions. These have been obtained by fitting the sum of fragment contributions to "real" 3D volume for a training set of about twelve thousand, mostly drug-like molecules. Three-dimensional molecular geometries for a training set were fully optimized by the semiempirical AM1 method.

Molecular Modeling
Homology modeling was carried out using the Molecular Operating Environment (MOE) suite [49]. Homology models of the hA1 and hA 3 were constructed. The crystallized structure of the hA 2A receptor (PDB: 3EML) was used as a template [48]. Protein sequence alignment of the 3 receptors (hA 1 , hA 2A and hA 3 ) used to generate the homology models was performed as previously described by Katritch et al. [50]. The alignment was made considering the highly conserved residues in the different TM helices. MOE software was used to generate the homology models [49]. Protein geometry was evaluated for the models taking into account Phi-Psi plots, rotamers, bond angles, bond lengths, atom clashes, dihedrals and contact energies. The presence of different conserved disulfide bridges was manually checked, such as the bridge between the corresponding Cys77 and Cys166 residues in the hA 2A . Induce Fit Docking Workflow in the Schrodinger package was used to optimize the final models [58]. Selective high affinity ligands (compounds coll_11 and jaco_mre3008_f20) [50] were used to adapt the protein pocket for the hA 1 and hA 3 , respectively. This procedure involved three steps: 1) Glide-based docking of the ligands using SP mode (standard-precision); 2) Protein pocket optimization using Prime and considering the residues within 5Å from the ligand poses; 3) Glide-based docking of the ligands in the refined pocket using XP mode (Extra-precision). As previously described [50], homology models were tested for their capability to discriminate ligands from decoys and between known subtype-selective compounds. ROC curves were performed, and the best models were selected for further molecular docking studies.

Molecular Docking of hA 1 and hA 3 ARs
Molecular docking studies using the hA 1 and hA 3 homology models, selected in the previous step, were carried out. Compounds were docked using Glide SP mode [52]. Ten poses for each ligand were collected and optimized using MM-GBSA in Prime [53], taking into account a flexible protein region defined by 5 Å from the ligand. Final binding modes were selected, taking into account the number of similar poses extracted from the calculations and geometrical correspondence to co-crystallized ligands in the hA 2A .

Conclusions
The current study was focused on the synthesis and the evaluation of binding affinity towards the four subtypes of human ARs of a selected series of methoxy and hydroxy coumarin-chalcone hybrids. Structure-activity relationship (SAR) studies of the new molecules highlighted that, in general, methoxy substitutions, as in the example of compounds 3 and 4, allow a superior hA 3 binding affinity and selectivity, whereas the hydroxy substitutions, as in the example of compounds 5-8, allow a modest hA 1 binding affinity. Substitutions at positions 5 and 7 of the coumarin scaffold proved to be essential for the potency and selectivity in both series of compounds. Compound 4, a methoxy derivative, and compound 7, a hydroxy derivative, proved to be the most potent compounds of the studied series, displaying a hA 3 K i = 2.49 µM and a hA 1 K i = 17.7 µM, respectively. Docking calculations allow an understanding the binding preference of the studied molecules. Finally, the theoretical values for the ADME properties show that all the coumarin-chalcone hybrids 1-8 do not break any of Lipinski's rules, being promising scaffolds for further compound optimization.