Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.

Correlation study between A3 adenosine receptor binding affinity and anti-renal interstitial fibrosis activity of truncated adenosine derivatives.

Truncated 4'-thionucleosides 1-4 and 4'-oxonucleosides 5-8 as potent and selective A3AR antagonists were synthesized from D-mannose and D-erythronic acid γ-lactone, respectively. These nucleosides were evaluated for their anti-fibrotic renoprotective activity in TGF-ß1-treated murine proximal tubular (mProx) cells. Their antagonistic activities for A3AR were proportional to their inhibitory activities against TGF-ß1-induced collagen I upregulation in mProx cells. This result suggests that the binding affinity of A3AR antagonists is closely correlated with their anti-fibrotic activity. Thus, A3AR antagonists might be novel therapeutic candidates for treating chronic kidney disease.

Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding.

Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

Structural refinement of pyrazolo[4,3-d]pyrimidine derivatives to obtain highly potent and selective antagonists for the human A3 adenosine receptor.

In previous research, we identified some 7-oxo- and 7-acylamino-substituted pyrazolo[4,3-d]pyrimidine derivatives as potent and selective human (h) A3 adenosine receptor (AR) antagonists. Herein we report on the structural refinement of this class of antagonists aimed at achieving improved receptor-ligand recognition. Hence, substituents with different steric bulk, flexibility and lipophilicity (Me, Ar, heteroaryl, CH2Ph) were introduced at the 5- and 2-positions of the bicyclic scaffold of both the 7-oxo and 7-amino derivatives, and acyl residues were appended on the 7-amino group of the latter. All the 2-phenylpyrazolo[4,3-d]pyrimidin-7-amines and 7-acylamines bearing a 4-methoxyphenyl- or a 2-thienyl group at the 5-position showed high hA3 affinity and selectivity. In particular, the 2-phenyl-5-(2-thienyl)-pyrazolo[4,3-d]pyrimidin-7-(4-methoxybenzoyl)amine 25 (Ki = 0.027 nM) is one of the most potent and selective hA3 antagonists reported so far. By using an in silico receptor-driven approach the obtained binding data were rationalized and the molecular bases of the observed hA3 AR affinities were critically described.

Exploring the 7-oxo-thiazolo[5,4-d]pyrimidine core for the design of new human adenosine A3 receptor antagonists. Synthesis, molecular modeling studies and pharmacological evaluation.

A new series of 5-methyl-thiazolo[5,4-d]pyrimidine-7-ones bearing different substituents at position 2 (aryl, heteroaryl and arylamino groups) was synthesized and evaluated in radioligand binding assays to determine their affinities at the human (h) A1, A2A, and A3 adenosine receptors (ARs). Efficacy at the hA(2B) and antagonism of selected ligands at the hA3 were also assessed through cAMP experiments. Some of the new derivatives exhibited good to high hA3AR affinity and selectivity versus all the other AR subtypes. Compound 2-(4-chlorophenyl)-5-methyl-thiazolo[5,4-d]pyrimidine-7-one 4 was found to be the most potent and selective ligand of the series (K(I) hA3 = 18 nM). Molecular docking studies of the reported derivatives were carried out to depict their hypothetical binding mode in our hA3 receptor model.

5'-C-Ethyl-tetrazolyl-N(6)-substituted adenosine and 2-chloro-adenosine derivatives as highly potent dual acting A1 adenosine receptor agonists and A3 adenosine receptor antagonists.

A series of N(6)-substituted-5'-C-(2-ethyl-2H-tetrazol-5-yl)-adenosine and 2-chloro-adenosine derivatives was synthesized as novel, highly potent dual acting hA1AR agonists and hA3AR antagonists, potentially useful in the treatment of glaucoma and other diseases. The best affinity and selectivity profiles were achieved by N(6)-substitution with a 2-fluoro-4-chloro-phenyl- or a methyl- group. Through an in silico receptor-driven approach, the molecular bases of the hA1- and hA3AR recognition and activation of this series of 5'-C-ethyl-tetrazolyl derivatives were explained.

A facile and novel synthesis of N(2)-, C(6)-substituted pyrazolo[3,4-d]pyrimidine-4 carboxylate derivatives as adenosine receptor antagonists.

An efficient synthetic procedure was adopted to synthesize a series of new molecules containing the pyrazolo[3,4-d]pyrimidine (PP) scaffold, which have been evaluated as promising human adenosine receptor (AR) antagonists. The effect of substitutions at the N(2), C(4) and C(6) positions of PPs on the affinity and selectivity towards the adenosine receptors were explored. Most of the pyrazolo[3,4-d]pyrimidine-4-carboxylates displayed from moderate to good affinity at the human A3AR (hA3AR), as indicated by the low micromolar range of Ki values (Ki hA3AR = 0.7-34 µM). In particular, compounds 60 and 62 displayed good affinity at the hA3AR (60, Ki hA3AR = 2.2 µM and 62, Ki hA3AR = 2.9 µM) and selectivity towards the other AR subtypes (60, >46-fold selective and 62, >34-fold selective, respectively). In view of these results, these novel PP analogues were docked both in the crystallographic structure of the hA2AAR and in a homology model of the hA3AR in order to support the structure activity relationship (SAR) analysis. These preliminary results demonstrated that pyrazolo[3,4-d]pyrimidine can be considered a promising scaffold to obtain new molecules with potent hA3AR antagonist activity.

1,2,4-triazolo[1,5-a]quinoxaline derivatives and their simplified analogues as adenosine A3 receptor antagonists. Synthesis, structure-affinity relationships and molecular modeling studies.

The 1,2,4-triazolo[1,5-a]quinoxaline (TQX) scaffold was extensively investigated in our previously reported studies and recently, our attention was focused at position 5 of the tricyclic nucleus where different acyl and carboxylate moieties were introduced (compounds 2-15). This study produced some interesting compounds endowed with good hA3 receptor affinity and selectivity. In addition, to find new insights about the structural requirements for hA3 receptor-ligand interaction, the tricyclic TQX ring was destroyed yielding some 1,2,4-triazole derivatives (compounds 16-23). These simplified compounds, though maintaining the crucial structural requirements for adenosine receptor-ligand interaction, have a very low hA3 adenosine receptor affinity, the only exception being compound 23 (1-[3-(4-methoxyphenyl)-1-phenyl-1H-1,2,4-triazol-5-yl]-3-phenylurea) endowed with a Ki value in the micro-molar range and high hA3 selectivity versus both hA1 and hA2A AR subtypes. Evaluation of the side products obtained in the herein reported synthetic pathways led to the identification of some new triazolo[1,5-a]quinoxalines as hA3AR antagonists (compounds 24-27). These derivatives, though lacking the classical structural requirements for the anchoring at the hA3 receptor site, show high hA3 affinity and in some case selectivity versus hA1 and hA2A subtypes. Molecular docking of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical binding mode to our model of hA3 receptor.

Synthesis and anti-renal fibrosis activity of conformationally locked truncated 2-hexynyl-N(6)-substituted-(N)-methanocarba-nucleosides as A3 adenosine receptor antagonists and partial agonists.

Truncated N(6)-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4'-thioadenosines. Hydrophobic N(6) and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N(6)-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f-4i did not differ significantly, with Ki values of 7.8-16.0 nM. N(6)-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-ß1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-ß1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 µM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.

Synthesis of novel pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives: potent and selective adenosine A3 receptor antagonists.

A series of novel pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives 5 was prepared from 2-amino-3-cyano-4-trifluoromethyl-6-phenylpyridine 1 in two steps via formation of iminoether 3 followed by reaction with different aroylhydrazides 4. Representative products 5 were evaluated for their affinity towards all four subtypes of human adenosine receptors. Compounds 2-(3-fluorophenyl)-8-phenyl-10-(trifluoromethyl)pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5b), 2-(furan-2-yl)-8-phenyl-10-(trifluoromethyl)pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5d), and 2-(furan-2-yl)-5-methyl-8-phenyl-10-(trifluoromethyl)pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5j) showed high affinity for the A3 receptors, with Ki values of 8.1, 10.4, and 12.1 nM, respectively, and were >1000-fold selective versus all other adenosine receptor subtypes.

Organoruthenium antagonists of human A3 adenosine receptors.

Human A3 adenosine receptor (A3AR) is a membrane-bound G protein-coupled receptor implicated in a number of severe pathological conditions, including cancer, in which it acts as a potential therapeutic target. To derive structure-activity relationships on pyrazolo-triazolo-pyrimidine (PTP)-based A3AR antagonists, we developed a new class of organometallic inhibitors through replacement of the triazolo moiety with an organoruthenium fragment. The objective was to introduce by design structural diversity into the PTP scaffold in order to tune their binding efficacy toward the target receptor. These novel organoruthenium antagonists displayed good aquatic stability and moderate binding affinity toward the hA3 receptor in the low micromolar range. The assembly of these complexes through a template-driven approach with selective ligand replacement at the metal center to control their steric and receptor-binding properties is discussed.

Water-soluble pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines as human A3 adenosine receptor antagonists.

A relevant problem of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus, an attractive scaffold for the preparation of adenosine receptor antagonists, is the low water solubility. We originally functionalized the C(5) position with a salifiable 4-pyridylcarbamoyl moiety that conferred good water solubility at low pH (<4.0) but poor solubility at physiologic pH, indicative of the dissociation of the pyridinium species. Here we replaced the pyridin-4-yl moiety with a 1-(substituted)piperidin-4-yl ring to exploit the higher basicity of this nucleus and for the the possibility to generate stable, water-soluble salts. The hydrochloride salt of the 1-(cyclohexylmethyl)piperidin-4-yl derivative (10, K(i)(hA(3)) = 9.7 nM, IC(50)(hA(3)) = 30 nM, K(i)(hA(1)/hA(3)) = 351, K(i)(hA(2A)/hA(3)) > 515, IC(50)(hA(2B)) > 5 µM) showed a solubility of 8 mg/mL at physiological pH and gave a stable aqueous system suitable for intravenous infusion. Molecular modeling studies were helpful in rationalizing the available structure-activity relationships and the selectivity profile of the new ligands.

Discovery of novel A3 adenosine receptor ligands based on chromone scaffold.

A project focused on the discovery of new chemical entities (NCEs) as AR ligands that incorporate a benzo-γ-pyrone [(4H)-1-benzopyran-4-one] substructure has been developed. Accordingly, two series of novel chromone carboxamides placed at positions C2 (compounds 2-13) and C3 (compounds 15-26) of the γ-pyrone ring were synthesized using chromone carboxylic acids (compounds 1 or 14) as starting materials. From this study and on the basis of the obtained structure-activity relationships it was concluded that the chromone carboxamide scaffold represent a novel class of AR ligands. The most remarkable chromones were compounds 21 and 26 that present a better affinity for A3AR (Ki = 3680 nM and Ki = 3750 nM, respectively). Receptor-driven molecular modeling studies provide information on the binding/selectivity data of the chromone. The data so far acquired are instrumental for future optimization of chromone carboxamide as a selective A3AR antagonist.

Recent advances on A3 adenosine receptor antagonists by QSAR tools.

Adenosine receptors (ARs) are widespread on virtually every human organ/tissue, and have long been considered promising therapeutic targets in a wide range of conditions, ranging from cerebral diseases to cancer, including inflammatory disorders. The knowledge acquired up to date in relation to ARs, in particular regarding the molecular biology of the A3 AR has provided a solid basis that led to the proposal of this receptor as a novel therapeutic target enabling the rational design and development of potent and selective A3 AR ligands. This review attempts to summarize the most recent developments in the A3 research field, focusing in particular on Quantitative Structure-Activity Relationships (QSAR) based studies that supported so far the design of new, potent and selective human A3 AR antagonists. In addition, a classical QSAR modeling study carried out on two series of pyrazolo-triazolopyrimidine derivatives is presented as a case study. Specifically, a systematic evaluation of linear and non-linear models along with a variety of structure representations and feature selection tools is reported. The combination of these techniques (neural networks to capture non-linear relationships in the data and feature selection to prevent over-fitting) was found to produce QSAR models with good overall accuracy and robustness, as well as predictivity on external data. Moreover, the study indicated that the antagonist activity of these derivatives is largely explained by electrostatic, steric and hydrogen-bonding factors, highlighting the role of the size, shape and type of inhibitor in forming effective blocking of the A3 AR subtype. The developed QSAR models could then be usefully employed to design new compounds selectively active towards the A3 adenosine receptor.

Highly potent and selective fluorescent antagonists of the human adenosine A3 receptor based on the 1,2,4-triazolo[4,3-a]quinoxalin-1-one scaffold.

The adenosine-A(3) receptor (A(3)AR) is a G protein-coupled receptor that shows promise as a therapeutic target for cancer, glaucoma, and various autoimmune inflammatory disorders, and as such, there is a need for molecular probes to study this receptor. Here, we report a series of fluorescent ligands containing different linkers and fluorophores based around a 1,2,4-triazolo[4,3-a]quinoxalin-1-one antagonist. One of these conjugates (19) displayed high affinity for the A(3)AR (pK(D) = 9.36 ± 0.12) and is >650-fold selective over other adenosine receptor subtypes. Confocal microscopy revealed clear, displaceable membrane labeling of CHO-A(3) cells with 19, with no detectable labeling of CHO-A(1) cells under identical conditions. This fluorescent ligand was also able to specifically label the A(3)AR in HEK293T cells containing a mixed adenosine receptor population. The subtype specificity, along with its excellent imaging properties, make 19 an ideal tool for studying A(3)AR distribution and organization, particularly in the presence of other adenosine receptor subtypes.

Structure-activity relationships of truncated C2- or C8-substituted adenosine derivatives as dual acting A2A and A3 adenosine receptor ligands.

Truncated N(6)-substituted-4'-oxo- and 4'-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A(2A) and A(3) adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-catalyzed cross-coupling reactions were utilized for functionalization of the C2 position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a hydrophobic C2 substituent were required for high affinity at the hA(2A)AR, but hydrophobic C8 substitution abolished binding at the hA(2A)AR. However, most of synthesized compounds displayed medium to high binding affinity at the hA(3)AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA(2A)AR agonists. C2 substitution probed geometrically through hA(2A)AR docking was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA(2A)AR agonist and hA(3)AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases.

New 2-heterocyclyl-imidazo[2,1-i]purin-5-one derivatives as potent and selective human A3 adenosine receptor antagonists.

A series of 4-allyl/benzyl-7,8-dihydro-8-methyl/ethyl-2-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-imidazo[2,1-i]purin-5(4H)-ones has been synthesized and evaluated in radioligand binding assays to determine their affinities at the human A(1), A(2A), and A(3) adenosine receptors. Efficacy at the hA(2B) AR and antagonism of selected ligands at the hA(3) AR were also assessed through cAMP experiments. All of the synthesized molecules exhibited high affinity at the hA(3) AR (K(i) values ranging from 1.46 to 44.8 nM), as well as remarkable selectivity versus A(1), A(2A), and A(2B) AR subtypes. Compound (R)-4-allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one (R-33) was found to be the most potent and selective ligand of the series (K(i) hA(3) = 1.46 nM, K(i) hA(2A)/K(i) hA(3) > 3425; IC(50) hA(2B)/K(i) hA(3) > 3425; K(i) hA(1)/K(i) hA(3) = 1729). Molecular modeling studies were helpful in rationalizing the available structure-activity relationships along with the selectivity profiles of the new series of ligands.

The identification of the 2-phenylphthalazin-1(2H)-one scaffold as a new decorable core skeleton for the design of potent and selective human A3 adenosine receptor antagonists.

Following a molecular simplification approach, we have identified the 2-phenylphthalazin-1(2H)-one (PHTZ) ring system as a new decorable core skeleton for the design of novel hA(3) adenosine receptor (AR) antagonists. Interest for this new series was driven by the structural similarity between the PHTZ skeleton and both the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (TQX) and the 4-carboxamido-quinazoline (QZ) scaffolds extensively investigated in our previously reported studies. Our attention was focused at position 4 of the phthalazine nucleus where different amido and ureido moieties were introduced (compounds 2-20). Some of the new PHTZ compounds showed high hA(3) AR affinity and selectivity, the 2,5-dimethoxyphenylphthalazin-1(2H)-one 18 being the most potent and selective hA(3) AR antagonist among this series (K(i) = 0.776 nM; hA(1)/hA(3) and hA(2A)/hA(3) > 12000). Molecular docking studies on the PHTZ derivatives revealed for these compounds a binding mode similar to that of the previously reported TQX and QZ series, as was expected from the simplification approach.

Pyrimidine derivatives as potent and selective A3 adenosine receptor antagonists.

Two regioisomeric series of diaryl 2- or 4-amidopyrimidines have been synthesized and their adenosine receptor affinities were determined in radioligand binding assays at the four human adenosine receptors (hARs). Some of the ligands prepared herein exhibit remarkable affinities (K(i) < 10 nm) and, most noticeably, the absence of activity at the A(1), A(2A), and A(2B) receptors. The structural determinants that support the affinity and selectivity profiles of the series were highlighted through an integrated computational approach, combining a 3D-QSAR model built on the second generation of GRid INdependent Descriptors (GRIND2) with a novel homology model of the hA(3) receptor. The robustness of the computational model was subsequently evaluated by the design of new derivatives exploring the alkyl substituent of the exocyclic amide group. The synthesis and evaluation of the novel compounds validated the predictive power of the model, exhibiting excellent agreement between predicted and experimental activities.

Design, synthesis, and binding of homologated truncated 4'-thioadenosine derivatives at the human A3 adenosine receptors.

We synthesized homologated truncated 4'-thioadenosine analogues 3 in which a methylene (CH(2)) group was inserted in place of the glycosidic bond of a potent and selective A(3) adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A(3) adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.