Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A2A Receptor.

In this study, we determined the crystal structure of an engineered human adenosine A2A receptor bound to a partial agonist and compared it to structures cocrystallized with either a full agonist or an antagonist/inverse agonist. The interaction between the partial agonist, belonging to a class of dicyanopyridines, and amino acids in the ligand binding pocket inspired us to develop a small library of derivatives and assess their affinity in radioligand binding studies and potency and intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of the derivatives retained the partial agonist profile, whereas other ligands turned into inverse agonists. We rationalized this remarkable behavior with additional computational docking studies.

New 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives. Evaluation of different moieties on the 6-aryl ring to obtain potent and selective human A2A adenosine receptor antagonists.

In this work, further structural investigations on the 8-amino-2-phenyl-6-aryl-1,2,4-triazolo[4,3-a]pyrazin-3-one series were carried out to achieve potent and selective human A2A adenosine receptor (AR) antagonists. Different ether and amide moieties were attached at the para-position of the 6-phenyl ring, thus leading to compounds 1-9 and 10-18, respectively. Most of these moieties contained terminal basic rings (pyrrolidine, morpholine, piperidine and substituted piperazines) which were thought to confer good physicochemical and drug-like properties. Compounds 11-16, bearing the amide linker, possessed high affinity and selectivity for the hA2A AR (Ki = 3.6-11.8 nM). Also derivatives 1-9, featuring an ether linker, preferentially targeted the hA2A AR but with lower affinity, compared to those of the relative amide compounds. Docking studies, carried out at the hA2A AR binding site, highlighted some crucial ligand-receptor interactions, particularly those provided by the appended substituent whose nature deeply affected hA2A AR affinity.

Antioxidant-Conjugated 1,2,4-Triazolo[4,3-a]pyrazin-3-one Derivatives: Highly Potent and Selective Human A2A Adenosine Receptor Antagonists Possessing Protective Efficacy in Neuropathic Pain.

New 8-amino-6-aryl-1,2,4-triazolo[4,3-a]pyrazin-3-ones were designed to obtain dual antioxidant-human A2A adenosine receptor (hA2A AR) antagonists. Two sets of compounds were synthesized, the first featuring phenol rings at the 6-position, the second bearing the lipoyl and 4-hydroxy-3,5-di-tertbut-benzoyl residues appended by different linkers on the 6-phenyl ring. Several new triazolopyrazines (1-21) were potent and selective hA2A AR antagonists (Ki = 0.17-54.5 nM). Compounds 11, 15, and 21, featuring antioxidant moieties, and compound 12, lacking the antioxidant functionality, reduced oxaliplatin-induced toxicity in microglia cells, the most active being the lipoyl-derivative 15 and the (4-hydroxy-3,5-di-tert-butyl)benzoyl-analogue 21 which were effective in reducing the oxygen free radical level. The lipoyl-derivative 15 was also able to revert oxaliplatin-induced neuropathy in the mouse. In vivo efficacy of 15 makes it a promising neuroprotective agent in oxidative stress-related diseases.

Modifications on the Amino-3,5-dicyanopyridine Core To Obtain Multifaceted Adenosine Receptor Ligands with Antineuropathic Activity.

A new series of amino-3,5-dicyanopyridines (1-31) was synthesized and biologically evaluated in order to further investigate the potential of this scaffold to obtain adenosine receptor (AR) ligands. In general, the modifications performed have led to compounds having high to good human (h) A1AR affinity and an inverse agonist profile. While most of the compounds are hA1AR-selective, some derivatives behave as mixed hA1AR inverse agonists/A2A and A2B AR antagonists. The latter compounds (9-12) showed that they reduce oxaliplatin-induced neuropathic pain by a mechanism involving the alpha7 subtype of nAchRs, similar to the nonselective AR antagonist caffeine, taken as the reference compound. Along with the pharmacological evaluation, chemical stability of methyl 3-(((6-amino-3,5-dicyano-4-(furan-2-yl)pyridin-2-yl)sulfanyl)methyl)benzoate 10 was assessed in plasma matrices (rat and human), and molecular modeling studies were carried out to better rationalize the available structure-activity relationships.

Novel 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives as potent human adenosine A1 and A2A receptor antagonists. Evaluation of their protective effect against ß-amyloid-induced neurotoxicity in SH-SY5Y cells.

In this work, an enlarged series of 1,2,4-triazolo[4,3-a]pyrazin-3-ones was designed to target the human (h) A2A adenosine receptor (AR) or both hA1 and hA2A ARs. The novel 8-amino-1,2,4-triazolopyrazin-3-one derivatives 1-25 featured a phenyl or a benzyl pendant at position 2 while different aryl/heteroaryl substituents were placed at position 6. Two compounds (8 and 10) endowed with high affinity (Ki = 7.2 and 10.6 nM) and a complete selectivity for the hA2A AR were identified. Moreover, several derivatives possessed nanomolar affinity for both hA1 and hA2A ARs (both Ki < 20 nM) and different degrees of selectivity versus the hA3 AR. Two selected compounds (10 and 25) demonstrated ability in preventing ß-amyloid peptide (25-35)-induced neurotoxicity in SH-SY5Y cells. Results of docking studies at the hA2A and hA1 AR crystal structures helped us to rationalize the observed affinity data and to highlight that the steric hindrance of the substituents at the 2- and 6-position of the bicyclic core affects the binding mode in the receptor cavity.

Novel human adenosine receptor antagonists based on the 7-amino-thiazolo[5,4-d]pyrimidine scaffold. Structural investigations at the 2-, 5- and 7-positions to enhance affinity and tune selectivity.

This paper describes the synthesis of novel 7-amino-thiazolo[5,4-d]pyrimidines bearing different substituents at positions 2, 5 and 7 of the thiazolopyrimidine scaffold. The synthesized compounds 2-27 were evaluated in radioligand binding (A1, A2A and A3) and adenylyl cyclase activity (A2B and A2A) assays, in order to evaluate their affinity and potency at human adenosine receptor subtypes. The current study allowed us to support that affinity and selectivity of 7-amino-thiazolo[5,4-d]pyrimidine derivatives towards the adenosine receptor subtypes can be modulated by the nature of the groups attached at positions 2, 5 and 7 of the bicyclic scaffold. To rationalize the hypothetical binding mode of the newly synthesized compounds, we also performed docking calculations in human A2A, A1 and A3 structures.

Structure-activity relationship studies and pharmacological characterization of N5-heteroarylalkyl-substituted-2-(2-furanyl)thiazolo[5,4-d]pyrimidine-5,7-diamine-based derivatives as inverse agonists at human A2A adenosine receptor.

This paper describes the synthesis and characterization of N5-(hetero)arylalkyl-substituted-thiazolo [5,4-d]pyrimidine-5,7-diamine derivatives (4-19) as novel human (h) A2A adenosine receptor (AR) inverse agonists. Competition binding and cyclic AMP assays indicate that the examined compounds behave as hA2A AR inverse agonists showing binding affinity values in the nanomolar or subnanomolar range. Notably, compounds 4, 5, 6 and 11 showed two affinity values for the hA2A ARs with the highest (KH) falling in the femtomolar range and the lowest (KL) of the nanomolar order. In addition, in cyclic AMP assays, compounds 4, 5, 6 and 11 exhibited potency (IC50) values in the picomolar range. This study has confirmed that 2-(2-furanyl)thiazolo [5,4-d]pyrimidine-5,7-diamine-based derivatives represent a unique new class of hA2A AR inverse agonists.

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists.

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A1, A2A, A2B and A3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA1 and hA2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A1/A2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA1 Ki = 10.2 nM; hA2A Ki = 4.72 nM) and behaved as a potent A1/A2A antagonist/inverse agonist (hA1 IC50 = 13.4 nM; hA2A IC50 = 5.34 nM).

Development of novel pyridazinone-based adenosine receptor ligands.

With the aim of finding new adenosine receptor (AR) ligands, a preliminary investigation focusing on the thieno[2,3-d]pyridazin-5(4H)-one scaffold was undertaken. The synthesized compounds 1-11 were evaluated for their binding at hA1, hA2A and hA3 ARs and efficacy at hA2B subtype in order to determine the affinity at the human adenosine receptor subtypes. Small structural changes on this scaffold highly influenced affinity; compound 5 (5-ethyl-7-(thiazol-2-yl)thieno[2,3-d]pyridazin-4(5H)-one) emerged as the best of this series. The simplicity of the synthetic process, the capability of the scaffold to be easily decorated, together with the predicted ADME properties confirm the role of these compounds as promising hits. A molecular docking investigation at the hA1AR crystal structure was performed to rationalize the SARs of the herein reported thienopyridazinones.

The aminopyridine-3,5-dicarbonitrile core for the design of new non-nucleoside-like agonists of the human adenosine A2B receptor.

A new series of amino-3,5-dicyanopyridines (3-28) as analogues of the adenosine hA2B receptor agonist BAY60-6583 (compound 1) was synthesized. All the compounds that interact with the hA2B adenosine receptor display EC50 values in the range 9-350 nM behaving as partial agonists, with the only exception being the 2-{[4-(4-acetamidophenyl)-6-amino-3,5-dicyanopyridin-2-yl]thio}acetamide (8) which shows a full agonist profile. Moreover, the 2-[(1H-imidazol-2-yl)methylthio)]-6-amino-4-(4-cyclopropylmethoxy-phenyl)pyridine-3,5-dicarbonitrile (15) turns out to be 3-fold more active than 1 although less selective. This result can be considered a real breakthrough due to the currently limited number of non-adenosine hA2B AR agonists reported in literature. To simulate the binding mode of nucleoside and non-nucleoside agonists at the hA2B AR, molecular docking studies were performed at homology models of this AR subtype developed by using two crystal structures of agonist-bound A2A AR as templates. These investigations allowed us to represent a hypothetical binding mode of hA2B receptor agonists belonging to the amino-3,5-dicyanopyridine series and to rationalize the observed SAR.

Structural investigations on coumarins leading to chromeno[4,3-c]pyrazol-4-ones and pyrano[4,3-c]pyrazol-4-ones: New scaffolds for the design of the tumor-associated carbonic anhydrase isoforms IX and XII.

Human carbonic anhydrases (hCAs, EC 4.2.1.1) IX and XII are overexpressed in a wide variety of cancers and are considered available drug targets for anti-tumor therapy since their inhibition has been shown to reduce tumor growth and metastasis. A set of coumarin derivatives (1-10) and several 1-aryl and 2-aryl-substituted chromeno[4,3-c]pyrazol-4-ones (11-37) and pyrano[4,3-c]pyrazol-4-ones (38-39) were synthesized and tested against the tumor-associated hCAs IX and XII and the cytosolic isoforms hCAs I and II. Several compounds were potent (Ki < 41 nM) and selective inhibitors of the hCA IX (13, 14, 19, 21, 25, 31, 33, 37 and 39), some derivatives (6, 11 and 17) were active against both hCA IX and XII isoforms (Ki = 5.6-9.6 nM), while none were effective against the off-target cytosolic hCAs I and II. Some selected inhibitors (6, 11, 13, 19, 21, 25, 31 and 39) showed activity as antiproliferative agents on HT-29 colon cancer cell lines both in normoxic and hypoxic conditions. This finding led us to hypothesize for these derivatives more than one mechanism of action, involving hCAs IX and XII inhibition in hypoxia and other not identified target(s) in normoxia.

The 1,2,4-Triazolo[4,3-a]pyrazin-3-one as a Versatile Scaffold for the Design of Potent Adenosine Human Receptor Antagonists. Structural Investigations to Target the A2A Receptor Subtype.

In this work, we describe the identification of the 1,2,4-triazolo[4,3-a]pyrazin-3-one as a new versatile scaffold for the development of adenosine human (h) receptor antagonists. The new chemotype ensued from a molecular simplification approach applied to our previously reported 1,2,4-triazolo[4,3-a]quinoxalin-1-one series. Hence, a set of novel 8-amino-2-aryl-1,2,4-triazolopyrazin-3-one derivatives, featured by different substituents on the 2-phenyl ring (R) and at position 6 (R6), was synthesized with the main purpose of targeting the hA2A adenosine receptor (AR). Several compounds possessed nanomolar affinity for the hA2A AR (Ki = 2.9-10 nM) and some, very interestingly, also showed high selectivity for the target. One selected potent hA2A AR antagonist (12, R = H, R6 = 4-methoxyphenyl) demonstrated some ability to counteract MPP+-induced neurotoxicity in cultured human neuroblastoma SH-SY5Y cells, a widely used in vitro Parkinson's disease model. Docking studies at hAR structures were performed to rationalize the observed affinity data.

3-Hydroxy-1H-quinazoline-2,4-dione as a New Scaffold To Develop Potent and Selective Inhibitors of the Tumor-Associated Carbonic Anhydrases IX and XII.

In this paper, we describe the discovery of the 3-hydroxyquinazoline-2,4-dione as a useful scaffold to obtain potent inhibitors of the tumor-associated human carbonic anhydrases (hCAs) IX and XII. A set of derivatives (1-29), bearing different substituents on the fused benzo ring (Cl, NO2, NH2, CF3, ureido, amido, heterocycles), were synthesized, and several of them showed nanomolar activity in inhibiting the hCA IX and XII isoforms, while they were ineffective against the cytosolic enzymes hCAs I and II. Some selected compounds were tested for their antiproliferative activity against HT-29 colon cancer cell lines. After 48 h of treatment with the lower dose (30 µM), derivatives 12, 14, 15, and 19 were significantly active, inducing a mortality by about 50% in both normoxia and hypoxia. This finding led us to hypothesize for these compounds more than one mechanism of action involving both CAs IX and XII and other not yet identified target(s).

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles.

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15-24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.[Formula: see text].

Imidazo[1,2-a]pyrazin-8-amine core for the design of new adenosine receptor antagonists: Structural exploration to target the A3 and A2A subtypes.

The imidazo[1,2-a]pyrazine ring system has been chosen as a new decorable core skeleton for the design of novel adenosine receptor (AR) antagonists targeting either the human (h) A3 or the hA2A receptor subtype. The N8-(hetero)arylcarboxyamido substituted compounds 4-14 and 21-30, bearing a 6-phenyl moiety or not, respectively, show good hA3 receptor affinity and selectivity versus the other ARs. In contrast, the 8-amino-6-(hetero)aryl substituted derivatives designed for targeting the hA2A receptor subtype (compounds 31-38) and also the 6-phenyl analogues 18-20 do not bind the hA2A AR, or show hA1 or balanced hA1/hA2A AR affinity in the micromolar range. Molecular docking of the new hA3 antagonists was carried out to depict their hypothetical binding mode to our refined model of the hA3 receptor. Some derivatives were evaluated for their fluorescent potentiality and showed some fluorescent emission properties. One of the most active hA3 antagonists herein reported, i.e. the 2,6-diphenyl-8-(3-pyridoylamino)imidazo[1,2-a]pyrazine 29, tested in a rat model of cerebral ischemia, delayed the occurrence of anoxic depolarization caused by oxygen and glucose deprivation in the hippocampus and allowed disrupted synaptic activity to recover.

Design, Synthesis, and Pharmacological Characterization of 2-(2-Furanyl)thiazolo[5,4-d]pyrimidine-5,7-diamine Derivatives: New Highly Potent A2A Adenosine Receptor Inverse Agonists with Antinociceptive Activity.

In this study, we describe the design and synthesis of new N5-substituted-2-(2-furanyl) thiazolo[5,4-d]pyrimidine-5,7-diamines (2-18) and their pharmacological characterization as A2A adenosine receptor (AR) antagonists by using in vitro and in vivo assays. In competition binding experiments two derivatives (13 and 14) emerged as outstanding ligands showing two different affinity values (KH and KL) for the hA2A receptor with the high affinity KH value in the femtomolar range. The in vitro functional activity assays, performed by using cyclic AMP experiments, assessed that they behave as potent inverse agonists at the hA2A AR. Compounds 13 and 14 were evaluated for their antinociceptive activity in acute experimental models of pain showing an effect equal to or greater than that of morphine. Overall, these novel inverse agonists might represent potential drug candidates for an alternative approach to the management of pain.

Exploring the 2- and 5-positions of the pyrazolo[4,3-d]pyrimidin-7-amino scaffold to target human A1 and A2A adenosine receptors.

A new series of 7-aminopyrazolo[4,3-d]pyrimidine derivatives (1-31) were synthesized to evaluate some structural modifications at the 2- and 5-positions aimed at shifting affinity towards the human (h) A2A adenosine receptor (AR) or both hA2A and hA1 ARs. The most active compounds were those featured by a 2-furyl or 5-methylfuran-2-yl moiety at position 5, combined with a benzyl or a substituted-benzyl group at position 2. Several of these derivatives (22-31) displayed nanomolar affinity for the hA2A AR (Ki=3.62-57nM) and slightly lower for the hA1 ARs, thus showing different degrees (3-22 fold) of hA2A versus hA1 selectivity. In particular, the 2-(2-methoxybenzyl)-5-(5-methylfuran-2-yl) derivative 25 possessed the highest hA2A and hA1 AR affinities (Ki=3.62nM and 18nM, respectively) and behaved as potent antagonist at both these receptors (cAMP assays). Its 2-(2-hydroxybenzyl) analog 26 also showed a high affinity for the hA2A AR (Ki=5.26nM) and was 22-fold selective versus the hA1 subtype. Molecular docking investigations performed at the hA2A AR crystal structure and at a homology model of the hA1 AR allowed us to represent the hypothetical binding mode of our derivatives and to rationalize the observed SARs.

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.