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].

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.

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.

1,2,4-Benzothiadiazine-1,1-dioxide derivatives as ionotropic glutamate receptor ligands: synthesis and structure-activity relationships.

Ionotropic glutamate receptor (iGluR) modulators, specially AMPA receptor antagonists, are potential tools for numerous therapeutic applications in neurological disorders, including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, epilepsy, chronic pain, and neuropathology ensuing from cerebral ischemia or cardiac arrest. In this work, the synthesis and binding affinities at the Gly/NMDA, AMPA, and kainic acid (KA) receptors of a new series of 1,2,4-benzothiadiazine-1,1-dioxide derivatives are reported. The results show that 1,2,4-benzothiadiazine-1,1-dioxide is a new scaffold for obtaining iGluR ligands. Moreover, this work has led us to the 7-(3-formylpyrrol-1-yl)-6-trifluoromethyl substituted compound 7, which displays the highest AMPA receptor affinity and high selectivity versus the Gly/NMDA (90-fold) and KA (46-fold) receptors.

Fluorescent ligands for adenosine receptors.

Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.

Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as adenosine receptor antagonists: synthesis, structure-affinity relationships and molecular modeling studies.

A number of 5-oxo-pyrazolo[1,5-c]quinazolines (series B-1), bearing at position-2 the claimed (hetero)aryl moiety (compounds 1-8) but also a carboxylate group (9-14), were designed as hA(3) AR antagonists. This study produced some interesting compounds endowed with good hA(3) receptor affinity and high selectivity, being totally inactive at all the other AR subtypes. In contrast, the corresponding 5-ammino derivatives (series B-2) do not bind or bind with very low affinity at the hA(3) AR, the only exception being the 5-N-benzoyl compound 19 that shows a hA(3)K(i) value in the high µ-molar range. Evaluation of the synthetic intermediates led to the identification of some 5(3)-(2-aminophenyl)-3(5)-(hetero)arylpyrazoles 20-24 with modest affinity but high selectivity toward the hA(3) AR subtype. Molecular docking of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical binding mode to our model of hA(3) receptor.

Synthesis, structure-affinity relationships, and molecular modeling studies of novel pyrazolo[3,4-c]quinoline derivatives as adenosine receptor antagonists.

This paper reports the study of new 2-phenyl- and 2-methylpyrazolo[3,4-c]quinolin-4-ones (series A) and 4-amines (series B), designed as adenosine receptor (AR) antagonists. The synthesized compounds bear at the 6-position various groups, with different lipophilicity and steric hindrance, that were thought to increase human A(1) and A(2A) AR affinities and selectivities, with respect to those of the parent 6-unsubstituted compounds. In series A, this modification was not tolerated since it reduced AR affinity, while in series B it shifted the binding towards the hA(1) subtype. To rationalize the observed structure-affinity relationships, molecular docking studies at A(2A)AR-based homology models of the A(1) and A(3) ARs and at the A(2A)AR crystal structure were carried out.

Remote control of movement disorders using a photoactive adenosine A2A receptor antagonist.

G protein-coupled adenosine receptors are promising therapeutic targets for a wide range of neuropathological conditions, including Parkinson's disease (PD). However, the ubiquity of adenosine receptors and the ultimate lack of selectivity of certain adenosine-based drugs have frequently diminished their therapeutic use. Photopharmacology is a novel approach that allows the spatiotemporal control of receptor function, thus circumventing some of these limitations. Here, we aimed to develop a light-sensitive caged adenosine A2A receptor (A2AR) antagonist to photocontrol movement disorders. We synthesized MRS7145 by blocking with coumarin the 5-amino position of the selective A2AR antagonist SCH442416, which could be photoreleased upon violet light illumination (405 nm). First, the light-dependent pharmacological profile of MRS7145 was determined in A2AR-expressing cells. Upon photoactivation, MRS7145 precluded A2AR ligand binding and agonist-induced cAMP accumulation. Next, the ability of MRS7145 to block A2AR in a light-dependent manner was assessed in vivo. To this end, A2AR antagonist-mediated locomotor activity potentiation was evaluated in brain (striatum) fiber-optic implanted mice. Upon irradiation (405 nm) of the dorsal striatum, MRS7145 induced significant hyperlocomotion and counteracted haloperidol-induced catalepsy and pilocarpine-induced tremor. Finally, its efficacy in reversing motor impairment was evaluated in a PD animal model, namely the hemiparkinsonian 6-hydroxydopamine (6-OHDA)-lesioned mouse. Photo-activated MRS7145 was able to potentiate the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (l-DOPA). Overall, MRS7145 is a new light-operated A2AR antagonist with potential utility to manage movement disorders, including PD.

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).

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.

7-Amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives: structural investigations at the 5-position to target human A1 and A(2A) adenosine receptors. Molecular modeling and pharmacological studies.

In previous research, several 7-amino-2-arylpyrazolo[4,3-d]pyrimidine derivatives were identified as highly potent and selective antagonists at the human A3 adenosine receptor. Structure-activity relationship studies highlighted that affinity and selectivity depended on the nature of the substituents at the 5- and 7-positions of the pyrazolo[4,3-d]pyrimidine scaffold. In particular, small lipophilic residues at the 5-position and a free amino group at position 7 afforded compounds able to bind all four human (h) adenosine receptors. Hence, to shift affinity toward the hA1 and/or hA(2A) subtypes, alkyl and arylalkyl chains of different length were appended at position 5 of the 2-phenylpyrazolo[4,3-d]pyrimidin-7-amine. Among the new compounds, a dual hA1/hA(2A) receptor antagonist was identified, namely the 5-(3-phenylpropyl) derivative 25, which shows high affinity both at human A1 (K(i) = 5.31 nM) and A(2A) (K(i) = 55 nM) receptors. We also obtained some potent and selective antagonists for the A1 receptor, such as the 5-(3-arylpropyl)-substituted compounds 26-31, whose affinities fall in the low nanomolar range (K(i) = 0.15-18 nM). Through an in silico receptor-driven approach, the obtained binding data were rationalized and the molecular bases of the hA1 and hA(2A) AR affinity and selectivity of derivatives 25-31 are explained.

Characterization by flow cytometry of fluorescent, selective agonist probes of the A(3) adenosine receptor.

Various fluorescent nucleoside agonists of the A3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N(6) position and rigid A3AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA3AR expressed in whole CHO cells and membranes. By FCM, binding was A3AR-mediated (blocked by A3AR antagonist, at least half through internalization), with t1/2 for association 38min in mA3AR-HEK293 cells; 26.4min in sucrose-treated hA3AR-CHO cells (Kd 31nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA3AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A3AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A3AR.

4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6 receptor.

4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions (N4 and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R.

2-Arylpyrazolo[4,3-d]pyrimidin-7-amino derivatives as new potent and selective human A3 adenosine receptor antagonists. Molecular modeling studies and pharmacological evaluation.

On the basis of our previously reported 2-arylpyrazolo[4,3-d]pyrimidin-7-ones, a set of 2-arylpyrazolo[4,3-d]pyrimidin-7-amines were designed as new human (h) A3 adenosine receptor (AR) antagonists. Lipophilic groups with different steric bulk were introduced at the 5-position of the bicyclic scaffold (R5 = Me, Ph, CH2Ph), and different acyl and carbamoyl moieties (R7) were appended on the 7-amino group, as well as a para-methoxy group inserted on the 2-phenyl ring. The presence of acyl groups turned out to be of paramount importance for an efficient and selective binding at the hA3 AR. In fact, most of the 7-acylamino derivatives showed low nanomolar affinity (Ki = 2.5-45 nM) and high selectivity toward this receptor. A few selected pyrazolo[4,3-d]pyrimidin-7-amides were effective in counteracting oxaliplatin-induced apoptosis in rat astrocyte cell cultures, an in vitro model of neurotoxicity. Through an in silico receptor-driven approach the obtained binding data were rationalized and the molecular bases of the observed hA3 AR affinity and hA3 versus hA2A AR selectivity were explained.

3-Hydroxy-1H-quinazoline-2,4-dione derivatives as new antagonists at ionotropic glutamate receptors: molecular modeling and pharmacological studies.

Based on our 3-hydroxy-7-chloroquinazoline-2,4-dione derivatives, previously reported as antagonists at ionotropic glutamate receptors, we synthesized new 3-hydroxyquinazoline-2,4-diones bearing a trifluoromethyl group at the 7-position and different groups at position 6. Glycine/NMDA, AMPA and kainate receptor binding data showed that the 7-trifluoromethyl residue increased AMPA and kainate receptor affinity and selectivity, with respect to the 7-chlorine atom. Among the probed 6-substituents, the 6-(1,2,4-triazol-4-yl) group (compound 8) was the most advantageous for AMPA receptor affinity and selectivity. Derivative 8 demonstrated to be effective in decreasing neuronal damage produced by oxygen and glucose deprivation in organotypic rat hippocampal slices and also showed anticonvulsant effects in pentylenetetrazole-induced convulsions. The previously reported kainate receptor antagonist 6-(2-carboxybenzoyl)-amino-7-chloro-3-hydroxyquinazoline-2,4-dione 3 prevented the failure of neurotransmission induced by oxygen and glucose deprivation in the CA1 region of rat hippocampal slices.