Marco Betti

7-Amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives: Structural investigations at the 5-position to target human A1 and A2A 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.

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.

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 Parkinsons 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).

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.

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.

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

Abstract 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.

The aminopyridine-3,5-dicarbonitrile core for the design of new non-nucleoside-like agonists of the human adenosine A 2B 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.