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Some pyrazolotriazolopyrimidines bearing different heteroarylcarbamoylamino moieties at the N5-position are described. We previously reported the synthesis of a water soluble compound with high potency and selectivity versus the human A3 adenosine receptor as antagonist, and herein we present an enlarged series of compounds related to the previously mentioned one. These compounds showed A3 adenosine receptor affinity in the nanomolar range and different levels of selectivity evaluated in radioligand binding assays at human A1, A2A, A2B, and A3 adenosine receptors. In particular, the effect of the heteroaryl substituents at the N5 position has been analyzed. This study allows us to recognize that the presence of a pyridinium moiety in this position not only increases water solubility but also improves or retains potency and selectivity at the human A3 adenosine receptors. In contrast, replacement of pyridine with different heterocycles produces loss of affinity and selectivity at the human A3 adenosine receptors. A molecular modeling study has been carried out with the aim to explain these various binding profiles.

A combined target-based and ligand-based drug design approach has been carried out to define a novel pharmacophore model of the human A3 receptor antagonists. High throughput molecular docking and comparative molecular field analysis (CoMFA) have been used in tandem to assemble a new target based pharmacophore model.

The combination of molecular electrostatic potential (MEP) surface properties (autocorrelation vectors) with the conventional partial least squares (PLS) analysis has been used for the prediction of the human A3 receptor antagonist activities. Three-hundred-fifty-eight structurally diverse human A3 receptor antagonists have been utilized to generate a novel ligand-based three-dimensional structure-activity relationship. Remarkably, our chemical library includes all 21 important chemical classes of human A3 antagonists currently discovered, and it represents the largest molecular collection used to generate a general human A3 antagonist structure-activity relationship.

A new, highly potent, selective, and water-soluble antagonist of the hA3 adenosine receptor was synthesized and tested in binding and functional assays. Compound 4 (5-[[(4- pyridyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo- [4,3-e]1,2,4-triazolo[1,5-c]pyrimidine hydrochloride) displayed high water solubility (15 mM) and the highest affinity (Ki ) 0.01 nM) and selectivity for the hA3 versus A1, A2A, and A2B receptors (>10000-fold) ever reported. A Schild analysis of the antagonism by 4 of agonist-induced inhibition of cAMP production in CHO cells expressing the hA3 receptor indicated a KB value of 0.20 nM.

It is accepted that neoplastic diseases are related to gene alteration or oncogene activation. In particular, DNA minor groove binding drugs have been extensively studied through the years in order to influence the regulation of gene expression by means of specific interactions with DNA based moieties. In this field, analogues of naturally occurring antitumour agents, such as CC-1065 and/or the duocarmycins, represent a new class of highly potent antineoplastic compounds, currently under investigation. CC-1065 and duocarmycins represent a class of exceptionally potent antitumour antibiotics that derive their biological effects from the reversible, stereoelectronically-controlled sequence selective alkylation of DNA. All natural compounds showed a cytotoxicity against leukaemia L1210 cell lines in the range 10 - 220pM but while CC-1065 showed a good antitumour activity in an in vivo model (optimal dose from 10 - 100 μg/g), duocarmycins showed weak antitumour activity. Despite its potency, CC-1065 cannot be used in humans due to eventual fatality. For this reason many scientists have focused their attention on this class of compounds, in order to obtain new derivatives with equal in vitro potency but a better profile in in vivo models. This effect is accompanied by dramatic changes in the morphology of hepatic mitochondria. On this basis, the recent developments on SARs for this class of compounds and their possible use as therapeutic agents are reviewed, with particular emphasis on recent patent literature and, finally, a conclusive opinion will be given on this topic.

A new series of 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-d-ribofuranuronamide-bearing N-arylureas or N-arylcarboxamido groups at the purine 6 position and N-arylureas combined with halogens or alkynyl chains at the 2 position have been synthesized and tested for affinity at A(1) and A(2A) adenosine receptors in rat brain membranes and at cloned rat A(3) receptors expressed in CHO cells. The derivatives contained the 5′ substituent found in the potent, nonselective agonist 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-d-ribofuranuronamide (NECA). While the carboxamido derivatives (9–13) showed affinity for A(1) receptors, the urea derivatives (30–45) showed different degrees of affinity and selectivity for the A(3) adenosine receptor subtype. In particular the derivative bearing a p-sulfonamidophenyl-urea at the 6 position, 31 showed a high affinity (K(i) = 9 nM) and selectivity for the A(3) receptors compared to that of the reference compound 1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-β-d-ribofuranuronamide (IB-MECA). Furthermore, the importance of the stereochemistry in the interaction of these ligands at the rat A(3) adenosine receptors has been evaluated by introducing a chiral chain at the 6 position. The introduction of halogens or alkynyl chains at the purine 2 position of selected ureas did not give the expected enhancement of potency at A(2A) and/or A(3) receptors but rather showed a dramatic reduction of A(2A) affinity, resulting in compounds with good A(2A)/A(3) selectivity. For example, the 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)-6-(4-methoxyphenylurea) derivative 61 showed the capability to bind simultaneously to A(1) and A(3) receptor subtypes, excluding the A(2A) receptor. Compound 31 was shown to be an agonist, 9-fold more potent than NECA, at A(3) receptors in rat RBL-2H3 mast cell membranes through stimulation of binding of [(35)S]GTP-γ-S.