Rosaria Volpini

Adenosine deaminase: Functional implications and different classes of inhibitors

Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency desease (ADA‐SCID), in which both B‐cell and T‐cell development is impaired. ADA‐SCID has been the first disorder to be treated by gene therapy, using polyethene glycol‐modified bovine ADA (PEG‐ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inibitors have been designed and synthesized, classified as ground‐state and transition‐state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury.

New substituted 9-alkylpurines as adenosine receptor ligands

In the present study an investigation of the structure-activity relationships in 9-ethylpurine derivatives, aimed at preparing A1, A2A, A2B, and A3 selective adenosine receptor antagonists, was undertaken. Our synthetic approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine (9), obtained from 2-amino-6-chloropurine (5); 9-ethyl-6-iodo-9H-purine (11), 8-bromo-9-ethyl-adenine (3) and 8-bromo-9-ethyl-6-iodo-9H-purine (13), obtained from 9-ethyl-adenine (2). The synthesized compounds were tested in in vitro radioligand binding assays at A1, A2A, and A3 human adenosine receptor subtypes. Due to the lack of a suitable radioligand the affinity of the 9-ethyladenine derivatives at A2B adenosine receptors was determined in adenylyl cyclase experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological profile at A1 and A2A receptors whereas some differences were found for the A3 and the A2B subtypes. 8-Bromo-9-ethyladenine (3) showed higher affinity for all receptors in comparison to the parent compound 2, and the highest affinity in the series for the A2A and A2B subtypes (Ki = 0.052 and 0.84 microM, respectively). Analyzing the different substituents, a phenethoxy group in 2-position (10a) gave the highest A2A versus A2B selectivity (near 400-fold), whereas a phenethylamino group in 2- and 6-position (10b and 12b, respectively) improved the affinity at A2B receptors, compared to the parent compound 2. The presence of a hexynyl substituent in 8-position led to a compound with good affinity at the A3 receptor (4d, Ki = 0.62 microM), whereas (ar)alkynyl groups are detrimental for the potency at the A2B subtype. These differences give raise to the hope that further modifications will result in the development of currently unavailable leads with good affinity and selectivity for A2B adenosine receptors.

Determination of ink photoinitiators in packaged beverages by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry

A new analytical method, using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) techniques, was developed for the determination in packaged food beverages of five ink photoinitiator residues: 2-isopropylthioxanthone (ITX), benzophenone, 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB), 1-hydroxycyclohexyl-1-phenyl ketone (IRGACURE 184) and ethyl-4-dimethylaminobenzoate (EDAB). Samples were extracted from selected beverages (milk, fruit juices and wine) and relative packagings, using n-hexane and dichloromethane, respectively, purified on solid-phase extraction (SPE) silica gel cartridges, and then analyzed in GC/MS and LC/MS. The recovery percentages, obtained spiking the beverage samples at concentrations of 4 and 10 microgl(-1) with a standard mixture of photoinitiators, were in the range 42-108% (milk), 50-84% (wine), and 48-109% (fruit juices). The repeatability of the method was assessed in all cases by the % of correlation value, that was lower than 19%. The lowest limits of detection (LODs) and limits of quantification (LOQs), obtained using GC/MS, were in the range 0.2-1 and 1-5 microgl(-1), respectively. The method was applied to the analysis of forty packaged food beverages (milk, fruit juices and wine samples). The most significant contamination was that of benzophenone, found in all samples in a concentration range of 5-217mugl(-1). Its presence was confirmed by an LC/Atmospheric-Pressure PhotoIonization (APPI)/MS/MS analysis. The photoinitiator (EHDAB) was found in eleven out of forty beverages in a concentration range of 0.13-0.8 microgl(-1). Less important was the ITX contamination, found in three out of forty samples in a range 0.2-0.24 microgl(-1). The work proposes a new method to analyze ink photoinitiator residues in polycoupled carton packaging and in contained food beverages.

A new ethyladenine antagonist of adenosine A2A receptors: Behavioral and biochemical characterization as an antiparkinsonian drug

Adenosine A(2A) receptor antagonists have emerged as an attractive non-dopaminergic target in clinical trials aimed at evaluating improvement in motor deficits in Parkinsons disease (PD). Moreover, preclinical studies suggest that A(2A) receptor antagonists may slow the course of the underlying neurodegeneration of dopaminergic neurons. In this study, we evaluated the efficacy of the new adenosine A(2A) receptor antagonist 8-ethoxy-9-ethyladenine (ANR 94) in parkinsonian models of akinesia and tremor. In addition, induction of the immediate early gene zif-268, and neuroprotective and anti-inflammatory effects of ANR 94 were evaluated. ANR 94 was effective in reversing parkinsonian tremor induced by the administration of tacrine. ANR 94 also counteracted akinesia (stepping test) and sensorimotor deficits (vibrissae-elicited forelimb-placing test), as well as potentiating l-dopa-induced contralateral turning behavior in 6-hydroxydopamine (6-OHDA) lesion model of PD. Potentiation of motor behavior in 6-OHDA-lesioned rats was not associated with increased induction of the immediate early gene zif-268 in the striatum, suggesting that ANR 94 does not induce long-term plastic changes in this structure. Finally, in a subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, ANR 94 protected nigrostriatal dopaminergic neurons from degeneration and counteracted neuroinflammatory processes by contrasting astroglial (glial fibrillary acidic protein, GFAP) and microglial (CD11b) activation. A(2A) receptor antagonism represents a uniquely realistic opportunity for improving PD treatment, since A(2A) receptor antagonists offer substantial symptomatic benefits and possibly disease-modifying activity. The characterization of ANR 94 may represent a further therapeutic opportunity for the treatment of PD with this new class of drugs.

Recent developments in adenosine A2A receptor ligands.

The development of potent and selective agonists and antagonists of adenosine receptors (ARs) has been a target of medicinal chemistry research for several decades, and recently the US Food and Drug Administration has approved Lexiscan, an adenosine derivative substituted at the 2 position, for use as a pharmacologic stress agent in radionuclide myocardial perfusion imaging. Currently, some other adenosine A(2A) receptor (A(2A)AR) agonists and antagonists are undergoing preclinical testing and clinical trials. While agonists are potent antiinflammatory agents also showing hypotensive effects, antagonists are being developed for the treatment of Parkinsons disease.However, since there are still major problems in this field, including side effects, low brain penetration (for the targeting of CNS diseases), short half-life, or lack of in vivo effects, the design and development of new AR ligands is a hot research topic.This review presents an update on the medicinal chemistry of A(2A)AR agonists and antagonists, and stresses the strong need for more selective ligands at the human A(2A)AR subtype, in particular in the case of agonists.

Structure-activity relationships of adenine and deazaadenine derivatives as ligands for adenine receptors, a new purinergic receptor family

Adenine derivatives bearing substituents in the 2-, N(6)-, 7-, 8-, and/or 9-position and a series of deazapurines were synthesized and investigated in [(3)H]adenine binding studies at the adenine receptor in rat brain cortical membrane preparations (rAde1R). Steep structure-activity relationships were observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the 9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of compounds was additionally investigated in binding studies at human embryonic kidney (HEK293) cells, which also express a high-affinity adenine binding site. Structure-affinity relationships at the human cell line were similar to those at the rAde1R, but not identical. In particular, N(6)-acetyladenine (25, K(i) rat: 2.85 microM; K(i) human: 0.515 microM) and 8-aminoadenine (33, K(i) rat: 6.51 microM; K(i) human: 0.0341 microM) were much more potent at the human as compared to the rat binding site. The new AdeR ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine receptor family.

8-Bromo-9-alkyl adenine derivatives as tools for developing new adenosine A2A and A2B receptors ligands.

Importance of making available selective adenosine receptor antagonists is boosted by recent findings of adenosine involvement in many CNS dysfunctions. In the present work a series of 8-bromo-9-alkyl adenines are prepared and fully characterized in radioligand binding assays or functional cyclase experiments in respect to their interaction with all the four adenosine receptor subtypes. Results show that the presence of the bromine atom in 8-position of 9-substituted adenines promotes in general the interaction with the adenosine receptors, in particular at the A(2A) subtype. The present study also demonstrates that adenine derivatives could be a good starting point to obtain selective adenosine A(2B) receptor antagonists.

Characterization of potent ligands at human recombinant adenosine receptors

The four adenosine receptor subtypes have been stably transfected into Chinese hamster ovary (CHO) cells allowing for comparative studies in a similar cellular background, using radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). We are currently using the transfected CHO cells for extensive screening of nucleosides and purine derivatives of our library. Screening of a number of 2‐alkynyl analogs of 5′‐N‐ethylcarboxamidoadenosine (NECA) indicated that introduction of particular substituents, such as the racemic 2‐phenylhydroxypropynyl group, led to a highly potent, nonselective agonist at A1, A2A, and A3 subtypes (PHPNECA, Ki in the low nanomolar range at the three subtypes). In the A2B functional assay, it has been found that PHPNECA (EC50 A2B = 0.88 μM) is threefold more potent than NECA. This article is the first report in which the introduction of a bulky group in the 2‐position of NECA led to a compound that is active as an agonist at the human A2B subtype. On the other hand, the presence of a phenyl ring conjugated to the triple bond as in phenylethynylNECA (PENECA) enhanced selectivity for the A3 subtype. In the purine series (potential antagonists), 8‐bromo‐9‐ethyladenine (8‐BEA) showed good affinity toward all adenosine receptor subtypes (Ki A1 = 0.28 μM, Ki A2A = 0.052 μM, Ki A2B = 0.84 μM, Ki A3 = 27.8 μM). On the other hand, the introduction of alkynyl chains in the 8‐position resulted in an increased affinity at the A3 receptor (8‐hexynyl‐9‐ethyladenine, 8‐HEEA, Ki A3 = 0.62 μM and 8‐phenylethynyl‐9‐ethyladenine, 8‐PEEA, Ki A3 = 0.086 μM). Drug Dev. Res. 45:176–181, 1998.

Adenosine receptor modeling: what does the A2A crystal structure tell us?

For a long time, there have been no experimentally determined structural data for any adenosine receptor (AR) and the only approach available for making structure/function correlations about these proteins has been homology modeling. While the early attempts to model these receptors followed the crystallization of bacteriorhodopsin, the cryo-microscopy studies of bovine and frog rhodopsin, and the modeling of a Calpha-template for the TM helices in the rhodopsin family of GPCRs, the crystallization of bovine rhodopsin by Palczewski was of extreme importance as it first provided the crystal structure of an eukaryotic GPCR to be used as template for more realistic homology models. Since then, rhodopsin-based modeling became the routine approach to develop AR structural models that proved to be useful for interpretation of site-directed mutagenesis data and for molecular docking studies. The recently reported crystal structures of the adrenergic beta1 and beta2 receptors only partially confirmed the structural features showed by bovine rhodopsin, raising a question about which template would have been better for further modeling of ARs. Such question remained actually not-answered, due to the publication in late 2008 of the crystal structure of human adenosine A(2A) receptor (AA(2A)R). Since its publication, this structure has been used for ligands docking analysis and has provided a high similarity template for homology modeling of the other AR subtypes. Still, the AA(2A)R crystal structure allows to verify the hypotheses that were made on the basis of the previously reported homology modeling and molecular docking.

Medicinal Chemistry of Adenosine A2A Receptor Agonists

The search for potent and selective A(2A) adenosine receptor agonists has been particularly fruitful in the early nineties. A series of 2-amino, 2-alkoxy, 2-alkythio-, 2-alkynyl-, and 2-alkenyl-derivatives of adenosine (Ado, 1) and N-ethylcarboxamidoadenosine (NECA, 30) have been synthesized and tested mainly on different model of rat A(1) and A(2A) receptor subtypes. From these studies some ligands, such as CGS 21680 (33), HENECA (42), and (S)-PHPNECA (46b), showed to possess high A(2A) affinity combined with good A(2A) vs A(1) selectivity. More detailed characterization of these ligands at the four cloned human adenosine receptor subtypes revealed that none of the prototypical adenosine receptor agonists exhibits at the same time high affinity and selectivity for the human A(2A)AR subtype. Both NECA and CGS 21680, which are avalaible as radioligands for this subtype, have lower affinity at human than at rat receptor. The 2-alkynylNECA derivatives HENECA an PHPNECA showed high affinity also at human A(3) receptors. In particular, (S)-PHPNECA displayed K(i)s in the low nanomolar range at A(1), A(2A), and A(3)subtypes and an EC(50) of 220 nM at human A(2B) receptor. On the other hand, it is now well known that the coronary vasodilation induced by Ado in different species is mediated by activation of A(2A)AR and a compound capable of producing coronary vasodilation through activation of A(2A)AR, but that is devoid of A(1)- and A(1)-agonist activity would have advantage over Ado for use in myocardial perfusion imaging studies. Other potential therapeutic applications of selective A(2A)AR agonists are as anti-aggregatory, anti-inflammatory, anti-psychotic, and anti-Huntingtons disease agents. This review is aimed at presenting a complete overview of the medicinal chemistry development of A(2A) adenosine receptor agonists and at stressing the strong need for more selective ligands at A(2A) human subtype.