Original article
N6-1,3-Diphenylurea derivatives of 2-phenyl-9-benzyladenines and 8-azaadenines: Synthesis and biological evaluation as allosteric modulators of A2A adenosine receptors

https://doi.org/10.1016/j.ejmech.2007.10.021Get rights and content

Abstract

Some 1-[4-(9-benzyl-2-phenyl-9H-purin-6-ylamino)-phenyl]-3-phenyl-urea derivatives and some 1-[4-(9-benzyl-2-phenyl-9H-8-azapurin-6-ylamino)-phenyl]-3-phenyl-urea derivatives were synthesised and evaluated for their interaction with adenosine receptors. It was found that some of these compounds can act as positive enhancers of agonist and antagonist radioligands for the A2A adenosine receptors. This evidence was also strengthened by functional data. Other compounds can act as negative modulators.

Furthermore these compounds show inhibitory properties for A1 and A3 adenosine receptors.

Graphical abstract

  1. Download : Download full-size image
It was found that some of these compounds can act as positive enhancers of agonist and antagonist radioligands for A2A adenosine receptors; other compounds can act as negative modulators.

Introduction

Allosteric modulation of G protein-coupled receptors is a relatively novel and unexplored pharmacological concept. Classically, the mechanism of action for receptor ligands consists either of mimicking or inhibiting the action of the endogenous signalling molecules, leading to the traditional classification of agonists as well as antagonists/inverse agonists, respectively. The desired effect is exerted through competition at the binding site for the endogenous neurotransmitter or hormone. Another kind of action elicited on the receptors by chemical compounds is the allosteric modulation. Allosteric modulators act at sites distinct from the agonist binding site, and their effect is evident only in the presence of an exogenously added agonist because they do not have an action “per se” but modulate the action of the naturally occurring hormone or neurotransmitter.

The presence of allosteric sites on a receptor provided a new target for drug discovery [1] and, in particular, allosteric modulators for adenosine receptors have potential therapeutic advantages over orthosteric ligands [2]. Two of the four subtypes of adenosine receptors have been reported to be allosterically regulated (A1, A3). For both A1 and A3 receptors, some allosteric modulators that are relatively selective have been developed and characterized [2]. For example, PD 81,723 showed selectivity toward the A1 [3], while 3-(2-pyridinyl)isoquinoline derivatives [4] and a group of 1H-imidazo-[4,5-c]quinolines showed selectivity toward the A3 subtype [5]. No A2A receptor selective allosteric modulators have been reported [2]. Only very few compounds can be found in the literature that act as allosteric modulators of A2A adenosine receptors: {4-methyl-7-[(methylamino)carbonyl]oxy}-2H-1-benzopyran-2-one (PD 120,918) [6] was reported to enhance agonist radioligand binding to the rat striatal A2A adenosine receptor, but without functional enhancement. Like other G protein-coupled receptors, A2A adenosine receptors are allosterically modulated by sodium ions, and by the potassium sparing diuretic, amiloride [7]. Also the compound SCH-202676 and other 2,3,5-substituted-[1,2,4]-thiadiazoles displayed peculiar displacement characteristics of both radiolabelled agonist and antagonist binding to A2A receptors [8], but further studies have suggested that thiadiazoles act rather as sulfhydryl modifying agents than as allosteric modulators [9].

In this study, we present a synthesis of new purine- and 8-azapurine-N6-1,3-diphenylurea derivatives and their biological characterization at the adenosine receptors with particular regard to allosteric modulation of the A2A subtype.

Section snippets

Chemistry

The novel substituted 8-azaadenine derivatives 1116 and substituted adenine derivatives 1722 were synthesised as shown in Schemes 1a,b and 2. The 9-benzyl-8-azahypoxanthine 3 was prepared following a known two-step reaction (Scheme 1a) in the presence of sodium ethoxide: the first step was the 1,3 dipolar addition reaction of benzylazide 1 and cyanoacetamide to give 1-benzyl-4-carbamoyl-5-amino-1H-1,2,3-triazole 2, which was not isolated; then, in the same flask, ethyl benzoate was added to

Biochemistry

Compounds 1122 (see Table 1) were initially tested on adenosine A1, A2A and A3 receptors in equilibrium radioligand displacement experiments. For tests involving the A1 receptors both a radiolabelled agonist ([3H]CCPA) and a radiolabelled antagonist ([3H]DPCPX) were used, while the agonist radioligand [3H]CGS21680 and the antagonist radioligand [3H]ZM241385 were used in the experiments involving A2A receptors. In the case of A3 receptors only the agonist radioligand [3H]NECA was used as a

Pharmacology

Since the activation of adenosine A2A receptors in “endothelium intact” rat isolated aorta induces vasorelaxing effects, mediated through the release of endothelial nitric oxide, this experimental model is widely used as a functional tool for the pharmacological characterization of drugs acting on this receptor subtype. In this study, the influence of the selected compound 14 on the vasorelaxing responses evoked by the adenosine agonist CGS21680 was evaluated on rat aortic rings with intact

Results and discussion

All compounds assayed on the A1 adenosine receptor at a final concentration of 10 μM inhibited [3H]CCPA binding, in particular, in a significant way, compounds 11, 12, 17 and 18 which have p-NO2 or p-NH2 phenylamine as substituent on the N6 of the adenine or the 8-azaadenine nucleus; the other compounds, having a bulkier substituent in the same position (1316 and 1922), showed much lower activity. It may be worth specifying here that, with the term “inhibition” we mean a decrease of signal

Conclusions

A quite diverse library of potential ligands for adenosine receptors was synthesised and assayed in both equilibrium radioligand binding assays and dissociation kinetic experiments with the aim of explaining in detail the interaction mechanisms by which the above ligands exert their actions. On the basis of analysis of the initial experiments, it may be concluded that the new compounds 1122 bind to the adenosine A1, A2A and A3 receptors. Their behaviour at A1 and A3 receptors is an inhibitory

Chemistry

Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. IR spectra in Nujol mulls were recorded on a Mattson Genesis series FTIR spectrometer. 1H NMR spectra were recorded on a Bruker AC 200 spectrometer in δ units from TMS as an internal standard; the compounds were dissolved in DMSO-d6. Mass spectra data were obtained with a Hewlett–Packard GC/MS system 5988. TLC was performed on precoated silica gel F254 plates (Merck). Microanalyses (C H N) were carried out on a

Acknowledgment

This research was supported by the Italian MIUR (Ministero Istruzione Università Ricerca).

References (15)

  • G. Biagi et al.

    Farmaco

    (2001)
  • G. Biagi et al.

    Bioorg. Med. Chem.

    (2005)
  • A. Christopoulos

    Nat. Rev. Drug Discov.

    (2002)
  • Z.-G. Gao et al.

    Mini-Rev. Med. Chem.

    (2005)
  • R.F. Bruns et al.

    Mol. Pharmacol.

    (1990)
  • Z.-G. Gao et al.

    Mol. Pharmacol.

    (2001)
  • Z.-G. Gao et al.

    Mol. Pharmacol.

    (2002)
There are more references available in the full text version of this article.

Cited by (25)

  • Adenosine and inflammation: What's new on the horizon?

    2014, Drug Discovery Today
    Citation Excerpt :

    The use of positive allosteric modulators appears to be a promising way, because these compounds, by potentiating the effect of endogenous adenosine, can lead to site-specific and event-specific responses mainly in inflammatory contexts, where adenosine is released in large quantities, with a decrease of unintended systemic adverse effects [19]. Currently, allosteric modulators are available mainly for A1 and A3 receptors [48,50–55], even though a research group has also developed A2A receptor allosteric enhancers [56] (Fig. 3). On the basis of current knowledge, adenosine A1 receptors appear to mediate pro- and anti-inflammatory activities [5].

  • Analysis of the infrared and Raman spectra of the symmetrically substituted 1,3-diphenylurea and 1,3-diphenylacetone (dibenzyl ketone)

    2012, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
    Citation Excerpt :

    The calculated vibrational wavenumbers of the near-planar cis–cis conformation of 1,3-diphenylurea were calculated at the B3LYP/6-311G** level of calculation and listed in Table 4, while those of the gauche–gauche 1,3-diphenylacetone are given in Table 5 and both are compared to the observed ones in the infrared and Raman spectra shown in Figs. 2 and 3. The interesting structural [8,9] and biological [10–13] properties of 1,3-diphenylurea and the chemical properties of 1,3-diphenylacetone [14–18] prompted the investigation of their structural stabilities and vibrational spectra in the present study. In an early study the conformational stability of gaseous 1,3-diphenylurea as a model anti-viral drug was investigated by combined ultra-violet, IR ion-dip spectroscopy, quantum chemical computations (DFT-B3LYP/6-31+G*), and single-point MP2 calculations using basis sets ranging from 6-31+G* to 6-311++G** [1].

  • Allosteric modulators of rhodopsin-like G protein-coupled receptors: Opportunities in drug development

    2012, Pharmacology and Therapeutics
    Citation Excerpt :

    The compounds structurally resemble known orthosteric A2A antagonists. Compound 32 was further evaluated and found to significantly enhance the vasorelaxing effects of an A2A agonist in rat aortic rings at a concentration of 10 μM (Giorgi et al., 2008). PAMs for A2AARs could be very useful as novel anti-inflammatory drugs, since the use of direct A2A agonists is limited by their strong hypotensive side-effects (El-Tayeb et al., 2011).

  • Allosteric modulation of adenosine receptors

    2011, Biochimica et Biophysica Acta - Biomembranes
    Citation Excerpt :

    In the equilibrium displacement studies both [3H]ZM241385 and [3H]CGS21680 were used; 29 at 10 μM increased the specific binding of the two radioligands. In a kinetic dissociation study this compound slowed the dissociation of [3H]ZM241385 from the adenosine A2A receptor, while a similar experiment on the agonist radioligand was not reported [42]. In a functional study on rat aortic rings 29 shifted the dose–response curve of CGS21680 significantly to the left.

  • Allosteric Modulation of Purine and Pyrimidine Receptors

    2011, Advances in Pharmacology
    Citation Excerpt :

    Sodium ions (high concentrations of NaCl) rather decreased the dissociation rate of the antagonist [3H]ZM241385 from the A2A AR in a concentration-dependent manner. Recently, a 2-phenyl-9-benzyl-8-azaadenine derivative 13 (Fig. 1C) was reported to be a PAM of both agonist and antagonist radioligand binding at the A2A AR, and it increased the potency of an A2A AR agonist to induced relaxation of rat aortic rings (Giorgi et al., 2008). Addex Pharmaceuticals is developing PAMs of the A2A AR for treatment of inflammatory diseases, such as psoriasis and osteoarthritis, but so far the compounds remain in the preclinical phase (http://www.addexpharma.com/press-releases/press-release-details/article/addex-rd-day-highlights-broadened-therapeutic-potential-of-allosteric-modulation-platform-to-includ/).

  • Allosteric Modulators of Adenosine Receptors

    2023, Topics in Medicinal Chemistry
View all citing articles on Scopus
View full text