KF26777 (2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride), a new potent and selective adenosine A3 receptor antagonist

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Abstract

We investigated the biochemical and pharmacological properties of a new adenosine A3 receptor antagonist, KF26777 (2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride). This compound was characterized using N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide ([125I]AB-MECA) or [35S]guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding to membranes from human embryonic kidney 293 (HEK293) cells expressing human adenosine A3 receptors. KF26777 showed a Ki value of 0.20±0.038 nM for human adenosine A3 receptors labeled with [125I]AB-MECA and possessed 9000-, 2350- and 3100-fold selectivity vs. human adenosine A1, A2A and A2B receptors, respectively. The inhibitory mode of binding was competitive. KF26777 inhibited the binding of [35S]GTPγS stimulated by 1 μM 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA). The IC50 value was 270±85 nM; the compound had no effect on basal activity. Dexamethasone treatment for HL-60 cells, human promyelocytic leukemia, up-regulated functional adenosine A3 receptors expression, and resulted in the enhanced elevation of intracellular Ca2+ concentration ([Ca2+]i) via the adenosine A3 receptor. KF26777 antagonized this [Ca2+]i mobilization induced by Cl-IB-MECA, with a KB value of 0.42±0.14 nM. These results indicate that KF26777 is a highly potent and selective antagonist of the human adenosine A3 receptor.

Introduction

Adenosine modulates many cellular functions via G protein-coupled receptors. Adenosine receptors are classified into A1, A2A, A2B and A3 receptors, which is based on cDNA cloning, affinities for agonists and antagonists and the mechanism of signal transduction Linden et al., 1991, Zhou et al., 1992, Palmer and Stiles, 1995. Activation of adenosine A3 receptors inhibits adenylyl cyclase and stimulates phospholipase C (Abbracchio et al., 1995) and D (Ali et al., 1996). The adenosine A3 receptor cDNAs have been cloned from multiple species. The homology of amino acid sequence in adenosine A3 receptors between rat (Zhou et al., 1992) and human (Salvatore et al., 1993) is only 74%. The difference between rat and human receptors seems to contribute to a marked difference in the affinity of ligands to these adenosine A3 receptors (Linden, 1994). Also, the tissue distribution of the adenosine A3 receptor varies across species: the rat mRNAs are detected in testis, lung, heart, kidney, brain and circulating inflammatory cells (Zhou et al., 1992); human mRNAs are widespread but the most abundant expression is in the lung, liver (Salvatore et al., 1993) and eosinophils (Walker et al., 1997).

Compared with adenosine A1 and A2A receptors, activation of adenosine A3 receptors requires a relatively high concentration of adenosine, which occurs during hypoxic stress and cellular damage such as ischemia and inflammation. The effect of the adenosine A3 receptor on brain ischemia is complicated: chronic preadministration of an adenosine A3 receptor agonist had a highly neuroprotective postischemic effect, but acute administration of the same agonist during ischemia exacerbated histological and functional damage (von Lubitz et al., 1994). With respect to inflammation, function of adenosine A3 receptors is controversial. In lung of patients with airway inflammation, the mRNA level of adenosine A3 receptors increased (Walker et al., 1997). In addition, stimulation of adenosine A3 receptors induces bronchospasm (Meade et al., 1996), enhances antigen-dependent degranulation (Ramkumar et al., 1993) and protects rat basophilic leukemia RBL-2H3 cells from apoptosis (Gao et al., 2001). Conversely, adenosine A3 receptor activation inhibits production of tumor necrosis factor-α (TNF-α) and inflammatory cytokines Hasko et al., 1998, McWhinney et al., 1996, Szabo et al., 1998, chemotaxis (Walker et al., 1997), degranulation and superoxide anion release (Ezeamuzie and Philips, 1999). In adenosine A3 receptor knockout mice, an adenosine A3 receptor selective agonist, 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA) exhibited no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells, and also the ability of Cl-IB-MECA to inhibit lipopolysaccharide-induced TNF-α production in vivo was decreased remarkably in contrast to wild type mice (Salvatore et al., 2000). These data support the finding that adenosine A3 receptors play a role in both anti- and proinflammatory responses. Thus, it is not clear whether an adenosine A3 receptor agonist or antagonist is a potential target for the treatment of brain ischemia and inflammatory diseases. In order to elucidate the physiological function of adenosine A3 receptors and to clarify the therapeutic benefit of drugs that act on the receptor, potent and selective adenosine A3 receptor antagonists are required.

Although several classes of adenosine A3 receptor antagonists have been reported including dihydropyridines, triazoloquinazolines, flavonoids, pyridines, isoquinolines and triazolonaphthyridines (Baraldi et al., 2000), the majority of these antagonists are not characterized sufficiently. We have identified a novel imidazopurine derivative, KF26777, 2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride (Fig. 1), as a potent and selective adenosine A3 receptor antagonist, and we report here the biochemical and pharmacological characterization of KF26777.

Section snippets

Materials

N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide ([125I]AB-MECA, specific activity, 74 TBq/mmol) was purchased from Amersham (Buckinghamshire, England). [3H]N6-cyclohexyladenosine ([3H]CHA, 0.925–1.85 TBq/mmol), [3H]-2-[p-(2-carboxyethyl)phenylamino]-5′-N-ethylcarboxamidoadenosine ([3H]CGS21680, 1.11–2.22 TBq/mmol), 8-[dipropyl-2,3,4,5-3H(N)]cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, 2.96–4.44 TBq/mmol) and [35S]guanosine 5′-O-(3-thiotriphosphate) ([35S]GTPγS, 46.2 TBq/mmol) were from

High affinity and selectivity to human adenosine A3 receptors of KF26777

As reported previously (Salvatore et al., 1993), [125I]AB-MECA bound with high affinity to membranes prepared from HEK293 cells expressing cloned human adenosine A3 receptors (clone HS-21a). KF26777 potently inhibited the [125I]AB-MECA binding to human adenosine A3 receptors in a dose dependent manner. The Ki value was 0.20±0.038 nM. Affinities of binding to other human adenosine receptor subtypes by KF26777 was much lower than that to the adenosine A3 receptors. The Ki values of KF26777 for

Discussion

Compound KF26777 is a potent and selective antagonist for human adenosine A3 receptors as evidenced by following. First, KF26777 showed a high affinity binding to human adenosine A3 receptors with a Ki value of 0.20 nM (Table 1). Scatchard analysis indicated that the binding mode of KF26777 was competitive, while that of I-ABOPX is noncompetitive (Fig. 2). Second, it was shown that KF26777 possessed about 9000-, 2350- and 3100-fold selectivity for the adenosine A3 receptors over the adenosine A1

Acknowledgements

We are grateful to Dr. Joel Linden for the supply of cell lines expressing each adenosine receptor subtypes, Dr. John A. Salmon for helpful discussion and Ms. Taeko Suzuki and Ms. Mayumi Ono for their excellent technical assistance.

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