Selective A2B Antagonists Research Articles

PEG-B-PLA-adenosine prevents osteoarthritis progression

Purpose: In a previous study we demonstrated that adenosine plays an important role in maintaining homeostasis in the joint. Adenosine has a short half-life (1-4 seconds in whole blood) limiting its therapeutic potential. Previously we showed that intrarticular injection of adenosine in a liposomal formulation prevents and reverses osteoarthritis (OA) progression in a post-traumatic OA (PTOA) animal model. Here we adopt a different strategy in prolonging the therapeutic half-life of adenosine by conjugating adenosine to the biodegradable carrier poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles. Methods: Six targeted polymeric (PEG-b-PLA) nanoparticles were synthetized by binding PLA-b-PEG400-N3 and PLA-b-PEG2000 block copolymers to adenosine at the 3’,4’-OH, 5’-OH, and 6-NH2 positions with an acetylene group. The efficacy of the new molecules to stimulate cAMP production was tested in RAW264.7 murine macrophage cells. PTOA was induced in Sprague Dawley rats (n=3 for each group) following non-surgical rupture of anterior cruciate ligament (ACL). Rats were treated with intra-articular injections of 100 ul of a suspension containing PLA-PEG2000-3,4-OH-adenosine (Nano-Ade; 1 mg/kg), or PLA-PEG2000 nanoparticles (Nano) or with saline for 6 weeks. The animals received the first injection 7 days after ACL rupture and injections were performed every 10 days. Knee swelling in the rats was measured before every injection. At the end of the experiment rats were sacrificed and both legs were collected for immunohistochemistry and microcomputed tomography (μCT) analysis. The capacity of the various nanoparticle preparations to bind to and activate adenosine receptors was studied as the capacity to stimulate cAMP accumulation and inhibit IL-1-stimulated IL-6, MMP13, col10a1 and NFkB translocation to the nucleus in primary murine chondrocytes. Results: Only adenosine-conjugated nanoparticles in which PEG2000 was bound to adenosine on the 3’,4’ hydroxyl groups stimulated cAMP increase (163% vs control; p<0.05) and this increase were blocked by selective antagonists of both adenosine A2A and A2B receptors. Intra-articular injection of the Nano-Ade in PTOA rats diminished swelling in affected knees (p<0.001 vs Nano). We observed that Nano-Ade diminished the IL-1β-stimulated expression of IL-6 mRNA expression (from 214±107 to 79±89 vs Control group), an effect that was partially reversed by A2AR (ZM241385 and SCH58261, 1μM each) and A2BR (PSB1115) antagonists. In contrast, the Nano-Ade diminished IL-1β-stimulated MMP13 and Collagen-10 mRNA and protein expression via stimulation of A2BR since the effect was reversed by the selective A2B antagonist but not by the A2AR-selective antagonist. Nano-Ade also diminished IL-1β stimulated activation of NF-kB, a central intracellular signal for inflammation (67% decrease vs IL-1β treated cells; p<0.001). In rats treated with Nano-Ade there was markedly reduced fibrillation of the cartilage surface (H&E staining) and less proteoglycan loss (Safranin-O staining) resulting in a significantly decreased OARSI score (Nano=2.99±1.20, Nano-Ade=0.88±0.69; p<0.03 vs Nano). Analysis of μCT scanned images showed an increase in cartilage volume in Nano-Ade treated rats (143% vs Nano; p=0.023). We also found that Nano-Ade reduced RNA expression for MMP-13 (83% reduction compare to IL-1β stimulated cells; p<0.001) and Collagen-10 (81% reduction compare to IL-1β stimulated cells; p<0.01) in primary murine chondrocytes stimulated with Il-1. Conclusions: Taken together these results demonstrate that the adenosine-functionalized particles bind to and activate adenosine A2A and A2B receptors on murine cells. Activation of adenosine receptors by PLA-PEG2000-3,4-OH-adenosine prevents OA progression and reduce pro-inflammatory mediators in murine chondrocytes.

Adenosine signaling in reserpine-induced depression in rats

A single, 6mg/kg intraperitoneal injection of reserpine increased floating time during forced swim testing 24h after administration in rats in five experiments. Although such behavioral depression traditionally is attributed to drug-induced depletion of brain monoamines, we examined the potential contribution of adenosine signaling, which is plausibly activated by reserpine treatment and contributes to behavioral depression in other paradigms. Whereas peripheral administration of the highly selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.5, 1.0, or 5.0mg/kg i.p.) 15min before swim testing failed to improve performance in reserpine-treated rats, swim deficits were completely reversed by 7mg/kg of the nonselective receptor antagonist caffeine. Performance deficits were also reversed by the nonselective A2 antagonist 3,7-dimethylxanthine (0, 0.5, 1.0mg/kg i.p.), and the highly selective A2A receptor antagonist (CSC: 8-(3chlorostyral)caffeine) (0.01, 0.1, or 1.0mg/kg i.p.) in a dose-dependent manner. The highly selective A2B antagonist alloxazine had no beneficial effect on swim performance at any dose under study (0.1, 1.0, and 5.0mg/kg i.p.).

Interactions of PPAR-alpha and adenosine receptors in hypoxia-induced angiogenesis

Hypoxia and adenosine are known to upregulate angiogenesis; however, the role of peroxisome proliferator-activated receptor alpha (PPARα) in angiogenesis is controversial. Using transgenic Tg(fli-1:EGFP) zebrafish embryos, interactions of PPARα and adenosine receptors in angiogenesis were evaluated under hypoxic conditions. Epifluorescent microscopy was used to assess angiogenesis by counting the number of intersegmental (ISV) and dorsal longitudinal anastomotic vessel (DLAV) at 28h post-fertilization (hpf). Hypoxia (6h) stimulated angiogenesis as the number of ISV and DLAV increased by 18-fold (p<0.01) and 100±8% (p<0.001), respectively, at 28hpf. Under normoxic and hypoxic conditions, WY-14643 (10μM), a PPARα activator, stimulated angiogenesis at 28hpf, while MK-886 (0.5μM), an antagonist of PPARα, attenuated these effects. Compared to normoxic condition, adenosine receptor activation with NECA (10μM) promoted angiogenesis more effectively under hypoxic conditions. Involvement of A2B receptor was implied in hypoxia-induced angiogenesis as MRS-1706 (10nM), a selective A2B antagonist attenuated NECA (10μM)-induced angiogenesis. NECA- or WY-14643-induced angiogenesis was also inhibited by miconazole (0.1μM), an inhibitor of epoxygenase dependent production of eicosatrienoic acid (EET) epoxide. Thus, we conclude that: activation of PPARα promoted angiogenesis just as activation of A2B receptors through an epoxide dependent mechanism.

Interdependence of PPARá/cytochrome P‐450 epoxygenase and adenosine receptors in hypoxia‐induced angiogenesis

Hypoxia, adenosine and cytochrome P‐450 epoxygenase (CYP epoxygenase) are known to upregulate angiogenesis; however, the role of peroxisome proliferator‐activated receptor alpha (PPARα) in angiogenesis is controversial. Epifluorescent microscopy was used to assess angiogenesis by counting the number of intersegmental (ISV) and dorsal longitudinal anastomotic vessels (DLAV) at 28 hours post‐fertilization (hpf). Under normoxic as well as hypoxic conditions, WY‐14643 (10 μM), an agonist of PPARα, stimulated angiogenesis while MK‐886 (0.5 μM), an antagonist of PPARα, blunted these effects. NECA (10 μM), an agonist of adenosine receptor promoted angiogenesis more effectively under hypoxic conditions as ISV increased by 85% (p&lt;0.001) and DLAV by 100 % (p&lt;0.001) under hypoxic condition. MRS‐1706 (10 nM), a selective A2B antagonist, abolished NECA‐induced increase in angiogenesis. NECA‐ or WY‐14643‐induced angiogenesis was also inhibited by PPOH (25 or 50 μM), an inhibitor of epoxygenase. Moreover, MK‐886 (0.5 μM) inhibited NECA‐induced adenosine receptor mediated angiogenesis and MRS‐1706 (10 nM) inhibited WY‐14643‐ induced PPARα‐mediated angiogenesis under hypoxic conditions. We conclude that hypoxia promoted PPARα as well as adenosine receptor‐mediated angiogenesis and activation of PPARα promoted angiogenesis just as activation of A2B receptors through epoxide‐dependent mechanism.

Discovery of benzothiazole-based adenosine A2B receptor antagonists with improved A2A selectivity

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A(2B) antagonists, due to its excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to impart superior potency) was prepared and evaluated for their selectivity against the A(2A) and A(1) receptors. This work resulted in the identification of compound 30, with excellent potency and high selectivity against both A(2A) and A(1) receptors.

Adenosinergic Modulation Of The Imidazoline I1‐Receptor‐Dependent Hypotensive Effect Of Ethanol In Acute Renal Failure

We recently reported that ethanol lowers blood pressure (BP) in rats with acute renal failure (ARF) via facilitation of sympathoinhibitory pathways of central imidazoline I1‐receptors. This study investigated whether adenosine receptor subtypes modulate the ethanol‐I1‐receptor interaction. The effect of selective blockade of adenosine A1, A2A, or A2B receptors on hemodynamic responses to consecutive administration of moxonidine, I1‐receptor agonist, and ethanol was determined in the glycerol model (10 ml/kg, i.m.) of ARF. Ethanol (1 g/kg i.v.) elicited significant decreases and increases in BP and heart rate (HR), respectively, in ARF rats. Prior exposure to moxonidine abolished the hypotensive but not the tachycardic effect of ethanol. The ability of moxonidine to abolish ethanol hypotension disappeared after selective blockade of adenosine A1 or A2A receptors with 8‐Cyclopentyl‐1,3‐dipropylxanthine and 8‐(3‐chlorostyryl) caffeine, respectively. In contrast, the moxonidine‐ethanol interaction was not affected in presence of alloxazine, a selective A2B antagonist or 8‐phenyltheophylline, a nonselective adenosine receptor antagonist. These findings suggest that alterations caused by moxonidine in adenosine A1 and A2A receptor signaling function to offset the I1‐receptor‐dependent hypotensive action of ethanol in ARF rats.

AMP579 binds to adenosine A2b receptors thus resolving longstanding mystery of its cardioprotective signaling

The mixed A1/A2a adenosine agonist AMP579 given at reperfusion is very protective in animal models of myocardial infarction; however, neither A1‐ nor A2a‐selective agonists can duplicate its protection despite blocking studies indicating that the protection came from an A2‐type receptor. We recently noted that A2b‐selective agonists are very protective and wondered whether AMP579 might also be an A2b agonist. AMP579 (500nM) for 1h starting at reperfusion protected isolated rabbit hearts exposed to 30 min‐regional ischemia/2h‐reperfusion (12.9±2.2% infarction of risk zone vs 32.0±1.9% in control hearts). A highly selective A2b antagonist PSB1115 (500nM) given for the first 15 min of reperfusion blocked AMP579's protection (32.2±3.1% infarction). We then confirmed A2b binding of AMP579 in human embryonic kidney cells transfected with human A2b receptors in which A2b occupation causes phosphorylation of ERK. AMP579 induced ERK phosphorylation with an EC50 of 250nM. These studies resolve a longstanding mystery related to AMP579's mechanism of protection, and further support the primacy of adenosine A2b receptors in the cardioprotective signaling that can be implemented at reperfusion. Supported by HL‐20648

3D‐Pharmacophore Models for Selective A2A and A2B Adenosine Receptor Antagonists.

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3D-Pharmacophore Models for Selective A2Aand A2BAdenosine Receptor Antagonists

Three-dimensional pharmacophore models were generated for A2A and A2B adenosine receptors (ARs) based on highly selective A2A and A2B antagonists using the Catalyst program. The best pharmacophore model for selective A2A antagonists (Hypo-A2A) was obtained through a careful validation process. Four features contained in Hypo-A2A (one ring aromatic feature (R), one positively ionizable feature (P), one hydrogen bond acceptor lipid feature (L), and one hydrophobic feature (H)) seem to be essential for antagonists in terms of binding activity and A2A AR selectivity. The best pharmacophore model for selective A2B antagonists (Hypo-A2B) was elaborated by modifying the Catalyst common features (HipHop) hypotheses generated from the selective A2B antagonists training set. Hypo-A2B also consists of four features: one ring aromatic feature (R), one hydrophobic aliphatic feature (Z), and two hydrogen bond acceptor lipid features (L). All features play an important role in A2B AR binding affinity and are essential for A2B selectivity. Both A2A and A2B pharmacophore models have been validated toward a wide set of test molecules containing structurally diverse selective antagonists of all AR subtypes. They are capable of identifying correspondingly high potent antagonists and differentiating antagonists between subtypes. The results of our study will act as a valuable tool for retrieving structurally diverse compounds with desired biological activities and designing novel selective adenosine receptor ligands.

Recent Progress in the Development of Adenosine Receptor Ligands as Antiinflammatory Drugs

Adenosine receptors belong to the family of G protein-coupled receptors. Four distinct subtypes are known, termed A(1), A(2A), A(2B) and A(3). Adenosine is an important signaling molecule which is released under inflammatory conditions. It can show antiinflammatory as well as proinflammatory activities, and the contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. Agonists selective for adenosine A(1) receptors show antinociceptive activity and are active in animal models of neuropathic and inflammatory pain. Adenosine A(2A) receptor agonists are potent antiinflammatory drugs. A(2A)-selective antagonists have shown antihyperalgesic activity in animal models of inflammatory pain. For A(2B)agonists as well as A(2B) antagonists antiinflammatory activity has been postulated. Selective A(2B) antagonists were shown to decrease (inflammatory) pain, and are promising candidates for the treatment of asthma. Adenosine A(3) receptor agonists appear to be proinflammatory, while there is evidence for an antiinflammatory effect of A(3) antagonists. There are some contradictory findings, and A(3) agonists are being developed for the treatment of inflammatory diseases such as arthritis. Indirect mechanisms increasing the extracellular concentration of adenosine using adenosine kinase inhibitors, adenosine deaminase inhibitors or adenosine uptake inhibitors, or increasing the potency of adenosine at the A(1) receptor subtype by allosteric modulators lead to potent antinociceptive and antiinflammatory activity. The advantage of indirectly acting drugs may be their site- and event-specific action since they are only active where adenosine has been released. In the past decade considerable progress has been made towards the identification of novel lead structures and the development of potent and selective ligands for all four adenosine receptor subtypes. A large number of patents has recently been filed and the field is finally in the process of translating many years of basic science into therapeutic application. This review article will focus on compounds published or patented within the past three years.

Adenosine Augments IL-10 Production by Macrophages through an A2B Receptor-Mediated Posttranscriptional Mechanism

Adenosine receptor ligands have anti-inflammatory effects and modulate immune responses by up-regulating IL-10 production by immunostimulated macrophages. The adenosine receptor family comprises G protein-coupled heptahelical transmembrane receptors classified into four types: A1, A2A, A2B, and A3. Our understanding of the signaling mechanisms leading to enhanced IL-10 production following adenosine receptor occupancy on macrophages is limited. In this study, we demonstrate that adenosine receptor occupancy increases IL-10 production by LPS-stimulated macrophages without affecting IL-10 promoter activity and IL-10 mRNA levels, indicating a posttranscriptional mechanism. Transfection experiments with reporter constructs containing sequences corresponding to the AU-rich 3'-untranslated region (UTR) of IL-10 mRNA confirmed that adenosine receptor activation acts by relieving the translational repressive effect of the IL-10 3'-UTR. By contrast, adenosine receptor activation failed to liberate the translational arrest conferred by the 3'-UTR of TNF-alpha mRNA. The IL-10 3'-UTR formed specific complexes with proteins present in cytoplasmic extracts of RAW 264.7 cells. Adenosine enhanced binding of proteins to a region of the IL-10 3'-UTR containing the GUAUUUAUU nonamer. The stimulatory effect of adenosine on IL-10 production was mediated through the A(2B) receptor, because the order of potency of selective agonists was 5'-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) = 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS-21680). Also, the selective A2B antagonist, alloxazine, prevented the effect of adenosine. Collectively, these studies identify a novel pathway in which activation of a G protein-coupled receptor augments translation of an anti-inflammatory gene.

Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype.

We previously reported that adenosine A2B receptor activation stimulates angiogenesis. Because hypoxia is a potent stimulus for the release of both adenosine and angiogenic factors, we tested the hypothesis that hypoxia alters the expression of adenosine receptors toward an "angiogenic" phenotype. We used human umbilical vein endothelial cells (HUVECs) and bronchial smooth muscle cells (BSMCs) because, under normoxic conditions, adenosine does not release vascular endothelial growth factor (VEGF). HUVECs expressed a characteristic A2A phenotype (the selective A2A agonist CGS21680 was as potent as the nonselective agonist 5'-N-ethylcarboxamidoadenosine [NECA] in generating cAMP). Hypoxia (4.6% O2, 3 hours) decreased A2A mRNA from 1.56+/-0.3% to 0.16+/-0.01% of beta-actin expression but increased A2B mRNA from 0.08+/-0.01% to 0.27+/-0.05%. Consistent with changes in receptor expression, CGS21680 failed to increase cAMP in hypoxic HUVECs, whereas NECA remained active (A2B phenotype), and NECA increased VEGF release from 9.5+/-1.0 to 14.2+/-1.2 pg/mL (P<0.05), indicating that increased A2B receptors were functionally coupled to upregulation of VEGF. Hypoxia had similar effects on BSMCs, increasing A2B mRNA by 2.4+/-0.3-fold, from 0.42+/-0.04% to 1.00+/-0.13% of beta-actin. Whereas NECA had no effect on VEGF release in normoxic BSMCs, it increased VEGF release in hypoxic BSMCs, from 74.6+/-9.6 to 188.3+/-16.7 pg/mL (P<0.01), and a selective A2B antagonist, CVT-6694, inhibited this increase. A2B receptors activated a VEGF reporter made unresponsive to hypoxia by mutating its hypoxia-inducible factor-1 (HIF-1) binding element, indicating a mechanism independent of HIF-1. In conclusion, hypoxia modulates the expression of adenosine receptors in human endothelial and smooth muscle cells toward an A2B"angiogenic" phenotype.

Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma.

Adenosine provokes bronchoconstriction in asthmatics through acute activation of mast cells, but its potential role in chronic inflammation has not been adequately characterized. We hypothesized that adenosine up-regulates Th2 cytokines in mast cells, thus promoting IgE synthesis by B lymphocytes. We tested this hypothesis in human mast cells (HMC-1) expressing A(2A), A(2B), and A(3) adenosine receptors. The adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA) (10 microM) increased mRNA expression of IL-1beta, IL-3, IL-4, IL-8, and IL-13, but not IL-2 and IFN-gamma. Up-regulation of IL-4 and IL-13 was verified using RT-PCR and ELISA; 10 microM NECA increased IL-13 concentrations in HMC-1 conditioned medium 28-fold, from 7.6 +/- 0.3 to 215 +/- 4 pg/ml, and increased IL-4 concentrations 6-fold, from 19.2 +/- 0.1 to 117 +/- 2 pg/ml. This effect was mediated by A(2B) receptors because neither the selective A(2A) agonist 2-p-(2-carboxyethyl)phenethylamino-NECA nor the selective A(3) agonist N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine reproduced it, and the selective A(2B) antagonist 3-isobutyl-8-pyrrolidinoxanthine prevented it. Constitutive expression of CD40 ligand on HMC-1 surface was not altered by NECA. Human B lymphocytes cocultured for 12 days with NECA-stimulated HMC-1 produced 870 +/- 33 pg IgE per 10(6) B cells, whereas lymphocytes cocultured with nonstimulated HMC-1, or cultured alone in the absence or in the presence of NECA, produced no IgE. Thus, we demonstrated induction of IgE synthesis by the interaction between adenosine-stimulated mast cells and B lymphocytes, and suggest that this mechanism is involved in the amplification of the allergic inflammatory responses associated with asthma.

Expression and function of A2B adenosine receptors in the U87MG tumor cells

AbstractAdenosine is released in increased amounts during hypoxic conditions and is a potential mediator of angiogenesis. The aims of this study were to determine the effect of adenosine on the release of angiogenic factors from the glioblastoma cell line U87MG and the subtypes of adenosine receptors that mediate this effect of adenosine. The adenosine analog NECA increased the release of IL‐8 to 160±13% of control at 24 h. As a positive control, cobalt (100 µM), a metal ion capable of inducing hypoxia‐like effects, increased the release of IL‐8 to 362±99% of control at 24 h. Using real‐time RT‐PCR, it was found that U87MG cells expressed mRNA transcripts of the A1, A2A, and A2B adenosine receptors. The functional presence of A2B adenosine receptors in this cell line was confirmed using pharmacological approaches. NECA stimulated cAMP accumulations with an EC50 value of 2.93±0.91 µM and this effect of NECA was inhibited by the selective A2B antagonist IPDX. In addition, NECA also caused an increase in the intracellular calcium concentration. Furthermore, cobalt increased the level of A2B mRNA to 187±16% and 259±37% of the vehicle control at 5 h and 24 h, respectively. In conclusion, these results suggest that A2B adenosine receptors are upregulated during hypoxia and may play an important role in mediating the effect of adenosine to increase the release of angiogenic factor IL‐8 by tumor cells. Drug Dev. Res. 58:405–411, 2003. © 2003 Wiley‐Liss, Inc.

Mast cell-mediated stimulation of angiogenesis: cooperative interaction between A2B and A3 adenosine receptors.

Adenosine is released during tissue injury, ischemia and tumor growth, and promotes angiogenesis. Because mast cells accumulate in the proximity of new blood vessel development, we examined if they may contribute to adenosine-induced angiogenesis. We found that HMC-1 human mast cells express A2A, A2B, and A3 adenosine receptors. The adenosine agonist NECA (100 micromol/L) increased interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and angiopoietin-2 mRNA expression. NECA-induced secretion of IL-8 and VEGF was verified by ELISA. A2B receptors mediate VEGF and IL-8 secretion because neither CGS21680 (selective A2A agonist) nor IB-MECA (selective A3 agonist) produced this effect, and it was inhibited by the selective A2B antagonist IPDX but not by the selective A2A antagonist SCH58261 or the selective A3 antagonist MRS1191. In contrast, the selective A3 agonist IB-MECA (EC50 1 nmol/L) stimulated angiopoietin-2 expression. Conditioned media from NECA-activated HMC-1 stimulated human umbilical vein endothelial cell proliferation and migration, and induced capillary tube formation. Capillary formation induced by mast cell-conditioned media was maximal if both HMC-1 A2B and A3 receptors were activated, whereas activation of A2B receptor alone was less effective. Thus, adenosine A2B and A3 receptors act in a functional cooperative fashion to promote angiogenesis by a paracrine mechanism involving the differential expression and secretion of angiogenic factors from human mast cells.

Anilide derivatives of an 8-phenylxanthine carboxylic congener are highly potent and selective antagonists at human A(2B) adenosine receptors.

No highly selective antagonists of the A(2B) adenosine receptor (AR) have been reported; however such antagonists have therapeutic potential as antiasthmatic agents. Here we report the synthesis of potent and selective A(2B) receptor antagonists. The structure-activity relationships (SAR) of 8-phenyl-1, 3-di-(n-propyl)xanthine derivatives in binding to recombinant human A(2B) ARs in HEK-293 cells (HEK-A(2B)) and at other AR subtypes were explored. Various amide derivatives of 8-[4-[[carboxymethyl]oxy]phenyl]-1,3-di-(n-propyl)xanthine, 4a, were synthesized. A comparison of aryl, alkyl, and aralkyl amides demonstrated that simple anilides, particularly those substituted in the para-position with electron-withdrawing groups, such as nitro, cyano, and acetyl, bind selectively to human A(2B) receptors in the range of 1-3 nM. The unsubstituted anilide 12 had a K(i) value at A(2B) receptors of 1.48 nM but was only moderately selective versus human A(1)/A(2A) receptors and nonselective versus rat A(1) receptors. Highly potent and selective A(2B) antagonists were a p-aminoacetophenone derivative 20 (K(i) value 1.39 nM) and ap-cyanoanilide 27 (K(i) value 1.97 nM). Compound 27 was 400-, 245-, and 123-fold selective for human A(2B) receptors versus human A(1)/A(2A)/A(3) receptors, respectively, and 8.5- and 310-fold selective versus rat A(1)/A(2A) receptors, respectively. Substitution of the 1,3-dipropyl groups with 1,3-diethyl offered no disadvantage for selectivity, and high affinities at A(2B) receptors were maintained. Substitution of the p-carboxymethyloxy group of 4a and its amides with acrylic acid decreased affinity at A(2B) receptors while increasing affinity at A(1) receptors. 1, 3-Di(cyclohexylmethyl) groups greatly reduced affinity at ARs, although the p-carboxymethyloxy derivative 9 was moderately selective for A(2B) receptors. Several selective A(2B) antagonists inhibited NECA-stimulated calcium mobilization in HEK-A(2B) cells.

Acyl-Hydrazide Derivatives of a Xanthine Carboxylic Congener (XCC) as Selective Antagonists at Human A2B Adenosine Receptors.

The structure–activity relationships (SAR) of 8-phenyl-1,3-dipropylxanthine derivatives in binding to recombinant human A2B adenosine receptors were explored, in order to identify selective antagonists. Based on the finding of receptor selectivity in MRS 1204, containing an N-hydroxysuccinimide ester attached through the p-position of the 8-phenyl substituent [Jacobson et al. (1999): Drug Dev. Res., 47:45–53], a hydrazide and its more stable imide derivatives were synthesized. The hydrazide of XCC (8-[4-[[[carboxy]methyl]oxy]phenyl]-1,3-dipropylxanthine) was acylated with a variety of mono- and dicarboxylic acids. Ki values were determined in the adenosine receptor binding assays. At recombinant human A2B receptors expressed in membranes of HEK-293 cells, antagonist radioligands used were the xanthine 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-oxyacetate-1-propyl-xanthine) and the nonxanthine antagonist [3H]ZM 241385 ([3H]4-(2-[7-amino-2-{furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino-ethyl)phenol). The initial screening utilized rat A1/A2A receptors and human A3 receptors, and selected compounds were examined at the human A1/A2A subtypes. A 1,2-dimethylmaleimide derivative, 14 (MRS 1595), bound to human A2B receptors with a Ki of 19 nM and proved to be selective vs. human A1/A2A/A3 receptors by 160-, 100-, and 35-fold, respectively. Enprofylline (3-propylxanthine) is slightly selective for A2B receptors, suggesting removal of the 1-propyl group; however, combination of the 1-H-3-Pr and 8-phenyl substituents eliminated the selectivity. Other potent and moderately selective A2B antagonists were a tetrahydrophthaloyl derivative 18b (MRS 1614, Ki value 10 nM) and amino acid conjugates of the XCC-hydrazide, i.e., the glutarimide 24b (MRS 1626, Ki value 13 nM), and protected dipeptide 27 (MRS 1615, Ki value 11 nM). Drug Dev. Res. 47:178–188, 1999.