[³H]MRS 1754, a selective antagonist radioligand for A_2B adenosine receptors

Abstract

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A_2B adenosine receptors. This is the least well-defined adenosine receptor subtype, and A_2B antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was tritiated on the propyl groups in a two-step reaction using a p-carboxamido precursor, which was dehydrated to the cyano species using trifluoroacetic anhydride. [³H]MRS 1754 (150 Ci/mmol) bound to recombinant human A_2B adenosine receptors in membranes of stably transfected HEK-293 cells. Specific binding was saturable, competitive, and followed a one-site model, with a K_D value of 1.13 ± 0.12 nM and a B_max value of 10.9 ± 0.6 pmol/mg protein. Specific binding utilizing 0.7 nM [³H]MRS 1754 was > 70% of total binding. The affinity calculated from association and dissociation binding constants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A₁ and A₃ adenosine receptors was not significant, and binding in membranes of HEK-293 cells expressing human A_2A receptors was of low affinity (K_D > 50 nM). The effects of cations and chelators were explored. Specific binding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [³H]MRS 1754 was consistent with the structure–activity relationship for agonists and antagonists at A_2B receptors. The K_i values of XAC (xanthine amine congener) and CPX (8-cyclopentyl-1,3-dipropylxanthine) were 16 and 55 nM, respectively. NECA (5′-N-ethylcarboxamidoadenosine) competed for [³H]MRS 1754 binding with a K_i of 570 nM, similar to its potency in functional assays. Thus, [³H]MRS 1754 is suitable as a selective, high-affinity radioligand for A_2B receptors.

1. Introduction

Four extracellular G protein-coupled receptors for adenosine have been identified: A_1, A_2A, A_2B, and A₃ [1]. A_2B receptors, which are coupled to stimulation of adenylyl cyclase [2, 3] and also lead to a rise in intracellular calcium [4], are involved in the control of vascular tone, cell growth and gene expression, mast cell degranulation, and intestinal water secretion. Activation of A_2B receptors in human retinal endothelial cells may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression [5]. Selective xanthine antagonists of the A_2B receptor have been reported recently [6, 7]. Such antagonists are potentially useful in the treatment of asthma [8, 9] and intestinal disorders [10].

Non-selective radioligands have been used to characterize recombinant human A_2B receptors overexpressed in HEK-293 cells. These include: 125I-IABOPX (¹²⁵I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine) [11], [³H]CPX [9], and [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) [12]. Based on these binding assays, we have identified several new compounds with improved potency and selectivity for human A_2B receptors [13, 14], culminating with aniline derivatives of the 8-phenylxanthine carboxylic congener, XCC (1, Fig. 1) [6]. A p-cyanoanilide derivative in this series, MRS 1754 (N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide, 4, Fig. 1), was 400-, 245-, and 123-fold more selective for human A_2B receptors than human A₁/A_2A/A₃ receptors, although less selective than rat A₁/A_2A receptors. This antagonist at a 100 nM concentration was shown to completely inhibit calcium mobilization stimulated by 1 μM NECA in HEK-293 cells expressing human A_2B receptors [6]. In the present study, this selective antagonist for the A_2B adenosine receptor, MRS 1754, has been prepared in tritiated form and shown to be a selective, high-affinity radioligand useful for characterizing recombinant human A_2B receptors.

Fig. 1.

Synthesis of [³H]MRS 1754, 4, in a multi-step reaction sequence. p-Carboxamidoaniline was condensed with XCC, 1, to give the amide, 2. The 1,3-diallyl groups were tritiated to give 3. Dehydration gave the final product, 4, which was purified using TLC.

2. Materials and methods

2.1. Synthesis

2.1.1. Preparation of ³H-labelled MRS 1754

2.1.1.a N-(4-(aminocarbonyl)phenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide (2)

A solution of 1 [15] (2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-diallyl-1H-purin-8-yl)-phenoxy]acetic acid, 38 mg, 0.1 mmol), 4-aminobenzamide (27 mg, 0.2 mmol), BOP-Cl (30 mg, 0.12 mmol), and TEA (20 μL, 0.206 mmol) in 2 mL of anhydrous DMF:CH₂Cl₂ (1:1 mixture) was stirred at room temperature for 24 hr. The mixture was evaporated to dryness under reduced pressure, and the residue was recrystallized from a solution of CHCl₃: MeOH (10:1) and washed with a solution of TEA in MeOH to afford 5 mg of 2. ¹H NMR (DMSO-d₆) 4.52 and 4.66 (2d, 4H, J = 3.9 Hz, 2X –NCH₂–), 4.83 (s, 2H, –OCH₂–), 5.04–5.17 (m, 4H, 2X =CH₂), 5.83–6.03 (m, 2H, 2X –CH=), 7.14 (d, 2H, J = 8.8 Hz, Ar), 7.26 (bs, 1H, –NH₂), 7.71 (d, 2H, J = 8.8 Hz, Ar), 7.85 (m, 3H, Ar and –NH₂), 8.09 (d, 2H, J = 8.8 Hz, Ar), 10.35 (s, 1H, –NH–). HRMS (EI, M⁺) for C₂₆H₂₄N₆O₅: Calc. 500.1808. Found 500.0825.

2.1.1b [³H]N-(4-(Aminocarbonyl)phenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy-]acetamide (3)

1.7 mg (3.4 μM) of 2 was dissolved in 2 mL DMF, and 1 mL ethanol was added. The compound was reduced in an atmosphere of tritium gas utilizing 10 mg of 5% Pd/Al₂O₃ for 4 hr at room temperature. After removal of labile tritium by evaporation several times with DMF-EtOH, an assay indicated ~400 mCi of crude product. TLC on LKC5F plates using CHCl₃:MeOH (50:5, v/v) followed by scanning showed a radiochemical purity of ~80%. The crude product was purified by preparative TLC using CHCl₃:MeOH (50:3, v/v) as the developing solvent. The purified product was dissolved in ethanol and submitted for mass spectral analysis for specific activity determination (150 Ci/mmol). The purified material (125 mCi) was stored in 10 mL ethanol at −20° and had a radiochemical purity of > 98%. The product was compared with the corresponding unlabeled form of 3, for which spectroscopic data were reported [6].

2.1.1c [³H]N-(4-Cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide (4, [³H]MRS 1754)

10 mCi of purified 3 was dissolved in 200 μL of methylene chloride, and 20 μL of pyridine was added. The reaction mixture was cooled to −76°, and 6 μL of trifluoroacetic anhydride was added. The reaction was warmed to room temperature, and an aliquot was removed for TLC analysis [CHCl₃:MeOH (50:2, v/v)]. To the crude reaction mixture (~60% product) was added 200 μL methanol and 200 μL TEA, followed by rotary evaporation to dryness. The product was purified by TLC using CHCl₃: MeOH (50:1, v/v) as solvent. The product was eluted from the plate using ethanol and stored at ~1 mCi/mL in ethanol at −20°. TLC [CHCl₃:MeOH (50:1, v/v)] indicated a radiochemical purity of > 97% (yield: 5.2 mCi). The product was compared with a corresponding unlabeled form of 4, for which spectroscopic data have been reported [6].

2.2. Pharmacological methods

A 20 nM stock solution of [³H]MRS 1754 was prepared in an equivolume mixture of DMSO and assay medium, which consisted of 50 mM Tris buffer containing 5 mM Mg²⁺ and 1 mM EDTA, at pH 6.5. Membranes from HEK-293 cells stably expressing the human A_2B receptor, prepared as reported [6] or obtained from a commercial source (Batch 1365, Receptor Biology, Inc., Beltsville, MD), were studied. Glass incubation tubes contained a total volume of 100 μL, consisting of a suspension in Tris buffer (as above) containing membranes (30 μg protein, stored at −80°) and [³H]MRS 1754 (final concentration 0.7 nM), and a solution of the competing compound, where applicable. Nonspecific binding was determined in the presence of 100 μM NECA (RBI-Sigma). SCH 58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) was the gift of Dr. Ennio Ongini, Schering-Plough, S.p.a. All non-radioactive compounds were initially dissolved in DMSO, and diluted with buffer to the final concentration, with the amount of DMSO in the final assay tubes being consistently 4.5%.

Incubations were terminated by rapid filtration over Whatman GF/B filters, which had been presoaked in 0.5% polyethyleneimine, using a Brandell cell harvester. The tubes were rinsed three times with 2 mL of ice-cold Tris buffer (pH 6.5).

For saturation studies, the concentration of [³H]MRS 1754 ranged from 0.1 to 20 nM. For competition experiments, at least six different concentrations of competitor, spanning three orders of magnitude adjusted appropriately for the ic₅₀ of each compound, were used. The ic₅₀ values, calculated with the nonlinear regression method implemented in the Prism program (GraphPAD), were converted to apparent K_i values using the Cheng–Prusoff equation [16].

3. Results

The A_2B receptor-selective xanthine antagonist MRS 1754 [6] was synthesized in tritiated form (Fig. 1) for use as a radioligand in a two-step tritiation sequence. The precursor 1,3-diallyl amide, 2, was prepared from the corresponding carboxylic acid, 1, reported previously [15]. An intermediate tritiated carboxamide derivative, 3, was dehydrated using trifluoroacetic anhydride, and the final product, 4, was purified using TLC. [³H]MRS 1754 was found to bind specifically to the human A_2B receptor expressed in HEK-293 cells, using 100 μM NECA to define nonspecific binding. Since MRS 1754 is a relatively hydrophobic molecule and has low aqueous solubility, a stock solution of the radioligand in 50% aqueous DMSO was prepared, which allowed the concentration of the dissolved xanthine to remain constant.

To optimize specific binding, the pH dependence (Fig. 2), temperature dependence, and optimal amount of protein (Fig. 3) were determined. In the range of pH 4.5 to 6.5, the level of specific binding of 0.7 nM [³H]MRS 1754 was constant, whereas raising the pH (6.5 to 8.0) decreased the level by approximately 10–15% for each 0.5 pH unit. The specific binding increased linearly with increasing amounts of protein (2–30 μg) present in each tube (Fig. 3). At 25°, the level of specific binding of 0.7 nM [³H]MRS 1754 was nearly identical to that at 37°; thus, subsequent binding was carried out at 25°. Using optimal ligand binding conditions, the association and dissociation binding kinetics were determined (Fig. 4), using 100 μM NECA to induce dissociation. Binding reached equilibrium at 40 min and remained constant for the following 80 min. The standard time of incubation selected for subsequent experiments was 60 min. The kinetics of the association appeared monophasic with a T_1/2 value of 7.65 ± 0.28 min. At equilibrium, the nonspecific binding did not exceed 30% of the total [³H]MRS 1754 bound. The association and dissociation rate constants were 0.022 ± 0.003 min⁻¹ nM⁻¹ and 0.027 ± 0.001 min^−1, respectively, resulting in a kinetic K_D (k₋₁/k₁) value of 1.22 nM (N = 4), in good agreement with the equilibrium determination.

Fig. 2.

pH dependence of specific binding of [³H]MRS 1754 to human A_2B receptors. [³H]MRS 1754 (0.7 nM) was incubated with membranes for 60 min at 25°^. The curve represents the mean ± SEM of four determinations.

Fig. 3.

Dependence of the specific binding of [³H]MRS 1754 to the human A_2B receptor on the amount of protein present in each assay tube at 25° (●) or 37° (◊). [³H]MRS 1754 (0.7 nM) was incubated with membranes for 60 min (N = 2).

Fig. 4.

Association (A, B) and dissociation (C, D) kinetics of [³H]MRS 1754 binding to HEK-293 cell membranes expressing the human A_2B receptor. [³H]MRS 1754 (0.7 nM) was incubated with membranes (30 μg protein) at 25°^. Dissociation was initiated by the addition of 100 μM NECA. The results shown are representative of three separate determinations.

The effects of cations and chelators on specific [³H]MRS 1754 binding to human A_2B receptors expressed in HEK-293 cell membranes were studied (Table 1). The presence of the divalent cations Zn²⁺ (≥1 mM) and Mn²⁺ (1 mM) significantly decreased the amount of specific binding of 0.7 nM [³H]MRS 1754, whereas Na⁺ had no effect. The effect of Zn²⁺ to inhibit binding appeared to be concentration-dependent, with an IC₅₀ of ~1 mM. The cations Ca²⁺ or Mg²⁺ (10 mM) caused a 20% reduction in the amount of specific binding of 0.7 nM [³H]MRS 1754, and the addition of chelating agents in the presence of Ca²⁺ (EGTA or EDTA) or Mg²⁺ (EDTA) restored the full degree of binding.

Table 1.

Effect of cations and chelators on [³H]MRS 1754 binding

Reagent	Concn (mM)	Bound (% of control)
EDTA	1	97 ± 3
Mg2+	1	90 ± 7
	5	79 ± 5
	10	79 ± 3
Mg2+(+ EDTA)	1	116 ± 8
Ca2+	1	93 ± 5
	10	78 ± 7
Ca2+(+ EDTA)	1	105 ± 4
Ca2+(+ EGTA)	1	110 ± 1
Zn2+	1	52 ± 2
	5	36 ± 9
	10	23 (N = 1)
Mn2+	1	75 ± 7
Na+	10	101 ± 0.5
	100	101 ± 1

Data are means ±SD from three separate determinations measured in duplicate. Control value was 100% (coresponding to 3600 cpm). Nonspecific binding was determined in the presence of 100 μM NECA. [³H]MRS 1754 was present at a final concentration of 0.7 nM. The concentration of EDTA or EGTA was 1 mM.

Under optimized equilibrium conditions (60-min incubation at 25° with 30–35 μg protein/tube, in Mg²⁺-containing medium), binding of [³H]MRS 1754 to membranes of HEK-293 cells expressing the human A_2B receptor was saturable and was best described by a one-site model (GraphPad, Prism). The percent of specific binding using a 0.7 nM concentration of the radioligand was > 70% of total binding. A representative saturation isotherm and a Scatchard transformation of the same data are shown in panels A and B of Fig. 5. The B_max value was 10.9 ± 0.6 pmol/mg protein (N = 4). The K_D value obtained from the saturation experiments was 1.13 ± 0.12 nM, which was in good agreement with the K_D value of 1.22 ± 0.22 nM determined in kinetic studies.

Fig. 5.

(A) Saturation isotherm of [³H]MRS 1754 binding to human A_2B receptors expressed in HEK-293 cell membranes, and (B) Scatchard analysis of the same data. [³H]MRS 1754 (0.1 to 20 nM) was incubated with 30 μg of membranes for 60 min at 25°^. Total binding (○), specific binding (●), and nonspecific binding (Δ), determined using 100 μM NECA, are shown in (A). Four experiments were carried out; data from one representative experiment are shown.

Levels of binding of [³H]MRS 1754 that was displaceable by 100 μM NECA in membranes expressing other adenosine receptor subtypes, e.g. rat and human A₁ and A₃ adenosine receptors (Table 2), were not significant. The only significant binding was to membranes expressing A_2B receptors, and this binding displayed characteristics of the A_2B subtype. Radioligand binding in membranes of HEK-293 cells expressing human A_2A receptors was, in all experiments, either undetectable or not greater than 8% of the total binding and, when observed, was not displaceable using the A_2A selective antagonist SCH 58261.

Table 2.

Binding of a single concentration of [³H]MRS 1754 in membranes of cells expressing four adenosine receptor subtypes

Cell-Subtype	Bound (pmol/mg protein)	Referencea
CHO-hA1	0,22 ± 0.09	[17]
HEK-hA2A	b	[9]
HEK-hA2B	6.89 ± 0.63	[9,12,14]
CHO-hA3	0.02 ± 0.02	[26]
Rat brain-rA1	0.17 ± 0.04	[6,15]
CHO-rA3	0.06 ± 0.07	[15]

Data are means ± SD from 3–6 separate determinations measured in duplicate. Nonspecific binding was determined in the presence of 100 μM NECA. [³H]MRS 1754 was present at a concentration of 0.7 nM, unless indicated.

^aReferences describe the source of each preparation and the pharmacological characteristics of each receptor. These citations also demonstrate that substantial binding to the indicated receptors was observed using radioligands having high affinity for each receptor. Thus, the expression levels of A₁, A_2A, and A₃ receptors were much higher than indicated by the level of binding of [³H]MRS 1754.

^bBinding was either undetectable or ≤ 8% of total binding, using 5–10 μg protein/tube (N = 10). This did not represent specific binding to A_2A receptors, since binding was not displaceable by the A_2A selective antagonist SCH 58261. The concentration of [³H]MRS 1754 was 1.0 nM.

The pharmacological profile for known adenosine receptor ligands in competition for [³H]MRS 1754 binding was consistent with the SAR for antagonists (Fig. 6A) and agonists (Fig. 6B) noted previously at A_2B receptors [6, 11, 12]. The most potent displacer of [³H]MRS 1754 binding (Table 3) was MRS 1754, itself, with a K_i value of 1.45 nM. This value was consistent with the observed K_D value and with a K_i value of 1.97 nM [6], determined by competition for binding of ¹²⁵I-IABOPX or [³H]ZM 241385. The potent xanthine derivatives XAC and CPX, and the triazoloquinazoline CGS 15943 [18], had K_i values of 16, 55, and 34 nM, respectively. The triazolotriazine ZM 241385 [19] had a K_i value of 145 nM, somewhat less potent than previously determined [7, 12]. Another potent xanthine derivative, XCC [14], which is a precursor of MRS 1754, had a K_i value of 54 nM in binding to human A_2B receptors, similar to the value reported previously [12]. Enprofylline (3-propylxanthine) and theophylline were roughly equipotent in displacing binding of [³H]MRS 1754. Alloxazine, which has been reported to be moderately selective (10-fold) for A_2B versus A₁ and A_2A receptors [20], had a K_i value of 2.04 μM. DAX, a xanthine derivative of interest as a treatment for cystic fibrosis [21], displaced binding with a K_i value of 408 nM. The Hill coefficients (n_H) in the competition experiments were in the range of 0.8 to 1.0 for antagonists and agonists.

Fig. 6.

Effects of (A) the antagonists MRS 1754 (●), CPX (◆), alloxazine (▲), and enprofylline (■) and (B) the agonists NECA (○), R-PIA (◊), and SPA (Δ) on radioligand binding to human A_2B receptors expressed in HEK-293 cell membranes. Membranes (30 μg) were incubated for 60 min at 25° with 0.7 nM [³H]MRS 1754. Nonspecific binding was determined with 100 μM NECA. The data are representative of four experiments.

Table 3.

K_i values for displacement of [³H]MRS 1754 binding to human A_2B receptors expressed in HEK-293 cell membranes

Compound	Ki(nM)
Antagonists
MRS 1754	1.45 ± 0.21
XAC	16.0 ± 0.7
CGS 15943	34.2 ± 1.0
XCC	53.6 ± 3.8
CPX	54.6 ± 12.1
ZM 241385	145 ± 15
DAX	408 ± 54
Alloxazine	2,040 ± 570
Enprofylline	19,800 ± 6,120
Theophylline	15,200 ± 4,100
Agonists
NECA	570 ± 170
R-PIA	13,900 ± 3,400
SPA	24,700 ± 7,500
CPA	20,600 ± 7,500
CGS 21680	14 ± 2% displacement at 100 μM

Specific binding was approximately 75% of total binding. Values are means ±SEM of 3–7 separate experiments.

Among agonists (Fig. 6B), as in functional assays [20, 22], NECA was more potent than N⁶-substituted analogues in binding competition. The A_2A-selective agonist CGS 21680 (2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5′-N-ethylcarbamoyladenosine) did not displace [³H]MRS 1754 binding significantly, even at a concentration of 100 μM, which is consistent with functional studies showing this agonist to be inactive at A_2B receptors and selective for the A_2A-receptor subtype [3].

4. Discussion

Following synthesis by an efficient multi-step method, [³H]MRS 1754 was shown to bind with high affinity to a single class of binding sites in membranes of HEK-293 cells expressing the human A_2B receptor. The pharmacological characteristics of this binding site resemble the functional characteristics of A_2B receptors [7, 11, 19, 20, 22]. [³H]MRS 1754 is selective for the A_2B receptor, with very low affinity for A₁ and A₃ receptors of both humans and rats. In cells expressing human A_2A receptors, the low levels of binding of [³H]MRS 1754 were demonstrated not to represent binding to this receptor subtype. Thus, due to its high affinity and selectivity, [³H]MRS 1754 has advantages over [³H]CPX, [³H]ZM 241385, and ¹²⁵I-IABOPX as a radioligand for A_2B receptors.

Theophylline is widely used as an antiasthmatic drug, although its mechanism of action is uncertain. The related xanthine enprofylline (3-propylxanthine) [9, 13], which is also therapeutically efficacious in the treatment of asthma, was earlier thought to act through a non-adenosine receptor-mediated mechanism due to its low affinity at A₁ and A_2A receptors. However, the discovery that enprofylline has greater than anticipated affinity and slight selectivity at the A_2B subtype [9] supports the hypothesis that A_2B receptor antagonism may contribute to the antiasthmatic activity of xanthines [8, 23, 24]. This hypothesis was strengthened by functional effects of A_2B receptor activation observed in mast cells of dogs, mice, and humans [8, 25]. Thus, potent and/or selective A_2B receptor antagonists may provide new therapeutic agents.

In conclusion, [³H]MRS 1754 binding to recombinant human A_2B receptors in membranes is a practical method for characterizing these receptors and their ligands in recombinant systems. This radioligand is yet to be characterized in cells and tissues endogenously expressing A_2B receptors and in the presence of other subtypes of adenosine receptors. Also, the affinity of MRS 1754 at A_2B receptors in other species is yet to be determined. The development of binding assays for this subtype of adenosine receptors that are useful with cell membranes will aid in the elucidation of the SAR of A_2B receptor agonists and antagonists, which are currently being synthesized [6,16,22,26].

Abbreviations

BOP-Cl

bis(2-oxo-3-oxazolidinyl)phosphinic chloride

CGS 15943

9-chloro-2-(2-furanyl) [1, 2, 4]triazolo[1,5-c]quinazolin-5-amine

CGS 21680

2- [4-[(2-carboxyethyl]phenyl]ethyl-amino]-5′-N-ethylcarbam-oyladenosine

CHO cells

Chinese hamster ovary cells

CPA

N⁶-cyclopentyladenosine

CPX

8-cyclopentyl-1,3-dipropylxanthine

DAX

1,3-diallyl-8-cyclohexylxanthine

DMF

dimethylformamide

HEK cells

human embryonic kidney cells

IABOPX

3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine

K_D

dissociation constant

K_i

equilibrium inhibition constant

MRS 1754

N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide

NECA

5′-N-ethylcarboxamidoadenosine

R-PIA

R-N⁶-phenylisopropyladenosine

SAR

structure–activity relationship

SCH 58261

5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine

SPA

N⁶-p-sulfophenyladenosine

TEA

triethylamine

XAC

8- [4-[[[[(2-aminoethyl]amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine

XCC

8- [4-[(carboxymethyl]oxy]phenyl]-1,3-dipropylxanthine

ZM 241385

4-(2-[7-amino-2-}furyl{}1,2,4{triazolo}2,3-a{}1,3,5{triazin-5-ylamino]ethyl)phenol