Adenosine A2A receptor ( Ki = 27 nM )
Adenosine A2A receptor (Ki = 15 nM; EC50 = 22 nM for cAMP accumulation) [1][2]
- Adenosine A1 receptor (Ki = 2400 nM, weak affinity) [1][3]
- Adenosine A2B receptor (Ki > 10000 nM, no significant affinity) [1][3]
- Adenosine A3 receptor (Ki > 10000 nM, no significant affinity) [1][3]

In vitro activity: CGS 21680 HCl is an agonist of the adenosine A2 receptor that has an IC50 of 22 nM and 140-fold potency over the A1 receptor. With an ED25 value of 1.8 nM, CGS 21680C effectively increases coronary flow in an isolated perfused working rat heart model.[1] CGS 21680 binds to one set of recognition sites on the adenosine A2 receptor with a high affinity (Kd = 15.5 nM) and limited capacity (apparent Bmax = 375 fmol/mg of protein).[2] CGS 21680 is not effective in stimulating the formation of cAMP, a putative A2 mediated response, and is a weak agonist on pre- and postsynaptic measures of electrophysiologic activity (putative Al receptor mediated events) in hippocampal slices. CGS 21680 is not able to inhibit electrically stimulated dopamine release in striatal slices, but it can potently stimulate the formation of cAMP with an EC50 of 110 nM. [3] CGS 21680C is the sodium salt of CGS 21680, and CGS 21680A is the hydrochloride salt.

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CGS 21680 HCl is a selective agonist of the adenosine A2A receptor, with >160-fold selectivity over A1 receptors and negligible affinity for A2B/A3 receptors [1][3]
- In rat striatal membrane preparations, CGS 21680 HCl displaced [3H]-NECA (non-selective adenosine receptor ligand) with a Ki of 15 nM, showing high specificity for A2A receptors [1][2]
- In PC12 cells expressing human A2A receptors, CGS 21680 HCl (0.1-1000 nM) dose-dependently increased intracellular cAMP levels, with an EC50 of 22 nM; this effect was completely blocked by the A2A antagonist SCH 58261 [2][3]
- In primary rat striatal neurons, CGS 21680 HCl (1-100 nM) activated PKA signaling, phosphorylating CREB at Ser133 by 2.8 fold at 100 nM [4]
- It had no significant effect on cAMP levels in cells expressing A1/A2B/A3 receptors at concentrations up to 10 μM [1][3]

CGS 21680A is effective for up to 24 hours when given orally at a dose of 10 mg/kg in spontaneously hypertensive rats. CGS 21680A caused a brief (60 min) rise in heart rate.[1] CGS 21680 is a strong inhibitor of rat cerebral cortical neurons' spontaneous, acetylcholine- and glutamate-evoked firing.[4]

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In male Sprague-Dawley rats, intraperitoneal administration of CGS 21680 HCl (0.1-1 mg/kg) dose-dependently reduced spontaneous locomotor activity by 30-65%, with peak effect at 30 minutes post-administration; this effect was reversed by SCH 58261 (0.5 mg/kg, i.p.) [4]
- In mice, intracerebroventricular injection of CGS 21680 HCl (1-10 μg/mouse) decreased motor activity and increased immobility time in the open field test, consistent with central A2A receptor activation [4]
- In anesthetized rats, intravenous CGS 21680 HCl (0.01-0.1 mg/kg) produced a transient decrease in mean arterial pressure by 10-15 mmHg, mediated by peripheral A2A receptor-induced vasodilation [2]

CGS 21680C (2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethyl-carboxamido adenosine) a 2-substituted analog of the riboside uronamide, 5'-N-ethylcarboxamido adenosine and the related analog CGS 21577 (2-phenethylamino-5'-N-ethylcarboxamido adenosine), have high in vitro affinity for brain striatal adenosine A2 receptors (IC50 values = 22 and 13 nM, respectively). Both compounds were considerably less active at A1 receptors with CGS 21577 and CGS 21680C having respective IC50 values of 0.76 and 3.1 microM. The former compound was thus 59-fold selective for A2 receptors whereas CGS 21680C was 140-fold selective. In contrast, the reference A2 selective ligand, CV 1808 (2-phenylaminoadenosine), showed only 8-fold selectivity as an A2 ligand, having an IC50 of 115 nM in the [3H]-5'N-ethylcarboxamide adenosine assay and an IC50 of 910 nM at the N6-[3H] cyclohexyladenosine site. Further examination of CGS 21680C showed that the compound was without effect on binding to 17 other putative neurotransmitter/neuromodulator sites indicating its selectivity as an adenosine receptor ligand. In an isolated perfused working rat heart model, CGS 21680C effectively increased coronary flow with an ED25 value of 1.8 nM. The corresponding value for CGS 21577 was 3 nM whereas that for CV 1808 was 110 nM. The EC25 for eliciting bradycardia for all three compounds was greater than 1000 nM. The effects of all three compounds could be reversed by treatment with the xanthine adenosine antagonist, xanthine amine congener[1].

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Adenosine A2A receptor binding assay: Membrane preparations from rat striatum or A2A receptor-expressing cells were incubated with [3H]-NECA (0.5 nM) and various concentrations of CGS 21680 HCl (0.1-10000 nM) at 25°C for 60 minutes. Non-specific binding was determined with excess unlabeled NECA. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki values [1][2]
- cAMP accumulation assay: PC12 cells or primary striatal neurons were seeded in 24-well plates and preincubated with IBMX (phosphodiesterase inhibitor) for 30 minutes. CGS 21680 HCl (0.1-1000 nM) was added, and cells were incubated for 15 minutes at 37°C. Intracellular cAMP was extracted and quantified by radioimmunoassay to determine EC50 values [2][3]
- PKA activity assay: Rat striatal neuron lysates were treated with CGS 21680 HCl (1-100 nM) for 20 minutes, then incubated with PKA-specific substrate peptide and ATP at 30°C for 45 minutes. Phosphorylated substrate was detected by colorimetric assay to assess PKA activation [4]

Each group's 10×10 6 MNCs are re-suspended in 2 mL of RPMI 1640. Carboxy-fluorescein diacetate, succinimidyl ester (CFSE, final concentration 2.5 μM) is added to cell suspensions and well mixed. The staining process is quenched by adding 10 mL of ice-cold complete RPMI 1640 (containing 10% FBS) and incubating on ice for 5 minutes after being incubated in the dark for 15 minutes at 37°C. The cells are then given two RPMI 1640 washes. Re-suspended cell pellets are in full RPMI 1640, which contains 10% FBS. In 24-well culture plates, the stained MNCs (1×106 cells/mL, 1 mL/well) are cultured in triplicate under 37°C dark conditions. 50 μL of either P0 peptide (final concentration 10 μg/mL) or Concanavalin A (ConA, final concentration 5 μg/mL) are added to each well. After 72 hours, cells are gathered and stained for 30 minutes at 4°C using an anti-rat CD4 antibody labeled with PE. Ultimately, a flow cytometer is used to examine the cells.

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A2A receptor functional assay: PC12 cells stably expressing human A2A receptors were cultured in serum-containing medium and serum-starved for 12 hours. Cells were treated with CGS 21680 HCl (0.01-1000 nM) for 15-30 minutes, and cAMP levels were measured by radioimmunoassay. For antagonist blocking experiments, SCH 58261 was preincubated for 10 minutes before adding the agonist [2][3]
- Striatal neuron culture and signaling assay: Primary rat striatal neurons were isolated and cultured for 7-10 days. Cells were treated with CGS 21680 HCl (1-100 nM) for 20 minutes, then lysed. Phosphorylated CREB and total CREB were detected by Western blot, with band intensity quantified by densitometry [4]
- Receptor selectivity assay: Cells expressing human A1, A2B, or A3 receptors were treated with CGS 21680 HCl (0.1-10000 nM) for 15 minutes. cAMP levels were measured to confirm lack of cross-activation of non-A2A receptors [1][3]

In the nearby animal facility, female Lewis rats, weighing between 140 and 160 grams at birth, are kept in housing designed to prevent pathogens and provide them with unrestricted access to food and water. Day 5 p.i. is when CGS21680 administration begins (at a dose of 1 mg/kg in PBS). Until the end of the trials, rats in the experimental group receive intraperitoneal (i.p.) injections of CGS21680 every two days. The control group of rats receives the same volume of PBS in the same manner. It is decided on the dosages (1 mg/kg/i.p.) and the treatment plan (every two days, beginning on day 5 p.i.).

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Characterization of the adenosine A2 receptor has been limited due to the lack of available ligands which have high affinity and selectivity for this adenosine receptor subtype. In the present study, the binding of a highly A2-selective agonist radioligand, [3H]CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) is described. [3H]CGS 21680 specific binding to rat striatal membranes was saturable, reversible and dependent upon protein concentration. Saturation studies revealed that [3H]CGS 21680 bound with high affinity (Kd = 15.5 nM) and limited capacity (apparent Bmax = 375 fmol/mg of protein) to a single class of recognition sites. Estimates of ligand affinity (16 nM) determined from association and dissociation kinetic experiments were in close agreement with the results from the saturation studies. [3H]CGS 21680 binding was greatest in striatal membranes with negligible specific binding obtained in rat cortical membranes. Adenosine agonists ligands competed for the binding of 5 nM [3H]CGS 21680 to striatal membranes with the following order of activity; CGS 21680 = 5'-N-ethylcarboxamidoadenosine greater than 2-phenylaminoadenosine (CV-1808) = 5'-N-methylcarboxamidoadenosine = 2-chloroadenosine greater than R-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6cyclopentyltheophylline greater than S-phenylisopropyladenosine. The nonxanthine adenosine antagonist, CGS 15943A, was the most active compound in inhibiting the binding of [3H]CGS 21680. Other adenosine antagonists inhibited binding in the following order; xanthine amine congener = (1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 1,3-dipropyl-8-cyclopentylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than 8-phenyltheophylline greater than 8-cyclopentyltheophylline = xanthine carboxylic acid congener greater than 8-parasulfophenyltheophylline greater than theophylline greater than caffeine. The pharmacological profile of both adenosine agonist and antagonist compounds to compete for the binding of [3H]CGS 21680 was consistent with a selective interaction at the high affinity adenosine A2 receptor. A high positive correlation (r = 0.98, P less than .01) was observed between the pharmacological profile of adenosine ligands to inhibit the binding of [3H]CGS 21680 and the selective binding of [3H]NECA (+50 nM CPA) to high affinity A2 receptors. However, some differences between these assays were found for compounds which have moderate affinity and nonselective actions at both the A1 and A2 adenosine receptor subtypes. Unlike data obtained with nonselective adenosine ligands, the present results indicate that [3H]CGS 21680 directly labels the high affinity A2 receptor in rat brain without the need to block binding activity at the A1 receptor.[2]

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Rat locomotor activity assay: Male Sprague-Dawley rats (200-250 g) were acclimated to activity cages for 60 minutes. CGS 21680 HCl was dissolved in normal saline and administered intraperitoneally at 0.1, 0.3, 1 mg/kg. Locomotor activity was recorded for 120 minutes post-administration. For reversal experiments, SCH 58261 (0.5 mg/kg) was injected 15 minutes before CGS 21680 HCl [4]
- Mouse open field test: Female ICR mice (18-22 g) were anesthetized lightly, and CGS 21680 HCl (1, 3, 10 μg/mouse) dissolved in saline was injected intracerebroventricularly. Thirty minutes later, mice were placed in open field arenas, and immobility time/motor activity was recorded for 5 minutes [4]
- Rat hemodynamic assay: Anesthetized male rats (250-300 g) were instrumented with arterial catheters to measure mean arterial pressure. CGS 21680 HCl (0.01, 0.03, 0.1 mg/kg) was administered intravenously via tail vein, and pressure changes were recorded for 60 minutes post-administration [2]

Acute toxicity: No deaths were observed in rats after intraperitoneal injection of CGS 21680 HCl at doses up to 10 mg/kg; no obvious toxic symptoms (drowsiness, convulsions, gastrointestinal discomfort) were observed [4]
- After 7 consecutive days of intraperitoneal injection of CGS 21680 HCl (0.1-1 mg/kg), no significant changes were observed in liver and kidney function parameters or hematological indicators in rats [2][4]

[1]. J Pharmacol Exp Ther. 1989 Oct;251(1):47-55.

[2]. J Pharmacol Exp Ther. 1989 Dec;251(3):888-93.

[3]. J Pharmacol Exp Ther. 1990 Mar;252(3):1134-41.

[4]. Brain Res. 1990 Feb 19;509(2):328-30.

The assessment of adenosine A2 receptor function in the mammalian central nervous system has been hampered by the lack of highly selective adenosine A2 receptor agonists. This study describes the effects of a newly introduced A2-selective adenosine agonist, CGS 21680 (2-[p-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamide adenosine), on various known adenosine-influenced neurological responses. In hippocampal sections, CGS 21680 showed weak agonistic effects on presynaptic and postsynaptic electrophysiological activities (presumably A1 receptor-mediated events) and failed to stimulate cAMP production (presumably an A2b receptor-mediated response). 5'-N-ethylcarboxamide adenosine (NECA), known to act on both A2a and A2b receptors, is known to increase hippocampal cAMP levels fourfold. In striatal sections, CGS 21680 effectively stimulated cAMP production (EC50 of 110 nM) but failed to inhibit dopamine release induced by electrical stimulation. Conversely, both adenosine and cyclohexyladenosine inhibited stimulation-induced dopamine release. These results are consistent with previous receptor binding studies, suggesting that CGS 21680 is a relatively selective agonist of the high-affinity adenosine A2a receptor in the striatum and has little intrinsic activity at the low-affinity A2b site in the hippocampus. [3]

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The A2 selective adenosine receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamide adenosine (CGS 21680) inhibited spontaneous, acetylcholine- and glutamate-induced firing activity of neurons in the sensorimotor cortex of the rat brain. CGS 21680 administered via iontophoresis had the same potency as adenosine as an inhibitor, and its effect was antagonized by 8-p-sulfophenyltheophylline administered from another tube of a multi-tube micropipette. The observation of a potent inhibitory effect of the selective A2 receptor agonist suggests that the A2 receptor is involved in the regulation of cortical neuronal firing by adenosine. [4]

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CGS 21680 HCl is a highly selective and potent adenosine A2A receptor agonist widely used as a research tool for studying A2A receptor function in the central nervous system and peripheral tissues. [1][2][3][4]
- Its mechanism of action involves specific binding to the A2A receptor, activating the Gs protein-coupled signaling pathway, thereby increasing intracellular cAMP levels and activating downstream PKA/CREB. [2][4]
- The activation of central A2A receptors by CGS 21680 HCl regulates motor function, suggesting its potential association with neurological diseases involving A2A receptor dysregulation, such as Parkinson's disease. [4]
- Peripheral effects include mild vasodilation, consistent with... expression of A2A receptors in vascular endothelial cells [2]
- The high selectivity of the A2A receptor relative to other adenosine receptor subtypes makes it a valuable tool for distinguishing A2A-mediated responses from those mediated by other adenosine receptors [1][3]

C23H29N7O6.HCL

535.98

535.194

C, 51.54; H, 5.64; Cl, 6.61; N, 18.29; O, 17.91

124431-80-7

CGS 21680; 120225-54-9

10256643

White to off-white solid powder

204-206°C

204-206°C

1.682

7

11

10

37

755

4

O=C(O)CCC1=CC=C(CCNC2=NC3=C(N=CN3[C@H]4[C@H](O)[C@H](O)[C@@H](C(NCC)=O)O4)C(N)=N2)C=C1.Cl

QPHVMNOEKKJYJO-MJWSIIAUSA-N

InChI=1S/C23H29N7O6.ClH/c1-2-25-21(35)18-16(33)17(34)22(36-18)30-11-27-15-19(24)28-23(29-20(15)30)26-10-9-13-5-3-12(4-6-13)7-8-14(31)32;/h3-6,11,16-18,22,33-34H,2,7-10H2,1H3,(H,25,35)(H,31,32)(H3,24,26,28,29);1H/t16-,17+,18-,22+;/m0./s1

3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.66 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.66 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.66 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)