bovine A2a ( Ki = 2.0 nM ); rat A2a ( Ki = 2.3 nM )
SCH-58261 is a potent and selective antagonist of the adenosine A2a receptor (A2aAR); it shows low nanomolar affinity for rat and bovine striatal A2aAR, with 50- to 100-fold selectivity over A1 adenosine receptor (rat and bovine brain respectively) and no affinity for A3 adenosine receptor or other receptors at concentrations up to 1 μM. pA2 values are 7.9 (rabbit platelet aggregation assay) and 9.5 (porcine coronary artery relaxation assay). [1]

In vitro activity: SCH 58261 causes the effects of CGS 21680 to be competitively antagonistic, thereby inhibiting rabbit platelet aggregation and pig coronary artery relaxation.[1]

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1. Receptor binding and selectivity: SCH-58261 exhibits high affinity for A2aAR in rat and bovine brain tissue binding studies (low nanomolar range) and significant selectivity over A1AR (50-100-fold). It does not bind to A3AR or other receptors at concentrations ≤1 μM. Saturation experiments confirm competitive antagonism at rat A1 and A2aAR. [1]

2. Functional antagonism: SCH-58261 competitively antagonizes the A2aAR-selective agonist CGS21680-induced effects, including inhibition of rabbit platelet aggregation (pA2=7.9) and relaxation of porcine coronary arteries (pA2=9.5). It fails to antagonize A2bAR-mediated vasorelaxation in guinea pig aorta (300 nM) and weakly inhibits A1AR-mediated negative chronotropy in rat atria (300 nM). [1]

3. Protection against oxygen-glucose deprivation (OGD)-induced astrocyte dysfunction: 200 nM SCH-58261 reverses OGD-mediated glutamate uptake impairment in astrocytes, elevates EAAT2 and PPARγ protein levels, and suppresses Ying Yang 1 (YY1) expression. It acts synergistically with A1AR agonist CCPA to enhance EAAT2 expression and reduce intracellular glutamate accumulation. [4]

4. Protection against intermittent hypoxia-induced PC12 cell injury: SCH-58261 increases cell viability, suppresses PKC and SUR1 (KATP channel subunit) expression, and decreases Cleaved-Caspase 3 levels in PC12 cells exposed to intermittent hypoxia. It inhibits the A2a-PKC-KATP signaling pathway-mediated apoptosis. [5]

SCH58261 (0.01 mg/kg, i.p.) decreases demyelination and levels of TNF-α, Fas-L, PAR, Bax expression, and JNK MAPK activation in mice with spinal cord injury. The neurological deficit is improved with long-term SCH58261 administration.[2]
SCH58261 (2 mg/kg, i.p.) ameliorates the motor disturbance and 6-OHDA-induced bradykinesia in rats with Parkinson's disease.[3]

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1. Neuroprotection in mouse spinal cord injury (SCI) model: [2]

- Systemic administration (0.01 mg/kg, intraperitoneal injection at 1, 6, and 10 hours post-SCI) reduces demyelination, TNF-α, Fas-L, PAR, and Bax expression, and inhibits JNK MAPK activation in spinal cord tissue 24 hours post-SCI.
- Chronic administration via osmotic minipump for 10 days improves neurological deficits (assessed by BMS motor score) up to 10 days post-SCI.
- Direct intraspinal injection exerts neuroprotective effects (reduces edema and white matter damage), while A2aAR agonist CGS21680 is ineffective when centrally administered.
- Reduces SCI-induced upregulation of A2aAR expression in spinal cord neurons, astrocytes, and oligodendrocytes.
2. Improvement of motor disorders in rat Parkinson's disease (PD) model: [3]

- Rat PD model is induced by unilateral injection of 8 μg 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNc).
- Intraperitoneal injection of SCH-58261 (2 mg/kg) acutely improves 6-OHDA-induced bradykinesia and balance disturbances, as evidenced by reduced elapsed time in the beam traversal test (p<0.001) at 10, 30, and 60 minutes post-administration.

Male Sprague-Dawley rats weighing 250–300 g are used to harvest the cortex and striatum of the rat brain. Within 10 mm after an animal is sacrificed, the frontal cortex and striatum of cow brains are taken from a nearby abattoir. [3H]CHA and [3H]CGS 21680 are used in the A1 and A2a ADO receptor binding assays. (3H) 2-[4-phenethylamino]-(2-carboxyethyl) as radioligands, 5'-N-ethylcarboxamidoadenosinel in this case. Using [125I]AB-MECA as the radioligand, a binding assay is carried out on CHO cells that have been stable transfected with the rat brain A3 ADO receptor. Saturation experiments are performed on rat brain tissues in the absence or presence of varying concentrations of SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]) in order to ascertain the type of inhibition (competitive or noncompetitive) at A1 ADO ([3H]CHA, 0.125-64 nM) and A2a ADO receptors ([3HJCGS 21680, 1-128 aM).[1,5-c]triazolo[1,2,4-]pyrimidine}. SCH 58261's affinity for a number of neurotransmitter binding sites, including mu-opioid, benzodiazepine, N-methyl-D-aspartate, D1 and D2 dopamine receptors, 5-HT1 and 5-HT2 receptors, M1 and M2 muscarinic receptors, and alpha-1, alpha-2, and beta-1 adrenoceptors, is determined using standard procedures.

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1. Adenosine receptor binding assay: [1]

Membrane preparations are isolated from rat and bovine brain tissues (striatum for A2aAR-rich samples). Different concentrations of SCH-58261 are incubated with the membrane preparations and a radiolabeled adenosine receptor ligand. After incubation, unbound ligand is removed by filtration, and the radioactivity of the bound ligand is measured to determine the binding affinity (Ki) of SCH-58261 for A2aAR and A1AR. Saturation binding experiments are performed to confirm the competitive nature of the antagonism.
2. Functional antagonism assay (porcine coronary artery relaxation): [1]

Porcine coronary artery rings are prepared and mounted in an organ bath with physiological buffer. The rings are pre-contracted with a vasoconstrictor, and then the A2aAR agonist CGS21680 is added to induce relaxation. SCH-58261 is added at different concentrations before CGS21680 administration, and the relaxation response is recorded. The pA2 value is calculated based on the concentration-response curves to evaluate the antagonistic potency.
3. Functional antagonism assay (rabbit platelet aggregation): [1]

Rabbit platelets are isolated and suspended in platelet-rich plasma. CGS21680 is used to inhibit platelet aggregation induced by a aggregating agent. SCH-58261 is pre-incubated with the platelets at various concentrations, and the aggregation rate is measured using a platelet aggregometer. The pA2 value is determined to assess the antagonistic activity of SCH-58261. [1]

The PC12 cells were randomly divided into 8 groups: A2a agonist group (CGS21680), A2a antagonist group (SCH58261), PKC inhibitor group (Chelerythrine, CHE), solvent control group (DMSO), intermittent hypoxia group (IH), persistent hypoxic group (SH), air-simulated control group (AC), and normal control group (Control). Cells for A2a agonist group, A2a antagonist group, PKC inhibitor group, solvent control group and intermittent hypoxia group (IH) were placed in hypoxic cell culture chambers. PC12 cells were seeded to a 96-well plate at a density of 5000/well and placed at 37 °C in a 5% CO₂ cell incubator for 24 h.
After attachment, cells were placed in an IH chamber which was controlled by regulating the ration of gaseous mixture that mimicked different levels of hypoxia exposure. The A2a receptor agonist CGS21680 (100 u M), the A2a receptor antagonist SCH58261(200 u M), and the PKC antagonist CHE(200 u M) were dissolved in 0.1% DMSO solution in different concentrations. These various treated-cells were incubated in IH chamber and exposed to 9 h-deoxygenation-reoxygenation cycle of 5% oxygen for 60 min. and 20% oxygen for 30 min.. For sustained hypoxia (SH), this group was treated with 5% oxygen for 6 h. Some cells were placed in a normal incubator at the exposure level of 5% CO2 for 9 h as the control group. In the room air (AC) group, cells were treated with room air for 9 h to assess the effect of frequent replacement of the gas on cells. The PC12 cells model was identified by the morphological changes using microscope as well as MTT assay which is an indicator of cellular viability. Cells were treated with 0.5 mg/ml of MTT solution for 2 h, and then solubilized in dimethyl sulfoxide. Optical density was measured by a spectrophotometer at a wavelength of 570 nm.[5]

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In the NSCLC cell line H1975, SCH 58261 reduces cell viability in a concentration-dependent manner.

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1. Astrocyte OGD injury and protection assay: [4]

Primary astrocytes are cultured and identified by immunofluorescence (S100b and GRAFP staining). Cells are exposed to OGD conditions for specified times, with or without 200 nM SCH-58261 (alone or combined with 30 nM CCPA). After treatment, Western blot is used to detect EAAT2, PPARγ, and YY1 protein levels; intracellular glutamate concentration is measured with a glutamate assay kit; co-immunoprecipitation (Co-IP) is performed to analyze the interaction between A1AR and A2aAR.
2. PC12 cell intermittent hypoxia injury and protection assay: [5]

PC12 cells are subjected to intermittent hypoxia cycles to induce injury. Cells are treated with SCH-58261 at appropriate concentrations, with or without PKC antagonist CHE. Cell viability is measured by a viability assay; Western blot is used to detect PKC, KATP channel subunits (Kir6.2, SUR1), and Cleaved-Caspase 3 expression levels.
3. A2aAR expression and localization assay (spinal cord cells): [2]

Spinal cord tissues from sham and SCI mice are processed into single-cell suspensions or sections. Double immunofluorescence staining is performed using antibodies against A2aAR (Texas red-labeled) and cell-specific markers (NeutroTracer green for neurons, GFAP for astrocytes, IBA1 for microglia, OSP for oligodendrocytes). The co-localization of A2aAR with different cell types is observed under a fluorescence microscope. [2]

Dissolved in Tween 80 (1 %) and administered (0.01 mg/kg, i.p.) twice/day for 7 days.
Rat SCH58261, systemically administered (0.01 mg/kg intraperitoneal. 1, 6 and 10 hours after SCI), reduced demyelination and levels of TNF-α, Fas-L, PAR, Bax expression and activation of JNK mitogen-activated protein kinase (MAPK) 24 hours after SCI. Chronic SCH58261 administration, by mini-osmotic pump delivery for 10 days, improved the neurological deficit up to 10 days after SCI. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia and oligodendrocytes. Soon after SCI (24 hours), these receptors showed enhanced expression in neurons. Both the A2A agonist and antagonist, administered intraperitoneally, reduced expression of the A2A receptor, ruling out the possibility that the neuroprotective effects of the A2A agonist are due to A2A receptor desensitization. When the A2A antagonist and agonist were centrally injected into injured SC, only SCH58261 appeared neuroprotective, while CGS21680 was ineffective.[2]

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In order to induce experimental model of Parkinson's disease, 6-hydoxydopamine (8 μg/rat) was injected unilaterally into the SNc. After three weeks as a recovery period, 6-OHDA-induced bradykinesia and balance disturbances were assessed by using beam traversal test 10, 30 and 60 minutes after intraperitoneal injections of the drugs (caffeine, SCH58261).[3]

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1. Mouse SCI model and drug administration: [2]

- Animals: Male mice (strain not specified) are used to establish SCI models via T5-T8 laminectomy and extradural compression of the spinal cord.
- Drug administration protocols:
- Short-term systemic administration: SCH-58261 (0.01 mg/kg) is administered via intraperitoneal injection at 1, 6, and 10 hours post-SCI; the control group receives vehicle.
- Chronic administration: SCH-58261 is delivered via osmotic minipump for 10 days post-SCI.
- Central administration: SCH-58261 is directly injected into the injured spinal cord.
- Sample collection and detection: Mice are sacrificed 24 hours or 10 days post-SCI. Spinal cord tissues are harvested for histological staining (Luxol fast blue, Weigert's, Oil red O, H&E), Western blot (A2aAR, phospho-JNK MAPK, β-actin), and immunofluorescence (A2aAR, cell-specific markers). Hind limb motor function is evaluated by BMS motor score daily.
2. Rat PD model and drug administration: [3]

- Animals: Male Wistar rats are used to establish PD models via unilateral intra-SNc injection of 8 μg 6-hydroxydopamine (6-OHDA).
- Recovery period: Rats are allowed 3 weeks of recovery after 6-OHDA injection.
- Drug administration: SCH-58261 (2 mg/kg) is administered via intraperitoneal injection; caffeine (30 mg/kg) is used as a positive control.
- Behavioral testing: Beam traversal test is performed at 10, 30, and 60 minutes post-administration to assess bradykinesia and balance disturbances. [3]

[1]. J Pharmacol Exp Ther. 1996 Feb;276(2):398-404.

[2]. AJ Neuroinflammation. 2011 Apr 12:8:31.

[3]. Acta Cir Bras. 2016 Feb;31(2):133-7.

[4]. PPAR Res. 2020 Mar 6:2020:2410264.

[5]. Brain Res Bull. 2019 Aug:150:118-126.

LSM-3822 belongs to the triazolopyrimidine class of compounds. We performed in vitro pharmacological characterization on a novel, highly selective A2a adenosine receptor antagonist, SCH 58261 [7-(2-phenylethyl)-5-amino-2-(2-furanyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine]. In binding studies on rat and bovine brain tissues, SCH 58261 exhibited low nanomolar affinity for striatal A2a adenosine receptors and good selectivity for A2a adenosine relative to A1 adenosine (approximately 50 to 100 times higher in rat and bovine brains, respectively). At concentrations up to 1 μM, SCH 58261 showed no affinity for A3 adenosine receptors or other receptors. Saturation experiments on rat A1 and A2a adenosine receptors indicated that this antagonistic effect is competitive. In two functional experiments, such as inhibition of rabbit platelet aggregation and porcine coronary artery dilation, SCH 58261 competitively antagonized the effects induced by the A2a adenosine selective agonist CGS 21680 (2-[4-(2-carboxyethyl)-phenethyl-amino]-5'-N-ethylcarboxamide adenosine). Specifically, the compound had pA2 values of 7.9 and 9.5, respectively. SCH 58261 (300 nM) failed to antagonize 5'-N-ethylcarboxamide adenosine-induced dilation of isolated guinea pig aorta, a response mediated by A2b adenosine receptors. Similarly, at the same concentration, the compound weakly inhibited the negative chronotropic effects of A1 adenosine induced by 2-chloro-N6-cyclopentyl adenosine in isolated rat atria. These data suggest that SCH 58261 is a potent and selective non-xanthine A2a adenosine antagonist that competes with the biological responses mediated by this receptor subtype. This compound is of great significance for studying the biological function of the A2a adenosine receptor and deserves further attention to elucidate the therapeutic potential of A2a antagonists. [1]

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Background: Permanent dysfunction following spinal cord injury (SCI) stems from both mechanical damage and secondary tissue responses involving inflammation. Increased adenosine and glutamate release shortly after SCI is one of the sequelae leading to dysfunction. The role of adenosine A2A receptors in central ischemia/trauma remains to be elucidated. Our previous studies have shown that systemic administration of the adenosine A2A receptor selective agonist CGS21680 after spinal cord injury can protect tissues from damage, improve motor dysfunction, and reduce inflammatory responses. This study aimed to investigate the effects of systemic administration of the adenosine A2A receptor antagonist SCH58261 after spinal cord injury on the above indicators. We further verified whether the main mechanism of action of the agonist and antagonist is in the peripheral or central regions. Methods: Spinal cord injury was induced in mice by performing a four-segment T5-T8 laminectomy followed by epidural compression to expose the spinal cord. Three administration regimens were employed: short-term intraperitoneal injection, long-term osmotic pump administration, and direct spinal injection. Results showed that systemic administration (0.01 mg/kg, intraperitoneal injection, 1, 6, and 10 hours post-spinal cord injury) of SCH58261 reduced demyelination 24 hours post-spinal cord injury and decreased the expression of TNF-α, Fas-L, PAR, Bax, and the activation of JNK mitogen-activated protein kinase (MAPK). Long-term (10 days) continuous osmotic pump administration of SCH58261 improved neurological deficits within 10 days post-spinal cord injury. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia, and oligodendrocytes. Shortly after spinal cord injury (24 hours), the expression of these receptors in neurons is enhanced. Intraperitoneal injection of both A2A agonists and antagonists reduced the expression of A2A receptors, ruling out the possibility that the neuroprotective effect of A2A agonists was caused by A2A receptor desensitization. When A2A antagonists and agonists were centrally injected into the damaged spinal cord, only SCH58261 showed a neuroprotective effect, while CGS21680 was ineffective. Conclusion: Our results indicate that A2A antagonists exert spinal cord injury protection by acting on central A2A receptors. Blocking A2A receptors may reduce excitotoxicity. Conversely, the neuroprotective effect provided by A2A agonists may be mainly attributed to peripheral effects. [2] Objective: To investigate the role of adenosine A2A receptors in 6-hydroxydopamine (6-OHDA)-induced dyskinesia in rats. Methods: To establish a Parkinson's disease experimental model, 6-hydroxydopamine (8 μg/rat) was injected unilaterally into the substantia nigra pars compacta (SNc). After a three-week recovery period, 6-OHDA-induced bradykinesia and balance disturbances were assessed using the balance beam test at 10, 30, and 60 minutes following intraperitoneal injections of the drugs (caffeine and SCH58261). Results: The results showed that 6-OHDA (8 μg/animal, intra-SNc injection) induced dyskinesia in Parkinson's disease and prolonged the completion time of the balance beam test (p<0.001). Intraperitoneal injections of caffeine (30 mg/kg) and SCH58261 (2 mg/kg) shortened beam dwell time (p<0.01 and p<0.001, respectively). We found that acute administration of caffeine and SCH58261 improved 6-OHDA-induced bradykinesia and dyskinesia. Conclusion: Adenosine A2AR antagonists can improve 6-OHDA-induced dyskinesia, and this effect appears to be mediated by inhibition of A2A presynaptic receptors in the substantia nigra pars compacta. [3] Obstructive sleep apnea-hypopnea syndrome (OSAHS) is associated with multiple systemic diseases. Central nervous system complications have been reported to lead to neurocognitive impairment, especially in children with OSAHS. Chronic intermittent hypoxia is considered the main pathophysiological mechanism of OSAHS. Adenosine plays an important role in cellular function through its interaction with its receptor. The A2a receptor has been identified as a factor involved in neuroprotection. However, the role of the adenosine A2a receptor in intermittent hypoxia-induced cell damage has not been fully elucidated. This study aimed to explore the potential mechanism by which A2a receptor-mediated intermittent hypoxia causes damage to PC12 cells. We found that CGS21680 activation of the A2a receptor reduced cell viability and increased the expression of protein kinase C (PKC) as well as the ATP-sensitive potassium channel (KATP) subunits Kir6.2 and SUR1. SCH58261 inhibition of the A2a receptor increased cell viability, inhibited the expression levels of PKC and SUR1, and ultimately exerted a protective effect in PC12 cells. In addition, we observed that the PKC antagonist CHE downregulated the expression of Kir6.2 and SUR1 and increased cell viability. Furthermore, we found that A2a receptor activation-induced cell damage was associated with increased Cleaved-Caspase 3 expression, while inhibition of A2a receptor or PKC reduced Cleaved-Caspase 3 expression. In summary, our results indicate that A2a receptor plays a role in PC12 cell damage induced by intermittent hypoxia exposure by activating PKC to induce KATP expression and by apoptosis mediated through the A2a-PKC-KATP signaling pathway. [5]

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1. Chemical properties: SCH-58261 is a non-xanthine heterocyclic compound with the chemical structure 7-(2-phenylethyl)-5-amino-2-(2-furanyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine. [1]
2. Mechanism of action: [2][4][5]
- In spinal cord injury (SCI): It acts on central A2a receptors, reduces excitotoxicity, inhibits neuroinflammation (reduces TNF-α and Fas-L levels), and inhibits apoptosis signaling (reduces Bax, activates Bcl-2, and inhibits JNK MAPK).
- In astrocytes: It regulates the formation of A1AR-A2aAR heterodimers, upregulates PPARγ transcription by inhibiting the recruitment of the YY1-HDAC1 complex to the PPARγ promoter, thereby increasing EAAT2 expression and glutamate uptake.
- In PC12 cells: It inhibits the A2a-PKC-KATP signaling pathway and reduces intermittent hypoxia-induced apoptosis (reduces Cleaved-Caspase 3). 3. Therapeutic potential: By targeting the pathological processes mediated by A2aAR, it is expected to be used to treat spinal cord injury, Parkinson's disease, and neurocognitive dysfunction associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). [2][3][5] 4. Research value: Due to its high efficiency and selectivity, it can serve as a valuable tool for studying the biological effects of A2aAR. [1]

C18H15N7O

345.36

345.134

C, 62.60; H, 4.38; N, 28.39; O, 4.63

160098-96-4

176408

White to yellow solid powder

1.54g/cm3

1.807

3.14

1

6

4

26

488

0

O1C([H])=C([H])C([H])=C1C1=NN2C(N([H])[H])=NC3=C(C([H])=NN3C([H])([H])C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])C2=N1

UTLPKQYUXOEJIL-UHFFFAOYSA-N

InChI=1S/C18H15N7O/c19-18-22-16-13(11-20-24(16)9-8-12-5-2-1-3-6-12)17-21-15(23-25(17)18)14-7-4-10-26-14/h1-7,10-11H,8-9H2,(H2,19,22)

4-(furan-2-yl)-10-(2-phenylethyl)-3,5,6,8,10,11-hexazatricyclo[7.3.0.02,6]dodeca-1(9),2,4,7,11-pentaen-7-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 2.08 mg/mL (6.02 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 20.8 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: ≥ 2.08 mg/mL (6.02 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 4%DMSO + 40%PEG300 + 4%Tween 80 52%ddH2O: 2.0mg/ml (5.79mM)

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