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| Targets |
The primary molecular target of (RS)-AMPA monohydrate is the AMPA subtype of ionotropic glutamate receptors (AMPARs), which it activates as a potent agonist without interfering with binding sites for kainic acid or NMDA receptors.
AMPA receptors (GluA1, GluA2, GluA3, GluA4, also called GluR1‑4). (RS)-AMPA monohydrate is a potent and selective agonist of AMPA receptors, which are a subclass of ionotropic glutamate receptors. It does not activate kainate or NMDA receptors. By binding to the agonist binding site, it opens the receptor channel, allowing Na+ influx and depolarization of the postsynaptic membrane. |
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| ln Vitro |
(RS)-AMPA monohydrate (10-3-10-4 M) causes the brainstem and spinal cord of cultivated rats to become depolarized. Although the effects of (RS)-AMPA monohydrate on depolarization differ greatly amongst neurons, it appears that the effects are dose-dependent. Applying (RS)-AMPA monohydrate at 10-5 M resulted in a very little depolarization (3–7 mV), however at 10–4 M, the depolarization's magnitude varied from 4–33 mV. The rate at which spontaneously firing neurons fire is also increased by (RS)-AMPA monohydrate, and quiet cells occasionally have short action potential bursts. Without impacting NMDA receptors, (RS)-AMPA monohydrate depolarizes the body by activating glutamate/quilamate receptors [1].
In cultured rat spinal and brainstem neurons, (RS)-AMPA monohydrate (10⁻⁵-10⁻⁴ M) induces dose-dependent depolarizations (3-7 mV at 10⁻⁵ M; 4-33 mV at 10⁻⁴ M), increases discharge rates of spontaneously firing neurons, and evokes short action potential bursts in silent cells by activating glutamate/quisqualate receptors without affecting NMDA receptors. In vitro, (RS)-AMPA monohydrate (10-⁴ to 10-3 M) leads to depolarization of cultured rat spinal and brainstem neurons. The depolarizing effect is dose‑dependent and is blocked by selective AMPA receptor antagonists (e.g., NBQX or CNQX). It does not interfere with the binding of kainic acid or NMDA to their respective receptors. In brain slice preparations, (RS)-AMPA induces fast excitatory postsynaptic currents (EPSCs) that are blocked by GYKI 52466 (a non‑competitive AMPA antagonist). |
| ln Vivo |
In vivo, (RS)-AMPA monohydrate is administered intracerebroventricularly (ICV) or by local injection into brain regions (e.g., hippocampus, striatum) to induce seizures, excitotoxicity, or to study synaptic transmission. It has been used to model epilepsy (AMPA‑induced seizures) and neuronal damage. The compound is not used therapeutically.
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| Enzyme Assay |
Binding assays for (RS)-AMPA monohydrate are typically performed using radiolabeled [³H]AMPA with rat brain membrane preparations or recombinant AMPA receptors; a standard saturation binding protocol involves incubating membranes with varying concentrations of [³H]AMPA (e.g., 5-100 nM) at 0-4°C for 60 minutes, with non-specific binding determined in the presence of excess unlabeled AMPA or L-glutamate.
For standard cell‑free AMPA receptor binding assays, rat brain synaptic membranes (cortex, hippocampus) are prepared. Membranes (200‑300 ug protein) are washed extensively to remove endogenous glutamate. Membranes are incubated with 5‑10 nM [3H]AMPA (specific activity 30‑60 Ci/mmol) and varying concentrations of unlabeled (RS)-AMPA (0.01‑1000 uM) in 50 mM Tris‑HCl buffer (pH 7.2) containing 100 mM KSCN (to enhance binding) for 30‑60 min at 4degC. Non‑specific binding is determined in the presence of 1 mM L‑glutamate. Bound radioligand is separated by rapid filtration through GF/B filters. The IC50 is calculated, and the Ki for the racemate is about 0.1‑1 uM. For selectivity, parallel assays are performed with [3H]kainate or [3H]MK‑801 (NMDA). |
| Cell Assay |
Cellular assays for (RS)-AMPA monohydrate are conducted using primary neuronal cultures; a representative protocol involves dissociating embryonic rat brainstem cells (E14), culturing them for 7-10 days, then treating with (RS)-AMPA monohydrate at concentrations ranging from 10⁻⁵ to 10⁻⁴ M for 3 days at different developmental stages, followed by cell survival assessment via counting gamma-enolase-positive neurons.
For cellular assays, primary rat cortical or hippocampal neurons (DIV 10‑14) are seeded in 96‑well plates (50,000 cells/well) in Neurobasal/B27 medium. For calcium imaging, neurons are loaded with Fluo‑4 AM (2.5 uM) in HBSS/HEPES for 60 min at 37degC. Cells are washed and then stimulated with (RS)-AMPA (0.1‑1000 uM) in the presence or absence of cyclothiazide (100 uM, to block desensitization). Fluorescence is measured. The EC50 for AMPA‑induced calcium influx is approximately 10‑50 uM. For electrophysiology (whole‑cell patch‑clamp), neurons or HEK‑293 cells expressing AMPA receptors are voltage‑clamped at -70 mV. (RS)-AMPA (0.1‑1000 uM) is applied, and the inward current is recorded. The EC50 is 10‑100 uM. For viability assays (excitotoxicity), neurons are exposed to (RS)-AMPA (100‑500 uM) for 15‑30 min at 37degC, then returned to conditioned medium for 24 h. Viability is measured by LDH release. (RS)-AMPA causes concentration‑dependent excitotoxicity. |
| Animal Protocol |
In vivo studies are performed in male C57BL/6 mice (20‑30 g) or Sprague‑Dawley rats (200‑300 g). (RS)-AMPA monohydrate is dissolved in sterile saline or artificial CSF (pH 7.4) and administered by intracerebroventricular (ICV) injection (0.1‑10 ug/animal in 5‑10 uL) or by intrastriatal injection (1‑10 ug in 1‑2 uL). For seizure studies, the compound is injected ICV, and animals are observed for 30‑60 min. Seizure severity is scored on the Racine scale (1‑5). The latency to first myoclonic jerk, clonic seizure, and tonic‑clonic seizure is recorded. For excitotoxicity studies, AMPA (2‑5 ug) is injected directly into the striatum of rats, and at 3‑7 days post‑injection, the lesion volume is measured by Nissl or TUNEL staining. For PK/PD studies, blood and brain samples are collected at multiple time points after ICV injection, and AMPA levels are measured by HPLC or LC‑MS/MS. (RS)-AMPA is rapidly cleared from the CNS (half‑life ~30‑60 min). For reversal studies, the selective AMPA antagonist NBQX (10‑30 mg/kg IP) is given 30 min before AMPA to confirm receptor specificity.
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| ADME/Pharmacokinetics |
(RS)-AMPA monohydrate (MW 204.18, C7H10N2O4·H2O) is a zwitterionic molecule. It is soluble in water (10 mg/mL). As a polar amino acid derivative, (RS)-AMPA does not readily cross the BBB. For CNS studies, it is administered directly into the brain (ICV or intraparenchymal). After ICV injection, the compound is distributed throughout the CSF and the brain via diffusion. The half‑life in the brain is short (30‑60 min) due to diffusion and clearance into the blood. (RS)-AMPA is not metabolized significantly; it is excreted unchanged in urine. Storage: at 4degC, protected from light.
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| Toxicity/Toxicokinetics |
(RS)-AMPA is a research chemical and is not used clinically. At ICV doses of 0.1‑10 ug in mice, it is well‑tolerated, but at higher doses (>10 ug), it induces severe seizures and mortality. When injected into the brain, it causes excitotoxic neuronal death. The compound is not cytotoxic in cell culture at concentrations <100 uM. Standard safety precautions for neuroactive substances should be followed.
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| References |
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| Additional Infomation |
(RS)-AMPA monohydrate (CAS 76463-67-7) is a potent and selective agonist of AMPA receptors. It is used as a research tool to study fast excitatory neurotransmission, long‑term potentiation (LTP), long‑term depression (LTD), excitotoxicity, and epilepsy. The (S)‑enantiomer (L‑AMPA) is the active isomer. The compound has no clinical indications and is not FDA‑approved. Storage: 2‑8degC, desiccated.
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| Molecular Formula |
C7H12N2O5
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| Molecular Weight |
204.180582046509
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| Exact Mass |
204.074
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| CAS # |
76463-67-7
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| Related CAS # |
(S)-AMPA;83643-88-3;(RS)-AMPA;77521-29-0;(RS)-AMPA hydrobromide;171259-81-7
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| PubChem CID |
53393722
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
14
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| Complexity |
284
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C(C)=C(C(N1)=O)CC(C(=O)O)N.O
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| InChi Key |
HFISYNCCKQHIAM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C7H10N2O4.H2O/c1-3-4(6(10)9-13-3)2-5(8)7(11)12;/h5H,2,8H2,1H3,(H,9,10)(H,11,12);1H2
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| Chemical Name |
2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid;hydrate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O: 5 mg/mL (24.49 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 7.14 mg/mL (34.97 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.8976 mL | 24.4882 mL | 48.9764 mL | |
| 5 mM | 0.9795 mL | 4.8976 mL | 9.7953 mL | |
| 10 mM | 0.4898 mL | 2.4488 mL | 4.8976 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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