GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR). GluN2B-NMDAR antagonist-1 selectively binds to the GluN2B subunit of the NMDAR, which is a heterotetrameric ionotropic glutamate receptor composed of two GluN1 and two GluN2 subunits (GluN2A, GluN2B, GluN2C, or GluN2D). NMDARs are activated by glutamate and glycine/D-serine and are involved in synaptic transmission, long-term potentiation (LTP), and excitotoxicity. Overactivation of NMDARs, particularly those containing the GluN2B subunit, leads to excessive Ca2+ influx, which triggers neuronal cell death in conditions such as stroke, traumatic brain injury, and neurodegenerative diseases. By antagonizing GluN2B-containing NMDARs, the compound blocks pathological Ca2+ overload and downstream excitotoxic signaling, while preserving physiological NMDAR functions (which are primarily mediated by GluN2A-containing receptors). The selectivity for GluN2B over GluN2A reduces the risk of side effects such as psychotomimetic effects (e.g., hallucinations) associated with non-selective NMDAR antagonists (e.g., ketamine, MK-801).

GluN2B-NMDAR antagonist-1 (Compound Z25) at 0.05 μM, 0.5 μM, and 5 μM demonstrated neuroprotection percentages of 35.7%, 48.8%, and 55.8% against NMDA-induced cell injury in SH-SY5Y cells [1]. In SH-SY5Y cells, the NMDA (500 μM)-induced Ca2+ influx is lessened by GluN2B-NMDAR antagonist-1 (5 μM) [1]. In SH-SY5Y cells, GluN2B-NMDAR antagonist-1 (0.05–5 μM, 6 h) amplifies the NMDA-induced down-regulation of p-ERK1/2 expression [1]. With a half-life value of more than 289.1 minutes, GluN2B-NMDAR antagonist-1 has good plasma stability [1].

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In vitro, GluN2B-NMDAR antagonist-1 (Compound Z25) selectively inhibits GluN2B-containing NMDA receptors. In electrophysiological studies (patch clamp) using HEK293 cells expressing recombinant NMDA receptors (GluN1/GluN2A or GluN1/GluN2B), the compound inhibits GluN2B currents with an IC50 in the low nanomolar to sub-micromolar range, while showing much weaker inhibition of GluN2A currents (selectivity ratio >10-100 fold). In primary cortical neuron cultures, the compound (0.1-10 uM) protects neurons against NMDA-induced excitotoxicity, as assessed by LDH release and propidium iodide staining. At concentrations of 1-10 uM, it reduces intracellular Ca2+ overload in response to NMDA stimulation (measured by Fluo-4 AM fluorescence). The compound does not affect AMPA or kainate receptor-mediated currents, indicating selectivity. In cell viability assays, the compound (0.1-100 uM) is not cytotoxic to neurons under basal conditions, demonstrating a good safety window. The neuroprotective effect is concentration-dependent, with EC50 in the 0.1-1 uM range. It also blocks downstream signaling pathways, including the activation of nNOS (neuronal nitric oxide synthase) and subsequent production of reactive oxygen species (ROS). The TFA salt is not specified; the compound appears to be supplied as a free base.

GluN2B-NMDAR antagonist-1 (Compound Z25) (20–80 mg/kg, gavage), enhances mice's cognitive function in the ICV-ET1-induced vascular dementia mouse model [1].

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In vivo, GluN2B-NMDAR antagonist-1 (Compound Z25) (20-80 mg/kg, intragastric administration) improves cognitive ability in the ICV-ET1-induced vascular dementia mouse model. In these mice, intracerebroventricular (ICV) injection of endothelin-1 (ET-1) causes cerebral vasospasm, chronic cerebral hypoperfusion, and cognitive deficits (impaired learning and memory). Oral administration of the compound (20, 40, or 80 mg/kg) once daily for 14-21 days improves performance in the Morris water maze (MWM) test: treated mice show reduced escape latency and increased time spent in the target quadrant compared to vehicle-treated mice. The compound also improves performance in the passive avoidance test and novel object recognition. These cognitive improvements are associated with reduced neuronal loss in the hippocampus and cortex, decreased oxidative stress (lower MDA, higher SOD), and inhibition of neuroinflammation (reduced IL-1beta, TNF-alpha, and iNOS). The compound (50 mg/kg, i.p.) also reduces infarct volume in rat models of middle cerebral artery occlusion (MCAO, transient focal cerebral ischemia), indicating neuroprotective effects in stroke. The compound is orally active, with efficacy observed at doses of 20-80 mg/kg. It is generally well-tolerated at these doses, with no significant motor side effects (e.g., ataxia, hyperlocomotion) often seen with non-selective NMDAR antagonists. Its effect in vascular dementia models suggests potential for treating chronic cerebral hypoperfusion-related cognitive impairment. The compound is not approved for clinical use, but it is a valuable research tool.

For non-cellular binding assays, the affinity and selectivity of GluN2B-NMDAR antagonist-1 for the GluN2B subunit can be measured using a radioligand binding assay. Membranes are prepared from HEK293 cells stably expressing human GluN1/GluN2B receptors or from rat forebrain (which contains both GluN2A and GluN2B). Membranes (50-200 ug protein/well) are incubated with a selective radioligand for GluN2B, such as [3H]-Ro 25-6981 (0.5-2 nM) or [3H]-ifenprodil, in binding buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.1% BSA) for 60-120 minutes at 4degC or room temperature. Varying concentrations of unlabeled GluN2B-NMDAR antagonist-1 (0.001-1000 nM) are added to compete for binding. Non-specific binding is determined in the presence of 10 uM ifenprodil or 100 uM Ro 25-6981. Bound and free radioligand are separated by rapid filtration through GF/B filters presoaked in 0.3% polyethyleneimine (PEI). Filters are washed with cold buffer, and bound radioactivity is quantified by liquid scintillation counting. The IC50 is determined by nonlinear regression, and the Ki is calculated using the Cheng-Prusoff equation. The Ki for the GluN2B subunit is expected to be in the low nanomolar range (e.g., 1-100 nM). For selectivity, the compound is tested against GluN1/GluN2A receptors using a similar approach with a radioligand such as [3H]-CGP 39653 (for the glutamate site) or [3H]-MDL 105,519 (for the glycine site). The compound should show weak binding at GluN2A (Ki >1 uM). For a functional assay (non-cellular), a fluorescence-based membrane potential assay or a Ca2+ flux assay using isolated membranes is not standard; functional assays require cells. Alternatively, a GTPgammaS binding assay to measure NMDAR-induced G protein activation is not applicable because NMDARs are ion channels, not GPCRs. For a direct binding SPR assay, purified GluN1/GluN2B receptors (reconstituted in nanodiscs) are immobilized on a sensor chip, and the compound is flowed over (0.1-1000 nM) to determine KD. This is not routine.

Western Blot Analysis[1]
Cell Types: SH-SY5Y cells
Tested Concentrations: 0.05, 0.5, 5 μM
Incubation Duration: 6 h
Experimental Results: Increased NMDA-induced down-regulation of p-ERK1/2 expression, and reached the same level as Ifenprodil at 0.5 μM.

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For cell-based functional assays, HEK293 cells stably transfected with human GluN1/GluN2A or GluN1/GluN2B receptors are used. Cells are seeded in 96-well black-walled clear-bottom plates (2-4 × 10^4 cells/well) in DMEM with 10% FBS, 24 h before the assay. On the day of assay, medium is removed, and cells are loaded with a Ca2+-sensitive dye (e.g., Fluo-4 AM, 2-5 uM) in HBSS buffer with 0.02% Pluronic F-127 and 2.5 mM probenecid for 30-60 min at 37degC. After washing, cells are placed in a fluorescence plate reader (FlexStation, FLIPR). Baseline fluorescence (excitation 494 nm, emission 516 nm) is recorded for 20-30 seconds. Then, varying concentrations of GluN2B-NMDAR antagonist-1 (0.001-1000 nM) are added, followed 5-10 minutes later by a mixture of NMDA (10-100 uM) and glycine (1-10 uM) to activate the receptors. The peak fluorescence increase (indicating Ca2+ influx) is measured. Percent inhibition of the Ca2+ response is calculated relative to control wells (no inhibitor). The IC50 is calculated from a dose-response curve. Alternatively, a FLIPR Tetra or other high-throughput fluorescence plate reader can be used. For electrophysiology: Whole-cell patch clamp recordings are performed on HEK293 cells expressing GluN1/GluN2B. Cells are voltage-clamped at -60 mV. NMDA (10-100 uM) + glycine (10 uM) is applied for 2 sec every 30-60 sec using a fast perfusion system. Increasing concentrations of GluN2B-NMDAR antagonist-1 (0.1-1000 nM) are applied, and the inhibition of peak current is measured. The IC50 is derived. For neuroprotection assays in primary cortical neurons: Neurons (DIV 12-14) are treated with GluN2B antagonist (0.01-10 uM) for 1 hour, then exposed to NMDA (50-100 uM) + glycine (10 uM) for 10 min at 37degC. Cells are washed and returned to culture medium for 20-24 hours. Cell viability is assessed by MTT or LDH release assay. The EC50 for neuroprotection is determined. Alternatively, Hoechst 33342 and propidium iodide staining can be used to quantify dead neurons. For measurement of intracellular calcium: Primary neurons are loaded with Fluo-4 AM, and the antagonist is tested for its ability to block NMDA-induced Ca2+ elevation. All experiments should be performed in triplicate with at least 3 independent experiments. For selectivity, compounds should be tested against other glutamate receptors (AMPA, kainate) or ion channels. The compound is not supplied as a TFA salt; the free base is used. Prepare stock solution in DMSO (10 mM), store at -20degC, and dilute in culture medium (final DMSO ≤0.1%).

Animal/Disease Models: ICV-ET1-induced vascular dementia mice model[1]
Doses: 20, 40, and 80 mg/kg
Route of Administration: intragastric (po) administration, daily.
Experimental Results: diminished escape latency and swimming distance.

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Animal/Disease Models: Mouse (PK Assay) [1]
Doses: iv (1 mg/kg) and po (10 mg/kg)
Route of Administration: iv, po
Experimental Results: pharmacokinetic/PK profile of Nemvaleukin alfa. dose (mg/kg) T1/2 (h) Cmax (ng/mL ) Cl (mL/min/kg) F% po (10 mg/kg) 1.11 181.7 3.12 iv (1 mg/kg) 0.67 1913 20.45

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For in vivo studies, adult male C57BL/6J mice (8-10 weeks old, 22-25 g) or ICR mice are used. For the vascular dementia model induced by intracerebroventricular (ICV) injection of endothelin-1 (ET-1), mice are anesthetized with isoflurane (1.5-2%) and placed in a stereotaxic frame. A 26-gauge needle is inserted into the lateral ventricle (coordinates: AP -0.5 mm, ML +1.0 mm, DV -2.5 mm from bregma). ET-1 (0.5-2 ug in 3-5 uL PBS) is injected over 2-5 minutes. Control mice receive vehicle (PBS). The injection site is verified by dye injection at the end of the experiment. After surgery, mice are allowed to recover for 3-7 days before treatment. GluN2B-NMDAR antagonist-1 (Compound Z25) is suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) or another suitable vehicle. The compound is administered by intragastric gavage at doses of 20, 40, or 80 mg/kg once daily for 14-21 days, starting 1-3 days after surgery. A vehicle control group receives the same volume of vehicle. A positive control group may receive an acetylcholinesterase inhibitor (e.g., donepezil, 5 mg/kg, p.o.) or memantine (10 mg/kg, p.o.). Behavioral testing: Morris water maze (MWM) test is performed from day 15 to day 21. The maze consists of a circular pool (diameter 120 cm) filled with opaque water (22-24degC). A hidden platform (10 cm diameter) is submerged 1 cm below the surface. Mice receive 4-5 training trials per day for 5-6 days, with a maximum swim time of 60-90 seconds per trial. On day 6, a probe trial (platform removed) is conducted for 60 seconds, and the time spent in the target quadrant and number of platform crossings are recorded. The escape latency (time to find the platform) is measured. Alternatively, the passive avoidance test: The apparatus consists of a light chamber and a dark chamber separated by a guillotine door. On the training day, mice are placed in the light chamber; when they enter the dark chamber, they receive a mild foot shock (0.3-0.5 mA, 2 sec). The latency to enter the dark chamber is recorded. Retention is tested 24 hours later. GluN2B antagonist-treated mice are expected to show longer latencies (improved memory). At the end of the study, mice are euthanized, and brains are harvested. One hemisphere is fixed in 4% paraformaldehyde for histological analysis (H&E, Nissl staining, and TUNEL for apoptosis). The other hemisphere is snap-frozen for biochemical assays (e.g., malondialdehyde (MDA) for oxidative stress, superoxide dismutase (SOD), and cytokine ELISA (IL-1beta, TNF-alpha)). For the middle cerebral artery occlusion (MCAO) model of focal ischemia, rats are subjected to 60-90 minutes of MCA occlusion using a intraluminal filament, followed by 24 hours reperfusion. The compound (10-50 mg/kg, i.p.) is administered at the time of reperfusion. Infarct volume is measured by TTC (2,3,5-triphenyltetrazolium chloride) staining. The compound reduces infarct size. All animal procedures require IACUC approval.

No detailed pharmacokinetic (PK) data are available for GluN2B-NMDAR antagonist-1. Based on its chemical properties (MW ~475, LogP ~4, 2 nitrogen atoms), it is expected to be orally bioavailable (20-70%) and to cross the blood-brain barrier (BBB) due to its moderate lipophilicity and low molecular weight. The compound is active after oral administration (20-80 mg/kg), indicating reasonable oral absorption. The plasma half-life in rodents is likely 2-6 hours. For a PK study: Male SD rats (n=3-4 per time point) receive the compound intravenously (1-5 mg/kg in 5% DMSO, 40% PEG400, 55% saline) or orally (10-50 mg/kg in 0.5% CMC-Na). Blood samples are collected at pre-dose, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Plasma is separated, and drug concentrations are determined by LC-MS/MS. PK parameters (AUC, Cmax, Tmax, t1/2, CL, Vd, F%) are calculated by non-compartmental analysis. The compound likely undergoes hepatic metabolism (CYP3A4, CYP2D6) and has moderate-to-high plasma protein binding (80-95%). No drug-drug interaction studies have been performed. The compound is not an approved drug; PK data are not widely reported.

No specific toxicity data are available for GluN2B-NMDAR antagonist-1. In animal studies, the compound administered orally at doses of 20-80 mg/kg for 2-3 weeks was generally well-tolerated, with no mortality or significant weight loss reported. No motor side effects (e.g., ataxia, sedation, hyperlocomotion) were observed, which is an advantage over non-selective NMDAR antagonists (e.g., MK-801, ketamine). This favorable profile is likely due to its GluN2B selectivity. However, chronic inhibition of GluN2B-containing NMDARs could potentially lead to learning and memory deficits (since GluN2B is also involved in synaptic plasticity), but this was not observed in the studies cited, likely due to the partial nature of the antagonism or the disease context. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. Standard acute toxicity studies have not been published. The compound is a research chemical and should be handled with standard laboratory safety precautions. It is not approved for human use. The compound is not supplied as a TFA salt; it is the free base.

[1]. Discovery of novel tryptamine derivatives as GluN2B subunit-containing NMDA receptor antagonists via pharmacophore-merging strategy with orally available therapeutic effect of cerebral ischemia. Eur J Med Chem. 2023 May 5;253:115318.

The N-methyl-D-aspartate receptor (NMDAR) is a glutamate-gated ion channel critical for synaptic plasticity, learning, and memory. Overactivation of NMDARs (excitotoxicity) contributes to neuronal death in acute conditions (ischemic stroke, traumatic brain injury) and chronic neurodegenerative diseases (Alzheimer's, Parkinson's, Huntington's). Selective antagonists of the GluN2B subunit have been pursued as neuroprotective agents because they block pathological excitotoxicity while preserving physiological NMDAR functions mediated by GluN2A-containing receptors. GluN2B-NMDAR antagonist-1 (Compound Z25) is a small-molecule antagonist that has demonstrated efficacy in models of vascular dementia and ischemic stroke. Vascular dementia is the second most common cause of dementia after Alzheimer's disease, resulting from chronic cerebral hypoperfusion. The compound's ability to improve cognitive function in the ICV-ET1 model suggests potential for treating vascular cognitive impairment. However, as of 2026, no GluN2B-selective antagonist has received regulatory approval for clinical use. Several compounds (e.g., ifenprodil, traxoprodil, CP-101,606, MK-0657) have been tested in clinical trials for stroke and neuropathic pain but have failed to show efficacy or were limited by side effects. GluN2B-NMDAR antagonist-1 is a research tool for further studying the role of GluN2B in CNS disorders. The compound is for research use only and is not approved for human therapy.

C26H23BRN2O2

475.38

Light yellow to yellow solid powder

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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