GluN1/GluN2A (NR1/NR2A)-containing N-methyl-D-aspartate receptors (NMDARs).
Selectivity: Shows >300-fold selectivity for GluN1/GluN2A over GluN1/GluN2B receptors [1].
In functional assays, it is inactive at GluN1/GluN2B and GluN1/GluN2D receptors up to 30 µM [1][2].

Compound 1, TCN 201, has a pIC50 of <4.3 and 6.8, respectively, making it more selective for GluN1/GluN2A NMDAR than GluN1/GluN2B NMDAR [1]. In oocytes, TCN 201 (10 μM) only modestly suppresses GluN1/GluN2B NMDAR-mediated currents [2]. In oocytes, TCN 201 (10-30 μM) exhibits a more powerful antagonistic effect on NMDAR-mediated responses, which is dependent on isoform and glycine [2]. In oocytes, NMDAR-mediated responses are not entirely blocked by TCN 201 (0.1-100 μM) [2]. In rat cortical neurons, TCN 201 (10 μM) antagonistic activity against NMDAR-mediated currents is inversely linked with its ifenprodil sensitivity [2]. In chick retina, cortical spreading depression (CSD) is inhibited by TCN 201 (1-9 μM) [3].

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Recombinant GluN1/GluN2A Receptor Antagonism: At human recombinant NR1/NR2A receptors expressed in U-2 OS cells, TCN-201 (referred to as compound 1) completely inhibited (92-100%) the receptor with a pIC50 of 6.8 (IC50 ≈ 158 nM) in a FLIPR/Ca2+ assay. It showed no activity at NR1/NR2B receptors up to 50 µM (pIC50 <4.3) [1].
- Potency in Electrophysiology: In whole-cell patch-clamp experiments on HEK 293T cells expressing human NR1/NR2A receptors, TCN-201 inhibited the current induced by 30 µM NMDA in the presence of 3 µM glycine with an IC50 of 109 nM (pIC50 = 7.0 ± 0.1) and a Hill slope of 1.4 ± 0.3 [1].
- Mechanism of Action Studies (FLIPR): In U-2 OS cells expressing NR1/NR2A, the inhibitory effect of TCN-201 (and compounds 2, 3, 6, 13) was surmounted by the addition of 1 mM glycine, but not by 1 mM L-glutamate. This indicates a glycine-dependent mechanism [1].
- Antagonism in Recombinant Receptors (Xenopus Oocytes): In TEVC recordings from X. laevis oocytes expressing GluN1/GluN2A receptors, TCN-201 antagonized NMDAR-mediated responses. The degree of block is dependent on the concentration of the GluN1-site co-agonist, glycine or D-serine, but is independent of the glutamate concentration. For example, with 30 µM glutamate and 3 µM glycine, the inhibition was ~82%, while with 30 µM glutamate and 30 µM glycine, the inhibition was ~51% [2].
- IC50 Determination (Xenopus Oocytes): Using TEVC, the IC50 of TCN-201 at GluN1/GluN2A receptors was dependent on the GluN1 agonist concentration. With glutamate fixed at 30 µM, the IC50 values were: 0.446 µM (Hill slope 1.42) for 3 µM glycine; 0.746 µM (Hill slope 1.49) for 10 µM glycine; and 3.89 µM (Hill slope 1.17) for 30 µM glycine. Using D-serine, the IC50 values were: 0.326 µM (Hill slope 1.57) for 3 µM D-serine; 0.816 µM (Hill slope 1.33) for 10 µM D-serine; and 1.92 µM (Hill slope 1.17) for 30 µM D-serine [2].
- Native NMDAR Antagonism (Cortical Neurons): In rat cortical neurons, TCN-201 (10 µM) produced minimal block (5 ± 2%) of NMDA-evoked currents in young cultures (DIV 9-10) where GluN2B expression predominates. In older cultures (DIV 15-18), it produced a greater block (16 ± 3%). In neurons transfected with GluN2A, the block was 47 ± 4%. The extent of TCN 201 block was negatively correlated (R² = 0.91) with the block produced by the GluN2B-selective antagonist, ifenprodil [2].
- Selectivity Profile: TCN-201 showed high specificity in a panel of more than 30 targets, including ion channels (TRPV4, hERG, hNaV1.5), receptors (adenosine, adrenergic, cannabinoid, dopamine, histamine, acetylcholine, serotonin, neurokinin, opioid, vasopressin), enzymes (cyclooxygenase, phosphodiesterase), and transporters (norepinephrine, serotonin) when tested at 10 µM concentration [1].

Rats with CSD exhibit an insufficient response to TCN-201 (10 mg/kg; intraperitoneal injection) regarding the blood oxygenation level-dependent (BOLD) response [4].

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In contrast to in vitro findings, a previous study using BOLD fMRI in rats showed that TCN-201 did not alter CSD propagation features. The discrepancy may be attributed to different recording methods: BOLD fMRI is largely based on brain-specific hemodynamic responses, whereas intrinsic optical signal is based on neuronal activity of chick retina. Whether TCN-201 could alter CSD in vivo using an electrophysiology approach requires further investigation [3].

Radioligand Displacement in Rat Cortical Membranes: To determine the binding site, single concentration (10 µM) displacement binding assays were performed on rat brain cortical membranes. TCN-201 displaced the glutamate site antagonist [3H]CGP 39653 by 36%. It showed low displacement (<25%) of the glycine site antagonist [3H]MDL 105,519 (24%), the pore blocker [3H]TCP (11%), and the NR2B NTD ligand [3H]ifenprodil (16%) [1].
- [3H]CGP 39653 Displacement Curve: In competition binding experiments against [3H]CGP 39653 in rat brain cortical membranes, TCN-201 partially inhibited specific binding with a maximal displacement of 44 ± 3% and a pIC50 of 6.5 ± 0.1 (Hill slope 0.8 ± 0.1, n=5) [1].

FLIPR/Ca2+ Assay in Recombinant U-2 OS Cells: Human osteosarcoma (U-2 OS) cells were transiently transduced with BacMam vectors containing NR1 and NR2A (or NR2B) NMDAR subunits. Cells were seeded in 384-well plates. The next day, they were loaded with the cytoplasmic calcium indicator Fluo-4 AM (2 µM) in an assay buffer containing probenecid (2.5 mM) to prevent dye efflux. After washing to remove ketamine, the cell plate was placed into a FLIPR instrument. Antagonist TCN-201 (or other compounds) was added, and fluorescence (excitation 488 nm, emission 540 nm) was monitored for 5 minutes. The response was normalized to the maximal response evoked by 30 µM (+)-MK-801. For mode-of-action studies, a second addition of 1 mM glycine or L-glutamate was performed 5 minutes after compound addition, and fluorescence was measured for an additional 5 minutes [1].
- FLIPR/Ca2+ Assay in Rat Cortical Neurons: Cortical neurons from embryonic day 18/19 Sprague-Dawley rats were cultured for 15 days. On the assay day, cultures were washed and loaded with Fluo-4 AM (2 µM) and probenecid (2.5 mM). After washing, the cells were transferred to a FLIPRTETRA. To assess antagonist activity, cells were first exposed to different concentrations of test drugs (e.g., TCN-201) for 10 minutes, then to a submaximal concentration of NMDA (EC50 10–20 µM) for 3 minutes. Ca2+ responses were quantified as area-under-the-curve and normalized to the NMDA-evoked response [1].
- Whole-Cell Patch-Clamp Electrophysiology in HEK 293T Cells: Human embryonic kidney (HEK) 293T cells were transiently transfected with human NMDAR subunits (NR1/NR2A, NR1/NR2B, or NR1/NR2D) and an EGFP plasmid for selection. Whole-cell voltage-clamp recordings were performed at a holding potential of -60 mV. Currents were evoked by rapidly exchanging the extracellular solution to one containing NMDA (30 µM) and glycine (3 µM) for 5 seconds every 60 seconds. To test antagonists, TCN-201 was co-applied with the agonists. For concentration-response curves (CRC), increasing concentrations of TCN-201 (0.03 to 3 µM) were applied cumulatively [1].
- Two-Electrode Voltage-Clamp (TEVC) in Xenopus Oocytes: X. laevis oocytes were injected with cRNA for rat GluN1 and GluN2A or GluN2B subunits. TEVC recordings were performed at room temperature at a holding potential of -30 to -40 mV. The bath solution contained BaCl2 and EDTA to chelate Zn2+. For antagonist experiments, TCN-201 was co-applied with glutamate (3-30 µM) and glycine or D-serine (3-30 µM). For Schild analysis, two-point concentration-response curves for glycine were constructed in the absence and presence of increasing concentrations of TCN-201 (0.3, 1, 3, 10 µM) [2].

Male chicks (aged 8-28 days) were used. The posterior eyeball was positioned in a chamber and perfused with Ringer's solution at 0.5 ml/min. Tissue was stabilized for at least 30 minutes before CSD induction at constant temperature of 32°C. Retinal spreading depression (RSD) was induced by ejection of 1 μl of 0.1 μM KCl. Ten repeated CSD episodes were induced with 20-minute intervals for tissue recovery. The retina was illuminated for 25 ms at 1 Hz using a high-power LED spotlight (625 nm peak wavelength). Reflected light was simultaneously recorded with a monochrome camera. Image sequences were taken at 1 Hz over a 3-minute period starting when CSD was elicited [3].
For TCN-201 testing, concentrations of 1, 3, and 9 μM were used. DMSO was used as the vehicle control, with maximum concentration of 0.1% DMSO not affecting CSD in chick retina. Ten CSD episodes were induced per experiment: initial Ringer's control; low concentration of drug or vehicle; medium concentration; high concentration; post-treatment with Ringer's control (drug removal). For each test sequence, the perfusion medium was changed immediately after the end of the 2nd, 4th, 6th, and 8th CSD recordings [3].

[1]. Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors. J Pharmacol Exp Ther. 2010 Dec; 335(3): 636-44.

[2]. TCN 201 selectively blocks GluN2A-containing NMDARs in a GluN1 co-agonist dependent but non-competitive manner. Neuropharmacology. 2012 Sep; 63(3): 441-9.

[3]. NR2A contributes to genesis and propagation of cortical spreading depression in rats. Sci Rep. 2016 Mar 22;6:23576.

[4]. Involvement of NMDA receptor subtypes in cortical spreading depression in rats assessed by fMRI. Neuropharmacology. 2015 Jun; 93:164-70.

Background and Novelty: TCN-201 (originally named compound 1) was identified from a high-throughput screening (HTS) campaign of approximately 2 million compounds. It is a sulfonamide derivative. To the authors' knowledge, it was among the first described compounds with a high degree of selectivity for NR1/NR2A over NR1/NR2B receptors [1].
- Mechanism of Action (Proposed): The mechanism is non-competitive and allosteric. Schild analysis showed a deviation from linearity at higher antagonist concentrations, consistent with an allosteric non-competitive mechanism. It is hypothesized to bind to a site near both glutamate and glycine binding sites, potentially at the dimer interface between the GluN1 and GluN2 agonist binding domains, leading to an acceleration of glycine/D-serine unbinding from the GluN1 subunit. The inhibition is surmountable by glycine but not by glutamate [1][2].
- Potency Comparison: TCN-201 is approximately 30-times more potent than the related compound TCN-213 (also known as compound 13). The pA2 value for TCN-201 is 7.15, compared to 5.69 for TCN-213 [2].
- Experimental Limitations: The utility of TCN-201 is limited by two factors: 1) its ability to block GluN2A-containing NMDARs is strongly dependent on the concentration of the GluN1-site agonist (glycine or D-serine), and it will not produce complete block at saturating concentrations of these agonists. 2) It possesses low solubility in physiological salt solutions, limiting the concentrations that can be tested [2].

C21H17N3O4FSCL

461.89378

461.061

852918-02-6

852918-02-6;

4787937

White to off-white solid powder

1.4±0.1 g/cm3

1.625

4.13

3

6

6

31

719

0

FYIBXBFDXNPBSF-UHFFFAOYSA-N

InChI=1S/C21H17ClFN3O4S/c22-18-12-17(10-11-19(18)23)31(29,30)24-13-14-6-8-16(9-7-14)21(28)26-25-20(27)15-4-2-1-3-5-15/h1-12,24H,13H2,(H,25,27)(H,26,28)

N-[[4-(benzamidocarbamoyl)phenyl]methyl]-3-chloro-4-fluorobenzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO : ~250 mg/mL (~541.25 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.50 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 (4.50 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 20.8 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: ≥ 2.08 mg/mL (4.50 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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 (Please use freshly prepared in vivo formulations for optimal results.)