Target: AMPA-type glutamate receptors [2]
Subunit-specific affinity: High affinity for GluR1 (Kd ~3-4 nM) and GluR2 (Kd ~7-12 nM) subunits; Lower affinity for GluR3 (Kd ~150-800 nM) and GluR4 (Kd ~150-800 nM) subunits. [2]
AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor, a subtype of ionotropic glutamate receptor. (S)-5-Fluorowillardiine acts as an agonist. [1][2][3]
Binding Affinity (KD) for Recombinant Homomeric Receptors (at 0°C): GluR1 flop: 2.9 ± 0.17 nM; GluR1 flip: 4.6 ± 0.54 nM; GluR2 flop: 8.4 ± 0.77 nM; GluR2 flip: 19 ± 2 nM (with a low-affinity component of 242 nM, 73%); GluR3 flop: 200 ± 31 nM; GluR3 flip: 178 ± 16 nM; GluR4 flop: 164 ± 29 nM; GluR4 flip: 245 ± 96 nM (with a low-affinity component of 99 nM, 88%). [2]
Binding Affinity (KD) for Native Rat Brain Receptors (at 25°C): High-affinity component: 22 ± 8% of sites, KD approx. 22 nM; Low-affinity component (dominant, ~92% of sites): KD approx. 964 nM. [2]
Binding Affinity (KD) for Solubilized Rat Brain Receptors: Complex, with multiple components showing affinities between 2 and 650 nM, reflecting the mixture of GluR1-4 subunits. [2]

- Binding Characteristics: In rat brain membranes, [³H](S)-5-fluorowillardiine binding revealed two distinct affinity components (high: ~20 nM, ~8% of sites; low: ~1 µM, ~92% of sites). Truncation of the C-terminus or solubilization of the receptors altered the binding profile, indicating the influence of the membrane environment. [2]
- Modulation of Binding by Allosteric Modulators: In the presence of KSCN (50 mM), the low-affinity component of [³H]FW binding increased >4-fold, but two binding components persisted. The AMPA receptor positive modulators (e.g., CX614, D1, LY392098) increased [³H]FW binding (e.g., D1 increased binding by 234±13% in the absence of KSCN, EC50 17 µM). The negative modulator cyclothiazide (CTZ) decreased binding (maximum reduction ~60%). The non-competitive antagonist GYKI 53655 caused a small increase in binding (~10%). [1][2]
- Subunit Selectivity: [³H]FW exhibited high affinity for GluR1 and GluR2 (KD 3-19 nM) but much lower affinity (by 20-100 fold) for GluR3 and GluR4 (KD 160-600 nM), indicating a large preference for receptors containing GluR1 and GluR2 subunits. [2]
- Excitotoxicity in NSC-34 Cells: In mouse neuroblastoma x spinal cord (NSC-34) cells, (S)-5-fluorowillardiine induced a dose-dependent decrease in cell viability, with maximal ~50% cell death at 500 µM after 72 hours. The rank order of potency for excitotoxins was (S)-AMPA > (S)-5-FW > L-Glutamate. The excitotoxic effect was blocked by the AMPA receptor antagonist CNQX (10 µM restored viability to ~85% of control). [3]
- Mechanism of Action: FW acts as a relatively specific AMPA receptor agonist to induce Ca²⁺ influx, leading to excitotoxic cell death in motor neuron-like NSC-34 cells. [3]

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The binding of [³H]Fluorowillardine to rat brain membranes exhibited high- and low-affinity components, with Kd values of approximately 20 nM and 1 μM, respectively, and low-affinity sites accounted for about 90% of all binding sites. [2]
When used to displace the antagonist [³H]CNQX binding to rat brain membranes, unlabeled Fluorowillardine displayed two affinity components with Kd values of 32 nM and 1.5 μM. [2]
Binding studies with solubilized receptors from rat brain indicated multiple affinity components for [³H]Fluorowillardine, with Kd values between 2 and 650 nM. [2]
The binding of [³H]Fluorowillardine to brain membranes is greatly skewed in favor of GluR1 and GluR2 subunits due to its subunit preference. [2]

(S)-(-)-5-Fluorowillardiine is identified as an excitotoxic neurotoxin when used in vivo, which consequently limits its application in intact animal studies . Because of this toxicity, it is rarely used in living animal models for therapeutic or functional studies. Its primary value lies in its application as a precise pharmacological probe to selectively stimulate AMPA receptors in isolated tissue preparations or in vitro systems . As a discovery agent, it remains an investigative tool rather than a therapeutic candidate .

Binding assays were performed to characterize the interaction of [³H]Fluorowillardine with AMPA receptors. For saturation binding, rat brain membranes were incubated with varying concentrations of [³H]Fluorowillardine (e.g., 3–2000 nM) for 60 minutes at 25°C (or 0°C). Incubations were terminated by centrifugation. The membrane pellets were rinsed, dissolved, and radioactivity was measured by scintillation counting. Nonspecific binding was determined in the presence of 5 mM L-glutamate. Data were analyzed using nonlinear regression to determine Kd and Bmax values. [2]
For competition binding experiments, the displacement of [³H]CNQX (40 nM) by unlabeled Fluorowillardine was measured in rat brain membranes at 25°C. Membranes were incubated with [³H]CNQX and varying concentrations of Fluorowillardine. Binding was terminated by centrifugation, and samples were processed as above. Displacement curves were fitted to determine IC50 values, which were corrected using the Cheng-Prusoff equation to estimate Kd values. [2]
Studies on recombinant homomeric AMPA receptors (GluR1-4) expressed in HEK293 cells were conducted. Permeabilized cells were incubated with [³H]Fluorowillardine at 0°C. Binding was terminated by filtration through glass fiber filters, followed by rapid washing with chilled buffer containing thiocyanate to minimize dissociation. Nonspecific binding was defined with 5 mM glutamate. [2]

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Binding assays for (S)-(-)-5-Fluorowillardiine are typically performed using rat brain membrane preparations or recombinant homomeric AMPA receptors expressed in HEK293 cells . A standard saturation binding protocol involves incubating membranes with varying concentrations of [³H]Fluorowillardiine (e.g., 3–2000 nM) for 60 minutes at 25°C (or at 0°C) . Incubations are terminated by centrifugation, followed by rinsing of membrane pellets. The pellets are then dissolved, and radioactivity is measured by scintillation counting . Nonspecific binding is determined in the presence of 5 mM L-glutamate. Data analysis is performed using nonlinear regression to determine Kd and Bmax values . For competition binding experiments, displacement of [³H]CNQX (40 nM) by unlabeled Fluorowillardiine is measured under similar conditions .

- Excitotoxicity Assay in NSC-34 Cells: Cells were plated in 96-well plates (1x10⁴ cells/well) and differentiated for 24 hours. For the assay, culture medium was replaced with DMEM without FCS to eliminate Ca²⁺ buffering. (S)-5-Fluorowillardiine was serially diluted in this medium and applied to wells. After a 72-hour incubation, cell viability was assessed using an MTT reduction assay. A dose-response study determined that 500 µM FW caused approximately 50% cell death, which was used as the "set point" for subsequent experiments. [3]
- Antagonist Rescue Assay: NSC-34 cells were co-treated with (S)-5-Fluorowillardiine (500 µM) and serial dilutions of the AMPA receptor antagonist CNQX (2.5, 5, and 10 µM). After 72 hours, cell viability was measured by MTT assay. CNQX at 10 µM significantly protected cells, restoring viability to approximately 85% of control levels. [3]
- NSC-34 Cell Line Characterization: Western blot analysis confirmed that NSC-34 cells express neuronal markers (β-tubulin III, NeuN, NF150) and motor neuron markers (ChAT, p75, nAChR), as well as all four AMPA receptor subunits (GluR1-4). The level of GluR2 was lower compared to other subunits. [3]

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The literature [2] does not describe cell-based functional assays (e.g., viability, signaling) using (S)-(-)-5-Fluorowillardine as a modulator. The compound was used solely as a radioligand in binding assays with cell membrane preparations.

No standardized in vivo animal treatment protocol for (S)-(-)-5-Fluorowillardiine is available in the published literature. Due to its identified excitotoxic neurotoxicity, this compound is rarely, if ever, used in intact animal models for therapeutic or pharmacodynamic evaluation . Its usage is virtually exclusive to in vitro preparations designed to study AMPA receptor function in isolated systems, such as brain slices or primary neuronal cultures. Consequently, no analgesic, anti-cancer, or other therapeutic efficacy data from animal models is available for this compound.

Specific pharmacokinetic parameters, such as half-life, volume of distribution, clearance, and bioavailability, have not been characterized or reported for (S)-(-)-5-Fluorowillardiine. As a research tool compound and not a therapeutic candidate, detailed ADME (Absorption, Distribution, Metabolism, Excretion) profiling has not been performed. Its physicochemical properties have been computationally predicted: LogP value is -1.54, and LogD (pH 7.4) is -4.26, indicating high hydrophilicity . The polar surface area is 113 Ų . The compound has a molecular weight of 217.15 g/mol and follows Lipinski's Rule of 5 with one violation .

In the context of the documented research, (S)-5-Fluorowillardiine is used as a tool to induce excitotoxic cell death in vitro. In NSC-34 cells, a 72-hour exposure to 500 µM FW resulted in approximately 50% cell death, demonstrating its neurotoxic potential via AMPA receptor overstimulation. No in vivo toxicity data (e.g., LD50) are provided. [3]

[1]. Effects of thiocyanate and AMPA receptor ligands on (S)-5-fluorowillardiine, (S)-AMPA and (R,S)-AMPA binding. Eur J Pharmacol. 1997 Jun 25;329(2-3):213-21.

[2]. Use of [3H]fluorowillardiine to study properties of AMPA receptor allosteric modulators. Brain Res. 2006 Mar 3;1076(1):25-41.

[3]. Antisense peptide nucleic acid targeting GluR3 delays disease onset and progression in the SOD1 G93A mouse model of familial ALS. J Neurosci Res. 2004 Aug 15;77(4):573-82.

3-(5-fluorouracil-1-yl)-L-alanine is an alanine derivative, namely L-alanine with a 5-fluorouracil-1-yl substituent at the 3 position. It is an AMPA receptor agonist that is more effective and selective than AMPA itself (with stronger agonistic effects on hGluR1 and hGluR2 receptors, with Ki values of 14.7 nM, 25.1 nM and 1820 nM, respectively). It is an organofluorine compound belonging to the non-protein L-α-amino acid family and is also a derivative of L-alanine. Its function is related to uracil.

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(S)-(-)-5-fluviradiine is a radiolabeled agonist used as a tool compound for studying the pharmacology of AMPA receptors and the role of allosteric modulators. [2]
It has a higher affinity than [³H]AMPA and is less affected by the ionized liquid anion thiocyanate, thus it is a practical ligand suitable for binding experiments under various conditions. [2]
Its significant subunit selectivity (high for GluR1/2, low for GluR3/4) means that the binding data obtained from the native meninges primarily reflect the drug's effects on receptors containing GluR1 and GluR2. [2]
This study highlights the importance of optimizing filtration experimental conditions (e.g., adding thiocyanate to the wash buffer, cooling buffer, minimizing wash time) to reduce radioligand dissociation loss, especially at low-affinity receptor sites. [2]

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- Pharmacological Tool: (S)-5-Fluorowillardiine is a valuable research tool for studying AMPA receptor pharmacology, including allosteric modulation and subunit-specific functions. Its binding profile is distinct from AMPA, showing a much larger affinity difference between GluR1/2 and GluR3/4. [2]
- Use in ALS Research: The excitotoxic effect of FW on NSC-34 cells was used to validate the efficacy of an antisense PNA targeting the AMPA receptor subunit GluR3. Pre-treatment with the antisense PNA protected NSC-34 cells from FW-induced cell death, confirming that GluR3 downregulation can mitigate excitotoxicity. This served as a proof-of-concept for a potential ALS therapy. [3]
- Experimental Considerations for Binding Assays: For [³H]FW binding assays, the centrifugation method is recommended over filtration to avoid loss of low-affinity binding sites. If filtration is necessary, the wash buffer must contain KSCN (50 mM) and be kept at 0°C to minimize ligand dissociation. [2]
- Inactive Enantiomer: The enantiomer (R)-5-fluorowillardiine is considered essentially inactive, used as a negative control in some pharmacological studies. [2]

C7H8N3O4F

217.15452

217.05

140187-23-1

(S)-(-)-5-Fluorowillardiine hydrochloride;1321546-70-6

126569

Typically exists as solid at room temperature

1.64 g/cm3

235ºC

1.601

-4.4

3

6

3

15

354

1

OC([C@@H](N)CN1C=C(F)C(NC1=O)=O)=O

DBWPFHJYSTVBCZ-BYPYZUCNSA-N

InChI=1S/C7H8FN3O4/c8-3-1-11(2-4(9)6(13)14)7(15)10-5(3)12/h1,4H,2,9H2,(H,13,14)(H,10,12,15)/t4-/m0/s1

(2S)-2-amino-3-(5-fluoro-2,4-dioxopyrimidin-1-yl)propanoic acid

5-Fluorowillardiine; (S)-5-FLUOROWILLARDIINE; (alphaS)-alpha-Amino-5-fluoro-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid; (S)-alpha-Amino-5-fluoro-3,4-dihydro-2,4-dioxo-1(2H)-pyrimmidinepropanoic acid;

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.)