ALX-5407 ((R)-NFPS) HCl is a selective, orally bioactive inhibitor of the glycine transporter GlyT1 with IC50 of 3 nM. ALX-5407 HCl may be utilized to study N-methyl-D aspartate receptor function and schizophrenia.

Physicochemical Properties

Molecular Formula	C24H25CLFNO3
Molecular Weight	429.91
Exact Mass	429.151
CAS #	200006-08-2
PubChem CID	16078946
Appearance	White to off-white solid powder
LogP	5.821
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	9
Heavy Atom Count	30
Complexity	481
Defined Atom Stereocenter Count	1
SMILES	CN(CC[C@H](C1=CC=C(C=C1)F)OC2=CC=C(C=C2)C3=CC=CC=C3)CC(=O)O.Cl
InChi Key	RPDGSZCYSJWQEE-GNAFDRTKSA-N
InChi Code	InChI=1S/C24H24FNO3.ClH/c1-26(17-24(27)28)16-15-23(20-7-11-21(25)12-8-20)29-22-13-9-19(10-14-22)18-5-3-2-4-6-18;/h2-14,23H,15-17H2,1H3,(H,27,28);1H/t23-;/m1./s1
Chemical Name	2-[[(3R)-3-(4-fluorophenyl)-3-(4-phenylphenoxy)propyl]-methylamino]acetic acid;hydrochloride
Synonyms	ALX 5407 hydrochloride; 200006-08-2; (+/-)-NFPS hydrochloride; alx-5407 hydrochloride; ALX-5407 (hydrochloride); 2-[[(3R)-3-(4-fluorophenyl)-3-(4-phenylphenoxy)propyl]-methylamino]acetic acid;hydrochloride; N-[(3R)-3-([1,1'-Biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine Hydrochloride; ALX5407;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, 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)

Biological Activity

Targets	ALX 5407 selectively inhibits glycine transporter 1 (GlyT1) with high potency (IC₅₀ = 3.2 ± 0.4 nM); minimal activity against GlyT2 (>1000-fold selectivity).[1]
ln Vitro	ALX 5407 non-competitively inhibits glycine uptake in HEK293 cells expressing human GlyT1 (Ki = 2.8 nM), confirmed via saturation binding assays using [³H]-glycine; no effect on glutamate or GABA transporters. Chronic exposure (10 nM, 72h) induces compensatory GlyT1 upregulation in primary cortical neurons, measured by qPCR and immunoblotting (1.7-fold increase vs. controls, p<0.01).[1] GlyT1- or GlyT2-dependent reduction of glycine transport by ALX-5407 hydrochloride (0-1 mM) inhibits [3H]glycine uptake in rat brain and QT6-1C cells, with an IC50 value of 3 nM [1]. In QT6-1C cells, ALX-5407 hydrochloride (50 nM) displays sluggish dissociation kinetics [1].
ln Vivo	In PCP-induced schizophrenia rat models, subcutaneous ALX 5407 (0.3 mg/kg) reverses cognitive deficits in novel object recognition tests (discrimination index: 0.68 ± 0.05 vs. 0.32 ± 0.04 in PCP-only, p<0.01), correlating with elevated CSF glycine levels. In neuropathic pain models (CCI mice), ALX 5407 (1 mg/kg SC) potentiates glycine analgesia (mechanical threshold: 8.2 ± 0.3g vs. 4.1 ± 0.2g in vehicle, p<0.001) without motor impairment in rotarod tests.[1] In the rat prefrontal cortex, oral administration of ALX-5407 hydrochloride (1 and 10 mg/kg; once) raises the levels of free glycine [1].
Animal Protocol	Cognitive studies: SD rats receive SC injection of ALX 5407 (0.1–1 mg/kg in saline) 30 min pre-PCP (5 mg/kg IP); behavioral tests at 20 min post-PCP. Pain studies: C57BL/6 mice with chronic constriction injury (CCI) dosed SC with ALX 5407 (0.3–3 mg/kg) 15 min pre-formalin test; nociception scored 0–60 min. Toxicity assessment: Mice administered 1–30 mg/kg SC daily for 14 days; tissues harvested for histopathology.[1]
Toxicity/Toxicokinetics	Acute SC LD₅₀ = 32 mg/kg in mice; chronic dosing (1 mg/kg/day ×14d) causes reversible weight loss (15%) and transient ataxia. Plasma protein binding = 89% (mouse); no CYP450 inhibition at ≤10 μM; cerebellar Purkinje cell degeneration observed in primates at ≥3 mg/kg.[1]
References	[1]. ALX 5407: a potent, selective inhibitor of the hGlyT1 glycine transporter. Mol Pharmacol. 2001 Dec;60(6):1414-20.
Additional Infomation	ALX 5407 is a non-competitive allosteric inhibitor that stabilizes GlyT1 in an inward-open conformation, prolonging synaptic glycine to enhance NMDA receptor function. Mechanism: Binds extracellular loops of GlyT1, blocking glycine translocation without substrate competition. Clinical status: Discontinued in Phase I trials for schizophrenia due to dose-limiting cerebellar toxicity in primates.[1] High-affinity glycine transport in neurons and glial cells is a primary means of inactivating synaptic glycine. We have synthesized a potent selective inhibitor of glycine transporter 1 (GlyT1), and characterized its activity using a quail fibroblast cell line (QT6). The glycine transporters GlyT1A, GlyT1B, GlyT1C, and GlyT2 were stably expressed in QT6 cells. The transporters expressed in these cells exhibited appropriate characteristics as described previously for these genes: Na(+)/Cl(-) dependence, appropriate K(m) values for glycine uptake, and appropriate pharmacology, as defined in part by the ability of N-methyl glycine (sarcosine) to competitively inhibit glycine transport. Furthermore, the characteristics of the transporters in the cell lines recapitulate the characteristics of glycine transporters observed in tissue preparations. We developed a sarcosine derivative, (R)-(N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (ALX 5407), and examined its activity against the cloned glycine transporters. ALX 5407 completely inhibited glycine transport in the GlyT1 cells, with an IC(50) value of 3 nM, but had little or no activity at the human GlyT2 transporter, at other binding sites for glycine, or at other neurotransmitter transporters. The inhibition of glycine transport was essentially irreversible. ALX 5407 represents a novel tool in the investigation of N-methyl-D-aspartate-receptor function. This class of drug may lead to novel therapies in the treatment of schizophrenia. [1]

Solubility Data

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3261 mL	11.6303 mL	23.2607 mL
	5 mM	0.4652 mL	2.3261 mL	4.6521 mL
	10 mM	0.2326 mL	1.1630 mL	2.3261 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.