5-HT1A Receptor
Befiradol hydrochloride targets the serotonin 5-HT1A receptor. It is a selective agonist with exceptional selectivity vs. other receptors and binding sites. By activating 5-HT1A receptors, it modulates neurotransmitter release and produces anxiolytic and antidepressant-like effects. Its high selectivity makes it a valuable tool for studying 5-HT1A receptor function.

F13640 (befiradol) is a novel 5-HT(1A) receptor agonist with exceptional selectivity vs. other receptors and binding sites[1]. F13640 activates both 5-HT(1A) autoreceptors and postsynaptic 5-HT(1A) receptors in prefrontal cortex with a similar potency. Both activities are likely involved in the analgesic properties of the compound.

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In vitro, Befiradol hydrochloride acts as a selective 5-HT1A receptor agonist. Its activity is typically assessed by measuring its ability to inhibit forskolin-stimulated cAMP accumulation or stimulate [35S]GTPγS binding in cells expressing 5-HT1A receptors. The compound's exceptional selectivity for 5-HT1A over other receptors makes it a valuable tool for studying 5-HT1A receptor function and signaling pathways.

Befiradol (F13640; NLX-112) raises the discharge rate of 80% of mPFC pyramidal neurons in the same dose range (ED50=0.62 μg/kg, iv) and decreases the activity of dorsal raphe serotonergic neurons at 0.2-18.2 μg/kg, iv (cumulative doses; ED50=0.69 μg/kg, iv). Subsequent injection of the 5-HT1A receptor antagonist (±)WAY100635 reverses both effects. Befiradol (F13640; NLX-112) (0.04 -0.63 mg/kg, ip) dose-dependently reduces extracellular 5-HT in the mPFC and hippocampal regions in microdialysis experiments. Similarly, Befiradol (F13640; NLX-112) (0.01-2.5 mg/kg, ip) raises extracellular DA in mPFC in a dose-dependent manner. a result reliant on the mPFC's postsynaptic 5-HT1A receptors being activated. In a concentration-dependent manner, local perfusion of Befiradol in mPFC (1-1,000 μM) likewise raises extracellular DA. Befiradol's local and systemic effects can be avoided by administering (±)WAY100635 beforehand[1].

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In vivo, Befiradol hydrochloride has anxiolytic activity and has been shown to inhibit mutant ATXN3 aggregation. As a selective 5-HT1A receptor agonist, it produces anxiolytic and antidepressant-like effects by activating central 5-HT1A receptors and modulating neurotransmitter release. It is used in research to study the role of 5-HT1A receptors in anxiety, depression, and neurodegenerative diseases.

For non-cell-based receptor binding assays, Befiradol hydrochloride can be evaluated using membrane preparations from cells expressing human 5-HT1A receptors. Radioligand binding displacement experiments are performed using a suitable radiolabeled ligand such as [3H]-8-OH-DPAT. Membrane homogenates are incubated with increasing concentrations of the test compound and a fixed concentration of the radioligand. Bound radioligand is separated from free by rapid filtration. Non-specific binding is determined in the presence of excess unlabeled 8-OH-DPAT. Ki values are calculated from displacement curves using nonlinear regression analysis.

For in vitro cellular assays, cells expressing human 5-HT1A receptors are cultured in appropriate media. For functional assays, the inhibition of forskolin-stimulated cAMP accumulation is measured. Cells are treated with various concentrations of Befiradol hydrochloride, and cAMP levels are measured using ELISA or HTRF-based detection. The compound's ability to inhibit cAMP accumulation is assessed, and EC50 values are calculated from dose-response curves. For [35S]GTPγS binding assays, membrane preparations are incubated with GDP, [35S]GTPγS, and various concentrations of the compound, and bound [35S]GTPγS is measured by filtration.

Rats were anaesthetized with chloral hydrate (400–500 mg kg−1, i.p.) or isoflurane. A guide cannula with a dummy probe was stereotaxically implanted into the mPFC, stereotaxic coordinates: AP +3.0 mm, L +0.8 mm, DV −1.7 mm, or the hippocampus: AP −4.8 mm, L +4.6 mm, DV −4.6 mm, from bregma and skull surface. Following surgery and recovery from anesthesia, animals were returned to their home cages. At the end of the day, each rat was placed in a microdialysis cage. On the following day, the dummy probe was replaced by a microdialysis probe (3 mm length, 0.5 mm diameter; CMA, Microdialysis AB). The probe was continuously perfused (1.1 μl min−1) with artificial CSF (aCSF) containing 1 μM citalopram for the measure of 5-HT. At least 2 h after probe insertion, samples were collected every 20 min with the first four samples used for baseline. For the experiment with systemic administration of the compounds, saline or (±)WAY100635 were injected s.c., followed, 40 min later, by i.p. administration of saline or F13640. For the experiments with local perfusion, saline was injected s.c. and 40 min later, F13640 was added to the perfusion medium for the concentration–response experiment. For the antagonism, (±)WAY100635 (or aCSF) was delivered through the dialysis probe and 40 min later, F13640 was added to the perfusion medium. Samples were collected for 140 min after administration or beginning of the perfusion of the agonist. At the end of the experiment, rats were killed by anesthetic overdose (pentobarbital 160 mg kg−1, i.p.) and the brain was removed, frozen and cut in a cryomicrotome (Jung Frigocut 2800) to verify the placement of the probe.[1]

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For in vivo animal studies, Befiradol hydrochloride can be administered to rodents via intraperitoneal injection or oral gavage. In models of anxiety (e.g., elevated plus maze), behavioral responses are assessed. In models of depression, the forced swim test may be used. In models of neurodegenerative disease, its effects on protein aggregation and motor function are evaluated. Dosing regimens vary depending on the specific model.

NLX-112 (F13640, befiradol) exhibits nanomolar affinity, high selectivity, and complete agonist potency for the 5-HT1A receptor. NLX-112 has demonstrated efficacy in rat, marmoset, and rhesus monkey models of levodopa-induced dyskinesia (LID) in Parkinson's disease and showed clinical efficacy in a Phase IIa proof-of-concept study for this indication. This study investigated the pharmacodynamics, pharmacokinetics (PK), and 5-HT1A receptor occupancy in the brain of NLX-112 in rats, as well as the PK characteristics in the presence and absence of levodopa. Within the tested dose range (0.04, 0.16, and 0.63 mg/kg, intraperitoneal injection), total and free NLX-112 exposures in plasma, cerebrospinal fluid, and striatal extracellular fluid were dose-proportional. Exposure to NLX-112 increases rapidly (Tmax 0.25–0.5 h), and its half-life in the brain is approximately three times that in plasma (1.1 h and 3.6 h, respectively). At a previously demonstrated pharmacologically relevant dose of 0.16 mg/kg intraperitoneally, which induces anti-levodopa-induced dyskinesia (LID) in Parkinson's disease rats, NLX-112 concentrations in the brain ranged from 51–63 ng/g over 0.15 to 1 hour. In miniature PET imaging experiments, NLX-112 showed a dose-dependent reduction in 18F-F13640 (i.e., 18F-NLX-112)-labeled 5-HT1A receptors in the cingulate gyrus and striatum (areas associated with motor control and emotion), with labeling almost completely inhibited at a dose of 0.63 mg/kg intraperitoneally. Co-administration of levodopa (6 mg/kg subcutaneously, the dose used to induce levodopa-induced dyskinesia in Parkinson's disease rats) with NLX-112 (0.16 mg/kg intraperitoneally) did not alter the pharmacokinetic parameters of either NLX-112 or levodopa in rat plasma and brain tissue. This study demonstrates that the pharmacokinetic profile of NLX-112 meets the "drug-ready" parameters for its central nervous system indication, and the results provide brain concentration and 5-HT1A receptor binding parameters related to the compound's anti-dyskinesia activity. https://pubmed.ncbi.nlm.nih.gov/39096379/

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Befiradol hydrochloride has a molecular weight of 430.32 and a molecular formula of C20H23Cl2F2N3O. It is a selective 5-HT1A receptor agonist. The compound should be stored at -20°C for long-term stability. It is soluble in DMSO and can be formulated for both in vitro and in vivo administration. It is for research use only and is not intended for human consumption.

The toxicity profile of Befiradol hydrochloride has not been extensively reported. As a 5-HT1A receptor agonist, potential adverse effects may include modulation of serotonergic signaling. The compound is for research use only and is not intended for human consumption. Standard toxicological evaluation would include acute and repeated-dose toxicity studies.

[1]. In vivo electrophysiological and neurochemical effects of the selective 5-HT1A receptor agonist, F13640, at pre- and postsynaptic 5-HT1A receptors in the rat. Psychopharmacology (Berl). 2012 May;221(2):261-72.

Reason: F13640 (befilradol) is a novel 5-HT(1A) receptor agonist with excellent selectivity for other receptors and binding sites. The drug has shown analgesic activity in animal models and is currently being developed for human use. [1] Objective: Given that the serotonergic system may play a dual role in pain, namely by modulating ascending spinal cord signals and emotional processing in the cortical limbic region, we investigated the in vivo activity of F13640 in cell-somatic dendritic autoreceptors and postsynaptic 5-HT(1A) heteroreceptors in the medial prefrontal cortex (mPFC). [1] Methods: In vivo single-cell recording and intracerebral microdialysis in rats. [1] Results: F13640 reduced the activity of dorsal raphe nucleus serotonergic neurons at intravenous doses of 0.2–18.2 μg kg(-1) (cumulative dose; ED(50) = 0.69 μg kg(-1), intravenous) and increased the firing rate of 80% of mPFC pyramidal neurons. Within the same dose range (ED50 = 0.62 μg kg⁻¹, intravenous), F13640 also affected neurons. Subsequent administration of the 5-HT1A receptor antagonist (±)WAY100635 reversed both effects. Microdialysis studies showed that F13640 (0.04–0.63 mg kg⁻¹, intraperitoneal) dose-dependently reduced extracellular 5-HT levels in the hippocampus and medial prefrontal cortex (mPFC). Similarly, F13640 (0.01–2.5 mg kg⁻¹, intraperitoneal) dose-dependently increased extracellular dopamine (DA) levels in the mPFC, an effect dependent on activation of postsynaptic 5-HT1A receptors in the mPFC. Local perfusion of F13640 (1–1000 μM) into the mPFC also increased extracellular DA levels in a concentration-dependent manner. Pre-administration of (±)WAY100635 blocked the systemic and local effects of F13640. [1] Conclusion: These results suggest that, following systemic administration, F13640 can activate 5-HT(1A) autoreceptors and postsynaptic 5-HT(1A) receptors in the prefrontal cortex with similar potency. Both of these activities may be related to the analgesic properties of the compound.

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Befiradol hydrochloride (NLX-112 hydrochloride; F 13640 hydrochloride, CAS 2436760-81-3) is a selective 5-HT1A receptor agonist. It has anxiolytic activity and inhibits mutant ATXN3 aggregation. It activates central 5-HT1A receptors, modulating neurotransmitter release and producing anxiolytic and antidepressant-like effects. It is available for research purposes only.

C20H23CL2F2N3O

430.31892991066

429.118

2436760-81-3

Befiradol;208110-64-9; 208110-65-0

135397148

Typically exists as white to off-white solids at room temperature

2

5

5

28

502

0

CC1=CN=C(C=C1)CNCC2(CCN(CC2)C(=O)C3=CC(=C(C=C3)F)Cl)F.Cl

MEFWJLIGVNWMAB-UHFFFAOYSA-N

InChI=1S/C20H22ClF2N3O.ClH/c1-14-2-4-16(25-11-14)12-24-13-20(23)6-8-26(9-7-20)19(27)15-3-5-18(22)17(21)10-15;/h2-5,10-11,24H,6-9,12-13H2,1H3;1H

(3-chloro-4-fluorophenyl)-[4-fluoro-4-[[(5-methylpyridin-2-yl)methylamino]methyl]piperidin-1-yl]methanone;hydrochloride

Befiradol hydrochloride; Befiradol (hydrochloride); Befiradol hydrochloride (208110-64-9 free base); NLX-112 hydrochloride; 2436760-81-3; F 13640 hydrochloride; F 13640 (hydrochloride)

2934.99.9001

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.

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

DMSO: 125 mg/mL (290.48 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.83 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 (4.83 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 (4.83 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.)