5-HT1A Receptor
Befiradol targets the 5-HT1A receptor, a serotonin receptor subtype that is widely expressed in the central nervous system. 5-HT1A receptors are G protein-coupled receptors that mediate the inhibitory effects of serotonin. They are involved in the regulation of mood, anxiety, cognition, and motor control. Befiradol is a highly selective and potent 5-HT1A receptor full agonist. It has >1000-fold selectivity for the 5-HT1A receptor compared to other receptor types. By activating 5-HT1A receptors, Befiradol modulates neurotransmission in brain regions involved in motor control and may reduce the dyskinesia associated with L-DOPA therapy in Parkinson's disease.

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 studies have characterized Befiradol as a highly selective and potent 5-HT1A receptor agonist. It has >1000-fold selectivity for the 5-HT1A receptor compared to other receptor types. The compound's activity at the 5-HT1A receptor has been confirmed in receptor binding and functional assays. Befiradol has been investigated for its potential to treat L-DOPA-induced dyskinesia in Parkinson's disease patients. These in vitro studies establish Befiradol as a potent and selective tool for studying 5-HT1A receptor function.

F13640 reduced the activity of dorsal raphe serotonergic neurons at 0.2-18.2 μg kg(-1), i.v. (cumulative doses; ED(50) = 0.69 μg kg(-1), i.v.) and increased the discharge rate of 80% of mPFC pyramidal neurons in the same dose range (ED(50) = 0.62 μg kg(-1), i.v.). Both effects were reversed by the subsequent administration of the 5-HT(1A) receptor antagonist (±)WAY100635. In microdialysis studies, F13640 (0.04-0.63 mg kg(-1), i.p.) dose-dependently decreased extracellular 5-HT in the hippocampus and mPFC. Likewise, F13640 (0.01-2.5 mg kg(-1), i.p.) dose-dependently increased extracellular DA in mPFC, an effect dependent on the activation of postsynaptic 5-HT(1A) receptors in mPFC. Local perfusion of F13640 in mPFC (1-1,000 μM) also increased extracellular DA in a concentration-dependent manner. Both the systemic and local effects of F13640 were prevented by prior (±)WAY100635 administration[1].

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In vivo studies have demonstrated that Befiradol has effects on motor function and dyskinesia. It has been investigated in clinical trials for the treatment of Parkinson's disease patients who exhibit L-DOPA-induced dyskinesia. Befiradol is currently under investigation in clinical trial NCT05148884 (Study to Assess the Safety, Tolerability and Preliminary Efficacy of NLX-112 Versus Placebo in L-dopa-induced Dyskinesia). The compound's ability to activate 5-HT1A receptors is believed to modulate dopaminergic signaling and reduce dyskinesia. Befiradol has a maximum clinical trial phase of II.

The in vitro receptor binding assays for Befiradol measure its affinity for the 5-HT1A receptor. In a typical assay, membranes are prepared from cells expressing the human 5-HT1A receptor and incubated with a radiolabeled ligand that binds to the 5-HT1A receptor, such as [3H]8-OH-DPAT. Increasing concentrations of unlabeled Befiradol are added to compete with the radiolabeled ligand for binding. After incubation, the bound and free ligand are separated by filtration, and the radioactivity is measured. The Ki is determined from the competition curve. To assess selectivity, the compound is tested against a panel of other receptors. Functional assays measure the compound's ability to activate 5-HT1A receptor-mediated signaling, such as the inhibition of cAMP accumulation or the activation of G protein-coupled inwardly rectifying potassium channels (GIRKs).

In vitro cell-based assays for Befiradol are used to study its effects on 5-HT1A receptor signaling. A common assay involves measuring the inhibition of forskolin-stimulated cAMP accumulation in cells expressing the 5-HT1A receptor. Cells are treated with Befiradol, and the levels of cAMP are measured using a competitive immunoassay. The compound's ability to inhibit cAMP accumulation is assessed. Other functional assays measure the activation of GIRK channels or the modulation of neurotransmitter release. These cell-based assays confirm that Befiradol is a functional agonist at the 5-HT1A receptor.

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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In vivo animal experiments for Befiradol have been conducted in animal models of Parkinson's disease and dyskinesia. In a typical study, Befiradol is administered to animals, and its effects on motor function and L-DOPA-induced dyskinesia are assessed. The compound's ability to reduce dyskinesia without affecting the anti-parkinsonian effects of L-DOPA is evaluated. Befiradol has been investigated in clinical trials for the treatment of L-DOPA-induced dyskinesia. However, specific preclinical protocols for Befiradol are not detailed in the available literature.

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 has a molecular weight of 393.86 g/mol and a molecular formula of C20H22ClF2N3O. It has a predicted boiling point of 536.9±50.0 °C, a density of 1.30, and a pKa of 8.01±0.20. The compound is supplied as a white to off-white solid. It is soluble in DMSO. For storage, it is recommended to keep the compound at -20°C. Pharmacokinetic properties such as absorption, distribution, metabolism, and excretion (ADME) have been studied in the context of clinical development. Befiradol is a small molecule drug with a maximum clinical trial phase of II.

Detailed toxicity data for Befiradol is not provided in standard product descriptions. As a compound that has been investigated in clinical trials, its safety profile has been evaluated. However, specific toxicity data, such as LD50 or organ toxicity, are not detailed in the available literature. In clinical trials, Befiradol has been studied for its safety and tolerability in patients with L-DOPA-induced dyskinesia. As with all research chemicals, standard laboratory safety precautions should be followed when handling Befiradol.

[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.

Serotonin 5-HT1 Receptor Agonists: Endogenous compounds and drugs that specifically stimulate serotonin 5-HT1 receptors. This heading contains agonists of one or more specific 5-HT1 receptor subtypes.

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Mechanism: F13640 (befiladox) is a novel 5-HT(1A) receptor agonist with extremely high selectivity for other receptors and binding sites. It has shown analgesic activity in animal models and is currently under development for human use.
Objective: Given the potential dual role of the serotonergic system 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 on cell-somatic dendritic autoreceptors and postsynaptic 5-HT(1A) heteroreceptors in the medial prefrontal cortex (mPFC). Methods: Single-cell recording and intracerebral microdialysis were used in rats. Results: Intravenous injection of F13640 (0.2–18.2 μg kg⁻¹) reduced the activity of dorsal raphe nucleus serotonergic neurons (cumulative dose; ED₅₀ = 0.69 μg kg⁻¹) and increased the firing rate of 80% of medial prefrontal cortex (mPFC) pyramidal neurons within the same dose range (ED₅₀ = 0.62 μg kg⁻¹). Subsequent administration of the 5-HT₁A receptor antagonist (±)WAY100635 reversed both effects. Microdialysis studies showed that F13640 (intraperitoneal injection, 0.04–0.63 mg kg⁻¹) dose-dependently reduced extracellular 5-HT levels in the hippocampus and mPFC. Similarly, F13640 (0.01–2.5 mg kg⁻¹, intraperitoneal injection) increased extracellular dopamine (DA) levels in the medial prefrontal cortex (mPFC) in a dose-dependent manner, an effect dependent on the activation of postsynaptic 5-HT₁A receptors in the mPFC. Local perfusion of F13640 (1–1000 μM) in the mPFC also increased extracellular DA levels in a concentration-dependent manner. Pre-administration of (±)WAY100635 blocked the systemic and local effects of F13640. Conclusion: These results suggest that, following systemic administration, F13640 activates 5-HT₁A autoreceptors and postsynaptic 5-HT₁A receptors in the prefrontal cortex with similar potency. Both activities may be related to the analgesic properties of the compound. [1]

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Befiradol (NLX-112, F13640) is a research compound that has been investigated for the treatment of L-DOPA-induced dyskinesia in Parkinson's disease patients. It is a highly selective and potent 5-HT1A receptor full agonist with >1000-fold selectivity for the 5-HT1A receptor compared to other receptor types. Befiradol is a small molecule drug with a maximum clinical trial phase of II. It is currently under investigation in clinical trial NCT05148884. Befiradol's mechanism of action involves activating 5-HT1A receptors, which modulates neurotransmission and may reduce dyskinesia. The compound is used as a research tool to study 5-HT1A receptor function and its role in motor control and dyskinesia.

C20H22CLF2N3O

393.86

393.142

C, 60.99; H, 5.63; Cl, 9.00; F, 9.65; N, 10.67; O, 4.06

208110-64-9

Befiradol hydrochloride;2436760-81-3

9865384

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

1.3

4.245

1

5

5

27

502

0

O=C(C1=CC=C(F)C(Cl)=C1)N2CCC(CNCC3=NC=C(C)C=C3)(F)CC2

PKZXLMVXBZICTF-UHFFFAOYSA-N

InChI=1S/C20H22ClF2N3O/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

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

F-13640; F 13640; NLX-112; Befiradol (free base); Befiradol [INN]; RAT9OHA1YH; F13640; CHEMBL45305;F13640

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)

DMSO : ~102 mg/mL (~258.98 mM)

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.)