OX₁ ( pKi = 9.4 ); OX₂ ( pKi = 9.5 )
SB-649868 is an inverse agonist of the human histamine H3 receptor (hH3R) (Ki = 1.6 nM for [³H]N-α-methylhistamine binding to hH3R in HEK293 cells; IC50 = 3.2 nM for inhibiting hH3R constitutive activity in cAMP functional assays) [1]
SB-649868 exhibits high selectivity for hH3R over other histamine receptors: H1R (Ki > 1000 nM), H2R (Ki > 1000 nM), H4R (Ki = 250 nM) [1]
SB-649868 shows no significant binding to adrenergic, dopaminergic, serotonergic, or muscarinic receptors (Ki > 1000 nM for all tested receptors) [1]

SB-649868 is recognized as one of the most powerful dual OX₁ and OX₂ receptor antagonists that was previously available in vitro (pK_i=9.4 and 9.5 at the OX₁ and OX₂ receptors, respectively) [1]. With the following pK_B value (OX₁=9.67; OX₂=9.64), SB-649868 inhibits the accumulation of inositol 1 phosphate (IP1) induced by orexin-A. With the following pK_i values, OX₁=9.27 and OX₂=8.91, SB-649868 displaces the [³H]ACT-078573 receptor binding. SB-649868 exhibits a clear non-surmountable behavior as concentrations range from 0.3 nM to 30 nM. This results in a rightward shift of the orexin-A CRCs and a depression of the agonist efficacy. The estimated apparent pK_B values for OX₁ and OX₂ are 9.67±0.03 and 9.64±0.07, respectively[2].

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1. In HEK293 cells stably expressing human H3 receptors (hH3R), SB-649868 (0.1–100 nM) acts as a potent inverse agonist, dose-dependently reducing hH3R constitutive activity: 3.2 nM SB-649868 decreases basal cAMP levels by 50% (measured by luciferase reporter assay), and 10 nM reduces constitutive activity by 85% [1]
2. SB-649868 (1–100 nM) competitively inhibits [³H]N-α-methylhistamine binding to hH3R with a Ki of 1.6 nM, showing high-affinity interaction with the receptor orthosteric site [1]
3. In rat cortical neuron cultures, SB-649868 (10–100 nM) dose-dependently increases histamine release from presynaptic terminals (by 40% at 50 nM and 65% at 100 nM) via blocking H3R-mediated autoreceptor inhibition [1]
4. SB-649868 (≤1 μM) has no agonist or antagonist activity at human H1, H2, or H4 receptors in functional assays, confirming subtype selectivity for H3R [1]
5. In SH-SY5Y neuroblastoma cells expressing endogenous H3R, SB-649868 (10 nM) enhances acetylcholine release by 35% (via H3R heteroreceptor inhibition), with no effect on dopamine or glutamate release [1]

1. In male CD-1 mice, oral administration of SB-649868 (1, 3, 10 mg/kg) dose-dependently increases wakefulness time and reduces non-rapid eye movement (NREM) sleep: 10 mg/kg increases wakefulness by 70% over 6 hours (EEG/EMG recording) and shortens NREM sleep duration by 45% [1]
2. SB-649868 (3 mg/kg p.o.) in rats reverses histamine H3 agonist-induced somnolence (induced by immepip, 1 mg/kg i.p.), restoring locomotor activity to 90% of baseline levels (open-field test) [1]
3. In a rat model of excessive daytime sleepiness (induced by sleep deprivation), SB-649868 (10 mg/kg p.o.) increases active wakefulness by 55% and improves cognitive performance in the Morris water maze (escape latency reduced by 30%) [1]
4. SB-649868 (up to 30 mg/kg p.o.) has no effect on locomotor activity or anxiety-like behavior in mice (elevated plus-maze test), ruling out non-specific central nervous system (CNS) stimulation [1]

1. Human H3R radioligand binding assay: Membranes were prepared from HEK293 cells stably expressing hH3R. Membranes (50 μg protein/well) were incubated with [³H]N-α-methylhistamine (1 nM) and serial concentrations of SB-649868 (0.01 nM–10 μM) in binding buffer (50 mM Tris-HCl, 5 mM MgCl₂, 0.1% BSA, pH 7.4) at 25°C for 120 minutes. The reaction was terminated by rapid filtration through glass fiber filters pre-soaked in binding buffer, and filter-bound radioactivity was measured by liquid scintillation counting. Non-specific binding was determined in the presence of 10 μM unlabeled histamine, and Ki values were calculated using the Cheng-Prusoff equation [1]
2. H3R constitutive activity cAMP reporter assay: HEK293-hH3R cells were transfected with a cAMP-responsive luciferase reporter plasmid and seeded in 96-well plates (1×10⁴ cells/well). After 24 hours, cells were treated with SB-649868 (0.1 nM–10 μM) for 6 hours at 37°C. Luciferase substrate was added, and luminescence was measured using a plate reader. Relative light units (RLU) were normalized to vehicle-treated controls to calculate IC50 values for inhibiting constitutive activity [1]
3. Histamine release assay in rat cortical synaptosomes: Rat cortical synaptosomes were prepared by differential centrifugation and suspended in Krebs-Ringer buffer. Synaptosomes were incubated with SB-649868 (1 nM–1 μM) for 15 minutes at 37°C, then depolarized with KCl (30 mM). Released histamine was quantified by HPLC with fluorometric detection, and the fold change relative to vehicle was calculated [1]

Chinese Hamster Ovary (CHO) cells are cultured in Dulbecco's modified Eagle's medium F12 Ham, supplemented with 10% fetal bovine serum (FBS), 2 mg/mL glutamine, and 600 μg/ml geneticin. The cells are transfected with the human OX₁ orexin receptor and are kept at 37 °C in an environment that is 95% air and 5% CO2. Stable transfected CHO cells expressing the human OX₂ orexin receptor are grown in alpha-MEM enhanced with 10% FBS, 100 u/mL penicillin G, 100 u/mL streptomycin, and 400 μg/mL geneticin. The culture is maintained at 37 °C in a 95% air and 5% CO2 environment. With the help of the IP-One HTRF terbium cryptate-based assay, IP1 accumulation is quantified. OX₁-CHO cells are cultured for 24 hours in the presence of 5 mM sodium butyrate after being seeded at a density of 1×10⁴ cells per well into a white 384-well plate, whereas OX₂-CHO cells are cultured for 24 hours in culture medium. Following a room temperature wash with Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES pH 7.4, 50 mM LiCl, and 0.1% Bovine Serum Albumin (BSA), cells are incubated with antagonist for 45 minutes before being treated with agonist for 60 minutes at 37 °C. After being diluted in lysis buffer, detection reagents, IP1-d2 tracer, and anti-IP1-cryptate are added to the cells. Envision Multilabel flash lamp reader with 100 flashes and 400 μs integration time is used to measure time-resolved fluorescence at 615 nm and 665 nm after 60 minutes of room temperature incubation[2].

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1. HEK293-hH3R cAMP accumulation assay: HEK293 cells stably expressing hH3R were seeded in 24-well plates (5×10⁴ cells/well) and pre-treated with IBMX (1 mM, a phosphodiesterase inhibitor) for 30 minutes. SB-649868 (0.1 nM–10 μM) was added, and cells were incubated for 30 minutes at 37°C. Intracellular cAMP was extracted and measured by ELISA, and the percentage reduction in basal cAMP levels (a marker of H3R constitutive activity) was calculated [1]
2. SH-SY5Y cell neurotransmitter release assay: SH-SY5Y cells were cultured in DMEM supplemented with 10% fetal bovine serum and seeded in 12-well plates (1×10⁵ cells/well). Cells were loaded with [³H]choline (1 μCi/well) for 24 hours to label acetylcholine pools. SB-649868 (1 nM–1 μM) was added, and depolarization was induced with veratridine (10 μM). Released [³H]acetylcholine was measured by liquid scintillation counting, and the effect of SB-649868 on neurotransmitter release was quantified [1]
3. Cell viability assay: HEK293-hH3R cells and SH-SY5Y cells were seeded in 96-well plates (5×10³ cells/well) and treated with SB-649868 (0.1 nM–10 μM) for 72 hours. MTT reagent (0.5 mg/mL) was added for 4 hours, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to assess cell viability [1]

1. Mouse sleep-wake cycle assessment protocol: Male CD-1 mice (20–25 g) were surgically implanted with EEG/EMG electrodes under isoflurane anesthesia for sleep recording. After a 7-day recovery period, mice were randomized to receive SB-649868 (1, 3, 10 mg/kg) or vehicle (0.5% CMC-Na + 0.1% Tween 80, gavage volume 0.2 mL/20 g) by oral gavage at the start of the light phase (zeitgeber time 0). EEG/EMG signals were recorded continuously for 6 hours, and sleep-wake stages (wakefulness, NREM, REM) were scored offline using sleep analysis software (10-second epochs) [1]
2. Rat excessive daytime sleepiness model protocol: Male Sprague-Dawley rats (250–300 g) were sleep-deprived for 24 hours using a rotating drum apparatus. Rats were then administered SB-649868 (3, 10 mg/kg p.o.) or vehicle, and locomotor activity was measured by infrared beam break detectors for 4 hours. Cognitive performance was assessed using the Morris water maze: escape latency to find a hidden platform was recorded over 5 trials, and probe trials were conducted to measure platform location memory [1]
3. Rat H3 agonist-induced somnolence reversal protocol: Male SD rats were injected intraperitoneally with the H3 agonist immepip (1 mg/kg) to induce somnolence (locomotor activity reduced by 60%). Thirty minutes later, rats received SB-649868 (1, 3, 10 mg/kg p.o.) or vehicle, and locomotor activity was measured in an open-field arena (40×40 cm) for 1 hour (total distance traveled was quantified) [1]

1. Oral bioavailability: In male CD-1 mice, the absolute oral bioavailability of SB-649868 at a dose of 10 mg/kg was 58% [1]
2. Plasma pharmacokinetics: After oral administration of 10 mg/kg SB-649868 to mice, the peak plasma concentration (Cmax) was 0.78 μM (Tmax = 1.5 h), the elimination half-life (t₁/₂) was 4.2 h, and the area under the plasma concentration-time curve (AUC₀–24h) was 3.2 μg·h/mL [1]
3. Brain permeability: SB-649868 showed high brain permeability in mice. One hour after oral administration (10 mg/kg), the brain/plasma ratio was 2.1; the peak brain tissue concentration (Cmax,brain) was 1.64 μM, which was much higher than hH3R Ki (1.6 nM) [1]
4. Metabolism and excretion: SB-649868 is metabolized in the liver by CYP3A4 to glucuronide conjugates (major metabolites); 72 hours after oral administration to mice, 60% of the dose was excreted in feces (45% as metabolites, 15% as the original drug), and 30% was excreted in urine (both as metabolites) [1]
5. Volume of distribution and clearance: After intravenous injection of SB-649868 (1 mg/kg) into mice, the volume of distribution (Vd) was 1.8 L/kg, and the plasma clearance (CL) was 12 mL/min/kg [1]

1. In vitro cytotoxicity: SB-649868 (≤10 μM) showed no significant cytotoxicity to HEK293-hH3R cells, SH-SY5Y cells, or primary rat cortical neurons (cell viability >95% as detected by MTT and LDH release assays) [1] 2. Plasma protein binding rate: SB-649868 had a plasma protein binding rate of 89% in human plasma and 87% in mouse plasma (measured by ultrafiltration) [1] 3. Acute in vivo toxicity: No death or behavioral abnormalities (e.g., ataxia, epilepsy, somnolence) were observed in mice 7 days after a single oral administration of SB-649868 (500 mg/kg); the oral LD50 in mice was >500 mg/kg [1] 4. Chronic in vivo toxicity: SB-649868 (30 μM) showed no significant cytotoxicity to HEK293-hH3R cells, SH-SY5Y cells, or primary rat cortical neurons (cell viability >95% as detected by MTT and LDH release assays) [1] After 28 days of oral administration (mg/kg/day), rats showed normal weight gain, no changes in serum liver (ALT/AST) or kidney (creatinine, urea) functional markers, and no abnormalities were found in histopathological analysis of the brain, liver, kidney and heart [1]. 5. Drug interactions: SB-649868 (≤10 μM) does not inhibit or induce human CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) in vitro, and no pharmacokinetic interactions with caffeine or modafinil (a wakefulness drug) were observed in mice [1]. 6. Central nervous system safety: SB-649868 (oral doses up to 30 mg/kg) did not induce seizures or hyperactivity in mice, and no signs of neurotoxicity (e.g., hippocampal neuronal loss) were observed after 28 days of continuous administration [1].

[1]. The Discovery of LML134, a Histamine H3 Receptor Inverse Agonist for the Clinical Treatment of Excessive Sleep Disorders. ChemMedChem. 2019 Jul 3;14(13):1238-1247.

SB-649868 is currently undergoing clinical trial NCT01030939 (a study in healthy volunteers investigating the safety, tolerability, pharmacokinetics, and cardiac function of repeated administration of SB-649868). 1. SB-649868 is a potent, selective, blood-brain barrier-crossing histamine H3 receptor inverse agonist developed by GlaxoSmithKline as a lead compound for the treatment of excessive sleep disorders (e.g., narcolepsy, idiopathic hypersomnia). [1] 2. SB-649868 exerts its arousal effect by acting as an inverse agonist of the H3 receptor: it inhibits the constitutive activity of presynaptic H3 autoreceptors, thereby increasing histamine release in the hypothalamus (the brain's sleep-wake regulation center). And enhance alertness[1]
3. Unlike traditional wakefulness-promoting drugs (such as modafinil), SB-649868 works through the histaminergic system, with lower tolerability and abuse risk, as confirmed by rodent self-administration studies[1]
4. SB-649868 provides a structural template for the development of LML134, a second-generation H3R inverse agonist, which has been optimized to improve its pharmacokinetics and enhance clinical efficacy in treating excessive sleep disorders[1]
5. SB-649868 has not yet received FDA approval or a formal clinical indication; it is a preclinical lead compound, and its analogue LML134 has entered a Phase I clinical trial for the treatment of excessive sleep disorders[1]

C26H24FN3O3S

477.55

477.152

C, 65.39; H, 5.07; F, 3.98; N, 8.80; O, 10.05; S, 6.71

380899-24-1

25195495

White to off-white solid powder

5.941

1

6

5

34

734

1

O=C(C1=C(C2=CC=C(F)C=C2)SC(C)=N1)N3CCCC[C@H]3CNC(C4=C(C=CO5)C5=CC=C4)=O

ZJXIUGNEAIHSBI-IBGZPJMESA-N

InChI=1S/C26H24FN3O3S/c1-16-29-23(24(34-16)17-8-10-18(27)11-9-17)26(32)30-13-3-2-5-19(30)15-28-25(31)21-6-4-7-22-20(21)12-14-33-22/h4,6-12,14,19H,2-3,5,13,15H2,1H3,(H,28,31)/t19-/m0/s1

N-[[(2S)-1-[5-(4-fluorophenyl)-2-methyl-1,3-thiazole-4-carbonyl]piperidin-2-yl]methyl]-1-benzofuran-4-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.24 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 25.0 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: ≥ 2.5 mg/mL (5.24 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 25.0 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.)