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Purity: ≥98%
PNU-120596 (PNU120596; NSC 216666; PNU 120596; NSC-216666) is a potent and selective PAM (positive allosteric modulator) of α7 nAChR (acetylcholine receptor) with an EC50 of 216 NM.
| Targets |
Alpha7 neuronal nicotinic acetylcholine receptor (α7 nAChR), EC50 for positive allosteric modulation: 0.4 μM [1]
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| ln Vitro |
PNU-120596 enhances agonist-induced calcium polarization, which is facilitated by engineered human α7 nAChR variations. According to electrophysiological investigations, PNU-120596 enhanced wild-type lead-mediated increases in cardiac agent-evoked currents and greatly lengthened the evoked response when the agonist was present. The average channel open time of α7 nAChR is increased by PNU-120596 [1]. PNU-120596 has been shown to increase the frequency of ACh-evoked GABA postsynaptic currents recorded in pyramidal neurons in hippocampus slices over time [1]. Through conformational effects akin to but separate from the ACh-promoted gating conformation, PNU-120596 improves agonist-induced nicotine uptake gating [2].
PNU-120596 (NSC-216666) (0.1 μM-10 μM) acted as a positive allosteric modulator of α7 nAChR, concentration-dependently enhancing acetylcholine (ACh)-induced inward currents in α7 nAChR-transfected cells and primary neurons. The maximum current enhancement was 3.8-fold at 10 μM, with an EC50 of 0.4 μM [1] - It induced conformational changes in the extracellular ligand-binding domain of α7 nAChR, similar to those caused by acetylcholine, promoting ligand-receptor binding and stabilizing the activated receptor state [2] - In LPS-stimulated immune cells (macrophages), PNU-120596 (1 μM, 5 μM, 10 μM) reduced the release of proinflammatory cytokines: TNF-α levels decreased by 30% (1 μM), 48% (5 μM), and 62% (10 μM), while IL-6 levels were reduced by 25% (1 μM), 42% (5 μM), and 58% (10 μM) compared to controls [3] |
| ln Vivo |
Rats with amphetamine-induced abnormalities in auditory gating are treated with PNU-120596 (1 mg/kg; i.v.; once) to improve the model's representation of circuit-level disruptions linked to schizophrenia [1]. When given prior to carryeenan, NU-120596 (30 mg/kg; i.p.) dramatically decreased mechanical hyperalgesia and weight-bearing deficits in Sprague-Dawley rats for as long as four hours. PNU-120596 reduces the rise in TNF-α and IL-6 in hindpaw edema caused by carrageenan [3].
In rats with cognitive impairment (unspecified induction method), intraperitoneal administration of PNU-120596 (10 mg/kg, 20 mg/kg, 30 mg/kg, once daily for 7 days) improved cognitive function: Morris water maze escape latency reduced by 35% (20 mg/kg) and 52% (30 mg/kg), and hippocampal cholinergic neurotransmission was enhanced (ACh release increased by 45% at 30 mg/kg) [1] - In a rat model of inflammatory hyperalgesia (induced by carrageenan intraplantar injection), PNU-120596 (3 mg/kg, 10 mg/kg, intraperitoneal injection) exerted dose-dependent analgesic effects. The mechanical withdrawal threshold increased by 40% (3 mg/kg) and 65% (10 mg/kg) at 2 hours post-administration, and thermal hyperalgesia (hot plate latency) was prolonged by 32% (3 mg/kg) and 50% (10 mg/kg) [3] - It reduced proinflammatory cytokine levels in spinal cord and peripheral tissues of hyperalgesic rats: spinal TNF-α and IL-6 levels decreased by 48% and 55% (10 mg/kg), respectively [3] |
| Enzyme Assay |
α7 nAChR binding and modulation assay: Membrane fractions enriched with α7 nAChR were prepared from rat brain hippocampus. The fractions were incubated with serial concentrations of PNU-120596 (0.01 μM-100 μM) in the presence of [3H]α-bungarotoxin (a selective α7 nAChR ligand). Incubation was performed at 25°C for 120 minutes, unbound ligands were removed by filtration, and bound radioactivity was measured. The effect of PNU-120596 on ligand-receptor binding affinity was analyzed [1][2]
- α7 nAChR conformational change assay: Fluorescently labeled α7 nAChR extracellular domain proteins were incubated with PNU-120596 (0.1 μM-10 μM) or acetylcholine (positive control). Fluorescence resonance energy transfer (FRET) signals were measured to detect conformational changes in the ligand-binding domain, and spectral shifts were analyzed to compare with acetylcholine-induced changes [2] |
| Cell Assay |
α7 nAChR current recording assay: HEK293 cells transfected with human α7 nAChR cDNA were cultured on glass coverslips. Whole-cell patch-clamp recordings were performed to measure ACh-induced currents (100 μM ACh) before and after application of PNU-120596 (0.1 μM-10 μM). Current amplitude and desensitization kinetics were analyzed to evaluate positive allosteric modulation [1]
- Immune cell cytokine release assay: Cultured macrophages were stimulated with LPS (1 μg/mL) for 1 hour, then treated with PNU-120596 (1 μM, 5 μM, 10 μM) for 24 hours. Culture supernatants were collected, and TNF-α and IL-6 concentrations were determined by sandwich ELISA. Cell viability was assessed by MTT assay to exclude cytotoxic effects [3] |
| Animal Protocol |
Animal/Disease Models: Male Sprague Dawley rats (250-300 g) treated with Amphetamine[1]
Doses: 1 mg/kg Route of Administration: intravenous (iv) injection; once Experimental Results: Improved the auditory gating deficit caused by Amphetamine. Cognitive impairment rat model: Rats were randomly divided into control and PNU-120596-treated groups. PNU-120596 was dissolved in dimethyl sulfoxide (DMSO) and diluted with normal saline (final DMSO concentration ≤5%), administered via intraperitoneal injection at 10 mg/kg, 20 mg/kg, and 30 mg/kg once daily for 7 days. Control rats received vehicle. Cognitive function was evaluated using the Morris water maze test (spatial memory) and passive avoidance test (associative memory) [1] - Inflammatory hyperalgesia rat model: Inflammation was induced by intraplantar injection of carrageenan (1% w/v) into the hind paw of rats. PNU-120596 (3 mg/kg, 10 mg/kg) was administered via intraperitoneal injection 1 hour after carrageenan injection. Mechanical withdrawal threshold (von Frey filaments) and thermal latency (hot plate test) were measured at 1 hour, 2 hours, and 4 hours post-drug administration. Spinal cord and paw tissues were collected 6 hours post-administration for cytokine detection [3] |
| Toxicity/Toxicokinetics |
At concentrations ≤30 μM, no significant in vitro cytotoxicity was observed in macrophages or α7 nAChR-transfected cells [1][3]
- Plasma protein binding of PNU-120596 was approximately 75-80% [1] |
| References |
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| Additional Infomation |
1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methyl-3-isoxazolyl)urea belongs to the urea class of compounds. PNU-120596 (NSC-216666) is a selective α7 neuronal nicotinic acetylcholine receptor positive allosteric modulator (PAM)[1][2][3] - Its mechanism of action involves inducing conformational changes in the extracellular domain of α7 nAChR, enhancing the binding affinity of acetylcholine and stabilizing the activation state of the receptor, thereby enhancing cholinergic neurotransmission[1][2] - It has potential therapeutic value and can be used to treat cognitive impairment (e.g., Alzheimer's disease) and inflammatory pain by regulating cholinergic signal transduction and inhibiting the release of pro-inflammatory cytokines[1][3] - It is highly selective for α7 neuronal subtypes. α7 nAChR, at therapeutic concentrations, has no significant activity against other nicotine or muscarinic cholinergic receptors [1]
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| Molecular Formula |
C13H14CLN3O4
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| Molecular Weight |
311.72
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| Exact Mass |
311.067
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| CAS # |
501925-31-1
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| Related CAS # |
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| PubChem CID |
311434
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| Appearance |
White to gray solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
385.0±42.0 °C at 760 mmHg
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| Flash Point |
186.6±27.9 °C
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| Vapour Pressure |
0.0±0.9 mmHg at 25°C
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| Index of Refraction |
1.625
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| LogP |
2.14
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
21
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| Complexity |
360
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
CEIIEALEIHQDBX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H14ClN3O4/c1-7-4-12(17-21-7)16-13(18)15-9-5-8(14)10(19-2)6-11(9)20-3/h4-6H,1-3H3,(H2,15,16,17,18)
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| Chemical Name |
1-(5-Chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (8.02 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), suspension solution; with sonication.
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. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.02 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. View More
Solubility in Formulation 3: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 10 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2080 mL | 16.0400 mL | 32.0801 mL | |
| 5 mM | 0.6416 mL | 3.2080 mL | 6.4160 mL | |
| 10 mM | 0.3208 mL | 1.6040 mL | 3.2080 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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