α7 nAChR/α7 nicotinic acetylcholine receptor
PNU-282987 S enantiomer hydrochloride targets α7 nicotinic acetylcholine receptor (α7 nAChR) as a full agonist, with a Ki value of 12 nM ([¹²⁵I]-α-bungarotoxin binding assay) and an EC₅₀ value of 0.3 μM (ion channel activation assay in Xenopus oocytes) [1]

PNU-282987 (free base) (Compound C7) has a Ki of 27 nM and removes R7-selective anti-methylaconine (MLA) from brain homogenates [1]. PNU-282987: PNU-282987, with an IC50 value of 4541 nM, likewise has an inhibitory effect on the 5-HT3 receptor[1]. EC50 for α7 nAChR pyramiding agent activity is 154 nM [1].

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PNU-282987 S enantiomer hydrochloride exhibited high affinity for α7 nAChR, displacing [¹²⁵I]-α-bungarotoxin binding to rat brain membranes with >95% inhibition at 1 μM, showing 8-fold higher affinity than its R enantiomer [1]
- In Xenopus oocytes expressing human α7 nAChR: The compound induced full ion channel activation, with a maximal response 1.2-fold that of acetylcholine and 10-fold higher potency than the R enantiomer [1]
- It displayed excellent selectivity for α7 nAChR over other nAChR subtypes: Ki > 10 μM for α4β2, α3β4, and α1βγδ nAChRs, and no significant binding to muscarinic receptors (M1-M5) at concentrations up to 10 μM [1]

Compound C7 (iv; 1, 3 mg/kg) PNU-282987 (free base) reverses gating defects [1]. In a diluted and MLA-blockable manner, PNU-282987 (30 μM) stimulates currents in hippocampal neurons [1].

Brain homogenate binding assays ([3 H]-MLA, [3 H]-cytisine, [3 H]-GR65630): [1]
Male Sprague-Dawley rats (300-350 g) were sacrificed by decapitation and the brains (whole brain minus cerebellum) were dissected quickly, weighed and homogenized in 9 volumes/g wet weight of ice-cold 0.32 M sucrose using a rotating pestle on setting 50 (10 up and down strokes). The homogenate was centrifuged at 1,000 x g for 10 minutes at 40° C. The supernatant was collected and centrifuged at 20,000 x g for 20 minutes at 40° C. The resulting pellet was resuspended to a protein concentration of 1 - 8 mg/ml. Aliquots of 5 ml homogenate were frozen at -80° C until needed for the assay. On the day of the assay, aliquots were thawed at room temperature and diluted with Kreb’s - 20 mM HEPES buffer pH 7.0 (at room temperature) containing 4.16 mM NaHCO3, 0.44 mM KH2PO4, 127 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl2, and 0.98 mM MgCl2, so that 25 - 150 mg protein are added per test tube. Protein concentration was determined by the Bradford method using bovine serum albumin as the standard. For α7, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 µM MLA, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 3 nM [3 H]-MLA (25 Ci/mmol). For α4, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 mM (-)-nicotine, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 1.0 nM [3 H]-cytisine. For 5-HT3, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 µM ICS-205930, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 0.45 nM [3 H]-GR65630. For all binding assays, 0.4 ml homogenate was added to test tubes containing buffer, test compound and radioligand, and was incubated in a final volume of 0.5 ml for 1 h at 25°. The incubations were terminated by rapid vacuum filtration through Whatman GF/B glass filter paper mounted on a 48 well Brandel cell harvester. Filters were pre-soaked in 50 mM Tris HCl pH 7.0 - 0.05 % polyethylenimine. The filters were washed two times with 5 ml aliquots of cold 0.9% saline and then counted for radioactivity by liquid scintillation spectrometry. The inhibition constant (Ki) was calculated from the concentration dependent inhibition of radioligand binding obtained by fitting the data to the Cheng-Prusoff equation. PNU-282987 had a Ki in this assay of 27 ± 1 nM (n = 48).
PNU-282987 did not significantly displace tritiated cytisine from rat brain homogenates at 1 µM (inhibition = 14 ± 4 %, n=13) With respect to the 5-HT3 receptor, PNU-282987 displaced tritiated GR-65630 with a Ki of 1,662 ± 331 nM (n=10)

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α7 nAChR radioligand binding assay: Rat brain membranes enriched in α7 nAChR were prepared and incubated with [¹²⁵I]-α-bungarotoxin and serial dilutions of PNU-282987 S enantiomer hydrochloride at 25°C for 2 hours. Bound radioligand was separated from free ligand by filtration through glass fiber filters, and radioactivity was quantified to calculate the Ki value [1]
- α7 nAChR functional assay: Xenopus oocytes were injected with cRNA encoding human α7 nAChR and cultured at 18°C for 48–72 hours. The oocytes were placed in a recording chamber, and PNU-282987 S enantiomer hydrochloride (0.01–10 μM) was applied. Membrane potential was clamped at -70 mV, and inward currents were recorded using two-electrode voltage clamp to determine EC₅₀ and agonist efficacy [1]
- Enantioselectivity assay: Membranes and oocytes were treated with equal concentrations of S enantiomer, R enantiomer, and racemic mixture of PNU-282987 to compare their binding affinity and functional activity [1]

α7 nAChR activation assay in Xenopus oocytes: Defolliculated Xenopus oocytes were injected with human α7 nAChR cRNA. After incubation, the oocytes were exposed to PNU-282987 S enantiomer hydrochloride at various concentrations, and ion currents were measured to assess receptor activation. The S enantiomer’s activity was compared to other stereoisomers to confirm enantioselectivity [1]

Patch-clamp electrophysiology: Cultured neurons were prepared according to Brewer . Briefly, Sprague- Dawley rats (postnatal day 3) were killed by decapitation and their brains were removed and placed in ice cold Hibernate-A medium. Hippocampal regions were gently removed, cut into small pieces and placed in Hibernate-A medium with 1 mg/ml papain for 60 min at 35°C. After digestion, the tissues were washed several times in Hibernate-A media and transferred to a 50 ml conical tube containing 6 ml Hibernate-A medium with 2% B-27 supplement. Neurons were dissociated by gentle trituration and plated onto poly-D-lysine/laminin coated coverslips at a density of 300 – 700 cells/mm2 , and transferred to 24-well tissue culture plates containing warmed culture medium composed of Neurobasal-A medium, B-27 supplement (2%), L-glutamine (0.5 mM), 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml Fungizone. Cells were maintained in a humidified incubator at 37°C and 6% CO2 for 1 – 2 weeks. The medium was changed after 24 h and then approximately every three days thereafter. Patch pipettes were pulled from borosilicate capillary glass using a Flaming/Brown micropipette puller and filled with an internal pipette solution composed of (in mM): CsCH3SO3 (126), CsCl (10), NaCl (4), MgCl2 (1), CaCl2 (0.5), EGTA (5), HEPES (10), ATP-Mg (3), GTP-Na (0.3), phosphocreatin (4), pH 7.2. The resistances of the patch pipettes when filled with internal solution ranged between 3 – 6 M•. All experiments were conducted at room temperature. Cultured cells were continuously superfused with an external bath solution containing (in mM): NaCl (140), KCl (5), CaCl2 (2), MgCl2 (1), HEPES (10), glucose (10), bicuculline (0.01), CNQX (0.005), D-AP-5 (0.005) tetrodotoxin (0.0005), pH 7.4. Compounds were dissolved in water or DMSO and diluted into the external bath solution containing a final DMSO concentration of 0.1% and delivered via a multibarrel fast perfusion system. Whole-cell currents were recorded using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Analog signals were filtered at 1/5 the sampling frequency, digitized, stored, and measured using pCLAMP software (Axon Instruments). All data are reported as mean ± SEM. [1]

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Auditory gating assay. Experiments were performed on Male Sprague-Dawley rats (weighing 250 to 300 gm) under chloral hydrate anesthesia (400 mg/kg, IP). The femoral artery and vein were cannulated for monitoring arterial blood pressure and administration of drugs or additional doses of anaesthetic, respectively. Unilateral hippocampal field potential (EEG) was recorded by a metal monopolar macroelectrode placed into the CA3 region (co-ordinates: 3.0 – 3.5 mm posterior from the bregma, 2.6 –3.0 mm lateral and 3.8 – 4.0 mm ventral; Paxinos and Watson, 19863 ). Field potentials were amplified, filtered (0.1 – 100 Hz), displayed and recorded for on-line and off-line analysis (Spike3). Quantitative EEG analysis was performed by means of Fast Fourier Transformation (Spike3). The auditory stimulus consisted of a pair of 10 ms, 5 KHz tone bursts with a 0.5 s delay between the first “conditioning” stimulus and second “test” stimulus. Auditory evoked responses were computed by averaging of responses to 50 pairs of stimuli presented with an interstimulus interval of 10 s. Percentage of gating was determined by the formula: (1 - test amplitude/conditioning amplitude) x 100. Amphetamine (D-amphetamine sulfate, 0.3-1 mg/kg, IV) was administered in order to disrupt sensory gating. Recordings of evoke potentials commenced 5 min after amphetamine administration, and only rats showing gating deficit exceeding 20 % were used for subsequent evaluation of α7 nAChR agonists or vehicle. Statistical significance was determined by means of two-tailed paired Student’s t-test.

[1]. Bodnar AL, Discovery and structure-activity relationship of quinuclidine benzamides as agonists of alpha7 nicotinic acetylcholine receptors. J Med Chem. 2005 Feb 24;48(4):905-8.

We used chimeric receptors to test the agonistic activity of a series of benzamide compounds on α7 nicotinic acetylcholine receptors (nAChR) in a functional, cell-based high-throughput assay. The results showed that quinine-ring benzamide compounds have α7 nAChR agonistic activity. The structure-activity relationship of these compounds differed from that of the 5-HT3 receptor (a structural homolog of α7 nAChR). Among them, the most active compound, PNU-282987, was also shown to activate native α7 nAChR in cultured rat neurons and reverse amphetamine-induced gating defects in rat neurons. [1] In summary, we used parallel synthesis to explore the structure-activity relationship of tertiary amine-containing aromatic amide compounds as α7 nAChR agonists. Among the five structurally different amine compounds tested, only the 3-aminoquinine ring produced active analogs. The R enantiomers were more active than the corresponding S enantiomers. In the benzamide moiety, the small substituent at the para position yielded the most active analog. The most potent analog in this series is 4-chlorobenzamide, namely PNU-282987. Multiple experiments have confirmed that the α7-5HT3 chimerism assay can predict the activity of natural α7 nAChR. PNU-282987 displaced MLA from rat brain homogenate at a Ki value of 27 nM and induced currents in rat hippocampal neurons in a concentration-dependent and MLA-blocking manner. We also tested this compound in a rat model of impaired sensory gating. Treatment of rats with impaired gating with PNU-282987 reversed the gating deficits. These results suggest that (R)-3-aminoquinine cycloaromatic amides (e.g., PNU-282897) can serve as templates for the search for α7 nAChR agonists, which may contribute to the treatment of cognitive and attentional deficits in schizophrenia. [1]

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PNU-282987 S enantiomer hydrochloride is the pharmacologically active stereoisomer of PNU-282987, a synthetic quinine-ring benzamide derivative. [1]
- Its mechanism of action involves positive binding to α7 nAChR, promoting receptor oligomerization and cation channel opening, and possessing full agonist potency. [1]
- The S configuration is crucial for the high affinity and potency of α7 nAChR because the R enantiomer has very low activity (Ki > 96 nM, EC₅₀ > 3 μM) [1]
- It is a valuable tool compound for studying the function of α7 nAChR (especially stereoselectivity studies) and has potential applications in the study of neurological diseases [1]

C14H18CL2N2O

301.211521625519

300.079

C, 55.83; H, 6.02; Cl, 23.54; N, 9.30; O, 5.31

128311-08-0

PNU-282987;123464-89-1;PNU-282987 S enantiomer free base;737727-12-7;PNU-282987 free base;711085-63-1

18645636

Typically exists as solid at room temperature

2

2

2

19

307

1

C1CN2CCC1[C@@H](C2)NC(=O)C3=CC=C(C=C3)Cl.Cl

HSEQUIRZHDYOIX-BTQNPOSSSA-N

InChI=1S/C14H17ClN2O.ClH/c15-12-3-1-11(2-4-12)14(18)16-13-9-17-7-5-10(13)6-8-17;/h1-4,10,13H,5-9H2,(H,16,18);1H/t13-;/m1./s1

N-[(3S)-1-azabicyclo[2.2.2]octan-3-yl]-4-chlorobenzamide;hydrochloride

(S)-4-Chloro-N-(quinuclidin-3-yl)benzamide hydrochloride; (S)-PNU-282987 (hydrochloride); PNU-282987 S enantiomer hydrochloride; Benzamide, N-1-azabicyclo[2.2.2]oct-3-yl-4-chloro-, monohydrochloride, (S)-; N-[(3S)-1-Azabicyclo[2.2.2]octan-3-yl]-4-chlorobenzamide;hydrochloride; SCHEMBL9601902;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)