α7 nAChR/α7 nicotinic acetylcholine receptor
In rat brain homogenate, PNU-282987 (compound C7) has a Ki of 27 nM and replaces the R7-selective antagonist methylaconitine (MLA) [1]. With an EC50 of 154 nM, PNU-282987 exhibits α7 nAChR agonist activity [1]. Moreover, PNU-282987 blocks 5-HT3 receptors with an IC50 of 4541nM[1].

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Several assays were used to validate that the chimera assay could be used to identify agonists of native receptors. In a binding assay, PNU-282987 displaced the α7 selective antagonist methyllycaconitine (MLA) from rat brain homogenates with a Ki of 27 nM. Also, when applied to cultured rat hippocampal neurons, PNU-282987 evoked a rapidly desensitizing inward whole-cell current that was concentration-dependent and blockable by MLA, consistent with opening of the α7 receptor [1]

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The selectivity of PNU-282987 over related receptors was also evaluated. In particular researchers were concerned with agonism of the neuromuscular junction form of the receptor (α1β1γδ) and the predominant ganglionic nAChR (α3β4). Activation of these receptors was shown to cause many of the undesirable effects of nonspecific agonists such as epibatidine and nicotine. 15 PNU-282987 showed no detectable agonist activity up to 100 μM and negligible antagonist activity (IC50 ≥ 60 μM) at both receptor subtypes. Further, PNU-282987 did not significantly displace tritiated cytisine from rat brain homogenates at 1 μM (14% inhibition), suggesting a high selectivity over the α4β2 subtype. 16 With respect to the 5-HT3 receptor, PNU-282987 displaced tritiated GR-65630 with a Ki of 1662 nM, 17 translating into a selectivity of about 62-fold for α7 compared to the high selectivity of 1 for the 5-HT3R (over 500-fold). In a cell-based FLIPR assay, PNU-282987 was found to be a functional antagonist of the 5-HT3 receptor (IC50 = 4541 nM). Broader selectivity of PNU-282987 was evaluated in a screen of 32 receptors, ion channels, and enzymes at Cerep (Rueil-Malmaison, France). At a test concentration of 1 μM, PNU-282987 produced <30% inhibition of specific binding or enzyme activity at all targets except the 5-HT3 receptor[1].

在大鼠脑匀浆中，PNU-282987（化合物 C7）的 Ki 为 27 nM，可替代 R7 选择性拮抗剂甲基乌头碱 (MLA) [1]。 PNU-282987 的 EC50 为 154 nM，具有 α7 nAChR 激动剂活性 [1]。此外，PNU-282987 还能阻断 5-HT3 受体，IC50 为 4541nM[1]。

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使用了几种试验来验证嵌合体试验可用于鉴定天然受体的激动剂。在结合试验中，PNU-282987以27 nM的Ki从大鼠脑匀浆中置换了α7选择性拮抗剂甲基乌头碱（MLA）。此外，当应用于培养的大鼠海马神经元时，PNU-282987诱发了一种快速脱敏的内向全细胞电流，该电流具有浓度依赖性，可被MLA阻断，与α7受体的开放一致[1]

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还评估了PNU-282987对相关受体的选择性。研究人员特别关注神经肌肉接头形式的受体（α1β1γδ）和主要神经节nAChR（α3β4）的激动作用。这些受体的激活被证明会导致非特异性激动剂如依巴替丁和尼古丁的许多不良反应。15 PNU-282987在两种受体亚型上均未检测到高达100μM的激动剂活性，拮抗剂活性可忽略不计（IC50≥60μM）。此外，PNU-282987在1μM下没有显著取代大鼠脑匀浆中的氚化金雀花碱（14%的抑制率），表明其对α4β2亚型具有高选择性。16关于5-HT3受体，PNU-282987以1662 nM的Ki取代了氚化的GR-65630，17与5-HT3R的高选择性1（超过500倍）相比，α7的选择性约为62倍。在基于细胞的FLIPR测定中，发现PNU-282987是5-HT3受体的功能性拮抗剂（IC50=4541nM）。在Cerep（法国Rueil-Malmaison）的32个受体、离子通道和酶的筛选中评估了PNU-282987的更广泛选择性。在1μM的测试浓度下，PNU-282987对除5-HT3受体外的所有靶点的特异性结合或酶活性产生了<30%的抑制[1]。

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PNU-282987 在基于细胞的FLIPR实验中，被鉴定为α7-5HT₃嵌合受体的强效激动剂，EC₅₀为154 nM。在结合实验中，它能从大鼠脑匀浆中置换选择性α7拮抗剂甲基牛扁碱（MLA），K_i为27 nM。在原代培养的大鼠海马神经元中，PNU-282987 能以浓度依赖性方式（0.3-30 μM）诱发快速脱敏的内向全细胞电流，且该电流可被10 nM MLA阻断，证实了其对天然α7 nAChR的激活作用。
选择性分析表明，PNU-282987（浓度高达100 μM）对神经肌肉接头型（α1β1γδ）和神经节型（α3β4）nAChR亚型没有可检测的激动剂活性，拮抗活性也很弱（IC₅₀ ≥ 60 μM）。在1 μM浓度下，它不能显著置换大鼠脑匀浆中的[³H]cytisine（抑制率14%），表明对α4β2 nAChR亚型具有高选择性。在针对32种受体、离子通道和酶的广谱筛选中（测试浓度为1 μM），PNU-282987 对除5-HT₃受体外的所有靶点产生的抑制率均低于30%。[1]

PNU-282987（化合物 C7）（iv；1、3 mg/kg）可以逆转门控缺陷[1]。在大鼠海马神经元中，PNU-282987 (30 μM) 以浓度依赖性和 MLA 可阻断的方式刺激电流 [1]。

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研究人员还在感觉门控受损的大鼠模型中测试了PNU-282987，该模型已用已知的α7部分激动剂GTS-21进行了验证。18全身给药d-苯丙胺（0.3或1mg/kg，iv）显著扰乱了麻醉大鼠的听觉门控，因为条件反射同时降低，测试反应相应增加。随后给予α7 nAChR激动剂PNU-282987（iv，1或3 mg/kg，n=10）显著逆转了苯丙胺诱导的门控缺陷（图4）。相比之下，在对照组大鼠中应用赋形剂并没有使苯丙胺诱导的门控缺陷正常化（n=9）。此外，PNU-282987（1mg/kg）对麻醉大鼠的正常门控（n=4）没有显著影响。

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静脉全身给予 PNU-282987 （1 或 3 mg/kg）能显著逆转麻醉大鼠由右旋苯丙胺（0.3 或 1 mg/kg，静脉注射）诱导的听觉感觉门控缺陷。相反，给予载体不能使缺陷恢复正常。此外，PNU-282987 （1 mg/kg）对麻醉大鼠正常的听觉门控没有显著影响。[1]

脑匀浆结合试验（[3H]-MLA，[3H]-金雀花碱，[3H]-GR65630）：[1]
通过断头处死雄性Sprague-Dawley大鼠（300-350g），快速解剖大脑（全脑减去小脑），称重并在50℃下使用旋转杵在9体积/g湿重的0.32M冰冷蔗糖中均质化（10次上下击打）。将匀浆在40°C下以1000 x g离心10分钟。收集上清液，在40°C.下以20000 x g离心20分钟。将所得沉淀重新悬浮至蛋白质浓度为1-8mg/ml。将5ml匀浆的等分试样在-80°C.下冷冻，直至需要进行分析。在测定当天，将等分试样在室温下解冻，并用含有4.16 mM NaHCO3、0.44 mM KH2PO4、127 mM NaCl、5.36 mM KCl、1.26 mM CaCl2和0.98 mM MgCl2的Kreb's-20 mM HEPES缓冲液pH 7.0（在室温下）稀释，从而每个试管中添加25-150 mg蛋白质。以牛血清白蛋白为标准，采用Bradford法测定蛋白质浓度。对于α7，在放射性配体之前添加1µM MLA的情况下平行孵育的组织中测定了非特异性结合，在竞争研究中，在添加约3 nM[3H]-MLA（25 Ci/mmol）之前，将化合物以越来越高的浓度添加到试管中。对于α4，在放射性配体之前添加1 mM（-）-尼古丁的情况下平行孵育的组织中测定了非特异性结合，在竞争研究中，在添加约1.0 nM[3H]-金雀花碱之前，将化合物以越来越高的浓度添加到试管中。对于5-HT3，在放射性配体之前添加1µM ICS-205930的情况下，在平行孵育的组织中测定非特异性结合，在竞争研究中，在添加约0.45 nM[3H]-GR65630之前，将化合物以越来越高的浓度添加到试管中。对于所有结合试验，将0.4 ml匀浆加入含有缓冲液、试验化合物和放射性配体的试管中，并在25°下以0.5 ml的最终体积孵育1小时。通过安装在48孔Brandel细胞采集器上的Whatman GF/B玻璃滤纸进行快速真空过滤，终止培养。过滤器预先浸泡在50 mM Tris-HCl pH 7.0-0.05%聚乙烯亚胺中。用5ml等分的0.9%冷盐水洗涤过滤器两次，然后通过液体闪烁光谱法计算放射性。抑制常数（Ki）是通过将数据拟合到Cheng-Prusoff方程中获得的放射性配体结合的浓度依赖性抑制来计算的。PNU-282987在该测定中的Ki为27±1nM（n=48）
PNU-282987在1µM时没有显著置换大鼠脑匀浆中的氚化金雀花碱（抑制率=14±4%，n=13）。就5-HT3受体而言，PNU-28298.7置换了氚化GR-65630，Ki为1662±331 nM（n=10）

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使用大鼠脑匀浆进行放射性配体结合实验。该实验测量PNU-282987与选择性α7拮抗剂[³H]甲基牛扁碱（MLA）竞争结合的能力。将匀浆与[³H]MLA及不同浓度的测试化合物共同孵育。通过过滤分离结合放射性，并根据竞争曲线计算K_i值。[1]

α7/5-HT3嵌合体、5-HT3、神经肌肉接头（α1β1γδ）和神经节（α3β4）nAChRs的功能性高通量筛选：[1]
α7/5-HT3嵌合体和5-HT3受体在SH-EP1细胞中稳定表达。TE671和SH-SY5Y细胞分别用作神经肌肉接头和神经节nAChRs的内源性来源。所有功能性高通量筛选均使用荧光成像板读数器进行钙通量测定。转染的SH-EP1细胞在含有非必需氨基酸的最低必需培养基（MEM）中生长，该培养基补充了10%胎牛血清、L-谷氨酰胺、100单位/ml青霉素/链霉素、250 ng/ml真菌素、400µg/ml Hygromycin-B和800µg/ml Geneticin。根据已发表的方法培养TE671和SH-SY5Y细胞。所有细胞均在37°C、6%CO2的培养箱中生长。在分析前两天，细胞被胰蛋白酶消化，并以每孔26×104个细胞的密度铺在96孔板中，板壁深色，底部透明。将细胞装载在2 mM钙绿-1、在无水二甲亚砜中制备的AM和20%普朗尼克F-127的1:1混合物中。将该试剂直接添加到每个孔的生长培养基中，以达到2µM钙绿-1，AM的最终浓度。然后将细胞在37°C的染料中孵育一小时，然后用4个周期的Bio-Tek洗板机洗涤。每个循环被编程为用Mark改良的Earle平衡盐溶液（MMEBSS）洗涤每个孔四次，该溶液由以下成分组成（单位为mM）：CaCl2（4）、MgSO4（0.8）、NaCl（20）、KCl（5.3）、D-葡萄糖（5.6）、Tris-HPES（20），N-甲基-D-葡糖胺（120），pH 7.4。第三个循环后，让细胞在37°C下孵育至少10分钟。每个孔中MMEBSS的最终体积为100µl。FLIPR被设置为使用500 mW的功率在488 nm处激发钙绿，并读取525 nm以上的荧光发射。使用0.5秒的曝光时间照射每个孔。使用2.0或1.2的F-stop集检测荧光。具体来说，在基线记录30秒后，使用3倍原液中的50µL将测试化合物添加到96孔板的每个孔中。在每个实验中，使用4个孔作为溶剂对照。PNU-282987基于细胞的FLIPR检测数据：α7/5-HT3受体嵌合体（EC50=178±5 nM（n=70））。5-羟色胺3受体功能拮抗剂（IC50=4541±413 nM，n=46）。α3：在高达100µM的浓度下没有可检测到的激动剂活性（n=69），对于拮抗剂活性，IC50≥60µM（n=70）。

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使用荧光成像板读数仪（FLIPR）实验评估化合物对α7-5HT₃嵌合受体的激动剂活性。表达嵌合受体的SHEP细胞预先装载钙敏感荧光染料。测量加入化合物后细胞内钙流的变化，作为受体激活的指标，并根据浓度-反应曲线确定EC₅₀值。
使用全细胞膜片钳电生理学技术在原代培养的大鼠海马神经元上评估化合物对天然α7 nAChR的功能活性。将神经元电压钳制，记录由浴槽施加PNU-282987所诱发的内向电流。这些电流可被共同施加的MLA所阻断，从而确认了对α7 nAChR的特异性。
使用基于FLIPR的功能实验在适当的细胞系上测定化合物对5-HT₃受体的拮抗活性，测量PNU-282987对标准激动剂反应的抑制作用。[1]

Animal/Disease Models: Rat[1]
Doses: 1, 3 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Dramatically reversed amphetamine-induced gating defects.

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\n\nPatch-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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\n\nAuditory 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]

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The auditory sensory gating effect was evaluated in anesthetized rats. Rats were anesthetized and prepared for recording auditory evoked potentials. A sensory gating deficit was induced by intravenous administration of D-amphetamine (0.3 or 1 mg/kg). PNU-282987 (1 or 3 mg/kg) was administered intravenously following amphetamine. Auditory evoked responses (conditioning and test pulses) were recorded, and the ratio of the test to conditioning response amplitude (T/C ratio) was calculated to assess gating. Lower T/C ratios indicate better sensory gating. Control animals received vehicle instead of the test compound.[1]

[1]. Discovery and structure-activity relationship of quinuclidine benzamides as agonists of alpha7 nicotinic acetylcholine receptors. J Med Chem.

PNU-282987 is a potent alpha7 nicotinic acetylcholine receptor (nAChR) agonist. PNU-282987 is also a functional antagonist of the 5-HT3 receptor. PNU-282987 can be used for the research of central and peripheral nervous systems.

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PNU-282987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide) is the most potent compound from a series of quinuclidine benzamides discovered as α7 nAChR agonists. The (R)-enantiomer of 3-aminoquinuclidine is the preferred configuration for α7 activity, which is opposite to the SAR for 5-HT₃ receptor antagonism within the same chemical class. Small para-substituents (e.g., Cl, F, OMe, Me) on the benzamide ring are critical for potency; increased steric bulk at this position drastically reduces activity. The compound serves as a selective pharmacological tool and a potential template for developing agents to treat cognitive and attentional deficits associated with conditions like schizophrenia, based on its ability to reverse sensory gating deficits in a rodent model.[1]

C14H18CL2N2O

301.21

300.079

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

123464-89-1

737727-12-7 (S enantiomer); 128311-08-0 (S enantiomer hydrochloride); PNU-282987 free base;711085-63-1

11243536

White to off-white solid powder

1.3±0.1 g/cm3

431.5±30.0 °C at 760 mmHg

214.8±24.6 °C

0.0±1.0 mmHg at 25°C

1.612

2.49

32.34

2

2

2

19

307

1

O=C(N[C@H]1CN2CCC1CC2)C3=CC=C(Cl)C=C3.[H]Cl

HSEQUIRZHDYOIX-ZOWNYOTGSA-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-;/m0./s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: 1 mg/mL (3.32 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 10.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1 mg/mL (3.32 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 10.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 50 mg/mL (166.00 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。