ADX71743

mGlu7/metabotropic glutamate receptor 7

ADX71743 的内部细胞系的 IC50 为 300 nM。当 ADX71743（3 μM；20 分钟）在高频刺激 (HFS) 之前进行预处理时，LTP 诱导几乎完全被阻断 [1]。 ADX71743 (0.1, 10 μM) 促进 L-AP4 诱导的抑制的浓度依赖性逆转，并逆转 L-AP4 引起的突触传递抑制。 10 μM 和 0.1 μM ADX71743 可以分别逆转 10% 和 11% 的 L-AP4 效应 [2]。 ADX71743 的 EC80（IC50 为 22 nM）拮抗谷氨酸，而 L-AP4 的 EC80（IC50 为 125 nM）也具有同样的作用 [2]。

在较低剂量（50 和 100 毫克/千克）下，ADX71743（50、100 和 150 毫克/千克；SC）可将埋藏大理石的数量显着降低至接近最大值 [2]。在小鼠中，ADX71743（小鼠 12.5、100 mg/kg，大鼠 100 mg/kg；SC）的 Cmax 为 1380、12766 ng/ml（12.5 mg/kg 和 100 mg/kg），T1/2 为 0.68， 0.40 小时 [2]。

在八种代谢型谷氨酸（mGlu）受体亚型中，只有mGlu7在成年动物海马Schaffer侧支（SC）-CA1突触的突触前表达。结合III组mGlu受体激动剂对该突触传递的抑制作用，mGlu7被认为是负责调节SC末端谷氨酸释放的主要自身受体。然而，mGlu7选择性药理学工具的缺乏阻碍了这一假设的直接检验。我们使用了一种新型的选择性mGlu7负变构调节剂（NAM）ADX71743和一种新描述的III组mGlu受体激动剂LSP4-2022，来阐明mGlu7在调节成年C57BL/6J雄性小鼠海马CA1区传递中的作用。有趣的是，尽管mGlu7激动剂抑制SC-CA1 EPSPs，但我们没有发现刺激SC-CA1传入神经激活mGlu7的证据。然而，LSP4-2022也降低了CA1锥体细胞中诱发的单突触IPSC，与对SC-CA1 EPSP的影响相反，ADX71743逆转了SC传入子高频刺激降低IPSC振幅的能力。此外，阻断mGlu7可阻止SC-CA1突触LTP的诱导，mGlu7的激活可增强次最大LTP。总之，这些数据表明，mGlu7在CA1区的抑制性突触中起着异质受体的作用，通过刺激谷氨酸能传入物激活mGlu7的主要作用是去抑制，而不是减少兴奋性传递。此外，这种mGlu7介导的去抑制是SC-CA1突触诱导LTP所必需的，这表明mGlu7可以作为治疗认知障碍的新治疗靶点[1]。

cAMP[2]
如前所述（Chruścicka等人，2015），用重组细胞系进行了均匀时间分辨荧光（HTRF）cAMP动态2测定。简而言之，收集稳定表达mGlu7受体的HEK 293 T-REx细胞，并将其悬浮在Hanks HEPES缓冲液中。将细胞悬浮液加入含有5μM毛喉素（终浓度）的化合物溶液中。在37°C下孵育5分钟后，加入裂解缓冲液中的5μl cAMP-d2缀合物，并通过自动移液系统与10μl细胞悬浮液混合。接下来，加入5μl抗cAMP隐窝结合物，1小时后读取620和665nm处的荧光。结果显示为665nm与620nm的比值乘以104。检测到的信号与样品中cAMP的浓度成反比。ADX71743或MMPIP的拮抗活性以其EC80浓度下L-Glu活性抑制的百分比表示。使用Prism 7.03版分析来自ADX71743或MMPIP的剂量反应数据。每个实验进行三次（n=3），每个数据点一式三份。

Animal/Disease Models: Adult male C57Bl6/J mice (24-30 g) and SD (SD (Sprague-Dawley)) rats (250-350 g) [2]
Doses: mice 12.5, 100 mg/kg, rats 100 mg/kg (drug Metabokinetic analysis)
Route of Administration: SC
Experimental Results: T1/2 in mice were 0.68, 0.40 hrs (hrs (hours)), Cmax were 1380, 12766ng/ml, 12.5mg/kg and 100mg/kg. The T1/2 of 100 mg/kg in rats is 1.5 hrs (hrs (hours)) and the Cmax is 16800 ng/ml.

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ADX71743 was dissolved in small amount of DMSO and then titrated in 20% captisol [Front Mol Neurosci. 2018 Sep 20;11:316.]

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Pharmacokinetic Studies[Front Mol Neurosci. 2018 Sep 20;11:316.]
The method described below was successfully applied to a pharmacokinetic study of ADX71743 and MMPIP in mouse (Albino Swiss) after i.p. injection. Compound ADX71743 and MMPIP were administered to mice at 10 mg/kg i.p. At 0.25, 0.50, 1.0, 2.0, 4.0, 6.0 h, the mice were anesthetized, and the blood was collected from the portal vein to the tubes containing 5% EDTA. The mice were then perfused with 0.1M PBS to remove remaining blood from the body, and the brains were taken out for the analysis. Blood was centrifuged at 2000 rpm for 10 min at 4°C, and the plasma was collected and frozen at -80°C for further analysis. [2]
Plasma and tissue samples from all drug-treated animals were thawed at room temperature prior to use. Standard protocol of sample preparation: 200 μl acetonitrile was added to the eppendorfs with 50 μl of studied plasma samples or tissue homogenate. Samples were mixed for 5 min on a mixer at 25°C and 1400 rpm. Tubes were then centrifuged at 2000 × g for 15 min at 4°C. About 180 μl of each supernatant was transferred into a plate well. Finally, each sample was injected into the column.

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MK-801-Induced Hyperactivity[Front Mol Neurosci. 2018 Sep 20;11:316.]
The locomotor activity was recorded individually for each animal in OPTO-M3 locomotor activity cages linked online to a compatible PC activity, as described previously by Woźniak et al., 2016b. Each cage (13 cm × 23 cm × 15 cm) was surrounded with an array of photocell beams. Interruptions of these photobeams resulted in horizontal activity defined as ambulation counts. The mice were placed in the locomotor activity cages for acclimatization for 30 min Then, MMPIP (10, 15 mg/kg) or ADX71743 (5, 10 mg/kg) were administered i.p. Both drugs were given 30 min prior to MK-801 injection (0.35 mg/kg, i.p.). The locomotor activity was measured for 60 min immediately after MK-801 administration.

The pharmacokinetic analysis of ADX71743 in mice and rats revealed that it is bioavailable after s.c. administration and is brain penetrant (cerebrospinal fluid concentration/total plasma concentration ratio at C(max) = 5.3%). [2]

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The concentration of ADX71743 and MMPIP in mouse plasma and brain are shown in Table ​Table11. Cmax was evident in brain and plasma 0.25 h after injection of ADX71743, and 0.5 h after MMPIP administration. Figure ​Figure33 represents comparison between ADX71743 and MMPIP concentrations in the brain in selected time points after administration.[Front Mol Neurosci. 2018 Sep 20;11:316.]
Data presented in Table ​Table22 showed that ADX71743 and MMPIP had different cytochrome P450 inhibition profile. Weak inhibition (IC50 > 10μM) of cytochrome P450 was observed in case of 1A2, 2B6, 2C9, 2D6 isoforms for both NAM mGluR7 standards. Mild inhibition (3.3 < IC50 < 10) of isoform 2C19 was determined for ADX71743 standard, while strong inhibition (IC50 < 1.1) was observed only for MMPIP in case of isoform 3A4 as well as 2C19.[Front Mol Neurosci. 2018 Sep 20;11:316.]

[1]. Activation of Metabotropic Glutamate Receptor 7 Is Required for Induction of Long-Term Potentiation at SC-CA1 Synapses in the Hippocampus. J Neurosci. 2015 May 13;35(19):7600-15.

[2]. ADX71743, a Potent and Selective Negative Allosteric Modulator of Metabotropic Glutamate Receptor 7: In Vitro and in Vivo Characterization. Pharmacol Exp Ther. 2013 Mar;344(3):624-36.

Metabotropic glutamate receptor 7 (mGlu(7)) has been suggested to be a promising novel target for treatment of a range of disorders, including anxiety, post-traumatic stress disorder, depression, drug abuse, and schizophrenia. Here we characterized a potent and selective mGlu(7) negative allosteric modulator (NAM) (+)-6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one (ADX71743). In vitro, Schild plot analysis and reversibility tests at the target confirmed the NAM properties of the compound and attenuation of L-(+)-2-amino-4-phosphonobutyric acid-induced synaptic depression confirmed activity at the native receptor. The pharmacokinetic analysis of ADX71743 in mice and rats revealed that it is bioavailable after s.c. administration and is brain penetrant (cerebrospinal fluid concentration/total plasma concentration ratio at C(max) = 5.3%). In vivo, ADX71743 (50, 100, 150 mg/kg, s.c.) caused no impairment of locomotor activity in rats and mice or activity on rotarod in mice. ADX71743 had an anxiolytic-like profile in the marble burying and elevated plus maze tests, dose-dependently reducing the number of buried marbles and increasing open arm exploration, respectively. Whereas ADX71743 caused a small reduction in amphetamine-induced hyperactivity in mice, it was inactive in the mouse 2,5-dimethoxy-4-iodoamphetamine-induced head twitch and the rat conditioned avoidance response tests. In addition, the compound was inactive in the mouse forced swim test. These data suggest that ADX71743 is a suitable compound to help unravel the physiologic role of mGlu(7) and to better understand its implication in central nervous system diseases. Our in vivo tests using ADX71743, reported here, suggest that pharmacological inhibition of mGlu(7) is a valid approach for developing novel pharmacotherapies to treat anxiety disorders, but may not be suitable for treatment of depression or psychosis.[2]

C17H19NO2

269.338264703751

269.141578

C, 75.81; H, 7.11; N, 5.20; O, 11.88

1431641-29-0

53391766

White to off-white solid powder

3.8

43.1Ų

0

3

2

20

370

0

O1C(CC)=NC2C(CC(C3C=CC(C)=CC=3C)CC1=2)=O

CPKZCQHJDFSOJT-UHFFFAOYSA-N

InChI=1S/C17H19NO2/c1-4-16-18-17-14(19)8-12(9-15(17)20-16)13-6-5-10(2)7-11(13)3/h5-7,12H,4,8-9H2,1-3H3

6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydro-5H-1,3-benzoxazol-4-one

(+/-)-ADX71743; CHEMBL4174742; 6-(2,4-Dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one; 6-(2,4-dimethylphenyl)-2-ethyl-4,5,6,7-tetrahydro-1,3-benzoxazol-4-one; 6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydro-5H-1,3-benzoxazol-4-one; (+)-6-(2,4-Dimethylphenyl)-2-ethyl-6,7-dihydro-4(5H)-benzoxazolone;

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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