Rat 5-HT1B Receptor (IC50 = 47 nM)

NAS181 对已研究的所有其他受体表现出极低的亲和力 (Ki>3000 nM)，例如多巴胺 D1 和 D2、α1-、α2- 和 β- 肾上腺素受体、5-HT2A、5-HT2C、5-HT6、和 5-HT7 和 5-HT2[1]。在预加载的大鼠枕叶皮质切片中，NAS181 (10-1000 nM) 剂量依赖性地增加 K+ 刺激的 [3H]-5-HT 释放 [1]。

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在寻找新的5-羟色胺（5-HT）受体拮抗剂的过程中，发现化合物（R）-（+）-2-[[[3-（吗啉甲基）-2H-色烯-8-基]氧基]甲基]吗啉甲磺酸盐（R）-25/NAS-181是一种选择性大鼠5-羟色胺1B（r5-HT1B）受体拮抗药。结合谱显示，与牛5-HT1B（Ki=630nM；n=1）受体相比，r5-HT1B（Ki-47+/-5nM；n=3）受体的偏好是13倍。该化合物对所检测的其他单胺能受体的亲和力非常低。r5-HT1B受体拮抗作用通过增强K+刺激的[3H]-5-HT从体外灌流的大鼠脑片中的释放来证明，这种作用被向灌流液中添加5-HT所拮抗。[1]
（R）-25/NAS-181对小牛尾状膜5-HT1D受体（主要构成同源牛5-HT1B受体）的亲和力比大鼠r5-HT1B受体低10倍以上。（R） -25对所有其他受试受体的亲和力都很低，包括5-HT2A、5-HT2C、5-HT6和5-HT7、α1-、α2-和β肾上腺素受体，以及多巴胺D1和D2（表2）。[1]
（R）-25/NAS-181的r5-HT1B拮抗特性特征在于K+刺激的[3H]-5-HT从预加载的大鼠枕皮质切片中释放。如表3（R）所示，-25在10-1000nM的浓度范围内剂量依赖性地增强[3H]-5-HT的释放。此外，（R）-25引起的释放增强在1000nM时被拮抗，但在100nM未标记的5-HT进入灌流液时没有被拮抗（表4），这表明这两种化合物之间存在竞争。[1]

NAS181（1-10 mg/kg；皮下注射）可剂量依赖性地增加额叶、腹侧海马皮层和 VHipp 中的乙酰胆碱 (ACh) 释放量[1]。在四个大鼠大脑区域（纹状体、额叶皮质、海马和下丘脑）中，NAS181（20 mg/kg；皮下注射）使 5-HT 周转率增加约 40%[1]。给予 NAS181（3 mg/kg；皮下注射）的大鼠表现出湿狗奶昔数量显着增加[1]。

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（R） 皮下注射20mg/kg的-25（NAS-181）可增强大鼠四个脑区（下丘脑、海马、纹状体和额叶皮层）的5-HT代谢，用3-羟基苯肼抑制脱羧酶后，5-HTP的积累量约为40%。在3 mg/kg sc（R）-25下，大鼠湿狗奶昔的数量显著增加，这是一种5-HT2A/5-HT2C反应，在用色氨酸羟化酶抑制剂对氯苯丙氨酸预处理后，5-HT的耗竭消除了这种反应。这些观察结果表明，（R）-25通过抑制末端r5-HT1B自身受体，增加了5-HT的周转和5-HT的突触浓度。[1]

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本研究旨在探讨5-羟色胺（1B）受体拮抗剂NAS-181（（R）-（+）-2-（3-吗啉代甲基-2H-色烯-8-基）氧甲基吗啉甲磺酸盐）对大鼠脑内胆碱能、谷氨酸能和GABA能神经传递的影响。通过微透析监测自由运动大鼠不同组额叶皮层（FC）和腹侧海马（VHipp）中乙酰胆碱、谷氨酸和GABA的细胞外水平。NAS-181（1、5或10 mg/kg，皮下注射）引起ACh水平的剂量依赖性增加，在注射后80分钟达到对照组的500%（FC）和230%（VHipp）的最大值。相反，皮下注射10或20mg/kg剂量的NAS-181对这些区域的Glu和GABA的基础细胞外水平没有影响。目前的数据表明，FC和VHipp（与认知功能密切相关的大脑结构）中的ACh神经传递受这些区域胆碱能末端5-HT（1B）异质受体的紧张抑制控制[2]。

放射性配体结合研究。[1]
按照Jackson等人27的描述（括号中的放射性配体和组织）确定了以下受体的亲和力：5-HT1A（[3H]OH-DPAT，大鼠海马）、α1-肾上腺素受体（[3H]哌唑嗪，大鼠皮层）、α2-肾上腺素受体（[3H]RX821002（（1,4-[6,7-3H]苯并二恶烷-2-甲氧基-2-基）-2-咪唑啉盐酸盐，大鼠皮质）、β-肾上腺素受体（~3H]二氢阿普洛尔，大鼠大脑皮层）、多巴胺D1（[3H]1CH-23390，大鼠纹状体）；多巴胺D2（[3H]raclopride、细胞（LtkhD2A）膜）。如Johansson等人所述，测定了以下受体：28 5-HT2A（[3H]酮色林，大鼠皮层）、5-HT2C（[3H]mesulergine，大鼠皮质）、5-HT6（[3H]-5-HT，细胞（CHOr5-HT6）膜）、5-HT7（[3H'-5-HT，电池（CHOr5-T7）膜）。[125I]在60μM异丙肾上腺素存在的情况下，碘氰基吡啶洛尔被用作大鼠大脑皮层膜中r5-HT1B受体的配体，以避免与Hoyer等人所述的β肾上腺素受体结合。根据Heuring和Peroutka的方法，在8-OH-DPAT（100 nM）和美苏利精氨酸（100 nM）存在下，29小牛尾状体膜用于用[3H]-5-HT测定5-HT1D受体，以避免结合5-HT1A和5-HT2C受体；在这三种测定中使用了30μM的5-羟色胺来确定特异性结合。如Ross所述，用[3H]DTG（N，N'-二（邻甲苯基）胍）测定与整个大鼠脑膜中σ受体的结合。

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钾刺激预加载[3H]-5-HT的大鼠皮质切片的[3H]溢出。[1]
使用了Rényi等人描述的方法。将预加载[3H]-5-HT的枕叶皮层切片（0.3×0.3mm）用新鲜氧化的Krebs-Henseleit缓冲液（pH 7.4）进行超灌注，该缓冲液含有2.5μM西酞普兰，流速为0.4mL/min。洗涤50分钟后，收集4分钟的组分，62分钟后，给予含有25mM KCl的缓冲溶液4分钟，然后用原始缓冲液进行超灌注。将切片在缓冲液中以适当浓度用试验化合物超灌72分钟。从98分钟开始，第二次加入25 mM KCl和试验化合物4分钟，然后继续超灌，并在122分钟停止。通过液体闪烁计数各组分中的放射性和切片中的剩余放射性，并确定每个组分的释放百分比。测定第二次刺激（S2）后在试验化合物存在下的释放率与第一次刺激（S1）后的释放率，并将其表示为不含试验化合物的对照中相应比率的百分比。为了评估内在活性，试验化合物和5-HT在同一溶液中给药。

Animal/Disease Models: Adult male SD (Sprague-Dawley) rats (250-300 g)
Doses: 1, 5, 10 mg/kg
Route of Administration: Sc in the scruff of the neck
Experimental Results: Increased the ACh release in the frontal cortex, reaching the maximal value of 500% of the control group within 80 min after the injection of the highest dose. Increased the ACh releases in VHipp with a maximum of 230% of the control values at 80 min after the injection of the highest dose.

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On the day of the experiment, the animals were placed in the CMA/120 system for freely moving animals (CMA/Microdialysis) for an initial 2 h in order to habituate to the new environment. A CMA/12 microdialysis probe (8 mm shaft, 2 mm membrane length) was inserted into the guide cannula of the operated animals. The probes were perfused at a constant flow rate of 1 μl/min with the artificial cerebrospinal fluid (aCSF) containing 148 mM NaCl, 4 mM KCl, 0.8 mM MgCl2, 1.4 CaCl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.2 and 0.5 μM neostigmine. Following a 120-min stabilization period, the samples were collected every 20 min using a CMA/170 refrigerated fraction collector (CMA/Microdialysis). The first 4 samples were taken for determination of basal extracellular levels of ACh, Glu and GABA, thereafter, the drug (NAS-181) was injected subcutaneously (s.c.) in the scruff of the neck. The fractions were collected for an additional 3 h. [2]

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5-HT Turnover in Various Brain Regions. [1]
The rate of 5-HT turnover in hypothalamus, hippocampus, frontal cortex, and striatum in rats was determined as the accumulation of 5-hydroxytryptophan (5-HTP) after treatment with the 5-HTP decarboxylase inhibitor 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015), 100 mg/kg sc. The test compound was injected sc 30 min before NSD 1015, and the rats were killed 30 min after. The various brain regions were rapidly dissected out, frozen on dry ice, and stored at −70 °C. 5-HTP, 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) were analyzed by HPLC.

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Determination of Wet Dog Shake Behavior. [1]
The number of wet dog shakes, including whole body shake and head shake, was determined during 60 min, starting 5 min after the injection of the test compound. Groups of 8−10 rats were used and compared with saline-treated rats (n = 30).

[1]. (R)-(+)-2-[[[3-(Morpholinomethyl)-2H-chromen-8-yl]oxy]methyl] morpholine methanesulfonate: a new selective rat 5-hydroxytryptamine1B receptor antagonist. J Med Chem. 1998 May 21;41(11):1934-42.

[2]. Effects of the 5-HT1B receptor antagonist NAS-181 on extracellular levels of acetylcholine, glutamate and GABA in the frontal cortex and ventral hippocampus of awake rats: a microdialysis study. Eur Neuropsychopharmacol. 2007 Sep;17(9):580-6.

The receptor profile of (R)-25 shows that this compound is a selective r5-HT1B receptor ligand with moderate to high potency. Since (R)-25 has low affinity for the 5-HT1D receptor in calf caudate, which mainly appears to consist of the bovine 5-HT1B receptor, the receptor profile of (R)-25 differs from that of 2‘-methyl-4‘-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127,935) which has high affinity for the two homologous forms of the 5-HT1B receptor and also has high affinity for the 5-HT1D receptor. The affinity of (R)-25 for the latter receptor type remains to be elucidated.
The potentiation of the K+-stimulated [3H]-5-HT release from superfused slices of rat occipital cortex and the competition between (R)-25 and 5-HT in this in vitro model showed that (R)-25 is an antagonist at this receptor. Moreover, (R)-25 markedly increased the 5-HTP accumulation in all four brain regions examined in 3-hydroxybenzylhydrazine-treated rats and increased the 5-HIAA/5-HT ratio in the brain even more. These findings suggest that the 5-HT turnover was increased in these 5-HT terminal regions, supposedly due to the increased 5-HT release from the terminals. The induction of wet dog shake behavior by (R)-25 is in strong accordance with this notion since the response was abolished by depletion of 5-HT in the brain with pCPA. These observations indicate that, under normal conditions, the terminal 5-HT1B receptors have a functional role in determining the amount of 5-HT released from the terminals.
Since (R)-25 is a selective r5-HT1B receptor antagonist, this compound may become a valuable tool for studies of the functional role of the r5-HT1B receptors in rodents.[1]

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These reports provided a rationale to examine the effects of the 5-HT1B receptor antagonist NAS-181 on extracellular Glu and GABA levels by microdialysis sampling in awake rats. Systemic administration of NAS-181 at doses up to 20 mg/kg s.c. did not affect extracellular hippocampal and cortical Glu and GABA concentrations as shown in Figure 3, Figure 4. A possible explanation of this finding is that, under in vivo conditions, glutamatergic and GABA-ergic neurons are only under weak control of inhibitory 5-HT1B heteroreceptors. Another possible explanation is related to technical limitations of microdialysis to monitor neuronally released pools of Glu and GABA. The sources of extracellular Glu and GABA are both of neuronal and glial origin and a probability of sampling neuronally derived Glu and GABA by microdialysis was questioned recently (Timmerman and Westerink, 1997). To our knowledge, there are only few microdialysis studies addressing the modulatory role of 5-HT1B receptors on Glu and GABA release. Thus, Srkalovic et al. (1994) observed reduced basal Glu levels following administration of the 5-HT1B receptor agonist TMFPP, whereas a non-selective 5-HT receptor antagonist metergoline significantly increased extracellular Glu levels in the suprachiasmatic nucleus. In another study, veratridine-evoked increase in extracellular Glu and Asp levels in the rat FC were attenuated by coperfusion with CP-93,129 (Golembiowska and Dziubina, 2002). However, this microdialysis study, as well as, the above mentioned studies involving electrophysiological recording was carried-out at conditions of extensive stimulation and with 5-HT1B receptor agonists, which makes it difficult to draw conclusions on possible tonic modulatory activity of 5-HT1B receptors over the glutamatergic and GABA-ergic neurons in the rat brain. Nevertheless, the results of the present study indicate that the potent stimulatory effect of NAS-181 on ACh release is unlikely to be mediated via disinhibition of GABA-ergic afferents or interneurons in the FC and VHipp areas, rather there is a direct inhibitory control of ACh neurotransmission via 5-HT1B heteroreceptors at the cholinergic terminals.
In conclusion, the present microdialysis study shows that systemic administration of the 5-HT1B receptor antagonist NAS-181 causes marked and dose-dependent increases in extracellular ACh levels both in the frontal cortex and ventral hippocampus of awake rats. The concentrations of Glu and GABA are unaffected. These results suggest that ACh neurotransmission is under tonic inhibitory control by 5-HT1B heteroreceptors. Information on the specific involvement of the 5-HT1B heteroreceptors located at cholinergic terminals or at cell body levels in the rat basal forebrain requires further studies using a dual-probe microdialysis approach. [2]

C21H34N2O10S2

538.63

538.165

1217474-40-2

NAS-181;205242-62-2; 205242-61-1

45073445

Off-white to light yellow solid powder

178

3

12

5

35

545

1

CS(=O)(=O)O.CS(=O)(=O)O.C1CO[C@H](CN1)COC2=CC=CC3=C2OCC(=C3)CN4CCOCC4

WMRMIRRDPBKNMY-ZEECNFPPSA-N

InChI=1S/C19H26N2O4.2CH4O3S/c1-2-16-10-15(12-21-5-8-22-9-6-21)13-25-19(16)18(3-1)24-14-17-11-20-4-7-23-17;2*1-5(2,3)4/h1-3,10,17,20H,4-9,11-14H2;2*1H3,(H,2,3,4)/t17-;;/m1../s1

methanesulfonic acid;(2R)-2-[[3-(morpholin-4-ylmethyl)-2H-chromen-8-yl]oxymethyl]morpholine

NAS-181; 1217474-40-2; 205242-62-2; (2R)-2-[[[3-(4-Morpholinylmethyl)-2H-1-benzopyran-8-yl]oxy]methyl]morpholine dimethanesulfonate; NAS-181 dimesylate; methanesulfonic acid;(2R)-2-[[3-(morpholin-4-ylmethyl)-2H-chromen-8-yl]oxymethyl]morpholine; NAS181; (2R)-2-[[[3-(4-Morpholinylmethyl)-2H-1-benzopyran-8-yl]oxy]methyl]morpholinedimethanesulfonate;

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)

DMSO: 62.5 mg/mL (116.04 mM)

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。
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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)
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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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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：
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