β adrenergic receptor

SR59230A（100 nM-50 μM；24 小时）能够以剂量依赖性方式降低 Neuro-2A、BE(2)C 和 SK-N-BE(2) NB 细胞系的细胞活力[3]。细胞活力测定[3] 细胞系：三种不同的神经母细胞瘤 (NB) 细胞系，一种小鼠 (Neuro-2A) 和两种人类 (SK-N-BE(2)、BE(2)C) 浓度：100 nM，1 μM、5 μM、10 μM 和 50 μM 孵育时间：24 小时 结果：细胞活力以剂量依赖性方式降低，浓度限制超过 1 µM 对 Neuro-2A 细胞和 5 µM 对 SK-N 细胞有显着影响-BE(2) 和 BE(2)C)。

MDMA (20 mg/kg) 会产生缓慢发展的体温过高，注射后 130 分钟达到最高 1.8°C。 SR59230A (0.5 mg/kg) 对 MDMA 缓慢发展的高热产生小幅但显着的减弱作用。 SR59230A (5 mg/kg) 显示出对 MDMA 的显着且显着的早期低温反应[4]。动物模型：雄性C-57BL6J野生型小鼠（22-35克）[4] 剂量：0.5或5 mg/kg 给药方式：皮下注射；在皮下注射 MDMA (20 mg/kg) 前 30 分钟给药。结果：MDMA对温度的调节作用涉及α1-肾上腺素受体拮抗作用。

SS对映体3-（2-乙基苯氧基）-1-[（1S）-1,2,3,4-四氢决萘-1-基氨基]-（2S）-2-丙醇草酸盐（SR 59230A）被认为是第一种β3-肾上腺素能受体拮抗剂。本工作表明，SR 59230A不同于其非活性RR对映体（SR 59483），在体外拮抗典型的β3-肾上腺素能反应，即SR 58611A，乙基-[（7s）-7-[[（2R）-2-（3-氯苯基）-2-羟基乙基]氨基]-5,6,7,8-四氢萘-2-基]氧乙酸盐盐酸盐或（-）-4-（3-叔丁基氨基-2-羟基丙氧基）苯并咪唑-2-酮（CGP 12177）刺激的大鼠棕色脂肪组织膜中cAMP的合成，pKB值分别为8.87+/-0.12和8.20+/-0.15[1]。

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竞争性放射性配体结合[4]
竞争结合试验在5ml聚丙烯试管中进行，一式两份。100µL膜等分液与100µL [3H]-prazosin (2 nmol·L−1；比活性：85 Cinmol−1)，100µL未标记的测试配体（浓度从1 nmol·L−1到0.1 mmol·L−1），孵育缓冲液（载体）或酚托拉明（10µmol·L−1）。实验在25℃下进行30分钟。在30分钟的孵育期后，用真空过滤分离结合的和游离的放射性配体。在所有试管中加入5 mL冷水洗涤缓冲液（Tris-HCl 50 mmol·L−1,EDTA 5 mmol·L−1:pH 7.4, 4oC），终止实验。这是随后通过Whatman GF/C玻璃纤维过滤器使用布兰德尔呼叫收割机快速过滤。然后用5毫升冷水洗涤缓冲液洗涤过滤器和试管四次。每个过滤器被放置在一个标准的聚丙烯闪烁瓶和5毫升有机液体闪烁介质添加到每个瓶。小瓶放置过夜，然后在LKB 1214机架Beta计数器上计数。

细胞系：三种不同的神经母细胞瘤 (NB) 细胞系，一种小鼠 (Neuro-2A) 和两种人类 (SK-N-BE(2)、BE(2)C) 浓度：100 nM、1 μM、5 μM、10 μM 和 50 μM 孵育时间：24 小时 结果：细胞活力以剂量依赖性方式降低，浓度限制超过 1 µM 对 Neuro-2A 细胞和 5 µM 对 SK-N-BE(2) 和BE(2)C)。

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MTT试验[3]
使用MTT法评估肿瘤细胞的活力。用不同浓度的SR59230A处理NB细胞24 h，然后在37℃MTT中保持1 h，然后用等体积的DMSO裂解。用分光光度计在570nm处测定溶解染料的吸光度。

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Neurosphere试验[3]
对于神经球形成实验，24孔板涂有1.2%用95%乙醇稀释的聚（2-羟乙基甲基丙烯酸酯）。然后，将细胞（5.000/孔）接种于由DMEM:F12添加2% B27, 1% N2, 20 ng/ml FGF和20 ng/ml EGF组成的Neurosphere基本培养基中。24 h后，分别用1 μM SR59230A和1 μM BRL37344单独或与1 μM ABC294640和10 μM CYM5520联合作用细胞。一旦形成，球体被破坏，细胞重新镀上第二次传代（P2）。7天后，使用ImageJ软件（美国国立卫生研究院）计数神经球并测量直径大小。然后破坏神经球并染色进行流式细胞术分析。

Male C-57BL6J wild-type mice (22-35 g)
0.5 or 5 mg/kg
Injected s.c.; administered 30 min prior to the injection s.c. of MDMA (20 mg/kg).

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Tumor syngeneic model [3]
Female NCI A/JCr mice 4-weeks-old were used. Neuro-2A cells were subcutaneously implanted in A/J recipient mice by injecting 1 × 106 cells in 100 µl of PBS in the right flank. When Neuro-2A cells formed a palpable tumor (about 6 days), treatments started. The treatments were administrated twice a day for SR59230A and Vehicle, and once a day for ABC294640 and CYM5520. SR59230A was delivered at 10 mg/kg of physiological solution via intraperitoneal (i.p.); ABC294640 was delivered at 30 mg/kg in 0,375% of Polysorbate 80 in PBS via per os (p.o); CYM5520 was delivered at 5 mg/kg in 3.6% DMSO in PBS via i.p. Tumor growth rate was evaluated by measuring tumor mass with a caliber, and tumor mass volume calculated as Volume = [(length × width)2/2]. Mice were sacrificed after 8 days of treatment.

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Animals were injected s.c. with the β3-adrenoceptor antagonist SR59230A (0.5 or 5 mg·kg−1) or SR59230A (5 mg·kg−1) plus prazosin (0.1 mg·kg−1). Antagonists or vehicle were administered 30 min prior to the injection s.c. of vehicle (1 mL·kg−1) or MDMA (20 mg·kg−1).[4]

[1]. Functional studies of the first selective beta 3-adrenergic receptor antagonist SR 59230A in rat brown adipocytes.Mol Pharmacol. 1996 Jan;49(1):7-14.

[2]. Involvement of β3-adrenergic receptors in the control of food intake in rats.Braz J Med Biol Res. 2011 Nov;44(11):1141-7.

[3]. β3-adrenoreceptor blockade reduces tumor growth and increases neuronal differentiation in neuroblastoma via SK2/S1P2 modulation.Oncogene. 2020 Jan;39(2):368-384.

[4]. Role of alpha 1- and beta 3-adrenoceptors in the modulation by SR59230A of the effects of MDMA on body temperature in the mouse. Br J Pharmacol. 2009 Sep;158(1):259-66.

The SS-enantiomer 3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahy dronaphth-1-ylaminol]-(2S)-2-propanol oxalate (SR 59230A) is proposed to be the first beta 3-adrenergic receptor antagonist. The present work shows that SR 59230A, unlike its inactive RR-enantiomer (SR 59483), antagonized a typical beta 3-adrenergic response in vitro, i.e., SR 58611A, the ethyl-[(7s)-7-[[(2R)-2-(3- chlorophenyl)-2-hydroxethyl]amino]-5,6,7,8-tetrahydronaphth- 2- yl]oxyacetate hydrochloride- or (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP 12177)-stimulated synthesis of cAMP in rat brown adipose tissue membranes, with pKB values of 8.87 +/- 0.12 and 8.20 +/- 0.15. In addition, SR 59230A had no antagonistic effect on forskolin-induced cAMP accumulation in rat interscapular brown adipose tissue. SR 59230A, in contrast to the selective beta 1- and beta 2-adrenoceptor antagonists (+/-)[2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]- 3-[4(1-methyl-4-trifluoromethyl-2-imidazolyl)-phenoxy]-2 propanol and erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan- 2-ol-hydrochloride did not counteract the cAMP production induced by (-)-isoprenaline or norepinephrine (NE) in rat brain areas rich in beta 1- or beta 2-adrenoceptors, such as frontal cortex and cerebellum. Moreover, in proliferating brown fat cells, in which the beta 1-adrenoceptor is the only beta-adrenergic subtype coupled to cAMP production, SR 59230A did not modify the production of cAMP induced by NE, whereas CGP 12177 did. In confluent brown fat cells, in which the beta 3-adrenoceptor is the functional beta-adrenergic subtype coupled to adenylyl cyclase, SR 59230A antagonized the NE-induced cAMP accumulation and glycerol release without affecting their basal values, whereas CGP 12177, which per se stimulated cAMP accumulation and glycerol release, did not change the NE-induced increase of either parameter. Finally, SR 59230A concentration-dependently counteracted the NE-stimulated synthesis of uncoupling protein gene in confluent brown fat cells, which is considered mainly a result of selective stimulation of beta 3-adrenoceptors. These results provide evidence that the new selective beta 3-adrenoceptor antagonist can contribute considerably to functional characterization of the beta 3-adrenoceptors.[1]

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This study examined the food intake changes evoked by intracerebroventricular (icv) injection of a selective agonist (BRL37344, 2 and 20 nmol) or antagonist (SR59230A, 10 and 50 nmol) of β3-adrenergic receptors in 24-h fasted rats (adult male Wistar rats, 200-350 g, N = 6/treatment). The animals were also pretreated with saline icv (SAL) or SR59230A (50 nmol) followed by BRL37344 (20 nmol) or SAL in order to determine the selectivity of the effects evoked by BRL37344 on food intake or the selectivity of the effects evoked by SR59230A on risk assessment (RA) behavior. The highest dose of BRL37344 (N = 7) decreased food intake 1 h after the treatment (6.4 ± 0.5 g in SAL-treated vs 4.2 ± 0.8 g in drug-treated rats). While both doses of SR59230A failed to affect food intake (5.1 ± 1.1 g for 10 nmol and 6.0 ± 1.8 g for 50 nmol), this treatment reduced the RA frequency (number/30 min) (4 ± 2 for SAL-treated vs 1 ± 1 for 10 nmol and 0.5 ± 1 for 50 nmol SR59230A-treated rats), an ethological parameter related to anxiety. While pretreatment with SR59230A (7.0 ± 0.5 g) abolished the hypophagia induced by BRL37344 (3.6 ± 0.9 g), BRL37344 suppressed the reduction in RA frequency caused by SR59230A. These results show that the hypophagia caused by BRL37344 is selectively mediated by β3-adrenergic receptors within the central nervous system. Moreover, they suggest the involvement of these receptors in the control of anxiety.[2]

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Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P2) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P2 signaling. [3]

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Background and purpose: We have investigated the ability of the beta(3)-adrenoceptor antagonist 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A) to affect the hyperthermia produced by methylenedioxymethamphetamine (MDMA) in conscious mice and whether alpha(1)-adrenoceptor antagonist actions are involved. Experimental approach: Mice were implanted with temperature probes under anaesthesia, and allowed 2 week recovery. MDMA (20 mg x kg(-1)) was administered subcutaneously 30 min after vehicle or test antagonist and effects on body temperature monitored by telemetry. Key results: Following vehicle, MDMA produced a slowly developing hyperthermia, reaching a maximum increase of 1.8 degrees C at 130 min post injection. A low concentration of SR59230A (0.5 mg x kg(-1)) produced a small but significant attenuation of the slowly developing hyperthermia to MDMA. A high concentration of SR59230A (5 mg x kg(-1)) revealed a significant and marked early hypothermic reaction to MDMA, an effect that was mimicked by the alpha(1)-adrenoceptor antagonist prazosin. Functional and ligand binding studies revealed actions of SR59230A at alpha(1)-adrenoceptors. Conclusions and implications: 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride in high concentrations modulates the hyperthermic actions of MDMA in mice in two ways: by blocking an early alpha(1)-adrenoceptor-mediated component to reveal a hypothermia, and by a small attenuation of the later hyperthermic component which may possibly be beta(3)-adrenoceptor-mediated (this seen with the low concentration of SR59230A). Hence, the major actions of SR59230A in modulating the actions of MDMA on temperature involve alpha(1)-adrenoceptor antagonism.[4]

C23H29NO6

415.4795

415.199

C, 66.49; H, 7.04; N, 3.37; O, 23.10

1932675-95-0

SR59230A;174689-39-5; (2R)-SR59230A; 1932675-95-0; SR59230A hydrochloride; 1135278-41-9; 174689-38-4

White to off-white solid powder

116Ų

CCC1=CC=CC=C1OC[C@@H](CN[C@H]2CCCC3=CC=CC=C23)O.C(=O)(C(=O)O)O

XTBQNQMNFXNGLR-VDWUQFQWSA-N

InChI=1S/C21H27NO2.C2H2O4/c1-2-16-8-4-6-13-21(16)24-15-18(23)14-22-20-12-7-10-17-9-3-5-11-19(17)20;3-1(4)2(5)6/h3-6,8-9,11,13,18,20,22-23H,2,7,10,12,14-15H2,1H3;(H,3,4)(H,5,6)/t18-,20+;/m1./s1

(2R)-1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydronaphthalen-1-yl]amino]propan-2-ol;oxalic acid

(2R)-SR59230A; (2R)-SR-59230A; 1932675-95-0;

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)

DMSO: 31.25 mg/mL (75.21 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。
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；
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