β adrenergic receptor

Hyperthermia caused by MDMA (20 mg/kg) develops gradually and reaches a maximum of 1.8°C 130 minutes after injection. SR59230A (0.5 mg/kg) causes the gradually escalating hyperthermia in response to MDMA to be slightly but significantly attenuated. SR59230A (5 mg/kg) indicates that MDMA causes a notable and early hypothermic response[4].

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 [1].

---

Competitive radioligand binding [4]
Competition binding assays were carried out in duplicate in 5 mL polypropylene test tubes. Membrane aliquots of 100 µL were incubated with 100 µL of [3H]-prazosin (2 nmol·L−1; Specific activity: 85 Cinmol−1), and 100 µL of unlabelled test ligand (concentrations from 1 nmol·L−1 to 0.1 mmol·L−1), incubation buffer (vehicle) or phentolamine (10 µmol·L−1). Assays were performed at 25oC for 30 min. Following the 30 min incubation period, bound and free radioligand were separated by vacuum filtration. The assays were terminated by the addition of 5 mL ice-cold wash buffer (Tris-HCl 50 mmol·L−1, EDTA 5 mmol·L−1: pH 7.4 at 4oC) to all tubes. This was followed by rapid filtration through Whatman GF/C glass fibre filters using a Brandell Call Harvester. Filters and tubes were then washed four times with 5 mL of ice-cold wash buffer. Each filter was placed in a standard polypropylene scintillation vial and 5 mL of organic liquid scintillation medium was added to each vial. The vials were left overnight before being counted on a LKB 1214 Rack Beta counter.

Cell Line: Three different neuroblastoma (NB) cell lines, one murine (Neuro-2A) and two human (SK-N-BE(2), BE(2)C)
Concentration: 100 nM, 1 μM, 5 μM, 10 μM, and 50 μM
Incubation Time: 24 hours
Result: Reduced cell viability in a dose-dependent manner, with significant effect at a concentration limit over 1 µM for Neuro-2A cells and 5 µM for SK-N-BE(2) and BE(2)C).

---

MTT assay [3]
Viability of tumor cells was evaluated using an MTT assay. NB cells were treated for 24 h with different concentration of SR59230A and then maintained in MTT for 1 h at 37 °C before lysis with an equal volume of DMSO. The absorbance of the solubilized dye was evaluated at 570 nm using a spectrophotometer.

---

Neurosphere assay[3]
For neurosphere formation assay, 24-well plates were coated with 1,2% of Poly(2-hydroxyethyl methacrylate) diluted in 95% ethanol. Then, cells were plated (5.000/well) in Neurosphere basic medium composed of DMEM:F12 supplemented with 2% B27, 1% N2, 20 ng/ml FGF and 20 ng/ml EGF. After 24 h, cells were treated with 1 μM SR59230A and 1 μM BRL37344 alone, or in combination with 1 μM ABC294640 and 10 μM CYM5520. Once formed, spheres were disrupted and cells re-plated for a second passage (P2). After 7 days, neurosphere were counted and the diameter size measured using the ImageJ software (National Institutes of Health, U.S.). Neurosphere were then disrupted and stained for a flow cytometry analysis.

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).

---

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.

---

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]

---

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]

---

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]

---

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

174689-39-5

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

9888075

White to off-white solid powder

542.6ºC at 760mmHg

281.9ºC

1.27E-11mmHg at 25°C

3.202

4

7

8

30

433

2

O([H])[C@]([H])(C([H])([H])OC1=C([H])C([H])=C([H])C([H])=C1C([H])([H])C([H])([H])[H])C([H])([H])N([H])[C@]1([H])C2=C([H])C([H])=C([H])C([H])=C2C([H])([H])C([H])([H])C1([H])[H].O([H])C(C(=O)O[H])=O

XTBQNQMNFXNGLR-MKSBGGEFSA-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-;/m0./s1

1-(2-Ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol oxalate

SR 59230A; SR-59230A; (S)-1-(2-Ethylphenoxy)-3-(((S)-1,2,3,4-tetrahydronaphthalen-1-yl)amino)propan-2-ol oxalate; 3-(2-ethylphenoxy)-1-(1,2,3,4-tetrahydronaphth-1-ylamino)-2-propanol oxalate; Z4G2GB3YHU; 2-Propanol, 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-, (2S)-, ethanedioate (1:1); MFCD00940163; SR59230A; SR-59230A oxalate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)