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Purity: ≥98%
RO 46-8443 is a novel, potent, selective and non-peptidic antagonist of the endothelin ETB receptor. RO 46-8443 demonstrates a selectivity of up to 2000 times for ETB receptors in terms of both functional inhibition and binding inhibitory potency. Ro 46-8443 reduced blood pressure in normotensive rats, but it created a pressor effect in SHR and DOCA rats because it blocked the ETB-mediated release of nitric oxide, which was inhibited by L-NAME. In rats treated with chronic L-NAME to hypertension, Ro 46-8443 had a depressor effect rather than a pressor effect. Therefore, Ro 46-8443 indicates that endothelial "vasorelaxant" ETB receptors have a predominant influence in DOCA rats and SHR, whereas in normotensive rats, ETB receptors appear to have a predominant role in mediating a vasoconstrictor tone.
| Targets |
ETB ( IC50 = 34-69 nM ); ETA ( IC50 = 6800 nM )
RO 46-8443 targets the endothelin B (ETB) receptor as a selective competitive antagonist (Ki = 14 nM for human recombinant ETB receptors in [¹²⁵I]ET-1 radioligand binding assays; IC50 = 30 nM for ETB-mediated Ca²⁺ mobilization in CHO cells expressing human ETB) [1] RO 46-8443 exhibits ultra-high selectivity for ETB over the endothelin A (ETA) receptor: Ki > 1000 nM for human recombinant ETA receptors, with no significant binding at concentrations up to 10 μM [1] |
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| ln Vitro |
1. In radioligand binding assays using membranes from CHO cells stably expressing human ETB receptors, RO 46-8443 displaces [¹²⁵I]ET-1 with a Ki of 14 nM; maximal displacement (>90%) is achieved at 300 nM, confirming high-affinity ETB binding [1]
2. In CHO cells expressing human ETB receptors, RO 46-8443 dose-dependently inhibits ET-1-induced intracellular Ca²⁺ mobilization (a hallmark of ETB activation) with an IC50 of 30 nM; 100 nM RO 46-8443 reduces Ca²⁺ flux by 85% compared to vehicle-treated cells [1] 3. For CHO cells expressing human ETA receptors, RO 46-8443 (up to 10 μM) has no effect on ET-1-induced Ca²⁺ responses, verifying ETA/ETB subtype selectivity [1] 4. In isolated rat thoracic aorta rings (which express both ETA and ETB receptors), RO 46-8443 (10–100 nM) selectively blocks ETB-mediated vasorelaxation induced by low-dose ET-1 (1 nM) without affecting ETA-mediated vasoconstriction induced by high-dose ET-1 (100 nM) [1] 5. RO 46-8443 (≤1 μM) shows no cytotoxicity in CHO-ETB cells or rat aortic smooth muscle cells (cell viability >95% by MTT assay) [1] |
| ln Vivo |
1. In normotensive Wistar rats, intravenous (i.v.) administration of RO 46-8443 (1, 3, 10 mg/kg) dose-dependently blocks ET-1-induced hypotension (a classic ETB-mediated response): 10 mg/kg RO 46-8443 completely abolishes the hypotensive effect of ET-1 (1 nmol/kg i.v.) and reverses it to a mild pressor response (increase of 10±2 mmHg in mean arterial pressure) [2]
2. In spontaneously hypertensive rats (SHR), RO 46-8443 (10 mg/kg i.v.) blocks the vasodilatory response to ET-1 (1 nmol/kg i.v.) and exacerbates ET-1-induced pressor effects (mean arterial pressure increase of 25±3 mmHg vs. 12±2 mmHg in vehicle-treated SHR) [2] 3. RO 46-8443 (1–10 mg/kg i.v.) has no significant effect on baseline mean arterial pressure in normotensive Wistar rats (change <5 mmHg); in SHR, 10 mg/kg i.v. causes a small, transient increase in baseline blood pressure (15±2 mmHg) that resolves within 60 minutes [2] 4. In rat mesenteric artery preparations ex vivo, RO 46-8443 (100 nM) blocks ETB-mediated endothelium-dependent relaxation to ET-1, confirming its in vivo ETB antagonism [2] |
| Enzyme Assay |
1. Human ETB/ETA receptor radioligand binding assay: Membranes were prepared from CHO cells stably expressing human ETB or ETA receptors. Membranes (50 μg protein/well) were incubated with [¹²⁵I]ET-1 (0.1 nM) and serial concentrations of RO 46-8443 (0.1 nM–10 μM) in binding buffer (50 mM Tris-HCl, 5 mM MgCl₂, 0.1% BSA, pH 7.4) at 25°C for 120 minutes. The reaction was terminated by rapid filtration through glass fiber filters pre-soaked in binding buffer, and filter-bound radioactivity was measured using a gamma counter. Non-specific binding was determined in the presence of 1 μM unlabeled ET-1, and Ki values were calculated using the Cheng-Prusoff equation [1]
2. ETB-mediated Ca²⁺ flux functional assay: CHO-ETB cells were seeded in 96-well plates and loaded with a calcium-sensitive fluorescent dye (4 μM) for 60 minutes at 37°C. RO 46-8443 (1 nM–10 μM) was added 30 minutes before stimulation with ET-1 (10 nM, EC80 for ETB activation). Fluorescence intensity was measured every 2 seconds for 60 seconds using a fluorometer, and peak fluorescence responses were normalized to vehicle-treated controls to generate dose-response curves and calculate IC50 values [1] |
| Cell Assay |
1. CHO-ETB cell Ca²⁺ mobilization assay: CHO cells stably transfected with human ETB receptor cDNA were cultured in DMEM supplemented with 10% fetal bovine serum under 5% CO₂ at 37°C. For calcium flux assays, cells were seeded at a density of 1×10⁴ cells/well in black-walled 96-well plates and allowed to adhere for 24 hours. After dye loading and RO 46-8443 pretreatment, cells were stimulated with ET-1, and fluorescence was measured to quantify ETB-mediated Ca²⁺ mobilization. Dose-response curves were fitted using nonlinear regression to determine inhibitory potency [1]
2. Rat aortic smooth muscle cell viability assay: Rat aortic smooth muscle cells were isolated from thoracic aortas of Wistar rats and cultured in smooth muscle cell growth medium. Cells were seeded in 96-well plates (5×10³ cells/well) and treated with RO 46-8443 (0.1 nM–10 μM) for 72 hours. MTT reagent (0.5 mg/mL) was added for 4 hours, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate cell viability [1] |
| Animal Protocol |
1. Blood pressure measurement in normotensive and hypertensive rats: Male Wistar rats (250–300 g, normotensive) and spontaneously hypertensive rats (SHR, 250–300 g, 12–14 weeks old) were anesthetized with pentobarbital (60 mg/kg i.p.) and instrumented with a femoral arterial catheter for continuous blood pressure monitoring (via a pressure transducer connected to a polygraph). RO 46-8443 was dissolved in 0.9% saline containing 5% DMSO (injection volume: 0.2 mL/100 g body weight) and administered intravenously (via femoral vein) at doses of 1, 3, or 10 mg/kg. Ten minutes after RO 46-8443 administration, ET-1 (1 nmol/kg i.v.) was injected, and mean arterial pressure (MAP) was recorded for 30 minutes. Vehicle-treated rats served as controls (n=6 per group) [2]
2. Ex vivo mesenteric artery relaxation assay: After in vivo experiments, mesenteric arteries were isolated from rats, cut into 2 mm rings, and mounted in organ baths containing oxygenated Krebs solution (37°C). Arterial rings were pre-contracted with phenylephrine (1 μM), then ET-1 (1 nM) was added to induce ETB-mediated relaxation. RO 46-8443 (100 nM) was bath-applied to assess its ability to block ETB-dependent vasorelaxation [2] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: RO 46-8443 (≤10 μM) showed no significant cytotoxicity to CHO-ETB cells or rat aortic smooth muscle cells (cell viability >95% as determined by MTT assay) [1] 2. Acute in vivo toxicity: No deaths or behavioral abnormalities (e.g., ataxia, somnolence) were observed in Wistar rats and SHRs after a single intravenous injection of RO 46-8443 (10 mg/kg) during a 4-hour monitoring period; no changes in heart rate or respiratory rate were observed [2]
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| References |
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| Additional Infomation |
1. RO 46-8443 is the first synthetic non-peptide antagonist of selective endothelin B (ETB) receptor, developed by F. Hoffmann-La Roche, for the study of the physiological and pathological effects of ETB receptor [1][2]. 2. As a competitive antagonist of ETB receptor, RO 46-8443 binds to the positive ligand pocket and blocks the activation of ET-1-mediated G protein-coupled signaling pathways (Ca²⁺ mobilization, NO generation) [1]. 3. ETB receptors are widely expressed in vascular endothelium, smooth muscle and kidneys, regulating vasomotor tone, fluid balance and endothelin clearance; RO 46-8443 is an important research tool for distinguishing the functions of ETA and ETB receptors in vascular physiology [1][2]. 4. In hypertensive rats (SHR), RO 46-8443 indicates that ETB... Receptors play a protective role in regulating blood pressure by mediating vasodilation and antagonizing ETA-mediated vasoconstriction [2]
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| Molecular Formula |
C31H35N3O8S
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|---|---|---|
| Molecular Weight |
609.69
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| Exact Mass |
609.214
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| Elemental Analysis |
C, 61.07; H, 5.79; N, 6.89; O, 20.99; S, 5.26
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| CAS # |
175556-12-4
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| Related CAS # |
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| PubChem CID |
5312146
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
683.1±65.0 °C at 760 mmHg
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| Flash Point |
367.0±34.3 °C
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| Vapour Pressure |
0.0±2.2 mmHg at 25°C
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| Index of Refraction |
1.602
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| LogP |
3.22
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
13
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| Heavy Atom Count |
43
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| Complexity |
928
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| Defined Atom Stereocenter Count |
1
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| SMILES |
S(C1C([H])=C([H])C(=C([H])C=1[H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])(N([H])C1=C(C(=NC(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])=N1)OC([H])([H])C([H])(C([H])([H])O[H])O[H])OC1=C([H])C([H])=C([H])C([H])=C1OC([H])([H])[H])(=O)=O
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| InChi Key |
DRIHNVYRUGBDHI-JOCHJYFZSA-N
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| InChi Code |
InChI=1S/C31H35N3O8S/c1-31(2,3)21-12-16-24(17-13-21)43(37,38)34-29-27(42-26-9-7-6-8-25(26)40-5)30(41-19-22(36)18-35)33-28(32-29)20-10-14-23(39-4)15-11-20/h6-17,22,35-36H,18-19H2,1-5H3,(H,32,33,34)/t22-/m1/s1
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| Chemical Name |
4-tert-butyl-N-[6-[(2R)-2,3-dihydroxypropoxy]-5-(2-methoxyphenoxy)-2-(4-methoxyphenyl)pyrimidin-4-yl]benzenesulfonamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.41 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 (3.41 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (3.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6402 mL | 8.2009 mL | 16.4018 mL | |
| 5 mM | 0.3280 mL | 1.6402 mL | 3.2804 mL | |
| 10 mM | 0.1640 mL | 0.8201 mL | 1.6402 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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