β adrenergic receptor
rac-Bufuralol hydrochloride targets β1 and β2 adrenergic receptors as a non-selective beta-adrenoreceptor antagonist. It also exhibits partial agonist activity at these receptors. The compound is a specific substrate for CYP2D6, an enzyme responsible for metabolizing a large number of therapeutic drugs. Its aromatic ring and basic nitrogen structure make it particularly useful for assessing CYP2D6 activity in vitro and in vivo.

Bufuralol (Ro 3-4787) hydrochloride, which is specific to CYP2D6 substrates and has an aromatic ring and basic nitrogen, is frequently used to assess CYP2D6 activity [3].

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In vitro, Bufuralol hydrochloride is widely used in the characterization of CYP2D6 activity. It serves as a probe substrate for CYP2D6-mediated 1'-hydroxylation, allowing for the functional characterization of enzymatic activity in various CYP2D6 variants. The compound has been used in baculovirus-mediated expression systems to study wild-type and variant CYP2D6 enzymes. In vitro studies have shown that most CYP2D6 variants exhibit significantly reduced catalytic activities compared to the wild-type.

The metabolism of bufuralol (Ro 3-4787) hydrochloride mediated by NADPH displays biphasic kinetics that are less efficient than those seen in monkeys' guts and in monkeys when cumene hydroperoxide (CuOOH) is present, which is consistent with that in the liver. The findings of the observations agree with [4].

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In vivo, Bufuralol hydrochloride is an orally bioactive compound that can be used to assess CYP2D6 activity in living organisms. The metabolism of bufuralol mediated by NADPH displays biphasic kinetics. The compound can decrease isoproterenol-induced peripheral resistance. As a non-cardioselective β-adrenoceptor antagonist with partial agonist activity, it produces effects consistent with beta-blockade, including reduction of heart rate and cardiac output.

Cytochromea P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with Bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high-performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for Bufuralol 1'-hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O-demethylation than those of the wild-type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild-type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations.[1]

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Metabolic phenotype can be affected by multiple factors, including allelic variation and interactions with inhibitors. Human CYP2D6 is responsible for approximately 20% of cytochrome P450-mediated drug metabolism but consists of more than 100 known variants; several variants are commonly found in the population, whereas others are quite rare. Four CYP2D6 allelic variants-three with a series of mutations distal to the active site (*34, *17-2, *17-3) and one ultra-metabolizer with mutations near the active site (*53), along with reference *1 and an active site mutant of *1 (Thr309Ala)-were expressed, purified, and studied for interactions with the typical substrates dextromethorphan and Bufuralol and the inactivator SCH 66712. We found that *34, *17-2, and *17-3 displayed reduced enzyme activity and NADPH coupling while producing the same metabolites as *1, suggesting a possible role for Arg296 in NADPH coupling. A higher-activity variant, *53, displayed similar NADPH coupling to *1 but was less susceptible to inactivation by SCH 66712. The Thr309Ala mutant showed similar activity to that of *1 but with greatly reduced NADPH coupling. Overall, these results suggest that kinetic and metabolic analysis of individual CYP2D6 variants is required to understand their possible contributions to variable drug response and the complexity of personalized medicine.[2]

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For non-cell-based receptor binding assays, rac-Bufuralol hydrochloride can be evaluated using membrane preparations from tissues expressing β1 and β2 adrenergic receptors. Radioligand binding displacement experiments are performed using a radiolabeled beta-antagonist such as [3H]-dihydroalprenolol. Membrane homogenates are incubated with increasing concentrations of the test compound and a fixed concentration of the radioligand at room temperature for 60-90 minutes. Bound radioligand is separated from free by rapid filtration through GF/B filters. Nonspecific binding is determined in the presence of excess unlabeled propranolol. Ki values are calculated from displacement curves using nonlinear regression analysis.

For in vitro cellular assays, insect microsomes containing expressed CYP2D6 proteins are incubated with Bufuralol at 37°C for 20 minutes. After termination, the metabolites (1'-hydroxybufuralol) are extracted and detected using high-performance liquid chromatography. For functional beta-adrenoreceptor assays, cells expressing β1 or β2 receptors are cultured in appropriate media. cAMP accumulation assays are performed where cells are treated with various concentrations of the compound and cAMP levels are measured using ELISA or HTRF-based detection. Partial agonist activity is assessed by measuring the compound's ability to both stimulate and inhibit receptor signaling.

Observations were made in eight subjects who exercised before and at 1, 2, 4, 6, 8 and 24 h after the double-blind oral administration of placebo, bufuralol 7.5, 15, 30, 60 and 120 mg and propranolol 40 and 160 mg. The exercise heart rate remained constant after placebo. Bufuralol 7.5 mg and propranolol 40 mg reduced exercise heart rate up to 6 and 8 h respectively after dosing but bufuralol 15, 30, 60 and 120 mg and propranolol 160 mg were still active at 24 h. The lowest exercise heart rate occurred at 2 h after all active treatments. Bufuralol 60 and 120 mg produced similar reduction in exercise tachycardia as propranolol 40 mg but less than propranolol 160 mg. Plasma levels of bufuralol and its two major metabolites were measured. The peak plasma concentrations of bufuralol occurred at 1.5 h after 7.5 mg and at 2 h after the other doses of bufuralol. In six subjects the plasma elimination half-life of bufuralol was 2.61 +/- 0.18 h and in the other three subjects 4.85 +/- 0.35 h. There was a corresponding longer time to peak concentration and plasma elimination half-life of the two metabolites in these three subjects. These findings show that bufuralol is a potent beta-adrenoceptor antagonist with partial agonist activity. It has a long duration of action and there is bimodal metabolism of the drug in man

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For in vivo animal studies, rac-Bufuralol hydrochloride is typically administered orally to rodents. The compound's metabolism is studied by collecting blood samples at various time points post-administration and analyzing for parent compound and metabolite concentrations. In cardiovascular studies, hemodynamic parameters including heart rate and blood pressure are monitored. The compound's partial agonist activity may result in less pronounced bradycardia compared to full beta-blockers. Dosing regimens vary depending on the specific experimental objectives.

Metabolites/Metabolites: Known metabolites of ibuprofen include 1',2'-vinylbuprofen, 4-hydroxybuprofen, and 6-hydroxybuprofen. Eight subjects were observed exercising 1, 2, 4, 6, 8, and 24 hours after receiving a double-blind oral dose of placebo, ibuprofen 7.5, 15, 30, 60, and 120 mg, and propranolol 40 and 160 mg. Exercise heart rate remained stable after placebo administration. Ibuprofen 7.5 mg and propranolol 40 mg reduced exercise heart rate at 6 and 8 hours after administration, respectively, but ibuprofen 15, 30, 60, and 120 mg and propranolol 160 mg remained effective at 24 hours. Exercise heart rate reached its lowest value 2 hours after all active treatments. Ibuprofen 60 mg and 120 mg were similar to propranolol 40 mg in reducing exercise-induced tachycardia, but less effective than propranolol 160 mg. The plasma concentrations of ibuprofen and its two major metabolites were determined. Peak plasma concentrations of ibuprofen 7.5 mg were reached 1.5 hours after administration, while peak plasma concentrations of other doses were reached 2 hours after administration. The plasma elimination half-life of ibuprofen was 2.61 ± 0.18 hours in 6 subjects and 4.85 ± 0.35 hours in 3 other subjects. The peak concentration times and plasma elimination half-lives of the two metabolites were also prolonged in these 3 subjects. These results suggest that ibuprofen is a potent β-adrenergic receptor antagonist with partial agonist activity. It has a long duration of action and exhibits bimodal metabolism in humans. [1]

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rac-Bufuralol hydrochloride is an orally bioactive compound. It has a molecular weight of 291.82 and is soluble in appropriate organic solvents. As a CYP2D6 probe substrate, its pharmacokinetic profile is influenced by CYP2D6 activity, making it useful for phenotyping individuals or animal models for CYP2D6 metabolic capacity. The compound's metabolism via 1'-hydroxylation is a well-characterized pathway used in drug-drug interaction studies and pharmacogenetic research.

The oral LD50 in rats was 750 mg/kg. Behavioral effects included: seizures or impact on the epilepsy threshold; ataxia; and respiratory depression in the lungs, pleura, or respiration. (Drug Research, 27(1410), 1977 [PMID:20114]). The subcutaneous LD50 in rats was 1400 mg/kg. Behavioral effects included: seizures or impact on the epilepsy threshold; ataxia; and respiratory depression in the lungs, pleura, or respiration. Drug Research, 27(1410), 1977 [PMID:20114]
Oral LD50 in mice: 177 mg/kg. Behavioral studies: seizures or impact on the epilepsy threshold; ataxia; lung, pleural, or respiratory: respiratory depression. Arzneimittel-Forschung. Drug Research, 27(1410), 1977 [PMID:20114]
Intraperitoneal LD50 in mice: 88 mg/kg. Behavioral studies: seizures or impact on the epilepsy threshold; ataxia; lung, pleural, or respiratory: respiratory depression. Arzneimittel-Forschung. Drug Research, 27(1410), 1977 [PMID:20114]
Intravenous LD50 in mice: 29700 ug/kg Behavior: seizures or effects on the epileptic threshold; Behavior: ataxia; Lungs, pleura, or respiration: respiratory depression. Arzneimittel-Forschung. Drug Research, 27(1410), 1977 [PMID:20114]

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The toxicity profile of rac-Bufuralol hydrochloride has not been extensively reported in the available literature. As a beta-adrenoreceptor antagonist, potential adverse effects may include bradycardia, hypotension, and bronchoconstriction, particularly in individuals with asthma or COPD. The compound is for research use only and not for human consumption. Standard toxicological evaluation would include acute and repeated-dose toxicity studies, as well as assessment of cardiovascular and respiratory effects.

[1]. Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro. Basic Clin Pharmacol Toxicol. 2016 Mar;118(3):190-9.

[2]. CYP2D6 Allelic Variants *34, *17-2, *17-3, and *53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712. Drug Metab Dispos. 2018 Aug;46(8):1106-1117.

[3]. (+)-bufuralol 1'-hydroxylation activity in human and rhesus monkey intestine and liver. Biochem Pharmacol. 1995 Oct 26;50(9):1521-5.

rac-Bufuralol hydrochloride (Bufuralol hydrochloride; Ro 3-4787 hydrochloride) is a potent, non-selective, orally bioactive beta-adrenoreceptor antagonist with partial agonist activity. It is widely used as a substrate for CYP2D6 probes in drug metabolism research. The compound is a non-specific β-receptor inhibitor with affinity for both β1 and β2 receptors. It is used in the characterization of CYP2D6 activity and in pharmacogenetic studies to understand the impact of CYP2D6 polymorphisms on drug metabolism.

C16H24CLNO2

297.82

297.15

C, 64.53; H, 8.12; Cl, 11.90; N, 4.70; O, 10.74

60398-91-6

Bufuralol;54340-62-4; 59652-29-8 (HCl); 60398-91-6 (racemic HCl)

151573

Off-white to light brown solid powder

393.2ºC at 760 mmHg

143-146ºC

191.6ºC

4.609

3

3

5

20

287

0

CCC1=CC=CC2=C1OC(=C2)C(CNC(C)(C)C)O.Cl

KJBONRGCLLBWCJ-UHFFFAOYSA-N

InChI=1S/C16H23NO2.ClH/c1-5-11-7-6-8-12-9-14(19-15(11)12)13(18)10-17-16(2,3)4;/h6-9,13,17-18H,5,10H2,1-4H3;1H

2-(tert-butylamino)-1-(7-ethyl-1-benzofuran-2-yl)ethanol;hydrochloride

Bufuralol hydrochloride; 60398-91-6; Bufuralol HCl; 59652-29-8; Angium; bufuralol, hydrochloride; Bufuralol (hydrochloride); Bufurolol hydrochloride;

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)

Ethanol: 15 mg/mL (50.37 mM)
DMSO: 10 mg/mL (33.58 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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