| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
3-Hydroxyphenazepam is a GABAA receptor agonist. It inhibits synaptosomal transporter-mediated [3H]GABA uptake. As a benzodiazepine metabolite, it potentiates the effect of the neurotransmitter GABA (gamma-aminobutyric acid) at GABAA receptors, increasing chloride ion channel opening frequency, leading to neuronal hyperpolarization and CNS depression. This results in anxiolytic, sedative, hypnotic, and muscle relaxant effects.
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| ln Vitro |
In vitro, 3-Hydroxyphenazepam inhibits synaptosomal transporter-mediated [3H]GABA uptake. This activity suggests it enhances GABAergic neurotransmission by preventing GABA reuptake. The compound has been studied in brain synaptosome preparations to characterize its effects on the GABA transporter system. The concentration required for GABA uptake inhibition has been characterized, with activity in the low micromolar range.
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| ln Vivo |
In vivo, 3-Hydroxyphenazepam is the bioactive metabolite of Cinazepam, a GABAA receptor agonist prodrug. The ratio of the concentration of phenazepam and 3-hydroxyphenazepam in brain and blood was determined in mice, reported to be around 1.1-1.3 for phenazepam and 0.83-0.93 for 3-hydroxyphenazepam. As an active metabolite, it is responsible for the sedative, anxiolytic, and anticonvulsant effects observed after phenazepam administration.
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| Enzyme Assay |
For in vitro enzyme assays, 3-Hydroxyphenazepam can be used to study GABA transporter inhibition. Synaptosomal preparations are isolated from rat brain homogenates by differential centrifugation. The synaptosomes are resuspended in assay buffer (e.g., HEPES-buffered Krebs-Ringer solution). [3H]GABA is added as a tracer, and varying concentrations of 3-Hydroxyphenazepam (e.g., 0.1-100 uM) are added to the reaction. After incubation at 37degC, samples are filtered to separate synaptosomes from free [3H]GABA, and radioactivity is counted. IC50 values are calculated to determine GABA uptake inhibition potency.
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| Cell Assay |
For in vitro cellular assays, neuronal cell lines or primary cultured neurons can be used. Cells are plated in 96-well plates and cultured in neurobasal medium. After reaching appropriate confluence, cells are treated with 3-Hydroxyphenazepam at various concentrations (e.g., 0.01-100 uM). GABAA receptor activation can be measured by patch-clamp electrophysiology, calcium imaging with fluorescent indicators (e.g., Fluo-4 AM for chloride-dependent changes), or by measuring chloride ion influx using fluorescent probes such as MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide).
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| Animal Protocol |
For in vivo animal studies, 3-Hydroxyphenazepam is typically administered to rodents via intraperitoneal injection (e.g., 0.1-10 mg/kg) or oral gavage. Behavioral tests can include the elevated plus maze (anxiolysis), rotarod (sedation/muscle relaxation), or open field test (locomotor activity). Blood and brain tissue are collected at various time points for LC-MS analysis to correlate plasma and brain concentrations with behavioral effects. The ratio of phenazepam to 3-hydroxyphenazepam in brain and blood can be quantified.
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| ADME/Pharmacokinetics |
3-Hydroxyphenazepam has a plasma half-life of approximately 20-60 hours in humans, typical of long-acting benzodiazepine metabolites. The compound is eliminated primarily by hepatic metabolism (glucuronidation) and renal excretion of conjugates. The brain-to-blood ratio in mice is approximately 0.8-0.9, indicating good brain penetration. Volume of distribution is large (∼2-4 L/kg) due to lipophilicity, and protein binding is high (∼90-95%).
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| Toxicity/Toxicokinetics |
3-Hydroxyphenazepam is a benzodiazepine metabolite with toxicity similar to other benzodiazepines. Acute overdose can cause CNS depression, sedation, ataxia, confusion, and respiratory depression in severe cases. Chronic use may lead to physical dependence and withdrawal syndrome upon discontinuation. As a research chemical, the toxicological properties have not been fully investigated, but it is harmful if swallowed. Use appropriate procedures to prevent direct contact with skin or eyes and prevent inhalation.
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| References |
[1]. Borisova T, et, al. GABAA receptor agonist cinazepam and its active metabolite 3-hydroxyphenazepam act differently at the presynaptic site. Eur Neuropsychopharmacol. 2021 Apr;45:39-51.
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| Additional Infomation |
Benzodiazepine active metabolites in the urine of rats and mice
3-Hydroxyphenazepam is not an approved drug but an active metabolite of phenazepam and Cinazepam. Phenazepam is a long-acting benzodiazepine approved in some countries (Russia) for the treatment of anxiety, alcohol withdrawal, and sleep disorders. 3-Hydroxyphenazepam is used as a research standard for forensic toxicology, drug metabolism studies, and analytical method development. It is also used to study the pharmacology of benzodiazepine metabolites and GABAergic neurotransmission. |
| Molecular Formula |
C15H10BRCLN2O2
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|---|---|
| Molecular Weight |
365.61
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| Exact Mass |
363.961
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| CAS # |
70030-11-4
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| PubChem CID |
125820
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.7±0.1 g/cm3
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| Boiling Point |
544.5±50.0 °C at 760 mmHg
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| Flash Point |
283.1±30.1 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.712
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| LogP |
2.46
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
21
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| Complexity |
445
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| Defined Atom Stereocenter Count |
0
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| SMILES |
BrC=1C=CC2=C(C1)C(C3=CC=CC=C3Cl)=NC(O)C(N2)=O
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| InChi Key |
KRJKJUWAZOWXNV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H10BrClN2O2/c16-8-5-6-12-10(7-8)13(19-15(21)14(20)18-12)9-3-1-2-4-11(9)17/h1-7,15,21H,(H,18,20)
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| Chemical Name |
7-bromo-5-(2-chlorophenyl)-3-hydroxy-1,3-dihydro-1,4-benzodiazepin-2-one
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7352 mL | 13.6758 mL | 27.3515 mL | |
| 5 mM | 0.5470 mL | 2.7352 mL | 5.4703 mL | |
| 10 mM | 0.2735 mL | 1.3676 mL | 2.7352 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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