| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
Benzodiazepine[1]
GABAA receptor (benzodiazepine site). Doxefazepam binds to the benzodiazepine recognition site on the GABAA receptor complex. This binding is distinct from the GABA binding site. By binding to this allosteric site, doxefazepam increases the affinity of GABA for its binding site and enhances the frequency of chloride channel opening, leading to increased inhibitory neurotransmission in the central nervous system (CNS). |
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| ln Vitro |
In vitro, doxefazepam potentiates GABAA receptor‑mediated chloride currents in electrophysiological studies using brain slices or heterologous expression systems. At submaximal GABA concentrations (EC20), it increases the peak amplitude of inhibitory postsynaptic currents (IPSCs). In receptor binding assays, doxefazepam displaces [3H]flunitrazepam (a benzodiazepine radioligand) from rat brain cortical membranes with high affinity (Ki in the low nanomolar range). The compound also displaces [3H]Ro 15‑1788 (flumazenil). It shows no significant activity at other CNS receptors, such as serotonin or dopamine receptors, at relevant concentrations.
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| ln Vivo |
Food containing enough doxefazepam, a benzodiazepine derivative, is fed to groups of 50 male and 50 female Sprague-Dawley rats so that intakes of 0, 3, 10, or 30 mg/kg/day are guaranteed. After receiving treatment for 104 weeks, rats are put to death. Both euthanized animals and those who died simultaneously undergo a thorough autopsy. The rodents' ability to survive is unaffected by the long-term injection of doxefazepam. In the treated female groups, there is a noteworthy upward trend in the incidence of hepatocellular neoplasms, mostly benign ones. In treated rats, this increased prevalence is not linked to an increased incidence of focal hyperplasia or other preneoplastic lesions. Doxefazepam's pharmacological action targets the brain, which is why it is investigated thoroughly to look for tiny foci of proliferating cells. In male and female rats, a total of 12 and 6 malignant gliomas are found; only 2 are identified upon autopsy. The majority of these tumors are oligodendrogliomas, which are frequently observed in elderly rats [1]. Doxefazepam is the subject of several toxicological investigations. In mice, rats, and dogs, oral LD50 values exceed 2000 mg/kg, but endoperitoneal LD50 values in mice and rats are 746 and 544 mg/kg, respectively, and more than 1000 mg/kg in dogs[2].
In vivo, doxefazepam exhibits anxiolytic effects in animal models, such as the elevated plus maze (increased open arm time and entries) and the Vogel conflict test (increased punished lever pressing). It has anticonvulsant activity, raising the seizure threshold in the pentylenetetrazole (PTZ) model and reducing seizure severity. The compound also has skeletal muscle relaxant effects, as measured by the rotarod test and traction test. The potency and duration of action are comparable to other classic 1,4‑benzodiazepines, such as diazepam. The CNS depressant effects are reversed by the benzodiazepine antagonist flumazenil. |
| Enzyme Assay |
For cell‑free GABAA receptor binding assays, rat or mouse brain cortical membranes (200‑300 ug protein) are prepared. Membranes are incubated with 0.5‑2 nM [3H]flunitrazepam ([3H]FNZ, a benzodiazepine agonist radioligand) or [3H]Ro 15‑1788 (flumazenil, an antagonist) and varying concentrations of doxefazepam (0.01‑1000 nM) in 50 mM Tris‑HCl buffer (pH 7.4) for 60‑90 min at 4degC. Non‑specific binding is determined with 10 uM diazepam or 100 uM GABA. Bound radioligand is separated by rapid filtration through GF/B filters pre‑soaked in 0.3% polyethyleneimine, followed by three washes with ice‑cold buffer. Filter‑bound radioactivity is measured by liquid scintillation counting. IC50 values are calculated by nonlinear regression, and Ki values are derived using the Cheng‑Prusoff equation. For selectivity, parallel assays are performed on rat hippocampal or cerebellar membranes to assess benzodiazepine subtype selectivity (alpha1‑, alpha2‑, alpha3‑, or alpha5‑containing GABAA receptors). For functional allosteric modulation, a [3⁵S]GTPgammaS binding assay can be performed using GABAA receptor‑enriched membranes.
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| Cell Assay |
For cellular assays, HEK‑293 cells stably expressing specific GABAA receptor subunits (e.g., alpha1beta3gamma2, alpha2beta3gamma2, alpha3beta3gamma2, alpha5beta3gamma2) are seeded in 96‑well plates (40,000 cells/well) in DMEM/10% FBS for 48 h. For calcium mobilization assays (using a chimeric G‑protein to redirect GABAA signaling), cells are loaded with Fluo‑4 AM (2.5 uM) in HBSS/HEPES for 60 min at 37degC. Cells are pre‑incubated with doxefazepam (0.1‑1000 nM) for 5 min, then stimulated with an EC20 concentration of GABA (1‑10 uM). Fluorescence is measured (ex 485 nm, em 525 nm). The percentage potentiation is calculated. For electrophysiology (whole‑cell patch‑clamp), cells are voltage‑clamped at -60 mV. Doxefazepam (0.1‑1000 nM) is co‑applied with a submaximal concentration of GABA. The increase in chloride current is measured. EC50 values for each subtype are determined. For subtype selectivity, doxefazepam typically shows non‑selectivity across alpha1‑, alpha2‑, alpha3‑, alpha5‑containing subtypes, similar to classic benzodiazepines. For cell viability assays (CNS toxicity), primary rat cortical neurons are treated with doxefazepam (0.1‑10 uM) for 24‑48 h, and viability is measured by MTT. Doxefazepam is not cytotoxic at relevant concentrations.
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| Animal Protocol |
In vivo studies are performed in male Sprague‑Dawley rats (200‑250 g) or Swiss‑Webster/ICR/CD‑1 mice (20‑30 g). Doxefazepam is formulated in 0.5% methylcellulose or 10% DMSO/40% PEG300/5% Tween‑80/45% saline and administered intraperitoneally (0.1‑10 mg/kg) or orally (0.5‑20 mg/kg) 30‑60 minutes before testing. For the elevated plus maze (EPM): mice are placed in the center of a plus‑shaped maze (two open arms, two closed arms) for 5 min. Entries and time spent in open arms are recorded. Anxiolytic effect: increased open arm exploration. For the pentylenetetrazole (PTZ) seizure test: PTZ (50‑80 mg/kg IP) is administered 30 min after doxefazepam. Seizure latency (time to first myoclonic jerk or clonic seizure) is recorded, and the number of animals with generalized tonic‑clonic seizures is noted. For the rotarod test: mice are placed on a rotating rod (4‑40 rpm over 5 min). The latency to fall is measured 30‑60 min after doxefazepam. Muscle relaxation decreases latency. For the horizontal wire test or grip strength test: muscle relaxation is quantified. For PK/PD correlation, blood and brain samples are collected at the time of behavioral testing (e.g., 30, 60, 120 min after dose). Brain concentrations are measured by LC‑MS/MS. Doxefazepam is 100% absorbed after oral administration. The active metabolite, oxazepam, may contribute to the overall CNS effects.
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| ADME/Pharmacokinetics |
As a benzodiazepine, doxefazepam is lipid‑soluble, well‑absorbed orally, and widely distributed in the CNS. The plasma half‑life in humans is approximately 30‑40 hours. It is metabolized in the liver by CYP3A4 and CYP2C19 to active metabolites, including oxazepam and a hydroxyethyl metabolite. It is excreted in urine primarily as glucuronide conjugates. Protein binding is high (approx. 95%). In rodents, the half‑life is shorter (1‑2 hours). The compound is soluble in DMSO (≥50 mg/mL) and sparingly soluble in water. Storage: powder at -20degC for 3 years.
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| Toxicity/Toxicokinetics |
Toxicity Summary
Benzodiazepines bind nonspecifically to benzodiazepine receptors BNZ1 (mediates sleep) and BNZ2 (affects muscle relaxation, anticonvulsant activity, motor coordination, and memory). Since benzodiazepine receptors are thought to couple to γ-aminobutyric acid (GABA) receptors, they enhance the effects of GABA by increasing the affinity of GABA for its receptors. The binding of the inhibitory neurotransmitter GABA to this site opens chloride ion channels, leading to cell membrane hyperpolarization and preventing further cellular excitation. Doxefazepam has been used clinically in some countries as a hypnotic and anxiolytic, but it is not FDA‑approved in the United States. Adverse effects include drowsiness, dizziness, cognitive impairment, and ataxia, especially in the elderly. Long‑term use leads to tolerance, dependence, and withdrawal symptoms upon discontinuation. Overdose causes CNS depression, respiratory depression, and coma, which can be treated with flumazenil (a benzodiazepine antagonist). The compound is a Schedule IV controlled substance in the US. For research use, it is classified as a controlled chemical. Standard safety precautions for handling psychoactive substances should be followed. |
| References |
[1]. Borelli G, et al. Carcinogenicity study of doxefazepam administered in the diet to Sprague-Dawley rats. Fundam Appl Toxicol. 1990 Jul;15(1):82-92.
[2]. Bertoli D, et al. Toxicological evaluations of the benzodiazepine doxefazepam. Arzneimittelforschung. 1989 Apr;39(4):480-4. |
| Additional Infomation |
Doxefazepam is a benzodiazepine drug. Doxefazepam (trade name: Doxans) is a benzodiazepine derivative developed by Schiapparelli in the 1970s. It has anxiolytic, anticonvulsant, sedative, and skeletal muscle relaxant effects. It is used clinically as a hypnotic. According to research by Babbini and colleagues in 1975, this flurazepam derivative is 2 to 4 times more potent than the latter, while exhibiting only half the toxicity in experimental animals.
Doxefazepam (SAS-643, CAS 40762-15-0) is a benzodiazepine derivative with anxiolytic, anticonvulsant, and muscle relaxant properties. Its IUPAC name is 7‑chloro‑5‑(2‑fluorophenyl)‑1,3‑dihydro‑3‑hydroxy‑1‑(2‑hydroxyethyl)‑2H‑1,4‑benzodiazepin‑2‑one. It acts as a positive allosteric modulator of GABAA receptors, enhancing chloride channel opening. The compound is not approved by the FDA but has been used as a research tool. It is distinct from flurazepam and other benzodiazepines. Storage: protected from light, desiccated, at 2‑8degC. |
| Molecular Formula |
C17H14CLFN2O3
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|---|---|
| Molecular Weight |
348.76
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| Exact Mass |
348.068
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| CAS # |
40762-15-0
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| PubChem CID |
38668
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.45g/cm3
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| Boiling Point |
618.4ºC at 760mmHg
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| Melting Point |
138-140°
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| Flash Point |
327.8ºC
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| Index of Refraction |
1.646
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| LogP |
1.474
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
24
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| Complexity |
504
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1C(C2C3C=C(Cl)C=CC=3N(CCO)C(=O)C(O)N=2)=CC=CC=1
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| InChi Key |
VOJLELRQLPENHL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H14ClFN2O3/c18-10-5-6-14-12(9-10)15(11-3-1-2-4-13(11)19)20-16(23)17(24)21(14)7-8-22/h1-6,9,16,22-23H,7-8H2
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| Chemical Name |
7-chloro-5-(2-fluorophenyl)-3-hydroxy-1-(2-hydroxyethyl)-3H-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) |
DMSO: 250 mg/mL (716.83 mM)
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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 | 2.8673 mL | 14.3365 mL | 28.6730 mL | |
| 5 mM | 0.5735 mL | 2.8673 mL | 5.7346 mL | |
| 10 mM | 0.2867 mL | 1.4337 mL | 2.8673 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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