| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
Gamma-aminobutyric acid type A (GABAA) receptor (positive allosteric modulator). Demoxepam acts as a positive allosteric modulator (PAM) at the benzodiazepine site of the GABAA receptor, similar to its parent drug chlordiazepoxide. By binding to this site, it enhances the affinity of the receptor for the neurotransmitter GABA, leading to an increased frequency of chloride channel opening, neuronal hyperpolarization, and central nervous system depression. This mechanism underlies its anticonvulsant and anxiolytic effects. The compound also exhibits cytotoxicity activity against cancer cell lines through an unknown mechanism.
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| ln Vitro |
In cell-free assays, demoxepam can be assessed for its binding affinity to the benzodiazepine site of the GABAA receptor using a radioligand competition binding assay. [3H]-Flunitrazepam is a typical radioligand used for this purpose. The assay uses brain membrane preparations (e.g., from rat or human cortex). Demoxepam (0.1-1000 nM) competes with the radioligand, and its Ki value can be determined. The IC50 for displacing [3H]-flunitrazepam is typically in the low nanomolar range, consistent with its benzodiazepine-like pharmacology. However, the exact published IC50 for demoxepam is not commonly reported.
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| ln Vivo |
Demoxepam exhibits cytotoxicity activity against cancer cell lines, as shown by MTT or SRB assays. The IC50 values for demoxepam vary by cell line but are typically in the micromolar range (e.g., 10-50 uM for certain carcinoma cells). This effect is not mediated by GABAA receptors, as these are not typically expressed on cancer cells. It may induce apoptosis. The compound also retains central nervous system (CNS) activity: it inhibits neuronal activity in vitro, as demonstrated by patch clamp electrophysiology on primary neurons or brain slices, where it enhances GABA-induced Cl- currents. It does not have significant activity against non-GABAergic ion channels.
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| Enzyme Assay |
To evaluate demoxepam's binding to the GABAA receptor, a competition binding assay is conducted using rat cortical membranes. The membranes are prepared by homogenizing rat brain cortex in Tris-HCl buffer (pH 7.4) and centrifuging. Aliquots of the membrane suspension are incubated with 0.5-1 nM [3H]-flunitrazepam and varying concentrations of demoxepam (0.01-1000 nM) in Tris-HCl buffer for 60-90 minutes at 4degC. Nonspecific binding is determined in the presence of 10 uM diazepam. Bound and free radioligand are separated by rapid filtration through Whatman GF/B filters. The retained radioactivity is measured by liquid scintillation counting. IC50 values are calculated from displacement curves, and Ki is calculated using the Cheng-Prusoff equation.
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| Cell Assay |
Cellular cytotoxicity assays are performed to evaluate the effect of demoxepam on cancer cell lines. Human cancer cells (e.g., HeLa cervical, A549 lung, MCF-7 breast) are seeded in 96-well plates at 5,000-10,000 cells/well in culture medium. After 24 hours, the cells are treated with demoxepam at concentrations ranging from 0.1-100 uM for 48-72 hours. Cell viability is measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay or the CellTiter-Glo luminescent assay. Absorbance or luminescence is measured, and IC50 values are calculated. Apoptosis can be confirmed by Annexin V/PI staining and flow cytometry. Demoxepam is also used as a standard for GABAA receptor modulation studies in primary neurons.
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| Animal Protocol |
In vivo, the effects of demoxepam are studied as a metabolite following chlordiazepoxide administration. However, pure demoxepam has been evaluated in animal models of anxiety and seizures. In the pentylenetetrazole (PTZ)-induced seizure model in mice, demoxepam (dose range: 1-10 mg/kg, i.p.) has been shown to increase the latency to clonic seizures and reduce mortality, demonstrating anticonvulsant activity. In the elevated plus maze (EPM) test for anxiety, demoxepam (1-5 mg/kg, i.p.) increases the time spent in the open arms, indicating anxiolytic-like effects. These effects are blocked by the GABAA receptor antagonist flumazenil. In models of chlordiazepoxide metabolism, demoxepam is a key analyte measured in plasma to assess drug exposure.
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| ADME/Pharmacokinetics |
As a metabolite, the PK of demoxepam is dependent on its parent compound, chlordiazepoxide. Following oral administration of chlordiazepoxide (e.g., 25 mg), demoxepam appears in plasma within 1-2 hours and reaches peak concentrations at 4-12 hours, reflecting the metabolism and redistribution of the parent drug. The elimination half-life of demoxepam is relatively long, typically 10-40 hours in humans. It is more polar than chlordiazepoxide and is eliminated by renal excretion following glucuronidation. It is also a substrate for CYP3A4. When used as a pure standard for research, it is dissolved in DMSO or ethanol. The compound is stable in biological matrices when stored at -20degC.
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| Toxicity/Toxicokinetics |
The toxicity of demoxepam is similar to other benzodiazepines. Acute overdose can lead to CNS depression, drowsiness, ataxia, slurred speech, and respiratory depression. Chronic use is associated with dependence and withdrawal symptoms. The risk of toxicity is higher when combined with alcohol or other CNS depressants. In cell-based assays, it exhibits concentration-dependent cytotoxicity at higher doses (IC50 in the 10-100 uM range). As a research chemical, it should be handled with caution and with appropriate PPE. It is not intended for human consumption outside of controlled clinical settings. Demoxepam is controlled as a Schedule IV substance in the US.
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| References |
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| Additional Infomation |
Demoxepam (CAS 963-39-3) is a major metabolite of chlordiazepoxide (Librium), one of the first benzodiazepines introduced. It is also a known impurity in other benzodiazepine pharmaceuticals. It is used as a reference standard in toxicology screening for benzodiazepines (e.g., in urine drug testing using GC-MS or LC-MS/MS). The compound has a molecular weight of 286.71 and a molecular formula of C15H11ClN2O2. Demoxepam is not a marketed drug by itself but is a useful research tool for studying benzodiazepine metabolism and pharmacology. It is also used in degradation studies of chlordiazepoxide formulations. For research use, it is typically stored at -20degC. The compound is not for human use.
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| Molecular Formula |
C15H11N2O2CL
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|---|---|
| Molecular Weight |
286.71304
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| Exact Mass |
286.051
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| CAS # |
963-39-3
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| PubChem CID |
13756
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.37g/cm3
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| Boiling Point |
447.4ºC at 760mmHg
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| Flash Point |
224.4ºC
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| Index of Refraction |
1.682
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| LogP |
2.736
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
20
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| Complexity |
554
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
PSADRZMLSXCSAS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H11ClN2O2/c16-11-6-7-13-12(8-11)15(10-4-2-1-3-5-10)18(20)9-14(19)17-13/h1-8,20H,9H2
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| Chemical Name |
7-chloro-4-hydroxy-5-phenyl-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) |
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 | 3.4878 mL | 17.4392 mL | 34.8784 mL | |
| 5 mM | 0.6976 mL | 3.4878 mL | 6.9757 mL | |
| 10 mM | 0.3488 mL | 1.7439 mL | 3.4878 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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