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Purity: ≥98%
5-methoxyflavone, a flavonoid analog, is a novel DNA polymerase-ß inhibitor and neuroprotective agent against ß-amyloid toxicity. 5-Methoxyflavone was confirmed to be an inhibitor of DNA pol-β activity using cultured primary neurons, a valuable model for examining the neuroprotective effects of putative DNA pol-β inhibitors because treatment with Aβ causes DNA replication and cell death in these neurons. 5-methoxyflavone decreased the number of S-phase neurons and the subsequent apoptotic death induced by Aβ, which is consistent with the inhibition of DNA pol-β. 5-Methoxyflavone is the first flavonoid compound that, as opposed to having broad antioxidant and anti-inflammatory effects, is able to stop neurodegeneration through a specific molecular mechanism.
| Targets |
DNA polymerase β
DNA Polymerase beta (pol-β) [1] GABAₐ receptors, adenosine receptors, glycine receptors, NMDA receptors [2] Death receptors (DR4, DR5), mitochondrial apoptotic pathway-related proteins (cFLIP, Mcl-1, BAX, Bid), caspase-8, caspase-3 [3] |
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| ln Vitro |
In vitro activity: 5-Methoxyflavone (compound 1) has been identified as a potential compound that can inhibit DNA pol-β in multiple instances and prevent the initiation of the cell cycle and subsequent neuronal apoptosis in primary neuronal cultures challenged with Aβ. On 92TAg cells, 5-methoxyflavone (10–30 μM) can greatly increase the toxicity of MMS. 5-Polymerase activity on a gapped substrate is significantly reduced by 1- or 10-methoxyflavone[1]. 5-Methoxyflavone (5-MF, 0-100 μg/mL) causes the proapoptotic protein BAX to increase and the antiapoptotic proteins cFLIP and Mcl-1 to decrease over time. 5-MF induces TRAIL-R1 (DR4) and TRAIL-R2 (DR5) in a manner that is dependent on time[2]. 1. 5-methoxyflavone was validated as a specific inhibitor of DNA pol-β activity (identified via in silico screening of ZINC database with 35+ million compounds, followed by pharmacological evaluation); it reduced the number of S-phase primary neurons and apoptotic death triggered by β-amyloid (Aβ), via inhibiting neuronal ectopic DNA replication (a core feature of degenerating neurons in AD/PD). [1] 2. 5-methoxyflavone (50, 100, 150 mg/kg, i.p. in in vivo studies; no in vitro concentration data for CNS effects) exhibited sedative-hypnotic potential via GABAₐ, adenosine, glycine, and NMDA receptors (in silico docking confirmed H-bond interactions with these receptors); pretreatment with picrotoxin/bicuculline (GABAₐ antagonists), caffeine (adenosine antagonist), glycine/NMDA reversed its hypnotic effect (no in vitro cell-based CNS activity data). [2] 3. 5-methoxyflavone (IC₂₀ concentration) was cytotoxic to human leukemic MOLT-4 cells (MTT assay, 24 h treatment, p < 0.05 vs control) but not to U937 cells; it enhanced TRAIL-induced apoptosis in MOLT-4 cells by upregulating DR4/DR5, downregulating cFLIP/Mcl-1, cleaving Bid, upregulating BAX, activating caspase-8/-3, and increasing ROS production (flow cytometry for annexin V/PI, DiOC₆, DCFH₂-DA; immunoblotting for apoptotic proteins; fluorometric assay for caspase activity); it increased mitochondrial transmembrane potential (ΔΨm) in MOLT-4 cells treated with TRAIL (p < 0.05 vs TRAIL alone); it was cytotoxic to PBMCs (MTT assay, 24 h treatment, p < 0.05 vs control). [3] |
| ln Vivo |
5-Methoxyflavone (100, 150 mg/kg, i.p) dramatically reduces the amount of time it takes to lose the righting reflex. 5. A notable and dose-dependent decrease in spontaneous locomotor activity is shown by methylflavone (50, 100, and 150 mg/kg, i.p.). 5. The rearing response is decreased by methylflavone (50, 100 mg/kg, i.p.). 5-The grooming response was totally eliminated by methylflavone (100, 125, and 150 mg/kg, i.p. ), just like in animals treated with diazepam[3].
1. 5-methoxyflavone (50, 100, 150 mg/kg, i.p.) showed dose-dependent sedative-hypnotic effects in mice: reduced spontaneous locomotor activity in open field test (F (530) = 87.17, P < 0.001); decreased latency to sleep and increased sleep duration after pentobarbitone/ether administration (p < 0.001); exhibited dose-dependent myorelaxant effects in inclined plane, horizontal wire, and rotarod tests; pretreatment with picrotoxin/bicuculline/caffeine/glycine/NMDA attenuated/abolished its hypnotic effect. [2] |
| Enzyme Assay |
Human DNA Pol-β Assay [1]
DNA pol-β inhibition was evaluated with a specific DNA pol-β assay kit. The assay was carried out strictly following the manufacturer’s instructions. Briefly, a reaction mixture containing a gapped DNA template, dNTPs (both supplied with the kit), and DNA pol-β was incubated for 30 min at RT with or without the compound to be tested. The formation of repaired duplexes, which, unlike gapped DNA, selectively incorporate a fluorescent dye in the presence of Reagent U (both supplied by the kit) was determined by measuring at 535 nm with the excitation wavelength at 485 nm in a fluorimeter. OA (50 μM) was used as a positive control for inhibition. Activity was evaluated as fluorescent signal to no-enzyme-background ratio. 1. DNA pol-β inhibition assay: A panel of 9 putative DNA pol-β inhibitors (including 5-methoxyflavone) identified via in silico screening were subjected to pharmacological evaluation; the enzyme activity of purified DNA pol-β was measured with specific substrates (details not specified) to validate inhibitory activity, and only 5-methoxyflavone was confirmed as a DNA pol-β inhibitor (no specific reaction conditions/quantification methods reported). [1] 2. In silico receptor binding assay: Molecular docking simulations were performed to assess the binding affinity of 5-methoxyflavone to GABAₐ, adenosine, glycine, and NMDA receptors; the binding sites and H-bond interactions between 5-methoxyflavone and each receptor were analyzed (no experimental enzyme/receptor activity assay for CNS targets). [2] 3. Caspase activity assay: MOLT-4 cells treated with 5-methoxyflavone (IC₂₀) for various times (3/6/12/18/24 h) with/without TRAIL (IC₂₀, 24 h) were lysed; caspase-8 activity was measured using IETD-AFC substrate, and caspase-3 activity using DEVD-AFC substrate; fluorescence intensity was quantified (n-fold increase vs control, p < 0.05 vs control) to determine enzyme activation. [3] |
| Cell Assay |
The MTT assay is used to assess the cell viability after treating MOLT-4, U937, or PBMC cells with DMF, TMF, PMF, 5-MF, or 2'-MF at different concentrations for 24 hours.
1. Primary neuron assay: Cultured primary neurons were treated with Aβ to induce ectopic DNA replication and death; 5-methoxyflavone was added to the culture medium, and the number of S-phase neurons was quantified (method not specified); apoptotic cell death was assessed (method not specified) to confirm neuroprotective effects via DNA pol-β inhibition. [1] 2. Leukemic cell viability assay: MOLT-4/U937/PBMC cells were plated in 96-well plates and treated with various concentrations of 5-methoxyflavone for 24 h; MTT reagent was added, and absorbance was measured to calculate cell viability (p < 0.05 vs control for MOLT-4/PBMC); for combination treatment, MOLT-4 cells were pretreated with 5-methoxyflavone (IC₂₀) for 24 h, then with various TRAIL concentrations for 24 h, and viability was measured (p < 0.05 vs single agent). [3] 3. Apoptosis assay (flow cytometry): MOLT-4 cells treated with 5-methoxyflavone (IC₂₀) for 3/6/12/18/24 h with/without TRAIL (IC₂₀, 24 h) were stained with annexin V-FITC/PI; early/late apoptotic/necrotic cells were quantified by flow cytometry; mitochondrial transmembrane potential (ΔΨm) was measured by DiOC₆ staining and flow cytometry (p < 0.05 vs control/TRAIL alone); ROS production was detected by DCFH₂-DA staining and flow cytometry (p < 0.05 vs control, H₂O₂ as positive control). [3] 4. Immunoblotting assay for apoptotic proteins: MOLT-4 cells treated with 5-methoxyflavone (IC₂₀) for various times with/without TRAIL (IC₂₀, 24 h) were lysed; whole-cell extracts were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against DR4, DR5, cFLIP, Mcl-1, BAX, Bid; band intensity was quantified by densitometry (p < 0.05 vs control). [3] |
| Animal Protocol |
1. Sedative-hypnotic assay in mice: Mice were randomly divided into groups and administered 5-methoxyflavone (50/100/150 mg/kg) via intraperitoneal injection (vehicle/dissolution formula not specified); spontaneous locomotor activity was measured in open field test (duration/frequency not specified); sleep latency/duration were recorded after pentobarbitone/ether administration (dose of pentobarbitone/ether not specified); myorelaxant effects were evaluated in inclined plane (angle/duration not specified), horizontal wire (latency to fall not specified), and rotarod tests (speed/duration not specified); for mechanism studies, mice were pretreated with picrotoxin/bicuculline/caffeine/glycine/NMDA (doses/routes not specified) before 5-methoxyflavone administration, and hypnotic effects were assessed. [2] |
| Toxicity/Toxicokinetics |
1. 5-Methoxyflavonoids exhibit cytotoxicity to PBMCs (MTT assay, 24 hours of treatment, p < 0.05 compared to the control group); (LD50, hepatotoxicity, nephrotoxicity, plasma protein binding rate, and drug interactions) are described in reference [3]. [3]
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| References |
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| Additional Infomation |
5-Methoxyflavonoids are ether compounds belonging to the flavonoid class.
1. 5-Methoxyflavonoids are the first anti-Aβ toxic flavonoid neuroprotective agents with a clear mechanism (DNA polymerase β inhibition) rather than general antioxidant/anti-inflammatory properties; neuronal cell cycle reactivation (DNA polymerase β-mediated ectopic DNA replication) is the core feature of neurodegenerative changes in Alzheimer's disease/Parkinson's disease. [1] 2. 5-Methoxyflavonoids exert sedative-hypnotic effects through multiple central nervous system receptors (GABAₐ, adenosine, glycine, NMDA), and computer simulation docking has confirmed their hydrogen bond interactions; it has the potential to become a novel sedative-hypnotic agent and muscle relaxant. [2] 3. 5-Methoxyflavonoids regulate TRAIL-induced apoptosis of TRAIL-resistant MOLT-4 leukemia cells through death receptors (DR4/DR5) and mitochondrial pathways; its effect is weaker than that of 2'-methoxyflavonoids, but it has a synergistic effect with TRAIL and can induce apoptosis of leukemia cells. [3] |
| Molecular Formula |
C16H12O3
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| Molecular Weight |
252.26
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| Exact Mass |
252.079
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| CAS # |
42079-78-7
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| Related CAS # |
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| PubChem CID |
94525
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| Appearance |
Yellow solid powder
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| Density |
1.24g/cm3
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| Boiling Point |
422.5ºC at 760mmHg
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| Melting Point |
131-133°C
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| Flash Point |
201.1ºC
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| Index of Refraction |
1.548
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| LogP |
3.468
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
19
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| Complexity |
368
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C(=C([H])C(C2C(=C([H])C([H])=C([H])C1=2)OC([H])([H])[H])=O)C1C([H])=C([H])C([H])=C([H])C=1[H]
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| InChi Key |
XRQSPUXANRGDAV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H12O3/c1-18-13-8-5-9-14-16(13)12(17)10-15(19-14)11-6-3-2-4-7-11/h2-10H,1H3
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| Chemical Name |
5-methoxy-2-phenylchromen-4-one
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| Synonyms |
5-Methoxyflavone
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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 |
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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) |
DMSO: 50~125 mg/mL (198.2~495.5 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (8.25 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 (8.25 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (8.25 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 | 3.9642 mL | 19.8208 mL | 39.6416 mL | |
| 5 mM | 0.7928 mL | 3.9642 mL | 7.9283 mL | |
| 10 mM | 0.3964 mL | 1.9821 mL | 3.9642 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.
J Nat Prod.2015 Nov 25;78(11):2704-11. |
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