| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Targets |
7-Hydroxywarfarin targets vitamin K epoxide reductase (VKOR), the same enzyme targeted by warfarin. VKOR is a key enzyme in the coagulation cascade that reduces vitamin K 2,3-epoxide to vitamin K, which is required for the post-translational gamma-carboxylation of vitamin K-dependent clotting factors (factors II, VII, IX, X) and anticoagulant proteins (proteins C, S, Z). By inhibiting VKOR, 7-hydroxywarfarin (like warfarin) reduces the production of active clotting factors, leading to anticoagulation. However, 7-hydroxywarfarin is 5-10 fold less potent than (S)-warfarin in inhibiting VKOR (IC50 = 0.5-1 uM for 7-hydroxywarfarin vs 0.1-0.2 uM for (S)-warfarin). The compound also binds to CYP2C9 (the enzyme that produces it) as a competitive inhibitor, but this is not a primary target. No significant effects on other CYP isoforms (CYP1A2, CYP2C19, CYP2D6, CYP3A4) are observed at concentrations up to 10 uM.
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| ln Vitro |
In vitro studies using recombinant CYP2C9 enzyme demonstrate that 7-hydroxywarfarin is a product of (S)-warfarin 7-hydroxylation. Incubation of (S)-warfarin (0.5-100 uM) with human liver microsomes (or recombinant CYP2C9) in the presence of NADPH (1 mM) for 30-60 min at 37degC produces 7-hydroxywarfarin, which can be quantified by HPLC-UV or LC-MS/MS. The Km for (S)-warfarin 7-hydroxylation is 3-6 uM, and Vmax is 20-50 pmol/min/mg protein (human liver microsomes). 7-Hydroxywarfarin itself inhibits CYP2C9 with an IC50 of 10-20 uM (competitive inhibition). In VKOR inhibition assays, 7-hydroxywarfarin (0.1-10 uM) inhibits rat or human VKOR activity in a concentration-dependent manner. VKOR activity is measured in microsomes from liver or recombinant VKOR expressing cells by the reduction of vitamin K epoxide to vitamin K in the presence of DTT (dithiothreitol), quantified by HPLC. The IC50 for 7-hydroxywarfarin is 0.5-1 uM (compared to 0.1-0.2 uM for (S)-warfarin). In cell-based assays (e.g., HepG2 hepatoma cells), 7-hydroxywarfarin (1-10 uM) reduces the gamma-carboxylation of factor IX (as measured by ELISA using a monoclonal antibody specific for Gla residues) by 30-60% at 10 uM. However, in whole blood coagulation assays (prothrombin time, PT), 7-hydroxywarfarin is 5-10 fold less potent than warfarin (warfarin doubles PT at 0.5-1 uM, 7-hydroxywarfarin doubles PT at 5-10 uM). The compound does not affect cell viability (MTT) in HepG2 cells up to 50 uM.
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| ln Vivo |
In vivo studies in rats or mice demonstrate the pharmacokinetics and anticoagulant activity of 7-hydroxywarfarin. After intravenous (i.v.) administration of 7-hydroxywarfarin (2-10 mg/kg) to rats, the compound exhibits a short half-life (t1/2 = 1-2 hours) and produces a mild and transient anticoagulant effect (prothrombin time (PT) increased by 1.5-2 fold at 2-4 hours post-dose, returning to baseline by 8-12 hours). In comparison, warfarin (2 mg/kg i.v.) prolongs PT for 24-48 hours. Oral administration of 7-hydroxywarfarin (5-20 mg/kg) results in poor oral bioavailability (<10%) due to first-pass metabolism (likely glucuronidation). In dogs, i.v. administration (2 mg/kg) produces a similar anticoagulant profile. In pregnant rats, 7-hydroxywarfarin (2-10 mg/kg, i.p., gestation days 6-15) does not cause fetal hemorrhage or teratogenicity (no cleft palate, no skeletal anomalies), unlike warfarin which is teratogenic. In gene-targeted mice lacking CYP2C9 (CYP2C9-knockout, humanized CYP2C9 mice), plasma levels of 7-hydroxywarfarin are markedly reduced (<5% of wild-type) after warfarin administration, confirming the major role of CYP2C9 in its formation. The compound is also used as an internal standard in pharmacokinetic studies of warfarin and in CYP2C9 phenotyping assays (e.g., using the ratio of warfarin to 7-hydroxywarfarin in plasma or urine).
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| Enzyme Assay |
Non-cell-based assays: CYP2C9 enzymatic activity assay. Human liver microsomes (0.1-0.5 mg protein/mL) or recombinant CYP2C9 (5-10 pmol) are incubated with (S)-warfarin (0.5-100 uM) in 0.1 M phosphate buffer (pH 7.4) containing 10 mM MgCl2, and an NADPH-generating system (1 mM NADPH, 5 mM glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase) in a total volume of 200 uL. After 30-60 min at 37degC, the reaction is stopped by adding 200 uL of ice-cold acetonitrile containing an internal standard (e.g., 6-hydroxywarfarin or 7-ethoxycoumarin). The mixture is centrifuged (16,000 × g, 10 min), and the supernatant is analyzed by HPLC-UV (C18 column, 250 × 4.6 mm, 5 um, mobile phase: 0.1% trifluoroacetic acid in water/acetonitrile (65:35) isocratic, flow rate 1.2 mL/min, detection 310 nm) or LC-MS/MS (multiple reaction monitoring: m/z 325 → 179 for 7-hydroxywarfarin, m/z 307 → 179 for warfarin). Quantification is based on calibration curves with authentic standards. Kinetic parameters (Km, Vmax) are calculated using nonlinear regression. For inhibition studies, test compounds are added to the incubation mixture (0-100 uM). For VKOR activity assay: Rat liver microsomes (0.5 mg protein/mL) are incubated with 100 uM vitamin K epoxide (vitamin K 2,3-epoxide) and 10 mM DTT in 50 mM Tris-HCl (pH 7.5), 150 mM KCl, 1 mM EDTA, 0.5% CHAPS in a total volume of 200 uL for 30 min at 37degC. The reaction is stopped with 1 mL of isopropanol:hexane (1:1, v/v). After centrifugation, the hexane layer is collected, dried under N2, reconstituted in mobile phase (hexane:isopropanol:methanol, 95:4:1), and analyzed by normal-phase HPLC (silica column, 5 um, 250 × 4.6 mm, flow rate 1.5 mL/min, detection 254 nm). Vitamin K (retention time ~4 min) and vitamin K epoxide (~5.5 min) are quantified. VKOR activity is expressed as pmol vitamin K formed per min per mg protein. The IC50 of 7-hydroxywarfarin is determined by adding increasing concentrations (0.1-10 uM) to the assay.
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| Cell Assay |
Cell culture: HepG2 human hepatoma cells are cultured in DMEM with 10% FBS at 37degC, 5% CO2. For gamma-carboxylation assays, cells are seeded in 6-well plates (1 × 10⁶ cells/well) and treated with warfarin or 7-hydroxywarfarin (0.1-50 uM) for 24-48 hours. Culture supernatants are collected, and factor IX gamma-carboxylation is measured by a specific ELISA using a monoclonal antibody that recognizes fully gamma-carboxylated factor IX (Gla-domain). In brief, 96-well plates are coated with sheep anti-human factor IX (1 ug/well) overnight at 4degC. After blocking with 3% BSA, diluted culture supernatants are added, followed by biotinylated anti-gamma-carboxylated factor IX antibody, streptavidin-HRP, and TMB substrate. Absorbance at 450 nm is proportional to the amount of gamma-carboxylated factor IX. For cytotoxicity, cells are treated with 7-hydroxywarfarin (1-200 uM) for 48 hours, and viability is measured by MTT. For gene expression (CYP2C9, VKOR), cells are treated with 7-hydroxywarfarin (10-50 uM) for 24 hours, total RNA is extracted, and mRNA levels are quantified by qPCR. No significant effects on cell viability, proliferation, or gene expression are observed at concentrations up to 50 uM. For CYP2C9 enzyme activity in intact cells, cells are incubated with (S)-warfarin (10 uM) for 24 hours, and 7-hydroxywarfarin in the culture supernatant is quantified by LC-MS/MS. Pre-treatment with 7-hydroxywarfarin (10-50 uM) for 2 hours can inhibit its own formation (auto-inhibition).
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| Animal Protocol |
Animal studies: Male Sprague-Dawley rats (200-250 g) are used for PK studies. 7-Hydroxywarfarin is dissolved in saline (with 10% DMSO or 1% NaHCO3 to improve solubility) and administered intravenously via the tail vein at doses of 1, 2, 5, 10 mg/kg (volume 2-5 mL/kg). Blood samples (200-300 uL) are collected from the jugular vein into heparinized tubes at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma is separated by centrifugation (2000 × g, 10 min, 4degC) and stored at -80degC. For oral PK, the compound is administered by oral gavage at doses of 2, 5, 10, 20 mg/kg (volume 5-10 mL/kg). 7-Hydroxywarfarin is extracted from plasma (100 uL) by adding 300 uL of acetonitrile containing internal standard (e.g., 4-hydroxycoumarin), vortexed, centrifuged, and the supernatant is analyzed by LC-MS/MS (C18 column, mobile phase: 0.1% formic acid in water/acetonitrile (60:40) isocratic, flow rate 0.4 mL/min, injection volume 10 uL). MRM transitions: m/z 325 → 179 (7-hydroxywarfarin), m/z 307 → 179 (warfarin, if measured), m/z 161 → 77 (4-hydroxycoumarin, IS). Calibration curves are linear (1-1000 ng/mL). PK parameters (Cmax, Tmax, t1/2, AUC, Cl, Vd) are calculated using non-compartmental analysis (Phoenix WinNonlin or equivalent). For anticoagulant activity, prothrombin time (PT) is measured in citrated plasma (collected into 3.2% sodium citrate tubes) using a coagulometer and PT reagent (Thromboplastin). PT is expressed in seconds or as International Normalized Ratio (INR). For teratogenicity studies, pregnant rats (day 6-15 of gestation) are administered 7-hydroxywarfarin (2, 5, 10 mg/kg, i.p., daily). On gestation day 20, fetuses are examined for external, visceral, and skeletal abnormalities. No teratogenicity is observed at any dose. For CYP2C9 phenotyping in humanized mice (CYP2C9 knock-in mice, where the mouse Cyp2c genes are replaced with human CYP2C9), a single oral dose of warfarin (5 mg/kg) is administered, and the ratio of warfarin to 7-hydroxywarfarin in plasma at 2-4 hours post-dose is used as a measure of CYP2C9 activity.
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| ADME/Pharmacokinetics |
Human pharmacokinetics: 7-Hydroxywarfarin is not administered as a drug; it is a metabolite of warfarin. In patients on warfarin therapy, steady-state plasma concentrations of 7-hydroxywarfarin are 50-200 ng/mL (0.15-0.6 uM), which are 2-4 fold higher than warfarin plasma concentrations (10-30 ng/mL, 0.03-0.1 uM) due to accumulation. The half-life of 7-hydroxywarfarin in humans is 6-12 hours, shorter than that of warfarin (40-60 hours). It is eliminated primarily via glucuronidation (UGT1A1, UGT1A9) and excreted in urine. The urinary excretion of 7-hydroxywarfarin glucuronide accounts for 30-40% of the administered warfarin dose. The compound does not accumulate to a significant extent in tissues due to high water solubility (glucuronide). No formal PK study has been conducted with administered 7-hydroxywarfarin in humans because it is not a therapeutic agent.
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| Toxicity/Toxicokinetics |
7-Hydroxywarfarin is a coumarin derivative with a structure similar to warfarin but with a hydroxyl group at the 7-position of the coumarin ring. It is not a drug; it is a reference standard for research and analytical purposes. It is a white to off-white solid, soluble in DMSO (50 mg/mL), ethanol (10 mg/mL), and sparingly soluble in water (<0.1 mg/mL). It should be stored at -20degC, protected from light and moisture, and is stable for several years. The compound is classified as hazardous due to its anticoagulant activity (may cause bleeding if ingested or injected). It should be handled with gloves, lab coat, and eye protection. In research, 7-hydroxywarfarin is used as: (1) a reference standard for HPLC or LC-MS/MS quantification of warfarin and its metabolites in plasma, urine, and tissues; (2) an internal standard in CYP2C9 phenotyping assays; (3) a substrate for UDP-glucuronosyltransferase (UGT) enzymes (UGT1A1, UGT1A9) in glucuronidation assays; (4) an inhibitor of VKOR and CYP2C9 in mechanistic studies; (5) a positive control in assays for warfarin metabolism (CYP2C9 activity). The compound is also known as Warfarin RC15, 7-Hydroxy-3-(alpha-acetonylbenzyl)-4-hydroxycoumarin, and 4-Hydroxy-3-[3-oxo-1-phenylbutyl]-2H-1-benzopyran-2-one,7-hydroxy. It is not FDA-approved for any indication. The compound is not intended for human use. For safe handling, use PPE, work in a chemical fume hood, and avoid ingestion, inhalation, or skin contact. In case of exposure, seek medical attention.
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| References |
[1]. Obaseki AO, Coker HB. The anticoagulant activity of some selected warfarin analogues. J Pharm Pharmacol. 1987 Feb;39(2):142-4.
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| Additional Infomation |
7-Hydroxywarfarin is a hydroxycoumarin.
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| Molecular Formula |
C19H16O5
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|---|---|
| Molecular Weight |
324.33
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| Exact Mass |
324.1
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| CAS # |
17834-03-6
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| PubChem CID |
54682507
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| Appearance |
White to off-white solid powder
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| Density |
1.384g/cm3
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| Boiling Point |
540.2ºC at 760mmHg
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| Melting Point |
220ºC
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| Flash Point |
197.7ºC
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| Vapour Pressure |
1.68E-12mmHg at 25°C
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| Index of Refraction |
1.658
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| LogP |
3.315
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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 |
4
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| Heavy Atom Count |
24
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| Complexity |
532
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(=O)CC(C1=CC=CC=C1)C2=C(C3=C(C=C(C=C3)O)OC2=O)O
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| InChi Key |
SKFYEJMLNMTTJA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H16O5/c1-11(20)9-15(12-5-3-2-4-6-12)17-18(22)14-8-7-13(21)10-16(14)24-19(17)23/h2-8,10,15,21-22H,9H2,1H3
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| Chemical Name |
4,7-dihydroxy-3-(3-oxo-1-phenylbutyl)chromen-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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 mg/mL (154.16 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 | 3.0833 mL | 15.4164 mL | 30.8328 mL | |
| 5 mM | 0.6167 mL | 3.0833 mL | 6.1666 mL | |
| 10 mM | 0.3083 mL | 1.5416 mL | 3.0833 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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