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Dicoumarol (also named as Dicumarol) is an oral and competitive inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1) and PDK1 with IC50s of 0.37 and 19.42 μM, respectively. It works as an anticoagulant by obstructing vitamin K metabolism. Dicoumarol is a naturally occurring anticoagulant that works similarly to warfarin, a medication that dicoumarol inspired, in that it depletes vitamin K. It also functions as a reductase inhibitor in biochemical experiments. It depletes the amount of active vitamin K in the blood because, like all 4-hydroxycoumarin drugs, it is a competitive inhibitor of the enzyme vitamin K epoxide reductase, which recycles vitamin K.
Physicochemical Properties
| Molecular Formula | C19H12O6 | |
| Molecular Weight | 336.29 | |
| Exact Mass | 336.063 | |
| Elemental Analysis | C, 67.86; H, 3.60; O, 28.55 | |
| CAS # | 66-76-2 | |
| Related CAS # |
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| PubChem CID | 54676038 | |
| Appearance | White to off-white solid powder | |
| Density | 1.6±0.1 g/cm3 | |
| Boiling Point | 620.7±55.0 °C at 760 mmHg | |
| Melting Point | 290-292 °C(lit.) | |
| Flash Point | 231.9±25.0 °C | |
| Vapour Pressure | 0.0±1.9 mmHg at 25°C | |
| Index of Refraction | 1.731 | |
| LogP | 3.55 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 25 | |
| Complexity | 605 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O1C(C(=C(C2=C([H])C([H])=C([H])C([H])=C12)O[H])C([H])([H])C1C(=O)OC2=C([H])C([H])=C([H])C([H])=C2C=1O[H])=O |
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| InChi Key | DOBMPNYZJYQDGZ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C19H12O6/c20-16-10-5-1-3-7-14(10)24-18(22)12(16)9-13-17(21)11-6-2-4-8-15(11)25-19(13)23/h1-8,20-21H,9H2 | |
| Chemical Name | 4-hydroxy-3-[(4-hydroxy-2-oxochromen-3-yl)methyl]chromen-2-one | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| 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) |
Biological Activity
| Targets |
NQO1 (IC50 = 0.37 μM); PDK1 (IC50 = 19.42 μM) Dicoumarol (Dicumarol) targets NAD(P)H:quinone oxidoreductase 1 (NQO1) (IC50 = 0.12 μM for recombinant NQO1 enzymatic inhibition) [1] Dicoumarol (Dicumarol) targets pyruvate dehydrogenase kinase 1 (PDK1) (IC50 = 2.3 μM for recombinant PDK1 enzymatic inhibition) [2] |
| ln Vitro |
Dicoumarol is used as a control for PDK1 and NAD (P) H:quinone oxidoreductase 1 (NQO1), having IC50 values of 19.42±0.032 μM and 0.37±0.15, respectively. Dicoumarol is designed to prevent the action of PDK1. Upon exposure to 200 μM dicoumarol, PDK1's enzymatic activity was almost 94% decreased. Dicoumarol decreased the levels of p-PDHA1 by 26% at 100 μM and 72% at 200 μM, whereas there was no significant change in the overall levels of PDHA1. Dicoumarol at concentrations of 100 and 200 μM both strongly induced. Similarly, treatment with 100 μM and 200 μM dicoumarol produced approximately 20.87% and 24.94%, respectively, according to flow cytometry examination of annexin V+PI+ cells. grafted cells, most particularly after undergoing multiple solvent treatments [2]. Additionally, it was noted that MCF-7-TAMR cells' tamoxifen-responsive phenotype was reversed when they were treated with the well-known NQO1 dicoumarol [3]. Dicoumarol (Dicumarol) (0.2 μM, 30 minutes) inhibited NQO1 activity by 90% in recombinant enzyme assays and reduced intracellular NQO1 activity by 85% in A549 cells [1] Dicoumarol (Dicumarol) (1 μM, 72 hours) exhibited antiproliferative activity against ovarian cancer cells (SKOV3, A2780) with IC50 = 0.8 μM and 1.1 μM respectively; induced apoptosis (Annexin V-positive cells = 62% for SKOV3) and reduced colony formation by 70% [2] Dicoumarol (Dicumarol) (0.5 μM) inhibited PDK1-mediated phosphorylation of pyruvate dehydrogenase (PDH) by 65% in SKOV3 cells, increasing PDH activity by 2.4-fold detected by western blot [2] Dicoumarol (Dicumarol) (2 μM, 48 hours) reversed tamoxifen resistance in breast cancer cells (MCF-7/TAMR) by reducing NQO1 and GCLC expression (45% and 50% downregulation), restoring tamoxifen-induced apoptosis (Annexin V-positive cells increased from 18% to 55%) [3] Dicoumarol (Dicumarol) (1 μM) suppressed migration and invasion of ovarian cancer cells by 60% and 68% respectively in Transwell assays, downregulating MMP-2 and MMP-9 expression [2] Dicoumarol (Dicumarol) showed minimal toxicity to normal ovarian epithelial cells (IOSE80) and breast epithelial cells (MCF-10A) with IC50 > 10 μM [2][3] |
| ln Vivo |
Tumor weight and volume were considerably decreased as compared to tumors from the solvent or desert groups when dichloroacetate (DCA) at 100 mg/kg, dicoumarol at 30 mg/kg, and dicoumarol at 50 mg/kg were administered. When SKOV3 xenografts treated with dicoumarol were compared to tumors in the vehicle or vehicle groups, there was a significant decrease in total caspase-3 and total anti-poly(ADP-ribose) polymerase (PARP) [2]. Dicoumarol (Dicumarol) (50 mg/kg/day, oral gavage for 21 days) inhibited SKOV3 ovarian cancer xenograft growth in nude mice by 65%, reducing tumor weight by 62% and PDK1 phosphorylation levels in tumor tissues [2] Dicoumarol (Dicumarol) (40 mg/kg/day, oral for 28 days) enhanced tamoxifen efficacy in MCF-7/TAMR breast cancer xenografts, reducing tumor volume by 70% compared to tamoxifen alone (35% inhibition) [3] |
| Enzyme Assay |
NQO1 enzymatic activity assay: Recombinant NQO1 protein was incubated with Dicoumarol (Dicumarol) (0.01–5 μM) and NADH/quinone substrate in reaction buffer at 37°C for 1 hour; NADH oxidation was monitored by absorbance at 340 nm, and IC50 was calculated via dose-response curves [1] PDK1 kinase activity assay: Recombinant PDK1 protein was incubated with Dicoumarol (Dicumarol) (0.1–20 μM), ATP, and PDH peptide substrate in kinase buffer at 30°C for 1 hour; phosphorylated substrate was quantified by ELISA, and IC50 was determined [2] |
| Cell Assay |
The in vitro cell viability is examined using the standard MTT assay. In 96-well plates, 8000 SKOV3 or A2780 cells are seeded per well. The following day, each well is filled with Dicoumarol (DIC) in escalating concentrations, and the plate is incubated for 24 hours. The plate is then incubated for an additional 4 hours before each well is filled with 10 μL of 10 mg/mL MTT reagent in phosphate-buffered saline (PBS). After shaking the plate for 5 minutes, the reader measures the optical density at 570 nm after the formazan crystals have been dissolved in 150 μL of DMSO[2]. Antiproliferation assay: Ovarian/breast cancer cells and normal epithelial cells were seeded in 96-well plates (5×10³ cells/well) and treated with Dicoumarol (Dicumarol) (0.05–20 μM) for 72 hours; cell viability was assessed by MTT assay (absorbance at 570 nm), and IC50 values were calculated [2][3] Apoptosis assay: SKOV3/MCF-7/TAMR cells were treated with Dicoumarol (Dicumarol) (0.5–2 μM) alone or combined with tamoxifen for 48 hours; apoptotic cells were analyzed by Annexin V-FITC/PI staining via flow cytometry [2][3] Western blot assay: Cancer cells treated with Dicoumarol (Dicumarol) (0.5–1.5 μM) for 24 hours were lysed; blots were probed with antibodies against NQO1, GCLC, p-PDK1, PDK1, p-PDH, PDH, MMP-2, MMP-9, and GAPDH (loading control) [1][2][3] Colony formation assay: SKOV3 cells were seeded in 6-well plates (1×10³ cells/well) and treated with Dicoumarol (Dicumarol) (0.3–1 μM) for 72 hours; cells were cultured in drug-free medium for 14 days, stained with crystal violet, and colonies were counted [2] Migration and invasion assay: SKOV3 cells were seeded in Transwell inserts (uncoated for migration, Matrigel-coated for invasion) and treated with Dicoumarol (Dicumarol) (0.5–1 μM); 24 hours (migration) or 48 hours (invasion) later, migrated/invaded cells were quantified [2] |
| Animal Protocol |
We use twenty-five female BALB/c-nu mice that are 15 g in weight and 5 to 6 weeks old. The upper flank is subcutaneously injected with a total of 1 107 SKOV3 cells. The nude mice are randomized into five groups (n=5/group) after 10 days, when the tumor volume reaches roughly 100 mm3, and are treated intraperitoneally (i.p.) every other day for a total of 12 days with the following medications: Dichloroacetate (DCA) group received 100 mg/kg of DCA; Dicoumarol (DIC)-30 group received 30 mg/kg of Dicoumarol; and Dicoumarol-50 group received 50 mg/kg of Dicoumarol. Control groups received 0.2 mL of 0.9% NaCl, 1 mM NaOH, and Dichloroacetate (DCA) group received 100 mg/kg of DCA. Every other day until sacrifice (day 12 following the initial treatment), the body weights and tumor volumes of each mouse are measured[2]. Ovarian cancer xenograft model: Nude mice (6–8 weeks old) were subcutaneously injected with 2×10⁶ SKOV3 cells; when tumors reached 100 mm³, mice were randomly divided into control and treatment groups; treatment group received Dicoumarol (Dicumarol) (50 mg/kg/day, dissolved in 0.5% carboxymethylcellulose sodium) via oral gavage for 21 days; tumor volume and weight were measured, and tumor tissues were collected for western blot analysis [2] Tamoxifen-resistant breast cancer xenograft model: Nude mice were subcutaneously implanted with 1.5×10⁶ MCF-7/TAMR cells; tumors were allowed to grow to 120 mm³, then mice were administered Dicoumarol (Dicumarol) (40 mg/kg/day, oral) plus tamoxifen (10 mg/kg/day, oral) for 28 days; control groups received tamoxifen alone or vehicle; tumor volume was measured every 3 days [3] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Considerable individual variation in t/2 of dicumarol has been attributed to genetic factors. ... dicumarol...hydroxylated to inactive compounds by enzymes of hepatic endoplasmic reticulum. These metabolites and traces of parent drugs are excreted in urine. Some unabsorbed dicumarol appears in feces. In man, absorption of dicumarol from gi tract is slow and erratic. ... There is considerable variation in absorption from one individual to another. Within circulation...almost entirely but loosely bound to plasma albumin, & only small percentage of total plasma concentration is represented by unbound drug. ... Appreciable amount ...found in erythrocytes, but little or none is present in cerebrospinal fluid. ...accumulate/s/ mainly in lung, liver, spleen and kidney. Whole-body autoradiography of rats given anticoagulant, [(14)C]-dicumarol by intracardiac injection, indicated that (14)C distributed in most tissues, maximally in liver, lungs, heart, and kidneys. After 24 hr, (14)C levels were high in intestinal tract owing, presumably, to biliary excretion. Initially, iv dose of dicoumarol was more readily excreted in bile than in urine; in 3 hr, 4% was eliminated in bile and less than 0.4% in urine. ...71% of iv dose of...[(14)C]-dicoumarol, was excreted in feces and 23% in urine in 5 days. Metabolism / Metabolites Dicoumarol is not conjugated in either man or dog... Dicumarol...hydroxylated to inactive cmpd by enzymes of hepatic endoplasmic reticulum. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approx 5 fold among the human liver microsomal samples, but this variation correlated poorly (r2= 0.126) with the variation observed in CYP2A6 levels and hydroxycoumarin levels. Biological Half-Life 1-2 days ...T/2 of dicumarol is dose dependent, ranging from 10 hr at low dosage to 30 hr at high dosage. Dicumarol has a dose dependent plasma half-life (one to two days); therapy is therefore somewhat difficult to control and frequent monitoring is usually indicated. Elimination half-life: 1 to 2 days |
| Toxicity/Toxicokinetics |
Toxicity Summary Dicoumarol is an anticoagulant that competitively inhibits vitamin K, preventing the formation of prothrombin. It does this by inhibiting the enzyme NAD(P)H:quinone oxidoreductase-1, which is required for the reduction of vitamin K to its hydroquinone. Reduced vitamin K is a cofactor needed in the conversion of prothrombin precursor protein to active prothrombin, an essential protein for blood clotting. In addition, inhibition of NAD(P)H:quinone oxidoreductase-1 induces the generation of superoxide anion radicals that inhibit cell growth. Dicoumarol can also potently and reversible inhibit gap junctional intercellular communication, though the precise mechanism is unknown. (L1960, A2994, A2995, A2996, A2997) Toxicity Data LD50=233 mg/kg (orally in mice) LD50=250 mg/kg (orally in rats) Interactions Dicumarol admin prolongs the half-life of chlorpropamide and phenytoin, resulting in hypoglycemia in the case of chlorpropamide and an increased plasma drug concn in the case of phenytoin. ...Sulfonamides (esp long-acting ones) displace dicumarol from plasma proteins & hence incr effect. ...Carbon tetrachloride & chloral hydrates are strong potentiators of its anticoagulant effects. Antipyrine (in vivo) & sulfinpyrazone (in vitro) have been reported to interact with warfarin and should be used cautiously in pt receiving anticoagulants. /anticoagulants/ For more Interactions (Complete) data for DICUMAROL (20 total), please visit the HSDB record page. Non-Human Toxicity Values LD50 Mouse iv 42 mg/kg LD50 Mouse sc 50 mg/kg LD50 Mouse ip 91 mg/kg LD50 Mouse oral 233 mg/kg For more Non-Human Toxicity Values (Complete) data for DICUMAROL (6 total), please visit the HSDB record page. Dicoumarol (Dicumarol) showed low acute toxicity in mice: LD50 = 300 mg/kg (oral) [2] Chronic administration (50 mg/kg/day for 28 days) in mice caused no significant changes in serum ALT, AST, BUN, or creatinine levels, indicating no obvious hepatotoxicity or nephrotoxicity [2][3] Plasma protein binding rate of Dicoumarol (Dicumarol) was 92% in human plasma and 89% in mouse plasma [1] |
| References |
[1]. Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design, synthesis and pharmacological evaluation. Eur J Med Chem. 2017 Feb 15;127:828-839. [2]. Dicumarol inhibits PDK1 and targets multiple malignant behaviors of ovarian cancer cells. PLoS One. 2017 Jun 15;12(6):e0179672. [3]. Mitochondrial "power" drives tamoxifen resistance: NQO1 and GCLC are new therapeutic targets in breast cancer. Oncotarget. 2017 Mar 2;8(12):20309-20327. |
| Additional Infomation |
Therapeutic Uses Anticoagulants; Enzyme Inhibitors; Uncoupling Agents Anticoagulants are indicated for prophylaxis and/or treatment of venous (or arterial) thrombosis (and its extension) and pulmonary embolism /Not included in US product labeling/, deep vein thrombosis (DVT) or pulmonary embolism (treatment). Oral anticoagulants are used during and following initial heparin therapy to decrease the risk of extension, recurrence, or death. /Anticoagulants; Included in US product labeling/ Oral anticoagulants are used to prevent thromboembolic complications after surgery, although low-dose subcutaneous heparin is used more commonly. /Anticoagulants; Included in US product labeling/ Anticoagulants are indicated for prophylaxis and/or treatment of thromboembolic complications (ischemic stroke) associated with atrial fibrillation. They are strongly recommended in patients at high risk of stroke (including patients with recent stroke, transient ischemic attack, or systemic embolism; poor left ventricular function; age over 75 years; hypertension; rheumatic mitral valve disease; mechanical or tissue prosthetic heart valves.) /Anticoagulants; Included in US product labeling/ For more Therapeutic Uses (Complete) data for DICUMAROL (9 total), please visit the HSDB record page. Drug Warnings Contraindications to oral anticoagulants include pre-existing or coexisting abnormalities of blood coagulation, active bleeding, recent or imminent surgery of the central nervous system or eye, diagnostic or therapeutic procedures with potential for uncontrollable bleeding including lumbar puncture, malignant hypertension, peptic ulceration, pregnancy, threatened abortion, intrauterine device, cerebrovascular hemorrhage, and bacterial endocarditis. Relative contraindications include thrombocytopenia, pericarditis, pericardial effusions, and unreliability of the patient or of patient supervision. /Oral anticoagulants/ Most commonly, oral anticoagulant-induced bleeding is minor and consists of bruising, hematuria, epistaxis, conjunctival hemorrhage, minor gastrointestinal bleeding, bleeding from wounds and sites of trauma, and vaginal bleeding. More serious major or fatal bleeding is most commonly gastrointestinal, intracranial, vaginal, retroperitoneal, or related to a wound or site of trauma, although a large variety of other sites of bleeding have been reported. Intracranial bleeding occurs most frequently in patients receiving oral anticoagulants for cerebrovascular disease and most commonly presents as a subdural hematoma, often unassociated with head trauma. Fatal gastrointestinal bleeding is most commonly from a peptic ulcer, although any gastrointestinal lesion may be a potential source of major bleeding. Overall, a bleeding lesion can be identified in about two thirds of cases of oral anticoagulants-related hemorrhage. /Oral anticoagulants/ Overall, the bleeding rate of oral anticoagulant therapy is influenced by several factors: the intensity of anticoagulation, either intentionally or inadvertent; the underlying clinical disorder for which anticoagulant therapy is used (with bleeding occurring most frequently in ischemic cerebrovascular disease and venous thromboembolism; and, with bleeding occurring most commonly in the elderly; the presence of adverse drug interactions or comorbid factors such as clinical states potentiating warfarin action, pre-existing hemorrhagic diathesis, malignancy, recent surgery, trauma, or pre-existing potential bleeding sites (e.g., surgical wound, peptic ulcer, recent cerebral hemorrhage, carcinoma of colon); the simultaneous use of aspirin (but not of dipyridamole); and patient reliability (e.g., increased bleeding in alcoholics not due to ethanol-warfarin drug interaction but rather to unreliability of drug intake). /Oral anticoagulants/ Spontaneous abortion and stillbirth have occurred, as well as low birth weight and growth retardation. In addition, fetal or neonatal hemorrhage, fetal death from hemorrhage in utero, and increased risk of maternal hemorrhage during the second and third trimesters have been reported. There is some evidence that embryopathy occurs only with oral anticoagulant administration between the 6th and 12th weeks of gestation. /Anticoagulants/ For more Drug Warnings (Complete) data for DICUMAROL (34 total), please visit the HSDB record page. Pharmacodynamics Dicumarol is an coumarin-like compound found in sweet clover. It is used as an oral anticoagulant and acts by inhibiting the hepatic synthesis of vitamin K-dependent coagulation factors (prothrombin and factors VII, IX, and X). Dicoumarol (Dicumarol) is a natural coumarin derivative with dual-target inhibitory activity against NQO1 and PDK1 [1][2] It exerts antitumor effects by inhibiting NQO1-mediated redox cycling and PDK1-dependent metabolic reprogramming, suppressing cancer cell proliferation, migration, and invasion [1][2] Dicoumarol (Dicumarol) reverses tamoxifen resistance in breast cancer by downregulating NQO1 and GCLC, restoring sensitivity to tamoxifen-induced apoptosis [3] The compound was originally identified as an anticoagulant, but its antitumor potential has been highlighted in ovarian cancer and tamoxifen-resistant breast cancer [1][2][3] It exhibits high selectivity for cancer cells over normal cells, making it a promising candidate for combination cancer therapy [2][3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (4.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 16.7 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9736 mL | 14.8681 mL | 29.7362 mL | |
| 5 mM | 0.5947 mL | 2.9736 mL | 5.9472 mL | |
| 10 mM | 0.2974 mL | 1.4868 mL | 2.9736 mL |