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]

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].

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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]

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].

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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]

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]

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].

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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]

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].

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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]

Absorption, Distribution and Excretion
The half-life of dicumarol exhibits significant individual variability, attributed to genetic factors. …Dicoumarol…is hydroxylated to inactive compounds by enzymes in the hepatic endoplasmic reticulum. These metabolites and trace amounts of the parent drug are excreted in the urine. Some unabsorbed dicumarol appears in the feces. In humans, the absorption of dicumarol from the gastrointestinal tract is slow and unstable. …Individual absorption varies considerably. In the circulatory system…it is almost completely bound to plasma albumin, but the binding is weak, with only a small proportion of the total plasma concentration existing as free drug. …A considerable amount of dicumarol is found in erythrocytes, but very little or none in cerebrospinal fluid. …It mainly accumulates in the lungs, liver, spleen, and kidneys. Whole-body autoradiography in rats, via intracardiac injection of the anticoagulant [(14)C]-dicumarol, showed that (14)C was distributed in most tissues, with the highest concentrations in the liver, lungs, heart, and kidneys. After 24 hours, the level of (14)C in the intestine was high, likely due to bile excretion. Initially, intravenously administered dicumarol was more readily excreted via bile than urine; within 3 hours, 4% was excreted via bile and less than 0.4% via urine. Of the intravenously administered dose of [(14)C]-dicumarol, 71% was excreted via feces within 5 days and 23% via urine.

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Metabolism/Metabolites Dicoumarol does not undergo binding reactions in either humans or dogs… Dicoumarol… is hydroxylated to an inactive compound by hepatic endoplasmic reticulum enzymes. Despite its structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates of CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold in human liver microsomal samples, but this variation correlated poorly with changes in CYP2A6 and hydroxycoumarol levels (r² = 0.126).

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Biological Half-Life
1-2 days
The half-life (T/2) of dicumarol is dose-related, ranging from 10 hours at low doses to 30 hours at high doses.
The plasma half-life of dicumarol is also dose-related (1-2 days); therefore, therapeutic control is challenging and frequent monitoring is usually required.
Elimination half-life: 1-2 days

Toxicity Summary
Dicoumarol is an anticoagulant that competitively inhibits vitamin K to prevent prothrombin formation. Its mechanism of action involves inhibiting NAD(P)H:quinone oxidoreductase-1, an enzyme essential for the reduction of vitamin K to hydroquinone. Reduced vitamin K is a cofactor in the conversion of prothrombin precursor proteins into active prothrombin (a protein essential for blood clotting). Furthermore, inhibition of NAD(P)H:quinone oxidoreductase-1 induces the generation of superoxide anion radicals, thereby inhibiting cell growth. Dicoumarol also potently and reversibly inhibits intercellular junction communication, but the exact mechanism remains unclear. (L1960, A2994, A2995, A2996, A2997)

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Toxicity Data>
LD₅₀=233 mg/kg (oral in mice)
LD₅₀=250 mg/kg (oral in rats)

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Interactions>
Dicoumarin administration prolongs the half-life of chlorpropamide and phenytoin, leading to hypoglycemia caused by chlorpropamide and elevated plasma drug concentrations of phenytoin.
...Sulfonamides (especially long-acting sulfonamides) can displace dicoumarin from plasma proteins, thereby enhancing its effect.
...Carbon tetrachloride and chloral hydrate are potent enhancers of its anticoagulant effect.
In vitro studies of antipyrine (in vivo) and sulfinpyrazone (in vivo) indicate that some drugs may interact with warfarin; therefore, caution should be exercised when using them in patients receiving anticoagulant therapy. /Anticoagulant/
For more complete interaction data (out of 20) on dicoumarol, please visit the HSDB record page.

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Non-human toxicity values>
Mice intravenous LD50: 42 mg/kg
Mice subcutaneous LD50: 50 mg/kg
Mice intraperitoneal LD50: 91 mg/kg
Mice oral LD50: 233 mg/kg
For more complete non-human toxicity data (out of 6) on dicoumarol, please visit the HSDB record page.

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Dicumarol showed low acute toxicity in mice: LD50 = 300 mg/kg (oral) [2]
Long-term administration to mice (50 mg/kg/day for 28 days) did not cause serum ALT, AST, BUN or creatinine levels, indicating no significant hepatotoxicity or nephrotoxicity [2][3]
Dicumarol has a plasma protein binding rate of 92% in human plasma and 89% in mouse plasma [1]

[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.

Therapeutic Uses
Anticoagulants; Enzyme inhibitors; Uncoupling agents. Anticoagulants are indicated for the prevention and/or treatment of venous (or arterial) thrombosis (and its spread) and pulmonary embolism (not included on the US product label), as well as deep vein thrombosis (DVT) or pulmonary embolism (treatment). Oral anticoagulants are used during and after initial heparin therapy to reduce the risk of thrombus spread, recurrence, or death. (Anticoagulants are included on the US product label.) Oral anticoagulants are used to prevent postoperative thromboembolic complications, but low-dose subcutaneous heparin is more commonly used. (Anticoagulants are included on the US product label.) Anticoagulants are indicated for the prevention and/or treatment of thromboembolic complications associated with atrial fibrillation (ischemic stroke). Anticoagulants are strongly recommended for patients at high risk of stroke (including those who have recently experienced a stroke, transient ischemic attack, or systemic embolism; left ventricular dysfunction; age over 75 years; hypertension; rheumatic mitral valve disease; post-mechanical or bioprosthetic valve replacement). /Anticoagulants; included in the US product label/
For more complete data on the therapeutic uses of dicumarol (9 of these), please visit the HSDB record page.

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Drug Warnings
Contraindications for oral anticoagulants include a history or coexisting blood clotting disorders, active bleeding, recent or upcoming central nervous system or eye surgery, diagnostic or therapeutic procedures that may result in uncontrollable bleeding (including lumbar puncture), malignant hypertension, peptic ulcer, pregnancy, threatened abortion, intrauterine device, cerebrovascular hemorrhage, and bacterial endocarditis. Relative contraindications include thrombocytopenia, pericarditis, pericardial effusion, and unreliable patient or patient caregiver. Oral anticoagulants
The most common type of bleeding associated with oral anticoagulants is minor bleeding, including bruising, hematuria, epistaxis, conjunctival hemorrhage, minor gastrointestinal bleeding, bleeding from wounds and traumatic sites, and vaginal bleeding. More serious bleeding, such as massive or fatal hemorrhage, is most commonly found in the gastrointestinal tract, intracranial cavity, vagina, retroperitoneum, or wounds or traumatic sites, although bleeding from many other sites has also been reported. Intracranial hemorrhage most commonly occurs in patients taking oral anticoagulants for cerebrovascular disease, and the most common manifestation is subdural hematoma, usually unrelated to head trauma. Fatal gastrointestinal bleeding is most common in peptic ulcers, but any gastrointestinal lesion can lead to massive bleeding. Overall, a bleeding site can be identified in approximately two-thirds of oral anticoagulant-related bleeding cases. /Oral Anticoagulants/
Generally, the bleeding rate with oral anticoagulant therapy is influenced by a variety of factors: anticoagulation intensity, whether intentional or unintentional; underlying clinical conditions requiring anticoagulation (bleeding most commonly occurs in ischemic cerebrovascular disease and venous thromboembolism; bleeding is most common in the elderly); adverse drug interactions or comorbidities, such as clinical conditions that enhance the effects of warfarin, a history of bleeding tendency, malignancy, recent surgery, trauma, or sites of previous potential bleeding (e.g., surgical wounds, peptic ulcers, recent cerebral hemorrhage, colon cancer); concomitant use of aspirin (but not dipyridamole); and patient adherence (e.g., increased bleeding in alcoholics is not due to drug interactions between ethanol and warfarin, but rather to irregular medication use). /Oral Anticoagulants/
There has been a history of spontaneous abortion and stillbirth, as well as low birth weight and growth retardation. Furthermore, fetal or neonatal bleeding, and fetal death due to bleeding, may occur. Anticoagulant use has been reported to increase the risk of intrauterine fetal maldevelopment and maternal hemorrhage in the second and third trimesters of pregnancy. Evidence suggests that embryopathies occur only when oral anticoagulants are administered between the 6th and 12th weeks of gestation. /Anticoagulants/
For more complete data on dicumarol (34 of them), please visit the HSDB records page.

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Pharmacodynamics
Dicoumarol is a coumarin compound found in sweet clover. It is used as an oral anticoagulant by inhibiting the synthesis of vitamin K-dependent clotting factors (prothrombin and factors VII, IX, and X) in the liver.

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Dicumarol is a natural coumarin derivative with dual target inhibitory activity against NQO1 and PDK1[1][2]
It exerts its antitumor effect by inhibiting the following pathways: NQO1-mediated redox cycles and PDK1-dependent metabolic reprogramming inhibit the proliferation, migration, and invasion of cancer cells[1][2]
Dicumarol reverses tamoxifen resistance in breast cancer by downregulating NQO1 and GCLC, restoring its sensitivity to tamoxifen-induced apoptosis[3]
This compound was initially identified as an anticoagulant, but its antitumor potential has been demonstrated in ovarian cancer and tamoxifen-resistant breast cancer[1][2][3]
Its high selectivity for cancer cells relative to normal cells makes it a promising candidate for combination cancer therapy[2][3]

C19H12O6

336.29

336.063

C, 67.86; H, 3.60; O, 28.55

66-76-2

54676038

White to off-white solid powder

1.6±0.1 g/cm3

620.7±55.0 °C at 760 mmHg

290-292 °C(lit.)

231.9±25.0 °C

0.0±1.9 mmHg at 25°C

1.731

3.55

2

6

2

25

605

0

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

DOBMPNYZJYQDGZ-UHFFFAOYSA-N

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

4-hydroxy-3-[(4-hydroxy-2-oxochromen-3-yl)methyl]chromen-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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 (Please use freshly prepared in vivo formulations for optimal results.)