The specific molecular target is not specified in the provided texts. In melanoma cells, treatment with 4-Hydroxycoumarin leads to decreased papillin expression, reduced translocation of papillin to focal adhesions, and reduced activation of FAK and Rac-1 [3].
In B16-F10 melanoma cells, 4-Hydroxycoumarin decreases tyrosine phosphorylation of several proteins in a concentration-dependent manner [2].

At the junction of the hepatitis C virus (HCV) NS3 protein's protease and helicase domains, a unique, highly conserved binding site was discovered. 4-Phenoxybenzylamine, a novel family of direct-acting antiviral medications, binds to this allosteric site and stabilizes the inactive conformation of the NS3 protein, inhibiting its action [1].

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In B16-F10 melanoma cells, 4-Hydroxycoumarin (50, 160, or 500 µM) disorganized the actin cytoskeleton, impairing the formation of stress fibers and lamellipodia, and induced cell shrinkage. This effect was concentration-dependent and selective for tumor cells, as it did not produce significant changes in B82 fibroblasts. The effect was reversible after removal of the compound [2].
4-Hydroxycoumarin produced a concentration-dependent reduction in the adhesion of B16-F10 cells to extracellular matrix proteins. At 500 µM, adhesion to fibronectin or vitronectin was reduced by approximately half. Adhesion to collagen type IV was reduced to one-fifth, and adhesion to laminin was reduced to one-tenth of the control value [2].
4-Hydroxycoumarin inhibited the random motility (migration) of B16-F10 cells in a wound healing assay. Concentrations of 50 and 160 µM partially inhibited migration, while 500 µM totally inhibited migration [2].
4-Hydroxycoumarin had no appreciable effect on cell viability of B16-F10 cells or B82 fibroblasts at concentrations from 50 to 500 µM after 24 hours of exposure. It also did not modify actin expression in either cell line [2].
In B16-F10 cells, 4-Hydroxycoumarin decreased tyrosine phosphorylation of several proteins in a concentration-dependent manner (50-500 µM). This effect correlated with cytoskeletal disorganization, impaired adhesion, and reduced migration [2].
In vitro treatment of B16-F10 melanoma cells with 4-Hydroxycoumarin decreases their metastatic capability [3].
4-Hydroxycoumarin (500 µM) is neither cytostatic nor cytotoxic and does not affect long-term survival of B16-F10 cells [3].
4-Hydroxycoumarin (500 µM) is not toxic to cultured hepatocytes [3].
4-Hydroxycoumarin (500 µM) is not toxic to proximal tubular LLC-PK1 cells in vitro [3].

Oral administration of 4-Hydroxycoumarin (10, 20, or 40 mg/kg/day) to C57BL/6 mice for 7 days prior to intravenous injection of B16-F10 cells reduced the number of pulmonary experimental metastases by >85% [3].
In C57BL/6 mice bearing a subcutaneous B16-F10 tumor, oral administration of 4-Hydroxycoumarin at 10 mg/kg/day reduced tumor size from day 22 and significantly increased mean survival time (P = 0.02). Higher doses (20 or 40 mg/kg/day) did not affect tumor size or survival. The positive control cyclophosphamide (200 mg/kg, single i.p. dose) reduced tumor size from day 16 but did not alter survival time [3].
In the same subcutaneous tumor model, 4-Hydroxycoumarin at 20 or 40 mg/kg/day reduced the number of spontaneous lung metastases by 50% (mean 5.5 ± 1.5 and 5.8 ± 4.8, respectively, vs. 11.9 ± 6.0 in control). At 10 mg/kg/day, the reduction was not statistically significant (7.0 ± 1.7) [3].
Prophylactic administration of 4-Hydroxycoumarin prior to intravenous injection of melanoma cells reduces metastases number, indicating it affects early steps of experimental metastasis rather than metastatic growth [3].

Cell viability was estimated using the MTT (3-(4,5-dimethyltiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay. The assay is based on the reduction of the soluble tetrazolium salt by mitochondria of viable cells. The product, an insoluble colored formazan, was dissolved in dimethyl sulfoxide and measured spectrophotometrically at 570 nm. The amount of reduced formazan is proportional to the number of viable cells [2].
For morphology and F-actin content analysis, cells treated in labtek chambers were fixed with 4% formaldehyde in PBS and permeated with 0.1% triton X-100 diluted in PBS. The polymerized actin was stained with phalloidin conjugated to a fluorophore. Cell morphology and F-actin were examined using fluorescence microscopy [2].
Cell adhesion to extracellular matrix proteins (human fibronectin (5 µg/ml), human vitronectin (1 µg/ml), mouse type IV collagen (20 µg/ml), or mouse laminin (10 µg/ml)) was performed. Proteins were adsorbed to 96-well microplates overnight at 4°C. Wells were blocked with heat-denatured bovine serum albumin (10 mg/ml in PBS). Treated cells were detached using non-enzymatic methods, resuspended, and 10⁴ cells were added to coated wells to adhere for 30 min at 37°C. Non-adherent cells were removed by washing, and remaining cells were fixed and stained with crystal violet. The absorbance of the solubilized dye was measured at 595 nm. Unspecific adhesion was estimated using wells coated only with BSA (<4%) and was subtracted [2].
Cell migration was investigated by a wound healing assay. After overnight incubation, experimental wounds were made by dragging a cell scraper across cell cultures. Cultures were rinsed with PBS, and serum-free medium containing either vehicle, 4-HC, or cytochalasin D was added. Cultures were photographed immediately (t = 0) and 24 h later using an inverted microscope [2].
Analysis of protein tyrosine phosphorylation: Treated cells were lysed in cold lysis buffer. After a 15 min incubation on ice, insoluble material was removed by centrifugation. Total protein (40 µg) was loaded in each line, separated by SDS-PAGE, and translocated onto nylon membranes. Cell extracts were probed with an anti-phosphotyrosine (PY) antibody (clone PY20, 1:500) or anti-actin (clone C11, 1:2000) and then subjected to enhanced chemiluminescence [2].

For the experimental metastasis assay, 4-Hydroxycoumarin (10, 20, or 40 mg/kg/day) was administered via oral gavage to male C57BL/6 mice for 7 days. The vehicle was 0.2% methylcellulose. On day 7, each mouse was injected into the tail vein with 8 × 10⁵ untreated B16-F10 cells. Mice were euthanized 2 weeks later, and lungs were excised to count pulmonary metastatic tumors [3].
For the survival/spontaneous metastasis assay, male C57BL/6 mice were injected subcutaneously with 2 × 10⁵ B16-F10 cells. After 2 weeks, mice with a tumor between 3-6 mm in diameter were selected. Mice were distributed into groups: control (0.2% methylcellulose), 4-Hydroxycoumarin (10, 20, or 40 mg/kg/day, oral gavage), and cyclophosphamide (200 mg/kg, single i.p. dose, positive control). Tumor size was estimated using the formula: (major axis) × (minor axis)² × 0.52. The day of death was registered to generate Kaplan-Meier graphs. Upon death, lungs were excised to quantify the number of pulmonary spontaneous metastases [3].
For toxicological evaluation, healthy mice were orally dosed with 4-Hydroxycoumarin (10, 20, or 40 mg/kg/day) for 60 days. Control mice received the vehicle (0.2% methylcellulose) [3].
For electron microscopy, intracardiac perfusion of mice was carried out with Zamboni's fixer (0.3% picric acid in 6.2% neutral buffered formaldehyde solution). Fixation of dissected organs (kidneys, livers, lungs) was completed by immersion in the same fixative for 24 h at 4°C. Samples were then postfixed in 1% osmium tetroxide, dehydrated, and embedded. Ultrafine sections of 60 nm thickness were obtained and analyzed with a transmission electron microscope [3].
Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured in citrated plasma of healthy mice that received 4-Hydroxycoumarin (10 mg/kg/day) for 30 days. PT was determined using the Quick method. aPTT was evaluated by addition of an activator reagent; the mixture was incubated for 120 s at 37°C, and then clot formation was induced with 0.02 M CaCl₂. In both methods, the time to clot formation was recorded [3].

The pharmacokinetics of 4-Hydroxycoumarin is unknown [3].
The lack of anticoagulant effect 48 h after a long-term administration of 4-Hydroxycoumarin (10 mg/kg/day for 30 days, PT returned to control levels: 11.8 ± 0.5 s vs. 11.2 ± 0.7 s in control) suggests that this compound is rapidly eliminated [3].
In vivo, the closely structurally related compound coumarin (1,2-benzopyrone) has a short half-life (approximately 1 h in humans) and is rapidly metabolized into hydroxylated derivatives, including 7-hydroxycoumarin (major) and 4-Hydroxycoumarin (minor) [2].

4-Hydroxycoumarin (20 or 40 mg/kg/day for 30-60 days) produced dose- and time-dependent hepatic damage in C57BL/6 mice, including cytoplasmic changes in hepatocytes surrounding the portal vein. At 10 mg/kg/day, no hepatic histological alterations were observed. Serum γ-glutamyltransferase (γ-GT) levels were not affected by any dose [3].
In kidneys, 4-Hydroxycoumarin (20 or 40 mg/kg/day) increased blood urea nitrogen (BUN) levels above the normal limit (7-30 mg/dl) from the third week. Histological analysis revealed metaplasia of the parietal layer of the capsule of Bowman and loss of microvilli in cuboidal epithelial cells of proximal convoluted tubules. Creatinine clearance rate (CCR) and urinary N-acetyl-β-D-glucosaminidase (NAG) levels were unaffected, indicating no functional damage to glomeruli or tubular cells [3].
In lungs, 4-Hydroxycoumarin at all evaluated doses (10, 20, 40 mg/kg/day) induced hyperplasia and loss of apical projections of Clara cells with leakage of its apical dome-shaped region from day 30. Type II alveolar cells showed lamellar bodies that were partially empty or with vacuolization [3].
4-Hydroxycoumarin (10 mg/kg/day for 30 days) produced a pronounced anticoagulant effect, increasing aPTT two-fold (117.7 ± 21.2 s vs. 62.8 ± 3.2 s in control) and PT >15-fold (>200 s vs. 13.3 ± 1.0 s in control). This effect was totally lost 48 hours after the last administration [3].
4-Hydroxycoumarin can be biotransformed into a 3,4-epoxide in the liver of C67BL/6 mice, which may be responsible for toxicity [3].

[1]. Discovery of an allosteric mechanism for the regulation of HCV NS3 protein function. Nat Chem Biol. 2012 Nov;8(11):920-5.

4-Hydroxycoumarin-based compounds are important among heterocyclic structures due to their biological and pharmaceutical activities. This review provides an overview of recent applications of 4-Hydroxycoumarin in multicomponent reactions for the synthesis of various heterocyclic compounds during 2015-2018. The most significant reactivity of 4-hydroxycoumarin is the nucleophilicity of the carbon atom at position 3, where reactions like Mannich reaction and coupling reaction take place [1].
The in vitro effects of 4-Hydroxycoumarin (disorganization of actin cytoskeleton, reduced adhesion, and motility) on melanoma cells suggest that it might be useful to prevent metastasis and could be used as an adjuvant therapy for melanoma [2].
The antimetastatic effect of 4-Hydroxycoumarin in vivo supports the hypothesis that coumarin may act as a prodrug, with its hydroxylated metabolites (like 4-HC) being the active agents [2][3].
The identification of the molecular mechanisms of the antimetastatic and antineoplastic actions of 4-Hydroxycoumarin may lead to the development of structurally and/or mechanistically related agents useful in the prevention of metastasis. The narrow therapeutic index of 4-Hydroxycoumarin limits its possible use as an adjuvant in melanoma therapy [3].

C13H13NO

199.2484

199.099

107622-80-0

2760343

Colorless to light yellow liquid

1.1±0.1 g/cm3

319.6±25.0 °C at 760 mmHg

149.5±16.4 °C

0.0±0.7 mmHg at 25°C

1.595

3.08

1

2

3

15

169

0

O(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])N([H])[H]

CCAZAGUSBMVSAR-UHFFFAOYSA-N

InChI=1S/C13H13NO/c14-10-11-6-8-13(9-7-11)15-12-4-2-1-3-5-12/h1-9H,10,14H2

(4-phenoxyphenyl)methanamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~12.5 mg/mL (~62.74 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (6.27 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 12.5 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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Solubility in Formulation 2: 1.25 mg/mL (6.27 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (6.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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