1. Metabolic Enzyme/Protease NF-κB Immunology/Inflammation Autophagy Apoptosis
  2. HIF/HIF Prolyl-Hydroxylase Reactive Oxygen Species NO Synthase Autophagy Apoptosis
  3. Ethyl 3,4-dihydroxybenzoate

Ethyl 3,4-dihydroxybenzoate  (Synonyms: Protocatechuic acid ethyl ester)

Cat. No.: HY-W016409 Purity: 99.96%
Handling Instructions Technical Support

Ethyl 3,4-dihydroxybenzoate (Protocatechuic acid ethyl ester) is an orally effective, blood-brain barrier-permeable, competitive prolyl hydroxylase (PHD) inhibitor that inhibits the hydroxylation modification of hypoxia-inducible factor (HIF) by PHD. Ethyl 3,4-dihydroxybenzoate stabilizes HIF-1α by inhibiting PHD, activates downstream pathways to induce autophagy and apoptosis of tumor cells, and regulates inflammatory responses, inhibits the NF-κB pathway, improves vascular permeability, and promotes osteoblast differentiation. Ethyl 3,4-dihydroxybenzoate has anti-tumor, anti-hypoxic injury, and bone metabolism regulation effects. It can also be used in the research of cardiovascular protection (such as reducing myocardial ischemic damage), bone tissue engineering (promoting osteogenesis/inhibiting osteoclast differentiation), and prevention and treatment of high-altitude cerebral edema.

For research use only. We do not sell to patients.

Ethyl 3,4-dihydroxybenzoate Chemical Structure

Ethyl 3,4-dihydroxybenzoate Chemical Structure

CAS No. : 3943-89-3

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10 mM * 1 mL in DMSO
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500 mg In-stock
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Customer Review

Based on 1 publication(s) in Google Scholar

Other Forms of Ethyl 3,4-dihydroxybenzoate:

Top Publications Citing Use of Products

1 Publications Citing Use of MCE Ethyl 3,4-dihydroxybenzoate

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  • Biological Activity

  • Purity & Documentation

  • References

  • Customer Review

Description

Ethyl 3,4-dihydroxybenzoate (Protocatechuic acid ethyl ester) is an orally effective, blood-brain barrier-permeable, competitive prolyl hydroxylase (PHD) inhibitor that inhibits the hydroxylation modification of hypoxia-inducible factor (HIF) by PHD. Ethyl 3,4-dihydroxybenzoate stabilizes HIF-1α by inhibiting PHD, activates downstream pathways to induce autophagy and apoptosis of tumor cells, and regulates inflammatory responses, inhibits the NF-κB pathway, improves vascular permeability, and promotes osteoblast differentiation. Ethyl 3,4-dihydroxybenzoate has anti-tumor, anti-hypoxic injury, and bone metabolism regulation effects. It can also be used in the research of cardiovascular protection (such as reducing myocardial ischemic damage), bone tissue engineering (promoting osteogenesis/inhibiting osteoclast differentiation), and prevention and treatment of high-altitude cerebral edema[1][2][3][4].

Cellular Effect
Cell Line Type Value Description References
BV-2 IC50
> 10 μM
Compound: 25
Antiinflammatory activity in C57BL6/J mouse BV2 cells assessed as inhibition of LPS-induced nitric oxide production after 24 hrs by Griess assay
Antiinflammatory activity in C57BL6/J mouse BV2 cells assessed as inhibition of LPS-induced nitric oxide production after 24 hrs by Griess assay
[PMID: 26110443]
MDCK CC50
> 169.99 μM
Compound: 3d
Cytotoxicity against MDCK cells after 72 hrs by MTT assay
Cytotoxicity against MDCK cells after 72 hrs by MTT assay
[PMID: 24803363]
MDCK IC50
16.11 μM
Compound: 3d
Antiviral activity against Influenza A virus (A/Vietnam/1194/2004(H5N1)) infected in MDCK cells assessed as inhibition of viral replication after 2 to 3 days by crystal violet staining-based plaque reduction assay
Antiviral activity against Influenza A virus (A/Vietnam/1194/2004(H5N1)) infected in MDCK cells assessed as inhibition of viral replication after 2 to 3 days by crystal violet staining-based plaque reduction assay
[PMID: 24803363]
In Vitro

1. Apoptosis experiment of esophageal cancer cells:
Ethyl 3,4-dihydroxybenzoate (50 μg/mL; 24-72 h) induces S phase cell cycle arrest, loss of mitochondrial membrane permeability and caspase-dependent apoptosis in KYSE 170 and EC109 esophageal squamous cell carcinoma cells, and upregulates the expression of NDRG1, BNIP3, Beclin and other proteins[1].
2. Myocardial protection experiment:
Ethyl 3,4-dihydroxybenzoate (1 mM; 15 min, pretreatment) can activate NOS in isolated cardiomyocytes through KATP channels, increase mitochondrial ROS levels, and exert myocardial protection[2].
3. Osteocyte differentiation assay:
Ethyl 3,4-dihydroxybenzoate (5 mg/mL; 3-14 d) significantly increases alkaline phosphatase (ALP) activity, type I collagen deposition and mineralizes nodule formation in MC3T3-E1 osteoblast precursor cells, and inhibits TRAP-positive multinucleate cell differentiation and TRAP enzyme activity in RAW264.7 osteoclasts in a dose-dependent manner (2-4 mg/mL; 6 d)[3].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Apoptosis Analysis[1]

Cell Line: KYSE 170 and EC109 esophageal squamous cell carcinoma cells
Concentration: 50 μg/mL
Incubation Time: 24 h, 36 h, 72 h
Result: Induced S-phase cell cycle arrest, mitochondrial membrane permeabilization, and caspase-dependent apoptosis in KYSE 170 and EC109 cells.
Upregulated autophagy- and apoptosis-related proteins, including NDRG1, BNIP3, and Beclin, with significant reduction in cell viability by 50-70% compared to controls.

Cell Differentiation Assay[3]

Cell Line: MC3T3-E1 preosteoblasts and RAW264.7 monocyte cells
Concentration: 5 mg/mL (MC3T3-E1); 1-4 mg/mL (RAW264.7)
Incubation Time: 3-14 days (MC3T3-E1); 6 days (RAW264.7)
Result: In MC3T3-E1 cells, significantly increased alkaline phosphatase (ALP) activity (2-fold at day 10), collagen type I deposition (1.5-fold at day 14), and mineralized nodule formation (34% increase at week 5).
In RAW264.7 cells, at 2-4 mg/mL dose-dependently reduced TRAP-positive multinucleated osteoclast formation by 54-77% and decreased TRAP enzyme activity, indicating inhibition of osteoclast differentiation.
In Vivo

1. In vivo bone tissue engineering experiment:
Ethyl 3,4-dihydroxybenzoate (5 mg/bead, 50 mg/bead; subcutaneous injection after Alginate gel embedding; single dose; 5-8 weeks) promotes the osteogenic differentiation of human mesenchymal stem cells, increases the expression of type I collagen and osteocalcin, and induces the formation of new bone tissue in the nude mouse subcutaneous transplantation model[3].
2. High-altitude cerebral edema prevention and treatment experiment:
Ethyl 3,4-dihydroxybenzoate (50-100 mg/kg; intraperitoneal injection; once daily; 3 days) significantly reduces brain water content and vascular permeability in the rat acute hypobaric hypoxia model, down-regulates pro-inflammatory factors such as TNF-α and IL-6, and up-regulates antioxidant proteins such as HIF-1α and HO-1[4].
3. Myocardial ischemia protection experiment:
Ethyl 3,4-dihydroxybenzoate (75 mg/kg; intraperitoneal injection; once daily; 3 days) reduces infarct area and inhibited NF-κB-mediated inflammatory response in the rat myocardial ischemia-reperfusion model by activating the NOS-NO pathway and mitochondrial KATP channel[4].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model: Subcutaneous bone tissue engineering model (Balb/c nude mice) (6 weeks old), subcutaneous transplantation of alginate-encapsulated hMSCs[3]
Dosage: 5 mg/bead or 50 mg/bead, incorporated into alginate gel
Administration: Single subcutaneous injection into the back, observed for 5-8 weeks
Result: Promoted osteogenic differentiation of human mesenchymal stem cells (hMSCs), as shown by von Kossa and Alizarin Red staining for calcium deposition.
Increased expression of collagen type I and osteocalcin in the transplanted tissue, with dose-dependent enhancement (50 mg/bead showing stronger effects).
Resulted organized new bone formation with reduced fibrous tissue compared to control groups.
Animal Model: Myocardial ischemia-reperfusion injury model (Sprague-Dawley rats) (180±20 g), myocardial ischemia-reperfusion injury model[4]
Dosage: 75 mg/kg, dissolved in DMSO/saline
Administration: Intraperitoneal injection, daily for 3 days, followed by 30-min coronary artery occlusion and 120-min reperfusion
Result: Significantly reduced infarct size by 68% compared to controls, associated with increased nitric oxide (NO) production, activation of mitochondrial ATP-sensitive potassium (mitoKATP) channels, and downregulation of nuclear factor-κB (NF-κB)-dependent pro-inflammatory cytokines (TNF-α, IL-6). Western blot analysis showed upregulation of hypoxia-inducible factor-1α (HIF-1α) and heme oxygenase-1 (HO-1), while immunohistochemistry revealed reduced neutrophil infiltration in the ischemic myocardium.
Animal Model: Acute hypobaric hypoxia model (Sprague-Dawley rats) (180±20 g), acute hypobaric hypoxia at 9144 m for 5 h[4]
Dosage: 75 mg/kg, dissolved in DMSO/saline
Administration: Intraperitoneal injection, daily for 3 days prior to hypoxia exposure
Result: Reduced brain water content (edema index) by 10% and vascular permeability (sodium fluorescein leakage) by 49% compared to hypoxic controls.
Decreased levels of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) and increased anti-inflammatory IL-10.
Upregulated HIF-1α, HO-1, and metallothionein-1 (MT-1) expression, while NF-κB nuclear translocation and vascular cell adhesion molecule-1 (VCAM-1) expression were significantly attenuated.
Molecular Weight

182.18

Formula

C9H10O4

CAS No.
Appearance

Solid

Color

White to off-white

SMILES

O=C(OCC)C1=CC=C(O)C(O)=C1

Structure Classification
Initial Source
Shipping

Room temperature in continental US; may vary elsewhere.

Storage

4°C, stored under nitrogen

*In solvent : -80°C, 6 months; -20°C, 1 month (stored under nitrogen)

Solvent & Solubility
In Vitro: 

DMSO : 100 mg/mL (548.92 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 5.4892 mL 27.4461 mL 54.8923 mL
5 mM 1.0978 mL 5.4892 mL 10.9785 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

  • Molarity Calculator

  • Dilution Calculator

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Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

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In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

    Solubility: ≥ 2.5 mg/mL (13.72 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
  • Protocol 2

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

    Solubility: ≥ 2.5 mg/mL (13.72 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

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(per animal)

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Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
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+
%
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Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration: mg/mL
Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).

*In solvent : -80°C, 6 months; -20°C, 1 month (stored under nitrogen)

The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation

Purity: 99.96%

References

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
DMSO 1 mM 5.4892 mL 27.4461 mL 54.8923 mL 137.2307 mL
5 mM 1.0978 mL 5.4892 mL 10.9785 mL 27.4461 mL
10 mM 0.5489 mL 2.7446 mL 5.4892 mL 13.7231 mL
15 mM 0.3659 mL 1.8297 mL 3.6595 mL 9.1487 mL
20 mM 0.2745 mL 1.3723 mL 2.7446 mL 6.8615 mL
25 mM 0.2196 mL 1.0978 mL 2.1957 mL 5.4892 mL
30 mM 0.1830 mL 0.9149 mL 1.8297 mL 4.5744 mL
40 mM 0.1372 mL 0.6862 mL 1.3723 mL 3.4308 mL
50 mM 0.1098 mL 0.5489 mL 1.0978 mL 2.7446 mL
60 mM 0.0915 mL 0.4574 mL 0.9149 mL 2.2872 mL
80 mM 0.0686 mL 0.3431 mL 0.6862 mL 1.7154 mL
100 mM 0.0549 mL 0.2745 mL 0.5489 mL 1.3723 mL
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Ethyl 3,4-dihydroxybenzoate
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