| Targets |
Epianhydrotetracycline belongs to the "class 2" tetracycline analogs, characterized by a mode of action entirely distinct from clinically used tetracyclines (class 1). While class 1 tetracyclines (e.g., tetracycline, doxycycline) inhibit bacterial protein synthesis by binding to the 30S ribosomal subunit, EATC fails to inhibit cell-free protein synthesis in vitro or is a very poor inhibitor . Instead, class 2 compounds rapidly inhibit the in vivo incorporation of precursors into DNA and RNA, suggesting that the cytoplasmic membrane is their primary site of action rather than the ribosome . Studies examining the chemical reactivity of bases in 16S rRNA to dimethyl sulfate confirmed that EATC does not affect ribosomal base reactivity, further supporting its non-ribosomal mechanism .
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| ln Vitro |
4-Epihydrotetracycline hydrochloride (0–20 mg/L; 96 h) resulted in a significant rise in ROS levels, MDA concentration, and PC content in the concentration range of 5-20.0 mg/L [1]. -Dose-dependent effects on SOD activity are observed with epihydrotetracycline hydrochloride (0–20 mg/L; 96 h), which first increases and subsequently decreases. The maximum SOD activity is achieved at a dosage of 2.50 mg/L [1]. 4-EpianHydrotetracycline HydroHClide (0–20 mg/L; 96 h) causes the cells to get disinfected [1]. 4-EpianHydrotetracycline HydroHClide (0-100 mg/L) kills zebrafish; the LC50 value at 96 hours is 29.13 mg/liter. Zebrafish have increased independent stability when exposed to 4-EpianHydrotetracycline HydroHClide; at 96 hours, the EC50 value is 8.57 mg/L [2]. 4-Due to its delayed jump action, epihydrotetracycline hydrochloride (10–20 mg/L) reduces clouding. MIC values of 2, 1, and 64 mg/L (5-20.0 mg/L) for Shewanella spp., Escherichia coli, and Pseudomonas aeruginosa, respectively, are observed when epihydrotetracycline hydrochloride (0–20 mg/L) is used. [2].
In vitro studies have demonstrated that Epianhydrotetracycline exhibits moderate antibacterial activity against a range of bacterial species. It is active against Pseudomonas, Agrobacterium, Moraxella, Bacillus, and Escherichia coli, with MIC₅₀ values ranging from 0.75 to 16 mg/L . More specifically, EATC shows inhibitory effects against Shewanella (MIC: 2 mg/L), E. coli (MIC: 1 mg/L), and Pseudomonas aeruginosa (MIC: 64 mg/L) . Despite its antibacterial activity, it is significantly less potent than the parent compound tetracycline. EATC also induces oxidative stress in vitro, significantly enhancing reactive oxygen species levels, malondialdehyde concentration, and protein carbonyl content in a dose-dependent manner . |
| ln Vivo |
In vivo toxicological studies in animal models have revealed that EATC exhibits significant biological activity primarily characterized by its toxicity. In zebrafish embryo models, EATC exposure for 96 hours produces lethal effects with an LC₅₀ of 29.13 mg/L and teratogenic effects with an EC₅₀ (malformation) of 8.57 mg/L . The compound also induces dose-dependent apoptosis, with 66.67% of embryos showing apoptosis at a concentration of 20.0 mg/L . Additional in vivo effects include reduced heart rate (at 10-20 mg/L) and delayed hatching (at 5-20 mg/L) in zebrafish embryos . In chick embryo studies, EATC showed significantly higher toxicity compared to tetracycline hydrochloride .
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| Enzyme Assay |
Methodology for Ribosomal Binding Assessment (16S rRNA Chemical Reactivity Assay): To assess whether Epianhydrotetracycline binds to the bacterial ribosome (30S subunit), a chemical probing assay using dimethyl sulfate (DMS) can be employed as described by Rasmussen et al. (1991) . Purified 70S ribosomes from E. coli are incubated with EATC (typically at 100 µM) in binding buffer containing 10 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 60 mM NH₄Cl, and 10 mM 2-mercaptoethanol for 15 minutes at 37°C. DMS is added to modify unprotected adenine (A) and cytosine (C) residues in 16S rRNA. The reaction is stopped with 2-mercaptoethanol, and rRNA is extracted. Primer extension analysis is performed using reverse transcriptase to identify positions of DMS modification. The absence of EATC-induced protection patterns compared to tetracycline (class 1) confirms that EATC does not bind to the ribosome.
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| Cell Assay |
Apoptosis analysis [1]
Cell Types: Zebrafish embryos Tested Concentrations: 0, 1.25, 2.5, 5, 10 and 20 mg/L Incubation Duration: 96 hrs (hours) Experimental Results: Dose-dependent induction of apoptosis, 66.67% of zebrafish embryos appeared Apoptosis. This phenomenon occurred at a dose of 20.0 mg/L. Methodology for Oxidative Stress and Apoptosis Assessment in Zebrafish Embryos: The in vitro cellular effects of Epianhydrotetracycline can be assessed using zebrafish embryo model . Zebrafish embryos at 6-8 hours post-fertilization are placed in 24-well plates (10-20 embryos per well) and exposed to various concentrations of EATC (0, 1.25, 2.5, 5, 10, 20 mg/L) for 96 hours . After exposure, embryos are homogenized in ice-cold phosphate-buffered saline. Supernatants are collected for biochemical assays: reactive oxygen species levels measured using DCFH-DA fluorescent probe (excitation 485 nm, emission 528 nm), malondialdehyde concentration determined by thiobarbituric acid reactive substances assay, and protein carbonyl content measured by DNPH derivatization. Superoxide dismutase activity is assessed using commercial kits. For apoptosis analysis, embryos are stained with acridine orange (5 μg/mL) for 30 minutes, washed, and observed under a fluorescence microscope to quantify apoptotic cells . |
| Animal Protocol |
Methodology for Acute and Sub-Acute Toxicity Study in Mice: The in vivo toxicity of Epianhydrotetracycline (anhydro-4-epi-tetracycline) has been evaluated in mice as described in Acta Pharmaceutica Sinica (1966) . For acute toxicity studies, male mice (18-22 g) are administered EATC intravenously via tail vein at various doses to determine the LD₅₀. For sub-acute toxicity studies, mice receive EATC at doses of 1 mg/day intravenously or 12 mg/day orally over a treatment period. Body weight is monitored daily. Urinalysis is performed using test strips or microscopy to detect proteinuria, glucosuria, and other abnormalities. At the conclusion of the study, animals are euthanized, and kidneys are harvested for histopathological examination (hematoxylin and eosin staining) to assess pathological changes in renal convoluted tubules . Toxicity parameters are compared across tetracycline, 4-epitetracycline, anhydrotetracycline, and EATC.
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| ADME/Pharmacokinetics |
As a tetracycline degradation product, EATC is not administered therapeutically and therefore has not been extensively characterized pharmacokinetically. Based on its structural similarity to tetracycline and other degradation products, EATC is expected to be capable of entering the cytoplasm of bacterial cells, as demonstrated by its ability to inhibit macromolecular synthesis in vivo . The compound is known to be soluble in ethanol, methanol, DMF, DMSO, and water . For research purposes, the compound is stored as a solid at -20°C for long-term stability, with solvent solutions stable at -80°C for up to one year . The compound is supplied as the hydrochloride salt (CAS: 4465-65-0) to enhance aqueous solubility.
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| Toxicity/Toxicokinetics |
Epianhydrotetracycline is considered a highly toxic tetracycline degradation product . In acute toxicity studies in mice, the intravenous LD₅₀ of anhydro-4-epi-tetracycline (EATC) is 193 mg/kg, compared to 160.7 mg/kg for tetracycline and 85.8 mg/kg for 4-epitetracycline . However, in sub-acute toxicity studies, EATC exhibits the most severe toxic effects of all tested tetracycline derivatives. Intravenous administration of just 1 mg/day or oral administration of 12 mg/day causes severe diabetic urine, proteinuria, and serious pathological changes in the renal convoluted tubules . Research indicates that EATC may be the chief compound responsible for Fanconi syndrome (a rare kidney disorder characterized by dysfunction of proximal tubular reabsorption) associated with ingestion of outdated tetracycline preparations . In zebrafish embryo studies, EATC shows dose-dependent developmental toxicity with an EC₅₀ of 8.57 mg/L for malformation . The toxicity mechanism involves induction of oxidative stress (increased ROS, MDA, protein carbonyl) and activation of apoptotic pathways . Due to its high toxicity, the presence of EATC in tetracycline pharmaceutical preparations is strictly controlled.
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| References |
[1]. Effects of 4-epianhydrotetracycline on oxidative stress in zebrafish (Danio rerio) embryos. Sci Total Environ. 2021 Nov 20;796:149047.
[2]. Developmental toxicity and transcriptome analysis of 4-epianhydrotetracycline to zebrafish (Danio rerio) embryos. Sci Total Environ. 2020 Sep 10;734:139227. [3]. Toxicity of tetracycline and its transformation products to a phosphorus removing Shewanella strain. Chemosphere. 2020 May;246:125681. |
| Exact Mass |
462.119
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|---|---|
| Elemental Analysis |
C, 57.09; H, 5.01; Cl, 7.66; N, 6.05; O, 24.19
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| CAS # |
4465-65-0
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| PubChem CID |
54682545
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| Appearance |
Typically exists as solid at room temperature
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| Boiling Point |
618.6ºC at 760 mmHg
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| Melting Point |
220ºC
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| Flash Point |
327.9ºC
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| LogP |
1.958
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
32
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| Complexity |
855
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| Defined Atom Stereocenter Count |
3
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| InChi Key |
SPFAOPCHYIJPHJ-MOMXNFOMSA-N
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| InChi Code |
InChI=1S/C22H22N2O7.ClH/c1-8-9-5-4-6-12(25)13(9)17(26)14-10(8)7-11-16(24(2)3)18(27)15(21(23)30)20(29)22(11,31)19(14)28;/h4-6,11,16,25-26,29,31H,7H2,1-3H3,(H2,23,30);1H/t11-,16+,22-;/m0./s1
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| Chemical Name |
(4R,4aS,12aR)-4-(dimethylamino)-1,10,11,12a-tetrahydroxy-6-methyl-3,12-dioxo-4a,5-dihydro-4H-tetracene-2-carboxamide;hydrochloride
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| Synonyms |
4-Epianhydrotetracycline hydrochloride; 4465-65-0; EATC; Epianhydrotetracycline (hydrochloride); 06Y7HM8DBA; Epianhydrotetracycline, 4-Epianhydrotetracycline HCl
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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.) |
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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