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Anhydrotetracycline hydrochloride is a tetracycline biosynthetic precursor that is a potent competitive broad-spectrum tetracycline destructase enzymes inhibitor. Anhydrotetracycline hydrochloride is an effector for tetracycline controlled gene expression systems in eukaryotic cells.
Anhydrotetracycline hydrochloride (ATC) is the hydrochloride salt form of anhydrotetracycline, with the molecular formula C₂₂H₂₃ClN₂O₇ and a molecular weight of 462.88 g/mol. It is a tetracycline biosynthetic precursor and a degradation product of tetracycline, characterized by the aromatization of the B ring of the tetracycline core structure. Unlike the parent compound tetracycline, which is a potent antibiotic, anhydrotetracycline hydrochloride exhibits no direct antibacterial activity. Instead, it is a powerful effector molecule for tetracycline repressor (TetR) and reverse tetracycline repressor (revTetR) systems in eukaryotic cells, making it a key tool for tetracycline-controlled gene expression systems (Tet-ON/Tet-OFF). The compound appears as a yellow powder and should be stored at -20°C for long-term stability.| Targets |
Anhydrotetracycline hydrochloride acts as an effector molecule that modulates the DNA-binding activity of the tetracycline repressor (TetR) and its engineered variant reverse tetracycline repressor (revTetR). In the absence of the effector, TetR binds tightly to its palindromic tetO operator DNA sequence, blocking transcription of downstream genes. Upon binding of anhydrotetracycline to TetR, the repressor undergoes a conformational change that causes it to dissociate from the DNA, enabling gene expression. Conversely, in revTetR systems, anhydrotetracycline acts as a corepressor, enhancing DNA-binding and inhibiting transcription. Additionally, anhydrotetracycline hydrochloride is a competitive broad-spectrum inhibitor of tetracycline destructase enzymes (Tet(50), Tet(X), Tet(X)_3), which are bacterial enzymes that degrade tetracycline antibiotics.
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| ln Vitro |
In vitro studies have extensively characterized the activity of anhydrotetracycline hydrochloride. It binds to the TetR with approximately 35-fold higher affinity compared to tetracycline, with an IC₅₀ near 0.4 μM. In β-galactosidase assays, TetR inhibited β-gal expression to nearly 1%, while approximately 100% expression was achieved in the presence of 0.4 µM ATC. For the revTetR system, the presence of 0.4 µM ATC resulted in a 5-fold decrease of β-gal activity. Anhydrotetracycline was much more effective than tetracycline in inactivating transcriptional transactivator (tTA), completely abolishing tTA-mediated luciferase activity at concentrations as low as 3 ng/mL. Treatment of NIH3T3-HER2 cells with anhydrotetracycline hydrochloride (10 ng/mL) for 3 days resulted in downregulation of the HER2 gene to below the detection limit. Treatment of N1 cells with the compound (10-200 nM) for 7 days significantly reduced the expression level of the Top10-driven GUS gene.
AnHydrotetracycline HCl can inhibit the degradation of Tet(50) (IC50=210 μM), Tet(X) (IC50=41 μM), and Tet(X)_3 (IC50=3 μM) of tetracyclines. AnHydrotetracycline salticide inhibits the degradation of Tet(50) (IC50=210 μM), Tet(X) (IC50=75 μM), and Tet(X)_3 (IC50=26 μM) of chlortetracycline. AnHydrotetracycline HCl can inhibit the degradation of Tet(50) (IC50=120 μM), Tet(X) (IC50=41 μM), and Tet(X)_3 (IC50=7 μM) of demeclocycline [1]. AnHydrotetracycline HCl can inhibit the degradation of Tet(50) (IC50=210 μM), Tet(X) (IC50=41 μM), and Tet(X)_3 (IC50=3 μM) of tetracyclines. AnHydrotetracycline salticide inhibits the degradation of Tet(50) (IC50=210 μM), Tet(X) (IC50=75 μM), and Tet(X)_3 (IC50=26 μM) of chlortetracycline. AnHydrotetracycline HydroHCl can inhibit the degradati |
| ln Vivo |
In vivo studies have demonstrated significant biological activity of anhydrotetracycline hydrochloride in animal models. Treatment of mice bearing NIH3T3-HER2 cell-based tumor xenografts with anhydrotetracycline hydrochloride (10 mg/kg; subcutaneous injection) resulted in tumor regression of more than 95% within 7 days. Similarly, intraperitoneal administration of the compound (10 mg/kg) induced downregulation of ERBB2 mRNA and protein, leading to a rapid reduction in tumor volume. In bacterial infection models, anhydrotetracycline acts synergistically with tetracycline to inhibit the growth of E. coli expressing tetracycline destructase enzymes, functionally rescuing tetracycline antibiotic activity. The fractional inhibitory concentration index (FICI) for the combination is 0.1875, indicating strong synergy. The compound effectively rescues tetracycline from Tet(X6)-mediated inactivation.
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| Enzyme Assay |
Methodology for Tetracycline Destructase Inhibition Assay: The inhibitory activity of anhydrotetracycline hydrochloride against tetracycline destructase enzymes is determined using a spectrophotometric assay. The enzyme (e.g., Tet(50), Tet(X), Tet(X)_3) is incubated with varying concentrations of anhydrotetracycline (0.1 μM to 10 mM) in reaction buffer containing the tetracycline substrate. The reaction is initiated by addition of the tetracycline substrate, and tetracycline consumption is monitored by measuring the change in absorbance at 400 nm over time. Reaction velocity is calculated from the linear portion of the absorbance decay curve. IC₅₀ values are determined by fitting dose-response data using non-linear regression analysis. For each enzyme-inhibitor pair, reactions are performed in triplicate, and results are expressed as mean ± standard deviation. The synergistic effect can be evaluated using minimum inhibitory concentration (MIC) assays and calculating the fractional inhibitory concentration index (FICI) by checkerboard titration.
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| Cell Assay |
Methodology for Gene Expression Modulation in Mammalian Cells: The ability of anhydrotetracycline hydrochloride to modulate gene expression in Tet-controlled systems is assessed using established mammalian cell lines expressing tetracycline-regulated reporter genes. NIH3T3-HER2 cells or N1 cells are cultured in appropriate medium (e.g., DMEM with 10% FBS) at 37°C with 5% CO₂. Anhydrotetracycline hydrochloride is added to the culture medium at concentrations ranging from 10 ng/mL to 200 nM for specified durations (e.g., 3 days for HER2 downregulation studies; 7 days for GUS gene expression studies). Following treatment, cells are harvested for analysis. Gene expression levels are quantified by RT-qPCR (mRNA) or Western blotting (protein). For reporter gene assays, enzymatic activity (e.g., GUS or luciferase) is measured using fluorogenic or chemiluminescent substrates. For β-galactosidase assays, cells transfected with TetR or revTetR systems are treated with varying concentrations of ATC (e.g., 0.4 µM) to assess repressor activity.
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| Animal Protocol |
Methodology for Tumor Xenograft Regression Study in Mice: The in vivo efficacy of anhydrotetracycline hydrochloride is evaluated using a mouse xenograft model. Female immunocompromised mice (e.g., nude mice) are subcutaneously implanted with NIH3T3-HER2 cells (or other tetracycline-responsive cell lines). Once tumors reach a measurable size (e.g., approximately 100-200 mm³), animals are randomly assigned to treatment groups (n≥5 per group). Anhydrotetracycline hydrochloride is administered at a dose of 10 mg/kg body weight via subcutaneous injection or intraperitoneal injection, typically once daily. Control animals receive vehicle alone (e.g., saline). Tumor volumes are measured every 2-3 days using calipers, and body weights are monitored to assess toxicity. After the treatment period (typically 7-14 days), animals are euthanized, and tumors are excised for further analysis (e.g., histology, gene expression analysis). For bacterial infection models, mice infected with tetracycline destructase-expressing E. coli are treated with tetracycline alone or in combination with anhydrotetracycline, and bacterial burden is assessed.
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| ADME/Pharmacokinetics |
The compound is known to be active in vivo following both subcutaneous and intraperitoneal administration, as demonstrated by tumor regression studies in mice at a dose of 10 mg/kg. Its ability to modulate gene expression in vivo and rescue tetracycline antibiotic activity suggests adequate bioavailability and tissue distribution. In cell culture studies, anhydrotetracycline demonstrated high functional stability, with the concentration that began to affect HeLa cell growth rate (>3 μg/mL) being more than a thousand-fold above the effective concentration (3 ng/mL). The compound is soluble in water and acetonitrile. According to EDQM guidelines, long-term storage conditions are +5°C ± 3°C.
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| Toxicity/Toxicokinetics |
The material safety data sheet (MSDS) indicates no identified hazards, with a signal word of "None". However, EDQM classification assigns a UN Code of 3077 (Environmentally hazardous substance, solid, n.o.s.), and a Shipping Group of A1A. In cell culture studies, the concentration at which prolonged presence of anhydrotetracycline began to affect the growth rate of HeLa cells (>3 μg/mL) was more than a thousand-fold above the effective concentration (3 ng/mL), indicating a wide therapeutic window in vitro. The compound is for research use only and not intended for human or veterinary use. As with all research chemicals, standard laboratory safety precautions should be followed when handling this compound.
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| References |
| Molecular Formula |
C22H22N2O7.HCL
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|---|---|
| Molecular Weight |
462.88022
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| Exact Mass |
462.119
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| CAS # |
13803-65-1
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| Related CAS # |
Anhydrotetracycline;1665-56-1
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| PubChem CID |
54710409
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| Appearance |
Yellow to brown solid powder
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| Boiling Point |
618.6ºC at 760 mmHg
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| Melting Point |
222.8ºC
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| Flash Point |
327.9ºC
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| Vapour Pressure |
3.66E-16mmHg at 25°C
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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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| SMILES |
CC1=C2C=CC=C(C2=C(C3=C1C[C@H]4[C@@H](C(=O)C(=C([C@]4(C3=O)O)O)C(=O)N)N(C)C)O)O.Cl
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| InChi Key |
SPFAOPCHYIJPHJ-WPJNXPDPSA-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 |
(4S,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 |
Anhydrotetracycline hydrochloride; 13803-65-1; 2-Naphthacenecarboxamide, 4-(dimethylamino)-1,4,4a,5,12,12a-hexahydro-3,10,11,12a-tetrahydroxy-6-methyl-1,12-dioxo-, hydrochloride (1:1), (4S,4aS,12aS)-; OVG14H105R; (4S,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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| 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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
H2O : ~170 mg/mL (~367.27 mM)
DMSO : ~100 mg/mL (~216.04 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.49 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1604 mL | 10.8019 mL | 21.6039 mL | |
| 5 mM | 0.4321 mL | 2.1604 mL | 4.3208 mL | |
| 10 mM | 0.2160 mL | 1.0802 mL | 2.1604 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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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