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Isochlortetracycline is classified as a microbiologically inactive metabolite of chlortetracycline. Unlike its parent compound chlortetracycline, which binds to the 30S ribosomal subunit to inhibit bacterial protein synthesis, isochlortetracycline exhibits minimal to no antibacterial activity. Research indicates that the conversion of chlortetracycline to isochlortetracycline involves the formation of an anhydro intermediate, resulting in a structural rearrangement that eliminates its ability to bind effectively to the bacterial ribosome. Consequently, isochlortetracycline does not have a defined therapeutic target in bacteria. Its primary relevance lies in its role as a degradation product and metabolite, making it an important marker compound for studying the stability, metabolism, and environmental fate of chlortetracycline.
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| ln Vitro |
In vitro studies have demonstrated that isochlortetracycline is devoid of significant antimicrobial activity. Early metabolic studies in rats and dogs using C14-labeled chlortetracycline found that while 33-70% of the administered microbiological activity was recovered in excreta, the compound isochlortetracycline, when present, did not contribute to this activity. This lack of antibacterial effect is attributed to the structural rearrangement that occurs during its formation from chlortetracycline, which disrupts the ability to bind to the bacterial 30S ribosomal subunit. In analytical chemistry, isochlortetracycline serves as a useful reference standard for high-performance liquid chromatography (HPLC) methods, as it can be generated post-column by alkaline treatment of chlortetracycline and detected via its fluorescence properties. When analyzed by HPLC, isochlortetracyc
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| ln Vivo |
In vivo studies confirm that isochlortetracycline is a major metabolite of chlortetracycline in animals. Research in laying hens fed with chlortetracycline demonstrated that isochlortetracycline and its 4-epimer are the principal metabolites excreted into eggs, appearing in substantial amounts in both egg white and yolk. The ratio of 4-epimer to parent compound was found to be lower in egg white and higher in yolk. In rat studies, following intravenous administration of C14-labeled chlortetracycline, small amounts of a blue-fluorescing material corresponding to isochlortetracycline were observed in the excreta. The formation of isochlortetracycline occurs via an acid- or base-catalyzed degradation pathway involving an anhydro intermediate. As an inactive metabolite, isochlortetracycline does not contribute to the therapeutic efficacy of chlortetracycline administration but serves as a marker for drug degradation and metabolic transformation.
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| Enzyme Assay |
Methodology for HPLC-FLD Analysis and Post-Column Derivatization: Isochlortetracycline is commonly generated and analyzed using post-column alkaline derivatization from chlortetracycline in a cell-free system. The chromatographic separation is achieved on a polystyrene-divinylbenzene copolymer column (PRP-1 column, 15 cm × 4.6 mm) with a mobile phase consisting of acetonitrile:0.2% perchloric acid (27:73, v/v) at a flow rate of 1.0 mL/min. Following separation, isochlortetracycline is formed on-line by the addition of 25% NaOH (w/v) at a flow rate of 0.2 mL/min into the column eluent via a post-column reaction coil. The reaction coil consists of 9 meters of Teflon tubing (1/16 inch o.d., 0.3 mm i.d.) knitted into a six-sided coil. The fluorescent derivative is detected at an excitation wavelength of 355 nm and emission wavelength greater than 389 nm. This method is linear for detection between 0.02 μg/mL and 4 μg/mL and can detect chlortetracycline (via conversion to isochlortetracycline) in milk at concentrations as low as 0.04 μg/mL.
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| Cell Assay |
Methodology for Microbiological Activity Testing (Inactive Metabolite Confirmation): To confirm the lack of antibacterial activity of isochlortetracycline, standard microbiological agar diffusion assays can be employed. Bacterial test strains such as Bacillus cereus or Staphylococcus aureus are cultured overnight and then inoculated onto Mueller-Hinton agar plates. Paper disks containing isochlortetracycline (at varying concentrations, e.g., 1-100 μg/mL) are placed onto the inoculated agar surface, alongside positive control disks containing chlortetracycline. After incubation at 37°C for 18-24 hours, zones of inhibition are measured. While chlortetracycline produces clear inhibition zones, isochlortetracycline exhibits no measurable zone of inhibition, confirming its microbiological inactivity. Alternatively, broth microdilution assays can be performed in 96-well plates using bacterial suspensions at ~5 × 10⁵ CFU/mL with serial dilutions of isochlortetracycline (0.03-64 μg/mL), incubated at 37°C for 18-24 hours, followed by optical density measurement at 600 nm to assess bacterial growth.
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| Animal Protocol |
Methodology for Metabolic Study in Laying Hens: To study the formation of isochlortetracycline as a metabolite, laying hens are administered chlortetracycline (CTC) via feed or drinking water. Following a medication period, eggs are collected daily and separated into egg white and yolk. Sample preparation involves homogenization of egg white or yolk, followed by solid-phase extraction using cyclohexyl-bonded reversed-phase cartridges for cleanup and concentration. The extracted samples are then analyzed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) to quantify chlortetracycline, isochlortetracycline, and their 4-epimers. Research has confirmed that isochlortetracycline and its 4-epimer are the principal metabolites in eggs after CTC feeding, appearing in substantial amounts in both fractions. The ratio of 4-epimer to parent compound was found to be lower in egg white and higher in yolk, with no change during medication with TC or CTC.
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| ADME/Pharmacokinetics |
Isochlortetracycline is not administered as a therapeutic agent but is formed in vivo as a degradation and metabolic product of chlortetracycline (CTC). Chlortetracycline itself is approximately 30% bioavailable after oral administration, with 50-55% protein binding and extensive metabolism (about 75%) in the gastrointestinal tract and liver. The elimination half-life of chlortetracycline ranges from 5.6 to 9 hours, with 60% excreted renally and >10% via biliary excretion. In rat studies using C14-labeled chlortetracycline, 76-97% of the administered radioactivity was recovered in urine and feces within 48-72 hours, but only 33-70% of the administered microbiological activity was recovered, indicating the formation of inactive metabolites such as isochlortetracycline and 4-epichlortetracycline. Small amounts of blue-fluorescing material corresponding to isochlortetracycline were observed in the excreta of some animals. In laying hens, isochlortetracycline accumulates in eggs, appearing in both egg white and yolk.
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| Toxicity/Toxicokinetics |
The toxicological profile of isochlortetracycline has not been extensively characterized independently, as it is primarily studied as a metabolite of chlortetracycline. Available data indicate that isochlortetracycline is microbiologically inactive, meaning it lacks the antibacterial activity of its parent compound. This reduced biological activity generally correlates with a lower potential for antibiotic-related adverse effects such as gastrointestinal flora disruption or selection for antibiotic resistance. However, as a tetracycline degradation product, its presence in food products such as eggs raises considerations for food safety monitoring. Analytical methods have been developed specifically to detect isochlortetracycline residues in animal-derived foods, with detection limits as low as 20 ng/g in tissues. Chlortetracycline, the parent compound, is known to inhibit bone and tooth mineralization in growing and unborn animals, cause tooth discoloration (yellow or brown), and impair liver and kidney function with long-term use. Allergic reactions to tetracyclines are rare. The significance of isochlortetracycline from a toxicological perspective lies primarily in its role as a marker for chlortetracycline degradation and metabolism, rather than as a direct toxicant.
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| References |
[1]. Evaluation of the stability of chlortetracycline in granular premixes by monitoring its conversion into degradation products. J Pharm Biomed Anal. 2005 Sep 15;39(3-4):523-30.
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| Molecular Formula |
C22H23CLN2O8
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| Molecular Weight |
478.88
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| Exact Mass |
514.091
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| CAS # |
514-53-4
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| PubChem CID |
54678405
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.61g/cm3
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| Boiling Point |
743.1ºC at 760mmHg
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| Melting Point |
231-236ºC (dec.)
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| Flash Point |
403.2ºC
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| LogP |
2.07
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
33
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| Complexity |
966
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| Defined Atom Stereocenter Count |
5
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| SMILES |
CN(C)C1C2CC(CC(=O)C2(O)C(=O)C(C(N)=O)=C1O)C1(C)OC(=O)c2c1c(Cl)ccc2O |c:18|
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| InChi Key |
ZDCFZNSICAQKSV-AXVXPIMKSA-N
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| InChi Code |
InChI=1S/C22H23ClN2O8/c1-21(15-10(23)4-5-11(26)13(15)20(31)33-21)8-6-9-16(25(2)3)17(28)14(19(24)30)18(29)22(9,32)12(27)7-8/h4-5,8-9,16,26,29,32H,6-7H2,1-3H3,(H2,24,30)/t8-,9-,16-,21-,22-/m0/s1
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| Chemical Name |
(4S,4aS,6S,8aR)-6-[(1S)-7-chloro-4-hydroxy-1-methyl-3-oxo-2-benzofuran-1-yl]-4-(dimethylamino)-1,8a-dihydroxy-3,8-dioxo-4a,5,6,7-tetrahydro-4H-naphthalene-2-carboxamide
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| Synonyms |
Isochlortetracycline; 514-53-4; Isoaureomycin; 7-Chloroisotetracycline; CO2NHD53YP;
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0882 mL | 10.4410 mL | 20.8821 mL | |
| 5 mM | 0.4176 mL | 2.0882 mL | 4.1764 mL | |
| 10 mM | 0.2088 mL | 1.0441 mL | 2.0882 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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