Tetracyclines epimerization

T. D. Sokoloski, L. A. Mitscher, J. V. Juvarkar, and B. Hoener, Rate and proposed mechanism of anhydro-tetracycline epimerization in acid solution,/. Pharm. Sci. 66, 1159-1165 (1977). 

The tetracycline molecule (1) presents a special challenge with regard to the study of stmcture—activity relationships. The difficulty has been to devise chemical pathways that preserve the BCD ring chromophore and its antibacterial properties. The labiUty of the 6-hydroxy group to acid and base degradation (12,13), plus the ease of epimerization (23) at position 4, contribute to chemical instabiUty under many reaction conditions. 

Under acidic conditions, dehydration to an anhydrotetracycline [20154-34-1] (8), C22H22N20y, occurs under basic ones, ring C opens to an isotetracycline [3811-31-2] (9), C22H24N20g. The anhydrotetracyclines, such as (8), appear to exhibit a mode of antibacterial action, but it is unlike that of tetracycline (24). Epimerization (23,25,26) at C-4 occurs in a variety of solvents within the pH range 2—6, particularly in acetic acid (25). A number of anions (27) facihtate this reaction. The reverse process, from 4-epitetracycline [79-85-6] C22H24N20g, to tetracycline, is promoted by chelation with ions such as calcium and magnesium (28). 

Treatment of 14 with hydrogen and a catalyst converts it to a mixture of epimeric 6-deoxy-5-oxytetracyclines (15 and 16), each of which is active as an antibiotic. The more active isomer has the natural tetracycline configuration of the methyl group at Ce and is in clinical use as a 6-deoxyoxytetracycline (15). ... 

All of these methods fail to show the presence of the epimeric form of the tetracyclines and in most instances streaking of the spots is a problem. A basic improvement in the paper chromatography of these antibiotics was achieved by Selzer and Wright (47) and Kelly and Bryske (48) when they reported methods for the pretreatment of the paper with com-plexing agents to bind the metallic ions which may be present. 

Most of the methods in which the paper is treated with a chelating agent are capable of showing a separation of some of the tetracycline drugs from each other and from their respective epimeric forms. They are also capable of revealing the presence of common degradation compounds of these drugs. 

Tetracyclines commonly comprise a plethora of related substances and degradation products, resulting from dehydration and epimerization reactions, and separating all of these constituents has proven to be quite a challenge. The necessity of adding ethylenediamine... 

The first three of these agents to be discovered, tetracycline (1)chlortetracycline (2), and oxytetracycline (3), are subject to two major modes of degradation under conditions occurring during their isolation, purification, formulation, and administration. These are dehydration and epimerization. Each of these reactions leads to inactivation of the antibiotic thus, considerable effort has been expended in attempts to prevent or minimize these reactions. 

They are approximately in agreement with values reported in literature, but the spectrum there was obtained with a HA-100 (23). The nmr shifts appear to be general regardless of solvent. Similar shifts are obtained in pyridine, trifluoroacetic acid and dimethyl sulfoxide. Particularly notable is the substantial downfield shift in the resoncance due to the C 4 proton when the tetracyclines epime-rize at C 4. This is especially convenient as both isomers are usually available and can be distinguished easily in this way. NMR was used to monitor the epimerization of TC since the dimethylamino resonance of TC and its C 4 epimer differ by 0.1 ppm (24). Formation of anhydrotetracycline could also be readily detected through NMR (23). 

Chemical instability reactions appear with or without microbiological contribution through reactions such as hydrolysis, oxidation, isomerization, and epi-merization. Interactions between ingredients and ingredients with container closure materials are established as the principal causes of these reactions [1], for instance, the hydrolysis of cefotaxime sodium, the oxidation of vitamin C, the isomerization of epinephrine, and the epimerization of tetracycline [7],... 

Dong, C. Xie, H. Shuang, S. Liu, C. Determination of tetracycline and 4-epimeric tetracycline by TLC-fluores-cence scanning densitometry. Anal Lett. 1999, 32, 1121-1130. 

Gyanchandani, N. D., McGilveray, I. J. and Hughes, D. W. Improved thin-layer chromatographic identification of tetracyclines and their degradation products application to an epimerization study. J. Pharm. Sci. 59 224-228, 1970. 

Another instability leading to a dramatic decrease of antibacterial action, to which all clinically used tetracyclines are subject, is epimerization of the natural 4-a-dimethy-lamino group A to the p-epimer B (Eq. 6.11). Under acidic conditions a 1 2 equilibrium is established in solution within a day. This occurs in a variety of solvents, especially acetic acid. Anions also tend to support this process. Divalent ions that chelate tetracyclines, particularly Ca2+, facilitate the reversal of the epimerization from the epi to the natural isomer. 

Tetracyclines (TCs) may degrade under extremes of pH with strong acids as well as alkali through epimerization. 

The nitroimidazoles, sulfonamides, and tetracyclines all present analytical challenges because of metabolism and/or chemical degradation. In the case of the nitroimidazoles, this is further complicated by the relatively low requirements for detection. Method development therefore has to take into account both metabolites as additional target compounds and low detection limits. Sulfonamide analysis has to take into account the potential for conversion of N -acetyl metabolites back to the parent compound. In contrast, in the analysis of honey, deconjugation is regarded as necessary to accurately determine sulfonamide concentrations. The facile, reversible formation of epimers is of particular concern in the analysis of those tetracyclines that can epimerize in the 4 position. Protein and metal binding are other issues that have to be overcome for successful tetracycline residue determination. 

Spisso B, de Aratljo M, Monteiro M, et al., A hquid chromatography-tandem mass spectrometry confirmatory assay for the simultaneous determination of several tetracyclines in milk considering keto-enol tau-tomerism and epimerization phenomena. Anal. Chim. Acta 2009 656 72-84. 

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