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Patent / 1967

Tetracycline-N,N'-dibenzylideneethylenediamine complexes for use in isolating tetracyclines

  • Ivan Villax
US 3280188
Tetracycline, oxytetracycline, chlortetracycline, and dimethylchlortetracycline are prepd. and isolated by adding PhCH:NCH2N:CHPh (I) to the culture medium during fermentation by a streptomyces tetracycline-producing microorganism, and then acidifying at a pH of 0.5-1.8 with an acid. The broth is filtered and the pH adjusted to 5 with NH3 in the event it contains enough alk. earth metal ions to form the I complex, or if not the pH is adjusted with an alk. earth metal hydroxide. I is added in the amt. of 0.5 mole per mole of the tetra-, oxy- or chlorotetracycline present and at least 1.5 moles per mole of demethylchlortetracycline. The addn. of the base is made until a pH of 8.0 to 9.5. The I complex is then pptd. The complexes can be used as such for pharmaceutical purposes or as feed supplements for animals, or by adjusting the pH of the suspension to the isoelec. point of the tetracycline with an acid, the pure base is crystd. The following example illustrates the process: 300 l. of a sterile culture medium of the compn.; corn steep liquor (50%) 28, CaCO3 12, starch 38, (NH4Cl) 1.5, MnSO4.4H2O 0.05, ZnCl2 0.02, CoCl2.6H2O 0.005, peanut meal 25, and lard oil 25 g./l. of tap water (pH 6.7 after sterilization) are inoculated with a spore suspension of Streptomyces aureofaciens. To the medium are added 100 mg./l. at the beginning and 200 mg./l. of I after 56 and 112 hrs. of fermentation. After 130 hrs. of fermentation, 2.8g./l. of demethylchlortetracycline are obtained. To the acidified filtrate 170 g. of I are added, NH4OH to pH 5.0, and 170 g. of I 1.5g./l. EDTA and 0.3g./l. of Na2SO3 (antioxidant). Upon raising the pH to 8.7, the complex precipitates and is further washed and purified. The final product is composed of: 52.8% of demethylchlortetracycline as anhydrous base (the remainder I, Ca, and water), and corresponds to the formula: demethylchlortetracycline2I Ca3 in the anhydrous state. The yield is 79%; sp. rotation, [.alpha.]D = -180 (c = 1 in 0.1 N HCl); uv spectrum max. 232 (E 1% 1 cm. = 503), 366/368 (E 1% 1 cm. = 171).