In recent years, the demand for reference standards of pharmaceutical impurities has been increasing, as they are essential for ensuring the quality and safety of medicines. Pimecrolimus, a macrolactam with immunomodulatory activity, is used in the treatment of inflammatory skin diseases. It is obtained through a semi-synthetic approach from ascomycin (FK520), during the fermentation of which, in addition to the main active substance, various by-products are formed. Since these are considered impurities in the final product, quality control requires determination of their content using appropriate reference standards. Traditional approaches to their preparation rely on purification of selected side fractions of the final active substance, which are time-consuming and, for some impurities, ineffective.Therefore, within the scope of this master’s thesis, a semisynthetic approach was developed starting from biosynthetic impurities of FK520.
From fermentation broths obtained using modified Streptomyces ascomycinicus H076 strains, which predominantly produce ethyl FK520 impurities, four biosynthetic impurities were isolated by liquid–liquid extraction; 19-ethyl-19-desmethyl FK520, 21-ethyl-21-desmethyl FK520, 27-ethyl-27-desmethyl FK520, and 17-desmethyl FK520. An average yield of 82% was achieved. Subsequent steps included removal of nonpolar impurities, purification by preparative liquid chromatography, and solid-phase extraction. An industrial selective chlorination process of ascomycin using N-chlorosuccinimide in the presence of triphenylphosphine was transferred to laboratory scale. Relative yields were evaluated, optimal reagent ratios were determined, and reaction rates were monitored at different FK520 concentrations. The highest relative yield of 75 % was obtained. Higher concentrations of the chlorinating agent accelerated the reaction and increased the formation of the dehydrated form of pimecrolimus. The optimized procedure was subsequently applied to the isolated impurities, achieving comparable conversions to the final products in the range of 63–78 %. After the reaction, the products were extracted, further purified, and crystallized. The identity and UV purity of the crystallized products were evaluated using liquid chromatography coupled with UV and MS. Four samples of target pimecrolimus impurities were prepared, with UV purity exceeding 80 %, in several cases, the combined content of the main form and its tautomers exceeded 90 %.
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