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Pojava sve veće otpornosti bakterija na postojeće antibiotike predstavlja ozbiljan zdravstveni problem pa se stoga u području farmaceutske i medicinske kemije ulažu veliki napori u razvoj novih antibiotika. Makrozoni predstavljaju nove konjugate azitromicina i derivata tiosemikarbazida koji pokazuju izvrsnu antibakterijsku aktivnost in vitro na susceptibilne i rezistentne bakterijske sojeve. Proces razvoja novih antibiotika sastoji se od mnogo koraka, pri čemu je izuzetno važna identifikacija farmaceutskih onečišćenja i novosintetiziranih spojeva – potencijalnih antibiotika. U tu svrhu vrlo često se koriste vezani analitički sustavi pomoću kojih se postiže učinkovitije i brže pročišćavanje te identifikacija spojeva u odnosu na klasične tehnike off-line. U ovom diplomskom radu provedena je izolacija i strukturna karakterizacija novosintetiziranih 3-trifluormetoksifenil- i 3-(3-fenilpropil)-makrozona te njihovih dijastereomera, kao i nekih onečišćenja iz reakcijskih smjesa pomoću sustava LC-SPE/krioNMR. Za strukturnu karakterizaciju izoliranih komponenata korištene su jedno- i dvodimenzijske tehnike NMR te spektrometrija masa. Na temelju analize prikupljenih podataka predložene su strukture izoliranih spojeva. Increasing bacterial resistance to existing antibiotics is a serious health problem, and great efforts are being made to discover and develop new antibiotics to overcome this problem. Macrozones are novel conjugates of azithromycin and thiosemicarbazides that show very good in vitro activity against susceptible and resistant bacterial strains. Development of new antibiotics is a multistep process and one of the most important steps is the identification of pharmaceutical impurities and newly synthesized compounds – potential antibiotics. For this purpose, hyphenated systems are very oftenly used to achieve more efficient and faster purification and identification of compounds compared to off-line techniques. In this study, isolation and structural characterization of newly synthesized 3-trifluoromethoxyphenyl- and 3-(3-phenylpropyl)-macrozones and their diastereoisomers, as well as some impurities from reaction mixtures, have been performed using hyphenated system LC-SPE/cryo-NMR. Oneand two-dimensional NMR techniques and mass spectrometry were used for structural characterization of isolated components. Based on the analysis of collected data, the structures of isolated compounds were proposed.

Makrozoni su novi konjugati azitromicina i derivata tiosemikarbazida koji pokazuju zadovoljavajuće antibakterijsko djelovanje in vitro na odabrane Gram-pozitivne i Gram-negativne bakterije. Zbog sve učestalije pojave bakterija rezistentnih na makrolide, nastavljaju se istraživanja s ciljem razvoja novih oblika makrolidnih antibiotika koji će biti djelotvorni na otporne bakterijske sojeve. Izolacija i određivanje strukture komponenata reakcijske smjese 9a-(2-feniletil)makrozona u ovome radu postignuta je primjenom vezanog sustava LC-SPE/NMR. Istraženi su uvjeti za uspješnu izolaciju i zadržavanje analita na SPE-nosačima. Makrozon je pokazao dobro zadržavanje na nepolarnoj nepokretnoj fazi SPE-nosača baziranoj na silika-materijalu, dok se veći broj polarnih onečišćenja zadržao bolje na divinilbenzenskim polimernim SPE-nosačima. Uz to, većina polarnih onečišćenja uspješno je ekstrahirana koristeći pritom veći dodatni protok vodene pokretne faze. Na temelju snimljenih jedno- i dvodimenzijskih spektara NMR, te analizom spektara MS, predložene su strukture izoliranih komponenata. Macrozones are new conjugates of azithromycin analogues, 9a-(γ-aminopropyl) derivatives, and thiosemicarbazides which show good in vitro activity against selected Gram-positive and Gram-negative bacterial strains. Due to the increasing number of macrolide-resistant bacteria, a search for new antibiotics continues, aimed at discovery of more effective antimicrobial agents. In this work, isolation and determination of the 9a-(2-phenylethyl)macrozone reaction mixture components was achieved using the LC-SPE/NMR system. Several parameters for successful separation and extraction of analytes were optimised. It was found that silica-bonded phase gave satisfactory results for the main compound, macrozone, however, for more polar compounds divinylbenzene polymer phase was better. Additionally, extraction efficiency for more polar impurities was obtained by using higher make-up flow. Structures of the isolated components were proposed on the basis of the recorded one- and two-dimensional NMR spectra and the MS spectra, respectively.