Azithromycin is a macrolide antibiotic used for the treatment of numerous bacterial infections. Structural modifications of macrolide ring expand the biological activity of macrolides, which is very important for the field of medicinal chemistry. In order to reconstruct the existing macrocyclic skeleton, the synthesis, optimization of reaction conditions and spectroscopic characterization of new macrocyclic azithromycin diastereoisomers were performed. The final targets were synthesized in three reaction steps. Azithromycin seco-amide derivatives 3a and 3b were obtained by 9a-amidations from azithromycin seco-ester 1a and N-Fmoc-(R)-3-amino-3-(3-chlorophenyl)-propionic acid 2a or N-Fmoc-(S)-3-amino-3-(3-chlorophenyl) propionic acid 2b. The synthesis of derivatives 3a and 3b was tested under various reactions conditions. The chosen conditions included base DIPEA and reagent HATU in DMF. By hydrolysis of derivatives 3a and 3b, azithromycin seco-acid derivatives 4a and 4b were prepared using LiOH×H2O in water and THF. Macrocyclic azithromycin amide derivatives 5a and 5b were obtained by intramolecular 13-amidations in flow reactor with extensive optimization of the reaction conditions. Flow amidations were tested by using different reagents. Reagent HATU enabled fast and complete conversion, so this reagent was used in further reactions. Amidations in the flow reactor were performed at different concentrations of reactions, at different retention times, and at different temperatures. It was established that optimal conditions of intramolecular amidation included concentration of reaction of 12.5 mM, retention time of 5 min, and temperature of 75 ºC. Under these conditions, complete conversion was achieved with a minimal percentage of undesired dimeric by-product. Therefore, the final targets, macrocyclic azithromycin amide derivatives 5a and 5b, were synthesized under optimized conditions using DIPEA and HATU in DMF on a larger scale in the flow reactor. The structures of obtained diastereoisomers 5a and 5b were confirmed by 1D and 2D NMR spectroscopy, which also proved the conformational isomerism of derivatives 5a and 5b.