Unprecedented Epimerization of an Azithromycin Analogue: Synthesis, Structure and Biological Activity of 2′-Dehydroxy-5″-Epi-Azithromycin

Abstract

Certain macrolide antibiotics, azithromycin included, possess anti-inflammatory properties that are considered fundamental for their efficacy in the treatment of chronic inflammatory diseases, such as diffuse pan-bronchiolitis and cystic fibrosis. In this study, we disclose a novel azithromycin analog obtained via Barton–McCombie oxidation during which an unprecedented epimerization on the cladinose sugar occurs. Its structure was thoroughly investigated using NMR spectroscopy and compared to the natural epimer, revealing how the change in configuration of one single stereocenter (out of 16) profoundly diminished the antimicrobial activity through spatial manipulation of ribosome binding epitopes. At the same time, the anti-inflammatory properties of parent macrolide were retained, as demonstrated by inhibition of LPS- and cigarette-smoke-induced pulmonary inflammation. Not surprisingly, the compound has promising developable properties including good oral bioavailability and a half-life that supports once-daily dosing. This novel anti-inflammatory candidate has significant potential to fill the gap in existing anti-inflammatory agents and broaden treatment possibilities.

An investigation of the predominant structure of antibiotic azithromycin in chloroform solution through NMR and thermodynamic analysis

“…This fact is justified by the difficulty in assigning the C6-OH proton signal presented in previous work of Barber 28 (in the undifferentiated values of 9.2 ppm and 2.6 ppm) and Shoeb et al from 2021 29 with a chemical shift of 3.05 ppm. Thus, taking Kragol et al 52 as a reference, the value of 7.16 ppm is consistent with the experimental chemical shift presented in this work (7.29 ppm). The C2 0 -OH and C4 00 -OH protons are highlighted in the pink rectangle and C11-OH and C12-OH in the black rectangle in Fig.…”

“…Depending on the type of epimerization, the chemical shift value of the proton C6–OH is shown to be equivalent to 8.98 ppm for one type of ring formation and 7.16 ppm for the other type of ring conformation. 52 This shows the dependence of the type of formation from erythromycin affecting the C6–OH values as it changes the chemical environment and the geometry of adjacent atoms. This fact is justified by the difficulty in assigning the C6–OH proton signal presented in previous work of Barber 28 (in the undifferentiated values of 9.2 ppm and 2.6 ppm) and Shoeb et al from 2021 29 with a chemical shift of 3.05 ppm.…”

“…In another experimental paper on 1 H NMR chemical shifts (in CDCl 3 ) for AZM published by Lazarevski et al 25 no assignment was made for the C6-OH and C2 0 -OH protons. In a very recent study by Kragol et al using NMR spectroscopy 52 on a novel azithromycin analog, where an unprecedented epimerization on the cladinose sugar occurred, it was shown that the conformation of cladinose affects the mutual arrangement of the sugars, as well as their orientation with respect to the lactone ring. Depending on the type of epimerization, the chemical shift value of the proton C6-OH is shown to be equivalent to 8.98 ppm for one type of ring formation and 7.16 ppm for the other type of ring conformation.…”

Modification of Azithromycin to Mitigate its Arrest of Autophagy

“…Such a derivative would provide an invaluable non-steroidal anti-inflammatory that concentrates at the airways, without contributing to the selection of AZM-resistance bacteria (43). This has been examined by Kragol et al (2022), who synthesised a stereoisomer of AZM with immunomodulatory activity, although there was no further assessment to determine whether the suppressive effects on autophagy were altered (44). Indeed, given the wide use of AZM and its remarkable capacity to localise within the mammalian cell, surprisingly little research has been conducted to identify further off-target effects.…”

“…However, no studies have attempted to identify specific lysosomal ion channel or transporter binding domains that could inform SAR studies to address AZM-mediated suppression of autophagosome-lysosome fusion. The most promising nonantibiotic AZM derivative reported thus far (to our knowledge) was by Kragol et al (2022), who synthesized a stereoisomer of AZM that retained significant immunomodulatory activity, albeit the suppressive effects on autophagy were not examined (57). Nevertheless, the combination this outcome with ours for AZM-[O] provide promising evidence that the structural determinants of AZM’s antibacterial activity, and the off-target effects for autophagy and innate immunity, may be mutually exclusive.…”