Anti-infective macrozones: design, biological evaluation and structure-activity relationships: Original scientific article

Tomislav Jednačak; Višnja Stepanić; Iva Habinovec; Ivana Mikulandra; Kristina Smokrović; Hana Čipčić Paljetak; Mirjana Bukvić; Jelena Parlov Vuković; Ivan Grgičević; Leda Divjak; Klaus Zangger; Predrag Novak

doi:10.5599/admet.3139

Authors

Tomislav Jednačak University of Zagreb, Faculty of Science, Department of Chemistry https://orcid.org/0000-0003-1620-094X
Višnja Stepanić Division of Electronics, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia https://orcid.org/0000-0001-9518-4153
Iva Habinovec Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia https://orcid.org/0009-0009-9925-9293
Ivana Mikulandra Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia https://orcid.org/0009-0006-4237-9903
Kristina Smokrović Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia https://orcid.org/0000-0002-9077-5293
Hana Čipčić Paljetak Center for Translational and Clinical Research, School of Medicine, University of Zagreb, Šalata 3, HR-10000 Zagreb, Croatia https://orcid.org/0000-0002-8837-3156
Mirjana Bukvić Selvita, Prilaz baruna Filipovića 29, HR-10000 Zagreb, Croatia https://orcid.org/0000-0002-1035-7981
Jelena Parlov Vuković NMR Centre, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia https://orcid.org/0000-0002-8464-5599
Ivan Grgičević Labtim Adria d.o.o., Jaruščica 7A, HR-10020 Zagreb, Croatia https://orcid.org/0009-0009-5708-6363
Leda Divjak Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia https://orcid.org/0009-0006-3797-3799
Klaus Zangger Organic and Bioorganic Chemistry, Institute of Chemistry, University of Graz, Heinrichstraße 28 A-8010 Graz, Austria https://orcid.org/0000-0003-1682-1594
Predrag Novak Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia https://orcid.org/0000-0002-9303-8213

DOI:

https://doi.org/10.5599/admet.3139

Keywords:

Azithromycin conjugates, thiosemicarbazones, synthesis, biological activity, resistance

Abstract

Background and purpose: To discover novel compounds active against sensitive and resistant bacterial strains, a series of novel azithromycin-thiosemicarbazone conjugates, the macrozones, have been synthesized and their biological activity evaluated with corresponding (quantitative) structure-activity relationship ((Q)SAR) analyses conducted. Experimental approach: A systematic variation of thiosemicarbazone side-chains and coupling at positions 4''-, 3-, and 9a of the azithromycin scaffold has resulted in a novel class of bacterial ribosome inhibitors. Key results: Compared to azithromycin, the activity of 4''-macrozones has shown the greatest improvements against efflux-resistant S. pneumoniae and S. aureus, as well as very good activity of 4'' derivatives against E. faecalis. QSAR calculations indicate that the antibacterial activity of macrozones is primarily determined by the position of the thiosemicarbazone side chain. Among the conjugated derivatives, the 4''-substituted macrozones exhibit the highest overall activity against a range of sensitive and efflux-resistant Gram-positive bacteria, as well as against Gram-negative E. coli strains, while those substituted at 9a- and 3- positions are found to be less potent. The antibacterial activity of macrozones is favourably influenced by larger fractions of their cationic and zwitterionic forms, their capacity for hydrogen bond formation, and the extension of p-electron delocalization involving the thiosemicarbazone moiety. Conclusion: The results obtained provide a sound basis for guiding further medicinal chemistry efforts toward the discovery of more potent macrolide anti-infectives, with particular emphasis on resistant bacteria that pose a serious threat to human health.

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References

[1] N.A.N. Amdan, N.A. Shahrulzamri, R. Hashim, N.M. Jamil. Understanding the evolution of macrolides resistance: A mini review. Journal of Global Antimicrobial Resistance 38 (2024) 368-375. https://doi.org/10.1016/j.jgar.2024.07.016 DOI: https://doi.org/10.1016/j.jgar.2024.07.016

[2] G.P. Dinos. The macrolide antibiotic renaissance. British Journal of Pharmacology 174 (2017) 2967-2983. https://doi.org/10.1111/bph.13936 DOI: https://doi.org/10.1111/bph.13936

[3] D.N. Wilson. Ribosome-targeting antibiotics and mechanism of bacterial resistance. Nature Reviews Microbiology 12 (2014) 35-48. https://doi.org/10.1038/nrmicro3155 DOI: https://doi.org/10.1038/nrmicro3155

[4] S. Ðokić, G. Kobrehel, N. Lopotar, B. Kamenar, A. Nagl, D. Mrvoš. Erythromycin series. Part 13. Synthesis and structure elucidation of 10-dihydro-10-deoxo-11-methyl-11-azaerythromycin A. Journal of Chemical Research (S) 12 (1988) 152-153. https://doi.org/10.1002/chin.198842362 DOI: https://doi.org/10.1002/chin.198842362

[5] C.-X. Ma, Y. Li, W.-T. Liu, Y. Li, F. Zhao, X.-T. Lian, J. Ding, S.-M. Liu, X.-P. Liu, B.-Z. Fan, L.-Y. Liu, F. Xue, J. Li, J.-R. Zhang, Z. Xue, X.-T. Pei, J.-Z. Lin, J.-H. Liang. Synthetic macrolides overcoming MLSBK-resistant pathogens. Cell Discovery 10 (2024) 75. https://doi.org/10.1038/s41421-024-00702-y DOI: https://doi.org/10.1038/s41421-024-00702-y

[6] A. Janas, K. Pyta, M. Gdaniec, P. Przybylski. An approach to modify 14-membered lactone macrolide antibiotic scaffolds. The Journal of Organic Chemistry 87 (2022) 3758-3761. https://doi.org/10.1021/acs.joc.1c02799 DOI: https://doi.org/10.1021/acs.joc.1c02799

[7] A.G. Myers, R.B. Clark. Discovery of macrolide antibiotics effective against multi-drug resistant Gram-negative pathogens. Accounts of Chemical Research 54 (2021) 1635-1645. https://doi.org/10.1021/acs.accounts.1c00020 DOI: https://doi.org/10.1021/acs.accounts.1c00020

[8] B.-Z. Fan, H. Hiasa, W. Lv, S. Brody, Z.-Y. Yang, C. Aldrich, M. Cushman, J.-H. Liang. Design, synthesis and structure-activity relationships of novel 15-membered macrolides: Quinolone/quinoline-containing sidechains thetered to the C-6 position of azithromycin acylides. European Journal of Medicinal Chemistry 193 (2020) 112222. https://doi.org/10.1016/j.ejmech.2020.112222 DOI: https://doi.org/10.1016/j.ejmech.2020.112222

[9] M. Yan, L. Xu, Y. Wang, J. Wan, Chenchen, Q. Li, R. Wang. Synthesis and antibacterial activity of 11,12-cyclic carbonate 4″-O-aralkylacetylhydrazineacyl azithromycin derivatives. Bioorganic Chemistry 94 (2020) 103475. https://doi.org/10.1016/j.bioorg.2019.103475 DOI: https://doi.org/10.1016/j.bioorg.2019.103475

[10] A. Janas, P. Przybylski. 14- and 15-membered lactone macrolides and their analogues and hybrids: Structure, molecular mechanism of action and biological activity. European Journal of Medicinal Chemistry 182 (2019) 111662. DOI: https://doi.org/10.1016/j.ejmech.2019.111662 DOI: https://doi.org/10.1016/j.ejmech.2019.111662

[11] I.B. Seiple, Z. Zhang, P. Jakubec, A. Langlois-Mercier, P.M. Wright, D.T. Hog, K. Yabu, S. Rao Allu, T. Fukuzaki, P.N. Carlsen, Y. Kitamura, X. Zhou, M.L. Condakes, F.T. Szczypiński, W.D. Green, A.G.A. Myers. Platform for the discovery of new macrolide antibiotics. Nature 533 (2016) 338-345. https://doi.org/10.1038/nature17967 DOI: https://doi.org/10.1038/nature17967

[12] Y. Wang, C. Cong, W.C. Chai, R. Dong, L. Jia, D. Song, Z. Zhou, S. Ma. Synthesis and antibacterial activity of novel 4″-O-(1-aralkyl-1,2,3-triazol-4-methyl-carbamoyl) azithromycin analogs. Bioorganic & Medicinal Chemistry Letters 27 (2017) 3872-3877. https://doi.org/10.1016/j.bmcl.2017.06.044 DOI: https://doi.org/10.1016/j.bmcl.2017.06.044

[13] D. Pavlović, S. Mutak. Synthesis and antibacterial evaluation of novel 4''-glycyl linked quinolyl-azithromycins with potent activity against macrolide-resistant pathogens. Bioorganic & Medicinal Chemistry 24 (2016) 1255-1267. https://doi.org/10.1016/j.bmc.2016.01.055 DOI: https://doi.org/10.1016/j.bmc.2016.01.055

[14] A. Janas, P. Pecyna, M. Gajecka, F. Bartl, P. Przybylski. Synthesis and antibacterial activity of new N-alkylammonium and carbonate-triazole derivatives within desosamine of 14- and 15-membered lactone macrolides. ChemMedChem 15 (2020) 1529-1551. https://doi.org/10.1002/cmdc.202000273 DOI: https://doi.org/10.1002/cmdc.202000273

[15] X.-M. Li, W. Lv, S.-Y. Guo, Y.-X. Li, B.-Z. Fan, M. Cushman, F.-S. Kong, J. Zhang, J.-H. Liang. Synthesis and structure-bactericidal activity relationships of non-ketolides: 9-oxime clarithromycin 11,12-cyclic carbonate featured with three-to eight-atom-length spacers at 3-OH. European Journal of Medicinal Chemistry 171 (2019) 235-254. https://doi.org/10.1016/j.ejmech.2019.03.037 DOI: https://doi.org/10.1016/j.ejmech.2019.03.037

[16] S. Alihodžić, M. Bukvić, I.J. Elenkov, A. Hutinec, S. Koštrun, D. Pešić, G. Saxty, L. Tomašković, D. Žiher. Chapter three - Current trends in macrocyclic drug discovery and beyond-Ro5. Progress in Medicinal Chemistry 57 (2018) 113-233. https://doi.org/10.1016/bs.pmch.2018.01.002 DOI: https://doi.org/10.1016/bs.pmch.2018.01.002

[17] B. Arsic, A. Awan, R.J. Brennan, J.A. Aguilar, R. Ledder, A.J. McBain, A.C. Regan, J. Barber. Theoretical and experimental investigation on clarithromycin, erythromycin A and azithromycin and descladinosyl derivatives of clarithromycin and azithromycin with 3-O substitution as anti-bacterial agents. Medicinal Chemistry Communications 5 (2014) 1347-1354. https://doi.org/10.1039/C4MD00220B DOI: https://doi.org/10.1039/C4MD00220B

[18] F. Schlünzen, R. Zarivach, J. Harms, A. Bashan, A. Tocilj, R. Albrecht, A. Yonath, F. Franceschi. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature 413 (2001) 814-821. https://doi.org/10.1038/35101544 DOI: https://doi.org/10.1038/35101544

[19] J.L. Hansen, J.A. Ippolito, N. Ban, P. Nissen, P.B. Moore, T.A. Steitz. The structures of four macrolide antibiotics bound to the large ribosomal subunit. Molecular Cell 10 (2002) 117-128. https://doi.org/10.1016/S1097-2765(02)00570-1 DOI: https://doi.org/10.1016/S1097-2765(02)00570-1

[20] J.A. Dunkle, L. Xiong, A.S. Mankin, J.H.D. Cate. Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proceedings of the National Academy of Sciences 107 (2010) 17152-17157. https://doi.org/10.1073/pnas.1007988107 DOI: https://doi.org/10.1073/pnas.1007988107

[21] E.V. Aleksandrova, C.-X. Ma, D. Klepacki, F. Alizadeh, N. Vázquez-Laslop, J.-H. Liang, Y.S. Polikanov, A.S. Mankin. Macrolones target bacterial ribosomes and DNA gyrase and can evade resistance mechanisms. Nature Chemical Biology 20 (2024) 1680-1690. https://doi.org/10.1038/s41589-024-01685-3 DOI: https://doi.org/10.1038/s41589-024-01685-3

[22] P. Novak, J. Barber, A. Čikoš, B. Arsić, J. Plavec, G. Lazarevski, P. Tepeš, N. Košutić-Hulita. Free and bound state structures of 6-O-methyl homoerithromycins and epitope mapping of their interactions with ribosomes. Bioorganic & Medicinal Chemistry 17 (2009) 5857-5867. https://doi.org/10.1016/j.bmc.2009.07.013 DOI: https://doi.org/10.1016/j.bmc.2009.07.013

[23] S. Glanzer, S.A. Pulido, S. Tutz, G.E. Wagner, M. Kriechbaum, N. Gubensäk, J. Trifunović, M. Dorn, W.M.F. Fabian, P. Novak, J. Reidl, K. Zangger. Structural and functional implications of the interaction between macrolide antibiotics and bile acids. Chemistry - A European Journal 21 (2015) 4350-4358. https://doi.org/10.1002/chem.201406413 DOI: https://doi.org/10.1002/chem.201406413

[24] S. Kosol, E. Schrank, M. Bukvić Krajačić, G.E. Wagner, N.H. Meyer, C. Göbl, G.N. Rechberger, K. Zangger, P. Novak. Probing the interactions of macrolide antibiotics with membrane-mimetics by NMR spectroscopy. Journal of Medicinal Chemistry 55 (2012) 5632-5636. https://doi.org/10.1021/jm300647f DOI: https://doi.org/10.1021/jm300647f

[25] I. Mikulandra, T. Jednačak, B. Bertoša, J. Parlov Vuković, I. Kušec, P. Novak. Interactions of aminopropyl-azithromycin derivatives, precursors in the synthesis of bioactive macrozones, with E. coli ribosome: NMR and molecular docking studies. Materials 14 (2021) 5561. https://doi.org/10.3390/ma14195561 DOI: https://doi.org/10.3390/ma14195561

[26] T. Jednačak, I. Mikulandra, K. Smokrović, A. Hloušek-Kasun, M. Kapustić, K. Delaš, I. Piantanida, M. Jurković, B. Bertoša, K. Zangger, P. Novak. Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies. Bioorganic Chemistry 147 (2024) 107338. https://doi.org/10.1016/j.bioorg.2024.107338 DOI: https://doi.org/10.1016/j.bioorg.2024.107338

[27] P. Rattanasuwan, P. Lertpongpipat, N. Hiranchatchawal, K. Wannaphueak, S. Pounghom, P. Thongkhao-on, M. Suwanthai, D. Sompradee, A. Saithongdee, C. Jaikang, P. Tajai. Metabolic insights into the warfarin-mango interaction: A pilot study integrating clinical observations and metabolomics. ADMET and DMPK 13(3) (2025) 2740. https://doi.org/10.5599/admet.2740 DOI: https://doi.org/10.5599/admet.2740

[28] N. Chabang, C. Wongwitayasombat, P. Tuchinda, B. Munyoo, N. Kangwanrangsan, S. Hongeng, B. Nutho, S. Charoensutthivarakul, P. Kanjanasirirat. New xanthone and chemical constituents from the aerial parts of Mallotus glomerulatus and their cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. ADMET and DMPK 13(5) (2025) 2901. https://doi.org/10.5599/admet.2901 DOI: https://doi.org/10.5599/admet.2901

[29] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, 15th Informational Supplement, CLSI document M100-S15, Wayne, Pennsylvania, 2005. https://clsi.org/shop/standards/m100/, access date: December 23, 2025

[30] D. Rogers, M. Hahn. Extended-connectivity fingerprints. Journal of Chemical Information and Modeling 50 (2010) 742-754. https://doi.org/10.1021/ci100050t DOI: https://doi.org/10.1021/ci100050t

[31] A. De la Peña, A. Krause, A. Rokitka, B. Stefanovic, B. Nelson, C. Hendrickson, D. Hoeft, D. Szot, D. Weiner, D. Miller, G. Fraczkiewicz, J. Rouse, J. Fiedler-Kelly, J. Mcnicholl, J.A. Dibella, J. Bartels, J.K. Paglia, J. Chauvin, J. Fohey, J. Cook, K. Morgan-Kehr, L. Luke, L. Lavange, L. Shoda, M. Alper, N. Musser, N. Miller, P. Kilford, R. Suderman, S. Suarez Sharp, S.Q. Siler, S. Evans, S. O’Connor, S. Chang, V. Lukacova, W.S. Woltosz, W. Frederick, X. Pepin, X. Zhang, Y.-N. Sun, SLP University+ Program, Simulations Plus Inc., 2021. https://www.simulations-plus.com/software/slp-university-program.

[32] J.J. Allaire, RStudio, Rev. 36, Posit PBC. https://posit.co/download/rstudio-desktop/, access date: December 23, 2025

[33] T. Sander, J. Freyss, M. von Korff, C. Rufener. DataWarrior: An open-source program for chemistry aware data visualization and analysis. Journal of Chemical Information and Modeling 55 (2015) 460-473. https://doi.org/10.1021/ci500588j DOI: https://doi.org/10.1021/ci500588j

[34] J.R. Quinlan, RuleQuest See5, Release 2.11a, RuleQuest Research, 2022. https://www.rulequest.com/see5-info.html, access date: December 21, 2025

[35] J.R. Quinlan, C4.5: Programs for Machine Learning, Morgan Kaufmann Publishers, San Mateo, California, 1993, ISBN: 1-55860-238-0. https://archive.org/details/c45programsforma0000quin/page/314/mode/2up, access date: December 21, 2025

[36] M. Bukvić Krajačić, M. Dumić, P. Novak, M. Cindrić, S. Koštrun, A. Fajdetić, S. Alihodžić, K. Brajša, N. Kujundžić. Discovery of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides active against efflux-mediated resistant Streptococcus pneumoniae. Bioorganic & Medicinal Chemistry Letters 21 (2011) 853-856. https://doi.org/10.1016/j.bmcl.2010.11.079 DOI: https://doi.org/10.1016/j.bmcl.2010.11.079

[37] M. Bukvić Krajačić, P. Novak, M. Cindrić, K. Brajša, M. Dumić, N. Kujundžić. Azithromycin-sulfonamide conjugates as inhibitors of resistant Streptococcus pyogenes strains. European Journal of Medicinal Chemistry 42 (2007) 138-145. https://doi.org/10.1016/j.ejmech.2006.08.008 DOI: https://doi.org/10.1016/j.ejmech.2006.08.008

[38] P. Fernandes, D. Pereira, P.B. Watkins, D. Bertrand. Differentiating the pharmacodynamics and toxicology of macrolide and ketolide antibiotics. Journal of Medicinal Chemistry 63 (2020) 6462-6473. https://doi.org/10.1021/acs.jmedchem.9b01159 DOI: https://doi.org/10.1021/acs.jmedchem.9b01159

[39] A. Nejabatdoust, H. Zamani, A. Salehzadeh, Functionalization of ZnO nanoparticles by glutamic acid and conjugation with thiosemicarbazide alters expression of efflux pump genes in multiple drug-resistant Staphylococcus aureus strains. Microbial Drug Resistance 25 (2019) 966-974. https://doi.org/10.1089/mdr.2018.0304 DOI: https://doi.org/10.1089/mdr.2018.0304

[40] I. Grgičević, I. Mikulandra, M. Bukvić, M. Banjanac, I. Habinovec, B. Bertoša, P. Novak. Discovery of macrozones, new antimicrobial thiosemicarbazone-based azithromycin conjugates: Design, synthesis and in vitro biological evaluation. International Journal of Antimicrobial Agents 56 (2020) 106147. https://doi.org/10.1016/j.ijantimicag.2020.106147 DOI: https://doi.org/10.1016/j.ijantimicag.2020.106147

[41] E.-W. Lee, M.N. Huda, T. Kuroda, T. Mizushima, T. Tsuchiya. EfrAB, an ABC multidrug efflux pump in Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 47 (2003) 3733-3738. https://doi.org/10.1128/aac.47.12.3733-3738.2003 DOI: https://doi.org/10.1128/AAC.47.12.3733-3738.2003