L-Ornithine derivatives with structural hetaryl and alkyl moiety: Synthesis and antibacterial activity

Objectives. Cationic amphiphiles and antimicrobial peptidomimetics are widely investigated as antibacterial agents due to their membrane-active mechanism of action. Particular attention is focused on the rational design of compounds in this class to achieve high antimicrobial activity. The aim of the present work is to synthesize bivalent cationic amphiphiles with L-ornithine as a branching element and evaluate the effectiveness of their antibacterial action. The compounds differ in terms of hydrophobicity due to the variation of N-terminal aliphatic amino acids in the polar block and alternation of dialkyl and alkyl-hetaryl radicals in the lipophilic block.
Methods. For the synthesis of nonpolar fragments of amphiphiles, methods for the alkylation of amines with alkyl bromides in the presence of carbonate salts were used. The formation of amide bonds of L-ornithine derivatives with amino acids was carried out using the carbodiimide method. For the reaction products recovery from the reaction mixture, column chromatography on silica gel and aluminum oxide activated Brockmann Grade II was used. The antimicrobial activity of the synthesized compounds against gram-positive B. subtilis 534 and gram-negative E. coli M17 bacterial strains was evaluated. Minimum inhibitory concentration (MIC) values were recorded using a serial microdilution method in a nutrient medium.
Results. Developed schemes for the preparation of bivalent cationic amphiphiles based on L-ornithine derivatives are presented. Differences in the structure of aliphatic amino acids (glycine, β-alanine, γ-aminobutyric acid (GABA)), in the length of alkyl radicals (C₈, C₁₂), or in the presence of an indole moiety, were used in the design of target compounds. The high antibacterial activity of the synthesized compounds was demonstrated. The most active compounds were lipoamino acids with terminal GABA residues and asymmetrical non-polar block (tryptamyl–dodecylamine). The MIC values were 0.39 μg/mL for gram-positive bacteria and 1.56 μg/mL for gram-negative bacteria. A GABA derivative with a symmetrical lipophilic moiety based on dioctylamine demonstrated activity with an MIC of 0.78 μg/mL against B. subtilis and 3.12 μg/mL against E. coli.
Conclusions. Nine new lipoamino acid cationic bivalent amphiphiles based on L-ornithine were synthesized. The structure of the obtained compounds was confirmed by nuclear magnetic resonance ¹H spectroscopy and mass spectrometry data. Leading compounds in antimicrobial activity against both gram-positive and gram-negative strains of bacteria were determined. The influence of the degree of lipophilicity in the asymmetric nonpolar block on the level of exhibited antimicrobial activity is demonstrated.

1. Francine P. Systems Biology: New Insight into Antibiotic Resistance. Microorganisms. 2022;10(12):2362. https://doi.org/10.3390/microorganisms10122362

2. Van Duin D., Paterson D.L. Multidrug-Resistant Bacteria in the Community: An Update. Infect. Dis. Clin. North Am. 2020;34(4):709–722. https://doi.org/10.1016/j.idc.2020.08.002

3. Mayegowda S.B., Ng M., Alghamdi S., Atwah B., Alhindi Z., Islam F. Role of Antimicrobial Drug in the Development of Potential Therapeutics. Evid. Based Complement. Alternat. Med. 2022;2022:2500613. https://doi.org/10.1155/2022/2500613

4. Bakka T.A., Strøm M.B., Andersen J.H., Gautun O.R. Synthesis and antimicrobial evaluation of cationic low molecular weight amphipathic 1, 2, 3-triazoles. Bioorg. Med. Chem. Lett. 2017;27(5):1119–1123. https://doi.org/10.1016/j.bmcl.2017.01.092

5. Taubes G. The bacteria fight back. Science. 2008;321(5887): 356–361. https://doi.org/10.1126/science.321.5887.356

6. Bortolotti A., Troiano C., Bobone S., Konai M.M., Ghosh C., Bocchinfuso G., Acharya Y., Santucci V., Bonacorsi S., Di Stefano C., Haldar J., Stella L. Mechanism of lipid bilayer perturbation by bactericidal membrane-active small molecules. Biochim. Biophys. Acta Biomembr. 2023;1865(1):184079. https://doi.org/10.1016/j.bbamem.2022.184079

7. Kristiansson E., Fick J., Janzon A., Grabic R., Rutgersson C., Weijdegård B., Söderström H., Larsson D.G. Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS One. 2011;6(2):e17038. https://doi.org/10.1371/journal.pone.0017038

8. Guo Y., Hou E., Wen T., Yan X., Han M., Bai L.P., Fu X., Liu J., Qin S. Development of Membrane-Active Honokiol/ Magnolol Amphiphiles as Potent Antibacterial Agents against Methicillin-Resistant Staphylococcus aureus (MRSA). J. Med. Chem. 2021;64(17):12903–12916. https://doi.org/10.1021/acs.jmedchem.1c01073

9. Schweizer L., Ramirez D., Schweizer F. Effects of Lysine N-ζ-Methylation in Ultrashort Tetrabasic Lipopeptides (UTBLPs) on the Potentiation of Rifampicin, Novobiocin, and Niclosamide in Gram-Negative Bacteria. Antibiotics. 2022;11(3):335. https://doi.org/10.3390/antibiotics11030335

10. Neubauer D., Jaśkiewicz M., Bauer M., Gołacki K., Kamysz W. Ultrashort Cationic Lipopeptides-Effect of N-Terminal Amino Acid and Fatty Acid Type on Antimicrobial Activity and Hemolysis. Molecules. 2020;25(2):257. https://doi.org/10.3390/molecules25020257

11. Dawgul M.A., Greber K.E., Bartoszewska S., Baranska-RybakW., Sawicki W., Kamysz W. In Vitro Evaluation of Cytotoxicity and Permeation Study on Lysine- and Arginine-Based Lipopeptides with Proven Antimicrobial Activity. Molecules. 2017;22(12):2173. https://doi.org/10.3390/molecules22122173

12. Wang M., Feng X., Gao R., Sang P., Pan X., Wei L., Lu C., WuC., CaiJ. Modular Design of Membrane-Active Antibiotics: From Macromolecular Antimicrobials to Small Scorpionlike Peptidomimetics. J. Med. Chem. 2021;64(14):9894–9905. https://doi.org/10.1021/acs.jmedchem.1c00312

13. RehbergN., SommerG.A., DrießenD., KruppaM., AdeniyiE.T., Chen S., Wang L., Wolf K., Tasch B.O.A., Ioerger T.R., Zhu K., Müller T.J.J., Kalscheuer R. Nature-Inspired (di)Azine-Bridged Bisindole Alkaloids with Potent Antibacterial in Vitro and in Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus. J. Med. Chem. 2020;63(21):12623–12641. https://doi.org/10.1021/acs.jmedchem.0c00826

14. Yu H.F., Qin X.J., Ding C.F., Wei X., Yang J., Luo J.R., Liu L., Khan A., Zhang L.C., Xia C.F., Luo X.D. Nepenthe-Like Indole Alkaloids with Antimicrobial Activity from Ervatamia chinensis. Org. Lett. 2018;20(13):4116–4120. https://doi.org/10.1021/acs.orglett.8b01675

15. Liu J., Li H., He Q., Chen K., ChenY., Zhong R., Li H., Fang S., Liu S., Lin S. Design, synthesis, and biological evaluation of tetrahydroquinoline amphiphiles as membrane-targeting antimicrobials against pathogenic bacteria and fungi. Eur. J. Med. Chem. 2022;243:114734. https://doi.org/10.1016/j.ejmech.2022.114734

16. Lawrence C.L., Okoh A.O., Vishwapathi V., McKenna S.T., Critchley M.E., Smith R.B. N-alkylated linear heptamethine polyenes as potent non-azole leads against Candida albicans fungal infections. Bioorg. Chem. 2020;102:104070. https://doi.org/10.1016/j.bioorg.2020.104070

17. Whitby L.R., Boyle K.E., Cai L., Yu X., Gochin M., Boger D.L. Discovery of HIV fusion inhibitors targeting gp41 using a comprehensive α-helix mimetic library. Bioorg. Med. Chem. Lett. 2012;22(8):2861–2865. https://doi.org/10.1016/j.bmcl.2012.02.062

18. Lee W.G., Gallardo-Macias R., Frey K.M., Spasov K.A., Bollini M., Anderson K.S., Jorgensen W.L. Picomolar inhibitors of HIV reverse transcriptase featuring bicyclic replacement of a cyanovinylphenyl group. J. Am. Chem. Soc. 2013;135(44):16705–16713. https://doi.org/10.1021/ja408917n

19. Pape V.F., Tóth S., Füredi A., Szebényi K., Lovrics A., Szabó P., Wiese M., Szakács G. Design, synthesis and biological evaluation of thiosemicarbazones, hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential to overcome multidrug resistance. Eur. J. Med. Chem. 2016;117:335–354. https://doi.org/10.1016/j.ejmech.2016.03.078

20. Özdemir A., Altintop M.D., Turan-Zitouni G., Çiftçi G.A., Ertorun İ., Alataş Ö., KaplancikliZ.A. Synthesis and evaluation of new indole-based chalcones as potential antiinflammatory agents. Eur. J. Med. Chem. 2015;89:304–309. https://doi.org/10.1016/j.ejmech.2014.10.056

21. Korotkin M.D., Filatova S.M., Denieva Z.G., Budanova U.A., Sebyakin Y.L. Synthesis of diethanolamine-based amino acid derivatives with symmetric and asymmetric radicals in their hydrophobic domain and potential antimicrobial activity. Tonk. Khim. Tekhnol. = Fine Chem. Technol. 2022;17(1): 50–64 (Russ., Eng.). https://doi.org/10.32362/2410-6593-2022-17-1-50-64 ]

22. Guseva M.K., Budanova U.A., Sebyakin Yu.L. Synthesis and evaluation of the antimicrobial activity of cationic amphiphiles based on bivalent diethylenetriamine derivatives. Pharm. Chem. J. 2023;56(12):1607–1612. https://doi.org/10.1007/s11094-023-02834-z [Original Russian Text: Guseva M.K., Budanova U.A., Sebyakin Yu.L. Synthesis and evaluation of the antimicrobial activity of cationic amphiphiles based on bivalent diethylenetriamine derivatives. Khimiko-Farmatsevticheskii Zhurnal. 2023;56(12):38–43 (in Russ.). https://doi.org/10.30906/0023-1134-2022-56-12-38-43 ]