Main chain cationic oligomerization (imidazolium) forming N-heterocyclic carbenes for efficient sterilization in complex environments

Abstract

Disclosed herein is a compound according to Formula Ia:. Also disclosed herein are pharmaceutical compositions comprising the above compounds, uses of the above compounds, a method of treating one or both of bacterial and fungal infections comprising the step of administering a pharmaceutically effective amount of the above compounds or the above pharmaceutical compositions, and an antibacterial and / or antifungal detergent composition comprising the above compound and a surfactant.

Description

Technical Field

[0001] This disclosure generally relates to antibiotics, and more specifically to main-chain cationic oligoimidazolium, which is used for effective sterilization in complex environments. Background Technology

[0002] The listing or discussion of prior published literature in this specification does not necessarily imply an admission that such literature is part of the prior art or is common general knowledge.

[0003] Antibiotics have long been indispensable tools for treatment and prevention in biomedical and agricultural applications, underpinning numerous modern biomedical interventions, including chemotherapy and surgery. However, the growing global crisis of bacterial resistance in almost all classes of antibiotics casts a shadow over these medical advances. This crisis is exacerbated by the slow progress in developing novel antimicrobial agents, a pressing challenge that has persisted for decades. Notably, approval for a new class of antibiotics for treating Gram-negative bacterial infections has been delayed for over sixty years. Traditional antibiotics typically work by targeting specific enzymes involved in conserved metabolic processes, inevitably leading to the emergence of resistant strains.

[0004] Against this backdrop, bacterial membranes have become one of the last frontiers in the search for novel antimicrobial drugs. Membrane-targeting formulations are promising due to their generally low likelihood of inducing resistance. Traditionally, antimicrobial polymers and peptides (AMPs) rely on disrupting the integrity of the bacterial cytoplasmic membrane to induce cell death. However, the development of classic cationic polymers and peptides targeting bacterial membrane permeability has been hampered by concerns about their toxicity and limited metabolic stability.

[0005] Antimicrobial peptides (AMPs), such as defensins, daptomycin, and scutellarin-2, typically kill bacteria through electrostatic interactions with the cell membrane, subsequently forming physical channels that ultimately lead to bacterial cell death. AMPs are highly attractive antimicrobial agents due to their unique bactericidal mechanism, distinct from antibiotics. However, the electrostatic interactions between AMPs and bacterial and mammalian membranes are relatively small, especially in the presence of salt and serum in physiological environments, resulting in low selectivity for eukaryotic cells and often high toxicity. Another approach utilizes important processes and mechanisms such as the electron transport chain (ETC), which reside on the bacterial cell membrane and are therefore potentially sensitive to selective targeted inhibition. The electron transport chain of mammalian counterparts, on the other hand, is hidden within the inner mitochondrial membrane within the cell. Furthermore, our understanding of the complex interactions between cationic agents and bacterial membranes is incomplete, and exploration of the structural diversity of cationic molecules is limited.

[0006] Therefore, there is a need for novel antibiotics that can effectively kill bacteria in complex environments. Summary of the Invention

[0007] The invention is described below with reference to the following numbers and embodiments.

[0008] 1. A compound according to formula Ia:

[0009] in: X - It is an anionic substance, selected from organic acids in the form of carboxylate salts, Br... - I - or Cl - ; Y represents OH, NH2, zwitterionic substances, or hydrazone; Each L represents independently: ; ; ;or , Each wavy line represents a connection point with the rest of the molecule. Or the compound represented by formula Ib:

[0010] Each L is independently selected from the list provided above; and

[0011] X - The definition is as described above. Or the compound represented by formula Ic:

[0012] in: R1 is selected from H, CH3, Cl, or CF3; One of R2 and R3 is H, CH3, Cl or CF3, and the other is H, or R2 and R3 together with the carbon atoms they are attached to form a benzene ring; n represents 6 or 8; X - The definition is as described above. Or the compound represented by formula Id:

[0013] Each L is independently selected from the list provided above; and

[0014] X - The definition is as described above, and

[0015] Solvates of compounds of formulas Ia-Id.

[0016] 2. The compound according to item 1, wherein the compound has the formula Ic.

[0017] 3. The compound according to item 2, wherein R1 is H.

[0018] 4. The compound according to item 2 or item 3, wherein one of R2 and R3 is H, CH3, Cl or CF3, and the other is H.

[0019] 5. The compound according to item 3, wherein one of R2 and R3 is H or CH3, and the other is H.

[0020] 6. The compound according to any one of items 2 to 5, wherein: n is 6; and / or X - Selected from Br - I - or Cl - And / or Y is OH.

[0021] 7. The compound according to any one of items 2 to 6, wherein: R1 is H; One of R2 and R3 is H or CH3, and the other is H; n is 6; and X - For Cl - .

[0022] 8. The compound according to item 1, wherein the compound has formula Ia or formula Ib.

[0023] 9. The compound according to item 8, wherein each L represents: ;or The wavy line represents the connection point with the rest of the molecule.

[0024] 10. The compound according to item 1, wherein the compound is a compound of formula Ia, wherein X - For Cl - And each L is .

[0025] 11. The compound according to item 1, wherein the compound is a compound of formula Ib, wherein X - For Cl - And each L is .

[0026] 12. A pharmaceutical composition comprising the compound of any one of items 1 to 11, and one or both of a pharmaceutically acceptable excipient and a carrier.

[0027] 13. Use in a medicament of any compound according to any one of entries 1 to 11 or of a pharmaceutical composition according to entry 12.

[0028] 14. The compound according to any one of items 1 to 11 or the pharmaceutical composition according to item 12 for the treatment of one or both of bacterial and fungal infections.

[0029] 15. Use of the compound according to any one of headings 1 to 11 or the pharmaceutical composition according to heading 12 in the preparation of a medicament for treating one or both of bacterial and fungal infections.

[0030] 16. A method for treating one or both of bacterial and fungal infections, comprising administering to a subject in need a pharmaceutically effective amount of a compound according to any one of entries 1 to 11 or a pharmaceutical composition according to entry 12.

[0031] 17. An antibacterial and / or antifungal detergent composition comprising: The compounds described in any of entries 1 to 11; and Surfactants.

[0032] 18. The antibacterial and / or antifungal detergent composition according to item 17, wherein the composition is in the form of a solid or liquid soap.

[0033] 19. The antibacterial and / or antifungal detergent composition according to item 18, wherein the composition is in the form of a shampoo. Attached Figure Description

[0034] Figure 1 shows structural diagrams of oligoimidazolium (OIM) analogs synthesized and bioevaluated in SAR studies, including (A) the OIM1-6 series, (B) the OIM1-8 series, (C) the biodegradable OIM1 series, and (D) the biodegradable OIM series.

[0035] Figure 2The synthetic route for the OIM1-6 derivative is shown. Reagents and conditions: (i) (3-bromopropyl)carbamate, NaH, tetrahydrofuran (THF), 0°C to 50°C, 18 hours, 71-76%; (ii) (3-bromopropyl)carbamate, NaOH, MeCN / H2O, 50°C, 81%; (iii) 1,4-dibromobutane, acetonitrile (MeCN), 80°C, 18 hours, 63-85%; (iv) NaH, 1,4-dibromobutane, THF, 0°C to 65°C, 18 hours, 66-9% 5%; (v) NaOH, MeCN / H2O, 50°C, 78%; (vi) MeCN, 80°C, 18-60 hours, 63-85%; (vii) 1,4-diiodobutane, MeCN / DMF or N-methyl-2-pyrrolidone (NMP) or dimethyl sulfoxide (DMSO), 80-120°C, 48 hours to 1 week; (viii) 33% by weight HBr in acetic acid solution, room temperature (rt), 18 hours; and (ix) Amberlyst AR26 OH resin, 10% HCl aqueous solution, in three steps, 18-59%.

[0036] Figure 3 The X-ray photoelectron spectroscopy (XPS) analysis results of compound 1 are shown, indicating that the amount of bromide ions present is negligible after ion exchange chromatography and dialysis.

[0037] Figure 4 The XPS analysis results for compound 2 are shown, indicating that the amount of bromide ions present is negligible after ion exchange chromatography and dialysis.

[0038] Figure 5 The XPS analysis results for compound 5 are shown, indicating that the amount of bromide ions present is negligible after ion exchange chromatography and dialysis.

[0039] Figure 6 The XPS analysis results of compound 13 are shown, indicating that the amount of bromide ions present after ion exchange chromatography and dialysis is negligible (a binding energy of 64-65 indicates the presence of bromine).

[0040] Figure 7 The XPS analysis results of compound 14 are shown, indicating that the amount of bromide ions present after ion exchange chromatography and dialysis is negligible (a binding energy of 64-65 indicates the presence of bromine).

[0041] Figure 8 The XPS analysis results of compound 16 are shown, indicating that the amount of bromide ions present after ion exchange chromatography and dialysis is negligible (a binding energy of 64-65 indicates the presence of bromine).

[0042] Figure 9Calibration curves for (A) OIM1-6-CH (1), (B) OIM1-6-C2(CH3) (2) and (C) OIM1-6-C4(CH3) (5) are shown using area and height plotted by liquid chromatography-mass spectrometry (LC-MS).

[0043] Figure 10 shows the cell viability of 3T3 fibroblasts after incubation with compounds (1-3) for 24, 48, and 72 hours, namely (A) OIM1-6-CH (1); (B) OIM1-6-C2(CH3) (2) and (C) OIM1-6-C2(Cl) (3). Human embryonic kidney (HEK) cells were also incubated with (D) OIM1-6-CH (1), (E) OIM1-6-C2(CH3) (2), and (F) OIM1-6-C2(Cl) (3) for 24, 48, and 72 hours. Cell survival rates of hepatocellular carcinoma (HepG2) cells after incubation with (G)OIM1-6-CH (1), (H)OIM1-6-C2(CH3) (2) and (I)OIM1-6-C2(Cl) (3) for 24 hours, 48 ​​hours and 72 hours.

[0044] Figure 11 shows the cell viability of 3T3 fibroblasts after incubation with compounds (4-6) for 24, 48, and 72 hours, namely: (A) OIM1-6-C2(CF3) (4); (B) OIM1-6-C4(CH3) (5) and (C) OIM1-6-C4(Cl) (6). Human embryonic kidney (HEK) cells after incubation with (D) OIM1-6-C2(CF3) (4), (E) OIM1-6-C4(CH3) (5) and (F) OIM1-6-C4(Cl) (6) for 24, 48, and 72 hours. Cell survival rates of hepatocellular carcinoma (HepG2) cells after incubation with (G)OIM1-6-C2(CF3)(4), (H)OIM1-6-C4(CH3)(5) and (I)OIM1-6-C4(Cl)(6) for 24 hours, 48 ​​hours and 72 hours.

[0045] Figure 12 shows the cell viability of 3T3 fibroblasts after incubation with compounds (7-9) for 24, 48, and 72 hours, namely (A) OIM1-6-C4(F) (7), (B) OIM1-6-(BZ) (8), and (C) OIM1-6-C4(CF3) (9). Human embryonic kidney (HEK) cells were also incubated with (D) OIM1-6-C4(F) (7), (E) OIM1-6-(BZ) (8), and (F) OIM1-6-C4(CF3) (9) for 24, 48, and 72 hours. Cell viability of hepatocellular carcinoma (HepG2) cells after incubation with (G)OIM1-6-C4(F)(7), (H)OIM1-6-(BZ)(8) and (I)OIM1-6-C4(CF3)(9) for 24 hours, 48 ​​hours and 72 hours.

[0046] Figure 13 shows the cell viability of 3T3 fibroblasts after incubation with compounds (10-12) for 24, 48, and 72 hours, namely (A) OIM1-8-CH (10), (B) OIM1-8-C2(CH3) (11), and (C) OIM1-8-C4(CH3) (12). Human embryonic kidney (HEK) cells after incubation with (D) OIM1-8-CH (10), (E) OIM1-8-C2(CH3) (11), and (F) OIM1-8-C4(CH3) (12) for 24, 48, and 72 hours. Cell survival rates of hepatocellular carcinoma (HepG2) cells after incubation with (G)OIM1-8-CH (10), OIM1-8-C2(CH3) (11) and (I)OIM1-8-C4(CH3) (12) for 24 hours, 48 ​​hours and 72 hours.

[0047] Figure 14 Cell viability of 3T3 fibroblasts (AB) after incubation with compounds (13-14) for 24, 48, and 72 hours is shown. Compounds (13-14) are: (A) OIM1-8-Bu-acetal (13) and (B) OIM1-8-Bu-PzAc (14). Cell viability of human embryonic kidney (HEK) cells after incubation with (C) OIM1-8-Bu-acetal (13) and (D) OIM1-8-Bu-PzAc (14) for 24, 48, and 72 hours is shown. Cell viability of hepatocellular carcinoma (HepG2) cells after incubation with (E) OIM1-8-Bu-acetal (13) and (F) OIM1-8-Bu-PzAc (14) for 24, 48, and 72 hours is shown.

[0048] Figure 15 shows the cell viability of 3T3 fibroblasts after incubation with compounds (15-16) for 24, 48, and 72 hours, namely (A) OIM1-12-6C-OH (15) and (B) OIM1-8-2D (16). Human embryonic kidney (HEK) cells after incubation with (C) OIM1-12-6C-OH (15) and (D) OIM1-8-2D (16) for 24, 48, and 72 hours. Hepatocellular carcinoma (HepG2) cells after incubation with (E) OIM1-12-6C-OH (15) and (F) OIM1-8-2D (16) for 24, 48, and 72 hours.

[0049] Figure 16 shows the cell viability of 3T3 fibroblasts after incubation with known antibiotics (A) gentamicin; (B) colistin and (C) ciprofloxacin for 24, 48, and 72 hours. Human embryonic kidney (HEK) cells after incubation with (D) gentamicin, (E) colistin and (F) ciprofloxacin for 24, 48, and 72 hours. Hepatocellular carcinoma (HepG2) cells after incubation with (G) gentamicin, (H) colistin and (I) ciprofloxacin for 24, 48, and 72 hours.

[0050] Figure 17 The synthetic route for the OIM1-8 derivatives is shown. Reagents and conditions: i) 1,4-dibromobutane (excess, 4 to 5 equivalents) acetonitrile (MeCN), 80 °C, 18 h, 55-68%; ii) 1d to 3e (1.0 equivalent) 10a to 12a (0.45 equivalent) acetonitrile + DMF (9:1); iii) 33 wt% HBr in acetic acid solution, room temperature, 18 h; and iv) Amberlyst AR26 OH resin, 10% aqueous HCl solution, 50-65%.

[0051] Figure 18The synthesis of OIM1-8 degradable derivatives is shown. Reagents and conditions: i) 1,4-Dibromobutane (excess, 4 to 5 equivalents) acetonitrile (MeCN), 80°C, 18 hours, 65%; ii) 1 g (1.0 equivalent), 1 d (2.5 equivalents) acetonitrile, 80°C, 18 hours, 62%; iii) 13a (1.0 equivalent), paraformaldehyde (0.5 equivalent) and concentrated H2SO4 (cat.), toluene, 130°C, 1.5 hours, 83%; iv) chloroacetyl chloride (2.1 equivalent), K2CO3 (2.5 equivalent), water + CHCl3 (1:1), 0°C to room temperature, 18 hours, 90%; v) 1 d (1.0 equivalent), 13b (5.0 equivalent), acetonitrile, 80°C, 3 hours, 50%; vi) 1 d (1.0 equivalent), 14b (5.0 equivalent), acetonitrile, 80°C, 3 hours, 68%; vii) For OIM1-8-Bu-acetal, 1h (2.1 equivalents), 13c (1.0 equivalents), acetonitrile + DMF (9:1); for OIM1-8-Bu-PzAc, 1h (2.1 equivalents), 14c (1.0 equivalents), acetonitrile + DMSO (1:1), overnight; and iv) Amberlyst AR26 OH resin, 10% HCl aqueous solution, 50-65%.

[0052] Figure 19 shows the synthetic routes (A and B) for the degradable OIM series OIM1-12-6C-OH(15). Reagents and conditions: (i) Boc-anhydride (2.3 equivalents), Mg(ClO4)2 (0.1 equivalents), DCM, reflux, 48 h, 64%; (ii) 15a (1 equivalent), imidazole 1a (1.1 equivalent), NaH (3 equivalents), anhydrous THF, 50 °C, 18 h, 71%; (iii) triphosgene (0.25 equivalents), pyridine (1.5 equivalents), DCM, 0 °C to room temperature, 18 h, 47%; (iv) 1d (1 equivalent), 15c (5 equivalents), MeCN, 85 °C, 3 h, 36%; (v) 15c (2.5 equivalents) (vi) 1d (2.5 equivalents), MeCN, 85°C, 3 hours, 75%; (vii) 1d (2.5 equivalents), MeCN, 95°C, 3 hours, 90%; (vii) 15c (5 equivalents), MeCN / DMF (2:1), 95°C, 3 hours, 97%; (viii) 1d (5 equivalents), MeCN / DMF (2:1), 95°C, 3 hours, 75-86%; and (ix) 15d (1 equivalent), 15h (2.01 equivalents), pure, 95°C, 2 hours, MeCN and EA wash, TFA / DCM (50%) 12 hours, Amberlyst A-26 OH type, 10% HCl, EA wash, Sephadex G-25 silica gel purification, 10%.

[0053] Figure 20 The synthetic route for the biodegradable OIM series OIM1-8-2D (16) is shown. Reagents and conditions: (i) chloroacetyl chloride (2.1 equivalents), K2CO3 (2.5 equivalents), water + CHCl3 (1:1), 0°C to room temperature, 18 h, 65% (16a); (ii) 1d (1.0 equivalents), 16a (5.0 equivalents), acetonitrile, 80°C, 3 h, 50%; (iii) 1e (2.1 equivalents), 16b (1.0 equivalents), acetonitrile + DMF + methanol (8:1:1), 95°C, overnight; (iv) 33% by weight HBr in acetic acid solution, room temperature, 2 h; (v) Amberlyst AR26OH resin, 10% aqueous HCl solution, three-step treatment, 55%.

[0054] Figure 21 The bactericidal effects of (A) OIM1-6-CH, (B) OIM1-6-C2(CH3), and (C) OIM1-6-C4(CH3) on Staphylococcus aureus LAC over time, tested at 1 to 8x MIC in MHB, are shown. The bactericidal effects of (D) OIM1-6-CH, (E) OIM1-6-C2(CH3), and (F) OIM1-6-C4(CH3) on Pseudomonas aeruginosa PAO1 over time, tested at 1 to 4x MIC in MHB, are also shown.

[0055] Figure 22 The following diagrams are shown: (A) Chemical structure of the simulated cationic oligomer; (B) Geometrically optimized structure of the simulated cationic oligomer; (C) Calculated Hirshfeld charge on the imidazolium ring in OIM.

[0056] Figure 23 The following studies illustrate: (A) Membrane depolarization of MRSA LAC treated with OIM1-6 compounds was studied using the DiSC3(5) assay, with bacitracin as an antibiotic control, and the treatment time was 1 hour. (B) Membrane permeability of LAC treated with OIM1-6 compounds was studied using the propidium iodide (PI) assay, with nisin as a positive control, and the treatment time was 1 hour.

[0057] Figure 24 The diagram shows that (A) OIMs with free C2-hydrogen are carbonic acid, which deprotonate in neutral water to form uncharged and hydrophobic N-heterocyclic carbene (NHC). II(B) Novel transport mechanism: (i) OIM carbonic acid is converted into an amphiphilic copolymer (composed of hydrophilic cations and hydrophobic NHC repeating units), which, compared with (ii) classical cationic polymers that form physical channels, can effectively transport the plasma membrane into the bacterial cytosol at a lower polymer threshold concentration. OIM binds to its intracellular targets (such as DNA, as previously studied) in the bacterial cytosol, leading to bacterial death, while classical cationic polymers are ineffective in killing bacteria.

[0058] Figure 25 shows the NHC formation study of OIM1-6-CH(1). (A) Through partial carbene ( II (B) Detection of NHC by reaction of monomer with D2O or AuCl(SMe2). (C) In D2O, the aqueous solution of OIM1-6-CH(1) changes over time at pH 6.63, pH 6.81, pH 7.16 and pH 8.21. 1H nuclear magnetic resonance (NMR) hydrogen-deuterium exchange. (C) Schematic diagram of the interaction between phosphatidylcholine / phosphatidylglycerol (PC / PG) liposomes containing NHC probes (carbazole dye and / or AuCl(SMe2)) and OIM-1-6-CH(1). (D) Fluorescence of carbazole embedded in the liposome bilayer in solution with OIM1-6-CH(1) or stable carbene (IPr). (EH) Evidence of OIM(1) forming NHC using AuCl(SMe2) embedded in the liposome bilayer and LC-MS / MS: (E) Total ion chromatogram (TIC) of Au(lipid)-OIM1-6-CH(1) obtained by LC-ESI-MS(+). (F) Extraction ion chromatograms (EIC) of (i) OIM1-6-CH (m / z 199.15) and (ii) Au-OIM1-6-CH (m / z 330.52) containing one Au atom (both at pH 7.4). (G) Identified chemical structures of EIC: (i) OIM1-6-CH(1) with m / z = 199.15 containing 4 cations and 2 NHCs; (ii) Au-OIM1-6-CH with m / z = 350.52 containing 1 Au-OIM1-6-CH, having 1 Au-containing cation, 2 imidazolium cations (without Au) and 3 NHCs. (H) (i) Mass spectrum (MS) of m / z = 199.15 (retention time 3.55 min), MS gap = 0.25, indicating z = 4 and total ion mass = 796.60; and (ii) Mass spectrum (MS) of m / z 330.52 (retention time 4.04 min), MS gap = 0.33, indicating z = 3 and total ion mass = 991.56. (I) Uptake of OIM derivatives into liposomes at pH 7.4 and pH 6.8. (J) (i) Carbazole is an NHC dye that becomes non-fluorescent in the presence of NHC due to hydrogen bonding; (ii) Reaction of NHC with (dimethyl sulfide) gold (I).

[0059] Figure 26 shows the confirmation of the peak at m / z 330.52 as Au-OIM-1-6-CH: (AD) hydrogen-deuterium exchange mass spectrometry experiments. (A) Structural assignments of (i) m / z 199.15 (OIM1-6-CH), (ii) m / z 330.52 (Au-OIM-1-6-CH), (iii) m / z 331.86 (deuterated Au-OIM-1-6-CH), and (iv) m / z 395.84 (2Au-OIM-1-6-CH). (B) Total ion chromatogram (TIC) and extracted ion chromatogram (EIC) of deuterated Au-OIM1-6-CH (m / z 331.86). (C) MS of non-deuterated Au-OIM1-6-CH (m / z 330.52). (D) MS analysis of deuterated Au-OIM1-6-CH (m / z 331.86). MS / MS analysis of selected ion at m / z 330.52: (E) before fragmentation and (F) after fragmentation. (G) Structural assignment of fragments.

[0060] Figure 27 shows the confirmation of the peak at m / z 330.52 as Au-OIM-1-6-CH: (AE) Hydrogen-deuterium exchange mass spectrometry experiment. (A) Total ion chromatogram (TIC) of Au-OIM-1-6-CH in d-methanol. (B) Extracted ion chromatogram (EIC) of deuterated Au-OIM-1-6-CH (m / z 331.86). (C) MS of deuterated Au-OIM-1-6-CH (m / z 331.86) and (D) MS of non-deuterated Au-OIM-1-6-CH (m / z 330.52). (E) Structural assignment of m / z 330.52 and m / z 331.86. (F) MS / MS analysis of the selected ion at m / z 330.52. (G) Structural assignment of fragments.

[0061] Figure 28 shows the NHC formation studies of OIM1-6-C2(CH3) (2) and OIM1-6-C4(CH3) (5). (A) Hydrogen-deuterium exchange over time in aqueous solutions at pH 6.63, pH 6.81, pH 7.16 and pH 8.21 and (B) Hydrogen-deuterium exchange over time in aqueous solutions at pH 6.63, pH 6.81, pH 7.16 and pH 8.21. (C) Structural assignments of Au-OIM1-6-C2 (m / z 537.32) and 2Au-OIM1-6-C2 (m / z 423.87) and (D) Structural assignments of Au-OIM1-6-C4 (m / z 537.32) and 2Au-OIM1-6-C4 (m / z 423.87). (E) Extraction ion chromatograms (EIC) of Au-(2) and 2Au-(2) at pH 7.4 and (G) pH 8.2; and (F) Extraction ion chromatograms (EIC) of Au-(5) and 2Au-(5) at pH 7.4 and (H) pH 8.2.

[0062] Figure 29 Computer simulations of the interaction between two forms of OIM1-6-CH(1) (cationic and NHC forms) and Staphylococcus aureus membrane-simulants are shown. (AB) The number of contacts between OIM1-6-CH(1) and Staphylococcus aureus membranes over simulation time: (A) cationic OIM-membrane system; and (B) OIM-NHC-membrane system. A single contact is defined as a minimum distance of less than 0.4 nm between polymer atoms and membrane atoms. (CD) Conformations of (1) with Staphylococcus aureus membrane-simulants: (C) cationic OIM-membrane system; and (D) OIM-NHC-membrane system. These conformations are taken from the final simulation frame of each simulation repetition.

[0063] Figure 30 shows (A) the method for determining the total and cytoplasmic uptake of compounds by bacteria. (i) Fluorescence of MRSA LAC stained with OIM-FITC / SYTO 9 after treatment with trypan blue (TB) and Triton X-100 (TX). TB dye cannot penetrate the intact plasma membrane; therefore, it quenches surface-bound fluorophores trapped in the cell wall and to some extent quenches fluorophores trapped on the bacterial membrane surface. The addition of 0.04% TX made the bacterial membrane permeable, allowing TB dye to enter the cytoplasm. At this point, TB dye could quench fluorophores in the cytoplasm, but not those partially / completely inserted into the membrane bilayer. Fluorescence signals were measured for 50,000 bacteria in all flow cytometry experiments. (B) Flow cytometry histograms of untreated LAC before TB treatment (unquenched) and after TB treatment (TB quenched). The dashed lines indicate the gating settings: bacteria with fluorescence intensity to the right of the gating are considered to have taken up the dye, and bacteria with fluorescence intensity to the left of the gating are considered not to have taken up the dye. (C) Flow cytometry histogram of untreated LAC stained with STYO 9 DNA dye. There was no significant difference in fluorescence intensity between unquenched and TB-quenched bacterial populations, but after TX treatment + TB quenching (TX + TB quenching), the fluorescence intensity decreased significantly (to the left of the gating), indicating that the TB dye had entered the cytoplasm and quenched the STYO 9 dye bound to DNA. (DF) Flow cytometry histogram of LAC 1 hour after treatment with 64 µg / mL of (iv) OIM1-6-CH (1), (E) OIM1-6-C2(CH3) (2), and (F) OIM1-6-C4(CH3) (5). The percentage of bacterial populations with membrane + cytoplasmic OIM uptake was obtained from the TB quenching histogram (to the right of the gating) and is shown as a bar chart in Figure 31. Following TX+TB quenching, the fluorescence intensity of (1) and (5) decreased significantly (to the left of the gating), while the fluorescence intensity of (2) did not decrease, indicating that (1) and (5) entered the cytoplasm, while (2) partially / completely inserted into the membrane bilayer. The bacterial population with cytoplasmic uptake of OIM was determined by the histogram difference between TB quenching and TX+TB quenching. (G) Histogram difference between TB quenching and TX+TB quenching, generated by Flowjo software, using compound (1) (Fig. 30D) as an example. The percentage of the bacterial population with cytoplasmic uptake is shown as a bar chart in Fig. 31.

[0064] Figure 31 shows the effect of NHC formation on OIM potency and bacterial uptake. (A) MIC of OIM and gentamicin against parental Staphylococcus aureus strains. MIC The tested concentration was as high as 4,096 µg / mL, MIC > 4,096 µg / mL was recorded as 8,192 µg / mL. (B) Percentage of LAC cells that took up OIM after 1 hour of treatment in TSB. (C) Mean fluorescence intensity (MFI) of cells that took up OIM after 1 hour of treatment in TSB. (D) MIC of OIM and gentamicin against wild-type LAC and respiratory defect mutants at different pH values. MIC The tested concentration was as high as 4,096 µg / mL, MIC > 4,096 µg / mL was recorded as 8,192 µg / mL. (E) Percentage of wild-type LAC and respiratory-deficient mutant cells that took up OIM1-6-CH(1) after treatment with TSB at pH 7.2 and pH 6.8 for 1 hour, using concentrations of 1x MIC at pH 7.2. (Wild-type LAC is 16 µg / mL, LAC Δ) menD It is 128 µg / mL, and LAC Δ hemB (256 µg / mL). (F) Sterilization kinetics of LAC treated with OIM1-6-CH (1) and OIM1-6-C4(CH3) (5) at 4 times the MIC values ​​(i.e., 16 µg / mL and 32 µg / mL, respectively) under aerobic, anaerobic and fermentation growth conditions.

[0065] Figure 32 shows (A) LAC treated with FITC-coupled OIM for 1 hour in MHB and (B) in TSB. Total OIM includes both surface-bound and internalized OIM portions unquenched by trypan blue quenching, with the internalized portion determined by trypan blue quenching. (C) LAC Δ after 1 hour of treatment with FITC-coupled OIM. menD (Traphanin Blue sudden death) and (D)LAC Δ hemB (Without trypan blue quenching). (E) In TSB at pH 7 and pH 6.8, LAC was used to couple FITC-conjugated OIM1-6-CH and OIM1-6-C4(CH3) at concentrations of 1x MIC at pH 7, respectively. (F) In TSB at pH 7 and pH 6.8, LAC Δ menD and LAC Δ hemB For FITC-coupled OIM1-6-CH (the concentration used was 1x MIC at pH 7 for each), ) intake.

[0066] Figure 33The evolution of resistance to OIM1-6-CH and OIM1-6-C4(CH3) is shown in (A) and (B) PAO1, respectively. (C) OCR of LAC after treatment with OIM1-6-CH, OIM1-6-C2(CH3), OIM1-6-C4(CH3), and HQNO, and the ΔC value of LAC are also shown. hemB and LAC Δ menD OCR of respiratory mutants. Fungicidal kinetics of Staphylococcus aureus LAC treated with (D)OIM1-6-CH or (E)OIM1-6-C4(CH3) at concentrations of 4 times their respective MIC values ​​(i.e., 16 µg / ml and 32 µg / ml, respectively) under aerobic, anaerobic and fermentation growth conditions.

[0067] Figure 34 The peaks at 8.8–8.9 ppm caused by deuterium exchange at (A) pH 7.16 and (B) pH 6.8 are shown to disappear. The extent of remaining protons at the C2 position is indicated above each spectrum. Deuterium exchange was observed to be faster at pH 7.16 than at pH 6.8.

[0068] Figure 35 The antimicrobial test results of OIM against Escherichia coli 8739 and Staphylococcus aureus 6538 in laundry applications are shown in four detergents (100 ppm sodium dodecylbenzene sulfonate (SDBS), 100 ppm sodium dodecyl sulfate (SDS), 50 ppm SDS + 50 ppm SDBS, and 389 ppm Japanese bagged detergent). (A) Schematic diagram of the antimicrobial application of OIM in laundry applications. The compounds tested are (B) polydiallyl dimethyl ammonium chloride (PDADMAC), (C) colistin, (D) OIM1-6-CH, (E) OIM1-6-C2(CH3), (F) OIM1-6-C4(CH3), (G) OIM1-8-Bu-2PzAc, and (H) OIM1-8-Bu-2Ac. The antimicrobial test was performed according to the standard test method ASTM-E2274. A minimum log reduction of 2 is required to pass the test.

[0069] Figure 36The in vivo efficacy of degradable OIM is demonstrated. (A) Mouse systemic infection model. (i) Protocol: Mice (n = 6 per group) were infected with Acinetobacter baumannii ATCC BAA 2803 by intraperitoneal (IP) injection of 300 μL of saline containing 5% mucin. Two hours post-infection, mice were administered a single dose of untreated PBS, 15 mg / kg imipenem, or 15 mg / kg OIM1-8-2D (16). Mice were sacrificed 26 hours post-infection, and bacterial counts were determined. (ii) Survival rate and (iii) reduction in the number of bacteria in the liver in the mouse systemic infection model. LOD: Limit of detection. p ≤ 0.0001, ns (not significant) p > 0.05 (one-way ANOVA). (B) Toxicity test of repeated administration (16) (15 mg / kg × 7-day dose). Body weight of mice after intraperitoneal injection of OIM1-8-2D (16) for 7 days at a daily dose of 15 mg / kg. (CE) Mastitis test in dairy cows. (C) Timeline and experimental setup of farm trials. (D) Bacterial counts in milk over time after bacterial challenge. Each symbol represents one milk sample from one teat (also known as one mammary fold). (E) Somatic cell counts (SCC) in milk samples after bacterial challenge. Each symbol represents a sample from one cow.

[0070] Figure 37 Detailed results of in vivo experiments in mice are shown. The reduction in bacterial logarithm in (A) kidney, (B) spleen, and (C) peritoneal fluid in a mouse model of systemic infection. LOD: Limit of detection. p ≤ 0.0001, ns (not significant) p > 0.05 (one-way ANOVA).

[0071] Figure 38 Images of nipples after 5 consecutive days of nipple soaking were shown in the safety test. No irritation was detected on the nipples treated with PIM1D.

[0072] Figure 39 The results of the Delvo test are shown for (A) the nipple surface and (B) the milk sample in the safety test.

[0073] Figure 40 The changes in milk composition (protein, fat and non-fat solids (SNF)) and somatic cell count (SCC) in PIM1D-treated nipples are shown during a 5-day safety test.

[0074] Figure 41The results of the bovine mastitis test are shown. (A) to (C): In vitro mastitis test: Escherichia coli, Staphylococcus aureus, and Streptococcus lactis were treated with (A) chlorhexidine, (B) OIM1-8-2D (16), and (C) PIM1D for 30 minutes in 18% milk at a concentration of 0.5%. Dashed lines indicate that at least log 10 The reduction was 5. (D) In ​​vivo mastitis test: Changes in milk composition of PIM1D-treated nipples after bacterial challenge. SNF represents non-fat solids. Detailed Implementation

[0075] Unexpectedly, three series of oligoimidazolium salts have been found to be particularly effective antibacterial and / or antifungal agents. Therefore, in a first aspect of the invention, a compound according to formula Ia is provided:

[0076] in: X - It is an anionic substance, selected from organic acids in the form of carboxylate salts, Br... - I - or Cl - ; Y represents OH, NH2, zwitterionic substances, or hydrazone; Each L represents independently: ; ; ;or , Each wavy line represents a connection point with the rest of the molecule. Or the compound represented by formula Ib:

[0077] Each L is independently selected from the list provided above; and

[0078] X - The definition is as described above. Or the compound represented by formula Ic:

[0079] in: R1 is selected from H, CH3, Cl, or CF3; One of R2 and R3 is H, CH3, Cl or CF3, and the other is H, or R2 and R3 together with the carbon atoms they are attached to form a benzene ring; n represents 6 or 8; X- The definition is as described above. Or the compound represented by formula Id:

[0080] Each L is independently selected from the list provided above; and

[0081] X - The definition is as described above, and

[0082] Solvates of compounds of formulas Ia-Id.

[0083] In the embodiments described herein, the word "comprising" can be interpreted as requiring the mentioned features but not limiting the presence of other features. Alternatively, the word "comprising" can also refer to situations where only the listed components / features are intended to be present (e.g., the word "comprising" can be replaced by the phrases "consisting of" or "substantially consisting of"). It is explicitly contemplated that both the broad and narrow interpretations can be applied to all aspects and embodiments of the invention. In other words, the word "comprising" and its synonyms can be replaced by the phrases "consisting of" or "substantially consisting of" or their synonyms, and vice versa.

[0084] The oligomers of the present invention referred to herein (in any aspect or embodiment of the invention) include such compounds themselves, tautomers of such compounds, and pharmaceutically acceptable solvates of such compounds.

[0085] As mentioned above, the oligomers of the present invention also encompass any solvates of the compounds and their salts. Preferred solvates are those formed by incorporating molecules of a non-toxic, pharmaceutically acceptable solvent (hereinafter referred to as a solvating solvent) into the solid-state structure (e.g., crystal structure) of the compounds of the present invention. Examples of such solvents include water, alcohols (such as ethanol, isopropanol, and butanol), and dimethyl sulfoxide. Solvates can be prepared by recrystallizing the compounds of the present invention with a solvent or a mixture of solvents containing a solvating solvent. In any given case, whether a solvate has been formed can be determined by analyzing the crystals of the compound using well-known and standard techniques such as thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and X-ray crystallography.

[0086] The solvate can be stoichiometric or non-stoichiometric. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates, and dihydrates.

[0087] For a more detailed discussion of solvates and the methods used to manufacture and characterize them, see Bryn. etal., Solid-State Chemistry of Drugs , Second Edition, published by SSCI, Incof West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0088] The oligomers of this invention may exist as regioisomers or exhibit tautomerism. All tautomer forms and mixtures thereof are included within the scope of this invention. For example, in compounds of formula Ic:

[0089] When R1 is H, R2 is a methyl group, and R3 is H, it can be represented as: However, the compound may have tautomers, where R1 is H, R3 is methyl, and R2 is H.

[0090] Therefore, in embodiments where R2 and R3 do not form a benzene ring together with the carbon atoms they are attached to, compounds of formula Ic can be represented as compounds of formula Ic′: ,in Rn can be H, CH3, Cl, or CF3. In this arrangement, the charge is distributed between the two nitrogen atoms and the carbon atom in between.

[0091] In the above aspects of the invention, the oligomers of the invention can be used in medical treatment methods. Therefore, according to other aspects of the invention, the following is provided: (a) Use of the oligomers of the present invention in pharmaceuticals; (b) The oligomers of the present invention are used to treat bacterial and / or fungal infections; (c) Use of the oligomers of the present invention in the preparation of medicaments for treating bacterial and / or fungal infections; and (d) A method for treating bacterial and / or fungal infections, the method comprising applying an effective amount of the oligomer of the present invention.

[0092] In the embodiments described herein, the oligomers of the present invention may be particularly useful in combating bacterial infections.

[0093] The term "bacterial infection" encompasses any disease or condition caused by microbial organisms inside or on the surface of a host. Examples of bacterial infections include, but are not limited to, those caused by mycobacteria (…). mycobacteria Pulmonary tuberculosis caused by Pseudomonas ( ) pseudomonas Burn wound infections caused by Staphylococcus aureus, etc.S. aureus Skin infections caused by Pseudomonas and Acinetobacter baumannii ( A. baumannii Fungal infections can cause wound infections, mastitis, and sepsis. The term "fungal infection" encompasses any disease or condition caused by fungal organisms inside or on the surface of the host. Examples of fungal infections include, but are not limited to, tinea pedis, tinea capitis, yeast infections, and tinea cruris.

[0094] A non-limiting list of bacteria that may be sensitive to the oligomers of this invention includes: *Acidithiobacillus fibrinolyticus* (… Acidothermus cellulyticus Acinetobacter baumannii ( Acinetobacter baumannii ), dental caries actinomycetes ( Actinomyces odontolyticus ), alkali-metal reducing bacteria ( Alkaliphilus metalliredigens ), Alkaliophilic bacteria ( Alkaliphilus oremlandii ), Arthrobacter chrysophagus ( Arthrobacter aurescens ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus clausti ( Bacillus clausii ), salt-resistant Bacillus ( Bacillus halodurans ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus pumilus ( Bacillus pumilus Bacillus subtilis ( Bacillus subtilis Bifidobacterium adolescentis ( ) Bifidobacterium adolescentis Bifidobacterium longum ( Bifidobacterium longum Burkholderia tsukia ( ), Burkholderia thailandensis ), glycolytic and pyrolytic cellulosic bacteria ( Caldicellulosiruptor saccharolyticus ), Hydrogen-producing carbon monoxide thermophilic bacteria ( Carboxydothermus hydrogenoformans Clostridium acetonebutanol ( Clostridium acetobutylicum Clostridium beyerridis ( Clostridium beijerinckii Clostridium botulinum ( Clostridium botulinum Clostridium cellulose ( ) Clostridium cellulolyticum Clostridium difficile ( Clostridium difficile Clostridium coccidioides ( Clostridium kluyveri Clostridium perfringens ( ), Clostridium difficile Clostridium leptum Clostridium perfringens ( ), Clostridium novii Clostridium novyi Clostridium perfringens ( ) Clostridium perfringens Clostridium tetani ( Clostridium tetani ), Clostridium pyrolyticum ( Clostridium thermocellum Corynebacterium diphtheriae ( Corynebacterium diphtheriae ), effective Corynebacterium ( Corynebacterium efficiens ), Corynebacterium glutamicum ( Corynebacterium glutamicum Corynebacterium yokaryotes ( Corynebacterium jeikeium ), Ureaplasma urealyticum ( Corynebacterium urealyticum ), Copenhagen desulfurization bacteria ( Desulfitobacterium hafniense ), reducing desulfurized enteroviruses ( Desulphosmaculum reducing Enterobacter cloacae () Enterobacter cloacae ), Enterococcus faecalis ( Enterococcus faecalis ), Enterococcus faecalis ( Enterococcus faecium ), Escherichia coli ( Escherichia coli ), Protruding belly eubacterium ( Eubacterium ventrally ), Siberian microbacteria ( Exiguobacterium sibiricum ), Dafenggou Delphiniformis ( Finegoldia magna ), Bacillus hygroscopicus ( Geobacillus kaustophilus ), heat-denitrified Bacillus subtilis ( Geobacillus thermodenitrificans ), genus Fasciola ( Janibacter sp. ), Radiation-resistant Animalcoccus ( Kineococcus radiotolerans ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Lactobacillus fermentum ( Lactobacillus fermentum Listeria monocytogenes ( Listeria monocytogenes Listeria monocytogenes ( ), harmless Listeria monocytogenes ( Harmless Listeria Listeria wilt ( Listeria welshmeri ), thermophilic acid bacteria ( Moorella thermoaceticus ), Mycobacterium avium ( Mycobacterium avium ), Mycobacterium bovis ( Mycobacterium bovis Mycobacterium chrysogenum ( ), Mycobacterium chrysogenum ( Mycobacterium gilvum Mycobacterium leprae ( Mycobacterium leprae ), Mycobacterium paratuberculosis ( Mycobacterium paratuberculosis ), Mycobacterium smegmatis ( Mycobacterium smegmatis ), Mycobacterium tuberculosis ( Mycobacterium tuberculosis ), Mycobacterium ulcerans ( Mycobacterium ulcerans ), Mycobacterium van Barenella ( Mycobacterium vanbaalenii ), Nocardia spp. Nocardioides sp. Nocardia anthrax ( ) Nocardia farcinica ), Bacillus subtilis of the Iping Ridge ( Oceanobacillus iheyensis ), heat propionate dark anaerobic sausage-shaped bacteria ( Pelotomaculum thermopropionic ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Rhodococcus spp. ( Rhodococcus sp. ), red sugar polysporum ( Saccharopolyspora erythraea Serratia depigmentosa ( ) Serratia decaying ), coagulase-negative staphylococcal species ( coagulase-negative Staphylococcus species Staphylococcus aureus ( Staphylococcus aureus ), methicillin-resistant Staphylococcus aureus (MRSA) methicillin resistant Staphylococcus aureus (MRSA) Staphylococcus epidermidis ( Staphylococcus epidermidis ), methicillin-resistant Staphylococcus epidermidis ( methicillin-resistant Staphylococcus epidermidis (MRSE) ), agalactococcus ( Streptococcus agalactiae Streptococcus glaucus ( ), Streptococcus glaucus Streptococcus gordonii Streptococcus suis ( ) Streptococcus mitis), oral streptococci ( Streptococcus oralis Streptococcus pneumoniae () Streptococcus pneumoniae ), Streptococcus sanguinis ( Streptococcus sanguinis Streptococcus suis ( Streptococcus suis Streptococcus lactis ( Streptococcus uberis ), avermectin streptomycin ( Streptomyces avermitilis Streptomyces azureus ( ), Streptomyces coelicolor ), ethanol-heat anaerobic rod-shaped bacteria ( Thermoanaerobacter ethanolicus ), Tengchong thermoanaerobic bacteria ( Thermoanaerobacter tengcongensis ) and their combinations. Specific bacteria will be discussed in the examples below.

[0095] To avoid ambiguity, in the context of this invention, the term "treatment" includes not only therapeutic or remissionary treatment for patients who require such treatment, but also preventive treatment and / or diagnosis for patients susceptible to related disease states.

[0096] The term "patient" includes references to mammalian patients (e.g., humans). As used herein, the terms "subject" or "patient" are recognized in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some embodiments, a subject is a subject requiring treatment or a subject with a disease or disorder. However, in other embodiments, a subject may be a healthy subject. The term does not indicate a specific age or sex. Therefore, it is intended to cover adult and newborn subjects, regardless of male or female.

[0097] The term "effective amount" refers to the amount of a compound that produces a therapeutic effect (e.g., sufficient to treat or prevent disease) in a patient receiving treatment. This effect can be objective (i.e., measurable by some test or biomarker) or subjective (i.e., the subject gives an indication of the effect or feels the effect).

[0098] Unless otherwise stated, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group, which may be substituted or unsubstituted. When the term "alkyl" refers to a C14 group... 1-6 When alkyl is used, the alkyl group can be ethyl, propyl (e.g., n-propyl or isopropyl), butyl (e.g., branched or straight-chain butyl), pentyl, hexyl, or more preferably methyl.

[0099] To avoid ambiguity, in the oligomers of the present invention, where two or more substituents may have the same properties, the actual properties of each substituent are not interdependent in any case.

[0100] In any of the embodiments of the invention that may be mentioned herein, n may be 6.

[0101] In embodiments of the invention that may be mentioned herein, X - Optional from Br - I - or Cl - .

[0102] In the embodiments of the invention that may be mentioned herein, Y may be OH.

[0103] The embodiments of the invention that may be mentioned include those relating to oligomers of the invention in which the compounds may have formula Ic. In these embodiments, one or more of the following may apply: (i) R1 can be H; (ii) One of R2 and R3 can be H, CH3, Cl or CF3, and the other can be H (e.g., one of R2 and R3 can be H or CH3, and the other can be H). (iii) n can be 6.

[0104] In specific embodiments of the invention that may be mentioned herein, when the compound is of formula Ic, then: R1 is H; One of R2 and R3 is H or CH3, and the other is H; n can be 6; and X - It can be Cl - .

[0105] In more specific embodiments of the invention that may be mentioned herein, the compound may have formula Ia or formula Ib. In these embodiments, each L may represent: ;or The wavy lines represent the connection points with the rest of the molecule.

[0106] In specific embodiments of the invention that may be mentioned herein, the compound may be a compound of formula Ia, wherein X - For Cl - And each L is .

[0107] In specific embodiments of the invention that may be mentioned herein, the compound may be a compound of formula Ib, wherein X - For Cl - And each L is .

[0108] It should be understood that the "L" groups in compounds of formulas Ia, Ib, and Id can confer biodegradability to these compounds. That is, these groups may be susceptible to action by enzymes or other biological mechanisms or organisms, thereby causing compounds of formulas Ia, Ib, and Id to decompose into two or more compounds (e.g., 2, 3, 4, 5, 6, etc.), limited only by the total number of "L" groups in the compound. This degradation can occur in vivo, allowing the compound to be metabolized and excreted, or in the environment (e.g., through the action of microorganisms in the environment, or through simple chemical degradation over time). As previously mentioned, in some cases, each "L" group may be selected from one or more possible options listed herein.

[0109] Other embodiments of the invention that may be mentioned include embodiments in which the oligomers of the invention are isotopically labeled. However, specific embodiments of the invention that may be mentioned include embodiments in which the oligomers of the invention are not isotopically labeled.

[0110] As used herein, the term "isotope-labeled" includes oligomers of the present invention wherein a non-natural isotope (or an isotope not naturally distributed) is present at one or more sites in the compound. Those skilled in the art will understand that "one or more sites in the compound" as used herein refers to one or more atoms in the oligomers of the present invention. Therefore, the term "isotope-labeled" includes compounds of the present invention that are enriched in isotopes at one or more sites in the oligomer.

[0111] The isotopic labeling or enrichment of the oligomers of the present invention can use any one of the radioactive or non-radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and / or iodine. Specific isotopes that may be mentioned in this regard include... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 35 S, 18 F, 37 CI, 77 Br、 82 Br and 125 I.

[0112] When the oligomers of the present invention are labeled or enriched with radioactive or non-radioactive isotopes, the oligomers of the present invention that may be mentioned include those compounds in which at least one atom exhibits an isotopic distribution, wherein the radioactive or non-radioactive isotope content of said atom is at least 10% higher than the natural level of said radioactive or non-radioactive isotope (e.g., 10% to 5000%, particularly 50% to 1000%, more particularly 100% to 500%).

[0113] As described above, the oligomers of the present invention can be used to treat bacterial and fungal infections. Therefore, a pharmaceutical composition comprising the oligomers of the present invention, and one or both of a pharmaceutically acceptable adjuvant and a carrier is also provided.

[0114] The oligomers of the present invention can be administered by any suitable route, but particularly by oral, intravenous, intramuscular, transdermal, subcutaneous, mucosal (e.g., sublingual or buccal), rectal, transdermal, nasal, pulmonary (e.g., tracheal or bronchial), local, or any other parenteral route, in the form of a pharmaceutically acceptable dosage form comprising the compound. Specific modes of administration that may be mentioned include oral, intravenous, transdermal, subcutaneous, nasal, intramuscular, or intraperitoneal administration.

[0115] The oligomers of the present invention are typically administered as pharmaceutical formulations in mixture with pharmaceutically acceptable adjuvants, diluents, or carriers, which can be selected with appropriate consideration of the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers can be chemically inert to the active compound and may not cause harmful side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations can be found, for example, in Remington. The Science and Practice of Pharmacy , 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenteral-acceptable aqueous solutions can be used, which are free of pyrogens and have the necessary pH, isotonicity, and stability. Suitable solutions will be well known to those skilled in the art, among which numerous methods are described in the literature. A brief overview of drug delivery methods can also be found, for example, in Langer, Science (1990) 249 , 1527.

[0116] Otherwise, those skilled in the art can routinely prepare suitable formulations using conventional techniques and / or in accordance with standards and / or recognized pharmaceutical practices.

[0117] The amount of the oligomers of the present invention in any pharmaceutical formulation used according to the present invention will depend on a variety of factors, such as the severity of the condition to be treated, the specific patient to be treated, and the compound used. However, those skilled in the art can routinely determine the content of the oligomers of the present invention in the formulation.

[0118] For example, solid oral dosage forms such as tablets or capsules may contain 1% to 99% (w / w) of the active ingredient; 0% to 99% (w / w) of a diluent or filler; 0% to 20% (w / w) of a disintegrant; 0% to 5% (w / w) of a lubricant; 0% to 5% (w / w) of a flow aid; 0% to 50% (w / w) of a granulator or binder; 0% to 5% (w / w) of an antioxidant; and 0% to 5% (w / w) of a colorant. Controlled-release tablets may also contain 0% to 90% (w / w) of a controlled-release polymer.

[0119] Parenteral preparations (such as solutions or suspensions for injection, or solutions for infusion) may contain 1% to 50% (w / w) of the active ingredient; 50% (w / w) to 99% (w / w) of a liquid or semi-solid carrier or excipient (such as a solvent like water); and 0% to 20% (w / w) of one or more other excipients, such as buffers, antioxidants, suspension stabilizers, tension modifiers, and preservatives.

[0120] Based on the disease to be treated, the patient, and the route of administration, the oligomers of the present invention can be administered to patients in need at different therapeutically effective doses.

[0121] However, in the context of this invention, the dosage administered to mammals (especially humans) should be sufficient to produce a therapeutic response in the mammal within a reasonable timeframe. Those skilled in the art will recognize that the exact dosage and choice of ingredients, as well as the most suitable delivery method, are also significantly influenced by factors such as the pharmacological properties of the formulation, the nature and severity of the condition being treated, the recipient's physical condition and mental acuity, the potency of the specific compound, the patient's age, condition, weight, sex, and response, and the stage / severity of the disease.

[0122] The administration method can be continuous or intermittent (e.g., by injection). The dosage can also be determined based on the time and frequency of administration. In the case of oral or parenteral administration, the dosage of the oligomer of the present invention can vary from about 0.01 mg to about 1000 mg per day.

[0123] In any case, medical practitioners or other professionals will be able to routinely determine the most suitable actual dose for an individual patient. The doses described above are merely examples of average cases; of course, in individual cases, higher or lower dose ranges may be required, which is also within the scope of this invention.

[0124] The various aspects of the invention described herein (e.g., the oligomers, methods, and uses described above) may have the following advantages: when treating the conditions described herein, they may be more convenient for doctors and / or patients, more effective, less toxic, more selective, have a wider range of activities, are more potent, have fewer side effects, or may have other useful pharmacological properties compared to similar compounds, combinations, methods (treatments), or uses known in the art for treating these or other conditions.

[0125] Other oligomers of the present invention can be prepared according to techniques well known to those skilled in the art, for example, as described in the Examples section below.

[0126] The compounds in this invention can be separated from their reaction mixtures using conventional techniques (e.g., recrystallization, column chromatography, preparative HPLC, etc.).

[0127] The oligomers of this invention have significant antibacterial activity, especially against pathogenic Gram-positive and Gram-negative bacteria, and therefore can also be used against bacteria in the skin flora, such as Corynebacterium sicca (…). Corynebacterium xerosis (Bacteria that cause body odor), as well as bacteria that fight yeast and mold. Therefore, they are also suitable for disinfecting skin and mucous membranes, as well as skin appendages (hair), and thus can also be used for disinfecting hands and wounds.

[0128] In view of the above, the oligomers of the present invention can be used as antibacterial active ingredients in personal care preparations such as shampoos, bath additives, hair care products, liquid and solid soaps (based on synthetic surfactants and salts of saturated and / or unsaturated fatty acids), lotions and creams, and other aqueous or alcoholic solutions, such as skin cleansers.

[0129] Therefore, an antibacterial and / or antifungal detergent composition comprising the oligomers and surfactants of the present invention is also provided. It should be understood that the composition may also contain other cosmetically acceptable carriers and / or adjuvants. The composition may in particular be in the form of a shampoo or a solid or liquid soap, but other compositions described above (e.g., other hair care products, lotions, and creams) are also contemplated.

[0130] The detergent composition may contain 0.01% to 15% by weight, for example 0.5% to 10% by weight, of the oligomer of the present invention. It should be understood that the detergent composition may contain more than one oligomer of the present invention.

[0131] Depending on the form of the detergent composition, in addition to the oligomers of the present invention, it may also contain other components such as chelating agents, colorants, fragrance oils, thickeners or curing agents (viscosity regulators), emollients, UV absorbers, skin protectants, antioxidants, and additives that improve mechanical properties (such as dicarboxylic acids and / or C). 14 -C 22 (Al, Zn, Ca and Mg salts of fatty acids), and optional preservatives.

[0132] Detergent compositions can be formulated as water-in-oil or oil-in-water emulsions, alcohol or alcohol-containing preparations, vesicle dispersions of ionic or nonionic amphiphilic lipids, gels, solid rods or aerosols.

[0133] As a water-in-oil or oil-in-water emulsion, the detergent composition may comprise 5% to 50% by weight of an oil phase, 5% to 20% by weight of an emulsifier, and 30% to 90% by weight of water. The oil phase may contain any oil suitable for cosmetic formulations, such as one or more hydrocarbon oils, waxes, natural oils, silicone oils, fatty acid esters, or fatty alcohols. Preferred mono- or polyols include ethanol, isopropanol, propylene glycol, hexanediol, glycerin, and sorbitol.

[0134] Detergent compositions can be provided in a variety of formulations. Suitable examples of compositions include, but are not limited to, skin care formulations (e.g., sheet or liquid soaps, soap-free detergents, or cleansing pastes), bath formulations (such as liquid compositions like foam baths, body washes, shower gels, or solid bath formulations), shaving formulations (such as shaving soaps, foaming shaving creams, non-foaming shaving creams, foams and gels, dry shaving pre-shaving formulations, aftershave lotions, or aftershave emulsions), and cosmetic hair care formulations (such as shampoo and conditioner formulations, hair care formulations such as…). Pretreatment preparations, conditioners, styling creams, styling gels, hair oils, shampoos, conditioning packs, deep conditioning products, hair structuring preparations (such as perming preparations for permanent perming (hot perm, mild perm, cold perm)), straightening preparations, liquid styling preparations, foams, hairsprays, bleaching preparations; such as hydrogen peroxide solutions, shine-enhancing shampoos, bleaching creams, bleaching powders, bleaching creams or oils, temporary, semi-permanent or permanent hair dyes, preparations containing self-oxidizing dyes, or natural hair dyes, such as stylosin or chamomile dyes).

[0135] For example, an antibacterial soap may have the following components: 0.01% by weight to 5% by weight of the oligomers of the present invention; 0.3% to 1% by weight of titanium dioxide; 1% to 10% by weight of stearic acid; and The remainder is soap base, such as sodium salts of tallow fatty acids and coconut fatty acids, or glycerin.

[0136] For example, a shampoo may have the following components: 0.01% by weight to 5% by weight of the oligomers of the present invention; 12.0% by weight of sodium dodecyl ether-2-sulfate; 4.0% by weight of cocamidopropyl betaine; 3.0% by weight of sodium chloride; and Add water to 100% by weight.

[0137] Other aspects and embodiments of the invention will now be discussed through the following non-limiting examples.

[0138] Example

[0139] Material

[0140] All chemicals and solvents were purchased from Fischer Scientific UK, Sigma-Aldrich, Merck Millipore, TCI Chemicals, and BLDpharm, and were not further purified before use.

[0141] Analytical techniques

[0142] Thin-layer chromatography (TLC)

[0143] Merck TLC silicone 60Å F 254 The plates were subjected to TLC. The TLC plates were visualized under ultraviolet light (256 & 366 nm).

[0144] Column chromatography

[0145] Using Davisil ® Column chromatography was performed using LC60A 40-63 micron silica gel (pore size 60 Å, 0.040-0.063 mm).

[0146] Nuclear magnetic resonance (NMR) spectroscopy

[0147] In the Bruker Avance DPX 300 ( 1 H and 13 C NMR at 300 MHz and 75.47 MHz respectively) or Bruker Avance III 400 ( 1 H and 13NMR spectra were recorded at 400.13 MHz and 101.62 MHz. Data processing was performed using TopSpin software (version 4.1.3), which compared the spectra with those obtained with residual solvent. Chemical shifts (δ) are expressed in parts per million (ppm), and coupling constants (δ) are expressed in ppm. J )for 1 H NMR is accurate to 0.01 Hz, for 13 C NMR is accurate to 0.1 Hz, and the following abbreviations are used to indicate peak multiplicity: s, singlet; d, doublet; t, triplet; q, quadruplet; qu, pentaplet; sext, hexaplet; m, multiplet; br, broad peak.

[0148] Mass spectrometry (MS)

[0149] Mass spectrometry analysis was performed on an ABI 4800 Proteomics Analyzer MALDI TOF / TOF mass spectrometer (Applied Biosystems).

[0150] Gel permeation chromatography (GPC)

[0151] Oligomers were characterized using Waters GPC with an aqueous superhydrogel column as the stationary phase and sodium acetate / acetic acid buffer (pH=4.5) as the mobile phase. All samples were dissolved in buffer solution to a final concentration of approximately 1 mg / mL and filtered through a 0.22 µm microfilter prior to analysis.

[0152] X-ray photoelectron spectroscopy (XPS)

[0153] Elemental analysis of anion exchange was achieved using an AXIS Supra spectrometer (Kratos Analytical, UK) equipped with a hemispherical analyzer and a monochromatic Al K-alpha source (1487 eV), performed at 15 mA and 15 kV. XPS spectra were obtained from a 700 × 300 μm... 2 The region was acquired at a 90° exit angle. Full elemental scans and high-resolution scans were performed using through energies of 160 eV and 20 eV, respectively. A bias voltage of 3.1 volts was applied to the sample to neutralize the charge accumulated on the sample surface.

[0154] Example 1. Synthesis of OIM1-6 derivatives

[0155] We synthesized a series of OIMs with precisely controllable molecular weights, suitable for the rigorous characterization required for mechanistic studies. We synthesized nine OIM1-6 derivatives, each containing six imidazole rings (1-9) (Figure 1A), including the parent OIM1-6-CH. These derivatives were used to investigate the effects of various substituents at the C2 or C4 positions of the imidazole moiety on antibacterial efficacy and cytotoxicity. The synthesis of these compounds (including the parent OIM1-6-CH(1)) was achieved through a stepwise synthetic strategy. Methods and chemical characterization of OIMs are as follows: Figure 2 And as described below. The parent compound OIM1-6-CH(1) has a hydrogen (H) on the C2 carbon (Figure 1A).

[0156] General Procedure 1: Alkylation of Imidazole Derivatives (1b-7b and 9b)

[0157] Under ice bath conditions, NaH (3.0 equivalents) was added fractionally to a solution of the desired imidazole derivatives (1a-9a) (1.0 equivalents) dissolved in tetrahydrofuran (THF). The reaction mixture was removed from the ice bath and treated with benzyl (3-bromopropyl)carbamate (1.0 equivalents). The reaction mixture was then heated to 50°C and stirred overnight. After the reaction was complete as indicated by TLC, the reaction mixture was filtered through a diatomaceous earth pad, the filtrate was concentrated onto silica gel under reduced pressure, and purified by silica gel column chromatography eluting with a hexane solution of 50-80% ethyl acetate (EtOAc) to give the desired product. The detailed synthesis procedure of 8b is described below.

[0158] The detailed synthetic steps for each compound (1b to 9b) are as follows: (3-(1 H -imidazol-1-yl)propyl)benzyl carbamate (1b) The title compound was synthesized as follows: using imidazole (1a) (3.00 g, 44.1 mmol) according to the method described in General Procedure 1, a pale yellow oil (8.34 g, 32.2 mmol, 73%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.50 – 7.24 (m, 6H), 7.17 (s, 1H), 6.90 (s, 1H), 5.04 (s, 2H), 3.97 (t, J = 6.9 Hz, 2H), 2.98 (q, J = 6.3 Hz, 2H), 1.84 (p, J = 6.7 Hz, 2H). 13C10 NMR (75 MHz, DMSO-d6) δ 156.1, 137.2, 137.1, 128.3, 127.7, 119.3, 65.3, 43.4, 37.4, 31.0. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 14 H 17 N3O2, m / z Calculated value (M+H) + = 260.1321; Measured value = 260.0104.

[0159] (3-(2-methyl-1) H -imidazol-1-yl)propyl)benzyl carbamate (2b)

[0160] The title compound was synthesized by following the method described in General Procedure 1, using 2-methyl-1 H -Imidazole (2a) (7.45 g, 90.8 mmol) yielded a pale yellow oil (14.8 g, 54.1 mmol, 60%). 1 H NMR (300MHz, DMSO-d6) δ 7.54 – 7.22 (m, 6H), 7.03 (s, 1H), 6.70 (s, 1H), 5.02 (s,2H), 3.85 (t, J = 7.0 Hz, 2H), 2.99 (q, J = 6.2 Hz, 2H), 2.23 (s, 3H), 1.78(p, J = 7.1 Hz, 2H). 13 C10 NMR (75 MHz, DMSO-d6) δ 156.0, 143.4, 136.9, 128.3, 127.7, 127.6, 126.1, 119.3, 65.1, 42.5, 37.1, 30.2, 12.4. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 15 H 19 N3O2, m / z Calculated value (M+H) + = 274.1477; Measured value = 274.0078.

[0161] (3-(2-chloro-1) H -imidazol-1-yl)propyl)benzyl carbamate (3b)

[0162] The title compound was synthesized by following the method described in General Procedure 1, using 2-chloro-1 H-Imidazole (3a) (1.00 g, 9.75 mmol) yielded a pale yellow oil (1.97 g, a mixture of C4-Me and C5-Me regioisomers, 6.71 mmol, 76%). 1 H NMR (400 MHz, DMSO-d6) δ 7.43 – 7.26 (m, 7H), 6.88 (s,1H), 5.02 (s, 2H), 3.94 (t, J = 7.1 Hz, 2H), 3.00 (app. q, J = 6.4 Hz), 1.82(app.qu, J = 6.9 Hz). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.2, 137.2, 130.1, 128.4, 127.8, 127.5, 122.3, 65.3, 43.6, 37.4, 30.0. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 14 H 16 ClN3O2, m / z Calculated value (M+H) + = 294.1004; Measured value = 293.9763.

[0163] (3-(2-(trifluoromethyl)-1 H -imidazol-1-yl)propyl)benzyl carbamate (4b)

[0164] The title compound was synthesized by following the method described in General Procedure 1, using 2-(trifluoromethyl)-1 H -Imidazole (4a) (5.00 g, 36.7 mmol) yielded a pale yellow oil (8.53 g, 26.1 mmol, 71%). 1 HNMR (400 MHz, DMSO-d6) δ 7.57 (s, 1H), 7.46 - 7.26 (m, 6H), 7.10 (s, 2H), 5.04 (s, 2H), 4.13 (t, J = 7.3 Hz, 2H), 3.05 (app. q, J = 6.4 Hz, 2H), 1.90(app.qu, J = 7.0 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 156.2, 137.2, 133.7 (q, J= 38.3 Hz), 128.3, 128.2, 128.0, 127.74, 126.4, 125.0, 119.1 (q, J =268.9 Hz), 65.3, 44.4, 37.4, 30.9. From NMR active F (CF3)-induced splitting. MALDI-TOF (HCCA matrix, reflector mode): for C 15 H 16 F3N3O2, m / z Calculated value (M+H) + = 328.1195; Measured value = 328.0313.

[0165] (3-(4-methyl-1) H -imidazol-1-yl)propyl)benzyl carbamate and (3-(5-methyl-1-yl) H -imidazol-1-yl)propane (5b) Benzyl carbamate as a single mixture.

[0166] The title compound was synthesized by following the method described in General Procedure 1, using 4-methyl-1 H -Imidazole (5a) (5.00 g, 60.9 mmol) yielded a pale yellow oil (11.0 g, a mixture of C4-Me and C5-Me regioisomers, 40.2 mmol, 66%). 1 H NMR (300 MHz, DMSO-d6) δ 7.51 - 7.45 (m, 1H), 7.42 –7.25 (m, 6H), 6.84 & 6.60 (s, 1H), 5.02 (s, 2H), 3.87 (t, J = 6.6 Hz, 2H), 2.97 (dq, J = 13.0, 6.5 Hz, 2H), 2.12 - 2.06 (m, 3H), 1.85 - 1.68 (m, 2H). 13 CNMR (75 MHz, DMSO-d6) δ 156.1, 137.1, 136.5, 136.4, 128.3, 127.7, 126.9, 115.5, 65.2, 43.2, 41.2, 37.4, 30.3, 13.6, 8.6. MALDI-TOF (HCCA matrix, reflector mode): for C 15 H 19 N3O2, m / z Calculated value (M+H) + = 274.1477; Measured value = 274.0112.

[0167] (3-(4-chloro-1) H -imidazol-1-yl)propyl)benzyl carbamate and (3-(5-chloro-1) H -imidazol-1-yl)propyl) Benzyl carbamate as a single mixture (6b)

[0168] The title compound was synthesized as follows: using 4-chloro-1, following the method described in General Procedure 1. H -Imidazole (6a) (1.00 g, 9.75 mmol) yielded an orange gel (1.57 g, a mixture of C4-Me and C5-Me regioisomers, 5.36 mmol, 66%). 1 H NMR (400 MHz, DMSO-d6) δ 7.81 – 7.56 (m, 1H), 7.43 –7.89 (m, 7H), 5.02 (s, 2H), 4.00 – 3.88 (m, 2H), 3.03 – 2.91 (m, 2H), 1.88 –1.77 (m, 2H). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.2, 137.7, 137.1, 136.4, 128.3, 127.8, 127.3, 125.2, 115.3, 65.3, 44.3, 31.9, 37.3, 30.6, 30.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 14 H 16 ClN3O2, m / z Calculated value (M+H) + = 294.1004; Measured value = 293.9888.

[0169] (3-(4-Fluoro-1) H -imidazol-1-yl)propyl)benzyl carbamate and (3-(5-fluoro-1-yl) H -imidazol-1-yl)propyl) Benzyl carbamate as a single mixture (7b)

[0170] The title compound was synthesized by following the method described in General Procedure 1, using 4-fluoro-1 H -Imidazole (7a) (680 mg, 7.90 mmol) yielded a pale yellow gel (1.62 g, a mixture of C4-Me and C5-Me regioisomers, 5.85 mmol, 56%). 1 H NMR (400 MHz, DMSO-d6) δ 7.42 – 7.26 (m, 7H), 6.91 –6.71 (m, 1H), 5.02 (s, 2H), 3.90 (t, J = 6.8 Hz, 2H), 2.96 (app. q, J = 6.4Hz), 1.82 (app.qu,J = 6.8 Hz). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.1, 155.1, 137.1, 130.9, 130.7, 128.3, 127.7, 97.8, 97.4, 65.3, 44.5, 37.3, 30.5. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 14 H 16 FN3O2, m / z Calculated value (M+H) + = 278.1299; Measured value = 277.9967.

[0171] (3-(1 H -benzo[ d Imidazol-1-yl)propyl)benzyl carbamate (8b)

[0172] Benzimidazole (8a) (391 mg, 3.31 mmol) was dissolved in MeCN (20 mL) and treated with a solution of NaOH (551 mg, 13.8 mmol) in water (5 mL). The reaction mixture was stirred at 50 °C for 2 hours. The reaction mixture was treated with a solution of (3-bromopropyl)carbamate (750 mg, 2.76 mmol) in MeCN (5 mL) and stirred at 50 °C for 18 hours. The organic layer (MeCN and water separated) was separated by pipetting, concentrated on silica gel under reduced pressure, and purified by silica gel column chromatography eluting with a solution of 20-50% ethyl acetate in hexane to give a colorless solid (688 mg, 2.22 mmol, 81%). 1 H NMR (400 MHz, DMSO-d6) δ 8.24 – 8.18 (m, 1H), 7.67 – 7.62 (m, 1H), 7.60 – 7.55 (m, 1H), 7.44 – 7.16 (m, 8H), 5.02 (s, 2H), 4.26 (t, J = 6.9 Hz, 2H), 3.00 (q, J = 6.4 Hz, 2H), 1.93 (app. qu, J = 6.9 Hz, 2H). 13C10 NMR (101MHz, DMSO-d6) δ 156.2, 144.0, 143.4, 141.9, 137.1, 133.7, 128.3, 127.8, 127.7, 122.2, 121.4, 119.4, 110.3, 65.3, 41.7, 37.6, 29.6. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 18 H 19 N3O2, m / z Calculated value (M+H) + = 310.1550; Measured value = 310.0454.

[0173] (3-(4-(trifluoromethyl)-1 H -imidazol-1-yl)propyl)benzyl carbamate and (3-(5-(trifluoromethyl)-1 H - Imidazol-1-yl)propyl)benzyl carbamate as a single mixture (9b)

[0174] The title compound was synthesized by following the method described in General Procedure 1, using 4-(trifluoromethyl)-1 H -Imidazole (9a) (5.00 g, 36.7 mmol) was used to give a colorless oil (9.13 g, a mixture of C4-Me and C5-Me regioisomers, 27.9 mmol, 76%). 1 H NMR (400 MHz, DMSO-d6) δ 7.85 (app. s, 2H),7.45 – 7.25 (m, 6H), 5.02 (s, 2H), 4.03 (t, J = 6.8 Hz, 2H), 2.97 (app. qu, J = 6.8 Hz). 13 C NMR (101 MHz, DMSO-d6) δ 156.2, 139.1, 137.1, 130.0 (q, J =37.7 Hz), 128.3, 128.0, 127.7, 126.6, 126.4, 122.1 (q, J = 266.4 Hz),120.44, 120.41, 65.3, 44.1, 37.2, 30.6. From NMR active F (CF3)-induced splitting. MALDI-TOF (HCCA matrix, reflector mode): for C 15 H 16 F3N3O2, m / z Calculated value (M+H)+ = 328.1195; Measured value = 328.0278.

[0175] General Procedure 2: Alkylation Steps of Compounds 1c-9c

[0176] Under argon protection, 1,4-dibromobutane (2.5 equivalents) was added to a stirred solution of the desired starting material (1b-9b) (1.0 equivalents) in anhydrous MeCN (1 mmol / mL). The reaction mixture was heated under reflux overnight and then cooled to room temperature. The reaction mixture was concentrated by rotary evaporation and purified by silica gel chromatography eluting with 0-15% methanol in ethyl acetate to give the desired alkylated brominated product.

[0177] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-1 H -Imidazole-3-onium bromide (1c)

[0178] The title compound was synthesized as follows: using 1b (3.00 g, 11.5 mmol) according to the method described in General Procedure 2, a colorless gel (4.12 g, 8.67 mmol, 75%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ9.39 (s, 1H), 7.88 (d, J = 3.4 Hz, 2H), 7.58 – 7.21 (m, 6H), 5.02 (s, 2H), 4.24 (q, J = 7.2 Hz, 4H), 3.56 (t, J = 6.4 Hz, 2H), 3.02 (q, J = 6.0 Hz, 2H), 2.05 – 1.86 (m, 4H), 1.86 – 1.72 (m, 2H). 13 C10 NMR (75 MHz, DMSO-d6) δ 156.2, 137.0, 136.2, 128.3, 127.76, 127.70, 122.45, 122.40, 65.3, 47.9, 46.5, 36.9, 34.1, 29.7, 28.7, 28.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 18 H 25 Br2N3O2, m / z Calculated value (M-Br) + = 394.1125; Measured value = 394.1405.

[0179] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-2-methyl-1 H -Imidazole-3-onium bromide （2c）

[0180] The title compound was synthesized as follows: using 2b (2.03 g, 7.42 mmol) according to the method described in General Procedure 2, a colorless gel (2.56 g, 5.23 mmol, 70%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ7.73 (s, 2H), 7.53 – 7.20 (m, 6H), 5.03 (s, 2H), 4.17-4.10 (m, 4H), 3.68 –3.46 (m, 2H), 3.05 (q, J = 6.0 Hz, 2H), 2.60 (s, 3H), 1.96-1.83 (m, 6H). 13 CNMR (75 MHz, DMSO-d6) δ 156.1, 143.9, 143.8, 137.0, 121.25, 121.21, 121.1, 65.3, 47.4, 46.6, 45.1, 37.1, 34.1, 28.9, 27.7, 25.9, 9.2. MALDI-TOF (HCCA matrix, reflector mode): for C 19 H 27 Br2N3O2, m / z Calculated value (M-Br) + = 408.1281; Measured value = 408.1255.

[0181] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-2-chloro-1H-imidazol-3-onium bromide （3c）

[0182] The title compound was synthesized as follows: following the method described in General Procedure 2, using 3b (1.50 g, 5.10 mmol), an orange gel (1.99 g, 3.90 mmol, 76%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ8.03 (s, 1H), 7.97 (s, 1H), 7.48 – 7.27 (m, 6H), 5.02 (s, 2H), 4.26-4.10 (m,4H), 3.57 (t, J = 6.2 Hz, 2H), 3.05 (q, J = 6.0 Hz, 2H), 2.00-1.79 (m, 6H). 13CNMR (101 MHz, DMSO-d6) δ 156.2, 137.10, 137.05, 131.2, 128.4, 127.9, 127.8, 124.1, 122.84, 122.81, 65.4, 49.0, 47.9, 46.4, 37.0, 34.2, 28.8, 28.7, 28.5, 27.4, 27.2. MALDI-TOF (HCCA matrix, reflector mode): for C 18 H 24 ClBrN3O2, m / z Calculated value (M-Br) + =428.0735; Measured value = 427.9606.

[0183] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-2-(trifluoromethyl)-1 H -imidazolium-3-onium Bromide (4c)

[0184] The title compound was synthesized as follows: using 4b (5.00 g, 15.3 mmol) according to the method described in General Procedure 2, a pale yellow oil (5.52 g, 10.2 mmol, 66%) was obtained. 1 H NMR (300 MHz, CDCl3): δ8.67 (s, 1H), 8.53 (s, 1H), 7.36 – 7.15 (m, 5H), 6.43 (br s, 1H), 4.99 (s,2H), 4.61 – 4.39 (m, 4H), 3.42 – 3.31 (m, 2H), 3.30 – 3.17 (m, 2H), 2.21 –1.85 (m, 6H). 13 C10 NMR (75 MHz, CDCl3): δ 157.9, 136.7, 128.5, 128.0, 127.9, 126.7, 66.5, 50.9, 50.4, 49.4, 37.5, 32.4, 30.4, 29.2, 29.0. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 19 H 24 F3Br2N3O2, m / z Calculated value (M-Br) + = 462.0999; Measured value = 462.0367.

[0185] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-methyl-1 H -Imidazole-3-onium bromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-methyl-1 H -Imidazole-3-onium bromide as a mono- A mixture (5c)

[0186] The title compound was synthesized as follows: using 5b (2.01 g, 7.35 mmol) according to the method described in General Procedure 2, a deep red gel (2.27 g, a mixture of C4-Me and C5-Me regioisomers, 4.64 mmol, 63%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.59 (s, 1H), 7.56 – 7.06 (m, 6H), 5.02 (s, 2H), 4.16 (t, J = 6.9 Hz, 4H), 3.57 (q, J = 6.8 Hz, 2H),3.13 – 2.93 (m, 2H), 2.28 (d, J = 9.8 Hz, 3H), 1.97 – 1.68 (m, 6H). 13 C10 NMR (75MHz, DMSO-d6) δ 156.2, 137.0, 135.7, 135.6, 130.8, 130.7, 128.3, 127.8, 127.7, 119.3, 119.2, 65.3, 48.5, 47.8, 46.4, 45.3, 43.9, 37.0, 34.1, 29.6, 28.7, 27.9, 27.4, 14.0, 8.6. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 19 H 27 Br2N3O2, m / z Calculated value (M-Br) + = 408.1281; Measured value = 408.1255.

[0187] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-4-chloro-1 H -Imidazole-3-onium bromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-chloro-1 H -Imidazole-3-onium bromide as a single mixture Compound (6c)

[0188] The title compound was synthesized as follows: using 6b (3.65 g, 12.5 mmol) according to the method described in General Procedure 2, a pale yellow oil (3.70 g, 7.27 mmol, 58%) was obtained. 1H NMR (400 MHz, DMSO-d6): δ9.38 (s, 1H), 8.15 (s, 1H), 7.45 – 7.28 (m, 6H), 5.02 (s, 2H), 4.25 – 4.12(m, 4H), 3.61 – 3.53 (m, 2H), 3.11 – 3.00 (m, 2H), 2.21 – 1.85 (m, 6H). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.2, 137.0, 136.9, 128.3, 127.8, 127.7, 120.5, 119.9, 65.4, 47.6, 46.2, 36.9, 34.1, 29.3, 28.7, 27.2. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 18 H 24 Br2ClN3O2, m / z Calculated value (M-Br) + = 428.0735; Measured value = 427.9743.

[0189] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-4-fluoro-1 H -Imidazole-3-onium bromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-fluoro-1 H -Imidazole-3-onium bromide as a single mixture Compound (7c)

[0190] The title compound was synthesized as follows: using 7b (1.03 g, 3.72 mmol) according to the method described in General Procedure 2, a pale yellow oil (668 mg, 1.35 mmol, 36%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ9.15 – 9.03 (m, 1H), 7.90 – 7.75 (m, 1H), 7.48 – 7.27 (m, 6H), 5.03 (s, 2H), 4.23 – 4.13 (m, 4H), 3.57 (t, J = 6.2 Hz, 2H), 3.04 (app q, J = 6.3 Hz, 2H),2.01 – 1.76 (m, 6H). 13C10 NMR (101 MHz, DMSO-d6) δ 156.2, 137.0, 131.1, 128.3, 127.8, 127.7, 102.9, 102.6, 65.4, 47.9, 44.9, 36.9, 34.0, 30.6, 29.3, 28.7, 27.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 18 H 24 FBr2N3O2, m / z Calculated value (M-Br) + =412.1030; Measured value = 412.0259.

[0191] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-1 H -benzo[ d Imidazole-3-onium bromide （8c）

[0192] The title compound was synthesized as follows: using 8b (675 mg, 2.18 mmol) according to the method described in General Procedure 2, a pale yellow oil (716 mg, 1.36 mmol, 62%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ9.89 – 9.81 (m, 1H), 8.16 – 8.03 (m, 2H), 7.74 – 7.66 (m, 2H), 7.48 – 7.25(m, 6H), 5.01 (s, 2H), 4.58 – 4.48 (m, 4H), 3.63 – 3.56 (m, 2H), 3.12 (app.q, J = 6.3 Hz, 2H), 2.13 – 2.00 (m, 4H), 1.94 – 1.86 (m, 2H). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.2, 142.3, 137.0, 131.11, 131.05, 128.3, 127.8, 127.7, 126.5, 113.6, 65.3, 45.8, 44.5, 37.3, 34.2, 28.9, 28.7, 27.3. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 22 H 27 Br2N3O2, m / z Calculated value (M-Br) + = 444.0214; Measured value = 444.1281.

[0193] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-4-(trifluoromethyl)-1 H -imidazolium-3-oniumBromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-(trifluoromethyl)-1 H -imidazolium-3-onium Bromides as a single mixture (9c)

[0194] The title compound was synthesized as follows: using 9b (4.50 g, 13.7 mmol) according to the method described in General Procedure 2, a pale yellow oil (6.08 g, 11.2 mmol, 85%) was obtained. 1 H NMR (300 MHz, CDCl3) δ10.72 (s, 1H), 8.15 (s, 1H), 7.41 – 7.33 (m, 5H), 6.47 (br s, 1H), 5.06 (s,2H), 4.53 – 4.41 (m, 4H), 3.31 – 3.23 (m, 2H), 2.22 – 1.98 (m, 8H). 13 C10 NMR (75MHz, CDCl3) δ 157.14, 136.5, 128.5, 127.9, 66.7, 50.7, 48.8, 37.3, 32.2, 30.1, 29.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 19 H 24 F3Br2N3O2, m / z Calculated value (M-Br) + =462.0999; Measured value = 462.0346.

[0195] General Procedure 3: Alkylation Steps of Bisimilar Compounds from Day 1 to Day 9

[0196] The desired imidazole analogues (1a-9a) (1.0 equivalent) were dissolved in THF (0.5 mmol / mL) and stirred in an ice bath for 10 min. NaH (2.5 equivalent) was added in portions to the reaction mixture, which was then removed from the ice bath and stirred for 1 h. 1,4-Dibromobutane (0.5 equivalent) was added, and the reaction mixture was stirred overnight at 50 °C. The resulting mixture was cooled to room temperature and filtered through a diatomaceous earth filter, washed with THF. The filtrate was dried by rotary evaporation and then dissolved in methanol. For 1d, 2d, and 5d: the methanol layer was washed three times with hexane and concentrated by rotary evaporation; no further purification was required to obtain the desired product. For 3d-4d and 6d-9d: the methanol layer was washed three times with hexane, concentrated by rotary evaporation, and purified by silica gel column chromatography eluting with a hexane solution of 33-100% EtOAc to obtain the desired product.

[0197] 1,4-double(1 H -imidazol-1-yl)butane (1d)

[0198] The title compound was synthesized as follows: using imidazole (1a) (8.00 g, 117.5 mmol) according to the method described in General Procedure 3, to give a colorless solid (10.2 g, 53.6 mmol, 91%). 1 H NMR (300 MHz, DMSO-d6) δ 7.61 (s, 2H), 7.14 (br s, 2H), 6.89 (br s, 2H), 3.98 – 3.73 (m, 4H), 1.64 – 1.59 (m, 4H). 13 C10 NMR (101 MHz, DMSO-d6) δ 137.27, 128.45, 119.31, 45.34, 27.73. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 10 H 14 N4, m / z Calculated value (M+H) + =191.1219; Measured value = 191.1296.

[0199] 1,4-Bis(2-methyl-1 H -imidazol-1-yl)butane(2d)

[0200] The title compound was synthesized by following the method described in General Procedure 3, using 2-methyl-1 H -Imidazole (2a) (10.0 g, 122 mmol) was used to give a colorless solid (11.8 g, 54.1 mmol, 89%). 1 H NMR (300 MHz, DMSO-d6) δ 7.01 (s, 2H), 6.71 (s, 2H), 3.86 (s, 4H), 2.25 (s, 6H), 1.61 (s, 4H). 13 C10 NMR (75 MHz, DMSO-d6) δ 143.8, 125.8, 119.6, 44.6, 27.1, 12.2. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 12 H 18 N4, m / z Calculated value (M+H) + = 219.1532; Measured value = 219.1009.

[0201] 1,4-Bis(2-chloro-1) H -imidazol-1-yl)butane (3d)

[0202] The title compound was synthesized as follows: Following the method described in General Procedure 3, 2-chloro-1 H-Imidazole (3a) (1.00 g, 9.75 mmol) was used to give a colorless solid (615 mg, 2.37 mmol, 49%). 1 H NMR (400 MHz, CDCl3) δ 6.97 (d, J = 1.2 Hz, 2H), 6.87 (d, J = 1.3 Hz, 2H), 3.97 – 3.90 (m, 4H), 1.81 – 1.74 (m, 4H). 13 C10 NMR (101 MHz, CDCl3) δ 131.9, 128.8, 120.7, 46.0, 27.3. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 10 H 12 Cl2N4, m / z Calculated value (M+H) + = 259.0512; Measured value = 258.9474.

[0203] 1,4-Bis(2-(trifluoromethyl)-1 H -imidazol-1-yl)butane (4d)

[0204] The title compound was synthesized by following the method described in General Procedure 3, using 2-(trifluoromethyl)-1 H -Imidazole (4a) (20.0 g, 146.9 mmol) yielded an orange oil (15.8 g, 48.5 mmol, 66%). 1 HNMR (300 MHz, CDCl3) δ 7.11 (s, 2H), 7.02 (s, 2H), 4.11 (s, 4H), 1.85 (s, 4H). 13 C10 NMR (75 MHz, CDCl3) δ 129.2, 122.9, 28.0, 46.5. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 12 H 12 F6N4, m / z Calculated value (M+H) + = 327.0966; Measured value = 327.0123.

[0205] 1,4-Bis(4-methyl-1 H -imidazol-1-yl)butane and the imidazol-CH3 associated regioisomers as a single mixture Compound (5d)

[0206] The title compound was synthesized as follows: following the method described in General Procedure 3, using 4-methyl-1 H-Imidazole (5a) (10.0 g, 122 mmol) was used to give a colorless solid (12.6 g, 57.7 mmol, 95%). 1 H NMR (300 MHz, DMSO-d6) δ 7.51 – 7.46 (m, 2H), 6.85 – 6.58 (m, 2H) 3.97 – 3.76 (m, 4H), 2.19 – 1.99 (m, 6H), 1.70 – 1.49 (m, 4H). 13 C10 NMR (75 MHz, DMSO-d6) δ 136.9, 136.3, 126.6, 125.9, 115.4, 45.2, 43.1, 27.6, 27.1, 13.5, 8.6. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 12 H 18 N4, m / z Calculated value (M+H) + = 219.1532; Measured value = 219.1009.

[0207] 1,4-Bis(4-chloro-1) H -imidazol-1-yl)butane and imidazol-Cl related regioisomers as a single mixture Object (6d)

[0208] The title compound was synthesized as follows: following the method described in General Procedure 3, using 4-chloro-1 H -Imidazole (6a) (3.00 g, 29.3 mmol) yielded a deep orange oil (2.92 g, 11.3 mmol, 77%). 1 H NMR (400MHz, DMSO-d6) δ 7.84 – 7.54 (m, 2H), 7.30 – 6.92 (m, 2H), 4.03 – 3.86 (m,4H), 1.72 – 1.54 (m, 4H). 13 C10 NMR (101 MHz, DMSO-d6) δ 136.3, 127.4, 125.0, 115.3, 46.1, 43.7, 27.2, 26.7. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 10 H 12 Cl2N4, m / z Calculated value (M+H) + = 259.0512; Measured value = 258.9255.

[0209] 1,4-Bis(4-fluoro-1) H -imidazol-1-yl)butane and imidazol-F associated regioisomers as a single mixture （7d）

[0210] The title compound was synthesized by following the method described in General Procedure 3, using 4-fluoro-1 H -Imidazole (7a) (1.74 g, 20.2 mmol) yielded a pale yellow solid (1.10 g, 4.84 mmol, 48%). 1 H NMR (400 MHz, DMSO-d6) δ 7.33 (br s, 2H), 6.86 (dd, J = 8.3 Hz, J = 1.7 Hz, 2H), 3.90 (t, J = 5.9 Hz, 4H), 1.62 (app. qu, J = 3.3 Hz, 4H). 13 C10 NMR (101 MHz, DMSO-d6) δ 157.4, 155.1, 130.8, 130.6, 97.8, 97.3, 46.3, 27.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 10 H 12 F2N4, m / z Calculated value (M+H) + = 227.1103; Measured value = 226.9815.

[0211] 1,4-bi( 1H- Benzo[ d Imidazol-1-yl)butane (8d)

[0212] Benzimidazole (8a) (4.10 g, 34.7 mmol) was dissolved in MeCN (80 mL) and treated with a solution of NaOH (5.56 g, 139 mmol) in water (20 mL). The reaction mixture was stirred at 50 °C for 2 hours. The reaction mixture was then treated with a solution of 1,4-dibromobutane (3.00 g, 13.9 mmol) in MeCN (10 mL), and the reaction mixture was stirred at 50 °C for 18 hours. The resulting suspension was washed with water and filtered, and the solid was dried to give a colorless solid (3.15 g, 10.8 mmol, 78%). 1 HNMR (400 MHz, DMSO-d6) δ 8.21 (s, 2H), 7.64 (d, J = 7.6 Hz, 2H), 7.57 (d, J =7.8 Hz, 2H), 7.64 (app. qu, J= 8.3 Hz, 4H), 4.27 (br s, 4H), §.78 (br s,4H). 13 C10 NMR (101 MHz, DMSO-d6) δ 143.9, 143.4, 133.7, 122.2, 121.4, 119.4, 110.4, 43.5, 26.7. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 18 H 18 N4, m / z Calculated value (M+H) + =291.1604; Measured value =291.0451.

[0213] 1,4-Bis(4-(trifluoromethyl)-1 H -imidazol-1-yl)butane and imidazol-CF3 related regioisomers as Single mixture (9d)

[0214] The title compound was synthesized by following the method described in General Procedure 3, using 4-(trifluoromethyl)-1 H -Imidazole (9a) (16.4 g, 100 mmol) yielded an orange oil (11.8 g, 36.2 mmol, 72%). 1 H NMR (300 MHz, MeOD) δ 7.41 – 7.89 (m, 4H), 4.06 – 4.12 (m, 4H), 1.76 (s, 4H). 13 CNMR (CDCl3, 75 MHz) δ 139.5, 120.8, 46.3, 27.7. MALDI-TOF (HCCA matrix, reflector mode): for C 12 H 12 F6N4, m / z Calculated value (M+H) + = 327.0966; Measured value = 327.0175.

[0215] General Procedure 4: Alkylation Steps for Compounds 1e-9e

[0216] At 80°C, the desired brominated starting materials 1c-9c (1 equivalent) were dissolved in anhydrous acetonitrile (0.5 mmol / mL), and the resulting product was added dropwise to the desired diimidazole starting materials 1d-9d (2.5 equivalents) dissolved in anhydrous MeCN (2 mmol / mL), which were being stirred. The resulting mixture was stirred under reflux overnight for several days. 1After the reaction was completed as monitored by ¹H NMR, the reaction mixture was concentrated by rotary evaporation. The residue was treated with a 1:1 mixture of water and chloroform:isopropanol (3:1) and transferred to a separatory funnel. The organic layer was removed, and the aqueous layer was washed five times with chloroform:isopropanol (3:1). The aqueous layer was concentrated under reduced pressure to obtain the desired product.

[0217] 1-(4-(1 H -imidazol-1-yl)butyl)-3-(4-(1-(3-(((benzyloxy)carbonyl)amino)propyl)-1 H -mi Azolium-3-onthium-3-yl)butyl)-1 H -Imidazole-3-onium dibromide (1e)

[0218] The title compound was synthesized as follows: using 1c (3.00 g, 6.34 mmol) according to the method described in General Procedure 4, a colorless hygroscopic gel (3.07 g, 4.61 mmol, 73%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 9.45 – 9.29 (m, 2H), 7.88 – 7.78 (m, 4H), 7.72 (s, 1H), 7.48 – 7.28 (m,6H), 7.20 (s, 1H), 6.92 (s, 1H), 5.02 (s, 2H), 4.20 (t, J = 6.9 Hz, 8H), 4.02(t, J = 6.5 Hz, 2H), 3.05-2.99 (m, 2H), 2.02 – 1.88 (m, 2H), 1.80-1.72 (m, 8H). 13 C10 NMR (75 MHz, DMSO-d6) δ 155.0, 136.0, 135.8, 135.1, 134.7, 127.2, 127.0, 126.68, 126.60, 121.3, 121.2, 118.1, 64.0, 47.0, 46.9, 45.4, 44.0, 43.8, 35.7, 28.5, 26.0, 25.3, 24.8. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 28 H 39 Br2N7O2, m / z Calculated value (M-2Br-H) + = 504.3082; Measured value = 504.3814.

[0219] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-2-methyl-3-(4-(2-methyl-1-(4-(2-methyl-1-methyl)) H -mi (azol-1-yl)butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -Imidazole-3-onium dibromide (2e)

[0220] The title compound was synthesized as follows: using 2c (2.50 g, 5.11 mmol) according to the method described in General Procedure 4, a colorless hygroscopic gel (2.45 g, 3.46 mmol, 68%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 7.88 – 7.66 (m, 4H), 7.52 – 7.23 (m, 6H), 7.16 (s, 1H), 6.87 (s, 1H), 5.03 (s, 2H), 4.21 – 4.11 (m, 8H), 3.96 (t, J = 6.6 Hz, 2H), 3.12 – 2.96 (m,2H), 2.68 – 2.60 (m, 6H), 2.29 (s, 3H), 1.91 (t, J = 6.6 Hz, 2H), 1.81 – 1.56(m, 8H). 13 C10 NMR (75 MHz, DMSO-d6) δ 154.7, 142.6, 142.5, 142.2, 135.6, 126.9, 126.4, 126.3, 123.4, 119.8, 119.8, 118.5, 63.9, 47.1, 45.6, 45.4, 43.7, 43.3, 35.7, 27.6, 25.2, 24.65, 24.3, 24.3, 10.9, 8.0, 7.9. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 31 H 45 Br2N7O2, m / z Calculated value (M-2Br-H) + = 546.3551; Measured value = 546.3480.

[0221] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-2-chloro-3-(4-(2-chloro-1-(4-(2-chloro-1) H -imidazole-1- (butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -Imidazole-3-onium dibromide (3e)

[0222] The title compound was synthesized as follows: using 3c (300 mg, 0.588 mmol) according to the method described in General Procedure 4, a pale yellow gel (375 mg, 0.488 mmol, 83%) was obtained. 1H NMR (400 MHz, DMSO-d6,) δ 8.13 – 7.93 (m, 4H), 7.49 – 7.2 (m, 7H), 6.93 (s, 1H), 5.01 (s, 2H), 4.29 – 4.11 (m, 8H), 4.06 – 3.93 (m, 2H), 3.12 – 2.99 (m, 2H), 2.02 – 1.68 (m, 10H). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.3, 137.1, 131.2, 131.1, 130.1, 128.4, 127.9, 127.8, 127.3, 122.9, 122.8, 122.4, 65.4, 48.2, 48.0, 46.4, 45.3, 44.7, 37.0, 28.5, 26.3, 25.4, 25.03, 25.00. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 28 H 36 Br2Cl3N7O2, m / z Calculated value (M-2Br-Cl+O) + = 588.2251; Measured value = 558.1045.7.

[0223] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-2-(trifluoromethyl)-3-(4-(2-(trifluoromethyl)-1-(4- (2-(trifluoromethyl)-1 H -imidazol-1-yl)butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -Imidazole-3-onium dibromide （4e）

[0224] The title compound was synthesized as follows: using 4c (5.00 g, 9.20 mmol) according to the method described in General Procedure 4, a pale yellow hygroscopic gel (4.92 g, 5.64 mmol, 61%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 8.35 – 8.15 (m, 4H), 7.60 – 7.11 (m, 7H), 5.02 (s, 2H), 4.50 –4.30 (m, 8H), 4.19 – 4.11 (m, 2H), 3.16 – 3.11 (m, 3H), 1.99 – 1.82 (m, 10H). 13 C10 NMR (75 MHz, DMSO-d6) δ 156.7, 128.8, 128.3, 128.2, 126.2, 65.8, 50.3, 49.0, 46.5, 27.4, 26.6. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR...31 H 36 Br2F9N7O2, m / z Calculated value (M-2Br-H) + =708.2703; Measured value = 708.2897.

[0225] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-5-methyl-3-(4-(4-methyl-1-(4-(5-methyl-1) H -mi (azol-1-yl)butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -Imidazole-3-onium dibromide and the imidazole-CH3 group are associated. Regioisomers as a single mixture (5e)

[0226] The title compound was synthesized as follows: using 5c ​​(2.50 g, 5.11 mmol) according to the method described in General Procedure 4, a deep red hygroscopic gel (2.15 g, 3.04 mmol, 59%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 9.32-9.28 (m, 2H), 7.62-7.58 (m, 3H), 7.47-7.45 (m, 1H), 7.40 –7.29 (m, 5H), 6.93 – 6.62 (m, 1H), 5.02 (s, 2H), 4.18-4.16 (m, 8H), 3.95-3.93(m, 2H), 3.05-2.98 (m, 2H), 2.28 (d, J = 8.3 Hz, 6H), 2.15-2.07 (m, 3H), 1.98– 1.91 (m, 2H), 1.81-1.70 (m, 8H). 13 C NMR (75 MHz, DMSO-d6) δ 156.1, 137.0,136.8, 136.3, 135.7, 137.6, 137.5, 130.8, 128.3, 127.8, 127.7, 125.6, 119.3,115.6, 65.3, 47.9, 46.4, 45.6, 45.5, 45.5, 40.3, 40.0, 39.7, 39.4, 39.2,38.9, 38.6, 36.9, 29.6, 27.1, 26., 25.8, 25.6, 25.3, 13.5, 8.6. MALDI-TOF (HCCA matrix, reflector mode): For C 31 H 45 Br2N7O2, m / z Calculated value (M-2Br-H) + = 546.3551; Measured value = 546.3480.

[0227] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-4-chloro-3-(4-(5-chloro-1-(4-(4-chloro-1) H -imidazole-1-(butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -Imidazole-3-onium dibromide and the regioisotropic association of imidazole-Cl The configuration is a single mixture (6e)

[0228] The title compound was synthesized as follows: using 6c (566 mg, 1.11 mmol) according to the method described in General Procedure 4, an orange gel (1.96 g, 0.941 mmol, 85%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ9.73 – 9.44 (m, 2H), 8.28 – 8.09 (m, 2H), 7.65 (s, 1H), 7.50 – 7.23 (m, 7H), 5.02 (s, 2H), 4.31 – 4.14 (m, 8H), 4.07 – 3.94 (m, 2H), 3.11 – 2.99 (m, 2H), 2.03 – 1.94 (m, 2H), 1.92 – 1.67 (m, 8H). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.3, 137.0, 136.9, 136.7, 136.4, 128.4, 127.8, 127.7, 120.6, 119.8, 115.3, 65.4, 49.1, 47.7, 46.5, 46.3, 46.0, 36.9, 29.4, 26.7, 26.0, 25.5, 25.4, 25.1, 25.0. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 28 H 36 Br2Cl3N7O2, m / z Calculated value (M-2Br-H) + =606.1912; Measured value = 606.1226.

[0229] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-4-fluoro-3-(4-(5-fluoro-1-(4-(4-fluoro-1) H -imidazole-1- (butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -Imidazole-3-onium dibromide and imidazole-F-related regioisomerism The body is a single mixture (7e)

[0230] The title compound was synthesized as follows: using 7c (656 mg, 1.33 mmol) according to the method described in General Procedure 4, a pale yellow hygroscopic gel (677 mg, 0.941 mmol, 71%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 9.26 – 9.10 (m, 2H), 8.01 – 7.78 (m, 3H), 7.49 – 7.28 (m, 6H), 6.89 (d, J= 8.2 Hz, 1H), 5.02 (s, 2H), 4.27 – 4.14 (m, 8H), 4.01 – 3.90 (m,2H), 3.09 – 2.99 (m, 2H), 1.91 – 1.63 (m, 10H). 13 C10 NMR (101 MHz, DMSO-d6) δ 156.2, 155.1, 147.4, 144.8, 137.0, 131.2, 130.9, 130.8, 130.7, 128.9, 128.3, 127.8, 127.7, 102.9, 102.8, 102.6, 97.8, 97.4, 65.4, 49.4, 49.2, 47.9, 46.1, 45.0, 36.8, 29.3, 26.7, 26.5, 25.9, 25.5, 25.0, 24.8. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 28 H 36 Br2F3N7O2, m / z Calculated value (M-2Br-H) + = 558.2800; Measured value = 558.2171.

[0231] 3-(4-(1 H -benzo[ d [Imidazol-1-yl)butyl)-1-(4-(3-(3-(((benzyloxy)carbonyl)amino)propyl)- 1 H -benzo[ d [Imidazol-3-onthium-1-yl)butyl)-1 H -benzo[ d Imidazole-3-onium dibromide (8e)

[0232] The title compound was synthesized as follows: using 8c (672 mg, 1.28 mmol) according to the method described in General Procedure 4, to obtain a colorless solid (753 mg, 0.923 mmol, 72%). 1 H NMR (400 MHz, DMSO-d6) δ10.27 – 9.78 (m, 2H), 8.82 (br s, 1H), 8.21 – 7.93 (m, 5H), 7.85 – 7.62 (m,6H), 7.50 – 7.42 (m, 1H), 7.41 – 7.25 (m, 6H), 4.98 (s, 2H), 4.63 – 4.49 (m,8H), 4.46 – 4.38 (m, 2H), 3.14 – 3.05 (m, 2H), 2.13 – 1.90 (m, 10H). 13C10 NMR (101 MHz, DMSO-d6) δ 156.2, 142.3, 142.2, 137.0, 131.0, 128.3, 127.8, 127.7, 126.5, 113.7, 65.3, 46.1, 44.5, 30.7, 26.0, 25.7, 25.4. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 40 H 45 Br2N7O2, m / z Calculated value (M-2Br-H) + = 654.3551; Measured value = 654.2896.

[0233] 1-(3-(((benzyloxy)carbonyl)amino)propyl)-4-(trifluoromethyl)-3-(4-(5-(trifluoromethyl)-1-(4- (4-(trifluoromethyl)-1 H -imidazol-1-yl)butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -Imidazole-3-onium dibromide Regioisomers associated with imidazolium-CF3 as a single mixture (9e)

[0234] The title compound was synthesized as follows: using 9c (5.85 g, 10.8 mmol) according to the method described in General Procedure 4, a pale yellow hygroscopic gel (6.00 g, 6.90 mmol, 64%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 9.89 – 9.75 (m, 2H), 8.77 (s, 2H), 7.90 (s, 2H), 7.36 – 7.45 (m, 6H), 5.03 (s, 2H), 4.40 – 4.20 (m, 8H), 4.17 – 4.05 (m, 2H), 3.12 – 3.03 (m, 2H), 1.91 – 1.81 (m, 10H). 13 C10 NMR (75 MHz, DMSO-d6) δ 156.7, 141.3, 139.6, 127.5, 128.8, 128.2, 126.2, 120.9, 65.9, 49.0, 48.4, 48.1, 46.2, 37.3, 29.8, 27.2, 26.3, 25.7. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 31 H 36 Br2F9N7O2, m / z Calculated value (M-2Br-H) + = 708.2703; Measured value = 708.2830.

[0235] General Procedure 5: Synthesis of Final Compounds 1, 2, 5 and 6

[0236] At room temperature, add 1,4-diiodobutane (0.5 equivalents) to a stirred solution of the desired starting materials 1e, 2e, 5e, and 6e (1.0 equivalents) in MeCN:DMF (9.5:0.5). Heat the reaction mixture to 90°C and maintain for 48 hours (1e, 2e, 5e) to 1 week (6e). Allow the reaction mixture to cool to room temperature. Separate the resulting gel, further mill three times with MeCN, and treat with a 33% (10 equivalents) solution of HBr in acetic acid, stirring the mixture overnight at room temperature. Treat the reaction mixture with excess EtOAc, separating the resulting precipitate / gel. Further mill the precipitate / gel with EtOAc, dissolve in water to a final concentration of 50-60 mM, and pass through an ion exchange resin containing Amberlyst. ® A-26 glass column is used to exchange countercharged ions for chloride ions. (Preparation of ion exchange resin column: 1 M HCl aqueous solution is passed through a column filled with Amberlyst...) ® A-26 (OH– form) glass column was used until the pH of the eluent reached the same value as the original solution, and then the resin was washed with water until the pH reached neutral. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 500-1000 Da, and dialyzed with 5 mL HCl in 5 L deionized water for 24 hours, changing the dialysate frequently every 2-3 hours. The resulting solution was concentrated by rotary evaporation and lyophilized to obtain the desired product.

[0237] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-1 H -imidazol-3-on-3-yl)butane (base)-1 H -imidazol-3-on-1-yl)butyl)-1 H 3-Imidazol-3-onium)octachloride (OIM1-6-CH,1)

[0238] The title compound was synthesized as follows: using 1e (1.00 g, 1.50 mmol) according to the method described in General Procedure 5, a pale brown hygroscopic solid (474 ​​mg, 0.443 mmol, 59%) was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 9.67 (br s, 6H), 8.59 (br s, 6H), 7.95 (br s, 12H), 4.70 – 4.05(m, 24H), 2.81 (br s, 4H), 2.24 (br s, 4H), 1.86 (br s, 20H). 13 C10 NMR (75 MHz, DMSO-d6) δ 136.4, 136.3, 122.5, 122.4, 48.0, 45.9, 35.4, 27.3, 25.9. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 44 H 76 Cl8N 14, m / z Calculated value (M-7H-8Cl) + = 793.5824; Measured value = 793.5392. GPC (aqueous phase) M n = 1025, M w = 1037, M p = 1044, PDI = 1.01. Figure 3 )

[0239] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-2-methyl-1 H -imidazol-3-on- 3-yl)butyl)-2-methyl-1 H -imidazol-3-onth-1-yl)butyl)-2-methyl-1 H -imidazolium-3-onium)octachloride (OIM1- 6-C2(CH3), 2)

[0240] The title compound was synthesized as follows: using 2e (1.50 g, 1.28 mmol) according to the method described in General Procedure 5, a pale brown hygroscopic solid (270 mg, 0.231 mmol, 36%) was obtained. 1 ¹H NMR (300 MHz, D₂O) δ 7.47 – 6.98 (m, 12H), 4.24 – 3.88 (m, 24H), 3.11 – 2.81 (m, 4H), 2.62 – 2.38 (18H), 2.20 – 1.93 (m, 4H), 1.75 (br s, 20H). 2 x -NH₃ was not observed due to deuterium exchange. + . 13 C10 NMR (75 MHz, D2O) δ 144.1, 143.9, 121.7, 121.1, 121.0, 120.9, 47.4, 47.3, 45.0, 45.0, 36.4, 26.8, 26.3, 26.0, 9.1, 8.99, 8.97. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 50 H 88 Cl8N 14 , m / z Calculated value (M-7H-8Cl) + = 877.6763; Measured value = 877.6971. GPC (aqueous phase) M n = 1002, M w = 1009, M p = 1016, PDI = 1.01. Figure 4 )

[0241] General Procedure 6: Synthesis of Final Compounds 3 and 7

[0242] At 90°C, 1,4-iodobutane (3 equivalents) was added to a stirred solution of the desired starting materials 3e and 7e (1.0 equivalents) in MeCN:DMF (1:0.2). The reaction mixture was stirred at 90°C for 24–48 hours, while simultaneously... 1 The reaction progress was monitored by ¹H NMR. The reaction mixture was cooled to room temperature, diluted with MeCN, and washed three times with hexane. The resulting solution was concentrated under reduced pressure to remove MeCN. The resulting solution was diluted with MeCN (1 mL) and added dropwise to another portion of 3e and 7e (1.5 equivalents). The reaction mixture was stirred at 90 °C for 48–60 h. The reaction mixture was cooled to room temperature and treated with excess EtOAc. The resulting precipitate / gel was separated, further ground with EtOAc, and treated with a 33% (10 equivalent) solution of HBr in acetic acid. The resulting mixture was stirred overnight at room temperature. The reaction mixture was treated with excess EtOAc, and the resulting precipitate / gel was separated. The precipitate / gel was further ground with EtOAc, dissolved in water to a final concentration of 50–60 mM, and passed through an ion exchange resin containing Amberlyst. ® A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 500-1000 Da, and dialyzed against a solution of 5 mL HCl in 5 L of deionized water for 24 hours, with the dialysate changed frequently every 2-3 hours. The resulting solution was concentrated by rotary evaporation and lyophilized to obtain the desired product.

[0243] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-2-chloro-1 H -imidazol-3-on-3- (butyl)-2-chloro-1 H -imidazol-3-onth-1-yl)butyl)-2-chloro-1 H -imidazolium-3-onium)octachloride (OIM1-6-C2) (Cl)，3）

[0244] The title compound was synthesized as follows: using 3e (250 mg, 0.325 mmol) according to the method described in General Procedure 6, a pale brown hygroscopic solid (110 mg, 0.086 mmol, 26%) was obtained. 1 ¹H NMR (400 MHz, D₂O) δ 7.79 – 7.31 (m, 12H), 4.34 – 3.93 (m, 24H), 3.08 – 2.96 (m, 4H), 2.28 – 2.15 (m, 4H), 1.99 – 1.62 (m, 20H). 2 x -NH₃ was not observed due to deuterium exchange. + . 13C10 NMR (101MHz, D2O) δ 135.6, 135.4, 122.91, 122.85, 122.7, 122.5, 120.2, 48.8, 48.5, 46.5, 46.2, 36.3, 27.3, 26.3, 26.2, 25.4, 25.3. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 44 H 70 Cl 14 N 14 , m / z Calculated value (M-7H-8Cl) + = 999.3456; Measured value = No expected ions found, possibly due to poor ionization and low peak intensity of chlorine isotopes. GPC (aqueous phase) M n = 855, M w = 863, M p =851, PDI = 1.01.

[0245] General Procedure 7: Synthesis of Final Compounds 4 and 9

[0246] Add 1,4-diiodobutane (0.5 equivalents) to a solution of the desired starting materials 4e and 9e (1.0 equivalents) in N-methyl-2-pyrrolidone (NMP) under stirring, and heat to 120°C for 72 hours. Allow the reaction mixture to cool to room temperature. Separate the resulting gel, mill three times with MeCN, and treat with a 33% (10 equivalents) solution of acetic acid in HBr, stirring the resulting mixture overnight at room temperature. Before post-treatment, use... 1 The reaction was confirmed to be complete by ¹H NMR. The reaction mixture was treated with excess EtOAc to separate the resulting precipitate / gel. The precipitate / gel was further ground with EtOAc, dissolved in water to a final concentration of 50-60 mM, and passed through an ion exchange resin containing Amberlyst. ® A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed against a solution of 5 mL HCl in 5 L of deionized water for 24 hours, with the dialysate changed frequently every 2-3 hours. The resulting solution was concentrated by rotary evaporation and then purified by Sephadex elution with deionized water. TM -G10 gel filtration chromatography purification (gel filtration chromatography preparation: Sephadex) TM G10 powder was suspended in DI water overnight to obtain a slurry. The slurry was packed into a glass column and eluted by gravity using DI water as the eluent. The pure fraction was collected and lyophilized to obtain the desired product.

[0247] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-2-(trifluoromethyl)-1 H -imidazol- 3-On-3-yl)butyl)-2-(trifluoromethyl)-1 H -imidazol-3-onth-1-yl)butyl)-2-(trifluoromethyl)-1 H -imidazole-3- Onium octachloride (OIM1-6-C2(CF3),4)

[0248] The title compound was synthesized as follows: using 4e (1.00 g, 1.15 mmol) according to the method described in General Procedure 7, a pale brown hygroscopic solid (223 mg, 0.150 mmol, 26%) was obtained. 1 ¹H NMR (400 MHz, D₂O) δ 7.98 – 7.83 (m, 8H), 7.61 – 7.42 (m, 4H), 4.63 – 4.16 (m, 24H), 3.21 – 3.05 (m, 4H), 2.43 – 2.25 (m, 4H), 2.15 – 1.86 (m, 20H). 2 x -NH₃ was not observed due to deuterium exchange. + . 13 C NMR (101 MHz, D2O) δ 135.6, 135.4, 130.9, 130.4, 125.7, 125.5, 122.7,122.5, 121.7, 121.1, 119.9, 117.7, 115.0, 69.6, 50.4, 48.9, 48.6, 48.2, 47.2,46.5, 36.4, 36.3, 27.4, 26.6, 26.4, 26.3, 26.23, 26.20, 25.9. 19 F NMR (376MHz, D2O) δ -58.4, -61.3. MALDI-TOF (HCCA matrix, reflector mode): for C 50 H 70 F 18 Cl8N 14 , m / z Calculated value (M-7H-8Cl) + =1201.5068; Measured value =1201.7402. GPC (aqueous phase) M n = 1004, M w = 1053, M p = 945, PDI = 1.05.

[0249] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-5-methyl-1 H -imidazol-3-on- 3-yl)butyl)-4-methyl-1 H -imidazol-3-onth-1-yl)butyl)-5-methyl-1 H -imidazolium-3-onium)octachloride and imidazolium The -CH3-linked regioisomers are a single mixture (OIM1-6-C4(CH3),5).

[0250] The title compound was synthesized as follows: using 5e (1.50 g, 1.28 mmol) according to the method described in General Procedure 5, a pale brown hygroscopic solid (314 mg, 0.269 mmol, 42%) was obtained. 1 ¹H NMR (300 MHz, D₂O) δ 8.88 – 8.65 (m, 6H), 7.39 – 7.19 (m, 6H), 4.35 – 4.03 (m, 24H), 3.17 – 3.00 (m, 4H), 2.41 – 2.14 (m, 22H), 2.04 – 1.76 (m, 20H). 2 x -NH₃ was not observed due to deuterium exchange. + . 13 C10 NMR (75 MHz, D2O) δ 134.7, 134.6, 13.5, 132.1, 131.9, 119.2, 119.1, 48.5, 47.7, 46.3, 46.1, 45.9, 45.8, 27.2, 26.8, 26.2, 26.1, 25.9, 25.7, 8.2. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 50 H 88 Cl8N 14 , m / z Calculated value (M-7H-8Cl) + = 877.6763; Measured value = 877.6996. GPC (aqueous phase) M n = 877, M w = 884, M p = 893, PDI = 1.01. Figure 5 )

[0251] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-5-chloro-1 H -imidazol-3-on-3- (butyl)-4-chloro-1 H -imidazol-3-onth-1-yl)butyl)-5-chloro-1 H -imidazolium-3-onium)octachloride and imidazolium-Cl group The regioisomers of the same compound are a single mixture (OIM1-6-C4(Cl),6).

[0252] The title compound was synthesized as follows: using 6e (300 mg, 0.39 mmol) according to the method described in General Procedure 5, to obtain an orange solid (145 mg, 0.112 mmol, 56%). 1¹H NMR (400 MHz, D₂O) δ 9.08 –8.97 (m, 6H), 7.77 – 7.67 (m, 6H), 4.41 – 4.23 (m, 24H), 3.20 – 3.06 (m, 4H), 2.38 – 2.24 (m, 4H), 2.05 – 1.90 (m, 20H). 2 x -NH₃ was not observed due to deuterium exchange. + . 13 C10 NMR (101 MHz, D2O) δ 135.7, 135.6, 122.7, 122.5, 119.7, 49.8, 47.5, 46.9, 46.8, 36.3, 27.2, 26.0, 25.6. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 44 H 70 Cl 14 N 14 , m / z Calculated value (M-7H-8Cl) + = 999.3456; Measured value = No expected ions found, possibly due to poor ionization and low peak intensity of chlorine isotopes. GPC (aqueous phase) M n = 692, M w = 701, M p = 715, PDI = 1.01.

[0253] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-5-fluoro-1 H -imidazol-3-on-3- (butyl)-4-fluoro-1 H -imidazol-3-onthiol-1-yl)butyl)-5-fluoro-1 H -imidazolium-3-onyl)octachloride and imidazolium-F-link The regioisomers of the same compound were used as a single mixture (OIM1-6-C4(F), 7).

[0254] The title compound was synthesized as follows: using 7e (191 mg, 0.107 mmol) according to the method described in General Procedure 6, a brown hygroscopic solid (51 mg, 0.043 mmol, 40%) was obtained. 1 H NMR (400 MHz, D2O) δ8.84 – 8.69 (m, 6H), 7.49 – 7.37 (m, 6H), 4.41 – 4.15 (m, 24H), 3.11 (t, J =7.9 Hz, 4H), 2.31 (app. qu, J = 7.7 Hz, 4H), 2.07 – 1.85 (m, 20H). 2 x -NH3 was not observed due to deuterium exchange. + . 13C NMR (101 MHz, D2O) δ 148.3, 145.6, 130.0, 102.8, 102.64, 102.57, 50.1, 47.8, 45.5, 45.4, 36.2, 27.0, 25.8, 25.5. 19 F NMR (376 MHz, D2O) δ -144.2, -144.49. MALDI-TOF (HCCA matrix, reflector mode): for C 50 H 88 Cl8N 14 , m / z Calculated value (M-7H-8Cl) + = 877.6763; Measured value = No expected ions found, possibly due to poor ionization. GPC (aqueous phase) M n =950, M w = 970, M p = 997, PDI = 1.02.

[0255] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-1 H -benzo[ d Imidazole-3-onyl- 3-(3-yl)butyl)-1 H -benzo[ d [Imidazol-3-onthium-1-yl)butyl)-1 H -benzo[ d Imidazole-3-onium)octachloride (OIM1- 6-Benzimazole, 8)

[0256] At 90 °C, 1,4-dibromobutane (13 µL, 0.107 mmol) was added to a stirred solution of the desired starting material 8e (178 mg, 0.218 mmol) in DMSO (5 mL). The reaction mixture was heated to 90 °C and maintained for 48 hours. The reaction mixture was cooled to room temperature and treated with excess EtOAc. The resulting precipitate / gel was separated, further ground with EtOAc, and treated with a 33% (2 mL) solution of HBr in acetic acid, with the mixture stirred overnight at room temperature. The reaction mixture was treated with excess EtOAc, and the resulting precipitate / gel was separated. The precipitate / gel was further ground with EtOAc, dissolved in a water / MeOH mixture to obtain a clear solution, and passed through an ion exchange resin containing Amberlyst. ® A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 500-1000 Da, and dialyzed against a solution of 5 mL HCl in 5 L of deionized water for 24 hours, with the dialysate changed frequently every 2-3 hours. The resulting solution was concentrated by rotary evaporation and lyophilized to give a colorless solid (38 mg, 0.027 mmol, 25%). 1H NMR (400 MHz, D2O) δ 8.44 –8.12 (m, 6H), 7.89 – 7.81 (m, 2H), 7.78 – 7.53 (m, 22H), 4.65 – 4.40 (m,24H), 3.17 (t, J = 7.8 Hz, 4H), 2.39 (app qu, J = 7.8 Hz, 4H), 2.12 – 1.92(m, 24H, -C H 2). No 2 x -NH3 was observed due to deuteration exchange. + . 13 C10 NMR (101 MHz, D2O) δ 140.9,140.1, 131.1, 130.9, 130.7, 127.4, 127.1, 126.9, 126.4, 115.1, 113.2, 113.1, 112.4, 46.7, 46.6, 44.2, 36.6, 26.7, 24.9, 24.6. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 68 H 88 Cl8N 14 , m / z Calculated value (M-7H-8Cl) + =1093.6763; Measured value =1093.6996.

[0257] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(3-aminopropyl)-5-(trifluoromethyl)-1 H -imidazol- 3-On-3-yl)butyl)-4-(trifluoromethyl)-1 H -imidazol-3-on-1-yl)butyl)-5-(trifluoromethyl)-1 H -imidazole-3- Onionium octachloride and imidazolyl-CF3 associated regioisomers as a single mixture (OIM1-6-C4(CF3), 9)

[0258] The title compound was synthesized as follows: using 7e (1.00 g, 1.15 mmol) according to the method described in General Procedure 7, a pale brown hygroscopic solid (155 mg, 0.104 mmol, 18%) was obtained. 1 ¹H NMR (400 MHz, D₂O) δ 9.36 – 9.12 (m, 6H), 8.40 – 8.25 (m, 6H), 4.55 – 4.16 (m, 24H), 3.19 – 3.10 (m, 4H), 2.42 – 2.29 (m, 4H), 2.19 – 1.83 (m, 20H). 2 x -NH₃ was not observed due to deuterium exchange. + . 13C NMR (101 MHz, D2O) δ 139.8, 139.4, 139.2, 125.1, 124.6, 119.6, 116.9,60.7, 49.7, 49.5, 48.44, 48.37, 47.4, 46.7, 36.2, 35.2, 28.0, 27.1, 26.7,26.0, 25.9, 25.7. 19 F NMR (376 MHz, D2O) δ -60.50, -60.52, -60.55, -60.56, -60.57, -60.61, -61.46, -61.49, -62.1, -62.2. MALDI-TOF (HCCA matrix, reflector mode): for C 50 H 70 F 18 Cl8N 14 , m / z Calculated value (M-7H-8Cl) + = 1201.5068; Measured value = 1199.5159. GPC (aqueous phase) M n =985, M w = 992, M p = 1000, PDI = 1.01.

[0259] Example 2. Synthesis of OIM1-6 derivatives

[0260] General Procedure 8: Synthesis of dibromo compounds 10a-12a

[0261] To a 1,4-dibromobutane (10 equivalents) solution stirred at 80 °C, 0.5 mmol / mL solutions of the desired diimidazole starting materials (1d, 2d, and 5d, 1.0 equivalents) in MeCN were added dropwise. The reaction mixture was stirred at 80 °C for 18 hours. For compounds 10a and 12a: the reaction mixture was cooled to room temperature, diluted with MeCN, transferred to a separatory funnel, and washed three times with hexane. The MeCN layer was concentrated by rotary evaporation and purified by silica gel column chromatography eluting with 10–30% MeOH in EtOAc solution to obtain the desired product. For compound 11a: the reaction mixture was cooled to room temperature and concentrated by rotary evaporation to obtain a colorless solid. The solid was ground three times with diethyl ether to obtain the desired product.

[0262] 1,1'-(butane-1,4-diyl)bis(3-(4-bromobutyl)-1 H (-Imidazol-3-onium)dibromide (10a)

[0263] The title compound was synthesized as follows: using 1 day (1.00 g, 5.26 mmol) according to the method described in General Procedure 8, a light brown gel (1.04 g, 1.67 mmol, 32%) was obtained. 1 H NMR (MeOD, 400 MHz) δ9.28 – 9.12 (m, 2H), 7.76 – 7.63 (m, 4H), 4.40 – 4.24 (m, 8H), 3.51 (t, J =6.50 Hz, 4H), 2.14 – 1.84 (m, 12H). 13 C10 NMR (MeOD, 101 MHz) δ 137.8, 124.1, 124.0, 50.31, 50.27, 33.4, 30.6, 29.9, 28.0. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 18 H 30 Br4N4, m / z Calculated value (M-2Br-H) + = 459.0753; Measured value = 459.0139.

[0264] 1,1'-(butane-1,4-diyl)bis(3-(4-bromobutyl)-2-methyl-1 H (-Imidazol-3-onium)dibromide (11a)

[0265] The title compound was synthesized as follows: following the method described in General Procedure 8, using 2 days (560 mg, 2.56 mmol), to obtain a colorless solid (1.28 g, 1.97 mmol, 77%). 1 H NMR (MeOD, 400 MHz) δ 7.66 –7.55 (m, 4H), 4.32 – 4.15 (m, 8H), 3.52 (t, J = 6.26 Hz, 4H), 2.77 – 2.65 (m, 6H), 2.07 – 1.83 (m, 12H). 13 C10 NMR (MeOD, 101 MHz) δ 145.7, 122.8, 48.8, 33.5, 30.6, 29.5, 27.7, 10.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 20 H 34 Br4N4, m / z Calculated value (M-2Br-H) + = 489.1047; Measured value = 489.0467.

[0266] 1,1'-(butane-1,4-diyl)bis(3-(4-bromobutyl)-4-methyl-1 H -imidazolium-3-onium) dibromide and imidazolium The azole-CH3 associated regioisomers as a single mixture (12a)

[0267] The title compound was synthesized as follows: following the method described in General Procedure 8, for 5 days (584 mg, 2.68 mmol), yielding a light brown gel (394 mg, 0.606 mmol, 23%). 1 H NMR (MeOD, 400 MHz) δ9.23 – 9.03 (m, 2H), 7.56 – 7.38 (m, 2H), 4.34 – 4.17 (m, 8H), 3.65 – 3.42(m, 4H), 2.43 – 2.34 (m, 6H), 2.11 – 1.85 (m, 12H). 13 C10 NMR (MeOD, 101 MHz) δ 136.7, 133.4, 133.3, 121.0, 120.93, 120.87, 62.1, 50.1, 47.5, 33.6, 33.5, 30.6, 30.2, 29.8, 29.4, 28.0, 27.9, 27.49, 27.45. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 20 H 34 Br4N4, m / z Calculated value (M-2Br-H) + = 489.1047; Measured value = 489.0489.

[0268] General Procedure 9: Synthesis of Final Compounds 10-12

[0269] At room temperature, the desired dibromo starting materials 10a-12a (1.0 equivalent) were dissolved in DMF (0.5 mmol / mL) under stirring. The desired starting materials 1e, 2e, and 5e (2 equivalents) were then added to a mixture of MeCN:DMF = 4 mL:0.5 mL. The reaction mixture was heated to 80 °C for 72 hours. The reaction mixture was then cooled to room temperature. The resulting gel was separated, further milled three times with MeCN, and treated with a 33% (10 equivalent) solution of HBr in acetic acid. The resulting mixture was stirred overnight at room temperature. Prior to post-treatment, [the following steps were performed]. 1 The reaction was confirmed to be complete by ¹H NMR. The reaction mixture was treated with excess EtOAc to separate the resulting precipitate / gel. The precipitate / gel was further ground with EtOAc, dissolved in water to a final concentration of 50-60 mM, and passed through an ion exchange resin containing Amberlyst. ®A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed against a solution of 1 mL HCl in 1 L deionized water for 4 hours, with the dialysate replaced at t=2 hours. The resulting solution was concentrated by rotary evaporation and lyophilized to obtain the desired product.

[0270] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(4-(3-(3-aminopropyl)-1 H -imidazol-3-on-1- (butyl)-1 H -imidazol-3-on-3-yl)butyl)-1 H -imidazol-3-on-1-yl)butyl)-1 H -imidazolium-3-onyl)decyl chloride Object (OIM1-8-CH, 10)

[0271] The title compound was synthesized as follows: using 10a (299 mg, 0.481 mmol) and 1e (641 mg, 0.962 mmol) according to the method described in General Procedure 9, a pale brown hygroscopic solid (220 mg, 0.157 mmol, 33%) was obtained. 1 H NMR (400 MHz, D2O) δ 8.95 – 8.78 (m, 8H), 7.62 – 7.46 (m, 16H), 4.35(t, J = 7.42, 4H), 4.32 – 4.19 (m, 28H), 3.12 – 3.05 (m, 4H), 2.30 (app. p, J = 7.72 Hz), 2.02 – 1.85 (m, 28H). No 2 x -NH3 was observed due to deuterium exchange. + . 13 C10 NMR (101 MHz, D2O) δ 135.6, 135.4, 122.7, 122.5, 48.9, 46.6, 36.4, 27.4, 26.3. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 58 H 98 Cl 10 N 14 , m / z Calculated value (M-9H-10Cl) + =1037.7512; Measured value =1037.7473. GPC (aqueous phase) M n = 1240, M w = 1264, M p = 1216, PDI = 1.02.

[0272] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(4-(3-(3-aminopropyl)-2-methyl-1 H -imidazol- 3-On-1-yl)butyl)-2-methyl-1 H -imidazol-3-on-3-yl)butyl)-2-methyl-1 H -imidazol-3-on-1-yl)butane 2-methyl-1-yl H -Imidazol-3-onium)decachloride (OIM1-8-C2(CH3), 11)

[0273] The title compound was synthesized as follows: using 10b (210 mg, 0.323 mmol) and 2e (457 mg, 0.646 mmol) according to the method described in General Procedure 9, a pale brown hygroscopic solid (124 mg, 0.082 mmol, 25%) was obtained. 1 H NMR (300 MHz, D2O) δ 7.49 – 7.37 (m, 16H), 4.26 (t, J = 7.58, 4H), 4.22 – 4.10 (m, 28H), 3.15 – 3.05 (m, 4H), 2.71 – 2.55 (m, 24H), 2.23 (app.p, J = 7.74 Hz, 4H), 1.95 – 1.82 (m, 28H). 2 x -NH3 was not observed due to deuterium exchange. + . 13 C10 NMR (101 MHz, D2O) δ 144.0, 121.1, 47.4, 25.9, 8.93. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 66 H 114 Cl 10 N 14 , m / z Calculated value (M-9H-10Cl) + = 1149.8764; Measured value = 1150.0909. GPC (aqueous phase) M n =1117, M w = 1135, M p = 1105, PDI = 1.02.

[0274] 3,3'-(butane-1,4-diyl)bis(1-(4-(3-(4-(1-(4-(3-(3-aminopropyl)-4-methyl-1 H -imidazol- 3-On-1-yl)butyl)-5-methyl-1 H -imidazol-3-on-3-yl)butyl)-4-methyl-1 H -imidazol-3-on-1-yl)butane 5-methyl-1-yl H -Imidazol-3-onium)decachloride (OIM1-8-C4(CH3), 12)

[0275] The title compound was synthesized as follows: using 10c (300 mg, 0.461 mmol) and 5e (653 mg, 0.922 mmol) according to the method described in General Procedure 9, to obtain a pale brown hygroscopic solid (115 mg, 0.076 mmol, 16%). 1¹H NMR (400 MHz, D₂O) δ 8.81 – 8.68 (m, 8H), 7.36 – 7.22 (m, 8H), 4.32 – 4.09 (m, 32H), 3.15 – 3.03 (m, 4H), 2.40 – 2.17 (m, 28H), 2.01 – 1.80 (m, 28H). 2 x -NH₃ was not observed due to deuterium exchange. + . 13 C10 NMR (101 MHz, D2O) δ 134.7, 132.0, 119.2, 48.7, 46.3, 46.0, 36.4, 27.3, 26.3, 26.2, 26.0, 26.9, 8.31. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 66 H 114 Cl 10 N 14 , m / z Calculated value (M-9H-10Cl) + =1149.8764; Measured value =1149.9030. GPC (aqueous phase) M n = 1006, M w = 1018, M p = 1007, PDI = 1.01.

[0276] Example 3. Synthesis of biodegradable OIM1 derivatives

[0277] Synthesis of 3-(4-bromobutyl)-1-butyl-1H-imidazol-3-onium (1g)

[0278] The title compound was synthesized as follows: using N-butylimidazole (5 g, 0.04 mol) according to the method described in General Procedure 2 (described in Example 1), a colorless gel (8.8 g, 65%) was obtained. 1 H NMR (400 MHz, MeOD)δ 9.14 (d, J = 5.9 Hz, 1H), 7.69 (ddt, J = 5.7, 3.7, 1.9 Hz, 2H), 4.39 – 4.16(m, 4H), 3.59 – 3.46 (m, 2H), 2.15 – 2.01 (m, 2H), 1.98 – 1.82 (m, 4H), 1.47– 1.31 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). 13C10 NMR (101 MHz, MeOD) δ 137.4, 124.0, 50.9, 50.2, 33.3, 33.2, 30.6, 29.9, 20.6, 13.8. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 11 H 20 Br2N2, m / z Calculated value (M-Br) + = 260.1985; Measured value = 260.9590.

[0279] 1-(4-(1H-imidazol-1-yl)butyl)-3-(4-(3-butyl-1H-imidazol-3-onthium-1-yl)butyl)-1H-imidazol Synthesis of azole-3-onium (1h)

[0280] The title compound was synthesized as follows: using 1 g (6.00 g, 0.019 mol) of the general procedure 4 (described in Example 1), a colorless hygroscopic gel (6.31 g, 62%) was obtained. 1 H NMR (400 MHz, DMSO) δ 9.50 – 9.06 (m, 2H), 7.91 – 7.70 (m, 5H), 7.21 (s, 1H), 6.95 (s, 1H), 4.21 (dt, J = 16.1, 5.1 Hz, 8H), 4.02 (t, J = 6.4 Hz, 2H), 1.96 – 1.50 (m,10H), 1.26 (dq, J = 14.6, 7.4 Hz, 2H), 0.99 – 0.82 (m, 3H). 13 C10 NMR (101 MHz, DMSO) δ 137.0, 136.0, 127.8, 122.5, 122.4, 119.4, 48.6, 48.2, 48.0, 45.3, 40.1, 31.2, 27.1, 26.4, 26.1, 26.0, 18.7, 13.2. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 21 H 34 Br2N6, m / z Calculated value (M-2Br-H) + = 369.5330; Measured value = 369.1999.

[0281] Synthesis of bis(3-bromopropoxy)methane (13b)

[0282] In a round-bottom flask equipped with a Dean-Stark apparatus, 3-bromo-1-propanol (3.2 mL, 3.57 mmol, 2 equivalents) and paraformaldehyde (0.5355 g, 1.78 mmol, 1 equivalent) were dissolved in toluene (10 mL). After adding concentrated sulfuric acid (1 drop, catalyst), the mixture was refluxed for 1.5 hours until approximately the desired amount of water was collected. Upon cooling, sodium bicarbonate (approximately 0.2 g) was added, and the mixture was then filtered. The filtrate was purified directly by rapid chromatography (95:5, PS / EtOAc) to give 13b (4.0 g, 83%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.68 (s, 1H), 3.68 (t, J =5.9 Hz, 2H), 3.53 (t, J = 6.5 Hz, 2H), 2.24 – 2.03 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 95.42 (s), 65.12 (s), 32.89 (s), 30.41 (s).

[0283] Synthesis of 1,1'-(piperazine-1,4-diyl)bis(2-chloroethane-1-one) (14b)

[0284] 100 mL of saturated K₂CO₃ was added to a 200 mL CHCl₃ solution of piperazine (5.00 g, 58.14 mmol). The reaction mixture was stirred and cooled to 0 °C in an ice bath. A CHCl₃ solution of 2-chloroacetyl chloride (19.71 g, 174.44 mmol) was added dropwise over 1 hour. The reaction mixture was warmed to room temperature and stirred for 2 hours. The organic phase was washed with HCl (2 × 100 mL, 1 M) and water (2 × 100 mL). The CHCl₃ layer was dried and evaporated under vacuum to give a white solid product (12.5 g, 90%). 1 H NMR (400MHz, CDCl3) δ 4.09 (s, 4H), 3.78 – 3.49 (m, 8H). 13 C NMR (101 MHz, CDCl3) δ165.3, 45.9 42.8, 40.7.

[0285] 1,1'-(butane-1,4-diyl)bis(3-(3-((3-bromopropoxy)methoxy)propyl)-1H-imidazol-3-onium) Synthesis of (13c)

[0286] The title compound was synthesized as follows: using 1d (1.00 g, 5.26 mmol) and 13b (7.63 g, 26.3 mmol) according to the method described in General Procedure 8 (described in Example 2), 13c (2.75 g, 68%) was obtained as a colorless gel. 1 H NMR (400 MHz, D2O) δ 8.80 (s, 2H), 7.48 (dt, J = 9.8, 1.9 Hz, 4H), 4.65 (s, 4H), 4.27 (t, J = 7.0 Hz, 4H), 4.21 (s, 4H), 3.67 (q, J = 5.7 Hz,4H), 3.63 – 3.55 (m, 4H), 3.48 (t, J = 6.4 Hz, 4H), 2.20 – 2.10 (m, 4H), 2.10 – 1.98 (m, 4H), 1.89 – 1.83 (m, 4H). 13 C10 NMR (101 MHz, D2O) δ 135.5, 122.8, 122.5, 94.9, 65.8, 64.6, 58.0, 48.91, 46.9, 46.7, 31.6, 31.0, 29.2, 26.3. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 24 H 42 Br4N4O4, m / z Calculated value (M-2Br-H) + = 610.4309; Measured value = 609.0820.

[0287] 1,1'-(butane-1,4-diyl)bis(3-(4-(2-chloroacetyl)piperazine-1-carbonyl)-1H-imidazol-3-onium) Synthesis of (14c)

[0288] The title compound was synthesized as follows: using 1d (1.00 g, 5.26 mmol) and 14b (6.28 g, 26.3 mmol) according to the method described in General Procedure 8 (described in Example 2), a colorless gel 14c (2.23 g, 68%) was obtained. 1 H NMR (400 MHz, D2O) δ 8.79 (s, 2H), 7.60 – 7.32 (m, 4H), 5.34 (d, J=8.3 Hz, 4H), 4.33 (s, 4H), 4.26 (s, 4H), 3.83 – 3.50 (m, 16H), 1.90 (d, J =2.9 Hz, 4H). 13 C10 NMR (101 MHz, D2O) δ 168.2, 165.6, 124.1, 122.0, 50.2, 48.97, 45.0, 43.8, 41.8, 41.2, 26.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 24 H 34 Cl4N8O4, m / z Calculated value (M-2Cl-2H) + =597.5409; Measured value =595.2284.

[0289] 1,1'-(butane-1,4-diyl)bis(3-(3-((3-(1-(4-(3-(4-(1-butyl-1H-imidazol-3-onthium-3- (butyl)-1H-imidazol-3-onthium-1-yl)butyl)-1H-imidazol-3-onthium-3-yl)propoxy)methoxy)propyl)-1H-imidazol Synthesis of azole-3-onium) octachloride (13, OIM1-8-Bu-acetal)

[0290] At room temperature, a solution of the desired starting material 1h (1.44 g, 2.70 mmol, 2.1 equivalent) dissolved in a mixture of MeCN:DMF = 18 mL: 2 mL was added to a stirred solution of dibromo starting material 13c (1 g, 1.29 mmol, 1.0 equivalent) in acetonitrile (10 mL). The reaction mixture was heated to 90 °C for 18 hours. The reaction mixture was then cooled to room temperature. The resulting gel was separated and further ground three times with MeCN / EtOAc (8:2) to separate the precipitate / gel. The precipitate / gel was dissolved in water to a final concentration of 50–60 mM and passed through an ion exchange resin containing Amberlyst. ® A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed against a solution of 1 mL HCl in 1 L of deionized water for 1 hour, with the dialysate changed at t = 0.5 hours. The resulting solution was concentrated by rotary evaporation and then lyophilized to obtain the desired product (1 g, 55%). 1 H NMR (400 MHz, D2O)δ 9.01 – 8.59 (m, 8H), 7.47 (tdd, J = 5.6, 3.7, 1.8 Hz, 16H), 4.66 (s, 4H), 4.31 – 4.11 (m, 32H), 3.61 (dd, J= 10.4, 4.4 Hz, 8H), 2.19 – 2.08 (m, 8H), 1.93 – 1.75 (m, 24H), 1.31 – 1.19 (m, 4H), 0.86 (t, J = 7.4 Hz, 6H). 13 C10 NMR (101 MHz, D2O) δ 135.5, 135.4, 135.3, 122.7, 122.6, 122.5, 122.5, 122.4, 122.2, 94.9, 64.6, 49.4, 48.9, 48.8, 46.9, 31.2, 29.3, 26.2, 18.8, 12.6. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 66 H 110 Cl8N 16 O4, m / z Calculated value (M-8Cl-8H) + =1183.6500; Measured value =1183.8577. GPC (aqueous phase) M n = 351, M w = 363, M p = 358, PDI = 1.03. Figure 6 )

[0291] 3,3'-(((2,2'-(butane-1,4-diylbis(1H-imidazol-3-onthium-1,3-diyl))bis(acetyl))bis(piperazine) Azine-4,1-diyl))bis(2-oxoethane-2,1-diyl))bis(1-(4-(3-(4-(1-butyl-1H-imidazol-3-onthium-3-yl)) Synthesis of butyl)-1H-imidazol-3-onthium-1-yl)butyl)-1H-imidazol-3-onthium)octachloride (14,OIM1-8-Bu-PzAc)

[0292] At room temperature, a solution of the desired starting material 1h (1.66 g, 2.70 mmol, 2.1 equivalent) dissolved in a mixture of MeCN:DMSO = 19 mL:1 mL was added to a stirred solution of dibromo starting material 14c (1 g, 1.49 mmol, 1.0 equivalent) in DMSO (10 mL). The reaction mixture was heated to 90 °C for 18 hours. The reaction mixture was then cooled to room temperature. The resulting gel was separated and further ground three times with MeCN / EtOAc (8:2) to separate the resulting precipitate / gel. The precipitate / gel was dissolved in water to achieve a final concentration of 50–60 mM and passed through an ion exchange resin containing Amberlyst. ®A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed against a solution of 1 mL HCl in 1 L deionized water for 2 hours, with the dialysate replaced at t = 1.0 hour. The resulting solution was concentrated by rotary evaporation and then lyophilized to obtain the desired product (1.39 g, 60%). 1 H NMR (400MHz, D2O) δ 8.85 – 8.75 (m, 8H), 7.53 -7.46 (m, 16H), 5.37 (d, J = 3.9 Hz, 8H), 4.33 – 4.11 (m, 24H), 3.72 (s, 8H), 3.64 (s, 8H), 1.97 – 1.75 (m, 12H), 1.32 – 1.19 (m, 12H), 0.86 (t, J = 7.4 Hz, 6H). 13 C NMR (101 MHz, D2O) δ 165.63(s), 137.17 (s), 135.33 (d, J = 16.3 Hz), 124.18 (s), 122.71 – 122.28 (m), 122.15 (d, J = 13.0 Hz), 50.32 (s), 49.42 (s), 49.07 (s), 48.80 (d, J = 9.6Hz), 43.89 (d, J = 13.2 Hz), 41.81 (d, J = 14.5 Hz), 31.19 (s), 26.39 – 25.96 (m), 18.77 (s), 12.63 (s). MALDI-TOF (HCCA matrix, reflector mode): for C 68 H 106 Cl8N 20 O4, m / z Calculated value (M-8Cl-8H) + = 1259.6680; Measured value = 1259.8456. GPC (aqueous phase) M n = 409, M w = 426,M p = 437, PDI = 1.04. Figure 7 )

[0293] Example 4. Synthesis of biodegradable OIM series (compounds 15-16)

[0294] 1-Bromo-3-( Uncle Dingyang base Synthesis of propane (15a)

[0295] To improve reactivity, anhydrous Mg(ClO4)2 was heated at 130°C for 2 hours under vacuum (0.1 Torr) before use. A CH2Cl2 solution (90 mL) of 10.0 g (71.95 mmol, 1 equivalent) of 3-bromo-1-propanol was added to a three-necked flask containing 1.6 g (7.20 mmol, 0.1 equivalent) of Mg(ClO4)2 and equipped with a condenser and magnetic stir bar. The mixture was stirred until the solution became clear. A CH2Cl2 solution (14 mL) of 32 g (143.8 mmol, 2 equivalent) of Boc2O was added to the reaction mixture, and bubbles were immediately observed. The reaction mixture was stirred under reflux for 48 hours. The reaction mixture was diluted with water and extracted with CH2Cl2. The organic layer was separated, dried over anhydrous Mg2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography eluting with a 5% EtOAc hexane solution to give the corresponding ether 15a (9.0 g, 64%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ = 3.54-3.47 (m, 4H), 2.06 (q, J = 8Hz, 2H), 1.21 (s, 9H). 13 C NMR (101MHz, CDCl3) δ = 72.73, 58.84, 33.57, 31.09, 27.53.

[0296] Synthesis of 1-(3-(tert-butoxy)propyl)-1H-imidazolium (15b)

[0297] The title compound was synthesized by using 15a (9 g, 46.1 mmol, 1 equivalent) and imidazole 1a (2.8 g, 50.7 mmol, 1.1 equivalent) as described in General Procedure 1 (described in Example 1) to obtain the desired target product 12b (6.0 g, 71%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ = 7.46 (s, 1H), 7.02 (s, 1H), 6.90 (s, 1H), 4.03 (t, J = 8Hz, 2H), 3.27 (t, J = 6Hz, 2H), 1.94 (t, J= 6Hz, 2H), 1.15 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ = 137.29, 129.17, 118.89, 72.83, 57.26, 43.81, 31.71, 27.47.

[0298] Synthesis of bis(3-bromopropyl) carbonate (15c)

[0299] Pyridine (12.80 g, 161.88 mmol, 1.5 equivalents) was added dropwise to a stirred solution of 3-bromopropanol (15 g, 107.92 mmol, 1 equivalent) in 200 mL of DCM at 0 °C. The reaction mixture was stirred for 10 minutes. After 10 minutes, triphosgene (8 g, 26.98 mmol, 0.25 equivalents) dissolved in 40 mL of DCM was added dropwise to the reaction mixture at 0 °C for 10 minutes, and then the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with ice water (100 mL) and stirred vigorously for 10 minutes, then transferred to a separatory funnel. The organic layer was separated, washed with 2% HCl (100 mL), and then washed with water (100 mL). The organic layer was dried over anhydrous Na₂SO₄ and filtered. The resulting filtrate was concentrated under reduced pressure to give the desired product, a colorless oil, 15c (15 g, 47%). 1 H NMR (400 MHz, CDCl3) δ 4.23 (t, J = 6Hz, 4H), 3.44 (t, J = 6Hz, 4H), 2.17(t, J = 6Hz, 4H). 13 C NMR (101 MHz, CDCl3) δ 154.75, 65.62, 31.61, 29.11.

[0300] 1,1'-(butane-1,4-diyl)bis(3-(3-(((3-bromopropoxy)carbonyl)oxy)propyl)-1H-imidazol-3- Synthesis of 15d-15-onium bromide

[0301] The title compound was synthesized as follows: using 1d (1.00 g, 5.25 mmol, 1 equivalent) and 15c (8.00 g, 26.28 mmol, 5 equivalent) according to the method described in General Procedure 8 (described in Example 2), the desired product, a pale yellow gelatinous substance 15d (1.5 g, 36%), was obtained. 1 H NMR (400 MHz, D2O) δ 8.88 (s, 2H), 7.55 – 7.53 (m, 4H), 4.35 – 4.19 (m, 16H), 3.52 (t,J = 6Hz, 4H), 2.28 – 2.24 (m, 4H), 2.21 – 2.15 (m, 4H), 1.91 – 1.88 (m, 4H). 13 C10 NMR (101 MHz, D2O) δ 155.39, 135.68, 122.76, 122.65, 66.67, 65.74, 65.34, 48.97, 46.79, 41.60, 30.91, 30.22, 28.44, 26.81, 26.31. ESI-MS negative ion mode; for C10 NMR... 24 H 38 Br5N4O6, m / z Calculated value [M + 3Br] - = 878.10; Measured value = 877.23.

[0302] 3-(3-(((3-bromopropoxy)carbonyl)oxy)propyl)-1-(3-(tert-butoxy)propyl)-1H-imidazol-3-onium Synthesis of bromide (15e)

[0303] The title compound was synthesized as follows: 15c (4.2 g, 13.7 mmol, 2.5 equivalents) was added dropwise to compound 15b (1 g, 5.50 mmol, 1 equivalent) at 85°C according to the method described in General Procedure 2 (described in Example 1) to give a colorless gel 15e (2 g, 75%). 1 H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 7.89-7.86 (m, 2H), 4.30-4.12 (m, 8H), 3.56 (t, J = 6Hz, 2H), 3.32 (t, J = 6Hz, 2H),2.19 – 2.12 (m, 4H), 2.00 (t, J = 6Hz, 2H), 1.09 (s, 9H). 13 C10 NMR (101 MHz, DMSO-d6) δ 154.67, 136.84, 123.09, 122.81, 72.79, 66.02, 65.09, 58.04, 48.98, 47.26, 46.53, 31.61, 31.08, 30.49, 29.08, 27.61. ESI-MS negative ion mode; for C10 NMR... 17 H 30 Br2N2O4, m / z Calculated value [M + 2Br]- = 566.14; Measured value = 565.42.

[0304] 1-(4-(1H-imidazol-1-yl)butyl)-3-(3-(((3-(1-(3-(3-(tert-butoxy)propyl)-1H-imidazol-3- On-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-onium bromide (15f)

[0305] The title compound was synthesized as follows: 1d (2 g, 10.30 mmol, 2.5 equivalents) was added dropwise to compound 15e (2 g, 4.10 mmol, 1 equivalent) at 90°C according to the method described in General Procedure 4 (described in Example 1) to obtain a yellow gel 15f (2.5 g, 90%). 1 ¹H NMR (400 MHz, D₂O) δ 8.85 (s, 3H, integral value decreases due to hydrogen exchange to deuterium), 7.68–7.45 (m, 6H), 7.12 (s, 1H), 6.97 (s, 1H), 4.31-4.23 (m,8H), 4.18-4.14 (m, 4H), 4.04 (t, J = 6Hz, 2H), 2.18 – 2.27 (m, 4H), 2.09-2.04(m, 2H), 1.81-1.74 (m, 4H), 1.12 (s, 9H). 13 C10 NMR (101 MHz, D2O) δ 155.24, 137.76, 127.52, 122.78, 122.56, 122.53, 122.50, 120.17, 74.72, 65.16, 58.10, 49.08, 48.95, 47.07, 46.50, 46.25, 29.76, 28.56, 28.49, 26.94, 26.55, 26.43, 10.1. ESI-MS negative ion mode; for C10 NMR... 27 H 44 Br3N6O4, m / z Calculated value [M + 3Br] - = 756.39; Measured value = 757.38.

[0306] 3-(3-(((3-bromopropoxy)carbonyl)oxy)propyl)-1-(4-(3-(3-(((3-(1-(3-(tert-butoxy)propyl) (yl)-1H-imidazol-3-onthium-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-onthium-1-yl)butyl)-1H-imidazol- Synthesis of 3-onium bromide (15g)

[0307] To a solution of the desired dibromoalkylating agent 15c (5.6 g, 18.50 mmol, 5 equivalents) in anhydrous MeCN (10 mL) solution, compound 15f (2.5 g, 3.70 mmol, equivalents) was added dropwise to a stirred liquid at 90 °C. If necessary, a few drops of DMF were added to ensure the reaction mixture was a clear solution. The reaction mixture was stirred at 90 °C for 3 hours. The reaction mixture was cooled to room temperature, and a gel-like precipitate formed. The solvent was decanted, and the precipitate was washed several times with ethyl acetate. The gel-like product was then dissolved in deionized water and washed several times with ethyl acetate to remove excess dibromoalkylating agent through a separatory funnel. The solution was then concentrated under reduced pressure to give 15 g (3.5 g, 97%) of a brown gel. 1 H NMR (400 MHz, D2O) δ 8.88 (s, 3H),7.54-7.51 (m, 6H), 4.34-4.16 (m, 20H), 3.50 (t, J = 6Hz, 2H), 3.44 (t, J =6Hz, 2H), 2.28 – 1.90 (m, 14H), 1.13 (s, 9H). 13 C10 NMR (101 MHz, DMSO-d6) δ 155.37, 155.26, 135.67, 122.83, 122.71, 122.61, 122.57, 74.75, 66.62, 65.31, 65.20, 65.07, 58.12, 48.94, 46.53, 46.49, 30.88, 30.19, 29.77, 28.59, 28.56, 28.42, 26.59, 26.29. ESI-MS negative ion mode; for C10 NMR... 34 H 56 Br5N6O7, m / z Calculated value [M + 4Br] - =1060.36; Measured value =1061.19.

[0308] 1-(4-(1H-imidazol-1-yl)butyl)-3-(3-(((3-(1-(4-(3-(3-(((3-(1-(3-(tert-butoxy))) propyl)-1H-imidazol-3-onthium-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-onthium-1-yl)butyl)-1H-imidazol-3-onthium-1-yl) Azazole-3-on-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-onium bromide (15h)

[0309] Add dropwise a solution of 15 g (3.5 g, 3.60 mmol, 1 equivalent) of anhydrous MeCN (5 mL) of the desired bromoimidazole analogue to a stirred MeCN (10 mL) solution of 1 day (3.40 g, 17.80 mmol, 5 equivalents) at 95 °C. If necessary, add a few drops of DMF to ensure the reaction mixture is a clear solution. Stir the reaction mixture at 95 °C for 3 hours. Concentrate the reaction mixture by rotary evaporation, add a 3:1 chloroform:isopropanol mixture to the reaction mixture, and then add deionized water (20 mL). Wash the aqueous layer with a 3:1 chloroform:isopropanol mixture (12 × 20 mL) and concentrate by rotary evaporation to give a yellow gel (4 g, 96%) for 15 hours. 1 ¹H NMR (400 MHz, D₂O) δ 7.66 (s, ¹H, integral value decreases due to hydrogen exchange to deuterium), 7.53–7.48 (m, 8H), 7.13 (s, 1H), 6.95 (s, 1H), 4.34-4.16 (m, 24H), 4.04(t, J = 6Hz, 2H), 3.71 (t, J = 6Hz, 2H), 2.28–2.20 (m, 8H), 2.10-2.04 (m,4H), 1.90-1.77 (m, 6H), 1.12 (s, 9H). 13 C10 NMR (101 MHz, D2O) δ 156.14, 155.24, 137.86, 135.65, 127.74, 122.78, 122.66, 122.56, 122.53, 122.38, 120.14, 74.73, 65.21, 65.09, 58.13, 49.09, 48.97, 48.92, 47.09, 46.68, 46.48, 46.19, 29.78, 28.60, 28.55, 27.00, 26.58, 26.56, 26.45, 26.29. ESI-MS negative ion mode; for C10 NMR... 44 H 70 Br5N 10 O7, m / z Calculated value [M + 5Br] - = 1250.63; Measured value = 1251.24.

[0310] 1,1'-(butane-1,4-diyl)bis(3-(3-((3-(1-(4-(3-(3-((3-(1-(4-(3-(3-(3-(1-(4-(3-(3-((3-(1-(4-(3-(3-((3-(1-(4-(3-(3-((3-())))))))))))))))))) (1-(3-hydroxypropyl)-1H-imidazol-3-onthium-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-onthium-1-yl)butyric acid (yl)-1H-imidazol-3-onthium-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-onthium-1-yl)butyl)-1H-imidazol- 3-On-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-onyl)chloride (15)

[0311] Dibromodiimidazole (400 mg, 5.00 mmol, 1 equivalent) was added to a stirred colloidal pentamer intermediate (1.2 g, 10.00 mmol, 2.01 equivalents) for 15 h under pure conditions at 99 °C, and the reaction mixture was stirred for 2 h. The reaction mixture was cooled to room temperature, and the resulting gel was washed with MeCN (3 × 10 mL) followed by EtOAc (2 × 10 mL). The tBu- group was deprotected with a 50% TFA DCM solution for 12 h, and the resulting gel was then washed with EtOAc followed by MeCN. The gel product was dissolved in deionized water to a final concentration of 50–60 mM and passed through an ion exchange resin containing Amberlyst. ® A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed against a solution of 1 mL HCl in 1 L deionized water for 1 hour, with the dialysate changed at t = 0.5 hours. The deionized water containing the product was concentrated by rotary evaporation, and the gel-like product was then dissolved in 5 mL of deionized water and purified by Sephadex-G25 silica gel, collecting the fraction. The final product was lyophilized to give 15 (200 mg, 10%), a pale yellow hygroscopic solid, as monitored by GPC and ESI-MS. 1 H NMR (400 MHz, D2O) δ 8.84 (s, 12H), 7.52 – 7.46 (m, 24H), 4.30 – 4.15 (m, 72H), 3.58 – 3.55(m, 4H), 2.26 – 2.20 (m, 23H), 2.07 – 1.87 (m, 24H). 13 C10 NMR (101 MHz, D2O) δ 155.29, 135.61, 126.27, 122.67, 122.50, 65.01, 57.94, 48.87, 46.67, 46.39, 31.57, 28.54, 26.23. ESI-MS negative ion mode; for C10 NMR... 104 H 162 Cl 13 N 24 O 20 , m / z Calculated value [M + 13Cl] - = 2529.44; Measured value = 2525.02; GPC (aqueous phase) M n = 1063, M w = 1103, M p= 1199, PDI = 1.04.

[0312] N , N Synthesis of '-(propane-1,3-diyl)bis(2-chloroacetamide) (16a)

[0313] 100 mL of saturated K₂CO₃ (23.3 g, 168.84 mmol) was added to a 200 mL solution of 1,3-propanediamine (5.00 g, 67.45 mmol, 1.0 equivalent) in CHCl₃. The reaction mixture was stirred and cooled to 0 °C in an ice bath. A CHCl₃ solution of 2-chloroacetyl chloride (19.0 g, 168.6 mmol, 2.5 equivalent) was added dropwise over 1 hour. The reaction mixture was warmed to room temperature and stirred for 2 hours. The organic phase was washed with HCl (2 × 100 mL, 1 M) and water (2 × 100 mL). The CHCl₃ layer was dried and evaporated under vacuum to give a white solid product (9.92 g, 65%). 1 H NMR (400 MHz, CDCl3) δ 7.18 (s, 2H), 4.09 (s, 4H), 3.39 (dd, J = 12.4, 6.3 Hz, 4H), 1.85 – 1.68 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 166.8, 42.6, 36.4, 29.4.

[0314] 1,1'-(butane-1,4-diyl)bis(3-(2-((3-(2-chloroacetamido)propyl)amino)-2-oxoethyl)- Synthesis of 1H-imidazol-3-onium (16b)

[0315] The title compound was synthesized by using 1d (1.00 g, 5.26 mmol, 1.0 equivalent) and 16a (5.97 g, 26.3 mmol, 5.0 equivalent) according to the method described in General Procedure 8 (described in Example 2) to obtain colorless gel 16b (1.86 g, 55%). 1 H NMR (400 MHz, D2O) δ 8.76 (d, J = 52.5 Hz, 2H, (the integral value decreases due to hydrogen exchange to deuterium), 7.46 (ddt, J = 24.0, 9.5, 1.8 Hz, 4H), 4.98 (d, J = 15.2Hz, 4H), 4.25 (s, 4H), 4.05 (s, 8H), 3.24 (dd, J = 11.5, 6.7 Hz, 8H), 1.77(ddd, J= 26.8, 20.4, 12.9 Hz, 8H). 13 C10 NMR (101 MHz, D2O) δ 169.6, 166.9, 136.9, 123.8, 122.7, 122.3, 50.8, 49.0, 42.4, 37.0, 35.0, 27.7, 26.4, 26.1. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 24 H 38 Cl2N8O4, m / z Calculated value (M-2Cl-2H) + =571.5040; Measured value =571.2546.

[0316] Mono(3,3'-(((((2,2'-(butane-1,4-diylbis(1H-imidazol-3-onthium-1,3-diyl))bis(acetyl)) bis(azanediyl))bis(propane-3,1-diyl))bis(azanediyl))bis(2-oxoethane-2,1-diyl))bis(1-(4- (1-(4-(1-(3-aminopropyl)-1H-imidazol-3-onthium-3-yl)butyl)-1H-imidazol-3-onthium-3-yl)butyl)-1H-imidazol- Synthesis of 3-onium)decachloride (16)

[0317] At room temperature, a solution of starting material 1e (2.17 g, 3.22 mmol, 2.1 equivalents) dissolved in a MeCN:DMF mixture of 18 mL:2 mL was added to a stirred methanol (5 mL) solution of dichloro starting material 16b (1 g, 1.55 mmol, 1.0 equivalent) at 80 °C. The resulting reaction mixture was heated to 95 °C for 18 hours. The reaction mixture was then cooled to room temperature (by crude...). 1 (H NMR confirmed the completion of the reaction). The resulting gel was separated and further ground three times with MeCN / EtOAc (8:2). The resulting gel was then treated with a 33% (10 equivalent) HBr acetic acid solution. The mixture was stirred at room temperature for 2 hours. The reaction mixture was treated with excess EtOAc, and the resulting gel was separated and dissolved in water to a final concentration of 50-60 mM. The mixture was then passed through an ion exchange resin containing Amberlyst. ® A glass column of A-26 was used to exchange the countercharged ions for chloride ions. The eluent containing the product was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed against a solution of 1 mL HCl in 1 L deionized water for 1 hour, with the dialysate changed at t = 0.5 hours. The resulting solution was concentrated by rotary evaporation and lyophilized to give the desired product 16 (1.8 g, 55%) as a white hygroscopic solid. 1 H NMR (400 MHz, D2O) δ 8.98 – 8.73 (m, 8H), 7.66 –7.29 (m, 16H), 5.03 (d, J = 9.8 Hz, 8H), 4.37 – 4.15 (m, 24H), 3.26 (t,J =6.9 Hz, 8H), 3.09 – 2.98 (m, 4H), 2.33 – 2.19 (m, 4H), 1.90 (d, J = 3.0 Hz, 20H), 1.74 (p, J = 6.9 Hz, 4H). 13 C10 NMR (101 MHz, D2O) δ 166.9, 136.9, 135.5, 123.8, 122.5, 50.9, 49.2, 48.5, 46.6, 37.1, 36.5, 27.6, 27.4, 27.2, 26.2. MALDI-TOF (HCCA matrix, reflector mode): for C10 NMR... 68 H 106 Cl8N 20 O4, m / z Calculated value (M-10Cl-8H) + =1239.6369; Measured value =1239.5843. GPC (aqueous phase) M n = 1322, M w = 1330, M p = 1347, PDI = 1.006. Figure 8 )

[0318] Example 5. Antibacterial properties of precisely targeted main-chain imidazolium oligomers

[0319] Bacterial culture

[0320] All strains were purchased from the American Type Culture Collection (ATCC). *Pseudomonas aeruginosa* PAO1, *Enterococcus faecalis* VRE583, and *Escherichia coli* 958 were purchased from the Singapore Centre for Environment and Life Sciences (SCELSE). Clinical isolates PAER (multidrug-resistant *Pseudomonas aeruginosa*), ACBAS (pan-drug-resistant *Acinetobacter baumannii*), AB-1 (multidrug-resistant *Acinetobacter baumannii*), KPNS (pan-drug-resistant *Klebsiella pneumoniae*), KPNR (carbapenem-resistant *Klebsiella pneumoniae*), ECOS (pan-drug-resistant *Escherichia coli*), ECOR (multidrug-resistant *Escherichia coli*), ECLOS (pan-drug-resistant *Enterobacter cloacae*), and CRE (carbapenem-resistant *Enterobacter cloacae*) were purchased from Tan Tock Seng Hospital (TTSH), Singapore. Klebsiella pneumoniae SGH10, Klebsiella pneumoniae BAK085, Klebsiella pneumoniae M7, Klebsiella pneumoniae SGH4, Acinetobacter baumannii X26, Acinetobacter baumannii X39, and Acinetobacter baumannii X40 were provided by the National University of Singapore. MRSA LAC, LAC LAC Δ menD and LAC Δ hemB As previously described (Pader, V). et al. , Infect. Immun. 2014, 82 (4337-4347), provided by Angelika Gründling of Imperial College London. Burkholderia thamnoides 700388 was provided by Samuel I. Miller of the University of Washington. All broth or agar media used in this study were purchased from Becton Dickinson. The strains were stored in 15% glycerol at -80°C.

[0321] Minimum inhibitory concentration (MIC)

[0322] The minimum inhibitory concentration (MIC) was determined using the standard broth microdilution method, with slight modifications (Wiegand, I). et al. , Nat. Protoc. 2008, 3 (163-175). In short, single colonies are picked and inoculated to obtain overnight cultures. Subcultures are prepared the following day and allowed to grow to the exponential phase. Two-fold serial dilutions of the test compound in MHB broth are prepared in 96-well plates, followed by the addition of a 5 × 10⁻⁶ concentration. 5 Exponential phase bacteria (CFU / mL). Vigorously shake the culture plate for 20 seconds to thoroughly mix the solution, then incubate the plate at 37°C for 18 hours. Record the OD. 600 Take readings and calculate the minimum concentration (MIC) that inhibits bacterial growth by 90%. 90 For the determination of MIC of Enterococcus faecalis 583 (VRE2), overnight and subcultured cultures were prepared in tryptone soybean broth (TSB) according to the above method, and the MIC was tested with and without 8 µg / mL ferric heme.

[0323] Cytotoxicity MTT assay

[0324] In vitro biocompatibility was investigated using mouse fibroblasts (3T3), human embryonic kidney (HEK293) cells, human hepatocellular carcinoma (HepG2) cells, and human alveolar basal epithelial adenocarcinoma cells (A549). Briefly, 3T3 and HepG2 cells were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin / streptomycin). HEK and A549 cells were cultured in medium supplemented with 15% FBS. All cells were incubated in a CO2 incubator at 37°C. When cell confluence was observed to reach 80% under a microscope, cells were treated with trypsin, concentrated, and counted using a hemocytometer. Then, 1×10⁻⁶ cells were cultured. 4 Cells were seeded per well in 96-well plates and allowed to grow for 24 hours, followed by treatment with different concentrations of compounds for 24 hours. Cell health was then qualitatively examined under a microscope and quantified using the MTT assay (3-[4,5-dimethylthiazolyl-2-yl]-2,5-diphenyltetrazolium bromide). Cell viability was calculated by comparing the absorbance of formazan formed by cells in treated and untreated wells. The compound concentration (IC50) that reduced cell growth by 50% compared to the untreated control group was determined. 50 The data represent the results of three independent experiments.

[0325] Time-based sterilization test

[0326] The bactericidal kinetics of OIM were determined by a time-bactericidal assay. Exponential phase bacteria (prepared according to the MIC protocol described above) were diluted to 5 x 10⁻⁶. 5 CFU / mL, continuously shaken at 37°C, and treated with different concentrations of OIM. Bacterial samples were aliquoted at specified time intervals (30 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h) and serially diluted with phosphate-buffered saline (PBS). The serially diluted samples were then spotted onto agar plates, incubated overnight, and colony counts were performed.

[0327] Computer simulation for calculating the Hirshfeld charge on the imidazolium ring of OIM

[0328] Computational studies were performed on five OIM compounds (1-3, 5-6) using Gaussian 16 software (Gaussian 16 Rev. C.01 (Wallingford, CT, 2016)). Oligomer models were constructed using GaussView 6.0, followed by geometric optimization and frequency analysis at the theoretical level of B3LYP / def2-TZVP / SMD (solvent = water) (Stephens, PJ). et al. , J. Phys. Chem. 1994, 98, 11623-11627;Weigend, F. & Ahlrichs, R., Phys. Chem. Chem. Phys. 2005, 7 , 3297-3305; Papajak, E. et al. , J. Chem. Theory Comput. 2011, 7 , 3027-3034; and Marenich, AV et al. , J. Phys. Chem. B 2009, 113 (6378-6396), and applied the Grimmes empirical dispersion correction em=gd3(bj) (Grimme, S). et al. , Chem. Rev. 2016, 116 (5105-5154) Figure 22 The natural bond orbitals (NBOs) of all atoms in each polymer were calculated (Weinhold, F. & Landis, CR, Chem. Educ. Res. Pract. 2001, 2 (91-104) and Mulliken charge. In addition, Multiwfn software (Lu, T. & Chen, F. Multiwfn, J. Comput. Chem. 2012, 33 (580-592) The Hirshfeld charge (Hirshfeld, FL, ...) is derived from the wavefunction calculated by DFT (converted from Gaussianchk to fchk using the formchk tool). Theor. Chim. Acta 1977, 44 , 129-138).

[0329] Detection of bacterial cell membrane depolarization and membrane integrity

[0330] The membrane depolarization activity of OIM was determined using the membrane potential-sensitive dye 3,3-dipropylthiocyanine iodide (DiSC3(5)). This determination followed the previously described protocol with slight modifications (Belley, A.). et al. , Antimicrob. Agents Chemother. 2009, 53 , 918-925; and Te Winkel, JD et al. , Front. Cell Dev. Biol. 2016, 4, 29). Precipitate the MRSA LAC from the exponential phase, wash twice with 5 mM HEPES and 5 mM glucose, and resuspend to 4 x 10⁻⁶. 7 CFU / mL. 0.5 µM DiSC3(5) was added to the bacteria, and 175 µL of bacteria were aliquoted into white 96-well plates. Fluorescence was monitored every 2 minutes with continuous oscillation using a Spark 10M microplate reader (Tecan, Switzerland) at excitation / emission wavelengths of 622 nm / 670 nm. The DiSC3(5) dye was absorbed into the bacterial membrane, causing the fluorescence signal to quench. Once a stable fluorescence reading was obtained, 25 µL of different concentrations of OIM were added, and fluorescence readings were recorded immediately over 1 hour. Bacitracin was used as a positive control. For the membrane integrity assay, LAC was prepared as described above, and 1 µM propidium iodide (PI) dye was added to the bacteria. The PI signal was monitored as a baseline reading before the addition of OIM and nisin (positive control), and then fluorescence readings were recorded over 1 hour using a Tecan microplate reader at excitation / emission wavelengths of 535 nm / 620 nm after the addition of the test compound.

[0331] pass 1 Hydrogen-deuterium exchange in OIM determined by H NMR.

[0332] According to previous research (Zhang, N). et al. , Advanced Science (Weinheim, Baden- Wurttemberg, Germany) 2021, 8 (e2100805), by adjusting the ratio of K2DPO4 (0.1 M stock concentration) and KD2PO4 (0.1 M stock concentration), a PBS buffer with the desired pH was prepared. All reactions were carried out in D2O with an ionic strength of 1.0 (KCl). In short, OIM was dissolved in PBS at a concentration of 10 mM (the concentration of the oligomer repeating unit), and 10 mM TMA (tetramethylammonium hydroxide) was used as an internal standard. The reaction was performed at 25°C using a Bruker 400 MHz nuclear magnetic resonance spectrometer. 1 Measurement of deuterium exchange by 1H NMR spectroscopy (Amyes, TL) et al. , J.Am.Chem.Soc. 2004, 126 ,4366-4374).

[0333] Carbazole probe captures NHC

[0334] (i) Preparation of carbazole (lipid)

[0335] Phosphatidylglycerol (PG; 10 mg / mL, 1.3 × 10⁻⁶)-5 mol / mL), phosphatidylcholine (PC; 10 mg / mL, 1.3 × 10⁻⁶ ... -5 mol / mL) and carbazole (2.2 mg / mL, 1.3 × 10 ... -5 Stock solutions were prepared by dissolving PC, PG, and carbazole in chloroform. A PC / PG / carbazole mixture with a molar ratio of 8:2:1 was prepared by mixing 0.8 mL of PC, 0.2 mL of PG, and 0.1 mL of carbazole stock solution in a round-bottom flask (25 mL). Chloroform was removed by rotary evaporation at 50 mbar and 20 °C for 20 min. The lipid membrane was hydrated with 1 mL of PBS buffer (pH 7.4). The suspension was then vortexed at maximum speed for 1.5 min, followed by sonication in an ice bath for 2 min. This vortex-sonication process was repeated three times, followed by extrusion of the solution 19 times through a 200 nm polycarbonate membrane using an Avanti mini extruder. The resulting liposome solution was dialyzed against water for 36 h using a dialysis tube with an MWCO of 2000 to remove free carbazole. The size distribution and zeta potential of the obtained liposomes were detected using a Malvern Nano Series Nano-ZS instrument (data not shown). All prepared liposome solutions were stored at 4 °C before use.

[0336] (ii) Fluorescence assay of carbazole (lipid)-OIM. OIM stock solutions (1, 2, and 5) were dissolved in H2O at a concentration of 10 mg / mL. The pH of the solution was adjusted to 7.4 with 1 M NaOH. IPr stock solution (50 mg / mL, dissolved in DMF) was prepared. Carbazole (lipid)-OIM was prepared by mixing 10 µL of carbazole (lipid), 100 µL of OIM stock solution, and 890 µL of PBS (pH 7.4). As a positive control, carbazole (lipid)-IPr was prepared by mixing 10 µL of carbazole (lipid), 10 µL of IPr stock solution, and 980 µL of PBS (pH 7.4). All mixtures were incubated at 37 °C for 36 h prior to fluorescence assay. Emission fluorescence spectra were obtained at an excitation wavelength of 295 nm.

[0337] AuCl(SMe 2 Probe captures NHC

[0338] (i) Preparation of Au (lipids)

[0339] Au (lipid) was prepared using the same protocol as carbazole (lipid), except that AuCl (SMe2) stock solution (3.82 mg / mL, 1.3 × 10⁻⁶) was used. -5The carbazole solution was replaced with a mol / mL solution of PC / PG / AuCl(SMe2), and the molar ratio of PC / PG / AuCl(SMe2) was 8:2:1. After vortex-sonication treatment, no extrusion was performed due to the sensitivity of AuCl(SMe2). Hydrophobic AuCl(SMe2) not trapped by the liposome bilayer was removed by centrifugation at 4000 g for 20 min at 10 °C. The resulting precipitate was washed once with water. Immediate removal of uncaptured AuCl(SMe2) after Au (lipid) preparation is crucial, as free AuCl(SMe2) molecules react with glycerol groups in PG to form Au nanoparticles, resulting in a purple solution unsuitable for further study.

[0340] (ii) Formation of Au-OIM in liposomes

[0341] The washed Au (lipid) particles were dispersed in 10 mL of 0.1× phosphate buffer (pH 7.4), and then OIM stock solution was added to a final concentration of 75 µg / mL. The mixture was incubated at 37 °C in the dark for 48 hours. The mixture was then lyophilized. The dried powder was dissolved in 1 mL of methanol. The addition of methanol disrupted the liposome structure, thereby releasing Au-OIM into the solution. The solution was centrifuged at 8000 g for 30 minutes at 10 °C to remove any Au nanoparticles. The resulting solution was subjected to chloroform-methanol-water extraction to remove lipids (Freeman, C). et al. , J. Am. Soc. Mass Spectrom. 2021, 32 , 2376-2385) and AuCl(SMe2), and then LC-MS determination was performed.

[0342] (iii) LC-MS characterization

[0343] Mass spectrometry analysis was performed using an Agilent Q-TOF / MS (6550 iFunnel) instrument. Electrospray ionization (ESI) mass spectrometry (MS) in positive ion mode was employed. All analyses were performed at 35 °C using a C18 column (50 mm × 2.1 mm id, 1.8 μm, Agilent). The mobile phase consisted of a linear gradient system of (A) water (0.1% formic acid) and (B) acetonitrile (0.1% formic acid). The mobile phase gradient conditions were as follows: 0–2 min, 95% A; 2–3.5 min, 95–30% A; 6.5–8.5 min, 30–95% A; 8.5–13.5 min, 95% A. The flow rate was 0.2 mL / min. The injection volume was 5 μL.

[0344] (iv) Structural confirmation of Au-(6) via deuterium-hydrogen exchange

[0345] Add a drop of D2O to the sample prepared as described above ((iii) LC-MS characterization) to replace the active hydrogen with deuterium, and then analyze it by LC-MS under the same conditions.

[0346] (v) Structural confirmation of Au-(6) using MS-MS

[0347] In the previous sample prepared as described above ((iii) LC-MS characterization), fragments of the target ion with m / z 330.52 (Au-OIM-(6)) were obtained by LC-MS with collision energy CID@10.

[0348] Liposome uptake of OIM

[0349] (i) Liposome membrane and nucleus uptake experiments

[0350] Liposomes were prepared using the same protocol as carbazole (liposomes), except that carbazole was not added. Liposome stock solution (10 mg / mL), OIM stock solution (10 mg / mL), and water were mixed to prepare 1 mL solutions, resulting in final concentrations of 100 ppm for liposomes and 5000 ppm for OIM. After incubation at 37°C for 20 hours, the solutions were filtered by tangential flow filtration (TFF) (MidiKross). ® Hollow fiber modules (MWCO 3kDa) were used to remove free OIM through 30 extrusion cycles with 0.2× PBS using a syringe. The resulting solution (approximately 1 mL) was divided into two aliquots. One aliquot was lyophilized to determine the total OIM uptake in the membrane and core. The lyophilized powder was dissolved in methanol, and lipids were removed by chloroform-methanol-water extraction (Freeman, C). et al. , J. Am. Soc. Mass Spectrom. 2021, 32 (2376-2385). The upper fraction (water / methanol phase) was collected for LC-MS analysis. Another fraction (approximately 0.5 mL) was added to 0.17 mL of ethanol (final ethanol concentration 25% v / v) and incubated at room temperature for 2 hours to disrupt membrane integrity and release OIM from the core. The OIM released from the core and the OIM trapped in the bilayer were separated by centrifugation at 4000 g for 30 minutes at 25 °C using a Vivaspin (MWCO 30 kDa), as the OIM released from the core would pass through the Vivaspin membrane, while the OIM trapped in the liposome bilayer would be retained on the membrane due to the size of the liposomes.

[0351] (ii) OIM calibration curve determined by LC-MS

[0352] To obtain accurate calibration curves, we prepared the lipid matrix using the same procedures as in the uptake experiments. Specifically, 200 μL of liposome stock solution (containing 0.4 mg PG and 1.6 mg PC) was lyophilized. Then, 16 mL of methanol was added to dissolve the liposomes, followed by 14.4 mL of deionized water, 16 mL of chloroform, and 46.4 μL of formic acid (final concentration 0.1% v / v) to remove lipids and avoid any damage to the C18 column. After centrifugation at 3000 rpm for 10 minutes, the supernatant (upper phase) was collected as the lipid matrix. Using this lipid matrix, standards of OIM1-6-CH (1), OIM1-6-C2(CH3) (2), and OIM1-6-C4(CH3) (5) at concentrations of 0, 1, 2, 3, and 4 ppm were prepared to plot calibration curves. These standards were then analyzed using an Agilent Q-TOF / MS (6550 iFunnel) instrument. The results are as follows: Figure 9 As shown.

[0353] The molecular dynamics of the interaction between the cationic and NHC forms of OIM1-6-CH and the model Staphylococcus aureus membrane. Mechanical simulation

[0354] (i) Membrane construction

[0355] Use CHARMM-GUI (Jo, S). et al. , J. Comput. Chem. 2008, 29 Epand, RM & Epand, RF) constructed a lipid bilayer representing the composition of Staphylococcus aureus membranes using membrane construction tools, comprising 58% phosphatidylglycerol (PG) and 42% cardiolipin (CL) (1859–1865). Biochim. Biophys. Acta 2009, 1788 , 289-294). PG and CL were constructed using DMPG (14:0 / 14:0) and TMCL2 (14:0, 14:0 / 14:0, 14:0) forms, respectively. The distribution of PG and CL lipids on the upper and lower lobes was symmetrically constructed. Menaquinone-8 (MQ8) molecules were constructed on the membrane model. The parameters of the membrane lipids were based on the CHARMM36 force field, and the parameters of MQs and OIM1-6-CH (1) were based on the CHARMM universal force field (Vanommeslaeghe, K. & MacKerell, AD, J. Chem. Inf. Model. 2012, 52 , 3144–3154; and Vanommeslaeghe, K. et al. , J. Chem. Inf. Model. 2012, 52, 3155–3168). The topological structure of the MQ8 molecule was modeled using CHARMM-GUI ligand reading (Kim, S.). et al. , J. Comput. Chem. 2017, 38 The membrane was generated using tools from 1879-1886, with a total lipid to total MQ8 ratio of 10:1. Equal amounts of MQ molecules were randomly and manually inserted into the upper and lower lobes of the constructed Staphylococcus aureus membrane. Subsequent manual coordinate adjustments and energy minimization steps were performed to eliminate structural conflicts between atoms. The membrane was constructed using TIP3P (Jorgensen, WL). et al. , J. Chem. Phys. 1983, 79 (926-935) Water molecules are solvated, and counter-charged ions are added to neutralize the system. The constructed Staphylococcus aureus membrane was prepared using GROMACS (Van Der Spoel, D. et al. , J. Comput. Chem. 2005, 26 Molecular dynamics (MD) simulations lasting 100 ns were performed using software 5.1.2 (Hess, B., 1701-1718). LINCS (Hess, B., J. Chem. Theory Comput. 2008, 4 The algorithm (116-122) constrains the bonds between heavy atoms and hydrogen to achieve a time step of 2 fs. Van der Waals interactions and short-range electrostatic interactions are calculated using a 1.2 nm cutoff value, while long-range electrostatic calculations employ the Particle Mesh Ewald method. A V-rescale thermostat is used to maintain the simulation temperature at 310 K (Bussi, G). et al. , J. Chem. Phys. 2007, 126 , 014101), and used Parrinello-Rahman (Parrinello, M. & Rahman, A., J. Appl. Phys. 1981, 52 (7182-7190) The pressure gauge maintains the pressure at 1 bar.

[0356] (ii) Simulation of OIM1-6-CH(1) on Staphylococcus aureus membrane

[0357] Using Discovery Studio 4.1 (Zhuo, S. et al. , Molecules 2020, 25Coordinates for the cationic and NHC forms of OIM1-6-CH(1) were constructed using the CHARMM-GUI ligand reading and modeling tool (Kim, S.). et al. , J. Comput. Chem. 2017, 38 The topology of (1) was obtained in 1879-1886. The NHC carbene bias was obtained by Gaussian 09 (Gaussian, Inc, Wallingford, CT, USA, 2009) in the triplet state as HF 6-31G and RESP (Bayly, CI) et al. , J. Phys. Chem. 1993, 97 The fitting level was calculated (10269-10280). OIM simulations on the membrane were performed using the last simulation frame of the previous 100 ns molecular dynamics simulation of Staphylococcus aureus membranes. Eight simulation systems of cationic OIM or OIM-NHC on Staphylococcus aureus membranes were established. A single oligomer molecule was placed 0.8 nm above the center of the upper leaflet of the membrane bilayer. Classical molecular dynamics simulations for 200 ns were performed on each system. The settings for the molecular dynamics simulations were similar to those described above.

[0358] (iii) Simulation analysis

[0359] The number of contacts between the OIM and the membrane was calculated as a function of simulation time to analyze the interaction behavior between the oligomer and the membrane. The contact number was calculated using GROMACS (Van Der Spoel, D. et al. , J. Comput. Chem. 2005, 26 The GMX Mindist tool (1701-1718) was used for simulation. A contact was defined as the minimum distance between OIM atoms and membrane atoms less than 0.4 nm. To further visualize the bonding between OIM and the membrane, PyMOL was used to visualize the last frame of each simulation system.

[0360] Results and Discussion

[0361] The OIM1-6 series (1-9) were tested against a range of Gram-positive and Gram-negative bacteria to determine their antibacterial efficacy (Table 1). OIM1-6-CH(1) has a hydrogen atom at the C2-carbon (denoted as C2-H), which shows good efficacy against a broad spectrum of bacteria, including Gram-positive pathogens (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli), with a minimum inhibitory concentration (MIC) of [missing value].90 Its efficacy against Enterobacter cloacae is relatively low (MIC). 90 = 16-32 µg / mL). Notably, OIM1-6-CH(1) is characterized by the acidity of its carbon and the presence of C(2)-H, exhibiting significant efficacy against the ESKAPE pathogen group, including Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae, with a geometric mean (Geo-MIC) of its minimum inhibitory concentration. 90 As low as 4.0 µg / mL.

[0362]

[0363] Interestingly, different ring substituents lead to different antibacterial efficacies. We also synthesized OIM1-6 derivatives with substitutions at the C2-carbon of the imidazolium moiety to investigate the structure-activity relationship (SAR) around the imidazolium moiety. We synthesized derivatives (2-4) with C2-hydrogens substituted by various substituents (i.e., the weak electron-donating group methyl-(-CH3), the electron-withdrawing group chlorine (-Cl), and the trifluoro group (-CF3)) (Fig. 1A). Compared with OIM1-6-CH (1), the C2-substituted derivatives (2-4) showed significantly reduced antibacterial efficacy. Among the three C2 derivatives, compounds (3, 4) containing the strong electron-withdrawing substituent C2-CF3 / -Cl maintained higher efficacy than (2). We hypothesize that compounds (3, 4) with the electron-withdrawing group C2-CF3 / -Cl increase the cation charge density at the C2-carbon. We also synthesized similar derivatives, but this time with substituents at the C4-position (5-9). Surprisingly, most of the C4-substituted derivatives, except for (9) with the -CF3 substituent, maintained their potency compared to (1). If the effect of the substituents were purely due to the cationic charge density around the C2-carbon, then the C4 derivatives (6-9) with electron-withdrawing groups should be more potent than the C4-CH3 (5) derivatives with electron-donating methyl groups, just as 3 and 4 are more potent than 2, but (6-9) are not significantly more potent than (5). We also expected that the C4-CF3 derivative (9) with the electron-withdrawing group -CF3 would make the C2-carbon more cationic, thus more potent than the C4-CH3 derivative (5), just as the C2-CF3 derivative (4) is more potent than the C2-CH3 derivative (2). However, the trend for (5) and (9) is opposite to that for (2) and (4). The cationic charge density on the imidazolium ring alone does not seem to explain the observed MIC. We hypothesize that C4-substituted compounds with acidic hydrogen on the C2 carbon dissociate at physiological pH to form N-heterocyclic carbene (NHC), which is hydrophobic, facilitates entry into the cell membrane, and affects bactericidal effects within the cell.

[0364] Derivatives (2-4) lost their carbonic acid properties because their C(2)-H atoms were replaced by various substituents, including both weak electron-donating groups (methyl-(-CH3)) and electron-withdrawing groups (chloro-(-Cl) and trifluoro-(-CF3)). Compared with the parent compound OIM1-6-CH(1), these derivatives exhibited significantly reduced potency. Specifically, the Geo-MIC of the three non-carbonic acid compounds (2-4) with C2-substituents... 90 The values ​​ranged from 118.5 µg / mL to 344.6 µg / mL, significantly higher than the Geo-MIC observed for compound (1). 90The value is 4.0 µg / mL. Simultaneously, we synthesized similar derivatives (5-8) that introduced a substituent at the C4-position, giving them carbonic acidity while retaining the C(2)-H atom. Surprisingly, the C4-substituted derivatives (5-8) maintained their potent antibacterial activity, Geo-MIC 90 The values ​​ranged from 4.4 µg / mL to 23.8 µg / mL. These values ​​were significantly lower than the recorded values ​​for its isomers (2–4), highlighting the crucial role of carbonic acid in maintaining antibacterial efficacy.

[0365] To investigate the effect of chain length on OIM efficacy, we synthesized oligomers (10, 11, and 12) containing eight repeating units with -CH3 substituents (Figs. 1B and 17). Interestingly, OIM1-8-C2(CH3)(11), lacking C2-H, exhibited reduced efficacy compared to the parent OIM1-8-CH(10), a phenomenon similar to that observed in the OIM1-6 series. The parent OIM1-8-CH(10) is characterized by the presence of a C(2)-H functional group, whose Geo-MIC... 90 At 3.4 µg / mL, it is an order of magnitude more potent than OIM1-8-C2(CH3)(11), which is not carbonic acid (Geo-MIC) due to the lack of C(2)-H atoms. 90 = 43.1 µg / mL). Furthermore, OIM1-8-C4(CH3)(12) with C(2)-H exhibited the same potency as (10), with its Geo-MIC 90 It is 3.4 µg / mL.

[0366] We also developed a series of biodegradable OIMs (13-14) using acetals and amide bonds respectively (Figures 1C, 6-7 and 18), which have good antibacterial efficacy against various bacteria (Table 1).

[0367] We further expanded our exploration by developing two biodegradable OIM compounds (15-16) with unsubstituted C(2)-H bonds, one using carbonate and the other using secondary diamide bonds (Figures 1C and 19-20). These biodegradable carbonates maintained strong antibacterial efficacy against a variety of bacterial strains, and their Geo-MIC... 90 The values ​​were 3.6 µg / mL and 4.9 µg / mL, respectively (Table 1). The OIM1-8 and degradable series further confirm the importance of carbonation in achieving antibacterial efficacy.

[0368] Further testing of the parent compound and the -CH3 series (compounds 1, 2, and 5) against clinically relevant multidrug-resistant (MDR) bacteria revealed that the parent compound and the C4-substituted methyl derivatives (i.e., 1 and 5) exhibited potent antimicrobial activity, while the C2 derivative (2) was ineffective (Table 4). We found that OIM1-6-CH exhibited potent antimicrobial activity against most MDR strains, with a MIC of [missing value]. 90 The concentration is 2-4 µg / mL. OIM1-6-C4 has slightly lower antibacterial efficacy, with a MIC of 2-4 µg / mL. 90 Typically 2-4 times higher than OIM1-6-CH, while OIM1-6-C2 was ineffective against all tested MDR bacteria (MIC). 90 = 32-512 µg / mL). We also tested the bactericidal kinetics of three OIM derivatives (1, 2, and 5) against Staphylococcus aureus LAC and Pseudomonas aeruginosa (PAO1). Figure 21 OIM1-6-CH and OIM1-6-C4(CH3) exhibit bactericidal activity and rapid bactericidal kinetics. At concentrations 4 times the MIC, these compounds can completely eradicate both bacterial strains within 1-2 hours. On the other hand, the efficacy of OIM1-6-C2(CH3) is significantly reduced.

[0369]

[0370] We also tested the biocompatibility of OIM1-6 compounds (1-9) using different eukaryotic cells and found that OIM1-6-CH generally showed no short-term toxicity (i.e., 24 hours) but exhibited long-term toxicity (i.e., 48 and 72 hours) in all tested cell lines (i.e., 3T3 fibroblasts, human embryonic kidney (HEK) cells, and HepG2 cells) (Table 5, Figures 10-16). The derivative (5) with a -CH3 group at the C4 position generally showed stronger long-term toxicity compared to OIM1-6-CH. Simultaneously, increasing the chain length of OIM from 6 repeating units (compounds 1, 2, and 5) to 8 repeating units (compounds 10-12) resulted in increased cytotoxicity. The biocompatibility of the degradable compounds (13-14) was improved compared to their parent OIM.

[0371]

[0372] We also tested the biocompatibility of the OIM compounds in various eukaryotic cell lines. Both the parent OIM1-6-CH(1) and OIM1-8-CH(10) carbonates exhibited low short-term (i.e., within 24 hours) toxicity. However, they showed high long-term (i.e., within 72 hours) toxicity in all tested cell lines, including 3T3 fibroblasts, human embryonic kidney (HEK) cells, and hepatocellular carcinoma (HepG2) cells (Tables 1-3, Figures 10-16). However, some substituted carbonate derivatives, such as C4-methyl (5, 12), C4-chloro (6) compounds, and degradable derivatives (15, 16), showed higher selectivity indices (Geo-IC) compared to their respective parent molecules, OIM1-6-CH(1) and OIM1-8-CH(10). 50 / Geo-MIC 90 ).

[0373] To gain a deeper understanding of these findings, we conducted computational studies to calculate the cationic charges on the parent compound (1) and its methyl- and chloro-substituted derivatives. Our computational analysis showed that the imidazolium ring of compound (1) carries a charge of 0.286 e. Compared to (1) ， The C2- and C4-substituted methyl derivatives, namely the C2-CH3(2) and C4-CH3(5) derivatives, exhibit similar charges on the imidazolium ring (0.276e and 0.289e, respectively). In contrast, the chlorinated derivatives (3) and (6) with C2-Cl and C4-Cl substituents, respectively, exhibit moderately increased charges on their imidazolium rings (0.306e and 0.324e, respectively). Figure 22 However, it is noteworthy that despite these differences in cation charge density caused by different substituents, the MICs of the C2-substituted (CH3- and Cl-) non-carbonate derivatives (2) and (3) were significantly lower than that of (1), while the C4-substituted carbonate derivatives (5) and (6) exhibited good MIC values. This suggests that the antibacterial efficacy of the carbonate derivatives (1), (5), and (6) is not solely related to the cation charge density on the imidazolium ring. Rather, their antibacterial activity appears to be influenced by other factors, which may be related to the presence or absence of the C(2)-H moiety in carbonate.

[0374] We selected three key compounds, namely the parent compound (1) and two CH3-substituted derivatives (compounds 2 and 5), for further mechanistic studies to explore the correlation with carbonic acid properties. We conducted comprehensive testing of these compounds against an expanded group of clinically relevant multidrug-resistant (MDR) bacteria. Interestingly, while both the parent OIM1-6-CH (1) and the C4-methyl-substituted derivative (5) exhibited potent antibacterial activity, the C2-methyl-substituted derivative (2) did not (Table 6), further confirming that its potent antibacterial activity is related to its carbonic acid properties. We conducted time-kinetic kinetic studies of these three OIMs (1, 2, and 5) against methicillin-resistant Staphylococcus aureus (MRSA) LAC strain and Pseudomonas aeruginosa PAO1. Figure 21 Notably, compounds (1) and (5) are both bactericides and exhibit rapid bactericidal kinetics, completely eradicating bacteria within 1–2 hours at concentrations 4–8 times their MIC. In contrast, compound (2) showed reduced potency, failing to eradicate bacteria even with prolonged exposure (20 hours) at 8 times its MIC. Subsequently, we conducted a series of comprehensive mechanistic studies to investigate the entry of OIM1–6 parent compound (1) and its methyl-substituted derivatives (2 and 5) into bacterial membranes mimicking liposomes and live MRSA bacteria, thereby elucidating the specific role of carbonic acid and the deprotonated NHC form generated during OIM entry.

[0375]

[0376] Example 6. OIM carbonate with deprotonated NHC crosses the plasma membrane of bacterial mimic liposomes and enters the liposome nucleus.

[0377] We used the DiSC3(5) and propidium iodide test ( Figure 23 The effects of these compounds on the bacterial membrane potential and physical integrity of MRSA were evaluated. The results showed that OIM1-6-CH and its C2 / C4-methyl-substituted derivatives (1-2 and 5) produced minimal perturbation to the bacterial membrane potential and physical membrane integrity of Staphylococcus aureus. Figure 23 This finding indicates that the antibacterial effects of these compounds differ significantly from those of typical cationic polymers, which typically form physical pores or holes in membranes.

[0378] We hypothesize that NHC exists transiently as Brønsted carbonic acid in polymers containing imidazolium ( Figure 24A) This contributes to and ultimately leads to its superior antibacterial efficacy. At any given moment, some (but not all) of the cationic repeating units in the imidazoline-containing polymer are converted into uncharged hydrophobic NHC, thus transforming the originally cationic hydrophilic homopolymer into an amphiphilic copolymer. Figure 24 A), this copolymer is able to cross the bacterial membrane at low threshold concentrations, which is a necessary prerequisite for the copolymer to enter the cytosol and reach its intracellular targets. Figure 24 (B) This unique entry mechanism benefits from the initial electrostatic attraction of cationic polymers to anionic bacterial membranes, in stark contrast to the physical disruption methods employed by classic cationic polymers, which may require higher charge concentration thresholds, leading to reduced potency and selectivity. This paper aims to demonstrate that short-chain OIMs can easily enter via the formation of NHCs.

[0379] We hypothesize that for OIM carbonic acid, i.e., compounds (1) and (5), at physiological pH and temperature in a buffered aqueous solution, a portion of the imidazolium repeating unit with dissociable C(2)-H can be converted to NHC ( II , Figure 24 To detect the formation of NHC, we used D2O solvent. 1 1H NMR spectroscopy was used to measure the hydrogen-deuterium (HD) exchange at the C(2) site of the imidazolium cation (Amyes, TL). et al. , J.Am.Chem.Soc. 2004, 126 (4366-4374) (Figure 25A). In this case, in OH In the presence of ions, oligoimidazolium will exhibit two monomeric forms (Figure 25C), namely, azoleonium cation ( I ) and azole-2-methylenecarbene ( II ), and will form copolymers (poly( I-II For compound (1), at pH 7.16 and pH 8.21, the C(2)-H... 1 A rapid decrease in ¹H NMR (Fig. 25B) indicates a faster HD exchange rate. Conversely, at more acidic pH values ​​(pH 6.81 and pH 6.63), the HD exchange rate slows down due to lower deuteroxide (base) concentrations (Fig. 25B). For OIM1-6-CH dissolved in PBS at pH 7.16, we observed the presence of C2- protons. 1 The weakening of the H NMR signal (approximately 8.80 ppm) confirmed its acidic nature and the formation of NHC (Figure 25B). These findings support the view that compound (1), as a carbopolyacid with an imidazolium repeating unit, is readily deprotonated at physiological pH, releasing protons to form an uncharged and hydrophobic residual NHC ( IIRepeating units. We hypothesize that these hydrophobic repeating units of OIM enable the newly formed cation-NHC copolymer to penetrate the anionic bacterial plasma membrane, which is impermeable to the initial hydrophilic polycations.

[0380] In bacterial membrane mimicry, the conversion of cationic OIM to NHC was captured using two probes (carbazole and gold(dimethyl sulfide) chloride (AuDMSCl)) (Figure 25C). We used liposomes to mimic bacterial membranes, encapsulating either a hydrophobic carbazole dye or AuDMSCl within a liposome bilayer. As shown in Figure 25D, when the carbazole dye was encapsulated in liposomes, the emission spectrum exhibited two structured emission peaks at 345 nm and 360 nm, with humps near 380 nm and 400 nm. This is consistent with the spectral characteristics of carbazole monomers (Guo, Y). et al. , RSC Adv. 2016, 6 (86989-86997), indicating that carbazole exists in the lipid bilayer as a monomer. A stable carbene 1,3-bis(2,6-diisopropylphenyl)-1,3-dihydro-2H-imidazolium-2-yl) (IPr) was used as a positive control. When IPr was added to the liquid phase of the liposome suspension, the carbazole emission intensity of the carbazole (lipid) + IPr (aq) mixture was significantly reduced (up to 70%) compared to carbazole (lipid). The formation of a complex between carbazole and NHC via hydrogen bonding causes the dye to lose its fluorescence. The significant decrease in emission intensity after adding IPr (aq) confirmed the formation of the [carbazole-IPr] complex, which is likely a hydrogen-bonded adduct between IPr and carbazole via C··HN bonds. Furthermore, we observed a 30% reduction in carbazole emission intensity for carbazole (lipid) + OIM1-6-CH, confirming the formation of NHC and its [carbazole-(OIM1-6-CH NHC)] complex in the lipid bilayer.

[0381] To demonstrate the presence of NHC in a bacterial mimic membrane, we attempted to capture OIM-NHC in anionic liposome bilayers using two NHC probes: carbazole and gold(dimethyl sulfide) chloride (AuCl(SMe2)) (Figure 25C). We constructed negatively charged liposomes mimicking bacterial plasma membranes and embedded NHC probes within the bilayer. These probe-lipid complexes are denoted as probe(lipid), such as carbazole(lipid) and Au(lipid). It has been reported that when stable NHC (such as 1,3-bis(2,6-diisopropylphenyl)-1,3-dihydro-2H-imidazolium-2-ylene(IPr)) binds to carbazole in a free state (e.g., in organic solvents), the resulting NHC-carbazole adduct (formed by hydrogen bonding between the carbene carbon with a lone pair electron and the R-NH of carbazole) leads to photoluminescence quenching (Kieser, JM). et al. , J.Am.Chem.Soc. 2019, 141 , 12055-12063). Our experiments showed that the addition of IPr (control) to the carbazole (lipid) aqueous solution significantly reduced the emission intensity of carbazole by about 70%, supporting the formation of the [carbazole + IPr NHC] complex (Fig. 25D). Similarly, when OIM1-6-CH (1) was introduced into the carbazole (lipid) solution, a small decrease in the emission intensity of carbazole was observed, confirming the presence of OIM-NHC in the membrane, which led to the formation of the [carbazole + OIM-NHC] complex in the lipid bilayer (Fig. 25C).

[0382] Furthermore, previous studies have shown that AuCl(SMe2) can react with NHC under mild alkaline conditions to form a covalently linked [AuCl(NHC)] complex (Nahra, F). et al. , Nat. Protoc. 2021, 16(1476-1493) (Fig. 25A). Hydrophobic AuCl(SMe2) was placed in a liposome bilayer to attempt to capture NHC generated by OIM. Then, an aqueous solution of OIM1-6-CH(1) was added to the Au(liposome) suspension. Due to the hydrophobicity of the AuDMSCl molecule, it was expected to remain in the liposome bilayer. When added to Au(liposome) in phosphate buffer at pH 7.4, the positively charged OIM1-6-CH was adsorbed onto the negatively charged glycerol phosphate-containing liposome surface by electrostatic attraction. If OIM1-6-CH penetrated the liposome and formed NHC in the liposome bilayer, the pre-encapsulated AuDMSCl may react with OIM1-6-CH NHC to form Au-OIM1-6-CH (Fig. 25J(i)), which can be detected by liquid chromatography-electron ionization mass spectrometry in positive ion mode (LC-ESI-MS(+)). The reaction products of Au (lipids) and OIM1-6 may form the reaction shown in Figure 25J(ii). Therefore, after incubation and removal of lipids, the reaction products were analyzed using liquid chromatography combined with electron ionization mass spectrometry in positive ion mode (LC-ESI-MS(+)). A major total ion chromatogram (TIC) peak was observed at elution time of 3.81 min, and two extracted ion chromatogram (EIC) peaks with observed m / z values ​​of 199.15 and 330.52 at 3.55 min and 4.04 min, respectively (Figures 25E and 25F(i)-(ii)). Based on the 0.25 unit mass spectrometric (MS) gap between adjacent ions in the isotopic cluster (Figure 25H(i)), the EIC peak at m / z = 199.15 was attributed to unreacted (excess) OIM1-6-CH (1) (Figure 25G(i)). The 0.25 unit gap between adjacent ions in the isotopic cluster indicates that the ion contains 4 charges (z = 4), thus the total ion mass is calculated to be 796.60, which corresponds to OIM1-6-CH containing 4 positive charges and 2 NHCs (Fig. 25J(ii)). Since MS of Au-OIM1-6-CH (Fig. 25H(ii)) shows a 0.33 unit gap between adjacent ions, the EIC peak at m / z = 330.52 is identified as Au-OIM1-6-CH (Fig. 25G(ii)). The assignment of these two structures is further confirmed by HD exchange of C(2)-H and structural analysis of the fragments (both using LC-MS / MS analysis) (Fig. 26). In the MS spectrum of Au-OIM1-6-H (Fig. 25H(ii)), the gap between adjacent ions is 0.33, which indicates that the ion contains 3 positive charges (z = 3), and the corresponding mass (m) of the total ion is 991.56, with the structure Au-OIM1-6-CH containing one Au atom (Fig. 25J(ii)).The structural assignment was confirmed by hydrogen-deuterium exchange and MS / MS analysis (Figure 27). In summary, the LC-MS peak at m / z 330.52 observed in the bacterial simulated liposome bilayer containing AuDMSCl confirmed that OIM1-6-CH can form carbenes in the hydrophobic membrane bilayer. We demonstrated that NHCs formed in the aprotic liposome bilayer for a sufficiently long time to react with the embedded Au (lipid) probes and form stable Au-OIM(1) within the membrane. These results provide conclusive evidence that some repeating units of OIMs are converted to the NHC state, allowing the resulting copolymer to enter the bilayer membrane, and that NHCs remain in the aprotic liposome bilayer for a sufficiently long time to react with the embedded Au (lipid) probes to form stable Au-OIM1 (lipid), thus confirming that OIM1-6-CH(1) forms NHCs and penetrates the membrane bilayer.

[0383] We also explored the possibility of NHC formation in two methyl-substituted derivatives, OIM1-6-C2(CH3)(2) and OIM1-6-C4(CH3)(5). Under pH 7.16, the H2O of the C(2)-H in compound (5) was... 1 The H NMR exchange rate was lower than that of compound (1), and exchange inhibition was observed at acidic pH values ​​(6.63 and 6.81) (Fig. 28A-B). Compound (2) lacked C(2)-H and did not undergo H-D exchange at either neutral or acidic pH values. At an alkaline pH of 8.21, compound (2) showed no reaction and no NHC formation, while NHC formation of compound (5) increased, reaching a stable state with 77% H-D exchange. We further investigated the effect of a more alkaline pH (8.2) on NHC formation of compounds (2) and (5) in an aprotic bilayer using an AuCl(SMe2) probe. Compound (5) (instead of compound (2)) exhibited NHC formation, and Au-OIM (5) was also separable in the membrane bilayer, although not as easily as compound (1) (Fig. 28C-H). This is consistent with the result that the methyl group on compound (5) has an electron-donating effect, which reduces the sensitivity of its C(2)-H to the deprotonation reaction, thus resulting in a weakened NHC formation compared to compound (1).

[0384] We further quantified (i) the total uptake of OIM by liposomes (including the liposome membrane and nucleus) and (ii) the uptake of OIM by the liposome nucleus alone (Fig. 25I). The latter indicates that OIM is readily taken up by bacterial cytosol, thereby achieving intracellular targeting. Our results showed that at physiological pH (7.4), the total uptake of compounds (1) and (5) was significantly higher than that of compound (2), and only compounds (1) and (5) entered the liposome nucleus, while compound (2) did not. At the acidic pH of 6.8, which inhibits carbene formation, the total uptake of all three compounds decreased significantly, and the nuclear uptake became negligible. Therefore, the degree of nuclear uptake of these three compounds is related to the ease of NHC formation. At physiological pH, compound (1) was the most likely of the three compounds to form NHC, and its nuclear uptake was the highest. In contrast, compound (2) could not penetrate the bilayer membrane due to its inability to form hydrophobic NHC, and therefore failed to enter the nucleus (Fig. 28A). Although compound (5) can also form NHC, the degree is lower than that of compound (1), and its nuclear uptake is in the middle among the three compounds.

[0385] To further verify our hypothesis that NHC formation initiates membrane entry, we performed computer simulations to analyze the interactions between the pure cationic form of OIM1-6-CH(1) and the pure NHC form (denoted as OIM and OIM-NHC, respectively) and a simulated Staphylococcus aureus membrane. The simulated OIM has six repeating cationic ring units, while OIM-NHC has six repeating NHC units. After being placed outside the constructed membrane surface, OIM-NHC formed a large number of stable contacts with the membrane, while the interaction between the cationic OIM and the membrane was limited ( Figure 29 AB). It is worth noting that cationic OIMs tend to bind to the membrane surface ( ). Figure 29 C), while OIM-NHC immediately penetrates and remains inside the hydrophobic interior of the membrane bilayer (C ...). Figure 29 D). These results support the view that the formation of NHC enhances the hydrophobicity of OIM, thereby facilitating its insertion into the bacterial membrane bilayer.

[0386] In summary, our study shows that the acidic hydrogen atom located on the C2-carbon of the imidazole ring in carbonates (1) and (5) can dissociate at physiological pH to form NHC. This phenomenon was detected by NMR in water and by embedded carbazole and AuCl(SMe2) probes in the liposome membrane bilayer. Compared with compound (5), compound (1) exhibits a stronger tendency to form NHC, with a pH of [missing value]. K aThe value was 21.32, lower than 22.15 for (5) (Table 7). Furthermore, computer simulations confirmed that the formation of NHC in carbonate (1) facilitates its entry into the membrane bilayer, as the formation of uncharged NHC increases the hydrophobicity of OIM. Therefore, the formation of NHC “hydrophobizes” OIM (1) and (5), thereby facilitating the oligomer’s entry into the membrane bilayer, a necessary prerequisite step for endocytosis into the liposome core. Conversely, the methyl substitution on the C2-carbon in compound (2) prevents the formation of non-carbonic carbene, thus hindering the entry of charged hydrophilic OIM into the bilayer and the liposome core.

[0387]

[0388] The acidity of various OIM carbonates (1, 5-9) may be an important factor in their good antibacterial effects. We... 1 H NMR was used to measure the p-values ​​of different OIMs (1, 5-9). K a Values ​​(Amyes, TL) et al. , J. Am. Chem. Soc. 2004, 126 (4366-4374), found that p of OIM1-6-CH(1) K a The value was 21.32 (Table 7), thus confirming (1) as carbonic acid. Among the tested derivatives, the weakest acidic carbonic acid was OIM1-6-C4(CH3) (5), p K a The value is 22.15, while the strongest acid is OIM1-6-C4(CF3) (9), p K a The value is 20.06, indicating that the acidity of OIM1-6-CH(1) is moderate (p). K a The value is 21.32). In (5), the electron-donating group -CH3 weakens the acidity of the C2 proton, resulting in its p K a The value is higher than (1). Conversely, electron-withdrawing groups (-Cl, -F, -Bz, and -CF3 in 6-9) enhance the acidity of the C2 proton, resulting in p in (6-9). K a The value decreased.

[0389] Example 7. Synthesis of FITC-conjugated OIM

[0390] General Program 10

[0391] Triethylamine (5 equivalents, 27 μL, 0.193 mmol) was added to a 1 mL solution of anhydrous dimethyl sulfoxide (DMSO) containing the desired starting materials 1, 2, and 5 (1.2 equivalents). The reaction mixture was stirred at room temperature for 10 minutes. Then, FITC (1 equivalent, 15 mg, 0.0385 mmol) dissolved in 0.5 mL of DMSO was added dropwise to the reaction mixture, and the mixture was stirred overnight at room temperature in the dark. The reaction mixture was diluted with water and transferred to a dialysis bag with a molecular weight cutoff of 500–1000 Da. Dialysis was performed with 1 mL HCl in 1 L of deionized water for 24 hours, with the dialysis water changed frequently every 2–3 hours. The resulting solution was concentrated by rotary evaporation and lyophilized to give the desired product, a mixture of unreacted starting materials, monoconjugated product, and diconjugated product. Based on the integration of signals in the 3.80–4.40 ppm and 6.00–6.70 ppm regions, the desired product was obtained using… 1 HNMR was used to estimate the dye conjugation percentage of each parent compound in these two regions, corresponding to the 12 x imidazolyl-N-CH2- in the parent OIM compound and the 2 x phenolic 3 x Ar-H in FITC, respectively. The 24:6 integration of the signals in the 3.80–4.40 ppm and 6.00–6.70 ppm regions was inferred as 100% conjugation (the ratio between the parent compound and the FITC dye was 1:1). Therefore, the dye conjugation percentage (expressed as a percentage) of each parent compound was obtained by calibrating the integration of the 3.80–4.40 ppm region signal to 24 and dividing the obtained integration value of the 6.00–6.70 ppm region signal by 6.

[0392] Example 8. Intracellular accumulation of OIM derivatives in bacteria is related to their carbonicity and the ease with which NHC is formed.

[0393] We then investigated the uptake of three OIMs (1, 2, and 5) in bacteria, particularly in the parental MRSA LAC strain and its respiratory-deficient mutant (with lower PMF).

[0394] OIM-FITC uptake test

[0395] The OIM-FITC uptake assay was performed according to the previously described method (Radlinski, LC). et al. , Cell Chem. Biol. 2019, 26 , 1355-1364; Avelar-Freitas, B. et al. , Braz. J. Med. Biol. Res. 2014, 47, 307-315; and Benincasa, M. et al. , Bio-protoc. 2016, 6 (e2038-e2038). Prepare exponential phase bacteria and dilute to 10 in TSB broth. 6 CFU / mL, then incubated with FITC-conjugated OIM (OIM-FITC molar ratio of 7.5%) at the desired concentration for 1 h in the dark. The cultures were centrifuged (10,000 rpm, 10 min) and washed twice with filter-sterilized PBS. The bacterial pellet was resuspended in 800 µL of PBS. Fluorescence of 150 µL of each sample was measured directly using the FITC channel of a flow cytometer (BD FACSVerse flow cytometer (3) laser), which provides data for unquenched uptake (Fig. 30). 300 µL of the remaining sample was quenched with 0.01% trypan blue (TB) dye before measurement, where fluorescence data provided total (i.e., bacterial membrane and cytosol) OIM uptake. Another 300 µL of the remaining sample was treated with 0.04% Triton X-100 (TX) for 15–20 min, followed by TB quenching, after which FITC fluorescence was measured, providing data for membrane fractional uptake. The histogram difference between total uptake and membrane-bound uptake provides the cytoplasmic uptake fraction. Detailed experimental procedures and flow cytometry histograms are shown in Figure 30. Bacteria not treated with OIM-FITC served as a negative control. The internalized OIM fraction was further validated by permeabilizing OIM-FITC-treated bacteria with 0.04% Triton X-100 for 15–20 minutes, followed by trypan blue quenching, and FITC fluorescence was subsequently measured. The fluorescence signal of cytoplasmic-internalized OIM-FITC in permeabilized bacteria was significantly reduced after quenching because trypan blue quenches cytoplasmic-internalized OIM-FITC; however, membrane-embedded OIM-FITC cannot be removed by trypan blue quenching. The former was observed in bacteria treated with SYTO 9, OIM1-6-CH-FITC, and OIM1-6-C4(CH3)-FITC, while the latter was observed in bacteria treated with OIM1-6-C2(CH3)-FITC. The internalized portion of OIM1-6-C2(CH3)-FITC is the difference between the trypan blue quenching fluorescence of unpermeable bacteria and the trypan blue quenching fluorescence of permeable bacteria.

[0396] Results and Discussion

[0397] Three OIM derivatives were labeled with FITC fluorescent dyes (as described in Example 7), and their uptake was quantified using flow cytometry (Figure 30). Given the high cell density required for flow cytometry experiments (10T), 6(CFU / mL), we measured MIC at higher cell densities. Values. To measure the internalized portion of FITC-OIM, we used 0.01% trypan blue (TB) dye to quench extracellularly bound FITC conjugated oligomers, as this dye cannot penetrate the cell membrane (Antonoplis, A). et al. , J. Am. Chem. Soc. 2018, 140 , 16140-16151; Avelar-Freitas, B. et al. , Braz. J. Med. Biol. Res. 2014, 47 , 307-315; and Benincasa, M. et al. , Bio-protoc. 2016, 6 e2038-e2038 ） To obtain fluorescence of the internalized portion without considering difficult-to-remove extracellular components (e.g., portions embedded in the plasma membrane), we treated the bacteria with TB-quenched FITC-OIM and subtracted the fluorescence from surfactant permeation (to release the internalized FITC-OIM) followed by TB-quenched FITC-OIM treatment.

[0398] The average MIC of the three compounds against the parental Staphylococcus aureus strain The values ​​were as follows: 12 μg / mL (1), >4096 μg / mL (2), and 48 μg / mL (5) (Fig. 31A), consistent with the MIC values ​​shown in Table 1. We assessed the percentage of LAC cell counts by (i) total uptake (into the membrane and cytosol) and (ii) cytoplasmic uptake. For compound (1), the percentage of Staphylococcus aureus strains exhibiting both cytoplasmic and total uptake was significant at all tested concentrations (Fig. 31B), indicating that a considerable proportion of bacteria had taken up compound (1) into their cytosol. This was supported by the higher mean fluorescence intensity (MFI) observed in cells treated with compound (1), which exhibited both cytoplasmic and total uptake (Fig. 31C). In the case of compound (5), the percentage of bacterial counts with cytoplasmic uptake was lower at low OIM concentrations, but higher at 32–64 μg / mL (its MIC). The MFI values ​​for total uptake and cytoplasmic uptake were significantly increased in cells treated with compound (5), confirming that compound (5) readily enters the cytoplasm within its effective concentration range. Conversely, for compound (2), the percentage of bacterial counts with cytoplasmic uptake was low at all tested concentrations, despite the correspondingly high MFI for this fraction, indicating that only a small fraction of cells took up compound (2). However, for those cells that did take up compound (2), they absorbed a significant amount of compound (2). The total number of cells showing total uptake of compound (2) was high, but the MFI for this fraction was low, indicating that many cells absorbed only a small amount of compound (2) adhering to their surfaces. When the results (Fig. 31B) were correlated with the MIC values ​​of compounds (1), (2), and (5) (Table 1), it was evident that there was a negative correlation between the MIC of the compounds and the percentage of LAC cell counts with cytoplasmic uptake. Specifically, compounds (1) and (5), which had low MIC values ​​and were carbonated, showed higher cytoplasmic uptake. Conversely, compound (2) has a high MIC value and is a non-carbonate compound, which shows low cytoplasmic uptake.

[0399] Among the three OIM1-6 derivatives, we observed the highest total uptake of OIM1-6-CH at low concentrations (4 and 8 μg / mL) (Figure 32A), while OIM1-6-C2(CH3) and OIM1-6-C4(CH3) showed low total uptake at these concentrations. Furthermore, at these low concentrations, OIM1-6-CH exhibited the highest internalization rate. However, the internalization percentage of OIM1-6-C4(CH3) increased significantly at 16 µg / mL, which is the MIC of OIM1-6-C4(CH3). (with 10) 6 (CFU / mL bacterial-related MIC value). Within the concentration range of 4-16 µg / mL, the amount of OIM1-6-C2(CH3) internalized into the cytosol is minimal (approximately 10%), but when the concentration reaches 128 µg / mL, its internalization amount increases significantly, which is also its MIC. Values. Similar trends were observed in the uptake of OIM by Staphylococcus aureus LAC tested in TSB medium: MIC values ​​for OIM1-6-CH and OIM1-6-C4(CH3) were also observed. Higher internalization rates were observed at concentrations of 8 µg / mL and 64 µg / mL, respectively (Figure 32B). For TSB medium, OIM1-6-C2(CH3) showed no MIC within the tested concentration range. In summary, at a fixed concentration, OIM1-6-CH exhibits the highest internalization efficiency, followed by OIM1-6-C4(CH3), both of which are carbonic acids capable of forming NHC. OIM-1-6-C2(CH3), lacking a dissociable C2 proton, shows poorer internalization, indicating that the presence of C2 hydrogen, which allows for NHC formation, is crucial for polymer internalization, and that polymer internalization is related to compound potency.

[0400] To better understand the relative contributions of these two factors (NHC formation and PMF) to the entry of compounds into bacterial cytosol, we conducted additional tests, as described below.

[0401] Under pH 7.2 conditions, the MRSA LAC parent strain was combined with a respiratory mutant lacking PMF (especially LAC Δ). menD and LAC Δ hemB When comparing (Fig. 31D), we observed that in the case of PMF absence (while NHC is still present), the MIC of (1) The concentration increased by 8-16 times. This result confirms the important role of PMF in antibacterial efficacy. Furthermore, under pH 7.2 conditions, the parental strain showed a significantly higher MIC compared to the mutant. The percentage of bacterial cell counts exhibiting total / cytoplasmic uptake was highest at the lower pH (Fig. 31E), supporting the view that cytoplasmic uptake is at least partially promoted by PMF. When comparing the MIC of the parental strain at different pH values ​​(7.2, 6.8, and 6.6), (1) At time (Fig. 31D), we observed MIC The concentration increased from 12 μg / mL to 64 μg / mL, a 5.3-fold increase, while the uptake decreased to some extent (Fig. 31E, first figure), but was still considerable, due to the high PMF content in the parent strain. This pH-dependent reduction in uptake confirms the importance of NHC formation in the uptake process, as acidic pH inhibits NHC formation, thereby reducing uptake and bactericidal effect. Therefore, the newly discovered NHC formation and PMF both play key roles in the antibacterial efficacy of (1) carbonic acid.

[0402] To further evaluate the importance of NHC in live bacteria, we used the MRSA LAC respiratory defect mutant to minimize the impact of PMF and evaluated the effect of pH on MIC. The effect. For the mutant treated with (1) (Fig. 31D), weakly acidic pH values ​​of 6.6 and 6.8 respectively affected the MIC The concentrations significantly increased to 1,024 µg / mL and ≥ 4,092 µg / mL. At these acidic pH values, even at very high concentrations exceeding 4,092 µg / mL, OIM(5) failed to kill the mutants. In contrast, the MIC of gentamicin was within the tested range. They showed only a weak dependence on pH (Figure 31D). Furthermore, at pH 6.8, the percentage of bacteria present in the cytoplasm and total uptake was significantly lower in both mutants than at pH 7.2, which is consistent with their MIC. The increase in values ​​is related (Fig. 31D). Therefore, in mutants with reduced PMF, inhibition of NHC formation greatly hinders intracellular uptake of OIM, resulting in poor bactericidal effect. This test at acidic pH further highlights the importance of NHC formation for (1) internalization.

[0403] To further assess the relative importance of NHC formation and PMF in the efficacy of OIM, we compared the bactericidal kinetics of compounds (1) and (5) against the parental LAC strain under oxidative, anoxic, and fermentation growth conditions (Fig. 31F). The parental LAC strain possessed an active electron transport chain (ETC) under both aerobic and anaerobic respiration conditions, but not under fermentation growth conditions. Although compound (1) exhibited rapid bactericidal kinetics under both oxidative and anoxic conditions, it still showed significant bactericidal activity under fermentation growth conditions, albeit with lower efficacy. This suggests that compound (1) can still achieve bactericidal activity in the absence of PMF or active ETC because it forms robust NHC, which promotes entry and internalization. On the other hand, OIM (5) showed poor bactericidal activity under both anaerobic and fermentation growth conditions, indicating its strong dependence on PMF, as it does not readily form NHC to promote entry.

[0404] In summary, these data indicate that (1) the formation of NHC from carbonic acid facilitates cytoplasmic uptake. Inhibition of NHC formation under acidic pH conditions leads to a significant reduction in antibacterial efficacy and cytoplasmic uptake. Furthermore, (1) is effective in killing bacteria even under low PMF conditions because it can utilize NHC for internalization. (2), being a non-carbonic compound, cannot form NHC, which hinders its ability to reach the bacterial cytosol, greatly weakening its antibacterial efficacy. OIM (5), due to its weak carbonicity (Fig. 27), has a weaker ability to form NHC and therefore relies more on PMF for intracellular uptake, as evidenced by its inability to kill low PMF mutants (Fig. 31D) and its ineffectiveness under fermentation growth conditions (Fig. 31F). In summary, these findings support the hypothesis that the formation of carbene from carbonic acid (1) and, to some extent, (5) promotes their entry into bacteria, thus giving them a potent MIC value.

[0405] Example 9. The binding of carbene-forming OIM1-6-CH to the bacterial surface and its intracellular endocytosis are independent of PMF.

[0406] Evolution of LAC and PAO1 resistance to OIM

[0407] Subcultures of *Staphylococcus aureus* LAC or *Pseudomonas aeruginosa* PAO1 were added to different concentrations of OIM, ranging from 0.5x MIC to 4x MIC according to the MIC protocol, with 8–10 independent replicates. After 18–24 hours, samples from each replicate that showed at least 50% growth at the highest OIM concentration were selected for the next round of subculture. The OIM concentration was adjusted based on the mutations obtained in the strain. Subcultures were repeated daily until the mutants acquired high resistance (MIC value ≥1024 µg / mL) or PAO1 was subcultured for 30 days. On each day of subculture, bacterial samples were stored at -80°C with 30% glycerol. At the end of the experiment, LAC mutants were streaked onto agar plates. Two colonies from each plate were selected for resistance stability testing. These LAC mutant strains were subcultured for 7 days without OIM, and the MIC was tested on days 1 and 7. Mutants exhibiting stable resistance were selected for whole-genome sequencing. Genomic DNA was extracted using the QIAamp DNA Mini Blood Mini Kit (with minor modifications). Cell walls were digested with 10 mg / mL lysozyme and 10 µg / mL lysostaphin, and bacteria were lysed using a stirrer before entering the standard operating procedure of the kit. The prepared DNA was sequenced using the Illumina Nextera DNA Library Preparation Kit. DNA sequencing (paired-end sequencing) was performed on an Illumina MiSeq instrument. Sequences were aligned to the genome of the parental strain *Staphylococcus aureus* LAC, and single nucleotide variants, small deletions, and insertions were identified using CLC Genomics Workbench software. Large deletions were identified by manual sequence comparison.

[0408] Oxygen consumption rate (OCR)

[0409] OCR was measured using a Seahorse XFe96 extracellular throughput analyzer (Seahorse Bioscience). Prior to use, the sensor cartridge was equilibrated according to the supplier's instructions, and the sample cartridge was coated with poly-D-lysine to promote bacterial adhesion. LAC passage cultures were grown to the exponential phase in M9 buffer (1x M9 salt solution, 2 mM MgSO4, 0.2% (w / v) glucose, 0.2% (w / v) casein amino acids, 0.2 µg / mL nicotinamide, 100 nM thiamine). Then, 100 µl of a 10% concentration of [unspecified ingredient] was added to each well of the sample cartridge (except for blank wells).6 CFU / mL bacteria. Centrifuge the sample cartridge at 1400xg for 10 minutes, then add 80 µl of M9 buffer to each well. Measure the baseline OCR and read three times before injecting OIM / antibiotic.

[0410] Time-sterilization test under anaerobic and fermentation conditions

[0411] TSB was used as the growth medium under aerobic, anaerobic, and fermentation growth conditions. Under anaerobic conditions, 100 mM sodium nitrate was added to the TSB. All liquid media, pipettes, and consumables used for anaerobic and fermentation growth conditions were equilibrated in an anaerobic chamber for at least 2 days. Overnight and subculture cultures were performed in an anaerobic chamber at 37°C. For the time-kill assay, 20 µl aliquots were taken at specified time points and serially diluted with 180 µl PBS. The diluted aliquots were spotted onto agar plates, and colonies were counted after overnight incubation.

[0412] Antagonism test

[0413] The antagonistic effect of PMF dissipators (CCCP, valamicin, and nigericin) on the OIM1 compound was tested using a checkerboard assay with Staphylococcus aureus LAC. Serial 2-fold dilutions of the PMF dissipators (starting at 0.5x MIC) and the OIM1 compound (starting at 64 µg / mL) were prepared in TSB. 25 µL of PMF dissipator (along the y-axis) and 25 µL of OIM1 compound (along the x-axis) were added to each well of a 96-well plate, followed by 50 µL of logarithmic growth phase bacteria (prepared according to the MIC protocol in Example 5). The solutions were thoroughly mixed, and the MIC was then determined according to the method described above in Example 5.

[0414] Results and Discussion

[0415] To understand the internalization mechanism of OIM oligomers, we evolved Staphylococcus aureus LAC and Pseudomonas aeruginosa PAO1 mutants to enhance their resistance to OIM1-6-CH and OIM1-6-C4(CH3). These mutants were obtained through sequential passage in media containing progressively increasing concentrations of antimicrobial OIM compounds. Figure 33(AB). Compared to the antibiotic control group, LAC evolved to have significantly enhanced resistance to OIM, while PAO1 evolved to have decreased resistance to OIM. We obtained LAC strains with stable enhanced resistance, and subsequently identified genomic mutations by whole-genome sequencing (Tables 8-9). The evolved LAC mutants were mutated in genes encoding ETC components and genes associated with cationic antimicrobial polymer (CAMP) resistance, confirming that impaired cytoplasmic uptake of bacteria and surface binding contribute to bacterial resistance.

[0416]

[0417] To investigate whether the ETC mutation in Staphylococcus aureus LAC is caused by a direct target of the ETC component of OIM, we subsequently used a respiration assay on the Seahorse XFe96 platform to study the oxygen consumption rate (OCR) of Staphylococcus aureus LAC (Lobritz, MA). et al. , Proc. Natl. Acad. Sci. USA 2015, 112 , 8173-8180; and Dwyer, DJ et al. , Proc. Natl. Acad. Sci. USA 2014, 111 (E2100-E2109). Unlike the respiratory agent 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), the OCR of LAC did not decrease immediately after the addition of OIM1-6-CH. Figure 33 (C) Therefore, OIM1-6-CH does not kill bacteria by blocking oxygen respiration. The decrease in OCR observed 30 minutes after treatment (i.e., after the first doubling time) is attributed to growth inhibition and death, rather than a direct effect on oxygen consumption. We also did not observe any disruption to OCR by OIM1-6-C2(CH3) and OIM1-6-C4(CH3). These results suggest that OIM does not directly target the oxidative phosphorylation pathway, and that the ETC mutation reduces respiration, thereby lowering the bacterial PMF in an attempt to reduce compound uptake.

[0418] In addition, we used respiratory defect mutants, particularly LAC strains that are unable to consume oxygen due to the lack of functional ETC. ΔmenD and LAC ΔhemB ( Figure 33C), to investigate the effect of PMF on OIM uptake. We also measured the surface binding and uptake of OIM1-FITC compounds in these respiratory-deficient mutants (in TSB), which had low PMF. Our results showed that all OIM1-dependent uptake of OIM1 in LAC was significantly improved compared to wild-type Staphylococcus aureus LAC treated in TSB (Figure 32B). ΔmenD and LAC ΔhemB Both binding and internalization were low (Figure 32C-D). After quenching surface-bound FITC-OIM with trypan blue, the internalization of OIM1-6-C2(CH3) and OIM1-6-C4(CH3) derivatives was also very low in these mutants. This indicates that these OIM derivatives have low interaction with their internal targets, which is consistent with their high MIC. 90 Related. However, the parent OIM1-6-CH in its MIC Even at a concentration of 128 µg / mL, significant intracellular uptake was still observed in these mutant strains. This suggests that, in addition to PMF-driven factors, another factor influences the internalization of OIM1-6-CH into LAC. ΔmenD and LAC ΔhemB It plays a role in mutant strains, indicating that the uptake of OIM1-6-CH is not entirely dependent on PMF.

[0419] We also determined the MIC of the compound against the respiratory mutant at pH 7.0. (LAC) ΔmenD and LAC Δ hemB Increased resistance to OIM1-6CH and OIM1-6-C4(CH3) (Table 10, pH 7). When using OIM1-6-CH, MIC... 90 The MIC increased from 2 µg / mL for the wild-type strain to 32 µg / mL for these mutants; while with OIM1-6-C4(CH3), the MIC increased from 4 µg / mL to 256-512 µg / mL, indicating that OIM requires PMF to be taken up and exert its antibacterial efficacy. OIM1-6-C2(CH3) was ineffective against both mutants.

[0420]

[0421] To further validate our hypothesis regarding the NHC dependence of OIM 1-6-CH uptake, we tested the MIC of OIM in slightly acidic TSB (pH 6.6 and pH 6.8) using Staphylococcus aureus LAC and respiratory mutants (Table 10). An acidic environment inhibits carbene formation of OIM. Figure 34This reduces the potency of our oligomers. Our results indeed show that, under slightly acidic pH conditions, OIM1-6-CH and OIM1-6-C4(CH3) significantly increased the MIC of wild-type LAC, while having minimal effect on the potency of gentamicin and daptomycin. Under slightly acidic pH conditions, the increase in MIC of OIM1-6-C4(CH3) was significantly greater than that of OIM1-6-CH (64-fold vs. 16-fold, respectively), indicating a greater dependence of NHC formation on the potency of OIM1-6-C4(CH3). We also observed that for LAC… ΔmenD and LAC ΔhemB The mutants showed significant ineffectiveness towards both OIM1-6-CH and OIM1-6-C4(CH3) with a slight decrease in pH, while having minimal effect on gentamicin and daptomycin. In TSB at pH 6.8, by adjusting their respective MICs... The uptake assays further confirmed the reduced potency of OIM1-6-CH and OIM1-6-C4(CH3) for wild-type LAC (Figure 32E). At pH 6.8, both surface binding and cytosolic endocytosis of OIM1-6-CH and OIM1-6-C4(CH3) were significantly reduced. At pH 6.8, LAC... ΔmenD and LAC ΔhemB Similar phenomena were observed in the uptake of OIM1-6-CH (Figure 32F). This suggests that carbene formation is crucial to the potency of OIM, and that inhibiting carbene by slightly acidic pH reduces the potency of our compound, especially for respiratory mutants with low / zero PMF.

[0422] To confirm the aforementioned carbene-dependent uptake, we measured the bactericidal kinetics of OIM1-6-CH and OIM1-6-C4(CH3) on LAC under aerobic, anaerobic, and fermentation conditions, respectively. Figure 33The PMF of bacteria differed under these three conditions; only under the first two conditions was the bacterial ETC active. Although OIM1-6-CH exhibited rapid bactericidal kinetics under both aerobic and anaerobic conditions, it still showed significant bactericidal activity under PMF-free fermentation conditions, despite slower bactericidal kinetics. This again demonstrates that OIM1-6-CH can still achieve bactericidal activity in the absence of active ETC / PMF. On the other hand, OIM1-6-C4(CH3) showed poor bactericidal activity under both anaerobic and fermentation conditions, indicating its strong dependence on PMF. For OIM1-6-CH, in the respiratory mutant, uptake occurred in the absence of PMF, and bactericidal activity was observed even in fermentation mode, suggesting that the uptake of OIM1-6-CH depends on a second mechanism besides PMF, most likely an NHC-related mechanism, which contributes to its intracellular accumulation and bactericidal activity. OIM1-6-C4(CH3) has a weaker ability to form NHC, therefore its intracellular uptake is more dependent on PMF. OIM1-6-C2(CH3), on the other hand, cannot form NHC, resulting in ineffective bacterial entry and thus poorer efficacy. In summary, these data indicate that carbene formation of OIM1-6-CH and OIM1-6-C4(CH3) promotes their internalization into bacteria, thereby achieving good MIC.

[0423] Example 10. Cationic OIM1-6-CH uniquely eradicates Escherichia coli and Staphylococcus aureus in anionic surfactant formulations.

[0424] Most cationic compounds are severely limited in their application due to their charge-neutralizing properties, making them unable to eradicate bacteria in anionic formulations. One such formulation is laundry detergent, which is primarily composed of anionic surfactants, along with builders, chelating agents, bleaching agents, enzymes, and stabilizers. These ingredients are carefully formulated to effectively remove dirt, grime, and stains from textiles. Here, we tested the antimicrobial performance of OIM and two commercially available antimicrobial compounds (polydiallyl dimethyl ammonium chloride (PDADMAC) and colistin) at a concentration of 100 ppm in commercial laundry detergent (specifically, 389 ppm Japanese bagged laundry detergent from Procter & Gamble) and three other analog detergents (100 ppm SDBS, 100 ppm SDS, and 50 ppm SDS plus 50 ppm SDBS). For a compound to pass the test, it must provide a reduction of at least 2 log against Escherichia coli 8739 and Staphylococcus aureus 6538.

[0425] Antibacterial test in laundry detergent

[0426] Antimicrobial testing was conducted according to the standard test method ASTM-E2274 (Li, X). et al., J. Mater. Chem. B 2018, 6 (4274-4292). In short, a piece of white cotton fabric (1.4 m × 2.8 m) was immersed in 5 L of an aqueous solution containing 2.5 mL of 0.5% Tween 80 and 2.5 g of sodium carbonate, and autoclaved at 121°C for 20 minutes. It was then rinsed with water and dried in an oven at 50°C for at least 24 hours. The fabric was cut into strips 5 cm wide and 15 g each. One end of each strip was perforated and fixed to the outer horizontal extension of a stainless steel shaft, and wrapped with sufficient tension around the three horizontal extensions. The ends of the strips were stapled, and the entire shaft was autoclaved at 121°C for 20 minutes in a container. Second-passaged Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538 cells were dispersed in 7 mL of 0.85% (w / w) sodium chloride solution, and the bacterial solution was adjusted to OD. 600 = 0.12. By using 3.8 mL of bacterial solution (OD) 600 = 0.12) was mixed with 0.2 mL of horse serum to prepare a stock inoculum. Subsequently, three 1-inch × 1.5-inch sterile fabric carriers were inoculated (in a single sterile petri dish) by adding 10 μL of water followed by 20 μL of the stock inoculum to the center of the fabric. The fabric carriers were dried in an oven at 37°C for 30 minutes. The inoculated fabric carriers were then inserted into a shaft, with two pieces of fabric sandwiched between the second and third layers, and one piece of fabric sandwiched between the third and fourth layers. A glass bottle was prepared containing 250 mL of water, 0.25 mL of hard water (5.903 wt% calcium chloride dihydrate and 2.721 wt% magnesium chloride hexahydrate, sterilized by filtration through a 0.45 μm syringe filter), 0.25 mL of Japanese bagged detergent (trade name P&G Ariel Bioscience Gel ball, composed of linear alkylbenzene sulfonate (LAS), alkyl ether sulfate, polyoxyethylene alkyl ether, and fatty acid ester) or modeling detergent (SDS, SDBS, SDS+SDBS), and 2.5 mL of OIM. The final concentrations of the Japanese bagged detergent, modeling detergent, and OIM were 389 ppm, 100 ppm, and 100 ppm, respectively. A wide-mouth bottle containing 250 mL of water, 0.25 mL of hard water, and 3.125 mL of 4% Tween 80 solution was used as a control. Place the shaft containing the inoculated fabric into a wide-mouthed bottle and invert for 10 minutes. Then transfer three inoculated fabrics to 30 mL of neutralizing agent (modified Letheen Broth) and vortex at maximum speed for 2 minutes. Continuously dilute 1 mL of the neutralizing agent solution with 0.85% sodium chloride to a final concentration of 10 mL. -4Take 1 mL of each dilution and inoculate it twice onto LB agar. Incubate the plates at 37°C for 24 hours, count the colonies and record the count as CFU / plate. Take the average of the two plates and multiply it by the dilution factor to obtain CFU / three fabric carriers. Then convert the average count to a log10 decrease value, i.e., log10 decrease value = log10(CFU / three fabric carriers of control) - log10(CFU / three fabric carriers of OIM).

[0427] Results and Discussion

[0428] like Figure 35 As shown in B, polycationic PDADMAC failed the test in all four detergents. Colistin passed the test in Escherichia coli 8739, but failed the test in Staphylococcus aureus 6538. Figure 35 C). It is understandable that colistin kills *E. coli* 8739 in detergents because of its membrane-disrupting ability against Gram-negative bacteria. Based on our newly discovered carbene mechanism, which distinguishes OIM from typical cationic polymers, OIM1-6-CH showed a reduction of more than 2 log in Japanese bagged detergents and three other model detergents. Figure 35 D). In contrast, OIM1-6-C2(CH3) barely passed the test against Escherichia coli 8739, but failed the test against Staphylococcus aureus 6538. Figure 35 E). Similarly, OIM1-6-C4(CH3) passed the test in SDS, SDS+SDBS and Japanese bagged detergent, but failed the test in SDBS. Figure 35 F). This can be explained by the fact that the antibacterial activity of OIM1-6-C2(CH3) and OIM1-6-C4(CH3) in detergents is mainly due to the formation of polyionic composite nanoparticles in anionic detergents. The poorer antibacterial activity of OIM1-6-C4(CH3) in SDBS may be due to the high hydrophobicity of SDBS. We believe that the antibacterial activity of OIM1-6-CH in detergents comes from its carbene mechanism, while the antibacterial activity of OIM1-6-C2(CH3) and OIM1-6-C4(CH3) in detergents comes from the polyionic composite nanoparticles formed in the detergent. We also tested two biodegradable OIMs, namely OIM1-8-Bu-2PzAc and OIM1-8-Bu-2Ac, which contain C2-protons but have degradable bonds between the imidazole rings. Figure 35 G showed that OIM1-8-Bu-2PzAc was able to pass through all four detergents. Except for its weaker antibacterial activity against E. coli 8739 in SDS+SDBS, OIM1-8-Bu-2Ac... Figure 35(H), exhibiting potent antibacterial activity under all other conditions. The results indicate that compounds containing an imidazolium rings with dissociable C2-protons exhibit better bactericidal activity in formulations containing anions.

[0429] Example 11. Degradable OIM is effective in a mouse model of systemic infection.

[0430] To evaluate the efficacy of our biodegradable compounds, we conducted experiments using a mouse intraperitoneal injection (IP) model.

[0431] Mouse toxicity and infection models

[0432] All experiments used 8-week-old female C57BL / 6J mice. Six mice were used in each experimental group. Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Nanyang Technological University (AUP# A20029). Toxicity of OIM1-8-2D(16) was assessed by intraperitoneal (IP) injection at 15 mg / kg daily for 7 days, with animal body weight measured daily for 14 consecutive days. For systemic infection, Acinetobacter baumannii ATCC BAA 2803 in the exponential phase was washed twice with PBS and resuspended in 5% mucin saline to 10 μL. 5 CFU / mL. Mice were inoculated with 300 µL of bacteria via intraperitoneal injection. Two hours post-infection, mice were administered 15 mg / kg OIM1-8-2D (16), 15 mg / kg imipenem, or PBS (untreated control) via intraperitoneal injection. For the quantitative antibacterial efficacy assay, mice were euthanized 26 hours post-infection, and bacterial counts were determined in ascites, liver, kidney, and spleen. For the survival assay, mice were monitored for up to 7 days post-infection.

[0433] Results and Discussion

[0434] In this model, mice were infected with a lethal dose of carbapenem-resistant Acinetobacter baumannii via intraperitoneal injection (IP). Subsequently, two hours post-infection, mice were treated with a single dose of 15 mg / kg OIM1-8-2D (16) or 15 mg / kg imipenem (control). Additionally, the control group received only PBS treatment (…). Figure 36 A(i)). A single dose (16) successfully rescued all mice and significantly reduced bacterial load in major organs, resulting in a 6-order-of-magnitude reduction in CFU. In contrast, untreated mice or mice treated with imipenem died of infection within 36 hours ( Figure 36A(ii) to (iii) and 37). To assess the in vivo tolerability of OIM, we administered 15 mg / kg daily for 7 consecutive days to mice (16), for a total dose of 105 mg / kg. Surprisingly, this dosing regimen did not result in a significant decrease in body weight in the mice ( Figure 36 B), which highlights the good biocompatibility of degradable OIM1-8-2D (16) and its excellent ability to eradicate bacteria in a mouse model of systemic infection.

[0435] Example 12. Eradication of bacteria to prevent mastitis in dairy cows by a biodegradable cationic OIM derivative (16) in animal studies.

[0436] We expanded our research scope to explore the potential applications of imidazolium-containing oligomers / polymers in the prevention of bovine mastitis, a major global problem affecting dairy herds.

[0437] In vitro mastitis test (BS EN 1656)

[0438] According to the recommendations of BS EN 1656 (British Standards Institution, 2019), Staphylococcus aureus ATCC 6538, Streptococcus lactis ATCC 19436, and Escherichia coli ATCC 10536 were used for testing. Second-passaged bacteria were streaked from tryptone soy agar (TSA) plates and inoculated into a tryptone-NaCl dilution (0.1% tryptone and 0.85% NaCl) at a concentration of 1.5 to 5 × 10⁻⁶. 8 CFU / mL. The test compound was dissolved in hard water at the desired concentration (1 L of water contains 0.119 g MgCl2, 0.277 g CaCl2, and 0.28 g NaHCO3). 20 µL of skim milk (10 g / L) was added to a 96-well plate, followed by 10 µL of bacterial test suspension. The plates were mixed and incubated at 30°C for 2 minutes. Then, 80 µL of the test compound was added and thoroughly mixed, followed by incubation at 30°C. At the desired time point, 20 µL of the product / milk / bacteria mixture was transferred to a new 96-well plate containing 160 µL of neutralizing agent (3% lecithin, 10% Tween 80 (w / v), and 0.3% sodium thiosulfate) and 20 µL of milliQ water, mixed thoroughly, and incubated at room temperature for 5 minutes to fully neutralize the compound. The mixture was then serially diluted 10-fold in tryptone NaCl dilution and inoculated onto TSA plates. After incubating at 37°C for 24 hours, the colonies were counted.

[0439] In vivo mastitis farm trial

[0440] (i) Farm conditions

[0441] The experiment was conducted at a dairy farm located in Xiaochengzi Village, Longjia Town, Jiutai District, Changchun City, Jilin Province. The mastitis research protocol was approved by Professor Li Qingjie, the chief researcher at Changchun University of Traditional Chinese Medicine (ethics protocol number 202 / 205). Detailed experimental methods are described in the Supplementary Methods section of the Mastitis Farm Trial below.

[0442] (ii) Experimental setup

[0443] The experimental procedure was designed according to the recommended protocol (Nickerson, S.). et al. (in NMC Annual Meeting Proceedings. 379-399 (Citeseer)). A 10-day adaptation period (day -10) was implemented before the start of the experiment. The safety test began on day 0. The first sampling was performed before the application of the nipple extract (t=day 0) to establish a baseline. Immediately after milking, the nipple extract, contained in a standard foam extract cup, was applied to the distal 25 mm of the nipple. The nipple extract was applied daily for 5 consecutive days. A second sampling was performed on day 5. The collected samples included: milk samples for quality and residue testing; and nipple surface samples for residue testing. The experimental results are described below in the in vivo mastitis safety test protocol and... Figures 38-40 The challenge trial began immediately after milking on day 5. A baseline was established by first sampling milk for bacterial CFU counts before bacterial exposure (t = day 5). Staphylococcus aureus ATCC 49525 (Wall, RJ) was tested immediately after each milking. et al. , Nat. Biotechnol. 2005, 23 (445-451) (concentration in TSB is 5 x 10⁻⁶) 7 (CFU / mL) The sample was applied to the nipple approximately 25 mm deep using a standard foam dip cup. The nipple dip was applied immediately after exposure to the bacterial suspension. Challenge was performed daily for 5 consecutive days. A second and third sampling was performed on days 8 and 10, respectively. Collected samples included milk samples for quality control and CFU counting.

[0444] (iii) Sampling procedures in mastitis challenge tests

[0445] (a) Milk Sampling. All milk samples were collected immediately before routine automated milking. Briefly, three to four streams of colostrum were discarded from each lactation parlor (procedure is described below in the Supplementary Methods for Mastitis Farm Trials for Pre-Milking Lactation), and the teat tip was disinfected with a cotton swab and a sample was collected. Approximately 10 mL of milk was collected from each teat daily from the start of the experiment. To determine milk quality (e.g., somatic cell count), milk samples were tested by a qualified testing laboratory within 24 hours. (b) Bacterial CFU Count. Milk samples were collected according to the procedure described above. The number of microorganisms in the milk was counted using a standard protocol. (c) Diagnostic Criteria for Infection. A new intramammary infection in a lactation parlor was diagnosed when the same bacterial species were isolated from the following: 1) two consecutive samples during the trial (>500 CFU / ml); 2) a single sample from a lactation parlor with clinical mastitis (>100 CFU / ml); or 3) three consecutive samples during the trial (>100 CFU / ml).

[0446] Supplementary methods for mastitis farm trials

[0447] Farm conditions

[0448] The experiment was conducted during the local winter, with outdoor temperatures ranging from -10 to 10°C. The farm housed over 500 dairy cows and was equipped with an automated milking system. The cows were housed in barns and milked in separate milking facilities with ambient temperatures between 5 and 10°C. Milking was routinely performed by skilled workers every day at 2 PM. The farm regularly soaked the teats in a commercially available iodine-containing product after milking, but this practice was discontinued 10 days before the start of the experiment to avoid carryover effects and interference with the farm's experimental results.

[0449] Preparation of product solution

[0450] The product stock solution was stored at 4°C. The product working solution was freshly prepared on the day of the experiment and diluted with sterile deionized water (at a ratio of 1 / 30) to achieve a final concentration of 0.05% active compound and 10% glycerol.

[0451] Breast preparation before milking

[0452] Pre-milking udder preparation includes moistening and cleaning the nipples with disposable, moist wipes (without disinfectant, one wipe per nipple) before milking. Pre-milking preparation is completed by expressing three streams of milk.

[0453] Sampling procedures in safety testing

[0454] (i) Adsorption of polymers on the skin

[0455] To determine polymer adsorption on the nipple skin, swab sampling was performed. After cleaning the nipples (procedure see Pre-milking Breast Preparation), swab sampling was conducted according to DIN 10113-1: 1997-07 (Scheib, S). et al. , Pathogens 2023, 12 (560) Samples were collected using both wet and dry swab techniques. A swab soaked in 0.25% sterile Ringer's solution was used to wipe around the nipple 1 cm from the nipple opening. The same procedure was then performed using a dry swab (ultrafine dry swab). The two swabs were then cut short and inserted into a test tube containing 2 mL of 0.25% sterile Ringer's solution. The amount of polymer residue in the solution was determined using the Delvo test kit. In short, 200 µl of test sample was added to a Delvo test ampoule and incubated at 63°C for 3 hours. The color change of the solid agar at the bottom of the Delvo test ampoule was recorded.

[0456] (ii) Milk sampling

[0457] Milk samples were collected according to the procedures described in the above-mentioned in vivo mastitis farm trial protocol. To determine the amount of polymer residues in the milk, the milk samples were sent to the laboratory where the polymer residues (Stead, S) were determined using the Delvo assay kit. et al. , Int. Dairy J. 2008, 18 (3-11). To determine milk quality (such as somatic cell count), milk samples are tested by a qualified testing laboratory within 24 hours.

[0458] Preparation of challenge cultures for mastitis challenge test

[0459] Staphylococcus aureus ATCC 49525 was used in farm trials (Wall, RJ) due to its close association with clinical bovine mastitis. et al. , Nat. Biotechnol. 2005, 23 (445-451). Single colonies were streaked from agar plates into tryptic soy broth (TSB) and incubated overnight at 37°C with shaking. The overnight culture was subcultured 1:100 in fresh TSB and incubated for another 3 hours to obtain exponentially growing bacteria. The bacteria were pelleted by centrifugation (3,000-4,000 g, 15 min), washed twice with 0.1% peptone, and diluted to approximately 5 × 10⁻⁶ in fresh TSB. 7 CFU / ml. Prepare TSB immediately before use. It contains approximately 5 x 10 CFU / ml. 7 A CFU / ml challenge suspension.

[0460] In vivo mastitis safety test

[0461] In the safety test of the application of PIM1D after milking, the following aspects were evaluated: (1) the irritation response of the PIM1D-treated nipple; (2) the polymer residue on the nipple surface and the milk sample of the PIM1D-treated nipple; and (3) changes in milk composition.

[0462] After applying PIM1D nipple soaking solution to milking daily for 5 consecutive days, we observed no irritation reactions (such as redness, edema, roughness, or additional damage) in the PIM1D-treated nipples. Figure 38 The cows did not exhibit any abnormal behaviors (such as restlessness, kicking, or rubbing their bellies), which are signs of teat irritation and itching. Furthermore, according to DelvoTest... 2 Confirmed (<10 ppm, detection limit), no polymer residue was detected on the nipple surface and in milk samples, indicating that the compound can be easily washed away during the pre-milking cleaning process. Figure 39 Furthermore, PIM1D-based teat extracts did not cause any significant changes in milk composition, including somatic cell count (SCC), protein, fat, and non-fat solids (SNF) content. Figure 40 ).

[0463] Results and Discussion

[0464] We first tested the in vitro bactericidal activity of our compounds in milk against pathogens that cause mastitis, specifically *Escherichia coli* 10536, *Staphylococcus aureus* 6538, and *Streptococcus lactis* 19436, according to the industry standard BS EN 1656 (British Standards Institution, 2019). An effective test compound was required to achieve a viable bacterial count of at least 5 log⁻¹ within 30 minutes or less. 10 Reduce the dosage. The control drug chlorhexidine performed poorly, failing to achieve the required 5 log [amount] against two Gram-positive bacteria. 10 Requirements for reducing quantity ( Figure 41 A). However, OIM1-8-2D (16) achieved the required 5 logs against Escherichia coli and Staphylococcus aureus within 30 minutes. 10 The amount was reduced, but the level against Streptococcus lactis only reached 4.77 log. 10 Reduction amount ( Figure 41 B) 。 We then explored a longer polymeric version of (16), namely PIM1D, which is a compound we previously reported (Zhong, W). et al. , Proc. Natl. Acad. Sci. USA 2020, 117(31376-31385). PIM1D achieved 5 log values ​​against all three bacteria. 10 Requirements for reducing quantity ( Figure 41 C). Moving from laboratory testing to real-world application, we conducted farm trials using PIM1D as an antibacterial agent in post-milking teat extracts to prevent mastitis infection in dairy cows. Figure 36 C). Prior to this, we assessed the safety of PIM1D-based teat dips and confirmed that they had no adverse effects on dairy cows. Figures 38-40 In mastitis challenge tests, we determined the occurrence of intramammary infection (IMI) based on bacterial counts (>500 CFU / mL) and somatic cell counts (SCC, >200,000 / mL) in milk samples (Nickerson, S). et al. in NMC Annual Meeting Proceedings. 379-399 (Citeseer)). Based on these two indicators, teat extract based on PIM1D successfully prevented mastitis infection in cows repeatedly exposed to Staphylococcus aureus e, while cows treated with the glycerol control group developed mastitis over time. Figure 36 DE).

[0465] Importantly, the PIM1D-based nipple extract did not affect the milk composition. Figure 41 D).

[0466] discuss

[0467] An engineered, biodegradable OIM derivative has demonstrated remarkable efficacy in a mouse model of sepsis infection, particularly against multidrug-resistant Gram-negative Acinetobacter baumannii strains. Furthermore, we have extended our findings to agriculture, where biodegradable OIM / PIM has been shown to act as an effective preventative against the globally prevalent problem of mastitis in dairy cows.

[0468] This disclosure highlights the crucial role of carbonic acid in the antibacterial properties of OIM, particularly those compounds containing deprotonable C(2)-H groups (1, 5-8, 10, 12, and 15-16). Carbonic acid (1, 5-8, 10, 12, and 15-16) exhibits potent antibacterial activity, while non-carbonic acid compounds lacking C(2)-H (2-4 and 11) show significantly reduced efficacy. Compared to classic advanced cationic polymers invented by us and others, OIM carbonic acid has a MIC approximately one order of magnitude lower and / or exhibits broad-spectrum efficacy with lower toxicity (Lam, SJ). et al. , Nat. Microbiol. 2016, 1 , 11;Chin, W. et al. , Nat. Commun. 2018, 9 , 14;Zhang, K. et al. , Nat. Commun. 2019, 10 , 4792).

[0469] pass 1 ¹H NMR showed that the C(2)-H in compounds (1) and (5) disappeared / deprotonated in neutral aqueous solution, confirming their carbonic properties. Using an AuCl(SMe2) probe and carbazole dye, NHC formed by OIM carbonic acid (1) was detected in the aprotic membrane bilayer, thus confirming that carbonic acid can effectively penetrate the bilayer membrane and form NHC.

[0470] Traditional cationic polymers cannot penetrate bacterial plasma membranes because the charged polymers are hydrophilic. When the cationic charge concentration exceeds a certain critical value, the cationic polymer forms pores or holes in the membrane, leading to cell death. Since this is a physical process, the threshold concentration is usually high, and the MIC value is usually tens of micrograms / mL. With the novel NHC-mediated entry into the membrane and cytosol, the entry process does not require the formation of pores or holes. Compared with (2), OIM carbonate (1 and 5) enters liposomes / bacterial nuclei with a lower cationic charge threshold concentration. Compared with (5) or (1), OIM (2) cannot enter the bacterial cytosol or liposome nucleus (see Figures 31B and 25I, respectively), and its MIC value is much higher because it relies solely on the physical membrane disruption process. Although OIM (1) may be more hydrophilic than the methyl-substituted derivatives (2) and (5), it can enter the membrane and nucleus of liposomes more efficiently because it can efficiently form NHC, thereby transporting it into the nucleus via the membrane. As OIM forms NHC, it transforms from an initially hydrophilic copolymer into an amphiphilic copolymer composed of hydrophilic imidazolium cationic repeating units and hydrophobic imidazolium-2-ylcarbene repeating units, thereby achieving efficient membrane penetration (see...). Figure 24 B). The positive charge of cationic OIMs enables them to exhibit electrostatic attraction to the negatively charged membrane surface of bacterial mimic liposomes, while the hydrophobic NHC repeating units facilitate OIM insertion into the liposome membrane, a fact supported by our simulation data (see [link to simulation]). Figure 29 This reveals a novel mechanism by which a cationic polymer (also carbonic acid) enters the membrane by forming NHC.

[0471] Regarding the transfer of OIM from the membrane to the liposome nucleus, it is noteworthy that the amounts of (1) and (5) entering the liposome nucleus are less than their amounts present in the membrane (Fig. 25I). In viable bacteria with functional PMF, the electric field across the membrane may favor the transfer of OIM-NHC to the cytosol. Alternatively, the acidic protons of quinone shuttles cyclically reduced in the redox active electron transport chain (ETC) may induce the quenching of OIM carbene to cationic OIM within the bacterial membrane, making it hydrophilic. This hydrophobic transition may prompt OIM to leave the hydrophobic membrane and enter the cytosol. Without being limited to theory, regardless of the precise transport mechanism from the membrane to the cytosol, this disclosure demonstrates that, with the assistance of NHC formation, carbonic acid successfully permeates into the bilayer membrane and exhibits high cytoplasmic uptake, ultimately reaching its intracellular target and rapidly killing the bacteria. Even under low PMF conditions, the formation of NHC from (1) readily promotes its internalization into the bacterial cytoplasm, as confirmed by experiments with respiratory-deficient mutants and fermenting bacteria.

[0472] Furthermore, our optimized degradable compounds (15) and (16) exhibited impressive minimum inhibitory concentrations (MICs) against multidrug-resistant bacteria, including the colistin-resistant Gram-negative Enterobacter cloacae 13047 with MRD. In a mouse model of sepsis infection, degradable OIM (16) demonstrated significant efficacy against multidrug-resistant Acinetobacter baumannii. Additionally, in farm trials, PIM1D was successfully used to prevent mastitis infection without any adverse effects observed in dairy cows.

[0473] Example 13. Study of different anti-charge ions

[0474] General steps for preparing compounds OIM1-8-2D containing different anti-charge ions

[0475] OIM1-8-2D(4Br - / 4Cl - The compound was treated with aqueous triethylamine (base, Et3N) to form an eight-membered N-heterocyclic carbene (NHC), and then quenched with an organic acid to generate a carboxylate imidazolium ionic liquid.

[0476]

[0477] Results and Discussion

[0478] Table 12 below summarizes the characteristics of OIM for various exchanges.

[0479]

[0480] in conclusion

[0481] In this paper, we investigated the mechanism by which main-chain imidazolium oligomers with precise molecular weights kill bacteria. Through stepwise synthesis, we prepared oligoimidazolium (OIM) derivatives with the same repeating unit but different substituents at the C2- and C4- positions of the imidazolium ring (Figure 1) and tested their antibacterial and toxic properties. Derivatives containing the C2-proton are generally effective antibacterial agents, while derivatives with C2-hydrogen substituents are generally not antibacterial. We found that the parent compound OIM1-6-H (1, Figure 1), containing C2-hydrogen, forms an N-heterocyclic carbene as carbonic acid in bacterial membrane mimicry at physiological pH and is readily taken up by bacteria, even in methicillin-resistant Staphylococcus aureus (MRSA) respiratory mutants lacking the functional electron transport chain (ETC). The extent of uptake and NHC formation can be modulated as shown by the methyl derivatives (compounds 2 and 5, Figure 1). Furthermore, the effectiveness of cationic OIMs (including degradable derivatives) with rapid bactericidal action and capable of forming NHC was demonstrated in complex real-world application environments, such as personal care products.

[0482] We demonstrate here that oligomers with an imidazolium ring containing a dissociable C2-proton (i.e., carbonic acid) form NHCs in hydrophobic bacterial mimic membrane bilayers. The ability of carbonic acid to form NHCs with unsubstituted C2-carbons is associated with good antibacterial properties. The formation of NHCs allows the polymer to easily enter the bacterial cytoplasm without the need for PMF, as confirmed by the uptake of OIM1-6-CH by respiratory-deficient mutants.

[0483] The ability to form NHC is related to the p-value of carbonic acid. K a Value-related. Has a high p-value. K a Carbonic acid with an electron-donating methyl substituent is less likely to form NHC, resulting in lower toxicity, but only a slight reduction in antibacterial properties. OIM1-6-CH exhibits excellent short-term (24 hours) toxicity, but may exhibit long-term (48-72 hours) toxicity due to diffusion. However, in OIM1-6-C4(CH3), where the carbene formation rate is lower, the toxicity can be suppressed.

[0484] This is a novel mechanism by which the polymer can enter and be absorbed by bacteria through the amphiphilic nature of a copolymer of cationic imidazoline and carbene, where imidazoline is cationic and hydrophilic, while carbene is hydrophobic. It exhibits a good minimum inhibitory concentration (MIC) against multidrug-resistant bacteria, including Gram-negative bacteria that are inherently resistant to antibiotics due to their outer membrane. Furthermore, it also shows a good MIC against colistin-resistant bacteria (Enterobacter cloacae 13047-MDR), confirming that its bactericidal mechanism differs from that of cationic peptides.

[0485] Furthermore, we demonstrate that the fission properties of imidazoline allow it to escape the binding of polyanions to OIMs, which would hinder the use of OIMs in many practical and physiological applications. We show that it rapidly kills both Gram-positive and Gram-negative bacteria in detergent formulations, giving this cationic compound a unique advantage. The diverse possibilities for modifying ring and side-chain structures provide unprecedented new properties for this class of carbene-forming cationic oligomers, enabling them to kill multidrug-resistant bacteria in complex environments. We term this novel family of compounds “cat-bene-biotics”.

[0486] AMPs and antimicrobial polymers (AMPs) are potential drug candidates for alternative antibiotics due to their broad-spectrum antimicrobial activity against multidrug-resistant bacteria. However, issues such as cytotoxicity and contamination are drawbacks of AMPs. We demonstrate that the oligomer OIM1-6-CH, with a hydrogen (H) atom at C2 and precisely six repeating imidazolium units, exhibits potent antimicrobial activity and low short-term eukaryotic toxicity. We also investigated the structure-activity relationship (SAR) around the imidazolium moiety through various substitutions at the C2 and C4 positions. The antimicrobial properties of the C4-substituted analogues indicate that the unsubstituted C2 carbon is essential for the optimal potency of the oligomer, while any substitution at the C2 position leads to a significant decrease in potency. We then demonstrate that at physiological pH, the acidic C2-hydrogen in the imidazolium structure can form N-heterocyclic carbene (NHC) in liposomes, while substitution at the C2 position prevents NHC formation. Using OIM1-6-CH and methyl-substituted OIM1-6-C2 / C4(CH3), we found that the unsubstituted C2 proton is crucial for the internalization of OIM into bacteria, which is related to their potency. While PMF facilitates intracellular uptake of OIM, OIM1-6-CH can still be internalized into the cytoplasm of respiratory-deficient mutants and fermentative bacteria via a second mechanism (i.e., NHC formation). Inhibiting NHC formation of OIM1-6-CH and OIM1-6-C4(CH3) at weakly acidic pH significantly reduces their potency against Staphylococcus aureus LAC and LAC respiratory-deficient mutants, highlighting the importance of carbene formation in the efficacy of our compounds. This additional mechanism helps maintain the excellent antimicrobial properties of OIM in the presence of high salt and surfactants, as demonstrated in the fabric tests in Example 10.

Claims

1. A compound according to formula Ia: in: X - It is an anionic substance, selected from organic acids in the form of carboxylate salts, Br... - I - or Cl - ; Y represents OH, NH2, zwitterionic substances, or hydrazone; Each L represents independently: ； ； ;or ， Each wavy line represents a connection point with the rest of the molecule. Or the compound represented by formula Ib: Each L is independently selected from the list provided above; and X - The definition is as described above. Or the compound represented by formula Ic: in: R1 is selected from H, CH3, Cl, or CF3; One of R2 and R3 is H, CH3, Cl or CF3, and the other is H, or R2 and R3 together with the carbon atoms they are attached to form a benzene ring; n represents 6 or 8; X - The definition is as described above. Or the compound represented by formula Id: Each L is independently selected from the list provided above; and X - The definition is as described above, and Solvates of compounds of formulas Ia-Id.

2. The compound according to claim 1, wherein the compound has the formula Ic.

3. The compound according to claim 2, wherein R1 is H.

4. The compound according to claim 2 or claim 3, wherein one of R2 and R3 is H, CH3, Cl or CF3, and the other is H.

5. The compound according to claim 3, wherein one of R2 and R3 is H or CH3, and the other is H.

6. The compound according to any one of claims 2 to 5, wherein: n is 6; and / or X - Selected from Br - I - or Cl - And / or Y is OH.

7. The compound according to any one of claims 2 to 6, wherein: R1 is H; One of R2 and R3 is H or CH3, and the other is H; n is 6; and X - It is Cl - .

8. The compound according to claim 1, wherein the compound has formula Ia or formula Ib.

9. The compound according to claim 8, wherein each L represents: ;or The wavy lines represent the connection points with the rest of the molecule.

10. The compound according to claim 1, wherein the compound is a compound of formula Ia, wherein X - For Cl - And each L is .

11. The compound according to claim 1, wherein the compound is a compound of formula Ib, wherein X - For Cl - And each L is .

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11, and one or both of a pharmaceutically acceptable adjuvant and a carrier.

13. Use of the compound according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 in a medicament.

14. The compound according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 for the treatment of one or both of bacterial and fungal infections.

15. Use of the compound according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating one or both of bacterial and fungal infections.

16. A method for treating one or both of bacterial and fungal infections, comprising administering to a subject in need a pharmaceutically effective amount of the compound according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12.

17. An antibacterial and / or antifungal detergent composition comprising: The compound according to any one of claims 1 to 11; and Surfactants.

18. The antibacterial and / or antifungal detergent composition according to claim 17, wherein the composition is in the form of a solid soap or a liquid soap.

19. The antibacterial and / or antifungal detergent composition according to claim 18, wherein the composition is in shampoo form.

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