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WO2024248739A1 - Main chain cationic oligo(imidazolium) forms n-heterocyclic carbene for effective bacterial killing in complex environment - Google Patents

Main chain cationic oligo(imidazolium) forms n-heterocyclic carbene for effective bacterial killing in complex environment Download PDF

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WO2024248739A1
WO2024248739A1 PCT/SG2024/050366 SG2024050366W WO2024248739A1 WO 2024248739 A1 WO2024248739 A1 WO 2024248739A1 SG 2024050366 W SG2024050366 W SG 2024050366W WO 2024248739 A1 WO2024248739 A1 WO 2024248739A1
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oim1
compound
nmr
mmol
oim
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PCT/SG2024/050366
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French (fr)
Inventor
Bee Eng Mary Chan
Kevin Pethe
Angelika GRÜNDLING
Chong Hui KOH
Mallikharjuna Rao LAMBU
Zhi Yuan KOK
Guangmin WEI
Madhu Babu TATINA
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Nanyang Technological University
Imperial College Innovations Limited
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Publication of WO2024248739A1 publication Critical patent/WO2024248739A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/54—1,3-Diazines; Hydrogenated 1,3-diazines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
    • A61K8/4946—Imidazoles or their condensed derivatives, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/02—Preparations for cleaning the hair
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
    • C08L79/04—Polycondensates having nitrogen-containing heterocyclic rings in the main chain; Polyhydrazides; Polyamide acids or similar polyimide precursors
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
    • C08G73/0605—Polycondensates containing five-membered rings, not condensed with other rings, with nitrogen atoms as the only ring hetero atoms
    • C08G73/0616—Polycondensates containing five-membered rings, not condensed with other rings, with nitrogen atoms as the only ring hetero atoms with only two nitrogen atoms in the ring

Definitions

  • the present disclosure generally relates to antibiotics, and more particularly relates to main chain cationic oligoimidazolium for effective bacterial killing in complex environments.
  • Antibiotics have long served as essential therapeutic and prophylactic tools for biomedical, as well as agricultural, applications, underpinning numerous modern biomedical interventions, including chemotherapy and surgical procedures.
  • the escalating global crisis of bacterial resistance to virtually all classes of antibiotics has cast a shadow over these medical advances. This crisis is further compounded by the sluggish pace of developing novel antimicrobial agents, a pressing challenge that has persisted for several decades.
  • the approval of new antibiotic classes for the treatment of Gram-negative bacterial infections has remained elusive for over six decades.
  • Conventional antibiotics typically operate by targeting specific enzymes involved in conserved metabolic processes, inevitably leading to the emergence of resistance strains.
  • the bacterial membrane represents one of the last frontiers in the quest for novel antibacterial drug.
  • Membrane-targeting agents hold promise as they are usually less susceptible to resistance development.
  • antimicrobial polymers and peptides AMPs
  • AMPs antimicrobial polymers and peptides
  • the advancement of classical cationic polymer and peptide agents that target bacterial membrane permeability has been hindered by concerns regarding their toxicity and limited metabolic stability.
  • Antimicrobial peptides such as defensins, daptomycin, magainin-2, etc. typically kill bacteria by interacting electrostatically with the cytoplasmic membrane, which is followed by formation of physical pores, eventually leading to bacterial cell death.
  • AMPs are attractive antimicrobial agents since they have a distinct mechanism of kill compared to antibiotics.
  • the relatively small differential electrostatic interaction between AMPs and bacterial membrane versus mammalian membrane, particularly in the presence of salt and serum in physiological environment result in low selectivity and often high toxicity towards eukaryotic cells.
  • ETC electron transport chain
  • X" is an anionic species selected from an organic acid in its carboxylate form, Br,
  • Y represents, OH, NH2, a zwitterionic species or a hydrazone group; each L independently represents:
  • each wiggly line represents a point of attachment to the rest of the molecule, or a compound according to formula lb: where each L is independently selected from the list provided above; and X' is as defined above, or a compound according to formula Ic:
  • Ri 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 to which they are attached form a benzene ring; n represents 6 or 8;
  • X- is as defined above, or a compound according to formula Id: where each L is independently selected from the list provided above;
  • X- is as defined above, and solvates thereof of compounds of formula la-ld.
  • X- is selected from Br, I- or Cl _ ;
  • Y is OH.
  • R 1 is H; one of R2 and R3 is H or CH3 and the other is H; n is 6; and
  • X- is Ch.
  • a pharmaceutical composition comprising a compound according to any one of Clauses 1 to 11 and one or both of a pharmaceutically acceptable adjuvant and carrier.
  • a method of treating one or both of a microbial and a fungal infection comprising the step of administering a pharmaceutically effective amount of a compound according to any one of Clauses 1 to 11 or a pharmaceutical composition according to Clause 12 to a subject in need thereof.
  • An antimicrobial and/or antifungal detergent composition comprising: a compound as described in any one of Clauses 1 to 11; and a surfactant. 18. The antimicrobial and/or antifungal detergent composition according to Clause 17, wherein the composition is in the form of a solid or liquid soap.
  • Fig. 1 depicts structural representation of oligoimidazolium (OIM) analogues synthesized and biologically evaluated in a SAR study for (A) OIM1-6 series, (B) OIM1-8 series, (C) degradable OIM1 series, and (D) degradable OIM series.
  • OIM oligoimidazolium
  • Fig. 2 depicts synthesis of OIM1-6 derivatives.
  • Reagents and conditions (i) benzyl (3- bromopropyl)carbamate, NaH, tetrahydrofuran (THF), 0 °C to 50 °C, 18 hours, 71-76%; (ii) benzyl (3-bromopropyl)carbamate, NaOH, MeCN/H 2 O, 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, 66-95%; (v) NaOH, MeCN/H 2 O, 50 °C, 78%; (vi) MeCN, 80 °C, 18-60 hours, 63-85%; (vii) 1,4-dii
  • Fig. 3 depicts X-ray photoelectron spectroscopy (XPS) analysis of compound 1 , indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis.
  • XPS X-ray photoelectron spectroscopy
  • Fig. 4 depicts XPS analysis of compound 2, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis.
  • Fig. 5 depicts XPS analysis of compound 5, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis.
  • Fig. 6 depicts XPS analysis of compound 13, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis (Binding energy at 64-65 indicates the presence of Bromine).
  • Fig. 7 depicts XPS analysis of compound 14, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis (Binding energy at 64-65 indicates the presence of Bromine).
  • Fig. 8 depicts XPS analysis of compound 16, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis (Binding energy at 64-65 indicates the presence of Bromine).
  • Fig. 9 depicts calibration curves of (A) OIM1-6-CH (1), (B) OIM1-6-C2(CH 3 ) (2) and (C) OIM1- 6-C4(CH3) (5) using area and height by liquid chromatography-mass spectrometry (LC-MS).
  • Fig. 10 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (1-3): (A) OIM1-6-CH (1); (B) OIM1-6-C2(CH 3 ) (2); and (C) OIM1-6-C2(CI) (3) for 24 hours, 48 hours and 72 hours.
  • HEK human embryonic kidney
  • HepG2 liver hepatocellular carcinoma
  • Fig. 11 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (4-6): (A) OIM1-6-C2(CF 3 ) (4); (B) OIM1-6-C4(CH 3 ) (5); and (C) OIM1-6-C4(CI) (6) for 24 hours, 48 hours and 72 hours.
  • HEK human embryonic kidney
  • HepG2 liver hepatocellular carcinoma
  • Fig. 12 depicts cell viability of 3T3 fibroblast cells determined after incubating with compounds (7-9) (A) OIM1-6-C4(F) (7), (B) OIM1-6-(BZ) (8), and (C) OIM1-6-C4(CF3) (9) for 24 hours, 48 hours and 72 hours.
  • Cell viability of Human Embryonic Kidney (HEK) cells determined after incubating with (D) OIM1-6-C4(F) (7), (E) OIM1-6-(BZ) (8), and (F) OIM1-6-C4(CF3) (9) for24 hours, 48 hours and 72 hours.
  • HEK Human Embryonic Kidney
  • HepG2 Liver Hepatocellular Carcinoma
  • Fig. 13 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (10-12):
  • HEK human embryonic kidney
  • HepG2 liver hepatocellular carcinoma
  • Fig. 14 depicts cell viability of 3T3 fibroblast cells (A-B) determined after incubating with compounds (13-14): (A) OIM1-8-Bu-Acetal (13); and (B) OIM1-8-Bu-PzAc (14) for 24 hours, 48 hours and 72 hours.
  • Cell viability of Human Embryonic Kidney (HEK) cells determined after incubating with (C) OIM1-8-Bu-Acetal (13), and (D) OIM1-8-Bu-PzAc (14) for 24 hours, 48 hours and 72 hours.
  • HEK Human Embryonic Kidney
  • HepG2 Liver Hepatocellular Carcinoma
  • Fig. 15 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (15-16): (A) OIM1-12-6C-OH (15); and (B) OIM1-8-2D (16) for 24 hours, 48 hours and 72 hours.
  • HEK human embryonic kidney
  • HepG2 liver hepatocellular carcinoma
  • Fig. 16 depicts cell viability of 3T3 fibroblast cells after incubation with known antibiotics: (A) gentamicin; (B) colistin; and (C) ciprofloxacin for 24 hours, 48 hours and 72 hours.
  • HEK human embryonic kidney
  • HepG2 liver hepatocellular carcinoma
  • Fig. 17 depicts synthesis of OIM1-8 derivatives. Reagents and conditions: i) 1,4- dibromobutane (excess, 4 to 5 equiv) acetonitrile (MeCN), 80 °C, 18 hours, 55-68%; ii) 1d to 3e (1.0 equiv.) 10a to 12a (0.45 equiv) acetonitrile+ DMF (9:1); iii) 33 wt. % HBr in acetic acid, rt, 18 hours; and iv) Amberlyst AR26 OH resin, 10% aq. HCI solution 50-65 %.
  • Fig. 18 depicts synthesis of OIM1-8 degradable derivatives.
  • Reagents and conditions i) 1 ,4- dibromobutane (excess, 4 to 5 equiv) acetonitrile (MeCN), 80 °C, 18 hours, 65%; ii) 1g (1.0 equiv), 1d (2.5 equiv.) acetonitrile, 80 °C, 18 hours, 62%; iii) 13a (1.0 equiv.), Paraformaldehyde (0.5 equiv.) & concentrated H2SO4 (cat.), Toluene, 130°C, 1.5 h, 83%; iv) Chloroacetylchloride (2.1 equiv), K2CO3 (2.5 equiv.), Water + CHCI3 (1:1), O °C-rt 18 hours, 90%; v) 1d (1.0 equiv.) 13b (5.0 equiv), Acet
  • Fig. 19 depicts (A and B) synthesis of degradable OIM series: OIM1-12-6C-OH (15).
  • Reagents and conditions Boc-anhydride (2.3 eq), Mg(CIO4)2 (0.1 eq), DCM, reflux, 48 hours, 64%;
  • Triphosgene (0.25 eq), Pyridine (1.5 eq), DCM, O °C-rt, 18 hours, 47%;
  • Fig. 20 depicts synthesis of degradable OIM series: OIM1-8-2D (16).
  • Reagents and conditions (i) Chloroacetylchloride (2.1 equiv ), K2CO3 (2.5 equiv.), water + CHCI3 (1 :1), 0 °C - rt 18 hours, 65% (16a); (ii) 1d (1.0 equiv.), 16a (5.0 equiv.), acetonitrile, 80 °C, 3 hours, 50%; (iii) 1e (2.1 equiv.), 16b (1.0 equiv.), acetonitrile + DMF + MeOH (8:1 :1), 95 °C overnight; (iv) 33 wt. % HBr in acetic acid, rt, 2 hours; (v) Amberlyst AR26 OH resin, 10% aq. HCI solution, 55% over three steps.
  • Fig. 21 depicts killing of S. aureus LAC over time in MHB in the presence of (A) OIM1-6-CH, (B) OIM1-6-C2(CH 3 ), (C) OIM1-6-C4(CH 3 ) tested from 1-to-8x MIC. Killing of P. aeruginosa PAO1 overtime in MHB with (D) OIM1-6-CH, (E) OIM1-6-C2(CH 3 ), (F) OIM1-6-C4(CH 3 ) tested from1-to-4x MIC.
  • Fig. 22 depicts (A) the chemical structures of the simulated cationic oligomers. (B) The geometry optimized structures of the simulated cationic oligomers. (C) Calculated Hirshfeld charges on the imidazolium rings of OIMs.
  • Fig. 23 depicts (A) membrane depolarization study of MRSA LAC treated with OIM1-6 compounds, with gramicidin as antibiotic control for 1 hour using DISC3(5) assay. (B) Membrane permeabilization study of LAC treated with OIM1-6 compounds, with nisin as positive control for 1 hour using propidium iodide (PI) assay.
  • Fig. 24 depicts (A) OIMs with free C2-hydrogens are carbon acids which deprotonate in neutral water to form N-heterocyclic carbenes (NHCs) (//) that are uncharged and hydrophobic.
  • N-heterocyclic carbenes N-heterocyclic carbenes
  • OIM carbon acid converts to an amphiphilic copolymer (made of hydrophilic cation and hydrophobic NHC repeats) to efficiently translocate the plasma membrane into bacterial cytosol at lower polymer threshold concentration, as compared with (ii) a classical cationic polymer that forms physical pores.
  • the OIMs bind with their intracellular target (DNA as previously studied) in bacterial cytosol resulting in bacteria death, as opposed to classical cationic polymer which is ineffective in killing the bacteria.
  • Fig. 25 depicts NHC formation studies with 0IM1-6-CH (1).
  • A Detection of NHC by the reaction of partial carbene (//) monomers with D2O or AuCI(SMe2).
  • B Hydrogen-deuterium exchange in 1 H nuclear magnetic resonance (NMR) of OIM1-6-CH (1) in aqueous solution as a function of time at pH 6.63, pH 6.81 , pH 7.16 and pH 8.21 in D2O.
  • C Schematic of interaction of phosphatidylcholines/phosphatidylglycerol (PC/PG) liposome containing NHC probes (carbazole dye and or AuCI(SMe 2 )) with OIM-1-6-CH (1).
  • PC/PG phosphatidylcholines/phosphatidylglycerol
  • Fig. 26 depicts confirmation of the peak m/z 330.52 to be Au-OIM-1-6-CH:
  • A-D Hydrogen deuterium exchange mass spectrometry experiment.
  • A Structure assignment 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).
  • Fig. 28 depicts NHC formation studies of OIM1-6-C2(CH 3 ) (2) and OIM1-6-C4(CH 3 ) (5).
  • Fig. 29 depicts computer simulation of interaction of 2 forms (cationic versus NHC forms) of OIM1-6-CH (1) with S. aureus membrane-mimic.
  • A-B Number of contacts between OIM1-6- CH (1) and S. aureus membrane as a function of simulation time: (A) cationic OIM-membrane system; and (B) OIM-NHC-membrane system. A contact was defined if the minimum distance between the polymer atom and membrane atom was less than 0.4 nm.
  • C-D Binding poses of (1) to S. aureus membrane-mimic: (C) cationic OIM-membrane system; and (D) OIM-NHC- membrane system. These poses were taken from the final simulation frame of each simulation repeat.
  • Fig. 30 depicts (A) method of determination of total and cytosolic uptakes of compounds by bacteria, (i) Illustration of florescence from OIM-fluorescein isothiocyanate (FITC)ZSYTO Sustained MRSA LAC upon Trypan Blue (TB) and Triton X-100 (TX) treatment.
  • TB dye is impermeable to intact plasma membrane; hence it will quench the surface-bound fluorophore trapped in the cell wall, and to some extend quench the fluorophore trapped on the surface of the bacterial membrane.
  • TX is added at 0.04% to permeabilize the bacteria membrane, allowing the entry of TB dye to the cytosolic.
  • TB dye could now quench the fluorophore in the cytosolic, however not the fluorophores that were partially/fully inserted into the membrane bilayer.
  • the fluorescence signals of 50,000 bacteria were determined.
  • B Flow cytometry histograms of untreated LAC before TB treatment (unquench) and after TB treatment (TB quench). Dotted line indicates the gating setting: bacteria with fluorescence intensity to the right of the gating is considered to have dye uptake, bacteria with fluorescence intensity to the left of the gating is considered to have no dye uptake.
  • C Flow cytometry histograms of untreated LAC stained with STYO 9 DNA dye.
  • the percentage of bacteria population with membrane+cytosolic OIM uptake is obtained from the TB quench histogram (right side of the gating) and illustrated as bar chart in Fig. 31 .
  • TX + TB quench Upon TX + TB quench, the fluorescence intensity dropped significantly (to the left of the gating) for (1) and (5) but not (2), indicating that (1) and (5) enter the cytosolic while the (2) is partially/fully inserted in the membrane bilayer.
  • the bacteria population that has cytosolic uptake of OIM is determined via histogram subtraction of TX + TB quench from TB quench.
  • G Histogram subtraction of TX + TB quench from TB quench generated by Flowjo software using compound (1) (Fig. 30D) as example.
  • the percentage of bacteria population with cytosolic uptake is illustrated as bar chart in Fig. 31.
  • Fig. 31 depicts effect of NHC formation on OIM’s potency and uptake into bacteria.
  • A MIC* of OIMs and gentamicin against S. aureus parental strain. MIC* is tested up to 4,096 pg/mL, MIC* > 4,096 pg/mL indicated as 8,192 pg/mL.
  • B The percentage of LAC cell count with OIM uptake treated in TSB for 1 hour.
  • C The mean fluorescence intensity (MFI) of cells with OIM uptake treated in TSB for 1 hour.
  • D MIC* of OIMs and gentamicin against wildtype LAC and respiration-deficient mutants at different pH.
  • MIC* is tested up to 4096 pg/mL, MIC* > 4096 pg/mL indicated as 8192 pg/mL.
  • E The cell count percentage of wildtype LAC and respiration-deficient mutants with OIM1-6-CH (1) uptake tested at pH 7.2 and pH 6.8 in TSB for 1 hour, at their respective 1x MIC* at pH 7.2. (wildtype LAC at 16 pg/mL, LAC menD at 128 pg/mL and LAC hemB at 256 pg/mL).
  • Fig. 32 depicts LAC treated with FITC conjugated OIM in (A) MHB and (B) TSB for 1 hour.
  • Total OIM includes the surface bound and internalized fraction of OIM with no Trypan Blue quenching, while the internalized fraction is established with Trypan Blue quenching.
  • C LAC menD and
  • D LAC hemB treated with FITC conjugated OIMs for 1 hour with and without Trypan Blue quenching.
  • E Uptake of FITC conjugated OIM1-6-CH and OIM1-6-C4(CH 3 ) by LAC at pH 7 and pH 6.8 in TSB at their respective 1x MIC* (pH 7).
  • F Uptake of FITC conjugated 0IM1-6-CH by LAC menD and LAC hemB at pH 7 and pH 6.8 in TSB at their respective 1x MIC* (pH 7).
  • Fig. 33 depicts resistance evolution of (A) LAC and (B) PAO1 against 0IM1-6-CH and OIM1- 6-C4(CH 3 ), respectively.
  • C OCR of LAC treated with OIM1-6 CH, OIM1-6 C2(CH 3 ), OIM1-6- C4(CH 3 ) and HQNO, as well as respiratory mutants of LAC AhemB and LAC AmenD. Killing kinetics of S. aureus LAC treated with (D) 0IM1-6-CH or (E) OIM1-6-C4(CH 3 ) in aerobic, anaerobic, and fermentative growth condition at their respective 4-fold MIC value (i.e. 16 pg/ml and 32 pg/ml respectively).
  • Fig. 34 depicts Deuterium exchange results in the disappearance of the peak at 8.8-8.9 ppm due to the C2-proton at (A) pH 7.16 and (B) pH 6.8. The extent of remained proton at C2 position is indicated above each spectrum. It is observed that deuterium exchange is faster at pH 7.16 than pH 6.8.
  • Fig. 35 depicts antibacterial test of OIMs against E. coli 8739 and S. aureus 6538 for the application of laundry use in 4 kinds of detergents (100 ppm of sodium dodecyl benzene sulfonate (SDBS), 100 ppm of sodium dodecyl sulfate (SDS), 50 ppm of SDS plus 50 ppm of SDBS, and 389 ppm of Japan pouch detergent).
  • SDBS sodium dodecyl benzene sulfonate
  • SDS sodium dodecyl sulfate
  • SDS sodium dodecyl sulfate
  • the tested compounds are (B) Polydiallyldimethylammonium chloride (PDADMAC), (C) colistin, (D) OIM1-6-CH, (E) OIM1-6-C2(CH 3 ), (F) OIM1-6-C4(CH 3 ), (G) OIM1-8-Bu-2PzAc, and (H) OIM1-8-Bu-2Ac.
  • Antibacterial test was performed according to standard test method ASTM-E2274. It requires at least 2 log reduction to pass the test.
  • Fig. 36 depicts in vivo efficacy of degradable OIMs.
  • A Murine systemic infection model
  • Fig. 38 depicts teat images after 5-day continuous application of teat dip in safety trial. No irritation responses were detected on PIM1 D-treated teats.
  • Fig. 39 depicts Delvo Test results of (A) teat surface and (B) milk samples in a safety trial.
  • Fig. 40 depicts milk composition changes (protein, fat and solids-not-fat (SNF)) as well as somatic cell count (SCC) in PIM1 D-treated teats during the 5-day safety trial.
  • SNF protein, fat and solids-not-fat
  • SCC somatic cell count
  • Fig. 41 depicts dairy mastitis testing results.
  • X' is an anionic species selected from an organic acid in its carboxylate form, Br, k or Cl-;
  • Y represents, OH, NH2, a zwitterionic species or a hydrazone group; each L independently represents: where each wiggly line represents a point of attachment to the rest of the molecule, or a compound according to formula lb: where each L is independently selected from the list provided above; and X' is as defined above, or a compound according to formula Ic: where:
  • Ri 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 to which they are attached form a benzene ring; n represents 6 or 8;
  • X- is as defined above, or a compound according to formula Id: where each L is independently selected from the list provided above;
  • X is as defined above, and solvates thereof of compounds of formula la-ld.
  • the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features.
  • the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention.
  • the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.
  • references herein in any aspect or embodiment of the invention include references to such compounds perse, to tautomers of such compounds, as well as to pharmaceutically acceptable solvates of such compounds.
  • oligomers of the invention are any solvates of the compounds and their salts.
  • Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent).
  • solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide.
  • Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent.
  • Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray crystallography.
  • TGA thermogravimetric analysis
  • DSC differential scanning calorimetry
  • X-ray crystallography X-ray crystallography
  • the solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.
  • the oligomers of the invention may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.
  • a compound of formula Ic where Ri is H, R 2 is methyl and R 3 is H, it may be represented as: as such, in embodiments where R 2 and R 3 do not together with the carbon atoms to which they are attached form a benzene ring, the compound of formula Ic may be drawn as a compound of formula lc’: Rn is H, CH3, Cl, or CF3. In this arrangement, the charge is distributed between the two nitrogen atoms and the carbon atom therebetween.
  • oligomers of the invention in the above-mentioned aspect of the invention may be utilised in a method of medical treatment.
  • the oligomer of the invention may be particularly useful with regard to microbial infections.
  • microbial infection covers any disease or condition caused by a microbial organism in or on a subject.
  • microbial infections include, but are not limited to, tuberculosis caused by mycobacteria, burn wound infections caused by pseudomonas etc., skin infections caused by S. aureus, wound infections caused by pseudomonas and A. baumannii, mastitis, and Sepsis.
  • fungal infection covers any disease or condition caused by a fungal organism in or on a subject. Examples of fungal infections include, but are not limited to, athlete’s foot, ringworm, yeast infections, and jock itch.
  • a non-limiting list of bacteria that may be susceptible to the oligomers of the invention include: Acidothermus cellulyticus, Acinetobacter baumannii, Actinomyces odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bifidobacterium adolescentis, Bifidiobacterium longum, Burkholderia thailandensis, Caldicellulosiruptor saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium cellulolyticum, Clostridium difficile, Clostridium
  • treatment includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.
  • patient and “patients” include references to mammalian (e.g. human) patients.
  • subject or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human.
  • the subject is a subject in need of treatment or a subject with a disease or disorder.
  • the subject can be a normal subject.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
  • alkyl refers to an unbranched or branched saturated hydrocarbyl radical, which may be substituted or unsubstituted.
  • alkyl refers to a C1-6 alkyl
  • the alkyl group may be ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl, hexyl or, more preferably, methyl.
  • n may be 6.
  • X' may be selected from Br,
  • Y may be OH.
  • Embodiments of the invention include those that relate to oligomers of the invention in which the compound may have formula Ic. In such embodiments, one or more of the following may apply:
  • R 1 may be H
  • R 2 and R 3 may be H, CH 3 , Cl, or CF 3 and the other may be H (e.g. one of R 2 and R 3 may be H or CH 3 and the other may be H);
  • R 1 is H; one of R 2 and R 3 is H or CH 3 and the other is H; n may be 6; and
  • X' may be Cl'.
  • each L may represent:
  • the compound may be a compound of formula la, wherein X' is Cl' and each L is
  • the compound may be a compound of formula lb wherein X' is Cl' and each L is
  • the “L” groups in the compounds of formula la, lb and Id may provide said compounds with biodegradeability. That is, these groups may be susceptible to the action of an enzyme or other biological mechanism or organism so as to split the compounds of formula la, lb and Id into two or more compounds (e g. 2, 3, 4, 5, 6, etc.), which is only limited by the total number of “L” groups present in said compounds. This degradation may occur in vivo, thereby allowing for the metabolism and excretion of said compounds or in the environment (e.g. through the action of microorganisms in the environment, or simple chemical degradation over time). As noted, each of the “L” groups may, in some cases, be selected from one or more of the possibilities listed herein. Further embodiments of the invention that may be mentioned include those in which the oligomer of the invention is isotopically labelled. However, particular embodiments of the invention that may be mentioned include those in which the oligomer of the invention is not isotopically labelled.
  • isotopically labelled when used herein includes references to oligomers of the invention in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound” will be understood by those skilled in the art to refer to one or more of the atoms of the oligomer of the invention. Thus, the term “isotopically labelled” includes references to compounds of the invention that are isotopically enriched at one or more positions in the oligomer.
  • the isotopic labelling or enrichment of the oligomer of the invention may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine.
  • a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine.
  • Particular isotopes that may be mentioned in this respect 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 l).
  • oligomer of the invention When the oligomer of the invention is labelled or enriched with a radioactive or nonradioactive isotope, oligomer of the invention that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or nonradioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.
  • the oligomers of the invention may be used in the treatment of microbial and fungal infections.
  • a pharmaceutical composition comprising the oligomer of the invention and one or both of a pharmaceutically acceptable adjuvant and carrier.
  • Oligomers of the invention may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form.
  • Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.
  • Oligomers of the invention will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable adjuvant diluent or carrier
  • Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
  • Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.
  • any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of oligomer of the invention in the formulation may be determined routinely by the skilled person.
  • a solid oral composition such as a tablet or capsule may contain from 1 to 99% (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment.
  • a controlled release tablet may in addition contain from 0 to 90% (w/w) of a release-controlling polymer.
  • a parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50% (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • a liquid or semisolid carrier or vehicle e.g. a solvent such as water
  • one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • oligomers of the invention may be administered at varying therapeutically effective doses to a patient in need thereof.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
  • Administration may be continuous or intermittent (e.g. by bolus injection).
  • the dosage may also be determined by the timing and frequency of administration.
  • the dosage can vary from about 0.01 mg to about 1000 mg per day of an oligomer of the invention.
  • the medical practitioner or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient.
  • the above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the aspects of the invention described herein may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.
  • oligomers of the invention may be prepared in accordance with techniques that are well known to those skilled in the art, for example as described hereinafter in the examples section.
  • Compounds of the invention may be isolated from their reaction mixtures using conventional techniques (e g. recrystallisation, column chromatography, preparative HPLC, etc.).
  • the oligomers of the invention exhibit a pronounced antimicrobial action, especially against pathogenic gram-positive and gram-negative bacteria and so may also act against bacteria of skin flora, e.g. Corynebacterium xerosis (bacteria that cause body odour), and also against yeasts and moulds. They are therefore also suitable in the disinfection of the skin and mucosa and also of integumentary appendages (hair), and so may also be suitable in the disinfection of the hands and of wounds.
  • Corynebacterium xerosis bacteria that cause body odour
  • yeasts and moulds are therefore also suitable in the disinfection of the skin and mucosa and also of integumentary appendages (hair), and so may also be suitable in the disinfection of the hands and of wounds.
  • the oligomers of the invention may be used as antimicrobial active ingredients in personal care preparations, for example 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, e.g. cleansing solutions for the skin.
  • personal care preparations for example 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, e.g. cleansing solutions for the skin.
  • an antimicrobial and/or antifungal detergent composition comprising an oligomer of the invention and a surfactant.
  • the composition may also contain additional cosmetically tolerable carriers and/or adjuvants.
  • Said composition may in particular be in the form of a shampoo or in the form of a solid or liquid soap, though other compositions as described hereinabove are also contemplated (e.g. other hair-care products, lotions and creams etc.).
  • the detergent composition may comprise from 0.01 to 15% by weight, such as from 0.5 to 10% by weight of an oligomer of the invention. It will be appreciated that more than one oligomer of the invention may form part of the detergent composition.
  • the detergent composition it will comprise, in addition to the oligomer of the invention, further constituents, for example sequestering agents, colourings, perfume oils, thickening or solidifying (consistency regulator) agents, emollients, UV absorbers, skin-protective agents, antioxidants, additives that improve mechanical properties, such as dicarboxylic acids and/or Al, Zn, Ca and Mg salts of C14-C22 fatty acids, and optionally preservatives.
  • further constituents for example sequestering agents, colourings, perfume oils, thickening or solidifying (consistency regulator) agents, emollients, UV absorbers, skin-protective agents, antioxidants, additives that improve mechanical properties, such as dicarboxylic acids and/or Al, Zn, Ca and Mg salts of C14-C22 fatty acids, and optionally preservatives.
  • the detergent composition may be formulated as a water-in-oil or oil-in-water emulsion, as an alcoholic or alcohol-containing formulation, as a vesicular dispersion of an ionic or non-ionic amphiphilic lipid, as a gel, a solid stick or as an aerosol formulation.
  • the detergent composition may comprise from 5 to 50 wt% of an oily phase, from 5 to 20 wt% of an emulsifier and from 30 to 90 wt% water.
  • the oily phase may contain any oil suitable for cosmetic formulations, e.g. one or more hydrocarbon oils, a wax, a natural oil, a silicone oil, a fatty acid ester or a fatty alcohol.
  • Preferred mono- or poly-ols are ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and sorbitol.
  • Detergent compositions may be provided in a wide variety of preparations.
  • suitable compositions include, but are not limited to skin-care preparations (e.g. skin-washing and cleansing preparations in the form of tablet-form or liquid soaps, soapless detergents or washing pastes), bath preparations, (e.g. liquid compositions such as foam baths, milks, shower preparations or solid bath preparations), shaving preparations (e.g. shaving soap, foaming shaving creams, non-foaming shaving creams, foams and gels, preshave preparations for dry shaving, aftershaves or after-shave lotions), cosmetic hair-treatment preparations (e.g. hair-washing preparations in the form of shampoos and conditioners, haircare preparations, e.g.
  • skin-care preparations e.g. skin-washing and cleansing preparations in the form of tablet-form or liquid soaps, soapless detergents or washing pastes
  • bath preparations e.g. liquid compositions such as foam baths, milks, shower preparations
  • pretreatment preparations hair tonics, styling creams, styling gels, pomades, hair rinses, treatment packs, intensive hair treatments, hair-structuring preparations, e.g. hair-waving preparations for permanent waves (hot wave, mild wave, cold wave), hairstraightening preparations, liquid hair-setting preparations, foams, hairsprays, bleaching preparations; e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams, bleaching powders, bleaching pastes or oils, temporary, semi-permanent or permanent hair colourants, preparations containing self-oxidising dyes, or natural hair colourants, such as henna or camomile).
  • hair-waving preparations for permanent waves hot wave, mild wave, cold wave
  • hairstraightening preparations liquid hair-setting preparations, foams, hairsprays
  • bleaching preparations e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams, bleaching powders, bleaching pastes or oils, temporary,
  • An antimicrobial soap may have, for example, the following composition:
  • stearic acid 1 to 10% by weight stearic acid; and the remainder being a soap base, e.g. the sodium salts of tallow fatty acid and coconut fatty acid or glycerol.
  • soap base e.g. the sodium salts of tallow fatty acid and coconut fatty acid or glycerol.
  • a shampoo may have, for example, the following composition: 0.01 to 5% by weight of an oligomer of the invention; 12.0% by weight sodium laureth-2-sulfate;
  • TLC was performed using Merck TLC Silica gel 60 A F 25 4 plates. TLC plate visualizations were conducted under UV light (256 & 366 nm).
  • NMR spectra were recorded on either a Bruker Avance DPX 300 ( 1 H and 13 C NMR at 300 MHz and 75.47 MHz respectively) or a Bruker Avance III 400 ( 1 H and 13 C NMR at 400.13 MHz and 101.62 MHz respectively).
  • the data was processed using TopSpin (version 4.1.3), which referenced the spectra to those of the residual solvents.
  • Mass spectra were recorded on a ABI 4800 Proteomics Analyzer MALDI TOF/TOF mass spectrometer (Applied Biosystems).
  • GPC Gel Permeation Chromatography
  • XPS X-ray photoelectron spectroscopy
  • Elemental analysis for anion exchange was achieved by performing X-ray photoelectron spectroscopy (XPS) using an AXIS Supra spectrometer (Kratos Analytical, UK) equipped with a hemispherical analyzer and a monochromatic Al K-alpha source (1487 eV) operated at 15 mA and 15 kV.
  • the XPS spectra were acquired from an area of 700 x 300 pm 2 with a take-off angle of 90°. Pass energy of 160 eV and 20 eV were used for the survey and high-resolution scans, respectively. A 3.1-volt bias was applied to the sample to neutralize charge build-up on the sample surface.
  • FIG. 1A A total of nine OIM1-6 derivatives each with six imidazolium rings (1-9) (Fig. 1A), including the parent OIM1-6-CH, were synthesized to study the effects of various substitutions at either the C2- or C4- position of the imidazolium moiety on antimicrobial potency and cytotoxicity.
  • the methods and chemical characterization of the OIMs are described in Fig. 2 and below.
  • the parent OIM1-6-CH (1) has a hydrogen (H) at the C2-carbon (Fig. 1A).
  • MALDI-TOF HCCA matrix, Reflector mode
  • Benzimidazole (8a) (391 mg, 3.31 mmol) was dissolved in MeCN (20 mL) and treated with 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 benzyl (3-bromopropyl) carbamate (750 mg, 2.76 mmol) in MeCN (5 mL) and the reaction mixture was stirred at 50°C for 18 hours.
  • 1,4 dibromobutane (2.5 equiv.) was added to a stirred solution of the required starting material (1b-9b) (1.0 equiv.) in dry MeCN (1 mmol/mL) under argon.
  • the reaction mixture was heated under reflux overnight and then cooled to rt.
  • the reaction mixture was concentrated under rotary evaporation and purified by silica gel chromatography eluting with 0-15% MeOH in EtOAc to afford the desired alkylated bromo-product.
  • MALDI-TOF HCCA matrix, Reflector mode
  • MALDI-TOF HCCA matrix, Reflector mode
  • MALDI-TOF HCCA matrix, Reflector mode
  • MALDI-TOF HCCA matrix, Reflector mode
  • Benzimidazole (8a) (4.10 g, 34.7 mmol) was dissolved in MeCN (80 mL) and treated with 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 treated with 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 filtered with water rinsing and the solids dried to afford a colorless solid (3.15 g, 10.8 mmol, 78%).
  • MALDI- TOF HCCA matrix, Reflector mode
  • MALDI-TOF HCCA matrix, Reflector mode
  • MALDI-TOF HCCA matrix, Reflector mode
  • the precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride.
  • Ionexchange resin column preparation 1 M HCI aqueous solution passed through a glass column packed with Amberlyst® A-26 (OH- form) until the pH of eluates reached the same value as the original solution, and then the resin was washed with water until a neutral pH was achieved).
  • the product-containing eluent was transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialysed against 5 ml_ HCI in 5 L of deionised water over 24 hours, with frequent changing of dialysis water every 2-3 hours.
  • the resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product.
  • the reaction mixture was allowed to cool to rt and treated with excess EtOAc.
  • the resulting precipitate/gum was isolated, further triturated with EtOAc, and treated with a solution of HBr in acetic acid (33%) (10 equiv.) and the resulting mixture was stirred at rt overnight.
  • the reaction mixture was treated with excess EtOAc and the resulting precipitate/gum was isolated.
  • the precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride.
  • the product-containing eluent was transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialyzed against 5 mL HCI in 5 L of deionized water over 24 hours, with frequent changing of dialysis water every 2-3 hours.
  • the resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product.
  • the precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride.
  • the product-containing eluent was transferred to a dialysis bag with Mw cut-off of 1000 Da and dialyzed against 5 mL HCI in 5 L of deionized water over 24 hours, with frequent changing of dialysis water every 2-3 hours.
  • the precipitate/gum was further triturated with EtOAc, dissolved in a water/MeOH mixture to give a clear solution and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride.
  • the product-containing eluent was transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialyzed against 5 mL HOI in 5 L of deionized water over 24 hours, with frequent changing of dialysis water every 2-3 hours.
  • the resulting solution was concentrated under rotary evaporation and lyophilized to afford a colorless solid (38 mg, 0.027 mmol, 25%).
  • the reaction mixture was treated with excess EtOAc and the resulting precipitate/gum was isolated.
  • the precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride.
  • the resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product.
  • 3-Bromo-1-propanol (3.2 ml_, 3.57mmol, 2 eq) and paraformaldehyde (0.5355 g, 1.78 mmol, 1 eq) were dissolved in toluene (10 ml_) in a round-bottom flask equipped with a Dean-Stark apparatus. Concentrated sulfuric acid (1 drop, cat.) was added and the mixture was refluxed for 1 ,5h, until approximately the expected amount of water has been collected. After cooling, sodium bicarbonate (approx. 0.2 g) was added and the mixture was filtered.
  • the precipitate/gum was dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 for exchanging counterion to chloride.
  • the resulting solution was concentrated under rotary evaporation and subjected to lyophilization to afford the desired product (1gm, 55%).
  • the reaction mixture was heated to 90 °C for 18 hours.
  • the reaction mixture was allowed to cool to rt.
  • the resulting gum was separated, further triturated three times with MeCN/EtOAc (8:2), the resulting precipitate/gum was isolated.
  • the precipitate/gum was dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 for exchanging counterion to chloride.
  • MALDI-TOF HCCA matrix, Reflector mode
  • Anhydrous Mg(CIC>4)2 was heated under vacuum at (0.1 Torr) at 130 °C for 2 h prior to use to enhance its reactivity.
  • the solution of 3-bromo-1 -propanol (10.0 g, 71.95 mmol, 1 equiv.) in CH 2 CI 2 (90 mL) was taken in a three necked flask equipped with condenser and magnetic stirred bar containing Mg(CIC>4)2 (1.6 g, 7.20 mmol, 0.1 equiv.) and stirred till the solution became clear.
  • the reaction mixture was cooled to room temperature, gummy precipitate formed decant the solvent and washed several times with ethyl acetate then the gummy product was dissolved into de-ionized water then wash with several times ethyl acetate to remove the excess dibromo alkylating reagent by separating funnel then concentrate the deionized water under reduced pressure to afford a brown gum 15g (3.5 g, 97%).
  • the gummy product was dissolved in deionized water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 for exchanging counterion to chloride.
  • the resulting gum was separated, and further triturated three times with MeCN/EtOAc (8:2), and the resulting gum was isolated and treated with a solution of HBr in acetic acid (33%) (10 equiv.) and the resulting mixture was stirred at rt for 2 hours.
  • the reaction mixture was treated with excess EtOAc and the resulting gum was isolated and dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride.
  • the resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product 16 white hygroscopic solid (1.8 g, 55%).
  • MICs were determined according to standard broth microdilution method with slight modification (Wiegand, I. et al., Nat. Protoc. 2008, 3, 163-175). Briefly, a single colony was picked and inoculated to obtain overnight culture. A subculture was prepared the next day and grown to exponential phase. A two-fold serial dilution of test compound in MHB broth was prepared in a 96-well plate, followed by addition of exponential phase bacteria at concentration of 5X 10 5 CFU/mL. The solution in the plate was thoroughly mixed by shaking the plates vigorously for 20 seconds, and the plate was subsequently incubated statically at 37 °C for 18 hours.
  • ODeoo readings were taken and the minimum concentration that inhibits bacterial growth by 90% (MICso) was calculated.
  • VRE2 E. faecalis 583
  • TLB tryptic soy broth
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • antibiotics penicillin/streptomycin
  • the killing kinetics of OIMs were determined via time-kill assay. Exponential phase bacteria (prepared via the MIC protocol above) were diluted to 5x10 5 CFU/mL and treated with varying concentrations of OIMs with constant shaking at 37 °C. Aliquots of bacterial samples were taken at specific time intervals (30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 24 hours) and serially diluted with phosphate buffered saline (PBS). The serially diluted samples were then spotted on agar plate and the number of colonies was counted after overnight incubation.
  • PBS phosphate buffered saline
  • Hirshfeld charges Hirshfeld, F. L., Theor. Chim. Acta 1977, 44, 129- 138
  • DFT calculated wavefunction Converted from Gaussian chk to fchk using the formchk utility
  • Multiwfn software Li, T. & Chen, F. Multiwfn, J. Comput. Chem. 2012, 33, 580-592
  • the membrane potential-sensitive dye 3,3'-Dipropylthiadicarbocyanine Iodide (DiSCs(5)) was used to determine the membrane depolarization activities of OIMs.
  • the assay was performed following a previously described protocol with minor modification (Belley, A. et al., Antimicrob. Agents Chemother. 2009, 53, 918-925; and Te Winkel, J. D. etal., Front. Cell Dev. Biol. 2016, 4, 29).
  • Exponential phase MRSA LAC was pelleted and washed twice with 5mM HEPES plus 5mM glucose and resuspended to 4x10 7 CFU/mL.
  • DiSCs(5) 0.5 pM was added to the bacteria and 175 pL of bacteria was aliquoted into a white 96 well plate. The fluorescence was monitored using a Spark 10M plate reader (Tecan, Switzerland) at Ex/Em of 622nm/670nm every 2 minutes with continuous shaking. The DiSCs(5) dye is absorbed into the bacterial membrane which results in the quenching of the fluorescence signal. Once a stable fluorescence reading was obtained, OIMs were added at varying concentrations in total volume of 25 pL, and the fluorescence reading was immediately recorded for 1 hour. Gramicidin was used as a positive control.
  • LAC membrane integrity assay
  • 1 pM propidium iodide (PI) dye was added to the bacteria.
  • the PI signal was monitored before the addition of OIMs and nisin (positive control) for baseline reading and subsequently recorded for 1 hour after the addition of test compounds at Ex/Em of 535nm/620nm using Tecan plate reader.
  • a mixture of PC/PG/carbazole at 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 solutions in a round-bottom flask (25 mL).
  • the chloroform was removed by rotary evaporation at 50 mbar, 20 °C for 20 min.
  • the lipid film was hydrated with 1 mL of PBS buffer (pH 7.4).
  • the suspension was then vortexed at top speed for 1.5 min, followed by 2 min of sonication in ice bath. This vortex-sonication process was performed three times, and the solution was then extruded 19 times through a 200 nm polycarbonate membrane with an Avanti mini-extruder.
  • the resulting liposome solution was dialyzed against water with MWCO 2000 tube for 36 hours to remove free carbazole.
  • the size distribution and zeta-potential of the resulting liposomes were checked with a Malvern Nano Series Nano-ZS instrument (data not shown). All prepared liposome solutions were stored at 4 °C prior to use.
  • Au(lipids) was prepared by the same protocol used for carbazole(lipids), replacing carbazole solution with the AuCI(SMe2) stock solution (3.82 mg/mL, 1.3 x 10 5 mol/mL), with PC/PG/AuCI(SMe2) molar ratio (8: 2: 1). After vortex-sonication, no extrusion was performed because of the sensitivity of AuCI(SMe2). Hydrophobic AuCI(SMe2) that was not trapped within liposome bilayer was removed by centrifugation at 4000 g for 20 min at 10 °C. The resulting pellets were washed with water once.
  • Au(lipids) pellets obtained after washing were dispersed in 10 ml_ of 0.1 x phosphate buffer (pH 7.4), followed by adding OIM stock solution at final concentration of 75 pg/mL. The mixture was incubated at 37 °C in the dark for 48 hours. Then the mixture was freeze dried. The dried powder was dissolved in 1 ml_ of methanol. The addition of methanol would break the liposome structure, thus releasing Au-OIM into the solution. The solution was centrifuged at 8,000 g for 30 min 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 AuCI(SMe 2 ) before LC-MS measurement.
  • Mass spectra were recorded by an Agilent Q-TOF/MS (6550 iFunnel) machine. Electrospray ionization (ESI) mass spectroscopy (MS) positive ion mode was used. C18 column (50 mm x 2.1 mm i.d., 1.8 pm, Agilent) was used at 35 °C for all the analyses.
  • the mobile phase consisted of a linear gradient system of (A) water (0.1 % formic acid) and (B) acetonitrile (0.1 % formic acid).
  • the gradient conditions of the mobile phase 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 pL.
  • Liposomes were prepared by the same protocol used for carbazole(lipids) with the exception that no carbazole was added. Liposome stock (10 mg/mL), OIM stock (10 mg/mL), and water were mixed to make 1 mL solution with final liposome and OIM concentration at 100 ppm and 5000 ppm, respectively. After incubation at 37 °C for 20 hours, free OIM was removed by Tangential Flow Filtration (TFF) (MidiKros® Hollow Fiber Module, MWCO 3kDa) with syringes through 30 cycles of extrusion with 0.2* concentration of PBS. The resulting solution (around 1 mL) was split into two equal portions.
  • TMF Tangential Flow Filtration
  • OIM released from the core and OIM trapped in the bilayer were separated by Vivaspin (MWCO 30 kDa) with centrifugation at 4000 g at 25 °C for 30 min, as OIM released from core would pass through the Vivaspin membrane while OIM trapped in the liposome bilayer would be retained at the membrane due to the size of the liposomes.
  • Vivaspin MWCO 30 kDa
  • lipid matrix To obtain an accurate calibration curve, we prepared lipid matrix with the same procedures used in the uptake experiments. Specifically, 200pL of liposome stock, containing 0.4 mg PG and 1.6 mg PC, was freeze-dried. After that, 16 mL methanol was added to dissolve the liposomes and then 14.4 mL DI water, 16 mL chloroform and 46.4 pL formic acid (final concentration 0.1 % v/v) were added to remove lipids to avoid any damage to the C18 column. After centrifugation at 3000 rpm for 10 min, the supernatant (top phase) was collected as the lipid matrix.
  • OIM1-6-CH (1), OIM1-6-C2(CH 3 ) (2) and OIM1-6-C4(CH 3 ) (5) were prepared with the lipid matrix at 0, 1 , 2, 3 and 4 ppm to build calibration curves. These standard samples were then tested with an Agilent Q-TOF/MS (6550 iFunnel) machine. Results are shown in Fig. 9. Molecular dynamics simulation of the interaction of cationic versus NHC forms of OIM1-6-CH with model S. aureus membrane
  • a 286-lipid membrane bilayer representative of the S. aureus membrane composition comprising 58% phosphatidylglycerol (PG) and 42% Cardiolipin (CL) (Epand, R. M. & Epand, R. F., Biochim. Biophys. Acta 2009, 1788, 289-294), was constructed using the CHARMM- GUI (Jo, S. et al., J. Comput. Chem. 2008, 29, 1859-1865) membrane builder tool.
  • DMPG 14:0/14:0
  • TMCL2 14:0,14:0/14:0,14:0
  • PG and CL lipids The distribution of PG and CL lipids on the upper and lower leaflets was symmetrically constructed.
  • Menaquinone-8 (MQ8) molecules were constructed on the membrane model. Parameters of membrane lipids were based on the CHARMM36 force field and parameters of the MQs and OIM1-6-CH (1) were based on the CHARMM General Force Field (Vanommeslaeghe, K. & MacKerell, A. D., J. Chem. Inf. Model. 2012, 52, 3144-3154; and Vanommeslaeghe, K. et al., J. Chem. Inf. Model. 2012, 52, 3155-3168).
  • An equal number of MQ molecules were manually inserted randomly into the upper and lower leaflet of the constructed S. aureus membrane. Manual adjustments of coordinates and energy minimization steps were subsequently performed to remove structural clashes between atoms.
  • the membranes were solvated with TIP3P (Jorgensen, W. L. et al., J. Chem. Phys. 1983, 79, 926-935) water molecules and counterions were added to neutralize the system.
  • aureus membranes were subjected to a 100 ns molecular dynamics (MD) simulation using GROMACS (Van Der Spoel, D. etal., J. Comput. Chem. 2005, 26, 1701-1718) 5.1.2 software.
  • the LINCS Hess, B., J. Chem. Theory Comput. 2008, 4, 116- 122
  • a 1.2 nm cutoff was used for Van der Waals interaction and short-range electrostatic interactions calculations, and Particle Mesh Ewald method was implemented for long range electrostatic calculations.
  • Simulation temperature was maintained at 310 K using a V-rescale thermostat (Bussi, G. etal., J. Chem.
  • Coordinates of the cationic and NHC forms of the OIM1-6-CH (1) were constructed using Discovery Studio 4.1 (Zhuo, S. et al., Molecules 2020, 25, 5649). Topologies of (1) were obtained from the CHARMM-GUI ligand reader and modeler (Kim, S. etal., J. Comput. Chem. 2017, 38, 1879-1886). Partial charges of the NHC carbene forms were calculated from Gaussian09 (Gaussian 09 (Gaussian, Inc, Wallingford, CT, USA, 2009)) in the triplet state at the level of HF 6-31G* and RESP (Bayly, C. I. et al., J. Phys. Chem.
  • the number of contacts between the OIM and the membrane as a function of simulation time was calculated to analyze the behavior of interactions between the oligomer and membrane. Contact number calculations were performed using the GROMACS (Van Der Spoel, D. et al., J. Comput. Chem. 2005, 26, 1701-1718) gmx mindist tool. A contact was defined if the minimum distance between the OIM atom and membrane atom was less than 0.4 nm. To further visualize the binding between OIM and the membrane, the last frame of each simulation system was visualized using pymol.
  • OIM1- 6-CH (1) characterized by its carbon acidity with the presence of a C(2)-H, exhibited substantial potency with a low geometric mean of minimum inhibitory concentration (Geo- MlCao) of 4.0 pg/mL against a panel of ESKAPE pathogens that includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae.
  • Table 1 Antibacterial and cytotoxicity of OIM series compounds (MICso, pg/mL).
  • Ciprofloxacin 1-2 4 ⁇ 1 ⁇ 1 ⁇ 1 ⁇ 1 ⁇ 1 ⁇ 1
  • IC50 half-maximal inhibitory concentration
  • OIM1-6 derivatives with substitution at the C2-carbon of the imidazolium moiety were also synthesized to study the structure-activity relationship (SAR) surrounding the imidazolium moiety.
  • Derivatives (2-4) (Fig. 1A) with C2-hydrogen replaced by various substituents, i.e., weakly electron-donating methyl- (-CH3), and electron-withdrawing chloro- (-CI) and trifluoro- (-CF3) groups, were made.
  • the C2-substituted derivatives (2-4) resulted in a significant loss in bacterial inhibition potency compared to 0IM1-6-CH (1).
  • the C4 derivatives (6-9) with electron withdrawing groups should have much better potency than the C4-CH 3 (5) derivative with the electron donating methyl group, just like 3, 4 are more potent than 2 but (6-9) were not obviously more potent than (5).
  • the trend of (5) versus (9) was reverse to that of (2) versus (4).
  • N-heterocyclic carbenes N-heterocyclic carbenes
  • the derivatives (2-4), devoid of carbon acid characteristic as their C(2)-H atoms were replaced by various substituents that encompass both weakly electron-donating (methyl- (-CH3)), and electron-withdrawing (chloro- (-CI) and trifluoro- (-CF3)) groups, exhibited markedly diminished potency when compared to the parent OIM1-6-CH (1).
  • the three non-carbon acid compounds (2-4) possessing C2-substituted groups displayed Geo-MIC90 values ranging from 118.5 pg/mL to 344.6 pg/mL, which were significantly higher than the Geo-MIC90 value of 4.0 pg/mL observed for compound (1).
  • OIM1-8-C2(CH3) without the C2-H (11) also had reduced potency compared with the parent OIM1-8-CH (10) which was similarly observed in the OIM1-6 series.
  • OIM1-8-C4(CH3) (12) possessing a C(2)-H, displayed potency identical to (10), with a Geo- MlCgo of 3.4 pg/mL.
  • OIM1-6-CH and OIM1-6-C4(CH3) are bactericidal and had fast kill kinetics. Both bacterial strains were completely eradicated within 1 -2h with these compounds at the concentration of 4x the MIC. On the other hand, OIM1-6-C2(CH3) had greatly reduced potency. Table 4. Antibacterial effect (MIC90, p.g/ml_) of selected OIM1-6 series compounds compared to the activity of antibiotics on a panel of pan-resistant bacteria and naturally antibiotic resistant bacteria.
  • the first MIC is in MHB, and the 2 nd MIC value is in MHB supplemented with hemin.
  • OIM1-6-CH generally had no short-term toxicity (i.e. 24 hours) but had long-term toxicity (i.e. 48 and 72 hours) against all cell lines tested, i.e. 3T3 fibroblast cells, human embryonic kidney (HEK) cells and HepG2 cells (Table 5, Figs. 10-16).
  • the derivative (5) with -CH 3 at C4 position generally showed improved long-term toxicity compared to OIM1- 6-CH.
  • increasing the chain length of the OIMs from 6 (in compounds 1, 2 and 5) to 8 repeating units (in 10-12) respectively resulted in increased cytotoxicity.
  • the biocompatibility of the degradable compounds (13-14) was improved compared to its parent OIM.
  • OIM1-6-CH (1) and OIM1-8-CH (10) carbon acids exhibited low short-term (i.e. within 24-hour) toxicity. However, they demonstrated elevated longer-term (i.e. 72-hour) toxicity against all tested cell lines, including 3T3 fibroblast cells, human embryonic kidney (HEK) cells and liver hepatocellular carcinoma (HepG2) cells (Tables 1-3, Figs. 10-16).
  • VRE2 E. faecalis 583 (VRE2) a 32-64 / 2 >512 / >512 128 / 8 8 / 4 16 / 4 >512 / >512/ >512
  • Geometric mean (Geo-MIC90) 3.5 265.0 10.1 4.9 7.4 26.9 20.4 a The first MIC values were tested in MHB, and the second MIC values (after the 7”) were tested in MHB supplemented with hemin.
  • Example 6 OIM carbon acids with deprotonated NHCs translocate plasma membrane of bacterial-mimicking liposomes and enter liposome cores
  • oligoimidazolium in the presence of OH ions will have two monomer forms (Fig. 25C), i.e. the azolium cation (/) and the azole-2-ylidene carbene (//) and will be a copolymer (poly(/-//)).
  • Fig. 25C the C(2)-H 1 H NMR rapidly decreased at pH 7.16 and pH 8.21 (Fig. 25B), indicative of a fast H-D exchange rate.
  • the H-D exchange rate slowed due to lower deuteroxide (base) concentration (Fig. 25B).
  • probe(lipids) such as carbozole(lipids) and Au(lipids).
  • NHC 1 ,3- Bis(2,6-diisopropylphenyl)-1 ,3-dihydro-2H-imidazol-2-ylidene (IPr)
  • IPr 1 ,3- Bis(2,6-diisopropylphenyl)-1 ,3-dihydro-2H-imidazol-2-ylidene
  • AuCI(SMe2) can react with NHC under mildly basic ambient conditions, resulting in the formation of the covalently linked [AuCI(NHC)] complex (Nahra, F. et al., Nat. Protoc. 2021 , 16, 1476-1493) (Fig. 25A).
  • the hydrophobic AuCI(SMe2) was placed in the liposome bilayer to attempt to capture NHCs generated from OIM. Then, OIM1-6-CH (1) aqueous solution was added into the Au(lipids) suspension. AuDMSCI molecules are expected to stay in the liposome bilayer because of their hydrophobic nature.
  • NHC formation “hydrophobizes" OIMs (1) and (5) to aid the oligomer entry into the membrane bilayer, an essential step preluding the internalization into the liposome core.
  • the methyl-substitution at the C2-carbon on compound (2) prevents carbene formation of the non-carbon acid, impeding the entry the charged hydrophilic OIM into both the bilayer and the liposome core.
  • the acidity of various OIM carbon acids (1, 5-9) may be an important factor contributing to good antibacterial efficacies.
  • the least acidic carbon acid is OIM1-6-C4(CH3) (5) with pK a 22.15 while the most acidic one is OIM1-6-C4(CF 3 ) (9) with pK a of 20.06 and OIM1-6-CH (1) has intermediate acidity (pK a 21.32).
  • the electron-donating -CH 3 in (5) makes the C2-proton less acidic resulting in a higher pK a than (1).
  • electron-withdrawing groups (-CI, -F, -Bz, and -CF 3 in 6-9) make the C2 proton more acidic, resulting in lower pK a in (6-9).
  • the reaction mixture was diluted with water, transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialyzed against 1 mL HCI in 1 L of deionized water over 24 hours, with frequent changing of dialysis water every 2- 3 hours.
  • the resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product as a mixture of the unreacted starting material, and mono- and bisconjugated products.
  • the percentage of dye conjugation per parent compound was estimated using 1 H NMR based on integrals of signals at the 3.80 -4.40 ppm and 6.00- 6.70 ppm region which corresponded to the 12 x imidazoyl-N-CH2- within the parent OIM compound and 3 x Ar-H of the 2 x phenolic group within FITC respectively.
  • 24:6 integrals of a signal at the 3.80 - 4.40 ppm and 6.00 - 6.70 ppm would infer 100% conjugation (1 :1 ratio between parent compound and FITC dye).
  • the percentage of dye conjugation per parent compound was obtained by calibrating the integrals of signals at 3.80 - 4.40 ppm to 24 and the obtained integral value of the signal at 6.00 - 6.70 ppm was divided by 6, expressed as a percentage.
  • OIM-FITC uptake experiment was conducted as described previously (Radlinski, L. C. 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).
  • Exponential phase bacteria were prepared and diluted to 10 s CFU/mL in TSB broth, then incubated with FITC-conjugated OIM (7.5% molar ratio of OIM-FITC) at desired concentrations for 1 hour in the dark.
  • the fluorescence signal of cytosolic - internalized OIM-FITC in permeabilized bacteria will be greatly reduced after quenching as Trypan Blue will be able to quench the cytosolic - internalized OIM-FITC, however the OIM-FITC that is embedded in the membrane could not be removed by TB quenching.
  • the former is observed for bacteria treated with SYTO 9, OIM1-6-CH-FITC and OIM1-6-C4(CHs)-FITC, while the latter is observed for OIM1-6-C2(CH3)-FITC.
  • the internalised portion for OIM1-6-C2(CH3)-FITC is then the subtraction of Trypan Blue quenched fluorescence of unpermeabilised with that of permeabilised treated bacteria.
  • the three OIMs derivatives were labelled with FITC fluorochrome (as described in Example 7), and their uptake was quantified using flow cytometry (Fig. 30). Given the higher cell density required for flow cytometry experiments (10 6 CFU/mL), we determined the MIC* values at the higher cell density.
  • FITC-OIM 0.01% Trypan Blue (TB) dye, which is membrane impermeable, was used to quench the extracellularly associated FITC-conjugated oligomers (Antonoplis, A. etal., J. Am. Chem. Soc. 2018, 140, 16140-16151; Avelar-Freitas, B. et al., Braz. J. Med. Biol.
  • the average MIC* value against the parental S. aureus strain for the three compounds were as follow: 12 pg/mL (1), >4096 pg/mL (2), and 48 pg/mL (5) (Fig. 31A), which align with the MIC values presented in Table 1.
  • Fig. 31A The average MIC* value against the parental S. aureus strain for the three compounds were as follow: 12 pg/mL (1), >4096 pg/mL (2), and 48 pg/mL (5) (Fig. 31A), which align with the MIC values presented in Table 1.
  • the percentage of S. aureus strain count exhibiting cytosolic uptake, as well as total uptake was significant at all tested concentrations (Fig. 31 B), indicating that a significant portion of bacteria had taken up (1) into their cytosol.
  • OIM1-6-CH had the highest total uptake at the low concentrations (4 and 8 pg/mL) (Fig. 32A) while OIM1-6-C2(CH3) and OIM1- 6-C4(CHs) had low total uptake at these concentrations. Further, OIM1-6-CH had the highest internalized fraction at these low concentrations. However, the internalized percent of OIM1- 6-C4(CH3) increased significantly at 16 pg/mL, which is the MIC* (MIC value correlating to 10 6 CFU/mL bacteria) of OIM1-6-C4(CH3).
  • the OIM1-6-C2(CH3) had the least amount (around 10%) of OIM internalized into the cytosol at 4-16 pg/mL but the internalized amount increased substantially at 128 pg/mL which is also its MIC* value. Similar trend was observed with the uptake of OIMs in S. aureus LAC tested in TSB medium: at the MIC* of OIM1-6-CH and OIM1- 6-C4(CH3) of 8 pg/mL and 64 pg/mL respectively, high internalised fractions were observed respectively (Fig. 32B). For TSB media, OIM1-6-C2(CH3) does not have MIC* in the concentration range tested.
  • OIM1-6-CH internalisation is most efficient followed by OIM1-6-C4(CH3) and both are carbon acids that can form NHC.
  • the internalisation of OIM-1-6-C2(CHs) without the dissociable C2 proton is poor, suggesting that the presence of C2 hydrogen allowing NHC formation is essential for polymer internalization that correlates with compound potency.
  • Example 9 The bacteria surface binding and intracellular internalisation of carbene- forming OIM1-6-CH is PMF independent
  • Sub-culture of S. aureus LAC or P. aeruginosa PAO1 was added to OIMs with varying concentration, starting from 0.5x MIC to 4x MIC based on the MIC protocol and was set up with 8-10 independent replicates. After 18-24 hours, each replicate with at least 50% growth in the highest concentration of OIMs were selected for the next passage. The OIMs concentration were adjusted based on the acquired mutations of the strains. The passage was repeated every day until the mutants acquired high resistance (MIC value was >1024 pg/mL) or until 30 days for PAO1. Bacteria samples were preserved with 30% glycerol in -80 °C on each day of the passage.
  • the LAC mutants were streaked out on agar plate. Two colonies from each plate were selected for resistance stability testing. These LAC mutant strains were passaged for 7 days without OIMs, and the MIC was tested at Day 1 and 7th respectively. Mutants that shown stable resistance were selected for whole genomic sequencing.
  • the genomic DNA was extracted using QIAamp DNA Mini Blood Mini Kitwith minor modifications.
  • the cell wall was digested with 10 mg/mL lysozyme and 10 pg/mL lysostaphin and the bacteria was lysed via beat beater before proceeding to the standard protocol of the kit.
  • DNA was prepared for sequencing by using an Illumina Nextera DNA Library Preparation Kit. DNA was sequenced on an Illumina MiSeq instrument (paired end sequencing). Sequences were mapped onto the genome of the parent strain S. aureus LAC, and CLC Genomics Workbench software was used to identify single nucleotide variations, small deletions, and insertions. Large deletions were identified by manual sequence comparison.
  • Oxygen consumption rate (OCR)
  • the OCR was measured using a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience).
  • the sensor cartridge was equilibrated per supplier instruction before use, and the sample cartridge was coated with poly-D-lysine for bacterial adhesion.
  • LAG sub-culture was grown in M9 buffer (1x M9 salt solution, 2 mM MgSO4, 0.2% (w/v) glucose, 0.2% (w/v) Casamino acids, 0.2 pg/mL Nicotinamide, 100 nM Thiamine) until exponential phase. Then, 100 pl of 10 6 CFU/mL bacteria were added into each well of the sample cartridge except the blank wells.
  • the sample cartridge was centrifuged at 1400xg for 10 min, then 80 pl M9 buffer was added into each well. Basal OCR was measured for 3 readings before OIMs/ antibiotics injection.
  • TSB was used as growth medium in aerobic, anaerobic and fermentation growth conditions.
  • 100 mM sodium nitrate was added into TSB.
  • All liquid media, pipettes and consumables used for anaerobic and fermentation growth conditions were equilibrated in the anaerobic chamber for at least 2 days. The overnight and subculture were grown at 37 °C in the anaerobic chamber.
  • 20 pl aliquots were removed at the indicated time points and serially diluted with 180 pl of PBS. Aliquots of the dilutions were spotted on agar plates and colonies were counted following overnight incubation.
  • the antagonism effect of PMF dissipating agents (CCCP, valinomycin and nigericin) on OIM1 compounds were tested with S. aureus LAC in a checkerboard assay.
  • Serial 2-fold dilutions of PMF dissipating agents (starting from 0.5x MIC) and OIM1 compounds (starting from 64 pg/mL) were prepared in TSB.
  • 25 pL of PMF dissipating agents (along y-axis) and 25 pL of OIM1 compounds (along x-axis) were added into each well of a 96-well plate, and 50 pL of log-phase bacteria (prepared as described in the MIC protocol in Example 5) was subsequently added.
  • the solutions were mixed thoroughly, and the MIC were determined as described above in Example 5.
  • Amino acid change fs refers to frameshift; * indicates stop codon.
  • OIM1-6-CH did not kill the bacteria through halting oxygen respiration.
  • the decrease in OCR observed 30 minutes post-treatment (i.e. after the first doubling time) was interpreted to be a consequence of growth inhibition and death rather than a direct effect on oxygen consumption.
  • Table 10 MIC of OIM series compounds and control antibiotics against S. aureus LAC and respiratory mutants in different pH.
  • OIM1-6-C4(CH 3 ) had increased more significantly than OIM1-6-CH (64-fold compared to 16-fold respectively) at the slightly acidic pH conditions, inferring greater dependence on the NHC formation for the potency of OIM1-6-C4(CH 3 ).
  • both OIM1-6-CH and OIM1-6- C4(CH 3 ) became significantly ineffective with slightly decreasing pH, while there was only minimal impact on gentamicin and daptomycin.
  • the reduced potency of OIM1-6-CH and OIM1-6-C4(CH 3 ) against the wildtype LAC was further confirmed by the uptake assay tested at pH 6.8 in TSB at their respective MIC* (Fig.
  • OIM1-6-C4(CH3) on the other hand, exhibited poor killing in both anaerobic and fermentative conditions, indicating that it is strongly PMF-dependent. Taking together for OIM1-6-CH, uptake can take place without PMF in respiration mutants and killing is observed even in fermentative mode, indicating that OIM1-6-CH uptake depends on a second mechanism apart from PMF, likely NHC-related, that contribute to its intracellular accumulation and killing.
  • OIM1-6-C4(CH 3 ) has weaker ability to form NHC and hence its stronger reliance on PMF for intracellular uptake, whereas the inability of OIM1-6-C2(CH 3 ) to form NHC render this compound ineffective to enter the bacteria and therefore has poor potency. Taken together, these data suggest that the carbene formation of OIM1-6-CH and OIM1-6-C4(CH 3 ) promotes their internalization into bacteria resulting in good MICs.
  • cationic compounds cannot eradicate bacteria in anion-containing formulations because of charge neutralization which severely limits their applications.
  • One such formulation is in laundry detergent which mainly consists of anionic surfactants, along with builders, chelators, bleaching agents, enzymes, and stabilizer that are meticulously formulated to remove dirt, soil, and stains effectively from textile.
  • Antibacterial test in laundry detergent Antibacterial test was performed according to standard test method ASTM-E2274 (Li, X. et al., J. Mater. Chem. B 2018, 6, 4274-4292). Briefly, a white cotton fabric (1.4 m x 2.8 m) was immersed in 5 L 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 a 50 °C oven for at least 24 hours. The cloth was cut into strips of 5 cm wide and 15 g each.
  • Japan pouch detergent marketed as P&G Ariel Bioscience Gel ball, composed of linear alkylbenzene sulfonate (LAS), alkyl ether sulfate, polyoxyethylene alkyl ether, and fatty acid ester
  • model detergents SDS, SDBS, SDS+SDBS
  • the final concentration of Japan pouch detergent, model detergent, and OIMs was 389 ppm, 100 ppm, 100 ppm, respectively.
  • the jar containing 250 mL of water, 0.25 mL of hard water, and 3.125 mL of 4 % Tween 80 solution was used as control.
  • the spindle with inoculated fabrics was placed into the jar and tumbled for 10 minutes, after which the three inoculated fabrics were transferred into 30 mL of neutralizer (Letheen Broth, Modified) and vortexed for two minutes at top speed.
  • 1 mL of neutralizer solution was serially diluted to 10' 4 with 0.85% NaCI.
  • 1 mL of each dilution was plated in duplicate in LB agar.
  • Iog10 reduction log10(CFU/three fabric carries of control) - log10(CFU/three fabric carries of OIM).
  • the polycation PDADMAC fail in all four detergents. Colistin pass in E. coli 8739 but failed in S. aureus 6538 (Fig. 35C). It is understandable that colistin kill E. coli 8739 in detergent because of its membrane disruption ability for Gram-negative bacteria. With our newly found carbene mechanism that distinguish OIMs from typical cationic polymers, OIM1-6-CH exhibit above 2 log reduction in Japan pouch and another three model detergents (Fig. 35D). In contrast, OIM1-6-C2(CH3) marginally pass when against E. coh 8739 but fail in S. aureus 6538 (Fig. 35E).
  • OIM1-6-C4(CH3) pass the test in SDS, SDS+SDBS and Japan pouch detergents, but fail in SDBS (Fig. 35F). It can be explained that OIM1-6-C2(CHs) and OIM1-6-C4(CH3) exhibit antibacterial activity in detergent mainly because of the polyion complex nanoparticles formed in anionic detergent. The poor antibacterial activity of OIM1-6- C4(CH3) in SDBS could be result from the higher hydrophobicity of SDBS. We suggest that the antibacterial activity of OIM1-6-CH in detergent is from its carbene mechanism, while the antibacterial activity of OIM1-6-C2(CH3) and OIM1-6-C4(CH3) in detergent is from the polyion complex nanoparticles formed in detergent.
  • OIM1- 8-Bu-2PzAc OIM1-8-Bu-2Ac
  • OIM1-8-Bu-2Ac which contain C2-proton but with degradable bond between imidazolium rings.
  • Fig. 35G shows that OIM1-8-Bu-2PzAc can pass all four detergents.
  • OIM1- 8-Bu-2Ac show potent antibacterial activity as well except in SDS+SDBS when against E. coh 8739 (Fig. 35H). The results indicate that compounds with imidazolium ring containing dissociable C2-proton display better bactericidal activity in anions-containing formulations.
  • Example 11 Degradable OIM is efficacious in a murine systemic infection model
  • IP intraperitoneal
  • mice were euthanized at 26 hours post-infection, and the bacteria counts in peritoneal fluid, livers, kidneys, and spleens were determined. For the survival test, mice were monitored over 7 days after infection. Results and discussion
  • mice were infected with a lethal dose of carbapenem-resistant A. baumannii through IP injection. Subsequently, they were treated with a single dose of 15 mg/kg OIM1-8- 2D (16) or 15 mg/kg imipenem (control) at two hours post-infection. Additionally, a control group received PBS only (Fig. 36A(i)). The administration of a single dose of (16) successfully rescued all the mice and led to a significant reduction in bacterial load in major organs, achieving a CFU reduction of 6 orders of magnitude. In contrast, untreated mice or those treated with imipenem succumbed to the infection within 36 hours (Figs. 36A(ii)-(iii) and 37).
  • mice To evaluate the in vivo tolerance of the OIM, we subjected the mice to seven successive daily doses of (16) at 15 mg/kg per day, totalling a combined dose of 105 mg/kg. Remarkably, this regimen did not result in any significant weight loss (Fig. 36B), underscoring the good biocompatibility of degradable OIM1-8-2D (16), coupled with its excellent ability to eradicate bacteria in a murine systemic infection model.
  • Example 12 A degradable cationic OIM derivative (16) eradicates bacteria to prevent dairy mastitis in animal trial
  • S. aureus ATCC 6538, S. uberis ATCC 19436 and E. coliATCC 10536 were used for the tests, as recommended by BS EN 1656 standard (Institution, B. S. (British Standards Institution, 2019)).
  • Bacteria of 2 nd subculture were streaked out from Tryptic Soy Agar (TSA) plates and inoculated into Tryptone NaCI diluent solution (0.1% tryptone and 0.85% NaCI) at 1.5 to 5 x 10® CFU/mL.
  • Test compounds were dissolved in hard water (0.119 g MgCh, 0.277 g CaCl2, 0.28 g NaHCOs in 1 L water) at desired concentration.
  • the trial was conducted in a dairy farm located at Jilin province, Changchun city, Jiutai district, Longjia town, Xiaochengzi village.
  • the mastitis protocol was approved by the Changchun University of Chinese Medicine (Ethics Protocol No. 202/205) to the Principal Investigator Professor Li Qingjie. Detailed experimental methods are described in the supplementary methodology of mastitis farm trial below.
  • a new intramammary infection in a quarter was diagnosed when the same bacterial species was isolated from 1) two consecutive samples during the trial (>500 CFU/ml); 2) a single sample from a quarter with clinical mastitis (> 100 CFU/ml); or 3) three consecutive samples during the trial (> 100 CFU/ml).
  • the trial was done in the local wintertime with outdoor temperature ranging from -10 to 10 °C.
  • the farm housed over 500 dairy cows and is equipped with an automated milking system. Cows were housed in the barn and were milked at a separate milking facility with ambient temperature of 5 to 10 °C. Milking is routinely done once per day at 2pm by a skilled worker. Post-milking teat dipping with iodine-based commercial product was practiced daily in the farm and was ceased 10-days prior to the start of the trial on the experimental cows to avoid carryover effect and disturbance on the farm trial results.
  • the product stock solution was stored at 4 °C.
  • the product working solution was prepared fresh on the day of the experiment by diluting the stock solution (1/30 ratio) in sterile DI water to achieve a final concentration of 0.05% active compound and 10% glycerol.
  • Pre-milking udder preparation consists of the use of single service water-moistened towels (free of sanitizer, one towel per teat) to wet and clean the teats prior to fore-stripping. Forestripping was accomplished by expressing three squirts of milk.
  • Swab sampling was carried out to determine the polymer adsorption on teat skin. After cleaning the teats (procedure stated in pre-milking udder preparation), the samples were taken using the wet and dry swab technique in accordance with DIN 10113-1 : 1997-07 (Scheib, S. et al., Pathogens 2023, 12, 560). A cotton wool swab moistened with sterile 0.25% Ringer’s solution was moved around the teat at a distance of 1 cm from the teat canal orifice. After that the same procedure was performed with a dry cotton wool swab (ultrafine, dry swab).
  • the milk samples were collected according to the procedure stated in the in vivo mastitis farm trial protocol above. To determine the polymer residual in milk, the milk samples were transferred to lab to determine polymer residuals using Delvo test kit (Stead, S. et al., Int. Dairy J. 2008, 18, 3-11). To determine the milk quality (e.g., somatic cell count), milk samples were tested by qualified testing labs within 24 hours.
  • Delvo test kit e.g., somatic cell count
  • S. aureus ATCC 49525 was used in farm trial due to its close relevance to clinical bovine mastitis (Wall, R. J. et al., Nat. Biotechnol. 2005, 23, 445-451).
  • a single colony was streaked out from agar plate into Trypticase Soy Broth (TSB) and incubated overnight under shaking at 37 °C. The overnight culture was sub-cultured 1 :100 into fresh TSB and incubated for 3 hours to obtain exponentially growing bacteria.
  • Bacteria were pelleted by centrifugation (3,000-4,000 g for 15 min), washed twice with 0.1% proteose-peptone and diluted to ⁇ 5 x 10 7 CFU/ml in fresh TSB.
  • the challenge suspension containing ⁇ 5 x 10 7 CFU/ml in TSB was prepared immediately before use.
  • the present disclosure underscores the pivotal role of carbon acidity in the antibacterial properties of OIMs, particularly those containing deprotonable C(2)-H groups (1, 5-8, 10, 12 and 15-16).
  • the carbon acids (1, 5-8, 10, 12 and 15-16) exhibit robust antibacterial efficacy, while non-carbon acids lacking C(2)-H (2-4, and 11) show considerably reduced effectiveness.
  • the OIM carbon acids have MICs that are about one order of magnitude lower and/or have broad spectrum potency, and with lower toxicity (Lam, S. J. et al., Nat. Microbiol. 2016, 1, 11 ; Chin, W. et al., Nat. Commun. 2018, 9, 14; and Zhang, K. et al., Nat. Commun. 2019, 10, 4792).
  • the classical cationic polymers cannot enter the bacterial plasma membrane as the charged polymers are hydrophilic. Above certain critical cationic charge concentrations, the cationic polymers form pores or holes in the membranes which then lead to cell death. As this is a physical process, the threshold concentrations are typically higher so that the MIC values are usually tens of microgram/mL. With the new NHC-mediated entry into the membrane and then cytosol, no pores or holes are needed for the entry into the membrane.
  • the OIM carbon acids (1 and 5) enters the liposome/bacterial core at lower cationic charge threshold concentration than (2). The OIM (2) cannot enter the bacterial cytosol or liposome core (Figs.
  • the OIM (1) though likely to be more hydrophilic than the methyl-substituted derivatives (2) and (5) can enter the membrane and core of liposomes more effectively, as it effectively forms NHC to enter the core through membrane translocation.
  • the OIM transforms with NHC formation from an initially hydrophilic to an amphiphilic copolymer which is composed of the hydrophilic imidazolium cation repeats and also the hydrophobic imidazol-2-yl carbene repeats, to achieve effective membrane entry (Fig. 24B).
  • the quantity of (1) and (5) entering the liposome core is smaller in comparison to their presence in the membranes (Fig. 25I).
  • the electric field across the membranes may favor the translocation of OIM-NHC into the cytosol.
  • the acidic protons of cycling reduced quinone shuttiers of redox-active electron transport chain (ETC) might induce the quenching of OIM carbene into cationic OIM within the bacterial membrane, rendering it hydrophilic. This shift in hydrophobicity could drive OIMs to exit the hydrophobic membranes and enter the cytosol.
  • the present disclosure demonstrates that carbon acids, aided by NHC formation, successfully infiltrate the bilayer membrane and exhibit high cytosolic uptake, ultimately reaching their intracellular targets, and hence resulting in rapid bacterial killing.
  • the facile NHC formation of (1) facilitates its internalization into the bacterial cytoplasm, even under conditions of low PMF, as evidenced by experiments with respiration-deficient mutants and fermentative bacteria.
  • our optimized degradable compounds (15) and (16) exhibit impressive minimum inhibitory concentrations (MICs) against multi-drug resistant bacteria, including the colistinresistant Gram-negative bacterium E. cloacae 13047 that is MDR.
  • MICs minimum inhibitory concentrations
  • the degradable OIM (16) demonstrated substantial efficacy against multi-drug resistant A. baumannii bacteria.
  • PIM1 D was successfully employed to prevent mastitis infection, with no adverse effects observed in the cows.
  • the OIM1-8-2D(4Br/4CI ) compound was treated with aqueous triethylamine (base, EtsN) to form the octa N-heterocyclic carbene (NHC) and then quenched with organic acids to generate the carboxylate imidazolium ionic liquids.
  • oligomers with the imidazolium ring containing dissociable C2-proton, i.e. carbon acids form NHCs in the hydrophobic bacterial-mimicking membrane bilayer.
  • the ability of the carbon acids to form NHC with unsubstituted C2-carbon is correlated with good antibacterial properties.
  • the formation of NHC enables the easy entry of the polymer into the bacterial cytoplasm without the need for a PMF as shown by the uptake of OIM1-6-CH into respiration defective mutants.
  • the ability to form NHC is correlated with the pK a values of the carbon acids. Carbon acids with higher pK a (with electron donating methyl substituent) makes the ability to form NHC more difficult resulting in lower toxicity but only slightly reduced antibacterial properties.
  • the toxicity of OIM1-6-CH at short term (24 hours) is excellent but it has toxicity longer term (48-72 hours) probably due to diffusion. However, the toxicity can be dampened in OIM1-6-C4(CH3) which has lower rate of carbene formation.
  • AMPs and antimicrobial polymers are potential pharmaceutical candidates to replace antibiotics as they are effective against a broad spectrum of multidrug resistant bacteria.
  • issues such as cytotoxicity and fouling are drawbacks of AMPs.
  • OIM1-6-CH which has a hydrogen (H) at the C2 carbon with exact 6 repeating imidazolium units has potent antibacterial activity with low short-term eukaryotic toxicity.
  • Structure-activity relationship (SAR) surrounding the imidazolium moiety was also studied by various substitutions at the C2 and C4 position.
  • OIM1-6-CH could still internalize into the cytosolic of respiration deficient mutants and fermentative bacteria via a second mechanism, which is the NHC formation.
  • Inhibiting the NHC formation of OIM1-6-CH and OIM1-6-C4(CH3) with slightly acidic pH greatly reduced their potency against S. aureus LAC and LAC respiratory mutants, conferring the importance of carbene formation in the efficacy of our compounds.
  • This additional mechanism aids to retain the excellent antibacterial properties of OIMs in the presence of high salts and surfactants as illustrated in the cloth test in Example 10.

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Abstract

Disclosed herein is a compound according to formula Ia: Also disclosed herein are a pharmaceutical composition comprising the compound as aforementioned, use of the compound as aforementioned, a method of treating one or both of a microbial and a fungal infection comprising the step of administering a pharmaceutically effective amount of the compound as aforementioned or the pharmaceutical composition as aforementioned, and an antimicrobial and/or antifungal detergent composition comprising the compound as aforementioned, and a surfactant.

Description

MAIN CHAIN CATIONIC OLIGO(IMIDAZOLIUM) FORMS N-HETEROCYCLIC CARBENE FOR EFFECTIVE BACTERIAL KILLING IN COMPLEX ENVIRONMENT
Field of Invention
The present disclosure generally relates to antibiotics, and more particularly relates to main chain cationic oligoimidazolium for effective bacterial killing in complex environments.
Background
The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Antibiotics have long served as essential therapeutic and prophylactic tools for biomedical, as well as agricultural, applications, underpinning numerous modern biomedical interventions, including chemotherapy and surgical procedures. However, the escalating global crisis of bacterial resistance to virtually all classes of antibiotics has cast a shadow over these medical advances. This crisis is further compounded by the sluggish pace of developing novel antimicrobial agents, a pressing challenge that has persisted for several decades. Notably, the approval of new antibiotic classes for the treatment of Gram-negative bacterial infections has remained elusive for over six decades. Conventional antibiotics typically operate by targeting specific enzymes involved in conserved metabolic processes, inevitably leading to the emergence of resistance strains.
In this context, the bacterial membrane represents one of the last frontiers in the quest for novel antibacterial drug. Membrane-targeting agents hold promise as they are usually less susceptible to resistance development. Conventionally, antimicrobial polymers and peptides (AMPs) depend on disrupting the integrity of bacterial cytoplasmic membranes to induce cell death. However, the advancement of classical cationic polymer and peptide agents that target bacterial membrane permeability has been hindered by concerns regarding their toxicity and limited metabolic stability.
Antimicrobial peptides (AMPs) such as defensins, daptomycin, magainin-2, etc. typically kill bacteria by interacting electrostatically with the cytoplasmic membrane, which is followed by formation of physical pores, eventually leading to bacterial cell death. AMPs are attractive antimicrobial agents since they have a distinct mechanism of kill compared to antibiotics. However, the relatively small differential electrostatic interaction between AMPs and bacterial membrane versus mammalian membrane, particularly in the presence of salt and serum in physiological environment, result in low selectivity and often high toxicity towards eukaryotic cells. An alternative approach is to exploit vital processes and "machineries" such as the electron transport chain (ETC), which are located at the bacterial cytoplasmic membrane and are therefore likely to be sensitive to selective targeted inhibition. The mammalian counterparts, on the other hand, have their ETCs hidden in the mitochondrial inner membrane inside the cells. Furthermore, our understanding of the intricate interactions between cationic agents and bacterial membrane remains incomplete, and the exploration of structural diversity in cationic molecules has been limited.
Therefore, there exists a need for new antibiotics for effective bacterial killing in complex environment.
Summary of Invention
The invention is described below with regard to the following numbered aspects and embodiments.
1. A compound according to formula la:
Figure imgf000004_0001
where:
X" is an anionic species selected from an organic acid in its carboxylate form, Br, |- or Of;
Y represents, OH, NH2, a zwitterionic species or a hydrazone group; each L independently represents:
Figure imgf000004_0002
Figure imgf000005_0001
where each wiggly line represents a point of attachment to the rest of the molecule, or a compound according to formula lb:
Figure imgf000005_0002
where each L is independently selected from the list provided above; and X' is as defined above, or a compound according to formula Ic:
Figure imgf000006_0001
where:
Ri 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 to which they are attached form a benzene ring; n represents 6 or 8;
X- is as defined above, or a compound according to formula Id:
Figure imgf000006_0002
where each L is independently selected from the list provided above; and
X- is as defined above, and solvates thereof of compounds of formula la-ld.
2. The compound according to Clause 1 , wherein the compound has formula Ic.
3. The compound according to Clause 2, wherein R1 is H.
4. The compound according to Clause 2 or Clause 3, wherein one of R2 and R3 is H, CH3,
Cl, or CF3 and the other is H. 5. The compound according to Clause 3, wherein one of R2 and R3 is H or CH3 and the other is H.
6. The compound according to any one of Clauses 2 to 5, wherein: n is 6; and/or
X- is selected from Br, I- or Cl_; and/or
Y is OH.
7. The compound according to any one of Clauses 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- is Ch.
8. The compound according to Clause 1, wherein the compound has formula la or formula lb.
9. The compound according to Clause 8, wherein each L represents:
Figure imgf000007_0001
, where the wiggly lines represent the points of attachment to the rest of the molecule. 10. The compound according to Clause 1, wherein the compound is a compound of
Figure imgf000008_0001
formula la, wherein X' is Cl' and each L is
11. The compound according to Clause 1 , wherein the compound is a compound of
Figure imgf000008_0002
formula lb wherein X' is Cl’ and each L is
12. A pharmaceutical composition comprising a compound according to any one of Clauses 1 to 11 and one or both of a pharmaceutically acceptable adjuvant and carrier.
13. Use of a compound according to any one of Clauses 1 to 11 or a pharmaceutical composition according to Clause 12 in medicine.
14. A compound according to any one of Clauses 1 to 11 or a pharmaceutical composition according to Clause 12 for use in the treatment of one or both of a microbial and a fungal infection.
15. Use of a compound according to any one of Clauses 1 to 11 or a pharmaceutical composition according to Clause 12 in the manufacture of a medicament to treat one or both of a microbial and a fungal infection.
16. A method of treating one or both of a microbial and a fungal infection comprising the step of administering a pharmaceutically effective amount of a compound according to any one of Clauses 1 to 11 or a pharmaceutical composition according to Clause 12 to a subject in need thereof.
17. An antimicrobial and/or antifungal detergent composition comprising: a compound as described in any one of Clauses 1 to 11; and a surfactant. 18. The antimicrobial and/or antifungal detergent composition according to Clause 17, wherein the composition is in the form of a solid or liquid soap.
19. The antimicrobial and/or antifungal detergent composition according to Clause 18, wherein the composition is in the form of a shampoo.
Drawings
Fig. 1 depicts structural representation of oligoimidazolium (OIM) analogues synthesized and biologically evaluated in a SAR study for (A) OIM1-6 series, (B) OIM1-8 series, (C) degradable OIM1 series, and (D) degradable OIM series.
Fig. 2 depicts synthesis of OIM1-6 derivatives. Reagents and conditions: (i) benzyl (3- bromopropyl)carbamate, NaH, tetrahydrofuran (THF), 0 °C to 50 °C, 18 hours, 71-76%; (ii) benzyl (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, 66-95%; (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 dimethylsulfoxide (DMSO), 80-120 °C, 48 hours to 1 week; (viii) 33 wt. % HBr in acetic acid, room temperature (rt), 18 hours; and (ix) Amberlyst AR26 OH resin, 10% aqueous (aq.) HCI solution 18-59% over three steps.
Fig. 3 depicts X-ray photoelectron spectroscopy (XPS) analysis of compound 1 , indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis.
Fig. 4 depicts XPS analysis of compound 2, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis.
Fig. 5 depicts XPS analysis of compound 5, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis.
Fig. 6 depicts XPS analysis of compound 13, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis (Binding energy at 64-65 indicates the presence of Bromine). Fig. 7 depicts XPS analysis of compound 14, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis (Binding energy at 64-65 indicates the presence of Bromine).
Fig. 8 depicts XPS analysis of compound 16, indicating insignificant presence of bromide ion after ion exchange chromatography and dialysis (Binding energy at 64-65 indicates the presence of Bromine).
Fig. 9 depicts calibration curves of (A) OIM1-6-CH (1), (B) OIM1-6-C2(CH3) (2) and (C) OIM1- 6-C4(CH3) (5) using area and height by liquid chromatography-mass spectrometry (LC-MS).
Fig. 10 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (1-3): (A) OIM1-6-CH (1); (B) OIM1-6-C2(CH3) (2); and (C) OIM1-6-C2(CI) (3) for 24 hours, 48 hours and 72 hours. Cell viability of human embryonic kidney (HEK) cells after incubation with (D) OIM1-6-CH (1), (E) OIM1-6-C2(CH3) (2), and (F) OIM1-6-C2(CI) (3) for 24 hours, 48 hours and 72 hours. Cell viability of liver hepatocellular carcinoma (HepG2) cells after incubation with (G) OIM1-6-CH (1), (H) OIM1-6-C2(CH3) (2), and (I) OIM1-6-C2(CI) (3) for 24 hours, 48 hours and 72 hours.
Fig. 11 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (4-6): (A) OIM1-6-C2(CF3) (4); (B) OIM1-6-C4(CH3) (5); and (C) OIM1-6-C4(CI) (6) for 24 hours, 48 hours and 72 hours. Cell viability of 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(CI) (6) for 24 hours, 48 hours and 72 hours. Cell viability of liver hepatocellular carcinoma (HepG2) cells after incubation with (G) OIM1-6-C2(CF3) (4), (H) OIM1-6-C4(CH3) (5), and (I) OIM1-6-C4(CI) (6) for 24 hours, 48 hours and 72 hours.
Fig. 12 depicts cell viability of 3T3 fibroblast cells determined after incubating with compounds (7-9) (A) OIM1-6-C4(F) (7), (B) OIM1-6-(BZ) (8), and (C) OIM1-6-C4(CF3) (9) for 24 hours, 48 hours and 72 hours. Cell viability of Human Embryonic Kidney (HEK) cells determined after incubating with (D) OIM1-6-C4(F) (7), (E) OIM1-6-(BZ) (8), and (F) OIM1-6-C4(CF3) (9) for24 hours, 48 hours and 72 hours. Cell viability of Liver Hepatocellular Carcinoma (HepG2) cells determined after incubating 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.
Fig. 13 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (10-12):
(A) OIM1-8-CH (10), (B) OIM1-8-C2(CH3) (11), and (C) OIM1-8-C4(CH3) (12) for 24 hours, 48 hours and 72 hours. Cell viability of 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 hours, 48 hours and 72 hours. Cell viability of liver hepatocellular carcinoma (HepG2) cells after incubation with (G) OIM1-8-CH (10), (H) OIM1-8-C2(CH3) (11), and (I) OIM1-8-C4(CH3) (12) for 24 hours, 48 hours and 72 hours.
Fig. 14 depicts cell viability of 3T3 fibroblast cells (A-B) determined after incubating with compounds (13-14): (A) OIM1-8-Bu-Acetal (13); and (B) OIM1-8-Bu-PzAc (14) for 24 hours, 48 hours and 72 hours. Cell viability of Human Embryonic Kidney (HEK) cells determined after incubating with (C) OIM1-8-Bu-Acetal (13), and (D) OIM1-8-Bu-PzAc (14) for 24 hours, 48 hours and 72 hours. Cell viability of Liver Hepatocellular Carcinoma (HepG2) cells determined after incubating with (E) OIM1-8-Bu-Acetal (13), and (F) OIM1-8-Bu-PzAc (14) for 24 hours, 48 hours and 72 hours.
Fig. 15 depicts cell viability of 3T3 fibroblast cells after incubation with compounds (15-16): (A) OIM1-12-6C-OH (15); and (B) OIM1-8-2D (16) for 24 hours, 48 hours and 72 hours. Cell viability of human embryonic kidney (HEK) cells after incubation with (C) OIM1-12-6C-OH (15), and (D) OIM1-8-2D (16) for 24 hours, 48 hours and 72 hours. Cell viability of liver hepatocellular carcinoma (HepG2) cells after incubation with (E) OIM1-12-6C-OH (15), and (F) OIM1-8-2D (16) for 24 hours, 48 hours and 72 hours.
Fig. 16 depicts cell viability of 3T3 fibroblast cells after incubation with known antibiotics: (A) gentamicin; (B) colistin; and (C) ciprofloxacin for 24 hours, 48 hours and 72 hours. Cell viability of human embryonic kidney (HEK) cells after incubation with (D) gentamicin, (E) colistin, and (F) ciprofloxacin for 24 hours, 48 hours and 72 hours. Cell viability of liver hepatocellular carcinoma (HepG2) cells after incubation with (G) gentamicin, (H) colistin, and (I) ciprofloxacin for 24 hours, 48 hours and 72 hours.
Fig. 17 depicts synthesis of OIM1-8 derivatives. Reagents and conditions: i) 1,4- dibromobutane (excess, 4 to 5 equiv) acetonitrile (MeCN), 80 °C, 18 hours, 55-68%; ii) 1d to 3e (1.0 equiv.) 10a to 12a (0.45 equiv) acetonitrile+ DMF (9:1); iii) 33 wt. % HBr in acetic acid, rt, 18 hours; and iv) Amberlyst AR26 OH resin, 10% aq. HCI solution 50-65 %.
Fig. 18 depicts synthesis of OIM1-8 degradable derivatives. Reagents and conditions: i) 1 ,4- dibromobutane (excess, 4 to 5 equiv) acetonitrile (MeCN), 80 °C, 18 hours, 65%; ii) 1g (1.0 equiv), 1d (2.5 equiv.) acetonitrile, 80 °C, 18 hours, 62%; iii) 13a (1.0 equiv.), Paraformaldehyde (0.5 equiv.) & concentrated H2SO4 (cat.), Toluene, 130°C, 1.5 h, 83%; iv) Chloroacetylchloride (2.1 equiv), K2CO3 (2.5 equiv.), Water + CHCI3 (1:1), O °C-rt 18 hours, 90%; v) 1d (1.0 equiv.) 13b (5.0 equiv), Acetonitrile, 80 °C, 3 hours, 50%; vi) 1d (1.0 equiv.) 14b (5.0 equiv), Acetonitrile, 80 °C, 3 hours, 68%; vii) for OIM1-8-Bu-acetal, 1 h (2.1 equiv ), 13c (1.0 equiv), acetonitrile + DMF (9:1); forOIM1-8-Bu-PzAc, 1 h (2.1 equiv.), 14c (1.0 equiv.), acetonitrile + DMSO (1 :1), overnight; and iv) Amberlyst AR26 OH resin, 10% aq. HCI solution 50-65 %.
Fig. 19 depicts (A and B) synthesis of degradable OIM series: OIM1-12-6C-OH (15). Reagents and conditions: Boc-anhydride (2.3 eq), Mg(CIO4)2 (0.1 eq), DCM, reflux, 48 hours, 64%; (ii) 15a (1 eq), Imidazole 1a (1.1 eq), NaH (3 eq), dry THF, 50 °C, 18 hours, 71%; (iii) Triphosgene (0.25 eq), Pyridine (1.5 eq), DCM, O °C-rt, 18 hours, 47%; (iv) 1d (1 eq), 15c (5 eq), MeCN, 85 °C, 3 hours, 36%; (v) 15c (2.5 eq), MeCN, 85 °C, 3 hours, 75%; (vi) 1d (2.5 eq), MeCN, 95 °C, 3 hours, 90%; (vii) 15c (5 eq), MeCN/DMF (2:1), 95 °C, 3 hours, 97%; (viii) 1d (5 eq), MeCN/DMF (2:1), 95 °C, 3 hours, 75-86%; and (ix) 15d (1 eq), 15h (2.01eq), Neat, 95 °C, 2 hours, MeCN & EA washings, TFA/DCM (50%) for 12 hours, Amberlyst A-26 OH form 10% HCI, EA washing, SephadexG-25 silica purification, 10%.
Fig. 20 depicts synthesis of degradable OIM series: OIM1-8-2D (16). Reagents and conditions: (i) Chloroacetylchloride (2.1 equiv ), K2CO3 (2.5 equiv.), water + CHCI3 (1 :1), 0 °C - rt 18 hours, 65% (16a); (ii) 1d (1.0 equiv.), 16a (5.0 equiv.), acetonitrile, 80 °C, 3 hours, 50%; (iii) 1e (2.1 equiv.), 16b (1.0 equiv.), acetonitrile + DMF + MeOH (8:1 :1), 95 °C overnight; (iv) 33 wt. % HBr in acetic acid, rt, 2 hours; (v) Amberlyst AR26 OH resin, 10% aq. HCI solution, 55% over three steps.
Fig. 21 depicts killing of S. aureus LAC over time in MHB in the presence of (A) OIM1-6-CH, (B) OIM1-6-C2(CH3), (C) OIM1-6-C4(CH3) tested from 1-to-8x MIC. Killing of P. aeruginosa PAO1 overtime in MHB with (D) OIM1-6-CH, (E) OIM1-6-C2(CH3), (F) OIM1-6-C4(CH3) tested from1-to-4x MIC.
Fig. 22 depicts (A) the chemical structures of the simulated cationic oligomers. (B) The geometry optimized structures of the simulated cationic oligomers. (C) Calculated Hirshfeld charges on the imidazolium rings of OIMs.
Fig. 23 depicts (A) membrane depolarization study of MRSA LAC treated with OIM1-6 compounds, with gramicidin as antibiotic control for 1 hour using DISC3(5) assay. (B) Membrane permeabilization study of LAC treated with OIM1-6 compounds, with nisin as positive control for 1 hour using propidium iodide (PI) assay. Fig. 24 depicts (A) OIMs with free C2-hydrogens are carbon acids which deprotonate in neutral water to form N-heterocyclic carbenes (NHCs) (//) that are uncharged and hydrophobic. (B) Novel mechanism of translocation: (i) OIM carbon acid converts to an amphiphilic copolymer (made of hydrophilic cation and hydrophobic NHC repeats) to efficiently translocate the plasma membrane into bacterial cytosol at lower polymer threshold concentration, as compared with (ii) a classical cationic polymer that forms physical pores. The OIMs bind with their intracellular target (DNA as previously studied) in bacterial cytosol resulting in bacteria death, as opposed to classical cationic polymer which is ineffective in killing the bacteria.
Fig. 25 depicts NHC formation studies with 0IM1-6-CH (1). (A) Detection of NHC by the reaction of partial carbene (//) monomers with D2O or AuCI(SMe2). (B) Hydrogen-deuterium exchange in 1H nuclear magnetic resonance (NMR) of OIM1-6-CH (1) in aqueous solution as a function of time at pH 6.63, pH 6.81 , pH 7.16 and pH 8.21 in D2O. (C) Schematic of interaction of phosphatidylcholines/phosphatidylglycerol (PC/PG) liposome containing NHC probes (carbazole dye and or AuCI(SMe2)) with OIM-1-6-CH (1). (D) Fluorescence of carbazole embedded in liposome bilayer with 0IM1-6-CH (1) or stable carbene (IPr) in solution. (E-H) Proof of NHC formation by OIM (1) using AuCI(SMe2) embedded in liposome bilayer and LC-MS/MS: (E) Total ion chromatogram (TIC) of Au(lipids)-OIM1-6-CH (1) obtained by LC-ESI-MS (+). (F) Extracted ion chromatogram (EIC) of (i) OIM1-6-CH (m/z 199.15), and (ii) Au-OIM1-6-CH containing one Au atom (m/z 330.52) (both at pH 7.4). (G) Assigned chemical structures of EIC of (I) m/z = 199.15 to be OIM1-6-CH (1) to be 4 cations and 2 NHCs, (ii) m/z = 350.52 to be 1Au-OIM1-6-CH with 1Au-containing cation, 2 imidazolium cations (without Au), and 3 NHCs. (H) Mass spectrum (MS) of (i) m/z = 199.15 (retention time 3.55 min) with MS gap = 0.25, indicating z = 4, and total ion mass = 796.60, and (ii) m/z 330.52 (retention time 4.04 min) with MS gap = 0.33, indicating z = 3, and total ion mass = 991 .56. (I) Uptake of OIM derivatives into liposome at pH 7.4 and pH 6.8. (J) (I) Carbazole is a NHC dye and becomes non-fluorescent with NHC present because of hydrogen bonding; (ii) Reaction of NHC with chloro(dimethyl sulfide)gold(l) .
Fig. 26 depicts confirmation of the peak m/z 330.52 to be Au-OIM-1-6-CH: (A-D) Hydrogen deuterium exchange mass spectrometry experiment. (A) Structure assignment 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 nondeuterated Au-OIMI-6-CH (m/z 330.52). (D) MS of deuterated Au-OIM1-6-CH (m/z 331.86). MS/MS analysis for selected ion of m/z 330.52 (E) before fragmentation and (F) after fragmentation. (G) Structure assignment of fragments. Fig. 27 depicts confirmation of the peak m/z 330.52 to be Au-OIM-1-6-CH: (A-E) Hydrogen deuterium exchange mass spectrometry experiment. (A) Total ion chromatograph (TIC) of Au- OIM1-6-CH in d-methanol. (B) Extracted ion chromatograph (EIC) of deuterated Au-OIM1-6- CH (m/z 331.86). (C) MS of deuterated Au-OIM1-6-CH (m/z 331.86) and (D) MS of nondeuterated Au-OIM1-6-CH (m/z 330.52). (E) Structure assignment of m/z 330.52 and m/z 331.86. (F) MS/MS analysis for selected ion of m/z 330.52. (G) Structure assignment of fragments.
Fig. 28 depicts NHC formation studies of OIM1-6-C2(CH3) (2) and OIM1-6-C4(CH3) (5). Hydrogen-deuterium exchange of (A) OIM1-6-C2(CH3) (2) and (B) OIM1-6-C4(CH3) (5) as a function of time at pH 6.63, pH 6.81 , pH 7.16 and pH 8.21 in aqueous solution. Structure assignments of (C) Au-OIM1-6-C2 (m/z 537.32) and 2Au-OIM1-6-C2 (m/z 423.87); and (D) Au-OIM1-6-C4 (m/z 537.32) and 2AU-OIM1-6-C4 (m/z 423.87). Extracted ion chromatogram (EIC) of Au-(2) and 2Au-(2) at (E) pH 7.4 and (G) pH 8.2; Au-(5) and 2Au-(5) at (F) pH 7.4 and (H) pH 8.2.
Fig. 29 depicts computer simulation of interaction of 2 forms (cationic versus NHC forms) of OIM1-6-CH (1) with S. aureus membrane-mimic. (A-B) Number of contacts between OIM1-6- CH (1) and S. aureus membrane as a function of simulation time: (A) cationic OIM-membrane system; and (B) OIM-NHC-membrane system. A contact was defined if the minimum distance between the polymer atom and membrane atom was less than 0.4 nm. (C-D) Binding poses of (1) to S. aureus membrane-mimic: (C) cationic OIM-membrane system; and (D) OIM-NHC- membrane system. These poses were taken from the final simulation frame of each simulation repeat.
Fig. 30 depicts (A) method of determination of total and cytosolic uptakes of compounds by bacteria, (i) Illustration of florescence from OIM-fluorescein isothiocyanate (FITC)ZSYTO Sustained MRSA LAC upon Trypan Blue (TB) and Triton X-100 (TX) treatment. TB dye is impermeable to intact plasma membrane; hence it will quench the surface-bound fluorophore trapped in the cell wall, and to some extend quench the fluorophore trapped on the surface of the bacterial membrane. TX is added at 0.04% to permeabilize the bacteria membrane, allowing the entry of TB dye to the cytosolic. TB dye could now quench the fluorophore in the cytosolic, however not the fluorophores that were partially/fully inserted into the membrane bilayer. For all flow cytometry experiments, the fluorescence signals of 50,000 bacteria were determined. (B) Flow cytometry histograms of untreated LAC before TB treatment (unquench) and after TB treatment (TB quench). Dotted line indicates the gating setting: bacteria with fluorescence intensity to the right of the gating is considered to have dye uptake, bacteria with fluorescence intensity to the left of the gating is considered to have no dye uptake. (C) Flow cytometry histograms of untreated LAC stained with STYO 9 DNA dye. The fluorescence intensity of unquench and TB quench bacteria population had no significant difference, however the intensity dropped significantly (to the left of the gating) after TX treatment + TB quenching (TX + TB quench), indicating that the TB dye has entered the cytosolic and quenched the DNA-bound STYO 9 dye. (D-F) Flow cytometry histograms of LAC treated with 64 pg/mL of (iv) 0IM1-6-CH (1), (E) OIM1-6-C2(CH3) (2) and (F) OIM1-6-C4(CH3) (5) for 1 hour. The percentage of bacteria population with membrane+cytosolic OIM uptake is obtained from the TB quench histogram (right side of the gating) and illustrated as bar chart in Fig. 31 . Upon TX + TB quench, the fluorescence intensity dropped significantly (to the left of the gating) for (1) and (5) but not (2), indicating that (1) and (5) enter the cytosolic while the (2) is partially/fully inserted in the membrane bilayer. The bacteria population that has cytosolic uptake of OIM is determined via histogram subtraction of TX + TB quench from TB quench. (G) Histogram subtraction of TX + TB quench from TB quench generated by Flowjo software using compound (1) (Fig. 30D) as example. The percentage of bacteria population with cytosolic uptake is illustrated as bar chart in Fig. 31.
Fig. 31 depicts effect of NHC formation on OIM’s potency and uptake into bacteria. (A) MIC* of OIMs and gentamicin against S. aureus parental strain. MIC* is tested up to 4,096 pg/mL, MIC* > 4,096 pg/mL indicated as 8,192 pg/mL. (B) The percentage of LAC cell count with OIM uptake treated in TSB for 1 hour. (C) The mean fluorescence intensity (MFI) of cells with OIM uptake treated in TSB for 1 hour. (D) MIC* of OIMs and gentamicin against wildtype LAC and respiration-deficient mutants at different pH. MIC* is tested up to 4096 pg/mL, MIC* > 4096 pg/mL indicated as 8192 pg/mL. (E) The cell count percentage of wildtype LAC and respiration-deficient mutants with OIM1-6-CH (1) uptake tested at pH 7.2 and pH 6.8 in TSB for 1 hour, at their respective 1x MIC* at pH 7.2. (wildtype LAC at 16 pg/mL, LAC menD at 128 pg/mL and LAC hemB at 256 pg/mL). (F) Killing kinetics of LAC treated with OIM1-6-CH (1) and OIM1-6-C4(CH3) (5) in aerobic, anaerobic, and fermentative growth conditions at their respective 4-fold MIC values (/.e., 16 pg/ml and 32 pg/ml, respectively).
Fig. 32 depicts LAC treated with FITC conjugated OIM in (A) MHB and (B) TSB for 1 hour. Total OIM includes the surface bound and internalized fraction of OIM with no Trypan Blue quenching, while the internalized fraction is established with Trypan Blue quenching. (C) LAC menD and (D) LAC hemB treated with FITC conjugated OIMs for 1 hour with and without Trypan Blue quenching. (E) Uptake of FITC conjugated OIM1-6-CH and OIM1-6-C4(CH3) by LAC at pH 7 and pH 6.8 in TSB at their respective 1x MIC* (pH 7). (F) Uptake of FITC conjugated 0IM1-6-CH by LAC menD and LAC hemB at pH 7 and pH 6.8 in TSB at their respective 1x MIC* (pH 7).
Fig. 33 depicts resistance evolution of (A) LAC and (B) PAO1 against 0IM1-6-CH and OIM1- 6-C4(CH3), respectively. (C) OCR of LAC treated with OIM1-6 CH, OIM1-6 C2(CH3), OIM1-6- C4(CH3) and HQNO, as well as respiratory mutants of LAC AhemB and LAC AmenD. Killing kinetics of S. aureus LAC treated with (D) 0IM1-6-CH or (E) OIM1-6-C4(CH3) in aerobic, anaerobic, and fermentative growth condition at their respective 4-fold MIC value (i.e. 16 pg/ml and 32 pg/ml respectively).
Fig. 34 depicts Deuterium exchange results in the disappearance of the peak at 8.8-8.9 ppm due to the C2-proton at (A) pH 7.16 and (B) pH 6.8. The extent of remained proton at C2 position is indicated above each spectrum. It is observed that deuterium exchange is faster at pH 7.16 than pH 6.8.
Fig. 35 depicts antibacterial test of OIMs against E. coli 8739 and S. aureus 6538 for the application of laundry use in 4 kinds of detergents (100 ppm of sodium dodecyl benzene sulfonate (SDBS), 100 ppm of sodium dodecyl sulfate (SDS), 50 ppm of SDS plus 50 ppm of SDBS, and 389 ppm of Japan pouch detergent). (A) Depiction of OIMs antibacterial application in laundry use. The tested compounds are (B) Polydiallyldimethylammonium 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. Antibacterial test was performed according to standard test method ASTM-E2274. It requires at least 2 log reduction to pass the test.
Fig. 36 depicts in vivo efficacy of degradable OIMs. (A) Murine systemic infection model, (i) Protocol: mice (n = 6 per group) were infected with A. baumannii ATCC BAA 2803 in 300 pL of 5% mucin saline via intraperitoneal (IP) injection. Mice were treated with single dose of PBS (untreated), 15 mg/kg imipenem or 15 mg/kg OIM1-8-2D (16) at 2 hours post-infection. Mice were sacrificed at 26 hours post-infection and bacteria numbers were determined, (ii) Survival rate and (iii) bacteria log reduction in liver in murine systemic infection model. LOD: limit of detection. ****p < 0.0001 and ns (not significant) p > 0.05 (one-way ANOVA). (B) Toxicity testing with repetitive dosing of (16) (15 mg/kg x 7 daily doses). Weight of mice treated with seven daily doses of 15mg/kg OIM1-8-2D (16) given via IP injections. (C-E) Diary mastitis test. (C) Timeline and experimental setup of the farm trial. (D) Bacteria count in milk over time upon bacteria challenge. Each symbol represents one milk sample from one teat (also known as one quarter). (E) Somatic cell count (SCC) in milk samples upon bacteria challenge. Each symbol represents the sample from one cow. Fig. 37 depicts detailed results of in vivo murine testing. Bacteria log reduction in (A) kidney, (B) spleen and (C) IP fluid in murine systemic infection model. LOD: limit of detection. ****p < 0.0001 and ns (not significant) p > 0.05. (one-way ANOVA).
Fig. 38 depicts teat images after 5-day continuous application of teat dip in safety trial. No irritation responses were detected on PIM1 D-treated teats.
Fig. 39 depicts Delvo Test results of (A) teat surface and (B) milk samples in a safety trial.
Fig. 40 depicts milk composition changes (protein, fat and solids-not-fat (SNF)) as well as somatic cell count (SCC) in PIM1 D-treated teats during the 5-day safety trial.
Fig. 41 depicts dairy mastitis testing results. (A) to (C): in vitro mastitis testing: treatment by (A) chlorhexidine; (B) OIM1-8-2D (16); and (C) PIM1 D in the presence of 18% milk at 0.5% concentration against E. coli, S. aureus and S. uberis for 30 minutes. The dotted line indicates the requirement of at least five-logio reduction. (D) In vivo mastitis testing: milk composition changes of PIM1 D treated teats upon bacteria challenge. SNF stands for solids-not-fat.
Description
It has been surprisingly found that three series of oligoimidazolium salts may be particularly good antimicrobial and/or anti-fungal agents. Thus, in a first aspect of the invention, there is provided a compound according to formula la:
Figure imgf000017_0001
where:
X' is an anionic species selected from an organic acid in its carboxylate form, Br, k or Cl-;
Y represents, OH, NH2, a zwitterionic species or a hydrazone group; each L independently represents:
Figure imgf000018_0001
where each wiggly line represents a point of attachment to the rest of the molecule, or a compound according to formula lb:
Figure imgf000018_0002
where each L is independently selected from the list provided above; and X' is as defined above, or a compound according to formula Ic:
Figure imgf000019_0001
where:
Ri 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 to which they are attached form a benzene ring; n represents 6 or 8;
X- is as defined above, or a compound according to formula Id:
Figure imgf000019_0002
where each L is independently selected from the list provided above; and
X" is as defined above, and solvates thereof of compounds of formula la-ld.
In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.
References herein (in any aspect or embodiment of the invention) to oligomers of the invention include references to such compounds perse, to tautomers of such compounds, as well as to pharmaceutically acceptable solvates of such compounds.
As mentioned above, also encompassed by oligomers of the invention are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray crystallography.
The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.
For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al. , Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.
The oligomers of the invention may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. For example, in a compound of formula Ic:
Figure imgf000021_0001
where Ri is H, R2 is methyl and R3 is H, it may be represented as:
Figure imgf000021_0003
as such, in embodiments where R2 and R3 do not together with the carbon atoms to which they are attached form a benzene ring, the compound of formula Ic may be drawn as a compound of formula lc’:
Figure imgf000021_0002
Rn is H, CH3, Cl, or CF3. In this arrangement, the charge is distributed between the two nitrogen atoms and the carbon atom therebetween.
The oligomers of the invention in the above-mentioned aspect of the invention may be utilised in a method of medical treatment. Thus, according to further aspects of the invention, there is provided:
(a) an oligomer of the invention for use in medicine;
(b) an oligomer of the invention for use in the treatment of a microbial and/or fungal infection;
(c) use of an oligomer of the invention for the preparation of a medicament for the treatment of a microbial and/or fungal infection; and
(d) a method of treatment of a microbial and/or fungal infection, which method comprises the administration of an effective amount of an oligomer of the invention.
In embodiments that may be mentioned herein, the oligomer of the invention may be particularly useful with regard to microbial infections.
The term “microbial infection” covers any disease or condition caused by a microbial organism in or on a subject. Examples of microbial infections include, but are not limited to, tuberculosis caused by mycobacteria, burn wound infections caused by pseudomonas etc., skin infections caused by S. aureus, wound infections caused by pseudomonas and A. baumannii, mastitis, and Sepsis. The term “fungal infection” covers any disease or condition caused by a fungal organism in or on a subject. Examples of fungal infections include, but are not limited to, athlete’s foot, ringworm, yeast infections, and jock itch.
A non-limiting list of bacteria that may be susceptible to the oligomers of the invention include: Acidothermus cellulyticus, Acinetobacter baumannii, Actinomyces odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bifidobacterium adolescentis, Bifidiobacterium longum, Burkholderia thailandensis, Caldicellulosiruptor saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium cellulolyticum, Clostridium difficile, Clostridium kluyveri, Clostridium leptum, Clostridium novyi, Clostridium perfringens, Clostridium tetani, Clostridium thermocellum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium jeikeium, Corynebacterium urealyticum, Desulfitobacterium hafniense, Desulfotomaculum reducens, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coll, Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna, Geobacillus kaustophilus, Geobacillus the rmodenitrificans, Janibacter sp., Kineococcus radiotolerans, Klebsiella pneumoniae, Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis, Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium paratuberculosis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii, Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis, Pelotomaculum thermopropionicum, Pseudomonas aeruginosa, Rhodococcus sp., Saccharopolyspora erythraea, Serratia marcescens, coagulase-negative Staphylococcus species, Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, methicillin resistant Staphylococcus epidermidis (MRSE), Streptococcus agalactiae, Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus suis, Streptococcus uberis, Streptomyces avermitilis, Streptomyces coelicolor, Thermoanaerobacter ethanolicus, Thermoanaerobactertengcongensis, and combinations thereof. Specific bacteria that may be mentioned herein are discussed in the examples below.
For the avoidance of doubt, in the context of the present invention, the term “treatment” includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.
The terms “patient” and “patients” include references to mammalian (e.g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect). Unless otherwise stated, the term “alkyl” refers to an unbranched or branched saturated hydrocarbyl radical, which may be substituted or unsubstituted. Where the term “alkyl” refers to a C1-6 alkyl, the alkyl group may be ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl, hexyl or, more preferably, methyl.
For the avoidance of doubt, in cases in which the identity of two or more substituents in a oligomer of the invention may be the same, the actual identities of the respective substituents are not in any way interdependent.
In embodiments of the invention that may be mentioned herein n may be 6.
In embodiments of the invention that may be mentioned herein, X' may be selected from Br, |- or Cl'.
In embodiments of the invention that may be mentioned herein, Y may be OH.
Embodiments of the invention that may be mentioned include those that relate to oligomers of the invention in which the compound may have formula Ic. In such embodiments, one or more of the following may apply:
(i) R1 may be H;
(ii) one of R2 and R3 may be H, CH3, Cl, or CF3 and the other may be H (e.g. one of R2 and R3 may be H or CH3 and the other may be H);
(iii) n may be 6.
In particular 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 may be 6; and
X' may be Cl'.
In more particular embodiments of the invention that may be mentioned herein, the compound may have formula la or formula lb. In such embodiments, each L may represent:
Figure imgf000025_0001
, where the wiggly lines represent the points of attachment to the rest of the molecule.
In particular embodiments of the invention that may be mentioned herein, the compound may be a compound of formula la, wherein X' is Cl' and each L is
Figure imgf000025_0002
In particular embodiments of the invention that may be mentioned herein, the compound may be a compound of formula lb wherein X' is Cl' and each L is
Figure imgf000025_0003
As will be appreciated, the “L” groups in the compounds of formula la, lb and Id may provide said compounds with biodegradeability. That is, these groups may be susceptible to the action of an enzyme or other biological mechanism or organism so as to split the compounds of formula la, lb and Id into two or more compounds (e g. 2, 3, 4, 5, 6, etc.), which is only limited by the total number of “L” groups present in said compounds. This degradation may occur in vivo, thereby allowing for the metabolism and excretion of said compounds or in the environment (e.g. through the action of microorganisms in the environment, or simple chemical degradation over time). As noted, each of the “L” groups may, in some cases, be selected from one or more of the possibilities listed herein. Further embodiments of the invention that may be mentioned include those in which the oligomer of the invention is isotopically labelled. However, particular embodiments of the invention that may be mentioned include those in which the oligomer of the invention is not isotopically labelled.
The term "isotopically labelled", when used herein includes references to oligomers of the invention in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the oligomer of the invention. Thus, the term "isotopically labelled" includes references to compounds of the invention that are isotopically enriched at one or more positions in the oligomer.
The isotopic labelling or enrichment of the oligomer of the invention may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine. Particular isotopes that may be mentioned in this respect include 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 35S, 18F, 37CI, 77Br, 82Br and 125l).
When the oligomer of the invention is labelled or enriched with a radioactive or nonradioactive isotope, oligomer of the invention that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or nonradioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.
As noted above, the oligomers of the invention may be used in the treatment of microbial and fungal infections. Thus, there is also provided a pharmaceutical composition comprising the oligomer of the invention and one or both of a pharmaceutically acceptable adjuvant and carrier.
Oligomers of the invention may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.
Oligomers of the invention will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.
Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.
The amount of the oligomer of the invention in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of oligomer of the invention in the formulation may be determined routinely by the skilled person.
For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99% (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90% (w/w) of a release-controlling polymer.
A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50% (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
Depending on the disorder, and the patient, to be treated, as well as the route of administration, oligomers of the invention may be administered at varying therapeutically effective doses to a patient in need thereof.
However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of an oligomer of the invention.
In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
The aspects of the invention described herein (e.g. the above-mentioned oligomers, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.
Other oligomers of the invention may be prepared in accordance with techniques that are well known to those skilled in the art, for example as described hereinafter in the examples section. Compounds of the invention may be isolated from their reaction mixtures using conventional techniques (e g. recrystallisation, column chromatography, preparative HPLC, etc.).
The oligomers of the invention exhibit a pronounced antimicrobial action, especially against pathogenic gram-positive and gram-negative bacteria and so may also act against bacteria of skin flora, e.g. Corynebacterium xerosis (bacteria that cause body odour), and also against yeasts and moulds. They are therefore also suitable in the disinfection of the skin and mucosa and also of integumentary appendages (hair), and so may also be suitable in the disinfection of the hands and of wounds.
Given the above, the oligomers of the invention may be used as antimicrobial active ingredients in personal care preparations, for example 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, e.g. cleansing solutions for the skin.
Thus, there is also provided an antimicrobial and/or antifungal detergent composition comprising an oligomer of the invention and a surfactant. It will be appreciated that the composition may also contain additional cosmetically tolerable carriers and/or adjuvants. Said composition may in particular be in the form of a shampoo or in the form of a solid or liquid soap, though other compositions as described hereinabove are also contemplated (e.g. other hair-care products, lotions and creams etc.).
The detergent composition may comprise from 0.01 to 15% by weight, such as from 0.5 to 10% by weight of an oligomer of the invention. It will be appreciated that more than one oligomer of the invention may form part of the detergent composition.
Depending upon the form of the detergent composition, it will comprise, in addition to the oligomer of the invention, further constituents, for example sequestering agents, colourings, perfume oils, thickening or solidifying (consistency regulator) agents, emollients, UV absorbers, skin-protective agents, antioxidants, additives that improve mechanical properties, such as dicarboxylic acids and/or Al, Zn, Ca and Mg salts of C14-C22 fatty acids, and optionally preservatives. The detergent composition may be formulated as a water-in-oil or oil-in-water emulsion, as an alcoholic or alcohol-containing formulation, as a vesicular dispersion of an ionic or non-ionic amphiphilic lipid, as a gel, a solid stick or as an aerosol formulation.
As a water-in-oil or oil-in-water emulsion, the detergent composition may comprise from 5 to 50 wt% of an oily phase, from 5 to 20 wt% of an emulsifier and from 30 to 90 wt% water. The oily phase may contain any oil suitable for cosmetic formulations, e.g. one or more hydrocarbon oils, a wax, a natural oil, a silicone oil, a fatty acid ester or a fatty alcohol. Preferred mono- or poly-ols are ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and sorbitol.
Detergent compositions may be provided in a wide variety of preparations. Examples of suitable compositions include, but are not limited to skin-care preparations (e.g. skin-washing and cleansing preparations in the form of tablet-form or liquid soaps, soapless detergents or washing pastes), bath preparations, (e.g. liquid compositions such as foam baths, milks, shower preparations or solid bath preparations), shaving preparations (e.g. shaving soap, foaming shaving creams, non-foaming shaving creams, foams and gels, preshave preparations for dry shaving, aftershaves or after-shave lotions), cosmetic hair-treatment preparations (e.g. hair-washing preparations in the form of shampoos and conditioners, haircare preparations, e.g. pretreatment preparations, hair tonics, styling creams, styling gels, pomades, hair rinses, treatment packs, intensive hair treatments, hair-structuring preparations, e.g. hair-waving preparations for permanent waves (hot wave, mild wave, cold wave), hairstraightening preparations, liquid hair-setting preparations, foams, hairsprays, bleaching preparations; e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams, bleaching powders, bleaching pastes or oils, temporary, semi-permanent or permanent hair colourants, preparations containing self-oxidising dyes, or natural hair colourants, such as henna or camomile).
An antimicrobial soap may have, for example, the following composition:
0.01 to 5% by weight of an oligomer of the invention;
0.3 to 1 % by weight titanium dioxide;
1 to 10% by weight stearic acid; and the remainder being a soap base, e.g. the sodium salts of tallow fatty acid and coconut fatty acid or glycerol.
A shampoo may have, for example, the following composition: 0.01 to 5% by weight of an oligomer of the invention; 12.0% by weight sodium laureth-2-sulfate;
4.0% by weight cocamidopropyl betaine;
3.0% by weight NaCI; and water to 100 wt%.
Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.
Examples
Materials
All chemicals and solvents were obtained from Fischer Scientific UK, Sigma-Aldrich, Merck Millipore, TCI Chemicals, and BLDpharm, and were used without further purification.
Analytical techniques
Thin layer chromatography (TLC)
TLC was performed using Merck TLC Silica gel 60 A F254 plates. TLC plate visualizations were conducted under UV light (256 & 366 nm).
Column chromatography
Column chromatography was carried out using Davisil® LC60A 40 - 63 micron chromatographic silica (pore size 60 A, 0.040-0.063 mm).
Nuclear magnetic resonance (NMR) spectroscopy
NMR spectra were recorded on either a Bruker Avance DPX 300 (1H and 13C NMR at 300 MHz and 75.47 MHz respectively) or a Bruker Avance III 400 (1H and 13C NMR at 400.13 MHz and 101.62 MHz respectively). The data was processed using TopSpin (version 4.1.3), which referenced the spectra to those of the residual solvents. Chemical shifts (6) were quoted in parts per million (ppm) and coupling constants (J) were reported to the nearest 0.01 Hz for 1H NMR and 0.1 Hz for 13C NMR along with peak multiplicities using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; qu, quintet; sext, sextet; m, multiplet, and br, broad.
Mass spectroscopy (MS)
Mass spectra were recorded on a ABI 4800 Proteomics Analyzer MALDI TOF/TOF mass spectrometer (Applied Biosystems).
Gel Permeation Chromatography (GPC) Oligomeric products were characterized by Waters GPC using a water phase ultra hydrogel column as the stationary phase and sodium acetate/acetic acid buffer (pH = 4.5) as the mobile phase. All samples were dissolved in the buffer solution with approx. 1 mg/mL final concentration and filtered through a 0.22 pm microfilter before sample analysis.
X-ray photoelectron spectroscopy (XPS)
Elemental analysis for anion exchange was achieved by performing X-ray photoelectron spectroscopy (XPS) using an AXIS Supra spectrometer (Kratos Analytical, UK) equipped with a hemispherical analyzer and a monochromatic Al K-alpha source (1487 eV) operated at 15 mA and 15 kV. The XPS spectra were acquired from an area of 700 x 300 pm2 with a take-off angle of 90°. Pass energy of 160 eV and 20 eV were used for the survey and high-resolution scans, respectively. A 3.1-volt bias was applied to the sample to neutralize charge build-up on the sample surface.
Example 1. Synthesis of OIM1-6 derivatives
We synthesized a series of OIMs with precisely controlled molecular weights amenable to rigorous characterizations required for mechanistic studies. A total of nine OIM1-6 derivatives each with six imidazolium rings (1-9) (Fig. 1A), including the parent OIM1-6-CH, were synthesized to study the effects of various substitutions at either the C2- or C4- position of the imidazolium moiety on antimicrobial potency and cytotoxicity. The synthesis of these compounds, including the parent OIM1-6-CH (1), were achieved via a step-by-step synthetic strategy. The methods and chemical characterization of the OIMs are described in Fig. 2 and below. The parent OIM1-6-CH (1) has a hydrogen (H) at the C2-carbon (Fig. 1A).
General Procedure 1: Alkylation of imidazole derivatives (1b-7b and 9b)
To a solution of the required imidazole derivative (1a-9a) (1.0 equiv.) dissolved in tetrahydrofuran (THF) under ice bath was added NaH (3.0 equiv) portion-wise. The reaction mixture was removed from the ice bath and treated with benzyl (3-bromopropyl)carbamate (1.0 equiv.). The reaction mixture was then heated to 50 °C and stirred overnight. After completion of the reaction shown by TLC, the reaction mixture was filtered through a celite pad, and the filtrate was concentrated under reduced pressure onto silica and purified by silica gel column chromatography eluting with 50-80% ethyl acetate (EtOAc) in hexane to afford the desired product. The detailed synthesis of 8b is described below.
The detailed synthesis steps of each compound (1b to 9b) are as follows: Benzyl (3-(1/7-imidazol-1-yl)propyl)carbamate (1b)
The title compound was synthesized following the method described in General Procedure 1 using imidazole (1a) (3.00 g, 44.1 mmol) to afford a pale-yellow oil (8.34 g, 32.2 mmol, 73%). 1H NMR (300 MHz, DMSO-de) 6 7.63 (s, 1 H), 7.50 - 7.24 (m, 6H), 7.17 (s, 1 H), 6.90 (s, 1 H), 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). 13C NMR (75 MHz, DMSO-de) 6 156.1 , 137.2, 137.1 , 128.3, 127.7, 119.3, 65.3, 43.4, 37.4, 31.0. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for C14H17N3O2 = 260.1321 ; measured = 260.0104.
Benzyl (3-(2-methyl-1/7-imidazol-1-yl)propyl)carbamate (2b)
The title compound was synthesized following the method described in General Procedure 1 using 2-methyl-1 /-/-imidazole (2a) (7.45 g, 90.8 mmol) to afford a pale-yellow oil (14.8 g, 54.1 mmol, 60%). 1H NMR (300 MHz, DMSO-de) 0 7.54 - 7.22 (m, 6H), 7.03 (s, 1 H), 6.70 (s, 1 H), 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). 13C NMR (75 MHz, DMSO-d8) 6 156.0, 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): m/z calculated (M+H)+ for C15H19N3O2 = 274.1477; measured = 274.0078.
Benzyl (3-(2-chloro-1/7-imidazol-1-yl)propyl)carbamate (3b)
The title compound was synthesized following the method described in General Procedure 1 using 2-chloro-1 /-/-imidazole (3a) (1.00 g, 9.75 mmol) to afford a pale-yellow oil (1.97 g as a mixture of C4-Me & C5-Me regioisomers, 6.71 mmol, 76%). 1H NMR (400 MHz, DMSO-de) 6 7.43 - 7.26 (m, 7H), 6.88 (s, 1 H), 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). 13C NMR (101 MHz, DMSO-de) 6 156.2, 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): m/z calculated (M+H)+ for C14H16CIN3O2 = 294.1004; measured = 293.9763.
Benzyl (3-(2-(trifluoromethyl)-1/-/-imidazol-1-yl)propyl)carbamate (4b)
The title compound was synthesized following the method described in General Procedure 1 using 2-(trifluoromethyl)-1 /-/-imidazole (4a) (5.00 g, 36.7 mmol) to afford a pale-yellow oil (8.53 g, 26.1 mmol, 71%). 1H NMR (400 MHz, DMSO-d8) 6 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). 13C NMR (101 MHz, DMSO-de) 6 156.2, 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. ‘Splitting by NMR active 19F (CF3). MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for C15H16F3N3O2 = 328.1195; measured = 328.0313. Benzyl (3-(4-methyl-1/7-imidazol-1-yl)propyl)carbamate and benzyl (3-(5-methyl-1/7-imidazol- 1-yl)propyl)carbamate as a single mixture (5b)
The title compound was synthesized following the method described in General Procedure 1 using 4-methyl-1H-imidazole (5a) (5.00 g, 60.9 mmol) to afford a pale-yellow oil (11.0 g as a mixture of C4-Me & C5-Me regio isomers, 40.2 mmol, 66%). 1H NMR (300 MHz, DMSO-de) 6 7.51 - 7.45 (m, 1 H), 7.42 - 7.25 (m, 6H), 6.84 & 6.60 (s, 1 H), 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). 13C NMR (75 MHz, DMSO-de) 6 156.1 , 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): m/z calculated (M+H)+ for C15H19N3O2 = 274.1477; measured = 274.0112.
Benzyl (3-(4-chloro-1/-/-imidazol-1-yl)propyl)carbamate and benzyl (3-(5-chloro-1H-imidazol- 1-yl)propyl)carbamate as a single mixture (6b)
The title compound was synthesized following the method described in General Procedure 1 using 4-chloro-1/-/-imidazole (6a) (1.00 g, 9.75 mmol) to afford an orange gum (1.57 g as a mixture of C4-Me & C5-Me regioisomers, 5.36 mmol, 66%). 1H NMR (400 MHz, DMSO-d6) 5 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). 13C NMR (101 MHz, DMSO-d6) 6 156.2, 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): m/z calculated (M+H)+ for Ci4Hi6CIN3O2 = 294.1004; measured = 293.9888.
Benzyl (3-(4-fluoro-1/-/-imidazol-1-yl)propyl)carbamate and benzyl (3-(5-fluoro-1/7-imidazol-1- yl)propyl)carbamate as a single mixture (7b)
The title compound was synthesized following the method described in General Procedure 1 using 4-fluoro-1H-imidazole (7a) (680 mg, 7.90 mmol) to afford a pale-yellow gum (1.62 g as a mixture of C4-Me & C5-Me regioisomers, 5.85 mmol, 56%). 1H NMR (400 MHz, DMSO-de) 6 7.42 - 7.26 (m, 7H), 6.91 - 6.71 (m, 1 H), 5.02 (s, 2H), 3.90 (t, J = 6.8 Hz, 2H), 2.96 (app. q, J = 6.4 Hz), 1.82 (app. qu, J = 6.8 Hz). 13C NMR (101 MHz, DMSO-d8) 6 156.1 , 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): m/z calculated (M+H)+ for C14H16FN3O2 = 278.1299; measured = 277.9967.
Benzyl (3-(1H-benzo[<dlimidazol-1-yl)propyl)carbamate (8b)
Benzimidazole (8a) (391 mg, 3.31 mmol) was dissolved in MeCN (20 mL) and treated with 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 benzyl (3-bromopropyl) carbamate (750 mg, 2.76 mmol) in MeCN (5 mL) and the reaction mixture was stirred at 50°C for 18 hours. The organic layer was isolated using pipetting (separation of MeCN and water occurred), concentrated under reduced pressure onto silica and purified by silica gel column chromatography eluting with 20-50% EtOAc in hexane to afford a colorless solid (688 mg, 2.22 mmol, 81%). 1H NMR (400 MHz, DMSO-de) 3 8.24 - 8.18 (m, 1 H), 7.67 - 7.62 (m, 1 H), 7.60 - 7.55 (m, 1 H), 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). 13C NMR (101 MHz, DMSO-ds) 6 156.2, 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): m/z calculated (M+H)+ for C18H19N3O2 = 310.1550; measured = 310.0454.
Benzyl (3-(4-(trifluoromethyl)-1H-imidazol-1-yl)propyl)carbamate and benzyl (3-(5- (trifluoromethyl)-1/-/-imidazol-1-yl)propyl)carbamate as a single mixture (9b)
The title compound was synthesized following the method described in General Procedure 1 using 4-(trifluoromethyl)-1H-imidazole (9a) (5.00 g, 36.7 mmol) to afford a colorless oil (9.13 g as a mixture of C4-Me & C5-Me regioisomers, 27.9 mmol, 76%). 1H NMR (400 MHz, DMSO- de) 3 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). 13C NMR (101 MHz, DMSO-d0) 5 156.2, 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. ‘Splitting by NMR active 19F (CF3). MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for C15H16F3N3O2 = 328.1195; measured = 328.0278.
General Procedure 2: Alkylation step for compounds 1c-9c
1,4 dibromobutane (2.5 equiv.) was added to a stirred solution of the required starting material (1b-9b) (1.0 equiv.) in dry MeCN (1 mmol/mL) under argon. The reaction mixture was heated under reflux overnight and then cooled to rt. The reaction mixture was concentrated under rotary evaporation and purified by silica gel chromatography eluting with 0-15% MeOH in EtOAc to afford the desired alkylated bromo-product.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-1H-imidazol-3-ium bromide (1c) The title compound was synthesized following the method described in General Procedure 2 using 1 b (3.00 g, 11.5 mmol) to afford a colorless gum (4.12 g, 8.67 mmol, 75%). 1H NMR (300 MHz, DMSO-de) 3 9.39 (s, 1 H), 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). 13C NMR (75 MHz, DMSO-de) 3 156.2, 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): m/z calculated (M-Br)+ for CisH25Br2N3O2 = 394.1125; measured = 394.1405. 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-2-methyl-1/-/-imidazol-3-ium bromide (2c)
The title compound was synthesized following the method described in General Procedure 2 using 2b (2.03 g, 7.42 mmol) to afford a colourless gum (2.56 g, 5.23 mmol, 70%). 1H NMR (300 MHz, DMSO-de) 6 7.73 (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). 13C NMR (75 MHz, DMSO-de) 6 156.1, 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): m/z calculated (M- Br)+ for Ci9H27Br2N3O2 = 408.1281 ; measured = 408.1255.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-2-chloro-1H-imidazol-3-ium bromide (3c)
The title compound was synthesized following the method described in General Procedure 2 using 3b (1.50 g, 5.10 mmol) to afford an orange gum (1.99 g, 3.90 mmol, 76%). 1 H NMR (400 MHz, DMSO-d6) 6 8.03 (s, 1 H), 7.97 (s, 1 H), 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). 13C NMR (101 MHz, DMSO-d6) 6 156.2, 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): m/z calculated (M-Br)+ for Ci8H24CIBrN3O2 = 428.0735; measured = 427.9606.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-2-(trifluoromethyl)-1/7-imidazol-3- ium bromide (4c)
The title compound was synthesized following the method described in General Procedure 2 using 4b (5.00 g, 15.3 mmol) to afford a pale-yellow oil (5.52 g, 10.2 mmol, 66%). 1H NMR (300 MHz, CDCIs): 6 8.67 (s, 1 H), 8.53 (s, 1 H), 7.36 - 7.15 (m, 5H), 6.43 (br s, 1 H), 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). 13C NMR (75 MHz, CDC ): 6 157.9, 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): m/z calculated (M-Br)+ for Ci9H24F3Br2N3O2 = 462.0999; measured = 462.0367.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-methyl-1H-imidazol-3-ium bromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-methyl-1/-/- imidazol-3-ium bromide as a single mixture (5c)
The title compound was synthesized following the method described in General Procedure 2 using 5b (2.01 g, 7.35 mmol) to afford a dark red gum (2.27 g as a mixture of C4-Me & C5-Me regioisomers, 4.64 mmol, 63%). 1H NMR (300 MHz, DMSO-de) 6 9.21 (s, 1H), 7.59 (s, 1 H), 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). 13C NMR (75 MHz, DMSO-ds) 6 156.2, 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): m/z calculated (M-Br)+ for Ci9H2?Br2N3O2 = 408.1281 ; measured = 408.1255.
1-(3-(((benzyloxy)carbonyl)amino)Dropyl)-3-(4-bromobutyl)-4-chloro-1/-/-imidazol-3-ium bromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-chloro-1/-/- imidazol-3-ium bromide as a single mixture (6c)
The title compound was synthesized following the method described in general procedure 2 using 6b (3.65 g, 12.5 mmol) to afford a yellow oil (3.70 g, 7.27 mmol, 58%). 1H NMR (400 MHz, DMSO-ds): 6 9.38 (s, 1H), 8.15 (s, 1 H), 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). 13C NMR (101 MHz, DMSO-d6) 6 156.2, 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): m/z calculated (M-Br)+ for Ci8H24Br2CIN3O2 = 428.0735; measured = 427.9743.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-4-fluoro-1H-imidazol-3-ium bromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5-fluoro-1/-/- imidazol-3-ium bromide as a single mixture (7c)
The title compound was synthesized following the method described in General Procedure 2 using 7b (1.03 g, 3.72 mmol) to afford a pale-yellow oil (668 mg, 1.35 mmol, 36%). 1H NMR (400 MHz, DMSO-ds) 6 9.15 - 9.03 (m, 1 H), 7.90 - 7.75 (m, 1 H), 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). 13C NMR (101 MHz, DMSO-ds) 6 156.2, 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): m/z calculated (M-Br)+ for CisH24FBr2N3O2 = 412.1030; measured = 412.0259.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-1H-benzo[cflimidazol-3-ium bromide (8c)
The title compound was synthesized following the method described in general procedure 2 using 8b (675 mg, 2.18 mmol) to afford a pale-yellow oil (716 mg, 1.36 mmol, 62%). 1H NMR (400 MHz, DMSO-ds) 6 9.89 - 9.81 (m, 1 H), 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). 13C NMR (101 MHz, DMSO-d8) 6 156.2, 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): m/z calculated (M-Br)+ for C22H27Br2N3O2 = 444.0214; measured = 444.1281.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-4-(trifluoromethyl)-1H-imidazol-3- ium bromide and 1-(3-(((benzyloxy)carbonyl)amino)propyl)-3-(4-bromobutyl)-5- (trifluoromethyl)- imidazol-3-ium bromide as a single mixture (9c)
Figure imgf000038_0001
The title compound was synthesized following the method described in General Procedure 2 using 9b (4.50 g, 13.7 mmol) to afford a pale-yellow oil (6.08 g, 11.2 mmol, 85%). 1H NMR (300 MHz, CDCh) 6 10.72 (s, 1H), 8.15 (s, 1 H), 7.41 - 7.33 (m, 5H), 6.47 (br s, 1 H), 5.06 (s, 2H), 4.53 - 4.41 (m, 4H), 3.31 - 3.23 (m, 2H), 2.22 - 1 .98 (m, 8H). 13C NMR (75 MHz, CDCh) 6 157.14, 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): m/z calculated (M-Br)+ for Ci9H24F3Br2N3O2 = 462.0999; measured = 462.0346.
General Procedure 3: Alkylation step for bis-imidazole compounds 1d-9d
The required imidazole analog (1a-9a) (1.0 equiv.) was dissolved in THF (0.5 mmol/mL) and stirred in an ice bath for 10 min. NaH (2.5 equiv.) was added portion wise to the reaction mixture and the reaction mixture was removed from the ice bath and stirred for 1 hour. 1,4- Dibromobutane (0.5 equiv.) was added, and the reaction mixture was stirred at 50 °C overnight. The resultant mixture was cooled to rt, and filtered through a pad of celite with THF washings. The filtrate was dried under rotary evaporation and subsequently dissolved in methanol. For 1d, 2d and 5d: The methanol layer was washed three times with hexane and concentrated under rotary evaporation to afford desired product without the need for further purification. For 3d-4d and 6d-9d: The methanol layer was washed three times with hexane, concentrated under rotary evaporation and purified by silica gel column chromatography eluting with 33- 100% EtOAc in hexane to afford the desired product.
1,4-di(1H-imidazol-1-yl)butane (1 d)
The title compound was synthesized following the method described in General Procedure 3 using imidazole (1a) (8.00 g, 117.5 mmol) to afford a colorless solid (10.2 g, 53.6 mmol, 91%). 1H NMR (300 MHz, DMSO-de) 6 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). 13C NMR (101 MHz, DMSO-de) 6 137.27, 128.45, 119.31 , 45.34, 27.73. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for C10H14N4 = 191.1219; measured = 191.1296.
1,4-bis(2-methyl-1H-imidazol-1-yl)butane (2d) The title compound was synthesized following the method described in General Procedure 3 using 2-methyl-1/-/-imidazole (2a) (10.0 g, 122 mmol) to afford a colorless solid (11.8 g, 54.1 mmol, 89%). 1H NMR (300 MHz, DMSO-d5) 6 7.01 (s, 2H), 6.71 (s, 2H), 3.86 (s, 4H), 2.25 (s, 6H), 1.61 (s, 4H). 13C NMR (75 MHz, DMSO-de) 0 143.8, 125.8, 119.6, 44.6, 27.1 , 12.2. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for CI2HISN4 = 219.1532; measured = 219.1009.
1.4-bis(2-chloro-1/-/-imidazol-1-yl)butane (3d)
The title compound was synthesized following the method described in General Procedure 3 using 2-chloro-1H-imidazole (3a) (1.00 g, 9.75 mmol) to afford a colorless solid (615 mg, 2.37 mmol, 49%). 1H NMR (400 MHz, CDCh) 6 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). 13C NMR (101 MHz, CDCh) 6 131.9, 128.8, 120.7, 46.0, 27.3. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for CIOHI2CI2N4 = 259.0512; measured = 258.9474.
1.4-bis(2-(trifluoromethyl)-1/-/-imidazol-1-yl)butane (4d)
The title compound was synthesized following the method described in General Procedure 3 using 2-(trifluoromethyl)-1/-/-imidazole (4a) (20.0 g, 146.9 mmol) to afford an orange oil (15.8 g, 48.5 mmol, 66%). 1H NMR (300 MHz, CDCh) 6 7.11 (s, 2H), 7.02 (s, 2H), 4.11 (s, 4H), 1.85 (s, 4H). 13C NMR (75 MHz, CDCh) 6 129.2, 122.9, 28.0, 46.5. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for CI2HI2FSN4 = 327.0966; measured = 327.0123.
1.4-bis(4-methyl-1/-/-imidazol-1-yl)butane and imidazoyl-CHs associated reoioisomers as a single mixture (5d)
The title compound was synthesized following the method described in General Procedure 3 using 4-methyl-1 /-/-imidazole (5a) (10.0 g, 122 mmol) to afford a colorless solid (12.6 g, 57.7 mmol, 95%). 1H NMR (300 MHz, DMSO-ds) 6 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). 13C NMR (75 MHz, DMSO-d6) 6 136.9, 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): m/z calculated (M+H)+ for Ci2HisN4 = 219.1532; measured = 219.1009.
1.4-bis(4-chloro-1/-/-imidazol-1-yl)butane and imidazoyl-CI associated regioisomers as a single mixture (6d)
The title compound was synthesized following the method described in General Procedure 3 using 4-chloro-1 /-/-imidazole (6a) (3.00 g, 29.3 mmol) to afford a dark orange oil (2.92 g, 11.3 mmol, 77%). 1H NMR (400 MHz, DMSO-de) 6 7.84 - 7.54 (m, 2H), 7.30 - 6.92 (m, 2H), 4.03 - 3.86 (m, 4H), 1.72 - 1.54 (m, 4H). 13C NMR (101 MHz, DMSO-d6) 6 136.3, 127.4, 125.0, 115.3, 46.1 , 43.7, 27.2, 26.7. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for CIOHI2CI2N4 = 259.0512; measured = 258.9255.
1.4-bis(4-fluoro-1H-imidazol-1-yl)butane and imidazoyl-F associated reqioisomers as a single mixture (7d)
The title compound was synthesized following the method described in General Procedure 3 using 4-fluoro-1H-imidazole (7a) (1.74 g, 20.2 mmol) to afford a pale-yellow solid (1.10 g, 4.84 mmol, 48%). 1H NMR (400 MHz, DMSO-de) 6 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,
Figure imgf000040_0001
3.3 Hz, 4H). 13C NMR (101 MHz, DMSO-ds) 6
157.4, 155.1, 130.8, 130.6, 97.8, 97.3, 46.3, 27.1. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for CWHI2F2N4 = 227.1103; measured = 226.9815.
1.4-bis(1H-benzorcflimidazol-1-yl)butane (8d)
Benzimidazole (8a) (4.10 g, 34.7 mmol) was dissolved in MeCN (80 mL) and treated with 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 treated with 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 filtered with water rinsing and the solids dried to afford a colorless solid (3.15 g, 10.8 mmol, 78%). 1H NMR (400 MHz, DMSO-de) 6 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). 13C NMR (101 MHz, DMSO-de) 6 143.9, 143.4, 133.7, 122.2, 121.4, 119.4, 110.4, 43.5, 26.7. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for C18H18N4 = 291.1604; measured = 291.0451.
1.4-bis(4-(trifluoromethyl)-1/-/-imidazol-1-yl)butane and imidazoyl-CFs associated reqioisomers as a single mixture (9d)
The title compound was synthesized following the method described in General Procedure 3 using 4-(trifluoromethyl)-1H-imidazole (9a) (16.4 g, 100 mmol) to afford as an orange oil (11.8 g, 36.2 mmol, 72%). 1H NMR (300 MHz, MeOD) 5 7.41 - 7.89 (m, 4H), 4.06 - 4.12 (m, 4H), 1.76 (s, 4H). 13C NMR (CDCb, 75 MHz) 6 139.5, 120.8, 46.3, 27.7. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M+H)+ for CI2HI2F6N4 = 327.0966; measured = 327.0175.
General Procedure 4: Alkylation step for compounds 1e-9e
The required bromo-starting material 1c-9c (1 equiv.) in dry MeCN (0.5 mmol/mL) was added dropwise to a stirred solution of the required bis-imidazole starting material 1d-9d (2.5 equiv.) in dry MeCN (2 mmol/mL) at 80 °C. The resulting mixture was stirred under reflux overnight for a few days. After completion of the reaction monitored by 1H NMR, the reaction mixture was concentrated under rotary evaporation, and the residue was treated with a 1:1 mixture of water and 3:1 chloroform: isopropanol and transferred into a separating funnel. The organic layer was removed and the aqueous layer was further washed with 3:1 chloroform:isopropanol five times. The aqueous layer was concentrated under reduced pressure to afford the desired product.
1-(4-(1/-/-imidazol-1-yl)butyl)-3-(4-(1-(3-(((benzyloxy)carbonyl)amino)propyl)-1H-imidazol-3- ium-3-yl)butyl)-1/7-imidazol-3-ium dibromide (1e)
The title compound was synthesized following the method described in General Procedure 4 using 1c (3.00 g, 6.34 mmol) to afford a colourless hygroscopic gum (3.07 g, 4.61 mmol, 73%). 1H NMR (300 MHz, DMSO-d6) 5 9.45 - 9.29 (m, 2H), 7.88 - 7.78 (m, 4H), 7.72 (s, 1 H), 7.48 - 7.28 (m, 6H), 7.20 (s, 1 H), 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). 13C NMR (75 MHz, DMSO- de) 6 155.0, 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): m/z calculated (M-2Br-H)+ for C28H39Br2N?O2 = 504.3082; measured = 504.3814.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-2-methyl-3-(4-(2-methyl-1-(4-(2-methyl-1H- imidazol-1-yl)butyl)-1H-imidazol-3-ium-3-yl)butyl)-1/-/-imidazol-3-ium dibromide (2e) The title compound was synthesized following the method described in General Procedure 4 using 2c (2.50 g, 5.11 mmol) to afford a colorless hygroscopic gum (2.45 g, 3.46 mmol, 68%). 1H NMR (300 MHz, DMSO-ds) 6 7.88 - 7.66 (m, 4H), 7.52 - 7.23 (m, 6H), 7.16 (s, 1 H), 6.87 (s, 1 H), 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). 13C NMR (75 MHz, DMSO-de) 6 154.7, 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): m/z calculated (M-2Br-H)+ for C3iH45Br2N?O2 = 546.3551 ; measured = 546.3480.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-2-chloro-3-(4-(2-chloro-1-(4-(2-chloro-1/7-imidazol- 1-yl)butyl)-1/-/-imidazol-3-ium-3-yl)butyl)-1H-imidazol-3-ium dibromide (3e)
The title compound was synthesized following the method described in General Procedure 4 using 3c (300 mg, 0.588 mmol) to afford a pale-yellow gum (375 mg, 0.488 mmol, 83%). 1H NMR (400 MHz, DMSO-de,) 6 8.13 - 7.93 (m, 4H), 7.49 - 7.2 (m, 7H), 6.93 (s, 1 H), 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). 13C NMR (101 MHz, DMSO-de) 6 156.3, 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): m/z calculated (M-2Br-CI+O)+ for C28H35Br2CI3N7O2 = 588.2251 ; measured = 558.1045.7
1-(3-(((benzyloxy)carbonyl)amino)propyl)-2-(trifluoromethyl)-3-(4-(2-(trifluoromethyl)-1-(4-(2- (trifluoromethyl)-1/7-imidazol-1-yl)butyl)-1/-/-imidazol-3-ium-3-yl)butyl)-1H-imidazol-3-ium dibromide (4e)
The title compound was synthesized following the method described in General Procedure 4 using 4c (5.00 g, 9.20 mmol) to afford a pale-yellow hygroscopic gum (4.92 g, 5.64 mmol, 61%). 1H NMR (300 MHz, DMSO-d8) 5 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, 3 H), 1.99 - 1.82 (m, 10 H). 13C NMR (75 MHz, DMSO-d8) 5 156.7, 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): m/z calculated (M-2Br-H)+ for C3iH38Br2FgN7O2 = 708.2703; measured = 708.2897.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-5-methyl-3-(4-(4-methyl-1-(4-(5-methyl-1H- imidazol-1-yl)butyl)-1/-/-imidazol-3-ium-3-yl)butyl)-1/7-imidazol-3-ium dibromide and imidazoyl-CHs associated regioisomers as a single mixture (5e)
The title compound was synthesized following the method described in General Procedure 4 using 5c (2.50 g, 5.11 mmol) to afford a dark red hygroscopic gum (2.15 g, 3.04 mmol, 59%). 1H NMR (300 MHz, DMSO-d8) 3 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, 1 H), 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). 13C NMR (75 MHz, DMSO-d8) 5 156.1 , 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): m/z calculated (M-2Br-H)+ for C3iH4sBr2N7O2 = 546.3551 ; measured = 546.3480.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-4-chloro-3-(4-(5-chloro-1-(4-(4-chloro-1H-imidazol- 1-yl)butyl)-1/-/-imidazol-3-ium-3-yl)butyl)-1/-/-imidazol-3-ium dibromide and imidazoyl-CI associated regioisomers as a single mixture (6e)
The title compound was synthesized following the method described in General Procedure 4 using 6c (566 mg, 1.11 mmol) to afford an orange gum (1.96 g, 0.941 mmol, 85%). 1H NMR (400 MHz, DMSO-d6) 5 9.73 - 9.44 (m, 2H), 8.28 - 8.09 (m, 2H), 7.65 (s, 1 H), 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). 13C NMR (101 MHz, DMSO-d6) 6 156.3, 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): m/z calculated (M-2Br-H)+ for C28H3SBr2Cl3N7O2 = 606.1912; measured = 606.1226.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-4-fluoro-3-(4-(5-fluoro-1-(4-(4-fluoro-1 H-imidazol-1- yl)butyl)- imidazol-3-ium-3-yl)butyl)-1/-/-imidazol-3-ium dibromide and imidazoyl-F associated regioisomers as a single mixture (7e)
The title compound was synthesized following the method described in General Procedure 4 using 7c (656 mg, 1.33 mmol) to afford a pale-yellow hygroscopic gum (677 mg, 0.941 mmol, 71%). 1H NMR (400 MHz, DMSO-de) 5 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, 1 H), 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). 13C NMR (101 MHz, DMSO-d6) 3 156.2, 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): m/z calculated (M-2Br-H)+ for C28H38Br2F3N7O2 = 558.2800; measured = 558.2171.
3-(4-(1H-benzorcf]imidazol-1-yl)butyl)-1-(4-(3-(3-(((benzyloxy)carbonyl)amino)propyl)-1/-/- benzo[cf]imidazol-3-ium-1-yl)butyl)-1/-/-benzo[cflimidazol-3-ium dibromide (8e)
The title compound was synthesized following the method described in General Procedure 4 using 8c (672 mg, 1.28 mmol) to afford a colorless solid (753 mg, 0.923 mmol, 72%). 1H NMR (400 MHz, DMSO-de) 6 10.27 - 9.78 (m, 2H), 8.82 (br s, 1 H), 8.21 - 7.93 (m, 5H), 7.85 - 7.62 (m, 6H), 7.50 - 7.42 (m, 1 H), 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). 13C NMR (101 MHz, DMSO-d6) 6
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): m/z calculated (M-2Br-H)+ for C4oH4sBr2N702 = 654.3551; measured = 654.2896.
1-(3-(((benzyloxy)carbonyl)amino)propyl)-4-(trifluoromethyl)-3-(4-(5-(trifluoromethyl)-1-(4-(4- (trifluoromethyl)-1H-imidazol-1-yl)butyl)-1/-/-imidazol-3-ium-3-yl)butyl)-1H-imidazol-3-ium dibromide and imidazoyl-CFs associated regioisomers as a single mixture (9e)
The title compound was synthesized following the method described in General Procedure 4 using 9c (5.85 g, 10.8 mmol) to afford a pale-yellow hygroscopic gum (6.00 g, 6.90 mmol, 64%). 1H NMR (300 MHz, DMSO-de) 6 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). 13C NMR (75 MHz, DMSO-de) 6 156.7, 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): m/z calculated (M-2Br-H)+ for CsiHseB^FgNyC = 708.2703; measured = 708.2830.
General Procedure 5: Synthesis of final compounds 1 , 2, 5 and 6
To a stirred solution of the required starting material 1e, 2e, 5e and 6e (1.0 equiv.) in MeCN:DMF (9.5:0.5) at rt was added 1 ,4-diiodobutane (0.5 equiv.). The reaction mixture was heated to 90 °C for 48 hours (1e, 2e, 5e) to 1 week (6e). The reaction mixture was allowed to cool to rt. The resulting gum was separated, further triturated three times with MeCN, and treated with a solution of HBr in acetic acid (33%) (10 equiv.) and the resulting mixture was stirred at rt overnight. The reaction mixture was treated with excess EtOAc and the resulting precipitate/gum was isolated. The precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride. (Ionexchange resin column preparation: 1 M HCI aqueous solution passed through a glass column packed with Amberlyst® A-26 (OH- form) until the pH of eluates reached the same value as the original solution, and then the resin was washed with water until a neutral pH was achieved). The product-containing eluent was transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialysed against 5 ml_ HCI in 5 L of deionised water over 24 hours, with frequent changing of dialysis water every 2-3 hours. The resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyl)-1/ -imidazol-3-ium-3-yl)butyl)-1/-/- imidazol-3-ium-1-yl)butyl)-1H-imidazol-3-ium) octachloride (OIM1-6-CH, 1)
The title compound was synthesized following the method described in General Procedure 5 using 1e (1.00 g, 1.50 mmol) to afford a pale brown hygroscopic solid (474 mg, 0.443 mmol, 59%). 1H NMR (300 MHz, DMSO-de) 5 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). 13C NMR (75 MHz, DMSO- de) 6 136.4, 136.3, 122.5, 122.4, 48.0, 45.9, 35.4, 27.3, 25.9. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M-7H-8CI)+ for C44H76CI8N14 = 793.5824; measured = 793.5392. GPC (Water Phase) Mn = 1025, Mw = 1037, Mp = 1044, PDI = 1.01. (Fig. 3)
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyl)-2-methyl-1/-/-imidazol-3-ium-3- yl)butyl)-2-methyl-1H-imidazol-3-ium-1-yl)butyl)-2-methyl-1/-/-imidazol-3-ium) octachloride
(OIM1-6-C2(CHe), 2)
The title compound was synthesized following the method described in General Procedure 5 using 2e (1.50 g, 1.28 mmol) to afford a pale brown hygroscopic solid (270 mg, 0.231 mmol, 36%). 1H NMR (300 MHz, D2O) 6 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 + not observed due to deuterium exchange. 13C NMR (75 MHz, D2O) 5 144.1, 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): m/z calculated (M-7H-8CI)+ for CSOHBBCIBNU = 877.6763; measured = 877.6971. GPC (Water Phase) Mn = 1002, Mw = 1009, Mp = 1016, PDI = 1.01. (Fig. 4)
General Procedure 6: Synthesis of final compounds 3 and 7
To a stirred solution of the required starting material 3e and 7e (1.0 equiv.) in MeCN:DMF (1 :0.2) at 90 °C was added 1 ,4 iodobutane (3 equiv.). The reaction mixture was stirred at 90 °C for24-48 hours with 1H NMR reaction monitoring. The reaction mixture was cooled to rt, 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 a separate portion of 3e and 7e (1 .5 equiv.). The reaction mixture was stirred at 90 °C for 48-60 hours. The reaction mixture was allowed to cool to rt and treated with excess EtOAc. The resulting precipitate/gum was isolated, further triturated with EtOAc, and treated with a solution of HBr in acetic acid (33%) (10 equiv.) and the resulting mixture was stirred at rt overnight. The reaction mixture was treated with excess EtOAc and the resulting precipitate/gum was isolated. The precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride. The product-containing eluent was transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialyzed against 5 mL HCI in 5 L of deionized water over 24 hours, with frequent changing of dialysis water every 2-3 hours. The resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyl)-2-chloro-1/-/-imidazol-3-ium-3- yl)butyl)-2-chloro-1H-imidazol-3-ium-1-yl)butyl)-2-chloro-1H-imidazol-3-ium) octachloride (OIM1-6-C2(CI), 3)
The title compound was synthesized following the method described in General Procedure 6 using 3e (250 mg, 0.325 mmol) to afford a pale brown hygroscopic solid (110 mg, 0.086 mmol, 26%). 1H NMR (400 MHz, D2O) 6 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 + not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 6 135.6, 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): m/z calculated (M-7H-8CI)+ for C44H7oCluNi4 = 999.3456; measured = desired ion not found, likely due to poor ionization combined with low peak intensity from chlorine isotopes. GPC (Water Phase) Mn = 855, Mw = 863, Mp = 851 , PDI = 1.01. General Procedure 7: Synthesis of final compounds 4 and 9
To a stirred solution of the required starting material 4e and 9e (1.0 equiv.) in N-methyl-2- pyrrolidone (NMP) was added 1 ,4-diiodobutane (0.5 equiv.) and heated to 120 °C for 72 hours. The reaction mixture was allowed to cool to rt. The resulting gum was separated, triturated three times with MeCN, treated with a solution of HBr in acetic acid (33%) (10 equiv.) and the resulting mixture was stirred at rt overnight. 1H NMR was used to confirm reaction completion before the work-up. The reaction mixture was treated with excess EtOAc and the resulting precipitate/gum was isolated. The precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride. The product-containing eluent was transferred to a dialysis bag with Mw cut-off of 1000 Da and dialyzed against 5 mL HCI in 5 L of deionized water over 24 hours, with frequent changing of dialysis water every 2-3 hours. The resulting solution was concentrated under rotary evaporation and purified by Sephadex™-G10 gel filtration chromatography eluting with deionized water (gel filtration chromatography preparation: SephadexTM-G10 powder was suspended in DI water overnight to give a slurry. The slurry was packed into a glass column and packed using DI water as eluent and gravitational elution). The pure fraction was collected and lyophilized to afford the desired product.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyl)-2-(trifluoromethyl)-1/-/-imidazol-3- ium-3-yl)butyl)-2-(trifluoromethyl)-1/-/-imidazol-3-ium-1-yl)butyl)-2-(trifluoromethyl)-1/-/- imidazol-3-ium) octachloride (OIM1-6-C2(CF3). 4)
The title compound was synthesized following the method described in General Procedure 7 using 4e (1.00 g, 1.15 mmol) to afford a pale brown hygroscopic solid (223 mg, 0.150 mmol, 26%). 1H NMR (400 MHz, D2O) 5 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 + not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 3 135.6, 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. 19F NMR (376 MHz, D2O) 6 -58.4, -61 .3. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M-7H-8CI)+ for CsoHyoFisCIgNu = 1201.5068; measured = 1201.7402. GPC (Water Phase) Mn = 1004, Mw = 1053, Mp = 945, PDI = 1.05.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyD-5-methyl-1/-/-imidazol-3-ium-3- yl)butyl)-4-methyl-1H-imidazol-3-ium-1-yl)butyl)-5-methyl-1H-imidazol-3-ium) octachloride and imidazoyl-CHs associated reqioisomers as a single mixture (OIM1-6-C4(CH3), 5) The title compound was synthesized following the method described in General Procedure 5 using 5e (1.50 g, 1.28 mmol) to afford a pale brown hygroscopic solid (314 mg, 0.269 mmol, 42%). 1H NMR (300 MHz, D2O) 5 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 + not observed due to deuterium exchange. 13C NMR (75 MHz, D2O) 5 134.7, 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): m/z calculated (M-7H-8CI)+ for CsoHssCIsN = 877.6763; measured = 877.6996. GPC (Water Phase) M„ = 877, Mw = 884, Mp = 893, PDI = 1.01. (Fig. 5)
3,3'-(butane-1.4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyl)-5-chloro-1H-imidazol-3-ium-3- yl)butyl)-4-chloro-1H-imidazol-3-ium-1-yl)butyl)-5-chloro-1H-imidazol-3-ium) octachloride and imidazoyl-CI associated reqioisomers as a single mixture (OIM1-6-C4(CI), 6)
The title compound was synthesized following the method described in General Procedure 5 using 6e (300 mg, 0.39 mmol) to afford an orange solid (145 mg, 0.112 mmol, 56%). 1H NMR (400 MHz, D2O) 6 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 + not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 5 135.7, 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): m/z calculated (M- 7H-8CI)+ for C44H 70CI14N14 - 999.3456; measured - desired ion not found, likely due to poor ionization combined with low peak intensity from chlorine isotopes. GPC (Water Phase) Mn = 692, Mw = 701, Mp = 715, PDI = 1.01.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyl)-5-fluoro-1/7-imidazol-3-ium-3- yl)butyl)-4-fluoro-1H-imidazol-3-ium-1-yl)butyl)-5-fluoro-1H-imidazol-3-ium) octachloride and imidazoyl-F associated reqioisomers as a single mixture (OIM1-6-C4(F), 7)
The title compound was synthesized following the method described in General Procedure 6 using 7e (191 mg, 0.107 mmol) to afford a brown hygroscopic solid (51 mg, 0.043 mmol, 40%). 1H NMR (400 MHz, D2O) 6 8.84 - 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 + not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 6 148.3, 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. 19F NMR (376 MHz, D2O) 6 -
144.2, -144.49. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M-7H-8CI)+ for CsoHssCIsNu = 877.6763; measured = desired ion not found, likely due to poor ionization. GPC (Water Phase) Mn = 950, Mw = 970, Mp = 997, PDI = 1.02. 3.3'-(butane-1.4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyr)-1/7-benzoMimidazol-3-ium-3- yl)butyl)-1H-benzo[cflimidazol-3-ium-1-yl)butyl)-1H-benzorcflimidazol-3-ium) octachloride (OIM1-6-Benzimidazole, 8)
To a stirred solution of the required starting material 8e (178 mg, 0.218 mmol) in DMSO (5 mL) at 90 °C was added 1 ,4-dibromobutane (13 pL, 0.107 mmol). The reaction mixture was heated to 90 °C for 48 hours. The reaction mixture was allowed to cool to rt and treated with excess EtOAc. The resulting precipitate/gum was isolated, further triturated with EtOAc, and treated with a solution of HBr in acetic acid (33%) (2 mL) and the resulting mixture was stirred at rt overnight. The reaction mixture was treated with excess EtOAc and the resulting precipitate/gum was isolated. The precipitate/gum was further triturated with EtOAc, dissolved in a water/MeOH mixture to give a clear solution and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride. The product-containing eluent was transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialyzed against 5 mL HOI in 5 L of deionized water over 24 hours, with frequent changing of dialysis water every 2-3 hours. The resulting solution was concentrated under rotary evaporation and lyophilized to afford a colorless solid (38 mg, 0.027 mmol, 25%). 1H NMR (400 MHz, D2O) 6 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, -CW2). 2 x -NH3 + not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 6 140.9, 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): m/z calculated (M-7H- 8CI)+ for C68H88ClsNi4 = 1093.6763; measured = 1093.6996.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(3-ammoniopropyl)-5-(trifluoromethyl)-1/7-imidazol-3- ium-3-yl)butyl)-4-(trifluoromethyl)-1/-/-imidazol-3-ium-1-yl)butyl)-5-(trifluoromethyl)-1H- imidazol-3-ium) octachloride and imidazoyl-CF3 associated reqioisomers as a single mixture (OIM1-6-C4(CF3). 9)
The title compound was synthesized following the method described in General Procedure 7 using 7e (1.00 g, 1.15 mmol) to afford a pale brown hygroscopic solid (155 mg, 0.104 mmol, 18%). 1H NMR (400 MHz, D2O) 6 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 + not observed due to deuterium exchange. 13C NMR (101 MHz,D2O) 6 139.8, 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. 18F NMR (376 MHz, D2O) 6 -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): m/z calculated (M-7H-8CI)+ for CSOHTOFISCISNU = 1201.5068; measured = 1199.5159. GPC (Water Phase) Mn = 985, Mw = 992, Mp = 1000, PDI = 1.01. Example 2. Synthesis of 0IM1-6 derivatives
Genera! Procedure 8: Synthesis of dibromo compounds 10a-12a
To a stirred solution of 1 ,4-dibromobutane (10 equiv.) at 80 °C was added dropwise a solution of the required bis-imidazole starting material 1d, 2d and 5d respectively (1.0 equiv.) in MeCN (0.5 mmol/mL). The reaction mixture was stirred at 80 °C for 18 hours. For compounds 10a and 12a: The reaction mixture was allowed to cool to rt, diluted with MeCN, transferred to a separating funnel, and washed three times with hexane. The MeCN layer was concentrated under rotary evaporation and purified by silica gel column chromatography eluting with 10- 30% MeOH in EtOAc to afford the desired product. For compound 11a: The reaction mixture was allowed to cool to and concentrated under rotary evaporation to afford a colourless solid. The solid was triturated three times with diethyl ether to afford the desired product.
1,T-(butane-1 ,4-diyl)bis(3-(4-bromobutyl)-1H-imidazol-3-ium) dibromide (10a)
The title compound was synthesized following the method described in General Procedure 8 using 1d (1.00 g, 5.26 mmol) to afford a pale brown gum (1.04 g, 1.67 mmol, 32%). 1H NMR (MeOD, 400 MHz) 5 9.28 - 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). 13C NMR (MeOD, 101 MHz) 5 137.8, 124.1 , 124.0, 50.31 , 50.27, 33.4, 30.6, 29.9, 28.0. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M- 2Br-H)+ for Ci8H3oBr4N4= 459.0753; measured = 459.0139.
1 , 1 '-(butane-1 ,4-diyl)bis(3-(4-bromobutyl)-2-methyl-1 /-/-imidazol-3-ium) dibromide (11a)
The title compound was synthesized following the method described in General Procedure 8 using 2d (560 mg, 2.56 mmol) to afford a colourless solid (1.28 g, 1.97 mmol, 77%). 1H NMR (MeOD, 400 MHz) 5 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). 13C NMR (MeOD, 101 MHz) 6 145.7, 122.8, 48.8, 33.5, 30.6, 29.5, 27.7, 10.1. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M-2Br- H)+ for C2oHs4Br4N4 = 489.1047; measured = 489.0467.
1 , 1 '-(butane-1 ,4-diyl)bis(3-(4-bromobutyl)-4-methyl-1 /7-imidazol-3-ium) dibromide and imidazoyl-CHs associated reqioisomers as a single mixture (12a)
The title compound was synthesized following the method described in General Procedure 8 using 5d (584 mg, 2.68 mmol) to afford a pale brown gum (394 mg, 0.606 mmol, 23%). 1H NMR (MeOD, 400 MHz) 5 9.23 - 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). 13C NMR (MeOD, 101 MHz) 5 136.7, 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): m/z calculated (M-2Br-H)+ for C20H34Br4N4 = 489.1047; measured = 489.0489.
General Procedure 9: Synthesis of final compounds 10-12
To a stirred solution of the required dibromo starting material 10a-12a (1.0 equiv.) in DMF (0.5 mmol/mL) at rt was added the required starting material 1e, 2e and 5e respectively (2 equiv.) in 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 allowed to cool to rt. The resulting gum was separated, further triturated three times with MeCN, and treated with a solution of HBr in acetic acid (33%) (10 equiv.) and the resulting mixture was stirred at rt overnight. 1H NMR was used to confirm reaction completion before the work-up. The reaction mixture was treated with excess EtOAc and the resulting precipitate/gum was isolated. The precipitate/gum was further triturated with EtOAc, dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride. The product-containing eluent was transferred to a dialysis bag with Mw cut-off of 1000 Da and dialysed against 1 mL HCI in 1 L of deionised water over 4 hours, with a single dialysis water change at t = 2 hours. The resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(4-(3-(3-ammoniopropyl)-1/-/-imidazol-3-ium-1-yl)butyl)- 1H-imidazol-3-ium-3-yl)butyl)-1H-imidazol-3-ium-1-yl)butyl)-1H-imidazol-3-ium) decachloride
(OIM1-8-CH, 10)
The title compound was synthesized following the method described in General Procedure 9 using 10a (299 mg, 0.481 mmol) and 1e (641 mg, 0.962 mmol) to afford a pale brown hygroscopic solid (220 mg, 0.157 mmol, 33%). 1H NMR (400 MHz, D2O) 5 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). 2 x -NHs+ not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 6 135.6, 135.4, 122.7, 122.5, 48.9, 46.6, 36.4, 27.4, 26.3. MALDI- TOF (HCCA matrix, Reflector mode): m/z calculated (M-9H-10CI)+ for CssHgsCIwNu = 1037.7512; measured = 1037.7473. GPC (Water Phase) Mn = 1240, Mw = 1264, Mp = 1216, PDI = 1.02.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(4-(3-(3-ammoniopropyl)-2-methyl-1/7-imidazol-3-ium- 1-yl)butyl)-2-methyl-1H-imidazol-3-ium-3-yl)butyl)-2-methyl-1H-imidazol-3-ium-1-yl)butyl)-2- methyl-1/-/-imidazol-3-ium) decachloride (OIM1-8-C2(CH3), 11)
The title compound was synthesized following the method described in General Procedure 9 using 10b (210 mg, 0.323 mmol) and 2e (457 mg, 0.646 mmol) to afford a pale brown hygroscopic solid (124 mg, 0.082 mmol, 25%).1H NMR (300 MHz, D2O) 57.49- 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 -NHs+ not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 5 144.0, 121.1 , 47.4, 25.9, 8.93. MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M-9H-10CI)+ for CBBHIUCIION = 1149.8764; measured =1150.0909. GPC (Water Phase) Mn =1117, Mw = 1135, Mp = 1105, PDI = 1.02.
3,3'-(butane-1 ,4-diyl)bis(1-(4-(3-(4-(1-(4-(3-(3-ammoniopropyl)-4-methyl-1/7-imidazol-3-ium- 1-yl)butyl)-5-methyl-1H-imidazol-3-ium-3-yl)butyl)-4-methyl-1H-imidazol-3-ium-1-yl)butyl)-5- methyl-1/-/-imidazol-3-ium) decachloride (OIM1-8-C4(CH3), 12)
The title compound was synthesized following the method described in General Procedure 9 using 10c (300 mg, 0.461 mmol) and 5e (653 mg, 0.922 mmol) to afford a pale brown hygroscopic solid (115 mg, 0.076 mmol, 16%). 1H NMR (400 MHz, D2O) 6 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 + not observed due to deuterium exchange. 13C NMR (101 MHz, D2O) 6 134.7, 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): m/z calculated (M-9H-10CI)+ for CBBHH4CIIONI4 = 1149.8764; measured = 1149.9030. GPC (Water Phase) Mn = 1006, Mw = 1018, Mp = 1007, PDI = 1.01.
Example 3. Synthesis of degradable OIM1 Derivatives
Synthesis of 3-(4-bromobutyl)-1-butyl-1H-imidazol-3-ium (1g)
The title compound was synthesized following the method described in General Procedure 2 (described in Example 1) using N-butyl imidazole (5g, 0.04mol) to afford a colorless gum (8.8 g, 65%). 1H NMR (400 MHz, MeOD) 6 9.14 (d, J = 5.9 Hz, 1 H), 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). 13C NMR (101 MHz, MeOD) 6 137.4, 124.0, 50.9, 50.2, 33.3, 33.2, 30.6, 29.9, 20.6, 13.8 . MALDI-TOF (HCCA matrix, Reflector mode): m/z calculated (M-Br)+ for CnH2oBr2N2 = 260.1985; measured = 260.9590
Synthesis of 1-(4-(1 H-imidazol-1-yl)butyl)-3-(4-(3-butyl-1 H-imidazol-3-ium-1-yl)butyl)-1 H- imidazol-3-ium (1h)
The title compound was synthesized following the method described in General Procedure 4 (described in Example 1) using 1g (6.00 g, 0.019 mol) to afford a colourless hygroscopic gum (6.31 g, 62%). 1H NMR (400 MHz, DMSO) 5 9.50 - 9.06 (m, 2H), 7.91 - 7.70 (m, 5H), 7.21 (s, 1H), 6.95 (s, 1 H), 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).13C NMR (101 MHz, DMSO) 5 137.0, 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): m/z calculated (M-2Br-H)+ for C2iH34Br2Ne = 369.5330; measured = 369.1999.
Synthesis of bis(3-bromoDropoxy)methane (13b)
3-Bromo-1-propanol (3.2 ml_, 3.57mmol, 2 eq) and paraformaldehyde (0.5355 g, 1.78 mmol, 1 eq) were dissolved in toluene (10 ml_) in a round-bottom flask equipped with a Dean-Stark apparatus. Concentrated sulfuric acid (1 drop, cat.) was added and the mixture was refluxed for 1 ,5h, until approximately the expected amount of water has been collected. After cooling, sodium bicarbonate (approx. 0.2 g) was added and the mixture was filtered. The filtrate was directly purified by flash chromatography (95:5, PS/EtOAc) to provide 13b (4.0 gm, 83%) as a pale-yellow oil. 1H NMR (400 MHz, CDCI3) 6 4.68 (s, 1 H), 3.68 (t, J = 5.9 Hz, 2H), 3.53 (t, J = 6.5 Hz, 2H), 2.24 - 2.03 (m, 2H). 13C NMR (101 MHz, CDCI3) 0 95.42 (s), 65.12 (s), 32.89 (s), 30.41 (s).
Synthesis of 1 ,T-(piperazine-1 ,4-diyl)bis(2-chloroethan-1-one) (14b)
100 ml_ of saturated K2CO3 was added to a solution of piperazine (5.00 g, 58.14 mmol) in CHCh (200 ml_). The reaction mixture was stirred and cooled to 0 °C in an ice bath. 2- Chloroacetyl chloride (19.71 g, 174.44 mmol) in CHCI3 was added dropwise over a period of 1 hour. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The organic phase was washed with HCI (2 x 100 ml_, 1 M) and water (2 x 100 mL). The CHCI3 layer was dried and evaporated in vacuo to obtain the product as white solid (12.5 gm, 90%). 1H NMR (400 MHz, CDCI3) 6 4.09 (s, 4H), 3.78 - 3.49 (m, 8H). 13C NMR (101 MHz, CDCI3) 6 165.3, 45.9 42.8, 40.7.
Synthesis of 1 ,T-(butane-1 ,4-diyl)bis(3-(3-((3-bromopropoxy)methoxy)propyl)-1 H-imidazol-3- ium) (13c)
The title compound was synthesized following the method described in General Procedure 8 (described in Example 2) using 1d (1.00 g, 5.26 mmol) and 13b (7.63 g, 26.3 mmol) to afford 13c as a colorless gum (2.75 g, 68%) H NMR (400 MHz, D2O) 6 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). 13C NMR (101 MHz, D2O) 6 135.5, 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): m/z calculated (M-2Br-H)+ for C24H42Br4N4O4 = 610.4309; measured =609.0820. Synthesis of 1 ,1 '-(butane- 1 ,4-diyl)bis(3-(4-(2-chloroacetyl)piperazine-1-carbonyl)-1 H- imidazol-3-ium) (14c)
The title compound was synthesized following the method described in General Procedure 8 (described in Example 2) using 1d (1.00 g, 5.26 mmol) and 14b (6.28 g, 26.3 mmol) to afford 14c as a colorless gum (2.23 g, 68%). 1H NMR (400 MHz, D2O) 6 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). 13C NMR (101 MHz, D2O) 6 168.2, 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): m/z calculated (M-2CI-2H) + for C24H34CI4N8O4 = 597.5409; measured =595.2284.
Synthesis of 1,T-(butane-1.4-diyl)bis(3-(3-((3-(1-(4-(3-(4-(1-butyl-1 H-imidazol-3-ium-3- yl)butyl)-1 H-imidazol-3-ium-1-yl)butyl)-1 H-imidazol-3-ium-3-yl)propoxy)methoxy)propyl)-1H- imidazol-3-ium)octachloride (13, OIM1-8-Bu-Acetal)
To a stirred solution of the dibromo starting material 13c (1 gm, 1.29 mmol, 1.0 equiv.) in acetonitrile (10 ml) at rt was added the required starting material 1h (1.44 gm, 2.70 mmol, 2.1 equiv.) in a mixture of MeCN:DMF = 18 mL:2ml_. The reaction mixture was heated to 90 °C for 18 hours. The reaction mixture was allowed to cool to rt. The resulting gum was separated, further triturated three times with MeCN/EtOAc (8:2), the resulting precipitate/gum was isolated. The precipitate/gum was dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 for exchanging counterion to chloride. The product-containing eluent was transferred to a dialysis bag with Mw cut-off of 1000 Da and dialysed against 1 mL HCI in 1 L of deionised water over 1 hour, with a single dialysis water change at t = 0.5 hour. The resulting solution was concentrated under rotary evaporation and subjected to lyophilization to afford the desired product (1gm, 55%). 1H NMR (400 MHz, D2O) 6 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). 13C NMR (101 MHz, D2O) 6 135.5, 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): m/z calculated (M-8CI-8H) + for Ce6HnoCl8Ni604= 1183.6500; measured =1183.8577. GPC (Water Phase) Mn = 351 , Mw = 363, Mp = 358, PDI = 1 .03. (Fig. 6)
Synthesis of 3,3'-(((2,2'-(butane-1 ,4-diylbis(1 H-imidazole-3-ium-1 ,3- diyl))bis(acetyl))bis(piperazine-4,1-diyl))bis(2-oxoethane-2,1-diyl))bis(1-(4-(3-(4-(1-butyl-1 H- imidazol-3-ium-3-yl)butyl)-1 H-imidazol-3-ium-1-yl)butyl)-1 H-imidazol-3-ium) octachloride (14, OIM1-8-Bu-PzAc) To a stirred solution of the dibromo starting material 14c (1 gm, 1.49 mmol, 1.0 equiv.) in DMSO (10 ml) at rt was added the required starting material 1h (1.66 gm, 2.70 mmol, 2.1 equiv.) in a mixture of MeCN:DMSO = 19 ml_:1 mL. The reaction mixture was heated to 90 °C for 18 hours. The reaction mixture was allowed to cool to rt. The resulting gum was separated, further triturated three times with MeCN/EtOAc (8:2), the resulting precipitate/gum was isolated. The precipitate/gum was dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 for exchanging counterion to chloride. The product-containing eluent was transferred to a dialysis bag with Mw cut-off of 1000 Da and dialysed against 1 mL HCI in 1 L of deionised water over 2 hours, with a single dialysis water change at t = 1.0 hour. The resulting solution was concentrated under rotary evaporation and subjected to lyophilization to afford the desired product (1.39 gm, 60%).1H NMR (400 MHz, D2O) 6 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). 13C NMR (101 MHz, D2O) 6 165.63 (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 = 9.6 Hz), 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): m/z calculated (M-8CI-8H) + for CesHweC N^Ch = 1259.6680; measured =1259.8456. GPC (Water Phase) Mn = 409, Mw = 426, Mp = 437, PDI = 1.04. (Fig. 7)
Example 4. Synthesis of degradable OIM series (compounds 15-16)
Synthesis of 1-bromo-3-(tert-butoxy)propane (15a)
Anhydrous Mg(CIC>4)2 was heated under vacuum at (0.1 Torr) at 130 °C for 2 h prior to use to enhance its reactivity. The solution of 3-bromo-1 -propanol (10.0 g, 71.95 mmol, 1 equiv.) in CH2CI2 (90 mL) was taken in a three necked flask equipped with condenser and magnetic stirred bar containing Mg(CIC>4)2 (1.6 g, 7.20 mmol, 0.1 equiv.) and stirred till the solution became clear. Boc2O (32 g, 143.8 mmol, 2 equiv.) in CH2CI2 (14 mL) was added to the reaction mixture and bubbling was observed immediately. The reaction mixture was stirred for 48 h under reflux. The reaction mixture was diluted with water and extracted with CH2CI2. The organic layer was separated, dried over anhydrous Mg2SC>4 and concentrated under reduced pressure. Purification of the crude product by silica gel column chromatography eluting with 5% EtOAc in hexane yielded the corresponding ether 15a (9.0 g, 64%) as colorless oil. 1H NMR (400 MHz, CDCh) 0 = 3.54-3.47 (m, 4H), 2.06 (q, J = 8Hz, 2H), 1.21 (s, 9H). 13C NMR (101 MHz, CDCh) 6 = 72.73, 58.84, 33.57, 31.09, 27.53.
Synthesis of 1-(3-(tert-butoxy)propyl)-1 H-imidazole (15b) The title compound was synthesized following the method described in General Procedure 1 (described in Example 1) using 15a (9 g, 46.1 mmol, 1 equiv.) and imidazole 1a (2.8 g, 50.7 mmol, 1.1 equiv.) to afford the desired product yielded 12b (6.0 g, 71%) as yellow color oil. 1H NMR (400 MHz, CDCb) 5 = 7.46 (s, 1 H), 7.02 (s, 1 H), 6.90 (s, 1 H), 4.03 (t, J = 8Hz, 2H), 3.27 (t, J = 6Hz, 2H), 1.94 (t, J = 6Hz, 2H), 1.15 (s, 9H). 13C NMR (101 MHz, CDCb) 6 = 137.29, 129.17, 118.89, 72.83, 57.26, 43.81 , 31.71 , 27.47.
Synthesis of bis(3-bromopropyl) carbonate (15c)
To a stirred solution of 3-bromopropanol (15g, 107.92 mmol, 1 equiv.) in DCM (200 ml_) was added dropwise pyridine (12.80 g, 161.88 mmol, 1.5 equiv.) at 0 °C. The reaction mixture was stirred for 10 mins. After 10 mins, triphosgene (8 g, 26.98 mmol, 0.25 equiv.) in 40 ml DCM was added dropwise to the reaction mixture at 0 °C for 10 mins then the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ice water (100 mL), vigorously stirred for 10 min and transferred to a separating funnel. The organic layer was isolated, further washed with 2% HCI (100 ml) then washed with water (100 ml). The organic layer was dried over anhydrous Na2SC>4 and filtered. The resulting filtrate was concentrated under reduced pressure to afford the desired product as a colorless oil 15c (15 g, 47%). 1H NMR (400 MHz, CDCb) 6 4.23 (t, J = 6Hz. 4H), 3.44 (t, J = 6Hz, 4H), 2.17 (t, J = 6Hz, 4H). 13C NMR (101 MHz, CDCb) 6 154.75, 65.62, 31.61 , 29.11.
Synthesis of 1 ,T-(butane-1 ,4-diyl)bis(3-(3-(((3-bromopropoxy)carbonyl)oxy)propyl)-1 H- imidazol-3-ium) bromide (15d)
The title compound was synthesized following the method described in General Procedure 8 (described in Example 2) using 1d (1.00 g, 5.25 mmol, 1 equiv.) and 15c (8.00 g, 26.28 mmol, 5 equiv.) to afford the desired product light yellow gum 15d (1.5 g, 36%). 1H NMR (400 MHz, D2O) 5 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). 13C NMR (101 MHz, D2O) 6 155.39, 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 Mode; m/z calculated [M + 3Br]' for Cz^sBrs^C = 878.10; measured = 877.23.
Synthesis of 3-(3-(((3-bromopropoxy)carbonyl)oxy)propyl)-1-(3-(tert-butoxy)propyl)-1 H- imidazol-3-ium bromide (15e)
The title compound was synthesized following the method described in General Procedure 2 (described in Example 1) using 15c (4.2 g, 13.7 mmol, 2.5 equiv.) at 85 °C was added dropwise into compound 15b (1g, 5.50 mmol, 1 eq) to afford a colorless gum 15e (2 g, 75%). 1H NMR (400 MHz, DMSO-de) 5 9.38 (s, 1 H), 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). 13C NMR (101 MHz, DMSO-d6) 6 154.67, 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 Mode; m/z calculated [M + 2Br]‘ for CiyHsoBrzNzCU = 566.14; measured = 565.42.
1-(4-(1 H-imidazol-1-yl)butyl)-3-(3-(((3-(1-(3-(tert-butoxy)propyl)-1 H-imidazol-3-ium-3- yl)propoxy)carbonyl)oxy)propyl)-1 H-imidazol-3-ium bromide (15f)
The title compound was synthesized following the method described in General Procedure 4 (described in Example 1) using 1d (2 g, 10.30 mmol, 2.5 equiv.) at 90 °C was added dropwise into compound 15e (2g, 4.10 mmol, 1 equiv.) to afford a yellow gum 15f (2.5 g, 90%). 1H NMR (400 MHz, D2O) 5 8.85 (s, 3H, Due to H exchange to Deuterium integration is less), 7.68-7.45 (m, 6H), 7.12 (s, 1 H), 6.97 (s, 1 H), 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). 13C NMR (101 MHz, D2O) 3 155.24, 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 Mode; m/z calculated [M + 3Br]' for C2?H44Br3NsO4 = 756.39; measured = 757.38.
Synthesis of 3-(3-(((3-bromopropoxy)carbonyl)oxy)propyl)-1-(4-(3-(3-(((3-(1-(3-(tert- butoxy)propyl)-1 H-imidazol-3-ium-3-yl)propoxy)carbonyl)oxy)propyl)-1 H-imidazol-3-ium-1- yl)butyl)-1 H-imidazol-3-ium bromide (15q)
To a stirred liquid of the desired dibromo-alkylating agent 15c (5.6 g, 18.50 mmol, 5 equiv.) at 90 °C was added dropwise into compound 15f (2.5 g, 3.70 mmol, equiv.) in dry MeCN (10 ml). A few drops of DMF were added, if required to ensure reaction mixture appeared as a clear solution. The reaction mixture was stirred at 90 °C for 3 hours. The reaction mixture was cooled to room temperature, gummy precipitate formed decant the solvent and washed several times with ethyl acetate then the gummy product was dissolved into de-ionized water then wash with several times ethyl acetate to remove the excess dibromo alkylating reagent by separating funnel then concentrate the deionized water under reduced pressure to afford a brown gum 15g (3.5 g, 97%). 1H NMR (400 MHz, D2O) 0 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). 13C NMR (101 MHz, DMSO-da) 6 155.37, 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 Mode; m/z calculated [M + 4Br]_ for C34H56Br5NeO7 = 1060.36; measured = 1061.19. 1-(4-(1 H-imidazol-1-yl)butyl)-3-(3-(((3-(1-(4-(3-(3-(((3-(1-(3-(tert-butoxy)propyl)-1 H-imidazol- 3-ium-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-ium-1-yl)butyl)-1 H-imidazol-3-ium-3- yl)propoxy)carbonyl)oxy)propyl)-1 H-imidazol-3-ium bromide (15h)
To a stirred solution of the desired bis-imidazole compound 1d (3.40 g, 17.80 mmol, 5 equiv.) in MeCN (10 ml_) was added dropwise the desired bromo-imidazolium analogue 15g (3.5 g, 3.60 mmol, 1 equiv.) in dry MeCN (5 mL) at 95 °C. A few drops of DMF were added, if required to ensure reaction mixture appeared as a clear solution. The reaction mixture was stirred at 95 °C for 3 hours. The reaction mixture was concentrated under rotary evaporation and 3:1 chloroform:isopropyl alcohol was added to the reaction mixture followed by de-ionised water (20 mL). The aqueous layer was washed with 3:1 chloroform to isopropyl alcohol (12 x 20 mL) and concentrated rotary evaporation to afford a yellow gum 15h (4 g, 96%). 1H NMR (400 MHz, D2O) 6 7.66 (s, 1 H, Due to H exchange to Deuterium integration is less), 7.53-7.48 (m, 8H), 7.13 (s, 1 H), 6.95 (s, 1 H), 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). 13C NMR (101 MHz, D2O) 6 156.14, 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 Mode; m/z calculated [M + 5Br]_ for C44H7oBr5Nio07 = 1250.63; measured = 1251.24.
1,T-(butane-1 ,4-diyl)bis(3-(3-(((3-(1-(4-(3-(3-(((3-(1-(4-(3-(3-(((3-(1-(3-hydroxypropyl)-1 H- imidazol-3-ium-3-yl)propoxy)carbonyl)oxy)propyl)-1 H-imidazol-3-ium-1-yl)butyl)-1 H-imidazol- 3-ium-3-yl)propoxy)carbonyl)oxy)propyl)-1H-imidazol-3-ium-1-yl)butyl)-1 H-imidazol-3-ium-3- yl)propoxy)carbonyl)oxy)propyl)-1 H-imidazol-3-ium)chloride (15)
To a stirred gummy pentamer intermediate 15h (1.2 g, 10.00 mmol, 2.01 equiv.) and dibromo bis-imidazole 15d (400 mg, 5.00 mmol, 1 equiv.) in neat condition at 99°C then the reaction mixture was stirred for 2 hours. The reaction mixture was allowed to cool to rt, the resulting gum was further washed with MeCN (3x10 ml) followed by EtOAc (2x10 ml). The tBu- group was deprotected using 50% TFA in DCM for 12 hours then the resulting gum was further washed with EtOAc followed by MeCN. The gummy product was dissolved in deionized water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 for exchanging counterion to chloride. The productcontaining eluent was transferred to a dialysis bag with an Mw cut-off of 1000 Da and dialyzed against 1 mL HCI in 1 L of deionized water over 1 hour, with a single dialysis water change at t = 0.5 hour. The product-containing de-ionized water was concentrated by rotary evaporation then the gummy product was dissolved in 5 ml of deionized water and passed through the Sephadex-g 25 silica purification to collect the fraction was monitored by GPC and ESI-MS and the resulting final product was subjected to lyophilization to afford a light-yellow hygroscopic solid 15 (200 mg, 10%). 1H NMR (400 MHz, D2O) 5 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). 13C NMR (101 MHz, D2O) 3 155.29, 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 Mode; m/z calculated [M + 13CI]’ for C104H162CI13N24OM = 2529.44; measured = 2525.02; GPC (Water Phase) Mn = 1063, Mw = 1103, Mp = 1199, PDI = 1.04.
Synthesis of /V,/V'-(propane-1 ,3-diyl)bis(2-chloroacetamide) (16a)
100 ml_ of saturated K2CO3 (23.3 g, 168.84 mmol) was added to a solution of 1 ,3-diamino propane (5.00 g, 67.45 mmol, 1.0 equiv.) in CHCI3 (200 ml_). The reaction mixture was stirred and cooled to 0 °C in an ice bath. 2-Chloroacetyl chloride (19. 0 g, 168.6 mmol, 2.5 equiv.)) in CHCI3 was added dropwise over a period of 1 hour. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The organic phase was washed with HCI (2 x 100 mL, 1 M) and water (2 x 100 ml_). The CHCh layer was dried and evaporated in vacuo to obtain the product as a white solid (9.92 g, 65%). 1H NMR (400 MHz, CDCI3) 6 7.18 (s, 2H), 4.09 (s, 4H), 3.39 (dd, J = 12.4, 6.3 Hz, 4H), 1.85 - 1.68 (m, 2H). 13C NMR (101 MHz, CDCh) 6 166.8, 42.6, 36.4, 29.4.
Synthesis of 1,T-(butane-1 ,4-diyl)bis(3-(2-((3-(2-chloroacetamido)propyl)amino)-2-oxoethyl)- 1H-imidazol-3-ium) (16b)
The title compound was synthesized following the method described in General Procedure 8 (described in Example 2) using 1d (1.00 g, 5.26 mmol, 1.0 equiv.) and 16a (5.97g, 26.3 mmol, 5.0 equiv.) to afford 16b as a colorless gum (1.86 g, 55%). 1H NMR (400 MHz, D2O) 6 8.76 (d, J = 52.5 Hz, 2H, Due to H exchange to Deuterium integration is less), 7.46 (ddt, J = 24.0, 9.5, 1.8 Hz, 4H), 4.98 (d, J = 15.2 Hz, 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). 13C NMR (101 MHz, D2O) 6 169.6, 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): m/z calculated (M-2CI-2H) + for C24H38Cl2N8O4 = 571.5040; measured =571.2546.
Synthesis of mono(3,3'-(((((2,2'-(butane-1 ,4-diylbis(1H-imidazole-3-ium-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-ammoniopropyl)-1 H-imidazol-3-ium-3-yl)butyl)-1 H-imidazol-3-ium-3- yl)butyl)-1 H-imidazol-3-ium)) decachloride (16)
To a stirred solution of the dichloro starting material 16b (1 g, 1.55 mmol, 1.0 equiv.) in methanol (5 ml) at rt was added to the starting material 1e (2.17 g, 3.22 mmol, 2.1 equiv.) in a mixture of MeCN: DMF = 18 mL:2mL at 80 °C. The resulting reaction mixture was heated to 95 °C for 18 hours. The reaction mixture was allowed to cool to rt (confirmed the reaction completion by crude 1H NMR). The resulting gum was separated, and further triturated three times with MeCN/EtOAc (8:2), and the resulting gum was isolated and treated with a solution of HBr in acetic acid (33%) (10 equiv.) and the resulting mixture was stirred at rt for 2 hours. The reaction mixture was treated with excess EtOAc and the resulting gum was isolated and dissolved in water to give a final concentration of 50-60 mM and was passed through a glass column containing ion-exchange resin Amberlyst® A-26 to exchange counterion to chloride. The product-containing eluent was transferred to a dialysis bag with an Mw cut-off of 1000 Da and dialyzed against 1 ml_ HCI in 1 L of deionized water over 1 hour, with a single dialysis water change at t = 0.5 hour. The resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product 16 white hygroscopic solid (1.8 g, 55%). 1H NMR (400 MHz, D2O) 3 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). 13C NMR (101 MHz, D2O) 6 166.9, 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): m/z calculated (M-10CI-8H) + for CesHioeCIsNaC = 1239.6369; measured =1239.5843. GPC (Water Phase) Mn = 1322, Mw = 1330, Mp = 1347, PDI = 1.006. (Fig. 8)
Example 5. Antibacterial properties of the precision main chain imidazolium oligomers
Bacterial culture
All bacterial strains were purchased from American Type Culture Collection (ATCC). Pseudomonas aeruginosa PAO1, Enterococcus faecalis VRE583, E. coli 958 were obtained from Singapore Center for Environmental and Life Sciences (SCELSE). Clinical isolates, PAER (multi-drug resistant P. aeruginosa), ACBAS (PAN-sensitive A. baumannii), AB-1 (multi-drug resistant A. baumannii), KPNS (PAN-sensitive K. pneumonia), KPNR (carbapenem resistant K. pneumoniae), ECOS (PAN-sensitive E. coli), ECOR (multi-drug resistant E. coli), ECLOS (PAN-sensitive E. cloacae), CRE (carbapenem resistant E. cloacae) were obtained from Tan Tock Seng Hospital (TTSH) Singapore. K. pneumoniae SGH10, K. pneumoniae BAK085, K. pneumoniae M7, K. pneumoniae SGH4, A. baumannii X26, A. baumannii X39, A. baumannii X40 were provided by National University of Singapore. MRSA LAC, LAC*, LAC AmenD and LAC AhemB have been described previously (Pader, V. et al., Infect. Immun. 2014, 82, 4337-4347) and were provided by Angelika Grundling, Imperial College London. B. thailandensis 700388 was provided by Samuel I. Miller, University of Washington. All broth or agar media used here were purchased from Becton Dickinson Company. The bacteria strains were stored in 15% glycerol at -80 °C. Minimum inhibitory concentration (MIC)
MICs were determined according to standard broth microdilution method with slight modification (Wiegand, I. et al., Nat. Protoc. 2008, 3, 163-175). Briefly, a single colony was picked and inoculated to obtain overnight culture. A subculture was prepared the next day and grown to exponential phase. A two-fold serial dilution of test compound in MHB broth was prepared in a 96-well plate, followed by addition of exponential phase bacteria at concentration of 5X 105 CFU/mL. The solution in the plate was thoroughly mixed by shaking the plates vigorously for 20 seconds, and the plate was subsequently incubated statically at 37 °C for 18 hours. ODeoo readings were taken and the minimum concentration that inhibits bacterial growth by 90% (MICso) was calculated. For MIC of E. faecalis 583 (VRE2), overnight culture and subculture was prepared as described above but in tryptic soy broth (TSB) broth and the MIC was tested without and with supplementation with 8 pg/ml_ hemin.
Cell cytotoxicity MTT assay
In vitro biocompatibility was investigated using mouse fibroblast cells (3T3), human embryonic kidney (HEK293) cells, human hepatocellular carcinoma (HepG2) cells, and adenocarcinomic human alveolar basal epithelial cells (A549). Briefly, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) was used to culture 3T3 and HepG2 cells. To culture HEK and A549 cells, 15% FBS supplementation was used. All cells were incubated at 37 °C in a CO2 incubator. When 80% confluence was observed under microscopy, cells were treated with trypsin, concentrated and counted using a haemocytometer. 1 *104 cells/well were then seeded into a 96-well plate, cells were grown for 24 hours and subsequently challenged for 24 hours with varied concentrations of compounds. Cell health condition was then qualitatively checked by microscopy and quantified using an MTT (3-[4, 5-dimethylthiazoyl-2-yl]-2, 5- diphenyl tetrazolium bromide) assay. The cell viability was calculated by comparing the absorbance of formazan formed by the cells in the challenge wells and in untreated wells. The compound concentration which caused 50% reduction of cell growth compared to untreated control (IC50), was determined. The data are representative of three independent experiments.
Time-kill assay
The killing kinetics of OIMs were determined via time-kill assay. Exponential phase bacteria (prepared via the MIC protocol above) were diluted to 5x105 CFU/mL and treated with varying concentrations of OIMs with constant shaking at 37 °C. Aliquots of bacterial samples were taken at specific time intervals (30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 24 hours) and serially diluted with phosphate buffered saline (PBS). The serially diluted samples were then spotted on agar plate and the number of colonies was counted after overnight incubation.
Computer simulation for calculation of Hirshfeld charges on the imidazolium rings of OIMs The computational study of the five OIM compounds (1-3, 5-6) was performed using Gaussian 16 software (Gaussian 16 Rev. C.01 (Wallingford, CT, 2016)). Models of the oligomers were built using GaussView 6.0 and subsequently subjected to geometry optimization and frequency analysis at the B3LYP/def2-TZVP/SMD (solvent = water) level of theory (Stephens, P. J. et a!., J. Phys. Chem. 1994, 98, 11623-11627; Weigend, F. & Ahlrichs, R„ Phys. Chem. Chem. Phys. 2005, 7, 3297-3305; Papajak, E. etal., J. Chem. Theory Comput. 2011 , 7, 3027- 3034; and Marenich, A. V. et al., J. Phys. Chem. B 2009, 113, 6378-6396), with Grimme's empirical dispersion correction em=gd3(bj) applied (Grimme, S. et al., Chem. Rev. 2016, 116, 5105-5154) (Fig. 22). Natural Bond Orbital (NBO) (Weinhold, F. & Landis, C. R., Chem. Educ. Res. Pract. 2001 , 2, 91-104) and Mulliken charges were calculated for all atoms in each polymer. Additionally, the Hirshfeld charges (Hirshfeld, F. L., Theor. Chim. Acta 1977, 44, 129- 138) were derived from the DFT calculated wavefunction (converted from Gaussian chk to fchk using the formchk utility) using Multiwfn software (Lu, T. & Chen, F. Multiwfn, J. Comput. Chem. 2012, 33, 580-592).
Bacterial cytoplasmic membrane depolarization and membrane integrity assay
The membrane potential-sensitive dye 3,3'-Dipropylthiadicarbocyanine Iodide (DiSCs(5)) was used to determine the membrane depolarization activities of OIMs. The assay was performed following a previously described protocol with minor modification (Belley, A. et al., Antimicrob. Agents Chemother. 2009, 53, 918-925; and Te Winkel, J. D. etal., Front. Cell Dev. Biol. 2016, 4, 29). Exponential phase MRSA LAC was pelleted and washed twice with 5mM HEPES plus 5mM glucose and resuspended to 4x107 CFU/mL. 0.5 pM of DiSCs(5) was added to the bacteria and 175 pL of bacteria was aliquoted into a white 96 well plate. The fluorescence was monitored using a Spark 10M plate reader (Tecan, Switzerland) at Ex/Em of 622nm/670nm every 2 minutes with continuous shaking. The DiSCs(5) dye is absorbed into the bacterial membrane which results in the quenching of the fluorescence signal. Once a stable fluorescence reading was obtained, OIMs were added at varying concentrations in total volume of 25 pL, and the fluorescence reading was immediately recorded for 1 hour. Gramicidin was used as a positive control. For membrane integrity assay, LAC was prepared as described above and 1 pM propidium iodide (PI) dye was added to the bacteria. The PI signal was monitored before the addition of OIMs and nisin (positive control) for baseline reading and subsequently recorded for 1 hour after the addition of test compounds at Ex/Em of 535nm/620nm using Tecan plate reader. Hydrogen-deuterium exchange of OIMs measured by 1H NMR
PBS buffers at desired pHs were prepared by adjusting the ratio of K2DPO4 (0.1 M stock concentration) and KD2PO4 (0.1 M stock concentration) according to previous study (Zhang, N. et al., Advanced Science (Weinheim, Baden- Wurttemberg, Germany) 2021 , 8, e2100805). All reactions were carried out in D2O at ionic strength = 1.0 (KCI). Briefly, OIMs were dissolved in PBS at 10 mM (concentration of oligomer repeat units) with 10 mM TMA (tetramethylammonium hydroxide) as internal standard. Deuterium exchange was measured by 1H NMR spectroscopy (Amyes, T. L. et al., J. Am. Chem. Soc. 2004, 126, 4366-4374) with a Bruker 400 MHz NMR at 25 °C.
NHC capture by carbazole probe
(i) Carbazole(lipids) preparation
Stock solutions of phosphatidylglycerol, (PG; 10 mg/mL, 1.3 x 105 mol/mL), phosphatidylcholines (PC; 10 mg/mL, 1.3 x 1Q-5 mol/mL) and carbazole (2.2 mg/mL, 1.3 x 10- 5 mol/mL) were prepared in chloroform. A mixture of PC/PG/carbazole at 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 solutions in a round-bottom flask (25 mL). The chloroform was removed by rotary evaporation at 50 mbar, 20 °C for 20 min. The lipid film was hydrated with 1 mL of PBS buffer (pH 7.4). The suspension was then vortexed at top speed for 1.5 min, followed by 2 min of sonication in ice bath. This vortex-sonication process was performed three times, and the solution was then extruded 19 times through a 200 nm polycarbonate membrane with an Avanti mini-extruder. The resulting liposome solution was dialyzed against water with MWCO 2000 tube for 36 hours to remove free carbazole. The size distribution and zeta-potential of the resulting liposomes were checked with a Malvern Nano Series Nano-ZS instrument (data not shown). All prepared liposome solutions were stored at 4 °C prior to use.
(ii) Fluorescence measurement of carbazole(lipids)-OIM. OIM stock solutions (1 , 2 and 5) were prepared in H2O at 10 mg/mL. The solution was adjusted to pH 7.4 with 1 M NaOH. Stock solution of IPr (50 mg/mL in DMF) was prepared. Carbazole(lipids)-OIM was prepared by mixing 10 pL of carbazole(lipids), 100 pL of OIM stock, and 890 pL of PBS (pH 7.4). Carbazole(lipids)-IPr, used as a positive control, was prepared by mixing 10 pL of carbazole(lipids), 10 pL of IPr stock, and 980 pL of PBS (pH 7.4). All the mixtures were incubated at 37 °C for 36 hours prior to fluorescence measurement. Emission fluorescence spectra were obtained at an excitation wavelength of 295 nm.
NHC capture by AuCI(SMe2) probe (i) Au(lipids) preparation
Au(lipids) was prepared by the same protocol used for carbazole(lipids), replacing carbazole solution with the AuCI(SMe2) stock solution (3.82 mg/mL, 1.3 x 105 mol/mL), with PC/PG/AuCI(SMe2) molar ratio (8: 2: 1). After vortex-sonication, no extrusion was performed because of the sensitivity of AuCI(SMe2). Hydrophobic AuCI(SMe2) that was not trapped within liposome bilayer was removed by centrifugation at 4000 g for 20 min at 10 °C. The resulting pellets were washed with water once. It was very important to remove untrapped AuCI(SMe2) immediately after Au(lipids) was prepared, since free AuCI(SMe2) molecules would react with glycerol groups of PG to form Au nanoparticles, leading to a purple solution that was not suitable for further study.
(ii) Formation of Au-OIM in liposome
Au(lipids) pellets obtained after washing were dispersed in 10 ml_ of 0.1 x phosphate buffer (pH 7.4), followed by adding OIM stock solution at final concentration of 75 pg/mL. The mixture was incubated at 37 °C in the dark for 48 hours. Then the mixture was freeze dried. The dried powder was dissolved in 1 ml_ of methanol. The addition of methanol would break the liposome structure, thus releasing Au-OIM into the solution. The solution was centrifuged at 8,000 g for 30 min 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 AuCI(SMe2) before LC-MS measurement.
(iii) LC-MS characterization
Mass spectra were recorded by an Agilent Q-TOF/MS (6550 iFunnel) machine. Electrospray ionization (ESI) mass spectroscopy (MS) positive ion mode was used. C18 column (50 mm x 2.1 mm i.d., 1.8 pm, Agilent) was used at 35 °C for all the analyses. The mobile phase consisted of a linear gradient system of (A) water (0.1 % formic acid) and (B) acetonitrile (0.1 % formic acid). The gradient conditions of the mobile phase 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 pL.
(iv) Structural confirmation of Au-(6) with deuterium-hydrogen exchange
The sample prepared as stated above ((iii) LC-MS characterization) was added a drop of D2O to replace the active hydrogen with deuterium, and then analyzed by LC-MS in the same conditions.
(v) Structural confirmation of Au-(6) with MS-MS The target ion m/z 330.52 (Au-OIM-(6)) in the previous sample prepared as stated above ((iii) LC-MS characterization) was targeted with collision energy CID @10 by LC-MS to get the fragments from the target ion.
OIM uptake in liposomes
(i) Uptake assay in liposome membrane and core
Liposomes were prepared by the same protocol used for carbazole(lipids) with the exception that no carbazole was added. Liposome stock (10 mg/mL), OIM stock (10 mg/mL), and water were mixed to make 1 mL solution with final liposome and OIM concentration at 100 ppm and 5000 ppm, respectively. After incubation at 37 °C for 20 hours, free OIM was removed by Tangential Flow Filtration (TFF) (MidiKros® Hollow Fiber Module, MWCO 3kDa) with syringes through 30 cycles of extrusion with 0.2* concentration of PBS. The resulting solution (around 1 mL) was split into two equal portions. One portion was freeze-dried to measure total uptake of OIM in the membrane and the core. The freeze-dried 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 top fraction (water/methanol phase) was collected for LC-MS measurement. The other portion (around 0.5 mL) was added into 0.17 mL ethanol (final ethanol concentration 25% v/v) and incubated at room temperature for 2 hours to damage the membrane’s integrity, thus releasing OIM in the core. OIM released from the core and OIM trapped in the bilayer were separated by Vivaspin (MWCO 30 kDa) with centrifugation at 4000 g at 25 °C for 30 min, as OIM released from core would pass through the Vivaspin membrane while OIM trapped in the liposome bilayer would be retained at the membrane due to the size of the liposomes.
(ii) Calibration curves of OIMs measured by LC-MS
To obtain an accurate calibration curve, we prepared lipid matrix with the same procedures used in the uptake experiments. Specifically, 200pL of liposome stock, containing 0.4 mg PG and 1.6 mg PC, was freeze-dried. After that, 16 mL methanol was added to dissolve the liposomes and then 14.4 mL DI water, 16 mL chloroform and 46.4 pL formic acid (final concentration 0.1 % v/v) were added to remove lipids to avoid any damage to the C18 column. After centrifugation at 3000 rpm for 10 min, the supernatant (top phase) was collected as the lipid matrix. OIM1-6-CH (1), OIM1-6-C2(CH3) (2) and OIM1-6-C4(CH3) (5) were prepared with the lipid matrix at 0, 1 , 2, 3 and 4 ppm to build calibration curves. These standard samples were then tested with an Agilent Q-TOF/MS (6550 iFunnel) machine. Results are shown in Fig. 9. Molecular dynamics simulation of the interaction of cationic versus NHC forms of OIM1-6-CH with model S. aureus membrane
(i) Membrane construction
A 286-lipid membrane bilayer representative of the S. aureus membrane composition, comprising 58% phosphatidylglycerol (PG) and 42% Cardiolipin (CL) (Epand, R. M. & Epand, R. F., Biochim. Biophys. Acta 2009, 1788, 289-294), was constructed using the CHARMM- GUI (Jo, S. et al., J. Comput. Chem. 2008, 29, 1859-1865) membrane builder tool. DMPG (14:0/14:0) and TMCL2 (14:0,14:0/14:0,14:0) forms of PG and CL were used, respectively. The distribution of PG and CL lipids on the upper and lower leaflets was symmetrically constructed. Menaquinone-8 (MQ8) molecules were constructed on the membrane model. Parameters of membrane lipids were based on the CHARMM36 force field and parameters of the MQs and OIM1-6-CH (1) were based on the CHARMM General Force Field (Vanommeslaeghe, K. & MacKerell, A. D., J. Chem. Inf. Model. 2012, 52, 3144-3154; and Vanommeslaeghe, K. et al., J. Chem. Inf. Model. 2012, 52, 3155-3168). Topologies of MQ8 molecules were generated using the CHARMM-GUI ligand reader and modeler (Kim, S. etal., J. Comput. Chem. 2017, 38, 1879-1886) tools, with adopted ratio of total lipid: total MQ8 = 10:1. An equal number of MQ molecules were manually inserted randomly into the upper and lower leaflet of the constructed S. aureus membrane. Manual adjustments of coordinates and energy minimization steps were subsequently performed to remove structural clashes between atoms. The membranes were solvated with TIP3P (Jorgensen, W. L. et al., J. Chem. Phys. 1983, 79, 926-935) water molecules and counterions were added to neutralize the system. The constructed S. aureus membranes were subjected to a 100 ns molecular dynamics (MD) simulation using GROMACS (Van Der Spoel, D. etal., J. Comput. Chem. 2005, 26, 1701-1718) 5.1.2 software. The LINCS (Hess, B., J. Chem. Theory Comput. 2008, 4, 116- 122) algorithm was used to constrain bonds between heavy atoms and hydrogen to enable a timestep of 2 fs. A 1.2 nm cutoff was used for Van der Waals interaction and short-range electrostatic interactions calculations, and Particle Mesh Ewald method was implemented for long range electrostatic calculations. Simulation temperature was maintained at 310 K using a V-rescale thermostat (Bussi, G. etal., J. Chem. Phys. 2007, 126, 014101) and 1 bar pressure using Parrinello-Rahman (Parrinello, M. & Rahman, A., J. Appl. Phys. 1981 , 52, 7182-7190) barostat.
(ii) Simulation of OIM1-6-CH (1) on S. aureus membranes
Coordinates of the cationic and NHC forms of the OIM1-6-CH (1) were constructed using Discovery Studio 4.1 (Zhuo, S. et al., Molecules 2020, 25, 5649). Topologies of (1) were obtained from the CHARMM-GUI ligand reader and modeler (Kim, S. etal., J. Comput. Chem. 2017, 38, 1879-1886). Partial charges of the NHC carbene forms were calculated from Gaussian09 (Gaussian 09 (Gaussian, Inc, Wallingford, CT, USA, 2009)) in the triplet state at the level of HF 6-31G* and RESP (Bayly, C. I. et al., J. Phys. Chem. 1993, 97, 10269-10280) fitting. The last simulation frame from the previous 100 ns molecular dynamics simulation run of S. aureus membranes was used for the simulation of the OIM on the membrane. Eight simulation systems were set up of the cationic OIM or OIM-NHC on S. aureus membrane. A single oligomer molecule was placed 0.8nm above the center of the upper leaflet of the membrane bilayer. Each system was subjected to classical molecular dynamics simulations for 200 ns. MD simulations were performed with settings similar to those described above.
(iii) Analysis of simulation
The number of contacts between the OIM and the membrane as a function of simulation time was calculated to analyze the behavior of interactions between the oligomer and membrane. Contact number calculations were performed using the GROMACS (Van Der Spoel, D. et al., J. Comput. Chem. 2005, 26, 1701-1718) gmx mindist tool. A contact was defined if the minimum distance between the OIM atom and membrane atom was less than 0.4 nm. To further visualize the binding between OIM and the membrane, the last frame of each simulation system was visualized using pymol.
Results and discussion
The OIM 1-6 series (1-9) were tested against a range of Gram-positive and Gram-negative bacteria to determine their antibacterial potency (Table 1). OIM1-6-CH (1) with a hydrogen at the C2-carbon (denoted as C2-H) had good potency as indicated by a low minimal inhibitory concentration (MICao) value against a broad spectrum of bacteria, including the Gram-positive pathogens Staphylococcus aureus and Enterococcus faecalis and the Gram-negative bacteria Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli, but was less potent (MICao = 16-32 pg/mL) against Enterobacter cloacae. Notably, OIM1- 6-CH (1), characterized by its carbon acidity with the presence of a C(2)-H, exhibited substantial potency with a low geometric mean of minimum inhibitory concentration (Geo- MlCao) of 4.0 pg/mL against a panel of ESKAPE pathogens that includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. Table 1. Antibacterial and cytotoxicity of OIM series compounds (MICso, pg/mL).
Ml Cao (pg/mL)
S. aureus E. faecium K. pneumoniae A. baumannii P. aeruginosa E. coli E. cloacae
No.
Oligomer ATCC 29213 ATCC 19434 ATCC 13883 ATCC 19606 PAO1 MG 1655 ATCC 13047
(+) (+) (-) (-) (-) (-) (-)
1 OIM1-6-CH 1-2 2 4 4 2-4 4 16-32
2 OIM1-6-C2(CH3) 256 >512 256 128 128 512 >512
3 OIM1-6-C2(CI) 16 >512 32 32 256 64-128 256-512
4 OIM1-6-C2(CF3) 32 >512 64 64 128 128 256
5 OIM1-6-C4(CH3) 2-4 8 16 16 8-16 8 64
6 OIM1-6-C4(CI) 2 4 4 8 4 4 8
7 OIM1-6-C4(F) 2 32 16 4 32 32 64 ®
8 OIM-1-6-(BZ) 8 64 8 64 64 8 32
9 OIM1-6-C4(CF3) 128 >512 512 256 512 256 >512
10 OIM1-8-CH 2 8 4 2-4 2 4 4
11 OIM1-8-C2 (CH3) 8 >512 32 16 32 32 64
12 OIM1-8-C4(CH3) 2 4-8 4 2-4 2 4 4
13 OIM1-8-Bu-Acetal 4 64 1 4 4 4 2
14 OIM1-8-Bu-PzAc 2 8-16 2 4 2 2 2
15 OIM1-12-6C-OH 4 4 1 4 2 4 4
16 OIM1-8-2D 2 4 2 4 16 8 8
17 Gentamicin <1 64 <1 8 <1 <1 <1
18 Colistin 128 >512 8 2 2 2 512
19 Ciprofloxacin 1-2 4 <1 <1 <1 <1 <1
Table 2. Antibacterial and cytotoxicity properties of OIM compounds.
Figure imgf000068_0001
Figure imgf000069_0001
a Geometric mean of half-maximal inhibitory concentration (IC50) is calculated from the IC50 of 3 cell lines: 3T3 fibroblast cells, human embryonic kidney (HEK) cells and liver hepatocellular carcinoma (HepG2) cells. The complete IC50 data are shown in Table 3, and the raw cytotoxicity data are shown in Figs. 10-16. b Selectivity index (SI) is the ratio of Geo-ICso of a specific timepoint to Geo-MICso.
Table 3. Half-maximal inhibitory concentration (IC50, pg/mL) of OIM compounds.
Figure imgf000069_0002
Figure imgf000070_0001
Interestingly, different ring substitutions led to different antibacterial potencies. OIM1-6 derivatives with substitution at the C2-carbon of the imidazolium moiety were also synthesized to study the structure-activity relationship (SAR) surrounding the imidazolium moiety. Derivatives (2-4) (Fig. 1A) with C2-hydrogen replaced by various substituents, i.e., weakly electron-donating methyl- (-CH3), and electron-withdrawing chloro- (-CI) and trifluoro- (-CF3) groups, were made. The C2-substituted derivatives (2-4) resulted in a significant loss in bacterial inhibition potency compared to 0IM1-6-CH (1). Amongst the 3 C2-derivatives, compounds (3, 4) containing highly electron-withdrawing C2-CF3/-CI substituents retain higher potency compared with (2). We hypothesized that compounds (3,4) with electron-withdrawing C2-CF3/-CI groups would increase the cationic charge density at the C2-carbon. We also made similar derivatives but this time with substituents at the C4-position (5-9). Surprisingly, most of the C4-substituted derivatives, except (9) with -CF3 substituent, retained potency compared with (1). If the effect of the substituents were purely due to cationic charge density around the C2-carbon, then the C4 derivatives (6-9) with electron withdrawing groups should have much better potency than the C4-CH3 (5) derivative with the electron donating methyl group, just like 3, 4 are more potent than 2 but (6-9) were not obviously more potent than (5). We would also have expected the C4-CF3 derivative (9) that has the electron-withdrawing -CF3 to make 02- carbon more cationic to be more potent than the C4-CH3 derivative (5), just like the C2-CF3 derivative (4) is more potent than C2-CH3 derivative (2). However, the trend of (5) versus (9) was reverse to that of (2) versus (4). It appears that the density of the cationic charge at the imidazolium ring alone cannot explain the observed MICs. We hypothesized that 04- substituted compounds with an acidic hydrogen at the C2-carbon can dissociate at physiological pH to form N-heterocyclic carbenes (NHCs) which is hydrophobic to aid membrane entry and bacteria killing is affected intracellularly.
The derivatives (2-4), devoid of carbon acid characteristic as their C(2)-H atoms were replaced by various substituents that encompass both weakly electron-donating (methyl- (-CH3)), and electron-withdrawing (chloro- (-CI) and trifluoro- (-CF3)) groups, exhibited markedly diminished potency when compared to the parent OIM1-6-CH (1). Specifically, the three non-carbon acid compounds (2-4) possessing C2-substituted groups displayed Geo-MIC90 values ranging from 118.5 pg/mL to 344.6 pg/mL, which were significantly higher than the Geo-MIC90 value of 4.0 pg/mL observed for compound (1). In parallel, we synthesized analogous derivatives (5-8) with substituents introduced at the C4-position instead, rendering them carbon acidity with the retention of C(2)-H atoms. Strikingly, the C4-substituted derivatives (5-8) maintained their potent antibacterial activity, boasting Geo-MICao of 4.4 to 23.8 pg/mL. These values were notably lower than those recorded for their isomers (2-4), underscoring the pivotal role of the carbon acid characteristic in preserving antibacterial potency. To investigate the influence of chain length on OIM potency, we synthesized oligomers containing eight repeat units with -CH3 substituents (10, 11, and 12) (Figs. 1 B and 17). Interestingly, OIM1-8-C2(CH3) without the C2-H (11) also had reduced potency compared with the parent OIM1-8-CH (10) which was similarly observed in the OIM1-6 series. The parent OIM1-8-CH (10) characterized by the presence of a C(2)-H functionality has Geo-MICgo of 3.4 pg/mL, which was one order of magnitude more potent than OIM1-8-C2(CH3) (11), which is not a carbon acid since it lacks the C(2)-H atom (Geo-MICgo = 43.1 pg/mL). Furthermore, OIM1-8-C4(CH3) (12), possessing a C(2)-H, displayed potency identical to (10), with a Geo- MlCgo of 3.4 pg/mL.
We had also developed degradable OIM series using acetal, and amide linkages respectively (13 - 14) (Figs. 1C, 6-7 and 18) that retains good potency against various bacteria (Table 1).
We further expanded our exploration to include the development of two degradable OIM compounds with unsubstituted C(2)-H using carbonate and secondary diamide linkages, respectively (15-16) (Figs. 10 and 19-20). These degradable carbon acids retained robust antibacterial potency against various bacterial strains, displaying Geo-MICgo of 3.6 pg/mL and 4.9 pg/mL, respectively (Table 1). The OIM1-8 and degradable series further confirm the importance of carbon acidity in achieving antibacterial potency.
Further testing of the parent and -CH3 series (compounds 1 , 2 and 5) with clinically relevant multi-drug resistant (MDR) bacteria show that the parent and the C4-substitued methyl derivative (i.e. 1 and 5), but not the C2-derivative (2), had potent antimicrobial activity (Table 4). We found that OIM1-6-CH was potent against the majority of MDR strains with MIC90 of 2- 4 pg/mL. OIM1-6-C4 had slightly poorer potency with MICgo generally of 2-4x higher than OIM1-6-CH while OIM1-6-C2 was ineffective (MIC90 = 32-512 pg/mL) against all the MDR bacteria tested. We also tested the killing kinetics of the three OIM derivatives (1 , 2 and 5) against S. aureus LAC and Pseudomonas aeruginosa (PAO1) (Fig. 21). OIM1-6-CH and OIM1-6-C4(CH3) are bactericidal and had fast kill kinetics. Both bacterial strains were completely eradicated within 1 -2h with these compounds at the concentration of 4x the MIC. On the other hand, OIM1-6-C2(CH3) had greatly reduced potency. Table 4. Antibacterial effect (MIC90, p.g/ml_) of selected OIM1-6 series compounds compared to the activity of antibiotics on a panel of pan-resistant bacteria and naturally antibiotic resistant bacteria.
MICgo (pg/mL) of polymer/antibiotics
OIM 1-6 CH OIM 1-6-C2 OIM 1-6-C4
Bacteria (Gram positive) (1) (CH3) (2) (CH3) (5) Colistin Gentamicin 2 512 4-8 256 1 2 128 4 64 <1 2 64 4 >512 <1 2 512 4 128 256
>512 / >512 /
32-64 / 2* 128 / 8* >512/ >512* >512* >512* 3) 1 32 1 128 <0.5 2 32 8 4 >512 2 128 8 2 >512 8 >512 >512 >512 64 2 256 8 4 1 4 1024 8 2 2 4 128 2 2 32 4 >512 16 2 0.5 4 >512 32 128 <1 2 256 2 2 >512 2 32 4 2 2 4 >512 32 2 >512 4 64 16 2 128 0 4 >512 32 256 2
Figure imgf000073_0001
*The first MIC is in MHB, and the 2nd MIC value is in MHB supplemented with hemin.
We also tested the biocompatibility of the OIM1-6 compounds (1-9) using different eukaryotic cells and showed that OIM1-6-CH generally had no short-term toxicity (i.e. 24 hours) but had long-term toxicity (i.e. 48 and 72 hours) against all cell lines tested, i.e. 3T3 fibroblast cells, human embryonic kidney (HEK) cells and HepG2 cells (Table 5, Figs. 10-16). The derivative (5) with -CH3 at C4 position generally showed improved long-term toxicity compared to OIM1- 6-CH. Meanwhile, increasing the chain length of the OIMs from 6 (in compounds 1, 2 and 5) to 8 repeating units (in 10-12) respectively resulted in increased cytotoxicity. The biocompatibility of the degradable compounds (13-14) was improved compared to its parent OIM.
Table 5. Cytotoxicity of OIM series compounds (IC50, pg/mL).
Figure imgf000074_0002
IC50 (pg/mL)
Figure imgf000074_0001
We also conducted biocompatibility tests for the OIM compounds across various eukaryotic cell lines. Both the parent OIM1-6-CH (1) and OIM1-8-CH (10) carbon acids exhibited low short-term (i.e. within 24-hour) toxicity. However, they demonstrated elevated longer-term (i.e. 72-hour) toxicity against all tested cell lines, including 3T3 fibroblast cells, human embryonic kidney (HEK) cells and liver hepatocellular carcinoma (HepG2) cells (Tables 1-3, Figs. 10-16). However, several substituted carbon acid derivatives, such as the C4-methyl (5, 12), C4- chloro (6) compounds, as well as the degradable derivatives (15, 16), displayed improved selectivity indices (Geo-ICso Geo-MICgo) compared to their respective parent molecules, i.e. OIM1-6-CH (1) and OIM1-8-CH (10).
To gain deeper insights into 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 revealed that the imidazolium ring of compound (1) had a charge of 0.286e. Both the C2- and C4-substituted methyl derivatives, i.e., C2-CH3 (2) and C4-CH3 (5) derivatives, displayed similar charges on the imidazolium rings (0.276e and 0.289e, respectively) when compared to (1). In contrast, the chloro-derivatives (3) and (6), featuring C2-CI and C4-CI substituents, respectively, exhibited moderately increased charges on their imidazolium rings (0.306e and 0.324e, respectively) (Fig. 22). However, it is noteworthy that despite these variations in cationic charge density with different substituents, the C2-substituted (CH3- and CI-) non carbon acid derivatives (2) and (3) had significantly poorer MICs compared to (1), while the C4-substituted carbon acids (5) and (6) exhibited good MICs values. This suggests that the antibacterial potency of the carbon acids (1), (5), and (6) is not solely correlated to the density of cationic charges on the imidazolium rings. Instead, their antimicrobial activity appears to be influenced by other factors, possibly related to the presence or absence of the carbon acid C(2)-H moiety.
We selected three key compounds for further mechanistic investigations into the relevance of carbon acidity, namely, the parent compound (1) and the two CH3-substituted derivatives (compounds 2 and 5). These compounds were subjected to comprehensive testing against an expanded panel of clinically relevant multi-drug resistant (MDR) bacteria. Interestingly, while both the parent OIM1-6-CH (1) and C4-methyl substituted derivative (5) exhibited potent antimicrobial activity, the C2-methyl substituted derivative (2) did not (Table 6), reinforcing the notion that their potent antimicrobial activities are correlated with their carbon acid characteristic. We conducted time-kill kinetics for these three OIMs (1, 2 and 5) against methicillin-resistant Staphylococcus aureus (MRSA) LAC strain and Pseudomonas aeruginosa PAO1 (Fig. 21). Notably, both (1) and (5) were bactericidal and displayed fast killing kinetics with complete eradication of the bacteria within 1-2 hours at concentrations 4- 8 times their MICs. In contrast, compound (2) displayed reduced potency, failing to eradicate the bacteria even with prolonged exposure (20 hours) at 8 times MIC. Subsequently, we conducted a comprehensive series of mechanistic studies to investigate the entry of the OIM1- 6 parent compound (1) and its methyl-substituted derivatives (2 and 5) into bacterial membrane-mimicking liposomes and live MRSA bacteria to elucidate the specific role of carbon acid and its resulting deprotonated form of NHC in the OIM entry process. Table 6. Antibacterial potency of OIM1-6 series compounds compare to the activity of antibiotics against a panel of multi-drug resistant (MDR) bacteria.
MICgo (pg/mL) of oligomers/antibiotics
OIM 1-6 CH OIM 1-6-C2OIM 1-6-C4OIM1-12-6C- OIM1-8-2D Bacteria (Gram positive) Colistin Gentamicin
(1) (CH3) (2) (CH3) (5) OH (15) (16)
S. aureus USA300 (MRSA) 2 512 4-8 2 32 256 1
S. aureus (LAC) 2 128 4 2 2 64 <1
S. aureus (LAC*) 2 64 4 8 8 >512 <1
S. aureus MRSA BAA 39 2 512 4 4 2 128 256
E. faecalis 583 (VRE2)a 32-64 / 2 >512 / >512 128 / 8 8 / 4 16 / 4 >512 / >512 >512/ >512
S. epidermis (MRSE 700563) 1 32 1 4 4 128 <0.5
Bacteria (Gram negative)
A. baumannii BAA 2803 2 32 8 4 2 4 >512
A. baumanii A - (MDR) 2 128 8 8 4 2 >512
B. thailandensis 700388 8 >512 >512 8 16 >512 64
E. coli NMT 1833 2 256 8 8 16 4 1
E. coli 958 (MDR) 4 1024 8 8 16 2 2
E. coli BAA2774 4 128 2 8 8 2 32
E. coli UTI89 4 >512 16 4 16 2 0.5
E. cloacae 13047-MDR 4 >512 32 4 8 128 <1
K. pneumoniae (KPN R) 2 256 2 2 4 2 >512
K. pneumoniae SGH 10 2 32 4 2 2 2 2
P. aeruginosa BAA2797 4 >512 32 8 16 2 >512
P. aeruginosa PAER 4 64 16 8 16 2 128
S. marcescens ATCC 13880 4 >512 32 8 16 256 2
Geometric mean (Geo-MIC90) 3.5 265.0 10.1 4.9 7.4 26.9 20.4 aThe first MIC values were tested in MHB, and the second MIC values (after the 7”) were tested in MHB supplemented with hemin.
Example 6. OIM carbon acids with deprotonated NHCs translocate plasma membrane of bacterial-mimicking liposomes and enter liposome cores
Using DiSC3(5) and propidium iodide assays (Fig. 23), we assessed the impact of these compounds on bacterial membrane potential and physical integrity in MRSA. We showed that OIM1-6-CH and the C2/C4-methyl substitution derivatives (1-2 and 5) induced minimal perturbation to the bacterial membrane potential and to the physical membrane integrity in S. aureus. (Fig. 23). This finding indicates that the antibacterial effects of these compounds differed significantly from those of classical cationic polymers which typically cause physical pores or holes in membranes.
We hypothesized that the transient presence of NHCs in imidazolium-containing polymers which act as Bronsted carbon acids (Fig. 24A), facilitates, and ultimately contributes to their exceptional antibacterial potency. The transformation of some (but not all) of the cationic repeats of imidazolium-containing polymers, at any single moment, to uncharged hydrophobic NHCs convert the initially cationic hydrophilic homopolymer to an amphiphilic copolymer (Fig. 24A) that enables copolymer translocation across bacterial membrane at low threshold concentrations, a step preluding the cytosol entry necessary to reach its intracellular target (Fig. 24B). This distinct mode of entry, facilitated by the initial electrostatic attraction of the cationic polymer to anionic bacterial membranes, stands in contrast to the physical disruption approach employed by classical cationic polymers, which likely requires higher charge concentration threshold that results in lower potency and selectivity. Herein, we seek to prove the facile entry of short OIMs via the NHC formation.
We hypothesized that for OIM carbon acids, i.e., compounds (1) and (5), a fraction of the imidazolium repeats with dissociable C(2)-H could convert to NHCs (//, Fig. 24) in buffered aqueous solution at physiological pH and temperature. To detect the NHC formation, we employed 1H NMR spectroscopy in D2O solvent to measure the hydrogen-to-deuterium (H-D) exchange at the C(2)-position of imidazolium cations (Amyes, T. L. et al., J. Am. Chem. Soc. 2004, 726, 4366-4374) (Fig. 25A). In that case, oligoimidazolium in the presence of OH ions will have two monomer forms (Fig. 25C), i.e. the azolium cation (/) and the azole-2-ylidene carbene (//) and will be a copolymer (poly(/-//)). For compound (1), the C(2)-H 1H NMR rapidly decreased at pH 7.16 and pH 8.21 (Fig. 25B), indicative of a fast H-D exchange rate. Conversely, at more acidic values (pH 6.81 and pH 6.63), the H-D exchange rate slowed due to lower deuteroxide (base) concentration (Fig. 25B). For OIM1-6-CH dissolved in PBS at pH = 7.16, we observed that the 1H NMR signal of the C2-proton (at around 8.80 ppm) decreased, corroborating its acidic property and NHC formation (Fig. 25B). These findings corroborate the notion that compound (1) functions as a carbon polyacid with imidazolium repeats that readily deprotonate at physiological pH, releasing protons to form the residual NHC (//) repeats which are uncharged and hydrophobic. We hypothesized that these hydrophobic repeat units of OIM enables the newly formed copolymer of cation and NHC to be able to penetrate the anionic bacterial plasma membrane which are otherwise impermeable to the initial hydrophilic polycation.
The transformation of cationic OIM to NHC in bacterial membrane mimics was captured by two probes (carbazole and chloro(dimethylsulfide)gold(l) (AuDMSCI) (Fig. 25C). We used liposome to mimic bacterial membrane and encapsulated the hydrophobic carbazole dye or AuDMSCI into the liposome bilayer. As shown in Fig. 25D, when the carbazole dye was encapsulated in liposomes, the emission spectrum exhibited two structured emission peaks at 345 and 360 nm, and humps at around 380 and 400 nm. This coincides with the spectral characteristic of carbazole monomers (Guo, Y. et a/., RSC Adv. 2016, 6, 86989-86997), indicating that carbazole exists as a monomer in the lipid bilayer. The stable carbene, 1 ,3- Bis(2,6-diisopropylphenyl)-1 ,3-dihydro-2H-imidazol-2-ylidene (IPr) was used as a positive control. When Ipr was added into the liquid phase of liposome suspension, the carbazole(lipids) + Ipr(aq) mixture exhibited a significant decrease (up to 70%) in carbazole emission when compared with carbazole(lipids). The hydrogen bonding of carbazole with a NHC to form a complex will make the dye non-fluorescent. The great decrease in emission upon the addition of Ipr(aq) corroborates the formation of a [carbazole-lpr] complex, which is likely a hydrogen bonded adduct between Ipr and carbazole involving C- HN bonding. Further, we observed that carbazole(lipids) + OIM1-6-CH displayed 30% decrease in carbazole emission, corroborating the formation of NHC and its [carbazole-(OIM1-6-CH NHC)] complex in the lipid bilayers.
To prove the presence of NHC in bacterial mimetic membranes, we endeavored to capture OIM-NHCs in the bilayer membrane of anionic liposomes using two NHC probes: carbazole and chloro(dimethylsulfide)gold(l) (AuCI(SMe2)) (Fig. 25C). We constructed negatively charged liposomes that mimic bacterial plasma membranes and embedded the NHC probes within the bilayer. This composite of probe within lipids is denoted as probe(lipids), such as carbozole(lipids) and Au(lipids). It has been reported that when a stable NHC, such as 1 ,3- Bis(2,6-diisopropylphenyl)-1 ,3-dihydro-2H-imidazol-2-ylidene (IPr), binds with carbazole which is fluorescent in the free state (such as in an organic solvent), the resulting NHC- carbazole adduct (which is formed from the hydrogen bonding of the carbene carbon that have lone pair electrons, with R-NH of carbazole) leads to photoluminescence quenching (Kieser, J. M. etal., J. Am. Chem. Soc. 2019, 141, 12055-12063). Our experiments demonstrated that the addition of IPr (control) to the carbazole(lipids) aqueous solution significantly reduced carbazole emission by approximately 70%, supporting the formation of the [carbazole+IPr NHC] complex (Fig. 25D). Likewise, when OIM1-6-CH (1) was introduced into the carbazole(lipids) solution, a decrease in carbazole emission, albeit smaller (approximately 30%), was observed, corroborating the presence of OIM-NHC in the membrane that leads to a [carbazole+OIM-NHC] complex within the lipid bilayer (Fig. 25C).
Furthermore, previous research has indicated that AuCI(SMe2) can react with NHC under mildly basic ambient conditions, resulting in the formation of the covalently linked [AuCI(NHC)] complex (Nahra, F. et al., Nat. Protoc. 2021 , 16, 1476-1493) (Fig. 25A). The hydrophobic AuCI(SMe2) was placed in the liposome bilayer to attempt to capture NHCs generated from OIM. Then, OIM1-6-CH (1) aqueous solution was added into the Au(lipids) suspension. AuDMSCI molecules are expected to stay in the liposome bilayer because of their hydrophobic nature. When added into the Au(lipids) at pH 7.4 in phosphate buffer, positively charged OIM1- 6-CH would absorb onto the negatively charged phosphoglycerol-containing liposome surface by electrostatic attraction. If OIM1-6-CH penetrates liposome and forms NHCs in liposome bilayer, the pre-encapsulated AuDMSCI may react with OIM1-6-CH NHCs to form Au-OIM1- 6-CH (Fig. 25 J(i)) which can be detected with liquid chromatography with electron ionization mass spectrometry in positive ion mode (LC-ESI-MS(+)). The product of Au(lipids) with OIM1- 6 may form the reaction shown in Fig. 25J(ii). Thus, following incubation and lipid removal, we analyzed the reaction product using /iquid chromatography coupled with electron /onization mass spectrometry in positive ion mode (LC-ESI-MS(+)). A major total ion chromatogram (TIC) peak at an elution time of 3.81 min, as well as 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, (Figs. 25E and 25F(i)-(ii)) were observed. The EIC peak of m/z = 199.15 was attributed to unreacted (excess) OIM1-6-CH (1) (Fig. 25G(i)) based on the mass spectrum (MS) gap of 0.25 units between adjacent ions in the isotope cluster (Fig. 25H(i)). The gap of 0.25 units between adjacent ions in the isotope cluster indicated that the ion contains 4 charges (z = 4), and therefore the mass of the total ion is calculated to be 796.60, which corresponds to OIM1- 6-CH containing 4 positive charges and 2 NHCs (Fig. 25J (ii)). The EIC peak with m/z = 330.52 was identified as Au-OIM1-6-CH (Fig. 25G(ii)) due to its MS (Fig. 25H(ii)) showing a gap of 0.33 between adjacent ions. The two structural assignments were further confirmed through H-D exchange of the C(2)-H and structural analysis of fragments, both analyzed with LC- MS/MS (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 of Au-OIM1-6-CH which contains one Au atom (Fig. 25J(ii)). The structure assignments were confirmed by hydrogen deuterium exchange and MS/MS analysis (Fig. 27). In summary, the observation of the m/z 330.52 LC-MS peak in bacterial-mimicking liposome bilayer containing AuDMSCI confirms that OIM1-6-CH can form carbene in the hydrophobic membrane bilayer. We showed that the NHCs formed persist long enough in the aprotic liposome bilayer to react with the embedded Au(lipids) probe to produce the stable Au-OIM(1) within the membrane. These results provided conclusive evidence that some repeat units of OIM transform into the NHC state, allowing the resulting copolymer to enter the bilayer membrane and the NHCs persist long enough in the aprotic liposome bilayer to react with the embedded Au(lipids) probe to form stable Au- OIM1 (lipids), corroborating that OIM1-6-CH (1) forms NHCs and penetrates the membrane bilayer.
We also explored the possibility of NHC formation in the two methyl-substituted derivatives, OIM1-6-C2(CH3) (2) and OIM1-6-C4(CH3) (5). Compound (5) showed lower H-D 1H NMR exchange rates of the C(2)-H compared to (1) at pH 7.16, with hindered exchange observed at acidic pH values (6.63 and 6.81) (Figs. 28A-B). Compound (2), lacking a C(2)-H, showed no H-D exchange at both neutral and acidic pHs. At the basic pH of 8.21 , (2) remained unresponsive and do not show NHC formation, while (5) exhibited increased NHC formation, plateauing at 77% of “H” being exchanged to “D”. We employed the AuCI(SMe2) probe to further investigate the impact of a more basic pH (8.2) on NHC formation of (2) and (5) within the aprotic bilayer membrane. The compound (5), but not (2), demonstrated NHC formation and the Au-OIM(5) was also isolable in the membrane bilayer, albeit less readily than (1) (Figs. 28C-H). This is consistent with the methyl-group on (5) exerting an electron-donating effect, making its C(2)-H less susceptible to deprotonation and thereby weakening its NHC formation as compared to (1).
We proceeded to quantify (i) the total OIM uptake into the liposomes (which includes the liposome membrane and core), and (ii) OIM uptake into the liposome core only (Fig. 25I). The latter is indicative of the OIM’s susceptibility to the bacterial cytosol uptake to achieve intracellular targeting. Our results showed that at physiological pH (7.4), both (1) and (5) exhibited significantly higher total uptake compared to (2), with only (1) and (5), but not (2), entering the liposome core. At the acidic pH of 6.8, at which carbene formation is inhibited, the total uptake of all three compounds was greatly reduced, with core uptake becoming negligible. Consequently, the extent of core uptake for these three compounds is correlated with the ease of NHC formation. At physiological pH, (1) which forms NHC most readily among the three compounds, displayed the highest core uptake. In contrast, (2) failed to enter the core, as it could not penetrate the bilayer membrane due to its inability to form the hydrophobic NHCs (Fig. 28A). The compound (5), while forming NHCs but to a lesser extent than (1), exhibited intermediate core uptake among the three compounds.
To further validate our hypothesis that NHC formation prime membrane entry, we conducted computer simulations to analyze the interaction of the purely cationic versus the purely NHC forms of OIM1-6-CH (1) (denoted as OIM and OIM-NHC respectively) with S. aureus mimetic membrane. The modeled OIM had six repeats of cationic rings while OIM-NHC had 6 repeats of NHC. After placement outside of the surface of the constructed membrane, the OIM-NHC formed numerous stable contacts with the membrane, whereas the cationic OIM had limited membrane interaction (Figs. 29A-B). Notably, the cationic OIM preferred binding towards the surface of the membrane (Fig. 29C), while OIM-NHC immediately penetrated and remained within the hydrophobic interior of the membrane bilayer (Fig. 29D). These results support the idea that NHC formation enhances the hydrophobicity of OIM, facilitating its insertion into the bacterial membrane bilayer.
In summary, our investigations have demonstrated that the acidic hydrogen located at the C2- carbon of imidazolium rings of the carbon acids (1) and (5) can dissociate at physiological pH, giving rise to the formation of NHC. This phenomenon was detected by NMR in water, and in the liposome membrane bilayer by the embedded carbazole and AuCI(SMe2) probes. Compound (1) exhibited a greater propensity for NHC formation compared to (5) and has a lower pKa of 21.32 than 22.15 (Table 7). Furthermore, computer simulations confirmed that NHC formation by the carbon acid (1) aids their entry into the membrane bilayer as formation of uncharged NHC increased the OIM hydrophobicity. Hence, NHC formation “hydrophobizes" OIMs (1) and (5) to aid the oligomer entry into the membrane bilayer, an essential step preluding the internalization into the liposome core. Conversely, the methyl-substitution at the C2-carbon on compound (2) prevents carbene formation of the non-carbon acid, impeding the entry the charged hydrophilic OIM into both the bilayer and the liposome core.
Table 7. pKa of OIM derivatives.
No Compound pD ex (M’1 S’1) kDO (M’1 S’1
Figure imgf000082_0001
1 OIM1-6-CH 7.56 7.58x104 1.16x104 4.84x103 21.32 6.92x1Q-15
5 OIM1-6-C4(CH3) 7.56 3.40x10’6 1.72x103 714.6 22.15 1.02 x1 O’15
6 OIM1-6-C4(CI) 6.58 1.83x10’4 2.68x104 1.12x104 20.95 1.70x10’15
7 OIM1-6-C4(F) 6.58 1.14x104 1.17x104 4.86x103 21.31 7.31 X10 18
8 OIM-1-6-(BZ) 6.26 1.13x10’4 3.42x104 1.43x104 20.84 1.05x10’15 9 OIM1-6-C4(CF3) 6.58 1.42x104 2.08x104 8.65x103 20.06
The acidity of various OIM carbon acids (1, 5-9) may be an important factor contributing to good antibacterial efficacies. We measured the pKa’s of different OIMs (1 , 5-9) via 1H NMR (Amyes, T. L. etal., J. Am. Chem. Soc. 2004, 126, 4366-4374) and found that the p a of OIM 1- 6-CH (1) was 21.32 (Table 7) confirming that (1) is a carbon acid. Amongst the derivatives tested, it is noted that the least acidic carbon acid is OIM1-6-C4(CH3) (5) with pKa 22.15 while the most acidic one is OIM1-6-C4(CF3) (9) with pKa of 20.06 and OIM1-6-CH (1) has intermediate acidity (pKa 21.32). The electron-donating -CH3 in (5) makes the C2-proton less acidic resulting in a higher pKa than (1). In contrast, electron-withdrawing groups (-CI, -F, -Bz, and -CF3 in 6-9) make the C2 proton more acidic, resulting in lower pKa in (6-9).
Example 7. Synthesis of FITC-conjugated OIMs
General Procedure 10
To a stirred solution of the required starting material 1, 2 and 5 (1.2 equiv.) in anhydrous dimethyl sulfoxide (DMSO (1 mL) was added triethylamine (5 equiv., 27 pL, 0.193 mmol) and the reaction mixture was stirred for 10 min at rt. The reaction mixture was subsequently treated dropwise with FITC (1 equiv., 15 mg, 0.0385 mmol) dissolved in DMSO (0.5 mL) and the reaction mixture was stirred at rt overnight in the dark. The reaction mixture was diluted with water, transferred to a dialysis bag with Mw cut-off of 500-1000 Da and dialyzed against 1 mL HCI in 1 L of deionized water over 24 hours, with frequent changing of dialysis water every 2- 3 hours. The resulting solution was concentrated under rotary evaporation and lyophilized to afford the desired product as a mixture of the unreacted starting material, and mono- and bisconjugated products. The percentage of dye conjugation per parent compound was estimated using 1H NMR based on integrals of signals at the 3.80 -4.40 ppm and 6.00- 6.70 ppm region which corresponded to the 12 x imidazoyl-N-CH2- within the parent OIM compound and 3 x Ar-H of the 2 x phenolic group within FITC respectively. 24:6 integrals of a signal at the 3.80 - 4.40 ppm and 6.00 - 6.70 ppm would infer 100% conjugation (1 :1 ratio between parent compound and FITC dye). As such, the percentage of dye conjugation per parent compound was obtained by calibrating the integrals of signals at 3.80 - 4.40 ppm to 24 and the obtained integral value of the signal at 6.00 - 6.70 ppm was divided by 6, expressed as a percentage.
Example 8. Intracellular accumulation of OIM derivatives in bacteria correlates with their carbon acidity and ease of NHC formation We then investigated the uptake of the three OIMs (1 , 2 and 5) into bacteria, specifically the parental MRSA LAC strain and its respiration-deficient mutants which have low PMF.
OIM-FITC uptake assay
The OIM-FITC uptake experiment was conducted as described previously (Radlinski, L. C. 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). Exponential phase bacteria were prepared and diluted to 10s CFU/mL in TSB broth, then incubated with FITC-conjugated OIM (7.5% molar ratio of OIM-FITC) at desired concentrations for 1 hour in the dark. Culture was centrifuged (10,000 rpm, 10 minutes) and washed twice with filter- sterilized PBS. Bacterial pellets were resuspended in 800 pL PBS. The fluorescence of 150 pL of each sample was directly measured with flow cytometer (BD FACSVerse Flow Cytometer (3) Laser) using the FITC channel, and this data gives the unquenched uptake data (Fig. 30). 300 pL of the remaining sample was quenched with 0.01% Trypan blue (TB) dye before measurement, in which the fluorescence data provides the total (i.e. bacterial membrane and cytosol) OIM uptake. Another 300 pL of the remaining sample was treated with 0.04% Triton X-100 (TX) for 15-20 min followed by TB quenching, after which the FITC fluorescence was measured, and the data provides the uptake in the membrane fraction. The histogram subtraction of the total uptake minus the uptake in the membrane faction provides the cytosolic uptake fraction. Detailed experimental method and the flow cytometry histograms are shown in Fig. 30. Bacteria without OIM-FITC treatment served as negative control. The internalized OIM fraction was further verified by permeabilizing the OIM-FITC treated bacteria using 0.04% Triton X-100 for 15-20 min followed by Trypan Blue quenching, and the FITC fluorescence was subsequently measured. The fluorescence signal of cytosolic - internalized OIM-FITC in permeabilized bacteria will be greatly reduced after quenching as Trypan Blue will be able to quench the cytosolic - internalized OIM-FITC, however the OIM-FITC that is embedded in the membrane could not be removed by TB quenching. The former is observed for bacteria treated with SYTO 9, OIM1-6-CH-FITC and OIM1-6-C4(CHs)-FITC, while the latter is observed for OIM1-6-C2(CH3)-FITC. The internalised portion for OIM1-6-C2(CH3)-FITC is then the subtraction of Trypan Blue quenched fluorescence of unpermeabilised with that of permeabilised treated bacteria.
Results and discussion
The three OIMs derivatives were labelled with FITC fluorochrome (as described in Example 7), and their uptake was quantified using flow cytometry (Fig. 30). Given the higher cell density required for flow cytometry experiments (106 CFU/mL), we determined the MIC* values at the higher cell density. To measure the internalised portion of FITC-OIM, 0.01% Trypan Blue (TB) dye, which is membrane impermeable, was used to quench the extracellularly associated FITC-conjugated oligomers (Antonoplis, A. etal., 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 the fluorescence due to the internalised portion without considering the extracellular part that is hard to remove (for example, that embedded in the plasma membrane), we subtract the fluorescence of FITC-OIM-treated bacteria that is TB-quenched by that of FITC-OIM-treated bacteria that is surfactant-permeabilised (to release the internalised FITC-OIM) and then quenched with TB.
The average MIC* value against the parental S. aureus strain for the three compounds were as follow: 12 pg/mL (1), >4096 pg/mL (2), and 48 pg/mL (5) (Fig. 31A), which align with the MIC values presented in Table 1. We assessed the percentage of LAC cell count with (i) total uptake (into both the membrane and the cytosol) and (ii) cytosolic uptake. For (1), the percentage of S. aureus strain count exhibiting cytosolic uptake, as well as total uptake, was significant at all tested concentrations (Fig. 31 B), indicating that a significant portion of bacteria had taken up (1) into their cytosol. This is supported by the high mean fluorescence intensity (MFI) observed in (1)-treated cells with both cytosolic and total uptakes (Fig. 31C). In the case of (5), the percentage of bacterial count with cytosolic and uptake was low at low OIM concentrations but increased substantially at 32-64 pg/mL (at its MIC* range). The MFI values in (5)-treated cells, demonstrating total and cytosolic uptakes, were also elevating, confirming that (5) also readily enters the cytosol at its effective concentration range. Conversely, for (2), the percentage of bacterial count with cytosolic uptake remained low at all tested concentrations, although the corresponding MFI of this fraction was high, indicating that only a small fraction of cells took up (2). However, for those that did, they absorbed substantial amounts of (2). The total number of cells showing total uptake of (2) was high, but this fraction displayed low MFI, indicating that many cells took up only a small amount of (2) that adhered on the surface. When correlating the results presented (Fig. 31 B) with the MIC values of (1), (2) and (5) (Table 1), it became apparent that there was an inverse correlation between the compound’s MIC and the percentage of LAC cell count with cytosolic uptake. Specifically, (1) and (5), which have low MIC values and are carbon acids, displayed high cytosolic uptake. Conversely, (2), which has a high MIC value and is a non-carbon acid, displayed a low cytosolic uptake.
Among the three OIM1-6 derivatives, we observed that OIM1-6-CH had the highest total uptake at the low concentrations (4 and 8 pg/mL) (Fig. 32A) while OIM1-6-C2(CH3) and OIM1- 6-C4(CHs) had low total uptake at these concentrations. Further, OIM1-6-CH had the highest internalized fraction at these low concentrations. However, the internalized percent of OIM1- 6-C4(CH3) increased significantly at 16 pg/mL, which is the MIC* (MIC value correlating to 106 CFU/mL bacteria) of OIM1-6-C4(CH3). The OIM1-6-C2(CH3) had the least amount (around 10%) of OIM internalized into the cytosol at 4-16 pg/mL but the internalized amount increased substantially at 128 pg/mL which is also its MIC* value. Similar trend was observed with the uptake of OIMs in S. aureus LAC tested in TSB medium: at the MIC* of OIM1-6-CH and OIM1- 6-C4(CH3) of 8 pg/mL and 64 pg/mL respectively, high internalised fractions were observed respectively (Fig. 32B). For TSB media, OIM1-6-C2(CH3) does not have MIC* in the concentration range tested. In summary, at a fixed concentration, OIM1-6-CH internalisation is most efficient followed by OIM1-6-C4(CH3) and both are carbon acids that can form NHC. The internalisation of OIM-1-6-C2(CHs) without the dissociable C2 proton is poor, suggesting that the presence of C2 hydrogen allowing NHC formation is essential for polymer internalization that correlates with compound potency.
To better understand the relative contribution of these two factors (NHC formation versus PMF) to compound uptake into the bacterial cytosol, we conducted additional tests, which are described below.
When comparing the MRSA LAC parental to the respiration-mutants lacking PMF, specifically LAC AmenD and LAC AhemB, at pH 7.2 (Fig. 31 D), we observed an increased MIC* of (1) by a factor of 8-16 when PMF was absent (while NHC was still present). This result confirms that PMF is an important factor in antibacterial efficacy. Furthermore, the percent of bacterial cell counts with total/cytosolic uptake at pH 7.2 at their MIC* was highest for the parental compared to the mutants (Fig. 31 E), supporting the notion that cytosolic uptake is, at least in part, facilitated by the PMF. When comparing the MIC* against the parental strain for (1) at different pHs (7.2 versus 6.8 and 6.6) (Fig. 31 D), we observed a 5.3-fold increase in MIC* from 12 pg/mL to 64 pg/mL, along with a decrease in uptake to some extent (Fig. 31 E, first panel), albeit still substantial since PMF is high in the parental strain. This pH-dependent reduction in uptake corroborates the importance of NHC formation in the uptake process, as acidic pH hinders NHC formation and subsequently reduces uptake and killing. Thus, both NHC formation, newly identified here, and PMF, play pivotal roles in the antibacterial potency of (1) carbon acid.
To further evaluate the importance of NHC in live bacteria, we employed the MRSA LAC respiration-deficient mutants to minimize the effect of PMF and assess the impact of pH on MIC*. For (1)-treated mutants (Fig. 31 D), the mildly acidic pHs of 6.6 and 6.8 dramatically increased MIC* to 1,024 pg/mL and > 4,092 pg/mL respectively. OIM (5) failed to kill the mutants at these acidic pHs, even at very high concentration exceeding 4,092 pg/mL. In contrast, the MIC* of gentamicin, exhibited only weakly dependent on pH in the tested range (Fig. 31 D). Additionally, the percentage of bacterial count with cytosolic and total uptakes for both mutants was significantly lower at pH 6.8 than at pH 7.2, which correlated with their increased MIC* values (Fig. 31 D). Therefore, inhibiting NHC formation greatly impeded the intracellular uptake of OIM in mutants with reduced PMF, resulting in poor killing. This test at acidic pH reinforced the importance of NHC formation for the internalization of (1).
To further evaluate the relative significance of NHC formation and PMF in OIM potency, we compared the killing kinetics of compounds (1) and (5) against the LAC parental strain under oxic, hypoxic, and fermentative growth conditions (Fig. 31 F). The parental LAC strain possesses an active electron transport chain (ETC) in aerobic and anaerobic respiration conditions, but not in fermentative growth condition. Although (1) exhibited rapid killing kinetics in both oxic and hypoxic conditions, there was still significant killing in the fermentative growth condition, albeit with lower potency. This demonstrates that in the absence of PMF or an active ETC, (1) can still achieve killing because of its robust NHC formation that promotes entry and internalization. OIM (5), on the other hand, exhibited poor killing in both anaerobic and fermentative growth conditions, indicating its strong reliance on PMF, as it forms NHC less readily to facilitate entry.
In summary, these data indicate that the NHC formation of (1) carbon acid aids cytosolic uptake. The suppression of the NHC formation at acidic pH resulted in a considerable reduction in antibacterial efficacy and reduced cytosolic uptake. Moreover, (1) can effectively kill bacteria even in conditions of low PMF, as it can utilize NHC for internalization. The inability of (2), a non-carbon acid, to form NHC impedes its ability to reach the bacterial cytosol, greatly impairing its antibacterial efficacy. OIM (5), characterized by a weaker ability to form NHC due to its reduced carbon acidity (Fig. 27), relies more heavily on PMF for intracellular uptake, as evidenced by its inability to kill mutants with low PMF (Fig. 31 D) and in fermentative growth conditions (Fig. 31F). Taken together, these findings support the hypothesis that carbene formation of carbon acids (1), and also to some extent for (5), promotes their entry into bacteria, resulting in their potent MIC values.
Example 9. The bacteria surface binding and intracellular internalisation of carbene- forming OIM1-6-CH is PMF independent
Resistance Evolution of LAC and PAO1 against OIMs
Sub-culture of S. aureus LAC or P. aeruginosa PAO1 was added to OIMs with varying concentration, starting from 0.5x MIC to 4x MIC based on the MIC protocol and was set up with 8-10 independent replicates. After 18-24 hours, each replicate with at least 50% growth in the highest concentration of OIMs were selected for the next passage. The OIMs concentration were adjusted based on the acquired mutations of the strains. The passage was repeated every day until the mutants acquired high resistance (MIC value was >1024 pg/mL) or until 30 days for PAO1. Bacteria samples were preserved with 30% glycerol in -80 °C on each day of the passage. At the end of the experiment, the LAC mutants were streaked out on agar plate. Two colonies from each plate were selected for resistance stability testing. These LAC mutant strains were passaged for 7 days without OIMs, and the MIC was tested at Day 1 and 7th respectively. Mutants that shown stable resistance were selected for whole genomic sequencing. The genomic DNA was extracted using QIAamp DNA Mini Blood Mini Kitwith minor modifications. The cell wall was digested with 10 mg/mL lysozyme and 10 pg/mL lysostaphin and the bacteria was lysed via beat beater before proceeding to the standard protocol of the kit. DNA was prepared for sequencing by using an Illumina Nextera DNA Library Preparation Kit. DNA was sequenced on an Illumina MiSeq instrument (paired end sequencing). Sequences were mapped onto the genome of the parent strain S. aureus LAC, and CLC Genomics Workbench software was used to identify single nucleotide variations, small deletions, and insertions. Large deletions were identified by manual sequence comparison.
Oxygen consumption rate (OCR)
The OCR was measured using a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience). The sensor cartridge was equilibrated per supplier instruction before use, and the sample cartridge was coated with poly-D-lysine for bacterial adhesion. LAG sub-culture was grown in M9 buffer (1x M9 salt solution, 2 mM MgSO4, 0.2% (w/v) glucose, 0.2% (w/v) Casamino acids, 0.2 pg/mL Nicotinamide, 100 nM Thiamine) until exponential phase. Then, 100 pl of 106 CFU/mL bacteria were added into each well of the sample cartridge except the blank wells. The sample cartridge was centrifuged at 1400xg for 10 min, then 80 pl M9 buffer was added into each well. Basal OCR was measured for 3 readings before OIMs/ antibiotics injection.
Time-kill assay in anaerobic and fermentative condition
TSB was used as growth medium in aerobic, anaerobic and fermentation growth conditions. For anaerobic condition, 100 mM sodium nitrate was added into TSB. All liquid media, pipettes and consumables used for anaerobic and fermentation growth conditions were equilibrated in the anaerobic chamber for at least 2 days. The overnight and subculture were grown at 37 °C in the anaerobic chamber. For time-kill testing, 20 pl aliquots were removed at the indicated time points and serially diluted with 180 pl of PBS. Aliquots of the dilutions were spotted on agar plates and colonies were counted following overnight incubation.
Antagonistic assay
The antagonism effect of PMF dissipating agents (CCCP, valinomycin and nigericin) on OIM1 compounds were tested with S. aureus LAC in a checkerboard assay. Serial 2-fold dilutions of PMF dissipating agents (starting from 0.5x MIC) and OIM1 compounds (starting from 64 pg/mL) were prepared in TSB. 25 pL of PMF dissipating agents (along y-axis) and 25 pL of OIM1 compounds (along x-axis) were added into each well of a 96-well plate, and 50 pL of log-phase bacteria (prepared as described in the MIC protocol in Example 5) was subsequently added. The solutions were mixed thoroughly, and the MIC were determined as described above in Example 5.
Results and discussion
To understand the mechanism of internalization of the OIM oligomers, we evolved S. aureus LAC and P. aeruginosa PAO1 mutants with increased resistance against OIM1-6-CH and OIM1-6-C4(CH3). Such mutants were obtained by continuous passage of the strain in medium with increasing concentrations of the antimicrobial OIM compounds (Figs. 33A-B). As compared to antibiotics control, LAC had evolved significance resistance whereas PAO1 had evolved low resistance against OIMs. LAC strains with a stable increase in resistance were obtained and subsequently genomic mutations were identified by whole genome sequencing (Tables 8-9). The evolved LAC mutants had mutations in genes coding for ETC components, and genes associated with cationic antimicrobial polymer (CAMP) resistance, corroborating impaired cytosolic uptake into bacteria and surface binding contributes to the bacterial resistance.
Table 8. Genetic variations in OIM1-6-CH (1) resistant LAC strains.
Amino acid
Day Pathway Type Genes Sample change
SNV menA Arg19Leu #2s aerobic
SNV menF Glu146Tyr #2big, #8big respiration
Day3 Deletion ndh2 #4 central Deletion galM Asn90fs #2big, #8big metabolism aerobic Deletion ndh2 #4
Day 10 respiration Deletion menB Asp76fs #2s central Deletion mqo Thr393delinsPro #2big, #4 metabolism ion transporter SNV cobl
Figure imgf000090_0001
CAMP SNV mprF Gly241Ser #2big, #4 cell wall SNV tarL Tyr274* #2s transcriptional
SNV codY lle113Asn #2s regulation aerobic Deletion ndh2 #2, #4 respiration central
Deletion mqo Thr393delinsPro #2, #4
Day 12 metabolism ion transporter
Figure imgf000090_0002
CAMP
SNV mprF Gly241Ser #2, #4
Amino acid change: fs refers to frameshift; * indicates stop codon.
Table 9. Genetic variations in OIM1-6-C4(CH3) (5) resistant LAC strains.
Day Pathway Type Genes Amino acid change Sample
Replacement qoxB His567fst aerobic respiration lnsertion ^oxB Glu566fst #4
Figure imgf000090_0003
surface protein SNV asp23 Mutation right after ORF #1big, #9big
SNV ebh Arg4111 Lys #4
SNV menB aerobic respiration Gln181Tyr #4
SNV menB
3 alcohol metabolism SNV adhE Thr373lle #4 virulance SNV sspB Val345Asp #4 aerobic respiration SNV menB Gln181 Lys #4
6 alcohol metabolism SNV adhE YP_492866.1: p. Thr373lle #4
CAMP SNV mprF Trp424Arg #4 Amino acid change: fs refers to frameshift; * indicates stop codon.
^Several amino acids are changed towards the end of QoxB - KEH amino acids mutated to NVFCNY.
To explore if the ETC mutations in S. aureus LAC were due to the direct target of the components of ETC of OIMs, we then studied the oxygen consumption rate (OCR) of S. aureus LAC using respirometry experiments on the Seahorse XFe96 platform (Lobritz, M. A. et al., Proc. Natl. Acad. Sei. U.S.A. 2015, 112, 8173-8180; and Dwyer, D. J. et al., Proc. Natl. Acad. Sci. U.S.A. 2014, 111, E2100-E2109). The OCR of LAC did not decrease immediately after addition of OIM1-6-CH (Fig. 33C) unlike the respiratory poison 2-heptyl-4- hydroxyquinoline-N-oxide (HQNO). Hence, OIM1-6-CH did not kill the bacteria through halting oxygen respiration. The decrease in OCR observed 30 minutes post-treatment (i.e. after the first doubling time) was interpreted to be a consequence of growth inhibition and death rather than a direct effect on oxygen consumption. We also did not observe OCR disruption for OIM 1- 6-C2(CH3) and OIM1-6-C4(CH3). These results infer that OIMs do not directly target the oxidative phosphorylation pathway and the ETC mutations lower the respiration and hence the PMF of the bacteria to attempt to lower the compound uptake.
Further, we used respiratory deficient mutants specifically LAC I menD and LAC AhemB which are unable to consume oxygen due to the lack of functioning ETC (Fig. 33C) to study the PMF influence on OIM uptake. We also measured the surface binding and uptake of the OIM1-FITC compound by these respiration mutants (in TSB), which had low PMF. Our result showed that all OIM1s had lower binding and internalization to both LAC menD and LAC AhemB (Figs. 32C-D) compared with wildtype S. aureus LAC treated in TSB (Fig. 32B). Upon quenching with trypan blue of surface-bound FITC-OIM, the internalization of the OIM1-6-C2(CH3) and OIM1-6-C4(CH3) derivatives into these mutants was also found to be very low, indicating that the interaction of these OIM derivatives with the internal target is low which correlates to their high MICso However, the parent OIM1-6-CH still showed a substantial intracellular uptake into these mutants at its MIC* concentration of 128 pg/mL. This suggests that besides the PMF driving force, another factor is at play for the internalization of OIM1-6-CH into the LAC menD and LAC AhemB mutants, indicating that OIM1-6-CH uptake is not purely PMF dependent.
We also measured the MIC of our compounds against the respiration mutants at pH 7.0. The LAC menD and LAC AhemB displayed increased resistance towards OIM1-6 CH and OIM1- 6-C4(CH3) (Table 10, at pH 7). Using OIM1-6-CH, the MICgo increased from 2 pg/mLfor the wildtype strain to 32 pg/mL with these mutants while with OIM1-6-C4(CH3), the MIC increased from 4 pg/mL to 256-512 pg/mL respectively, indicating that the OIMs need a PMF for uptake and antibacterial potency. OIM1-6-C2(CH3) remained ineffective against both mutants.
Table 10. MIC of OIM series compounds and control antibiotics against S. aureus LAC and respiratory mutants in different pH.
MICgo in TSB (pg/mL) stains pH OIM1-6-CH OIM1-6-C2 OIM1-6-C4
Gentamicin Daptomycin
(1) (CH3) (2) (CH3) (5)
LAC 2 >4096 4 2 4
LAC menD 7 32-64 >4096 512 16-32 4
LAC ArtemB 32 >4096 256 16 8
LAC 32 >4096 256 4 8
LAC AmenD 6.8 1024 >4096 >4096 16-32 4
LAC ArtemB 512-1024 >4096 >4096 32 8
LAC 32 >4096 256 4 8
LAC AmenD 6.6 2048 >4096 >4096 32 8
LAC hemB 4096 >4096 >4096 32-64 8
To further investigate our hypothesis of NHC-dependence of OIM1-6-CH uptake, we tested the MIC of OIMs in slightly acidic TSB (pH 6.6 and pH 6.8) using S. aureus LAC and the respiratory mutants (Table 10). Acidic environment would quench the carbene formation of OIM’s (Fig. 34) and hence would reduce the potency of our oligomers. Our results indeed showed significant increase in MIC’s of both OIM1-6-CH and OIM1-6-C4(CH3) against the wildtype LAC in slightly acidic pH while there is minimal impact on the potency of gentamicin and daptomycin. MIC of OIM1-6-C4(CH3) had increased more significantly than OIM1-6-CH (64-fold compared to 16-fold respectively) at the slightly acidic pH conditions, inferring greater dependence on the NHC formation for the potency of OIM1-6-C4(CH3). We also observed that against both the LAC menD and LAC AhemB mutants, both OIM1-6-CH and OIM1-6- C4(CH3) became significantly ineffective with slightly decreasing pH, while there was only minimal impact on gentamicin and daptomycin. The reduced potency of OIM1-6-CH and OIM1-6-C4(CH3) against the wildtype LAC was further confirmed by the uptake assay tested at pH 6.8 in TSB at their respective MIC* (Fig. 32E). The surface-bound and cytosolic internalization of both OIM1-6-CH and OIM1-6-C4(CH3) significantly reduced at pH 6.8. Similar phenomenon is also observed with the uptake of OIM1-6-CH by both LAC menD and LAC AhemB at pH 6.8 (Fig. 32F). This shows that carbene formation is important for the potency of OIMs and inhibited carbene via a slightly acidic pH decreases the efficacy of our compound, especially against respiration mutants with low/zero PMF.
To confirm the above carbene dependent uptake, we measured the killing kinetics of OIM1-6- CH and OIM1-6-C4(CH3) against LAC under aerobic, anaerobic, and fermentative condition respectively (Figs. 33D-E). The bacteria in these three conditions differ in their PMF; only in the first two conditions the bacteria have active ETC operating. Although OIM1-6-CH exhibited fast killing kinetics in both aerobic and anaerobic condition, there was still significant killing in the fermentation condition without PMF although the kinetics was slower. This again shows that in the absence of an active ETC/PMF, OIM1-6-CH can achieve killing. OIM1-6-C4(CH3), on the other hand, exhibited poor killing in both anaerobic and fermentative conditions, indicating that it is strongly PMF-dependent. Taking together for OIM1-6-CH, uptake can take place without PMF in respiration mutants and killing is observed even in fermentative mode, indicating that OIM1-6-CH uptake depends on a second mechanism apart from PMF, likely NHC-related, that contribute to its intracellular accumulation and killing. OIM1-6-C4(CH3) has weaker ability to form NHC and hence its stronger reliance on PMF for intracellular uptake, whereas the inability of OIM1-6-C2(CH3) to form NHC render this compound ineffective to enter the bacteria and therefore has poor potency. Taken together, these data suggest that the carbene formation of OIM1-6-CH and OIM1-6-C4(CH3) promotes their internalization into bacteria resulting in good MICs.
Example 10. Cationic OIM1-6-CH uniquely eradicate E. coll and S. aureus in anionic surfactant formulations
Most cationic compounds cannot eradicate bacteria in anion-containing formulations because of charge neutralization which severely limits their applications. One such formulation is in laundry detergent which mainly consists of anionic surfactants, along with builders, chelators, bleaching agents, enzymes, and stabilizer that are meticulously formulated to remove dirt, soil, and stains effectively from textile. Herein, we tested antibacterial properties of OIMs and two commercial antibacterial compounds (Poly diallyl dimethyl ammonium chloride (PDADMAC) and colistin) at the concentration of 100 ppm in the commercial laundry detergent (specifically, 389 ppm Japan pouch provided by Procter & Gamble (P&G)), and in another three model detergents (100 ppm of SDBS, 100 ppm of SDS, and 50 ppm of SDS plus 50 ppm of SDBS), respectively. For a compound to pass the test, it needs to provide at least 2 log reduction against E. coll 8739 and S. aureus 6538.
Antibacterial test in laundry detergent Antibacterial test was performed according to standard test method ASTM-E2274 (Li, X. et al., J. Mater. Chem. B 2018, 6, 4274-4292). Briefly, a white cotton fabric (1.4 m x 2.8 m) was immersed in 5 L 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 a 50 °C oven for at least 24 hours. The cloth was cut into strips of 5 cm wide and 15 g each. One end of the strip was pierced and secured onto the outer horizontal extension of a stainless-steel spindle and winded around the three horizontal extensions with sufficient tension. The end of the fabric strip was stapled and the whole spindle was autoclaved at 121 °C for 20 minutes inside a container. Second sub-cultured E. coli ATCC 8739 and S. aureus ATCC 6538 cells were dispersed in 7 mL of 0.85 wt% NaCI and this bacteria solution was adjusted to O.Dsoo = 0.12. Stock inoculum was prepared by mixing 3.8 mL of bacteria solution (O.Deoo = 0.12) with 0.2 mL of horse serum. After this, three sterile fabric carriers with dimension of 1 inch x 1.5 inch (in a single sterile Petri dish) were inoculated by adding 10 pL of water to the centre of fabric followed by 20 pL of stock inoculum. Fabric carriers were dried at 37 °C oven for 30 minutes. The inoculated fabric carriers were inserted into the spindle, with two fabrics between the second and the third layer and one fabric between the third and fourth layer. A glass gar containing 250 mL of water, 0.25 mL of hard water (5.903 wt% of calcium chloride dihydrate and 2.721 wt% of magnesium chloride hexahydrate, filter sterilized with 0.45 pm syringe filter), 0.25 mL of Japan pouch detergent (marketed as P&G Ariel Bioscience Gel ball, composed of linear alkylbenzene sulfonate (LAS), alkyl ether sulfate, polyoxyethylene alkyl ether, and fatty acid ester) or model detergents (SDS, SDBS, SDS+SDBS), and 2.5 mL of OIMs was prepared. The final concentration of Japan pouch detergent, model detergent, and OIMs was 389 ppm, 100 ppm, 100 ppm, respectively. The jar containing 250 mL of water, 0.25 mL of hard water, and 3.125 mL of 4 % Tween 80 solution was used as control. The spindle with inoculated fabrics was placed into the jar and tumbled for 10 minutes, after which the three inoculated fabrics were transferred into 30 mL of neutralizer (Letheen Broth, Modified) and vortexed for two minutes at top speed. 1 mL of neutralizer solution was serially diluted to 10'4 with 0.85% NaCI. 1 mL of each dilution was plated in duplicate in LB agar. The plates were incubated at 37 °C for 24 hours, and colonies were counted and recorded as CFU/plate. The duplicate plates were averaged and multiplied by the dilution factor to arrive at CFU/three fabric carries. The average count was then converted into Iog10 reduction by Iog10 reduction = log10(CFU/three fabric carries of control) - log10(CFU/three fabric carries of OIM).
Results and discussion
As shown in Fig. 35B, the polycation PDADMAC fail in all four detergents. Colistin pass in E. coli 8739 but failed in S. aureus 6538 (Fig. 35C). It is understandable that colistin kill E. coli 8739 in detergent because of its membrane disruption ability for Gram-negative bacteria. With our newly found carbene mechanism that distinguish OIMs from typical cationic polymers, OIM1-6-CH exhibit above 2 log reduction in Japan pouch and another three model detergents (Fig. 35D). In contrast, OIM1-6-C2(CH3) marginally pass when against E. coh 8739 but fail in S. aureus 6538 (Fig. 35E). Similarly, OIM1-6-C4(CH3) pass the test in SDS, SDS+SDBS and Japan pouch detergents, but fail in SDBS (Fig. 35F). It can be explained that OIM1-6-C2(CHs) and OIM1-6-C4(CH3) exhibit antibacterial activity in detergent mainly because of the polyion complex nanoparticles formed in anionic detergent. The poor antibacterial activity of OIM1-6- C4(CH3) in SDBS could be result from the higher hydrophobicity of SDBS. We suggest that the antibacterial activity of OIM1-6-CH in detergent is from its carbene mechanism, while the antibacterial activity of OIM1-6-C2(CH3) and OIM1-6-C4(CH3) in detergent is from the polyion complex nanoparticles formed in detergent. We also tested two biodegradable OIMs, OIM1- 8-Bu-2PzAc and OIM1-8-Bu-2Ac, which contain C2-proton but with degradable bond between imidazolium rings. Fig. 35G shows that OIM1-8-Bu-2PzAc can pass all four detergents. OIM1- 8-Bu-2Ac show potent antibacterial activity as well except in SDS+SDBS when against E. coh 8739 (Fig. 35H). The results indicate that compounds with imidazolium ring containing dissociable C2-proton display better bactericidal activity in anions-containing formulations.
Example 11. Degradable OIM is efficacious in a murine systemic infection model
To assess the efficacy of our degradable compound, we initiated experiments using a murine intraperitoneal (IP) model.
Murine toxicity and infection model
C57BL/6J female mice (8 weeks old) were used for all experiments. Six mice were used for each experimental group. The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Nanyang Technological University (AUP# A20029). The toxicity of OIM1-8-2D (16) was assessed by intraperitoneal (IP) injection, 15 mg/kg daily for 7 days, with daily measurement of animal weight for 14 days. For systemic infection, exponential phase A. baumannii ATCC BAA 2803 was washed twice with PBS and resuspended to 105 CFU/mL in 5% mucin saline solution. Mice were injected with 300 pL bacteria via IP route. At 2 hours post-infection, 15mg/kg OIM1-8-2D (16), 15mg/kg imipenem or PBS (untreated control) was administered to the mice via IP injection. For the quantitative antibacterial efficacy part of the study, mice were euthanized at 26 hours post-infection, and the bacteria counts in peritoneal fluid, livers, kidneys, and spleens were determined. For the survival test, mice were monitored over 7 days after infection. Results and discussion
In this model, mice were infected with a lethal dose of carbapenem-resistant A. baumannii through IP injection. Subsequently, they were treated with a single dose of 15 mg/kg OIM1-8- 2D (16) or 15 mg/kg imipenem (control) at two hours post-infection. Additionally, a control group received PBS only (Fig. 36A(i)). The administration of a single dose of (16) successfully rescued all the mice and led to a significant reduction in bacterial load in major organs, achieving a CFU reduction of 6 orders of magnitude. In contrast, untreated mice or those treated with imipenem succumbed to the infection within 36 hours (Figs. 36A(ii)-(iii) and 37). To evaluate the in vivo tolerance of the OIM, we subjected the mice to seven successive daily doses of (16) at 15 mg/kg per day, totalling a combined dose of 105 mg/kg. Remarkably, this regimen did not result in any significant weight loss (Fig. 36B), underscoring the good biocompatibility of degradable OIM1-8-2D (16), coupled with its excellent ability to eradicate bacteria in a murine systemic infection model.
Example 12. A degradable cationic OIM derivative (16) eradicates bacteria to prevent dairy mastitis in animal trial
We extended our investigations to explore the potential application of imidazolium-containing oligomer/polymer in preventing dairy mastitis, a significant global problem affecting dairy herds.
In vitro mastitis test (BS EN 1656)
S. aureus ATCC 6538, S. uberis ATCC 19436 and E. coliATCC 10536 were used for the tests, as recommended by BS EN 1656 standard (Institution, B. S. (British Standards Institution, 2019)). Bacteria of 2nd subculture were streaked out from Tryptic Soy Agar (TSA) plates and inoculated into Tryptone NaCI diluent solution (0.1% tryptone and 0.85% NaCI) at 1.5 to 5 x 10® CFU/mL. Test compounds were dissolved in hard water (0.119 g MgCh, 0.277 g CaCl2, 0.28 g NaHCOs in 1 L water) at desired concentration. 20 pL skimmed milk (10 g/L) was added into a 96 well plate, followed by addition of 10 pL bacteria test suspension. The plate was mixed and incubated at 30 °C for 2 min. Subsequently, 80 pL of test compound was added and mixed well, and then incubated at 30 °C. At the desired time points, 20 pL of the product/milk/bacteria mixture was transferred to a new 96-well plate containing 160 pL neutralizer (Lecithin 3%, Tween 80 10% (w/v), Sodium Thiosulphate 0.3%) and 20 pL milliQ water, mixed well and incubated at room temperature for 5 min to fully neutralize the compound. The mixture was then 10-fold serially diluted in Tryptone NaCI diluent solution and plated onto TSA plates. Colonies were counted after 24 hours incubation at 37 °C.
In vivo mastitis farm trial (i) Farm condition
The trial was conducted in a dairy farm located at Jilin Province, Changchun city, Jiutai district, Longjia town, Xiaochengzi village. The mastitis protocol was approved by the Changchun University of Chinese Medicine (Ethics Protocol No. 202/205) to the Principal Investigator Professor Li Qingjie. Detailed experimental methods are described in the supplementary methodology of mastitis farm trial below.
(ii) Experimental setup
The experiment procedures were designed in accordance with recommended protocols (Nickerson, S. et al. in NMC Annual Meeting Proceedings. 379-399 (Citeseer)). A 10-day acclimation period (Day-10) was implemented prior to the start of the experiment. Safety trial started on Day 0. First sampling was done before teat dip application (t = Day 0) to establish baseline. Teat dip filled in a conventional foam dip cup was applied to a distal 25 mm of teats immediately after milking. Teat dip was applied daily for 5 consecutive days. 2nd sampling was done on Day 5. Samples collected: milk sample for quality check and residual check; teat surface sample for residual check. The experimental results are described in the in vivo mastitis safety trial testing protocol below and Figs. 38-40. Challenge trial started on Day 5 immediately after milking. First sampling of bacterial CFU count in milk was done before bacteria exposure to establish the baseline (t = Day 5). S. aureus ATCC 49525 (Wall, R. J. et al., Nat. Biotechnol. 2005, 23, 445-451) (5x107 CFU/mL in TSB) was applied to the teats at a depth of approximately 25 mm in a conventional foam dip cup right after each milking. Teat dip was applied immediately after exposure to the bacteria suspension. Challenge was carried out daily for 5 consecutive days. 2nd and 3rd sampling were done on Day 8 and Day 10, respectively. Samples collected: milk sample for quality check and bacteria CFU count.
(iii) Sampling procedures in mastitis challenge trial
(a) Milk sampling. All milk samples were collected immediately prior to regular automated milking. Briefly, three or four streams of foremilk (procedure stated in pre-milking udder preparation in supplementary methodology of mastitis farm trial below) were discarded from each quarter before sanitizing teat ends with cotton swabs and collecting samples. Approximately 10 mL of milk samples from each teat were collected daily starting from the onset of the experiment. To determine the milk quality (e.g. somatic cell count), milk samples were passed and tested by qualified testing labs within 24 hours, (b) Bacteria CFU count. The milk samples were collected according to the procedures stated above. The number of microbes in milk was counted using standard protocol, (c) Criteria for diagnosing infections. A new intramammary infection in a quarter was diagnosed when the same bacterial species was isolated from 1) two consecutive samples during the trial (>500 CFU/ml); 2) a single sample from a quarter with clinical mastitis (> 100 CFU/ml); or 3) three consecutive samples during the trial (> 100 CFU/ml).
Supplementary methodology of mastitis farm trial
Farm Condition
The trial was done in the local wintertime with outdoor temperature ranging from -10 to 10 °C. The farm housed over 500 dairy cows and is equipped with an automated milking system. Cows were housed in the barn and were milked at a separate milking facility with ambient temperature of 5 to 10 °C. Milking is routinely done once per day at 2pm by a skilled worker. Post-milking teat dipping with iodine-based commercial product was practiced daily in the farm and was ceased 10-days prior to the start of the trial on the experimental cows to avoid carryover effect and disturbance on the farm trial results.
Preparation of product solution
The product stock solution was stored at 4 °C. The product working solution was prepared fresh on the day of the experiment by diluting the stock solution (1/30 ratio) in sterile DI water to achieve a final concentration of 0.05% active compound and 10% glycerol.
Pre-milking udder preparation
Pre-milking udder preparation consists of the use of single service water-moistened towels (free of sanitizer, one towel per teat) to wet and clean the teats prior to fore-stripping. Forestripping was accomplished by expressing three squirts of milk.
Sampling procedures in safety trial
(i) Polymer adsorption on skin
Swab sampling was carried out to determine the polymer adsorption on teat skin. After cleaning the teats (procedure stated in pre-milking udder preparation), the samples were taken using the wet and dry swab technique in accordance with DIN 10113-1 : 1997-07 (Scheib, S. et al., Pathogens 2023, 12, 560). A cotton wool swab moistened with sterile 0.25% Ringer’s solution was moved around the teat at a distance of 1 cm from the teat canal orifice. After that the same procedure was performed with a dry cotton wool swab (ultrafine, dry swab). Both swabs were shortened and inserted into one test tube containing 2 mL of the sterile 0.25% Ringer’s solution. The solution was used to determine polymer residuals using Delvo test Kit. Briefly, 200 pl of the test samples were added to the Delvo test ampoules and were incubated at 63 °C for 3 hours. The color change of the solid agar at the bottom of Delvo test ampoules was recorded. (ii) Milk sampling
The milk samples were collected according to the procedure stated in the in vivo mastitis farm trial protocol above. To determine the polymer residual in milk, the milk samples were transferred to lab to determine polymer residuals using Delvo test kit (Stead, S. et al., Int. Dairy J. 2008, 18, 3-11). To determine the milk quality (e.g., somatic cell count), milk samples were tested by qualified testing labs within 24 hours.
Preparation of challenge culture for mastitis challenge trial
S. aureus ATCC 49525 was used in farm trial due to its close relevance to clinical bovine mastitis (Wall, R. J. et al., Nat. Biotechnol. 2005, 23, 445-451). A single colony was streaked out from agar plate into Trypticase Soy Broth (TSB) and incubated overnight under shaking at 37 °C. The overnight culture was sub-cultured 1 :100 into fresh TSB and incubated for 3 hours to obtain exponentially growing bacteria. Bacteria were pelleted by centrifugation (3,000-4,000 g for 15 min), washed twice with 0.1% proteose-peptone and diluted to ~ 5 x 107 CFU/ml in fresh TSB. The challenge suspension containing ~ 5 x 107 CFU/ml in TSB was prepared immediately before use.
In vivo mastitis safety t at testing
In the safety test of post-milking teat dip application, evaluation of (1) irritation responses on PIM1 D treated teats, (2) polymer residuals on teat surface and milk samples of PIM1 D treated teats and (3) changes in milk compositions were performed.
Upon the daily application of post-milking PIM1D teat dip for 5 consecutive days, we observed no irritation response (e.g. redness, edema, roughness, additional lesion) for the PIM1 D- treated teats (Fig. 38). The cows also did not show abnormal behaviors (e.g., restless, kicking or rubbing at abdomen area), which are signs of irritating and itching teat skin. Moreover, no polymer residuals were detected on the teat surface and in milk samples as confirmed by Delvo Test2 (<10 ppm, limit of detection), indicating that the compound can be easily washed off during the pre-milking cleaning steps (Fig. 39). Besides, PIM1 D-based teat dip did not cause any significant changes in milk composition, including somatic cell count (SCC), protein, fat and solids not-fat (SNF) contents (Fig. 40).
Results and discussion
We first tested the in vitro bactericidal activity of our compounds in milk against mastitiscausing pathogens, specifically E. coli 10536, S. aureus 6538 and S. uberis 19436, following the industrial standard BS EN 1656 (Institution, B. S. (British Standards Institution, 2019)). Effective test compounds are required to achieve a minimum of 5 logic reduction of viable bacteria within 30 minutes or less. The control drug chlorhexidine exhibited poor performance, failing to achieve the required 5 logw reduction for the two Gram-positive bacteria (Fig. 41 A). However, OIM1-8-2D (16) demonstrated the required 5 logw reduction for E. coll and S. aureus in 30 minutes, falling just short with a 4.77 logw reduction for S. uberis (Fig. 41 B). We then explored the longer polymer version of (16), which is the PIM1 D, a compound we had previously reported (Zhong, W. et al., Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 31376-31385). PIM1 D met the 5 logw reduction requirement for all three bacteria (Fig. 41C). Moving from laboratory testing to practical application, a farm trial was conducted using PIM1 D as a postmilking teat dip antibacterial agent to prevent dairy mastitis infection (Fig. 36C). Prior to this, we assessed the safety of PIM1 D-based teat dip and confirmed that it did not cause no adverse effects on the cows (Figs. 38-40). In the mastitis challenge trial, we determined the onset of intramammary infection (IM I) based on bacteria count (>500 CFU/mL) and somatic cell count (SCC, > 200,000/mL) in milk samples (Nickerson, S. et al. in NMC Annual Meeting Proceedings. 379-399 (Citeseer)). According to both indicators, PIM I D-based teat dip successfully prevented mastitis infection upon repeated exposure to S. aureus e, whereas cows treated with glycerol control developed mastitis over time (Figs. 36D-E).
Importantly, the PIM1 D-based teat dip did not affect the milk composition (Fig. 41 D).
Discussion
An engineered degradable OIM derivative demonstrated outstanding efficacy in a murine model of sepsis infection, notably against multi-drug resistant Gram-negative Acinetobacter baumannii strain. Additionally, we have extended our findings to the agriculture domain, where a degradable OIM/PIM, act as effective prophylactic agents against diary mastitis, a pervasive global issue.
The present disclosure underscores the pivotal role of carbon acidity in the antibacterial properties of OIMs, particularly those containing deprotonable C(2)-H groups (1, 5-8, 10, 12 and 15-16). The carbon acids (1, 5-8, 10, 12 and 15-16) exhibit robust antibacterial efficacy, while non-carbon acids lacking C(2)-H (2-4, and 11) show considerably reduced effectiveness. Compared with classical advanced cationic polymers that we and others have invented, the OIM carbon acids have MICs that are about one order of magnitude lower and/or have broad spectrum potency, and with lower toxicity (Lam, S. J. et al., Nat. Microbiol. 2016, 1, 11 ; Chin, W. et al., Nat. Commun. 2018, 9, 14; and Zhang, K. et al., Nat. Commun. 2019, 10, 4792).
The disappearance/deprotonation of the C(2)-H of (1) and (5) in neutral aqueous water, was shown by 1H NMR, corroborating their carbon acidity. The NHCs formed from OIM carbon acid (1) are detectable within the aprotic membrane bilayer with the AuCI(SMe2) probe and carbazole dye, corroborating that the carbon acids efficiently penetrate bilayer membranes with NHC formation.
The classical cationic polymers cannot enter the bacterial plasma membrane as the charged polymers are hydrophilic. Above certain critical cationic charge concentrations, the cationic polymers form pores or holes in the membranes which then lead to cell death. As this is a physical process, the threshold concentrations are typically higher so that the MIC values are usually tens of microgram/mL. With the new NHC-mediated entry into the membrane and then cytosol, no pores or holes are needed for the entry into the membrane. The OIM carbon acids (1 and 5) enters the liposome/bacterial core at lower cationic charge threshold concentration than (2). The OIM (2) cannot enter the bacterial cytosol or liposome core (Figs. 31 B and 25I, respectively) as compared with (5) or (1) and has much higher MIC as it is solely dependent on the physical membrane disruption process. The OIM (1), though likely to be more hydrophilic than the methyl-substituted derivatives (2) and (5) can enter the membrane and core of liposomes more effectively, as it effectively forms NHC to enter the core through membrane translocation. The OIM transforms with NHC formation from an initially hydrophilic to an amphiphilic copolymer which is composed of the hydrophilic imidazolium cation repeats and also the hydrophobic imidazol-2-yl carbene repeats, to achieve effective membrane entry (Fig. 24B). The positively charged nature of the cationic OIMs caused their electrostatic attraction to the negatively charged membrane surface of bacterial-mimicking liposomes, while the hydrophobic NHC repeats aid the insertion of OIMs into the liposome membrane, as supported by our simulation data (Fig. 29). This reveals a novel mechanism for membrane entry of cationic polymers that are also carbon acids, through NHC formation.
Regarding the translocation of OIMs from the membrane into the liposome core, it is worth noting that the quantity of (1) and (5) entering the liposome core is smaller in comparison to their presence in the membranes (Fig. 25I). In live bacteria which has a functional PMF, the electric field across the membranes may favor the translocation of OIM-NHC into the cytosol. Alternatively, the acidic protons of cycling reduced quinone shuttiers of redox-active electron transport chain (ETC) might induce the quenching of OIM carbene into cationic OIM within the bacterial membrane, rendering it hydrophilic. This shift in hydrophobicity could drive OIMs to exit the hydrophobic membranes and enter the cytosol. Without wishing to be bound by theory, regardless of the precise transport mechanism from the membrane to the cytosol, the present disclosure demonstrates that carbon acids, aided by NHC formation, successfully infiltrate the bilayer membrane and exhibit high cytosolic uptake, ultimately reaching their intracellular targets, and hence resulting in rapid bacterial killing. The facile NHC formation of (1) facilitates its internalization into the bacterial cytoplasm, even under conditions of low PMF, as evidenced by experiments with respiration-deficient mutants and fermentative bacteria.
Moreover, our optimized degradable compounds (15) and (16) exhibit impressive minimum inhibitory concentrations (MICs) against multi-drug resistant bacteria, including the colistinresistant Gram-negative bacterium E. cloacae 13047 that is MDR. In murine infection sepsis model, the degradable OIM (16) demonstrated substantial efficacy against multi-drug resistant A. baumannii bacteria. Additionally, in a field trial, PIM1 D was successfully employed to prevent mastitis infection, with no adverse effects observed in the cows.
Example 13. Study of different counter-ions
General procedure for making compound OIM1-8-2D with different counter-ions
The OIM1-8-2D(4Br/4CI ) compound was treated with aqueous triethylamine (base, EtsN) to form the octa N-heterocyclic carbene (NHC) and then quenched with organic acids to generate the carboxylate imidazolium ionic liquids.
Table 11. OIMs with different counter-ions.
Figure imgf000102_0001
Results and discussion
The properties of the various exchanged OIMs are summarised in Table 12 below. Table 12. Bioassay results (MIC and MTT assays - performed as described in Example 5) of anion exchanged OIM1-8-2D.
Figure imgf000103_0002
a Geometric mean of half-maximal inhibitory concentration (ICso) is calculated from the IC50 of 3 cell lines: 3T3 fibroblast cells, human embryonic kidney (HEK) cells and liver hepatocellular carcinoma (HepG2) cells. b Selectivity index (SI) is the ratio of Geo-ICso of a specific timepoint to Geo-MICso.
5 pneumoniae (ATCC 13883); AB is A. baumannii (ATCC 19606); PA is
Figure imgf000103_0001
13047 (-).
Conclusion
Herein, we investigated the mechanisms by which precise main chain imidazolium oligomers with exact molecular weights kill bacteria. Derivatives of oligoimidazoliums (OIMs) with same repeat units but having different substituents at the C2- and Oppositions of the imidazolium ring (Fig. 1) were made via step-by-step synthesis and tested for their antibacterial and toxicity profiles. Derivatives having C2-protons were generally potent antimicrobials, while derivatives with C2-hydrogen replaced by substituents were generally non-antibacterial by comparison. The parent compound OIM1-6-H (1 , Fig. 1) with C2-hydrogen was found to act as carbon acids to form N-heterocyclic carbenes in bacterial membrane mimics at physiological pH and show substantial uptake into bacteria, even in the in respiration mutants of Methicillin-resistant Staphylococcus aureus (MRSA) without a functional electron transport chain (ETC). The degree of uptake and NHC formation may be tuned as shown with methyl-derivatives (compounds 2 and 5, Fig. 1). Further, the effectiveness of the fast-killing NHC-forming cationic OIMs, including degradable derivatives, in complex realistic application settings such as in consumer care products are shown.
We show here that oligomers with the imidazolium ring containing dissociable C2-proton, i.e. carbon acids, form NHCs in the hydrophobic bacterial-mimicking membrane bilayer. The ability of the carbon acids to form NHC with unsubstituted C2-carbon is correlated with good antibacterial properties. The formation of NHC enables the easy entry of the polymer into the bacterial cytoplasm without the need for a PMF as shown by the uptake of OIM1-6-CH into respiration defective mutants.
The ability to form NHC is correlated with the pKa values of the carbon acids. Carbon acids with higher pKa (with electron donating methyl substituent) makes the ability to form NHC more difficult resulting in lower toxicity but only slightly reduced antibacterial properties. The toxicity of OIM1-6-CH at short term (24 hours) is excellent but it has toxicity longer term (48-72 hours) probably due to diffusion. However, the toxicity can be dampened in OIM1-6-C4(CH3) which has lower rate of carbene formation.
This is a new mechanism of entry and uptake of a polymer by bacteria via the amphiphilic nature of the resulting copolymer of cationic imidazolium and carbene where the imidazolium is cationic and hydrophilic and the carbene is hydrophobic. It can achieve good MIC against multi-drug resistant bacteria also including gram-negative Bacteria that are intrinsically resistant to antibiotics because of their outer membranes. It can also achieve good MICs against colistin-resistant bacteria (E. cloacae 13047-MDR) corroborating that the mechanism of kill is distinct to that of cationic peptides. Further, we showed that the schizophrenic nature of imidazolium allows it to escape the trapping of OIM by polyanions which prevent the usage of the OIMs in many real world and physiological applications. We showed that it can kill both Gram-positive and Gram-negative bacteria in detergent formulation in a fast manner making such cationic compound uniquely advantageous. The diverse possibilities of modifying the ring and side chain structures offers to this class of carbene forming cationic oligomers new properties not possible before to kill multidrug resistant bacteria in complex environments. We shall call this new family of compounds “cat-bene-biotics”.
AMPs and antimicrobial polymers (AMPs) are potential pharmaceutical candidates to replace antibiotics as they are effective against a broad spectrum of multidrug resistant bacteria. However, issues such as cytotoxicity and fouling are drawbacks of AMPs. We show that the oligomer OIM1-6-CH which has a hydrogen (H) at the C2 carbon with exact 6 repeating imidazolium units has potent antibacterial activity with low short-term eukaryotic toxicity. Structure-activity relationship (SAR) surrounding the imidazolium moiety was also studied by various substitutions at the C2 and C4 position. The antibacterial properties from analogues of 04 substitution suggest that the unsubstituted C2 carbon is required for ideal potency of the oligomer, while any substitution at the 02 position led to significant loss in potency. We then showed that the acidic C2-hydrogen at the imidazolium structure could form N-heterocyclic carbene (NHC) in liposomes at physiological pH, while substitutions at the C2 position prevents the NHC formation. Using OIM1-6-CH and methyl-substituted OIM1-6-C2/C4(CH3), we found that unsubstituted C2 proton is essential for the internalization of the OIMs into bacteria which correlates to its potency. While the PMF aided the intracellular uptake of the OIMs, OIM1-6-CH could still internalize into the cytosolic of respiration deficient mutants and fermentative bacteria via a second mechanism, which is the NHC formation. Inhibiting the NHC formation of OIM1-6-CH and OIM1-6-C4(CH3) with slightly acidic pH greatly reduced their potency against S. aureus LAC and LAC respiratory mutants, conferring the importance of carbene formation in the efficacy of our compounds. This additional mechanism aids to retain the excellent antibacterial properties of OIMs in the presence of high salts and surfactants as illustrated in the cloth test in Example 10.

Claims

Claims
1. A compound according to formula la:
Figure imgf000106_0001
where:
X" is an anionic species selected from an organic acid in its carboxylate form, Br, |- or Cl"
Y represents, OH, NH2, a zwitterionic species or a hydrazone group; each L independently represents:
Figure imgf000106_0002
where each wiggly line represents a point of attachment to the rest of the molecule, or a compound according to formula lb:
Figure imgf000107_0001
where each L is independently selected from the list provided above; and
X" is as defined above, or a compound according to formula Ic:
Figure imgf000107_0002
where:
Ri 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 to which they are attached form a benzene ring; n represents 6 or 8;
X' is as defined above, or a compound according to formula Id:
Figure imgf000108_0001
where each L is independently selected from the list provided above; and X' is as defined above, and solvates of the compounds of formula la-ld.
2. The compound according to Claim 1, wherein the compound has 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" is 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- is Ch.
8. The compound according to Claim 1 , wherein the compound has formula la or formula lb.
9. The compound according to Claim 8, wherein each L represents:
Figure imgf000109_0001
, where the wiggly lines represent the points of attachment to the rest of the molecule.
10. The compound according to Claim 1 , wherein the compound is a compound of formula
Figure imgf000109_0002
la, wherein X' is Cl' and each L is
11 . The compound according to Claim 1 , wherein the compound is a compound of formula
Figure imgf000109_0003
lb wherein X' is 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 carrier.
13. Use of a compound according to any one of Claims 1 to 11 or a pharmaceutical composition according to Claim 12 in medicine.
14. A compound according to any one of Claims 1 to 11 or a pharmaceutical composition according to Claim 12 for use in the treatment of one or both of a microbial and a fungal infection.
15. Use of a compound according to any one of Claims 1 to 11 or a pharmaceutical composition according to Claim 12 in the manufacture of a medicament to treat one or both of a microbial and a fungal infection.
16. A method of treating one or both of a microbial and a fungal infection comprising the step of administering a pharmaceutically effective amount of a compound according to any one of Claims 1 to 11 or a pharmaceutical composition according to Claim 12 to a subject in need thereof.
17. An antimicrobial and/or antifungal detergent composition comprising: a compound as described in any one of Claims 1 to 11 ; and a surfactant.
18. The antimicrobial and/or antifungal detergent composition according to Claim 17, wherein the composition is in the form of a solid or liquid soap.
19. The antimicrobial and/or antifungal detergent composition according to Claim 18, wherein the composition is in the form of a shampoo.
PCT/SG2024/050366 2023-06-01 2024-06-03 Main chain cationic oligo(imidazolium) forms n-heterocyclic carbene for effective bacterial killing in complex environment WO2024248739A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6416770B1 (en) * 1998-01-26 2002-07-09 L'ORéAL S.A. Use of heterocyclic quaternary polyammonium polymers as protective agent for keratin fibres and cosmetic compositions
WO2019004940A1 (en) * 2017-06-30 2019-01-03 Agency For Science, Technology And Research Degradable imidazolium oligomer and polymer for antimicrobial applications
WO2021242174A1 (en) * 2020-05-26 2021-12-02 Nanyang Technological University Biodegradable polyimidazoliums and oligoimidazoliums

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6416770B1 (en) * 1998-01-26 2002-07-09 L'ORéAL S.A. Use of heterocyclic quaternary polyammonium polymers as protective agent for keratin fibres and cosmetic compositions
WO2019004940A1 (en) * 2017-06-30 2019-01-03 Agency For Science, Technology And Research Degradable imidazolium oligomer and polymer for antimicrobial applications
WO2021242174A1 (en) * 2020-05-26 2021-12-02 Nanyang Technological University Biodegradable polyimidazoliums and oligoimidazoliums

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