Biodegradable poly- and oligo-imidazoliums

Abstract

Disclosed herein are compounds in the form of polymers, oligomers, and defined molecules having repeating units, all of which incorporate a repeating unit formed from an imidazolium group and a biodegradable chain linked to an adjacent repeating unit. The compounds disclosed herein can have antimicrobial activity and thus can be used to treat microbial infections and / or to treat surfaces to prevent microbial infections. Also disclosed herein are methods of forming the compounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of polyimidazolium and oligoimidazolium and defined molecules with similar characteristics. These molecules all contain degradable, in particular biodegradable, moieties, enabling them to be broken down in the body. These molecules can be used to treat microbial infections or act as antimicrobial agents (for example, in personal care products or on surfaces). BACKGROUND

[0002] The listing or discussion of an allegedly 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.

[0003] The emergence and spread of multidrug-resistant (MDR) pathogens is a highly concerning problem worldwide. Recently, the World Health Organization (WHO) made a call to action to develop new antibacterial agents for the most problematic superbugs, including carbapenem-resistant Acinetobacter baumannii (CRE-AB), carbapenem-resistant Pseudomonas aeruginosa (CRE-PA), and carbapenem-resistant Enterobacteriaceae (CRE-EB) producing extended-spectrum beta-lactamases (ESBLs) (World Health Organization (WHO). Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. 2017).

[0004] Antimicrobial peptides (AMPs) are considered promising candidates for treating multidrug-resistant (MDR) bacteria. The basic design elements of AMPs include hydrophobic properties and regions of charged residues (typically cationic residues to enable interaction with the bacterial cell surface) to disrupt the bacterial cell membrane (Ganewatta, MS et al., Polymer 2015, 63, A1-A29). However, the development of AMPs is generally hampered by their poor pharmacokinetic properties, low stability in biological fluids, toxicity to mammalian cells due to poor selectivity, and typically high minimum inhibitory concentrations (MICs) compared to classical antibiotics. Clinically, however, naturally complex AMPs such as cyclic lipopeptides (e.g., polymyxins) are used to target difficult-to-treat Gram-negative bacterial infections. However, their high cost and toxicity currently limit their use as a last-resort alternative. Coitin, an antimicrobial peptide, has recently seen increased use as a last resort antibiotic due to its believed ability to kill bacteria by disrupting membrane integrity (Velkov, T. et al., J. Med. Chem. 2010, 53, 1898-1916). However, coitin requires intravenous administration and is nephrotoxic (Javan, AO et al., Eur. J. Clin. Pharmacol. 2015, 71, 801-810).

[0005] Besides peptides, synthetic polymers are widely used as disinfectants due to their high antimicrobial potency. Most of these polymers are synthesized via free radical polymerization (FRP), ring-opening polymerization (ROP), and post-functionalization, which typically involve multiple steps, are difficult to purify, use organic solvents, and are difficult to scale up. These antimicrobial polymers generally exhibit high toxicity and limited-range antimicrobial efficacy.

[0006] Therefore, there is a need to develop new AMP-like analogs with improved properties. Summary of the Invention

[0007] The aspects and embodiments of the invention will now be described with reference to the following numbered clauses.

[0008] 1. A polymer or oligomer or a pharmaceutically acceptable solvate thereof, comprising a first repeating unit, the first repeating unit comprising an imidazolium group and a biodegradable chain attached to an adjacent repeating unit.

[0009] 2. The polymer or oligomer as described in Clause 1, wherein the only repeating unit is the first repeating unit.

[0010] 3. The polymer or oligomer of clause 1, wherein the polymer or oligomer further comprises a second repeat unit comprising an imidazolium group and a non-biodegradable alkyl chain or another biodegradable alkyl chain connected to an adjacent repeat unit, optionally wherein the polymer or oligomer further comprises a second repeat unit comprising an imidazolium group and a non-biodegradable alkyl chain connected to an adjacent repeat unit.

[0011] 4. The polymer or oligomer of clause 3, wherein one or more of the following apply:

[0012] (a) the polymer or oligomer comprises 1 to 75 mol%, such as 5 to 60 mol%, such as 10 to 50 mol%, such as 20 to 30 mol% of the first repeat unit; and

[0013] (b) the repeat units of the polymer or oligomer are randomly distributed or the repeat units can be formed into blocks, optionally wherein the repeat units of the polymer or oligomer are randomly distributed.

[0014] 5. The polymer or oligomer of any one of the preceding clauses, wherein the biodegradable chain in the first repeat unit comprises one or more biodegradable functional groups, wherein the one or more biodegradable functional groups are selected from one or more of the group consisting of: urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0015] (ai) the one or more biodegradable functional groups are selected from one or more of the group consisting of: amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0016] (aii) the one or more biodegradable functional groups are selected from one or more of the group consisting of: carbamate, or more particularly, amide, ester, and carbonate; or

[0017] (aiii) the one or more biodegradable functional groups are amide.

[0018] 6. The polymer or oligomer of any one of the preceding clauses, wherein the number average molecular weight is 800 to 10,000 Daltons, such as 900 to 5,000 Daltons, such as 1,000 to 3,000 Daltons, such as 1,000 to 2,000 Daltons.

[0019] 7. The polymer or oligomer of any one of the preceding clauses, wherein the polymer or oligomer is of Formula I:

[0020]

[0021] wherein:

[0022] x is 0.01 to 1.0;

[0023] Y - is a counterion;

[0024] o is 0 to 10 (e.g., 0 to 6, such as 1 to 5);

[0025] p is 1 to 12;

[0026] q is 0 to 14 (e.g., 0 to 6);

[0027] r is 0 to 12;

[0028] D is a biodegradable functional group;

[0029] D’ is a biodegradable functional group or bond;

[0030] each R 1 is a branched or unbranched C 1-3 alkyl group or derivative thereof;

[0031] each t is 0, 1, or 2 (e.g., t is 0 or 1);

[0032] each t’ is 0, 1, or 2 (e.g., t’ is 0 or 1);

[0033] each R 2 is a branched or unbranched C 1-3 alkyl group or derivative thereof;

[0034] or a pharmaceutically acceptable solvate thereof.

[0035] 8. The polymer or oligomer of clause 7, wherein one or more of the following apply:

[0036] (bi) each D is selected from urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0037] (aa) each D is selected from one or more of the group consisting of amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0038] (ab) each D is selected from one or more of the group consisting of carbamate, or more particularly, amide, ester, and carbonate; or

[0039] (ac) each D is selected from one or more of the group consisting of carbonate and amide (e.g., each D is an amide);

[0040] (bii) each D’ is selected from the group consisting of a bond, urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0041] (ad) each D’ is selected from one or more of the group consisting of a bond, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0042] (ae) each D’ is selected from one or more of the group consisting of a bond, amide, ester, carbamate, and carbonate;

[0043] (af) each D’ is selected from one or more of the group consisting of a bond and amide;

[0044] (ag) each D’ is selected from one or more of the group consisting of amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0045] (ah) each D’ is selected from one or more of the group consisting of amide, ester, carbamate, and carbonate;

[0046] (ai) each D’ is amide;

[0047] (biii) Y - is selected from one or more of the group consisting of halogen, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ); - is selected from one or more of the group consisting of halogen, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - );

[0048] (biv) x is 0.01 to 1.0, such as 0.025 to 0.75, such as 0.05 to 0.6, such as 0.1 to 0.5, such as 0.2 to 0.3;

[0049] (bv) t and t’ are 0;

[0050] (bvi) p is 1 to 6; and

[0051] (bvii) r is 1 to 6.

[0052] 9. The polymer or oligomer of any one of the preceding clauses, wherein the polymer is selected from the group consisting of:

[0053]

[0054]

[0055] 10. A molecule, or a pharmaceutically acceptable solvate thereof, comprising:

[0056] a first block of oligomer repeat units, wherein each repeat unit comprises an imidazolium group and a non-biodegradable alkyl chain connected to an adjacent repeat unit;

[0057] a second block of oligomer repeat units, wherein each repeat unit comprises an imidazolium group and a non-biodegradable alkyl chain connected to an adjacent repeat unit; and

[0058] a linking group connecting the first block and the second block together, wherein the linking group comprises one or more biodegradable functional groups.

[0059] 11. The molecule of clause 10, wherein the one or more biodegradable functional groups are selected from one or more of the group consisting of: urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0060] (ci) the one or more biodegradable functional groups are selected from one or more of the group consisting of: amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0061] (cii) the one or more biodegradable functional groups are selected from one or more of the group consisting of: carbamate, or more particularly, amide, ester, and carbonate;

[0062] (ciii) the one or more biodegradable functional groups are selected from one or both of: amide and carbonate; or

[0063] (civ) the one or more biodegradable functional groups are amide.

[0064] 12. The molecule of clause 10 or clause 11, wherein the molecular weight is 1,000 Daltons to 5,000 Daltons, optionally wherein the molecular weight is 1,000 Daltons to 4,000 Daltons.

[0065] 13. The molecule of any one of clauses 10 to 12, wherein the molecule is of Formula II:

[0066]

[0067] wherein:

[0068] each m is independently 1 to 8 (e.g., 1 to 6);

[0069] each Y - is a counterion;

[0070] n' is 0 to 12;

[0071] each o' is independently selected from 0 to 20;

[0072] each p' is independently selected from 0 to 12 (e.g., 0 to 6);

[0073] each p" is independently selected from 0 to 12 (e.g., 0 to 6);

[0074] each T is independently a terminal functional group selected from amine, ammonium, guanidinium, biguanidinium, alkyl, and aryl;

[0075] each D is a biodegradable functional group, or a pharmaceutically acceptable solvate thereof.

[0076] 14. The molecule according to Clause 13, wherein one or more of the following apply:

[0077] (di) each D is independently selected from urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0078] (ba) each D is independently selected from one or more of the group consisting of amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0079] (bb) each D is independently selected from one or more of the group consisting of carbamate, or more particularly, amide, ester, and carbonate; or

[0080] (bc) each D is an amide;

[0081] (dii) Y - is selected from one or more of the group consisting of halogen, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ), optionally wherein Y - is selected from one or more of the group consisting of halogen, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ); and

[0082] (dii) p" is 0 to 6 (e.g., p" is 0).

[0083] 15. The molecule according to any one of Clauses 10 to 14, wherein the molecule is selected from the group consisting of:

[0084]

[0085]

[0086] 16. The polymer or oligomer or pharmaceutically acceptable solvate thereof according to any one of Clauses 1 to 9 and / or the molecule or pharmaceutically acceptable solvate thereof according to any one of Clauses 10 to 15, for use in medicine.

[0087] 17. Use of the polymer or oligomer or a pharmaceutically acceptable solvate thereof under any one of Clauses 1 to 9 and / or the molecule or a pharmaceutically acceptable solvate thereof under any one of Clauses 10 to 15 in the manufacture of a medicament for treating diseases including microbial infections.

[0088] 18. The polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of Clauses 1 to 9 and / or the molecule or a pharmaceutically acceptable solvate thereof according to any one of Clauses 10 to 15, for use in the treatment of diseases including microbial infections.

[0089] 19. A method of treating a disease including a microbial infection, comprising administering to a subject in need a therapeutically effective amount of a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of clauses 1 to 9 and / or a therapeutically effective amount of a molecule or a pharmaceutically acceptable solvate thereof according to any one of clauses 10 to 15.

[0090] 20. Use of the polymer or oligomer or molecule as described in Clause 17, the polymer or oligomer or molecule as described in Clause 18, and the method as described in Clause 19, wherein the microbial infection is an infected wound or cystic fibrosis.

[0091] 21. A disinfectant formulation comprising a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of Clauses 1 to 9 and / or a molecule or a pharmaceutically acceptable solvate thereof according to any one of Clauses 10 to 15.

[0092] 22. An article having a surface, wherein the surface is coated with a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of clauses 1 to 9 and / or a molecule or a pharmaceutically acceptable solvate thereof according to any one of clauses 10 to 15 to provide antimicrobial properties to the surface of the article, optionally wherein the article is a urinary catheter. Attached Figure Description

[0093] Figure 1 The chemical structures of polyimidazolium (PIM) synthesized and used in the experiment were determined. The number of repeating subunits of each PIM was estimated by gel permeation chromatography (GPC).

[0094] Figure 2Viability of (A) P. aeruginosa PAOl ; and (B) MRSA LAC* treated with PIMl (0.5-4 times MIC for each bacterial species) compared to controls with no PIMl added. Cells were incubated in MHB at 37°C and sampled at the times indicated. Cell numbers were determined as colony forming units (CFU) per mL by plating.

[0095] Figure 3 Iodide propidium (PI) staining of P. aeruginosa PAOl cells is depicted. (A) Control cells (no antibiotic); (B) cells treated with colistin (1 times MIC); (C) fluorescence microscopy images of cells treated with PIMl (1 times MIC); and (D) percentage of iodide propidium (PI) positive cells exposed to the indicated concentrations of PIMl (blue, left bars) or colistin (orange, right bars) as determined by flow cytometry. Cells were incubated in the presence of the indicated antibiotic for 1 h prior to microscopy or flow cytometry.

[0096] Figure 4 Relative levels of cell membrane potential (ΔΨ) of P. aeruginosa PAOl cells exposed to increasing concentrations of PIMl, the ionophore gramicidin, or the antibiotic gentamicin are depicted. Relative membrane potential was assessed by using the ΔΨ-sensitive fluorescent membrane probe DiS-C3-(5). An increase in DiS-C3-(5) fluorescence corresponds to dissipation of ΔΨ. The ionophore gramicidin is a control agent known to collapse ΔΨ, and uptake of the antibiotic gentamicin requires ΔΨ but does not dissipate ΔΨ. The relative dye fluorescence values shown are the average of four tests (from 2 runs, each with replicates) with (small) standard deviations 30 min after addition of the test compounds.

[0097] Figure 5 Uptake of PIMl -FITC conjugate by P. aeruginosa PAOl and the relationship between PIMl activity and membrane potential are depicted. (A) fluorescence microscopy images of control cells (without PIMl) stained with the membrane dye FM™ 4-64FX; (B) fluorescence microscopy images of cells treated with PIMl -FITC (1 times MIC) and stained with FM™ 4-64FX; (C) MIC of PIMl against P. aeruginosa in MHB with various pH adjusted 90 (μg / mL); and (D) MIC of PIMl against P. aeruginosa PAOl in the presence of valinomycin (left bars) or nigericin (right bars) 90 (μg / mL).

[0098] Figure 6The effect of metabolic state on PIM1 killing of P. aeruginosa PAOl is shown. (A) Survival of stationary phase (Sta) bacteria and log phase (Log) bacteria after exposure to PIM1, CST, or GEN 4-h; (B) effect of fumarate (15 mM) on survival of stationary phase bacteria. The same results for Sta-PIM1, Sta-CST, and Sta-GEN were used in A and B.

[0099] Figure 7 The evolution of antibiotic resistance in (A) P. aeruginosa PAOl and (B) MRSA LAC* is shown. P. aeruginosa was grown in MHB containing different concentrations of PIM1 or ciprofloxacin, and MRSA was grown in TSB containing different concentrations of PIM1 or ciprofloxacin. Bacteria showing visible growth at the highest antibiotic concentration were transferred daily. Data are reported as the highest antibiotic concentration at which growth was observed, and are given as fold increase in concentration relative to the MIC 90

[0100] Figure 8 PIM1 treatment of skin wound infection is shown. Wounds were infected with pan-antibiotic resistant P. aeruginosa PAER and treated with 5 mg / kg imipenem (P. aeruginosa PAER is imipenem resistant) or 0.1, 1, 5, or 10 mg / kg PIM1 4 h post-infection. Bacterial numbers were determined by plating and data for each individual mouse is reported. Horizontal lines indicate the mean and bars ± SD. *P < 0.05, **P < 0.01, and ns indicates P > 0.05.

[0101] Figure 9 PIM1 (but not PIM1D) has significant toxicity is shown. (A) Weight of mice treated with a single 6 mg / kg dose of PIM1 (day 0) or a daily dose of 15 mg / kg PIM1D for one week (days 0-6) by intraperitoneal (IP) injection. There were five mice in each group. (B) Alanine aminotransferase (ALT); (C) aspartate aminotransferase (AST), and (D) blood urea nitrogen (BUN) levels in the blood of mice treated with 15 mg / kg PIM1D for 7 days daily. Blood from mice given saline solution mock injections was drawn before the initial injection and 1 day later. Blood from PIM1D-treated mice was drawn 1 day, 3 days, and 7 days after the first injection dose. There were five mice in each group, and data for individual mice are shown as the mean and standard deviation.

[0102] Figure 10 ​A schematic of the synthesis of degradable PIM1D incorporating amides is shown: (A) synthesis scheme of degradable diamine A; and (B) synthesis scheme of degradable PIM1D incorporating amides (n is the actual number average degree of polymerization and x is the mole fraction of degradable repeat units. n is about 10 and x is 20-30%).

[0103] Figure 11 PIM1D is shown to be effective in IP sepsis models induced by P. aeruginosa PAO1, MDR P. aeruginosa (PAER), MDR A. baumannii, and methicillin-resistant S. aureus MRSA USA300. (A) P. aeruginosa PAO1; (B) MDR P. aeruginosa (PAER); (C) MDR A. baumannii (AB-1); and (D) methicillin-resistant S. aureus MRSA USA300 induced septicemia models. Colony forming unit (CFU) counts of liver. Kaplan-Meier curves represent (E) PAO1; (F) PAER; (G) AB-1; and (H) MRSA USA300 induced septicemia models. Geometric mean ± s.d., n = 5. One-way ANOVA; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

[0104] Figure 12 (A-C) P. aeruginosa PAO1; (D-F) MDR P. aeruginosa (PAER); (G-I) MDR A. baumannii (AB-1); and (J-L) methicillin-resistant S. aureus MRSA USA300 induced septicemia models are shown. CFU counts of kidney, spleen, and IP fluid. CFU counts of (A) kidney; (B) spleen; and (C) IP fluid in PAO1 induced septicemia models. CFU counts of (D) kidney; (E) spleen; and (F) IP fluid in PAER induced septicemia models. CFU counts of (G) kidney; (H) spleen; and (I) IP fluid in AB-1 induced septicemia models. CFU counts of (J) kidney; (K) spleen; and (L) IP fluid in MRSA USA300 induced septicemia models. *P < 0.05, **P < 0.01, and ns is not significant (two-tailed Student’s t-test).

[0105] Figure 13Blood biochemistry analyses depicting day 1, day 3, and day 7, in which mice received a single dosing, three consecutive dosings, and seven consecutive dosings of PIM1D (15 mg / kg) via IP injection, respectively. (A) Alanine aminotransferase (ALT); (B) aspartate aminotransferase (AST); (C) blood urea nitrogen (BUN); (D) creatinine (CRE); (E) total bilirubin (TBIL); (F) total protein (TP); (G) globulin (GLO); and (H) glucose (GLU). Blood biochemistry parameters from each mouse are shown as individual points with error bars representing the bias of each experimental group.

[0106] Figure 14 PIM1D is shown to be effective in neutropenic lung models using methicillin-resistant Staphylococcus aureus MRSA USA300 and Klebsiella pneumoniae ATCC 13883. (A) MRSA USA300 and (B) K. pneumoniae-induced neutropenic lung model CFU counts of the lungs. Kaplan-Meier curves representing (C) MRSA USA300 and (D) K. pneumoniae-induced neutropenic lung model mouse survival. Geometric mean ± s.d. One-way ANOVA; ns, not significant, *P < 0.05, **P < 0.01.

[0107] Figure 15 Synthesis of PIM1 bromide monomer is depicted.

[0108] Figure 16 Synthesis of PIM1-Br is depicted.

[0109] Figure 17 General synthesis of non-degradable backbone cationic PIMs is depicted.

[0110] Figure 18 Synthesis of TFA salts of diamide diamines (n = 4, 6, 8, 10, and 12) monomers is depicted.

[0111] Figure 19 General synthesis of degradable backbone cationic PIMs by (a) copolymerization of degradable and non-degradable diamines and (b) homopolymerization of degradable diamines is depicted.

[0112] Figure 20 Chemical structures of a series of PIMs (P1-P6) are depicted.

[0113] Figure 21Antibiofilm properties of PIM and benzalkonium chloride (BAC, reference) measured by minimum biofilm eradication concentration (MBEC) assay are depicted. Surviving bacterial counts of MRSA BAA39 on each microtiter plate peg after 4h treatment with PIM or BAC.

[0114] Figure 22 Antibiofilm properties of PIM and BAC (reference) measured by MBEC assay are depicted. Surviving bacterial counts of PAOl on each microtiter plate peg after 4h treatment with PIM or BAC.

[0115] Figure 23 Synthesis of 2+2 carbonate monomers is depicted.

[0116] Figure 24 Synthetic scheme of (a) carbonate monomers and (b) biodegradable PIM D2 incorporating carbonate is depicted.

[0117] Figure 25 Synthetic scheme of OIM1D-3C-6 and OIM1D-3C-8 is depicted.

[0118] Figure 26 Efficacy of OIM1D-3C-8 in (A) multidrug resistant Klebsiella pneumoniae; (B) methicillin-resistant Staphylococcus aureus induced neutropenic lung infection model; (C) change in mouse weight after addition of 20 mg / kg compound by intranasal delivery; and (D) OIM1D-3C-8 / OIM1D-3C-6 (2:1 wt.% and 1:1 wt.%) in multidrug resistant Klebsiella pneumoniae induced neutropenic lung infection model is depicted.

[0119] Figure 27 Schematic of various diamines with degradable linkers for biodegradable PIM synthesis (p = 1-12, q = 0-10) is depicted. DETAILED DESCRIPTION

[0120] Disclosed herein is a series of poly(alkylated imidazolium) (PIM) salts that contain one or more degradable moieties in the alkyl chain. Surprisingly, these PIM salts exhibit excellent broad-spectrum antimicrobial properties against a range of clinically important ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter) bacterial species, while having low toxicity to mammalian cells. Indeed, PIMs with higher molar fractions of degradable linker moieties show higher biocompatibility. Furthermore, it has been found that the polymers disclosed herein are active against both Gram-positive and Gram-negative bacteria.

[0121] The term "Gram-positive bacteria" refers to bacteria that have a cell wall with a large amount of peptidoglycan. Gram-positive bacteria are identified by their tendency to retain crystal violet and stain dark blue or purple in the Gram staining protocol.

[0122] The term "Gram-negative bacteria" refers to bacteria that have a thinner layer of peptidoglycan, which does not retain crystal violet stain in the Gram staining protocol, but rather retains a counterstain, typically safranin. Gram-negative bacteria stain red or pink in the Gram staining protocol.

[0123] Thus, in a first aspect of the application, a polymer or oligomer, or a pharmaceutically acceptable solvate thereof, is disclosed, comprising a first repeat unit, the first repeat unit comprising an imidazolium group and a biodegradable chain linked to an adjacent repeat unit.

[0124] In embodiments herein, the word "comprising" can be interpreted as meaning that the mentioned features are required, but the presence of other features is not excluded. Alternatively, the word "comprising" can also relate to cases where it is intended that only the recited components / features are present (e.g. the word "comprising" can be replaced by the phrase "consisting of" or "consisting essentially of"). It is explicitly envisaged that both the broad and the narrow interpretation can apply to all aspects and embodiments of the application. In other words, the word "comprising" and its synonyms can be replaced by the phrase "consisting of" or the phrase "consisting essentially of", and vice versa.

[0125] The phrase "consisting essentially of and its grammatical variants, can be interpreted herein as meaning a material that can have minor impurities. For example, the material can be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0126] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "a first repeat unit" includes multiple such repeat units, and so forth.

[0127] The term "biodegradable linkage" as used herein refers to a linking group that connects one imidazolium group to another. Such a biodegradable linkage can include one or more biodegradable functional groups.

[0128] Any suitable biodegradable functional group can be used herein. The term biodegradable functional group as used herein is intended to mean a functional group that can be cleaved in the environment and / or in vivo by chemical or biological materials present in the surrounding environment in which the oligomer, polymer or molecule of the present application can be found. Non-limiting examples of biodegradable functional groups that can be mentioned herein include ureas, carbamates, acetals, amides, esters, carbonates, urethanes, disulfides, anhydrides and hydrazones. The functional group can be susceptible to cleavage by chemicals or biological materials in the surrounding environment (for example, an ester can be cleaved due to acidic or basic conditions of the environment or due to the presence of enzymes). Such cleavage can occur in vivo or ex vivo, depending on the use and / or disposal mode of the materials disclosed herein. Examples of functional groups that can not be biodegradable include ether linkages.

[0129] In the mentioned embodiments of the polymer or oligomer of the first aspect of the present application, the only repeat unit can be the first repeat unit. However, in alternative embodiments of the first aspect of the present application, the polymer or oligomer can further comprise a second repeat unit comprising an imidazolium group and a non-biodegradable alkyl chain or another biodegradable alkyl chain linked to an adjacent repeat unit. In certain embodiments in which the polymer or oligomer can further comprise a second repeat unit, one or more of the following can apply:

[0130] (a) the polymer or oligomer can comprise 1 to 75 mol%, such as 5 to 60 mol%, such as 10 to 50 mol%, such as 20 to 30 mol% of the first repeat unit; and

[0131] (b) the repeat units of the polymer or oligomer can be randomly distributed or the repeat units can be formed into blocks, more particularly the repeat units of the polymer or oligomer can be randomly distributed. In the specific embodiments described above, the second repeat unit can be a second repeat unit comprising an imidazolium group and a non-biodegradable alkyl chain.

[0132] In embodiments of the first aspect of the application which can be mentioned herein, the biodegradable chain in the first repeat unit comprises one or more biodegradable functional groups, wherein the one or more biodegradable functional groups are selected from one or more of the group consisting of: urea, carbamate, acetal, amide, ester, carbonate, urethane, disulphide, anhydride and hydrazone, optionally wherein:

[0133] (ai) the one or more biodegradable functional groups can be selected from one or more of the group consisting of: amide, ester, carbonate, urethane, disulphide, anhydride and hydrazone;

[0134] (aii) the one or more biodegradable functional groups can be selected from one or more of the group consisting of: carbamate, or more particularly, amide, ester and carbonate; or

[0135] (aiii) the one or more biodegradable functional groups can be amide.

[0136] In embodiments of the first aspect of the application which can be mentioned herein, the number average molecular weight can be 800 to 10,000 Daltons, such as 900 to 5,000 Daltons, such as 1,000 to 3,000 Daltons, such as 1,000 to 2,000 Daltons.

[0137] In specific embodiments of the first aspect of the application which can be mentioned herein, the polymer or oligomer can have the formula I:

[0138]

[0139] wherein:

[0140] x is 0.01 to 1.0;

[0141] Y - is a counterion;

[0142] o is 0 to 10 (e.g. 0 to 6, such as 1 to 5);

[0143] p is 1 to 12;

[0144] q is 0 to 14 (e.g. 0 to 6);

[0145] r is 0 to 12;

[0146] D is a biodegradable functional group;

[0147] D’ is a biodegradable functional group or a bond;

[0148] each R 1 is a branched or unbranched C 1-3 alkyl group or a derivative thereof;

[0149] each t is 0, 1 or 2;

[0150] each t’ is 0, 1 or 2;

[0151] each R 2 is a branched or unbranched C 1-3 alkyl group or a derivative thereof;

[0152] or a pharmaceutically acceptable solvate thereof.

[0153] As used herein, the term “C 1-3 alkyl” can mean, for example, ethyl, propyl (e.g., n-propyl or isopropyl), or more preferably methyl. Derivatives of C 1-3 alkyl can mean substituted C

[0154] C 1-3 alkyl groups. Examples of substituted C 1-3 alkyl groups that can be mentioned herein include, but are not limited to, halogen (e.g., Br, CI, or more particularly F). A specific derivative that can be mentioned herein is CF3.

[0155] In embodiments of the application that relate to polymers or oligomers according to Formula I, one or more of the following apply:

[0156] (bi) each D can be selected from urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0157] (aa) each D can be selected from one or more of the group consisting of amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0158] (ab) each D can be selected from one or more of the group consisting of carbamate, or more particularly, amide, ester, and carbonate; or

[0159] (ac) each D can be selected from one or more of the group consisting of carbonate and amide (e.g., each D is amide);

[0160] (bii) each D’ can be selected from a bond, urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0161] (ad) Each D' may be selected from one or more of the following groups: bonds, amides, esters, carbonates, urethanes, disulfides, acid anhydrides, and hydrazones;

[0162] (ae) Each D' can be selected from one or more of the following groups: bond, amide, ester, carbamate and carbonate;

[0163] (af) Each D' may be selected from one or more of the following groups: bonds and amides;

[0164] (ag) Each D' may be selected from one or more of the following groups: amides, esters, carbonates, urethanes, disulfides, acid anhydrides, and hydrazones;

[0165] (ah) Each D' may be selected from one or more of the following groups: amides, esters, carbamates and carbonates;

[0166] (ai) Each D' can be an amide;

[0167] (biii)Y - One or more of the following groups can be selected: halogens, acetates, phosphates, sulfonates, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2). - ), where Y is chosen arbitrarily - One or more of the following groups can be selected: chloride, acetate, phosphate, sulfonate and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2) - );

[0168] (biv)x can be 0.01 to 1.0, such as 0.025 to 0.75, such as 0.05 to 0.6, such as 0.1 to 0.5, such as 0.2 to 0.3;

[0169] (bv)t and t' can be 0;

[0170] (bvi)p can be 1 to 6; and

[0171] (bvii)r can be 1 to 6.

[0172] As will be understood, any combination of the above variables is conceived.

[0173] It will be understood that D and D' can be the same or different. In a specific embodiment of the invention, D' can be a biodegradable functional group, such that the biodegradable chain has two biodegradable functional groups. However, in other embodiments (e.g., when D is a carbamate), D' can be a bond.

[0174] Embodiments of the application that can be mentioned include those in which the polymer or oligomer of the first aspect of the application (such as a polymer or oligomer of Formula I) is a compound selected from the following list:

[0175]

[0176]

[0177] When the polymer or oligomer comprises two repeat units, the amount of repeat units comprising one or more biodegradable functional groups can be 1 to 99 mol%, such as 5 to 95 mol%, such as 10 to 90 mol%, such as 20 to 80 mol%, such as 25 to 75 mol%, such as 50 mol%. In particular embodiments that can be mentioned herein, the amount of repeat units comprising one or more biodegradable functional groups can be 20 to 30 mol%.

[0178] For the avoidance of doubt, it is expressly contemplated that where multiple numerical ranges are recited herein in reference to the same characteristic, the endpoints of each range are intended to be combined in any order to provide further contemplated (and implicitly disclosed) ranges. Thus, for the ranges listed above (and for the first repeat unit generally), the following ranges are contemplated:

[0179] 1 to 5 mol%, 1 to 10 mol%, 1 to 20 mol%, 1 to 25 mol%, 1 to 30 mol%, 1 to 50 mol%, 1 to 60 mol%, 1 to 75 mol%, 1 to 80 mol%, 1 to 95 mol%, 1 to 99 mol%;

[0180] 5 to 10 mol%, 5 to 20 mol%, 5 to 25 mol%, 5 to 30 mol%, 5 to 50 mol%, 5 to 60 mol%, 5 to 75 mol%, 5 to 80 mol%, 5 to 95 mol%, 5 to 99 mol%;

[0181] 10 to 20 mol%, 10 to 25 mol%, 10 to 30 mol%, 10 to 50 mol%, 10 to 60 mol%, 10 to 75 mol%, 10 to 80 mol%, 10 to 95 mol%, 10 to 99 mol%;

[0182] 20 to 25 mol%, 20 to 30 mol%, 20 to 50 mol%, 20 to 60 mol%, 20 to 75 mol%, 20 to 80 mol%, 20 to 95 mol%, 20 to 99 mol%;

[0183] 25 to 30 mol%, 25 to 50 mol%, 25 to 60 mol%, 25 to 75 mol%, 25 to 80 mol%, 25 to 95 mol%, 25 to 99 mol%;

[0184] 30 to 50 mol%, 30 to 60 mol%, 30 to 75 mol%, 30 to 80 mol%, 30 to 95 mol%, 30 to 99 mol%;

[0185] 50 to 60 mol%, 50 to 75 mol%, 50 to 80 mol%, 50 to 95 mol%, 50 to 99 mol%;

[0186] 60 to 75 mol%, 60 to 80 mol%, 60 to 95 mol%, 60 to 99 mol%;

[0187] 75 to 80 mol%, 75 to 95 mol%, 75 to 99 mol%;

[0188] 80 to 95 mol%, 80 to 99 mol%; and

[0189] 95 to 99 mol%.

[0190] In the specific embodiments of (b) and (c) in the above table, the repeating unit comprising one or more biodegradable functional groups can be present in an amount of 50 mol%.

[0191] In embodiments of the application that can be mentioned herein, the polymers and oligomers in the above table can have a number average molecular weight of 960 to 3,000 Daltons, such as 966 to 2,800 Daltons.

[0192] In a second aspect of the application, there is disclosed a molecule or a pharmaceutically acceptable solvate thereof, comprising:

[0193] a first block of oligomer repeating units, wherein each repeating unit comprises an imidazolium group and a non-biodegradable alkyl chain connected to an adjacent repeating unit;

[0194] a second block of oligomer repeating units, wherein each repeating unit comprises an imidazolium group and a non-biodegradable alkyl chain connected to an adjacent repeating unit; and

[0195] a linking group connecting the first block and the second block together, wherein the linking group comprises one or more biodegradable functional groups.

[0196] In embodiments of the second aspect of the application that can be mentioned herein, the one or more biodegradable functional groups can be selected from one or more of the group consisting of urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0197] (ci) the one or more biodegradable functional groups can be selected from one or more of the group consisting of amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0198] (cii) the one or more biodegradable functional groups can be selected from one or more of the group consisting of carbamate, or more particularly, amide, ester, and carbonate;

[0199] (ciii) the one or more biodegradable functional groups can be selected from one or both of amide and carbonate; or

[0200] (civ) the one or more biodegradable functional groups can be amide.

[0201] In embodiments of the second aspect of the application, the molecular weight of the molecule can be 1,000 Daltons to 5,000 Daltons, optionally wherein the molecular weight is 1,000 Daltons to 4,000 Daltons.

[0202] In particular embodiments of the second aspect of the application that can be mentioned herein, the molecule can have the formula II:

[0203]

[0204] wherein:

[0205] each m is independently 1 to 8 (e.g., 1 to 6);

[0206] each Y - is a counterion;

[0207] n’ is 0 to 12;

[0208] each o’ is independently selected from 0 to 20;

[0209] each p’ is independently selected from 0 to 12 (e.g., 0 to 6);

[0210] each p” is independently selected from 0 to 12 (e.g., 0 to 6);

[0211] each T is independently a terminal functional group selected from amine, ammonium, guanidinium, bisguanidinium, alkyl, and aryl;

[0212] each D is a biodegradable functional group, or a pharmaceutically acceptable solvate thereof.

[0213] In embodiments of the application involving polymers or oligomers according to Formula II, one or more of the following can apply:

[0214] (di) each D can be independently selected from urea, carbamate, acetal, amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone, optionally wherein:

[0215] (ba) each D can be independently selected from one or more of the group consisting of amide, ester, carbonate, urethane, disulfide, anhydride, and hydrazone;

[0216] (bb) each D can be independently selected from one or more of the group consisting of carbamate, or more particularly, amide, ester, and carbonate; or

[0217] (bc) each D can be amide;

[0218] (dii) Y - may be selected from one or more of the group consisting of halogen, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ), optionally wherein Y - is selected from one or more of the group consisting of halogen, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ); and

[0219] (dii) p’” can be 0 to 6 (e.g., p” is 0).

[0220] Embodiments of the application that can be mentioned include those in which the molecule of the second aspect of the application is selected from the following list:

[0221]

[0222] References herein (in any aspect or embodiment of the application) to polymers, oligomers, and molecules herein (including polymers or oligomers of Formula I or molecules of Formula II) include references to such compounds per se, tautomers of such compounds, and pharmaceutically acceptable salts or solvates or pharmaceutically functional derivatives of such compounds.

[0223] Pharmaceutically acceptable salts that can be mentioned include acid addition salts and base addition salts. Such salts can be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of Formula I or Formula II with one or more equivalents of an appropriate acid or base, optionally in a solvent or medium in which the salt is insoluble, followed by removal of the solvent or the medium by standard techniques, for example by filtration or by freeze-drying. Salts can also be prepared by exchanging a counter-ion of a compound of Formula I or Formula II in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

[0224] Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids, e.g., hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulfuric acids, and organic acids, e.g., tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulfonic acids.

[0225] Examples of acid addition salts include those formed with acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acids (e.g., benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphor-sulfonic acid, (+)-(1 S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid (e.g., D-gluconic acid), glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), a-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, isethionic acid, lactic acid (e.g., (+)-L-lactic acid and (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-)-L-malic acid), malonic acid, (±)-DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g., (+)-L-tartaric acid), thiocyanic acid, undecylenic acid, and valeric acid.

[0226] Specific examples of salts are salts derived from mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid; from organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, arylsulfonic acids; and from metals, such as sodium, magnesium, or preferably potassium and calcium.

[0227] As will be appreciated, the polymers, oligomers and molecules described herein can already include a counterion, but such counterions can be replaced with a different counterion if desired. For example, the polymers, oligomers and molecules described herein can be subjected to an ion exchange column in order to replace one counterion with a different counterion.

[0228] As noted above, the polymers, oligomers and molecules described herein also encompass any solvate of the compounds and salts thereof. Preferred solvates are solvates formed by incorporation of molecules of a non-toxic pharmaceutically-acceptable solvent (hereinafter referred to as a solvating solvent) into the solid state structure (e.g. crystal structure) of a compound of the application. Examples of such solvents include water, alcohols such as ethanol, isopropanol and butanol, and dimethylsulphoxide. Solvates can be prepared by recrystallizing a compound of the application with a solvent or a mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given case can be determined by subjecting crystals of the compound to analysis using well-known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

[0229] Solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemi-hydrates, monohydrates and dihydrates.

[0230] For a more detailed discussion of solvates and methods for making and characterizing 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.

[0231] A "pharmaceutically functional derivative" of a polymer, oligomer or molecule described herein, as defined herein, includes ester derivatives and / or derivatives which have, or provide, the same biological function and / or activity as any relevant compound of the application. Thus, for the purposes of this application, this term also includes prodrugs of the polymers, oligomers and molecules described herein.

[0232] The term "prodrug" of a relevant polymer, oligomer or molecule described herein includes any compound that upon oral or parenteral administration is metabolized in vivo to form an active agent in experimentally detectable amounts and within a predetermined time (e.g. within the dosing interval of between 6 and 24 hours (i.e. one to four times a day)).

[0233] The prodrug polymers, oligomers, and molecules described herein can be prepared by modifying a functional group present on the compound such that the modification cleaves in vivo when such a prodrug is administered to a mammalian subject. The modification is typically achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include polymers, oligomers, and molecules described herein in which a hydroxyl, amino, thiol, carboxyl, or carbonyl group in a compound of Formula I or Formula II is bound to any group that can cleave in vivo to regenerate the free hydroxyl, amino, thiol, carboxyl, or carbonyl group, respectively.

[0234] Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, ester groups of carboxyl functional groups, N-acyl derivatives, and N-Mannich bases. General information on prodrugs can be found, for example, in Bundegaard, H. "Design of Prodrugs" p. I-92, Elsevier, New York-Oxford (1985).

[0235] The polymers, oligomers, and molecules described herein can contain double bonds and thus can exist as E (entgegen) and Z (zusammen) geometric isomers with respect to each individual double bond. All such isomers and mixtures thereof are included within the scope of the present application.

[0236] The polymers, oligomers, and molecules described herein can exist as positional isomers and can also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the present application.

[0237] The polymers, oligomers, and molecules described herein can contain one or more asymmetric carbon atoms and thus can exhibit optical and / or diastereomeric isomerism. Diastereomers can be separated using conventional techniques, e.g., chromatography or fractional crystallization. The various stereoisomers can be isolated by separation of a racemic or other mixture of the compounds using conventional techniques such as, for example, fractional crystallization or HPLC. Alternatively, the desired optical isomers can be manufactured by the synthesis of appropriate optically active starting materials (i.e., the 'chiral pool') under conditions that produce a product of only one optical activity (i.e., 'chiral pool' method); reaction of appropriate starting materials with 'chiral auxiliaries' that can be removed at a suitable stage; derivatization (i.e., resolution, including dynamic resolution), for example, with an acid of the same chirality, followed by separation of the diastereomeric derivatives by conventional means such as chromatography; or reaction with an all appropriate chiral reagents or chiral catalysts under conditions known to the skilled artisan. All stereoisomers and mixtures thereof are included within the scope of the present application.

[0238] For the avoidance of doubt, in the context of the present application, the term "treatment" includes reference to therapeutic or palliative treatment of a patient in need of such treatment, as well as prophylactic treatment and / or diagnosis of a patient susceptible to or otherwise at risk of a relevant disease state.

[0239] The terms "patient" and "patients" include reference to a mammalian (e.g., human) patient. The terms "subject" or "patient" as used herein are art-recognized and used interchangeably herein to refer to a mammal, including a 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 having a disease or condition. However, in other embodiments, the subject can be a normal subject. The term does not indicate a particular age or sex. Thus, adult and neonatal subjects, whether male or female, are intended to be encompassed.

[0240] The term "effective amount" refers to an amount of a compound that confers a therapeutic effect (e.g., sufficient to treat or prevent a disease) on the patient being treated. The effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives indication of effect by a self-reported outcome).

[0241] The term "halogen" when used herein includes reference to fluorine, chlorine, bromine and iodine.

[0242] Unless otherwise indicated, the term "aryl" when used herein includes reference to C 6-14 )aryl groups. Such groups can be monocyclic, bicyclic or tricyclic and have from 6 to 14 ring carbon atoms, at least one of which is aromatic. The point of attachment of the aryl group can be through any atom of the ring system. However, when the aryl group is bicyclic or tricyclic, they are attached to the remainder of the molecule through an aromatic ring. C 6-10 )aryl groups. Such groups can be monocyclic, bicyclic or tricyclic and have from 6 to 14 ring carbon atoms, at least one of which is aromatic. The point of attachment of the aryl group can be through any atom of the ring system. However, when the aryl group is bicyclic or tricyclic, they are attached to the remainder of the molecule through an aromatic ring. C 6-14 Aryl groups include phenyl, naphthyl and the like, such as 1,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Embodiments of the present application that can be mentioned include those wherein the aryl group is phenyl.

[0243] Unless otherwise indicated, the term "alkyl" refers to unbranched or branched, un-cyclic, saturated or unsaturated (as such, e.g., alkenyl or alkynyl) hydrocarbyl groups, which can be substituted or unsubstituted (with, e.g., one or more halogen atoms). Where the term "alkyl" refers to un-cyclic groups, they are preferably C 1-10 alkyl groups and more preferably, C 1-6alkyl (such as ethyl, propyl (e.g., n-propyl or isopropyl), butyl (e.g., branched or unbranched butyl), pentyl, or more preferably, methyl). Where the term "alkyl" is a cyclic group (which can be designated as the group "cycloalkyl"), it is preferably a C 3-12 cycloalkyl, and more preferably C 5-10 (eg. C 5-7 )cycloalkyl.

[0244] Further embodiments of the application that can be mentioned include those in which the polymers, oligomers, and molecules described herein are isotopically labeled. However, other specific embodiments of the application that can be mentioned include those in which the polymers, oligomers, and molecules described herein are not isotopically labeled.

[0245] The term "isotopically labeled," when used herein, includes reference to the polymers, oligomers, and molecules described herein in which a non-natural isotope (or non-natural distribution of isotopes) is present at one or more positions in the compound. Reference 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 polymers, oligomers, and molecules described herein. Thus, the term "isotopically labeled" includes reference to the polymers, oligomers, and molecules described herein that are isotopically enriched at one or more positions in the compound.

[0246] Isotopic labeling or enrichment of the polymers, oligomers, and molecules described herein can be with any radioactive or non-radioactive isotope of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine, and / or iodine. Particular isotopes that can be mentioned 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).

[0247] When the polymers, oligomers and molecules described herein are labelled or enriched with a radioactive or non-radioactive isotope, the polymers, oligomers and molecules described herein can be mentioned to include those in which at least one atom in the compound exhibits an isotopic distribution in which the radioactive or non-radioactive isotope of the atom in question is present at a level which exceeds the natural level of that radioactive or non-radioactive isotope by at least 10% (e.g. 10% to 5000%, particularly 50% to 1000%, and more particularly 100% to 500%).

[0248] The compounds disclosed herein can be particularly useful in the treatment of microbial infections. Accordingly, in a third aspect of the application, there is provided a polymer or oligomer according to the first aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof and / or a molecule according to the second aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof, for use in medicine.

[0249] Furthermore, in a fourth aspect of the application, there is provided:

[0250] (AAA) use of a polymer or oligomer according to the first aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof and / or a molecule according to the second aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof, in the manufacture of a medicament for the treatment of a disease comprising a microbial infection;

[0251] (AAB) use of a polymer or oligomer according to the first aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof and / or a molecule according to the second aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof, in the manufacture of a medicament for the treatment of a disease comprising a microbial infection; and

[0252] (AAC) a method of treating a disease comprising a microbial infection, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a polymer or oligomer according to the first aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof and / or a therapeutically effective amount of a molecule according to the second aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof.

[0253] In embodiments of the fourth aspect of the application, the microbial infection can involve an infected wound or cystic fibrosis.

[0254] The term "microbial infection" encompasses any disease or disorder caused by a microbial organism in or on a subject. Examples of microbial infections include, but are not limited to, tuberculosis caused by Mycobacterium, burn wound infection caused by Pseudomonas, skin infection caused by Staphylococcus aureus, wound infection caused by Pseudomonas and Acinetobacter baumanii, and sepsis. The term "fungal infection" encompasses any disease or disorder caused by a microbial organism in or on a subject. Examples of microbial infections include, but are not limited to, athlete's foot, ringworm, yeast infection, and jock itch.

[0255] A non-limiting list of bacteria that can be sensitive to the polymers and copolymers of the present application includes: Acidothermus cellulyticus, Actinomyces odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bifidobacterium adolescentis, Bifidiobacterium longum, 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 matellense, Corynebacterium minutissimum, Corynebacterium parvum, Corynebacterium striatum, Enterococcus faecalis, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus avium, Enterococcus raffinosus, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus saccharolyticus, Enterococcus solitarius, Enterococcus thailandicus, Enterococcus xiangfangensis, Enterococcus camellinus, Enterococcus columbae, Enterococcus dispar, Enterococcus flavescens, Enterococcus hermannii, Enterococcus hieni, Enterococcus holsaticus, Enterococcus hoveniae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus saccharolyticus, Enterococcus solitarius, Enterococcus thailandicus, Enterococcus xiangfangensis, Enterococcus camellinus, Enterococcus columbae, Enterococcus dispar, Enterococcus flavescens, Enterococcus hermannii, Enterococcus hieni, Enterococcus holsaticus, Enterococcus hoveniae,glutamicum), Corynebacterium jeikeium, Corynebacterium urealyticum, Desulfitobacterium hafniense, Desulfotomaculum reducens, Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna, Geobacillus kaustophilus, Geobacillus the rmodenitrificans, Janibacter sp., Kineococcus radiotolerans, Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua, Listeria The following bacteria are listed: *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*, and *Pelotomaculum*. thermopropionicum, Rhodococcus sp., SaccharopolysporaStaphylococcus 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, Streptomyces avermitilis, Streptomyces coelicolor, Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis, and combinations thereof.

[0256] As mentioned above, the polymers, oligomers and molecules of the application can be used to treat microbial and fungal infections. Thus, there is also provided a pharmaceutical composition comprising a polymer, oligomer or molecule of the application and a pharmaceutically acceptable carrier.

[0257] The polymers, oligomers or molecules of the application can be administered by any suitable route, but in particular can be administered orally, intravenously, intramuscularly, dermally, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. transorganally or transbronchially), 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 can be mentioned include oral, intravenous, dermal, subcutaneous, nasal, intramuscular or intraperitoneal administration.

[0258] As used herein, reference to polymers and oligomers of the application relates to polymers and oligomers of the first aspect of the application (and any technically reasonable combination of embodiments thereof), while reference to molecules of the application relates to polymers and oligomers of the second aspect of the application (and any technically reasonable combination of embodiments thereof).

[0259] The polymers, oligomers or molecules of the application will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier which can be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers can be chemically inert and non-toxic to the active compounds under the conditions of use, and can be selected from a variety of water- and non-water-soluble materials. Suitable pharmaceutical formulations can be found, for example, in Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenterally acceptable aqueous solutions can be used which are pyrogen-free and have the requisite pH, isotonicity and stability. Suitable solutions will be well known to the skilled person, with numerous methods described in the literature. A brief review of methods for drug delivery can also be found, for example, in Langer, Science (1990) 249, 1527.

[0260] Otherwise, the preparation of suitable formulations can be routinely achieved by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0261] The amount of the polymers, oligomers or molecules of the application in any pharmaceutical formulation used in accordance with the application will depend on a variety of factors such as the severity of the condition to be treated, the particular patient to be treated and the compound(s) used. In any case, the amount of the polymers, oligomers or molecules of the application in the formulation can be routinely determined by the skilled person.

[0262] For example, solid oral compositions such as tablets or capsules can contain from 1 to 99% (w / w) of the active ingredient; from 0 to 99% (w / w) of a 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 glidant; from 0 to 50% (w / w) of a granulating or binding agent; from 0 to 5% (w / w) of an antioxidant and from 0 to 5% (w / w) of a colorant. Controlled release tablets can additionally contain from 0 to 90% (w / w) of a release-controlling polymer.

[0263] Parenteral preparations such as solutions or suspensions for injection or solutions for infusion can comprise from 1 to 50% (w / w) of the active ingredient; and from 50% (w / w) to 99% (w / w) of a liquid or semi-solid carrier or vehicle (e.g. a solvent such as water); and from 0-20% (w / w) of one or more further excipients such as buffers, antioxidants, suspension stabilizers, tonicity adjusting agents and preservatives.

[0264] Depending on the condition and patient to be treated and the route of administration, the polymers, oligomers or molecules of the application can be administered to a patient in need thereof in different therapeutically effective doses.

[0265] However, in the context of the present application, the dose administered to a mammal, particularly a human, should be sufficient to effect a therapeutic response in the mammal over a reasonable time frame. One skilled in the art will recognize that the exact dose and composition and the selection of the most suitable delivery regimen will also be influenced by 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 and the potential of the particular compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0266] The administration can be continuous or intermittent (e.g. by bolus injection). The dosage can also be determined by the timing and frequency of administration. In the case of oral or parenteral administration, the dosage of the polymers or copolymers of the application can vary from about 0.01 mg to about 1000 mg per day.

[0267] In any case, the practicing physician or other skilled person will be able to determine routinely the actual dosage which would be most appropriate for an individual patient. The dosages mentioned above are exemplary of the average case; there can be individual instances where higher or lower dosages are merited, and such are within the scope of the application.

[0268] The aspects of the application described herein (e.g. the above-mentioned polymers, oligomers and molecules, methods and uses) can have the advantage that, in the treatment of the conditions described herein, they are more convenient for the physician and / or patient, more efficacious, less toxic, have a better selectivity, have a broader range of activity, are more potent, produce fewer side effects, or can have other useful pharmacological properties than similar compounds, combinations, methods (treatments) or uses known in the prior art for the treatment of these conditions or other aspects.

[0269] The polymers, oligomers or molecules of the application can be prepared according to techniques well known to the skilled person, for example as described in the Examples section below.

[0270] The polymers, oligomers or molecules of the application can be isolated from their reaction mixtures using conventional techniques (e.g. recrystallisation, column chromatography, preparative HPLC etc.).

[0271] In a fifth aspect of the application, there is provided a disinfectant formulation comprising a polymer or oligomer according to the first aspect of the application, or a pharmaceutically acceptable solvate thereof, and any technically reasonable combination of embodiments thereof, and / or a molecule according to the second aspect of the application, or a pharmaceutically acceptable solvate thereof, and any technically reasonable combination of embodiments thereof.

[0272] In view of the above, the polymers, oligomers or molecules of the application can be used as antimicrobial active ingredients in personal care formulations, such as disinfectants, 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, the disinfectant formulation mentioned above can refer to any of the formulations listed in this paragraph.

[0273] When used as a simple disinfectant composition (i.e. intended to be used as a disinfectant only), the disinfectant formulation composition can comprise from 0.01 to 20% by weight, such as from 0.5 to 10% by weight, of the polymer, oligomer or molecule of the application. It will be appreciated that more than one polymer, oligomer or molecule of the application can form part of the disinfectant composition.

[0274] The polymers, oligomers or molecules of the application exhibit pronounced antimicrobial action, in particular against pathogenic Gram-positive and Gram-negative bacteria, and thus also against the bacteria of the skin flora, e.g. Corynebacterium xerosis (a bacteria that causes body odour), and also against yeasts and moulds. They are thus also suitable for the disinfection of the skin and mucous membranes and also of the outer skin appendages (hair), and thus also for the disinfection of the hands and wounds.

[0275] Thus, there is also provided an antimicrobial and / or antifungal cleanser composition comprising a polymer, oligomer or molecule of the application and a surfactant. It will be appreciated that the composition can also comprise further cosmetically tolerable carriers and / or adjuvants. The composition can in particular be in the form of a shampoo or a solid or liquid soap, although other compositions as described above (e.g. other hair care products, lotions and creams etc.) are also contemplated.

[0276] The cleanser composition can comprise from 0.01 to 15% by weight, such as from 0.5 to 10% by weight, of the polymer or copolymer of the application. It will be appreciated that more than one polymer and copolymer of the application can form part of the cleanser composition.

[0277] Depending on the form of the cleanser composition, in addition to the polymer or copolymer of the present application, it will also include additional ingredients such as chelating agents, colorants, perfume oils, thickening or solidifying (consistency adjusting) agents, emollients, UV absorbers, skin protectants, antioxidants, additives to improve the mechanical properties (such as dicarboxylic acids, and / or C 14 -C 22 Al, Zn, Ca and Mg salts of fatty acids) and optionally a preservative.

[0278] The cleanser composition can be formulated as a water-in-oil or oil-in-water emulsion, an alcoholic or alcohol-containing preparation, a vesicular dispersion of ionic or non-ionic amphiphilic lipids, a gel, a solid stick or an aerosol preparation.

[0279] As a water-in-oil or oil-in-water emulsion, the cleanser composition can include 5 to 50 wt% of an oil phase, 5 to 20 wt% of an emulsifier and 30 to 90 wt% of water. The oil phase can comprise any oil suitable for cosmetic preparations, such as one or more hydrocarbon oils, waxes, natural oils, silicone oils, fatty acid esters or fatty alcohols. Preferred mono- or polyhydric alcohols are ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and sorbitol.

[0280] The cleanser composition can be provided in a variety of formulations. Examples of suitable compositions include, but are not limited to, skin care preparations (e.g., preparations for washing and cleansing the skin in the form of tablets or in the form of liquid soaps, soap-free cleansers 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 soaps, lathering shaving creams, non-lathering shaving creams, foams and gels, pre-shave preparations for dry shaving, after-shave lotions or aftershave lotion), cosmetic hair treatment preparations (e.g., hair-washing preparations in the form of shampoos and conditioners, hair-care preparations such as pre-treatment preparations, hair tonics, styling creams, styling gels, hair pomades, hair rinses, treatment packs, intensive hair treatments, hair-structuring preparations such as waving preparations for permanent waves (hot waves, mild waves, cold waves), hair straightening preparations, liquid hair-styling preparations, foams, hair sprays, bleaching preparations; e.g., hydrogen peroxide solutions, lightening shampoos, bleaching creams, bleaching powders, bleaching pastes or oils, temporary, semi-permanent or permanent hair colorants, preparations containing autoxidizable dyes, or natural hair colorants such as henna or chamomile).

[0281] The antimicrobial soap can have, for example, the following composition:

[0282] 0.01 to 5% by weight of the polymer, oligomer or molecule of the present application;

[0283] 0.3 to 1% by weight of titanium dioxide;

[0284] from 1 to 10% by weight of stearic acid; and

[0285] the remainder being a soap base, such as sodium salts of tallow fatty acids and coconut fatty acids or glycerol.

[0286] The shampoo can have, for example, the following composition:

[0287] from 0.01 to 5% by weight of the polymer, oligomer or molecule of the present application;

[0288] from 12.0% by weight of sodium lauryl ether-2-sulfate;

[0289] from 4.0% by weight of cocamidopropyl betaine;

[0290] from 3.0% by weight of NaCI; and

[0291] water to 100 wt%.

[0292] In a sixth aspect of the application, there is provided an article having a surface, wherein the surface is coated with a polymer or oligomer according to the first aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof and / or a molecule according to the second aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof to provide antimicrobial properties to the surface of the article, optionally wherein the article is a urinary catheter.

[0293] For example, the article according to the application can be a urinary catheter, wherein the surface has been coated with a polymer or oligomer according to the first aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof and / or a molecule according to the second aspect of the application or a pharmaceutically acceptable solvate thereof and any technically reasonable combination of embodiments thereof. Urinary tract infections can be caused by pathogenic bacteria, such as E. coli, and if left untreated, the infection can progress to a systemic infection that can even lead to death. By coating the urinary catheter with the compounds disclosed herein, bacterial infection can be prevented. As will be appreciated, additional components can be added to the coating to provide additional properties (for example, an anti-inflammatory agent can be coated onto the surface to prevent inflammation, etc.). As will be appreciated, the antimicrobial compounds disclosed herein can also be coated onto other medical devices.

[0294] Further aspects and embodiments of the application are described in the numbered statements below.

[0295] 1. A random copolymer having the following general structure:

[0296]

[0297] wherein D is a biodegradable fragment, which can be an amide, an ester, a carbonate, a urethane, a disulfide, an anhydride, a hydrazone;

[0298] Y - is a counterion, which can be chloride, acetate, phosphate, sulfonate, bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ) and

[0299] 0 < o < 6;

[0300] 1 < p < 6;

[0301] 0 < q < 6.

[0302] 2. The random copolymer according to statement 1, wherein x is between 0.10 and 0.50.

[0303] 3. The random copolymer according to statement 1 or 2, wherein the random copolymer has a molecular weight distribution of 1 KDa to 5 KDa.

[0304] 4. A molecule having the following general structure

[0305]

[0306] wherein D is a biodegradable fragment, which can be an amide, an ester, a carbonate, a urethane, a disulfide, an anhydride, a hydrazone;

[0307] Y - is a counterion, which can be chloride, acetate, phosphate, sulfonate, bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ) and

[0308] T is a terminal group, which can be an amine, an ammonium, a guanidinium, a biguanidinium, an alkyl, an aryl; and

[0309] 1 < m < 6;

[0310] 0 < n < 12;

[0311] 0 < o < 20;

[0312] 0 < p < 6.

[0313] 5. Use of the random copolymer according to any one of statements 1 to 3 or of the molecule according to statement 4 in medicine.

[0314] 6. Use of the random copolymer according to any one of statements 1 to 3 or of the molecule according to statement 4 for the treatment of a microbial infection.

[0315] 7. Use of a random copolymer of any one of statements 1 to 3 or a molecule of statement 4 in the manufacture of a medicament for treating a microbial infection in a subject in need thereof.

[0316] 8. A method of treating a subject having a microbial infection, comprising the step of administering to the subject a therapeutically effective amount of a random copolymer of any one of statements 1 to 3 or a molecule of statement 4, such that the microbial infection is treated.

[0317] Antibacterial biodegradable polyimidazolium and oligoimidazolium (plus defined molecules) are discussed here as the compounds of the present invention, which show good antibacterial activity against both Gram-positive and Gram-negative bacteria in vitro (e.g. polymer PIM1D and oligomer OIM1D-mC-6 (m=3, 8) - for additional details, see the experimental section below). In addition, the compounds of the present invention show good biocompatibility in vivo. For example, a single intraperitoneal injection of polymer PIM1D can rescue mice in a murine sepsis model induced by MDR P. aeruginosa and A. baumannii, while cumulative intraperitoneal injection of PIM1D for 7 days causes negligible toxicity. These findings identify the degradable compounds of the present invention as promising antibacterial candidates to address the current emerging drug resistance crisis.

[0318] Emerging multi-drug resistant bacterial pathogens pose a serious threat to human public health. Antimicrobial polymers have been widely explored as alternative therapeutics, but mostly failed due to their poor biocompatibility and high MIC values. Surprisingly, the compounds of the present invention maintain high antimicrobial activity while also being biodegradable in vivo, thereby reducing or eliminating problems associated with toxicity of non-degradable compounds in vivo, which are caused or exacerbated by their long-term residence in the body. For example, polymer PIM1D shows high antibacterial activity against even multi-drug resistant P. aeruginosa, A. baumannii and K. pneumoniae, which are on the WHO's top critical pathogens list. It is also effective against multi-drug resistant Gram-positive bacteria and against colistin-ineffective mycobacteria, demonstrating its broad antibacterial efficacy. This, together with its good biocompatibility, makes PIM1D an excellent antibacterial candidate. Similar properties are found for other compounds of the present invention.

[0319] Untreated bacterial sepsis is extremely deadly. The involvement of multidrug-resistant bacterial pathogens makes treatment even more problematic, as they are untreatable by most antibiotics. As described above and below, a single injection of PIM1D demonstrated excellent efficacy in rescuing mice with sepsis induced by MDR Pseudomonas aeruginosa PAER and MDR Acinetobacter baumannii AB-1. PIM1D also showed efficacy in treating murine sepsis caused by methicillin-resistant Staphylococcus aureus. Distal lung infections are difficult to treat and are often used to evaluate the efficacy of antimicrobial agents before entering clinical trials. PIM1D showed good efficacy in treating lung infections caused by Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus. Furthermore, only negligible toxicity was observed after seven consecutive intraperitoneal injections of PIM1D at a therapeutic dose of 15 mg / kg (with a cumulative dose of 105 mg / kg). The above highlights the potential of PIM1D (and other compounds of the present invention) in antimicrobial applications.

[0320] Further aspects and embodiments of the invention will now be described with reference to the following non-limiting examples.

[0321] Example

[0322] Materials

[0323] Unless otherwise specified, all chemicals used in the synthesis were purchased from Sigma-Aldrich Co. LLC. (St. Louis, USA) and used directly in the reactions. Commercial AR-grade solvents were used as is, as accepted from Merck, without further distillation. For column chromatography, industrial-grade solvents were used as is, as accepted from SG Labware Pte Ltd (Singapore), without any distillation. 1,4-Diaminobutane (diamine B), mucin, silica gel (35-70 mesh), silica gel 60 (100-200 mesh), A-26 OH resin and cation-adjusted Mueller Hinton Broth (CAMHB) were purchased from Merck & Co., USA. L-lysine and 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)) were purchased from Combi-Blocks, Inc. (San Diego, CA, USA). N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HC1) and 1-hydroxybenzotriazole (HOBt) were purchased from GL Biochem Ltd. (Shanghai, China). Cyclophosphamide was purchased from MedChemExpress LLC (Shanghai, China). Propidium iodide (PI) staining kit, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), and FM TM 4-64 FX was purchased from Thermo Fisher Scientific (MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar (MA, USA). Fluorescein isothiocyanate (FITC) was purchased from Biotium, Inc. (CA, USA). Zymosan standard was purchased from Polymer Standards Service (PA, USA). 1 K Dalton cut-off Spectra / Por® membranes were purchased from Spectrum Laboratories, Inc. (CA, USA). 6 Dialysis membranes were purchased from Repligen, USA. Muller Hinton Broth (MHB), Trypticase Soy Broth (TSB), Lysogeny Broth (LB), and agar (LB agar) were purchased from Becton Dickson, USA. Vancomycin and colistin were purchased from Chem-Impex International Inc., USA. Middlebrook 7H9 Broth medium was purchased from BD Difco. Bovine serum albumin fraction V was purchased from Roche.

[0324] Bacteria and growth conditions

[0325] Pseudomonas aeruginosa PAOl was provided by Scott Rice, Nanyang Technology University. Enterococcus faecalis VRE583 and Escherichia coli EC958 were obtained from the Singapore Center for Environmental and Life Sciences (SCELSE). Pan-resistant Pseudomonas aeruginosa PAER, pan-susceptible Acinetobacter baumannii ACBAS, multidrug-resistant Acinetobacter baumannii AB-1, pan-susceptible Klebsiella pneumoniae KPNS, carbapenem-resistant Klebsiella pneumoniae KPNR, pan-susceptible Escherichia coli ECOS, MDR Escherichia coli ECOR, pan-susceptible Enterobacter cloacae ECLOS, and carbapenem-resistant Enterobacter cloacae CRE were obtained from Tan Tock Seng Hospital (TTSH), Singapore. MRSA USA300 LAC, LAC derivative Staphylococcus aureus LAC*, and LAC menD mutant have been previously described (Pader, V. et al., Infect. Immun. 2014, 82, 4337-4347). Klebsiella pneumoniae SGH10, Klebsiella pneumoniae BAK085, Klebsiella pneumoniae M7, Klebsiella pneumoniae SGH4, multidrug-resistant Acinetobacter baumannii X26, extensively drug-resistant Acinetobacter baumannii X39, Acinetobacter baumannii X40 were provided by Dr. Yunn-Hwen Gan, National University of Singapore. Colistin-resistant Pseudomonas aeruginosa (PAK pmrB12) and Burkholderia thailandensis 700388 were provided by Samuel I. Miller, University of Washington School of Medicine. Mycobacterium abscessus (rough and smooth) and Mycobacterium smegmatis mc 2155Culture and testing were performed in the Kevin Pethe laboratory at the Lee Kong Chian school of medicine. M. bovis bacillus Calmette-Guérin was from our stock. All other bacteria were purchased from the American Type Culture Collection. Bacteria were grown at 37°C in Mueller Hinton broth (MHB) (Wiegand, I. et al., Nat. Protoc. 2008, 3, 163-175) under shaking, unless otherwise stated. S. aureus was grown on tryptone soya broth (TSB). Mycobacteria were grown in Middlebrook 7H9 broth medium supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% ADS supplement (which was made by dissolving bovine serum albumin fraction V (25 g), D-dextrose (10 g) and sodium chloride (4.05 g) in water (500 mL)). For mycobacterial growth inhibition assays, glycerol was not supplemented. For plating, we solidified lysate broth (LB) with 1.5% agar (LB agar) and plates were incubated at 37°C.

[0326] Analytical techniques

[0327] Bruker Avance DPX 300MHz NMR instrument 1 H nuclear magnetic resonance (NMR), 1 H- 1 H homonuclear correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC), 13 C NMR and distortionless enhancement by polarization transfer enhancement (DEPT-135) analysis, all samples were dissolved in deuterated solvents CDCl3, D2O, MeOD or DMSO-d6. In 1 In H NMR spectra, the chemical shift (δ) of the solvent residual peak of D2O was set at 4.79 and of DMSO-d6at 2.50 and in proton-decoupled 13 In C NMR spectra, the middle peak of the solvent residual peak of DMSO-d6was set at 39.52. Mass analysis was recorded on a MALDI-ToF ABI 4800. Molecular weight and number average molecular weight distribution (Mn) were determined by GPC equipped with two ultrahydrogel columns in series and a RI detector using a mixture of water / methanol (MeOH) / 0.5M acetic acid (AcOH) containing 0.5M sodium acetate as eluent (pH = 4.5, flow rate = 0.5 mL / min).w / M n Prior to sample analysis, all samples were dissolved in acetate buffer at approximately 1 mg / mL and filtered through a 0.22 μm microfilter. Merck silica gel 60 (100-200 mesh) was used as the stationary phase for column chromatography separation of the crude mixture. Thin-layer chromatography (TLC) was performed using Merck 60F254 pre-coated silica gel plates, and the plates were visualized using UV light, ninhydrin staining, or alkaline KMnO4 solution.

[0328] Preparation of acidified water for dialysis

[0329] Acidified water for dialysis was prepared by adding 1M hydrochloric acid (HCl, 3mL) to Millipore water (5L).

[0330] Procedure for loading the column with chloro anions

[0331] Pass a 10% aqueous HCl solution through a filling material... A glass column of A-26 (OH– form) was used until the pH of the eluent was the same as that of the original solution. The resin was then washed with water until neutral pH was reached. The process was carried out at room temperature using gravity as the driving force.

[0332] Procedure for gel filtration chromatography (GFC)

[0333] Sephadex TM -G25 powder was dissolved in deionized (DI) water and submerged overnight, during which the powder swelled into a slurry. Sephadex was then eluted using deionized (DI) water as the eluent and gravity elution. TM The slurry is filled into the glass column.

[0334] Comparative Example 1. Synthesis of main-chain alkylated polyimidazolium (PIM) chloride salt PIM0-5

[0335] An aqueous acidic solution (total 100 mmol) of a diamine selected from the following list was prepared by adding a diamine to water (25 mL) and cooling the reaction mixture in an ice-water bath. Then, a 37% HCl solution (200 mmol) was added to the reaction mixture to obtain an acidic diamine solution. The aqueous acidic solution of the diamine was maintained in an ice-water bath for 30 min. Then, a mixture of formaldehyde (8.12 g, 100 mmol) and glyoxal (14.51 g, 100 mmol) was added dropwise to the reaction mixture. The reaction mixture was refluxed at 80 °C for 4.5 h. During reflux, the solution changed from colorless to pale yellow. The solvent and unreacted monomers were removed by rotary evaporation to obtain a yellow viscous oil. The oil was diluted with water and dialyzed against acidified water at pH 3–4 (1-kDa Spectra / ). 6 dialysis membrane, Repligen, USA) for one day, with acidified water changed 3 times, to obtain water-soluble PIM0-5( Figure 1 ).

[0336] List of diamines used to synthesize PIM0-5

[0337] 1,3-diaminopropane - PIM0

[0338] 1,4-diaminobutane (diamine B) - PIM1

[0339] 1,6-diaminohexane - PIM2

[0340] 1,8-diaminooctane - PIM3

[0341] 1,5-diamino-2-methylpentane - PIM4

[0342] L-lysine - PIM5

[0343] PIM0

[0344] 1 H NMR (300 MHz, D20, 25 °C [ppm]): δ 8.98 (s, 1 H, imidazole-H), 7.61 (s, 2H, imidazole-H), 4.36 (t, 4H, -CH2-), 2.54 (m, 2H, -CH2-).

[0345] PIM1

[0346] 1 H NMR (300 MHz, D20, 25 °C [ppm]): δ 8.84 (s, 1 H, imidazole-H), 7.51 (s, 2H, imidazole-H), 4.24 (t, 4H, -CH2-), 1.91 (m, 4H, -CH2-).

[0347] PIM2

[0348] 1 H NMR (300 MHz, D20, 25 °C [ppm]): δ 8.77 (s, 1 H, imidazole-H), 7.47 (s, 2H, imidazole-H), 4.16 (t, 4H, -CH2-), 1.85 (m, 4H, -CH2-), 1.33 (m, 4H, -CH2-).

[0349] PIM3

[0350] 1H NMR (300 MHz, D20, 25 °C [ppm]): δ 8.76 (s, 1 H, imidazole-H), 7.47 (s, 2H, imidazole-H), 4.16 (t, 4H, -CH2-), 1.84 (m, 4H, -CH2-), 1.29 (m, 8H, -CH2-).

[0351] PIM4

[0352] 1 H NMR (300 MHz, D20, 25 °C [ppm]): δ 8.82 (s, 1 H, imidazole-H), 7.50 (s, 2H, imidazole-H), 4.19 (t, 2H, -CH2-), 3.98 (m, 1 H, -CH-), 2.15-1.78 (m, 4H, -CH2-), 1,46-1.15 (m, 2H, -CH2-), 0.84 (s, 3H, -CH3).

[0353] PIM5

[0354] 1 H NMR (300 MHz, D20, 25 °C [ppm]): δ 9.12-8.78 (m, 1 H, imidazole-H), 7.65-7.47 (m, 2H, imidazole-H), 5.13 (m, 1 H, -N-CH-), 4.21 (m, 2H, -CH2-), 2.33-2.18 (m, 2H, -CH2-), 1.95 (m, 2H, -CH2-); 1.26 (m, 2H, -CH2-).

[0355] Comparative Example 2. Synthesis of PIM1 -fluorescein isothiocyanate (FITC) conjugate (FITC-conjugated PIM1)

[0356] PIM1 (1 eq) was dissolved in 0.1 M sodium bicarbonate (NaHC03) in water (1 mL) and the reaction mixture was stirred for 30 min. After that, FITC (1 eq) was added to the reaction mixture and stirred overnight in the dark. The PIM1 -FITC conjugate was then dialyzed against acidified water (500-1000 Da cut-off dialysis membrane) for 2 days to remove salts and unreacted dye, with acidified water changed 3 times a day. The resulting conjugate was lyophilized to obtain the final PIM1 -FITC conjugate. A calibration curve was established using absorbance of PIM1 -FITC in PBS at 493 nm, and from the results obtained, the molar ratio of FITC to PIM1 was estimated to be about 15%.

[0357] Comparative Example 3. In vitro antibacterial and cytotoxic effects of PIM0-5

[0358] Bacterial growth-inhibition and killing assays

[0359] The minimum inhibitory concentration (MIC) was determined by a slight modification of the broth microdilution method (Wiegand, I. et al., Nat. Protoc. 2008, 3, 163). Overnight cultures of bacterial strains were subcultured and grown to mid-log phase, followed by an optical density (OD) check, then diluted to 1 x 10 6 Colony forming units (CFU) / mL were used as inoculum. Test compounds were prepared in DI water at a final concentration of 10.24 mg / mL and diluted to 1.024 mg / mL in fresh MHB. Two-fold dilution series of test compounds were prepared in MHB media in 96-well plates (final volume of 50 μL per well) achieving a concentration gradient from 512 μg / mL to 1 μg / mL and incubated at 37 °C for 10 min under shaking (orbital shaker at 225 rotations per minute (rpm)) before each well was inoculated with 50 μL of bacterial suspension, positive control (MHB media and bacterial suspension without polymer) and sterile control (MHB media only). Plates were mixed in a shaker incubator for 10 min and then incubated statically at 37 °C for 18 h. After that time, OD at 600 nm was measured (OD 600 ) for each well. For assays involving mycobacteria, compounds were serially diluted in two-fold steps and 2 μL of this dilution series was spotted in 96-well plates to which 200 μL of OD 600 0.005 (about 5 x 10 5 CFU / mL) of bacteria in log phase. For M. smegmatis, plates were incubated at 37 °C for 48 h and for M. bovis Calmette-Guerin, plates were incubated at 37 °C for 5 days. MIC was reported as the concentration of compound that inhibited bacterial growth by at least 90% (MIC 90 ). Agar plating was performed to confirm the inoculum bacterial concentration. Three independent experiments were performed for each compound and each bacterial strain was tested and the range of MIC values for each compound is reported.

[0360] Cytotoxicity assays for mammalian cells

[0361] PIM0-5 were tested for toxicity using the mouse embryonic fibroblast 3T3 cell line. Cytotoxicity was measured by using standard methods (International Organization for Standardization (2009) ISO 10993-5: Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity. ISO Geneva), 1-34). First, 3T3 cells were cultured in medium containing 89% DMEM, 10% FBS, and 1% antibiotics (penicillin / streptomycin). When 80% confluence in culture flasks was observed by microscopy, the cells were trypsinized, concentrated, and counted using a hemocytometer. 1 x 10 4 cells were seeded in each well of a 96-well plate. After incubating the 96-well plate for 24 h, test compounds at a concentration range of 128 μg / mL to 4 μg / mL were added to each well of the 96-well plate. After another 24 h of incubation, cell viability was qualitatively assessed by microscopy and quantified by MTT assay. Cell viability was assessed by comparing the absorbance of formazan in the wells with antimicrobial agent to the absorbance of formazan in the wells with untreated cells. IC 50 values were reported as the level of test compound that reduced the number of viable cells by 50%. The data presented are the average of triplicate measurements with a standard deviation of 10% or less.

[0362] LB agar plate count

[0363] The bacterial solution was serially diluted in PBS by a factor of 10. The diluted solution was dropped on solidified agar plates at 5 μL per drop. After drying in a biosafety hood, the plates were incubated in a 37 °C incubator for 18 h, then the bacterial colonies were counted and the respective dilution factor was recorded. Finally, the bacterial concentration was back-calculated.

[0364] Results and discussion

[0365] Table 1 shows the physical and biological properties of different batches of PIM1 in Pseudomonas aeruginosa PAER, Acinetobacter baumannii AB-1 (MDR), and Staphylococcus aureus USA300 (MRSA). All PIM chloride salts except PIM5 showed significant antimicrobial activity (Table 2). This can be because PIM5's carboxylated alkyl chain makes it the least hydrophobic in the series, and PIM5 is a zwitterion rather than a cation. PIM0 showed reduced activity due to its short alkyl chain being less hydrophobic than PIM1.

[0366] Table 1. Physical and biological properties of PIM1 in different batches of P. aeruginosa PAER, A. baumannii AB-1 (MDR), and S. aureus USA 300 (MRSA).

[0367]

[0368] Table 2. Antimicrobial and cytotoxic effects of PIM0-5.

[0369] MIC 90 or IC 50 (mg / ml) 1

[0370]

[0371] 1 The minimum PIM concentration required to inhibit bacterial growth by at least 90% (MIC 90 ) or to halve the viability of 3T3 cells (IC 50 ). Values are ranges of three independent experiments.

[0372] 2 Gram-positive bacterial strains were S. aureus ATCC 29213, E. faecium ATCC 19434, and Gram-negative bacterial strains were K. pneumoniae ATCC 13883, A. baumannii ATCC 19606, P. aeruginosa PAOl, E. coli ATCC 8739, and E. cloacae ATCC 13047.

[0373] 3 Mouse fibroblast 3T3 cells.

[0374] Unlike PIM2 and PIM3, PIM1 did not exhibit toxicity to 3T3 cells (Table 2). This can be due to the fact that PIM2 and PIM3 have alkyl chains that are two or four carbons longer, respectively, than PIM1. These results suggest that small differences in alkyl chain length can significantly affect mammalian cell toxicity.

[0375] Accordingly, PIM1 was selected for further study due to its potent antimicrobial activity against a range of pathogenic bacteria and the fact that it did not show measurable acute mammalian cell toxicity in our PIM screen (Table 2).

[0376] Comparative Example 4. In vitro antimicrobial activity and cytotoxicity of PIM1

[0377] PIM1 was screened for antibacterial activity against a wider variety of bacterial pathogens following the protocol outlined in Comparative Example 3. The cytotoxicity of PIM1 in HEK293, HepG2, and A549 cells was also determined as described in Comparative Example 3, except that DMEM supplemented with 15% FBS was used to culture HepG2, HEK293, and A549 cells. In addition, the antibacterial activity of PIM1 was compared to the commercial antibiotics colistin and polymyxin B.

[0378] Results and discussion

[0379] We found that PIM1 showed potent antibacterial activity against a variety of pan-antibiotic resistant Gram-positive and Gram-negative bacteria, including colistin-resistant B. thailandensis and P. aeruginosa mutants. We noted that PIM1 was also a potent anti-mycobacterial compound. In contrast, PIM1 had a broader spectrum of activity than colistin and polymyxin B, which are not particularly effective antibiotics against Gram-positive bacteria (Table 3). These findings suggest that PIM1 has a different mode of action than colistin. Finally, no toxicity was evident even at the highest levels of PIM1 when tested in four different mammalian cell lines (Table 4).

[0380] Table 3. Antibacterial effect of PIM1 compared to the activity of colistin on a panel of pan-resistant bacteria and naturally antibiotic-resistant bacteria.

[0381] MIC 90 (mg / mL) 1

[0382]

[0383] 1 Antimicrobial concentration that inhibited bacterial growth by at least 90%. Values are ranges of three independent experiments.

[0384] 2MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-resistant Enterococcus; MDR, multidrug-resistant; P. aeruginosa PAK pmrB-12 is a colistin-resistant mutant derived from P. aeruginosa PAK (Moskowitz, S. M. et al., J. Bacteriol. 2004, 186, 575-579); XDR, extensively drug-resistant (Magiorakos, A. P. et al., Clin. Microbiol. Infect. 2012, 18, 268-281); B. thailandensis 700388 is a natural colistin-resistant close relative of the emerging pathogen B. pseudomallei (Burkholderia pseudomallei is also colistin-resistant) (Olaitan, A. O. et al., Front. Microbiol. 2014, 5, 643).

[0385] 3 ND, not done.

[0386] Table 4. Comparison of PIM1, colistin, and polymyxin B cytotoxicity.

[0387]

[0388] 1 Antimicrobial concentration that induces 50% inhibition of mammalian cell viability. Values are the mean of triplicates with a standard deviation of less than 10%.

[0389] 2 ND, not done.

[0390] Comparative Example 5. Bactericidal properties of PIM1.

[0391] To determine whether PIM1 is bactericidal or bacteriostatic, we inoculated MHB with the model gram-negative pathogen P. aeruginosa PAOl or TSB with the gram-positive pathogen methicillin-resistant S. aureus LAC* from a log-phase culture and determined total CFU in samples over time in the presence of different concentrations of PIM1 after inoculation by plating for colony counts on LB agar. Two independent experiments were performed and results are mean ± SD.

[0392] Results and discussion

[0393] Bacterial growth was evident in the absence of PIM1 or in the presence of PIM1 at half the MIC level Figure 2). At twice the MIC, both P. aeruginosa and S. aureus were killed by PIM1. From these experiments, we concluded that PIM1 is bactericidal.

[0394] Comparative Example 6. Novel mode of action of antibacterial PIM1

[0395] Propidium iodide (PI) staining

[0396] P. aeruginosa PAOl was used in the PI experiments. Cells grown in MHB were harvested at mid-log phase and the cells were resuspended in fresh MHB. PIM1 or colistin (positive control) was added at the indicated concentrations. After 1 h incubation with the antimicrobial, the cell suspension was sampled to determine the number of cells by plating. The remaining cells were washed with PBS and stained with PI (15 pg / mL) according to the manufacturer’s protocol. An Attune NxT flow cytometer (Thermo Fisher Scientific, USA) was used to determine the percentage of cells that had taken up PI (dead cells). A Zeiss LSM800 confocal microscope was used to image cells on poly-lysine coated dishes (MatTek Corporation, USA).

[0397] Monitoring membrane potential

[0398] Membrane potential (ΔΨ) in P. aeruginosa was monitored using the membrane potential sensitive dye 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)) by using a previously reported procedure (Zhang, L. et al., Antimicrob. Agents Chemother. 2000, 44, 3317-3321). P. aeruginosa PAOl cells were harvested from mid-log phase cultures by centrifugation and suspended in 5 mM HEPES buffer containing 100 mM KC1 and 0.2 mM EDTA to permeabilize the outer membrane for entry of DiS-C3-(5). The bacterial suspension was then adjusted to OD 600 0.02 and DiS-C3-(5) was added (final concentration 1 mM). The cell suspension (180 pL) was then added to each well of a 96-well plate and test compounds were added to the wells as indicated to make a final mixture of 200 pL. Fluorescence in each well was measured every 2 min in a Spark 10M microtiter plate reader (Tecan, Switzerland) with an excitation of 622 nm and an emission of 670 nm. Data were collected 30 min after addition of test compounds. Two independent experiments were performed and the data here are the mean ± SD.

[0399] Cell uptake protocol

[0400] Cellular uptake of PIM1-FITC was monitored as described elsewhere (Radlinski, L.C. et al., Cell Chem. Biol. 2019, 26, 1355-1364) with slight modifications. Briefly, cells grown in MHB were harvested at mid-log phase and cells were suspended in fresh MHB containing 1 MIC of PIM1-FITC (MIC of PIM1-FITC is the same as PIM1) for 30 min. Cells were then harvested by centrifugation, washed once with PBS, and then fixed with 4% paraformaldehyde in PBS for 15 min. Fixed cells were washed twice with PBS, and then incubated with 5 pg / mL FM TM 4-64FX (Invitrogen TM , Thermo Fisher Scientific, USA) for 10 min on ice. Cells were washed again twice with PBS, and then mounted in glass slides using Fluoromount TM G (Merck & Co., USA) and subsequently imaged using Zeiss Super Resolution System ELYRA PS.1 with LSM800 system.

[0401] Results and discussion

[0402] PIMs were designed to have moderately hydrophobic alkyl chains with a cationic imidazolium moiety. Thus, like antimicrobial peptides (Velkov, T. et al., J. Med. Chem. 2010, 53, 1898-1916), the activity of PIMs can involve permeabilization of the cell membrane. Furthermore, as seen in Comparative Example 6, PIM1 has a mode of action different from colistin. To test this hypothesis, uptake of the fluorescent dye PI in P. aeruginosa treated with PIM1 and colistin was compared. Live cells with intact cell membranes exclude PI. If the membrane is permeabilized, PI can enter the cell. As expected, almost all cells treated with colistin were stained, but most cells treated with high concentrations of PIM1 excluded PI Figure 3 ). These results support the idea that PIM1 activity does not involve membrane disruption like colistin. To further support this idea, we monitored ΔΨ in P. aeruginosa using the lipophilic fluorescent dye DiS-C3-(5). While treatment with the proton ionophore gramicidin resulted in a sharp increase in DiS-C3-(5) fluorescence, indicating ΔΨ dissipation, PIM1 did not show such an effect Figure 4

[0403] ​Since PIM1 does not disrupt membranes and does not dissipate ΔΨ, we hypothesized that PIM1 can be taken up by cells. Therefore, cellular uptake of a fluorescent derivative of PIM1, PIM1-FITC, was synthesized in Comparative Example 2 and was employed to treat P. aeruginosa. As shown in Figure 1, Figure 5 A-B, PIM1-FITC enters cells. We hypothesize that, like cationic antibiotics (e.g., gentamicin (GEN)), association of PIM1 with cells and antimicrobial activity can depend on ΔΨ. If so, activity should be high when P. aeruginosa is in a basic environment, and activity should decrease in an acidic environment. In bacteria like P. aeruginosa, the proton motive force (PMF) remains relatively constant over a range of external pH values, as does the cytoplasmic pH (slightly basic). The total PMF is composed of ΔΨ and the pH gradient across the cell membrane (ΔpH). Thus, in a slightly basic environment, the cytoplasmic and external pH values are similar, and the PMF is primarily in the form of ΔΨ. In an acidic environment, the external pH is lower than the cytoplasmic pH, and the PMF is primarily in the form of ΔpH. In fact, the MIC of PIM1 depends on the external pH, and PIM1 shows poor antimicrobial activity at pH 5 Figure 5 C). These findings suggest that uptake of PIM1 is ΔΨ-dependent.

[0404] Comparative Example 7. Effect of valinomycin and nigericin on the MIC of PIM1 against P. aeruginosa

[0405] Valinomycin, nigericin, and PIM1 were dissolved in MHB. Stock solutions were added to wells in a microtiter plate to give a volume of 50 μΐ^, to which 50 μΐ^ of a log phase P. aeruginosa culture was added. MICs were determined as described in Comparative Example 3 90 .

[0406] Results and discussion

[0407] To gain further insight into the mode of action of PIM1, the effect of a potassium ionophore (valinomycin) and a sodium-potassium exchanger (nigericin) on PIM1 activity was investigated. At neutral pH, valinomycin decreases ΔΨ, and nigericin collapses ΔpH (Farha, M. A. et al., Chem. Biol. 2013, 20, 1168-1178). The results obtained are consistent with our hypothesis. The MIC of PIM1 against P. aeruginosa was increased by valinomycin treatment, but was not greatly affected by nigericin Figure 5 D). Combining the results obtained here and in Comparative Example 6, we infer that PIM1 is taken up by cells in a ΔΨ-dependent manner, but we cannot distinguish whether it exerts its antimicrobial effect at the cell membrane or in the cytoplasm.

[0408] Example 8. Effect of metabolic state on PIM1 killing of P. aeruginosa PAOl

[0409] In addition to obtaining stationary phase cells by overnight growth in MHB, we used a previously reported method (S. Meylan et al., Cell Chem. Biol. 2017, 24, 195-206) to determine the effect of PIM1 and other antibiotics on stationary phase P. aeruginosa PAOl survival and we compared PIM1 to GEN. The results were compared to those for P. aeruginosa PAOl harvested from MHB cultures in the mid-log growth phase. In addition, we tested the ability of an energy source to enhance PIM1 killing of stationary phase P. aeruginosa by adding fumarate (15 mM) to stationary phase cells.

[0410] Results and discussion

[0411] In general, antibiotics have limited activity against non-growing bacteria. This is evident for P. aeruginosa when comparing the bactericidal activity of an antibiotic such as GEN against stationary phase cells incubated in the presence and absence of an energy source (S. Meylan et al., Cell Chem. Biol. 2017, 24, 195-206; and K. R. Allison et al., Nature 2011, 473, 216-220). Based on our findings that PIM1 does not appear to disrupt membrane integrity and, like GEN, its activity requires ΔΨ, we hypothesize that its bactericidal activity against nutrient starved bacteria can be limited. In fact, stationary phase cells are much less sensitive to killing by PIM1 (or GEN as a control) than to killing by colistin (A). When fumarate is supplied as an energy source to stationary phase cells, the bactericidal activity of both PIM1 and GEN is restored (B). We infer that, like GEN and many other antibiotics, PIM1 will have limited utility as a bactericidal agent against non-growing bacteria. We also note that these experiments are consistent with our conclusion that PIM1 does not act by disrupting the cell membrane and that its activity requires ΔΨ. Figure 6 A). When fumarate is supplied as an energy source to stationary phase cells, the bactericidal activity of both PIM1 and GEN is restored (B). We infer that, like GEN and many other antibiotics, PIM1 will have limited utility as a bactericidal agent against non-growing bacteria. We also note that these experiments are consistent with our conclusion that PIM1 does not act by disrupting the cell membrane and that its activity requires ΔΨ. Figure 6 B). We infer that, like GEN and many other antibiotics, PIM1 will have limited utility as a bactericidal agent against non-growing bacteria. We also note that these experiments are consistent with our conclusion that PIM1 does not act by disrupting the cell membrane and that its activity requires ΔΨ.

[0412] Example 9. Laboratory evolution of PIM1 resistance.

[0413] Laboratory evolution mutation assay

[0414] Experiments on spontaneous PIM1 -resistance and ciprofloxacin resistance evolution involved serial passaging as described elsewhere (Ling, L. L. et al., Nature 2015, 517, 455-459). We used P. aeruginosa PAO1 grown in MHB or MRSA LAC* grown in TSB. The initial transfer inoculum was 10 7 cells / mL, using 2 mL test tubes and 96-well plates for P. aeruginosa and MRSA, respectively, in 1 mL or 100 μL with different amounts of antibiotic. The larger volume for P. aeruginosa experiments was to increase the cell number, as resistance did not arise in smaller culture volumes for this species. Bacterial growth was monitored at 24 h intervals. Transfers were made daily, and the inoculum for transfers (100-fold dilution) was from cultures with the highest antibiotic level that allowed growth to an OD 600 of at least 0.2. For P. aeruginosa, the experiment was run for 30 days. For MRSA LAC*, the experiment ended at day 15. Isolates of MRSA LAC* were obtained from the last transfer and stored as glycerol stocks at -80 °C for further studies.

[0415] Whole genome sequencing

[0416] Genomic DNA was isolated from PIM1 -resistant S. aureus mutants using standard procedures and DNA was prepared for sequencing by using the Illumina Nextera DNA Library Prep Kit. DNA was sequenced on an Illumina MiSeq instrument (paired end sequencing). Sequences were mapped onto the genome of the parental MRSA LAC* (Bowman, L. et al., J. Biol. Chem. 2016, 291, 26970-26986) and single nucleotide variations, small deletions and insertions were identified using the CLC Genomics Workbench software. Large deletions were identified by manual sequence comparison. DNA sequences have been deposited in the European Nucleotide Archive (ENA) and the accession number is PRJEB37791.

[0417] Results and discussion

[0418] To evaluate the potential of designer PIMs as therapeutic agents, and possibly gain further insight into the mechanism of action of PIMs, we performed repeated passaging experiments on P. aeruginosa and MRSA with gradually increasing concentrations of PIM1 or ciprofloxacin (control). For P. aeruginosa, ciprofloxacin-resistant mutants arose, but no PIM1 -resistant mutants arose Figure 7). The rate of emergence of PIM1 -resistant MRSA was similar to that of ciprofloxacin-resistant mutants.

[0419] To gain insight into the nature of the PIM resistance phenotype in our evolving MRSA population, we isolated bacteria from the final passage. Of the 21 isolates characterized, they all showed small colony variation (SCV) phenotype, 15 had PIM1 MICs that were 128-fold higher than the original strain, and the other 6 had PIM1 MICs that were 64-128-fold higher than the parent strain. We sequenced the genomes of the 15 isolates that showed MICs >128-fold higher than the unevolved strain (Shi, Z. et al., Polymer resistant Staphylococcus aureus strains. European Nucleotide Archive. Deposited April 14, 2020). All but one had mutations in genes required for methylnaphthoquinone biosynthesis (genes in the menA-F operon or in ispD). Several isolates also had mutations in genes known to confer resistance to cationic peptides, particularly in vraG or vraF, graR or graS, or fmtC (Falord, M. et al., PloS One 2011, 6, e21323; Joo, H.-S. et al., Biochim. Biophys. Acta 2015, 1848, 3055-3061; and Yang, S.-J. et al., Infect. Immun. 2012, 80, 74-81) (Table 5). The genes encoding methylnaphthoquinone synthesis were of particular interest because the relationship between methylnaphthoquinone and PIM1 activity can provide some clues about the mode of action of PIM1. We therefore compared the PIM1 sensitivity of a menD deletion mutant to that of its parent.

[0420] This menD mutant was unable to produce methylnaphthoquinone J. et al., Antimicrob. Agents Chemother. 2008, 52, 4017) and growth is restricted to fermentation. Like our evolved PIM1 -resistant isolates, this mutant has the SCV phenotype. This is the characteristic phenotype of methylnaphthoquinone synthesis mutants (Von Eiff, C. et al., J. Bacteriol. 2006, 188, 687). The menD mutant shows an 8-fold increase in PIM1 resistance compared to its parent (MIC of 16 μg / mL vs. 2 μg / mL for the parent). Thus, we believe that methylnaphthoquinone or a functional electron transport system is involved in the susceptibility of MRSA to PIM1, but other factors must also be involved in the very high PIM1 resistance of our evolved isolates. We infer that PIM1 directly interferes with the electron transport chain, which leads to the production of toxic reactive oxygen species, or that the uptake of PIM1 is hindered during fermentative growth and thus its antimicrobial activity is reduced.

[0421] Table 5. List of common associated mutations in the laboratory-evolved PIM1 -resistant S. aureus LAC* mutant.

[0422]

[0423] 1 All single base substitutions are non-synonymous mutations that either code for an amino acid substitution or a stop codon.

[0424] 2 Mutant 5114 is the only PIM1 -resistant mutant for which we did not identify a mutation in the methylnaphthoquinone biosynthesis genes.

[0425] Comparative Example 10. Efficacy of PIM1 treatment in animal infection

[0426] Mice were housed at room temperature with a 12 h light-dark cycle for one week prior to infection. Our skin infection model was as follows: wounds (approximately 5 mm in diameter) were created on the shaved back skin of female C57B / 6 mice (8-9 weeks old) by punch biopsy and log phase cells of P. aeruginosa PAER were introduced into the wounds (approximately 10 6 CFU in 10 μL PBS) by pipetting. The infected wounds were immediately covered with Tegaderm (3M, USA). At 4 h post-infection, antimicrobial (PIM1 and imipenem (Imp)) treatment was initiated by Tegaderm injection. Thereafter, another layer of Tegaderm was applied. After another 24 h, we removed a 1 -cm 2Square tissue samples of 1 cm x 1 cm were taken from the back of the mice and homogenized to determine cell number by plating. Our protocol was approved by the Institutional Care and Use Committee of Nanyang Technological University (NTU IACUC, protocol A0362).

[0427] Results and discussion

[0428] The ability of PIM1 to control carbapenem-resistant P. aeruginosa murine wound infections was evaluated. As expected, the number of Imp-resistant strains of P. aeruginosa increased in untreated or Imp-treated wounds over the next 24 h. The number of P. aeruginosa was slightly reduced when treated once with 0.1 mg / kg of PIM1 and substantially reduced by about four logs when treated once with 1 mg / kg or higher doses of PIM1 compared to untreated or Imp-treated wounds. Figure 8

[0429] Comparative Example 11. Toxicity of PIM1 treatment in animal infections

[0430] For the systemic infection model, we first evaluated the toxicity of PIM1 (IP injection, 6 mg / kg) in female BALB / c mice (8-9 weeks old) by tracking body weight over a 14-day period. Body weight was recorded daily for 5 days.

[0431] Results and discussion

[0432] The safety of PIM1 when delivered to mice by IP injection was tested and evidence of acute toxicity was found. We observed a decrease in body weight over a 5-day period following single dose administration Figure 9 A).

[0433] Example 1. Synthesis of degradable PIM1 D precursor (N,N'-(propane-1,3-diyl)bis(2- aminoacetamide)) (diamine A) Figure 10 A)

[0434] ​EDC.HC1 (14.58 g, 76.1 mmol) and HOBt (10.70 g, 79.14 mmol) were added to a solution of Boc-Gly-OH (8.0 g, 45.66 mmol) in dry DMF (25 mL) at 0 °C (ice water) under stirring over 30 min. 1,3-Diaminopropane (1.28 mL, 15.22 mmol) was added dropwise to the reaction mixture kept at 0 °C (ice water) over 10 min. Then, the reaction mixture was allowed to reach room temperature and continuously stirred for 48 h. Then, water (50 mL) was added and the product was extracted three times with ethyl acetate (EtOAc) or DCM (150 mL). The extract was washed three times with water (50 mL) and then once with brine (50 mL). The EtOAc or DCM layer was dried over anhydrous Na2S04(ca. 50 g). Then, the Na2S04was filtered off and the filtrate was concentrated by rotary evaporation (20 min at 50 °C, 120 rpm). The residue was dried under vacuum at room temperature overnight. The dried residue was dissolved in dry DCM (30 mL), then kept at 0 °C (ice water) and trifluoroacetic acid (TFA, 8 mL) was added dropwise over 10 min. After that, the reaction mixture was stirred at room temperature for 12 h. The crude product was concentrated by rotary evaporation at 50 °C for 10 min at 120 rpm. Then, toluene (50 mL) was added and the solution was further rotary evaporated at 50 °C for 30 min at 120 rpm. The residue was purified by silica gel 60 column chromatography eluting with the following consecutive eluents: (i) 30% methanol (MeOH) in dichloromethane (DCM, 500 mL) to remove impurities, then (ii) 2% TFA in MeOH (1000 mL) to obtain the degradable diammonium TFA salt A (3.0 g, 7.20 mmol).

[0435] 1 H NMR (300 MHz, DMSO-d6, 25 °C [ppm]): δ 8.55 (t, J = 5.4 Hz, 2H), 8.18 (br s, 6H), 3.53 (s, 4H), 3.14 (q, J = 6.3 Hz, 4H), 1.54-1.63 (m, 2H). 13 CNMR (75 MHz, DMSO-d6, 25 °C [ppm]): δ 166.14, 159.58 (-CO-CF3), 159.16 (-CO-CF3), 158.74 (-CO-CF3), 158.32 (-CO-CF3), 123.24 (-CF3), 119.29 (-CF3), 115.33 (-CF3), 111.38 (-CF3), 40.26, 36.73, 28.86.

[0436] Example 2. Synthesis of biodegradable PIM1D

[0437] To obtain diamine A, Et3N (1 mL) was added to a stirred solution of diammonium TFA salt A (400 mg, 0.96 mmol) in MeOH (4 mL) maintained at 0 °C (ice water). After stirring the reaction mixture at room temperature for 30 min, the volatiles were evaporated using a rotary evaporator and dried under vacuum at room temperature for 20 min to obtain degradable diamine A. The obtained diamine A was immediately used for a poly-Radziszewski reaction with diamine B to form biodegradable PIM1D.

[0438] PIM1D synthesis as follows Figure 10 The procedure described in section B was performed. A first mixture of glyoxal (40 wt%, 349 mg, 2.4 mmol) and formaldehyde (37 wt%, 195 mg, 2.4 mmol) in ice-cold AcOH and tetrahydrofuran (THF) (3:1.25 mL) at 0 °C (ice water) was prepared. A second solution comprising degradable diamine A (181 mg, 0.96 mmol) and non-degradable diamine B (127 mg, 1.44 mmol) in AcOH and THF (3:1.25 mL) at 0 °C (ice water) was also prepared. The first mixture was added dropwise to the second mixture over 10 min at 0 °C (ice water). The reaction mixture (which was pale yellow) was then heated to room temperature, and the reaction mixture turned brown. After allowing the reaction mixture to stand at room temperature for 24 h, the final reaction mixture (approximately 10 mL) was directly transferred to a 1 K Dalton-restricted Spectra / The polymer solution was dialyzed against a Repligen membrane (USA) and dialyzed against 5 L of acidified water (pH 3-4), with the acidified water replaced three times over a 24-hour period. The polymer solution in the dialysis bag was transferred to a round-bottom flask, and the water was evaporated using a rotary evaporator (70 °C, 1 h, 120 rpm) to obtain solid PIM1D in the round-bottom flask. To transfer PIM1D for freeze-drying, water (5 mL) was added to the polymer solution, and the concentrated PIM1D solution was poured into a small Falcon tube (15 mL) and then freeze-dried at -80 °C to obtain pure PIM1D. GPC and NMR characterization were performed to confirm the molecular weight and chemical structure of PIM1D.

[0439] Characterization

[0440] GPC showed a narrow distribution of the final PIM1D compound. In DMSO-d6 1Chemical shifts at 9.62 ppm and 7.81 ppm in the1H NMR spectrum confirmed the formation of the imidazolium ring, while signals at 1.59 to 5.06 ppm corresponded to the alkyl chains in PIM1D. The1H NMR spectrum in DMSO-d6 13 The13C NMR spectrum further confirmed the peak assignments: signals at 121.06 to 136.53 ppm indicated the formation of the imidazolium ring, signals at 25.78 to 52.77 ppm indicated the presence of the alkyl chains, and signals at 164.99 and 167.05 ppm indicated the presence of the amide carbonyl groups. These assignments were further confirmed by DEPT-135, COSY, and HMQC analyses. In the DEPT spectrum, the signals at 167.05 and 164.99 ppm corresponding to the carbonyl groups of the amide disappeared, and the signals corresponding to the C2-H, C4-H, and C5-H protons of the imidazolium ring showed positive phases, while the other signals of the CH2groups of the polymer chain showed negative phases. In the COSY spectrum, the correlations of the alkyl chains in the polymer chain were observed, indicating their adjacent positions. However, the signals at 5.07 ppm and 4.57 ppm did not show correlations, confirming that the two non-equivalent -CH2-CO- groups did not have adjacent protons, indicating that the -CH2-carbons of these groups were connected to the N atoms of the imidazolium ring. The HMQC spectrum further confirmed these assignments by showing the correlations of the protons and carbons in both the imidazolium ring and the alkyl chains of PIM1D. 13 The carbonyl group signals of the amide at 167.05 and 164.99 ppm in the13C NMR disappeared, and the signals corresponding to the C2-H, C4-H, and C5-H protons of the imidazolium ring showed positive phases, while the other signals of the CH2groups of the polymer chain showed negative phases. In the COSY spectrum, the correlations of the alkyl chains in the polymer chain were observed, indicating their adjacent positions. However, the signals at 5.07 ppm and 4.57 ppm did not show correlations, confirming that the two non-equivalent -CH2-CO- groups did not have adjacent protons, indicating that the -CH2-carbons of these groups were connected to the N atoms of the imidazolium ring. The HMQC spectrum further confirmed these assignments by showing the correlations of the protons and carbons in both the imidazolium ring and the alkyl chains of PIM1D.

[0441] Example 3. Optimization of reaction conditions for PIM1D synthesis

[0442] To optimize the effects of reaction conditions on the biological profile of PIM1D, specific reaction parameters in Example 2 were varied, including the feed ratio of diamine A to diamine B, reaction temperature, reaction time, etc. The biological profile of PIM1D was determined by its antibacterial activity and cell viability as described in Comparative Example 3.

[0443] Results and discussion

[0444] The molar ratio of diamine A to diamine B was varied (Table 6, entries 1-3) to optimize the percentage of degradable moieties (diamine A). The results showed that entry 3, in which the molar ratio of diamine A to diamine B in the feed was 2:3, produced an optimized PIM1D with good antibacterial efficacy and lowest mammalian cell toxicity (Tables 7-8, entry 3). Table 6, entries 1-2 (and corresponding entries 1-2 of Tables 7-8), with lower degradable diamine feed ratios, resulted in PIM1Ds that were more toxic but showed good antibacterial efficacy.

[0445] Table 6. Optimization of reaction conditions for the synthesis of PIM1D from diamine A and diamine B.

[0446]

[0447] The reactions and purifications were carried out under different conditions (as detailed in Table 1) according to the typical experimental procedure given for PIM1D synthesis. a Reactions were carried out at larger scale (4.8 mmol of aldehyde scale).bReactions were carried out with high dilution (AcOH (10 mL) for 2.4 mmol of aldehyde).cPolymers containing acetate counterions were obtained without the use of HC1 during dialysis.

[0448] Table 7. Antimicrobial activity of PIM1D synthesized under different reaction conditions.

[0449]

[0450] Table 8. Cell viability of PIM1D synthesized under different reaction conditions.

[0451]

[0452] Further reaction condition optimization was carried out by varying the solvent ratio, temperature, polymerization reaction time, dialysis membrane, and dialysis time (Table 6, entries 4-17). The resulting compounds showed Mw in the range of 1 KDa to 2 KDa, with narrow molecular weight distribution, and the final percentage of degradable diamine A (in the product) in the range of 17% to 30% (Table 6, entries 4-17). All these compounds showed good antimicrobial activity with MIC n against MDR P. aeruginosa and methicillin-resistant S. aureus in the range of 4-16 pg / mL (Table 7, entries 4-17). The biocompatibility was tested using 3T3 fibroblast and liver HepG2 cells, and the tested compounds (Table 8, entries 4-17) showed cell viability higher than 50% at all four concentrations (128 pg / mL to 1024 pg / mL). These results indicate that slight changes in the reaction conditions in PIM1D synthesis do not greatly affect its biological properties (Table 6-8, entries 4-17), and the biological profile of PIM1D is not sensitive to the molecular weight in the range of 1 KDa to 2 KDa. This tolerance to changes in reaction conditions will make the compounds easier to develop as commercial products, thus it has a large potential in various antimicrobial applications. 90

[0453] Example 4. In vitro antimicrobial activity and biocompatibility of PIM1D

[0454] ​PIM1D and colistin were tested against a larger panel of MDR gram-positive and gram-negative bacteria by following the protocol in Comparative Example 3. The in vitro biocompatibility of PIM1D and colistin was assessed by MTT test using 3T3, HEK293, HepG2 and A549 cells by following the protocol in Comparative Example 3.

[0455] Results and discussion

[0456] Table 9 shows the physical and biological properties of PIM1D in different batches in P. aeruginosa PAER, A. baumannii AB-1 (MDR) and S. aureus USA300 (MRSA). Surprisingly, PIM1D showed potent antibacterial activity against a larger panel of MDR gram-positive and gram-negative bacteria including the colistin-intrinsic resistant B. thailandensis 700388 (Table 10), MDR A. baumannii, P. aeruginosa and K. pneumoniae, which are the top priority key pathogens for new antibiotics by WHO (World Health Organization (WHO). Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. 2017). We noticed that PIM1D is also a potent anti-mycobacterial compound. Overall, we demonstrated that PIM1D is an effective antibacterial agent and has a broader spectrum of activity than colistin.

[0457] Table 9. Physical and biological properties of PIM1D in different batches in P. aeruginosa PAER, A. baumannii AB-1 (MDR) and S. aureus USA 300 (MRSA).

[0458]

[0459] Table 10. MIC of PIM1D against pathogens, mycobacteria and human cell lines 90 and cytotoxicity.

[0460]

[0461]

[0462] PIM1D showed IC 50 values greater than 1024 pg / mL, similar to the antibiotic control colistin (Table 10). Considering the MIC 90Values in the range of 8-16 pg / mL, it would have a therapeutic window of more than 50. Therefore, PIM1D has the potential to be developed as an antimicrobial agent.

[0463] Example 5. In vivo toxicity and antimicrobial efficacy of degradable PIM1D

[0464] In vivo toxicity study

[0465] The in vivo toxicity of PIM1D was assessed by monitoring the body weight and blood biomarkers of mice over a period of 14 days. BALB / c female mice (8-9 weeks old) were randomly grouped into two groups: saline control and PIM1D treatment groups. Each mouse in the PIM1D treatment group received PIM1D (15 mg / kg) by IP injection for seven consecutive days (cumulative dose of 105 mg / kg). The same volume of saline was injected intraperitoneally into the saline control group. On days 1, 3, and 7, mouse blood was drawn from the submandibular vein for blood biochemical assays using a Pointcare V3 blood chemistry analyzer (MNCHIP, Tianjin, China) according to the manufacturer’s protocol (Zhang, K. et al., Nat. Commun. 2019, 10, 4792). Similarly, mouse blood in the saline control group was collected and quantified for comparison. The mice were closely monitored until 14 days after the first injection. This protocol was approved by the Animal Ethics and Welfare Committee (AEWC, protocol AEWC-2018-07) of Ningbo University.

[0466] In vivo efficacy study

[0467] PIM1D was evaluated for in vivo efficacy using murine sepsis models. Experiments of mouse sepsis infection models of MDR Pseudomonas aeruginosa PAER and MDR Acinetobacter baumannii AB-1 were performed under the guidelines of protocols approved by the Nanyang Technological University Institutional Care and Use Committee (NTU IACUC). Experiments of mouse sepsis infection models of wild-type P. aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus MRSA USA300 were performed according to protocols reviewed and approved by the Animal Ethics and Welfare Committee (AEWC) of Ningbo University. BALB / c female mice (8-9 weeks old) were used to test the septic shock protection efficacy of all murine infection models. Exponential phase bacteria were washed twice with saline and resuspended in the same volume of saline. 300 pL of bacterial suspension with different concentrations in 5% mucin was introduced into each mouse by IP injection to first determine the lethal bacterial dose, and then the determined concentration was used for the following studies. The use of mucin is to render the mice immunocompromised, similar to hospitalized patients. Two hours post-infection, mice (5 per group) were treated with a single dose of test compound. Positive control and negative control groups of mice were injected with the same dose of antibiotic and the same amount of saline, respectively, at the same time point. Mice were monitored for survival for 7 days. In a separate group of mice, all mice were euthanized 26 h post-infection. Peritoneal wash was then performed by injecting PBS (2.0 mL) into the IP cavity followed by 1 min of abdominal massage. Then, approximately 0.5 mL of peritoneal fluid was recovered for CFU analysis. Bacterial loads in the spleen, liver, and kidney of the animals were also assessed. To check whether bacterial infection was established 2 h post-infection, mice receiving the same bacterial inoculum were sacrificed, and IP fluid as well as all organs (including kidney, liver, and spleen) were harvested to determine CFU. Experiments of sepsis caused by MRSA were similar to those of P. aeruginosa, except that mice were immunosuppressed by intraperitoneal injection of 150 mg / kg and 100 mg / kg of cyclophosphamide on day 4 and day 1 (Chin, W. et al., Nat. Commun. 2018, 9, 917). Treatments were given twice at 2 h and 26 h post-infection. Bacterial loads were from organs of mice sacrificed at 50 h post-infection. For non-treated groups, mice were sacrificed at 26 h or 50 h post-infection, whichever was closer to their time of death. For pre-treated groups, mice were sacrificed at 2 h post-infection. Bacterial levels were analyzed with one-way classification analysis of variance (ANOVA) and two-tailed Student’s t-test (Graphpad Prism, version 7 for Windows).

[0468] Results and discussion

[0469] Mice treated daily with PIM1D for seven days did not show significant weight loss Figure 9 A), and no signs of distress were observed. To gain further information about the potential of PIM1D toxicity when delivered by IP injection, we analyzed blood chemistry and found that many markers sensitive to drug toxicity were unchanged by the initial dosing or even after the last dose of PIM1D was delivered Figure 9 B-D). This is a significant improvement over PIM1, in which animals showed significant weight loss and toxic effects after dosing with the compound. Thus, the retention of broad-spectrum activity coupled with a reduction in toxicity makes PIM1D a promising antimicrobial compound.

[0470] In all sepsis models with different bacterial strains, when treatment was initiated 2 h post-infection, bacterial cells spread to all organs, including the kidney, liver, and spleen (see Figure 11 A-D, bacterial CFU counts in the "pre-treatment" group). For P. aeruginosa PAOl -induced septic shock, PIM1D treatment reduced the bacterial burden by more than 3 log in all organs (kidney, liver, and spleen) harvested compared to untreated controls Figure 12 A and Figure 12 A-C), and almost complete bacterial clearance was observed in the peritoneal space, demonstrating similar in vivo efficacy as the Imp antibiotic control Figure 12 C). Moreover, all mice treated with Imp or PIM1D survived the 7-day monitoring period without signs of distress, whereas untreated mice all died Figure 11 E).

[0471] Next, the in vivo efficacy of PIM1D was evaluated in MDR P. aeruginosa (PAER)-induced peritoneal shock. Mice receiving a single dose of PIM1D (15 mg / kg) 2 h post-infection survived at 100%, in contrast to the survival rate of zero for untreated controls or mice receiving the same dose of Imp Figure 11 F). Moreover, more than 99.9% bacterial reduction was found in all harvested organs, including the kidney, liver, and spleen, and almost complete bacterial eradication was shown in the peritoneal space compared to untreated controls or Imp controls Figure 11 B and Figure 12 D-F).

[0472] In a model of sepsis induced by MDR Acinetobacter baumannii (AB-1), better bacterial reduction was observed for mice treated with a single dose of PIM1D (15 mg / kg) than Imp control (15 mg / kg). About 99.9% bacterial removal was found in the harvested organs of mice treated with PIM1D compared to untreated controls, and more than 99.999% bacterial reduction was observed in the peritoneal space Figure 11 C and Figure 12 G-I). Furthermore, mice receiving PIM1D treatment showed 100% survival, compared to 80% survival for the Imp treated group and 0% survival for the untreated mouse group Figure 11 G).

[0473] Mouse blood collected from the submandibular vein on day 1, day 3, and day 7 was analyzed using a veterinary chemistry analyzer to assess ALT, AST, and BUN levels, among others. Mice receiving saline by IP injection daily were used as controls. No significant changes in ALT and AST levels, which are representative of liver toxicity, were found over 7 days, and negligible changes in BUN levels, which are representative of kidney toxicity, were observed Figure 13 A-H). These results demonstrate that the introduction of degradable moieties successfully reduced the in vivo toxicity of the PIM series while maintaining their antibacterial efficacy in vivo.

[0474] Example 6. In vivo efficacy of PIM1D in immunosuppressed mice

[0475] Immunosuppression was induced by IP injection of cyclophosphamide (150 mg / kg) on day 4 and cyclophosphamide (100 mg / kg) on day 1 into BALB / c female mice (8-9 weeks old) prior to introduction of infection. The animal study protocol was approved by the Ningbo University Animal Ethics and Welfare Committee. Mice were infected with methicillin-resistant Staphylococcus aureus MRSA USA300 by following the protocol in Example 5. Two separate IP injections of 15 mg / kg antibiotic (PIM1D and vancomycin) were given at 2 h and 26 h post-infection. Mouse organ collection and peritoneal washes were applied at 50 h post-infection to determine bacterial burden.

[0476] Results and discussion

[0477] The efficacy of PIM1D in MRSA-induced sepsis in immunosuppressed mice was assessed, further demonstrating the broad-spectrum antibacterial activity of PIM1D. More than 99% bacterial reduction was observed in all harvested organs of mice treated with PIM1D compared to untreated controls, and superior bacterial clearance was shown compared to vancomycin treatment Figure 11 D and Figure 12J-L). In the peritoneal space, a reduction of over 99.99% of bacteria was exhibited, similar to the vancomycin-treated control Figure 12 L). All mice survived for mice receiving PIM1D or vancomycin treatment, in contrast to 0% of mice surviving in the untreated group Figure 11 H). Thus, PIM1D protected immunosuppressed mice infected with MRSA USA300 from disease and reduced bacterial burden in affected organs.

[0478] Example 7. In vivo efficacy of PIM1D in neutropenic lung infection model

[0479] To demonstrate in vivo efficacy in treating distal infections, PIM1D was used to treat a neutropenic lung infection model caused by MRSA USA300 and Klebsiella pneumoniae (#13883).

[0480] Neutropenic lung infection model

[0481] Prior to the introduction of infection, immunosuppression was induced by cyclophosphamide (150 mg / kg) on day 4 and cyclophosphamide (100 mg / kg) on day 1 IP injection into BALB / c female mice (8-9 weeks old). Lung infection was established by intratracheal delivery of MRSA USA300 or Klebsiella pneumoniae (#13883). Infected mice were treated with 20 mg / kg of PIM1D-CA (a 1 : 1 wt% mixture of PIM1D and citric acid; citric acid was added to minimize the accompanying toxicity) or antibiotics (vancomycin or colistin) by intratracheal delivery at 2 h post-infection, while mice in the untreated group received PBS only. Mice were monitored for survival over a week. In separate experiments, mouse lungs were harvested and homogenized at 26 h post-infection, then plated to check bacterial burden. The animal study protocol was approved by the Animal Ethics and Welfare Committee of Ningbo University.

[0482] Results and discussion

[0483] In neutropenic lung infection induced by MRSA, a single treatment by intratracheal delivery of 20 mg / kg PIM1D-CA (a 1 : 1 wt.% mixture of PIM1D and citric acid) reduced bacterial burden by over 99.9% efficiency compared to mice that did not undergo any treatment Figure 14 A). Furthermore, PIM1D-CA treatment was also superior to vancomycin at the same treatment dose. In addition, infected mice treated with PIM1D-CA showed 100% survival, in contrast to zero survival rate for the infected control group and 40% survival rate for mice receiving vancomycin treatment Figure 14B), indicating the superior activity of PIM1D in treating neutropenic lung infection by MRSA.

[0484] Considering the broad-spectrum antibacterial activity of PIM1D, we also evaluated its efficacy in neutropenic lung infection by Klebsiella pneumoniae (#13883). A single intratracheal delivery of PIM1D-CA (20 mg / kg) reduced Klebsiella pneumoniae in the lungs of mice by over 99.9% compared to the infected control ( Figure 14 C), similar to colistin-treated mice. Moreover, both PIM1D-CA and colistin-treated mice survived for the week of monitoring, while the untreated mice did not survive Figure 14 D).

[0485] Advantages of PIM1D over PIM1

[0486] The results in Examples 1 to 7 surprisingly show that PIM1D not only shows no evidence of toxicity, but also retains significant antibacterial activity and shows efficacy in treating murine sepsis infection in vivo. Thus, together with its good biocompatibility, PIM1D is a superior antibacterial candidate over PIM1.

[0487] Comparative Example 12. Synthesis of PIM1 bromide (PIM1-Br) monomer

[0488] Imidazole (10.0 g, 146.9 mmol) was dissolved in THF. NaH (10.6 g, 440.7 mmol) was added to the solution in portions at 0 °C, and the reaction mixture was stirred at room temperature for 1 h. 1,4-Dibromobutane (63.5 g, 294.11 mmol) (2.0 eq) was added, and the reaction mixture was heated at reflux (50 °C) for 5 h Figure 15 ), to give PIM1-Br monomer (15.1 g, 46%) as an orange oil.

[0489] 1 H NMR (CDC13, 300 MHz): δ 3.10-1.23 (m, 4H, -CH2), 3.43 (t, 2H, -CH2), 4.06 (t, 2H, -CH2), 6.90 (s, 2H, imidazole H), 7.04 (s, 2H, imidazole H), 7.49 (s, 1H, imidazole C2-H).

[0490] Comparative Example 13. Autopolymerization approach for preparing PIM1-Br and the effect of reaction conditions on the autopolymerization reaction

[0491] PIM1-Br monomer prepared in Comparative Example 12 was dissolved in respective solvent selected from water, NMP and DMF at monomer: solvent volume ratio of 1:3. Polymerization was carried out under vigorous stirring and heating by immersing the reaction flask in an oil bath. After the pre-determined reaction time, the reaction mixture was diluted with DI water, dialyzed (MWCO 1000 Da) in DI water for 3 days and freeze-dried to obtain PIM-Br compounds Figure 16 ), which were characterized by GPC (Table 11).

[0492] Results and discussion

[0493] The effect of different reaction conditions on the self-polymerization of PIM1-Br was investigated using GPC. A summary of the GPC results is provided in Table 11.

[0494] Table 11. Self-polymerization of PIM1-Br under different reaction conditions.

[0495]

[0496] Comparative Example 14. Antimicrobial efficacy of PIM1-Br

[0497] The antimicrobial efficacy of PIM1-Br was investigated by measuring the MIC of the compound against different bacteria following the protocol in Comparative Example 3.

[0498] Results and discussion

[0499] Table 12. Summary of the antimicrobial efficacy of PIM1-Br.

[0500]

[0501] Comparative Example 15. Synthesis of non-degradable backbone cationic PIMs (P(ImC6) and P(ImC8)) Figure 17 )

[0502] A compound selected from 1,6-diaminohexane or 1,8-diaminooctane (100 mmol) in water (30 mL) was introduced into a three-necked flask with a stir bar. HCI (16.7 mL) was slowly added to the reaction mixture. After stirring at room temperature for 30 min, a mixture of 37% formaldehyde (100 mmol) and 40% glyoxal (100 mmol) was introduced. The reaction was refluxed at 100 °C for 12 h and the color of the reaction mixture gradually changed from colorless to light yellow. After removing part of the solvent and unreacted monomers by rotary evaporation, the crude product was dialyzed (1-KDa cut-off Spectra / Por® 6 dialysis membrane, Repligen, USA) against acidified water at pH 3-4 for one day. The product was freeze-dried and characterized by GPC (Table 13). 6 dialysis membrane, Repligen, USA) for one day. The product was freeze-dried and characterized by GPC (Table 13). 1P(ImC6) and P(ImC8) were characterized by H NMR and GPC analysis (Table 13).

[0503] P(ImC6)

[0504] 1 H NMR (300 MHz, D20): δ 8.77 (s, 1H, imidazole-H), 7.48 (s, 2H, imidazole-H), 4.18 (t, 4H), 1.76 (m, 4H), 1.30 (m, 8H).

[0505] P(ImC8)

[0506] 1 H NMR (300 MHz, D20): δ 8.77 (s, 1H, imidazole-H), 7.48 (s, 2H, imidazole-H), 4.18 (t, 4H), 1.76 (m, 4H), 1.30 (m, 8H).

[0507] List of abbreviations for non-degradable PIMs

[0508] P(ImC6) - P1

[0509] P(ImC8) - P2

[0510] Example 8. Synthesis of TFA salts of diamide diamines (n = 4, 6, 8, 10 and 12) monomers. Figure 18 ).

[0511] Diamide diamine (n=4) TFA salt

[0512] The diamide diamine (n = 4) TFA salt was prepared by following the protocol in Example 1 from diamine B (5.00 g, 56.72 mmol). The white solid was collected and dried to yield the diamide diamine (n = 4) TFA salt (48.1%, 11.73 g).

[0513] 1 H NMR (300 MHz, D20): δ 8.77 (s, 1H, imidazole-H), 7.48 (s, 2H, imidazole-H), 4.18 (t, 4H), 1.76 (m, 4H), 1.30 (m, 8H).

[0514] Diamide diamine (n=6) TFA salt

[0515] The diamide diamine (n = 6) TFA salt was prepared by following the protocol in Example 1 from 1,6-diaminohexane (5.00 g, 43.10 mmol) to yield the diamide diamine (n = 6) TFA salt as a white solid (41%, 4.80 g).

[0516] 1H NMR (300 MHz, DMSO-D6): δ 8.35 (t, J = 5.4 Hz, 2H), 8.05 (br s, 6H), 3.53 (s, 4H), 3.14 (q, J = 6.3 Hz, 4H), 1.54 - 1.63 (m, 2H).

[0517] Diamide diamine (n=8) TFA salt

[0518] The diamide diamine (n = 8) TFA salt was prepared by following the protocol in Example 1 from 1,8-diaminooctane (2.50 g, 21.55 mmol) to give diamide diamine (n = 8) TFA salt as a white solid (58.3%, 3.50 g).

[0519] 1 H NMR (300 MHz, DMSO-D6): δ 8.34 (t, J = 5.4 Hz, 2H), 8.04 (br s, 6H), 3.52 (s, 4H), 3.14 (q, J = 6.3 Hz, 4H), 1.42 - 1.26 (m, 12H).

[0520] Diamide diamine (n=10) TFA salt

[0521] The diamide diamine (n = 10) TFA salt was prepared by following the protocol in Example 1 from 1,10-diaminodecane (2.50 g, 21.55 mmol) to give diamide diamine (n = 10) TFA salt as an orange solid (46%, 3.80 g).

[0522] 1 H NMR (300 MHz, DMSO-D6): δ 8.39 (t, J = 5.4 Hz, 2H), 8.12 (br s, 6H), 3.52 (s, 4H), 3.10 (q, J = 6.3 Hz, 4H), 1.40 - 1.24 (m, 16H).

[0523] Diamide diamine (n=12) TFA salt

[0524] The diamide diamine (n = 12) TFA salt was prepared by following the protocol in Example 1 from 1,12-diaminododecane (5.00 g, 43.10 mmol) to give diamide diamine (n = 12) TFA salt as a white solid (45.3%, 5.50 g).

[0525] 1H NMR (300MHz, DMSO-D6): δ8.35 (t, J = 5.4Hz, 2H), 8.05 (brs, 6H), 3.51 (s, 4H), 3.10 (q, J = 6.3Hz, 4H), 1.39-1.23 (m, 22H).

[0526] Example 9. Synthesis of biodegradable main-chain cationic PIM (P(ImC6-co-ImC6D)-50%, P(ImC8-co-ImC8D)-50%), P(ImC6D) and P(ImC8D)).

[0527] P(ImC6-co-ImC6D)-50% and P(ImC8-co-ImC8D)-50% are copolymerized to form ( Figure 19 a), while P(ImC6D) and P(ImC8D) are homopolymerized into ( Figure 19 b).

[0528] P(ImC6-co-ImC6D)-50%

[0529] P(ImC6-co-ImC6D)-50% was prepared by using a diamide diamine (n=6) TFA salt and 1,6-diaminohexane according to the scheme in Example 2, having a molar fraction of 50% of degradable diamine.

[0530] 1 ¹H NMR (300MHz, D₂O): δ 8.85 (m, 1H, imidazole-H), 7.50 (m, 2H, imidazole-H), 5.00 (t, 2H), 4.22 (t, 2H), 3.23 (s, 2H), 1.88 (s, 2H), 1.49 (m, 4H).

[0531] P(ImC8-co-ImC8D)-50%

[0532] P(ImC8-co-ImC8D)-50% was prepared by using a diamide diamine (n=8) TFA salt and 1,8-diaminooctane according to the scheme in Example 2, having a molar fraction of 50% of degradable amines.

[0533] 1 ¹H NMR (300MHz, D₂O): δ 8.85 (m, 1H, imidazole-H), 7.51 (m, 2H, imidazole-H), 5.04 (d, 2H), 4.19 (m, 2H), 3.19 (m, 2H), 1.90 (s, 2H), 1.63–1.39 (m, 8H).

[0534] P(ImC6D)

[0535] P(ImC6D) was prepared by following the protocol in Example 2 from the diamide diamine (n = 6) TFA salt, except that no non-degradable amine was added. After dialysis, P(ImC6D) was obtained.

[0536] 1 H NMR (300 MHz, D20): δ 8.93 (s, 1H, imidazole-H), 7.54 (s, 2H, imidazole-H), 5.08 (s, 4H), 3.24 (s, 4H), 1.56-1.43 (m, 8H).

[0537] P(ImC8D)

[0538] P(ImC8D) was prepared by following the protocol in Example 2 from the diamide diamine (n = 8) TFA salt, except that no non-degradable amine was added. After dialysis, P(ImC8D) was obtained.

[0539] 1 H NMR (300 MHz, D20): δ 8.93 (s, 1H, imidazole-H), 7.54 (s, 2H, imidazole-H), 5.08 (s, 4H), 3.24 (s, 4H), 1.56-1.43 (m, 8H).

[0540] List of abbreviations for degradable PIMs

[0541] P(ImC6-co-ImC6D)-50% - P3

[0542] P(ImC6D) - P4

[0543] P(ImC8-co-ImC8D)-50% - P5

[0544] P(ImC8D) - P6

[0545] All PIMs prepared here and in Comparative Example 15 were characterized by H NMR and GPC (Table 13). Figure 20 ) by 1 H NMR and GPC (Table 13).

[0546] Table 13. Actual molar fraction of degradable diamine, Mw, Mn, and polydispersity (Mw / Mn) of PIMs. n , M w , and polydispersity (M n / M w ).

[0547]

[0548] Example 10. In vitro antimicrobial activity and cytotoxicity of P1-P6

[0549] The three types of PIM prepared were evaluated according to the scheme in Comparative Example 3. Figure 20 The antimicrobial activity against planktonic bacteria was measured by determining its MIC values ​​against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (BAA39) and Gram-negative strains of Staphylococcus aureus, as well as Pseudomonas aeruginosa O1 and Escherichia coli. Benzalkonium chloride (BAC) was used as a reference. The cytotoxicity of PIM against mouse embryonic fibroblast 3T3 cells was tested according to the MTT assay protocol in Comparative Example 3.

[0550] Results and discussion

[0551] PIMs with higher molar fractions of degradable linkers (100%) were less potent in killing bacteria than non-degradable PIMs (0% molar fraction of degradable linkers), as shown in Table 14. However, this trend was not evident for PIMs with longer alkyl linkers (P4, P5, and P6). Comparing the viability of cells treated with PIMs of different molar fractions of degradable linkers (0%, 50%, and 100%), we can see a trend of increasing biocompatibility with increasing fraction, which is contrary to the trend of antimicrobial activity against planktonic bacteria.

[0552] Table 14. MIC (μg / mL) values ​​of PIM and BAC (reference) for bacterial groups.

[0553]

[0554] Example 11. In vitro anti-biofilm activity of P1-P6

[0555] MBEC

[0556] MBEC was measured using a microtiter plate-based technique. Simply put, 160 μL of MRSA BAA39 or Pseudomonas aeruginosa O1 suspension (cell density ~10⁻⁶) was added. 7 CFU / mL was added to 96-well growth plates covered with caps containing MBEC pins. Biofilms grew on the pin caps after incubation at 37°C for 24–48 h. After removing airborne bacteria by washing twice with PBS, the caps containing biofilm were transferred to attack plates containing a 2-fold serial dilution of one of the P1–P6 solutions, with a total volume of 200 μL per well. Treatment was carried out at room temperature for 4 h. The pin caps were then washed again with PBS and transferred to recovery plates containing PBS (200 μL) per well. Viable biofilm bacteria were expelled from the pin caps by sonication for 30 ± 5 min, and the isolated bacteria were then serially diluted 10-fold in sterile PBS and plated onto agar plates. Colony counting was performed after incubation at 37°C for 24 h.

[0557] Results and discussion

[0558] As Figure 21 shown, the overall anti-biofilm efficacy against MRSAB AA 39 can be ordered as follows: P(ImC8) > P(ImC8-co-ImC8D)-50% ~ P(ImC6) > BAC. Similarly, for P. aeruginosa 01( Figure 22 ), the anti-biofilm efficacy order is: P(ImC8) > P(ImC8-co-ImC8D)-50% > P(ImC6) > BAC.

[0559] Example 12. Synthesis of degradable 2+2 carbonate monomer (Compound 4)

[0560] The synthesis of the carbonate monomer (Compound 4) involves compounds 1-3 and three steps Figure 23 ). We synthesized imidazole carboxylate (Compound 2) by reacting CDI with alcohol (Compound 1) to give Compound 2 in good yield. Carbonyl formation was then achieved by treating Compound 2 with CDI and Compound 1 in the presence of catalytic amount of NaOH to provide the desired boc-protected carbonate (Compound 3) in good yield. Boc deprotection was carried out in TFA in DCM to give the desired Compound 4 in good yield.

[0561] 1H-Imidazole-1-carboxylic acid 2-((tert-butoxycarbonyl)amino)ethyl ester (Compound 2)

[0562] In a 250 mL round bottom flask equipped with a dry N2inlet and a magnetic stirrer, dry toluene (150 mL) and 1,1'-carbonyldiimidazole (CDI, 10.0 g, 0.0310 mol) were added, followed by tert-butyl (2-hydroxyethyl)carbamate (Compound 1, 5.0 g, 0.0198 mol) and KOH (5.2 mg, 0.003 mol). The mixture was heated at 60 °C for 4 h with stirring. Formation of a clear solution was observed. The reaction mixture was cooled to room temperature. The solution was concentrated in vacuo, dissolved in DCM (200 mL), and washed with water (3 x 50 mL). The solution was dried over anhydrous Na2S04and concentrated in vacuo to give Compound 2 as a white solid (5.1 g, 62.1%).

[0563] 1 H NMR (300 MHz, DMSO-D6): δ 8.15 (s, 1H), 7.44 (s, 1H), 7.07 (s, 1H), 4.91 (br s, 1H), 4.47 (t, J = 5.2 Hz, 2H), 3.52 (q, J = 6.3 Hz, 2H), 1.44 (s, 9H).

[0564] Di-tert-butyl ((carbonylbis(oxy)bis(ethane-2,1-diyl)diaminocarboxylate (Compound 3)

[0565] In a 250 mL round bottom flask equipped with a dry N2inlet and a magnetic stirrer, dry toluene (150 mL) and CDI (6.3 g, 0.0389 mol) were added, followed by compound 2 (5.0 g, 0.019 mol), compound 1 (3.17 g, 0.0195 mol) and KOH (5.17 mg, 0.003 mol). The mixture was heated at 60 °C for 18 h under stirring. Formation of a clear solution was observed. The reaction mixture was cooled to room temperature. The solution was concentrated under vacuum, dissolved in DCM (200 mL) and washed with water (3 x 50 mL). The solution was dried over anhydrous Na2S04and concentrated under vacuum. The resulting crude product was purified by column chromatography (EtOAc:hexane 3:7) to yield compound 3 (4.80 g, 58.8%) as a white solid.

[0566] 1 H NMR (300 MHz, DMSO-D6): d 5.21 (br s, 2H), 4.29 (t, J = 5.1 Hz, 4H) 3.33 (s, 4H), 1.25 (s, 18H).

[0567] 2,2,2-trifluoroacetate (Compound 4) CO

[0568] In a 100 mL round bottom flask equipped with a dry N2inlet and a magnetic stirrer, compound 3 (4.0 g, 0.0389 mol) was dissolved in dry DCM (50 mL) and TFA (6 mL, excess) was added. The reaction mixture was stirred at room temperature for 18 h. Then, the reaction mixture was concentrated under reduced pressure to yield carbonate monomer 4 (3.60 g, 75%) as a white solid.

[0569] 1 H NMR (300 MHz, D2O): d 4.34 (t, J = 5.1 Hz, 4H), 3.27-3.24 (m, 4H). 13 C NMR (75 MHz, D2O): d 166.14, 159.58 (-CF3), 159.16 (-CF3), 158.74 (-CF3), 158.32 (-CF3), 154.56 (CO-O), 123.24 (-CF3), 119.29 (-CF3), 115.33 (-CF3), 111.38 (-CF3), 64.26, 38.23. CO -CF3), 159.16 (-CF3), 158.74 (-CF3), 158.32 (-CF3), 154.56 (CO-O), 123.24 (-CF3), 119.29 (-CF3), 115.33 (-CF3), 111.38 (-CF3), 64.26, 38.23. CO -CF3), 159.16 (-CF3), 158.74 (-CF3), 158.32 (-CF3), 154.56 (CO-O), 123.24 (-CF3), 119.29 (-CF3), 115.33 (-CF3), 111.38 (-CF3), 64.26, 38.23. CO -CF3), 159.16 (-CF3), 158.74 (-CF3), 158.32 (-CF3), 154.56 (CO-O), 123.24 (-CF3), 119.29 (-CF3), 115.33 (-CF3), 111.38 (-CF3), 64.26, 38.23. Figure 24 -CF3), 159.16 (-CF3), 158.74 (-CF3), 158.32 (-CF3), 154.56 (CO-O), 123.24 (-CF3), 119.29 (-CF3), 115.33 (-CF3), 111.38 (-CF3), 64.26, 38.23.

[0570] Example 13. Synthesis of biodegradable PIM D2 with carbonate linker

[0571] PIM D2-1-8 were prepared from compound 4 by following the protocol in Example 2 and controlling the stoichiometric ratio and concentration of the starting materials (Table 15). Figure 25

[0572] 1 H NMR (300 MHz, D20): δ 8.85 (m, 1H, imidazole-H), 7.50 (m, 2H, imidazole-H), 4.47 (s, 4H). 13 CNMR (75 MHz, D20): δ 154.29, 136.64, 122.96, 66.25, 48.31.

[0573] Table 15. Summary of polymerization conditions and molecular weight of biodegradable PIMs with carbonate incorporated (PIM D2).

[0574]

[0575] a Molar ratio is the ratio of diamine to aldehyde; b Concentration is the concentration of aldehyde; c S. aureus is S. aureus 29213.

[0576] From Table 15 we can see that the concentration of diamine has only a small effect on the molecular weight of the polymer, but the stoichiometric ratio of diamine to aldehyde shows a significant effect on the molecular weight of the polymer. The highest molecular weight obtained is PIM D2-5 with a molecular weight of 1522 g / mol and a narrow polydispersity of 1.08. With the same concentration of diamine and aldehyde, the molecular weight of the polymer increases with the increase of the number of repeating units. 1 H NMR and 13 C NMR spectra further confirm the chemical structure of biodegradable PIMs with carbonate incorporated.

[0577] Example 14. Stepwise synthesis of degradable oligoimidazoliums (OIM1D-3C-6 and OIM1D-3C-8)

[0578] Considering the good antibacterial activity and biocompatibility of PIM1D, stepwise synthesis was explored to make oligoimidazoliums with biodegradable amide linkers and well-defined molecular weight. Imidazoliums with three repeating units were prepared by a stepwise approach, which were connected together using N,N'-(alkane-1,3-diyl)bis(2-chloroacetamide) linkers to give the final degradable compounds, named OIM1D-3C-6 and OIM1D-3C-8 for the degradable linkers with three carbons and eight carbons in the alkyl chain, respectively. The synthesis was achieved in six steps 1,4-Bis(1H-imidazol-1-yl)butane (Compound 5) ​), and eight intermediate compounds (compounds 5-12) are required to obtain the final degradable oligoimidazoliums (OIM1D-3C-6 and OIM1D-3C-8). Compounds were characterized by NMR and MALDI-TOF where appropriate.

[0579] Compound 6

[0580] Compound 5 was prepared by following the protocol in Comparative Example 12 from imidazole (4.00 g, 0.058 mol 1 1 equivalent), except that the reaction mixture was heated at reflux (70 °C) overnight, and the product was purified by extraction with MeOH. The MeOH phase was washed with hexanes three times, and compound 5 was obtained as white solid crystals (10.2 g, 92%) by rotary evaporation.

[0581] 1 H NMR (300 MHz, DMSO-d6) δ 7.61 (s, 2H), 7.14 (br s, 2H), 6.89 (br s, 2H), 3.98 - 3.73 (m, 4H), 1.64 - 1.59 (m, 4H). MALDI-TOF (CHCA matrix, Reflector mode) C 10 H 14 N4: Calculated 190.1218 (M); Found 191.1296 (M + H).

[0582] Compound 7

[0583] Triethylamine (Et3N) (1.2 equivalents, 10.6 g, 0.105 mol) was added to a stirred solution of aminopropylimidazole (1.0 equivalent, 11.0 g, 0.088 mol) in DCM (110 mL) at 0 °C. CBzCl (1.1 equivalents, 16.5 g, 0.096 mol) was slowly added over a period of 10 min via syringe. The reaction mixture was allowed to stir and warm to room temperature overnight. The reaction was transferred to a separatory funnel, and the organic layer was extracted with 0.2 M HC1 (100 mL), then successively extracted with water (100 mL) four times. The organic layer was dried over anhydrous Na2S04, concentrated by rotary evaporation and subjected to silica gel chromatography to obtain compound 6 (20.5 g, 90%).

[0584] 1H NMR (300 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.50 - 7.24 (m, 6H), 7.17 (s, 1H), 6.90 (s, 1H), 5.04 (s, 2H), 3.97 (t, J = 6.9 Hz, 2H), 2.98 (q, J = 6.3 Hz, 2H), 1.84 (p, J = 6.7 Hz, 2H). 13 CNMR (75 MHz, DMSO-d6) δ 156.1, 137.2, 137.1, 128.3, 127.7, 119.3, 65.3, 43.4, 37.4, 31.0.

[0585] Compound 8

[0586] Compound 6 (3.00 g, 0.0115 mol, 1.0 equiv) was added to a stirring solution of 1,4-dibromobutane (4.5 mL, 0.0375 mol, 2.5 equiv) in dry ACN (10 mL) under an argon atmosphere. The reaction mixture was heated at 70 °C for 14 h, then cooled to room temperature. The solvent was removed by rotary evaporation under vacuum and silica gel chromatography eluting with EtOAc to 15% MeOH / EtOAc to give compound 7 as a white syrup (4.10 g, 76%).

[0587] 1 H NMR (300 MHz, DMSO-d6) δ 9.39 (s, 1H), 7.88 (d, J = 3.4 Hz, 2H), 7.58 - 7.21 (m, 6H), 5.02 (s, 2H), 4.24 (q, J = 7.2 Hz, 4H), 3.56 (t, J = 6.4 Hz, 2H), 3.02 (q, J = 6.0 Hz, 2H), 2.05 - 1.86 (m, 4H), 1.86 - 1.72 (m, 2H). 13 C NMR (75 MHz, DMSO-d6) δ 156.2, 137.0, 136.2, 128.3, 127.76, 127.70, 122.45, 122.40, 65.3, 47.9, 46.5, 36.9, 34.1, 29.7, 28.7, 28.1. MALDI-TOF (CHCA matrix, Reflector mode) C 18 H 25 Br2N3O2: Calculated 473.0314 (M); Found 394.1405 (M-Br).

[0588] Compound 9

[0589] Compound 5 (1.80 g, 0.009 mol, 1.5 equiv) was added to a stirred solution of compound 7 (3.0 g, 0.006 mol, 1.0 equiv) in dry ACN (10 mL) and the resulting mixture was heated at 70 °C under argon atmosphere overnight. After monitoring the end of the reaction by TLC, the solvent was removed under vacuum and the resulting mixture was subjected to flash silica gel (100-200 mesh) column chromatography (mobile phase EtOAc to MeOH; 10 to 50%) to give compound 8 (3.00 g, 72%) as a hygroscopic white solid.

[0590] 1 H NMR (300 MHz, DMSO-d6) δ 9.35 (t, J = 24.3 Hz, 2H), 7.88 - 7.78 (m, 4H), 7.72 (s, 1H), 7.48 - 7.28 (m, 6H), 7.20 (s, 1H), 6.92 (s, 1H), 5.02 (s, 2H), 4.20 (t, J = 6.9 Hz, 8H), 4.02 (t, J = 6.5 Hz, 2H), 3.05 - 2.99 (m, 2H), 2.02 - 1.88 (m, 2H), 1.80 - 1.72 (m, 8H). 13 C NMR (75 MHz, DMSO-d6) δ 155.0, 136.0, 135.8, 135.1, 134.7, 127.2, 127.0, 126.68, 126.60, 121.3, 121.2, 118.1, 64.0, 47.0, 46.9, 45.4, 44.0, 43.8, 35.7, 28.5, 26.0, 25.3, 24.8. MALDI-TOF (CHCA matrix, Reflector mode) C 28 H 39 Br2N7O2Calcd 663.1532 (M); Found (M-2Br-H) 504.3814.

[0591] Compound 10

[0592] To a solution of K2CO3(33 mmol, 3.3 equiv) in water / DCM (1:3, 18 mL) was added 1,3-diaminopropane (10.0 g, 1 equiv) at 0 °C. The resulting mixture was allowed to cool and then chloroacetyl chloride (22 mmol, 2.2 equiv) was added dropwise over a period of 1 h at 0 °C. After complete addition, the ice bath was removed and the mixture was allowed to stir at room temperature overnight. The desired product was extracted three times with DCM. Subsequently, the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 9 (82%, 24.5 g).

[0593] 1 H NMR (DMSO-d6) δ 8.59 (s, 2H), 4.05 (s, 4H), 3.09 (t, 4H), 1.55 - 1.62 (m, 2H).

[0594] Compound 11

[0595] Compound 10 was prepared based on the protocol for compound 9 from 1,8-diamino octane (1 equiv).

[0596] Compound 12

[0597] To a stirred solution of compound 8 (1.0 equiv) in ACN:DMF (9:1) was added compound 9 (0.5 equiv) at room temperature, followed by heating at 80 °C for 48 h. The reaction mixture was cooled to room temperature, and filtered and the resulting precipitate was collected as a hygroscopic sticky compound, which was further washed with ACN three times and lyophilized to give a crude mixture of compound 11 and impurities.

[0598] 1 H NMR δ (D20) 8.79 (s, 2H), 8.72 (s, 2H), 8.62 (s, 2H), 7.46 - 7.31 (m, 24H), 4.96 (s, 8H), 4.24 - 4.11 (m, 20H), 3.08 - 3.05 (m, H), 3.05-3.03 (m, 4H), 1.82 - 1.64 (m, 22H). 13 C NMR (75 MHz, DMSO-d6) δ 165.5, 156.8, 132.5, 131.9, 127.1, 126.3, 122.4, 121.15, 121.12, 65.4, 48.1, 47.4, 46.8, 35.7, 27.5, 26.4, 2.8.

[0599] OIM1D-3C-6

[0600] Compound 12 was prepared based on the protocol for compound 11 from compounds 8 and 10.

[0601] 1H NMR δ (DMSO-d6) 9.56 (s, 2H), 9.47 (s, 2H), 9.37 (s, 2H), 8.65 (s, 2H), 7.88 - 7.84 (br s, 12H), 7.49 (s, 2H), 7.10 - 7.32 (m, 10H), 5.05 (s, 4H), 5.02 (s, H), 4.08 - 4.06 (m, 20H), 3.04 - 3.01 (m, 8H), 1.97 - 1.82 (m, 20H), 1.26 - 1.15 (m, 14H).

[0602] OIM1D-3C-8

[0603] Compound 11 was dissolved in a solution of HBr in AcOH (33%) and the resulting mixture was stirred at room temperature for 3 h. EtOAc (2 mL) was added to precipitate the amine salt. The solvent was extracted and the resulting residue was retained. The resulting compound was dissolved in water (50-60 mM) and passed through a column containing loading chloride of A-26 (OH - form). The column was further washed with water until complete separation of the compound, which was then concentrated under vacuum. The obtained material was diluted with water and dialyzed (Mw-CO 500-1000 D) against acidified water (1 mL) for 1 day, with 6-7 changes of acidified water. The solution in the dialysis bag was poured into a Falcon tube and freeze-dried to obtain OIM1D-3C-6 (about 30%).

[0604] 1 H NMR δ (D2O) 8.79 (s, 4H), 8.74 (s, 2H), 7.46 - 7.41 (m, 12H), 4.96 (s, 4H), 4.19 - 4.15 (m, 20H), 3.17 (s, 4H), 2.90 (s, 4H), 2.18 (m, 4H), 1.88 - 1.65 (m, 18H).

[0605] OIM1D-3C-8 was prepared from compound 12 based on the protocol for OIM1D-3C-6. Results and discussion

[0606] 1 H NMR δ (D2O) 8.80 (s, 4H), 8.75 (s, 2H), 7.47 - 7.40 (m, 12H), 4.94 (s, 4H), 4.24 - 4.16 (m, 20H), 3.03 (t, 4H), 2.90 (t, 4H), 2.19 - 2.16 (m, 4H), 1.83 (br s, 16H), 1.42 - 1.40 (m, 4H), 1.48 - 1.46 (m, 8H).

[0607] ​​Example 15. In vitro biological profile of degradable OIM1D-3C-6 and OIM1D-3C-8

[0608] The in vitro biological profile of OIM1D-3C-6 and OIM1D-3C-8 was evaluated using the MIC and MTT experiments described in Comparative Example 3.

[0609] Results and discussion

[0610] OIM1D-3C-6 and OIM1D-3C-8 showed good antibacterial activity against S. aureus and methicillin-resistant S. aureus as well as E. coli, with MICs 90 ranging from 2-16 pg / mL (Table 16). OIM1D-3C-6 showed reduced antibacterial potency against P. aeruginosa PAOl, with a MIC 90 of 128 pg / mL. Both OIM1D-3C-6 and OIM1D-3C-8 showed good biocompatibility, with IC 50 values exceeding 1024 pg / mL, as determined using 3T3 fibroblasts by MTT testing.

[0611] Table 16. MICs of OIM1D-3C-6 and OIM1D-3C-8 against pathogens and human cell lines 90 .

[0612]

[0613] Thus, by modulating the degradable linker chain, the degradable functional group, the imidazolium repeat unit, and the end group, a library of biodegradable oligomeric imidazoliums with multifunctional functionalities can be constructed. This would be a good candidate for mechanistic studies, degradation rate studies, pharmacokinetic and pharmacodynamic studies in animal models.

[0614] Example 16. In vivo studies of degradable OIM1D-3C-6 and OIM1D-3C-8

[0615] The in vivo efficacy of OIM1D-3C-6 and OIM1D-3C-8 was evaluated using the neutropenic lung infection model described in Example 7, while their in vivo intranasal toxicity was determined as described below.

[0616] In vivo intranasal toxicity

[0617] OIM1D-3C-8 and OIM1D-3C-8 / OIM1D-3C-6 mixture at 20 mg / kg were delivered intranasally to randomly grouped mice (ICR, female). The mice were monitored daily for body weight and condition until 7 days after compound delivery.

[0618] Figure 26

[0619] Results show that with 10 mg / kg of OIM1D-3C-8, bacterial load was reduced by 60%, while 20 mg / kg OIM1D-3C-8 reduced bacterial load by about 2 log orders Figure 26 A), demonstrating the efficacy of OIM1D-3C-8 in reducing bacterial load in a lung infection model. In a neutropenic lung infection induced by methicillin-resistant Staphylococcus aureus, a reduction of about 2 log orders in bacterial load was also observed Figure 26 B), demonstrating the efficacy of OIM1D-3C-8 against Gram-positive bacterial infection. We then investigated the toxicity of OIM1D-3C-8 by intranasal delivery of OIM1D-3C-8 (20 mg / kg), followed by body weight monitoring. Results show that this led to a gradual decrease in body weight over time Figure 26 C).

[0620] To reduce the in vivo toxicity of OIM1D-3C-8, we mixed OIM1D-3C-8 with OIM1D-3C-6 in a 2:1 and 1:1 weight ratio. With this mixture, toxicity was successfully reduced, and negligible weight loss over time was observed Figure 26 C). We then evaluated the efficacy of these two mixtures, and found that for the OIM1D-3C-8 / OIM1D-3C-6 (2:1 wt.%) mixture, a reduction of about two log orders in bacterial load was observed ​ D) in a lung infection induced by MDR Klebsiella pneumoniae, similar to OIM1D-3C-8. In summary, we have demonstrated that the OIM1D-3C-8 / OIM1D-3C-6 (2:1 wt.%) mixture has good in vivo efficacy with limited toxicity, indicating its potential as a therapeutic agent in MDR bacterial infection.

Claims

1. A polymer or oligomer or a pharmaceutically acceptable solvate thereof, comprising a first repeating unit and a second repeating unit, the first repeating unit comprising an imidazolium group and a biodegradable chain attached to an adjacent repeating unit, and the second repeating unit comprising an imidazolium group and a non-biodegradable alkyl chain attached to an adjacent repeating unit, wherein the polymer or oligomer has Formula I: in: x is between 0.10 and 0.50; Y - The anti-charge ion is selected from one or more of the following groups: chloride, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide ion N(Tf)2. - ; o ranges from 0 to 6; p ranges from 1 to 6; q is between 0 and 6; r ranges from 1 to 6; D is a biodegradable functional group, where D is an amide group; D' is a biodegradable functional group, where each D' is an amide group; Or a pharmaceutically acceptable solvate thereof.

2. The polymer or oligomer according to claim 1, wherein one or more of the following are applicable: (a) The polymer or oligomer comprises 1 to 75 mol% of the first repeating unit; and (b) The repeating units of the polymer or oligomer are randomly distributed or the repeating units are formed as blocks.

3. The polymer or oligomer according to claim 1, wherein the number average molecular weight is 800 to 10,000 Daltons.

4. The polymer or oligomer of claim 1, wherein the polymer is selected from the group consisting of: 。 5. Use of the polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 4 in the manufacture of a medicament for treating diseases including microbial infections.

6. A disinfectant formulation comprising a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 4.

7. An article having a surface, wherein the surface is coated with a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 4 to provide antimicrobial properties to the surface of the article.

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