Biodegradable polyimidazolium and oligoimidazolium

Abstract

Disclosed herein are compounds in the form of polymers, oligomers, and defined molecules, all of which have repeating units that incorporate a repeating unit formed from an imidazolium group and a biodegradable chain that connects adjacent repeating units. The compounds disclosed herein may have antimicrobial activity and therefore may be used to treat microbial infections and / or to treat surfaces to prevent microbial infections. Methods for forming the compounds are also disclosed herein.

Description

[Technical Field]

[0001] Technical field This invention relates to the field of polyimidazolium and oligoimidazolium, as well as defined molecules having similar characteristics. All of these molecules contain a degradable (particularly biodegradable) moiety that allows for degradation in vivo. These molecules may be useful in treating microbial infections or may act as antimicrobial agents (e.g., on personal care products or surfaces). [Background technology]

[0002] Any list or discussion of previously published documents in this specification should not be considered an endorsement that such documents are part of the latest technology or general knowledge.

[0003] The emergence and spread of multidrug-resistant (MDR) pathogens is a major global concern. Recently, the World Health Organization (WHO) called for the development of new antimicrobial agents against the most problematic superbugs, including carbapenem-resistant Acinetobacter baumannii (CRE-AB), carbapenem-resistant Pseudomonas aeruginosa (CRE-PA), and extended-spectrum β-lactamase (ESBL)-producing carbapenem-resistant Enterobacteriaceae (CRE-EB) (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 the treatment of multidrug-resistant (MDR) bacteria. Basic design elements of AMPs include hydrophobicity and regions of charged residues (typically cationic residues that enable interaction with the bacterial cell surface) to disrupt bacterial cell membranes (Ganewatta, MS et al., Polymer c2015, 63, A1-A29). However, the development of AMPs is often hampered by poor pharmacokinetic properties, low stability in body fluids, toxicity to mammalian cells due to low selectivity, and generally higher minimum inhibitory concentrations (MICs) compared to conventional antibiotics. Nevertheless, naturally occurring complex AMPs, such as cyclic lipopeptides (e.g., polymyxins), are used clinically for difficult-to-treat Gram-negative bacterial infections. However, their high cost and toxicity significantly limit their use as a last resort alternative. Colistin, an antimicrobial peptide, is thought to kill bacteria by disrupting membrane integrity, and its use as a last-resort alternative antibiotic has recently increased (Velkov, T. et al., J. Med. Chem. 2010, 53, 1898-1916). However, colistin requires intravenous administration and is nephrotoxic (Javan, AO et al., Eur. J. Clin. Pharmacol. 2015, 71, 801-810).

[0005] In addition to peptides, synthetic polymers are widely used as disinfectants due to their high antimicrobial activity. Most of these polymers are synthesized by free radical polymerization (FRP), ring-opening polymerization (ROP), and post-functionalization, which often involve multiple steps, difficult purification, and the use of organic solvents, making them difficult to scale up. These antimicrobial polymers typically exhibit a high degree of toxicity with a limited range of antimicrobial effects.

[0006] Therefore, it is necessary to develop new AMP-like analogs with improved properties. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] World Health Organization (WHO) Global Priority List of Antibiotic-Resistant Bacteria 2017 to Guide Research, Discovery and Development of New Antibiotics [Non-Patent Document 2] Ganewatta,MSet al.,Polymer 2015,63,A1-A29 [Non-Patent Document 3] Velkov,T.et al., J.Med.Chem.2010,53,1898-1916 [Non-Patent Document 4] Javan,AOet al.,Eur.J.Clin.Pharmacol. 2015,71,801-810 [Overview of the project] [Means for solving the problem]

[0008] Summary of the Invention Herein, aspects and embodiments of the present invention will be described with reference to the following numbered sections.

[0009] Section 1. A polymer or oligomer or a pharmaceutically acceptable solvate thereof comprising a first repeating unit containing an imidazolium group and a biodegradable chain bonded to an adjacent repeating unit.

[0010] Section 2. The polymer or oligomer according to item 1, wherein only the aforementioned repeating unit is the first repeating unit.

[0011] Section 3. The polymer or oligomer according to claim 1, further comprising a second repeating unit comprising an imidazolium group and a non-biodegradable alkyl chain or a further biodegradable alkyl chain bonded to an adjacent repeating unit, and optionally, the polymer or oligomer further comprising a second repeating unit comprising an imidazolium group and a non-biodegradable alkyl chain bonded to an adjacent repeating unit.

[0012] Section 4. below: (a) The polymer or oligomer comprises, for example, 1 to 75 mol% of the first repeating units, such as 5 to 60 mol%, such as 10 to 50 mol%, such as 20 to 30 mol%, and (b) The repeating units of the polymer or oligomer are randomly distributed, or the repeating units are formed as blocks, and optionally the repeating units of the polymer or oligomer are randomly distributed. A polymer or oligomer as described in item 3, which falls under one or more of the following categories.

[0013] Section 5. The biodegradable chain in the first repeating unit comprises one or more biodegradable functional groups, and the one or more biodegradable functional groups are selected from one or more of the group consisting of urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride, and hydrazone, and optionally, (ai) The one or more biodegradable functional groups are selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (aii) The one or more biodegradable functional groups are selected from the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, or (aiii) The one or more biodegradable functional groups are amides. A polymer or oligomer as described in any one of items 1 to 4.

[0014] Section 6. The polymer or oligomer according to any one of claims 1 to 5, wherein the number average molecular weight is, for example, 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.

[0015] Section 7. The aforementioned polymer or oligomer is of formula I: [ka] [In the above formula I, x is between 0.01 and 1.0. Y - This is a counterion, o is a number from 0 to 10 (for example, 0 to 6, like 1 to 5). p is between 1 and 12. q is between 0 and 14 (for example, between 0 and 6), r is between 0 and 12. D is a biodegradable functional group, D' is a biodegradable functional group or bond. Each R 1 C is branched or unbranched 1-3 Alkyl or derivative thereof, Each t is 0, 1, or 2 (for example, t is 0 or 1), Each t' is 0, 1, or 2 (for example, t' is 0 or 1), Each R 2 C is branched or unbranched 1-3 [Alkyl or derivative thereof] A polymer or oligomer or pharmaceutically acceptable solvate thereof according to any one of claims 1 to 6, having the above.

[0016] Section 8. below: (bi) Each D is selected from urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride and hydrazone, optionally (aa) Each D is selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (ab) Each D is selected from one or more of the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, (ac) Each D is selected from one or more of the group consisting of carbonate esters and amides (for example, each D is an amide), (bii) Each D' is selected from bonded, urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride and hydrazone, optionally (ad) Each D' is selected from one or more of the group consisting of bonds, amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (ae) Each D' is selected from one or more of the group consisting of bonds, amides, esters, carbamates, and carbonate esters. (af) Each D' is selected from one or more of the group consisting of bonds and amides, (ag) Each D' is selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (ah) Each D' is selected from one or more of the group consisting of amides, esters, carbamates, and carbonate esters, (ai) Each D' is an amide, (biii) Y - This includes halo, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf)2 - ) Selected from one or more of the group consisting of, Y -is selected from one or more of the group consisting of chloro, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ) and (biv) x is from 0.01 to 1.0, such as from 0.025 to 0.75, such as from 0.05 to 0.6, such as from 0.1 to 0.5, such as from 0.2 to 0.3 (bv) t and t’ are 0 (bvi) p is from 1 to 6, and (bvii) r is from 1 to 6 The polymer or oligomer according to item 7, to which one or more of the above apply

[0017] Item 9 The polymer is

Chemical formula

[0018] Item 10 The first block of the repeating unit of the oligomer, where each repeating unit contains an imidazolium group and a non-biodegradable alkyl chain bonded to an adjacent repeating unit The second block of the repeating unit of the oligomer, where each repeating unit contains an imidazolium group and a non-biodegradable alkyl chain bonded to an adjacent repeating unit A linking group that binds together with the first block and the second block, where the linking group contains one or more biodegradable functional groups A molecule or its pharmaceutically acceptable solvate comprising

[0019] Item 11 The one or more biodegradable functional groups are selected from one or more of the group consisting of urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride, and hydrazone, optionally (ci) The one or more biodegradable functional groups are selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (cii) The one or more biodegradable functional groups are selected from the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, (ciii) The one or more biodegradable functional groups are selected from one or both of amides and carbonate esters, or (civ) The one or more biodegradable functional groups are amides. , the molecule described in item 10.

[0020] Section 12. The molecule according to item 10 or item 11, wherein the molecular weight is 1,000 daltons to 5,000 daltons, and optionally the molecular weight is 1,000 daltons to 4,000 daltons.

[0021] Section 13. The aforementioned molecule is given by formula II: [ka] [In the above formula II, Each m is independently between 1 and 8 (for example, between 1 and 6). Each Y - It is a counterion, n' is between 0 and 12. Each o' is independently selected from 0 to 20. Each p' is independently selected from 0 to 12 (for example, 0 to 6). Each p'' is independently selected from 0 to 12 (for example, 0 to 6). Each T is independently a terminal functional group selected from amine, ammonium, guanidinium, bisguanidinium, alkyl, and aryl. Each D is a biodegradable functional group or a pharmaceutically acceptable solvate thereof. A molecule having any one of the items 10 to 12.

[0022] Section 14. below: (di) Each D is independently selected from the group consisting of urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride, and hydrazone, and optionally, (ba) Each D is independently selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (bb) Each D is independently selected from one or more of the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, or (bc) Each D is an amide, (dii) Y - This includes halo, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf)2 - One or more of the group consisting of ) are selected, and Y is arbitrarily selected. - This includes chloro, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf)2 - Selected from one or more of the groups consisting of ), (dii) p'' is between 0 and 6 (p'' is 0), A molecule described in item 13, wherein one or more of the following apply.

[0023] Section 15. The aforementioned molecule, [ka] A molecule selected from the group consisting of any one of items 10 to 14.

[0024] Section 16. Polymers or oligomers described in any one of sections 1 to 9, or pharmaceutically acceptable solvates thereof, and / or molecules described in any one of sections 10 to 15, for use in medical applications.

[0025] Section 17. Use of polymers or oligomers or pharmaceutically acceptable solvates thereof as described in any one of items 1 to 9 and / or molecules or pharmaceutically acceptable solvates thereof as described in any one of items 10 to 15 in the manufacture of a pharmaceutical product for the treatment of a disease comprising a microbial infection.

[0026] Section 18. Polymers or oligomers or pharmaceutically acceptable solvates thereof as described in any one of sections 1 to 9 and / or molecules or pharmaceutically acceptable solvates thereof as described in any one of sections 10 to 15, for use in the treatment of diseases comprising microbial infections.

[0027] Section 19. A method for treating a disease comprising a microbial infection, comprising the step of administering to a subject in need a therapeutically effective amount of a polymer or oligomer or a pharmaceutically acceptable solvate thereof as described in any one of items 1 to 9 and / or a molecule or a pharmaceutically acceptable solvate thereof as described in any one of items 10 to 15.

[0028] Section 20. The polymer or oligomer or molecule described in item 17, the polymer or oligomer or molecule for use described in item 18, and the use of the method described in item 19, Use if the aforementioned microbial infection is an infected wound or cystic fibrosis.

[0029] Section 21. A preservative formulation comprising a polymer or oligomer or a pharmaceutically acceptable solvate thereof as described in any one of items 1 to 9, and / or a molecule or a pharmaceutically acceptable solvate thereof as described in any one of items 10 to 15.

[0030] Section 22. An article having a surface, wherein the surface is coated with a polymer or oligomer or a pharmaceutically acceptable solvate thereof as described in any one of items 1 to 9 and / or a molecule or a pharmaceutically acceptable solvate thereof as described in any one of items 10 to 15, thereby imparting antimicrobial properties to the surface of the article, and optionally the article is a urethral catheter. [Brief explanation of the drawing]

[0031] drawing [Figure 1] Chemical structures of polyimidazolium (PIM) synthesized and used in the experiment. The number of repeating subunits in each PIM was estimated by gel permeation chromatography (GPC). [Figure 2] Figure 2 shows the viability (growth capacity) of MRSA LACs* treated with PIM1 (0.5 to 4 times the MIC of each bacterial species) compared to (A) Pseudomonas aeruginosa PAO1 and (B) a control group without PIM1. Cells were incubated in MHB at 37°C and sampled at the indicated times. Cell counts were determined as colony-forming units (CFU) per mL by plate counting. [Figure 3] Figure 3 shows propidium iodide (PI) staining of Pseudomonas aeruginosa PAO1 cells. Fluorescence microscopy images of (A) control cells (no antibiotics), (B) cells treated with colistin (1x MIC), (C) cells treated with PIM1 (1x MIC), and (D) percentages of propidium iodide (PI)-positive cells exposed to PIM1 (blue, left bar) or colistin (orange, right bar) at concentrations indicated as determined by flow cytometry. Cells were incubated for 1 hour in the presence of the indicated antibiotics prior to microscopy or flow cytometry. [Figure 4]Figure 4 shows the relative levels of cell membrane potential (ΔΨ) of Pseudomonas aeruginosa PAO1 cells exposed to increasing concentrations of PIM1, the ionophore gramicidin, or the antibiotic gentamicin. Relative membrane potential was assessed using the ΔΨ-sensitive fluorescent membrane probe DiS-C3-(5). Increased DiS-C3-(5) fluorescence corresponds to ΔΨ dissipation. The ionophore gramicidin is a known regulator that disrupts ΔΨ, while the antibiotic gentamicin requires ΔΨ for uptake but does not dissipate it. The relative dye fluorescence values ​​shown 30 minutes after the addition of the test compound were the average of four tests (from two runs each) with a (small) standard deviation. [Figure 5] Figure 5 shows the uptake of the PIM1-FTIC conjugate by Pseudomonas aeruginosa PAO1 and the relationship between PIM1 activity and membrane potential. (A) Fluorescence microscopy image of control cells (without PIM1) stained with membrane dye FMTM4-64FX, (B) Fluorescence microscopy image of cells treated with PIM1-FITC (1x MIC) and stained with FMTM4-64FX, (C) MIC90 (μg / mL) of PIM1 against Pseudomonas aeruginosa in MHB at various pH levels, and (D) MIC90 (μg / mL) of PIM1 against Pseudomonas aeruginosa PAO1 in the presence of valinomycin (left bar) or nigericin (right bar). [Figure 6] Figure 6 shows the effect of metabolic status on Pseudomonas aeruginosa PAO1 killing by PIM1; (A) survival of stationary-phase (Sta) and logarithmic-phase (Log) bacteria after 4 hours of exposure to PIM1, CST, or GEN, and (B) effect of fumarate (15 mM) on the survival of stationary-phase bacteria. The same results for Sta-PIM1, Sta-CST, and Sta-GEN were used in A and B. [Figure 7]Figure 7 shows the evolution of antibiotic resistance in (A) Pseudomonas aeruginosa PAO1 and (B) MRSA LAC*. Pseudomonas aeruginosa was grown in MHB and MRSA 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 shown as a multiple of the concentration increase relative to the MIC90 on day 1. [Figure 8] Figure 8 shows PIM1 treatment for skin wound infections. Wounds infected with panantibiotic-resistant Pseudomonas aeruginosa PAER were treated with 5 mg / kg imipenem (Pseudomonas aeruginosa PAER is imipenem-resistant) or 0.1, 1, 5, or 10 mg / kg PIM1 for 4 hours. Bacterial counts were determined by plate counting, and data for individual mice are reported. Horizontal lines represent mean values, and bars represent ±SD. *P<0.05, **P<0.01, and ns indicate P>0.05. [Figure 9] Figure 9 shows that PIM1, not PIM1D, is clearly toxic; (A) Body weight of mice treated with a single 6 mg / kg PIM1 (day 0) or 15 mg / kg PIM1D administered intraperitoneally (IP) for 1 week (days 0-6). There were 5 mice in each group. (B) Alanine aminotransferase (ALT), (C) Aspartate aminotransferase (AST), (D) Blood nitrogen urea (BUN) levels in the blood of mice treated with 15 mg / kg PIM1D administered daily for 7 days. Blood of mice given mock injections of saline solution was collected immediately before and 1 day after the first injection. Blood of PIM1D-treated mice was collected 1, 3, and 7 days after the first injection. There were 5 mice in each group, and data for individual mice, as well as the mean and standard deviation, are shown. [Figure 10] Figure 10 shows a schematic diagram of the synthesis of amide-integrated degradable PIM1D: (A) synthesis scheme of degradable diamine A, and (B) synthesis scheme of amide-integrated degradable PIM1D (where n is the actual number-average degree of polymerization, x is the mole fraction of the degradable repeating unit, n is approximately 10, and x is 20-30%). [Figure 11]Figure 11 shows that PIM1D is effective in IP sepsis models induced by Pseudomonas aeruginosa PAO1, MDR Pseudomonas aeruginosa (PAER), MDR A. baumannii, and methicillin-resistant Staphylococcus aureus (MRSA USA300). Colony-forming unit (CFU) counts in the liver in the sepsis models were induced by (A) Pseudomonas aeruginosa PAO1, (B) MDR Pseudomonas aeruginosa (PAER), (C) MDR A. baumannii (AB-1), and (D) methicillin-resistant Staphylococcus aureus (MRSA USA300). Kaplan-Meier curves representing mouse survival in the sepsis models were induced by (E) PAO1, (F) PAER, (G) AB-1, and (H) MRSA USA300. Geometric mean ± standard deviation, n=5. One-way ANOVA, ns are not significant, *P<0.05, **P≦0.01, ***P≦0.001. [Figure 12] Figure 12 shows the CFU counts of the kidney, spleen, and IP fluid in a sepsis model. (A-C) Pseudomonas aeruginosa PAO1, (D-F) MDR Pseudomonas aeruginosa (PAER), (GI) MDR A. baumannii (AB-1), and (J-L) methicillin-resistant Staphylococcus aureus (MRSA USA300) were induced. CFU counts in (A) kidney, (B) spleen, and (C) IP fluid in a sepsis model were induced by PAO1. CFU counts in (D) kidney, (E) spleen, and (F) IP fluid in a sepsis model were induced by PAER. CFU counts in (G) kidney, (H) spleen, and (I) IP fluid in a sepsis model were induced by AB-1. CFU counts in (J) kidney, (K) spleen, and (L) IP fluid in a sepsis model were induced by MRSA USA300. *P<0.05, **P≦0.01, and ns are not significant (two-sided Student's t-test). [Figure 13]Figure 13 shows blood biochemical analyses on days 1, 3, and 7 after receiving a single, three-consecutive, and seven-consecutive dose 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 biochemical parameters from each mouse are shown as individual points with error bars representing the deviation for each experimental group. [Figure 14] Figure 14 shows the efficacy of PIM1D in a neutropenic lung model using methicillin-resistant Staphylococcus aureus (MRSA) USA300 and Klebsiella pneumoniae (ATCC) 13883. Lung CFU counts in the neutropenic lung model were induced by (A) MRSA USA300 and (B) Klebsiella pneumoniae. Kaplan-Meier curves representing mouse survival in the neutropenic lung model were induced by (C) MRSA USA300 and (D) Klebsiella pneumoniae. Geometric mean ± standard deviation, one-way ANOVA, ns is not significant, *P<0.05, **P<0.01. [Figure 15] Figure 15 shows the synthesis of PIM1 bromide monomer. [Figure 16] Figure 16 shows the synthesis of PIM1-Br. [Figure 17] Figure 17 shows a typical synthesis of non-degradable main-chain cationic PIMs. [Figure 18] Figure 18 shows the synthesis of TFA salts of diamidodiamine (n=4, 6, 8, 10, and 12) monomers. [Figure 19] Figure 19 shows the general synthesis of degradable main-chain cationic PIMs by (a) copolymerization of degradable and non-degradable diamines and (b) homopolymerization of degradable diamines. [Figure 20] Figure 20 shows the chemical structures of the series of PIMs (P1-P6). [Figure 21]Figure 21 shows the antibiofilm properties of PIM and benzalkonium chloride (BAC, reference) as measured by the Minimum Biofilm Eradication Concentration (MBEC) assay. Viable cell counts of MRSA BAA39 on each microtiter plate peg after 4 hours of treatment with PIM or BAC. [Figure 22] Figure 22 shows the antibiofilm properties of PIM and BAC (reference) as measured by MBEC assay. Viable PAO1 count on each microtiter plate peg after 4 hours of treatment with PIM or BAC. [Figure 23] Figure 23 shows the synthesis of the 2+2 carbonate monomer. [Figure 24] Figure 24 shows the synthesis schemes for (a) carbonate monomer and (b) carbonate-integrated biodegradable PIM D2. [Figure 25] Figure 25 shows the synthesis schemes for OIM1D-3C-6 and OIM1D-3C-8. [Figure 26] Figure 26 shows the efficacy of OIM1D-3C-8 in a neutropenic lung infection model induced by (A) multidrug-resistant Klebsiella pneumoniae and (B) methicillin-resistant Staphylococcus aureus, (C) changes in mouse body weight after administration of 20 mg / kg of the compound via intranasal delivery, and (D) the efficacy of OIM1D-3C-8 / OIM1D-3C-6 (2:1 mass%) and 1:1 mass%) in a neutropenic lung infection model induced by multidrug-resistant Klebsiella pneumoniae. [Figure 27] Figure 27 shows schematic diagrams of various diamines having a degradable linker for biodegradable PIM synthesis (p=1~12, q=0~10). [Modes for carrying out the invention]

[0032] explanation Disclosed herein are a series of poly(alkylated imidazolium) (PIM) salts, each containing one or more degradable moieties in its alkyl chain. Remarkably, these PIM salts exhibit excellent broad-spectrum antimicrobial properties against a clinically important range of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacillus baumannii, Pseudomonas aeruginosa, and Enterobacter) bacterial species, while exhibiting low toxicity to mammalian cells. In fact, PIMs with a higher mole fraction of the degradable linker moiety showed greater biocompatibility. Furthermore, the polymers disclosed herein have been found to be active against both Gram-positive and Gram-negative bacteria.

[0033] The term "Gram-positive bacteria" refers to bacteria that have a cell wall containing a large amount of peptidoglycan. Gram-positive bacteria are identified by their tendency to retain crystal violet and stain dark blue or violet in the Gram staining protocol.

[0034] The term "Gram-negative bacteria" refers to bacteria that do not retain crystal violet staining in the Gram staining protocol, but instead have a thinner peptidoglycan layer that retains counterstaining, typically safranin. Gram-negative bacteria stain red or pink in the Gram staining protocol.

[0035] Therefore, in a first aspect of the present invention, a polymer or oligomer or a pharmaceutically acceptable solvate thereof is disclosed, comprising a first repeating unit containing an imidazolium group and a biodegradable chain bonded to an adjacent repeating unit.

[0036] In the embodiments herein, the term “comprising” may be interpreted as requiring the mentioned features but not limiting the presence of other features. Alternatively, the term “comprising” may also relate to situations where only the enumerated configurations / features are intended to exist (for example, the term “comprising” may be replaced with the phrases “consisting of” or “consists essentially of”). It is explicitly intended that both the broader and narrower interpretations are applicable to all aspects and embodiments of the invention. In other words, the word “comprising” and its synonyms may be replaced with the phrases “consisting of” or “consists essentially of” or their synonyms, and vice versa.

[0037] The phrase "essentially made from" and its pseudoonym can be interpreted in this specification as referring to a material that may contain small amounts of impurities. For example, a material may have a purity of 90% or higher, such as 95% or higher, 97% or higher, 99% or higher, 99.9% or higher, 99.99% or higher, 99.999% or higher, or 100% or higher.

[0038] When used herein, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise. For example, a reference to "composition" includes mixtures of two or more such compositions, and a reference to "first repeating unit" includes multiple such repeating units, without prejudice that further (different) repeating units may exist.

[0039] As used herein, the term "biodegradeable chain" refers to a bonding group that connects one imidazolium group to another imidazolium group. This biodegradable chain may contain one or more biodegradable functional groups.

[0040] Any suitable biodegradable functional group may be used herein. As used herein, the term biodegradable functional group is intended to mean a functional group that can be cleaved in vivo and / or in vivo by chemical or biological substances present in the environment in which the oligomer, polymer, or molecule of the present invention finds itself. Non-limiting examples of biodegradable functional groups that may be referred to herein include urea, carbamates, acetals, amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. Such functional groups may be susceptible to cleavage by chemical or biological substances in the surrounding environment (for example, esters may be cleaved by acidic or basic conditions in their environment, or by the presence of enzymes). This cleavage may occur in vivo or ex vivo, depending on how the materials disclosed herein are used and / or disposed of. Examples of functional groups that may not be biodegradable include ether bonds.

[0041] In embodiments relating to the polymer or oligomer of the first aspect of the present invention, only the repeating unit may constitute the first repeating unit. However, in alternative embodiments of the first aspect of the present invention, the polymer or oligomer may further comprise a second repeating unit comprising an imidazolium group and a non-biodegradable alkyl chain or a further biodegradable alkyl chain bonded to an adjacent repeating unit. In particular embodiments where the polymer or oligomer may further comprise a second repeating unit, one or more of the following may apply: (a) The polymer or oligomer may consist of 1 to 75 mol% of first repeating units, such as 5 to 60 mol%, such as 10 to 50 mol%, such as 20 to 30 mol%, and (b) The repeating units of the polymer or oligomer may be randomly distributed, or the repeating units may be formed as blocks, more specifically the repeating units of the polymer or oligomer may be randomly distributed. In the particular embodiments described above, the second repeating unit may be a second repeating unit comprising an imidazolium group and a non-biodegradable alkyl chain.

[0042] In the first embodiment of the present invention as referred to herein, the biodegradable chain in the first repeating unit comprises one or more biodegradable functional groups, the one or more biodegradable functional groups being selected from one or more of the group consisting of urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride and hydrazone, optionally (ai)1 or more biodegradable functional groups may be selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (aii)1 or more biodegradable functional groups may be selected from the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, or (aiii) One or more biodegradable functional groups may be amides.

[0043] In embodiments of the first aspect of the present invention as referred to herein, the number-average molecular weight may be 800 to 10,000 daltons, such as 900 to 5,000 daltons, such as 1,000 to 3,000 daltons, or such as 1,000 to 2,000 daltons.

[0044] In specific embodiments of the first aspect of the present invention as referred to herein, the polymer or oligomer is of formula I: [ka] [In formula I, x is between 0.01 and 1.0. Y - It is a counterion, o is a number from 0 to 10 (for example, 0 to 6, like 1 to 5). p is between 1 and 12. q is between 0 and 14 (for example, between 0 and 6), r is between 0 and 12. D is a biodegradable functional group, D' is a biodegradable functional group or bond. Each R 1 C is branched or unbranched 1-3 Alkyl or derivative thereof, Each t is 0, 1, or 2. Each t' is 0, 1, or 2. Each R 2 C is branched or unbranched 1-3 [Alkyl or derivative thereof] May contain, or have pharmaceutically acceptable solvates thereof.

[0045] When used herein, "C 1-3 The term "alkyl" may refer to, for example, ethyl, propyl (e.g., n-propyl or isopropyl), or more preferably methyl. 1-3 Alkyl derivatives are substituted C 1-3 This may refer to alkyl groups. Substitutive C may be referred to herein. 1-3 Examples of alkyl groups include, but are not limited to, halos (e.g., Br, Cl, or more specifically F). A particular derivative that may be referred to herein is CF3.

[0046] In embodiments of the present invention relating to polymers or oligomers according to Formula I, one or more of the following apply: (bi) Each D may be selected from urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride and hydrazone, optionally (aa) Each D may be selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (ab) Each D may be selected from one or more of the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, (ac) Each D may be selected from one or more of the group consisting of carbonate esters and amides (for example, each D is an amide), (bii) Each D' may be selected from bonded, urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride and hydrazone, optionally (ad) Each D' may be selected from one or more of the group consisting of bonds, amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (ae) Each D' may be selected from one or more of the group consisting of bonds, amides, esters, carbamates, and carbonate esters. (af) Each D' may be selected from one or more of the group consisting of bonds and amides, (ag) Each D' may be selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (ah) Each D' may be selected from one or more of the group consisting of amides, esters, carbamates and carbonate esters. (ai) Each D' can be an amide, (biii) Y - This includes halo, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf)2 - ) may be selected from one or more of the group consisting of, Y - This includes chloro, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf)2 - ) may be selected from one or more of the group consisting of (biv) x can be a range of 0.01 to 1.0, such as 0.025 to 0.75, 0.05 to 0.6, 0.1 to 0.5, 0.2 to 0.3, etc. (bv) t and t' can be 0, (bvi) p can be 1 to 6, and (bvii) r can be 1 to 6.

[0047] To ensure understanding, any combination of the above variables is assumed.

[0048] It will be understood that D and D' may be the same or different. In certain embodiments of the present invention, D' may be a biodegradable functional group, such that the biodegradable chain has two biodegradable functional groups. However, in other embodiments (for example, when D is a carbamate), D' may be a bond.

[0049] Embodiments of the present invention that may be mentioned include polymers or oligomers of the first aspect of the present invention (such as polymers or oligomers of formula I), as listed: [ka] This includes compounds selected from the following.

[0050] If the polymer or oligomer contains two repeating units, the amount of repeating units containing one or more biodegradable functional groups may be 1 to 99 mol%, such as 5 to 95 mol%, 10 to 90 mol%, 20 to 80 mol%, 25 to 75 mol%, or 50 mol%. In certain embodiments that may be referenced herein, the amount of repeating units containing one or more biodegradable functional groups may be 20 to 30 mol%.

[0051] To avoid any ambiguity, it is explicitly intended that numerous numerical ranges relating to the same feature are cited herein, and that the endpoints of each range, when combined in any order, provide a more carefully considered (implicitly disclosed) range. Thus, for the ranges listed above (and generally for the first repeating unit), the following ranges are considered: 1-5 mol%, 1-10 mol%, 1-20 mol%, 1-25 mol%, 1-30 mol%, 1-50 mol%, 1-60 mol%, 1-75 mol%, 1-80 mol%, 1-95 mol%, 1-99 mol%, 5-10 mol%, 5-20 mol%, 5-25 mol%, 5-30 mol%, 5-50 mol%, 5-60 mol%, 5-75 mol%, 5-80 mol%, 5-95 mol%, 5-99 mol%, 10-20 mol%, 10-25 mol%, 10-30 mol%, 10-50 mol%, 10-60 mol%, 10-75 mol%, 10-80 mol%, 10-95 mol%, 10-99 mol%, 20-25 mol%, 20-30 mol%, 20-50 mol%, 20-60 mol%, 20-75 mol%, 20-80 mol%, 20-95 mol%, 20-99 mol%, 25-30 mol%, 25-50 mol%, 25-60 mol%, 25-75 mol%, 25-80 mol%, 25-95 mol%, 25-99 mol%, 30-50 mol%, 30-60 mol%, 30-75 mol%, 30-80 mol%, 30-95 mol%, 30-99 mol%, 50-60 mol%, 50-75 mol%, 50-80 mol%, 50-95 mol%, 50-99 mol%, 60-75 mol%, 60-80 mol%, 60-95 mol%, 60-99 mol%, 75-80 mol%, 75-95 mol%, 75-99 mol%, 80-95 mol%, 80-99 mol%, and 95-99 mol%.

[0052] In the specific embodiments of (b) and (c) in the table above, repeating units containing one or more biodegradable functional groups may be present in an amount of 50 mol%.

[0053] In embodiments of the present invention as referred to herein, the polymers and oligomers in the above table may have a number-average molecular weight of 960 to 3,000 daltons, such as 966 to 2,800 daltons.

[0054] A second aspect of the present invention discloses a molecule or a pharmaceutically acceptable solvate thereof comprising the following: The first block of the oligomeric repeating unit, where each repeating unit comprises an imidazolium group and a non-biodegradable alkyl chain bonded to an adjacent repeating unit, A second block of oligomeric repeating units, where each repeating unit comprises an imidazolium group and a non-biodegradable alkyl chain bonded to an adjacent repeating unit, and A linking group that connects both the first and second blocks, wherein the linking group contains one or more biodegradable functional groups.

[0055] In a second embodiment of the present invention as referred to herein, one or more biodegradable functional groups may be selected from one or more of the group consisting of urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride and hydrazone, optionally (ci) One or more biodegradable functional groups may be selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (cii) One or more biodegradable functional groups may be selected from the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, (ciii) One or more biodegradable functional groups may be selected from either or both amides and carbonate esters, (civ) One or more biodegradable functional groups may be amides.

[0056] In a second embodiment of the present invention, the molecular weight of the molecule may be between 1,000 daltons and 5,000 daltons, and optionally between 1,000 daltons and 4,000 daltons.

[0057] In a specific embodiment of a second aspect of the present invention as referred to herein, the molecule is formula II: [ka] [In formula II, Each m is independently between 1 and 8 (for example, between 1 and 6). Each Y - It is a counterion, n' is between 0 and 12. Each o' is independently selected from 0 to 20. Each p' is independently selected from 0 to 12 (for example, 0 to 6). Each p'' is independently selected from 0 to 12 (for example, 0 to 6). Each T is independently a terminal functional group selected from amine, ammonium, guanidinium, bisguanidinium, alkyl, and aryl. Each D is a biodegradable functional group. A potentially possessing, or a pharmaceutically acceptable solvate thereof.

[0058] In embodiments of the present invention relating to polymers or oligomers according to Formula II, one or more of the following may apply: (di) Each D may be independently selected from the group consisting of urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride and hydrazone, optionally (ba) Each D may be independently selected from one or more of the group consisting of amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. (bb) Each D may be independently selected from one or more of the group consisting of carbamates or, more specifically, amides, esters, and carbonate esters, (bc) Each D can be an amide, (dii) Y - This includes halo, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf)2 - One or more of the group consisting of ) are selected, and Y is arbitrarily selected. - This includes chloro, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf)2- ) may be selected from one or more of the group consisting of, (dii) p'' can be between 0 and 6.

[0059] Embodiments of the present invention that may be mentioned include those in which the molecule of the second aspect of the present invention is selected from the following list:

[0060] [ka]

[0061] References in this specification to polymers, oligomers, and molecules (including polymers or oligomers of formula I or molecules of formula II) (in any aspect or embodiment of the present invention) include references to such compounds themselves, tautomers of such compounds, and pharmaceutically acceptable salts or solvates of such compounds, or pharmaceutically functional derivatives of such compounds.

[0062] Pharmaceutically acceptable salts that can be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example, by the reaction of a free acid or free base of the compound of formula I or formula II with one equivalent or more of a suitable acid or base, and optionally, the salt insoluble in a solvent or medium, and then the solvent or medium is removed using standard techniques (e.g., by vacuum, lyophilization or filtration). Salts may also be prepared by exchanging a counterion of the compound of formula I or formula II in salt form with another counterion, for example, using a suitable ion exchange resin.

[0063] Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral and organic acids, as well as salts derived from metals such as sodium, magnesium, or preferably potassium and calcium.

[0064] Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acid (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, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, and cyclamic acid. , dodecyl sulfate, 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), α-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 Examples include acid addition salts formed from (e.g., (-)-L-malic acid), malonic acid, (±)-DL-mandelic acid, metaphosphate, 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-aminosalicylic 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.

[0065] Specific examples of salts include mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid; organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acid; and salts derived from metals such as sodium, magnesium, or preferably potassium and calcium.

[0066] As should be understood, the polymers, oligomers, and molecules described herein may already contain counterions, which can be exchanged for other ions as needed. For example, the polymers, oligomers, and molecules described herein may be subjected to an ion exchange column to replace one counterion with another.

[0067] As described above, any solvates of the compounds and their salts are also included in the polymers, oligomers, and molecules described herein. Preferred solvates are those formed by incorporating the molecule of the compound of the present invention into the solid structure (e.g., crystalline structure) of a non-toxic, pharmaceutically acceptable solvent (hereinafter referred to as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol, and butanol), and dimethyl sulfoxides. Solvates can be prepared by recrystallizing the compound of the present invention in a solvent or solvent mixture containing the solvating solvent. Whether a solvate has been formed in any example can be determined by analyzing the crystal of the compound using well-known standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray crystallography.

[0068] Solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, examples of which include hemihydrates, monohydrates, and dihydrates.

[0069] For a detailed description of solvates and the methods used for their preparation and characterization, see Bryn et al., Solid-State Chemistry of Drugs, 2nd edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0070] The “pharmaceutically functional derivatives” of the polymers, oligomers, and molecules described herein as defined herein include ester derivatives and / or derivatives that have or provide the same biological function and / or activity as any related compound of the present invention. Therefore, for the purposes of the present invention, this term also includes prodrugs of the polymers, oligomers, and molecules described herein.

[0071] The term “prodrug” for any related polymer, oligomer, or molecule as used herein includes any compound that, after oral or parenteral administration, is metabolized in vivo to form an activator in experimentally detectable amounts within a given time interval (e.g., between 6 and 24 hours, i.e., once to four times daily).

[0072] The prodrug polymers, oligomers, and molecules described herein can be prepared by modifying functional groups present in a compound such that the modification is cleaved in vivo when such a prodrug is administered to a mammalian subject. The modification is typically achieved by synthesizing the parent compound with the prodrug substituent. Prodrugs include the polymers, oligomers, and molecules described herein in which a hydroxyl, amino, sulfhydryl, carboxyl, or carbonyl group in a compound of formula I or formula II is bonded to any group that can be cleaved in vivo to regenerate a free hydroxyl, amino, sulfhydryl, carboxyl, or carbonyl group, respectively.

[0073] Examples of prodrugs, but not limited to these, include 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).

[0074] The polymers, oligomers, and molecules described herein may contain double bonds and therefore may exist as E (entgegen) and Z (zusammen) geometric isomers for each individual double bond. All such isomers and mixtures thereof are within the scope of the present invention.

[0075] The polymers, oligomers, and molecules described herein may exist as positional isomers and may also exhibit tautomerism. All tautomer forms and mixtures thereof are within the scope of the present invention.

[0076] The polymers, oligomers, and molecules described herein may contain one or more chiral carbon atoms and therefore may exhibit optical isomerism and / or diastereoisomerism. Diastereoisomers can be separated using conventional techniques, e.g., chromatography or fractional crystallization. Various stereoisomers can be isolated by conventional methods, e.g., fractional crystallization or HPLC, by separating racemic or other mixtures of the compound. Alternatively, a desired optical isomer can be produced by reacting a suitable optically active starting material with a "chiral auxiliary" that can then be removed at an appropriate step, followed by derivatization with, for example, a homochiral acid (i.e., separation including dynamic restructuring), by separating diastereomer derivatives by conventional means such as chromatography, e.g., by reaction with a suitable chiral reagent or chiral catalyst under conditions known to those skilled in the art (i.e., the "chiral pool" method). All stereoisomers and mixtures thereof are within the scope of the present invention.

[0077] To avoid any ambiguity, in the context of this invention, the term “treatment” includes references to therapeutic or palliative treatment for patients in need of such treatment, as well as to preventive treatment and / or diagnosis for patients susceptible to related disease conditions.

[0078] The terms “patient” and “patients” include references to mammalian patients (e.g., humans). The terms “subject” or “patient” as used herein are well-known in the art and are therefore used interchangeably herein, and refer to mammals including dogs, cats, rats, mice, monkeys, cattle, horses, goats, sheep, pigs, camels, and most preferably humans. In some embodiments, the subject is a subject requiring treatment or having a disease or disorder. In other embodiments, however, the subject may be a healthy subject. The terms are not intended to indicate a specific age or sex; therefore, adult and neonatal subjects, whether male or female, are intended to be covered.

[0079] The term "effective amount" refers to the amount of a compound that produces a therapeutic effect on a patient receiving treatment (for example, sufficient to treat or prevent a disease). The effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject exhibits or feels the effect).

[0080] The term "halo," as used herein, includes references to fluoro, chloro, bromo, and iodine.

[0081] Unless otherwise stated, the term "aryl" as used herein is C 6-14 (For example, C 6-10 ) Contains an aryl group. Such a group can be monocyclic, bicyclic, or tricyclic, and has 6 to 14 ring carbon atoms, at least one of which is aromatic. The bonding site of the aryl group can be via any atom in the ring system. However, if the aryl group is bicyclic or tricyclic, they are bonded to the rest of the molecule via the aromatic ring. C 6-14The aryl group includes, for example, phenyl and naphthyl groups such as 1,2,3,4-tetrahydronaphthyl, indanyl, indenyl, and fluorenyl. Embodiments of the present invention that may be mentioned include those in which the aryl is phenyl.

[0082] Unless otherwise stated, the term "alkyl(alkyl)" means an unbranched or branched, acyclic, saturated or unsaturated (forming, e.g., alkenyl or alkynyl) hydrocarbon radical which may be substituted (e.g., with one or more halo atoms) or unsubstituted. When the term "alkyl" refers to an acyclic group, it is preferably C 1-10 Alkyl, more preferably C 1-6 Alkyl (e.g., ethyl, propyl (e.g., n-propyl or isopropyl), butyl (e.g., branched or unbranched butyl), pentyl, or more preferably methyl). When the term "alkyl" refers to a cyclic group (sometimes the group "cycloalkyl" is specified), it is preferably C 3-12 It is a cycloalkyl, more preferably C 5-10 (For example C 5-7 It is a cycloalkyl compound.

[0083] Further embodiments of the present invention that may be mentioned include those in which the polymers, oligomers, and molecules described herein are isotope-labeled. However, other specific embodiments of the present invention that may be mentioned include those in which the polymers, oligomers, and molecules described herein are not isotope-labeled.

[0084] When used herein, the term “isotopically labeled” includes references to polymers, oligomers, and molecules described herein in which non-natural isotopes (or non-natural distributions of isotopes) are present at one or more positions in the compound. References to “one or more positions in the compound” herein will be understood by those skilled in the art to mean one or more atoms of the polymers, oligomers, and molecules described herein. Therefore, the term “isotopically labeled” includes references to polymers, oligomers, and molecules described herein in which isotopes are enriched at one or more positions in the compound.

[0085] The isotopic labeling or enrichment of polymers, oligomers, and molecules described herein may be by radioactive or non-radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine, and / or iodine. Certain isotopes that may be mentioned in this regard include: 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 35 S, 18 F, 37 Cl, 77 Br, 82 Br and 125 I is included.

[0086] If the polymers, oligomers, and molecules described herein are labeled or enriched with radioactive or non-radioactive isotopes, then the polymers, oligomers, and molecules that may be mentioned herein 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 of at least 10% (e.g., 10% to 5000%, particularly 50% to 1000%, more specifically 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.

[0087] The compounds disclosed herein may be particularly useful in the treatment of microbial infections. Accordingly, a third aspect of the present invention provides any technically suitable combination of polymers or oligomers according to the first aspect of the present invention or pharmaceutically acceptable solvates thereof and embodiments thereof, and / or polymers or oligomers according to the second aspect of the present invention or pharmaceutically acceptable solvates thereof and embodiments thereof for medical use.

[0088] Furthermore, a fourth aspect of the present invention provides the following: (AAA) Use of a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to the first aspect of the present invention, and a technically appropriate combination of its embodiments, and / or a molecule or a pharmaceutically acceptable solvate thereof according to the second aspect of the present invention, in the manufacture of a pharmaceutical product for treating a disease including a microbial infection. (AAB) Polymers or oligomers or pharmaceutically acceptable solvates thereof according to the first aspect of the present invention, and any technically reasonable combination of the embodiments thereof, for use in the treatment of diseases including microbial infections, and / or molecules or pharmaceutically acceptable solvates thereof according to the second aspect of the present invention, and any technically reasonable combination of the embodiments thereof, (AAC) A method for treating a disease including a microbial infection, comprising the step of administering to a subject in need of such treatment an effective amount of a polymer or oligomer according to a first aspect of the present invention or a pharmaceutically acceptable solvate thereof, and any technically appropriate combination thereof, and / or an effective amount of a molecule according to a second aspect of the present invention or a pharmaceutically acceptable solvate thereof, and any technically appropriate combination thereof.

[0089] In a fourth embodiment of the present invention, the microbial infection may be associated with an infected wound or cystic fibrosis.

[0090] The term "microbial infection" encompasses any disease or condition caused by microorganisms within or on the subject. Examples of microbial infections include, but are not limited to, tuberculosis caused by mycobacteria, burn infections caused by Pseudomonas species, skin infections caused by Staphylococcus aureus, wound infections caused by Pseudomonas species and Baumannii, and sepsis. The term "fungal infection" encompasses any disease or condition caused by microorganisms within or on the subject. Examples of microbial infections include, but are not limited to, athlete's foot, tinea, yeast infections, and jock itch.

[0091] A non-limiting list of bacteria that may be susceptible to the polymers and copolymers of the present invention includes: Acidothermus cellulose, Actinomyces odontrichus, Alkaliphyllus metalliredigens, Alkaliphyllus oremlandii, Acinetobacter aurescens, Bacillus amyloriquefaciens, Bacillus clauzii, Bacillus halodurans, Bacillus licheniformis, Bacillus pymirus, Bacillus subtilis, Bifidobacterium adrescentis, Bifidobacterium longum, and Caldicellulosiruptor accharoliticus. Clostridium saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium acetobutyricum, Clostridium baizelinky, Clostridium botulinum, Clostridium ceruloticum, Clostridium difficile, Clostridium cruiberi, Clostridium reptum, Clostridium nobyi, Clostridium perflingens, Clostridium tetani, Clostridium thermocerum, Corynebacterium Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium jeikeium, Corynebacterium urealyticum, Desulfitobacterium hafniense, Desulfotomaculum reducens, Eubacterium ventriosum, Ixobacterium sibiricum, Fingoldia magna, Geobacillus kaustophilus, Geobacillus rmodenitrificans, Janibacter sp.), Kineococcus radiotolerance, Lactobacillus fermentum, Listeria monocytogenes, Listeria inocure, Listeria welsimeri, Moorella thermosetica, Mycobacterium avium, Mycobacterium bovis, Mycobacterium gilvam, Mycobacterium leprae, Mycobacterium paratuberculosis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium bambareni, Nocardioides species, Nocardia farsinica, Oceanobacillus iheomaensis, Saccharopolispora erythraea, Coagulase negative Examples include various Staphylococcus species, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis (MRSE), Streptococcus agalactie, Streptococcus goldonii, Streptococcus mitis, Streptococcus orasis, Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus suisse, Streptomyces abermitilis, Streptomyces cericola, Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis, and combinations thereof.

[0092] As described above, the polymers, oligomers, and molecules of the present invention can be used in the treatment of microbial and fungal infections. Accordingly, pharmaceutical compositions comprising the polymers, oligomers, or molecules of the present invention and pharmaceutically acceptable carriers are also provided.

[0093] The polymers, oligomers, or molecules of the present invention may be administered by any suitable route in the form of pharmaceutically acceptable dosage forms or pharmaceutically appropriate preparations containing the compounds, particularly by oral, intravenous, intramuscular, cutaneous, subcutaneous, transmucosal (e.g., sublingual or buccal), rectal, transdermal, transnasal, pulmonary (e.g., trachea or bronchi), topical, or other parenteral routes. Specific modes of administration that may be mentioned may include oral, intravenous, cutaneous, subcutaneous, transnasal, intramuscular, or intraperitoneal administration.

[0094] As used herein, references to the polymers and oligomers of the present invention refer to the polymers and oligomers of the first aspect of the present invention (and any technically reasonable combination of its embodiments), while references to the molecules of the present invention refer to the polymers and oligomers of the second aspect of the present invention (and any technically reasonable combination of its embodiments).

[0095] The polymers, oligomers, or molecules of the present invention are generally administered as pharmaceutical formulations mixed with pharmaceutically acceptable adjuvants, diluents, or carriers, which can be selected considering the intended route of administration and standard pharmacokinetics. Such pharmaceutically acceptable carriers may be chemically inert to the active compound and may not have adverse side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations can be found, for example, in Remington, The Science and Practice of Pharmacy, 19th edition, Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenterally acceptable aqueous solutions that do not contain pyrogens and have the required pH, isotonicity, and stability may be used. Suitable solutions are well known to those skilled in the art, and numerous methods are described in the literature. A brief review of drug delivery methods can also be found, for example, in Langer, Science (1990) 249, 1527.

[0096] Otherwise, the preparation of appropriate formulations can be routinely achieved by those skilled in the art using routine techniques and / or by standard and / or accepted pharmaceutical practices.

[0097] The amount of the polymer, oligomer, or molecule of the present invention in any pharmaceutical formulation used in accordance with the present invention depends on various factors, such as the severity of the condition being treated, the specific patient being treated, and the compound used. In any case, the amount of the polymer, oligomer, or molecule of the present invention in a formulation can be routinely determined by those skilled in the art.

[0098] For example, a solid oral composition such as a tablet or capsule may contain 1-99% (w / w) of active ingredients; 0-99% (w / w) of diluent or filler; 0-20% (w / w) of disintegrant; 0-5% (w / w) of lubricant; 0-5% (w / w) of flow aid; 0-50% (w / w) of granulator or binder; 0-5% (w / w) of antioxidant; and 0-5% (w / w) of pigment. In addition, controlled-release tablets may contain 0-90% (w / w) of controlled-release polymer.

[0099] Parenteral formulations (e.g., solutions or suspensions for injection, or solutions for infusion) may contain 1–50% (w / w) of the active ingredient, 50% (w / w)–99% (w / w) of a liquid or semi-solid carrier or vehicle (e.g., a solvent such as water), and 0–20% (w / w) of one or more other excipients, such as buffers, antioxidants, suspension stabilizers, isotonic agents, and preservatives.

[0100] Depending on the disease, the patient being treated, and the route of administration, the polymers, oligomers, or molecules of the present invention can be administered to patients who require them in amounts effective for various therapies.

[0101] However, in the context of the present invention, the dose administered to mammals, particularly humans, should be sufficient to produce a therapeutic response in the mammal over a reasonable time frame. Those skilled in the art will recognize that the selection of the precise dose and composition, as well as the most appropriate delivery plan, is influenced, among other things, by the pharmacological properties of the formulation, the nature and severity of the condition being treated, as well as the physical condition, the mental acuity of the recipient, and the potency of the particular compound, the age, condition, weight, sex and response of the patient being treated, and the stage / severity of the disease.

[0102] Administration may be continuous or intermittent (e.g., by bolus injection). The dosage may also be determined by the timing and frequency of administration. For oral or parenteral administration, the dosage may vary from about 0.01 mg to about 1000 mg per day of the polymer or copolymer of the present invention.

[0103] In any case, general practitioners or other persons skilled in the art will be able to routinely determine the most appropriate actual dosage for individual patients. The above-mentioned dosages are examples of average cases, and of course, there may be individual cases where a higher or lower dose range is obtained, and such cases are within the scope of the present invention.

[0104] Aspects of the present invention described herein (e.g., the polymers, oligomers and molecules, methods and uses described above) may have the following advantages in the treatment of the conditions described herein, and may be more convenient for physicians and / or patients, more effective, less toxic, more selective, have a broader range of activity, be more potent, have fewer side effects, or have other useful pharmacological properties than similar compounds, combinations, methods (treatments) or uses known in the prior art for use in those conditions or other treatments.

[0105] The polymers, oligomers, or molecules of the present invention can be prepared according to techniques well known to those skilled in the art, for example, as described in the Examples section below.

[0106] The polymers, oligomers, or molecules of the present invention can be isolated from their reaction mixtures using conventional techniques (e.g., recrystallization, column chromatography, preparative HPLC, etc.).

[0107] A fifth aspect of the present invention provides a polymer or oligomer according to the first aspect of the present invention, or a pharmaceutically acceptable solvate thereof, and any technically valid combination of the embodiments thereof, and / or a molecule or pharmaceutically acceptable solvate according to the second aspect of the present invention, and any technically valid combination of the embodiments thereof.

[0108] Considering the above, the polymers, oligomers, or molecules of the present invention can be used as antimicrobial active ingredients in personal care preparations, such as preservatives, 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 and skin cleansing solutions. Thus, the disinfectant preparations mentioned above may refer to any of the preparations listed in this paragraph.

[0109] When used as a simple disinfectant composition (i.e., intended solely for use as a disinfectant), the disinfectant composition may contain 0.01 to 20% by mass of polymers, oligomers, or molecules, for example, 0.5 to 10% by mass. It will be understood that two or more polymers, oligomers, or molecules of the present invention may form part of the disinfectant composition.

[0110] The polymers, oligomers, or molecules of the present invention exhibit remarkable antibacterial activity, particularly against pathogenic Gram-positive and Gram-negative bacteria, and may therefore act against bacteria in the skin flora, such as Corynebacterium zeolicifolium (a bacterium that causes body odor), as well as against yeasts and fungi. Consequently, they are suitable for disinfecting the skin and mucous membranes, as well as external appendages (hair), and may also be suitable for disinfecting hands and wounds.

[0111] Accordingly, antimicrobial and / or antifungal detergent compositions comprising polymers, oligomers, or molecules and surfactants of the present invention are also provided. It will be understood that the compositions may also comprise additional cosmetically acceptable carriers and / or auxiliaries. The compositions may be in the form of shampoos, or solid or liquid soaps, but other compositions are also conceivable (e.g., other hair care products, lotions, and creams).

[0112] The detergent composition may contain, for example, 0.01 to 15% by mass of the polymer or copolymer of the present invention, such as 0.5 to 10% by mass. It will be understood that two or more polymers and copolymers of the present invention may form part of the detergent composition.

[0113] Depending on the form of the detergent composition, in addition to the polymer or copolymer of the present invention, it may contain further components, such as metal ion chelating agents, colorants, fragrances, thickeners or solidifying agents (consistency modifiers), skin emollients, UV absorbers, skin protectants, antioxidants, and additives, such as dicarboxylic acids and / or C 14 ~C 22 It will consist of adducts that improve the mechanical properties of fatty acids, such as Al, Zn, Ca, and Mg salts, and any preservatives.

[0114] The detergent composition can be formulated as an emulsion of water in oil or oil in water, as an alcohol or alcohol-containing formulation, as a vesicular dispersion of ionic or nonionic amphiphilic lipids, as a gel, as a solid, as a stick, or as an aerosol formulation.

[0115] As an oil-in-water or oil-in-water emulsion, the detergent composition may contain 5-50% by mass of oil phase, 5-20% by mass of emulsifier, and 30-90% by mass of water. The oil phase may contain any oil suitable for cosmetic formulation, such as one or more hydrocarbon oils, waxes, natural oils, silicone oils, fatty acid esters, or fatty alcohols. Preferred monools or polyols are ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol, and sorbitol.

[0116] Detergent compositions can be provided in a wide variety of formulations. Suitable compositions include, but are not limited to, skincare products (e.g., skin wash and cleansing products in tablet or liquid soap form, soapless detergents or washing pastes), bath products (e.g., liquid compositions such as foam baths, milks, shower products or solid bath products), shaving products (e.g., shaving soaps, foaming shaving creams, non-foaming shaving creams, foams and gels, pre-shave products for dry shaving, aftershaves or aftershave lotions), and cosmetic hair treatments (e.g., hair care products in the form of shampoos and conditioners). Products include hair preparations, such as pre-treatment preparations, hair tonics, styling creams, styling gels, pomades, hair rinses, treatment packs, intensive hair treatments, hair structure preparations, such as hair wave preparations for permanent waves (hot waves, mild waves, cold waves), hair straightening agents, liquid hair styling products, foams, hair sprays, bleaching agents, such as hydrogen peroxide, lightening shampoos, bleach creams, bleach powders, bleach pastes or oils, temporary, semi-permanent or permanent hair colorants, preparations containing self-oxidizing dyes, or natural hair colorants such as henna and chamomile.

[0117] Antibacterial soap may have, for example, the following composition: 0.01 to 5% by mass of the polymer, oligomer, or molecule of the present invention, 0.3 to 1 mass% titanium dioxide, 1-10% by mass of stearic acid, The remaining ingredients are soap bases such as sodium salts of animal fats and coconut fatty acids or glycerol.

[0118] A shampoo may have, for example, the following composition: 0.01 to 5% by mass of the polymer, oligomer, or molecule of the present invention 12.0% by mass of sodium laureth-2-sulfate, 4.0% by mass of cocamidopropyl betaine, 3.0 mass% NaCl, and Water up to 100 wt%.

[0119] In a sixth aspect of the present invention, an article having a surface is provided, the surface of which may be coated with a polymer or oligomer or pharmaceutically acceptable solvates thereof according to a first aspect of the present invention or any technically reasonable combination thereof, and / or a molecule or pharmaceutically acceptable solvate thereof according to a second aspect of the present invention or any technically reasonable combination thereof, providing antimicrobial properties to the surface of the article, wherein the article is optionally a urethral catheter.

[0120] For example, an article according to the present invention may be a urethral catheter whose surface is coated with a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to a first aspect of the present invention and any technically reasonable combination of its embodiments, and / or a molecule or a pharmaceutically acceptable solvate thereof according to a second aspect of the present invention and any technically reasonable combination of its embodiments. Urinary tract infections can be caused by pathogenic bacteria such as Escherichia coli, and if left untreated, the infection can develop into a systemic infection and even be fatal. By coating a urethral catheter with the compounds disclosed herein, bacterial infection can be prevented. As understood, additional components may be added to the coating to provide additional properties (e.g., coating the surface with an anti-inflammatory agent to prevent inflammation). As understood, the antimicrobial compounds disclosed herein can also be used to coat other medical devices.

[0121] Further aspects and embodiments of the present invention are described in the following numbered statements. 1. The following general structure: [ka] [In the general structural formula, D is a biodegradable fragment that may be an amide, ester, carbonate, urethane, disulfide, anhydride, or hydrazone. Y - This includes chloride, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ) It is a possible counterion, 0≦o≦6, 1 ≤ p ≤ 6, 0 ≤ q ≤ 6 A random copolymer having the following properties.

[0122] 2. A random copolymer as described in Statement 1, where x is between 0.10 and 0.50.

[0123] 3. A random copolymer according to statement 1 or 2, wherein the molecular weight distribution of the random copolymer is 1 kDa to 5 kDa.

[0124] 4. The following general structure: [ka] [In general structure, D is a biodegradable fragment that may be an amide, ester, carbonate, urethane, disulfide, anhydride, or hydrazone. Y - This includes chloride, acetate, phosphate, sulfonate, and bis((trifluoromethyl)sulfonyl)imide (N(Tf)2 - ) is a possible counterion, T is a terminal group that can be an amine, ammonium, guanidium, bisguanidium, alkyl, or aryl group. 1 ≤ m ≤ 6, 0≦n≦12, 0≦o≦20, [0 ≤ p ≤ 6] A molecule that possesses the following properties.

[0125] 5. Medical use of any random copolymer described in any of Statements 1-3, or any molecule described in Statement 4.

[0126] 6. Use of any random copolymer described in any one of statements 1-3, or any molecule described in statement 4, for use in the treatment of microbial infections.

[0127] 7. Use of any random copolymer described in any one of statements 1-3, or the molecule described in statement 4, in the manufacture of a pharmaceutical product for treating microbial infections in subjects requiring it.

[0128] 8. A method for treating a subject suffering from a microbial infection, comprising the step of administering a therapeutically effective amount to a subject, for the purpose of treating the microbial infection, a random copolymer described in any one of statements 1 to 3 or a molecule described in statement 4.

[0129] The antimicrobial biodegradable polyimidazoles and oligoimidazoles (and defined molecules) discussed herein collectively as compounds of the present invention exhibit good antimicrobial activity against both Gram-positive and Gram-negative bacteria in vitro (e.g., polymer PIM1D and oligomer OIM1D-mC-6(m=3,8)—see the experimental section below for details). Furthermore, the compounds of the present invention exhibit good in vivo biocompatibility. For example, a single intraperitoneal injection of polymer PIM1D was able to save mice in a mouse sepsis model induced by MDR Pseudomonas aeruginosa and Acinetobacter baumanni, while cumulative intraperitoneal injections of PIM1D over 7 days caused negligible toxicity. These findings indicate that the biodegradable compounds of the present invention are promising antimicrobial agent candidates to address the emerging drug resistance crisis.

[0130] Emerging multidrug-resistant bacterial pathogens pose a serious threat to human public health. Antimicrobial polymers have been widely investigated as alternative treatments, but have largely failed due to poor biocompatibility and high MIC values. Surprisingly, the compounds of the present invention retain high antimicrobial activity while simultaneously being biodegradable in vivo, thereby mitigating or eliminating problems associated with the toxicity of non-degradable compounds in the body, which are caused or exacerbated by the long-term presence of non-degradable compounds in the body. For example, polymer PIM1D showed high antimicrobial activity against multidrug-resistant Pseudomonas aeruginosa, A. baumannii, and Klebsiella pneumoniae, which are high on the WHO's list of important pathogens. It also demonstrated broad antimicrobial efficacy, even against multidrug-resistant Gram-positive bacteria and mycobacteria that are inactive to colistin treatment. This, along with its excellent biocompatibility, makes PIM1D an excellent antimicrobial candidate. Similar properties were found in other compounds of the present invention.

[0131] Bacterial sepsis is highly fatal if left untreated. The involvement of multidrug-resistant bacterial pathogens makes treatment even more problematic, as they are untreatable with most antibiotics. As shown in the examples above and below, a single injection of PIM1D demonstrated excellent efficacy in rescuing mice suffering from sepsis caused by MDR Pseudomonas aeruginosa PAER and MDR A. baumannii AB-1. PIM1D was also effective in treating sepsis in mice caused by methicillin-resistant Staphylococcus aureus. Distal lung infections are difficult to treat and are typically used to evaluate the efficacy of antimicrobial agents before moving to clinical studies. PIM1D demonstrated excellent efficacy in treating lung infections caused by Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus. Furthermore, only minimal toxicity was observed after seven consecutive intraperitoneal injections of PIM1D at a therapeutic dose of 15 mg / kg and a cumulative dose of 105 mg / kg. The above highlights the potential of PIM1D (and other compounds of the present invention) in antimicrobial applications.

[0132] Further aspects and embodiments of the present invention will now be described with reference to the following non-limiting examples. [Examples]

[0133] Examples material All chemicals used in the synthesis were purchased from Sigma-Aldrich Co. LLC (St. Louis, USA) and used directly in the reaction unless otherwise specified. Commercial AR-grade solvents were used as received from Merck without further distillation. For column chromatography, industrial-grade solvents obtained from SG Labware Pte Ltd (Singapore) were used as is without distillation. 1,4-diaminobutane (diamine B), mucin, silica gel (35-70 mesh), silica gel 60 (100-200 mesh), Amberlyst® A-26OH resin, and cation-modified Mueller-Hinton culture medium (or broth) (CAMHB) were purchased from Merck, USA. L-lysine and 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)) were purchased from Combi-Blocks, Inc. (San Diego, California, USA). N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl) 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 medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), and FM® 4-64FX were purchased from Thermo Fisher Scientific (Massachusetts, USA). 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar (Massachusetts, USA). Fluorescein isothiocyanate (FITC) was purchased from Biotium, Inc. (California, USA). Pullulan standards were purchased from Polymer Standards Service (Pennsylvania, USA). 1K Dalton cutoff Spectra / Por® 6 dialysis membrane was purchased from Repligen, USA.Mueller-Hilton culture medium (MHB), Trypticase soy culture medium (TSB), Lysogeny culture medium (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.

[0134] Bacteria and growth conditions Pseudomonas aeruginosa PAO1 was provided by Scott Rice of Nanyang Technological 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 A. 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 cloaca ECLOS, and carbapenem-resistant E. cloaca CRE were obtained from Tan Tok Seng Hospital, Singapore (TTSH). MRSA USA300 LAC, the LAC derivative Staphylococcus aureus LAC*, and the LAC menD variant 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 A. baumannii X26, broadly drug-resistant A. baumannii X39, and A. 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, Washington University School of Medicine. Mycobacterium abscesses (rough and smooth surfaces) and Mycobacterium smegmatis mc2155 were cultured and tested in Kevin Peet's laboratory at Liconchian Medical College. Mycobacterium bovine tuberculosis (Bacillus calmet-Guéran) is in our collection. All other bacteria were purchased from the American Type Culture Collection. Unless otherwise specified, bacteria were grown in Mueller-Hilton culture medium (MHB) (Wiegand, I. et al., Nat. Protoc. 2008, 3, 163-175) at 37°C with shaking. Staphylococcus aureus was grown in Trypticase soy culture medium (TSB).Mycobacteria were grown in Middlebrook 7H9 broth medium supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADS supplement (prepared by dissolving 25g of bovine serum albumin fraction V, 10g of D-dextrose, and 4.05g of sodium chloride in 500mL of water). Glycerol was not supplemented for the mycobacterial growth inhibition assay. For plating, the lysogenic culture (LB) was solidified with 1.5% agar (LB agar), and the plates were incubated at 37°C.

[0135] analysis technology All samples are 1 H nuclear magnetic resonance (NMR), 1 H- 1 H-isonuclear correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), 13 Prior to distortionless enhancement by 13C NMR and polarization transfer (DEPT-135) analysis using a Bruker Advanced DPX 300 MHz NMR spectrometer, the samples were dissolved in deuterated solvents CDCl3, D2O, MeOD, or DMSO-d6. 1 In the 1H NMR spectrum, the chemical shift (δ) of the solvent residual peak was set to 4.79 for D2O and 2.50 for DMSO-d6, indicating proton separation. 13In the 13C NMR spectrum, the central peak of the solvent residual peak of DMSO-d6 was set at 39.52. Mass spectrometry was recorded on a MALDI-ToF ABI4800. The molecular weight and number-average molecular weight distribution (Mw / Mn) were determined by GPC equipped with two ultra-hydrogel columns in series and a RI detector using a water / methanol (MeOH) / 0.5 M acetic acid (AcOH) mixture (54 / 23 / 23 v / v) containing 0.5 M sodium acetate as the eluent (pH = 4.5, flow rate = 0.5 mL / min). All samples were dissolved in acetate buffer at approximately 1 mg / mL and filtered through a 0.22 μm microfilter before sample analysis. 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 a Merck 60F254 pre-coated silica gel plate, and the plate was visualized using a UV lamp, chemical staining with ninhydrin, or a basic KMnO4 solution.

[0136] Preparation of acidic water for dialysis The acidified water for dialysis was prepared by adding 1 M hydrochloric acid (HCl, 3 mL) to Milli-Q water (5 L).

[0137] Procedure for loading a chloride anion column An aqueous 10% HCl solution was passed through a glass column filled with Amberlyst® A - 26 (OH-type) until the pH of the eluate became the same as that of the original solution. Next, the resin was washed with water until it reached a neutral pH. This process was carried out at room temperature using gravity as the driving force.

[0138] Gel filtration chromatography (GFC) procedure When Sephadex® - G25 powder was dissolved in deionized (DI) water and soaked overnight, the powder swelled to form a slurry. The Sephadex® slurry was packed into a glass column using deionized (DI) water as the eluent, and gravity elution was performed.

[0139] Comparative Example 1. Synthesis of Main Chain Alkylated Polyimidazolium (PIM) Chloride Salt, PIM0 - 5 An acidic aqueous solution (total 100 mmol) of diamine selected from the following list was prepared by adding 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 acidic aqueous solution of diamine was maintained in an ice water bath for 30 minutes. 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 hours. During reflux, the solution changed from colorless to yellowish. The solvent and unreacted monomers were removed by a rotary evaporator to obtain a yellow viscous oil. The oil was diluted with water and dialyzed against acidic water with a pH of 3 - 4 (1-KDa-cutoff Spectra / Por® 6 dialysis membrane, Repligen, USA) for 1 day, and the acidic water was changed 3 times to obtain water-soluble PIM0 - 5 (Figure 1).

[0140] List of diamines used in the synthesis of PIM0-5 1,3-Diaminopropane - PIM0 1,4-Diaminobutane (Diamine B) - PIM1 1,6-Diaminohexane - PIM2 1,8-Diaminooctane - PIM3 1,5-Diamino-2-methylpentane - PIM4 L-Lysine - PIM5 PIM0 1 H NMR (300 MHz, D2O, 25 °C [ppm]): δ 8.98 (s, 1H, imidazole-H), 7.61 (s, 2H, imidazole-H), 4.36 (t, 4H, -CH2-), 2.54 (m, 2H, -CH2-).

[0141] PIM1 1 H NMR (300 MHz, D2O, 25 °C [ppm]): δ 8.84 (s, 1H, imidazole-H), 7.51 (s, 2H, imidazole-H), 4.24 (t, 4H, -CH۲-), 1.91 (m, 4H, -CH2-).

[0142] PIM2 1 ¹H NMR (300MHz, D2O, 25℃ [ppm]): δ 8.77 (s, 1H, imidazole-H), 7.47 (s, 2H, imidazole-H), 4.16 (t, 4H, -CH2-), 1.85 (m, 4H, -CH2-), 1.33 (m, 4H, -CH2-).

[0143] PIM3 1 ¹H NMR (300MHz, D2O, 25℃ [ppm]): δ 8.76 (s, 1H, imidazole-H), 7.47 (s, 2H, imidazole-H), 4.16 (t, 4H, -CH2-), 1.84 (m, 4H, -CH2-), 1.29 (m, 8H, -CH2-).

[0144] PIM4 1 ¹H NMR (300MHz, D2O, 25℃ [ppm]): δ 8.82 (s, 1H, imidazole-H), 7.50 (s, 2H, imidazole-H), 4.19 (t, 2H, -CH2-), 3.98 (m, 1H, -CH-), 2.15-1.78 (m, 4H, -CH2-), 1.46-1.15 (m, 2H, -CH2-), 0.84 (s, 3H, -CH3).

[0145] PIM5 1 ¹H NMR (300MHz, D2O, 25℃ [ppm]): δ 9.12-8.78 (m, 1H, imidazole-H), 7.65-7.47 (m, 2H, imidazole-H), 5.13 (m, 1H, -N-CH-), 4.21 (m, 2H, -CH2-), 2.33-2.18 (m, 2H, -CH2-), 1.95 (m, 2H, -CH2-); 1.26 (m, 2H, -CH2-).

[0146] Comparative Example 2. Synthesis of PIM1-fluorescein isothiocyanate (FITC) complex (FITC-bound PIM1) One equivalent of PIM1 was dissolved in 0.1 M sodium bicarbonate (NaHCO3) in 1 mL of water, and the reaction mixture was stirred for 30 minutes. Then, one equivalent of FITC was added to the reaction mixture, and the mixture was stirred in the dark overnight. The PIM1-FITC complex was then dialyzed against acidic water (500-1000 Da cutoff dialysis membrane) for 2 days to remove salts and unreacted pigments, with the acidic water being changed three times a day. The resulting complex was freeze-dried to obtain the final PIM1-FITC complex. A calibration curve was created using the absorbance of PIM1-FITC at 493 nm in PBS, and from the results obtained, the molar ratio of FITC to PIM1 was estimated to be approximately 15%.

[0147] Comparative Example 3. In vitro antimicrobial and cytotoxic effects of PIM0-5 Bacterial growth inhibition and bactericidal assays The minimum inhibitory concentration (MIC) was determined by slightly modifying the microdilution method of the culture medium (Wiegand, I. et al., Nat. Protoc. 2008, 3,163). The bacterial strains were subcultured overnight and allowed to grow to the intermediate logarithmic (Log) stage. After checking the optical density (OD), 1 × 10⁶ per mL was used as inoculum. 6 The test compound was diluted to colony-forming units (CFU). The test compound was prepared in DI water at a final concentration of 10.24 mg / mL and diluted to 1.024 mg / mL in fresh MHB. A 2-fold dilution series of the test compound was prepared in MHB medium in 96-well plates (final volume 50 μL per well) to achieve a concentration gradient from 512 μg / mL to 1 μg / mL. Before inoculating each well with 50 μL of bacterial suspension, the positive control (bacterial suspension in MHB medium and without polymer) was incubated at 37°C for 10 minutes by shaking (orbital shaker at 225 rpm), sterilizing the control (MHB only) medium. After mixing the plates in a shaker incubator for 10 minutes, they were statically incubated at 37°C for 18 hours. Subsequently, OD (Optical Dioxide Disorder) at 600 nm was used. 600 ) was measured. In assays containing mycobacteria, the compound was sequentially diluted in 2-fold steps, and 2 μL of this dilution series was spotted into a 96-well plate, and OD600 is 0.005 (approximately 5 x 10) 5 200 μL of logarithmic-phase bacteria (CFU / mL) was added to each plate. These plates were incubated at 37°C for 48 hours for Smegma bacteria and at 37°C for 5 days for M. bovis bacillus carmetto-guerane. The MIC was defined as the concentration of the compound that inhibited bacterial growth by at least 90% (MIC). 90 It was reported as follows. Agar plate cultures were performed to confirm the concentration of the seeded bacteria. Three independent experiments were conducted for each compound, each bacterial strain was tested, and the range of MIC values ​​for each compound was reported.

[0148] Mammalian cell cytotoxicity assay The toxicity of PIM0-5 was tested using mouse embryonic fibroblast cell lines 3T3. Cytotoxicity was assessed using standard methods (International Organization for Standardization (2009) ISO 10993-5: Biological evaluation of medical devices - Part 5: Testing of in vitro cytotoxicity (ISO Geneva), 1-34). 3T3 cells were initially cultured in a medium containing 89% DMEM, 10% FBS, and 1% antibiotic (penicillin / streptomycin). After confirming that the confluence in the culture flask reached 80% under a microscope, the cells were treated with trypsin, concentrated, and counted using a hemocytometer. 1 × 10⁻⁶ 4 Cells were seeded into each well of a 96-well plate. After incubating the 96-well plate for 24 hours, test compounds were added to each well of the 96-well plate at concentrations ranging from 128 μg / mL to 4 μg / mL. After a further 24 hours of incubation, cell viability was qualitatively assessed by microscopy and quantified by the MTT assay. Cell viability was assessed by comparing the absorbance of formazan in the wells containing the antimicrobial agent with the absorbance of formazan in the wells containing untreated cells. The IC50 value was reported as the level of the test compound that reduced the number of viable cells by 50%. The data presented are the mean of three measurements, with a standard deviation of less than 10%.

[0149] LB Agar Plate Count The bacterial solution was serially diluted 10-fold with PBS. The diluted solution was dropped onto a solid agar plate at 5 μL per drop. After drying in a biosafety hood, the plate was incubated in an incubator at 37 °C for 18 hours, and then the bacterial colonies were counted and each dilution factor was recorded. Finally, the bacterial concentration was calculated backwards.

[0150] Results and Discussion Table 1 shows the physical and biological properties of various batches of PIM1 in Pseudomonas aeruginosa PAER, A. baumannii AB-1 (MDR), and Staphylococcus aureus USA300 (MRSA). All PIM chloride salts except PIM5 showed significant antibacterial activity (Table 2). This may be because the carboxylated alkyl chain of PIM5 is the least hydrophobic in the series and PIM5 is zwitterionic rather than cationic. PIM0 showed a decrease in activity because its short alkyl chain is less hydrophobic than that of PIM1.

[0151] Table 1. Physical and biological properties of various batches of PIM1 in Pseudomonas aeruginosa PAER, A. baumannii AB-1 (MDR) and Staphylococcus aureus USA300 (MRSA)

Table 1

[0152] Table 2. Antibacterial and cytotoxic effects of PIM0-5

Table 2

[0153] Unlike PIM2 and PIM3, PIM1 did not show toxicity to 3T3 cells (Table 2). This is likely because PIM2 and PIM3 have alkyl chains that are two or four carbon atoms longer than PIM1, respectively. These results suggest that even slight differences in alkyl chains can have a dramatic impact on the toxicity of mammalian cells.

[0154] Therefore, PIM1 was selected for further study due to its potent antimicrobial activity across the entire spectrum of pathogenic bacteria and the fact that it did not exhibit the acute mammalian cytotoxicity measurable by PIM screening (Table 2).

[0155] Comparative Example 4. In vitro antimicrobial activity and cytotoxicity of PIM1 PIM1 was used to screen for antimicrobial activity against a wider range of bacterial pathogens by following the protocol of Comparative Example 3. The cytotoxicity of PIM1 in HEK293, HepG2, and A549 cells was also measured as described in Comparative Example 3, except that DMEM supplemented with 15% FBS was used to culture the HepG2, HEK293, and A549 cells. Furthermore, the antimicrobial activity of PIM1 was compared with the commercially available antibiotics colistin and polymyxin B.

[0156] Results and Discussion PIM1 was found to exhibit potent antibacterial activity against a variety of panantibiotic-resistant Gram-positive and Gram-negative bacteria, including colistin-resistant Burkholderia tylandensis and Pseudomonas aeruginosa variants. It was also noted that PIM1 is a potent anti-mycobacterial compound. In comparison, PIM1 had a broader activity spectrum 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 mechanism of action than colistin. Finally, no toxicity was apparent even at the highest levels of PIM1 when tested in four different mammalian cell lines (Table 4).

[0157] Table 3. Antimicrobial efficacy of PIM1 compared to colistin activity against a panel of pan-resistant and naturally occurring antibiotic-resistant bacteria. [Table 3] 1 The concentration of an antimicrobial agent that inhibits bacterial growth by at least 90%. The value is within the range of three independent experiments. 2 MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-resistant enterococcus; MDR, multidrug-resistant; Pseudomonas aeruginosa PAKpmrB-12 is a colistin-resistant mutant derived from Pseudomonas aeruginosa PAK (Moskowitz, SM et al., J. Bacteriol. 2004, 186, 575-579); XDR, broadly drug-resistant (Magiorakos, AP et al., Clin. Microbiol. Infect. 2012, 18, 268-281); B. tyrandensis 700388 is a naturally colistin-resistant relative of the emerging pathogen Burkholderia pseudomallei (B. pseudomallei is also colistin-resistant) (Olaitan, AO et al., Front. Microbiol. 2014, 5, 643). 3 ND, not implemented.

[0158] Table 4. Comparison of the cytotoxicity of PIM1, colistin, and polymyxin B [Table 4] 1 Concentration of the antibacterial agent that induced a maximum 50% inhibition of the viability of mammalian cells. Values are the means of three with a standard deviation of less than 10%. 2 ND, not performed.

[0159] Comparative Example 5. Bactericidal properties of PIM1 To determine whether PIM1 is bactericidal or bacteriostatic, model Gram-negative pathogen Pseudomonas aeruginosa PAO1 in MHB or Gram-positive pathogen methicillin-resistant MRSA Staphylococcus aureus LAC from logarithmic-phase cultures * were inoculated and after inoculation was determined by plating on LB agar to count, the total CFU in the sample was determined over time in the presence of different concentrations of PIM1. Two independent experiments were performed and the results are mean ± SD.

[0160] Results and Discussion Bacterial growth was evident in the absence of PIM1 or in the presence of PIM1 at half the level of the MIC (Figure 2). At twice the MIC, both Pseudomonas aeruginosa and Staphylococcus aureus were bactericidal by PIM1. From these experiments, it was concluded that PIM1 is bactericidal.

[0161] Comparative Example 6. Novel mechanism of action of antibacterial PIM1 Propidium iodide (PI) staining Pseudomonas aeruginosa PAO1 was used in the PI experiment. Cells grown in MHB were harvested at logarithmic metaphase and resuspended in fresh MHB. PIM1 or colistin (positive control) was added at the indicated concentrations. After 1 hour incubation with the antimicrobial agent, the cell suspension was sampled and the cell count was determined by plate counting. The remaining cells were washed with PBS and stained with PI (15 μg / mL) according to the manufacturer's protocol. The percentage of cells incorporating PI (dead cells) was determined using Attune NxT flow cytometry (Thermo Fisher Scientific, USA). Polylysine was measured using a Zeiss LSM800 confocal microscope. Cells on a covered petri dish (MatTek Corporation, USA) were imaged.

[0162] Membrane potential monitoring Membrane potential (ΔΨ) in Pseudomonas aeruginosa was monitored using a membrane potential-sensitive dye, 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)), following a previously reported procedure (Zhang, L. et al., Antimicrob. Agents Chemother. 2000, 44, 3317-3321). Pseudomonas aeruginosa PAO1 cells were recovered from metaphase logarithmic cultures by centrifugation and suspension in 5 mM HEPES buffer containing 100 mM KCl and 0.2 mM EDTA, and the outer membrane was permeabilized for DiS-C3-(5) entry. The bacterial suspension was then OD (Oxygen-Draining). 600 The solution was adjusted to 0.02, and DiS-C3-(5) was added (final concentration 1 μM). Then, the cell suspension (180 μL) was added to each well of a 96-well plate, and the test compound was added to each well as indicated, resulting in a final mixture of 200 μL. Fluorescence in each well was measured every 2 minutes using a Spark 10M microtiter plate reader (Tecan, Switzerland) with excitation at 622 nm and emission at 670 nm. Data were collected 30 minutes after the addition of the test compound. Two independent experiments were performed, and the data here are mean ± SD.

[0163] Cell uptake protocol Intracellular uptake of PIM1-FITC was monitored with some modifications, as described elsewhere (Radlinski, LC et al., Cell Chem. Biol. 2019, 26, 1355-1364). Briefly, cells grown in MHB were harvested at logarithmic metaphase and suspended in fresh MHB containing PIM1-FITC at 1 MIC (the MIC for PIM1-FITC was the same as for PIM1) for 30 minutes. The cells were then harvested by centrifugation, washed once with PBS, and fixed in 4% paraformaldehyde in PBS for 15 minutes. After washing the fixed cells twice with PBS, they were incubated on ice for 10 minutes with 5 μg / mL FMTM 4-64FX (invitrogen®, Thermo Fisher Scientific, USA). The cells were washed twice again with PBS, then sealed on slides using Fluoromount® aqueous mounting medium (Merck & Co., USA), and subsequently imaged using a Zeiss Super Resolution System ELYRA PS.1 with an LSM 800 system.

[0164] Results and Discussion PIMs were designed to have a moderately hydrophobic alkyl chain with a cationic imidazolium moiety. Therefore, like antimicrobial peptides (Velkov, T. et al., J. Med. Chem. 2010, 53, 1898-1916), cell membrane permeability may be involved in the activity of PIMs. Furthermore, as seen in Comparative Example 6, PIM1 has a different mechanism of action than colistin. To test this hypothesis, the uptake of the fluorescent dye PI into PIM1-treated and colistin-treated Pseudomonas aeruginosa was compared. Viable cells with intact cell membranes excluded PI. If the membrane was permeabilized, PI could 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 view that PIM1 activity does not involve membrane disruption, unlike colistin. To further support this view, we monitored the ΔΨ of Pseudomonas aeruginosa using the lipophilic fluorescent dye DiS-C3-(5). Treatment with the proton ionophore gramicidin dramatically increased DiS-C3-(5) fluorescence, indicating ΔΨ dissipation, but PIM1 did not show such an effect (Figure 4).

[0165] Since PIM1 does not disrupt the membrane and does not dissipate ΔΨ, it was hypothesized that PIM1 could be taken up by cells. Therefore, the cellular uptake of PIM1-FITC, a fluorescent derivative of PIM1, was synthesized in Comparative Example 2 and used to treat Pseudomonas aeruginosa. As shown in Figures 5A-B, PIM1-FITC entered the cells. Similar to cationic antibiotics (such as gentamicin (GEN)), it was hypothesized that the association with cells and the antibacterial activity of PIM1 may depend on ΔΨ. If so, Pseudomonas aeruginosa should have higher activity in an alkaline environment and lower activity in an acidic environment. In bacteria like Pseudomonas aeruginosa, the proton-driven force (PMF) remains relatively constant across the range of external pH values, as does the cytoplasmic pH (weakly basic). Total PMF consists of ΔΨ and the pH gradient across the cell membrane (ΔpH). Therefore, in a weakly alkaline environment, the cytoplasmic and external pH values ​​are similar, and the PMF is mainly in the form (morphology) of ΔΨ. In an acidic environment, the external pH is lower than the cytoplasmic pH, and PMF primarily takes the form (morphology) of ΔpH. In fact, the MIC of PIM1 is dependent on the external pH, and PIM1 showed low antimicrobial activity at pH 5 (Figure 5C). These findings suggest that PIM1 uptake is ΔΨ-dependent.

[0166] Comparative Example 7. Effects of Valinomycin and Nigericin on the MIC of PIM1 against Pseudomonas aeruginosa Valinomycin, nigericin, and PIM1 were dissolved in MHB. The stock solution was added to the wells of a microtiter plate to make a total volume of 50 μL, and 50 μL of logarithmic-phase Pseudomonas aeruginosa culture was added. MIC 90 This was determined as described in Comparative Example 3.

[0167] Results and Discussion To gain further insight into the mechanism of action of PIM1, we investigated the effects of potassium ionophores (valinomycin) and sodium-potassium exchangers (nigericin) on PIM1 activity. At neutral pH, valinomycin reduced ΔΨ, while nigericin disrupted ΔpH (Farha, MA et al., Chem. Biol. 2013, 20, 1168-1178). The results were consistent with our hypothesis. The MIC of Pseudomonas aeruginosa PIM1 increased with valinomycin treatment but was not significantly affected by nigericin (Figure 5D). Combining these results with those from Comparative Example 6, we conclude that PIM1 is taken up by cells in a ΔΨ-dependent manner, but we cannot distinguish whether it exerts its antibacterial effect at the cell membrane or in the cytoplasm.

[0168] Comparative Example 8. Effect of metabolic status on the killing of Pseudomonas aeruginosa PAO1 by PIM1 Using a previously reported method (S. Meylan et al., Cell Chem. Biol. 2017, 24, 195-206), the effects of PIM1 and other antibiotics on the survival of Pseudomonas aeruginosa PAO1 were determined, however, quiescent cells were obtained by growing them overnight in MHB, and PIM1 and GEN were compared. The results were compared with those of Pseudomonas aeruginosa PAO1 isolated from MHB cultures in the metaphase logarithmic growth phase. Furthermore, the ability of fumarate (15 mM) as an energy source to enhance PIM1-induced death of quiescent Pseudomonas aeruginosa was tested by adding fumarate to quiescent cells.

[0169] Results and Discussion Generally, antibiotics have limited activity against non-growing bacteria. In the case of Pseudomonas aeruginosa, this is evident when comparing the bactericidal activity of antibiotics such as GEN against quiescent cells incubated in the presence and absence of an energy source (S. Meylan et al., Cell Chem. Biol. 2017, 24, 195-206; KR Allison et al., Nature 2011, 473, 216-220). PIM1 does not appear to disrupt membrane integrity, and based on our findings that, like GEN, its activity requires ΔΨ, we hypothesized that its bactericidal activity against nutrient-deficient bacteria might be limited. Indeed, quiescent cells were less susceptible to PIM1 cell death (or GEN death as a control) than colistin-induced cell death (Figure 6A). When fumarate was supplied to quiescent cells as an energy source, the bactericidal activity of both PIM1 and GEN was restored (Figure 6B). As is the case with GEN and many other antibiotics, we conclude that PIM1 has limited efficacy as a bactericide against non-growing bacteria. It should also be noted that these experiments are consistent with our conclusion that PIM1 does not act by disrupting the cell membrane and requires ΔΨ for activity.

[0170] Comparative Example 9. Laboratory evolution of PIM1 resistance Laboratory Evolutionary Mutation Assay Experiments on the evolution of spontaneous PIM1 resistance and ciprofloxacin resistance included continuous passage, as described elsewhere (Ling, LL et al., Nature 2015, 517, 455-459). This involved Pseudomonas aeruginosa PAO1 grown in MHB or MRSA LAC grown in TSB. * One of the following was used. The inoculum for the first transplant was 10 7Using 1 mL or 100 μL of various amounts of antibiotics in cells / mL, 2 mL test tubes and 96-well plates were used for each of Pseudomonas aeruginosa and MRSA. Increasing the volume of the experiment using Pseudomonas aeruginosa, the cell number increased because resistance did not appear when the culture volume of this species was small. Bacterial growth was monitored at 24-hour intervals. Transplantation was performed daily, and the inoculation material for transplantation (100-fold dilution) was from a culture containing the highest level of antibiotics that allowed growth up to at least 0.2 OD 600 The experiment for Pseudomonas aeruginosa lasted for 30 days. For MRSA LAC * the experiment ended in 15 days. The isolates of MRSA LAC * were obtained from the last transfer and stored as glycerol stocks at -80 °C for further research.

[0171] Whole-genome sequencing Genomic DNA was isolated from PIM1-resistant Staphylococcus aureus mutants using standard procedures, and DNA was prepared for sequencing using the Illumina Nextera DNA library preparation kit. The DNA was sequenced on an Illumina MiSeq instrument (paired-end sequencing). The sequences were mapped to the genome of the parental strain MRSA LAC * (Bowman, L. et al., J. Biol. Chem. 2016, 291, 26970-26986), and single nucleotide mutations, small deletions and insertions were identified using CLC Genomics Workbench software. Large deletions were identified by manual sequence comparison. The DNA sequences were deposited in the European Nucleotide Archive (ENA) with the accession number PRJEB37791.

[0172] Results and Discussion To evaluate the potential of PIM, a well-known therapeutic agent, and potentially gain further insight into its mechanism of action, repeated passage experiments were conducted against Pseudomonas aeruginosa and MRSA at increased concentrations of PIM1 or ciprofloxacin (control). In Pseudomonas aeruginosa, ciprofloxacin-resistant mutants appeared, but PIM1-resistant mutants did not (Figure 7). PIM1-resistant MRSA appeared at a similar rate to the appearance of ciprofloxacin-resistant mutants.

[0173] To gain insight into the nature of PIM resistance phenotypes in evolved MRSA populations, bacteria were isolated from the last passage. Of the 21 characterized isolates, all exhibited a small colony variant (SCV) phenotype, 15 had a PIM1 MIC of more than 128 times that of the initial strain, and the other 6 had a PIM1 MIC of 64–128 times that of the parental strain. The genomes of the 15 isolates showing a PIM1 MIC of more than 128 times that of the non-evolved strain were sequenced (Shi, Z. et al., Polymer resistance Staphylococcus aureus strains. European Nucleotide Archive. Deposited 14 April 2020.). All but one had mutations in genes required for menaquinone biosynthesis (either the menA-F operon or ispD gene). Some isolates also had mutations in genes known to confer resistance to cationic peptides, specifically 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; Yang, S.-J. et al., Infect. Immun. 2012, 80, 74-81) (Table 5). Genes encoding menaquinone synthesis were of particular interest because the relationship between menaquinone and PIM1 activity may provide clues about the mode of PIM1 activity. Therefore, the PIM1 sensitivity of menD deletion mutants was compared to that of their parents.

[0174] This menD mutant is unable to produce menaquinone (Lannergard, J. et al., Antimicrob. Agents Chemother. 2008, 52, 4017) and is growth-restricted against fermentation. Like our evolved PIM1-resistant isolate, this mutant has an SCV phenotype, which is a characteristic phenotype of menaquinone-synthesizing mutants (Von Eiff, C. et al., J. Bacteriol. 2006, 188, 687). The menD mutant showed an 8-fold increase in PIM1 resistance compared to its parent (MIC of 16 μg / mL compared to 2 μg / mL for the parent). Therefore, we believe that menaquinone or the functional electron transport system is involved in the susceptibility of MRSA to PIM1, but other factors must also be involved in the extremely high PIM1 resistance of the evolved isolate. We hypothesized that PIM1 either directly interferes with the electron transport chain leading to the generation of toxic reactive oxygen species, or that its uptake during fermentation growth is inhibited, resulting in a decrease in its antimicrobial activity.

[0175] Table 5. PIM1-resistant Staphylococcus aureus LACs that evolved in the laboratory. * List of common related mutations in mutants [Table 5] 1 All base substitutions were non-synonymous mutations that encoded either amino acid substitutions or stop codons. 2 Mutant 5114 is the only PIM1-resistant mutant in which no mutation in the menaquinone biosynthesis gene could be identified.

[0176] Comparative Example 10. Efficacy of PIM1 treatment in animal infectious diseases Mice were housed at room temperature in a 12-hour light-dark cycle for one week prior to infection. Our skin infection model was as follows: Punch biopsies were made from shaved dorsal skin wounds (approximately 5 mm in diameter) of female C57B / 6 mice (8-9 weeks old) to obtain log-phase cells of Pseudomonas aeruginosa PAER. These were introduced into the wound by pipetting (approximately 106 CFU in 10 μL PBS). The infected wound was immediately covered with Tegaderm (3M, USA). Four hours post-infection, antibiotic treatment (PIM1 and imipenem (Imp)) was initiated by injection via Tegaderm. Subsequently, another layer of Tegaderm was applied. After a further 24 hours, 1 cm from the center of the wound 2 Tissue samples were taken from all four sides, homogenized, and cell counts were determined by plate counting. Our protocol was approved by the Institutional Care and Use Committee of Nanyang University of Technology (NTU IACUC, Protocol A0362).

[0177] Results and Discussion The ability of PIM1 to control carbapenem-resistant Pseudomonas aeruginosa wound infections in mice was evaluated. As expected, Imp-resistant strains of Pseudomonas aeruginosa increased in number over the next 24 hours in untreated and Imp-treated wounds. Compared to untreated or Imp-treated wounds, the number of Pseudomonas aeruginosa decreased slightly after a single treatment with 0.1 mg / kg of PIM1 and approximately 4-logarithmically after a single treatment with doses of 1 mg / kg or higher of PIM1 (Figure 8).

[0178] Comparative Example 11. Toxicity of PIM1 treatment in animal infections In a systemic infection model, the toxicity of PIM1 (IP injection, 6 mg / kg) in female BALB / c mice (8-9 weeks old) was first evaluated by tracking body weight over 14 days. Body weight was recorded daily for 5 days.

[0179] Results and Discussion The safety of PIM1 when delivered to mice by IP injection was tested, and evidence of acute toxicity was found. Weight loss was observed over 5 days after a single dose (Figure 9A).

[0180] Example 1. Synthesis of the precursor of the degradable PIM1D (N,N'-(propane-1,3-diyl)bis(2-aminoacetamide)) (diamine A) (Figure 10A) EDC.HCl (14.58 g, 76.1 mmol) and HOBt (10.70 g, 79.14 mmol) were stirred in a solution of Boc-Gly-OH (8.0 g, 45.66 mmol) in anhydrous DMF (25 mL) at 0°C (ice water) for 30 minutes. 1,3-diaminopropane (1.28 mL, 15.22 mmol), kept at room temperature, was added dropwise to the reaction mixture, which was maintained at 0°C (ice water), over 10 minutes. The reaction mixture was then allowed to reach room temperature and stirred continuously for 48 hours. Next, water (50 mL) was added, and the product was extracted three times with ethyl acetate ( Depositphotos) or DCM (150 mL). The extract was washed three times with water (50 mL), and then once with brine (50 mL). The Depositphotos or DCM layer was dried with anhydrous Na2SO4 (approximately 50 g). Next, Na2SO4 was filtered off, and the filtrate was concentrated using a rotary evaporator (50°C for 20 minutes at 120 rpm). The residue was dried under vacuum at room temperature overnight. The dried residue was dissolved in anhydrous DCM (30 mL), and then trifluoroacetic acid (TFA, 8 mL) was added dropwise over 10 minutes while maintaining the temperature at 0°C (ice water). The reaction mixture was then stirred at room temperature for 12 hours. The crude product was concentrated using a rotary evaporator at 50°C for 10 minutes at 120 rpm. Next, toluene (50 mL) was added, and the solution was further evaporated using a rotary evaporator at 50°C for 30 minutes at 120 rpm. The residue is purified by silica gel 60 column chromatography using (i) a 30% methanol (MeOH) dichloromethane solution (DCM, 500 mL) followed by (ii) a 2% TFA MeOH solution (1000 mL) as sequential eluents to remove impurities and produce a degradable diammonium TFA salt A (3.0 g, 7.20 mmol).

[0181] 1 H NMR (300MHz, DMSO-d6, 25℃[ppm]): δ 8.55(t,J=5.4Hz,2H),8.18(brs,6H),3.53(s,4H),3.14(q,J=6.3Hz,4H),1.54-1.63(m,2H).13 C NMR(75MHz,DMSO-d6,25℃[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.

[0182] Example 2. Synthesis of degradable PIM1D 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). The reaction mixture was stirred at room temperature for 30 minutes, after which the volatile substances were evaporated using a rotary evaporator, and the mixture was vacuum-dried at room temperature for 20 minutes to obtain degradable diamine A. The obtained diamine A was immediately used in a poly-Radziszewski reaction with diamine B to form biodegradable PIM1D.

[0183] The synthesis of PIM1D was carried out as shown in Figure 10B. A first mixture of glyoxal (40 wt%, 349 mg, 2.4 mmol) and formaldehyde (37 wt%, 195 mg, 2.4 mmol) in glacial acetic acid and tetrahydrofuran (THF) (3:1.25 mL) was prepared at 0°C (ice water). A second solution containing 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 at 0°C (ice water) over 10 minutes. The reaction mixture (yellowish in color) was then warmed to room temperature, at which point it turned brown. After the reaction mixture was left at room temperature for 24 hours, the final reaction mixture (approximately 10 mL) was directly transferred to a 1 K Dalton cutoff Spectra / Por® 6 dialysis membrane (Repligen, USA) and dialyzed against 5 L of acidified water (pH=3~4), with the acidified water being changed three times over 24 hours. 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 hour, 120 rpm) to obtain solid PIM1D in the round-bottom flask. To transfer PIM1D for lyophilization, water (5 mL) was added to the polymer solution, and the concentrated PIM1D solution was decanted into a small Falcon tube (15 mL) and lyophilized at -80°C to obtain pure PIM1D. To confirm the molecular weight and chemical structure of PIM1D, characterization was performed by GPC and NMR.

[0184] Characterization GPC showed a narrow distribution of the final PIM1D compound in DMSO-d6. 1 Chemical shifts at 9.62 ppm and 7.81 ppm in the 1H NMR spectrum confirm the formation of an imidazolium ring, and signals at 1.59–5.06 ppm correspond to the alkyl chain in PIM1D in MSO-d6. 13The assignment of the peaks was further confirmed by 13C NMR spectroscopy: signals from 121.06–136.53 ppm indicate the formation of an imidazolium ring, signals from 25.78–52.77 ppm indicate the presence of an alkyl chain, and signals from 164.99 and 167.05 ppm indicate the presence of an amide carbonyl. These assignments were further confirmed by DEPT-135, COZY, and HMQC analysis. In the DEPT spectrum, 13 In 13C NMR, the carbonyl group signals of the amide, which appeared at 167.05 and 164.99 ppm, disappeared, and the signals corresponding to the C2-H, C4-H, and C5-H protons of the imidazolium ring showed a positive phase, while other signals of the CH2 group of the polymer chain showed a negative phase. In the COSY spectrum, correlations between the alkyl chains of the polymer chain were observed, indicating their adjacent positions. However, no correlation was observed between the signals at 5.07 ppm and 4.57 ppm, confirming that the two non-equivalent -CH2-CO- groups have no adjacent protons, indicating that the -CH2-carbons of these groups are bonded to the N atom of the imidazolium ring. The HMQC spectrum further supports the assignment by showing proton and carbon correlations in both the imidazolium ring and the alkyl chain of PIM1D.

[0185] Example 3. Optimization of reaction conditions for PIM1D synthesis To optimize the influence of reaction conditions on the biological profile of PIM1D, specific reaction parameters from Example 2 were modified, including the supply ratio of diamine A and diamine B, reaction temperature, and reaction time. The biological profile of PIM1D was determined by its antimicrobial activity and cell viability, as described in Comparative Example 3.

[0186] Results and Discussion The molar ratio of diamine A to diamine B (Table 6, entries 1-3) was varied to optimize the percentage of the degradable portion (diamine A). The results showed that entry 3 (where the molar ratio of diamine A to diamine B in the feed was 2:3) produced an optimized PIM1D with excellent antimicrobial activity and minimal mammalian cytotoxicity (Tables 7-8, entry 3). Table 6, entries 1-2 (and corresponding entries 1-2, Tables 7-8) yielded PIM1D with much higher toxicity due to the lower supply ratio of degradable diamine, but still demonstrated excellent antimicrobial activity.

[0187] Table 6. Optimization of reaction conditions for the synthesis of PIM1D from diamine A and diamine B. [Table 6] *Reactions and purifications under different conditions (detailed in Table 1) were carried out according to the typical experimental procedure given for PIM1D synthesis. a The reaction was carried out on a larger scale (4.8 mmol aldehyde scale). b The reaction was carried out at high dilution (10 mL of AcOH to 2.4 mmol of aldehyde). c A polymer containing acetate counterions was obtained during dialysis without the use of HCl.

[0188] Table 7. Antimicrobial activity of PIM1D synthesized under various reaction conditions. [Table 7]

[0189] Table 8. Cell viability of PIM1D synthesized under various reaction conditions. [Table 8]

[0190] Further optimization of reaction conditions was performed by varying the solvent ratio, temperature, polymerization reaction time, dialysis membrane, and dialysis time (Table 6, entries 4-17). The resulting compounds exhibited a narrow molecular weight distribution with Mn in the range of 1 kDa to 2 kDa, and the final proportion of degradable diamine A (in the product) was in the range of 17% to 30% (Table 6, entries 4-17). All of these compounds showed good antibacterial activity and MIC 90 The concentrations were mostly in the range of 4–16 μg / mL against both multidrug-resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (Table 7, entries 4–17). Biocompatibility was tested using 3T3 fibroblasts and liver HepG2 cells, and the tested compounds (Table 8, entries 4–17) showed cell viability of over 50% at all four concentrations (128 μg / mL–1024 μg / mL). These results indicate that slight changes in reaction conditions in PIM1D synthesis do not significantly affect its biological properties (Tables 6–8, entries 4–17), and that the biological profile of PIM1D is not affected by its molecular weight in the range of 1 KDa–2 KDa. This resistance to variations in reaction conditions facilitates the commercialization of the compound and holds great potential for various antimicrobial applications.

[0191] Example 4. In vitro antimicrobial activity and biocompatibility of PIM1D PIM1D and colistin were tested against a larger panel of MDR Gram-positive and Gram-negative bacteria according to the protocol of Comparative Example 3. The in vitro biocompatibility of PIM1D and colistin was evaluated by MTT tests using 3T3, HEK293, HepG2, and A549 cells according to the protocol of Comparative Example 3.

[0192] Results and Discussion Table 9 shows the physical and biological properties of various batches of PIM1D in Pseudomonas aeruginosa PAER, A. baumannii AB-1 (MDR), and Staphylococcus aureus USA300 (MRSA). Surprisingly, PIM1D showed potent antibacterial activity against colistin-resistant B. tyrandensis 700388 (Table 10), a critical pathogen for which the WHO has requested new antibiotics (World Health Organization (WHO) Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery and Development of New Antibiotics 2017), MDR A. baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as a larger panel of MDR Gram-positive and Gram-negative bacteria. Note that PIM1D is also a potent anti-mycobacterium compound. Overall, we demonstrated that PIM1D is an effective antibacterial agent with broader spectral activity than colistin.

[0193] Table 9. Physical and biological characteristics of various batches of PIM1D in Pseudomonas aeruginosa PAER, A. baumannii AB-1 (MDR), and Staphylococcus aureus USA300 (MRSA). [Table 9]

[0194] Table 10 MICs of PIM1D against pathogens, mycobacteria, and human cell lines 90 and cytotoxicity [Table 10]

[0195] PIM1D greater than 1024 μg / mL is an IC2 50 The values ​​shown are similar to those of colistin, the antibiotic control (Table 10). MIC of PIM1D against most bacterial strains. 90 Considering that the values ​​were in the range of 8-16 μg / mL, a large therapeutic window of over 50 can be obtained. Therefore, PIM1D has the potential to be developed as an antimicrobial agent.

[0196] Example 5. In vivo toxicity and antimicrobial efficacy of degradable PIM1D In vivo toxicity testing The in vivo toxicity of PIM1D was assessed by monitoring mouse body weight and blood biomarkers over 14 days. BALB / c female mice (8-9 weeks old) were randomly divided into two groups: a saline control group and a PIM1D treatment group. Each mouse in the PIM1D group received PIM1D (15 mg / kg) daily for 7 consecutive days via IP injection (cumulative dose 105 mg / kg). The saline control group received the same amount of saline intraperitoneally. On days 1, 3, and 7, mouse blood was collected from the submandibular vein and subjected to 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, blood from mice in the saline control group was collected and quantified for comparison. The mice's condition was closely monitored from the first injection until 14 days later. This protocol was approved by the Animal Ethics and Welfare Committee (AEWC, Protocol AEWC-2018-07) of Ningbo University.

[0197] In vivo efficacy trial The in vivo efficacy of PIM1D was evaluated using a mouse sepsis model. Experiments with mouse sepsis infection models of MDR Pseudomonas aeruginosa PAER and MDR A. baumannii AB-1 were conducted within the guidance of a protocol approved by the Institutional Care and Use Committee of Nanyang University of Technology (NTU IACUC). Experiments with mouse sepsis infection models of wild-type Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus MRSA USA300 were conducted according to a protocol reviewed and approved by the Animal Ethics and Welfare Committee (AEWC) of Ningbo University. The septic shock protective effect of all mouse infection models was tested using BALB / c female mice (8-9 weeks old). Bacteria in the exponential growth phase were washed twice with saline and resuspended in the same volume of saline. 300 μL of bacterial suspensions of various concentrations in 5% mucin were introduced into each mouse by IP injection to first determine the lethal bacterial load, and the determined concentration was used in subsequent studies. The use of mucin is intended for immunocompromised mice, as well as hospitalized patients. Two hours after infection, mice (5 per group) were treated with a single dose of the test compound. Positive and negative control groups of mice were injected with the same dose of antibiotic and the same volume of saline at the same time. The mice were monitored for survival for 7 days. In another group of mice, all mice were euthanized 26 hours after infection. Peritoneal lavage was then performed by injecting PBS (2.0 mL) into the IP cavity, followed by 1 minute of abdominal massage. Approximately 0.5 mL of ascites fluid was then collected for CFU analysis. Bacterial load was also assessed in the spleen, liver, and kidneys of the animals. To confirm whether bacterial infection was established two hours after infection, mice inoculated with the same bacteria were sacrificed, and the IP fluid and all organs (including kidneys, liver, and spleen) were collected to determine the CFU. Experiments concerning sepsis caused by MRSA were similar to those with Pseudomonas aeruginosa, except that mice were immunosuppressed by intraperitoneal injection of 150 mg / kg and 100 mg / kg of cyclophosphamide on days 4 and 1, respectively (Chin, W. et al., Nat. Commun. 2018, 9, 917). Treatment was administered twice, at 2 hours and 26 hours post-infection. Bacterial load was obtained from the organs of sacrificial mice 50 hours after infection.In the untreated group, mice were sacrificed at either 26 or 50 hours after infection, whichever was closer to the time of death. In the pre-treated group, mice were sacrificed 2 hours after infection. Bacterial levels were analyzed using one-way analysis of variance (ANOVA) and two-sided Student's t-test (Graphpad Prism for Windows, version 7).

[0198] Results and Discussion Mice treated with PIM1D daily for 7 days showed no significant weight loss (Figure 9A) and no signs of distress. To gain further information regarding the potential toxicity of PIM1D when delivered by IP injection, blood chemistry analysis revealed that many drug toxicity-sensitive markers remained unchanged until the first dose or even after the last dose of PIM1D was delivered (Figures 9B-D). This is a significant improvement over PIM1, in which animals showed significant weight loss and toxic effects after administration of the compound. Therefore, coupled with reduced toxicity and retained broad-spectrum activity, PIM1D is a promising antimicrobial compound.

[0199] In all sepsis models with different bacterial strains, bacterial cells spread to all organs, including the kidneys, liver, and spleen, within 2 hours post-infection when treatment was initiated (see bacterial CFU counts in the “pre-treatment” group in Figures 11A–D). In cases of Pseudomonas aeruginosa PAO1-induced septic shock, PIM1D treatment reduced the bacterial load by more than 3 Log orders in all collected organs (kidneys, liver, and spleen) compared to untreated controls (Figures 12A and 12A–C), and nearly complete bacterial clearance was observed in the peritoneal cavity, demonstrating in vivo efficacy similar to that of the Imp antibiotic control (Figure 12C). Furthermore, all mice treated with either Imp or PIM1D survived without signs of distress during the 7-day monitoring period, while all untreated mice died (Figure 11E).

[0200] Next, the in vivo efficacy of PIM1D in multidrug-resistant Pseudomonas aeruginosa (PAER)-induced peritoneal shock was evaluated. Mice treated with a single dose of PIM1D (15 mg / kg) had a 100% survival rate at 2 hours post-infection, compared to zero survival rates in untreated controls or mice treated with the same dose of Imp (Figure 11F). Furthermore, compared to untreated controls or Imp controls, a reduction of over 99.9% in bacteria was observed in all harvested organs (including kidneys, liver, and spleen), demonstrating near-complete eradication of bacteria in the peritoneal cavity (Figures 11B and 12D-F).

[0201] In a sepsis model induced by MDR A. baumannii (AB-1), mice treated with a single dose of PIM1D (15 mg / kg) showed superior bacterial reduction compared to Imp controls (15 mg / kg). Approximately 99.9% bacterial elimination was observed in PIM1D-treated mice compared to untreated controls in collected organs, and a bacterial reduction of over 99.999% was observed in the peritoneal cavity (Figure 11C and Figures 12G-I). Furthermore, PIM1D-treated mice showed 100% survival compared to 80% survival in the Imp-treated group and 0% survival in the untreated mouse group (Figure 11G).

[0202] Mouse blood samples collected from the submandibular vein on days 1, 3, and 7 were analyzed using a veterinary chemistry analyzer to evaluate ALT, AST, and BUN levels. Mice that received saline daily via IP injection were used as controls. Over the 7-day period, no significant changes were observed in ALT and AST levels, which indicate hepatic toxicity, and negligible changes were observed in BUN levels, which indicate nephrotoxicity (Figures 13A-H). These results indicate that the introduction of a degradable portion reduces the in vivo toxicity of the PIM series to a normal level and maintains its in vivo antibacterial activity.

[0203] Example 6. In vivo efficacy of PIM1D in immunosuppressed mice 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) before infection was introduced. The animal research protocol was approved by the Animal Ethics and Welfare Committee of Ningbo University. Mice were infected with methicillin-resistant Staphylococcus aureus (MRSA) USA300 according to the protocol of Example 5. Two separate IP injections of 15 mg / kg antibiotics (PIM1D and vancomycin) were administered 2 and 26 hours after infection. Organ harvesting and peritoneal lavage of the mice were performed 50 hours after infection to determine the bacterial load.

[0204] Results and Discussion The efficacy of PIM1D in MRSA-induced sepsis in immunosuppressed mice was evaluated, further demonstrating its broad-spectrum antimicrobial activity. Compared to untreated controls, mice treated with PIM1D showed a bacterial reduction of over 99% in all harvested organs, demonstrating superior bacterial clearance compared to vancomycin treatment (Figures 11D and 12J-L). In the peritoneal cavity, a bacterial reduction of over 99.99% was observed, similar to that of vancomycin-treated controls (Figure 12L). In mice treated with either PIM1D or vancomycin, all mice survived, in contrast to 0% survival in the untreated group (Figure 11H). Therefore, PIM1D protected immunosuppressed mice infected with MRSA USA300 from disease and reduced the bacterial load in affected organs.

[0205] Example 7. In vivo efficacy of PIM1D in a neutropenic lung infection model. To demonstrate its in vivo efficacy in treating distal infections, PIM1D was used to treat neutropenic pulmonary infection models caused by MRSA USA300 and Klebsiella pneumoniae (#13883).

[0206] Neutropenic lung infection model Immunosuppression was induced by intrapulmonary 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) before infection was introduced. Lung infection was established by intratracheal delivery of MRSA USA300 or Klebsiella pneumoniae (#13883). Infected mice were treated 2 hours post-infection by intratracheal delivery with 20 mg / kg PIM1D-CA (a mixture of PIM1D and citrate, 1:1 wt%, with citrate added to minimize associated toxicity) or an antibiotic (vancomycin or coristan), while untreated mice received only PBS. The survival of the mice was monitored for one week. In another experiment, the lungs of mice were collected 26 hours after infection, homogenized, and then plated to confirm bacterial load. The animal research protocol was approved by the Animal Ethics and Welfare Committee of Ningbo University.

[0207] Results and Discussion In neutropenic lung infections induced by MRSA, a single dose of 20 mg / kg PIM1D-CA (a 1:1 wt.% mixture of PIM1D and citrate) delivered intratracheally reduced bacterial infections by over 99.9% compared to mice without bacterial load (Figure 14A). Furthermore, PIM1D-CA treatment was superior to vancomycin at the same treatment dose. In addition, infected mice treated with PIM1D-CA showed a 100% survival rate compared to the infection control group (zero survival rate) and a 40% survival rate compared to vancomycin-treated mice (Figure 14B), demonstrating the superior activity of PIM1D in treating neutropenic lung infections caused by MRSA.

[0208] Considering the broad-spectrum antibacterial activity of PIM1D, its efficacy in neutropenic lung infections caused by Klebsiella pneumoniae (#13883) was also evaluated. Single intratracheal delivery of PIM1D-CA (20 mg / kg) reduced Klebsiella pneumoniae in the lungs of mice by more than 99.9% compared to infected controls, similar to colistin-treated mice (Figure 14C). Furthermore, both PIM1D-CA and colistin-treated mice survived within the one-week monitoring period, while untreated mice did not survive (Figure 14D).

[0209] Advantages of PIM1D over PIM1 The results of Examples 1-7 are remarkable, showing that PIM1D not only showed no evidence of toxicity but also retained significant antibacterial activity, demonstrating efficacy in treating septic infections in mice in vivo. Therefore, along with its excellent biocompatibility, PIM1D is a superior antibacterial candidate for PIM1.

[0210] Comparative Example 12. Synthesis of PIM1 bromide (PIM1-Br) monomer Imidazole (10.0 g, 146.9 mmol) was dissolved in THF. NaH (10.6 g, 440.7 mmol) was gradually added to the solution at 0°C, and the reaction mixture was stirred at room temperature for 1 hour. 1,4-dibromobutane (63.5 g, 294.11 mmol) (2.0 equivalents) was added, and the reaction mixture was heated under reflux (50°C) for 5 hours (Figure 15) to obtain PIM1-Br monomer as an orange oil (15.1 g, 46%).

[0211] 1 ¹H NMR (CDCl3, 300MHz): 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).

[0212] Comparative Example 13. Autopolymerization pathway for preparing PIM1-Br and the effect of reaction conditions on the autopolymerization reaction. The PIM1-Br monomer prepared in Comparative Example 12 was dissolved in a solvent selected from water, NMP, and DMF in a volume ratio (monomer:solvent) of 1:3. The polymerization reaction was carried out with vigorous stirring, and the reaction flask was heated in an oil bath. After the predetermined reaction time, the reaction mixture was diluted with deionized water, dialyzed with deionized water for 3 days (MWCO1000Da), and freeze-dried to obtain the PIM-Br compound characterized by GPC (Table 11) (Figure 16).

[0213] Results and Discussion The effects of various reaction conditions on the autopolymerization reaction of PIM1-Br were investigated using GPC. A summary of the GPC results is shown in Table 11.

[0214] Table 11. Autopolymerization of PIM1-Br under different reaction conditions. [Table 11]

[0215] Comparative Example 14. Antibacterial effect of PIM1-Br The antimicrobial effect of PIM1-Br was investigated according to the protocol of Comparative Example 3, and the MIC of the compound against different bacteria was measured.

[0216] Results and Discussion Table 12. Summary of the antibacterial effects of PIM1-Br [Table 12]

[0217] Comparative Example 15. Synthesis of non-degradable back-chain cationic PIMs, P(ImC6) and P(ImC8) (Figure 17) A compound selected from 1,6-diaminohexane or 1,8-diaminooctane (total 100 mmol) in water (30 mL) was introduced into a three-necked flask equipped with a stirring bar. HCl (16.7 mL) was slowly added to the reaction mixture. After stirring at room temperature for 30 minutes, a mixture of 37% formaldehyde (100 mmol) and 40% glyoxal (100 mmol) was introduced. The reaction mixture was refluxed at 100°C for 12 hours, during which the color of the reaction mixture gradually changed from colorless to yellowish. After removing some of the solvent and unreacted monomers using a rotary evaporator, the crude product was dialyzed for 1 day against acidic water, pH 3-4 (1-KDa cutoff Spectra / Por® 6 dialysis membrane, Repligen, USA). P(ImC6) and P(ImC8) were obtained. 1 The findings were characterized by 1H NMR and GPC analysis (Table 13).

[0218] P(ImC6) 1 ¹H NMR (300 MHz, D2O): 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).

[0219] P(ImC8) 1 ¹H NMR (300 MHz, D2O): δ 8.77 (s, 1H, imidazole-H), 7.48 (s, 2H, imidazole-H), 4.17 (t, 4H), 1.74 (m, 4H), 1.25 (m, 4H).

[0220] List of abbreviations for non-degradable PIMs P(ImC6)-P1 P(ImC8)-P2

[0221] Example 8. Synthesis of TFA salts of diamidodiamine (n=4, 6, 8, 10, and 12) monomers (Figure 18) Diamidodiamine (n=4) TFA salt The diamidediamine (n=4) TFA salt was prepared from diamine B (5.00 g, 56.72 mmol) according to the protocol of Example 1. The white solid was collected and dried to obtain the diamidediamine TFA salt (n=4) (48.1%, 11.73 g).

[0222] 1 H NMR(300MHz,D2O):δ 3.24(s,4H),2.68(s,4H),0.96(s,4H).

[0223] Diamidodiamine (n=6) TFA salt Diamidodiamine (n=6) TFA salts were prepared from 1,6-diaminohexane (5.00 g, 43.10 mmol) according to the protocol of Example 1, and the TFA salts of diamidodiamine (n=6) were obtained as a white solid (41%, 4.80 g).

[0224] 1 H NMR(300MHz,DMSO-D6):δ 8.35(t,J=5.4Hz,2H),8.05(brs,6H),3.53(s,4H),3.14(q,J=6.3Hz,4H),1.54-1.63(m,2H).

[0225] Diamidodiamine (n=8) TFA salt Diamidodiamine (n=8) TFA salts were prepared from 1,8-diaminooctane (2.50 g, 21.55 mmol) according to the protocol of Example 1, and the TFA salts of diamidodiamine (n=8) were obtained as a white solid (58.3%, 3.50 g).

[0226] 1 H NMR(300MHz,DMSO-D6):δ 8.34(t,J=5.4Hz,2H),8.04(brs,6H),3.52(s,4H),3.14(q,J=6.3Hz,4H),1.42-1.26(m,12H).

[0227] Diamidodiamine (n=10) TFA salt The diamidediamine (n=10) TFA salt was prepared from 1,10-diaminodecane (2.50 g, 21.55 mmol) according to the protocol of Example 1, and the TFA salt of diamidediamine (n=10) was obtained as an orange solid (46%, 3.80 g).

[0228] 1 H NMR(300MHz,DMSO-D6):δ 8.39(t,J=5.4Hz,2H),8.12(brs,6H),3.52(s,4H),3.10(q,J=6.3Hz,4H),1.40-1.24(m,16H).

[0229] Diamidodiamine (n=12) TFA salt The diamidediamine (n=12) TFA salt was prepared from 1,12-diaminododecane (5.00 g, 43.10 mmol) according to the protocol of Example 1, and the TFA salt of diamidediamine (n=12) was obtained as a white solid (45.3%, 5.50 g).

[0230] 1 H NMR (300MHz, DMSO-D6): δ 8.35(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).

[0231] Example 9. Synthesis of degradable main-chain cationic PIMs (P(ImC6-co-ImC6D)-50, P(ImC8-co-ImC8D)-50%), P(ImC6D), and P(ImC8D)). P(ImC6-co-ImC6D)-50% and P(ImC8-co-ImC8D)-50% were synthesized by copolymerization (Figure 19a), while P(ImC6D) and P(ImC8D) were synthesized by homopolymerization (Figure 19b).

[0232] P(ImC6-Co-ImC6D)-50% P(ImC6-co-ImC6D)-50% was prepared from diamidediamine (n=6) TFA salt and 1,6-diaminohexane with a mole fraction of 50% of the degradable diamine according to the protocol of Example 2 to obtain P(ImC6-co-ImC6D)-50%.

[0233] 1 ¹H NMR (300 MHz, 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).

[0234] P(ImC8-Co-ImC8D)-50% P(ImC8-co-ImC8D)-50% was prepared from diamidediamine (n=8) TFA salt and 1,8-diaminooctane with a mole fraction of 50% of the degradable amine according to the protocol of Example 2 to obtain P(ImC8-co-ImC8D)-50%.

[0235] 1 ¹H NMR (300 MHz, 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).

[0236] P(ImC6D) P(ImC6D) was prepared from a diamidediamine (n=6) TFA salt according to the protocol of Example 2, except that a non-degradable amine was not added. After dialysis, P(ImC6D) was obtained.

[0237] 1 ¹H NMR (300 MHz, D2O): 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).

[0238] P(ImC8D) P(ImC8D) was prepared from a diamidediamine (n=8) TFA salt according to the protocol of Example 2, except that a non-degradable amine was not added. After dialysis, P(ImC8D) was obtained.

[0239] 1 ¹H NMR (300 MHz, D2O): δ 8.93 (s, 1H, imidazole-H), 7.53 (s, 2H, imidazole-H), 5.00 (s, 4H), 3.24 (s, 4H), 1.51-1.28 (m, 12H).

[0240] List of abbreviations for degradable PIM P(ImC6-Co-ImC6D)-50%-P3 P(ImC6D)-P4 P(ImC8-ko-ImC8D)-50%-P5 P(ImC8D)-P6

[0241] All PIMs prepared here and in Comparative Example 15 (Figure 20) 1 Characterized by 1H NMR and GPC (Table 13).

[0242] Table 13. Actual mole fractions, Mn, Mw, and polydispersity (Mn / Mw) of degradable diamines in PIMs. [Table 13]

[0243] Example 10. In vitro antimicrobial activity and cytotoxicity of P1-P6 The antimicrobial activity of the three prepared PIMs (Figure 20) against planktonic bacteria was evaluated according to the protocol of Comparative Example 3, and the MIC values ​​were measured against methicillin-resistant Staphylococcus aureus BAA39 and Gram-positive and Gram-negative bacterial strains, Pseudomonas aeruginosa O1, and Escherichia coli. Benzalkonium chloride (BAC) was used as a reference. The cytotoxicity of PIMS was tested against mouse embryonic fibroblasts (3T3 cells) according to the MTT assay protocol of Comparative Example 3.

[0244] Results and Discussion As shown in Table 14, PIM with a high mole fraction of degradable linker (100%) had lower bactericidal activity than non-degradable PIM (molar fraction of degradable linker 0%). However, this trend was not evident in PIM with longer alkyl linkers (P4, P5, and P6). When comparing the viability of cells treated with PIM at different mole fractions of degradable linker (0%, 50%, and 100%), a trend of increasing biocompatibility was observed with increasing fraction. This is the opposite trend to that seen in antimicrobial activity against planktonic bacteria.

[0245] Table 14. MIC (μg / mL) values ​​of PIM and BAC (reference) for a bacterial panel. [Table 14]

[0246] Example 11. In vitro antibiofilm activity of P1-P6 MBEC MBEC was measured using a microtiter plate-based technique. Briefly, this involved measuring a 160 μL suspension of MRSA BAA39 or Pseudomonas aeruginosa O1 (approximately 10 7 Cell density (CFU / mL) was added to a 96-well growth plate covered with a lid containing MBEC PEGs. After incubation at 37°C for 24–48 hours, a biofilm was grown on the PEG lid. After washing twice with PBS to remove planktonic bacteria, the biofilm-bearing lid was transferred to a challenge plate containing one 2x serial dilution of P1–P6 solution, with a total volume of 200 μL per well. The treatment was carried out at room temperature for 4 hours. Subsequently, the PEG lid was washed again with PBS and transferred to a recovery plate containing PBS (200 μL) in each well. The surviving biofilm bacteria were removed from the PEG lid by sonication for 30 ± 5 minutes, and the isolated bacteria were serially diluted 10x in sterile PBS and spread onto agar plates. After incubation at 37°C for 24 hours, the colonies were counted.

[0247] Results and Discussion As shown in Figure 21, the overall effectiveness of antibiofilms against MRSA BAA39 can be ranked as follows: P(ImC8) > P(ImC8-Co-ImC8D)-50% ~ P(ImC6) > BAC. Similarly, for Pseudomonas aeruginosa O1 (Figure 22), the order of antibiofilm efficacy was P(ImC8) > P(ImC8-Co-ImC8D)-50% > P(ImC6) > BAC.

[0248] Example 12. Synthesis of a degradable 2+2 carbonate monomer (compound 4) The synthesis of the carbonate monomer (compound 4) involved compounds 1-3 and three steps (Figure 23). CDI was reacted with an alcohol (compound 1) to synthesize an imidazole carbonate ester (compound 2), yielding compound 2 in high yield. Subsequent carbonyl formation was achieved by treating compound 2 with CDI and compound 1 in the presence of a catalytic amount of NaOH, yielding the desired boc-protected carbonate (compound 3) in high yield. Boc deprotection was carried out with TFA in DCM to obtain the desired compound 4 in high yield.

[0249] 2-((tert-butoxycarbonyl)amino)ethyl 1H-imidazole-1-carboxylate (compound 2) In a 250 mL round-bottom flask equipped with a dry N2 inlet 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 hours with stirring. The 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 then washed three times with water (3 × 50 mL). The solution was dried over anhydrous Na₂SO₄ and concentrated under vacuum to obtain compound 2 as a white solid (5.1 g, 62.1%).

[0250] 1H NMR (300MHz, DMSO-D6): δ 8.15(s,1H),7.44(s,1H),7.07(s,1H),4.91(brs,1H),4.47(t,J=5.2Hz,2H),3.52(q,J=6.3Hz,2H),1.44(s,9H).

[0251] Di-tert-butyl((carbonylbis(oxy)bis(ethane-2,1-diyl)dicarbamate (compound 3) In a 250 mL round-bottom flask equipped with a dry N2 inlet 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 hours with stirring. The 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 three times with water (3 × 50 mL). The solution was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The resulting crude product was purified by column chromatography (Â:hexane 3:7) to obtain compound 3 as a white solid (4.80 g, 58.8%).

[0252] 1 H NMR (300MHz, DMSO-D6): δ 5.21 (brs, 2H), 4.29 (t, J=5.1Hz, 4H) 3.33 (s, 4H), 1.25 (s, 18H).

[0253] 2,2'-(carbonylbis(oxy)dietanaminonium 2,2,2-trifluoroacetate (compound 4)) In a 100 mL round-bottom flask equipped with a dry N2 inlet 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 hours. Next, the reaction mixture was concentrated under reduced pressure to obtain carbonate monomer 4 as a white solid (3.60 g, 75%).

[0254] 1 H NMR(300MHz,D2O):δ 4.34(t,J=5.1Hz,4H),3.27-3.24(m,4H). 13C NMR(75MHz,D2O):δ 166.14,159.58(- CO -CF3),159.16(- CO -CF3),158.74(- CO -CF3),158.32(- CO -CF3),154.56(CO-O),123.24(-CF3),119.29(-CF3),115.33(-CF3),111.38(-CF3),64.26,38.23.

[0255] Example 13. Synthesis of biodegradable PIM D2 having a carbonate linker. PIM D2-1-8 was prepared from compound 4 (Figure 24) by controlling the stoichiometric ratio and concentration of the starting materials according to the protocol of Example 2 (Table 15).

[0256] 1 ¹H NMR (300 MHz, D2O): δ 8.85 (m, 1H, imidazole-H), 7.50 (m, 2H, imidazole-H), 4.47 (s, 4H). 13 C NMR(75MHz,D2O):δ 154.29,136.64,122.96,66.25,48.31.

[0257] Table 15. Summary of polymerization conditions and molecular weights of biodegradable PIM (PIM D2) incorporating carbonate. [Table 15] a The molar ratio is the ratio of diamine to aldehyde. b The concentration is the concentration of aldehyde. c Staphylococcus aureus is Staphylococcus aureus 29213.

[0258] Table 15 shows that the diamine concentration had only a slight effect on the polymer molecular weight, but the diamine-to-aldehyde stoichiometric ratio had a significant effect on the polymer molecular weight. The highest molecular weight obtained was PIMD2-5, which has a molecular weight of 1522 g / mol and a narrow polydispersity of 1.08. The chemical structure of the biodegradable PIM with incorporated carbonate is: 1 H NMR spectrum and 13 Further verification was conducted using both 1C NMR spectra.

[0259] Example 14. Stepwise synthesis of degradable hexymidazolium (OIM1D-3C-6 and OIM1D-3C-8) Given the excellent antimicrobial activity and biocompatibility of PIM1D, stepwise synthesis was investigated to create oligoimidazolium with a biodegradable amide linker and a clearly defined molecular weight. Imidazorium having three repeating units was prepared stepwise and linked using an N,N'-(alkane-1,3-diyl)bis(2-chloroacetamide) linker to obtain the final degradable compounds. These are called OIM1D-3C-6 and OIM1D-3C-8, degradable linkers having three and eight carbon atoms in the alkyl chain, respectively. The synthesis was achieved in six steps (Figure 25), and eight intermediate compounds (compounds 5-12) were required to obtain the final degradable oligoimidazolium (OIM1D-3C-6 and OIM1D-3C-8). The compounds were characterized by NMR and MALDI-TOF, where applicable.

[0260] 1,4-di(1H-imidazol-1-yl)butane (compound 5) Compound 5 was prepared from imidazole (4.00 g, 0.058 mol 11 equivalents) according to the protocol of Comparative Example 12, except that the reaction mixture was heated overnight under reflux (70°C) and the product was purified by extraction with MeOH. The MeOH phase was washed three times with hexane, and white solid crystals of compound 5 (10.2 g, 92%) were obtained using a rotary evaporator.

[0261] 1H NMR(300MHz,DMSO-d6)δ 7.61(s,2H),7.14(brs,2H),6.89(brs,2H),3.98-3.73(m,4H),1.64-1.59(m,4H).MALDI-TOF(CHCA matrix,Reflector mode)C 10 H 14 N4:calc.190.1218(M);found 191.1296(M+H).

[0262] compound 6 Triethylamine (Et3N) (1.2 equivalents, 10.6 g, 0.105 mol) was added at 0°C to a stirred solution of aminopropylimidazole (1.0 equivalent, 11.0 g, 0.088 mol) in DCM (110 mL). CBzCl (1.1 equivalents, 16.5 g, 0.096 mol) was slowly added via syringe over 10 minutes. The reaction mixture was stirred and warmed overnight to room temperature. The reaction product was transferred to a separatory funnel, and the organic layer was extracted with 0.2 M HCl (100 mL), followed by four consecutive extractions with water (100 mL). The organic layer was dried over anhydrous Na2SO4, concentrated using a rotary evaporator, and subjected to silica gel chromatography to obtain compound 6 (20.5 g, 90%).

[0263] 1 H NMR(300MHz,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=6.9Hz,2H),2.98(q,J=6.3Hz,2H),1.84(p,J=6.7Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ 156.1,137.2,137.1,128.3,127.7,119.3,65.3,43.4,37.4,31.0.

[0264] compound 7 1,4-Dibromobutane (4.5 mL, 0.0375 mol, 2.5 equivalents) was added to a stirred solution of compound 6 (3.00 g, 0.0115 mol, 1.0 equivalent) in dry ACN (10 mL) under an argon atmosphere. The reaction mixture was heated at 70°C for 14 hours and then cooled to room temperature. The solvent was removed under vacuum using a rotary evaporator, and compound 7 was obtained as a white syrup (4.10 g, 76%) by silica gel chromatography eluting ethyl ethyl ethyl ethyl ester with 15% MeOH / ethyl ethyl ester.

[0265] 1 H NMR(300MHz,DMSO-d6)δ 9.39(s,1H),7.88(d,J=3.4Hz,2H),7.58-7.21(m,6H),5.02(s,2H),4.24(q,J=7.2Hz,4 H),3.56(t,J=6.4Hz,2H),3.02(q,J=6.0Hz,2H),2.05-1.86(m,4H),1.86-1.72(m,2H). 13 C NMR(75MHz,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:calc.473.0314(M);found 394.1405(M-Br).

[0266] compound 8 Compound 5 (1.80 g, 0.009 mol, 1.5 equivalents) was added to a stirred solution of Compound 7 (3.0 g, 0.006 mol, 1.0 equivalent) in dry ACN (10 mL), and the resulting mixture was heated overnight at 70°C under an argon atmosphere. After monitoring the completion 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 siRNA to MeOH; 10 to 50%) to obtain Compound 8 as a hygroscopic white solid (3.00 g, 72%).

[0267] 1 H NMR(300MHz,DMSO-d6)δ 9.35(t,J=24.3Hz,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=6.9Hz,8H),4.02(t,J=6.5Hz,2H),3.05-2.99(m,2H),2.02-1.88(m,2H),1.80-1.72(m,8H). 13 C NMR(75MHz,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 Br2N7O2calc.663.1532(M);found (M-2Br-H)504.3814.

[0268] compound 9 1,3-diaminopropane (10.0 g, 1 equivalent) was added at 0°C to a water / DCM (1:3, 18 mL) solution of K2CO3 (33 mmol, 3.3 equivalents). After the resulting mixture was allowed to cool, chloroacetyl chloride (22 mmol, 2.2 equivalents) was added dropwise over 1 hour at 0°C. After the addition was complete, the ice bath was removed and the mixture was stirred overnight at room temperature. 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 obtain compound 9 (82%, 24.5 g).

[0269] 1 H NMR(DMSO-d6)δ 8.59(s,2H),4.05(s,4H),3.09(t,4H),1.55-1.62(m,2H).

[0270] compound 10 Compound 10 was prepared from 1,8-diaminooctane (1 equivalent) based on the protocol for compound 9.

[0271] compound 11 A solution of compound 8 (1.0 equivalent) in ACN:DMF (9:1) was stirred at room temperature, and compound 9 (0.5 equivalent) was added. The mixture was heated at 80°C for 48 hours. The reaction mixture was cooled to room temperature, the resulting precipitate was filtered and collected as a hygroscopic gum-like compound. This was then washed three times with ACN and freeze-dried to obtain a crude mixture of compound 11 and impurities.

[0272] 1 H-NMR δ(D2O)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(75MHz,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.

[0273] compound 12 Compound 12 was prepared from compounds 8 and 10 based on the protocol for compound 11.

[0274] 1 H-NMR δ(DMSO-d6)9.56(s,2H),9.47(s,2H),9.37(s,2H),8.65(s,2H),7.88-7.84(brs,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).

[0275] OIM1D-3C-6 Compound 11 was dissolved in a 33% AcOH solution of HBr, and the resulting mixture was stirred at room temperature for 3 hours. Upon addition of 2 mL of ethyl acetate, the amine salt precipitated. The solvent was removed, and the resulting residue was retained. The obtained compound was dissolved in water (50-60 mM) and passed through a column containing Amberlyst® A-26 (OH- type) loaded with chloride. The column was further washed with water until the compound was completely isolated, and then concentrated under vacuum. The obtained substance was diluted with water and dialyzed against acidified water (1 mL) for 1 day (Mw-CO₂ 500-1000 D), with the acidified water being changed 6-7 times. The solution in the dialyze bag was decanted into a Falcon tube and lyophilized to obtain OIM1D-3C-6 (approximately 30%).

[0276] 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).

[0277] OIM1D-3C-8 IM1D-3C-8 was prepared from compound 12 according to the protocol for OIM1D-3C-6.

[0278] 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(brs,16H),1.42-1.40(m,4H),1.48-1.46(m,8H).

[0279] Example 15. In vitro biological profiles of degradable OIM1D-3C-6 and OIM1D-3C-8 The in vitro biological profiles of OIM1D-3C-6 and OIM1D-3C-8 were evaluated using the MIC and MTT experiments described in Comparative Example 3.

[0280] Results and Discussion OIM1D-3C-6 and OIM1D-3C-8 exhibit good antibacterial activity against both Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, as well as Escherichia coli, and MIC 90 The range was 2-16 μg / mL (Table 16). IM1D-3C-6 showed decreased antibacterial activity against Pseudomonas aeruginosa PAO1, and the MIC 90 The concentration was 128 μg / mL. Both OIM1D-3C-6 and IC were determined in an MTT test using 3T3 fibroblasts. 50 It showed good biocompatibility with a concentration exceeding 1024 μg / mL.

[0281] Table 16. MICs of OIM1D-3C-6 and OIM1D-3C-8 against pathogens and human cell lines. 90 [Table 16]

[0282] Therefore, by adjusting the degradable linker chain, degradable functional groups, imidazolium repeating units, and terminal groups, a library of biodegradable oligoimidazolium with versatile functions can be constructed. This would be a good candidate for mechanistic studies, degradation rate studies, pharmacokinetic and pharmacodynamic studies in animal models.

[0283] Example 16. In vivo testing of degradable OIM1D-3C-6 and OIM1D-3C-8. 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.

[0284] In vivo intranasal toxicity 20 mg / kg of OIM1D-3C-8 and the OIM1D-3C-8 / OIM1D-3C-6 mixture were delivered intranasally to randomly grouped mice (ICR, female). The mice's body weight and condition were monitored daily for up to 7 days after compound delivery.

[0285] Results and Discussion The results showed that 10 mg / kg of OIM1D-3C-8 reduced bacterial load by 60%, while 20 mg / kg of OIM1D-3C-8 reduced bacterial load by approximately 2 Log orders (Figure 26A), demonstrating the effectiveness of OIM1D-3C-8 in reducing bacterial load in a lung infection model. In neutropenic lung infections induced by methicillin-resistant Staphylococcus aureus, a reduction of approximately 2 Log orders in bacterial load was also observed (Figure 26B), demonstrating the effectiveness of OIM1D-3C-8 in combating Gram-positive bacterial infections. Next, the toxicity of OIM1D-3C-8 was investigated by intranasal delivery of OIM1D-3C-8 (20 mg / kg) followed by weight monitoring. The results showed that this resulted in a gradual decrease in body weight over time (Figure 26C).

[0286] To reduce the in vivo toxicity of OIM1D-3C-8, OIM1D-3C-8 and OIM1D-3C-6 were mixed in weight ratios of 2:1 and 1:1. Using these mixtures, toxicity was successfully reduced, and negligible weight loss over time was observed (Figure 26C). Next, the efficacy of these two mixtures was evaluated, and it was found that the OIM1D-3C-8 / OIM1D-3C-6 (2:1 wt.%) mixture resulted in a reduction of approximately 2 Log orders of bacterial load in lung infections induced by MDR K. Pneumonia, which is similar to OIM1D-3C-8 (Figure 26D). In summary, the OIM1D-3C-8 / OIM1D-3C-6 (2:1 wt.%) mixture demonstrated good in vivo efficacy with limited toxicity, suggesting potential as a therapeutic agent for MDR bacterial infections.

Claims

1. It comprises a first repeating unit containing an imidazolium group and a biodegradable chain, and a second repeating unit containing an imidazolium group and a non-biodegradable alkyl chain. Polymers or oligomers are of formula I: 【Chemistry 1】 [In the above formula I, x is between 0.025 and 0.

75. Y － It is a counterion, o is between 0 and 10. p is between 1 and 12. q is between 0 and 14. r is between 0 and 12. D is a biodegradable functional group selected from urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride, and hydrazone. D' is a biodegradable functional group or bond selected from bonds, urea, carbamates, acetals, amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones. Each R １ This refers to branched or unbranched, unsubstituted or substituted with one or more halo atoms of C. １－３ It is alkyl, Each t is 0, 1, or 2. Each t' is 0, 1, or 2. Each R ２ This refers to branched or unbranched, unsubstituted or substituted with one or more halo atoms of C. １－３ It is alkyl. A polymer or oligomer having the properties of a polymer or oligomer.

2. below: (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. A polymer or oligomer according to claim 1, which corresponds to one or more of the following.

3. The polymer or oligomer according to claim 1 or 2, wherein the polymer or oligomer has a number-average molecular weight of 800 to 10,000 Daltons.

4. Each D is selected from amides, esters, carbonate esters, urethanes, disulfides, anhydrides, and hydrazones, wherein the polymer or oligomer is according to any one of claims 1 to 3.

5. Each D is selected from one or more of the group consisting of carbamates, amides, esters, and carbonate esters, the polymer or oligomer according to any one of claims 1 to 3.

6. Each D' is selected from bond, amide, ester, carbonate ester, urethane, disulfide, anhydride, and hydrazone, the polymer or oligomer according to any one of claims 1 to 5.

7. Each D' is selected from one or more of the group consisting of bonded, amide, ester, carbamate, and carbonate ester polymers or oligomers according to any one of claims 1 to 5.

8. Y － This includes halo, acetate, phosphate, sulfonate and bis((trisfluoromethyl)sulfonyl)imide (N(Tf) ２ － A polymer or oligomer according to any one of claims 1 to 7, selected from one or more of the group consisting of ).

9. The polymer or oligomer according to any one of claims 1 to 8, wherein x is 0.05 to 0.

6.

10. The polymer or oligomer according to any one of claims 1 to 9, wherein t and t' are 0.

11. p is 1 to 6, the polymer or oligomer according to any one of claims 1 to 10.

12. The polymer or oligomer according to any one of claims 1 to 11, wherein r is 1 to 6.

13. The aforementioned polymer is 【Chemistry 2】 A polymer or oligomer according to any one of claims 1 to 12, selected from the group consisting of the following.

14. A polymer or oligomer according to any one of claims 1 to 13, or a pharmaceutically acceptable solvate thereof, for use in medical applications.

15. Use of a polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 13 in the manufacture of a pharmaceutical product for treating a disease comprising a microbial infection.

16. A polymer or oligomer or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 13, for use in the treatment of a disease comprising a microbial infection.

17. The use of the polymer or oligomer according to claim 15, wherein the microbial infection is an infected wound or cystic fibrosis.

18. The polymer or oligomer according to claim 16, wherein the microbial infection is an infected wound or cystic fibrosis.

19. A preservative formulation comprising a polymer or oligomer according to any one of claims 1 to 13 or a pharmaceutically acceptable solvate thereof.

20. An article having a surface, wherein the surface is coated with a polymer or oligomer or a pharmaceutically acceptable solvate thereof as described in any one of claims 1 to 13, thereby imparting antimicrobial properties to the surface of the article, and optionally the article is a urethral catheter.

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