CN111344333A

CN111344333A - Imidazox-quaternary ammonium copolymers as novel antibacterial and antifungal materials

Info

Publication number: CN111344333A
Application number: CN201880069489.4A
Authority: CN
Inventors: 张玉根; 袁媛
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2017-11-01
Filing date: 2018-09-28
Publication date: 2020-06-26
Also published as: WO2019088917A1; SG11202002936YA

Abstract

The invention relates to a quaternary ammonium imidazole of formula (I)

Copolymer (b):

wherein each group is defined in the specification. The invention also relates to a polymer having the following formula (II):

Description

Imidazox-quaternary ammonium copolymers as novel antibacterial and antifungal materials

Cross Reference to Related Applications

The present application claims priority to singapore application No. 10201709027U filed on 1/11/2017, the disclosure of which is incorporated herein by reference.

Technical Field

The present invention generally relates to polymers of formula (I) or formula (II). The invention also relates to compositions, pharmaceutical compositions, methods of making polymers of formula (I) or formula (II), uses thereof, and methods of use.

Background

The increasing threat of infectious diseases and global pandemics has underscored the importance of antibiotics and hygiene. Although many classes of antibiotics and antimicrobial agents have been developed and used, the emergence of antimicrobial agent resistance in patients and the environment has prompted the development of new antimicrobial materials that avoid or reduce the selection of resistant microbial strains.

Among the various types of antimicrobial agents, cationic compounds, such as Quaternary Ammonium Compounds (QACs), are currently the most valuable preservatives and disinfectants. QACs exert antibacterial activity against gram-positive and gram-negative bacteria and against some pathogenic species of fungi and protozoa. However, over the past few years, the development and use of QACs has diminished due to the emergence of antimicrobial resistance and potential toxicity to mammalian cells and ecosystems.

More recently, imidazoles

Salts have emerged as new alternatives to antimicrobial applications. Diimidazoles

The salts exhibit good antimicrobial activity and low toxicity to mammalian cells. Can be prepared by modifying imidazole with different functional groups

Or change the anion to adjust selectivity. A series of backbone imidazoles have been developed

Oligomers and polymers that exhibit high efficacy and high selectivity against a wide range of bacteria and fungi. These polymers and oligomers are designed to fully embody the basic characteristics of antimicrobial peptides, such as the amphiphilic structure of the cationic hydrophilic group and the hydrophobic portion.

Indeed, polycationic materials provide an effective solution to resistance and toxicity problems. Synthetic polymers targeting membranes of many pathogenic species are reported to have low sensitivity to developing resistance, unlike small molecule antibiotics and conventional low molecular weight QACs. In topical applications, cationic polymers have limited residual toxicity because they are more difficult to penetrate the skin. Compared to low molecular weight QACs, cationic polymers have a higher positive charge density, which promotes initial adsorption on negatively charged bacterial surfaces and disruption of cell membranes, resulting in significantly enhanced antibacterial activity. However, a disadvantage of some cationic polymers is that they can lead to hemolysis, which is one of the more harmful side effects of many cationic polymers.

Accordingly, there is a need for a new family of polymers having antimicrobial properties and which address or mitigate one or more of the above-mentioned disadvantages.

Disclosure of Invention

According to a first aspect, there is provided a polymer having the following formula (I):

wherein

L¹、L²And L³Independently selected from the group consisting of: optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted alkenylalkyl, optionally substituted alkylalkenyl, optionally substituted alkylalkenylalkyl, optionally substituted arylalkyl, optionally substituted arylalkenyl, optionally substituted arylalkynyl, optionally substituted alkylaryl, optionally substituted alkenylaryl, and optionally substituted alkynylaryl;

x is independently selected from halogen;

n, m and p are independently integers of at least 1;

q is 0 or an integer of at least 1;

a has the following structure:

wherein R is¹、R²、R³And R⁴Independently selected from optionally substituted alkyl, or R¹、R²、R³And R⁴Any two of which may together form at least one bridging group, and

x and y are independently integers of at least 1.

Advantageously, an ammonium-imidazole as defined herein

The copolymer can exhibit resistance against a wide range of microorganisms (microbes or microorganisms)And (4) microbial activity. More advantageously, the ammonium-imidazole

The copolymers may also exhibit antifungal activity against a wide range of fungal species. With mono-component imidazoles

Polymers or ammonium polymers in contrast to most ammonium-imidazoles

The copolymer may show much higher activity against fungi. More advantageously, the copolymer may be biocompatible and degradable, having non-resistant properties. These copolymers may be nonhemolytic.

According to another aspect, there is provided a composition comprising a polymer as defined herein, or a salt or hydrate thereof, and a carrier.

Advantageously, the ammonium-imidazole

The copolymer may not suffer from antimicrobial resistance. More advantageously, the ammonium-imidazole

The copolymers are useful against microorganisms that have developed resistance to conventional antimicrobial drugs.

According to another aspect, there is provided the use of a polymer as defined herein or a composition as defined herein as a non-therapeutic agent for killing or inhibiting the growth of microorganisms.

According to another aspect, there is provided a pharmaceutical composition comprising a polymer as defined herein, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

According to another aspect, there is provided a method for killing or inhibiting the growth of a microorganism, the method comprising administering to a subject a polymer as defined herein or a pharmaceutical composition as defined herein.

According to another aspect, there is provided a polymer as defined herein or a pharmaceutical composition as defined herein for use in killing or inhibiting the growth of a microorganism.

According to another aspect, there is provided the use of a polymer as defined herein or a pharmaceutical composition as defined herein in the manufacture of a medicament for killing or inhibiting the growth of a microorganism.

According to another aspect, there is provided a method for treating a microbial infection, the method comprising administering to a subject a polymer as defined herein or a pharmaceutical composition as defined herein.

According to another aspect, there is provided a polymer as defined herein or a pharmaceutical composition as defined herein for use as an antibiotic.

According to another aspect, there is provided the use of a polymer as defined herein or a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a microbial infection.

According to another aspect, there is provided a process for the preparation of a polymer as defined herein, comprising the steps of:

reacting a diamine having the structure:

wherein R is¹、R²、R³And R⁴Independently selected from optionally substituted alkyl, or R¹、R²、R³And R⁴Any two of which together form at least one bridging group; and x is an integer of at least 1;

with a diimidazole having the structure:

wherein L is⁴Selected from the group consisting of: o-phenylene, p-phenylene, m-phenylene, and vinylene;

and a dihalide having the structure:

wherein L is⁵Selected from the group consisting of: o-phenylene, p-phenylene, m-phenylene, and vinylene; and X is a halide;

under reaction conditions.

Advantageously, these copolymers can be easily and directly synthesized and are relatively low cost.

Definition of

The following words and terms used herein shall have the indicated meanings:

the term "polymer" as used herein refers to a macromolecule or macromolecule composed of multiple repeat units up to 30 identical repeat units in total, where the repeat units may be ammonium, imidazole, or the like

Any of the linkers of formula (I) (L)¹、L²And L³) Any of the linkers of formula (II) (L)⁴And L⁵) Or any combination thereof.

Unless otherwise specified, "alkyl" as a group or part of a group refers to a straight or branched chain aliphatic hydrocarbon group having 1 to 16 carbon atoms (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon atoms), preferably C, to be broadly construed₁-C₁₆Alkyl radical, C₁-C₁₂Alkyl, more preferably C₁-C₁₀Alkyl, most preferably C₁-C₆An alkyl group. Examples of suitable straight and branched alkyl substituents include, but are not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, pentyl (amyl), 1, 2-dimethylpropyl, 1-dimethylpropyl, pentyl (amyl), isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 3-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, undecyl, 2, 3-trimethyl-undecyl, dodecyl, 2-dimethyl-dodecyl.Alkyl, tridecyl, 2-methyl-tridecyl, tetradecyl, 2-methyl-tetradecyl, pentadecyl, 2-methyl-pentadecyl, hexadecyl, 2-methyl-hexadecyl, and the like. Alkyl groups may be optionally substituted with one or more groups as defined below under the term "optionally substituted".

"alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group which contains at least one carbon-carbon double bond and which may be straight or branched, preferably having 2 to 16 carbon atoms in the normal chain, for example 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms, preferably C₁-C₁₆Alkenyl radical, C₁-C₁₂Alkenyl, more preferably C₁-C₁₀Alkenyl, most preferably C₁-C₆An alkenyl group. The groups may contain multiple double bonds in the normal chain and are independently E or Z around each other in orientation. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, and nonenyl. The groups may be terminal groups or bridging groups. The bridging group may be an ethenylene group (ethylene or vinylene). An alkenyl group may be optionally substituted with one or more groups as defined below under the term "optionally substituted".

"alkynyl" as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched, preferably having from 2 to 16 carbon atoms in the normal chain, for example, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms, preferably C₁-C₁₆Alkynyl, C₁-C₁₂Alkynyl, more preferably C₁-C₁₀Alkynyl, most preferably C₁-C₆Alkynyl. Exemplary structures include, but are not limited to, ethynyl and propynyl. The groups may be terminal groups or bridging groups. Alkynyl groups may be optionally substituted with one or more groups as defined below under the term "optionally substituted".

"aryl" as a group or part of a group to be interpreted broadly means (i) preferably having 5 to 12 atoms per ring (e.g. 5, 6, 7)8, 9, 10, 11 or 12 carbon atoms) in a monocyclic or fused polycyclic aromatic carbocyclic ring (a ring structure having all carbon ring atoms), wherein the optional substitution may be di-or tri-substituted. Examples of aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety wherein phenyl and C_5-7Cycloalkyl or C_5-7Cycloalkenyl groups are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl, or indanyl. The groups may be terminal groups or bridging groups. Typically, aryl is C₆-C₁₈And (4) an aryl group. Aryl groups may be optionally substituted with one or more groups as defined below under the term "optionally substituted". Preferably, the group may include ortho-phenylene (ortho-phenylene group), para-phenylene (para-phenylene group), and meta-phenylene (meta-phenylene group), wherein it is used interchangeably with ortho-phenylene (o-phenylene group), para-phenylene (p-phenylene group), and meta-phenylene (m-phenylene group).

"alkylaryl" refers to alkyl-aryl groups in which the alkyl and aryl moieties are as defined herein. Alternatively, "arylalkyl" refers to aryl-alkyl groups in that order, wherein the aryl and alkyl portions are as defined herein. Preferred alkylaryl groups are C having 6 or 10 carbon atoms in the aryl group₁-C₄-alkylaryl. Preferred arylalkyl radicals are aryl-C having 6 or 10 carbon atoms in the aryl radical₁-C₄-an alkyl group. The groups may be terminal groups or bridging groups. If the group is a terminal group, it is bonded to the rest of the molecule through an aryl group. The alkyl portion of the alkylaryl or arylalkyl group can also be a terminating molecule.

"alkenylalkyl" refers to alkenyl-alkyl, wherein the alkenyl and alkyl moieties are as defined herein. Alternatively, "alkylalkenyl" refers to alkyl-alkenyl groups in that order, wherein the alkyl and alkenyl moieties are as defined herein. Preferred alkenylalkyl radicals are C having from 1 to 10 carbon atoms in the alkyl radical₂-C₆-alkenylalkyl. Preferred alkylalkenyls are alkyl-C having 1 to 10 carbon atoms in the alkyl group₂-C₆-alkenyl. The groups may be terminal groups or bridging groups. If it is notThe group is a terminal group, then it is bonded to the rest of the molecule through an aryl group.

"Alkylalkenylalkyl" refers to alkyl-alkenyl-alkyl groups in which the alkenyl and alkyl portions are as defined herein. Preferred alkylalkenylalkyl groups are alkyl groups C having 1 to 10 carbon atoms in the alkyl group₂-C₆-alkenylalkyl. Preferred alkylalkenylalkyl groups are alkyl-C having 1 to 10 carbon atoms in the alkyl group₂-C₆-alkenyl-alkyl. The groups may be terminal groups or bridging groups.

"alkenylaryl" refers to alkenyl-aryl, wherein the alkenyl and aryl moieties are as defined herein. Alternatively, "arylalkenyl" refers to aryl-alkenyl groups in that order, wherein the aryl and alkenyl moieties are as defined herein. Preferred alkenylaryl radicals are C having 6 or 10 carbon atoms in the aryl radical₂-C₆-alkenylaryl. Preferred arylalkenyl is aryl-C having 6 or 10 carbon atoms in the aryl radical₂-C₆-alkenyl. The groups may be terminal groups or bridging groups. If the group is a terminal group, it is bonded to the rest of the molecule through an aryl group. The alkenyl moiety of an alkenylaryl or arylalkenyl group may also be a terminating molecule.

"alkynylaryl" refers to alkynyl-aryl groups in which the alkynyl and aryl moieties are as defined herein. Alternatively, "arylalkynyl" refers to aryl-alkynyl groups in that order, wherein the aryl and alkynyl moieties are as defined herein. Preferred alkynylaryl radicals are C having 6 or 10 carbon atoms in the aryl radical₂-C₆-alkynylaryl. Preferred arylalkynyl groups are aryl-C having 6 or 10 carbon atoms in the aryl radical₂-C₆-alkynyl. The groups may be terminal groups or bridging groups. If the group is a terminal group, it is bonded to the rest of the molecule through an aryl group. The alkynyl moiety of the alkynylaryl or arylalkynyl can also be a terminating molecule.

A "bond" is a linkage between atoms in a compound or molecule. The bond may be a single bond, a double bond, or a triple bond.

"archaebacteria" refers to a single-cell microbial domain that does not have any nuclei or any other organelles within the cell.

"bacteria" refers to members of a large group of unicellular microorganisms that have cell walls but lack nuclear membranes or membrane-bound organelles and organized nuclei, including some that can cause disease. When a cell wall is present, the bacterium(s) is classified as Gram (Gram) positive or Gram negative. While many bacteria are aerobic, requiring the presence of oxygen to survive, other bacteria are anaerobic and can only survive in the absence of oxygen. Bacteria are any of a range of predominantly round, helical or rod-shaped unicellular prokaryotic microorganisms that generally live in soil, water, organic matter or in plant and animal bodies, making their own food (especially from sunlight).

"bridging group" means a group having 2 to 50 atoms (excluding hydrogen atoms), preferably 2 to 40 atoms, 2 to 30 atoms, 2 to 20 atoms (e.g., more preferably 2 to 20 carbon atoms), more preferably 2 to 16 carbon atoms (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon atoms) in the normal chain, C₁-C₁₆Alkyl radical, C₁-C₁₂Alkyl, more preferably C₁-C₁₀Alkyl, most preferably C₁-C₆An alkyl group. The alkyl group may be a divalent alkyl group, alkenyl group, alkynyl group, aryl group, but is not limited thereto. Exemplary alkyl groups include, but are not limited to, ethenylene (-CH)₂CH₂-), o-phenylene, p-phenylene or m-phenylene. The bridging group may be optionally substituted with one or more groups as defined below under the term "optionally substituted".

"fungus" refers to any member of the eukaryotic population, including microorganisms such as yeasts and molds.

"halide" or "halogen" means chlorine, fluorine, bromine or iodine.

"Microorganism (microbe)" or "Microorganism (microbe)", which are used interchangeably, refers to a microscopic (too small to be visible to the naked eye) organism. Microorganisms are often described as single cells or unicellular organisms.

"polydispersity index or value" or "dispersity index

"refers to a measure of the molecular mass distribution in a given polymer sample. Calculating of polymers

(PDI)：PDI＝M_w/M_nWherein M is_wIs the weight average molecular weight and M_nIs the number average molecular weight. M_nIs more sensitive to molecules of low molecular mass, whereas M_wIs more sensitive to high molecular mass molecules. Dispersability indicates the distribution of individual molecular masses among a batch of polymers.

Has a value equal to or greater than 1, but as the polymer chain approaches a uniform chain length,

close to one (1).

"protist" refers to any eukaryotic organism, or collection of organisms that are not animals, plants, or fungi. Protists do not form natural populations or clades because they exclude certain eukaryotes; however, as with algae or invertebrates, they are often grouped together for convenience. Although there are exceptions, they are primarily microscopic and unicellular, or consist of a single cell. The cells of protists are highly organized, with a nucleus and a specialized cellular machinery called organelle.

The term "xylylene" as used herein may be used interchangeably with "xylene".

The term "optionally substituted" as used herein means that the group to which the term refers may be unsubstituted or substituted with one or more groups independently selected from: alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkylalkenyl, heterocycloalkyl, cycloalkylheteroalkyl, cycloalkoxy, cycloalkenoxy, cycloamino, halo, carboxy, haloalkyl, haloalkynyl, alkyneOxy, heteroalkyl, heteroalkoxy, hydroxy, hydroxyalkyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyl, haloalkynyl, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamino, aminoalkyl, alkynylamino, acyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkoxycarbonyl, alkoxycycloalkyl, alkoxyheteroaryl, alkoxyheterocycloalkyl, alkenoyl, alkynoyl, acylamino, diamido, acyloxy, alkylsulfonyloxy, heterocycle, heterocycloalkenyl, heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkoxy, heterocycloalkyloxy, heterocyclylamino, haloheterocycloalkyl, alkylsulfinyl, alkylsulfonyl, haloalkynyl, haloalkenyloxy, nitro, amino, alkoxyheteroaryl, alkoxyheterocycloalkylalkyl, alkenoyl, alkoxyheteroaryl, alkoxyheterocycloalkyl, alkenoyl, alkylthionyl (alkylthionyl), alkylcarbonyloxy, alkylthio, acylthio, aminosulfonyl, phosphorus-containing groups (e.g., phosphono and phosphinyl), sulfinyl, sulfinylamino, sulfonyl, sulfonylamino, aryl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroalkyl, heteroarylamino, heteroaryloxy, arylalkenyl, alkenylaryl, arylalkyl, alkylaryl, alkylheteroaryl, aryloxy, arylsulfonyl, cyano, cyanate, isocyanate, -C (O) NH (alkyl), and-C (O) N (alkyl)₂. When the term "substituted" is used, the group to which the term refers may be substituted with one or more of the same groups described above.

The word "substantially" does not exclude "completely", e.g., a composition that is "substantially free" of Y may be completely free of Y. Where desired, the word "substantially" may be omitted from the definition of the invention.

Unless otherwise specified, the terms "comprising" and "comprise" and grammatical variations thereof are intended to mean "open" or "inclusive" such that they include the recited elements, but also allow for inclusion of additional, unrecited elements.

The term "about," as used herein in the context of concentrations of components of a formulation, generally means +/-5% of the stated value, more generally +/-4% of the stated value, more generally +/-3% of the stated value, more generally +/-2% of the stated value, even more generally +/-1% of the stated value, and even more generally +/-0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as a limitation on the scope of the disclosed ranges. Thus, the description of a range should be considered to have specifically disclosed all the possible subranges within that range as well as individual numerical values. For example, description of a range (e.g., 1 to 6) should be considered to have disclosed sub-ranges (e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) as well as individual numbers within the range (e.g., 1,2, 3, 4, 5, and 6). This applies regardless of the breadth of the range.

Certain embodiments may also be broadly and generically described herein. Each of the narrower species and subclass groupings falling within the general disclosure also form part of the present disclosure. This includes the generic description of embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Detailed Description

Illustrative, non-limiting embodiments of the polymers having the following formula (I) will now be disclosed.

The polymer may have the following formula (I) or a salt or hydrate thereof:

wherein

L¹、L²And L³Independently selected from the group consisting of: optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted alkenylalkyl, optionally substituted alkylalkenyl, optionally substituted alkylalkenylalkyl, optionally substituted arylalkyl, optionally substituted arylalkenylAlkynyl, optionally substituted alkylaryl, optionally substituted alkenylaryl and optionally substituted alkynylaryl;

x is independently selected from halogen;

n, m and p are independently integers of at least 1;

q is 0 or an integer of at least 1;

a has the following structure:

x and y are independently integers of at least 1.

In the polymer having the formula (I), L¹、L²And L³May be independently selected from the group consisting of: optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted alkenylalkyl, optionally substituted alkylalkenyl, optionally substituted alkylalkenylalkyl, optionally substituted arylalkyl, optionally substituted arylalkenyl, optionally substituted arylalkynyl, optionally substituted alkylaryl, optionally substituted alkenylaryl, and optionally substituted alkynylaryl; the carbon atoms of the alkyl group can range from 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, or preferably 1 to 6 carbon atoms. The carbon atoms of the alkenyl or alkynyl group can range from 2 to 20 carbon atoms, 2 to 16 carbon atoms, 2 to 12 carbon atoms, 2 to 10 carbon atoms, or preferably 2 to 6 carbon atoms. The carbon atoms of the aryl group can range from 5 to 18 carbon atoms, 5 to 12 carbon atoms, 5 to 10 carbon atoms, 6 to 18 carbon atoms, or preferably 6 to 12 carbon atoms. The aryl group may be selected from the group consisting of o-xylylene group, m-xylylene group, and p-xylylene group.

Alkenyl-alkyl may be C_2-6alkenyl-C_1-6An alkyl group. The alkyl-alkenyl radical may beTo be C_1-6alkyl-C_2-6An alkenyl group. The alkyl-alkenyl-alkyl group may be C_1-6alkyl-C_2-6alkenyl-C_1-6An alkyl group. The aryl-alkyl group may be C_6-12aryl-C_1-6An alkyl group. The alkyl-aryl group may be C_1-6alkyl-C_6-12And (4) an aryl group. The aryl-alkenyl group may be C_6-12aryl-C_2-6An alkenyl group. The alkenyl-aryl group may be C_2-6alkenyl-C_6-12And (4) an aryl group. Aryl-alkynyl may be C_6-12aryl-C_2-6Alkynyl. Alkynyl-aryl may be C_2-6alkynyl-C_6-12And (4) an aryl group. The alkyl-aryl-alkyl group may be C_1-6alkyl-phenyl-C_1-6An alkyl group. C_1-6The alkyl group may be methyl, ethyl, propyl, butyl, pentyl or hexyl. C_1-6alkyl-phenyl-C_1-6The phenyl group of the alkyl group may be selected from the group consisting of o-xylylene group, m-xylylene group, and p-xylylene group.

In the polymer having formula (I), X may be a halogen from group VII of the periodic table. x may be a halide or halogen selected from fluoride, chloride, bromide and iodide.

In the polymer having formula (I), n, m, and p may independently be an integer of at least 1, or 1 to 5,000, 1 to 1,000, 1 to 500, 1 to 100, preferably 1 to 50 (i.e., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50), more preferably 1 to 10, or most preferably 1 to 5). p may preferably be an integer of 1.

In the polymer having formula (I), q may be 0 or may be an integer of at least 1, or an integer of 1 to 10,000, 1 to 5,000, 1 to 1,000, 1 to 500, 1 to 100, 1 to 50, preferably 1 to 25 (i.e., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25). q may preferably be an integer of 1.

In the polymer having formula (I), a may have the following structure:

wherein R is¹、R²、R³And R⁴May be independently selected from optionally substituted alkyl, or R¹、R²、R³And R⁴Any two of which may together form at least one bridging group, and x and y may independently be integers of at least 1. The optionally substituted alkyl group may be optionally substituted C_1-18Alkyl, preferably optionally substituted C_1-12Alkyl or more preferably optionally substituted C_1-6An alkyl group. R¹、R²、R³And R⁴Any two of which may together form at least one bridging group. R¹And R²May together form at least one bridging group. R³And R⁴May together form at least one bridging group. The bridging group may be C₂-C₁₀Bridging group, preferably C₂-C₅A bridging group, or more preferably a vinylidene group. The bridging group may be optionally substituted.

x and y may independently be an integer of at least 1 or at least 1 to 10, 1 to 5, 1,2, 3, 4 or 5, or more preferably 1.

In the polymer having formula (I), a may be selected from the group consisting of the following structures:

in the polymer having the formula (I), A and L¹The molar ratio therebetween may be in the range of 1:5 to 5:1, in the range of 1:4 to 5:1, in the range of 1:3 to 5:1, in the range of 1:2 to 5:1, in the range of 1:1 to 5:1, in the range of 2:1 to 5:1, in the range of 3:1 to 5:1, in the range of 4:1 to 5:1, in the range of 1:5 to 4:1, in the range of 1:5 to 3:1, in the range of 1:5 to 2:1 or in the range of 1:5 to 1:1, in the range of 1:5 to 1:2, in the range of 1:5 to 1:3 or in the range of 1:5 to 1: 4.

In the polymer having the formula (I), (L)¹+L²) And L³In a molar ratio of about 1:5 to about 51, in the range of 1:4 to 5:1, in the range of 1:3 to 5:1, in the range of 1:2 to 5:1, in the range of 1:1 to 5:1, in the range of 2:1 to 5:1, in the range of 3:1 to 5:1, in the range of 4:1 to 5:1, in the range of 1:5 to 4:1, in the range of 1:5 to 3:1, in the range of 1:5 to 2:1 or in the range of 1:5 to 1:1, in the range of 1:5 to 1:2, in the range of 1:5 to 1:3 or in the range of 1:5 to 1: 4.

In the polymer having formula (I), the polymer may have a molecular weight in the following range: about 1,000 to about 20,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about 9,000, about 1,000 to about 8,000, about 1,000 to about 7,000, about 1,000 to about 6,000, about 1,000 to about 5,000, about 1,000 to about 4,000, about 1,000 to about 3,000, about 1,000 to about 2,000, about 2,000 to about 10,000, about 3,000 to about 10,000, about 4,000 to about 10,000, about 5,000 to about 10,000, about 6,000 to about 10,000, about 7,000 to about 10,000, about 8,000 to about 10,000, about 9,000 to about 10,000, about 10,000 to about 20,000, about 10,000 to about 15,000, or about 15,000 to about 20,000. When the polymer having formula (I) is synthesized in Dimethylformamide (DMF), the polymer may have a high molecular weight.

In the polymer having formula (I), the polymer may have a polydispersity value in the following range: about 1.2 to about 3.2, about 1.4 to about 3.2, about 1.6 to about 3.2, about 1.8 to about 3.2, about 2.0 to about 3.2, about 2.2 to about 3.2, about 2.4 to about 3.2, about 2.6 to about 3.2, about 2.8 to about 3.2, about 3.0 to about 3.2, about 1.2 to about 3.0, about 1.2 to about 2.8, about 1.2 to about 2.6, about 1.2 to about 2.4, about 1.2 to about 2.2, about 1.2 to about 2.0, about 1.2 to about 1.8, about 1.2 to about 1.6, or about 1.2 to about 1.4.

Advantageously, the polymer having formula (I) is characterized by a relatively low degree of polymerization (M)_w< 10,000) and high polydispersity values (1.3 < D < 3.1).

The polymer may have the following formula (II):

wherein

L⁴And L⁵Independently selected from optionally substituted alkenyl or optionally substituted aryl. The optionally substituted alkenyl group may be optionally substituted C_2-18Alkenyl, optionally substituted C_2-12Alkenyl or preferably optionally substituted C_2-6An alkenyl group. The optionally substituted alkenyl group may be a vinylene group. The optionally substituted aryl group may be optionally substituted C_6-18Aryl, optionally substituted C_6-12Aryl or preferably optionally substituted C₆And (4) an aryl group. Optionally substituted aryl may be selected from the group consisting of: ortho-phenylene (o-phenylene), para-phenylene (p-phenylene), and meta-phenylene (m-phenylene).

In the polymer having formula (II), X may be a halogen from group VII of the periodic table. X may be a halide or halogen selected from fluoride, chloride, bromide and iodide.

In a polymer having formula (II):

can be selected from the group consisting of:

in the polymer having formula (II), p and q may independently be integers of 1 to 20, 1 to 10, 1 to 5, 1,2, 3, 4 or 5, or more preferably 1.

The polymer having formula (I) or formula (II) may be selected from the group consisting of:

the molecular weight distribution of the polymer having formula (I) may be affected by the solubility of the polymer in the solvent. Due to the higher polarity of DMF, the solubility of the polymer of formula (I) in DMF may be higher than in THF.

Advantageously, when ammonium and imidazole

Ammonium polymers or imidazoles combined together rather than individually

When polymeric, the ammonium-imidazoles are as defined by the formula (I) or the formula (II)

The copolymer may exhibit a synergistic effect. Preferably, DABCO and imidazole

Can be used as ammonium-imidazole

The copolymers are combined together.

Advantageously, the ammonium-imidazoles as defined by the formula (I) formula (II)

The copolymers may exhibit antimicrobial activity against a wide range of microorganisms. More advantageously, the ammonium-imidazole

Polymers or ammonium polymers in contrast to most ammonium-imidazoles

The copolymer may show much higher activity against fungi.

Ammonium-imidazoles as defined by formula (I) or formula (II)

The Minimum Inhibitory Concentration (MIC) of the copolymer against bacteria may be in the following range: about 1 to about 200. mu.g/mL, about 1 to about 180. mu.g/mL, or about 1 to about 160. mu.g/mL.

Preferably, the ammonium-imidazole as defined by formula (I) or formula (II)

The Minimum Inhibitory Concentration (MIC) of the copolymer against bacteria (e.coli) may be in the following range: about 10 to about 150. mu.g/mL, about 10 to about 125. mu.g/mL, about 10 to about 100. mu.g/mL, about 10 to about 80. mu.g/mL, about 10 to about 60. mu.g/mL, about 10 to about 40. mu.g/mL, preferably about 10 to about 30. mu.g/mL or more preferably about 10 to about 16. mu.g/mL. Ammonium-imidazoles

The copolymer may preferably be a TMED-imidazole having a p-phenylene linker

Copolymers or DMP-imidazoles with p-phenylene linkers

A copolymer. More preferably, ammonium-imidazoles

The copolymer may be a DABCO-imidazole with a trans-butene linker

A copolymer.

Preferably, the ammonium-imidazole as defined by formula (I) or formula (II)

The Minimum Inhibitory Concentration (MIC) of the copolymer against bacteria (staphylococcus aureus) may be in the following range: about 5 to about 80. mu.g/mL, about 5 to about 60. mu.g/mL, about 5 to about 50. mu.g/mL, about 5 to about 40. mu.g/mL, about 5 to about 30. mu.g/mL, about 5 to about 20. mu.g/mL, preferably about 5 to about 16. mu.g/mL or more preferablyAbout 8. mu.g/mL is selected. Ammonium-imidazoles

The copolymer may preferably be a TMED-imidazole having a p-phenylene linker

Copolymers or DMP-imidazoles with p-phenylene linkers

A copolymer. More preferably, ammonium-imidazoles

The copolymer may be a DABCO-imidazole with a trans-butene linker

A copolymer.

Preferably, the ammonium-imidazole as defined by formula (I) or formula (II)

The Minimum Inhibitory Concentration (MIC) of the copolymer against bacteria (pseudomonas aeruginosa) may be in the following range: about 10 to about 80. mu.g/mL, about 10 to about 60. mu.g/mL, about 10 to about 40. mu.g/mL, about 10 to about 30. mu.g/mL, about 10 to about 25. mu.g/mL, about 10 to about 20. mu.g/mL, preferably about 31. mu.g/mL, or more preferably 16. mu.g/mL. Ammonium-imidazoles

The copolymer may preferably be a DMP-imidazole having a p-phenylene linker

A copolymer. More preferably, ammonium-imidazoles

The copolymer may be a DABCO-imidazole with a trans-butene linker

A copolymer.

Ammonium-imidazoles as defined by formula (I) or formula (II)

The Minimum Inhibitory Concentration (MIC) of the copolymer against fungal species may be in the following range: about 1 to about 200. mu.g/mL, about 1 to about 180. mu.g/mL, about 1 to about 160. mu.g/mL, or about 1 to about 140. mu.g/mL.

Preferably, the ammonium-imidazole as defined by formula (I) or formula (II)

The Minimum Inhibitory Concentration (MIC) of the copolymer against fungal species (candida albicans) may be in the following range: about 1 to about 130. mu.g/mL, about 1 to about 115. mu.g/mL, about 1 to about 100. mu.g/mL, about 1 to about 70. mu.g/mL, about 1 to about 40. mu.g/mL, about 1 to about 20. mu.g/mL, about 1 to about 10. mu.g/mL, preferably about 1 to about 8. mu.g/mL, or more preferably about 2. mu.g/mL. Ammonium-imidazoles

The copolymer may preferably be a DMP-based polymer having a trans-butene linker. More preferably, ammonium-imidazoles

The copolymer may be a DABCO-imidazole with a trans-butene linker

A copolymer.

Preferably, the ammonium-imidazole as defined by formula (I) or formula (II)

The Minimum Inhibitory Concentration (MIC) of the copolymer against a fungal species (fusarium solani) can be in the following range: about 20 to about 130. mu.g/mL, about 20 to about 100. mu.g/mL, about 20 to about 80. mu.g/mL, about 20 to about 60. mu.g/mL, about 20 to about 50. mu.g/mL, about 20 to about 40. mu.g/mL, about 20 to about 35. mu.g/mL, or more preferably about 31. mu.g/mL. Ammonium-imidazoles

The copolymer may preferably be a TMED-imidazole having a p-phenylene linker

A copolymer. More preferably, ammonium-imidazoles

The copolymer may be a DABCO-imidazole with a trans-butene linker

A copolymer.

Advantageously, the ammonium-imidazole

More advantageously, DABCO-imidazoles with trans-butene linkers

The copolymer may have a better Minimum Inhibitory Concentration (MIC) than conventional antimicrobial drugs. The Minimum Inhibitory Concentration (MIC) may range from about 1 to about 10 μ g/mL, preferably from about 1 to about 8 μ g/mL, or more preferably about 2 μ g/mL.

DABCO-imidazoles with trans-butene linkers

The Minimum Fungicidal Concentration (MFC) of the copolymer may be in the following ranges: about 50 to about 90. mu.g/mL, about 50 to about 80. mu.g/mL, about 50 to about 70. mu.g/mL, preferably about 55 to about 65. mu.g/mL, or more preferably 62. mu.g/mL. Ammonium-imidazoles as defined by formula (I) or formula (II)

The copolymer may have better stability than conventional antimicrobial drugs. Advantageously, the copolymer may be biocompatible and non-hemolytic.

More advantageously, these copolymers can be easily and directly synthesized and are relatively low cost.

Exemplary, non-limiting embodiments of compositions comprising the polymers as defined herein will now be disclosed.

The composition may comprise a polymer or salt or hydrate thereof, as defined herein, and a carrier.

Exemplary, non-limiting embodiments of the use of the polymers or compositions as defined herein will now be disclosed.

The use of a polymer as defined herein or a composition as defined herein may be as a non-therapeutic agent for killing or inhibiting the growth of microorganisms. Alternatively, the polymers may be used as therapeutic agents for killing or inhibiting the growth of microorganisms.

Illustrative, non-limiting embodiments of pharmaceutical compositions will now be disclosed.

The pharmaceutical composition may comprise a polymer as defined herein, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient.

The polymers may be administered alone or in combination with a pharmaceutically acceptable carrier, diluent or excipient in the form of a pharmaceutical composition.

The amount of polymer in the composition can be such that it is effective to measurably treat a microbial infection or disease. The amount of polymer in the composition can be such that it is effective to measurably treat a disease, disorder, or condition associated with a microbial infection. The amount of polymer in the composition can be such that it is effective to measurably treat a disease, disorder, or condition associated with a microbial infection from any microorganism. The compositions can be formulated for administration to a subject in need of such compositions. The compositions can be formulated for administration to a patient in need of such compositions.

Where polymers are used, they may be administered in any form or manner that provides the polymers with bioavailability. One skilled in the art of preparing formulations can readily select the appropriate form and mode of administration depending on the particular characteristics of the compound selected, the condition to be treated, the stage of the condition to be treated, and other relevant circumstances.

The pharmaceutically acceptable carrier or pharmaceutically acceptable excipient may be a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the polymer with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions may include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol or wool fat.

The composition or pharmaceutical composition as defined above may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition is administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present disclosure may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be employed include water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids (e.g., oleic acid and its glyceride derivatives) are useful in the preparation of injectables, which are pharmaceutically-acceptable natural oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersants commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants (e.g., Tweens, Spans, and other emulsifiers or bioavailability enhancers) commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

The pharmaceutically acceptable composition as defined above may be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. Certain sweetening, flavoring or coloring agents may also be added, if desired.

Pharmaceutical compositions for parenteral injection may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

If desired, and for more efficient distribution, the polymers can be incorporated into slow release or targeted delivery systems (e.g., polymer matrices, liposomes, and microspheres).

The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use.

Alternatively, a pharmaceutically acceptable composition as defined above may be administered in the form of a suppository for rectal administration. These may be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutically acceptable composition as defined above may also be administered topically, especially when the therapeutic target includes an area or organ (including diseases of the eye, skin or lower intestinal tract) that is readily accessible by topical application. Suitable topical formulations can be readily prepared for each of these areas or organs.

Topical application for the lower intestinal tract may be carried out in rectal suppository formulations (see above) or in suitable enema formulations. Topical transdermal patches may also be used.

For topical application, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of a compound as defined above may include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers can include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic applications, the pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or preferably as solutions in isotonic, pH adjusted sterile saline, with or without preservatives, such as benzalkonium chloride (benzalkonium chloride). Alternatively, for ophthalmic applications, the pharmaceutically acceptable compositions may be formulated in the form of an ointment (e.g., petrolatum).

The pharmaceutically acceptable composition as defined above may also be administered by nasal aerosol or inhalation. Such compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other conventional solubilizing or dispersing agents.

Most preferably, the pharmaceutically acceptable composition as defined above may be formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, a pharmaceutically acceptable composition as defined above may be administered without food. In other embodiments, a pharmaceutically acceptable composition as defined above may be administered with food.

The amount of compound that can be combined with the carrier material to produce a single dosage composition can vary depending upon the host treated, the particular mode of administration. Preferably, the compositions should be formulated such that a dose of inhibitor of 0.01-100mg/kg body weight/day can be administered to a patient receiving these compositions.

It will also be understood that the specific dose and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination and the judgment of the treating physician and the severity of the particular disease undergoing therapy. The amount of the polymer of the present disclosure in the composition will also depend on the particular compound in the composition.

Exemplary, non-limiting embodiments of methods for killing or inhibiting the growth of microorganisms will now be disclosed.

A method for killing or inhibiting the growth of a microorganism, which method may comprise administering to a subject a polymer as defined herein or a pharmaceutical composition as defined herein.

The subject may be a cell. The subject may be a human or animal body. The cells may be present in an in vitro cell culture. The cell may be from a cell line. The cell line may be an immortalized cell line, a genetically modified cell line or a primary cell line. The cells may be from a tissue of a subject. The cell may be within a subject.

Methods of killing or inhibiting the growth of microorganisms may be non-therapeutic methods, wherein the polymer may be formulated as a disinfectant, sanitizer, or surface cleaner. The polymers are useful in creating a corrosion-resistant environment.

Exemplary non-limiting embodiments of a polymer or pharmaceutical composition as defined herein will now be disclosed.

The polymer as defined herein or the pharmaceutical composition as defined herein may be used to kill or inhibit the growth of microorganisms. The polymer as defined herein or the pharmaceutical composition as defined herein may be used to kill or inhibit the growth of microorganisms.

Exemplary non-limiting embodiments of the use of the polymer or pharmaceutical composition as defined herein will now be disclosed.

There is provided the use of a polymer as defined herein or a pharmaceutical composition as defined herein in the manufacture of a medicament for killing or inhibiting the growth of a microorganism.

The microorganism may be a bacterium, archaea, fungus, protist, animal, plant, or any mixture thereof.

The microorganism may be a disease causing microorganism. The disease may be a microbial infection or a microbial disease. The disease causing microorganism may be selected from the group consisting of: escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Fusarium solani (Fusarium solani), and Candida albicans (Candida albicans).

Exemplary, non-limiting embodiments of methods for treating microbial infections will now be disclosed.

A method for treating a microbial infection may comprise the step of administering to a subject a polymer as defined above or a pharmaceutical composition as defined above. The polymer or pharmaceutical composition can treat microbial infections or diseases caused by microorganisms.

The polymer as defined above or the pharmaceutical composition as defined above may be used as an antibiotic. Antibiotics can be used to treat microbial infections or diseases. Antibiotics can be used to treat microbial infections or diseases.

There is provided the use of a polymer as defined above or a pharmaceutical composition as defined above in the manufacture of a medicament for the treatment of a microbial infection.

The microbial infection or disease may be caused by bacteria, archaea, fungi, protists, animals, plants or any mixture thereof. Microbial infections or diseases can also result.

The microorganism may be selected from the group consisting of: escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Fusarium solani, and Candida albicans.

The method for inhibition or the method for treatment may be an in vitro method. The method for inhibition or the method for treatment may also be an in vivo method.

Exemplary, non-limiting embodiments of a process for preparing a polymer having the following formula (II) will now be disclosed.

A method for preparing a polymer as defined herein may comprise the steps of:

reacting a diamine having the structure:

with a diimidazole having the structure:

and a dihalide having the structure:

under reaction conditions.

The above method has the following structure

The diamine of (A) can be selected from

And

group (d) of (a). The diamine may not be limited to these examples.

The above method has the following structure

Can be selected from

And

group (d) of (a). The bisimidazole may not be limited to these examples.

The above method has the following structure

Can be selected from

And

and the group consisting of. The dihalide may not be limited to these examples. The X substituent may be iodide or fluoride.

X of the dihalide may be a halide or halogen from group VII of the periodic table. x may be a halide or halogen selected from fluoride, chloride, bromide and iodide.

The reaction conditions of the above process may include the temperature at which the reaction is stirred, the solvent in which the reaction is stirred, or the time period or duration required for the reaction to complete.

The temperature of the reaction may be an elevated temperature. The elevated temperature may be in the following range: about 60 ℃ to about 100 ℃, about 65 ℃ to about 100 ℃, about 70 ℃ to about 100 ℃, about 75 ℃ to about 100 ℃, about 80 ℃ to about 100 ℃, about 85 ℃ to about 100 ℃, about 90 ℃ to about 100 ℃, about 95 ℃ to about 100 ℃, about 60 ℃ to about 95 ℃, about 60 ℃ to about 90 ℃, about 60 ℃ to about 85 ℃, about 60 ℃ to about 80 ℃, about 60 ℃ to about 75 ℃, about 60 ℃ to about 70 ℃ or about 60 ℃ to about 65 ℃. The temperature may preferably be in the range of about 70 to 90 ℃.

The time period or duration of the reaction may be in the following ranges: about 18 hours to 60 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, about 60 hours, preferably about 18 hours, or more preferably about 24 hours.

The solvent for the reaction may be an organic solvent. The solvent may be a polar solvent or a non-polar solvent. The solvent may preferably be a polar organic solvent. The polar organic solvent may be selected from the group consisting of: tetrahydrofuran (THF), Dimethylformamide (DMF), acetone, dimethyl sulfoxide (DMSO), acetonitrile, dichloromethane, and ethyl acetate (EtOAc). The polar organic solvent is preferably Tetrahydrofuran (THF), Dimethylformamide (DMF) or any mixture thereof.

The yields of the various copolymers obtained by the above process or reaction may be in the following ranges: about 45% to about 95%, about 49% to about 95%, about 55% to about 95%, about 60% to about 95%, about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, or about 45% to about 49%.

The diamine and the diimidazole may be contacted in a molar ratio in the following range: in the range of about 1:5 to about 5:1, in the range of 1:4 to 5:1, in the range of 1:3 to 5:1, in the range of 1:2 to 5:1, in the range of 1:1 to 5:1, in the range of 2:1 to 5:1, in the range of 3:1 to 5:1, in the range of 4:1 to 5:1, in the range of 1:5 to 4:1, in the range of 1:5 to 3:1, in the range of 1:5 to 2:1 or in the range of 1:5 to 1:1, in the range of 1:5 to 1:2, in the range of 1:5 to 1:3 or in the range of 1:5 to 1: 4. Preferably, the molar ratio may be 1:3, 1:1 or 3: 1.

The diimidazole and dihalide may be contacted in a molar ratio in the following range: in the range of about 1:6 to about 5:6, in the range of 1:5 to 5:6, in the range of 1:4 to 5:6, in the range of 1:3 to 5:6, in the range of 1:2 to 5:6, in the range of 1:1 to 5:6, in the range of 2:1 to 5:6, in the range of 3:1 to 5:6, in the range of 4:1 to 5:6, in the range of 5:2 to 5:6, in the range of 5:3 to 5:6, in the range of 5:4 to 5:6, in the range of 5(1) (5) to 5:6, in the range of 1:5 to 4:6, in the range of 1:5 to 3:6, in the range of 1:5 to 2:6 or in the range of 1:5 to 1:6, in the range of 1:5 to 5: 1), in the range of 1:5 to 5:6, in the range of 1:5 to 5: 1:5, in the range of 1:5 to 5:6, in the range of 1:5 to 5: 5, in the range of 1:5 to 5, In the range of 1:5 to 5:2 or in the range of 1:5 to 5: 1. Preferably, the molar ratio may be 1:4, 1:2 or 3: 4.

The diimidazole, diamine and dihalide may be contacted in any combination other than those described above, in the molar ratios as defined above.

Brief description of the drawings

The drawings illustrate the disclosed embodiments and serve to explain the principles of the disclosed embodiments. It is to be understood, however, that the drawings are designed solely for the purposes of illustration and not as a definition of the limits of the invention.

FIG. 1 shows a schematic view of a

FIG. 1 shows the antifungal activity of IBN-132-3 against Candida albicans. Fig. 1A shows the killing efficacy against candida albicans after 48 hours of treatment with low concentrations (from 0.5MIC to 4MIC, MIC 2 μ g/mL) of IBN-132-3. FIG. 1B shows colony forming units of Candida albicans after 24 hours or 48 hours of treatment with different concentrations (μ g/mL) of IBN-132-3(T), amphotericin B (A), and fluconazole (F). Candida albicans grown in yeast mold broth was used as a control. Circles indicate that no colonies were observed. Data are presented as mean ± standard deviation of triplicates. P < 0.05; p < 0.01.

FIG. 2

FIG. 2 shows the resistance acquisition against Staphylococcus aureus (FIG. 2A) and Candida albicans (FIG. 2B) in the presence of sub-MIC levels of copolymer and antibiotic.

FIG. 3

FIG. 3 is a series of Scanning Electron Microscopy (SEM) images of Candida albicans treated with copolymer (125. mu.g/mL) for 24 hours at room temperature. FIG. 3A is a control, FIG. 3B is IBN-131-2, FIG. 3C is IBN-132-3, and FIG. 3D is IBN-212. The cell wall is destroyed after exposure. Candida albicans treated with YMB was used as a control. The scale bar represents 5 μm.

FIG. 4

FIG. 4 shows a range of antimicrobial activities of the copolymers against biofilms. Cell viability maintained in biofilms of Candida albicans (FIG. 4A), Escherichia coli (FIG. 4B), Staphylococcus aureus (FIG. 4C) and Pseudomonas aeruginosa (FIG. 4D) treated with different copolymers for 24 hours. Data are expressed as mean ± standard deviation of quadruplicate. Statistical significance relative to control, P < 0.05; p < 0.01.

Examples

Non-limiting examples of the present invention will be described in further detail by reference to specific examples, which should not be construed as limiting the scope of the invention in any way.

List of abbreviations used

ACN: acetonitrile

AcOEt: ethyl acetate

AcOH: acetic acid

NH₄Cl: ammonium chloride

AUC: area under curve

Saline water: saturated aqueous NaCl solution

bs: wide signal (broad peak)¹H NMR

At cat: catalyst and process for preparing same

Cs₂CO₃: cesium carbonate

CH₂Cl₂Or DCM: methylene chloride (Methylene chloride or Dichloromethane)

DABCO: 1, 4-diazabicyclo [2.2.2] -octane

DBU: 1, 8-diazabicycloundec-7-enes

DI: deionized water

DMF: n, N-dimethylformamide

DMSO, DMSO: dimethyl sulfoxide

DMSO-d₆: deuterated dimethyl sulfoxide

DMP: 1, 4-dimethylpiperazine

Ether: ether (A)

EtOH: ethanol

H: hour(s)

HPLC: high pressure liquid chromatography

IPA: isopropanol (2-propanol)

KOH: potassium hydroxide

L: lifting of wine

LC-MS: liquid chromatography-mass spectrometry

Me: methyl radical

MeOH: methanol

m.p.: melting Point

min: minute (min)

MS: mass spectrometry

Et₃N: triethylamine

Na₂CO₃: sodium carbonate

NaHCO₃: sodium bicarbonate

NaH: sodium hydride

NaOH: sodium hydroxide

Na₂SO₄: sodium sulfate

NMR: nuclear magnetic resonance

PBS: phosphate buffered saline

Rt: at room temperature

TEA: triethylamine

THF: tetrahydrofuran (THF)

TLC: thin layer chromatography

TMED: tetramethylethane-1, 2-diamine

Materials and methods

All anhydrous solvents were purchased from Sigma-Aldrich (st.louis, Missouri, u.s.a.) and used without further purification. All other reagents were used as received unless otherwise noted in the experimental text below.

Analytical Thin Layer Chromatography (TLC) was carried out using Merck 60F-254 silica gel plates, by UV light (254nm) and/or with 20% KMwO₄w/v in H₂The plates were heated to visualize after staining with the solution in O. Flash column chromatography was performed on Kieselgel 60(0.040-0.063mm) supplied by Merck (Burlington, Massachusetts, u.s.a.).

Recording on a Bruker AV-400(400MHz) spectrometer¹H and¹³c Nuclear Magnetic Resonance (NMR) spectrum. Chemical shifts (δ) are reported in parts per million (ppm) with the residual solvent peak of tetramethylsilane used as an internal standard at 0.00 ppm. Reported in the following order¹Chemical shift, multiplicities (br ═ broad, s ═ singlet, D ═ doublet, t ═ triplet, q ═ quartet and M ═ multiplet), integrals and assignments the molecular weights of the polymers and copolymers were determined using a Waters 2695 Gel Permeation Chromatograph (GPC) equipped with two serially connected ultrahydrogel columns (300mm × 7.8.8 mm) and a Waters 2414 differential refractometer detector (Waters, Massachusetts, u.s.a.). the details of the mobile phases used were as follows: 54/23/23 (v/v/v%) 0.5M aqueous sodium acetate/methanol/acetic acid, flow rate 1.0 mL/min. a series of poly (ethylene glycol) standards of different molecular weights (633: 20,600) (Polymer Standard, Rhode Island, u.s.a.) were used for calibration and the Mw curves were subsequently calculated from the calibration D.

Example 1

Structural design:

by incorporating bis-quaternary ammonium components into the polyimidazole

The copolymers are synthesized in the chain to enhance structural diversity and achieve optimal antibacterial/antifungal activity. Here, the selection includes 1, 4-diazabicyclo [2.2.2]Three α, omega-tertiary diamines of octane (DABCO), tetramethylethane-1, 2-diamine (TMED) and 1, 4-Dimethylpiperazine (DMP) to form bis-quaternary ammonium diamines and diimidazoles randomly linked by benzyl or allyl dihalide compounds (butene or xylylene linkers, scheme 1)And (6) connecting. By utilizing the structural design, various ammonium-imidazoles are synthesized

A random copolymer.

Scheme 1. atactic ammonium-imidazoles

And (3) structural design of the copolymer.

Synthesis of diimidazoles

Scheme 2. Synthesis procedure for diimidazoles

1, 4-bis (N-imidazol-1-ylmethyl) benzoic acid (1 a.) NaOH (0.5g,12.5mmol) was added to a solution of imidazole (0.9g,13.0mmol) in Dimethylsulfoxide (DMSO), and the resulting suspension was stirred at 90 ℃ for 2 hours, α' -dichloro-p-xylene (0.99g,5.7mmol) was added to the residue, the resulting solution was stirred at 40 ℃ for 1 hour, the solvent was removed under vacuum, the product was extracted with Dichloromethane (DCM), and 1a was obtained in quantitative yield after removal of the solvent.¹H NMR(CDCl₃)：δ7.55(s,2H),7.13(s,4H),7.10(s,2H),6.89(s,2H),5.12(s,4H)。MS(GC-MS)m/z238(M⁺)。

1, 2-bis (N-imidazol-1-ylmethyl) benzene (1b) the desired specified product (1b) was synthesized in quantitative yield using α '-dichloro-o-xylene instead of α' -dichloro-p-xylene in the reaction.

Example 2

Polymer and ammonium-imidazole

Synthesis of copolymer

General procedure for the Synthesis of polymers and copolymers

By mixing diimidazole αThe omega-diamine and dihalide linker are mixed in a polar organic solvent (e.g. THF and DMF) at 70 to 90 ℃ for at least 24 hours to effect the ammonium-imidazole-linker

And (3) synthesizing a copolymer. For each copolymer, moderate to good yields of 49% to 95% were obtained. Six copolymer samples (IBN-111, IBN-112, IBN-121, IBN-122, IBN-131 and IBN-132) with different linkers were synthesized using DABCO as the diamine (scheme 3). In addition, the amount of DABCO to diimidazole ratio in the selected copolymers (IBN-131 and IBN-132) was adjusted to achieve higher antimicrobial activity.

An example of a specific procedure for the synthesis of copolymer IBN-132-3(THF) is provided below:

DABCO (0.336g,3.0mmol) and 1b (0.238g,1.0mmol) were dissolved in 5mL THF then a solution of

trans

1,4 dibromo-2-butene (0.856g,4.0mmol) in DMF (5mL) was added dropwise at room temperature after the addition was complete, the reaction mixture was heated to 70 ℃ and stirred for 24 hours the copolymer was obtained by centrifugation (5000rpm, 5 minutes) and washed with acetone (3 × 15mL) to give a white powder in 85% yield.

1, 4-diazabicyclo [2.2.2] s are also synthesized using methods similar to those described above]Octane (DABCO) alone (IBN-110, IBN-120 and IBN-130). Individual polymers (IBN-110, IBN-120 and IBN-130) were used as comparative polymers for ammonium-imidazole

The copolymers were tested.

The remaining polymers (IBN-110, IBN-120 and IBN-130) and copolymers (IBN-111, IBN-112, IBN-121, IBN-122, IBN-131 and IBN-132) can be synthesized according to the respective diamine, diimidazole and aryl/alkyl linkers as shown in the final compound and molar ratios as described in the following scheme (scheme 3).

Scheme 3. DABCO-based polymers and DABCO-imidazoles

The structure of the copolymer.

IBN-110：¹H NMR(400MHz,DMSO-d₆)：δ7.56-7.80(m,4nH,PhH),4.50-5.08(m,4nH,DABCO-CH₂-Ph),2.94-4.19(d,12nH,DABCO N⁺-CH₂-)。

IBN-111：¹H NMR(400MHz,DMSO-d₆)：δ9.30-9.73(m,2nH,Im C2H),6.90-7.94(m,16nH,Im C4H,Im C5H,PhH),5.18-5.62(m,8nH,Ph-CH₂-Im),4.52-4.96(m,4nH,Ph-CH₂-DABCO),2.93-3.97(m,12nH,DABCO N⁺-CH₂-)。

IBN-112：¹H NMR(400MHz,DMSO-d₆)：δ9.19-9.68(m,2nH,Im C2H),6.91-7.94(m,16nH,Im C4H,Im C5H,PhH),5.34-5.75(m,8nH,Ph-CH₂-Im),4.50-5.13(m,4nH,Ph-CH₂-DABCO),2.93-3.99(m,12nH,DABCO N⁺-CH₂-)。

IBN-120：¹H NMR(400MHz,DMSO-d₆)：δ7.40-8.10(m,4nH,PhH),4.67-5.69(m,4nH,DABCO-CH₂-Ph),3.09-4.11(m,12nH,DABCO N⁺-CH₂-)。

IBN-121：¹H NMR(400MHz,DMSO-d₆)：δ9.56-9.86(m,2nH,Im C2H),6.99-8.04(m,16nH,Im C4H,Im C5H,PhH),5.42-5.77(m,8nH,Ph-CH₂-Im),4.92-5.28(m,4nH,Ph-CH₂-DABCO),2.98-4.06(m,12nH,DABCO N⁺-CH₂-)。

IBN-122：¹H NMR(400MHz,DMSO-d₆)：δ9.53-9.88(m,2nH,Im C2H),7.01-7.93(m,16nH,Im-C4H,Im-C5H,PhH),4.62-5.88(m,12nH,Ph-CH₂-Im,Ph-CH₂-DABCO),2.98-4.06(m,12nH,DABCO-N⁺-CH₂-)。

IBN-130：¹H NMR(400MHz,DMSO-d₆)：δ6.16-6.50(m,2nH,-CH₂-CH＝CH-CH₂-),4.30-4.64(s,4nH,-CH₂-CH＝CH-CH₂-),3.00-4.11(m,12nH,DABCO N⁺-CH₂-)。

IBN-131-2：¹H NMR(400MHz,DMSO-d₆)：δ9.61-9.68(m,2nH,Im C2H),7.86-7.99(m,4nH,Im C4H,Im C5H),7.56(m,4nH,PhH),6.00-6.44(m,4nH,-CH＝CH-),5.53-5.57(m,4nH,Ph-CH₂-Im),4.39-5.04(m,8nH,-CH₂-C＝C-CH₂-),3.09-4.14(m,12nH,DABCO N⁺-CH₂). The NMR data of the copolymers IBN-131-1 and IBN-131-3 were identical to that of IBN-131-2.

IBN-132-3：¹H NMR(400MHz,DMSO-d₆): δ 9.16-9.54(m,2nH, Im C2H),7.64-8.06(m,4nH, Im C4H, Im C5H),7.33-7.49(m,4nH, PhH),6.93-7.12(d, Im-C4H, Im C5H terminal), 6.02-6.41(m,8nH, -CH ═ CH-),5.33-5.79(m,4nH, Ph-CH ═ CH-),5.33-5.79₂-Im),4.37-5.06(m,16nH,-CH₂-C＝C-CH₂-),3.08-4.10(m,36nH,DABCO N⁺-CH₂-). The NMR data for the copolymers IBN-132-1 and IBN-132-2 were identical to IBN-132-3.

For the second set of copolymers, tetramethylethane-1, 2-diamine (TMED) was used as diamine and the same general procedure as described above was used and based on the molar ratios and respective diamine, diimidazole and aryl linkers as shown in the following scheme (scheme 4), copolymers IBN-211 and IBN-212 with p-xylylene linkers were obtained. When an o-xylylene or trans-butene linker is used, instead two small molecules (IBN-220 and IBN-230) are formed.

Scheme 4. TMED-based polymers, Small molecules and TMED-imidazoles

The structure of the copolymer.

IBN-210：¹H NMR(400MHz,DMSO-d₆)：δ7.71-7.97(m,4nH,PhH),4.89(s,4nH,Ph-CH₂-TMED),4.34(s,4nH,N⁺-CH₂-CH₂-N⁺),3.10-3.27(d,12nH,N⁺-CH₃)。

IBN-211：¹H NMR(400Hz,DMSO-d₆)：δ9.51-9.65(m,2nH,Im C2H),6.94-7.87(m,16nH,Im C4H,Im C5H,PhH),5.20-5.58(m,8nH,Ph-CH₂-Im),4.89(s,4nH,Ph-CH₂-TMED),4.34(s,4nH,N⁺-CH₂-CH₂-N⁺),3.08-3.25(d,12nH,N⁺-CH₃)。

IBN-212：¹H NMR(400Hz,DMSO-d₆)：δ9.56-9.59(m,2nH,Im C2H),6.90-7.98(m,16nH,Im C4H,Im C5H,PhH),5.38-5.74(m,8nH,Ph-CH₂-Im),4.89(s,4nH,Ph-CH₂-TMED),4.34(s,4nH,N⁺-CH₂-CH₂-N⁺),3.09-3.27(m,12nH,N⁺-CH₃)。

1, 4-Dimethylpiperazine (DMP) is less reactive than DABCO and TMED. Thus, when L1 is ortho-phenylene, no polymer is formed. According to the general procedure as described above, and based on the molar ratios as shown in the following scheme (scheme 5) and the respective diamine, diimidazole and aryl linkers, polymers with p-xylylene and trans-butene linkers were obtained. However, their yields are lower than if they had analogues of DABCO or TMED diamine.

Scheme 5 DMP-based polymers and ammonium-imidazoles

The structure of the copolymer.

IBN-310：¹H NMR(400MHz,D₂O)：δ7.50-7.90(m,4nH,PhH),4.85-5.03(m,4nH,Ph-CH₂-DMP),3.51-4.23(m,8nH,N⁺-CH₂-CH₂-N⁺),3.06-3.30(m,6nH,N⁺-CH₃)。

IBN-330：¹H NMR(400MHz,D₂O)：δ6.63(d,2nH,,-CH＝CH-),4.52(d,

4nH,-CH₂-C＝C-CH₂-),3.81-4.34(s,8nH,N⁺-CH₂-CH₂-N⁺),3.19-3.40(m,6nH,N⁺-CH₃)。

Example 3 molecular weight of polymers and copolymers

Molecular weight determination

Molecular weights of polymers and copolymers were determined using a Waters 2695 Gel Permeation Chromatograph (GPC) equipped with two serially connected ultra hydrogel columns (300mm × 7.8.8 mm) and a Waters 2414 differential refractometer detector (Massachusetts, U.S.A..) the details of the mobile phase used were 54/23/23 (v/v/v%) 0.5M aqueous sodium acetate/methanol/acetic acid at a flow rate of 1.0mL/min, calibration was performed using a series of poly (ethylene glycol) standards (Polymer Standard Service, Rhode Island, U.S.A.) of different molecular weights (633-20,600), then Mw and D were calculated from the calibration curves.

The molecular weights of the polymers and copolymers were measured using Gel Permeation Chromatography (GPC). Like many step-growth polymers, these compounds are characterized by a relatively low degree of polymerization (M)_w< 10,000) and high dispersibility values (1.3 < D < 3.1) (Table 1). The distribution of molecular weight is influenced by the solubility of the polymer in the solvent. Generally, the solubility of the polymer in DMF is higher than in THF due to the higher polarity of DMF. Therefore, most polymers synthesized in DMF have a higher molecular weight.

TABLE 1 molecular weights of polymers and copolymers.

Example 4 antimicrobial and antifungal Studies

Minimum inhibitory concentration

Staphylococcus aureus (ATCC 6538, gram positive), Escherichia coli (ATCC 8739, gram negative), Pseudomonas aeruginosa (ATCC 9027, gram negative) and Candida albicans (ATCC 10231, fungus) were used as representative microorganisms to stimulate imidazole

The antimicrobial function of the salt. All bacteria and fungi were stored frozen at-80 ℃ and in Mueller Hinton broth (MHB, BD Sing) before the experimentapore) at 37 ℃ overnight, fungi were grown in yeast mold broth (YMB, BD Singapore) at 22 ℃ overnight, sub-samples of these cultures were grown for an additional 3 hours and diluted to give an optical density (O.D.) value at 600nm of 0.07, which corresponds to 3 × 10 for bacteria⁸CFU mL^-1And 10 of fungi⁶CFU mL^-1(McFarland's Standard 1; confirmation by plate count).

The polymer was added at 4mg mL^-1Dissolved in MHB or YMB and the Minimum Inhibitory Concentration (MIC) was determined by microdilution assay in each well of a 96-well plate, a bacterial solution (100. mu.L, 3 × 10)⁸CFU mL^-1) With 100. mu.L of polymer solution (typically at 4mg mL)^-1To 2. mu.g mL^-1Range, serial two-fold dilution). The plate was incubated at 37 ℃ for 24 hours at a constant shaking speed of 300 rpm. Except that the fungal solution is about 10 in YMB⁶CFU mL^-1And MIC measurements against candida albicans were similar to bacteria, except plates were incubated at room temperature.

The minimum inhibitory concentration is considered to be the concentration of antimicrobial oligomer/polymer where less than 50% of microbial growth is observed with a microplate reader (TECAN). The medium solution containing only microbial cells was used as a control (100% microbial growth). The assay was performed in 4 replicates and the experiment was repeated at least twice.

Antifungal Activity and Minimum Fungicidal Concentration (MFC)

IBN-132-3(THF) was dissolved in YMB (2. mu.g/mL to 62. mu.g/mL, serial two-fold dilutions). One hundred microliters of each solution was placed in a 96-well microplate. One hundred microliters of candida albicans suspension (10) was then added⁶CFU/ml) was added to each well. Fungi grown in YMB were used as controls. For MFC, the antibiotics amphotericin B and fluconazole were also tested as positive controls. The 96-well plate was kept at room temperature in an incubator under constant shaking. After the desired period of incubation, the respective cell suspensions (100 μ Ι _ were collected, serially diluted at 1:10, and 100 μ Ι _ of each dilution was plated on two nutrient agar plates (Luria-Bertani broth with 1.5% agar). After 48 hours incubation, Colony Forming Units (CFU) were countedNumber, and calculate CFU/mL accordingly.

Table 2. antimicrobial activity of the control under the conditions of the present study.

Resistance study

Drug resistance was induced by repeated treatments of staphylococcus aureus or candida albicans with the copolymer and control antibiotics. First, the MIC of the test copolymer against Staphylococcus aureus or Candida albicans was determined by broth microdilution. Serial passages (sub-MIC of staphylococcus aureus at this passage is 1/2 for MIC and sub-MIC of candida albicans at this passage is 1/8 for MIC) were then initiated by transferring suspensions of the microorganisms grown at sub-MIC of the copolymer for another MIC determination. After 24 hours incubation, cells grown at sub-MIC of test compound/antibiotic were again transferred and MIC was determined. MIC test against staphylococcus aureus or candida albicans for 15 passages.

Drug resistance behavior was assessed by recording changes normalized to the MIC at the first passage. The conventional antibiotic amphotericin B was used as a control against candida albicans and Norfloxacin (Norfloxacin) was used as a control against staphylococcus aureus.

In vitro preparation of biofilms

Fungal Candida albicans cells were cultured in YMB at room temperature. Bacterial cells were cultured in MHB at 37 ℃. All microorganisms were grown overnight to reach mid-log phase of growth. The concentration of the microorganism was adjusted so that the O.D. value at 600nm was 0.07. The bacterial solution is then diluted 10³Multiple to achieve 3 × 10⁵Initial load of CFU/mL, and Candida albicans cells used without further dilution 100. mu.L of the microbial solution was added to each well of a 96 flat bottom well plate and the fungus was incubated at room temperature and the bacteria at 37 ℃ under constant shaking after 24 hours, the medium in each well was discarded and 100. mu.L of 3 × 10 was added to the wells⁵CFU/mL fresh microbial suspension. This process was repeated daily for 7 days to allow biofilm formation. For candida albicans, after 24 hours, fresh medium without fungi was added to replace the microbial solution. Further incubation was continued for 2 days. After incubation for 7 or 3 days, the biofilm formed in the wells was washed three times with Phosphate Buffered Saline (PBS) before further analysis.

Biofilm sensitivity assay using XTT reduction assay

Using (2-methoxy-4-nitro-5-sulfo-phenol) -2H-tetrazole

-5-formanilide (XTT) reduction assay to quantify viable microorganisms on the surface of each well by measuring mitochondrial enzyme activity in viable cells. In this assay, mitochondrial dehydrogenase of live microbial cells reduces XTT to an orange formazan derivative, and changes in o.d. readings are recorded to analyze the viability of cells on the surface. Polymer/copolymer solutions (100 μ L, different concentrations) were added to the wells containing the biofilm. Antibiotic solution and pure medium were used as controls. After 24 hours of incubation, wells were washed once with PBS and then submerged in a mixed solution of 100. mu.L PBS, 10. mu.L XTT (1mg/mL) and 2. mu.L menadione (0.4 mM). After incubation for 4 hours at 37 ℃, the absorbance of the sample at 490nm was measured using a microplate reader with 600nm as the reference wavelength. Using the formula [ (OD)_490nm-OD_600nm)_{Polymer and method of making same}]/[(OD_490nm-OD_600nm)_Control]× 100% relative cell viability was calculated the data are expressed as mean ± standard deviation of 4 replicates per concentration.

Hemolysis study

Fresh rat Red Blood Cells (RBCs) were diluted with PBS buffer to give RBC stock suspensions (4% by volume of blood cells). An aliquot of 100. mu.L of RBC suspension was mixed with 100. mu.L of a copolymer solution (4mg mL-¹To 2. mu.g mL-¹Serial two-fold dilutions in PBS) were mixed. After incubation at 37 ℃ for 1 hour, the mixture was centrifuged at 2000rpm for 5 minutes. An aliquot (100 μ L) of the supernatant was transferred to a 96-well plate. Determination as a function of hemoglobin release by measuring the absorbance of the supernatant at 576nm using a microplate readerHemolytic activity. Control solution containing only PBS was used as reference for 0% hemolysis. The absorbance of erythrocytes lysed with 0.5% Triton-X was taken as 100% hemolysis. Data are expressed as mean and standard deviation of four replicates and the test is repeated twice.

Hemolysis [% OD [ ]_{576nm (Polymer)}-OD_576nm(PBS)]/[OD_{576nm(Triton-X)}-OD_576nm(PBS)]×100％

Scanning Electron Microscopy (SEM) Observation

Candida albicans cells grown in YMB without or with 125. mu.g/mL copolymer for 24 hours were collected (10)⁶CFU/mL) and centrifuged at 3000rpm for 5 minutes. The pellet was washed twice with PBS buffer. The samples were then fixed with glutaraldehyde (2.5%) for 4 hours, followed by washing with Deionized (DI) water. Dehydration was performed using a series of ethanol/water solutions (35%, 50%, 75%, 90%, 95% and 100%). The dehydrated sample was fixed on a copper tape. After 2 days of drying, the samples were further coated with platinum for imaging with a JEOL JSM-7400f (japan) field emission scanning electron microscope operating with an accelerating voltage at 3 keV.

Statistical analysis

Data are expressed as mean ± standard deviation of mean (s.d. indicated by error bar). Student's t-test was used to determine significance between groups. Differences of p < 0.05 were considered statistically significant.

Example 5 DABCO-imidazole

Copolymer

DABCO-imidazoles

Antimicrobial activity of the copolymer

Evaluation of DABCO-imidazole

Antimicrobial activity of the copolymer against four different and clinically relevant microorganisms: staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicansAnd (5) carrying out pearl fungus. Their Minimum Inhibitory Concentrations (MIC) against the four microorganisms are presented in table 3. All DABCO polymers and DABCO-imidazoles

The copolymers all exhibit antimicrobial activity against the tested microorganisms. Interestingly, the structure of the linker affects the antimicrobial activity. In general, polymers and copolymers containing trans-butenyl linkers are the most active materials. Polymers and copolymers having an ortho-xylyl linker are more reactive than those having a para-xylyl linker. Thus, the order of antimicrobial activity of the polymer is trans-butene linker > o-xylylene linker > p-xylylene linker.

DABCO-imidazoles with trans-butene linkers

The copolymers showed excellent antifungal activity (table 3). MICs for Candida albicans were all less than 10. mu.g/mL. They are compared with DABCO polymer IBN-130 or imidazole

Polymer PIM-45 was more effective, meaning when DABCO and imidazole were used

When combined together, have a synergistic effect. The copolymers synthesized in THF, which generally have lower molecular weights than the polymers synthesized in DMF, show higher antifungal activity and lower toxicity (table 3, entries 15 to 20). In particular, IBN-132-3(DABCO with diimidazole) synthesized in THF

The ratio of (3) is the most active antifungal compound, with a MIC of 2. mu.g/mL. No significant hemolysis was observed at the highest concentration of IBN-132-3 of 2000. mu.g/mL.

Table 3 Minimum Inhibitory Concentrations (MIC) of DABCO-based polymers and copolymers.^a

^aFor about 3 × 10⁸CFU/mL bacteria or 10⁶MIC of CFU/mL fungal test. E.C. (escherichia coli), s.a. (staphylococcus aureus), p.a. (pseudomonas aeruginosa), c.a. (candida albicans), F.S. (fusarium solani).

^bTo 10⁵MIC of CFU/mL bacterial assay.

The killing efficacy against candida albicans after 48 hours treatment with different concentrations of IBN-132-3(THF) is shown in fig. 1A. IBN-132-3 inhibited the growth of Candida albicans at low concentrations, even below MIC. At half MIC (1. mu.g/mL), Candida albicans grew slower than the control. As the concentration increased, the inhibition increased and a 3 log reduction was observed at the 8 μ g/mL concentration. The antifungal activity of IBN-132-3 was compared to two conventional antibiotics currently used in clinical therapy (amphotericin B (AmB)) and fluconazole (FIG. 1B and Table 4). The growth of candida albicans in the presence of fluconazole was not significantly different from the untreated control, which means that fluconazole was not active against our test strain. The MIC of IBN-132-3 was 2. mu.g/mL, which was lower than that of AmB (4. mu.g/mL for 24 hours treatment). Although the Minimum Fungicidal Concentration (MFC) (16. mu.g/mL) for 24 hours of AmB treatment was lower than IBN-132-3 (62. mu.g/mL), the concentration of Candida albicans increased after 48 hours due to the poor stability of amphotericin B.

TABLE 4 comparison of MIC and MFC between IBN-132-3 and antibiotics.

Example 6-TMED-imidazole

And DMP-imidazoles

Copolymer

TMED-imidazoles

Copolymers and DMP-imidazoles

Antimicrobial activity of the copolymer

The MIC of the copolymers with TMED or DMP units was also tested and the results are shown in tables 5 and 6. Both the obtained polymers and copolymers show good antimicrobial activity. Two small molecules (IBN-220 and IBN-230) were inactive.

TABLE 5 Minimum Inhibitory Concentration (MIC) for TMED-based polymers and copolymers.^a

^bTo 10⁵MIC of CFU/mL bacterial assay.

TABLE 6 Minimum Inhibitory Concentration (MIC) of DMP-based polymers/copolymers^a

^bTo 10⁵MIC of CFU/mL bacterial assay.

Example 7

Study of drug resistance

By serial passage of staphylococcus aureus or candida albicans treated at sub-MIC levels for each polymer,the possibility of bacterial/fungal cells developing resistance after repeated exposure to the copolymer was investigated. MIC values were measured after each passage. For comparison, antibiotics (norfloxacin and amphotericin B) were also tested. As shown in fig. 2A, the MIC of norfloxacin against staphylococcus aureus increased at passage 4. By passage 15, the MIC value for norfloxacin increased to 124-fold the original MIC value. In contrast, the MICs of IBN-131-2, IBN-132-3 and IBN-212(DMF) remained unchanged throughout the 15 passages. The results show that bacteria are paired with DABCO-imidazole, compared to norfloxacin

Copolymer and TMED-imidazole

The copolymer has a much lower tendency to develop resistance.

Resistance to antifungal agents is much less studied than resistance to bacteria. However, the current increase in fungal infections has intensified the search for innovative, safer and more effective agents against fungal infections. As shown in FIG. 2B, the MICs of IBN-131-2, IBN-132-3 and IBN-212 for Candida albicans remained constant for 15 passages of sub-MIC dose treatment, indicating that Candida albicans failed to develop resistance to the copolymer on consecutive 15 days of treatment. The MIC of amphotericin B increased at passage 4 and three-fold at passage 5, indicating that resistance can be induced when the fungus is repeatedly treated with amphotericin B.

Scanning Electron Microscopy (SEM) Observation

The antifungal mechanism was studied by visualizing typical cell structures with or without treatment via Scanning Electron Microscopy (SEM). The morphological changes of candida albicans after treatment with the copolymer are shown in fig. 3. The cell wall of the copolymer-treated candida albicans was disrupted compared to the control intact cell wall (fig. 3A), and lysed after 24 hours exposure (fig. 3B-3D). Given that the copolymer acts through an association mechanism, it requires a proper balance of hydrophobic and hydrophilic regions to kill the fungus. The copolymer integrates outside the cell, resulting in membrane destabilization and lysis. This membrane cleavage mechanism may be responsible for the reduced efficacy of drug resistance.

Biofilm sensitivity

Individual organisms in biofilms are embedded in a matrix of slimy extracellular polymers and often exhibit very different phenotypes from planktonic cells. In particular, the bacteria/fungi in biofilms are much more resistant to antimicrobial agents than their planktonic counterparts. Therefore, drug therapy for biofilms is sometimes ineffective. Candida biofilms have been reported to be resistant to several clinically important antifungal agents, including amphotericin B and fluconazole. Herein, the activity of the copolymers against biofilms was also tested.

As shown in fig. 4, all the copolymers tested exhibited strong antifungal and antimicrobial activity against candida albicans (fig. 4A) biofilms and three bacteria [ e. For example, 80% of Candida albicans was eradicated within 24 hours after a single treatment with 31. mu.g/mL IBN-132-3. In contrast, candida albicans treated with the same concentration of amphotericin B showed no significant difference compared to the untreated control. Similarly, 87% of E.coli were eradicated within 24 hours after a single treatment with 125. mu.g/mL of IBN-212 (FIG. 4B). The results demonstrate that the copolymer is effective against biofilms.

The above examples show that these ammonium-imidazoles

The copolymers have excellent antimicrobial activity against a wide range of microorganisms and related biofilms, and may have the necessary degradability and non-resistance. Ammonium-imidazoles containing trans butenyl linkers

Copolymers are probably the most reactive materials. Ammonium-imidazoles can be tuned by using different ratios of monomers and linkers with different hydrophobicity and flexibility

Activity of the copolymer.

INDUSTRIAL APPLICABILITY

The polymer as defined above may be used as a composition associated with a carrier or a pharmaceutical composition associated with a pharmaceutically acceptable carrier. The polymer or composition as defined above may be used as a non-therapeutic agent for killing or inhibiting the growth of microorganisms. Polymers as defined above are useful in many applications due to their ability to inhibit microbial growth.

Polymers or pharmaceutical compositions as defined above may be used in a number of applications due to their ability to inhibit the growth of microorganisms, or for the treatment of microbial infections or diseases, wherein the polymer or the pharmaceutical composition is administered to a subject. The polymer or pharmaceutical composition as defined above may be used to kill or inhibit the growth of microorganisms. The polymer or pharmaceutical composition as defined above may be used for the preparation of a medicament for killing or inhibiting the growth of microorganisms.

The polymer or pharmaceutical composition as defined above may be used for the treatment of microbial infections or diseases. The polymer or pharmaceutical composition as defined above may also be used as an antibiotic. The polymer or pharmaceutical composition as defined above may be used for the preparation of a medicament for the treatment of a microbial infection or disease. The microbial infection or disease may be caused by a microorganism selected from the group consisting of: bacteria, archaea, fungi, protists, animals, plants, or any mixture thereof.

The polymer as defined above may exhibit antimicrobial activity against a test microorganism (bacteria), such as staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa. The polymer as defined above may exhibit antifungal activity against fungal species (e.g., candida albicans and fusarium solani).

Novel ammonium-imidazoles as defined hereinbefore

The copolymer may have a tunable degradation profile (profile) under different conditions, which will be eliminated in agriculture and in the environmentHas a wide range of applications in toxicity. Bacteria and fungi may have shown a lower tendency to develop resistance to the copolymer than conventional antibiotics. Among them, DABCO-imidazole

The copolymer exhibits excellent antifungal activity and biocompatibility. Advantageously, these copolymers are easy to synthesize and relatively low cost, having wide application in medical, agricultural and environmental disinfection. The copolymers are also useful in topical wound treatment, as preservatives or disinfectants for consumer care and personal care products.

It will be apparent that various other modifications and improvements of this invention will be apparent to those skilled in the art upon reading the foregoing disclosure and it is intended that all such modifications and improvements be within the scope of the appended claims.

Claims

1. A polymer, or a salt or hydrate thereof, having the following formula (I):

wherein

x is independently selected from halogen;

n, m and p are independently integers of at least 1;

q is 0 or an integer of at least 1;

a has the following structure:

x and y are independently integers of at least 1.

2. The polymer of claim 1, wherein n and m are independently integers from 1 to 50.

3. The polymer of claim 1, wherein x and y are independently integers from 1 to 5.

4. The polymer of claim 3, wherein x and y are independently 1.

5. The polymer of any of the preceding claims, wherein R¹、R²、R³And R⁴Together form at least one C₂To C₅A bridging group.

6. The polymer of claim 5, wherein R¹And R²Together form a vinylidene group.

7. The polymer of claim 6, wherein R³And R⁴Together form a vinylidene group.

8. The polymer of any one of claims 5 to 7, wherein A is selected from the group consisting of the following structures:

9. the polymer of any of the preceding claims, wherein A and L¹In the range of 1:5 to 5: 1.

10. The polymer of any of the preceding claims wherein (L)¹+L²) And L³In the range of about 1:5 to about 5: 1.

11. The polymer of any of the preceding claims, wherein the polymer has a molecular weight in the range of about 1000 to about 10,000.

12. The polymer of any of the preceding claims, wherein the polymer has a polydispersity value in the range of about 1.2 to about 3.2.

13. The polymer of any of the preceding claims, having the following formula (II):

wherein

L⁴And L⁵Independently selected from optionally substituted alkenyl or optionally substituted aryl.

14. The polymer of claim 13, wherein L⁴And L⁵Independently selected from the group consisting of ortho-phenylene, para-phenylene, meta-phenylene, and vinylene.

15. The polymer of any of the preceding claims, wherein X is Cl or Br.

16. The polymer of any one of claims 13 to 15, wherein the moiety in formula (II)

Selected from the group consisting of:

17. the polymer of any of the preceding claims, wherein p and q are independently integers from 1 to 5.

18. The polymer of claim 17, wherein p and q are independently 1.

19. The polymer of any one of the preceding claims, selected from the group consisting of:

20. a composition comprising the polymer of any one of the preceding claims, or a salt or hydrate thereof, and a carrier.

21. Use of a polymer according to any one of claims 1 to 19 or a composition according to claim 20 as a non-therapeutic agent for killing or inhibiting the growth of microorganisms.

22. A pharmaceutical composition comprising the polymer of any one of claims 1-19, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

23. A method for killing or inhibiting the growth of a microorganism, the method comprising administering to a subject the polymer of any one of claims 1-19 or the pharmaceutical composition of claim 22.

24. The polymer of any one of claims 1 to 19 or the pharmaceutical composition of claim 22 for use in killing or inhibiting the growth of a microorganism.

25. Use of a polymer according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 22 in the manufacture of a medicament for killing or inhibiting the growth of a microorganism.

26. The method, polymer, pharmaceutical composition or use according to any one of claims 21 or 23 to 25, wherein the microorganism is a bacterium, archaebacteria, fungus, protist, animal, plant or any mixture thereof.

27. A method for treating a microbial infection, the method comprising administering to a subject a polymer according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 22.

28. The polymer of any one of claims 1 to 19, or the pharmaceutical composition of claim 22, for use as an antibiotic.

29. Use of a polymer according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 22 in the manufacture of a medicament for the treatment of a microbial infection.

30. The method, polymer, pharmaceutical composition or use according to any one of claims 27 to 29, wherein the microbial infection is caused by a bacterium, archaea, fungus, protist, animal, plant or any mixture thereof.

31. A process for preparing the polymer of any one of claims 13 to 19, comprising the steps of:

reacting a diamine having the structure:

with a diimidazole having the structure:

and a dihalide having the structure:

under reaction conditions.

32. The method of claim 31, wherein the diamine and the diimidazole are contacted at a molar ratio in the range of about 1:5 to about 5: 1.

33. The process as set forth in claim 31 wherein the diimidazole and dihalide are contacted in a molar ratio in the range of about 1:6 to about 5: 6.

34. The method of any one of claims 31-33, wherein the reaction is carried out in a polar organic solvent, wherein the polar solvent is Tetrahydrofuran (THF), Dimethylformamide (DMF), or any mixture thereof.

35. The method of any one of claims 31-34, wherein the reaction is conducted at a temperature in the range of about 60 ℃ to about 100 ℃.

36. The method of any one of claims 31-35, wherein the reaction is carried out for at least 18 hours.