WO2024129836A1 - Biscationic quaternary phosphonium compounds as soft antimicrobial agents - Google Patents

Biscationic quaternary phosphonium compounds as soft antimicrobial agents Download PDF

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Publication number
WO2024129836A1
WO2024129836A1 PCT/US2023/083797 US2023083797W WO2024129836A1 WO 2024129836 A1 WO2024129836 A1 WO 2024129836A1 US 2023083797 W US2023083797 W US 2023083797W WO 2024129836 A1 WO2024129836 A1 WO 2024129836A1
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compound
alkyl
group
formula
antimicrobial composition
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PCT/US2023/083797
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French (fr)
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Kevin MINBIOLE
William WUEST
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Villanova University
Emory University
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Publication of WO2024129836A1 publication Critical patent/WO2024129836A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
    • A01N57/22—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds containing aromatic radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/54—Quaternary phosphonium compounds
    • C07F9/5449—Polyphosphonium compounds

Definitions

  • the disclosure relates to antimicrobial compositions and related methods generally. More particularly, the disclosed subject matter relates to a biscationic quaternary phosphonium compound, a composition comprising the biscationic quaternary phosphonium compound, the methods of making the same, and the methods of using the same as antimicrobial agents.
  • BAC benzalkonium chloride
  • QACs act by electrostatically adhering to bacterial membranes via their cationic head and subsequently disrupting the membrane with the insertion of their lipophilic tail. This class of disinfectants has enjoyed tremendous success and usage in a wide number of antimicrobial applications.
  • bacteria are increasingly displaying resistance to known QACs, taking advantage of a multitude of resistance mechanisms, including upregulation of antimicrobial efflux pumps, alterations to the bacterial membrane, and enzymatic degradation of QACs.
  • Notable pathogens such as A. baumannii and P. aeruginosa have developed sufficient resistance mechanisms towards common disinfectants that they are renewing the problem of bacterial infections in hospital, domestic and commercial settings.
  • the present disclosure provides a biscationic quaternary phosphonium compound, an antimicrobial composition comprising such a compound, the method of making such a compound, the method of making such an antimicrobial composition, and the methods of using such a compound or composition for antimicrobial use.
  • the biscationic quaternary phosphonium compound comprises amide substitution.
  • the compound or the composition provided in the disclosure has an ability to kill or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, to attenuate the severity of a microbial infection, or to prevent or inhibit formation of a biofilm or eradicate pre-established biofilms (i.e. antibiofilm use).
  • an antimicrobial composition comprising a compound having the formula wherein:
  • Li and L2 each is a linking group selected from the group consisting of a C1-10 alkyl, a C1-10 cycloalkyl, an aryl, and any combination thereof
  • each of Ri, R2, R3, and R4 is selected from the group consisting of a C1-12 alkyl, a C1-12 cycloalkyl, and an aryl group, which is unsubstituted or optionally substituted with a functional group selected from the group consisting of -OH, -OR’, -NH2, -NHR’, -NR’ 2, -SH, -SR’, -O-C(O)R’, -C(O)OR’, -C(O)R’, -C(O)NR’R’, -CF3, -OCF3, halogen, and any combination thereof;
  • R’ is H or a Ci-4 alkyl; each of Rs, R6, R7, and Rs is H or a C1-20 alkyl, wherein at least one of Rs and Re is a C1-20 alkyl and at least one of R7 and Rs is a C1-20 alkyl,
  • the present disclosure provides a composition or a product described herein.
  • the antimicrobial composition is a disinfectant configured to be applied to a surface of an object in need thereof.
  • the antimicrobial composition is a pharmaceutical composition configured to be administrated to a subject in need thereof.
  • the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an active ingredient.
  • the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an additive for stabilizing the pharmaceutical composition.
  • the present disclosure provides a compound as described herein, a method of making such a compound, and a method of using such a compound.
  • the present disclosure provides a method of making the antimicrobial composition as described herein.
  • a method comprises preparing the compound having the formula (I) or (II) as described.
  • the method may further comprise mixing an effective amount of the compound having the formula (I) or (II) and a carrier.
  • the present disclosure also provides different products as described herein, which comprise the antimicrobial composition.
  • the present disclosure provides a method of killing, preventing, or inhibiting microbial growth.
  • the method comprises applying the antimicrobial composition to a surface of an object in need thereof.
  • such a method comprises administrating the antimicrobial composition to a subject in need thereof.
  • the antimicrobial composition or the compound is used to kill, prevent, or inhibit growth of at least one group of microorganisms such as bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm, or disperse or eradicate a pre-established biofilm.
  • the compound and the composition provided in the disclosure have significant advantages. They have excellent abilities to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, and can attenuate the severity of a microbial infection, and can also prevent or inhibit formation of a biofilm or eradicate pre-established biofilms.
  • a biscationic quaternary phosphonium compound comprising amide substitution has good stability, particularly in neutral and mildly acidic conditions.
  • Such a compound undergoes hydrolysis under basic condition and decomposes at the phosphonium center, leading to inactive phosphine oxide products. So the compounds and the compositions are environmental-friendly and can be easily degraded through a process such as water treatment.
  • FIG. 1 shows existing ester- and amide-containing antimicrobial quaternary ammonium compounds (QACs).
  • FIG. 2 shows some two types of exemplary compounds in accordance with some embodiments.
  • FIG. 3 shows ORTEP (Oak Ridge Thermal Ellipsoid Plot) diagrams showing the molecular structure of an exemplary compound, P3P-8A,8A, which is provided in accordance with some embodiment, as determined by x-ray diffraction.
  • ORTEP Oil Ridge Thermal Ellipsoid Plot
  • FIGS. 4A-4D show the profiles of an exemplary compound, P3P-9A, 9A, together with sodium hypophosphate pentahydrate as an internal standard (IS), monitored by 31 P NMR (nuclear magnetic resonance) spectroscopy at different pH conditions for different periods of time to show whether or not the exemplary compound undergoes decomposition.
  • FIGS. 4A, 4B, 4C, and 4D show the results at pH of 4, 6, 7, and 10, respectively.
  • FIGS. 5A-5D show the profiles of an exemplary compound, P6P-9A, 9A, together with sodium hypophosphate pentahydrate as an internal standard (IS), monitored by 31 P NMR (nuclear magnetic resonance) spectroscopy at different pH conditions for different periods of time to show whether or not the exemplary compound undergoes decomposition.
  • FIGS. A, 5B, 5C, and 5D show the results at pH of 4, 6, 7, and 10, respectively.
  • FIG. 6A illustrates the change in bioactivity before and after decomposition, from P3P-12A,12A to hydrolysis product, P3P-oxide.
  • the internal standard (IS) used was sodium hypophosphate pentahydrate.
  • the internal standard (IS) used was sodium hypophosphate pentahydrate.
  • FIG. 8 shows the species in HRMS (ESI+) total ion chromatograms of extracted P3P-12A,12A and P3P-12A,12A decomposition products.
  • FIG. 9 illustrates a proposed water-treatment plant breakdown strategy using the soft QPCs provided in the present disclosure and related chemical reactions verified by the experimental results of 31 P NMR (nuclear magnetic resonance).
  • the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” It is intended that any component, element, attribute, or step that is positively recited herein may be explicitly excluded in the claims, whether such components, elements, attributes, or steps are listed as alternatives or whether they are recited in isolation.
  • antimicrobial refers to an ability to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, or to attenuate the severity of a microbial infection.
  • the antimicrobial compounds or compositions of the present invention are compounds or compositions that may be used for cleaning or sterilization, or may be used in the treatment of disease and infection.
  • the applications may include both in vitro and in vivo antimicrobial uses.
  • Applying” an antimicrobial composition may include administrating a composition into a human or animal subject.
  • biofilm refers to a film formed by a group of microorganisms adhered together.
  • antibiofilm refers to an ability to kill, disperse and/or eradicate a pre-established biofilm.
  • alkyl refers to a straight chain, cyclic, branched or unbranched saturated or unsaturated hydrocarbon chain containing 1-25 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n- septyl, n-octyl, n-nonyl, cyclo-hexyl, and the like.
  • the alkyl may be straight or branched, and can be optionally substituted.
  • a C1-12 alkyl refers to an alkyl group having a number of carbon atoms selected from 1 to 12. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,” respectively).
  • Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1 -pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2- butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3- methyl- 1-butynyl, and the like.
  • aryl refers to a group that contains any carbon-based aromatic group including, but not limited to, phenyl, benzyl, and the like. The aryl can be optionally substituted.
  • optionally substituted means that group in question may be unsubstituted or it may be substituted one or several times, such as 1 to 3 times or 1 to 5 times.
  • an alkyl group that is “optionally substituted” with 1 to 5 chloro atoms may be unsubstituted, or it may contain 1, 2, 3, 4, or 5 chlorine atoms.
  • Substituted chemical moieties include one or more substituents that replace hydrogen.
  • microorganism refers to a small (often, but not always, microscopic) organism that is typically, but not exclusively, single cellular, and includes organisms from the kingdoms bacteria, archaea, protozoa, and fungi.
  • the present disclosure is primarily directed to microorganisms that are pathogenic and capable of causing disease. Such microbes may be detected from a sample.
  • microorganism includes bacteria and fungi capable of causing disease, particularly disease in humans and other mammals and animals in need of treatment.
  • object refers to any article having a solid surface.
  • An object may also include a body part of a subject.
  • a composition to be applied to a surface of an object may be in a form of liquid or aerosol.
  • a “subject” refers any animal, preferably a human being such as a patient, livestock, rodent, monkey, or domestic pet. In some embodiments, the subject may be exhibiting symptoms of, at risk of, or diagnosed with a disease or condition by analysis of a sample.
  • sample can refer to a tissue sample, cell sample, a fluid sample, and the like.
  • a sample may be taken from a host subject.
  • the tissue sample can include hair, buccal swabs, blood, saliva, semen, muscle, or tissue from any internal organ.
  • the fluid may be, but is not limited to, urine, blood, ascites, pleural fluid, spinal fluid, semen, wound exudates, sputum, fecal matter, saliva, and the like.
  • the body tissue can include, but is not limited to, skin, muscle, endometrial, uterine, and cervical tissue.
  • a sample in the context of the present disclosure, is primarily a biological sample (e.g., from a living host) the sample may also be an environmental sample suspected of contamination by microbes, such as a water sample, food sample, soil sample, and the like.
  • a liquid sample and some solid samples may be used as a test sample without modification for testing directly, if a solid sample is to be made into liquid form for testing and/or a liquid sample is to be diluted, a test sample may be made by reconstituting, dissolving, or diluting the sample in a fluid such as water, buffered saline, and the like.
  • the terms “administration” or “administrating” is understood to encompass any type of application to a subject including in vitro or in vivo administrations.
  • the types of administration include, are not limited to, topical, oral, parenteral, rectal, intramuscular injection, inhalation, intrathecal, sublingual, nasal, transdermal, and any combination thereof.
  • the composition to be administrated may be in a form of a solid dose form such as a tablet or a capsule, a liquid form, or an aerosol.
  • the terms “prevent” and “preventing” include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity is reduced. [0046] As used herein, the terms “treat” and “treating” are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.
  • ⁇ ективное amount refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below.
  • the therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will induce a particular response in target cells, e.g., reduction of platelet adhesion and/or cell migration.
  • the specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
  • linking group refers to any variety of molecular arrangements that can be used to bridge molecular moieties together.
  • this disclosure contemplates derivatives of compounds disclosed herein.
  • the term “derivative” refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue.
  • the derivative may be structurally similar because it is lacking one or more atoms, substituted, a salt, or alternative salt, an ester, in different hydration/oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur atom, or replacing an amino group with a hydroxy group.
  • Example substituents may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, amine, and ester.
  • cell culture or “growth medium” or “media” refers to a composition that contains components that facilitate cell maintenance and growth through protein biosynthesis, such as vitamins, amino acids, inorganic salts, a buffer, and a fuel, e.g., acetate, succinate, a saccharide/disaccharide/polysaccharide, medium chain fatty acids, and/or optionally nucleotides.
  • a fuel e.g., acetate, succinate, a saccharide/disaccharide/polysaccharide, medium chain fatty acids, and/or optionally nucleotides.
  • Typical components in a growth medium include amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and others); vitamins such as retinol, carotene, thiamine, riboflavin, niacin, biotin, folate, and ascorbic acid; carbohydrate such as glucose, galactose, fructose, or maltose; inorganic salts such as sodium, calcium, iron, potassium, magnesium, zinc; serum; and buffering agents. Additionally, a growth medium may contain phenol red as a pH indication.
  • MIC minimum inhibitory concentration
  • bacteria for example, the Gram-positive Staphylococcus aureus and Enterococcus faecalis and the Gram-negative Escherichia coli and Pseudomonas aeruginosa SNQYQ determined by standard methods. See also P.A. Wayne, Methods for Dilution Antimicrobial Tests for Bacteria that Grow Aerobically; Approved Standard, Ninth Edition, 2012, CLSI Document M07-A9, Vol. 32, No. 2.
  • the minimum biofilm eradication concentration (MBEC)” of a compound is defined as the lowest concentration of compound dosed against a previously established bacterial biofilm that leads to a clear well (optical density of less than 0.1) when the treated biofilm is regrown in fresh media, indicating >95% clearance of bacteria.
  • a regrowth assay was used to establish the MBEC of a compound to evaluate the antibiofilm activity.
  • Examples of abbreviations used in the present disclosure include: QAC, quaternary ammonium compound; QPC, quaternary phosphonium compound; AMR, antimicrobial resistance; BAC, benzalkonium chloride; WWTP, wastewater treatment plant; NMR, nuclear magnetic resonance; dppp, l,3-bis(diphenylphosphino)propane; dpph, 1,6- bis(diphenylphosphino)hexane; MSSA, methicillin-susceptible Staphylococcus aureus, CA- MRSA, community-acquired methicillin-resistant Staphylococcus aureus,' HA-MRSA, hospital- acquired methicillin-resistant Staphylococcus aureus, MIC, minimum inhibitory concentration; RBC, red blood cell; TSB, tryptic soy broth; OD, Optical density; MHB, Mueller-Hinton broth; DMSO, dimethyl sulfoxide; and PBS,
  • the present disclosure provides a biscationic quaternary phosphonium compound, an antimicrobial composition comprising such a compound, the method of making such a compound, the method of making such an antimicrobial composition, and the methods of using such a compound or composition for antimicrobial use.
  • the compound or the composition provided in the disclosure has an ability to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, to attenuate the severity of a microbial infection, or to prevent or inhibit formation of a biofilm or eradicate pre- established biofilms (i.e. antibiofilm use).
  • Quaternary ammonium compounds serve as a first line of defense against infectious pathogens. As resistance to QACs emerges in the environment, the development of next-generation disinfectants is of utmost priority for human health. Balancing antibacterial potency with environmental considerations is required to effectively counter the development of bacterial resistance.
  • bisQPCs biscationic quaternary phosphonium compounds
  • These compounds termed soft QPCs, comprise hydrolyzable amide moi eties in their side chains to promote decomposition under environmental conditions. Strong bioactivity against a panel of bacterial pathogens such as seven bacterial pathogens as described herein was observed, highlighted by single-digit micromolar activity for compounds P6P-12A,12A and P3P-12A,12A.
  • One objective of the present disclosure was to develop novel antimicrobial agents that balance strong protection against emerging pathogens, while presenting degradability during environmental exposure.
  • FIG. 2 show some exemplary compounds in accordance with some embodiments.
  • the present disclosure provides a biscationic quaternary phosphonium compound with amide substitution, an antimicrobial composition comprising such a compound, and the method of making such an antimicrobial compound or composition, and the method of using such a compound or composition for antimicrobial use.
  • the compound or the composition provided in the disclosure has an ability to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, or to attenuate the severity of a microbial infection.
  • the compound or the composition provided in the disclosure has an ability to inhibit or eradicate pre-established biofilms (i.e. antibiofilm use) formed by the microorganisms.
  • an antimicrobial composition comprising a compound having the formula wherein:
  • Li and L2 each is a linking group selected from the group consisting of a C1-10 alkyl, a C1-10 cycloalkyl, an aryl, and any combination thereof
  • each of Ri, R2, R3, and R4 is selected from the group consisting of a C1-12 alkyl, a C1-12 cycloalkyl, and an aryl group, which is unsubstituted or optionally substituted with a functional group selected from the group consisting of -OH, -OR’, -NH2, -NHR’, -NR’ 2, -SH, -SR’, -O-C(O)R’, -C(O)OR’, -C(O)R’, -C(O)NR’R’, -CF3, -OCF3, halogen, and any combination thereof;
  • R’ is H or a C1-4 alkyl; each of Rs, Re, R7, and Rs is H or a C1-20 alkyl, wherein at least one of Rs and Re is a C1-20 alkyl and at least one of R7 and Rs is a C1-20 alkyl,
  • X is used for illustration only. It can be monovalent anions or monovalent negative ions such as fluoride ion (F"), chloride ion (Cl"), bromide ion (Br ), and iodide (I ). X may also represent multi -valent anions such as carbonate and sulfate [0066]
  • the present disclosure provides a composition or a product described herein.
  • the antimicrobial composition is a disinfectant configured to be applied to a surface of an object in need thereof.
  • the antimicrobial composition is a pharmaceutical composition configured to be administrated to a subject in need thereof.
  • the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an active ingredient.
  • the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an additive for stabilizing the pharmaceutical composition.
  • Li is a C2- 10 alkyl.
  • Li is an alternating structure comprising alkyl and cycloalkyl, for example, an alternating structure comprising a C2-10 alkyl and a C4-6 cycloalkyl.
  • alkyl for the linking groups can be understood as alkylene group, which have two connecting bonds.
  • Li comprises an aryl group such as a phenyl group, or a combination of an aryl group and one or more alkyl.
  • Li may be a combination of - alkyl-Ph-alkyl-.
  • L2 may be a C1-6 alkyl in some embodiments.
  • the linking group Li or L2 is a C1-6 alkyl such as -CH2-, - CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, or- CH2CH2CH2CH2CH2CH2-.
  • Ri, R2, R3, or R4 each is a C1-6 alkyl or a cyclohexyl such as cyclohexyl, or a phenyl group.
  • Ri, R2, R3, and R4 are the same, Rs and R7 are the same, and Rs and Rs are the same.
  • the compound has a symmetrical structure.
  • Rs and R7 are the same and are an alkyl having a formula CnH 2n+i, wherein n is an integer in the range of from 2 to 20, and Rs and Rs are the same and are H or methyl.
  • the compound having the formula (I) has a formula:
  • Ri, R2, R3, and R4 are a same group R, which may be selected from a C1-6 alkyl, a C1-6 cycloalkyl, and phenyl.
  • the value of n can be in a range of 2 to 20, for example, from 6 to 16, from 2 to 12, from 4 to 12, from 6 to 12, from 8 to 12, from 10 to 12, or any other suitable ranges. In some embodiments, n is in a range of from 5 to 12. Li is a
  • C2-10 alkyl and L2 is a C1-6 alkyl.
  • X is any suitable group for anions.
  • the value of m depends on the valence of the anions. For example, when X a halogen, a tosylate, an acetate, or a hydrocarbonate, y is 1, and m is 2. When X is a carbonate or a sulfate, y is 2, and m is 1. In some embodiments, X is a halogen selected from F, Cl, Br, and I, and m is 2.
  • the present disclosure provides a compound as described herein, a method of making such a compound, and a method of using such a compound.
  • Examples of a suitable compound include, but are not limited to the compounds from Cl to C 14 and related analogs as shown below: and analogs of Compounds Cl to C 14 wherein -Ph is replaced with methyl, ethyl, or cyclohexyl.
  • each of these compounds comprises 2 Br , as counter ions.
  • exemplary compounds made include at least two series of compounds, each of which has a formula being :
  • Li is a C3 alkyl or a Ce alkyl, respectively, and L2 is -CH2-.
  • the compounds having the formula (V) or (VI) are abbreviated in a format of P3P-#A, #A or P6P-#A,#A, respectively.
  • the numbers of “3” and “6” represents the number of the carbon atoms in the linking group Li.
  • the number sign (#) represents the sum of n, the number of carbon atoms in the linking group L2 plus two (corresponding to the amide group).
  • the letter “A” represents amide.
  • Ri, R2, Rt, and R4 are the same (R), and are phenyl.
  • Compound (Cl) having the following structure: is coded as P3P-8A,8A or ph P3P-8A,8A, or ph P3P-8A,8A, Cl.
  • the present disclosure provides a method of making the antimicrobial composition as described herein.
  • a method comprises preparing the compound having the formula (I) or (II) as described.
  • the compound having the formula (I) can be synthesized as illustrated in Scheme 1.
  • the compound having the formula (I) can be prepared by the alkylation reaction of a suitable bisphosphine starting compound having the formula (VII) with a suitable chemical having L2 linking group and amide function groups.
  • a suitable electrophile bearing a suitable group such as an alkyl, or a cycloalkyl, or a phenyl backbone as well as a leaving group, can be alkylated under substitution conditions, which can optimize solvent conditions and temperature; two equivalents of the electrophile would be utilized.
  • the method may further comprise mixing an effective amount of the compound having the formula (I) or (II) and a carrier.
  • a suitable carrier may include, but are not limited to, a solvent, a carrier, an additive, any other suitable ingredient, or combinations thereof.
  • the present disclosure also provides an antimicrobial composition
  • an antimicrobial composition comprising a compound having the formula (I) or (II) as described, and a carrier such as a solvent.
  • the antimicrobial composition can also comprise other ingredients and additives.
  • the content of the compound having the formula (I) or (II) can be in any suitable concentration.
  • such a concentration can be in the range from 0.01 pM to 100 pM, for example, from 0.1 pM to 10 pM.
  • the content of the compound having the formula (I) or (II) may be at a concentration of from 0.1 wt.% to 5 wt.
  • % for example, in the range of from 0.2 wt.% to 2.5 wt. %.
  • the carrier include but are not limited to a solvent.
  • other additives include but are not limited to surfactants, anti-foaming agents, anti-freezing agents, gelling agents, and combinations thereof.
  • the antimicrobial composition may also comprise a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient suitable for a solid preparation such as tablets or capsules can be, for example, binders (e.g., acacia, gelatin, dextrin, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone), solvents, dispersion media, diluents (e.g., lactose, sucrose, mannitol, com starch, potato starch, calcium phosphate, calcium citrate, crystalline cellulose), lubricants (e.g., magnesium stearate, calcium stearate, stearic acid, talc, anhydrous silicic acid), disintegrants (e.g., corn starch, potato starch, carboxymethylcellulose, carboxymethylcellulose calcium, alginic acid), and wetting agents (e.g., sodium laurylsulfate).
  • binders e.g., a
  • a pharmaceutically acceptable carrier or excipient suitable for a liquid preparation can be, for example, aqueous vehicles (e.g., water), suspending agents (e.g., acacia, gelatin, methyl cellulose, carboxymethylcellulose sodium, hydroxymethyl-cellulose, aluminum stearate gel), surfactants (e.g., lecithin, sorbitan monooleate, glycerin monostearate), and non-aqueous vehicles (e.g., glycerin, propylene glycol, vegetable oil).
  • aqueous vehicles e.g., water
  • suspending agents e.g., acacia, gelatin, methyl cellulose, carboxymethylcellulose sodium, hydroxymethyl-cellulose, aluminum stearate gel
  • surfactants e.g., lecithin, sorbitan monooleate, glycerin monostearate
  • non-aqueous vehicles e.g., glycerin, propylene glycol, vegetable oil
  • liquid preparations may contain preservatives (e.g., p-hydroxybenzoic acid methyl ester, p-hydroxybenzoic acid propyl ester), flavors, and/or coloring agents.
  • preservatives e.g., p-hydroxybenzoic acid methyl ester, p-hydroxybenzoic acid propyl ester
  • flavors e.g., p-hydroxybenzoic acid propyl ester
  • coloring agents e.g., p-hydroxybenzoic acid methyl ester, p-hydroxybenzoic acid propyl ester
  • the antimicrobial composition in this disclosure can be formulated to be in any suitable form, including but not limited to liquid, gel and paste.
  • the present disclosure also provides different products as described herein, which comprise the antimicrobial composition.
  • the present disclosure provides a method of killing, preventing, or inhibiting microbial growth.
  • the method comprises applying the antimicrobial composition to a surface of an object in need thereof.
  • a suitable method include but are not limited to pouring, spraying, any other suitable methods and any combinations thereof.
  • such a method comprises administrating the antimicrobial composition to a subject in need thereof.
  • the method of administration may include in vitro or in vivo administration.
  • the types of administration include, are not limited to, topical, oral, parenteral, rectal, intramuscular injection, inhalation, intrathecal, sublingual, nasal, transdermal, and any combination thereof.
  • the antimicrobial composition or the compound is used to kill, prevent, or inhibit growth of at least one group of microorganisms such as bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm, or disperse or eradicate a pre-established biofilm.
  • this disclosure relates to methods of treating or preventing diseases or conditions comprising administering an effective amount of a composition comprising the quaternary phosphonium compound disclosed herein to a subject in need thereof.
  • the disclosure contemplates that a quaternary phosphonium compound disclosed herein may be used in antimicrobial applications optionally in combination with other antimicrobial agents for prevention of disease onset and treatment.
  • the quaternary phosphonium compound disclosed herein may be used in medical device coatings (medical implants and tools, IV catheters), wound dressings (embedded in gauze bandages), wound rinses (i.e. surgical rinses), wound-vacuum systems, whole body baths (e.g., in combo with bleach baths for treatment of skin flares for atopic dermatitis/eczema), soaps, personal care products (body washes, lotions, soaps) for high-risk patients or for populations with high risk of exposure (e.g. athletes using common sports equipment in gym), and veterinary applications (e.g.
  • this disclosure relates to methods of treating or preventing a microbial infection comprising administering to a subject in need thereof an effective amount of a quaternary phosphonium compound as disclosed herein.
  • the microbial infection is a bacterial, fungal, pest, or viral infection.
  • this disclosure relates to methods of treating or preventing bacterial infections comprising administering or contacting the skin of a subject with formula comprising a quaternary phosphonium compound as disclosed herein to a subject in need thereof.
  • the formula is administered in combination with another antibiotic agent.
  • this disclosure provides a method of using quaternary phosphonium compounds disclosed herein for treating or preventing an Acinetobacter baumannii infection, other bacterial infection, other multidrug resistant bacteria, or other microbial infection by administering an effective amount of quaternary phosphonium compounds disclosed herein to a subject in need thereof.
  • the quaternary phosphonium compound is co-administered with an antibiotic selected from the group comprising of sulfonamides, diaminopyrimidines, quinolones, beta-lactam antibiotics, cephalosporins, tetracyclines, nitrobenzene derivatives, aminoglycosides, macrolide antibiotics, polypeptide antibiotics, nitrofuran derivatives, nitroimidazoles, nicotinic acid derivatives, polyene antibiotics, imidazole derivatives or glycopeptide, cyclic lipopeptides, glycylcyclines and oxazolidinones.
  • an antibiotic selected from the group comprising of sulfonamides, diaminopyrimidines, quinolones, beta-lactam antibiotics, cephalosporins, tetracyclines, nitrobenzene derivatives, aminoglycosides, macrolide antibiotics, polypeptide antibiotics, nitrofur
  • these antibiotics include but are not limited to sulphadiazine, sulphones - [dapsone (DDS) and para-aminosalicylic (PAS)], sulfanilamide, sulfamethizole, sulfamethoxazole, sulphapyridine, trimethoprim, pyrimethamine, nalidixic acids, norfloxacin, ciprofloxacin, cinoxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, ofloxacin, pefloxacin, sparfloxacin, trovafloxacin, penicillins (amoxicillin, ampicillin, azlocillin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, hetacillin
  • the subject is diagnosed with a bacterial infection.
  • the subject is diagnosed with bacteremia, pneumonia, staphylococcal food poisoning, necrotizing pneumonia, necrotizing fasciitis, scalded skin syndrome, post-operation bacterial infection, medical device bacterial infection, bacterial infection of the skin, soft tissue bacterial infection, or toxic shock syndrome.
  • this disclosure provides methods of treating or preventing a toxin-mediated bacterial infection comprising administering an effective amount of a quaternary phosphonium compound as disclosed herein to a subject in need thereof, including a subject at risk of, exhibiting symptoms of, or diagnosed with scalded skin syndrome (esp. in neonates), abscesses, necrotizing fasciitis, sepsis, or atopic dermatitis (eczema).
  • scalded skin syndrome esp. in neonates
  • abscesses necrotizing fasciitis
  • sepsis sepsis
  • atopic dermatitis eczema
  • this disclosure provides methods of treating or preventing bacterial infections or acne comprising administering to a subject in need thereof or contacting the skin of a subject in need thereof with a formula comprising of a quaternary phosphonium compound as disclosed herein. In some embodiments, the formula is administered in combination with another antibiotic.
  • the subject is at risk of a bacterial infection due to being diagnosed with an abscess, furuncle, cellulitis, folliculitis, atopic dermatitis, psoriasis, impetigo, septic arthritis, brain abscess, burn wound, venous ulcer, diabetic foot ulcer, surgical wound, carbuncle, or meningitis.
  • a flow of wound rinse/irrigation solution can be applied across an open wound surface to achieve wound hydration, to remove deeper debris, and to assist with the visual examination.
  • the disclosure relates to methods of irrigating a wound using a solution comprising a quaternary phosphonium compound as disclosed herein.
  • this disclosure provides methods of using quaternary phosphonium compounds disclosed herein for killing microbes, preventing or inhibiting microbe growth, preventing a biofdm formation, or preventing the spread of an Acinetobacter baumannii infection, other bacterial infection, other multidrug resistant bacteria, or other microbial infection by sanitizing a surface, e.g., by contacting the surface with a solid (e.g., powder), liquid, or spray composition, with a quaternary phosphonium compound disclosed herein in an effective amount.
  • this disclosure provides methods of preventing cellular infections comprising applying a quaternary phosphonium compound disclosed herein on top of or inside a cell growth medium.
  • this disclosure provides methods of preventing plant microbial infections comprising applying a quaternary phosphonium compound disclosed herein to the exterior, leaf, seed, or stem of a plant. In some embodiments, this disclosure provides methods of preventing plant microbial infections comprising applying a quaternary phosphonium compound disclosed herein on top of or into soil, dirt, sand, or other medium from which roots of the plant reside.
  • compositions and devices comprising a quaternary phosphonium compound disclosed herein.
  • this disclosure provides soaps and disinfectant products comprising a quaternary phosphonium compound disclosed herein.
  • Contemplated topical formulations for skin flares i.e., for atopic dermatitis or other infections related to a disrupted skin barrier
  • another drug such as a topical steroid, anti-inflammatory agent, and promoter of skin barrier function or skin moisturizer.
  • this disclosure provides a container configured to create a liquid spray comprising a quaternary phosphonium compound disclosed herein.
  • this disclosure provides a pharmaceutical formulation comprising a quaternary phosphonium compound disclosed herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical formulation is in the form of a lotion, liquid, or gel.
  • the pharmaceutical formulation is in the form of a particle, bead, tablet, capsule, pill, or injectable solution.
  • the injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as water, dimethyl sulfoxide, mannitol, 1,3 -butanediol, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable non-toxic, parenterally-acceptable diluents or solvents such as water, dimethyl sulfoxide, mannitol, 1,3 -butanediol, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • the pharmaceutical formulation can also include any type of pharmaceutically acceptable excipients, additives, or vehicles.
  • diluents or fillers such as dextrates, dicalcium phosphate, calcium sulphate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose, inositol, powdered sugar, bentonite, microcrystalline cellulose, or hydroxypropyl methylcellulose, may be added to the composition to increase the bulk of the composition.
  • the formulation is a directly compressible composition comprising a quaternary phosphonium compound disclosed herein but no excipients, additives, or vehicles.
  • the disclosure provides a pharmaceutical or cosmetic formulation comprising a quaternary phosphonium compound disclosed herein and a pharmaceutically acceptable excipient or cosmetically acceptable excipient.
  • the disclosure provides a liquid or gel formulation optionally further comprising an antibacterial agent, a topical steroid, an anti-inflammatory agent, a promoter of skin barrier function, a skin moisturizer, or combinations thereof.
  • the antibacterial agent is daptomycin, linezolid, vancomycin, nafcillin, cefazolin, dicloxacillin, clindamycin, rifampin, or sulfamethoxazole-trimethoprim (Bactrim).
  • the disclosure provides a wound dressings or wound rinse comprising a quaternary phosphonium compound disclosed herein wherein the wound dressing comprises an absorbent pad and optionally an adhesive.
  • the disclosure provides disinfectant sprays or wipes formulation for surfaces and fomites comprising a quaternary phosphonium compound disclosed herein.
  • this disclosure provides a medical device coated with a quaternary phosphonium compound as disclosed herein.
  • the medical device is a screw, pin, plate, rod, disk, needle, catheter, tube, stent, pacemaker, defibrillators (ICDs), artificial hip or knee joint/implant, breast implant, intra-uterine device, ear tube, contact lens, or implantable pump.
  • ICDs defibrillators
  • this disclosure provides a surgical tool coated with a quaternary phosphonium compound as disclosed herein.
  • the surgical tool is a forceps, tweezers, scalpel, knife, scissors, retractor, needle, gauze, sponge, suction, staple, stapler, clip, laparoscopic instrument, electrosurgical cauterizer, ultrasonic device, camera, camera lens, fiber optic cable, insufflator, needle, bronchoscope, cystoscope, saw, or robotic arm.
  • the disclosure provides a wound dressing comprising a quaternary phosphonium compound as disclosed herein wherein the wound dress comprises an absorbent pad and optionally an adhesive optionally in combination with another antibiotic agent.
  • the wound dressing is a foam or compression dressing or a cover dressing such as wraps, gauze and tape.
  • the wound dressing comprises alginate or collagen.
  • the wound dressing is a hydrocolloid dressing, e.g., carboxy-methylcellulose and gelatin optionally in a polyurethane foam or film, optionally comprising one or more agents selected from pectin, a polysaccharide, and an adhesive.
  • the wound dressing is a hydrogel.
  • Hydrogels are polymers that contain a high content of hydroxy and/or carboxyl containing monomers or salts thereof, e.g., vinyl alcohol, acrylic acid, 2-hydroxyethylmethacrylate, ethylene glycol dimethacrylate monomers, which are co-polymers to provide varying degrees of hydration. Due to the hydrophilic monomers, the hydrogels typically absorb water.
  • Contemplated hydrogel dressings include: amorphous hydrogel, which are a free-flowing gel that are typically distributed in tubes, foil packets and spray bottles; an impregnated hydrogel, which are typically saturated onto a gauze pad, nonwoven sponge ropes and/or strips; or a sheet hydrogel which are gel held together by a fiber mesh.
  • the disclosure provides a wound rinse comprising a quaternary phosphonium compound as disclosed herein optionally containing normal saline, sterile water, a detergent, a surfactant, a preservative, or iodine.
  • the disclosure provides a kit comprising a container comprising a quaternary phosphonium compound as disclosed herein optionally comprising a second container comprising a rinse solution or containing surgical device or tool, normal saline, sterile water, a detergent, a surfactant, a preservative, iodine, hydrogen peroxide, or sodium hypochlorite or other compound disclosed herein.
  • the disclosure provides a cosmetic formulation comprising a quaternary phosphonium compound as disclosed herein and cosmetically acceptable excipient or additive.
  • the disclosure relates to a solid or liquid soap or lotion comprising a quaternary phosphonium compound as disclosed herein and a fatty acid.
  • additives can be selected from the group consisting of oily bodies, surfactants, emulsifiers, fats, waxes, pearlescent waxes, bodying agents, thickeners, superfatting agents, stabilizers, polymers, silicone compounds, lecithins, phospholipids, biogenic active ingredients, deodorants, antimicrobial agents, antiperspirants, film formers, antidandruff agents, swelling agents, insect repellents, hydrotropes, solubilizers, preservatives, perfume oils and dyes.
  • additives are selected from the group consisting of surfactants, emulsifiers, fats, waxes, stabilizers, deodorants, antiperspirants, antidandruff agents, and perfume oils.
  • this disclosure provides a cell growth medium comprising a quaternary phosphonium compound disclosed herein.
  • the solvent used was chloroform-c/ (CDCI3), using the residual solvent peak as an internal reference of 7.26 ppm for ’H NMR and 77.16 ppm for 13 C NMR.
  • Accurate mass spectrometry data was acquired on an AB Sciex 5600 TripleTOF using electrospray ionization in positive mode.
  • Samples were analyzed using 400 MHz JEOL spectrophotometer, specifically focusing on 31 P NMR (162 Hz Larmor frequency for 31 P NMR).
  • the internal standard was comprised of 2 mg/mL solution of sodium hypophosphite pentahydrate (NaH2PO2*5H2O) in deionized water.
  • NaH2PO2*5H2O sodium hypophosphite pentahydrate
  • the prepared library of compounds was assessed for performance as antibacterial disinfectants by determining the minimum inhibitory concentration (MIC) for each against a panel of bacteria.
  • the bacteria selected for the study were chosen to evaluate the broad spectrum antimicrobial performance of the QPCs, and therefore, included both Gram-positive and Gramnegative bacterial strains, including those with known antimicrobial resistance mechanisms.
  • These bacteria include the Gram-positive strains of methicillin-susceptible Staphylococcus aureus [MSSA; SH1000], community-acquired methicillin-resistant Staphylococcus, aureus [CA-MRSA; USA 300-0114], hospital-acquired methicillin-resistant Staphylococcus aureus [HA-MRSA; ATCC 33591], an Enterococcus faecalis [OG1RF], and the Gram-negative strains of Escherichia coli [MC4100], Acinetobacter baumannii [ATCC 17498] and Pseudomonas aeruginosa [PAO1],
  • 106 CFU/mL and 100 pL were inoculated into each well of a U-bottom 96-well plate containing 100 pL of compound solution. Plates were incubated statically at 37 °C for 48 hours upon which wells were evaluated visually for bacterial growth. The MIC was determined as the lowest concentration of compound resulting in no bacterial growth visible to the naked eye, based on the highest value in three independent experiments. Aqueous DMSO controls were conducted as appropriate for each compound.
  • RBC lysis assays were performed on mechanically defibrinated sheep blood (Hemostat Labs: DSB030). An aliquot of 1.5 mL blood was placed into a microcentrifuge tube and centrifuged at 3,800 rpm for ten minutes. The supernatant was removed, and the cells were resuspended with 1 mL of phosphate-buffered saline (PBS). The suspension was centrifuged as described above, the supernatant was removed, and cells were resuspended 4 additional times in 1 mL PBS. The final cell suspension was diluted twenty -fold with PBS.
  • PBS phosphate-buffered saline
  • Mitochondrial toxicity was evaluated using a Promega Mitochondrial ToxGloTM kit.
  • Human hepatocellular carcinoma cells HepG2
  • RPML1640 medium containing 10% FBS at 37 °C and 5% CO2.
  • Cells were seeded at a density of 2,500 cells/well in 384 well tissue culture plates in either glucose (10 mM) or galactose (10 mM) supplemented media and were incubated overnight to allow for cell adherence. Cells were rinsed and replaced with serum-free media prior to experimentation.
  • the Mitochondrial ToxGloTM assay was performed in accordance with manufacturer instructions.
  • X-ray intensity data for P3P-8A,8A were collected on a Rigaku XtaLAB Synergy-S diffractometer using an HyPix-6000HE HPC area detector.
  • the intensity data were integrated using CrysAlisPro, which produced a listing of unaveraged F 2 and o(F 2 ) values.
  • the structure solution was determined using SHELXT and refinement was conducted using SHELXLwith anisotropic refinement of the thermal parameters for non-hydrogen atoms. Hydrogen atoms were placed using a riding model and refined isotropically.
  • the inventors successfully synthesized the soft QPCs described herein, including 14 amide-based soft QPCs as described above and illustrated in FIG .2, varying the distance between the phosphonium cations and alkyl chain lengths.
  • the amide-bearing lipophilic group was constructed by exposing long-chained amines to chloroacetyl chloride.
  • N-alkyl-2-chloroacetamide building blocks were then reacted with two bisphosphine nucleophiles: l,3-bis(diphenylphosphino)propane (dppp) and 1,6- bis(diphenylphosphino)hexane (dpph), which are readily available.
  • the bisalkylation reactions were successful under standard SN2 conditions (acetonitrile, reflux, 24 h, argon atmosphere), affording the 14 QPCs in good yields (40-98% as shown in Scheme 3). Purification of the crude products was accomplished via trituration with cyclohexane. [0182] As described above, a naming system using a PzP format is used.
  • the number “3” comes from one carbon atoms plus one carbon and one nitrogen in the carbonyl group.
  • the addition of the letter A reflects the inclusion of amide functionality in the chain, resulting in a format of PzP-#A,#A.
  • FIG. 3 shows ORTEP diagrams for P3P-8A,8A (CCDC Refence Number 2196105). Bond lengths for select P-C, N-C, O-C and C-C bonds are labelled in A and the O-C- C and angle for the amide is labeled in degrees. Thermal ellipsoids are shown at the 50% probability level. Hydrogens and co-crystalized solvents are omitted for clarity.
  • FIG. 6A illustrates the change in bioactivity before and after decomposition.
  • FIGS. 6B-7B show the profiles of an exemplary compound, P3P-12A,12A, as monitored by 31 P NMR spectroscopy after exposure at pH of 7 and 10, respectively, for a period of time over 24 hours.
  • FIGS. 7A-7B show the profiles of an exemplary compound, P6P-12A,12A, as monitored by 31 P NMR spectroscopy after exposure at pH of 7 and 10, respectively, for a period of time over 24 hours.
  • both P3P-12A,12A and P6P-12A,12A remained unchanged over a 24-hour period (FIG. 6B and FIG. 7A).
  • both P3P-12A,12A and P6P-12A,12A completely decomposed within 1 h and 5 h respectively (FIG. 6C and FIG. 7B).
  • the species in the HRMS (ESI+) Total Ion Chromatogram of extracted P3P-12A,12A decomposition products include Species (A) having a chemical formula C11H24NCE, with m/z 186.1853 (100.0%), 187.1886 (11.9%); and Species (B) having a chemical formula C27H27O2P2+ with m/z: 445.1481 (100.0%), 446. 1515 (29.2%), 447.1548 (4. 1%).
  • the species in the HRMS (ESI+) Total Ion Chromatogram of extracted P6P-12A,12A decomposition products include Species (A) having a chemical formula Ci 1H24NO with m/z 186.1853 (100.0%), 187.1886 (11.9%); Species (C) having a chemical formula C3oH3302P2 + with m/z: 445.1481 (100.0%), 446.1515 (29.2%), 447.1548 (4.1%).
  • the major phosphorus decomposition product was identified to be a corresponding phosphine oxide as shown in FIG. 6A and as shown in FIG. 8 except that the phosphine oxide is not ionized as tested in mass spectroscopy.
  • Both the P3P- and P6P-phosphine oxides were prepared according to previous literature reports and were confirmed to be a match in both the mass and elution time observed in the LCMS for the decomposition products.
  • the compounds provided in the present disclosure may be used as an disinfectant applied to a surface, or an ingredient or a stabilizer for a pharmaceutical composition.
  • the compounds can be degraded in a water treatment process.
  • the compound and the reaction shown in FIG. 9 are for illustration only, and the uses can be applicable to any compound provided in the present disclosure.
  • the bioactivities of the compounds were assessed via minimum inhibitory concentration (MIC) and hemolysis (lysis20) assays, wherein the latter was used as a proxy for cytotoxicity.
  • MIC minimum inhibitory concentration
  • lysis20 hemolysis assays
  • the inventors previously synthesized best-in-class bisQPC (P6P-10,10) and two commercial QACs (benzalkonium chloride [BAC;
  • the compound P6P-10, 10 has the following chemical structure:
  • the compounds were each screened against a panel of seven bacterial strains, including four gram-positive strains (methicillin-susceptible Staphylococcus aureus [MSSA; SH1000], community-acquired methicillin-resistant Staphylococcus aureus [CA-MRSA; USA 300-0114], hospital-acquired methicillin-resistant Staphylococcus aureus [HA-MRSA; ATCC 33591]), and Enterococcus faecalis [OG1RF], as well as three gram-negative strains (Escherichia coli [MC4100], Acinetobacter baumannii [ATCC 17948], an Pseudomonas aeruginosa [PAO1]).
  • Escherichia coli [MC4100] Acinetobacter baumannii [ATCC 17948], an Pseudomonas aeruginosa [PAO1]
  • RBC red blood cell
  • Table 2 shows antimicrobial activity (MIC) and hemolysis (lysis2o) of the prepared bisQPCs compared to commercially available QACs against gram-positive strains methicillin-susceptible A aureus (MSSA), community-acquired methicillin-resistant S. aureus (CA-MRSA) and hospital-acquired methicillin-resistant 5. aureus (HA-MRSA), and E. faecalis as well as gram-negative strains E. coli, A. baumannii, and P. aeruginosa.
  • MSSA methicillin-susceptible A aureus
  • CA-MRSA community-acquired methicillin-resistant S. aureus
  • H-MRSA hospital-acquired methicillin-resistant 5.
  • E. faecalis E. faecalis as well as gram-negative strains E. coli, A. baumannii, and P. aeruginosa.
  • P6P-12A,12A has improved performance over BAC, particularly against gramnegative pathogens, with 4-16x greater activity.
  • P6P-12A,12A The potency of P6P-12A,12A is narrowly improved compared to DDAC and approaches the performance of the inventors’ best amphiphile prepared to date, P6P-10,10.
  • the top exemplary compounds P3P-12A,12A and P6P-12A, 12A also performed well against resistant gram-positive S. aureus strains as well, with no increase in MIC values for either HA- or CA-MRSA, as compared to MS SA.
  • the lysis2o values of the hydrolysis products i.e., the phosphine oxides of P3P and P6P
  • the hydrolysis products i.e., the phosphine oxides of P3P and P6P
  • the lysis2o values of the hydrolysis products were found to be >125 pM, reflecting only modest toxicity in this assay; antibacterial activity was likewise not observed at concentrations tested.
  • the present disclosure provides the novel phosphonium -based compounds such as a series of 14 exemplary biscationic amphiphilic structures above as soft amphiphiles.
  • Multiple compounds with amide-bearing side chains demonstrated single-digit micromolar antimicrobial activity, highlighted by the most effective compounds in this series, P6P-12A,12A and P3P-12A,12A.
  • the compound and the composition provided in the disclosure have significant advantages. They have excellent abilities to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, and can attenuate the severity of a microbial infection, and can also prevent or inhibit formation of a biofilm or eradicate pre-established biofilms.
  • a biscationic quaternary phosphonium compound comprising amide substitution has good stability, particularly in neutral and mildly acidic conditions.
  • Such a compound undergoes hydrolysis and decomposes at the phosphonium center, leading to inactive phosphine oxide products. So the compounds and the compositions are environmental-friendly and can be easily degraded through a process such as water treatment. The uses of these compounds or compositions will greatly reduce the chance for microorganisms to develop resistance to these compounds.

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Abstract

A compound having formula (I) and an antimicrobial composition comprising a compound having the formula (I) or (II) are provided: L1 and L2 each is a linking group selected from a C1-10 alkyl, a C1-10 cycloalkyl, an aryl, and any combination thereof. Each of R1, R2, R3, and R4 is a C1-12 alkyl, a C1-12 cycloalkyl, or an aryl group, which may be optionally substituted with a functional group such as -OH, -OR', -NH2, - NHR', -NR'2, -SH, -SR', -O-C(O)R', -C(O)OR', -C(O)R', -C(O)NR'R', -CF3, -OCF3, or halogen. R' is H or a C1-4 alkyl. Each of R5, R6, R7, and Rs is H or a C1-20 alkyl, at least one of R5 and R6 is a C1-20 alkyl, and at least one of R7 and R8 is a C1-20 alkyl. X is a halogen, a tosylate, a hydrocarbonate, a carbonate, a sulfate, or an acetate, m and y are integers being 1 or 2, wherein m * y =2.

Description

BISCATIONIC QUATERNARY PHOSPHONIUM COMPOUNDS AS SOFT ANTIMICROBIAL AGENTS
PRIORITY CLAIM AND CROSS-REFERENCE
[0001] This application claims the priority benefit of U.S. Provisional Application No.
63/387,730, filed December 16, 2022, which application is expressly incorporated by reference herein in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under R35 GM119426 awarded by the National Institute of General Medical Sciences, and under CHE- 1827930 and CHE-2018399 by the National Science Foundation. The government has certain rights in the invention.
FIELD OF THE INVENTION
[0003] The disclosure relates to antimicrobial compositions and related methods generally. More particularly, the disclosed subject matter relates to a biscationic quaternary phosphonium compound, a composition comprising the biscationic quaternary phosphonium compound, the methods of making the same, and the methods of using the same as antimicrobial agents.
BACKGROUND
[0004] The relentless emergence of SARS-CoV-2 variants and the looming threat of future pandemics highlights the urgent need to develop potent disinfectants. To address the spread and transmission of SARS-CoV-2, the Center for Disease Control and Prevention (CDC) and Environmental Protection Agency (EP A) have maintained a registry of disinfectants to combat current and future outbreaks. Quaternary ammonium compounds (QACs), which represent the largest share of active agents in the list, have served as effective antiseptics and disinfectants for many decades. [0005] For approximately 100 years, quaternary ammonium compounds (QACs) have been employed as successful tools to mitigate the transfer of pathogenic bacteria. Over much of this time, society has heavily relied on just a handful of these disinfecting amphiphilic compounds, with the most visible commercial disinfectant of this class being benzalkonium chloride (BAC), a mixture of long-chained alkyl dimethylbenzylammonium chlorides.
[0006] QACs act by electrostatically adhering to bacterial membranes via their cationic head and subsequently disrupting the membrane with the insertion of their lipophilic tail. This class of disinfectants has enjoyed tremendous success and usage in a wide number of antimicrobial applications.
[0007] However, bacteria are increasingly displaying resistance to known QACs, taking advantage of a multitude of resistance mechanisms, including upregulation of antimicrobial efflux pumps, alterations to the bacterial membrane, and enzymatic degradation of QACs. Notable pathogens such as A. baumannii and P. aeruginosa have developed sufficient resistance mechanisms towards common disinfectants that they are renewing the problem of bacterial infections in hospital, domestic and commercial settings.
[0008] The paucity of structural diversity amongst the select number of QACs in commercial use has further driven QAC cross-resistance via these mechanisms, attenuating the efficacy of many disinfectants. Moreover, the presence of persistent QACs in the environment at subinhibitory concentrations has been demonstrated to drive the spread of antibiotic resistance, in addition to QAC resistance.
[0009] Concurrent with this rise in bacterial resistance, robust QACs are being shown to negatively impact the environment. During the COVID-19 pandemic, the use of antimicrobial materials rose to unprecedented levels and usage remains elevated. It was reported that 75% of QACs are released into wastewater treatment plants (WWTPs), and the remainder is directly released into the environment. In most cases, WWTPs can remove the bulk of QACs through absorption into activated sludge.
[0010] However, studies show that residual QAC concentrations of 20 - 300 pg/L have been found in surface water, even after treatment. QACs are still found in aquatic environments, especially at higher concentrations when downstream from municipal/industrial wastewater treatment plants and hospitals. The most commonplace QAC, benzalkonium chloride (BAC), possesses an environmental half-life of nine months due to its chemical stability and slow biodegradation. Long-term environmental exposure to QACs has shown growth inhibition and lethal effects in most aquatic organisms. In addition, increased antimicrobial resistance has emerged as these compounds persist in the environment.
[0011] The need for novel antimicrobial agents that balance strong protection against emerging pathogens, while presenting limited stability during environmental exposure, is of paramount importance.
SUMMARY
[0012] This Summary is provided to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.
[0013] The present disclosure provides a biscationic quaternary phosphonium compound, an antimicrobial composition comprising such a compound, the method of making such a compound, the method of making such an antimicrobial composition, and the methods of using such a compound or composition for antimicrobial use. The biscationic quaternary phosphonium compound comprises amide substitution. The compound or the composition provided in the disclosure has an ability to kill or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, to attenuate the severity of a microbial infection, or to prevent or inhibit formation of a biofilm or eradicate pre-established biofilms (i.e. antibiofilm use).
[0014] In accordance with some embodiments, the present disclosure provides an antimicrobial composition comprising a compound having the formula
Figure imgf000005_0001
wherein:
Li and L2 each is a linking group selected from the group consisting of a C1-10 alkyl, a C1-10 cycloalkyl, an aryl, and any combination thereof, each of Ri, R2, R3, and R4 is selected from the group consisting of a C1-12 alkyl, a C1-12 cycloalkyl, and an aryl group, which is unsubstituted or optionally substituted with a functional group selected from the group consisting of -OH, -OR’, -NH2, -NHR’, -NR’ 2, -SH, -SR’, -O-C(O)R’, -C(O)OR’, -C(O)R’, -C(O)NR’R’, -CF3, -OCF3, halogen, and any combination thereof;
R’ is H or a Ci-4 alkyl; each of Rs, R6, R7, and Rs is H or a C1-20 alkyl, wherein at least one of Rs and Re is a C1-20 alkyl and at least one of R7 and Rs is a C1-20 alkyl,
X is selected from the group consisting of a halogen, a tosylate, a hydrocarbonate, a carbonate, a sulfate, and an acetate; and m and y are integers being 1 or 2, wherein m * y =2.
[0015] The present disclosure provides a composition or a product described herein. In some embodiments, the antimicrobial composition is a disinfectant configured to be applied to a surface of an object in need thereof. In some embodiments, the antimicrobial composition is a pharmaceutical composition configured to be administrated to a subject in need thereof. The pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an active ingredient. In some embodiments, the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an additive for stabilizing the pharmaceutical composition.
[0016] In another aspect, the present disclosure provides a compound as described herein, a method of making such a compound, and a method of using such a compound.
[0017] In another aspect, the present disclosure provides a method of making the antimicrobial composition as described herein. In some embodiments, such a method comprises preparing the compound having the formula (I) or (II) as described. The method may further comprise mixing an effective amount of the compound having the formula (I) or (II) and a carrier.
[0018] In another aspect, the present disclosure also provides different products as described herein, which comprise the antimicrobial composition. [0019] In another aspect, the present disclosure provides a method of killing, preventing, or inhibiting microbial growth. For example, in some embodiments, the method comprises applying the antimicrobial composition to a surface of an object in need thereof. In some embodiments, such a method comprises administrating the antimicrobial composition to a subject in need thereof. The antimicrobial composition or the compound is used to kill, prevent, or inhibit growth of at least one group of microorganisms such as bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm, or disperse or eradicate a pre-established biofilm.
[0020] The compound and the composition provided in the disclosure have significant advantages. They have excellent abilities to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, and can attenuate the severity of a microbial infection, and can also prevent or inhibit formation of a biofilm or eradicate pre-established biofilms. Surprisingly, such a biscationic quaternary phosphonium compound comprising amide substitution has good stability, particularly in neutral and mildly acidic conditions. Such a compound undergoes hydrolysis under basic condition and decomposes at the phosphonium center, leading to inactive phosphine oxide products. So the compounds and the compositions are environmental-friendly and can be easily degraded through a process such as water treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Like reference numerals denote like features throughout specification and drawings.
[0022] FIG. 1 shows existing ester- and amide-containing antimicrobial quaternary ammonium compounds (QACs).
[0023] FIG. 2 shows some two types of exemplary compounds in accordance with some embodiments. [0024] FIG. 3 shows ORTEP (Oak Ridge Thermal Ellipsoid Plot) diagrams showing the molecular structure of an exemplary compound, P3P-8A,8A, which is provided in accordance with some embodiment, as determined by x-ray diffraction.
[0025] FIGS. 4A-4D show the profiles of an exemplary compound, P3P-9A, 9A, together with sodium hypophosphate pentahydrate as an internal standard (IS), monitored by 31P NMR (nuclear magnetic resonance) spectroscopy at different pH conditions for different periods of time to show whether or not the exemplary compound undergoes decomposition. FIGS. 4A, 4B, 4C, and 4D show the results at pH of 4, 6, 7, and 10, respectively.
[0026] FIGS. 5A-5D show the profiles of an exemplary compound, P6P-9A, 9A, together with sodium hypophosphate pentahydrate as an internal standard (IS), monitored by 31P NMR (nuclear magnetic resonance) spectroscopy at different pH conditions for different periods of time to show whether or not the exemplary compound undergoes decomposition. FIGS. A, 5B, 5C, and 5D show the results at pH of 4, 6, 7, and 10, respectively.
[0027] FIG. 6A illustrates the change in bioactivity before and after decomposition, from P3P-12A,12A to hydrolysis product, P3P-oxide.
[0028] FIG. 6B shows the profiles of an exemplary compound, P3P-12A,12A, as monitored by 31P NMR spectroscopy after exposure to pH = 7 buffer for a period of time over 24 hours. FIG. 6C shows the profiles of an exemplary compound, P3P-12A,12A, as monitored by 31P NMR spectroscopy after exposure to pH = 10 buffer for a period of time over 24 hours. The internal standard (IS) used was sodium hypophosphate pentahydrate.
[0029] FIG. 7A shows the profiles of an exemplary compound, P6P-12A,12A, as monitored by 31P NMR spectroscopy after exposure to pH = 7 buffer for a period of time over 24 hours. FIG. 7B shows the profiles of an exemplary compound, P6P-12A,12A, as monitored by 31P NMR spectroscopy after exposure to pH = 10 buffer for a period of time over 24 hours. The internal standard (IS) used was sodium hypophosphate pentahydrate.
[0030] FIG. 8 shows the species in HRMS (ESI+) total ion chromatograms of extracted P3P-12A,12A and P3P-12A,12A decomposition products.
[0031 ] FIG. 9 illustrates a proposed water-treatment plant breakdown strategy using the soft QPCs provided in the present disclosure and related chemical reactions verified by the experimental results of 31P NMR (nuclear magnetic resonance). DETAILED DESCRIPTION
[0032] For purposes of the description hereinafter, it is to be understood that the embodiments described below may assume alternative variations and embodiments. It is also to be understood that the specific articles, compositions, and/or processes described herein are exemplary and should not be considered as limiting.
[0033] In the present disclosure, the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” or “a structure” is a reference to one or more of such compounds or structures and equivalents thereof known to those skilled in the art, and so forth.
[0034] When values are expressed as approximations, by use of the antecedent “about” or “approximately,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” preferably refers to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” preferably (but not always) refers to a value of 7.2% to 8.8%, inclusive.
[0035] Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, “2-5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” It is intended that any component, element, attribute, or step that is positively recited herein may be explicitly excluded in the claims, whether such components, elements, attributes, or steps are listed as alternatives or whether they are recited in isolation.
[0036] The term “antimicrobial” refers to an ability to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, or to attenuate the severity of a microbial infection. The antimicrobial compounds or compositions of the present invention are compounds or compositions that may be used for cleaning or sterilization, or may be used in the treatment of disease and infection. The applications may include both in vitro and in vivo antimicrobial uses. “Applying” an antimicrobial composition may include administrating a composition into a human or animal subject.
[0037] The term “biofilm” as used herein refers to a film formed by a group of microorganisms adhered together. The term “antibiofilm” as used herein refers to an ability to kill, disperse and/or eradicate a pre-established biofilm.
[0038] The term “alkyl” as used herein refers to a straight chain, cyclic, branched or unbranched saturated or unsaturated hydrocarbon chain containing 1-25 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n- septyl, n-octyl, n-nonyl, cyclo-hexyl, and the like. The alkyl may be straight or branched, and can be optionally substituted. “A C1-12 alkyl” as used herein refers to an alkyl group having a number of carbon atoms selected from 1 to 12. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,” respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1 -pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2- butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3- methyl- 1-butynyl, and the like.
[0039] The term “aryl” as used herein refers to a group that contains any carbon-based aromatic group including, but not limited to, phenyl, benzyl, and the like. The aryl can be optionally substituted.
[0040] The term “optionally substituted” means that group in question may be unsubstituted or it may be substituted one or several times, such as 1 to 3 times or 1 to 5 times. For example, an alkyl group that is “optionally substituted” with 1 to 5 chloro atoms, may be unsubstituted, or it may contain 1, 2, 3, 4, or 5 chlorine atoms. Substituted chemical moieties include one or more substituents that replace hydrogen.
[0041] The term “microorganism” or “microbe” as used herein refers to a small (often, but not always, microscopic) organism that is typically, but not exclusively, single cellular, and includes organisms from the kingdoms bacteria, archaea, protozoa, and fungi. The present disclosure is primarily directed to microorganisms that are pathogenic and capable of causing disease. Such microbes may be detected from a sample. In embodiments, microorganism includes bacteria and fungi capable of causing disease, particularly disease in humans and other mammals and animals in need of treatment.
[0042] The term “object” used herein refers to any article having a solid surface. An object may also include a body part of a subject. A composition to be applied to a surface of an object may be in a form of liquid or aerosol.
[0043] A “subject” refers any animal, preferably a human being such as a patient, livestock, rodent, monkey, or domestic pet. In some embodiments, the subject may be exhibiting symptoms of, at risk of, or diagnosed with a disease or condition by analysis of a sample. The term “sample” can refer to a tissue sample, cell sample, a fluid sample, and the like. A sample may be taken from a host subject. The tissue sample can include hair, buccal swabs, blood, saliva, semen, muscle, or tissue from any internal organ. The fluid may be, but is not limited to, urine, blood, ascites, pleural fluid, spinal fluid, semen, wound exudates, sputum, fecal matter, saliva, and the like. The body tissue can include, but is not limited to, skin, muscle, endometrial, uterine, and cervical tissue. While a sample, in the context of the present disclosure, is primarily a biological sample (e.g., from a living host) the sample may also be an environmental sample suspected of contamination by microbes, such as a water sample, food sample, soil sample, and the like. Although a liquid sample and some solid samples may be used as a test sample without modification for testing directly, if a solid sample is to be made into liquid form for testing and/or a liquid sample is to be diluted, a test sample may be made by reconstituting, dissolving, or diluting the sample in a fluid such as water, buffered saline, and the like.
[0044] As used herein, the terms “administration” or “administrating” is understood to encompass any type of application to a subject including in vitro or in vivo administrations. For example, the types of administration include, are not limited to, topical, oral, parenteral, rectal, intramuscular injection, inhalation, intrathecal, sublingual, nasal, transdermal, and any combination thereof. The composition to be administrated may be in a form of a solid dose form such as a tablet or a capsule, a liquid form, or an aerosol.
[0045] As used herein, the terms “prevent” and “preventing” include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity is reduced. [0046] As used herein, the terms “treat” and “treating” are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.
[0047] The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below.
The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of platelet adhesion and/or cell migration. The specific dose will vary depending on, for example, the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
[0048] A “linking group” refers to any variety of molecular arrangements that can be used to bridge molecular moieties together.
[0049] In some embodiments, this disclosure contemplates derivatives of compounds disclosed herein. As used herein, the term “derivative” refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, substituted, a salt, or alternative salt, an ester, in different hydration/oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur atom, or replacing an amino group with a hydroxy group.
[0050] The term “substituted” refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are “substituents.” The molecule may be multiply substituted. In the case of an oxo substituent (“=O”), two hydrogen atoms are replaced. Example substituents may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, amine, and ester.
[0051] The terms, “cell culture” or “growth medium” or “media” refers to a composition that contains components that facilitate cell maintenance and growth through protein biosynthesis, such as vitamins, amino acids, inorganic salts, a buffer, and a fuel, e.g., acetate, succinate, a saccharide/disaccharide/polysaccharide, medium chain fatty acids, and/or optionally nucleotides. Typical components in a growth medium include amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and others); vitamins such as retinol, carotene, thiamine, riboflavin, niacin, biotin, folate, and ascorbic acid; carbohydrate such as glucose, galactose, fructose, or maltose; inorganic salts such as sodium, calcium, iron, potassium, magnesium, zinc; serum; and buffering agents. Additionally, a growth medium may contain phenol red as a pH indication.
[0052] The term “minimum inhibitory concentration (MIC)” means the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. MIC values against bacteria, for example, the Gram-positive Staphylococcus aureus and Enterococcus faecalis and the Gram-negative Escherichia coli and Pseudomonas aeruginosa SNQYQ determined by standard methods. See also P.A. Wayne, Methods for Dilution Antimicrobial Tests for Bacteria that Grow Aerobically; Approved Standard, Ninth Edition, 2012, CLSI Document M07-A9, Vol. 32, No. 2.
[0053] The term “the minimum biofilm eradication concentration (MBEC)” of a compound is defined as the lowest concentration of compound dosed against a previously established bacterial biofilm that leads to a clear well (optical density of less than 0.1) when the treated biofilm is regrown in fresh media, indicating >95% clearance of bacteria. A regrowth assay was used to establish the MBEC of a compound to evaluate the antibiofilm activity.
[0054] Examples of abbreviations used in the present disclosure include: QAC, quaternary ammonium compound; QPC, quaternary phosphonium compound; AMR, antimicrobial resistance; BAC, benzalkonium chloride; WWTP, wastewater treatment plant; NMR, nuclear magnetic resonance; dppp, l,3-bis(diphenylphosphino)propane; dpph, 1,6- bis(diphenylphosphino)hexane; MSSA, methicillin-susceptible Staphylococcus aureus, CA- MRSA, community-acquired methicillin-resistant Staphylococcus aureus,' HA-MRSA, hospital- acquired methicillin-resistant Staphylococcus aureus, MIC, minimum inhibitory concentration; RBC, red blood cell; TSB, tryptic soy broth; OD, Optical density; MHB, Mueller-Hinton broth; DMSO, dimethyl sulfoxide; and PBS, phosphate-buffered saline.
[0055] The present disclosure provides a biscationic quaternary phosphonium compound, an antimicrobial composition comprising such a compound, the method of making such a compound, the method of making such an antimicrobial composition, and the methods of using such a compound or composition for antimicrobial use. The compound or the composition provided in the disclosure has an ability to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, to attenuate the severity of a microbial infection, or to prevent or inhibit formation of a biofilm or eradicate pre- established biofilms (i.e. antibiofilm use).
[0056] Quaternary ammonium compounds (QACs) serve as a first line of defense against infectious pathogens. As resistance to QACs emerges in the environment, the development of next-generation disinfectants is of utmost priority for human health. Balancing antibacterial potency with environmental considerations is required to effectively counter the development of bacterial resistance.
[0057] To address this challenge, a series of novel biscationic quaternary phosphonium compounds (bisQPCs) provided in the present disclosure has been prepared as amphiphilic disinfectants through straightforward, high-yielding alkylation reactions. These compounds, termed soft QPCs, comprise hydrolyzable amide moi eties in their side chains to promote decomposition under environmental conditions. Strong bioactivity against a panel of bacterial pathogens such as seven bacterial pathogens as described herein was observed, highlighted by single-digit micromolar activity for compounds P6P-12A,12A and P3P-12A,12A. Hydrolysis experiments in pure water and buffers of varying pH revealed surprising decomposition of the soft QPCs under basic conditions at the phosphonium center, leading to inactive phosphine oxide products; QPC stability (> 24 h) was maintained in neutral solutions. The results of this present disclosure unveil soft QPCs as a potent and environmentally conscious new class of bisQPC disinfectants. The terms “soft” before QPCs used herein refers to that a compound comprises at least one hydrolyzable unit.
[0058] One objective of the present disclosure was to develop novel antimicrobial agents that balance strong protection against emerging pathogens, while presenting degradability during environmental exposure.
[0059] Over the past decade, the inventors’ collaborative efforts have led to the preparation and biological assessment of over 700 novel quaternary ammonium compounds (QACs). The inventors’ groups investigated the assembly of novel hydrolyzable amphiphiles aiming to develop a library of antimicrobial agents with tuned instability to mitigate their environmental persistence. The inventors’ work added to a growing literature of soft QACs, joining the efforts of Bodor, who coined the term “soft antimicrobials,” as well as Haidar, Ahlstrom, and others (FIG. 1). The compounds reported by Bodor and Haidar are shown in FIG. 1 (top above “Bodor” and “Haidar,” respectively).
[0060] Exploiting varying multicationic QAC architectures, the inventors synthesized 40 QACs bearing either ester or amide linkers with encouraging antimicrobial and stability data as reported in 2017. These QACs have two general structures as shown in FIG. 1 (bottom above “Minbiole and Wuesf ’). While the ester-containing QACs were generally short-lived based on the inventors’ results, the stability of the amide-containing QACs displayed a direct correlation to the over-all pH of the sample. Under acidic conditions, the amides immediately decomposed, but stability of over 24 h was apparent in deionized water and buffered solutions of pH > 7. Importantly, these amide-based QACs displayed potent antimicrobial activity, with minimum inhibitory concentra-tion (MIC) values at single-digit micromolar values.
[0061] More recently, the inventors’ efforts have turned to the investigation of quaternary phosphonium compounds (QPCs) as an alternative set of antimicrobial agents to QACs with diverse architectures and potent broad-spectrum activities. Initial results have shown that biscationic QPCs (bisQPCs) exhibit high potency against both gram-positive and gram-negative pathogenic bacterial strains with the ability to evade QAC resistance mechanisms while still allowing for straightforward construction. Due to the lack of decomposition studies on such a disinfectant class, the inventors turned their focus to the construction of a library of soft antimicrobial QPCs (soft QPCs) as provided in the present disclosure.
[0062] Accordingly, the inventors prepared a series of bisQPCs that bear amide linkages in the side chains, expecting that amide hydrolysis would lead to the formation of non- amphiphilic residues, and evaluated these compounds for bioactivity, toxicity, and stability in varying buffer solutions, facilitating comparison to the analogous soft QACs previously reported. FIG. 2 show some exemplary compounds in accordance with some embodiments.
[0063] The present disclosure provides a biscationic quaternary phosphonium compound with amide substitution, an antimicrobial composition comprising such a compound, and the method of making such an antimicrobial compound or composition, and the method of using such a compound or composition for antimicrobial use. The compound or the composition provided in the disclosure has an ability to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, or to attenuate the severity of a microbial infection. The compound or the composition provided in the disclosure has an ability to inhibit or eradicate pre-established biofilms (i.e. antibiofilm use) formed by the microorganisms.
[0064] In accordance with some embodiments, the present disclosure provides an antimicrobial composition comprising a compound having the formula
Figure imgf000016_0001
wherein:
Li and L2 each is a linking group selected from the group consisting of a C1-10 alkyl, a C1-10 cycloalkyl, an aryl, and any combination thereof, each of Ri, R2, R3, and R4 is selected from the group consisting of a C1-12 alkyl, a C1-12 cycloalkyl, and an aryl group, which is unsubstituted or optionally substituted with a functional group selected from the group consisting of -OH, -OR’, -NH2, -NHR’, -NR’ 2, -SH, -SR’, -O-C(O)R’, -C(O)OR’, -C(O)R’, -C(O)NR’R’, -CF3, -OCF3, halogen, and any combination thereof;
R’ is H or a C1-4 alkyl; each of Rs, Re, R7, and Rs is H or a C1-20 alkyl, wherein at least one of Rs and Re is a C1-20 alkyl and at least one of R7 and Rs is a C1-20 alkyl,
X is selected from the group consisting of a halogen, a tosylate, a hydrocarbonate, a carbonate, a sulfate, and an acetate; and m and y are integers being 1 or 2, wherein m * y =2.
[0065] In the present disclosure, X is used for illustration only. It can be monovalent anions or monovalent negative ions such as fluoride ion (F"), chloride ion (Cl"), bromide ion (Br ), and iodide (I ). X may also represent multi -valent anions such as carbonate and sulfate [0066] The present disclosure provides a composition or a product described herein. In some embodiments, the antimicrobial composition is a disinfectant configured to be applied to a surface of an object in need thereof. In some embodiments, the antimicrobial composition is a pharmaceutical composition configured to be administrated to a subject in need thereof. The pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an active ingredient. In some embodiments, the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an additive for stabilizing the pharmaceutical composition.
[0067] In the compound having the formula (I) or (II), in some embodiments, Li is a C2- 10 alkyl. In some embodiments, Li is an alternating structure comprising alkyl and cycloalkyl, for example, an alternating structure comprising a C2-10 alkyl and a C4-6 cycloalkyl. The term “alkyl” for the linking groups can be understood as alkylene group, which have two connecting bonds. In some embodiments, Li comprises an aryl group such as a phenyl group, or a combination of an aryl group and one or more alkyl. For example, Li may be a combination of - alkyl-Ph-alkyl-.
[0068] L2 may be a C1-6 alkyl in some embodiments.
[0069] In some embodiments, the linking group Li or L2 is a C1-6 alkyl such as -CH2-, - CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, or- CH2CH2CH2CH2CH2CH2-.
[0070] In some embodiments, Ri, R2, R3, or R4 each is a C1-6 alkyl or a cyclohexyl such as cyclohexyl, or a phenyl group.
[0071] In some embodiments, Ri, R2, R3, and R4 are the same, Rs and R7 are the same, and Rs and Rs are the same. The compound has a symmetrical structure.
[0072] In some embodiments, Rs and R7 are the same and are an alkyl having a formula CnH 2n+i, wherein n is an integer in the range of from 2 to 20, and Rs and Rs are the same and are H or methyl.
[0073] In some embodiments, the compound having the formula (I) has a formula:
Figure imgf000017_0001
[0074] The compound having formula (II) has a formula:
Figure imgf000018_0001
[0075] In the formula (III) or (IV), Ri, R2, R3, and R4 are a same group R, which may be selected from a C1-6 alkyl, a C1-6 cycloalkyl, and phenyl. The value of n can be in a range of 2 to 20, for example, from 6 to 16, from 2 to 12, from 4 to 12, from 6 to 12, from 8 to 12, from 10 to 12, or any other suitable ranges. In some embodiments, n is in a range of from 5 to 12. Li is a
C2-10 alkyl and L2 is a C1-6 alkyl.
[0076] X is any suitable group for anions. The value of m depends on the valence of the anions. For example, when X a halogen, a tosylate, an acetate, or a hydrocarbonate, y is 1, and m is 2. When X is a carbonate or a sulfate, y is 2, and m is 1. In some embodiments, X is a halogen selected from F, Cl, Br, and I, and m is 2.
[0077] In another aspect, the present disclosure provides a compound as described herein, a method of making such a compound, and a method of using such a compound.
[0078] Examples of a suitable compound include, but are not limited to the compounds from Cl to C 14 and related analogs as shown below:
Figure imgf000018_0002
Figure imgf000019_0001
Figure imgf000020_0001
and analogs of Compounds Cl to C 14 wherein -Ph is replaced with methyl, ethyl, or cyclohexyl.
[0079] In the compounds above, the counter ions 2C1’ may be replaced with other suitable counter ions as described. For example, in some embodiments, each of these compounds comprises 2 Br , as counter ions.
[0080] In the present disclosure, exemplary compounds made include at least two series of compounds, each of which has a formula being :
Figure imgf000020_0002
P6P-#A,#A
(VI).
[0081] In these compound, Li is a C3 alkyl or a Ce alkyl, respectively, and L2 is -CH2-. The compounds having the formula (V) or (VI) are abbreviated in a format of P3P-#A, #A or P6P-#A,#A, respectively. The numbers of “3” and “6” represents the number of the carbon atoms in the linking group Li. The number sign (#) represents the sum of n, the number of carbon atoms in the linking group L2 plus two (corresponding to the amide group). The letter “A” represents amide. In the formulas (V) and (VI), Ri, R2, Rt, and R4 are the same (R), and are phenyl. So these compounds can be also abbreviated in a format of phP3P-#A, #A or pllP6P- #A,#A, respectively. The phenyl group as shown can be also replaced with other groups such as methyl, ethyl and cyclohexyl. “Me”, “Et” and “Cy” represent methyl, ethyl and cyclohexyl, respectively. In the general formulas, “Me”, “Et” or “Cy” may be added in the left side of a formula. [0082] Therefore, a compound abbreviation may be in a format of RPLiP, (n+L2+2)A,
(n+L2+2)A, X, for the compounds having formula (III) or (IV), respectively. “Li” and “L2” represent the number of the carbon atoms in the respective linking group or the name of the linking group. X and n are the same as those in the formulas.
[0083] For example, Compound (Cl) having the following structure:
Figure imgf000021_0001
is coded as P3P-8A,8A or phP3P-8A,8A, or phP3P-8A,8A, Cl.
[0084] In another aspect, the present disclosure provides a method of making the antimicrobial composition as described herein. In some embodiments, such a method comprises preparing the compound having the formula (I) or (II) as described. The compound having the formula (I) can be synthesized as illustrated in Scheme 1.
[0085] Scheme 1
Figure imgf000021_0003
[0086] The compound having the formula (II) can be synthesized as illustrated in
Schedule 2.
[0087] Scheme 2
Figure imgf000021_0002
(VII) (II)
[0088] As shown in Schemes 1 and 2, the compound having the formula (I) can be prepared by the alkylation reaction of a suitable bisphosphine starting compound having the formula (VII) with a suitable chemical having L2 linking group and amide function groups. A suitable electrophile, bearing a suitable group such as an alkyl, or a cycloalkyl, or a phenyl backbone as well as a leaving group, can be alkylated under substitution conditions, which can optimize solvent conditions and temperature; two equivalents of the electrophile would be utilized.
[0089] The method may further comprise mixing an effective amount of the compound having the formula (I) or (II) and a carrier. Examples of a suitable carrier may include, but are not limited to, a solvent, a carrier, an additive, any other suitable ingredient, or combinations thereof.
[0090] In some embodiments, the present disclosure also provides an antimicrobial composition comprising a compound having the formula (I) or (II) as described, and a carrier such as a solvent. The antimicrobial composition can also comprise other ingredients and additives. The content of the compound having the formula (I) or (II) can be in any suitable concentration. For example, in some embodiments, such a concentration can be in the range from 0.01 pM to 100 pM, for example, from 0.1 pM to 10 pM. In some embodiments, the content of the compound having the formula (I) or (II) may be at a concentration of from 0.1 wt.% to 5 wt. %, for example, in the range of from 0.2 wt.% to 2.5 wt. %. Examples of the carrier include but are not limited to a solvent. Examples of other additives include but are not limited to surfactants, anti-foaming agents, anti-freezing agents, gelling agents, and combinations thereof.
[0091] The antimicrobial composition may also comprise a pharmaceutically acceptable carrier or excipient. A pharmaceutically acceptable carrier or excipient suitable for a solid preparation such as tablets or capsules can be, for example, binders (e.g., acacia, gelatin, dextrin, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone), solvents, dispersion media, diluents (e.g., lactose, sucrose, mannitol, com starch, potato starch, calcium phosphate, calcium citrate, crystalline cellulose), lubricants (e.g., magnesium stearate, calcium stearate, stearic acid, talc, anhydrous silicic acid), disintegrants (e.g., corn starch, potato starch, carboxymethylcellulose, carboxymethylcellulose calcium, alginic acid), and wetting agents (e.g., sodium laurylsulfate).
[0092] A pharmaceutically acceptable carrier or excipient suitable for a liquid preparation, such as solutions or suspensions, can be, for example, aqueous vehicles (e.g., water), suspending agents (e.g., acacia, gelatin, methyl cellulose, carboxymethylcellulose sodium, hydroxymethyl-cellulose, aluminum stearate gel), surfactants (e.g., lecithin, sorbitan monooleate, glycerin monostearate), and non-aqueous vehicles (e.g., glycerin, propylene glycol, vegetable oil). Moreover, liquid preparations may contain preservatives (e.g., p-hydroxybenzoic acid methyl ester, p-hydroxybenzoic acid propyl ester), flavors, and/or coloring agents. The antimicrobial composition in this disclosure can be formulated to be in any suitable form, including but not limited to liquid, gel and paste.
[0093] The present disclosure also provides different products as described herein, which comprise the antimicrobial composition.
[0094] Method of Use
[0095] In another aspect, the present disclosure provides a method of killing, preventing, or inhibiting microbial growth. For example, in some embodiments, the method comprises applying the antimicrobial composition to a surface of an object in need thereof. Examples of a suitable method include but are not limited to pouring, spraying, any other suitable methods and any combinations thereof.
[0096] In some embodiments, such a method comprises administrating the antimicrobial composition to a subject in need thereof. The method of administration may include in vitro or in vivo administration. For example, the types of administration include, are not limited to, topical, oral, parenteral, rectal, intramuscular injection, inhalation, intrathecal, sublingual, nasal, transdermal, and any combination thereof.
[0097] The antimicrobial composition or the compound is used to kill, prevent, or inhibit growth of at least one group of microorganisms such as bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm, or disperse or eradicate a pre-established biofilm. [0098] In some embodiments, this disclosure relates to methods of treating or preventing diseases or conditions comprising administering an effective amount of a composition comprising the quaternary phosphonium compound disclosed herein to a subject in need thereof. [0099] In some embodiments, the disclosure contemplates that a quaternary phosphonium compound disclosed herein may be used in antimicrobial applications optionally in combination with other antimicrobial agents for prevention of disease onset and treatment. In some embodiments, the quaternary phosphonium compound disclosed herein may be used in medical device coatings (medical implants and tools, IV catheters), wound dressings (embedded in gauze bandages), wound rinses (i.e. surgical rinses), wound-vacuum systems, whole body baths (e.g., in combo with bleach baths for treatment of skin flares for atopic dermatitis/eczema), soaps, personal care products (body washes, lotions, soaps) for high-risk patients or for populations with high risk of exposure (e.g. athletes using common sports equipment in gym), and veterinary applications (e.g. anti-infectives for companion animals, race horses, etc.) [0100] In some embodiments, this disclosure relates to methods of treating or preventing a microbial infection comprising administering to a subject in need thereof an effective amount of a quaternary phosphonium compound as disclosed herein. In some embodiments, the microbial infection is a bacterial, fungal, pest, or viral infection.
[0101] In some embodiments, this disclosure relates to methods of treating or preventing bacterial infections comprising administering or contacting the skin of a subject with formula comprising a quaternary phosphonium compound as disclosed herein to a subject in need thereof. In some embodiments, the formula is administered in combination with another antibiotic agent. [0102] In some embodiments, this disclosure provides a method of using quaternary phosphonium compounds disclosed herein for treating or preventing an Acinetobacter baumannii infection, other bacterial infection, other multidrug resistant bacteria, or other microbial infection by administering an effective amount of quaternary phosphonium compounds disclosed herein to a subject in need thereof. In further embodiments, the quaternary phosphonium compound is co-administered with an antibiotic selected from the group comprising of sulfonamides, diaminopyrimidines, quinolones, beta-lactam antibiotics, cephalosporins, tetracyclines, nitrobenzene derivatives, aminoglycosides, macrolide antibiotics, polypeptide antibiotics, nitrofuran derivatives, nitroimidazoles, nicotinic acid derivatives, polyene antibiotics, imidazole derivatives or glycopeptide, cyclic lipopeptides, glycylcyclines and oxazolidinones. In further embodiments, these antibiotics include but are not limited to sulphadiazine, sulphones - [dapsone (DDS) and para-aminosalicylic (PAS)], sulfanilamide, sulfamethizole, sulfamethoxazole, sulphapyridine, trimethoprim, pyrimethamine, nalidixic acids, norfloxacin, ciprofloxacin, cinoxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, ofloxacin, pefloxacin, sparfloxacin, trovafloxacin, penicillins (amoxicillin, ampicillin, azlocillin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, hetacillin, oxacillin, mezlocillin, penicillin G, penicillin V, piperacillin), cephalosporins (cefacetrile, cefadroxil, cefalexin, cephaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefonicid, ceforanide, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefotetan, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefoperazone, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefepime), moxalactam, carbapenems (imipenem, ertapenem, meropenem) monobactams (aztreonam), oxytetracycline, chlortetracycline, clomocycline, demeclocycline, tetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, chloramphenicol, amikacin, gentamicin, framycetin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, colistin, bacitracin, tyrothricin, nitrofurantoin, furazolidone, metronidazole, tinidazole, isoniazid, pyrazinamide, ethionamide, nystatin, amphotericin-B, hamycin, miconazole, clotrimazole, ketoconazole, fluconazole, rifampicin, lincomycin, clindamycin, spectinomycin, fosfomycin, loracarbef, polymyxin B, polymyxin B Sulphate, procaine, ramoplanin, teicoplanin, vancomycin, and/or nitrofurantoin.
[0103] In some embodiments, the subject is diagnosed with a bacterial infection. In some embodiments, the subject is diagnosed with bacteremia, pneumonia, staphylococcal food poisoning, necrotizing pneumonia, necrotizing fasciitis, scalded skin syndrome, post-operation bacterial infection, medical device bacterial infection, bacterial infection of the skin, soft tissue bacterial infection, or toxic shock syndrome.
[0104] In some embodiments, this disclosure provides methods of treating or preventing a toxin-mediated bacterial infection comprising administering an effective amount of a quaternary phosphonium compound as disclosed herein to a subject in need thereof, including a subject at risk of, exhibiting symptoms of, or diagnosed with scalded skin syndrome (esp. in neonates), abscesses, necrotizing fasciitis, sepsis, or atopic dermatitis (eczema).
[0105] In some embodiments, this disclosure provides methods of treating or preventing bacterial infections or acne comprising administering to a subject in need thereof or contacting the skin of a subject in need thereof with a formula comprising of a quaternary phosphonium compound as disclosed herein. In some embodiments, the formula is administered in combination with another antibiotic. [0106] In some embodiments, the subject is at risk of a bacterial infection due to being diagnosed with an abscess, furuncle, cellulitis, folliculitis, atopic dermatitis, psoriasis, impetigo, septic arthritis, brain abscess, burn wound, venous ulcer, diabetic foot ulcer, surgical wound, carbuncle, or meningitis.
[0107] A flow of wound rinse/irrigation solution can be applied across an open wound surface to achieve wound hydration, to remove deeper debris, and to assist with the visual examination. In some embodiments, the disclosure relates to methods of irrigating a wound using a solution comprising a quaternary phosphonium compound as disclosed herein.
[0108] In some embodiments, this disclosure provides methods of using quaternary phosphonium compounds disclosed herein for killing microbes, preventing or inhibiting microbe growth, preventing a biofdm formation, or preventing the spread of an Acinetobacter baumannii infection, other bacterial infection, other multidrug resistant bacteria, or other microbial infection by sanitizing a surface, e.g., by contacting the surface with a solid (e.g., powder), liquid, or spray composition, with a quaternary phosphonium compound disclosed herein in an effective amount. [0109] In some embodiments, this disclosure provides methods of preventing cellular infections comprising applying a quaternary phosphonium compound disclosed herein on top of or inside a cell growth medium.
[0110] In some embodiments, this disclosure provides methods of preventing plant microbial infections comprising applying a quaternary phosphonium compound disclosed herein to the exterior, leaf, seed, or stem of a plant. In some embodiments, this disclosure provides methods of preventing plant microbial infections comprising applying a quaternary phosphonium compound disclosed herein on top of or into soil, dirt, sand, or other medium from which roots of the plant reside.
[0111] Compositions and Devices
[0112] In some embodiments, this disclosure provides compositions and devices comprising a quaternary phosphonium compound disclosed herein.
[0113] In some embodiments, this disclosure provides soaps and disinfectant products comprising a quaternary phosphonium compound disclosed herein. Contemplated topical formulations for skin flares (i.e., for atopic dermatitis or other infections related to a disrupted skin barrier) may be combined with another drug such as a topical steroid, anti-inflammatory agent, and promoter of skin barrier function or skin moisturizer. In some embodiments, this disclosure provides a container configured to create a liquid spray comprising a quaternary phosphonium compound disclosed herein.
[0114] In some embodiments, this disclosure provides a pharmaceutical formulation comprising a quaternary phosphonium compound disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical formulation is in the form of a lotion, liquid, or gel. In some embodiment, the pharmaceutical formulation is in the form of a particle, bead, tablet, capsule, pill, or injectable solution. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as water, dimethyl sulfoxide, mannitol, 1,3 -butanediol, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
[0115] The pharmaceutical formulation can also include any type of pharmaceutically acceptable excipients, additives, or vehicles. For example, diluents or fillers, such as dextrates, dicalcium phosphate, calcium sulphate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose, inositol, powdered sugar, bentonite, microcrystalline cellulose, or hydroxypropyl methylcellulose, may be added to the composition to increase the bulk of the composition.
[0116] In some embodiments, the formulation is a directly compressible composition comprising a quaternary phosphonium compound disclosed herein but no excipients, additives, or vehicles.
[0117] In some embodiments, the disclosure provides a pharmaceutical or cosmetic formulation comprising a quaternary phosphonium compound disclosed herein and a pharmaceutically acceptable excipient or cosmetically acceptable excipient. In some embodiments, the disclosure provides a liquid or gel formulation optionally further comprising an antibacterial agent, a topical steroid, an anti-inflammatory agent, a promoter of skin barrier function, a skin moisturizer, or combinations thereof. In some embodiments, the antibacterial agent is daptomycin, linezolid, vancomycin, nafcillin, cefazolin, dicloxacillin, clindamycin, rifampin, or sulfamethoxazole-trimethoprim (Bactrim). [0118] In some embodiments, the disclosure provides a wound dressings or wound rinse comprising a quaternary phosphonium compound disclosed herein wherein the wound dressing comprises an absorbent pad and optionally an adhesive.
[0119] In some embodiments, the disclosure provides disinfectant sprays or wipes formulation for surfaces and fomites comprising a quaternary phosphonium compound disclosed herein.
[0120] In some embodiments, this disclosure provides a medical device coated with a quaternary phosphonium compound as disclosed herein. In some embodiments, the medical device is a screw, pin, plate, rod, disk, needle, catheter, tube, stent, pacemaker, defibrillators (ICDs), artificial hip or knee joint/implant, breast implant, intra-uterine device, ear tube, contact lens, or implantable pump.
[0121] In some embodiments, this disclosure provides a surgical tool coated with a quaternary phosphonium compound as disclosed herein. In some embodiments, the surgical tool is a forceps, tweezers, scalpel, knife, scissors, retractor, needle, gauze, sponge, suction, staple, stapler, clip, laparoscopic instrument, electrosurgical cauterizer, ultrasonic device, camera, camera lens, fiber optic cable, insufflator, needle, bronchoscope, cystoscope, saw, or robotic arm. [0122] In some embodiments, the disclosure provides a wound dressing comprising a quaternary phosphonium compound as disclosed herein wherein the wound dress comprises an absorbent pad and optionally an adhesive optionally in combination with another antibiotic agent. In some embodiments, the wound dressing is a foam or compression dressing or a cover dressing such as wraps, gauze and tape. In some embodiments, the wound dressing comprises alginate or collagen. In some embodiments, the wound dressing is a hydrocolloid dressing, e.g., carboxy-methylcellulose and gelatin optionally in a polyurethane foam or film, optionally comprising one or more agents selected from pectin, a polysaccharide, and an adhesive.
[0123] In some embodiments, the wound dressing is a hydrogel. Hydrogels are polymers that contain a high content of hydroxy and/or carboxyl containing monomers or salts thereof, e.g., vinyl alcohol, acrylic acid, 2-hydroxyethylmethacrylate, ethylene glycol dimethacrylate monomers, which are co-polymers to provide varying degrees of hydration. Due to the hydrophilic monomers, the hydrogels typically absorb water. Contemplated hydrogel dressings include: amorphous hydrogel, which are a free-flowing gel that are typically distributed in tubes, foil packets and spray bottles; an impregnated hydrogel, which are typically saturated onto a gauze pad, nonwoven sponge ropes and/or strips; or a sheet hydrogel which are gel held together by a fiber mesh.
[0124] In some embodiments, the disclosure provides a wound rinse comprising a quaternary phosphonium compound as disclosed herein optionally containing normal saline, sterile water, a detergent, a surfactant, a preservative, or iodine.
[0125] In some embodiments, the disclosure provides a kit comprising a container comprising a quaternary phosphonium compound as disclosed herein optionally comprising a second container comprising a rinse solution or containing surgical device or tool, normal saline, sterile water, a detergent, a surfactant, a preservative, iodine, hydrogen peroxide, or sodium hypochlorite or other compound disclosed herein.
[0126] In some embodiments, the disclosure provides a cosmetic formulation comprising a quaternary phosphonium compound as disclosed herein and cosmetically acceptable excipient or additive. In some embodiments, the disclosure relates to a solid or liquid soap or lotion comprising a quaternary phosphonium compound as disclosed herein and a fatty acid. In some embodiments, additives can be selected from the group consisting of oily bodies, surfactants, emulsifiers, fats, waxes, pearlescent waxes, bodying agents, thickeners, superfatting agents, stabilizers, polymers, silicone compounds, lecithins, phospholipids, biogenic active ingredients, deodorants, antimicrobial agents, antiperspirants, film formers, antidandruff agents, swelling agents, insect repellents, hydrotropes, solubilizers, preservatives, perfume oils and dyes. In some embodiments, additives are selected from the group consisting of surfactants, emulsifiers, fats, waxes, stabilizers, deodorants, antiperspirants, antidandruff agents, and perfume oils.
[0127] In some embodiments, this disclosure provides a cell growth medium comprising a quaternary phosphonium compound disclosed herein.
[0128] EXAMPLES
[0129] A series of biscationic quaternary phosphonium compounds have been prepared. Such compounds have powerful antimicrobial activities. The examples described below are for the purpose of illustration only.
[0130] 1. Exemplary Compounds and Their Syntheses
[0131] Reagents and solvents were used from Sigma- Aldrich, Acros Organics, TCI Chemicals, and ThermoFisher Scientific without further purification. Reactions containing phosphorus starting materials were carried out under an argon atmosphere, with reagent grade solvents and magnetic stirring. All yields refer to spectroscopically pure compounds. 1 H. 13C, and 31P NMR spectra were measured with a 400 MHz or 500 MHz JEOL spectrophotometer, and chemical shifts were reported on a 8-scale (ppm) downfield from TMS or 85% H3PO4. Coupling constants were calculated in hertz (Hz). The solvent used was chloroform-c/ (CDCI3), using the residual solvent peak as an internal reference of 7.26 ppm for ’H NMR and 77.16 ppm for 13C NMR. Accurate mass spectrometry data was acquired on an AB Sciex 5600 TripleTOF using electrospray ionization in positive mode.
[0132] Synthesis of soft QPCs:
[0133] Preparation of P3P-8A,8A
Figure imgf000030_0001
[0134] To a solution of l,3-bis(diphenylphosphino)propane (0.357 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-pentylacetamide (0.295 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (~15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P3P-8A,8A as a white, powdery solid (0.533 g, 83.3%); ’HNMR (400 MHz, chloroform-c/), 8 8.88 (t, J= 6.3 Hz, 2H), 8.06-8.01 (m, 8H), 7.73-7.69 (m, 4H), 7.65- 7.61 (m, 8H), 4.57 (d, J = 14.5 Hz, 4H), 3.74 (m, 4H), 2.93 (q, J = 6.7 Hz, 4H), 2.13 (m, 2H), 1.31-1.09 (m, 12H), 0.81 (t, J = 7.0 Hz, 6H); 13C{’H} NMR (100.6 MHz, chloroform-c/), 8 162.5 (t, J= 2.9 Hz), 134.9, 133.5 (quint, J = 4.8 Hz), 130.2, 117.7 (d, J= 86.2 Hz), 40.1, 30.0 (d, J = 53.0 Hz), 29.0, 28.7, 23.4 (dd, J= 16.9, 53.9 Hz), 22.3, 16.2, 14.0; ^P H] NMR (162.0 MHz, chloroform-c/) 8 24.04. HRMS (ESI+): 334.1834, C41H54N2O2P2 [M-2C1]2+ requires 334.1825. [0135] Preparation of P3P-9 A, 9A
Figure imgf000030_0002
[0136] To a solution of l,3-bis(diphenylphosphino)propane (0.355 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-hexylacetamide (0.320 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (~15 mL) at 45 °C for 1 hour and then isolated by vacuum fdtration, resulting in P3P-9A,9A as a white, powdery solid (0.446 g, 67.5%); JH NMR (400 MHz, chloroform-;/), 8 8.88 (t, J= 6.0 Hz, 2H), 8.06-8.01 (m, 8H), 7.71-7.68 (m, 4H), 7.64- 7.59 (m, 8H), 4.58 (d, J = 14.3 Hz, 4H), 3.73 (m, 4H), 2.92 (q, J = 7.0 Hz, 4H), 2.11 (m, 2H), 1.27-1.12 (m, 16H), 0.82 (t, J = 7.2 Hz, 6H); 13C{JH} NMR (100.6 MHz, chloroform-;/), 8 162.5 (t, ./ - 2,4 Hz), 134.9, 133.5 (quint, J= 5.3 Hz), 130.2, 117.7 (d, J= 86.2 Hz), 40.2, 31.4, 30.0 (d, J = 52.5 Hz), 29.0, 26.6, 23.7 (dd, J= 16.9 Hz), 22.6, 16.2, 14.1; 31P{1H} NMR (162.0 MHz, chloroform-;/) 824.19. HRMS (ESI+): 348.1991, C43H58N2O2P2 [M-2C1]2+ requires 348.1982.
[0137] Preparation of P3P-10A,10A
Figure imgf000031_0001
[0138] To a solution of l,3-bis(diphenylphosphino)propane (0.365 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-hepylacetamide (0.362 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24.5 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (~15 mL) at 45 °C for 1 hour and then isolated by vacuum fdtration, resulting in P3P-10A,10A as a white, powdery solid (0.590 g, 81.8%); 'H NMR (400 MHz, chloroform-;/), 8 8.87 (t, J= 5.7 Hz, 2H), 8.06-8.01 (m, 8H), 7.73-7.70 (m, 4H), 7.65-7.60 (m, 8H), 4.57 (d, J = 14.6 Hz, 4H), 3.73 (m, 4H), 2.92 (q, J = 6.1 Hz, 4H), 2.11 (m, 2H), 1.30-1.16 (m, 20H), 0.84 (t, J = 7.0 Hz, 6H); 13C{JH} NMR (100.6 MHz, chloroform- d), 8 162.4 (t, J = 2.4 Hz), 134.9, 133.5 (quint, J= 5.3 Hz), 130.2, 117.8 (d, J= 86.2 Hz), 40.2, 31.8, 30.0 (d, J= 53.0 Hz) 29.1, 28.9, 26.9, 23.4 (dd, J= 16.9 Hz) 22.6, 16.2, 14.2; 31P{1HJ NMR (162.0 MHz, chloroform-;/) 8 24.10. HRMS (ESI+): 362.2152, C45H62N2O2P2 [M-2C1]2+ requires 362.2138.
[0139] Preparation of P3P-11A,11A
Figure imgf000032_0001
[0140] To a solution of l,3-bis(diphenylphosphino)propane (0.361 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-octylacetamide (0.371 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P3P-11A,11A as a white, powdery solid (0.589 g, 83.3%); rH NMR (400 MHz, chloroform-t/), 8 8.87 (t, J= 5.7 Hz, 2H), 8.06-8.01 (m, 8H), 7.73-7.69 (m, 4H), 7.65-7.60 (m, 8H), 4.57 (d, J = 14.3 Hz, 4H), 3.73 (m, 4H), 2.92 (q, J = 6.2 Hz, 4H), 2.11 (m, 2H), 1.28-1.15 (m, 24H), 0.85 (t, J = 6.9 Hz, 6H); 13C{JH} NMR (100.6 MHz, chloroform- d), 8 162.4 (t, J = 2.4 Hz), 134.9, 133.5 (quint, J= 4.8 Hz), 130.2, 117.7 (d, J = 86.2 Hz), 40.2, 31.9, 30.0 (d, J= 52.5 Hz), 29.3, 29.2, 29.1, 27.0, 23.5 (dd, J= 17.8, 53.5 Hz), 22.7, 16.2, 14.2; 31P{ JH} NMR (162.0 MHz, chloroform-t/) 8 24.09. HRMS (ESI+): 376.2301, C47H66N2O2P2 [M- 2C1]2+ requires 376.2295.
[0141] Preparation of P3P-12A,12A
Figure imgf000032_0002
[0142] To a solution of l,3-bis(diphenylphosphino)propane (0.357 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-nonylacetamide (0.393 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 20 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P3P-12A,12A as a yellow, flaky solid (0.458 g, 62.1%); ’HNMR (400 MHz, chloroform-c/), 8 8.87 (t, J= 5.5 Hz, 2H), 8.06-8.01 (m, 8H), 7.73-7.69 (m, 4H), 7.65- 7.60 (m, 8H), 4.57 (d, J = 14.6 Hz, 4H), 3.74 (m, 4H), 2.92 (q, J = 6.2 Hz, 4H), 2.12 (m, 2H), 1.28-1.16 (m, 28H), 0.86 (t, J = 6.9 Hz, 6H); 13C{JH} NMR (100.6 MHz, chloroform-r/), 8 162.4 (t, J= 2.9 Hz), 134.9, 133.5 (quint, J = 4.8 Hz), 130.2, 117.7 (d, J= 86.2 Hz), 40.2, 31.9, 30.0 (d, J= 53.0 Hz), 29.5, 29.33, 29.28, 29.1, 27.0, 23.4 (dd, J= 17.8, 54.0 Hz), 22.7, 16.2, 14.2; 31P{1H] NMR (162.0 MHz, chloroform-^/) 8 24.08. HRMS (ESI+): 390.2458, C49H70N2O2P2 [M- 2C1]2+ requires 390.2451.
[0143] Preparation of P3P-13A,13A
Figure imgf000033_0001
[0144] To a solution of l,3-bis(diphenylphosphino)propane (0.378 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-decylacetamide (0.434 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 20 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P3P-13A,13A as a white, powdery solid (0.628 g, 77.9%); 'H NMR (400 MHz, chloroform-;/), 8 8.87 (t, J= 5.7 Hz, 2H), 8.06-8.01 (m, 8H), 7.73-7.68 (m, 4H), 7.65-7.60 (m, 8H), 4.57 (d, J - 14.4 Hz, 4H), 3.74 (m, 4H), 2.92 (q, J - 6.8 Hz, 4H), 2.13 (m, 2H), 1.25-1.16 (m, 32H), 0.86 (t, J = 5.7 Hz, 6H); 13C{1H} NMR (100.6 MHz, chloroform- d), > 162.4 (t, J = 2.4 Hz), 134.9, 133.5 (quint, J= 4.8 Hz), 130.3, 117.7 (d, J= 86.2 Hz), 40.2, 32.0, 30.0 (d, J= 53.0 Hz), 29.63, 29.58, 29.4, 29.3, 29.1, 27.0, 23.4 (dd, J= 16.9, 53.5 Hz), 22.8, 16.2, 14.2; 31P{1H} NMR (162.0 MHz, chloroform-;/) 8 24.04. HRMS (ESI+): 404.2620, C51H74N2O2P2 [M-2C1]2+ requires 404.2608.
[0145] Preparation of P3P-15A,15A
Figure imgf000033_0002
[0146] To a solution of l,3-bis(diphenylphosphino)propane (0.354 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-dodecylacetamide (0.469 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24.5 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P3P-15A,15A as a white, powdery solid (0.752 g, 93.5%); 'H NMR (400 MHz, chloroform-;/), 8 8.88 (t, J= 6.0 Hz, 2H), 8.06-8.01 (m, 8H), 7.72-7.68 (m, 4H), 7.64-7.60 (m, 8H), 4.58 (d, J = 14.4 Hz, 4H), 3.74 (m, 4H), 2.92 (q, J = 6.2 Hz, 4H), 2.13 (m, 2H), 1.28-1.15 (m, 40H), 0.86 (t, J = 6.8 Hz, 6H); 13C{1H} NMR (100.6 MHz, chloroform- d), b 162.4 (t, J = 2.4 Hz), 134.9, 133.5 (quint, J= 4.3 Hz), 130.3, 117.7 (d, J= 86.2 Hz), 40.2, 32.0, 30.0 (d, J= 53.9 Hz), 29.73, 29.71, 29.69, 29.6, 29.4, 29.3, 29.1, 27.0, 23.4 (dd, J= 16.9, 53.9 Hz), 22.8, 16.2, 14.2; 31P{1H} NMR (162.0 MHz, chloroform-;/) 8 24.08. HRMS (ESI+): 432.2929, C55H82N2O2P2 [M-2C1]2+ requires 432.2921.
[0147] Preparation of P6P-8A,8A
Figure imgf000034_0001
[0148] To a solution of l,6-bis(diphenylphosphino)hexane (0.354 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-pentylacetamide (0.283 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P6P-8A,8A as a white, powdery solid (0.238 g, 38.9%); 'H NMR (400 MHz, chloroform-^), 8 9.33 (t, J= 5.3 Hz, 2H), 8.01-7.96 (m, 8H), 7.75-7.71 (m, 4H), 7.66- 7.61 (m, 8H), 4.70 (d, J = 14.1 Hz, 4H), 3.18 (m, 4H), 3.02 (q, J = 6.88 Hz, 4H), 1.62 (s, 8H), 1.33-1.11 (m, 12H), 0.80 (t, J = 7.0 Hz, 6H); 13C{JH} NMR (100.6 MHz, chloroform-;/), 8 162.4 (d, J= 5.3 Hz), 134.8 (d, J= 2.9 Hz), 133.5 (d, J= 10.1 Hz), 130.2 (d, J = 12.5 Hz), 118.4 (d, J = 84.8 Hz), 40.1, 30.0 (d, J= 52.0 Hz), 29.1, 28.9 (d, J= 16.9 Hz), 28.8, 22.7 (d, J= 51.1 Hz), 22.3, 21.2, 14.1; 31P{1H} NMR (162.0 MHz, chloroform -d) 8 25.53. HRMS (ESI+): 355.2070, C44H60N2O2P2 [M-2C1]2+ requires 355.2060.
[0149] Preparation of P6P-9A,9A
Figure imgf000034_0002
[0150] To a solution of l,6-bis(diphenylphosphino)hexane (0.356 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-hexylacetamide (0.296 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24.5 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 C for 1 hour and then isolated by vacuum filtration, resulting in P6P-9A,9A as a white, powdery solid (0.584 g, 91.9%); 'H NMR (400 MHz, chloroform-6?), 8 9.30 (t, J= 6.0 Hz, 2H), 8.00-7.96 (m, 8H), 7.74-7.70 (m, 4H), 7.65- 7.61 (m, 8H), 4.70 (d, J = 14.3 Hz, 4H), 3. 17 (m, 4H), 3.01 (q, J = 7.0 Hz, 4H), 1.61 (s, 8H), 1.29-1.12 (m, 16H), 0.81 (t, J = 7.2 Hz, 6H); ^C H} NMR (100.6 MHz, chloroform-6?), 8 162.7 (d, J= 5.3 Hz), 134.7 (d, J = 2.9 Hz), 133.5 (d, J= 10.1 Hz), 130.2 (d, J= 12.5 Hz), 118.4 (d, J = 84.8 Hz), 40.1, 31.5, 29.9 (d, J= 52.5 Hz), 29.0, 28.7 (d, J= 16.9 Hz), 26.7, 22.6, 22.5 (d, J = 51.1 Hz), 21.1, 14.1; 31P{1H} NMR (162.0 MHz, chloroform-6?) 825.54. HRMS (ESI+): 369.2236, C46H64N2O2P2 [M-2C1]2+ requires 369.2216.
[0151] Preparation of P6P- 10 A, 10A
Figure imgf000035_0001
[0152] To a solution of l,6-bis(diphenylphosphino)hexane (0.370 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-heptylacetamide (0.329 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24.5 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (~15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P6P-10A,10A as a white, powdery solid (0.322 g, 47.2%); 'H NMR (400 MHz, chloroform-6?), 8 9.33 (t, J= 5.4 Hz, 2H), 7.99-7.95 (m, 8H), 7.73-7.69 (m, 4H), 7.64-7.60 (m, 8H), 4.69 (d, J - 14.3 Hz, 4H), 3.16 (m, 4H), 3.00 (q, J - 6.2 Hz, 4H), 1.61 (s, 8H), 1.29-1.15 (m, 20H), 0.83 (t, J = 6.9 Hz, 6H); 13C{1H} NMR (100.6 MHz, chloroform-t/). 8 162.7 (d, J= 5.3 Hz), 134.7 (d, J= 2.9 Hz), 133.5 (d, J= 10.1 Hz), 130.1 (d, J= 13.0 Hz), 118.4 (d, J= 84.8 Hz), 40.1, 31.8, 29.9 (d, J= 53.0 Hz), 29.01, 28.95, 28.7 (d, J= 16.9 Hz), 26.9, 22.6 (d, J= 51.1 Hz), 22.7, 21.1 , 14.2; 31P{1H] NMR (162.0 MHz, chloroform -6?) 8 25.53. HRMS (ESI+): 383.2388, C48H68N2O2P2 [M-2C1]2+ requires 383.2373.
[0153] Preparation of P6P-11A,11A
Figure imgf000035_0002
[0154] To a solution of l,6-bis(diphenylphosphino)hexane (0.366 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-octylacetamide (0.345 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (~15 mL) at 45 °C for 1 hour and then isolated by vacuum fdtration, resulting in P6P-11A,11A as a white, powdery solid (0.444 g, 66.6%); rH NMR (400 MHz, chloroform-^, 8 9.24 (t, J= 5.7 Hz, 2H), 8.01-7.96 (m, 8H), 7.74-7.71 (m, 4H), 7.65-7.61 (m, 8H), 4.70 (d, J = 14.6 Hz, 4H), 3.18 (m, 4H), 3.01 (q, J = 6.1 Hz, 4H), 1.62 (s, 8H), 1.28-1.14 (m, 24H), 0.83 (t, J = 7.0 Hz, 6H); ^C H] NMR (100.6 MHz, chloroform-;/), 6 162.8 (d, J = 5.3 Hz), 134.7 (d, J= 2.9 Hz), 133.5 (d, J= 10.1 Hz), 130.1 (d, J= 13.0 Hz), 118.4 (d, J = 84.8 Hz), 40.2, 31.9, 29.9 (d, J = 52.5 Hz), 29.27, 29.23, 29.1, 28.7 (d, J= 16.9 Hz), 27.0, 22.6 (d, J= 51.1 Hz), 22.7, 21.1, 14.2; 31P{1H} NMR (162.0 MHz, chloroform-;/) 8 25.61. HRMS (ESI+): 397.2538, C50H72N2O2P2 [M-2C1]2+ requires 397.2529.
[0155] Preparation of P6P- 12 A, 12A
Figure imgf000036_0001
[0156] To a solution of l,6-bis(diphenylphosphino)hexane (0.355 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-nonylacetamide (0.393 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 21 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (~15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P6P-12A,12A as an orange, flaky solid (0.684 g, 97.9%); 'H NMR (400 MHz, chloroform-;/), 8 9.24 (t, J= 6.0 Hz, 2H), 8.00-7.94 (m, 8H), 7.73-7.69 (m, 4H), 7.64- 7.59 (m, 8H), 4.69 (d, J = 14.3 Hz, 4H), 3.16 (m, 4H), 3.00 (q, J = 6.2 Hz, 4H), 1.60 (s, 8H), 1.28-1.13 (m, 28H), 0.84 (t, J = 6.9 Hz, 6H); ^C H} NMR (100.6 MHz, chloroform-;/), 8 162.8 (d, J= 5.3 Hz), 134.7 (d, J= 2.9 Hz), 133.5 (d, J= 10.1 Hz), 130.1 (d, J= 13.0 Hz), 118.4 (d, J = 85.3 Hz), 40.2, 31.9, 29.9 (d, J= 52.5 Hz), 29.5, 29.34, 29.31, 29.1, 28.7 (d, J= 16.9 Hz), 27.0, 22.6 (d, J= 51.1 Hz), 22.7, 21.1, 14.2; 31P{1H} NMR (162.0 MHz, chloroform-;/) 825.58. HRMS (ESI+): 411.2694, C52H76N2O2P2 [M-2C1]2+ requires 411.2686.
[0157] Preparation of P6P-13AJ3A
Figure imgf000037_0001
[0158] To a solution of l,6-bis(diphenylphosphino)hexane (0.369 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-decylacetamide (0.391 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 20 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 C for 1 hour and then isolated by vacuum filtration, resulting in P6P-13A,13A as a white, powdery solid (0.496 g, 66.3%); 'l l NMR (400 MHz, chloroform-t/), 5 9.31 (t, J= 5.6 Hz, 2H), 8.02-7.96 (m, 8H), 7.74-7.70 (m, 4H), 7.65-7.61 (m, 8H), 4.70 (d, J = 14.4 Hz, 4H), 3.17 (m, 4H), 3.01 (q, J = 6.3 Hz, 4H), 1.62 (s, 8H), 1.27-1.15 (m, 32H), 0.85 (t, J = 6.9 Hz, 6H); ^CfH) NMR (100.6 MHz, chloroform-^, 8 162.8 (d, J= 5.3 Hz), 134.7 (d, J= 2.9 Hz), 133.5 (d, J= 10.1 Hz), 130.1 (d, J= 12.5 Hz), 118.4 (d, J= 84.8 Hz), 40.2, 32.0, 29.9 (d, .7= 53.0 Hz), 29.64, 29.59, 28.4, 29.3, 29.1, 28.7 (d, J= 16.9 Hz), 27.0, 22.6 (d, J= 51.1 Hz), 22.8, 21.1, 14.2; 31P{1H} NMR (162.0 MHz, chloroform- ) 5 25.55. HRMS (ESI+): 425.2861, C54H80N2O2P2 [M-2C1]2+ requires 425.2842. [0159] Preparation of P6P-15A,15A
Figure imgf000037_0002
[0160] To a solution of l,6-bis(diphenylphosphino)hexane (0.355 g, 1.00 mmol) in acetonitrile (5 mL) was added 2-chloro-N-dodecylacetamide (0.427 g, 2.10 mmol). The solution was flushed with argon, heated to reflux, and stirred for 24.5 hours. After cooling to room temperature, the excess solvent was removed from the flask using rotary evaporation. The resulting solid was triturated with cyclohexane (-15 mL) at 45 °C for 1 hour and then isolated by vacuum filtration, resulting in P6P-15A,15A as a white, powdery solid (0.659 g, 86.2%); rH NMR (400 MHz, chloroform^/), 5 9.31 (t, J= 5.4 Hz, 2H), 8.02-7.96 (m, 8H), 7.74-7.70 (m, 4H), 7.65-7.61 (m, 8H), 4.70 (d, J = 14.4 Hz, 4H), 3.18 (m, 4H), 3.01 (q, J = 6.2 Hz, 4H), 1.62 (m, 8H), 1.27-1.14 (m, 40H), 0.85 (t, J = 6.9 Hz, 6H); 13C{JH} NMR (100.6 MHz, chloroform- d), 5 162.8 (d, J= 5.3 Hz), 134.7 (d, J= 3.4 Hz), 133.5 (d, J= 10.1 Hz), 130.1 (d, J= 12.5 Hz), 118.4 (d, J= 85.3 Hz), 40.2, 32.0, 29.9 (d, J= 52.5 Hz), 29.73, 29.71, 29.70, 29.6, 29.4, 29.3, 29.1, 28.7 (d, .7 = 16.9 Hz), 27.0, 22.6 (d, ,7= 51.1 Hz), 22.8, 21.1, 14.2; 31P{1H} NMR (162.0 MHz, chloroform-c/) 825.56. HRMS (ESI+): 453.3169, C58H88N2O2P2 [M-2C1]2+ requires 453.3155.
[0161] The synthesis schemes, structures, and yields of the exemplary compounds above are summarized in Scheme 3.
[0162] Scheme 3
Figure imgf000038_0001
[0163] 2. Testing methods:
[0164] Decomposition Studies
[0165] Samples were analyzed using 400 MHz JEOL spectrophotometer, specifically focusing on 31P NMR (162 Hz Larmor frequency for 31P NMR). The internal standard was comprised of 2 mg/mL solution of sodium hypophosphite pentahydrate (NaH2PO2*5H2O) in deionized water. To a 5 mb sample vial, 10 mg of each compound and 0.5 mL of the internal standard was added. The sample was vial was placed under hot running water for roughly 5 mins, and consecutively mixed on a Fischer Digital Vortex Mixer for 30 seconds. This process was repeated until all the material was in solution. A 0.5 mL aliquot of buffer was added to each sample vial and was promptly transferred to an NMR tube and then placed in the NMR for analysis. Data was collected at 5 minutes, 1 hour, 5 hours and 24 hours for each sample. This procedure was performed with buffers of pH = 4, 6, 7, and 10. The organic components were extracted with dichloromethane and then characterized via HRMS; the decomposition products were determined by mass. [0166] Biological Assays
[0167] For all biological assays, laboratory strains of methicillin-susceptible Staphylococcus aureus MSSA (SH1000), Enterococcus faecalis (OG1RF), Escherichia coli (MC4100), Pseudomonas aeruginosa (PAO1), Acinetohacter baumannii (ATCC 17948), community-acquired methicillin-resistant Staphylococcus aureus CA-MRSA (USAS 00-0114), and hospital-acquired methicillin-resistant Staphylococcus aureus HA-MRSA (ATCC 33591) were grown with shaking at 37 °C overnight from freezer stocks in 5 mL of the indicated media: SH1000, OG1RF, MC4100, USA300-0114, and PAO1 were grown in BD™ Mueller-Hinton broth (MHB), whereas ATCC 33591 was grown in BD™ tryptic soy broth (TSB). Optical density (OD) measurements were obtained using a SpectraMax iD3 plate reader (Molecular Devices, United States).
[0168] Minimum Inhibitory Concentration (MIC)
[0169] The prepared library of compounds was assessed for performance as antibacterial disinfectants by determining the minimum inhibitory concentration (MIC) for each against a panel of bacteria. The bacteria selected for the study were chosen to evaluate the broad spectrum antimicrobial performance of the QPCs, and therefore, included both Gram-positive and Gramnegative bacterial strains, including those with known antimicrobial resistance mechanisms. These bacteria include the Gram-positive strains of methicillin-susceptible Staphylococcus aureus [MSSA; SH1000], community-acquired methicillin-resistant Staphylococcus, aureus [CA-MRSA; USA 300-0114], hospital-acquired methicillin-resistant Staphylococcus aureus [HA-MRSA; ATCC 33591], an Enterococcus faecalis [OG1RF], and the Gram-negative strains of Escherichia coli [MC4100], Acinetobacter baumannii [ATCC 17498] and Pseudomonas aeruginosa [PAO1],
[0170] Compounds were serially diluted two-fold from stock solutions (1.0 mM) to yield twelve 100 pL test concentrations, wherein the starting concentration of DMSO was 2.5%. Overnight 5. aureus, E. faecalis, E. coli, P. aeruginosa, A. baumannii, USA300-0114 (CA- MRSA), and ATCC 33591 (HA-MRSA) cultures were diluted to ca. 106 CFU/mL in MHB or TSB and regrown to mid-exponential phase, as determined by optical density recorded at 600 nm (OD600). All cultures were then diluted again to ca. 106 CFU/mL and 100 pL were inoculated into each well of a U-bottom 96-well plate containing 100 pL of compound solution. Plates were incubated statically at 37 °C for 48 hours upon which wells were evaluated visually for bacterial growth. The MIC was determined as the lowest concentration of compound resulting in no bacterial growth visible to the naked eye, based on the highest value in three independent experiments. Aqueous DMSO controls were conducted as appropriate for each compound.
[0171] Red Blood Cell (RBC) Lysis Assay (lysis2o)
[0172] The potential cytotoxicity of the compounds was also considered with the evaluation of hemolysis (lysis20) assays with 20% lysis of red blood cells serving as an indicator of potentially toxic compounds.
[0173] RBC lysis assays were performed on mechanically defibrinated sheep blood (Hemostat Labs: DSB030). An aliquot of 1.5 mL blood was placed into a microcentrifuge tube and centrifuged at 3,800 rpm for ten minutes. The supernatant was removed, and the cells were resuspended with 1 mL of phosphate-buffered saline (PBS). The suspension was centrifuged as described above, the supernatant was removed, and cells were resuspended 4 additional times in 1 mL PBS. The final cell suspension was diluted twenty -fold with PBS.
[0174] Compounds were serially diluted with PBS two-fold from stock solutions (1.0 mM) to yield 100 pL of twelve test concentrations on a flat-bottom 96-well plate (Corning, 351172), wherein the starting concentration of DMSO was 2.5%. To each of the wells, 100 pL of the twenty-fold suspension dilution was then inoculated. The concentration of DMSO in the first well was 2.5%, resulting in DMSO-induced lysis at all concentrations >63 pM. TritonX (1% by volume) served as a positive control (100% lysis marker) and sterile PBS served as a negative control (0% lysis marker). Samples were then placed in an incubator at 37 °C and shaken at 200 rpm. After 1 hour, the samples were centrifuged at 3,800 rpm for ten minutes.
[0175] The absorbance of the supernatant was measured with a UV spectrometer at a 540 nm wavelength. The concentration inducing 20% RBC lysis was then calculated for each compound based upon the absorbance of the TritonX and PBS controls. Aqueous DMSO controls were conducted as appropriate for each compound.
[0176] Mitochondrial Toxicity Assay
[0177] Mitochondrial toxicity was evaluated using a Promega Mitochondrial ToxGlo™ kit. Human hepatocellular carcinoma cells (HepG2) and were cultured in RPML1640 medium containing 10% FBS at 37 °C and 5% CO2. Cells were seeded at a density of 2,500 cells/well in 384 well tissue culture plates in either glucose (10 mM) or galactose (10 mM) supplemented media and were incubated overnight to allow for cell adherence. Cells were rinsed and replaced with serum-free media prior to experimentation. The Mitochondrial ToxGlo™ assay was performed in accordance with manufacturer instructions. Cells were incubated with test compounds for 90 minutes prior to assay per the manufacturer’s protocol. Cells were incubated for 30 minutes with a cell impermeable, fluorogenic substrate. Cell integrity was measured by subsequent fluorescence (Ex/Em 485/525 nm) produced by necrosis-associated protease activity upon the substrate. Lysis buffer was then added, and net ATP levels were determined by luminescence measurement from a luciferase reporter. Compounds were serially diluted twofold from stock solutions (1.0 mM) to yield twelve 100 pL test concentrations, wherein the starting concentration of DMSO was 0.5%. Mitochondrial toxicity, per the manufacturer guidelines, was defined as a greater than 20% decrease in the ATP measure with a less than 20% increase in cytotoxicity.
[0178] Single Crystal X-ray Crystallography
[0179] X-ray intensity data for P3P-8A,8A (CCDC reference number 2196105) were collected on a Rigaku XtaLAB Synergy-S diffractometer using an HyPix-6000HE HPC area detector. The data collection employed Cu-Ka radiation (X = 1.54184 A) and data was collected at a temperature of 100 K. The intensity data were integrated using CrysAlisPro, which produced a listing of unaveraged F2 and o(F2) values. The structure solution was determined using SHELXT and refinement was conducted using SHELXLwith anisotropic refinement of the thermal parameters for non-hydrogen atoms. Hydrogen atoms were placed using a riding model and refined isotropically.
[0180] 3. Results
[0181] The inventors successfully synthesized the soft QPCs described herein, including 14 amide-based soft QPCs as described above and illustrated in FIG .2, varying the distance between the phosphonium cations and alkyl chain lengths. In the first step of the synthesis, the amide-bearing lipophilic group was constructed by exposing long-chained amines to chloroacetyl chloride. The resulting N-alkyl-2-chloroacetamide building blocks were then reacted with two bisphosphine nucleophiles: l,3-bis(diphenylphosphino)propane (dppp) and 1,6- bis(diphenylphosphino)hexane (dpph), which are readily available. The bisalkylation reactions were successful under standard SN2 conditions (acetonitrile, reflux, 24 h, argon atmosphere), affording the 14 QPCs in good yields (40-98% as shown in Scheme 3). Purification of the crude products was accomplished via trituration with cyclohexane. [0182] As described above, a naming system using a PzP format is used. The number z reflects the number of carbons linking the two phosphorus atoms (m = 3, 6; P3P or P6P with propyl or hexyl connections for Li, respectively). The length of a chain containing the amide group is identified by the total number of atoms in the chain (#), which includes the number of alkyl carbons (n) plus three atoms reflecting the acetamide residue (total # = n + 3) when L2 has one carbon atom. The number “3” comes from one carbon atoms plus one carbon and one nitrogen in the carbonyl group. Finally, the addition of the letter A reflects the inclusion of amide functionality in the chain, resulting in a format of PzP-#A,#A.
[0183] Surprisingly, synthetic investigations using N-alkyl-2-bromoacetamide building blocks led to unpredictable production of impurities in addition to the desired product. The similarity in structural features of the impurities to the product as determined by JH NMR spectroscopy is suspected to arise from counterion modification. Attempts to avoid the formation of these impurities or separate these impurities from the desired products were unsuccessful in the inventors’ trials performed.
[0184] To obtain further structural insight into these compounds, colorless needle crystals of P3P-8A,8A suitable for single crystal X-ray analysis were grown by layering of diethyl ether onto a solution of the compound in acetonitrile at room temperature. Table 1 shows the single crystal X-ray crystallography data of this compound, while FIG. 3 shows ORTEP diagrams. Detailed crystallographic data for this structure can be obtained free of charge by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK.
[0185] Table 1.
Figure imgf000042_0001
Figure imgf000043_0001
[0186] FIG. 3 shows ORTEP diagrams for P3P-8A,8A (CCDC Refence Number 2196105). Bond lengths for select P-C, N-C, O-C and C-C bonds are labelled in A and the O-C- C and angle for the amide is labeled in degrees. Thermal ellipsoids are shown at the 50% probability level. Hydrogens and co-crystalized solvents are omitted for clarity.
[0187] Interestingly, in the solid state, the two nonpolar groups are disposed in a nearly parallel trajectory, and two of the aromatic rings are nearly coplanar, as shown in FIG. 3.
Evaluation of the experimental bond angles and lengths for the amide groups present in P3P- 8A,8A demonstrates a structure which follows suit with a simple model amide, formamide. This structural similarity indicates the amide is unperturbed by the proximity of the phosphonium center and, by extension, should maintain the expected hydrolyzable functionality of the amide. [0188] The stability of these compounds was assessed through exposure of P3P-9A,9A and P6P-9A,9A to deionized water as well as buffered solutions of pH 4, 6, 7, and 10. [0189] These conditions were selected to reflect different environmental conditions, including the variable pH of soil. Decomposition was determined using 31P NMR spectroscopy through the qualitative loss of QPC product signal. Solutions were prepared at a concentration of 10 mg/mL of sample with 1 mg/mL of sodium hypophosphate pentahydrate (NaH2PO2 AH2O) used as a non-reactive phosphorus-containing internal standard.
[0190] It was hypothesized that the soft QPCs would degrade rapidly under acidic conditions due to expected reactivity at the amide and similarly to the fate of soft QACs, while displaying relative stability in neutral and basic conditions. However, instead of observing the decomposition of the QPCs at pH = 4, both P3P-9A,9A and P6P-9A,9A immediately precipitated out of solution leaving no observable 31P NMR signals for product or decomposition products, as shown in FIG. 4A and FIG. 5A corresponding to Compounds P3P-9A,9A and P6P- 9A,9A, respectively. 'H NMR spectroscopy and LCMS analysis confirmed the precipitate was the corresponding intact P3P-9A,9A and P6P-9A,9A products: no decomposition products were observed from amide hydrolysis.
[0191] Referring to FIGS. 4B-4C and FIGS. 5B-5C corresponding to Compounds P3P- 9A,9A and P6P-9A,9A, respectively, at the more mildly acidic conditions of pH = 6 and neutral conditions of pH = 7 both P3P-9A,9A and P6P-9A,9A remained unchanged over the 24 h testing window.
[0192] Referring to FIG. 4D, under basic conditions of pH = 10, P3P-9A,9A showed the emergence of a new phosphorus signal at the 5 h mark which continued to develop over 24 h. Referring to FIG. 5D, interestingly, and in contrast to P3P-9A,9A and the inventors’ previous amide-containing amphiphile results, a ~24-hour stability was observed for P6P-9A,9A at pH = 10. Over the course of a week the samples of P6P-9A,9A at pH = 10 began to form a precipitate which was identified by LCMS as the oxidized dpph.
[0193] In light of these observations for P3P-9A,9A and P6P-9A,9A the stability studies were repeated for P3P-12A,12A and P6P-12A,12A under neutral (pH = 7) and basic (pH = 10) conditions. FIG. 6A illustrates the change in bioactivity before and after decomposition. FIGS. 6B-7B show the profiles of an exemplary compound, P3P-12A,12A, as monitored by 31P NMR spectroscopy after exposure at pH of 7 and 10, respectively, for a period of time over 24 hours. FIGS. 7A-7B show the profiles of an exemplary compound, P6P-12A,12A, as monitored by 31P NMR spectroscopy after exposure at pH of 7 and 10, respectively, for a period of time over 24 hours.
[0194] In neutral conditions, both P3P-12A,12A and P6P-12A,12A remained unchanged over a 24-hour period (FIG. 6B and FIG. 7A). Under basic conditions (e.g., at pH of 10), both P3P-12A,12A and P6P-12A,12A completely decomposed within 1 h and 5 h respectively (FIG. 6C and FIG. 7B).
[0195] In support of this 31P NMR data, LCMS analysis indicated that the soft QPCs underwent hydrolysis at the phosphonium center, rather than at the amide; the amide side chain was in fact observed completely intact. The reaction is illustrated in FIG. 6A. The species from mass spectroscopy are illustrated in FIG. 8. Referring to FIG. 8, the species in the HRMS (ESI+) Total Ion Chromatogram of extracted P3P-12A,12A decomposition products include Species (A) having a chemical formula C11H24NCE, with m/z 186.1853 (100.0%), 187.1886 (11.9%); and Species (B) having a chemical formula C27H27O2P2+ with m/z: 445.1481 (100.0%), 446. 1515 (29.2%), 447.1548 (4. 1%). The species in the HRMS (ESI+) Total Ion Chromatogram of extracted P6P-12A,12A decomposition products include Species (A) having a chemical formula Ci 1H24NO with m/z 186.1853 (100.0%), 187.1886 (11.9%); Species (C) having a chemical formula C3oH3302P2+ with m/z: 445.1481 (100.0%), 446.1515 (29.2%), 447.1548 (4.1%).
[0196] The major phosphorus decomposition product was identified to be a corresponding phosphine oxide as shown in FIG. 6A and as shown in FIG. 8 except that the phosphine oxide is not ionized as tested in mass spectroscopy. Both the P3P- and P6P-phosphine oxides were prepared according to previous literature reports and were confirmed to be a match in both the mass and elution time observed in the LCMS for the decomposition products.
[0197] Referring to FIG. 9, the compounds provided in the present disclosure may be used as an disinfectant applied to a surface, or an ingredient or a stabilizer for a pharmaceutical composition. The compounds can be degraded in a water treatment process. The compound and the reaction shown in FIG. 9 are for illustration only, and the uses can be applicable to any compound provided in the present disclosure.
[0198] The behavior of these compounds at different pH levels suggests at least two possible treatment avenues to remediate persistence of these QPCs in aqueous waste streams: filtration of precipitated products at low pH and decomposition at higher pH. Prior reports using photocatalytic decomposition methods have indicated a greater susceptibility for QPC degradation compared to QACs, suggesting a third potential decomposition pathway for these materials outside of the scope tested herein.
[0199] The bioactivities of the compounds were assessed via minimum inhibitory concentration (MIC) and hemolysis (lysis20) assays, wherein the latter was used as a proxy for cytotoxicity. In addition to the soft bisQPC compounds, the inventors’ previously synthesized best-in-class bisQPC (P6P-10,10) and two commercial QACs (benzalkonium chloride [BAC;
70% benzyldimethyldodecylammonium chloride and 30% benzyldimethyltetradecylammonium chloride] and didecyldimethylammonium chloride [DDAC]) were used as controls for comparison. The compound P6P-10, 10 has the following chemical structure:
Figure imgf000046_0001
P6P-10,10
[0200] To determine the MIC values, the compounds were each screened against a panel of seven bacterial strains, including four gram-positive strains (methicillin-susceptible Staphylococcus aureus [MSSA; SH1000], community-acquired methicillin-resistant Staphylococcus aureus [CA-MRSA; USA 300-0114], hospital-acquired methicillin-resistant Staphylococcus aureus [HA-MRSA; ATCC 33591]), and Enterococcus faecalis [OG1RF], as well as three gram-negative strains (Escherichia coli [MC4100], Acinetobacter baumannii [ATCC 17948], an Pseudomonas aeruginosa [PAO1]). To evaluate the hemolysis activities, the compound concentrations resulting in 20% red blood cell (RBC) lysis (ly sis2o) were determined. The results of the MIC and hemolysis assays are reported in Table 2.
[0201] Table 2 shows antimicrobial activity (MIC) and hemolysis (lysis2o) of the prepared bisQPCs compared to commercially available QACs against gram-positive strains methicillin-susceptible A aureus (MSSA), community-acquired methicillin-resistant S. aureus (CA-MRSA) and hospital-acquired methicillin-resistant 5. aureus (HA-MRSA), and E. faecalis as well as gram-negative strains E. coli, A. baumannii, and P. aeruginosa. [0202] Table 2
Figure imgf000047_0001
[0203] Several interesting bioactivity trends emerge for these soft QPCs. Strong antimicrobial activities were observed for many compounds, most notably P3P-12A,12A and P6P-12A, 12A. The strongest determinant of biological activity in the amphiphilic structures was the length of the non-polar side chain, wherein amphiphiles with twelve-carbon side chains displayed the most potent activities. There is a small but measurable advantage in the bioactivity of the 6-carbon linker (P6P) over the 3-carbon linker (P3P), as P6P-12A, 12A boasts MICs between 2-8 pM against the entire panel of bacteria and compares quite favorably against the three controls. P6P-12A,12A has improved performance over BAC, particularly against gramnegative pathogens, with 4-16x greater activity. The potency of P6P-12A,12A is narrowly improved compared to DDAC and approaches the performance of the inventors’ best amphiphile prepared to date, P6P-10,10. The top exemplary compounds P3P-12A,12A and P6P-12A, 12A also performed well against resistant gram-positive S. aureus strains as well, with no increase in MIC values for either HA- or CA-MRSA, as compared to MS SA.
[0204] Next, probing the cytotoxicity of the molecules, the lysis2o concentration for each compound was also determined. A general correlation between bioactivity and toxicity is observed, with the compounds having the most effective bioactivity also having a strong RBC lysis. However, a few compounds indicated a more measured balance between bioactivity and RBC lysis, which could indicate that it may be possible to tailor these compounds further to have high antimicrobial efficacy with lower risks of toxicity. For example, P6P-10A,10A did not demonstrate strong RBC lysis (125 pM) yet maintained low micromolar activity against the bacterial panel. Therefore, to optimize the balance between antimicrobial activity and low mammalian cytotoxicity, compounds of somewhat shorter chain length (# = 9-10) and/or compound mixtures may be used.
[0205] Further, it should be noted that the lysis2o values of the hydrolysis products (i.e., the phosphine oxides of P3P and P6P) were found to be >125 pM, reflecting only modest toxicity in this assay; antibacterial activity was likewise not observed at concentrations tested. These noticeable bioactivity differences between the bisQPCs and the decomposition products are viewed as desirable trends towards the goal of mitigating the development of antimicrobial resistance mechanisms due to environmental persistence.
[0206] In addition to RBC lysis, mitochondrial toxicity was assessed. Quaternary phosphonium species are known to target the mitochondria of human cells. Due to this known activity, the two of the best-in-class soft QPCs, P6P-12A,12A and P3P-12A,12A, were tested for mitochondrial toxicity in human hepatocellular carcinoma cells (HepG2). Pleasingly, no mitochondrial toxicity was observed for the compounds tested.
[0207] As illustrated and described above, the present disclosure provides the novel phosphonium -based compounds such as a series of 14 exemplary biscationic amphiphilic structures above as soft amphiphiles. Multiple compounds with amide-bearing side chains demonstrated single-digit micromolar antimicrobial activity, highlighted by the most effective compounds in this series, P6P-12A,12A and P3P-12A,12A. These compounds demonstrate desirable soft characteristics, with stabilities of at least 24 h in deionized water and pH = 7 buffer, as would be intended during product use as a disinfectant, but hydrolyze upon longer exposure to basic conditions. With observed basic hydrolysis at the phosphonium center, these compounds are clearly susceptible to environmental breakdown. The observed hydrolysis products, for example, P3P-oxide and P6P-oxide, showed no bioactivity or toxicity at the levels tested, indicating complete deactivation of the bisQPCs under hydrolysis conditions. With designed instability, options for toxicity minimization, and a broad suite of antimicrobial activity, the prepared soft QPCs represent a promising class of soft antimicrobial structures.
[0208] The compound and the composition provided in the disclosure have significant advantages. They have excellent abilities to kill, prevent, or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, and can attenuate the severity of a microbial infection, and can also prevent or inhibit formation of a biofilm or eradicate pre-established biofilms. Surprisingly, such a biscationic quaternary phosphonium compound comprising amide substitution has good stability, particularly in neutral and mildly acidic conditions. Such a compound undergoes hydrolysis and decomposes at the phosphonium center, leading to inactive phosphine oxide products. So the compounds and the compositions are environmental-friendly and can be easily degraded through a process such as water treatment. The uses of these compounds or compositions will greatly reduce the chance for microorganisms to develop resistance to these compounds.
[0209] Although the subject matter has been described in terms of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be construed broadly, to include other variants and embodiments, which may be made by those skilled in the art.

Claims

What is claimed is:
1. An antimicrobial composition comprising a compound having a formula
Figure imgf000050_0001
wherein:
Li and L2 each is a linking group selected from the group consisting of a C1-10 alkyl, a Ci- 10 cycloalkyl, an aryl, and any combination thereof, each of Ri, R2, R3, and R4 is selected from the group consisting of a C1-12 alkyl, a C1-12 cycloalkyl, and an aryl group, which is unsubstituted or optionally substituted with a functional group selected from the group consisting of -OH, -OR’, -NH2, -NHR’, -NR’ 2, -SH, -SR’, -O- C(O)R’, -C(O)OR’, -C(O)R’, -C(O)NR’R’, -CF3, -OCF3, halogen, and any combination thereof;
R’ is H or a C1-4 alkyl; each of Rs, Re, R7, and Rs is H or a C1-20 alkyl, wherein at least one of Rs and Re is a C1-20 alkyl and at least one of R7 and Rs is a C1-20 alkyl,
X is selected from the group consisting of a halogen, a tosylate, a hydrocarbonate, a carbonate, a sulfate, and an acetate; and m and y are integers being 1 or 2, wherein m * y =2.
2. The antimicrobial composition of claim 1, wherein Li is a C2-10 alkyl.
3. The antimicrobial composition of claim 1, wherein Li is an alternating structure comprising a C2-10 alkyl and a C4-6 cycloalkyl.
4. The antimicrobial composition of claim 1, wherein L2 is a C1-6 alkyl.
5. The antimicrobial composition of claim 1, wherein each of Ri, R2, Rs, and R4 is a C1-6 alkyl or a cyclohexyl, or a phenyl.
6. The antimicrobial composition of claim 1, wherein Ri, R2, R3, and R4 are the same, Rs and R7 are the same, and Rs and Rs are the same.
7. The antimicrobial composition of claim 1, wherein Rs and R7 are the same and are an alkyl having a formula CnH 2n+i, wherein n is an integer in the range of from 2 to 20, and Rs and Rs are the same and are H or methyl.
8. The antimicrobial composition of claim 7, wherein the compound having the formula (I) has a formula:
Figure imgf000051_0001
the compound having formula (II) has a formula:
Figure imgf000051_0002
wherein Ri, R2, R3, and R4 are a same group R selected from the group consisting of a C1-6 alkyl, a C1-6 cycloalkyl, and phenyl.
9. The antimicrobial composition of claim 8, wherein n is in a range of from 5 to 12.
10. The antimicrobial composition of claim 8, wherein Li is a C2-10 alkyl and L2 is a C1-6 alkyl.
11 The antimicrobial composition of claim 1, wherein X is a halogen selected from F, Cl, Br, and I, m is 2, and y is 1.
12. The antimicrobial composition of claim 1, wherein the compound is selected from the group consisting of:
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
and analogs of Compounds Cl to C 14 wherein -Ph is replaced with methyl, ethyl, or cyclohexyl.
13. The antimicrobial composition of claim 1, wherein the antimicrobial composition is a disinfectant configured to be applied to a surface of an object in need thereof.
14. The antimicrobial composition of claim 1, wherein the antimicrobial composition is a pharmaceutical composition configured to be administrated to a subject in need thereof.
15. The antimicrobial composition of claim 14, wherein the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an active ingredient.
16. The antimicrobial composition of claim 14, wherein the pharmaceutical composition comprises an effective amount of the compound having the formula (I) or (II) as an additive for stabilizing the pharmaceutical composition.
17. A method of making the antimicrobial composition of claim 1 comprising preparing the compound having the formula (I) or (II).
18. The method of claim 17, further comprising mixing an effective amount of the compound having the formula (I) or (II) and a carrier.
19. A method of killing, preventing, or inhibiting microbial growth, comprising applying the antimicrobial composition of claim 1 to a surface of an object in need thereof.
20. A method of killing, preventing, or inhibiting microbial growth, comprising administrating the antimicrobial composition of claim 1 to a subject in need thereof.
21. A compound for antimicrobial use having having a formula
Figure imgf000055_0001
wherein:
Li and L2 each is a linking group selected from the group consisting of a C1-10 alkyl, a Ci- 10 cycloalkyl, an aryl, and any combination thereof, each of Ri, R2, R3, and R4 is selected from the group consisting of a C1-12 alkyl, a C1-12 cycloalkyl, and an aryl group, which is unsubstituted or optionally substituted with a functional group selected from the group consisting of -OH, -OR’, -NH2, -NHR’, -NR’ 2, -SH, -SR’, -O- C(O)R’, -C(O)OR’, -C(O)R’, -C(O)NR’R’, -CF3, -OCF3, halogen, and any combination thereof;
R’ is H or a C1-4 alkyl; each of Rs, Re, R7, and Rs is H or a C1-20 alkyl, wherein at least one of Rs and Re is a C1-20 alkyl and at least one of R7 and Rs is a C1-20 alkyl,
X is selected from the group consisting of a halogen, a tosylate, a hydrocarbonate, a carbonate, a sulfate, and an acetate; and m and y are integers being 1 or 2, wherein m * y =2.
22. The compound of claim 21, wherein Li is a C2-10 alkyl.
23. The compound of claim 21, wherein Li is an alternating structure comprising a C2-10 alkyl and a C4-6 cycloalkyl.
24. The compound of claim 21, wherein L2 is a C1-6 alkyl.
25. The compound of claim 21, wherein each of Ri, R2, R3, and R4 is a C1-6 alkyl or a cyclohexyl, or a phenyl.
26. The compound of claim 21, wherein Ri, R2, R3, and R4 are the same, Rs and R7 are the same, and Re and Rs are the same.
27. The compound of claim 21, wherein Rs and R7 are the same and are an alkyl having a formula CnH 2n+i, wherein n is an integer in the range of from 2 to 20, and Re and Rs are the same and are H or methyl.
28. The compound of claim 27, wherein the compound having the formula (I) has a formula:
Figure imgf000056_0001
the compound having formula (II) has a formula:
Figure imgf000056_0002
wherein Ri, R2, R3, and R4 are a same group R selected from the group consisting of a C1-6 alkyl, a C1-6 cycloalkyl, and phenyl.
29. The compound of claim 28, wherein n is in a range of from 5 to 12.
30. The compound of claim 28, wherein Li is a C2-10 alkyl and L2 is a C1-6 alkyl.
31. The compound of claim 21, wherein X is a halogen selected from F, Cl, Br, and I, m is 2, and y is 1.
32. The compound of claim 21, wherein the compound is selected from the group consisting of:
Figure imgf000057_0001
Figure imgf000058_0001
and analogs of Compounds Cl to C 14 wherein -Ph is replaced with methyl, ethyl, or cyclohexyl.
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