CA2972079A1 - A potent synthetic inorganic antibacterial with activity against drug-resistant pathogens - Google Patents
A potent synthetic inorganic antibacterial with activity against drug-resistant pathogens Download PDFInfo
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- CA2972079A1 CA2972079A1 CA2972079A CA2972079A CA2972079A1 CA 2972079 A1 CA2972079 A1 CA 2972079A1 CA 2972079 A CA2972079 A CA 2972079A CA 2972079 A CA2972079 A CA 2972079A CA 2972079 A1 CA2972079 A1 CA 2972079A1
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- phosphopyricin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
A novel inorganic antibiotic, phosphopyricin, has antibiotic activity against the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). We show that this potent antibiotic is bactericidal, and exhibits low toxicity in an acute dose assay in mice. As a synthetic compound that does not occur naturally, phosphopyricin would be evolutionarily foreign to microbes, thereby slowing the evolution of resistance. In addition, it degrades in light, and will break down in the general environment where strong selective pressures imposed by antibiotic residuals are known to accelerate antibiotic resistance. Phosphopyricin represents an innovation in antimicrobials, having a synthetic core, and a photosensitive chemical architecture that would reduce accumulation in the environment.
Description
I
A POTENT SYNTHETIC INORGANIC ANTIBACTERIAL WITH ACTIVITY AGAINST
DRUG-RESISTANT PATHOGENS
BACKGROUND OF THE INVENTION
Widespread and increasing antibiotic resistance among the "ESKAPE"
pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.)1 has become a critical problem for healthcare. Organic natural products, which have provided the core set of current therapeutics, have been central to our ability to control these multi-drug resistant pathogens; however, microbes have been harnessing antibiotics for competition for billions of years, and as a result, mechanisms for resisting and tolerating antibiotics have been evolving for just as long3.4. For each new antimicrobial natural product discovered, at least one mechanism of resistance is already present in the general environment, greatly accelerating the emergence of antibiotic resistance5. In fact, over evolutionary time, microbes have likely encountered most naturally occurring organic and inorganic molecules in the environment, and have evolved specific strategies to contend with those that are immediately toxic3. Compounding the antibiotic resistance problem are the high concentrations of anthropogenically-generated antibiotic residuals that are deposited into the environment, resulting in strong selective pressures for the evolution of resistance".
One possible strategy for overcoming these obstacles and regaining some ground on the antibiotic resistance problem is to develop synthetic antimicrobials whose chemical architectures do not occur naturally, and would thus be evolutionarily foreign to bacteria. In addition, if such antimicrobials degraded rapidly in the general environment, strong selective pressure for the evolution of resistance would be reduced significantly. Organophosphorus compounds remain an untapped pool of chemical architectures that could possess novel chemistries and differential binding affinities to diverse microbiological targets. Also, because phosphorus has a similar electronegativity to carbon, the chemistry of low valent phosphorus often resembles that of carbon, making it highly amenable for syntheses of compounds for biological
A POTENT SYNTHETIC INORGANIC ANTIBACTERIAL WITH ACTIVITY AGAINST
DRUG-RESISTANT PATHOGENS
BACKGROUND OF THE INVENTION
Widespread and increasing antibiotic resistance among the "ESKAPE"
pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.)1 has become a critical problem for healthcare. Organic natural products, which have provided the core set of current therapeutics, have been central to our ability to control these multi-drug resistant pathogens; however, microbes have been harnessing antibiotics for competition for billions of years, and as a result, mechanisms for resisting and tolerating antibiotics have been evolving for just as long3.4. For each new antimicrobial natural product discovered, at least one mechanism of resistance is already present in the general environment, greatly accelerating the emergence of antibiotic resistance5. In fact, over evolutionary time, microbes have likely encountered most naturally occurring organic and inorganic molecules in the environment, and have evolved specific strategies to contend with those that are immediately toxic3. Compounding the antibiotic resistance problem are the high concentrations of anthropogenically-generated antibiotic residuals that are deposited into the environment, resulting in strong selective pressures for the evolution of resistance".
One possible strategy for overcoming these obstacles and regaining some ground on the antibiotic resistance problem is to develop synthetic antimicrobials whose chemical architectures do not occur naturally, and would thus be evolutionarily foreign to bacteria. In addition, if such antimicrobials degraded rapidly in the general environment, strong selective pressure for the evolution of resistance would be reduced significantly. Organophosphorus compounds remain an untapped pool of chemical architectures that could possess novel chemistries and differential binding affinities to diverse microbiological targets. Also, because phosphorus has a similar electronegativity to carbon, the chemistry of low valent phosphorus often resembles that of carbon, making it highly amenable for syntheses of compounds for biological
2 applications8. Phosphorus-containing antibiotics have been synthesized previously, including fosfomycin8, clindamycinw, and torezolid11; however, these molecules are organophosphates that may be susceptible to existing antibiotic resistance mechanisms.
In contrast to organophosphates, phosphine derivatives remain underexplored as potential antimicrobials12,13. These compounds provide a large pool of potentially biologically active molecules with vast structural diversity, mainly due to the ease with which they can be modified chemically". Phosphines also provide a simple means of introducing metal complexes into biologically active molecules, via coordination of the lone pair, further expanding the range of inorganic groups that can be explored.
Phosphine derivatives have been shown to have potential as anti-cancer drugs13, and even to have potential as antibiotics12. Some have been developed into resistance protein inhibitors, including avibactam, clavulanic acid, tazobactam, and sulbactam18-18. One recent study synthesized phosphine derivatives of ciprofloxacin and norfloxacin, yielding an additional class of broad-spectrum antibiotics effective against P. aeruginosa, S. aureus, K. pneumoniae, and E. co/P.9. The use of phosphorus-containing functional groups can yield compounds that bind strongly to certain enzymes where a carbon or nitrogen analog would not. Non-phosphate, organophosphorus compounds therefore have enormous capacity to provide new chemical architectures for the development of next-generation antibiotics.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a compound comprising the structure of Formula (I):
nO R' /¨ R = aryl,heteroaryl, OC ______ ,W
alkynyl, vinyl, allyl.
0 RS alkyl, aryl (I)
In contrast to organophosphates, phosphine derivatives remain underexplored as potential antimicrobials12,13. These compounds provide a large pool of potentially biologically active molecules with vast structural diversity, mainly due to the ease with which they can be modified chemically". Phosphines also provide a simple means of introducing metal complexes into biologically active molecules, via coordination of the lone pair, further expanding the range of inorganic groups that can be explored.
Phosphine derivatives have been shown to have potential as anti-cancer drugs13, and even to have potential as antibiotics12. Some have been developed into resistance protein inhibitors, including avibactam, clavulanic acid, tazobactam, and sulbactam18-18. One recent study synthesized phosphine derivatives of ciprofloxacin and norfloxacin, yielding an additional class of broad-spectrum antibiotics effective against P. aeruginosa, S. aureus, K. pneumoniae, and E. co/P.9. The use of phosphorus-containing functional groups can yield compounds that bind strongly to certain enzymes where a carbon or nitrogen analog would not. Non-phosphate, organophosphorus compounds therefore have enormous capacity to provide new chemical architectures for the development of next-generation antibiotics.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a compound comprising the structure of Formula (I):
nO R' /¨ R = aryl,heteroaryl, OC ______ ,W
alkynyl, vinyl, allyl.
0 RS alkyl, aryl (I)
3 wherein:
"W" is tungsten;
"C" is carbon;
"0" is oxygen;
"P" is phosphorus;
R is selected from substituted aryl, alkynyl, vinyl, cyclic vinyl, substituted vinyl, ally!, cyclic ally1 and substituted ally!: and R' is alkyl or aryl.
The compound may comprise the structure of Formula (11a) or (11b):
0 0 Ph 0 Ph C 0 ph /Ph OC¨W __ P OC __ W ___ P
/ /
0 cl 0 cl (11a) (fib) R
N S
= __ Ph X = H, OMe, LLJ NEt2, CH3, Ph X
The compound may comprise the structure of Formula (III):
"W" is tungsten;
"C" is carbon;
"0" is oxygen;
"P" is phosphorus;
R is selected from substituted aryl, alkynyl, vinyl, cyclic vinyl, substituted vinyl, ally!, cyclic ally1 and substituted ally!: and R' is alkyl or aryl.
The compound may comprise the structure of Formula (11a) or (11b):
0 0 Ph 0 Ph C 0 ph /Ph OC¨W __ P OC __ W ___ P
/ /
0 cl 0 cl (11a) (fib) R
N S
= __ Ph X = H, OMe, LLJ NEt2, CH3, Ph X
The compound may comprise the structure of Formula (III):
4 0 Ph /C / ph OC¨W
(Ill) According to another aspect of the invention, there is provided use of a compound as described above for treating a bacterial infection.
According to another aspect of the invention, there is provided a method of treating a bacterial infection comprising administering to an individual in need of such treatment an effective amount of a compound as described above.
According to a further aspect of the invention, there is provided a method of preparing a medicament for treating a bacterial infection comprising admixing a compound as described above with a suitable excipient.
According to yet another aspect of the invention, there is provided a compound as described above for treating a bacterial infection.
The individual may be an animal, for example a mammal, for example, a human, although as will be appreciated by one of skill in the art, uses such as for example veterinary uses are also contemplated and are within the scope of the invention.
Furthermore, as discussed herein, given the wide range of bacterial targets sensitive to the compounds of the invention, the compounds may also be used for the treatment of bacterial infections of plants, that is, for the treatment or elimination of plant pathogens.
According to another aspect of the invention, there is provided an antibacterial composition comprising a compound as described above.
According to another aspect of the invention, there is provided the use of a compound as described herein in an antibacterial composition.
According to another aspect of the invention, there is provided a method of
(Ill) According to another aspect of the invention, there is provided use of a compound as described above for treating a bacterial infection.
According to another aspect of the invention, there is provided a method of treating a bacterial infection comprising administering to an individual in need of such treatment an effective amount of a compound as described above.
According to a further aspect of the invention, there is provided a method of preparing a medicament for treating a bacterial infection comprising admixing a compound as described above with a suitable excipient.
According to yet another aspect of the invention, there is provided a compound as described above for treating a bacterial infection.
The individual may be an animal, for example a mammal, for example, a human, although as will be appreciated by one of skill in the art, uses such as for example veterinary uses are also contemplated and are within the scope of the invention.
Furthermore, as discussed herein, given the wide range of bacterial targets sensitive to the compounds of the invention, the compounds may also be used for the treatment of bacterial infections of plants, that is, for the treatment or elimination of plant pathogens.
According to another aspect of the invention, there is provided an antibacterial composition comprising a compound as described above.
According to another aspect of the invention, there is provided the use of a compound as described herein in an antibacterial composition.
According to another aspect of the invention, there is provided a method of
5 preparing an antibacterial composition comprising admixing a compound as described herein with a suitable excipient.
As will be appreciated by one of skill in the art, the antibacterial composition may be a pharmaceutical composition or a medicament for treatment of an animal, for example, a mammal, for example a human. Alternatively, the antibacterial composition may be used for the treatment of a non-human animal, for example but by no means limited to a livestock animal, a domestic animal or a wild animal.
The antibacterial composition may be formulated for agricultural use, that is, as an anti-bacterial agent against plant pathogens.
The antibacterial composition may be a disinfectant, for example but by no means limited to a spray, a gel, a powder or a solution for use in the environment, that is, ex vivo, for example, on surfaces that are to be cleaned, for example, kitchen or food preparation surfaces, floors, exterior body surfaces such as hands and the like, that is, anything that may need or benefit from disinfection prior to human contact.
The antibacterial composition may be used as a general disinfectant for surfaces, for example, on medical devices and/or medical instruments prior to contact with or insertion into patients.
As discussed herein, the compounds of the invention will break down under direct light in between about 8-16 hours. As discussed herein, that is sufficient time for the compounds of the invention to exert their effects on bacteria. Also, if it's maintained in the dark during the disinfection process, it maintains its activity.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Development of phosphopyricin (9bWC3) by derivatization.
Elimination of the phosphirene ring of antimicrobial compound 9aWC2 yielded compound 9bWC2, both of which had similar minimum inhibitory concentrations against Staphylococcus aureus K1-7. Substitution of the C2-bonded pyrrole of 9bWC2 with a thiophene, phenyl or indole reduced activity. Replacement of the C2-bonded pyrrole of 9bWC2 with a C3-bonded pyrrole resulted in phosphopyricin (9bWC3), and increased potency against S. aureus. Phosphopyricin had similar activity against the
As will be appreciated by one of skill in the art, the antibacterial composition may be a pharmaceutical composition or a medicament for treatment of an animal, for example, a mammal, for example a human. Alternatively, the antibacterial composition may be used for the treatment of a non-human animal, for example but by no means limited to a livestock animal, a domestic animal or a wild animal.
The antibacterial composition may be formulated for agricultural use, that is, as an anti-bacterial agent against plant pathogens.
The antibacterial composition may be a disinfectant, for example but by no means limited to a spray, a gel, a powder or a solution for use in the environment, that is, ex vivo, for example, on surfaces that are to be cleaned, for example, kitchen or food preparation surfaces, floors, exterior body surfaces such as hands and the like, that is, anything that may need or benefit from disinfection prior to human contact.
The antibacterial composition may be used as a general disinfectant for surfaces, for example, on medical devices and/or medical instruments prior to contact with or insertion into patients.
As discussed herein, the compounds of the invention will break down under direct light in between about 8-16 hours. As discussed herein, that is sufficient time for the compounds of the invention to exert their effects on bacteria. Also, if it's maintained in the dark during the disinfection process, it maintains its activity.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Development of phosphopyricin (9bWC3) by derivatization.
Elimination of the phosphirene ring of antimicrobial compound 9aWC2 yielded compound 9bWC2, both of which had similar minimum inhibitory concentrations against Staphylococcus aureus K1-7. Substitution of the C2-bonded pyrrole of 9bWC2 with a thiophene, phenyl or indole reduced activity. Replacement of the C2-bonded pyrrole of 9bWC2 with a C3-bonded pyrrole resulted in phosphopyricin (9bWC3), and increased potency against S. aureus. Phosphopyricin had similar activity against the
6 Gram-positive Enterococcus faecium KO2G0810 and S. mutans. Substitution of the tungsten pentacarbonyl group of phosphopyricin with either a molybdenum pentacarbonyl group or an oxide abolished activity (< 1024 pg/ml).
Figure 2. Properties of phosphopyricin. a. Minimum inhibitory concentration of phosphopyricin against Staphylococcus aureus K1-7. b. Activity of phosphopyricin against S. aureus K1-7 following exposure to 24 hours of light, as compared to dark and isopropanol (iso) controls. c. Recovery of S. aureus K1-7 from culture exposed to phosphopyricin or 9bWO2, relative to the antibiotic-free control. *p < 10-5;
**p < 10-6.
d. Optical density of S. enterica incubated in the presence of either phosphopyricin with EDTA (PP EDTA), EDTA alone (EDTA), phosphopyricin alone (PP), or with neither (culture control). Letters above error bars (s.d.) indicate statistically significantly different groups (p < 0.005).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
The acronymously named "ESKAPE" pathogens represent a group of bacteria that continue to pose a serious threat to human health, not only due to their propensity for repeated emergence, but also due to their ability to "eskape"
antibiotic treatment1.2.
The evolution of multi-drug resistance in these pathogens alone has greatly outpaced the development of new therapeutics, necessitating an alternative strategy for antibiotic development that considers the evolutionary mechanisms driving antibiotic resistance. As described herein, we synthesized a novel inorganic antibiotic, phosphopyricin, which has antibiotic activity against the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus
Figure 2. Properties of phosphopyricin. a. Minimum inhibitory concentration of phosphopyricin against Staphylococcus aureus K1-7. b. Activity of phosphopyricin against S. aureus K1-7 following exposure to 24 hours of light, as compared to dark and isopropanol (iso) controls. c. Recovery of S. aureus K1-7 from culture exposed to phosphopyricin or 9bWO2, relative to the antibiotic-free control. *p < 10-5;
**p < 10-6.
d. Optical density of S. enterica incubated in the presence of either phosphopyricin with EDTA (PP EDTA), EDTA alone (EDTA), phosphopyricin alone (PP), or with neither (culture control). Letters above error bars (s.d.) indicate statistically significantly different groups (p < 0.005).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
The acronymously named "ESKAPE" pathogens represent a group of bacteria that continue to pose a serious threat to human health, not only due to their propensity for repeated emergence, but also due to their ability to "eskape"
antibiotic treatment1.2.
The evolution of multi-drug resistance in these pathogens alone has greatly outpaced the development of new therapeutics, necessitating an alternative strategy for antibiotic development that considers the evolutionary mechanisms driving antibiotic resistance. As described herein, we synthesized a novel inorganic antibiotic, phosphopyricin, which has antibiotic activity against the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus
7 faecium (VRE). As a synthetic compound that does not occur naturally, phosphopyricin would be evolutionarily foreign to microbes, thereby slowing the evolution of resistance. In addition, it degrades in light, and will break down in the general environment where strong selective pressures imposed by antibiotic residuals are known to accelerate antibiotic resistance. Phosphopyricin represents an innovation in bactericidals, having a synthetic core, and a photosensitive chemical architecture that would reduce accumulation in the environment.
Synthetic organic antibiotics based on conserved cores have expanded the pool of effective antibiotics in the short term31, yet their inherent common structure has enabled microbes to acquire new resistance mechanisms that target the common backbone, or to evolve resistance through incremental mutations of existing genetic determinants32,33. Here we demonstrate that organophosphorus compounds, a group that has not been evaluated for antimicrobial activity in any systematic fashion, have the capacity to yield new architectures for clinically relevant pathogens. In addition, non-phosphate organophosphorus compounds have the incredible advantage over existing antibiotics in that these compounds do not occur naturally in the environment, such that resistance to these foreign compounds would not have already evolved.
Although microbes have been shown to possess specific strategies for utilizing, metabolizing, or simply contending with phosphate compounds in the environment34, the evolution of resistance to these synthetic organophosphorus compounds would be slowed considerably, since microbes are unlikely able to metabolize these xenobiotics 35'36. The exploration of synthetic organophosphorus compounds has the potential to make a significant impact for antimicrobial discovery, and to move the field in a new direction that considers not only molecular mechanisms of resistance, but also the evolutionary pressures that drive it.
According to an aspect of the invention, there is provided a compound comprising the structure of Formula (I):
Synthetic organic antibiotics based on conserved cores have expanded the pool of effective antibiotics in the short term31, yet their inherent common structure has enabled microbes to acquire new resistance mechanisms that target the common backbone, or to evolve resistance through incremental mutations of existing genetic determinants32,33. Here we demonstrate that organophosphorus compounds, a group that has not been evaluated for antimicrobial activity in any systematic fashion, have the capacity to yield new architectures for clinically relevant pathogens. In addition, non-phosphate organophosphorus compounds have the incredible advantage over existing antibiotics in that these compounds do not occur naturally in the environment, such that resistance to these foreign compounds would not have already evolved.
Although microbes have been shown to possess specific strategies for utilizing, metabolizing, or simply contending with phosphate compounds in the environment34, the evolution of resistance to these synthetic organophosphorus compounds would be slowed considerably, since microbes are unlikely able to metabolize these xenobiotics 35'36. The exploration of synthetic organophosphorus compounds has the potential to make a significant impact for antimicrobial discovery, and to move the field in a new direction that considers not only molecular mechanisms of resistance, but also the evolutionary pressures that drive it.
According to an aspect of the invention, there is provided a compound comprising the structure of Formula (I):
8 rs /¨ R = aryl,heteroaryl, OC _______ W
c/ alkynyl, vinyl, ally!.
R' = alkyl, aryl (I) wherein:
"W" is tungsten "C" is carbon "0" is oxygen "P" is phosphorus R is selected from substituted aryl (including, but not limited to aninsolyl, anininyl, tolyl, napththyl), heteroaryl (including, but not limited to pyrolyl, thienyl, indolyl, pyrazolyl, imidazolyl), alkynyl (C2-C6, phenylalkynyl), vinyl, cyclic vinyl and substituted vinyl (C2-C6), allyl, cyclic allyl and substituted ally' (C3-C7);
and R' is alkyl or aryl (Ph, C1-C6 alkyl, C3-C6 cyclic alkyl).
As discussed herein, the R, P, and W(C0)5 moieties are essential to the function of the compound. For example, variation of R changes the spectrum of antibacterial activity (le different R groups result in antimicrobial activity against different groups of microbes) and increases or decreases MIC against specific microbes, while removal or replacement of W(C0)5 eliminates antimicrobial activity.
R' is less essential to function, and variation of R' can be used to alter properties of the compound, for example, solubility and membrane permeability, as discussed below.
According to another aspect of the invention, there is provided a compound comprising the structure of Formula (11a) or (11b):
c/ alkynyl, vinyl, ally!.
R' = alkyl, aryl (I) wherein:
"W" is tungsten "C" is carbon "0" is oxygen "P" is phosphorus R is selected from substituted aryl (including, but not limited to aninsolyl, anininyl, tolyl, napththyl), heteroaryl (including, but not limited to pyrolyl, thienyl, indolyl, pyrazolyl, imidazolyl), alkynyl (C2-C6, phenylalkynyl), vinyl, cyclic vinyl and substituted vinyl (C2-C6), allyl, cyclic allyl and substituted ally' (C3-C7);
and R' is alkyl or aryl (Ph, C1-C6 alkyl, C3-C6 cyclic alkyl).
As discussed herein, the R, P, and W(C0)5 moieties are essential to the function of the compound. For example, variation of R changes the spectrum of antibacterial activity (le different R groups result in antimicrobial activity against different groups of microbes) and increases or decreases MIC against specific microbes, while removal or replacement of W(C0)5 eliminates antimicrobial activity.
R' is less essential to function, and variation of R' can be used to alter properties of the compound, for example, solubility and membrane permeability, as discussed below.
According to another aspect of the invention, there is provided a compound comprising the structure of Formula (11a) or (11b):
9 0 0 Ph 0 Ph C co / Ph Ph OC¨W __ P OC¨W __ P
C/
\R
0 cl OC c (11a) (11b) R =
N
___________________ Ph 401 X = H, OMe, NEt2, CH3, Ph X
=
According to another aspect of the invention, there is provided a compound comprising the structure of Formula (Ill):
0 Ph / ph OC¨W _________________ P
OC c (III) As will be appreciated by one of skill in the art, many modifications can be made to a compound according to Formula (1), Formula (Ha), Formula (11b) or Formula (III) which will alter and/or improve a beneficial property thereof. For example, water solubility of phosphopyricin can be improved by introducing ionic groups such as sulfonate (S03-) or quaternary ammonium (NR3+) groups onto the functionally non-
C/
\R
0 cl OC c (11a) (11b) R =
N
___________________ Ph 401 X = H, OMe, NEt2, CH3, Ph X
=
According to another aspect of the invention, there is provided a compound comprising the structure of Formula (Ill):
0 Ph / ph OC¨W _________________ P
OC c (III) As will be appreciated by one of skill in the art, many modifications can be made to a compound according to Formula (1), Formula (Ha), Formula (11b) or Formula (III) which will alter and/or improve a beneficial property thereof. For example, water solubility of phosphopyricin can be improved by introducing ionic groups such as sulfonate (S03-) or quaternary ammonium (NR3+) groups onto the functionally non-
10 essential auxiliary phenyl rings.
NMe3 SO3 0 0 R = aryl,heteroaryl, Cs- alkynyl, vinyl, ally1 Ph OC W ___________ P OC W
0 c 0 r.
As discussed above, for example, the R' group can be modified. As such, many additional modifications to the compounds of Formula (1), Formula (11a), Formula (11b) and Formula (Ill), for example, to improve stability, improve solubility, improve permeability and the like, will be readily apparent to one of skill in the art.
As discussed herein, the compounds of Formula (I), Formula (11a), Formula (11b) and Formula (111) are light-labile. Specifically, the tungsten complex is susceptible to light-induced ligand dissociation, leading to a coordinatively unsaturated complex that is susceptible to oxidation, leading to decomposition and loss of antibacterial activity. In that absence of light, the complex remains coordinatively saturated, rendering it less susceptible to oxidation and decomposition, meaning that it will persist in biological systems long enough to be an effective antimicrobial. As will be appreciated by one of skill in the art, the presence of pharmaceutical compounds in the environment, particularly in water, is an on-going concern. Furthermore, when these pharmaceutical compounds are antibiotics, their presence in the environment can lead to development of bacterial resistance. However, because the compounds of the invention are light-labile, while they are effective when in the body in the patient, they quickly decompose within the environment, as discussed herein.
As discussed herein, the compounds of the invention will break down under direct light in between about 8-16 hours. As discussed herein, that is sufficient time for the compounds of the invention to exert their effects on bacteria. Also, if it's maintained in the dark during the disinfection process, it maintains its activity.
While not wishing to be bound to a particular theory or hypothesis, the
NMe3 SO3 0 0 R = aryl,heteroaryl, Cs- alkynyl, vinyl, ally1 Ph OC W ___________ P OC W
0 c 0 r.
As discussed above, for example, the R' group can be modified. As such, many additional modifications to the compounds of Formula (1), Formula (11a), Formula (11b) and Formula (Ill), for example, to improve stability, improve solubility, improve permeability and the like, will be readily apparent to one of skill in the art.
As discussed herein, the compounds of Formula (I), Formula (11a), Formula (11b) and Formula (111) are light-labile. Specifically, the tungsten complex is susceptible to light-induced ligand dissociation, leading to a coordinatively unsaturated complex that is susceptible to oxidation, leading to decomposition and loss of antibacterial activity. In that absence of light, the complex remains coordinatively saturated, rendering it less susceptible to oxidation and decomposition, meaning that it will persist in biological systems long enough to be an effective antimicrobial. As will be appreciated by one of skill in the art, the presence of pharmaceutical compounds in the environment, particularly in water, is an on-going concern. Furthermore, when these pharmaceutical compounds are antibiotics, their presence in the environment can lead to development of bacterial resistance. However, because the compounds of the invention are light-labile, while they are effective when in the body in the patient, they quickly decompose within the environment, as discussed herein.
As discussed herein, the compounds of the invention will break down under direct light in between about 8-16 hours. As discussed herein, that is sufficient time for the compounds of the invention to exert their effects on bacteria. Also, if it's maintained in the dark during the disinfection process, it maintains its activity.
While not wishing to be bound to a particular theory or hypothesis, the
11 inventors believe that the mode of action of phosphopyricin involves competitive binding of molybdopterin, which normally binds molybdenum to form an integral protein cofactor, molybdenum cofactor. By taking the place of molybdenum, phosphopyricin may be interfering with key metabolic processes at multiple levels. In this scenario, bacteria that utilize molybdenum as a cofactor would be susceptible to phosphopyricin, unlike those bacteria and members of the Archaea that normally utilize tungsten (most often extremophires, such as Desulfovibrio, Thermococcus, Methanobacterium, Pyrococcus). Because of the universality of its target, phosphopyricin is a general antibacterial that is active against a broad range of bacteria. Consistent with this, biological activity was demonstrated for several Gram-positive bacteria within the class Bacilli including species of Staphylococcus (order BadHales), and both Streptococcus and Enterococcus (order Lactobacillales), suggesting that there would be biological activity against other clinically relevant pathogens that are also within these orders such as Bacillus (anthrax) and Listeria.
Similarly, the demonstration that Salmonella can be made susceptible in the presence of EDTA is strongly suggestive that the target is present in Gram-negatives and that the antibiotic would also be effective against a broad range of other pathogens such as E. coil, Klebsiella and Enterobacter, which exhibit extended-spectrum resistance against several major classes of antibiotics.
According to another aspect of the invention, there is provided a method of treating a bacterial infection in an individual in need of such treatment comprising administering to an individual who has a bacterial infection or who is suspected of having a bacterial infection an effective amount of a compound as set forth in Formula (I), Formula (11a), Formula (11b) or Formula (III).
As will be appreciated by one of skill in the art, an individual in need of such treatment is a person who is known to be or is suspected of suffering from a bacterial infection. As will be appreciated by one of skill in the art, symptoms associated with bacterial infections vary greatly but are well known to those of skill in the art.
As discussed above, while not wishing to be bound to a particular theory or hypothesis, the inventors believe that the mode of action of phosphopyricin involves
Similarly, the demonstration that Salmonella can be made susceptible in the presence of EDTA is strongly suggestive that the target is present in Gram-negatives and that the antibiotic would also be effective against a broad range of other pathogens such as E. coil, Klebsiella and Enterobacter, which exhibit extended-spectrum resistance against several major classes of antibiotics.
According to another aspect of the invention, there is provided a method of treating a bacterial infection in an individual in need of such treatment comprising administering to an individual who has a bacterial infection or who is suspected of having a bacterial infection an effective amount of a compound as set forth in Formula (I), Formula (11a), Formula (11b) or Formula (III).
As will be appreciated by one of skill in the art, an individual in need of such treatment is a person who is known to be or is suspected of suffering from a bacterial infection. As will be appreciated by one of skill in the art, symptoms associated with bacterial infections vary greatly but are well known to those of skill in the art.
As discussed above, while not wishing to be bound to a particular theory or hypothesis, the inventors believe that the mode of action of phosphopyricin involves
12 competitive binding of molybdopterin, which normally binds molybdenum to form an integral protein cofactor, molybdenum cofactor. By taking the place of molybdenum, phosphopyricin may be interfering with key metabolic processes at multiple levels. In this scenario, bacteria that utilize molybdenum as a cofactor would be susceptible to phosphopyricin.
Accordingly, the bacterial infection treated by a compound as set forth in Formula (1), Formula (11a), Formula (11b) or Formula (III) may be caused by any bacteria, as discussed herein.
For example, the bacteria may be Gram-positive bacteria selected from for example but by no means limited to the class Bacilli including species of Staphylococcus (order Bacillales), Streptococcus and Enterococcus (order Lactobacillales), Bacillus (anthrax) and Listeria.
Alternatively, the bacteria may be Gram-negative bacteria, for example, but by no means limited to Salmonella, E. coli, Klebsiella and Enterobacter.
As discussed above, the compounds described herein have wide applicability and have been demonstrated to have effectiveness against a wide variety of bacteria.
As such, it is to be understood that specific bacterial strains identified herein are for illustrative purposes.
The individual may be an animal, for example a mammal, for example, a human, although as will be appreciated by one of skill in the art, uses such as for example veterinary uses are also contemplated and are within the scope of the invention.
Furthermore, as discussed herein, given the wide range of bacterial targets sensitive to the compounds of the invention, the compounds may also be used for the treatment of bacterial infections of plants, that is, for the treatment or elimination of plant pathogens.
According to another aspect of the invention, there is provided an antibacterial composition comprising a compound as described above.
According to another aspect of the invention, there is provided the use of a compound as described herein in an antibacterial composition.
Accordingly, the bacterial infection treated by a compound as set forth in Formula (1), Formula (11a), Formula (11b) or Formula (III) may be caused by any bacteria, as discussed herein.
For example, the bacteria may be Gram-positive bacteria selected from for example but by no means limited to the class Bacilli including species of Staphylococcus (order Bacillales), Streptococcus and Enterococcus (order Lactobacillales), Bacillus (anthrax) and Listeria.
Alternatively, the bacteria may be Gram-negative bacteria, for example, but by no means limited to Salmonella, E. coli, Klebsiella and Enterobacter.
As discussed above, the compounds described herein have wide applicability and have been demonstrated to have effectiveness against a wide variety of bacteria.
As such, it is to be understood that specific bacterial strains identified herein are for illustrative purposes.
The individual may be an animal, for example a mammal, for example, a human, although as will be appreciated by one of skill in the art, uses such as for example veterinary uses are also contemplated and are within the scope of the invention.
Furthermore, as discussed herein, given the wide range of bacterial targets sensitive to the compounds of the invention, the compounds may also be used for the treatment of bacterial infections of plants, that is, for the treatment or elimination of plant pathogens.
According to another aspect of the invention, there is provided an antibacterial composition comprising a compound as described above.
According to another aspect of the invention, there is provided the use of a compound as described herein in an antibacterial composition.
13 According to another aspect of the invention, there is provided a method of preparing an antibacterial composition comprising admixing a compound as described herein with a suitable excipient.
As will be appreciated by one of skill in the art, the antibacterial composition may be a pharmaceutical composition or a medicament for treatment of an animal, for example, a mammal, for example a human.
Alternatively, the antibacterial composition may be used for the treatment of a non-human animal, for example but by no means limited to a livestock animal, a domestic animal or a wild animal.
The antibacterial composition may be formulated for agricultural use, that is, as an anti-bacterial agent against plant pathogens. As will be appreciated by one of skill in the art, in these embodiments, the antibacterial composition may be formulated for administration as a spray, for example as a hand-held spray or pump spray for administration to individual plants or as a spray from a tank, tanker, sprayer or similar .. agricultural equipment.
The antibacterial composition may be a disinfectant, for example but by no means limited to formulated as a spray, a solution, a gel or a powder for use in the environment, that is, ex vivo, for example, on surfaces that are to be cleaned, for example, kitchen or food preparation surfaces, floors, exterior body surfaces such as hands and the like, that is, anything that may need or benefit from disinfection prior to human contact.
The antibacterial composition may be used as a general disinfectant for surfaces, for example, on medical devices and/or medical instruments prior to contact with or insertion into patients.
As used herein, an effective amount refers to an amount of the compound that is sufficient to reduce at least one of the symptoms associated with the bacterial infection, for example, reducing colony forming units per ml or reducing severity of symptoms associated with the bacterial infection. For example, the daily adult dose for treating Gram-positive infections using approved antibiotics having a MIC
< 16 ug/mL range between 200-2500 mg. It is further noted that an effective amount can
As will be appreciated by one of skill in the art, the antibacterial composition may be a pharmaceutical composition or a medicament for treatment of an animal, for example, a mammal, for example a human.
Alternatively, the antibacterial composition may be used for the treatment of a non-human animal, for example but by no means limited to a livestock animal, a domestic animal or a wild animal.
The antibacterial composition may be formulated for agricultural use, that is, as an anti-bacterial agent against plant pathogens. As will be appreciated by one of skill in the art, in these embodiments, the antibacterial composition may be formulated for administration as a spray, for example as a hand-held spray or pump spray for administration to individual plants or as a spray from a tank, tanker, sprayer or similar .. agricultural equipment.
The antibacterial composition may be a disinfectant, for example but by no means limited to formulated as a spray, a solution, a gel or a powder for use in the environment, that is, ex vivo, for example, on surfaces that are to be cleaned, for example, kitchen or food preparation surfaces, floors, exterior body surfaces such as hands and the like, that is, anything that may need or benefit from disinfection prior to human contact.
The antibacterial composition may be used as a general disinfectant for surfaces, for example, on medical devices and/or medical instruments prior to contact with or insertion into patients.
As used herein, an effective amount refers to an amount of the compound that is sufficient to reduce at least one of the symptoms associated with the bacterial infection, for example, reducing colony forming units per ml or reducing severity of symptoms associated with the bacterial infection. For example, the daily adult dose for treating Gram-positive infections using approved antibiotics having a MIC
< 16 ug/mL range between 200-2500 mg. It is further noted that an effective amount can
14 be determined through routine experimentation and is within the scope of the invention.
For example, an effective amount may be an amount that is sufficient to reduce the severity of at least one symptom associated with a bacterial infection, for example, reducing fever, reducing colony forming units per ml, reducing swelling, reducing pain, reducing redness, reducing shaking, improving low blood pressure, reducing severity of conjunctivitis, reducing irritation, reducing nausea, reducing general feeling of discomfort and reducing congestion.
The invention will now be further elucidated by way of examples; however, the invention is not necessarily limited by or to the examples.
We used metal-mediated electrophilic substitution 20-22 to rapidly generate a diverse library of synthetic organophosphorus compounds containing carbon-phosphorus bonds. One compound in the library, 9aWC2, a tungsten phosphine complex (Figure 1), showed inhibitory activity against Staphylococcus aureus K1-7 at a minimum inhibitory concentration (MIC) of 4 ¨ 8 pg/mL. This promising candidate antimicrobial was modified to eliminate the synthetically challenging phosphirene ring, yielding compound 9bWC2, which exhibited similar potency towards S. aureus (MIC
of 4¨ 8 pg/mL) (Figure 1). To identify features of 9bWC2 that were essential for antimicrobial activity, we systematically varied chemical side groups, beginning with substitution of the C2-bound pyrrole with 02-bound thiophene, which resulted in loss of antimicrobial activity against S. aureus (11bW, MIC > 1024 pg/mL) (Figure 1).
Substitution of pyrrole with indole resulted in slightly reduced potency against S.
aureus (10bW, MIC of 8¨ 16 pg/mL), while substitution of the pyrrole with a phenyl group, significantly reduced potency against S. aureus (12bW, 256¨ 512 pg/mL), but increased the spectrum of activity to include Gram-negative, Klebsiella sp.
and Salmonella sp. (MIC of 256 ¨ 512 pg/mL) (Figure 1). We then replaced the 02-bound pyrrole ring with a C3-bound pyrrole (9bW03), resulting in an increased potency against S. aureus, from a MIC of 4 ¨ 8 pg/ml to 2 ¨4 pg/ml (Figure 2A). An evaluation of the potency of this compound against the Gram-positive Enterococcus faecium KO2G0810 and S. mutans revealed that both were also inhibited at MICs of 2 ¨ 4
For example, an effective amount may be an amount that is sufficient to reduce the severity of at least one symptom associated with a bacterial infection, for example, reducing fever, reducing colony forming units per ml, reducing swelling, reducing pain, reducing redness, reducing shaking, improving low blood pressure, reducing severity of conjunctivitis, reducing irritation, reducing nausea, reducing general feeling of discomfort and reducing congestion.
The invention will now be further elucidated by way of examples; however, the invention is not necessarily limited by or to the examples.
We used metal-mediated electrophilic substitution 20-22 to rapidly generate a diverse library of synthetic organophosphorus compounds containing carbon-phosphorus bonds. One compound in the library, 9aWC2, a tungsten phosphine complex (Figure 1), showed inhibitory activity against Staphylococcus aureus K1-7 at a minimum inhibitory concentration (MIC) of 4 ¨ 8 pg/mL. This promising candidate antimicrobial was modified to eliminate the synthetically challenging phosphirene ring, yielding compound 9bWC2, which exhibited similar potency towards S. aureus (MIC
of 4¨ 8 pg/mL) (Figure 1). To identify features of 9bWC2 that were essential for antimicrobial activity, we systematically varied chemical side groups, beginning with substitution of the C2-bound pyrrole with 02-bound thiophene, which resulted in loss of antimicrobial activity against S. aureus (11bW, MIC > 1024 pg/mL) (Figure 1).
Substitution of pyrrole with indole resulted in slightly reduced potency against S.
aureus (10bW, MIC of 8¨ 16 pg/mL), while substitution of the pyrrole with a phenyl group, significantly reduced potency against S. aureus (12bW, 256¨ 512 pg/mL), but increased the spectrum of activity to include Gram-negative, Klebsiella sp.
and Salmonella sp. (MIC of 256 ¨ 512 pg/mL) (Figure 1). We then replaced the 02-bound pyrrole ring with a C3-bound pyrrole (9bW03), resulting in an increased potency against S. aureus, from a MIC of 4 ¨ 8 pg/ml to 2 ¨4 pg/ml (Figure 2A). An evaluation of the potency of this compound against the Gram-positive Enterococcus faecium KO2G0810 and S. mutans revealed that both were also inhibited at MICs of 2 ¨ 4
15 pg/mL, and spot testing on bacterial overlay plates containing all three Gram-positive bacteria revealed no spontaneously resistant mutants. This compound, 9bWC3, was named phosphopyricin (Figure 1). Substitution of the tungsten pentacarbonyl group of phosphopyricin with molybdenum pentacarbonyl (9bMoC3) or oxygen (9b0C3) abolished detectable antibiotic activity (below 1024 pg/mL), suggesting an important role for the W(C0)5 fragment, and the P-bound N-heterocycle.
We observed two important properties of 9bWC2 and phosphopyricin (9bWC3). Firstly, we noted that potency of 9bWC2 decreased significantly upon exposure to light. After 4 hours of fluorescent light exposure at 92.7 pmol/s .m2, the M1C of 9bWC2 for S. aureus K1-7 increased from 4 ¨8 pg/mL to 8¨ 16 pg/mL, suggesting photolytic degradation. Like its parent compound, phosphopyricin activity was also markedly reduced after 24 hours in continuous fluorescent light at the same intensity (Figure 28), suggesting that phosphopyricin would photodegrade gradually in the general environment. Secondly, we determined that both 9bWC2 and phosphopyricin were bactericidal. Bacteriostatic agents are able to arrest bacterial growth, while bactericidal agents compromise bacterial cell viability and eradicate >99.9% of a given inoculum 23. Approximately 5 x 105 cfu of S. aureus K1-7 was exposed to either 50 pg/mL phosphopyricin or 9bWC2 for 24 hours in 1 mL 10 mM
MgSO4 buffer, and the viability of the bacteria subsequently assessed.
Relative to the antibiotic-free control, viable cell number was reduced by 99.9993% for 9bWC2, and 100% for phosphopyricin (Figure 2C). Given this potency, we assessed its activity against the Gram-negative Pseudomonas aeruginosa, S. enter/ca subsp.
Typhimurium 14028, Cronobacter sp. 12202, Enterobacter sp. TX1, Klebsiella sp.
B011499, and Acinetobacter baummanii ATCC17978, and all were shown to be resistant (>1024 pg/mL). The outer membrane of Gram-negative strains can act as a barrier to foreign agents, including antimicrobials, but can be destabilized with compounds like the chelating agent, ethylenediaminetetraacetic acid (EDTA) 24'25. For example, the Gram-negative S. enter/ca subsp. Typhimurium 14028 becomes sensitive to the Gram-positive-specific antibiotic, nisin, in the presence of EDTA, which destabilizes the outer membrane, thereby overcoming physical exclusion of the
We observed two important properties of 9bWC2 and phosphopyricin (9bWC3). Firstly, we noted that potency of 9bWC2 decreased significantly upon exposure to light. After 4 hours of fluorescent light exposure at 92.7 pmol/s .m2, the M1C of 9bWC2 for S. aureus K1-7 increased from 4 ¨8 pg/mL to 8¨ 16 pg/mL, suggesting photolytic degradation. Like its parent compound, phosphopyricin activity was also markedly reduced after 24 hours in continuous fluorescent light at the same intensity (Figure 28), suggesting that phosphopyricin would photodegrade gradually in the general environment. Secondly, we determined that both 9bWC2 and phosphopyricin were bactericidal. Bacteriostatic agents are able to arrest bacterial growth, while bactericidal agents compromise bacterial cell viability and eradicate >99.9% of a given inoculum 23. Approximately 5 x 105 cfu of S. aureus K1-7 was exposed to either 50 pg/mL phosphopyricin or 9bWC2 for 24 hours in 1 mL 10 mM
MgSO4 buffer, and the viability of the bacteria subsequently assessed.
Relative to the antibiotic-free control, viable cell number was reduced by 99.9993% for 9bWC2, and 100% for phosphopyricin (Figure 2C). Given this potency, we assessed its activity against the Gram-negative Pseudomonas aeruginosa, S. enter/ca subsp.
Typhimurium 14028, Cronobacter sp. 12202, Enterobacter sp. TX1, Klebsiella sp.
B011499, and Acinetobacter baummanii ATCC17978, and all were shown to be resistant (>1024 pg/mL). The outer membrane of Gram-negative strains can act as a barrier to foreign agents, including antimicrobials, but can be destabilized with compounds like the chelating agent, ethylenediaminetetraacetic acid (EDTA) 24'25. For example, the Gram-negative S. enter/ca subsp. Typhimurium 14028 becomes sensitive to the Gram-positive-specific antibiotic, nisin, in the presence of EDTA, which destabilizes the outer membrane, thereby overcoming physical exclusion of the
16 antibiotic from the cell 24. We incubated S. enterica subsp. Typhimurium 14028 for 24 hours at 37 C in the presence of 32 pg/mL phosphopyricin, with and without 1.5 mM
EDTA. Bacterial titres were reduced significantly when EDTA was combined with phosphopyricin, as compared to cultures containing only phosphopyricin (Tukey's HSD, p < 2 x 10-12) (Figure 2D). This suggests that the target(s) of phosphopyricin are also present in Gram-negative bacteria, and that the spectrum of activity is determined by the ability of the compound to cross the bacterial cell wall.
Thus, the spectrum of activity of phosphopyricin can be expanded with additives that increase bacterial cell wall permeability. Phosphopyricin contains a transition metal, tungsten, which could bind competitively to essential proteins where other co-factors, like molybdenum cofactor, would normally bind 26.
Methods Bacterial isolates and culturing conditions Staphylococcus aureus K1-7, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecium K02G0810, Cronobacter sp. 12202, Enterobacter sp. TX1, Klebsiella sp. B011499, Klebsiella sp. G4061350, Acinetobacter baumannii ATCC17978, and Streptococcus mutans UAlS9:wt were cultured in lysogeny broth (LB, BD Biosciences) at 37 C, except for E. faecium K02G0810, which was cultured .. in brain-heart infusion (BHI, BD Biosciences) medium at 37 C.
Chemical synthesis and derivatization Compounds 9aWC2, 9bWC2, 9bWC3, 10bW, and 11bW were synthesized using published procedures 2o-22. To synthesize 0=PPh2(04H3NH) (9b0), compound [W(C0)5{PPh2(C4H3NH)}] (9bW, 60 mg, 0.104 mmol, 2 isomers) and dppe (43.6 mg, 0.110 mmol) were dissolved in THF (2 mL) and irradiated with UV for 2 h, resulting in a color change from colorless to yellow. The solvent was removed under reduced pressure, and the residue was purified by flash chromatography (silica gel, 50/50 v/v diethyl ether/petroleum ether). After purification, the free phosphine oxide .. {0=PPh2(C4H3NH)} (9b0, 2 isomers) was obtained as a white powder; Yield: 17 mg,
EDTA. Bacterial titres were reduced significantly when EDTA was combined with phosphopyricin, as compared to cultures containing only phosphopyricin (Tukey's HSD, p < 2 x 10-12) (Figure 2D). This suggests that the target(s) of phosphopyricin are also present in Gram-negative bacteria, and that the spectrum of activity is determined by the ability of the compound to cross the bacterial cell wall.
Thus, the spectrum of activity of phosphopyricin can be expanded with additives that increase bacterial cell wall permeability. Phosphopyricin contains a transition metal, tungsten, which could bind competitively to essential proteins where other co-factors, like molybdenum cofactor, would normally bind 26.
Methods Bacterial isolates and culturing conditions Staphylococcus aureus K1-7, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecium K02G0810, Cronobacter sp. 12202, Enterobacter sp. TX1, Klebsiella sp. B011499, Klebsiella sp. G4061350, Acinetobacter baumannii ATCC17978, and Streptococcus mutans UAlS9:wt were cultured in lysogeny broth (LB, BD Biosciences) at 37 C, except for E. faecium K02G0810, which was cultured .. in brain-heart infusion (BHI, BD Biosciences) medium at 37 C.
Chemical synthesis and derivatization Compounds 9aWC2, 9bWC2, 9bWC3, 10bW, and 11bW were synthesized using published procedures 2o-22. To synthesize 0=PPh2(04H3NH) (9b0), compound [W(C0)5{PPh2(C4H3NH)}] (9bW, 60 mg, 0.104 mmol, 2 isomers) and dppe (43.6 mg, 0.110 mmol) were dissolved in THF (2 mL) and irradiated with UV for 2 h, resulting in a color change from colorless to yellow. The solvent was removed under reduced pressure, and the residue was purified by flash chromatography (silica gel, 50/50 v/v diethyl ether/petroleum ether). After purification, the free phosphine oxide .. {0=PPh2(C4H3NH)} (9b0, 2 isomers) was obtained as a white powder; Yield: 17 mg,
17 62%. 31P{1H} NMR (CDCI3): 516.7 [0=PPh2(2-C4H3NH)] and 18.2 [0=PPh2(3-C4H3NH)].
The synthesis of [W(C0)6{PPh3}] (12bW) was carried out by first adding (CH3)3N0=2H20 (66.3 mg, 0.597 mmol) to a solution of [W(C0)61 (200 mg, 0.568 mmol) in acetonitrile (15 mL), in small portions, over 5 min. The resulting yellow solution was stirred for 40 min, and the solvent was removed under reduced pressure.
The residue was dissolved in toluene (2 mL) and the solvent was again removed under reduced pressure. The crude [W(C0)6CCH3CNI1 was dissolved in THE (15 mL), PPh3 (164 mg, 0.625 mmol) was added, and the mixture was heated at 50 C for h. The solvent was removed under reduced pressure, and the residue was purified by flash chromatography (silica gel, 10/90 diethyl ether/petroleum ether). The white product was crystallized by cooling a saturated hexane/diethylether solution to -20 C.
Yield: 35%. IR (vCO, CH2Cl2, cm-1): 2071(w), 1944(vs). 31P{1H} NMR (CDCI3): 6 21.6 (1Jpw = 244 Hz).
To synthesize [Mo(CO)s{PPh2(C4H3NH)}] (9bMo), a solution of Mo(C0)6 (2.00 g, 7.58 mmol, MW= 264.01 g/mol) and PPh2CI (1.36 mL, 9.09 mmol) in dry toluene (50 mL) was heated under reflux for 90 minutes resulting in a colour change from yellow to amber colour. The volume was reduced under vacuum to -10 mL, and 10 mL of Petroleum ether was added. The solution was filtered and anhydrous alumina (1 g) was added, the mixture was shaken and then filtered. The filtrate was evacuated to yield [Mo(C0)6(PPh2C1)] as a pale yellow powder. Yield: 2.25 g (65%).
31P{1H} NMR
= 6123, IR ( vCO, CH2Cl2, cm-1): 2081, 1969, 2020 cm-1. Silver trifluoromethanesulfonate (90 mg, 0.350 mmol) was added to a solution of [Mo(CO)6(PPh2C1)] (80 mg, 0.175 mmol) in 0H2Cl2 (2 mL). The solution was stirred for 3 hours, and then filtered to remove AgCl. Pyrrole (24 pt, 0.350 mmol) was added 24 L, the solution was stirred for 5 minutes and then the solvent was evaporated under reduced pressure. The residue was purified using column chromatography (silica gel, 10:90 v/v diethyl ether/petroleum ether) to yield 30 mg of a white powder, which was shown to be [Mo(C0)6(PPh2(C4H3NH)] (2 isomers). Yield: 37%. 31P{1H} NMR
(CD0I3):
6 15.8, and 15. Yield = 37%. 31P{1H} NMR = 615.8; 615. For bioassays, powdered
The synthesis of [W(C0)6{PPh3}] (12bW) was carried out by first adding (CH3)3N0=2H20 (66.3 mg, 0.597 mmol) to a solution of [W(C0)61 (200 mg, 0.568 mmol) in acetonitrile (15 mL), in small portions, over 5 min. The resulting yellow solution was stirred for 40 min, and the solvent was removed under reduced pressure.
The residue was dissolved in toluene (2 mL) and the solvent was again removed under reduced pressure. The crude [W(C0)6CCH3CNI1 was dissolved in THE (15 mL), PPh3 (164 mg, 0.625 mmol) was added, and the mixture was heated at 50 C for h. The solvent was removed under reduced pressure, and the residue was purified by flash chromatography (silica gel, 10/90 diethyl ether/petroleum ether). The white product was crystallized by cooling a saturated hexane/diethylether solution to -20 C.
Yield: 35%. IR (vCO, CH2Cl2, cm-1): 2071(w), 1944(vs). 31P{1H} NMR (CDCI3): 6 21.6 (1Jpw = 244 Hz).
To synthesize [Mo(CO)s{PPh2(C4H3NH)}] (9bMo), a solution of Mo(C0)6 (2.00 g, 7.58 mmol, MW= 264.01 g/mol) and PPh2CI (1.36 mL, 9.09 mmol) in dry toluene (50 mL) was heated under reflux for 90 minutes resulting in a colour change from yellow to amber colour. The volume was reduced under vacuum to -10 mL, and 10 mL of Petroleum ether was added. The solution was filtered and anhydrous alumina (1 g) was added, the mixture was shaken and then filtered. The filtrate was evacuated to yield [Mo(C0)6(PPh2C1)] as a pale yellow powder. Yield: 2.25 g (65%).
31P{1H} NMR
= 6123, IR ( vCO, CH2Cl2, cm-1): 2081, 1969, 2020 cm-1. Silver trifluoromethanesulfonate (90 mg, 0.350 mmol) was added to a solution of [Mo(CO)6(PPh2C1)] (80 mg, 0.175 mmol) in 0H2Cl2 (2 mL). The solution was stirred for 3 hours, and then filtered to remove AgCl. Pyrrole (24 pt, 0.350 mmol) was added 24 L, the solution was stirred for 5 minutes and then the solvent was evaporated under reduced pressure. The residue was purified using column chromatography (silica gel, 10:90 v/v diethyl ether/petroleum ether) to yield 30 mg of a white powder, which was shown to be [Mo(C0)6(PPh2(C4H3NH)] (2 isomers). Yield: 37%. 31P{1H} NMR
(CD0I3):
6 15.8, and 15. Yield = 37%. 31P{1H} NMR = 615.8; 615. For bioassays, powdered
18 organophosphorus compounds were resuspended in either 100% isopropanol (9aWC2, 9bWC2, 9bWC3, 9b0C3, 10bW), dimethylsulfoxide (11bW) or a 50:50 isopropanol-tetrahydrofuran (v/v) solution (9bMoC3).
Minimum inhibitory concentration and bactericidal assays The minimum inhibitory concentrations (MIC) of each compound was assessed in 96-well plates by serial dilution of a 10 mg/mL stock solution of the organophosphorus compound in LB or BHI medium from 1 pg/mL to 1024 pg/mL (one per column). A no-compound (solvent-only) control was used in the final column of the 12-column plate. Approximately 1 x 105 cfu of bacteria was added to each well to a .. final volume of 200 pL, the microplate covered with sterile, breathable rayon film (VVVR International), and incubated at 30 C with shaking at 220 RPM for 24 hours.
To evaluate bacteriostatic or bactericidal activity, an overnight culture of S.
aureus K1-7 in LB medium was diluted to yield 1 mL suspensions of 5 x 106 cfu in 10 mM MgSO4. Each suspension was treated with a final concentration of 50 pg/mL
of .. either 9bWC2 or 9bWC3 (in isopropanol). Control treatments were supplemented with an equal volume of isopropanol. Cultures were shaken at 220 RPM for 24 hours at 21 C, and bacterial survivorship assessed by colony enumeration of serial dilutions plated on LB.
EDTA Assay To evaluate the effects of phosphopyricin on Gram-negative bacteria, we adapted the protocol of Stevens et al. 24. Salmonella typhimurium 14028 was cultured in BHI broth at 37 C overnight, and 1 mL of bacterial culture (Olisoonm =
¨1.25) was transferred to a 1.5 mL rnicrofuge tube and bacteria pelleted by centrifugation (1 minute at 12.0 x 1000 min-lx g). The supernatant was removed, and cells were re-.. suspended in 1 mL 10 mM MgSO4. Using a 96-well plate, 1 x 10 cfu were incubated in BHI broth containing 1.5 mM EDTA solution, 32 pg/mL phosphopyricin, or both at 37 C for 24 hours. S. typhimurium cells grown in BHI broth alone served as a no-treatment control.
Photolysis assay
Minimum inhibitory concentration and bactericidal assays The minimum inhibitory concentrations (MIC) of each compound was assessed in 96-well plates by serial dilution of a 10 mg/mL stock solution of the organophosphorus compound in LB or BHI medium from 1 pg/mL to 1024 pg/mL (one per column). A no-compound (solvent-only) control was used in the final column of the 12-column plate. Approximately 1 x 105 cfu of bacteria was added to each well to a .. final volume of 200 pL, the microplate covered with sterile, breathable rayon film (VVVR International), and incubated at 30 C with shaking at 220 RPM for 24 hours.
To evaluate bacteriostatic or bactericidal activity, an overnight culture of S.
aureus K1-7 in LB medium was diluted to yield 1 mL suspensions of 5 x 106 cfu in 10 mM MgSO4. Each suspension was treated with a final concentration of 50 pg/mL
of .. either 9bWC2 or 9bWC3 (in isopropanol). Control treatments were supplemented with an equal volume of isopropanol. Cultures were shaken at 220 RPM for 24 hours at 21 C, and bacterial survivorship assessed by colony enumeration of serial dilutions plated on LB.
EDTA Assay To evaluate the effects of phosphopyricin on Gram-negative bacteria, we adapted the protocol of Stevens et al. 24. Salmonella typhimurium 14028 was cultured in BHI broth at 37 C overnight, and 1 mL of bacterial culture (Olisoonm =
¨1.25) was transferred to a 1.5 mL rnicrofuge tube and bacteria pelleted by centrifugation (1 minute at 12.0 x 1000 min-lx g). The supernatant was removed, and cells were re-.. suspended in 1 mL 10 mM MgSO4. Using a 96-well plate, 1 x 10 cfu were incubated in BHI broth containing 1.5 mM EDTA solution, 32 pg/mL phosphopyricin, or both at 37 C for 24 hours. S. typhimurium cells grown in BHI broth alone served as a no-treatment control.
Photolysis assay
19 Glass vials containing compound stock solution of 9bWC3 in isopropanol at a concentration of 10 mg/mL were placed in a light chamber at 21 C, and subjected to either 8, 16 or 24 hours of 92.7 pmol/s.m2 continuous fluorescent light generated by three, 60 cm Philips F20112/CW, 20-watt bulbs. The amount of light was measured with a Li-cor Quantum / Radiometer / Photometer (LI-189). Control vials were wrapped in aluminum foil and placed in the same conditions. Following treatment, compounds were spotted on overlay media plates containing S. aureus K1-7, as prepared previously 37.
Statistical analysis For the EDTA assay, SPSS Statistics was used to perform a one-way ANOVA, which identified a statistically significant difference between groups (F(3,14) =
374.102, p = 1.3x10-13). A Tukey post hoc test revealed statistically significant differences in growth between the phosphopyricin with EDTA treatment (0D600nrn =
0.407 +/- 0.077) and the other three treatment groups: only EDTA (0D600nm =
1.236 +/- 0.021), only phosphopyricin (0D600nm = 1.4420 +/- 0.053), and no treatment (0D600nm = 1.260 +/- 0.037); p < 1.3x10-11. Levene's test of homogeneity showed no statistically significant differences between variances across treatments (1.421, p =
0.279).
Statistical evaluation of bactericidal activity of 9bWC2 and 9bWC3 (phosphopyricin) was carried out with SPSS Statistics using the Mann-Whitney test, which indicated statistically significant differences between the control group (supplemented with isopropanol) and both 9bWC2 (U = 0, p < 2 x 106) and phosphopyricin (U = 0, p < 3 x 10-7).
The scope of the claims should not be limited by the preferred embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.
Statistical analysis For the EDTA assay, SPSS Statistics was used to perform a one-way ANOVA, which identified a statistically significant difference between groups (F(3,14) =
374.102, p = 1.3x10-13). A Tukey post hoc test revealed statistically significant differences in growth between the phosphopyricin with EDTA treatment (0D600nrn =
0.407 +/- 0.077) and the other three treatment groups: only EDTA (0D600nm =
1.236 +/- 0.021), only phosphopyricin (0D600nm = 1.4420 +/- 0.053), and no treatment (0D600nm = 1.260 +/- 0.037); p < 1.3x10-11. Levene's test of homogeneity showed no statistically significant differences between variances across treatments (1.421, p =
0.279).
Statistical evaluation of bactericidal activity of 9bWC2 and 9bWC3 (phosphopyricin) was carried out with SPSS Statistics using the Mann-Whitney test, which indicated statistically significant differences between the control group (supplemented with isopropanol) and both 9bWC2 (U = 0, p < 2 x 106) and phosphopyricin (U = 0, p < 3 x 10-7).
The scope of the claims should not be limited by the preferred embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.
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2 Pendleton, J. N., Gorman, S. P. & Gilmore, B. F. Clinical relevance of the ESKAPE pathogens. Expert Review of Anti-Infective Therapy 11, 297-308, doi:10.1586/eri.13.12 (2013).
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Microbiology and Molecular Biology Reviews 74, 417, doi:10.1128/mmbr.00016-10 (2010).
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Claims (15)
1. A compound comprising the structure of Formula (I):
wherein:
"W" is tungsten;
"C" is carbon;
"O" is oxygen;
"P" is phosphorus;
R is selected from substituted aryl, alkynyl, vinyl, cyclic vinyl, substituted vinyl, allyl, cyclic allyl and substituted ally]; and R' is alkyl or aryl.
wherein:
"W" is tungsten;
"C" is carbon;
"O" is oxygen;
"P" is phosphorus;
R is selected from substituted aryl, alkynyl, vinyl, cyclic vinyl, substituted vinyl, allyl, cyclic allyl and substituted ally]; and R' is alkyl or aryl.
2. The compound according to claim 1 wherein the compound comprises the structure of Formula (IIa) or (IIb):
3. The compound according to claim 1 wherein the compound comprises the structure of Formula (III):
4. Use of a compound according to any one of claims 1 to 3 for treating a bacterial infection.
5. The use according to claim 4 wherein the bacterial infection is of an animal.
6. The use according to claim 5 wherein the bacterial infection is of a human.
7. The use according to claim 4 wherein the bacterial infection is of a plant.
8. A method of treating a bacterial infection comprising administering to an individual in need of such treatment an effective amount of a compound according to any one of claims 1-3.
9. A method of preparing a medicament for treating a bacterial infection comprising admixing a compound according to any one of claims 1 to 3 with a suitable excipient.
10. A compound according to any one of claims 1 to 3 for treating a bacterial infection.
11. An antibacterial composition comprising a compound according to any one of claims 1 to 3.
12. Use of a compound according to any one of claims 1 to 3 in an antibacterial composition.
13. A method of preparing an antibacterial composition comprising admixing a compound according to any one of claims 1 to 3 with a suitable excipient.
14. The method according to claim 13 wherein the excipient is an excipient suitable for agricultural use.
15. The method according to claim 13 wherein the excipient is suitable for formulation of a spray.
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