Antimicrobial composition

Abstract

The present disclosure relates to organosulfur compounds and compositions including the same. In particular, the present disclosure relates to bis (4-fluorophenyl) disulfides, bis (4-chlorophenyl) disulfides, bis (4-chlorophenyl) thiosulfides, bis (4-chlorophenyl) thiosulfides, bis (4-fluorophenyl) thiosulfides, bis (4-chlorophenyl) thiosulfides, bis (4-chlorophenyl) thiosulfates, bis (4-fluorophenyl) thiosulfates, and diisopropyl thiosulfates. Such compositions are useful for the treatment of microbial infections, in particular bacterial, fungal, protozoal or algal infections. Compositions comprising the compounds may also be used for cleaning, disinfecting or agricultural use.

Description

Technical Field

[0001] The present disclosure relates to organic sulfur compounds and compositions comprising the same. In particular, the present disclosure relates to bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and diisopropyl thiosulfate. Such compositions are used for treating microbial infections, particularly bacterial, fungal, protozoal, or algal infections. Compositions comprising the compounds for cleaning, disinfection, or agricultural use are also provided. Background Art

[0002] Microorganisms, such as bacteria and fungi, are almost ubiquitous and exist in a wide variety of forms. Most microorganisms are harmless and are in fact indispensable for life on Earth and are essential for plant, animal, and human health. For example, the microbiota in the human and animal gut, where bacteria and fungi coexist symbiotically with their host, is called the gut flora. In addition, bacteria are naturally present on the skin and are part of the immune system. Another example is soil biology, which consists mostly of bacteria, fungi, and protozoa. Another example is aquatic biology and wastewater treatment, where protozoa play an important role in improving water clarity. Some bacteria, fungi (including yeasts), protozoa, and algae can cause pathogen infections, for example, in animals or humans. These pathological infections can lead to diseases and illnesses in the infected individuals. Plants are also vulnerable to microbial infections.

[0003] Therefore, there is a need for antimicrobial compounds and alternative therapies for microbial infections. Summary of the Invention

[0004] The present disclosure provides the following preferred embodiments.

[0005] 1. A compound for treating or preventing microbial infections, preferably bacterial or fungal infections, or a composition comprising at least one such compound, the compound being selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate.

[0006] 2. A method for treating or preventing microbial infections in an individual, comprising administering to the individual in need thereof a compound, or a composition comprising at least one said compound, wherein the compound is selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate.

[0007] 3. A composition comprising a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate, wherein the composition is a pharmaceutical composition, a pesticide composition, or a food composition.

[0008] 4. An article having a surface at least partially coated with a compound or a composition comprising at least one said compound, wherein the compound is selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate, preferably, wherein the article is a medical device or a surgical device.

[0009] 5. A cleaning or disinfecting composition comprising

[0010] (i) a compound selected from: bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate; and

[0011] (ii) a surfactant.

[0012] 6. An in vitro method comprising applying a cleaning or disinfecting composition to a surface, wherein the composition comprises a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate.

[0013] 7. The method according to embodiment 6, wherein the method is a method for sanitizing or disinfecting the surface.

[0014] 8. Use of a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds, as a disinfectant, a sanitizing agent or an antimicrobial agent in food.

[0015] 9. A cleaning or disinfecting product comprising a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds.

[0016] 10. An agricultural composition comprising a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate and di-isopropyl thiosulfate, and an agricultural excipient, carrier and / or solvent.

[0017] 11. A method for preventing or treating an infection of a plant or a plant part, comprising contacting the plant or the plant part with a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds, thereby preventing or treating the infection.

[0018] 12. Use of a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds, for preventing or treating an infection of a plant or a plant part.

[0019] 13. The use or method according to embodiment 11 or 12, wherein the compound or composition is directly applied to the plant, the seeds of the plant or the soil of the plant or the seeds.

[0020] 14. Use or method according to any one of embodiments 11 - 13, wherein the plant is selected from the group consisting of Begoniaceae, Solanaceae, Amaranthaceae, Rosaceae, and Brassicaceae, preferably, wherein the plant is selected from the group consisting of Begonia, tomato, potato, beet, strawberry, Cabbage, apple, Orchidaceae, Chrysanthemum, Fabaceae, Cucurbitaceae, Pisum, Vitis, Vaccinia, and Lactuca. Brief description of the drawings:

[0022] Figure 1 : Average MIC90 values for each compound for each strain. These values were determined during the MIC assay and then averaged for each strain. Each data point is the average MIC90 for each strain. The labels at the data points represent the strain names: Escherichia coli = Ec, Klebsiella pneumoniae = Kp, Streptococcus agalactiae = Sag, Lactococcus garvieae = Lg, Streptococcus lactis = Su, Streptococcus suis = Ss, Staphylococcus aureus = Sau, Acinetobacter baumannii = Ab, Micrococcus luteus = Ml, Staphylococcus epidermidis = Se, Pseudomonas aeruginosa = Pa, Moraxella viscosa (M.viscosa) = MV, Candida albicans = Ca. For bis - 4 - chlorophenyl disulfide and Lg, Kp, Su, Sag, Ec, Ss, Sau, and Ab, the MIC90 is represented as 1000 μM, but is actually > 1000 μM because the MIC90 value could not be detected within the test range. The same is true for bis(4 - fluorophenyl) disulfide, Pa, Ab, Kp, Ec, and Lg, but its true MIC90 value is higher than 4000 μM, and for bis(4 - chlorophenyl) thiosulfate and Ec, Kp, and Ab, the MIC90 value > 1000 μM.

[0023] Figure 2 : Shows the cytotoxic effects of the emulsifier Tween - 80 and DMSO (without antimicrobial (AM) compounds) at the concentrations used on the Caco - 2 cell line.

[0024] Figure 3 : Shows the cytotoxic effects of the emulsifier Tween - 80 and DMSO (without antimicrobial (AM) compounds) at the concentrations used on the HepG2 cell line.

[0025] Figure 4: Cytotoxicity of emulsified AM2 on Caco-2 cells.

[0026] Figure 5 : Cytotoxicity of emulsified AM2 on HepG2 cells.

[0027] Figure 6 : Cytotoxicity of emulsified AM3 on Caco-2 cells.

[0028] Figure 7 : Cytotoxicity of emulsified AM3 on HepG2 cells.

[0029] Figure 8 : Cytotoxicity of emulsified AM6 on Caco-2 cells.

[0030] Figure 9 : Cytotoxicity of emulsified AM6 on HepG2 cells.

[0031] Figure 10 : Cytotoxicity of emulsified AM8-A on Caco-2 cells. AM8-A was emulsified with 30-fold less Tween 80 and DMSO was not added.

[0032] Figure 11 : Cytotoxicity of emulsified AM8-A on HepG2 cells. AM8-A was emulsified with 30-fold less Tween 80 and DMSO was not added.

[0033] Figure 12 : Cytotoxicity of emulsified AM8-C on Caco-2 cells. AM8-C was emulsified with 30-fold less Tween 80 and DMSO was not added.

[0034] Figure 13 : Cytotoxicity of emulsified AM8-C on HepG2 cells. AM8-C was emulsified with 30-fold less Tween 80 and DMSO was not added.

[0035] Figure 14 : After incubation with AM2, bis(p-chlorophenyl) disulfide; AM3, bis(p-fluorophenyl) disulfide; AM6, bis(p-chlorophenyl) thiosulfate; AM8, bis(isopropyl) thiosulfate and QQ2, diisopropyl thiosulfate for 7 days, the colony diameter of the fungus Pythium aphanidermatum was measured. The larger the colony diameter, the smaller the growth inhibition.

[0036] Figure 15 : After incubation with AM2, AM3, AM6, AM8 and QQ2 for 7 days, the colony diameter of the fungus Phytophthora cinnamiomi was measured.

[0037] Figure 16 : After incubation with AM2, AM3, AM6, AM8, and QQ2 for 7 days, the colony diameter of the fungus Fusarium oxysporum was measured.

[0038] Figure 17 : After incubation with AM2, AM3, AM6, AM8, and QQ2 for 7 days, the colony diameter of the fungus Sclerotinia sclerotiorum was measured.

[0039] Figure 18 : After incubation with AM2, AM3, AM6, AM8, and QQ2 for 7 days, the colony diameter of the fungus Rhizoctonia solani was measured.

[0040] Figure 19 : After incubation with AM2, AM3, AM6, AM8, and QQ2 for 7 days, the colony diameter of the fungus Botrytis cinerea was measured.

[0041] Figure 20 : The raw data of the colony diameter 3 days after inoculation.

[0042] Figure 21 : The raw data of the colony diameter 7 days after inoculation.

[0043] Figure 22 : The raw data of the colony diameter 10 days after inoculation.

[0044] Figure 23 : The raw data of the colony diameter 14 days after inoculation.

[0045] Figure 24 : Effect of daily exposure to AM-8 on dAMR Staphylococcus aureus in mature biofilms: Seven-day-old mature Staphylococcus aureus LUH14616 biofilms in 96-well polystyrene plates were exposed daily to different concentrations of AM-8 or (as a control, its diluent (1% DMSO)) for up to 4 days and exposed to PBS containing 2% v / v BHI. After each day, viable cell counts of the biofilms were performed.

[0046] Figure 25 : Effects of AM-2 and AM-8 on persister cells of AMR Staphylococcus aureus:

[0047] The Staphylococcus aureus LUH14616 biofilm that matures in seven days was exposed to 10-fold the minimum bactericidal concentration (MBC) of rifampicin and ciprofloxacin for up to three days. Next, the biofilm was exposed to different concentrations of AM-2 or AM-8 for 24 hours, or exposed to 10xMBC rifampicin / ciprofloxacin (to demonstrate the antibiotic tolerance of persister bacteria). The experiment was repeated three times.

[0048] Figure 26 : AM-8 hardly produces / no resistance in AMR Staphylococcus aureus LUH14616: Staphylococcus aureus LUH146161 was subsequently exposed to a series of AM-8 concentrations. For this purpose, two independently synthesized batches of AM-8 with a minimum purity of 96.3% were used. A total of 19 passages were carried out. As a control, Staphylococcus aureus LUH14616 was exposed to a range of doses of rifampicin, and when resistance occurred, the antibiotic concentration was adjusted to the range of antibiotic concentrations of the above AM-8. The results are expressed as fold increase, that is, the ratio of the MIC after each passage to the MIC at the start of the experiment. The circles and squares in the figure coincide.

[0049] AM2 = bis(4-chlorophenyl) disulfide

[0050] AM3 = bis(4-fluorophenyl) disulfide

[0051] AM6 = bis(4-chlorophenyl) thiosulfate

[0052] AM8 = diisopropyl thiosulfate. AM8-A and AM8-C correspond to two different batches of synthesized AM8. Detailed implementation mode

[0053] The present disclosure provides new uses and methods, as well as compositions comprising one or more compounds selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate. The compounds are also described herein as "the compounds of the present invention". Preferably, the compounds are selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, and di-isopropyl thiosulfate. Preferably, the compounds are selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, and di-isopropyl thiosulfate.

[0054] In a preferred embodiment, the compound is bis(4-fluorophenyl) disulfide. In a preferred embodiment, the compound is bis(4-chlorophenyl) disulfide. In a preferred embodiment, the compound is bis(4-chlorophenyl) thiosulfite. In a preferred embodiment, the compound is bis(4-fluorophenyl) thiosulfite. In a preferred embodiment, the compound is bis(4-chlorophenyl) sulfate. In a preferred embodiment, the compound is bis(4-fluorophenyl) sulfate. In a preferred embodiment, the compound is di-isopropyl sulfate.

[0055] It should be understood that the terms "sulphide" and "sulfide" are used interchangeably herein.

[0056] Methods for preparing the compounds described herein are known in the art. In some embodiments, the compounds are commercially available or can be prepared as described in Example 1.

[0057] In some embodiments, there is provided a composition wherein at least 50%, preferably at least 90% by weight of the active ingredient is a compound of the present invention. In some embodiments, there is provided a composition wherein the only active ingredient is a compound of the present invention, optionally comprising a further antibacterial agent and / or an anti-inflammatory agent. In some embodiments, there is provided a composition wherein at least 50%, preferably at least 90% by weight of the active ingredient is a compound of the present invention as disclosed herein. In some embodiments, there is provided a composition wherein the only active ingredient is a compound of the present invention, optionally comprising a further antibacterial agent and / or an anti-inflammatory agent.

[0058] Microbial infection

[0059] The compounds and compositions comprising the same disclosed herein can be used for treating or preventing infections. For example, specific uses are for treating or preventing respiratory infections, intestinal infections, chest infections, breast infections, skin infections, bladder infections, ear infections, systemic infections, joint infections, brain infections. Infections suitable for treatment also include, for example, bacterial prostatitis, bacterial vaginosis, biliary tract infections, chronic sinusitis, chronic lung diseases, dental caries, endocarditis, kidney stones, laryngitis, lung infections in cystic fibrosis, gingivitis, mastitis, middle ear infections, non-social (bloodstream) infections, obstructive lung diseases, osteomyelitis, otitis media, periodontitis, pneumonia, prostatitis, rhinosinusitis, sinusitis, tonsillitis, tuberculosis, urinary tract infections and wound infections.

[0060] As used herein, "infection" refers to an infection by a pathogen that can cause a disease, for example. Specifically, such infections are bacterial, fungal (including yeast), protozoal, or algal infections. Preferably, the infection is a microbial infection. In a preferred embodiment, the infection is a bacterial infection. In a preferred embodiment, the infection is a fungal infection (including yeast infection). In a preferred embodiment, the infection is a protozoal infection. In a preferred embodiment, the infection is an algal infection.

[0061] Treatment or prevention

[0062] As used herein, "treatment of an infection" refers to reducing the severity and / or duration of the infection and / or reducing the severity and / or duration of the symptoms of the infection. Preferably, the treatment results in the restoration of the health of the individual. Preferably, the individual has fewer or shorter-lasting disease symptoms. As used herein, "prevention of an infection" refers to preventing or delaying the occurrence of an infection or one or more symptoms associated with the infection.

[0063] The compounds disclosed herein can be used to treat acute infections. Acute infections can be characterized by the growth of microorganisms (such as bacteria) in a planktonic state, while chronic infections are generally associated with the presence of biofilms. In some embodiments, acute infections are characterized by an infection (or infection symptoms) lasting less than 6 months.

[0064] The compounds disclosed herein can also be used to treat chronic and / or persistent infections. The terms "persistent infection" and "chronic infection" are generally used interchangeably, but are based on different mechanisms. Persistent infections are generally controlled by the immune defense, but can be activated when this immune defense weakens. Persistent infections are generally asymptomatic and are only clinically visible when the immune defense is unable to control the pathogen. Although persistent infections are generally asymptomatic, methods for detecting such persistent infections are well known to those skilled in the art, including, for example, detecting microorganisms in patient samples (such as blood or urine). In chronic infections, the pathogen remains in a group of cell / tissue compartments (such as joints or lung tissue). The patient always has disease symptoms, although these symptoms may be milder than those in the acute phase of the infection.

[0065] In some embodiments, the infection is a chronic wound infection. In some embodiments, the wounds treated with the compounds of the present invention include, for example, Staphylococcus aureus; Streptococcus; Gram-negative bacteria, such as Treponema, Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa; or yeast / fungi, such as Candida (Candida albicans), Cladosporidium herbarum, Trichosporum, Rhodosporidium, and Malassezia.

[0066] In some embodiments, the infection is caused by one or more microorganisms selected from: Acinetobacter baumannii, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Piscirikettsia salmonis, Renibacterium salmoninarum, Staphylococcus aureus, Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Streptococcus agalactiae, Escherichia coli, Clostridium perfringens, Myxobacteria, Micrococcus luteus, Lactococcus garvieae, Candida albicans, Haemophilus influenzae, Streptococcus suis, Streptococcus suis type 2, Streptococcus dysgalactiae, Arcanobacterium pyogenes, Treponema, Yersinia pestis, Streptococcus dysgalactiae subspecies equisimilis, Serratia marcescens, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, Legionella pneumophila, Fusobacterium necrophorum, Corynebacterium pseudotuberculosis, Streptococcus spp., Porphyromonas gingivalis, Pseudomonas aeruginosa, Enterococcus faecalis, Neisseria gonorrhoeae, Salmonella enterica and Pseudomonas aeruginosa. Preferably, the infection is caused by Candida albicans.

[0067] In preferred embodiments, the bacterial infection is caused by one or more bacteria selected from Acinetobacter baumannii, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Piscirikettsia salmonis, Renibacterium salmoninarum, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Clostridium perfringens, Myxococcus stipitatus, Micrococcus luteus, Lactococcus garvieae, Candida albicans, Haemophilus influenzae, Streptococcus suis type 2, Streptococcus dysgalactiae and Arcanobacterium pyogenes.

[0068] In certain embodiments, the bacterial infection is caused by Gram-negative bacteria. In certain embodiments, the bacterial infection is caused by Gram-positive bacteria. In certain embodiments, the bacterial infection is caused by multi-drug resistant bacteria. In certain embodiments, the bacterial infection is a methicillin-resistant Staphylococcus aureus (MRSA)-associated infection or a Staphylococcus epidermidis (e.g., MRSE)-associated infection.

[0069] In a preferred embodiment, the bacterium causing the infection is Escherichia coli. Preferably, the bacterial infection is a recurrent urinary tract infection, a catheter-associated urinary tract infection or a biliary tract infection.

[0070] In a preferred embodiment, the bacterium causing the infection is Pseudomonas aeruginosa. Preferably, the bacterial infection is a cystic fibrosis lung infection, a chronic wound infection, a catheter-associated urinary tract infection, chronic rhinosinusitis, chronic otitis media, bronchiectasis, chronic obstructive pulmonary disease or contact lens-related keratitis.

[0071] In a preferred embodiment, the bacterium causing the infection is Staphylococcus aureus. Preferably, the bacterial infection is chronic osteomyelitis, chronic rhinosinusitis, endocarditis, chronic otitis media or an (orthopedic) implant.

[0072] In a preferred embodiment, the bacterium causing the infection is Staphylococcus epidermidis. Preferably, the bacterial infection is a central venous catheter, an orthopedic implant, or chronic osteomyelitis.

[0073] In a preferred embodiment, the bacterium causing the infection is Streptococcus pneumoniae. Preferably, the bacterial infection is a nasopharyngeal infection, chronic rhinosinusitis, chronic otitis media, or a chronic obstructive pulmonary disease infection.

[0074] In a preferred embodiment, the bacterium causing the infection is Streptococcus pyogenes. Preferably, the bacterial infection is an oral and nasopharyngeal infection, recurrent tonsillitis.

[0075] In some embodiments, the fungal infection is caused by a fungus selected from the group consisting of Absidia, Actinomucor, Aspergillus, Botrytis, Candida, Centrospora, Cephalosporium, Diatrype, Chaetomium, Chaetomium, Cladosporium, Colletotrichum, Conidiobolus, Didymella, Fusarium (including Fusarium oxysporum), Geotrichum, Guignardia, Helminthosporium, Histoplasma, Lecythophora, Malassezia, Nectria, Nocardia, Oospora, Ophiobolus, Paecilomyces, Paracoccidioides brasiliensis, Penicillium, Phymatotrichum, Phytophthora, Pythium, Piedraia hortai, Rhizoctonia, Rhizopus, Rhodosporidium, Saccharomyces, Sclerotinia, Sclerotinia, Torulopsis, and Trichophyton. In a preferred embodiment, the fungal infection is caused by the genus Fusarium. In a preferred embodiment, the fungal infection is caused by Fusarium oxysporum.

[0076] In some embodiments, the protozoal infection is caused by a protozoan selected from the group consisting of Plasmodium, Entamoeba, Giardia, Toxoplasma, Cryptosporidium, Trichomonas, Trypanosoma, Leishmania, Acanthamoeba, Naegleria, Balantidium, Babesia, and Cyclospora. Preferably, the protozoal infection is caused by a protozoan of the genus Plasmodium.

[0077] In some embodiments, the algal infection is caused by microalgae selected from the group consisting of Prototheca, Helicosporidium, Chlorella, and Desmodesmus. Preferably, the algal infection is caused by algae of the genus Prototheca. More preferably, the algal infection is a breast infection.

[0078] The determination of acute and chronic infections is known to practitioners. For example, according to the Mayo Clinic, having four or more yeast infections in a year indicates a chronic yeast infection, while having two or more bladder infections in six months indicates a chronic bladder infection (also known as recurrent urinary tract infection).

[0079] The most common method for treating bacterial pathological infections is the use of antibiotics. Current antibiotics mainly act through growth-dependent mechanisms and target rapidly dividing bacteria. However, non-replicating or slower-growing bacteria (such as dormant persister cells) exhibit high levels of antibiotic tolerance and / or resistance, leading to persistent and recurrent infections. The compounds disclosed herein are applicable to infections including antibiotic-resistant bacteria, antibiotic-tolerant bacteria, and antibiotic-persistent bacteria. The compounds disclosed herein are also suitable as a second-line therapy, or more precisely, for individuals who do not respond to previous treatments (such as antimicrobial treatments) or in whom the disease recurs, for example, within one year or six months.

[0080] As shown in Example 5, the compounds disclosed herein also result in little or no resistance. This is an advantage of antibiotic treatment.

[0081] In some embodiments, microbial infections also include infections caused by microorganisms attached to indwelling devices (e.g., medical implants, catheters, etc.). In some embodiments, the compounds and compositions disclosed herein can be used to treat and prevent infections of implanted medical devices (e.g., joint prostheses and heart valves further disclosed herein).

[0082] In some embodiments, the compounds and compositions disclosed herein can also be used to prevent or reduce inflammation caused by infection. Inflammation is part of the complex biological response of body tissues to harmful stimuli (such as pathogens) and is a protective response involving immune cells and molecular mediators. The function of inflammation is to eliminate pathogens.

[0083] In a preferred embodiment, treating an individual with the compounds disclosed herein or a composition comprising the same compounds can prevent or reduce clinical inflammation in an animal (e.g., a cow). Preferably, the treatment can prevent or reduce (clinical) inflammation of the udder. In another embodiment, treating an individual with the compounds and compositions disclosed herein can prevent or reduce (clinical) inflammation in a human. For example, the treatment can prevent or alleviate skin inflammation, preferably prevent eczema.

[0084] In addition to the compounds described herein, other anti-inflammatory drugs can be administered to inhibit the inflammatory response and reduce tissue damage. In a preferred embodiment, the treatment (treatment and prophylaxis) disclosed herein further comprises administering an anti-inflammatory agent. Anti-inflammatory agents include, for example, non-steroidal anti-inflammatory agents (cox / lox inhibitors) such as ibuprofen, paracetamol, aspirin, diclofenac, ketoprofen, tolmetin, etodolac and fenoprofen. Natural anti-inflammatory agents can also be used, such as curcumin, ginger, spirulina, capsicum, cinnamon, cloves, sage, rosemary, black pepper, natural aspirin, frankincense, sanguinaria and / or green tea. In some embodiments, the methods and uses disclosed herein include combination therapy of the therapeutic organosulfur compounds disclosed herein with an anti-inflammatory agent. The compounds can be administered together or separately. In some embodiments, a composition comprising the therapeutic organosulfur compound and an anti-inflammatory agent disclosed herein is provided.

[0085] In some embodiments, the method comprises administering to an individual in need a composition comprising a compound disclosed herein, preferably for the treatment or prophylaxis of an infection (in particular a bacterial, fungal, yeast, protozoal or algal infection). In some embodiments, the composition can be administered to an individual to treat (e.g., a therapeutic agent) or prevent (e.g., a prophylactic agent) a disease or disorder or an infection. In some embodiments, the individual has or is at risk of having a microbial infection.

[0086] The composition can be administered to any individual, particularly an animal. Preferably, the animal is a ruminant (such as cattle and goats), more preferably cattle. In some embodiments, the animal is not a dairy cow. Preferably, the animal is a non-ruminant, such as a monogastric animal, a rodent, a non-human primate, a pig, a horse, a dog, a cat or a bird. In a preferred embodiment, the animal is a human. In some embodiments, the animal is a non-human animal. In some embodiments, the animal is an aquatic animal, such as a fish, a mollusk and a crustacean. Preferably, the animal is a mammal or a bird.

[0087] Without wishing to be bound by theory, the present disclosure provides that the compositions disclosed herein can have beneficial effects after a single administration. In a preferred embodiment, the effect is achieved by providing a single oral administration of the composition disclosed herein. Such oral administration can be, for example, an extended-release tablet providing a compound disclosed herein.

[0088] The present disclosure also provides multiple administrations. For example, the composition can be provided more than once a day, daily, weekly or monthly. In an exemplary embodiment, the composition can be provided once a day for a week or until symptoms resolve. Since the compounds disclosed herein produce little to no drug resistance, they can be discontinued once symptoms resolve or multiple treatments are required.

[0089] The actual dosage levels of the pharmaceutical preparations described herein may vary to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and / or materials used in combination, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition.

[0090] Those skilled in the art know that smaller animals require a larger drug dose per body weight because of their higher metabolic rate. Nair and Jacob (J Basic Clin Pharm. Mar-May 2016;7(2):27–31) and Holliday et al. (1967 The Relation of Metabolic Rate to Body Weight and Organ Size. A Review. Pediat. Res. 1:185-195) reviewed dose conversions between animals and between humans and animals.

[0091] In some embodiments of the methods and uses disclosed herein, a compound disclosed herein is provided to a human at a level of at least 5 mg / day (e.g., by oral administration). Preferably, a compound is provided at a level of at least 10 mg / day. In some embodiments, a compound disclosed herein is provided to a human at a dose between 0.1 mg / kg and 100 mg / kg. Such an amount of the compound is particularly useful when the compound is provided systemically (e.g., orally). Those skilled in the art will recognize that lower amounts may be used when administered topically (e.g., on the skin, gums, wounds). The compositions disclosed herein are preferably provided for at least one week or until symptoms resolve. Although such compositions may be provided several times (e.g., once a week, once a month, twice a year, etc.), prophylactic and therapeutic effects are observed after a single use.

[0092] In some embodiments, a composition is provided that comprises a compound disclosed herein and one or more other pharmaceutical agents, such as antibiotics (e.g., antibacterial, antiviral, antifungal), anti-inflammatory agents, antipyretics, and analgesics.

[0093] In some embodiments, a compound disclosed herein is used in combination with another antibacterial agent (e.g., an antifungal drug or an antibiotic). As will be understood by those skilled in the art, the combination of an antibacterial agent with a compound described herein can reduce the dose and / or frequency of administration of the antibacterial agent.

[0094] Exemplary antimicrobials that can be used in combination therapy include antifungals such as miconazole, ketoconazole, econazole, terbinafine, ciclopirox, tolnaftate, sertaconazole, sulconazole, amphotericin b, chloroxylenol, chloroiodoquine, butenafine, naftifine, nystatin, and clotrimazole. Exemplary antibiotics include penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems.

[0095] The present disclosure provides compositions comprising the compounds disclosed herein and an antimicrobial. As will be understood by a skilled practitioner, the compounds and the antimicrobial can also be provided separately. In some embodiments, the compounds and the antimicrobial therapy overlap. In some embodiments, treatment with the compounds of the invention precedes the antimicrobial therapy.

[0096] In some embodiments, the compositions disclosed herein are provided as a food composition or a food product or a functional food product.

[0097] As used herein, the term "food" refers to any liquid or solid substance intended for ingestion to provide nutritional support and energy to a subject / organism. Food includes any type of food or animal feed, as well as functional foods. For example, a food composition can be incorporated into food, or it can be applied to the surface of food (e.g., in spray or liquid form). Food can also be immersed in the food composition. In some embodiments, the food composition prevents or inhibits spoilage of the food by microorganisms. In some embodiments, the food composition inhibits the growth of microorganisms and / or kills microorganisms on the food. A person skilled in the art can determine appropriate concentrations to achieve the desired inhibition of or killing of microbial growth.

[0098] As used herein, the term "functional food" refers to those foods that are prepared not only for their nutritional properties, but also to achieve a specific function, such as improving health or reducing the risk of contracting a disease. Such functional foods can also be referred to as dietary supplements or (animal) food additives. To this end, bioactive compounds such as minerals, vitamins, fatty acids, bacteria with beneficial effects, dietary fiber, and antioxidants, etc. are added thereto. Such foods can be in any suitable form for oral administration, such as in liquid, gel, powder, pill, tablet, or gel capsule form.

[0099] Functional foods may also include animal digests, such as any material produced by chemical and / or enzymatic hydrolysis of clean and undigested animal tissues. Functional foods may be those that also include dried brewer's yeast, for example, the dried inactivated agent as a by-product of the brewing industry. Animal digests and dried brewer's yeast have been found to enhance the palatability of functional foods. When present in functional foods, animal digests account for about 10% to about 90% of the functional food, while dried brewer's yeast accounts for about 1% to about 30% of the functional food.

[0100] In some embodiments, the present disclosure provides a composition comprising a compound of the invention and at least one pharmaceutically acceptable carrier, diluent, and / or excipient. (See, e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (ed.) Mack Publishing Company, April 1997). As used herein, the term "pharmaceutically acceptable" refers to those compositions, or combinations of agents, materials, or compositions and / or their dosage forms within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. In addition, the term "pharmaceutically acceptable diluent or carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, used to transport or convey a peptide from one organ or body part to another.

[0101] The pharmaceutical composition can be administered by any suitable route and manner. As is known to those skilled in the art, the route and / or manner of administration will vary depending on the desired result. The pharmaceutical composition can be formulated according to conventional procedures for administration by any route, such as parenterally, topically (including ophthalmic), orally, sublingually, transdermally, or by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intracoronary, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. The preferred routes are oral or topical administration.

[0102] The composition can be in any suitable form, such as liquid, semi-solid, and solid dosage forms. The composition can be tablets, capsules, powders, granules, lozenges, creams, or liquid formulations (especially for skin or ocular administration), such as sterile parenteral solutions or suspensions, or sprays, aerosols, or other conventional inhalation methods. The pharmaceutical compositions of the present invention include pharmaceutical compositions suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. In a particular embodiment, the composition is a topical composition in the form of a cream, gel, ointment, lotion, foam, suspension, spray, aerosol, or powder aerosol. The composition is particularly suitable for skin administration. Suitable compositions also include oral care compositions, such as toothpaste, dentifrice, tooth powder, tooth gel, subgingival gel, mouthwash / gargle, artificial saliva, denture products, oral sprays, lozenges, oral tablets, and chewing gums.

[0103] The present disclosure also provides a cleaning or disinfecting product comprising one or more of the compounds disclosed herein or the compositions disclosed herein. In some embodiments, the product is a solution. The solution includes a cleaning solution, a sanitizing solution, and a disinfecting solution. In some embodiments, the product is an article, such as a rag, cloth, pad, or sponge.

[0104] The present invention also provides a cleaning, sanitizing, or disinfecting composition comprising the compound of the present invention. Preferably, the composition further comprises at least one surfactant. Suitable surfactants include anionic surfactants, cationic surfactants, nonionic surfactants, or amphoteric surfactants. In some embodiments, the cleaning or disinfecting composition comprises an anionic surfactant. Anionic surfactants include, for example, alkyl sulfates (such as sodium lauryl sulfate, sodium laureth sulfate, ammonium lauryl sulfate, ammonium laureth sulfate, sodium myristyl sulfate), sulfonates (such as perfluorooctane sulfonate, perfluorobutane sulfonate), alkyl ether phosphates, alkyl aryl ether phosphates, carboxylates (such as sodium stearate, perfluorooctanoate).

[0105] In some embodiments, the cleaning or disinfecting composition comprises a cationic surfactant. Suitable cationic surfactants include, for example, quaternary ammonium compounds and their salts (such as cetyltrimethylammonium bromide, benzalkonium chloride, benzethonium chloride, dimethyldioctadecylammonium chloride, dioctadecyldimethylammonium bromide, cetylpyridinium chloride).

[0106] In some embodiments, the cleaning or disinfecting composition comprises a nonionic surfactant. Suitable nonionic surfactants include, for example, fatty alcohol ethoxylates, alkylphenol ethoxylates, ethoxylated amines, fatty acid amides (such as cocoamide monoethanolamine, cocoamide diethanolamine), poloxamers, polyethylene glycols, glycerol fatty acid esters (such as glycerol monostearate, glycerol monolaurate), alkyl polyglycosides (such as decyl glucoside, lauryl glucoside, octyl glucoside), sorbitan esters (such as sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate), and polysorbates (such as Tween 20, Tween 80).

[0107] Suitable amphoteric surfactants include alkylamidopropylamine oxides, alkyldimethylamine oxides, betaines (such as lauryl betaine, cocoamidopropyl betaine), dodecyldimethylamine oxide, and myristamine oxide

[0108] In some embodiments, the composition comprises at least 0.1 wt% surfactant.

[0109] The present disclosure also provides in vitro methods that include applying the compositions disclosed herein to a surface. Preferably, the method is a method of cleaning, sanitizing, or disinfecting. More preferably, the method is a method of sanitizing or disinfecting. The compositions used in the method include, for example, cleaning compositions, sanitizing compositions, and disinfecting compositions. In some embodiments, the method includes contacting microorganisms adhered to the surface with the compositions disclosed herein.

[0110] Any surface can be treated with the compounds or compositions disclosed herein to coat such surface. The surface can be, for example, sprayed, dipped, wiped, or soaked in the composition. Surfaces include glass, metal, porous and non-porous surfaces. It also relates to the external and internal surfaces of equipment that may be contaminated, such as equipment in the food industry or medical equipment in hospitals and healthcare facilities, as well as plumbing systems (such as sink drains), countertops, building materials, plumbing systems, clean rooms. Surfaces also refer to the internal or external surfaces of pipes, such as drain pipes, as well as surfaces in swimming pools, water tanks (such as for aquaculture), purification filters, toilets, sinks, greenhouses. Surfaces also include water, such as water from drinking troughs.

[0111] In some embodiments, the surface is the surface of a medical device, such as a prosthesis (hip implant, dental implant, prosthetic joint, voice prosthesis, penile prosthesis), mechanical heart valve, cardiac pacemaker, arteriovenous shunt, scleral buckle, catheter (e.g., central venous catheter, intravascular catheter, urinary catheter, Hickman catheter, peritoneal dialysis catheter, endotracheal catheter), tympanostomy tube, tracheostomy tube, surgical suture, bone anchor, bone screw, intraocular lens, contact lens, intrauterine device, aortic femoral graft or vascular graft. Other medical devices include abdominal drain, biliary stent, breast implant, cardiac pacemaker, cerebrospinal fluid shunt, contact lens, defibrillator, denture, electrodialyzer, endotracheal tube, indwelling urinary catheter, intrauterine device, intravenous catheter, joint prosthesis, mechanical heart valve, nephrostomy tube, orthopedic implant, peritoneal dialysis catheter, artificial heart valve, allograft orthopedic device, tissue filler, urethral stent, vascular prosthesis, ventilator-associated pneumonia, ventricular assist device, ventricular assist device, ventricular shunt and voice prosthesis.

[0112] In some embodiments, the surface is the surface of a surgical instrument, such as forceps, tweezers, scissors, skin hook, tube, needle, retractor, curette, drill, chisel, file or saw.

[0113] The present disclosure also provides an article having a surface at least partially coated with a compound of the present invention or a composition disclosed herein. In some embodiments, the article is a medical device or a surgical instrument.

[0114] In some embodiments, the in vitro method is a method for cleaning the surface. As used herein, the term "cleaning" refers to the removal of visible dirt (e.g., organic and inorganic materials), grime, debris and / or other impurities from the surface.

[0115] In some embodiments, the in vitro method is a method for sanitizing the surface. As used herein, the terms "sanitizing", "sanitization" or "sanitize" refer to reducing the number of microorganisms to a level considered safe according to public health standards or requirements. Sanitization may not necessarily eliminate all microorganisms on the treated surface. Such standards and requirements are well known to those skilled in the art.

[0116] In some embodiments, the in vitro method is a method of disinfecting the surface. The terms "disinfecting", "disinfection", or "disinfect" as used herein refer to the destruction and / or irreversible inactivation of pathogenic microorganisms and other types of microorganisms (except bacterial spores). The terms "inactivating" or "inactivation" as used herein refer to rendering microorganisms unable to grow / replicate. In some embodiments, the disinfection method eliminates all pathogenic microorganisms and other types of microorganisms. In some embodiments, the disinfectant is a chemical sterilant. The chemical sterilant is a disinfectant for long-term exposure that can kill spores. In some embodiments, the disinfectant is a high-level disinfectant, an intermediate-level disinfectant, or a low-level disinfectant. High-level disinfectants kill all microorganisms except a large number of bacterial spores. Intermediate-level disinfectants can kill mycobacteria, bacteria, most viruses, and most fungi, but not necessarily bacterial spores. Low-level disinfectants can kill most bacteria, some fungi, and some viruses. As will be understood by those skilled in the art, in some embodiments, the use of a surface disinfection method or a disinfection composition does not require the destruction or inactivation of all microorganisms or all types of microorganisms to produce a useful effect.

[0117] Hygiene treatment and disinfection result in a reduction in the number of specific microorganisms or colony-forming units (CFUs). The efficiency of hygiene treatment or disinfection is typically described by a logarithmic reduction. The terms "n-log reduction" or "n-logarithmic reduction" as used herein refer to the percentage of specific microorganisms reduced / killed / inactivated by a disinfection or hygiene treatment method. A 1-log reduction means a 90% reduction (i.e., 10 times smaller) of the target microorganism from the original level; a 2-log reduction means a 99% reduction (i.e., 100 times smaller) of the target microorganism from the original level; a 3-log reduction means a 99.9% reduction (i.e., 1000 times smaller) of the target microorganism; and so on.

[0118] In some embodiments, the disinfection provides at least a 3-log reduction, preferably at least a 5-log reduction, more preferably at least a 6-log reduction. In a preferred embodiment, the disinfection provides at least a 6-log reduction.

[0119] In some embodiments, the hygiene treatment provides at least a 1-log reduction, at least a 2-log reduction, at least a 3-log reduction, at least a 5-log reduction, at least a 6-log reduction. In a preferred embodiment, the hygiene treatment provides at least a 3-log reduction.

[0120] In some embodiments, the cleaning or disinfecting composition is contacted with the treated surface for a minimum contact time sufficient. In some embodiments, the cleaning or disinfecting composition is contacted with the treated surface for at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 25 minutes, at least 30 minutes, at least 1 hour, at least 2 hours.

[0121] Hygienic treatment and disinfection can provide residual or persistent efficacy against microorganisms. In some embodiments, the residual efficacy provided by the hygienic treatment or disinfection lasts for at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least one week, at least two weeks, at least one month.

[0122] In some embodiments, the residual efficacy provided by the hygienic treatment or disinfection lasts for at most 2 hours, at most 6 hours, at most 12 hours, at most 24 hours, at most 48 hours, at most 72 hours, at most one week, at most two weeks, at most one month.

[0123] The present disclosure also provides the use of the compounds or compositions disclosed herein as disinfectants, sanitizing agents or antimicrobial agents in food.

[0124] In some embodiments, the use of an antimicrobial agent in food prevents or inhibits spoilage and / or degradation of the food by microorganisms. In some embodiments, the use of the antimicrobial agent inhibits the growth of microorganisms on the food and / or kills the microorganisms. A person skilled in the art can determine the appropriate concentration of the antimicrobial agent to obtain the desired growth inhibition or killing of microorganisms in the food.

[0125] Furthermore, the present disclosure provides an agricultural composition comprising one or more of the compounds of the present invention and an agriculturally acceptable excipient and / or carrier. Such carriers and solvents are known to those skilled in the art and do not cause unacceptable damage to the plant or its environment, and / or are not dangerous to the user or other persons who may be exposed. For example, an agriculturally acceptable carrier can be a solid carrier, a gel carrier, a liquid carrier, a suspension or an emulsion. A non-limiting example of a solvent is water.

[0126] In some embodiments, the composition comprises at least 40 wt%, preferably at least 50 wt% of one or more of the compounds of the present invention. In some embodiments, the composition comprises at least 60 wt%, preferably at least 80 wt%, more preferably at least 95 wt% of one or more of the compounds of the present invention.

[0127] The present disclosure includes a method for preventing or treating infection of a plant or a plant part, comprising contacting the plant or the plant part with the compound of the present invention or the composition disclosed herein.

[0128] The present disclosure includes using the compounds of the present invention or the compositions disclosed herein for preventing or treating infections of plants or plant parts.

[0129] One skilled in the art can determine whether the compounds of the present invention or the compositions disclosed herein prevent or treat plant infections. Different bacteria and fungi attack different plants and plant parts and cause different symptoms. For example, bacteria or fungi may cause changes in quality characteristics, such as changes in the color, shape, size, hardness, etc. of the plant or plant part; rot, wounds; or wilting, depending on the genus or species of the plant. Generally, one skilled in the art, such as a farmer or grower, knows which plants are associated with which quality characteristics. The compounds or compositions disclosed herein have a beneficial effect on plants or plant parts. Specifically, the compounds or compositions disclosed herein prevent or treat infections of plants or plant parts. In some embodiments, compared to an infected plant, the compounds or compositions disclosed herein can improve one or more quality characteristics.

[0130] In some embodiments, the infection of the plant or plant part is caused by bacteria or fungi. In some embodiments, the infection of the plant or plant part is caused by one or more bacteria selected from the group consisting of: Xanthomonas spp., Erwinia amylovora, Rhizobium spp., Clavibacter michiganensis, Agrobacterium radiobacter, Burkholderia spp., Pseudomonadota spp., Pseudomonas spp. (preferably Pseudomonas syringae), Phytoplasma spp., and Spiroplasma spp.

[0131] In some embodiments, the infection of the plant or plant part is caused by one or more bacteria selected from the group consisting of Acidovorax, Bacillus, Dickeya, Pectobacterium, Pantoea, Burkholderia, Erwinia, Ralstonia, Rhizobium, Streptomyces, Clavibacter, Xylella, Vitis vinifera, and Agrobacterium.

[0132] In some embodiments, the infection of the plant or plant part is caused by one or more bacteria selected from the group consisting of Pseudomonas syringae pathovars, Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas oryzae pv. Oryzae, Xanthomonas campestris pathovars, Xanthomonas axonopodis pathovars, Erwinia amylovora, Xylella fastidiosa, Dickeya dadantii, Dickeva solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum.

[0133] In some embodiments, the infection of the plant or plant part is caused by one or more bacteria selected from the group consisting of Xanthomonas campestris, Erwinia amylovora, Rhizobium spp., Clavibacter michiganensis, and Agrobacterium radiobacter.

[0134] In some embodiments, the infection of the plant or plant part is caused by a fungus selected from one or more of the following: Pythium ultimum, Fusarium oxysporum, Fusarium solani, Phytophthora cactorum, Rhizoctonia spp. (such as Rhizoctonia solani), Oidium spp., Uncinula spp., Erysiphe spp., Fusarium spp., Thielaviopsis spp., Verticillium spp., Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago spp., Phakospora pachyrhizi, Puccinia spp., and Armillaria spp.

[0135] In some embodiments, the infection of the plant or plant part is caused by oomycetes, such as Pythium spp. and Phytophthora spp. In some embodiments, the infection of the plant or plant part is caused by Phytomyxea spp.

[0136] The compound or composition can be applied to the plant or plant part (including cuttings, newly emerged seedlings, and established vegetation, including roots and above-ground parts, such as leaves, stems, flowers, fruits, branches, limbs, roots, etc.), plant seeds (e.g., before germination), or the surrounding soil, particularly the rhizosphere of the plant. As used herein, the term rhizosphere refers to the soil region adjacent to the roots of a living plant. The width of the rhizosphere is typically within 100 millimeters from the root surface.

[0137] In some embodiments, the compound of the present invention or the composition comprising the compound is directly applied to the plant, the seeds of the plant, or the soil of the plant or seeds.

[0138] As used herein, the term "plant" encompasses crops, ornamental plants, trees, grasses, annuals, perennials, or any other common cultivated members of the plant kingdom. As used herein, the term "crop" includes plant species of commercial value that are grown and cultivated for commercial purposes. Thus, crops include floral and non-floral plants, perennials and annuals, trees, shrubs, vegetable plants, fruit trees, turf, and ground cover plants.

[0139] In some embodiments, the plant belongs to a family selected from the group consisting of Begoniaceae (Begoniaceae), nightshades (Solanaceae), amaranths (Amaranthaceae), roses (Rosaceae), crucifers (Brassicaceae or Cruciferae), orchids (Orchidaceae), composite (Asteraceae or Compositae), Fabaceae, cucumber (Cucurbitaceae), Vitis, and Vaccinia.

[0140] In some embodiments, the plant belongs to a family selected from the group consisting of Begoniaceae (Begoniaceae), nightshades (Solanaceae), amaranths (Amaranthaceae), roses (Rosaceae), crucifers (Brassicaceae or Cruciferae).

[0141] In a preferred embodiment, the plant belongs to the family Begoniaceae. Preferably, the plant belongs to the genus Begonia.

[0142] In a preferred embodiment, the plant belongs to the family Solanaceae. Preferably, the plant belongs to the genus Solanum. Preferably, the plant is selected from tomato (S. lycopersicum), potato (S. tuberosum), eggplant (S. melongena), and pepino (S. muricatum). More preferably, the plant is tomato (S. lycopersicum) or potato (S. tuberosum).

[0143] In a preferred embodiment, the plant belongs to the Amaranthaceae family. Preferably, the plant belongs to the Beta genus. Preferably, the plant is selected from Beta vulgaris (Altissima group), spinach beet or Swiss chard (B. vulgaris, Cicla group), Swiss chard (B. vulgaris, Flavescens group), beetroot (B. vulgaris, Conditiva group), and beet (B. vulgaris, Crassa group). More preferably, the plant is Beta vulgaris (Altissima group).

[0144] In a preferred embodiment, the plant belongs to the Rosaceae family. Preferably, the plant belongs to the Fragaria or Malus genus.

[0145] In a preferred embodiment, the plant belongs to the Brassicaceae (or Cruciferae) family. Preferably, the plant belongs to the Brassica genus.

[0146] In a preferred embodiment, the plant belongs to the Orchidaceae family. In an exemplary embodiment, the plant is an ornamental plant, such as an orchid, particularly of the Phalaenopsis genus, or another flowering plant, such as from the Cymbidium genus.

[0147] In a preferred embodiment, the plant belongs to the Asteraceae family (Asteraceae or Compositae). Preferably, the plant belongs to the Chrysanthemum, Aster, or Lactuca genus.

[0148] In a preferred embodiment, the plant belongs to the Fabaceae family. Preferably, the plant belongs to the Pisum or Phaseolus genus.

[0149] In a preferred embodiment, the plant belongs to the Cucurbitaceae family. Preferably, the plant belongs to the Cucumis or Cucurbita genus. Preferably, the plant belongs to the Cucumis genus and is selected from Cucumis sativus, melon, and gherkin (C. anguria). In other preferred embodiments, the plant belongs to the Cucurbita genus and is selected from the group consisting of C. pepo (especially zucchini) and pumpkins (C. argyrosperma, C. digitata, C. maxima, and C. moschata).

[0150] In a preferred embodiment, the plant belongs to the Vitaceae family.

[0151] In a preferred embodiment, the plant belongs to the Vacciniaceae family.

[0152] In a preferred embodiment, the plant is selected from the group consisting of Begonia, tomato, potato, beet, strawberry, cabbage, apple, Orchidaceae, chrysanthemum, Leguminosae, Cucurbitaceae, Pisum, Vitis, Vaccinia, and Lactuca.

[0153] In a preferred embodiment, the plant is selected from Begonia, tomato, potato, beet, strawberry, cabbage, and apple.

[0154] The compounds or compositions disclosed herein can be administered singly or multiple times. For example, the composition can be provided daily, weekly, monthly, or annually. In an exemplary embodiment, the compound or composition can be provided once daily for one week or until the compound or composition becomes effective.

[0155] In some embodiments, the compositions disclosed herein are provided in the form of a spray solution. When the composition is sprayed onto the plant, the solution can be deposited on the plant part (e.g., leaf) in the form of droplets, and the small surface-to-volume ratio of the droplets causes evaporation, which may result in the composition remaining on the plant part (e.g., leaf) in the form of a residue. This effect can be reduced by adding a wetting agent to the composition.

[0156] The amount of the compound or composition administered will depend on a variety of factors, including the method of administration, the time of administration, the decomposition rate of the specific compound used, the duration of treatment, pesticidal treatment, compounds and / or materials used in combination, age, weight, general health status, and previous treatment, as well as similar factors well known in the agricultural field. A horticulturist, plant grower, or farmer with ordinary skill in the art can readily determine the effective amount of the compound or composition required.

[0157] It is clear to those skilled in the art that lower concentrations / amounts of the compounds disclosed herein can be administered to slow-growing plants, such as cacti and succulents. It is also clear to those skilled in the art that the concentration / amount in water and the frequency of administration depend on the plant species, subspecies, cultivar, hybrid, variety. In addition, it is clear to those skilled in the art that the dose that a plant can tolerate depends on the growth stage and size of the plant. In addition, it is clear to those skilled in the art that the concentration / amount in water and the frequency of administration depend on the growth conditions, such as light, temperature, evaporation, nutrient concentration, and pH value in the root substrate, air flow, and the application of other pesticides. In addition, it is clear to those skilled in the art that the concentration / amount in water and the frequency of administration depend on the time of application, day, night, and seasonal and weather conditions.

[0158] As used herein, "comprising" and variations thereof are used in their non-limiting sense to mean including the item(s) following the word, but not excluding items not specifically mentioned. Additionally, the verb "consisting of" may be replaced with "consisting essentially of", which means that the compounds or auxiliary compounds defined herein may contain additional ingredients in addition to the specifically identified ingredients, provided that such additional ingredients do not alter the unique characteristics of the invention.

[0159] As used herein, the articles "a" and "an" refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one element or more than one element.

[0160] When used in conjunction with a numerical value (approximate 10, about 10), the word "approximate" or "about" preferably means that the value can be 10 times the given value or less than 1% of that value.

[0161] The compounds and compositions disclosed herein can be used as therapies and therapeutic treatments, and thus can be used as pharmaceuticals and in methods for preparing pharmaceuticals. In some embodiments, the present disclosure provides methods that are not for the treatment of the human or animal body and / or do not include methods for modifying the genetic identity of the human germ line.

[0162] wherein the cells are not human germ cells.

[0163] All patents and literature references cited in this specification are hereby incorporated herein by reference in their entirety.

[0164] The invention is further explained in the following examples. These examples do not limit the scope of the invention, but are only for the purpose of illustrating the invention.

[0165] Examples

[0166] Example 1: Synthesis of bis(p-chlorophenyl) thiosulfate, bis(p-fluorophenyl) thiosulfate, and bis(isopropyl) thiosulfate on a 50 g scale.

[0167] Scope. In this example, the synthesis of bis(p-chlorophenyl) thiosulfate (Compound 1a), bis(p-fluorophenyl) thiosulfate (Compound 1b), and bis(isopropyl) thiosulfate (Compound 2) is described.

[0168] Experimental design. The synthesis of the compounds started with a 10 g pilot run to evaluate the reaction. Based on the yields obtained and the observations, further scale-up or more detailed studies were conducted.

[0169] Synthesis of Bis(4-chlorophenyl) Thiosulfate (Compound 1a)

[0170] The first attempt was carried out according to the 10 g scale proposal, using the oxidation conditions found in the literature (ACS Catal. (2020), 10, pp. 8765 - 8779).

[0171] Scheme 1. Synthesis of compound 1a:

[0172]

[0173] In the first test reaction where the mixture was kept at room temperature for 48 h, compound 1a was obtained as a mixture with the starting material and compounds 3a and 7.

[0174] Scheme 2. Product formation during the reaction

[0175]

[0176] The crude compound 1a was analyzed by LCMS - 24 at UV 265 nm. The analysis was performed using an Agilent 1290 Infinity II series (with UV detector), an ELSD 1290 infinity II detector, and an Agilent 6135 mass detector equipped with a Waters XSelect CSH C18 2.5 μm 2.1×50 mm (PN: 186006101) column. Mobile phase A: 10 mM ammonium bicarbonate, pH 9.5 (aqueous solution), and mobile phase B was acetonitrile. UV detection was carried out at 265 nm. The main peak eluted at Rt = 3.86 min. Mass spectrometry analysis of the main peak (Rt = 3.86 min) showed a mixture of compound 1a [M + 18] and compound 3a.

[0177] The crude product was washed with hot water to remove 4 - chlorobenzenesulfonic acid, and this treatment also removed other minor impurities. The starting material remained in the mixture. Subsequently, the filter cake was washed with pentane to remove the starting material, giving the pure compound.

[0178] After the first 10 g test, a second batch of tests was carried out on a 50 g scale. After purification, the amounts of the two batches did not reach the required amount.

[0179] Thereafter, a second synthesis was carried out with 50 g of the substrate. The batches were combined and compound 1a (50 g) was shipped with a purity of 98%. The purity was determined by LCMS - 5CL.M (sequential method) (Waters Acquity HSS T3 (2.1x75 mm; 1.8 μm; RRHD 1200 bar, mobile phase A: 10 mM NH4OAc (water / methanol / acetonitrile 900 / 60 / 40); mobile phase B: 10 mM NH4OAc (water / methanol / acetonitrile 100 / 540 / 360).

[0180] Synthesis of Bis(4-fluorophenyl) Thiosulfate (Compound 1b)

[0181] Bis(p-fluorophenyl) thiosulfate is synthesized by oxidizing 4-fluorothiophenol using N-chlorosuccinimide as shown in the following figure.

[0182]

[0183] Under an air atmosphere at room temperature, 10 mmol (1.28 g) of 4-fluorothiophenol (Compound [I]) and 15 mmol (4 g) of N-chlorosuccinimide (Compound [II]) were placed in a round-bottom flask, approximately 20 mL of acetonitrile was added, and the mixture was stirred and reacted at room temperature. After half an hour, the reaction was completed. Immediately after the reaction ended, the mixture contained (4-fluorophenyl) thiosulfate (Compound 1b or [III] as shown in the scheme), bis(4-fluorophenyl) thiosulfite (Compound [II]), and bis(4-fluorophenyl) disulfide (Compound [V]). Then the reaction solution was transferred to a 250 mL separatory funnel and extracted with ethyl acetate (30 mL × 3). The upper organic phases were combined, dried, filtered, and distilled to obtain the crude product. The crude product was transferred to a round-bottom flask, slightly heated, and ethyl acetate was added until the crude product was completely dissolved. Then, it was cooled sufficiently to separate out the solid, and the product was filtered to obtain bis(p-fluorophenyl) thiosulfate. The yield was 70% (1.00 g). The product was a white solid with a melting point of 69 - 70 °C.

[0184] The product was identified by NMR and was in line with the NMR characteristics of bis(p-fluorophenyl) thiosulfate: 1H NMR (400 MHz, chloroform-d) δ 7.58 - 7.55 (m, 2H), 7.36 - 7.32 (m, 2H), 7.12 - 7.08 (m, 2H), 7.06 - 7.02 (m, 2H).

[0185] The molecular weight also corresponded to that of this compound (determined by GC-MS): M = 286 daltons.

[0186] Synthesis of Bis(isopropyl) Thiosulfate (Compound 2)

[0187]

[0188] The synthesis of Compound 2 was started on a 95 g (disulfide 1) scale. First, 2.1 molar equivalents (equivalent: 129 g) of hydrogen peroxide were added at <45 °C, but a mixture of 2 and 2-sulfoxide was produced, and some 2-sulfone (or other by-products) may also have been produced. After 24 hours, the reaction was still not complete, so it was pushed by adding more hydroxide peroxide (0.5 + 1.0 equivalent); a slight exothermic reaction was observed.

[0189] The reaction was followed by NMR (400 MHz, chloroform-d).

[0190] After 48 h at room temperature, the reaction was stopped and concentrated in vacuo. The crude material (190.9 g) containing some AcOH and other impurities was directly applied onto silica gel (600 g) and eluted in heptane with a gradient of 0% EtOAc to 20% EtOAc to give 37 g of the desired compound 2 (bis(4-isopropyl) thiosulfate) (yield 33%).

[0191] The reaction was repeated on a 50 g scale to obtain the desired amount with a purity of 96.3%. The purity was determined by HPLC-MS. The molecular weight also corresponded to this compound (including NH4 present in the eluent): M = 200 Da.

[0192] Example 2: MIC values of some (potentially pathogenic) animal-associated microorganisms

[0193] Example 2a: MIC, MBEC and MBIC determinations

[0194] The MIC determinations in this example measure the growth inhibition of planktonic microorganisms. As shown herein, many of the tested compounds have an effect on planktonic bacteria.

[0195] Table 3: Compounds used in this study, their CAS numbers and suppliers.

[0196] Compound CAS Number Supplier Di-n-propyl Disulfide 629-19-6 Sigma Aldrich Diisopropyl Disulfide 4253-89-8 Acros Bis(4-fluorophenyl) Disulfide 405-31-2 Fischer Scientific Bis(4-chlorophenyl) Disulfide 1142-19-4 Thermo Scientific Di(n-propyl) Thiosulfite 1948-52-3 Organic synthesis Bis(4-chlorophenyl) Thiosulfite 6481-72-7 Organic synthesis Bis(4-fluorophenyl) Thiosulfite 61169-14-0 Organic synthesis Di(n-propyl) Thiosulfate 1113-13-9 Syncom Diisopropyl Thiosulfate 10027-69-7 Organic synthesis Bis(4-chlorophenyl) Thiosulfate 1146-44-7 Organic synthesis Bis(4-fluorophenyl) Thiosulfate 2905-15-9 Organic synthesis

[0197] 1.1 Dissolution and Dilution of Compounds

[0198] In an Eppendorf tube, weigh 20 mg of the compound shown in Table 3. Record the exact weight and calculate the volume of solvent required to achieve an 80 mM dilution. Add half of the volume first, i.e., add Tween 80 and let it stand for 5 minutes. In the second step, add the other half volume of DMSO and resuspend to assist the dilution process. Invert the closed tube 5 - 10 times and then vortex thoroughly for at least 30 seconds. If crystals are still visible after this process, place the tube in an oscillator at 37 °C and shake at 150 rpm for at least 30 minutes (this indicates whether the dissolution time exceeds 2 hours). The dissolved and transparent dilution has a concentration of 80 mM and is diluted 10 - fold with cation - adjusted Mueller Hinton Broth 2 (MHB - II) for MIC determination (Method 1.2) to reach a working concentration of 8 mM (0.5 mL is required for one strain). For bis(4 - chlorophenyl) thiosulfate, a low - concentration stock solution of 2 mM needs to be prepared. To do this, weigh the required amount of the compound and add 2% v / v Tween 80 and 2% v / v DMSO of the final volume of the stock solution to the substance. Heat this mixture in a water bath set at 40 °C and shake occasionally. When all the substance has dissolved, fill up the volume with growth medium to the final volume. This 2 mM bis(4 - chlorophenyl) thiosulfate stock solution is used for the MIC plate and is diluted two - fold, so that the test range of bis(4 - chlorophenyl) thiosulfate is from 1 mM to 0.002 mM.

[0199] For MBEC determination (Method 1.3), the solution needs to be diluted to a final concentration of 2 mM in 0.9% saline solution and then further diluted in saline to 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM. Add these dilutions as treatment solutions to the 96 - well plates with grown biofilms (see Method 3) (0.5 mL is required for one strain). The dilutions must remain transparent.

[0200] Table 4. List of microorganisms used in this study.

[0201] Test Microorganisms Strain Number Staphylococcus epidermidis ATCC 35984 Pseudomonas aeruginosa ATCC 27853 Escherichia coli ATCC 25922 Klebsiella pneumoniae ATCC 13883 Streptococcus agalactiae ATCC 13813 Lactococcus garvieae Isolate 2 Streptococcus uberis ATCC 27985 Streptococcus suis type 2 21-018358-002 Staphylococcus aureus (MRSA) LUH14616 Acinetobacter baumannii RUH875 Micrococcus luteus ATCC 4698

[0202] *: Kindly provided by Leids Universitair Medisch Centrum (LUMC), Albinusdreef 2, 2333ZA, the Netherlands

[0203] 1.2 MIC Determination

[0204] The microorganisms shown in Table 4 were inoculated onto tryptic soy agar (TSA) plates and incubated overnight at 37°C. After incubation, 3 - 5 well - separated colonies with the same morphology were selected from the TSA plates and resuspended in 2 mL of 0.9% saline solution with 3 - 6 glass beads and vortexed. The optical density (OD TM ) of the bacterial suspension at 600 nm was measured using a spectrophotometer (Evolution 600 201 / 220 UV - Vis spectrophotometer, Thermo Fisher Scientific), and diluted to an OD 600 of 0.0008, corresponding to 10 6 CFU / mL.

[0205] The MIC assay was performed in flat - bottom 96 - well microtiter plates. 50 μL of MHB - II was added to columns 2 - 11, and 100 μL was added to column 12 for a sterility control. 100 μL of an 8 mM test compound solution (Method 1) was added to column 1. A two - fold dilution series of the MHB - II solution was achieved by resuspending 50 μL of the 8 mM solution in column 1 into column 2. This step was repeated until column 10. The respective concentrations of Tween 80 and DMSO in column 1 were 2.5% and were diluted two - fold throughout the plate. 50 μL of the bacterial suspension was added to columns 1 - 11. The microtiter plate was sealed with a sticky polyethylene film (Diversified Biotech) for the microplate and incubated at 37°C for 24 hours. After incubation, the OD was measured at 600 nm using a Varioskan (Thermo Fisher Scientific). After normalizing the obtained OD values to the growth control in column 11, the lowest concentrations that resulted in a 50% (MIC50) and 90% (MIC90) reduction in the final OD600 of the bacterial culture were calculated. This assay was repeated three times in three independent experiments.

[0206] 1.3 MBEC Determination

[0207] Frozen aliquots of Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 were inoculated into 20 mL of tryptic soy broth (TSB) in a 100 - mL conical flask and incubated overnight at 37°C and 150 rpm (Infors). The OD TM of the overnight culture was measured using a spectrophotometer (Evolution 600 201 / 220 UV - Vis spectrophotometer, Thermo Fisher Scientific), and diluted to an OD 600 of 0.2, corresponding to 10 8 CFU / mL. The OD600 A cell suspension with a concentration of 0.2 was inoculated into a U-bottom 96-well plate, with 100 μL pipetted into each well of columns 1 - 11, and 100 μL of culture medium was placed in column 12 as a sterile control. The 96-well plate was sealed with adhesive polyethylene film to seal the microplate and incubated at 37 °C for 48 hours under static conditions. After the biofilm growth stage, 100 μL of the treatment solution (Method 1) was added to the vials, with treatment concentrations of 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM, and the concentrations of Tween 80 and DMSO were 0.63%, 0.32%, 0.16%, and 0.08% respectively. The same concentration was applied to four different wells of the same bacterial strain, and four replicate experiments were conducted for each concentration and strain. Under static conditions, it was incubated at 37 °C for 20 hours. After treatment, the supernatant was carefully removed, and the wells with biofilm were carefully rinsed once with 200 μL of 0.9% saline solution. 200 μL of saline solution was added and the biofilm was thoroughly suspended. After complete homogenization, 20 μL was used for the first dilution in 180 μL of 0.9% saline solution, which was prepared in a second plate. This dilution was continued until the dilution step of 10E - 5, and 100 μL of the 10E - 4 and 10E - 5 dilutions were placed on TSA plates. The TSA plates were incubated at 37 °C for 24 hours. The colonies on the plates were counted, and the colony-forming units per milliliter of the assigned biofilm, their mean value, and the log reduction compared to the untreated samples from the same microplate were calculated. The reduction in colony-forming units is closely related to the thickness of the biofilm and thus to the number of bacteria present in the biofilm. Therefore, a significant reduction in colony-forming units in the biofilm was defined as a log reduction value of ≥1 or greater. This assay was repeated twice in two independent experiments.

[0208] 1.4 MBIC Determination

[0209] Frozen aliquots of Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 were inoculated onto tryptic soy agar (TSA) plates and incubated overnight at 37 °C. After incubation, 3 - 5 well-isolated colonies with the same morphology were selected from the TSA plates and resuspended in 2 mL of 0.9% saline solution with 3 - 6 glass beads and vortexed. The optical density of the bacterial suspension was measured at 600 nm (OD TM using a spectrophotometer (Evolution 600 ) 201 / 220 UV-Visible Spectrophotometer, Thermo Fisher Scientific), and diluted to OD 600It was 0.2. The MBIC assay was carried out in a 96-well microtiter plate. 50 μL of TSB was added to columns 2 - 11, and 100 μL was added to column 12 for the sterility control. 100 μL of an 8 mM test compound solution (Method 1) was added to column 1. A two-fold dilution series of the TSB solution was achieved by resuspending 50 μL of the 8 mM solution in column 1 into column 2. This step was repeated until column 10. The respective concentrations of Tween 80 and DMSO in column 1 were 2.5%, and they were diluted two-fold throughout the plate. 50 μL of the bacterial suspension was added to columns 1 - 11. The microtiter plate was sealed with a sticky polyethylene film (Diversified Biotech) for the microplate and incubated at 37 °C for 48 hours. After incubation, the supernatant was carefully removed, and the wells with biofilms were rinsed once with 100 μL of 0.9% saline solution. 100 μL of 0.1 M HCl was added and incubated at room temperature for 1 hour to fix the biofilm. After incubation, the HCl was removed, 100 μL of crystal violet (0.1% v / v aqueous solution) was added and incubated at room temperature for 30 minutes. The unbound crystal violet was removed, and the wells were rinsed once with 100 μL of deionized water. 100 μL of 30% acetic acid was added and incubated at 37 °C and 150 rpm for 1 hour. The solution was resuspended and transferred to a flat-bottom 96-well plate, and the absorbance at 540 nm was measured with a spectrophotometer. This assay was repeated twice in two independent experiments.

[0210] Results and Discussion. Tables 5 and 6 show the test results. In Tables 5 and 6, many organic sulfur compounds exhibited low MIC values and could be used as antibacterial compounds.

[0211]

[0212]

[0213] Example 2b: MIC50, MIC50, and MBIC against animal and human pathogens

[0214] Some compounds were selected from Example 2a for further antibacterial studies. Their selection was based on antibacterial effects and stability after synthesis. The selected compounds were bis(4-chlorophenyl) disulfide, bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, and diisopropyl thiosulfate, and they were tested against various suspected human, animal, and plant pathogens, bacteria, and fungi.

[0215] Scope. The microbial growth inhibitory activities of organic sulfur compounds were measured in this study. Some of these compounds were obtained commercially (bis(4-chlorophenyl) disulfide and bis(4-fluorophenyl) disulfide). Other compounds were synthesized upon request (Table 7; Organic synthesis). The measured microbial growth inhibitory (MIC) and bactericidal (MBC) capabilities were compared with the measurements of the biofilm eradicating compound di-n-propyl thiosulfate.

[0216] Study design. The microbial growth inhibitory activities of some organically synthesized (Table 7; Organic synthesis) and commercially obtained organic sulfur compounds were measured in this study.

[0217] Due to the low solubility of these compounds in water, the concentration at which the compounds could be dissolved with the aid of a solvent to make the concentration suitable for bioassays was first evaluated. Thereafter, minimum inhibitory concentration (MIC) determinations were performed in triplicate on the stock solutions of the compounds with various bacteria from the Gram-positive and Gram-negative bacterial groups and the yeast - Candida albicans in a 2-fold dilution range. The concentration showing less than 10% growth after 24 h was considered the MIC90 value, while the concentration showing 50% growth after 24 h compared to the bacterial growth control was considered the MIC50 value. Escherichia coli and Staphylococcus aureus were used as representatives of Gram-negative and Gram-positive bacteria, respectively. In addition, the yeast Candida albicans was also tested. After MIC determinations of diisopropyl thiosulfate and di-n-propyl thiosulfate, the minimum bactericidal concentration was determined to study whether growth inhibition (bacteriostasis) also meant killing of the cells (bactericidal).

[0218] Table 7. Compounds used in this study, their CAS numbers and suppliers.

[0219]

[0220] In Table 8, the strains and the sources of the strains are provided.

[0221] Table 8. List of strains, strain characteristics and sources of the strains.

[0222] Strain Strain Number Gram-positive / Negative Strain Supplier Escherichia coli ATCC 25922 - LGC Standards Klebsiella pneumoniae ATCC 13883 - LGC Standards Streptococcus agalactiae ATCC 13813 + LGC Standards Lactococcus garvieae Isolate 2 + Isolated from the field Streptococcus uberis ATCC 29785 + LGC Standards Streptococcus suis (type 2) 21-018358-002 + Isolated from the field Staphylococcus aureus (MRSA) LUH 14616 + LUMC Acinetobacter baumannii RUH875 - LUMC Micrococcus luteus ATCC 4698 + LGC Standards Staphylococcus epidermidis ATCC 35984 + LGC Standards Pseudomonas aeruginosa ATCC 27853 - LGC Standards Actinomyces viscosus Isolate 12064 - Norwegian Veterinary Institute Candida albicans Y01-19 n.a. Groton, Connecticut, USA

[0223] LUMC: Leiden University Medical Centre, Leiden, the Netherlands

[0224] n.a.: not applicable

[0225] The emulsion of the test compound was prepared as shown in Table 9. In an Eppendorf tube, 10 - 20 mg of the test compound was weighed. Subsequently, the required amount of Tween 80 was added, and then an appropriate amount of DMSO was added. After vigorous shaking, the dissolved and transparent dilution was diluted 10-fold with Mueller Hinton broth (MHB) for MIC determination. For Candida albicans, Sabourad broth was used; for Candida glutinosa, TSB containing 2% NaCl was used. The stock solution was used for the determination and diluted in a 2-fold dilution series.

[0226] Table 9. Composition of the test solution, Tween 80, and DMSO. MIC determination was performed using the medium Mueller Hinton broth (MHB).

[0227]

[0228] The minimum bactericidal concentration (MBIC) was determined as follows: A frozen aliquot of the target cells was inoculated onto a tryptic soy agar (TSA) plate and incubated overnight at 37 °C. For Candida albicans, Sabourad agar was used. Moraxella viscosa was inoculated into liquid TSB supplemented with 2% NaCl and incubated for two days under static conditions at 15 °C. After incubation, 3 - 5 well-separated colonies with the same morphology were selected from the plate and resuspended in 2 mL of 0.9% saline solution, 3 - 6 glass beads were added and vortexed. For Moraxella viscosa, the pre-culture was diluted 10-fold in sterile softened water. The optical density (OD600) of the bacterial suspension was measured at 600 nm and diluted to an OD600 of 0.0008, equivalent to 10 6CFU / mL. MIC determinations were performed in flat-bottom 96-well microtiter plates. Fifty microliters of the appropriate growth medium, Sabouraud broth for Candida albicans, TSB 2% NaCl for Actinomyces viscosus, and MHB for the remaining strains were added to columns 2 - 11, and 100 μL was added to column 12 for a sterility control. One hundred microliters of an 8 mM test compound solution (Method 1) was added to column 1. A two-fold dilution series of the solution in the growth medium was achieved by resuspending 50 μL of the stock solution from column 1 into column 2. This step was repeated until column 10. The concentrations of Tween 80 and DMSO in column 1 were each half of the stock solution concentration and were further two-fold diluted across the plate. Fifty microliters of the cell suspension was added to columns 1 - 11. The microtiter plates were sealed with a sticky polyethylene film (Diversified Biotech) and incubated at 37 °C for 24 h. After incubation, the OD at 600 nm was measured using a Varioskan (Thermo Fisher Scientific). After normalizing the obtained OD values to the growth control in column 11, the lowest concentrations that resulted in a 50% (MIC50) and 90% (MIC90) reduction in the final OD600 of the culture were calculated. This determination was repeated three times in three independent experiments. The mean values of the MIC90 and MIC50 values were taken and the standard deviation was calculated. Thereafter, the mean and standard deviation for each strain and compound were calculated in a bar graph to compare the antibacterial activity of the compounds against different strains ( Figure 1 ).

[0229] In GraphPad, the technical replicate values (four per experiment) in the plate were plotted against the concentrations used to provide information on replicate spread (results not shown here).

[0230] Conclusions and discussion. The results of the MIC determinations ( Figure 1 ) indicated that most of the test compounds had growth inhibitory effects on the strains in this study.

[0231] Bis(4-chlorophenyl) disulfide shows low solubility. By adding solvents such as Tween 80 and DMSO, a stock solution of 2 mM can be prepared in the culture medium. This allows a maximum test concentration of 1 mM. Similarly, for bis(4-chlorophenyl) thiosulfate, the calculated solubility is 0.013 mM. Therefore, after using the solvent, the maximum test concentration may be 0.575 mM. If the actual growth inhibition concentration (MIC90) is higher than these maximum determined concentrations, it will not be detected in this assay. Bis(4-chlorophenyl) disulfide was observed to have inhibitory effects on Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Lactococcus garvieae, Streptococcus lactis, Streptococcus suis type 2, Staphylococcus aureus (MRSA), and Acinetobacter baumannii, while bis(4-chlorophenyl) thiosulfate also has inhibitory effects on Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. The solubilities of dipropyl thiosulfate and diisopropyl thiosulfate are 5.6 mM and 9.5 mM respectively. Therefore, the assay results of 4 mM are still within the theoretical solubility range.

[0232] In addition, the between-batch differences of diisopropyl thiosulfate are small, and the MIC90 values of the same species are within a similar range ( Figure 1 , where columns A and C of diisopropyl thiosulfate represent batch A and batch C respectively). The sensitivity of Candida albicans is significantly different compared to other tested species. Candida albicans seems to have higher sensitivity, and thus lower MIC90 values. The conclusion is that all compounds exhibit growth inhibitory effects, which depend on the microorganism.

[0233] The minimum bactericidal concentration (MBC) of two compounds, namely diisopropyl thiosulfate and dipropyl thiosulfate, was determined. MBC is the lowest concentration of the compound that does not show any colonies on the agar plate inoculated from the MIC assay and incubated at the optimal growth temperature for 20 hours, depending on the strain. This assay was performed three times independently for each strain of Escherichia coli, Staphylococcus aureus, and Candida albicans. Therefore, the standard deviation can also be calculated (Table 10).

[0234] Table 10. MBC [uM] values of diisopropyl thiosulfate and dipropyl thiosulfate against Escherichia coli, Staphylococcus aureus, and Candida albicans. These values were determined by performing three independent MBC assays (n = 3). These values are represented by the standard deviation of different experiments.

[0235]

[0236] Conclusion and discussion. It can be observed that dipropyl thiosulfate has the lowest MBC value among all species and thus has the highest bactericidal activity.

[0237] MIC assay ( Figure 1)Provides information on growth inhibition within a 24-hour time window in the presence of a compound. If exposure to the compound is stopped, (viable) cells may resume growth. By performing an MBC assay, the bactericidal effect of the compound against a specific strain can be determined. Di-n-propyl thiosulfate and di-isopropyl thiosulfate also exhibited bactericidal activity (MBC) against Escherichia coli and Candida albicans at growth inhibitory concentrations (MIC90). For Staphylococcus aureus, the MBC values of these two compounds were higher than the MIC90 values, thus showing bacteriostatic effects. This means that viable cells were still present at the MIC90 concentration. The bactericidal effect (killing of pathogens) occurred with increasing concentration.

[0238] Example 3

[0239] AM-8 eradicates Staphylococcus aureus LUH 14616 biofilm

[0240] In addition to antimicrobial-resistant (AMR) bacteria, biofilms also reduce the efficacy of antibiotics against clinical infections (Sharma D, Misba L, Khan AU. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob Resis Infect Control 8;76, 2019). Biofilms are communities of bacteria residing within an extracellular polymeric matrix (self-produced) that protects the bacteria from the action of, for example, antibiotics (review, Penesyan A, Paulsen IT, Kjelleberg S, Gillings MR. Three faces of biofilms: a microbial lifestyle, a nascent multicellular organism, and an incubator for diversity. NPJ Biofilms Microbiomes. 2021 7(1):80. Doi: 10.1038 / s41522-021-00251-2).

[0241] Scope. The aim of this in vitro experiment was to evaluate the efficacy of AM-8 (di-isopropyl thiosulfate) against AMR Staphylococcus aureus in clinically relevant biofilms.

[0242] Experimental design.

[0243] Biofilm eradication assay. The assay for assessing the reduction of bacterial numbers in mature biofilms was the same as previously described (Scheper HJ, Wubbolts JM, Verhagen JAM, de Visser AW, van der Wal RJP, Visser LG, de Boer MJG, and Nibbering PH. SAAP-148 eradicates MRSA persisters within mature biofilm models simulating prosthetic joint infection. Front. Microbiol. 12:625952. Doi:10.3389 / fmicb.2021.625952. eCollection 2021), with one modification, namely that the biofilm was exposed to AM-8 daily for 4 consecutive days. Briefly, approximately 1x10 7 CFU log-phase bacteria were cultured in BHI and sealed in 96-well flat-bottom polystyrene microplates with airtight seals for 7 days. Next, the mature biofilms were washed twice with PBS and then exposed to AM-8 or the diluent of this compound as a control for 24 hours. Thereafter, the biofilms were washed and then exposed to AM-8 / its diluent again for one day, or - if the daily exposure was terminated - sonicated for microbiological quantification of the viable bacterial count. The results were expressed as the number of surviving bacteria during the exposure. The experiment was repeated three times.

[0244] Results. Since mature biofilms more closely mimic clinical infections than 24-hour immature biofilms (Cazander G, van de Veerdonk MC, Vandenbroucke-Grauls CM, Schreurs MW, Jukema GN. Maggot excretions inhibit biofilm formation on biomaterials. Clin Orthop Relat Res; 468(10):2789-96, 2010), and antibiotics have little effect on the bacteria in these bacterial communities, the ability of AM-8 to eradicate Staphylococcus aureus LUH14616 in 7-day mature biofilms was evaluated. The results of the dose-range study showed that the biofilm-reducing effect of a single dose of AM-8 increased over time but did not completely eradicate the bacteria within the biofilm (results not shown). Therefore, the possibility that multiple daily exposures to AM-8 were more effective than a single dose against the Staphylococcus aureus LUH14616 biofilm was investigated. The results showed that with an increasing number of daily exposures, the AM-8 dose-dependently and significantly reduced the number of bacteria in the mature biofilm ( Figure 24)。In summary, using a lower dose of AM-8 multiple times a day is more effective than using a higher dose of this compound once.

[0245] Conclusion. AM-8 can eliminate all bacteria within the biofilm, which is almost impossible for antibiotics to do (see also Example 4).

[0246] Example 4: Anti-persister experiment

[0247] Approximately 80% of clinical infections are associated with biofilms. A biofilm is a structured microbial community embedded in a 3D extracellular matrix (Penesyan et al. (2021). Three faces of biofilms: a microbial lifestyle, anascent multicellular organism, and an incubator for diversity. NPJ Biofilms Microbiomes 7:80). This matrix provides protection for the entire bacterial community from environmental stressors, including antibiotics and effector molecules of the immune system. Additionally, bacteria within biofilms are highly heterogeneous, including tolerant and persister bacteria, and thrive in different local microenvironments. Persister bacteria are bacterial cells that are temporarily in a state of slow or no growth and reduced metabolism. They occur both randomly and in response to environmental cues (such as antibiotics) and are typically present at detectable levels within biofilms (Prax M, and Bertram R. (2014). Metabolic aspects of bacterial persisters. In: Frontiers in Cellular and Infection Microbiology (Vol. 4, Issue OCT). Frontiers Research Foundation). Previous studies have reported that this slow or non-growing hypometabolic state is associated with antibiotic tolerance but not antibiotic resistance (Martínez JL, and Rojo F. (2011). Metabolic regulation of antibiotic resistance. In FEMS Microbiology Reviews 35(5), 768–789). Persisters are likely responsible for re-infections in patients (Lewis K. (2010) Persister cells. Annu Rev Microbiol. 64:57-72). Bacteria within biofilms have been reported to be up to 1,000 times more resistant to multiple antibiotics than planktonic bacteria (Sharma D, Misba L, Khan AU (2019). Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob Resis Infect Control 8:76).

[0248] Range. In this experiment, the susceptibility of persister bacteria to the antibacterial compound bis(4-chlorophenyl) disulfide was determined.

[0249] Experimental design. The ability of AM-2 (bis(4-chlorophenyl) disulfide) and AM-8 to reduce the number of bacteria in mature biofilms has been evaluated as previously described (Scheper et al (2021) Scheper H, Wubbolts JM, Verhagen JAM, de Visser AW, van der Wal RJP, Visser LG, de Boer MJG, and Nibbering PH. SAAP-148 eradicates MRSA persisters within mature biofilm models simulating prosthetic joint infection. Front. Microbiol. 12:625952. Doi: 10.3389 / fmicb.2021.625952. eCollection 2021), with one modification, namely that the biofilms were exposed to the compounds daily for up to 4 days continuously. Briefly, antimicrobial-resistant (AMR) Staphylococcus aureus LUH14616 was cultured to mid-logarithmic phase in tryptic soy broth (TSB; Oxoid Ltd, Basingstoke, UK) at 37 °C and 200 rpm, centrifuged at 3,000 rpm for 10 minutes, and then resuspended in brain heart infusion broth (BHI; Oxoid Ltd, Basingstoke, UK). Next, approximately 1×10 7 CFU bacteria in 96-well flat-bottomed polystyrene microtiter plates were cultured and sealed with an air-permeable seal for 7 days. Thereafter, the biofilms were washed and exposed to high-dose (10-fold minimum inhibitory concentration, i.e., the lowest concentration that results in a lack of visible growth; MIC) rifampicin and ciprofloxacin (both from Sigma-Aldrich) for 3 days. Subsequently, the biofilms were washed twice with PBS to remove the antibiotics and bacterial cells in the suspension, and then exposed to increasing concentrations of AM-2 (in PBS supplemented with 1% DMSO and 1% Tween-80) or 10×MIC antibiotics for 24 hours (as a control). Finally, the released bacteria were removed by washing, and the biofilms were sonicated to evaluate the number of viable bacteria within the biofilms. To account for potentially slow-growing bacteria, we re-analyzed the bacterial plates after one week. The results are expressed as the number of surviving bacteria (CFU / ml).

[0250] Results. Since persister bacteria are difficult to eliminate with antibiotics and may cause reinfection (Lewis, 2020), we evaluated the effects of AM-2 and AM-8 on persister bacteria in mature AMR Staphylococcus aureus biofilms exposed to antibiotics by sonication. The results showed that the highest dose of AM-2 was effective against persister bacteria( Figure 25 ). The conclusion is that AM-2 is effective against bacteria in the persistent state. In this state, bacteria are difficult to treat with traditional antibiotics.

[0251] Example 5. Repeated treatment

[0252] Current antibiotics mainly act through growth-dependent mechanisms and target rapidly dividing bacteria. However, repeated use of antibiotic treatment to control persistent and recurrent infections may lead to antibiotic resistance. In this example, it was demonstrated that Staphylococcus aureus LUH14616 did not develop significant antibiotic resistance after repeated exposure to diisopropyl thiosulfate.

[0253] Scope. In this example, the possibility of inducing resistance by diisopropyl thiosulfate (AM-8) in AMR Staphylococcus aureus LUH 14616 was investigated using two independently synthesized batches of materials.

[0254] Experimental design. Potential resistance to AM-8 was evaluated as described by Habets and Brockhurst (2012) (Therapeutic antimicrobial peptides may compromise natural immunity. Biol Lett 8:416 - 418). Briefly, log-phase bacteria (Staphylococcus aureus LUH14616) were diluted to a concentration of 2×10 6 CFU / ml in RPMI-mod. Next, 50 μl of this bacterial suspension was mixed with 50 μl of an AM-8 dilution or (as a comparator) rifampicin in a V-bottom 96-well plate. Thereafter, the plates were sealed with plastic wrap and incubated in a shaking incubator at 37 °C (200 rpm) for 24 h. The plates were then centrifuged at 2,000 rpm for 5 min and growth inhibition was determined. To initiate the resistance development test, 100 μl containing 2×10 6The mid-logarithmic bacterial suspension at CFU / ml was mixed with 150 μl of RPMI-mod, and 5 μl of this diluted bacterial suspension was applied to the wells with V-bottoms, which contained a dilution range of AM-8 or antibiotics (various concentrations below and above the MIC) in RPMI-mod (RPMI 1640 (Sigma-Aldrich) modified with 20 mM Hepes and L-glutamine and without sodium bicarbonate; also referred to as RPMI-mod). After incubation for 24 hours in a shaking incubator at 37 °C (200 rpm), the growth inhibition was determined. 100 μl of the bacterial suspension in the 0.5×MIC well was mixed with 150 μl of RPMI-mod, and 5 μl of the diluted bacterial suspension was used to inoculate a new 96-well plate containing a series of dilutions of AM-8 or rifampicin, and then these mixtures were incubated as described above. If drug resistance occurred, the concentration range of AM-8 and / or antibiotics was adjusted accordingly. This was repeated 19 times. The results were expressed as the fold increase in the MIC compared to the MIC at the start of the experiment.

[0255] Results. The ability of two batches (four in each batch) of AM-8 to induce drug resistance in Staphylococcus aureus LUH14616 was evaluated. The results showed that during 19 passages, the MIC of AM-8 hardly increased (occasionally increased by 2-fold) ( Figure 26 ). For rifampicin, a rapid increase in the MIC was observed in the 4th - 5th passages of Staphylococcus aureus LUH14616, with a 10-fold increase. After that, the MIC further increased to 146-fold after the 10th passage and reached an increase of >292-fold after the last passage.

[0256] Conclusion. Compared with rifampicin, AM-8 did not increase the MIC during 19 passages, indicating that the development of drug resistance to AM-8 did not occur.

[0257] Example 6: Cytotoxicity Test on Caco-2 and HepG2 Cell Lines

[0258] To prevent health damage to infected individuals, the cytotoxicity of antibacterial compounds used to control infections should be acceptable during use. This example shows that the exposure of Caco-2 and HepG2 cell lines to bis(4-chlorophenyl) disulfide, bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, and diisopropyl thiosulfate has cytotoxic effects on these cell lines.

[0259] Range. As a standard test, cytotoxicity was measured on Caco-2 and HepG2 cell lines. The cytotoxicity of the cell lines was determined by measuring the amount of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) converted by mitochondrial dehydrogenase after exposure to different concentrations of the antibacterial compound. In live cells, dehydrogenase converts soluble MTT into water-insoluble purple formazan crystals that cannot cross the cell membrane. The higher the metabolic activity of the cells, the more purple crystals are formed and the lower the cytotoxicity of the antibacterial compound.

[0260] Experimental design. This experiment used the Caco-2 cell line (colorectal adenocarcinoma cells) and HepG2 (derived from the human liver) from ATCC. The intestinal Caco-2 cell line was cultured in 75 cm 2 culture flasks. Minimum Essential Medium (MEM) (catalog number 31095029, ThermoFischer Scientific), supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FCS) (Gibco), 1% (v / v) non-essential amino acids (Gibco), 1% sodium pyruvate (Gibco), and penicillin (100 U / mL) / streptomycin (100 μg / mL), was used as the culture medium for growing the cells. The cells were kept in an incubator providing optimal humidity and temperature (cultured for 72 hours in a humidified atmosphere of 95% air and 5% CO2 at 37 °C). Caco-2 cells were seeded in flat-bottom 96-well plates for the experiment.

[0261] HepG2 adherent cells were isolated from the human liver and showed epithelial morphology. The culture conditions of the cells were followed according to the standard protocol. The HepG2 cell line was cultured in 75 cm 2 culture flasks. Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) + Glutamax, supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FCS) (Gibco), and penicillin (100 U / mL) / streptomycin (100 μg / mL) were used as the culture medium for growing the cells. The cells were kept in an incubator providing optimal humidity and temperature (a humidified environment of 95% air and 5% CO2 at 37 °C for 72 hours).

[0262] Cells were cultured on flat-bottom 96-well plates to reach the appropriate density required for the experiment. Each cell line was exposed to emulsified test compounds (8 concentrations ranging from 7.8 μM to 1 mM) with increasing concentrations in standard medium (Catalog 31331028, ThermoFischer Scientific). Notably, all experiments were conducted in FBS (Catalog A3840101, ThermoFischer Scientific) and antibiotic-free medium. The ratio of Tween-80 and DMSO used for emulsifying the antimicrobial compounds in the concentration assay was between 0.007% and 1%. The cells were incubated with the emulsified test compounds for 24 hours and assayed according to the standard protocol. At the end of the experiment, the medium (cell supernatant) was removed, and lysis buffer (0.04 M hydrochloric acid, propan-2-ol solution) was added to each well to dissolve the purple formazan crystals. In the last step, the absorbance was recorded at 595 nm using a multi-well reader. Control experiments were conducted in the same manner for the two cell lines using the emulsifier Tween 80 and DMSO without the antimicrobial compound. The results of all experiments were expressed as the mean ± standard error of the mean (SEM) of three independent experiments (three wells per condition). One-way ANOVA and Bonferroni post hoc tests were used to statistically evaluate the differences between the results.

[0263] Results.

[0264] In Figure 2 - 3 control experiments were performed on two cell lines (Caco-2 and HepG2) in which the cytotoxic effects of the applied emulsifier Tween 80 and DMSO concentrations were tested, where no antimicrobial (AM) compounds were present. Exposure of the Caco-2 and HepG2 cell lines to increasing concentrations of the emulsifier showed cytotoxic effects. In Figure 4 - 13 the effects of emulsified antimicrobial compounds on the cytotoxicity of Caco-2 and HepG2 cell lines are shown. DMSO 5% (labeled D 5%) was used as a positive control for cytotoxicity. The lowest X-axis represents the concentrations of Tween-80 and DMSO used for emulsifying the antimicrobial compounds present in the assay, ranging from 0.007% to 1%. The following antimicrobial compounds were tested and labeled: AM2, bis(4-chlorophenyl) disulfide; AM3, bis(4-fluorophenyl) disulfide; AM6, bis(4-chlorophenyl) thiosulfate, AM8, diisopropyl thiosulfate. AM8-A and AM8-C correspond to two different batches of the synthesized AM8.

[0265] Since the emulsifier also exhibits cytotoxic effects, which are additive to the cytotoxic effects of the antimicrobial agent, the effects of the emulsifier should be subtracted from the total cytotoxic effects ( Figure 2 - 3)。The exception is AM8 (diisopropyl thiosulfate, Figure 10 - 13 ):since this antimicrobial compound requires almost no emulsifier. In particular, compared to other AM compounds, DMSO is not used when emulsifying AM8 and the amount of Tween-80 used is 30 times less.

[0266] Based on the obtained results, it was concluded that all the compounds exhibited a slight cytotoxic effect and when Caco-2 and HepG2 cells were exposed to diisopropyl thiosulfate (AM8, Figure 10 - 13 ), the cytotoxicity was the lowest.

[0267] Example 7: Treatment of infected plant crops

[0268] Example 7a

[0269] In this example, the antibacterial activity of the compounds of the present invention against plants and plant parts was tested.

[0270] For this purpose, plants of the families Begoniaceae, Solanaceae, Amaranthaceae, Rosaceae and Brassicaceae were selected. Infections caused by various bacteria and fungi were tested. Specifically, the antibacterial activity of the compounds of the present invention against pathogenic fungi selected from Pythium ultimum, Fusarium oxysporum, Fusarium solani, Phytophthora cactorum, Rhizoctonia solani was tested; and against pathogenic bacteria selected from Xanthomonas campestris, Erwinia amylovora, Rhizobium, Corynebacterium michiganense, Agrobacterium radiobacter. Table 11 below gives an exemplary overview of the infected plants.

[0271] Table 11: Exemplary overview of plants infected with the fungi / bacteria to be tested

[0272] Fungus / Bacteria Plant Pythium ultimum Begonia Fusarium oxysporum Tomato, Potato Fusarium solani Beet Phytophthora cactorum Strawberry Rhizoctonia solani Potato Xanthomonas Cabbage Erwinia amylovora Apple

[0273] Example 7b. Inhibition of Antimicrobial Compounds against Fungal and Bacterial Plant Pathogens

[0274] Many crops of the families Begoniaceae, Solanaceae, Amaranthaceae, Rosaceae and Brassicaceae are infected with plant pathogens of the fungal genera Pythium, Fusarium, Phytophthora, Rhizoctonia and the bacterial genera Xanthomonas, Erwinia, Rhizobium, Corynebacterium and Agrobacterium. This example shows that the above-mentioned antimicrobial compounds can inhibit the growth of fungal and bacterial plant pathogens and can be used as effective control agents.

[0275] Range. In this example, the antibacterial activity of the compounds of the present invention against plant pathogens was tested. Di-n-propyl thiosulfate (QQ2) was used as a reference compound.

[0276] Specifically, the antibacterial activities of bis(p-chlorophenyl) disulfide (AM2), bis(p-fluorophenyl) disulfide (AM3), bis(p-chlorophenyl) thiosulfate (AM6), and diisopropyl thiosulfate (AM8) of the present invention were tested against pathogenic fungi selected from Pythium aphanidermatum, Phytophthora cinnamomi, Fusarium oxysporum, Sclerotinia sclerotiorum, Rhizoctonia solani, and Botrytis cinerea.

[0277] Experimental design. The effects of the compounds on fungal growth were tested on agar plates with different dilutions of the compounds. The fungi were inoculated in the middle of the agar plates. The diameters of the colonies were measured at different time intervals.

[0278] The fungi were pre-cultured on the agar media shown in Table 12.

[0279] Table 12. List of fungi tested in this example. PDA, potato dextrose agar medium; OA, oat agar; MEA, malt extract agar medium

[0280] Pathogen Test Medium Remarks Pythium aphanidermatum PDA Pythium aphanidermatum PDA authenticated collection: isolated from chrysanthemum and identified by DNA sequencing Phytophthora cinnamomi PDA CBS277.55 Fusarium oxysporum PDA Authenticated collection: isolated from spinach and identified by DNA sequencing Sclerotinia sclerotiorum OA Authenticated collection: isolated from chicory Rhizoctonia solani MEA CBS 323.84 Botrytis cinerea OA Authenticated collection: isolated from tomato

[0281] The fungi were pre-cultured on appropriate agar media, such as potato dextrose agar or malt extract agar at 24°C. After culturing at 24°C for 3, 7, 10, and 14 days, the diameters of the fungal colonies were measured on the agar plates. Since the diameter of the agar plate is 8 cm, this is the maximum colony size.

[0282] The agar media were prepared with different dilutions of bis(p-chlorophenyl) disulfide (AM2), bis(p-fluorophenyl) disulfide (AM3), bis(p-chlorophenyl) thiosulfate (AM6), and diisopropyl thiosulfate (AM8).

[0283] When the liquid agar medium was still warm, the test compounds were added to the agar medium to prevent the compounds from decomposing due to heat. 50-fold concentrated stock solutions of the antibacterial compounds at each dilution were prepared in 50% Tween-80 and 50% DMSO (so that the DMSO and Tween 80 concentrations were the same in all agar plates). In the agar plates, the following concentrations of the antibacterial compounds were used for each strain: 1.0 mM, 0.50 mM, 0.25 mM, 0.125 mM, and 0.063 mM, in the corresponding media shown in Table 12. Each concentration was tested 4 times in petri dishes with a diameter of 8 cm, and each petri dish contained 11 ml of agar medium.

[0284] Thereafter, the plates were inoculated in the middle of the petri dishes. Culturing was carried out at 24°C. The colony diameters were measured at t = 3, 7, 10, and 14 days after inoculation. Since the diameter of the petri dish is 8 cm, this diameter is the maximum diameter measured.

[0285] Results Figure 14 - 19 showed the effects of various antimicrobial compounds after 7 days of incubation: on day 7 of incubation, the differences in growth inhibition were clearly visible. The larger the colony diameter, the smaller the growth inhibition. Figure 14 Pythium ( Figure 15 ) did not show any significant growth at the tested concentrations of antimicrobial compounds on day 7 after inoculation (although growth inhibition was observed at earlier time points), while the growth of other fungi was inhibited. Phytophthora cinnamomi ( Figure 16 ), Fusarium oxysporum ( Figure 17 ) and Sclerotinia sclerotiorum ( Figure 15 - 16 ) were the most sensitive to the antimicrobial compounds. In addition, the growth inhibition depended on the antimicrobial compound applied and the fungus applied. In addition, the growth inhibition of Phytophthora cinnamomi and Fusarium oxysporum was concentration-dependent. In particular, the higher the concentration of the antimicrobial compound, the greater the growth inhibition. Figure 20 - 23 showed the raw experimental data at t = 3, 7, 10 and 14. The tested antimicrobials were labeled as follows: product 2, bis(4-chlorophenyl) disulfide (AM2); product 3, bis(4-fluorophenyl) disulfide (AM3); product 6, bis(4-chlorophenyl) thiosulfate (AM6); product 8, diisopropyl thiosulfate (AM8); product Q, di-n-propyl thiosulfate (QQ2).

[0286] Conclusion. This example clearly shows that bis(p-chlorophenyl) disulfide, bis(p-fluorophenyl) disulfide, bis(p-chlorophenyl) thiosulfate and diisopropyl thiosulfate significantly inhibited the growth of plant pathogens, indicating their suitability for the control of microbial pests in crops. Some examples of pests in plants and crops are listed in Table 13, and if pests occur, they can be treated.

[0287] Table 13: Exemplary overview of plants affected by the tested fungi.

[0288] Fungus / Bacteria Plant Pythium ultimum Begonia Fusarium oxysporum Tomato, Potato Fusarium solani Beet Phytophthora cactorum Strawberry Rhizoctonia solani Potato Xanthomonas campestris Cabbage Erwinia amylovora Apple

Claims

1. A compound for treating or preventing microbial infections, preferably bacterial or fungal infections, or a composition comprising at least one such compound, said compound being selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, and di-isopropyl thiosulfate.

2. A method of treating or preventing a microbial infection in an individual, comprising administering to the individual in need thereof a compound or a composition comprising at least one such compound, said compound being selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, and diisopropyl thiosulfate.

3. A composition comprising a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, and di-isopropyl thiosulfate, wherein said composition is a pharmaceutical composition, a pesticide composition, or a food composition.

4. An article having a surface at least partially coated with a compound or a composition comprising at least one such compound, said compound being selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, and di-isopropyl thiosulfate, preferably, wherein said article is a medical device or a surgical device.

5. A cleaning or disinfecting composition comprising (i) a compound selected from: bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, and di-isopropyl thiosulfate; and (ii) a surfactant.

6. An in vitro method comprising applying a cleaning or disinfecting composition to a surface, wherein said composition comprises a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite, and di-isopropyl thiosulfate.

7. The method according to claim 6, wherein the method is a method of sanitizing or disinfecting the surface.

8. Use of a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds, as a disinfectant, a sanitizing agent or an antimicrobial agent in food.

9. A cleaning or disinfecting product comprising a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds.

10. An agricultural composition comprising a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite and diisopropyl thiosulfate, and an agricultural excipient, carrier and / or solvent.

11. A method for preventing or treating an infection of a plant or a plant part, comprising contacting the plant or the plant part with a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds, thereby preventing or treating the infection.

12. Use of a compound selected from bis(4-fluorophenyl) disulfide, bis(4-chlorophenyl) disulfide, bis(4-chlorophenyl) thiosulfate, bis(4-fluorophenyl) thiosulfate, bis(4-chlorophenyl) thiosulfite, bis(4-fluorophenyl) thiosulfite and di-isopropyl thiosulfate, or a composition comprising at least one of said compounds, for preventing or treating an infection of a plant or a plant part.

13. The use or method according to claim 11 or 12, wherein the compound or composition is directly applied to the plant, the seeds of the plant or the soil of the plant or the seeds.

14. The use or method according to any one of claims 11-13, wherein the plant is selected from the group consisting of Begoniaceae, Solanaceae, Amaranthaceae, Rosaceae and Brassicaceae, and preferably, wherein the plant is selected from the group consisting of Begonia, tomato, potato, beet, strawberry, cabbage, apple, Orchidaceae, chrysanthemum, Leguminosae, Cucurbitaceae, Pisum, Vitis, Vaccinia and Lactuca.

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