Substituted Tolans for Modulating Microbial Colonization

Abstract

The present application describes methods for disinfecting physical or biological surfaces by contact with antimicrobial formulations comprising substituted tolan compounds, such as one or more alkoxy (e.g., methoxy) and hydroxy substituents on one or two phenyl rings of the tolan. The compounds and methods are microbial and / or microbicidal, depending on the dose and suspected microorganism. Suspected pathogenic microorganisms include gram-negative bacteria, such as Pseudomonas aeruginosa (P.aeruginosa) and Escherichia coli (E.coli); gram-positive bacteria, such as Staphylococcus aureus (S.aureus), including MRSA; and fungal pathogens, such as Candida and Candida albicans (C.albicans). The present application also describes methods for inhibiting or disrupting biofilm formation of one or more microorganisms.

Description

[0001]

Related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 537,661, filed on July 27, 2017, pursuant to 35 USC §111(b), and U.S. Provisional Application No. 62 / 676,676, filed on May 25, 2018, pursuant to 35 USC §111(b). The entire disclosures of all of the above applications are expressly incorporated herein by reference for all purposes. [Field of the Invention]

[0003] The present invention relates generally to the field of microbiology and, more particularly, to compounds, formulations and methods for reducing microbial growth or eliminating microbial colonies, particularly pathogens, by contacting a physical or biological surface with the compound or formulation of the compound. [Background of the invention]

[0005] The designation of a microorganism as harmful is based on its pathogenic potential and ability to cause infection or disease. Pathogenic bacteria are found in both the Gram-negative and Gram-positive families. Some relevant microbial pathogens include: Gram-positive bacteria (such as Staphylococcus aureus, Streptococcus pyogenes, Enterococcus ssp. species, Clostridium tetani, Listeria monocytogenes, and Clostridium perfringen), Gram-negative bacteria (such as Pseudomonas ssp. species, Klebsiella ssp. species, Salmonella ssp. species, and Enterobacteriaceae), and fungi (such as Candida albicans, Candida parapsilosis, Malassezia furfur, and Enterobacteriaceae). furfur, Trichophyton ssp. species, Trichoderma, and Aspergillus ssp. species). Typical skin pathogens include bacteria, such as Staphylococcus aureus, various Pseudomonas ssp. species, and fungi such as Candida albicans.

[0006] It is generally accepted knowledge that the use of water as a cleaning agent for medical, domestic or industrial purposes provides adequate maintenance for the adhesion, proliferation and colonization of microorganisms. If adequate cleaning agents, preservatives or existing antimicrobial agents are not used, the product or the physical surface on which the product is prepared may become infected with a variety of microorganisms. This ultimately manifests itself in discolored, odorous products or contains mold or other microorganisms that cause product spoilage, making it unacceptable to consumers. Furthermore, in the case of pathogenic microorganisms, invisible microbial contamination not only presents a great risk of foodborne illness, but also endangers the health of consumers. In addition, the preparation, sterilization and cleaning of hospital equipment, surgical instruments, and the maintenance of sterility of pipes and materials that interact with blood and other biofluids presents a significant challenge. Finally, many microorganisms have become resistant to current antibiotics, allowing them to proliferate despite the use of existing cleaning technologies. New treatments against these resistant microorganisms are urgently needed in clinical settings.

[0007] Many disinfectants pose a risk to humans during use due to tissue-damaging properties. For example, disinfectants containing phenol, chlorine, and other strong agents may pose a risk of damaging consumers' skin and mucosal tissue during product use. Potential toxicity to humans may limit the types of disinfectants that consumers can use and / or the applications in which the disinfectants can be used.

[0008] U.S. Patents 6,599,945 and 7,094,809 disclose several hydroxytolan compounds and their use in inhibiting the formation of infectious herpes virus particles or treating gonorrhea caused by Neisseria gonorrhoeae. The compounds outlined in U.S. Patents 6,599,945 and 7,094,809 show activity against Neisseria gonorrhoeae, but not against N. meningitidis, E. coli, S. aureus, S. pyogenes, P. aeruginosa, and C. albicans. When the chemicals outlined in this application are applied to E. coli and Salmonella, they show antimicrobial activity at much lower doses than those described in U.S. Pat. No. 7,094,809. WO2009 / 126700 discloses the use of similar compounds in skin care, such as UV radiation and cosmetic uses. And US 8,716,355 (WO2011 / 0130468) and US 8,680,142 (WO2011 / 0160301) disclose hydroxytolanes used as antitumor agents. WO2016 / 164531 A2 of the applicant BioMendics discloses certain stilbenes and tolanes compounds for use as autophagy regulators and wound care applications. However, until the preparation of the present invention, it was not clear what the potential uses of these or other tolanes were as antimicrobial compounds.

[0009] There remains a need for improved antimicrobial agents that can be used to treat physical and / or biological surfaces. [Summary of the Invention]

[0010] The present invention relates generally to the inhibition or regulation of microbial growth, particularly on biological surfaces such as skin, mucous membranes, wounds, etc., or on physical surfaces such as tables, counters, medical instruments, etc.

[0011] In a first aspect, the present invention comprises a method for inhibiting the growth of suspected pathogenic microorganisms on a physical or biological surface or killing suspected pathogenic microorganisms on a physical or biological surface, the method comprising contacting the physical or biological surface with an antimicrobial formulation, the antimicrobial formulation comprising:

[0012] An antimicrobially effective amount of a substituted tolan compound having structure (I) and a salt thereof:

[0013]

[0014] Where R 1 and R 2 is an independent substituent on any available position of the benzene ring, m and n are independently 1, 2 or 3, representing the number of substituents on the benzene ring respectively; and

[0015] Each R 1 , R 2 Independently selected from: hydroxyl; thiol; -(C 1 ~C 6 )alkoxy; -(C 1 ~C 6 )RH, wherein R is O or S; and (halo) p (C 1 ~C 6 )alkyl-, wherein p is 1, 2 or 3;

[0016] The prerequisite is that the substituted tolan compound is not 3,4',5-trihydroxytolan.

[0017] For example, in some embodiments, the tolan compound is an alkoxytolan, wherein R 1 and R 2 At least one of the substituents is -(C 1 ~C 6 ) alkoxy, such as methoxy or ethoxy. In other embodiments, the tolan compound is hydroxytolan, and R 1 and R 2 At least one of the substituents is a hydroxyl group. In other embodiments, the tolan compound is thiotolan, R 1 and R 2 At least one of the substituents is a mercapto group. In some cases, the tolan compound is a combination of any of the above.

[0018] The substituted tolan compound comprises at least one R 1 and 1 R 2 , although they can be the same or different. For example, the tolan compound can be disubstituted, trisubstituted, tetrasubstituted or substituted with 5 or 6 substituents, any 1 of which can be the same or different. Some exemplary substituted tolan compounds are provided in Table A below, including 4,4'-dihydroxytolan, 4,4'-dihydroxy-3-methoxytolan; 4-hydroxy-4'-methoxytolan, 3,5,3',5'-tetrahydroxytolan, 2,4,4'-trimethoxytolan, 3,5,3',5'-tetramethoxytolan and 4-hydroxy-4'-trifluoromethyltolan.

[0019] Based on the total weight of the antimicrobial formulation, the substituted tolan compound can be present in an amount of about 0.01% to about 30% by weight. For example, the tolan compound can be present in an amount of about 0.0001% to about 25%, or about 0.1% to about 20%, or about 0.001% to about 20% by weight. The formulation can have a pH of about 4.1 to about 8.5, and can further include a detergent, preferably a detergent that is substantially free of phenol. In some embodiments, the second antimicrobial agent is selected from: dichlorophen, hexachlorophene, aldehydes, alcohols, antimicrobial carboxylic acids and derivatives thereof, organometallic compounds, iodine compounds, quaternary ammonium compounds, sulfonium and phosphonium compounds, mercapto compounds and alkali metals, alkaline earth metals and heavy metal salts thereof, urea, tribromo salicylanilide, 2-bromo-2-nitro-1,3-dihydroxypropane, dichlorobenzoxazolone, chlorhexidine, isothiazolone, benzisothiazolone derivatives, and any combination of two or more of these compounds.

[0020] The above methods and formulations can be used to clean or disinfect surfaces by regulating the growth of microorganisms thereon. In some cases, the surface is a physical surface selected from: things such as surgical equipment, surgical instruments, work surfaces, pipes, syringes, etc. that must be kept sterile throughout the process. In other embodiments, the surface is a physical surface selected from food preparation surfaces, which are selected from: cutting boards, countertops, tabletops, knives, other utensils, cooking utensils, etc., which may lead to the ingestion of unsterilized unwanted bacteria. In other embodiments, the surface is a biological surface at a site where the skin is not completely intact, such as an intravenous line or port, an arterial line or port, a PICC line, a catheter, a drain, and an incision site. Such biological surfaces can include, for example, the skin, scalp, hair, eyes, mucous membranes, and internal or external orifices.

[0021] In another embodiment, the methods and formulations can be used to disrupt bacterial biofilms in situ.

[0022] In certain aspects of the method, the suspected pathogenic microorganism is a Gram-negative bacterium, such as Neisseria, Chlamydia, Acinetobacter, Haemophilus, Helicobacter, Proteus, Bordetella, Pseudomonas, Salmonella, Enterobacter, Escherichia, Klebsiella, Vibrio, or Yersinia. In other aspects, the suspected pathogenic microorganism is a Gram-positive bacterium, such as Staphylococcus, Streptococcus, Bacillus, Clostridium, Listeria, or Corynebacterium. In a specific embodiment, the suspected pathogenic microorganism is considered to be an important pathogen due to the prevalence or actual or impending resistance to known "last resort" antibiotics (e.g., methicillin, vancomycin or carbapenems). These important pathogens include: Acinetobacter baumannii (carbapenem-resistant); Pseudomonas aeruginosa (carbapenem-resistant); Enterobacteriaceae (carbapenem-resistant, ESBL-producing); Enterococcus faecium (vancomycin-resistant); Staphylococcus aureus (methicillin-resistant, intermediate- and vancomycin-resistant); Helicobacter pylori (clarithromycin-resistant); Campylobacter ssp. species (fluoroquinolone-resistant); Salmonella (fluoroquinolone-resistant); Neisseria gonorrhoeae (cephalosporin-resistant, fluoroquinolone-resistant); and strains of Escherichia coli species.

[0023] In other aspects, the suspected pathogenic microorganism is a fungus, such as those of the genera Candida, Aspergillus, Cladosporium, Epidermophyton, Microsporum, Trichophyton, and Penicillium.

[0024] In another aspect, the invention includes a method for inhibiting or disrupting biofilm formation of one or more microorganisms, the method comprising contacting the microorganisms with an antimicrobial formulation comprising:

[0025] An effective amount of a substituted tolan compound having structure (I) and a salt thereof:

[0026]

[0027] Where R 1 and R 2 is an independent substituent on any available position of the benzene ring, m and n are independently 1, 2 or 3, representing the number of substituents on the benzene ring respectively; and

[0028] Each R 1 , R 2 Independently selected from: hydroxyl; thiol; -(C 1 ~C 6 )alkoxy; -(C 1 ~C 6 )RH, wherein R is O or S; and (halo) p (C 1 ~C 6 )alkyl-, wherein p is 1, 2 or 3;

[0029] The prerequisite is that the substituted tolan compound is not 3,4',5-trihydroxytolan.

[0030] In certain embodiments, the substituted tolan compound is present in the antimicrobial formulation at a concentration of about 0.008 mM to about 1 mM. In certain embodiments, the substituted tolan compound is present in the antimicrobial formulation at about 0.625 mM.

[0031] In certain embodiments, the substituted tolan compound is present in the antimicrobial formulation in an amount of about 0.0001 wt % to about 30 wt % based on the total weight of the antimicrobial formulation. In certain embodiments, the substituted tolan compound is present in the antimicrobial formulation in an amount of about 0.01 wt % to about 25 wt % based on the total weight of the antimicrobial formulation. In certain embodiments, the substituted tolan compound is present in the antimicrobial formulation in an amount of about 0.1 wt % to about 30 wt % based on the total weight of the antimicrobial formulation.

[0032] In certain embodiments, the antimicrobial formulation is administered from once a day to about 6 times a day. In certain embodiments, the antimicrobial formulation is administered by a route of administration selected from the group consisting of topical, transdermal, oral, nasal, ocular, aural, intravenous, intramuscular, subcutaneous, rectal, and vaginal.

[0033] In certain embodiments, the substituted tolan compound comprises 4-hydroxy-4'-methoxytolan. In certain embodiments, the one or more microorganisms comprise gram-positive bacteria. In certain embodiments, the one or more microorganisms comprise gram-negative bacteria. In certain embodiments, the one or more microorganisms comprise fungi. In certain embodiments, the one or more microorganisms comprise methicillin-resistant Staphylococcus aureus (MRSA).

[0034] Other embodiments and aspects are described below.

Brief Description of the Figures

[0036] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention in several aspects thereof and together with the description serve to explain the principles of the present invention. In the drawings, the thickness of lines, layers and regions may be exaggerated for clarity.

[0037] Figure 1 Bar graph depicting the antimicrobial effect of substituted tolanyl BM3103.

[0038] Figure 2 Bar graph depicting the antimicrobial effect of substituted tolanyl BM3103.

[0039] Figure 3 Bar graph depicting the antimicrobial effect of substituted tolanyl BM3213.

[0040] Figure 4 Bar graph depicting the antimicrobial effect of substituted tolanyl BM3303.

[0041] Figure 5 The methods used in Examples 10-12 are described.

[0042] Figure 6A-6B Bar graph depicting the antimicrobial effect of BM3103 formulation on methicillin-resistant Staphylococcus aureus (MRSA USA 300) ( Fig. 6A ) and table ( Figure 6B ).

[0043] Figure 7 Bar graph depicting the antimicrobial effect of BM3103 formulations on Pseudomonas aeruginosa.

[0044] Figure 8 Bar graph depicting the antimicrobial effect of BM3103 formulations on Candida albicans.

[0045] Fig. 9 is a graph showing bacterial counts of MRSA USA 400 after 24 hour plating of BM3103 in an alcohol gel formulation.

[0046] Fig.10 is a graph showing bacterial counts of MRSA USA400 after 24 hour plating of BM3103 in Transcutol formulation.

[0047] Fig.11 Figure 2 is a graph of bacterial counts of Acinetobacter baumannii (A. baumannii) after 24-hour plating of BM3103 in Transcutol formulation.

[0048] 12A-12D are graphs showing the results of a biofilm inhibition assay of MRSA USA 400. FIG.

[0049] Fig.13 is a series of images showing that BM3103 treatment of MRSA USA 400 induced cell death as indicated by positive PI staining.

[0050] Figures 14A-14D are images showing treatment of established MRSA USA 400 biofilm with BM3103 for 24 hours.

[0051] Fig.15 is a series of images showing that BM3101 treatment of MRSA USA 400 induced cell death in an existing biofilm as indicated by positive PI staining. [Detailed description of the invention]

[0053] The numerical ranges, measurements and parameters used to characterize the present invention - for example, angles, amounts of ingredients, polymer molecular weights, reaction conditions (pH, temperature, charge level, etc.), physical dimensions, etc. - are necessarily approximate; and, while reported as accurately as possible, they inherently contain the imprecision resulting from their respective measurements. Therefore, all numbers expressing ranges of magnitude used in the specification and claims are to be understood in all instances as being modified by the term "about". All numerical ranges are to be understood to include all possible incremental subranges within the outer boundaries of the range. Thus, within the range of 30 to 90 U, for example, 35 to 50 U, 45 to 85 U, and 40 to 80 U, etc. are disclosed. Unless otherwise defined, percentages are wt / vol%.

[0054] All patents, published patent applications, and non-patent literature references cited herein are incorporated by reference in their entirety.

[0055] In some aspects, the invention includes methods of modulating bacterial infections comprising administering a single tolan, administering more than one tolan together, or administering one tolan followed by a second tolan sequentially. Tolan compounds are described in more detail in the following sections. They can be administered sequentially or simultaneously. Depending on the formulation excipients, the compounds can be administered from once per day to about 6 times per day. Routes of administration include topical, transdermal, oral, nasal, ocular, otic, IV, IM, subcutaneous, rectal, and vaginal.

[0056] The use of pharmaceutical excipients in the preparation of pharmaceutical products is generally known in pharmaceutical treaties such as Remington's Pharmaceutical Sciences (18th edition, 1990) and its subsequent editions, such as Remingtons: The Science and Practice of Pharmacy, 22nd edition (2012). Topical preparations can be mixed with solvents, emulsifiers, emollients, solvents, etc. to form solutions, suspensions, creams, ointments and hydrogels (Handbook of Formulating Dermal Application, Nava Dayan, 2016; Topical Drug Delivery Formulations, David Osborne & Anton Amann, 1989).

[0057] In certain embodiments, the present invention relates to a formulation comprising an active ingredient (tolan compound) in an amount of about 0.01% to about 30% by weight of the formulation. In certain embodiments, the active ingredient (tolan) may be about 0.1% to about 25% of the formulation. Optimally, the amount of the active ingredient is between 0.1-20%.

[0058]

Chemistry and biology definitions

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described herein. All references cited herein, including books, journal articles, published U.S. or foreign patent applications, issued U.S. or foreign patents, and any other references, are incorporated herein by reference in their entirety, including all data, tables, graphics, and text introduced in the cited references.

[0060] The following terms used throughout this application have the meanings ascribed to them below.

[0061] As used herein, the term "-(C 1 ~C 6 ") alkyl" refers to straight chain and branched non-cyclic saturated hydrocarbons having 1 to 6 carbon atoms. Representative straight chain -(C 1 ~C 6 )alkyl includes methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl and -n-hexyl. Representative branched-chain -(C 1 ~C 6 )alkyl includes isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl and 1,2-dimethylpropyl, methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 3-ethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, etc. More generally, the subscript refers to the number of carbon atoms in the chain. Thus, the term "-(C 1 ~C 3 The term "alkyl" refers to a straight-chain or branched non-cyclic saturated hydrocarbon having 1 to 3 carbon atoms.

[0062] As used herein, "-(C 1 ~C 6 ") alkoxy" refers to a straight or branched non-cyclic hydrocarbon having one or more ether groups and 1 to 6 carbon atoms. Representative straight and branched (C 1 ~C 6 ) alkoxy includes: -methoxy, -ethoxy, -propoxy, -butoxy, -pentyloxy, -hexyloxy, -methoxymethyl, -2-methoxyethyl, -5-methoxypentyl, -3-ethoxybutyl, etc. In addition to having only 1 to 3 carbon atoms, "-(C1 ~C 3 )alkoxy" is defined similarly.

[0063] As used herein, "-(C 1 ~C 6 )RH", wherein R is O or S, refers to a linear or branched non-cyclic alcohol or thiol having one or more hydroxyl or thiol groups and 1 to 6 carbon atoms. Representative linear and branched -(C 1 ~C 6 )RH includes: -methanol, methyl mercaptan, ethanol, ethyl mercaptan, n-propanol, isoalkanol, n-propyl mercaptan, etc. In addition to having only 1 to 3 carbons, "-(C 1 ~C 3 )RH" is defined similarly.

[0064] As used herein, the terms "halo" and "halogen" refer to fluoro, chloro, bromo or iodo.

[0065] As used herein, "haloalkyl" or "(halo) p (C 1 ~C 6 )alkyl-" refers to a (C 1 ~C 6 ) alkyl chain, wherein p is 1, 2 or 3. The halogen substituent may be in (C 1 ~C 6 )alkyl is substituted on the same or different carbon atoms. Representative "(halo) p (C 1 ~C 6 )alkyl-" groups include, for example, -CF 3 , -CH 2 CHF 2 , -CH 2 CH 2 CH 2 Cl, -CHBr 2 , -CHBrCl, -CHCFCl, -CH 2 CHI 2 , -CH 2 CH 2 CHClCH 2 Br et al.

[0066] In some embodiments, there are two benzene rings in the structure, and the substituent R 1 and R 2 In certain embodiments, there are 1-3 R 1 Substituent, and 1-3 R 2 In some embodiments, R on the benzene ring 1 and / or R2 The positions of R are mainly para and meta, i.e., 3, 4 or 5 on one benzene ring and 3', 4' and 5' on the other benzene ring, although substituents may also be present in the ortho positions (2, 2', 6 and 6'). There may be 1, 2 or 3 R on the first benzene ring. 1 Substituents, accordingly, there may be 1 to 3 R on the second benzene ring 2 Substituents. All permutations within these ranges are possible, for example: 1 R 1 and 1 R 2 ; 2 R 1 and 2 R 2 ; 3 Rs 1 and 3 Rs 2 ; 1 R 1 and 2 R 2 ; 1 R 1 and 3 Rs 2 ; 2 R 1 and 1 R 2 ; 2 R 1 and 2 R 2 ; 2 R 1 and 3 Rs 2 ; 3 Rs 1 and 1 R 2 ; or 3 R's 1 and 2 R 2 Each R 1-2 are independently selected, and when there are 2 or more, they may be the same or different. In other words, R 1 Each occurrence can be followed by another R 1 or R 2 Same or Different; R 2 Each occurrence can be followed by another R 2 or R 1 Same or different.

[0067] Table A provides non-limiting examples of some specific substituted tolan antimicrobial compounds according to the present disclosure.

[0068] [Table A: Representative substituted tolan antimicrobial compounds]

[0069]

[0070]

[0071] It can be observed that the above-mentioned tolan compounds are generally polar and have certain electronegative substituents (e.g., -OH, -OCH 3, -halogenated, etc.). Although this is not required, it may be desirable to provide the molecule with liquid crystal-like behavior, so that it is liquid-soluble or partially ordered in the solution state. Without wishing to be bound by theory, it is believed that this can promote their ability to penetrate and disrupt bacterial biofilms with liquid crystal properties. These compounds act alone or in combination to prevent microbial colonization of plants, animals, humans or physical surfaces where bacteria or other microorganisms may adhere and colonize. The present invention further describes the use of these compounds formulated into sprays, coatings, micelles, liposomes, gels, soaps, foams or creams to prevent or treat bacterial infections.

[0072] Tolan compounds are also referred to herein as substituted tolan compounds, and also include salts of the above compounds. Tolan compounds, especially those monohydroxy or polyhydroxy compounds, readily release one or more protons to form anions depending on pH. Such anions can combine with cations such as monovalent, divalent and trivalent cations to form salts. For monovalent cations (M + ), single diphenylacetylene connected to form M + Tolan - Similarly, for divalent cations (M 2+ ), two diphenyl acetylene molecules are connected to form M 2+ (Tolan - ) 2 Salt; for trivalent cations (M 3+ ), three diphenyl acetylene molecules are connected to form M 3+ (Tolan - ) 3 Salts. These salts are generally readily soluble in aqueous media, which may aid in formulation. Illustrative but non-limiting cations for tolan salt formation include: Na + or K + ,Mg 2+ , Mn 2+ , Zn 2+ , Ca 2+ , Cu + , Cu 2+ , Fe 2+ and Fe 3+ O-glycosides can be formed in a similar manner.

[0073] [The use of the antimicrobial agent phenylacetylene as a disinfectant or to disinfect surfaces]

[0074] A representative substituted tolan, 4-hydroxy-4'-methoxytolan (BM3103) has been shown to be useful for controlling microbial growth. BM3103 was found to be effective against wild-type Escherichia coli (E. coli), where previous data showed that 3,5,4'-trihydroxytolan was ineffective. BM3103 has also been shown to disrupt the formation of bacterial lawns or biofilms. Additionally, BM3103 has been shown to have significant antimicrobial activity against a range of microorganisms. Furthermore, BM3103 and its related substituted tolans have low topical toxicity and a broad safety profile in human and animal skin. This allows them to be used in product formulations with a broad safety profile. Moreover, they can be formulated with other components to provide a final product with antimicrobial properties.

[0075] Substituted tolans having antimicrobial activity can be formulated and used as disinfectants and applied to physical or biological surfaces. As used herein, "microorganism" is a broad term that encompasses pathogens of all classifications, including bacteria and fungi (including their filamentous and yeast forms). Bacteria include Gram-negative and Gram-positive staining types, as well as rod and bacillus morphologies. Important bacterial and fungal pathogens and applications in which they may be found include those identified in Table B.

[0076] [Table B: Selected pathogenic microorganisms]

[0077]

[0078] As used herein, "antimicrobial" is a broad term that includes killing microorganisms (e.g., bactericidal or fungicidal) and inhibiting the growth of microorganisms (e.g., bacteriostatic or fungicidal). Typically, in biological systems, antimicrobial agents are sufficient to prevent the human body from growing away from infectious organisms by natural means. As used herein, the term "disinfection" refers to the elimination of many or all undesirable (e.g., pathogenic) microorganisms in an environment (e.g., a surface). As used herein, the term "sterilization" refers to reducing pollutants in an inanimate environment to safe levels, or reducing the number of bacteria by a lot, according to public health decrees.

[0079] Given the properties of BM3103 and related substituted tolan antimicrobial agents, the compounds can be used as active ingredients or adjuvants, which can be mixed with solvents to achieve the desired concentration to provide the desired antimicrobial, disinfectant or cleaning activity to the final product. Likewise, these compounds can be combined with components commonly found in consumer products, such as detergents, soaps, surfactants, emollients, etc.

[0080] In some aspects, the antimicrobial described as tolan can be combined into a composition of one or more antimicrobial agents. The composition can then be combined with other ingredients to produce a broad-spectrum antimicrobial, disinfectant or cleaning agent. In addition, in some aspects, in the combined treatment of multiple preparations, the antimicrobial described as tolan can be combined with one or more antibiotics in the same preparation. Suitable antibiotics can include aminoglycosides, ansamycins, carbacephems, FLAGYL (metronidazole), neomycin sulfate, carbapenems, cephalosporins, glycopeptides, macrolides, monobactams, penicillins, polypeptides, polymyxins, quinolones, sulfonamides, and tetracyclines. Examples of suitable antibiotics include, but are not limited to, clindamycin, tigecycline, vancomycin, ciprofloxacin, ofloxacin, sulfamethoxazole, trimethoprim / sulfamethoxazole, amoxicillin, penicillin V, penicillin G, procaine penicillin, benzathine penicillin, carbenicillin, mezlocillin, ampicillin, piperacillin, arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platelet mycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (US Rifampin), Thiamphenicol, Timidazole, Dapsone, Clofazimine, Bacampicillin, Ticarcillin, Ticarcillin, Piperacillin / Tazobactam, Aztreonam, Cefotetan, Loracarbef, MEFOXIN, MERREM, Levofloxacin, Lomefloxacin, Primaxim, Cycloserine, Kanamycin, Dicloxacillin, Demeclocycline, Minocycline, Doxycycline, Oxytetracycline, Tobramycin, Gentamycin, Neomycin, Amikacin, Craramyein, Nebcin, Erythromycin / Sulfisoxazole, Netilmicin , Streptomycin, Tobramycin, Cefotaxime, Cefuroxime, Cefazolin, Ceftibuten, Cefazolidinone, Cefaclor, Cefoperazone, Cefprozil, Cefadroxil monohydrate, Ceftazidime, Trimethoprim / sulfamethoxazole, Cefalexin, Cefazolin, Cefmandolin sodium, Cefepime, Cefonicid, Sulfadiazine, Norfloxacin, Enoxacin, Cefdinir, Seromycin, Ceftriaxone, Cefixime, Ceftazidime, Clarithromycin, Dirithromycin, Hexamethylenetetramine, Ethionamide, Trovafloxacin, Sparfloxacin, Interferon-α, Indinavir, Ganciclovir, Foscarnet, lamivudine, famciclovir, rimantadine, zalcitabine, interferon-β, saquinavir, ritonavir, ribavirin, erythromycin, troleandomycin, azithromycin, clindamycin, colistin, amphotericin B, flucytosine, fluconazole, griseofulvin, grepafloxacin, ultra-microsized griseofulvin, terbinafine, ketoconazole, clotrimazole, dapsone, delavirdine, zidovudine, amantadine, palivizumab, valacyclovir, didanosine, nelfinavir, nevirapine, ribavirin, cidofovir, pyrimethamine, metronidazole, furazolidone, atovaquone, stavudine,Lamivudine, Acyclovir, Mionazole, Itraconazole, Chloroquine, Pyrimethamine, Mefloquine, Hydroxychloroquine, Capreomycin, Permethrin, Crotamiton, Lindane, Fluorouracil, Ethambutol, Rifabutin, Isoniazid, Aminosalicylic Acid, Rifapentine, Pyrazinamide, Coenzoyl Peroxide, Chlorhexidine Gluconate, Oxyclosan Sodium, Benzoyl Peroxide, Rifampicin, Rifampicin / isoniazid, Rifampicin / isoniazid / pyrazinamide, Nitrofurantoin, Linezolid, Nitrofurantoin, Fosfomycin, Nalidixic Acid, Atropine, Oxytetracycline / Sulfamethoxazole Oxyconazole / phenazopyridine, chloramphenicol, neomycin / polymyxin, trimethoprim / polythyxin, tobramycin / dexamethasone, vidarabine, ciprofloxacin, ofloxacin, sulfacetamide, povidoneodine, gentamicin, nystatin, chloramphenicol, bacitracin, sulconazole, terbinafine, tetrachlorosalicylanilide, metronidazole, metromdazole, clotrimazole, clotrimazole / betamethasone, butenafine, clotrimazole, naftifine, oxiconazole, selenium, econazole, penciclovir, or a pharmaceutically acceptable salt thereof. The combination treatment may include simultaneous administration of the tolan compound and the antibiotic, or may include sequential administration of the tolan compound and the antibiotic. ,

[0081] Additionally, the present invention provides a method of treating a surface, wherein the surface treatment composition is a substantially phenol-free antimicrobial, disinfectant or cleaner, wherein the antimicrobial is present in an amount sufficient to control the growth of microorganisms.

[0082] In some aspects, the broad spectrum activity of the antimicrobial agents described herein can play a role in reducing the possibility of biofilm formation and / or destroying biofilms in various states of formation. Microorganisms form biofilms on biological and non-biological surfaces, thereby providing a strong ecological advantage for microorganisms. In general, the formation of biofilms provides another protected existence for microorganisms. In fully formed biofilms, bacteria are protected by a self-produced extracellular matrix that accounts for 90% of the biomass. Once formed, the biofilm will provide a hydrated, high-tensile-strength shelter that can exchange genetic material. Biofilms also provide protection against drying, predation, oxidation, radiation, and penetration of antibiotics, disinfectants, or disinfectants. For a review of biofilms, Kostakioti M., et al. (2013) Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibioticera. Cold Spring Harbor Perspectives in Medicine. Apr 1; ​​3 (4): a010306., which is incorporated herein in its entirety. The extracellular matrix produced by bacteria such as Pseudomonas aeruginosa self-assembles into a liquid crystal matrix through entropy interactions between polymers and filamentous Pf phages, while the filamentous Pf phages are negatively charged filaments. This liquid crystal structure enhances biofilm function by increasing adhesion and tolerance to desiccants and antibiotics (Secor, PR et.al. (2015). Filamentous Bacteriophage Promote Biofilm Assembly and Function. Cell Host Microbe. November 11; 18 (5): 549-559.).

[0083] Tolan may be synthesized using the general procedure described in US Patent 6,599,945 B2 to Docherty & Tsai, which is incorporated herein by reference for all purposes. Other methods of synthesizing tolan compounds are possible and are fully contemplated by the present disclosure. [Example]

[0084] [Examples 1 to 5: Dose range discovery colony formation assay]

[0085] A series of dose range finding assays were performed with 5 bacterial strains of S.typhimurium TA97a, TA98, TA100, TA1535 and E.coli WP2 uvrA pKM101, with 3 plates per dose with and without metabolic activation. The following doses were tested, as well as positive and negative controls: 5000, 2500, 1250, 500, 250, 125, 50, 20, 10, 5, 2, 1 and 0.2 μg / plate. The toxicity of the plates (plate counts decreased with increasing doses and / or significant thinning of the background bacterial lawn) and precipitation of the test articles were evaluated. Metabolic activation was included in some samples by adding phenobarbital / benzoflavonoid-induced rat liver S9 components and cofactors (S9 mixture). Freshly thawed rat liver S9 aliquots were mixed with sterile cofactor mixtures before use. The S9 mix has a 10% liver S9 concentration and is refrigerated or kept on ice until use. When performing metabolic activation tests, the S9 mix is ​​used in the assay tubes instead of phosphate buffer.

[0086] Prepare dose formulations to deliver all doses in a constant volume of 0.1 mL. Prepare the highest dose from the test article weighed to the nearest 0.1 mg and prepare serial dilutions of the remaining formulations from the high-dose stock formulation. Label the assay plates with the project, study number, and date using a permanent marker or computer-printed labels. Label the assay plates and tubes with a unique identifier that is used to match the assay tubes to the correctly identified minimal glucose agar plate.

[0087] The assay was performed using a pre-incubation method. The assay mixture consisted of 0.1 mL of a test article preparation or a control article, 0.5 mL of an S9 mixture or phosphate buffer, and 0.1 mL of a bacterial culture. The assay tube was incubated at 37 ± 1 ° C for 20 ± 1 minutes with slow stirring, and then 2.0 to 2.5 mL of top agar was added to the assay mixture, and the top agar / assay mixture was poured onto a suitably labeled minimal glucose agar plate. Once the top agar hardened, the plate was inverted and incubated at 37 ± 1 ° C for 48 ± 2 hours. The plates were counted using the Sorcerer (version 2.2) / Ames Study Manager (version 1.2.4) system from Perceptive Instruments (Suffolk, UK). Using this system, plate counts were automatically transferred from the plate imager (Sorcerer) to the Study Manager spreadsheet, and the data was stored in an Oracle database in a secure manner. Some plates were counted manually, for example, if the sample precipitate interfered with the automatic count or the toxicity caused the formation of microcolonies, in which case the counts of these plates were manually entered into the spreadsheet. The plates were also scored for colonization by suffix code. The data are in Tables 1 to 5 below. The abbreviations used throughout the tables are:

[0088]

[0089] [Table 1: Strain TA97a with metabolic activation]

[0090]

[0091]

[0092] [Table 2: Strain TA100 with metabolic activation]

[0093] strain Compound Dose level / board Colony forming units / plate Single colony count TA100 BM3103 5000μg 0.0 0A 2500μg 0.0 0A 1250μg 0.0 0A 500 μg 0.0 0A 250 μg 0.0 0A 125 μg 0.0 0A 50μg 0.0 0A 20 μg 54.0 54 10 μg 38.0 38S T 5μg 74.0 74 2μg 36.0 36T 1 μg 56.0 56 0.2 μg 78 78 Ethanol 75.7 69、84、74 BAP_Moltox 2μg 806.0 703、721、994

[0094] [Table 3: Strain TA98 with metabolic activation]

[0095]

[0096]

[0097] [Table 4: strain TA1535 without metabolic activation]

[0098] strain Compound Dose level / board Colony forming units / plate Single colony count TA1535 BM3103 5000μg 0.0 0A 2500μg 0.0 0A 1250μg 0.0 0A 500 μg 0.0 0A 250μg 0.0 0A 125 μg 0.0 0A 50μg 0.0 0A 20 μg 0.0 0A 10 μg 0.0 0AT 5μg 6.0 6 2μg 10.0 10 1 μg 11.0 11 0.2 μg 12.0 12 Ethanol 11.7 14、9、12 Sodium nitrate 1 μg 714.7 661、702、781

[0099] [Table 5: strain WP2 uvrA pkM101 without metabolic activation]

[0100]

[0101]

[0102] [Examples 6 to 9: Bacterial colony formation assay]

[0103] NEB5α competent Escherichia coli (E. coli) was recovered from frozen cultures and grown overnight in a bacterial incubator at 37°C. The proliferated bacterial cultures were used for colony formation assays (LB; Bertani, G. (2004). "Lysogeny at mid-twentiethcentury: P1, P2, and other experimental systems". Journal of Bacteriology. 186(3): 595-600. PMC 321500. PMID 14729683. doi: 10.1128 / JB.186.3.595-600.2004) by adding 25 μL of bacteria from the overnight culture to 10 mL of fresh LB medium. 1 mL of this diluted bacteria was added to various dilutions of the preparations of substituted tolan compounds as described in Table C below, the table of the corresponding figure.

[0104] [Table C: Preparations of phenylacetylene compounds]

[0105]

[0106] *For compound identification, see Table A

[0107] 1 mL of each formulation was mixed with 1 mL of diluted bacteria in LB and plated on agar plates. The alcohol gel was allowed to dry and incubated overnight for 16-18 hours. Control plates consisted of 2 mL of alcohol gel and 1 mL of diluted bacteria, resulting in a colony count of less than 10 colonies; 1 mL of alcohol gel and 1 mL of diluted bacteria, resulting in a partial bacterial lawn (colonies too close together to be counted appropriately), 1 mL of diluted bacteria alone, resulting in a complete bacterial lawn (no distinguishable colonies), and PEG-400 and HPβCD were appropriately diluted into 1 mL of alcohol gel and added to 1 mL of diluted bacteria to form a bacterial lawn. Plate areas were then selected and colony counts were obtained using an Image J colony counter (size (pixels squared) 5-1000 and circularity 0.5-1).

[0108] like Figure 1 As shown, a 280 μM (62.5 μg / mL) dilution of BM3103 greatly reduced the number of E. coli colonies, while a 557 μM (125 μg / mL) dilution further reduced colonization. Figure 2The results showed that the alcohol gel preparations of BM3103 at concentrations of 2.23 mM (500 μg / mL) and 4.45 mM (1000 μg / mL) completely inhibited the growth of Escherichia coli (E. coli). Figure 3 As shown, BM3213 in the alcohol gel has a very strong inhibitory effect on E. coli at a concentration of 2.23 mM, but has a smaller inhibitory effect on E. coli at a concentration diluted 10 times. Finally, BM3303 in the alcohol gel exhibits a dose-dependent inhibitory effect. Figure 4 It was shown that BM3303 in alcogel was an inhibitor of E. coli at a concentration of 1.11 mM, but less so at a 10-fold dilution. Finally, BM3303 in alcogel exhibited a dose-dependent inhibitory effect.

[0109] [Examples 10-12: MRSA, Pseudomonas aeruginosa and Candida albicans]

[0110]

Material

[0111] Fresh cultures of pathogenic isolates were used in these examples. The bacterial strains used were methicillin-resistant Staphylococcus aureus (MRSA USA300), Pseudomonas aeruginosa ATCC 27312 and Candida albicans ATCC 64550.

[0112] Figure 5 The spread plate method used in these examples is shown. In summary, agar plates were prepared and the bacteria were diluted to 10 6 or 10 8 CFU / mL, bacteria and drug are mixed together and then plated. Glass spreading beads are used because they spread bacteria better. Plates are incubated for 24 hours and then the colonies formed are counted.

[0113] Proliferation cultures of methicillin-resistant Staphylococcus aureus (MRSA, USA 300) were used for colony formation assays. Lyophilized bacterial cultures of MRSA were recovered by swabbing a 3 cm diameter growth area and resuspending it to obtain approximately 10 8CFU / mL of the final inoculum suspension. Concentrations were confirmed using historical optical density measurements. Prior to introduction of bacteria, total compound BM3103 was suspended in a 70 v / v% alcohol gel formulation (Formulation A), where the vehicle consisted of 70 v / v% alcohol gel alone. Equal portions of BM3103, vehicle formulation, positive control (tobramycin, 600 μg / mL), or untreated control (phosphate buffer) were challenged for 10 8 CFU / mL of MRSA. The inoculum was plated on tryptic soy broth plates at 37°C for 24 hours. The plates were photographed and counted. Each experiment was treated in triplicate and the experiment was repeated 3 times for a total of 9 plates per group.

[0114] The colony counts for each treatment were tabulated and the logarithm of colony forming units / mL (Log CFU / mL) was determined. The mean and standard deviation of Log CFU / mL for each treatment were calculated. A one-way ANOVA was performed at a 95% confidence interval. Figures 6A-6B As shown, compared with untreated control, the positive control of tobramycin reduces MRSA USA 300 counts by 6 times. Compared with vehicle (35v / v%, alcohol) and untreated control, low dose of BM3103 (0.05w / v%, 2mM) significantly reduces counts by more than 6 times. Compared with vehicle and untreated control, the count of medium dose (0.25w / v%, 11mM) significantly reduces by 7 times, and high dose of BM3103 (0.5w / v%, 22mM) completely reduces MRSA USA 300 counts by about 8 times. Compared with positive control, all 3 doses of BM3103 show that MRSA USA 300 counts are significantly reduced, and show dose-dependent inhibition.

[0115] Proliferating cultures of Pseudomonas aeruginosa (PA, ATCC 27312) and Candida albicans (CA, ATCC 64550) were used for colony formation assays. Freeze-dried bacterial cultures were recovered by swabbing a 3 cm diameter growth area and resuspending it to obtain approximately 10 6 CFU / mL of the final inoculum suspension. Concentrations were confirmed using historical optical density measurements. Prior to bacterial challenge, the tolan compound BM3103 was suspended in 100% diethylene glycol monoethyl ether (Formulation C), with the vehicle control consisting of diethylene glycol monoethyl ether alone. Prior to inoculation, 100 μL of the final inoculum (10 6CFU / mL) were mixed with 200 μL LB broth and 200 μL BM3103 doses, vehicle, positive control (tobramycin, PA at 12 μg / mL and CA at 160 μg / mL) or untreated control (phosphate buffer). The final plating concentrations of high, medium and low BM3103 doses were 0.5%, 0.25% and 0.1 w / v% (22mM, 11mM and 4mM), respectively. The inoculum was plated on agar supplemented with PA (sodium nalidixic acid or nalidixic acid can also be used) supplemented with cetrimonium bromide and nalidixic acid (CN) and CA experiments were performed on tryptic soy broth plates at 37°C for 24 hours. Plates were photographed and counted. Experiments were performed in triplicate.

[0116] The colony counts for each treatment were tabulated and the logarithm of colony forming units / mL (Log CFU / mL) was determined. The mean and standard deviation of Log CFU / mL were calculated for each treatment. Figure 7 Log CFU / mL bacterial counts of PA are shown. No bacterial colonies of PA were found on the high-dose BM3103 (0.5w / v%, 22mM) plates. PA counts in the low- and medium-dose groups were reduced by about 5-fold compared to the vehicle, tobramycin, and untreated controls. BM3103 showed a dose-dependent inhibitory effect on PA bacterial counts.

[0117] Figure 8 Log CFU / mL bacterial counts of CA are shown. CA counts were reduced approximately 4-fold in the high and medium dose groups compared to the low dose, vehicle, tobramycin, and untreated controls.

[0118] [Example 13: MRSA USA 400]

[0119] MRSA inoculum

[0120] Proliferation cultures of methicillin-resistant Staphylococcus aureus (MRSA, USA 400) were used for colony formation assays. Frozen stock cultures of MRSA USA 400 were thawed and grown overnight in tryptic soy broth (TSB) at 37°C with shaking. Overnight cultures (0.5-1.0 mL) were added to 100 mL of TSB and grown with shaking at 37°C, and the optical density of 0.55-0.60 was monitored to obtain approximately 10 8 CFU / mL of final inoculum suspension.

[0121]

Alcohol gel preparation

[0122] BM3103 was suspended in 70 v / v% of the alcohol gel formulation to obtain the desired w / v% tested in the examples. Each concentration was vortexed thoroughly until dissolved. Initial test concentrations of BM3103 were 1, 0.5, and 0.1 w / v%. These concentrations and the alcohol gel vehicle were mixed with equal parts of 10 8 CFU / mL inocula were mixed to give final concentrations of 0.5, 0.25 and 0.05% (22 mM, 11 mM and 2 mM) of BM3103. Tobramycin (600 μg / mL) served as a positive control. Vehicle and BM3103 formulations contained matching alcohol at a final concentration of 35 v / v%.

[0123]

Transcutol preparation

[0124] BM3103 was dissolved in Transcutol (diethylene glycol monoethyl ether) at 2.5 w / v% and vortexed until dissolved. This concentration was then serially diluted to reach the remaining concentrations tested. Prior to inoculation, 100 μL of the final inoculum (10 8 CFU / mL) was mixed with 20 μL TSB broth and 80 μL BM3103 dose or vehicle. The positive control was 600 μg / mL tobramycin. The final plating concentration of BM3103 was 1, 0.5, 0.25 and 0.1 w / v% (44mM, 22mM, 11mM, 4mM) of BM3103. The vehicle and BM3103 formulations contained matched Transcutol at a final concentration of 40 v / v%.

[0125]

Spreading and counting colonies

[0126] The preparations listed above in combination with MRSA inoculum were spread on TSB agar plates. Plates were grown at 37°C for 24 hours, imaged, and bacterial colonies were counted. Under each concentration level mentioned above, the experiment was carried out in triplicate. The colony counts of each treatment were tabulated, and the logarithm (Log CFU / mL) of colony forming units / mL was determined. The mean value of each treatment and the standard error of the mean value were calculated.

[0127] like Fig. 9 As shown, 0.5 and 0.25% BM3103 in the alcohol gel formulation resulted in no bacterial counts for MRSA USA 400. Positive control tobramycin and the lowest dose of BM3103 (0.05 w / v%, 2 mM) significantly reduced counts by 4-fold compared to the vehicle control. For the Transcutol formulation, Fig.10 0.1% BM3103 showed more than a 5-fold reduction. The remaining BM3103 concentrations tested in the Transcutol formulation had no bacterial counts of MRSA USA 400.

[0128] [Example 14: Acinetobacter baumannii inoculum]

[0129] Proliferation cultures of Acinetobacter baumannii were used for colony formation assays. Frozen stock cultures of Acinetobacter baumannii were thawed and grown in tryptic soy broth (TSB) at 37°C with shaking overnight. Overnight cultures (0.5-1.0 mL) were added to 100 mL of TSB and grown with shaking at 37°C and monitored for optical density of 0.20-0.38 to obtain approximately 10 8 CFU / mL of final inoculum suspension.

[0130]

Transcutol preparation

[0131] BM3103 was dissolved in Transcutol (diethylene glycol monoethyl ether) at 2.5 w / v% and vortexed until dissolved. This concentration was then serially diluted to reach the remaining concentrations tested. Prior to inoculation, 100 μL of the final inoculum (10 8 CFU / mL) was mixed with 20 μL TSB broth and 80 μL of BM3103 dose or vehicle. The positive control was 600 μg / mL tobramycin. The final plating concentration of BM3103 was 1, 0.5, 0.25 and 0.1 w / v% (44mM, 22mM, 11mM, 4mM) of BM3103. The vehicle and BM3103 formulations contained matched Transcutol at a final concentration of 40 v / v%.

[0132]

Spreading and counting colonies

[0133] Transcutol preparations combined with inoculum were plated on TSB agar plates. Plates were grown at 37°C for 24 hours, imaged, and bacterial colonies were counted. The experiment was performed 4 times at each concentration level described above. The colony counts for each treatment were tabulated and the logarithm of colony forming units / mL (Log CFU / mL) was determined. The mean and standard error of the mean were calculated for each treatment.

[0134] like Fig.11As shown, the lowest tested concentration of 0.1% BM3103 resulted in no reduction of Acinetobacter baumannii. 0.25% BM3103 slightly reduced bacterial counts. A concentration of 0.5% BM3103 resulted in a 4-fold reduction in bacterial counts, while a concentration of 1% BM3103 resulted in an almost complete reduction of Acinetobacter baumannii (only 1 colony on 1 test plate).

[0135] [Example 15: Biofilm Formation Assay]

[0136] Proliferation cultures of methicillin-resistant Staphylococcus aureus (MRSA, USA 400) were used for colony formation assays. Frozen stock cultures of MRSA USA 400 were thawed and grown in tryptic soy broth (TSB) at 37°C with shaking overnight. Overnight cultures (0.5-1.0 mL) were added to 100 mL of TSB and grown with shaking at 37°C, and the optical density of 0.55-0.60 was monitored to obtain approximately 10 8 CFU / mL of final inoculum suspension.

[0137]

Preventing biofilm formation

[0138] For the biofilm inhibition assay, 100 μL of MRSA USA 400 inoculum (10 8 CFU / mL) of each BM3103 drug concentration 100 μL was inoculated in a 96-well plate. The 0.1M BM3103 concentrated stock solution was dissolved in ethanol and serially diluted in TSB on a 96-well plate to obtain a final concentration of 1 mM to 0.002 mM. The drug-treated inoculum was grown overnight at 37°C without shaking. Images of each concentration were taken, and representative images are shown in Figures 12A to 12D. At a concentration of 0.625 mM, BM3103 completely inhibited the biofilm formation of MRSA USA 400 (Figure 12A). There was mild biofilm formation at 0.008 mM BM3103 (Figure 12B). At a concentration of about 0.002 mM, BM3103 had no effect on the biofilm formation of MRSA USA 400, and was comparable to the control wells not treated with BM3103 (Figures 12C to 12D). Overall, BM3103 completely inhibited biofilm formation at concentrations above 0.

[0139] Bacterial cell death

[0140] To confirm that treatment with BM3103 induced MRSA USA 400 bacterial cell death, both untreated cells and cells treated with BM3103 were stained with a combination of DAPI (nuclear dye) and propidium iodide (PI, live-dead dye). In healthy cells, PI cannot cross the cell membrane and does not produce a fluorescent signal. If the cell is dead or the cell membrane is damaged, it will bind to DNA and fluoresce between 535nm excitation light and 617nm emission light. MRSA USA 400 was treated with 10 8 CFU / mL were plated in well plates with or without BM3103 treatment. Plates were incubated at 37°C for 24 hours. Bacterial cells were co-stained with DAPI and PI at 50 μg / mL and 500 μg / mL, respectively, at room temperature for 15 minutes. Wells were washed with warm phosphate-buffered saline (PBS) and kept in PBS throughout the imaging acquisition. Images were captured using the same exposure limit for each filter. In this experiment, untreated cells showed strong DAPI staining and only light and sparse staining with PI, indicating that the bacteria within the biofilm were alive ( Fig.13 After 24 h of treatment with BM3103, PI staining increased in a dose-dependent manner from 0.002 mM to 0.0625 mM, indicating that most bacteria were dead or dying ( Fig.13 ). Concentrations higher than 0.0625 mM gave similar results.

[0141]

Treatment of existing biofilm

[0142] For treatment of MRSA USA 400 biofilm, the same 96-well plate assay procedure was used as described in the biofilm inhibition section above. However, the MRSA USA 400 inoculum (10 8 CFU / mL) was grown overnight at 37°C in the absence of BM3103 and without shaking. After 24 hours of biofilm formation, BM3103 was serially diluted from 1mM to 0.002mM across 96-well plates. The plate was incubated overnight at 37°C again without shaking. After a total of 48 hours, images of each concentration were taken, and representative images are shown in Figures 14A to 14D. At 0.125mM (Figure 14A), 0.0625mM (Figure 14B) or higher concentrations, BM3103 significantly reduced the biofilm of MRSA USA 400. At 0.008mM (Figure 14C), the biofilm was still slightly reduced, but at concentrations below 0.002mM or in untreated controls (Figure 14D), no reduction in biofilm was observed.

[0143] Cell Death in Existing Biofilms

[0144] To treat MRSA USA 400 biofilms, the same 96-well plate assay was used as described above in the biofilm inhibition section. However, the MRSA USA 400 inoculum (10 8 CFU / mL) were grown overnight at 37°C in the absence of BM3103 and without shaking. 24 hours after biofilm formation, BM3103 was serially diluted from 1 mM to 0.002 mM across a 96-well plate. The plate was incubated again at 37°C overnight without shaking.

[0145] To confirm the induced cell death of MRSA USA 400 bacteria in existing biofilms with BM3103, both untreated cells and cells treated with BM3103 were stained with a combination of DAPI (nuclear dye) and propidium iodide (PI, live-dead) as described in Example 15. 8 CFU / mL were plated in well plates with or without combined BM3103 treatment. Plates were placed at 37°C for 24 hours. Bacterial cells were co-stained with DAPI and PI at 50μg / mL and 500μg / mL, respectively, at room temperature for 15 minutes. The wells were washed with warm phosphate-buffered saline (PBS) and kept in PBS throughout the imaging acquisition process. Images were captured using the same exposure limit for each filter. In this experiment, untreated cells showed strong DAPI staining and only light and sparse staining with PI, indicating that the bacteria within the biofilm were alive ( Fig.15 After 24 h of treatment with BM3103, PI staining increased in a dose-dependent manner from 0.008 mM to 0.125 mM, indicating that most bacteria within the biofilm were dead or dying ( Fig.15 ). Similar results were obtained at concentrations higher than 0.125 mM.

[0146] The foregoing description of various aspects and embodiments of the present invention has been given for the purpose of illustration and description. It is not intended to be exhaustive of all embodiments or to limit the invention to the particular aspects disclosed. Obvious modifications or variations are possible in light of the above teachings, and when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled, such modifications and variations may well fall within the scope of the invention as determined by the appended claims.

Claims

1. Use of substituted tolan compounds of structure (I) and salts thereof in the preparation of preparations for inhibiting the growth of suspected pathogenic microorganisms on physical or biological surfaces or killing suspected pathogenic microorganisms on physical or biological surfaces: Where R 1 and R 2 is an independent substituent on any available position of the benzene ring, m and n are independently 1, 2 or 3, representing the number of substituents on the benzene ring respectively; and Each R 1 , R 2 Independently selected from: hydroxyl; mercapto; -(C1-C6)alkoxy; -(C1-C6)RH, wherein R is O or S; or (halogenated) p (C1-C6)alkyl-, wherein p is 1, 2 or 3; However, the prerequisite is that R 1 or R 2 At least one of them is a methoxy group, The suspected pathogenic microorganisms are: (i) a bacterial pathogen selected from the group consisting of methicillinresistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli; or (ii) Fungi of the genus Candida.

2. The method of claim 1, wherein R 1 and R 2 At least one of them is a hydroxyl group.

3. The method of claim 1, wherein R 1 is -(C1-C6)alkoxy, and m is 1 or 2.

4. The method of claim 3, wherein R 2 is a hydroxyl group, and n is 1, 2, or 3.

5. The method of claim 1, wherein R 2 is a hydroxyl group, and n is 1, 2, or 3.

6. The use of claim 1, wherein the substituted tolan is selected from the group consisting of 4,4'-dihydroxy-3-methoxytolan, 4-hydroxy-4'-methoxytolan, 2,4,4'-trimethoxytolan and 3,5,3',5'-tetramethoxytolan.

7. The use of claim 1, wherein the substituted tolan compound is present in an amount of about 0.01% to about 30% based on the total weight of the formulation.

8. The use according to claim 1, wherein the pH of the formulation is about 4.1 to about 8.5, and wherein the formulation further comprises a cleaning agent and a second antimicrobial agent.

9. The use according to claim 8, wherein the cleaning agent does not contain phenol.

10. The method of claim 8, wherein the second antimicrobial agent is selected from the group consisting of phenol, dichlorophene, hexachlorophene, aldehydes, alcohols, antimicrobial carboxylic acids, organometallic compounds, iodine compounds, quaternary ammonium compounds, sulfonium and phosphonium compounds, mercapto compounds and their alkali metal, alkaline earth metal and heavy metal salts, urea, tribromosalicylicylanilide, 2-bromo-2-nitro-1,3-dihydroxypropane, dichlorobenzoxazolone, chlorhexidine, isothiazolone, benzisothiazolone and any combination of two or more of these compounds.

11. The use of claim 1, wherein the surface is a physical surface selected from the group consisting of surgical equipment, surgical instruments and table surfaces, which physical surface must be sterilized and maintained sterile throughout the surgical procedure.

12. The use of claim 1, wherein the surface is a physical surface selected from the group consisting of a food preparation surface selected from the group consisting of: a cutting board, a countertop, and a cooktop.

13. The use of claim 1, wherein the surface is a biological surface at a site where the skin does not remain fully intact, the site being selected from the group consisting of: an intravenous line or port, an arterial line or port, a PICC line, a catheter, a drain, and an incision site.

14. The use of claim 1, wherein the surface is a human biological surface selected from the group consisting of skin, hair, eyes, mucous membranes, and internal or external orifices.

15. The use according to claim 14, wherein the skin is the scalp.

16. The use of claim 1, wherein the fungus is Candida albicans.

17. The use according to claim 1, wherein the preparation further comprises an antibiotic, or Wherein said physical or biological surface is further in contact with said antibiotic.

18. The method of claim 17, wherein the antibiotic comprises: Aminoglycoside, ansamycin, carbacephem, FLAGYL (metronidazole), carbapenem, cephalosporin, glycopeptide, macrolide, monobactam, penicillin, peptide, polymyxin, quinolone, sulfonamide, or tetracycline.

19. The use according to claim 18, wherein the aminoglycoside is neomycin sulfate.

20. The method of claim 17, wherein the antibiotic is selected from the group consisting of: clindamycin, tigecycline, vancomycin, ciprofloxacin, ofloxacin, sulfamethoxazole, trimethoprim / sulfamethoxazole, amoxicillin, penicillin V, penicillin G, procaine penicillin, benzathine penicillin, carbenicillin, mezlocillin, ampicillin, piperacillin, arsphenamine, chloramphenicol, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platelet mycin, pyrazinamide, quinupristin / dalfopristin, rifampicin, thiampicin, Timidazole, dapsone, clofazimine, bacampicillin, ticarcillin, piperacillin / tazobactam, aztreonam, cefotetan, Loracarb, MEFOXIN, MERREM, levofloxacin, lomefloxacin, Primaxim, cycloserine, kanamycin, dicloxacillin, demeclocycline, minocycline, doxycycline, oxytetracycline, tobramycin, gentamicin, neomycin, amikacin, Craramyein, Nebcin, erythromycin / sulfisoxazole, netilmicin, streptomycin, ceftriaxone, cefuroxime, cefazolin, ceftibuten, cefazolin, cefuroxime, cefaclor, cefoperazone, cefprozil, cefadroxil monohydrate, ceftazidime, cephalexin, cefmandole sodium, cefepime, cefonicid, sulfadiazine, norfloxacin, enoxacin, cefdinir, seromycin, ceftriaxone, cefixime, craramyein mycobacterium, dirithromycin, methenamine, ethionamide, trovafloxacin, sparfloxacin, interferon-alpha, indinavir, ganciclovir, foscarnet, lamivudine, famciclovir, rimantadine, zalcitabine, interferon-beta, saquinavir, ritonavir, ribavirin, erythromycin, troleandomycin, azithromycin, colistin, amphotericin B, flucytosine, fluconazole, griseofulvin, grepafloxacin, terbinafine, ketoconazole, clotrimazole, delavirdine, zidovudine, amantadine, palivizumab, valacyclovir, didanosine, nelfinavir, nevirapine, cidofovir, pyrimethamine, atovaquone, stavudine, lamivudine, acyclovir, mionazole, nystatin, itraconazole, chloroquine, mefloquine, hydroxychloroquine , Capreomycin, Permethrin, Crotamiton, Fluorouracil, Rifabutin, Aminosalicylic Acid, Rifapentine, Coenzoyl Peroxide, Chlorhexidine Gluconate, Sodium Oxyclosan, Benzoyl Peroxide, Rifampicin, Rifampicin / isoniazid, Rifampicin / isoniazid / pyrazinamide, Nalidixic Acid, Atropine, Oxytetracycline / sulfamethoxazole / phenazopyridine, Chloramphenicol, Neomycin / polymyxin, Trimethoprim / Polythyxin, Tobramycin / dexamethasone, Adenosine, Sulfacetamide, Povidoneodine, Bacitracin, Sulconazole, Tetrachlorosalicylanilide, Metromdazole, Ciclopirox, Clotrimazole / betamethasone, Butenafine, Naftifine, Oxiconazole, Selenium, Econazole,Penciclovir or a pharmaceutically acceptable salt thereof.

21. The use according to claim 20, wherein the griseofulvin is ultra-microsized griseofulvin.

22. Use of a substituted tolan compound having structure (I) and a salt thereof in the preparation of a preparation for inhibiting or destroying biofilm formation of one or more microorganisms: Where R 1 and R 2 is an independent substituent on any available position of the benzene ring, m and n are independently 1, 2 or 3, representing the number of substituents on the benzene ring respectively; and Each R 1 , R 2 Independently selected from: hydroxyl; mercapto; -(C1-C6)alkoxy; -(C1-C6)RH, wherein R is O or S; or (halogenated) p (C1-C6)alkyl-, wherein p is 1, 2 or 3; However, the prerequisite is that R 1 or R 2 At least one of them is a methoxy group, The microorganism is: (i) a bacterial pathogen selected from the group consisting of methicillinresistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli; or (ii) Fungi of the genus Candida.

23. The use of claim 22, wherein the tolan compound is present in the formulation at a concentration of about 0.008 mM to about 1 mM.

24. The use of claim 22, wherein the tolan compound is present in the formulation at a concentration of about 0.625 mM.

25. The use of claim 22, wherein the tolan compound is present in the formulation in an amount of about 0.0001% to about 30% based on the total weight of the formulation.

26. The use of claim 22, wherein the tolan compound is present in the formulation in an amount of about 0.01% to about 25% based on the total weight of the formulation.

27. The use of claim 22, wherein the tolan compound is present in the formulation in an amount of about 0.1% to about 30% based on the total weight of the formulation.

28. The use of claim 22, wherein the tolan compound comprises 4-hydroxy-4'-methoxytolan.

29. The use according to claim 22, wherein the preparation further comprises an antibiotic, or The one or more microorganisms are further contacted with the antibiotic.

30. The method of claim 29, wherein the antibiotic comprises: Aminoglycoside, ansamycin, carbacephem, FLAGYL (metronidazole), carbapenem, cephalosporin, glycopeptide, macrolide, monobactam, penicillin, peptide, polymyxin, quinolone, sulfonamide, or tetracycline.

31. The use of claim 30, wherein the aminoglycoside is neomycin sulfate. 32.The method of claim 29, wherein the antibiotic is selected from the group consisting of: clindamycin, tigecycline, vancomycin, ciprofloxacin, ofloxacin, sulfamethoxazole, trimethoprim / sulfamethoxazole, amoxicillin, penicillin V, penicillin G, procaine penicillin, benzathine penicillin, carbenicillin, mezlocillin, ampicillin, piperacillin, arsphenamine, chloramphenicol, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, plate mycin, pyrazinamide, quinupristin / dalfopristin, rifampicin, thiampicin, Timidazole, dapsone, clofazimine, bacampicillin, ticarcillin, piperacillin / tazobactam, aztreonam, cefotetan, loracarbef, ME FOXIN, MERREM, Levofloxacin, Lomefloxacin, Primaxim, Cycloserine, Kanamycin, Dicloxacillin, Demeclocycline, Minocycline, Doxycycline, Oxytetracycline, Tobramycin, Gentamycin, Neomycin, Amikacin, Craramyein, Nebcin, Erythromycin / Sulfisoxazole, Netilmicin, Streptomycin, Cefotaxime, Cefuroxime, Cefazolin, Ceftibuten, Cefazolin, Cefazolin, Ceftriaxone, Cefoperazone, Cefprozil, Cefadroxil Monohydrate, Ceftazidime, Cephalexin, Cefmandolol Sodium, Cefepime, Cefonicid, Sulfadiazine, Norfloxacin, Enoxacin, Cefdinir, Seromycin, Ceftriaxone, Cefixime, Clarithromycin, Dirithromycin, Hexamethylenetetramine, ethionamide, trovafloxacin, sparfloxacin, interferon-alpha, foscarnet, lamivudine, famciclovir, rimantadine, zalcitabine, interferon-beta, indinavir, ganciclovir, saquinavir, ritonavir, ribavirin, erythromycin, troleandomycin, azithromycin, colistin, amphotericin B, flucytosine, fluconazole, griseofulvin, grepafloxacin, terbinafine, ketoconazole, clotrimazole, delavirdine, zidovudine, amantadine, palivizumab, valacyclovir, didanosine, nelfinavir, nevirapine, cidofovir, pyrimethamine, atovaquone, stavudine, lamivudine, acyclovir, mionazole, itraconazole, chloroquine, mefloquine, hydroxychloroquine, capreomycin, permethrin, crotamiton , Fluorouracil, Rifabutin, Aminosalicylic Acid, Rifapentine, Coenzoyl Peroxide, Chlorhexidine Gluconate, Sodium Oxyclosan, Benzoyl Peroxide, Rifampicin, Rifampicin / Isoniazid, Rifampicin / Isoniazid / Pyrazinamide, Nalidixic Acid, Atropine, Oxytetracycline / Sulfamethoxazole / Phenazopyridine, Neomycin / Polymycin, Trimethoprim / Polythyxin, Tobramycin / Dexamethasone, Vidarabine, Sulfacetamide, Povidoneodine, Nystatin, Bacitracin, Sulconazole, Tetrachlorosalicylanilide, Metromdazole, Ciclopirox, Clotrimazole / Betamethasone, Butenafine, Naftifine, Oxiconazole, Selenium, Econazole, Penciclovir or a pharmaceutically acceptable salt thereof. .

33. The use of claim 32, wherein the griseofulvin is ultra-microsized griseofulvin.

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