Biofilm destruction

Abstract

The present invention relates to a method for dispersing a biofilm containing Gram-negative bacteria, wherein the method comprises the step of exposing said biofilm to an epoxyglienone compound or a salt thereof. For example, a method for treating an infection comprising the step of locally administering an epoxyglienone compound into or on an established biofilm containing Gram-negative bacteria, either locally or by injection, to destroy the structure of said biofilm, and a method for preventing the formation of a biofilm containing Gram-negative bacteria or dispersing a biofilm containing a Gram-negative biofilm formed on a medical device are also described.

Description

Technology Field

[0001] The present invention relates to a method for dispersing a biofilm containing Gram-negative bacteria, wherein the method comprises the step of exposing said biofilm to an epoxytiglienone compound or a salt thereof. For example, a method for treating an infection comprising the step of locally administering an epoxytiglienone compound into or on an established biofilm containing Gram-negative bacteria, either locally or by injection, to destroy the structure of said biofilm, and a method for preventing the formation of a biofilm containing Gram-negative bacteria or dispersing a biofilm containing a Gram-negative biofilm formed on a medical device are also described. Background Technology

[0002] Gram-negative bacteria are associated with various infections of the respiratory, genitourinary, gastrointestinal, and circulatory systems. They are also a major factor in the persistence of chronic wounds, osteomyelitis, and infections at surgical sites, medical devices, and post-transplantation. Severe infections caused by Gram-negative bacteria can result in significant morbidity and mortality, particularly in immunocompromised patients. The most important clinical Gram-negative pathogen is Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Acinetobacter Baumannii( Acinetobacter baumannii ) and members of the Enterobacteriaceae, especially Escherichia coli ( Escherichia coli ) and Klebsiella pneumoniae( Klebsiella pneumoniae It is. Other examples of medically relevant Gram-negative bacteria are Neisseria (which causes gonorrhea and meningitis). Neisseria ) species, Haemophilus influenzae( Hemophilus influenzae), Legionella pneumophila( Legionella pneumophila ), Yersinia Festis( Yersinia pestis ), Proteus mirabilis( Proteus mirabilis ) and Salmonella( Salmonella Includes ) species.

[0003] The treatment of Gram-negative bacterial infections involves (a) the emergence and spread of multidrug resistance to most or all conventional antibiotics among the many microorganisms mentioned above (Ho et al. 2010; Doi et al. (b) and the ability to form difficult-to-handle, aggregated communities called biofilms have become increasingly problematic due to both (Cepas 2017) and (b) the ability to form such communities (Cepas 2017) et al. 2019). The management of difficult-to-manage Gram-negative biofilms presents complex and challenging clinical problems, particularly those with limited treatment options. Current strategies in many clinical situations involve removing biofilms (if feasible) by aggressive physical methods (e.g., debridement) coupled with high-dose and often prolonged antibacterial chemotherapy as the standard of treatment (Hoiby). et al. 2015). However, complete eradication of biofilms using the above method is often unsuccessful, leading to recurrence and selection for increased levels of bacterial resistance. The above vicious chronic cycle further weakens the efficacy of antibiotics and, in many cases, facilitates the more widespread use of 'last resort' antibiotics, which have significant side effects.

[0004] There is an urgent need for new agents to treat Gram-negative biofilm infections. Traditional approaches to treating bacterial infections are antibiotic-based and have focused on the direct targeting, inhibition, or killing of causative pathogens (using the concepts of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC); however, the complex nature of biofilms implies the need for new therapeutic strategies.

[0005] A biofilm is a bacterial community embedded within a protective self-producing matrix of extracellular polymeric material (EPS) formed from polysaccharides, proteins, lipids, and nucleic acids (RNA and extracellular DNA). When living in said biofilm, the inhabiting bacteria have significantly greater resistance to antibiotics compared to their planktonic free-living counterparts (Hoiby et al. (2010), largely protected from the host's immune system. Much of the aforementioned resistance of biofilms can be attributed to EPS, which provides a formidable physical barrier against the penetration of antibiotics and cellular effectors of the immune system (Gunn et al. 2016). The structure of the biofilm as a dense polymeric 'mesh' of EPS and bacteria also means that it is very resistant to removal from strongly attached tissue and material surfaces.

[0006] In contrast to current treatment standards that rely critically on antibiotics, various 'non-antibiotic' strategies for treating Gram-negative biofilms have been proposed and are currently under investigation. These include interference with signaling networks in biofilms, targeting of biofilm adhesion, and disruption of the biofilm's EPS matrix (Koo et al. 2017). Unlike conventional antibiotics, the above strategy also targets bacterial 'virulence factors' rather than bacterial growth, and thus induces low selective pressure, which has the potential benefit of minimizing the development of resistance.

[0007] Among the aforementioned 'non-antibiotic' strategies, disrupting the integrity of the EPS matrix of established biofilms is particularly attractive. By making the EPS matrix more permeable, deaggregating bacteria, and interfering with pathogen signaling networks, this strategy theoretically makes individual bacteria more accessible to the immune system or / or enhances the action of antibiotics (Gunn et al. 2016; Fleming & Rumbaugh 2017). By utilizing the above approach, various therapies involving monoclonal antibodies and polymers targeting enzymes (proteases, DNAases, glycoside hydrolases), peptides, and specific components of the matrix (e.g., exopolysaccharides and eDNA) are under investigation (Koo et al. 2017).

[0008] Epoxyglienone is a small molecule with diverse biological activities. It is a potent anti-tumor compound, possesses anti-parasitic properties, and stimulates immune and other cellular responses that promote the healing of acute and chronic wounds. Previously, epoxyglienone was reported to have direct antibiotic activity against various Gram-positive bacteria (WO2007 / 070985 and WO2014 / 169356). However, in a recent study to more specifically evaluate the antibiotic properties of epoxyglienone compounds against pathogenic strains of three Gram-negative pathogens (E. coli, P. aruginosa, and A. baumannii), no antibiotic effect was found, and it was impossible to establish the minimum inhibitory concentration (MIC) in the conventional planktonic culture system used for routine antibiotic screening.

[0009] The present invention is based on a subsequent unexpected discovery that epoxyglienone has a potent 'non-antibiotic' effect on established biofilms of various Gram-negative bacteria, despite having no direct antibiotic activity against at least partially planktonic Gram-negative bacteria. The problem to be solved

[0010] In a first aspect of the present invention, a method for dispersing a biofilm comprising Gram-negative bacteria is provided, said method comprising the step of exposing said biofilm to an epoxyglienone compound of formula (I) or a salt thereof:

[0011]

[0012] As mentioned above,

[0013] R1 is hydrogen and C 1-6 Selected from alkyls;

[0014] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0015] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0016] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0017] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0018] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0019] R8 is hydrogen and C 1-6 Selected from alkyls.

[0020] In another aspect of the present invention, a method for treating a bacterial infection comprising a biofilm containing Gram-negative bacteria is provided, the method comprising the step of topically administering an epoxyglienone compound of formula (I), or a pharmaceutically acceptable salt thereof, to said bacterial infection:

[0021]

[0022] As mentioned above,

[0023] R1 is hydrogen and C 1-6 Selected from alkyls;

[0024] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0025] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0026] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0027] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0028] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0029] R8 is hydrogen and C 1-6 Selected from alkyls.

[0030] In a further aspect of the present invention, a method is provided to prevent the formation of a biofilm containing Gram-negative bacteria on a medical device or to disperse a biofilm containing Gram-negative bacteria, said method comprising an epoxyglienone compound of formula (I). , or includes the step of applying a pharmaceutically acceptable salt thereof to the medical device, wherein the compound of formula (I) is as follows:

[0031]

[0032] As mentioned above,

[0033] R1 is hydrogen and C 1-6 Selected from alkyls;

[0034] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0035] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0036] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0037] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0038] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6Selected from alkynylaryls;

[0039] R8 is hydrogen and C 1-6 Selected from alkyls. Brief explanation of the drawing

[0040] Figure 1 provides confocal laser scanning microscope (CLSM) images showing biofilm disruption of E. coli IR57 after treatment with compounds 1, 4, and 6 compared to comparative compound CC-1, untreated, and ethanol equivalent (blank) controls. The upper panel of each image group is an overhead view, and the smaller lower panel is a cross-sectional view. Figure 2a shows the biomass volume or bio-volume (μm) quantified and confirmed by COMSTAT image analysis after treating E. coli IR57 biofilms with compounds 1, 4, and 6 compared to comparative compound CC-1, untreated, and ethanol equivalent (blank) controls. 3 / ㎛ 2 It provides ). The reduction in bio-volume is evident in biofilms treated with the compounds and was significant upon treatment with compound 1 (p<0.05). In Figure 2b, there was no significant difference in the dead / living bacteria ratio between any compound and control treatments, which demonstrates that the destruction of E. coli IR57 biofilm density and bio-volume in the experiment was not directly associated with antibiotic activity. Figure 3 provides a graph of the mean change rate (MSD) of 200 nm FluoSpheres® as a function of time (in seconds) within the E. coli IR57 biofilm structure. The disruption and alteration of the E. coli IR57 biofilm structure are illustrated by the substantially higher mean change rate of FluoSphere® particles after treatment with compounds 1, 4, and 6 compared to untreated and ethanol equivalent (blank) controls. Figure 4 provides a graph of increased biofilm creep compliance (demonstrating) reduced resistance to mechanical deformation of the biofilm medium derived from MSD versus lag time as shown in Figure 3 after treating E. coli IR57 biofilms with compounds 1, 4, and 6 compared to untreated and ethanol equivalent (blank) controls. Figure 5a provides CLSM images comparing compounds 1, 4, 6 and comparative compound CC-1 with untreated and ethanol equivalent (blank) controls when applied to the biofilm of A. baumannii 7789. In each image, the top panel is an overhead view, and the small bottom panel is a cross-sectional view. Figure 5b is a CLSM image comparing compounds 1, 4, 6 and comparative compound CC-1 with untreated and ethanol equivalent (blank) controls when applied to the biofilm of P. aruginosa PAO1. In each image, the top panel is an overhead view, and the small bottom panel is a cross-sectional view. Figure 6a shows the biomass volume or bio-volume (μm) of A. Baumannii 7789 biofilms quantified and confirmed by COMSTAT image analysis after treatment with compounds 1, 4, 6 and comparative compound CC-1, compared to untreated and ethanol equivalent (blank) controls. 3 / ㎛ 2 It provides ). A significant reduction in bio-volume was evident in biofilms treated with Compounds 1, 4, and Comparative Compound CC-1 (p<0.05). Figure 6b shows the biofilm biomass volume or bio-volume (μm) quantified and confirmed by COMSTAT image analysis after treating P. aruginosae PAO1 biofilms with Compounds 1, 4, 6, and Comparative Compound CC-1, compared to untreated and ethanol equivalent (blank) controls. 3 / ㎛ 2Compound 4 alone had a significant effect on biofilm bio-volume compared to other treatments (P<0.05). In Figures 6c and 6d, there were no significant differences in the dead / living bacteria ratio quantified and confirmed by COMSTAT image analysis after treating A. baumannii 7789 and P. aruginosa PAO1 biofilms with Compounds 1, 4, 6 and Comparative Compound CC-1 compared to untreated and ethanol equivalent (blank) controls. Figure 7 shows compounds 1, 4, and 6 compared with the non-control treatment group and the positive control of 70% isopropanol, E. coli IR57, P. aeruginosa PAO1, and S. aureus ( aureus Data on cell membrane permeability when applied to planktonic cells of )(1004A; MRSA) are provided. Only Gram-negative strains (E. coli IR57 and P. aruginosa PAO1) showed a significant increase in cell permeability when treated with compounds 1 and 4 at concentrations ≥512 μg / ml, unlike Gram-positive strains (MRSA 1004A), which demonstrated significant cell permeability at concentrations as low as 32 μg / ml. Figure 8 provides representative images showing the induction of NETosis and necrosis in neutrophils treated with six concentrations of Compound 4 at two times, 3 and 6 hours after treatment. NETosis / necrosis was first observed at 3 hours after treatment at the two highest concentrations (50 and 500 μM) and at 6 hours after treatment at the four lowest concentrations. At 500 μM, intracellular chromatin condensed, suggesting that necrosis had occurred. At 50 μM, the chromatin was more diffuse, suggesting that NETosis had occurred. Figure 9 provides a graph showing the mean values ​​and standard deviations for the in vitro release of human defense peptide LL-37 from neutrophils at 3 hours after the application of compound 4 at four therapeutically relevant concentrations. The release of LL-37 from neutrophils increased in a concentration-dependent manner. Specific details for implementing the invention

[0041] definition

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Any method and material similar or equivalent to that described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0043] The articles "han" and "hana" are used in this specification to indicate one or more (i.e., at least one) of the grammatical objects of said articles. For example, "han element" means one element or one or more elements.

[0044] As used herein, the term “about” indicates a content, level, value, dimension, size, or amount that varies by 25%, 20%, 15%, or 10% of the reference content, level, value, dimension, size, or amount.

[0045] Throughout this specification, unless the context otherwise requires, the words “include,” “include,” and “include” will be understood to mean that a mentioned step or element or group of steps or elements is included, but any other step or element or group of steps or elements is excluded.

[0046] The term "alkyl" refers to an optionally substituted linear and branched hydrocarbon group having 1 to 8 carbon atoms. Where appropriate, the alkyl group may have a -C1-C6 alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement, for example. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, 1-methylpropyl, 2,2-dimethylethyl, pentyl, 2-methylbutyl, 3-methylbutyl, 1,2-dimethylpropyl, 2,3-dimethylpropyl, 3,3-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl and 3-ethylbutyl, heptyl, 1-methylhexyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 1-ethylpentyl, 2-ethylpentyl, 3-ethylpentyl, 1,1-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,5-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 1,1-dimethylpentyl, 2,2-dimethylpentyl, octyl, 1-methylheptyl, 2-methylheptyl, 3-methylheptyl, 4-methylheptyl, 5-methylheptyl, 1-ethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 1,1-dimethylhexyl, 1,2-dimethylhexyl, 1,3-dimethylhexyl, 1,4-dimethylhexyl, 1,5-dimethylhexyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 1,1-dimethylhexyl, 2,2-dimethylhexyl, etc. Includes.

[0047] The term “alkenyl” refers to an optionally substituted unsaturated linear or branched hydrocarbon having 2 to 8 carbon atoms and at least one double bond. Where appropriate, the alkenyl group may have a C2-C6 alkenyl comprising an alkenyl group having a specific number of carbon atoms, e.g., 2, 3, 4, 5, or 6 carbon atoms, arranged in a linear or branched configuration. Non-limiting examples of alkenyl groups include ethenyl, propenyl, 1-methylethenyl, butenyl, 1-methylprop-1-enyl, 1-methylprop-1-enyl, 1-ethylethenyl, fenthenyl, 1-methylbut-1-enyl, 2-methylbut-1-enyl, 2-methylbut-2-enyl, 1,2-dimethylprop-1-enyl, 1,2-dimethylprop-2-enyl, hexenyl, 1-methylpent-1-enyl, 2-methylpent-1-enyl, 3-methylpent-1-enyl, 1-ethylbut-1-enyl, 2-ethylbut-1-enyl, 1-methylpent-2-enyl, 2-methylpent-2-enyl, 3-methylpent-2-enyl, 4-methylpent-2-enyl, 1-ethylbut-2-enyl, 2-ethylbut-2-enyl, 1,2-dimethylbut-2-enyl, 1,3-dimethylbut-2-enyl, 2,3-dimethylbut-2-enyl, 1-methylpent-3-enyl, 2-methylpent-3-enyl, 3-methylpent-3-enyl, 4-methylpent-3-enyl, 1-ethylbut-3-enyl, 2-ethylbut-3-enyl, 3-ethylbut-3-enyl, 1,2-dimethylbut-3-enyl, 1,3-dimethylbut-3-enyl, 1,1-dimethylbut-3-enyl, 2,3-dimethylbut-3-enyl, 2,2-dimethylbut-3-enyl, hex-1,3-diene, 1-heptenyl, 2-Heptenyl, 3-Heptenyl, 4-Heptenyl, 5-Heptenyl, 6-Heptenyl, 2,4-Heptadienyl, 2,6-Heptadienyl, 2,4,6-Heptatrienyl, 1-Methylhexa-1-enyl, 2-Methylhexa-1-enyl, 3-Methylhexa-1-enyl, 4-Methylhexa-1-enyl, 5-Methylhexa-1-enyl, 1-Methylhexa-2-enyl, 2-Methylhexa-2-enyl, 3-Methylhexa-2-enyl, 4-Methylhexa-2-enyl, 5-Methylhexa-2-enyl, 1-Methylhexa-3-enyl, 2-Methylhexa-3-enyl, 3-Methylhexa-3-enyl, 4-Methylhexa-3-enyl, 5-methylhexa-3-enyl,1-methylhexa-4-enyl, 2-methylhexa-4-enyl, 3-methylhexa-4-enyl, 4-methylhexa-4-enyl, 5-methylhexa-4-enyl, 1-methylhexa-5-enyl, 2-methylhexa-5-enyl, 3-methylhexa-5-enyl, 4-methylhexa-5-enyl, 5-methylhexa-5-enyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 2,4-octadienyl, 2,6-octadienyl, 2,4,6-octatrienyl, 1-methylhepta-1-enyl, 2-methylhepta-1-enyl, 3-methylhepta-1-enyl, 4-methylhepta-1-enyl, 5-methylhepta-1-enyl, 1-methylhepta-2-enyl, 2-methylhepta-2-enyl, 3-methylhepta-2-enyl, 4-methylhepta-2-enyl, 5-methylhepta-2-enyl, 1-methylhepta-3-enyl, 2-methylhepta-3-enyl, 3-methylhepta-3-enyl, 4-methylhepta-3-enyl, 5-methylhepta-3-enyl, 1-methylhepta-4-enyl, 2-methylhepta-4-enyl, 3-methylhepta-4-enyl, 4-methylhepta-4-enyl, 5-methylhepta-4-enyl, 1-methylhepta-5-enyl, 2-methylhepta-5-enyl, 3-methylhepta-5-enyl, 4-methylhepta-5-enyl, Includes 5-methylhepta-5-enyl, etc.

[0048] The term “alkynyl” refers to an optionally substituted unsaturated linear or branched hydrocarbon having 2 to 8 carbon atoms and at least one triple bond. Where appropriate, the alkynyl group may have a C2-C6 alkynyl comprising an alkynyl group having a specific number of carbon atoms, for example, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement. Non-limiting examples include ethinyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methylprop-2-inyl, 1-fentinyl, 2-fentinyl, 3-fentinyl, 4-fentinyl, 1-methylbut-2-inyl, 1,1-dimethylprop-2-inyl, 3-methylbut-1-inyl, 1-hexinyl, 2-hexinyl, 3-hexinyl, 4-hexinyl, 5-hexinyl, 3-methylpent-1-inyl, 4-methylpent-1-inyl, 1-methylpent-2-inyl, 4-methylpent-2-inyl, 3,3-dimethylbut-1-inyl, 1,1-dimethylbut-2-inyl, 1,2-dimethylbut-3-enyl, 1,1-dimethylbut-3-enyl, 2,2-dimethylbut-3-enyl, 1-heptinyl, 2-heptinyl, 3-heptinyl, 4-heptinyl, 5-heptinyl, 6-heptinyl, 3-methylhex-1-enyl, 4-methylhex-1-enyl, 1-methylhex-2-enyl, 4-methylhex-2-enyl, 3,3-dimethylpent-1-enyl, 1,1-dimethylpent-2-enyl, 1,2-dimethylpent-3-enyl, 1,1-dimethylpent-3-enyl, 2,2-dimethylpent-3-enyl, 1-octinyl, 2-octinyl, 3-octinyl, 4-octinyl, 5-octinyl, Includes 6-octinyl, 7-octinyl, 3-methylhept-1-inyl, 4-methylhept-1-inyl, 1-methylhept-2-inyl, 4-methylhept-2-inyl, 3,3-dimethylhex-1-inyl, 1,1-dimethylhex-2-inyl, 1,2-dimethylhex-3-inyl, 1,1-dimethylhex-3-inyl, 2,2-dimethylhex-3-inyl, etc.

[0049] The terms "cycloalkyl" and "carbocyclic" refer to optionally substituted saturated or unsaturated mono-cyclic hydrocarbon groups. Where appropriate, the cycloalkyl group may have a specific number of carbon atoms, for example, C3-C6 cycloalkyl is a carbocyclic group having 3, 4, 5, or 6 carbon atoms. Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.

[0050] "Aryl" refers to C6-C atoms, each having up to 7 atoms in its ring. 14 It refers to a membered monocyclic, bicyclic, or tricyclic carbocyclic ring system, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and biphenyl. The aryl may include 1 to 3 benzene rings. If two or more aromatic rings are present, the rings may be fused together so that adjacent rings share a common bond.

[0051] Each aryl, alkenyl, alkynyl, cycloalkyl, or aryl is C as an individual entity or part of a larger entity 1-6 Alkyl, C 2-6 Alkenyl, C 3-6 Cycloalkyl, oxo(=O), -OH, -SH, C 1-6 Alkyl O-, C 2-6 Alkenyl O-, C 3-6 Cycloalkyl O-, C 1-6 Alkyl S-, C 2-6 Alkenyl S-, C 3-6 Cycloalkyl S-, -CO2H, -CO2C 1-6 Alkyl, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 Alkyl)2, -NH(phenyl), -N(phenyl)2, -CN, -NO2, -halogen, CF3, -OCF3, -SCF3, -CHF2, -OCHF2, -SCHF2, -phenyl, -C 1-6Alkylphenyl, -Ophenyl, -C(O)phenyl, -C(O)C 1-6 It may be optionally substituted with one or more optional substituents selected from the group consisting of alkyls. In particular, examples of suitable substituents for the cycloalkyl and aryl groups in R6 and R7 are methyl, ethyl, propyl, isopropyl, butyl, sec- Butyl, tert -butyl, vinyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylthio, ethylthio, propylthio, isopropylthio, butylthio, hydroxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, fluoro, chloro, bromo, iodo, cyano, nitro, -CO2H, -CO2CH3, -C(O)CH3, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl, difluoromethoxy, difluoromethylthio, amino, methylamino, dimethylamino, phenyl, phenoxy, phenylcarbonyl, benzyl, and acetyl are included without limitation.

[0052] The above-mentioned epoxyglienone compound may be in the form of a pharmaceutically acceptable salt. However, it will be recognized that a non-pharmaceutically acceptable salt is also within the scope of the present invention, as it may be useful as an intermediate in the preparation of a pharmaceutically acceptable salt, useful during storage or transport, or useful in a non-pharmaceutical setting. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carboxylic acid, boric acid, sulfamic acid, and hydrobromide, or salts of pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, maleic acid, citric acid, lactic acid, mucinous acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0053] Basic salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium.

[0054] Basic nitrogen-containing groups can be quaternized with preparations such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chloride, bromide, and iodide; dialkyl sulfates, e.g., dimethyl and diethyl sulfates; and others.

[0055] It will also be recognized that the above-mentioned epoxytiglienone compound has an asymmetric center and thus may exist in one or more stereoisomer forms. Accordingly, the present invention also relates to compounds in isomer forms substantially purified at one or more asymmetric centers, for example, to greater than about 90% ee, for example, greater than about 95% or 97% ee or greater than 99% ee, as well as mixtures thereof comprising racemic mixtures. Such isomers may be obtained by isolation from natural sources, for example by asymmetric synthesis using chiral intermediates, or by chiral resolution. The compounds of the present invention may exist as geometric isomers. The present invention also relates to compounds or mixtures thereof in substantially purified cis (Z) or trans (E) forms.

[0056] The compounds of the present invention may be obtained by isolation from a plant or part of a plant, by derivatization of the isolated compounds, or by derivatization of related compounds. The isolation and derivatization procedures may be found in WO 2007 / 070985 and WO 2014 / 169356.

[0057] The term "6,7-epoxythiglienone compound" refers to a compound having the following carbon cyclic structure:

[0058]

[0059] The above compound has a tricyclo[9.3.0.0]tetradecane system with a fused cyclopropane ring added to a 6-membered ring. The epoxide is fused to a 7-membered ring at the 6,7-position, and the 5-membered ring has a 1,2-en-3-one structure.

[0060] Method of the present invention

[0061] The present invention provides a method for dispersing a biofilm containing Gram-negative bacteria, the method comprising the step of exposing the biofilm to an epoxyglienone compound of formula (I) or a salt thereof:

[0062]

[0063] As mentioned above,

[0064] R1 is hydrogen and C 1-6 Selected from alkyls;

[0065] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0066] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0067] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0068] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0069] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0070] R8 is hydrogen and C 1-6 Selected from alkyls.

[0071] The biofilm containing the above Gram-negative bacteria may be in any situation suitable for treatment. The biofilm may be on an inanimate surface or on or within a biological system. In some embodiments, the biofilm is on an inanimate surface, for example, a bench in medical, laboratory, or food manufacturing, or a manufacturing surface such as a surgical table or floor, a laboratory bench, a kitchen bench or floor, or manufacturing equipment for pharmaceuticals, nutritional supplements, cosmetics, or personal care products. In some embodiments, the inanimate surface may be a medical device, for example, a medical device, a surgical instrument, a catheter, a prosthesis, and an implant. In some embodiments, the biofilm infects the subject, for example, a wound such as a surgical wound or a burn. In some embodiments, the biofilm infects the subject at the site where a medical device, for example, a catheter or an implant, was used.

[0072] In some embodiments, the biofilm comprises only Gram-negative bacteria. In some embodiments, the biofilm comprises only one species of Gram-negative bacteria. In other embodiments, the biofilm comprises one or more species of Gram-negative bacteria. In some embodiments, the biofilm comprises a population of Gram-negative and Gram-positive bacteria.

[0073] As used herein, the term "dispersing" means that the biofilm is degraded so that at least some of the bacteria are released from the matrix of the extracellular polymeric material (EPS) and are presumed to be in a planktonic state. In a planktonic state, the bacteria are accessible to the action of the immune system and / or antibiotics.

[0074] In some embodiments, the biofilm containing the Gram-negative bacteria is Pseudomonas species, Acinetobacter species, Aeromonas ( Aeromonas ) species, Bacteroides( Bacteroides ) species, Bordetella( Bordetella ) species, Borrelia( Borrelia) species, Burgholderia( Burkholderia ) species, Citrobacter( Citrobacter ) species, Compylobacter( Compylobacter ) species, Esserician species, Enterobacter( Enterobacter ) species, Flavobacterium( Flavobacterium ) species, Foucault bacterium( Fusobacterium ) species, Klebsiella species, Leptospira( Leptospira ) species, Neisseria species, Helicobacter( Helicobacter ) species, Haemophilus species, Legionella species, Moraxella( Moraxella ) bell, Yersinia Species, Oligella Oligella ) species, Pantoea( Pantoea ) species, Porphyromonas( Porphyromonas ) species, Prevotella( Prevotella ) species, Proteus species, Raultella( Raoutella ) species, Salmonella species, Serratia( Serratia ) species, Shigella( Shigella ) species, Sphingomonas( Sphingomonas ) species, Stenotopomonas ( Stenotophomonas ) species, Treponema ( Treponema ) species, Veilonella( Veillonella ) species and Vibrio ( Vibrio It comprises one or more Gram-negative bacteria selected from species, particularly Pseudomonas species, Acinetobacter species, Ecericia species, Klebsiella species, Neisseria species, Haemophilus species, Legionella species, Yersinia species, Proteus species and Salmonella species, particularly Pseudomonas species, Acinetobacter species, Ecericia species and Klebsiella species. In some embodiments, the Gram-negative bacteria are particularly good biofilm-forming bacteria, and Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonoriae ( gonorrhoeae ), Neisseria meningitidis( meningitidis ), Haemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica( enterocolitica ), Salmonella enterica( enterica), Salmonella bongori( bongori ), Proteus mirabillis, Enterobacter cloacae( cloacae ), Serratia Marcescens( marcescens ), Bacteroides fragilis( fragilis ), Foucault necroforum( necrophorum ), Burkholderia Sepacian( cepacian ) and Prevotella intermedia( intermedia ), in particular Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonoriae, Neisseria meningitidis, Haemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica and Salmonella bongori, in particular Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae are selected.

[0075] In another aspect of the present invention, the biofilm described above, dispersed by the method, is present on or within a bacterial infection of a target. Accordingly, the present invention comprises a method for treating a bacterial infection comprising a biofilm containing Gram-negative bacteria, wherein the method comprises an epoxyglienone compound of formula (I) in the bacterial infection, or includes the step of locally administering a pharmaceutically acceptable salt thereof:

[0076]

[0077] As mentioned above,

[0078] R1 is hydrogen and C 1-6 Selected from alkyls;

[0079] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0080] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0081] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0082] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0083] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0084] R8 is hydrogen and C 1-6 Selected from alkyls.

[0085] In some embodiments, the infection is an acute infection. In other embodiments, the infection is a chronic infection. In some embodiments, the infection is a postoperative infection or an infection at the site of insertion or use of a medical device, for example, the insertion site of a catheter such as an intravenous catheter or a urinary catheter, or an implant site such as a dental implant or a cochlear implant.

[0086] The term “topical administration” means that the compound of Formula (I) is applied directly to a biofilm. In some embodiments, the topical administration may be a local application such as using a lotion, cream, ointment, foam, suspension, liquid wash, or spray. In other embodiments, the topical administration may be achieved during surgery where the infected tissue is exposed or delivered by an injection guided by imaging technology, for example, endoscopic ultrasound or stereotactic imaging. In certain embodiments, the topical administration is to a biofilm containing an infection located in an area accessible from the outside of the body, for example, on the skin, over an external wound, inside the mouth or nose, inside the ear or external auditory canal, anus / rectum, vagina, urethra, or around the milk canal of livestock.

[0087] In some embodiments, the dispersion of the biofilm is accompanied by stimulation of the immune system, so that the immune system can resolve the infection without the need for antibiotic administration by stimulating, for example, the expression of chemokines / cytokines accompanying the recruitment of host-defense peptides or neutrophils / granulocytes in stromal and dermal cell types, and by inducing potent antimicrobial defense of resident and recruited innate immune cells, including the release of reactive oxygen species, extracellular networks, and broad-spectrum antimicrobial peptides.

[0088] In another embodiment, the dispersion of the biofilm is accompanied by the co-administration of an antibiotic. In some embodiments, the compound of formula (I) or its salt is administered in combination with an antibiotic to which Gram-negative bacteria are susceptible when in a planktonic state. Thus, when the biofilm is dispersed, the planktonic Gram-negative bacteria are exposed to the antibacterial activity of the antibiotic.

[0089] Suitable antibiotics for Gram-negative bacteria include third-generation cephalosporins such as ceftazidime and ceftiofur; fourth-generation cephalosporins such as cefepim; aminoglycosides such as streptomycin, neomycin, gentamicin, amikacin, tobramycin, and plasmomycin; monocyclic beta-lactams such as aztreonam; beta-lactam inhibitor combinations such as piperacillin / tazobactam, amoxicillin / clavulanic acid, cefepim / AAI101, aztreonam / avibactam, ceftaroline / avibactam, imipenem / relebactam, and meropenem / RPX7009; beta-lactams such as penicillin, cephalosporin, S649266, and BAL30072; and carbapenems such as imipenem, doripenem, ertapenem, and meropenem. Polymyxin-E antibiotics such as colistin; quinolones / fluoroquinolones such as ciprofloxacin, fleroxacin, norfloxacin, enrofloxacin, marbofloxacin, pinafloxacin, lascufloxacin, avarofloxacin, nadifloxacin, delafloxacin, nemonoxacin, and zabofloxacin; sulfonamides such as cotrimoxazole; tetracyclines / glycylcyclines such as tigecycline, eravacycline, and omadacycline; topoisomerase inhibitors such as ETX0914 and GSK2140944; and other antibiotics such as chloramphenicol and fosfomycin; are included, but are not limited to.

[0090] The term "combination" is intended to mean that the compound of formula (I) and the antibiotic are active in the biofilm, so that the biofilm is destroyed and planktonic bacteria are exposed to the antibiotic. In some embodiments, the combination of the compound of formula (I) and the antibiotic is administered in a single composition. In other embodiments, the compound of formula (I) and the antibiotic are administered simultaneously or sequentially in separate compositions.

[0091] Objects that can be treated with bacterial infections are mammals, birds, aquatic animals, such as fish, or reptiles. In some embodiments, the object is a human, a laboratory animal, e.g., a primate, mouse, rat, or rabbit, a companion animal, e.g., a dog or cat, a working animal, e.g., a horse, donkey, etc., a domestic animal, e.g., a cow, bull, pig, sheep, goat, deer, llama, alpaca, etc., or a captive wild animal, e.g., a lion, leopard, cheetah, elephant, zebra, antelope, giraffe, koala, kangaroo, and a reptile, e.g., a crocodile, lizard, snake, etc., a bird, particularly poultry, e.g., a chicken, duck, goose, quail, or pheasant, or a captive bird, e.g., a budgerigar or canary, a cockatoo, a parakeet, a macaw, a parrot, etc., or a fish, particularly a captive fish, e.g., a farmed fish (salmon, trout, barramundi, etc.) or a tropical fish (zebrafish, guppy, Siamese fighting fish, clownfish, cardinal tetra, etc.), a dolphin, a whale, etc., located in a zoo or wildlife park. These are things. In a specific embodiment, the object is a human or a pet.

[0092] "Effective dose" refers to the amount required at least partially to achieve the desired reaction, e.g., biofilm dispersion within the infected site. The said amount varies depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. The effective dose of the 6,7-epoxyglienone compound is expected to fall within a relatively broad range that can be determined through routine testing. The effective dose for human patients may be, for example, in the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dose, or 0.1 ng to 1 g per cm² of body surface area per dose. The said dose is preferably in the range of 1 mg to 1 g per kg of body weight per dose or 1 mg to 1 g per cm² of body surface area per dose, e.g., 1 mg to 1 g per kg of body weight per dose, or 1 mg to 1 g per cm² of body surface area per dose. In one embodiment, the dosage is in the range of 1 ng to 500 mg per kg of body weight per dosage or 0.1 ng to 500 mg per cm² of surface area per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage or 1 mg to 250 mg per cm² of body surface area per dosage. In yet another embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage or 1 mg to 100 mg per cm² of body surface area per dosage, for example up to 50 mg per kg of body weight per dosage or 50 mg per cm² of body surface area per dosage. In another embodiment, the dosage is in the range of 1 mg to 1 mg per kg of body weight per dosage or 1 mg to 1 mg per cm² of body surface area per dosage.

[0093] In a further aspect of the present invention, the biofilm described above dispersed by the method is present or potentially present on a medical device. Accordingly, the present invention comprises a method for preventing the formation of a biofilm containing Gram-negative bacteria on a medical device or for dispersing a biofilm containing Gram-negative bacteria on a medical device; said method comprising an epoxyglienone compound of formula (I), or includes the step of applying a pharmaceutically acceptable salt thereof to a medical device; the compound of formula (I) is as follows:

[0094]

[0095] As mentioned above,

[0096] R1 is hydrogen and C 1-6 Selected from alkyls;

[0097] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0098] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0099] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0100] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0101] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0102] R8 is hydrogen and C 1-6 Selected from alkyls.

[0103] In some embodiments, the medical device is coated with a composition comprising a compound of formula (I) or a salt thereof to prevent the formation of a biofilm containing Gram-negative bacteria. In other embodiments, the medical device is washed or coated with a composition comprising a compound of formula (I) to disperse a biofilm containing Gram-negative bacteria and remove it from the device.

[0104] As used herein, the term “medical device” refers to a device used on a human or animal body for therapeutic benefit, which has a physical or mechanical effect on said body or is used to measure or monitor function in said body. Suitable medical devices include indwelling medical devices, e.g., urinary catheters, vascular access devices, endotracheal tubes, tracheostomy tubes, bowel supply tubes, and wound drainage tubes; invasive medical devices, e.g., centerlines, mechanical heart valves, pacemakers, prosthetic or replacement joints, pins, rods, screws, and plates for fixing fractured bones; catheters, e.g., urinary catheters, venous catheters, Swan-Ganz catheters, Quinton catheters, intrauterine catheters, drainage catheters, and pigtail catheters; and prostheses, dental prostheses including, e.g., limb prostheses, dentures, occlusions, and dental implants. It includes implants, such as cochlear implants, coronary artery stents, contraceptive implants, cosmetic implants, and dental implants. As used herein, medical devices also include medical equipment, such as syringes, speculas, blood pressure monitors, scanners, ultrasound probes, etc.; as well as surgical instruments, such as scalpels, forceps, clamps, retractors, lancets, endoscopes, and calipers.

[0105] In some embodiments, the cleaning or coating includes a disinfectant, a sterilizing agent, or an antibiotic.

[0106] The present invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating bacterial infections including biofilms containing Gram-negative bacteria, said drug being adapted for topical administration to bacterial infections:

[0107]

[0108] As mentioned above,

[0109] R1 is hydrogen and C 1-6 Selected from alkyls;

[0110] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0111] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0112] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0113] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0114] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0115] R8 is hydrogen and C 1-6 Selected from alkyls.

[0116] In another aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in treating a bacterial infection comprising a biofilm containing Gram-negative bacteria, wherein the compound of formula (I) is as follows, and said compound is intended for topical administration for a bacterial infection:

[0117]

[0118] As mentioned above,

[0119] R1 is hydrogen and C 1-6 Selected from alkyls;

[0120] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0121] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0122] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0123] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0124] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0125] R8 is hydrogen and C 1-6Selected from alkyls.

[0126] 6,7-Epoxythiglienone compound

[0127] The compound useful in the method of the present invention is the compound of formula (I), or its salt, in particular a pharmaceutically acceptable salt:

[0128]

[0129] As mentioned above,

[0130] R1 is hydrogen and C 1-6 Selected from alkyls;

[0131] R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0132] R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenil, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl;

[0133] R4 and R5 independently contain hydrogen and C 1-6 Selected from alkyls;

[0134] R6 is hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8Cycloalkyl, -C(O) aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 Selected from alkynylaryls;

[0135] R7 is hydroxyl, -OC 1-6 Alkyl, -OC 2-6 Alkenil, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, -OC(O)C 2-6 alkynyl, -C(O)C 3-8 Cycloalkyl, -C(O)C 1-6 Alkyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkenyl C 3-8 Cycloalkyl, -C(O)C 2-6 Alkinyl C 3-8 Cycloalkyl, -OC(O) aryl, -OC(O)C 1-6 Alkylaryl, -C(O)C 2-6 alkenylaryl and -C(O)C 2-6 Selected from alkynylaryls;

[0136] R8 is hydrogen and C 1-6 Selected from alkyls.

[0137] In a specific implementation of expression (I), one or more of the following apply:

[0138] R1 is -C 1-3 Selected from alkyls, in particular -CH3;

[0139] R2 is -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Alkynyl; especially -OC(O)C 3-6 Alkyl and -OC(O)C 3-6 Selected from alkenyl;

[0140] R3 is -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Alkynyl; especially -OC(O)C3-6 Alkyl, -OC(O)C 3-6 Alkenyl and -OC(O)C 3-6 Selected from alkynyl;

[0141] R4 and R5 are independently -C 1-3 Selected from alkyls; in particular, all are methyls;

[0142] R6 is hydrogen, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 Alkynyl and -C(O) aryl; in particular selected from hydrogen, -C(O)CH3, -C(O)CH2CH3, -C(O)CH(CH3)2 and -C(O)CH2CH2CH3;

[0143] R7 is hydroxyl, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl and -OC(O)C 2-6 Selected from alkynes; in particular hydroxyls;

[0144] R8 is C 1-3 It is an alkyl, especially a methyl.

[0145] In some embodiments, the compound of formula (I) has stereochemistry as shown in formula (II) below:

[0146]

[0147] In some embodiments, the epoxide at the 6,7-position lies above the plane of the ring system. In other embodiments, the epoxide at the 6,7-position lies below the plane of the ring system. In some embodiments, the R2 group at the 12-position S And, in another embodiment, the R2 at the 12th position R am.

[0148] In some embodiments of Formula (I), the alkyl or alkenyl group of R2 and / or R3 is a branched alkyl or alkenyl group. In other embodiments of Formula (I), the alkyl or alkenyl group of R2 and / or R3 is a linear alkyl or alkenyl group.

[0149] In some embodiments, the alkyl or alkenyl group of R2 and / or R3 has a chain length of intermediate hydrophobicity, e.g., C4, C5, or C6.

[0150] In some embodiments, R6 is an acyl group, e.g., acetyl (-C(O)CH3), -C(O)CH2CH3, -C(O)CH(CH3)2, or -C(O)CH2CH2CH3. In some embodiments, R6 is hydrogen.

[0151] In a specific embodiment, the epoxyglienone compound is selected from the following compounds, or their salts, in particular pharmaceutically acceptable salts:

[0152] 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 1);

[0153] 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 2);

[0154] 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 3);

[0155] 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 4);

[0156] 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13-pentahydroxy-20-acetyloxy-1-tiglien-3-one (compound 5);

[0157] 12-propanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 6);

[0158] 12,13-dithigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 7);

[0159] 12-(2-methylbutanoyl)-13-tigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 8);

[0160] 12-butanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 9);

[0161] 12-(3,3-dimethylbut-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 10);

[0162] 12-hex-2,4-dienoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 11);

[0163] 12-tigloyl-13-(2-methylpropanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 12);

[0164] 12-but-2-enoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 13);

[0165] 12-tigloyl-13-butanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 14);

[0166] 12,13-dibutanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 15);

[0167] 12,13-dipentanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 16);

[0168] 12,13-di-(2 E ,4 E )-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 17);

[0169] 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 18);

[0170] 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 19);

[0171] 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 20);

[0172] 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 21).

[0173] In a specific embodiment, the 6,7-epoxyglienone compound is selected from compounds 1, 2, 3, 4 and 6.

[0174] The above epoxyglienone compound can be obtained by extraction or semi-synthetically from an extracted compound. Methods for obtaining the epoxyglienone compound are provided in WO2007 / 070985 and WO2014 / 169356, the contents of which are incorporated by reference.

[0175] composition

[0176] A 6,7-epoxythiglienone compound or its salt may be used as is, but it may be more convenient to use it in the form of a composition with a carrier, a diluent, and / or an excipient. In some embodiments, the composition may be a liquid useful for immersing a medical device. In other embodiments, the composition may be a coating composition useful for coating a medical device. In yet another embodiment, the composition may be a pharmaceutical composition suitable for administration to a patient.

[0177] The dosage form and ratio for pharmaceutical use and composition will be readily determined by a person skilled in the art.

[0178] The 6,7-epoxytiglianone compound is formulated for topical administration directly onto or into a biofilm. In some embodiments, the 6,7-epoxytiglianone compound is formulated for topical administration in the form of a gel, ointment, lotion, cream, or transdermal patch that can be applied directly onto a biofilm. In other embodiments, for example, when the composition is injected internally to make topical contact with a biofilm, the epoxytiglianone compound is formulated for injection.

[0179] In some embodiments, suitably, the composition is a pharmaceutical composition(s) and comprises a pharmaceutically acceptable excipient or an acceptable excipient. “Pharmaceutically acceptable excipient” means a solid or liquid filler, diluent, or encapsulating material that can be safely used. Depending on the specific route of administration, various carriers well known in the art may be used. The carrier or excipient may be selected from the group comprising sugars, starch, cellulose and its derivatives, cyclodextrin, malt, gelatin or other gelling agents, polymers, talc, calcium sulfate, vegetable oils, synthetic oils, alcohols and / or polyols, alginic acid, phosphate buffered solutions, emulsifying agents, isotonic saline, and pyrogen-free water.

[0180] The liquid formulation comprises solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, the injectable liquid formulation may be formulated with or without a buffer in dimethyl sulfoxide (DMSO), a solution in aqueous 1,2-propanediol, an aqueous solution of gamma cyclodextrin or 2-hydroxypropyl-beta-cyclodextrin, a saline solution, or a polyethylene glycol solution. The preferred pH range is 3.0 to 4.5. Suitable buffers buffer the formulation to a pH of 3.5 to 4.5 and include, but are not limited to, acetate buffers and citrate buffers.

[0181] Accordingly, a composition of the 6,7-epoxyglienone compound may be formulated for administration (e.g., by injection, e.g., by bolus injection at the site of a biofilm infection) and may be provided in ampoules, pre-filled syringes, as a unit dose in small volume fluids, or in multi-dose containers with added preservatives. The composition may take the form of a suspension, solution, gel, or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient may be in powder form to be reconstituted before use into a suitable vehicle, e.g., a sterile pyrogen-free water, obtained by aseptic isolation of a sterile solid or by lyophilization from a solution.

[0182] Pharmaceutical compositions of 6,7-epoxystiglylenone compounds suitable for administration may be provided in separate units, such as syringes, vials, tubes, or sachets, each containing a predetermined amount of one or more pharmaceutically active 6,7-epoxystiglylenone compounds, as powders or granules, or as solutions or suspensions in aqueous liquids, cyclodextrin solutions, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions, or as solutions or suspensions in creams or gels, or as suspensions of micro- or nano-particles incorporating 6,7-epoxystiglylenone compounds, wherein the micro- or nano-particles may include, but are not limited to, silica or polylactide micro- or nano-particles. Such compositions may be prepared by any pharmaceutical method, but all methods include the step of linking one or more pharmaceutically active compounds of the present invention with a carrier constituting one or more necessary components. Generally, the above composition is prepared by uniformly and closely mixing the formulation of the present invention with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, molding the product in a desired manner.

[0183] For topical administration to the epidermis or other organs, the compound according to the present invention may be formulated as a gel, ointment, emulsion, paste, cream, or lotion, or as a transdermal patch. A gel may be prepared by adding a suitable thickener to an aqueous / alcoholic composition of the compound. Suitable thickeners or gelling agents, such as polyvinyl carboxypolymer carbomer 940, are known in the art. Ointments and creams may be formulated with an aqueous or oily base, for example, with the addition of a suitable thickener and / or gelling agent. A lotion may be formulated with an aqueous or oily base and will also generally contain one or more emulsifying agents, stabilizers, dispersants, suspending agents, thickeners, or chromogenic agents.

[0184] Formulations suitable for topical administration also include solutions or suspensions that can be administered topically in the form of a bath or soaking solution or spray, or absorbed into a dressing.

[0185] Liquid formulations suitable for soaking or cleaning medical devices may also include solutions or suspensions in solvents, such as water and alcohol or mixtures thereof. The solutions or suspensions may also contain other components such as emulsifying agents, surfactants, disinfectants, bactericides, antibiotics, colorants, etc.

[0186] The coating is a suitable polymeric coating known in the art capable of incorporating the compound of Formula (I), such that the surface of the coating is accessible and sufficient to prevent the formation of a biofilm containing Gram-negative bacteria. Suitable coatings include hydroxyapatite, calcium phosphate, biophosphonates, bioactive ceramics, polyhydroxyalkanoates, mesoporous materials, hydrogels, drug-eluting coatings such as polymer coatings, calcium phosphate, chitosan, collagen, and bone grafts / cancellous bone.

[0187] Examples

[0188] The compounds of the present invention may be obtained by isolating from a plant or part of a plant, derivatizing an isolated compound, or derivatizing a related compound. The isolation and derivatization procedures may be found in WO 2007 / 070985 and WO 2014 / 169356.

[0189] Example 1: Epoxy-tigilane does not have direct antibiotic activity against Gram-negative bacteria in a planktonic culture system.

[0190] The effects of five epoxyglienones (compounds 1, 2, 3, 4, and 6) and two comparison compounds (epoxyglienones with longer carbon chains at the C12 position) on six human pathogenic bacteria (two Gram-positive and four Gram-negative species) were measured in a conventional planktonic culture system. The antimicrobial activity of each epoxyglienone against each bacterial strain was determined by performing the Minimum Inhibitory Concentration (MIC) assay (a standard assay introduced to define and quantify antibiotic activity).

[0191] The Gram-positive bacteria used in this study were methicillin-resistant Staphylococcus aureus (MRSA) 1004A and Streptococcus pyogenes ( Streptococcus pyogenes The Gram-negative bacteria used in this study were Pseudomonas aeruginosa PAO1, Escherichia coli IR57 (V7), Klebsiella pneumoniae, and Acinetobacter baumannii 7789 (V19). MIC analysis was performed on the planktogenic cultures of each bacterium grown in Mueller-Hinton broth using the standard broth dilution method described by Jorgensen et al. (1999) with epoxyglienone dissolved in ethanol. The results are shown in Table 1 below.

[0192] Minimum inhibitory concentrations (µg / ml) determined for compounds 1, 2, 3, 4, and 6 and comparison compounds CC-1 and CC-2 against 2 Gram-positive bacteria and 4 Gram-negative bacteria. C = confluent growth, i.e., MIC could not be determined at any concentration tested. The data provided represent the average of three repeated tests. bacteria Compound number Comparative Compounds* 1 2 3 4 6 CC-1 CC-2 Gram-positive tumor S. Aureus 256 256 256 512 C 8 16 S. Phyogenes 128 128 128 256 C 8 8 Gram-negative species E. coli C C C C C C C P. Aeruginosa C C C C C C C K. Nummoniae C C C C C C C A. Baumannny C C C C C C C

[0193] The comparative compound is an epoxytiglienone having a longer carbon chain at the C12 position. CC-1 is 12-(2,4-decadienoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one, and CC-2 is 12 R -(2,4-Decadienoil)-13 R -(2-methylbutanoyl)-6,7-epoxy-4 R ,5 R ,9 S ,12 R ,13 R ,20-hexahydroxy-1-tiglian-3-one.

[0194] The above results demonstrated that the direct antimicrobial properties of epoxyglienone in planktonic cultures are limited to Gram-positive bacteria. For any Gram-negative bacteria tested, the MIC value for any compound could not be determined, and none of them were affected (bacteria grew fully at concentrations of the tested compounds exceeding 512 µg / ml).

[0195] Example 2: Epoxytiglienone destroyed the established biofilm of Gram-negative Escherichia coli.

[0196] E. coli ( E. coli The effects of administering compounds 1, 4, and 6 and comparative compound CC-1 on the disruption of established in vitro biofilms were investigated using the method described by Powell et al. (2018).

[0197] After growing a biofilm of Escherichia coli IR57 in Muller-Hinton (MH) broth for 24 hours in 96-well glass-bottom plates, the 50% supernatant was replaced with fresh MH broth with or without the added epoxytiglienone compound at a final concentration of 256 µg / ml (vehicle was ethanol). An ethanol equivalent blank was used as an additional control treatment. Subsequently, the plates were incubated at 37°C for an additional 24 hours.

[0198] Subsequently, the biofilm was imaged using a confocal laser scanning microscope (CLSM), followed by staining each well with Live / Dead® Baclight stain added with phosphate-buffered saline (PBS), and then imaged using a z-stack CLSM. The generated images were analyzed by COMSTAT software to determine (i) the biofilm biomass volume or bio-volume (㎛). 3 / ㎛ 2 ) and (ii) generated measurements of the dead / living bacteria ratio.

[0199] CLSM demonstrated that there were distinct differences among the treatments regarding the distribution of bacteria in the biofilm. Compounds 1, 4, and 6 induced significant changes in the distribution and density of bacteria in the biofilm compared to untreated and ethanol equivalent (blank) controls (Fig. 1); this was quantified and confirmed by COMSTAT image analysis. A significant decrease in bio-volume was evident in the biofilm treated with Compound 1 (p<0.05; Fig. 2a). Comparative Compound CC-1 had no distinct effect on biofilm distribution and density or bio-volume. Interestingly, there was no significant difference in the dead / living bacteria ratio between any epoxytiglienone and the two control treatments (Fig. 2b), which demonstrates that the effect of epoxytiglienone on biofilm density and bio-volume in the experiment was independent of direct antibiotic activity.

[0200] Example 3: Epoxytiglienone disrupts the extracellular matrix and increases the diffusion of particles through the established biofilm of Gram-negative E. coli bacteria.

[0201] The effects of three epoxyglienones (compounds 1, 4, and 6) on the assembly and permeability of established E. coli biofilms were evaluated using multiple particle tracking (MPT). MPT is a recently described technique that enables the simultaneous tracking of micron-sized particles through biofilms using a microscope, from which diffusion-based parameters of particles embedded within the extracellular polymeric matrix (EPS) of the biofilm can be determined (Cao et al. 2016). MPT measurements also enable the calculation of micro-rheological properties of biofilm structures after treatment with test compounds.

[0202] As described in Example 2 above, an E. coli biofilm was established and epoxytiglienone and control treatment groups were applied. 24 hours after the application of the treatment, the biofilm was stained with SYTO9® and 0.0025% negatively charged carboxylate-modified FluoSpheres® (200 nm) added to the biofilm, and incubated for an additional 2 hours. Subsequently, the movement of FluoSphere® particles within the biofilm was captured on video using an epifluorescence microscope equipped with a high frame rate camera (33 ms). After tracking the particle trajectories using ImageJ software (Mosaic), the following three parameters of 200 nm FluoSphere® particles were calculated: (i) ensemble diffusion coefficient (Deff), (ii) ensemble mean change (MSD), and (iii) creep compliance (a measure of resistance to mechanical deformation derived from MSD versus delay time). Each treatment was repeated three times.

[0203] All three tested epoxyglienone compounds increased the diffusion of particles through established E. coli biofilms by 80 to 420 times compared to the control treatment group (Table 2).

[0204] Diffusion coefficient for 200 nm negatively charged carboxylate-modified FluoSpheres® particles in E. coli IR57 biofilms treated with epoxyglienone compound Diffusion coefficient (Deff) (㎠.s -1 ×10 -9 )±standard deviation Compound 4 0.8449±0.1250 Compound 1 0.6018±0.0901 Compound 6 0.1607±0.0104 Non-control treatment group 0.002±0.0014 Ethanol alone control group 0.0155±0.0052

[0205] Significant destruction and alteration of the structure of the E. coli biofilm matrix by epoxytiglienone was demonstrated by a substantially higher average rate of change of FluoSphere® particles after treatment (Fig. 3), and reduced resistance to mechanical deformation in the treated biofilm was evident from the increase in creep compliance of the epoxytiglienone-treated biofilm compared to the control (Fig. 4).

[0206] Example 4: Epoxytiglienone significantly reduced biomass in established biofilms of two different Gram-negative pathogens, P. aeruginosa and A. baumannii.

[0207] The effect of epoxyglienone (compounds 1, 4, 6 and comparative compound CC-1) on the disruption of established in vitro biofilms was investigated in two different species of Gram-negative bacteria (Pseudomonas aeruginosa PA01 and Acinetobacter baumannii) using the method described in Example 2 above. Cell penetration after treatment with compounds 1, 4, and 6 was also determined using SYTOX™ Green nucleic acid staining, along with a non-control treatment group and a positive control of 70% isopropanol.

[0208] Consistent with the results of the E. coli study above, epoxyglienone compounds significantly altered the distribution and density of bacteria in A. baumannii biofilms compared to untreated and ethanol equivalent (blank) controls (Fig. 5a). This was also reflected in the effect when measured by bio-volume for A. baumannii biofilms (Fig. 6a), where a significant decrease was observed for compounds 1, 4, and CC-1. For P. arruginosa, only compound 4 had a significant effect on biofilm distribution (Fig. 5b) and bio-volume compared to the other treatment groups (Fig. 6b). The dead / living bacteria ratio was also evaluated as in Example 2 using E. coli, and there was no difference between the treatment groups for A. baumannii or P. arruginosa (Figs. 6c and 6d). Cell permeability studies on Escherichia coli and P. aruginosa bacteria (Fig. 7) showed a significant increase in permeability only when treated with compounds 1 and 4 at concentrations ≥512 µg / ml, which are greater than the compound concentration used in the biofilm disruption assay (256 µg / ml). This further confirms that the effect of epoxyglienone on biofilm density and bio-volume in the above experiment was independent of any direct antibiotic activity killing bacteria inside the biofilm.

[0209] Example 5: Epoxytiglienone resolves established biofilms in vivo in a mouse model of chronic biofilm infection.

[0210] The effect of administering Compound 4 on biofilm infection in vivo was investigated in a diabetic murine model of chronic biofilm infection (Zhao et al. 2010). In this study, the inventors used the method described by Dhall et al. (2014) in which bacterial biofilm infection occurs naturally after the creation of a wound.

[0211] Briefly, db / dbDiabetic mice (> 6 months of age) were housed under non-sterile conditions for 4–5 weeks, after which a wound was created on the back of each mouse using a 6 mm diameter incisional punch biopsy. Subsequently, a catalase inhibitor was administered intraperitoneally (1 g / kg aminotriazole), and a glutathione peroxidase (GPx) inhibitor was administered topically near the margins of the wound site (1 g / kg mercaptosuccinic acid), followed by dressing with Tegaderm. After 24 hours, a biofilm was clearly visible at the wound sites of all mice. The mice were divided into two groups (7 mice per group) and treated with Compound 4 (0.3 mg / ml in a hydrogel vehicle) or a control group (hydrogel vehicle alone). The Tegaderm dressing was then replaced. Two additional administrations of Compound 4 or the control group (vehicle alone) were performed on days 8 and 15. Antibiotic or other antimicrobial treatments were not applied during this study.

[0212] The presence of biofilms and the surface area of ​​the wounds were evaluated throughout the course of this study. For histological and histochemical analysis of the wound sites, biopsies were taken from mice treated with Compound 4 and vehicle alone (control) between 21 and 28 days post-injury. In the control group, only 1 out of 7 wounds healed within the aforementioned period, and fibrous scabs and bacterial infiltration were clearly present at the wound sites. In infected wounds treated with epoxyglienone, complete wound healing was evident in 6 out of 7 mice treated with Compound 4 (along with complete re-epithelialization, resolution of inflammation, and absence of bacteria within the dermis).

[0213] Example 6: In vitro treatment of human adult keratinocytes and fibroblasts with epoxyglienone induces upregulation of chemokines / cytokines associated with neutrophil recruitment.

[0214] In addition to disrupting the structure of biofilms containing Gram-negative bacteria, the effects of epoxyglienone on the regulation of genes associated with the host response to bacterial infection were investigated in vitro using human adult epithelial keratinocytes (HEKa) and human adult dermal fibroblasts (HDF) in microarray studies.

[0215] For the above study, HEKa was cultured in EpiLife® medium supplemented with Supplement S7 (all products from Life Technologies, Carlsbad, CA, USA), and HDF was cultured in Medium 106 supplemented with LSGS (Low Serum Growth Supplement) (all products from Life Technologies). Subsequently, cells were treated with vehicle or 170 nM Compound 4 for 0, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours. RNA was extracted using the Qiagen RNeasy Mini Kit and biotinized using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX, USA). Labeled RNA was hybridized to a human HT-12 v4 BeadChip array (Illumina Inc, San Diego, CA, USA) and scanned according to standard Illumina protocols. Data were extracted using GenomeStudio (Illumina) with default analysis settings and without standardization methods. The generated data was loaded into GeneSpring GX (Agilent, Santa Clara, CA, USA). The representation values ​​were standardized using displacement standardization along with the default settings.

[0216] In vitro for both HDF and HEKa, treatment with compound 4 (170 nM) resulted in two key chemokines / cytokines associated with PMNL recruitment within 2 to 4 hours ( IL8, CXCL1induced a significant upregulation of ). In HEKa, compound 4 induced host defense peptides ( at 8 to 72 hours after treatment) DEFB2, DEFB3, DEFB4, RNASE7 Significantly increased the production of ).

[0217] Example 7: Epoxytiglienone treatment induces necrosis / necrosis and releases the antimicrobial peptide cathelicidin LL-37 in vitro from isolated human neutrophils.

[0218] Neutrophils are the most abundant white blood cells in the blood and constitute the first line of host defense against infectious pathogens. The core of their function is the ability to be recruited to the site of infection, recognize microorganisms, and subsequently be activated to target pathogens through a combination of phagocytic and cytotoxic mechanisms. Among the mechanisms used by neutrophils to kill pathogens are (i) the generation of reactive oxygen species, (ii) the release of nuclear chromatin components (coated with histones, proteases, and granular and cytoplasmic proteins) to immobilize and capture pathogens (the process of netosis), and (iii) the release of antimicrobial peptides.

[0219] After confirming the effects of epoxytiglienone compounds on the upregulation of chemokine and cytokine production associated with neutrophil recruitment in HDF and HEKa (see Example 6 above), the effects of compound 4 on two aspects of neutrophil function, namely the induction of necrosis / necrosis and the release of antimicrobial peptides, were investigated.

[0220] For the above analysis method, neutrophils were isolated from fresh blood of healthy human donors by lysis of the red blood cell pellet obtained by Ficoll-Paque precipitation. The neutrophils (∼4×10⁶) 6 Cells ( / mL) were incubated in complete culture medium with 10 μg / mL dihydroethidium (DHE) (Sigma-Aldrich) at 37°C for 15 minutes, and an equivalent amount of unstained cells were tested as an unstained control.

[0221] The necrosis / necrosis assay followed the method fully described by Brinkmann et al. (2010). Isolated neutrophils were plated into 96-well plates (RPMI1640, 10% FCS) and incubated with 1:50,000 dilutions of Hoescht (10 mg / mL) and Sytox® Green (5 mM). Compound 4 was added at six different concentrations (0.005, 0.05, 0.5, 5, 50, 500 μM), and the cells were incubated at 37°C and 5% CO2. Vehicle-treated controls were included in all assays, and each assay was repeated three times. Hoescht / Sytox® Green fluorescence images were recorded for each well at 3 and 6 hours.

[0222] Neutrophil necrosis and necrosis were first observed at 3 hours after treatment with compound 4 at the two highest concentrations (50 μM and 500 μM) and were evident at 6 hours at four low concentrations (0.005 μM - 5 μM) (Fig. 8).

[0223] To investigate the release of antimicrobial peptides by neutrophils in response to treatment with Compound 4, isolated neutrophils were incubated with the vehicle or Compound 4 at four concentrations (62.5, 125, 250, and 500 μM). At 3 hours, the cell culture supernatant was removed and tested for LL-37 content using an LL-37-targeted ELISA kit (Hycult Biotech). The ELISA readings were standardized against a vehicle-only control to determine the fold increase in LL-37 release.

[0224] 3 hours after treatment with compound 4, the release of LL-37 from neutrophils increased in a concentration-dependent manner (Fig. 9). At concentrations of compound 4 ranging from 125 to 500 μM, LL-37 release was 3 to 5 times higher than that of the control treatment group (Fig. 9).

[0225] Data from the above examples show that at therapeutically relevant concentrations in vitro, compound 4 induces the transition of suicidal neutrophil necrosis to necrosis, thereby inducing the release of potent antimicrobial defense peptide LL-37.

[0226] The data from Examples 6 and 7 above also demonstrate that, in addition to the direct effect of disrupting the structure of Gram-negative biofilms, epoxyglienone can also induce a local innate immune response in both migratory and resident myeloid cells (e.g., neutrophils) in dermal or stromal cell types. This response suggests that the effect of epoxyglienone alone may be suitable for resolving many biofilm infections without the need for conventional antibiotics.

[0227] References

[0228] If any prior art literature is cited in this specification, it should be understood that such citation does not constitute an acknowledgment that such published literature forms part of the common knowledge of the art in Australia or any other country.

[0229] Brinkmann V et al. 2010. Neutrophil extracellular traps: How to generate and visualize. Them. J. Vis. Exp. 36: e 1724.

[0230] Cao, H. et al . (2016) Revealing region-specific biofilm viscoelastic properties by means of a micro-rheological approach. npj Biofilms and Microbiomes , 2(1), pp. 1-7.

[0231] Cepas V et al.2019. Relationship between biofilm formation and antimicrobial resistance in Gram-negative bacteria. Microb. Drug Resist. 25: 72-79.

[0232] Dhall S et al. 2014. "Generating and Reversing Chronic Wounds in Diabetic Mice by Manipulating Wound Redox Parameters." J. Diabetes Res. 562625.

[0233] Doi et al. 2017. Gram-negative bacterial infections: Research priorities, accomplishments and future directions. Clin Infect Dis. 64 (S1): S30-S35

[0234] Fleming D & Rumbaugh KP 2017. Approaches to dispersing medical biofilms. Microorganisms 2017 5, 15.

[0235] Gunn JS et al. 2016. What's on the outside matters: the role of extracellular polymeric substance of Gram-negative biofilms in evading host immunity and as a target for therapeutic intervention . J. Biol. Chem. 291: 12538-12546.

[0236] Hoiby et al. 2010. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents 35: 322-332.

[0237] Hoiby N et al. 2015. ESCMID guideline for the diagnosis and treatment of biofilm infections. Clin. Microbiol. Infect. 21 (Suppl. 1): S1-S25.

[0238] Ho J et al. 2010. Multiresistant Gram-negative infections: a global perspective. Curr. Opin. Infect. Dis. 23: 546-53.

[0239] Jorgensen JJH et al. 1999. Antibacterial susceptibility tests: dilution and disk diffusion methods. In Murray PR et al. (Eds.), Manual of Clinical Microbiology (pp 1526-1543). Washington, DC: ASM Press.

[0240] Koo H et al. 2017. Targeting microbial biofilms: current and prospective therapeutic strategies. Nat. Rev. Microbiol. 15: 740-755.

[0241] Powell et al. 2018 Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides. NPJ Biofilms Microbiomes . 4: 13.

[0242] Zhao et al. 2010. Delayed wound healing in diabetic (db / db) mice with Pseudomonas aeruginosabiofilm challenge: a model for the study of chronic wounds. Wound Repair Regen. 18: 467-477.

Claims

Claim 1 A pharmaceutical composition for treating bacterial infections comprising biofilms containing Gram-negative bacteria, wherein the pharmaceutical composition is for topical administration and comprises an epoxyglienone compound of formula (II) or a pharmaceutically acceptable salt thereof: In the above, R1 is hydrogen and C 1-6 Selected from alkyl; R2 is -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl; R3 is -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl; R4 and R5 are independently hydrogen and C 1-6 Selected from alkyls; R6 is hydrogen and -C(O)C 1-6 Selected from alkyl; R7 is hydroxyl; R8 is hydrogen and C 1-6 Selected from alkyls. Claim 2 A pharmaceutical composition according to claim 1, wherein the topical administration is a topical administration. Claim 3 A pharmaceutical composition according to claim 1, wherein one or more of the following are applied: i) said infection is a postoperative infection; ii) said infection is an infection at the insertion site of a medical device; iii) said infection is an infection at the implantation site of an implant; or iv) said infection is a chronic bacterial infection. Claim 4 The pharmaceutical composition of claim 1, wherein the administration is combined with an antibiotic to which Gram-negative bacteria are susceptible when in a planktonic state. Claim 5 A pharmaceutical composition according to claim 1, wherein one or more of the following are applied: i) R1 is C 1-3 ii) it is alkyl and R2 is -OC(O)C 1-7 Alkyl and -OC(O)C 2-7 Selected from alkenyl; iii) R3 is -OC(O)C 1-7 Alkyl and -OC(O)C 2-7 Selected from alkenyls; iv) R4 and R5 are each methyl; and v) R8 is C 1-3 It is an alkyl. Claim 6 In claim 1, R2 is -OC(O)C 3-6 Alkyl and -OC(O)C 3-6 A pharmaceutical composition selected from alkenyls. Claim 7 In claim 1, R3 is -OC(O)C 3-6 Alkyl and -OC(O)C 3-6 A pharmaceutical composition selected from alkenyls. Claim 8 In claim 1, R6 is A pharmaceutical composition selected from hydrogen and -C(O)CH3. Claim 9 A pharmaceutical composition according to claim 1, wherein the alkyl or alkenyl group of R2 and / or R3 is a branched alkyl or alkenyl group. Claim 10 A pharmaceutical composition according to claim 1, wherein the alkyl or alkenyl group of R2 and / or R3 is a linear alkyl or alkenyl group. Claim 11 The pharmaceutical composition of claim 1, wherein the compound of formula (II) is selected from the following compounds or pharmaceutically acceptable salts thereof: 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 1); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 2); 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 3); 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 4); 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13-pentahydroxy-20-acetyloxy-1-tiglien-3-one (compound 5); 12-propanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 6);12,13-dithigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 7);12-(2-methylbutanoyl)-13-tigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 8);12-butanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 9);12-(3,3-dimethylbut-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 10);12-hex-2,4-dienoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 11);12-tigloyl-13-(2-methylpropanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 12);12-but-2-enoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 13); 12-tigloyl-13-butanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 14); 12,13-dibutanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 15); 12,13-dipentanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 16);12,13-di-(2; E ,4 E )-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 17); 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 19); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 20); and 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 21) . Claim 12 In claim 1, the biofilm comprising the Gram-negative bacteria comprises Pseudomonas species, Acinetobacter species, Aeromonas species, Bacteroides species, Bordetella species, Borrelia species, Burgholderia species, Citrobacter species, Compylobacter species, Escherichia species, Enterobacter species, Flavobacterium species, Fucobacterium species, Klebsiella species, Leptospira species, Neisseria species, Helicobacter species, Haemophilus species, Legionella species, Moraxella species, Yersinia species, Oligella species, Pantoea species, Porphyromonas species, Prevotella species, Proteus species, Raultella species, Salmonella species, Serratia species, Shigella species, Sphingomonas species, Stenothopomonas species, Treponema species, Veilonella species, and A pharmaceutical composition comprising at least one Gram-negative bacterium selected from Vibrio species. Claim 13 A pharmaceutical composition according to claim 12, wherein the Gram-negative bacteria are selected from Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonoriae, Neisseria meningitidis, Haemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica, Salmonella bongori, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Fucobacterium necrophorum, Burgholderia cepacian, and Prevotella intermedia. Claim 14 A method for preventing the formation of a biofilm containing Gram-negative bacteria on an inanimate surface or medical device, comprising the step of applying an epoxyglienone compound of formula (II) or a pharmaceutically acceptable salt thereof to an inanimate surface or medical device, wherein the epoxyglienone compound of formula (II) is of the following method: In the above, R1 is hydrogen and C 1-6 Selected from alkyl; R2 is -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl; R3 is -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl and -OC(O)C 2-7 Selected from alkynyl; R4 and R5 are independently hydrogen and C 1-6 Selected from alkyls; R6 is hydrogen and -C(O)C 1-6 Selected from alkyl; R7 is hydroxyl; R8 is hydrogen and C 1-6 Selected from alkyls. Claim 15 Claim 14, wherein the method is a method for preventing or dispersing the formation of a biofilm containing Gram-negative bacteria on a medical device. Claim 16 The method of claim 15, wherein the medical device is a surgical instrument, a catheter, or a medical implant. Claim 17 In claim 15, a method to which one of the following is applied: i) the medical device is a catheter; or ii) the medical device is a dental implant. Claim 18 In claim 14, a method in which one or more of the following are applied: i) R1 is C 1-3 ii) it is alkyl and R2 is -OC(O)C 1-7 Alkyl and -OC(O)C 2-7 Selected from alkenyl; iii) R3 is -OC(O)C 1-7 Alkyl and -OC(O)C 2-7 Selected from alkenyls; iv) R4 and R5 are each methyl; and v) R8 is C 1-3 It is an alkyl. Claim 19 In claim 14, R2 is -OC(O)C 3-6 Alkyl and -OC(O)C 3-6 A method selected from alkenyl. Claim 20 In claim 14, R3 is -OC(O)C 3-6 Alkyl and -OC(O)C 3-6 A method selected from alkenyl. Claim 21 In claim 14, R6 is A method selected from hydrogen and -C(O)CH3. Claim 22 The method of claim 14, wherein the alkyl or alkenyl group of R2 and / or R3 is a branched alkyl or alkenyl group. Claim 23 The method of claim 14, wherein the alkyl or alkenyl group of R2 and / or R3 is a linear alkyl or alkenyl group. Claim 24 In claim 14, the compound of formula (II) is selected from the following compounds or pharmaceutically acceptable salts thereof: 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 1); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 2); 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 3); 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 4); 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13-pentahydroxy-20-acetyloxy-1-tiglien-3-one (compound 5); 12-propanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 6);12,13-dithigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 7);12-(2-methylbutanoyl)-13-tigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 8);12-butanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 9);12-(3,3-dimethylbut-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 10);12-hex-2,4-dienoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 11);12-tigloyl-13-(2-methylpropanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 12);12-but-2-enoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 13); 12-tigloyl-13-butanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 14); 12,13-dibutanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 15); 12,13-dipentanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 16);12,13-di-(2; E ,4 E )-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 17); 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 19); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 20); and 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 21) . Claim 25 In claim 14, the biofilm comprising the Gram-negative bacteria comprises Pseudomonas species, Acinetobacter species, Aeromonas species, Bacteroides species, Bordetella species, Borrelia species, Burgholderia species, Citrobacter species, Compylobacter species, Escherichia species, Enterobacter species, Flavobacterium species, Fucobacterium species, Klebsiella species, Leptospira species, Neisseria species, Helicobacter species, Haemophilus species, Legionella species, Moraxella species, Yersinia species, Oligella species, Pantoea species, Porphyromonas species, Prevotella species, Proteus species, Raultella species, Salmonella species, Serratia species, Shigella species, Sphingomonas species, Stenothopomonas species, Treponema species, Veilonella species, and A method comprising at least one Gram-negative bacterium selected from Vibrio species. Claim 26 The method of claim 25, wherein the Gram-negative bacteria are selected from Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonoriae, Neisseria meningitidis, Haemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica, Salmonella bongori, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Fucobacterium necrophorum, Burgholderia cepacian, and Prevotella intermedia.

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