Biofilm destruction

Abstract

The present invention relates to a method for dispersing biofilms containing Gram-negative bacteria, comprising exposing the biofilm to an epoxytiglienone compound or a salt thereof. Methods for treating infections comprising local administration, e.g., topical or by injection, of an epoxytiglienone compound into or onto an established biofilm containing Gram-negative bacteria to disrupt the structure of the biofilm, and methods for preventing biofilms containing Gram-negative bacteria, including the formation or dispersal of biofilms, including Gram-negative biofilms formed on medical devices, are also described.

Description

[Technical Field]

[0001] The present invention relates to a method for dispersing biofilms containing Gram-negative bacteria, comprising exposing the biofilm to an epoxytiglienone compound or a salt thereof. Methods for treating infections comprising local administration, e.g., topical or by injection, of an epoxytiglienone compound into or onto an established biofilm containing Gram-negative bacteria to disrupt the structure of the biofilm, and methods for preventing biofilms containing Gram-negative bacteria, including the formation or dispersal of biofilms, including Gram-negative biofilms formed on medical devices, are also described. [Background technology]

[0002] Gram-negative bacteria are associated with a variety of respiratory, genitourinary, gastrointestinal, and circulatory system infections. They are also a major factor in chronic wounds, osteomyelitis, and the persistence of surgical site, medical device, and post-transplant infections. Serious infections caused by Gram-negative bacteria can cause significant morbidity and mortality, especially in immunocompromised patients. The most important clinical Gram-negative pathogens are Pseudomonas aeruginosa, Acinetobacter baumannii, and members of the Enterobacteriaceae family, particularly Escherichia coli and Klebsiella pneumoniae. Other medically relevant examples of Gram-negative bacteria include Neisseria species (which cause gonorrhea and meningitis), Hemophilus influenzae, Legionella pneumophila, Yersinia pestis, Proteus mirabilis, and Salmonella species.

[0003] Treating Gram-negative bacterial infections is becoming increasingly problematic due to both: (a) the emergence and spread of multidrug resistance to most or all conventional antibiotics among many of these microorganisms (Ho et al. 2010; Doi et al. 2017), and (b) their ability to form intractable, cohesive communities called biofilms (Cepas et al. 2019). Managing intractable Gram-negative bacterial biofilms, in particular, poses a complex and challenging clinical problem due to limited treatment options. Current strategies in many clinical settings involve biofilm removal (when feasible) through aggressive physical methods (e.g., debridement) combined with high-dose, often long-term antimicrobial chemotherapy as standard of care (Hoiby et al. 2015). However, these methods often fail to completely eradicate biofilms, leading to relapse and selection for increasing levels of bacterial resistance. This chronic, vicious cycle further erodes antibiotic effectiveness and promotes the more widespread use of "last resort" antibiotics, many of which have significant side effects.

[0004] New drugs are urgently needed to treat Gram-negative bacterial biofilm infections. While traditional approaches to treating bacterial infections are antibiotic-based and have focused on directly targeting and inhibiting or killing the causative pathogen (using the concepts of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)), the complex nature of biofilms means that new treatment strategies are needed.

[0005] Biofilms are bacterial communities embedded in a protective, self-produced matrix of extracellular polymeric substances (EPS) formed from polysaccharides, proteins, lipids, and nucleic acids (RNA and extracellular DNA). Resident bacteria living in biofilms are significantly more resistant to antibiotics than planktonic, free-living bacteria (Hoiby et al. 2010) and are largely protected from the host immune system. Much of this resistance of biofilms can be attributed to the EPS, which presents a formidable physical barrier to the penetration of antibiotics and immune cellular effectors (Gunn et al. 2016). The structure of biofilms as dense polymeric "meshes" of EPS and bacteria also means that they are highly resistant to removal from the surfaces of tissues and materials to which they are tightly attached.

[0006] In contrast to the current standard of care, which relies critically on antibiotics, a series of "non-antibiotic" strategies for treating Gram-negative bacterial biofilms have been proposed and are currently under investigation. These include interfering with biofilm signaling networks, targeting biofilm adhesion, and disrupting the biofilm EPS matrix (Koo et al. 2017). Unlike traditional antibiotics, these strategies target bacterial "virulence factors" rather than bacterial growth, thus inducing low selective pressure and thus offering the potential advantage of minimizing the development of resistance.

[0007] Among these "non-antibiotic" strategies, disrupting the integrity of the EPS matrix of established biofilms is particularly attractive. By making the EPS matrix more permeable, degrading bacteria, and interfering with virulence signaling networks, this strategy theoretically makes individual bacteria more accessible to the immune system and / or to enhance antibiotic action (Gunnett et al. 2016; Fleming & Rumbaugh 2017). Using this approach, a variety of treatments are under investigation, including enzymes (proteases, DNases, glycoside hydrolases), peptides, monoclonal antibodies, and polymers that target specific components of the matrix (e.g., exopolysaccharides and eDNA) (Koo et al. 2017).

[0008] Epoxytiglienones are small molecules with diverse biologically active properties. They are potent antitumor compounds, possess antiparasitic properties, and stimulate immune and other cellular responses that promote the healing of acute and chronic wounds. Epoxytiglienones have previously been reported to have direct antibiotic activity against a range of Gram-positive bacteria (WO 2007 / 070985 and WO 2014 / 169356). However, recent studies to more specifically evaluate the antibiotic properties of epoxytiglienones against pathogenic strains of three Gram-negative pathogens (E. coli, P. aeruginosa, and A. baumannii) found no antibiotic effect and failed to establish minimum inhibitory concentrations (MICs) in conventional planktonic culture systems used for routine antibiotic screening. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention is based, at least in part, on the subsequent unexpected discovery that, despite the lack of direct antibiotic activity against planktonic Gram-negative bacteria, epoxytiglienone has potent "non-antibiotic" effects against a range of established biofilms of Gram-negative bacteria. [Means for solving the problem]

[0010] According to a first aspect of the present invention, there is provided a method for dispersing a biofilm containing Gram-negative bacteria, the method comprising: [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 Alkynyl C 3-8 Cycloalkyl, C(O)aryl, C(O)C 1-6 Alkylaryl, -C(O)C 2-6Alkenylaryl and -OC(O)C 2-6 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a salt thereof.

[0011] According to another aspect of the present invention, there is provided a method of treating a bacterial infection involving a biofilm comprising Gram-negative bacteria, the method comprising: [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 Alkynyl 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 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a pharmaceutically acceptable salt thereof, to the bacterial infection.

[0012] According to a further aspect of the present invention, there is provided a method for preventing a biofilm containing Gram-negative bacteria from forming on a medical device or for dispersing a biofilm containing Gram-negative bacteria on a medical device, the method comprising: [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 3-8Cycloalkyl, -C(O)C 2-6 Alkynyl 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 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a pharmaceutically acceptable salt thereof, onto the medical device. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 provides confocal laser scanning microscopy (CLSM) images of biofilm disruption of E. coli IR57 after treatment with compounds 1, 4, and 6, compared with the comparator compound CC-1, untreated, and ethanol-matched (blank) controls. The top panel of each group of images is a top view, and the smaller bottom panels are cross-sectional views.

[0014] [Figure 2]Figure 2 provides the following: A: Biofilm biomass or biomass (μm / μm) quantified and confirmed by COMSTAT image analysis after treatment of E. coli IR57 biofilms with compounds 1, 4, and 6, compared with the comparator compound CC-1, untreated, and ethanol equivalent (blank) controls. A reduction in biomass was evident in biofilms treated with these compounds, with a significant reduction in biomass for compound 1 treatment (p < 0.05). B: There was no significant difference in the dead / live bacteria ratio between the compounds and the control treatment, indicating that disruption of E. coli IR57 biofilm density and biomass in these experiments was not related to direct antibiotic activity.

[0015] [Figure 3] Figure 3 shows a graph of the mean square displacement (MSD) over time (in seconds) of 200 nm FluoSpheres® within the E. coli IR57 biofilm structure. Disruption and alteration of the E. coli IR57 biofilm structure is indicated by significantly higher mean square displacement of FluoSphere® particles after treatment with compounds 1, 4, and 6 compared to untreated and ethanol equivalent (blank) controls.

[0016] [Figure 4] Figure 4 shows a graph of the increase in creep compliance of biofilms, indicating a decrease in the resistance to mechanical deformation of the biofilm medium resulting from the MSD and lag time, as shown in Figure 3 , after treatment with compounds 1, 4, and 6 compared to untreated and ethanol equivalent (blank) controls.

[0017] [Figure 5]Figure 5 provides the following: A: CLSM images of compounds 1, 4, 6, and comparator compound CC-1 applied to A. baumannii 7789 biofilms compared to untreated and ethanol-equivalent (blank) controls. The top panel of each image is a top view, and the small bottom panel is a cross-section. B: CLSM images of compounds 1, 4, 6, and comparator compound CC-1 applied to P. aeruginosa PAO1 biofilms compared to untreated and ethanol-equivalent (blank) controls.

[0018] [Figure 6-1] Figure 6-1 provides the following: A: Biofilm biomass or biomass (μm3 / μm2) quantified and confirmed by COMSTAT image analysis after treatment of A. baumannii 7789 biofilms with compounds 1, 4, 6, and comparator compound CC-1 compared to untreated and ethanol-equivalent (blank) controls. A significant decrease in biomass was evident in biofilms treated with compounds 1, 4, and comparator compound CC-1 (p < 0.05). B: Biofilm biomass or biomass (μm3 / μm2) quantified and confirmed by COMSTAT image analysis after treatment of P. aeruginosa PAO1 biofilms with compounds 1, 4, 6, and comparator compound CC-1 compared to untreated and ethanol-equivalent (blank) controls. Only compound 4 significantly affected biofilm biomass compared to the other treatments (p < 0.05).

[0019] [Figure 6-2] Figure 6-2 provides the following: C and D: There were no significant differences in the DEAD / LIVE bacterial ratios, as quantified and confirmed by COMSTAT image analysis, after treatment of A. baumannii 7789 and P. aeruginosa PAO1 biofilms with compounds 1, 4, 6, and comparator compound CC-1 compared to untreated and ethanol equivalent (blank) controls.

[0020] [Figure 7]Figure 7 shows cell membrane permeability data for compounds 1, 4, and 6 applied to planktonic cells of E. coli IR57, P. aeruginosa PAO1, and S. aureus (1004A; MRSA) compared to untreated and 70% isopropanol positive controls. Unlike the Gram-positive strain (MRSA 1004A), the Gram-negative strains (E. coli IR57 and P. aeruginosa PAO1) showed significant increases in cell permeability at concentrations as low as 32 μg / mL only when treated with compounds 1 and 4 at concentrations above 512 μg / mL.

[0021] [Figure 8] Figure 8 shows representative images demonstrating the induction of NETosis and necrosis in neutrophils treated with six concentrations of Compound 4 at two, three, and six hours post-treatment. NETosis / necrosis was first observed at 3 hours post-treatment at the two highest concentrations (50 and 500 μM) and at 6 hours at the four lower concentrations. At 500 μM, chromatin within the cells was condensed, indicating necrosis had occurred. At 50 μM, chromatin was more diffuse, indicating NETosis had occurred.

[0022] [Figure 9] Figure 9 shows a graph of the mean and standard deviation of the in vitro release of the human protective peptide LL-37 from neutrophils 3 hours after application of four therapeutically relevant concentrations of Compound 4. The release of LL-37 from neutrophils increased in a concentration-dependent manner. DETAILED DESCRIPTION OF THE INVENTION

[0023] Definition: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0024] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0025] As used herein, the term "about" refers to a quantity, level, value, dimension, size, or amount that varies by as much as 25%, 20%, 15%, or 10% from a reference amount, level, value, dimension, size, or amount.

[0026] Throughout this specification, unless necessary, the terms "comprise", "comprises", and "comprising" will be understood to mean the inclusion of a stated step or element, or group of steps or elements, with any other step or element or group of steps or elements, and not the exclusion of other steps.

[0027] The term "alkyl" refers to optionally substituted straight-chain and branched hydrocarbon groups having 1 to 8 carbon atoms. Where appropriate, the alkyl group can have a specified number of carbon atoms, e.g., -C1-C6 alkyl, including alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement. 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.

[0028] The term "alkenyl" refers to an optionally substituted unsaturated straight-chain or branched hydrocarbon having from 2 to 8 carbon atoms and having at least one double bond. Where appropriate, an alkenyl group can have a specified number of carbon atoms, e.g., C2-C6 alkenyl, including alkenyl groups having 2, 3, 4, 5, or 6 carbon atoms in a straight-chain or branched arrangement. Non-limiting examples of alkenyl groups include ethenyl, propenyl, 1-methylethenyl, butenyl, 1-methylprop-1-enyl, 1-methylprop-1-enyl, 1-ethylethenyl, pentenyl, 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-ethylbu-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, Hexa-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-methylhex-1-enyl, 2-methylhex-1-enyl, 3-methylhex-1-enyl, 4-methylhex-1-enyl, 5-methylhex-1-enyl, 1-methylhex-2-enyl, 2-methylhex-2-enyl, 3-methylhex-2-enyl, 4-methylhex-2-enyl, 5-methylhex-2-enyl, 1-methylhex-3-enyl, 2-methylhex-3-enyl, 3-methylhex-3-enyl, 4-methylhex-3-enyl, 5-methylhex-3-enyl, 1-methylhex-4-enyl, 2-methylhex-4-enyl, 3-methylhex-4-enyl, 4-methylhex-4-enyl, 5-methylhex-4-enyl, 1-methylhex-5-enyl, 2-methylhex-5-enyl, 3-methylhex-5-enyl, 4-methylhex-5-enyl, 5-methylhex-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-methylhept-1-enyl, 2-methylhept-1-enyl, 3-methylhept-1-enyl, 4-methylhept-1-enyl, 5-methylhept-1-enyl, 1-methylhept-2-enyl, 2-methylhept-2-enyl, 3-methylhept-2-enyl, 4-methylhept-2-enyl, 5-methylhept-2-enyl, 1-methylhept-3-enyl, 2-methylhept-3-enyl, 3-methylhept-3-enyl, 4-methylhepta -3-enyl, 5-methylhept-3-enyl, 1-methylhept-4-enyl, 2-methylhept-4-enyl, 3-methylhept-4-enyl, 4-methylhept-4-enyl, 5-methylhept-4-enyl, 1-methylhept-5-enyl, 2-methylhept-5-enyl, 3-methylhept-5-enyl, 4-methylhept-5-enyl, 5-methylhept-5-enyl, etc.

[0029] The term "alkynyl" refers to an optionally substituted unsaturated straight or branched chain hydrocarbon having 2 to 8 carbon atoms and having at least one triple bond. Where appropriate, alkynyl groups can have a specified number of carbon atoms, e.g., C2-C6 alkynyl, including alkynyl groups having 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement. Non-limiting examples include: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methylprop-2-ynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methylbut-2-ynyl, 1,1-dimethylpropynyl-2-ynyl, 3-methylbut-1-ynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 6-hexynyl, 7-hexynyl, 8-hexynyl, 9-hexynyl, 10-hexynyl, 11-hexynyl, 12-hexynyl, 13-hexynyl, 14-hexynyl, 15-hexynyl, 16-hexynyl, 17-hexynyl, 18-hexynyl, 19-hexynyl, 20-hexynyl, 21-hexynyl, 22-hexynyl, 23-hexynyl, 24-hexynyl, 25-hexynyl, 26-hexynyl, 27-hexynyl, 28-hexynyl, 29-hexynyl, 30-hexynyl, 31-hexynyl, 32-hexynyl, 33-hexynyl, 34-hexynyl, 35-hexynyl, 36-hexynyl, 37-hexyn xynyl, 3-methylpent-1-ynyl, 4-methylpent-1-ynyl, 1-methylpent-2-ynyl, 4-methylpent-2-ynyl, 3,3-dimethylbut-1-ynyl, 1,1-dimethylbut-2-ynyl, 1,2-dimethylbut-3-ynyl, 1,1-dimethylbut-3-ynyl, 2,2-dimethylbut-3-ynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl butynyl, 6-heptynyl, 3-methylhex-1-ynyl, 4-methylhex-1-ynyl, 1-methylhex-2-ynyl, 4-methylhex-2-ynyl, 3,3-dimethylpent-1-ynyl, 1,1-dimethylpent-2-ynyl, 1,2-dimethylpent-3-ynyl, 1,1-dimethylpent-3-ynyl, 2,2-dimethylpent-3-ynyl, 1-octynyl, 2-octynyl, 3-octynyl octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl, 3-methylhept-1-ynyl, 4-methylhept-1-ynyl, 1-methylhept-2-ynyl, 4-methylhept-2-ynyl, 3,3-dimethylhex-1-ynyl, 1,1-dimethylhex-2-ynyl, 1,2-dimethylhex-3-ynyl, 1,1-dimethylhex-3-ynyl, 2,2-dimethylhex-3-ynyl, and the like.

[0030] The terms "cycloalkyl" and "carbocyclic" refer to an optionally substituted saturated or unsaturated monocyclic hydrocarbon group. Where appropriate, the cycloalkyl group has a specified number of carbon atoms, e.g., C3-C6 cycloalkyl is a carbocyclic group having 3, 4, 5, or 6 carbon atoms. Non-limiting examples can include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like.

[0031] "Aryl" refers to a C6-C6 alkyl group having up to seven atoms in each ring, with at least one ring being aromatic. 14 "(Aryl)" refers to a 1-membered monocyclic, bicyclic, or tricyclic carbocyclic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and biphenyl. An aryl may contain 1 to 3 benzene rings. When two or more aromatic rings are present, the rings may be fused together so that adjacent rings share a common bond.

[0032] Each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, whether an individual entity or part of a larger entity, may be optionally substituted with one or more optional substituents selected from the group consisting of: C 1-6 Alkyl, C2 -6 Alkenyl, C 3-6 Cycloalkyl, oxo(=O), OH, -SH, C 1-6 Alkyl O-, C 2-6 Alkenyl O-, C 3-6 Cycloalkyl S-, C 1-6 Alkenyl S-, C 3-6 CycloalkylS-, 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 1-6 Alkylphenyl, Ophenyl, -C(O)phenyl, -C(O)C1-6 Examples of suitable substituents, particularly for the cycloalkyl and aryl groups of R and R, include, but are not limited to, 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, COH, COCH, -C(O)CH, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl, difluoromethoxy, difluoromethylthio, amino, methylamino, dimethylamino, phenyl, phenoxy, phenylcarbonyl, benzyl, and acetyl.

[0033] The epoxytiglienone compound can be in the form of a pharmaceutically acceptable salt.However, it will be understood that non-pharmaceutical acceptable salts are also included within the scope of the present invention, as they may be useful as intermediates in the preparation of pharmaceutically acceptable salts, or may be useful during storage or transportation, or may be useful in non-pharmaceutical environments. 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, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid, 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, mucilage, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzonesulfonic acid, salicyclicsulfanilic acid, aspartic acid, glutamic acid, edetic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0034] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium.

[0035] Basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl and diethyl sulfates; and others.

[0036] It will be appreciated that epoxytiglienone compounds may have asymmetric centers and therefore may exist in multiple stereoisomeric forms.Therefore, the present invention also relates to compounds with one or more asymmetric centers, for example, substantially pure isomeric forms with an ee of about 90% or more, for example, about 95%, 97%, or 99% or more, and mixtures thereof, including racemates.Such isomers can 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 can exist as geometric isomers.The present invention also relates to substantially pure cis (Z) or trans (E) forms of compounds or mixtures thereof.

[0037] The compounds of the present invention can be obtained by isolation from plants or plant parts, or by derivatization of isolated compounds, or by derivatization of related compounds. Isolation and derivatization procedures are described in WO 2007 / 070985 and WO 2014 / 169356.

[0038] The term "6,7-epoxytiglienone compound" refers to a compound having the following carbocyclic structure: [ka]

[0039] The compound has a tricyclo[9.3.0.0]tetradecane system with a fused cyclopropane ring attached to a six-membered ring. The epoxide is fused to the seven-membered ring at the 6 and 7 positions, and the five-membered ring has a 1,2-en-3-one structure.

[0040] The method of the present invention: The present invention provides a method for dispersing a biofilm comprising Gram-negative bacteria, wherein the method comprises dispersing a biofilm comprising a compound of formula (I): [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 Alkynyl C3-8 Cycloalkyl, C(O)aryl, C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl and -OC(O)C 2-6 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a salt thereof.

[0041] Biofilms containing Gram-negative bacteria may be present in any context suitable for treatment. Biofilms may be present on inanimate surfaces or on or within biological systems. According to some embodiments, the biofilm is present on an inanimate surface, such as a medical, laboratory, food preparation, or manufacturing surface, such as a surgical bed or floor, a laboratory bench, a kitchen bench or floor, or manufacturing equipment for pharmaceuticals, dietary supplements, cosmetics, or personal care products. According to some embodiments, the inanimate surface may be a medical device, such as a medical instrument, a surgical instrument, a catheter, a prosthesis, or an implant. According to some embodiments, the biofilm is present in an infected wound, such as a surgical wound or a burn. According to some embodiments, the biofilm is present in a subject at the site of application of a medical device, such as a catheter or an implant.

[0042] According to some embodiments, the biofilm comprises only gram-negative bacteria. According to some embodiments, the biofilm comprises only one species of gram-negative bacteria. According to other embodiments, the biofilm comprises two or more species of gram-negative bacteria. According to some embodiments, the biofilm comprises a population of gram-negative and gram-positive bacteria.

[0043] As used herein, the term "dispersal" refers to the breakdown of a biofilm such that at least a portion of the bacteria are released from the extracellular polymeric substance (EPS) matrix and assume a planktonic state, where they become accessible to the immune system and / or the action of antibiotics.

[0044] According to some embodiments, the biofilm comprising gram-negative bacteria comprises one or more gram-negative bacteria selected from: Pseudomonas spp., Acinetobacter spp., Aeromonas spp., Bacteroides spp., Bordetella spp., Borrelia spp., Burkholderia spp., Citrobacter spp., Compylobacter spp., Escherichia spp., Enterobacter spp., Flavobacterium spp., Fusobacterium spp., Klebsiella spp., Leptospira spp., Neisseria spp., Helicobacter spp., Hemophilus spp., Legionella spp., Moraxella spp., Yersinia spp., Oligella spp., Pantoea spp., Porphyromonas spp., Prevotella spp., Proteus spp., Raoutella spp., Salmonella spp., Serratia spp., Shigella spp., Sphingomonas spp. Species, Stenotophomonas sp., Treponema sp., Veillonella sp. and Vibrio sp., especially Pseudomonas sp., Acinetobacter sp., Escherichia sp., Klebsiella sp., Neisseria sp., Hemophilus sp., Legionella sp., Yersinia sp., Proteus sp. species and Salmonella species, more specifically Pseudomonas species, Acinetobacter species, Escherichia species, and Klebsiella species.According to some embodiments, the Gram-negative bacteria are particularly good biofilm formers and are selected from: Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica, Salmonella bongori, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Fusobacterium necrophorum, Burkholderia cepacian and Prevotella intermedia, in particular Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica and Salmonella bongori, more specifically Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae.

[0045] According to another aspect of the invention, the biofilm dispersed by this method is present in a bacterial infection on or in a subject. Accordingly, the invention includes a method of treating a bacterial infection comprising a biofilm comprising Gram-negative bacteria, the method comprising treating the bacterial infection with a compound of formula (I): [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 Alkynyl 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 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -OC 2-6 Alkynyl, -OC(O)C 1-6Alkyl, 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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a pharmaceutically acceptable salt thereof.

[0046] According to some embodiments, the infection is an acute infection. According to other embodiments, the infection is a chronic infection. According to some embodiments, the infection is a post-operative infection or an infection at the site of insertion or use of a medical device, for example, an insertion site of a catheter such as an intravenous catheter or a urinary catheter, or at the site of an implant, for example, a dental implant and a hip implant.

[0047] The term "topical administration" means that the compound of formula (I) is applied directly to the biofilm. According to some embodiments, topical administration may be local administration, such as by use of a lotion, cream, ointment, foam, suspension, liquid wash, or spray. According to other embodiments, topical administration may be achieved during surgery, where infected tissue is exposed, or delivered by injection guided by imaging techniques, such as endoscopic ultrasound or stereotactic imaging. According to certain embodiments, topical administration is to biofilms containing infections located in sites accessible from the outside of the body, such as the skin, wounds, mouth or nose, ear or ear canal, anus / rectum, vagina, urethra, or milk tract of livestock.

[0048] According to some embodiments, biofilm dispersal is accompanied by stimulating the immune system to resolve the infection without the need for antibiotic administration, for example, by stimulating the expression of host defense peptides in stromal and cutaneous cell types and chemokines / cytokines involved in neutrophil / granulocyte recruitment, and by inducing potent antimicrobial defenses of resident and recruited innate immune cells, including the release of reactive oxygen species, extracellular nets, and broad-spectrum antimicrobial peptides.

[0049] In other embodiments, dispersal of the biofilm involves the simultaneous administration of an antibiotic. In some embodiments, the compound of formula (I) or a salt thereof is administered in combination with an antibiotic to which the Gram-negative bacteria are susceptible when in planktonic state. Thus, once the biofilm is dispersed, the planktonic Gram-negative bacteria are exposed to the antibacterial activity of the antibiotic.

[0050] Antibiotics suitable for Gram-negative bacteria include, but are not limited to: third generation cephalosporins such as ceftazidime and ceftiofur; fourth generation cephalosporins such as cefepime; aminoglycosides such as streptomycin, neomycin, gentamicin, amikacin, tobramycin, plazomycin; monocyclic beta-lactams such as aztreonam; beta-lactam inhibitor combinations, e.g., piperacillin / tazobactam, amoxicillin / clavulanic acid, cefepime / AAI101, aztreonam / avibactam, ceftaroline / avibactam, imipenem / relebactam and meropenem / RPX7009; beta-lactams such as penicillins, cephalosporins, S649266 and BAL30072; carbapenems such as imipenem, doripenem, ertapenem, and meropenem; polymyxin-E antibiotics such as colistin; Quinolones / fluoroquinolones such as ciprofloxacin, fleroxacin, norfloxacin, enrofloxacin, marbofloxacin, finafloxacin, lascufloxacin, abalofloxacin, nadifloxacin, delafloxacin, nemonoxacin, and zavofloxacin; 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.

[0051] The term "combination" means that the compound of formula (I) and the antibiotic are active in the biofilm, resulting in disruption of the biofilm and exposure of planktonic bacteria to the antibiotic. According to some embodiments, the combination of the compound of formula (I) and the antibiotic is administered in a single composition. According to other embodiments, the compound of formula (I) and the antibiotic are administered simultaneously or sequentially in separate compositions.

[0052] Subjects that may be infected with bacteria and treated include mammals, birds, aquatic animals such as fish, or reptiles. According to some embodiments, the subject is a human, a laboratory animal, such as a primate, a mouse, rat or rabbit, a companion animal such as a dog or cat, a working animal, such as a horse, donkey, or farm animal, such as a cow, bull, pig, sheep, goat, deer, llama, alpaca, or captive wild animal, such as an animal in a zoo or wildlife park, for example a lion, leopard, cheetah, elephant, striped duiker, antelope, giraffe, koala, kangaroo, reptile, such as a crocodile, lizard, snake, or bird, particularly poultry such as a chicken, duck, goose, quail, pheasant, or captive bird, such as a badger or canary, cockatoo, parakeet, macaw, parrot, or fish, particularly captive fish, such as aquacultured fish (such as salmon, trout, barramundi) or tropical fish (such as zebra fish, guppy, Siamese fighting fish, clown fish, cardinal tetra, etc.), dolphin, whale, or the like. In certain embodiments, the subject is a human or a companion animal.

[0053] By "effective amount," we mean the amount necessary to at least partially achieve a desired response, such as dispersal of a biofilm within an infected area. The amount will vary depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the composition formulation, an evaluation of the medical condition, and other relevant factors. Effective amounts of 6,7-epoxytiglienone compounds are expected to fall within a relatively broad range that can be determined through routine testing. For example, an effective dose relevant to a human patient may be approximately 0.1 ng / kg to 1 g / kg of body weight per dose, or 1 cm of body surface area per dose. 2 The dosage may range from 0.1 ng to 1 g per kg of body weight or cm of body surface area. 2 in the range of 1 μg to 1 g per dose, e.g., 1 mg to 1 g per kg of body weight or per cm of body surface area per dose 2 According to one embodiment, the dosage ranges from 1 mg to 1 g per dose per kg of body weight, or from 0.1 ng to 500 mg per dose per cm of surface area. 2According to another embodiment, the dosage ranges from 1 mg to 250 mg per kg of body weight or from 1 mg to 250 mg per cm of surface area. 2 According to yet another embodiment, the dosage ranges from 1 mg to 100 mg per kg of body weight or from 1 mg to 100 mg per cm of surface area. 2 range, e.g., up to 50 mg per dose / kg of body weight, or 50 mg per dose / cm of surface area 2 According to yet another embodiment, the dosage ranges from 1 μg to 1 mg per kg of body weight per dose, or from 1 μg to 1 mg per cm of surface area per dose. 2 The range is.

[0054] According to a further aspect of the present invention, the biofilm dispersed by this method is present or potentially present on a medical device. Thus, the present invention includes a method for preventing a biofilm containing Gram-negative bacteria from forming on a medical device, or a method for dispersing a biofilm containing Gram-negative bacteria on a medical device, wherein the method comprises: [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 Alkynyl 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 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a pharmaceutically acceptable salt thereof to a medical device.

[0055] According to some embodiments, medical devices are coated with a composition comprising a compound of formula (I) or a salt thereof to prevent the formation of biofilms containing Gram-negative bacteria. According to other embodiments, medical devices are washed or coated with a composition comprising a compound of formula (I) to disperse biofilms containing Gram-negative bacteria and remove them from the device.

[0056] As used herein, the term "medical device" refers to a device used on the human or animal body for therapeutic benefit, having a physical or mechanical effect on the body, or used to measure or monitor bodily functions. Suitable medical devices include: indwelling medical devices such as urinary catheters, vascular access devices, endotracheal tubes, tracheotomies, enteral feeding tubes, and wound drains; invasive medical devices such as central venous catheters, mechanical heart valves, pacemakers, prosthetic or artificial joints, pins, rods, screws, and plates for fixing fractured bones; catheters such as urinary catheters, intravenous catheters, Swan-Ganz catheters, Quinton catheters, intrauterine catheters, drainage catheters, and pigtail catheters; prostheses such as limb prostheses, dentures, obturators, dental prostheses including dental implants; implants such as cochlear implants, coronary stents, contraceptive implants, cosmetic implants, and dental implants. Medical devices as used herein include: medical instruments, such as syringes, speculum, sphygmomanometers, scanners, ultrasound probes, and the like; and surgical instruments, such as scalpels, forceps, clamps, retractors, lancets, endoscopes, calipers.

[0057] According to some embodiments, the cleaning agent or coating comprises a disinfectant, antiseptic, or antibiotic.

[0058] The present invention also relates to the use of a compound of formula (I): [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 Alkynyl 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 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -OC 2-6 Alkynyl, -OC(O)C 1-6 Alkyl, OC(O)C 2-6 Alkenyl, -OC(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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a pharmaceutically acceptable salt thereof.

[0059] According to another aspect, the present invention provides a compound of formula (I) for use in the treatment of a bacterial infection, including a biofilm comprising Gram-negative bacteria, wherein said compound of formula (I) has the following formula (I): [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; R6 is hydrogen, -C 1-6 Alkyl, -C2-6 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 Alkynyl 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 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a pharmaceutically acceptable salt thereof, and said compound is for topical administration to a bacterial infection.

[0060] 6,7-Epoxytiglienone Compounds: Compounds useful in the methods of the present invention have the following formula (I): [ka] [Wherein R1 is hydrogen and C 1-6 alkyl; R2 is -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 alkenyl, and —OC(O)C2-7 alkynyl; R3 is -OH, -OC 1-8 Alkyl, -OC 2-8 Alkenyl, -OC 2-8 Alkynyl, -OC(O)C 1-7 Alkyl, -OC(O)C 2-7 Alkenyl, and -OC(O)C 2-7 alkynyl; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; 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 Alkynyl 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 alkynylaryl; R7 is hydroxy, -OC 1-6 Alkyl, -OC2-6 alkenyl, -OC 2-6 Alkynyl, -OC(O)C1-6 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 Alkynyl 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 alkynylaryl; R8 is hydrogen or C 1-6 or a salt thereof, particularly a pharmaceutically acceptable salt thereof.

[0061] According to certain embodiments of formula (I), one or more of the following apply: R1 is -C 1-3 alkyl, in particular -CH3; 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 alkenyl; R3 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, -OC(O)C 3-6 Alkenyl and -OC(O)C 3-6 alkynyl; R4 and R5, particularly when both are methyl, may be -C 1-3 independently selected from alkyl; R6 is hydrogen, C(O)C 1-6 Alkyl, C(O)C2-6 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; R7 is hydroxyl, OC(O)C 1-6 Alkyl, OC(O)C 2-6 Alkenyl, and -CO)C 2-6 alkenyl, in particular hydroxyl; R8 is C 1-3 Alkyl, especially methyl.

[0062] According to some embodiments, the compound of formula (I) has the following formula (II): [ka] It has the stereochemistry as shown in

[0063] According to some embodiments, the epoxides at the 6 and 7 positions are above the plane of the ring system. According to other embodiments, the epoxides at the 6 and 7 positions are below the plane of the ring system. According to some embodiments, the R2 group at the 12 position is S, and according to other embodiments, the R2 group at the 12 position is R.

[0064] According to some embodiments of Formula (I), the alkyl or alkenyl group of R2 and / or R3 is a branched alkyl or alkenyl group. According to other embodiments of Formula (I), the alkyl or alkenyl group of R2 and / or R3 is a linear alkyl or alkenyl group.

[0065] According to some embodiments, the alkyl or alkenyl groups of R2 and / or R3 have a moderate degree of hydrophobicity, for example, a chain length of C4, C5, or C6.

[0066] According to some embodiments, R6 is an acyl group, such as acetyl (-C(O)CH3), C(O)CH2CH3, -C(O)CH(CH3)2, or -C(O)CH2CH2CH3. According to some embodiments, R6 is hydrogen.

[0067] According to certain embodiments, the epoxytiglienone compound is selected from: 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-ditigloyl-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-Hexa-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-(2E,4E)-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 17); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 18); 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 19); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiaen-3-one (compound 20); and 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 21); or a salt thereof, particularly a pharmaceutically acceptable salt thereof.

[0068] According to a particular embodiment, the 6,7-epoxytiglienone compound is selected from compounds 1, 2, 3, 4 and 6.

[0069] Epoxytiglienone compounds can be obtained by extraction, or can be obtained semi-synthetically from extracted compounds.Methods for obtaining epoxytiglienone compounds are provided in WO 2007 / 070985 and WO 2014 / 169356, the contents of which are incorporated by reference.

[0070] Composition: Although the 6,7-epoxytiglienone compound or its salt can be used alone, it may be more convenient to use them in the form of a composition together with a carrier, diluent, and / or excipient. According to some embodiments, the composition may be a solution useful for immersing a medical device. According to other embodiments, the composition may be a coating composition useful for coating a medical device. According to still other embodiments, the composition may be a pharmaceutical composition suitable for administration to a patient.

[0071] The dosage form and pharmaceutical use and proportion of the composition can be readily determined by one skilled in the art.

[0072] The 6,7-epoxytiglienone compound is formulated for topical administration onto or to a biofilm. According to some embodiments, the 6,7-epoxytiglienone compound is formulated for topical administration in the form of a gel, ointment, lotion, cream, or transdermal patch that can be applied directly to the biofilm. According to other embodiments, the epoxytiglienone compound is formulated for injection, for example, by injecting the composition internally to locally contact the biofilm.

[0073] In some embodiments, the composition is suitably a pharmaceutical composition and includes a pharmaceutically acceptable excipient or excipients. A "pharmaceutically acceptable excipient" refers to a solid or liquid filler, diluent, or encapsulating substance that can be safely used. Depending on the particular route of administration, various carriers well known in the art can be used. These carriers or excipients can be selected from the group including: sugars, starches, cellulose and its derivatives, cyclodextrins, malt, gelatin or other gelling agents, polymers, talc, calcium sulfate, vegetable oils, synthetic oils, alcohols and / or polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and pyrogen-free water.

[0074] Liquid preparations include solutions, suspensions, and emulsions, such as water or water-pyrogenic glycol solutions. For example, injectable liquid preparations can be formulated as solutions in 1,2-propanediol in water, dimethyl sulfoxide (DMSO), gamma cyclodextrin or 2-hydroxypropyl-β-cyclodextrin in water, saline, or polyethylene glycol, with or without buffer. The preferred pH range is 3.0-4.5. Suitable buffers buffer the preparation at a pH between 3.5 and 4.5 and include, but are not limited to, acetate and citrate buffers.

[0075] Thus, the 6,7-epoxytiglienone compound composition can be formulated for administration (e.g., by injection, e.g., bolus injection at the biofilm infection site) and presented in unit dosage form in ampoules, prefilled syringes, small insufflators, or multi-dose containers with added preservatives. The composition can take the form of a suspension, solution, gel, or emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient can be obtained by aseptic isolation of a sterile solid or lyophilization from a solution for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0076] Pharmaceutical compositions of 6,7-epoxytiglienone compounds suitable for administration can be presented in discrete units, such as syringes, vials, tubes, or sachets, each containing a predetermined amount of one or more pharmaceutically active 6,7-epoxytiglienone compounds as a powder or granules, or as a solution or suspension in an aqueous liquid, a cyclodextrin solution, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion, or as a solution or suspension in a cream or gel, or as a suspension of microparticles or nanoparticles incorporating the 6,7-epoxytiglienone compounds, including, but not limited to, silica or polylactide microparticles or nanoparticles. Such compositions can be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more pharmaceutically active compounds of the present invention with the carrier, which constitutes one or more necessary ingredients. Generally, compositions are prepared by uniformly and intimately admixing the agent of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired presentation.

[0077] For topical administration to the epidermis or other organs, the compounds according to the present invention can be formulated as gels, ointments, emulsions, pastes, creams, or lotions, or as transdermal patches. Gels can be prepared using suitable thickeners, which can be added to aqueous / alcoholic compositions of the compounds. Suitable thickeners or gelling agents, such as polyvinyl carboxypolymer Carbomer 940, are known in the art. Ointments and creams can be formulated, for example, with an aqueous or oily base, with the addition of suitable thickeners and / or gelling agents. Lotions can be formulated with an aqueous or oily base, and generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners, or colorants.

[0078] Formulations suitable for topical administration also include solutions or suspensions which can be applied topically in the form of a bath or soak or spray, or which can be absorbed into a dressing.

[0079] 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 include other ingredients such as emulsifiers, surfactants, preservatives, disinfectants, antibiotics, colorants, etc.

[0080] The coating may be any suitable polymeric coating known in the art that can incorporate a compound of formula (I) such that the coating is accessible at the surface of the coating 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, and polymer coatings, calcium phosphate, chitosan, collagen, and drug-eluting coatings such as bone graft / cancellous bone.

[0081] References: Where a prior art publication is referenced herein, it should be understood that such reference is not an admission that the publication forms part of the general knowledge in the art in Australia or anywhere else. Brinkmann V et al. 2010. Neutrophil extracellular traps: How to generate and visualize. Them. J. Vis. Exp. 36: e 1724. 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. Cepas V et al. 2019. Relationship between biofilm formation and antimicrobial resistance in Gram-negative bacteria. Microb. Drug Resist. 25: 72-79. Dhall S et al. 2014. “Generating and Reversing Chronic Wounds in Diabetic Mice by Manipulating Wound Redox Parameters.” J. Diabetes Res. 562625. Doi et al. 2017. Gram-negative bacterial infections: Research priorities, accomplishments and future directions. Clin Infect Dis. 64 (S1): S30-S35 Fleming D & Rumbaugh KP 2017. Approaches to dispersing medical biofilms. Microorganisms 2017 5, 15. 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. Hoiby et al. 2010. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents 35: 322-332. Hoiby N et al. 2015. ESCMID guideline for the diagnosis and treatment of biofilm infections. Clin. Microbiol. Infect. 21 (Suppl. 1): S1-S25. Ho J et al. 2010. Multiresistant Gram-negative infections: a global perspective. Curr. Opin. Infect. Dis. 23: 546-53. 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. Koo H et al. 2017. Targeting microbial biofilms: current and prospective therapeutic strategies. Nat. Rev. Microbiol. 15: 740-755. Powell et al. 2018 Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides. NPJ Biofilms Microbiomes. 4: 13. Zhao et al. 2010. Delayed wound healing in diabetic (db / db) mice with Pseudomonas aeruginosa biofilm challenge: a model for the study of chronic wounds. Wound Repair Regen. 18: 467-477. [Example]

[0082] The compounds of the present invention can be obtained by isolation from plants or plant parts, or by derivatization of isolated compounds, or by derivatization of related compounds. Isolation and derivatization procedures are described in WO 2007 / 070985 and WO 2014 / 169356.

[0083] Example 1: Epoxytigilan has no direct antibiotic activity against Gram-negative bacteria in planktonic culture systems: The effects of five epoxytiglienones (compounds 1, 2, 3, 4, and 6) and two comparator compounds (epoxytiglienones with a long carbon chain at the C12 position) on six human pathogens (two Gram-positive and four Gram-negative bacteria) were measured in a conventional planktonic culture system. Minimum inhibitory concentration (MIC) assays (standard assays used to define and quantify antibiotic activity) were performed to determine the antibacterial activity of each epoxytiglienones against each bacterial strain.

[0084] The Gram-positive bacteria used in this study were methicillin-resistant Staphylococcus aureus (MRSA) 1004A and 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 assays were performed on planktonic cultures of each bacteria grown in Mueller-Hinton medium using the standard broth dilution method described by Jorgensen et al. (1999) using epoxytiglienone dissolved in ethanol. The results are shown in Table 1 below.

[0085] [Table 1]

[0086] The results showed that the direct antibacterial properties of epoxytiglienone in planktonic cultures were limited to Gram-positive bacteria. Although the MIC values ​​of the compound could not be determined for any of the tested Gram-negative bacteria, they were all unaffected (confluent bacterial growth at concentrations of the test compound above 512 μg / mL).

[0087] Example 2: Epoxytiglienone disrupts established biofilms of Gram-negative E. coli: The effects of administration of compounds 1, 4, and 6 and the comparator compound CC-1 on disruption of established in vitro biofilms of Escherichia coli (E. coli) were investigated using the method described by Powell et al. (2018).

[0088] E. coli IR57 biofilms were cultured in 96-well glass-bottom plates in Mueller-Hinton (MH) medium for 24 hours, after which 50% of the supernatant was replaced with fresh MH medium (vehicle: ethanol) supplemented with or without epoxytiglienone compounds to a final concentration of 256 μg / mL. An ethanol-equivalent blank was used as an additional control treatment. The plates were then incubated at 37°C for an additional 24 hours.

[0089] Biofilms were then imaged using confocal laser scanning microscopy (CLSM) after staining with Live / Dead® Baclight stain, with phosphate-buffered saline (PBS) added to each well before imaging with z-stack CLSM. The resulting images were analyzed with COMSTAT software to determine (i) biofilm biomass or biomass (μm 3 / μm 2 ) and (ii) measurements of the DEAD / LIVE bacteria ratio were generated.

[0090] CLSM showed significant differences between treatments in the distribution of bacteria within the biofilm. Compounds 1, 4, and 6 caused significant changes in the distribution and density of bacteria within the biofilm compared to untreated and ethanol equivalent (blank) controls (Figure 1); this was quantified and confirmed with COMSTAT image analysis. The significant reduction in the amount of bacteria was This was evident in biofilms treated with compound 1 (p < 0.05; Figure 2A). The comparator compound CC-1 had no apparent effect on biofilm distribution and density or biomass. Interestingly, there were no significant differences in the DEAD / LIVE bacterial ratios (Figure 2B) between either of the epoxytiglienone and the two control treatments, indicating that the effect of epoxytiglienone on biofilm density and biomass in these experiments is independent of direct antibiotic activity.

[0091] Example 3: Epoxytiglienone disrupts the extracellular matrix and increases particle diffusion through established biofilms of Gram-negative E. coli bacteria: The effects of three epoxytiglienones (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 uses microscopy to simultaneously track micron-sized particles through a biofilm, allowing for the determination of diffusion-based parameters of particles embedded within the biofilm's extracellular polymeric matrix (EPS) (Cao et al. 2016). MPT measurements also allow for the calculation of microrheological properties of the biofilm structure after treatment with test compounds.

[0092] E. coli biofilms were established and treated with epoxytiglienone and control treatments as described in Example 2 above. Twenty-four hours after treatment application, biofilms were stained with SYTO9®, and 0.0025% negatively charged, carboxylic acid-modified FluoSpheres® (200 nm) were added to the biofilms and incubated for an additional 2 hours. The movement of FluoSpheres® particles within the biofilms was then captured on video using an epifluorescence microscope equipped with a high-frame-rate camera (33 ms). ImageJ software (Mosaic) was used to track particle trajectories before calculating three parameters for the 200 nm FluoSpheres® particles: (i) ensemble diffusion coefficient (Deff), (ii) ensemble mean-squared displacement (MSD), and (iii) creep compliance (a measure of resistance to mechanical deformation derived from the MSD versus lag time). Each treatment was replicated three times.

[0093] All three epoxytiglienone compounds tested increased particle diffusion through established E. coli biofilms by 80–420-fold compared to the control treatment (Table 2 ).

[0094] [Table 2]

[0095] The significant disruption and alteration of the structure of the E. coli biofilm matrix by epoxytiglienone was indicated by the substantially higher mean square displacement values ​​of FluoSpheres® particles after treatment (Figure 3), and the decreased resistance of the treated biofilms to mechanical deformation was evident in the increased creep compliance of the epoxytiglienone-treated biofilms compared to the control (Figure 4).

[0096] Example 4: Epoxytiglienone significantly reduces the biomass of established biofilms of two other Gram-negative pathogens, P. aeruginosa and A. baumannii: The effect of epoxytiglienones (compounds 1, 4, 6 and comparator compound CC-1) on the disruption of established in vitro biofilms was further investigated in two other Gram-negative bacteria (Pseudomonas aeruginosa PA01 and Acinetobacter baumannii 7789) using the methods described above in Example 2. Cell permeability after treatment with compounds 1, 4, and 6 was also determined using SYTOX® Green Nucleic Acid Stain with an untreated control and a 70% isopropanol positive control.

[0097] Consistent with the results of the E. coli study, the epoxytiglienone compounds significantly altered bacterial distribution and density within A. baumannii biofilms compared to untreated and ethanol-equivalent (blank) controls (Figure 5A). This was also reflected in the effects measured on biomass in A. baumannii biofilms (Figure 6A), with significant reductions observed for compounds 1, 4, and CC-1. For P. aeruginosa, only compound 4 significantly affected biofilm distribution (Figure 5B) and biomass compared to the other treatments (Figure 6B). The DEAD / LIVE bacterial ratio was also assessed, and similar to Example 2 using E. coli, no differences were observed between either A. baumannii or P. aeruginosa treatments (Figure 6C and D). Cell permeability studies of E. coli and P. aeruginosa (Figure 7) showed a significant increase in permeability only when treated with compounds 1 and 4 at concentrations above 512 μg / mL, which is greater than the compound concentration (256 μg / mL) used in the biofilm disruption assay. This further confirms that the effect of epoxytiglienone on biofilm density and biomass in these experiments is independent of direct antibiotic activity in killing bacteria within the biofilm.

[0098] Example 5: Epoxytiglienone disintegrates established biofilms in vivo in a mouse model of chronic biofilm infection: The effect of compound 4 administration on biofilm infection in vivo was studied in a diabetic mouse model of chronic biofilm infection (Zhao et al. 2010). This study used the method described by Dhall et al. (2014), in which bacterial biofilm infection develops spontaneously after wound creation.

[0099] Briefly, db / db diabetic mice (>6 months old) were housed under non-sterile conditions for 4–5 weeks, after which a 6 mm diameter excision punch biopsy was created on the back of each mouse. Next, a catalase inhibitor (1 g / kg aminotriazole) and a glutathione peroxidase (GPx) inhibitor (1 g / kg mercaptosuccinic acid) were administered topically around the edge of the wound site before bandaging with Tegaderm. After 24 h, biofilms were observed at the wound site in all mice. Mice were divided into two groups (7 mice per group) and treated with either Compound 4 (0.3 mg / mL in hydrogel vehicle) or control treatment (hydrogel vehicle only). The Tegaderm dressing was then replaced. Two additional doses of Compound 4 or control (vehicle only) were administered on days 8 and 15. No antibiotics or other antimicrobial treatments were applied during the study.

[0100] The presence of biofilm and wound surface area were assessed over the course of the study. Biopsies were taken from mice treated with Compound 4 and vehicle alone (control) from 21 to 28 days after wounding for histological and histochemical analysis of the wound site. Only one of seven wounds in the control group healed within this period, with fibrous sloughing and the presence of bacterial infiltration clearly evident at the wound site. In infected wounds treated with epoxytiglienone, complete wound healing was evident in six of seven mice treated with Compound 4 (with complete re-epithelialization, resolution of inflammation, and absence of bacteria within the dermis).

[0101] Example 6: Epoxytiglienone treatment of human adult keratinocytes and fibroblasts in vitro induces upregulation of chemokines / cytokines involved in neutrophil recruitment: In addition to disrupting the structure of biofilms containing Gram-negative bacteria, the effect of epoxytiglienone on the regulation of genes involved in the host response to bacterial infection was investigated in vitro in microarray studies using human adult epidermal keratinocytes (HEKa) and human adult dermal fibroblasts (HDF).

[0102] In these studies, HEKa were cultured in EpiLife® medium supplemented with supplement S7 (both from Life Technologies, Carlsbad, CA, USA), and HDFs were cultured in medium 106 (both from Life Technologies) supplemented with LSGS (low serum growth supplement). Cells were then 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 a Qiagen RNeasy mini kit and biotinylated using Illumina TotalPrep RNA Amplification (Ambion, Austin, TX, USA). Labeled RNA was hybridized to HumanHT-12v4 BeadChip Arrays (Illumina Inc, San Diego, CA, USA) and scanned according to standard Illumina protocols. Data were extracted in GenomeStudio (Illumina) using default analysis settings and no normalization methods. The resulting data were imported into GeneSpring GX (Agilent, Santa Clara, CA, USA). Expression values ​​were normalized using quantile normalization with default settings.

[0103] In both HDFs and HEKa in vitro, treatment with compound 4 (170 nM) induced significant upregulation of two key chemokines / cytokines (IL8, CXCL1) involved in PMNL recruitment within 2-4 hours. In HEKa, compound 4 significantly upregulated the production of host defense peptides (DEFB2, DEFB3, DEFB4, RNASE7) within 8-72 hours after treatment.

[0104] Example 7: Epoxytiglienone treatment induces NETosis / necrosis and release of the antimicrobial peptide cathelicidin LL-37 from isolated human neutrophils in vitro: Neutrophils are the most abundant white blood cells in the blood and constitute the first line of host defense against infectious pathogens. Central to their function is their ability to be recruited to sites of infection, recognize microorganisms, and then become activated and kill pathogens through a combination of phagocytic and cytotoxic mechanisms. These mechanisms used by neutrophils to kill pathogens include: (i) generation of reactive oxygen species, (ii) shedding of nuclear chromatin content (coated with histones, proteases, granular, and cytoplasmic proteins) to immobilize and capture pathogens (a process known as NETosis), and (iii) release of antimicrobial peptides.

[0105] After identifying the effect of epoxytiglienone compound in upregulating the production in HDFs and HEKa of chemokines and cytokines involved in neutrophil recruitment (see Example 6 above), we investigated the effect of compound 4 on two aspects of neutrophil function: NETosis / necrosis and antimicrobial peptide release.

[0106] In these assays, neutrophils were isolated from fresh blood of healthy human donors by lysing the red blood cell pellet obtained by Ficoll-Paque sedimentation. Neutrophils (approximately 4 x 10 6 Cells / mL) were incubated with 10 µg / mL dihydroethidium (DHE) (Sigma-Aldrich) in complete medium for 15 min at 37 °C, along with an aliquot of unstained cells to be tested as an unstained control.

[0107] The NETosis / necrosis assay followed the method fully described by Brinkmann et al. (2010). Isolated neutrophils were plated in 96-well plates (RPMI 1640, 10% FCS) and incubated with a 1:50,000 dilution of Hoescht (10 mg / mL) and Sytox® Green (5 mM). Compound 4 was added at six concentrations (0.005, 0.05, 0.5, 50, and 500 μM), and the cells were incubated at 37°C under 5% CO2. Vehicle-treated controls were included in all assays, with three replicates for each assay. Hoescht / Sytox® Green fluorescence images were recorded for each well at 3 and 6 hours.

[0108] Neutrophil NETosis and necrosis were first observed at 3 h after treatment with the two highest concentrations of compound 4 (50 μM and 500 μM) and were evident at 6 h with the four lower concentrations (0.005 μM–5 μM; Figure 8 ).

[0109] To examine antimicrobial peptide release by neutrophils in response to treatment with Compound 4, isolated neutrophils were incubated with either vehicle or Compound 4 at four concentrations (62.5, 125, 250, and 500 μM). After 3 h, cell culture supernatants were removed and tested for LL-37 content using an ELISA kit for LL-37 (HycultBiotech). ELISA readings were normalized to the vehicle-only control to determine the fold increase in LL-37 release.

[0110] LL-37 release from neutrophils 3 hours after treatment with compound 4 increased in a concentration-dependent manner (Figure 9). At concentrations of 125–500 μM compound 4, LL-37 release was 3–5 times higher than that of the control treatment (Figure 9).

[0111] The data from this example demonstrate that at therapeutically relevant concentrations in vitro, compound 4 induces the transition of suicidal neutrophil NETosis to necrosis, resulting in the release of the potent antimicrobial defense peptide LL-37.

[0112] The data in Examples 6 and 7 above indicate that in addition to its direct effect of disrupting Gram-negative biofilm structure, epoxytiglienone may also induce local innate immune responses in both migratory / resident myeloid cells (e.g., neutrophils) and skin and stromal cell types. Such responses suggest that the effects of epoxytiglienone alone may be sufficient to resolve many biofilm infections without the need for conventional antibiotics.

Claims

1. A pharmaceutical composition for treating a bacterial infection involving a biofilm containing Gram-negative bacteria, the pharmaceutical composition being for topical administration, and comprising a compound of the following formula (II): 【Chemistry 1】 [In the formula, R １ is hydrogen and C １－６ alkyl; R ２ is -OC(O)C １－７ Alkyl, —OC(O)C ２－７ alkenyl, and —OC(O)C2-7 alkynyl; R ３ is -OC(O)C １－７ Alkyl, —OC(O)C ２－７ Alkenyl, and —OC(O)C ２－７ alkynyl; R ４ and R ５ is hydrogen and C １－６ independently selected from alkyl; R ６ is hydrogen and -C(O)C １－６ alkyl; R ７ is hydroxy; and R ８ is hydrogen or C １－６ alkyl] or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition as described in claim 1 or a pharmaceutical composition as described in claim 1, wherein the local administration is a topical administration.

3. The following: i) the infection is a post-operative infection; ii) the infection is an infection at the site of insertion of a medical device; iii) the infection is an infection at the implant site; or iv) the infection is a chronic bacterial infection; 3. The pharmaceutical composition according to claim 1, wherein any one of the following is applied.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the administration is performed in combination with an antibiotic that acts on the gram-negative bacteria in a planktonic state.

5. The following: i) R 1 is C 1-3 alkyl; ii) R 2 is selected from —OC(O)C 1-7 alkyl and —OC(O)C 2-7 alkenyl; iii) R 3 is selected from —OC(O)C 1-7 alkyl and —OC(O)C 2-7 alkenyl; iv) R 4 and R 5 are each methyl; and v) R 8 is C 1-3 alkyl; The pharmaceutical composition according to any one of claims 1 to 4, wherein one or more of the following applies:

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein R 2 is selected from -OC(O)C 3-6 alkyl and -OC(O)C 3-6 alkenyl.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein R 3 is selected from -OC(O)C 3-6 alkyl and -OC(O)C 3-6 alkenyl.

8. The pharmaceutical composition according to claim 1, wherein R 6 is selected from hydrogen and —C(O)CH.

9. The following: i) the alkyl or alkenyl group of R 2 and / or R 3 is a branched alkyl or alkenyl group; or ii) the alkyl or alkenyl group of R 2 and / or R 3 is a straight-chain alkyl or alkenyl group; The pharmaceutical composition according to any one of claims 1 to 8, wherein any one of the following is applied.

10. The compound of formula (II) 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-ditigloyl-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-Hexa-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-(2E,4E)-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 17); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 18); 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 19); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 20); and 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 21); The pharmaceutical composition according to any one of claims 1 to 9, wherein the compound is selected from the group consisting of benzodiazepine, benzodiazepine, benzophenone, ...

11. The biofilm containing Gram-negative bacteria is Pseudomonas species, Acinetobacter species, Aeromonas species, Bacteroides species, Bordetella species, Borrelia species, Burkholderia species, Citrobacter species, Compylobacter species, Escherichia species, Enterobacter species, Flavobacterium species, Fusobacterium species. Species, Klebsiella sp., Leptospira sp., Neisseria sp., Helicobacter sp., Hemophilus sp., Legionella sp., Moraxella sp., Yersinia sp., Oligella sp., Pantoea sp., Porphyromonas sp., Prevotella sp., Proteus sp., Raoutella sp., Salmonella sp., Serratia sp., Shigella Species, Sphingomonas sp., Stenotophomonas 11. The pharmaceutical composition according to claim 1, comprising at least one Gram-negative bacterium selected from the group consisting of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica, Salmonella bongori, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Fusobacterium necrophorum, Burkholderia cepacian and Prevotella intermedia.

12. In the manufacture of a medicament for the treatment of a bacterial infection, including a biofilm comprising Gram-negative bacteria, a compound of formula (II): 【Chemistry 2】 [In the formula, R １ is hydrogen and C １－６ alkyl; R ２ is -OC(O)C １－７ Alkyl, —OC(O)C ２－７ alkenyl, and —OC(O)C2-7 alkynyl; R ３ is -OC(O)C １－７ Alkyl, —OC(O)C ２－７ Alkenyl, and —OC(O)C ２－７ alkynyl; R ４ and R ５ is hydrogen and C １－６ independently selected from alkyl; R ６ is hydrogen and -C(O)C １－６ alkyl; R ７ is hydroxy; and R ８ is hydrogen or C １－６ alkyl] or a pharmaceutically acceptable salt thereof, wherein the medicament is for topical administration.

13. 13. The use according to claim 12, wherein the local administration is a topical administration.

14. below: i) the infection is a post-operative infection; ii) the infection is an infection at the site of insertion of a medical device; iii) the infection is an infection at the implant site; or iv) the infection is a chronic bacterial infection; 14. The use according to claim 12 or 13, wherein one of the following applies:

15. The use according to any one of claims 12 to 14, wherein the administration is carried out in combination with an antibiotic that acts on the gram-negative bacteria in a planktonic state.

16. The following: i) R 1 is C 1-3 alkyl; ii) R 2 is selected from —OC(O)C 1-7 alkyl and —OC(O)C 2-7 alkenyl; iii) R 3 is selected from —OC(O)C 1-7 alkyl and —OC(O)C 2-7 alkenyl; iv) R 4 and R 5 are each methyl; and v) R 8 is C 1-3 alkyl; The use according to any one of claims 12 to 15, wherein one or more of the following applies:

17. The use according to any one of claims 12 to 16, wherein R 2 is selected from -OC(O)C 3-6 alkyl and -OC(O)C 3-6 alkenyl.

18. The use according to any one of claims 12 to 17, wherein R 3 is selected from -OC(O)C 3-6 alkyl and -OC(O)C 3-6 alkenyl.

19. The use according to any one of claims 12 to 18, wherein R 6 is selected from hydrogen and -C(O)CH.

20. The following: i) the alkyl or alkenyl group of R 2 and / or R 3 is a branched alkyl or alkenyl group; or ii) the alkyl or alkenyl group of R 2 and / or R 3 is a straight-chain alkyl or alkenyl group; The use according to any one of claims 12 to 19, wherein any one of the following applies.

21. The compound of formula (II) 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-ditigloyl-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-Hexa-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-(2E,4E)-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 17); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 18); 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 19); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 20); and 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 21); The use according to any one of claims 12 to 20, wherein the compound is selected from the group consisting of benzodiazepine, benzodiazepine, benzophenone, ...

22. The biofilm containing Gram-negative bacteria is Pseudomonas species, Acinetobacter species, Aeromonas species, Bacteroides species, Bordetella species, Borrelia species, Burkholderia species, Citrobacter species, Compylobacter species, Escherichia species, Enterobacter species, Flavobacterium species, Fusobacterium species. Species, Klebsiella sp., Leptospira sp., Neisseria sp., Helicobacter sp., Hemophilus sp., Legionella sp., Moraxella sp., Yersinia sp., Oligella sp., Pantoea sp., Porphyromonas sp., Prevotella sp., Proteus sp., Raoutella sp., Salmonella sp., Serratia sp., Shigella Species, Sphingomonas sp., Stenotophomonas 22. The use according to any one of claims 12 to 21, comprising at least one Gram-negative bacterium selected from the species Treponema, Veillonella and Vibrio, in particular the Gram-negative bacterium is selected from Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica, Salmonella bongori, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Fusobacterium necrophorum, Burkholderia cepacian and Prevotella intermedia.

23. 1. A method for preventing or dispersing the formation of a biofilm comprising gram-negative bacteria on an inanimate surface or on a medical device, the method comprising: 【Transformation 3】 [In the formula, R １ is hydrogen and C １－６ alkyl; R ２ is -OC(O)C １－７ Alkyl, —OC(O)C ２－７ alkenyl, and —OC(O)C2-7 alkynyl; R ３ is -OC(O)C １－７ Alkyl, —OC(O)C ２－７ Alkenyl, and —OC(O)C ２－７ alkynyl; R ４ and R ５ is hydrogen and C １－６ independently selected from alkyl; R ６ is hydrogen and -C(O)C １－６ alkyl; R ７ is hydroxy; and R ８ is hydrogen or C １－６ alkyl] or a pharmaceutically acceptable salt thereof onto said inanimate surface or medical device.

24. 24. The method of claim 23, wherein the method is for preventing or dispersing the formation of a biofilm comprising gram-negative bacteria on a medical device.

25. 25. The method of claim 24, wherein the medical device is a surgical instrument, a catheter, or a medical implant.

26. below: i) the medical device is a catheter; or ii) the medical device is a dental implant; 26. The method according to claim 24 or 25, wherein any one of the following applies:

27. below: i) R １ But C １－３ is alkyl; ii) R ２ But -OC(O)C １－７ Alkyl and —OC(O)C ２－７ alkenyl; iii) R ３ is -OC(O)C １－７ Alkyl and —OC(O)C ２－７ alkenyl; iv) R ４ and R ５ are each methyl; and v) R ８ is C １－３ is alkyl; The method according to any one of claims 23 to 26, wherein one or more of the following applies:

28. R ２ But -OC(O)C ３－６ Alkyl and —OC(O)C ３－６ The method of any one of claims 23 to 27, wherein the alkyl group is selected from the group consisting of alkenyl and methyl.

29. R ３ But -OC(O)C ３－６ Alkyl and —OC(O)C ３－６ The method of any one of claims 23 to 28, wherein the alkyl group is selected from the group consisting of alkenyl and methyl.

30. R ６ The method of any one of claims 23 to 29, wherein is selected from hydrogen and -C(O)CH.

31. below: i) R ２ and / or R ３ is a branched alkyl or alkenyl group; or ii) R ２ and / or R ３ wherein the alkyl or alkenyl group is a straight-chain alkyl or alkenyl group; The method according to any one of claims 23 to 30, wherein any one of the following applies.

32. The compound of formula (II) is 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-ditigloyl-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-Hexa-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-(2E,4E)-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 17); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 18); 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 19); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 20); and 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-thiliaen-3-one (compound 21); or a pharmaceutically acceptable salt thereof.

33. The biofilm containing Gram-negative bacteria is Pseudomonas species, Acinetobacter species, Aeromonas species, Bacteroides species, Bordetella species, Borrelia species, Burkholderia species, Citrobacter species, Compylobacter species, Escherichia species, Enterobacter species, Flavobacterium species, Fusobacterium species, Klebsiella sp., Leptospira sp., Neisseria sp., Helicobacter sp., Hemophilus sp., Legionella sp., Moraxella sp., Yersinia sp., Oligella sp., Pantoea sp., Porphyromonas sp., Prevotella sp., Proteus sp., Raoutella sp., Salmonella sp., Serratia sp., Shigella Species, Sphingomonas sp., Stenotophomonas 33. The method according to any one of claims 23 to 32, comprising at least one Gram-negative bacterium selected from the species Treponema, Veillonella and Vibrio, in particular the Gram-negative bacterium is selected from Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica, Salmonella bongori, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Fusobacterium necrophorum, Burkholderia cepacian and Prevotella intermedia.

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