Novel antibiotics with quorum sensing inhibitor activity

Abstract

The present invention relates to paecilopenone compounds that can be isolated from the fungus Alaharph Aspergillus sp., which inhibit bacterial quorum sensing, biofilm formation and virulence factor production. Paecilopenone can be used as a medicament, alone or in combination with other antimicrobial agents, in particular as a medicament for the treatment and prophylaxis of microbial infections, such as infections caused by multidrug resistant bacteria.

Description

Technical Field

[0001] The present invention relates to the fields of medicine, microbiology and organic chemistry. In particular, the present invention relates to compounds obtainable from fungi having bacterial quorum sensing inhibitor activity, antimicrobial activity, methods for their production and their use in treating microbial infections. Background Art

[0002] Bacterial infections are a serious threat to human and animal life. Antibiotics have been developed to combat bacterial infections. The first antibiotic drug, penicillin, was discovered in 1928 and proved to be very effective in combating bacterial infections. Penicillin has literally saved millions of lives. Many more antibiotics have been discovered since then. However, resistance to existing antibiotics is developing and the rapid spread of bacterial infections is exacerbated by the increasing globalization process. So-called superbugs are emerging, which are resistant to almost all currently known antibiotics. These bacteria now pose a serious threat to human life. Methicillin-resistant Staphylococcus aureus or MRSA, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, pathogenic Streptococcus pneumoniae are notorious bacterial strains that are resistant to known antimicrobial agents. Furthermore, bacterial species such as Mycobacterium tuberculosis are (re)emerging as causes of disease in humans.

[0003] Therefore, there is an increasing need in the art for new antibiotics, preferably new antibiotics useful for combating bacterial infections that are resistant to most or all currently known antibiotics. A potential target for alternative treatment is quorum sensing (QS). Via QS, bacteria sense the density of a bacterial population. When the density is high, the QS pathway is activated and the bacteria begin to cooperate. QS is involved in the pathways that regulate virulence factor production and biofilm formation, described in Bassler, BL and Losick, R. Bacterially Speaking. (2006) Cell 125, pp. 237–246. Bacteria in biofilms are less sensitive to immune responses and antibiotic treatment. Thus, inhibition of QS may lead to increased sensitivity to antibiotics and faster clearance of bacteria, as described in Bjarnsholt, T. et al., Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. (2005) Microbiology 151, pp. 373–383 and Christensen, LD et al., Impact of Pseudomonas aeruginosa quorum sensing of biofilm persistence in an in vivo intraperitoneal foreign-body infection model. (2007) Microbiology 153, pp. 2312-2320. In addition, inhibition of virulence factor production can prevent bacteria from damaging host tissues, as described in Defoirdt, T. Quorum-Sensing Systems as Targets for Antivirulence Therapy. (2018) Trends Microbiol. 26, 313–328. QS inhibition does not kill bacteria, but weakens them, which makes it possible to fight them with the immune system or in combination with other antibiotics. The object of the present invention is to provide such novel antibiotics, methods for their production and their use in the treatment of bacterial infections. Summary of the invention

[0004] The present invention relates to paecilomycone derivatives for use in the treatment or prevention of microbial infections. The present invention may also be expressed as a method for the treatment or prevention of microbial infections, the method comprising administering a composition comprising a paecilomycone derivative to a subject in need thereof. The present invention may also be expressed as the use of a paecilomycone derivative for the manufacture of a medicament for use in the treatment or prevention of microbial infections. Related to the use according to the first aspect is the use of a paecilomycone derivative for preventing or reducing biofilm formation or bacterial proliferation. The present invention further relates to the use of a paecilomycone derivative for inhibiting quorum sensing or biofilm formation.

[0005] Surprisingly, the inventors have found that the pyralidone derivatives having the phenalene structure (1) as defined below show significantly increased quorum sensing inhibition compared to funalenone, which is structurally closely related to the pyralidone derivatives but has a methoxy group at position C2 instead of a methoxy group at position C7. The pyralidone derivatives have been described by Lu et al. in J. Agric. Food Chem. 2014, 62, 11917-11923, while fullenone has been described by Inokoshi et al. in J Antibiotics 1999, 52, 1095-1100. The inventors of the present invention were the first to demonstrate the antibacterial effect of the pyralidone derivatives. DETAILED DESCRIPTION

[0006] The present invention focuses on the quaternary ketone derivatives having structure (1), in particular their quorum sensing inhibitory activity. This is reflected in various aspects of the present invention, which are defined as uses, methods and compositions below. It will be understood by those skilled in the art that each aspect of the compound is applicable to all aspects of the present invention, just as the preferred embodiments of one aspect are also applicable to the related aspects of the present invention. Compound

[0007] The present invention relates to a penicillin derivative having a phenazone structure (1):

[0008] Herein, each of C1, C2, C3, C4 and C9 is independently selected from C═O, C—OH, C—OR 2 , C=S, C–SH, C–SR 2 , C–NH2 or C–N(R 2 )2, wherein the carbon atom is part of a phenazone ring structure. Each R 2 C alone 1-6Alkyl, C(Y)–C 1-6 Alkyl, C(Y)–Y–C 1-6 Alkyl and C(Y)–N(H) 0-2 (C 1-6 alkyl) 0-2 , wherein each Y is independently O or S. Preferably, all Y are O. In this context, these alkyl groups are preferably C 1-4 Alkyl, more preferably C 1-2 Preferably, at least C2, more preferably each of C1, C2, C3, C4 and C9 is individually selected from C=O, C-OH or C-NH2. Further preferably, C2 is C=O or C-OH. Preferably there is an odd number of C=O moieties, such as 1, 3 or 5, preferably 1 or 3. In a preferred embodiment, one of C1 and C9 is C=O, and / or both C2 and C3 are C=O or neither is C=O.

[0009] The substituent on C7 is represented by XR 1 In this paper, R 1 Selected from monocyclic aromatic groups, C 1-6 Alkyl, C(O)–C 1-6 Alkyl, C(O)–O–C 1-6 Alkyl and C(O)–N(H) 0-2 (C 1-6 alkyl) 0-2 Preferably, R 1 It is C 1-4 Alkyl, more preferably methyl or ethyl, most preferably R 1 X is S or O, preferably X is O. In a particularly preferred embodiment, XR 1 =OCH3.

[0010] In a preferred embodiment: C1 is selected from C=O, C-OH or C-NH2; C2 is C=O or C-OH; C3 is C=O or C-OH; C4 is C-OH; and C9 is C=O or C-OH. In this context, preferably one of C1 and C9 is C=O, and / or both C2 and C3 are C=O or neither is C=O.

[0011] In a particularly preferred embodiment, the compound is paecilomyces ketone A, paecilomyces ketone B or paecilomyces ketone C or a mixture thereof. In the context of the present invention, paecilomyces ketone A, paecilomyces ketone B and paecilomyces ketone C are collectively referred to as "pecilomyces ketones". The structures of these compounds are as follows: Figure 8 In a preferred embodiment, the compound is paecilomyces ketone A, wherein XR 1=OCH3; C1=C2=C3=C4=C-OH; C5=C=O. In a preferred embodiment, the compound is paecilomyces ketone B, wherein XR 1 =OCH3; C1=C2=C3=C=O, C4=C5=C-OH. In a preferred embodiment, the compound is paecilomyces ketone C, wherein XR 1 =OCH3; C1=C-NH2; C2=C3=C4=C-OH, C5=C=O. In a most preferred embodiment, the compound is paecilomyces pyrrolidone A.

[0012] Also envisioned in the present invention are salts, esters and prodrugs of the compounds according to the invention, preferably pharmaceutically acceptable salts, esters and prodrugs of these compounds, and more preferably pharmaceutically acceptable salts of these compounds.

[0013] The compound may also be present in a mixture of several compounds having the phenalene structure (1). Such a mixture of compounds preferably comprises at least one of paecilomycinone A, paecilomycinone B and paecilomycinone C, preferably a mixture of two or even all three thereof. Most preferably, the mixture comprises at least paecilomycinone A, preferably in combination with one or both of paecilomycinone B and paecilomycinone C.

[0014] Preferably, the compound according to the invention is in isolated form. When the compound is a mixture of compounds having the phenalene structure (1), they can be isolated together as a mixture. The compound according to the invention can be obtained by synthesis or by isolation from a natural source, for example as a (secondary) metabolite from Aspergillus allahabadii (available as CBS164.63).

[0015] The compounds of the present invention have quorum sensing inhibitor activity. Quorum sensing inhibitor activity can be evaluated by those skilled in the art, preferably by quorum sensing assays as exemplified herein, wherein Chromobacterium violaceum is used as a screening platform for evaluating the effect on quorum sensing. When quorum sensing is activated, Chromobacterium violaceum bacteria produce violaceum, which is a purple compound. Chromobacterium violaceum bacteria are grown overnight (o / n) at 27°C in TSB medium (3% w / v tryptic soy broth in H2O). The bacteria are diluted 1000x in TSB medium and grown until OD600=0.5-0.7. The bacteria are resuspended in TSB medium containing the compound and incubated on an orbital shaker at 27°C for 20h. The culture medium is aspirated and the precipitated violaceum is dissolved in 96% ethanol. In order to avoid interference due to cell turbidity, the sample is centrifuged again and the dissolved violaceum is transferred to a new plate. The amount of violacein was assessed spectrophotometrically by evaluating the OD at 562 nm. 562 Less than the OD of the control bacteria 562 If the OD value is 80%, the sample is scored as inhibitory. 562 The signal reduction is due to the inhibition of quorum sensing rather than just the reduced viability of the bacteria, and the viability assay can be performed in parallel with the quorum sensing assay. In order to evaluate the viability of bacteria, Chromobacterium violaceum bacteria were grown overnight (o / n) at 27°C in TSB medium. The bacteria were diluted 1000x in TSB medium and grown until OD600=0.5-0.7. The bacteria were resuspended in TSB medium containing the compound and incubated at 27°C for 20h. The bacteria were collected by centrifugation at 3000rpm for 10min. The supernatant was aspirated and resazurin (150μg / mL in saline) was added. The plate was incubated for another 45min at 27°C, and fluorescence was measured by excitation at 540nm and emission at 590nm. If the fluorescence signal is less than 70% of the fluorescence signal of the control untreated bacteria, the sample is scored as having an effect on the viability of the bacteria.

[0016] The compound preferably has quorum sensing inhibitor activity in Pseudomonas aeruginosa (strain PAO1). The quorum sensing activity in PAO1 cells can be assessed by a person skilled in the art, preferably by a quorum sensing reporter assay as exemplified herein. The PAO1 reporter is expressed under the control of a lasB promoter, a rhlA promoter, a pqsA promoter or a constitutive promoter, P A1 / 04 / 03Green fluorescent protein was expressed under the control of . The reporter bacteria were inoculated in AB minimal medium supplemented with 0.5% glucose and 0.5% casamino acids. The culture was diluted to OD450 = 0.1-0.2 in normal saline and then added to AB medium with a certain concentration range of compounds. GFP fluorescence (excitation at 485nm, emission at 535nm) and absorbance (600nm) were measured every 15min at 34°C for 15h. The IC50 value was calculated based on the maximum slope of GFP / OD. If the IC50 of the reporter line is less than half of the IC50 of the control bacteria, the compound is scored as a quorum sensing inhibitor.

[0017] The compounds of the present invention may have antimicrobial or antibiotic activity, more preferably antibacterial activity. Antimicrobial or antibacterial activity can be assessed by suitable assays known to those skilled in the art, preferably by growth inhibition assays as exemplified herein. Preferably, growth inhibition is assessed by culturing bacteria cultured overnight (o / n) in Mueller-Hinton broth (MHB) at 37°C in the presence or absence of an antibacterial compound. The assay is visually read, and if the bacteria have grown significantly, the sample is scored as non-inhibitory. If the culture broth is completely clear, the sample is scored as inhibitory.

[0018] The antimicrobial effects of the compounds of the invention can also be exemplified by growth kinetics assays, in which the effects of the compounds of interest on the growth of bacterial isolates in liquid culture are assessed in time, as known to those skilled in the art. Briefly, bacteria are inoculated in AB minimal medium or King's A medium supplemented with 0.5% glucose and 0.5% casamino acids and incubated overnight (o / n) at 37°C. Overnight (o / n) cultures are diluted in saline until OD 600 =0.1-0.2. With or without antimicrobial agents, the diluted culture (150 μL) was transferred to a honeycomb microplate containing 150 μL AB minimal medium or King's A medium supplemented with 0.5% glucose and 0.5% casamino acids to a total volume of 300 μL. At 34°C, on Bioscreen C (Growth CurvesAb Co., Ltd.), the absorbance at 600 nm was measured every 15 min for 24 h. If the slope of the growth curve decreases or there is a lag time in bacterial growth, the compound is understood herein to have antibacterial activity.

[0019] The compounds of the present invention may have antibacterial activity against Gram-positive bacteria or against Gram-negative bacteria or against both. In one embodiment, the compounds of the present invention have antibacterial activity against Gram-positive bacteria, preferably bacteria belonging to a genus selected from the group consisting of Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus, Enterococcus and Clostridium, or more particularly, bacteria belonging to a species selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Corynebacterium diphtheria, Listeria monocytogenes, Enterococcus faecalis, faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, and Clostridium tetani, as assessed in an assay as defined herein. Even more preferably, the compounds of the present invention have antibacterial activity against the following strains: Staphylococcus epidermidis (08A1071), methicillin-resistant Staphylococcus aureus 476 (S0101) (available from ATCC BA-1721), methicillin-resistant Staphylococcus aureus (MRSA) ST8: USA300 (S1474) (available from NARSA Collection NRS482), Enterococcus faecium (15A623), Enterococcus faecium (16D030), preferably against at least 2, 3, 4, 5 or all of these strains, as assessed in an assay as defined herein. Most preferably, the compounds of the present invention have antibacterial activity against methicillin-resistant Staphylococcus, preferably methicillin-resistant Staphylococcus aureus.Even more preferably, the compounds of the invention have antibacterial activity against methicillin-resistant Staphylococcus aureus strain ST8:USA300 (S1474) as assessed in an assay as defined herein.

[0020] Alternatively or additionally, the compounds of the invention have antibacterial activity against Gram-negative bacteria, preferably bacteria belonging to a genus selected from the group consisting of Acinetobacter, Actinobacillus, Bordetella, Brucella, Campylobacter, Chromobacterium, Cyanobacteria, Enterobacter, Erwinia, Escherichia, Franciscella, Helicobacter, ter, Hemophilus, Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio, Yersinia, or, more particularly, a bacterium belonging to the species selected from the group consisting of Klebsiella pneumoniae pneumoniae) (e.g. MR44), Acinetobacter calcoaceticus (e.g. 15A600), Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Chromobacterium violaceum and Escherichia coli, as assessed in an assay as defined herein. More preferably, the compounds of the invention have antibacterial activity against the following strains: Escherichia coli (available from ATCC, No. 25922), Klebsiella pneumoniae (MR44), Pseudomonas aeruginosa (available from ATCC, No. 27853), Pseudomonas aeruginosa (PAO1), Acinetobacter baumanii (12-5112), Acinetobacter calcoaceticus (15A600, available from NCCB, No. 100514), Chromobacterium violaceum, preferably against at least 2, 3, 4, 5, 6 or all of these strains, as assessed in an assay as defined herein.

[0021] Preferably, when using a bacterium as defined herein, a compound is understood herein as having antibacterial activity if a sample comprising at most 250 μM, 125 μM, 63 μM, 31 μM, 16 μM, 8 μM, 4 μM, 2 μM or preferably at most 1 μM of the compound is scored in an assay as defined herein. More preferably, a compound is understood herein as having antibacterial activity if the compound has at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or preferably 100% of the paecilomycin activity, as assessed in an assay as defined herein using a bacterium as defined herein.

[0022] Compounds according to the present invention can be prepared synthetically or can be separated from natural sources. In one embodiment, the method for producing the compound preferably includes step (a) in culture medium and preferably under conditions conducive to the production of the compound, culturing the fungus of Aspergillus (Aspergillus) fungal family, and (b) optionally recovering the compound. Preferably, the fungus of Aspergillus fungal family is a fungus producing a compound according to the present invention. The fungus producing the compound according to the present invention is understood herein as when growing in a standard culture medium (e.g., potato glucose broth (PDB) + 2% glucose) for culturing fungi, producing a fungus with one or more of the following characteristics in the culture medium: (a) the compound has antimicrobial activity in the assay as defined above; (b) ethyl acetate can be used to extract the compound from the culture medium; (c) the compound has a molecular weight of about 289, 287 or 286; (d) the compound has a UV maximum absorbance at 213, 254 or 207nm; and other features defined herein or determined in the examples herein.

[0023] Preferably, the fungus is a fungus belonging to the genus Aspergillus, more preferably, the fungus is a fungus of the species Aspergillus allahabadii, and most preferably, the fungus is Aspergillus allahabadii strain CBS164.63, or strains derived from these fungi, e.g. by genetic modification and / or mutagenesis and selection.

[0024] The culture medium for cultivating fungi is a culture medium that at least comprises a suitable carbon source and further comprises other nutrients and components that support fungal growth. Suitable culture mediums for fungal fermentation are well known in the art. Suitable carbon sources include, for example, glucose, syrup, dextrin, sucrose, starch, molasses, etc. can be used. The culture medium will further include nitrogen sources, such as soybean meal, wheat germ, corn steep liquor, cottonseed cake, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. Other culture medium components can be ions that can produce sodium, potassium, calcium, magnesium, cobalt, chloride ion, phosphorus ion, sulfide ion, etc.

[0025] In step (a) of the method, the fungus preferably ferments (at least a portion of) the carbon source into a compound according to the invention. Preferably, the fungus is cultured at a temperature of less than 30° C. in step (a). More preferably, the fermentation (in step a) is carried out at a temperature of no more than 29° C., 28° C., 27° C. or 26° C., and the temperature is at least 20° C., 21° C., 22° C., 23° C. or 24° C., most preferably, the temperature is about 25° C. The fermentation is preferably an aerobic fermentation, especially a submerged aeration fermentation is preferred.

[0026] The method for producing the compound further preferably comprises step (b), wherein the compound is recovered, purified and / or separated. The compound is preferably recovered from the culture medium by solvent extraction, optionally after removing biomass from the culture medium. The technician will know suitable organic solvents for extracting the compound from the culture medium and / or from the isolated mycelium, such as ethyl acetate, chloroform, methylene chloride, ether, ethyl acetate, butyl acetate, methanol, ethanol, propanol, butanol or a combination thereof. The preferred solvent for solvent extraction is ethyl acetate. After concentrating the extract, the compound of the present invention can be further purified and separated, for example, by using a commonly used chromatographic adsorbent or a carrier material. Pharmaceutical composition

[0027] The compounds according to the invention are typically comprised in a pharmaceutical composition, wherein the compound is an active ingredient. An active ingredient is understood herein as an ingredient having quorum sensing inhibitory activity.

[0028] In addition to active ingredients, the pharmaceutical composition preferably further comprises a pharmaceutically acceptable carrier, excipient and / or vehicle. Therefore, the compound according to the present invention can be formulated into a pharmaceutical composition by preparing with an additive such as a pharmaceutically or physiologically acceptable excipient, carrier and vehicle. Suitable pharmaceutically or physiologically acceptable excipients, carriers and vehicles include processing agents and drug delivery modifiers and reinforcing agents, such as calcium phosphate, magnesium stearate, talcum, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidone, low melting wax, ion exchange resins, etc., and any two or more combinations thereof. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey (1991) and "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, Philadelphia, 20th Edition (2003) and 21st Edition (2005), which are incorporated herein by reference.

[0029] The pharmaceutical composition may comprise a unit dose formulation, wherein the unit dose is a dose sufficient to have a therapeutic effect. The unit dose may be sufficient as a single dose to have a therapeutic effect. Alternatively, the unit dose may be a dose that is periodically administered during the treatment of an infectious disease.

[0030] The pharmaceutical composition containing the compound of the present invention can be in any form suitable for the intended method of administration, including, for example, solutions, suspensions or emulsions. Liquid carriers are typically used to prepare solutions, suspensions and emulsions. Liquid carriers considered for use in the practice of the present invention include, for example, water, saline, one or more pharmaceutically acceptable organic solvents, pharmaceutically acceptable oils or fats, etc., and mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives, such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickeners, viscosity modifiers, stabilizers, etc. Suitable organic solvents include, for example, monohydric alcohols (such as ethanol) and polyols (such as glycols). Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, etc. For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate, isopropyl myristate, etc. The composition of the present invention may also be in the form of microparticles, microcapsules, liposome capsules, etc., and any combination of two or more thereof.

[0031] Sustained or controlled release delivery systems can be used, such as diffusion-controlled matrix systems or erodible systems, such as described, for example, in Lee, "Diffusion-Controlled Matrix Systems", pp. 155-198 and Ron and Langer, "Erodible Systems", pp. 199-224, such as described, for example, in "Treatise on Controlled Drug Delivery", ed. A. Kydonieus, Marcel Dekker, Inc., New York 1992. The matrix can be, for example, a biodegradable material that can spontaneously degrade in situ and in vivo, such as by hydrolysis or enzymatic cleavage (e.g., by proteases). The delivery system can be, for example, a naturally occurring or synthetic polymer or copolymer, such as in the form of a hydrogel. Exemplary polymers having cleavable bonds include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates), and poly(phosphazenes).

[0032] The compounds of the present invention can be administered enterally, orally, parenterally, sublingually, by inhalation (e.g., as aerosols or sprays), rectally or topically in dosage unit formulations containing conventional nontoxic pharmaceutically or physiologically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoresis, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e.g., via nasal mucosa), subdural, rectal, gastrointestinal, etc., and directly to specific or affected organs or tissues. In order to be delivered to the central nervous system, spinal cord and epidural administration or administration to the ventricles can be used. Topical administration can also involve the use of transdermal administration, such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. These compounds are mixed with pharmaceutically acceptable carriers, adjuvants and vehicles suitable for the desired route of administration. Oral administration is a preferred route of administration, and formulations suitable for oral administration are preferred formulations. The compounds described for use herein may be in solid form, liquid form, aerosol form, or in tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, enemas, colon lavages, emulsions, dispersions, food premixes, and in other suitable forms. These compounds may also be administered as liposome formulations. These compounds may also be administered as prodrugs, wherein the prodrugs are converted into therapeutically effective forms in the subject being treated. Additional methods of administration are known in the art.

[0033] Injectable preparations (e.g., sterile injectable aqueous or oily suspensions) can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, for example, as solutions in propylene glycol. Acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are generally used as solvents or suspension media. For this purpose, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids, such as oleic acid, are used in injectable preparations.

[0034] Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycol which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

[0035] Solid dosage forms for oral administration can include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound can be blended with at least one inert diluent (e.g., sucrose, lactose or starch). Such dosage forms can also include other substances in addition to the inert diluent, such as lubricants (e.g., magnesium stearate). In the case of capsules, tablets and pills, the dosage form can also include a buffer. Tablets and pills can be prepared with enteric coatings in addition.

[0036] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art (such as water). Such compositions may also include adjuvants (such as wetting agents, emulsifying and suspending agents, cyclodextrins) as well as sweeteners, flavoring agents and aromatics.

[0037] The compounds of the present invention can also be administered in the form of liposomes. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by monolayer or multilayer hydrated liquid crystals dispersed in an aqueous medium. Any nontoxic, physiologically acceptable and metabolizable lipids capable of forming liposomes can be used. In addition to the compounds of the present invention, the compositions of the present invention in the form of liposomes can also contain stabilizers, preservatives, excipients, etc. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods for forming liposomes are known in the art. See, for example, editor Prescott, Methods in Cell Biology [Cell Biology Methods], Volume XIV, Academic Press [Academic Press], New York, New York, pages 33 and below (1976).

[0038] The compounds according to the present invention are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular pharmaceutical composition employed; the metabolic stability and duration of action of the composition; the age, weight, general health, sex and diet of the patient; the mode and time of administration; the rate of excretion; the combination of drugs; the severity of the particular disorder or condition; and the subject being treated. Typically, the therapeutically effective daily dose is from about 0.001 mg / kg to about 100 mg / kg, preferably, from about 0.01 mg / kg to about 50 mg / kg, more preferably, from about 1 mg / kg to about 25 mg / kg.

[0039] For veterinary use, the dosage is generally in the range of 1 to 200 mg, preferably 5 to 100 mg / kg body weight / day, while varying according to the purpose of administration (for treatment or for prevention), the species and size of the animal, the type of pathogenic organism and the severity of the symptoms.

[0040] The pharmaceutical composition of the present invention intended, for example, for parenteral or oral administration should contain an amount of the compound of the present invention such that a suitable dosage will be obtained. Typically, the amount of the compound is at least 0.01% of the pharmaceutical composition of the present invention. When intended for oral administration, the amount may vary between 0.1% to about 70% of the weight of the composition. Preferred oral pharmaceutical compositions contain about 4% to about 50% of the compound of the present invention. Preferred pharmaceutical compositions and preparations according to the present invention are prepared so that the parenteral dosage unit contains a compound of the present invention by weight between 0.01% and 10% before dilution. application

[0041] The compounds according to the present invention are suitable for use as medicines, in particular in the treatment or prevention of microbial infections. The medicine can be a medicine for both human and veterinary purposes. Therefore, the present invention also relates to compounds according to the present invention for use in the treatment or prevention of microbial infections. Preferably, the compound is used for use in the treatment or prevention of microbial infections in human or animal subjects. The microbial infection is preferably a bacterial infection. In an alternative embodiment, the compounds according to the present invention are used in cosmetics or non-therapeutic methods, which are used to treat or prevent microbial infections and / or for disinfecting human or animal subjects and their body parts. Therefore, the above-mentioned pharmaceutical composition comprising the compound according to the present invention as an active ingredient can also be or be used as a cosmetic composition or a disinfectant.

[0042] In one embodiment, the compounds according to the invention are used to treat or prevent infections caused by multi-drug resistant bacteria. Multi-drug resistant bacteria are understood herein as bacterial strains that are resistant to more than one type of antibiotic and are therefore more difficult (if not impossible) to treat. Well-known examples of multi-drug resistant bacteria include, for example, methicillin-resistant Staphylococcus aureus (MRSA) and Enterobacteriaceae that produce extended-spectrum beta-lactamases (ESBLs).

[0043] Compounds according to the present invention can be active for Gram-positive bacteria, Gram-negative bacteria or both. In one embodiment, compounds according to the present invention are used to treat or prevent infections caused by Gram-positive bacteria, particularly pathogenic Gram-positive bacteria. The infection of Gram-positive bacteria that can be treated and / or prevented using compounds or compositions according to the present invention includes bacteria belonging to the genus selected from: Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus, Enterococcus and Clostridium, or more particularly, bacteria belonging to the genus selected from: Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Corynebacterium diphtheriae, Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens and Clostridium tetani. Good results were obtained when the compounds or compositions according to the invention were used for the treatment and / or prevention of infections caused by methicillin-resistant Staphylococcus, preferably methicillin-resistant Staphylococcus aureus.

[0044] In an alternative embodiment, the compounds according to the invention are used to treat or prevent infections caused by Gram-negative bacteria, particularly pathogenic Gram-negative bacteria. Infections with Gram-negative bacteria that can be treated and / or prevented using the compounds or compositions according to the invention include bacteria belonging to the genus selected from the group consisting of Acinetobacter, Actinomyces, Bordetella, Brucella, Campylobacter, Cyanobacteria, Enterobacter, Erwinia, Escherichia, Francisella, Helicobacter, Haemophilus, Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella, Serratia marcescens, Shigella, Treponema, Vibrio, Yersinia, or more particularly, bacteria belonging to the species selected from the group consisting of Klebsiella pneumoniae (e.g. MR44), Acinetobacter calcoaceticus (e.g. 15A600), Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Chromobacterium violaceum and Escherichia coli. In an alternative preferred embodiment, the Gram-negative bacterium belongs to the genus: Pseudomonas or Chromobacterium, more preferably to the species: Pseudomonas aeruginosa or Chromobacterium violaceum.

[0045] In a preferred embodiment, the compounds according to the invention are used for the treatment and / or prevention of infections caused by Mycobacterium or Mycoplasma (e.g. Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans and Mycoplasma pneumonia).

[0046] In other words, the present invention relates to a method for treating or preventing microbial infection, the method comprising administering an effective amount of a compound according to the present invention to a subject in need thereof. Typically, the subject suffers from a microbial infectious disease. The subject may be a human or an animal. Microbial infection may be an infection of bacteria as defined above. The present invention according to this aspect may also be described as the use of a compound according to the present invention for the manufacture of a medicament for use in treating or preventing microbial infection.

[0047] The compounds according to the invention can be used in a method for treating or preventing microbial infections or diseases selected from the group consisting of, for example, bacterial infections of wounds including surgical wounds, lung infections (e.g., tuberculosis, cystic fibrosis), skin infections, and systemic bacterial infections. For example, diseases that can be treated or prevented by the antimicrobial compounds of the invention include, but are not limited to, anthrax, cystic fibrosis, food poisoning, folliculitis, furuncle, carbuncle, erysipelas, cellulitis, lymphangitis / lymphadenitis, paronychia, subcutaneous abscesses, hidradenitis, acne tufts, infectious atherosclerosis, perianal abscesses, mastitis, superficial secondary infections after trauma, burns or surgical trauma, pharyngitis, acute bronchitis, tonsillitis, chronic bronchitis , bronchiectasis, diffuse panbronchiolitis, secondary infection of chronic respiratory diseases, pneumonia, pyelonephritis, cystitis, prostatitis, epididymitis, urethritis, cholecystitis, cholangitis, bacillary dysentery, enteritis, adnexitis, intrauterine infection, vestibular glanditis, blepharitis, hordeolum, dacryocystitis, tarsal adenitis, corneal ulcer, otitis media, sinusitis, periodontal disease, pericoronitis, jawitis, peritonitis, endocarditis, septicemia, meningitis and skin infections.

[0048] In a preferred embodiment, the treatment or prevention according to the present invention involves a combination therapy in which the compound according to the present invention is combined with a second (or additional) active agent (typically an antibiotic agent). Such combination therapy with two (or more) active components is well known in the art and can be performed in any manner known to the skilled person.

[0049] With regard to the uses defined above, the present invention also relates to the use of the compounds according to the present invention for preventing or reducing biofilm formation. In other words, the present invention relates to a method for preventing or reducing biofilm formation, the method comprising contacting a compound according to the present invention with a subject or article in which biofilm formation occurs or may occur. Such uses or methods may be medical or non-medical. As found by the inventors, the quorum sensing inhibitory activity of the compounds according to the present invention negatively affects bacterial biofilm formation, so as to prevent or reduce such biofilm formation. In this article, biofilm is typically a bacterial biofilm.

[0050] With regard to the uses defined above, the present invention also relates to the use of the compounds according to the present invention for preventing or reducing bacterial proliferation. In other words, the present invention relates to a method for preventing or reducing bacterial proliferation, the method comprising contacting a compound according to the present invention with a subject or article in which bacterial proliferation occurs or may occur. Such uses or methods may be medical or non-medical. As discovered by the inventors, the quorum sensing inhibitory activity of the compounds according to the present invention negatively affects bacterial proliferation, so that such bacterial proliferation is prevented or reduced.

[0051] With regard to the uses defined above, the present invention also relates to the use of the compounds according to the present invention for inhibiting bacterial quorum sensing, bacterial biofilm formation and virulence factor production. In other words, the present invention relates to a method for inhibiting bacterial quorum sensing, bacterial biofilm formation and virulence factor production, the method comprising contacting a compound according to the present invention with a subject or article in need. Such uses or methods may be medical or non-medical. As discovered by the inventors, the quorum sensing inhibitory activity of the compounds according to the present invention negatively affects bacterial quorum sensing, bacterial biofilm formation and virulence factor production, so that the situation is prevented or reduced.

[0052] In the context of the present invention, the compounds according to the invention can also be used as antifouling agents. In this case, the reduction of biofilm formation on the surface leads to a reduction in its fouling. This is particularly relevant in the case of ship hulls, which often suffer from fouling caused by biofilms. Therefore, in one embodiment, the use according to the invention is for reducing the fouling of surfaces, preferably hulls. Alternatively, the effect of the compounds according to the invention in reducing biofilm formation can also be used in agriculture. It is known that biofilm formation reduces crop yields. In one embodiment, the compounds according to the invention are added to soil to reduce biofilm formation therein and increase the yield of crops grown in the soil. Therefore, in one embodiment, the use according to the invention is for increasing crop yields.

[0053] With regard to the uses defined above, the present invention also relates to the use of the compounds according to the invention as antimicrobial agents, preferably antibacterial agents. Such uses may be medical or non-medical. As discovered by the inventors, the quorum sensing inhibitory activity of the compounds according to the invention negatively affects the bacterial growth rate, making the compounds have an antimicrobial effect.

[0054] Therefore, the present invention further relates to an antimicrobial composition, preferably a disinfectant, comprising a compound according to the present invention as an active ingredient. Disinfectants are antimicrobial agents specifically for inanimate objects. Antimicrobial compositions are particularly useful for reducing or preventing biofilm formation, as found in processing and / or medical equipment. The antimicrobial composition or disinfectant of the present invention may be in liquid, solid or semi-liquid or semi-solid form. This aspect of the present invention may also be described as the use of a compound of the present invention in an antimicrobial composition, preferably a disinfectant, wherein the use is as an antimicrobial agent. Preferably, the antimicrobial composition or disinfectant is used to control gram-positive bacteria, gram-negative bacteria or both.

[0055] In one embodiment, the antimicrobial composition is used to control Gram-positive bacteria, in particular Gram-positive bacteria belonging to the genus selected from Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus, Enterococcus and Clostridium, or more particularly, belonging to the species selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Corynebacterium diphtheriae, Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens and Clostridium tetani. Most preferably, methicillin-resistant Staphylococcus, preferably methicillin-resistant Staphylococcus aureus.

[0056] In an alternative embodiment, the compounds according to the invention are used to control Gram-negative bacteria, in particular Gram-negative bacteria belonging to the genera selected from the group consisting of Acinetobacter, Actinomyces, Bordetella, Brucella, Campylobacter, Cyanobacteria, Enterobacter, Erwinia, Escherichia, Francisella, Helicobacter, Haemophilus, Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella, Serratia marcescens, Shigella, Treponema, Vibrio, Yersinia, or, more particularly, bacteria belonging to the species selected from the group consisting of Klebsiella pneumoniae (e.g. MR44), Acinetobacter calcoaceticus (e.g. 15A600), Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Chromobacterium violaceum and Escherichia coli. In an alternative preferred embodiment, the Gram-negative bacterium belongs to the genus: Pseudomonas or Chromobacterium, more preferably to the species: Pseudomonas aeruginosa or Chromobacterium violaceum.

[0057] Preferably, the antimicrobial composition or pharmaceutical composition according to the present invention further comprises a pharmaceutically acceptable carrier and / or another active ingredient selected from the group consisting of: bacteriophage, bacteriostat, bactericide, antibiotic, surfactant and / or enzyme. The antibiotic can be any antibiotic known in the art, including antibiotics and chemotherapeutic agents. The enzyme includes but is not limited to enzymes that help decompose biofilms (e.g., biofilms found in processing and / or medical devices), such as but not limited to polysaccharide depolymerases and proteases. The surfactant can be used to wet the surface so that the active ingredients of the present invention (including the compounds of the present invention) are properly distributed on different surfaces, and can be used to dissolve and remove dirt so that microorganisms (preferably bacteria) can access the active ingredients. Suitable surfactants include but are not limited to polysorbate (tween) 80, 20 and 81 and Dobanols (Shell Chemical Co. RTM). The antimicrobial composition or disinfectant of the present invention can further include surface disinfectants known in the art, such as but not limited to benzoic acid and PBT, preferably surface disinfectants compatible with the compounds of the first aspect of the present invention.

[0058] Preferably, the antimicrobial composition is used in a method for controlling microbial contamination in processing equipment or medical equipment. In this method, the antimicrobial composition is contacted with processing equipment or medical equipment. Preferably, the method for controlling microbial contamination includes reducing the count of bacteria (typically pathogenic bacteria), first preventing their growth and / or preventing biofilm formation on processing or medical equipment. Preferably, the method is controlled for cleaning and sterilizing medical equipment. In this article, medical equipment can refer to fiberscopes, like intragastric cameras, laparoscopes, thoracoscopes and arthroscopes, and medical supplies, like catheters and tubes, which have long catheters or hollow parts and tend to be repeatedly adopted by being introduced into the human body. The method of the present invention encompasses the application of the compound to different physical locations on processing or medical equipment by a variety of ways, including but not limited to blending, spraying or direct application according to the compound of the first aspect of the present invention or the composition of the sixth aspect of the present invention. Optionally, the method of the present invention can be combined with any sterilization method or disinfectant known in the art, such as ultrasonic cleaning, irradiation or heat sterilization, by immersing the device in a disinfectant solution such as ethanol, ammonium, iodine and / or aldehyde disinfectants, or by using gas sterilization, by maintaining the device in a closed atmosphere such as formaldehyde gas or ethylene oxide gas.

[0059] In this document and its claims, the verb "to comprise" and its conjugations are used in its non-limiting sense to mean including the items following the word, but not excluding items not specifically mentioned. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires the presence of one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one".

[0060] All patents and literature references cited in this specification are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 : Preparative HPLC chromatogram (x axis: time in min): The extract was fractionated using TFA as a modifier. The black box is the active fraction.

[0062] Figure 2 : Preparative HPLC chromatogram (x axis: time in min): The active fractions were subsequently fractionated using NH4OAc as a modifier. The black box is the active fraction.

[0063] Figure 3: Analytical HPLC chromatogram of the active fraction in TFA (x axis: time in min). In addition to the largest peak (peicyllone A), there are two further peaks (peicyllone B and picyllone C). UV-VIS spectra of the corresponding peaks are included (x axis: wavelength in nm).

[0064] Figure 4 : LC-MS data of active compounds (x axis = m / z). (Top) ESI (+) spectrum showing a peak at 289 [M+H], (Bottom) ESI (-) spectrum showing a peak at 287 [MH].

[0065] Figure 5 : LC-MS data (x-axis = m / z) of the peak corresponding to paecilomyces lysine B. (Top) ESI (+) spectrum showing the peak at 287 [M+H], (Bottom) ESI (-) spectrum showing the peak at 285 [MH].

[0066] Figure 6 : LC-MS data (x-axis = m / z) of the peak corresponding to paecilomyces cerevisiae C. (Top) ESI (+) spectrum showing the peak at 288 [M+H], (Bottom) ESI (-) spectrum showing the peak at 286 [MH].

[0067] Figure 7 : Chemical structure of paecilomyces ketone.

[0068] Figure 8 : Quorum sensing inhibition in Chromobacterium violaceum after treatment with paecilomyces tinctorius. Experiments were performed in triplicate and error bars represent mean ± SEM.

[0069] Fig. 9 : Quorum sensing inhibition in Pseudomonas aeruginosa (PAO1) reporter after treatment with paecilomycin. Calculated IC 50 The values ​​were 49.8 μM (lasB-GFP), 233.8 μM (rhlA-GFP), 31.5 μM (pqsA-GFP) and 143.2 μM (WT-GFP). The experiments were performed in triplicate. The maximum slope of the mean RFU / OD is plotted.

[0070] Fig.10 : Growth curves of Pseudomonas aeruginosa in AB minimal medium after treatment with different concentrations of paecilomycinone (grey curve) versus untreated growth curve (black curve). The experiment was performed in triplicate and the error bars represent mean ± SEM.

[0071] Figure 11: No inhibition of quorum sensing in response to the closely related fullerenone. (A) Chemical structure of fullerenone, which is very similar to penicillinone, but with a different methoxy position. (B) Quorum sensing activity of Chromobacterium violaceum and assessment of bacterial viability in response to treatment with fullerenone. Experiments were performed in triplicate and error bars represent mean ± SEM. (C) Quorum sensing inhibition in Pseudomonas aeruginosa (PAO1) reporter after treatment with fullerenone. Experiments were performed in triplicate. The maximum slope of the mean RFU / OD is plotted.

[0072] Fig.12 : Inhibition of biofilm formation in PAO1 after treatment with paecilomycin. Experiments were performed with biological replicates (triplicates) containing technical replicates (triplicates). The mean of the biological replicates was used for analysis. Error bars represent mean ± SEM. One-way ANOVA (corrected for multiple comparisons with Dunnett's test) was performed to determine statistical significance. Values ​​were compared to DMSO-treated controls (**, P < 0.005; ***, P < 0.001).

[0073] Fig.13 : Inhibition of pyocyanin synthesis in PAO1 after treatment with paecilomycin. The figure represents a representative experiment performed in triplicate. Error bars represent mean ± SEM. One-way ANOVA (corrected for multiple comparisons with Dunnett's test) was performed to determine statistical significance. Values ​​were compared to DMSO-treated controls (***, P < 0.001; ****, P < 0.0001).

[0074] Fig.14 : Growth curves of Pseudomonas aeruginosa in King's A medium after treatment with different concentrations of paecilomycinone (grey curve) versus untreated growth curve (black curve). The experiment was performed in triplicate and the error bars represent mean ± SEM.

[0075] Fig.15 : Activation of rhamnolipid synthesis after treatment with paecilomyces lysine. Experiments were performed with biological replicates (triplicates) containing technical replicates (triplicates). The mean of the biological replicates was used for analysis. Error bars represent mean ± SEM. One-way ANOVA (corrected for multiple comparisons with Dunnett's test) was performed to determine statistical significance. Values ​​were compared to DMSO-treated controls (*, P < 0.05).

[0076] Figure 16: Antibiotic susceptibility microdilution test with different concentrations of paecilomyces lysine in MHB medium in different Gram-negative and Gram-positive bacterial strains. Bacterial growth was measured at 595 nm and 620 nm every 15 min for 24 h.

[0077] Fig.17 Antibiotic susceptibility microdilution testing was performed in AB minimal medium with different concentrations of paecilomyces lysate in different Gram-negative bacteria. Bacterial growth was measured at 595 nm and 620 nm every 15 min for 24 h.

[0078] Fig.18 : Cell viability assay (resazurin assay) of HepG2 cells after treatment with paecilomycinone. Experiments were performed in triplicate and error bars represent mean ± SEM. Examples

[0079] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Fungal growth

[0080] The fungus Aspergillus allahabadii strain CBS164.63 (available from Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; www.wi.knaw.nl) was cultured on malt extract agar (MEA) plates at 25°C for 7 days. The fungus was then inoculated in 3 flasks containing 50 ml of potato dextrose broth (PDB) + 2% glucose. Each flask was incubated at different temperatures -15°C, 25°C or 25°C with 100 rpm orbital shaking for 7 days. After 7 days, the fungal culture medium was filter sterilized (0.22 μm pore filter). The filtrate was tested on Chromobacterium violaceum as follows. Quorum sensing assay

[0081] The purple bacterium was grown overnight (o / n) in tryptic soy broth at 27°C. The bacteria were diluted in TSB and grown until OD600 = 0.5-0.7. The bacteria were diluted 1:1000, and 50 μL of bacterial suspension was transferred to a 96-well plate containing 50 μL of fungal filtrate, extract or purified compound within a certain concentration range. The purified compounds of formula 1, 2 and 3 were tested in triplicate by 2-fold dilution. The plate was incubated at 27°C for 20h while shaking at 180rpm. The plate was centrifuged at 3000rpm for 10min to precipitate purple bacterin. The supernatant was aspirated and the precipitate was resuspended in 200 μL of 96% ethanol. The plate was centrifuged again at 3000rpm for 10min, and 100 μL of supernatant containing purple bacterin was transferred to a new 96-well plate, and OD was measured on an ASYS expert microplate reader. 562To quantify the production of purple bacteriocin. To measure the viability of the bacteria, the plates grown in parallel with the above plates were centrifuged at 3000 rpm for 10 min. The supernatant was aspirated and resazurin (150 μg / mL in saline) was added. The plates were incubated at 27°C for another 45 min, after which fluorescence was measured on a PHERAstar microplate reader (BMG Labtech) using an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Purification of compounds with quorum sensing inhibitor activity

[0082] Based on the first results, 60 jars of 50 ml PDB + 2% glucose were inoculated with the fungus Aspergillus alahabadi strain CBS164.63 and incubated at 25°C for 7 days with 100 rpm orbital shaking. The culture medium was filter sterilized as described and extracted 3 times with EtOAc. The EtOAc was evaporated and the residue was dissolved in 2 ml DMSO. The extract was then graded on a preparative HPLC (see Table 5 for equipment information) Prep using a C18 Reprosil column with UV-detection between 214 nm and 254 nm. The buffer used is described in Table 1, and the buffer B gradient was run as described in Table 2. Table 1: HPLC buffers Buffer A Buffer B 100% MQ 100% acetonitrile 0.1% trifluoroacetic acid 0.1% trifluoroacetic acid Table 2: HPLC gradient

[0083] The fractions were dried overnight (o / n) in a speed-vac. The fraction residues were dissolved in DMSO and tested for quorum sensing activity on Chromobacterium violaceum in serial dilutions starting from 40-fold. Fraction 20 showed inhibition of violacein production in Chromobacterium violaceum, which was consistent with UV detection of HPLC fractionation ( Figure 1 ).

[0084] To purify this fraction, the remaining fraction was dried overnight (o / n) in a speed-vac and dissolved in DMSO. The sample was then fractionated using the preparative HPLC described above. The buffer used for this purification run is described in Table 3 and the buffer B gradient is described in Table 4. Table 3: HPLC buffers Buffer A Buffer B 95%MQ 20%MQ 5% acetonitrile 80% acetonitrile 10 mM ammonium acetate 10 mM ammonium acetate Table 4: HPLC gradient Time (min) Buffer B percentage 0-5 0 5-45 0-100 45-50 100 50-55 100-0 55-60 0

[0085] The fractions were dried overnight (o / n) in a speed-vac. The fraction residues were dissolved in DMSO and tested for quorum sensing activity on Chromobacterium violaceum in serial dilutions starting from 40-fold. Fraction 1 showed inhibition of violacein production in Chromobacterium violaceum, which was consistent with UV detection of HPLC fractionation ( Figure 2 ). Identification of compounds with quorum sensing activity

[0086] The sample was run on analytical HPLC (see Table 6 for equipment information) and the compound was found to be 85% pure ( Figure 3 ). Using LC-MS ( Figure 4 ) (see Table 5 for equipment information) and high resolution mass spectrometry analysis of fraction 20.1. The calculated monoisotopic mass is 289.5937 and the molecular formula is predicted to be C 15 H 12 O6. UV-VIS was also measured, showing a characteristic UV-VIS spectrum (213(100), 238sh, 277sh, 388(40))( Figure 3 A). However, analytical HPLC showed that a single fraction of preparative HPLC produced three peaks, wherein the relative abundances of peaks A, B and C were 85%, 5% and 10%, respectively. The relative abundances of the three peaks were pH dependent. The three peaks contained a nominal mass of 288 (peak A, Figure 3 ), 286 (peak B, Figure 5 ) and 287 (peak C, Figure 6 ) related compounds, and (217(90), 254(100), 331(50)) (peak B, Figure 3 B) and (207(100), 259(50), 378(50))(Peak C, Figure 3 C) UV-VIS spectrum. Structural Elucidation of Bioactive Compounds

[0087] The entire fraction 20.1 was subsequently evaporated in a speed-vac and the residue was dissolved in 400 μl of DMSO-d6. The yield of the fraction was about 5 mg dry weight (from 3 L fungal culture). Measurement 1 H-NMR spectrum and 13 C-NMR spectrum. Although not all carbons were detected, the NMR data (like the UV-VIS spectrum) showed high similarity to that of paecilomyces quinolinone, which was reported by Lu et al. in J. Agric. Food Chem. 2014, 62, 11917-11923. δ H 6.72(5H) and δ H 2.74(11H3) and δ H6.44(8H) and δ H 3.98 (10H3) confirmed the position of the methoxy group. These results show that peak A ( Figure 4 ) contains pseudopaecilomycin A. The molecular weights and UV-VIS spectra of peaks B and C indicate that these peaks represent pseudopaecilomycin B and C, respectively. Figure 7 ). The different penicillins cannot be separated by HPLC, and the relative concentration of each penicillin is pH dependent. Therefore, the combination of the three penicillins will be referred to as penicillins. Penicillins inhibit quorum sensing, biofilm formation, and virulence factor production

[0088] Quorum sensing was evaluated in the Gram-negative bacteria Chromobacterium violaceum. Chromobacterium violaceum produces a purple pigment, violacein, upon quorum sensing activation, as described by Skogman et al. in A. Flavones as quorum sensing inhibitors identified by a newly optimized screening platform using chromobacterium violaceum as reporter bacteria. (2016) Molecules 21, 1211. Paecilomyces tinctorius strongly inhibited violacein production, with an IC 50 72.5μM( Figure 8 Viability was also determined to distinguish between the effects of penicillin on quorum sensing and effects on bacterial growth, which could be erroneously interpreted as an effect on violacein production. No toxic effects on violacein were detected until a concentration of 1 mM ( Figure 8 ). In conclusion, at concentrations that do not affect cell viability, paecilomyces pyrrolidone is an effective quorum sensing inhibitor in Chromobacterium violaceum.

[0089] To test the potential of penicillin as a quorum sensing inhibitor in more clinically relevant bacteria, the effect on Pseudomonas aeruginosa (PAO1) was tested. For this purpose, bacterial reporter strains expressing GFP when the quorum sensing pathway is activated were used, including lasB-GFP, rhlA-GFP, pqsA-GFP, and WT-GFP constitutively expressing GFP as a control. To correct for potential effects on bacterial growth, OD600 was measured in parallel to GFP fluorescence intensity, and RFU / OD was plotted. Penicillin inhibited GFP expression in lasB-GFP reporter and pqsA-GFP reporter, with the IC of lasB 50 is 49.8 μM, and the IC 5031.5μM ( Fig. 9 ). In the control WT-GFP bacteria, paecilomyces pyrrolidone affected GFP expression, and its IC 50 The rhlA reporter was insensitive to paecilomyces pyrrolidone, with an IC 50 is 233.8μM, the IC 50 Actually higher than the IC of WT-GFP 50 These results indicate that quorum sensing in the clinically relevant Pseudomonas aeruginosa (PAO1) bacteria is inhibited by paecilomyces pyrrolidone, regardless of the effect on bacterial growth. Treatment with high concentrations (62.5 μM and higher) of paecilomyces pyrrolidone affected PAO1 growth rate but not maximum bacterial density ( Fig.10 ).

[0090] Fullerenone is closely related to penicillin A, B, and C ( Fig.11A ). The overall structure is very similar, but the methoxy group position in fullerone is different compared to penicillinone. We tested commercially available fullerone (Bio-connect, Huissen, The Netherlands, AG-CN2-0137-M001) for quorum sensing inhibitor activity in Chromobacterium violaceum bacteria and found that treatment with up to 1 mM fullerone did not inhibit violaceum production ( Fig.10 B) Fullerenone also did not affect cell viability in an assay performed in parallel with the quorum sensing assay ( Fig. 11B We also tested the effect of fullerenone on quorum sensing in the PAO1 reporters lasB-GFP, rhlA-GFP, pqsA-GFP, and as a control WT-GFP, and found no effect on quorum sensing at fullerenone concentrations up to 500 μM ( Fig. 11C We conclude that fullerenone does not affect quorum sensing, which is surprising because the highly related fulerenones A, B, and C do inhibit quorum sensing.

[0091] Since quorum sensing has downstream effects on biofilm maturation in P. aeruginosa, as described by Jakobsen et al., Bacterial biofilm control by perturbation of bacterial signaling processes. (2017) Int. J. Mol. Sci. 18, 1970, we tested the effect of penicillin on biofilm formation of WT PAO1 bacteria. Penicillin had a significant inhibitory effect on biofilm formation starting from 62.5 μM ( Fig.12 ). This effect is unlikely to be due to an effect on bacterial growth. Although bacterial growth rate was reduced in the presence of 62.5 μM or more of paecilomycin, bacterial density after 24 h was indistinguishable between treated and untreated bacteria. These results suggest that paecilomycin affects biofilm formation in PAO1 bacteria through mechanisms other than simply affecting bacterial growth.

[0092] Another downstream effect of quorum sensing in P. aeruginosa is the production of virulence factors, as described in Rutherford and Bassler. Bacterial quorum sensing: Its role in virulence and possibilities for its control. (2012) Cold Spring Harb. Perspect. Med. 2, 1–25. The effects of penicillin on various virulence factors were evaluated. Penicillin induced a strong and significant inhibition of pyocyanin production, with a maximum inhibition of 88% and an IC of 1. 50 The lowest concentration tested, 1.95 μM, showed a significant reduction in pyocyanin production ( Fig.13 Pyocyanin production was determined in King's A medium. To exclude accidental effects on PAO1 growth due to the medium, growth kinetics were also determined in King's A medium. The initial slopes of the PAO1 bacterial density curves were similar in the control and paecilomycin-treated samples, indicating that bacterial growth was similar ( Fig.14 ). However, in samples treated with high concentrations (from 31.3 μM) of pyrimidine, bacterial density did not reach the same level. However, the difference in bacterial density after 24 h of treatment was relatively small and could not explain the large reduction in pyocyanin production in response to pyrimidine (see Fig.13 and 14). These results indicate that pyocyanin production in PAO1 bacteria was significantly affected in response to paecilomyces lysine treatment.

[0093] Rhamnolipid production was increased in PAO1 cells in response to paecilomyces lysine. The increase was modest, up to 33%, and the variability between individual samples measured three times was large ( Fig.15 ). This effect was statistically significant only after treatment with 31 μM of paecilomyces pyrrolidone. These results indicate that paecilomyces pyrrolidone induces a modest increase in rhamnolipid production in PAO1 bacteria. Bacterial growth kinetics in the presence of paecilomyces pyrrolidone

[0094] After the structure of pseudopaecilomycin was elucidated and the quorum sensing inhibitory activity was established, we studied the growth kinetics of several bacterial strains in the presence of 1, 2, 4, 8, 16, 32, 63, 125 or 250 μM pseudopaecilomycin to determine whether the compound has antibacterial activity. In the presence of the compound, the bacteria were cultured at 37 ° C for 24 h, and the OD600 was measured every 15 min for 24 h. The bacteria tested were two strains of Enterococcus faecium (15A623 and 16D30), MRSAUSA300, MSSA, Staphylococcus epidermidis, Acinetobacter baumannii, Acinetobacter calcoaceticus, Enterobacter cloacae (E. cloacae) complex, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa (ATCC 27853) and Pseudomonas aeruginosa (PAO1) (Figure 16). Based on these growth curves, we can conclude that penicillin inhibits the growth of Enterococcus faecium (15A623 and 16D30), MRSAUSA300, MSSA, Staphylococcus epidermidis, and Klebsiella pneumoniae in MHB medium.

[0095] A similar assay was performed, with the bacteria grown in AB minimal medium. The bacteria tested in this medium were Acinetobacter baumannii, Acinetobacter calcoaceticus, Enterobacter cloacae complex, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa (ATCC 27853), and Pseudomonas aeruginosa (PAO1) ( Fig.17 ).

[0096] Based on these growth curves, we can conclude that penicillin affects the growth of A. baumannii, A. calcoaceticus, E. cloacae complex, E. coli, K. pneumoniae, P. aeruginosa (ATCC 27853), and P. aeruginosa (PAO1) in AB minimal medium. Cytotoxicity

[0097] The cytotoxicity of penicillin on eukaryotic cells was tested using human liver-derived HepG2 cells. The viability of HepG2 cells was evaluated after treatment with a range of concentrations of penicillin for 24 h. At high concentrations, penicillin was toxic to HepG2 cells with an IC 50 The results showed that the viability of HepG2 cells was not different from that of untreated control cells at concentrations up to 125 μM of paecilomycin. These results show that paecilomycin is not cytotoxic to human cells at concentrations that effectively inhibit quorum sensing and / or inhibit bacterial growth, indicating that there is a concentration range for treating bacterial infections. Table 5: Devices

Claims

1. A compound having structure (1): in: - Each of C1, C2, C3, C4 and C9 is independently selected from C═O, C—OH, C—OR 2 , C=S, C–SH, C–SR 2 , C–NH2 or C–N(R 2 )2, wherein the carbon atom is part of a phenalene ring structure, and wherein each R 2 C alone 1-6 Alkyl, C(Y)–C 1-6 Alkyl, C(Y)–Y–C 1-6 Alkyl and C(Y)–N(H) 0-2 (C 1-6 alkyl) 0-2 , wherein each Y is individually O or S; -R 1 Selected from monocyclic aromatic groups, C 1-6 Alkyl, C(O)–C 1-6 Alkyl, C(O)–O–C 1-6 Alkyl and C(O)–N(H) 0-2 (C 1-6 alkyl) 0-2 ; -X is S or O, For use in the treatment or prevention of microbial infections.

2. The compound for use according to claim 1, wherein C1, C2, C3, C4 and C9 are independently selected from C=O, C-OH or C-NH2.

3. Compound for use according to claim 1 or 2, wherein there is an odd number of C=O moieties, preferably 1 or 3.

4. The compound for use according to any one of claims 1 to 3, wherein one of C1 and C9 is C=O, and / or both C2 and C3 are C=O or neither is C=O.

5. The compound for use according to any one of claims 1 to 4, wherein the compound is selected from the group consisting of paecilomycin A, paecilomycin B, paecilomycin C and mixtures thereof.

6. The compound for use according to any one of claims 1 to 5, wherein XR 1 It is OCH3.

7. The compound for use according to any one of claims 1 to 6, wherein the microbial infection is a bacterial infection.

8. The compound for use according to claim 7, wherein the bacterial infection is an infection caused by multidrug-resistant bacteria.

9. The compound for use according to claim 7 or 8, wherein the bacterial infection is an infection caused by Gram-positive bacteria or Gram-negative bacteria, preferably an infection caused by Gram-negative bacteria.

10. A compound for use according to any one of claims 1 to 9, wherein the treatment is a combination treatment of the compound of structure (1) with a second active ingredient, preferably wherein the second active ingredient is an antibiotic.

11. Use of a compound according to any one of claims 1 to 6 for preventing or reducing bacterial biofilm formation.

12. Use of a compound according to any one of claims 1 to 6 for preventing or reducing bacterial proliferation.

13. Use of a compound according to any one of claims 1 to 6 for inhibiting bacterial quorum sensing, biofilm formation and virulence factor production.

14. Use of a compound according to any one of claims 1 to 6 as an antimicrobial agent.

15. An antimicrobial composition comprising a compound according to any one of claims 1 to 6.

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