Antimicrobial compounds, methods for their preparation and use

Abstract

The present disclosure relates to a method for biosynthesizing an antimicrobial compound of formula (I), comprising culturing microbial cells engineered to overexpress fatty acyl-CoA synthase (FAS) or RedD, and isolating the antimicrobial compound of formula (I) produced by the microbial cells, wherein biosynthesis of the antimicrobial compound of formula (I) is upregulated by at least about two-fold compared to a tetramic acid analog derived from a wild-type microbial cell. The disclosure also relates to compounds of formula (I) and their uses in treating microbial diseases or conditions.

Description

[Technical Field]

[0001] The present invention relates generally to antimicrobial compounds, their preparation methods and their uses. [Background technology]

[0002] Natural products (NPs) are highly diverse 3D molecules with high efficacy in medical, agricultural, and food applications. An estimated 50% of commercially available non-biologic drugs are NPs, NP-derived products, or NP mimetics. Actinomycetes have enormous biosynthetic potential to produce novel, bioactive natural compounds, but these microorganisms are often silent under laboratory conditions. For example, the model streptomycete organism Streptomyces coelicolor was thought to have only three NPs based on classical screening. However, genomics and further engineering have now revealed that it can produce 17 unique NPs and more than 20 potential biosynthetic gene clusters (BGCs). With the accumulation of genomic information, the diversity and number of identified BGCs have also rapidly increased, with an estimated 33K putative BGCs discovered in a 1.1K bacterial genome. Silenced gene clusters have recently become a significant natural resource of chemical diversity that can be exploited. To harness these resources, various strategies exist to activate and upregulate NP biosynthesis. As genetic manipulation becomes increasingly easier, genetic-based strategies may be an efficient route to activate silent gene clusters.

[0003] However, even with various genetic strategies, it remains difficult to identify useful NPs, to biosynthesize specific NPs, and to derivatize NPs with specific functional groups and stereochemistry.

[0004] It is desired to overcome or ameliorate at least one of the above problems. Summary of the Invention

[0005] The present invention provides methods for producing tetramic acids and the tetramic acids themselves. Specifically, we demonstrate the application of genetically-based activation to upregulate the production of two tetramic acid compounds, compound 1 (BE 54476-A) and compound 2 (BE 54476-B) (Figure 1). Surprisingly, these upregulated compounds not only possessed antibacterial activity against Staphylococcus aureus but also exhibited bioactivity against Acinetobacter baumannii. These compounds are believed to be the first examples of tetramic acid-type compounds with Gram-negative bioactivity (Figure 1).

[0006] The present invention relates to a compound of formula (I): [ka] wherein R is optionally substituted alkyl, comprising: a) culturing microbial cells engineered to overexpress fatty acyl-CoA synthase (FAS) or RedD; b) isolating the antimicrobial compound of formula (I) produced by the microbial cells; wherein the biosynthesis of the antimicrobial compound of formula (I) is upregulated by at least about two-fold compared to the tetramic acid analog derived from a wild-type microbial cell.

[0007] This method allows the antimicrobial compound of formula (I) to be upregulated so that it can be extracted in at least sufficient amounts.

[0008] In some embodiments, the microbial cell is engineered by introducing a FAS expression cassette or a RedD expression cassette into the genome of the microbial cell.

[0009] In some embodiments, the FAS expression cassette or the RedD expression cassette is integrated into the genome of the microbial cell by an integrative plasmid.

[0010] In some embodiments, the microbial cell is a bacterium, hi some embodiments, the bacterium is a Streptomyces species.

[0011] In some embodiments, the method further comprises the step of inoculating a seed medium with engineered microbial cells prior to the culturing step (step a).

[0012] In some embodiments, the seed medium is SV2 seed medium.

[0013] In some embodiments, the engineered microbial cells are inoculated for at least 4 days.

[0014] In some embodiments, the engineered microbial cells are cultured in a medium comprising glycerol and / or starch, hi some embodiments, the medium is CA07LB (glycerol-based) and / or CA10LB (starch-based).

[0015] In some embodiments, the engineered microbial cells are cultured for at least 7 days.

[0016] In some embodiments, the engineered microbial cells are co-cultured with an inducer strain, which in some embodiments is a mycolic acid bacterium, such as a Rhodococcus species.

[0017] In some embodiments, the volume ratio of inducer strain to engineered microbial cells is about 1:2 to about 1:5, in some embodiments, the volume ratio of inducer strain to engineered microbial cells is about 1:3.

[0018] In some embodiments, the method further comprises freeze-drying the culture prior to the isolating step (step b).

[0019] In some embodiments, the antimicrobial compound of Formula (I) is isolated from a microbial cell culture medium.

[0020] In some embodiments, the antimicrobial compound of Formula (I) is an antibacterial compound.

[0021] In some embodiments, the antimicrobial compounds of Formula (I) are characterized by antibacterial activity against gram-positive and gram-negative bacteria.

[0022] In some embodiments, the Gram-positive bacterium is S. aureus.

[0023] In some embodiments, the antimicrobial compounds of Formula (I) have a minimum inhibitory concentration (MIC) against Gram-positive bacteria of less than about 20 μM, less than about 15 μM, or less than about 8 μM. 50 ) is characterized by.

[0024] In some embodiments, the antimicrobial compounds of Formula (I) have a minimum lethal concentration (MBC) against Gram-positive bacteria of less than about 80 μM, less than about 65 μM, or less than about 22 μM. 50 ) is characterized by.

[0025] In some embodiments, the gram-negative bacterium is A. baumannii.

[0026] In some embodiments, the antimicrobial compounds of Formula (I) have a minimum inhibitory concentration (MIC) against Gram-negative bacteria of less than about 20 μM, less than about 10 μM, or less than about 7 μM. 50 ) is characterized by.

[0027] In some embodiments, the antimicrobial compounds of Formula (I) have an IC against cancer cells of less than about 50 μM, less than about 35 μM. 50 It is characterized by:

[0028] The present invention relates to a compound of formula (I): [ka] Also provided is an antimicrobial compound of the formula: where R is optionally substituted alkyl.

[0029] It has been discovered that the absence of a methyl group in the tetramic acid moiety may confer biological activity against gram-negative bacteria.

[0030] In some embodiments, R is optionally substituted methyl, optionally substituted ethyl, or optionally substituted propyl.

[0031] In some embodiments, the antimicrobial compound of Formula (I) has the formula (Ia): [ka] where R is optionally substituted alkyl.

[0032] In some embodiments, the antimicrobial compound is: [ka] is selected from.

[0033] The present invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0034] The present invention also provides a method of treating a microbial disease or condition, comprising administering to a subject in need thereof an antimicrobial compound of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0035] In some embodiments, the microbial disease or condition is selected from diseases or conditions caused by S. aureus and / or A. baumannii. Such diseases or conditions can be skin and soft tissue infections (such as abscesses), blood infections, pneumonia, bone and joint infections, urinary tract infections, pneumonia, or open wound infections.

[0036] Embodiments of the invention will now be described, by way of non-limiting example, with reference to the drawings, in which: [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 shows the structures of selected known tetramic acid-containing natural products, as well as Compound 1 and Compound 2. [Figure 2] Figure 1 shows activation in Streptomyces sp. A58051. (A) Yields of compound 1 and compound 2 for various mutants under CA10LB and CA07LB fermentation. A100020 and A100023 are FAS-overexpressing strains. A100292 is a RedD-overexpressing strain. (B) Total metabolites produced in RedD, FAS mutants compared to WT. The number of metabolites is shown in parentheses in each section; metabolites in overlapping sections are produced under two or more regulators. Only metabolite signals with a base peak abundance of at least 10 are considered here (a list of metabolites is provided in the supplement). (C) Comparison of unique metabolites produced in wild-type (WT) FAS and RedD-integrated strains under CA10LB medium conditions. [Figure 3] Figure 10. Co-culture fermentation: fold change over wild type in CA07LB for mutants alone, mutants in co-culture with inducer strain Rhodococcus sp. T5718. [Figure 4] 3A-3C show the chemical structures of tetramic acid analogs, Compound 1 and Compound 2 (the relative configurations of the stereocenters were determined by NOESY correlation as shown in 3C). (B) Selected COSY and HMBC correlations of Compound 1 and Compound 2. (C) Selected NOESY correlations of Compound 1 and Compound 2. [Figure 5] FIG. 1 shows the integration of an overexpression cassette of SCO6196 (an AMP-binding domain-containing protein) into Streptomyces sp. A58051. [Figure 6]FIG. 1 shows dose response curves for K. aerogenes (ATCC™ 13048™), P. aeruginosa (ATCC™ 9027™), and A. fumigatus (ATCC™ 46645™). A) Compound 1, B) Compound 2. [Figure 7] Dose response curves for S. aureus Rosenbach (ATCC™ 25923™) (SA25923). A) Compound 1, B) Compound 2. [Figure 8] Figure 1 shows dose response curves for A. baumannii (ATCC™ 19606™) (ACB19606). A) Compound 1, B) Compound 2. [Figure 9] Figure 1 shows dose response curves for A549 human lung cancer cells (ATCC™ CCL-185™) (A549). A) Compound 1, B) Compound 2. DETAILED DESCRIPTION OF THE INVENTION

[0038] Antimicrobial compounds refer to agents that can be used primarily against microorganisms by killing them and / or inhibiting their further growth. Microorganisms include bacteria, protozoa, algae, and fungi. Of course, antibacterial compounds refer to agents that can be used primarily against bacteria by killing them or inhibiting their further growth.

[0039] The present invention relates to a compound of formula (I): [ka] wherein R is optionally substituted alkyl, comprising the steps of: a) culturing microbial cells engineered to overexpress fatty acyl-CoA synthase (FAS) or RedD; b) isolating the antimicrobial compound of formula (I) produced by the microbial cells; The present invention provides a method comprising:

[0040] The microbial cells may contain a cellular or enzymatic pathway that produces intracellular triacylglycerol. In some embodiments, the microbial cells are configured to upregulate the intracellular triacylglycerol of the microbial cells. This method can upregulate the antimicrobial compound of Formula (I) so that it can be extracted in at least sufficient amounts.

[0041] Fatty acyl-CoA synthase (FAS) or RedD acts to upregulate the intracellular triacylglycerol pool of microbial cells, thereby upregulating the production of secondary metabolites, which increases the production of the antimicrobial compound of formula (I).

[0042] Microbial cells can be characterized by an enzymatic pathway that synthesizes tetramic acids and / or analogs (including compounds of Formula (I)). For example, the enzymatic pathway can include polyketide synthases, cyclases, reductases, and / or nonribosomal peptide synthetases. In some embodiments, microbial cells are engineered to synthesize compounds of Formula (I) via an enzymatic pathway. However, the yield of the compound can be low. Therefore, microbial cells can be engineered to increase the yield of tetramic acids. In some embodiments, microbial cells are engineered by introducing a fatty acyl-CoA synthase (FAS) expression cassette or a RedD expression cassette into the microbial cell. In some embodiments, microbial cells are engineered by introducing a fatty acyl-CoA synthase (FAS) expression cassette or a RedD expression cassette into the genome of the microbial cell. An expression cassette is a separate component of vector DNA that consists of genes and regulatory sequences that are expressed by the transfected cell. Upon successful transformation, the expression cassette directs the cellular machinery to make RNA and protein(s). Some expression cassettes are designed for modular cloning of protein-coding sequences, so that the same cassette can be easily modified to produce different proteins. The expression cassette (and its transgene) may simply be incorporated into the microbial cell and transcribed and / or translated, or the expression cassette may be integrated into the genome of the microbial cell. In this regard, as long as FAS and RedD are expressed, an antimicrobial compound may be produced. As used herein, "expression cassette" and "overexpression cassette" are used interchangeably.

[0043] In some embodiments, FAS or RedD is overexpressed. In some embodiments, the FAS overexpression cassette or RedD overexpression cassette is integrated into the genome of the microbial cell by an integrative plasmid. Integrative plasmids are most often suicide vectors, i.e., vectors that cannot replicate in the target host and must either integrate or disappear; therefore, any plasmid that can be efficiently introduced into the recipient can be used.

[0044] In some embodiments, the integrating plasmid is pSET152.

[0045] In some embodiments, the microbial cell is a bacterium. In some embodiments, the bacterium is a Streptomyces species. In some embodiments, the bacterium is an actinomycete. Other microbial cells, such as heterologous hosts, can also be used. Heterologous expression refers to the expression of a gene or portion of a gene in a host organism that does not naturally possess that gene or gene fragment. Insertion of a gene in a heterologous host can be accomplished by recombinant DNA techniques.

[0046] In some embodiments, the method further comprises inoculating a seed medium with the engineered microbial cells prior to the culturing step (step a). In some embodiments, the seed medium is SV2 seed medium. SV2 seed medium may contain 15 g / L glucose, 15 g / L glycerol, 15 g / L soy peptone, 1 g / L calcium carbonate, and may have a pH of 7.0.

[0047] In some embodiments, the engineered microbial cells are inoculated for at least 4 days. In other embodiments, the engineered microbial cells are inoculated for at least 5 or 6 days. Inoculation introduces the microorganisms into an environment where they can grow and multiply.

[0048] In some embodiments, engineered microbial cells are cultured in a medium containing glycerol and / or starch. It is believed that glycerol can be channeled to glycerol-3-phosphate and TAG, thereby increasing flux to compound production. In some embodiments, the medium is CA07LB (glycerol-based) and / or CA10LB (starch-based).

[0049] Microbiological culture or microbial culture is the process of multiplying microbial organisms by growing them in a defined culture medium under controlled laboratory conditions.

[0050] In some embodiments, the engineered microbial cells are cultured for at least 7 days, hi other embodiments, the engineered microbial cells are cultured for at least 8, 9, or 10 days.

[0051] In some embodiments, the engineered microbial cells are co-cultured with an inducer strain. The inducer strain is a substance capable of activating the growth of the engineered microbial cells. For example, mycolic acids can be used to activate the growth of the engineered microbial cells. In some embodiments, the inducer strain is a mycolic acid-containing bacterium, such as a Rhodococcus species.

[0052] In some embodiments, the volume ratio of inducer strain to engineered microbial cells is about 1:2 to about 1:5. In some embodiments, the volume ratio of inducer strain to engineered microbial cells is about 1:3.

[0053] In some embodiments, the method further comprises freeze-drying the culture prior to the isolation step (step b). In this manner, the engineered microbial cells can be lyophilized into a powder.

[0054] In some embodiments, the antimicrobial compounds are isolated from the microbial cell culture medium. In some embodiments, the antimicrobial compounds are extracted from the culture medium using an organic solvent. In some embodiments, the antimicrobial compounds are extracted from the culture medium using methanol.

[0055] In some embodiments, the antimicrobial compound is upregulated at least about 2-fold compared to a tetramic acid analog derived from a wild-type microorganism. The tetramic acid analog derived from a wild-type microorganism can be BE-54476. In other embodiments, the upregulation is at least about 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, or 20-fold.

[0056] In some embodiments, the antimicrobial compound is an antibacterial compound.

[0057] In some embodiments, the antimicrobial compound is characterized by antibacterial activity against Gram-positive and Gram-negative bacteria. In some embodiments, the Gram-positive bacteria is S. aureus. In some embodiments, the Gram-negative bacteria is A. baumannii.

[0058] In some embodiments, the antimicrobial compound has a minimum inhibitory concentration (MIC) against Gram-positive bacteria of less than about 20 μM. 50 In some embodiments, the antimicrobial compound has a minimum inhibitory concentration (MIC) against Gram-positive bacteria of less than about 15 μM. 50 In some embodiments, the antimicrobial compound has a minimum inhibitory concentration (MIC) against Gram-positive bacteria of less than about 8 μM. 50 ) is characterized by.

[0059] In some embodiments, the antimicrobial compound has a minimum lethal concentration (MBC) for Gram-positive bacteria of less than about 80 μM. 50 In some embodiments, the antimicrobial compound has a minimum lethal concentration (MBC) for Gram-positive bacteria of less than about 65 μM. 50In some embodiments, the antimicrobial compound has a minimum lethal concentration (MBC) for Gram-positive bacteria of less than about 22 μM. 50 ) is characterized by.

[0060] In some embodiments, the antimicrobial compound has a minimum inhibitory concentration (MIC) against Gram-negative bacteria of less than about 20 μM. 50 In some embodiments, the antimicrobial compound has a minimum inhibitory concentration (MIC) against Gram-negative bacteria of less than about 10 μM. 50 In some embodiments, the antimicrobial compound has a minimum inhibitory concentration (MIC) against Gram-negative bacteria of less than about 7 μM. 50 ) is characterized by.

[0061] In some embodiments, the antimicrobial compounds of Formula (I) have an IC against cancer cells of less than about 50 μM. 50 In some embodiments, the antimicrobial compound of Formula (I) has an IC against cancer cells of less than about 35 μM. 50 It is characterized by:

[0062] In some embodiments, the compound of formula (I): [ka] wherein R is optionally substituted alkyl, comprising the steps of: a) incorporating a fatty acyl-CoA synthase (FAS) expression cassette or a RedD expression cassette into a microbial cell to form an engineered microbial cell; b) culturing the engineered microbial cells; c) isolating the antimicrobial compounds produced by the microbial cells; A method is provided, comprising:

[0063] In some embodiments, the compound of formula (I): [ka] wherein R is optionally substituted alkyl, comprising the steps of: a) integrating a fatty acyl-CoA synthase (FAS) expression cassette or a RedD expression cassette into the genome of a microbial cell to form an engineered microbial cell; b) culturing the engineered microbial cells; c) isolating the antimicrobial compounds produced by the microbial cells; A method is provided, comprising:

[0064] In some embodiments, the compound of formula (I): [ka] wherein R is optionally substituted alkyl, comprising the steps of: a) integrating a fatty acyl-CoA synthase (FAS) expression cassette or a RedD expression cassette into the genome of a microbial cell to form an engineered microbial cell; b) culturing the engineered microbial cells; c) freeze-drying the engineered microbial cells of step b); d) isolating the antimicrobial compounds produced by the microbial cells; A method is provided, comprising:

[0065] The present invention also provides a method for the large-scale production of antimicrobial compounds. Using this method in the laboratory, antimicrobial compounds can be isolated at a scale of at least about 2 mg / L or 3 mg / L. Under co-culture conditions, up to a 10-fold improvement in compound production can be observed, corresponding to a yield of up to about 20 mg / L. The engineered microbial cells may also be co-cultured with an inducer strain, such as a Rhodococcus species.

[0066] In some embodiments, the method comprises: a) culturing microbial cells engineered to overexpress fatty acyl-CoA synthase (FAS) or RedD; b) isolating the antimicrobial compound of formula (I) produced by the microbial cells; wherein the microbial cells are cultured in co-culture in the presence of an inducer strain.

[0067] The present invention provides [ka] Also provided are antimicrobial compounds of formula (I), including: wherein R is optionally substituted alkyl.

[0068] It has been discovered that the absence of a methyl group in the tetramic acid moiety may confer biological activity against gram-negative bacteria.

[0069] In some embodiments, R is optionally substituted methyl, optionally substituted ethyl, or optionally substituted propyl. In some embodiments, R is methyl, ethyl, or propyl.

[0070] As used herein, "optionally substituted" means that the group is hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino ... It is understood to mean that the "optionally substituted amino" group may or may not be further substituted or fused (to form a fused polycyclic group) with one or more groups selected from siloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclylamino, and unsymmetrical disubstituted amines having different substituents selected from alkyl, aryl, heteroaryl, heterocyclyl, etc. For example, an "optionally substituted amino" group can include amino acid and peptide residues.

[0071] In some embodiments, the optional substituents on R are one or more groups selected from hydroxyl, acyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, cyano, halogen, nitro, phosphorylamino, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, and heterocyclyloxy.

[0072] In some embodiments, the antimicrobial compound of Formula (I) has the formula (Ia): [ka] where R is optionally substituted alkyl.

[0073] In some embodiments, the antimicrobial compound is: [ka] is selected from.

[0074] The present invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0075] The present invention also provides a method of treating a microbial disease or condition, comprising administering to a subject in need thereof an antimicrobial compound of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0076] The present invention provides the use of an antimicrobial compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for the treatment of a microbial disease or condition.

[0077] The present invention also provides an antimicrobial compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in the treatment of a microbial disease or condition.

[0078] In some embodiments, the microbial disease or condition is selected from a disease or condition caused by S. aureus and / or A. baumannii. Such a disease or condition can be a skin or soft tissue infection (such as an abscess), a blood infection, pneumonia, a bone or joint infection, a urinary tract infection, pneumonia, or an open wound infection.

[0079] The present invention also provides a method of treating cancer, comprising administering to a subject in need thereof an antimicrobial compound of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0080] The present invention provides the use of an antimicrobial compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for treating cancer.

[0081] The present invention also provides an antimicrobial compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in the treatment of cancer.

[0082] In some embodiments, the cancer is lung cancer.

[0083] The compounds of the present invention can be administered to a subject as their pharmaceutically acceptable salts. 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, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0084] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium. In particular, the present invention includes within its scope cationic salts, such as sodium or potassium salts, or alkyl esters (e.g., methyl, ethyl) of the phosphate group.

[0085] Basic nitrogen-containing groups can 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 sulfate and diethyl sulfate; and the like.

[0086] It will be understood that any compound that is a prodrug of a compound of Formula (I) is within the scope and spirit of the present invention. Thus, the compounds of the present invention can be administered to a subject in the form of a pharmaceutically acceptable prodrug. The term "prodrug" is used in its broadest sense and encompasses derivatives that are converted to the compounds of the present invention in vivo. Such derivatives will be readily apparent to those skilled in the art. Other documents generally describing prodrugs (and their preparation) include "Design of Prodrugs," 1985, H. Bundgaard (Elsevier); "The Practice of Medicinal Chemistry," 1996, Camille G. Wermuth et al., Chapter 31 (Academic Press); and "A Textbook of Drug Design and Development," 1991, Bundgaard et al., Chapter 5 (Harwood Academic Publishers). For example, an amine moiety can be quarternized or converted to an amide moiety, or a hydroxyl moiety can be converted to an ester moiety.

[0087] The compounds of the present invention may be in crystalline form either as free compounds or as solvates (e.g., hydrates), and both forms are intended to be within the scope of the present invention. Methods of solvation are generally known in the art.

[0088] The compounds of the present invention, or pharmaceutically acceptable salts, solvates, or prodrugs thereof, are administered to a patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially achieving a desired effect, or delaying the onset of macular degeneration, inhibiting its progression, or completely halting or reversing its onset or progression.

[0089] As used herein, the term "effective amount" refers to an amount of a compound that, when administered according to a desired dosing regimen, results in a desired therapeutic activity. Administration can be at intervals of minutes, hours, days, weeks, months, or years, or continuously over any one of these time periods. A suitable dosage can be within the range of about 0.1 ng / kg to 1 g / kg of body weight per administration, e.g., 1 mg to 1 g / kg of body weight per administration. In one embodiment, the dosage can be within the range of 1 mg to 500 mg / kg of body weight per administration. In another embodiment, the dosage can be within the range of 1 mg to 250 mg / kg of body weight per administration. In yet another embodiment, the dosage can be within the range of 1 mg to 100 mg / kg of body weight per administration, e.g., up to 50 mg / kg of body weight per administration.

[0090] Suitable dosages and administration regimens can be determined by the attending physician and may vary depending on the severity of the condition and the general age, health and weight of the patient being treated.

[0091] The compounds of the present invention can be administered in a single dose or in continuous administration. While it is possible for the active ingredient to be administered alone, it is preferable to provide it as a composition, preferably a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carrier, diluent, or excipient. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, skin penetration agents, surfactants, isotonic and absorption agents, and the like. It will be understood that the compositions of the present invention may also contain other supplementary physiologically active agents.

[0092] Carriers must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0093] The compound can be injected directly into the eye, particularly the vitreous body of the eye. The compound or composition of the present invention can be administered to the vitreous body of the eye using any intravitreal or transscleral administration method. For example, the compound or composition can be administered to the vitreous body of the eye by intravitreal injection. Intravitreal injection typically involves administering a total amount of 0.1 ng to 10 mg of the compound of the present invention or a pharmaceutically acceptable salt, solvate, or prodrug per injection.

[0094] Injectables for such use can be prepared in conventional forms, either as liquid solutions or suspensions, or solid forms suitable for preparation as a solution or suspension in liquid prior to injection, or as emulsions. Carriers can include, for example, water, saline (e.g., normal saline (NS), phosphate-buffered saline (PBS), balanced salt solution (BSS)), Ringer's lactate, dextrose, glycerol, ethanol, etc., and, if desired, small amounts of auxiliary substances such as wetting or emulsifying agents, buffers, etc. can be added. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. For example, the compound or composition can be dissolved in a pharmaceutically effective carrier and injected into the vitreous of the eye using a temporary approach (e.g., about 3 mm to about 4 mm posterior to the limbus of the human eye to avoid damage to the lens) through a thin-gauge hollow needle (e.g., a 30-gauge, ½-inch, or ⅜-inch needle).

[0095] Those skilled in the art will appreciate that other means of injecting and / or administering a compound or composition into the vitreous of the eye can also be used. These other means can include, for example, intravitreal medical delivery devices. These devices and methods can include, for example, intravitreal pharmaceutical delivery devices and biodegradable polymeric delivery members that are inserted into the eye for long-term delivery of pharmaceuticals. These devices and methods can also include transscleral delivery devices.

[0096] Other modes of administration, including topical or intravenous administration, may also be possible. For example, a solution or suspension of the compounds or compositions of the present invention can be formulated as eye drops or a membrane eye patch that is applied directly to the surface of the eye. Topical application typically involves administering the compounds of the present invention in amounts of 0.1 ng to 10 mg.

[0097] The compounds or compositions of the invention may also be suitable for intravenous administration. For example, the compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, may be administered intravenously at a dose of up to 16 mg / m 2 can be administered intravenously at a dose of

[0098] The compounds or compositions of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary, or paste. In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt, solvate, or prodrug thereof may be administered orally.

[0099] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder (e.g., an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets can optionally be coated or scored, and can be formulated to provide slow or controlled release of the active ingredient therein, using, for example, hydroxypropyl methylcellulose in various proportions to provide the desired release profile. Tablets can optionally be enteric coated to provide release in parts of the gut other than the stomach.

[0100] The compounds or compositions of the invention may be suitable for topical administration in the mouth, including lozenges comprising the active ingredient in a flavored base, usually sucrose and gum arabic or gum tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and gum arabic; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0101] The compounds or compositions of the present invention may be suitable for topical administration to the skin and may include the compound dissolved or suspended in any suitable carrier or base, and may take the form of a lotion, gel, cream, paste, ointment, etc. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The compounds of the present invention may also be administered using a transdermal patch.

[0102] The compounds or compositions of the present invention may be suitable for parenteral administration, including aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bactericides, and solutes which render the compounds or compositions isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compounds or compositions may be presented in single-dose or multi-dose hermetically sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind described above.

[0103] Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

[0104] It should be understood that the compositions of the present invention may contain, in addition to the active ingredients specifically mentioned above, other agents conventionally used in the art for the type of composition in question. For example, those suitable for oral administration may contain additional agents such as binders, sweeteners, thickeners, flavorings, disintegrants, coating agents, preservatives, lubricants, and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid, or agar. Suitable flavoring agents include peppermint oil, wintergreen oil, cherry, orange, or raspberry flavor. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac, or gluten. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methylparaben, propylparaben, or sodium bisulfite. Suitable lubricating agents include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. [Example]

[0105] A detailed description of the operation of the present invention is provided below. In the following embodiments, the present invention is described in connection with some conditions for consistency to illustrate the present invention. However, those skilled in the art will understand that the present invention is not limited to such.

[0106] Strain activation by integration of multifunctional regulators Streptomyces sp. A58051 was obtained from the Agency for Science, Technology and Research (AIST) of Singapore. *The gene was derived from the Natural Organism Library (NOL) of the National Institute for Standardization (STAR). Based on genome analysis, it is 77.6% similar to Streptomyces durhamensis (GCA_000725475.1). To perturb and activate secondary metabolites in Streptomyces sp. A58051, we first used a genetic engineering strategy to introduce multifunctional regulators into its genome. In this study, mutants were engineered with overexpression cassettes containing either fatty acyl-CoA synthase (FAS, SCO6196, https: / / www.ncbi.nlm.nih.gov / gene / 1101637) or the specific pathway regulatory protein (SARP), RedD (SLIV_09220, https: / / www.ncbi.nlm.nih.gov / protein / 672368967). FAS has previously been shown to upregulate the intracellular triacylglycerol pool in Streptomyces, resulting in upregulation of secondary metabolite production. While SARPs are often pathway-specific, RedD and similar homologs have been observed to have broad effects on biosynthetic gene clusters within Streptomyces. In overexpression mutants of both FAS and RedD, upregulation of two tetramic acids, Compound 1 and Compound 2, was observed (Figure 1). To compare differences between the wild-type and mutant strains, cheminformatics was used to identify the 10 observed in base peak chromatograms (BPCs) from liquid chromatography-mass spectrometry (LC-MS). 5Unique metabolites in the extracts were classified based on peaks with base peak abundances above 1000 m / z. These peaks were characterized primarily by their base peak m / z and retention time (full details on the process are provided in the Supplementary Information). Overexpression of FAS (A100020) and RedD (A100292) appeared to nearly double the number of unique metabolite species from 16 to 28 (Figure 2b), with FAS having the greatest impact on the chemical profile. Eleven new metabolites could arise from FAS overexpression (Figure 2c).

[0107] The upregulation of tetramic acid compounds was also found to be medium-dependent. Among the mutant strains, CA10LB (starch-based) fermentation significantly increased yields by 3- to 15-fold over wild-type production under the same conditions. Meanwhile, only a 2-fold increase was observed in CA07LB (glycerol-based) fermentation (Figure 2). Depending on the fermentation medium conditions and mutants, this strategy resulted in a 2- to 15-fold change in the production of 1 and 2, including an increase in the production ratio of 2 (Figure 2). Coculture with the mycolic acid bacterium Rhodococcus sp. T5718 could further triple the yield of this native strain (Figure 3).

[0108] Isolation and structural characterization of compound 1 and compound 2 A100020 was cultured in 1 L of CA07LB liquid medium at 28°C for 10 days. The obtained MeOH extract was collected as fraction C. 18 HPLC separation gave two new tetramic acid derivatives, Compound 1 and Compound 2 (Figure 4).

[0109] Compound 1 was isolated as a brown amorphous powder and showed (-)-HRESI MS data (m / z 362.1968 [M−H] - , C 20 Analysis of H8O5N (calculated value, 362.1973) reveals that the molecular formula is C 20 H 29 It was assigned to O5N. 1 The H NMR spectrum shows four methyl groups (δ H 0.75, 1.04, 1.63 and 3.26), three methylene (δ H1.15 / 1.65, 1.29 and 3.75), and eight methine protons (δ H The signals corresponding to the α- and β-glucans were 1.39, 2.12, 2.57, 2.76, 2.99, 3.43, 4.06 and 5.61. 13 The C NMR spectrum and edited HSQC spectrum showed 20 resonances, including four methyl, three methylene, eight methine, and five unprotonated carbons. The UV absorption maxima at 220 nm and 285 nm are typical of the tetramic acid moiety, which is at δ C Characteristic broad peaks at 104.5, 178.2, 196.0 and 201.8 13 This was further supported by the C NMR signal. 13 The C peak was likely due to keto-enol tautomerism of the tetramic acid moiety. The remaining structure of 1 was assigned based on COSY and HMBC data (Figure 4B). Analysis of the COSY spectrum yielded the fragments H-5 / H-6 / H2-7 / H-8 / H-9 / H-10 / H-11 / H-2 / H-3 and H3-15 / H-8 (Figure 4B). These data, along with HMBC correlations from H-5 to C-6, C-7 and C-11, and H-10 to C-6, established the partial structure of decalin. Furthermore, the H-3 / H2-12 / H3-13 fragments were identified. 1 H- 1 The H COSY spin system and the HMBC cross peak from H2-12 to C-3 confirmed the location of the CH3-CH2- substituent at C-3. C HMBC correlation to the olefinic carbon at 135.4 located the methyl group at C-4. The position of the methoxy at C-10 is δ H δ from the singlet methyl resonance at 3.75 C The presence of a tetramic acid moiety at C-2 was evident by HMBC correlation to carbon dioxide at 88.7. H 3.75) to C-2' and C-4', and H-2 (δ H This was further confirmed by consideration of the deshielded chemical shifts of 1 and 2 (δ 4.06). (16, 17) Finally, the molecular formula of 1 and the chemical shifts of CH-9 (δ H / δC 3.43 / 75.1), the hydroxy group was located at C-9.

[0110] The relative configuration of 1 was confirmed by NOESY data (Figure 4C) and 1 H- 1 The assignment was based on analysis of H-coupling constants and comparison with reported NMR data of similar decaline-containing tetramic acid compounds. For example, the decaline ring junction signal H-11 (δ H 2.76) and H-6(δ H The chemical shifts of H-2 and H-9 and H-7 were consistent with those of other cis-decalin tetramic acid analogues compared to the trans-fused congeners. The large coupling constant of 9.8 Hz between H-9 and H-10 indicated that these protons were axially oriented. ax NOESY correlation (δ H The peaks (1.15, ddd (12.3, 12.3, 12.3)) suggested that these protons were on the same side of the molecule (α-orientation). Finally, additional NOESY cross-peaks between H-6 and H-8 and between H-3 and H-11 supported the β-configuration of these protons. Thus, the structure of 1 was established as a new tetramic acid derivative.

[0111] Compound 2 was identified by (-)-HRESIMS as having the molecular formula C 20 H 31 It was assigned to O5N. 1 H NMR data and 13 The C NMR data and UV spectrum were similar to those of 1. Detailed analysis of the NMR and MS data of 2 suggested that compound 2 has an additional -CH2- group. 2D NMR data analysis showed that 2 has a propyl moiety at C-3 instead of the ethyl group of 1. This is due to the δ H Further support was provided by the triplet methyl (13-CH3) HMBC correlation of 0.81 to C-12 and C-13, and the COSY correlation of 13-CH3 / H2-13 / H2-12 / H-3. 1 H-1 Analysis of the H-coupling constants assigned the same relative configuration to 2 as previously determined for 1.

[0112] [Table 1]

[0113] Biological activity of Compound 1 and Compound 2 Compounds 1 and 2 were tested for antimicrobial activity against a panel of microorganisms consisting of Gram-positive and Gram-negative bacteria, as well as one fungal strain: A. baumannii (ATCC™ 19606™), K. aerogenes (ATCC™ 13048™), P. aeruginosa (ATCC™ 9027™), S. aureus Rosenbachii (ATCC™ 25923™), and A. fumigatus (ATCC™ 46645™).

[0114] Both compounds were found to be inactive against the Gram-negative bacteria K. aerogenes (ATCC™ 13048™) and P. aeruginosa (ATCC™ 9027™) and the fungal strain A. fumigatus (ATCC™ 46645™) (Table 2, Figure 6). However, Compound 1 and Compound 2 showed activity against S. aureus Rosenbachii (ATCC™ 25923™) (Table 2, Figure 7), with Compound 1's minimum inhibitory concentration (MIC) exceeding that of Compound 1. 50 ) was 14.5 μM, and the minimum lethal concentration (MBC 50 ) was 65.5 μM, and the MIC of compound 2 50 7.5 μM, MBC 50 Interestingly, the compound was also found to have activity against the Gram-negative strain A. baumannii (ATCC™ 19606™) (Table 2, Figure 8), with an MIC of Compound 1 of 2.2 μM. 50 MIC of compound 2 was 9.8 μM. 50The IC was 6.9 μM. In antimicrobial testing against A. baumannii, no MBC activity was observed, likely due to different resistance mechanisms in these Gram-negative bacteria. Separately, both compounds were also tested for cytotoxicity against the human lung cancer cell line A549 (ATCC™ CCL-185™), with IC 50 were 34.4 μM and 46.3 μM, respectively (Table 2, Figure 9).

[0115] [Table 2]

[0116] Essay The emergence of infectious diseases, coupled with the declining efficacy of antibiotics due to multidrug resistance, is a persistent international problem. Therefore, in addition to corrective measures for appropriate antibiotic use, new agents to replace current state-of-the-art antibiotics against infectious diseases are needed. In recent years, the emergence of multidrug-resistant Acinetobacter baumannii has also raised concerns, especially as it has become resistant to last-resort antibiotics. Here, we demonstrate the use of gene activation to upregulate a novel tetramic acid compound with remarkable biological activity against A. baumannii.

[0117] Other structurally similar tetramic acid analogs (Figure 1) have been reported to exhibit antimicrobial activity, primarily against Gram-positive bacteria. For example, equisetin has been described as active against Staphylococcus erythraea and Staphylococcus aureus, ascosalipyrrolidone A has shown activity against Bacillus megaterium, Mycoptypha microsporosum, and Microbotyryum violaceum, and BU-4514N and the fungal metabolite altercetin have shown inhibitory activity against several Gram-positive bacteria. Notably, in comparison to compounds 1 and 2, the similar compound, antibiotic BE-54476, has been reported to have antibacterial activity against Gram-positive bacteria such as Bacillus subtilis, Enterococcus faecalis, and S. aureus, as well as antitumor activity. Surprisingly, removal of the methyl group (1 and 2) was sufficient to demonstrate significant bioactivity against Acinetobacter baumannii, one of the first examples of Gram-negative bioactivity within a similar scaffold. Compound 1's chemical structure differs from that of compound 2 at C-3, featuring an ethyl group as opposed to the propyl moiety of compound 2. However, bioactivity testing demonstrated that both compounds possess similar activity, including antibacterial activity against Gram-positive S. aureus and Gram-negative A. baumannii, as well as cytotoxicity against human lung cancer A549 cells. This indicates that the difference in chemical structure between compounds 1 and 2 does not significantly affect their antimicrobial and cytotoxic activities. Demonstrating their uniqueness, compounds 1 and 2 were not observed elsewhere in high-resolution MS scans among approximately 2K actinomycetes in the NOL collection.

[0118] Finally, we demonstrated multiple strategies to increase tetramic acid yield, including genetic engineering and medium optimization. Overexpression of either RedD or FAS improved tetramic acid yield, enabling the isolation of the compound. In the wild-type strain, the medium played a major role in improving yield. However, this difference was reduced in the overexpression mutant. FAS was previously demonstrated to enable Streptomyces to utilize the intracellular triacylglycerol pool to promote natural product biosynthesis. In the wild-type strain, glycerol-based fermentation was shown to be 2- to 4-fold more productive than non-glycerol medium. We hypothesized that glycerol would be channeled to glycerol-3-phosphate and TAG, thereby increasing flux toward tetramic acid production. However, in the presence of FAS overexpression, this pathway toward acyl-CoA was no longer limiting, and as a result, the yield difference across the medium was observed to be less significant.

[0119] Overall, a combination of strategies has been used to perturb the regulation of antibiotic production. In this study, seemingly unrelated regulators were sufficient to increase production of low-yield but bioactive compounds. This increase allowed the discovery of new tetramic acid analogs with novel activity against A. baumannii.

[0120] Materials and Methods Overexpression cassette The overexpression FAS cassette is * -FAS (accession code: WP_011030732). The overexpression RedD cassette is * -RedD (accession code: AIJ12849.1). *is a strong constitutive promoter. The integrative plasmid was obtained by inserting the overexpression cassette into pSET152. Integration was mediated by the attP site of Streptomyces phage ΦC31. The completed plasmid was conjugated into Streptomyces sp. A58051 from the Natural Organism Library Collection (SIFBI, NPL), and the integrated mutants were screened and sequenced (Supplementary Figure 1). The FAS mutants are designated A100020 and A100023, and the RedD integrated mutant is designated A100292 (Figure 2).

[0121] Fermentation and Extraction Wild-type Streptomyces and the edited mutants were grown on ISP2 plates (10 g / L malt extract broth, 4 g / L Bacto yeast extract, 4 g / L glucose, 20 g / L Bacto agar) for 5 days at 30° C. Three 5 mm diameter agar plugs from the culture plates were then used to inoculate four 250 mL Erlenmeyer flasks containing 50 mL each of SV2 seed medium (15 g / L glucose, 15 g / L glycerol, 15 g / L soy peptone, 1 g / L calcium carbonate, pH 7.0) and incubated at 30° C. for 4 days with shaking at 200 rpm.

[0122] A 2.5 mL volume of the homogenized seed culture was then inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium: CA07LB (glycerol 15 g / L, oatmeal 30 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 5 g / L, disodium hydrogen phosphate dodecahydrate 5 g / L, magnesium chloride hexahydrate 1 g / L) or CA10LB (soluble starch 20 g / L, soybean flour 15 g / L, potassium dihydrogen phosphate 3 g / L, disodium hydrogen phosphate dodecahydrate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, trace salt solution 1 mL / L, iron(II) heptahydrate 2 g / L, manganese chloride tetrahydrate 2 g / L, zinc sulfate heptahydrate 2 g / L, copper(II) sulfate pentahydrate 2 g / L, cobalt(II) chloride hydrate 2 g / L, pH 7.2). All cultures were fermented for 9 days at 30°C with shaking at 200 rpm in a 50 mm throw.

[0123] After the incubation period, the cultures were freeze-dried. The freeze-dried samples were extracted overnight with methanol. The extracted mixture was passed through a cellulose filter (Whatman Grade 4, 1004-185), and the filtrate was dried using a rotary evaporator.

[0124] Large-scale fermentation and extraction The A100020 mutant was grown on Bennet agar (Himedia, M694) plates for 5 days at 28°C. Three 5 mm diameter agar plugs from the culture plates were then used to inoculate 4 x 250 mL Erlenmeyer flasks each containing 50 mL of SV2 seed medium and incubated at 28°C for 4 days with shaking at 200 rpm.

[0125] A volume of 2.5 mL of the homogenized seed culture was then inoculated into 250 mL Erlenmeyer flasks containing 50 mL of fermentation medium CA07LB. All cultures were fermented for 10 days at 28°C with shaking at 200 rpm with a 50 mm shaking diameter.

[0126] For coculture fermentation with Rhodococcus sp. T5718 (Natural Organism Library, Agency for Science, Technology and Research), the inducer strain and seed culture of A100020 (FAS mutant strain) were inoculated into 50 mL of CA07LB at a volume ratio of 1:3 (0.8 mL:2.5 mL) with shaking at 200 rpm at 30°C for 7 days.

[0127] After the incubation period, the culture was harvested and freeze-dried. The freeze-dried culture was extracted with methanol overnight. The extracted mixture was passed through a cellulose filter (Whatman Grade 4, 1004-185), and the filtrate was dried using a rotary evaporator.

[0128] Mutant-to-mutant analysis The extract was analyzed on an Agilent 1290 Infinity LC System coupled to an Agilent 6540 accurate mass quadrupole time-of-flight (QTOF) mass spectrometer. Five microliters of the extract was analyzed on a Waters Acquity UPLC BEH C column. 18 The injection was performed on a 2.1 x 50 mm, 1.7 μm column. The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% formic acid. The analysis was performed using an 8-minute gradient elution from 2% B to 100% B at a flow rate of 0.5 mL / min. Both MS and MS / MS data were acquired in positive and / or negative electrospray ionization (ESI) mode. Typical QTOF operating parameters were as follows: sheath gas nitrogen, 12 L / min at 325 °C; drying gas nitrogen flow, 12 L / min at 350 °C; nebulizer pressure, 50 psi; nozzle voltage, 1.5 kV; capillary voltage, 4 kV. Positive ion mode lock masses were the purine ion at m / z 121.0509 and the HP-0921 ion at m / z 922.0098.

[0129] Data analysis Compounds were detected based on peaks identified in base peak chromatograms (BPCs) from LC-MS data. ESI (+ve / -ve) mass spectral data at the apex of the detected peaks were sampled and corrected for background noise by subtracting the nearest baseline mass spectrum. An in-house algorithm was developed and used to detect peaks from the BPC data and perform background correction for the peaks. The detected peaks were then characterized by four key parameters: (1) base peak m / z, (2) retention time, (3) molecular ion peak, and (4) the number of m / z peaks with abundances greater than 50,000. Peaks were considered identical if they met two criteria: (1) base peak m / z within 0.02 m / z and (2) retention time within 0.2 min.

[0130] Compound isolation The dried extracts from 1 L of fermentation were combined and partitioned with 240 mL of a 1:1:1 ratio of CHCl / MeOH / H0. The aqueous MeOH layer was washed with 80 mL of CHCl (×2) and dried under reduced pressure using a Buchi rotary evaporator. The dried crude extract (6 g) was resuspended by maceration in 20 mL of MeOH, sonicated for 5 minutes, and centrifuged to separate the insoluble and soluble fractions. The supernatant was transferred to a 50 mL round-bottom flask and dried using a Buchi rotary evaporator. The dried concentrated sample (427 mg) was dissolved in 2.5 mL of MeOH and centrifuged. The supernatant was then purified under the following conditions: solvent A: HO + 0.1% HCOOH, solvent B: MeCN + 0.1% HCOOH, flow rate: 30 mL / min, gradient conditions: 90:10 isocratic for 5 min, followed by 10% to 45% solvent B for 15 min, 45% to 75% solvent B for 38 min, 75% to 100% solvent B for 2 min, and finally 100% solvent B isocratic for 12 min. 18 Purification by reverse phase preparative HPLC gave 3.5 mg of compound 1 and 2.3 mg of compound 2.

[0131] General Chemistry Laboratory Procedures A JASCO P-2000 digital polarimeter was used for specific rotation measurements. NMR spectra were recorded on a Bruker DRX-400 NMR spectrometer equipped with a cryoprobe and a 5 mm BBI ( 1 H, G-COSY, multiplicity-edited G-HSQC and G-HMBC spectra) or BBO ( 13 C spectra) were collected using a probe head. 1 H NMR and 13 The C NMR chemical shifts are δ H 3.31 ppm and δ C The residual solvent peak of MeOH-d4 at 49.0 ppm was used as the reference. The LC / MS Purification System was an Agilent 1260 Infinity Preparative-Scale, and the LC / MS Purification System was an Agilent 6130B single quadrupole mass spectrometer. The LC / MS Purification System was an Agilent 5 Prep-C system. 18Preparative HPLC experiments were performed using a 100 x 30 mm, 5 μm, 100 Å column. HPLC-LCMS was performed using an Agilent UHPLC 1290 Infinity coupled to an Agilent 6540 accurate mass quadrupole time-of-flight (QTOF) mass spectrometer equipped with a splitter and ESI source. The HPLC-LCMS was performed using an Acquity UPLC BEH C under standard gradient conditions from 98% water with 0.1% formic acid to 100% acetonitrile with 0.1% formic acid over 8.6 minutes. 18 Analyses were performed using a 2.1 x 50 mm, 1.7 μm column at a flow rate of 0.5 mL / min. All chromatographic, specific rotation and UV solvents were Fisher Chemical HPLC or LCMS grade.

[0132] Chemical structure data Compound 1. Brown amorphous powder; [α] 23 D +50.8(c0.5, MeOH);UV(MeCN / H2O) λ max (%)220(100%), 285(77%)nm;(-)-HRESIMS:m / z 362.1968[MH] - (C 20 H 28 Calculated O5N value, 362.1973); 1 H NMR and 13 C NMR data, see Table 1.

[0133] Compound 2: Brown amorphous powder; [α] 23 D +28(c0.2, MeOH);UV(MeCN / H2O) λmax(%)222(100%), 286(83%)nm;(-)-HRESIMS:m / z 376.2132[MH] - (C 20 H 30 calculated O5N value, 376.2129); 1 H NMR and 13 C NMR data, see Table 1.

[0134] Biological assays The minimum inhibitory concentration (MIC) and minimum bactericidal / fungicidal concentration (MBC / MFC) of isolated compounds against a panel of microbial pathogens were determined using the microbroth dilution method, which follows Clinical Laboratory Standards Institute (CLSI) guidelines with minor modifications. Antibacterial assays were performed using Acinetobacter baumannii (ATCC 19606), Klebsiella aerogenes (ATCC 13048), Pseudomonas aeruginosa (ATCC 9027), and Staphylococcus aureus Rosenbach (ATCC 25923) at 5 × 10 5 Antifungal assays were performed using Aspergillus fumigatus (ATCC™ 46645™) at 2.5 x 10 cells / mL. 4 The assay was performed at spores / mL. Standard inhibitors, gentamicin (Gibco) and amphotericin (Sigma-Aldrich), were used as assay controls for the antibacterial and antifungal assays, respectively. For MIC determination, bacterial cells were incubated with the isolated compounds for 24 hours at 37°C, and for fungal spores, they were incubated for 72 hours at 25°C. The optical density at 600 nm was then measured using a microplate reader (Tecan Infinite™ M1000 Pro) to assess the inhibitory effect of the compounds on microbial growth. Five microliters of the treated culture was then transferred to fresh medium in a 384-well microtiter plate, and the MBC / MFC was subsequently evaluated. The plates were incubated under the same conditions, and the MBC / MFC was determined by measuring the optical density at 600 nm. All assays were performed in triplicate to ensure reproducibility.

[0135] For mammalian cell cytotoxicity assays, A549 human lung carcinoma cells (ATCC™ CCL-185™) were cultured at 3.3 × 10 4Cells were seeded at 1000 cells / mL into 384-well microplates. Cells were treated with compounds for 72 hours and incubated at 37°C in the presence of 5% CO2. Puromycin (Sigma-Aldrich), a standard inhibitor, was used as an assay control for cytotoxicity tests. To assess the cytotoxic effect of compounds on cells, the microplates were incubated with PrestoBlue™ cell viability reagent (ThermoFisher Scientific, USA) for 2 hours, and then fluorescence was read at excitation 560 nm and emission 590 nm. IC for antimicrobial and cytotoxic activity 50 Analysis of the values ​​was performed with the GraphPad Prism program (GraphPad Software, CA).

[0136] It will be understood that many further modifications and permutations of various aspects of the described embodiments are possible, and accordingly, the described aspects are intended to embrace all such alterations, modifications and variations that fall within the spirit and scope of the appended claims.

[0137] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0138] Throughout this specification and the appended claims, unless the context requires otherwise, the phrase "consisting essentially of," and variations such as "consists essentially of," are understood to indicate that the recited element(s) are essential or required elements of the invention. This phrase permits the presence of other unrecited elements that do not materially affect the characteristics of the invention, but excludes additional unspecified elements that affect the basic and novel characteristics of the method being defined.

[0139] The reference in this specification to any prior publication (or information derived therefrom) or any known matter is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

Claims

1. Equation (I): 【Chemistry 1】 A method for biosynthesizing an antimicrobial compound of (wherein R is an optionally substituted alkyl group), a) A step of culturing microbial cells that have been manipulated to overexpress fatty acid acyl-CoA synthase (FAS) or RedD, b) A step of isolating the antimicrobial compound of formula (I) produced by the microbial cells, Includes, A method for upregulating the biosynthesis of the antimicrobial compound of formula (I) by at least twofold compared to a tetramic acid analog derived from wild-type microbial cells.

2. The microbial cells are manipulated by introducing a FAS expression cassette or a RedD expression cassette into the genome of the microbial cells, or The method according to claim 1, wherein the FAS expression cassette or RedD expression cassette is incorporated into the genome of the microbial cell by an embedded plasmid.

3. The method according to claim 1 or 2, wherein the microbial cell is a bacterium.

4. The method according to claim 1, further comprising the step of inoculating the manipulated microbial cells into a seed medium before the culture step (step a).

5. The method according to claim 4, wherein the manipulated microbial cells are inoculated for at least four days.

6. The method according to claim 1, wherein the manipulated microbial cells are cultured in a medium containing glycerol and / or starch.

7. The method according to claim 1, wherein the manipulated microbial cells are cultured for at least 7 days.

8. The method according to claim 1, wherein the manipulated microbial cells are co-cultured with an inducer strain.

9. The method according to claim 7 or 8, wherein the volume ratio of the inducer strain to the manipulated microbial cells is 1:2 to 1:

5.

10. The method according to claim 1, further comprising the step of freeze-drying the culture before the isolation step (step b).

11. The method according to claim 1, wherein the antimicrobial compound of formula (I) is isolated from a microbial cell culture medium.

12. The antimicrobial compound of formula (I) is a) Minimum inhibitory concentration (MIC 50) for Gram-positive bacteria less than 20 μM, b) Minimum lethal concentration for Gram-positive bacteria less than 80 μM (MBC 50), c) Minimum inhibitory concentration (MIC 50) for Gram-negative bacteria less than 20 μM, and d) IC50 for cancer cells less than 50 μM The method according to claim 1, characterized by at least one of the following:

13. The method according to claim 12, wherein the Gram-negative bacterium is A. baumannii.

14. Equation (I): 【Chemistry 2】 An antimicrobial compound of the form (wherein R is an optionally substituted alkyl group).

15. The antimicrobial compound of formula (I) is formula (Ia): 【Transformation 3】 The antimicrobial compound according to claim 14, which is a compound of (wherein R is an optionally substituted alkyl group). 【Request Item 16】 【Chemistry 4】 An antimicrobial compound selected from, according to claim 14 or 15.

17. A pharmaceutical composition comprising the compound of formula (I) described in claim 14, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

18. A pharmaceutical product comprising the compound of formula (I) described in claim 14.

19. A pharmaceutical composition comprising the compound of (I) according to claim 14, used for the treatment of a microbial disease or pathological condition.

20. Use of an antimicrobial compound of formula (I) according to claim 14 or 15, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in the manufacture of a pharmaceutical for treating a microbial disease or condition.

21. The pharmaceutical composition according to claim 19, wherein the microbial disease or condition is selected from diseases or conditions caused by S. aureus and / or A. baumannii.

22. The pharmaceutical composition according to claim 19, wherein the microbial disease or condition is selected from skin and soft tissue infections, blood infections, pneumonia, bone and joint infections, urinary tract infections, pneumonia, or infections due to open wounds.

23. The use according to claim 20, wherein the microbial disease or condition is selected from diseases or conditions caused by S. aureus and / or A. baumannii.

24. The use according to claim 20, wherein the microbial disease or condition is selected from skin and soft tissue infections, blood infections, pneumonia, bone and joint infections, urinary tract infections, pneumonia, or infections due to open wounds.

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